Synthesis of constrained complexing agents

Abstract

Described herein are improved methods for the synthesis of tethered complexing agents. Compounds, including salts and solvates thereof, as well as compositions related to the improved methods are also described.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of U.S. Patent Application No. 63 / 272,547, filed October 27, 2021, the disclosure of which is incorporated herein by reference in its entirety for all purposes.

[0002] This application relates to methods of preparing restraining complexing agents (RESCA) and RESCA compounds, as well as compositions prepared therewith. [Background technology]

[0003] Proteins that bind to molecules important in diagnosis, disease monitoring, and visualization ("marker-binding proteins") may be associated or conjugated with a detectable label (e.g., a radioactive or fluorescent label) that allows visualization of the molecule, and thus its location and amount (qualitative or quantitative). In some cases, the detectable label is directly associated or conjugated to the marker-binding protein, while in other cases, the detectable label is attached to the marker protein via a linking molecule ("linker") that may be non-cleavable or cleavable (thus allowing for controlled association of the marker-binding protein with its detectable label). Linkers may be associated with the detectable label in a variety of ways, including direct conjugation to chelation, which is suitable for some metal-containing detectable labels. However, linkers may be incompatible with certain marker proteins, may be inefficient, or may require conditions for binding to the marker protein that require multiple reagents.

[0004] Restrictive complexing agents (RESCA) are used to bind biomolecules 18 F (fluorine-18) is a class of chelator molecules that have been successfully used to facilitate PET (positron emission tomography) imaging, which may be useful as a diagnostic and / or therapeutic tool for a variety of diseases and conditions. 18 The properties of F, especially its moderate half-life, are 89Zr (zirconium-89) and 124 Compared with other radionuclides such as I (iodine-124), this makes it an attractive option for imaging techniques. 18 Aluminum monofluoride (Al) as a source of F 18 F} 2+ ) is used 18 Compared to previously known methods of F labeling, RESCA allows for faster labeling in aqueous media and at lower temperatures. 18 Despite the potential applications of RESCA, the challenging low-yield synthesis has led to the development of RESCA-Al 18 F labeling strategies are a potential barrier to the widespread development and use of F labeling strategies. An improved process for synthesizing RESCA was developed using RESCA-Al 18 It will be useful for the continued development of F labeling methods and their various applications. Summary of the Invention

[0005] The present application provides methods for preparing RESCA compounds, RESCA compounds and compositions, and methods of use thereof.

[0006] In some embodiments, formula (I) TIFF2024539994000001.tif48170 formula (I) A process for preparing a compound of the formula (I), a diastereomer thereof, a salt thereof, a solvate thereof, or a combination of any of the foregoing, comprising the steps of: The method is: (a) Formula (Ia): TIFF2024539994000002.tif16170 Formula (Ia) or a diastereomer thereof, is reacted with a compound of formula (Ib): TIFF2024539994000003.tif21170 formula (Ib) in the presence of a reducing agent in a solvent to obtain a compound of formula (Ic): TIFF2024539994000004.tif40170(Ic) or a diastereomer thereof; (b) reacting a compound of formula (Ic), or a salt thereof, with a compound of formula (Id): TIFF2024539994000005.tif12170 expression (Id) in the presence of a base in a solvent, Formula (Ie): TIFF2024539994000006.tif50170 formula (Ie) or a diastereomer thereof; (c) reacting the compound of formula (Ie) with a base in a solvent to obtain a compound of formula (If): TIFF2024539994000007.tif43170 expression (If) or a diastereomer thereof, comprising forming a compound of formula (1-3): (d) reacting a compound of formula (If) with a compound of formula (Ig): TIFF2024539994000008.tif25170 formula (Ig) in the presence of a carboxyl activating agent in a solvent to obtain a compound of formula (Ih): TIFF2024539994000009.tif45170 type (Ih) or a diastereomer thereof, comprising forming a compound (1-4): (e) a step (1-5) comprising reacting a compound of formula (Ih) with an acid to form a compound of formula (I), a diastereomer thereof, a salt thereof, a solvate thereof, or a combination of any of the foregoing; During the ceremony, R 1 and R 2 are each independently C1-C6 alkyl; R 3 is a halo; R 4 , R 5 , R 6 and R 7 is each independently halo or hydrogen.

[0007] In some embodiments, the solvent in step (1-1) comprises an alcohol. In some embodiments, the solvent is ethanol. In some embodiments, the reducing agent in step (1-1) is a hydride. In some embodiments, the reducing agent is NaBH(OAc)3.

[0008] In some embodiments, step (1-1) further comprises purifying the compound of Formula (Ic) by column chromatography.

[0009] In some embodiments, the solvent in step (1-2) is a polar organic solvent. In some embodiments, the solvent is CH3CN. In some embodiments, the base in step (1-2) is an organic base. In some embodiments, the organic base is an amine base. In some embodiments, the amine base is N,N-diisopropylethylamine (DIPEA).

[0010] In some embodiments, step (1-2) further comprises purifying the compound of formula (1e) by column chromatography.

[0011] In some embodiments, the solvent in step (1-3) is an aqueous solvent. In some embodiments, the solvent further comprises tetrahydrofuran (THF). In some embodiments, the base in step (1-3) is KOH.

[0012] In some embodiments, the solvent in step (1-4) is a halogenated hydrocarbon. In some embodiments, the solvent is CH2Cl2. In some embodiments, the carboxyl activating reagent is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI).

[0013] In some embodiments, the acid in step (1-5) is trifluoroacetic acid.

[0014] In some embodiments, steps (1-5) further comprise purifying the compound of Formula (Ih) by preparative high performance liquid chromatography.

[0015] In some embodiments of any of the preceding formulas, R 1 is methyl. In some embodiments, R 2 is tert-butyl. In some embodiments, R 3 is Br. In some embodiments, R 4 , R 5 , R 6 and R 7 are each F. In some embodiments, the compound of Formula (I) is Compound 1. TIFF2024539994000010.tif48170 Compound 1

[0016] In some embodiments, formula (I) TIFF2024539994000011.tif48170 formula (I) A process for preparing a compound of the formula (I), a diastereomer thereof, a salt thereof, a solvate thereof, or a combination of any of the foregoing, comprising the steps of: The method is as follows: (a) Formula (Ia): TIFF2024539994000012.tif16170 formula (Ia) with a compound of formula (Ib): TIFF2024539994000013.tif21170 formula (Ib) in the presence of a reducing agent in a solvent to obtain a compound of formula (Ic): Step (2-1) comprising forming a compound of formula Ic: (b) reacting a compound of formula (Ic) with an acid to produce a salt of formula (Ic) (2-2): (c) reacting a salt of formula (Ic) with a compound of formula (Id): TIFF2024539994000015.tif12170 expression (Id) in the presence of a base in a solvent, Formula (Ie): TIFF2024539994000016.tif48170 formula (Ie) or a diastereomer thereof, or a salt thereof, comprising forming a compound of formula (2-3): (d) reacting the compound of formula (Ie) with a base in a solvent to obtain a compound of formula (If): TIFF2024539994000017.tif46170 expression (If) or a diastereomer or a salt thereof, comprising forming a compound of formula (2-4): (e) reacting a compound of formula (If) or a salt thereof with a compound of formula (Ig): TIFF2024539994000018.tif25170 formula (Ig) in the presence of a carboxyl activating agent in a solvent to obtain a compound of formula (Ih): TIFF2024539994000019.tif45170 formula (Ih) or a diastereomer or a salt thereof, and (f) a step (2-6) comprising reacting a compound of formula (Ih) with an acid to form a compound of formula (I), a diastereomer thereof, a salt thereof, a solvate thereof, or a combination of any of the foregoing; During the ceremony, R 1 and R 2 are each independently C1-C6 alkyl; R 3 is a halo; R 4 , R 5 , R 6 and R 7 is each independently halo or hydrogen.

[0017] In some embodiments, the solvent in step (2-1) comprises an alcohol. In some embodiments, the alcohol is ethanol. In some embodiments, the reducing agent in step (2-1) comprises a hydride. In some embodiments, the reducing agent is NaBH(OAc)3.

[0018] In some embodiments, the acid in step (2-2) is orotic acid. In some embodiments, step (2-2) provides a salt of Formula (Ic) having the following structure: TIFF2024539994000020.tif45170 Formula (Ic) Orotic acid

[0019] In some embodiments, step (2-2) comprises crystallizing the salt of Formula (Ic) or a diastereomer thereof as an orotate salt.

[0020] In some embodiments, the acid in step (2-2) is fumaric acid. In some embodiments, step (2-2) provides a salt of Formula (Ic) having the following structure: TIFF2024539994000021.tif45170 Formula (Ic) Fumaric acid

[0021] In some embodiments, step (2-2) further comprises crystallizing the salt of Formula (Ic) or a diastereomer thereof as a fumarate salt.

[0022] In some embodiments, the base in step (2-3) is KCO. In some embodiments, the solvent in step (2-3) comprises toluene. In some embodiments, the solvent in step (2-3) comprises water.

[0023] In some embodiments, the base in step (2-4) is KOH. In some embodiments, the solvent in step (2-4) comprises THF. In some embodiments, the solvent in step (2-4) comprises water.

[0024] In some embodiments, step (2-4) provides the salt of Formula (If) as an HCl salt, and step (2-4) further comprises crystallizing the salt of Formula (If).

[0025] In some embodiments, the solvent in step (2-5) is a halogenated hydrocarbon. In some embodiments, the halogenated hydrocarbon is CH2Cl2. In some embodiments, the carboxyl activating reagent in step (2-5) is EDCI.

[0026] In some embodiments, the solvent in step (2-6) is dioxane. In some embodiments, the acid in step (2-6) is HCl.

[0027] In some embodiments, step (2-6) provides a salt of Formula (I), or a solvate thereof, having the following structure: TIFF2024539994000022.tif48170 Formula (I-1)HCl

[0028] In some embodiments, step (2-6) further comprises crystallizing the salt of formula (I). In some embodiments in any of the preceding formulas, R 1 is methyl. In some embodiments, R 2 is tert-butyl. In some embodiments, R 3 is Br. In some embodiments, R 4 , R 5 , R 6 and R 7 is F.

[0029] In some embodiments, the solvate salt of Formula (I) has the structure: TIFF2024539994000023.tif48170 Compound 2

[0030] In some embodiments, a composition comprising one or more diastereomers of a compound of formula (I), a salt thereof, or a solvate thereof. TIFF2024539994000024.tif48170 formula (I) (produced by the methods described herein) are provided herein.

[0031] In some embodiments, the composition comprises at least about 90% (w / w) of the trans diastereomer of the compound of formula (I). In some embodiments, the trans diastereomer has the following structure: TIFF2024539994000025.tif48170 formula (I-1)

[0032] In some embodiments, R 4 , R 5 , R 6 and R 7 is F.

[0033] In some embodiments, a salt of Formula (I) or a solvate thereof is provided, the salt of Formula (I) having the structure: TIFF2024539994000026.tif48170 Formula (I-1)HCl (produced by the methods described herein). In some embodiments, R 4 , R 5 , R 6 and R 7 is F.

[0034] In some embodiments, the formula (Ie): TIFF2024539994000027.tif50170 formula(Ie) Provided herein is a compound of the formula: (In the formula, R 1 and R 2 are each independently C1-C6 alkyl.

[0035] In some embodiments, the compound of formula (Ic): TIFF2024539994000028.tif40170(Ic) Provided herein is a compound of the formula: (In the formula, R 1 is C1-C6 alkyl.

[0036] In some embodiments, provided herein are compositions comprising a complex comprising a compound prepared using the methods described herein and a metal halide. In some embodiments, the metal halide is {Al 18 F} 2+ In some embodiments, at least 90% (w / w) of the compounds in the composition have the following structure: TIFF2024539994000029.tif48170 formula (I-1) In some embodiments, R 4 , R 5 , R 6 and R 7 is F. In some embodiments, the composition further comprises a polypeptide, and the complex is bound to the polypeptide. In some embodiments, the polypeptide is an antibody or an antibody fragment. In some embodiments, the antibody fragment is selected from the group consisting of VHH, scFv, minibody, Fab, Fab', and Fv fragments. In some embodiments, the polypeptide is a non-antibody scaffold protein. In some embodiments, the non-antibody scaffold protein is selected from the group consisting of affibody, affilin, noctin, fibronectin, anticalin, atrimer, avimer, FN3 scaffold, fynomer, Kunitz domain, pronectin, OBody, and DARPin. In some embodiments, the composition is a pharmaceutical composition suitable for administration to a subject. In some embodiments, the pharmaceutical composition is suitable for intravenous administration. In some embodiments, the subject is a human.

[0037] In some embodiments, the polypeptide and 18 1. A method for preparing a labeling agent comprising a F radionuclide, comprising the steps of: (a) contacting a polypeptide with a compound prepared using the methods described herein under conditions that allow binding of the conjugate to the polypeptide to provide a conjugate; and (b) removing the complex by 18 Provided herein is a method comprising contacting a labeling agent with an aluminum fluoride complex comprising F to provide the labeling agent.

[0038] In some embodiments, a crystalline salt of a compound having the structure: TIFF2024539994000030.tif38170 Compound 1c Orotic acid Salt, (a) an X-ray powder diffraction pattern having characteristic peaks at about 7.5° 2-theta, about 13.9° 2-theta, about 16.6° 2-theta, about 23.4° 2-theta, and about 25.6° 2-theta; (b) an X-ray powder diffraction pattern substantially identical to the pattern for “orotic acid type A” in Figure 1 ; (c) Thermogravimetric analysis thermogram showing a mass loss of approximately 3.3% between 20 and 150 °C; (d) Thermogravimetric analysis thermogram substantially the same as that shown in Figure 2; (e) Differential scanning calorimetry thermogram showing an endothermic peak at about 172.8°C; (f) A differential scanning calorimetry thermogram substantially the same as that shown in Figure 2; (g) Has a characteristic peak at about 5.68 ppm 1 H nuclear magnetic resonance spectrum; or (h) Substantially the same as shown in Figure 3 1 Provided herein is a crystalline salt having at least one H nuclear magnetic resonance spectrum.

