Synthesis of restrained complexing agents

Abstract

This article describes improved methods for the synthesis of constrained complexes. Compounds and compositions related to these improved methods are also described, including their salts and solvates.

Description

[Technical Field]

[0001] This application relates to a method for preparing a restrained compound (RESCA) and RESCA compounds and compositions prepared therefrom. [Previous Technology]

[0002] Proteins that bind to molecules important in diagnostics, disease surveillance, and visualization (“marker-binding proteins”) may associate with or attach to detectable markers (e.g., radiolabels or fluorescent markers), which allow visualization of the molecules to determine their location and quantity (qualitative or quantitative). In some cases, the detectable marker associates or attaches directly to the marker-binding protein, while in others, the detectable marker attaches to the marker protein via linker molecules (“linkers”), which may be non-cleavable or cleavable (thus allowing controlled association between the marker-binding protein and its detectable marker). Linkers can be associated with detectable markers in a variety of ways, including direct binding to chelates, the latter being suitable for some metal-containing detectable markers. However, linker binding to a marker protein may require conditions incompatible with the specific marker protein, or be inefficient, or require multiple reagents.

[0003] Restricted chelating agents (RESCAs) are a class of chelating molecules that have been successfully used to label biomolecules with 18F (fluorine-18) and facilitate PET (positron emission tomography) imaging, which can be used as a diagnostic and / or therapeutic tool for various diseases and conditions. The properties of 18F, particularly its moderate half-life, make it an attractive option for imaging techniques compared to other radionuclides such as 89Zr (zirconium-89) and 124I (iodine-124). Using aluminum monofluoride ({Al 18F}²⁺) as the source of 18F has become a strategy that allows 18F labeling via RESCA in an aqueous medium at lower temperatures compared to previously known 18F labeling methods. Despite the potential applications of RESCA, the laborious and low-yield synthesis is a potential obstacle to the widespread development and use of the RESCA-Al 18F labeling strategy. The improved RESCA synthesis method will be useful for the continued development of the RESCA-Al 18F labeling method and its various applications. [Summary of the Invention]

[0004] This application provides a method for preparing RESCA compounds, RESCA compounds and their components and methods of use.

[0005] In some embodiments, this document provides a method for preparing a compound of formula (I): a non-mirror isomer of formula (I), a salt thereof, a solvate thereof, or a combination thereof, wherein the method comprises: (a) step (1-1), which comprises reacting a compound of formula (Ia): formula (Ia) or a non-mirror isomer thereof with a compound of formula (Ib): formula (Ib) in a solvent and in the presence of a reducing agent to form a compound of formula (Ic): formula (Ic) or a non-mirror isomer thereof; (b) step (1-2), which comprises reacting a compound of formula (Ic) or a salt thereof with a compound of formula (Id): formula (Id) in the presence of a base and in a solvent to form a compound of formula (Ie): formula (Ie) or a non-mirror isomer thereof; (c) step (1-3), which comprises reacting a compound of formula (Ie) with a base in a solvent to form a compound of formula (If): formula (If) (d) Step (1-4), comprising reacting compound (If) with compound (Ig):Ig in a solvent and in the presence of a carboxyl activator to form compound (Ih):Ih or a non-mirror isomer thereof; and (e) Step (1-5), comprising reacting compound (Ih) with an acid to form compound (I), a non-mirror isomer thereof, a salt thereof, a solvate thereof, or a combination thereof, wherein R1 and R2 are each independently C1-C6 alkyl; R3 is a halogroup; and R4, R5, R6 and R7 are each independently a halogroup or hydrogen.

[0006] 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.

[0007] In some embodiments, step (1-1) further includes purifying compound (Ic) by column chromatography.

[0008] In some embodiments, the solvent in steps (1-2) is a polar organic solvent. In some embodiments, the solvent is CH3CN. In some embodiments, the base in steps (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).

[0009] In some embodiments, steps (1-2) further include purifying the compound of formula (Ie) by column chromatography.

[0010] In some embodiments, the solvent in steps (1-3) is an aqueous solvent. In some embodiments, the solvent further comprises tetrahydrofuran (THF). In some embodiments, the base in steps (1-3) is KOH.

[0011] In some embodiments, the solvent in steps (1-4) is a halogenated hydrocarbon. In some embodiments, the solvent is CH₂Cl₂. In some embodiments, the carboxyl activator is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI).

[0012] In some embodiments, the acid in steps (1-5) is trifluoroacetic acid.

[0013] In some embodiments, steps (1-5) further include purifying the compound of formula (Ih) by preparative high performance liquid chromatography.

[0014] In some embodiments of any of the foregoing formulas, R1 is methyl. In one embodiment, R2 is tributyl. In some embodiments, R3 is Br. In some embodiments, R4, R5, R6 and R7 are each F. In some embodiments, the compound of formula (I) is compound 1: Compound 1

[0015] In some embodiments, this document provides a method for preparing a compound of formula (I): a non-mirror isomer of formula (I), a salt thereof, a solvate thereof, or a combination thereof, wherein the method comprises: (a) step (2-1), comprising reacting compound (Ia) with compound (Ib) in a solvent and in the presence of a reducing agent to form compound (Ic) of formula (Ic); (b) step (2-2), comprising reacting compound (Ic) with an acid to produce a salt of formula (Ic); (c) step (2-3), comprising reacting the salt of formula (Ic) with compound (Id) in the presence of a base and in a solvent to form compound (Ie) of formula (Ie), a non-mirror isomer thereof, or a salt thereof; (d) Step (2-4) comprises reacting compound (Ie) with a base in a solvent to form compound (2f): of formula (If), a non-mirror isomer thereof, or a salt thereof; and (e) Step (2-5) comprises reacting compound (If) or a salt thereof with compound (Ig): of formula (Ig) in a solvent and in the presence of a carboxyl activator to form compound (Ih): a non-mirror isomer thereof, or a salt thereof; and (f) Step (2-6) comprises reacting compound (Ih) with an acid to form compound (I), a non-mirror isomer thereof, a salt thereof, a solvate thereof, or a combination thereof, wherein R1 and R2 are each independently C1-C6 alkyl; R3 is a halogroup; and R4, R5, R6 and R7 are each independently a halogroup or hydrogen.

[0016] 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.

[0017] In some embodiments, the acid in step (2-2) is orotate. In some embodiments, step (2-2) provides a salt of formula (Ic) having the following structure: formula (Ic) orotate.

[0018] In some embodiments, step (2-2) includes crystallizing the salt of formula (Ic) or a non-mirror isomer thereof into orotic acid salt.

[0019] 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: formula (Ic) fumarate.

[0020] In some embodiments, step (2-2) further comprises crystallizing the salt of formula (Ic) or its non-mirror isomer into fumarate.

[0021] In some embodiments, the base in step (2-3) is K₂CO₃. In some embodiments, the solvent in step (2-3) comprises toluene. In some embodiments, the solvent in step (2-3) comprises water.

[0022] In some embodiments, the base in steps (2-4) is KOH. In some embodiments, the solvent in steps (2-4) comprises THF. In some embodiments, the solvent in steps (2-4) comprises water.

[0023] In some embodiments, step (2-4) provides the salt of formula (If) as an HCl salt, and wherein step (2-4) further comprises crystallizing the salt of formula (If).

[0024] In some embodiments, the solvent in steps (2-5) is a halogenated hydrocarbon. In some embodiments, the halogenated hydrocarbon is CH₂Cl₂. In some embodiments, the carboxyl activator in steps (2-5) is EDCI.

[0025] In some embodiments, the solvent in steps (2-6) is dialkyl. In some embodiments, the acid in steps (2-6) is HCl.

[0026] In some embodiments, step (2-6) provides a salt of formula (I) or a solvate thereof having the following structure: Formula (I-1) HCl

[0027] In some embodiments, steps (2-6) further comprise crystallizing the salt of formula (I). In some embodiments of any of the foregoing formulas, R1 is methyl. In one embodiment, R2 is tributyl. In some embodiments, R3 is Br. In some embodiments, R4, R5, R6, and R7 are F.

[0028] In some embodiments, the salt of the solvate of formula (I) has the following structure: Compound 2

[0029] In some embodiments, this document provides a composition comprising one or more non-mirror isomers of a compound of formula (I), its salts or solvates thereof: formula (I) which are produced by the methods described herein.

[0030] In some embodiments, the composition comprises at least about 90% (by weight) of the trans-non-mirror image isomer of the compound of formula (I). In some embodiments, the trans-non-mirror image isomer has the following structure: Formula (I-1)

[0031] In some embodiments, R4, R5, R6 and R7 are F.

[0032] In some embodiments, a salt of formula (I) or a solvate thereof is provided, wherein the salt of formula (I) has the following structure: formula (I-1) HCl, which is produced by the method described herein. In some embodiments, R4, R5, R6 and R7 are F.

[0033] In some embodiments, this document provides compounds of formula (Ie): non-mirror isomers of formula (Ie) or salts thereof, wherein R1 and R2 are each independently C1-C6 alkyl.

[0034] In some embodiments, this document provides compounds of formula (Ic): non-mirror isomers of formula (Ic) or salts thereof, wherein R1 is a C1-C6 alkyl group.

[0035] In some embodiments, this document provides a composition comprising a complex comprising a compound prepared using the methods described herein and a metal halide. In some embodiments, the metal halide is {Al 18F} 2+. In some embodiments, at least 90% (by weight) of the compound in the composition has the following structure: Formula (I-1) In some embodiments, R4, R5, R6, and R7 are F. In some embodiments, the composition further comprises a polypeptide, wherein the complex binds 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, microantibodies, 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: affinity antibodies, affilin, knottin, reticulin, anticarrier protein, atrimer, high-affinity multimer, FN3 scaffold, fynomer, Kunitz domain, pronectin, OBody, and DARPin. In some embodiments, the composition is a pharmaceutical composition suitable for administration to an individual. In some embodiments, the pharmaceutical composition is suitable for intravenous administration. In some embodiments, the individual is a human.

[0036] In some embodiments, this document provides a method for preparing a labeling reagent comprising a polypeptide and an 18F radionuclide, the method comprising: (a) contacting the polypeptide with a compound prepared using the method described herein, provided that the complex is allowed to bind to the polypeptide to provide a conjugate; and (b) contacting the conjugate with an aluminum fluoride complex comprising 18F to provide a labeling reagent.

