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WO2024050016A1 - Compositions et procédés d'inhibition et de dégradation ciblées de protéines dans une cellule d'insecte - Google Patents

Compositions et procédés d'inhibition et de dégradation ciblées de protéines dans une cellule d'insecte Download PDF

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Publication number
WO2024050016A1
WO2024050016A1 PCT/US2023/031716 US2023031716W WO2024050016A1 WO 2024050016 A1 WO2024050016 A1 WO 2024050016A1 US 2023031716 W US2023031716 W US 2023031716W WO 2024050016 A1 WO2024050016 A1 WO 2024050016A1
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Prior art keywords
optionally substituted
alkyl
ethoxy
hydroxy
oxy
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PCT/US2023/031716
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English (en)
Inventor
Jason D. Speake
Christopher C. LADNER
Hamilton D. Dickson
John G. Catalano
Kelly D. D. BROCCIO
Melina M. R. LILLICH
Samuel G. GATTIS
Steven C. BREMMER
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Oerth Bio Llc
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Publication of WO2024050016A1 publication Critical patent/WO2024050016A1/fr

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Classifications

    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
    • A01P7/00—Arthropodicides
    • A01P7/04—Insecticides
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
    • A01N43/72—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with nitrogen atoms and oxygen or sulfur atoms as ring hetero atoms
    • A01N43/74—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with nitrogen atoms and oxygen or sulfur atoms as ring hetero atoms five-membered rings with one nitrogen atom and either one oxygen atom or one sulfur atom in positions 1,3
    • A01N43/78—1,3-Thiazoles; Hydrogenated 1,3-thiazoles
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
    • A01N43/72—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with nitrogen atoms and oxygen or sulfur atoms as ring hetero atoms
    • A01N43/80—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with nitrogen atoms and oxygen or sulfur atoms as ring hetero atoms five-membered rings with one nitrogen atom and either one oxygen atom or one sulfur atom in positions 1,2
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
    • A01N43/90—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having two or more relevant hetero rings, condensed among themselves or with a common carbocyclic ring system
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N53/00—Biocides, pest repellants or attractants, or plant growth regulators containing cyclopropane carboxylic acids or derivatives thereof

Definitions

  • compositions comprising one or more protein targeting moieties, one or more ligase targeting moieties, and a linker covalently bonded to the one or more protein targeting moieties and the one or more ligase targeting moieties, as well as methods for the use of such compositions.
  • compositions for controlling insect infestations in plants have typically been in the form of chemical insecticides.
  • chemical insecticides are generally not selective.
  • applications of chemical insecticides intended to control insect pests in crop plants can exert their effects on non-target insects (including pollinators) and other invertebrates.
  • Chemical insecticides often persist in the environment and may be slow to degrade, thus potentially accumulating in the food chain.
  • the use of persistent chemical insecticides can result in the development of resistance in the target insect species.
  • Biological pest control agents such as Bacillus thuringiensis strains expressing pesticidal polypeptides, have been applied to crop plants with satisfactory results, thus offering an alternative or complement to chemical pesticides.
  • the expression of Cry proteins in transgenic plants has provided efficient protection against certain insect pests, and transgenic plants expressing such proteins have been commercialized, allowing farmers to reduce or eliminate applications of chemical insect control agents.
  • transgenic plants expressing Cry proteins have been shown to be extremely effective, insect pests have developed resistance against the Cry proteins expressed in certain transgenic plants. Therefore, there remains a need to identify new and effective pest control agents.
  • Compounds include bifunctional compounds that target an insect protein of interest to a cellular pathway. In some instances, the pathway leads to degradation or inhibition of the insect protein.
  • the bifunctional compounds can be selected from proteolysis targeting chimeras (PROTACs), molecular glues, phosphatase recruiting chimeras (PhoRCs), deubiquitinase-targeting chimeras (DUBTAC), ribonuclease targeting chimeras (RIBOTACs), autophagy-targeting chimeras (AUTACs), autophagosome- tethering compounds (ATTECs), lysosome-targeting chimeras (LYTACs), and Chaperone- mediated Protein Degradation/Degrader (CHAMP) molecules.
  • PROTACs proteolysis targeting chimeras
  • PhoRCs phosphatase recruiting chimeras
  • DUBTAC deubiquitinase-targeting chimeras
  • RIBOTACs ribon
  • the methods utilize Proteolysis Targeting Chimeras (PROTACs) to direct degradation or inhibition of a protein of interest in an insect.
  • PROTACs are a class of bifunctional molecules that utilize the endogenous protein homeostasis machinery to degrade a protein of interest (POI).
  • POI protein of interest
  • PROTACs of the invention induce proximity between an insect ligase and a POI by forming a ternary complex that results in the ubiquitination and subsequent degradation of the POI by the proteasome.
  • the PROTAC bifunctional compounds comprise a binding ligand for a ubiquitin ligase and a second binding ligand for a protein of interest (POI), the target protein, to mediate ubiquitin transfer to, and degradation of, the protein of interest through the proteasome.
  • the first binding ligand is also referred to as a ligase targeting moiety (LTM) while the second binding ligand is referred to as a protein targeting moiety (PTM).
  • LTM ligase targeting moiety
  • PTM protein targeting moiety
  • the compounds disclosed herein include enantiomers, diastereomers, stereoisomers or agriculturally acceptable salts thereof.
  • the provided compounds, compositions, and methods are advantageous as smaller amounts of the PROTAC compositions are needed for administration to control insects.
  • the bifunctional compounds provided herein can include more than one ligase binding moiety, more than one insect protein targeting moiety, and more than one linker.
  • the bifunctional compounds of the invention can be provided in compositions comprising suitable agricultural carriers for delivery directly to the insect pests, and to the locus of the insect pests, including plants, surfaces, and areas where they are ready for contact by insects.
  • Compositions may further include insects, insect cells, plants, plant cells, plant parts and treated surfaces comprising the bifunctional compounds provided herein.
  • the disclosure is to a compound or a salt or a solvate thereof.
  • the compound includes one or more protein targeting moieties that each independently bind a protein that is expressed by an insect cell.
  • the compound further includes one or more ligase targeting moieties that each independently bind a ubiquitin ligase that is functional in the insect cell.
  • the compound further includes a linker covalently bonded to the one or more protein targeting moieties and the one or more ligase targeting moieties.
  • the disclosure is to a composition including a compound as disclosed herein, or a salt or solvate thereof. In some embodiments, the composition further includes an agriculturally acceptable carrier.
  • the disclosure is to a method of controlling a level of a target protein in an insect cell. The method includes contacting the insect cell with an effective amount of a compound as disclosed herein, or with an effective amount of a composition as disclosed herein. BRIEF DESCRIPTION OF THE DRAWINGS [0015]
  • FIG.1 presents a table and an image of a gel showing that FKBP substrate is ubiquitinated in vitro through sfVHL mediated by dTAGV-1 PROTAC in presence of E1 & E2.
  • FIG.2 presents graphs showing the FKBP-GFP to RFP signal ratio when treated with dTAG-V1 (dose curve) and Bortezomib (1 or 10 ⁇ M).
  • FIG.3 presents microscopy images showing the data plotted in the graphs of FIG.2.
  • the images are of dTAG-V1 treated Sf9 cells expressing FKBP-GFP-P2A-RFP observed in Brightfield, green fluorescent protein and red fluorescent protein excitation wavelengths.
  • DETAILED DESCRIPTION Compounds, compositions, and methods for inhibiting and modulating protein levels in an insect are provided.
  • the bifunctional compounds are PROTACs, and methods comprise their use in targeting and modulating the levels of proteins of interest (POI) in an insect cell.
  • the PROTAC compositions function to recruit endogenous proteins to an insect ubiquitin ligase enzyme for ubiquitination and subsequent degradation or inhibition.
  • the compounds may be designed to target any protein of interest present in an insect cell.
  • the compounds may be provided in agricultural compositions.
  • heterobifunctional molecules depend on non-UPS pathways to degrade a protein of interest (POI). These classes include autophagy-targeting chimeras (AUTACs), autophagosome-tethering compounds (ATTECs), lysosome-targeting chimeras (LYTACs), molecular glues, and antibody-based PROTACs (AbTACs).
  • AUTACs autophagy-targeting chimeras
  • ATTECs autophagosome-tethering compounds
  • LYTACs lysosome-targeting chimeras
  • AbTACs antibody-based PROTACs
  • AUTACs and ATTECs are also macroautophagy degradation-targeting chimeras (MADTACs).
  • MADTACs macroautophagy degradation-targeting chimeras
  • AUTECs and ATTECs focus on intracellular triggering of targeted protein degradation, while LYTACs and AbTACs trigger intercellular degradation via an extracellular process.
  • AUTACs link a warhead for the POI to a guanine derivative that tags the protein for degradation by the autophagy machinery.
  • ATTECs link a POI warhead to a ligand that binds to the autophagy protein LC3 (microtubule-associated protein 1 light chain 3 ⁇ ) glue, thereby bypassing the ubiquitin pathways by directly tethering the POI to the autophagosome.
  • LYTACs bind a membrane-bound POI and the extracellular domains of a lysosome-shuttling receptor, which then drags the POI into the lysosome for degradation.
  • AbTACs are bispecific antibodies that recruit membrane bound E3 ligases to a membrane POI for degradation by the lysosome degradation pathway.
  • Molecular glues are small molecules that act as adhesives by making two proteins bind each other. Molecular glues engage the E3 ligase allowing that complex to recruit its target. CHAMPs are bifunctional molecules similar to PROTACs. CHAMPs recruit the POI via a chaperone protein complex, leading to the ubiquitination of the POI and then the degradation of the POI via the proteasome system. Chaperones can interact with a diverse collection of E3 ligases.
  • the key chaperone HSP90 (enabling the use of CHAMPs for human therapeutics) is highly conserved in insects: e.g., 81% (bitscore of 1,131) between humans and fall armyworm (Spodoptera frugiperda) with 14 out of 15 key amino acid residues perfectly identical in the binding site being used to bind to HSP90.
  • compositions and methods that relate to the surprising and unexpected discovery that levels of target proteins expressed in an insect cell may be controlled with compounds comprising one or more protein targeting moieties (PTM) that each independently bind a target protein, one or more ligase targeting moieties (LTM) that each independently bind a ubiquitin ligase that is functional in an insect cell and a linker covalently bonded to the one or more protein targeting moieties and the one or more ligase targeting moieties.
  • PTM protein targeting moieties
  • LTM ligase targeting moieties
  • An E3 ubiquitin ligase protein (e.g., Von Hippel-Lindau E3 ubiquitin ligase (VHL) or Cereblon (CRBN)) ubiquitinates a target protein once it and the target protein are placed in proximity by a bifunctional or chimeric construct that binds the E3 ubiquitin ligase protein and the target protein. Accordingly, the present disclosure provides such compounds and compositions comprising an E3 ubiquitin ligase binding moiety coupled to a protein target binding moiety, which result in the ubiquitination of a chosen target protein and leads to degradation of the target protein by the proteasome.
  • VHL Von Hippel-Lindau E3 ubiquitin ligase
  • CRBN Cereblon
  • the present disclosure provides compounds that comprise a ligand, e.g., a small molecule ligand (i.e., having a molecular weight of below 2,000 Daltons, 1,000 Daltons, 500 Daltons, or 200 Daltons) that is capable of binding to a ubiquitin ligase, such as VHL or CRBN.
  • a ligand e.g., a small molecule ligand (i.e., having a molecular weight of below 2,000 Daltons, 1,000 Daltons, 500 Daltons, or 200 Daltons) that is capable of binding to a ubiquitin ligase, such as VHL or CRBN.
  • the compounds also comprise a moiety that is capable of binding to target protein in such a way that the target protein is placed in proximity to the ubiquitin ligase to effect degradation (and/or inhibition) of that protein.
  • the terms “including,” “comprising,” “having,” “containing,” and variations thereof, are inclusive and open-ended and do not exclude additional, unrecited elements or method steps beyond those explicitly recited.
  • the phrase “consisting of” is closed and excludes any element, step, or ingredient not explicitly specified.
  • the phrase “consisting essentially of” limits the scope of the described feature to the specified materials or steps and those that do not materially affect the basic and novel characteristics of the disclosed feature.
  • the term “alkyl,” by itself or as part of another substituent refers to a straight or branched, saturated, aliphatic radical having the number of carbon atoms indicated.
  • a branched alkyl may include one or branches having a geminal, vicinal, and/or isolated pattern.
  • an alkyl may include gem-methyl groups.
  • Alkyl may include any number of carbons, such as C1-2, C1-3, C1-4, C1-5, C1-6, C1-7, C1-8, C1-9, C1-10, C2-3, C2-4, C2-5, C2-6, C3-4, C3-5, C 3-6 , C 4-5 , C 4-6 and C 5-6 .
  • C 1-6 alkyl includes, but is not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, hexyl, etc.
  • Alkyl may also refer to alkyl groups having up to 20 carbons atoms, such as, but not limited to heptyl, octyl, nonyl, decyl, etc. Alkyl groups may be substituted or unsubstituted.
  • substituted alkyl groups may be substituted with one or more groups selected from halo, hydroxy, amino, alkylamino, amido, acyl, nitro, cyano, and alkoxy.
  • hydroxyalkyl or alkylhydroxy refer to an alkyl group, as defined above, where at least one of the hydrogen atoms is replaced with a hydroxy group.
  • alkylhydroxy groups can have any suitable number of carbon atoms, such as C 1-6 .
  • Exemplary alkylhydroxy groups include, but are not limited to, hydroxy-methyl, hydroxyethyl (where the hydroxy is in the 1- or 2-position), hydroxypropyl (where the hydroxy is in the 1-, 2- or 3-position), hydroxybutyl (where the hydroxy is in the 1-, 2-, 3- or 4-position), hydroxypentyl (where the hydroxy is in the 1-, 2-, 3-, 4- or 5-position), hydroxyhexyl (where the hydroxy is in the 1-, 2-, 3-, 4-, 5- or 6-position), 1,2-dihydroxyethyl, and the like.
  • heteroalkyl refers to an alkyl group of any suitable length and having from 1 to 3 heteroatoms such as N, O and S. Additional heteroatoms may also be useful, including, but not limited to, B, Al, Si and P. The heteroatoms may also be oxidized, such as, but not limited to, -S(O)- and -S(O)2-.
  • heteroalkyl may include ethers, thioethers and alkyl-amines. The heteroatom portion of the heteroalkyl may replace a hydrogen of the alkyl group to form a hydroxy, thio, or amino group.
  • the heteroatom portion may be the connecting atom, or be inserted between two carbon atoms.
  • Heteroalkyl groups may be substituted or unsubstituted.
  • the term “alkylene,” by itself or as part of another substituent, refers to a straight or branched, saturated, aliphatic radical having the number of carbon atoms indicated, and linking at least two other groups, i.e., a divalent hydrocarbon radical.
  • the two moieties linked to the alkylene may be linked to the same atom or different atoms of the alkylene group.
  • a straight chain alkylene may be the bivalent radical of -(CH 2 ) n - , where n is 1, 2, 3, 4, 5 or 6.
  • a branched alkylene may include one or branches having a geminal, vicinal, and/or isolated pattern.
  • an alkylene may include gem-methyl groups.
  • Representative alkylene groups include, but are not limited to, methylene, ethylene, propylene, isopropylene, butylene, isobutylene, sec-butylene, pentylene and hexylene.
  • Alkylene groups may be substituted or unsubstituted.
  • alkoxy refers to a substituted alkyl group having an oxygen atom that connects the alkyl group to the point of attachment: alkyl-O-.
  • alkoxy groups may have any suitable number of carbon atoms, such as C 1-6 .
  • Alkoxy groups include, for example, methoxy, ethoxy, propoxy, isopropoxy, butoxy, 2butoxy, isobutoxy, secbutoxy, tertbutoxy, pentoxy, hexoxy, etc.
  • halogen refers to fluorine, chlorine, bromine and iodine.
  • haloalkyl refers to a substituted alkyl, as defined above, where some or all of the hydrogen atoms are replaced with halogen atoms.
  • haloalkyl groups may have any suitable number of carbon atoms, such as C1-6.
  • haloalkyl includes trifluoromethyl, fluoromethyl, etc.
  • perfluoro may be used to define a compound or radical where all the hydrogens are replaced with fluorine.
  • perfluoromethane includes 1,1,1trifluoromethyl.
  • haloalkoxy refers to an alkoxy group where some or all of the hydrogen atoms are substituted with halogen atoms.
  • haloalkoxy groups may have any suitable number of carbon atoms, such as C1-6.
  • the alkoxy groups may be substituted with 1, 2, 3, or more halogens.
  • halogens for example by fluorine
  • the compounds are persubstituted, for example, perfluorinated.
  • Haloalkoxy includes, but is not limited to, trifluoromethoxy, 2,2,2- trifluoroethoxy, perfluoroethoxy, etc.
  • amino refers to a moiety –NR 2 , wherein each R group is H or alkyl.
  • alkyl amine or “alkylamino” refer to a substituted alkyl group as defined above, having one or more amino groups.
  • the amino groups may be primary, secondary or tertiary.
  • the alkyl amine may be further substituted with a hydroxy group to form an amino-hydroxy group.
  • Alkyl amines useful in the present disclosure include, but are not limited to, ethyl amine, propyl amine, isopropyl amine, ethylene diamine and ethanolamine.
  • the amino group may link the alkyl amine to the point of attachment with the rest of the compound, be at the omega position of the alkyl group, or link together at least two carbon atoms of the alkyl group.
  • alkyl amines are useful in the present disclosure.
  • the term “amido” refers to a moiety –NRC(O)R or –C(O)NR 2 , wherein each R group is H or alkyl.
  • acyl refers to an RC(O) group, where the R group is alkyl.
  • cycloalkyl by itself or as part of another substituent, refers to a saturated or partially unsaturated, monocyclic, fused bicyclic or bridged polycyclic ring assembly containing from 3 to 12 ring atoms, or the number of atoms indicated. Cycloalkyl may include any number of carbons, such as C3-6, C4-6, C5-6, C3-8, C4-8, C5-8, C6-8, C3-9, C3-10, C3-11, and C 3-12 .
  • Saturated monocyclic cycloalkyl rings include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cyclooctyl.
  • Saturated bicyclic and polycyclic cycloalkyl rings include, for example, bicyclo[1.1.1]pentane, norbornane, [2.2.2] bicyclooctane, decahydronaphthalene and adamantane. Cycloalkyl groups may also be partially unsaturated, having one or more double or triple bonds in the ring.
  • Representative cycloalkyl groups that are partially unsaturated include, but are not limited to, cyclobutene, cyclopentene, cyclohexene, cyclohexadiene (1,3- and 1,4-isomers), cycloheptene, cycloheptadiene, cyclooctene, cyclooctadiene (1,3-, 1,4- and 1,5-isomers), norbornene, and norbornadiene.
  • exemplary groups include, but are not limited to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl.
  • exemplary groups include, but are not limited to cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl. Cycloalkyl groups may be substituted or unsubstituted.
  • substituted cycloalkyl groups may be substituted with one or more groups selected from halo, hydroxy, amino, alkylamino, amido, acyl, nitro, cyano, and alkoxy.
  • lower cycloalkyl refers to a cycloalkyl radical having from three to seven carbons including, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl.
  • aryl by itself or as part of another substituent, refers to an aromatic ring system having any suitable number of carbon ring atoms and any suitable number of rings.
  • Aryl groups may include any suitable number of carbon ring atoms, such as C6, C7, C8, C 9 , C 10 , C 11 , C 12 , C 13 , C 14 , C 15 or C 16 , as well as C 6-10 , C 6-12 , or C 6-14 .
  • Aryl groups may be monocyclic, fused to form bicyclic (e.g., benzocyclohexyl) or tricyclic groups, or linked by a bond to form a biaryl group.
  • Representative aryl groups include phenyl, naphthyl and biphenyl. Other aryl groups include benzyl, having a methylene linking group.
  • aryl groups have from 6 to 12 ring members, such as phenyl, naphthyl or biphenyl. Other aryl groups have from 6 to 10 ring members, such as phenyl or naphthyl. Some other aryl groups have 6 ring members, such as phenyl.
  • Aryl groups may be substituted or unsubstituted. Unless otherwise specified, “substituted aryl” groups may be substituted with one or more groups selected from halo, hydroxy, amino, alkylamino, amido, acyl, nitro, cyano, and alkoxy.
  • aralkyl refers to a substituted alkyl, as defined above, where some or all of the hydrogen atoms are replaced with an aryl group as defined above. As for the unsubstituted portion of the alkyl group, aralkyl groups may have any suitable number of carbon atoms, such as C 1-6.
  • heteroaryl by itself or as part of another substituent, refers to a monocyclic or fused bicyclic or tricyclic aromatic ring assembly containing 5 to 16 ring atoms, where from 1 to 5 of the ring atoms are a heteroatom such as N, O or S.
  • heteroatoms may also be useful, including, but not limited to, B, Al, Si and P.
  • the heteroatoms may be oxidized to form moieties such as, but not limited to, -S(O)- and -S(O)2-.
  • Heteroaryl groups may include any number of ring atoms, such as C 5-6 , C 3-8 , C 4-8 , C 5-8 , C 6-8 , C 3-9 , C 3-10 , C 3-11 , or C 3-12 , wherein at least one of the carbon atoms is replaced by a heteroatom.
  • heteroaryl groups may be C 5-8 heteroaryl, wherein 1 to 4 carbon ring atoms are replaced with heteroatoms; or C5-8 heteroaryl, wherein 1 to 3 carbon ring atoms are replaced with heteroatoms; or C 5-6 heteroaryl, wherein 1 to 4 carbon ring atoms are replaced with heteroatoms; or C 5-6 heteroaryl, wherein 1 to 3 carbon ring atoms are replaced with heteroatoms.
  • the heteroaryl group may include groups such as pyrrole, pyridine, imidazole, pyrazole, triazole, tetrazole, pyrazine, pyrimidine, pyridazine, triazine (1,2,3-, 1,2,4- and 1,3,5-isomers), thiophene, furan, thiazole, isothiazole, oxazole, and isoxazole.
  • the heteroaryl groups may also be fused to aromatic ring systems, such as a phenyl ring, to form members including, but not limited to, benzopyrroles such as indole and isoindole, benzopyridines such as quinoline and isoquinoline, benzopyrazine (quinoxaline), benzopyrimidine (quinazoline), benzopyridazines such as phthalazine and cinnoline, benzothiophene, and benzofuran.
  • Other heteroaryl groups include heteroaryl rings linked by a bond, such as bipyridine. Heteroaryl groups may be substituted or unsubstituted.
  • substituted heteroaryl groups may be substituted with one or more groups selected from halo, hydroxy, amino, alkylamino, amido, acyl, nitro, cyano, and alkoxy.
  • the heteroaryl groups may be linked via any position on the ring.
  • pyrrole includes 1-, 2- and 3-pyrrole
  • pyridine includes 2-, 3- and 4-pyridine
  • imidazole includes 1-, 2-, 4- and 5-imidazole
  • pyrazole includes 1-, 3-, 4- and 5-pyrazole
  • triazole includes 1-, 4- and 5- triazole
  • tetrazole includes 1- and 5-tetrazole
  • pyrimidine includes 2-, 4-, 5- and 6- pyrimidine
  • pyridazine includes 3- and 4-pyridazine
  • 1,2,3-triazine includes 4- and 5-triazine
  • 1,2,4-triazine includes 3-, 5- and 6-triazine
  • 1,3,5-triazine includes 2-triazine
  • thiophene includes 2- and 3- thiophene
  • furan includes 2- and 3-furan
  • thiazole includes 2-, 4- and 5-thiazole
  • isothiazole includes 3-, 4- and 5-isothiazole
  • oxazole includes 2-, 4- and
  • heteroaryl groups include those having from 5 to 10 ring members and from 1 to 3 ring atoms including N, O or S, such as pyrrole, pyridine, imidazole, pyrazole, triazole, pyrazine, pyrimidine, pyridazine, triazine (1,2,3-, 1,2,4- and 1,3,5-isomers), thiophene, furan, thiazole, isothiazole, oxazole, isoxazole, indole, isoindole, quinoline, isoquinoline, quinoxaline, quinazoline, phthalazine, cinnoline, benzothiophene, and benzofuran.
  • N, O or S such as pyrrole, pyridine, imidazole, pyrazole, triazole, pyrazine, pyrimidine, pyridazine, triazine (1,2,3-, 1,2,4- and 1,
  • heteroaryl groups include those having from 5 to 8 ring members and from 1 to 3 heteroatoms, such as pyrrole, pyridine, imidazole, pyrazole, triazole, pyrazine, pyrimidine, pyridazine, triazine (1,2,3-, 1,2,4- and 1,3,5-isomers), thiophene, furan, thiazole, isothiazole, oxazole, and isoxazole.