[0039] In some embodiments, a crystalline salt of a compound having the structure: TIFF2024539994000031.tif38170 Compound 1c Orotic acid Salt, (a) an X-ray powder diffraction pattern having characteristic peaks at about 7.2° 2-theta, about 14.0° 2-theta, about 15.2° 2-theta, about 22.7° 2-theta, and about 25.0° 2-theta; (b) X-ray powder diffraction pattern substantially identical to the pattern for “orotic acid type C” in Figure 1 ; (c) Thermogravimetric analysis thermogram showing a mass loss of approximately 2.6% between 20 and 150 °C; (d) Thermogravimetric analysis thermogram substantially the same as that shown in Figure 4 ; (e) a differential scanning calorimetry thermogram showing an exothermic peak at about 161.7°C and an endothermic peak at about 182.9°C; (f) Differential scanning calorimetry thermogram substantially the same as that shown in Figure 4; (g) Has a characteristic peak at about 5.69 ppm 1 H nuclear magnetic resonance spectrum; and (h) Substantially the same as shown in Figure 5 1 Provided herein is a crystalline salt having at least one H nuclear magnetic resonance spectrum.

[0040] In some embodiments, a crystalline salt of a compound having the structure: TIFF2024539994000032.tif38170 Compound 1c Orotic acid Salt, (a) an X-ray powder diffraction pattern having characteristic peaks at about 0.3° 2-theta, about 7.8° 2-theta, about 9.7° 2-theta, about 13.9° 2-theta, about 16.7° 2-theta, about 21.4° 2-theta, and about 23.4° 2-theta; (b) X-ray powder diffraction patterns substantially identical to either the “wet sample” or the “dry sample” shown in Figure 6 ; (c) Thermogravimetric analysis thermogram showing a mass loss of approximately 4.8% between 20 and 150 °C; (d) Thermogravimetric analysis thermogram substantially the same as that shown in Figure 7 ; (e) a differential scanning calorimetry thermogram showing an exothermic peak at about 178.1°C and an endothermic peak at about 185.6°C; (f) Differential scanning calorimetry thermogram substantially the same as that shown in Figure 7; (g) Has a characteristic peak at about 5.68 ppm 1 H nuclear magnetic resonance spectrum; and (h) Substantially the same as shown in Figure 8 1 Provided herein is a crystalline salt having at least one H nuclear magnetic resonance spectrum.

[0041] In some embodiments, a crystalline salt of a compound having the structure: TIFF2024539994000033.tif41170 Compound 1c Fumaric acid Salt, (a) an X-ray powder diffraction pattern having characteristic peaks at about 7.9° 2-theta, about 15.8° 2-theta, about 18.6° 2-theta, about 19.3° 2-theta, about 20.1° 2-theta, about 21.0° 2-theta, and about 24.7° 2-theta; (b) X-ray powder diffraction patterns substantially identical to either the “wet sample” or the “dry sample” shown in Figure 9 ; (c) Thermogravimetric analysis thermogram showing a mass loss of approximately 2.4% between 20 and 140 °C; (d) Thermogravimetric analysis thermogram substantially the same as that shown in Figure 10; (e) Differential scanning calorimetry thermogram showing an endothermic peak at about 154.7°C; (f) A differential scanning calorimetry thermogram substantially the same as that shown in Figure 10; (g) Has a characteristic peak at about 6.39 ppm 1 H nuclear magnetic resonance spectrum; and (h) Substantially the same as shown in FIG. 11 1 Provided herein is a crystalline salt having at least one H nuclear magnetic resonance spectrum.

[0042] In some embodiments, a crystalline solvate of a compound having the structure: TIFF2024539994000034.tif48170 Compound 2 The solvate of the salt is (a) an X-ray powder diffraction pattern having characteristic peaks at about 6.8° 2-theta, about 17.6° 2-theta, about 20.3° 2-theta, about 22.2° 2-theta, and about 23.3° 2-theta; and (b) Provided herein is a crystalline solvate having at least one X-ray powder diffraction pattern substantially the same as that shown in FIG.

[0043] In some embodiments, (i) a compound prepared using the methods provided herein; or (ii) Provided herein are tethered complexing agents, including the crystallizing salts or solvates described herein.

[0044] In some embodiments, (i) a compound prepared using the methods described herein, or (ii) Provided herein are labeling moieties that include a detectable label that includes a metal halide attached or chelated to a linker that includes a crystallizing salt or solvate as described herein.

[0045] In some embodiments, (i) a compound prepared using the methods described herein, or (ii) Provided herein is a label-conjugated marker binder protein comprising a detectable label comprising a metal halide linked to the marker binder protein by a linker comprising a crystallizing salt or solvate as described herein.

[0046] In some embodiments, (i) a compound prepared using the methods described herein, or (ii) exposing the marker binder protein to a label-bound marker binder protein comprising a detectable label comprising a metal halide linked to the marker binder protein by a linker comprising a crystallizing salt or solvate as described herein; the marker binder protein binds to the molecule; and Methods are provided herein that include detecting the label. [Brief description of the drawings]

[0047] [Figure 1] FIG. 1 is a collection of XRPD (X-ray powder diffraction) patterns of a sample of compound 1c orotic acid compound.

[0048] [Diagram 2] FIG. 2 shows the TGA (thermogravimetric analysis) and DSC (differential scanning calorimetry) thermograms of compound 1c orotic acid (orotic acid type A).

[0049] [Diagram 3] FIG. 3 is a 1H NMR (nuclear magnetic resonance) spectrum of compound 1c orotic acid (orotic acid type A).

[0050] [Figure 4] FIG. 4 shows the TGA and DSC thermograms of compound 1c orotic acid (orotic acid type C).

[0051] [Diagram 5] FIG. 5 is the 1H NMR spectrum of compound 1c orotic acid (orotic acid type C).

[0052] [Figure 6] FIG. 6 is a collection of XRPD patterns of sample compound 1c orotic acid (reformulated orotic acid type A).

[0053] [Figure 7] FIG. 7 shows the TGA and DSC thermograms of compound 1c orotic acid (reformulated orotic acid type A).

[0054] [Figure 8] FIG. 8 is the 1H NMR spectrum of compound 1c orotic acid (reprepared orotic acid type A).

[0055] [Figure 9] FIG. 9 is a collection of XRPD patterns of sample compound 1c fumaric acid (fumaric acid type A).

[0056] [Figure 10] FIG. 10 shows the TGA and DSC thermograms of compound 1c fumaric acid (fumaric acid type A).

[0057] [Figure 11] FIG. 11 is the 1H NMR spectrum of compound 1c fumaric acid (fumaric acid type A).

[0058] [Figure 12] FIG. 12 is a collection of XRPD patterns of sample compound 1c fumaric acid (reconstituted fumaric acid type A).

[0059] [Figure 13] FIG. 13 shows the TGA and DSC thermograms of compound 1c fumaric acid (reconstituted fumaric acid type A).

[0060] [Figure 14] FIG. 14 is the 1H NMR spectrum of compound 1c fumaric acid (reprepared fumaric acid type A).

[0061] [Figure 15] FIG. 15 is a collection of XRPD patterns of samples of Compound 2 compound. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0062] The disclosed synthesis method includes several improvements over known methods of RESCA synthesis (WO 2016 / 065435). Compared to previously reported methods, the number of steps required is reduced, less expensive reagents are used, and / or yields are improved. In some embodiments, the use of column chromatography is eliminated in some or all of the steps of the synthesis. In some embodiments, the synthesis retains some or all of the above-mentioned advantages when the method is carried out on a larger scale than previously reported methods.

[0063] The disclosed method uses a different synthetic route than the previously known methods for the synthesis of RESCA. The first step of the improved method is to prepare a compound of formula (Ic): TIFF2024539994000035.tif40170 formula (Ic) or a salt thereof, providing at least one point of distinction of the method disclosed herein from previous methods. The improved synthetic route eliminates the need for palladium catalyst. In some embodiments (e.g., Example 3 below), the product of some steps can be crystallized, eliminating the need for more complex purification of intermediates.

[0064] I. Definition As used herein, "C1-C xThe term "alkyl", alone or in combination, means a straight-chain or branched alkyl moiety having 1 to x carbon atoms, where x is an integer. Thus, the term "C1-C6 alkyl", alone or in combination, means a straight-chain or branched alkyl moiety having 1 to 6 carbon atoms. Some examples of straight-chain and branched C1-C6 alkyl groups, when present at the terminal end of the molecule, are, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, 2-methylbutyl, 2,2-dimethylpropyl, n-hexyl, 2-methylpentyl, 3-methylpentyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl, and 3,3-dimethylpropyl. When the two portions of the molecule are linked by a C1-C6 alkyl group (i.e., C1-C6 contains a divalent "alkylene" portion), examples include -CH2-, -CH2CH2-, -CH(CH3)-, CH2CH2CH2-, CH(C2H5)-, and -C(CH3)2-.

[0065] As used herein, "halogen" refers to fluorine, chlorine, bromine, or iodine. The term "halo" refers to a halogen substituent, i.e., fluoro, chloro, bromo, or iodo.

[0066] As used herein, the term "halogenated hydrocarbon" refers to a hydrocarbon compound or moiety substituted at least once with a halogen moiety. Some halogenated hydrocarbons are useful as solvents for chemical synthesis. Examples of the term "halogenated hydrocarbon" include, but are not limited to, dichloromethane (also known as CH2Cl2 or DCM) and chloroform (also known as CHCl3).

[0067] As used herein, the term "equivalent" means molar equivalent, unless otherwise specified.

[0068] As used herein, the term "chiral" refers to molecules that are not superimposable on their mirror image, and the term "achiral" refers to molecules that are superimposable on their mirror image.

[0069] As used herein, the term "stereoisomers" refers to compounds that have identical chemical formulae (i.e., the same number of each type of atom), but differ with regard to the arrangement of the atoms or groups in physical space.

[0070] As used herein, the term "diastereomer" refers to a stereoisomer with two or more chiral centers and whose molecules are not mirror images of one another. Diastereomers may have different physical properties, such as melting points, boiling points, spectral properties, and reactivities.

[0071] As used herein, the term "enantiomer" refers to a molecule that is chiral and is the mirror image of another molecule. Two molecules that are mirror images of each other are "enantiomers." Enantiomers are not superimposable on each other.

[0072] Stereochemical definitions and conventions used herein generally follow S.P. Parker, ed., McGraw-Hill Dictionary of Chemical Terms (1984) McGraw-Hill Book Company, New York; and Eliel, E. and Wilen, S., "Stereochemistry of Organic Compounds", John Wiley & Sons, Inc., New York, 1994. Compounds in this application may contain asymmetric or chiral centers and therefore may exist as different stereoisomers. All stereoisomers of compounds listed herein, including but not limited to diastereomers, enantiomers, atropisomers, and mixtures thereof (e.g., racemic mixtures), are intended to be included in this application.

[0073] One or more compounds of the present application may exist in unsolvated as well as solvated forms with pharma- ceutically acceptable solvents such as water, ethanol, and the like, and the present application is intended to encompass both solvated and unsolvated forms. "Solvate" refers to a physical association of a compound of the present application with one or more solvent molecules. This physical association includes varying degrees of ionic and covalent bonding, including hydrogen bonding. In certain embodiments, a solvate will be capable of isolation, such as when one or more solvent molecules are incorporated into the crystal lattice of a crystalline solid. "Solvate" encompasses both solution-phase and isolatable solvates. Non-limiting examples of suitable solvates include solvates of ethanol, water, 1,4-dioxane, hydrochloric acid, and the like. "Hydrate" is a solvate in which the solvent molecule is H2O.

[0074] It is understood that embodiments described herein as "comprising" certain features mean that embodiments "consisting of" and / or "consisting essentially of" those features are contemplated.

[0075] References herein to a value or parameter "about" refer to the normal error range of the respective value, which is readily known to those skilled in the art. References herein to a value or parameter "about" include (and describe) aspects directed to the value or parameter itself. For example, a description that refers to "about X" includes the description of "X".

[0076] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0077] As used herein, the term "and / or", e.g., the phrase "A and / or B", is intended to include both A and B; A or B; A (alone); and B (alone). Similarly, as used herein, the term "and / or", e.g., "A, B, and / or C", is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B, or C; A and B; B and C; A (alone); B (alone); and C (alone).

[0078] II. Synthesis method Provided herein are methods for the synthesis of compounds capable of chelating metal halides. In some embodiments, the compounds have the formula (I): TIFF2024539994000036.tif48170 formula (I) or a diastereomer thereof, a salt thereof, a solvate thereof, or a combination of any of the foregoing. (In the formula, R 4 , R 5 , R 6 and R 7 are each independently halo or hydroxy.

[0079] In some embodiments, the compound of formula (I) has the structure: TIFF2024539994000037.tif48170 formula (I-1) or a salt thereof, a solvate thereof, or a solvate of a salt thereof.

[0080] In some embodiments, the compound of formula (I) is Compound 1: TIFF2024539994000038.tif48170 compound 1, or a salt thereof, a solvate thereof, or a solvate of a salt thereof.

