WO2025117878A1

WO2025117878A1 - Compounds and methods for promoting autophagy

Info

Publication number: WO2025117878A1
Application number: PCT/US2024/057948
Authority: WO
Inventors: Erik Lee MEREDITH; John Kelly; Daniel W. BAIRD
Original assignee: Casma Therapeutics Inc
Current assignee: Casma Therapeutics Inc
Priority date: 2023-12-01
Filing date: 2024-11-29
Publication date: 2025-06-05
Anticipated expiration: 2026-06-01

Abstract

The present disclosure provides compounds and uses thereof, wherein the compound is of formula I: or a pharmaceutically acceptable salt thereof, wherein: A1 and A2 are each moieties that bind to or associate with FIP200; and B is a linker moiety.

Description

COMPOUNDS AND METHODS FOR PROMOTING AUTOPHAGY

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to United States Provisional Application No. 63/605,364, filed December 1, 2023, the entirety of which is incorporated herein by reference.

BACKGROUND

[0002] Macroautophagy is a cellular mechanism for the removal of aggregated proteins and cellular waste from the cell. See Turco, et al. , Molecular Cell, 74:330-346 (2019). The autophagy process includes the formation of a double-membrane autophagosome around said aggregated proteins and cellular waste that is then delivered to the lysosome, where the material is then degraded. See Fujioka and Noda, Current Opin. Cell Bio., 69:23-29 (2021). The autophagy process prevents the accumulation of toxic materials, which are associated with many diseases, such as neurodegeneration, cancer, and aging. See Ohnstad, et al., The EMBO Journal (2020)39:el04948.

SUMMARY

[0003] The present disclosure encompasses, among other things, the surprising discovery that bifunctional small molecule compounds can be used to promote autophagy, and thereby have use in the treatment of a variety of diseases, disorders, and conditions, such as those described herein. In particular, the present disclosure provides particular’ bifunctional compounds that promote the aggregation of particular cellular machinery, such as the FIP200 scaffold protein, which leads to the formation of the autophagosome. See Turco, et al., Molecular Cell, 74:330-346 (2019). By providing compounds that lead to dimers, trimers, and tetramers of aggregated FIP200 proteins, the compounds of the present disclosure can be used to promote autophagy in cellular systems, and therefore be useful in the treatment of a variety of diseases, disorders, and conditions.

[0004] For example, in some embodiments, the present disclosure provides a compound represented by formula I:

A¹-B-A² or a pharmaceutically acceptable salt thereof, wherein:

A¹ and A² are each moieties that bind to or associate with FIP200; and B is a linker moiety.

[0005] In some embodiments, a compound described herein is a compound of formula I, wherein A¹ and A² are each independently selected from formula II- 1, II-2, II-3, or II-4:

each G¹ is independently an optionally substituted C₆-C₁₂ aryl or an optionally substituted 5- to 6-membered heteroaryl; each X¹ is independently S, N(R³), O- optionally substituted C₁-C₆ aliphatic, or optionally substituted C₃-C₆ cycloaliphatic; each X² is independently C(R³) or N, provided that, when X² is N, then X¹ is optionally substituted C₁-C₆ aliphatic, or optionally substituted C₃-C₆ cycloaliphatic; when a bond between X³ and X⁴ is a single bond, then X³ is N(R^2a), and X⁴ is C(O) when a bond between X³ and X⁴ is a double bond, then X³ is C(R^2b) and X⁴ is C(R³), or