[0037] In some embodiments, a crystalline salt of a compound having the following structure is provided: Compound 1c orotate, wherein the salt has at least one of the following: (a) an X-ray powder diffraction pattern having characteristic peaks at about 7.5° 2-θ, about 13.9° 2-θ, about 16.6° 2-θ, about 23.4° 2-θ, and about 25.6° 2-θ; (b) an X-ray powder diffraction pattern substantially identical to the "Type A orotate" pattern in FIG. 1; (c) a thermogravimetric analysis (TGA) chromatogram showing a mass loss of about 3.3% between 20°C and 150°C; (d) a thermogravimetric analysis chromatogram substantially identical to that shown in FIG. 2; (e) a differential scanning calorimetry (DSC) chromatogram showing an endothermic peak at about 172.8°C; (f) a chromatogram similar to that in FIG. 2. (g) A differential scanning calorimetry thermal analysis chromatogram that is substantially the same as shown in Figure 3; or (h) a 1H NMR spectrum with a characteristic peak at approximately 5.68 ppm; or (h) a 1H NMR spectrum that is substantially the same as shown in Figure 3.

[0038] In some embodiments, a crystalline salt of a compound having the following structure is provided: Compound 1c orotate, wherein the salt has at least one of the following: (a) an X-ray powder diffraction pattern having characteristic peaks at about 7.2° 2-θ, about 14.0° 2-θ, about 15.2° 2-θ, about 22.7° 2-θ, and about 25.0° 2-θ; (b) an X-ray powder diffraction pattern substantially identical to the "Type C orotate" pattern in FIG1; (c) a thermogravimetric analysis (TGA) chromatogram showing a mass loss of about 2.6% between 20°C and 150°C; (d) a thermogravimetric analysis chromatogram substantially identical to that shown in FIG4; (e) a differential scanning calorimetry (DSC) chromatogram showing an exothermic peak at about 161.7°C and an endothermic peak at about 182.9°C; (f) (g) A differential scanning calorimetry thermal analysis plot that is substantially the same as shown in Figure 4; and (h) a 1H NMR spectrum with a characteristic peak at approximately 5.69 ppm; and (h) a 1H NMR spectrum that is substantially the same as shown in Figure 5.

[0039] In some embodiments, a crystalline salt of a compound having the following structure is provided: Compound 1c orotic acid salt wherein the salt has at least one of the following: (a) an X-ray powder diffraction pattern having characteristic peaks at about 7.3° 2-θ, about 7.8° 2-θ, about 9.7° 2-θ, about 13.9° 2-θ, about 16.7° 2-θ, about 21.4° 2-θ and about 23.4° 2-θ; (b) an X-ray powder diffraction pattern substantially the same as that of the "wet sample" or "dry sample" shown in FIG. 6; (c) a thermogravimetric analysis chromatogram showing a mass loss of about 4.8% between 20°C and 150°C; (d) a thermogravimetric analysis chromatogram substantially the same as that shown in FIG. 7; (e) an exothermic peak showing a peak at about 178.1°C and a peak at about 185.6°C. (f) Differential scanning calorimetry (DSC) chromatogram of the endothermic peak at (p); (g) 1H NMR spectrum with a characteristic peak at approximately 5.68 ppm; and (h) 1H NMR spectrum with a characteristic peak at approximately 5.68 ppm.

[0040] In some embodiments, a crystalline salt of a compound having the following structure is provided: Compound 1c fumarate, wherein the salt has at least one of the following: (a) an X-ray powder diffraction pattern having characteristic peaks at about 7.9° 2-θ, about 15.8° 2-θ, about 18.6° 2-θ, about 19.3° 2-θ, about 20.1° 2-θ, about 21.0° 2-θ, and about 24.7° 2-θ; (b) an X-ray powder diffraction pattern substantially the same as that of the "wet sample" or "dry sample" shown in FIG. 9; (c) a thermogravimetric analysis (TGA) chromatogram showing a mass loss of about 2.4% between 20°C and 140°C; (d) a thermogravimetric analysis chromatogram substantially the same as that shown in FIG. 10; (e) a chromatogram showing a mass loss of about 154.7°C. (f) Differential scanning calorimetry (DSC) chromatogram of the endothermic peak at (p); (g) chromatogram of the same differential scanning calorimetry (DSC) chromatogram as shown in Figure 10; (h) ¹H NMR spectrum with a characteristic peak at approximately 6.39 ppm; and (f) ¹H NMR spectrum of the same differential scanning calorimetry (DSC) chromatogram as shown in Figure 11.

[0041] In some embodiments, a crystalline solvate of a salt of a compound having the following structure is provided: Compound 2 wherein the solvate of the salt has at least one of the following: (a) an X-ray powder diffraction pattern having characteristic peaks at about 6.8° 2-θ, about 17.6° 2-θ, about 20.3° 2-θ, about 22.2° 2-θ and about 23.3° 2-θ; and (b) an X-ray powder diffraction pattern substantially the same as that shown in FIG. 15.

[0042] In some embodiments, this document provides a constrained complex comprising (i) a compound prepared using the methods provided herein; or (ii) a crystalline salt or solvate as described herein.

[0043] In some embodiments, this document provides a marker portion comprising a detectable marker comprising a metal halide bound to or chelated to a linker comprising (i) a compound prepared using the methods provided herein; or (ii) a crystalline salt or solvate as described herein.

[0044] In some embodiments, this document provides a label-binding marker-binding protein comprising a detectable marker comprising a metal halide linked to the marker-binding protein by a linker comprising (i) a compound prepared using the methods provided herein; or (ii) a crystalline salt or solvate as described herein.

[0045] In some embodiments, this document provides a method for visualizing a molecule, the method comprising exposing the molecule to a label-bound label-conjugate protein, the label-conjugate protein comprising a detectable label comprising a metal halide linked to the label-conjugate protein by a linker comprising (i) a compound prepared using the methods provided herein; or (ii) a crystalline salt or solvate as described herein, wherein the label-conjugate protein is bound to the molecule; and a detectable label.

Implementation Method

[0047] Cross-reference to related applications

[0048] This application claims priority and interest in U.S. Application No. 63 / 272,547, filed on October 27, 2021, which is incorporated herein by reference in its entirety for all purposes.

[0049] The disclosed synthetic method incorporates several improvements over the known RESCA synthetic method (WO 2016 / 065435). Compared to previously reported methods, it reduces the number of required steps, uses less expensive reagents, and / or improves the yield. In some embodiments, the use of column chromatography has been excluded in some or all of the synthetic steps. In some embodiments, when the method is carried out on a larger scale than previously reported methods, the synthesis retains some or all of the aforementioned benefits.

[0050] The disclosed method uses a synthetic route different from previously known RESCA synthesis methods. The first step of the improved method is the synthesis of a compound of formula (Ic): formula (Ic) or a salt thereof, which provides at least one key point distinguishing the method disclosed herein from previous methods. The improved synthetic route eliminates the need for a palladium catalyst. In some embodiments (e.g., Example 3, as shown below), the products of some steps can be crystallized to eliminate the need for more complex purification of intermediates. I. Definitions

[0051] As used herein, the term "C1-Cx alkyl" alone or in combination means a straight-chain or branched alkyl moiety having 1 to x carbon atoms, where x is an integer. Therefore, 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 moiety, if present at the end of a molecule, include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, dibutyl, 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 two parts of a molecule are connected by C1-C6 alkyl groups (i.e., C1-C6 includes a divalent "alkylene" part), examples include, but are not limited to, -CH2-, -CH2CH2-, -CH(CH3)-, -CH2CH2CH2-, -CH(C2H5)-, and -C(CH3)2-.

[0052] As used herein, "halogen" means fluorine, chlorine, bromine or iodine. The term "halogen group" means a halogen substituent, namely a fluorine group, a chlorine group, a bromine group or an iodine group.

[0053] As used herein, the term "halogenated hydrocarbon" refers to a hydrocarbon compound or part thereof that has been partially substituted with a halogen at least once. Some halogenated hydrocarbons can be used as solvents in chemical synthesis. The term "halogenated hydrocarbon" includes, for example but not limited to, dichloromethane (also known as CH₂Cl₂ or DCM) and chloroform (also known as CHCl₃).

[0054] Unless otherwise stated, as used herein, the term “equivalent” means molar equivalent.

[0055] As used herein, the term “chiral” refers to a molecule that cannot be superimposed on its mirror image, while the term “chiral” refers to a molecule that can be superimposed on its mirror image.

[0056] As used herein, the term “stereoisomer” refers to a compound having the same chemical formula (i.e., the same number of atoms of each type) but different arrangements of atoms or groups in physical space.

[0057] As used herein, the term "non-mirror image isomer" refers to a stereoisomer having two or more chiral centers and whose molecules are not mirror images of each other. Non-mirror image isomers may have different physical properties (e.g., melting point, boiling point, spectral properties, and reactivity).

[0058] As used herein, the term "mirror image isomer" refers to a chiral molecule that is a mirror image of another molecule. Two molecules that are mirror images of each other are called "mirror image isomers". Mirror image isomers cannot be superimposed on each other.

[0059] The stereochemical definitions and conventions used herein generally follow those of SP Parker, 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. The compounds in this application may contain asymmetric or chiral centers and thus exist as different stereoisomers. All stereoisomers of the compounds cited herein, including but not limited to non-mirror image isomers, mirror image isomers, configurational isomers, and mixtures thereof (e.g., racemic mixtures), are included in this application.

[0060] One or more compounds of this application may exist in both solvated and solvated forms with pharmaceutically acceptable solvents such as water and ethanol, and this application is intended to include both solvated and solvated forms. "Solvate" means the physical association of a compound of this application with one or more solvent molecules. This physical association involves varying degrees of ionic and covalent bonding, including hydrogen bonding. In some embodiments, the solvate will be separable, such as when one or more solvent molecules are incorporated into the lattice of a crystalline solid. "Solvate" includes both solution phases and separable solvates. Non-limiting examples of suitable solvates include solvates of ethanol, water, 1,4-dimethylamine, hydrochloric acid, etc. "Hydrate" is a solvate in which the solvent molecule is H₂O.

[0061] It should be understood that the embodiments described herein as "comprising" certain features imply that the embodiments are "composed" and / or "substantially composed of" those features that have been considered.

[0062] References to "about" a value or parameter herein refer to the general range of error of various values ​​that are readily known to those skilled in the art to which this invention pertains. In this document, a value or parameter referring to "about" includes (and describes) the state of that value or parameter itself. For example, a description referring to "about X" includes a description of "X".

[0063] As used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural indicators unless the context clearly indicates otherwise.

[0064] As used herein, the term "and / or" such as "A, B and / or C" is intended to include both A and B; A or B; A (alone); and B (alone). Similarly, as used herein, the term "and / or" such as "A, B and / or C" is intended to cover each of the following embodiments: A, B and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone). II. Synthesis Method

[0065] This document provides a method for chelating metal halides with compounds. In some embodiments, the compound is a compound of formula (I): a non-mirror isomer of formula (I), its salt, its solvate, or a combination thereof, wherein R4, R5, R6 and R7 are each independently a halogen group of hydrogen.