  • heteroatoms such as pyrrole, pyridine, imidazole, pyrazole, triazole, pyrazine, pyrimidine, pyridazine, triazine (1,2,3-, 1,2,4- and 1,3,5-isomers), thiophene, furan, thiazole, isothiazole, oxazole, and isoxazole.
  • heteroaryl groups include those having from 9 to 12 ring members and from 1 to 3 heteroatoms, such as indole, isoindole, quinoline, isoquinoline, quinoxaline, quinazoline, phthalazine, cinnoline, benzothiophene, benzofuran and bipyridine.
  • heteroaryl groups include those having from 5 to 6 ring members and from 1 to 2 ring heteroatoms including N, O or S, such as pyrrole, pyridine, imidazole, pyrazole, pyrazine, pyrimidine, pyridazine, thiophene, furan, thiazole, isothiazole, oxazole, and isoxazole.
  • heterocyclyl refers to a saturated heterocyclyl ring system having from 3 to 15 ring members, a partially unsaturated non-aromatic ring, or a partially unsaturated, non-aromatic multiple-ring system in which one or more of the carbon atoms are each independently replaced with the same or different heteroatom such as N, O and S. Additional heteroatoms may also be useful, including, but not limited to, B, Al, Si and P. The heteroatoms may be oxidized to form moieties such as, but not limited to, -S(O)- and -S(O) 2 -.
  • Heterocyclyl groups may include any number of ring atoms, such as, C 3-6 , C4-6, C5-6, C3-8, C4-8, C5-8, C6-8, C3-9, C3-10, C3-11, C3-12, or C3-15, wherein at least one of the carbon atoms is replaced by a heteroatom. Any suitable number of carbon ring atoms may be replaced with heteroatoms in the heterocyclyl groups, such as 1, 2, 3, or 4, or 1 to 2, 1 to 3, 1 to 4, 2 to 3, 2 to 4, or 3 to 4.
  • the heterocyclyl group may include groups such as aziridine, azetidine, pyrrolidine, piperidine, azepane, azocane, quinuclidine, pyrazolidine, imidazolidine, piperazine (1,2-, 1,3- and 1,4-isomers), oxirane, oxetane, tetrahydrofuran, oxane (tetrahydropyran), oxepane, thiirane, thietane, thiolane (tetrahydrothiophene), thiane (tetrahydrothiopyran), oxazolidine, isoxazolidine, thiazolidine, isothiazolidine, dioxolane, dithiolane, morpholine, thiomorpholine, dioxane, or dithiane.
  • groups such as aziridine, azetidine, pyrrolidine, piperidine, azepan
  • heterocyloalkyl rings of heterocyclyl groups may also be fused to aromatic or non-aromatic rings to form members including, but not limited to, indoline.
  • Heterocyclyl groups thus include partially unsaturated ring systems containing one or more double bonds, including fused ring systems with one aromatic ring and one non- aromatic ring, but not fully aromatic ring systems.
  • Examples include dihydroquinolines, e.g., 3,4- dihydroquinoline, dihydroisoquinolines, e.g., 1,2-dihydroisoquinoline, tetrahydroquinolines, e.g., 1,2,3,4-tetrahydroquinoline, tetrahydroisoquinoline, dihydroimidazole, tetrahydroimidazole, etc., isoindoline, isoindolones (e.g., isoindolin-1-one), isatin, dihydrophthalazine, quinolinone, spiro[cyclopropane-1,1'-isoindolin]-3'-one, and the like.
  • dihydroquinolines e.g., 3,4- dihydroquinoline
  • dihydroisoquinolines e.g., 1,2-dihydroisoquinoline
  • tetrahydroquinolines e.g., 1,2,3,4-t
  • aziridine may be 1- or 2-aziridine
  • azetidine may be 1- or 2- azetidine
  • pyrrolidine may be 1-, 2- or 3-pyrrolidine
  • piperidine may be 1-, 2-, 3- or 4-piperidine
  • pyrazolidine may be 1-, 2-, 3-, or 4- pyrazolidine
  • imidazolidine may be 1-, 2-, 3- or 4-imidazolidine
  • piperazine may be 1-, 2-, 3- or 4-piperazine
  • tetrahydrofuran may be 1- or 2-tetrahydrofuran
  • oxazolidine may be 2-, 3-, 4- or 5- oxazolidine
  • isoxazolidine may be 2-, 3-, 4- or 5-isoxazolidine
  • thiazolidine may be 2-, 3-, 4- or 5-thiazolidine
  • isothiazolidine may be 2-, 3-, 4- or 5- isothiazolidine
  • morpholine may be 2-, 3- or 4-morpholine.
  • nitro refers to the moiety –NO 2 .
  • cyano refers to a carbon atom triple-bonded to a nitrogen atom (i.e., the moiety –C ⁇ N).
  • sulfonyl refers to a moiety –SO 2 R, wherein the R group is alkyl, haloalkyl, aryl, or halogen. “Alkylsulfonyl” refers to a sulfonyl moiety wherein the R group is alkyl.
  • “Sulfonyl halide” refers to a sulfonyl moiety wherein the R group is halogen.
  • the term “salt” refers to acid or base salts of the compounds of the present disclosure.
  • An “agriculturally acceptable salt” is one that is compatible with other ingredients of a formulation composition containing the compound, and that is not deleterious to a recipient thereof. It is thus understood that the agriculturally acceptable salts are non-toxic.
  • salts are mineral acid (hydrochloric acid, hydrobromic acid, phosphoric acid, and the like) salts, organic acid (acetic acid, propionic acid, glutamic acid, citric acid and the like) salts, and quaternary ammonium (methyl iodide, ethyl iodide, and the like) salts.
  • Salts of the acidic compounds of the present disclosure are salts formed with bases, namely cationic salts such as alkali and alkaline earth metal salts, such as sodium, lithium, potassium, calcium, magnesium, as well as ammonium salts, such as ammonium, trimethyl-ammonium, diethylammonium, and tris-(hydroxymethyl)-methyl-ammonium salts.
  • bases namely cationic salts such as alkali and alkaline earth metal salts, such as sodium, lithium, potassium, calcium, magnesium, as well as ammonium salts, such as ammonium, trimethyl-ammonium, diethylammonium, and tris-(hydroxymethyl)-methyl-ammonium salts.
  • acid addition salts such as of mineral acids, organic carboxylic and organic sulfonic acids, e.g., hydrochloric acid, methanesulfonic acid, maleic acid
  • a basic group such as pyridyl
  • the neutral forms of the compounds may be regenerated by contacting the salt with a base or acid and isolating the parent compound in the conventional manner.
  • the parent form of the compound differs from the various salt forms in certain physical properties, such as solubility in polar solvents, but otherwise the salts are equivalent to the parent form of the compound for the purposes of the present disclosure.
  • the term “solvate” refers to a compound that is complexed to at least one solvent molecule.
  • the compounds of the present disclosure may be complexed with from 1 to 10 solvent molecules.
  • the solvent is water and the solvate is a hydrate.
  • the term “agriculturally acceptable carrier” refers to a substance that aids the administration of an active agent to and absorption by an agricultural crop and may be included in the compositions of the present disclosure without causing a significant adverse toxicological effect on the agricultural crop.
  • An agriculturally acceptable carriers is thus compatible with the other ingredients of the formulation and not deleterious to the environment or organism (e.g., plant) to which it is applied.
  • Non-limiting examples of agriculturally acceptable carriers include water, NaCl, normal saline solutions, normal sucrose, normal glucose, binders, fillers, disintegrants, lubricants, coatings, and the like.
  • One of skill in the art will recognize that other agriculturally acceptable carriers are useful in the present disclosure.
  • the term “degradation efficiency” refers to a measure of an ability of a molecule (e.g., a targeted protein degrader) to induce degradation of a particular substance (e.g., a targeted protein).
  • the degradation efficiency of a molecule can be measured in terms of, for example, a half-maximal degradation concentration (DC50) or a maximal degradation (Dmax).
  • the half-maximal degradation concentration (DC50) of a molecule is the concentration of the molecule at which degradation of 50% of the targeted substance has been induced.
  • the maximal degradation (D max ) of a molecule is the maximal observed percent of the targeted substance that the molecule induces the degradation of.
  • a PROTAC is a bifunctional compound having a binding ligand that binds a protein in an insect cell, where this binding ligand is referred to herein as a protein targeting moiety (PTM).
  • PTM protein targeting moiety
  • L linker
  • LTM ligase targeting moiety
  • PROTACs may be used to inhibit or degrade essentially any protein of interest in an insect (e.g., proteins required for the insect and/or arachnid to maintain its normal physiological and biochemical functions, proteins that limit the insect's and/or arachnid’s ability to feed, grow, or survive, or proteins involved in processes such as development, metabolism, or neurotransmission).
  • the provided PROTACs may be designed by considering multiple factors, including the type of ligase being targeted, the selected configuration of the PROTAC linker, the choice of the POI ligand and its binding site, the choice of the ligase ligand and its binding site, and the nature of the protein-protein interaction interface between the ligase and the POI.
  • the half-maximal inhibitory concentration (IC 50 ) values of the LTMs and the bifunctional compounds, described herein, are measures of the binding affinity exhibited by the LTMs and bifunctional compounds, respectively.
  • the IC50 values may be determined according to any method known in the art such as, for example, a fluorescent polarization assay.
  • the LTM exhibits a binding affinity to the E3 ubiquitin ligase (e.g., VHL or CRBN) with an IC50 of less than about 1 mM.
  • the LTMs described herein demonstrate an activity with an IC 50 of less than about 500 ⁇ M, less than 200 ⁇ M, less than about 100 ⁇ M, less than about 50 ⁇ M, less than about 10 ⁇ M, less than about 5 ⁇ M, or less than about 1 ⁇ M.
  • the bifunctional compounds described herein exhibit a binding affinity to the E3 ubiquitin ligase (e.g., VHL or CRBN) and/or the target protein with an IC 50 of less than about 100 ⁇ M, less than about 50 ⁇ M, less than about 10 ⁇ M, less than about 1 ⁇ M, less than about 500 nM, less than about 100 nM, less than about 50 nM, less than about 10 nM, less than about 1 nM, less than about 500 pM, less than about 100 pM, less than about 50 pM, less than about 10 pM, less than about 1 pM, less than about 0.5 pM, less than about 0.1 pM, less than about 0.05 pM, less than about 0.01 pM, less than about 0.005 pM, or less than about 0.001 pM.
  • E3 ubiquitin ligase e.g., VHL or CRBN
  • the D Max of the bifunctional compounds described herein may be determined according to any method known in the art such as, for example, a western blot analysis.
  • the bifunctional compounds have a D Max greater than or equal to 80%.
  • the bifunctional compounds have a D Max greater than 30%, greater than 50%, greater than 75%, or greater than or equal to 80%.
  • the bifunctional compounds have a DMax greater than 50%.
  • the bifunctional compounds have a D Max greater than 75%.
  • the DC50 value of the bifunctional compounds described herein may be determined according to any method known in the art such as, for example, a western blot analysis.
  • the DC 50 value of the bifunctional compounds is less than about 10 ⁇ M, less than about 1 ⁇ M, less than about 500 nM, less than about 100 nM, less than about 50 nM, less than about 100 nM, less than about 50 nM, less than about 10 nM or less than about 2.5 nM.
  • the DC50 value of the bifunctional compounds is less than 10 nM. In some embodiments described herein, the DC50 value of the bifunctional compounds is less than 2.5 nM.
  • the bifunctional compounds have a D Max greater than 30%, greater than 50%, greater than 75%, or greater than or equal to 80% and the DC50 value of the bifunctional compounds is less than about 10 ⁇ M, less than about 1 ⁇ M, less than about 500 nM, less than about 100 nM, less than about 50 nM, less than about 100 nM, less than about 50 nM, less than about 10 nM or less than 2.5 nM.
  • the bifunctional compound includes compounds having a DC 50 of ⁇ about 2.5 nM, wherein the DC 50 is optionally determined as described herein.
  • the bifunctional compound includes compounds having a DC50 that is ⁇ about 2.5 nM and ⁇ about 10 nM, wherein the DC50 is optionally determined as described herein. In some embodiments described herein, the bifunctional compound includes compounds having a DC 50 of ⁇ about 2.5 nM and ⁇ about 30 nM, wherein the DC50 is optionally determined as described herein. In some embodiments described herein, the bifunctional compound includes compounds having a DC 50 of ⁇ about 30 nM, wherein the DC 50 is optionally determined as described herein. In some embodiments described herein, a compound or compounds having a DC50 of ⁇ about 30 nM is or are excluded.
  • the DC 50 value of the bifunctional compounds described herein can be determined according to any method known in the art, such as, for example, a western blot analysis.
  • the bifunctional compound includes compounds having a DMax of ⁇ about 75% degraded, wherein the DMax is optionally determined as described herein.
  • the bifunctional compound includes compounds having a D Max that is > about 50% degraded and ⁇ about 75% degraded, wherein the DMax is optionally determined as described herein.
  • the bifunctional compound includes compounds having a D Max of ⁇ about 30% degraded, wherein the D Max is optionally determined as described herein.
  • a compound or compounds having a DMax of ⁇ about 30 % degraded is or are excluded, wherein the D Max may be determined as described herein.
  • a compound or compounds having a DMax of ⁇ about 50 % degraded is or are excluded, wherein the DMax may be determined as described herein.
  • the DMax value of the bifunctional compounds described herein can be determined according to any method known in the art, such as, for example, a western blot analysis or as described herein. 1.
  • Insect ubiquitin ligases and ligase targeting moieties LTMs
  • cheminformatics and computational chemistry approaches e.g., ligand conformational sampling, PROTAC conformational sampling, 3D docking, protein- protein docking, molecular dynamics, machine learning
  • ligand conformational sampling e.g., PROTAC conformational sampling, 3D docking, protein- protein docking, molecular dynamics, machine learning
  • Such structures may then be optimized through rational medicinal chemistry optimization. See, for example, Ishida, T. and Ciulli, A. (2021) SLAS Discovery 26(4) 484-502.
  • one or more known ubiquitin ligase binders are used in the compounds and methods provided herein. Such binders are disclosed in U.S.
  • Other E3 ligase ligands include those described in M.
  • the ubiquitin ligase is Cereblon
  • the ligase targeting moiety is an N-substituted 1,3-dioxoisoindolinyl moiety, which is optionally substituted with one or more substituents independently selected from the group consisting of C1-6 alkyl, halo, hydroxy, amino, C 1-6 alkylamino, C 1-6 amido, C 1-6 acyl, nitro, cyano, and C 1-6 alkoxy.
  • the ubiquitin ligase is VHL.
  • the von Hippel-Lindau tumor suppressor (VHL) is an E3 ubiquitin ligase.
  • VHL comprises the substrate recognition subunit/E3 ubiquitin ligase complex VCB, which includes elongins B and C, and a complex including Cullin-2 and Rbx1.
  • the primary substrate of VHL is Hypoxia Inducible Factor 1 ⁇ (HIF-1 ⁇ ), a transcription factor.
  • HIF-1 ⁇ Hypoxia Inducible Factor 1 ⁇
  • targeting moieties may be selected from those disclosed in US Patent Nos.10,730,870; 10,071,164; 10,730,862; 10,772,962; and 11,242,344; all of which are herein incorporated by reference.
  • the VHL targeting moiety has a structure according to the formula I), wherein R 5a and R 5b may each indepen en, hydroxy, amine, haloalkyl, optionally substituted alkyl, optionally substituted alkoxy, optionally substituted hydroxyl alkyl, optionally substituted alkylamine, optionally substituted amide, optionally substituted alkyl-amide, optionally substituted alkyl-cyano, optionally substituted alkyl-phosphate, optionally substituted aryl, optionally substituted alkyl-aryl, optionally substituted heteroalkyl, optionally substituted alkyl-heterocyclyl, optionally substituted alkoxy-heterocyclyl, COR 14 , alkyl-COR 14 , CONR 15a R 15b , NHCOR 14 , NHCH 3 COR 14 , or -X-L 1 .
  • R 5a and R 5b are combined with the carbon atom to which they are attached to form an optionally substituted 3- to 5-membered cycloalkyl, heterocyclyl, spirocycloalkyl, or spiroheterocyclyl, wherein the spiroheterocyclyl is not epoxide or aziridine.
  • Each R 6 may independently be C 1-6 alkyl, halogen, C 1-6 haloalkyl, hydroxy, C 1-6 alkoxy, C 1-6 haloalkoxy, C 3-8 cycloalkyl, C 6-12 aryl, C 5-12 heteroaryl, C 3-15 heterocyclyl, cyano, nitro, NR 15a R 15b , OR 14 , CONR 15a R 15b , NR 15a COR 15b , SO2NR 15a R 15b , NR 15a SO2R 15b , or -X-L 1 , wherein the alkyl, haloalkyl, alkoxy, cycloalkyl, aryl, heteroaryl, and heterocyclyl are optionally substituted with 1 to 4 R 6a groups.
  • R 8 may be an optionally substituted aryl, optionally substituted heteroaryl, or .
  • R 9 and R 10 may independently be hydrogen, optionally substituted alkyl, optionally sub stituted cycloalkyl, optionally substituted hydroxyalkyl, optionally substituted heteroaryl, haloalkyl, or .
  • R 9 and R 10 may be combined with the carbon atom to which they are attached to form an optionally substituted cycloalkyl.
  • R 2 and R 3 may each independently be hydrogen, C 1-6 alkyl, or C 1-6 hydroxyalkyl.
  • R 2 and R 3 may be combined with the carbon to which they are attached to form a C3-8 cycloalkyl, C6-12 aryl, or C5-12 heteroaryl, wherein the cycloalkyl, aryl and heteroaryl are optionally substituted with 1 to 4 R 2a groups.
  • R 4 may be hydrogen, C 1-6 alkyl, C 1-6 hydroxyalkyl, or -X-L 1 .
  • R 11 may be an optionally substituted heterocyclyl, optionally substituted alkoxy, optionally substituted heteroaryl, optionally substituted ary R 1 may be hydrogen, optionally optionally substituted (cycloalkyl)alkylcarbonyl, optionally substituted aralkylcarbonyl, optionally substituted arylcarbonyl, optionally substituted (heterocyclyl)carbonyl, optionally substituted aralkyl, - C(O)R 1a , or -C(O)-X-L 1 .
  • R 1a may be C 1-6 alkyl, C 3-8 cycloalkyl, C 6-12 aryl, or C 5-12 heteroaryl, wherein the cycloalkyl, aryl, and heteroaryl are optionally substituted with 1 to 4 R 1b groups.
  • Each R 1b , R 2a , and R 6a may independently be C 1-6 alkyl, cyano, halogen, C 1-6 haloalkyl, hydroxy, C 1-6 alkoxy, C 1-6 haloalkoxy, or -X-L 1 .
  • R 12 may be hydrogen or optionally substituted alkyl.
  • Each R 13 may independently be hydrogen, halogen, optionally substituted alkoxy, cyano, optionally substituted alkyl, haloalkyl, haloalkoxy, or -X-L 1 .
  • Each R 14 may independently be hydrogen, OH, O-C 1-6 alkyl, optionally substituted alkyl, or NR 15a R 15b .
  • R 15a and R 15b may each independently be hydrogen, optionally substituted alkyl, or optionally substituted cycloalkyl, or are combined with the nitrogen atom to which they are attached to form a 4- to 6-membered heterocyclyl.
  • R 16 is hydroxy, a group that can be metabolized to hydroxy, or sulfonyl halide.
  • A may be optionally substituted phenyl, optionally substituted napthyl, or an optionally substituted 5- to 10- membered heteroaryl.
  • X may be a bond, CH2, NH, NMe, O, or S.
  • L 1 is a site of attachment to the linker.
  • the subscripts n and p may each independently be an integer from 0 to 4.
  • At least one -X-L 1 is present in the compound of Formula (I) when the compound is a bifunctional compound, e.g., PROTAC, having the general structure LTM-L 1 -PTM.
  • the VHL targeting moiety has a structure according to the formula a) wherein R 1 may be hydrogen, C 1-6 1 4 )-X-L .
  • R may be hydrogen, C 1-6 alkyl, C1-6 hydroxyalkyl, or -X-L 1 .
  • R 5a may be hydrogen, C1-6 alkyl, halogen, C1-6 haloalkyl, C1-6 alkyl-amide, hydroxy, C1-6 alkoxy, C1-6 haloalkoxy or -X-L 1 .
  • Each R 6 may independently be C1-6 alkyl, halogen, C 1-6 haloalkyl, hydroxy, C 1-6 alkoxy, C 1-6 haloalkoxy, C 3-8 cycloalkyl, C 6-12 aryl, C5-12 heteroaryl, OR 14 , or -X-L 1 , wherein the cycloalkyl, aryl, and heteroaryl are optionally substituted with 1 to 4 R 6a groups.
  • Other features of formula (Ia) are as described above for formula (I).
  • R 1 is -C(O)R 1a or -C(O)-X-L 1 .
  • R 1 is - C(O)R 1a .
  • R 1a is C 1-6 alkyl or C 3-8 cycloalkyl, wherein the cycloalkyl is optionally substituted with 1 to 4 R 1b groups. In some embodiments, R 1a is C 1-6 alkyl or cyclopropyl, wherein the cyclopropyl is optionally substituted with 1 to 4 R 1b groups. In some embodiments, R 1a is cyclopyropyl optionally substituted with 1 to 4 R 1b groups. In some embodiments, R 1a is fluorocyclopropyl. In some embodiments, R 1a is C 1-6 alkyl. In some embodiments, R 1a is methyl. In some embodiments, R 1 is -C(O)-X-L 1 .
  • R 2 and R 3 are each independently C1-6 alkyl. In some embodiments, R 2 and R 3 are each methyl.
  • R 4 is C1-6 alkyl or -X-L 1 . In some embodiments, R 4 is C1-6 alkyl. In some embodiments, R 4 is methyl. In some embodiments, R 4 is -X-L 1 . In some embodiments, R 4 is -SL 1 .
  • R 5a is hydrogen, C 1-6 alkyl, hydroxy, C 1-6 alkoxy or -X-L 1 . In some embodiments, R 5a is hydrogen, methyl or -X-L 1 .
  • each R 6 is independently C 1-6 alkyl, hydroxy, C 1-6 alkoxy, C 6-12 aryl, C 5-12 heteroaryl, or -X-L 1 , wherein the aryl and heteroaryl are optionally substituted with 1 to 4 R 6a groups.
  • each R 6 is independently C5-12 heteroaryl and -X-L 1 , wherein the heteroaryl is optionally substituted with 1 to 4 R 6a groups.
  • each R 6 is independently thiazole or -X-L 1 , wherein the thiazole is optionally substituted with 1 to 4 R 6a groups.
  • one R 6 is thiazole optionally substituted with 1 to 4 R 6a groups. In some embodiments, one R 6 is thiazole optionally substituted with 1 to 4 C1-6 alkyl groups. In some embodiments, one R 6 is methylthiazole. In some embodiments, one R 6 is -X-L 1 . In some embodiments, one R 6 is -O-L 1 . In some embodiments, one R 6 is methoxy. In some embodiments, n is 2 and one R 6 is methylthiazole and one R 6 is methoxy. In some embodiments, n is 2 and one R 6 is methylthiazole and one R 6 is -X-L 1 . [0074] In some embodiments, R 16 is hydroxy.
  • R 16 is sulfonyl halide. In some embodiments, R 16 is sulfonyl fluoride. [0075] In some embodiments, subscript n is 1 or 2. In some embodiments, subscript n is 1. In some embodiments, subscript n is 2. [0076] In some embodiments, the VHL targeting moiety has a structure according to the formula b) wherein R 2 , R 3 , R 4 , R 5a , R 6 , R 11 , an e. [0077] In some embodiments, the VHL targeting moiety has a structure that is , , , , , , , 23
  • a linker (L) is generally used to connect the PTM to the compound of Formula I.
  • L is a bond (i.e., absent).
  • L is a chemical linker.
  • the linker is a connector with a linear non-hydrogen atom number in the range of 1 to 20, e.g., in the range of 1 to 12, in the range of 3 to 14, in the range of 5 to 16, in the range of 7 to 18, or in the range of 9 to 20.
  • the connector may contain functional groups including, but not limited to, ethers, amides, alkanes and alkyl groups, alkenes and alkenyl groups, alkynes and alkynyl groups, ketones, hydroxyls, carboxylic acids, thioethers, sulfoxides, and sulfones.
  • the linker may contain aryl groups, heteroaryl groups, cyclic groups (including heterocyclic and carbocyclic monocycles, bicycles, tricycles).
  • Substitution including, but not limited to, one or more of halo, such as Cl, F, Br, I, hydroxy, alkyl, amino, alkylamino, amido, acyl, nitro, cyano, and alkoxy may be included in the linker.
  • suitable linker moieties include, but are not limited to, those disclosed in WO 2013/106643, WO 2015/160845, WO 2016/149668, WO 2016/197032, WO 2017/011371, WO 2017/011590, WO 2018/144649, and WO 2019/148055, which are incorporated herein by reference in their entirety.