[0081] The methods described herein have chemical and / or commercial advantages over previously disclosed synthetic methods. In some embodiments, the yield of the reaction is higher than the reported yields of compound 1 or compound 2 (shown below) from previously known methods (e.g., WO 2016 / 065435). In some embodiments, the methods described herein provide products in high yields. In some embodiments, the yield is at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 98%, or at least about 99%. In some embodiments, the yield is about 20% to about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 98%, about 99%, or about 100%. In some embodiments, the yield is about 30% to about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 98%, about 99%, or about 100%. In some embodiments, the yield is about 40% to about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 98%, about 99%, or about 100%. In some embodiments, the yield is about 50% to about 60%, about 70%, about 80%, about 90%, about 95%, about 98%, about 99%, or about 100%. In some embodiments, the yield is about 60% to about 70%, about 80%, about 90%, about 95%, about 98%, about 99%, or about 100%. In some embodiments, the yield is about 70% to about 80%, about 90%, about 95%, about 98%, about 99% and about 100%. In some embodiments, the yield is about 80% to about 90%, about 95%, about 98%, about 99% or about 100%. In some embodiments, the yield is about 90% to about 95%, about 98%, about 99% or about 100%. In some embodiments, the yield is about 95% to about 98%, about 99% or about 100%. In some embodiments, the yield is about 98% to about 99% or about 100%. In some embodiments, the yield is about 99% to about 100%. In certain embodiments, the yield is about 20% to about 30%.In other specific embodiments, the yield is about 50% to about 70%, e.g., 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69% and 70%.

[0082] In some embodiments, the method provides a product with higher purity than the previously disclosed method. In some embodiments, the method provides a composition comprising a single diastereomer of formula (I) (e.g., Compound 1 or Compound 2) with a purity of at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%. In some embodiments, the purity is about 97.5A%, 98A%, and 99A% (area%) as determined by high performance liquid chromatography (HPLC).

[0083] In some embodiments, the reagents used in the present method are less expensive than those used in the previously disclosed methods. In some embodiments, the disclosed method uses reagents that are less expensive and often more environmentally friendly than those used in the previously disclosed methods. However, importantly, the method does not require a palladium catalyst.

[0084] In some embodiments, a compound of formula (I) (e.g., Compound 1 or Compound 2) may be prepared on a large scale. In some embodiments, "large scale" refers to the use of at least 100, 250, 500, 750, or 1000 grams of starting material, intermediate, or reagent in any step of the process. In some embodiments, "large scale" refers to the use of at least 1, 10, 25, 50, or 100 kg of starting material, intermediate, or reagent in any step of the process.

[0085] In some embodiments, the method provides a product with improved yield and / or higher purity than previously disclosed methods. In some embodiments, the method provides a product with improved yield and / or higher purity at a larger scale than previously known methods. Any of the yields, purities, and scales may be combined together as if each combination of a particular yield, a particular purity, and a particular scale were specifically and individually recited.

[0086] A. First approach In some embodiments, formula (I) TIFF2024539994000039.tif48170 formula (I) (In the formula, R 4 , R 5 , R 6 and R 7 are each independently halo (e.g., F) or hydrogen), a diastereomer thereof, a salt thereof, a solvate thereof, or a combination of any of the foregoing, comprising the steps of: A method is provided herein, comprising one or more of steps (1-1) to (1-5). An exemplary synthesis scheme is shown in Scheme 1 of Example 1.

[0087] Step (1-1) is a step of producing a compound of formula (Ia): TIFF2024539994000040.tif16170 formula (Ia) or a diastereomer thereof, is reacted with a compound of formula (Ib): TIFF2024539994000041.tif21170 formula (Ib) in the presence of a reducing agent in a solvent to obtain a compound of formula (Ic): TIFF2024539994000042.tif40170 formula (Ic) or a diastereomer thereof, 1 is C1-C6 alkyl).

[0088] In some embodiments, about 0.5 to about 2 equivalents of the compound of formula (Ib) are used relative to the amount of the compound of formula (Ia). In some embodiments, about 1 equivalent is used.

[0089] In some embodiments, the compound of Formula (Ia) is a stereoisomer selected from the group consisting of: TIFF2024539994000043.tif33170

[0090] In some embodiments, the solvent in step (1-1) comprises an alcohol. In some embodiments, the solvent is methanol, ethanol, n-propanol, or isopropanol. In some embodiments, the solvent is ethanol.

[0091] In some embodiments, the reducing agent in step (1-1) comprises a hydride. In some embodiments, the reducing agent comprises NaBH(OAc)3, NaBH4CN, and NaBH4. In some embodiments, the reducing agent is NaBH(OAc)3. In some embodiments, 1 to 2 equivalents of the reducing agent are used relative to the amount of the compound of formula (Ia) (i.e., the molar ratio of the reducing agent to the compound is 1:1 to 2:1). In some embodiments, about 1.5 to about 2 equivalents of the reducing agent are used. In some embodiments, about 1.8 to about 2 equivalents of the reducing agent are used. In some embodiments, about 1.9 equivalents of the reducing agent are used.

[0092] In some embodiments, step (1-1) is carried out at a temperature maintained at about 20° C. to about 70° C. for a duration of about 1 to about 3 hours. In some embodiments, the temperature is maintained at about 30° C. to about 60° C. In some embodiments, the temperature is about 40° C. to about 50° C. In some embodiments, the duration is about 2 hours. In some embodiments, step (1-1) is carried out at a temperature maintained at about 40° C. to about 50° C. for a duration of about 2 hours.

[0093] In some embodiments, step (1-1) further comprises purifying the compound of formula (Ic). In some embodiments, the purification uses a silica gel column. In some embodiments, the purification uses a solvent mixture. In some embodiments, the solvent mixture comprises dichloromethane (DCM) and methanol (MeOH). In some embodiments, the HPLC comprises varying the ratio of DCM to MeOH over time from 100:0 to 30:1 (v / v). In some embodiments, the MeOH comprises NH3. In some embodiments, the NH3 is present in MeOH at a concentration of 7 M.

[0094] In some embodiments, step (1-1) does not include purification of the compound of Formula (Ic).

[0095] In some embodiments, the compound of Formula (Ic) is a compound of one of the following formulas: TIFF2024539994000044.tif50170

[0096] Step (1-2) is a step of reacting a compound of formula (Ic) or a diastereomer thereof with a compound of formula (Id) TIFF2024539994000045.tif12170 expression (Id) in the presence of a base and a solvent, Formula (Ie): TIFF2024539994000046.tif50170 formula (Ie) or a diastereomer thereof. (In the formula, R 1 and R 2 are each independently C1-C6 alkyl; R 3 is a halo.)

[0097] In some embodiments, about 2 to about 5 equivalents of the compound of formula (Id) are used relative to the amount of the compound of formula (Ic). In some embodiments, about 3 to about 4 equivalents are used. In some embodiments, 3.5 equivalents are used.

[0098] In some embodiments, the solvent in step (1-2) comprises a polar organic solvent. In some embodiments, the solvent comprises CH3CN, tetrahydrofuran (THF), MeTHF, acetone, methyl ethyl ketone, isopropyl acetate, N,N-dimethylformamide (DMF), N-methyl-2-pyrrolidone (NMP) or dimethyl sulfoxide (DMSO). In some embodiments, the solvent is CH3CN.

[0099] In some embodiments, the base in step (1-2) is an organic base. In some embodiments, the base is an amine base. In some embodiments, the base is triethylamine, tributylamine, N-methylmorpholine, N,N-diisopropylethylamine (DIPEA) or 1,8-diazabicyclo[5.4.0]undec-7-ene. In some embodiments, the solvent is DIPEA. In some embodiments, about 4 to about 12 equivalents of base are used relative to the amount of the compound of formula (Ic). In some embodiments, about 6 to about 10 equivalents of base are used. In some embodiments, about 7 to about 9 equivalents of base are used. In some embodiments, about 8 equivalents of base are used.

[0100] In some embodiments, step (1-2) is carried out in CH3CN in the presence of DIPEA. In some such embodiments, about 8 equivalents of DIPEA are used.

[0101] In some embodiments, step (1-2) is carried out at a temperature maintained at about 20° C. to about 70° C. for a duration of about 12 to about 24 hours. In some embodiments, the temperature is maintained at about 30° C. to about 60° C. In some embodiments, the temperature is about 40° C. to about 50° C. In some embodiments, the duration is between about 16 and about 20 hours. In some embodiments, the duration is about 18 hours. In some embodiments, step (1-2) is carried out at a temperature maintained at about 40° C. to about 50° C. for a duration of about 18 hours.

[0102] In some embodiments, step (1-2) further comprises purifying the compound of formula (Ie). In some embodiments, step (1-2) further comprises purifying the compound of formula (Ie) by column chromatography. In some embodiments, the purification uses a silica gel column. In some embodiments, the purification uses a solvent mixture comprising ethyl acetate and n-heptane. In some embodiments, the purification comprises changing the ratio of ethyl acetate to n-heptane over time from 100:0 to 1:5 (v / v).

[0103] In some embodiments, step (1-2) does not include purification of the compound of Formula (Ie).

[0104] In some embodiments, the compound of formula (Ie) is a compound of the following formula: TIFF2024539994000047.tif60170

[0105] Step (1-3) is reacting a compound of formula (Ie) or a diastereomer thereof with a base in a solvent to obtain a compound of formula (If): TIFF2024539994000048.tif46170 formula (1f) or a diastereomer thereof, wherein each R 2 is C1-C6 alkyl.

[0106] In some embodiments, the solvent in step (1-3) comprises water. In some embodiments, the solvent comprises an organic solvent. In some embodiments, the solvent is a mixture of water and an organic solvent. In some embodiments, the solvent comprises tetrahydrofuran (THF), MeTHF, methyl tertiary-butyl ether, cyclopentyl methyl ether, dichloromethane, and isopropyl acetate. In some embodiments, the solvent comprises THF. In some embodiments, the solvent is a mixture of water and THF. In some embodiments, a ratio of organic solvent to water is used. In some embodiments, the ratio of organic solvent to water is about 4:1 to 1:1. In some embodiments, the ratio is about 10:3 to about 6:3. In some embodiments, the ratio is about 8:3. All ratios in the above embodiments are equally applicable to the embodiment where the ratio is the ratio of THF to water.

[0107] In some embodiments, the base in step (1-3) is a strong base. In some embodiments, the base is KOH. In some embodiments, about 1 to about 3 equivalents of base are used relative to the amount of the compound of formula (Ie). In some embodiments, about 2 equivalents of base are used.

[0108] In some embodiments, step (1-3) is carried out in a mixture of water and THF in the presence of KOH. In some such embodiments, about 2 equivalents of KOH are used.

[0109] In some embodiments, the compound of formula (If) is a compound of one of the following formulae: TIFF2024539994000049.tif53170

[0110] In some embodiments, step (1-3) is carried out at a temperature maintained at about -20°C to about 30°C for a duration of about 18 to about 30 hours. In some embodiments, the temperature is maintained at about -10°C to about 20°C. In some embodiments, the temperature is about 0°C to about 10°C. In some embodiments, the duration is about 16 to about 20 hours. In some embodiments, the duration is about 18 hours. In some embodiments, step (1-3) is carried out at a temperature maintained at about 0°C to about 10°C for a duration of about 18 hours.

[0111] Step (1-4) is a step of reacting a compound of formula (If) or a diastereomer thereof with a compound of formula (Ig): TIFF2024539994000050.tif25170 formula (Ig) in the presence of a carboxyl activating agent in a solvent to obtain a compound of formula (Ih): TIFF2024539994000051.tif45170 formula(Ih) or a diastereomer thereof. (In the formula, R 2 are independently C1-C6 alkyl; R 4 , R 5 , R 6 and R 7 are each independently halo or hydrogen.

[0112] In some embodiments, the compound of formula (Ih) is a compound of one of the following formulas: TIFF2024539994000052.tif49170

[0113] In some embodiments, about 0.8 to about 1.6 equivalents of the compound of formula (Ig) are used relative to the amount of the compound of formula (If). In some embodiments, about 1.0 to about 1.4 equivalents are used. In some embodiments, about 1.2 equivalents are used.

[0114] In some embodiments, the solvent in step (1-4) is a halogenated hydrocarbon. In some embodiments, the solvent is selected from the group consisting of 1,2-dichloroethane, CH2Cl2, and CHCl3. In some embodiments, the solvent is CH2Cl2 (DCM or dichloromethane).

[0115] In some embodiments, the carboxyl activating reagent in steps (1-4) comprises a carbodiimide. In some embodiments, the carboxyl activating reagent is 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDCI).

[0116] In some embodiments, steps (1-4) are carried out in DCM in the presence of EDCI.

[0117] In some embodiments, step (1-4) is carried out at a temperature maintained at about 0° C. to about 50° C. for a duration of about 8 to about 24 hours. In some embodiments, the temperature is maintained at about 10° C. to about 40° C. In some embodiments, the temperature is about 20° C. to about 30° C. In some embodiments, the duration is about 12 to about 20 hours. In some embodiments, the duration is about 16 hours. In some embodiments, step (1-4) is carried out at a temperature maintained at about 20° C. to about 30° C. for a duration of about 10 hours. In some embodiments, step (1-4) is carried out at a temperature maintained at about 20° C. to about 30° C. for a duration of about 16 hours.

[0118] Step (1-5) comprises reacting a compound of formula (Ih) or a diastereomer thereof with an acid to form a compound of formula (I) or a diastereomer thereof. In some embodiments, the acid comprises HCl, HBr, H2SO4, methanesulfonic acid, p-toluenesulfonic acid, or trifluoroacetic acid. In some embodiments, the acid is trifluoroacetic acid.

[0119] In some embodiments, step (1-5) further comprises purifying the compound of formula (I). In some embodiments, step (1-5) further comprises purifying the compound of formula (I) by preparative high performance liquid chromatography (HPLC). In some embodiments, HPLC is performed with a 98:2 ratio of 0.01% TFA in water to acetonitrile.

[0120] In some embodiments, steps (1-5) do not include purification of the compound of Formula (I).