[0066] In some embodiments, the compound of formula (I) is a compound having the following structure: formula (I-1) or its salt, its solvate or a solvate of its salt.

[0067] In some embodiments, the compound of formula (I) is compound 1: compound 1, or a salt thereof, a solvate thereof, or a solvate thereof.

[0068] The method described herein has chemical and / or commercial advantages compared to previously disclosed synthetic methods. In some embodiments, the yield of this reaction is greater than that of compound 1 or compound 2 (as shown below) in previously known methods (e.g., WO 2016 / 065435). In some embodiments, the method described herein provides the product in high yield. 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 between about 20% and 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 between about 30% and 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 between about 40% and 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 between about 50% and about 60%, about 70%, about 80%, about 90%, about 95%, about 98%, about 99%, or about 100%. In some embodiments, the yield is between about 60% and about 70%, about 80%, about 90%, about 95%, about 98%, about 99%, or about 100%. In some embodiments, the yield is between about 70% and about 80%, about 90%, about 95%, about 98%, about 99%, or about 100%. In some embodiments, the yield is between about 80% and about 90%, about 95%, about 98%, about 99%, or about 100%. In some embodiments, the yield is between about 90% and about 95%, about 98%, about 99%, or about 100%. In some embodiments, the yield is between about 95% and about 98%, about 99%, or about 100%. In some embodiments, the yield is between about 98% and about 99% or about 100%. In some embodiments, the yield is between about 99% and about 100%. In a particular embodiment, the yield is between about 20% and about 30%.In other specific embodiments, the yield is about 50% to about 70%, such as about 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, and 70%.

[0069] In some embodiments, the method provides a product with higher purity than previously disclosed methods. In some embodiments, the method provides a composition comprising a single non-mirror image isomer of formula (I) (e.g., compound 1 or compound 2) having 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 97.5 Å%, 98 Å%, and 99 Å (area%), as determined using high-performance liquid chromatography (HPLC).

[0070] In some embodiments, the reagents used in this method are cheaper than those used in previously disclosed methods. In some embodiments, the reagents used in the disclosed method are generally cheaper and more environmentally friendly than those used in previously disclosed methods; however, importantly, the method does not require a palladium catalyst.

[0071] In some embodiments, the compound of formula (I) (e.g., compound 1 or compound 2) can be prepared on a large scale. In some embodiments, "large scale" means using at least 100 g, 250 g, 500 g, 750 g, or 1000 g of starting material, intermediate, or reagent in any step of the method. In some embodiments, "large scale" means using at least 1 kg, 10 kg, 25 kg, 50 kg, or 100 kg of starting material, intermediate, or reagent in any step of the method.

[0072] In some embodiments, this method provides a product with improved yield and / or higher purity than previously disclosed methods. In some embodiments, this method provides a product with improved yield and / or higher purity on a larger scale than previously disclosed methods. Any of the yield, purity, and scale may be combined, as each combination of a particular yield, particular purity, and particular scale is specifically and separately listed. A. First Method

[0073] In some embodiments, this document provides a method for preparing a compound of formula (I), a non-mirror isomer of formula (I), a salt thereof, a solvate thereof, or a combination thereof, wherein R4, R5, R6, and R7 are each independently a halogen (e.g., F) or hydrogen, and wherein the method comprises one or more of steps (1-1) to (1-5). An exemplary synthetic scheme is shown in Scheme 1 of Example 1.

[0074] Step (1-1) comprises reacting compound (Ia) or a non-mirror isomer of it with compound (Ib) in a solvent and in the presence of a reducing agent to form compound (Ic) or a non-mirror isomer of it, wherein R1 is a C1-C6 alkyl group.

[0075] In some embodiments, a compound of formula (Ib) is used in an amount between about 0.5 equivalents and about 2 equivalents compared to the amount of compound of formula (Ia). In some embodiments, about 1 equivalent is used.

[0076] In some embodiments, the compound of formula (Ia) is a stereoisomer selected from the group consisting of:

[0077] 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.

[0078] 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, a reducing agent of 1 to 2 equivalents is used compared to the amount of the compound of formula (Ia) (i.e., the molar ratio of reducing agent to compound is between 1:1 and 2:1). In some embodiments, a reducing agent of about 1.5 to about 2 equivalents is used. In some embodiments, a reducing agent of about 1.8 to about 2 equivalents is used. In some embodiments, a reducing agent of about 1.9 equivalents is used.

[0079] In some embodiments, step (1-1) is performed at a temperature maintained between about 20°C and about 70°C for a duration between about 1 hour and about 3 hours. In some embodiments, the temperature is maintained between about 30°C and about 60°C. In some embodiments, the temperature is between about 40°C and about 50°C. In some embodiments, the duration is about 2 hours. In some embodiments, step (1-1) is performed at a temperature maintained between about 40°C and about 50°C for a duration of about 2 hours.

[0080] In some embodiments, step (1-1) further comprises purifying the compound of formula (Ic). In some embodiments, purification is performed using a silicone column. In some embodiments, purification is performed using a solvent mixture. In some embodiments, the solvent mixture comprises dichloromethane (DCM) and methanol (MeOH). In some embodiments, HPLC comprises changing the ratio of DCM to MeOH from 100:0 to 30:1 (v / v) over time. In some embodiments, MeOH comprises NH3. In some embodiments, NH3 is present in MeOH at a concentration of 7M.

[0081] In some embodiments, step (1-1) does not include the purified (Ic) compound.

[0082] In some embodiments, the compound of formula (Ic) is a compound having one of the following formulas:

[0083] Step (1-2) comprises reacting compound of formula (Ic) or a non-mirror isomer thereof with compound of formula (Id) in the presence of a base and a solvent to form compound of formula (Ie) or a non-mirror isomer thereof, wherein R1 and R2 are each independently C1-C6 alkyl; and R3 is a halogroup.

[0084] In some embodiments, a compound of formula (Id) is used in amounts between about 2 equivalents and about 5 equivalents compared to the amount of compound of formula (Ic). In some embodiments, about 3 equivalents to about 4 equivalents are used. In some embodiments, 3.5 equivalents are used.

[0085] In some embodiments, the solvent in steps (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.

[0086] In some embodiments, the base in steps (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-diacerbicyclo[5.4.0]undec-7-ene. In some embodiments, the base is DIPEA. In some embodiments, a base between about 4 equivalents and about 12 equivalents is used compared to the amount of the compound of formula (Ic). In some embodiments, a base between about 6 equivalents and about 10 equivalents is used. In some embodiments, a base between about 7 equivalents and about 9 equivalents is used. In some embodiments, about 8 equivalents of the base is used.

[0087] In some embodiments, steps (1-2) are performed in CH3CN in the presence of DIPEA. In some such embodiments, approximately 8 equivalents of DIPEA are used.

[0088] In some embodiments, steps (1-2) are performed at a temperature maintained between about 20°C and about 70°C for a duration between about 12 hours and about 24 hours. In some embodiments, the temperature is maintained between about 30°C and about 60°C. In some embodiments, the temperature is between about 40°C and about 50°C. In some embodiments, the duration is between about 16 hours and about 20 hours. In some embodiments, the duration is about 18 hours. In some embodiments, steps (1-2) are performed at a temperature maintained between about 40°C and about 50°C for a duration of about 18 hours.

[0089] In some embodiments, steps (1-2) further comprise purifying the (Ie) compound. In some embodiments, steps (1-2) further comprise purifying the (Ie) compound by column chromatography. In some embodiments, purification uses a silicone column. In some embodiments, purification uses a solvent mixture comprising ethyl acetate and n-heptane. In some embodiments, purification comprises changing the ratio of ethyl acetate to n-heptane from 100:0 to 1:5 (v / v) over time.

[0090] In some embodiments, steps (1-2) do not include the purified (Ie) compound.

[0091] In some embodiments, the compound of formula (Ie) is a compound having one of the following formulas:

[0092] Step (1-3) comprises reacting a compound of formula (Ie) or a non-mirror isomer thereof with a base in a solvent to form a compound of formula (If): formula (If) or a non-mirror isomer thereof, wherein each R2 is a C1-C6 alkyl group.

[0093] In some embodiments, the solvent in steps (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 tributyl 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, an organic solvent to water ratio is used. In some embodiments, the organic solvent to water ratio is between about 4:1 and 1:1. In some embodiments, the ratio is between about 10:3 and about 6:3. In some embodiments, the ratio is about 8:3. All ratios in the above embodiments also apply to embodiments where the ratio is a THF to water ratio.

[0094] In some embodiments, the base in steps (1-3) is a strong base. In some embodiments, the base is KOH. In some embodiments, about 1 equivalent to about 3 equivalents of the base are used compared to the amount of the compound of formula (Ie). In some embodiments, about 2 equivalents of the base are used.

[0095] In some embodiments, steps (1-3) are 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.

[0096] In some embodiments, the compound of formula (If) is a compound having one of the following formulas:

[0097] In some embodiments, steps (1-3) are performed at a temperature maintained between about -20°C and about 30°C for a duration between about 18 hours and about 30 hours. In some embodiments, the temperature is maintained between about -10°C and about 20°C. In some embodiments, the temperature is between about 0°C and about 10°C. In some embodiments, the duration is between about 16 hours and about 20 hours. In some embodiments, the duration is about 18 hours. In some embodiments, steps (1-3) are performed at a temperature maintained between about 0°C and about 10°C for a duration of about 18 hours.

[0098] Step (1-4) comprises reacting compound of formula (If) or a non-mirror isomer thereof with compound of formula (Ig) in a solvent and in the presence of a carboxyl activating agent to form compound of formula (Ih) or a non-mirror isomer thereof, wherein each R2 is independently C1-C6 alkyl; and R4, R5, R6 and R7 are each independently a halogroup or hydrogen.

[0099] In some embodiments, the compound of formula (Ih) is a compound having one of the following formulas:

[0100] In some embodiments, the compound of formula (Ig) is used in amounts between about 0.8 equivalents and about 1.6 equivalents compared to the amount of the compound of formula (Ig). In some embodiments, about 1.0 equivalents to about 1.4 equivalents are used. In some embodiments, about 1.2 equivalents are used.

[0101] In some embodiments, the solvent in steps (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).

[0102] In some embodiments, the carboxyl activator in steps (1-4) comprises carbodiimide. In some embodiments, the carboxyl activator is 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDCI).

[0103] In some embodiments, steps (1-4) are performed in the DCM in the presence of EDCI.

[0104] In some embodiments, steps (1-4) are performed at a temperature maintained between about 0°C and about 50°C for a duration between about 8 hours and about 24 hours. In some embodiments, the temperature is maintained between about 10°C and about 40°C. In some embodiments, the temperature is between about 20°C and about 30°C. In some embodiments, the duration is between about 12 hours and about 20 hours. In some embodiments, the duration is about 16 hours. In some embodiments, steps (1-4) are performed at a temperature maintained between about 20°C and about 30°C for a duration of about 10 hours. In some embodiments, steps (1-4) are performed at a temperature maintained between about 20°C and about 30°C for a duration of about 16 hours.