N; each R¹ is independently selected from halogen, optionally substituted C₁-C₆ aliphatic, and optionally substituted -O-C₁-C₆ aliphatic; or two instances of R¹ come together with the atoms to which they arc attached to form a n optionally substituted C₆-C₁₂ aryl ring, an optionally substituted C₄-C₆ cycloaliphatic ring, a 5- to 6- membered heteroaryl ring comprising 1 to 3 heteroatoms selected from N, O, and S, or an optionally substituted 5- to 6-membered heterocyclic ring comprising 1 to 3 heteroatoms selected from N, O, and S; each R^2a is independently optionally substituted C₁-C₆ aliphatic; each R^2b is independently hydrogen, optionally substituted C₁-C₆ aliphatic, optionally substituted -N(R³)-C₁-C₆ aliphatic, or optionally substituted -O-C₁-C₆ aliphatic; each R³ is independently selected from hydrogen, halogen, and optionally substituted Ci- C₆ aliphatic; each R⁴ is independently selected from optionally substituted C₆-C₁₂ aryl, optionally substituted 5- to 12- membered heteroaryl ring comprising 1 to 3 heteroatoms selected from N, O, and S, optionally substituted 4- to 12-membered heterocyclyl comprising 1 to 3 heteroatoms selected from N, O, and S, and optionally substituted C₁-C₆ aliphatic; each R⁵ is independently an optionally substituted 4- to 6-membered heterocyclic ring, an optionally substituted 5- to 6-membered heteroaryl ring, an optionally C₃-C₆ cycloaliphatic ring, an optionally substituted C₆-C₁₂ aryl, or an optionally substituted C₁-C₆ aliphatic; each L¹ is independently a bond, -C(O)-, -S(O)-, -S(O)₂-, or -NR³-; each n is independently 0, 1, 2, 3, 4, 5, or 6; each X⁵, X⁶, and X⁷ are independently selected from the group consisting of N and CH; each R⁷ is independently hydrogen, an optionally substituted -O-C₁-C₆ aliphatic, - S(O)₂R³, optionally substituted C₁-C₆ aliphatic, an optionally substituted 4- to 12- membered heterocycle ring comprising 1 to 4 heteroatoms selected from N, O, and S, an optionally substituted C₆-C₁₂ aryl, or an optionally substituted 5- to 12-membered heteroaryl comprising 1 to 4 heteroatoms selected from N, O, and S each G² is independently an optionally substituted C₆-C₁₂ aryl, optionally substituted 4- to 12-membered heterocycle comprising 1 to 4 heteroatoms selected from N, O, and S, optionally substituted 5- to 6-membered heteroaryl comprising 1 to 4 heteroatoms selected from N, O, and S, or optionally substituted C₃-C₆ cycloaliphatic; each G³ is independently an optionally substituted 4- to 12- membered heterocycle ring comprising 1 to 4 heteroatoms selected from N, O, and S, an optionally substituted 5- to 6-membered heteroaryl ring comprising 1 to 4 heteroatoms selected from N, O, and S, an optionally substituted C₃-C₆ cycloaliphatic ring or optionally substituted C₆-C₁₂ aryl; each L² is independently a bond, -NR³-C(O)-, -C(O)-NR³-, optionally substituted C₁-C₆ aliphatic, optionally substituted 4- to 6-membered heterocyclic, or optionally substituted C₃-C₆ cycloaliphatic; each L³ is independently -NR³-, -O-, -C(O)-, -NR³-C(O)-, -NR³-S(O)₂-, -C(O)-NR³-, - S(O)₂ NR³-, -NR³-C(O)-NR³-, optionally substituted C₁-C₆ aliphatic, or an optionally substituted C₃-C₆ cycloaliphatic ring;