[0121] In some embodiments, step (1-5) is carried out at a temperature maintained at about 0° C. to about 50° C. for a duration of about 8 to about 24 hours. In some embodiments, the temperature is maintained at about 10° C. to about 40° C. In some embodiments, the temperature is about 20° C. to about 30° C. In some embodiments, the duration is about 12 to 20 hours. In some embodiments, the duration is about 16 hours. In some embodiments, step (1-5) is carried out at a temperature maintained at about 20° C. to about 30° C. for a duration of about 16 hours.

[0122] In some embodiments, the compound of formula (I) is a compound of one of the following formulas: TIFF2024539994000053.tif49170

[0123] B. Second Approach In some embodiments, the present invention comprises a method for producing a pharmaceutical composition comprising formula (I): TIFF2024539994000054.tif53170 formula (I) The present invention provides a method for preparing a compound of the formula: (In the formula, R 4 , R 5 , R 6 , and R 7are each independently halo (e.g., F) or hydrogen), the method includes one or more of steps (2-1) to (2-6). An exemplary synthesis scheme is shown in Scheme 2 of Example 3. One advantage of the second approach is that the crystallization step eliminates the need for an expensive and time-consuming purification step such as silica gel chromatography.

[0124] Step (2-1) is a process for producing a compound of formula (Ia): TIFF2024539994000055.tif16170 Formula (Ia) or a diastereomer thereof, is reacted with a compound of formula (Ib): TIFF2024539994000056.tif21170 formula (Ib) in the presence of a reducing agent in a solvent to obtain a compound of formula (Ic): TIFF2024539994000057.tif40170 formula (Ic) or a diastereomer thereof, 1 is C1-C6 alkyl).

[0125] In some embodiments, about 0.5 to about 3 equivalents of the compound of formula (Ia) are used relative to the amount of the compound of formula (Ib). In some embodiments, about 1 to about 2 equivalents are used. In some embodiments, about 1.3 equivalents are used.

[0126] In some embodiments, the compound of formula (Ia) comprises, consists of, or consists essentially of one stereoisomer. In some embodiments, the compound of formula (Ia) is a mixture of stereoisomers. In some embodiments, the compound of formula (Ia) is a racemic mixture. In some embodiments, the compound of formula (Ia) is selected from the group consisting of: TIFF2024539994000058.tif33170 In some embodiments, the compound of Formula (Ia) is a mixture comprising two or more of the three stereoisomers shown above.

[0127] In some embodiments, the solvent in step (2-1) comprises an alcohol. In some embodiments, the solvent is methanol, ethanol, n-propanol, or isopropanol. In some embodiments, the solvent is ethanol.

[0128] In some embodiments, the reducing agent in step (2-1) comprises a hydride. In some embodiments, the reducing agent comprises NaBH(OAc)3. In some embodiments, the reducing agent is NaBH(OAc)3. In some embodiments, about 1 to about 5 equivalents of hydride are used relative to the amount of compound of Formula (Ib). In some embodiments, about 2 to about 4 equivalents are used. In some embodiments, about 3 equivalents are used.

[0129] In some embodiments, step (2-1) is carried out in ethanol in the presence of NaBH(OAc). In some embodiments, 3 equivalents of NaBH(OAc) are used.

[0130] In some embodiments, step (2-1) is carried out at a temperature maintained at about 20° C. to about 65° C. for a duration of about 1 to about 3 hours. In some embodiments, the temperature is maintained at about 30° C. to about 55° C. In some embodiments, the temperature is about 40° C. to about 45° C. In some embodiments, the duration is about 2 hours. In some embodiments, step (2-1) is carried out at a temperature maintained at about 40° C. to about 45° C. for a duration of about 2 hours.

[0131] In some embodiments, the compound of Formula (Ic) is a compound of one of the following formulas: TIFF2024539994000059.tif56170

[0132] Step (2-2) comprises reacting a compound of formula (Ic) or a diastereomer thereof with an acid to produce a salt of formula (Ic) or a diastereomer thereof. In some embodiments, the acid is an organic acid. In some embodiments, the acid is selected from the group consisting of fumaric acid, L-tartaric acid, and orotic acid.

[0133] In some embodiments, the acid is orotic acid. In some embodiments, the salt of Formula (Ic) or a diastereomer thereof has the following structure: TIFF2024539994000060.tif43170 Formula (Ic) Orotic acid. In some embodiments, the salt of Formula (I) is compound 1c orotic acid: TIFF2024539994000061.tif38170 Compound 1c Orotic acid. In some embodiments, step (2-2) comprises crystallizing a salt of formula (Ic) or a diastereomer thereof. In some embodiments, step (2-2) comprises crystallizing an orotate salt of formula (Ic) or a diastereomer thereof. In some embodiments, a salt of formula (Ic) or a diastereomer thereof is crystallized from acetone, 2-methyl-tetrahydrofuran, or a mixture of acetonitrile and water (e.g., 19:1 CH3CN and HO).

[0134] In some embodiments, the acid is fumaric acid. In some embodiments, the salt of Formula (Ic) or a diastereomer thereof has the following structure: TIFF2024539994000062.tif45170 Formula (Ic) Fumaric acid. In some embodiments, the salt of Formula (Ic) is compound 1c fumaric acid: TIFF2024539994000063.tif41170 Compound 1c Fumaric acid. In some embodiments, step (2-2) further comprises crystallizing the salt of Formula (Ic) or a diastereomer thereof as a salt of fumaric acid. In some embodiments, the salt of Formula (Ic) or a diastereomer thereof is crystallized from acetone.

[0135] Step (2-3) is reacting a salt of formula (Ic) or a diastereomer thereof with a salt of formula (Id): TIFF2024539994000064.tif12170 expression (Id) in the presence of a base and a solvent, Formula (Ie): TIFF2024539994000065.tif48170 formula (Ie) or a diastereomer thereof. (In the formula, R 1 and R 2 are each independently C1-C6 alkyl; R 3 is a halo.)

[0136] In some embodiments, about 2 to about 5 equivalents of the compound of formula (Id) are used relative to the amount of the salt of formula (Ic). In some embodiments, about 3 to about 4 equivalents are used. In some embodiments, about 3.5 equivalents are used.

[0137] In some embodiments, the base in step (2-3) comprises a carbonate. In some embodiments, the base comprises K2CO3. In some embodiments, the base is K2CO3. In some embodiments, about 1 to about 5 equivalents of base are used relative to the salt of Formula (Ic), or a diastereomer thereof. In some embodiments, about 2 to about 4 equivalents are used. In some embodiments, about 3 equivalents are used.

[0138] In some embodiments, the solvent in step (2-3) comprises water. In some embodiments, the solvent comprises an organic solvent. In some embodiments, the solvent comprises toluene. In some embodiments, the solvent is a mixture of water and an organic solvent. In some embodiments, the solvent is a mixture of water and toluene. In some embodiments, water and toluene are used in a ratio of about 2:1 to about 1:2. In some embodiments, the ratio is about 1:1.

[0139] In some embodiments, step (2-3) is carried out in a mixture of water and toluene in the presence of K2CO3. In some such embodiments, the water and toluene are in a ratio of about 1:1. In some such embodiments, about 3 equivalents of K2CO3 are used. In some such embodiments, the ratio of water to toluene is about 1:1 and about 3 equivalents of K2CO3 are used.

[0140] In some embodiments, step (2-3) is carried out at a temperature maintained at about 25° C. to about 65° C. for a duration of about 8 to about 24 hours. In some embodiments, the temperature is maintained at about 35° C. to about 55° C. In some embodiments, the temperature is maintained at about 45° C. In some embodiments, the duration is between about 12 and about 20 hours. In some embodiments, the duration is about 16 hours. In some embodiments, step (2-3) is carried out at a temperature maintained at about 45° C. for a duration of about 16 hours.

[0141] In some embodiments, the compound of formula (Ie) is a compound of the following formula: TIFF2024539994000066.tif60170

[0142] Step (2-4) is reacting a compound of formula (Ie) or a diastereomer thereof with a base in a solvent to obtain a compound of formula (If): TIFF2024539994000067.tif46170 formula (1f) or a diastereomer thereof, or a salt thereof.

[0143] In some embodiments, the base in step (2-4) is a strong base. In some embodiments, the base is KOH. In some embodiments, about 1 to about 4 equivalents of KOH are used relative to the amount of compound of Formula (Ie). In some embodiments, about 2 to about 3 equivalents are used. In some embodiments, about 2.5 equivalents are used.

[0144] In some embodiments, the solvent in step (2-4) comprises THF. In some embodiments, the solvent comprises water. In some embodiments, the solvent is a mixture comprising water and THF.

[0145] In some embodiments, step (2-4) is carried out in the presence of KOH in a mixture of water and THF. In some such embodiments, 2.5 equivalents of KOH are used.

[0146] In some embodiments, step (2-4) is carried out at a temperature maintained between about 0° C. and 5° C. for a duration of about 20 to about 60 hours. In some embodiments, the temperature was maintained between about 0° C. and 5° C. for a period of about 30 to about 40 hours. In some embodiments, the temperature was maintained at about 0° C. to 5° C. for a duration of about 20 hours.

[0147] In some embodiments, step (2-4) provides an HCl salt of formula (If) or a diastereomer thereof. In some embodiments, step (2-4) comprises crystallizing the salt of formula (If) or a diastereomer thereof. In some embodiments, the salt of formula (If) has the structure: TIFF2024539994000068.tif43170 formula (1f) HCl or a diastereomer thereof.

[0148] In some embodiments, the HCl salt of formula (If) has the following structure: TIFF2024539994000069.tif46170

[0149] In some embodiments, the compound of formula (If) or a salt thereof includes a compound of one of the following formulas or a salt thereof: TIFF2024539994000070.tif54170

[0150] Step (2-5) is reacting a compound of formula (If), a diastereomer thereof, a salt thereof, or a diastereomer of a salt thereof with a compound of formula (Ig): TIFF2024539994000071.tif25170 formula (Ig) in the presence of a carboxyl activating agent in a solvent to obtain a compound of formula (Ih): TIFF2024539994000072.tif45170 type (Ih) or a diastereomer thereof, or a salt thereof.

[0151] In some embodiments, about 1 to about 1.5 equivalents of the compound of formula (Ig) are used relative to the amount of the compound of formula (If). In some embodiments, about 1.2 equivalents are used.

[0152] In some embodiments, the solvent in step (2-5) is a halogenated hydrocarbon. In some embodiments, the solvent is selected from the group consisting of CH2Cl2 or CHCl3. In some embodiments, the solvent is CH2Cl2.

[0153] In some embodiments, the carboxyl activating reagent in step (2-5) is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI). In some embodiments, about 1 to about 2 equivalents of EDCI are used relative to the amount of the compound of formula (If). In some embodiments, about 1.5 equivalents are used.

[0154] In some embodiments, step (2-5) is carried out in CH2Cl2 in the presence of EDCI.

[0155] In some embodiments, step (2-5) is carried out for a duration of about 2 to about 4 hours at a temperature maintained at about 0° C. to about 5° C. In some embodiments, the duration is about 3 hours. In some embodiments, step (2-5) is carried out for a duration of about 3 hours at a temperature maintained at about 0° C. to about 5° C.

[0156] In some embodiments, the compound of formula (Ih) is a compound of one of the following formulas: TIFF2024539994000073.tif49170

[0157] Step (2-6) comprises reacting a compound of formula (Ih) or a diastereomer thereof, a salt thereof, or a diastereomer of the salt thereof with an acid to form a compound of formula (I) or a salt thereof. In some embodiments, step (2-6) comprises reacting a compound of formula (Ih) with an acid to form a salt of a compound of formula (I).

[0158] In some embodiments, the compound of formula (I) is a compound of one of the following formulas or a salt thereof: TIFF2024539994000074.tif46170

[0159] In some embodiments, the acid in step (2-6) is a mineral acid. In some embodiments, the acid in step (2-6) is hydrochloric acid. In some embodiments, the salt of Formula (I) or a solvate thereof is an HCl salt. In some embodiments, the salt of Formula (I) or a solvate thereof has the structure: TIFF2024539994000075.tif48170 Formula (I)HCl In some embodiments, the salt of Formula (I) or a solvate thereof has the structure: TIFF2024539994000076.tif48170 has the formula (I-1) HCl.

[0160] In some embodiments, step (2-6) is carried out in dioxane. In some embodiments, step (2-6) is carried out at a temperature maintained at about 45° C. to about 70° C. for a duration of about 2 to about 4 hours. In some embodiments, the temperature is maintained at about 55° C. to about 60° C. In some embodiments, the duration is about 3 hours. In some embodiments, step (2-6) is carried out in dioxane at a temperature maintained at about 55° C. to about 60° C. for about 3 hours.

[0161] In some embodiments, the compound of formula (I), or a salt, solvate, or salt of a solvate thereof, is compound 2: TIFF2024539994000077.tif56170 Compound 2. In some embodiments, the compound of formula (I) is a diastereomer of compound 2.

[0162] In some embodiments of any of the foregoing methods (including those described in the "First Approach" and "Second Approach" subsections), R 1 is a straight chain C1-C6 alkyl. In some embodiments, R 1 is C1-C3 alkyl. In some embodiments, R 1 is a C1-C3 straight chain alkyl. In some embodiments, R 1 is methyl, ethyl, isopropyl, n-propyl, t-butyl, or sec-butyl. 1 is methyl, ethyl, or isopropyl. In some embodiments, R 1 is methyl or ethyl. In some embodiments, R 1 is methyl.