[0105] Steps (1-5) comprise reacting a compound of formula (Ih) or a non-mirror isomer thereof with an acid to form a compound of formula (I) or a non-mirror isomer 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.

[0106] In some embodiments, steps (1-5) further comprise purifying compound (I). In some embodiments, steps (1-5) further comprise purifying compound (I) by preparative high-performance liquid chromatography (HPLC). In some embodiments, the HPLC is performed using a 0.01% TFA aqueous solution and acetonitrile at a ratio of 98:2.

[0107] In some embodiments, steps (1-5) do not include the purified compound (I).

[0108] In some embodiments, steps (1-5) are performed at a temperature maintained between about 0°C and about 50°C for a duration between about 8 hours and about 24 hours. In some embodiments, the temperature is maintained between about 10°C and about 40°C. In some embodiments, the temperature is between about 20°C and about 30°C. In some embodiments, the duration is between about 12 hours and 20 hours. In some embodiments, the duration is about 16 hours. In some embodiments, steps (1-5) are performed at a temperature maintained between about 20°C and about 30°C for a duration of about 16 hours.

[0109] In some embodiments, the compound of formula (I) is a compound having one of the following formulas: B. Second method

[0110] In some embodiments, a method is provided for preparing a compound of formula (I): a non-mirror isomer of formula (I), a salt thereof, a solvate thereof, or a combination thereof, wherein R4, R5, R6, and R7 are each independently a halogen (e.g., F) or hydrogen, wherein the method comprises one or more of steps (2-1) to (2-6). An exemplary synthetic scheme is shown in scheme 2 of Example 3. One advantage of the second method is that the crystallization step does not require expensive and time-consuming purification steps, such as silica gel chromatography.

[0111] Step (2-1) comprises reacting compound (Ia) or a non-mirror isomer of it with compound (Ib) in a solvent and in the presence of a reducing agent to form compound (Ic) or a non-mirror isomer of it, wherein R1 is a C1-C6 alkyl group.

[0112] In some embodiments, about 0.5 equivalents to about 3 equivalents of compound (Ia) are used compared to the amount of compound (Ib). In some embodiments, about 1 equivalent to about 2 equivalents are used. In some embodiments, about 1.3 equivalents are used.

[0113] In some embodiments, the compound of formula (Ia) comprises, consists of, or is substantially composed of a 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: In some embodiments, the compound of formula (Ia) is a mixture comprising two or more of the three stereoisomers shown above.

[0114] 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.

[0115] 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 equivalent to about 5 equivalents of the hydride are used compared to the amount of the compound of formula (Ib). In some embodiments, about 2 equivalents to about 4 equivalents are used. In some embodiments, about 3 equivalents are used.

[0116] In some embodiments, step (2-1) is performed in ethanol in the presence of NaBH(OAc)3. In some embodiments, 3 equivalents of NaBH(OAc)3 are used.

[0117] In some embodiments, step (2-1) is performed at a temperature maintained between about 20°C and about 65°C for a duration between about 1 hour and about 3 hours. In some embodiments, the temperature is maintained between about 30°C and about 55°C. In some embodiments, the temperature is between about 40°C and about 45°C. In some embodiments, the duration is about 2 hours. In some embodiments, step (2-1) is performed at a temperature maintained between about 40°C and about 45°C for a duration of about 2 hours.

[0118] In some embodiments, the compound of formula (Ic) is a compound having one of the following formulas:

[0119] Step (2-2) comprises reacting a compound of formula (Ic) or a non-mirror isomer thereof with an acid to produce a salt of formula (Ic) or a non-mirror isomer thereof. In some embodiments, the acid is an organic acid. In some embodiments, the acid system is selected from the group consisting of fumaric acid, L-tartaric acid, and orotic acid.

[0120] In some embodiments, the acid is orotic acid. In some embodiments, the salt of formula (Ic) or a non-mirror image isomer thereof has the following structure: orotic acid salt of formula (Ic). In some embodiments, the salt of formula (Ic) is compound 1c orotic acid salt: compound 1c orotic acid salt. In some embodiments, step (2-2) comprises crystallizing the salt of formula (Ic) or a non-mirror image isomer thereof. In some embodiments, step (2-2) comprises crystallizing the orotic acid salt of formula (Ic) or a non-mirror image isomer thereof. In some embodiments, the salt of formula (Ic) or a non-mirror image isomer thereof is crystallized from acetone, 2-methyltetrahydrofuran or acetonitrile to a water mixture (e.g., 19:1 CH3CN and H2O).

[0121] In some embodiments, the acid is fumaric acid. In some embodiments, the salt of formula (Ic) or its non-mirror isomer has the following structure: formula (Ic) fumarate. In some embodiments, the salt of formula (Ic) is compound 1c fumarate: compound 1c fumarate. In some embodiments, step (2-2) further comprises crystallizing the salt of formula (Ic) or its non-mirror isomer to form fumarate. In some embodiments, the salt of formula (Ic) or its non-mirror isomer is crystallized from acetone.

[0122] Step (2-3) comprises reacting a salt of formula (Ic) or a non-mirror isomer of formula (Id) with a compound of formula (Id) in the presence of a base and a solvent to form a compound of formula (Ie), a non-mirror isomer of formula (Ie), wherein R1 and R2 are each independently C1-C6 alkyl; and R3 is a halogroup.

[0123] In some embodiments, a compound of formula (Id) is used in amounts between about 2 equivalents and about 5 equivalents compared to the amount of salt of formula (Ic). In some embodiments, about 3 equivalents to about 4 equivalents are used. In some embodiments, about 3.5 equivalents are used.

[0124] In some embodiments, the base in steps (2-3) comprises a carbonate. In some embodiments, the base comprises K₂CO₃. In some embodiments, the base is K₂CO₃. In some embodiments, about 1 equivalent to about 5 equivalents of base are used compared to the amount of the salt of formula (Ic) and its non-mirror isomers. In some embodiments, about 2 equivalents to about 4 equivalents are used. In some embodiments, about 3 equivalents are used.

[0125] In some embodiments, the solvent in steps (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 between about 2:1 and about 1:2. In some embodiments, the ratio is about 1:1.

[0126] In some embodiments, steps (2-3) are carried out in a mixture of water and toluene in the presence of K₂CO₃. In some such embodiments, the ratio of water to toluene is 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.

[0127] In some embodiments, steps (2-3) are performed at a temperature maintained between about 25°C and about 65°C for a duration between about 8 hours and about 24 hours. In some embodiments, the temperature is maintained between about 35°C and about 55°C. In some embodiments, the temperature is maintained at about 45°C. In some embodiments, the duration is between about 12 hours and about 20 hours. In some embodiments, the duration is about 16 hours. In some embodiments, steps (2-3) are performed at a temperature maintained at 45°C for a duration of about 16 hours.

[0128] In some embodiments, the compound of formula (Ie) is a compound having one of the following formulas:

[0129] Step (2-4) involves reacting a compound of formula (Ie) or a non-mirror isomer thereof with a base in a solvent to form a compound of formula (If): formula (If), a non-mirror isomer thereof, or a salt thereof.

[0130] In some embodiments, the base in steps (2-4) is a strong base. In some embodiments, the base is KOH. In some embodiments, KOH is used in amounts between about 1 equivalent and about 4 equivalents compared to the amount of the compound of formula (Ie). In some embodiments, about 2 equivalents to about 3 equivalents are used. In some embodiments, about 2.5 equivalents are used.

[0131] In some embodiments, the solvent in steps (2-4) comprises THF. In some embodiments, the solvent comprises water. In some embodiments, the solvent is a mixture comprising water and THF.

[0132] In some embodiments, steps (2-4) are carried out in a mixture of water and THF in the presence of KOH. In some such embodiments, 2.5 equivalents of KOH are used.

[0133] In some embodiments, steps (2-4) are performed at a temperature maintained between about 0°C and 5°C for a duration between about 20 hours and about 60 hours. In some embodiments, the temperature is maintained between about 0°C and 5°C for a duration between about 30 hours and about 40 hours. In some embodiments, the temperature is maintained between about 0°C and 5°C for a duration of about 20 hours.

[0134] In some embodiments, step (2-4) provides an HCl salt of formula (If) or a non-mirror isomer thereof. In some embodiments, step (2-4) comprises crystallizing the salt of formula (If) or a non-mirror isomer thereof. In some embodiments, the salt of formula (If) has the following structure: HCl of formula (1f) or a non-mirror isomer thereof.

[0135] In some embodiments, the HCl salt of formula (If) has the following structure:

[0136] In some embodiments, the compound of formula (If) or a salt thereof comprises a compound or a salt thereof having one of the following formulas:

[0137] Step (2-5) involves reacting a compound of formula (If), a non-mirror isomer thereof, a salt thereof, or a non-mirror isomer thereof with a compound of formula (Ig): formula (Ig) in a solvent and in the presence of a carboxyl activating agent to form a compound of formula (Ih): a non-mirror isomer thereof, or a salt thereof.

[0138] In some embodiments, a compound of formula (Ig) is used in an amount between about 1 equivalent and about 1.5 equivalents compared to the amount of the compound of formula (If). In some embodiments, about 1.2 equivalents are used.

[0139] In some embodiments, the solvent in steps (2-5) is a halocarbon. In some embodiments, the solvent is selected from the group consisting of CH2Cl2 or CHCl3. In some embodiments, the solvent is CH2Cl2.

[0140] In some embodiments, the carboxyl activator in steps (2-5) is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI). In some embodiments, EDCI in amounts between about 1 equivalent and about 2 equivalents is used compared to the amount of the compound of formula (If). In some embodiments, about 1.5 equivalents is used.

[0141] In some embodiments, steps (2-5) are performed in CH2Cl2 in the presence of EDCI.

[0142] In some embodiments, steps (2-5) are performed at a temperature maintained between about 0°C and about 5°C for a duration between about 2 hours and about 4 hours. In some embodiments, the duration is about 3 hours. In some embodiments, steps (2-5) are performed at a temperature maintained between about 0°C and about 5°C for a duration of about 3 hours.

[0143] In some embodiments, the compound of formula (Ih) is a compound having one of the following formulas:

[0144] Step (2-6) comprises reacting a compound of formula (Ih), or a non-mirror isomer thereof, a salt thereof, or a non-mirror isomer 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 compound of formula (I).

