Rapafucin derivative compounds and methods of use thereof

Abstract

The present disclosure provides macrocycles generated from the immunophilin ligand family of natural products FK506 and rapamycin. The generation of a Rapafucin library comprising FK506 and rapamycin binding domains should have great potential as new lead compounds for the development of drugs for the treatment of diseases.

Description

[0001] Cross-reference to related applications

[0002] This application claims the benefit of priority to U.S. Serial No. 62 / 909,008, filed October 1, 2019, pursuant to 35 USC §119(e), the entire contents of which are incorporated herein by reference.

[0003] Government-supported statement

[0004] This invention was completed with government support, based on CA174428 granted by the National Institutes of Health. The government holds certain rights to this invention.

[0005] Background Information

[0006] Macrocyclic natural products FK506 and rapamycin are approved immunosuppressive drugs with important biological activities. Both have been shown to inhibit T cell activation, although through different mechanisms. Furthermore, rapamycin has been shown to possess potent antiproliferative activity. FK506 and rapamycin share an unusual mode of action; they function by recruiting the abundantly and ubiquitously expressed cellular protein prolyl cis-trans isomerase FKBP, and the binary complex subsequently binds to and allosterically inhibits their target proteins calcineurin and mTOR, respectively. Structurally, FK506 and rapamycin share a similar FKBP-binding domain but differ in their effector domains. In both FK506 and rapamycin, nature has taught us to convert the effector domain of FK506 to that of rapamycin, which can change the target from calcineurin to mTOR. The generation of macrocyclic Rapafucin libraries containing the binding domains of FK506 and rapamycin should hold great potential as novel lead compounds for the development of drugs to treat diseases.

[0007] With the completion of the sequencing and annotation of the human genome, a complete catalog of all human proteins encoded in the genome is now available. However, the functions of most of these proteins remain unknown. One way to elucidate the functions of these proteins is to search for small molecule ligands that specifically bind to proteins of interest and disrupt their biochemistry and cellular function. Therefore, a major challenge for chemical biologists today is to discover novel small molecule probes against new proteins to facilitate the elucidation of their functions. Recent advances in protein chip development have provided exciting new opportunities for simultaneously screening chemical libraries targeting almost the entire human proteome. A single chip in the form of a glass slide is sufficient to present the complete proteome in a repeating array. Recently, protein chips with 17,000 human proteins presented on a single slide have been produced. A major advantage of screening using human protein chips is that the fully presented proteome can be queried at once in a small volume of assay buffer (<3 mL). However, screening human protein chips with most (if not all) of existing chemical libraries is not yet feasible due to the lack of a universal readout for detecting the binding of ligands to proteins on these chips. While artificial tags can be added to individual compounds in synthetic libraries, the added tags themselves often interfere with the activity of the ligands. Therefore, there remains a need for new compounds and methods for screening chemical libraries targeting the human proteome.

[0008] Overview

[0009] This disclosure relates to libraries of Rapafucin compounds, methods for preparing these compounds, and methods of using them. This disclosure also relates to DNA-encoded libraries of heterocyclic molecules, and more specifically to DNA-encoded libraries of heterocyclic compounds based on the natural product FK506 and the immunoavidin ligand family of rapamycin.

[0010] This article also provides a macrocyclic compound of formula (XIV) or a pharmaceutically acceptable salt, solvate, or stereoisomer thereof:

[0011]

[0012] Each n, m, and p can be an integer selected independently from 0 to 5.

[0013] Each of R1, R2, and R3 can be independently selected from the following groups: H, F, Cl, Br, CF3, CN, N3, -N(R 12 )2、-N(R 12 )3、-CON(R 12 2. NO2, OH, OCH3, methyl, ethyl, propyl, -COOH, -SO3H, -PO(OR) 12)2、-OPO(OR 12 )2、-(CH2) q COOH、-O-(CH2) q COOH、-S-(CH2) q COOH、-CO-(CH2) q COOH、-NR 12 -(CH2) q COOH、-(CH2) q SO3H、-O-(CH2) q SO3H、-S-(CH2) q SO3H、-CO-(CH2) q SO3H、-NR 12 -(CH2) q SO3H、-(CH2) q N(R 12 )2、-O-(CH2) q N(R 12 )2、-S-(CH2) q N(R 12 )2、-CO-(CH2) q N(R 12 )2、-(CH2) q N(R 12 )3、-O-(CH2) q N(R 12 )3、-S-(CH2) q N(R 12 )3、-CO-(CH2) q N(R 12 )3、-NR 12 -(CH2) q N(R 12 )3、-(CH2) q CON(R 12 )2、-O-(CH2) q CON(R 12 )2、-S-(CH2) q CON(R 12 )2、-CO-(CH2) q CON(R 12 )2、-(CH2) q PO(OR 12 )2、-O(CH2) q PO(OR 12 )2、-S(CH2) q PO(OR 12 )2、-CO(CH2) q PO(OR12 )2、-NR 12 (CH2) q PO(OR 12 2、-(CH2) q OPO(OR 12 2、-O(CH2) q OPO(OR 12 2、-S(CH2) q OPO(OR 12 2、-CO(CH2) q OPO(OR 12 )2 and -NR 12 (CH2) q OPO(OR 12 )2.

[0014] In one aspect, q can be an integer selected from 0 to 5. Each of R4, R5, R6, R7, R9, and R... 11 It can be independently selected from the following groups: H, methyl, ethyl, propyl, and isopropyl.

[0015] In another aspect, each R8 and R 10 It can be independently selected from the following groups: H, halogen, hydroxyl, C. 1-20 Alkyl, N3, NH2, NO2, CF3, OCF3, OCHF2, COC 1-20 Alkyl, CO2C 1-20 Alkyl groups, 5- or 6-membered cyclic structures formed with adjacent nitrogen atoms, -N(R) 12 )2、-N(R 12 3. -CON(R) 12 )2、-COOH、-SO3H、-PO(OR 12 )2、-OPO(OR 12 2、-(CH2) q COOH, -O-(CH2) q COOH, -S-(CH2) q COOH, -CO-(CH2) q COOH, -NR 12 -(CH2) q COOH, -(CH2) q SO3H, -O-(CH2) q SO3H, -S-(CH2) q SO3H, -CO-(CH2) q SO3H, -NR 12 -(CH2) q SO3H, -(CH2) qN(R 12 )2, -O-(CH2) q N(R 12 )2, -S-(CH2) q N(R 12 )2, -CO-(CH2) q N(R 12 )2, -(CH2) q N(R 12 )3, -O-(CH2) q N(R 12 )3, -S-(CH2) q N(R 12 )3, -CO-(CH2) q N(R 12 )3, -NR 12 -(CH2) q N(R 12 )3, -(CH2) q CON(R 12 )2, -O-(CH2) q CON(R 12 )2, -S-(CH2) q CON(R 12 )2, -CO-(CH2) q CON(R 12 )2, -(CH2) q PO(OR 12 )2, -O(CH2) q PO(OR 12 )2, -S(CH2) q PO(OR 12 )2, -CO(CH2) q PO(OR 12 )2, -NR 12 (CH2) q PO(OR 12 )2, -(CH2) q OPO(OR 12 )2, -O(CH2) q OPO(OR 12 )2, -S(CH2) q OPO(OR 12 )2, -CO(CH2) q OPO(OR 12 )2 and -NR 12 (CH2) q OPO(OR 12 )2.

[0016] Each R12 It can be independently selected from the following groups: H, methyl, ethyl, propyl, and isopropyl.

[0017] The conditions are R2, R3, R8, and R. 10 At least one of them is selected from -N(R) 12 )2、-N(R 12 )3、-CON(R 12 )2、-COOH、-SO3H、-PO(OR 12 )2、-OPO(OR 12 2、-(CH2) q COOH, -O-(CH2) q COOH, -S-(CH2) q COOH, -CO-(CH2) q COOH, -NR 12 -(CH2) q COOH, -(CH2) q SO3H, -O-(CH2) q SO3H, -S-(CH2) q SO3H, -CO-(CH2) q SO3H, -NR 12 -(CH2) q SO3H, -(CH2) q N(R 12 2、-O-(CH2) q N(R 12 2、-S-(CH2) q N(R 12 2、-CO-(CH2) q N(R 12 2、-(CH2) q N(R 12 3、-O-(CH2) q N(R 12 3. -S-(CH2) q N(R 12 3. -CO-(CH2) q N(R 12 3. -NR 12 -(CH2) q N(R 12 3、-(CH2) q CON(R 12 2、-O-(CH2) q CON(R 12 2、-S-(CH2) q CON(R 122、-CO-(CH2) q CON(R 12 2、-(CH2) q PO(OR 12 2、-O(CH2) q PO(OR 12 2、-S(CH2) q PO(OR 12 2、-CO(CH2) q PO(OR 12 )2、-NR 12 (CH2) q PO(OR 12 2、-(CH2) q OPO(OR 12 2、-O(CH2) q OPO(OR 12 2、-S(CH2) q OPO(OR 12 2、-CO(CH2) q OPO(OR 12 )2 and -NR 12 (CH2) q OPO(OR 12 )2.

[0018] In some respects, R1 can be H, R2 can be H, R3 can be -O-CH2COOH, and p can be 1. In some respects, compound 1593 having the following structure is disclosed herein:

[0019]

[0020] This document also discloses a pharmaceutical composition comprising an effective amount of the compound of formula (XIV) and a pharmaceutically acceptable carrier. This document also discloses a method of treating a subject for a disease, which may include administering an effective amount of the compound of formula (XIV). In some aspects, the disease may be selected from acute kidney injury, cerebral ischemia, hepatic ischemia-reperfusion injury, and organ transplant transport solution. In some aspects, the compound may be administered intravenously.

[0021] This paper also discloses a method for synthesizing macrocyclic compounds, comprising attaching a linker having an amine-terminated structure to a resin; sequentially reacting the linker-modified resin with different amino acids to obtain a peptide-modified resin; removing the resin to obtain a peptide intermediate; subjecting the peptide intermediate to reversed-phase chromatography to obtain a pure diastereomer of the peptide intermediate; reacting the pure diastereomer of the peptide intermediate with an FKBP-binding domain (FKBD); and performing a macrocyclization reaction via olefin metathesis or lactamation. In some aspects, four amino acids are used to obtain a tetrapeptide intermediate. In some aspects, an R stereoisomer is obtained. Brief description of the attached diagram

[0023] Figure 1 Urea levels in a rat renal ischemia-reperfusion model were shown after 24 hours of continuous administration. Dipyridamole (DPA) was administered at 10 mg / kg; compound 1593 was administered at 12 mg / kg or 4 mg / kg; and compound 1594 was administered at 4 mg / kg.

[0024] Figure 2 Creatinine levels in a rat renal ischemia-reperfusion model are shown after 24 hours of continuous administration. Dipyridamole (DPA) was administered at 10 mg / kg; compound 1593 was administered at 12 mg / kg or 4 mg / kg; and compound 1594 was administered at 4 mg / kg.

[0025] Figure 3 The levels of kidney injury molecule-1 (KIM-1) in a rat model of renal ischemia-reperfusion after 24 hours of administration are shown. Dipyridamole (DPA) was administered at 10 mg / kg; compound 1593 was administered at 12 mg / kg or 4 mg / kg; and compound 1594 was administered at 4 mg / kg.

[0026] Figure 4 The levels of neutrophil gelatinase-associated lipocalin-1 (NGAL-1) in a rat model of renal ischemia-reperfusion after 24 hours of administration are shown. Dipyridamole (DPA) was administered at 10 mg / kg; compound 1593 was administered at 12 mg / kg or 4 mg / kg; and compound 1594 was administered at 4 mg / kg.

[0027] Detailed Explanation

[0028] Nature is a rich source of bioactive small molecules, which exhibit a dazzling array of cellular activities thanks to billions of years of evolution. Rapamycin and FK506 belong to a family of macrocyclic natural products with unique structures and unusual modes of action. Upon entering the cell, these two compounds form binary complexes with FKBP12 and other members of the FKBP family. The FKBP12-rapamycin complex can then bind to mTOR and block its kinase activity on downstream substrates such as p70S6K and 4E-BP, while the FKBP12-FK506 complex interacts with calcineurin, a protein phosphatase whose inhibition prevents calcium-dependent signaling and T cell activation. The ability of rapamycin and FK506 to bind FKBP gives them numerous advantages as small molecule probes in biology and as drugs in medicine. First, the binding of both rapamycin and FK506 to FKBP significantly increases their effective size, allowing for allosteric blockade of the substrate's active site with mTOR or calcineurin via indirect disruption of protein-protein interactions. Second, the abundant and ubiquitous expression of intracellular FKBP serves to enrich rapamycin and FK506 within intracellular compartments and maintain their stability. Third, as macrocycles, FK506 and rapamycin are able to interact with proteins more extensively than smaller molecules, regardless of their ability to bind FKBP. Finally, but not least, the high levels of FKBP expression in blood cells make them suitable reservoirs and carriers for efficient in vivo delivery. Therefore, it is not surprising that both rapamycin and FK506 have become widely used drugs in their natural forms without further chemical modification.

[0029] Both rapamycin and FK506 can be divided into two structural and functional domains: an FKBP-binding domain (FKBD) and an effector domain mediating interactions with mTOR or calcineurin. The FKBD structures of rapamycin and FK506 are very similar, but their effector domains differ due to their exclusive target specificities. The existence of separable and modular structural domains of FK506 and rapamycin has been widely used to generate novel analogues of both, including dimerizing chemical inducers and a large number of rapamycin analogues (known as rapalogs), to alter the specificity of rapamycin to the mutant FKBP-rapamycin-binding domain for mTOR and to improve the toxicity and solubility profiles of rapamycin. The existence of two different FKBD-containing macrocycles with different target specificities also raises the interesting question of whether replacing the effector domain of rapamycin or FK506 can further expand the target repertoire of the resulting macrocycles. In their pioneering work, Chakraborty and colleagues synthesized several rapamycin-peptide hybrid molecules that maintained a high affinity for FKBP but did not exhibit biological activity. Recently, we and others independently attempted to explore this possibility by preparing larger libraries of FKBD-containing macrocycles. In one study, a much larger library of FKBD-containing macrocycles was prepared using synthetic mimics of FKBD, but the resulting macrocycles suffered a significant loss of binding affinity for FKBP12, likely due to a lack of bioactive compounds from the library. Using natural FKBD extracted from rapamycin, we also observed a significant loss of FKBP binding affinity during macrocycle formation (see below).

[0030]

[0031] Scheme 1. The structures of rapamycin and FK506 are highlighted in the FKBD section.

[0032] The Rapafucin library is synthesized as described in WO2017 / 136708. Rapadocin compounds and analogues are disclosed in WO2017 / 136717 for the inhibition of human balance nucleoside transporter 1 (ENT1). Rapaglutin and analogues are disclosed in WO2017 / 136731 for use as inhibitors of cell proliferation and for cancer treatment. Approximately 45,000 compounds were generated, and continued screening of the library, as described in WO2018 / 045250, identified several compounds as inhibitors of MIF nuclease activity. All these references are incorporated herein by reference.

[0033] In our continued efforts to explore the potential of using FKBD-containing macrocycles to target novel proteins, we attempted to optimize and successfully identify FKBDs that, when incorporated into macrocycles, allow for a significant retention of binding affinity for FKBP12. We also established a simplified synthetic route for the parallel synthesis of large numbers of FKBD-containing macrocycles.

[0034] The following are some abbreviations used in this disclosure. 2-MeTHF refers to 2-methyltetrahydrofuran; DMF refers to dimethylformamide; DMSO refers to dimethyl sulfoxide; DCM refers to dichloromethane; HATU refers to 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate; DIEA refers to N,N-diisopropylethylamine; TFA refers to trifluoroacetic acid; Fmoc refers to fluorenylmethyloxycarbonyl; MeOH refers to methanol; EtOAc refers to ethyl acetate; MgSO4 refers to magnesium sulfate; COMU-PF6 refers to (1-cyano-2-ethoxy-2-oxoethyleneaminooxy)dimethylamino-morpholino-carbomonium hexafluorophosphate; CAN refers to acetonitrile; Oxyma refers to ethyl cyanohydroxyimino; LC-MS refers to liquid chromatography-mass spectrometry; T3P refers to n-propanephosphonic anhydride; SPPS refers to solid-phase peptide synthesis.

[0035] The following explanations of terms and methods are provided to better describe this disclosure and to guide those skilled in the art in practicing it. Unless the context clearly indicates otherwise, the singular terms “a,” “an,” and “the” include plural indicators. Similarly, unless the context clearly indicates otherwise, the word “or” is intended to include “and.” The term “comprises” means “includes.” Thus, “comprises A or B” means “includes A, B, or A and B” without excluding additional elements. The term “about” will be understood by those skilled in the art. Whether or not the term “about” is explicitly used, each quantity given herein refers to an actual given value, and it is also intended to refer to an approximation of such a given value that would be reasonably inferred by those skilled in the art.

[0036] It should also be understood that all base or amino acid sizes and all molecular weight or molecular mass values ​​given for nucleic acids or peptides are approximate and are provided for descriptive purposes. Although similar or equivalent methods and materials to those described herein may be used in practice or testing of the contents of this disclosure, suitable methods and materials are described below.

[0037] Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Definitions of commonly used terms in molecular biology can be found in: Benjamin Lewin, Genes V, Oxford University Press, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8).

[0038] Unless otherwise indicated, the nomenclature of substituents not explicitly defined herein is achieved by naming the terminal portion of the functional group, followed by the adjacent functional group toward the attachment point. Those skilled in the art will recognize that the above definitions are not intended to include prohibited substitution patterns (e.g., methyl groups substituted with five different groups, pentavalent carbons, and similar substitution patterns). Such prohibited substitution patterns are readily recognized by those skilled in the art. All publications, patent applications, patents, and other references mentioned herein are incorporated herein by reference in their entirety. All sequences provided in the disclosed Genbank accession numbers are incorporated herein by reference. In case of conflict, this specification, including explanations of terminology, shall prevail. Furthermore, the materials, methods, and examples are illustrative only and are not intended to be limiting.

[0039] An alkyl group refers to a monovalent group derived from an alkane by removing a hydrogen atom from any carbon atom. It includes straight-chain and branched groups having from 1 to 12 carbon atoms, typically from 1 to about 10 carbon atoms, or from 1 to about 6 carbon atoms in some embodiments, or having 1, 2, 3, or 4 carbon atoms in other embodiments. Examples of straight-chain alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, n-butyl, n-pentyl, and n-hexyl groups. Examples of branched alkyl groups include, but are not limited to, isopropyl, isobutyl, sec-butyl, and tert-butyl groups. Alkyl groups may be substituted or unsubstituted. Representative substituted alkyl groups may be monosubstituted or substituted more than once, such as, but not limited to, monosubstituted, disubstituted, or trisubstituted. As used herein, unless otherwise stated, the term alkyl refers to both cyclic and acyclic groups.

[0040] The term "cyclic hydrocarbon group" or "cycloalkyl group" refers to a monovalent group derived from cycloalkanes by removing a hydrogen atom from a ring carbon atom. A cycloalkyl group is a saturated or partially saturated non-aromatic structure having a monocyclic or polycyclic structure, including discrete, fused, bridged ring systems and spirocyclic systems, having 3 to 14 carbon atoms, or in some embodiments 3 to 12, 3 to 10, 3 to 8, 3, 4, 5, 6, or 7 carbon atoms. Cycloalkyl groups may be substituted or unsubstituted. Representative substituted cycloalkyl groups can be monosubstituted or substituted more than once, such as, but not limited to, monosubstituted, disubstituted, or trisubstituted. Examples of monocyclic cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl groups. Examples of polycyclic systems include, but are not limited to, bicyclic [4.4.0]decane, bicyclic [2.2.1]heptane, spiro[2.2]pentane, and the like. (Cyclic)oxy refers to -O-cyclic hydrocarbon group. (Cyclic)thio refers to -S-cyclic hydrocarbon group. The term also covers oxidized forms of sulfur, such as -S(O)-cyclic hydrocarbon group or -S(O)2-cyclic hydrocarbon group.

[0041] An alkenyl group refers to a straight-chain, branched, or cycloalkenyl group as defined above, having one or more double bonds between two carbon atoms. An alkenyl group may have 2 to about 12 carbon atoms, or in some embodiments from 1 to about 10 carbon atoms, or in other embodiments from 1 to about 6 carbon atoms, or in other embodiments 1, 2, 3, or 4 carbon atoms. An alkenyl group may be substituted or unsubstituted. Representative substituted alkenyl groups may be monosubstituted or substituted more than once, such as, but not limited to, monosubstituted, disubstituted, or trisubstituted. Examples of alkenyl groups include, but are not limited to, vinyl, allyl, -CH=CH(CH3), -CH=C(CH3)2, -C(CH3)=CH2, cyclopentenyl, cyclohexenyl, butadienyl, pentadienyl, and hexadienyl, among others.

[0042] An alkynyl group refers to a straight-chain, branched, or cyclic alkynyl group as defined above, having one or more triple bonds between two carbon atoms. An alkynyl group may have 2 to about 12 carbon atoms, or in some embodiments from 1 to about 10 carbon atoms, or in other embodiments from 1 to about 6 carbon atoms, or in other embodiments 1, 2, 3, or 4 carbon atoms. The alkynyl group may be substituted or unsubstituted. Representative substituted alkynyl groups may be monosubstituted or substituted more than once, such as, but not limited to, monosubstituted, disubstituted, or trisubstituted. Exemplary alkynyl groups include, but are not limited to, ethynyl, propynyl, and -C≡C(CH3), and others.

[0043] Aryl groups are cyclic aromatic hydrocarbons including monocyclic and polycyclic compounds (including polycyclic compounds containing individual and / or fused aryl groups). Aryl groups can contain from 6 to about 18 ring carbons, or in some embodiments from 6 to 14 ring carbons, or even in other embodiments from 6 to 10 ring carbons. Aryl groups also include heteroaryl groups, which are aromatic cyclic compounds containing five or more ring members, where one or more ring carbon atoms are replaced by heteroatoms such as, but not limited to, N, O, and S. Aryl groups can be substituted or unsubstituted. Representative substituted aryl groups can be monosubstituted or substituted more than once, such as, but not limited to, monosubstituted, disubstituted, or trisubstituted. Aryl groups include, but are not limited to, phenyl groups, biphenylenyl groups, triphenylenyl groups, naphthyl groups, anthraceneyl groups, and pyrene groups. Aryloxy groups refer to -O-aryl. Arylthio groups refer to -S-aryl, where the aryl group is as defined herein. The term also covers oxidized forms of sulfur, such as -S(O)-aryl or -S(O)2-aryl. Heteroaryloxy refers to -O-heteroaryl. Heteroarylthio refers to -S-heteroaryl. The term also covers oxidized forms of sulfur, such as -S(O)-heteroaryl or -S(O)2-heteroaryl.

[0044] Suitable heterocyclic groups include cyclic groups having atoms of at least two different elements as members of their rings, one or more of which are heteroatoms, such as, but not limited to, N, O, or S. Heterocyclic groups may include 3 to about 20 ring members, or in some embodiments 3 to 18, or about 3 to 15, 3 to 12, 3 to 10, or 3 to 6 ring members. The ring system in the heterocyclic group may be unsaturated, partially saturated, and / or saturated. Heterocyclic groups may be substituted or unsubstituted. Representative substituted heterocyclic groups may be monosubstituted or substituted more than once, such as, but not limited to, monosubstituted, disubstituted, or trisubstituted. Exemplary heterocyclic groups include, but are not limited to, pyrrolidinyl groups, tetrahydrofuryl groups, dihydrofuryl groups, tetrahydrothienyl groups, tetrahydrothiopyranyl groups, piperidinyl groups, morpholinyl groups, thiomorpholinyl groups, thioxanyl groups, piperazine groups, azetidinyl groups, aziridinyl groups, imidazoalkyl groups, pyrazolyl groups, thiazoalkyl groups, and tetrahydrothienyl groups. The following groups are listed: thiophenyl, tetrahydrofuranyl, dioxolyl, furanyl, thiophene, pyrrole, imidazole, pyrazolyl, pyrazolinyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, thiazolinyl, oxetanyl, thietanyl, homopiperidyl, oxepanyl, thiepanyl, and oxazapyridine. oxazepinyl group, diaza basal groups, thiazolidinium Heterocyclic groups include 1,2,3,6-tetrahydropyridyl, indololinyl, 2H-pyranyl, 4H-pyranyl, dioxanepentyl, dioxanyl, purine, quinazinyl, terpineyl, phthalazinyl, pteridineyl, and benzothiazolyl. Heterocyclic oxy groups refer to -O-heterocyclic groups. Heterocyclic thio groups refer to -S-heterocyclic groups. The term also covers oxidized forms of sulfur, such as -S(O)-heterocyclic groups or -S(O)2-heterocyclic groups.

[0045] A polycyclic group or polycyclic aromatic hydrocarbon group refers to two or more rings in which two or more carbon atoms are shared by two adjacent rings; these are called "fused rings." If the rings are connected by a single shared carbon atom, they are called "spiral" ring systems. Rings connected by non-adjacent atoms are called "bridged" rings. Polycyclic groups can be substituted or unsubstituted. Representative polycyclic groups can be substituted once or more.

[0046] Halogen groups include F, Cl, Br, and I; nitro groups refer to -NO2; cyano groups refer to -CN; isocyano groups refer to -N≡C; epoxy groups encompass structures in which an oxygen atom is directly attached to two adjacent or non-adjacent carbon atoms in a carbon chain or ring system, which is essentially a cyclic ether structure. Epoxides are cyclic ethers with a triatomic ring.

[0047] An alkoxy group is a substituted or unsubstituted alkyl group as defined above, bonded to an oxygen single bond. Alkoxy groups may be substituted or unsubstituted. Representative substituted alkoxy groups may be substituted once or more. Exemplary alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy, butoxy, pentoxy, hexoxy, isopropoxy, sec-butoxy, tert-butoxy, cyclopropoxy, cyclobutoxy, cyclopentoxy, and cyclohexoxy.

[0048] Thiols refer to -SH. Thiocarbonyl groups refer to (=S). Sulfonyl groups refer to -SO2-alkyl, -SO2-substituted alkyl, -SO2-cyclic hydrocarbon, -SO2-substituted cyclic hydrocarbon, -SO2-aryl, -SO2-substituted aryl, -SO2-heteroaryl, -SO2-substituted heteroaryl, -SO2-heterocyclic, and -SO2-substituted heterocyclic groups. Sulfonylamino groups refer to -NR. a SO2 alkyl, -NR a SO2-substituted alkyl group, -NR a SO2 cyclic hydrocarbon group, -NR a SO2 replaced by cyclic hydrocarbon groups, -NR a SO2 aryl, -NR a SO2 replaced by aryl and -NR a SO2 heteroaryl, -NR a SO2 replaced by heteroaryl groups, -NR a SO2 heterocyclic group, -NR a SO2 is replaced by heterocyclic groups, where each R a Independently, as defined in this article.

[0049] The carboxyl group refers to -COOH or its salt. Carboxylate esters refer to -C(O)O-alkyl, -C(O)O-substituted alkyl, -C(O)O-aryl, -C(O)O-substituted aryl, -C(O)O-cyclic hydrocarbon, -C(O)O-substituted cyclic hydrocarbon, -C(O)O-heteroaryl, -C(O)O-substituted heteroaryl, -C(O)O-heterocyclic, and -C(O)O-substituted heterocyclic. The amino group (of a carboxylate ester) refers to -NR. a -C(O)O-alkyl, -NR a -C(O)O- substituted alkyl group, -NR a -C(O)O-aryl, -NR a -C(O)O- substituted aryl group, -NR a -C(O)O-cyclic hydrocarbon group, -NR a -C(O)O- substituted cyclic hydrocarbon groups, -NR a -C(O)O-heteroaryl, -NR a -C(O)O- substituted heteroaryl groups, -NR a -C(O)O- heterocyclic groups and -NR a -C(O)O- substituted heterocyclic group, where R a As described in this article, the (carboxylate) oxygen group refers to -OC(O)O-alkyl, -OC(O)O-substituted alkyl, -OC(O)O-aryl, -OC(O)O-substituted aryl, -OC(O)O-cyclic hydrocarbon, -OC(O)O-substituted cyclic hydrocarbon, -OC(O)O-heteroaryl, -OC(O)O-substituted heteroaryl, -OC(O)O-heterocyclic, and -OC(O)O-substituted heterocyclic. Oxygenation refers to (=O).

[0050] The terms "amine" and "amino" refer to derivatives of ammonia in which one or more hydrogen atoms have been replaced by substituents, including but not limited to alkyl groups, alkenyl groups, aryl groups, and heterocyclic groups. The urethane group refers to –O(C=O)NR1R2, where R1 and R2 are independently hydrogen, aliphatic groups, aryl groups, or heterocyclic groups.

[0051] The amino carbonyl group refers to -C(O)N(R) b )2, where each R b Independently selected from hydrogen, alkyl, substituted alkyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, heteroaryl, substituted heteroaryl, heterocyclic, substituted heterocyclic. Furthermore, each R b It can optionally be linked to the nitrogen bound thereto to form a heterocyclic group or a substituted heterocyclic group, provided that the two R groups are... bNot all of them are hydrogen. Aminocarbonyl alkyl refers to -alkyl C(O)N(R) b )2, where each R b Independently selected from hydrogen, alkyl, substituted alkyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, heteroaryl, substituted heteroaryl, heterocyclic, substituted heterocyclic. Furthermore, each R b It can optionally be linked to the nitrogen bound thereto to form a heterocyclic group or a substituted heterocyclic group, provided that the two R groups are... b Not all of them are hydrogen. The amino group (amino carbonyl amino) refers to -NR. a C(O)N(R b )2, where R a and each R b As defined in this article, amino dicarbonylamino refers to -NR. a C(O)C(O)N(R b )2, where R a and each R b As defined in this article, aminocarbonyloxy group refers to -OC(O)N(R) b )2, where each R b Independently, as defined herein, the aminosulfonyl group refers to -SO2N(R b )2, where each R b Independently, as defined in this article.

[0052] Imino refers to -N=R c , where R c It can be selected from hydrogen, aminocarbonylalkoxy, substituted aminocarbonylalkoxy, aminocarbonylalkylamino and substituted aminocarbonylalkylamino.

[0053] The term "optionally substituted" means that the preceding group may or may not be substituted. When substituted, the substituents of the "optionally substituted" group may include, but are not limited to, one or more substituents independently selected from, or in combination with, the following groups or a specifically designated group: lower alkyl, lower alkenyl, lower alkynyl, lower alkanoyl, lower heteroalkyl, lower heterocyclic hydrocarbon, lower haloalkyl, lower haloalkenyl, lower haloalkynyl, lower perhaloalkyl, lower perhaloalkoxy, lower cyclic hydrocarbon, phenyl, aryl, aryloxy, lower alkoxy, lower haloalkoxy, oxo The following groups are substituents: lower acyloxy groups, carbonyl groups, carboxyl groups, lower alkyl carbonyl groups, lower carboxyl esters, lower formamide groups, cyano groups, hydrogen, halogens, hydroxyl groups, amino groups, lower alkylamino groups, arylamino groups, acylamino groups, nitro groups, thiols, lower alkyl thiols, lower haloalkyl thiols, lower perhaloalkyl thiols, aryl thiols, sulfonates, sulfonic acids, trisubstituted silyl groups, N3, SH, SCH3, C(O)CH3, CO2CH3, CO2H, pyridyl, thiophene, furanyl, lower carbamates, and lower ureas. Two substituents can be linked together to form a fused five-, six-, or seven-membered carbon ring or heterocycle consisting of zero to three heteroatoms, such as forming methylenedioxy or ethylenedioxy. The optionally substituted group can be unsubstituted (e.g., -CH2CH3), fully substituted (e.g., -CF2CF3), monosubstituted (e.g., -CH2CH2F), or substituted at any level between fully substituted and monosubstituted (e.g., -CH2CF3). When a substituent is described without a limitation on substitution, both the substituted and unsubstituted forms are covered. When the substituent is defined as "substituted," the substituted form is specifically intended. Additionally, optional substituents of different groups of specific parts may be defined as needed; in these cases, the optional substitution will be as defined, usually immediately following the phrase "optionally substituted...".

[0054] Pharmaceutically acceptable salts of the compounds described herein include, for example, conventional non-toxic salts or quaternary ammonium salts derived from compounds of non-toxic organic or inorganic acids. Such conventional non-toxic salts include, for example, salts derived from inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, aminosulfonic acid, phosphoric acid, and nitric acid, and similar inorganic acids; and salts prepared from organic acids such as acetic acid, propionic acid, succinic acid, glycolic acid, stearic acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, palmitic acid, maleic acid, hydroxymaleic acid, phenylacetic acid, glutamic acid, benzoic acid, salicylic acid, sulfanilic acid, 2-acetoxybenzoic acid, fumaric acid, toluenesulfonic acid, methanesulfonic acid, ethanedisulfonic acid, oxalic acid, hydroxyethanesulfonic acid, and similar organic acids. In other cases, the described compounds may contain one or more acidic functional groups and are therefore capable of forming pharmaceutically acceptable salts with pharmaceutically acceptable bases. These salts can also be prepared in situ during the manufacturing process of the application medium or dosage form, or by reacting a purified compound in its free acid form with a suitable base, such as a pharmaceutically acceptable hydroxide, carbonate, or bicarbonate of a metal cation, with ammonia, or with a pharmaceutically acceptable primary, secondary, or tertiary organic amine alone. Representative alkali metal or alkaline earth metal salts include lithium, sodium, potassium, calcium, magnesium, and aluminum salts, and similar salts. Representative organic amines that can be used to form base addition salts include ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, and similar organic amines.

[0055] The term “treatment” is used interchangeably with the term “method of treatment” in this document and refers to both of the following: 1) therapeutic treatments or measures that cure, alleviate, reduce or relieve symptoms of a diagnosed pathological condition, disease, or disorder and / or stop the progression of a diagnosed pathological condition, disease, or disorder; and 2) preventive / protective measures. Individuals requiring treatment may include individuals who already have a specific medical condition or disorder, as well as individuals who may eventually develop a disorder (i.e., individuals requiring preventive measures).

[0056] As used herein, the term "subject" refers to any individual or patient to whom the methods of this subject are applied. Typically, a subject is a human being, although, as those skilled in the art will understand, a subject may be an animal.

[0057] The terms “therapeutic effective amount,” “effective dose,” “therapeutic effective dose,” “effective amount,” or similar terms refer to an amount of the subject compound that will elicit the biological or medical response sought in a tissue, system, animal, or human by administration of said compound. Typically, the response is an improvement in a patient’s symptoms or a desired biological outcome. Such an amount should be sufficient to inhibit MIF activity.

[0058] This document also discloses pharmaceutical compositions comprising compounds having the structure of formula (I). The term "pharmaceuticalally acceptable carrier" refers to a non-toxic carrier that can be administered to a patient together with the compounds of this disclosure without impairing the pharmacological activity of the compounds of this disclosure. Pharmaceutically acceptable carriers that can be used in these compositions include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffering substances such as phosphates, glycine, sorbic acid, potassium sorbate, mixtures of saturated plant fatty acid metaglycerides, water, salts or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethyl cellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, and lanolin.

[0059] Pharmaceutically acceptable carriers that can be used in the pharmaceutical compositions disclosed herein include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffering substances such as phosphates, glycine, sorbic acid, potassium sorbate, mixtures of saturated plant fatty acid metaglycerides, water, salts or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethyl cellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, lanolin, and self-emulsifying drug delivery systems (SEDDS) such as α-tocopherol, polyethylene glycol 1000 succinate, or other similar polymer delivery matrices.

[0060] In pharmaceutical compositions containing only the compounds described herein as active ingredients, the method of administering these compositions may additionally include the step of administering additional agents or therapies to a subject. Such therapies include, but are not limited to, therapies for anemia, diabetes, hypertension, cholesterol, neuropharmacology, cardiovascular function, inflammation, immune function, and blood cell production; hormones and antagonists, drugs affecting gastrointestinal function, chemotherapeutic agents for microbial diseases, and / or chemotherapeutic agents for oncological diseases. Other pharmacological therapies may include any other drugs or biologics found in any drug class. For example, other drug classes may include allergy / cold / ENT therapies, analgesics, anesthetics, anti-inflammatory agents, antimicrobial agents, antiviral agents, asthma / pulmonary therapies, cardiovascular therapies, dermatological therapies, endocrine / metabolic therapies, gastrointestinal therapies, cancer therapies, immunological therapies, neurological therapies, ophthalmic therapies, psychotic therapies, or rheumatic therapies. Other examples of agents or therapies that can be administered in conjunction with the compounds described herein include matrix metalloproteinase inhibitors, lipoxygenase inhibitors, cytokine antagonists, immunosuppressants, cytokines, growth factors, immunomodulators, prostaglandins, or anti-angiogenic compounds.

[0061] As used herein, the term “therapeutic effective amount” refers to an amount of an active compound or pharmaceutical agent that elicits a biological or medical response in a tissue, system, animal, individual, or human sought by an investigator, veterinarian, physician, or other clinician, including one or more of the following: (1) prevention of disease; for example, prevention of disease, condition, or disorder in an individual who may be susceptible to disease, condition, or disorder but has not yet experienced or presented with a pathology or symptom; (2) suppression of disease; for example, suppression of disease, condition, or disorder in an individual who is experiencing or presenting with a pathology or symptom (i.e., prevention of further development of pathology and / or symptom); and (3) improvement of disease; for example, improvement of disease, condition, or disorder in an individual who is experiencing or presenting with a pathology or symptom (i.e., reversal of pathology and / or symptom).

[0062] As used herein, the terms “combination,” “combined,” and related terms refer to the simultaneous or sequential administration of a therapeutic agent according to this disclosure. For example, the described compound may be administered simultaneously or sequentially with another therapeutic agent in a single unit dosage form or together with a single unit dosage form. Thus, this disclosure provides a single unit dosage form comprising the described compound, an additional therapeutic agent, and a pharmaceutically acceptable carrier, adjuvant, or mediator. When a patient or individual is simultaneously exposed to two agents, the two or more agents are generally considered to be administered “combined.” In many embodiments, the two or more agents are considered to be administered “combined” when a patient or individual simultaneously shows treatment-relevant levels of the agents in a specific target tissue or sample (e.g., in the brain, in serum, etc.).

[0063] When the compounds of this disclosure are administered in combination with other agents, they may be administered to the patient sequentially or simultaneously. Alternatively, the pharmaceutical or preventive compositions according to this disclosure may comprise ivermectin or any other compound described herein in combination with another therapeutic or preventive agent. Additional therapeutic agents typically administered to treat a particular disease or condition may be referred to as “agents suitable for the disease or condition being treated.”

[0064] The compounds used in the compositions and methods of this disclosure can also be modified to enhance selective biological properties by adding appropriate functional groups. Such modifications are known in the art and include modifications that increase biological penetration into a given biological system (e.g., blood, lymphatic system, or central nervous system), increase oral bioavailability, increase solubility to allow administration by injection, alter metabolism, and / or change excretion rates.

[0065] According to a preferred embodiment, the compositions of this disclosure are formulated for administration to a subject or patient, such as a mammal, preferably a human. Such pharmaceutical compositions are used to improve, treat, or prevent any of the diseases described herein in the subject.

[0066] The agents disclosed herein are typically administered as pharmaceutical compositions comprising an active therapeutic agent, i.e., and a variety of other pharmaceutically acceptable components. See Remington's Pharmaceutical Science (15th edition, Mack Publishing Company, Easton, Pa., 1980). Preferred forms depend on the intended mode of administration and therapeutic application. Depending on the desired formulation, the composition may also comprise a pharmaceutically acceptable nontoxic carrier or diluent, defined as a medium commonly used to formulate pharmaceutical compositions for administration to animals or humans. Diluents are selected so as not to affect the biological activity of the composition. Examples of such diluents are distilled water, physiological phosphate-buffered saline, Ringer's solution, dextran solution, and Hank's solution. Furthermore, pharmaceutical compositions or formulations may also comprise other carriers, adjuvants, or nontoxic, non-therapeutic, non-immunogenic stabilizers and analogues.

[0067] In some embodiments, this disclosure provides pharmaceutically acceptable compositions comprising a therapeutically effective amount of one or more of the described compounds formulated with one or more pharmaceutically acceptable carriers (additives) and / or diluents for the treatment of diseases described herein, including but not limited to stroke, ischemia, Alzheimer's disease, ankylosing spondylitis, arthritis, osteoarthritis, rheumatoid arthritis, psoriatic arthritis, asthma, atherosclerosis, Crohn's disease, colitis, dermatitis, diverticulitis, fibromyalgia, hepatitis, irritable bowel syndrome, systemic lupus erythematosus, nephritis, ulcerative colitis, and Parkinson's disease. While the described compounds may be administered alone, it is preferred that they be administered as pharmaceutical formulations (compositions) as described herein. The described compounds can be formulated similarly to other drugs for administration in any convenient manner for use in human or veterinary medicine.