  • linker design facilitates binding to the target protein and the ubiquitin ligase, influencing degradation efficiency as well as having a significant influence on the overall molecular properties of the PROTAC.
  • the linker structure contributes to achieving appropriate biological function by providing a beneficial combination of cellular uptake, ternary complex geometry, stability, and aqueous solubility.
  • the choice of linker may be informed by, for example, the E3 ligase ligand, the target-binding ligand, and both the identity and attachment positions of the linker.
  • Linkers as disclosed herein may be tested with ligase targeting moieties and protein targeting moieties in various combinations.
  • the linker has a structure -L 2 L 3 -.
  • the L 2 and L 3 components of the linker structure may each independently be a bond, a divalent polymer moiety, or C1-30 alkylene, wherein one or more carbon atoms in each C 1-30 alkylene is optionally and independently replaced by O, C(O), S, or NR 7 .
  • One or more groupings of adjacent carbon atoms in each C1-30 alkylene may optionally and independently be replaced by –NR 7 (CO) or (CO)NR 7 .
  • One or more groupings of adjacent carbon atoms in each C1-30 alkylene may be optionally and independently replaced by a 4- to 8-membered, divalent carbocycle or a 4- to 8-membered, divalent heterocycle having one to four heteroatoms selected from O, S, and N.
  • L is a C 1-6 alkylene diradical linker. In some embodiments, L is a methylene linker. In some embodiments, L is an ethylene linker. In some embodiments, L is a - CH2(OCH2CH2)j- diradical linker, where subscript j is an integer ranging from 1 to 10. In some embodiments, L is -CH 2 (OCH 2 CH 2 ) j -.
  • L is oxy-C 1-6 alkylene (e.g., oxymethylene, oxyethylene, or oxypropylene), optionally substituted with C 1-6 alkyl (e.g., isopropyl) or C3-8 cycloalkyl.
  • L is -NH2CH2CH2- or -C(O)NHCH2CH2-.
  • R L1 and R L2 are each independently optionally linked to other groups to form cycloalkyl and/or heterocyclyl moiety.
  • R L1 and R L2 also are each independently optionally substituted with 1-4 R L5 groups.
  • L comprises one or more ethylene glycol diradical moieties, one or more of which is optionally replaced by a moiety independently selected from: .
  • L comprises one or more ethylene glycol diradical moieties, one or more of which is optionally replaced by a moiety independently selected from: .
  • ompound having two reactive groups including, but not limited to, aldehydes, carboxylates, activated esters, amines, alcohols, and ionizable CH bonds
  • examples of such compounds include, but are not limited to the following.
  • the linker L 1 in any of the formulae provided herein is selected from the groups listed below, wherein “R 1 ” represents the point of attachment to the PTM, and “R 2 ” represents the point of attachment to the LTM, e.g., to the X of one or more of R 1 , R 1b , R 2a , R 4 , R 5a , R 5b , R 6 , R 6a , or R 13 .
  • Target proteins of interest and protein targeting moieties are a protein that is essential to the growth, development, reproduction, and/or survival of the target insect pest.
  • Target proteins include proteins required for the insect and/or arachnid to maintain its normal physiological and biochemical functions. Inhibition or degradation of the target protein limits the insect's and/or arachnid’s ability to feed, grow, or survive.
  • insect and/or arachnid proteins that may targeted by the provided compounds, compositions, and methods include essential proteins, proteins involved in processes such as development, metabolism, or neurotransmission, and proteins that are targets of existing insecticides and/or arachnids.
  • the target protein functions in pathways required for one or more cellular functions such as transcription, translation, formation of the cytoskeleton, cell-cycle, metabolism (anabolism or catabolism), endocytosis, intracellular and intercellular transport, calcium binding, nucleus import and export, nucleic acid binding, signal peptidase-protein binding, the proteasome, vesicle transport, neuro-transmission, water-balance, ion-balance, splicing, mitosis, meiosis, chromosome organization, stability or integrity, micro RNAs, siRNAs, posttranslational protein modifications, electron transport, apoptosis, membrane integrity, and cell adhesion.
  • cellular functions such as transcription, translation, formation of the cytoskeleton, cell-cycle, metabolism (anabolism or catabolism), endocytosis, intracellular and intercellular transport, calcium binding, nucleus import and export, nucleic acid binding, signal peptidase-protein binding, the proteasome, vesicle transport
  • insect and/or arachnid proteins that may be targeted by the provided compounds, compositions, and methods include essential proteins, proteins involved in processes such as development, metabolism, or neurotransmission, and proteins that are targets of existing insecticides.
  • the following targets and pathways may be targeted with the insect control PROTACs disclosed herein; examples of exemplary target binders associated with each target are given in parenthesis: (1) Acetylcholinesterase targeted by Carbamates (e.g., Carbofuran, Carbosulfan, Methomyl) or Organophosphates (e.g., Acephate, Chlorpyrifos, Phorate); (2) GABA-gated chloride channel targeted by Cyclodiene Organochlorines (e.g., Chlordane, Endosulfan) or Phenylpyrazoles (e.g., Ethiprole, Fipronil); (3) Sodium channel targeted by Pyrethroids and Pyrethrins (e.g., Bifenthr
  • target proteins of interest include juvenile hormone receptor, Bromodomain Containing 3 (BRD3), Cholone O-Acetyltransferase (ChAT), Dihydrofolate Reductase (DHFR), FK506 binding proteins (FKBP), Geranylgeranyl diphosphate synthase 1 (GGPS1), 3-Hydroxy- 3Methylglutaryl-CoA Reductase (HMGCR), Inosine-5'-monophosphate dehydrogenase (IMPDH), Juvenile hormone acid O-methyltranserase (JHAMT), lysine-tRNA ligase (KRS1), MET Proto-Oncogene, Receptor Tyrosine Kinase (MET), Thioredoxin reductases (TXNRD), N- myristoyl transferase (NMT), SWI/SNF Related, Matrix Associated, Actin Dependent Regulator of Chromatin, Subfamily A, Member 2 (
  • Target binders include those selected from, but not limited to, analogues and derivatives of compounds such as Abamectin, Acephate, Acequinocyl, Acetamiprid, Acrinathrin, Acynonapyr, Afidopyropen, Alanycarb, Aldicarb, Allethrin, Amitraz, Azinphos-methyl, Azocyclotin, Benfuracarb, Benomyl, Bensultap, Benzpyrimoxan, Bifenazate, Bifenthrin, Bioallethrin S-cyclopentenyl, Bioresmethrin, Bistrifluron, BPMC, Broflanilide, Bromopropylate, Buprofezin, Cadusafos, Carbaryl, Carbofuran, Carbosulfan, Cartap, Chlorantraniliprole, Chlorethoxyfos, Chlorfenapyr, Chlorfenvin
  • WDS Ligands include, but are not limited to: * R 23 X 2 X 3 O 2 1 Formula 2 wherein the symbol * indicates o a linker, for example any of the linkers described in Section B.2.
  • R 21 is C6-C10 aryl or C5-C10 heteroaryl.
  • R 22 is heterocycloalkyl, which contains one or more nitrogen atoms.
  • R 23 is selected from C 6 -C 10 aryl, C 5 -C 10 heteroaryl, or heterocycloalkyl, heterocycloalkenyl.
  • R 24 , R 25 , R 26 , R 27 , R 28 , R 29 , R 30 , and R 31 are independently selected from H, C 1 -C 8 alkyl, C 1 -C 8 haloalkyl, C 1 -C 8 hydroxyalkyl, C 3 - C 10 cycloalkyl, C 3 -C 10 heterocyclyl, C(O)C 1 -C 8 alkyl, C(O)C 1 -C 8 haloalkyl, C(O)C 1 -C 8 hydroxyalkyl, C(O)C3-C10 cycloalkyl, and C(O)C3-C10 heterocyclyl, or R 26 and R 27 ; R 30 and R 31 together with the nitrogen atom to which they are connected can independently form 3-10 membered heterocyclyl rings.
  • R 32 , R 33 , and R 34 are independently selected from H, C 1 -C 8 alkyl, C1-C8 haloalkyl, C1-C8 alkoxyalkyl, C1-C8 hydroxyalkyl, C3-C8 cycloalkyl, C3-C7 heterocycloalkyl, C6-C10 aryl, C5-C10 heteroaryl, C(O)C1-C8 alkyl, C(O)C1-C8 haloalkyl, C(O)C1- C 8 hydroxyalkyl, C(O)C 1 -C 8 alkoxyalkyl, C(O)C 3 -C 10 cycloalkyl, C(O)C 3 -C 10 heterocyclyl, C(O)C6-C10 aryl, C(O)C5-C10 heteroaryl, C1-C8 alkyleneC3-C10 cycloalkyl, C1-C8 alkyleneC3-
  • R 35 and R 36 are independently selected from H, C 1 -C 8 alkyl, C 1 -C 8 haloalkyl, C 1 -C 8 alkoxyalkyl, C 1 -C 8 hydroxyalkyl, C 3 -C 10 cycloalkyl, C 3 -C 10 heterocycloalkyl, C 6 -C 10 aryl, C 5 -C 10 heteroaryl, C(O)C1-C8 alkyl, C(O)C1-C8 haloalkyl, C(O)C1-C8 hydroxyalkyl, C(O)C1-C8 alkoxyalkyl, C(O)C 3 -C 10 cycloalkyl, C(O)C 3 -C 10 heterocycloalkyl, C(O)C 6 -C 10 aryl, C(O)C 5 -C 10 heteroaryl, C(O)OC 1 -C 8 alkyl, C(O)OC 1 -C 8 halo
  • R 37 is selected from C 3 -C 10 cycloalkyl, C 3 -C 10 heterocycloalkyl, C 6 -C 10 aryl, C 5 -C 10 heteroaryl, C(O)C 1 -C 8 alkyl, C(O)C 1 -C 8 haloalkyl, C(O)C 1 -C 8 hydroxyalkyl, C(O)C 1 -C 8 alkoxyalkyl, C(O)C3-C10 cycloalkyl, C(O)C3-C10 heterocycloalkyl, C(O)C6-C10 aryl, and C(O)C 5 -C 10 heteroaryl.
  • X 1 , X 2 , and X 3 are independently selected from CR 38 , and N.
  • R 38 is selected from H, F, Cl, C 1-8 alkyl, C 1 -C 8 haloalkyl, C 1 -C 8 alkoxyalkyl, or C 3 -C 8 cycloalkyl.
  • R 21 has a structure of:
  • R 22 has a structure of: [0094] In some aspects of Formula 2, R 23 has a structure of: wherein e symo n caes a connec on o a ner, or exampe any o e ners described in Section B.2.
  • R 39 is selected from a bond, C 1 -C 8 alkyl, C 1 -C 8 haloalkyl, OR 40 , SR 40 , SO 2 R 40 , NR 41 R 42 , R 43 , C1- C8 alkyleneR 43 , C2-C8 alkenyleneR 43 , OC1-C8 alkyleneR 43 , SC1- C8 alkyleneR 43 , C1-C8 alkyleneOR 40 , C1-C8 alkyleneSR 40 , C1-C8 alkyleneNR 41 R 42 , OC1-C8 alkyleneOR 40 , OC 1 -C 8 alkyleneSR 40 , OC 1 -C 8 alkyleneNR 41 R 42 , SC 1 -C 8 alkyleneOR 40 , SC 1 -C 8 alkyleneSR 40 , SC 1 -C 8 alkyleneNR 41 R 42 , C(O)OR 40 , C(S)OR 40 , C(O) NR 41 R 42
  • R 40 is selected from H, C1-C8 alkyl, C1-C8 haloalkyl, C1-C8 alkoxyalkyl, C1-C8 hydroxyalkyl, C3- C 8 cycloalkyl, C 3 -C 7 heterocycloalkyl, C 6 -C 10 aryl, C 5 -C 10 heteroaryl, C(O)C 1 -C 8 alkyl, C(O)C 1 - C 8 haloalkyl, C(O)C 1 -C 8 hydroxyalkyl, C(O)C 1 -C 8 alkoxyalkyl, C(O)C 3 -C 10 cycloalkyl, C(O)C 3 - C10 heterocyclyl, C(O)C6-C10 aryl, C(O)C5-C10 heteroaryl, C1-C8 alkyleneC3-C10 cycloalkyl, C1- C8 alkyleneC3-C10 heterocyclo
  • R 41 and R 42 are independently selected from H, C 1 -C 8 alkyl, C 1 -C 8 haloalkyl, C 1 -C 8 alkoxyalkyl, C1-C8 hydroxyalkyl, C3-C10 cycloalkyl, C3-C10 heterocycloalkyl, C6-C10 aryl, C5-C10 heteroaryl, C(O)C1-C8 alkyl, C(O)C1-C8 haloalkyl, C(O)C1-C8 hydroxyalkyl, C(O)C1-C8 alkoxyalkyl, C(O)C 3 -C 10 cycloalkyl, C(O)C 3 -C 10 heterocycloalkyl, C(O)C 6 -C 10 aryl, C(O)C 5 -C 10 heteroaryl, C(O)OC 1 -C 8 alkyl, C(O)OC 1 -C 8 haloalkyl, C
  • R 43 is selected from C 3 -C 10 cycloalkyl, C 3 -C 10 heterocycloalkyl, C 6 -C 10 aryl, C 5 -C 10 heteroaryl, C(O)C 1 -C 8 alkyl, C(O)C 1 -C 8 haloalkyl, C(O)C 1 -C 8 hydroxyalkyl, C(O)C 1 -C 8 alkoxyalkyl, C(O)C3-C10 cycloalkyl, C(O)C3-C10 heterocycloalkyl, C(O)C6-C10 aryl, and C(O)C5-C10 heteroaryl.
  • Additional WDS Ligands include, but are not limited to: wherein the symbol * indicates a connection to a linker, for example any of the linkers described in Section B.2.
  • X 4 , X 5 , and X 6 are independently selected from null, CR 56 , and N, wherein R 56 , at each occurrence, is independently selected from hydrogen, halogen, cyano, nitro, optionally substituted C 1 -C 8 alkyl, optionally substituted C 1 -C 8 haloalkyl, optionally substituted C 1 - C8 alkoxy, optionally substituted C1-C8 haloalkoxy, optionally substituted C1-C8 alkylamino, optionally substituted C1-C8 haloalkylamino, optionally substituted C1-C8 alkoxycarbonyl, optionally substituted C 1 -C 8 haloalkoxycarbonyl, optionally substituted C 1 -C 8 alkylaminocarbonyl, optionally substituted
  • X 4 , X 5 , and X 6 are CR 56.
  • X 4 and X 5 are CR 56 ; and X 6 is N.
  • R 56 is selected from hydrogen, halogen, optionally substituted C1-C8 alkyl, optionally substituted C1-C8 alkoxy, optionally substituted C1- C 8 alkylamino, optionally substituted C 1 -C 8 alkoxycarbonyl, optionally substituted C 3 - C 8 carbocyclyl, and optionally substituted C 4 -C 8 heterocyclyl.
  • R 56 is selected from H, F, Cl, Br, CH3, CH3O, and CH3O(CO)-.
  • R 56 is H.
  • A1 is selected from null, optionally substituted C 1 -C 8 alkylene, optionally substituted C1-C8 alkylamino, optionally substituted C1-C8 alkyleneamino, optionally substituted C2-C8 alkenylene, optionally substituted C2-C8 alkenyleneamino, optionally substituted C2-C8 alkynylene, and optionally substituted C 2 -C 8 alkynyleneamino. [0099] In some aspects of Formulae 3A, 3B and 3C, A1 is selected from null, and optionally substituted C1-C8 alkylene.
  • A1 is null. [0100] In some aspects of Formulae 3A, 3B and 3C, A1 is CH 2 . In some aspects of Formulae 3A, 3B and 3C, R 51 is selected from selected from null, carbocyclyl, heterocyclyl, aryl, and heteroaryl, which are optionally substituted with one or more substituents independently selected from hydrogen, halogen, oxo, CN, NO2, optionally substituted C1-C8 alkyl, optionally substituted C 2 -C 8 alkenyl, optionally substituted C 2 -C 8 alkynyl, optionally substituted C 1 -C 8 alkoxyC 1 - C8alkyl, optionally substituted C1-C8alkylaminoC1-C8alkyl, optionally substituted C1-C8alkoxy, optionally substituted C1-C8alkylamino, optionally substituted 4-10 membered heterocyclyl
  • R 51 is selected from aryl and heteroaryl, which are optionally substituted with one or more substituents independently selected from hydrogen, halogen, oxo, CN, NO2, optionally substituted C1-C8 alkyl, optionally substituted C2- C 8 alkenyl, optionally substituted C 2 -C 8 alkynyl, optionally substituted C 1 -C 8 alkoxyC 1 -C 8 alkyl, optionally substituted C 1 -C 8 alkylaminoC 1 -C 8 alkyl, optionally substituted C 1 -C 8 alkoxy, optionally substituted C1-C8alkylamino, optionally substituted 4-10 membered heterocyclylC1- C 8 alkyl, optionally substituted 3-10 membered carbocyclylC 1 -C 8 alkyl, optionally substituted 4- 10 membered heterocyclyloxy, optionally substituted 3-10 membered carbocyclylC 1 -C 8 alkyl, optionally substituted 4-
  • R 52 is selected from null, hydrogen, optionally substituted C 1 -C 8 alkyl, optionally substituted C 1 -C 8 haloalkyl, optionally substituted C 3 - C8 cycloalkyl, optionally substituted C4-C8 heterocycloalkyl, optionally substituted aryl, and optionally substituted heteroaryl. [0102] In some aspects of Formulae 3A, 3B and 3C, R 52 is selected from null, hydrogen, optionally substituted C1-C8 alkyl, optionally substituted C3-C8 cycloalkyl, and optionally substituted C3-C8 cycloalkylC1-C8 alkyl.
  • R 53 is selected from null, hydrogen, and optionally substituted C 1 -C 8 alkyl. [0103] In some aspects of Formulae 3A, 3B and 3C, R 53 is selected from null, hydrogen, methyl, methylene, ethyl, ethylene, isopropyl, and cyclopropyl.
  • R 54 is selected from null, hydrogen, halogen, optionally substituted C 1 -C 8 alkyl, optionally substituted C 1 -C 8 alkoxy, optionally substituted C 1 -C 8 alkylamino, optionally substituted C3-C8 cycloalkyl, optionally substituted C3-C8 cycloalkoxy, optionally substituted C3- C 8 cycloalkylamino, optionally substituted C 4 -C 8 heterocyclyl, optionally substituted C 2 -C 8 alkenylene, optionally substituted C 2 -C 8 alkynylene, optionally substituted aryl, optionally substituted aryloxy, optionally substituted arylamino, and optionally substituted heteroaryl.
  • R 53 and R 54 together with the atoms to which they are connected optionally form a 5-membered carbocyclyl ring, 6-membered carbocyclyl ring, 5- membered heterocyclyl ring, or 6-membered heterocyclyl ring. [0104] In some aspects of Formulae 3A, 3B and 3C, R 53 and R 54 , together with the atoms to which they are connected optionally form a 5-membered carbocyclyl ring.
  • R 55 at each occurrence, is independently selected from hydrogen, halogen, optionally substituted C1-C8 alkyl, optionally substituted C1-C8 alkoxy, optionally substituted C 1 -C 8 alkylamino, optionally substituted C 3 -C 8 carbocyclyl, and optionally substituted C 4 -C 8 heterocyclyl. [0105] In some aspects of Formulae 3A, 3B and 3C, R 55 is hydrogen.
  • suitable PTMs include those known in the art and described in, for example, Doroshow et al, Annals of Oncology, 2017, 28, 1776-1787; Perez-Salviaa and Esteller Epigenetics, 2017, 12, 323-339; Klein, RMD Open, 2018, 4:e000744; Ocana et al, Oncotarget, 2017, 8, 71285-71291; Hogg et al, Blood, 2017, 130, 2537; U.S.
  • the BRD3 binders include those selected from, but not limited to, olinone (CAS No.1770789-37-1), JQ1 (CAS No.1268524-70-4), Apabetalone/RVX-208/RVX000222 (CAS No.1044870-39-4), PF-1 (1403764-72-6), Mivebresib/ABBV-075 (CAS No.1445993-26-9), BAY1238097 (CAS No.1564268-08-1), BI 894999 (CAS No.1660117-38-3), BMS-986158 (CAS No.1800340-40-2), CPI-0610 (CAS No.
  • RVX2135 (CAS No.1253733-17-3), BAY-299 (CAS No.2080306-23-4), GSK1324726A/I-BET726 (CAS No.1300031-52-0), Molibresib/I-BET762/GSK525762 (CAS No.1260907-17-2), RVX297 (CAS No.1044871-04-6), SF1126 (CAS No.936487-67-1), INCB054329 (CAS No.1628607-64-6), INCB057643 (CAS No.1820889-23-3), LY294002 (CAS No.154447-36-6), AZD5153 (CAS No.1869912-40-2), MT-1 (CAS No.2060573-82-0), and MS645 (CAS No.2250091-96-2) and analogues and derivatives thereof.
  • Suitable synthetic routes are depicted in the schemes herein.
  • Those skilled in the art will recognize if a stereocenter exists in the compounds disclosed herein. Accordingly, the present disclosure includes both possible stereoisomers (unless specified in the synthesis) and includes not only racemic compounds but the individual enantiomers and/or diastereomers as well.
  • a compound When a compound is desired as a single enantiomer or diastereomer, it may be obtained by stereospecific synthesis or by resolution of the final product or any convenient intermediate. Resolution of the final product, an intermediate, or a starting material may be affected by any suitable method known in the art. See, for example, "Stereochemistry of Organic Compounds" by E. L. Eliel, S. H. Wilen, and L.
  • the compounds of the present disclosure may be prepared in a number of ways well known to those skilled in the art of organic synthesis.
  • compounds of the present disclosure may be synthesized using the methods described below, together with synthetic methods known in the art of synthetic organic chemistry, or variations thereon as appreciated by those skilled in the art. Preferred methods include but are not limited to those methods described herein.
  • Non-limiting examples of protecting groups e.g., N-terminal protecting groups which may be employed for synthesis of the compounds provided herein include Fmoc, Boc, allyloxycarbonyl (Alloc), benzyloxycarbonyl (Z), and photolabile protecting groups.
  • Sidechain protecting groups include, but are not limited to, Fmoc; Boc; cyclohexyloxycarbonyl (Hoc); allyloxycarbonyl (Alloc); mesityl-2-sulfonyl (Mts); 4-(N-methylamino)butanoyl (Nmbu); 2,4- dimethylpent-3-yloxycarbonyl (Doc); 1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)-3-ethyl (Dde); 1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)-3-methylbutyl (ivDde); 4-methyltrityl (Mtt).
  • a base may be used to activate or complete the activation of amino acids prior to exposing the amino acids to reaction partners. Any suitable base may be used.
  • the base is a Lewis base.
  • the base is a non-nucleophilic bases, such as triisopropylethylamine, N,N-diisopropylethylamine, certain tertiary amines, or collidine, that are non-reactive to or react slowly with protected peptides to remove protecting groups.
  • the base has a sufficient pKa to allow for deprotonation of the amino acid carboxylic acid.
  • a coupling agent may be used to form a bond with the carboxylate moiety of an amino acid to facilitate the coupling reaction and the formation of an amide bond.
  • the coupling agent may be used to form activated amino acids before combination with a coupling partner (e.g., an amine) to produce the peptide product. Any suitable coupling agent may be used.
  • the coupling agent is a carbodiimide, a guanidinium salt, a phosphonium salt, or a uronium salt.
  • carbodiimides include, but are not limited to, ⁇ , ⁇ '- dicyclohexylcarbodiimide (DCC), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), and the like.
  • phosphonium salts include, but are not limited to, (benzotriazol-l- yloxy)tripyrrolidino-phosphonium hexafluorophosphate (PyBOP); bromotris(dimethylamino)phosphonium hexafluorophosphate (BroP); and the like.
  • guanidinium/uronium salts include, but are not limited to, O-(benzotriazol-l-yl)- ⁇ , ⁇ , ⁇ ', ⁇ '- tetramethyluronium hexafluorophosphate (HBTU); 2-(7-aza-lH-benzotriazole-l- yl)-l, l,3,3- tetramethyluronium hexafluorophosphate (HATU); l-[(l-(cyano-2-ethoxy-2- oxoethylideneaminooxy) dimethylaminomorpholino)] uronium hexafluorophosphate (COMU); and the like.
  • HBTU O-(benzotriazol-l-yl)- ⁇ , ⁇ , ⁇ ', ⁇ '- tetramethyluronium hexafluorophosphate
  • HATU 2-(7-aza-lH-benzotriazole-l- yl)
  • peptide bond formation may be conducted with a carboxylic acid (e.g., RC(O)OH), a base, and a coupling agent as described above, activated acid derivatives (e.g., RC(O)X, wherein X is a leaving group) may also be employed.