[0145] In some embodiments, the compound of formula (I) is a compound having one of the following formulas or a salt thereof:

[0146] In some embodiments, the acid in steps (2-6) is an inorganic acid. In some embodiments, the acid in steps (2-6) is hydrochloric acid. In some embodiments, the salt of formula (I) or its solvate is an HCl salt. In some embodiments, the salt of formula (I) or its solvate has the following structure: formula (I) HCl or its non-mirror image isomer. In some embodiments, the salt of formula (I) or its solvate has the following structure: formula (I-1) HCl

[0147] In some embodiments, steps (2-6) are carried out in dimethyl methacrylate. In some embodiments, steps (2-6) are carried out at a temperature maintained between about 45°C and about 70°C for a duration between about 2 hours and about 4 hours. In some embodiments, the temperature is maintained between about 55°C and about 60°C. In some embodiments, the duration is about 3 hours. In some embodiments, steps (2-6) are carried out in dimethyl methacrylate at a temperature maintained between 55°C and about 60°C for about 3 hours.

[0148] In some embodiments, the compound of formula (I), or its salt, solvate, or a salt of its solvate, is compound 2: compound 2. In some embodiments, the compound of formula (I) is a non-mirror isomer of compound 2.

[0149] In some embodiments of any of the foregoing methods (including the methods described in the "First Method" and "Second Method" sections), R1 is a straight-chain C1-C6 alkyl group. In some embodiments, R1 is a C1-C3 alkyl group. In some embodiments, R1 is a C1-C3 straight-chain alkyl group. In some embodiments, R1 is methyl, ethyl, isopropyl, n-butyl, tert-butyl, or dibutyl. In some embodiments, R1 is methyl, ethyl, or isopropyl. In some embodiments, R1 is methyl or ethyl. In some embodiments, R1 is methyl.

[0150] In some embodiments of any of the foregoing methods (including the methods described in the "First Method" and "Second Method" sections), R2 is a C3-C6 branched alkyl group. In some embodiments, R2 is methyl, ethyl, isopropyl, n-butyl, dibutyl, or tert-butyl. In some embodiments, R2 is ethyl, isopropyl, or tert-butyl. In some embodiments, R2 is isopropyl or tert-butyl. In some embodiments, R2 is tert-butyl.

[0151] In some embodiments of any of the foregoing methods (including the methods described in the "First Method" and "Second Method" sections), R3 is F, Cl, or Br. In some embodiments, R3 is Cl or Br. In some embodiments, R3 is Br.

[0152] In some embodiments of any of the foregoing methods (including the methods described in the "First Method" and "Second Method" sections), R4, R5, R6, and R7 are each independently a halogroup or hydrogen. In some embodiments, R4, R5, R6, and R7 are each independently Cl or F. In some embodiments, R4, R5, R6, and R7 are each F. III. Composition, Labeling Reagent, and Method of Use

[0153] Furthermore, this document discloses compositions comprising the compounds and intermediates disclosed herein. In particular, it considers compositions of stereoisomers of compounds of formulas (I), (Ic), (Ie), (If), and (Ih) (including salts, solvates, and solvates of salts of any of the foregoing). In some embodiments, this document provides a composition comprising one or more non-mirror image isomers of a compound of formula (I), its salt, or a solvate thereof: Formula (I) In some embodiments, the compound of formula (I) is prepared using the methods described in Part II-A. In some embodiments, the compound of formula (I) is prepared using the methods described in Part II-B. In some embodiments, R4, R5, R6, and R7 are each F.

[0154] In some embodiments, the composition comprises compound 1, its salt, or a solvate thereof: Compound 1 In some embodiments, the composition comprises or substantially comprises, or consists of or substantially consists of, a single non-mirror image isomer of compound 1. 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% (by weight) a trans-non-mirror image isomer of compound 1, or a salt, solvate, or a solvate of a salt. 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% (by weight) a trans-non-mirror image isomer of compound 2: Compound 2

[0155] In some embodiments, the composition further comprises a detectable marker. In some embodiments, the detectable marker is a radioactive marker, such as 18F. In some embodiments, the detectable marker is part of a metal halide. In some embodiments, the metal halide is {AlF}²⁺. In some embodiments, the metal halide is {Al¹⁸F}²⁺. In some embodiments, the marker (e.g., the metal halide) forms a complex with a linker. In some embodiments, the linker is a compound of formula (I) (e.g., compound 1 or compound 2). In some embodiments, the marker is chelated by the linker.

[0156] In some embodiments, the composition further comprises a polypeptide to which a compound of formula (I) (e.g., compound 1 or compound 2) is bound. In some embodiments, the composition comprises a polypeptide bound to a complex comprising a compound of formula (I) (e.g., compound 1 or compound 2) and a metal halide (e.g., {Al 18F} 2+). In some embodiments, the polypeptide specifically binds to a target molecule such as a marker. In some embodiments, the polypeptide is a polypeptide or protein that binds to a marker. 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, microantibodies, 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: affinity antibodies, afenine, knotting proteins, reticulin, anticarrier proteins, atresomes, high-affinity multimers, FN3 scaffolds, fenomomo, Koonitz domains, proton-binding proteins, OBody, and DARPin.

[0157] Furthermore, this document provides a composition comprising a marker-binding protein, a linker, and a marker. In some embodiments, the composition further comprises a marker-binding protein linked to a marker via a linker. In some embodiments, the linker is compound 1. In some embodiments, the linker is compound 2. In some embodiments, the marker is a metal halide. In some embodiments, the metal halide is {Al 18F} 2+. In some embodiments, the linker chelates the marker. In some embodiments, the linker chelates the metal halide.

[0158] The term "antibody" as used 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., single-arm antibodies), four-chain antibodies (such as IgG antibodies), heavy-chain-only antibodies (HcAbs), and antibody fragments thereof, provided 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.

[0159] Antibody fragments comprise a portion of an antibody, preferably an antigen-binding or variable region of the antibody. Examples of antibody fragments include single-domain antibodies (including VHH antibodies), Fab, Fab', F(ab')2 and Fv fragments, dimers, linear antibodies (see U.S. Patent 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.

[0160] In some embodiments, the composition is suitable for administration to an individual. In some embodiments, the composition is suitable for intravenous administration. In some embodiments, the individual is a mammal. In some embodiments, the individual is a human.

[0161] Furthermore, this document provides methods for preparing labeling reagents using any of the compounds described herein. Some related methods can be found in WO2021 / 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 by reference in their entirety. In some embodiments, the reagent is a label-binding protein. In some embodiments, this document provides a method for preparing a labeling reagent comprising a polypeptide and an 18F radionuclides, the method comprising: (a) contacting the polypeptide with a compound (e.g., compound 1) or composition prepared using the method described herein, under conditions allowing the complex to bind to the polypeptide to provide a conjugate; and (b) contacting the conjugate with an aluminum fluoride complex comprising 18F (e.g., {Al 18F}²⁺) to provide the labeling reagent. In some embodiments, the compound binds to lysine residues of the polypeptide. In some embodiments, the conjugate 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 comprise methionine and / or N-acetylglucosamine. In some embodiments, the conjugate is contacted with the aluminum fluoride complex in the presence of methionine and N-acetylglucosamine. In some embodiments, the method comprises purifying the labeling reagent from the reaction mixture comprising the conjugate and aluminum fluoride using a desalting column. In some embodiments, the desalination column is equilibrated with a buffer comprising histidine, methionine, N-acetylstryptophan, and / or sucrose.

[0162] Further provided are labeling reagents prepared using the methods described herein and methods for using such labeling reagents, for example, in vivo imaging, diagnosis, prognosis, or monitoring. In some embodiments, the labeling reagent is used for in vivo detection of a target molecule via the specific binding of a peptide to the target molecule. In some embodiments, the target molecule is detected by at least one of the following: immunoPET (positron emission tomography), SPECT (single-photon emission computed tomography), MRI (magnetic resonance imaging) (also known as NMR (nuclear magnetic resonance)), near-infrared (NIR), or Cerenkov luminescence imaging (CLI).

[0163] The term "detection" is intended to include determining the presence or absence of a substance or quantifying the amount of a substance (e.g., a target molecule). Therefore, this term refers to the use of the materials, compositions, and methods of this application for qualitative and quantitative determination. Generally, the specific technique used for detection is not critical to the practice of the methods in this application. For example, "detection" of the methods described herein may include observing the presence or absence of a target molecule or changes in the amount of the target molecule. When compared to a control, detection may include quantifying any change (increase or decrease) in any value between 10% and 90%, or between 30% and 60%, or exceeding 100%. Detection may include quantifying any change in any value between 2 and 10 times (inclusive) or more (e.g., 100 times). IV. Compounds

[0164] Furthermore, this document discloses intermediates for the synthetic methods described herein. In some embodiments, the compound is a compound of formula (Ie): a non-mirror isomer of formula (Ie) or a salt thereof, wherein R1 and R2 are each independently C1-C6 alkyl.

[0165] In some embodiments, the compound of formula (Ie) or a salt thereof has the structure of one of the following formulas: In some embodiments, the compound has the formula: Formula (Ie-1)

[0166] In some embodiments, the compound is of formula (Ic): formula (Ic) or its non-mirror isomers or salts, wherein R1 is a C1-C6 alkyl group.

[0167] In some embodiments, the compound of formula (Ic) or its salt has the structure of one of the following formulas: In some embodiments, the compound has the formula: Salt of formula (Ic-1)

[0168] discloses a salt of a specific intermediate or a solvate thereof.

[0169] In some embodiments, the salt is orotic acid salt of compound 1c: compound 1c orotic acid salt is orotic acid salt of compound (Ic), wherein R1 is methyl.

[0170] In some embodiments, the salt is compound 1c fumarate: compound 1c fumarate is a fumarate of formula (Ic), wherein R1 is methyl.

[0171] In some embodiments, the salt is compound 2: compound 2 is a solvate of the salt of compound 1.

[0172] In some embodiments, compound 1c orotate has at least one of the following: (a) XRPD (X-ray powder diffraction) spectrum with characteristic peaks at 7.5° 2θ, about 13.9° 2θ, about 16.6° 2θ, about 23.4° 2θ, and about 25.6° 2θ; (b) XRPD spectrum substantially the same as one of those shown in Figure 1; (c) TGA (thermogravimetric analysis) thermal analysis spectrum showing a mass loss of about 3.3% between 20°C and 150°C; (d) TGA thermal analysis spectrum substantially the same as that shown in Figure 2; (e) DSC (differential scanning calorimetry) thermal analysis spectrum showing an endothermic peak at about 172.8°C; (f) DSC thermal analysis spectrum substantially the same as that shown in Figure 2; (g) 1H NMR (nuclear magnetic resonance). The spectrum, with its characteristic peak located at approximately 5.68 ppm; or the (h) 1H NMR spectrum, which is essentially the same as that shown in Figure 3.

