Boronic acid compounds, compositions, and methods

Abstract

Compositions and methods comprising: 1) a compound having one or more substituted or unsubstituted boronic acid moieties (boronic acid compounds); and 2) one or more sulfur-containing compounds. Preferred compositions can exhibit reduced degradation over extended storage periods.

Description

Cross-reference of related applications

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 165,479, filed on 24 March 2021, the contents of which are incorporated in their entirety by reference. [Background technology]

[0002] Various boronic acid compounds, including bortezomib and other boronic acid-containing pharmaceuticals, exhibit significant degradation after synthesis, presenting clear challenges for their use. See, for example, Patent Document 1 (incorporated by reference).

[0003] A specific problem with boronic acid compounds was oxidative instability. At physiological pH, various boronic acid compounds can be oxidized by reactive oxygen species. As a result, bortezomib (Velcade®) is supplied as a lyophilized powder that needs to be reconstituted before administration, and the resulting solution is only sufficiently stable for about 8 hours. [Prior art documents] [Patent Documents]

[0004] [Patent Document 1] U.S. Patent No. 9,180,093 [Overview of the project] [Means for solving the problem]

[0005] The inventors have discovered a novel composition comprising a compound (boronic acid compound) having one or more substituted or unsubstituted boronic acid moieties that can exhibit enhanced stability over a long period of time.

[0006] More specifically, the inventors have found that boronic acid compounds, when mixed with sulfur-containing compounds, exhibit significantly reduced degradation over long storage periods. For example, see the comparative results shown in the following examples.

[0007] The inventors have also found that when a sulfur-containing compound is included in the reaction process of a boronic acid compound, the synthesis of the boronic acid compound can be carried out in higher yield and / or with increased purity.

[0008] The present composition and method are particularly useful for use with therapeutic boronic acid compounds. In one embodiment, a pharmaceutical composition is provided comprising a) a therapeutic boronic acid compound; and b) one or more sulfur-containing compounds.

[0009] A variety of sulfur-containing compounds can be used, including compounds containing one or more sulfides, sulfoxides, sulfites (or sulfites), sulfoxylates, or other sulfur-containing moieties.

[0010] In certain embodiments, suitable and preferred sulfur-containing compounds include one or more sulfide groups (e.g., (CH2) n -SR; where R is hydrogen or C 1-6 It may contain an alkyl group where n is 1 to 6. In other embodiments, a sulfur-containing compound is preferred that contains one or more sulfite groups (e.g., a sulfite salt (e.g., a sodium salt), a bisulfite salt (e.g., an acetone or sodium salt), a metabisulfite salt (e.g., a sodium salt), a metabisulfite salt (e.g., a sodium or potassium salt), or a hydrosulfite salt (e.g., a sodium salt)), one or more formaldehyde sulfoxylate groups and / or one or more thiourea groups.

[0011] In certain embodiments, sulfur-containing compounds having molecular weights of about 2000 daltons or less, or 1500 daltons or less, or 1000, 900, 800, 700, 600, 500, 400, 300, 250, or 200 daltons or less are preferred. In certain embodiments, sulfur-containing compounds having molecular weights from about 150 daltons to about 200, 300, 400, 500, or 1000 daltons are preferred. In certain embodiments, one or more nonpolymeric sulfur-containing compounds may be used. In other embodiments, polymeric sulfur-containing compounds may be used.

[0012] In certain embodiments, preferred sulfur-containing compounds include those designated as generally recognized as safe (GRAS) by the U.S. Food and Drug Administration, such as N-acetylmethionine and / or L-glutathione.

[0013] In certain embodiments, the composition and method may further contain one or more sulfur-containing compounds in combination with one or more stabilizer compounds that do not contain sulfides or other sulfur-functional groups. For example, in a particular preferred system, the composition comprises a) a therapeutic boronic acid compound; b) one or more sulfur-containing compounds; and c) one or more sulfur-free stabilizer compounds (e.g., 5-dihydroxybenzoic acid or a salt thereof, ascorbic acid or a salt thereof, and / or histidine).

[0014] A wide variety of boronic acid compounds can be used in this composition and method. As referred to herein, boronic acid compounds or other similar terms include: 1) compounds having one or more boronic acid moieties having an unsubstituted OH group (e.g., a moiety of formula RB(OH)2; where R is a non-hydrogen substituent, such as an optionally substituted alkyl or an optionally substituted carbocyclic aryl); 2) one or more substituted boronic acid moieties (e.g., one or more boronic acid ester moieties, e.g., formula RB(OR 1 )(OR 2 ) part; in the formula, R, R 1 and R 2 Each of these may be a non-hydrogen substituent such as an alkyl group which may be optionally substituted, or a carbocyclic aryl group which may be optionally substituted, and R 1 and R 2 This includes compounds having (one of which may be hydrogen); references to boronic acid compounds herein include boronic acid salts.

[0015] In certain preferred embodiments, the boronic acid compound comprises one or more boronic acid moieties having an unsubstituted -OH group, such as a moiety of formula RB(OH)2 (wherein R is a non-hydrogen substituent).

[0016] As described above, in certain embodiments, the boronic acid compound may be therapeutically active, as proposed or confirmed by in vitro or in vivo results, for the treatment of any of the broad range of diseases and disorders, including, for example, cancer, bacterial infections, viral infections, and other diseases or disorders. Therapeutically active boronic acid compounds may also be used for diagnostic purposes, including imaging.

[0017] In a preferred embodiment, a method is provided for treating a subject suffering from a disease or disorder related to the expression of fibroblast-activating protein (FAP), the method comprising administering a pharmaceutical composition disclosed herein to the subject.

[0018] In certain preferred embodiments, the therapeutic boronic acid compound may comprise one or more carbocyclic groups or heteroaromatic groups.

[0019] In certain preferred embodiments, the therapeutic boronic acid compound may be a proteasome inhibitor.

[0020] In certain embodiments, the therapeutic boronic acid compound may be bortezomib, ixazomib, or vaborbactam.

[0021] In certain embodiments, therapeutic boronic acid compounds may be radiopharmaceutical compounds used for diagnostic purposes or in the treatment of diseases or disorders.

[0022] This composition may be in various forms, including fluid and solid compositions. In certain embodiments, the composition may be an aqueous formulation, including those adapted for injection or other parenteral administration. In other embodiments, the composition may be in solid form, including those adapted for oral administration.

[0023] In preferred embodiments, the composition (including pharmaceutical compositions) exhibits stability over a long period after preparation. For example, for certain preferred aqueous compositions comprising 1) a boronic acid compound and 2) one or more sulfur compounds, the boronic acid compound exhibits radiochemical purity of at least or up to 90%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% for 3, 4, 5, 6, 7, 8, 9, 10, 12, or 14 days or longer after preparation of the composition. The radiochemical purity of the boronic acid compound can preferably be determined by chromatography, such as HPLC.

[0024] Preferably, one or more sulfur compounds are present in the composition in a stabilizing effective amount. The preferred amount of one or more sulfur compounds can vary widely, but may be at least 0.01N, or 0.5N, 0.1N, 0.2N, 0.3N, 0.4N, 0.5N or more. The mass ratio (weight:weight ratio) of 1) one or more sulfur compounds to 2) one or more boronic acid compounds in the composition can vary widely, but may preferably be, for example, 1:10 to 10:1.

[0025] The appropriate and preferred amount of boronic acid compounds in a composition (including pharmaceutical compositions) can vary widely. For example, in certain embodiments, one or more boronic acid compounds may be present in amounts of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.2, 1.4, 1.6, 1.8, 2.0, 2.5, or 3.0 mg or more per 1 mL of a fluid (e.g., aqueous) composition. The appropriate and preferred amount of boronic acid compounds in a composition is also described in the following examples.

[0026] In a further embodiment, a method for synthesizing a boronic acid compound is provided, which appropriately comprises reacting a boronic acid precursor compound in the presence of one or more sulfur compounds to provide a boronic acid compound. The precursor compound may be a reagent compound that produces the boronic acid compound.

[0027] In another embodiment, a method is provided for preparing a composition containing a boronic acid compound (including a pharmaceutical composition containing a therapeutic boronic acid compound). In one embodiment, such a method comprises 1) mixing a boronic acid compound (which may be a therapeutic boronic acid compound) and 2) one or more sulfur compounds. In certain embodiments, one or more stabilizer compounds that do not contain sulfur functionality may also be mixed with the boronic acid compound and one or more sulfur compounds. The multiple compounds may be adequately mixed in aqueous and / or one or more organic solvents, but in certain embodiments, an aqueous formulation is preferred.

[0028] In certain embodiments, the boronic acid compound may be associated with a sulfur compound before mixing. For example, the boronic acid compound may be synthesized in the presence of one or more sulfur compounds, which are present together with the boronic acid compound in the reaction mixture. This reaction mixture may then be mixed with one or more sulfur compounds, which may be the same as or different from the sulfur compounds present in the reaction mixture.

[0029] Alternatively, the boronic acid compound may not contain any or substantially any sulfur compounds before being mixed with one or more sulfur compounds.

[0030] In a further embodiment, a packaged preparation or kit containing a boronic acid compound (including a therapeutic boronic acid compound) is provided. The packaged preparation or kit may include 1) the boronic acid compound, 2) one or more sulfur compounds, and optionally, 3) instructions for use of the boronic acid compound, including for diseases or disorders such as cancer, bacterial infections, or viral infections. Preferably, the packaged preparation or kit contains a therapeutically effective amount of the therapeutic boronic acid compound. The instructions may be in the form of a written document, preferably including a packaging label.

[0031] Other aspects of the present invention are disclosed below. [Brief explanation of the drawing]

[0032] [Figure 1] The tumor growth curve for 177Lu-6522 is shown. [Figure 2] The survival curve for 177Lu-6522 is shown. [Figure 3] This shows the accumulation and retention of 68Ga-6555 over time after administration. [Figure 4] This study shows the stabilizing effects of N-acetyl-L-methionine (NAM), sodium ascorbate (SA), and L-methionine as a function of the remaining percentage (%) of [177Lu]-6555. [Modes for carrying out the invention]

[0033] The following definitions provide definitions for some of the terms that are frequently used herein. These terms have their defined and preferred meanings in each case of their use in the remainder of this specification. As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural references unless the content expressly indicates otherwise. The following definitions are provided for the terms alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, aralkyl, heteroaryl, heteroaralkyl, alkenyl, and alkynyl. These terms have their defined and preferred meanings in each case of their use in the remainder of this specification.

[0034] As used herein, the term "SPECT" is an abbreviation for single-photon emission computed tomography.

[0035] As used herein, the term "PET" is an abbreviation for positron emission tomography.

[0036] As used herein, the term "CT" is an abbreviation for computed tomography.

[0037] As used herein, the term "MRI" is an abbreviation for magnetic resonance imaging.

[0038] As used herein, the term "SIRT" is an abbreviation for selective internal radiation therapy.

[0039] As used herein, the term "EDTA" is an abbreviation for ethylenediaminetetraacetic acid.

[0040] As used herein, the term "DOTA" is an abbreviation for 1,4,7,10-tetraazacyclododecane-1,4,7,10-N,N',N'',N'''-tetraacetic acid.

[0041] As used herein, the term "DOTAGA" is an abbreviation for 1,4,7,10-tetraazacyclododecane,1-(glutaric acid)-4,7,10-triacetic acid.

[0042] As used herein, the term "DTPA" is an abbreviation for diethylenetriaminepentaacetic acid.

[0043] As used herein, the terms metal “chelating agent” or “chelator” refer to a polydentate ligand that forms two or more distinct coordination bonds with a single central atom (in particular, a radioisotope).

[0044] As used herein, the term “therapeutic effective dose” includes, within its meaning, a non-toxic but sufficient amount of the compound or composition used in the present invention to provide the desired therapeutic effect. The exact amount required varies from subject to subject depending on factors such as the species being treated, the age, weight and general condition of the subject, comorbidities, the severity of the condition being treated, the specific drug administered, and the method of administration. Therefore, in any given case, a suitable “effective dose” can be determined by a person skilled in the art using only routine methods.

[0045] The term "alkyl" refers to a saturated linear or branched carbon chain. Preferably, the chain contains 1 to 10 carbon atoms, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms, such as methyl, ethyl, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, hexyl, pentyl, or octyl. The alkyl group may be substituted.

[0046] The term "heteroalkyl" refers to a saturated linear or branched carbon chain. Preferably, the chain contains 1 to 9 carbon atoms, i.e., 1, 2, 3, 4, 5, 6, 7, 8, or 9 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, hexyl, pentyl, octyl, etc., which are interrupted one or more times by the same or different heteroatoms, for example, 1, 2, 3, 4, or 5 times. Preferably, the heteroatom is selected from O, S, and N, and is, for example, -O-CH3, -S-CH3, -CH2-O-CH3, -CH2-O-CH2-CH3, -CH2-S-CH3, -CH2-S-CH2-CH3, -CH2-,CH2-O-CH3, -CH2-CH2-O-CH2-CH3, -CH2-CH2-S-CH3, -CH2-CH2-S-CH2-CH3, etc. The heteroalkyl group may not be substituted.

[0047] The terms “cycloalkyl” and “heterocycloalkyl,” when used alone or in combination with other terms, represent the cyclic versions of “alkyl” and “heteroalkyl,” respectively, unless otherwise specified, preferably with 3, 4, 5, 6, 7, 8, 9, or 10 atoms forming the ring, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, etc. The terms “cycloalkyl” and “heterocycloalkyl” also mean that their bicyclic, tricyclic, and polycyclic versions are included. The term “heterocycloalkyl” preferably means a 5-membered saturated ring (at least one member being an N, O, or S atom, and possibly containing one further O or one further N); a 6-membered saturated ring (at least one member being an N, O, or S atom, and possibly containing one further O or one further N or two further N atoms); or a 9-membered or 10-membered saturated bicyclic ring (at least one member being an N, O, or S atom, and possibly containing one further N atom, two further N or three further N atoms). The "cycloalkyl" and "heterocycloalkyl" groups may be substituted. Furthermore, in the case of heterocycloalkyl groups, the heteroatom can occupy a position where the heterocycle is bonded to the rest of the molecule. Examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, spiro[3,3]heptyl, spiro[3,4]octyl, spiro[4,3]octyl, spiro[3,5]nonyl, spiro[5,3]nonyl, spiro[3,6]decyl, spiro[6,3]decyl, spiro[4,5]decyl, spiro[5,4]decyl, bicyclo[2.2.1]heptyl, bicyclo[2.2.2]octyl, and adamantyl.Examples of heterocycloalkyls include 1-(1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-morpholinyl, 3-morpholinyl, 1,8-diazo-spiro-[4,5]decyl, 1,7-diazo-spiro-[4,5]decyl, 1,6-diazo-spiro-[4,5]decyl, 2,8-diazo-spiro[4,5]decyl, 2,7-diazo-spiro[4,5]decyl, 2,6-diazo-spiro[4,5]decyl, 1,8-diazo-spiro-[5,4]decyl, 1,7-diazo-spirotetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl, 1-piperazinyl, and 2-piperazinyl.

[0048] The term "aryl" preferably refers to an aromatic monocyclic ring containing six carbon atoms, an aromatic bicyclic ring system containing ten carbon atoms, or an aromatic tricyclic ring system containing fourteen carbon atoms. Examples include phenyl, naphthyl, or anthracenyl. The aryl group may be substituted.

[0049] The term "aralkyl" refers to the alkyl moiety substituted with an aryl group, where alkyl and aryl have the meanings outlined above. An example is the benzyl radical. Preferably, in this context, the alkyl chain contains 1 to 8 carbon atoms, i.e., 1, 2, 3, 4, 5, 6, 7, or 8 carbon atoms, such as methyl, ethylmethyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butenyl, tert-butyl, pentyl, hexyl, pentyl, and octyl. The aralkyl group may be substituted in the alkyl and / or aryl moieties of the group.

[0050] The term "heteroaryl" preferably means a 5 or 6-membered aromatic monocyclic ring in which at least one carbon atom is substituted by one, two, three or four (in the case of a 5-membered ring) or one, two, three, four or five (in the case of a 6-membered ring) heteroatoms, preferably selected from O, N, and S; an aromatic bicyclic ring system in which one, two, three, four, five, or six carbon atoms out of eight, nine, ten, eleven, or twelve carbon atoms are substituted by one or different heteroatoms, preferably selected from O, N, and S; or an aromatic tricyclic ring system in which one, two, three, four, five, or six carbon atoms out of thirteen, fourteen, fifteen, or sixteen carbon atoms are substituted by one or different heteroatoms, preferably selected from O, N, and S. Examples include oxazolyl, isoxazolyl, 1,2,5-oxadiazolyl, 1,2,3-oxadiazolyl, pyrrolyl, imidazolyl, pyrazolyl, 1,2,3-triazolyl, thiazolyl, isothiazolyl, 1,2,3-thiadiazolyl, 1,2,5-thiadiazolyl, pyridinyl, pyrimidinyl, pyrazinyl, 1,2,3-triazinyl, 1,2,4-triazinyl, 1,3,5-triazinyl, 1-benzofuranyl, 2-benzofuranyl, indoyl, isoindoyl These include isoindoyl, benzothiophenyl, 2-benzothiophenyl, 1H-indazolyl, benzimidazolyl, benzoxazolyl, indoxazinyl, 2,1-benzoxazolyl, benzothiazolyl, 1,2-benzoisothiazolyl, 2,1-benzoisothiazolyl, benzotriazolyl, quinolinyl, isoquinolinyl, quinoxalinyl, quinazolinyl, quinolinyl, 1,2,3-benzotriazinyl, or 1,2,4-benzotriazinyl.

[0051] The term "heteroaralkyl" refers to the alkyl portion substituted with a heteroaryl group, where alkyl and heteroaryl have the meanings outlined above. Examples include 2-alkylpyridinyl, 3-alkylpyridinyl, or 2-methylpyridinyl. Preferably, in this context, the alkyl chain contains 1 to 8 carbon atoms, i.e., 1, 2, 3, 4, 5, 6, 7, or 8 carbon atoms, such as methyl, ethylmethyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butenyl, tert-butyl, pentyl, hexyl, pentyl, and octyl.

[0052] The heteroaralkyl group may be substituted in the alkyl and / or heteroaryl portion of the group.

[0053] The terms "alkenyl" and "cycloalkenyl" refer to olefin unsaturated carbon atoms comprising a chain or ring having one or more double bonds. Examples include propenyl and cyclohexenyl. Preferably, the alkenyl chain contains 2 to 8 carbon atoms, i.e., 2, 3, 4, 5, 6, 7, or 8 carbon atoms, such as ethenyl, 1-propenyl, 2-propenyl, isopropenyl, 1-butenyl, 2-butenyl, 3-butenyl, isobutenyl, sec-butenyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, hexenyl, pentenyl, and octenyl. Preferably, the cycloalkenyl ring contains 3 to 8 carbon atoms, i.e., 3, 4, 5, 6, 7, or 8 carbon atoms, such as 1-cyclopropenyl, 2-cyclopropenyl, 1-cyclobutenyl, 2-cyclobutenyl, 1-cyclopentenyl, 2-cyclopentenyl, 3-cyclopentenyl, cyclohexenyl, cyclopentenyl, and cyclooctenyl.

[0054] The term "alkynyl" refers to an unsaturated carbon atom comprising a chain or ring having one or more triple bonds. An example is the propargyl radical. Preferably, the alkynyl chain contains 2 to 8 carbon atoms, i.e., 2, 3, 4, 5, 6, 7, or 8 carbon atoms, such as ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, 3-butynyl, 1-pentynyl, 2-pentynyl, 3-pentynyl, 4-pentynyl, hexynyl, pentynyl, or octinyl.

[0055] In one embodiment, carbon atoms or hydrogen atoms in alkyl, heteroalkyl, cycloalkyl, aryl, aralkyl, alkenyl, cycloalkenyl, and alkynyl radicals may be independently substituted with one or more elements selected from the group consisting of O, S, and N, or they may be substituted with groups containing one or more elements selected from the group consisting of O, S, and N.

[0056] The embodiments include alkoxy, cycloalkoxy, aryloxy, aralkoxy, alkenyloxy, cycloalkenyloxy, alkynyloxy, alkylthio, cycloalkylthio, arylthio, aralkylthio, alkenylthio, cycloalkenylthio, alkynylthio, alkylamino, cycloalkylamino, arylamino, aralkylamino, alkenylamino, cycloalkenylamino, and alkynylamino radicals.

[0057] Other embodiments include hydroxyalkyl, hydroxycycloalkyl, hydroxyaryl, hydroxyaralkyl, hydroxyalkenyl, hydroxycycloalkenyl, hydroxyalinyl, mercaptoalkyl, mercaptocycloalkyl, mercaptoaryl, mercaptoaralkyl, mercaptoalkenyl, mercaptocycloalkenyl, mercaptoalkynyl, aminoalkyl, aminocycloalkyl, aminoaryl, aminoaralkyl, aminoalkenyl, aminocycloalkenyl, and aminoalkynyl radicals.

[0058] In another embodiment, the hydrogen atoms in the alkyl, heteroalkyl, cycloalkyl, aryl, aralkyl, alkenyl, cycloalkenyl, and alkynyl radicals may be independently substituted by one or more halogen atoms. One of these radicals is a trifluoromethyl radical.

[0059] When two or more radicals or two or more residues can be selected independently of each other, the term "independently" means that the radicals or residues may be the same or different.

[0060] As used herein, a phrase defining a length range limitation, such as "1 to 6," means any integer from 1 to 6, i.e., 1, 2, 3, 4, 5, and 6. In other words, any range defined by two explicitly stated integers means that any integer defining the limitation and any integer included in the range are disclosed.

[0061] As used herein, the term "halo" refers to a halogen residue selected from the group consisting of F, Br, I, and Cl. Preferably, the halogen is F.

[0062] As used herein, the term “linker” means any chemically suitable linker. Preferably, the linker is not cleaved at all or is only slowly cleaved under physiological conditions. Therefore, preferably, the linker does not contain recognition sequences for proteases or recognition structures for other degrading enzymes. Since the compounds of the present invention are preferably administered systemically, allowing extensive access to all compartments of the body, and subsequently concentrated wherever tumors are located in the body, it is preferable that the linker is selected so as not to be cleaved at all or only slowly in the blood. Cleavage is considered slow if less than 50% of the linker is cleaved 2 hours after administration of the compound to a human patient. A suitable linker may include, for example, substituted alkyl, heteroalkyl, cycloalkyl, cycloheteroalkyl, aryl, heteroaryl, aralkyl, heteroaralkyl, alkenyl, heteroalkenyl, cycloalkenyl, cycloheteroalkenyl, alkynyl, sulfonyl, amines, ethers, thioethers, phosphines, phosphoramidates, carboxamides, esters, imide esters, amidines, thioesters, sulfonamides, 3-thiopyrrolidine-2,5-dione, carbamates, ureas, guanidines, thioureas, disulfides, oximes, hydrazines, hydrazides, hydrazones, diaza bonds, triazoles, triazolines, tetrazines, platinum complexes, and amino acids or combinations thereof. Preferably, the linker includes or consists of 1,4-piperazine, 1,3-propane, and phenol ethers or combinations thereof.

[0063] The expression "optionally substituted" refers to a group in which one, two, three, or more hydrogen atoms may be independently replaced by each substituent.

[0064] As used herein, the term "amino acid" refers to any organic acid containing one or more amino substituents, for example, an α-, β- or γ-amino derivative of an aliphatic carboxylic acid.

[0065] The term "conventional amino acid" refers to the 20 naturally occurring amino acids, and includes all stereoisomers thereof, namely D-, L-, D- and L-amino acids.

[0066] As used herein, the term "N-containing aromatic or non-aromatic monocyclic or bicyclic heterocycle" refers to a cyclic saturated or unsaturated hydrocarbon compound containing at least one nitrogen atom as a constituent of the cyclic chain.

[0067] As used herein, the term "radioactive moiety" refers to a molecular assembly that carries a radionuclide. The nuclide is bound by either a covalent bond or a coordinate bond that remains stable under physiological conditions. Examples of radioactive moieties include [131I]-3-iodobenzoic acid or 68GaDOTA.

[0068] As used herein, "fluorescent isotope" emits electromagnetic radiation after excitation by electromagnetic radiation of a shorter wavelength.

[0069] As used herein, "radioisotope" or "radioactive isotope" is a radioactive isotope of an element (encompassed by the term "radionuclide") that emits α-, β-, β+ and γ-radiation. Exemplary radioactive isotopes are discussed below, for example, 18 F, 43 K, 44 Sc, 47 Sc, 51 Cr, 51 Mn, 52 Mn, 57 Co, 58 Co, 59 Fe, 64 Cu, 67 Cu, 67 Ga, 68 Ga, 71 Ge, 72 As, 72Se、 75 Br、 76 Br、 77 As、 77 Br、 81 Rb、 83 Sr、 86 Y、 88 Y、 89 Sr、 89 Zr、 90 Y、 97 Ru、 99m Tc、 100 Pd、 101m Rh、 103 Pd、 105 Rh、 109 Pd、 110 In、 111 Ag、 111 In、 113 In、 119 Sb 121 Sn、 123 I、 124 I、 125 I、 127 Cs、 128 Ba、 129 Cs、 131 Cs、 131 I、 139 La、 140 La、 142 Pr、 143 Pr、 149 Pm、 149 Tb、 151 Eu、 152 Tb、 153 Eu、 153 Sm、 159 Gr、 Tb、 161 Tb、 165 Dy、 166 Dy、 166 Ho、 169 Eu、 175 Yb、 177 Lu、 186 Re、 188 Re、 189 Re、 191 Os、 193 Pt、 194 Ir、 197 Hg、 198 Au、 199 Ag、 199 Au、 201 Tl、 203 Pb、211 At, 212 Bi, 212 Pb, 213 Bi, 225 Ac, 226 Th, and 227 Th are included.