  • carboxylic acid e.g., RC(O)OH
  • base e.g., a base
  • a coupling agent e.g., activated acid derivatives (e.g., RC(O)X, wherein X is a leaving group) may also be employed.
  • activated acid derivatives e.g., RC(O)X, wherein X is a leaving group
  • Activated acid derivatives include, but are not limited to, anhydrides (including symmetric, mixed, or cyclic anhydrides), activated esters (e.g., p-nitrophenyl esters, pentafluorophenyl esters, N-succinimidyl esters, and the like), acylazoles (e.g., acylimidazoles, prepared using carbonyl diimidazole, and the like), acyl azides, and acid halides (e.g., acid chlorides).
  • anhydrides including symmetric, mixed, or cyclic anhydrides
  • activated esters e.g., p-nitrophenyl esters, pentafluorophenyl esters, N-succinimidyl esters, and the like
  • acylazoles e.g., acylimidazoles, prepared using carbonyl diimidazole, and the like
  • acyl azides e.g., acid chlorides
  • Non-limiting examples of support materials for solid-phase peptide synthesis include polystyrene (e.g., microporous polystyrene resin, mesoporous polystyrene resin, macroporous polystyrene resin; including commercially available Wang resins, Rink amide resins, and trityl resins), glass, polysaccharides (e.g., cellulose, agarose), polyacrylamide resins, polyethylene glycol, or copolymer resins (e.g., comprising polyethylene glycol, polystyrene, etc.).
  • the solid support may have any suitable form factor.
  • the solid support may be in the form of beads, particles, fibers, or in any other suitable form factor. D.
  • the disclosure provides methods for controlling a level of a target protein that is expressed in an insect cell.
  • the target protein can be, for example, any of the insect proteins of interest described in Section B.3.
  • the methods generally include contacting the insect cell with an effective amount of any of the bifunctional compounds, e.g., PROTAC compound, described herein.
  • the bifunctional compound can have one or more protein targeting moieties covalently bonded via a linker to one or more ligase targeting moieties, where the ligase targeting moieties can have the chemical structure of any of the formulas provided herein.
  • the ligase targeting moiety has a structure according to any one of the formulas described in Section B.1, the linker is any of those described in Section B.2, and/or the protein targeting moiety is any of those described in Section B.3.
  • the compound contacted with the insect cell includes one or more compounds having any of the structures set forth in Tables 1-3 of the Examples. [0117]
  • the effective amount of the compound contacted with the insect cell in the provided methods for controlling a target protein level can be a pesticidally effective amount, referred to herein as an effective amount.
  • An “effective amount” is an amount of a compound that causes the death of at least one pest (i.e., insect) or that noticeably reduces pest growth, feeding, or normal physiological development.
  • an effective amount of a provided compound can cause an insect growth, feeding, and/or development decrease of about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 85%, 90%, 95% or greater.
  • contacting an insect cell with an effective amount of a provided compound controls a level of a target protein by inducing degradation of the target protein within the insect cell.
  • the amount of the compound contacted with the insect cell may induce degradation of the target protein, where the degradation has an effective degradation efficiency.
  • An effective degradation efficiency may be one having a DC50 and/or Dmax satisfying a particular cutoff. Exemplary values of DC 50 and D max indicating effective degradation efficiency can be any of those described in Section B of this disclosure.
  • the bifunctional compounds disclosed herein display activity against a variety of insect pests, which may include economically important agronomic, forest, greenhouse, nursery, ornamentals, food and fiber, public and animal health, domestic and commercial structure, household, and stored product pests.
  • Insect pests include insects selected from the orders Coleoptera, Diptera, Hymenoptera, Lepidoptera, Mallophaga, Homoptera, Hemiptera, Orthoptera, Thysanoptera, Dermaptera, Isoptera, Anoplura, Siphonaptera, Trichoptera, etc., particularly Lepidoptera.
  • Insects of the order Coleoptera affected by the provided compounds include weevils from the families Anthribidae, Bruchidae, and Curculionidae (including, but not limited to: Anthonomus grandis (boll weevil); Lissorhoptrus oryzophilus (rice water weevil); Sitophilus granarius (granary weevil); S. oryzae (rice weevil); Hypera punctata (clover leaf weevil); Cylindrocopturus adspersus (sunflower stem weevil); Smicronyx fulvus (red sunflower seed weevil); S.
  • Anthonomus grandis boll weevil
  • Lissorhoptrus oryzophilus rice water weevil
  • Sitophilus granarius granary weevil
  • S. oryzae rice weevil
  • Hypera punctata clover leaf weevil
  • Cylindrocopturus adspersus unsunflower stem
  • sordidus (gray sunflower seed weevil); Sphenophorus maidis (maize billbug)); flea beetles, cucumber beetles, rootworms, leaf beetles, potato beetles, and leafminers in the family Chrysomelidae (including, but not limited to: Leptinotarsa decemlineata (Colorado potato beetle); Diabrotica virgifera virgifera (western corn rootworm); D. barberi (northern corn rootworm); D.
  • immaculata (southern masked chafer, white grub); Rhizotrogus majalis (European chafer); Phyllophaga crinita (white grub); Ligyrus gibbosus (carrot beetle)); carpet beetles from the family Dermestidae; wireworms from the family Elateridae, Eleodes spp., Melanotus spp.; Conoderus spp.; Limonius spp.; Agriotes spp.; Ctenicera spp.; Aeolus spp.; bark beetles from the family Scolytidae; and beetles from the family Tenebrionidae.
  • Insects of the order Lepidoptera affected by the provided compounds include, but are not limited to, armyworms, cutworms, loopers, and heliothines in the family Noctuidae Spodoptera frugiperda (fall armyworm); S. exigua (beet armyworm); S. litura (tobacco cutworm, cluster caterpillar); Mamestra configurata (bertha armyworm); M. brassicae (cabbage moth); Agrotis ipsilon (black cutworm); A. orthogonia (western cutworm); A.
  • subterranea subterranea (granulate cutworm); Alabama argillacea (cotton leaf worm); Trichoplusia ni (cabbage looper); Pseudoplusia includens (soybean looper); Anticarsia gemmatalis (velvetbean caterpillar); Hypena scabra (green cloverworm); Heliothis virescens (tobacco budworm); Pseudaletia unipuncta (armyworm); Athetis mindara (rough skinned cutworm); Euxoa messoria (darksided cutworm); Earias insulana (spiny bollworm); E.
  • vittella spotted bollworm
  • H. zea corn earworm or cotton bollworm
  • Melanchra picta zebra caterpillar
  • Egira Xylomyges
  • curialis citrus cutworm
  • saccharalis (surgarcane borer); Eoreuma loftini (Mexican rice borer); Ephestia elutella (tobacco (cacao) moth); Galleria mellonella (greater wax moth); Herpetogramma licarsisalis (sod webworm); Homoeosoma electellum (sunflower moth); Elasmopalpus lignosellus (lesser cornstalk borer); Achroia grisella (lesser wax moth); Loxostege sticticalis (beet webworm); Orthaga thyrisalis (tea tree web moth); Maruca testulalis (bean pod borer); Plodia interpunctella (Indian meal moth); Scirpophaga incertulas (yellow stem borer); Udea rubigalis (celery leaftier); and leafrollers, budworms, seed worms, and fruit worms in the family Tortricid
  • variana Eastern blackheaded budworm
  • Archips argyrospila fruit tree leaf roller
  • A. rosana European leaf roller
  • other Archips species Adoxophyes orana (summer fruit tortrix moth); Cochylis hospes (banded sunflower moth); Cydia latiferreana (filbertworm); C. pomonella (coding moth); Platynota flavedana (variegated leafroller); P.
  • stultana omnivorous leafroller
  • Lobesia botrana European grape vine moth
  • Spilonota ocellana eyespotted bud moth
  • Endopiza viteana grape berry moth
  • Eupoecilia ambiguella vine moth
  • Bonagota salubricola Brainzilian apple leafroller
  • Grapholita molesta oriental fruit moth
  • Suleima helianthana unsunflower bud moth
  • Argyrotaenia spp. and Choristoneura spp.
  • Insects in the order Lepidoptera affected by the provided compounds further include, but are not limited to, Alsophila pometaria (fall cankerworm); Anarsia lineatella (peach twig borer); Anisota senatoria (orange striped oakworm); Antheraea pernyi (Chinese Oak Silkmoth); Bombyx mori (Silkworm); Bucculatrix thurberiella (cotton leaf perforator); Colias eurytheme (alfalfa caterpillar); Datana integerrima (walnut caterpillar); Dendrolimus sibiricus (Siberian silk moth), Ennomos subsignaria (elm spanworm); Erannis tiliaria (linden looper); Euproctis chrysorrhoea (browntail moth); Harrisina americana (grapeleaf skeletonizer); Hemileuca oliviae (range caterpillar); Hyphantria cunea
  • fiscellaria lugubrosa (Western hemlock looper); Leucoma salicis (satin moth); Lymantria dispar (gypsy moth); Manduca quinquemaculata (five spotted hawk moth, tomato hornworm); M.
  • sexta tomato hornworm, tobacco hornworm
  • Operophtera brumata winter moth
  • Paleacrita vernata spring cankerworm
  • Papilio cresphontes giant swallowtail, orange dog
  • Phryganidia californica California oakworm
  • Phyllocnistis citrella citrus leafminer
  • Phyllonorycter blancardella spotted tentiform leafminer
  • Pieris brassicae large white butterfly
  • P. rapae small white butterfly
  • Immature insects of the order Diptera affected by the provided compounds include leafminers such as Agromyza parvicornis (corn blotch leafminer); midges (including, but not limited to, Contarinia sorghicola (sorghum midge); Mayetiola destructor (Hessian fly); Sitodiplosis mosellana (wheat midge); Neolasioptera murtfeldtiana, (sunflower seed midge)); fruit flies (Tephritidae), Oscinella frit (frit flies); maggots (including, but not limited to: Delia platura (seedcorn maggot); D.
  • leafminers such as Agromyza parvicornis (corn blotch leafminer); midges (including, but not limited to, Contarinia sorghicola (sorghum midge); Mayetiola destructor (Hessian fly); Sitodiplosis mosellana (wheat midge); Neolasioptera mur
  • Anopheles spp. including, but not limited to, An. gambiae, An. quadrimaculatus and An. stephensi
  • Culex spp. black flies Prosimulium spp.
  • Simulium spp. biting midges, sand flies, sciarids, and other Nematocera.
  • insects of the orders Hemiptera and Homoptera affected by the provided compounds include insects such as, but not limited to, adelgids from the family Adelgidae, plant bugs from the family Miridae, cicadas from the family Cicadidae, leafhoppers, Empoasca spp.; from the family Cicadellidae, planthoppers from the families Cixiidae, Flatidae, Fulgoroidea, Issidae and Delphacidae, treehoppers from the family Membracidae, psyllids from the family Psyllidae, whiteflies from the family Aleyrodidae, aphids from the family Aphididae, phylloxera from the family Phylloxeridae, mealybugs from the family Pseudococcidae, scales from the families Asterolecanidae, Coccidae, Dactylopi
  • Agronomically important members from the order Homoptera affected by the provided compounds further include, but are not limited to: Acyrthisiphon pisum (pea aphid); Aphis craccivora (cowpea aphid); A. fabae (black bean aphid); A. gossypii (cotton aphid, melon aphid); A. maidiradicis (corn root aphid); A. pomi (apple aphid); A.
  • spiraecola spirea aphid
  • Aulacorthum solani foxglove aphid
  • Chaetosiphon fragaefolii strawberry aphid
  • Diuraphis noxia Russian wheat aphid
  • Dysaphis plantaginea Rosy apple aphid
  • Eriosoma lanigerum woolly apple aphid
  • Brevicoryne brassicae cabbage aphid
  • Hyalopterus pruni mealy plum aphid
  • Lipaphis erysimi turningip aphid
  • Metopolophium dirrhodum cereal aphid
  • Macrosiphum euphorbiae potato aphid
  • Myzus persicae peach-potato aphid, green peach aphid
  • Nasonovia ribisnigri lettuce aphid
  • root aphids and gall aphids Rhopalosiphum maidis (corn leaf aphid); R. padi (bird cherry-oat aphid); Schizaphis graminum (greenbug); Sipha flava (yellow sugarcane aphid); Sitobion avenae (English grain aphid); Therioaphis maculata (spotted alfalfa aphid); Toxoptera aurantii (black citrus aphid); and T. citricida (brown citrus aphid); Adelges spp.
  • nigropictus (rice leafhopper); Nilaparvata lugens (brown planthopper); Peregrinus maidis (corn planthopper); Sogatella furcifera (white-backed planthopper); Sogatodes orizicola (rice delphacid); Typhlocyba pomaria (white apple leafhopper); Erythroneoura spp. (grape leafhoppers); Magicicada septendecim (periodical cicada); Icerya purchasi (cottony cushion scale); Quadraspidiotus perniciosus (San Jose scale); Planococcus citri (citrus mealybug); Pseudococcus spp.
  • Agronomically important species from the order Hemiptera affected by the provided compounds include, but are not limited to: Acrosternum hilare (green stink bug); Anasa tristis (squash bug); Blissus leucopterus leucopterus (chinch bug); Corythuca gossypii (cotton lace bug); Cyrtopeltis modesta (tomato bug); Dysdercus suturellus (cotton stainer); Euschistus servus (brown stink bug); E.
  • variolarius one-spotted stink bug
  • Graptostethus spp. complex of seed bugs
  • Leptoglossus corculus leaf-footed pine seed bug
  • Lygus lineolaris tarnished plant bug
  • L. Hesperus Western tarnished plant bug
  • L. pratensis common meadow bug
  • L. rugulipennis European tarnished plant bug
  • Lygocoris pabulinus common green capsid
  • Nezara viridula (southern green stink bug); Oebalus pugnax (rice stink bug); Oncopeltus fasciatus (large milkweed bug); Pseudatomoscelis seriatus (cotton fleahopper).
  • Insects included in the order Hemiptera include: Calocoris norvegicus (strawberry bug); Orthops campestris; Plesiocoris rugicollis (apple capsid); Cyrtopeltis modestus (tomato bug); Cyrtopeltis notatus (suckfly); Spanagonicus albofasciatus (whitemarked fleahopper); Diaphnocoris chlorionis (honeylocust plant bug); Labopidicola allii (onion plant bug); Pseudatomoscelis seriatus (cotton flea hopper); Adelphocoris rapidus (rapid plant bug); Poecilocapsus lineatus (four-lined plant bug); Nysius ericae (false chinch bug); Nysius raphanus (false chinch bug); Nezara viridula (Southern green stink bug); Eurygaster spp.; Coreida
  • Insects of the order Acari (mites) affected by the provided compounds include Aceria tosichella (wheat curl mite); Petrobia latens (brown wheat mite); spider mites and red mites in the family Tetranychidae, Panonychus ulmi (European red mite); Tetranychus urticae (two spotted spider mite); (T. mcdanieli (McDaniel mite); T. cinnabarinus (carmine spider mite); T.
  • turkestani strawberry spider mite
  • flat mites in the family Tenuipalpidae, Brevipalpus lewisi citrus flat mite
  • rust and bud mites in the family Eriophyidae and other foliar feeding mites and mites important in human and animal health i.e., dust mites in the family Epidermoptidae, follicle mites in the family Demodicidae, grain mites in the family Glycyphagidae, ticks in the order Ixodidae.
  • Ixodes scapularis (deer tick); I.
  • Insect pests of the order Thysanura affected by the provided compounds include Lepisma saccharina (silverfish); Thermobia domestics (firebrat).
  • Additional arthropod pests include: spiders in the order Araneae such as Loxosceles reclusa (brown recluse spider); and the Latrodectus mactans (black widow spider); and centipedes in the order Scutigeromorpha such as Scutigera coleoptrata (house centipede).
  • the pesticidal activity of the compositions disclosed herein may be tested in insect pests in their early developmental stages, e.g., as larvae or other immature forms.
  • bioassay techniques are known to one skilled in the art. General procedures include addition of the experimental compound or organism to the diet source in an enclosed container. Bioassays may be performed as described in Czapla and Lang (1990) J.
  • Measurable indicia of pesticidal activity include, but are not limited to, changes in mortality, weight loss, attraction, repellency, and other behavioral and physical changes after feeding and exposure for an appropriate length of time. See, for example US Patent No.7,619,064.
  • E. COMPOSITIONS [0130]
  • the bifunctional compounds disclosed herein may be formulated into compositions for use. Such compositions comprise one or more provided bifunctional compounds, and one or more agriculturally acceptable carriers, surfactants or application-promoting adjuvants customarily employed in the art of formulation.
  • the term "agriculturally- acceptable carrier” covers all adjuvants, inert components, dispersants, surfactants, tackifiers, binders, etc.
  • Suitable carriers and adjuvants may be solid or liquid and correspond to the substances ordinarily employed in formulation technology, e.g., natural or regenerated mineral substances, solvents, dispersants, wetting agents, tackifiers, binders or the like.
  • the compositions may comprise more than one type of bifunctional compound, as well as additional agents, such as insecticides, fertilizers, herbicides, and plant supplements.
  • the formulations may be prepared into edible "baits” or fashioned into pest "traps” to permit feeding or ingestion by a target pest of the pesticidal formulation.
  • compositions may be applied to the environment of an insect pest by, for example, spraying, atomizing, dusting, scattering, coating or pouring, introducing into or on the soil, introducing into irrigation water, by seed treatment or general application or dusting at the time when the pest has begun to appear or before the appearance of pests as a protective measure.
  • the provided compositions may be mixed with grain to protect the grain during storage. It is generally important to obtain good control of pests in the early stages of plant growth, as this is the time when the plant may be most severely damaged.
  • Provided embodiments include granular forms of the composition comprising an agrochemical such as, for example, an herbicide, an insecticide, a fertilizer, an inert carrier, and the like.
  • the methods include application to the insect pests, application to the plant or plant locus, e.g., leaf application, seed coating and soil application, or application to other surfaces in the environment in which the insect is present. The number of applications and the rate of application depend on the intensity of infestation by the corresponding pest.
  • the composition is formulated as a powder, dust, pellet, granule, spray, emulsion, colloid, solution or the like.
  • the composition is prepared by desiccation, lyophilization, homogenization, extraction, filtration, centrifugation, sedimentation, or concentration.
  • the bifunctional compounds is present in a concentration of from about 1% to about 99% by weight, e.g., from about 1 wt% to about 60 wt%, from about 10 wt% to about 70 wt%, from about 20 wt% to about 80 wt%, from about 30 wt% to about 90 wt%, or from about 40 wt% to about 100 wt%.
  • the composition is applied to any plant species for control of a pest.
  • Plants of interest include grain plants that provide seeds of interest, oil-seed plants, leguminous plants, vegetables, ornamentals, trees, turfgrass, and the like.
  • Seeds of interest include grain seeds, such as corn, wheat, barley, rice, sorghum, rye, millet, etc.
  • Oil-seed plants include cotton, soybean, safflower, sunflower, Brassica, maize, alfalfa, palm, coconut, flax, castor, olive etc.
  • Leguminous plants include beans and peas. Beans include guar, locust bean, fenugreek, soybean, garden beans, cowpea, mungbean, lima bean, fava bean, lentils, chickpea, etc.
  • compositions and compounds may be applied to the surface of the plant. Some of the bifunctional compounds may be taken up by the plant. In any event, the compounds are accessible to insect pest by feeding on the plants.
  • plants of interest include, but are not limited to, corn (Zea mays), Brassica sp. (e.g., B. napus, B. rapa, B.
  • juncea particularly those Brassica species useful as sources of seed oil, alfalfa (Medicago sativa), rice (Oryza sativa), rye (Secale cereale), sorghum (Sorghum bicolor, Sorghum vulgare), millet (e.g., pearl millet (Pennisetum glaucum), proso millet (Panicum miliaceum), foxtail millet (Setaria italica), finger millet (Eleusine coracana)), sunflower (Helianthus annuus), safflower (Carthamus tinctorius), wheat (Triticum aestivum), soybean (Glycine max), tobacco (Nicotiana tabacum), potato (Solanum tuberosum), peanuts (Arachis hypogaea), cotton (Gossypium barbadense, Gossypium hirsutum), sweet potato (Ipomoea batatus), cassaya (Manihot esculent
  • Embodiment 1 A method of controlling a level of a target protein that is expressed in an insect cell, the method comprising contacting the insect cell with an effective amount of a compound, the compound comprising: one or more protein targeting moieties (PTM) that each independently bind the target protein; one or more ligase targeting moieties (LTM) that each independently bind a ubiquitin ligase that is functional in the insect cell; and a linker (L) covalently bonded to the one or more protein targeting moieties and the one or more ligase targeting moieties.
  • PTM protein targeting moieties
  • LTM ligase targeting moieties
  • L linker
  • Embodiment 2 An embodiment of embodiment 1, wherein the ubiquitin ligases that the one or more ligase targeting moieties bind to comprise the von Hippel-Lindau tumor suppressor (VHL) or cereblon.
  • Embodiment 3 An embodiment of embodiment 1 or 2, wherein at least one of the one or more ligase targeting moieties has a structure according to the formula: I), wherein R 5a and R 5b are each independ hydroxyl, amine, haloalkyl, optionally substituted alkyl, optionally substituted alkoxy, optionally substituted hydroxyl alkyl, optionally substituted alkylamine, optionally substituted amide, optionally substituted alkyl-amide, optionally substituted alkyl-cyano, optionally substituted alkyl-phosphate, optionally substituted aryl, optionally substituted alkyl-aryl, optionally substituted heteroalkyl, optionally substituted alkyl-heterocyclyl, optional
  • Embodiment 4 An embodiment of embodiment 3, wherein at least one of the one or more ligase targeting moieties has a structure according to the formula: wherein R 1 is hydrogen, C1-6 alkyl, is hydrogen, C1-6 alkyl, C1-6 hydroxyalkyl, or -X-L 1 ; R 5a is hydr 6 haloalkyl, C 1-6 alkyl-amide, hydroxy, C1-6 alkoxy, C1-6 haloalkoxy or -X-L 1 ; and each R 6 is independently C1-6 alkyl, halogen, C1-6 haloalkyl, hydroxy, C1-6 alkoxy, C1-6 haloalkoxy, C3-8 cycloalkyl, C6-12 aryl, C5-12 heteroaryl, OR 14 , or -X-L 1 , wherein the cycloalkyl, aryl, and heteroaryl are optionally substituted with 1 to 4 R 6a groups.
  • R 1 is hydrogen, C1-6 alkyl, is hydrogen, C
  • Embodiment 5 An embodiment of embodiment 3 or 4, wherein R 1 is -C(O)R 1a or - C(O)-X-L 1 ; and R 1a is C 1-6 alkyl or C 3-8 cycloalkyl, wherein the cycloalkyl is optionally substituted with 1 to 4 R 1b groups.
  • Embodiment 6 An embodiment of any one of embodiments 3-5, wherein R 2 and R 3 are each independently C1-6 alkyl.
  • Embodiment 7 An embodiment of any one of embodiments 3-6, wherein R 4 is C 1-6 alkyl or -X-L 1 .
  • Embodiment 8 An embodiment of any one of embodiments 3-7, wherein R 5a is hydroxy or methyl.
  • Embodiment 9 An embodiment of any one of embodiments 3-8, wherein each R 6 is independently C5-12 heteroaryl or -X-L 1 , wherein the heteroaryl is optionally substituted with 1 to 4 R 6a groups; and each R 6a is independently C 1-6 alkyl.
  • Embodiment 10 An embodiment of embodiment 9, wherein each R 6 is independently thiazole or -X-L 1 , wherein the thiazole is optionally substituted with 1 to 4 R 6a groups.
  • Embodiment 11 An embodiment of any one of embodiments 3-10, wherein subscript n is 1 or 2.
  • Embodiment 12 An embodiment of any one of embodiments 3-11, wherein at least one of the one or more ligase targeting moieties has a structure according to the formula: b).
  • Embodiment 13 An embo t 12, wherein at least one of the one or more ligase targeting moieties has the structure: N S 6 R 5a R HN O O H X N 1 N L O OH , , , , , 104
  • Embodime n at least one of the one or more ligase targeting moieties comprises an N-substituted 1,3,-dioxoisoindolinyl moiety optionally substituted with one or more substituents that are each independently C1-6 alkyl, halogen, hydroxy, amino, C 1-6 alkylamino, C 1-6 amido, C 1-6 acyl, nitro, cyano, or C 1-6 alkoxy.
  • Embodiment 15 An embodiment of any one of embodiments 1-14, wherein the linker has a linear non-hydrogen atom number from 1 to 20.