[0173] In some embodiments, compound 1c orotate has at least one of the following: (a) an XRPD (X-ray powder diffraction) spectrum with characteristic peaks at 7.2° 2θ, about 14.0° 2θ, about 15.2° 2θ, about 22.7° 2θ, and about 25.0° 2θ; (b) an XRPD spectrum substantially identical to one of those shown in Figure 1; (c) a TGA (thermogravimetric analysis) chromatogram showing a mass loss of about 2.6% between 20°C and 150°C; (d) a TGA chromatogram substantially identical to that shown in Figure 4; (e) a DSC (differential scanning calorimetry) chromatogram showing an exothermic peak at about 161.7°C and an endothermic peak at about 182.9°C; (f) a DSC chromatogram substantially identical to that shown in Figure 4; (g) The 1H NMR (nuclear magnetic resonance) spectrum has a characteristic peak at approximately 5.69 ppm; and the (h) 1H NMR spectrum is essentially the same as that shown in Figure 5.

[0174] In some embodiments, compound 1c orotate has at least one of the following: (a) an XRPD (X-ray powder diffraction) spectrum with characteristic peaks at 7.3° 2θ, about 7.8° 2θ, about 9.7° 2θ, about 13.9° 2θ, about 16.7° 2θ, about 21.4° 2θ, and about 23.4° 2θ; (b) an XRPD spectrum substantially identical to one of those shown in Figure 6; (c) a TGA (thermogravimetric analysis) thermal analysis plot showing a mass loss of about 4.8% between 20°C and 150°C; (d) a TGA thermal analysis plot substantially identical to that shown in Figure 7; (e) a DSC (differential scanning calorimetry) thermal analysis plot showing an exothermic peak at about 178.1°C and an endothermic peak at about 185.6°C; (f) a DSC The thermal analysis spectrum is essentially the same as that shown in Figure 7; (g) 1H NMR (nuclear magnetic resonance) spectrum with a characteristic peak at approximately 5.68 ppm; and (h) 1H NMR spectrum, which is essentially the same as that shown in Figure 8.

[0175] In some embodiments, compound 1c fumarate has at least one of the following: (a) an XRPD (X-ray powder diffraction) spectrum with characteristic peaks at 7.9° 2θ, about 15.8° 2θ, about 18.6° 2θ, about 19.3° 2θ, about 20.1° 2θ, about 21.0° 2θ, and about 24.7° 2θ; (b) an XRPD spectrum substantially identical to one of those shown in Figure 9; (c) a TGA (thermogravimetric analysis) thermal analysis spectrum showing a mass loss of about 2.4% between 20°C and 140°C; (d) a TGA thermal analysis spectrum substantially identical to that shown in Figure 10; (e) a DSC (differential scanning calorimetry) thermal analysis spectrum showing an endothermic peak at about 154.7°C; (f) a DSC thermal analysis spectrum similar to that shown in Figure 10. The results are essentially the same as those shown in Figure 11; (g) 1H NMR (nuclear magnetic resonance) spectrum with a characteristic peak at approximately 6.39 ppm; and (h) 1H NMR spectrum, which is essentially the same as those shown in Figure 11.

[0176] In some embodiments, the crystalline salt of compound 1c fumarate has at least one of the following: (a) an XRPD (X-ray powder diffraction) spectrum with characteristic peaks at 7.9° 2θ, about 15.8° 2θ, about 18.6° 2θ, about 19.3° 2θ, about 20.1° 2θ, about 21.0° 2θ, and about 24.7° 2θ; (b) an XRPD spectrum substantially the same as one of those shown in Figure 12; (c) a TGA (thermogravimetric analysis) thermal analysis spectrum showing a mass loss of about 1.9% between 20°C and 140°C; (d) a TGA thermal analysis spectrum substantially the same as that shown in Figure 13; (e) a DSC (differential scanning calorimetry) thermal analysis spectrum showing an endothermic peak at about 156.2°C; (f) a DSC The thermal analysis spectrum is essentially the same as that shown in Figure 13; (g) 1H NMR (nuclear magnetic resonance) spectrum with a characteristic peak at approximately 6.39 ppm; and (h) 1H NMR spectrum, which is essentially the same as that shown in Figure 14.

[0177] In some embodiments, compound 2 has at least one of the following: (a) an XRPD (X-ray powder diffraction) spectrum with characteristic peaks at 6.8° 2θ, about 17.6° 2θ, about 20.3° 2θ, about 22.2° 2θ, and about 23.3° 2θ; and (b) an XRPD spectrum substantially the same as that shown in Figure 15. Examples

[0178] The following abbreviations are used throughout the example: A% - Area percentage based on HPLC analysis; HPLC - High performance liquid chromatography; 1H NMR - Proton nuclear magnetic resonance; MeOH - Methanol; EtOH - Ethanol; EtOAc - Ethyl acetate; DMSO - Dimethyl sulfoxide; NaBH(OAc)3-triethoxyborohydride; DIPEA - N,N-diisopropylethylamine; MTBE - Methyl tributyl ether. Example 1: Small-scale palladium-free synthesis scheme 1 for compound 1.

[0179] Step 1a-1: The synthesis of compound 1c – methyl-2-(4-((((1R,2R)-2-aminocyclohexyl)-amino)methyl)phenyl)acetate was carried out over 15 minutes by adding methyl 2-(4-methoxyphenyl)acetate (compound 1b, 310 g, 1.74 mol) and NaBH(OAc)3 (730 g, 3.31 mol) in batches 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, while maintaining the temperature below 60 °C. The resulting mixture was stirred for 2 hours and then cooled to 20°C. The residual solids were removed by filtration, and the mother liquor was concentrated under vacuum and then purified by silica gel chromatography (3% MeOH 7 M NH 3 in CH 2Cl 2) to give compound 1c (240 g, 0.87 mol, yield 50%, 96 A% HPLC). 1H NMR (400 MHz, DMSO- d 6): 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). 13C 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 for C16H24N2O2[M+H]+ is 277.1911, experimental value is 277.1912.

[0180] Steps 1a-2: Synthesis of di-tert-butyl-2,2'-(((1R,2R)-2-((2-(tert-butoxy)-2-oxyethyl)(4-(2-methoxy-2-oxyethyl)benzyl)amino)cyclohexyl)azandiyl) 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 hours. The solvent was changed from CH3CN to EtOAc (2.5 L, 11 mL / g), and the organic solvent was washed with H2O (3400 mL, 2.1 mL / g), dried over Na2SO4, filtered, the solvent was removed under vacuum, and purified by silica gel chromatography (20% EtOAc in n-heptane) to give compound 1e (400 g, 0.65 mol, yield 79%, 98 A% HPLC). 1H NMR (400 MHz, DMSO- d 6): 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 – 1.56 (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, CDCl 3): δ 172.1, 171.6, 139.1, 132.3, 129.4, 128.9, 80.2, 63.6, 60.9, 54.3, 53.2, 52.7, 51.9, 40.9, 29.3, 28.4, 28.1, 25.8, 25.7.The calculated HRMS (m / z) for C 34H 54N 2O 8[M+H] + is 619.3953, while the experimental value is 619.3952.

[0181] Steps 1a-3: Synthesis of compound 1f – 2-(4-((((1R,2R)-2-(bis(2-(tri-butoxy)-2-(-oxyethyl)amino)cyclohexyl)(2-(tri-butoxy)-2-(-oxyethyl)amino)methyl)phenyl)acetic acid A solution of compound 1e (353 g, 0.60 mol) in THF (3 L, 8 mL / g) at 0°C was mixed with 1 M KOH aqueous solution (67.5 g, 1.2 mol, in 1.2 L H₂O) while maintaining the temperature below 10°C. The resulting mixture was stirred for 24 hours, and then the pH was adjusted to 5 to 6 with 1 M HCl aqueous solution while maintaining the temperature below 10°C. The solvent was changed from THF to CH₂Cl₂ (3.8 L, 10 mL / g), and the layers were separated. The organic solvent was washed with 25% NaCl aqueous solution (1.85 L, 5 mL / g), dried over Na₂SO₄, and concentrated under vacuum at 40 °C to give compound 1f (327 g, 0.54 mol, 95% yield, 98% HPLC). 1H NMR (400 MHz, CDCl 3): 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, CDCl 3): δ 172.6, 171.4, 170.5 165.2, 137.3, 131.7, 130.4, 127.9, 84.6, 82.2, 65.2, 60.9, 58.5, 55.9, 49.8, 41.5, 31.1, 28.1, 27.9, 26.0, 25.4, 24.5, 24.2.The calculated HRMS (m / z) for C 33H 52N 2O 8[M+H] + is 605.3796, while the experimental value is 605.3790.

[0182] Steps 1a-4: Synthesis of di-tert-butyl diacetate of compound 1h – 2,2'-(((1R,2R)-2-((2-(tri-butoxy)-2-yloxyethyl)(4-(2-yloxy-2-(2,3,5,6-tetrafluorophenoxy)ethyl)benzyl)amino)cyclohexyl)-azinediyl)diacetate. EDCI (70 g, 0.45 mol) was added to a mixture of compound 1f (180 g, 0.30 mol) and 2,3,5,6-tetrafluorophenol (59 g, 0.36 mol) in CH2Cl2 (1.8 L, 10 mL / g) at 25 °C, and the mixture was stirred for 16 hours. The mixture was cooled to 0°C, washed with 1 M NaHCO3 aqueous solution (31.8 L, 10 mL / g) and 25% NaCl aqueous solution (900 mL, 5 mL / g), and the organic layer was dried over Na2SO4, filtered, and the solvent was removed under vacuum to give compound 1h (216 g, yield 96%, 93 A% HPLC). 1H NMR (400 MHz, CDCl 3): 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, CDCl 3): 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. The calculated HRMS (m / z) for C 39H 52F 4N 2O 8[M+H] + is 753.3733, while the experimental value is 753.3744.

[0183] Steps 1a-5: Synthesis of compound 1 – 2,2'-(((1R,2R)-2-((carboxymethyl)(4-(2-oxy-2-(2,3,5,6-tetrafluorophenoxy)ethyl)benzyl)amino)cyclohexyl)azandiyl)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 hours. The solution was concentrated under vacuum at 40 °C and purified by preparative HPLC to give compound 1 (95 g, 59% yield, 0.16 mol, 99% HPLC, 99% ee). 1H NMR (400 MHz, CD 3OD): 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, CD 3OD): 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. The calculated HRMS (m / z) for C 27H 28F 4N 2O 8[M+H] + is 585.1855, while the experimental value is 585.1854. Example 2: Large-scale palladium-free synthesis of compound 1. Scheme 1 was applied to the following synthesis.