* represents a point of attachment to moiety B; and wherein: when A¹ or A² is a moiety of formula II- 3 then R⁶ is a bond, optionally substituted C₁-C₆ aliphatic, optionally substituted 2- to 6-membered heteroaliphatic, optionally substituted 4- to 6-membered heterocycle comprising one 1 to 3 heteroatoms selected from N, O, and S, optionally substituted C₆-C₁₂ aryl, or optionally substituted C₃-C₆ cycloaliphatic; and R⁸ is independently halogen, -OR³, -C(O)N(R³)₂, -C(O)OR³, optionally substituted C₁-C₆ aliphatic, optionally substituted C₃-C₆ cycloaliphatic, optionally substituted C₆-C₁₂ aryl; and when A¹ or A² is of formula II-4, then R⁶ is H, halogen, optionally substituted C₁-C₆ aliphatic, optionally substituted 2- to 6-membered heteroaliphatic, optionally substituted 4- to 6-membered heterocycle comprising one 1 to 3 heteroatoms selected from N, O, and S, optionally substituted C₆-C₁₂ aryl, or optionally substituted C₃-C₆ cycloaliphatic; and R⁸ is a bond, -O-, -C(O)NR³-, -C(O)OR³, optionally substituted C₁-C₆ aliphatic, optionally substituted C₃-C₆ cycloaliphatic, or optionally substituted C₆-C₁₂ aryl.

[0006] In some embodiments, the present disclosure provides a method of treating a disease, disorder, or condition in a subject comprising administering to the subject a compound or composition described herein.

[0007] In some embodiments, the present disclosure provides a method of promoting autophagy in a subject comprising administering to the subject a compound or composition described herein. BRIEF DESCRPTION OF THE DRAWING

[0008] FIG. 1 is an illustration of forms of FIP200 dimers, including a dimer, trimer, and tetramer.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

[0009] Provided herein are compounds and compositions useful for the promotion of autophagy, which can be used for the treatment of a variety of diseases, disorders, and conditions. In particular, the present disclosure encompasses an insight, among other things, that small molecule compounds can be used to aggregate precursors to the generation of the autophagosome to thereby promote autophagy.

[0010] Accordingly, in some embodiments, the present disclosure provides a compound represented by formula I:

A¹-B-A²

I or a pharmaceutically acceptable salt thereof, wherein A¹ and A² are each moieties that bind to or associate with FIP200, and B is a linker moiety. Classes and subclasses of A¹, B, and A² are described herein.

Compounds and Definitions

[0011] Compounds of this disclosure include those described generally above and are further illustrated by the classes, subclasses, and species disclosed herein. As used herein, the following definitions shall apply unless otherwise indicated. For purposes of this disclosure, the chemical elements are identified in accordance with the Periodic Table of Elements, CAS version, Handbook of Chemistry and Physics, 75^th Ed. Additionally, general principles of organic chemistry are described in “Organic Chemistry”, Thomas Sorrell, University Science Books, Sausalito: 1999, and “March’s Advanced Organic Chemistry”, 5^th Ed., Ed.: Smith, M.B. and March, J., John Wiley & Sons, New York: 2001, the entire contents of which are hereby incorporated by reference. [0012] Unless otherwise stated, structures depicted herein are meant to include all stereoisomeric (e.g., enantiomeric or diastcrcomcric) forms of the structure, as well as all geometric or conformational isomeric forms of the structure. For example, the R and S configurations of each stereocenter are contemplated as part of the disclosure. Therefore, single stereochemical isomers, as well as enantiomeric, diastereomeric, and geometric (or conformational) mixtures of provided compounds are within the scope of the disclosure. For example, in some cases, Table 1 shows one or more stereoisomers of a compound, and unless otherwise indicated, represents each stereoisomer alone and/or as a mixture. Unless otherwise stated, all tautomeric forms of provided compounds are within the scope of the disclosure.

[0013] Unless otherwise indicated, structures depicted herein are meant to include compounds that differ only in the presence of one or more isotopically enriched atoms. For example, compounds having the present structures including replacement of hydrogen by deuterium or tritium, or replacement of a carbon by ¹³C- or ¹⁴C-enriched carbon are within the scope of this disclosure.

[0014] About or approximately: As used herein, the term "approximately" or "about," as applied to one or more values of interest, refers to a value that is similar to a stated reference value. In general, those skilled in the art, familial- within the context, will appreciate the relevant degree of variance encompassed by "about" or "approximately" in that context. For example, in some embodiments, the term "approximately" or "about" may encompass a range of values that are within (i.e., ±) 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of the referred value.