[0070] The term "radiopharmaceutical" is used in the context of the present invention to refer to a biologically active compound modified by a radioisotope. In particular, intercalating substances can be used to deliver radioactivity in direct proximity to DNA (for example, the Hoechst-33258 131 I-carrying derivative).

[0071] The terms "chelating agent" or "chelate" are used interchangeably in the context of the present invention to refer to a molecule, often an organic molecule, and often a Lewis base, having two or more unshared electron pairs available for donation to a metal ion. The metal ion usually coordinates to the chelating agent with two or more electron pairs. The terms "bidentate chelating agent", "tridentate chelating agent", and "tetradentate chelating agent" refer to chelating agents having, respectively, two, three, and four electron pairs readily available for simultaneous donation to the metal ion coordinated by the chelating agent. Usually, the electron pairs of a chelating agent form coordinate bonds with a single metal ion, but in certain instances, the chelating agent may form coordinate bonds with multiple metal ions, and various bonding modes are possible.

[0072] The term "fluorescent dye" (also referred to herein as "fluorescent moiety", "fluorophore" or "fluorochrome") is used in the context of the present invention to refer to a compound that, after excitation by electromagnetic radiation, emits, for example, visible light or infrared light of a suitable shorter wavelength. It is understood by those skilled in the art that each fluorescent dye has a predetermined excitation wavelength. All fluorescent moieties are encompassed by this term. The specific examples of fluorescent moieties described herein are illustrative and are not meant to limit the fluorescent moieties for use with the targeting molecules disclosed herein.

[0073] In the context of this invention, the term "contrast agent" is used to refer to a compound that enhances the contrast of a structure or fluid in medical imaging. Enhancement is achieved by absorbing electromagnetic radiation or altering the electromagnetic field.

[0074] As used herein, the term "paramagnetism" refers to paramagnetism induced by unpaired electrons in a medium. Paramagnetic materials induce a magnetic field when an external magnetic field is applied. Unlike diamagnetism, the direction of the induced magnetic field is the same as the external magnetic field, and unlike ferromagnetism, the magnetic field is not maintained in the absence of the external magnetic field.

[0075] As used herein, the term “nanoparticles” preferably refers to spherical particles having a diameter of 1 to 100 nanometers. Depending on the composition, nanoparticles may have evaluable magnetic, optical, or physicochemical properties. Furthermore, surface modification is achievable for many types of nanoparticles. The term “pharmaceutically acceptable salt” refers to a salt of the compound of the present invention. Suitable pharmaceutically acceptable salts of the compound of the present invention include, for example, acid addition salts that can be formed by mixing a solution of choline or a derivative thereof with a solution of a pharmaceutically acceptable acid such as hydrochloric acid, sulfuric acid, fumaric acid, maleic acid, succinic acid, acetic acid, benzoic acid, citric acid, tartaric acid, carbonic acid, or phosphoric acid. Furthermore, if the compound of the present invention has an acidic moiety, suitable pharmaceutically acceptable salts thereof include alkali metal salts (e.g., sodium or potassium salts); alkaline earth metal salts (e.g., calcium or magnesium salts); and salts formed with suitable organic ligands (e.g., ammonium, quaternary ammonium, and amine cations formed using counter anions such as halides, hydroxides, carboxylic acids, sulfuric acid, phosphoric acid, nitric acid, alkyl sulfonic acid, and aryl sulfonic acid).

[0076] Examples of pharmaceutically acceptable salts include, but are not limited to, acetates, adipines, alginates, ascorbicates, aspartates, benzenesulfons, benzoates, bicarbonates, bisulfates, tartrates, borates, bromides, butyrates, calcium edetate, camphorates, camphor sulfons, camsylates, carbonates, chlorides, citrates, clavulanates, cyclopentanepropionates, diglucons, dihydrochlorides, dodecyl sulfate, edetates. Edisylate, estolate, esylate, ethanesulfonate, formate, fumarate, gluceptate, glucoheptonate, gluconate, glutamate, glycerophosphate, glycolylarsanilate, hemisulfate, heptanoate, hexanoate, hexyl resorcinate e) Hydravamin, hydrobromide, hydrochloride, hydroiodide, 2-hydroxyethanesulfonic acid, hydroxynaphthoate, iodide, isothiocyanate, lactate, lactobionate, laurate, lauryl sulfate, malate, maleate, malonate, mandelate, mesylate, methanesulfonate, methyl sulfate, mucate, 2-naphthalenesulfonate, napsylate, nicotinate, nitrate, N-methylglucamine ammonium salt, oleate, Oxalates, pamoates (embonates), palmitates, pantothenates, pectinates, persulfates, 3-phenylpropionates, phosphates / diphosphates, picrinates, pivalates, polygalacturonates, propions, salicylates, stearates, sulfates, basic acetates, succinates, tannates, tartrates, theoclates, tosylates, triethiodides, undecanoates, valersates, etc. (e.g., Berge, SM)(See, et al., “Pharmaceutical Salts”, Journal of Pharmaceutical Science, 1977, 66, 1-19). Certain specific compounds of the present invention contain both basic and acidic functional groups that enable the compound to be converted to either a base-addition salt or an acid-addition salt.

[0077] The neutral form of a compound can be regenerated by contacting the salt with a base or acid and isolating the parent compound in the conventional manner. The parent form of a compound differs from the various salt forms in certain physical properties, such as solubility in polar solvents, but otherwise, the salt is equivalent to the parent form of the compound for the purposes of this invention.

[0078] In addition to salt forms, the present invention provides compounds in prodrug form. Prodrugs of the compounds described herein are compounds that readily undergo chemical changes under physiological conditions to provide the compound of formula (I). Prodrugs are active or inactive compounds that, after administration to a patient, are chemically modified into the compound of the present invention by in vivo physiological actions such as hydrolysis and metabolism. Furthermore, prodrugs can be converted into the compound of the present invention by chemical or biochemical methods in an ex vivo environment. For example, a prodrug can be slowly converted into the compound of the present invention when placed in a transdermal patch reservoir with a suitable enzyme. The aptitudes and techniques involved in the preparation and use of prodrugs are well known to those skilled in the art. For a general consideration of prodrugs including esters, see Svensson and Tunek Drug Metabolism Reviews 16.5 (1988) and Bundgaard Design of Prodrugs, Elsevier (1985).

[0079] Examples of masked carboxylate anions include various esters such as alkyl (e.g., methyl, ethyl), cycloalkyl (e.g., cyclohexyl), aralkyl (e.g., benzyl, p-methoxybenzyl), and alkylcarbonyloxyalkyl (e.g., pivaloyloxymethyl). Amines are masked as arylcarbonyloxymethyl substituted derivatives, which are cleaved by esterases in vivo to release the free drug and formaldehyde (Bungaard J. Med. Chem. 2503(1989)). In addition, drugs containing acidic NH groups such as imidazole, imide, and indole are masked with N-acyloxymethyl groups (Bundgaard Design of Prodrugs, Elsevier (1985)).

[0080] Hydroxyl groups are masked as esters and ethers. EP00039051 (Sloan and Little, Apr. 11, 1981) discloses Mannich base hydroxamic acid prodrugs, their preparation and use.

[0081] Certain compounds of the present invention may exist in unsolvated forms as well as solvated forms, including hydrated forms. In general, the solvated forms are equivalent to the unsolvated forms and are intended to be encompassed within the scope of the present invention.

[0082] Certain compounds of the present invention may exist in multiple crystalline or amorphous forms. In general, all physical forms are equivalent for the uses contemplated by the present invention and are intended to be within the scope of the present invention.

[0083] Certain compounds of the present invention possess asymmetric carbon atoms (optical centers) or double bonds; all racemates, diastereomers, geometric isomers and individual isomers are intended to be encompassed within the scope of the present invention.

[0084] The compounds of the present invention may also contain unnatural proportions of atomic isotopes at one or more of the atoms that constitute such compounds. For example, the compounds may be, for example, tritium ( 3 H), iodine-125 ( 125 I) or carbon-14 ( 14 C) radiolabeled with radioisotopes. All isotopic variations of the compounds of the present invention, whether radioactive or not, are intended to be encompassed within the scope of the present invention.

[0085] As used in the present application, the term "pharmaceutical composition" refers to a substance and / or a combination of substances used for the identification, prevention or treatment of a tissue condition or disease. The pharmaceutical composition is formulated to be suitable for administration to a patient for preventing and / or treating a disease. Furthermore, the pharmaceutical composition refers to the combination of an active agent with an inert or active carrier that renders the composition suitable for therapeutic use. The pharmaceutical composition may be formulated for oral, parenteral, topical, inhalative, rectal, sublingual, transdermal, subcutaneous or vaginal application routes according to its chemical and physical properties. Pharmaceutical compositions include solid, semi-solid, liquid, and transdermal therapeutic systems (TTS). The solid composition is selected from the group consisting of tablets, coated tablets, powders, granules, pellets, capsules, effervescent tablets or transdermal therapeutic systems. Also included are liquid compositions selected from the group consisting of solutions, syrups, infusions, extracts, solutions for intravenous administration, solutions for infusion, or solutions of the carrier systems of the present invention. Semi-solid compositions that can be used in the context of the present invention include emulsions, suspensions, creams, lotions, gels, globules, buccal tablets and suppositories.

[0086] "Pharmaceutically acceptable" means approved by a federal or state government regulatory agency, or listed in the United States Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.

[0087] As used herein, the term “carrier” refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic agent is administered. Such pharmaceutical carriers may be sterile liquids, such as saline solutions, and oils (of petroleum, animal, plant, or synthetic origin, including peanut oil, soybean oil, mineral oil, sesame oil, etc.). When a pharmaceutical composition is administered intravenously, physiological saline is a preferred carrier. Physiological saline, as well as aqueous dextrose and glycerol solutions, can also be used as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, wheat flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, and ethanol. If desired, the composition may also contain small amounts of wetting or emulsifying agents or pH buffers. Examples of suitable drug carriers are described in "Remington's Pharmaceutical Sciences" by E.W. Martin.

[0088] As used herein, the term “fibroblast-activating protein (FAP)” is also known as “seplase.” Both terms may be used interchangeably herein. Fibroblast-activating protein is a homodimer integral protein having dipeptidyl peptidase IV (DPPIV)-like folding, characterized by an α / β-hydrolase domain and an eight-bladed β-propeller domain.

[0089] "Medical imaging" means any technique used to visualize internal regions of the human or animal body for diagnostic, research, or therapeutic purposes. For example, boronic acid compounds can be detected (and quantified) by radioscintigraphy, magnetic resonance imaging (MRI), computed tomography (CT scan), nuclear imaging, positron emission tomography (PET) including metallic tomography contrast agents, optical imaging (such as fluorescence imaging including near-infrared fluorescence (NIRF) imaging), bioluminescence imaging, or a combination thereof. The functional portion is, optionally, a contrast agent for X-ray imaging. Agents useful for enhancing such techniques are materials that enable visualization of specific gene loci, organs, or disease sites within the body and / or result in some improvement in the quality of images produced by imaging techniques, providing improved or easier interpretation of those images. Such agents are referred to herein as contrast agents, and their use facilitates the distinction between different parts of an image by increasing the "contrast" between different regions of the image. Therefore, the term "contrast agent" encompasses agents used to improve the quality of images that can be produced even in the absence of such agents (for example, in the case of MRI), as well as agents that are a prerequisite for image production (for example, in the case of nuclear imaging).

[0090] Sulfur compounds As discussed, a variety of sulfur-containing compounds can be used, including compounds containing one or more sulfides, sulfoxides, sulfites, sulfoxylates, thiourea, or other sulfur-containing moieties.

[0091] In certain embodiments, suitable and preferred sulfur-containing compounds include one or more sulfide groups (e.g., -(CH2) n -SR; where R is hydrogen or C 1-6It may include an alkyl group where n is an integer from 0 to 6 or greater, typically 1 to 6). Particularly preferred sulfur compounds containing a sulfide functional group include, for example, methionine, N-acetylmethionine, and L-glutathione.

[0092] Furthermore, suitable and preferred sulfur-containing compounds may contain one or more sulfite groups (e.g., sulfites such as sodium sulfite). Suitable sulfite-containing sulfur compounds for use in this composition and method include bisulfites (e.g., sodium or other salts), metabisulfites (e.g., sodium or potassium or other salts), or hyposulfites (or hydrosulfites) (e.g., sodium or other salts).

[0093] Furthermore, suitable and preferred sulfur-containing compounds may also include other sulfur-functional compounds such as formaldehyde sulfoxylate compounds and thiourea compounds.

[0094] More specifically, preferred sulfur-containing compounds may include the following compounds, or compounds containing one or more of the following groups: Sulfides (thioethers), including alkyl sulfides, carbocyclic aryl sulfides, and other aromatic sulfides; Sulfoxides, including alkyl sulfoxides, carbocyclic aryl sulfoxides, and other aromatic sulfoxides; Sulfites containing sodium sulfite; Formaldehyde sulfoxylate compounds (hydroxymethyl sulfinates); Metabisulfites, including potassium metabisulfite and sodium metabisulfite; Bisulfite (or hydrogen sulfite); Thiourea; and / or Sodium hypochlorite, a type of sulfite.

[0095] Furthermore, preferred sulfur compounds for use in this composition and method exhibit desired solubility in aqueous formulations, thereby facilitating the use of aqueous compositions containing sulfur compounds for, for example, injection or other administration. For example, the sulfur compounds are preferably water-soluble at concentrations of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.2, 1.4, 1.6, 1.8, or 2 mg / mL or higher. Water solubility can be determined by visual inspection of the aqueous formulation in which no particles are detected.

[0096] Preferred sulfur compounds also include, in addition to the sulfur-containing group present in the compound, one or more halogens (F, Cl, Br, or I); haloalkyls, for example, haloC 1-12 Alkyl (where halo can be F, Cl, Br or I); amide; amine; alkylamine, e.g., RN(R) 1 )-R 2 (In the formula, R, R 1 and R 2 One, two, or each the same or different C 1-12 It is alkyl, R, R 1 and R 2 It may contain one or more polar moieties, such as one or two hydrogen atoms; keto; carboxy(-COOH); hydroxyl; and others. Such additional polar moieties can increase the water solubility of the sulfur compound.

[0097] Furthermore, certain sulfur compounds may be empirically evaluated as preferable for use in the present composition, method, and kit. Therefore, for example, using protocols such as those described in any of Examples 1 to 6 below, sulfur compounds miscible with boronic acid compounds in the composition may be evaluated for their desired stabilizing or degradation-reducing effect on boronic acid compounds compared to a control (i.e., a boronic acid compound under the same conditions but in the absence of the sulfur compound being evaluated).

[0098] In particular, certain sulfur compounds can be evaluated using boronic acid compounds by the procedure described in Example 1 below, which involves an aqueous admixture of the evaluated sulfur compound and the associated boronic acid compound maintained at 45°C. An evaluated sulfur compound can be considered suitable or preferred for the uses disclosed herein, for example, when the purity of the boronic acid compound is greater than 85%, more preferably 90%, 95% or greater than 95% in the evaluation after 4 days of the aqueous admixture. The purity of the boronic acid compound can be evaluated as described in Example 1 below or by other methods including HPLC.

[0099] As discussed, in certain embodiments, sulfur-containing compounds having a molecular weight of about 2000 Daltons or less, or 1500 Daltons or less, or 1000, 900, 800, 700, 600, 500, 400, 300, 250 or 200 Daltons or less are preferred. In certain embodiments, sulfur-containing compounds having a molecular weight from about 150 Daltons up to about 200, 300, 400, 500 or 1000 Daltons are preferred. In certain aspects, one or more non-polymeric sulfur-containing compounds can be utilized. In other aspects, polymeric sulfur-containing compounds can be used.

[0100] In certain embodiments, preferred sulfur-containing compounds include those designated as Generally Recognized As Safe (GRAS) by the U.S. Food and Drug Administration, such as N-acetylmethionine and / or L-glutathione.

[0101] As discussed, in certain embodiments, the present compositions and methods may further comprise one or more sulfur-containing compounds in combination with one or more stabilizer compounds that do not contain sulfide or other sulfur functionalities.

[0102] For example, in a particular preferred system, the composition may comprise: a) a boronic acid compound such as a therapeutic boronic acid compound; b) one or more sulfur-containing compounds; and c) one or more non-sulfur-functionalized stabilizer compounds, such as 5-dihydroxybenzoic acid or a salt thereof, ascorbic acid or a salt thereof (including sodium ascorbate), histidine, melatonin, ethanol, and / or Se-methionine.

[0103] In a further preferred embodiment, the composition may comprise: 1) a boronic acid compound such as a therapeutic boronic acid compound; 2) one or more sulfur compounds such as a sulfide compound and / or sulfite (or sulfite) compound (e.g., N-acetylmethionine and / or sodium metabisulfite); and 3) one or more sulfur-free stabilizer compounds, such as gentisic acid or ascorbic acid, or salts thereof (e.g., sodium ascorbate and sodium gentisic acid).

[0104] A particularly preferred mixture of a sulfur-containing compound and a non-sulfur-containing stabilizer is an aqueous formulation containing 5 mM N-acetylmethionine, 5 mM sodium metabisulfite, 5 mM sodium ascorbate, and 2 mM sodium gentisate at a suitable pH of 4.0 to 4.5.

[0105] A generally preferred stabilizer for use in this composition and method that does not contain sulfur atoms or sulfur-containing moieties may have a molecular weight of about 2000 daltons or less, or 1500 daltons or less, or 1000, 900, 800, 700, 600, 500, 400, 300, 250, or 200 daltons or less. In certain embodiments, sulfur-containing compounds having a molecular weight of about 150 daltons to about 200, 300, 400, 500, or 1000 daltons are preferred. In certain embodiments, preferred stabilizers that do not contain sulfur atoms or sulfur-containing moieties include stabilizers generally recognized as safe (GRAS) by the U.S. Food and Drug Administration, such as gentisic acid, sodium ascorbate, and sodium gentisic acid.

[0106] Sulfur compounds can be suitably used in a wide range of amounts in this composition. The optimal amount can be easily determined empirically, for example, by evaluating the desired stabilizing or degradation-reducing effect of the associated boronic acid compound using a protocol such as those described in any of Examples 1 to 6 below.

[0107] As discussed, in certain embodiments, a suitable amount of one or more sulfur compounds in the composition may be, for example, at least 0.01N, or 0.05N, 0.1N, 0.2N, 0.3N, 0.4N, 0.5N or more.

[0108] Generally, the mass ratio (weight:weight ratio) of one or more sulfur compounds in a composition (e.g., an aqueous composition) to one or more boronic acid compounds present in the composition is preferably 1:10 to 10:1. For solid compositions that may be used for oral administration, a relatively high amount of one or more sulfur compounds may be preferred.

[0109] Boronic acid compounds In one embodiment, preferred boronic acid compounds include bortezomib, ixazomib, and babolbactam, as well as their salts and formulations. The structures of these compounds are as follows: [ka]

[0110] Further preferred boronic acid compounds (including therapeutic boronic acid compounds) are compounds having the structure represented by formula I, or pharmaceutically acceptable salts thereof: [ka] [In the formula, R represents a radioactive moiety, chelating agent, fluorescent moiety, photoacoustic reporting molecule, Raman-active reporting molecule, contrast agent, detectable nanoparticles, or enzyme; R1 represents (C1~C6) alkyl; R2 is -B(-Y 1 )(-Y 2 ) or -CN represents; Y 1 and Y 2 These may independently represent an -OH group, or together with the boron atom to which they are bonded, they may represent a group hydrolyzable to boronic acid, or together with the boron atom to which they are bonded, they may form a 5- to 8-membered ring hydrolyzable to boronic acid; R3 represents H or (C1-C6) alkyl; R4 is either absent or each independently represents one, two, or three substituents selected from the group consisting of (C1-C6) alkyl, -OH, -NH2, and halogen; X represents either O or S; [L represents a joint or linker.]

[0111] In certain preferred embodiments, the compound of formula I comprises one or more radioactive isotopes.

[0112] In certain preferred embodiments, the compound of formula I comprises one or more therapeutic radioisotopes.

[0113] In certain preferred embodiments, the compound of formula I comprises one or more diagnostic radioisotopes.

[0114] In certain preferred embodiments, R is the radioactive portion.

[0115] In certain preferred embodiments, R is a chelating agent.

[0116] In a further preferred embodiment, R is a chelating agent, and the compound of formula I comprises one or more radioisotopes. In certain aspects of such embodiments, one or more radioisotopes may be therapeutic radioisotopes. In other specific aspects of such embodiments, one or more radioisotopes may be diagnostic radioisotopes.

[0117] In a further preferred embodiment, R is a chelating agent comprising one or more complexed radioactive isotopes. In certain aspects of such embodiments, one or more radioactive isotopes may be therapeutic radioactive isotopes. In other specific aspects of such embodiments, one or more radioactive isotopes may be diagnostic radioactive isotopes.

[0118] In certain preferred embodiments, R1 represents -CH3 or -CH2CH3, and more preferably -CH3.

[0119] In certain preferred embodiments, R2 is -B(-Y 1 )(-Y 2 It represents ), and more preferably represents -B(OH)2.

[0120] In certain preferred embodiments, R3 represents H.

[0121] In certain preferred embodiments, R4 is absent.

[0122] In certain preferred embodiments, X represents O.

[0123] In certain preferred embodiments, the compound is represented by the following formula II or III, or a pharmaceutically acceptable salt thereof: [ka] [ka] [In the formula, R and L are as defined above.]

[0124] In certain preferred embodiments, the compound of formula II or III comprises one or more radioactive isotopes.

[0125] In certain preferred embodiments, the compound of formula II or III comprises one or more therapeutic radioisotopes.

[0126] In certain preferred embodiments, the compound of formula II or III comprises one or more diagnostic radioisotopes.

[0127] In certain preferred embodiments of the compound of formula II or III, R is the radioactive moiety.

[0128] In certain preferred embodiments of the compound of formula II or III, R is a chelating agent.

[0129] In a further preferred embodiment of the compound of formula II or III, R is a chelating agent, and the compound of formula II or III contains a radioisotope. In certain aspects of such embodiments, one or more radioisotopes may be therapeutic radioisotopes. In other specific aspects of such embodiments, one or more radioisotopes may be diagnostic radioisotopes.

[0130] In a further preferred embodiment, R is a chelating agent comprising one or more complexed radioisotopes. In certain aspects of such embodiments, one or more radioisotopes may be therapeutic radioisotopes. In other specific aspects of such embodiments, one or more radioisotopes may be diagnostic radioisotopes.

[0131] In certain embodiments, the boronic acid compound may have a structure represented by formula IV, or a pharmaceutically acceptable salt thereof: [ka] [In the formula, R, R1, R2, R3, R4, X, and L are as defined above; n represents an integer between 2 and 6.

[0132] In certain preferred embodiments, the compound of formula IV comprises one or more radioisotopes.

[0133] In certain preferred embodiments, the compound of formula IV comprises one or more therapeutic radioisotopes.

[0134] In certain preferred embodiments, the compound of formula IV comprises one or more diagnostic radioisotopes.

[0135] In certain preferred embodiments of the compound of formula IV, R is the radioactive moiety.

[0136] In certain preferred embodiments of the compound of formula IV, R is a chelating agent.

[0137] In a further preferred embodiment of the compound of formula IV, R is a chelating agent, and the compound of formula IV contains a radioisotope. In certain aspects of such embodiments, one or more radionuclides may be therapeutic radioisotopes. In other specific aspects of such embodiments, one or more radioisotopes may be diagnostic radioisotopes.

[0138] In a further preferred embodiment, R is a chelating agent comprising one or more complexed radioisotopes. In certain aspects of such embodiments, one or more radioisotopes may be therapeutic radioisotopes. In other specific aspects of such embodiments, one or more radioisotopes may be diagnostic radioisotopes.

[0139] In certain embodiments, the boronic acid compound is having a structure represented by formula V, or a pharmaceutically acceptable salt thereof: [ka] [In the formula, R, R1, R2, R3, R4, X, and L are as defined above; R5 represents the part of the molecule that modifies its pharmacokinetics and / or biodistribution; n represents an integer between 1 and 6.

[0140] In certain preferred embodiments, the compound of formula V comprises one or more radioisotopes.

[0141] In certain preferred embodiments, the compound of formula V comprises one or more therapeutic radioisotopes.

[0142] In certain preferred embodiments, the compound of formula V comprises one or more diagnostic radioisotopes.

[0143] In certain preferred embodiments of the compound of formula V, R is the radioactive moiety.

[0144] In certain preferred embodiments of the compound of formula V, R is a chelating agent.

[0145] In a further preferred embodiment of the compound of formula V, R is a chelating agent, and the compound of formula V contains a radioisotope. In certain aspects of such embodiments, one or more radioisotopes may be therapeutic radioisotopes. In other specific aspects of such embodiments, one or more radioisotopes may be diagnostic radioisotopes.

[0146] In a further preferred embodiment, R is a chelating agent comprising one or more complexed radioisotopes. In certain aspects of such embodiments, one or more radionuclides may be therapeutic radioisotopes. In other specific aspects of such embodiments, one or more radionuclides may be diagnostic radioisotopes.

[0147] The boronic acid compounds used in this composition, method, or kit may contain radioisotopes to enable imaging and / or radiotherapy.

[0148] Suitable radioisotopes may include radioactive halogens, radioactive metals, or metalloid isotopes. Preferably, the radioisotopes are water-soluble metal cations and halogens.