[0068] As described in detail, the pharmaceutical compositions of this disclosure can be specifically formulated for administration in solid or liquid form, including those suitable for: oral administration, such as enemas (aqueous or non-aqueous solutions or suspensions), tablets, such as tablets intended for oral absorption, sublingual absorption, and systemic absorption, bolus, powder, granules, pastes for application to the tongue; parenteral administration, such as as a sterile solution or suspension or a sustained-release formulation via subcutaneous, intramuscular, intravenous, or epidural injection; local administration, such as as creams, ointments, or controlled-release patches or sprays applied to the skin, lungs, or mouth; vaginal or rectal administration, such as as pessaries, creams, or foams; sublingual administration; ocular administration; transdermal administration; or nasal administration, lung administration, and administration to other mucosal surfaces.

[0069] Wetting agents, emulsifiers and lubricants such as sodium lauryl sulfate and magnesium stearate, as well as colorants, releasing agents, coating agents, sweeteners, flavoring agents and perfuming agents, preservatives and antioxidants may also be present in the composition.

[0070] Examples of pharmaceutically acceptable antioxidants include: water-soluble antioxidants such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, and the like; oil-soluble antioxidants such as ascorbate palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, α-tocopherol, and the like; and metal chelating agents such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

[0071] Formulations used according to this disclosure include those suitable for oral, nasal, topical (including sublingual and sublingual), rectal, vaginal, and / or parenteral administration. Formulations may be conveniently presented in unit dosage forms and may be prepared by any method well known in the pharmaceutical field. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will vary depending on the host being treated and the specific mode of administration. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be the amount of the compound that produces the therapeutic effect. Typically, this amount will be in the range of about 1% to about 99% of the active ingredient. In some embodiments, this amount will be in the range of about 5% to about 70%, about 10% to about 50%, or about 20% to about 40%.

[0072] In some embodiments, the formulations described herein comprise excipients selected from the group consisting of cyclodextrins, liposomes, micelle forming agents such as bile acids, and polymer carriers such as polyesters and polyanhydrides; and the compounds of this disclosure. In some embodiments, the previously mentioned formulations make the compounds described in this disclosure orally bioavailable.

[0073] Methods for preparing formulations or compositions comprising the described compounds include the step of associating the compounds of this disclosure with a carrier and optionally one or more auxiliary components. Generally, formulations can be prepared by uniformly and tightly associating the compounds of this disclosure with a liquid carrier or a subdivided solid carrier or both, and then shaping the product if necessary.

[0074] Pharmaceutical compositions can be in the form of sterile injectable articles, for example, as sterile injectable aqueous or oily suspensions. These suspensions can be formulated using suitable dispersants or wetting agents (such as, for example, Tween 80, Cremophor RH40, and Cremophor E1) and suspending agents according to techniques known in the art. Sterile injectable articles can also be sterile injectable solutions or suspensions in non-toxic, parenteral-acceptable diluents or solvents, such as solutions in 1,3-butanediol. Acceptable media and solvents that can be used include mannitol, water, Ringer's solution, and isotonic sodium chloride solution. Furthermore, sterile, non-volatile oils are routinely used as solvents or suspension media. For this purpose, any mild, non-volatile oil can be used, including synthetic monoglycerides or diglycerides. Fatty acids such as oleic acid and its glycerol derivatives can be used to prepare injectables, as can natural, pharmaceutically acceptable oils such as olive oil or castor oil (especially in their polyoxyethylated form). These oil solutions or suspensions may also contain long-chain alcohol diluents or dispersants, such as those described in Pharmacopeia Helvetica, or similar alcohols. Other commonly used surfactants such as Tween, Span, and other emulsifiers, or bioavailability enhancers typically used to manufacture pharmaceutically acceptable solid, liquid, or other dosage forms, may also be used for formulation purposes.

[0075] In some cases, slowing the absorption of drugs administered subcutaneously or intramuscularly can be desirable to prolong the effect of the drug. This can be achieved by using liquid suspensions of crystalline or amorphous materials with poor water solubility. The absorption rate of the drug then depends on its dissolution rate, which in turn can depend on the crystal size and crystal form. Alternatively, delayed absorption of parenteral drug forms can be achieved by dissolving or suspending the drug in an oil medium.

[0076] Injectable depot formulations are prepared by forming a microcapsule matrix of the described compound in a biodegradable polymer such as polylactide-polyglycolic acid. The rate of drug release can be controlled depending on the drug-to-polymer ratio and the properties of the specific polymer used. Other examples of biodegradable polymers include poly(orthoester) and poly(anhydride). Depot-type injectable formulations are also prepared by encapsulating the drug in liposomes or microemulsions that are compatible with body tissues.

[0077] The pharmaceutical compositions disclosed herein can be administered orally in any orally acceptable dosage form, including but not limited to capsules, tablets, and aqueous suspensions and solutions. In the case of tablets for oral use, common carriers include lactose and corn starch. Lubricants, such as magnesium stearate, are also typically added. For oral administration in capsule form, useful diluents include lactose and dried corn starch. When aqueous suspensions and solutions, as well as propylene glycol, are administered orally, the active ingredient is combined with an emulsifier and a suspending agent. If desired, certain sweeteners and / or flavoring agents and / or coloring agents may be added.

[0078] The formulations suitable for oral administration described herein may be in the form of capsules, pouches, pills, tablets, lozenges (using a flavored basis, typically sucrose and gum arabic or tragacanth), powders, granules, or as solutions or suspensions in aqueous or non-aqueous liquids, or as oil-in-water or water-in-oil liquid emulsions, or as elixirs or syrups, or as pasteurees (using an inert basis, such as gelatin and glycerin, or sucrose and gum arabic) and / or as mouthwashes and the like, each comprising a predetermined amount of the compound of this disclosure as an active ingredient. The compounds described herein may also be administered as granules, electuals, or pastes.

[0079] In solid dosage forms (capsules, tablets, pills, sugar-coated pills, powders, granules, and the like) intended for oral administration, the active ingredient is mixed with one or more pharmaceutically acceptable carriers, such as sodium citrate or calcium hydrogen phosphate, and / or any of the following: fillers or extenders, such as starch, lactose, sucrose, glucose, mannitol, and / or silicic acid; binders, such as, for example, carboxymethyl cellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose, and / or arbutin. Lagerstroemia indica gum; humectants such as glycerin; disintegrants such as agar, calcium carbonate, potato starch or cassava starch, alginate, certain silicates and sodium carbonate; solution blocking agents such as paraffin; absorption enhancers such as quaternary ammonium compounds; wetting agents such as, for example, cetyl alcohol, glyceryl monostearate and nonionic surfactants; absorbents such as kaolin and bentonite clay; lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate and mixtures thereof; and colorants. In the case of capsules, tablets and pills, the pharmaceutical composition may also contain a buffer. Similar types of solid compositions may also be used as fillers in soft-shell gelatin capsules and hard-shell gelatin capsules using excipients such as lactose or milk sugar and high molecular weight polyethylene glycol and the like.

[0080] Tablets can be prepared by compression or molding, optionally with one or more excipients. Compressed tablets can be prepared using binders (e.g., gelatin or hydroxypropyl methylcellulose), lubricants, inert diluents, preservatives, disintegrants (e.g., sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surfactants, or dispersants. Molded tablets can be prepared in a suitable machine in which a mixture of powdered compounds is wetted with an inert liquid diluent. If a solid carrier is used, the article can be in tablet form, in powder or pellet form placed in a hard gelatin capsule, or in the form of a sugar tablet or lozenge. The amount of solid carrier will vary, for example, from about 25 mg to 800 mg, preferably from about 25 mg to 400 mg. When a liquid carrier is used, the article can be in the form of, for example, syrups, emulsions, soft gelatin capsules, sterile injectable liquids such as ampoules, or non-aqueous liquid suspensions. When the composition is in capsule form, any conventional encapsulation is suitable, for example, using the previously mentioned carrier in a hard gelatin capsule shell.

[0081] Tablets and other solid dosage forms such as sugar-coated pills, capsules, pellets, and granules can optionally be obtained or prepared with coatings and shells, such as enteric coatings and other coatings well known in pharmaceutical formulation techniques. They can be optionally or additionally formulated to provide slow or controlled release of the active ingredient therein, for example using varying proportions of hydroxypropyl methylcellulose to provide a desired release profile, other polymer matrices, liposomes, and / or microspheres. They can be formulated for rapid release, for example, by lyophilization. They can be sterilized, for example, by filtration through a bacteria-retaining filter or by incorporating a sterilizing agent in the form of a sterile solid composition that can be dissolved in sterile water or some other sterile injectable medium just before use. These compositions may also optionally contain an opacifying agent and can be compositions that release the active ingredient only or preferentially in a portion of the gastrointestinal tract, optionally in a delayed manner. Examples of encapsulation compositions that can be used include polymeric substances and waxes. If appropriate, the active ingredient may also be in a microencapsulated form together with one or more of the excipients described above.

[0082] Liquid dosage forms for oral administration of the compounds disclosed herein include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and elixirs. In addition to the active ingredient, the liquid dosage form may contain inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizers, and emulsifiers, such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butanediol, oils (particularly cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil, and sesame oil), glycerol, tetrahydrofuran methanol, fatty acid esters of polyethylene glycol and sorbitol, and mixtures thereof.

[0083] In addition to inert diluents, oral compositions may also contain adjuvants such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents, coloring agents, flavoring agents and preservatives.

[0084] In addition to the active compound, the suspension may contain suspending agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and dehydrated sorbitol esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar and tragacanth gum, and mixtures thereof.

[0085] The pharmaceutical compositions of this disclosure can also be administered in the form of suppositories for rectal use. These compositions can be prepared by mixing the compounds of this disclosure with a suitable non-irritating excipient that is solid at room temperature but liquid at rectal temperature and thus melts in the rectum to release the active ingredient. Such materials include, but are not limited to, cocoa butter, beeswax, and polyethylene glycol.

[0086] Topical application of the pharmaceutical compositions of this disclosure is particularly useful when the desired treatment involves areas or organs that are easily accessible through local application. For local application to the skin, the pharmaceutical composition should be formulated with a suitable ointment containing an active ingredient suspended or dissolved in a carrier. Carriers for the local application of the compounds of this disclosure include, but are not limited to, mineral oil, liquid petroleum, white petroleum, propylene glycol, polyoxyethylene polyoxypropylene compounds, emulsified waxes, and water. Alternatively, the pharmaceutical composition can be formulated with a suitable lotion or cream containing an active compound suspended or dissolved in a carrier. Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl ester wax, cetearyl alcohol, 2-octyldodecanool, benzyl alcohol, and water. The pharmaceutical compositions of this disclosure can also be locally applied to the lower intestine via rectal suppository formulations or as a suitable enema formulation. Topically applied transdermal patches are also included in this disclosure.

[0087] The pharmaceutical compositions disclosed herein can be administered via nasal aerosol or inhalation. Such compositions are prepared according to techniques well known in the field of pharmaceutical formulation and can be prepared as solutions in saline using benzyl alcohol or other suitable preservatives, absorption enhancers for improving bioavailability, fluorocarbons, and / or other solubilizers or dispersants known in the art.

[0088] For ophthalmic applications, the pharmaceutical composition may be formulated as a micronized suspension in isotonic, pH-adjusted sterile saline, with or without a preservative such as benzalkonium chloride, or preferably as a solution in isotonic, pH-adjusted sterile saline. Alternatively, for ophthalmic applications, the pharmaceutical composition may be formulated in an ointment such as petrolatum.

[0089] Transdermal patches offer the additional advantage of providing controlled delivery of the compounds of this disclosure into the body. Such dosage forms can be prepared by dissolving or dispersing the compounds in a suitable medium. Absorption enhancers can also be used to increase the flux of the compounds across the skin. The rate of such flux can be controlled by providing a rate-controlled membrane or by dispersing the compounds in a polymer matrix or gel.

[0090] Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions of this disclosure include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like) and suitable mixtures thereof, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Suitable flowability can be maintained, for example, by using coating materials such as lecithin, by maintaining the desired particle size in the case of dispersions, and by using surfactants.

[0091] Such compositions may also contain adjuvants such as preservatives, wetting agents, emulsifiers, and dispersants. In some embodiments, the inclusion of one or more antibacterial and / or antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and the like, may be desirable. Optionally or additionally desirable, the inclusion of isotonic agents, such as sugars, sodium chloride, and the like, in the composition may be desirable. Furthermore, prolonged absorption of injectable drug forms can be achieved by including agents that delay absorption, such as aluminum monostearate and gelatin.

[0092] In some embodiments, the described compound or pharmaceutical product is administered orally. In other embodiments, the described compound or pharmaceutical product is administered intravenously. Alternative routes of administration include sublingual, intramuscular, and transdermal administration.

[0093] When the compounds described herein are administered to humans and animals as pharmaceuticals, they may be given on their own or as pharmaceutical compositions comprising, for example, 0.1% to 99.5% (more preferably 0.5% to 90%) of an active ingredient in combination with a pharmaceutically acceptable carrier.

[0094] The products described herein can be administered orally, parenterally, topically, or rectally. They are, of course, administered in a form suitable for the relevant route of administration. For example, they are administered in tablet or capsule form via injection, inhalation, eye wash, ointment, suppository, etc.; via injection, infusion, or inhalation; topically via lotion or ointment; and rectally via suppository. Oral administration is preferred.

[0095] Such compounds can be administered to humans and other animals for therapeutic purposes via any suitable route of administration, including oral, nasal (e.g., via a spray), rectal, vaginal, parenteral, intracerebrospinal, and topical (e.g., via powder, ointment, or drops, including sublingual and sublingual administration).

[0096] Regardless of the chosen route of administration, the compounds described herein and / or pharmaceutical compositions of this disclosure, which can be used in a suitable hydrated form, are formulated into pharmaceutically acceptable dosage forms using conventional methods known to those skilled in the art.

[0097] The actual dose level of the active ingredient in the pharmaceutical composition of this disclosure can be varied to obtain an amount of active ingredient that is effective for achieving the desired therapeutic response for a particular patient, composition, and administration mode, while being non-toxic to the patient.

[0098] The terms “administration of” and / or “administering” should be understood as meaning the provision of a therapeutically effective amount of a pharmaceutical composition to a subject in need of treatment. Routes of administration may be enteric, local, or parenteral. Therefore, routes of administration include, but are not limited to, intradermal, subcutaneous, intravenous, intraperitoneal, intraarterial, intrasheath, intracapsular, intraorbital, intracardiac, intradermal, percutaneous, transtracheal, subepidermal, intra-articular, subcapsular, subarachnoid, intraspinal, and intrasternal administration, oral, sublingual, sublingual, rectal, vaginal, nasal / ocular administration, as well as infusion, inhalation, and nebulization.

[0099] Crystal structures of the FKBP-FK506-calcineurin and FKBP-rapamycin-TOR complexes reveal that both FK506 and rapamycin can be divided into two functional domains: an "FKBP-binding domain (FKBD)" and an "effect domain," which mediate their interactions with calcineurin and TOR, respectively. While extensive protein-protein interactions exist between FKBP and calcineurin in their ternary complex, far fewer interactions are observed between FKBP and TOR, suggesting that the key role of FKBP in inhibiting TOR via rapamycin is its binding to the drug's FKBD and presentation of its effector domain to TOR.

[0100] A structural comparison of FK506 and rapamycin reveals that they share a nearly identical FKBD, but each possesses a distinct effector domain. By exchanging the effector domain of FK506 with that of rapamycin, the target can be changed from calcineurin to TOR, which has no sequence, functional, or structural similarity to each other. Furthermore, other proteins can be targeted by grafting novel structures onto the FKBDs of both FK506 and rapamycin. Therefore, generating new compounds with novel target specificity can be achieved by grafting a sufficiently large combinatorial library onto the FKBD, combined with a proteome-wide screen, in which each compound in the library is tested against every protein in the human proteome.

[0101]

[0102] In some embodiments, this document provides a macrocyclic compound according to formula (I), the macrocyclic compound comprising FKBD, an effector domain, a first linker and a second linker, wherein FKBD, the effector domain, the first linker and the second linker together form a macrocycle.

[0103] In some embodiments, this document provides macrocyclic compounds according to formula (II) or optically pure stereoisomers thereof or pharmaceutically acceptable salts thereof.

[0104]

[0105] B can be CH2, NH, NMe, O, S, or S(O)2; X can be O, NH, or NMe; E can be CH or N; n is an integer selected from 0 to 4; m is an integer selected from 1 to 10. AA in the formula represents natural and non-natural amino acids, each of which can be selected from Table 4 below.

[0106] In some implementations, m can be 1. In some implementations, m can be 2. In some implementations, m can be 3. In some implementations, m can be 4. In some implementations, m can be 5. In some implementations, m can be 6. In some implementations, m can be 7. In some implementations, m can be 8. In some implementations, m can be 9. In some implementations, m can be 10. In a specific implementation, m is 3 or 4.

[0107] Each R 1 Choose from the following groups: H, halogen, hydroxyl, C 1-20 Alkyl, N3, NH2, NO2, CF3, OCF3, OCHF2, COC 1-20 Alkyl and CO2C 1-20 Alkyl group. R 2 Choose C freely 6-15 Aryl and C 1-10 The group consisting of heteroaryl groups, optionally substituted with: H, halogen, hydroxyl, N3, NH2, NO2, CF3, C 1-10 Alkyl, substituted C 1-10 Alkyl, C 1-10 Alkoxy groups, substituted C 1-10 Alkoxy, acyl, acylamino, acyloxy, acyl C 1-10 Alkoxy, amino, substituted amino, aminoacyl, aminocarbonyl C 1-10 Alkyl, aminocarbonylamino, aminodicarbonylamino, aminocarbonyloxy, aminosulfonyl, C 6-15 Aryl, substituted C 6-15 Aryl, C 6-15 Aryloxy group, substituted C 6-15 aryloxy group, C 6-15 aryl thio group, substituted C 6-15 Arylthio, carboxyl, carboxyl ester, (carboxyl ester)amino, (carboxyl ester)oxy, cyano, C 3-8 Cyclic hydrocarbon group, substituted C 3-8 Cyclic hydrocarbon group, (C 3-8 Cycloalkyl)oxy group, substituted (C) 3-8 Cycloalkyl)oxy group, (C 3-8 Cyclic hydrocarbon group) thio group, substituted (C) 3-8 Cyclic hydrocarbon group) sulfide group, C 1-10 heteroaryl, substituted C 1-10 heteroaryl, C 1-10 Heteroaryl groups, substituted C 1-10 heteroaryloxy, C 1-10 Heteroaryl thiols, substituted C 1-10 heteroaryl thio, C 2-10Heterocyclic group, substituted C 2-10 Heterocyclic group, C 2-10 Heterocyclic oxygen group, substituted C 2-10 Heterocyclic oxygen, C 2-10 Heterocyclic thio group, substituted C 2-10 Heterocyclic thio, imino, oxo, sulfonyl, sulfonylamino, thiol, C 1-10 Alkyl thio group, substituted C 1-10 Alkyl thio and thiocarbonyl.

[0108] V is Z is a key. Where R 3 and R 4 Each is independently selected from the following groups: hydrogen, hydroxyl, halogen, alkyl, alkoxy, cycloalkyl, cyano, alkylthio, amino, alkylamino, and dialkylamino; K is O, CHR 5 CR 5 , N and NR 5 , where R 5 It is hydrogen or alkyl.

[0109] L 1 L 2 or L 3 Each of them can be selected from the group of structures shown in Table 1 below.

[0110] Table 1. Connector base structure.

[0111]

[0112]

[0113]

[0114]

[0115]

[0116]

[0117]

[0118]

[0119]

[0120]

[0121]

[0122]

[0123] *Each R 20 and R 21 Independently select from the following groups: hydrogen, hydroxyl, OR 22 NR 23 R 24 alkyl, arylalkyl Where R N It is aryl, alkyl, or arylalkyl; wherein R 22 R 23 and R 24 Each is either hydrogen or alkyl.

[0124] In some implementations, the FKBD-containing portion may have a structure according to formula (III) or an optically pure stereoisomer or a pharmaceutically acceptable salt thereof before being incorporated into the macrocycle.

[0125]

[0126] Where L is selected from the structures in Table 1; A is CH2, NH, O or S; each X is independently O, NH or NMe; E is CH or N; Indicates a single or double bond. n is an integer selected from 0 to 4.

[0127] Each R 1 Choose from the following groups: H, halogen, hydroxyl, C 1-20 Alkyl, N3, NH2, NO2, CF3, OCF3, OCHF2, COC 1-20 Alkyl and CO2C 1-20 Alkyl group. R 2 Choose from the following groups: H, halogen, hydroxyl, C 1-20 Alkyl, N3, NH2, NO2, CF3, OCF3, OCHF2, COC 1-20 Alkyl and CO2C 1-20 Alkyl group. R 3 Choose C freely 6-15 Aryl and C 1-10 The group consisting of heteroaryl groups, optionally substituted with: H, halogen, hydroxyl, N3, NH2, NO2, CF3, C 1-10 Alkyl, substituted C 1-10 Alkyl, C 1-10 Alkoxy groups, substituted C 1-10 Alkoxy, acyl, acylamino, acyloxy, acyl C 1-10 Alkoxy, amino, substituted amino, aminoacyl, aminocarbonyl C 1-10 Alkyl, aminocarbonylamino, aminodicarbonylamino, aminocarbonyloxy, aminosulfonyl, C6-15 Aryl, substituted C 6-15 Aryl, C 6-15 Aryloxy group, substituted C 6-15 aryloxy group, C 6-15 aryl thio group, substituted C 6-15 Arylthio, carboxyl, carboxyl ester, (carboxyl ester)amino, (carboxyl ester)oxy, cyano, C 3-8 Cyclic hydrocarbon group, substituted C 3-8 Cyclic hydrocarbon group, (C 3-8 Cycloalkyl)oxy group, substituted (C) 3-8 Cycloalkyl)oxy group, (C 3-8 Cyclic hydrocarbon group) thio group, substituted (C) 3-8 Cyclic hydrocarbon group) sulfide group, C 1-10 heteroaryl, substituted C 1-10 heteroaryl, C 1-10 Heteroaryl groups, substituted C 1-10 heteroaryloxy, C 1-10 Heteroaryl thiols, substituted C 1-10 heteroaryl thio, C 2-10 Heterocyclic group, substituted C 2-10 Heterocyclic group, C 2-10 Heterocyclic oxygen group, substituted C 2-10 Heterocyclic oxygen, C 2-10 Heterocyclic thio group, substituted C 2-10 Heterocyclic thio, imino, oxo, sulfonyl, sulfonylamino, thiol, C 1-10 Alkyl thio group, substituted C 1-10 Alkyl thio and thiocarbonyl.

[0128] V is Z is a key. Where R 4 and R 5 Each is independently selected from the following groups: hydrogen, hydroxyl, halogen, alkyl, alkoxy, cycloalkyl, cyano, alkylthio, amino, alkylamino, and dialkylamino; K is O, CHR 6 CR 6 , N and NR 6 , where R 6 It is hydrogen or alkyl.

[0129] In some implementations, the FKBD-containing portion may have a structure according to formula (IV) or an optically pure stereoisomer or a pharmaceutically acceptable salt thereof before being incorporated into the macrocycle.

[0130]

[0131] Where L is selected from the structures in Table 1; A is CH2, NH, O, or S; each X is independently O or NH; E is CH or N; each R 1 Choose from the following groups: H, halogen, hydroxyl, C 1-20 Alkyl, N3, NH2, NO2, CF3, OCF3, OCHF2, COC 1-20 Alkyl and CO2C 1-20 Alkyl; each R 2 Choose from the following groups: H, halogen, hydroxyl, N3, NH2, NO2, CF3, C 1-10 Alkyl, substituted C 1-10 Alkyl, C 1-10 Alkoxy groups, substituted C 1-10 Alkoxy, acyl, acylamino, acyloxy, acyl C 1-10 Alkoxy, amino, substituted amino, aminoacyl, aminocarbonyl C 1-10 Alkyl, aminocarbonylamino, aminodicarbonylamino, aminocarbonyloxy, aminosulfonyl, C 6-15 Aryl, substituted C 6-15 Aryl, C 6-15 Aryloxy group, substituted C 6-15 aryloxy group, C 6-15 aryl thio group, substituted C 6-15 Arylthio, carboxyl, carboxyl ester, (carboxyl ester)amino, (carboxyl ester)oxy, cyano, C 3-8 Cyclic hydrocarbon group, substituted C 3-8 Cyclic hydrocarbon group, (C 3-8 Cycloalkyl)oxy group, substituted (C) 3-8 Cycloalkyl)oxy group, (C 3-8 Cyclic hydrocarbon group) thio group, substituted (C) 3-8 Cyclic hydrocarbon group) sulfide group, C 1-10 heteroaryl, substituted C 1-10 heteroaryl, C 1-10 Heteroaryl groups, substituted C 1-10 heteroaryloxy, C 1-10 Heteroaryl thiols, substituted C 1-10 heteroaryl thio, C 2-10 Heterocyclic group, substituted C 2-10 Heterocyclic group, C 2-10 Heterocyclic oxygen group, substituted C 2-10 Heterocyclic oxygen, C 2-10 Heterocyclic thio group, substituted C 2-10 Heterocyclic thio, imino, oxo, sulfonyl, sulfonylamino, thiol, C 1-10 Alkyl thio group, substituted C 1-10Alkyl thio and thiocarbonyl; n is an integer selected from 0 to 4; and m is an integer selected from 0 to 5.

[0132] In some embodiments, the Rapafucin compound of this disclosure may have a structure according to formula (V) or an optically pure stereoisomer thereof or a pharmaceutically acceptable salt thereof.

[0133]

[0134] Where L is selected from the groups in Table 1; A is CH2, NH, NMe, O, S(O)2 or S; each X is independently O, NMe or NH; E is CH or N.

[0135] R 1 R 2 R 3 and R 4 Each of these can be independently selected from the following groups: H, halogen, hydroxyl, N3, NH2, NO2, CF3, OCF3, OCHF2, COC 1-20 Alkyl, CO2C 1-20 Alkyl, C 3-8 Cyclic hydrocarbon group, C 2-8 alkenyl, C 2-8 alkynyl group, C 1-10 Alkoxy, C 6-15 Aryl, C 6-15 aryloxy group, C 6-15 Arylthio, C 2-10 Carboxyl group, C 1-10 Alkylamino, thiol, C 1-10 Alkyl thio, C 1-10 Alkyl disulfides, C 6-15 Arylthio, C 1-10 heteroaryl thio, (C 3-8 Cyclic hydrocarbon group) sulfide group, C 2-10 Heterocyclic thio group, sulfonyl group, C 1-10 Alkylsulfonyl, amide, C 1-10 Alkyl amide, selenol, C 1-10 Alkyl selenol, C 6-15 Arylselenool, C 1-10 heteroaryl selenools, (C 3-8 Cyclic alkyl group) selenool, C 2-10 Heterocyclic selenools, guanidinos, C 1-10 Alkylguanidine, urea, C 1-10 Alkyl urea, ammonium, C 1-10 Alkylammonium, cyano, C 1-10 Alkyl cyano, C 1-10 Alkyl nitro, adamantane, phosphonate, C 1-10 Alkyl phosphonates and C6-15 Arylphosphonates, each of which may optionally be substituted with: H, halogen, hydroxyl, N3, NH2, NO2, CF3, C 1-20 Alkyl, substituted C 1-20 Alkyl, C 1-10 Alkoxy groups, substituted C 1-10 Alkoxy, acyl, acylamino, acyloxy, acyl C 1-10 Alkoxy, amino, substituted amino, aminoacyl, aminocarbonyl C 1-10 Alkyl, aminocarbonylamino, aminodicarbonylamino, aminocarbonyloxy, aminosulfonyl, C 6-15 Aryl, substituted C 6-15 Aryl, C 6-15 Aryloxy group, substituted C 6-15 aryloxy group, C 6-15 aryl thio group, substituted C 6-15 Arylthio, carboxyl, carboxyl ester, (carboxyl ester)amino, (carboxyl ester)oxy, cyano, C 3-8 Cyclic hydrocarbon group, substituted C 3-8 Cyclic hydrocarbon group, (C 3-8 Cycloalkyl)oxy group, substituted (C) 3-8 Cycloalkyl)oxy group, (C 3-8 Cyclic hydrocarbon group) thio group, substituted (C) 3-8 Cyclic hydrocarbon group, thio group, halogenated group, hydroxyl group, C 1-10 heteroaryl, substituted C 1-10 heteroaryl, C 1-10 Heteroaryl groups, substituted C 1-10 heteroaryloxy, C 1-10 Heteroaryl thiols, substituted C 1-10 heteroaryl thio, C 2-10 Heterocyclic group, substituted C 2-10 Heterocyclic group, C 2-10 Heterocyclic oxygen group, substituted C 2-10 Heterocyclic oxygen, C 2-10 Heterocyclic thio group, substituted C 2-10 Heterocyclic thio, imino, oxo, sulfonyl, sulfonylamino, thiol, C 1-10 Alkyl thio group, substituted C 1-10 Alkyl thio and thiocarbonyl.

[0136] Or any R 4 With any R 3 A ring structure is formed, and the ring structure is selected from C. 2-10 Heterocyclic groups and C 1-10 The group consisting of heteroaryl groups, optionally substituted with: H, halogen, hydroxyl, N3, NH2, NO2, CF3, C1-10 Alkyl, substituted C 1-10 Alkyl, C 1-10 Alkoxy groups, substituted C 1-10 Alkoxy, acyl, acylamino, acyloxy, acyl C 1-10 Alkoxy, amino, substituted amino, aminoacyl, aminocarbonyl C 1-10 Alkyl, aminocarbonylamino, aminodicarbonylamino, aminocarbonyloxy, aminosulfonyl, C 6-15 Aryl, substituted C 6-15 Aryl, C 6-15 Aryloxy group, substituted C 6-15 aryloxy group, C 6-15 aryl thio group, substituted C 6-15 Arylthio, carboxyl, carboxyl ester, (carboxyl ester)amino, (carboxyl ester)oxy, cyano, C 3-8 Cyclic hydrocarbon group, substituted C 3-8 Cyclic hydrocarbon group, (C 3-8 Cycloalkyl)oxy group, substituted (C) 3-8 Cycloalkyl)oxy group, (C 3-8 Cyclic hydrocarbon group) thio group, substituted (C) 3-8 Cyclic hydrocarbon group, thio group, halogenated group, hydroxyl group, C 1-10 heteroaryl, substituted C 1-10 heteroaryl, C 1-10 Heteroaryl groups, substituted C 1-10 heteroaryloxy, C 1-10 Heteroaryl thiols, substituted C 1-10 heteroaryl thio, C 2-10 Heterocyclic group, substituted C 2-10 Heterocyclic group, C 2-10 Heterocyclic oxygen group, substituted C 2-10 Heterocyclic oxygen, C 2-10 Heterocyclic thio group, substituted C 2-10 Heterocyclic thio, imino, oxo, sulfonyl, sulfonylamino, thiol, C 1-10 Alkyl thio group, substituted C 1-10 Alkyl thio and thiocarbonyl.

[0137] n is an integer selected from 0 to 4; m is an integer selected from 0 to 5; each p is an integer independently selected from 0 to 2; q is an integer selected from 1 to 10.

[0138] In some implementations, q can be 1. In some implementations, q can be 2. In some implementations, q can be 3. In some implementations, q can be 4. In some implementations, q can be 5. In some implementations, q can be 6. In some implementations, q can be 7. In some implementations, q can be 8. In some implementations, q can be 9. In some implementations, q can be 10. In a specific implementation, q is 3 or 4.

[0139] In some embodiments, the Rapafucin compound of this disclosure may have a structure according to formula (VI) or an optically pure stereoisomer thereof or a pharmaceutically acceptable salt thereof.

[0140]

[0141] Each L 1 L 2 or L 3 The linker structures can be independently selected from those in Table 1. Each AA1, AA2, AA3, or AA4 can be independently selected from the amino acid monomers shown in Table 3 below. X can be CH2, NH, O, or S; Y can be O, NH, or N-alkyl; E can be CH or N; n is an integer selected from 0 to 4. The amino acid can be NC-linked or CN-linked.

[0142] Each R 1 Choose from the following groups: H, halogen, hydroxyl, C 1-20 Alkyl, N3, NH2, NO2, CF3, OCF3, OCHF2, COC 1-20 Alkyl and CO2C 1-20 Alkyl group. R 2 Choose C freely 6-15 Aryl and C 1-10 The group consisting of heteroaryl groups, optionally substituted with: H, halogen, hydroxyl, N3, NH2, NO2, CF3, C 1-10 Alkyl, substituted C 1-10 Alkyl, C 1-10 Alkoxy groups, substituted C 1-10 Alkoxy, acyl, acylamino, acyloxy, acyl C 1-10 Alkoxy, amino, substituted amino, aminoacyl, aminocarbonyl C 1-10 Alkyl, aminocarbonylamino, aminodicarbonylamino, aminocarbonyloxy, aminosulfonyl, C 6-15 Aryl, substituted C 6-15 Aryl, C 6-15 Aryloxy group, substituted C 6-15 aryloxy group, C 6-15 aryl thio group, substituted C6-15 Arylthio, carboxyl, carboxyl ester, (carboxyl ester)amino, (carboxyl ester)oxy, cyano, C 3-8 Cyclic hydrocarbon group, substituted C 3-8 Cyclic hydrocarbon group, (C 3-8 Cycloalkyl)oxy group, substituted (C) 3-8 Cycloalkyl)oxy group, (C 3-8 Cyclic hydrocarbon group) thio group, substituted (C) 3-8 Cyclic hydrocarbon group) sulfide group, C 1-10 heteroaryl, substituted C 1-10 heteroaryl, C 1-10 Heteroaryl groups, substituted C 1-10 heteroaryloxy, C 1-10 Heteroaryl thiols, substituted C 1-10 heteroaryl thio, C 2-10 Heterocyclic group, substituted C 2-10 Heterocyclic group, C 2-10 Heterocyclic oxygen group, substituted C 2-10 Heterocyclic oxygen, C 2-10 Heterocyclic thio group, substituted C 2-10 Heterocyclic thio, imino, oxo, sulfonyl, sulfonylamino, thiol, C 1-10 Alkyl thio group, substituted C 1-10 Alkyl thio and thiocarbonyl.

[0143] V is Z is a key. Where R 3 and R 4 Each is independently selected from the following groups: hydrogen, hydroxyl, halogen, alkyl, alkoxy, cycloalkyl, cyano, alkylthio, amino, alkylamino, and dialkylamino; K is O, CHR 5 CR 5 , N and NR 5 , where R 5 It is hydrogen or alkyl.

[0144] Synthetic routes for Rapafucin. Several methods exist for the synthesis of Rapafucin, including both solid-phase and solution-phase synthesis. These methods can lead to modifications of the linker group and / or effector domains, including alkylation, amide bond formation, double bond metathesis, oxadiazole formation, triazole formation, dithiol formation, sulfone formation, Diels-Alder cycloaddition, and others.

[0145] We employ solid-phase peptide synthesis to assemble peptide effector domains. Pre-assembled FKBDs, capped at one end with a carboxylic acid and at the other with an olefin, are then coupled with peptides that retain the chain on the beads. To facilitate the purification of the newly formed macrocycle, a coupled macrocyclization and cyclic release strategy is employed, whereby macrocyclization is accompanied by the simultaneous release of the macrocyclic product from the solid beads. Those skilled in the art can envision different macrocyclization methods for synthesizing the Rapafucin molecules of this disclosure. In some embodiments, ring-closed metathesis (RCM) / cyclic release is used. In some embodiments, macrocyclic lactamation can be used for the efficient parallel synthesis of different Rapafucins. A cis-C6 linker can be used to construct Rapafucin libraries. A combination of moderate temperature and catalyst loading (140 °C, 30 mol% Hoveyda–Grubbs II catalyst) is used for the subsequent large-scale synthesis of Rapafucin libraries.

[0146] Other ring-closing methods can be used to synthesize the Rapafucin molecule disclosed herein. Exemplary methods may include, but are not limited to, ammonolysis, chemical enzymatic methods, click chemistry, macrocyclization by using a ring contraction with an auxiliary group, macrocyclization mediated by a sulfur-containing group, macrocyclization via cycloaddition, macrocyclization via a Wittiga or Wittig-like reaction, macrocyclization from a multicomponent reaction, metal-assisted macrocyclization, macrocyclization via a CN bond, macrocyclization via a CO bond, alkylation with or without metal assistance, intramolecular cyclopropanation, oxidative coupling of aromatic hydrocarbons, side-chain cyclization, and oxidative coupling of aromatic hydrocarbons. Each of these macrocyclization methods can be carried out in a solid or solution phase. Macrocyclization reactions by using a ring contraction with an auxiliary group may include, but are not limited to, the use of hydroxybenzaldehyde, the use of hydroxynitrophenol, and the use of nitrovinylphenol. Macrocyclization reactions mediated by sulfur-containing groups can include, but are not limited to, thiazoline formation with O-to-N acyl transfer, transesterification with S-to-N acyl transfer, ring tautomerization with S-to-N acyl transfer, Staudinger ligation ring contraction, dithiol-ene macrocyclization, thiol-ene macrocyclization, thioalkylation, and disulfide formation. Macrocyclization reactions via cycloaddition can include, but are not limited to, phosphorene-azide linkage and oxadiazole grafting. Metal-assisted macrocyclization can include, but is not limited to, C / C bond formation, Suzuki coupling, Sonogashira coupling, Tasuji-Trost reaction, Glaser-Hay coupling, and nickel-catalyzed macrocyclization. Macrocyclization reactions via CN bonds can include, but are not limited to, Ullmann coupling and Buchwald-Hartwig animation. Macrocyclization reactions via CO bonds can include, but are not limited to, Chan-Lam-Evans coupling, CH activation, and Ullmann coupling. Macrocyclization reactions via alkylation can include enol chemistry, Williamson etherification, Mitsunobu reaction, aromatic nucleophilic substitution (SNAr), and Friedel-Crafts type alkylation.

[0147] In some implementations, the Rapafucin molecule can be cyclized using the methods described in Marsault, E., & Peterson, ML (eds.). (2017). Practical Medicinal Chemistry with Macrocycles: Design, Synthesis, and Case Studies, which is incorporated herein by reference in its entirety. Some non-limiting examples of macrocyclization methods are shown in Table 2 below, where each n can be an integer selected independently from 0 to 10.

[0148] Table 2. Other macrocyclization methods that can be used for the synthesis of Rapafucin.

[0149]

[0150]

[0151]

[0152]

[0153]

[0154]

[0155]

[0156]

[0157]

[0158]

[0159]

[0160]

[0161]

[0162]

[0163] In some embodiments, the Rapafucin compound of this disclosure may have a structure according to formula (VII) or an optically pure stereoisomer thereof or a pharmaceutically acceptable salt thereof.

[0164]

[0165] Each T1 or T2 can be independently selected from the terminal structures outlined in Table 2 above prior to macrocyclization. Each L1, L2, or L3 can be independently selected from the linker structures in Table 1. Each AA can be independently selected from the amino acid monomers shown in Table 3 below. X can be CH2, NH, O, or S; Y can be O, NH, or N-alkyl; E can be CH or N; n is an integer selected from 0 to 4. The amino acid can be NC-linked or CN-linked.

[0166] In some implementations, m can be 1. In some implementations, m can be 2. In some implementations, m can be 3. In some implementations, m can be 4. In some implementations, m can be 5. In some implementations, m can be 6. In some implementations, m can be 7. In some implementations, m can be 8. In some implementations, m can be 9. In some implementations, m can be 10. In a specific implementation, m is 3 or 4.

[0167] Each R 1 Choose from the following groups: H, halogen, hydroxyl, C 1-20 Alkyl, N3, NH2, NO2, CF3, OCF3, OCHF2, COC 1-20 Alkyl and CO2C 1-20 Alkyl group. R 2 Choose C freely 6-15 Aryl and C 1-10 The group consisting of heteroaryl groups, optionally substituted with: H, halogen, hydroxyl, N3, NH2, NO2, CF3, C 1-10 Alkyl, substituted C 1-10 Alkyl, C 1-10 Alkoxy groups, substituted C 1-10 Alkoxy, acyl, acylamino, acyloxy, acyl C 1-10 Alkoxy, amino, substituted amino, aminoacyl, aminocarbonyl C 1-10 Alkyl, aminocarbonylamino, aminodicarbonylamino, aminocarbonyloxy, aminosulfonyl, C 6-15 Aryl, substituted C 6-15 Aryl, C 6-15 Aryloxy group, substituted C 6-15 aryloxy group, C 6-15 aryl thio group, substituted C 6-15 Arylthio, carboxyl, carboxyl ester, (carboxyl ester)amino, (carboxyl ester)oxy, cyano, C 3-8 Cyclic hydrocarbon group, substituted C 3-8 Cyclic hydrocarbon group, (C 3-8 Cycloalkyl)oxy group, substituted (C) 3-8 Cycloalkyl)oxy group, (C3-8 Cyclic hydrocarbon group) thio group, substituted (C) 3-8 Cyclic hydrocarbon group) sulfide group, C 1-10 heteroaryl, substituted C 1-10 heteroaryl, C 1-10 Heteroaryl groups, substituted C 1-10 heteroaryloxy, C 1-10 Heteroaryl thiols, substituted C 1-10 heteroaryl thio, C 2-10 Heterocyclic group, substituted C 2-10 Heterocyclic group, C 2-10 Heterocyclic oxygen group, substituted C 2-10 Heterocyclic oxygen, C 2-10 Heterocyclic thio group, substituted C 2-10 Heterocyclic thio, imino, oxo, sulfonyl, sulfonylamino, thiol, C 1-10 Alkyl thio group, substituted C 1-10 Alkyl thio and thiocarbonyl.