[0184] Step 1b-1: Synthesis of Compound 1c – Methyl-2-(4-((((1R,2R)-2-aminocyclohexyl)-amino)methyl)phenyl)acetate Compound 1b (2.20 kg, 12.35 mol, 1 equivalent), Compound 1a (1.41 kg, 12.35 mol, 1 equivalent), and EtOH (44 L, 20 mL / g) were added to a 100 L jacketed reactor. The mixture temperature was adjusted to 40°C to 50°C. NaBH(OAc)3 (5.28 kg, 24.91 mol, 2 equivalents) was added in portions to the mixture at 40°C to 50°C. The reaction mixture was then stirred at 40°C to 50°C for 2 hours. The reaction was monitored by HPLC (6 A% of Compound 1b, 77 A% of Compound 1c). The temperature of the reaction mixture was adjusted to 20°C to 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 vacuum below 45°C. The crude oily product was purified by silicone column chromatography (DCM / MeOH (7M NH3) = 100 / 0 to 30 / 1 (v / v)). The fraction containing the product was collected and concentrated to dryness under vacuum below 45°C. 1.75 kg of oily compound 1c was obtained with a purity of 95.4% HPLC and a yield of 51%.

[0185] Step 1b-2: Synthesis of compound 1e – di-tertiary-butyl-2,2'-(((1R,2R)-2-((2-(tertiary-butoxy)-2-oxyethyl)(4-(2-methoxy-2-oxyethyl)benzyl)amino)cyclohexyl)azandiyl) diacetate. Compound 1c (1.70 kg, 6.15 mol, 1 equivalent), CH3CN (17 L, 10 mL / g) and diisopropylethylamine (DIPEA, 6.40 kg, 49.52 mol, 8.0 equivalent) were added to a 50 L jacketed reactor. The temperature of the mixture was adjusted to 40°C to 50°C. Then, compound 1d (4.20 kg, 21.53 mol, 3.5 equivalent) was added to the mixture in three batches over three hours at 40°C to 50°C. The reaction mixture was stirred at 40-50°C for 18 hours. The reaction was monitored by HPLC (0 A% of compound 1c, 92 A% of compound 1e). The mixture was concentrated to dryness under vacuum 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 vacuum at 45°C. The crude product was purified by silicone column chromatography (ethyl acetate / n-heptane = 0 / 100 to 1 / 5 (v / v)). The fraction containing the product was collected and concentrated to dryness under vacuum. 2.75 kg of oily compound 1e was obtained with a purity of 97.4% HPLC and a yield of 72%.

[0186] Step 1b-3: Synthesis of Compound 1f – 2-(4-((((1R,2R)-2-(bis(2-(tri-butoxy)-2-(-oxyethyl)amino)cyclohexyl)(2-(tri-butoxy)-2-(-oxyethyl)amino)methyl)phenyl)acetic acid Compound 1e (2.60 kg, 4.20 mol, 1 equivalent) and THF (26 L, 10 mL / g) were added to a 50 L jacketed reactor. The temperature of the reaction mixture was adjusted to 0°C to 10°C. Then, at 0°C to 10°C, an aqueous solution of KOH (8.6 mol, 2 equivalent, 0.48 kg KOH in H₂O (8.4 L), 3 mL / g) was added to the reaction mixture in 4 portions over 4 hours. The reaction mixture was stirred at 0°C to 10°C for 24 hours. The reaction was monitored by HPLC (1 A% compound 1e, 95 A% compound 1f). The pH of the reaction mixture was adjusted to 5 to 6 using 1 M HCl aqueous solution (10 kg) at 0 to 10 °C. The mixture was then concentrated under vacuum below 40 °C to remove THF. 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 silicone mat (2.50 kg), and the filter cake was washed with DCM (8 L, 3.1 mL / g). The filtrates were combined and concentrated to dryness under vacuum at 45 °C. 2.20 kg of a pale yellow solid, compound 1f, was obtained with a purity of 98 A% by HPLC, in a yield of 87%.

[0187] Step 1b-4: Synthesis of di-tert-butyl diacetate of compound 1h – 2,2'-(((1R,2R)-2-((2-(tert-butoxy)-2-yloxyethyl)(4-(2-yloxy-2-(2,3,5,6-tetrafluorophenoxy)ethyl)benzyl)amino)cyclohexyl)-azalkyldiyl)diacetate. Compound 1f (2.00 kg, 3.31 mol, 1 equivalent), compound 1g (0.72 kg, 4.34 mol, 1.3 equivalent), and DCM (21 L, 10.5 mL / g) were added to a 50 L jacketed reactor. The reaction mixture temperature was adjusted to 20°C to 30°C. EDCI (0.81 kg, 4.23 mol, 1.3 equivalent) was added to the reaction mixture in portions. The reaction mixture was cooled to 20°C to 30°C and stirred for 10 hours. The reaction was monitored by HPLC (2 A% compound 1f, 98 A% compound 1h). The reaction mixture was cooled to 0°C to 10°C and then washed five times with 0.5 M NaHCO3 aqueous solution (16 kg) at 0°C to 10°C. The two layers were separated, and the organic layer (0.3 A% SM4, 93 A% D) was monitored by HPLC. The organic layer was dried over Na2SO4 (2 kg), filtered through a silicone mat (2 kg), and the filter cake was washed with DCM (17 L, 8.5 mL / g). The filtrates were combined and concentrated under vacuum at below 35°C to approximately 4 kg to obtain crude compound 1h (2.49 kg) with a purity of 95 A% and a yield of 100%.

[0188] 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)azandiyl)diacetic acid Crude compound 1h (2.49 kg) and TFA (41 kg, 10 mL / g) were added to a 50 L jacketed reactor. The reaction mixture was adjusted to 20°C to 30°C and stirred for 16 hours. The reaction was monitored by HPLC (0 A% compound 1h, 94 A% compound 1). The reaction mixture was then concentrated under vacuum below 45°C. 6 kg of crude compound 1 was obtained as an oil (1.93 kg) with a purity of 91 A% HPLC. Crude compound 1 was purified by preparative HPLC (mobile phase A: 0.01% TFA aqueous solution (v / v); mobile phase B: acetonitrile), and the purified product was lyophilized to obtain a white solid of compound 1 with a purity of 98% HPLC and a yield of 51% (through two steps). (Note: Compound 1 in the preparative HPLC fraction is unstable and needs to be lyophilized immediately. The residual TFA in the solid is 0.1 equivalents of E.) Example 3: Large-scale synthesis scheme 2 for compound 2

[0189] Step 2-1: Synthesis of Compound 1c – Methyl-2-(4-((((1R,2R)-2-aminocyclohexyl)-amino)methyl)phenyl)acetate 50 g of Compound 1b and 1500 mL of EtOH were added to a 3 L jacketed reactor. The mixture was heated to 45°C to 50°C. Then, Compound 1a (41.66 g, 1.3 equivalents) was added. The mixture was stirred at 45°C to 50°C for 10 minutes. Then, NaBH(OAc)3 (178.42 g, 3.0 equivalents) was added. The mixture was stirred at 45°C to 50°C for 2 hours. The reaction was monitored by HPLC (77 A% Compound 1c, 8 A% Compound 1b). The reaction mixture was filtered and washed with 150 mL of EtOH. Add 1000 mL of water to the filtrate and concentrate at 30-35°C to remove EtOH. Add another 500 mL of water and 500 mL of MeTHF, and stir the mixture for 10 minutes, then separate the layers. Adjust the pH of the aqueous phase to 9.1 with NH3•H2O and extract twice with 250 mL of DCM. Combine the DCM layers, dry over Na2SO4, and filter to give 882 g of compound 1c as a DCM solution, with a corrected yield of 66% (89.3% A, analytically 5.78% by weight).

[0190] Step 2-2: Synthesis of orotic acid salt of compound 1c. 500 mL of MeCN was added to the DCM solution of compound 1c described above. 2,4-dichloro-1H-pyrimidin-6-carboxylic acid (28.79 g, 1.0 equivalent) was added to the solution. The reaction was stirred at 20°C to 25°C for 16 hours. The mixture was then filtered, and the filter cake was washed with 100 mL of MeCN. The filter cake was dried under vacuum at 30°C to 35°C for 20 hours. 79.29 g of orotic acid salt of compound 1c was obtained, with a corrected yield of 64% from the two steps of compound 2b (purity 97.1% HPLC, analytical grade 98.2% by weight). 1H 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).

[0191] Steps 2-3: Synthesis of di-tert-butyl-2,2'-(((1R,2R)-2-((2-(tert-butoxy)-2-oxyethyl)(4-(2-methoxy-2-oxyethyl)benzyl)amino)cyclohexyl)azanyl)diacetate. Orotic acid salt of compound 1c (135.70 g, analytical weight 95.8%), 1300 mL toluene, and 1300 mL water were added to a 5 L jacketed reactor. Then, K₂CO₃ (124.63 g, 3.0 equivalent) was added, and the mixture was stirred at 20°C to 25°C for 0.5 hours. Compound 2d (205.22 g, 3.5 equivalent) was added. The mixture was heated to 40°C to 45°C and stirred at 40°C to 45°C for 16 hours. The reaction was monitored by HPLC (93.8% A, B, no residual compound 1c). The reaction mixture was filtered to remove orotic acid, and the filter cake was washed with 390 mL of toluene. The layers were separated. The organic layer was washed three times with an aqueous solution of NaOH (1 equivalent, 12.0 g NaOH in 1300 mL 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 oily compound 1e was obtained with a purity of 96.6% A by HPLC, an analytical yield of 81% by weight, and a corrected yield of 90%. Residual toluene was 8.0% by weight, and residual compound 2d was 5.2% by weight. 1H 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).

[0192] Steps 2-4: Synthesis of Compound 1f Hydrochloride – 2-(4-((((1R,2R)-2-(bis(2-(tri-butoxy)-2-(-oxyethyl)amino)cyclohexyl)(2-(tri-butoxy)-2-(-oxyethyl)amino)methyl)phenyl)acetic acid 185.19 g of Compound 1e (81 wt% as determined by QNMR) was added to a 5 L jacketed reactor, along with 1500 mL of THF. The mixture was cooled to 0°C to 5°C. Then, an aqueous solution of KOH (2.5 equivalents of 34 g KOH in 450 mL of water) was slowly added to the reaction mixture at 0°C to 5°C. The reaction mixture was stirred at 0°C to 5°C for 40 hours. The reaction was monitored by HPLC (95.2 Å of Compound 1f, no Compound 1e remaining). The mixture was adjusted to pH 2.0 with approximately 700 mL of 1 M HCl aqueous solution (4.5 v). The mixture was extracted with 1500 mL of DCM. The organic layer was distilled and exchanged twice with DCM (750 mL DCM each time). A solution of 509.4 g of compound 1f in DCM was obtained, with a corrected yield of 107% (analytical percentages: 30.7 wt%, residual THF: 5.5 wt%, residual toluene: 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 seeds were added. The mixture was stirred at 25 °C for 21 hours. 1400 mL of MTBE (9 v) was added over 8 hours at 25 °C. The mixture was stirred at 25 °C for 40 hours. The mixture was filtered, and the filter cake was washed three times with 940 mL of DCM:MTBE = 1:4, v: v. The filter cake was dried at 25 °C for 24 hours. 154.2 g of compound 1f hydrochloride was obtained with a purity of 98.0 A% by HPLC, an analytical yield of 90.2% by weight, and a corrected yield of 90% from compound 1e.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, 2H), 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).