  • Embodiment 16 An embodiment of any one of embodiments 1-15, wherein the linker has a structure –L 2 -L 3 -, wherein L 2 and L 3 are each independently a bond, a divalent polymer moiety, or C1-30 alkylene, wherein one or more carbon atoms in each C1-30 alkylene are optionally and independently replaced by O, C(O), S, or NR 7 ; one or more groupings of adjacent carbon atoms in each C 1-30 alkylene are optionally and independently replaced by -NR 7 (CO)- or -(CO)NR 7 -; and one or more groupings of adjacent carbon atoms in each C1-30 alkylene are optionally and independently replaced by a 4- to 8-membered, divalent carbocycle or a 4- to 8-membered, divalent heterocycle having one to four heteroatoms selected from O, S, and N; and each R 7 is independently hydrogen or C1-6 alkyl.
  • Embodiment 17 An embodiment of any one of embodiments 1-16, wherein the linker comprises one or more ethylene glycol diradical moieties, one or more of which is optionally replaced by the moiety: , , , or . 105
  • Embodiment 18 An embodiment of any one of embodiments 1-17, wherein at least one of the one or more protein targeting moieties has the structure: , wher
  • Embodiment 19 An embodiment of any one of embodiments 1-18, wherein the proteins that the one or more protein targeting moieties bind to are essential to growth, development, reproduction, or survival of the insect.
  • Embodiment 20 An embodiment of embodiment 19, wherein the proteins comprise acetyl CoA carboxylase, acetylcholinesterase, GABA-gated chloride channels, sodium channels, nicotinic acetylcholine receptors, glutamate-gated chloride channels, chordontal organ TRPV channels, chitin synthase, mitochondrial ATP synthase, ecdysone receptors, octopamine receptors, voltage-dependent sodium channels, ryanodine receptors, calcium-activated potassium channels, juvenile hormone receptors, Bromaindomain Containing 3 (BRD3), chitin acetyltransferase, Cholone O-Acetyltransferase (ChAT), Dihydrofolate Reductase (DHFR), FK506 binding proteins (FKBP), Geranylgeranyl diphosphate synthase 1 (GGPS1), 3-Hydroxy- 3Methylgluta
  • Embodiment 21 An embodiment of embodiment 20, wherein the proteins comprise Bromaindomain Containing 3 (BRD3) or Will Die Slowly (WDS).
  • Embodiment 22 An embodiment of any one of embodiments 1-21, wherein the insect cell is a cell of an insect that is a member of the order Lepidoptera, Coleoptera, Diptera, Hymenoptera, Mallophaga, Homoptera, Hemiptera, Orthoptera, Thysanoptera, Dermaptera, Isoptera, Anoplura, Siphonaptera, or Trichoptera.
  • Embodiment 23 An embodiment of embodiment 22, wherein the insect is a member of the order Lepidoptera.
  • Embodiment 24 A composition comprising the compound of any one of embodiments 1-23, or a salt or solvate thereof, and an agriculturally acceptable carrier.
  • Embodiment 25 A method of controlling a level of a target protein in an insect cell, the method comprising contacting the insect cell with an effective amount of the compound of any one of embodiments 1-23, or with an effective amount of the composition of embodiment 24.
  • Embodiment 26 An embodiment of embodiment 25, wherein the controlling of the level of the target protein comprises degrading the target protein.
  • Embodiment 27 An embodiment of embodiment 25 or 26, wherein the insect cell is a cell of an insect, and wherein the contacting of the insect cell with an effective amount of the compound or composition comprises applying the compound or composition to an environment in which the insect is present.
  • Embodiment 28 An embodiment of embodiment 27, wherein the compound or composition is applied to an insect pest, a plant, a seed, the soil, or other surface in the environment in which the insect is present.
  • Table 1 comprises bifunctional compounds having VHL ligase targeting moieties and FKBP protein targeting moieties. The compounds set forth in Table 1 were synthesized as described in detail below in Examples A1-A44. Table 1 Example Structure
  • Table 3 comprises bifunctional compounds having VHL ligase targeting moieties and BRD3 protein targeting moieties.
  • the compounds set forth in Table 3 were synthesized as described in detail below in Examples C1-C56.
  • the reaction mixture was stirred at 25 °C for 1 hour.
  • the reaction mixture was diluted with DCM (8 mL) and H2O (5 mL).
  • the aqueous phase was extracted with DCM (4 mL x 3).
  • the combined organic layers were washed with brine (10 mL x 3), dried over Na 2 SO 4 , filtered, and concentrated in vacuo.
  • the residue was re-dissolved in DCM (5 mL) and washed with 2N HCl aqueous solution (2 mL).
  • Step 1 [0 , y y y y y y y . n-2- one (200 mg, 509.57 ⁇ mol; prepared using general methods described herein for the preparation of (5s,8s)-4-(benzyloxy)-8-hydroxy-3-mesityl-1-oxaspiro[4.5]dec-3-en-2-one) and tert-butyl 2- [2-[2-(2-bromoethoxy)ethoxy]-ethoxy]acetate (333.48 mg, 1.02 mmol) in toluene (4 mL) were added KOH (142.96 mg, 2.55 mmol) and TBAB (82.14 mg, 254.79 ⁇ mol). The mixture was stirred at 50 °C for 12 hours.
  • the pH of the reaction mixture was adjusted to 5-6 by addition of 2 M aqueous HCl and the resulting mixture extracted with EtOAc (20 mL x 3). The combined organic layers were washed with brine (20 mL), dried over anhydrous Na 2 SO 4 , filtered, and concentrated under reduced pressure to give a residue.
  • Step 2 To a mixture of 2-(2-(2-(2-(((5r,8r)-4-(benzyloxy)-3-mesityl-2-oxo-1-oxaspiro[4.5]dec- 3-en-8-yl)oxy)ethoxy)ethoxy)acetic acid (30 mg, 51.49 ⁇ mol) in THF (5 mL) were added 10% Pd/C (5 mg) and 20% Pd(OH) 2 /C (5 mg) under N 2 . The mixture was degassed, purged with H 2 (3x), and stirred at 25 °C under H 2 (15 psi, balloon) for 0.5 hours.
  • Step 3 [ , S)- 1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (23.44 mg, 48.73 ⁇ mol, HCl salt) and 2-(2-(2-(2-(((5r,8r)-4-hydroxy-3-mesityl-2-oxo-1-oxaspiro[4.5]dec-3-en-8- yl)oxy)ethoxy)ethoxy)ethoxy)acetic acid (20 mg, 40.60 ⁇ mol) in DCM (1 mL) were added 50% T 3 P (38.76 mg, 60.91 ⁇ mol, 36.22 ⁇ L) and DIPEA (20.99 mg, 162.42 ⁇ mol, 28.29 ⁇ L).
  • Step 1 [0 y y y y y y y y y y . g, . 7 mmol, 35.25 mL) in THF (300 mL) was added 60% NaH (4.51 g, 112.79 mmol) at 0 °C under N2. The resulting mixture was stirred at 0 °C for 1 hour. The mixture was treated with tert-butyl 2-bromoacetate (20 g, 102.54 mmol, 15.15 mL) and stirred at 25 °C for 12 hours. The mixture was added to H 2 O (500 mL) and extracted with EtOAc (500 mL x 68 x).
  • n-2- one 200 mg, 509.57 ⁇ mol; see Example 7, Step 10
  • tert-butyl 2-[2-[2-[2-(2- bromoethoxy)ethoxy]ethoxy]ethoxy]acetate 283.78 mg, 764.36 ⁇ mol
  • KOH 142.96 mg, 2.55 mmol
  • TBAB 82.14 mg, 254.79 ⁇ mol
  • the mixture was stirred at 50 °C for 12 hours.
  • the pH of the reaction mixture was adjusted to 3-4 by addition of 2 M aqueous HCl aqueous.
  • EtOAc (20 mL) and H 2 O (20 mL) were added and layers were separated.
  • Step 1 [01 ] o a so u on o -[ -[ -( - y roxye oxy)e oxy]e oxy]e ano ( . g, .26 mmol, 45.08 mL) in DCM (50 mL) was added TsCl (5 g, 26.23 mmol) and Et 3 N (3.98 g, 39.34 mmol, 5.48 mL) at 0 °C. The resulting mixture was stirred at 25 °C for 16 hours. The reaction mixture was washed with H2O (60 mL), 1M aqueous HCl (60 mL x 2), sat.
  • H2O 60 mL
  • 1M aqueous HCl 60 mL x 2
  • Step 2 To a solution of 2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethyl 4- methylbenzenesulfonate (6 g, 17.22 mmol) in DCM (50 mL) was added PPTS (216.38 mg, 861.05 ⁇ mol) and 3,4-dihydro-2H-pyran (1.74 g, 20.67 mmol, 1.89 mL) at 0 °C. The resulting mixture was stirred at 25 °C for 16 hours. The reaction mixture was concentrated in vacuo.
  • Step 6 A mixture of tert-butyl 14-(((5r,8r)-4-(benzyloxy)-3-mesityl-2-oxo-1-oxaspiro[4.5]dec- 3-en-8-yl)oxy)-3,6,9,12-tetraoxatetradecan-1-oate (160 mg, 187.45 ⁇ mol, 80% purity), 10% Pd/C (20 mg) and 20% Pd(OH)2/C (20 mg) in EtOH (5 mL) was stirred at 25 °C for 2 hours under a H 2 atmosphere (15 Psi).
  • Step 1 [0 ] o a m xure o ( s, s)- -( enzy oxy)- - y roxy- -mes y - -oxasp ro[ . ] ec- -en-2- one (300 mg, 764.36 ⁇ mol; see Example 7, Step 10), tert-butyl 2-[2-[2-[2-[2-(2- bromoethoxy)ethoxy]ethoxy]-ethoxy]ethoxy]acetate (476.18 mg, 1.15 mmol) and TBAB (98.56 mg, 305.74 ⁇ mol) in toluene (5 mL) was added KOH (214.42 mg, 3.82 mmol).
  • the mixture was stirred at 50°C under N2 for 15 hours.
  • the mixture was poured into water (50 mL) and the pH adjusted to 3-4 by addition of aqueous HCl.
  • the mixture was extracted with EtOAc (50 mL).
  • the organic phase was washed with brine (50 mL x 2), dried over anhydrous Na2SO4, filtered, and concentrated at reduced pressure.
  • Step 2 182 [0189] To a solution of 17-(((5s,8s)-4-(benzyloxy)-3-mesityl-2-oxo-1-oxaspiro[4.5]dec-3-en- 8-yl)oxy)-3,6,9,12,15-pentaoxaheptadecanoic acid (53 mg, 79.01 ⁇ mol) in THF (3 mL) was added 10% Pd/C (10 mg) under N 2 . The mixture was degassed and purged with H 2 (3x). The mixture was stirred at 30 °C under 15 psi of H2 (balloon) for 2 hours. LCMS indicated remaining starting material.
  • Step 3 [0 90] o a m xture o 7-(((5s,8s)- - ydroxy-3-mes ty - -oxo- -oxaspro[ .5]dec-3-en-8- yl)oxy)-3,6,9,12,15-pentaoxaheptadecanoic acid (50 mg, crude), (2S,4R)-1-[(2S)-2-amino-3,3- dimethyl-butanoyl]-4-hydroxy-N-[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2- carboxamide (62.13 mg, 129.16 ⁇ mol, HCl salt) and DIPEA (44.52 mg, 344.43 ⁇ mol, 59.99 ⁇ L) in DCM (2 mL) was added 50% T3P (82.19 mg, 129.16 ⁇ mol, 76.82
  • the mixture was stirred at 30 °C under N 2 for 1 hour.
  • the mixture was diluted with DCM (20 mL), washed with 0.1M aqueous HCl (20 mL) and brine (20 mL x 2).
  • the organic phase was dried over anhydrous Na2SO4, filtered, and concentrated to dryness at reduced pressure.
  • Step 1 [0 , y y y y y y p . n-2- one (300.00 mg, 764.36 ⁇ mol; prepared using general methods described herein for the preparation of (5s,8s)-4-(benzyloxy)-8-hydroxy-3-mesityl-1-oxaspiro[4.5]dec-3-en-2-one), tert- butyl 2-[2-[2-[2-[2-(2-bromo-ethoxy)ethoxy]ethoxy]ethoxy]acetate (476.18 mg, 1.15 mmol) and TBAB (98.56 mg, 305.74 ⁇ mol) in toluene (10 mL) was added KOH (214.42 mg, 3.82 mmol).
  • the mixture was stirred at 50 °C under N 2 for 12 hours.
  • the mixture was poured into water (50 mL) and the pH adjusted to 3-4 by addition of aqueous HCl.
  • the mixture was extracted with EtOAc (50 mL).
  • the organic phase was washed with brine (50 mL x 2), dried over anhydrous Na 2 SO 4 , filtered, and concentrated in vacuo.
  • Step 2 To a mixture of 17-(((5r,8r)-4-(benzyloxy)-3-mesityl-2-oxo-1-oxaspiro[4.5]dec-3-en-8- yl)oxy)-3,6,9,12,15-pentaoxaheptadecanoic acid (26.00 mg, 38.76 ⁇ mol), (2S,4R)-1-((S)-2- amino-3,3-dimethylbutanoyl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5- yl)phenyl)ethyl)pyrrolidine-2-carboxamide (27.97 mg, 58.14 ⁇ mol, HCl salt) and DIPEA (20.04 mg, 155.04 ⁇ mol, 27.00 ⁇ L) in DCM (2 mL) was added 50% T 3 P (37.00 mg, 58.14 ⁇ mol, 34.58 ⁇ L).
  • the mixture was degassed and purged with H 2 (3x). The mixture was stirred at 30 °C under 15 psi H 2 (balloon) for 1 hour at which point LCMS indicated remaining starting material. The mixture was treated with 20% Pd(OH)2/C (10 mg) and maintained under H2 as above for 15 hours. The mixture was filtered, and the filtrate concentrated in vacuo.
  • Step 1 To a stirr , y y 0 g, 561.08 mmol) in MeOH (1 L) was added SOCl2 (133.50 g, 1.12 mol, 81.40 mL) dropwise at 0 °C over 1 hour. The mixture was stirred at 25° C for 15 hours at which point the mixture had turned clear. The mixture was concentrated in vacuo. The residue was diluted with saturated aqueous NaHCO 3 (1 L) and extracted with EtOAc (1L).
  • Step 2 To a stirred solution of 2-[2-[2-[2-[2-(2- hydroxyethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethanol (14.47 g, 51.27 mmol) in THF (100 mL) was added 60% NaH (1.13 g, 28.20 mmol) at 0 °C under N 2 . The mixture was stirred at 0 °C for 30 minutes. Tert-butyl 2-bromoacetate (5 g, 25.63 mmol, 3.79 mL) was then added. The mixture was stirred at 25 °C for another 15 hours. The mixture was diluted with water (500 mL) and extracted with EtOAc (500 mL x 2).
  • Step 3 [0 hydroxyethoxy)ethoxy]ethoxy]ethoxy]-ethoxy]ethoxy]acetate (4 g, 10.09 mmol) and CBr 4 (5.02 g, 15.13 mmol) in DCM (40 mL) was added PPh3 (3.97 g, 15.13 mmol) portion-wise at 0 °C. The mixture was stirred at 25 °C under N 2 for 15 hours. The mixture was diluted with DCM (160 mL) and washed with brine (200 mL). The organic phase was dried over anhydrous Na 2 SO 4 , filtered, and concentrated in vacuo.
  • Step 5 To a s y y y y y y y y y y y y y y carbonitrile (10 g, 32.95 mmol) in EtOH (150 mL) at -40 °C was bubbled HCl gas (45 g, 1.23 mol, 44.12 mL). After stirring at 25 °C for 15 hours, the mixture was concentrated in vacuo. The residue was suspended in DME (100 mL) and treated with 1 M aqueous HCl (49.43 mL). The mixture was stirred at 25 °C for another 2 hours, mixed with saturated NaHCO 3 aqueous solution (500 mL) and extracted with EtOAc (500 mL).
  • Step 7 [0200] T , piro[4.5]dec- 3-en-2-one (4.08 g, 10.40 mmol) and K2CO3 (2.87 g, 20.79 mmol) in DMF (40 mL) at 25 °C was added ethyl iodide (2.43 g, 15.59 mmol, 1.25 mL). The mixture was stirred at 30 °C under N2 for 15 hours and then poured into water (500 mL) and extracted with EtOAc (500 mL). The organic phase was washed with brine (500 mL x 2), dried over anhydrous Na 2 SO 4 , filtered, and concentrated in vacuo.
  • Step 8 [0201] o a so u on o ( s, s)- -( enzyoxy)- -e oxy- -mes y - -oxasp ro[ . ] ec-3-en-2- one (3.6 g, 8.56 mmol) in MeOH (40 mL) under N2 was added 10% Pd/C (0.3 g) and 20% Pd(OH)2/C (0.3 g). The mixture was purged with H2 (3x) and stirred at 40 °C under 15 psi of H2 (balloon) for 15 hours.
  • Step 9 A mixture of (5s,8s)-4-ethoxy-8-hydroxy-3-mesityl-1-oxaspiro[4.5]dec-3-en-2-one (1 g, 3.03 mmol) and KOH (849.01 mg, 15.13 mmol) in dimethylacetamide (10 mL) was stirred at 130 °C for 2 hours and cooled to 25 °C. The mixture was combined with the reaction mixture from a similar reaction, poured into water, extracted with EtOAc (250 mL), and the organic layer discarded. The pH of the aqueous phase was adjusted to 3-4 by addition of 2 M aqueous HCl and then extracted with EtOAc (250 mL).
  • ec-3-en-2-one (2.2 g, 7.28 mmol) and K2CO3 (2.01 g, 14.55 mmol) in DMF (22 mL) at 25°C was added benzyl bromide (1.49 g, 8.73 mmol, 1.04 mL). The mixture was stirred at 25 °C for 15 hours, diluted with water (150 mL) and extracted with EtOAc (150 mL). The organic phase was washed with brine (150 mL x 2), dried over anhydrous Na2SO4, filtered and concentrated in vacuo.
  • Step 11 To a stirred mixture of (5s,8s)-4-(benzyloxy)-8-hydroxy-3-mesityl-1-oxaspiro[4.5]dec- 3-en-2-one (200 mg, 509.57 ⁇ mol), tert-butyl 2-[2-[2-[2-[2-[2-(2- bromoethoxy)ethoxy]ethoxy]ethoxy]ethoxy]acetate (351.12 mg, 764.36 ⁇ mol), and KOH (142.95 mg, 2.55 mmol) in xylene (2 mL) was added TBAB (32.85 mg, 101.91 ⁇ mol) at 25 °C.
  • Step 1 To a mixture of (5r,8r)-4-(benzyloxy)-8-hydroxy-3-mesityl-1-oxaspiro[4.5]dec-3-en-2- one (300.00 mg, 764.36 ⁇ mol; prepared using general methods described herein for the preparation of (5s,8s)-4-(benzyloxy)-8-hydroxy-3-mesityl-1-oxaspiro[4.5]dec-3-en-2-one), tert- butyl 2-[2-[2-[2-[2-[2-[2-[2-[2-(2-bromoethoxy)ethoxy]ethoxy]ethoxy]ethoxy]acetate (526.68 mg, 1.15 mmol), and KOH (214.44 mg, 3.82 mmol) in toluene (10 mL) was added TBAB (98.56 mg, 305.74 ⁇ mol) at 25 °C.
  • the mixture was degassed, and purged with H2 (3x), and stirred at 30 °C under 15 psi H2 (balloon) for 1 hour.
  • LCMS indicated that substantial starting material remained.
  • the mixture was treated with 20% Pd(OH)2 (10 mg) and subjected to 15 psi H2 as described above for an additional 15 hours.
  • Step 1 To a solution of (5s,8s)-4-(benzyloxy)-3-mesityl-8-(piperidin-4-yloxy)-1- oxaspiro[4.5]dec-3-en-2-one (220 mg, 373.11 ⁇ mol, crude, TFA salt; see Example 11, Step 5) and tert-butyl 2-[2-(2-bromoethoxy)ethoxy]acetate (158.47 mg, 559.66 ⁇ mol) in DMF (4 mL) were added NaI (22.37 mg, 149.24 ⁇ mol) and K 2 CO 3 (154.70 mg, 1.12 mmol).
  • Step 2 [ oxaspiro[4.5]dec-3-en-8-yl)oxy)piperidin-1-yl)ethoxy)ethoxy)acetate (120 mg, 177.03 ⁇ mol) in THF (5 mL) were added 20% Pd/C (50 mg) and 20% Pd(OH) 2 /C (50 mg) under N 2 . The mixture was degassed, purged with H2 (3x), and stirred at 25 °C for 1 hour under 15 psi H2 (balloon).
  • Step 3 [0 212] To a solution of tert-butyl 2-(2-(2-(4-(((5s,8s)-4-hydroxy-3-mesityl-2-oxo-1- oxaspiro[4.5]dec-3-en-8-yl)oxy)piperidin-1-yl)ethoxy)ethoxy)acetate (90 mg, 153.13 ⁇ mol, crude) in DCM (4 mL) was added TFA (770.00 mg, 6.75 mmol, 0.5 mL).
  • Step 4 196 [0213] To a solution of 2-(2-(2-(4-(((5s,8s)-4-hydroxy-3-mesityl-2-oxo-1-oxaspiro[4.5]dec-3- en-8-yl)oxy)-piperidin-1-yl)ethoxy)ethoxy)acetic acid (80 mg, 150.48 ⁇ mol, crude product) and (2S,4R)-1-((S)-2-amino-3,3-dimethylbutanoyl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5- yl)phenyl)ethyl)pyrrolidine-2-carboxamide (86.87 mg, 195.38 ⁇ mol) in DCM (3 mL) were added DIPEA (58.34 mg, 451.44 ⁇ mol, 78.63 ⁇ L) and 50% T 3 P (143.64 mg, 225.72 ⁇ mol, 134.24 ⁇ L) at
  • Step 1 To a solution of (5r,8r)-4-(benzyloxy)-3-mesityl-8-(piperidin-4-yloxy)-1- oxaspiro[4.5]dec-3-en-2-one (250 mg, 423.99 ⁇ mol, crude, TFA salt; prepared from (5r,8r)-4- (benzyloxy)-8-hydroxy-3-mesityl-1-oxaspiro[4.5]dec-3-en-2-one using the general methods described in Example 11) and tert-butyl 2-[2-(2-bromoethoxy)ethoxy]acetate (180.08 mg, 635.98 ⁇ mol) in DMF (4 mL) were added NaI (25.42 mg, 169.59 ⁇ mol) and K 2 CO 3 (175.80 mg, 1.27 mmol).
  • Step 2 [0 5] o a so ut on o tert-buty -( -( -( -( -((((5r,8r)- -(benzy oxy)-3-mes ty - -oxo- - oxaspiro[4.5]dec-3-en-8-yl)oxy)piperidin-1-yl)ethoxy)ethoxy)acetate (300 mg, 442.57 ⁇ mol, crude) in THF (10 mL) were added 20% Pd/C (0.1 g) and 20% Pd(OH)2 (0.1 g) under N2. The mixture was degassed and purged with H2 (3x).
  • Step 3 To a solution of tert-butyl 2-(2-(2-(4-(((5r,8r)-4-hydroxy-3-mesityl-2-oxo-1- oxaspiro[4.5]dec-3-en-8-yl)oxy)piperidin-1-yl)ethoxy)ethoxy)acetate (300 mg, 510.43 ⁇ mol, crude) in DCM (10 mL) was added TFA (3.08 g, 27.01 mmol, 2 mL).
  • Step 1 [02 , - - y y - - y y- - y - y - y - - p .5]dec- 3-en-2-one (1 g, 2.55 mmol; see Example 7, Step 10) in THF (15 mL) was added t-BuOK (1 mol/L, 2.80 mL) at 25 °C. After 1 hour, 2-chloro-4-fluoro-pyridine (402.16 mg, 3.06 mmol) was added. The mixture was stirred at 50 °C for 12 hours, quenched with saturated aqueous NH4Cl aqueous (10 mL), and diluted with EtOAc (20 mL) and H 2 O (5 mL).
  • Step 2 200 To a mixture of (5s,8s)-4-(benzyloxy)-8-((2-chloropyridin-4-yl)oxy)-3-mesityl-1- oxaspiro[4.5]dec-3-en-2-one (4 g, 7.94 mmol) in AcOH (100 mL) was added 10% Pd/C (1 g). The mixture was stirred at 60 °C for 24 hours under H 2 (50 psi).
  • Step 3 [0 0] o a st rred m xture o (5s,8s)- - ydroxy-3-mes ty-8-(p per d n- -y oxy)- - oxaspiro[4.5]dec-3-en-2-one (3.25 g, 7.29 mmol, AcOH salt) and (Boc) 2 O (1.91 g, 8.75 mmol, 2.01 mL) in DCM (60 mL) at 0 °C was added Et3N (1.48 g, 14.59 mmol, 2.03 mL).
  • Step 5 [0 222] A mixture of tert-butyl 4-(((5s,8s)-4-(benzyloxy)-3-mesityl-2-oxo-1-oxaspiro[4.5]dec- 3-en-8-yl)oxy)-piperidine-1-carboxylate (2.4 g, 4.17 mmol) in DCM (80 mL) and TFA (15 mL) was stirred at 0 °C for 0.5 hours.