[0168] V is Z is a key. Where R 3 and R 4 Each is independently selected from the following groups: hydrogen, hydroxyl, halogen, alkyl, alkoxy, cycloalkyl, cyano, alkylthio, amino, alkylamino, and dialkylamino; K is O, CHR 5 CR 5 , N and NR 5 , where R 5 It is hydrogen or alkyl.

[0169] Table 3 below shows the FKBD portion with connecting bases before incorporation into the Rapafucin macrocyclic structure.

[0170] Table 3. FKBD / Connector Base Section Used in This Disclosure.

[0171]

[0172]

[0173]

[0174]

[0175]

[0176]

[0177]

[0178]

[0179]

[0180] *The FKBD is reduced and cyclized via lactamation.

[0181] Table 4 below shows the amino acid monomers used in the synthesis of the Rapafucin macrocyclic compound in this disclosure.

[0182] Table 4. Monomers used in this disclosure.

[0183]

[0184]

[0185]

[0186]

[0187]

[0188]

[0189]

[0190]

[0191]

[0192]

[0193]

[0194]

[0195]

[0196]

[0197]

[0198]

[0199]

[0200]

[0201]

[0202]

[0203]

[0204]

[0205]

[0206]

[0207]

[0208]

[0209]

[0210]

[0211]

[0212]

[0213]

[0214]

[0215] Option 2. Hydroxyl groups for peptide synthesis / linking in RbAsp, dD, D and SbAsp.

[0216] The monomers RbAsp, dD, D, and SbAsp have more than one hydroxyl group. In some embodiments, the hydroxyl group used as a linking point with adjacent residues in each of these monomers is illustrated in Scheme 2 above. In some embodiments, another hydroxyl group in these monomers may be used as a linking point with adjacent residues.

[0217] In some embodiments, this document discloses compounds of formula VIII or pharmaceutically acceptable salts or solvates thereof.

[0218]

[0219] In some implementations, R can be R 1 R 2 R 3 R 4 and R 5 Each can be independently selected from hydrogen, hydroxyl, alkoxy, cyano, alkylthio, amino, and alkylamino groups, as well as... in It can be a resin; where A 1 A 2 A3 A 4 and A 5 One, two, three, or four can be N or P, and the rest are CH; where B 1 B 2 B 3 and B 4 One, two, three, or four of them can be O, N, or S, and the remainder can be CH or CH2; among them It can be a single key or a double key.

[0220] In some implementations, X1 can be O or NR. 6 Y can be -C(O)- or X2 can be (CH2) m O, OC(O), NR 6 NR 6 C(O); Z can be W can be O, CH, CH2, or CR. 9 or CR 10 R 11 L1 and L2 can each be independently a direct bond, a substituted or unsubstituted -(C1-C6)alkyl-, or a substituted or unsubstituted -(CH2). n O(C1-C6)alkyl-, substituted or unsubstituted -(CH2) n C(O) (C1-C6)alkyl-, substituted or unsubstituted -(CH2) n C(O)O(C1-C6)alkyl-, substituted or unsubstituted-(CH2) n OC(O)(C1-C6)alkyl-, substituted or unsubstituted-(CH2) n NH(C1-C6)alkyl-, substituted or unsubstituted -(CH2) n C(O)NH(C1-C6)alkyl-, substituted or unsubstituted-(CH2) n S(C1-C6)alkyl-, substituted or unsubstituted -(CH2) n C(O)(CH2) n S(C1-C6)alkyl-, substituted or unsubstituted -(C2-C6)alken-, substituted or unsubstituted -(CH2) n O(C2-C6) alkenyl-, substituted or unsubstituted -(CH2) n C(O)(C2-C6)alkenyl-, substituted or unsubstituted -(CH2) n C(O)O(C2-C6)alkenyl-, substituted or unsubstituted-(CH2)n OC(O)(C2-C6)alkenyl-, substituted or unsubstituted-(CH2) n NH(C2-C6) alkenyl-, substituted or unsubstituted -(CH2) n C(O)NH(C2-C6)alkenyl-, substituted or unsubstituted -(CH2) n S(C2-C6) alkenyl-, substituted or unsubstituted -(CH2) n C(O)(CH2) n S(C2-C6)alkenyl-, substituted or unsubstituted -(C2-C6)ynyl-, substituted or unsubstituted -(CH2) n O(C2-C6) ynyl group -, substituted or unsubstituted -(CH2) n C(O)(C2-C6) ynyl group -, substituted or unsubstituted -(CH2) n C(O)O(C2-C6) ynyl group -, substituted or unsubstituted -(CH2) n OC(O)(C2-C6) ynyl group -, substituted or unsubstituted -(CH2) n NH(C2-C6) ynyl group -, substituted or unsubstituted -(CH2) n C(O)NH(C2-C6) ynyl group -, substituted or unsubstituted -(CH2) n S(C2-C6) ynyl group -, substituted or unsubstituted -(CH2) n C(O)(CH2) n S(C2-C6) ynyl-, substituted or unsubstituted -(C1-C6) alkyl-NR 18 - (CH2) that has been substituted or not substituted n O(C1-C6)alkyl-NR 18 - (CH2) that has been substituted or not substituted n C(O)(C1-C6)alkyl-NR 18 - (CH2) that has been substituted or not substituted n C(O)O(C1-C6)alkyl-NR 18 - (CH2) that has been substituted or not substituted n OC(O)(C1-C6)alkyl-NR 18 - (CH2) that has been substituted or not substituted n NH(C1-C6)alkyl-NR 18 - (CH2) that has been substituted or not substituted n C(O)NH(C1-C6)alkyl-NR 18- (CH2) that has been substituted or not substituted n S(C1-C6)alkyl-NR 18 - (CH2) that has been substituted or not substituted n C(O)(CH2) n S(C1-C6)alkyl-NR 18 - Substituted or unsubstituted -(C2-C6)alkenyl-NR 18 - (CH2) that has been substituted or not substituted n O(C2-C6)alkenyl-NR 18 - (CH2) that has been substituted or not substituted n C(O)(C2-C6)alkenyl-NR 18 - (CH2) that has been substituted or not substituted n C(O)O(C2-C6)alkenyl-NR 18 - (CH2) that has been substituted or not substituted n OC(O)(C2-C6)alkenyl-NR 18 - (CH2) that has been substituted or not substituted n NH(C2-C6)alkenyl-NR 18 - (CH2) that has been substituted or not substituted n C(O)NH(C2-C6)alkenyl-NR 18 - (CH2) that has been substituted or not substituted n S(C2-C6)alkenyl-NR 18 - (CH2) that has been substituted or not substituted n C(O)(CH2) n S(C2-C6)alkenyl-NR 18 - Substituted or unsubstituted -(C2-C6)-ynyl-NR 18 - (CH2) that has been substituted or not substituted n O(C2-C6) ynyl-NR 18 - (CH2) that has been substituted or not substituted n C(O)(C2-C6)alkynyl-NR 18 - (CH2) that has been substituted or not substituted n C(O)O(C2-C6)alkynyl-NR 18 - (CH2) that has been substituted or not substituted n OC(O)(C2-C6) ynyl-NR 18 - (CH2) that has been substituted or not substituted n NH(C2-C6) alkyne-NR18 - (CH2) that has been substituted or not substituted n C(O)NH(C2-C6)alkynyl-NR 18 - (CH2) that has been substituted or not substituted n S(C2-C6) ynyl-NR 18 - (CH2) that has been substituted or not substituted n C(O)(CH2) n S(C2-C6) ynyl-NR 18 - Substituted or unsubstituted -(C1-C6)alkyl-C(O) - Substituted or unsubstituted -(CH2) n O(C1-C6)alkyl-C(O)-, substituted or unsubstituted -(CH2) n C(O) (C1-C6)alkyl-C(O)-, substituted or unsubstituted -(CH2) n C(O)O(C1-C6)alkyl-C(O)-, substituted or unsubstituted -(CH2) n OC(O)(C1-C6)alkyl-C(O)-, substituted or unsubstituted -(CH2) n NH(C1-C6)alkyl-C(O)-, substituted or unsubstituted -(CH2) n C(O)NH(C1-C6)alkyl-C(O)-, substituted or unsubstituted -(CH2) n S(C1-C6)alkyl-C(O)-, substituted or unsubstituted -(CH2) n C(O)(CH2) n S(C1-C6)alkyl-C(O)-, substituted or unsubstituted -(C2-C6)alkenyl-C(O)-, substituted or unsubstituted -(CH2) n O(C2-C6) alkenyl-C(O)-, substituted or unsubstituted -(CH2) n C(O)(C2-C6)alkenyl-C(O)-, substituted or unsubstituted -(CH2) n C(O)O(C2-C6)alkenyl-C(O)-, substituted or unsubstituted -(CH2) n OC(O)(C2-C6)alkenyl-C(O)-, substituted or unsubstituted -(CH2) n NH(C2-C6)alkenyl-C(O)-, substituted or unsubstituted -(CH2) n C(O)NH(C2-C6)alkenyl-C(O)-, substituted or unsubstituted -(CH2) nS(C2-C6)alkenyl-C(O)-, substituted or unsubstituted -(CH2) n C(O)(CH2) n S(C2-C6)alkenyl-C(O)-, substituted or unsubstituted -(C2-C6)ynyl-C(O)-, substituted or unsubstituted -(CH2) n O(C2-C6) ynyl group -C(O)-, substituted or unsubstituted -(CH2) n C(O)(C2-C6) ynyl group -C(O)-, substituted or unsubstituted -(CH2) n C(O)O(C2-C6) ynyl group -C(O)-, substituted or unsubstituted -(CH2) n OC(O)(C2-C6) ynyl group -C(O)-, substituted or unsubstituted -(CH2) n NH(C2-C6) ynyl group -C(O)-, substituted or unsubstituted -(CH2) n C(O)NH(C2-C6) ynyl group -C(O)-, substituted or unsubstituted -(CH2) n S(C2-C6) ynyl group -C(O)-, substituted or unsubstituted -(CH2) n C(O)(CH2) n S(C2-C6) ynyl-C(O)-, -O-, -NH-, -S-, -S(O)-, -SO2-, -Si- and -B-, wherein each alkyl group, alkenyl group and ynyl group may optionally be substituted with the following: alkyl, alkoxy, amino, hydroxyl, mercapto, halogen, carboxyl, oxo, cyano, nitro or trifluoromethyl.

[0221] L3 can be a direct bond, a substituted or unsubstituted -(C1-C6)alkyl-, or a substituted or unsubstituted -(CH2). n O(C1-C6)alkyl-, substituted or unsubstituted -(CH2) n C(O) (C1-C6)alkyl-, substituted or unsubstituted -(CH2) n C(O)O(C1-C6)alkyl-, substituted or unsubstituted-(CH2) n OC(O)(C1-C6)alkyl-, substituted or unsubstituted-(CH2) n NH(C1-C6)alkyl-, substituted or unsubstituted -(CH2) n C(O)NH(C1-C6)alkyl-, substituted or unsubstituted-(CH2) n S(C1-C6)alkyl-, substituted or unsubstituted -(CH2)n C(O)(CH2) n S(C1-C6)alkyl-, substituted or unsubstituted -(C2-C6)alken-, substituted or unsubstituted -(CH2) n O(C2-C6) alkenyl-, substituted or unsubstituted -(CH2) n C(O)(C2-C6)alkenyl-, substituted or unsubstituted -(CH2) n C(O)O(C2-C6)alkenyl-, substituted or unsubstituted-(CH2) n OC(O)(C2-C6)alkenyl-, substituted or unsubstituted-(CH2) n NH(C2-C6) alkenyl-, substituted or unsubstituted -(CH2) n C(O)NH(C2-C6)alkenyl-, substituted or unsubstituted -(CH2) n S(C2-C6) alkenyl-, substituted or unsubstituted -(CH2) n C(O)(CH2) n S(C2-C6)alkenyl-, substituted or unsubstituted -(C2-C6)ynyl-, substituted or unsubstituted -(CH2) n O(C2-C6) ynyl group -, substituted or unsubstituted -(CH2) n C(O)(C2-C6) ynyl group -, substituted or unsubstituted -(CH2) n C(O)O(C2-C6) ynyl group -, substituted or unsubstituted -(CH2) n OC(O)(C2-C6) ynyl group -, substituted or unsubstituted -(CH2) n NH(C2-C6) ynyl group -, substituted or unsubstituted -(CH2) n C(O)NH(C2-C6) ynyl group -, substituted or unsubstituted -(CH2) n S(C2-C6) ynyl group -, substituted or unsubstituted -(CH2) n C(O)(CH2) n S(C2-C6) ynyl-, substituted or unsubstituted -(C1-C6) alkyl-NR 18 - (CH2) that has been substituted or not substituted n O(C1-C6)alkyl-NR 18 - (CH2) that has been substituted or not substituted n C(O)(C1-C6)alkyl-NR 18 - (CH2) that has been substituted or not substitutedn C(O)O(C1-C6)alkyl-NR 18 - (CH2) that has been substituted or not substituted n OC(O)(C1-C6)alkyl-NR 18 - (CH2) that has been substituted or not substituted n NH(C1-C6)alkyl-NR 18 - (CH2) that has been substituted or not substituted n C(O)NH(C1-C6)alkyl-NR 18 - (CH2) that has been substituted or not substituted n S(C1-C6)alkyl-NR 18 - (CH2) that has been substituted or not substituted n C(O)(CH2) n S(C1-C6)alkyl-NR 18 - Substituted or unsubstituted -(C2-C6)alkenyl-NR 18 - (CH2) that has been substituted or not substituted n O(C2-C6)alkenyl-NR 18 - (CH2) that has been substituted or not substituted n C(O)(C2-C6)alkenyl-NR 18 - (CH2) that has been substituted or not substituted n C(O)O(C2-C6)alkenyl-NR 18 - (CH2) that has been substituted or not substituted n OC(O)(C2-C6)alkenyl-NR 18 - (CH2) that has been substituted or not substituted n NH(C2-C6)alkenyl-NR 18 - (CH2) that has been substituted or not substituted n C(O)NH(C2-C6)alkenyl-NR 18 - (CH2) that has been substituted or not substituted n S(C2-C6)alkenyl-NR 18 - (CH2) that has been substituted or not substituted n C(O)(CH2) n S(C2-C6)alkenyl-NR 18 - Substituted or unsubstituted -(C2-C6)-ynyl-NR 18 - (CH2) that has been substituted or not substituted n O(C2-C6) ynyl-NR 18- (CH2) that has been substituted or not substituted n C(O)(C2-C6)alkynyl-NR 18 - (CH2) that has been substituted or not substituted n C(O)O(C2-C6)alkynyl-NR 18 - (CH2) that has been substituted or not substituted n OC(O)(C2-C6) ynyl-NR 18 - (CH2) that has been substituted or not substituted n NH(C2-C6) alkyne-NR 18 - (CH2) that has been substituted or not substituted n C(O)NH(C2-C6)alkynyl-NR 18 - (CH2) that has been substituted or not substituted n S(C2-C6) ynyl-NR 18 - (CH2) that has been substituted or not substituted n C(O)(CH2) n S(C2-C6) ynyl-NR 18 - Substituted or unsubstituted -(C1-C6)alkyl-C(O) - Substituted or unsubstituted -(CH2) n O(C1-C6)alkyl-C(O)-, substituted or unsubstituted -(CH2) n C(O) (C1-C6)alkyl-C(O)-, substituted or unsubstituted -(CH2) n C(O)O(C1-C6)alkyl-C(O)-, substituted or unsubstituted -(CH2) n OC(O)(C1-C6)alkyl-C(O)-, substituted or unsubstituted -(CH2) n NH(C1-C6)alkyl-C(O)-, substituted or unsubstituted -(CH2) n C(O)NH(C1-C6)alkyl-C(O)-, substituted or unsubstituted -(CH2) n S(C1-C6)alkyl-C(O)-, substituted or unsubstituted -(CH2) n C(O)(CH2) n S(C1-C6)alkyl-C(O)-, substituted or unsubstituted -(C2-C6)alkenyl-C(O)-, substituted or unsubstituted -(CH2) n O(C2-C6) alkenyl-C(O)-, substituted or unsubstituted -(CH2) nC(O)(C2-C6)alkenyl-C(O)-, substituted or unsubstituted -(CH2) n C(O)O(C2-C6)alkenyl-C(O)-, substituted or unsubstituted -(CH2) n OC(O)(C2-C6)alkenyl-C(O)-, substituted or unsubstituted -(CH2) n NH(C2-C6)alkenyl-C(O)-, substituted or unsubstituted -(CH2) n C(O)NH(C2-C6)alkenyl-C(O)-, substituted or unsubstituted -(CH2) n S(C2-C6)alkenyl-C(O)-, substituted or unsubstituted -(CH2) n C(O)(CH2) n S(C2-C6)alkenyl-C(O)-, substituted or unsubstituted -(C2-C6)ynyl-C(O)-, substituted or unsubstituted -(CH2) n O(C2-C6) ynyl group -C(O)-, substituted or unsubstituted -(CH2) n C(O)(C2-C6) ynyl group -C(O)-, substituted or unsubstituted -(CH2) n C(O)O(C2-C6) ynyl group -C(O)-, substituted or unsubstituted -(CH2) n OC(O)(C2-C6) ynyl group -C(O)-, substituted or unsubstituted -(CH2) n NH(C2-C6) ynyl group -C(O)-, substituted or unsubstituted -(CH2) n C(O)NH(C2-C6) ynyl group -C(O)-, substituted or unsubstituted -(CH2) n S(C2-C6) ynyl group -C(O)-, substituted or unsubstituted -(CH2) n C(O)(CH2) n S(C2-C6)alkynyl-C(O)-, wherein each alkyl group, alkenyl group and alkynyl group may optionally be substituted with the following: alkyl, alkoxy, amino, hydroxyl, mercapto, halogen, carboxyl, oxo, cyano, nitro or trifluoromethyl.

[0222] Each m can independently be an integer selected from 0, 1, 2, 3, 4, 5, and 6; each n can independently be an integer selected from 0, 1, 2, 3, 4, 5, and 6; R 6 It is hydrogen or alkyl; R 7 and R 8Each group is independently selected from hydrogen, hydroxyl, alkyl, alkoxy, cyano, alkylthio, amino, alkylamino, and OPG, where OPG is a protecting group; R 9 R 10 and R 11 Each group is independently selected from hydrogen, hydroxyl, alkyl, alkoxy, cyano, alkylthio, amino and alkylamino, and OPG, where OPG is a protecting group.

[0223] The effector domain can have equation (A):

[0224]

[0225] R 12 R 14 R 16 and R 18 Each of these can be independently hydrogen, a substituted or unsubstituted alkyl group, a substituted or unsubstituted alkenyl group, a substituted or unsubstituted alkynyl group, a substituted or unsubstituted cycloalkyl group, a substituted or unsubstituted perfluoroalkyl group, a substituted or unsubstituted alkoxy group, a substituted or unsubstituted alkylamino group, a substituted or unsubstituted aryl group, a substituted or unsubstituted alkylaryl group, a substituted or unsubstituted heteroalkyl group, a substituted or unsubstituted heterocyclic alkyl group, a substituted or unsubstituted heteroaryl group, a substituted or unsubstituted heteroalkylaryl group, or (CH2). n CN, (CH2) n CF3, (CH2) n C2F5.

[0226] R 13 R 15 and R 17 Each of these can be a side chain of a naturally occurring amino acid and its modified form, including but not limited to the D-amino acid configuration, or hydrogen, halogen, amino, cyano, nitro, trifluoromethyl, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted perfluoroalkyl, substituted or unsubstituted alkoxy, substituted or unsubstituted alkylamino, substituted or unsubstituted alkylthio, substituted or unsubstituted aryl, substituted or unsubstituted alkylaryl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heterocyclic, substituted or unsubstituted heteroaryl, substituted or unsubstituted heteroalkylaryl, substituted or unsubstituted (CH2). n -Aryl, substituted or unsubstituted (CH2) n -Heteroaryl, (CH2) n CN, (CH2) nCF3, (CH2) n C2F5, (CH2) n OR 19 (CH2) n C(O)R 19 (CH2) n C(O)OR 19 (CH2) n OC(O)R 19 (CH2) n NR 20 R 21 (CH2) n C(O)NR 20 R 21 (CH2) n NR 22 C(O)R 19 (CH2) n NR 22 C(O)OR 19 (CH2) n NR 22 C(O)NR 20 R 21 (CH2) n SR 19 (CH2) n S(O) j NR 20 R 21 (CH2) n NR 22 S(O) j R 19 Or -(CH2) n NR 22 S(O) j NR 20 R 21 .

[0227] R 12 and R 13 R 14 and R 15 R 16 and R 17 They can be covalently linked to form substituted or unsubstituted 5-, 6-, or 7-membered heterocycles. Each k can independently be an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. Each j can independently be an integer selected from 0, 1, and 2. R 19 R 20 R 21 and R 22Each can be hydrogen, halogen, amino, cyano, nitro, trifluoromethyl, alkyl, alkenyl, alkynyl, cycloalkyl, perfluoroalkyl, alkoxy, alkylamino, alkylthio, aryl, alkylaryl, heteroalkyl, heterocyclic, heteroaryl, or heteroalkylaryl.

[0228] Or R 19 and R 22 As described above, and R 20 and R 21 Together with the N atom to which they are attached, they form substituted or unsubstituted 5-, 6-, or 7-membered heterocyclic hydrocarbon groups or substituted or unsubstituted 5-membered heteroaryl groups, wherein for R 13 R 15 and R 17 Each of the listed groups may optionally be independently substituted by one to three groups selected from the following: halogen, amino, cyano, nitro, trifluoromethyl, alkyl, alkenyl, ynyl, cycloalkyl, perfluoroalkyl, alkoxy, alkylamino, alkylthio, aryl, alkylaryl, heteroalkyl, heterocyclic, heteroaryl, heteroalkylaryl, (CH2) n CN, (CH2) n CF3, (CH2) n C2F5, (CH2) n OR 19 (CH2) n C(O)R 19 (CH2) n C(O)OR 19 (CH2) n OC(O)R 19 (CH2) n NR 20 R 21 (CH2) n C(O)NR 20 R 21 (CH2) n NR 22 C(O)R 19 (CH2) n NR 22 C(O)OR 19 (CH2) n NR 22 C(O)NR 20 R 21 (CH2) n SR 19 (CH2) n S(O) j NR 20 R 21 (CH2) nNR 22 S(O) j R 19 Or -(CH2) n NR 22 S(O) j NR 20 R 21 .

[0229] Alternatively, the effector domain can have equation (B):

[0230]

[0231] Each k can be an integer independently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; R 23 It can be hydrogen or alkyl; X3 can be substituted or unsubstituted -(C1-C2) 30 Alkyl-, alkenyl-, ynyl-, wherein each carbon is individually assumed to be one of the following redox states: CH2, CH-OH, C(O);

[0232] Alternatively, the effector domain can have equation (C):

[0233]

[0234] X4 can be substituted or unsubstituted -(C1-C 30 Alkyl-, alkenyl-, alkynyl-, wherein each carbon is individually assumed to be one of the following redox states: CH2, CH-OH, C(O).

[0235] Or the effector domain has equation (D):

[0236]

[0237]

[0238] R 24 and R 25 Each is either hydrogen or alkyl; X5 can be substituted or unsubstituted -(C1-C2) 30 Alkyl-, alkenyl-, alkynyl-, wherein each carbon is individually assumed to be one of the following redox states: CH2, CH-OH, C(O).

[0239] Alternatively, the effector domain can be equation (E):

[0240]

[0241] X6 can be substituted or not substituted -(C1-C 30Alkyl-, alkenyl-, alkynyl-, wherein each carbon is individually assumed to be one of the following redox states: CH2, CH-OH, C(O).

[0242] In some implementations, L3 is not Where R 26 It is hydrogen or alkyl.

[0243] In some implementations, R is not Where R 3 It is hydrogen, hydroxyl, or OPG, where PG is a protecting group or in It is a resin; where R 2 It is hydrogen, hydroxyl, or alkoxy; and R is wherein 1 R 4 and R 5 Each is independently hydrogen or unsubstituted, as specified by chemical bonding; among which It is either a single bond or a double bond.

[0244] In some embodiments, L1 and L2 are independently not direct bonds, substituted or unsubstituted -(C1-C6)alkyl-, substituted or unsubstituted -(CH2). n O(C1-C6)alkyl-, substituted or unsubstituted -(CH2) n C(O)-, substituted or unsubstituted -(CH2) n C(O) (C1-C6)alkyl-, substituted or unsubstituted -(CH2) n C(O)O(C1-C6)alkyl-, substituted or unsubstituted-(CH2) n NH(C1-C6)alkyl-, substituted or unsubstituted -(CH2) n C(O)NH(C1-C6)alkyl-, substituted or unsubstituted-(CH2) n S(C1-C6)alkyl-, substituted or unsubstituted -(CH2) n C(O)(CH2) n S(C1-C6)alkyl-, substituted or unsubstituted -(C2-C6)alken-, substituted or unsubstituted -(CH2) n O(C2-C6) alkenyl-, substituted or unsubstituted -(CH2) n C(O)(C2-C6)alkenyl-, substituted or unsubstituted -(CH2) n C(O)O(C2-C6)alkenyl-, substituted or unsubstituted-(CH2) nNH(C2-C6) alkenyl-, substituted or unsubstituted -(CH2) n C(O)NH(C2-C6)alkenyl-, substituted or unsubstituted -(CH2) n S(C2-C6) alkenyl-, substituted or unsubstituted -(CH2) n C(O)(CH2) n S(C2-C6)alkenyl-, substituted or unsubstituted -(C2-C6)ynyl-, substituted or unsubstituted -(CH2) n O(C2-C6) ynyl group -, substituted or unsubstituted -(CH2) n C(O)(C2-C6) ynyl group -, substituted or unsubstituted -(CH2) n C(O)O(C2-C6) ynyl group -, substituted or unsubstituted -(CH2) n NH(C1-C6) ynyl group -, substituted or unsubstituted -(CH2) n C(O)NH(C2-C6) ynyl group -, substituted or unsubstituted -(CH2) n S(C2-C6) ynyl group -, substituted or unsubstituted -(CH2) n C(O)(CH2) n S(C2-C6)alkynyl-, wherein each alkyl group, alkenyl group and alkynyl group may optionally be substituted with alkyl, alkoxy, amino, carboxyl, cyano, nitro or trifluoromethyl.

[0245] In some implementations, the effector domain is a compound of formula (F).

[0246]

[0247] R 12 R 14 R 14 '、R 16 and R 27 Each is independently neither hydrogen nor alkyl, and R 13 R 14 R 14 'and R 16Each of the following is independently not hydrogen, halogen, amino, cyano, nitro, trifluoromethyl, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted perfluoroalkyl, substituted or unsubstituted alkoxy, substituted or unsubstituted alkylamino, substituted or unsubstituted alkylthio, substituted or unsubstituted aryl, substituted or unsubstituted alkylaryl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heterocyclic, substituted or unsubstituted heteroaryl, substituted or unsubstituted heteroalkylaryl, (CH2) n CN, (CH2) n CF3, (CH2) n C2F5, (CH2) n OR 19 (CH2) n C(O)R 19 (CH2) n C(O)OR 19 (CH2) n OC(O)R 19 (CH2) n NR 20 R 21 (CH2) n C(O)NR 20 R 21 (CH2) n NR 22 C(O)R 19 (CH2) n NR 22 C(O)OR 19 (CH2) n NR 22 C(O)NR 20 R 21 (CH2) n S(O) j NR 20 R 21 (CH2) n NR 22 S(O) j R 19 Or -(CH2) n NR 22 S(O) j NR 20 R 21 n is an integer selected from 0, 1, 2, 3, 4, 5, and 6; j is an integer selected from 0, 1, and 2.

[0248] R 19 R20 R 21 and R 22 Each of the following is independently hydrogen, halogen, amino, cyano, nitro, trifluoromethyl, alkyl, alkenyl, alkynyl, cycloalkyl, perfluoroalkyl, alkoxy, alkylamino, alkylthio, aryl, alkylaryl, heteroalkyl, heterocyclic, heteroaryl or heteroalkylaryl, or R 19 and R 22 As described above, and R 20 and R 21 Together with the N atom to which they are attached, they form substituted or unsubstituted 5-, 6-, or 7-membered heterocyclic hydrocarbon groups or substituted or unsubstituted 5-membered heteroaryl groups.

[0249] For R 13 R 15 and R 17 Each of the listed groups may optionally be independently substituted by one to three groups selected from the following: halogen, amino, cyano, nitro, trifluoromethyl, alkyl, alkenyl, ynyl, cycloalkyl, perfluoroalkyl, alkoxy, alkylamino, alkylthio, aryl, alkylaryl, heteroalkyl, heterocyclic, heteroaryl, heteroalkylaryl, (CH2) n CN, (CH2) n CF3, (CH2) n C2F5, (CH2) n OR 19 (CH2) n C(O)R 19 (CH2) n C(O)OR 19 (CH2) n OC(O)R 19 (CH2) n NR 20 R 21 (CH2) n C(O)NR 20 R 21 (CH2) n NR 22 C(O)R 19 (CH2) n NR 22 C(O)OR 19 (CH2) n NR 22 C(O)NR 20 R 21 (CH2) n SR 19 (CH2) n S(O) j NR 20R 21 (CH2) n NR 22 S(O) j R 19 Or -(CH2) n NR 22 S(O) j NR 20 R 21 .

[0250] In some implementations, L3 in formula (VII) is -CH2CH2–, and R is R 1 R 4 R 5 and R 6 Each is hydrogen; R 2 and R 3 Each is a methoxy group; m = 0; Y is X2 is O or NR 6 C(O); L1 is –CH2-C(O)- or -(CH2)2C(O)-; Z is L2 is –OCO-CH=CH-(CH2)2N(Me)-. In some embodiments, X2 is O, and L1 is -CH2-C(O)-. In some embodiments, X2 is NR. 6 C(O), and L1 is -(CH2)2C(O)-.

[0251] In some implementations, the effector domain can be of equation (G).

[0252]

[0253] Where R 12 R 14 R 14′ and R 16 Each of the following is independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted perfluoroalkyl, substituted or unsubstituted alkoxy, substituted or unsubstituted alkylamino, substituted or unsubstituted aryl, substituted or unsubstituted alkylaryl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heterocyclic, substituted or unsubstituted heteroaryl, substituted or unsubstituted heteroalkylaryl, (CH2) n CN, (CH2) n CF3, (CH2) n C2F5.

[0254] R 13 R15 R 15 'and R 17 Each of these can be a side chain of a naturally occurring amino acid and its modified form, including but not limited to the D-amino acid configuration, or hydrogen, halogen, amino, cyano, nitro, trifluoromethyl, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted perfluoroalkyl, substituted or unsubstituted alkoxy, substituted or unsubstituted alkylamino, substituted or unsubstituted alkylthio, substituted or unsubstituted aryl, substituted or unsubstituted alkylaryl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heterocyclic, substituted or unsubstituted heteroaryl, substituted or unsubstituted heteroalkylaryl, substituted or unsubstituted (CH2). n -Aryl, substituted or unsubstituted (CH2) n -Heteroaryl, (CH2) n CN, (CH2) n CF3, (CH2) n C2F5, (CH2) n OR 19 (CH2) n C(O)R 19 (CH2) n C(O)OR 19 (CH2) n OC(O)R 19 (CH2) n NR 20 R 21 (CH2) n C(O)NR 20 R 21 (CH2) n NR 22 C(O)R 19 (CH2) n NR 22 C(O)OR 19 (CH2) n NR 22 C(O)NR 20 R 21 (CH2) n SR 19 (CH2) n S(O) j NR 20 R 21 (CH2) n NR 22 S(O) j R19 Or -(CH2) n NR 22 S(O) j NR 20 R 21 .

[0255] R 12 and R 13 R 14 and R 15 R 14 'and R 15 '、R 16 and R 17 They can be covalently linked to form substituted or unsubstituted 5-, 6-, or 7-membered heterocycles.

[0256] In some embodiments, this document discloses a method for screening compounds for treating cancer using a heterozygous cyclic library of the immunoavidin ligand family based on the natural product FK506 and rapamycin. In some embodiments, this document discloses a method for screening compounds for treating autoimmune diseases using a heterozygous cyclic library of the immunoavidin ligand family based on the natural product FK506 and rapamycin.

[0257] Macrocyclic natural products FK506 and rapamycin are approved immunosuppressive drugs with important biological activities. Both have been shown to inhibit T cell activation, each with a different mechanism. Furthermore, rapamycin has been shown to have potent antiproliferative activity. FK506 and rapamycin share an unusual mode of action; they act by recruiting the abundantly and ubiquitously expressed cellular protein prolyl cis-trans isomerase FKBP, and the binary complex subsequently binds to and allosterically inhibits their target proteins calcineurin and mTOR, respectively. Structurally, FK506 and rapamycin share a similar FKBP-binding domain but differ in their effector domains. In both FK506 and rapamycin, nature has taught us to convert the effector domain of FK506 to that of rapamycin, which can change the target from calcineurin to mTOR. The generation of macrocyclic Rapafucin libraries containing the binding domains of FK506 and rapamycin should have great potential as novel lead compounds for the development of drugs to treat diseases.

[0258] There are various methods for generating compound libraries for the development and screening of compounds that may be useful in treating diseases. One such method is the development of encoded libraries, and particularly libraries in which each compound includes an amplifiable tag. Such libraries include DNA-encoded libraries, where the DNA tags identifying library members can be amplified using molecular biology techniques such as polymerase chain reaction (PCR). The use of such methods for generating macrocyclic Rapafucin libraries containing both FK506-like binding domains and rapamycin-like binding domains has not yet been demonstrated. Therefore, there remains a need for DNA-encoded Rapafucin libraries containing macrocyclic FK506-like binding domains and rapamycin-like binding domains.

[0259] In one aspect, this document provides a labeled macrocyclic compound comprising: an FK506-binding protein-binding domain (FKBD); an effector domain; a first linker region; and a second linker region; wherein the FKBD, the effector domain, the first linker region, and the second linker region together form a macrocycle; and wherein at least one of the FKBD, the effector domain, the first linker region, and the second linker region can be operatively linked to one or more oligonucleotides (D), said oligonucleotides (D) being capable of identifying the structure of at least one of the FKBD, the effector domain, the first linker region, and the second linker region.

[0260] In some embodiments, this document provides macrocyclic compounds labeled with formula (IX):

[0261]

[0262] In some embodiments, h, i, j, and k are each independently an integer from 0 to 20, provided that at least one of h, i, j, and k is not 0; and D is an oligonucleotide capable of identifying at least one of FKBD, effector domain, linker region A, or linker region Z, wherein a solid line connecting FKBD, effector domain, linker region A, and / or linker region Z indicates an operable link, and a wavy line indicates an operable link. In some embodiments, the oligonucleotide (D) can be operablely linked to at least one of FKBD, effector domain, linker region A, or linker region Z.

[0263] In some embodiments, this document provides macrocyclic compounds of formula (X) labeled with the following formula: (1) A pharmaceutically acceptable salt, solvate, or stereoisomer thereof.

[0264]

[0265] In some embodiments, ring A is a 5- to 10-membered aryl, cycloalkyl, heteroaryl, or heterocyclic alkyl group optionally substituted with 1 to 17 substituents, each of which is independently selected from the group consisting of: hydrogen, hydroxyl, halogen, alkyl, alkoxy, cyano, haloalkyl, haloalkoxy, alkylthio, oxo, amino, alkylamino, dialkylamino, in It is a resin; J is independently selected from the following group each time it appears: -C(O)NR 6 -,

[0266]

[0267] Where R 6 Each is hydrogen, alkyl, arylalkyl, Where R N R is aryl, alkyl, or arylalkyl; R' is hydrogen, alkyl, arylalkyl, or haloalkyl; D is an oligonucleotide each time it appears; L b and L c Each time it appears, it is independently selected from the following groups: bond, -O-, -S-, -OC(O)-, -C(O)O-, -(CH2). n C(O)-、-(CH2) n C(O)C(O)-、-(CH2) n NR 5 C(O)C(O)-、-NR 5 (CH2) n C(O)C(O)-, optionally substituted (CH2) n C 1-6 Alkylene (CH2) n - Optionally substituted (CH2) n C(O)C 1-6 Alkylene (CH2) n - Optionally substituted (CH2) n NR 5 C 1-6 Alkylene (CH2) n - Optionally substituted (CH2) n C(O)NR 5 C 1-6 Alkylene (CH2) n - Optionally substituted (CH2) n NR 5 C(O)C 1-6 Alkylene (CH2) n - Optionally substituted (CH2) n C(O)OC1-6 Alkylene (CH2) n - Optionally substituted (CH2) n OC(O)C 1-6 Alkylene (CH2) n - Optionally substituted (CH2) n OC 1-6 Alkylene (CH2) n - Optionally substituted (CH2) n NR 5 C 1-6 Alkylene (CH2) n - Optionally substituted (CH2) n -SC 1-6 Alkylene (CH2) n - and optionally substituted (CH2CH2O) n Each alkylene group is optionally substituted by one or two independently selected groups from the group consisting of: halo, hydroxy, haloalkyl, haloalkoxy, alkyl, alkoxy, amino, carboxyl, cyano, nitro, NHFmoc; wherein each R 5 Independently hydrogen, alkyl, arylalkyl, Where R N It is aryl, alkyl, or arylalkyl; X is O, S, or NR. 8 , where R 8 It is hydrogen, hydroxyl, OR 9 NR 10 R 11 alkyl, arylalkyl Where R N It is aryl, alkyl, or arylalkyl; wherein R 9 R 10 and R 11 Each is independently hydrogen or alkyl; V 1 and V 2 Each independently is W is Wherein ring B is a 4- to 10-membered heterocyclic hydrocarbon group optionally substituted with 1 to 10 substituents, each of which is selected from the group consisting of: hydrogen, hydroxyl, halogen, alkyl, alkoxy, cyano, haloalkyl, haloalkoxy, alkylthio, oxo, amino, alkylamino, dialkylamino, arylalkyl, Where R 12 It is aryl, alkyl, or arylalkyl; wherein R 13 It is hydrogen, hydroxyl, OR 16 NR 17 R 18 alkyl, arylalkyl Where R N It is aryl, alkyl, or arylalkyl; R 14 and R 15 Each is independently hydrogen, hydroxyl, halogen, alkyl, alkoxy, haloalkyl, haloalkoxy, aryl, arylalkyl, or heteroaryl; Z is a bond, Where R 16 and R 17 Each is independently selected from the following groups: hydrogen, hydroxyl, halogen, alkyl, alkoxy, cycloalkyl, cyano, alkylthio, amino, alkylamino, and dialkylamino; K is O, CHR 18 CR 18 , N or / and NR 18 , where R 18 It is hydrogen or alkyl;

[0268] L a L 1 L 2 L 3 L 4 L 5 L 6 L 7 and L 8 Each is an independent key, -O-, -NR 19 -、-SO-、-SO2-、(CH2) n -、 Alternatively, the linking groups may be selected from those in Table 1; wherein the ring C is a 5- to 6-membered heteroaryl group optionally substituted with 1 to 4 substituents, each of which is independently selected from the group consisting of: hydrogen, hydroxyl, halogen, alkyl, alkoxy, haloalkyl, haloalkoxy, cyano, alkylthio, amino, alkylamino, dialkylamino, and Each R 19 R 20 and R 21 Independently select from the following groups: hydrogen, hydroxyl, OR 22 NR 23 R 24 alkyl, arylalkyl Where R N It is aryl, alkyl, or arylalkyl; wherein R 22 R 23 and R 24 Each is independently either hydrogen or alkyl;

[0269] n is 0, 1, 2, 3, 4, 5, or 6; where the effector structural domain has equation (Xa):

[0270]

[0271] In some implementations, each k a k b k c k d k e k f k g k h and k i Independently, it is either 0 or 1; each X a X b X c X d X e X f X g X h and X i Independently, it is a bond, -S-, -SS-, -S(O)-, -S(O)2-, substituted or unsubstituted -(C1-C3)alkylene-, -(C2-C4)alkenylene-, -(C2-C4)ynylene- or Wherein ring E is phenyl or 5-6-membered heteroaryl or heterocyclic hydrocarbon group; wherein each w is independently 0, 1 or 2; each R 1 R 1a R 1b R 1c R 1d R 1e R 1f R 1g R 1h R 1i and R 4 It is independently hydrogen, alkyl, arylalkyl, or NR 25 , where R 25 It is hydrogen, hydroxyl, OR 26 NR 27 R 28 alkyl, arylalkyl Where R N It is aryl, alkyl, or arylalkyl; wherein R 26 R 27 and R 28 Each is independently hydrogen or alkyl; each R 2 R 3 R 2a R 3a R 2b R 3b R 2c R 3c R 2d R 3d R 2e R 3e R2f R 3f R 2g R 3g R 2h R 3h R 2i and R 3i Independently selected from the group consisting of: hydrogen, halogen, amino, cyano, nitro, haloalkyl, optionally substituted alkyl, optionally substituted alkoxy, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted cyclic hydrocarbon, optionally substituted heterocyclic hydrocarbon, optionally substituted alkylamino, optionally substituted dialkylamino, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted arylalkyl, optionally substituted heteroarylalkyl, and...