[0193] Steps 2-5: Synthesis of Compound 1h Hydrochloride – 2,2'-(((1R,2R)-2-((2-(tri-butoxy)-2-yloxyethyl)(4-(2-yloxy-2-(2,3,5,6-tetrafluorophenoxy)ethyl)benzyl)amino)cyclohexyl)-azinediyl)diacetic acid di-tri-butyl ester 33.26 g of Compound 1f hydrochloride (analytical weight 90.2%) and Compound 1 g (9.89 g, 1.2 equivalents) were added to a 1 L jacketed reactor. The mixture was degassed three times with N2. 300 mL of DCM was added to the mixture. The mixture was cooled to 0°C to 5°C under N2 protection. 13.31 g, 1.4 equivalents, of EDCI was added. The mixture was stirred at 0°C to 5°C for 20 hours. The reaction was monitored by HPLC (1 g of 89.2 A% compound, 1 f of no remaining compound). 300 mL of water was added, and the mixture was heated to 20-25°C. The layers were separated. The KF in the organic layer was 0.38%. The organic layer was recirculated through an Al₂O₃ filter cartridge (Al₂O₃, 75.3 g) for 5 hours to reduce the KF to 0.07%. The organic layer was concentrated to approximately 120 mL to obtain a solution of compound 1h hydrochloride in DCM (37.35 g, purity 94.7 A%).

[0194] Steps 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)azandiyl)diacetic acid The above-obtained compound hydrochloride solution was added to a 1 L jacketed reactor under N2 protection. 4N HCl / dimethylalkanes (370 mL, 29.8 equivalents, pre-dried by molecular sieves) were added. The mixture was heated to 55°C to 60°C and stirred at 55°C to 60°C for 3 hours. The reaction was monitored by HPLC (92.8% of compound 2, no remaining compound for 2 hours). The reaction was cooled to 20°C to 25°C, filtered, and the filter cake was washed with 50 mL of DCM. The filter cake was dried under vacuum at 45°C to 50°C for 16 hours. 26.35 g of compound 2 was obtained with a purity of 97.5% A (HPLC, analytical grade 79.9 wt%) and a corrected yield of 70%. Residual chloride was 5.2 wt%. E:HCl = 1:1. Residual diane was 11 wt%. E:diane = 1:0.9. 1H NMR (400MHz, DMSO-d 6) 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). Example 4: Salt screening and characterization of crystallization products.

[0195] Starting with oily compound 1c, a total of 51 salt screening experiments were conducted using 17 acids in three solvent systems (see Table 1 below). Five crystallization matching results were obtained: type A fumarate; type A L-tartrate; and types A, B, and C orotates. The L-tartrate matching result was considered to degrade over time in air. Fumarate and orotates were characterized by X-ray powder diffraction (XRPD), thermogravimetric analysis (TGA), differential scanning calorimetry (DSC), and nuclear magnetic resonance (NMR) (see Figures 1 to 5 and Figures 9 to 11). Three crystalline samples from the 1,5-naphthalenedisulfonic acid, gallic acid, and gentianic acid systems were presumed to be degradation products. Based on these properties, type A fumarate and type A orotate were re-prepared using slurry at a scale of approximately 200 mg and characterized using the same technological advancements described above (see Figures 6-8 and 12-15). The dried XRPD samples were dried under vacuum for at least 2 hours. Table 1. Salt screening results. Salt / solvent system acetone 2-MeTHF CH3CN / H2O (19:1, v / v) blank sample gel* gel* gel* HCl gel* gel* gel* Citric acid gel* gel* gel* Maleic acid gel* gel* gel* fumaric acid Type A fumarate Amorphous gel* L-maleic acid gel* Amorphous** gel* L-tartaric acid Amorphous Amorphous Type A L-tartrate** adipic acid gel* gel* gel* Succinic acid gel* gel* gel* Viscous acid Amorphous Amorphous gel* p-Toluenesulfonic acid gel* gel* gel* 4-Aminobenzoic acid gel* gel* gel* gallic acid Degradation products** gel* gel* whey acid Orotic A Orotic acid type B Orotic A 1,5-Naphthalenedisulfonic acid Degradation products* gel* gel* benzenesulfonic acid gel* gel* gel* Cinnamon acid gel* gel* gel* *: A clear solution was obtained from the slurry at 5°C, therefore slow evaporation was carried out at room temperature. **: Obtained from slurry at 5℃ [Simplified Explanation of the Diagram]

[0046] Figure 1 is a collection of XRPD (X-ray powder diffraction) spectra of the compound sample of compound 1c orotic acid salt. Figure 2 shows the TGA (thermogravimetric analysis) and DSC (differential scanning calorimetry) thermal analysis spectra of compound 1c orotic acid salt (type A orotic acid salt). Figure 3 shows the 1H NMR (nuclear magnetic resonance) spectrum of compound 1c orotic acid salt (type A orotic acid salt). Figure 4 shows the TGA and DSC thermal analysis spectra of compound 1c orotic acid salt (type C orotic acid salt). Figure 5 shows the 1H NMR spectrum of compound 1c orotic acid salt (type C orotic acid salt). Figure 6 is a collection of XRPD spectra of sample compound 1c orotic acid salt (reconstituted type A orotic acid salt). Figure 7 shows the TGA and DSC thermal analysis spectra of compound 1c orotic acid salt (reconstituted type A orotic acid salt). Figure 8 shows the 1H NMR spectrum of compound 1c orotic acid salt (reconstituted type A orotic acid salt). Figure 9 shows a collection of XRPD spectra of sample compound 1c fumarate (type A fumarate). Figure 10 shows the TGA and DSC thermal analysis chromatograms of compound 1c fumarate (type A fumarate). Figure 11 shows the 1H NMR spectrum of compound 1c fumarate (type A fumarate). Figure 12 shows a collection of XRPD spectra of sample compound 1c fumarate (reconstituted type A fumarate). Figure 13 shows the TGA and DSC thermal analysis chromatograms of compound 1c fumarate (reconstituted type A fumarate). Figure 14 shows the 1H NMR spectrum of compound 1c fumarate (reconstituted type A fumarate). Figure 15 is a collection of XRPD spectra of compound 2.

Claims

1. A method for preparing a compound of formula (I) and its non-mirror isomers, salts, solvates, or combinations thereof, wherein the method comprises: (a) step (1-1), comprising reacting a compound of formula (Ia) : formula (Ia) or its non-mirror isomers with a compound of formula (Ib) : formula (Ib) in a solvent and in the presence of a reducing agent to form a compound of formula (Ic) : formula (Ic) or its non-mirror isomers; (b) step (1-2), comprising reacting the compound of formula (Ic) or its salts with a compound of formula (Id) : formula (Id) in the presence of a base and in a solvent to form a compound of formula (Ie) : formula (Ie) or its non-mirror isomers; (c) step (1-3), comprising reacting the compound of formula (Ie) with a base in a solvent to form a compound of formula (If) : formula (If) (d) Step (1-4), comprising reacting the compound of formula (If) with the compound of formula (Ig): Formula (Ig) in a solvent and in the presence of a carboxyl activating agent to form the compound of formula (Ih): Formula (Ih) or a non-mirror isomer thereof; and (e) Step (1-5), comprising reacting the compound of formula (Ih) with an acid to form the compound of formula (I), a non-mirror isomer thereof, a salt thereof, a solvate thereof, or a combination thereof, wherein R1 and R2 are each independently C1-C6 alkyl; R3 is a halogroup; and R4, R5, R6 and R7 are each independently a halogroup or hydrogen.

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

3. The method of claim 1 or 2, wherein the reducing agent in step (1-1) is a hydride.

4. The method of claim 1, wherein the solvent in step (1-3) is an aqueous solvent.

5. The method of claim 4, wherein the solvent in steps (1-3) further comprises tetrahydrofuran (THF).

6. The method of claim 1, wherein the compound of formula (I) is compound 1: compound 1.

7. A method for preparing a compound of formula (I) that is a non-mirror image of itself, a salt thereof, a solvate thereof, or a combination thereof, wherein the method comprises: (a) step (2-1), comprising reacting compound (Ia) and compound (Ib) in a solvent in the presence of a reducing agent to form compound (Ic); (b) step (2-2), comprising reacting compound (Ic) with an acid to produce a salt of formula (Ic); and (c) step (2-3), comprising reacting the salt of formula (Ic) with compound (Id) in the presence of a base in a solvent to form compound (Ie) that is a non-mirror image of itself, or a salt thereof. (d) Step (2-4), which involves reacting the compound of formula (Ie) with a base in a solvent to form the compound of formula (If): formula (If) or its non-mirror isomer or salt thereof; and (e) step (2-5), which comprises reacting the compound of formula (If) or a salt thereof with the compound of formula (Ig): formula (Ig) in a solvent and in the presence of a carboxyl activating agent to form the compound of formula (Ih): formula (Ih) or a non-mirror isomer thereof, or a salt thereof; And (f) step (2-6), which comprises reacting the compound of formula (Ih) with an acid to form the compound of formula (I), its non-mirror isomer, its salt, its solvate, or any combination thereof, wherein R1 and R2 are each independently C1-C6 alkyl; R3 is a halogroup; and R4, R5, R6 and R7 are each independently a halogroup or hydrogen.

8. The method of claim 7, wherein the solvent in step (2-1) comprises an alcohol.

9. The method of claim 7, wherein the reducing agent in step (2-1) comprises a hydride.

10. The method of claim 7, wherein step (2-2) provides a salt of formula (Ic) having the following structure: orotate of formula (Ic).

11. The method of claim 10, wherein step (2-2) comprises crystallizing the salt of formula (Ic) or a non-mirror isomer thereof into an orotate salt.

12. The method of claim 7, wherein step (2-2) provides a salt of formula (Ic) having the following structure: formula (Ic) fumarate.

13. The method of claim 7, wherein the solvent in step (2-3) comprises toluene or water.

14. The method of claim 7, wherein the base in step (2-4) is KOH.

15. The method of claim 7, wherein the solvent in step (2-4) comprises THF or water.

16. A compound of formula (Ie), a non-mirror isomer thereof, or a salt thereof, wherein R1 and R2 are each independently C1-C6 alkyl.

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