[0015] Administering: As used herein, the term "administering" or "administration" typically refers to the administration of a composition to a subject to achieve delivery of an agent that is, or is included in, a composition to a target site or a site to be treated. Those of ordinary skill in the art will be aware of a variety of routes that may, in appropriate circumstances, be utilized for administration to a subject, for example a human. For example, in some embodiments, administration may be ocular-, oral, parenteral, topical, etc. In some particular embodiments, administration may be bronchial (e.g., by bronchial instillation), buccal, dermal (which may be or comprise, for example, one or more of topical to the dermis, intradermal, interdermal, transdermal, etc.), enteral, intra-arterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, within a specific organ (e.g., intrahepatic), mucosal, nasal, oral, rectal, subcutaneous, sublingual, topical, tracheal (e.g., by intratracheal instillation), vaginal, vitreal, etc. In some embodiments, administration may be parenteral. In some embodiments, administration may be oral. In some particular embodiments, administration may be intravenous. In some particular embodiments, administration may be subcutaneous. In some embodiments, administration may involve only a single dose. In some embodiments, administration may involve application of a fixed number of doses. In some embodiments, administration may involve dosing that is intermittent (e.g., a plurality of doses separated in time) and/or periodic (e.g., individual doses separated by a common period of time) dosing. In some embodiments, administration may involve continuous dosing (e.g., perfusion) for at least a selected period of time. In some embodiments, administration may comprise a prime- and-boost protocol. A prime-and-boost protocol can include administration of a first dose of a pharmaceutical composition (e.g., an immunogenic composition, e.g., a vaccine) followed by, after an interval of time, administration of a second or subsequent dose of a pharmaceutical composition (e.g., an immunogenic composition, e.g., a vaccine). In the case of an immunogenic composition, a prime-and-boost protocol can result in an increased immune response in a patient.

[0016] Agent-. As used herein, the term “agent”, may refer to a compound, molecule, or entity of any chemical class including, for example, a small molecule, polypeptide, nucleic acid, saccharide, lipid, metal, or a combination or complex thereof. In some embodiments, the term “agent” may refer to a compound, molecule, or entity that comprises a polymer. In some embodiments, the term may refer to a compound or entity that comprises one or more polymeric moieties. In some embodiments, the term “agent” may refer to a compound, molecule, or entity that is substantially free of a particular polymer or polymeric moiety. In some embodiments, the term may refer to a compound, molecule, or entity that lacks or is substantially free of any polymer or polymeric moiety.

[0017] Aliphatic: The term “aliphatic” refers to a straight-chain (i.e., unbranched) or branched, substituted or unsubstituted hydrocarbon chain that is completely saturated or that contains one or more units of unsaturation, or a monocyclic hydrocarbon or bicyclic hydrocarbon that is completely saturated or that contains one or more units of unsaturation, but which is not aromatic (also referred to herein as “cycloaliphatic”), that has a single point or more than one points of attachment to the rest of the molecule. Unless otherwise specified, aliphatic groups contain 1-12 aliphatic carbon atoms. In some embodiments, aliphatic groups contain 1-6 aliphatic carbon atoms (e.g., C_1-6). In some embodiments, aliphatic groups contain 1 -5 aliphatic carbon atoms (e.g., C_1-5). In other embodiments, aliphatic groups contain 1-4 aliphatic carbon atoms (e.g., C_1-4). In still other embodiments, aliphatic groups contain 1-3 aliphatic carbon atoms (e.g., C1-3), and in yet other embodiments, aliphatic groups contain 1-2 aliphatic carbon atoms (e.g., C_1-2). Suitable aliphatic groups include, but are not limited to, linear or branched, substituted or unsubstituted alkyl, alkenyl, or alkynyl groups and hybrids thereof. A preferred aliphatic group is C_1-6 alkyl.