[0163] In some embodiments of any of the foregoing methods (including those described in the "First Approach" and "Second Approach" subsections), R 2 is a C3-C6 branched alkyl. In some embodiments, R 2 is methyl, ethyl, isopropyl, n-propyl, sec-butyl, or t-butyl. In some embodiments, R 2 is ethyl, isopropyl, or t-butyl. In some embodiments, R 2 is isopropyl or t-butyl. In some embodiments, R 2 is t-butyl.

[0164] In some embodiments of any of the foregoing methods (including those described in the "First Approach" and "Second Approach" subsections), R 3 is F, Cl or Br. In some embodiments, R 3 is Cl or Br. In some embodiments, R 3 is Br.

[0165] In some embodiments of any of the foregoing methods (including those described in the "First Approach" and "Second Approach" subsections), R 4 , R 5 , R 6 and R 7 are each independently halo or hydrogen. 4 , R 5 , R 6 and R 7 are each independently Cl or F. In some embodiments, R 4 , R 5 , R 6 and R 7 are F, respectively.

[0166] III. Compositions, Labeling Agents and Methods of Use Also disclosed herein are compositions and intermediates comprising the compounds disclosed herein.In particular, compositions of stereoisomers of the compounds of formula (I), (Ic), (Ie), (If) and (Ih) are contemplated, including any of the salts, solvates and solvates of salts described above.In some embodiments, compositions are provided that comprise one or more diastereomers of the compound of formula (I), its salt or its solvate. TIFF2024539994000078.tif48170 formula (I) In some embodiments, compounds of formula (I) are prepared using the methods described in Section II-A. In some embodiments, compounds of formula (I) are prepared using the methods described in Section II-B. In some embodiments, R 4 , R 5 , R 6 and R7 are F, respectively.

[0167] In some embodiments, the composition comprises Compound 1, a salt thereof, or a solvate thereof. TIFF2024539994000079.tif48170 Compound 1 In some embodiments, the composition comprises, consists of, or consists essentially of a single diastereomer. In some embodiments, the composition comprises at least about 80%, at least about 90%, at least about 95%, at least about 97%, or at least about 99% (w / w) of the trans diastereomer of Compound 1, or a salt, solvate, or solvate of a salt thereof. In some embodiments, the composition comprises at least about 80%, at least about 90%, at least about 95%, at least about 97%, or at least about 99% (w / w) of the trans diastereomer of Compound 2. TIFF2024539994000080.tif48170 Compound 2

[0168] In some embodiments, the composition further comprises a detectable label. In some embodiments, the detectable label is 18 In some embodiments, the detectable label is a radioactive label such as {AlF}. In some embodiments, the detectable label is a portion of a metal halide. In some embodiments, the metal halide is {AlF} 2+ In some embodiments, the metal halide is {Al 18 F} 2+ In some embodiments, the label (e.g., a metal halide) is complexed with the linker. In some embodiments, the linker is a compound of formula (I) (e.g., Compound 1 or Compound 2). In some embodiments, the label is chelated by the linker.

[0169] In some embodiments, the composition further comprises a polypeptide, and the compound of Formula (I) (e.g., Compound 1 or Compound 2) is bound to the polypeptide. In some embodiments, the composition comprises a compound of Formula (I) (e.g., Compound 1 or Compound 2) and a metal halide (e.g., {Al 18 F} 2+ In some embodiments, the polypeptide specifically binds to a target molecule, such as a marker molecule. In some embodiments, the polypeptide is a marker-binding polypeptide or protein. In some embodiments, the polypeptide is an antibody or antibody fragment. In some embodiments, the antibody fragment is selected from the group consisting of VHH, scFv, minibody, Fab, Fab', and Fv fragments. In some embodiments, the polypeptide is a non-antibody scaffold protein. In some embodiments, the non-antibody scaffold protein is selected from the group consisting of affibody, affilin, noctin, fibronectin, anticalin, atrimer, avimer, FN3 scaffold, fynomer, Kunitz domain, pronectin, OBody, and DARPin.

[0170] Also provided herein are compositions comprising a marker binding protein, a linker, and a label. In some embodiments, the composition further comprises a marker binding protein linked to a label by a linker. In some embodiments, the linker is Compound 1. In some embodiments, the linker is Compound 2. In some embodiments, the label is a metal halide. In some embodiments, the metal halide is {Al 18 F} 2+ In some embodiments, the linker chelates a label. In some embodiments, the linker chelates a metal halide.

[0171] The term "antibody" herein is used in the broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, monovalent antibodies (e.g., one-arm antibodies), four-chain antibodies (such as IgG antibodies), heavy-chain-only antibodies (HcAbs), and antibody fragments thereof, so long as they exhibit the desired antigen-binding activity. The term "four-chain antibody" is used interchangeably herein and refers to an antibody or antigen-binding fragment having two heavy chains and two light chains.

[0172] "Antibody fragment" includes a portion of an antibody, preferably an antigen-binding region or a variable region of an antibody. Examples of antibody fragments include single domain antibodies (including VHH antibodies), Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies (see U.S. Pat. No. 5,641,870, Example 2; Zapata et al., Protein Eng. 8(10):1057-1062

[1995] ); single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

[0173] In some embodiments, the composition is suitable for administration to a subject. In some embodiments, the composition is suitable for intravenous administration. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human.

[0174] Also provided is a method of preparing a labeling agent using any one of the compounds described herein. Some related methods can be found in WO 2021 / 046159 and Cleeren, F., Lecina, J., Bridoux, J. et al., Direct fluorine-18 labeling of heat-sensitive biomolecules for positron emission tomography imaging using the Al18F-RESCA method, Nat Protoc, 13, 2330-2347 (2018), both of which are incorporated herein in their entirety. In some embodiments, the agent is a marker-binding protein. In some embodiments, the polypeptide and 18A method of preparing a labeling agent comprising a F radionuclide, comprising: (a) contacting a polypeptide with a compound (e.g., Compound 1) or any one of the compositions prepared using the methods described herein under conditions that permit binding of the conjugate to the polypeptide to provide a conjugate; and (b) contacting the conjugate with 18 F (e.g., {Al 18 F} 2+ ) to provide a labeling agent. In some embodiments, the compound binds to a lysine residue of the polypeptide. In some embodiments, the complex is contacted with the aluminum fluoride complex in the presence of one or more antioxidant compounds. In some embodiments, the one or more antioxidant compounds include methionine and / or N-acetyl-tryptophan. In some embodiments, the complex is contacted with the aluminum fluoride complex in the presence of methionine and N-acetyl-tryptophan. In some embodiments, the method includes purifying the labeling agent from a reaction mixture comprising the complex and aluminum fluoride by a desalting column. In some embodiments, the desalting column is equilibrated with a buffer comprising histidine, methionine, N-acetyl-tryptophan, and / or sucrose. In some embodiments, the desalting column is equilibrated with a buffer comprising histidine, methionine, N-acetyl-tryptophan, and sucrose.

[0175] Further provided are the labeling agent prepared using the methods described herein, and the method of using the labeling agent, for example, for in vivo imaging, diagnosis, prognosis, or monitoring.In some embodiments, the labeling agent is used for the in vivo detection of target molecule through the specific binding of polypeptide to target molecule.In some embodiments, the target molecule is detected through at least one of immunoPET (positron emission tomography), SPECT (single photon emission computed tomography), NMR (nuclear magnetic resonance), near infrared (NIR), or MRI (magnetic resonance imaging), also known as Cerenkov luminescence imaging (CLI).

[0176] The term "detecting" is intended to include determining the presence or absence of a substance or quantifying the amount of a substance (e.g., a target molecule). Thus, the term refers to the use of the materials, compositions and methods of the present application for qualitative and quantitative measurements. In general, the particular technique used for detection is not critical to the practice of the methods in the present application. For example, "detecting" according to the methods described herein may include observing the presence or absence of a target molecule or a change in the level of a target molecule. Detection may include quantifying any value between 10% and 90%, or any value between 30% and 60%, or more than 100% change (increase or decrease) as compared to a control. Detecting may include quantifying any value change of 2-fold to 10-fold, or more, e.g., 100-fold.

[0177] IV.Compound Also disclosed herein are intermediates of the synthetic methods described herein. In some embodiments, the compound has the formula (Ie): TIFF2024539994000081.tif50170 formula(Ie) or a diastereomer thereof, or a salt thereof. (In the formula, R 1 and R 2 are each independently C1-C6 alkyl.

[0178] In some embodiments, the compound of formula (Ie) or a salt thereof has a structure according to one of the following formulas: TIFF2024539994000082.tif61170 In some embodiments, the compound has the formula: TIFF2024539994000083.tif50170 formula(Ie-1)

[0179] In some embodiments, the compound has formula (Ic): TIFF2024539994000084.tif40170 formula (Ic) or a diastereomer or salt thereof, 1 is C1-C6 alkyl).

[0180] In some embodiments, the compound of formula (Ic) or a salt thereof has a structure according to one of the following formulas: In some embodiments, the compound has the formula: TIFF2024539994000086.tif40170 has the formula (Ic-1).

[0181] salt Salts or solvates of certain intermediates are disclosed.

[0182] In some embodiments, the salt is compound 1c orotic acid. TIFF2024539994000087.tif38170 Compound 1c Orotic acid Compound 1c orotic acid is R 1 is the orotate salt of the compound of formula (Ic) wherein

[0183] In some embodiments, the salt is compound 1c fumaric acid. TIFF2024539994000088.tif41170 Compound 1c Fumaric acid Compound 1c Fumaric acid is R 1 is the fumarate salt of compound of formula (Ic) wherein

[0184] In some embodiments, the salt is Compound 2. TIFF2024539994000089.tif48170 Compound 2 Compound 2 is a solvate of a salt of Compound 1.

[0185] In some embodiments, compound 1c orotic acid is (a) an XRPD (X-ray powder diffraction) pattern having characteristic peaks at about 7.5° 2-theta, about 13.9° 2-theta, about 16.6° 2-theta, about 23.4° 2-theta, and about 25.6° 2-theta; (b) An XRPD pattern substantially similar to that shown in Figure 1 ; (c) TGA (thermogravimetric analysis) thermogram showing a mass loss of approximately 3.3% between 20 and 150 °C; (d) TGA thermogram substantially the same as that shown in Figure 2 ; (e) DSC (differential scanning calorimetry) thermogram showing an endothermic peak at approximately 172.8°C; (f) DSC thermogram substantially the same as that shown in Figure 2 ; (g) Has a characteristic peak at about 5.68 ppm 1 H NMR (nuclear magnetic resonance) spectrum; or (h) Substantially the same as shown in Figure 3 1 H NMR spectrum.

[0186] In some embodiments, compound 1c orotic acid is (a) an XRPD (X-ray powder diffraction) pattern having characteristic peaks at about 7.2° 2-theta, about 14.0° 2-theta, about 15.2° 2-theta, about 22.7° 2-theta, and about 25.0° 2-theta; (b) An XRPD pattern substantially similar to that shown in Figure 1 ; (c) TGA (thermogravimetric analysis) thermogram showing a mass loss of about 2.6% between 20 and 150 °C; (d) TGA thermogram substantially the same as that shown in Figure 4 ; (e) DSC (differential scanning calorimetry) thermogram showing an exothermic peak at about 161.7°C and an endothermic peak at about 182.9°C; (f) DSC thermogram substantially the same as that shown in Figure 4 ; (g) Has a characteristic peak at about 5.69 ppm 1 H NMR (nuclear magnetic resonance) spectrum; and (h) Substantially the same as shown in Figure 5 1 H NMR spectrum.

[0187] In some embodiments, compound 1c orotic acid is (a) an XRPD (X-ray powder diffraction) pattern having characteristic peaks at about 7.3° 2-theta, about 7.8° 2-theta, about 9.7° 2-theta, about 13.9° 2-theta, about 16.7° 2-theta, about 21.4° 2-theta, and about 23.4° 2-theta; (b) XRPD pattern substantially the same as that shown in Figure 6 ; (c) TGA (thermogravimetric analysis) thermogram showing a mass loss of about 4.8% between 20 and 150 °C; (d) TGA thermogram substantially the same as that shown in Figure 7 ; (e) DSC (differential scanning calorimetry) thermogram showing an exothermic peak at about 178.1°C and an endothermic peak at about 185.6°C; (f) DSC thermogram substantially the same as that shown in Figure 7 ; (g) Has a characteristic peak at about 5.68 ppm 1 H NMR (nuclear magnetic resonance) spectrum; and (h) Substantially the same as shown in Figure 8 1 H NMR spectrum.

[0188] In some embodiments, compound 1c orotic acid is a) an XRPD (X-ray powder diffraction) pattern having characteristic peaks at about 7.9° 2-theta, about 15.8° 2-theta, about 18.6° 2-theta, about 19.3° 2-theta, about 20.1° 2-theta, about 21.0° 2-theta, and about 24.7° 2-theta; (b) An XRPD pattern substantially the same as that shown in Figure 9 ; (c) TGA (thermogravimetric analysis) thermogram showing a mass loss of approximately 2.4% between 20 and 140 °C; (d) TGA thermogram substantially the same as that shown in Figure 10 ; (e) DSC (differential scanning calorimetry) thermogram showing an endothermic peak at approximately 154.7°C; (f) DSC thermogram substantially the same as that shown in Figure 10 ; (g) Has a characteristic peak at about 6.39 ppm 1 H NMR (nuclear magnetic resonance) spectrum; and (h) Substantially the same as shown in FIG. 11 1 H NMR spectrum.