[0149] Examples of radioactive isotopes include: 18 F, 43 K, 44 Sc, 47 Sc, 51 Cr, 51 Mn, 52 Mn, 57 Co, 58 Co, 59 Fe, 64 Cu, 67 Cu, 67 Ga, 68 Ga, 71 Enjoy, 72 As, 72 Se, 75 Br, 76 Br, 77 As, 77 Br, 81 Rb, 83 Sr, 86 Y,88 Y, 89 Sr, 89 Zr, 90 Y, 97 Ru, 99m Tc, 100 Pd, 101m Rh, 103 Pd, 105 Rh, 109 Pd, 110 In, 111 Ag, 111 In, 113 In, 119 Sb, 121 Sn, 123 I, 124 I, 125 I, 127 Cs, 128 Ba, 129 Cs, 131 Cs, 131 I, 139 La, 140 La, 142 Pr, 143 Pr, 149 Pm, 149 Tb, 151 Eu, 152 Tb, 153 Eu, 153 Sm, 159 Gr, 161 Tb, 165 Dy, 166 Dy, 166 Ho, 169 Eu, 175 Yb, 177 Lu, 186 Re, 188 Re, 189 Re, 191 Os, 193 Pt, 194 Ir, 197 Hg, 198 Au, 199 Ag, 199 Au, 201 Tl, 203 Pb, 211 At, 212 Bi, 212 Pb, 213 Bi, 225 Ac, 226 Th, and 227 Th are exemplified.

[0150] Diagnostic radioisotopes can be suitably used for diagnostic imaging, and in particular, 18 F, 64 Cu, 68 Ga, 89 Zr, 111 In, 123 I, 124 I, 99m Tc may be included. Therapeutic radioisotopes can be appropriately used in a variety of treatments, including treating cancer, for example, among others, 90 Y, 131 I, 161 Tb, 177 Lu, 211 At, 212 Pb, 225 Ac, 227 Th may be included.

[0151] In certain embodiments, radioisotopes are intended to enable imaging by SPECT imaging and / or PET imaging, among others. Single-photon emission tomography (SPECT) is a nuclear medicine tomography technique that uses gamma rays and can provide true 3D information. The information is often presented as cross-sectional slices across the patient. The gamma-ray emission of the isotope allows us to see where the radiolabeled material has accumulated in the patient's body. Such a true 3D representation can be useful in tumor imaging. Positron emission tomography (PET) is a nuclear medicine imaging technique that produces 3D images and has higher sensitivity than conventional SPECT imaging. The system detects pairs of gamma rays indirectly emitted by a positron-emitting radionuclide (tracer) introduced into the body. A 3D image of the tracer concentration in the body is then constructed by computer analysis, and 3D imaging is often achieved with the help of computed tomography (CT) X-ray scans performed on the patient in the same session on the same machine. Positron-emitting isotopes can also be used in conjunction with CT to provide 3D imaging of the anatomical distribution of labeled medical devices.

[0152] In a particular embodiment, the radioactive isotope is 44 Sc, 47 Sc, 51 Cr, 51 Mn, 52 Mn, 57 Co, 58 Co, 59 Fe, 64 Cu, 67 Cu, 86 Y, 88 Y, 89 Zr, 90 Y, 97 Ru, 99m Tc, 100 Pd, 101m Rh, 103 Pd, 105 Rh, 109 Pd, 111 Ag, 177 Lu, 186 Re, 188 Re, 189 Re, 191 Os, 193 Pt, 194 Ir, 197 Hg, 198 Au, 199 Ag, and 199 Au, 225 Ac, 226 Th or 227 These are transition metals such as Th. In certain embodiments, preferably, the radioactive isotopes are 44 Sc, 47 Sc, 64 Cu, 89 Zr, 90 Y, 99m Tc, 177 Lu, 186 Re, 188 Re, 225 Ac, 226 Th or 227 It is Th.

[0153] In certain embodiments, radioactive isotopes are 43 K, 81 Rb, 83 Sr, 89 Sr, 127 Cs, 128 Ba, 129 Cs and131 These are s-block metals such as Cs.

[0154] In a particular embodiment, the radioactive isotope is 67 Ga, 68 Ga, 71 Enjoy, 72 As, 72 Se, 77 As, 110 In, 111 In, 113 In, 119 Sb, 121 Sn, 201 Tl, 203 Pb, 212 Bi, 212 Pb, and 213 Bi and other radioactive isotopes are found in groups 13-16 of the periodic table. In certain embodiments, preferred radioactive isotopes include: 68 Ga, 111 In, 212 Pb or 213 It contains Bi.

[0155] In a particular embodiment, the radioactive isotope is 18 F, 75 Br, 76 Br, 77 Br, 123 I, 124 I, 125 I, 131 I and 211 These are halogens such as At. In certain embodiments, preferred radioactive isotopes include: 18 F, 123 I, 124 I, 131 I or 211 At is included.

[0156] In a particular embodiment, the radioactive isotope is 139 La, 140 La, 142 Pr, 143 Pr, 149 PM, 151 EU, 153 EU, 153 Sm, 159 Gr, 149 Tb,152 Tb, 161 Tb, 165 Dy, 166 Dy, 166 Ho and 169 EU, 175 These are lanthanides such as Yb. In certain embodiments, preferred radioactive isotopes include 149 Tb, 152 Tb or 161 Tb is included.

[0157] In a particular embodiment, the radioactive isotope is 225 Ac, 226 Th and 227 These are actinides such as Th. In certain embodiments, preferred radioactive isotopes include: 225 Ac or 227 The word "th" is included.

[0158] The radiolabeled material of the present invention may also optionally include a combination of at least two radioisotopes to enable imaging and / or therapy. The radioisotope combination may optionally be selected from, for example, Ga-68 and Lu-177; F-18 and Lu-177; In-111 and Lu-177; Ga-68 and Y-90; F-18 and Y-90; In-111 and Y-90; Ga-68 and Ac-225; F-18 and Ac-225; In-111 and Ac-225.

[0159] The present invention may further include the use of at least one non-radioactive, non-toxic carrier metal. For example, the carrier metal may be selected from Bi and Fe. For example, the non-radioactive carrier metal may enable MRI imaging (e.g., Fe) or X-ray contrast imaging (e.g., Bi). A further example of a carrier metal is trivalent bismuth, which further provides X-ray contrast to microspheres, making them imageable by CT.

[0160] In certain embodiments, R is a chelating agent or chelating moiety, for example, a chelator for a radioactive metal or paramagnetic ion, including a radioactive isotope.

[0161] The chelating agent may include any chelator known in the art; see, for example, Parus et al., “Chemistry and bifunctional chelating agents for binding (177)Lu,” Curr Radiopharm. 2015; 8(2):86-94; Wangler et al., “Chelating agents and their use in radiopharmaceutical sciences,” Mini Rev Med Chem. 2011 October; 11(11):968-83; Liu, “Bifunctional Coupling Agents for Radiolabeling of Biomolecules and Target-Specific Delivery of Metallic Radionuclides,” Adv Drug Deliv Rev. 2008 September; 60(12): 1347-1370.

[0162] Specific examples are shown in Table 1. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5]

[0163] In certain preferred embodiments, R may be a DOTA bonded via any of its four carboxylic acid groups.

[0164] In certain embodiments, the chelator includes a radioactive isotope chelated with it.

[0165] In certain embodiments, the chelator includes a paramagnetic material chelated with it. Examples of paramagnetic ions include chromium(III), manganese(II), iron(III), iron(II), cobalt(II), nickel(II), copper(II), neodymium(III), samarium(III), ytterbium(III), gadolinium(III), vanadium(II), terbium(III), dysprosium(III), holmium(III), erbium(III), or combinations of these paramagnetic ions.

[0166] If a portion is a detectable label, it may be a fluorescent portion.

[0167] In some embodiments, the fluorescent moiety is selected from fluorescent proteins, fluorescent peptides, fluorescent dyes, fluorescent materials, or combinations thereof.

[0168] In certain embodiments, R is a fluorescent dye, which may be selected from the group consisting of, for example, xanthenes, acridines, oxazines, cyanines, styryl dyes, coumarins (such as coumarin 343, methoxycoumarin, and dialkylaminocoumarin), porfins, metal-ligand complexes, fluorescent proteins, nanocrystals, perylenes, boron-dipyrromethenes, and phthalocyanines, as well as conjugates and combinations of these types of dyes. Specific examples of fluorescent labels include organic dyes, such as cyanine, fluorescein and fluorescein derivatives, rhodamine and rhodamine derivatives, Alexa Fluor, Dylight fluor (e.g., DyLight547 and Dylight647), Hylight fluor (e.g., HiLyte Fluor 647, HiLyte Fluor 680, and HiLyte Fluor 750), IRDye (e.g., IR Dye 800, IRDye 800CW, IRDye 800RS, and IRDye 700DX), Dy fluro (e.g., Dy677, Dy676, Dy682, Dy752, and Dy780), VivoTag fluor (e.g., VivoTag-680, VivoTag-S680, and VivoTag-S750), ATTO Dye, and BODIPY fluor (BODIPY FL, BODIPY Examples include, but are not limited to, R6G, BODIPY TMR, BODIPY TR, BODIPY 530 / 550, BODIPY 558 / 568, BODIPY 564 / 570, BODIPY 576 / 589, BODIPY 581 / 591, BODIPY 630 / 650, and BODIPY 650 / 665, etc., carbocyanin, indocarbocyanin, oxacarbocyanin, thiacarbocyanin, merocyanine, polymethine, boron-dipyromethane (BODIPY) dyes, ADS780WS, ADS830WS, and ADS832WS, as well as other fluorophores known to those skilled in the art.

[0169] Further examples include the fluorescent parts Cy3, Cy5, Cy5.5 (also known as Cy5++), CY2, CY7, CY7.5, fluorescein isothiocyanate (FITC), 4',5'-dichloro-2',7'-dimethoxy-fluorescein, naphthofluorescein, 2',4',5',7'-tetra-bromosulfone-fluorescein, tetramethylrhodamine isothiocyanate (TRITC), phycoerythrin, Cy7, fluorescein (FAM), Cy3, Cy3.5 (also known as Cy3++), Texas Red, Texas Red-X, Marina Blue, Oregon Green 488, Oregon Green 500, Oregon Green 514, Pacific Blue, PyMPO, AMCA, AMCA-S, Cascade Blue, Cascade Yellow, DM-NERF, Eosin, Erythrosin, FAM, LightCycler fluor (LightCycler-Red 640, and LightCycler Red 705, etc.), Tetramethylrhodamine (TMR), Rhodamine, Rhodamine derivative (ROX), Hexachlorofluorescein (HEX), Rhodamine 6G (R6G), Carboxy-X-Rhodamine, Lisamin Rhodamine B, Pyrene, Rhodamine B, Rhodamine 6G, Rhodamine Green, Rhodamine Red, Rhodameal Green, Tetramethylrhodamine, Carboxytetramethylrhodamine, Rhodamine derivative JA133, Alexa Fluorescent Dye (Alexa Fluor 350, Alexa Fluor 488, Alexa Fluor 546, Alexa Fluor 633, Alexa Fluor 555, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor (700, AlexaFluor 750, and AlexaFluor 790, etc.), 4',Fluorescent transition metal complexes such as 6-diamidino-2-phenylindole (DAPI), propidium iodide, AMCA, Spectrum Green, Spectrum Orange, Spectrum Aqua, Lissamine, and europium may be selected. Available fluorescent compounds also include fluorescent proteins such as GFP (green fluorescent protein), enhanced GFP (EGFP), blue fluorescent protein and derivatives (BFP, EBFP, EBFP2, Azurite, mKalama1), cyan fluorescent protein and derivatives (CFP, ECFP, Cerulean, CYPet), and yellow fluorescent protein and derivatives (YFP, Citrine, Venus, YPet). See also WO2008 / 142571, WO2009 / 056282, and WO99 / 22026 (all incorporated by reference).

[0170] In certain embodiments, the detectable portion is a biological fluorophore (such as a fluorescent polypeptide or peptide), including but not limited to GFP (e.g., EBFP, EBFP2, Azurite, mKalamal, ECFP, Cerulean, CYpet, YFP, Citrine, Venus, Ypet) and green fluorescent protein (GFP) derivatives of R-phycoerythrin.

[0171] In certain embodiments, R is a photoacoustic reporter molecule. Exemplary photoacoustic reporter molecules include indocyanine green (ICG), Alexa Fluor 750, Evans Blue, BHQ3, QXL680, IRDye880CW, MMPSense 680, methylene blue, PPCy-C8, and Cypate-C18.

[0172] In certain embodiments, the detectable portion is a detectable nanoparticle selected from the group consisting of plasmon nanoparticles, quantum dots, nanodiamonds, polypyrrole nanoparticles, copper sulfide nanoparticles, graphene nanosheets, iron oxide-gold core-shell nanoparticles, Gd2O3 nanoparticles, single-walled carbon nanotubes, dye-filled perfluorocarbon nanoparticles, and superparamagnetic iron oxide nanoparticles.

[0173] In a particular embodiment, the detectable portion includes quantum dots.

[0174] In a particular embodiment, the detectable portion includes an infrared-emitting quantum dot.

[0175] In certain embodiments, the detectable portion is a Raman-active reporter molecule, such as a single-walled carbon nanotube (SWNT) or a surface-enhanced Raman scattering (SERS) agent. An example of a SERS agent is a metal nanoparticle labeled with a Raman-active reporter molecule. In certain examples, there are fluorescent dyes that can also be used as Raman-active reporter molecules, such as Cy3, Cy5, rhodamine, and chalcogenopyryllium dyes.

[0176] Examples of enzyme labels R include, but are not limited to, horseradish peroxidase (HRP), alkaline phosphatase (AP), glucose oxidase, and β-galactosidase. In some embodiments, the agent of the present invention is an imaging agent selected to be useful as part of a method for performing image-guided surgery, such as for resection, dissection, ablation, removal, or stent placement or placement of other in situ devices. For example, the agent can be administered to a patient (human or veterinary subject) in an amount sufficient to preferentially localize to the target tissue of surgery, and the surgeon can detect the presence or absence of the imaging agent during the surgical procedure. In this regard, the detectable portion may preferably be optically detectable, for example, fluorescence or other optically active portion. Such an imaging agent can be advantageously used in an operating room, where the surgical field may be irradiated with sufficient electromagnetic radiation to make detectable portions, such as fluorophores or quantum dots, detectable, either directly by the surgeon or via monitoring means (e.g., screen / monitor). Exemplary uses of such imaging agents generally include endoscopic and laparoscopic surgical procedures in which the presence (or absence) of the imaging agent can be optically observed by an endoscope, laparoscope, or percutaneous means.

[0177] In certain embodiments, image-guided surgery may be image-guided robot-assisted surgery.

[0178] In certain embodiments, the boronic acid compound is represented by general formula IIb, where R is as defined above and X is C or N. In certain preferred embodiments of IIb, R is a chelator. [ka]

[0179] Outstanding boronic acid compounds include the following: [ka] [ka]

[0180] Particularly preferred boronic acid compounds include the following: [ka]

[0181] Furthermore, compounds 6555, 6952, and 6522 containing radionuclides are particularly preferred. For example, the following boronic acid compounds of formulas A, B, and C are preferred: [ka] [In the formulas A, B, and C, M is a radioactive isotope or a metal.]

[0182] In certain embodiments, in formulas A, B, and / or C, M is a diagnostic radioactive isotope.

[0183] In certain embodiments, in formulas A, B, and / or C, M is a therapeutic radioactive isotope.

[0184] In certain embodiments, in formulas A, B, and / or C, M is Ga-67, Ga-68, Lu-177, or Y-90.

[0185] In certain embodiments, in formulas A, B and / or C, M is an s-block metal, for example, 43 K, 81 Rb, 83 Sr, 89 Sr, 127 Cs, 128 Ba, 129 Cs, and 131 Examples include Cs.

[0186] In certain embodiments, in equations A, B, and / or C, M is located in groups 13 through 16 of the periodic table, for example, 67 Ga, 68 Ga, 71 Enjoy, 72 As, 72 Se, 77 As, 110 In, 111 In, 113 In, 119 Sb, 121 Sn, 201 Tl, 203 Pb, 212 Bi, 212 Pb, and 213 Bi, especially, 68 Ga, 111 In, 212 Pb, or 213 Examples include Bi.

[0187] In certain embodiments, in formulas A, B and / or C, M is a halogen, for example, 18 F, 75 Br, 76 Br, 77 Br, 123 I, 124 I, 125 I, 131 I, and 211 At, especially, 18 F, 123 I, 124 I, 131 I, or 211 Examples include "at," etc.

[0188] In certain embodiments, in formulas A, B and / or C, M is a lanthanide, for example, 139 La, 140 La, 142 Pr, 143 Pr, 149 PM, 151 EU, 153 EU, 153 Sm, 159 Gr, 149 Tb, 152 Tb, 161 Tb, 165 Dy, 166 Dy, 166Ho, and 169 EU, 175 Yb, especially 149 Tb, 152 Tb, or 161 Examples include the trombone.

[0189] In certain embodiments, in formulas A, B and / or C, M is an actinide, for example, 225 Ac, 226 Th, and 227 Th, especially, 225 Ac or 227 It is Th.

[0190] Particularly preferred are such compounds that have formed complexes with one or more radioactive isotopes, including the following: [ka] [ka]

[0191] In certain preferred embodiments of formula V, R5 is a half-life extension moiety, such as a non-proteinogenic half-life extension moiety, e.g., a water-soluble polymer such as polyethylene glycol (PEG) or discrete PEG, hydroxyethyl starch (HES), lipids, branched or unbranched acyl groups, branched or unbranched C8-C30 acyl groups, branched or unbranched alkyl groups, and branched or unbranched C8-C30 alkyl groups; as well as a proteinogenic half-life extension moiety, e.g., serum albumin, transferrin, adonectin (e.g., albumin-bound or pharmacokinetically extending (PKE) adonectin), an Fc domain, and an unstructured polypeptide such as XTEN or PAS polypeptide (e.g., a conformationally disordered polypeptide sequence composed of amino acids Pro, Ala, and / or Ser), and any of the aforementioned fragments.

[0192] For example, to provide any of the compounds of formulas I to V above, in which R is a radioactive moiety or R is a chelating agent, and a radioisotope or metal forms a complex with the chelating agent, the compounds of the present invention that form a complex with a radioisotope or metal can be easily prepared. For example, an aqueous mixture of 1) a radioisotope reagent, e.g., a radioisotope halide reagent, and 2) a precursor compound such as a compound having a chelating moiety; is reacted appropriately with stirring for a time and temperature sufficient for the radioisotope to form a complex with the precursor compound. Exemplary incorporated reaction times and temperatures are described in the following examples and may preferably include reaction times of 5 to 60 minutes and reaction temperatures up to or above 90°C.

[0193] The reaction mixture may appropriately contain one or more stabilizing compounds, such as organic stabilizers, for example, 2,5-dihydroxybenzoic acid or its salts, ascorbic acid or its salts, methionine, histidine, melatonin, N-acetylmethionine, or ethanol, with N-acetylmethionine being preferred in certain embodiments. Preferred stabilizers include those generally recognized as safe (GRAS) under U.S. Food and Drug Administration standards.

[0194] In certain embodiments, sulfur-containing stabilizer compounds, including compounds containing one or more sulfide moieties such as N-acetylmethionine and reduced L-glutathione, are preferred stabilizers to be included in radionuclide reagent / precursor compound mixtures during integration reactions.

[0195] A wide variety of polymers and other molecules can be linked to boronic acid compounds to modify the biological properties of the resulting boronic acid compounds and / or to provide them with novel biological properties. These polymers can be linked to boronic acid compounds via naturally encoded amino acids, via unnaturally encoded amino acids, via any functional substituents on natural or unnatural amino acids, or via any substituents or functional groups attached to natural or unnatural amino acids. The molecular weight of the polymers is not limited but may be in a broad range including, but not limited to, about 100 Da to about 100,000 Da or more. The molecular weight of the polymer may be approximately 100 Da to approximately 100,000 Da, such as 100,000 Da, 95,000 Da, 90,000 Da, 85,000 Da, 80,000 Da, 75,000 Da, 70,000 Da, 65,000 Da, 60,000 Da, 55,000 Da, 50,000 Da, 45,000 Da, 40,000 Da, 35,000 Da, 30,000 Da, and 25,000 This includes, but is not limited to, Da, 20,000Da, 15,000Da, 10,000Da, 9,000Da, 8,000Da, 7,000Da, 6,000Da, 5,000Da, 4,000Da, 3,000Da, 2,000Da, 1,000Da, 900Da, 800Da, 700Da, 600Da, 500Da, 400Da, 300Da, 200Da, and 100Da. In some embodiments, the molecular weight of the polymer is about 100Da to about 50,000Da. In some embodiments, the molecular weight of the polymer is about 100Da to about 40,000Da. In some embodiments, the molecular weight of the polymer is about 1,000Da to about 40,000Da. In some embodiments, the molecular weight of the polymer is approximately 5,000 Da to approximately 40,000 Da. In some embodiments, the molecular weight of the polymer is approximately 10,000 Da to approximately 40,000 Da.

[0196] For this purpose, various methods have been developed, including PEGylation, polysialylation, HESation, glycosylation, or recombinant PEG analogs fused to flexible, hydrophilic amino acid chains (500-600 amino acids) (see Chapman, (2002) Adv Drug Deliv Rev. 54. 531-545; Schlapschy et al., (2007) Prot Eng Des Sel. 20, 273-283; Contermann (2011) Curr Op Biotechnol. 22, 868-876; Jevsevar et al., (2012) Methods Mol Biol. 901, 233-246).

[0197] Examples of polymers include polyalkyl ethers and their alkoxy-capped analogs (e.g., polyoxyethylene glycol, polyoxyethylene / propylene glycol, and their methoxy or ethoxy-capped analogs, particularly polyoxyethylene glycol, the latter also known as polyethylene glycol or PEG); discrete PEG (dPEG); polyvinylpyrrolidone; polyvinyl alkyl ethers; polyoxazolines, polyalkyloxazolines, and polyhydroxyalkyloxazolines; polyacrylamides, polyalkylacrylamides, and polyhydroxyalkylacrylamides (e.g., polyhydroxypropyl methacrylamide and its derivatives); polyhydroxyalkyl acrylates; polysialic acids and their analogs; hydrophilic peptide sequences; and polysaccharides and their derivatives, including dextran and dextran derivatives. Derivatives, e.g., carboxymethyl dextran, dextran sulfate, aminodextran; cellulose and its derivatives, e.g., carboxymethylcellulose, hydroxyalkylcellulose; chitin and its derivatives, e.g., chitosan, succinyl chitosan, carboxymethyl chitin, carboxymethyl chitosan; hyaluronic acid and its derivatives; starch; alginates; chondroitin sulfate; albumin; pullulan and carboxymethyl pullulan; polyamino acids and their derivatives, e.g., polyglutamic acid, polylysine, polyaspartic acid, polyasparaginamide; maleic anhydride copolymers, e.g., styrene maleic anhydride copolymer, divinyl ethyl ether maleic anhydride copolymer, etc.; polyvinyl alcohol; copolymers thereof, terpolymers thereof; mixtures thereof, and derivatives thereof.

[0198] The selected polymer may be water-soluble so that the boronic acid compound it binds to does not precipitate in an aqueous environment such as a physiological environment. The water-soluble polymer may be in any structural form, including but not limited to linear, forked, or branched. Typically, water-soluble polymers are poly(alkylene glycols), such as poly(ethylene glycol) (PEG), but other water-soluble polymers may also be used. PEG is used as an example to illustrate some embodiments of this disclosure. For the therapeutic use of boronic acid compounds, the polymer may be pharmaceutically acceptable.

[0199] The term "PEG" is widely used to encompass any polyethylene glycol molecule, regardless of size or modification at the PEG terminus, and can be represented as linked to a boronic acid compound by the following formula: XO-(CH2CH2O) n -CH2CH2- or XO-(CH2CH2O) n - [wherein n is 2 to 10,000, and X is H or a terminal modification (e.g., C1-C4 alkyl, protecting group or terminal functional group)]. In some cases, the PEG used in the polypeptides of this disclosure is terminated at one end with hydroxy or methoxy, i.e., X is H or CH3 ("methoxyPEG").

[0200] The number of water-soluble polymers linked to the boronic acid compound (i.e., the degree of PEGylation or glycosylation) can be adjusted to provide changes (including, but not limited to, increases or decreases) in the pharmacological, pharmacokinetic, or pharmacodynamic properties of the resulting boronic acid compound, such as the in vivo half-life. In some embodiments, the half-life of the resulting boronic acid compound is increased by at least about 10, 20, 30, 40, 50, 60, 70, 80, 90 percent, 2 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 11 times, 12 times, 13 times, 14 times, 15 times, 16 times, 17 times, 18 times, 19 times, 20 times, 25 times, 30 times, 35 times, 40 times, 50 times, or at least about 100 times compared to the unmodified polypeptide.

[0201] Another variant of polymer systems useful for modifying the PK or other biological properties of the resulting boronic acid compounds is the use of unstructured hydrophilic amino acid polymers, which are functional analogs of PEG. The inherent biodegradability of the polypeptide platform makes it an attractive alternative to PEG, potentially a more beneficial alternative. Another advantage is the precise molecular structure of the recombinant molecule, in contrast to the polydispersity of PEG. Unlike HSA and Fc peptide fusions, where the three-dimensional folding of the fusion partner must be maintained, recombinant fusions to unstructured partners can often be subjected to higher temperatures or harsher conditions such as HPLC purification.

[0202] One more advanced polypeptide in this class is called XTEN(Amunix), which is 864 amino acids long and composed of six amino acids (A, E, G, P, S, and T). See Schellenberger et al. “A recombinant polypeptide extends the in vivo half-life of peptides and proteins in a tunable manner” 2009 Nat Biotechnol. 27(12):1186-90. This is made possible by the biodegradable properties of the polymer, which is much larger than that of the typically used 40 kDa PEG and consequently gives a longer half-life extension. Fusion of XTEN to boronic acid compounds should result in a half-life extension of the final boronic acid compound that is 60 to 130 times longer than that of the unmodified polypeptide.

[0203] A second polymer based on similar conceptual considerations is PAS (XL-Protein GmbH). Schlapschy et al. “PASYlation: a biological alternative to PEGylation for extending the plasma half-life of pharmaceutically active proteins” 2013 Protein Eng Des Sel. 26(8):489-501. Random coil polymers consist of an even more limited set of just three small uncharged amino acids: proline, alanine, and serine.