  • Step 6 To a solution of (5s,8s)-4-(benzyloxy)-3-mesityl-8-(piperidin-4-yloxy)-1- oxaspiro[4.5]dec-3-en-2-one (260 mg, 440.95 ⁇ mol, TFA salt) and tert-butyl 2-[2-[2-(2- bromoethoxy)ethoxy] ethoxy] acetate (173.14 mg, 529.14 ⁇ mol) in DMF (6 mL) at 25 o C was added K 2 CO 3 (121.88 mg, 881.89 ⁇ mol) and NaI (13.22 mg, 88.19 ⁇ mol).
  • Step 7 [0 ] o a m x ure o tert- u y -( -( -( -( -( -(((( s, s)- -( enzyoxy)- -mes y - -oxo- - oxaspiro[4.5]dec-3-en-8-yl)oxy)piperidin-1-yl)ethoxy)ethoxy)ethoxy)acetate (250 mg, 346.30 ⁇ mol) in THF (5 mL) were added 10% Pd/C (40 mg) and 20% Pd(OH)2/C (40 mg). The reaction mixture was stirred at 25 °C for 10 mins under 15 psi H 2 .
  • Step 8 A mixture of tert-butyl 2-(2-(2-(2-(4-(((5s,8s)-4-hydroxy-3-mesityl-2-oxo-1- oxaspiro[4.5]dec-3-en-8-yl)oxy)piperidin-1-yl)ethoxy)ethoxy)acetate (200 mg, 316.56 ⁇ mol) in TFA (1 mL) and DCM (3 mL) was stirred at 25 °C for 1 hour and then concentrated in vacuo to afford 2-(2-(2-(2-(4-(((5s,8s)-4-hydroxy-3-mesityl-2-oxo-1-oxaspiro[4.5]dec-3-en-8- yl)oxy)piperidin-1-yl)ethoxy)ethoxy)ethoxy)acetic acid (180 mg, crude) as yellow oil which was used to the next step without further purification.
  • Step 1 oxaspiro[4.5]dec-3-en-2-one (247.95 mg, 420.51 ⁇ mol, TFA salt; prepared from (5r,8r)-4- (benzyloxy)-8-hydroxy-3-mesityl-1-oxaspiro[4.5]dec-3-en-2-one using the general methods described in Example 11) and tert-butyl 2-[2-[2-(2-bromoethoxy)ethoxy]ethoxy]acetate (206.39 mg, 630.76 ⁇ mol) in DMF (2 mL) were added K2CO3 (174.35 mg, 1.26 mmol) and NaI (63.03 mg, 420.51 ⁇ mol).
  • Step 2 To a solution of tert-butyl 2-(2-(2-(2-(4-(((5r,8r)-4-(benzyloxy)-3-mesityl-2-oxo-1- oxaspiro[4.5]dec-3-en-8-yl)oxy)piperidin-1-yl)ethoxy)ethoxy)acetate (203 mg, 281.20 ⁇ mol) in THF (4 mL) was added 10% Pd/C (20 mg) and 20% Pd(OH) 2 (20 mg) under N 2 . The mixture was degassed and purged with H 2 (3x), then stirred under 15 psi H 2 (balloon) at 25°C for 1 hour.
  • Step 3 [0 y , y y y oxaspiro[4.5]dec-3-en-8-yl)oxy)piperidin-1-yl)ethoxy)ethoxy)ethoxy)acetate (150 mg, 237.42 ⁇ mol, crude) in DCM (1.5 mL) was added TFA (770.00 mg, 6.75 mmol, 0.5 mL). The mixture was stirred at 25 °C for 1 hour and evaporated to dryness.
  • Step 1 To a mixture of (5s,8s)-4-(benzyloxy)-3-mesityl-8-(piperidin-4-yloxy)-1- oxaspiro[4.5]dec-3-en-2-one (250 mg, 423.99 ⁇ mol, TFA salt; see Example 11, Step 5) and tert- butyl 2-[2-[2-[2-(2-bromoethoxy)ethoxy]ethoxy]ethoxy]acetate (157.41 mg, 423.99 ⁇ mol) in DMF (3 mL) were added K2CO3 (175.80 mg, 1.27 mmol) and NaI (63.55 mg, 423.99 ⁇ mol).
  • Step 2 oxaspiro[4.5]dec-3-en-8-yl)oxy)piperidin-1-yl)-3,6,9,12-tetraoxatetradecanoate (230 mg, 300.27 ⁇ mol) in THF (30 mL) were added 10% Pd/C (40 mg) and 20% Pd(OH)2/C (40mg) under N2. The mixture was degassed, purged with H 2 (3x), and stirred at 25 °C for 0.2 hour under 15 psi H 2 (balloon). The mixture was filtered, and the filtrate concentrated to dryness at reduced pressure.
  • Step 3 To a mixture of tert-butyl 14-(4-(((5s,8s)-4-hydroxy-3-mesityl-2-oxo-1- oxaspiro[4.5]dec-3-en-8-yl)oxy)piperidin-1-yl)-3,6,9,12-tetraoxatetradecanoate (180 mg, 266.33 ⁇ mol) in DCM (4 mL) was added TFA (3.08 g, 27.01 mmol, 2 mL) at 0 °C.
  • Step 4 1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (133.74 mg, 278.02 ⁇ mol, HCl salt) and 14-(4-(((5s,8s)-4-hydroxy-3-mesityl-2-oxo-1-oxaspiro[4.5]dec-3-en-8- yl)oxy)piperidin-1-yl)-3,6,9,12-tetraoxatetradecanoic acid (170 mg, 231.68 ⁇ mol, TFA salt) in DCM (6 mL) were added 50% T3P (221.15 mg, 347.52 ⁇ mol, 206.68 ⁇ L) and DIPEA (119.77 mg, 926.73 ⁇ mol, 161.42 ⁇ L).
  • EXAMPLE A-14 SYNTHESIS OF (2S,4R)-1-((S)-2-(TERT-BUTYL)-17-(4-(((5S,8S)-4- HYDROXY-3-MESITYL-2-OXO-1-OXASPIRO[4.5]DEC-3-EN-8-YL)OXY)PIPERIDIN- 1-YL)-4-OXO-6,9,12,15-TETRAOXA-3-AZAHEPTADECANOYL)-4-HYDROXY-N-((S)- 1-(4-(4-METHYLTHIAZOL-5-YL)PHENYL)ETHYL)PYRROLIDINE-2- CARBOXAMIDE A.
  • Step 1 y y y y y y y y oxaspiro[4.5]dec-3-en-2-one (250 mg, 423.99 ⁇ mol, TFA salt; prepared from (5r,8r)-4- (benzyloxy)-8-hydroxy-3-mesityl-1-oxaspiro[4.5]dec-3-en-2-one using the general methods described in Example 11), tert-butyl 2-[2-[2-[2-[2-(2-bromoethoxy)ethoxy]ethoxy]ethoxy]acetate (236.12 mg, 635.98 ⁇ mol) , NaI (63.55 mg, 423.99 ⁇ mol) and K2CO3 (351.59 mg, 2.54 mmol) in DMF (5 mL).
  • Step 4 A mixture of 14-(4-(((5r,8r)-4-hydroxy-3-mesityl-2-oxo-1-oxaspiro[4.5]dec-3-en-8- yl)oxy)piperidin-1-yl)-3,6,9,12-tetraoxatetradecanoic acid (477 mg, 650.07 ⁇ mol, TFA salt), (2S,4R)-1-[(2S)-2-amino-3,3-dimethyl-butanoyl]-4-hydroxy-N-[(1S)-1-[4-(4-methylthiazol-5- yl)phenyl]ethyl]pyrrolidine-2-carboxamide (406.53 mg, 845.09 ⁇ mol, HCl salt), 50% T 3 P (517.10 mg, 1.63 mmol, 483.27 ⁇ L) and DIPEA (840.17 mg, 6.50 mmol, 1.13 mL) in THF (10 mL) was stirred at
  • Step 1 To a mixture of (5s,8s)-4-(benzyloxy)-3-mesityl-8-(piperidin-4-yloxy)-1- oxaspiro[4.5]dec-3-en-2-one (220 mg, 373.11 ⁇ mol, TFA salt; see Example 11, Step 5) in DMF (5 mL) were added tert-butyl 2-[2-[2-[2-[2-(2- bromoethoxy)ethoxy]ethoxy]ethoxy]ethoxy]acetate (201.45 mg, 485.04 ⁇ mol), K 2 CO 3 (154.70 mg, 1.12 mmol) and NaI (55.93 mg, 373.11 ⁇ mol).
  • Step 2 oxaspiro[4.5]dec-3-en-8-yl)oxy)piperidin-1-yl)-3,6,9,12,15-pentaoxaheptadecanoate (300 mg, 370.36 ⁇ mol) in THF (10 mL) were added 10% Pd/C (50 mg) and 10% Pd(OH) 2 (50 mg) under N2. The mixture was degassed, purged with H2 (3x), and stirred at 25 °C for 1 hour under 15 psi H2 (balloon).
  • Step 3 To a mixture of tert-butyl 17-(4-(((5s,8s)-4-hydroxy-3-mesityl-2-oxo-1- oxaspiro[4.5]dec-3-en-8-yl)oxy)piperidin-1-yl)-3,6,9,12,15-pentaoxaheptadecanoate (250 mg, 347.27 ⁇ mol, crude) in DCM (3 mL) was added TFA (4.62 g, 40.52 mmol, 3 mL).
  • Step 4 213 To a mixture of 17-(4-(((5s,8s)-4-hydroxy-3-mesityl-2-oxo-1-oxaspiro[4.5]dec-3-en-8- yl)oxy)piperidin-1-yl)-3,6,9,12,15-pentaoxaheptadecanoic acid (220 mg, 282.84 ⁇ mol, crude, TFA salt) in THF (5 mL) were added (2S,4R)-1-[(2S)-2-amino-3,3-dimethyl-butanoyl]-4- hydroxy-N-[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (204.09 mg, 424.26 ⁇ mol, HCl salt), 50% T 3 P (539.97 mg, 848.53 ⁇ mol, 504.65 ⁇ L) and DIPEA (292.44 mg, 2.
  • Step 1 To a mixture of (5r,8r)-4-(benzyloxy)-3-mesityl-8-(piperidin-4-yloxy)-1- oxaspiro[4.5]dec-3-en-2-one (238 mg, 403.64 ⁇ mol, TFA salt; prepared from (5r,8r)-4- (benzyloxy)-8-hydroxy-3-mesityl-1-oxaspiro[4.5]dec-3-en-2-one using the general methods described in Example 11) and tert-butyl 17-bromo-3,6,9,12,15-pentaoxaheptadecanoate (201.16 mg, 484.36 ⁇ mol) in DMF (5 mL) were added K2CO3 (167.35 mg, 1.21 mmol) and NaI (6.05 mg, 40.36 ⁇ mol).
  • the mixture was stirred at 35 °C for 12 hours.
  • the reaction mixture was diluted with water (20 mL) and EtOAc (30 mL) and the phases separated.
  • the aqueous phase was extracted with EtOAc (30 mL x 3).
  • the combined organic phases were washed with brine (20 mL x 3), dried over Na2SO4, filtered, and concentrated in vacuo.
  • Step 2 [ ] o a m xure o er - u y -( -((( r, r)- -( enzy oxy)- -mes y- -oxo- - oxaspiro[4.5]dec-3-en-8-yl)oxy)piperidin-1-yl)-3,6,9,12,15-pentaoxaheptadecanoate (220 mg, 271.60 ⁇ mol) in THF (6 mL) were added 10% Pd(OH) 2 /C (50 mg) and 10% Pd/C (100 mg) under N2.
  • EXAMPLE A-17 SYNTHESIS OF (2S,4R)-4-HYDROXY-1-((S)-2-(2-(2-(4-(2-(((5S,8S)-4- HYDROXY-3-MESITYL-2-OXO-1-OXASPIRO[4.5]DEC-3-EN-8- YL)OXY)ETHYL)PIPERAZIN-1-YL)ETHOXY)ACETAMIDO)-3,3- DIMETHYLBUTANOYL)-N-((S)-1-(4-(4-METHYLTHIAZOL-5- YL)PHENYL)ETHYL)PYRROLIDINE-2-CARBOXAMIDE A.
  • Step 1 y y y y y y y y oxaspiro[4.5]dec-3-en-2-one (465.75 mg, 752.80 ⁇ mol, TFA salt; see Example 19, Step 3) and tert-butyl 2-(2-bromoethoxy)acetate (150 mg, 627.34 ⁇ mol) in DMF (2 mL) were added NaI (9.40 mg, 62.73 ⁇ mol) and K2CO3 (260.11 mg, 1.88 mmol). The mixture was stirred at 50 °C for 12 hours. The reaction mixture was diluted with H2O (20 mL) and extracted with EtOAc (40 mL x 3).
  • Step 4 To a mixture of (2S,4R)-1-[(2S)-2-amino-3,3-dimethyl-butanoyl]-4-hydroxy-N-[(1S)- 1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (111.74 mg, 232.28 ⁇ mol, HCl salt) and 2-(2-(4-(2-(((5s,8s)-4-hydroxy-3-mesityl-2-oxo-1-oxaspiro[4.5]dec-3-en-8- yl)oxy)ethyl)piperazin-1-yl)ethoxy)acetic acid (100 mg, 193.56 ⁇ mol) in DCM (1 mL) were added 50% T3P (184.76 mg, 290.35 ⁇ mol, 172.68 ⁇ L) and DIPEA (100.06 mg, 774.26 ⁇ mol, 134.86 ⁇ L).
  • the mixture was stirred at 25 °C for 12 hours and 40 °C for 4 hours.
  • the reaction mixture was diluted with H 2 O (20 mL) and extracted with EtOAc (20 mL x 3). The combined organic layers were washed with brine (20 mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to give a residue.
  • Step 1 To a mixture of (5r,8r)-4-(benzyloxy)-3-mesityl-8-(2-(piperazin-1-yl)ethoxy)-1- oxaspiro[4.5]dec-3-en-2-one (244 mg, 394.39 ⁇ mol, TFA salt; see Example 20, Step 2) and tert- butyl 2-(2-bromoethoxy)acetate (113.16 mg, 473.26 ⁇ mol) in DMF (3 mL) were added K2CO3 (163.52 mg, 1.18 mmol) and NaI (5.91 mg, 39.44 ⁇ mol). The mixture was stirred at 25 °C for 12 hours.
  • Step 2 - y - - - - - - , - - y y - - y , - - y y - - y - - - - oxaspiro[4.5]dec-3-en-8-yl)oxy)ethyl)piperazin-1-yl)ethoxy)acetate (175 mg, 264.01 ⁇ mol) in THF (10 mL) were added 10% Pd/C (20 mg) and 20% Pd(OH) 2 /C (20 mg) under N 2 . The mixture was degassed, purged with H 2 (3x), and stirred at 25 °C for 0.5 hour under 15 psi H 2 (balloon).
  • Step 3 To a mixture of tert-butyl 2-(2-(4-(2-(((5r,8r)-4-hydroxy-3-mesityl-2-oxo-1- oxaspiro[4.5]dec-3-en-8-yl)oxy)ethyl)piperazin-1-yl)ethoxy)acetate (150.00 mg, 261.90 ⁇ mol) in DCM (6 mL) was added TFA (5.33 g, 46.75 mmol, 3.46 mL) at 0 °C.
  • Step 4 )- 1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (137.30 mg, 285.42 ⁇ mol, HCl salt) and 2-(2-(4-(2-(((5r,8r)-4-hydroxy-3-mesityl-2-oxo-1-oxaspiro[4.5]dec-3-en-8- yl)oxy)ethyl)piperazin-1-yl)ethoxy)acetic acid (150 mg, 237.85 ⁇ mol, TFA salt) in DCM (5 mL) were added 50% T3P (227.04 mg, 356.78 ⁇ mol, 212.19 ⁇ L) and DIPEA (122.96 mg, 951.40 ⁇ mol, 165.71 ⁇ L).
  • EXAMPLE A-19 SYNTHESIS OF (2S,4R)-4-HYDROXY-1-((S)-2-(2-(2-(2-(4-(2-(((5S,8S)- 4-HYDROXY-3-MESITYL-2-OXO-1-OXASPIRO[4.5]DEC-3-EN-8- YL)OXY)ETHYL)PIPERAZIN-1-YL)ETHOXY)ETHOXY)ACETAMIDO)-3,3- DIMETHYLBUTANOYL)-N-((S)-1-(4-(4-METHYLTHIAZOL-5- YL)PHENYL)ETHYL)PYRROLIDINE-2-CARBOXAMIDE A.
  • Step 1 To a solut xylate (20 g, 86.84 mmol) and PPh3 (25.06 g, 95.53 mmol) in DCM (100 mL) was added a solution of CBr4 (31.68 g, 95.53 mmol) in DCM (50 mL) at 0 °C. The mixture was stirred at 25 °C for 12 hours and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (silica gel, gradient elution from 10 to 50% EtOAc in petroleum ether) to give tert-butyl 4-(2-bromoethyl)piperazine-1-carboxylate (13.9 g) as a white solid.
  • Step 3 [025 , oxaspiro[4.5]dec-3-en-8-yl)oxy)ethyl)piperazine-1-carboxylate (2.00 g, 3.31 mmol) in DCM (20 mL) was dropwise added TFA (4 mL) at 0 °C. The mixture was stirred at 25 °C for 1 hour. The reaction mixture was concentrated under reduced pressure to give (5s,8s)-4-(benzyloxy)-3- mesityl-8-(2-(piperazin-1-yl)ethoxy)-1-oxaspiro[4.5]dec-3-en-2-one (3.2 g, crude, TFA salt) as a brown solid.
  • HNMR CDCl 3 , 400MHz.
  • Step 4 To a mixture of (5s,8s)-4-(benzyloxy)-3-mesityl-8-(2-(piperazin-1-yl)ethoxy)-1- oxaspiro[4.5]dec-3-en-2-one (200 mg, 323.27 ⁇ mol, TFA salt) in DMF (5 mL) were added tert- butyl 2-[2-(2-bromoethoxy)ethoxy]acetate (137.30 mg, 484.90 ⁇ mol), K 2 CO 3 (134.04 mg, 969.80 ⁇ mol) and NaI (48.45 mg, 323.27 ⁇ mol). The mixture was stirred at 50 °C for 12 hours.
  • Step 5 oxaspiro[4.5]dec-3-en-8-yl)oxy)ethyl)piperazin-1-yl)ethoxy)ethoxy)acetate (110 mg, 155.61 ⁇ mol) in DCM (2 mL) was added TFA (3.08 g, 27.01 mmol, 2 mL).
  • Step 6 To a mixture of 2-(2-(4-(2-(((5s,8s)-4-(benzyloxy)-3-mesityl-2-oxo-1- oxaspiro[4.5]dec-3-en-8-yl)oxy)ethyl)piperazin-1-yl)ethoxy)ethoxy)acetic acid (100 mg, 153.66 ⁇ mol, crude) in DCM (5 mL) were added (2S,4R)-1-[(2S)-2-amino-3,3-dimethyl-butanoyl]-4- hydroxy-N-[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (73.92 mg, 153.66 ⁇ mol, HCl salt), 50% T 3 P (146.67 mg, 460.97 ⁇ mol, 137.08 ⁇ L) and DIPEA (158.87 mg, 1.23
  • EXAMPLE A-20 SYNTHESIS OF (2S,4R)-4-HYDROXY-1-((S)-2-(2-(2-(2-(4-(2-(((5R,8R)- 4-HYDROXY-3-MESITYL-2-OXO-1-OXASPIRO[4.5]DEC-3-EN-8- YL)OXY)ETHYL)PIPERAZIN-1-YL)ETHOXY)ETHOXY)ACETAMIDO)-3,3- DIMETHYLBUTANOYL)-N-((S)-1-(4-(4-METHYLTHIAZOL-5- YL)PHENYL)ETHYL)PYRROLIDINE-2-CARBOXAMIDE A.
  • Step 1 [02 , y y y y y y p . -2- one (500 mg, 1.27 mmol; prepared using general methods described herein for the preparation of (5s,8s)-4-(benzyloxy)-8-hydroxy-3-mesityl-1-oxaspiro[4.5]dec-3-en-2-one), tertbutyl 4-(2- bromoethyl)piperazine-1-carboxylate (934 mg, 3.19 mmol), TBAB (205 mg, 635.92 ⁇ mol) and KOH (357 mg, 6.36 mmol) in xylene (10 mL) was stirred at 60 °C for 10 hours under microwave irradiation.
  • Step 2 [026 , oxaspiro[4.5]dec-3-en-8-yl)oxy)ethyl)piperazine-1-carboxylate (650 mg, 1.07 mmol) in DCM (5 mL) was added TFA (3.08 g, 27.01 mmol, 2 mL) at 0 °C.
  • Step 3 oxaspiro[4.5]dec-3-en-2-one (244 mg, 394.39 ⁇ mol, TFA) and tert-butyl 2-(2-(2- bromoethoxy)ethoxy)acetate (134.01 mg, 473.26 ⁇ mol) in DMF (2 mL) were added K2CO3 (218.03 mg, 1.58 mmol) and NaI (5.91 mg, 39.44 ⁇ mol). The mixture was stirred at 25° C for 12 hours. To the reaction mixture were added water (10 mL) and EtOAc (20 mL) and the phases separated. The aqueous phase was extracted with EtOAc (30 mL x 3).
  • Step 5 O O O TFA, DCM, 2 o O O OH N O O 5 C, 1 h O N O O
  • Step 1 To a mixture of tert-butyl 4-(2-hydroxyethyl) piperazine-1-carboxylate (1 g, 4.34 mmol) in DCM (10 mL) was added Rh(OAc) 2 (96 mg, 434.40 ⁇ mol). The mixture was degassed and purged with nitrogen (3x). Then a solution of ethyl 2-diazoacetate (991 mg, 8.69 mmol) in DCM (10 mL) was added slowly at 0 °C. The mixture was then stirred at 25 °C for 12 hours under nitrogen. The mixture was diluted with water (50 mL) and extracted with DCM (20 mL x 2).
  • Step 2 [0269] T o a mixture of tert-butyl 4-[2-(2-ethoxy-2-oxo-ethoxy)ethyl]piperazine-1-carboxylate (600 mg, 1.90 mmol) in DCM (6 mL) was added TFA (3.08 g, 27.01 mmol, 2 mL) at 0 °C. The mixture was then stirred at 0 °C for 2 hours. The mixture was concentrated to give ethyl 2-(2- piperazin-1-ylethoxy)acetate (0.63 g, crude, TFA salt) as a brown oil. HNMR: CDCl 3 , 400MHz.
  • Step 3 To a solution of (5s,8s)-4-(benzyloxy)-8-(2-(2-bromoethoxy)ethoxy)-3-mesityl-1- oxaspiro[4.5]dec-3-en-2-one (300.00 mg, 551.99 ⁇ mol; prepared from (5s,8s)-4-(benzyloxy)-8- hydroxy-3-mesityl-1-oxaspiro[4.5]dec-3-en-2-one using general methods described herein for the preparation of (5r,8r)-4-(benzyloxy)-8-(2-(2-bromoethoxy)ethoxy)-3-mesityl-1- oxaspiro[4.5]dec-3-en-2-one) and ethyl 2-(2-piperazin-1-ylethoxy)acetate (364.64 mg, 1.10 mmol, TFA salt) in DMF (3 mL) were added K2CO3 (381.44 mg, 2.76 mmol) and NaI
  • Step 6 [0 73] o a st rred m xture o -( -( -( -( -( -( -((((5s,8s)- - ydroxy-3-mes ty- -oxo- - oxaspiro[4.5]dec-3-en-8-yl)oxy)ethoxy)ethyl)piperazin-1-yl)ethoxy)acetic acid (0.2 g, 356.71 ⁇ mol), (2S,4R)-1-[(2S)-2-amino-3,3-dimethylbutanoyl]-4-hydroxy-N-[(1S)-1-[4-(4- methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (257.39 mg, 535.07 ⁇ mol, HCl salt) in THF (2 mL) was added 50% T3P (680.99 mg, 1.07 m
  • Step 1 O O O O O O O N O O O N O HN O N o a so u o o , - - e y o y - - - - o oe o y e o y - - es y - - oxaspiro[4.5]dec-3-en-2-one (180 mg, 331.19 ⁇ mol; see Example 24, Step 1) and ethyl 2-(2- piperazin-1-ylethoxy)acetate (218.79 mg, 662.39 ⁇ mol, TFA salt) in DMF (2 mL) were added K2CO3 (228.87 mg, 1.66 mmol) and NaI (49.64 mg, 331.19 ⁇ mol).