[0189] In some embodiments, the crystalline salt of compound 1c fumaric acid is (a) an XRPD (X-ray powder diffraction) pattern having characteristic peaks at about 7.9° 2-theta, about 15.8° 2-theta, about 18.6° 2-theta, about 19.3° 2-theta, about 20.1° 2-theta, about 21.0° 2-theta, and about 24.7° 2-theta; (b) an XRPD pattern substantially the same as that shown in Figure 12; (c) TGA (thermogravimetric analysis) thermogram showing a mass loss of approximately 1.9% between 20 and 140 °C; (d) TGA thermogram substantially the same as that shown in Figure 13; (e) DSC (differential scanning calorimetry) thermogram showing an endothermic peak at approximately 156.2°C; (f) DSC thermogram substantially the same as that shown in Figure 13 ; (g) Has a characteristic peak at about 6.39 ppm 1 H NMR (nuclear magnetic resonance) spectrum; and (h) Substantially the same as shown in FIG. 14 1 H NMR spectrum.

[0190] In some embodiments, compound 2 is (a) an XRPD (X-ray powder diffraction) pattern having characteristic peaks at about 6.8° 2-theta, about 17.6° 2-theta, about 20.3° 2-theta, about 22.2° 2-theta, and about 23.3° 2-theta; and (b) has at least one XRPD pattern substantially the same as that shown in FIG. 15; EXAMPLES

[0191] The following abbreviations are used in the examples: A% - Area% based on HPLC analysis HPLC - High Performance Liquid Chromatography 1 H NMR - Proton Nuclear Magnetic Resonance MeOH - Methanol EtOH - Ethanol EtOAc - Ethyl acetate DMSO - Dimethyl sulfoxide NaBH(OAc)3-Sodium triacetoxyborohydride DIPEA-N,N-Diisopropylethylamine MTBE: Methyl tertiary-butyl ether Example 1: Small scale palladium-free synthesis of compound 1 Scheme 1. TIFF2024539994000090.tif97170

[0192] Step 1a-1: Synthesis of compound 1c-methyl-2-(4-((((1R,2R)-2-aminocyclohexyl)-amino)methyl)phenyl)acetate To a mixture of (1R,2R)-(-)-trans-1,2-diaminocyclohexane (compound 1a, 200 g, 1.75 mol) in EtOH (6 L, 30 mL / g) at 50° C., methyl 2-(4-formylphenyl)acetate (compound 1b, 310 g, 1.74 mol) was added in portions over 15 min while maintaining the temperature below 60° C. The resulting mixture was stirred for 2 h, then cooled to 20° C., residual solids were removed by filtration, and the mother liquor was concentrated under reduced pressure and then purified by silica gel chromatography (3% MeOH 7M NH3 in CH2Cl2) to give compound 1c (240 g, 0.87 mol, 50% yield, 96A% HPLC). 1 H NMR(400 MHz,DMSO-d6):7.28(d,J=8.1 Hz,2H),7.19(d,J=8.1 Hz,2H),3.80(d,J=13.5 Hz,1H),3.64(s,2H),3.61(s,3H),3.59(d,J=13.6 Hz,1H),2.29-2.23(m,1H),2.05-1.93(m,2H),1.79-1.73(m,1H),1.68-1.54(m,2H),1.27-0.85(m,4H).13 C NMR(101 MHz,DMSO-d6):δ 172.0,140.8,132.7,129.4,128.3,63.5,55.3,52.0,50.5,40.4,36.1,31.2,25.5,25.4.HRMS(m / z) calculated value C 16 H 24 N2O2[M+H] + 277.1911, measured value 277.1912.

[0193] Step 1a-2: Synthesis of compound 1e-di-tert-butyl-2,2'(((1R,2R)-2-((2-(tert-butoxy)-2-oxoethyl)(4-(2-methoxy-2-oxoethyl)benzyl)amino)cyclohexyl)azanediyl)diacetate Compound 1d (556 g, 2.84 mol) was added to a mixture of compound 1c (225 g, 0.81 mol) and DIPEA (842 g, 6.50 mol) in CH3CN (2.3 L, 10 mL / g) at 50°C and stirred for 18 h. CH3CN was solvent-displaced with EtOAc (2.5 L, 11 mL / g), and the organic solvent was then washed with H2O (3 x 400 mL, 2.1 mL / g), dried over Na2SO4, filtered, and the solvent was removed under reduced pressure, and purified by silica gel chromatography (20% EtOAc in n-heptane) to give compound 1e (400 g, 0.65 mol, 79% yield, 98A% HPLC). 1 H NMR(400 MHz,DMSO-d6):7.33(d,J=8.1 Hz,2H),7.16(d,J=8.1 Hz,2H),4.50(s,2H),3.90(d,J=13.5 Hz,1H),3.60(s,3H),3.56(d,J=13.5 Hz,1H),3.41,3.34(ABq,J=16.0 Hz,4H),3.27,3.19(ABq,J=16.6 Hz,2H),2.75-2.65(m,1H),2.56-2.50(m,1H),1.95-1.87(m,2H),1.66-1.5 6(m,2H),1.39(s,18H),1.35(s,9H),1.30-1.27(m,1H),1.06-0.97(m,3H). 13C NMR(101 MHz, CDCl3):δ HRMS (m / z) calculated value C 34 H 54 N2O8[M+H] + 619.3953, measured value 619.3952.

[0194] Step 1a-3: Synthesis of compound 1f-2-(4-((((1R,2R)-2-(bis(2-(tert-butoxy)-2-oxoethyl)amino)cyclohexyl)(2-(tert-butoxy)-2-oxoethyl)amino)methyl)phenyl)acetic acid To a solution of compound 1e (353 g, 0.60 mol) in THF (3 L, 8 mL / g) at 0° C. was added 1M aqueous KOH (67.5 g, 1.2 mol in 1.2 L of HO) while maintaining the temperature below 10° C. The resulting mixture was stirred for 24 h, then the pH was adjusted to 5-6 with 1M aqueous HCl while maintaining the temperature below 10° C. Solvent displacement from THF to CHCl (3.8 L, 10 mL / g) was then performed, and the layers were separated and the organic solvent was washed with 25% aqueous NaCl (1.85 L, 5 mL / g), dried over NaSO, filtered, and concentrated under reduced pressure at 40° C. to give compound 1f (327 g, 0.54 mol, 95% yield, 98A% HPLC). 1 H NMR(400 MHz,CDCl3):9.10(s,1H),7.38(d,J=7.9 Hz,2H),7.22(d,J=7.8 Hz,2H),4.09(d,J=13.4 Hz,1H),3.83(d,J=13.4 Hz,1H),3.61(s,2H),3.48(s,2H),3.35(s,4H),2.76-2.65(m,2H),2.14(d,J=12.2 Hz,1H),2.00(d,J=9.7 Hz,1H),1.73-1.64(m,2H),1.43(s,18H),1.40(s,9H),1.28-1.18(m 1H),1.13-1.03(m,3H). 13C NMR(101 MHz,CDCl3):δ 172.6,171.4,170.5 HRMS(m / z) Calculated value C 33 H 52 N2O8[M+H] + 605.3796, measured value 605.3790.

[0195] Step 1a-4: Synthesis of compound 1h-di-tert-butyl 2,2'-(((1R,2R)-2-((2-(tert-butoxy)-2-oxoethyl)(4-(2-oxo-2-(2,3,5,6-tetrafluorophenoxy)ethyl)benzyl)amino)cyclohexyl)-azanediyl)diacetate To a mixture of compound 1f (180 g, 0.30 mol) and 2,3,5,6-tetrafluorophenol (59 g, 0.36 mol) in CHCl (1.8 L, 10 mL / g) at 25° C., EDCI (70 g, 0.45 mol) was added and stirred for 16 h. The mixture was cooled to 0° C., washed with 1 M aqueous NaHCO (3×1.8 L, 10 mL / g) and 25% aqueous NaCl (900 mL, 5 mL / g), the organic layer was dried over NaSO, filtered, and the solvent was removed under reduced pressure to give compound 1h (216 g, 96% yield, 93A% HPLC). 1 H NMR(400 MHz,CDCl3):7.39(d,J=7.8 Hz,2H),7.21(d,J=7.9 Hz,2H),6.95-6.86(m,1H),3.97(d,J=13.6 Hz,1H),3.87(s,2H),3.62(d,J=13.6 Hz,1H),3.42,3.37(ABq,J=16.8 Hz,4H),3.30,3.20(ABq,J=16.8 Hz,2H),2.70-2.60(m,1H),2.52-2.45(m,1H),2.01-1.93(m,2H),1.65-1.57(m,2H),1.36(s,18H),1.34(s,9H),1.21-0.94(m,4H). 13C NMR (101 MHz, CDCl3): δ 172.0,171.6,167.5,146.0(dtd,J=248.5,11.9,4.2 Hz),140.6(dddd,J=250.8,15.2,4.6,2.1 Hz),140.0,130.5,129.7,128.9,103.2(t,J=22.8 Hz),80.2,63.6,61.0,54.2,53.4,53.1,52.7,39.9,29.2,28.4,28.1,25.8,25.7.HRMS(m / z) Calculated value C 39 H 52 F4N2O8[M+H] + 753.3733, measured value 753.3744.

[0196] Step 1a-5: Synthesis of compound 1-2,2'-(((1R,2R)-2-((carboxymethyl)(4-(2-oxo-2-(2,3,5,6-tetrafluorophenoxy)ethyl)benzyl)amino)cyclohexyl)azanediyl)diacetic acid Compound 1h (210 g, 0.28 mol) was dissolved in TFA (2.1 L, 10 mL / g) at 20° C. and stirred for 4 h. The solution was concentrated under reduced pressure at 40° C. and purified by preparative HPLC to give compound 1 (95 g, 59% yield, 0.16 mol, 99A% HPLC, 99% ee). 1 H NMR(400 MHz,CD3OD):7.67(d,J=8.2 Hz,2H),7.44(d,J=8.0,2H),7.43-7.34(m,1H),4.59(br s,1H),4.09(s,3H),3.94-3.33(br s,3H),3.52(d,J=16.4 Hz,2H),3.28-3.06(m,3H),2.29(d,J=12.3 Hz,1H),2.19(d,J=10.5 Hz,1H),1.92-1.77(m,2H),1.62-1.40(m,1H),1.38-1.19(m,3H). 13C NMR(101 MHz,CD3OD):δ 172.2,171.9,169.7,167.5,146.1(dtd,J=247.5,12.2,2.8 Hz),140.5(ddm,J=250,16.2 Hz),130.9,130.7,129.9,129.4,103.4(t,J=22.2 Hz),62.4,61.1,50.9,40.0,38.9,25.1,24.2,24.0.HRMS(m / z) Calculated value C 27 H 28 F4N2O8[M+H] + 585.1855, measured value 585.1854.

[0197] Example 2: Large-Scale Palladium-Free Synthesis of Compound 1 Scheme 1 applies to the synthesis of the following: Step 1b-1: Synthesis of compound 1c-methyl-2-(4-((((1R,2R)-2-aminocyclohexyl)-amino)methyl)phenyl)acetate To a 100 L jacketed reactor, compound 1b (2.20 kg, 12.35 mol, 1 equiv.), compound 1a (1.41 kg, 12.35 mol, 1 equiv.) and EtOH (44 L, 20 mL / g) were added. The temperature of the mixture was adjusted to 40-50° C. NaBH(OAc)3 (5.28 kg, 24.91 mol, 2 equiv.) was added portionwise to the mixture at 40-50° C. The reaction mixture was then stirred at 40-50° C. for 2 h. The reaction was monitored by HPLC (6 A% compound 1b, 77 A% compound 1c). The temperature of the reaction mixture was adjusted to 20-30° C. The reaction mixture was then filtered and the filter cake was washed with EtOH (5 L, 2.3 mL / g). The filtrate was concentrated to dryness under reduced pressure below 45° C. The crude oily product was purified by silica gel column chromatography using DCM / MeOH (7M NH3) = 100 / 0 to 30 / 1 (v / v). The product-containing fractions were collected and concentrated to dryness under reduced pressure below 45 °C. 1.75 kg of compound 1c was obtained as an oily product with an HPLC purity of 95.4 A% in 51% yield.

[0198] Step 1b-2: Synthesis of compound 1e-di-tert-butyl-2,2'(((1R,2R)-2-((2-(tert-butoxy)-2-oxoethyl)(4-(2-methoxy-2-oxoethyl)benzyl)amino)cyclohexyl)azanediyl)diacetate To a 50 L jacketed reactor, compound 1c (1.70 kg, 6.15 mol, 1 equiv), CH3CN (17 L, 10 mL / g) and diisopropylethylamine (DIPEA, 6.40 kg, 49.52 mol, 8.0 equiv) were added. The temperature of the mixture was adjusted to 40-50 °C. Compound 1d (4.20 kg, 21.53 mol, 3.5 equiv) was then added to the mixture in three portions at 40-50 °C for 3 h. The reaction mixture was stirred at 40-50 °C for 18 h. The reaction was monitored by HPLC (0 A% compound 1c, 92 A% compound 1e). The mixture was concentrated to dryness under reduced pressure below 45 °C. Ethyl acetate (17 L, 10 mL / g) was then added to the mixture. The mixture was washed three times with H2O (5 L, 3 mL / g). The organic layer was separated and dried over Na2SO4 (2.50 kg). The mixture was then filtered and the filter cake was washed with ethyl acetate (4 L, 2.4 mL / g). The filtrates were combined and concentrated to dryness under reduced pressure below 45°C. The crude product was purified by silica gel column chromatography using ethyl acetate / n-heptane = 0 / 100 to 1 / 5 (v / v). The product-containing fractions were collected and concentrated to dryness under reduced pressure. 2.75 kg of compound 1e was obtained as an oil with HPLC purity of 97.4 A% in 72% yield.