[0272] Or, the effector domain may have the following formula (Xb):

[0273]

[0274] Among them AA 1 AA 2 ... and AA r Each of these is a natural or non-natural amino acid residue; and r is 3, 4, 5, 6, 7, 8, 9, or 10;

[0275] Or, the effector domain may have the following formula (Xc):

[0276]

[0277] Each t is independently an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; R 29 It is hydrogen, hydroxyl, OR 30 NR 31 R 32 alkyl, arylalkyl Where R N It is aryl, alkyl, or arylalkyl; wherein R 30 R 31 and R 32 Each is independently hydrogen or alkyl; X 3 It is a substituted or unsubstituted -(C1-C6)alkylene-, -(C2-C6)alkenyl-, -(C2-C6)ynylene- or Wherein ring E is phenyl or 5-6-membered heteroaryl or heterocyclic hydrocarbon group; wherein each w is independently 0, 1 or 2;

[0278] Or, the effector domain may have the following formula (Xd):

[0279]

[0280] Where X 4 It is a substituted or unsubstituted -(C1-C6)alkylene-, -(C2-C6)alkenyl-, -(C2-C6)ynylene- or Wherein ring E is phenyl or 5-6-membered heteroaryl or heterocyclic hydrocarbon group; wherein each w is independently 0, 1 or 2;

[0281] Or, the effector domain may have the following formula (Xe):

[0282]

[0283] Where R 33 R 34 R 35 and R 36 Each is either hydrogen or alkyl; X 5 It is a substituted or unsubstituted -(C1-C6)alkylene-, -(C2-C6)alkenyl-, -(C2-C6)ynylene- or Wherein ring E is phenyl or 5-6-membered heteroaryl or heterocyclic hydrocarbon group; wherein each w is independently 0, 1 or 2;

[0284] Or, the effector domain may have the following formula (Xf):

[0285]

[0286] X 6 It is a substituted or unsubstituted -(C1-C6)alkylene-, -(C2-C6)alkenyl-, -(C2-C6)ynylene- or Wherein ring E is phenyl or a 5- or 6-membered heteroaryl or heterocyclic hydrocarbon group; wherein each w is independently 0, 1, or 2; the condition is that when R is L is ethylene, X is O, and W is... V is Z is -L 6 -L 7 -L 8 -yes Then -L 1 -L 2 -L 3 -L 4 -L 5 -no And ring A is at least one Replacement, or R 2 R 3 R 2a R 3a R 2b R3b R 2c R 3c R 2d R 3d R 2e R 3e R 2f R 3f R 2g R 3g R 2h R 3h R 2i and R 3i At least one of them is or L a L 1 L 2 L 3 L 4 L 5 L 6 L 7 and L 8 At least one of them is by at least one The substituted ring C, or at least one of the linking groups selected from Table 1, is substituted by at least one replace.

[0287] In another aspect, this document provides a compound library containing more than one macrocyclic compound labeled differently according to any of the foregoing. In some embodiments, this document provides a compound library containing at least about 10 2 A number of macrocyclic compounds labeled differently according to any of the above. In some embodiments, this document provides a compound library containing approximately 10 2 Seed to about 10 10 A macrocyclic compound labeled according to any of the above.

[0288] In another aspect, this document provides a method for preparing a library of labeled macrocyclic compounds as disclosed herein, the method comprising synthesizing more than one macrocyclic compound labeled differently according to any of the foregoing.

[0289] In another aspect, this document provides a method for preparing labeled macrocyclic compounds as disclosed herein, the method comprising operatively linking at least one oligonucleotide (D) to at least one of an FKBD, an effector domain, a first linker region, and a second linker region, and forming a macrocycle comprising the FKBD, the effector domain, the first linker region, and the second linker region.

[0290] In some embodiments, this document provides a method for preparing a labeled macrocyclic compound as disclosed herein, the method comprising operably linking the macrocyclic compound to at least one oligonucleotide (D), the macrocyclic compound comprising an FKBD, an effector domain, a first linker region, and a second linker region, wherein the FKBD, the effector domain, the first linker region, and the second linker region together form a macrocycle; and wherein at least one oligonucleotide (D) is recognizable by the structure of at least one of the FKBD, the effector domain, the first linker region, and the second linker region.

[0291] In another aspect, a method for preparing labeled macrocyclic compounds includes: taking a compound of formula (XI):

[0292]

[0293] Compounds operably linked to formula (XII):

[0294] Q′-L c -D

[0295] Formula (XII).

[0296] In some implementation schemes, and Each occurrence is independent of: key, -O-, -NR 19 -、-SO-、-SO2-、-(CH2) n -、 Alternatively, the linking groups may be selected from those in Table 1, wherein the ring C is a 5- to 6-membered heteroaryl group optionally substituted with 1 to 4 substituents, each of which is independently selected from the group consisting of: hydrogen, hydroxyl, halogen, alkyl, alkoxy, haloalkyl, haloalkoxy, cyano, alkylthio, amino, alkylamino, dialkylamino; wherein R 19 Choose from the following groups: hydrogen, hydroxyl, OR 22 NR 23 R 24 alkyl, arylalkyl Where R N It is aryl, alkyl, or arylalkyl; wherein R 22 R 23 and R 24 Each is independently hydrogen or alkyl; Q and Q' are each independently selected from the group consisting of: N3, -C≡CH, NR 6 R 7 ,-COOH,-ONH2,-SH,-NH2, -(C=O)R',

[0297] Where R 6and R 7 Each is independently hydrogen, alkyl, arylalkyl, Where R N It is aryl, alkyl, or arylalkyl; and R' is hydrogen, alkyl, arylalkyl, or haloalkyl; L b and L c Each time it appears, it is independently selected from the following groups: bond, -O-, -S-, -OC(O)-, -C(O)O-, -(CH2). n C(O)-、-(CH2) n C(O)C(O)-、-(CH2) n NR 5 C(O)C(O)-、-NR 5 (CH2) n C(O)C(O)-, optionally substituted (CH2) n C 1-6 Alkylene-(CH2) n - Optionally substituted (CH2) n C(O)C 1-6 Alkylene-(CH2) n - Optionally substituted (CH2) n NR 5 C 1-6 Alkylene-(CH2) n - Optionally substituted (CH2) n C(O)NR 5 C 1-6 Alkylene-(CH2) n - Optionally substituted (CH2) n NR 5 C(O)C 1-6 Alkylene-(CH2) n - Optionally substituted (CH2) n C(O)OC 1-6 Alkylene-(CH2) n - Optionally substituted (CH2) n OC(O)C 1-6 Alkylene-(CH2) n - Optionally substituted (CH2) n OC 1-6 Alkylene-(CH2) n - Optionally substituted (CH2) n NR 5 C 1-6 Alkylene-(CH2) n - Optionally substituted (CH2) n -SC 1-6Alkylene-(CH2) n - and optionally substituted (CH2CH2O) n Each alkylene group is optionally substituted by one or two independently selected groups from the group consisting of: halo, hydroxy, haloalkyl, haloalkoxy, alkyl, alkoxy, amino, carboxyl, cyano, nitro, NHFmoc; wherein each R 5 Independently, it is hydrogen, alkyl, arylalkyl, Where R N It is aryl, alkyl, or arylalkyl;

[0298] D is an oligonucleotide; h, i, j, and k are each independent integers from 0 to 20, provided that at least one of h, i, j, and k is not 0; n is an integer from 1 to 5; m is an integer from 1 to 5.

[0299] In another aspect, this paper provides a method for preparing labeled macrocyclic compounds, the method comprising: a compound of formula (X):

[0300]

[0301] Compounds operably linked to formula (XII):

[0302] Q′-L c -D

[0303] Formula (XII),

[0304] Ring A is a 5- to 10-membered aryl, cycloalkyl, heteroaryl, or heterocyclic alkyl group optionally substituted with 1 to 17 substituents, each of which is independently selected from the group consisting of: hydrogen, hydroxyl, halogen, alkyl, alkoxy, cyano, haloalkyl, haloalkoxy, alkylthio, oxo, amino, alkylamino, dialkylamino. in It is resin;

[0305] L b and L c Choose independently from the following groups: bond, -O-, -S-, -OC(O)-, -C(O)O-, -(CH2) n C(O)-、-(CH2) n C(O)C(O)-、-(CH2) n NR 5 C(O)C(O)-、-NR 5 (CH2) n C(O)C(O)-, optionally substituted (CH2) n C 1-6 Alkylene-(CH2)n - Optionally substituted (CH2) n C(O)C 1-6 Alkylene-(CH2) n - Optionally substituted (CH2) n NR 5 C 1-6 Alkylene-(CH2) n - Optionally substituted (CH2) n C(O)NR 5 C 1-6 Alkylene-(CH2) n - Optionally substituted (CH2) n NR 5 C(O)C 1-6 Alkylene-(CH2) n - Optionally substituted (CH2) n C(O)OC 1-6 Alkylene-(CH2) n - Optionally substituted (CH2) n OC(O)C 1-6 Alkylene-(CH2) n - Optionally substituted (CH2) n OC 1-6 Alkylene-(CH2) n - Optionally substituted (CH2) n NR 5 C 1-6 Alkylene-(CH2) n - Optionally substituted (CH2) n -SC 1-6 Alkylene-(CH2) n - and optionally substituted (CH2CH2O) n Each alkylene group is optionally substituted by one or two independently selected groups from the group consisting of: halo, hydroxy, haloalkyl, haloalkoxy, alkyl, alkoxy, amino, carboxyl, cyano, nitro, NHFmoc; wherein each R 5 Independently, it is hydrogen, alkyl, arylalkyl, Where R N It is aryl, alkyl, or arylalkyl;

[0306] Q and Q' are independently chosen from the following groups: -N3, -C≡CH, NR 6 R 7 ,-COOH,-ONH2,-SH,-NH2, -(C=O)R',

[0307] Where R 6 and R 7 Each is independently hydrogen, alkyl, arylalkyl, Where R N It is aryl, alkyl, or arylalkyl; and R' is hydrogen, alkyl, arylalkyl, or haloalkyl; X is O, S, or NR. 8 , where R 8 It is hydrogen, hydroxyl, OR 9 NR 10 R 11 alkyl, arylalkyl Where R N It is aryl, alkyl, or arylalkyl; wherein R 9 R 10 and R 11 Each is independently hydrogen or alkyl; V 1 and V 2 Each independently is W is Wherein ring B is a 4- to 10-membered heterocyclic hydrocarbon group optionally substituted with 1 to 10 substituents, each of which is selected from the group consisting of: hydrogen, hydroxyl, halogen, alkyl, alkoxy, cyano, haloalkyl, haloalkoxy, alkylthio, oxo, amino, alkylamino, dialkylamino, arylalkyl, Where R 12 It is aryl, alkyl, or arylalkyl; wherein R 13 It is hydrogen, hydroxyl, OR 16 NR 17 R 18 alkyl, arylalkyl Where R N It is aryl, alkyl, or arylalkyl; R 14 and R 15 Each of them is independently hydrogen, hydroxyl, halogen, alkyl, alkoxy, haloalkyl, haloalkoxy, aryl, arylalkyl, or heteroaryl;

[0308] Z is a key. Where R 16 and R 17 Each is independently selected from the following groups: hydrogen, hydroxyl, halogen, alkyl, alkoxy, cycloalkyl, cyano, alkylthio, amino, alkylamino, and dialkylamino; K is O, CHR 18 CR 18 , N and NR 18 , where R 18 It is hydrogen or alkyl.

[0309] L a L 1 L2 L 3 L 4 L 5 L 6 L 7 and L 8 Each is an independent key, -O-, -NR 19 -、-SO-、-SO2-、-(CH2) n -、 Alternatively, the linking groups may be selected from those in Table 1; wherein the ring C is a 5- to 6-membered heteroaryl group optionally substituted with 1 to 4 substituents, each of which is independently selected from the group consisting of: hydrogen, hydroxyl, halogen, alkyl, alkoxy, haloalkyl, haloalkoxy, cyano, alkylthio, amino, alkylamino, dialkylamino, and Where R 19 Choose from the following groups: hydrogen, hydroxyl, OR 22 NR 23 R 24 alkyl, arylalkyl Where R N It is aryl, alkyl, or arylalkyl; wherein R 22 R 23 and R 24 Each is independently either hydrogen or alkyl;

[0310] n is 0, 1, 2, 3, 4, 5, or 6; where the effector structural domain has equation (Xa):

[0311]

[0312] Each k a k b k c k d k e k f k g k h and k i Independently, it is either 0 or 1; each X a X b X c X d X e X f X g X h and X i Independently, it is a bond, -S-, -SS-, -S(O)-, -S(O)2-, substituted or unsubstituted -(C1-C3)alkylene-, -(C2-C4)alkenylene-, -(C2-C4)ynylene- or Wherein ring E is phenyl or 5-6-membered heteroaryl or heterocyclic hydrocarbon group; wherein each w is independently 0, 1 or 2; each R 1 R 1a R 1b R 1c R 1d R 1e R 1f R 1g R 1h R 1i and R 4 It is independently hydrogen, alkyl, arylalkyl, or NR 25 , where R 25 It is hydrogen, hydroxyl, OR 26 NR 27 R 28 alkyl, arylalkyl Where R N It is aryl, alkyl, or arylalkyl; wherein R 26 R 27 and R 28 Each is independently hydrogen or alkyl; each R 2 R 3 R 2a R 3a R 2b R 3b R 2c R 3c R 2d R 3d R 2e R 3e R 2f R 3f R 2g R 3g R 2h R 3h R 2i and R 3i Independently selected from the group consisting of: hydrogen, halogen, amino, cyano, nitro, haloalkyl, optionally substituted alkyl, optionally substituted alkoxy, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted cyclic hydrocarbon, optionally substituted heterocyclic hydrocarbon, optionally substituted alkylamino, optionally substituted dialkylamino, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted arylalkyl, optionally substituted heteroarylalkyl, and...

[0313] Or, the effector domain may have the following formula (Xb):

[0314]

[0315] Among them AA1 AA 2 ... and AA r Each of these is a natural or non-natural amino acid residue; and r is 3, 4, 5, 6, 7, 8, 9, or 10;

[0316] Or, the effector domain may have the following formula (Xc):

[0317]

[0318] Each t is independently an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; R 29 It is hydrogen, hydroxyl, OR 30 NR 31 R 32 alkyl, arylalkyl Where R N It is aryl, alkyl, or arylalkyl; wherein R 30 R 31 and R 32 Each is independently hydrogen or alkyl; X 3 It is a substituted or unsubstituted -(C1-C6)alkylene-, -(C2-C6)alkenyl-, -(C2-C6)ynylene- or Wherein ring E is phenyl or 5-6-membered heteroaryl or heterocyclic hydrocarbon group; wherein each w is independently 0, 1 or 2;

[0319] Or, the effector domain may have the following formula (Xd):

[0320]

[0321] X 4 It is a substituted or unsubstituted -(C1-C6)alkylene-, -(C2-C6)alkenyl-, -(C2-C6)ynylene- or Wherein ring E is phenyl or 5-6-membered heteroaryl or heterocyclic hydrocarbon group; wherein each w is independently 0, 1 or 2;

[0322] Or, the effector domain may have the following formula (Xe):

[0323]

[0324] R 33 R 34 R 35 and R 36 Each is either hydrogen or alkyl; X 5 It is a substituted or unsubstituted -(C1-C6)alkylene-, -(C2-C6)alkenyl-, -(C2-C6)ynylene- or Wherein ring E is phenyl or 5-6-membered heteroaryl or heterocyclic hydrocarbon group; wherein each w is independently 0, 1 or 2;

[0325] Or, the effector domain may have the following formula (Xf):

[0326]

[0327] X 6 It is a substituted or unsubstituted -(C1-C6)alkylene-, -(C2-C6)alkenyl-, -(C2-C6)ynylene- or Wherein ring E is phenyl or a 5-6 membered heteroaryl or heterocyclic hydrocarbon group; wherein each w is independently 0, 1 or 2; and the condition is that when ring A is At that time, L a It is ethylene, X is O, W is V 1 yes V 2 yes Z is -L 6 -L 7 -L 8 -yes And -L 1 -L 2 -L 3 -L 4 -L 5 -no D is an oligonucleotide; wherein the ring A is bounded by at least one... Replacement, or R 2 R 3 R 2a R 3a R 2b R 3b R 2c R 3c R 2d R 3d R 2e R 3e R 2f R 3f R 2g R 3g R 2h R 3h R 2i and R 3i At least one of them is or L a L 1 L 2 L 3 L 4 L 5 L 6L 7 and L 8 At least one of them is by at least one The substituted ring C, or at least one of the linking groups selected from Table 1, is substituted by at least one replace.

[0328] This article also provides a macrocyclic compound of formula (XIV) or a pharmaceutically acceptable salt, solvate, or stereoisomer thereof:

[0329]

[0330]

[0331] Each n, m, and p can be an integer selected independently from 0 to 5;

[0332] Each of R1, R2, and R3 can be independently selected from the following groups: H, F, Cl, Br, CF3, CN, N3, -N(R 12 )2、-N(R 12 )3、-CON(R 12 2. NO2, OH, OCH3, methyl, ethyl, propyl, -COOH, -SO3H, -PO(OR) 12 )2、-OPO(OR 12 2、-(CH2) q COOH, -O-(CH2) q COOH, -S-(CH2) q COOH, -CO-(CH2) q COOH, -NR 12 -(CH2) q COOH, -(CH2) q SO3H, -O-(CH2) q SO3H, -S-(CH2) q SO3H, -CO-(CH2) q SO3H, -NR 12 -(CH2) q SO3H, -(CH2) q N(R 12 2、-O-(CH2) q N(R 12 2、-S-(CH2) q N(R 12 2、-CO-(CH2) q N(R 12 2、-(CH2) q N(R 12 3、-O-(CH2)q N(R 12 3. -S-(CH2) q N(R 12 3. -CO-(CH2) q N(R 12 3. -NR 12 -(CH2) q N(R 12 3、-(CH2) q CON(R 12 2、-O-(CH2) q CON(R 12 2、-S-(CH2) q CON(R 12 2、-CO-(CH2) q CON(R 12 2、-(CH2) q PO(OR 12 2、-O(CH2) q PO(OR 12 2、-S(CH2) q PO(OR 12 2、-CO(CH2) q PO(OR 12 )2、-NR 12 (CH2) q PO(OR 12 2、-(CH2) q OPO(OR 12 2、-O(CH2) q OPO(OR 12 2、-S(CH2) q OPO(OR 12 2、-CO(CH2) q OPO(OR 12 )2 and -NR 12 (CH2) q OPO(OR 12 )2.

[0333] q can be an integer selected from 0 to 5; each of R4, R5, R6, R7, R9 and R 11 It can be independently selected from the following groups: H, methyl, ethyl, propyl, and isopropyl.

[0334] Each R8 and R 10 It can be independently selected from the following groups: H, halogen, hydroxyl, C. 1-20 Alkyl, N3, NH2, NO2, CF3, OCF3, OCHF2, COC 1-20Alkyl, CO2C 1-20 Alkyl groups, 5- or 6-membered cyclic structures formed with adjacent nitrogen atoms, -N(R) 12 )2、-N(R 12 )3、-CON(R 12 )2、-COOH、-SO3H、-PO(OR 12 )2、-OPO(OR 12 2、-(CH2) q COOH, -O-(CH2) q COOH, -S-(CH2) q COOH, -CO-(CH2) q COOH, -NR 12 -(CH2) q COOH, -(CH2) q SO3H, -O-(CH2) q SO3H, -S-(CH2) q SO3H, -CO-(CH2) q SO3H, -NR 12 -(CH2) q SO3H, -(CH2) q N(R 12 2、-O-(CH2) q N(R 12 2、-S-(CH2) q N(R 12 2、-CO-(CH2) q N(R 12 2、-(CH2) q N(R 12 3、-O-(CH2) q N(R 12 3. -S-(CH2) q N(R 12 3. -CO-(CH2) q N(R 12 3. -NR 12 -(CH2) q N(R 12 3、-(CH2) q CON(R 12 2、-O-(CH2) q CON(R 12 2、-S-(CH2) q CON(R 12 2、-CO-(CH2) q CON(R 12 2、-(CH2)q PO(OR 12 2、-O(CH2) q PO(OR 12 2、-S(CH2) q PO(OR 12 2、-CO(CH2) q PO(OR 12 )2、-NR 12 (CH2) q PO(OR 12 2、-(CH2) q OPO(OR 12 2、-O(CH2) q OPO(OR 12 2、-S(CH2) q OPO(OR 12 2、-CO(CH2) q OPO(OR 12 )2 and -NR 12 (CH2) q OPO(OR 12 )2,

[0335] Each R 12 It can be independently selected from the following groups: H, methyl, ethyl, propyl, and isopropyl.

[0336] The conditions are R2, R3, R8, and R. 10 At least one of them is selected from -N(R) 12 )2、-N(R 12 )3、-CON(R 12 )2、-COOH、-SO3H、-PO(OR 12 )2、-OPO(OR 12 2、-(CH2) q COOH, -O-(CH2) q COOH, -S-(CH2) q COOH, -CO-(CH2) q COOH, -NR 12 -(CH2) q COOH, -(CH2) q SO3H, -O-(CH2) q SO3H, -S-(CH2) q SO3H, -CO-(CH2) q SO3H, -NR 12 -(CH2) q SO3H, -(CH2) q N(R12 )2, -O-(CH2) q N(R 12 )2, -S-(CH2) q N(R 12 )2, -CO-(CH2) q N(R 12 )2, -(CH2) q N(R 12 )3, -O-(CH2) q N(R 12 )3, -S-(CH2) q N(R 12 )3, -CO-(CH2) q N(R 12 )3, -NR 12 -(CH2) q N(R 12 )3, -(CH2) q CON(R 12 )2, -O-(CH2) q CON(R 12 )2, -S-(CH2) q CON(R 12 )2, -CO-(CH2) q CON(R 12 )2, -(CH2) q PO(OR 12 )2, -O(CH2) q PO(OR 12 )2, -S(CH2) q PO(OR 12 )2, -CO(CH2) q PO(OR 12 )2, -NR 12 (CH2) q PO(OR 12 )2, -(CH2) q OPO(OR 12 )2, -O(CH2) q OPO(OR 12 )2, -S(CH2) q OPO(OR 12 )2, -CO(CH2) q OPO(OR 12 )2 and -NR 12 (CH2) q OPO(OR 12 )2.

[0337] In yet another aspect, this document provides a method for identifying one or more compounds that bind to a biological target, the method comprising: (a) incubating the biological target with at least a portion of a library of compounds according to claim 2 containing more than one different labeled macrocyclic compounds to prepare at least one bound compound and at least one unbound compound among the more than one different labeled macrocyclic compounds; (b) removing at least one unbound compound; and (c) sequencing each of the oligonucleotides (D) of the at least one bound compound.

[0338] In some embodiments, the DNA-encoded library may be a single-pharmacophore library, wherein only one chemical moiety can be attached to a single strand of DNA, as described, for example, in Neri & Lerner, Annu. Rev. Biochem. (2018) 87:5.1-5.24, which is incorporated herein by reference in its entirety. In some embodiments, the DNA-encoded library may be a dual-pharmacophore library, wherein two separate molecules can be attached to a double strand of DNA, as described, for example, in Mannocci et al., Chem. Commun. (2011) 47:12747-53, which is incorporated herein by reference in its entirety.

[0339] In another aspect, this document provides a method for preparing a library of labeled macrocyclic compounds, the method comprising synthesizing more than one different labeled macrocyclic compound. In some embodiments, each of the more than one different labeled macrocyclic compounds comprises a macrocyclic compound operably linked to at least one oligonucleotide (D). In some embodiments, each of the more than one different labeled macrocyclic compounds in the compound library comprises a macrocyclic compound operably linked to at least one oligonucleotide (D). In some embodiments, the macrocyclic compound comprises an FKBD, an effector domain, a first linker region, and a second linker region. In some embodiments, the FKBD, the effector domain, the first linker region, and the second linker region together form a macrocycle. In some embodiments, each of the at least one oligonucleotide (D) is recognizable as having at least one of the FKBD, the effector domain, the first linker region, and the second linker region of each of the more than one different labeled macrocyclic compound. In some embodiments, each of the more than one different labeled macrocyclic compounds in the compound library comprises a compound of formula (A) (as defined above). In some embodiments, each compound in the compound library of more than one differently labeled macrocyclic compounds comprises a compound of formula (I) (as defined herein). In some embodiments, each compound in the compound library of more than one differently labeled macrocyclic compounds may be a reaction product operatively linking a compound of formula (B) (as defined herein) with a compound of formula (C) (as defined herein). In some embodiments, each compound in the compound library of more than one differently labeled macrocyclic compounds may be a reaction product operatively linking a compound of formula (B′) (as defined herein) with a compound of formula (C) (as defined herein).

[0340] In some implementations, the method for synthesizing the compound library can be selected from the group consisting of: split-and-pool method, DNA templated library synthesis (DTS), encoding self-assembly chemistry (ESAC) library synthesis, DNA-recorded library synthesis, DNA-directed library synthesis, DNA routing, and 3-D proximity-based library synthesis (YoctoReactor). As will be appreciated by those skilled in the art, various techniques for synthesizing libraries of labeled macrocyclic compounds are described below: for example, Neri & Lerner, Annu. Rev. Biochem. (2018) 87: 5.1–5.24; Roman et al., SLAS Discov. (2018) 23(5): 387-396; Lim, C&EN, (2017) 95(29): 10–10; Halford, C&EN, (2017) 95(25): 28-33; Estevez, Tetrahedron: Asymmetry. (2017) 28: 837–842; Neri, Chembiochem. (2017) 4; 18(9): 827-828; Yuen & Franzini, Chembiochem. (2017) 4; 18(9): 829-836; Skopic et al., Chem Sci.(2017)1;8(5):3356-3361; Shi et al.; Bioorg MedChem Lett.(2017)1;27(3):361-69; Zimmermann&Neri, Drug Discov Today.(2016)21(11):1828-1834; Satz et al., Bioconjug Chem.(2015)19;26(8):1623-32; Ding et al.,ACSComb Sci.(2016)10;18(10):625-629; Arico-Muendel, MedChemComm, (2016)7(10):1898-1909; Skopic, MedChemComm, (2016)7(10):1957-1965; Satz, CS Comb.Sci.(2016)18(7):415–424;Tian et al., MedChemComm,(2016)7(7):1316-1322;Salamon et al., ACS Chem Biol.(2016)19;11(2):296-307;Satz et al., Bioconjug Chem.(2015)19;26(8):1623-32;Connors et al., Curr Opin Chem Biol.(2015)26:42-7;Blakskjaer et al., Curr Opin Chem Biol.(2015)26:62-71;Scheuermann & Neri, Curr Opin Chem Biol.(2015)26:99-103;Franzini et al., Angew Chem Int Ed Engl.(2015)23;54(13):3927-31;Franzini et al., Bioconjug Chem.(2014)20;25(8):1453-61;Franzini, Neri & Scheuermann, Acc Chem Res.(2014)15;47(4):1247-55;Mannocci et al., Chem. Commun.(2011)47:12747-53;Kleiner et al., Chem SocRev.(2011)40(12):5707–17;Clark,Curr Opin Chem Biol.(2010)14(3):396-403;Mannocci et al.,Proc Natl Acad Sci US A.(2008)18;105(46):17670-75;Buller et al.,Bioorg Med Chem Lett.(2008)18(22):5926-31;Scheuermann et al.,Bioconjugate Chem.(2008)19:778–85;Zimmerman et al.,ChemBioChem(2017)18(9):853-57;andCuozzo et al.,ChemBioChem(2017),18(9):864-71, each of which is incorporated herein by reference in its entirety.

[0341] In some embodiments, the method for synthesizing a library of labeled macrocyclic compounds includes DNA-recorded library synthesis, wherein coding and library synthesis are performed separately, as described, for example, in Shi et al., Bioorg Med Chem Lett. (2017) 1; 27(3):361-369; Kleiner et al., Chem Soc Rev. (2011) 40(12):5707–17. In some embodiments, DNA-recorded library synthesis includes splitting and merging methods, as described, for example, Krall, Scheuermann & Neri, Angew Chem. Int. Ed Engl. (2013) 28; 52(5):1384-402; Mannocci et al., Chem. Commun. (2011) 47:12747-53; and Morgan et al., US7,989,395, each of which is incorporated herein by reference in its entirety. In some embodiments, the splitting and merging method includes sequentially chemically linking an oligonucleotide tag to an initial oligonucleotide (or headpiece), which can be covalently linked to a chemically generated entity via sequential splitting and merging steps. In some embodiments, during each splitting step, a chemical synthesis step may be performed concurrently with the oligonucleotide linking step.

[0342] In some embodiments, the library can be synthesized via a series of split and merge cycles, wherein an initial oligonucleotide (or head fragment) can react with a first set of structural units (e.g., more than one FKBD structural unit). For each structural unit in the first set (e.g., each FKBD structural unit), an oligonucleotide (D) can be attached to the initial oligonucleotide (or head fragment), and the resulting products can be merged (or mixed) and subsequently split into separate reactions. Subsequently, in some embodiments, a second set of structural units (e.g., more than one effector domain structural unit) can be added, and an oligonucleotide (D) can be attached to each structural unit in the second set. In some embodiments, each oligonucleotide (D) identifies a different structural unit.

[0343] In some embodiments, methods for synthesizing libraries of labeled macrocyclic compounds include DNA-directed library synthesis, wherein the DNA both encodes and templates the library synthesis, as described, for example, in Kleiner et al., Bioconjugate Chem. (2010) 21, 1836–41; and Shi et al., Bioorg Med Chem Lett. (2017) 1; 27(3): 361–369, each of which is incorporated herein by reference in its entirety. In some embodiments, DNA-directed library synthesis includes DNA templated synthesis (DTS) methods, as described, for example, in Mannocci et al., Chem. Commun. (2011) 47:12747-53; Franzini, Neri & Scheuermann, Acc Chem Res. (2014) 15; 47(4):1247-55; and Mannocci et al., Chem. Commun. (2011) 47:12747-53, each of which is incorporated herein by reference in its entirety. In some embodiments, the DTS method includes DNA oligonucleotides that not only encode but also guide library construction. See Buller et al., Bioconjugate Chem. (2010) 21, 1571–80, which is incorporated herein by reference in its entirety. In some embodiments, different structural units can be incorporated into a molecule using DNA-linking reagents that can be forced to approach each other by base pairing between their DNA tags. See Gartner et al., Science (2004) 305:1601-05, which is incorporated herein by reference in its entirety. In some embodiments, a library of long oligonucleotides may be synthesized first as a template for a DNA-encoded library. In some embodiments, the oligonucleotides may be subjected to sequence-specific chemistry by immobilization on a resin labeled with complementary DNA sequences. See Wrenn & Harbury, Annu. Rev. Biochem. (2007) 76:331-49, which is incorporated herein by reference in its entirety.

[0344] In some implementations, DNA-guided library synthesis includes 3-D proximity-based library synthesis, also known as YoctoReactor technology, which is described, for example, in Blakskjaer et al., Curr Opin Chem Biol. (2015) 26:62-7, which is incorporated herein by reference in its entirety.

[0345] In some embodiments, methods for synthesizing libraries of labeled macrocyclic compounds include the synthesis of libraries encoding self-assembly chemistry (ESAC) libraries, also known as dual-pharmacophore DNA-encoded chemical libraries, as described, for example, in Mannocci et al., Chem. Commun. (2011) 47:12747-53; Melkko et al., Nat. Biotechnol. (2004) 22(5):568-74; Scheuermann et al., Bioconjugate Chem. (2008) 19:778–85; and Neri et al., US8,642,215, each of which is incorporated herein by reference in its entirety. In some embodiments, the library of labeled macrocyclic compounds synthesized by ESAC synthesis includes, for example, a non-covalent assembly of complementary oligonucleotide sub-libraries, wherein each sub-library may contain a first oligonucleotide attached to a first structural unit, wherein the first oligonucleotide contains a coding domain that identifies the first structural unit and a hybridization domain that self-assembles into a second oligonucleotide attached to a second structural unit, wherein the second oligonucleotide contains a coding domain that identifies the second structural unit and a hybridization domain that self-assembles into the first oligonucleotide.

[0346] In some embodiments, the method of synthesizing a library of labeled macrocyclic compounds includes DNA routing, as described, for example, in Clark, Curr Opin Chem Biol. (2010) 14(3):396-403, which is incorporated herein by reference in its entirety.

[0347] In some embodiments, oligonucleotide ligation may be performed using one of several methods that will be understood by those skilled in the art, such as those described in Zimmermann & Neri, Drug Discov. Today. (2016) 21(11):1828-1834; and Keefe et al., Curr Opin Chem Biol. (2015) 26:80-88, each of which is incorporated herein by reference in its entirety. In some embodiments, oligonucleotide ligation may be enzymatic ligation. In some embodiments, oligonucleotide ligation may be chemical ligation.

[0348] In some embodiments, ligation involves base-pairing a short complementary “adaptor” oligonucleotide with a single-stranded oligonucleotide at either end of the ligation site, allowing single-stranded DNA tags to be ligated in each cycle. See Clark et al., Nat. Chem. Biol. (2009) 5:647-54, which is incorporated herein by reference in its entirety. In some embodiments, oligonucleotide ligation involves utilizing a 2-base overhang at the 3' end of the head fragment and the DNA tag of each structural unit to form a sticky end for ligation. In some embodiments, the sequence of the overhang may be cycle-dependent and not cycle-dependent, such that any DNA tag can be ligated to any DNA tag from a previous cycle, without ligating to a truncated sequence. See the above literature. In some embodiments, the oligonucleotide ligation step may utilize oligonucleotides that have the opposite significance for subsequent cycles, having a small overlapping region in which the two oligonucleotides are complementary. In some embodiments, instead of ligation, DNA polymerase may be used to fill in the remainder of the complementary sequence, producing a double-stranded oligonucleotide containing two tags. In some embodiments, oligonucleotide ligation may be chemical. While not wishing to be bound by theory, it is thought that chemical linkages could allow for greater flexibility regarding solution conditions and reduce the necessary buffer exchange steps. See Keefe et al., Curr Opin Chem Biol. (2015) 26:80-88, which is incorporated herein by reference in its entirety.

[0349] In some embodiments, this document provides a method for identifying one or more compounds that bind to a biological target, the method comprising: (a) incubating the biological target with at least a portion of a library of more than one differently labeled macrocyclic compounds to prepare at least one bound compound and at least one unbound compound from the more than one differently labeled macrocyclic compounds; (b) removing the at least one unbound compound; and (c) sequencing each of at least one oligonucleotide (D) of the at least one bound compound. In some embodiments, each of the more than one differently labeled macrocyclic compounds in the compound library comprises a macrocyclic compound operatively linked to at least one oligonucleotide (D). In some embodiments, the macrocyclic compound comprises an FKBD, an effector domain, a first linker region, and a second linker region. In some embodiments, the FKBD, effector domain, first linker region, and second linker region together form a macrocycle. In some embodiments, each of the at least one oligonucleotide (D) can identify at least one of the FKBD, effector domain, first linker region, and second linker region of each of the more than one differently labeled macrocyclic compounds. In some embodiments, the compound library contains more than one compound of a different labeled macrocyclic compound, each comprising a compound of formula (A) (as defined above). In some embodiments, the compound library contains more than one compound of a different labeled macrocyclic compound, each comprising a compound of formula (I) (as defined above).As will be appreciated by those skilled in the art, various techniques for synthesizing libraries of labeled macrocyclic compounds are described below: for example, Kuai et al., SLAS Discov. (2018) 23(5): 405-416; Brown et al., Annu. Rev. Biochem. (2018) 87: 5.1–5.24; Roman et al., SLAS Discov. (2018) 23(5): 387-396; Amigo et al., SLAS Discov. (2018) 23(5): 397-404; Shi et al., Bioconjug Chem. (2017) 20; 28(9): 2293-2301; Machutta et al., Nat Commun. (2017) 8: 16081; Li et al., Chembiochem. (2017) 4; 18(9): 848-852; Satz et al., ACS Comb Sci.(2017)10;19(4):234-238; Denton&Krusemark, MedChemComm,(2016)7(10):2020-2027; Eidam&Satz , MedChemComm, (2016) 7(7):1323-1331; Bao et al., Anal. Chem., (2016) 88(10):5498–5506; Decurtins et al., Nat Protoc.(2016)11(4):764-80; Harris et al., J. Med. Chem. (2016) 59(5):2163-78; Satz, ACS Chem Biol. (2016) 16; 10(10):2237-45; Chan et al., Curr Opin Chem Biol. (2015) 26:55-61; Franzini et al., ChemCommun. (2015) 11; 51(38):8014-16; and Buller et al., Bioorg Med Chem Lett. (2010) 15; 20(14):4188-92. Each of these is incorporated herein by reference in its entirety.

[0350] In some embodiments, the incubation step may be performed under conditions where at least one of more than one differently labeled macrocyclic compounds suitable for the compound library binds to the biological target. Those skilled in the art will understand what conditions would be considered suitable for the binding of at least one of more than one differently labeled macrocyclic compounds from the compound library to the biological target.

[0351] In some embodiments, identifying one or more compounds that bind to a biological target includes a binding-wash-elution procedure for molecular selection, as described, for example, in Ding et al., ACS Med. Chem. Lett. (2015) 7; 6(8): 888-93, which is incorporated herein by reference in its entirety. In some embodiments, the incubation step (a) includes contacting more than one labeled compound from the compound library with the target protein, wherein the target protein may be immobilized on a substrate (e.g., resin). In some embodiments, the removal step (b) includes washing the substrate to remove at least one unbound compound. In some embodiments, the sequencing step (c) includes sequencing at least one oligonucleotide (D) to identify which of the more than one labeled compound binds to the target protein.

[0352] In some embodiments, identifying one or more compounds that bind to a biological target involves utilizing unmodified, unfixed target proteins. Such methods, employing a link-crosslink-purification strategy, are described, for example, in Shi et al., Bioconjug. Chem. (2017) 20; 28(9): 2293-2301, which is incorporated herein by reference in its entirety. In some embodiments, other methods for identifying one or more compounds that bind to a biological target may be utilized. Such methods will be obvious to those skilled in the art, and examples of such methods are described, for example, in Machutta et al., Nat. Commun. (2017) 8:16081; Chan et al., Curr. Opin. Chem. Biol. (2015) 26:55-61; Lim, C&EN, (2017) 95(29):10; Amigo et al., SLAS Discov. (2018) 23(5):397-404; Tian et al., MedChemComm. (2016) 7(7):1316-1322; Satz, CS Comb. Sci. (2016) 18(7):415–424, each of which is incorporated herein by reference in its entirety.

[0353] Tables 5-7 below illustrate all the Rapafucin compounds synthesized and characterized in this disclosure. In some embodiments, this disclosure does not include Rapafucin compounds having an AA2 such as dmPhe. In some embodiments, this disclosure does not include Rapafucin compounds having an AA2 such as dPro, dHoPro, or G. In some embodiments, this disclosure does not include Rapafucin compounds having an AA1 such as G, mG, Pro, and dPro.

[0354]

[0355] Tables 5-7 below show all Rapafucin molecules in this disclosure, with structural portions shown according to formula (XIII). Examples of chemical structures derived from formula (XIII) for compound 1 are shown below. In the case of amino acid monomers and FKBD, a dehydration reaction occurs, which produces peptide bonds. An example where monomer 4 is not specified is Rapafucin comprising FKBD with a linker group and only 3 monomers.

[0356]

[0357]

[0358]

[0359] Table 5. Rapafucin compounds 1 to 578 as disclosed in this disclosure.

[0360]

[0361]

[0362]

[0363]

[0364]

[0365]

[0366]

[0367]

[0368]

[0369]

[0370]

[0371]

[0372]

[0373]

[0374]

[0375]

[0376] Table 6. Rapafucin compounds 579 to 877 as described in this disclosure.

[0377]

[0378]

[0379]

[0380]

[0381]

[0382]

[0383]

[0384]

[0385] Table 7. Rapafucin compounds 878 to 1604 as disclosed in this disclosure.

[0386]

[0387]

[0388]

[0389]

[0390]

[0391]

[0392]

[0393]

[0394]

[0395]

[0396]

[0397]

[0398]

[0399]

[0400]

[0401]

[0402]

[0403]

[0404]

[0405] In treatment, the dosage of the agent is optionally in the range of about 0.0001 mg / kg of subject weight to about 100 mg / kg of subject weight, about 0.01 mg / kg of subject weight to about 5 mg / kg of subject weight, about 0.15 mg / kg of subject weight to about 3 mg / kg of subject weight, about 0.5 mg / kg of subject weight to about 2 mg / kg of subject weight, and about 1 mg / kg of subject weight to about 2 mg / kg of subject weight. In other embodiments, the dosage is in the range of about 100 mg / kg of subject weight to about 5 g / kg of subject weight, about 500 mg / kg of subject weight to about 2 mg / kg of subject weight, and about 750 mg / kg of subject weight to about 1.5 g / kg of subject weight. For example, depending on the type and severity of the disease, a dosage of about 1 μg / kg to 15 mg / kg (e.g., 0.1 mg / kg-20 mg / kg) is a candidate dosage for administration to the patient, whether by a single or multiple individual administrations or by continuous infusion. Typical daily doses range from about 1 μg / kg to 100 mg / kg or more, depending on the factors mentioned above. For repeated administration over several days or longer, treatment continues until the desired suppression of disease symptoms is achieved, depending on the condition. However, other dosing regimens can also be useful. Unit doses can range, for example, from about 5 mg to 500 mg, such as 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, and 300 mg. Progression of treatment is monitored using routine techniques and assays.