[0204] In a particular preferred embodiment of formula V, R5 is a polyethylene glycol polymer.

[0205] In a particular preferred embodiment of formula V, R5 is a polyethylene glycol polymer and n is 1.

[0206] The present invention also provides pharmaceutical compositions comprising at least one compound of any of formulas I to V and optionally a pharmaceutically acceptable carrier and / or excipient. In certain embodiments, the pharmaceutical compositions are intended for use in the diagnosis or treatment of diseases characterized by overexpression of fibroblast-activating protein (FAP) in animal, preferably human, subjects.

[0207] Suitable pharmaceutically acceptable vehicles include non-toxic buffers such as phosphates, citrates, and other organic acids; salts such as sodium chloride; antioxidants including ascorbic acid and methionine; preservatives such as octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, alkylparabens such as phenol, butyl or benzyl alcohol, methyl or propylparaben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol; low molecular weight polypeptides (e.g., less than about 10 amino acid residues); serum albumin Examples of such substances include, but are not limited to, proteins such as methyl esters, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; carbohydrates such as monosaccharides, disaccharides, glucose, mannose, or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes such as Zn-protein complexes; and nonionic surfactants such as TWEEN® or polyethylene glycol (PEG). (Remington: The Science and Practice of Pharmacy, 22nd Edition, 2012, Pharmaceutical Press, London.)

[0208] The pharmaceutical compositions of this disclosure may be administered by any number of methods for either topical or systemic treatment. Administration may include topical administration by epidermal or transdermal patch, ointment, lotion, cream, gel, drop, suppository, spray, liquid and powder; pulmonary administration by inhalation or blowing of powder or aerosol, including nebulizer, intratracheal and intranasal; oral administration; or parenteral administration, including intravenous, intra-arterial, intratumoral, subcutaneous, intraperitoneal, intramuscular (e.g., by injection or infusion), or intracranial (e.g., intrathecal or intraventricular).

[0209] Typical administration of the radiopharmaceutical of the present invention may be by intravenous injection or other parenteral administration.

[0210] Therapeutic formulations can be in unit dosage forms. Such formulations include tablets, pills, capsules, powders, granules, liquids or suspensions in water or a non-aqueous medium, or suppositories. In solid compositions such as tablets, the main active ingredient is mixed with a pharmaceutical carrier. Conventional tableting components include corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, calcium diphosphate, or gum, and diluents (e.g., water). These can be used to form solid pre-formulation compositions containing a homogeneous mixture of the compounds of this disclosure or non-toxic, pharmaceutically acceptable salts thereof. These solid pre-formulation compositions are then subdivided into the unit dosage forms of the types described above. Tablets, pills, etc., of formulations or compositions can be coated or otherwise formulated to provide dosage forms that offer the benefit of long-lasting action. For example, tablets or pills may include an inner composition coated with an outer component. Furthermore, the two components may be separated by an enteric coating that resists disintegration and allows the inner component to pass through the stomach intact or delays its release. Various materials can be used for such enteric coatings or coatings, including numerous polymer acids and mixtures of polymer acids with materials such as shellac, cetyl alcohol, and cellulose acetate.

[0211] Kit and method Yet another aspect of the present invention provides a kit comprising, or consisting of, at least one compound of any of formulas I to V or formulas A, B, or C, and instructions for use for the diagnosis or treatment of a disease.

[0212] A further aspect of the present invention provides a method for diagnosing, imaging, or reducing tissue expressing or overexpressing FAP in an animal (preferably a human patient), comprising administering to the animal a pharmaceutical composition disclosed herein, which comprises a composition containing at least one compound of any of formulas I to V or formulas A, B, or C.

[0213] In some embodiments, the tissue overexpressing FAP is a tumor, particularly a solid tumor. In some embodiments, the tumor is selected from the group consisting of: colorectal tumors, pancreatic tumors, lung tumors, ovarian tumors, liver tumors, breast tumors, kidney tumors, prostate tumors, neuroendocrine tumors, gastrointestinal tumors, melanoma, cervical tumors, bladder tumors, glioblastoma, and head and neck tumors. In some embodiments, the tumor is a colorectal tumor. In some embodiments, the tumor is an ovarian tumor. In some embodiments, the tumor is a lung tumor. In some embodiments, the tumor is a pancreatic tumor. In some embodiments, the tumor is a melanoma. In some embodiments, the tumor is a bladder tumor. In some embodiments, the tumor is a prostate tumor. Further examples include the target Affimer drug (Affimer Agent) is used to treat osteosarcoma, rhabdomyosarcoma, neuroblastoma, kidney cancer, leukemia, renal transitional cell carcinoma, bladder cancer, Wilm's cancer, ovarian cancer, pancreatic cancer, breast cancer (including triple-negative breast cancer), prostate cancer, bone cancer, lung cancer (e.g., small cell lung cancer or non-small cell lung cancer), stomach cancer, colorectal cancer, cervical cancer, synovial sarcoma, head and neck cancer, squamous cell carcinoma, multiple myeloma, renal cell carcinoma, retinoblastoma, hepatocellular carcinoma, hepatocellular carcinoma, and melanoma. It can be used to treat patients suffering from cancers such as noma, rhabdoid tumor of the kidney, Ewing's sarcoma, chondrosarcoma, brain cancer, glioblastoma, meningioma, pituitary adenoma, vestibular schwannoma, primitive neuroectoderm tumor, medulloblastoma, astrocytoma, undifferentiated astrocytoma, oligodendroglioma, ependymoma, choroid plexus papilloma, polycythemia vera, thrombocythemia, idiopathic myelofibrosis, soft tissue sarcoma, thyroid cancer, endometrial cancer, carcinoid cancer, or liver cancer, breast cancer, or gastric cancer. In some embodiments of this disclosure, the cancer is metastatic cancer, for example, metastatic cancer of the various cancers described above.

[0214] In some embodiments, in addition to administering the boronic acid compounds described herein, the method or procedure further comprises administering at least one additional immune response stimulant. In some embodiments, the additional immune response stimulants include, but are not limited to, colony-stimulating factors (e.g., granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage colony-stimulating factor (M-CSF), granulocyte colony-stimulating factor (G-CSF), stem cell factor (SCF)), interleukins (e.g., IL-1, IL-2, IL-3, IL-7, IL-12, IL-15, IL-18), checkpoint inhibitors, antibodies that block immunosuppressive function (e.g., anti-CTLA-4 antibody, anti-CD28 antibody, anti-CD3 antibody), Toll-like receptors (e.g., TLR4, TLR7, TLR9), or members of the B7 family (e.g., CD80, CD86). The additional immune response stimulant may be administered before, concurrently with, and / or after the administration of the boronic acid compounds. Pharmaceutical compositions comprising boronic acid compounds and immune response stimulants are also provided. In some embodiments, the immune response stimulant comprises one, two, three, or more immune response stimulants.

[0215] In some embodiments, in addition to administering the boronic acid compounds described herein, the method or procedure further includes administering at least one additional therapeutic agent. The additional therapeutic agent may be administered before, simultaneously with, and / or after the administration of the boronic acid compounds. Pharmaceutical compositions comprising the boronic acid compounds and the additional therapeutic agents are also provided. In some embodiments, the at least one additional therapeutic agent comprises one, two, three, or more additional therapeutic agents.

[0216] Combination therapy, which uses two or more therapeutic agents, often involves drugs that act by different mechanisms of action, although this is not mandatory. Combination therapy using drugs with different mechanisms of action can produce additive or synergistic effects. Combination therapy can enable lower doses of each drug than those used in monotherapy, thereby reducing toxic side effects and / or increasing the therapeutic index of boronic acid compounds. Combination therapy can reduce the likelihood of drug-resistant cancer cells developing. In some embodiments, combination therapy includes a therapeutic agent that affects the immune response (e.g., enhances or activates the response) and a therapeutic agent that affects tumor / cancer cells (e.g., inhibits or kills them).

[0217] In some embodiments of the methods described herein, the combination of the boronic acid compounds described herein with at least one additional therapeutic agent results in additive or synergistic effects. In some embodiments, the combination therapy enhances the therapeutic index of the boronic acid compound. In some embodiments, the combination therapy enhances the therapeutic index of the additional therapeutic agent. In some embodiments, the combination therapy results in a reduction of the toxicity and / or side effects of the boronic acid compound. In some embodiments, the combination therapy results in a reduction of the toxicity and / or side effects of the additional therapeutic agent.

[0218] Useful classes of therapeutic agents include, for example, antitubulins, auristatins, DNA minor groove binders, DNA replication inhibitors, alkylating agents (e.g., cisplatin, mononuclear (platinum), dinuclear (platinum), and trinuclear platinum complexes, and platinum complexes such as carboplatin), anthracyclines, antibiotics, antifolic acids, antimetabolites, chemotherapeutic sensitizers, duocalmycin, etoposide, fluorinated pyrimidines, ionophores, lexitropsin, nitrosourea, platinol, purine antimetabolites, puromycin, radiosensitizers, steroids, taxanes, topoisomerase inhibitors, vinca alkaloids, etc. In some embodiments, the second therapeutic agent is an alkylating agent, antimetabolite, mitotic inhibitor, topoisomerase inhibitor, or angiogenesis inhibitor.

[0219] Therapeutic agents that can be administered in combination with the boronic acid compounds described herein include chemotherapeutic agents. Therefore, in some embodiments, the method or procedure involves administering the boronic acid compounds of this disclosure in combination with chemotherapeutic agents, or in combination with a cocktail of chemotherapeutic agents. Treatment with boronic acid compounds may occur before, simultaneously with, or after the administration of chemotherapeutic agents. Combination administration may include simultaneous administration of a single pharmaceutical formulation or separate formulations, or sequential administration in any order, within a time frame that allows all active agents to exert their biological activity simultaneously. The preparation and administration schedule for such chemotherapeutic agents may be used to be determined according to the manufacturer's instructions or empirically by those skilled in the art. Such preparation and administration schedules for chemotherapy are also described in The Chemotherapy Source Book, 4th Edition, 2008, MC Perry, Editor, Lippincott, Williams & Wilkins, Philadelphia, Pa.

[0220] Useful chemotherapeutic agents in this disclosure include alkylating agents, e.g., thiotepa and cyclophosphamide (CYTOXAN); alkyl sulfonates, e.g., busulfan, improsulfan and pigosulfan; aziridines, e.g., benzodopa, carbocone, meturedopa and uredopa; ethyleneimines and methylmelamines (including altoretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylolmelamine). ); Nitrogen mustards, e.g., chlorambucil, chlornafadin, colophosphamide, estramustine, ifosfamide, mechloretamine, mechloretamine oxide hydrochloride, melphalan, nobuenvicin, fenesterine, prednimustine, trophosphamide, uracil mustard; Nitrosoureas, e.g., carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; Antibiotics, e.g., acrasinomycin, actinomycin, ausramycin, azaserine, bleomycin, ca Cutinomycin, calicheamicin, carabicin, carminomycin, cardinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcelomycin, mitomycin, mycophenolic acid, nogaramycin, olibomycin, peplomycin, potophyllomycin, puromycin, queramycin, rhodorubicin, streptonigrin, streptozocin, tuberculosis Lucidin, ubenimex, dinostatin, zolubicin; antimetabolites, e.g., methotrexate and 5-fluorouracil (5-FU); folic acid analogs, e.g., denopterin, methotrexate, pteropterin, trimethrexate; purine analogs, e.g., fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs, e.g., ancitabine, azacitidine, 6-azauridine, carmofur, cytosine arabinoside, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU;Androgens, e.g., carsterone, dromostanolone propionate, epithiostanol, mepitiostane, testolactone; anti-adrenal agents, e.g., aminoglutethimide, mitotane, trilostane; folic acid supplements, e.g., folic acid; acegraton; aldofamide glycosides; aminolevulinic acid; amsacrin; bestrabusil; bisantren; edatraxate; defofamine; demecolsin; diaziquan; elformitin; eriptinium acetate; etogluside ;Gallium nitrate;Hydroxyurea;Lentinan;Lonidamin;Mitoguazone;Mitoxantrone;Mopidamole;Nitracrine;Pentostatin;Fenamet;Pirarubicin;Podophyllic acid;2-Ethylhydrazide;Procarbazine;PSK;Lazoxane;Schizophyllan;Spirogermanium;Tenuazonic acid;Triadicone, 2,2',2"-Trichlorotriethylamine;Urethane;Vindesine;Dacarbazine;Mannomustine;Mitobronitol;Mitractol;Pipobromane;Gacitosine;Arabinoside (Ara- C); Taxoids, e.g., paclitaxel (TAXOL) and doxetaxel (TAXOTERE); Chlorambucil; Gemcitabine, 6-thioguanine; Mercaptopurine; Platinum analogs, e.g., cisplatin and carboplatin; Vinblastine; Platinum; Etoposide (VP-16); Ifosfamide; Mitomycin C; Mitoxantrone; Vincristine; Vinorelbine; Navelbine; Novantrone; Teniposide; Daunomycin; Aminopterin; Ibandronate; CPT-11; Topoisomerase inhibitors R Examples of chemotherapeutic agents include, but are not limited to, FS2000; difluoromethylolnithine (DMFO); retinoic acid; esperamicin, capecitabine (XELODA), and any pharmaceutically acceptable salts, acids, or derivatives of any of the above. Other chemotherapeutic agents include, for example, antiestrogens such as tamoxifen, raloxifen, aromatase inhibitors 4(5)-imidazoles, 4-hydroxytamoxifen, trioxyfen, keoxyfen, LY117018, onapristone, and toremifene (FARESTON);Furthermore, antihormone agents that act to modulate or inhibit the hormonal effects on tumors include, for example, antiandrogens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; as well as pharmaceutically acceptable salts, acids, or derivatives of any of the above; in certain embodiments, the additional therapeutic agent is cisplatin. In certain embodiments, the additional therapeutic agent is carboplatin.

[0221] In some embodiments of the methods described herein, the chemotherapeutic agent is a topoisomerase inhibitor. A topoisomerase inhibitor is a chemotherapeutic agent that interferes with the action of a topoisomerase enzyme (e.g., topoisomerase I or II). Topoisomerase inhibitors include, but are not limited to, doxorubicin HCl, daunorubicin citrate, mitoxantrone HCl, actinomycin D, etoposide, topotecan HCl, teniposide (VM-26), and irinotecan, as well as any pharmaceutically acceptable salts, acids, or derivatives thereof. In some embodiments, an additional therapeutic agent is irinotecan.

[0222] In some embodiments, the chemotherapeutic agent is an antimetabolite. An antimetabolite is a chemical substance that is similar in structure to a metabolite required for a normal biochemical reaction but is sufficiently different to interfere with one or more normal cellular functions, such as cell division. Antimetabolites include, but are not limited to, gemcitabine, fluorouracil, capecitabine, methotrexate sodium, laritrexed, pemetrexed, tegafur, cytosine arabinoside, thioguanine, 5-azacitidine, 6-mercaptopurine, azathioprine, 6-thioguanine, pentostatin, fludarabine phosphate, and cladribine, as well as any pharmaceutically acceptable salts, acids, or derivatives thereof. In some embodiments, the additional therapeutic agent is gemcitabine.

[0223] In some embodiments of the methods described herein, the chemotherapeutic agent is an antimitotic agent, including but not limited to a tubulin-binding agent. In some embodiments, the agent is a taxane. In some embodiments, the agent is paclitaxel or docetaxel, or a pharmaceutically acceptable salt, acid, or derivative of paclitaxel or docetaxel. In some embodiments, the agent is paclitaxel (TAXOL), docetaxel (TAXOTERE), albumin-conjugated paclitaxel (nab-paclitaxel; ABRAXANE), DHA-paclitaxel, or PG-paclitaxel. In certain alternative embodiments, the antimitotic agent includes vinca alkaloids such as vincristine, vinblastine, vinorelbine, or vindesine, or pharmaceutically acceptable salts, acids, or derivatives thereof. In some embodiments, the antimitotic agent is a kinesin Eg5 inhibitor or a mitotic kinase inhibitor such as Aurora A or Plk1. In some embodiments, the additional therapeutic agent is paclitaxel. In some embodiments, the additional therapeutic agent is nab-paclitaxel.

[0224] In some embodiments of the methods described herein, additional therapeutic agents include drugs such as small molecules. For example, a treatment may include the co-administration of a boronic acid compound of the Disclosure with a small molecule that acts as an inhibitor of tumor-associated antigens, including but not limited to EGFR, HER2 (ErbB2), and / or VEGF. In some embodiments, the boronic acid compound of the Disclosure is administered in combination with a protein kinase inhibitor selected from the group consisting of gefitinib (IRESSA), erlotinib (TARCEVA), sunitinib (SUTENT), lapatanib, vandetanib (ZACTIMA), AEE788, CI-1033, cediranib (RECENTIN), sorafenib (NEXAVAR), and pazopanib (GW786034B). In some embodiments, additional therapeutic agents include mTOR inhibitors.

[0225] In some embodiments of the methods described herein, the additional therapeutic agent is a small molecule that inhibits the cancer stem cell pathway. In some embodiments, the additional therapeutic agent is an inhibitor of the Notch pathway. In some embodiments, the additional therapeutic agent is an inhibitor of the Wnt pathway. In some embodiments, the additional therapeutic agent is an inhibitor of the BMP pathway. In some embodiments, the additional therapeutic agent is an inhibitor of the Hippo pathway. In some embodiments, the additional therapeutic agent is an inhibitor of the mTOR / AKR pathway. In some embodiments, the additional therapeutic agent is an inhibitor of the RSPO / LGR pathway.

[0226] In some embodiments of the methods described herein, the additional therapeutic agent includes biological molecules such as antibodies. For example, the treatment may include the combined administration of the boronic acid compounds of this disclosure with antibodies against tumor-associated antigens, including but not limited to antibodies that bind to EGFR, HER2 / ErbB2, and / or VEGF. In some embodiments, the additional therapeutic agent is an antibody specific to a cancer stem cell marker. In some embodiments, the additional therapeutic agent is an antibody that binds to a component of the Notch pathway. In some embodiments, the additional therapeutic agent is an antibody that binds to a component of the Wnt pathway. In some embodiments, the additional therapeutic agent is an antibody that inhibits the cancer stem cell pathway. In some embodiments, the additional therapeutic agent is an inhibitor of the Notch pathway. In some embodiments, the additional therapeutic agent is an inhibitor of the Wnt pathway. In some embodiments, the additional therapeutic agent is an inhibitor of the BMP pathway. In some embodiments, the additional therapeutic agent is an antibody that inhibits β-catenin signaling. In some embodiments, the additional therapeutic agent is an antibody that is an angiogenesis inhibitor (e.g., an anti-VEGF or VEGF receptor antibody). In some embodiments, additional therapeutic agents include bevacizumab (AVASTIN), ramucirumab, trastuzumab (HERCEPTIN), pertuzumab (OMNITARG), panitumumab (VECTIBIX), nimotuzumab, zaltumumab, or cetuximab (ERBITUX).

[0227] In some embodiments of the methods described herein, the additional therapeutic agent is an antibody that modulates the immune response. In some embodiments, the additional therapeutic agent is an anti-PD-1 antibody, an anti-LAG-3 antibody, an anti-CTLA-4 antibody, an anti-TIM-3 antibody, or an anti-TIGIT antibody.

[0228] Furthermore, treatment with the boronic acid compounds described herein may include combined treatment with one or more other biological molecules such as cytokines (e.g., lymphokines, interleukins, tumor necrosis factors, and / or growth factors), or may be accompanied by surgical resection of the tumor, removal of cancer cells, or any other treatment deemed necessary by the treating physician. In some embodiments, the additional therapeutic agent is an immune response stimulant.

[0229] In some embodiments of the methods described herein, boronic acid compounds can be combined with growth factors selected from the group consisting of adrenomedullin (AM), angiopoietin (Ang), BMP, BDNF, EGF, erythropoietin (EPO), FGF, GDNF, G-CSF, GM-CSF, GDF9, HGF, HDGF, IGF, migration stimulant, myostatin (GDF-8), NGF, neurotrophin, PDGF, thrombopoietin, TGF-α, TGF-β, TNF-α, VEGF, P1GF, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-12, IL-15, and IL-18.

[0230] In some embodiments of the methods described herein, an additional therapeutic agent is an immune response stimulant. In some embodiments, the immune response stimulant is selected from the group consisting of granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage colony-stimulating factor (M-CSF), granulocyte colony-stimulating factor (G-CSF), interleukin-3 (IL-3), interleukin-12 (IL-12), interleukin-1 (IL-1), interleukin-2 (IL-2), B7-1 (CD80), B7-2 (CD86), 4-1BB ligand, anti-CD3 antibody, anti-CTLA-4 antibody, anti-TIGIT antibody, anti-PD-1 antibody, anti-LAG-3 antibody, and anti-TIM-3 antibody.

[0231] In some embodiments of the methods described herein, the immune response stimulant is selected from the group consisting of PD-1 activity modulators, PD-L2 activity modulators, CTLA-4 activity modulators, CD28 activity modulators, CD80 activity modulators, CD86 activity modulators, 4-1BB activity modulators, OX40 activity modulators, KIR activity modulators, Tim-3 activity modulators, LAG3 activity modulators, CD27 activity modulators, CD40 activity modulators, GITR activity modulators, TIGIT activity modulators, CD20 activity modulators, CD96 activity modulators, IDO1 activity modulators, cytokines, chemokines, interferons, interleukins, lymphokines, members of the tumor necrosis factor (TNF) family, and immunostimulatory oligonucleotides.

[0232] In some embodiments of the methods described herein, the immune response stimulant is selected from the group consisting of PD-1 antagonists, PD-L2 antagonists, CTLA-4 antagonists, CD80 antagonists, CD86 antagonists, KIR antagonists, Tim-3 antagonists, LAG3 antagonists, TIGIT antagonists, CD20 antagonists, CD96 antagonists, and / or IDO1 antagonists.

[0233] In some embodiments of the methods described herein, the PD-1 antagonist is an antibody that specifically binds to PD-1. In some embodiments, the antibody that binds to PD-1 is KEYTRUDA (MK-3475), pizilizumab (CT-011), nivolumab (OPDIVO, BMS-936558, MDX-1106), MEDI0680 (AMP-514), REGN2810, BGB-A317, PDR-001, or STI-A1110. In some embodiments, the antibody that binds to PD-1 is an antibody described in PCT Publication WO2014 / 179664 and identified, for example, as APE2058, APE1922, APE1923, APE1924, APE1950, or APE1963, or an antibody containing the CDR region of any of these antibodies. In other embodiments, the PD-1 antagonist is a fusion protein containing PD-L2, for example, AMP-224. In other embodiments, the PD-1 antagonist is a peptide inhibitor, for example, AUNP-12.

[0234] In some embodiments, the CTLA-4 antagonist is an antibody that specifically binds to CTLA-4. In some embodiments, the antibody that binds to CTLA-4 is ipilimumab (YERVOY) or tremelimumab (CP-675,206). In some embodiments, the CTLA-4 antagonist is a CTLA-4 fusion protein, such as KAHR-102.

[0235] In some embodiments, the LAG3 antagonist is an antibody that specifically binds to LAG3. In some embodiments, antibodies that bind to LAG3 include IMP701, IMP731, BMS-986016, LAG525, and GSK2831781. In some embodiments, the LAG3 antagonist includes a soluble LAG3 receptor, such as IMP321.

[0236] In some embodiments, the KIR antagonist is an antibody that specifically binds to KIR. In some embodiments, the antibody that binds to KIR is lirilumab.

[0237] In some embodiments, the immune response stimulant is selected from the group consisting of CD28 agonists, 4-1BB agonists, OX40 agonists, CD27 agonists, CD80 agonists, CD86 agonists, CD40 agonists, and GITR agonists. In some embodiments, the OX40 agonist includes an OX40 ligand or its OX40 binding moiety. For example, the OX40 agonist may be MEDI6383. In some embodiments, the OX40 agonist is an antibody that specifically binds to OX40. In some embodiments, the antibody that binds to OX40 is MEDI6469, MEDI0562, or MOXR0916 (RG7888). In some embodiments, the OX40 agonist is a vector capable of expressing the OX40 ligand (e.g., an expression vector such as an adenovirus or a virus). In some embodiments, the OX40 expression vector is Delta-24-RGDOX or DNX2401.

[0238] In some embodiments, the 4-1BB(CD137) agonist is a binding molecule such as antikarin. In some embodiments, antikarin is PRS-343. In some embodiments, the 4-1BB agonist is an antibody that specifically binds to 4-1BB. In some embodiments, the antibody that binds to 4-1BB is PF-2566 (PF-05082566) or urelumab (BMS-663513).

[0239] In some embodiments, the CD27 agonist is an antibody that specifically binds to CD27. In some embodiments, the antibody that binds to CD27 is varylumab (CDX-1127).

[0240] In some embodiments, the GITR agonist comprises a GITR ligand or its GITR-binding moiety. In some embodiments, the GITR agonist is an antibody that specifically binds to GITR. In some embodiments, the antibody that binds to GITR is TRX518, MK-4166, or INBRX-110.

[0241] In some embodiments, the immune response stimulant includes, but is not limited to, cytokines such as chemokines, interferons, interleukins, lymphokines, and members of the tumor necrosis factor (TNF) family. In some embodiments, the immune response stimulant includes immunostimulatory oligonucleotides such as CpG dinucleotides.

[0242] In some embodiments, the immune response stimulant includes, but is not limited to, anti-PD-1 antibody, anti-PD-L2 antibody, anti-CTLA-4 antibody, anti-CD28 antibody, anti-CD80 antibody, anti-CD86 antibody, anti-4-1BB antibody, anti-OX40 antibody, anti-KIR antibody, anti-Tim-3 antibody, anti-LAG3 antibody, anti-CD27 antibody, anti-CD40 antibody, anti-GITR antibody, anti-TIGIT antibody, anti-CD20 antibody, anti-CD96 antibody, or anti-IDO1 antibody.