[0018] Alkyl: The term “alkyl”, used alone or as part of a larger moiety, refers to a saturated, optionally substituted straight or branched chain hydrocarbon group having (unless otherwise specified) 1-12, 1-10, 1-8, 1-6, 1-4, 1-3, or 1-2 carbon atoms (e.g., C_1-12, C1-10, C_1-8, C_1-6, C_1-4, C_1- ₃, or C_1-2). Exemplary alkyl groups include methyl, ethyl, propyl, butyl, pentyl, hexyl, and heptyl. [0019] Alkylene: The term “alkylene” is refers to a bivalent alkyl group. In some embodiments, “alkylene” is a bivalent straight or branched alkyl group. In some embodiments, an "alkylene chain" is a polymethylene group, i.e., -(CH₂)_n-, wherein n is a positive integer, e.g., from 1 to 6, from 1 to 4, from 1 to 3, from 1 to 2, or from 2 to 3. An optionally substituted alkylene chain is a polymethylene group in which one or more methylene hydrogen atoms is optionally replaced with a substituent. Suitable substituents include those described below for a substituted aliphatic group and also include those described in the specification herein. It will be appreciated that two substituents of the alkylene group may be taken together to form a ring system. In certain embodiments, two substituents can be taken together to form a 3- to 7-membered ring. The substituents can be on the same or different atoms. The suffix “-ene” or “-enyl” when appended to certain groups herein are intended to refer to a bifunctional moiety of said group. For example, “-ene” or “-enyl”, when appended to “cyclopropyl” becomes “cyclopropylene” or

“cyclopropylenyl” and is intended to refer to a bifunctional cyclopropyl group, e.g.,

[0020] Alkenyl: The term “alkenyl”, used alone or as part of a larger moiety, refers to an optionally substituted straight or branched chain or cyclic hydrocarbon group having at least one double bond and having (unless otherwise specified) 2-12, 2-10, 2-8, 2-6, 2-4, or 2-3 carbon atoms(e.g., C_2-12, C_2-10, C_2-8, C_2-6, C_2-4, or C_2-3). Exemplary alkenyl groups include ethenyl, propenyl, butenyl, pentenyl, hexenyl, and heptenyl. The term “cycloalkenyl” refers to an optionally substituted non-aromatic monocyclic or multicyclic ring system containing at least one carbon-carbon double bond and having about 3 to about 10 carbon atoms. Exemplary monocyclic cycloalkcnyl rings include cyclopcntcnyl, cyclohcxcnyl, and cyclohcptcnyl.

[0021] Alkynyl: The term “alkynyl”, used alone or as part of a larger moiety, refers to an optionally substituted straight or branched chain hydrocarbon group having at least one triple bond and having (unless otherwise specified) 2-12, 2-10, 2-8, 2-6, 2-4, or 2-3 carbon atoms (e.g., C_2-12, C_2-10, C_2-8, C_2-6, C_2-4, or C_2-3). Exemplary alkynyl groups include ethynyl, propynyl, butynyl, pentynyl, hexynyl, and heptynyl.

[0022] Aryl: The term “aryl” refers to monocyclic and bicyclic ring systems having a total of six to fourteen ring members (e.g., C₆-C₁₄), wherein at least one ring in the system is aromatic and wherein each ring in the system contains three to seven ring members. In some embodiments, an “aryl” group contains between six and twelve total ring members (e.g., C₆-C₁₂). The term “aryl” may be used interchangeably with the term “aryl ring”. In certain embodiments, “aryl” refers to an aromatic ring system which includes, but not limited to, phenyl, biphenyl, naphthyl, anthracyl and the like, which may bear one or more substituents. Unless otherwise specified, “aryl” groups are hydrocarbons. In some embodiments, an “aryl” ring system is an aromatic ring (e.g., phenyl) that is fused to a non-aromatic ring (e.g., cycloalkyl). Examples of aryl rings include that are fused include