[0406] In some embodiments, the agent is administered to a human patient at an effective amount (or dose) of less than about 1 μg / kg, for example, from about 0.35 μg / kg to about 0.75 μg / kg or from about 0.40 μg / kg to about 0.60 μg / kg. In some embodiments, the dose of the agent is about 0.35 μg / kg, or about 0.40 μg / kg, or about 0.45 μg / kg, or about 0.50 μg / kg, or about 0.55 μg / kg, or about 0.60 μg / kg, or about 0.65 μg / kg, or about 0.70 μg / kg, or about 0.75 μg / kg, or about 0.80 μg / kg, or about 0.85 μg / kg, or about 0.90 μg / kg, or about 0.95 μg / kg, or about 1 μg / kg. In various embodiments, the absolute dose of the agent is about 2 μg / subject to about 45 μg / subject, or about 5 μg / subject to about 40 μg / subject, or about 10 μg / subject to about 30 μg / subject, or about 15 μg / subject to about 25 μg / subject. In some embodiments, the absolute dose of the agent is about 20 μg, or about 30 μg, or about 40 μg.

[0407] In various embodiments, the dosage of the agent can be determined by the weight of the human patient. For example, for pediatric human patients weighing from about 0 kg to about 5 kg (e.g., about 0 kg, or about 1 kg, or about 2 kg, or about 3 kg, or about 4 kg, or about 5 kg), the absolute dose of the agent is about 2 μg; or for pediatric human patients weighing from about 6 kg to about 8 kg (e.g., about 6 kg, or about 7 kg, or about 8 kg), it is about 3 μg; or for pediatric human patients weighing from about 9 kg to about 13 kg (e.g., 9 kg, or about 10 kg, or about 11 kg, or about 12 kg, or about 13 kg), it is about 5 μg; or for pediatric human patients weighing from about 14 kg to about 20 kg (e.g., about 14 kg, or about 16 kg, or about 18 kg, or about 20 kg), it is about 8 μg; or for pediatric human patients weighing from about 21 kg to about 30 kg (e.g., about 21 kg, or about 23 kg, or about 25 kg, or about 27 kg, or about 30 kg), it is about 2 μg. The dosage is approximately 12 μg for patients; or approximately 13 μg for pediatric human patients weighing approximately 31 kg to approximately 33 kg (e.g., approximately 31 kg, or approximately 32 kg, or approximately 33 kg); or approximately 20 μg for adult human patients weighing approximately 34 kg to approximately 50 kg (e.g., approximately 34 kg, or approximately 36 kg, or approximately 38 kg, or approximately 40 kg, or approximately 42 kg, or approximately 44 kg, or approximately 46 kg, or approximately 48 kg, or approximately 50 kg); or approximately 30 μg for adult human patients weighing approximately 51 kg to approximately 75 kg (e.g., approximately 51 kg, or approximately 55 kg, or approximately 60 kg, or approximately 65 kg, or approximately 70 kg, or approximately 75 kg); or approximately 45 μg for adult human patients weighing more than approximately 114 kg (e.g., approximately 114 kg, or approximately 120 kg, or approximately 130 kg, or approximately 140 kg, or approximately 150 kg).

[0408] In some embodiments, the agent according to the methods provided herein is administered subcutaneously (sc), intravenously (iv), intramuscularly (im), intranasally, or locally. Administration of the agent described herein can be independent of the method and can be once to four times daily, once to four times monthly, once to six times annually, or once every two, three, four, or five years. Administration can continue for a duration of one day, one month, two months, three months, six months, one year, two years, or three years, and can even continue for the lifetime of a human patient. Dosage can be administered as a single dose or divided into multiple doses. In some embodiments, the agent is administered about once to about three times (e.g., once, twice, or three times).

[0409] The following embodiments are provided to further illustrate the advantages and features of this disclosure, but are not intended to limit the scope of this disclosure. While these embodiments are typical examples of those that can be used, other procedures, methods, or techniques known to those skilled in the art may be used alternatively. Example

[0410] General experiments used in the synthesis. Reagents used in the synthesis. Piperidine and N,N-diisopropylethylamine (DIPEA) were purchased from Alfa Aesar. Anhydrous pyridine was purchased from Acros. The solid support resin with 2-chlorotriphenylmethyl chloride (catalog #: 03498) was purchased from Chem-Impex. HATU was purchased from ChemImpex. Fmoc-protected amino acid structural units were purchased from ChemImpex, Novabiochem, or GL Biochem. Dichloromethane (DCM or CH2Cl2), methanol (MeOH), hexane, ethyl acetate (EtOAc), 1,2-dichloroethane (DCE, anhydrous), N,N'-dimethylformamide (DMF, anhydrous), second-generation Hoveyda-Grubbs catalyst, and all other chemical reagents were purchased from Sigma-Aldrich.

[0411] Instruments used for synthesis and purification. NMR spectroscopy was recorded using Burker-400 and Burker-500. High-performance liquid chromatography (HPLC) analysis was performed using an Agilent LC-MS system (Agilent 1260 series, mass detector 6120 quadrupole). Orbital oscillations of solid-phase reactions were performed on a Mettler-Toledo Bohdan MiniBlock system (30 mg–200 mg resin in SiliCycle tubes) or a VWR Mini oscillator (0.2 g–2 g resin in a plastic syringe with a sintering plate). Reagents were added using adjustable Rainin 8-channel pipettes from the MiniBlock system. Microwave reactions were performed using a Biotage Initiator Plus or Multiwave Pro from Anton Parr with 24-well silicon carbide blocks. Compound purification at scales of 0.05 g–50 g was performed using a Teledyne Isco CombiFlash RF 200 or Biotage Isolera One system followed by a Heidolph rotary evaporator. Purification at scales of 1 mg–50 mg was performed using an Agilent HPLC system. The purification was carried out via a vacuum manifold (Sigma-Aldrich, Visiprep). TMRapafucin mixtures from a library of 45,000 compounds were purified in high-throughput mode using an SPE column (Biotage, 460-0200-C, ISOLUTE, SI 2 g / 6 mL) on an SPE vacuum manifold (disposable liner, 12 ports). The mixtures were then dried overnight in a custom-designed box (50 cm × 50 cm × 15 cm) that allowed rapid air flow inside to remove the solvent. High-throughput weighing of the compounds in the library was performed using a Mettler-Toledo analytical balance connected to a computer with a custom-coded electronic spreadsheet (with an RS232 port Sartorious Entris line).

[0412] FKBD Example 1

[0413] 4-((3-((R)-1-(((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutyryl)piperidin-2-carbonyl)oxy)-3-(3,4-dimethoxyphenyl)propyl)phenyl)amino)-4-oxobutyric acid (aFKBD)

[0414]

[0415] Scheme 3. Synthesis of intermediate 5 ((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutyryl)piperidine-2-carboxylic acid).

[0416] 2-Allyl-1-(tert-butyl)(S)-piperidine-1,2-dicarboxylate (2). Cs₂CO₃ (2.90 g) was added to a solution of N-Boc homoproline 1 (6.30 g) in DMF (40 mL). The resulting suspension was stirred at room temperature for 5 min, followed by the addition of allyl bromide (6.3 g). After stirring at room temperature for 2 h, the suspension was filtered through a diatomaceous earth mat, washed with EtOAc (50 mL), and washed with HCl (1 M, 50 mL × 3). The organic layer was dried over Na₂SO₄ and co-evaporated with toluene (30 mL × 2). The crude product (8.10 g) was collected as a yellow oil and was sufficiently pure for use in the next step without further purification. The crude product (8.10 g) and TFA (4.3 g) were thoroughly mixed in dichloromethane (20 mL) and stirred at room temperature for 0.5 h. 2-Allyl 1-(tert-butyl)(S)-piperidine-1,2-dicarboxylate 2 (3.00 g) was collected as a yellow oil and was pure enough to be used in the next step without further purification.

[0417] (S)-1-(4-hydroxy-3,3-dimethyl-2-oxobutyryl)piperidine-2-carboxylic acid allyl ester (3). Compound 2 (3.0 g), dihydro-4,4-dimethyl-2,3-furandione (2.1 g) and DMAP (20 mg) were dissolved in toluene (20 mL), and the reaction was refluxed in an oil bath (120 °C) for 14 h. After solvent removal, the residue was purified by column chromatography (80-200 mesh) on EtOAc / hexane (1 / 3). Compound 3 (3.50 g) was collected as a yellow oil. 1 H NMR (500MHz, CDCl3) δ6.04–5.80(m,1H),5.36(d,J=17Hz,1H),5.31–5.25(m,2H),4.68(s,2H),3.76–3.56(m,2H),3.50(d ,J=13Hz,1H),3.40(s,1H),3.20(t,J=13Hz,1H),2.37(d,J=13Hz,1H),1.84–1.61(m,3H),1.61–1.34(m,2H),1.24(s,6H). 13 C NMR (126MHz, CDCl3) δ205.9,170.1,168.1,131.4,119.2,69.3,66.3,51.6,49.5,44.2,26.3,24.8,21.3,21.2,21.0.

[0418] (S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutyryl)piperidine-2-carboxylic acid allyl ester (4) was added dropwise in an ice bath for 30 min to a mixture of compound 3 (3.50 g) and N,N-diisopropylethylamine (2.0 mL) in 50 mL of dry CH2Cl2 (20 mL). After the addition, the reaction was allowed to continue stirring at room temperature for 30 min, followed by quenching with a saturated NaHCO3 solution (20 mL). The organic phase was washed with water, dried over Na2SO4, concentrated, and purified by column chromatography (EtOAc:hexane = 1:5) to provide product 4 (2.21 g) as a yellow oil. 1HNMR(500MHz,CDCl3)δ6.39(dd,J=17,1.5Hz,1H),6.08(dd,J=17,11Hz,1H),5.91(ddt,J=17,11,6Hz,1H),5.84(dd,J=11, 1.5Hz,1H),5.35(ddd,J=17,2.5,1.5Hz,1H),5.28-5.25(m,1H),5.26(ddd,J=11,2.5,1.5Hz,1H),4.66(ddd,J=6,4,2.5Hz ,2H),4.37(d,J=11Hz,1H),4.27(d,J=11Hz,1H),3.52(dd,J=13,1.5Hz,1H),3.23(td,J=13,3Hz,1H),2.34(d,J=14Hz,1H) ,1.84–1.76(m,1H),1.76–1.67(m,1H),1.67–1.60(m,1H),1.59–1.47(m,1H),1.47–1.38(m,1H),1.36(s,3H),1.35(s,3H). 13 C NMR (126MHz, CDCl3) δ 204.8, 169.8, 166.7, 165.5, 131.5, 131.2, 128.0, 118.9, 69.5, 69.3, 66.0, 51.3, 46.7, 43.9, 26.4, 24.9, 22.2, 21.5, 21.1. For [M+H]+C18H25NO6, calculated value: 352.1760, observed value: 352.1753.

[0419] (S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutyryl)piperidine-2-carboxylic acid (5). Compound 4 (4.2 g), Pd(PPh3)4 (230 mg), and N-methylaniline (2.5 mL) were dissolved in THF (40 mL) and stirred at room temperature for 6 h. The reaction mixture was then diluted with EtOAc (80 mL) and washed with HCl (1 M, 50 mL × 3). The organic phase was separated, dried over Na2SO4, filtered, and concentrated. The crude product was purified by column chromatography (200-400 mesh), wherein byproducts were eluted with 2% MeOH in dichloromethane, followed by elution of the desired product with 3% MeOH and 0.1% AcOH in dichloromethane. Compound 5 (2.55 g) was collected as a white solid (66%). 1H NMR(500MHz, CDCl3)δ9.96(s,1H),6.39(d,J=17Hz,1H),6.08(dd,J=17,10Hz,1H),5.85(d,J=10Hz,1H),5.30(s,1H),4.55-4.30(m,1H),4.32( d,J=6Hz,2H),3.53(d,J=12Hz,1H),3.24(t,J=12Hz,1H),2.35(d,J=13H z,1H),1.91–1.60(m,2H),1.60–1.42(m,2H),1.36(s,3H),1.34(s,3H). 13 C NMR (126MHz, CDCl3) δ 204.7, 175.3, 166.8, 165.7, 131.4, 127.8, 69.6, 69.5, 51.2, 46.7, 44.0, 26.2, 24.9, 22.1, 21.8, 21.1. For [M+H]+C15H21NO6, calculated value: 312.1447, observed value: 312.1444.

[0420]

[0421] Synthesis of Scheme 4.aFKBD.

[0422] (E)-1-(3-aminophenyl)-3-(3,4-dimethoxyphenyl)prop-2-en-1-one (6). NaOH (0.2 g in 2 mL of water) was added to a solution of a mixture of 3,4-dimethoxybenzaldehyde (5.10 g) and 3-aminoacetophenone (4.15 g) in EtOH (20 mL, 95%). The reaction mixture was stirred at room temperature for 6 h, and a slurry of yellow precipitate formed. The reaction mixture was then diluted with EtOAc (40 mL) and washed with water (30 mL × 3). After concentration, crude product 6 (9.0 g) was sufficiently pure for the next step.

[0423] 1-(3-Aminophenyl)-3-(3,4-Dimethoxyphenyl)prop-1-one (7). Pd / C (10%, 1.61 g) was added to a solution of α,β-unsaturated ketone 6 (crude, 9.0 g) in MeOH (20 mL). The reaction vessel was repeatedly flushed with hydrogen using a hydrogen balloon and high vacuum. The reaction mixture was stirred at room temperature for 1 h, then filtered through a diatomaceous earth mat. A longer reaction time would result in the formation of undesirable byproducts. The filtrate was concentrated and subjected to column chromatography (50 g silica gel), eluted with EtOAc / CH2Cl2 / hexane (1 / 3 / 3 to 1 / 1 / 1). 7 (2.48 g) was collected as a yellow oil. 1H NMR (500MHz, CDCl3) δ7.36–7.16(m,3H,ar),6.92–6.71(m,4H,ar),3.86(s,3H,OCH3),3.85 (s,3H,OCH3),3.81(s,2H,NH2),3.23(t,J=7.5Hz,2H,COCH2),2.99(t,J=7.4Hz,2H,ArCH2). 13 C NMR (126MHz, CDCl3) δ 199.66 (C=O), 148.90 (ar), 147.38 (ar), 146.82 (ar), 138.03 (ar), 134.03 (ar), 129.49 (ar), 120.19 (ar), 119.61 (ar), 118.44 (ar), 113.91 (ar), 111.87 (ar), 111.35 (ar), 55.98 (OCH3), 55.87 (OCH3), 40.80 (COCH2), 29.91 (ArCH2). For [M+H]+C17H19NO3, calculated value: 286.1443, observed value: 286.1436.

[0424] 4-((3-(3-(3,4-dimethoxyphenyl)propionyl)phenyl)amino)-4-oxobutyric acid (9). Aniline 7 (3.50 g), succinic anhydride (1.0 g), and DMAP (61 mg) were mixed in dichloromethane (30 mL). After stirring at room temperature for 3 h, the reaction mixture was washed with HCl (1 M, 30 mL × 4). The crude product (3.80 g) was collected as a white solid and used directly in the next step without further purification. Cs2CO3 (1.86 g) was added to the solution of the above crude product (3.80 g) in DMF (20 mL). The resulting suspension was stirred at room temperature for 10 min, followed by the addition of allyl bromide (1.50 mL). The reaction mixture was stirred for an additional 2 h. The white precipitate was filtered off using a diatomaceous earth mat. EtOAc (40 mL) and H2O (40 mL) were added to the filtrate. After stirring for 10 minutes, the product was precipitated. Product 9 (2.11 g) was obtained by filtration, air-dried to a grayish-white solid, and used in the next step without further purification.

[0425] (R)-1-(3-(4-(allyloxy)-4-oxobutyrylamino)phenyl)-3-(3,4-dimethoxyphenyl)propyl(S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutyryl)piperidine-2-carboxylate (10). Alcohol 9 (1.65 g) and formic acid 5 (1.26 g, for synthesis, see Example 1 of FKBD) were dissolved in a mixture of THF (anhydrous, 5 mL) and dichloromethane (anhydrous, 10 mL). Benzoyl chloride (0.60 mL), Et3N (1.0 mL), and DMAP (18 mg) were added sequentially, and the resulting suspension was stirred at room temperature for 2 h. Without further treatment, the mixture was subjected to column chromatography (80-200 mesh) using EtOAc / hexane (1 / 2 → 1 / 1). 10 (2.50 g) was collected as a yellow foam. ¹H NMR (500 MHz, CDCl₃) δ 8.08 (s, ¹H), 7.65 (d, J = 8 Hz, ¹H), 7.46 (s, ¹H), 7.28 (t, J = 8 Hz, ¹H), 7.01 (d, J = 8 Hz, ¹H), 6.77 (d, J = 9 Hz, ¹H), 6.69 (d, J = 5 Hz, ¹H), 6.67 (s, ¹H), 6.39 (dd, J = 17, 1.5Hz,1H),6.06(dd,J=17,10.5Hz,1H),5.90(ddt,J=17,10.5,6Hz,1H),5.83(dd,J=10.5 ,1.5Hz,1H),5.79(ddd,J=10.5,8,3.5Hz,1H),5.31(dd,J=17,1.5Hz,2H),5.31(d,J=6Hz, 1H),5.22(dd,J=10.5,1.5Hz,1H),4.60(dt,J=6,1.5Hz,2H),4.33(d,J=0.7Hz,2H),3.86( s,3H),3.85(s,3H),3.46(d,J=14Hz,1H),3.09(dd,J=18,8Hz,1H),2.78(t,J=6Hz,2H),2. 70(t,J=6Hz,2H),2.62–2.48(m,2H),2.36(d,J=14Hz,1H),2.30–2.16(m,1H),2.13–2.00( m, 1H), 1.74 (d, J = 10.5Hz, 2H), 1.62 (d, J = 12Hz, 1H), 1.42 (d, J = 12.6Hz, 1H), 1.36 (s, 6H).13C NMR (126MHz, CDCl3) δ205.6,172.6,169.8,169.3,166.2,165.6,148.9,147.3,140.7,138.6,133.5,132.0,131.5,129.2,127.8,122.0,120 .2,119.3,118.4,117.2,111.7,111.3,76.5,69.2,65.5,55.9,55.9,5 1.3,46.8,44.1,38.1,31.9,31.1,29.3,26.1,25.1,22.0,21.9,20.9.

[0426] 4-((3-((R)-1-(((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutyryl)piperidin-2-carbonyl)oxy)-3-(3,4-dimethoxyphenyl)propyl)phenyl)amino)-4-oxobutyric acid (aFKBD). 10 (2.50 g), Pd(PPh3)4 (100 mg), and N-methylaniline (1.0 mL) were thoroughly mixed in THF (20 mL) at room temperature for 5 h. The reaction mixture was then diluted with EtOAc (50 mL) and washed with HCl (1 M, 50 mL × 3). The organic phase was dried over Na2SO4, filtered, and concentrated. The crude product was purified by column chromatography (200-400 mesh), in which byproducts were eluted with 2% MeOH in dichloromethane, followed by elution of the desired product with 3% MeOH and 0.05% AcOH in dichloromethane. aFKBD (2.25 g) was collected as a grayish-white foam. 1H NMR (500MHz, CDCl3) δ8.40(s,1H),7.62(s,1H),7.48(s,1H),7.26(t,J=7.5Hz,1H),7.01(d,J=7.5Hz,1H),6.97(dd,J=16,7Hz,1H ),6.86–6.74(m,1H),6.74–6.58(m,2H),5.85–5.68(m,2H),5.39–5.24(m,1H),4.29(q,J=11Hz,2H),3.86(s,3H),3.84(s,3H),3.4 6(d,J=13Hz,1H),3.13(t,J=13Hz,1H),2.74(d,J=5.5Hz,2H),2.69(d,J=5.5Hz,2H),2.63–2.48(m,2H),2.36(d,J=13Hz,1H),2.3 0–2.15(m,1H),2.15–1.99(m,1H),1.85(d,J=6Hz,1H),1.75(d,J=12Hz,1H),1.63(d,J=13Hz,1H),1.55–1.38(m,2H),1.34(s,6H). 13 C NMR (126MHz, CDCl3) δ205.6,176.8,170.4,169.4,166.4,166.1,148.9,147.3,145.9,140.7,138.5,133.5,129.2,122.1,121.9,1 20.2,119.5,117.4,111.8,111.4,76.6,69.0,55.9,55.8,51.4,46.8,44.1,38.1,31.6,31.1,29.3,26.2,25.0,21.8,20.9,18.1. For the HRMS of [M+H]+C36H44O2N11, the calculated value is 681.3023 and the observed value is 681.3018.

[0427] FKBD Example 2

[0428] 2-(3-((R)-1-(((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutyryl)piperidin-2-carbonyl)oxy)-3-(3,4-dimethoxyphenyl)propyl)phenoxy)acetic acid (eFKBD)

[0429]

[0430] Synthesis of Scheme 5.eFKBD.

[0431] (R)-1-(3-(2-(tert-butoxy)-2-oxoethoxy)phenyl)-3-(3,4-dimethoxyphenyl)propyl(S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutyryl)piperidine-2-carboxylate (6). Alcohol 4 (3.8 g, 1.0 equivalent) was added to a round-bottom 42 flask under argon protection. For its synthesis, see Liu et al. (2014). Angew.Chem.Int.Ed. 53:10049-55), formic acid 5 (4.1 g, 1.2 equivalents, see FKBD Example 1 for synthesis) and DMAP (134 mg, 0.1 equivalents) were dissolved in a mixture of THF (anhydrous, 35 mL) and dichloromethane (anhydrous, 35 mL). Et3N (4.7 mL) and benzoyl chloride (2.17 mL, 2.62 g, 1.7 equivalents) were added dropwise sequentially using a syringe, and the resulting suspension was stirred at room temperature for 2 h. The reaction was monitored by TLC. When complete conversion was achieved, the reaction mixture was diluted with 500 mL of EtOAc and washed with 5% HCl and saturated NaHCO3. The organic phase was washed with brine and dried over Na2SO4. The solvent was then removed, and the product was purified by column chromatography (80-200 mesh) using EtOAc / hexane (1 / 10 to 1 / 3). 6 (5.3 g, 69%) was collected as a pale yellow foam. 1 H NMR(500MHz, CDCl3)δ7.26(d,J=8Hz,1H),6.97(d,J=8.5Hz,1H),6.93–6.89(m,1H),6.86–6.81(m,1H),6.78(d,J=8.5Hz,1H),6.71–6.64(m,2H), 6.38(dd,J=17,1.5Hz,1H),6.06(dd,J=17,10.5Hz,1H),5.82(dd,J=10.5,1.5Hz,1H),5.78(dd,J=8,6Hz,1H),5.29(d,J=5Hz,1H),4.53(s,2H),4. 36(d,J=11Hz,1H),4.27(d,J=11Hz,1H),3.86(s,3H),3.84(s,3H),3.48( d,J=13Hz,1H),3.17(td,J=13,3.0Hz,1H),2.67–2.44(m,2H),2.37(d,J= 14Hz,1H),2.32–2.18(m,1H),2.14–1.99(m,1H),1.83–1.65(m,2H),1.65 –1.56(m,1H),1.50-1.43(m,2H),1.48(s,9H),1.35(s,3H),1.35(s,3H). 13C NMR (126MHz, CDCl3) δ204.8,169.4,167.8,166.4,165.4,158.1,148.9,147.3,141.3,133.4,131.2,129.7,127.9,120.2,119.8, 114.2,113.2,111.7,111.3,82.3,76.7,69.2,65.7,55.9,55.8,51.4,46.6,44.0,37.9,31.2,28.0,26.4,25.0,22.1,21.6,21.1. For the HRMS of [M+H]+C38H49NO11, the calculated value is 696.3384, and the observed value is 696.3386.

[0432] 2-(3-((R)-1-(((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutyryl)piperidin-2-carbonyl)oxy)-3-(3,4-dimethoxyphenyl)propyl)phenoxy)acetic acid (eFKBD). Compound 6 (5.3 g, 1.0 equivalent) was dissolved in 60 mL of dichloromethane in a round-bottom flask under Ar protection. TFA (17 mL, 11.4 g, 13 equivalent) was then added in 3 parts by syringe over 3.5 h while stirring at room temperature. The reaction was monitored by TLC. When complete conversion was achieved, the solvent and TFA were removed under vacuum. The product was purified by column chromatography (80-200 mesh) using EtOAc / hexane (1 / 5 → 1 / 1). eFKBD (4.6 g, 96%) was collected as a pale yellow foam. 1 H NMR (500MHz, CDCl3) δ7.28 (dd, J=3.5Hz, 3.5Hz, 1H), 6.88 (d, J=8.5Hz, 1H), 6.83-6.81 (m, 2H), 6.80-6.78 (m, 1H) ,6.69-6.67(m,2H),6.37(d,J=8.5Hz,1H),6.05-6.02(m,1H),5.83-5.72(m,2H),5.30-5.28(dd,J=10,5Hz,1H), 4.67(dd,J=10,5Hz,1H),4.17(dd,J=10,6Hz,2H),3.48-3.45(m,1H),3.24-3.22(m,1H),2.61-2.55(m,2H),2.38 (m,1H),2.23(m,1H),2.04(m,1H),1.79(m,1H),1.62(m,1H),1.33(m,1H),1.30(m,1H),1.25(s,3H),1.24(s,3H). 13C NMR (126MHz, CDCl3) δ 204.6, 169.2, 166.7, 165.7, 157.9, 149.0, 147.5, 141.7, 131.4, 129.9, 127.9, 120.0, 115.4, 111.8, 111.4, 111.1, 69.3, 65.2, 60.5, 55.9, 51.7, 44.1, 38.0, 31.4, 22.1, 21.1, 14.2. For [M+H]+C34H42NO11, calculated value: 640.2758, observed value: 640.2761.

[0433] FKBD Example 3

[0434] 4-(3-((R)-1-((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutyryl)piperidin-2-carbonyloxy)-3-morpholinopropyl)phenylamino)-4-oxobutyric acid (Raa1)

[0435]

[0436]

[0437] Scheme 6. Synthesis of the Raa1 FKBD portion.

[0438] 1-(3-Nitrophenyl)prop-2-en-1-one (2). Paraformaldehyde (36 g, 120 mmol) was added to a stirred solution of 1-(3-nitrophenyl)ethyl ketone 1 (20 g, 120 mmol), N-methylaniline trifluoroacetate (26.8 g, 120 mmol), and TFA (1.4 g, 12 mmol) in THF (300 mL). The resulting reaction was heated to reflux for 16 h. The solvent was removed under vacuum, and the residue was diluted with water (100 mL) and EA (200 mL). The organic extract was dried over Na2SO4 and concentrated under vacuum to provide compound 2 (14.2 g, crude) as a yellow solid, which was used in the next step without purification. [M+H] + =178.1.

[0439] 3-Morpholino-1-(3-nitrophenyl)prop-1-one (3). Morpholine (2.95 g, 33.9 mmol) was added to a solution of 2 (12 g, 33.9 mmol, crude) in DMF (30 mL), followed by the addition of 4-methylbenzenesulfonic acid (5.83 g, 33.9 mmol). After stirring at room temperature for 5 h, the reaction was quenched with H2O (50 mL) and extracted with EA (100 mL × 3). The organic extract was dried over Na2SO4 and concentrated under vacuum to give a crude product, which was further purified by column chromatography (SiO2, methanol / DCM = 0–10% as eluent) to give compound 3 (6.6 g, 74%) as a yellow oil. [M+H] + =265.2.

[0440] 1-(3-Aminophenyl)-3-morpholinopropyl-1-one (4). At room temperature, 10% Pd / C (wetting, 840 mg) was added to a solution of 3 (4.2 g, 15.9 mmol) in THF (20 mL). The resulting reaction mixture was hydrogenated with H2 (g) at room temperature for 8 h. The reaction mixture was then filtered and concentrated under vacuum to provide crude compound 4 (3.46 g, crude) as a yellow oil, which was used directly in the next step. [M+H] + =235.1.

[0441] 4-(3-(3-morpholinopropionyl)phenylamino)-4-oxobutyrate tert-butyl ester (6). At room temperature, DIPEA (5.55 g, 43 mmol) was added to a solution of 4 (5.05 g, 21.5 mmol) and 4-tert-butoxy-4-oxobutyric acid 5 (4.86 g, 27.95 mmol) in DMF (20 mL), followed by HATU (10.62 g, 27.95 mmol). The resulting reaction mixture was stirred at room temperature for 2 h. The reaction was quenched with H2O (50 mL) and extracted with EA (100 mL × 3). The organic extract was dried over Na2SO4 and concentrated under vacuum to give a crude product, which was further purified by column chromatography (SiO2, methanol / DCM = 0–5% as eluent) to provide compound 6 (4.3 g, 51%) as a yellow solid. [M+H] + =391.0.

[0442] (R)-tert-butyl-4-(3-(1-hydroxy-3-morpholinopropyl)phenylamino)-4-oxobutyrate (7). At -20 °C, a solution of ketone 6 (4.1 g, 10.5 mmol) in anhydrous THF (40 mL) was added to (+)DIPChloride (42 mmol) in heptane (1.7 M, 24.7 mL). The resulting reaction mixture was stirred at -20 °C until 6 was completely converted, and quenched with 2,2'-(ethane-1,2-diylbis(oxy))diethylamine (7 g, 47.25 mmol) until an insoluble complex was formed. After stirring at room temperature for an additional 30 min, the suspension was filtered through a diatomaceous earth pad and concentrated under vacuum to give a crude product, which was further purified by column chromatography (SiO2, CH3OH / EA = 0–5% as eluent) to give compound 7 (1.0 g, 24%) as a grayish-white solid. [M+H] + =393.0.

[0443] (S)-((R)-1-(3-(4-tert-butoxy-4-oxobutyrylamino)phenyl)-3-morpholinopropyl)1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutyryl)piperidine-2-carboxylate (9). Solutions of 7 (1.0 g, 2.55 mmol) and 8 (952 mg, 3.06 mmol) in anhydrous DCM (25 mL) were cooled to -20 °C under an argon atmosphere, followed by the addition of a solution of DCC (630 mg, 3.06 mmol) in anhydrous DCM (2 mL), and then a solution of 4-(dimethylamino)pyridine (DMAP, 31 mg, 0.255 mmol). The resulting white suspension was stirred at -20 °C for 2 h. The reaction mixture was then filtered, and the filtrate was dried over Na₂SO₄ and concentrated under vacuum to give a crude product, which was further purified by column chromatography (SiO₂, CH₃OH / DCM = 0–5% as eluent) to give compound 9 as a white solid (1.3 g, 76%). [M+H + =686.0.

[0444] 4-(3-((R)-1-((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutyryl)piperidin-2-carbonyloxy)-3-morpholinopropyl)phenylamino)-4-oxobutyric acid (RAa-1)

[0445] At room temperature, add 2 mL of TFA to a solution of 9 (1.3 g, 1.9 mmol) in DCM (10 mL). Stir the resulting mixture at room temperature for 3 h. The reaction mixture is then directly loaded into a silica gel fast column (CH3OH / DCM = 0–5% as eluent) to provide RAa-1 (620 mg, 51%) as a white solid.

[0446] FKBD Example 4

[0447] 4-(3-((R)-1-((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutyryl)piperidin-2-carbonyloxy)-4-morpholinobutyl)phenylamino)-4-oxobutyric acid (Raa2)

[0448]

[0449] Scheme 7. Synthesis of the Raa2 FKBD portion.

[0450] 3-(3-Nitrobenzoyl)-dihydrofuran-2(3H)-one (3). LiHMDS (1M, 77mL, 77mmol in THF) was added to a stirred solution of dihydrofuran-2(3H)-one 2 (6.02g, 70mmol) in anhydrous THF (60mL) at -78°C, and the mixture was stirred for 2 h under an argon atmosphere. Then, a solution of 3-nitrobenzoyl chloride 1 (6.5g, 35mmol) in anhydrous THF (10mL) was added at -78°C. The resulting reaction mixture was slowly heated to room temperature and stirred at room temperature for 16 h. The mixture was then treated with saturated NH4Cl. 含水 The reaction was quenched (20 mL), and extracted with EA (100 mL × 3). The organic extract was dried over Na₂SO₄ and concentrated under vacuum to provide compound 3 (8.5 g, crude) as a yellow oil, which was used directly in the next step without purification. [M+H] + =236.1.

[0451] 4-Bromo-1-(3-nitrophenyl)but-1-one (4). A solution of 3 (25.9 g, 110 mmol, crude product) in 40% HBr (150 mL) was heated to 70 °C for 2 h. The reaction mixture was cooled to room temperature and precipitated with saturated NaHCO3. 3含水 Adjust the pH to 5-6 and extract with EA (200 mL × 3). Dry the organic extract with Na2SO4 and concentrate under vacuum to give a crude product, which is further purified by column chromatography (SiO2, EA / PE = 0–10% as eluent) to give compound 4 as a yellow oil (18.5 g, 74% in 2 steps).

[0452] 4-Morphyrin-1-(3-nitrophenyl)but-1-one (5). At room temperature, K₂CO₃ (8.64 g, 62.5 mmol) was added to a solution of 4 (8.5 g, 31.25 mmol) and morpholine (2.72 g, 31.25 mmol) in CH₃CN (100 mL). The resulting reaction mixture was heated to reflux for 2 h. The reaction mixture was then filtered and concentrated under vacuum to give a crude product, which was further purified by column chromatography (SiO₂, methanol / DCM = 0–5% as eluent) to give compound 5 (4.6 g, 53%) as a yellow oil. [M+H] + =279.2.

[0453] 1-(3-Aminophenyl)-4-morpholinobut-1-one (6). At room temperature, 10% Pd / C (wetted, 1.18 g) was added to a solution of 5 (5.9 g, 21.2 mmol) in THF (60 mL). The resulting reaction mixture was hydrogenated with H2 (g) at room temperature for 10 h. The reaction mixture was then filtered and concentrated under vacuum to provide crude compound 6 (4.8 g, crude) as a yellow solid, which was used directly in the next step. [M+H] + =249.0.

[0454] At room temperature, DIPEA (5.0 g, 38.7 mmol) was added to a solution of 6 (4.8 g, 19.35 mmol) and 4-tert-butoxy-4-oxobutyric acid 7 (4.86 g, 27.95 mmol) in DMF (15 mL), followed by HATU (9.56 g, 25.15 mmol). The resulting reaction mixture was stirred at room temperature for 2 h. The reaction was quenched with H2O (50 mL) and extracted with EA (100 mL × 3). The organic extract was dried over Na2SO4 and concentrated under vacuum to give a crude product, which was further purified by column chromatography (SiO2, methanol / DCM = 0–5% as eluent) to give compound 8 (6.6 g, 84%) as a yellow solid. [M+H] + =405.0.

[0455] (R)-tert-butyl-4-(3-(1-hydroxy-4-morpholinobutyl)phenylamino)-4-oxobutyrate (9). At -20 °C, a solution of ketone 8 (5.0 g, 12.4 mmol) in heptane (1.7 M, 29 mL) was added to (+)-DIPChloride (49.6 mmol) in anhydrous THF (20 mL). The resulting reaction mixture was stirred at -20 °C until 8 was completely converted, then quenched with 2,2'-(ethane-1,2-diylbis(oxy))diethylamine (8.3 g, 55.8 mmol) until an insoluble complex was formed. After stirring at room temperature for an additional 30 min, the suspension was filtered through a diatomaceous earth pad and concentrated under vacuum to give a crude product, which was further purified by column chromatography (SiO2, CH3OH / EA = 0–5% as eluent) to give compound 9 (2.5 g, 50%) as a grayish-white solid. [M+H] + =407.3.

[0456] (S)-((R)-1-(3-(4-tert-butoxy-4-oxobutyrylamino)phenyl)-4-morpholinobutyl)1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutyryl)piperidine-2-carboxylate (11). Under an argon atmosphere, solutions of 9 (2.45 g, 6.05 mmol) and 10 (2.29 g, 7.38 mmol) in anhydrous DCM (40 mL) were cooled to -20 °C, followed by the addition of a solution of DCC (1.52 g, 7.38 mmol) in anhydrous DCM (5 mL), and then a solution of 4-(dimethylamino)pyridine (DMAP, 75 mg, 0.615 mmol) in anhydrous DCM (1 mL). The resulting white suspension was stirred at -20 °C for 2 h. The reaction mixture was then filtered, and the filtrate was dried over Na₂SO₄ and concentrated under vacuum to give a crude product, which was further purified by column chromatography (SiO₂, CH₃OH / DCM = 0–5% as eluent) to give compound 11 (3 g, 69%) as a white solid. [M+H] + =700.0.

[0457] 4-(3-((R)-1-((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutyryl)piperidin-2-carbonyloxy)-4-morpholinobutyl)phenylamino)-4-oxobutyric acid (Raa2). At room temperature, 2 mL of TFA was added to a solution of 11 (1.0 g, 1.42 mmol) in DCM (10 mL). The resulting mixture was stirred at room temperature for 2 h. The reaction mixture was directly loaded into a silica gel fast column (CH3OH / DCM = 0–5% as eluent) to provide Raa2 (550 mg, 60%) as a white solid.

[0458] FKBD Example 5

[0459] 4-(3-((R)-3-(4-(((9H-fluoren-9-yl)methoxy)carbonyl)piperazin-1-yl)-1-((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutyryl)piperidine-2-carbonyloxy)propyl)phenylamino)-4-oxobutyric acid (Raa3)

[0460]

[0461] Scheme 8. Synthesis of the Raa3 FKBD portion.

[0462] 4-(3-(3-nitrophenyl)-3-oxopropyl)piperazine-1-carboxylic acid tert-butyl ester (3). DIPEA (3.64 g, 28.2 mmol) was added to a solution of 1 (10 g, 28.2 mmol, crude) in DMF (20 mL), followed by the addition of 2 (5.24 g, 28.2 mmol). After stirring at room temperature for 2 h, the reaction was quenched with H2O (100 mL) and extracted with EA (100 mL × 3). The organic extract was dried over Na2SO4 and concentrated under vacuum to give a crude product, which was further purified by column chromatography (SiO2, methanol / DCM = 0–10% as eluent) to provide compound 3 (6.1 g, 60%) as a yellow oil. [M+H] + =364.2.

[0463] 4-(3-(3-aminophenyl)-3-oxopropyl)piperazine-1-carboxylic acid tert-butyl ester (4). At room temperature, 10% Pd / C (wetted, 1.22 g) was added to a solution of 3 (6.1 g, 15.9 mmol) in THF (50 mL). The resulting reaction mixture was hydrogenated with H2 (g) at room temperature for 8 h. The reaction mixture was then filtered and concentrated under vacuum to provide crude compound 4 (5.5 g, crude) as a brown solid, which was used directly in the next step. [M+H] +=334.3.

[0464] 4-(3-(3-(4-tert-butoxy-4-oxobutyrylamino)phenyl)-3-oxopropyl)piperazine-1-carboxylic acid tert-butyl ester (6). At room temperature, DIPEA (5.04 g, 38.99 mmol) was added to a solution of 4 (5.2 g, 15.6 mmol) and 4-tert-butoxy-4-oxobutyric acid 5 (3.53 g, 20.27 mmol) in DMF (35 mL), followed by HATU (7.71 g, 20.27 mmol). The resulting reaction mixture was stirred at room temperature for 4 h. The reaction was quenched with H2O (50 mL) and extracted with EA (100 mL × 3). The organic extract was dried over Na2SO4 and concentrated under vacuum to give a crude product, which was further purified by column chromatography (SiO2, PE / EA = 0–50% as eluent) to provide compound 6 (4.3 g, 56%) as a yellow solid. [M+H] + =490.4.

[0465] (R)-tert-butyl-4-(3-(3-(4-tert-butoxy-4-oxobutyrylamino)phenyl)-3-hydroxypropyl)piperazine-1-carboxylate (7). At -20 °C, (+)DIPChloride (38.8 mmol) in heptane (1.7 M, 23 mL) was added to a solution of ketone 6 (3.8 g, 7.76 mmol) in anhydrous THF (30 mL). The resulting reaction mixture was stirred at -20 °C until 6 was completely converted, and quenched with 2,2'-(ethane-1,2-diylbis(oxy))diethylamine (6.32 g, 42.68 mmol) until an insoluble complex was formed. After stirring at room temperature for another 30 min, the suspension was filtered through a diatomaceous earth pad and concentrated under vacuum to give a crude product, which was further purified by column chromatography (SiO2, CH3OH / EA = 0–5% as eluent) to give compound 7 (1.9 g, 51%) as a grayish-white solid. [M+H] + =492.3.