[0243] In some embodiments, the boronic acid compounds disclosed herein may be used alone or in conjunction with radiotherapy.

[0244] In some embodiments, the boronic acid compounds disclosed herein may be used alone or in conjunction with targeted therapies. Examples of targeted therapies include hormone therapy, signaling inhibitors (e.g., EGFR inhibitors such as cetuximab (Erbitux) and erlotinib (Tarceva)); HER2 inhibitors (e.g., trastuzumab (Herceptin) and pertuzumab (Perjeta)); BCR-ABL inhibitors (e.g., imatinib (Gleevec) and dasatinib (Sprycel)); ALK inhibitors (e.g., crizotinib (Xalkori) and ceritinib (Zykadia)); BRAF inhibitors (e.g., These include vemurafenib (Zelboraf) and dabrafenib (Tafinlar), gene expression modulators, apoptosis inducers (e.g., bortezomib (Velcade) and carfilzomib (Kyprolis)), angiogenesis inhibitors (e.g., bevacizumab (Avastin) and ramucirumab (Cyramza)), and toxin-conjugated monoclonal antibodies (e.g., brentuximab vedotin (Adcetris) and ad-trastuzumab emtansine (Kadcyla)).

[0245] In some embodiments of this disclosure, boronic acid compounds are administered, for example, as part of a pharmaceutical composition in association with STING agonists. Cyclic dinucleotides (CDNs), cyclic di-AMPs (produced by Listeria monocytogenes and other bacteria), and their analogues, cyclic di-GMP and cyclic GMP-AMP, are recognized by host cells as pathogen-associated molecular patterns (PAMPs) that bind to pathogen-recognition receptors (PRRs) known as Stimulator of Interferon Genes (STING). STING is an adapter protein in the cytoplasm of host mammalian cells that activates the TANK-binding kinase (TBK1)-IRF3 and NF-κB signaling axis, resulting in the induction of IFN-β and other gene products that potently activate innate immunity. STING is now recognized as a component of the host cytosolic surveillance pathway that senses infection by intracellular pathogens, responds by inducing IFN-α production, and leads to the development of an acquired defense pathogen-specific immune response consisting of both antigen-specific CD4+ and CD8+ T cells and pathogen-specific antibodies. U.S. Patent Nos. 7,709,458 and 7,592,326 (both incorporated by reference); WO2007 / 054279, WO2014 / 093936, WO2014 / 179335, WO2014 / 189805, WO2015 / 185565, WO2016 / 096174, WO2016 / 145102, WO2017 / 027645, WO2017 / 027646, and WO2017 / 075477 (all incorporated by reference); and Yan et al., Bioorg. Med. Chem Lett. 18:5631-4, 2008.

[0246] In some embodiments of this disclosure, the boronic acid compounds of this disclosure are administered in conjunction with an Akt inhibitor. Exemplary AKT inhibitors include GDC0068 (also known as GDC-0068, ipatasertib, and RG7440), MK-2206, perifosine (also known as KRX-0401), GSK690693, AT7867, tricilibine, CCT128930, A-674563, PHT-427, Akti-1 / 2, afuresertib ( Also known as GSK2110183), AT13148, GSK2141795, BAY1125976, uprosertib (also known as GSK2141795), AKT inhibitor VIII (1,3-dihydro-1-[1-[[4-(6-phenyl-1H-imidazo[4,5-g]quinoxaline-7-yl)phenyl]m-ethyl]-4-piperidinyl]-2H-benzimidazole-2-one), Akt inhibitor X (2-chloro-N,N-diethyl) -10H-phenoxazine-10-butanamine, monohydrochloride), MK-2206 (8-(4-(1-aminocyclobutyl)phenyl)-9-phenyl-[1,2,4]triazolo[3,4-f][-1,6]naphthirizine-3(2H)-one), uprosertib (N-((S)-1-amino-3-(3,4-difluorophenyl)propan-2-yl)-5-chloro-4-(4-chloro-1-methyl-1H-pyrazole-5-yl)furan-2-carboxamide), Patasertib (ipatasertib) ((S)-2-(4-chlorophenyl)-1-(4-((5R,7R)-7-hydroxy-5-methyl-6,7-dihydro-5H-cyclopenta[d]pyrimidine-4-yl)piperazin-1-yl)-3-(isopropylamino)propan-1-one), AZD5363 (4-piperidinecarboxamide, 4-amino-N-[(1S)-1-(4-chlorophenyl)-3-hydroxypropyl]-1-(7H-pyrrolo[2,This includes 3-d]p-irimidine-4-yl), perifosine, GSK690693, GDC-0068, trisilbine, CCT128930, A-674563, PF-04691502, AT7867, miltefosine, PHT-427, honokiol, trisilibine phosphate, and KP372-1A (10H-indeno[2,1-e]tetrazolo[1,5-b][1,2,4]triazine-10-one), and Akt inhibitor IX (CAS98510-80-6). Further Akt inhibitors include ATP competitive inhibitors, such as isoquinoline-5-sulfonamides (e.g., H-8, H-89, NL-71-101), azepane derivatives (e.g., (-)-valanol derivatives), aminoflazanes (e.g., GSK690693), heterocyclic compounds (e.g., 7-azaindole, 6-phenylpurine derivatives, pyrrolo[2,3-d]pyrimidine derivatives, CCT128930, 3-aminopyrrolidine, anilinotriazole derivatives, spiroindoline derivatives, AZD5363, A-674563). , A-443654), phenylpyrazole derivatives (e.g., AT7867, AT13148), thiophenecarboxamide derivatives (e.g., Afuresertib (GSK2110183), 2-pyridimyl-5-amidethiophene derivative (DC120), uprosertib (GSK2141795); allosteric inhibitors, e.g., 2,3-diphenylquinoxaline analogs (e.g., 2,3-diphenylquinoxaline derivatives, triazolo[3,4-f][1,6) Naphthyridine-3(2H)-one derivative (MK-2206), alkyl phospholipids (e.g., Edelfosine (1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine, ET-18-OCH3), Ilmofosine (BM41.440), Miltefosine (Hexadecylphosphocholine, HePC), Perifosine (D-21266), Erucylphosphocholine (ErPC), Erufosine (ErPC3, Erucylphosphohomocholine), Indole-3-Cal Binol analogs (e.g., indole-3-carbinol, 3-chloroacetylindole, diindolylmethane, diethyl 6-methoxy-5,7-dihydroindoro[2,3-b]carbazole-2,10-dicarboxylate (SR13668), OSU-A9), sulfonamide derivatives (e.g., PH-316, PHT-427), thiourea derivatives (e.g., PIT-1, PIT-2, DM-PIT-1, N-[(1-methyl-1H-pyrazole-4-yl)carbonyl]-N'-(3-bromophenyl)-thiourea), purine derivatives (e.g., tricilibine (TCN, NSC)) 154020), Trisilibine monophosphate active analog (TCN-P), 4-amino-pyrido[2,3-d]pyrimidine derivative API-1, 3-phenyl-3H-imidazo[4,5-b]pyridine derivative, ARQ 092), BAY 1125976, 3-methylxanthine, quinoline-4-carboxamide, 2-[4-(cyclohexa-1,3-dien-1-yl)-1H-pyrazole-3-yl]phenol, 3-oxo-chylic acid, 3α- and 3β-acetoxy-chylic acid, acetoxy-chylic acid; and irreversible inhibitors, such as natural products, antibiotics, lactokinomycin, frenolicin B, carafungin, medelmycin, Boc-Phe-vinyl ketone, 4-hydroxynonenal (4-HNE), 1,6-naphthilidinone derivatives, and imidazo-1,2-pyridine derivatives.

[0247] In some embodiments of the present disclosure, the boronic acid compounds of the present disclosure are administered in association with MEK inhibitors. Exemplary MEK inhibitors include AZD6244 (Selumetinib), PD0325901, GSK1120212 (Trametinib), U0126-EtOH, PD184352, RDEA119 (Rafametinib), PD98059, BIX02189, MEK162 (Binimetinib), AS-703026 (Pimasertib), SL-327, BIX02188, AZD8330, TAK-733, cobimetinib, and PD318088.

[0248] In some embodiments of the present disclosure, the boronic acid compounds of the present disclosure are administered in association with both anthracyclines such as doxorubicin and cyclophosphamides (including pegylated liposomal doxorubicin).

[0249] In some embodiments of the present disclosure, the boronic acid compounds of the present disclosure are administered in association with both an anti-CD20 antibody and an anti-CD3 antibody, or with a bispecific CD20 / CD3 conjugate (including CD20 / CD3 BiTE).

[0250] In some embodiments of the present disclosure, the boronic acid compounds of the present disclosure are administered in association with a CD73 inhibitor, a CD39 inhibitor, or both. These inhibitors may be CD73 or CD39 conjugates (such as antibodies, antibody fragments, or antibody mimetic compounds) that inhibit ectonucleosidase activity. The inhibitors may be small molecule inhibitors of ectonucleosidase activity, such as 6-N,N-diethyl-β-γ-dibromomethylene-D-adenosine-5'-triphosphate trisodium salt hydrate, PSB069, or PSB06126.

[0251] In some embodiments of the present disclosure, the boronic acid compounds of the present disclosure are administered in conjunction with poly-ADP-ribose polymerase (PARP). Exemplary PARP inhibitors include olaparib, niraparib, rucaparib, talazoparib, veliparib, CEP9722, MK4827, and BGB-290.

[0252] In some embodiments of this disclosure, the boronic acid compounds of this disclosure are administered in association with an oncolytic virus. An exemplary oncolytic virus is talimodine laherparepvec.

[0253] In some embodiments of the present disclosure, the boronic acid compounds of the present disclosure are administered in association with a CSF-1 antagonist, such as a drug that binds to CSF-1 or CSF1R and inhibits the interaction of CSF-1 with CSF1R on macrophages. Exemplary CSF-1 antagonists include emactuzumab and FPA008.

[0254] In some embodiments of the present disclosure, the boronic acid compounds of the present disclosure are administered in association with an anti-CD38 antibody. Exemplary anti-CD38 antibodies include daratumumab and isatuximab.

[0255] In some embodiments of the present disclosure, the boronic acid compounds of the present disclosure are administered in association with an anti-CD40 antibody. Exemplary anti-CD40 antibodies include selicrelumab and dacetuzumab.

[0256] In some embodiments of the present disclosure, the boronic acid compounds of the present disclosure are administered in conjunction with an anaplastic lymphoma kinase (ALK) inhibitor. Exemplary ALK inhibitors include alectinib, crizotinib, and ceritinib.

[0257] In some embodiments of the present disclosure, the boronic acid compounds of the present disclosure are administered in association with a multi-kinase inhibitor or an anti-angiogenic inhibitor that inhibits one or more selected from the group consisting of family members of VEGFR, PDGFR, and FGFR. Exemplary inhibitors include axitinib, cediranib, linifanib, motesanib, nintedanib, pazopanib, ponatinib, regorafenib, sorafenib, sunitinib, tivozanib, vatalanib, LY2874455, or SU5402.

[0258] In some embodiments of the present disclosure, the boronic acid compounds of the present disclosure are administered in conjunction with one or more vaccines intended to stimulate an immune response to one or more predetermined antigens. The antigens may be administered directly to an individual, or they may be expressed in the individual from, for example, autologous or allogeneic tumor cell vaccines (e.g., GVAX), dendritic cell vaccines, DNA vaccines, RNA vaccines, virus-based vaccines, bacterial or yeast vaccines (e.g., Listeria monocytogenes or Saccharomyces cerevisiae). See, for example, Guo et al., Adv. Cancer Res. 2013; 119: 421-475; Obeid et al., Semin Oncol. 2015 August; 42(4): 549-561. The target antigen may also be a fragment or fusion polypeptide containing an immunoactive portion of the antigens listed in the table.

[0259] In some embodiments of this disclosure, the boronic acid compounds of this disclosure are administered in association with one or more antiemetics, including but not limited to: casopitant (GlaxoSmithKline), netupitant (MGI-Helsinn), and other NK-1 receptor antagonists; palonosetron (marketed as Aloxi by MGI Pharma); aprepitant (marketed as Emend by Merck and Co.; Rahway, NJ); diphenhydramine (marketed as Benadryl by Pfizer; New York, NY); hydroxyzine (marketed as Atarax by Pfizer; New York, NY); metoclopramide (marketed as Reglan by AH Robins Co.; Richmond, Va.); lorazepam (marketed as Ativan by Wyeth; Madison, NJ); alprazolam (marketed as Xanax by Pfizer; New New York, NY), haloperidol (marketed as Haldol by Ortho-McNeil; Raritan, NJ), droperidol (Inapsine), dronabinol (marketed as Marinol by Solvay Pharmaceuticals, Inc.; Marietta, Ga.), dexamethasone (marketed as Decadron by Merck and Co.; Rahway, NJ), methylprednisolone (marketed as Medrol by Pfizer; New York, NY), prochlorperazine (marketed as Compazine by Glaxosmithkline; Rearch Triangle Park, NC.), granisetron (marketed as Kytril by Hoffmann-La Roche Inc.; Nutley, NJ), ondansetron (marketed as Zofran by Glaxosmithkline; Rearch Triangle Park, NC.), drasetron (marketed as Anzemet by Sanofi-Aventis; New York, NY). Tropicetron (sold as Navoban by Novartis; East Hanover, NJ).

[0260] Other side effects of cancer treatment include red and white blood cell deficiencies. Therefore, in some embodiments of this disclosure, boronic acid compounds are administered in conjunction with agents that treat or prevent such deficiencies, such as filgrastim, PEG-filgrastim, erythropoietin, epoetin-α, or darbepoetin-α.

[0261] In some embodiments, boronic acid compositions are administered in conjunction with anti-cancer radiotherapy. For example, in some embodiments of this disclosure, the radiotherapy is external beam radiation therapy (EBT) (a method for delivering a beam of high-energy X-rays to the site of a tumor). The beam is produced outside the patient's body (e.g., by a linear accelerator) and targets the tumor site. These X-rays can destroy cancer cells, and careful treatment planning allows for the preservation of surrounding normal tissue. No radioactive source is placed inside the patient's body. In some embodiments of this disclosure, the radiotherapy is proton beam therapy (a type of conformal irradiation therapy in which protons are bombarded with diseased tissue instead of X-rays). In some embodiments of this disclosure, the radiotherapy is conformal external beam radiation therapy (a procedure that uses advanced techniques to tailor the radiotherapy to the individual's body structure). In some embodiments of this disclosure, the radiotherapy is brachytherapy (temporary placement of radioactive material inside the body, typically used to deliver an additional dose or boost of radiation to a particular area). [Examples]

[0262] The following non-restrictive examples are illustrative.

[0263] Examples 1-3: In the following Examples 1-3, Tables 1, 2, and 3 summarize the effectiveness of sulfur compounds in stabilizing bortezomib against oxidation and decomposition reactions. The following procedures were used for each of the following Examples 1-3.

[0264] Sample preparation: 100 mg of API (active pharmaceutical ingredient) was suspended in 100 mL of water or another solvent in a different ratio, and thoroughly mixed using a vortex mixer and sonicator (gently warmed if necessary) to prepare a 1 mg / mL solution A. 10 mg of each excipient was weighed into a vial, 1 mL of solution A was added, and thoroughly mixed to obtain test solution B (10:1 excipient / API). 100 μL of solution B was taken and diluted to 1 mL with solution A to obtain test solution C (1:1 excipient / API). 100 μL of solution C was taken and diluted to 1 mL with solution A to obtain test solution D (0.1:1 excipient / API). For the solid powder form of bortezomib containing the excipients, the method described in U.S. Patent Application Publication 2005 / 0282742 was adopted. 280 mg of API was dissolved in 112 mL of tert-butanol and diluted with water to 280 mL to obtain solution E. 80 mg of each excipient was weighed into a vial, 8 mL of solution E was added, and after thorough mixing, the mixture was freeze-dried to obtain the test powder (excipient / API in a 10:1 ratio).

[0265] LC-MS analysis: The analysis was performed on a Hewlett Packard LC / MSD system equipped with a UV detector (monitoring at 254 nm) using an Agilent Eclipsc Plus C18 RP-HPLC column (4.6 x 50 mm, 1.8 μm) with solvent gradients A) water (0.1% TFA) and B) acetonitrile (0.08% TFA) at 0.5 mL / min. Typically, the eluent gradient was 2% B for the first 3 minutes, then 2% to 98% B for 6 minutes, and maintained at 98% B for the next 5 minutes.

[0266] Example 1 : Aqueous formulations containing bortezomib in various weight ratios along with several sulfur-containing compounds were evaluated for their stability over time based on the purity of bortezomib. The results are shown in Table 2 below, which shows the results for specific sulfur compounds in aqueous solution at 45°C.

[0267] [Table 2-1] [Table 2-2]

[0268] Example 2 : Aqueous formulations containing bortezomib in various weight ratios with several sulfur-containing compounds were evaluated for their stability over time based on the purity of bortezomib in aqueous solution (1 mg / mL, 50°C). The results are shown in Table 3 below, which shows the results for specific sulfur compounds and several sulfur-free compounds at 50°C.

[0269] [Table 3]

[0270] Example 3 : Aqueous formulations containing bortezomib with several sulfur-containing compounds in various weight ratios were evaluated for their stability over time based on the purity of bortezomib at 1 mg / mL in water, propylene glycol (PG), or butylene glycol (BG) at 50°C. The results are shown in Table 4 below, where the table shows the results for specific sulfur compounds in aqueous, non-aqueous, and mixed solvent systems. Note that N-acetyl-L-methionine provides essentially complete protection for bortezomib, but in the absence of N-acetyl-L-methionine, bortezomib undergoes 67% degradation.

[0271] [Table 4]

[0272] Example 4 : The effect of N-acetylmethionine (NAM) on the stability of compound 6522 (the structure of compound 6522 is shown immediately below) at 80°C in the presence of the free radical generator AAPH2,2'-azobis-(2-amidopropane) hydrochloride. The results are shown in Table 5 below. [ka]

[0273] [Table 5]

[0274] Example 5 : The effect of 5 nM N-acetylmethionine (NAM) on the stability of compound 6522 (structure of compound 6522 shown above) at 60°C in the presence of AAPH2,2'-azobis-(2-amidopropane) hydrochloride as a function of pH. The results are shown in Table 6 below.

[0275] [Table 6]

[0276] Example 6 : The effects of specified sulfur-containing compounds and other stabilizer compounds (including sulfur-free stabilizer compounds), as well as mixtures of sulfur-containing and sulfur-free stabilizer compounds, on boronic acid compound 6555 (structure shown above) at various pH values. The results are shown in Tables 7, 8, and 9 below.

[0277] [Table 7-1] [Table 7-2]

[0278] [Table 8-1] [Table 8-2]

[0279] [Table 9-1] [Table 9-2] [Table 9-3] [Table 9-4] [Table 9-5]

[0280] Example 7: Synthesis of compounds 4613B and 4613C [ka]

[0281] Experiment Section Reagents obtained from commercial sources were used without further purification. L-boroPro-pn was synthesized using the previously described synthesis method (TS. J. Coutts et al. J. Med. Chem. 1996, 39, 2087 - 2094). All target compounds were purified by RP-HPLC using a Varian semi-preparative system with a Discovery C18 569226-URP-HPLC column. The mobile phase was typically prepared by mixing water (0.1% TFA) with acetonitrile (0.08% TFA) in a gradient concentration. Purity determined by HPLC analysis was over 95%. Mass spectra and HPLC retention times were recorded using an Eclipse Plus C18 RP-HPLC column (4.6 × 50 mm, 1.8 μm) with a solvent gradient of A) water (0.1% TFA) and B) acetonitrile (0.08% TFA) at 0.5 mL / min on a Hewlett Packard HPLC / MSD system equipped with a UV detector (monitoring at 215 nm). Unless otherwise noted, all HPLC retention times are given for an eluent gradient of 2% B for the first 3 minutes, followed by a 2% to 98% B gradient over 6 minutes, and maintained for the next 5 minutes. 1 HC NMR spectra were recorded using a Bruker Avance 300 MHz NMR spectrometer with a 5 mm inverse multinuclear probe. Chemical shifts are reported in ppm(δ) relative to DSS (in D2O).

[0282] Synthesis of Compound 2 To a stirred solution of N-Boc-D-Ala-OH (1, 1.9 g, 10 mmol) in anhydrous DMF (40 mL), L-boroPro-pn.HCl (3.0 g, 10.5 mmol), HATU (4.0 g, 10.5 mmol), and DIEA (4.0 mL, 23 mmol) were added under ice bath cooling. The resulting mixture was stirred at room temperature for 2 hours and then concentrated under vacuum. The residue was dissolved in ethyl acetate (150 mL) and washed sequentially with 0.1N KHSO4 (3 × 40 mL), NaHCO3 aqueous solution (3 × 40 mL), and brine (30 mL). The organic phase was dried over anhydrous MgSO4, filtered, and evaporated under vacuum to obtain N-Boc-D-Ala-L-boroPro-pn, which was then added to a 4N HCl solution in dioxane (30 mL) under ice bath cooling. The resulting mixture was stirred at room temperature for 2 hours and then concentrated in a vacuum. The residue was co-evaporated with dichloromethane (3 × 30 mL) in a vacuum and completely dried. Compound 2 was thus obtained as a white powder (3.3 g, 92% in 2 steps).

[0283] Synthesis of compound 3860 Compound 2 (375 mg, 1.05 mmol), HATU (400 mg, 10.5 mmol), and DIEA (0.40 mL, 2.3 mmol) were added to a stirred solution of 6-(N'-Boc-hydrazino)-nicotinic acid (253 mg, 1 mmol) in anhydrous DMF (4 mL) under ice bath cooling. The resulting mixture was stirred at room temperature for 2 hours and then concentrated under vacuum. The residue was dissolved in dichloromethane (50 mL) and washed sequentially with aqueous solution of NaHCO3 (3 × 10 mL) and brine (10 mL). The organic phase was dried over anhydrous MgSO4, filtered, and evaporated under vacuum to obtain compound 3a. This was then dissolved in dry dichloromethane (5.0 mL) and cooled to -78°C while adding BCl3 (1 M in dichloromethane, 5.0 mL) dropwise. The mixture was stirred at -78°C for 1 hour and then concentrated under vacuum. The residue was partitioned between ether (5 mL) and water (5 mL). The aqueous phase was washed twice with an additional ether (2 × 5 mL), concentrated under reduced pressure, and further purified by semi-preparative RP-HPLC to obtain compound 3860 as a white powder (280 mg, 65%). LC-MS (ESI +)m / z (relative intensity): 322.1 ([M+H] + ,95);304.1([M-H2O+H] + ,100);tr=7.4 minutes.

[0284] Synthesis of compound 4613B Compound 3860 (11 mg, 0.03 mmol) was added at room temperature to a stirred solution of IRDye 800CW NHS ester (11.7 mg, 0.01 mmol) in phosphate buffer (10 mL) at pH 7.8. The pH was adjusted with 5% NaHCO3 as needed. The resulting mixture was stirred at the same temperature for 3 hours and purified by semi-preparative RP-HPLC to obtain compound 4613B as a cottony green powder (11 mg, 84%). LC-MS (ESI) + )m / z (relative intensity): 1288.1 ([M-H2O+H] + ,25),635.8([(M-2xH2O) / 2+H] + ,100);tr=7.7 minutes. 1 HNMR(D2O): δ1.10-1.35(m,17H),1.50-2.02(m,14H),2.20-2.80(m,6H),2.88-2.93(m,3H),3.52-3.55(m,2H),3.88-3.9 1(m,4H),4.58-4.61(m,1H),6.00-6.09(m,1H),7.12-7.21(m,5H),7.67-7.76(m,9H),8.25(d,J=9.3Hz,1H),8.40(s,1H).

[0285] Synthesis of compound 4634 Compound 4634 was obtained by reacting 6-(N'-Boc-hydrazino)-benzoic acid with compound 2 in a manner similar to the preparation of 3860. LC-MS (ESI) + )m / z (relative intensity): 605.5 ([2x(M-H2O)+H] + ,100),303.3([M-H2O+H] + ,67);tr=7.7 minutes.

[0286] Synthesis of compound 4613C Compound 4613C was obtained by reacting IRDye 800CW NHS ester with 4634 in a manner similar to that used to prepare 4613B from 3860. LC-MS (ESI) + )m / z (relative intensity): 1287.6 ([M-H2O+H] + ,88),635.6([(M-2xH2O) / 2+H] + ,100);tr=7.9 minutes.

[0287] Example 8: Synthesis of compounds 4536B, 6481, and 5183 [ka]

[0288] Synthesis of compound 4536B To a stirred solution of tri-tert-butyl 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (57 mg, 0.10 mmol) in anhydrous DMF (1 mL), HBTU (40 mg, 0.105 mmol) and DIEA (40 μl, 0.23 mmol) were added under ice bath cooling. The resulting mixture was stirred at room temperature for 15 minutes. Compound 4634 (40 mg, 0.11 mmol) was added to the solution and stirred for a further 2 hours. The mixture was purified by semi-preparative RP-HPLC, dried, and then redissolved in dichloromethane (0.5 mL). TFA (2 mL) was added, and the reaction mixture was stirred overnight at room temperature. After removal of TFA and dichloromethane, water (2 mL) was added, and the resulting mixture was stirred at room temperature for 1 hour to obtain the crude product, which was directly purified by semi-preparative RP-HPLC to obtain 85 mg of compound 4536B as a white powder. LC-MS (ESI + )m / z (relative intensity): 689.2 ([M-H2O+H] + ,100);tr=7.4 minutes. 1 H NMR(D2O): δ1.43(d,J=7.0Hz,3H),1.65-1.71(m,1H),2.00-2.15(m,3H),2.85-3.90(m,26H),6.93-7.00(m,2H),7.71-7.78(m,2H).