[0023] Biological sample: As used herein, the term “biological sample” typically refers to a sample obtained or derived from a biological source (c.g., a tissue or organism or cell culture) of interest, as described herein. In some embodiments, a source of interest comprises an organism, such as an animal or human. In some embodiments, a biological sample is or comprises biological tissue or fluid. In some embodiments, a biological sample may be or comprise bone marrow; blood; blood cells; ascites; tissue or fine needle biopsy samples; cell-containing body fluids; free floating nucleic acids; sputum; saliva; urine; cerebrospinal fluid, peritoneal fluid; pleural fluid; feces; lymph; gynecological fluids; skin swabs; vaginal swabs; oral swabs; nasal swabs; washings or lavages such as a ductal lavages or broncheoalveolar lavages; aspirates; scrapings; bone marrow specimens; tissue biopsy specimens; surgical specimens; feces, other body fluids, secretions, and/or excretions; and/or cells therefrom, etc. In some embodiments, a biological sample is or comprises cells obtained from an individual. In some embodiments, obtained cells are or include cells from an individual from whom the sample is obtained. In some embodiments, a sample is a “primary sample” obtained directly from a source of interest by any appropriate means. For example, in some embodiments, a primary biological sample is obtained by methods selected from the group consisting of biopsy (e.g., fine needle aspiration or tissue biopsy), surgery, collection of body fluid (e.g., blood, lymph, feces etc.), etc. In some embodiments, as will be clear from context, the term “sample” refers to a preparation that is obtained by processing (e.g., by removing one or more components of and/or by adding one or more agents to) a primary sample. For example, filtering using a semi-permeable membrane. Such a “processed sample” may comprise, for example, nucleic acids or proteins extracted from a sample or obtained by subjecting a primary sample to techniques such as amplification or reverse transcription of mRNA, isolation and/or purification of certain components, etc.

[0024] Bridged bicyclic: As used herein, the term “bridged bicyclic” refers to any bicyclic ring system, i.e. carbocyclic or heterocyclic, saturated or partially unsaturated, having at least one bridge. As defined by IUPAC, a “bridge” is an unbranched chain of atoms or an atom or a valence bond connecting two bridgeheads, where a “bridgehead” is any skeletal atom of the ring system which is bonded to three or more skeletal atoms (excluding hydrogen). In some embodiments, a bridged bicyclic group has 7-12 ring members and 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. Such bridged bicyclic groups are well known in the art and include those groups set forth below where each group is attached to the rest of the molecule at any substitutable carbon or nitrogen atom. Unless otherwise specified, a bridged bicyclic group is optionally substituted with one or more substituents as set forth for aliphatic groups. Additionally or alternatively, any substitutable nitrogen of a bridged bicyclic group is optionally substituted. Exemplary bridged bicyclics include:

[0025] Carrier: As used herein, the term “carrier” refers to a diluent, adjuvant, excipient, or vehicle with which a composition is administered. In some exemplary embodiments, carriers can include sterile liquids, such as, for example, water and oils, including oils of petroleum, animal, vegetable or synthetic origin, such as, for example, peanut oil, soybean oil, mineral oil, sesame oil and the like. In some embodiments, carriers are or include one or more solid components.

[0026] Combination therapy: As used herein, the term “combination therapy” refers to those situations in which a subject is simultaneously exposed to two or more therapeutic regimens (e.g., two or more therapeutic agents or modality (ies)). In some embodiments, the two or more regimens may be administered simultaneously; in some embodiments, such regimens may be administered sequentially (e.g., all “doses” of a first regimen are administered prior to administration of any doses of a second regimen); in some embodiments, such agents are administered in overlapping dosing regimens. In some embodiments, “administration” of combination therapy may involve administration of one or more agent(s) or modality(ies) to a subject receiving the other agent(s) or modality(ies) in the combination. For clarity, combination therapy does not require that individual agents be administered together in a single composition (or even necessarily at the same time), although in some embodiments, two or more agents, or active moieties thereof, may be administered together in a combination composition, or even in a combination compound (e.g., as part of a single chemical complex or covalent entity).