[0466] 4-((R)-3-((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutyryl)piperidine-2-carbonyloxy)-3-(3-(4-tert-butoxy-4-oxobutyrylamino)phenyl)propyl)piperazine-1-carboxylic acid tert-butyl ester (9). Under an argon atmosphere, solutions of 7 (1.03 g, 2.1 mmol) and 8 (784 mg, 2.52 mmol) in anhydrous DCM (20 mL) were cooled to -20 °C, followed by the addition of a solution of DCC (865 mg, 4.2 mmol) in anhydrous DCM (2 mL), and then a solution of 4-(dimethylamino)pyridine (DMAP, 26 mg, 0.21 mmol). The resulting white suspension was stirred at -20 °C for 2 h. The reaction mixture was then filtered, and the filtrate was dried over Na₂SO₄ and concentrated under vacuum to give a crude product, which was further purified by column chromatography (SiO₂, CH₃OH / DCM = 0–5% as eluent) to provide compound 9 (1.2 g, 72%) as a yellow solid. [M+H] + =784.9.

[0467] 4-(3-((R)-1-((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutyryl)piperidin-2-carbonyloxy)-3-(piperazin-1-yl)propyl)phenylamino)-4-oxobutyric acid (10). At room temperature, TFA (3 mL) was added to a solution of 9 (1.2 g, 1.9 mmol) in DCM (6 mL). The resulting mixture was stirred at room temperature for 3 h. The reaction mixture was concentrated under vacuum to provide compound 10 (1.1 g, crude) as a yellow solid. [M+H] + =628.9.

[0468] 4-(3-((R)-3-(4-(((9H-fluoren-9-yl)methoxy)carbonyl)piperazin-1-yl)-1-((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutyryl)piperidin-2-carbonyloxy)propyl)phenylamino)-4-oxobutyric acid (Raa3). At room temperature, Na2CO3 (369 mg, 3.48 mmol) was added to a solution of 10 (1.1 g, 1.74 mmol) in DMF (4 mL), followed by the addition of FmocChloride (450 mg, 1.74 mmol). The resulting reaction mixture was stirred at room temperature for 30 min. The reaction was quenched with H2O (10 mL) and extracted with EA (30 mL × 3). The organic extract was dried over Na2SO4 and concentrated under vacuum to give a crude product, which was further purified by column chromatography (SiO2, methanol / DCM = 0–5% as eluent) to give Raa3 (680 mg, 46%) as a white solid.

[0469] FKBD Example 6

[0470] 4-(3-((R)-4-(4-(((9H-fluoren-9-yl)methoxy)carbonyl)piperazin-1-yl)-1-((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutyryl)piperidine-2-carbonyloxy)butyl)phenylamino)-4-oxobutyric acid (Raa4)

[0471]

[0472] Scheme 9. Synthesis of the Raa4 FKBD portion.

[0473] 4-(4-(3-nitrophenyl)-4-oxobutyl)piperazine-1-carboxylic acid tert-butyl ester (3). At room temperature, K₂CO₃ (10.7 g, 77.2 mmol) was added to solutions of 1 (10.5 g, 38.6 mmol) and 2 (7.2 g, 38.6 mmol) in CH₃CN (100 mL). The resulting reaction mixture was heated to reflux for 2 h. The reaction mixture was then filtered and concentrated under vacuum to give a crude product, which was further purified by column chromatography (SiO₂, methanol / DCM = 0–5% as eluent) to give compound 3 (8.3 g, 57%) as a yellow solid. [M+H] + =378.0.

[0474] 4-(4-(3-aminophenyl)-4-oxobutyl)piperazine-1-carboxylic acid tert-butyl ester (4). At room temperature, 10% Pd / C (wetted, 1.66 g) was added to a solution of 3 (8.3 g, 22 mmol) in THF (60 mL). The resulting reaction mixture was hydrogenated with H2 (g) at room temperature for 10 h. The reaction mixture was then filtered and concentrated under vacuum to provide crude compound 4 (7.4 g, crude) as a yellow solid, which was used directly in the next step. [M+H] + =348.3.

[0475] 4-(3-(4-morpholinobutyryl)phenylamino)-4-oxobutyrate tert-butyl ester (6). At room temperature, DIPEA (5.5 g, 42.6 mmol) was added to a solution of 4 (7.4 g, 21.3 mmol) and 4-tert-butoxy-4-oxobutyric acid 5 (4.82 g, 27.6 mmol) in DMF (15 mL), followed by HATU (10.5 g, 27.69 mmol). The resulting reaction mixture was stirred at room temperature for 2 h. The reaction was quenched with H2O (50 mL) and extracted with EA (100 mL × 3). The organic extract was dried over Na2SO4 and concentrated under vacuum to give a crude product, which was further purified by column chromatography (SiO2, methanol / DCM = 0–5% as eluent) to provide compound 6 (8.5 g, 79%) as a yellow solid. [M+H] + =504.0.

[0476] (R)-tert-butyl 4-(4-(3-(4-tert-butoxy-4-oxobutyrylamino)phenyl)-4-hydroxybutyl)piperazine-1-carboxylate (7). At -20 °C, (+)DIPChloride (35.6 mmol) in heptane (1.7 M, 21 mL) was added to a solution of ketone 6 (4.5 g, 8.9 mmol) in anhydrous THF (20 mL). The resulting reaction mixture was stirred at -20 °C until 6 was completely converted, and then quenched with 2,2'-(ethane-1,2-dimethylbis(oxy))diethylamine (5.9 g, 40.0 mmol) until an insoluble complex was formed. After stirring at room temperature for another 30 min, the suspension was filtered through a diatomaceous earth pad and concentrated under vacuum to give a crude product, which was further purified by column chromatography (SiO2, methanol / EA = 0–5% as eluent) to provide compound 7 (2.5 g, 55%) as a grayish-white solid. [M+H] + =506.0.

[0477] 4-((R)-4-((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutyryl)piperidine-2-carbonyloxy)-4-(3-(4-tert-butoxy-4-oxobutyrylamino)phenyl)butyl)piperazine-1-carboxylic acid tert-butyl ester (9). Solutions of 7 (2.3 g, 4.5 mmol) and 8 (1.68 g, 5.4 mmol) in anhydrous DCM (30 mL) were cooled to -20 °C under an argon atmosphere, followed by the addition of a solution of DCC (1.11 g, 5.4 mmol) in anhydrous DCM (5 mL), and then a solution of 4-(dimethylamino)pyridine (DMAP, 55 mg, 0.615 mmol) in anhydrous DCM (1 mL). The resulting white suspension was stirred at -20 °C for 2 h. The reaction mixture was then filtered, and the filtrate was dried over Na₂SO₄ and concentrated under vacuum to give a crude product, which was further purified by column chromatography (SiO₂, methanol / DCM = 0–5% as eluent) to give compound 9 (2.9 g, 80%) as a white solid. [M+H] + =799.5.

[0478] 4-(3-((R)-1-((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutyryl)piperidin-2-carbonyloxy)-4-(piperazin-1-yl)butyl)phenylamino)-4-oxobutyric acid (10). At room temperature, 3 mL of TFA was added to a solution of 9 (2.9 g, 3.6 mmol) in DCM (10 mL). The resulting mixture was stirred at room temperature for 4 h. The reaction mixture was directly loaded into a silica gel fast column (CH3OH / DCM = 0–5% as eluent) to provide compound 10 (2.6 g, crude) as a yellow solid, which was directly used in the next step. [M+H] + =643.4.

[0479] 4-(3-((R)-4-(4-(((9H-fluoren-9-yl)methoxy)carbonyl)piperazin-1-yl)-1-((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutyryl)piperidin-2-carbonyloxy)butyl)phenylamino)-4-oxobutyric acid (Raa4). At room temperature, Na2CO3 (343 mg, 3.24 mmol) was added to a solution of 10 (1.2 g, 1.62 mmol) in DMF (2 mL), followed by the addition of FmocChloride (419 mg, 1.62 mmol). The resulting reaction mixture was stirred at room temperature for 30 min. The reaction was quenched with H2O (10 mL) and extracted with EA (30 mL × 3). The organic extract was dried over Na2SO4 and concentrated under vacuum to give a crude product, which was further purified by column chromatography (SiO2, methanol / DCM = 0–5% as eluent) to give Raa4 (570 mg, 40%) as a white solid.

[0480] FKBD Example 7

[0481] 4-(5-((R)-1-((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutyryl)piperidin-2-carbonyloxy)-3-(3,4-dimethoxyphenyl)propyl)pyridin-3-ylamino)-4-oxobutyric acid (Raa5)

[0482]

[0483]

[0484] Scheme 10. Synthesis of the Raa5 FKBD portion.

[0485] 1-(5-aminopyridin-3-yl)acetone (2). At room temperature, 10% Pd / C (wetting, 8.0 g) was added to a solution of 1 (13 g, 78.3 mmol) in THF (100 mL). The resulting reaction mixture was stirred at room temperature under H2 (g) for 10 h. The reaction mixture was then filtered and concentrated under vacuum to give a crude product, which was further purified by column chromatography (SiO2, methanol / DCM = 0–5% as eluent) to give compound 2 (10 g, 94%) as a yellow solid. [M+H] + =137.0.

[0486] (E)-1-(5-aminopyridin-3-yl)-3-(3,4-dimethoxyphenyl)prop-2-en-1-one (4). At 0 °C, LiOH·H₂O (2 g, 47.8 mmol) was added to solutions of 2 (6.5 g, 47.8 mmol) and 3 (7.9 g, 47.8 mmol) in CH₃OH (60 mL). The resulting reaction mixture was stirred at room temperature for 3 h. The solvent was removed under vacuum, and the residue was diluted with DCM and H₂O. The organic extract was dried over Na₂SO₄ and concentrated under vacuum to give a crude product, which was further purified by column chromatography (SiO₂, methanol / DCM = 0–5% as eluent) to provide compound 4 (1.8 g, 13%) as a yellow solid. [M+H] + =285.0.

[0487] (E)-tert-butyl-4-(5-(3-(3,4-dimethoxyphenyl)acryloyl)pyridin-3-ylamino)-4-oxobutyrate (6). At room temperature, Et3N (12.7 g, 12.6 mmol) was added to a solution of 4 (1.8 g, 6.3 mmol) and 4-tert-butoxy-4-oxobutyric acid 5 (1.1 g, 6.3 mmol) in DCM (35 mL), followed by the addition of T3P (50% in EtOAc, 8.0 g, 12.6 mmol). The resulting reaction mixture was stirred at room temperature for 1 h. The reaction was quenched with H2O (20 mL) and extracted with DCM (40 mL × 2). The organic extract was dried over Na₂SO₄ and concentrated under vacuum to give a crude product, which was further purified by column chromatography (SiO₂, methanol / DCM = 0–5% as eluent) to provide compound 6 (1.88 g, 68%) as a yellow solid. [M+H] + =440.9.

[0488] 4-(5-(3-(3,4-dimethoxyphenyl)propionyl)pyridin-3-ylamino)-4-oxobutyrate tert-butyl ester (7). At room temperature, 10% Pd / C (wetting, 380 mg) was added to a solution of 6 (1.88 g, 4.27 mmol) in THF (50 mL) and methanol (5 mL). The resulting reaction mixture was hydrogenated with H2 (g) at room temperature for 4 h. The reaction mixture was then filtered and concentrated under vacuum to give a crude product, which was further purified by column chromatography (SiO2, methanol / DCM = 0–5% as eluent) to give compound 7 (1.34 g, 71%) as a brown solid. [M+H] + =442.9.

[0489] (R)-tert-butyl 4-(5-(3-(3,4-dimethoxyphenyl)-1-hydroxypropyl)pyridin-3-ylamino)-4-oxobutyrate (8). At -20 °C, a solution of ketone 7 (1.34 g, 3.0 mmol) in anhydrous THF (20 mL) was added to (+) DIPChloride (12.0 mmol) in heptane (1.7 M, 7.05 mL). The resulting reaction mixture was stirred at -20 °C until 7 was completely converted, and then quenched with 2,2'-(ethane-1,2-diylbis(oxy))diethylamine (2.0 g, 13.5 mmol) until an insoluble complex was formed. After stirring at room temperature for another 30 min, the suspension was filtered through a diatomaceous earth pad and concentrated under vacuum to give a crude product, which was further purified by column chromatography (SiO2, CH3OH / EA = 0–5% as eluent) to give compound 8 (0.99 g, 74%) as a white solid. [M+H] + =445.0.

[0490] (S)-((R)-1-(5-(4-tert-butoxy-4-oxobutyrylamino)pyridin-3-yl)-3-(3,4-dimethoxyphenyl)propyl)1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutyryl)piperidine-2-carboxylate (10). Solutions of 8 (990 mg, 2.22 mmol) and 9 (827 mg, 2.66 mmol) in anhydrous DCM (20 mL) were cooled to -20 °C under an argon atmosphere, followed by the addition of a solution of DCC (548 mg, 2.66 mmol) in anhydrous DCM (2 mL), and then a solution of 4-(dimethylamino)pyridine (DMAP, 27 mg, 0.22 mmol) in anhydrous DCM (1 mL). The resulting white suspension was stirred at -20 °C for 2 h. The reaction mixture was then filtered, and the filtrate was dried over Na₂SO₄ and concentrated under vacuum to give a crude product, which was further purified by column chromatography (SiO₂, CH₃OH / DCM = 0–5% as eluent) to give compound 10 (1.3 g, 79%) as a white solid. [M+H] + =738.0.

[0491] 4-(5-((R)-1-((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutyryl)piperidin-2-carbonyloxy)-3-(3,4-dimethoxyphenyl)propyl)pyridin-3-ylamino)-4-oxobutyric acid (Raa5). At room temperature, 5 mL of TFA was added to a solution of 10 g (1.3 g, 1.76 mmol) in DCM (10 mL). The resulting mixture was stirred at room temperature for 2 h. The reaction mixture was directly loaded into a silica gel fast column (CH3OH / DCM = 0–5% as eluent) to provide Raa5 (960 mg, 80%) as a white solid.

[0492] FKBD Example 8

[0493] 4-(6-((R)-1-((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutyryl)piperidin-2-carbonyloxy)-3-(3,4-dimethoxyphenyl)propyl)pyridin-2-ylamino)-4-oxobutyric acid (Raa6)

[0494]

[0495] Scheme 11. Synthesis of the Raa6 FKBD portion.

[0496] (E)-1-(6-aminopyridin-2-yl)-3-(3,4-dimethoxyphenyl)prop-2-en-1-one (3). At room temperature, LiOH·H2O (1.74 g, 41.35 mmol) was added to solutions of 1 (3.75 g, 27.57 mmol) and 2 (4.58 g, 27.57 mmol) in CH3OH (40 mL). The resulting reaction mixture was stirred at room temperature for 3 h. The solvent was removed under vacuum, and the residue was diluted with DCM and H2O. The organic extract was dried over Na2SO4 and concentrated under vacuum to give a crude product, which was further purified by column chromatography (SiO2, methanol / DCM = 0–5% as eluent) to provide compound 3 (3.2 g, 41%) as a yellow solid. [M+H] + =285.0.

[0497] (E)-tert-butyl-4-(6-(3-(3,4-dimethoxyphenyl)acryloyl)pyridin-2-ylamino)-4-oxobutyrate (5). At 0 °C, POCl3 (2.58 g, 16.89 mmol) was added to a solution of 3 (3.2 g, 11.26 mmol) in pyridine (10 mL) and 4-tert-butoxy-4-oxobutyric acid 4 (2.35 g, 13.5 mmol). The resulting reaction mixture was stirred at 0 °C for 15 min. The reaction was quenched with H2O (20 mL) and extracted with EA (30 mL × 3). The organic extract was dried over Na2SO4 and concentrated under vacuum to give a crude product, which was further purified by column chromatography (SiO2, methanol / DCM = 0–5% as eluent) to provide compound 5 (2.45 g, 49%) as a yellow solid. [M+H] + =440.9.

[0498] 4-(6-(3-(3,4-dimethoxyphenyl)propionyl)pyridin-2-ylamino)-4-oxobutyrate tert-butyl ester (6). At room temperature, 10% Pd / C (wetting, 500 mg) was added to a solution of 5 (2.45 g, 5.56 mmol) in THF (30 mL). The resulting reaction mixture was hydrogenated with H2 (g) at room temperature for 4 h. The reaction mixture was then filtered and concentrated under vacuum to give a crude product, which was further purified by column chromatography (SiO2, methanol / DCM = 0–5% as eluent) to provide compound 6 (1.5 g, 61%) as a yellow solid. [M+H] + =443.3.

[0499] (R)-tert-butyl 4-(6-(3-(3,4-dimethoxyphenyl)-1-hydroxypropyl)pyridin-2-ylamino)-4-oxobutyrate (7). At -20 °C, (+)-DIPChloride (12.64 mmol) in heptane (1.7 M, 7.5 mL) was added to a solution of ketone 6 (1.4 g, 3.16 mmol) in anhydrous DCM (20 mL). The resulting reaction mixture was stirred at -20 °C until 7 was completely converted, and then quenched with 2,2'-(ethane-1,2-diylbis(oxy))diethylamine (2.1 g, 14.22 mmol) until an insoluble complex was formed. After stirring at room temperature for an additional 30 min, the suspension was filtered through a diatomaceous earth pad and concentrated under vacuum to give a crude product, which was further purified by column chromatography (SiO2, CH3OH / EA = 0–5% as eluent) to provide compound 7 (1.0 g, 71%) as a white solid. [M+H] + =445.3.

[0500] (S)-((R)-1-(6-(4-tert-butoxy-4-oxobutyrylamino)pyridin-2-yl)-3-(3,4-dimethoxyphenyl)propyl)1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutyryl)piperidine-2-carboxylate (9). Solutions of 7 (1.0 g, 2.24 mmol) and 8 (836 mg, 2.69 mmol) in anhydrous DCM (20 mL) were cooled to -20 °C under an argon atmosphere, followed by the addition of a solution of DCC (554 mg, 2.69 mmol) in anhydrous DCM (2 mL), and then a solution of 4-(dimethylamino)pyridine (DMAP, 27 mg, 0.22 mmol) in anhydrous DCM (1 mL). The resulting white suspension was stirred at -20 °C for 2 h. The reaction mixture was then filtered, and the filtrate was dried over Na₂SO₄ and concentrated under vacuum to give a crude product, which was further purified by column chromatography (SiO₂, CH₃OH / DCM = 0–5% as eluent) to give compound 9 (0.38 g, 23%) as a white solid. [M+H] + =738.4.

[0501] 4-(6-((R)-1-((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutyryl)piperidin-2-carbonyloxy)-3-(3,4-dimethoxyphenyl)propyl)pyridin-2-ylamino)-4-oxobutyric acid (Raa6). At room temperature, 2 mL of TFA was added to a solution of 9 (0.38 g, 1.76 mmol) in DCM (5 mL). The resulting mixture was stirred at room temperature for 2 h. The reaction mixture was directly loaded into a silica gel fast column (CH3OH / DCM = 0–5% as eluent) to provide Raa6 (310 mg, 89%) as a white solid.

[0502] FKBD Example 9

[0503] 4-((6-((R)-(((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutyryl)piperidin-2-carbonyl)oxy)-3-(3,4-dimethoxyphenyl)propyl)pyrazin-2-yl)amino)-4-oxobutyric acid (Raa7)

[0504]

[0505] Scheme 12. Synthesis of the Raa7 FKBD portion.

[0506] 6-(1-Butoxyvinyl)pyrazine-2-amine (3). At room temperature, Pd(AcO)₂ (0.8 g, 3.7 mmol) and DPPF (4.12 g, 7.4 mmol) were added to a solution of 1 (16 g, 124 mmol) in ethylene glycol (150 mL). The mixture was degassed with Ar₂, and then 2 and Et₃N were injected sequentially. The reaction mixture was heated to reflux and the reaction continued for 1.5 h. The product mixture was poured into water (300 mL) and extracted with DCM (100 mL x 3). The organic phases were combined and washed with brine (100 mL x 3). The mixture was filtered and concentrated to give 3 (12 g, 50%) as a white solid. [M+H] + =194.

[0507] 1-(6-Aminopyrazin-2-yl)ethyl-1-one (4). 5% HCl (20 ml) was added to a solution of 3 (12 g, 62 mmol) in DCM (50 ml). The reaction mixture was stirred at room temperature for 0.5 h. The product mixture was poured into water (200 ml), and the pH was adjusted to 8–9 with K₂CO₃ (containing water). Extraction was performed with DCM (50 ml * 6), the organic phases were combined, and the mixture was concentrated to give a crude product. Purification was performed by silica gel chromatography (PE / EA = 20–30% as eluent) to give product 4 (2.9 g, 34%) as a yellow solid. [M+H] + =138.

[0508] (E)-1-(5-aminopyrazin-2-yl)-3-(3,4-dimethoxyphenyl)prop-2-en-1-one (6). LiOH (1.74 g, 42 mol) and 5 (3.43 g, 21 mmol) were added to a solution of 4 (2.9 g, 21 mmol) in MeOH (20 mL). The reaction mixture was stirred at 40 °C for 1 h. The product mixture was poured into water (200 mL), filtered until no more precipitate formed, the solid cake was washed with water, and then almost no MeOH was removed. The product was dried to give product 6 (3.8 g, 64.5%) as a yellow solid. [M+H] + =286.

[0509] (E)-4-((6-(3-(3,4-dimethoxyphenyl)acryloyl)pyrazin-2-yl)amino-4-oxobutyrate tert-butyl ester (8). At 0 °C, POCl3 (6.12 g, 400 mmol) was added to a solution of 8 (3.8 g, 133 mmol) and 7 (4.64 g, 266 mmol) in pyridine (100 ml). The reaction mixture was stirred at 0 °C for 30 min. The product mixture was poured into water (300 ml), extracted with DCM (100 ml * 3), the organic phases were combined and washed with brine (100 ml * 5). The product was dried over Na2SO4, filtered, and concentrated to give a crude product. Purification was performed by silica gel chromatography (MeOH / DCM = 1–2% as eluent) to give product 8 (5 g, 68%) as a yellow solid. [M+H] + =442.

[0510] 4-((6-(3-(3,4-dimethoxyphenyl)propionyl)pyrazin-2-yl)amino)-4-oxobutyrate tert-butyl ester (9). Pd / C (500 mg, 10%) was added to a solution of 8 (5.0 g, 113 mmol) in THF, the reaction mixture was degassed with H2*5, and stirred at room temperature for 4 h. The mixture was filtered and the filtrate was concentrated to give the crude product. Purification was performed by silica gel chromatography (MeOH / DCM = 1–2% as eluent) to give product 9 (2.0 g, 40%) as a yellow solid. [M+H] + =444.

[0511] (R)-4-((6-(3-(3,4-dimethoxyphenyl)-1-hydroxyphenyl)pyrazin-2-yl)amino)-4-oxobutyrate tert-butyl ester (11). At -20°C, DIPC1 (14.5 g, 450 mmol) was added to a solution of 9 (2.0 g, 45 mmol) in DCM (50 ml), and degassed with Ar2. The reaction mixture was stirred at -20°C for 5 h. Quenched with 10 (6.75 g, 455 mmol). The product mixture was directly concentrated, and the brown residue was purified by silica gel chromatography (MeOH / DCM = 2–5% as eluent) to give product 11 (1.0 g, 50%) as a yellow solid. [M+H] + =446.

[0512] (R)-1-(6-(4-tert-butoxy)-4-oxobutyrylamino)pyrazin-2-yl)-3-(3,4-dimethoxyphenyl(S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutyryl)piperidine-2-carboxylate (13). At -20°C, 12 (1.05 g, 34 mmol) was added to a solution of 11 (1.0 g, 22 mmol) in DCM (30 ml), and degassed with Ar2. DCC (0.7 g, 34 mmol) and DMAP (0.03 g, 2.2 mmol) were then injected sequentially into DCM. The reaction mixture was stirred at -20 °C for 1 h. The solid cake was filtered and washed with DCM (20 ml), the filtrates were combined and evaporated to give the crude product. Purification was performed by silica gel chromatography (MeOH / DCM = 1–2% as eluent) to give product 13 (1.8 g, 85%) as a yellow solid. [M+H] + =739.

[0513] 4-((6-((R)-(((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutyryl)piperidin-2-carbonyl)oxy)-3-(3,4-dimethoxyphenyl)propyl)pyrazin-2-yl)amino)-4-oxobutyric acid (Raa7). TFA (20 mL) was added to a solution of 13 (1.8 g, 24 mmol) in DCM (20 mL). The reaction mixture was stirred at room temperature for 2 h. The product mixture was directly concentrated, and the yellow residue was purified by silica gel chromatography (MeOH / DCM = 1–2% as eluent) to give product Raa7 (500 mg, 30%) as a pale yellow solid.

[0514] FKBD Example 10

[0515] 4-((3-((R)-1-(((S)-4-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutyryl)morpholine-3-carbonyl)oxy)-3-(3,4-dimethoxyphenyl)propyl)phenyl)amino)-4-oxobutyric acid (Raa8)

[0516]

[0517] Scheme 13. Synthesis of the Raa8 FKBD portion.

[0518] (E)-3-(3,4-dimethoxyphenyl)-1-(3-nitrophenyl)prop-2-en-1-one (3). At 0 °C, NaOH (15 g) was added to a solution of 3,4-dimethoxybenzaldehyde 1 (60 g, 360 mmol) and 1-(3-nitrophenyl)ethyl-1-one 2 (59.6 g, 360 mmol) in MeOH (1100 mL). The resulting solution was stirred at room temperature for 10 h. The precipitate was collected to give compound 3 (97 g, 86%) as a yellow solid. [M+Na] + =336.1.

[0519] 1-(3-Aminophenyl)-3-(3,4-Dimethoxyphenyl)prop-1-one (4). A solution of 3 (32 g, 110 mmol) in THF (120 mL) and 10% Pd / C (10 g) was hydrogenated with H2 for 8 h at room temperature. The reaction mixture was then filtered and concentrated. The residue was purified by silica gel rapid column chromatography (AcOEt / PE 1:3) to give compound 4 (24 g, 76%) as a white solid. [M+H] + =286.2.

[0520] 4-((3-(3-(3,4-dimethoxyphenyl)propionyl)phenyl)amino)-4-oxobutyrate tert-butyl ester (5). 4-tert-butoxy-4-oxobutyric acid (8.8 g, 50 mmol), DIPEA (13.6 g, 105 mmol), and HATU (19.2 g, 50 mmol) were added to a solution of 4 (12.0 g, 42 mmol) in DCM (30 mL). The mixture was stirred at room temperature for 16 h, and the product was purified by silica gel rapid column chromatography (AcOEt / PE 1:2) to give compound 5 (16 g, 79%) as a white solid. [M+Na] + =464.0.

[0521] (R)-4-((3-(3-(3,4-dimethoxyphenyl)-1-hydroxypropyl)phenyl)amino)-4-oxobutyrate tert-butyl ester (6). A solution of ketone 5 (11.9 g, 26.9 mmol) in dry THF (120 mL) at -20 °C was treated with a solution of (+)-DIPChloride (135 mmol) in heptane (1.7 M, 79 mL) at -20 °C. The resulting mixture was reacted at -20 °C until 5 was completely converted, and then quenched with 2,2′-(ethylenedioxy)diethylamine (20 g) until an insoluble complex was formed. After stirring at room temperature for an additional 30 min, the suspension was filtered through a diatomaceous earth mat and concentrated. The crude compound was purified by silica gel rapid column chromatography (AcOEt / PE 3:1) to give compound 6 (7.9 g, 66%, ee 97%) as a pale yellow oil. [M+Na] + =466.3.

[0522] (R)-1-(3-(4-(tert-butoxy)-4-oxobutyrylamino)phenyl)-3-(3,4-dimethoxyphenyl)propyl(S)-4-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutyryl)morpholine-3-carboxylate (8). Solutions of 6 (2.36 g, 5.32 mmol) and 7 (2 g, 6.38 mmol) in CH2Cl2 (10 mL) were cooled to -20 °C under an argon atmosphere, followed by the addition of a solution of DCC (1.65 g, 7.98 mmol) in CH2Cl2 (5 mL), and then a solution of 4-(dimethylamino)pyridine (DMAP, 65 mg, 0.53 mmol) in CH2Cl2 (2 mL). The resulting white suspension was allowed to be stirred at -20 °C for 2 h. The reaction mixture was then filtered, evaporated, and the crude compound was purified by silica gel rapid column chromatography (AcOEt / PE 2:1) to give compound 8 (2.5 g, 64%) as a pale yellow oil. [M+Na] + =761.4.

[0523] 4-((3-((R)-1-(((S)-4-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutyryl)morpholin-3-carbonyl)oxy)-3-(3,4-dimethoxyphenyl)propyl)phenyl)amino)-4-oxobutyric acid (Raa8). A solution of Raa8 (2.5 g, 3.45 mmol) in 12 mL of CH2Cl2 was treated at 0 °C with a solution of 40% TFA in 12 mL of CH2Cl2. The mixture was allowed to react at room temperature until complete conversion. The reaction mixture was directly loaded into a silica gel fast column (AcOEt / PE / AcOH 1:2:0.5%) to provide Raa8 (815 mg, 38%) as a pale yellow solid.

[0524] FKBD Example 11

[0525] 4-((3-((R)-1-(((S)-4-(((9H-fluoren-9-yl)methoxy)carbonyl)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutyryl)piperazine-2-carbonyl)oxy)-3-(3,4-dimethoxyphenyl)propyl)phenyl)amino)-4-oxobutyric acid (Raa9)

[0526]

[0527] Scheme 14. Synthesis of the Raa9 FKBD portion.

[0528] 1-((9H-fluorene-9-yl)methyl)3-((R)-1-(3-(4-(tert-butoxy)-4-oxobutyrylamino)phenyl)-3-(3,4-dimethoxyphenyl)propyl)(S)-4-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutyryl)piperazine-1,3-dicarboxylate (3). Under an argon atmosphere, solutions of 1 (1.35 g, 3.04 mmol) and 2 (1.95 g, 3.65 mmol) in CH2Cl2 (10 mL) were cooled to -20 °C, followed by the addition of a solution of DCC (940 mg, 4.56 mmol) in CH2Cl2 (5 mL), and then a solution of 4-(dimethylamino)pyridine (DMAP, 37 mg, 0.3 mmol) in CH2Cl2 (2 mL). The resulting white suspension was allowed to be stirred at -20 °C for 3 h. The reaction mixture was then filtered, evaporated, and the crude compound was purified by silica gel rapid column chromatography (DCM / MeOH 96:4) to give compound 3 (3.0 g, quantified) as a white solid. [M+Na] + =981.6.

[0529] 4-((3-((R)-1-(((S)-4-(((9H-fluoren-9-yl)methoxy)carbonyl)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutyryl)piperazine-2-carbonyl)oxy)-3-(3,4-dimethoxyphenyl)propyl)phenyl)amino)-4-oxobutyric acid (Raa9). A solution of 3 (1.5 g, 1.56 mmol) in 12 mL of CH2Cl2 was treated with a solution of 40% TFA in 12 mL of CH2Cl2 at 0 °C. The mixture was allowed to react at room temperature until complete conversion. The reaction mixture was directly loaded into a silica gel fast column (AcOEt / PE / AcOH 1:2:0.5%) to provide Raa9 (1.4 g, 99%) as a white solid.

[0530] FKBD Example 12

[0531] (S)-((R)-1-(3-(4-tert-butoxy-4-oxobutyrylamino)phenyl)-3-(3,4-dimethoxyphenyl)propyl)1-(4-acryloyloxy)-3,3-dimethyl-2-oxobutyryl)-4-methylpiperazine-2-carboxylate (Raa10)

[0532]

[0533] Scheme 15. Synthesis of the Raa10 FKBD portion.

[0534] (S)-1-(9H-fluorene-9-yl)methyl 3-((R)-1-(3-(4-tert-butoxy-4-oxobutyrylamino)phenyl)-3-(3,4-dimethoxyphenyl)propyl)4-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutyryl)piperazine-1,3-dicarboxylate (2). At 0 °C, TBAF (3.2 mL, 1.0 M, 3.18 mmol) was added to a solution of 1 (1.3 g, 1.35 mmol) in DMF (5 mL). The resulting solution was heated to room temperature for 5 h. After this time, the reaction mixture was washed with NaHCO3 (containing water, 50 mL x 3) and NaCl (containing water, 50 mL x 3). The organic phase was concentrated. The reaction mixture was purified on silica using DCM / MEOH = 50 / 1 to give 2 (800 mg, 80%) as a colorless oil. [M+H] + =738.4.

[0535] (S)-((R)-1-(3-(4-tert-butoxy-4-oxobutyrylamino)phenyl)-3-(3,4-dimethoxyphenyl)propyl)1-(4-acryloyloxy)-3,3-dimethyl-2-oxobutyryl)-4-methylpiperazine-2-carboxylate (Raa10). A solution of 2 (800 mg, 1.08 mmol) in CHOOH (1.6 mL) was treated with an aqueous solution of formaldehyde (37% in water, 0.8 mL, 1.3 mmol) and stirred at 50 °C for 1 h. After this time, the reaction mixture was purified with DCM / MeOH = 100 / 1, giving 3 (400 mg, 50%) as a colorless oil. [M+H] + =751.9.

[0536] FKBD Example 13

[0537] (S)-4-(3-((R)-1-((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutyryl)piperidin-2-carbonyloxy)-3-(3,4-dimethoxyphenyl)propyl)phenylamino)-3-hydroxy-4-oxobutyric acid (Raa11)

[0538]

[0539] Scheme 16. Synthesis of the Raa11 FKBD portion.

[0540] 3-(3-(3,4-dimethoxyphenyl)propionyl)phenylcarbamate tert-butyl ester (2). Add (Boc)₂O (9.75 g, 44.68 mmol) to a solution of 1-(3-aminophenyl)-3-(3,4-dimethoxyphenyl)prop-1-one 1 (8.5 g, 29.79 mmol) in 1,4-dioxane (85 mL). Heat the resulting solution to 100 °C for 3 h. Evaporate the solvent, and the residue (10.3 g, crude product) was used directly in the next step without purification. [M+Na] + =408.

[0541] (R)-tert-butyl-3-(3-(3,4-dimethoxyphenyl)-1-hydroxypropyl)phenylcarbamate (3). A solution of ketone 2 (10 g, crude) in dry THF (200 mL) at -20 °C was treated with a solution of (+)-DIPChloride in heptane (1.7 M, 76.2 mL) at -20 °C. The resulting mixture was reacted at -20 °C until 2 was completely converted, and then quenched with 2,2′-(ethylenedioxy)diethylamine (23.1 g) until an insoluble complex was formed. After stirring at room temperature for an additional 30 min, the suspension was filtered through a diatomaceous earth mat and concentrated. The crude compound was purified by silica gel rapid column chromatography (AcOEt / PE 1:3) to give compound 3 (8.3 g, 80%) as a pale yellow oil. [M+Na] + =410.

[0542] (S)-((R)-1-(3-(tert-butoxycarbonylamino)phenyl)-3-(3,4-dimethoxyphenyl)propyl)1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutyryl)piperidine-2-carboxylate (5). Under an argon atmosphere, solutions of 3 (8.3 g, 21.42 mmol) and 4 (8 g, 25.7 mmol) in CH2Cl2 (100 mL) were cooled to -20 °C, followed by the addition of a solution of DCC (5.3 g, 25.7 mmol) in CH2Cl2 (5 mL), and then a solution of 4-(dimethylamino)pyridine (DMAP, 318 mg, 2.6 mmol) in CH2Cl2 (2 mL). The resulting white suspension was allowed to be stirred at -20 °C for 2 h. The reaction mixture was then filtered, evaporated, and the crude compound was purified by silica gel rapid column chromatography (AcOEt / PE 1:3) to give compound 5 (12 g, 83%) as a pale yellow oil. [M+Na] + =703.3.

[0543] (S)-((R)-1-(3-aminophenyl)-3-(3,4-dimethoxyphenyl)propyl)1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutyryl)piperidine-2-carboxylate (6). Add 6 ml of TFA to a solution of 5 g (7.34 mmol) in DCM (30 ml). Stir the mixture at 35 °C for 6 h. Evaporate the solvent, and the residue (5.0 g, crude product) was used directly in the next step without purification. [M+H] + =580.8.

[0544] (S)-4-(3-((R)-1-((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutyryl)piperidin-2-carbonyloxy)-3-(3,4-dimethoxyphenyl)propyl)phenylamino)-3-hydroxy-4-oxobutyric acid (Raa11). Add 7 (400 mg, 3.4 mmol) and DMAP (25 mg, 0.2 mmol) to a solution of 6 (1.0 g, crude) in DCM (20 mL). Allow the mixture to react at room temperature until complete conversion. Charge the reaction mixture directly into a silica gel fast column (DCM / MeOH = 10:1) to provide Raa11 (450 mg, 38%) as a white solid.

[0545] FKBD Example 14

[0546] (S)-4-(3-((R)-1-((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutyryl)piperidin-2-carbonyloxy)-3-(3,4-dimethoxyphenyl)propyl)phenylamino)-2-hydroxy-4-oxobutyric acid (Raa12)

[0547]

[0548] Scheme 17. Synthesis of the Raa12 FKBD portion.

[0549] The synthesis of 6 is the same as that of Raa11.

[0550] (S)-((R)-1-(3-((S)-4-(allyloxy)-3-hydroxy-4-oxobutyrylamino)phenyl)-3-(3,4-dimethoxyphenyl)propyl)1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutyryl)piperidine-2-carboxylate (8). 7 (1.2 g, 6.9 mmol), DIPEA (1.33 g, 0.32 mmol), and HATU (1.96 g, 5.16 mmol) were added to a solution of 6 (2 g, 3.44 mmol) in DMF (30 ml). The mixture was stirred at room temperature for 3 h, then diluted with EtOAc. The organic layer was washed with brine, dried over Na2SO4, and concentrated under vacuum. The residue was purified by silica gel column chromatography (DCM / MeOH 10:1) to give product 8 (800 mg, 32%) as a yellow oil. [M+H] + =737.

[0551] (S)-4-(3-((R)-1-((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutyryl)piperidin-2-carbonyloxy)-3-(3,4-dimethoxyphenyl)propyl)phenylamino)-2-hydroxy-4-oxobutyric acid (Raa12). N-methylaniline (232 mg, 2.17 mmol) and Pd(PPh3)4 (115 mg, 0.1 mmol) were added to a solution of N2 (800 mg, 1.09 mmol) in 100 mL of THF. The mixture was allowed to react at room temperature under a N2 atmosphere until complete conversion. The reaction mixture was directly loaded into a silica gel fast column (DCM / MeOH 10:1) to provide Raa12 (120 mg, 16%) as a white solid.

[0552] FKBD Example 15

[0553] (S)-3-(((9H-fluorene-9-yl)methoxy)carbonylamino)-4-(3-((R)-1-((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutyryl)piperidine-2-carbonyloxy)-3-(3,4-dimethoxyphenyl)propyl)phenylamino)-4-oxobutyric acid (Raa13)

[0554]

[0555] Scheme 18. Synthesis of the FKBD portion of Raa13.

[0556] (S)-tert-butyl 3-(((9H-fluorene-9-yl)methoxy)carbonylamino)-4-(3-(3-(3,4-dimethoxyphenyl)propionyl)phenylamino)-4-oxobutyrate (3). Solutions of 1 (4.0 g, 14.03 mmol) and 2 (7.0 g, 16.8 mmol) in DCM (150 mL) were treated with DIPEA (8 mL, 42.1 mmol) and HATU (8.0 g, 21.1 mmol) at 0 °C, with stirring allowed at room temperature for 15 h. After this time, the reaction mixture was washed with H2O and extracted with AcOEt (50 mL * 3). The organic phase was dried over Na2SO4 and concentrated. The residue was purified by silica gel rapid column chromatography (AcOEt / PE 1:10) to give compound 3 (9 g, 90%) as a brown oil. [M+Na] + =700.9.

[0557] (S)-tert-butyl 3-(((9H-fluorene-9-yl)methoxy)carbonylamino)-4-(3-((R)-3-(3,4-dimethoxyphenyl)-1-hydroxypropyl)phenylamino)-4-oxobutyrate (4). A solution of ketone 3 (4.7 g, 6.9 mmol) in dry THF (130 mL) at -20 °C was treated with a solution of (+)-DIPChloride (27.7 mmol) in heptane (1.7 M, 16.3 mL) at -20 °C. The resulting mixture was reacted at -20 °C until 3 was completely converted, and then quenched with 2,2′-(ethylenedioxy)diethylamine (2.8 mL) until an insoluble complex was formed. After stirring at room temperature for an additional 30 min, the suspension was filtered through a diatomaceous earth mat and concentrated. The crude compound was purified by silica gel rapid column chromatography (AcOEt / PE1:10) to give compound 4 (1.7 g, 40%) as a pale yellow oil. [M+Na] + =702.8.

[0558] (S)-((R)-1-(3-((S)-2-(((9H-fluoren-9-yl)methoxy)carbonylamino)-4-tert-butoxy-4-oxobutyrylamino)phenyl)-3-(3,4-dimethoxyphenyl)propyl)1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutyryl)piperidine-2-carboxylate (6). Under an argon atmosphere, solutions of 4 (1.7 g, 2.5 mmol) and 5 (1.2 g, 3.75 mmol) in CH2Cl2 (50 mL) were cooled to -20 °C, followed by the addition of a solution of DCC (0.78 g, 3.75 mmol) in CH2Cl2 (5 mL), and then a solution of 4-(dimethylamino)pyridine (DMAP, 30 mg, 0.25 mmol) in CH2Cl2 (2 mL). The resulting white suspension was allowed to be stirred at -20°C for 2 hours. The reaction mixture was then filtered, evaporated, and the crude compound was purified by silica gel rapid column chromatography (AcOEt / PE 1:5) to give compound 6 (1.0 g, 50%) as a pale yellow oil.