[0289] Synthesis of compound 6481 Compound 4536B (6 mg) was dissolved in water (1.0 mL). CuCl2 (1.0 M in water, 20 μl) was added. The resulting mixture was stirred for half an hour and then purified by semi-preparative HPLC elution with 10% to 50% B (solvent A: 0.05% TFA aqueous solution; solvent B: acetonitrile). The desired fraction was collected and freeze-dried to obtain 4 mg of compound 6481 as a blue-green powder. LC-MS (ESI) + )m / z (relative intensity): 750.9 ([M-H2O+H] + ,49),745.7([M-H2O-H] + ,29),377.5([(M-2xH2O) / 2+H] + ,100);tr=7.4 minutes.

[0290] Synthesis of compound 5183 Compound 4536B (6 mg) was dissolved in water (1.0 mL). GdCl3 (1.0 M in water, 20 μl) was added. The resulting mixture was adjusted to pH 6 with 1N NH3 / H2O, stirred for half an hour, and then purified by semi-preparative HPLC eluting with 10% to 50% B (solvent A: 0.05% TFA aqueous solution; solvent B: acetonitrile). The desired fraction was collected and freeze-dried to obtain 4 mg of compound 5183 as a white powder. LC-MS (ESI) + )m / z (relative intensity): 843.9 ([M-H2O+H] + ,32),421.8([(M-2xH2O) / 2+H] + ,100); tr=9.1 minutes (0-3 minutes: 5% B; 3-9 minutes: 5-15% B; 9-14 minutes: 15-25% B).

[0291] Example 9: Synthesis of compounds 6486S~6489S and 6486~6489 [ka]

[0292] Synthesis of compound 6487S Compound 4634 was first coupled with N-Boc-Gly-OH, and then the Boc protection was removed under the same conditions as the preparation of compound 2 from Boc-D-Ala-OH and boroPro-pn.HCl; this was then coupled with tri-tert-butyl 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, and all -OtBu ester protection was removed under the same conditions as the preparation of compound 4536B from 4634 to obtain compound 6487S as a white powder. LC-MS(ESI + )m / z (relative intensity): 746.4 ([M-H2O+H] + ,100);tr=7.4 minutes.

[0293] Synthesis of compound 6487 Compound 6487 was prepared as a blue-green powder from compound 6487 by the same method used to prepare compound 6481 from 4536B. LC-MS (ESI + )m / z (relative intensity): 807.5 ([M-H2O+H] + ,30),802.9([M-H2O-H] + ,100);tr=7.7 minutes.

[0294] Synthesis of compound 6486S Compound 6486S was prepared as a white powder by the same method used to prepare 6487S. LC-MS (ESI + )m / z (relative intensity): 803.3 ([M-H2O+H] + ,100);tr=7.6 minutes.

[0295] Synthesis of compound 6486 Compound 6486 was prepared from compound 6486S as a blue-green powder by the same method used to prepare 6481 from 4536B. LC-MS (ESI + )m / z (relative intensity): 862.7 ([M-H2O+H] + ,100);tr=7.7 minutes.

[0296] Synthesis of compound 6488S Compound 6488S was prepared as a white powder by the same method used to prepare 6487S. LC-MS (ESI + )m / z (relative intensity): 762.8 ([M-H2O+H] + ,100);tr=7.4 minutes.

[0297] Synthesis of compound 6488 Compound 6488 was prepared from compound 6488S as a blue-green powder by the same method used to prepare compound 6481 from 4536B. LC-MS (ESI + )m / z (relative intensity): 822.3 ([M-H2O+H] + ,100);tr=7.7 minutes.

[0298] Synthesis of compound 6489S Compound 6489S was prepared as a white powder by the same method used to prepare 6487S. LC-MS (ESI + )m / z (relative intensity): 774.4 ([M-H2O+H] + ,100);tr=7.5 minutes.

[0299] Synthesis of compound 6489 Compound 6489 was prepared from compound 6489S as a blue-green powder by the same method used to prepare compound 6481 from 4536B. LC-MS (ESI + )m / z (relative intensity): 836.8 ([M-H2O+H] + ,100),832([M-H2O-H] + ,63);tr=7.6 minutes.

[0300] Example 10: Synthesis of compounds 6572 and 6572CU [ka]

[0301] Example 11: Synthesis of compounds 6521-6522 and 6521CU-6522CU : [ka]

[0302] Example 12: Synthesis of compounds 6549 and 6551 : [ka]

[0303] Example 13: Synthesis of compounds 6555 and 6556 : [ka]

[0304] Example 14: Synthesis of compounds 6508-6509 and 6508CU-6509CU : [ka]

[0305] Table 10 shows the compounds from Examples 7 to 14. [Table 10-1] [Table 10-2] [Table 10-3] [Table 10-4] [Table 10-5] [Table 10-6]

[0306] Example 15: Synthesis of GHK analogs 6415 and 6433 [ka]

[0307] Synthesis of compound 6415 Compound 6415 was prepared as a white powder by steps 5 through 7 of the following steps, as shown in Scheme 4. LC-MS (ESI +)m / z (relative intensity): 767.2 ([M-H2O+H] + ,100);tr=7.6 minutes.

[0308] Synthesis of compound 6433 Compound 6415 (29 mg) was dissolved in water (0.2 mL). Cu(OAc)2 (0.3 M in water, 103 μl) was added. The resulting mixture was stirred for half an hour and directly freeze-dried to obtain 11 mg of compound 6433 as a green-blue powder (11 mg). LC-MS (ESI) + )m / z (relative intensity): 829.2 ([M-H2O+H] + ,100); tr=7.5 min (Note: The solvent for this LCMS was plain. No TFA was added).

[0309] Example 16: In vitro assay Biological materials: For the in vitro IC50 determination assay, recombinant human DPPIV, DPP9, FAP, and PREP were purchased from R&D Systems, and DPP8 was purchased from Biomol International. The buffer systems used were A (25 mM Tris, pH 8.0), B (50 mM Tris, pH 7.5), C (50 mM Tris, 140 mM NaCl, pH 7.5), D (25 mM Tris, 250 mM NaCl, pH 7.5), and E (20 mM Tris, 20 mM KCl, pH 7.4). The fluorescence-generating substrates were Gly-Pro-AMC, Z-GlyPro-AMC, or Suc-Gly-Pro-AMC, or an N-terminally blocked FAP-specific substrate, purchased from Bachem. The cell culture medium was RPMI 1640 without phenol red, supplemented with 2 mM glutamine, 10 mM HEPES, 1 mM sodium pyruvate, 4500 mg / L glucose, 100 IU / mL penicillin, and 100 μg / mL streptomycin. Substrate specificity assay: The peptide library (0.21 mM) was incubated with 1 nM FAP in buffer E at 37°C for 24 hours. This reaction was quenched by adding 1.2 N HCl. Samples were analyzed by reverse-phase HPLC-MS on a Thermo Finnigan LCQ Duo, and peaks in the resulting base peak chromatograms were quantified. Relative cleavage values ​​were determined by comparing the quenched abundance of intact peptides with that in the initial library.

[0310] In vitro enzyme IC50 assays. Enzyme activity of DPPIV, DPP8, DPP9, FAP, and PREP was measured at 25°C using a Molecular Devices M2e multi-detection microtiter plate reader, monitoring fluorescence at excitation wavelength 380 nm and emission wavelength 460 nm. The substrate was either H-Gly-Pro-AMC for the DPPIV, DPP8, and DPP9 assays, or Z-Gly-Pro-AMC for the FAP and PREP assays. The reaction mixture consisted of 25 μM substrate, enzyme, buffer A (DPPIV and DPP9), buffer B (DPP8), buffer C (FAP), or buffer D (PREP), and an appropriate amount of inhibitor (10 -4 ~10 -11 The solution contained 210 μL of inhibitor (in the M range) in total volume. The final enzyme concentrations were 0.1, 0.8, 0.4, 1.2, and 0.6 nM for DPPIV, DPP8, DPP9, FAP, and PREP, respectively. The IC50 value is defined as the concentration of the inhibitor required to reduce enzyme activity by 50% after pre-incubating with the enzyme at 25°C for 10 minutes before substrate addition. Inhibitor stock solutions (100 mM) were prepared for compounds 1 and 20 in either pH 2.0 HCl solution or DMSO. The solutions prepared in pH 2.0 were pre-incubated at 25°C for 4 hours and then diluted. Immediately before the start of the experiment, the 100 mM stock was diluted in appropriate assay buffer for 10 minutes. -3 Further dilution was performed using medium (M), from which 1:10 serial dilutions were prepared. All inhibitors were tested in triplicates.

[0311] [Table 11-1] [Table 11-2] [Table 11-3] [Table 11-4] [Table 11-5] Table 11-6 Table 11-7 Table 11-8 Table 11-9

[0312] Example 17: Exemplary ligand synthesized by a similar method

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[0313] Example 18: Synthesis of DOTA-PNP Scheme 10 [ka]

[0314] Experiment Section Reagents obtained from commercial sources were used without further purification. All target compounds were purified by RP-HPLC using a Varian semi-preparative system with a Discovery C18 569226-U RP-HPLC column. The mobile phase for semi-preparative HPLC was typically prepared by mixing water (4.8 mM HCl) with acetonitrile in a gradient concentration. Mass spectra and HPLC retention times were recorded on a Hewlett Packard HPLC / MSD system with an Eclipse Plus C18 RP-HPLC column (4.6 × 50 mm, 1.8 μm), a solvent gradient of A) water (0.1% TFA) and B) acetonitrile, at 0.5 mL / min, and a UV detector (monitoring at 215 nm). Unless otherwise noted, all HPLC retention times are given for an eluent gradient of 2% B for the first 3 minutes, then 2% to 98% B over 6 minutes, and maintaining this for the next 6 minutes.

[0315] DOTA-PNP synthesis DOTA-PNP was synthesized using the previously described synthesis method (Walter Mier, etc. Bioconjugate Chem., 2005, 16, 237 - 240. TS. J. Coutts et al. J. Med. Chem. 1996, 39, 2087 - 2094). DOTA (AstaTech, BN21603; 500 mg, 1.24 mmol) was dissolved in 10 mL of water. A solution of 1.24 mmol of 4-nitrophenol (TCI America, N022025G) in 8 mL of acetonitrile was added. A solution of 255 mg (1.24 mmol) of N,N'-dicyclohexylcarbodiimide in 8 mL of pyridine was added dropwise with vigorous stirring. The reaction mixture was stirred for 90 minutes and concentrated to dryness under reduced pressure. The residue was dissolved in a 20% aqueous acetonitrile solution. The suspension was filtered to remove N,N'-dicyclohexylurea, and the filtrate was purified by a semi-preparative Discovery C18 569226-U RP-HPLC column (21.2 mm × 25 cm, 5 μm) equipped with a UV detector (monitoring at 215 nm). A gradient elution system was used, utilizing mobile phase A (4.8 mM HCl) and mobile phase B (acetonitrile). The gradient was performed at a flow rate of 20 mL / min, starting with 98% A and 2% B for 5 minutes; increasing to 70% A and 30% B over 15 minutes; and holding for a further 5 minutes. The combined fraction was directly lyophilized to obtain DOTA-PNP as a white powder (4 × HCl salt, 250 mg, 30%). LC-MS (ESI) + )m / z (relative intensity): 526.1 ([M+H] + ,100);tr=7.7 minutes.

[0316] supporting material Mass spectra and HPLC retention times were recorded on a Hewlett Packard HPLC / MSD system equipped with a UV detector (monitoring at 215 nm) using a ZORBAX Eclipse Plus C18 RP-HPLC column (4.6 × 50 mm, 1.8 μm) with a solvent gradient of A) water (0.1% TFA) and B) acetonitrile at 0.5 mL / min. The eluent gradient was 2% B for the first 3 minutes, then 2% to 98% B over 6 minutes, and maintained for the next 5 minutes (0–3 min: 2% B; 3–9 min: 2–98% B; 9–15 min: 98% B). MS was performed in positive mode. Data were analyzed using Agilent Chemstation software.

[0317] Example 19: Synthesis of 6555 / 6555LU / 6555GA Scheme 11 [ka]

[0318] Solubility and storage After lyophilization, the target compounds 6555, 6555LU, or 6555GA are readily soluble in water (solubility > 50 mg / mL). In aqueous solution at approximately pH 3, the inventors observed no signs of degradation during the HPLC purification and subsequent lyophilization processes. For long-term storage, the target compounds should be kept in solid form in a freezer at temperatures below -15°C. For short-term storage, a refrigerator (+4°C) should suffice.

[0319] Experiment Section Reagents obtained from commercial sources were used without further purification. L-boroPro-pn was synthesized using the previously described synthesis method (TS. J. Coutts et al. J. Med. Chem. 1996, 39, 2087 - 2094). All target compounds were purified by RP-HPLC using a Varian semi-preparative system with a Discovery C18 569226-U RP-HPLC column. The mobile phase for semi-preparative HPLC was typically prepared by mixing water (0.1% TFA) with acetonitrile in a gradient concentration. Mass spectra and HPLC retention times were recorded on a Hewlett Packard HPLC / MSD system equipped with a UV detector (monitoring at 215 nm) using an Eclipse Plus C18 RP-HPLC column (4.6 × 50 mm, 1.8 μm) with a solvent gradient of A) water (0.1% TFA) and B) acetonitrile at 0.5 mL / min. Unless otherwise specified, all HPLC retention times are given for an eluent gradient where the first 3 minutes are in 2% B, followed by a 6-minute transition from 2% to 98% B, and then maintained for the next 6 minutes.

[0320] Synthesis of Intermediate 1 To a stirred solution of N-Boc-D-Ala-OH (Aldrich, 15048-25G; 1.9g, 10 mmol) in anhydrous DMF (40 mL), L-boroPro-pn.HCl (3.0 g, 10.5 mmol), HATU (4.0 g, 10.5 mmol), and DIEA (4.0 mL, 23 mmol) were added under ice bath cooling. The resulting mixture was stirred at room temperature for 2 hours and then concentrated under vacuum. The residue was dissolved in ethyl acetate (150 mL) and washed sequentially with 0.1N KHSO4 (3 × 40 mL), NaHCO3 (3 × 40 mL) aqueous solution, and brine (30 mL). The organic phase was dried over anhydrous MgSO4, filtered, and evaporated under vacuum to obtain N-Boc-D-Ala-L-boroPro-pn, which was purified by silica gel flash chromatography eluted with ethyl acetate / hexane; and then added to a solution of 4N HCl in dioxane (30 mL) under ice water cooling. The resulting mixture was stirred at room temperature for 2 hours and then concentrated under vacuum. The residue was co-evaporated with dichloromethane (3 × 30 mL) under vacuum and completely dried. Compound 1 was thus obtained as a white powder (3.3 g, 92% in 2 steps).

[0321] Synthesis of Intermediate 2 Compound 1 (750 mg, 2.1 mmol), HATU (800 mg, 2.1 mmol), and DIEA (0.80 mL, 4.6 mmol) were added to a stirred solution of 4-[(tert-butoxycarbonylamino)methyl]benzoic acid (TCI, B4305; 505 mg, 2 mmol) in anhydrous DMF (8 mL) under ice bath cooling. The resulting mixture was stirred at room temperature for 2 hours and then concentrated under vacuum. The residue was dissolved in dichloromethane (100 mL) and washed sequentially with 0.1 N KHSO4 (3 × 15 mL), NaHCO3 aqueous solution (3 × 15 mL), and brine (10 mL). The organic phase was dried over anhydrous MgSO4, filtered, and evaporated under vacuum to obtain 4-(N-Boc-aminomethyl-PhCO-D-Ala-L-boroPro-pn), which was purified by silica gel flash chromatography eluted with ethyl acetate / hexane; then added to a solution of 4N HCl in dioxane (10 mL) under ice water cooling. The resulting mixture was stirred at room temperature for 2 hours and then concentrated under vacuum. The residue was co-evaporated with dichloromethane (3 × 20 mL) under vacuum and completely dried. Thus, compound 2 was obtained as a white powder (830 mg, 85% in 2 steps). LC-MS (ESI + )m / z (relative intensity): 453.7 ([M+H] + ,100);tr=9.0 minutes.

[0322] Synthesis of compound 6555 (Method I) To a stirred solution of DOTA-(OtBu)3 (AstaTech, 67012, CAS: 137076-54-1; 172 mg, 0.3 mmol) in anhydrous DCM (3 mL), intermediate compound 2 (162 mg, 0.33 mmol), PyBOP (172 mg, 0.33 mmol), and DIEA (0.12 mL, 0.69 mmol) were added under ice bath cooling. The resulting mixture was stirred at room temperature for 3 hours, then diluted further with dichloromethane (30 mL), and sequentially washed with 5% citric acid (3 × 5 mL), NaHCO3 aqueous solution (3 × 5 mL), and brine (5 mL). The organic phase was dried over anhydrous MgSO4, filtered, and evaporated under vacuum to obtain a crude intermediate, which was then redissolved in dichloromethane (1.5 mL) and TFA (6 mL). The resulting mixture was stirred overnight at room temperature. After removing TFA and dichloromethane, water (9 mL) was added, and the resulting mixture was stirred at room temperature for 1 hour. Then, phenylboronic acid (48 mg, 0.39 mmol), acetonitrile (3 mL), and TBME (18 mL) were added. The resulting mixture was stirred at room temperature for 3 hours, and the separated aqueous phase was washed with further TBME. The aqueous phase was slightly concentrated under vacuum and purified by a semi-preparative Discovery C18 569226-U RP-HPLC column (21.2 mm × 25 cm, 5 μm) equipped with a UV detector (monitoring at 215 nm). A gradient elution system was used with mobile phase A (0.1% TFA) and mobile phase B (acetonitrile). The gradient was performed at a flow rate of 20 mL / min, starting with 95% A and 5% B for 5 minutes; increasing to 70% A and 30% B over 20 minutes; then increasing to 2% A and 98% B over 1 minute, and holding for a further 5 minutes. The combined fraction was directly freeze-dried to obtain 6555 as a white powder (4×TFA salt, 130 mg, 37% across 3 steps). LC-MS (ESI + )m / z (relative intensity): 688.0 ([M-H2O+H] + ,100),345.4(63);tr=7.6 minutes.

[0323] Synthesis of compound 6555 (Method II) To a stirred solution of DOTA-PNP (synthesized in-house, 204 mg, 0.30 mmol) and intermediate compound 2 (189 mg, 0.33 mmol) in anhydrous DMF (4 mL), TEA (360 μl, 2.07 mmol) was added under ice bath cooling. The resulting mixture was stirred overnight at room temperature. The reaction mixture was then concentrated under vacuum. Water (9 mL) was added, and the pH was further adjusted to approximately 1.5 with 1N TFA. Phenylboronic acid (48 mg, 0.39 mmol), acetonitrile (3 mL), and TBME (18 mL) were added. The resulting mixture was stirred at room temperature for 3 hours and post-treated as described above to obtain 6555 as a white powder (4 × TFA salt, 140 mg, 40% in 2 steps).

[0324] Synthesis of compound 6555LU Compound 6555 (10 mg, 8.6 μmol) was added to a solution of LuCl3 (18 mg, 64 μmol) in acetate buffer (0.23 M, pH 5.2, 3 mL). The resulting mixture was stirred at 90°C for 23 minutes and then purified using a semi-preparative Discovery C18 569226-U RP-HPLC column (21.2 mm × 25 cm, 5 μm) with a UV detector (monitoring at 215 nm). A gradient elution system was used, utilizing mobile phase A (0.05% TFA in water) and mobile phase B (acetonitrile). The gradient was started at a flow rate of 20 mL / min with 95% A and 5% B for 5 minutes; increased to 70% A and 30% B over 20 minutes; then increased to 2% A and 98% B over 1 minute, and held for another 5 minutes. The combined fraction was directly freeze-dried to obtain 6555 LU as a white powder (4×TFA salt, 5 mg, 44%). LC-MS (ESI + m / z (relative intensity): 859.5 (100); tr = 15.2 mins (see attached LCMS and conditions).

[0325] Synthesis of compound 6555GA Compound 6555 (10 mg, 8.6 μmol) was added to a solution of GaCl3 (12 mg, 66 μmol) in acetate buffer (0.23 M, pH 5.2, 4 mL). The resulting mixture was stirred at 90°C for 23 minutes and then purified using a semi-preparative Discovery C18 569226-U RP-HPLC column (21.2 mm × 25 cm, 5 μm) with a UV detector (monitoring at 215 nm). A gradient elution system was used, utilizing mobile phase A (0.05% TFA in water) and mobile phase B (acetonitrile). The gradient was started at a flow rate of 20 mL / min with 95% A and 5% B for 5 minutes; increased to 70% A and 30% B over 20 minutes; then increased to 2% A and 98% B over 1 minute, and held for another 5 minutes. The combined fraction was directly freeze-dried to obtain 6555GA as a white powder (4×TFA salt, 7 mg, 66%). LC-MS (ESI + m / z (relative intensity): 754.4 (100); tr = 16.9 mins (see attached LCMS and conditions).

[0326] supporting material Compound 6555 LC-MS spectrum of compound 6555: LC-MS was performed using a Hewlett Packard HPLC / MSD system equipped with a UV detector (monitoring at 215 nm) and a ZORBAX Eclipse Plus C18 RP-HPLC column (4.6 × 50 mm, 1.8 μm). A gradient elution system was used, utilizing mobile phase A (0.1% TFA) and mobile phase B (acetonitrile). The gradient was started at a flow rate of 0.5 mL / min, beginning with 98% A and 2% B for 3 minutes; increasing to 2% A and 98% B over 6 minutes; and maintaining this for a further 5 minutes. Finally, the gradient parameters were returned to the initial starting conditions. MS was performed in positive mode. Data were analyzed using Agilent Chemstation software.

[0327] Compounds with 6555LU LC-MS was performed using a Hewlett Packard HPLC / MSD system equipped with a UV detector (monitored at 215 nm) and a Luna C18, 4.6 mm × 150 mm, 3.0 μm, 100A column. A gradient elution system was used, utilizing mobile phase A (50 mM AcONH4) and mobile phase B (acetonitrile). The gradient was started at a flow rate of 1.0 mL / min, with 98% A and 2% B for 5 minutes; increased to 74% A and 26% B over 15 minutes; and then increased to 2% A and 98% B over 5 minutes. Finally, the gradient parameters were returned to the initial starting conditions. MS was performed in negative mode. Data were analyzed using Agilent Chemstation software.

[0328] 6555GA compound LC-MS was performed using a Hewlett Packard HPLC / MSD system equipped with a UV detector (monitoring at 215 nm) and a Luna C18, 4.6 mm × 150 mm, 3.0 μm, 100A column. A gradient elution system was used, utilizing mobile phase A (50 mM AcONH4) and mobile phase B (acetonitrile). The gradient was started at a flow rate of 1.0 mL / min with 98% A and 2% B for 5 minutes; increased to 74% A and 26% B over 15 minutes; and then increased to 2% A and 98% B over 5 minutes. Finally, the gradient parameters were returned to the initial starting conditions. MS was performed in negative mode. Data were analyzed using Agilent Chemstation software.

[0329] Example 20: Synthesis of 6952 / 6952LU / 6952GA Scheme 12 [ka]

[0330] Solubility and storage After lyophilization, the target compounds 6952, 6952LU, or 6952GA are readily soluble in water (solubility > 50 mg / mL). In aqueous solution at approximately pH 3, the inventors observed no signs of degradation during the HPLC purification and subsequent lyophilization processes. For long-term storage, the target compounds should be kept in solid form in a freezer at temperatures below -15°C. For short-term storage, a refrigerator (+4°C) should suffice.

[0331] Experiment Section Reagents obtained from commercial sources were used without further purification. L-boroPro-pn was synthesized using the previously described synthesis method (TS. J. Coutts et al. J. Med. Chem. 1996, 39, 2087 - 2094). All target compounds were purified by RP-HPLC using a Varian semi-preparative system with a Discovery C18 569226-U RP-HPLC column. The mobile phase for semi-preparative HPLC was typically prepared by mixing water (0.1% TFA) with acetonitrile in a gradient concentration. Mass spectra and HPLC retention times were recorded on a Hewlett Packard HPLC / MSD system equipped with a UV detector (monitoring at 215 nm) using an Eclipse Plus C18 RP-HPLC column (4.6 × 50 mm, 1.8 μm) with a solvent gradient of A) water (0.1% TFA) and B) acetonitrile, at 0.5 mL / min. Unless otherwise specified, all HPLC retention times are given for an eluent gradient where the first 3 minutes are in 2% B, followed by a 6-minute transition from 2% to 98% B, and then maintained for the next 6 minutes.

[0332] Synthesis of Intermediate 1 To a stirred solution of N-Boc-D-Ala-OH (Aldrich, 15048-25G; 1.9g, 10 mmol) in anhydrous DMF (40 mL), L-boroPro-pn.HCl (3.0 g, 10.5 mmol), HATU (4.0 g, 10.5 mmol), and DIEA (4.0 mL, 23 mmol) were added under ice bath cooling. The resulting mixture was stirred at room temperature for 2 hours and then concentrated under vacuum. The residue was dissolved in ethyl acetate (150 mL) and washed sequentially with 0.1N KHSO4 (3 × 40 mL), NaHCO3 aqueous solution (3 × 40 mL), and brine (30 mL). The organic phase was dried over anhydrous MgSO4, filtered, and evaporated under vacuum to obtain N-Boc-D-Ala-L-boroPro-pn, which was purified by silica gel flash chromatography eluted with ethyl acetate / hexane; and then added to a solution of 4N HCl in dioxane (30 mL) under ice water cooling. The resulting mixture was stirred at room temperature for 2 hours and then concentrated under vacuum. The residue was co-evaporated with dichloromethane (3 × 30 mL) under vacuum and completely dried. Compound 1 was thus obtained as a white powder (3.3 g, 92% in 2 steps).