  • Step 3 y , y y y oxaspiro[4.5]dec-3-en-8-yl)oxy)ethoxy)ethyl)piperazin-1-yl)ethoxy)acetate (50 mg, 84.93 ⁇ mol, crude) in EtOH (1 mL) and H 2 O (0.2 mL) was added LiOH . H 2 O (17.82 mg, 424.64 ⁇ mol). The mixture was stirred at 25 °C for 1 hour. The mixture was concentrated under reduced pressure to remove EtOH. The pH of the reaction mixture was adjusted to 3-4 by addition of 2 M aqueous HCl.
  • Step 1 To a mixture of 2-(2-hydroxyethoxy)ethanol (21.76 g, 205.07 mmol, 19.43 mL) in THF (300 mL) was added 60% NaH (4.51 g, 112.79 mmol) at 0 °C under N 2 . After 1 hour at 0 °C the mixture was treated with tert-butyl 2-bromoacetate (20 g, 102.54 mmol, 15.15 mL) and stirred at 25 °C for 12 hours. The mixture was poured into water (200 mL) and extracted with EtOAc (200 mL x 3).
  • Step 2 [0279] T y y y y y y y y g, .62 mmol) in DCM (50 mL) was added carbon tetrabromide (9.03 g, 27.24 mmol), followed by PPh 3 (7.14 g, 27.24 mmol). The mixture was stirred at 25 °C for 12 hours and then concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (silica gel, gradient elution from 0 to 5% EtOAc in petroleum ether) to give tert-butyl 2-[2-(2- bromoethoxy)ethoxy]acetate (0.86 g, 3.04 mmol, 22.30% yield) as a yellow oil.
  • Step 4 oxaspiro[4.5]dec-3-en-2-one (200.00 mg, 367.99 ⁇ mol; prepared from (5s,8s)-4-(benzyloxy)-8- hydroxy-3-mesityl-1-oxaspiro[4.5]dec-3-en-2-one using general methods described herein for the preparation of (5r,8r)-4-(benzyloxy)-8-(2-(2-bromoethoxy)ethoxy)-3-mesityl-1- oxaspiro[4.5]dec-3-en-2-one), tert-butyl 2-[2-(2-piperazin-1-ylethoxy)ethoxy]acetate (159.18 mg, 551.99 ⁇ mol), K 2 CO 3 (152.58 mg, 1.10 mmol) and NaI (55.16 mg, 367.99 ⁇ mol) in DMF (2 mL) was stirred under N2 at 50 °C for 12 hours.
  • Step 5 To a solution of tert-butyl 2-(2-(2-(4-(2-(2-(((5s,8s)-4-(benzyloxy)-3-mesityl-2-oxo-1- oxaspiro[4.5]dec-3-en-8-yl)oxy)ethoxy)ethyl)piperazin-1-yl)ethoxy)ethoxy)acetate (200 mg, 266.33 ⁇ mol) in DCM (2 mL) at 0 °C was added TFA (1.54 g, 13.51 mmol, 1 mL).
  • Step 6 oxaspiro[4.5]dec-3-en-8-yl)oxy)ethoxy)ethyl)piperazin-1-yl)ethoxy)ethoxy)acetic acid (330 mg, 474.92 ⁇ mol) and (2S,4R)-1-[(2S)-2-amino-3,3–dimethyl–butan oyl]-4-hydroxy-N-[(1S)-1-[4- (4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (316.72 mg, 712.38 ⁇ mol) in DCM (5 mL) at 0°C were added 50% T3P (453.33 mg, 712.38 ⁇ mol, 423.67 ⁇ L) and DIPEA (368.27 mg, 2.85 mmol, 496.32 ⁇ L).
  • Step 1 A mixture of (5r,8r)-4-(benzyloxy)-8-hydroxy-3-mesityl-1-oxaspiro[4.5]dec-3-en-2- one (650 mg, 1.66 mmol; prepared using general methods described herein for the preparation of (5s,8s)-4-(benzyloxy)-8-hydroxy-3-mesityl-1-oxaspiro[4.5]dec-3-en-2-one), 1-bromo-2-(2- bromoethoxy)ethane (1.15 g, 4.97 mmol, 622.59 ⁇ L), KOH (465 mg, 8.29 mmol) and TBAB (534 mg, 1.66 mmol) in toluene (5 mL) was stirred at 40 °C for 5 hours under microwave irradiation.
  • Step 5 To a mixture of 2-(2-(4-(2-(2-(((5r,8r)-4-hydroxy-3-mesityl-2-oxo-1- oxaspiro[4.5]dec-3-en-8-yl)oxy)ethoxy)ethyl)piperazin-1-yl)ethoxy)ethoxy)acetic acid (56 mg, 77.91 ⁇ mol) and (2S,4R)-1-[(2S)-2-amino-3,3-dimethyl-butanoyl]-4-hydroxy-N-[(1S)-1-[4-(4- methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (45 mg, 93.55 ⁇ mol, HCl salt) in DCM (3 mL) were added DIPEA (50 mg, 386.87 ⁇ mol, 67.39 ⁇ L) and 50% T3P (74.37 mg, 116.87 ⁇
  • Step 1 To a solution of 2-[2-(2-hydroxyethoxy)ethoxy]ethanol (15.40 g, 102.54 mmol, 13.75 mL) in THF (100 mL) was added NaH (2.26 g, 56.39 mmol, 60% purity) at 0 °C under N2. After 0.5 hours, tert-butyl 2-bromoacetate (10 g, 51.27 mmol, 7.58 mL) was added to the mixture at 0 °C. The resulting mixture was stirred at 25 °C for 12 hours under N 2 . The mixture was added to water (500 mL) and extracted with EtOAc (500 mL x 2).
  • Step 2 [0291] .8 g, 18.16 mmol) and CBr4 (6.02 g, 18.16 mmol) in DCM (80 mL) was added PPh3 (4.76 g, 18.16 mmol) at 0 °C. The mixture was stirred at 25 °C for 12 hours. The reaction mixture was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (silica gel, gradient elution from 8:1 to 5:1 petroleum ether:EtOAc) to give tert- butyl 2-[2-[2-(2-bromoethoxy)ethoxy]ethoxy]acetate (2.6 g, 7.95 mmol, 43.75% yield) as a colorless oil.
  • Step 7 To a stirred mixture of 2-(2-(2-(4-(2-(2-((((5s,8s)-4-hydroxy-3-mesityl-2-oxo-1- oxaspiro[4.5]dec-3-en-8-yl)oxy)ethoxy)ethyl)piperazin-1-yl)ethoxy)ethoxy)acetic acid (160 mg, 182.48 ⁇ mol, di-TFA salt) and (2S,4R)-1-[(2S)-2-amino-3,3-dimethyl-butanoyl]-4- hydroxy-N-[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (131.67 mg, 273.71 ⁇ mol, HCl salt) in DCM (3 mL) were added DIPEA (141.50 mg, 1.09 mmol, 190.70 ⁇ L) and 50% T
  • the mixture was stirred at 30 °C for 3 hours.
  • the reaction mixture was diluted with DCM (20 mL), washed with water (20 mL) and brine (20 mL x 2).
  • the organic phase was dried over anhydrous Na2SO4, filtered, and concentrated at reduced pressure.
  • Step 1 To a mixture of (5r,8r)-4-(benzyloxy)-8-(2-(2-bromoethoxy)ethoxy)-3-mesityl-1- oxaspiro[4.5]dec-3-en-2-one (90 mg, 165.60 ⁇ mol; see Example 24, Step 1) and tert-butyl 2-[2- [2-(2-piperazin-1-ylethoxy)ethoxy]ethoxy]acetate (71.57 mg, 215.28 ⁇ mol) in DMF (3 mL) were added K2CO3 (68.66 mg, 496.79 ⁇ mol) and NaI (2.48 mg, 16.56 ⁇ mol).
  • Step 2 o- 1-oxaspiro[4.5]dec-3-en-8-yl)oxy)ethoxy)ethyl)piperazin-1-yl)ethoxy)ethoxy)acetate (70 mg, 88.05 ⁇ mol) in THF (10 mL) were added 10% Pd/C (10 mg) and 20% Pd(OH)2/C (10 mg) under N 2 . The mixture was degassed, purged with H 2 (3x), and stirred at 25 °C for 4.5 hours under 15 psi H 2 (balloon).
  • Step 3 To a mixture of tert-butyl 2-(2-(2-(4-(2-(2-((((5r,8r)-4-hydroxy-3-mesityl-2-oxo-1- oxaspiro[4.5]dec-3-en-8-yl)oxy)ethoxy)ethyl)piperazin-1-yl)ethoxy)ethoxy)acetate (60 mg, 85.12 ⁇ mol) in DCM (4 mL) at 0 °C was added TFA (3.08 g, 27.01 mmol, 2 mL).
  • EXAMPLE A-27 SYNTHESIS OF (2S,4R)-4-HYDROXY-1-((S)-2-(2-(2-(2-(4-(2-(2-(2-(2-(2-(2-(2-(((5S,8S)-4-HYDROXY-3-MESITYL-2-OXO-1-OXASPIRO[4.5]DEC-3-EN-8- YL)OXY)ETHOXY)ETHOXY)ETHYL)PIPERAZIN-1- YL)ETHOXY)ETHOXY)ACETAMIDO)-3,3-DIMETHYLBUTANOYL)-N-((S)-1-(4-(4- METHYLTHIAZOL-5-YL)PHENYL)ETHYL)PYRROLIDINE-2-CARBOXAMIDE A.
  • Step 1 yl- 1-oxaspiro[4.5]dec-3-en-2-one (200.00 mg, 340.40 ⁇ mol; prepared from (5s,8s)-4-(benzyloxy)- 8-hydroxy-3-mesityl-1-oxaspiro[4.5]dec-3-en-2-one according to Example 28, Step 3), tert-butyl 2-[2-(2-piperazin-1-ylethoxy)ethoxy]acetate (147.25 mg, 510.60 ⁇ mol; see Example 23, Step 3), K 2 CO 3 (141.14 mg, 1.02 mmol) and NaI (51.02 mg, 340.40 ⁇ mol) in DMF (2 mL) was stirred under N2 at 50 °C for 12 hours.
  • Step 3 oxaspiro[4.5]dec-3-en-8-yl)oxy)ethoxy)ethoxy)ethyl)piperazin-1-yl)ethoxy)ethoxy)acetic acid (250 mg, 293.11 ⁇ mol, TFA salt) and (2S,4R)-1-[(2S)-2-amino-3,3- dimethyl-butanoyl]-4- hydroxy-N-[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (195.47 mg, 439.66 ⁇ mol) in DCM (5 mL) were added DIPEA (227.29 mg, 1.76 mmol, 306.32 ⁇ L) and 50% T 3 P (279.78 mg, 439.66 ⁇ mol, 261.48 ⁇ L).
  • Step 1 To a mixture of (5s,8s)-4-(benzyloxy)-8-hydroxy-3-mesityl-1-oxaspiro[4.5]dec-3-en-2- one (10 g, 25.48 mmol; see Example 7, Step 10), 4-nitrobenzoic acid (5.54 g, 33.12 mmol) and PPh3 (10.69 g, 40.77 mmol) in THF (150 mL) was added diethyl azodicarboxylate (7.10 g, 40.77 mmol, 7.41 mL) dropwise at 0 °C. The mixture was then stirred at 25 °C for 12 hours under N 2 .
  • Step 2 [0306] A m ixture of (5r,8r)-4-(benzyloxy)-3-mesityl-2-oxo-1-oxaspiro[4.5]dec-3-en-8-yl 4- nitrobenzoate (13.5 g, 24.93 mmol) and K2CO3 (6.89 g, 49.82 mmol) in MeOH (150 mL) was stirred at 25 °C for 2 hours and then concentrated to give a residue. The residue was diluted with water (200 mL), and then extracted with EtOAc (50 mL x 2). The combined organic phases were washed with brine (200 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to give the crude product.
  • Step 3 [0307 -en-2- one (500 mg, 1.27 mmol), 1,2-bis(2-bromoethoxy)ethane (879 mg, 3.19 mmol), TBAB (205 mg, 635.92 ⁇ mol) and KOH (357 mg, 6.36 mmol) in toluene (5 mL) was stirred at 50 °C for 4 hours under microwave irradiation. The mixture was diluted with water (50 mL) and extracted with EtOAc (20 mL x 2). The combined organic phases were washed with brine (50 mL), dried over Na2SO4, filtered, and concentrated to give a residue.
  • Step 4 A mixture of (5r,8r)-4-(benzyloxy)-8-(2-(2-(2-bromoethoxy)ethoxy)ethoxy)-3-mesityl- 1-oxaspiro[4.5]dec-3-en-2-one (270 mg, 321.68 ⁇ mol, 70% purity), tert-butyl 2-[2-(2-piperazin- 1-ylethoxy)ethoxy]acetate (278 mg, 482.00 ⁇ mol, 50% purity; see Example 23, Step 3), K2CO3 (133 mg, 962.33 ⁇ mol) and NaI (48 mg, 320.23 ⁇ mol) in DMF (5 mL) was stirred at 40 °C for 12 hours at which point LCMS indicated unreacted starting material remaining.
  • the reaction mixture was treated with additional portions of tert-butyl 2-[2-(2-piperazin-1- ylethoxy)ethoxy]acetate (278 mg, 482.00 ⁇ mol, 50% purity), K 2 CO 3 (89 mg, 643.97 ⁇ mol) and NaI (48 mg, 320.23 ⁇ mol) and the mixture stirred at 50 °C for an additional 4 hours .
  • the mixture was diluted with water (50 mL) and extracted with EtOAc (20 mL x 2). The combined organic phases were washed with brine (50 mL x 3), dried over Na2SO4, filtered, and concentrated to give a residue.
  • Step 5 [ ] o a m xure o er - u y -( -( -( -( -( -( -( -( -( -(((( r, r)- -( enzy oxy)- -mes y - -oxo- 1-oxaspiro[4.5]dec-3-en-8-yl)oxy)ethoxy)ethoxy)ethyl)piperazin-1-yl)ethoxy)ethoxy)acetate (180 mg, 226.41 ⁇ mol) in THF (10 mL) were added 20% Pd(OH) 2 /C (50 mg) and 10% Pd/C (50 mg) under N2.
  • Step 1 To a soluti on of 5,5-dimethylcyclohexane-1,3-dione (2 g, 14.27 mmol) in DMSO (40 mL) were added 4-chloro-2-iodo-1-methyl-benzene (5.40 g, 21.40 mmol), CuI (271.72 mg, 1.43 mmol), K 2 CO 3 (5.92 g, 42.80 mmol) and L-Proline (328.52 mg, 2.85 mmol). The reaction mixture was degassed and purged with N2 three times, and stirred at 120°C for 36 hours under N 2 atmosphere.
  • Step 2 2-(5-chloro-2-methyl-phenyl)-3-hydroxy-5,5-dimethyl-cyclohex-2-en-1-one (500 mg, 1.89 mmol), (4-benzyloxyphenyl)boronic acid (1.29 g, 5.67 mmol), K2CO3 (783.05 mg, 5.67 mmol) and XPhos Pd G3 (159.86 mg, 188.86 ⁇ mol) were combined in a microwave tube with dimethylacetamide (10 mL). The sealed tube was purged with N 2 for several minutes and heated at 150°C for 5 hours in a microwave reactor. The reaction mixture was partitioned between EtOAc (20 mL) and water (150 mL).
  • Step 3 To a mixture of 2-[5-(4-benzyloxyphenyl)-2-methyl-phenyl]-3-hydroxy-5,5-dimethyl- cyclohex-2-en-1-one (470 mg, 1.14 mmol) in DMF (10 mL) was added iodoethane (266.55 mg, 1.71 mmol, 136.69 ⁇ L) and K2CO3 (314.93 mg, 2.28 mmol). The mixture was stirred at 35°C for 2 hours. The reaction was partitioned between EtOAc (50 mL) and water (100 mL). The aqueous phase was extracted with EtOAc (20 mL ⁇ 3).
  • Step 4 To a mixture of 2-[5-(4-benzyloxyphenyl)-2-methyl-phenyl]-3-ethoxy-5,5-dimethyl- cyclohex-2-en-1-one (500 mg, 1.13 mmol) in MeOH (10 mL) and THF (3 mL) was added Pd/C (150 mg, 5% purity) under the N 2 . The mixture was degassed and purged with H 2 three times and stirred at 25 °C for 1.5 hours under H2 balloon (15 psi).
  • EXAMPLE A-30 SYNTHESIS OF 3-ETHOXY-5,5-DIMETHYL-2-[2-METHYL-5-[4-(4- PIPERIDYLOXY)PHENYL]PHENYL]CYCLOHEX-2-EN-1-ONE (INTERMEDIATE (INT.) 2) A.
  • Step 1 To a mixture of 3-ethoxy-2-[5-(4-hydroxyphenyl)-2-methyl-phenyl]-5,5-dimethyl- cyclohex-2-en-1-one (INT 1) (1 g, 2.85 mmol) and tert-butyl 4-hydroxypiperidine-1-carboxylate (861.44 mg, 4.28 mmol) in toluene (10 mL) were added tributylphosphine (1.15 g, 5.71 mmol, 1.41 mL) and TMAD (982.68 mg, 5.71 mmol). The mixture was degassed and purged with N2 three times, then stirred at 120 °C for 12 hours.
  • Step 2 To a mixture of tert-butyl 4-[4-[3-(2-ethoxy-4,4-dimethyl-6-oxo-cyclohexen-1-yl)-4- methyl-phenyl]-phenoxy]piperidine-1-carboxylate (1.05 g, 1.97 mmol, 99.9% purity in CH2Cl2 (2 mL) was added TFA (6.47 g, 56.73 mmol, 4.20 mL). The mixture was stirred at 25°C for 0.5 hour.
  • EXAMPLE A-31 SYNTHESIS OF (2S)-2-[[2-[3-(4 HYDROXYPHENYL)PROPANOYLAMINO]-2-METHYL-PROPANOYL]AMINO]-3- METHYL-N-[(1S)-2-METHYL-1-(PYRROLIDINE-1- CARBONYL)PROPYL]BUTANAMIDE A.
  • Step 1 [0318] o a so ut on o -[ -( - ydroxyet oxy)et oxy]et ano ( 5.
  • Step 2 To a solution of tert-butyl 2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]acetate (4.8 g, 18.16 mmol) and CBr4 (6.02 g, 18.16 mmol) in CH2Cl2 (80 mL) was added PPh3 (4.76 g, 18.16 mmol) at 0 °C. The mixture was stirred at 25 °C for 12 hours.
  • Step 3 [0 - y- - - - y yp y - - y -p y - , - y - cyclohex-2-en-1-one (Int.1) (200 mg, 570.70 ⁇ mol) in DMAC (5 mL) were added NaI (85.54 mg, 570.70 ⁇ mol), tert-butyl 2-[2-[2-(2-bromoethoxy)ethoxy]ethoxy]acetate (224.09 mg, 684.83 ⁇ mol) and K2CO3 (157.75 mg, 1.14 mmol). The mixture was stirred at 50 °C for 12 hours.
  • reaction mixture was diluted with H2O (40 mL) and extracted with Ethyl acetate (60 mL x 3). The combined organic layers were washed with brine (80 mL x 3), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure.
  • Step 4 To a solution of tert-butyl 2-[2-[2-[2-[4-[3-(2-ethoxy-4,4-dimethyl-6-oxo-cyclohexen- 1-yl)-4-methyl-phenyl]phenoxy]ethoxy]ethoxy]acetate (140 mg, 234.60 ⁇ mol) in CH 2 Cl 2 (2 mL) was dropwise added TFA (0.4 mL). The mixture was stirred at 25 °C for 1 hour.
  • Step 5 [0 - - - - - - - - - y- , - y - - - y - -y - - methylphenyl]-phenoxy]ethoxy]ethoxy]acetic acid (200 mg, 369.93 ⁇ mol), DIPEA (191.24 mg, 1.48 mmol, 257.73 ⁇ L) and (2S,4R)-1-[(2S)-2-amino-3,3-dimethyl-butanoyl]-4- hydroxy-N-[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (266.93 mg, 554.89 ⁇ mol, HCl salt) in CH 2 Cl 2 (2 mL) was slowly added T 3 P (353.11 mg, 554.89 ⁇ mol, 330.01 ⁇ L, 50% purity).
  • the mixture was stirred at 35 °C for 0.5 hour.
  • the reaction mixture was diluted with H 2 O (10 mL) and extracted with Ethyl acetate (30 mL x 3). The combined organic layers were dried over anhydrous Na 2 SO 4 , filtered and concentrated under reduced pressure.
  • EXAMPLE A-35 SYNTHESIS OF (2S)-2-[[2-[3-(4- HYDROXYPHENYL)PROPANOYLAMINO]-2-METHYL-PROPANOYL]AMINO]-3- METHYL-N-[(1S)-2-METHYL-1-(PYRROLIDINE-1- CARBONYL)PROPYL]BUTANAMIDE A.
  • Step 1 [0330] y y y . g, . , . mL) in THF (300 mL) was added NaH (4.51 g, 112.79 mmol, 60% purity) at 0°C under N2 and stirred at 0°C for 1 hour.
  • tert-butyl 2-bromoacetate (20 g, 102.54 mmol, 15.15 mL) and stirred at 25°C for 12 hours.
  • the mixture was poured into water (200 mL) and extracted with EtOAc (200 mL x 3). The organic phase was washed with brine (200 mL), dried over anhydrous Na2SO4, filtered, and concentrated in vacuo.
  • Step 3 [03 , piperidyloxy)phenyl]phenyl]cyclohex-2-en-1-one (INT 2) (0.154 g, 281.23 ⁇ mol, TFA) in DMAC (2 mL) were added tert-butyl 2-[2-(2-bromo-ethoxy)ethoxy]acetate (119.45 mg, 421.85 ⁇ mol), NaI (21.08 mg, 140.62 ⁇ mol) and K 2 CO 3 (116.60 mg, 843.69 ⁇ mol). The mixture was stirred at 50°C for 16 hours. EtOAc (20 mL) and water (20 mL) were added, and layers were separated. The aqueous phase was extracted with EtOAc (10 mL ⁇ 3).
  • Step 5 [0 , - methyl-phenyl]phenoxy]-1-piperidyl]ethoxy]ethoxy]acetic acid (110 mg, 189.75 ⁇ mol, crude) in DCM (5 mL) were added (2S,4R)-1-[(2S)-2-amino-3,3-dimethyl-butanoyl]-4-hydroxy-N-[(1S)- 1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (182.55 mg, 379.49 ⁇ mol, HCl salt), T 3 P (181.12 mg, 284.62 ⁇ mol, 169.27 ⁇ L, 50% purity) and DIPEA (98.09 mg, 758.98 ⁇ mol, 132.20 ⁇ L).
  • Step 1 [0342] yl- cyclohex-2-en-1-one (562 mg, 1.60 mmol), tert-butyl 4-(2-bromoethyl)piperazine-1-carboxylate (940.38 mg, 3.21 mmol), NaI (240.38 mg, 1.60 mmol), K 2 CO 3 (443.27 mg, 3.21 mmol) in DMF (10 mL) was degassed and purged with N 2 , and then the mixture was stirred at 50 °C for 16 hours under N2 atmosphere. The reaction mixture was quenched with water (200 mL) and extracted with CH2Cl2 (50 mL x 3).
  • Step 2 269 [0343] A mixture of tert-butyl 4-[2-[4-[3-(2-ethoxy-4,4-dimethyl-6-oxo-cyclohexen-1-yl)-4- methyl-phenyl]-phenoxy]ethyl]piperazine-1-carboxylate (0.627 g, 1.08 mmol, 97% purity) in TFA (2 mL) and CH 2 Cl 2 (2 mL) was degassed and purged with N 2 , and then the mixture was stirred at 25 °C for 3 hours under N2 atmosphere.
  • Step 4 A mixture of tert-butyl 2-[2-[2-[4-[2-[4-[3-(2-ethoxy-4,4-dimethyl-6-oxo-cyclohexen- 1-yl)-4-methyl-phenyl]phenoxy]ethyl]piperazin-1-yl]ethoxy]ethoxy]acetate (317 mg, 476.79 ⁇ mol) in TFA (5 mL) and CH2Cl2 (5 mL) was degassed and purged with N2, and then the mixture was stirred at 25 °C for 1 hour under N 2 atmosphere.