[0559] (S)-3-(((9H-fluorene-9-yl)methoxy)carbonylamino)-4-(3-((R)-1-((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutyryl)piperidin-2-carbonyloxy)-3-(3,4-dimethoxyphenyl)propyl)phenylamino)-4-oxobutyric acid (Raa13). A solution of 6 (1.0 g, 1.02 mmol) in 10 mL of CH2Cl2 was treated with a solution of 40% TFA in 10 mL of CH2Cl2 at 0 °C. The mixture was allowed to react at room temperature until complete conversion. The reaction mixture was directly loaded into a silica gel fast column (DCM / MeOH = 50 / 1) to provide Raa13 (401 mg, 42%) as a white solid.

[0560] FKBD Example 16

[0561] (S)-2-(((9H-fluorene-9-yl)methoxy)carbonylamino)-4-(3-((R)-1-((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutyryl)piperidine-2-carbonyloxy)-3-(3,4-dimethoxyphenyl)propyl)phenylamino)-4-oxobutyric acid (Raa14).

[0562]

[0563] Synthesis of the FKBD portion of Scheme 19. Raa14.

[0564] (S)-tert-butyl-2-(((9H-fluorene-9-yl)methoxy)carbonylamino)-4-(3-(3-(3,4-dimethoxyphenyl)propionyl)phenylamino)-4-oxobutyrate (3). Solutions of 1 (4.0 g, 14.03 mmol) and 2 (7.0 g, 16.8 mmol) in DCM (150 mL) were treated with DIPEA (8 mL, 42.1 mmol) and HATU (8.0 g, 21.1 mmol) at 0 °C, with stirring allowed at room temperature for 15 h. After this time, the reaction mixture was washed with H2O and extracted with AcOEt (50 mL * 3). The organic phase was dried over Na2SO4 and concentrated. The residue was purified by silica gel rapid column chromatography (AcOEt / PE 1:10) to give compound 3 (9 g, 90%) as a brown oil. [M+Na] + =700.9.

[0565] (S)-tert-butyl 2-(((9H-fluorene-9-yl)methoxy)carbonylamino)-4-(3-((S)-3-(3,4-dimethoxyphenyl)-1-hydroxypropyl)phenylamino)-4-oxobutyrate (4). A solution of ketone 3 (4.0 g, 5.9 mmol) in dry THF (80 mL) at -20 °C was treated with a solution of (+)-DIPChloride (23.6 mmol) in heptane (1.7 M, 14.0 mL) at -20 °C. The resulting mixture was reacted at -20 °C until 3 was completely converted, and then quenched with 2,2′-(ethylenedioxy)diethylamine (2.8 mL) until an insoluble complex was formed. After stirring at room temperature for an additional 30 min, the suspension was filtered through a diatomaceous earth mat and concentrated. The crude compound was purified by silica gel rapid column chromatography (AcOEt / PE1:10) to give compound 4 (2.0 g, 50%) as a pale yellow oil. [M+Na] + =702.8.

[0566] (S)-((R)-1-(3-((S)-3-(((9H-fluoren-9-yl)methoxy)carbonylamino)-4-tert-butoxy-4-oxobutyrylamino)phenyl)-3-(3,4-dimethoxyphenyl)propyl)1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutyryl)piperidine-2-carboxylate (6). Under an argon atmosphere, solutions of 4 (2.0 g, 2.9 mmol) and 5 (1.2 g, 3.82 mmol) in CH2Cl2 (50 mL) were cooled to -20 °C, followed by the addition of a solution of DCC (0.91 g, 4.11 mmol) in CH2Cl2 (5 mL), and then a solution of 4-(dimethylamino)pyridine (DMAP, 35 mg, 0.29 mmol) in CH2Cl2 (2 mL). The resulting white suspension was allowed to be stirred at -20°C for 2 hours. The reaction mixture was then filtered, evaporated, and the crude compound was purified by silica gel rapid column chromatography (AcOEt / PE 1:5) to give compound 6 (1.0 g, 50%) as a pale yellow oil.

[0567] (S)-2-(((9H-fluorene-9-yl)methoxy)carbonylamino)-4-(3-((R)-1-((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutyryl)piperidin-2-carbonyloxy)-3-(3,4-dimethoxyphenyl)propyl)phenylamino)-4-oxobutyric acid (Raa14). A solution of 6 (1.0 g, 1.02 mmol) in 10 mL of CH2Cl2 was treated with a solution of 40% TFA in 10 mL of CH2Cl2 at 0 °C. The mixture was allowed to react at room temperature until complete conversion. The reaction mixture was directly loaded into a silica gel fast column (DCM / MeOH = 50 / 1) to provide Raa14 (367 mg, 42%) as a white solid.

[0568] FKBD Example 17

[0569] (2S,3S)-4-((3-((R)-1-(((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutyryl)piperidin-2-carbonyl)oxy)-3-(3,4-dimethoxyphenyl)propyl)phenyl)amino)-2,3-dihydroxy-4-oxobutyric acid (Raa15)

[0570]

[0571]

[0572] Synthesis of the FKBD portion of Scheme 20. Raa15.

[0573] (3R,4S)-3,4-dihydroxydihydrofuran-2,5-dione (2). At 25°C, 2,2,2-trifluoroacetic anhydride (27.9 g, 133.2 mmol) was added to a solution of (2R,3S)-2,3-dihydroxysuccinic acid 1 (10 g, 66.6 mmol) in DCM (100 mL). The resulting solution was stirred at room temperature for 12 h. The mixture was concentrated under vacuum. The crude product was washed with petroleum ether (100 mL) to provide 2 (6 g, 68%) as a white solid.

[0574] (2S,3S)-4-((3-((R)-1-(((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutyryl)piperidin-2-carbonyl)oxy)-3-(3,4-dimethoxyphenyl)propyl)phenyl)amino)-2,3-dihydroxy-4-oxobutyric acid (Raa15). A mixture of 6 (2 g, 3.4 mmol), 2 (0.896 g, 6.8 mmol), and DMAP (80 mg, 0.68 mmol) in THF (60 mL) was stirred at 50 °C for 6 h. The mixture was filtered and concentrated under vacuum. The resulting residue was purified by preparative HPLC to provide Raa15 (476 mg, 19%) as a white solid.

[0575] FKBD Example 18

[0576] 3-((((9H-fluorene-9-yl)methoxy)amino)-4-((3-((R)-1-(((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutyryl)piperidin-2-carbonyl)oxy)-3-(3,4-dimethoxyphenyl)propyl)phenyl)amino)-2-hydroxy-4-oxobutyric acid (Raa16)

[0577]

[0578] Scheme 21. Synthesis of the Raa16 FKBD portion.

[0579] 2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-3-hydroxysuccinic acid (2). 250 ml of 10% NaCO3 (containing water) was added to a solution of 1 (10 g, 67.1 mmol) in 1,4-dioxane (150 ml), and then FmocCl was added dropwise in 1,4-dioxane (150 ml) at 0 °C. The reaction mixture was stirred at 0 °C for 10 min, and then raised to room temperature and stirred for another 4 h. The product mixture was poured into water (500 ml) and extracted three times with EA (200 ml). The aqueous layer was adjusted to pH 2-3 with 2 M HCl and then extracted three times with DCM (200 ml). The organic layers were combined, washed three times with brine (200 ml), dried over Na2SO4, filtered, and concentrated to give product 2 (22 g, 88%) as a white solid. [M+Na] + =394.

[0580] 2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-2-(2,2-dimethyl-5-oxo-1,3-dioxolane-4-yl)acetic acid (4). 3 (14 g, 135 mmol) and PTSA (0.46 g, 2.7 mmol) were added to a solution of 2 (5 g, 13.5 mmol) in EA (50 mL). The reaction mixture was refluxed for 16 h. The product mixture was concentrated directly, and the brown residue was purified by silica gel chromatography (EA / PE = 10–50% as eluent) to give 4 (3.8 g, 68.6%) as a white solid. [M+Na] + =434.

[0581] (1R)-1-(3-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-2-(2,2-dimethyl-5-oxo-1,3-dioxolane-4-yl)acetamido)phenyl)-3-(3,4-dimethoxyphenyl(2S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutyryl)piperidine-2-carboxylate (6). HATU (3 g, 8 mmol) and DIEA (1.38 g, 10.8 mmol) were added to a solution of 4 (2.2 g, 5.4 mmol) in DMF (150 ml). Finally, 5 (2.6 g) was added. 4.5 mmol). The reaction mixture was stirred at room temperature for 1 h. The product mixture was poured into water (300 ml), extracted with DCM (100 ml * 3), the organic phases were combined and washed with brine (100 ml * 5). The mixture was dried over Na₂SO₄, filtered, and concentrated to give the crude product. Purification was performed by silica gel chromatography (methanol / DCM = 0–2% as eluent) to give compound 6 (3.7 g, 71%) as a white solid. [M + Na] + =996.

[0582] 3-((((9H-fluorene-9-yl)methoxy)amino)-4-((3-((R)-1-(((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutyryl)piperidin-2-carbonyl)oxy)-3-(3,4-dimethoxyphenyl)propyl)phenyl)amino)-2-hydroxy-4-oxobutyric acid (Raa16). THF (40 mL) was added to a solution of 6 (3.7 g, 38 mmol) in THF / H₂O (10 mL / 10 mL). The reaction mixture was stirred at room temperature for 1 h. The product mixture was directly evaporated, and the residue was purified by silica gel chromatography (HCOOH / DCM = 0–5% as eluent) to give compound Raa16 (500 mg, 14%) as a pale yellow solid.

[0583] FKBD Example 19

[0584] 2-(3-((R)-1-(((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutyryl)piperidine-2-carbonyl)oxy)-3-(3,4,5-trimethoxyphenyl)propyl)phenoxy)acetic acid (Rae1)

[0585]

[0586]

[0587] Scheme 22. Synthesis of the Rae1 FKBD portion.

[0588] (E)-1-(3-hydroxyphenyl)-3-(3,4,5-trimethoxyphenyl)prop-2-en-1-one (3). At 0 °C, a solution of 10% aqueous NaOH (41 mL, 4.1 g, 101.9 mmol) was added to a solution of 3,4,5-trimethoxybenzaldehyde 1 (5 g, 25.5 mmol) and 3′-hydroxyacetophenone 2 (3.47 g, 25.5 mmol) in EtOH (50 mL). The resulting solution was heated to 65 °C for 2 h. The solvent was evaporated, and the residue (5.5 g, crude product) was used directly in the next step without purification. [M+H] + =314.9.

[0589] 3-(1-Hydroxy-3-(3,4,5-trimethoxyphenyl)propyl)phenol (4). A solution of 3 (5.5 g, crude) in THF (40 mL) and 10% Pd / C (2 g) was hydrogenated with H2 for 4 h at room temperature. The reaction mixture was then filtered and concentrated to a solid (5.96 g, crude). [M+H-H2O] + =300.9.

[0590] 2-(3-(1-hydroxy-3-(3,4,5-trimethoxyphenyl)propyl)phenoxy)tert-butyl acetate (5). A solution of 4 (5.96 g, 18.8 mmol, crude) and K2CO3 (3.12 g, 22.6 mmol) in DMF (30 mL) was treated with tert-butyl bromoacetate (3.68 g, 18.8 mmol) and stirred at room temperature for 5 h. After this time, the reaction mixture was poured into ice, and a yellow solid precipitated. The crude product was purified by preparative HPLC to give 5 (4.65 g, 42% (3 steps)) as a yellow solid. [M+Na] + =454.8.

[0591] 2-(3-(3-(3,4,5-trimethoxyphenyl)propionyl)phenoxy)tert-butyl acetate (6). A solution of 5 (4.65 g, 10.75 mmol) in CH2Cl2 (110 mL) was treated with Dys-Martin periodine (11.4 g, 26.88 mmol) and allowed to stir at room temperature for 3 h, followed by quenching with a solution of 10% aqueous NaS2O3. The solution was extracted twice with CH2Cl2. The combined organic layers were washed with saturated NaHCO3, brine, dried over Na2SO4, and concentrated under vacuum. The crude compound was purified by silica gel rapid column chromatography (AcOEt / PE 1:3) to give compound 6 (4.5 g, 97%) as a white solid. [M+Na] + =453.2.

[0592] (R)-2-(3-(1-hydroxy-3-(3,4,5-trimethoxyphenyl)propyl)phenoxy)tert-butyl acetate (7). A solution of ketone 6 (3.98 g, 9.25 mmol) in dry THF (40 mL) at -20 °C was treated with a solution of (+)-DIPChloride (18.5 mmol) in heptane (1.7 M, 10.88 mL) at -20 °C. The resulting mixture was reacted at -20 °C until 6 was completely converted, and then quenched with 2,2′-(ethylenedioxy)diethylamine (2.8 mL) until an insoluble complex was formed. After stirring at room temperature for an additional 30 min, the suspension was filtered through a diatomaceous earth mat and concentrated. The crude compound was purified by silica gel rapid column chromatography (AcOEt / PE 1:3) to give compound 7 (2.8 g, 70%, ee>99%) as a pale yellow oil. [M+Na] + =455.2.

[0593] (R)-1-(3-(2-(tert-butoxy)-2-oxoethoxy)phenyl)-3-(3,4,5-trimethoxyphenyl)propyl(S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutyryl)piperidine-2-carboxylate (9). Solutions of 7 (1.85 g, 4.3 mmol) and 8 (2 g, 6.4 mmol) in CH2Cl2 (15 mL) were cooled to -20 °C under an argon atmosphere, followed by the addition of a solution of DCC (1.3 g, 6.4 mmol) in CH2Cl2 (5 mL), and then a solution of 4-(dimethylamino)pyridine (DMAP, 52.3 mg, 0.43 mmol) in CH2Cl2 (2 mL). The resulting white suspension was allowed to be stirred at -20 °C for 2 h. The reaction mixture was then filtered, evaporated, and the crude compound was purified by silica gel rapid column chromatography (AcOEt / PE 1:2) to give compound 9 (2.5 g, 80%) as a pale yellow oil. [M+Na] + =748.4.

[0594] 2-(3-((R)-1-(((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutyryl)piperidin-2-carbonyl)oxy)-3-(3,4,5-trimethoxyphenyl)propyl)phenoxy)acetic acid (Rae1). A solution of 9 g (2.5 g, 3.44 mmol) in 11.5 mL of CH2Cl2 was treated with a solution of 40% TFA in 11.5 mL of CH2Cl2 at 0 °C. The mixture was allowed to react at room temperature until complete conversion. The reaction mixture was directly loaded into a silica gel fast column (AcOEt / PE / AcOH 1:2:0.5%) to provide Rae1 (969 mg, 42%) as a white solid.

[0595] FKBD Example 20

[0596] 2-(3-((R)-1-(((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutyryl)piperidine-2-carbonyl)oxy)-3-(2,3,4-trimethoxyphenyl)propyl)phenoxy)acetic acid (Rae2)

[0597]

[0598] Scheme 23. Synthesis of the Rae2 FKBD portion.

[0599] (E)-1-(3-hydroxyphenyl)-3-(2,3,4-trimethoxyphenyl)prop-2-en-1-one (3). At 0 °C, a solution of 10% aqueous NaOH (41 mL, 4.1 g, 101.9 mmol) was added to a solution of 2,3,4-trimethoxybenzaldehyde 1 (5 g, 25.5 mmol) and 3′-hydroxyacetophenone 2 (3.47 g, 25.5 mmol) in EtOH (30 mL). The resulting solution was heated to 65 °C for 3 h. The solvent was evaporated, and the residue was purified by silica gel rapid column chromatography (AcOEt / PE 1:5) to give compound 3 (6.6 g, 83%) as a yellow oil. [M+H] + =314.9.

[0600] 3-(1-Hydroxy-3-(2,3,4-trimethoxyphenyl)propyl)phenol (4). A solution of 3 (6.6 g, 21 mmol) in THF (30 mL) and 10% Pd / C (3 g) was hydrogenated with H₂ for 16 h at room temperature. The reaction mixture was then filtered and concentrated to a colorless oil (8 g, crude product). [M + H₂O] + =301.0.

[0601] 2-(3-(1-hydroxy-3-(2,3,4-trimethoxyphenyl)propyl)phenoxy)tert-butyl bromoacetate (5). A solution of 4 (8 g, 25 mmol, crude) in DMF (30 mL) and K2CO3 (4.19 g, 30 mmol) was treated with tert-butyl bromoacetate (5.92 g, 30 mmol) and stirred at room temperature for 6 h. After this time, the reaction mixture was quenched by H2O and extracted twice with EtOAc. The combined organic layers were washed with brine, dried over Na2SO4, and concentrated under vacuum. The crude compound was purified by silica gel rapid column chromatography (AcOEt / PE 1:3) to give compound 6 (8.2 g, 90% (2 steps)) as a colorless oil. [M+H-H2O-tBu] + =358.8.

[0602] 2-(3-(3-(2,3,4-trimethoxyphenyl)propionyl)phenoxy)tert-butyl acetate (6). A solution of 5 (5.75 g, 13.29 mmol) in CH2Cl2 (30 mL) was treated with Dys-Martin periodine (11.28 g, 26.59 mmol) and allowed to be stirred at room temperature for 2 h, followed by quenching with a solution of 10% aqueous NaS2O3. The solution was extracted twice with CH2Cl2. The combined organic layers were washed with saturated NaHCO3 brine, dried over Na2SO4, and concentrated under vacuum. The crude compound was purified by silica gel rapid column chromatography (AcOEt / PE 1:3) to give compound 6 (5 g, 87%) as a yellow oil.

[0603] (R)-2-(3-(1-hydroxy-3-(2,3,4-trimethoxyphenyl)propyl)phenoxy)tert-butyl acetate (7). A solution of ketone 6 (5 g, 11.61 mmol) in dry THF (50 mL) at -20 °C was treated with a solution of (+)-DIPChloride (23.23 mmol) in heptane (1.7 M, 13.66 mL) at -20 °C. The resulting mixture was reacted at -20 °C until 6 was completely converted, and then quenched with 2,2′-(ethylenedioxy)diethylamine (3.4 mL) until an insoluble complex was formed. After stirring at room temperature for an additional 30 min, the suspension was filtered through a diatomaceous earth mat and concentrated. The crude compound was purified by silica gel rapid column chromatography (AcOEt / PE 1:3) to give compound 7 (4 g, 80%, ee 83%) as a pale yellow oil. [M+H-H2O-tBu] + =358.9.

[0604] (R)-1-(3-(2-(tert-butoxy)-2-oxoethoxy)phenyl)-3-(3,4,5-trimethoxyphenyl)propyl(S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutyryl)piperidine-2-carboxylate (9). Solutions of 7 (2 g, 4.62 mmol) and 8 (2.16 g, 6.93 mmol) in CH2Cl2 (23 mL) were cooled to -20 °C under an argon atmosphere, followed by the addition of a solution of DCC (1.43 g, 6.93 mmol) in CH2Cl2 (5 mL), and then a solution of 4-(dimethylamino)pyridine (DMAP, 57 mg, 0.46 mmol) in CH2Cl2 (2 mL). The resulting white suspension was allowed to be stirred at -20 °C for 2 h. The reaction mixture was then filtered, evaporated, and the crude compound was purified by silica gel rapid column chromatography (AcOEt / PE 1:2) to give compound 9 (2.13 g, 64%) as a pale yellow oil. [M+Na]+ =748.4.

[0605] 2-(3-((R)-1-(((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutyryl)piperidin-2-carbonyl)oxy)-3-(2,3,4-trimethoxyphenyl)propyl)phenoxy)acetic acid (Rae2). A solution of 9 g (2.13 g, 2.93 mmol) in 12 mL of CH2Cl2 was treated with a solution of 40% TFA in 12 mL of CH2Cl2 at 0 °C. The mixture was allowed to react at room temperature until complete conversion. The reaction mixture was directly loaded into a silica gel fast column (AcOEt / PE / AcOH 1:2:0.5%) to provide Rae2 (508 mg, 25%) as a pale yellow solid.

[0606] FKBD Example 21

[0607] 2-(3-((R)-1-(((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutyryl)piperidine-2-carbonyl)oxy)-3-(2,4,5-trimethoxyphenyl)propyl)phenoxy)acetic acid (Rae3)

[0608]

[0609] Scheme 24. Synthesis of the Rae3 FKBD portion.

[0610] (E)-1-(3-hydroxyphenyl)-3-(2,4,5-trimethoxyphenyl)prop-2-en-1-one (3). At 0 °C, a solution of 10% aqueous KOH (15 mL, 5.1 g, 91.84 mmol) was added to a solution of 2,4,5-trimethoxybenzaldehyde 1 (4.5 g, 22.96 mmol) and 3′-hydroxyacetophenone 2 (3.1 g, 22.96 mmol) in EtOH (50 mL). The resulting solution was heated to 60 °C for 4 h. The solvent was evaporated, and the residue was purified by silica gel rapid column chromatography (AcOEt / PE 1:3) to give compound 3 (5.9 g, 82%) as a yellow oil. [M+H] + =315.1.

[0611] (E)-2-(3-(3-(2,4,5-trimethoxyphenyl)acryloyl)phenoxy)tert-butyl bromoacetate (4). A solution of 3 (7.4 g, 23.6 mmol) and K2CO3 (3.9 g, 28.3 mmol) in DMF (200 mL) was treated with tert-butyl bromoacetate (5.5 g, 28.3 mmol) and stirred at room temperature for 4 h. After this time, the reaction mixture was quenched by H2O and extracted twice with EtOAc. The combined organic layers were washed with brine, dried over Na2SO4, and concentrated under vacuum. The crude compound was purified by silica gel rapid column chromatography (AcOEt / PE 1:3) to give compound 4 (8 g, 80%) as a colorless oil. [M+H] + =429.3.

[0612] 2-(3-(3-(2,4,5-trimethoxyphenyl)propionyl)phenoxy)tert-butyl acetate (5). A solution of 4 (8 g, 18.69 mmol) in THF (200 mL) and 10% Pd / C (1 g) was hydrogenated with H2 for 8 h at room temperature. The reaction mixture was then filtered and concentrated. The residue was purified by silica gel rapid column chromatography (AcOEt / PE 1:3) to give compound 5 (6 g, 75%) as a colorless oil. [M+Na] + =453.2.

[0613] (R)-2-(3-(1-hydroxy-3-(2,4,5-trimethoxyphenyl)propyl)phenoxy)tert-butyl acetate (6). A solution of ketone 5 (6 g, 13.95 mmol) in dry THF (60 mL) at -20 °C was treated with a solution of (+)-DIPChloride (41.86 mmol) in heptane (1.7 M, 24.6 mL) at -20 °C. The resulting mixture was reacted at -20 °C until 5 was completely converted, and then quenched with 2,2′-(ethylenedioxy)diethylamine (5.9 mL) until an insoluble complex was formed. After stirring at room temperature for an additional 30 min, the suspension was filtered through a diatomaceous earth mat and concentrated. The crude compound was purified by silica gel rapid column chromatography (AcOEt / PE 1:1) to give compound 6 (5.5 g, 92%, ee>99%) as a pale yellow oil. [M+Na] + =455.2.

[0614] (R)-1-(3-(2-(tert-butoxy)-2-oxoethoxy)phenyl)-3-(2,4,5-trimethoxyphenyl)propyl(S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutyryl)piperidine-2-carboxylate (8). Solutions of 6 (1.85 g, 4.28 mmol) and 7 (2 g, 6.42 mmol) in CH2Cl2 (10 mL) were cooled to -20 °C under an argon atmosphere, followed by the addition of a solution of DCC (1.33 g, 6.42 mmol) in CH2Cl2 (5 mL), and then a solution of 4-(dimethylamino)pyridine (DMAP, 52 mg, 0.43 mmol) in CH2Cl2 (2 mL). The resulting white suspension was allowed to be stirred at -20 °C for 2 h. The reaction mixture was then filtered, evaporated, and the crude compound was purified by silica gel rapid column chromatography (AcOEt / PE 1:1) to give compound 8 (2.35 g, 76%) as a pale yellow oil. [M+Na] + =747.9.

[0615] 2-(3-((R)-1-(((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutyryl)piperidin-2-carbonyl)oxy)-3-(2,4,5-trimethoxyphenyl)propyl)phenoxy)acetic acid (Rae3). A solution of 8 (2.35 g, 3.24 mmol) in 12 mL of CH2Cl2 was treated with a solution of 40% TFA in 12 mL of CH2Cl2 at 0 °C. The mixture was allowed to react at room temperature until complete conversion. The reaction mixture was directly loaded into a silica gel fast column (AcOEt / PE / AcOH 1:2:0.5%) to provide Rae3 (815 mg, 37%) as a pale yellow solid.

[0616] FKBD Example 22

[0617] 2-(3-((R)-1-(((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutyryl)piperidine-2-carbonyl)oxy)-3-(2,3,5-trimethoxyphenyl)propyl)phenoxy)acetic acid (Rae4)

[0618]

[0619] Scheme 25. Synthesis of the Rae4 FKBD portion.

[0620] (E)-1-(3-hydroxyphenyl)-3-(2,3,5-trimethoxyphenyl)prop-2-en-1-one (3). At 0 °C, a solution of 10% aqueous NaOH (50 mL, 122.4 mmol) was added to a solution of 2,3,5-trimethoxybenzaldehyde 1 (6 g, 30.6 mmol) and 1-(3-hydroxyphenyl)ethyl-1-one 2 (4.2 g, 30.6 mmol) in EtOH (50 mL). The resulting solution was stirred at room temperature for 12 h. The solution was adjusted to pH 4 by dropwise addition of 4 M aqueous HCl at 0 °C, producing a large amount of yellow solid. The mixture was then filtered and the solid was washed with water (50 mL) to provide 3 (5.5 g, 57%) as a yellow solid. [M+H] + =315.2.

[0621] (E)-2-(3-(3-(2,3,5-trimethoxyphenyl)acryloyl)phenoxy)tert-butyl bromoacetate (4). A solution of 3 (5.5 g, 17.4 mmol) and K2CO3 (4.82 g, 34.9 mmol) in DMF (40 mL) was treated with tert-butyl bromoacetate (4.06 g, 20.9 mmol) and stirred at room temperature for 12 h. After this time, the reaction mixture was poured into ice, and a yellow solid precipitated. The mixture was filtered and the solid was washed with water (30 mL). The crude product was washed with petroleum ether (50 mL) to give 4 (7 g, 93%) as a yellow solid. [M+H] + =428.8.

[0622] 2-(3-(3-(2,3,5-trimethoxyphenyl)propionyl)phenoxy)tert-butyl acetate (5). A solution of 4 (7 g, 11.68 mmol) in THF (100 mL) and 10% Pd / C (1 g) was hydrogenated with H2 for 4 h at room temperature. The reaction mixture was then filtered and concentrated. The crude product was purified by column chromatography on silica gel to give 5 (3.5 g, 50%) as a yellow oil. [M+Na] + =452.9.

[0623] (R)-2-(3-(1-hydroxy-3-(2,3,5-trimethoxyphenyl)propyl)phenoxy)tert-butyl acetate (6). A solution of ketone 5 (3.5 g, 8.14 mmol) in dry THF (30 mL) at -20 °C was treated with a solution of (+)-DIPChloride (16.2 mmol) in heptane (1.7 M, 9.5 mL) at -20 °C. The resulting mixture was reacted at -20 °C until 6 was completely converted, and then quenched with 2,2′-(ethylenedioxy)diethylamine (2.4 g) until an insoluble complex was formed. After stirring at room temperature for an additional 30 min, the suspension was filtered through a diatomaceous earth mat and concentrated. The crude compound was purified by silica gel rapid column chromatography (AcOEt / PE 1:4) to give compound 6 (2.2 g, 63%, ee 97% relative to the racemic mixture) as a pale yellow oil. [M+Na] + =454.9.

[0624] (R)-1-(3-(2-(tert-butoxy)-2-oxoethoxy)phenyl)-3-(2,3,5-trimethoxyphenyl)propyl(S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutyryl)piperidine-2-carboxylate (8). Solutions of 6 (2.2 g, 5.09 mmol) and 8 (1.89 g, 6.1 mmol) in CH2Cl2 (15 mL) were cooled to -20 °C under an argon atmosphere, followed by the addition of a solution of DCC (1.36 g, 6.6 mmol) in CH2Cl2 (5 mL), and then a solution of 4-(dimethylamino)pyridine (62 mg, 0.5 mmol) in CH2Cl2 (2 mL). The resulting white suspension was allowed to be stirred at -20 °C for 2 h. The reaction mixture was then filtered, evaporated, and the crude compound was purified by silica gel rapid column chromatography (AcOEt / PE1:2) to give compound 8 (1.8 g, 49%) as a pale yellow oil. [M+Na] + =748.4.

[0625] 2-(3-((R)-1-(((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutyryl)piperidin-2-carbonyl)oxy)-3-(2,3,5-trimethoxyphenyl)propyl)phenoxy)acetic acid (Rae4). A solution of 8 g (1.8 g, 2.48 mmol) in 10 mL of CH2Cl2 was treated with a solution of 40% TFA in 10 mL of CH2Cl2 at 0 °C. The mixture was allowed to react at room temperature until complete conversion. The reaction mixture was directly loaded into a silica gel fast column (AcOEt / PE / AcOH 1:3:0.5%) to provide Rae4 (652 mg, 39%) as a pale yellow solid.

[0626] FKBD Example 23

[0627] 2-(3-((R)-1-(((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutyryl)piperidine-2-carbonyl)oxy)-3-(2,3,6-trimethoxyphenyl)propyl)phenoxy)acetic acid (Rae5)

[0628]

[0629] Scheme 26. Synthesis of the Rae5 FKBD portion.

[0630] (E)-1-(3-hydroxyphenyl)-3-(2,3,6-trimethoxyphenyl)prop-2-en-1-one (3). At 0 °C, a solution of 40% aqueous KOH (15 mL, 5.7 g, 101.92 mmol) was added to a solution of 2,3,6-trimethoxybenzaldehyde 1 (5 g, 25.48 mmol) and 3′-hydroxyacetophenone 2 (3.47 g, 25.48 mmol) in EtOH (40 mL). The resulting solution was reacted at room temperature for 4 h. The solvent was evaporated, and the residue was purified by silica gel rapid column chromatography (AcOEt / PE 1:2) to give compound 3 (4 g, 50%) as a yellow oil. [M+H] + =315.2.

[0631] (E)-2-(3-(3-(2,3,6-trimethoxyphenyl)acryloyl)phenoxy)tert-butyl bromoacetate (4). A solution of 3 (3.5 g, 11.15 mmol) and K2CO3 (1.85 g, 13.37 mmol) in DMF (60 mL) was treated with tert-butyl bromoacetate (2.6 g, 13.37 mmol) and stirred at room temperature for 4 h. After this time, the reaction mixture was quenched by H2O and extracted twice with EtOAc. The combined organic layers were washed with brine, dried over Na2SO4, and concentrated under vacuum. The crude compound was purified by silica gel rapid column chromatography (AcOEt / PE 1:4) to give compound 4 (4.7 g, 98%) as a yellow oil. [M+H] + =429.0.

[0632] 2-(3-(3-(2,3,6-trimethoxyphenyl)propionyl)phenoxy)tert-butyl acetate (5). A solution of 4 (4.6 g, 10.75 mmol) in THF (70 mL) and 10% Pd / C (0.5 g) was hydrogenated with H2 for 4 h at room temperature. The reaction mixture was then filtered and concentrated. The residue was purified by silica gel rapid column chromatography (AcOEt / PE 1:3) to give compound 5 (2.9 g, 63%) as a colorless oil. [M+Na] + =453.3.

[0633] (R)-2-(3-(1-hydroxy-3-(2,3,6-trimethoxyphenyl)propyl)phenoxy)tert-butyl acetate (6). A solution of ketone 5 (2.9 g, 6.7 mmol) in dry THF (30 mL) at -20 °C was treated with a solution of (+)-DIPChloride (13.48 mmol) in heptane (1.7 M, 7.9 mL) at -20 °C. The resulting mixture was reacted at -20 °C until 5 was completely converted, and then quenched with 2,2′-(ethylenedioxy)diethylamine (1.96 mL) until an insoluble complex was formed. After stirring at room temperature for an additional 30 min, the suspension was filtered through a diatomaceous earth mat and concentrated. The crude compound was purified by silica gel rapid column chromatography (AcOEt / PE 1:5) to give compound 6 (2.4 g, 83%, ee>99%) as a pale yellow oil. [M+Na] + =454.9.

[0634] (R)-1-(3-(2-(tert-butoxy)-2-oxoethoxy)phenyl)-3-(2,3,6-trimethoxyphenyl)propyl(S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutyryl)piperidine-2-carboxylate (8). Under an argon atmosphere, solutions of 6 (1.46 g, 3.45 mmol) and 7 (1.6 g, 5.17 mmol) in CH2Cl2 (18 mL) were cooled to -20 °C, followed by the addition of a solution of DCC (1.065 g, 5.17 mmol) in CH2Cl2 (5 mL), and then a solution of 4-(dimethylamino)pyridine (DMAP, 43 mg, 0.35 mmol) in CH2Cl2 (2 mL). The resulting white suspension was allowed to be stirred at -20 °C for 2 h. The reaction mixture was then filtered, evaporated, and the crude compound was purified by silica gel rapid column chromatography (AcOEt / PE1:1) to give compound 8 (1.7 g, 68%) as a pale yellow oil.

[0635] 2-(3-((R)-1-(((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutyryl)piperidin-2-carbonyl)oxy)-3-(2,3,6-trimethoxyphenyl)propyl)phenoxy)acetic acid (Rae5). A solution of 8 g (1.7 g, 2.34 mmol) in 12 mL of CH2Cl2 was treated with a solution of 40% TFA in 12 mL of CH2Cl2 at 0 °C. The mixture was allowed to react at room temperature until complete conversion. The reaction mixture was directly loaded into a silica gel fast column (AcOEt / PE / AcOH 1:2:0.5%) to provide Rae5 (494 mg, 31%) as a pale yellow solid.

[0636] FKBD Example 24

[0637] 2-(3-((R)-1-(((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutyryl)piperidine-2-carbonyl)oxy)-3-(2-hydroxy-3,4-dimethoxyphenyl)propyl)phenoxy)acetic acid (Rae9)

[0638]

[0639] Scheme 27. Synthesis of the Rae9 FKBD portion.

[0640] 2-(tert-butoxy)-3,4-dimethoxybenzaldehyde (2). Under an Ar atmosphere, 1,1-di-tert-butoxy-N,N-dimethylmethylamine 2 (29.1 mL, 122 mmol) was added to a solution of 2-hydroxy-3,4-dimethoxybenzaldehyde 1 (2.77 g, 15.2 mmol) in anhydrous toluene (30 mL). The mixture was stirred at 80 °C for 6 h, and then the solvent was evaporated. The residue was purified by silica gel rapid column chromatography (AcOEt / PE1:1) to give compound 2 (2.965 g, 82%) as a yellow solid. [M+Na] + =261.1.

[0641] (E)-3-(2-(tert-butoxy)-3,4-dimethoxyphenyl)-1-(3-hydroxyphenyl)prop-2-en-1-one (4). At 0 °C, a solution of 40% aqueous KOH (6.98 g, 49.8 mmol) was added to a solution of 2 (2.965 g, 12.4 mmol) and 3′-hydroxyacetophenone 3 (2.03 g, 14.9 mmol) in EtOH (50 mL). The resulting solution was stirred at 60 °C for 4 h. The solution was poured into water and acidified to pH 4 with 1 M HCl aqueous solution, and extracted twice with EtOAc. The combined organic layers were washed with brine, dried over Na2SO4, and concentrated under vacuum. The crude compound was purified by silica gel rapid column chromatography (AcOEt / PE 1:1) to give compound 4 (3.2 g, 72%) as a yellow oil. [M+Na] + =378.9.

[0642] (E)-2-(3-(3-(2-(tert-butoxy)-3,4-dimethoxyphenyl)acryloyl)phenoxy)tert-butyl bromoacetate (5). A solution of 4 (3.2 g, 9 mmol) in DMF (30 mL) and K₂CO₃ (1.49 g, 10.8 mmol) was treated with tert-butyl bromoacetate (1.58 mL, 10.8 mmol) and stirred at room temperature for 5 h. After this time, the reaction mixture was quenched by H₂O and extracted twice with EtOAc. The combined organic layers were washed with brine, dried over Na₂SO₄ and concentrated under vacuum. The crude compound was purified by silica gel rapid column chromatography (AcOEt / PE 1:4) to give compound 5 (4 g, 95%) as a yellow oil. [M+Na] + =493.3.

[0643] 2-(3-(3-(2-(tert-butoxy)-3,4-dimethoxyphenyl)propionyl)phenoxy)tert-butyl acetate (6). A solution of 5 (4 g, 8.5 mmol) in THF (50 mL) and 10% Pd / C (0.8 g) was hydrogenated with H2 for 3 h at room temperature. The reaction mixture was then filtered and concentrated. The residue was purified by silica gel rapid column chromatography (AcOEt / PE 1:3) to give compound 6 (2.878 g, 72%) as a colorless oil. [M+Na] + =495.3.

[0644] (R)-2-(3-(3-(3-(tert-butoxy)-4,5-dimethoxyphenyl)-1-hydroxypropyl)phenoxy)tert-butyl acetate (7). A solution of ketone 6 (2.878 g, 6.1 mmol) in dry THF (30 mL) at -20 °C was treated with a solution of (+)-DIPChloride (24.4 mmol) in heptane (1.7 M, 14.3 mL) at -20 °C. The resulting mixture was reacted at -20 °C until 6 was completely converted, and then quenched with 2,2′-(ethylenedioxy)diethylamine (3.6 mL) until an insoluble complex was formed. After stirring at room temperature for an additional 30 min, the suspension was filtered through a diatomaceous earth mat and concentrated. The crude compound was purified by silica gel rapid column chromatography (AcOEt / PE 1:3) to give compound 7 (2 g, 70%, ee>99%) as a pale yellow oil. [M+Na] + =497.0.

[0645] (R)-1-(3-(2-(tert-butoxy)-2-oxoethoxy)phenyl)-3-(2-(tert-butoxy)-3,4-dimethoxyphenyl)propyl(S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutyryl)piperidine-2-carboxylate (9). Solutions of 7 (1.8 g, 3.793 mmol) and 8 (1.77 g, 5.69 mmol) in CH2Cl2 (13 mL) were cooled to -20 °C under an argon atmosphere, followed by the addition of a solution of DCC (1.17 g, 5.69 mmol) in CH2Cl2 (5 mL), and then a solution of 4-(dimethylamino)pyridine (DMAP, 46 mg, 0.379 mmol) in CH2Cl2 (2 mL). The resulting white suspension was allowed to be stirred at -20 °C for 2 h. The reaction mixture was then filtered, evaporated, and the crude compound was purified by silica gel rapid column chromatography (AcOEt / PE 1:3) to give compound 9 (2.5 g, 86%) as a pale yellow oil. [M+Na] + =790.4.

[0646] 2-(3-((R)-1-(((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutyryl)piperidine-2-carbonyl)oxy)-3-(2-hydroxy-3,4-dimethoxyphenyl)propyl)phenoxy)acetic acid (Rae9):

[0647] At 0°C, a solution of Rae9 (2.5 g, 3.26 mmol) in 12 mL of CH2Cl2 was treated with a solution of 40% TFA in 12 mL of CH2Cl2. The mixture was allowed to react at room temperature until complete conversion. The reaction mixture was directly loaded into a silica gel fast column (AcOEt / PE / AcOH 1:2:0.5%) to provide Rae9 (636 mg, 30%) as a pale yellow solid.

[0648] FKBD Example 25

[0649] 2-(3-((R)-1-(((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutyryl)piperidine-2-carbonyl)oxy)-3-(3-hydroxy-4,5-dimethoxyphenyl)propyl)phenoxy)acetic acid (Rae10)

[0650]

[0651]

[0652] Scheme 28. Synthesis of the Rae10 FKBD portion.

[0653] 3-(tert-butoxy)-4,5-dimethoxybenzaldehyde (2). Under an Ar atmosphere, 1,1-di-tert-butoxy-N,N-dimethylmethylamine 2 (29.1 mL, 122 mmol) was added to a solution of 3-hydroxy-4,5-dimethoxybenzaldehyde 1 (2.77 g, 15.2 mmol) in anhydrous toluene (30 mL). The mixture was stirred at 80 °C for 6 h, and then the solvent was evaporated. The residue was purified by silica gel rapid column chromatography (AcOEt / PE1:1) to give compound 2 (3.126 g, 86%) as a yellow solid. [M+H] + =239.0.