[0333] Synthesis of Intermediate 2 Compound 1 (750 mg, 2.1 mmol), HATU (800 mg, 2.1 mmol), and DIEA (0.80 mL, 4.6 mmol) were added to a stirred solution of trans-4-(tert-butoxycarbonylaminomethyl)cyclohexanecarboxylic acid (TCI, B3253; 515 mg, 2 mmol) in anhydrous DMF (8 mL) under ice bath cooling. The resulting mixture was stirred at room temperature for 2 hours and then concentrated under vacuum. The residue was dissolved in dichloromethane (100 mL) and washed sequentially with 0.1 N KHSO4 (3 × 15 mL), aqueous NaHCO3 (3 × 15 mL), and brine (10 mL). The organic phase was dried over anhydrous MgSO4, filtered, and evaporated under vacuum to obtain N-Boc protected compound 2, which was purified by silica gel flash chromatography eluted with ethyl acetate / hexane; and then added to a solution of 4 N HCl in dioxane (10 mL) under ice bath cooling. The resulting mixture was stirred at room temperature for 2 hours and then concentrated under vacuum. The residue was co-evaporated with dichloromethane (3 × 20 mL) under vacuum and completely dried. In this way, compound 2 was obtained as a white powder (890 mg, 90% in 2 steps). LC-MS (ESI + )m / z (relative intensity): 459.9 ([M+H] + ,100);tr=8.9 minutes.

[0334] Synthesis of compound 6952 (Method I) To a stirred solution of DOTA-(OtBu)3 (AstaTech, 67012, CAS: 137076-54-1; 172 mg, 0.3 mmol) in anhydrous DCM (3 mL), intermediate compound 2 (162 mg, 0.33 mmol), PyBOP (172 mg, 0.33 mmol), and DIEA (0.12 mL, 0.69 mmol) were added under ice bath cooling. The resulting mixture was stirred at room temperature for 3 hours, then diluted further with dichloromethane (30 mL), and sequentially washed with 5% citric acid (3 × 5 mL), NaHCO3 aqueous solution (3 × 5 mL), and brine (5 mL). The organic phase was dried over anhydrous MgSO4, filtered, and evaporated under vacuum to obtain a crude intermediate, which was then redissolved in dichloromethane (1.5 mL) and TFA (6 mL). The resulting mixture was stirred overnight at room temperature. After removing TFA and dichloromethane, water (9 mL) was added, and the resulting mixture was stirred at room temperature for 1 hour. Then, phenylboronic acid (48 mg, 0.39 mmol), acetonitrile (3 mL), and TBME (18 mL) were added. The resulting mixture was stirred at room temperature for 3 hours, and the separated aqueous phase was washed with further TBME. The aqueous phase was slightly concentrated under vacuum and purified using a semi-preparative Discovery C18 569226-U RP-HPLC column (21.2 mm × 25 cm, 5 μm) with a UV detector (monitoring at 215 nm). A gradient elution system was used with mobile phase A (0.1% TFA) and mobile phase B (acetonitrile). The gradient was started at a flow rate of 20 mL / min with 95% A and 5% B for 5 minutes; increased to 70% A and 30% B over 20 minutes; then increased to 2% A and 98% B over 1 minute, and held for another 5 minutes. The combined fraction was directly freeze-dried to obtain 6952 as a white powder (4×TFA salt, 123 mg, 35% in 3 steps). LC-MS (ESI + )m / z (relative intensity): 694.1 ([M-H2O+H] + ,100),348.9(29);tr=7.5 minutes.

[0335] Synthesis of compound 6952 (Method II) To a stirred solution of DOTA-PNP (synthesized in-house, 204 mg, 0.30 mmol) and intermediate compound 2 (162 mg, 0.33 mmol) in anhydrous DMF (4 mL), TEA (360 μl, 2.07 mmol) was added under ice bath cooling. The resulting mixture was stirred overnight at room temperature. The reaction mixture was then concentrated under vacuum. Water (9 mL) was added, and the pH was adjusted to approximately 1.5 with 1N TFA. Phenylboronic acid (48 mg, 0.39 mmol), acetonitrile (3 mL), and TBME (18 mL) were added. The resulting mixture was stirred at room temperature for 3 hours and post-treated as described above to obtain 6952 as a white powder (4 × TFA salt, 140 mg, 40% in 2 steps).

[0336] Synthesis of compound 6952LU Compound 6952 (10 mg, 8.6 μmol) was added to a solution of LuCl3 (18 mg, 64 μmol) in acetate buffer (0.23 M, pH 5.2, 3 mL). The resulting mixture was stirred at 90°C for 23 minutes and then purified using a semi-preparative Discovery C18 569226-U RP-HPLC column (21.2 mm × 25 cm, 5 μm) with a UV detector (monitoring at 215 nm). A gradient elution system was used, utilizing mobile phase A (0.05% TFA in water) and mobile phase B (acetonitrile). The gradient was started at a flow rate of 20 mL / min with 95% A and 5% B for 5 minutes; increased to 70% A and 30% B over 20 minutes; then increased to 2% A and 98% B over 1 minute, and held for another 5 minutes. The combined fraction was directly freeze-dried to obtain 6952 LU as a white powder (4×TFA salt, 5 mg, 44%). LC-MS (ESI + m / z (relative intensity): 865.5 (100); tr = 14.9 mins (see attached LCMS and conditions).

[0337] Synthesis of compound 6952GA Compound 6952 (10 mg, 8.6 μmol) was added to a solution of GaCl3 (12 mg, 66 μmol) in acetate buffer (0.23 M, pH 5.2, 4 mL). The resulting mixture was stirred at 90°C for 23 minutes and then purified using a semi-preparative Discovery C18 569226-U RP-HPLC column (21.2 mm × 25 cm, 5 μm) with a UV detector (monitoring at 215 nm). A gradient elution system was used, utilizing mobile phase A (0.05% TFA in water) and mobile phase B (acetonitrile). The gradient was started at a flow rate of 20 mL / min with 95% A and 5% B for 5 minutes; increased to 70% A and 30% B over 20 minutes; then increased to 2% A and 98% B over 1 minute, and held for another 5 minutes. The combined fraction was directly freeze-dried to obtain 6952GA as a white powder (4×TFA salt, 6 mg, 57%). LC-MS (ESI + m / z (relative intensity): 760.9 (100); tr = 16.1 min (see attached LCMS and conditions).

[0338] supporting material Compound 6952 : LC-MS was performed using a Hewlett Packard HPLC / MSD system equipped with a UV detector (monitoring at 215 nm) and a ZORBAX Eclipse Plus C18 RP-HPLC column (4.6 × 50 mm, 1.8 μm). A gradient elution system was used, utilizing mobile phase A (0.1% TFA) and mobile phase B (acetonitrile). The gradient was started at a flow rate of 0.5 mL / min with 98% A and 2% B for 3 minutes; increased to 2% A and 98% B over 6 minutes; and maintained for a further 5 minutes. Finally, the gradient parameters were returned to the initial starting conditions. MS was performed in positive mode. Data were analyzed using Agilent Chemstation software.

[0339] Compound 6952LU LC-MS was performed using a Hewlett Packard HPLC / MSD system equipped with a UV detector (monitoring at 215 nm) and a Luna C18, 4.6 mm × 150 mm, 3.0 μm, 100A column. A gradient elution system was used, utilizing mobile phase A (50 mM AcONH4) and mobile phase B (acetonitrile). The gradient was started at a flow rate of 1.0 mL / min with 98% A and 2% B for 5 minutes; increased to 74% A and 26% B over 15 minutes; and then increased to 2% A and 98% B over 5 minutes. Finally, the gradient parameters were returned to the initial starting conditions. MS was performed in negative mode. Data were analyzed using Agilent Chemstation software.

[0340] Compound 6952GA LC-MS was performed using a Hewlett Packard HPLC / MSD system equipped with a UV detector (monitoring at 215 nm) and a Luna C18, 4.6 mm × 150 mm, 3.0 μm, 100A column. A gradient elution system was used, utilizing mobile phase A (50 mM AcONH4) and mobile phase B (acetonitrile). The gradient was started at a flow rate of 1.0 mL / min with 98% A and 2% B for 5 minutes; increased to 74% A and 26% B over 15 minutes; and then increased to 2% A and 98% B over 5 minutes. Finally, the gradient parameters were returned to the initial starting conditions. MS was performed in negative mode. Data were analyzed using Agilent Chemstation software.

[0341] Example 21: In Vitro Assay: Dipeptidyl Peptidase IV, Fibroblast-Activating Protein, and Prolyl Oligo Peptidase The purpose of this assay is to determine the IC of various inhibitors against recombinant human dipeptidyl peptidase IV (DPPIV), fibroblast-activating protein (FAP), or prolyl oligopeptidase (PREP). 50 The task is to make a decision.

[0342] The assay is performed in the following steps: 1. Dissolve the compound in DMSO to a final concentration of 100 mM. From this, prepare a 1 mM stock solution using the following methods: 50 mM Tris, 140 mM NaCl buffer, pH 7.5 (FAP) / 25 mM Tris, 250 mM NaCl buffer, pH 7.5 / 140 mM NaCl buffer, pH 8.0 (PREP). 2. The previously prepared 1 mM compound stock is serially diluted (1:10) in one row of a 96-well plate in appropriate assay buffers (FAP: 50 mM Tris, 140 mM NaCl, pH 7.5 / PREP: 25 mM Tris, 0.25 M NaCl, pH 7.5 / DPPIV: 25 mM Tris, pH 8.0). 3. Prepare 20× substrate solutions (FAP and PREP: 2.5 mM Z-Gly-Pro-AMC in DMSO (VWR, catalog no. I-1145.0050BA) / DPPIV: 100 mM Gly-Pro-AMC in DMSO (VWR, catalog no. 100042-646)) by diluting the DMSO stock in an appropriate assay buffer. 4. Dilute the enzymes in appropriate assay buffer. The final enzyme concentrations should be 0.1, 1.2, and 0.6 nM for DPPIV, FAP, and PREP, respectively. Add 180 μL to each required well in columns 2–10. Column 1 (A, B, C) should be prepared as a control using 200 μL of appropriate assay buffer. Column 1 (D, E, F, G, H) should be prepared as an inhibitor-free control using 20 μL of appropriate assay buffer and 180 μL of enzyme. 5. If appropriate, add 20 μL of the target compound from the diluted plate prepared in step 2 to rows 2-10 of the assay plate. Each sample should be tested in triplicate form. Incubate at room temperature for 10 minutes, shaking the plate for the first 2 minutes. 6. Add 10 μL of the 20× substrate prepared in step 3 to each well, incubate at room temperature for 15 minutes, and shake the plate for the first 2 minutes. 7.λ ex :380, λ emRead the fluorescence at :460.

[0343] Table 12 summarizes the compounds (Group I) containing the DOTA / DOTAGA-[XXaa]n-DP core and the results of their in vivo assays. (DP core = [dAla / dSer / Gly]-[boroPro / Pro-nitrile], XXaa = α-amino acid) [Table 12-1] [Table 12-2] [Table 12-3] [Table 12-4] [Table 12-5] [Table 12-6] [Table 12-7] [Table 12-8] [Table 12-9] [Table 12-10] [Table 12-11] [Table 12-12] [Table 12-13] [Table 12-14] [Table 12-15] [Table 12-16]

[0344] Table 13 summarizes the compounds (group IA) containing a DOTA / DOTAGA-alkyl-[XXaa]n-DP core and the results of their in vivo assays. [Table 13-1] [Table 13-2] [Table 13-3]

[0345] Table 14 summarizes the compounds (Group II) containing the DOTA / DOTAGA-[XXaa]n-[aromatic]-DP core and the results of their in vivo assays. [Table 14-1] [Table 14-2] [Table 14-3] [Table 14-4] [Table 14-5] [Table 14-6] [Table 14-7] [Table 14-8] [Table 14-9]

[0346] Table 15 summarizes the compounds (Group IIA) containing a DOTA / DOTAGA-alkyl-[aromatic]-DP core and the results of their in vivo assays. [Table 15-1] [Table 15-2] [Table 15-3] [Table 15-4] [Table 15-5] [Table 15-6] [Table 15-7] [Table 15-8]

[0347] Table 16 summarizes the compounds (Group III) containing the DOTA / DOTAGA-[XXaa]n-[cycloalkyl]-DP core and the results of their in vivo assays. [Table 16-1] [Table 16-2] [Table 16-3]

[0348] Table 17 summarizes the compounds (Group IIIA) containing a DOTA / DOTAGA-alkyl-[cycloalkyl]-DP core and the results of their in vivo assays. [Table 17]

[0349] Table 18 summarizes the results of other compounds and their in vivo assays. [Table 18-1] [Table 18-2] [Table 18-3] [Table 18-4] [Table 18-5] [Table 18-6] [Table 18-7] [Table 18-8] [Table 18-9] [Table 18-10] [Table 18-11] [Table 18-12] [Table 18-13] [Table 18-14] [Table 18-15]

[0350] Example 22:[ 68 Preparation of Ga-6522 [ka]

[0351] The above radiopharmaceuticals [ 68 GaCl3-6522 can be prepared under the following conditions: 73 nmol of radiochemical precursor 6522 (Example 11 above), 0.5 M sodium acetate, 0.4 M N-acetylmethionine, and approximately 400 MBq of GaCl3 were heated at 90°C for 20 minutes with shaking at pH 4.0 and a total volume of 7.875 mL. The reaction mixture was diluted with 40 mL of water and purified using a C18 solid-phase extraction cartridge pre-prepared with ethanol and water. The product was eluted with 2 mL of ethanol, and the ethanol was evaporated. The evaporated product was diluted with 0.6 mL of 0.9% physiological saline, and the pH was adjusted to 5.0 by adding 70 μL of 1 M NaOH. The product was filtered by sterile filtration (Millex-GV, 0.22 μm).

[0352] Labeling efficiency was analyzed by instant thin-layer chromatography (iTLC) and was typically >90%. For iTLC analysis, 1 μL of the product was applied to a strip of iTLC-SG chromatography paper (Agilent, P / NSGI0001, 114 cm × 2.5 cm) and developed with 30% CH3CN / 70% 1M NH4OAc (6.5 cm solvent transfer) to release the product. 68 Ga and 68 Ga-colloid (Rf approximately 0) and [ 68Ga]-6522 and its associated impurities (Rf approximately 0.7) were evaluated. iTLC strips were analyzed using an Eckert & Ziegler AR-2000 Radio-TLC Imaging Scanner. Radiochemical purity was analyzed by high-performance chromatography (HPLC) and was typically >98%. Briefly, the product was analyzed using a Phenomenex Luna 3.0 μm C18(2), 100 Å, 150 mm × 4.6 mm column. Eluent A: 50 mM ammonium acetate aqueous solution, Eluent B: Acetonitrile. Gradient: 0-5 min 2% B; 5-20 min 2%-26% B; 20-25 min 26%-98% B; 25-26 min 98%-2% B; 26-30 min 2% B. Flow rate: 1.0 mL / min, Radio-HPLC detector: NaI (Eckert & Zeigler FC-1000), UV: 215 nm.

[0353] Example 23:[ 177 Preparation of Lu]-6522 [ka]

[0354] The above radiopharmaceuticals [ 177 Lu]-6522 can be prepared under the following conditions: 73 nmol / mL of radiochemical precursor 6522 (Example 11 above), 80 mM sodium acetate, 0.4 M N-acetylmethionine, and 7.8 GBq / mL 177 LuCl3 was heated at 70°C for 15 minutes with shaking at pH 4, total volume 0.26 mL. The reaction mixture was diluted with 2.34 mL of buffer to obtain the following final conditions: 8 mM sodium acetate, 0.2 M N-acetylmethionine, 6.5 mg / mL sodium ascorbate, and 0.1 mg / mL DTPA (pH 5). The product was filtered by sterile filtration (Millex-GV, 0.22 μm).

[0355] Labeling efficiency was analyzed by instant thin-layer chromatography (iTLC) and was typically >98%. For iTLC analysis, 1 μL of diluted labeling solution was applied to a strip of iTLC-SA chromatography paper (Agilent P / N A120B12, 114 × 2.5 mm) and developed in 0.1 M citrate buffer (8 cm solvent transfer) to release the label. 177 Lu(Rf>0.5) and [ 177 Lu]-6522 (Rf approximately 0) was evaluated. iTLC strips were analyzed using an Eckert & Ziegler AR-2000 Radio-TLC Imaging Scanner. Radiochemical purity was analyzed by high-performance chromatography (HPLC) and was typically >70%. Briefly, the product was analyzed using a Phenomenex Luna 3.0 μm C18(2), 100 Å, 150 mm × 4.6 mm column. Eluent A: 50 mM ammonium acetate aqueous solution, Eluent B: Acetonitrile. Gradient: 0-5 min 2% B; 5-20 min 2%-26% B; 20-25 min 26%-98% B; 25-26 min 98%-2% B; 26-30 min 2% B. Flow rate: 1.0 mL / min, Radio-HPLC detector: NaI (Eckert & Ziegler FC-1000), UV: 215 nm.

[0356] Example 24:[ 177 Additional preparation of Lu]-6522 [ka]

[0357] The above radiopharmaceuticals [ 177 Lu]-6522 can be prepared under the following conditions: approximately 58 μg / mL of compound 6522 (Example 11 above), 70 mM sodium acetate, 0.2 M N-acetylmethionine and 7.8 GBq / mL 177LuCl3 was heated at 90°C for approximately 15 minutes with shaking at pH 4 and a total volume of 1.27 mL. The reaction mixture was diluted with 17.43 mL of buffer to obtain the final conditions: 0.2 M sodium acetate, 0.2 M N-acetylmethionine, pH 6.

[0358] Radiochemical purity was analyzed by high-performance chromatography (HPLC) and was typically >85%. Briefly, 20 μL of diluted product was analyzed using a Luna C18(2) column. Eluent A: 50 mM ammonium acetate aqueous solution, Eluent B: acetonitrile, gradient 2% B (5 min), 2% to 26% B at 15 min, 98% B at 5 min, flow rate 1.1 mL / min, detector: NaI radio detector (Eckert & Ziegler), UV / Vis 215 nm.

[0359] Example 25:[ 177 Lu]-6555 [ka]

[0360] The above radiopharmaceuticals [ 177 Lu]-6555 can be prepared under the following conditions: 73 nmol / mL of radiochemical precursor 6555 (Example 19 above), 0.2 M sodium acetate, 10 mg / mL sodium ascorbate, 5 mg / mL gentisic acid, 0.1 M N-acetylmethionine, and 4.0 GBq / mL 177 LuCl3 was heated at 50°C for 40 minutes with shaking at pH 4.5 and a total volume of 0.5 mL. The reaction mixture was diluted with 4.5 mL of buffer to obtain the following final conditions: 20 mM sodium acetate, 0.2 M N-acetylmethionine, 6.5 mg / mL sodium ascorbate, 0.5 mg / mL gentisic acid, and 0.1 mg / mL DTPA, pH 5.

[0361] Labeling efficiency was analyzed by instant thin-layer chromatography (iTLC) and was typically >98%. For iTLC analysis, 1 μL of diluted labeling solution was applied to a strip of iTLC-SA chromatography paper (Agilent P / N A120B12, 114 × 2.5 mm) and developed in 0.1 M citrate buffer (8 cm solvent transfer) to release the label. 177 Lu(Rf>0.5) and [ 177 Lu]-6555 (Rf approximately 0) was evaluated. iTLC strips were analyzed using an Eckert & Ziegler AR-2000 Radio-TLC Imaging Scanner. Radiochemical purity was analyzed by high-performance chromatography (HPLC) and was typically >90%. Briefly, the product was analyzed using a Phenomenex Luna 3.0 μm C18(2), 100 Å, 150 mm × 4.6 mm column. Eluent A: 50 mM ammonium acetate aqueous solution, Eluent B: Acetonitrile. Gradient: 0-5 min 2% B; 5-20 min 2%-26% B; 20-25 min 26%-98% B; 25-26 min 98%-2% B; 26-30 min 2% B. Flow rate: 1.0 mL / min, Radio-HPLC detector: NaI (Eckert & Ziegler FC-1000), UV: 215 nm. Radiochemical purity remained >90% for 3 days at room temperature.

[0362] Example 26:[ 177 Lu]-6952 [ka]

[0363] The above radiopharmaceuticals [ 177Lu]-6952 can be prepared using the radiochemical precursor 6952 (Example 20 above) as described in Example 25. Labeling efficiency was analyzed by instant thin-layer chromatography (iTLC) and was typically >98%. For iTLC analysis, 1 μL of diluted labeling solution was applied to a strip of iTLC-SA chromatography paper (Agilent P / N A120B12, 114 × 2.5 mm) and developed in 0.1 M citrate buffer (8 cm solvent transfer) to release the label. 177 Lu(Rf>0.5) and [ 177 Lu]-6952 (Rf approximately 0) was evaluated. iTLC strips were analyzed using an Eckert & Ziegler AR-2000 Radio-TLC Imaging Scanner. Radiochemical purity was analyzed by high-performance chromatography (HPLC) and was typically >90%. Briefly, the product was analyzed using a Phenomenex Luna 3.0 μm C18(2), 100 Å, 150 mm × 4.6 mm column. Eluent A: 50 mM ammonium acetate aqueous solution, Eluent B: Acetonitrile. Gradient: 0-5 min 2% B; 5-20 min 2%-26% B; 20-25 min 26%-98% B; 25-26 min 98%-2% B; 26-30 min 2% B. Flow rate 1.0 mL / min, Radio-HPLC detector: NaI (Eckert & Ziegler FC-1000), UV 215 nm. Radiochemical purity remained >90% for 3 days at room temperature.

[0364] Example 27:[ 68 Ga]-6555 [ka]

[0365] The above radiopharmaceuticals [ 68GaCl3-6555 can be prepared under the following conditions: 73 nmol of radiochemical precursor 6555 (Example 19 above), 0.5 M sodium acetate, 0.4 M N-acetylmethionine, and approximately 1200 MBq of GaCl3 were heated at 90°C for 20 minutes with shaking at pH 4.0 and a total volume of 7.875 mL. The reaction mixture was diluted with 40 mL of water and purified using a C18 solid-phase extraction cartridge pre-prepared with ethanol and water. The product was eluted with 3 mL of ethanol, and the ethanol was evaporated. The evaporated product was diluted with 0.5 mL of phosphate-buffered saline, and the pH was adjusted to 5.0 by adding 70 μL of 1 M NaOH. The product was filtered by sterile filtration (Millex-GV, 0.22 μm).

[0366] Labeling efficiency was analyzed by instant thin-layer chromatography (iTLC) and was typically >95%. For iTLC analysis, 1 μL of the product was applied to a strip of iTLC-SG chromatography paper (Agilent, P / NSGI0001, 114 cm × 2.5 cm) and developed in 30% CH3CN / 70% 1M NH4OAc (6.5 cm solvent transfer) to release the product. 68 Ga and 68 Ga-colloid (Rf approximately 0) and [ 68Ga]-6555 and its associated impurities (Rf approximately 0.7) were evaluated. iTLC strips were analyzed using an Eckert & Ziegler AR-2000 Radio-TLC Imaging Scanner. Radiochemical purity was analyzed by high-performance chromatography (HPLC) and was typically >95%. Briefly, the product was analyzed using a Phenomenex Luna 3.0 μm C18(2), 100 Å, 150 mm × 4.6 mm column. Eluent A: 50 mM ammonium acetate aqueous solution, Eluent B: Acetonitrile. Gradient: 0-5 min 2% B; 5-20 min 2%-26% B; 20-25 min 26%-98% B; 25-26 min 98%-2% B; 26-30 min 2% B. Flow rate: 1.0 mL / min, Radio-HPLC detector: NaI (Eckert & Ziegler FC-1000), UV: 215 nm. Radiochemical purity remained >95% at room temperature for 4 hours.

[0367] Example 28:[ 68 Ga]-6952 [ka]

[0368] The above radiolabeled product [ 68 GaCl3-6952 was formed under the following conditions: 73 nmol of radiochemical precursor 6952 (Example 20 above), 0.5 M sodium acetate, 0.4 M N-acetylmethionine, and approximately 1200 MBq of GaCl3 were heated at 90°C for 20 minutes with shaking at pH 4.0 and a total volume of 7.875 mL. The reaction mixture was diluted with 40 mL of water and purified using a C18 solid-phase extraction cartridge pre-prepared with ethanol and water. The product was eluted with 2 mL of ethanol, and the ethanol was evaporated. The evaporated product was diluted with 0.5 mL of phosphate-buffered saline, and the pH was adjusted to 5.0 by adding 65 μL of 1 M NaOH. The product was filtered by sterile filtration (Millex-GV, 0.22 μm).

[0369] Labeling efficiency was analyzed by instant thin-layer chromatography (iTLC) and was typically >95%. For iTLC analysis, 1 μL of the product was applied to a strip of iTLC-SG chromatography paper (Agilent, P / NSGI0001, 114 cm × 2.5 cm) and developed with 30% CH3CN / 70% 1M NH4OAc (6.5 cm solvent transfer) to release the product. 68 Ga and 68 Ga-colloid (Rf approximately 0) and [ 68 Ga-6952 and its associated impurities (Rf approximately 0.7) were evaluated. iTLC strips were analyzed using an Eckert & Ziegler AR-2000 Radio-TLC Imaging Scanner. Radiochemical purity was analyzed by high-performance chromatography (HPLC) and was typically >95%. Briefly, the product was analyzed using a Phenomenex Luna 3.0 μm C18(2), 100 Å, 150 mm × 4.6 mm column. Eluent A: 50 mM ammonium acetate aqueous solution, Eluent B: Acetonitrile. Gradient: 0-5 min 2% B; 5-20 min 2%-26% B; 20-25 min 26%-98% B; 25-26 min 98%-2% B; 26-30 min 2% B. Flow rate: 1.0 mL / min, Radio-HPLC detector: NaI (Eckert & Ziegler FC-1000), UV: 215 nm. Radiochemical purity remained >95% at room temperature for 4 hours.