[0199] Step 1b-3: Synthesis of compound 1f-2-(4-((((1R,2R)-2-(bis(2-(tert-butoxy)-2-oxoethyl)amino)cyclohexyl)(2-(tert-butoxy)-2-oxoethyl)amino)methyl)phenyl)acetic acid To a 50 L jacketed reactor, compound 1e (2.60 kg, 4.20 mol, 1 equiv.) and THF (26 L, 10 mL / g) were added. The temperature of the reaction mixture was adjusted to 0-10 °C. Then, aqueous KOH solution (8.6 mol, 2 equiv., 0.48 kg KOH in HO 8.4 L, 3 mL / g) was added in 4 portions over 4 h at 0-10 °C. The reaction mixture was stirred at 0-10 °C for 24 h. The reaction was monitored by HPLC (1 A% compound 1e, 95 A% compound 1f). The reaction mixture was adjusted to pH 5-6 with 1 M aqueous HCl (10 kg) at 0-10 °C. The mixture was then concentrated to remove THF under reduced pressure below 40 °C. The reaction mixture was extracted twice with DCM (25 L, 9.5 mL / g). The organic layers were combined, dried over Na2SO4 (2.50 kg), filtered through a silica gel pad (2.50 kg), and the filter cake was then washed with DCM (8 L, 3.1 mL / g). The filtrates were combined and concentrated to dryness under reduced pressure below 45° C. 2.20 kg of compound 1f was obtained as a pale yellow solid with 98A% HPLC purity in 87% yield.

[0200] Step 1b-4: Synthesis of compound 1h-di-tert-butyl 2,2'-(((1R,2R)-2-((2-(tert-butoxy)-2-oxoethyl)(4-(2-oxo-2-(2,3,5,6-tetrafluorophenoxy)ethyl)benzyl)amino)cyclohexyl)-azanediyl)diacetate To a 50 L jacketed reactor, compound 1f (2.00 kg, 3.31 mol, 1 equiv), compound 1g (0.72 kg, 4.34 mol, 1.3 equiv) and DCM (21 L, 10.5 mL / g) were added. The temperature of the reaction mixture was adjusted to 20-30 °C. EDCI (0.81 kg, 4.23 mol, 1.3 equiv) was added in portions to the reaction mixture. The temperature of the reaction mixture was adjusted to 20-30 °C and stirred for 10 h. The reaction was monitored by HPLC (2 A% compound 1f, 98 A% compound 1h). The temperature of the reaction mixture was adjusted to 0-10 °C and then washed five times with 0.5 M aqueous NaHCO3 (16 kg) at 0-10 °C. The two layers were separated and the organic layer was monitored by HPLC (0.3 A% SM4, 93 A% D). The organic layer was dried over Na2SO4 (2 kg), filtered through a silica gel pad (2 kg), and the cake was washed with DCM (17 L, 8.5 mL / g). The filtrates were combined and concentrated under reduced pressure below 35° C. to approximately 4 kg to give crude compound 1h (2.49 kg) with a purity of 95A in 100% yield.

[0201] Step 1b-5: Synthesis of compound 1-2,2'-(((1R,2R)-2-((carboxymethyl)(4-(2-oxo-2-(2,3,5,6-tetrafluorophenoxy)ethyl)benzyl)amino)cyclohexyl)azanediyl)diacetic acid A 50 L jacketed reactor was charged with crude compound 1h (2.49 kg) and TFA (41 kg, 10 mL / g). The temperature of the reaction mixture was adjusted to 20-30° C. and stirred for 16 h. The reaction was monitored by HPLC (0 A% compound 1h, 94 A% compound 1). The reaction mixture was then concentrated under reduced pressure below 45° C. 6 kg of crude compound 1 was obtained as an oil (1.93 kg) with an HPLC purity of 91 A%. Crude compound 1 was purified by preparative HPLC (mobile phase A: 0.01% TFA in water (v / v); mobile phase B: acetonitrile) and the purified product was lyophilized to give compound 1 as a white solid with an HPLC purity of 98A in 51% yield over two steps. (Note: Compound 1 in the preparative HPLC fraction is not stable and must be immediately frozen for lyophilization. Residual TFA in the solid is 0.1 equivalents relative to E.) Example 3: Large-scale synthesis of compound 2 Scheme 2 TIFF2024539994000091.tif77170

[0202] Step 2-1: Synthesis of compound 1c-methyl-2-(4-((((1R,2R)-2-aminocyclohexyl)-amino)methyl)phenyl)acetate To a 3 L jacketed reactor, 50 g of compound 1b and 1500 mL of EtOH were added. The mixture was warmed to 45-50 °C. Compound 1a (41.66 g, 1.3 equiv.) was then added. The mixture was stirred at 45-50 °C for 10 min. NaBH(OAc)3 (178.42 g, 3.0 equiv.) was then added. The mixture was stirred at 45-50 °C for 2 h. The reaction was monitored by HPLC (77 A% compound 1c, 8 A% compound 1b). The reaction was filtered and washed with 150 mL of EtOH. The filtrate was added with 1000 mL of water and concentrated at 30-35 °C to remove EtOH. An additional 500 mL of water and 500 mL of MeTHF were added, the mixture was stirred for 10 min, and the layers were separated. The aqueous phase was adjusted to pH 9.1 with NH3·H2O and extracted twice with 250 mL of DCM. The DCM layers were combined, dried over Na2SO4, and filtered to give 882 g of compound 1c as a DCM solution in 66% corrected yield (89.3 A%, assay 5.78 wt%).

[0203] Step 2-2: Synthesis of orotate salt of compound 1c 500 mL of MeCN was added to the above DCM solution of compound 1c. 2,4-Dioxo-1H-pyrimidine-6-carboxylic acid (28.79 g, 1.0 equiv.) was added to the solution. The reaction was stirred at 20-25° C. for 16 h. The mixture was then filtered and the filter cake was washed with 100 mL of MeCN. The filter cake was dried at 30-35° C. under reduced pressure for 20 h. 79.29 g of the orotate salt of compound 1c was obtained from compound 2b in 64% corrected yield over two steps (97.1A% HPLC purity, assay 98.2 wt%). 1 H NMR(400MHz,DMSO-d6)δ=7.36(d,J=7.9 Hz,2H),7.18(d,J=7.9 Hz,2H),5.80(s,1H),3.86(d,J=13.4 Hz,1H),3.66-3.57(m,6H),2.78(dt,J=3.9,10.9 Hz,1H),2.39(dt,J=3.7,10.3 Hz,1H),2.13(br d,J=11.7 Hz,1H),2.02(br d,J=11.6 Hz,1H),1.66(br d,J=9.0 Hz,2H),1.44-1.28(m,1H),1.26-0.98(m,3H).

[0204] Step 2-3: Synthesis of compound 1e-di-tert-butyl-2,2'(((1R,2R)-2-((2-(tert-butoxy)-2-oxoethyl)(4-(2-methoxy-2-oxoethyl)benzyl)amino)cyclohexyl)azanediyl)diacetate To a 5 L jacketed reactor was added the orotate salt of compound 1c (135.70 g, assay 95.8 wt%), 1300 mL of toluene and 1300 mL of water. K2CO3 (124.63 g, 3.0 equiv.) was then added and the mixture was stirred at 20-25 °C for 0.5 h. Compound 2d (205.22 g, 3.5 equiv.) was added. The mixture was warmed to 40-45 °C and stirred at 40-45 °C for 16 h. The reaction was monitored by HPLC (93.8 A% B, no compound 1c remained). The reaction mixture was filtered to remove orotic acid and the cake was washed with 390 mL of toluene. The layers were separated. The organic layer was washed three times with aqueous NaOH (1 equiv., 12.0 g NaOH in 1300 mL of H2O) at 20-25 °C for 0.5 h to remove residual compound 2d. The organic layer was concentrated to dryness. 206.8 g of compound 1e was obtained as an oil in 90% corrected yield with an HPLC purity of 96.6A and an assay of 81% by weight. Residual toluene is 8.0% by weight and residual compound 2d is 5.2% by weight. 1 H NMR(400MHz,DMSO-d6)δ=7.34(d,J=7.9 Hz,2H),7.21-7.12(m,2H),3.91(d,J=13.6 Hz,1H),3.68-3.53(m,6H),3.47-3.31(m,4H),3.24(q,J=16.5 Hz,2H),2.75-2.63(m,1H),2.01-1.83(m,2H),1.60(br d,J=6.2 Hz,2H),1.48-1.32(m,27H),1.25-0.96(m,5H).

[0205] Step 2-4: Synthesis of compound 1f 2-(4-((((1R,2R)-2-(bis(2-(tert-butoxy)-2-oxoethyl)amino)cyclohexyl)(2-(tert-butoxy)-2-oxoethyl)amino)methyl)phenyl)acetic acid hydrochloride In a 5 L jacketed reactor, 185.19 g of compound 1e (81 wt%, assay by QNMR) and 1500 mL of THF were added. The reaction mixture was cooled to 0-5 °C. Then, aqueous KOH (2.5 equivalents of KOH in 450 mL of water, 34 g) was slowly added to the reaction mixture at 0-5 °C. The reaction mixture was stirred at 0-5 °C for 40 h. The reaction was monitored by HPLC (95.2 A% of compound 1f, no compound 1e remained). The mixture was adjusted to pH 2.0 with 1 M aqueous HCl (about 700 mL, 4.5 v). The mixture was extracted with 1500 mL of DCM. The organic layer was distilled and exchanged twice with DCM (750 mL of DCM each time). 509.4 g of compound 1f in DCM solution was obtained in 107% corrected yield. (Assay was 30.7 wt %, with THF remaining at 5.5 wt % and toluene remaining at 1.1 wt %). A solution of 508.9 g of C in DCM (1.7 v DCM) was added to a 2 L jacketed reactor. 230 mL of DCM (1.5 v) and 230 mL of MTBE (1.5 v) were added. The mixture was stirred at 25° C. until a clear solution was obtained. 230 mL of MTBE (1.5 v) was added to the mixture. 1.7 g of C seed crystals were added. The mixture was stirred at 25° C. for 21 h. 1400 mL of MTBE (9 v) was added over 8 h at 25° C. The mixture was stirred at 25° C. for 40 h. The mixture was filtered and the filter cake was washed three times with 940 mL (DCM:MTBE=1:4, v:v). The filter cake was dried at 25° C. for 24 h. 154.2 g of compound 1f hydrochloride salt with an HPLC purity of 98.0 A% and an assay of 90.2 wt% was obtained from compound 1e in a corrected yield of 90%. 1H NMR(400MHz,chloroform-d)δ=9.16-9.14(m,1H),8.97(br s,1H),7.65-7.58(m,2H),7.36-7.26(m,2H),4.73-4.55(m,2H),4.41-4.07(m,1H),4.00-3.81(m,1H),3.78-3.65(m,2 H),3.66-3.33(m,2H),3.25-3.03(m,2H),2.81-2.50(m,2H),2.20-1.70(m,3H),1.60-1.33(m,28H),1.26-1.12(m,2H)

[0206] Step 2-5: Synthesis of compound 1h-di-tert-butyl 2,2'-(((1R,2R)-2-((2-(tert-butoxy)-2-oxoethyl)(4-(2-oxo-2-(2,3,5,6-tetrafluorophenoxy)ethyl)benzyl)amino)cyclohexyl)-azanediyl)diacetate hydrochloride To a 1 L jacketed reactor, 33.26 g of compound 1f hydrochloride (assay 90.2 wt%) and compound 1g (9.89 g, 1.2 equiv.) were added. The mixture was degassed with N2 three times. 300 mL of DCM was added to the mixture. The mixture was cooled to 0-5 °C under N2 protection. EDCI (13.31 g, 1.4 equiv.) was added. The mixture was stirred at 0-5 °C for 20 h. The reaction was monitored by HPLC (89.2 A% of compound 1g, no compound 1f remained). 300 mL of water was added and the mixture was warmed to 20-25 °C. The layers were separated. The KF of the organic layer is 0.38%. The organic layer was recirculated through an Al2O3 filtration cartridge (Al2O3, 75.3 g) for 5 h, lowering the KF to 0.07%. The organic layer was concentrated to about 120 mL to give a solution of compound 1h hydrochloride in DCM (37.35 g, purity 94.7A%).

[0207] Step 2-6: Synthesis of compound 2-2,2'-(((1R,2R)-2-((carboxymethyl)(4-(2-oxo-2-(2,3,5,6-tetrafluorophenoxy)ethyl)benzyl)amino)cyclohexyl)azanediyl)diacetic acid In a 1 L jacketed reactor under N2 protection, the solution of compound 1h hydrochloride obtained above was added. 4N HCl / dioxane (370 mL, 29.8 equiv., pre-dried with molecular sieves) was added. The mixture was warmed to 55-60°C and stirred at 55-60°C for 3 h. The reaction was monitored by HPLC (92.8 A% compound 2, no compound 2h remained). The reaction was cooled to 20-25°C, filtered, and the filter cake was washed with 50 mL DCM. The filter cake was dried at 45-50°C under reduced pressure for 16 h. 26.35 g of compound 2 was obtained in 70% corrected yield with 97.5 A% HPLC purity and assay 79.9 wt%. Residual chloride is 5.2 wt%. E:HCl=1:1. Residual dioxane is 11 wt%. E:dioxane=1:0.9. 1 H NMR(400MHz,DMSO-d6)δ=7.95(tt,J=7.4,10.9 Hz,1H),7.78-7.57(m,2H),7.42(br d,J=7.9 Hz,1H),7.33-7.15(m,1H),4.50(br d,J=12.7 Hz,1H),4.54-4.42(m,1H),4.42-4.19(m,3H),3.89(br d,J=17.1 Hz,2H),3.66-3.50(m,9H),3.50-2.96(m,4H),2.23(br d,J=10.5 Hz,1H),2.07(br s,1H),1.72(br d,J=18.7 Hz,2H),1.55-1.12(m,4H).