  • Step 5 [0 o a so u o o - - - - - - - - -e o y- , - e y - -o o-cyc o e e - -yl)-4- methyl-phenyl]phenoxy]ethyl]piperazin-1-yl]ethoxy]ethoxy]acetic acid (600 mg, crude, TFA salt), (2S,4R)-1-[(2S)-2-amino-3,3-dimethyl-butanoyl]-4-hydroxy-N-[(1S)-1-[4-(4- methylthiazol-5-yl)-phenyl]ethyl]pyrrolidine-2-carboxamide (160.02 mg, 332.64 ⁇ mol, HCl salt) and DIPEA (286.60 mg, 2.22 mmol, 386.26 ⁇ L) in CH 2 Cl 2 (10 mL) was added T 3 P (2
  • Step 1 To a mixture of 3-ethoxy-2-[5-(4-hydroxyphenyl)-2-methyl-phenyl]-5,5-dimethyl- cyclohex-2-en-1-one (Int 1) (600 mg, 1.71 mmol) in DMF (10 mL) were added 1-bromo-2-(2- bromoethoxy)ethane (595.58 mg, 2.57 mmol, 321.94 ⁇ L), K 2 CO 3 (709.86 mg, 5.14 mmol) and NaI (256.63 mg, 1.71 mmol). The mixture was stirred at 40°C for 12 hours. The reaction was partitioned between EtOAc (20 mL) and water (20 mL).
  • Step 3 To a stirred solution of ethyl 2-[2-[4-[2-[2-[4-[3-(2-ethoxy-4,4-dimethyl-6-oxo- cyclohexen-1-yl)-4-methylphenyl]phenoxy]ethoxy]ethyl]piperazin-1-yl]ethoxy]acetate (180 mg, 282.66 ⁇ mol) in EtOH (1 mL) and H2O (0.5 mL) was added LiOH.H2O (59.30 mg, 1.41 mmol) at room temperature (25 °C). The mixture was stirred at 50°C for 0.5 hours. The mixture was concentrated in vacuo.
  • Step 1 [ - , - p y - - - , - y - -py , - zol- 2-yl)-2-hydroxybenzyl)acetamide (150 mg, 360.32 ⁇ mol) and tert-butyl 8-bromooctanoate (150.91 mg, 540.48 ⁇ mol) in DMF (2 mL) was added K2CO3 (99.60 mg, 720.63 ⁇ mol). The mixture was stirred at 70 °C for 12 h. To the mixture were added ethyl acetate (50 mL) and water (15 mL), and the layers were separated.
  • EXAMPLE B-4 SYNTHESIS OF (2S,4R)-1-((S)-2-(10-(4-(5-(2-FLUORO-5-(4-FLUORO- 2-(TRIFLUOROMETHYL)BENZAMIDO)-4-((3R,5S)-3,4,5-TRIMETHYLPIPERAZIN-1- YL)PHENYL)PYRIMIDIN-2-YL)PIPERAZIN-1-YL)-10-OXODECANAMIDO)-3,3- DIMETHYLBUTANOYL)-4-HYDROXY-N-((S)-1-(4-(4-METHYLTHIAZOL-5- YL)PHENYL)ETHYL)PYRROLIDINE-2-CARBOXAMIDE.
  • Step 1 ded NMM (341.26 mg, 3.37 mmol, 370.94 ⁇ L), EDCI (155.23 mg, 809.74 ⁇ mol), HOAt (18.37 mg, 134.96 ⁇ mol) and (2S,4R)-1-((S)-2-amino-3,3-dimethylbutanoyl)-4-hydroxy-N-((S)-1-(4-(4- methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (300 mg, 674.78 ⁇ mol). The mixture was stirred at 25 °C for 12 hours.
  • Step 2 yl)phenyl)ethyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-10- oxodecanoic acid (150 mg, 238.54 ⁇ mol) in DMF (3 mL) was added NMM (100.53 mg, 993.92 ⁇ mol, 109.27 ⁇ L), EDCI (45.73 mg, 238.54 ⁇ mol ), HOAt (5.41 mg, 39.76 ⁇ mol) and 4-fluoro- N-(4-fluoro-5-(2-(piperazin-1-yl)pyrimidin-5-yl)-2-((3R,5S)-3,4,5-trimethylpiperazin-1- yl)phenyl)-2-(trifluoromethyl)benzamide (124.45 mg, 198.78 ⁇ mol, HCl).
  • Step 1 [0 367] o a so ut on o -( -(tert-butoxycarbony )p peraz n- -y )acet c ac d (3 . mg, 1.32 mmol) in DMF (5 mL) was added NMM (511.89 mg, 5.06 mmol, 556.40 ⁇ L), EDCI (232.84 mg, 1.21 mmol), HOAt (27.55 mg, 202.43 ⁇ mol) and (2S,4R)-1-((S)-2-amino-3,3-dimethylbutanoyl)- 4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (450 mg, 1.01 mmol).
  • Step 3 To a solution of (2S,4R)-1-((S)-3,3-dimethyl-2-(2-(piperazin-1-yl)acetamido)butanoyl)- 4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (400 mg, 584.14 ⁇ mol, HCl) in DMF (3 mL) was added DIPEA (150.99 mg, 1.17 mmol, 203.49 ⁇ L) and tert-butyl 4-bromobutanoate (260.65 mg, 1.17 mmol). The mixture was stirred at 50 °C for 12 hours.
  • Step 1 To a solution of 2-(4-(tert-butoxycarbonyl)piperazin-1-yl)acetic acid (93.65 mg, 383.35 ⁇ mol) in DMF (3 mL) was added NMM (96.94 mg, 958.37 ⁇ mol, 105.37 ⁇ L), EDCI (73.49 mg, 383.35 ⁇ mol), HOAt (8.70 mg, 63.89 ⁇ mol) and 4-fluoro-N-(4-fluoro-5-(2-(piperazin-1- yl)pyrimidin-5-yl)-2-((3R,5S)-3,4,5-trimethylpiperazin-1-yl)phenyl)-2- (trifluoromethyl)benzamide (200 mg, 319.46 ⁇ mol, HCl).
  • Step 3 1- yl)pyrimidin-5-yl)-2-((3R,5S)-3,4,5-trimethylpiperazin-1-yl)phenyl)-2- (trifluoromethyl)benzamide (193 mg, 232.59 ⁇ mol, TFA) in DMF (3 mL) was added DIPEA (90.18 mg, 697.77 ⁇ mol, 203.49 ⁇ L) and tert-butyl 4-bromobutanoate (103.79 mg, 465.18 ⁇ mol) at 25 o C. The mixture was stirred at 80 °C for 12 hours. The mixture was diluted with H 2 O (6 mL) and extracted with ethyl acetate (3 mL x 3).
  • Step 1 [0 ] mxture o tert- uty -( - romop eny )acetate ( g, . mmo ), et y pent- - enoate (2.84 g, 22.13 mmol), Pd(OAc)2 (248.40 mg, 1.11 mmol), TBAB (3.57 g, 11.06 mmol) and NaHCO3 (3.72 g, 44.26 mmol, 1.72 mL) in DMF (30 mL) was degassed and purged with N2 for 3 times, and then the mixture was stirred at 90 °C for 12 hours under N 2 atmosphere.
  • Step 2 290 [0379] To a solution of ethyl (E)-5-(3-(2-(tert-butoxy)-2-oxoethyl)phenyl)pent-4-enoate 540 mg, 1.70 mmol) in MeOH (10 mL) was added Pd/C (200 mg, 187.93 ⁇ mol, 10% purity) under N 2 atmosphere. The mixture was degassed and purged with H 2 for three times. The mixture was stirred under H2 (50 Psi) at 25 °C for 12 hours. The reaction mixture was filtered through a pad of celite.
  • Step 3 [0 ] o a so ut on o et y -( -( -(tert- utoxy)- -oxoet y)p eny )pentanoate ( mg, 1.53 mmol) in CH2Cl2 (5 mL) was added TFA (7.68 g, 67.31 mmol, 5 mL). The resulting mixture was stirred at 25 °C for 1 hour. The mixture was concentrated in vacuo to give 2-(3-(5- ethoxy-5-oxopentyl)phenyl)acetic acid (480 mg, crude, TFA) as a colorless oil.
  • Step 4 To a solution of 2-(3-(5-ethoxy-5-oxopentyl)phenyl)acetic acid (400 mg, 1.06 mmol, TFA), (2S,4R)-1-((S)-2-amino-3,3-dimethylbutanoyl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5- yl)phenyl)ethyl)pyrrolidine-2-carboxamide (470.04 mg, 1.06 mmol) and NMM (320.81 mg, 3.17 mmol, 348.71 ⁇ L) in DMSO (10 mL) were added EDCI (304.02 mg, 1.59 mmol) and HOAt (215.86 mg, 1.59 mmol, 221.85 ⁇ L).
  • Step 1 [0 388] To a solution of 2-(3-bromophenyl)acetic acid (377.28 mg, 1.75 mmol) in DMF (10 mL) was added EDCI (310.45 mg, 1.62 mmol), NMM (682.52 mg, 6.75 mmol, 741.87 ⁇ L) and HOAt (36.74 mg, 269.91 ⁇ mol). (2S,4R)-1-((S)-2-amino-3,3-dimethylbutanoyl)-4-hydroxy-N- ((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (600 mg, 1.35 mmol) was added.
  • the mixture was stirred at 25 °C for 2 hours.
  • the reaction mixture was diluted with ethyl acetate (10 mL) and water (30 mL).
  • the mixture was extracted with ethyl acetate (10 mL*3).
  • the combined organic layer was washed with brine (20 mL), dried with anhydrous Na 2 SO 4 , filtered, and concentrated in vacuo.
  • Step 2 [0 , yl)-4- hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (220 mg, 342.88 ⁇ mol), methyl 2-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)acetate (189.36 mg, 685.77 ⁇ mol), Pd(dppf)Cl 2 (25.09 mg, 34.29 ⁇ mol), Na 2 CO 3 (72.68 mg, 685.77 ⁇ mol) in dioxane (2 mL) and H2O (0.4 mL) was degassed and purged with N2 for 3 times, and then the mixture was stirred at 90 °C for 2 hours under N2 atmosphere.
  • reaction mixture was diluted with CH 2 Cl 2 (10 mL) and water (10 mL). The mixture was extracted with CH 2 Cl 2 (10 mL*3). The combined organic layer was washed with brine (20 mL), dried with anhydrous Na2SO4, filtered, and concentrated in vacuo.
  • Step 1 To a solution of tert-butyl 4-(piperazin-1-ylmethyl)piperidine-1-carboxylate (0.8 g, 2.82 mmol), K2CO3 (800.00 mg, 5.79 mmol) in CH3CN (15 mL) was added ethyl 2- bromoacetate (560.00 mg, 3.35 mmol, 371.11 ⁇ L) at 20 °C. The reaction mixture was stirred at 20 °C for 2 hours. Ethyl acetate (30 mL) and water (40 mL) were added and layers were separated. The aqueous phase was extracted with Ethyl acetate (30 mL *2).
  • Step 3 302 To a solution of 2-(4-((1-(tert-butoxycarbonyl)piperidin-4-yl)methyl)piperazin-1- yl)acetic acid (300 mg, 878.62 ⁇ mol), NMM (266.61 mg, 2.64 mmol, 289.79 ⁇ L), HOAt (179.38 mg, 1.32 mmol) in DMF (8 mL) was added EDCI (252.65 mg, 1.32 mmol) at 20 °C and the mixture was stirred for 30 minutes.
  • Step 5 [0 ] o a so u on o ( , )- -(( )- , - me y - -( -( -(p per n- -y me y )p perazin-1- yl)acetamido)butanoyl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2- carboxamide (180 mg, 255.55 ⁇ mol, HCl), tert-butyl 2-bromoacetate (55 mg, 281.97 ⁇ mol) in DMF (5 mL) was added DIPEA (74.20 mg, 574.11 ⁇ mol) at 20 °C and stirred for 48 hours.
  • DIPEA 74.20 mg, 574.11 ⁇ mol
  • reaction mixture was diluted with H2O (30 mL).
  • the aqueous phase was extracted with Ethyl acetate (20 mL*5).
  • the combined organic layer was washed with brine (20 mL), dried over Na 2 SO 4 , filtered, and concentrated in vacuo.
  • Step 6 304 [0401] To a solution of tert-butyl 2-(4-((4-(2-(((S)-1-((2S,4R)-4-hydroxy-2-(((S)-1-(4-(4- methylthiazol-5-yl)phenyl)ethyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2- yl)amino)-2-oxoethyl)piperazin-1-yl)methyl)piperidin-1-yl)acetate (0.19 g, 242.95 ⁇ mol) in CH2Cl2 (5 mL) was added TFA (1.54 g, 13.46 mmol, 1 mL) at 25 °C and stirred for 12 hours.
  • Step 7 [0 402] To a solution of 2-(4-((4-(2-(((S)-1-((2S,4R)-4-hydroxy-2-(((S)-1-(4-(4-methylthiazol- 5-yl)phenyl)ethyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-2- oxoethyl)piperazin-1-yl)methyl)piperidin-1-yl)acetic acid (150 mg, 206.63 ⁇ mol), NMM (62.70 mg, 619.89 ⁇ mol), HOAt (33.75 mg, 247.95 ⁇ mol) in CH2CL2 (5 mL) was added EDCI (79.22 mg, 413.26 ⁇ mol) at 20 °C, the reaction mixture was stirred for 0.5 hours.
  • EXAMPLE B-22 SYNTHESIS OF (2S,4S)-1-((S)-2-(3-(2-(3-(4-(5-(2-FLUORO-5-(4- FLUORO-2-(TRIFLUOROMETHYL)BENZAMIDO)-4-((3R,5S)-3,4,5- TRIMETHYLPIPERAZIN-1-YL)PHENYL)PYRIMIDIN-2-YL)PIPERAZIN-1-YL)-3- OXOPROPOXY)ETHOXY)PROPANAMIDO)-3,3-DIMETHYLBUTANOYL)-4- HYDROXY-N-(4-(4-METHYLTHIAZOL-5-YL)BENZYL)PYRROLIDINE-2- CARBOXAMIDE.
  • Step 1 N To a solution of 1-bromo-2,4-difluoro-5-nitrobenzene (20 g, 84.04 mmol) in toluene (200 mL) was added K2CO3 (23.23 g, 168.08 mmol) at 50 °C for 30 min, and then (2S,6R)-1,2,6- trimethylpiperazine (10.77 g, 84.04 mmol) was added. The resulting mixture was stirred at 50 °C for 1 hour. LCMS showed the desired mass. After the reaction mixture was cooled to 0 °C. The reaction mixture was filtered through a pad of celite. The filtrate was concentrated in vacuo.
  • Step 2 [0408] o a so u o o , - - - o o- - uo o- - op e y - , , - e ylpiperazine (25 g, 72.21 mmol) in AcOH (120 mL) was added Fe (24.20 g, 433.28 mmol). The resulting mixture was stirred at 60 °C for 1 hour. LCMS showed the desired mass. After cooling to room temperature (25 °C), the mixture was adjusted pH to 9-12 with NaOH aqueous solution. The mixture was diluted with methanol (100 mL) and H2O (100 mL). Layers were separated.
  • Step 4 A mixture of N-(5-bromo-4-fluoro-2-((3R,5S)-3,4,5-trimethylpiperazin-1-yl)phenyl)-4- fluoro-2-(trifluoromethyl)benzamide (7 g, 13.83 mmol), tert-butyl 4-(4,4,5,5-tetramethyl-1,3,2- dioxaborolan-2-yl)-3,6-dihydropyridine-1(2H)-carboxylate (6.41 g, 20.74 mmol), Na2CO3 (2.93 g, 27.65 mmol) and Pd(dppf)Cl 2 (1.01 g, 1.38 mmol) in dioxane (70 mL)/ H 2 O (14 mL) was degassed and purged with N2 for 3 times, and then the mixture was stirred at 80 °C for 1 hour under N2 atmosphere.
  • the reaction mixture was poured into 100 mL of water and the solid was washed with ethyl acetate (100 mL x 3).
  • the aqueous phase was extracted with CH 2 Cl 2 / MeOH (10:1, 100 mL x 3).
  • Step 6 311 [0412] To a solution of tert-butyl piperazine-1-carboxylate (6.53 g, 35.08 mmol) and 2- chloropyrimidine-5-carbaldehyde (5 g, 35.08 mmol) in THF (150 mL) was added NaBH(OAc) 3 (11.15 g, 52.62 mmol). The reaction was stirred at 25 °C for 12 hours. The reaction mixture was filtered. The filtrated was concentrated in vacuo.
  • Step 9 To a solution of succinic acid (548.53 mg, 4.65 mmol) in DMF (5 mL) and CH 2 Cl 2 (10 mL) was added NMM (1.17 g, 11.61 mmol, 1.28 mL) , EDCI (534.28 mg, 2.79 mmol) and HOAt (63.22 mg, 464.51 ⁇ mol). Then the mixture was cooled to 0 °C. (2S,4R)-1-((S)-2-amino- 3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (1 g, 2.32 mmol) was added.
  • Step 10 To a solution of 4-(((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5- yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-4-oxobutanoic acid (80 mg, 150.76 ⁇ mol) in DMF (2 mL) was added HOAt (3.42 mg, 25.13 ⁇ mol), NMM (63.54 mg, 628.18 ⁇ mol, 69.06 ⁇ L) and EDCI (28.90 mg, 150.76 ⁇ mol) at 0 °C.
  • Step 1 [0 - - - - - - y - - y p y p p - - y cid (125 mg, 391.37 ⁇ mol), 4-fluoro-N-(4-fluoro-5-(2-(piperazin-1-yl)pyrimidin-5-yl)-2-((3R,5S)- 3,4,5-trimethylpiperazin-1-yl)phenyl)-2-(trifluoromethyl)benzamide (122.51 mg, 195.68 ⁇ mol, HCl) and NMM (118.76 mg, 1.17 mmol, 129.08 ⁇ L) in DMSO (5 mL) were added EDCI (150.05 mg, 782.73 ⁇ mol) and HOAt (106.54 mg, 782.73 ⁇ mol).
  • Step 2 [ , methylthiazol-5-yl)phenyl)ethyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2- yl)carbamoyl)piperidin-1-yl)phenyl)acetate (260 mg, 348.54 ⁇ mol) in CH2Cl2 (2 mL) was added TFA (2.30 g, 20.19 mmol, 1.5 mL). The resulting mixture was stirred at 25 °C for 1 hour. The mixture was concentrated in vacuo.
  • EXAMPLE B-44 SYNTHESIS OF (2S,4R)-1-((S)-2-(4-(4-(4-(5-(2-FLUORO-5-(4- FLUORO-2-(TRIFLUOROMETHYL)BENZAMIDO)-4-((3R,5S)-3,4,5- TRIMETHYLPIPERAZIN-1-YL)PHENYL)PYRIMIDIN-2-YL)PIPERAZINE-1- CARBONYL)PIPERIDIN-1-YL)BENZAMIDO)-3,3-DIMETHYLBUTANOYL)-4- HYDROXY-N-((S)-1-(4-(4-METHYLTHIAZOL-5- YL)PHENYL)ETHYL)PYRROLIDINE-2-CARBOXAMIDE.
  • EXAMPLE B-45 SYNTHESIS OF (2S,4R)-1-((2S)-2-(4-(3-(4-(5-(2-FLUORO-5-(4- FLUORO-2-(TRIFLUOROMETHYL)BENZAMIDO)-4-((3R,5S)-3,4,5- TRIMETHYLPIPERAZIN-1-YL)PHENYL)PYRIMIDIN-2-YL)PIPERAZINE-1- CARBONYL)PIPERIDIN-1-YL)BENZAMIDO)-3,3-DIMETHYLBUTANOYL)-4- HYDROXY-N-((S)-1-(4-(4-METHYLTHIAZOL-5- YL)PHENYL)ETHYL)PYRROLIDINE-2-CARBOXAMIDE.
  • EXAMPLE B-46 SYNTHESIS OF (2S,4R)-1-((2S)-2-(3-(3-(4-(5-(2-FLUORO-5-(4- FLUORO-2-(TRIFLUOROMETHYL)BENZAMIDO)-4-((3R,5S)-3,4,5- TRIMETHYLPIPERAZIN-1-YL)PHENYL)PYRIMIDIN-2-YL)PIPERAZINE-1- CARBONYL)PIPERIDIN-1-YL)BENZAMIDO)-3,3-DIMETHYLBUTANOYL)-4- HYDROXY-N-((S)-1-(4-(4-METHYLTHIAZOL-5- YL)PHENYL)ETHYL)PYRROLIDINE-2-CARBOXAMIDE.
  • EXAMPLE B-47 SYNTHESIS OF 1-(4-(4-(5-(2-FLUORO-5-(4-FLUORO-2- (TRIFLUOROMETHYL)BENZAMIDO)-4-((3R,5S)-3,4,5-TRIMETHYLPIPERAZIN-1- YL)PHENYL)PYRIMIDIN-2-YL)PIPERAZINE-1-CARBONYL)PHENYL)-I-((S)-1- ((2S,4R)-4-HYDROXY-2-(((S)-1-(4-(4-METHYLTHIAZOL-5- YL)PHENYL)ETHYL)CARBAMOYL)PYRROLIDIN-1-YL)-3,3-DIMETHYL-1- OXOBUTAN-2-YL)PIPERIDINE-3-CARBOXAMIDE.
  • EXAMPLE B-48 SYNTHESIS OF 1-(4-(4-(5-(2-FLUORO-5-(4-FLUORO-2- (TRIFLUOROMETHYL)BENZAMIDO)-4-((3R,5S)-3,4,5-TRIMETHYLPIPERAZIN-1- YL)PHENYL)PYRIMIDIN-2-YL)PIPERAZINE-1-CARBONYL)PHENYL)-N-((S)-1- ((2S,4R)-4-HYDROXY-2-(((S)-1-(4-(4-METHYLTHIAZOL-5- YL)PHENYL)ETHYL)CARBAMOYL)PYRROLIDIN-1-YL)-3,3-DIMETHYL-1- OXOBUTAN-2-YL)PIPERIDINE-4-CARBOXAMIDE.
  • EXAMPLE B-49 SYNTHESIS OF 1-[3-[4-[5-[2-FLUORO-5-[[4-FLUORO-2- (TRIFLUOROMETHYL)BENZOYL]AMINO]-4-[(3R,5S)-3,4,5- TRIMETHYLPIPERAZIN-1-YL]PHENYL]PYRIMIDIN-2-YL]PIPERAZINE-1- CARBONYL]PHENYL]-N-[(1S)-1-[(2S,4R)-4-HYDROXY-2-[[(1S)-1-[4-(4- METHYLTHIAZOL-5-YL)PHENYL]ETHYL]CARBAMOYL]PYRROLIDINE-1- CARBONYL]-2,2-DIMETHYL-PROPYL]PIPERIDINE-4-CARBOXAMIDE.
  • EXAMPLE B-50 SYNTHESIS OF 1-(3-(4-(5-(2-FLUORO-5-(4-FLUORO-2- (TRIFLUOROMETHYL)BENZAMIDO)-4-((3R,5S)-3,4,5-TRIMETHYLPIPERAZIN-1- YL)PHENYL)PYRIMIDIN-2-YL)PIPERAZINE-1-CARBONYL)PHENYL)-N-((S)-1- ((2S,4R)-4-HYDROXY-2-(((S)-1-(4-(4-METHYLTHIAZOL-5- YL)PHENYL)ETHYL)CARBAMOYL)PYRROLIDIN-1-YL)-3,3-DIMETHYL-1- OXOBUTAN-2-YL)PIPERIDINE-3-CARBOXAMIDE.
  • Step 1 A mixture of (2S,4R)-1-[(2S)-2-[(1-fluorocyclopropanecarbonyl)amino]-3,3-dimethyl- butanoyl]-4-hydroxy-N-[[2-hydroxy-4-(4-methylthiazol-5-yl)phenyl]methyl]pyrrolidine-2- carboxamide (150 mg, 281.62 ⁇ mol), tert-butyl 7-bromoheptanoate (89.62 mg, 337.95 ⁇ mol), KI (46.75 mg, 281.62 ⁇ mol) and Cs 2 CO 3 (275.27 mg, 844.87 ⁇ mol) in DMF (1.5 mL) was heated to 60 °C and stirred for 1 hour under N 2 .

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  • Life Sciences & Earth Sciences (AREA)
  • Plant Pathology (AREA)
  • Environmental Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Pest Control & Pesticides (AREA)
  • Health & Medical Sciences (AREA)
  • Agronomy & Crop Science (AREA)
  • Dentistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Chemical & Material Sciences (AREA)
  • Insects & Arthropods (AREA)
  • Plural Heterocyclic Compounds (AREA)

Abstract

La présente invention concerne des compositions, des composés et des procédés pour moduler les niveaux de protéines cibles dans des insectes et des cellules d'insectes. Les composés peuvent contenir une ou plusieurs fractions de ciblage de protéine qui se lient chacune indépendamment à une protéine qui est exprimée par une cellule d'insecte ; une ou plusieurs fractions de ciblage de ligase qui se lient chacune indépendamment à une ubiquitine ligase qui est fonctionnelle dans la cellule d'insecte ; et un lieur lié par covalence à ladite fraction de ciblage de protéine et à ladite fraction de ciblage de ligase ; ou un sel ou un solvate de celui-ci.
PCT/US2023/031716 2022-08-31 2023-08-31 Compositions et procédés d'inhibition et de dégradation ciblées de protéines dans une cellule d'insecte WO2024050016A1 (fr)

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