[0370] Example 29: In vivo in vivo distribution test Using a gamma counter, in a group of tumor-carrying male Fox Chase SCID mice inoculated with HEK-mFAP cell lines, [ 177 In vivo distribution studies were conducted using Lu]-6522 (also known as compound #2). In the design of this study, [ 177 Fifteen mice (average body weight 22.7 ± 1.4 g) injected with Lu]-6522 were used. 177Lu-6522 was administered intravenously (IV) at a dose of 175 μL, or 9.05 ± 0.70 MBq. Animals in each group (n=3-5) were sacrificed at specific time points, and blood and organs were collected by cardiac puncture 4, 24, 48, and 168 hours after injection. The organs were excised, weighed, and their activity was measured using a gamma counter (165.6-364.3 keV). Uptake in tumor and normal tissues was expressed as %ID / g.

[0371] material and method Animals and breeding Fox Chase SCID mouse strain code 236 was obtained for this study from Charles River Laboratories (Kingston, NY, USA). Animals were housed in groups of five until the start of the experiment. Animals were acclimatized for seven days prior to the start of the study. All animal experiments were approved by the University Health Network (UHN) Animal Care Committee and adhered to the ethical guidelines of the Canadian Council on Animal Care. Animals were housed under a 12-hour light / 12-hour dark schedule at a constant temperature (20°C) and 40% relative humidity, with free access to food and water.

[0372] The body weight of the animals was measured and recorded four days after inoculation with the HEK-mFAP cell line and monitored until the day of injection of the radioactive tracer. The animals were not fasted before administration. The body weight on the day of tracer administration is shown in Table 19.

[0373] [Table 19]

[0374] Cell culture and inoculation HEK-mFAP cells were cultured in RPMI 1640 (VWR, catalog number 45000-404) supplemented with the following: 1. 2 mM L-glutamine (VWR, catalog number 45000-676) 2. 10mM HEPES (VWR, catalog number 45000-690) 3. 1 mM sodium pyruvate (VWR, catalog number 45000-710) 4. 4500 mg / L glucose (VWR, catalog number 45001-116) 5. 1x penicillin-streptomycin (VWR, catalog number 45000-652) 6. 10% FBS (Thermo Fisher Scientific, catalog number 10082147)

[0375] Cells were cultured at 37°C under a 5% CO2 atmosphere. 7-9 week old male Fox Chase SCID (Charles River Laboratories, strain code 236) were fed 4 × 10¹⁶ cells in 100 μL of phenol red-free RPMI 1640 (VWR, catalog number 45000-410) (supplemented as described for growth medium, but without antibiotics or FBS). 6 Tumor xenografts were established by subcutaneous injection of cells into the right flank. Cells were inoculated at passage 9 with a survival rate >90%. Batch 1 involved inoculation of 16 mice, and Batch 2 involved inoculation of 15 mice.

[0376] Tumor volume and randomization of mice Compound #2([ 177 The average tumor volume of mice injected with Lu]-6522 was 51.8 ± 44.4 mm². 3 Therefore, an in vivo distribution study was performed 30 days after tumor cell inoculation. Tumor volume was given by V = length × width 2 The calculation was performed using a multiplier of 0.5. Table 20 shows the randomization of animals by tumor volume.

[0377] [Table 20]

[0378] [ 177 Lu]-6522 batch usage and quality It has a radiochemical purity of 85.98%. 177 One vial containing Lu]-6522 was used (prepared according to Example 24).

[0379] 9.05±0.70MBq[ 177 Syringes were prepared with the dose of Lu]-6522 (compound #2). The injected radioactivity was calculated by subtracting the decay-corrected residual activity in the syringe after injection from the decay-corrected activity in the syringe before injection. The injected radioactivity per animal and per group is summarized in Table 21.

[0380] Anesthesia, dose management Mice were anesthetized using isoflurane (Fresenius Kabi Canada Ltd.) (5% induction, 1.5-2% maintenance). A 27Ga catheter (27G winged infusion set, 15 cm long, SAI Infusion Technologies) was placed in the tail vein, and approximately 145-175 μL of tracer was manually injected. The actual dose administered to each animal is shown in Table 21. After injection, the catheter was flushed with 30 μL of saline.

[0381] [Table 21]

[0382] In vivo distribution test In vivo distribution studies were performed 4, 24, 48, and 168 hours (pi) after injection. Three to five mice were sacrificed at each time point, and tumor, blood, and normal tissue samples were collected, weighed, and their radioactivity was measured using a gamma counter. Uptake of tumors and normal tissue was expressed as the mean ± SEM of the percentage of administered radioactivity per gram (%ID / g).

[0383] Collection of gamma (γ) count data The radioactivity of organs / tissues was measured using a gamma counter (1480 WIZARD 3”, Perkin Elmer; counts 60 seconds per vial). Depending on the animal batch used, [ 177 Each time an organ was measured using the Lu]-6522 sample, the count was converted to activity using conversion factors obtained from standard samples of known volume and known radioactivity (MBq). Through this method, all activity values ​​are essentially corrected for decay with respect to injection time.

[0384] The percentage of administered radioactivity per organ (%ID) was calculated using the following formula: %ID = Attenuated organ activity [MBq] / administered radioactivity [MBq] × 100%

[0385] The percentage of administered radioactivity per gram of organ weight (%ID / g) for each organ was calculated using the following formula: %ID / g=%ID / organ weight[g]

[0386] Results and Discussion [ 177 The Lu]-6522 formulation was administered to a total of 15 male Fox Chase SCID mice. Ex vivo gamma counts of various organs were performed 4, 24, 48, and 168 hours after tracer administration (n=3-5).

[0387] The incorporation results for compound #2, expressed as %ID / g, are summarized in Table 19 below.

[0388] For compound #2, the highest tumor uptake, as well as the lowest radioactivity concentrations in blood and other normal tissues, were observed 4 hours after injection. Compound #2 showed high tumor uptake as early as 4 hours after injection, at 33.04 ± 5.29% ID / g. As shown in Table 19, the kidney showed uptake similar to compound #1, at 2.35 ± 0.51% ID / g after 4 hours, and its uptake decreased over time, falling to 0.17 ± 0.02% ID / g at measurement 7 days after injection.

[0389] Higher uptake was found in the kidneys compared to all other organs, suggesting that the primary excretion route is via the kidneys. High radioactivity was observed in the skin of mice 4 hours after injection, which may be due to the excretion of the compound in the urine and contamination of the mouse skin with radioactive urine. TIFF0007939699000149.tif108170

[0390] conclusion Compound #2([ 177 Lu)-6522) showed high localization and low uptake in tumor xenografts up to 168 hours after administration.

[0391] Example 30: Efficacy and survival test The purpose of this study is to evaluate tumor growth retardation and median survival, 177 The objective was to evaluate the therapeutic efficacy of a single injection of Lu]-6522.

[0392] [ 177 Lu]-6522 was provided at three concentrations, prepared for injection (80 μL / mouse), and the administered radioactivity was determined using a well counter (Capintec calibrated) #430x10.

[0393] [ 177 Lu]-6522 is provided as described in Example 24 above. The following treatment compositions were prepared: 1. Vehicle (selected formulation, 100 μL) 2. Precursor (compound 6522) (80 μL) 3.[ 177 Lu]-6522 15 MBq (80 μL) 4.[ 177 Lu]-6522 30 MBq (80 μL) 5.[ 177 Lu]-6522 60 MBq (80 μL)

[0394] A total of 30 HEK-mFAP tumor-carrying mice were used in the study. In male Fox Chase SCID mice (6-8 weeks old, Charles River Laboratories), tumor xenografts were established by subcutaneous injection of 5 million HEK-mFAP cells in 100 μL of PBS into the right flank.

[0395] The health status of the mice, including weight measurement, was checked weekly throughout the study period. Tumor growth was monitored weekly using calipas measurement (tumor volume = length × width). 2 (×0.5). Test endpoints included tumor size >2 cm in any dimension, tumor ulceration, mouse being mortal, and >15% body weight loss since the last measurement. Mice were housed in cages in groups of five with free access to food and water, at an ambient temperature of 20°C, 40%–50% humidity, and a 12-hour light / 12-hour dark cycle.

[0396] Mice were randomized into five groups, with n=6 mice / group. Treatment compositions (1-5 above) were administered intravenously via the tail vein using a catheter (equipped with a 30Ga needle). The administered radioactivity was determined using a well counter (Capintec calibrated). Tumor growth was monitored weekly using caliper measurements, and the mice were further tracked for survival.

[0397] result: Data were collected for tumor volume and survival analysis. No weight loss was observed in any of the treatment groups. ·[ 177Only treatment with a 60 MBq dose of Lu-6522 showed a statistically significant survival benefit compared to the vehicle or precursor group (see Figure 2). 177 All mice in the Lu]-6522 60MBq group survived for more than 50 days after treatment (see Figure 2). ·[ 177 Tumor growth retardation was observed in the Lu]-6522 15 MBq and 30 MBq groups (Figure 1), but this tumor growth retardation did not lead to a survival benefit (Figure 2). ·[ 177 In the Lu]-6522 60MBq group, tumors regressed until approximately 43 days after treatment, after which they began to regrow (see Figure 1). The trial concluded on the 57th day after the start of treatment.

[0398] Example 31: 68 PET imaging and in vivo distribution of Ga-6555 Part 1. Dynamic PET imaging. The purpose of this study is to investigate HEK-mFAP tumor-carrying mice. 68 The objective is to perform Ga-6555 PET / CT dynamic imaging to evaluate tumor uptake and retention over time, as well as nonspecific uptake. 68 PET imaging of Ga-6555 (prepared according to Example 27) was performed using a dedicated small animal PET / CT scanner (Siemens Multimodality Inveon, Siemens Medical Solutions USA, Inc.). Mice were anesthetized using 3% isoflurane / medical air inhalation before radiotracer injection and during the scan period. Warming was used to maintain a healthy core body temperature of the mice during the unconscious period. 68Following a bolus intravenous injection of Ga-6555 (average 8 MBq, range 7.7–8.1 MBq) via the lateral tail vein, dynamic radioscans were acquired in list-mode format over 60 minutes. The acquired data were then sorted into 0.5 mm sinogram bins and 19-hour frames for image reconstruction using FORE / 3D-OSEM-MAP. After PET acquisition, low-dose CT scans (80 kVp, 0.5 mA) were acquired for anatomical reference and to provide guidance for depicting selected tissue volumes of interest (VOIs). Reconstructed PET / CT images were analyzed using Siemens Inveon Research Workplace software. Radioactivity retention within selected tissues was obtained from the mean voxel intensity values ​​within the VOIs, and then converted to megabecquerels per milliliter using calibration factors determined for the Inveon PET System. These values ​​were then divided by the administered activity in megabecquerels and the animal's body weight to obtain Standardized Uptake Values ​​(SUVs) derived from the image VOIs. The inventors used the maximum SUV value within a VOI (SUVmax) as a quantitative imaging metric independent of tissue-specific variability. The PET images shown are axial, coronal, and sagittal sections with the mouse in a prone position.

[0399] 68 Uptake of Ga-6555 was observed in tumors and elimination organs (kidneys and bladder), and this was consistent across three mice. The time-versus-activity curve of 68Ga-6555 in tumors showed rapid accumulation (<5 mins) and retention in tumors, reaching a plateau at 60 mins. Data from one mouse are shown in Figure 3.

[0400] Part 2: In vivo distribution. The purpose of this study is to determine the distribution in HEK-mFAP tumor-carrying mice. 68 The objective was to evaluate the in vivo distribution of Ga-6555. HEK-mFAP tumor-carrying mice were used in the study (N=3). The mice contained approximately 8 MBq (in the range of 7.3-8.5 MBq). 68Ga-6555 (prepared according to Example 27; IV into the tail vein using a catheter fitted with a 30Ga needle) was injected. After a 50-minute uptake time (injection was performed under anesthesia using isoflurane inhalation and maintained under anesthesia for 50 minutes), the mice were euthanized (with CO2) and tissues were collected (blood, heart, lungs, liver, spleen, pancreas, stomach, small intestine, kidneys, muscle, femur, bone, skin, brain, and tumor via cardiac puncture). After resection, the radioactivity of gallium-68 in the tissue samples was counted using a Cobra-II Auto-Gamma counter (Packard Instruments, Meriden, CTA), the weight was measured, and the data was expressed as a percentage of administered radioactivity per gram (%ID / g).

[0401] The majority of the activity was located in tumors (10.1 mean %ID / g). The kidney had the next highest activity (1.37 mean %ID / g). All other selected tissues had low uptake levels, similar to muscle, which were considered background levels. TIFF0007939699000150.tif151146

[0402] Example 32: 68 PET imaging and in vivo distribution of Ga-6952 Prepared according to Example 28 68 The procedure was carried out using Ga-6952 according to Example 31.

[0403] Part 1: Dynamic PET imaging. Mice with approximately 8.6 MBq (range 7.6-10.0 MBq) 68 Ga-6952 was injected. 68 Ga-6952 uptake was observed in tumors and excretory organs (kidneys and bladder), and this was consistent across three mice. 68 The time-versus-activity curve of Ga-6952 in tumors showed rapid accumulation (<5 min) and retention in tumors, reaching a plateau at 60 min.

[0404] In vivo distribution. Mice were injected with approximately 8.6 MBq (ranging from 7.6 to 10.0 MBq). The majority of the activity was located in tumors (mean 8.8 %ID / g). The kidneys had the next highest activity (mean 2.18 %ID / g). All other selected tissues showed low uptake levels, similar to muscle, which were considered background levels. The data are shown below. TIFF0007939699000151.tif152147

[0405] Example 33: Treatment Protocol Human patients are selected for treatment after being diagnosed with metastatic cancer.

[0406] Treatment: In a 0.4N sterile aqueous solution of N-acetylmethionine [ 177 Lu]-6555 is administered by intravenous injection. The administration regimen may consist of one or more infusions of 7.4 GBq each, administered at intervals of at least two weeks.

[0407] Example 34: Additional Treatment Protocol Human patients are selected for treatment after being diagnosed with metastatic cancer.

[0408] Treatment: In a sterile aqueous solution containing 0.4N N-acetylmethionine [ 68 Administer Ga-6555 by intravenous injection. The administration regimen may include a single infusion of 185-370 MBq followed by PET-CT or PET-MRI imaging.

[0409] Example 35: Additional stabilization test Three stabilizers: 1) N-acetyl-L-methionine (NAM), 2) sodium ascorbate (SA), and 3) L-methionine [ 177 The effect on Lu]-6555 was evaluated. The results are shown in Figure 4.

[0410] The following aqueous mixtures were prepared and maintained with stirring for the indicated time and temperature: [177 LU]-6555: 2 mg / ml AAPH * :50mM AAPH The amount of stabilizer shown in Figure 4 pH=5.0, 80°C for 30 minutes * AAPH is 2,2'-azobis-(2-amidopropane) hydrochloride.

[0411] The remaining [ after the above 80°C / 30 minute treatment 177 The amount of Lu]-6555 is shown in Figure 4 and was determined using the following method: Instrumentation: Agilent 1290 UPLC / 6460 Triple Quad LC / MS Column: Zorbax Eclipse Plus C18, 2.1 × 50 mm, 1.8 μm (Agilent, 1200 bar) HPLC method: Mobile phase A: 0.1% TFA in water Mobile phase B: 0.08% TFA in ACN Flow rate: 0.8mL / min Gradient: 0-1 minute: 2%B; 6 minutes: 26%B. Finally, preferred embodiments of the present invention are described in separate sections. [Embodiment 1] (a) therapeutic boronic acid compounds; and (b) One or more sulfur compounds A pharmaceutical composition containing the following: [Embodiment 2] The pharmaceutical composition according to Embodiment 1, wherein the one or more sulfur compounds include one or more organic compounds containing sulfide moieties. [Embodiment 3] The pharmaceutical composition according to Embodiment 1 or 2, wherein the one or more sulfur compounds include compounds having a molecular weight of less than 1500. [Embodiment 4] The pharmaceutical composition according to Embodiment 1 or 2, wherein the one or more sulfur compounds include compounds having a molecular weight of less than 800. [Embodiment 5] The pharmaceutical composition according to Embodiment 1 or 2, wherein the one or more sulfur compounds include compounds having a molecular weight of less than 300. [Embodiment 6] The pharmaceutical composition according to any one of Embodiments 1 to 5, comprising one or more sulfur compounds, including a nonpolymeric compound. [Embodiment 7] The pharmaceutical composition according to any one of Embodiments 1 to 6, wherein the one or more sulfur compounds are generally recognized as safe (GRAS). [Embodiment 8] The pharmaceutical composition according to any one of Embodiments 1 to 7, wherein the one or more sulfur compounds include N-acetylmethionine and / or L-glutathione. [Embodiment 9] The pharmaceutical composition according to Embodiment 1, wherein the one or more sulfur compounds include a sulfide, a sulfoxide, or a sulfite group. [Embodiment 10] A pharmaceutical composition according to any one of Embodiments 1 to 7, further comprising one or more stabilizer compounds that do not contain sulfur compounds. [Embodiment 11] The pharmaceutical composition according to Embodiment 10, wherein the one or more stabilizer compounds are 2,5-dihydroxybenzoic acid (gentisic acid) or a salt thereof, ascorbic acid or a salt thereof, and / or histidine. [Embodiment 12] A pharmaceutical composition according to any one of embodiments 1 to 11, which is a fluid formulation. [Embodiment 13] A pharmaceutical composition according to any one of Embodiments 1 to 12, which is an aqueous formulation. [Embodiment 14] A pharmaceutical composition according to any one of Embodiments 1 to 13, adapted for parenteral administration. [Embodiment 15] A pharmaceutical composition according to any one of Embodiments 1 to 14, adapted for oral administration. [Embodiment 16] A pharmaceutical composition according to any one of Embodiments 1 to 15, for use in the diagnosis or treatment of a disease or disorder related to the expression of fibroblast-activating protein (FAP) in a subject. [Embodiment 17] The pharmaceutical composition according to any one of Embodiments 1 to 16, wherein the boronic acid compound is an anticancer agent. [Embodiment 18] The pharmaceutical composition according to any one of Embodiments 1 to 16, wherein the boronic acid compound is an antibacterial agent. [Embodiment 19] The pharmaceutical composition according to any one of Embodiments 1 to 16, wherein the boronic acid compound is an antiviral agent. [Embodiment 20] The boronic acid compound comprises one or more carbocyclic groups or heteroaromatic groups, as described in any of Embodiments 1 to 19. [Embodiment 21] The boronic acid compound is given by the following formula I:

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Claims

1. (a) therapeutic boronic acid compounds; and (b) One or more sulfur compounds selected from N-acetylmethionine and L-glutathione A pharmaceutical composition containing, The boronic acid compound is given by the following formula I: 【Chemistry 1】 [In the formula, R represents a radioactive moiety, a chelating agent (optionally containing chelated metal ions), a fluorescent moiety, a photoacoustic reporter molecule, a Raman-active reporter molecule, a contrast agent, detectable nanoparticles, or an enzyme; R1 represents an alkyl group (C1-C6); R2 represents -B(-Y1)(-Y2); Y1 and Y2 are either independently -OH groups, or together with the boron atoms to which they are bonded, they represent groups hydrolyzable to boronic acid, or together with the boron atoms to which they are bonded, they form 5- to 8-membered rings hydrolyzable to boronic acid; R3 represents H or (C1-C6) alkyl; R4 is either absent or each independently represents one, two, or three substituents selected from the group consisting of (C1-C6) alkyl, -OH, -NH2, and halogen; X represents O or S; [L represents a joint or linker] A pharmaceutical composition that is represented by or a pharmaceutically acceptable salt thereof.

2. The pharmaceutical composition according to claim 1, further comprising one or more stabilizer compounds that do not contain sulfur compounds.

3. The pharmaceutical composition according to claim 2, wherein the one or more stabilizer compounds are 2,5-dihydroxybenzoic acid (gentisic acid) or a salt thereof, ascorbic acid or a salt thereof, and / or histidine.

4. A pharmaceutical composition according to any one of claims 1 to 3, which is a fluid formulation.

5. A pharmaceutical composition according to any one of claims 1 to 3, which is an aqueous formulation.

6. A pharmaceutical composition according to any one of claims 1 to 5, adapted for parenteral administration.

7. The pharmaceutical composition according to any one of claims 1 to 5, adapted for oral administration.

8. A pharmaceutical composition according to any one of claims 1 to 7, for use in the diagnosis or treatment of a disease or disorder related to the expression of fibroblast-activating protein (FAP) in a subject.

9. The boronic acid compounds are given by the following formulas II to V: 【Chemistry 2】 [In the formula, R represents a radioactive moiety, a chelating agent (optionally containing chelated metal ions), a fluorescent moiety, a photoacoustic reporter molecule, a Raman-active reporter molecule, a contrast agent, detectable nanoparticles, or an enzyme; R １ is, (C １ ~C ６ ) Represents alkyl; R ２ is -B(-Y １ ) (-Y ２ ) represents; Y １ and Y ２ These may independently represent an -OH group, or together with the boron atom to which they are bonded, they may represent a group hydrolyzable to boronic acid, or together with the boron atom to which they are bonded, they may form a 5- to 8-membered ring hydrolyzable to boronic acid; R ３ represents H or (C １ to C ６ )alkyl; R ４ They either do not exist, or each exists independently (C １ ~C ６ ) alkyl, -OH, -NH ２ Represents one, two, or three substituents selected from the group consisting of , and halogens; R ５ This represents the part that modifies the pharmacokinetics and / or in vivo distribution of the molecule; X represents O or S; L represents a joint or linker; n represents an integer from 2 to 6 in equation IV, and an integer from 1 to 6 in equation V. A pharmaceutical composition according to any one of claims 1 to 8, which is represented by any one of the above, or a pharmaceutically acceptable salt thereof.

10. The boronic acid compound is the following formula A, B, or C: 【Transformation 3】 [In the formula, M is either absent, or a radioactive isotope or a metal.] It is represented by or a pharmaceutically acceptable salt thereof. The pharmaceutical composition according to claim 9, wherein the pharmaceutical composition comprises L-glutathione.

11. The pharmaceutical composition according to claim 9, wherein the boronic acid compound is represented by formula II.

12. The pharmaceutical composition according to claim 9, wherein the boronic acid compound is represented by formula III.

13. The pharmaceutical composition according to claim 9, wherein the boronic acid compound is represented by formula IV.

14. The pharmaceutical composition according to claim 9, wherein the boronic acid compound is represented by formula V.

15. The boronic acid compound is 【Chemistry 4】 【Transformation 5】 or 【Transformation 6】 It is represented by or a pharmaceutically acceptable salt thereof. The pharmaceutical composition according to claim 9, wherein the pharmaceutical composition comprises L-glutathione.

16. The pharmaceutical composition according to any one of claims 1 to 14, wherein the boronic acid compound forms a complex with a radioactive isotope.

17. The pharmaceutical composition according to any one of claims 1 to 14, wherein the boronic acid compound forms a complex with a metal ion.

18. The pharmaceutical composition according to claims 1 to 14, wherein the boronic acid compound forms a complex with a diagnostic radionuclide.

19. The pharmaceutical composition according to claims 1 to 14, wherein the boronic acid compound forms a complex with a therapeutic radionuclide.

20. The boronic acid compound is ４３ Sc, ４４ Sc, ５１ Mn, ５２ Mn, ６４ Cd, ６７ Ga, ６８ Ga, ８６ Y, ８９ Zr, ９４ｍ Tc, ９９ｍ Tc, １１１ In, １４９ Tb, １５２ Tb, １５５ Tb, ２０１ Tl, ２０３ Pb, １８ F, ７６ Br, ７７ Br, １２３ I, １２４ I, and １２５ A pharmaceutical composition according to any one of claims 1 to 14, which forms a complex with a radionuclide selected from I.

21. The pharmaceutical composition according to any one of claims 1 to 14, wherein the boronic acid compound forms a complex with a radionuclide selected from 47 Sc, 67 Cu, 89 Sr, 90 Y, 153 Sm, 149 Tb, 161 Tb, 177 Lu, 186 Re, 188 Re, 212 Pb, 213 Bi, 223 Ra, 225 Ac, 226 Th, 227 Th, 131 I, and 211 At.

22. The pharmaceutical composition according to any one of claims 1 to 14, wherein the boronic acid compound forms a complex with 177-Lu.

23. The pharmaceutical composition according to any one of claims 1 to 14, wherein the boronic acid compound forms a complex with 68-Ga.

24. The pharmaceutical composition according to any one of claims 1 to 14, wherein the boronic acid compound forms a complex with 225-Ac.

25. The pharmaceutical composition according to any one of claims 1 to 14, wherein the boronic acid compound forms a complex with 64-Cu.

26. The pharmaceutical composition according to any one of claims 1 to 25, wherein the boronic acid compound is present in the composition in a therapeutically effective amount.

27. The pharmaceutical composition according to any one of claims 1 to 26, wherein the one or more sulfur compounds are present in the composition in an amount of at least about 0.1 N.

28. The pharmaceutical composition according to any one of claims 1 to 26, wherein the mass ratio (weight:weight ratio) of 1) one or more sulfur compounds to 2) one or more boronic acid compounds is 1:10 to 10:

1.

29. The pharmaceutical composition according to any one of claims 1 to 28, wherein the boronic acid compound has a purity of at least 95% three days after preparation and storage at 25°C.

30. (a) The pharmaceutical composition according to any one of claims 1 to 29; and (b) Instructions for use of the composition A kit that includes this.

31. The kit according to claim 30, wherein the instructions for use are for the diagnosis or treatment of a disease or disorder associated with the overexpression of fibroblast-activating protein (FAP).

32. (i) The above instructions for use are for the treatment of cancer, or (ii) The above instructions for use are for the treatment of bacterial infections, or (iii) The kit according to claim 30 or 31, wherein the instructions for use are for the treatment of viral infections.

33. A pharmaceutical composition according to any one of claims 1 to 29, for treating a subject suffering from a neoplasm, a subject suffering from a tumor, a subject suffering from a bacterial infection, or a subject suffering from a viral infection.

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