[0208] Example 4: Salt screening and characterization of crystalline products Starting from oil-like compound 1c, a total of 51 salt screening experiments were performed using 17 acids in three solvent systems (see Table 1 below). Five crystalline hits were obtained: fumaric acid type A, L-tartrate type A, and orotic acid types A, B, and C. The L-tartrate hit was thought to decompose in air over time. The fumarate and orotic acid salts were characterized by X-ray power diffraction (XRPD), thermogravimetric analysis (TGA), differential scanning calorimetry (DSC), and nuclear magnetic resonance (NMR) (see Figures 1-5 and 9-11). The three crystalline samples from the 1,5-naphthalenedisulfonic acid, gallic acid, and gentisic acid systems were speculated to be decomposition products. Based on the properties, fumaric acid type A and orotic acid type A were reprepared by slurry on a ∼200 mg scale and recharacterized by the same techniques described above (see Figures 6-8 and 12-15). Dry XRPD samples were dried under vacuum for at least 2 hours. TIFF2024539994000092.tif130170

Claims

1. Formula (I): Formula (I) a diastereomer thereof, a salt thereof, a solvate thereof, or a combination of any of the foregoing, comprising: (a) Formula (Ia): Formula (Ia) or a diastereomer thereof, is reacted with a compound of formula (Ib): Formula (Ib) in the presence of a reducing agent in a solvent to obtain a compound of formula (Ic): (Ic) or a diastereomer thereof; (b) reacting the compound of formula (Ic) or a salt thereof with a compound of formula (Id): Formula (Id) in a solvent in the presence of a base, Formula (Ie): Formula (Ie) or a diastereomer thereof; (c) reacting the compound of formula (Ie) with a base in a solvent to obtain a compound of formula (If): Formula (If) or a diastereomer thereof: (d) reacting the compound of formula (If) with a compound of formula (Ig): Formula (Ig) in the presence of a carboxyl activating reagent in a solvent to obtain a compound of formula (Ih): Formula (Ih) or a diastereomer thereof: and (e) a step (1-5) comprising reacting said compound of formula (Ih) with an acid to form a compound of formula (I), a diastereomer thereof, a salt thereof, a solvate thereof, or a combination of any of the foregoing; During the ceremony, R １ and R ２ are each independently C １ -C ６ is alkyl; R ３ is a halo; R ４ , R ５ , R ６ and R ７ are each independently halo or hydrogen.

2. The method according to claim 1, wherein the solvent in step (1-1) comprises an alcohol.

3. The method according to claim 1, wherein the reducing agent in step (1-1) is a hydride.

4. The method according to claim 1, wherein the step (1-1) further comprises purifying the compound of formula (Ic) by column chromatography.

5. The method according to claim 1, wherein the solvent in step (1-2) is a polar organic solvent.

6. The method according to claim 1, wherein the base in step (1-2) is an organic base.

7. The method according to claim 1, wherein the step (1-2) further comprises purifying the compound of formula (1e) by column chromatography.

8. The method according to claim 1, wherein the solvent in step (1-3) is an aqueous solvent.

9. The method according to claim 8, wherein the solvent in step (1-3) further comprises tetrahydrofuran (THF).

10. The method according to claim 1, wherein the base in step (1-3) is KOH.

11. The method according to claim 1, wherein the solvent in step (1-4) is a halogenated hydrocarbon.

12. 2. The method of claim 1, wherein the carboxyl-activating reagent is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI).

13. The method according to claim 1, wherein the acid in step (1-5) is trifluoroacetic acid.

14. The method of claim 1, wherein the step (1-5) further comprises purifying the compound of formula (Ih) by preparative high performance liquid chromatography.

15. The compound of formula (I) is Compound 1: Compound 1 The method of claim 1, wherein

16. Formula (I): Formula (I) a diastereomer thereof, a salt thereof, a solvate thereof, or a combination of any of the foregoing, comprising: (a) Formula (Ia): Formula (Ia) with a compound of formula (Ib): Formula (Ib) in the presence of a reducing agent in a solvent to obtain a compound of formula (Ic): Formula (Ic) Step (2-1) comprising forming a compound of: (b) Step (2-2) comprising reacting a compound of formula (Ic) with an acid to produce a salt of formula (Ic): (c) reacting the salt of formula (Ic) with a compound of formula (Id): Formula (Id) in a solvent in the presence of a base, Formula (Ie): Formula (Ie) a step (2-3) of forming a compound of formula (I), a diastereomer thereof, or a salt thereof: (d) reacting the compound of formula (Ie) with a base in a solvent to obtain a compound of formula (If): Formula (If) a step (2-4) comprising forming a compound of the formula (I), a diastereomer thereof, or a salt thereof; and (e) reacting the compound of formula (If) or a salt thereof with a compound of formula (Ig): Formula (Ig) in the presence of a carboxyl activating reagent in a solvent to obtain a compound of formula (Ih): Formula (Ih) a step (2-5) comprising forming a compound of the formula (I), a diastereomer thereof, or a salt thereof: (f) a step (2-6) comprising reacting said compound of formula (Ih) with an acid to form a compound of formula (I), a diastereomer thereof, a salt thereof, a solvate thereof, or a combination of any of the foregoing; During the ceremony, R １ and R ２ are each independently C １ -C ６ is alkyl; R ３ is a halo; R ４ , R ５ , R ６ and R ７ are each independently halo or hydrogen.

17. The method according to claim 16, wherein the solvent in step (2-1) comprises an alcohol.

18. The method according to claim 16, wherein the reducing agent in step (2-1) comprises a hydride.

19. The step (2-2) is carried out by adding a compound having the following structure: Formula (Ic) orotate 17. The method of claim 16, wherein the salt has the formula (Ic):

20. The method according to claim 19, wherein the step (2-2) comprises crystallizing the salt of formula (Ic) or a diastereomer thereof as an orotate salt.

21. The step (2-2) is carried out by adding a compound having the following structure: Formula (Ic) Fumarate 17. The method of claim 16, wherein the salt has the formula (Ic):

22. The method according to claim 21, wherein the step (2-2) further comprises crystallizing the salt of formula (Ic) or a diastereomer thereof as a fumarate salt.

23. The base in the step (2-3) is K ２ CO ３ 17. The method of claim 16, wherein:

24. The method according to claim 16, wherein the solvent in step (2-3) comprises toluene or water.

25. The method according to claim 16, wherein the base in step (2-4) is KOH.

26. The method according to claim 16, wherein the solvent in step (2-4) comprises THF or water.

27. 17. The method of claim 16, wherein step (2-4) provides the salt of formula (If) as an HCl salt, and step (2-4) further comprises crystallizing the salt of formula (If).

28. The method according to claim 16, wherein the solvent in step (2-5) is a halogenated hydrocarbon.

29. The method according to claim 16, wherein the carboxyl-activating reagent in step (2-5) is EDCI.

30. The step (2-6) is carried out by adding a compound having the following structure: Formula (I-1) HCl 17. The method of claim 16, wherein the salt of formula (I), or a solvate thereof, is provided,

31. The method of claim 30, wherein step (2-6) further comprises crystallizing the salt of formula (I).

32. The salt of the solvate of formula (I) has the structure: Compound 2 17. The method of claim 16, comprising:

33. Formula (Ie): Formula (Ie) a compound of the formula (I), a diastereomer thereof, or a salt thereof, In the formula, R １ and R ２ are each independently C １ -C ６ A compound of formula (Ie), a diastereomer thereof, or a salt thereof, wherein:

34. Formula (Ic): Formula (Ic) a compound of the formula (I), a diastereomer thereof, or a salt thereof, In the formula, R １ is C １ -C ６ A compound of formula (Ic), a diastereomer thereof, or a salt thereof, wherein: R is alkyl;

35. Polypeptides and １８ 1. A method for preparing a labeling agent comprising an F radionuclide, comprising: (a) contacting the polypeptide with the compound prepared using the method of any one of claims 1 to 32 under conditions that allow binding of the complex to the polypeptide to provide a conjugate; and (b) reacting the conjugate with １８ with an aluminum fluoride complex containing F to provide said labeling agent.

36. 36. The method of claim 35, wherein the polypeptide is an antibody or antibody fragment.

37. 36. The method of claim 35, wherein the polypeptide is a non-antibody scaffold protein.

38. structure: Compound 1c orotate A crystalline salt of a compound having the formula: The salt is (a) an X-ray powder diffraction pattern having characteristic peaks at about 7.5° 2-theta, about 13.9° 2-theta, about 16.6° 2-theta, about 23.4° 2-theta, and about 25.6° 2-theta; (b) an X-ray powder diffraction pattern substantially the same as the pattern for "Orotate Type A" in Figure 1; (c) Thermogravimetric analysis thermogram showing a mass loss of about 3.3% between 20°C and 150°C; (d) a thermogravimetric analysis thermogram substantially the same as that shown in Figure 2; (e) a differential scanning calorimetry thermogram showing an endothermic peak at about 172.8°C; (f) a differential scanning calorimetry thermogram substantially the same as that shown in Figure 2; (g) Having a characteristic peak at about 5.68 ppm １ H nuclear magnetic resonance spectrum; or (h) Substantially the same as shown in FIG. １ H nuclear magnetic resonance spectrum A crystalline salt having at least one of the following: Figure 1 Figure 2 Figure 3

39. structure: Compound 1c orotate A crystalline salt of a compound having the formula: The salt is (a) an X-ray powder diffraction pattern having characteristic peaks at about 7.2° 2-theta, about 14.0° 2-theta, about 15.2° 2-theta, about 22.7° 2-theta, and about 25.0° 2-theta; (b) an X-ray powder diffraction pattern substantially the same as the pattern for "Orotate Type C" in Figure 1; (c) Thermogravimetric analysis thermogram showing a mass loss of about 2.6% between 20°C and 150°C; (d) a thermogravimetric analysis thermogram substantially the same as that shown in Figure 4; (e) a differential scanning calorimetry thermogram showing an exothermic peak at about 161.7°C and an endothermic peak at about 182.9°C; (f) a differential scanning calorimetry thermogram substantially the same as that shown in Figure 4; (g) Having a characteristic peak at about 5.69 ppm １ H nuclear magnetic resonance spectrum; and (h) Substantially the same as shown in FIG. １ H nuclear magnetic resonance spectrum A crystalline salt having at least one of the following: Figure 1 Figure 4 Figure 5

40. structure: Compound 1c orotate A crystalline salt of a compound having the formula: The salt is (a) an X-ray powder diffraction pattern having characteristic peaks at about 0.3° 2-theta, about 7.8° 2-theta, about 9.7° 2-theta, about 13.9° 2-theta, about 16.7° 2-theta, about 21.4° 2-theta, and about 23.4° 2-theta; (b) an X-ray powder diffraction pattern substantially the same as either the "wet sample" or the "dry sample" shown in Figure 6; (c) Thermogravimetric analysis thermogram showing a mass loss of about 4.8% between 20°C and 150°C; (d) a thermogravimetric analysis thermogram substantially the same as that shown in Figure 7; (e) a differential scanning calorimetry thermogram showing an exothermic peak at about 178.1°C and an endothermic peak at about 185.6°C; (f) a differential scanning calorimetry thermogram substantially the same as that shown in Figure 7; (g) Having a characteristic peak at about 5.68 ppm １ H nuclear magnetic resonance spectrum; and (h) Substantially the same as shown in FIG. １ H nuclear magnetic resonance spectrum A crystalline salt having at least one of the following: Figure 6 Figure 7 Figure 8

41. structure: Compound 1c fumarate A crystalline salt of a compound having the formula: The salt is (a) an X-ray powder diffraction pattern having characteristic peaks at about 7.9° 2-theta, about 15.8° 2-theta, about 18.6° 2-theta, about 19.3° 2-theta, about 20.1° 2-theta, about 21.0° 2-theta, and about 24.7° 2-theta; (b) an X-ray powder diffraction pattern substantially identical to either the "wet sample" or the "dry sample" shown in Figure 9; (c) Thermogravimetric analysis thermogram showing a mass loss of about 2.4% between 20°C and 140°C; (d) a thermogravimetric analysis thermogram substantially the same as that shown in Figure 10; (e) a differential scanning calorimetry thermogram showing an endothermic peak at about 154.7°C; (f) a differential scanning calorimetry thermogram substantially the same as that shown in Figure 10; (g) Having a characteristic peak at about 6.39 ppm １ H nuclear magnetic resonance spectrum; and (h) Substantially the same as shown in FIG. １ H nuclear magnetic resonance spectrum A crystalline salt having at least one of the following: Figure 9 Figure 10 Figure 11

42. structure: Compound 2 A crystalline solvate of a salt of a compound having The solvate of the salt is (a) an X-ray powder diffraction pattern having characteristic peaks at about 6.8° 2-theta, about 17.6° 2-theta, about 20.3° 2-theta, about 22.2° 2-theta, and about 23.3° 2-theta; and (b) An X-ray powder diffraction pattern substantially the same as that shown in FIG. A crystalline solvate having at least one of: Figure 15

43. A constraining complexing agent comprising a crystallized salt or solvate according to any one of claims 38 to 42.

44. 43. A label moiety comprising a detectable label comprising a metal halide attached or chelated to a linker comprising the crystallized salt or solvate of any one of claims 38 to 42.

45. 43. A label-binding marker binder protein comprising a detectable label comprising a metal halide linked to the marker binder protein by a linker comprising the crystallized salt or solvate of any one of claims 38 to 42.

46. 1. A method for visualizing a molecule, comprising: The molecule is a target-binding marker binder protein, (i) a compound prepared using the method of any one of claims 1 to 32; or (ii) A crystallized salt or solvate according to any one of claims 38 to 42. to a label-bound marker binder protein comprising a detectable label comprising a metal halide linked to said marker binder protein by a linker comprising exposing the molecule to a labeled-bound marker binder protein, wherein the marker binder protein binds to the molecule; and detecting the label. A method comprising:

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