[0654] (E)-3-(3-(tert-butoxy)-4,5-dimethoxyphenyl)-1-(3-hydroxyphenyl)prop-2-en-1-one (4). At 0 °C, a solution of 40% aqueous KOH (7.36 g, 52 mmol) was added to a solution of 2 (3.126 g, 13 mmol) and 3′-hydroxyacetophenone 3 (2.14 g, 15.7 mmol) in EtOH (30 mL). The resulting solution was stirred at 60 °C for 4 h. The solution was poured into water and acidified to pH 4 with 1 M HCl aqueous solution, and extracted twice with EtOAc. The combined organic layers were washed with brine, dried over Na2SO4, and concentrated under vacuum. The crude compound was purified by silica gel rapid column chromatography (AcOEt / PE 1:1) to give compound 4 (2.489 g, 54%) as a yellow oil. [M+H] + =357.0.

[0655] (E)-2-(3-(3-(3-(tert-butoxy)-4,5-dimethoxyphenyl)acryloyl)phenoxy)tert-butyl bromoacetate (5). A solution of 4 (2.489 g, 6.98 mmol) and K2CO3 (1.16 g, 8.38 mmol) in DMF (30 mL) was treated with tert-butyl bromoacetate (1.2 mL, 8.38 mmol) and stirred at room temperature for 5 h. After this time, the reaction mixture was quenched by H2O and extracted twice with EtOAc. The combined organic layers were washed with brine, dried over Na2SO4, and concentrated under vacuum. The crude compound was purified by silica gel rapid column chromatography (AcOEt / PE 1:4) to give compound 5 (3.1 g, 95%) as a yellow oil. [M+H] + =471.0.

[0656] 2-(3-(3-(3-(tert-butoxy)-4,5-dimethoxyphenyl)propionyl)phenoxy)tert-butyl acetate (6). A solution of 5 (3.1 g, 6.59 mmol) in THF (50 mL) and 10% Pd / C (0.5 g) was hydrogenated with H2 for 3 h at room temperature. The reaction mixture was then filtered and concentrated. The residue was purified by silica gel rapid column chromatography (AcOEt / PE 1:3) to give compound 6 (2.88 g, 93%) as a colorless oil. [M+Na] + =495.3.

[0657] (R)-2-(3-(3-(3-(tert-butoxy)-4,5-dimethoxyphenyl)-1-hydroxypropyl)phenoxy)tert-butyl acetate (7). A solution of ketone 6 (2.868 g, 6.07 mmol) in dry THF (30 mL) at -20 °C was treated with a solution of (+)-DIPChloride (12.1 mmol) in heptane (1.7 M, 7.1 mL) at -20 °C. The resulting mixture was reacted at -20 °C until 6 was completely converted, and then quenched with 2,2′-(ethylenedioxy)diethylamine (3.6 mL) until an insoluble complex was formed. After stirring at room temperature for an additional 30 min, the suspension was filtered through a diatomaceous earth mat and concentrated. The crude compound was purified by silica gel rapid column chromatography (AcOEt / PE 1:3) to give compound 7 as a pale yellow oil (2.03 g, 70%, ee > 99% relative to the racemic mixture). [M+Na] + =497.3.

[0658] (R)-1-(3-(3-(tert-butoxy)-2-oxoethoxy)phenyl)-3-(2-(tert-butoxy)-4,5-dimethoxyphenyl)propyl(S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutyryl)piperidine-2-carboxylate (9). Solutions of 7 (2.03 g, 4.3 mmol) and 8 (2 g, 6.4 mmol) in CH2Cl2 (43 mL) were cooled to -20 °C under an argon atmosphere, followed by the addition of a solution of DCC (1.3 g, 6.4 mmol) in CH2Cl2 (5 mL), and then a solution of 4-(dimethylamino)pyridine (DMAP, 52.3 mg, 0.43 mmol) in CH2Cl2 (2 mL). The resulting white suspension was allowed to be stirred at -20 °C for 2 h. The reaction mixture was then filtered, evaporated, and the crude compound was purified by silica gel rapid column chromatography (AcOEt / PE 1:3) to give compound 9 (2.5 g, 76%) as a pale yellow oil. [M+Na] + =790.3.

[0659] 2-(3-((R)-1-(((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutyryl)piperidine-2-carbonyl)oxy)-3-(3-hydroxy-4,5-dimethoxyphenyl)propyl)phenoxy)acetic acid (Rae10).

[0660] At 0 °C, a solution of 9 (2.5 g, 3.26 mmol) in 12 mL of CH2Cl2 was treated with a solution of 40% TFA in 12 mL of CH2Cl2. The mixture was allowed to react at room temperature until complete conversion. The reaction mixture was directly loaded into a silica gel fast column (AcOEt / PE / AcOH 1:2:0.5%) to provide Rae10 (1.334 g, 62%) as a pale yellow solid.

[0661] FKBD Example 26

[0662] 2-(3-((R)-1-(((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutyryl)piperidine-2-carbonyl)oxy)-3-(2-hydroxy-4,5-dimethoxyphenyl)propyl)phenoxy)acetic acid (Rae11)

[0663]

[0664] Scheme 29. Synthesis of the FKBD portion of Rae11.

[0665] 2-(tert-butoxy)-4,5-dimethoxybenzaldehyde (2). Under an Ar atmosphere, 1,1-di-tert-butoxy-N,N-dimethylmethylamine 2 (31.6 mL, 132 mmol) was added to a solution of 2-hydroxy-4,5-dimethoxybenzaldehyde 1 (3 g, 16.5 mmol) in anhydrous toluene (15 mL). The mixture was stirred at 80 °C for 6 h, and then the solvent was evaporated. The residue was purified by silica gel rapid column chromatography (AcOEt / PE 1:1) to give compound 2 (3.875 g, 99%) as a yellow solid. [M+Na] + =261.2.

[0666] (E)-3-(2-(tert-butoxy)-4,5-dimethoxyphenyl)-1-(3-hydroxyphenyl)prop-2-en-1-one (4). At 0 °C, a solution of 40% aqueous KOH (8.5 mL, 3.65 g, 65.2 mmol) was added to a solution of 2 (3.875 g, 16.3 mmol) and 3′-hydroxyacetophenone 3 (2.436 g, 17.9 mmol) in EtOH (50 mL). The resulting solution was stirred at 60 °C for 4 h. The solution was poured into water and acidified to pH 4 with 1 M HCl aqueous solution, and extracted twice with EtOAc. The combined organic layers were washed with brine, dried over Na2SO4, and concentrated under vacuum. The crude compound was purified by silica gel rapid column chromatography (AcOEt / PE 1:1) to give compound 4 (5.2 g, 90%) as a yellow oil. [M+H] + =357.2.

[0667] (E)-2-(3-(3-(2-(tert-butoxy)-4,5-dimethoxyphenyl)acryloyl)phenoxy)tert-butyl bromoacetate (5). A solution of 4 (5.2 g, 14.59 mmol) in DMF (50 mL) and K₂CO₃ (2.4 g, 17.5 mmol) was treated with tert-butyl bromoacetate (2.55 mL, 17.5 mmol) and stirred at room temperature for 5 h. After this time, the reaction mixture was quenched by H₂O and extracted twice with EtOAc. The combined organic layers were washed with brine, dried over Na₂SO₄ and concentrated under vacuum. The crude compound was purified by silica gel rapid column chromatography (AcOEt / PE 1:4) to give compound 5 (6 g, 88%) as a yellow oil. [M+H] + =471.0.

[0668] 2-(3-(3-(2-(tert-butoxy)-4,5-dimethoxyphenyl)propionyl)phenoxy)tert-butyl acetate (6). A solution of 5 (6 g, 12.75 mmol) in THF (70 mL) and 10% Pd / C (1 g) was hydrogenated with H2 for 4 h at room temperature. The reaction mixture was then filtered and concentrated. The residue was purified by silica gel rapid column chromatography (AcOEt / PE 1:3) to give compound 6 (4.5 g, 75%) as a colorless oil. [M+Na] + =495.3.

[0669] (R)-2-(3-(3-(2-(tert-butoxy)-4,5-dimethoxyphenyl)-1-hydroxypropyl)phenoxy)tert-butyl acetate (7). A solution of ketone 6 (4.5 g, 9.5 mmol) in dry THF (45 mL) at -20 °C was treated with a solution of (+)-DIPChloride (19 mmol) in heptane (1.7 M, 11.2 mL) at -20 °C. The resulting mixture was reacted at -20 °C until 6 was completely converted, and then quenched with 2,2′-(ethylenedioxy)diethylamine (2.8 mL) until an insoluble complex was formed. After stirring at room temperature for an additional 30 min, the suspension was filtered through a diatomaceous earth mat and concentrated. The crude compound was purified by silica gel rapid column chromatography (AcOEt / PE 1:3) to give compound 7 (3.2 g, 70%, ee > 99% relative to the racemic mixture) as a pale yellow oil. [M+Na] + =496.7.

[0670] (R)-1-(3-(2-(tert-butoxy)-2-oxoethoxy)phenyl)-3-(2-(tert-butoxy)-4,5-dimethoxyphenyl)propyl(S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutyryl)piperidine-2-carboxylate (9). Solutions of 7 (1.93 g, 4.07 mmol) and 8 (1.9 g, 6.103 mmol) in CH2Cl2 (43 mL) were cooled to -20 °C under an argon atmosphere, followed by the addition of a solution of DCC (1.26 g, 6.103 mmol) in CH2Cl2 (5 mL), and then a solution of 4-(dimethylamino)pyridine (DMAP, 50 mg, 0.407 mmol) in CH2Cl2 (2 mL). The resulting white suspension was allowed to be stirred at -20 °C for 2 h. The reaction mixture was then filtered, evaporated, and the crude compound was purified by silica gel rapid column chromatography (AcOEt / PE 1:3) to give compound 9 (2.1 g, 67%) as a pale yellow oil. [M+Na] + =790.4.

[0671] 2-(3-((R)-1-(((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutyryl)piperidine-2-carbonyl)oxy)-3-(2-hydroxy-4,5-dimethoxyphenyl)propyl)phenoxy)acetic acid (Rae11).

[0672] A solution of 9 (2.1 g, 2.73 mmol) in 12 mL of CH2Cl2 was treated with a solution of 40% TFA in 12 mL of CH2Cl2 at 0 °C. The mixture was allowed to react at room temperature until complete conversion. The reaction mixture was directly loaded into a silica gel fast column (AcOEt / PE / AcOH 1:2:0.5%) to provide Rae11 (638 mg, 23%) as a pale yellow solid.

[0673] FKBD Example 27

[0674] 2-(3-((R)-1-(((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutyryl)piperidine-2-carbonyl)oxy)-3-(3-fluoro-4,5-dimethoxyphenyl)propyl)phenoxy)acetic acid (Rae12)

[0675]

[0676] Synthesis of the FKBD portion of Scheme 30. Rae12.

[0677] (E)-3-(3-fluoro-4,5-dimethoxyphenyl)-1-(3-hydroxyphenyl)prop-2-en-1-one (3). At 0 °C, a solution of 10% aqueous NaOH (40 mL, 97.6 mmol) was added to a solution of 3-fluoro-4,5-dimethoxybenzaldehyde 1 (4.5 g, 24.4 mmol) and 1-(3-hydroxyphenyl)ethyl-1-one 2 (3.3 g, 24.4 mmol) in EtOH (60 mL). The resulting solution was stirred at room temperature for 12 h. The solution was adjusted to pH 4 by dropwise addition of 4 M aqueous HCl at 0 °C, producing a large amount of yellow solid. The mixture was then filtered and the solid was washed with water (50 mL) to provide 3 (4 g, 54%) as a yellow solid. [M+H] + =303.1.

[0678] (E)-2-(3-(3-(3-fluoro-4,5-dimethoxyphenyl)acryloyl)phenoxy)tert-butyl bromoacetate (4). A solution of 3 (4 g, 13.2 mmol) and K2CO3 (3.65 g, 26.4 mmol) in DMF (30 mL) was treated with tert-butyl bromoacetate (3.08 g, 15.8 mmol) and stirred at room temperature for 5 h. After this time, the reaction mixture was poured into ice, and a yellow solid precipitated. The mixture was filtered and the solid was washed with water (30 mL). The crude product was purified by column chromatography on silica gel (AcOEt / PE1:4) to give 4 (5.2 g, 94%) as a yellow solid. [M+Na] + =438.7.

[0679] 2-(3-(3-(3-fluoro-4,5-dimethoxyphenyl)-1-hydroxypropyl)phenoxy)tert-butyl acetate (5). A solution of 4 (5.2 g, 12.5 mmol) in THF (100 mL) and 10% Pd / C (1 g) was hydrogenated with H2 for 2 h at room temperature. The reaction mixture was then filtered and concentrated. The crude product was purified by column chromatography on silica gel to give 5 (5 g, 96%) as a yellow oil. [M+Na] + =443.2.

[0680] 2-(3-(3-(3-fluoro-4,5-dimethoxyphenyl)propionyl)phenoxy)tert-butyl acetate (6). A solution of 5 (5 g, 11.9 mmol) in CH2Cl2 (100 mL) was treated with Dys-Martin periodine (15.2 g, 36 mmol) and allowed to stir at room temperature for 2 h, followed by quenching with a solution of 10% aqueous NaS2O3. The solution was extracted twice with CH2Cl2. The combined organic layers were washed with saturated NaHCO3 brine, dried over Na2SO4, and concentrated under vacuum. The crude compound was purified by silica gel rapid column chromatography (AcOEt / PE 1:3) to give compound 6 (4 g, 80%) as a white solid. [M+Na] + =441.2.

[0681] (R)-2-(3-(3-(3-fluoro-4,5-dimethoxyphenyl)-1-hydroxypropyl)phenoxy)tert-butyl acetate (7). A solution of ketone 6 (4 g, 9.56 mmol) in dry THF (30 mL) at -20 °C was treated with a solution of (+)-DIPChloride (19.1 mmol) in heptane (1.7 M, 11.2 mL) at -20 °C. The resulting mixture was reacted at -20 °C until 6 was completely converted, and then quenched with 2,2′-(ethylenedioxy)diethylamine (2.8 g) until an insoluble complex was formed. After stirring at room temperature for an additional 30 min, the suspension was filtered through a diatomaceous earth mat and concentrated. The crude compound was purified by silica gel rapid column chromatography (AcOEt / PE 1:4) to give compound 6 (2.2 g, 55%, ee>99%) as a pale yellow oil. [M+Na] + =442.7.

[0682] (R)-1-(3-(2-(tert-butoxy)-2-oxoethoxy)phenyl)-3-(3-fluoro-4,5-dimethoxyphenyl)propyl(S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutyryl)piperidine-2-carboxylate (9). Solutions of 7 (2.2 g, 5.23 mmol) and 8 (1.80 g, 5.76 mmol) in CH2Cl2 (20 mL) were cooled to -20 °C under an argon atmosphere, followed by the addition of a solution of DCC (1.4 g, 6.79 mmol) in CH2Cl2 (10 mL), and then a solution of 4-(dimethylamino)pyridine (63 mg, 0.52 mmol) in CH2Cl2 (2 mL). The resulting white suspension was allowed to be stirred at -20 °C for 2 h. The reaction mixture was then filtered, evaporated, and the crude compound was purified by silica gel rapid column chromatography (AcOEt / PE 1:3) to give compound 9 (1.8 g, 48%) as a pale yellow oil. [M+Na] + =736.4.

[0683] 2-(3-((R)-1-(((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutyryl)piperidin-2-carbonyl)oxy)-3-(3-fluoro-4,5-dimethoxyphenyl)propyl)phenoxy)acetic acid (Rae12). A solution of 9 g (1.8 g, 2.52 mmol) in 10 mL of CH2Cl2 was treated with a solution of 40% TFA in 10 mL of CH2Cl2 at 0 °C. The mixture was allowed to react at room temperature until complete conversion. The reaction mixture was directly loaded into a silica gel fast column (AcOEt / PE / AcOH 1:3:0.5%) to provide Rae12 (590 mg, 35%) as a pale yellow solid.

[0684] FKBD Example 28

[0685] 2-(3-((R)-1-(((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutyryl)piperidine-2-carbonyl)oxy)-3-(2-fluoro-4,5-dimethoxyphenyl)propyl)phenoxy)acetic acid (Rae13)

[0686]

[0687]

[0688] Scheme 31. Synthesis of the FKBD portion of Rae13.

[0689] (E)-3-(2-fluoro-4,5-dimethoxyphenyl)-1-(3-hydroxyphenyl)prop-2-en-1-one (3). At 0 °C, a solution of 10% aqueous NaOH (40 mL, 97.6 mmol) was added to a solution of 2-fluoro-4,5-dimethoxybenzaldehyde 1 (4.5 g, 24.4 mmol) and 1-(3-hydroxyphenyl)ethyl-1-one 2 (3.3 g, 24.4 mmol) in EtOH (60 mL). The resulting solution was stirred at 65 °C for 6 h. The solution was adjusted to pH 4 by dropwise addition of 4 M aqueous HCl at 0 °C, producing a large amount of yellow solid. The mixture was then filtered and the solid was washed with water (50 mL) to provide 3 (7 g, 94%) as a yellow solid. [M+H] + =302.8.

[0690] 3-(3-(2-fluoro-4,5-dimethoxyphenyl)-1-hydroxypropyl)phenol (4). A solution of 3 (7 g, 23.1 mmol) in THF (150 mL) and 10% Pd / C (2 g) was hydrogenated with H₂ for 12 h at room temperature. The reaction mixture was then filtered and concentrated. The crude product was purified by column chromatography on silica gel to give 4 (7 g, 98%) as a yellow oil. [M+Na] + =328.8.

[0691] 2-(3-(3-(2-fluoro-4,5-dimethoxyphenyl)-1-hydroxypropyl)phenoxy)tert-butyl acetate (5). A solution of 4 (7 g, 23.1 mmol) in DMF (200 mL) and K₂CO₃ (7 g, 50.6 mmol) was treated with tert-butyl bromoacetate (6.7 g, 34.5 mmol) and stirred at room temperature for 24 h. After this time, the reaction mixture was poured into ice, and a yellow solid precipitated. The mixture was filtered and the solid was washed with water (300 mL). The crude product was purified by column chromatography on silica gel (AcOEt / PE1:6) to give 5 (8 g, 82%) as a yellow solid. [M+Na] + =443.2.

[0692] 2-(3-(3-(2-fluoro-4,5-dimethoxyphenyl)propionyl)phenoxy)tert-butyl acetate (6). A solution of 5 (8 g, 19 mmol) in CH2Cl2 (100 mL) was treated with Dys-Martin periodine (16 g, 38 mmol) and allowed to stir at room temperature for 2 h, followed by quenching with a solution of 10% aqueous NaS2O3. The solution was extracted twice with CH2Cl2. The combined organic layers were washed with saturated NaHCO3, brine, dried over Na2SO4, and concentrated under vacuum. The crude compound was purified by silica gel rapid column chromatography (AcOEt / PE 1:3) to give compound 6 (6.3 g, 78%) as a yellow solid. [M+Na] + =440.7.

[0693] (R)-2-(3-(3-(2-fluoro-4,5-dimethoxyphenyl)-1-hydroxypropyl)phenoxy)tert-butyl acetate (7). A solution of ketone 6 (6.3 g, 15.07 mmol) in dry THF (60 mL) at -20 °C was treated with a solution of (+)-DIPChloride (45.2 mmol) in heptane (1.7 M, 26.5 mL) at -20 °C. The resulting mixture was reacted at -20 °C until 6 was completely converted, and then quenched with 2,2′-(ethylenedioxy)diethylamine (6.6 g) until an insoluble complex was formed. After stirring at room temperature for an additional 30 min, the suspension was filtered through a diatomaceous earth mat and concentrated. The crude compound was purified by silica gel rapid column chromatography (AcOEt / PE 1:3) to give compound 7 (4.3 g, 68%, ee>99%) as a pale yellow oil. [M+Na] + =443.2.

[0694] (R)-1-(3-(2-(tert-butoxy)-2-oxoethoxy)phenyl)-3-(2-fluoro-4,5-dimethoxyphenyl)propyl(S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutyryl)piperidine-2-carboxylate (9). Solutions of 7 (1.6 g, 3.81 mmol) and 8 (1.77 g, 5.71 mmol) in CH2Cl2 (20 mL) were cooled to -20 °C under an argon atmosphere, followed by the addition of a solution of DCC (1.17 g, 5.71 mmol) in CH2Cl2 (10 mL), and then a solution of 4-(dimethylamino)pyridine (50 mg, 0.38 mmol) in CH2Cl2 (2 mL). The resulting white suspension was allowed to be stirred at -20 °C for 2 h. The reaction mixture was then filtered, evaporated, and the crude compound was purified by silica gel rapid column chromatography (AcOEt / PE 1:2) to give compound 9 (1.7 g, 62%) as a pale yellow oil. [M+Na] + =736.4.

[0695] 2-(3-((R)-1-(((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutyryl)piperidine-2-carbonyl)oxy)-3-(2-fluoro-4,5-dimethoxyphenyl)propyl)phenoxy)acetic acid (Rae13).

[0696] A solution of 9 g (1.7 g, 2.38 mmol) in 10 mL of CH₂Cl₂ was treated with a solution of 40% TFA in 10 mL of CH₂Cl₂ at 0 °C. The mixture was allowed to react at room temperature until complete conversion. The reaction mixture was directly loaded into a silica gel fast column (AcOEt / PE / AcOH 1:3:0.5%) to provide Rae13 (520 mg, 33%) as a pale yellow solid.

[0697] FKBD Example 29

[0698] 2-(3-((R)-1-(((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutyryl)piperidine-2-carbonyl)oxy)-3-(2-fluoro-3,4-dimethoxyphenyl)propyl)phenoxy)acetic acid (Rae14)

[0699]

[0700] Scheme 32. Synthesis of the FKBD portion of Rae14.

[0701] (E)-3-(2-fluoro-3,4-dimethoxyphenyl)-1-(3-hydroxyphenyl)prop-2-en-1-one (3). At 0 °C, a solution of 40% aqueous KOH (15.22 g, 108.70 mmol) was added to solutions 1 (5.0 g, 27.17 mmol) and 2 (4.10 g, 29.89 mmol) in EtOH (150 mL). The resulting solutions were heated to 35 °C for 2 h. The solvent was evaporated, and the residue (4.8 g 58%) was used directly in the next step without purification. [M+H] + =303.0.

[0702] (E)-tert-butyl 2-(3-(3-(2-fluoro-3,4-dimethoxyphenyl)acryloyl)phenoxy)acetate (4). A solution of 3 (5.0 g, 16.55 mmol, crude) and K2CO3 (2.74 g, 19.87 mmol) in DMF (40 mL) was treated with tert-butyl bromoacetate (3.9 g, 19.87 mmol) and stirred at room temperature for 5 h. After this time, the reaction mixture was poured into ice, and a yellow solid precipitated. The mixture was filtered and the solid was washed with water (30 mL). The crude product was purified by column chromatography on silica gel to give 4 (6.0 g, 80%) as a yellow solid. [M+Na] + =439.2.

[0703] 2-(3-(3-(2-fluoro-3,4-dimethoxyphenyl)propionyl)phenoxy)tert-butyl acetate (5). A solution of 4 (4.0 g, 9.62 mmol) in THF (150 mL) and 10% Pd / C (1.0 g) was hydrogenated with H2 for 4 h at room temperature. The reaction mixture was then filtered and concentrated. The crude product was purified by column chromatography on silica gel to give 5 (2.8 g, 70%) as a yellow oil. [M+Na] + =440.8.

[0704] (R)-2-(3-(3-(2-fluoro-3,4-dimethoxyphenyl)-1-hydroxypropyl)phenoxy)tert-butyl acetate (6). A solution of ketone 5 (2.8 g, 6.7 mmol) in dry THF (30 mL) at -20 °C was treated with a solution of (+)-DIPChloride (26.8 mmol) in heptane (1.7 M, 15.7 mL) at -20 °C. The resulting mixture was reacted at -20 °C until 6 was completely converted, and then quenched with 2,2′-(ethylenedioxy)diethylamine (3.96 g) until an insoluble complex was formed. After stirring at room temperature for an additional 30 min, the suspension was filtered through a diatomaceous earth mat and concentrated. The crude compound was purified by silica gel rapid column chromatography (AcOEt / PE 1:4) to give compound 6 (1.3 g, 46%, ee>99%) as a pale yellow oil. [M+Na] + =442.7.

[0705] (R)-1-(3-(2-(tert-butoxy)-2-oxoethoxy)phenyl)-3-(2-fluoro-3,4-dimethoxyphenyl)propyl(S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutyryl)piperidine-2-carboxylate (8). Under an argon atmosphere, solutions of 6 (1.3 g, 3.09 mmol) and 7 (1.25 g, 4.02 mmol) in CH2Cl2 (15 mL) were cooled to -20 °C, followed by the addition of a solution of DCC (0.83 g, 4.02 mmol) in CH2Cl2 (5 mL), and then a solution of 4-(dimethylamino)pyridine (40 mg, 0.31 mmol) in CH2Cl2 (2 mL). The resulting white suspension was allowed to be stirred at -20 °C for 2 h. The reaction mixture was then filtered, evaporated, and the crude compound was purified by silica gel rapid column chromatography (AcOEt / PE 1:2) to give compound 8 (1.4 g, 63%) as a pale yellow oil. [M+Na] + =736.3.

[0706] 2-(3-((R)-1-(((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutyryl)piperidine-2-carbonyl)oxy)-3-(2-fluoro-3,4-dimethoxyphenyl)propyl)phenoxy)acetic acid (Rae14).

[0707] At 0°C, a solution of 8 g (1.4 g, 1.96 mmol) of CH₂Cl₂ (10 mL) was treated with a solution of 40% TFA in CH₂Cl₂ (10 mL). The mixture was allowed to react at room temperature until complete conversion. The reaction mixture was directly loaded into a silica gel fast column (AcOEt / PE / AcOH 1:3:0.5%) to provide Rae14 (585 mg, 45%) as a pale yellow solid.

[0708] FKBD Example 30

[0709] 2-(3-((R)-1-(((S)-4-(((9H-fluoren-9-yl)methoxy)carbonyl)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutyryl)piperazine-2-carbonyl)oxy)-3-(3,4-dimethoxyphenyl)propyl)phenoxy)acetic acid (Rae16)

[0710]

[0711] Scheme 33. Synthesis of the FKBD portion of Rae16.

[0712] (E)-3-(3,4-dimethoxyphenyl)-1-(3-hydroxyphenyl)prop-2-en-1-one (3). At 0 °C, a solution of 40% aqueous KOH (44 mL, 20 g, 352.8 mmol) was added to a solution of 3,4-dimethoxybenzaldehyde 1 (17.6 g, 105.8 mmol) and 3′-hydroxyacetophenone 2 (12 g, 88.2 mmol) in EtOH (160 mL). The resulting solution was stirred at room temperature for 2 h, then poured into ice-H2O, acidified with 1 M HCl solution, and extracted with EtOAc. The combined organic layers were dried over Na2SO4 and concentrated under vacuum. The residue was recrystallized from EtOAc-PE to give a pale yellow powder (23 g, 92%). [M+H] + =285.2.

[0713] 3-(3-(3,4-dimethoxyphenyl)-1-hydroxypropyl)phenol (4). A solution of 3 (16 g, 56.3 mmol) in THF (150 mL) and 10% Pd / C (1.6 g) was hydrogenated with H₂ for 4 h at room temperature. The reaction mixture was then filtered and concentrated to a solid (16.3 g, quantitative). [M+Na] + =311.2.

[0714] 2-(3-(3-(3,4-dimethoxyphenyl)-1-hydroxypropyl)phenoxy)tert-butyl bromoacetate (5). A solution of 4 (16.3 g, 56.53 mmol) and K2CO3 (9.4 g, 67.83 mmol) in DMF (150 mL) was treated with tert-butyl bromoacetate (9.9 mL, 67.83 mmol) and stirred at room temperature for 5 h. After this time, the reaction mixture was poured into ice, and a yellow solid (20 g, 88%) precipitated. [M+Na] + =424.9.

[0715] 2-(3-(3-(3,4-dimethoxyphenyl)propionyl)phenoxy)tert-butyl acetate (6). A solution of 5 (20 g, 49.7 mmol) in CH2Cl2 (400 mL) was treated with Dys-Martin periodine (63 g, 149 mmol) and allowed to be stirred at room temperature for 3 h, followed by quenching with a solution of 10% aqueous NaS2O3. The solution was extracted twice with CH2Cl2. The combined organic layers were washed with saturated NaHCO3, brine, dried over Na2SO4, and concentrated under vacuum. The crude compound was purified by silica gel rapid column chromatography (AcOEt / PE 1:3) to give compound 6 (11 g, 55%) as a white solid. [M+Na] + =423.3.

[0716] (R)-2-(3-(3-(3,4-dimethoxyphenyl)-1-hydroxypropyl)phenoxy)tert-butyl acetate (7). A solution of ketone 6 (11.156 g, 27.9 mmol) in dry THF (100 mL) at -20 °C was treated with a solution of (+)-DIPChloride (83.6 mmol) in heptane (1.7 M, 49 mL) at -20 °C. The resulting mixture was reacted at -20 °C until 6 was completely converted, and then quenched with 2,2′-(ethylenedioxy)diethylamine (11.5 mL) until an insoluble complex was formed. After stirring at room temperature for an additional 30 min, the suspension was filtered through a diatomaceous earth mat and concentrated. The crude compound was purified by silica gel rapid column chromatography (AcOEt / PE 1:3) to give compound 7 (6.3 g, 58%, ee>99%) as a pale yellow oil. [M+Na] + =425.3.

[0717] 1-((9H-fluorene-9-yl)methyl)3-((R)-1-(3-(2-(tert-butoxy)-2-oxoethoxy)phenyl)-3-(3,4-dimethoxyphenyl)propyl)(S)-4-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutyryl)piperazine-1,3-dicarboxylate (9). Under an argon atmosphere, solutions of 7 (1.224 g, 3 mmol) and 8 (2.44 g, 4.56 mmol) in CH2Cl2 (10 mL) were cooled to -20 °C, followed by the addition of a solution of DCC (0.94 g, 4.56 mmol) in CH2Cl2 (5 mL), and then a solution of 4-(dimethylamino)pyridine (DMAP, 37 mg, 0.3 mmol) in CH2Cl2 (2 mL). The resulting white suspension was allowed to be stirred at -20 °C for 2 h. The reaction mixture was then filtered, evaporated, and the crude compound was purified by silica gel rapid column chromatography (AcOEt / PE 1:2) to give compound 9 (1.8 g, 70%) as a pale yellow oil. [M+Na] + =940.7.

[0718] 2-(3-((R)-1-(((S)-4-(((9H-fluorene-9-yl)methoxy)carbonyl)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutyryl)piperazine-2-carbonyl)oxy)-3-(3,4-dimethoxyphenyl)propyl)phenoxy)acetic acid (Rae16). A solution of 9 g (1.8 g, 1.96 mmol) in 11.5 mL of CH2Cl2 was treated with a solution of 40% TFA in 11.5 mL of CH2Cl2 at 0 °C. The mixture was allowed to react at room temperature until complete conversion. The reaction mixture was directly loaded into a silica gel fast column (AcOEt / PE / AcOH 1:2:0.5%) to provide Rae16 (964 mg, 57%) as a white solid.

[0719] FKBD Example 31

[0720] 2-(3-((R)-1-(((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutyryl)-4-methylpiperazine-2-carbonyl)oxy)-3-(3,4-dimethoxyphenyl)propyl)phenoxy)acetic acid (Rae17)

[0721]

[0722] Scheme 34. Synthesis of the FKBD portion of Rae17.

[0723] (R)-1-(3-(2-(tert-butoxy)-2-oxoethoxy)phenyl)-3-(3,4-dimethoxyphenyl)propyl(S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutyryl)piperazine-2-carboxylate (2). At 0 °C, TBAF (2.5 mL, 1.0 M, 2.55 mmol) was added to a solution of 1 (1.0 g, 1.09 mmol) in DMF (5 mL). The resulting solution was heated to room temperature for 5 h. After this time, the reaction mixture was diluted with DCM and washed with saturated aqueous NaHCO3 solution and brine. The organic layer was concentrated under vacuum, and the residue was purified by silica gel rapid column chromatography (DCM / MeOH 50:1) to give compound 2 (670 mg, 80%) as a colorless oil. [M+H] + =696.9.

[0724] (R)-1-(3-(2-(tert-butoxy)-2-oxoethoxy)phenyl)-3-(3,4-dimethoxyphenyl)propyl(S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutyryl)-4-methylpiperazine-2-carboxylate (3). A solution of 2 (670 mg, 0.96 mmol) in CHOOH (1.5 mL) was treated with an aqueous solution of formaldehyde (37% in water, 0.77 mL, 1.15 mmol) and stirred at 50 °C for 1 h. After this time, the reaction mixture was purified with DCM / MeOH / AcOH = 100 / 1 / 0.5% to give 3 (500 mg, 73%) as a colorless oil. [M+H] + =710.9.

[0725] 2-(3-((R)-1-(((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutyryl)-4-methylpiperazine-2-carbonyl)oxy)-3-(3,4-dimethoxyphenyl)propyl)phenoxy)acetic acid (Rae17). A solution of 9 g (0.5 g, 0.7 mmol) in HCOOH (40 mL) was heated to 40 °C for 2 h. The reaction mixture was directly loaded into a silica gel fast column (AcOEt / PE / AcOH 1:2:0.5%) to provide Rae17 (368.7 mg, 80%) as a white solid.

[0726] FKBD Example 32

[0727] 2-(3-((R)-1-(((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutyryl)piperidine-2-carbonyl)oxy)-3-(3,4-dimethoxyphenyl)propyl)-4-fluorophenoxy)acetic acid (Rae18)

[0728]

[0729]

[0730] Synthesis of the FKBD portion of Scheme 35. Rae18.

[0731] (E)-3-(3,4-dimethoxyphenyl)-1-(2-fluoro-5-hydroxyphenyl)prop-2-en-1-one (3). At 0 °C, a solution of 10% aqueous NaOH (50 mL, 120.4 mmol) was added to a solution of 3,4-dimethoxybenzaldehyde 1 (5 g, 30.1 mmol) and 1-(2-fluoro-5-hydroxyphenyl)ethyl-1-one 2 (4.6 g, 30.1 mmol) in EtOH (60 mL). The resulting solution was stirred at room temperature for 12 h. The solution was adjusted to pH 4 by dropwise addition of 4 M aqueous HCl at 0 °C, producing a large amount of yellow solid. The mixture was then filtered and the solid was washed with water (50 mL) to provide 3 (9 g, 99%) as a yellow solid. [M+H] + =303.2.

[0732] 3-(3,4-Dimethoxyphenyl)-1-(2-fluoro-5-hydroxyphenyl)prop-1-one (4). A solution of 3 (9 g, 29.8 mmol) in THF (200 mL) and 10% Pd / C (2 g) was hydrogenated with H₂ for 12 h at room temperature. The reaction mixture was then filtered and concentrated. The crude product was used in the next step without any further purification. [M+H] + =304.8.

[0733] 2-(3-(3-(3,4-dimethoxyphenyl)propionyl)-4-fluorophenoxy)tert-butyl bromoacetate (5). A solution of 4 (10 g, 32.8 mmol) in DMF (200 mL) and K2CO3 (9 g, 65.6 mmol) was treated with tert-butyl bromoacetate (7.7 g, 39.3 mmol) and stirred at room temperature for 8 h. After this time, the reaction mixture was poured into ice, and a yellow solid precipitated. The mixture was filtered and the solid was washed with water (300 mL). The crude product was purified by column chromatography on silica gel (AcOEt / PE 1:6) to give 5 (4 g, 32%, 2 steps) as a yellow oil. [M+Na] + =441.0.

[0734] (R)-2-(3-(3-(3,4-dimethoxyphenyl)-1-hydroxypropyl)-4-fluorophenoxy)tert-butyl acetate (6). A solution of ketone 5 (4 g, 9.56 mmol) in dry THF (30 mL) at -20 °C was treated with a solution of (+)-DIPChloride (28.68 mmol) in heptane (1.7 M, 16.8 mL) at -20 °C. The resulting mixture was reacted at -20 °C until 6 was completely converted, and then quenched with 2,2′-(ethylenedioxy)diethylamine (4.2 g) until an insoluble complex was formed. After stirring at room temperature for an additional 30 min, the suspension was filtered through a diatomaceous earth mat and concentrated. The crude compound was purified by silica gel rapid column chromatography (AcOEt / PE 1:3) to give compound 6 (2 g, 50%, ee 93%) as a pale yellow oil. [M+Na] + =442.7.

[0735] (R)-1-(5-(2-(tert-butoxy)-2-oxoethoxy)-2-fluorophenyl)-3-(3,4-dimethoxyphenyl)propyl(S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutyryl)piperidine-2-carboxylate (8). Under an argon atmosphere, solutions of 6 (2 g, 4.76 mmol) and 7 (2.22 g, 7.14 mmol) in CH2Cl2 (20 mL) were cooled to -20 °C, followed by the addition of a solution of DCC (1.47 g, 7.14 mmol) in CH2Cl2 (10 mL), and then a solution of 4-(dimethylamino)pyridine (60 mg, 0.47 mmol) in CH2Cl2 (2 mL). The resulting white suspension was allowed to be stirred at -20 °C for 2 h. The reaction mixture was then filtered, evaporated, and the crude compound was purified by silica gel rapid column chromatography (AcOEt / PE 1:2) to give compound 8 (1.8 g, 45%) as a pale yellow oil. [M+Na] + =736.3.

[0736] 2-(3-((R)-1-(((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutyryl)piperidin-2-carbonyl)oxy)-3-(3,4-dimethoxyphenyl)propyl)-4-fluorophenoxy)acetic acid (Rae18). A solution of 8 (1.7 g, 2.52 mmol) in 10 mL of CH2Cl2 was treated with a solution of 40% TFA in 10 mL of CH2Cl2 at 0 °C. The mixture was allowed to react at room temperature until complete conversion. The reaction mixture was directly loaded into a silica gel fast column (AcOEt / PE / AcOH 1:3:0.5%) to provide Rae18 (705 mg, 42%) as a white solid.

[0737] FKBD Example 33

[0738] 2-(3-((R)-1-(((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutyryl)piperidin-2-carbonyl)oxy)-3-(3,4-dimethoxyphenyl)propyl)-5-fluorophenoxy)acetic acid (Rae-19)

[0739]

[0740] Scheme 36. Synthesis of the FKBD portion of Rae19.

[0741] (E)-3-(3,4-dimethoxyphenyl)-1-(3-fluoro-5-hydroxyphenyl)prop-2-en-1-one (3). At 0 °C, a solution of 40% aqueous KOH (19.6 g, 140 mmol) was added to a solution of 3,4-dimethoxybenzaldehyde 1 (6.391 g, 38.5 mmol) and 1-(3-fluoro-5-hydroxyphenyl)ethyl-1-one 2 (5.39 g, 35 mmol) in EtOH (70 mL). The resulting solution was reacted at room temperature for 4 h. The yellow solid was filtered to give compound 3 (8.3 g, 78%). [M+H] + =303.0.

[0742] (E)-2-(3-(3-(3,4-dimethoxyphenyl)acryloyl)-5-fluorophenoxy)tert-butyl bromoacetate (4). A solution of 3 (8.3 g, 27.5 mmol) and K2CO3 (4.55 g, 32.9 mmol) in DMF (80 mL) was treated with tert-butyl bromoacetate (6.4 g, 32.9 mmol) and stirred at room temperature for 4 h. After this time, the reaction mixture was quenched by H2O and extracted twice with EtOAc. The combined organic layers were concentrated under vacuum and used for the next step without purification (11.11 g, 97%). [M+Na]+ =439.2.

[0743] 2-(3-(3-(3,4-dimethoxyphenyl)propionyl)-5-fluorophenoxy)tert-butyl acetate (5). A solution of 4 (11.11 g, 26.7 mmol) in THF (200 mL) and 10% Pd / C (1.11 g) was hydrogenated with H₂ for 4 h at room temperature. The reaction mixture was then filtered and concentrated. The residue was purified by silica gel rapid column chromatography (AcOEt / PE 1:1) to give compound 5 (4.2 g, 38%) as a colorless oil. [M+Na] + =440.7.

[0744] (R)-2-(3-(3-(3,4-dimethoxyphenyl)-1-hydroxypropyl)-5-fluorophenoxy)tert-butyl acetate (6). A solution of ketone 5 (4.2 g, 10 mmol) in dry THF (40 mL) at -20 °C was treated with a solution of (+)-DIPChloride (20 mmol) in heptane (1.7 M, 11.8 mL) at -20 °C. The resulting mixture was reacted at -20 °C until 5 was completely converted, and then quenched with 2,2′-(ethylenedioxy)diethylamine (2.9 mL) until an insoluble complex was formed. After stirring at room temperature for an additional 30 min, the suspension was filtered through a diatomaceous earth mat and concentrated. The crude compound was purified by silica gel rapid column chromatography (AcOEt / PE 1:5) to give compound 6 (2.94 g, 70%, ee 98% relative to the racemic mixture) as a pale yellow oil. [M+Na] + =44...

Claims

1. A compound or a pharmaceutically acceptable salt thereof, wherein the compound is compound 1593, the structure of which is as follows: 。 2. A pharmaceutical composition comprising an effective amount of the compound of claim 1 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

3. Use of the compound of claim 1 or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the prevention of renal ischemia-reperfusion injury in a subject.

4. The use according to claim 3, wherein the drug is administered intravenously.

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