[0027] Composition: Those skilled in the art will appreciate that the term “composition” may be used to refer to a discrete physical entity that comprises one or more specified components. In general, unless otherwise specified, a composition may be of any form - e.g., gas, gel, liquid, solid, etc. [0028] Cycloaliphatic. As used herein, the term “cycloaliphatic” refers to a monocyclic C_3-8 hydrocarbon or a bicyclic C_6-10 hydrocarbon that is completely saturated or that contains one or more units of unsaturation, but which is not aromatic, that has a single point or more than one points of attachment to the rest of the molecule.

[0029] Cycloalkyl-. As used herein, the term “cycloalkyl” refers to an optionally substituted saturated ring monocyclic or polycyclic system of about 3 to about 10 ring carbon atoms. Exemplary monocyclic cycloalkyl rings include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl.

[0030] Dosage form or unit dosage form: Those skilled in the art will appreciate that the term “dosage form” may be used to refer to a physically discrete unit of an active agent (e.g., a therapeutic or diagnostic agent) for administration to a subject. Typically, each such unit contains a predetermined quantity of active agent. In some embodiments, such quantity is a unit dosage amount (or a whole fraction thereof) appropriate for administration in accordance with a dosing regimen that has been determined to correlate with a desired or beneficial outcome when administered to a relevant population (i.e., with a therapeutic dosing regimen).

[0031] Dosing regimen or therapeutic regimen: Those skilled in the ait will appreciate that the terms “dosing regimen” and “therapeutic regimen” may be used to refer to a set of unit doses (typically more than one) that are administered individually to a subject, typically separated by periods of time. In some embodiments, a given therapeutic agent has a recommended dosing regimen, which may involve one or more doses. In some embodiments, a dosing regimen comprises a plurality of doses each of which is separated in time from other doses. In some embodiments, individual doses are separated from one another by a time period of the same length; in some embodiments, a dosing regimen comprises a plurality of doses and at least two different time periods separating individual doses. In some embodiments, all doses within a dosing regimen are of the same unit dose amount. In some embodiments, different doses within a dosing regimen are of different amounts. In some embodiments, a dosing regimen comprises a first dose in a first dose amount, followed by one or more additional doses in a second dose amount different from the first dose amount. In some embodiments, a dosing regimen comprises a first dose in a first dose amount, followed by one or more additional doses in a second dose amount same as the first dose amount. In some embodiments, a dosing regimen is correlated with a desired or beneficial outcome when administered across a relevant population (i.e., is a therapeutic dosing regimen). [0032] Excipient: As used herein, the term “excipient” refers to a non -therapeutic agent that may be included in a pharmaceutical composition, for example, to provide or contribute to a desired consistency or stabilizing effect. Suitable pharmaceutical excipients include, for example, starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.

[0033] Halogen: The term “halogen” means F, Cl, Br, or I.

[0034] Heteroaliphatic : The term “heteroaliphatic” or “heteroaliphatic group”, as used herein, denotes an optionally substituted hydrocarbon moiety having, in addition to carbon atoms, from one to five heteroatoms, that may be straight-chain (i.e., unbranched), branched, or cyclic (“heterocyclic”) and may be completely saturated or may contain one or more units of unsaturation, but which is not aromatic. The term “heteroatom” refers to nitrogen, oxygen, or sulfur, and includes any oxidized form of nitrogen or sulfur, and any quatemized form of a basic nitrogen. The term “nitrogen” also includes a substituted nitrogen. Unless otherwise specified, heteroaliphatic groups contain 1-10 carbon atoms wherein 1-3 carbon atoms are optionally and independently replaced with heteroatoms selected from oxygen, nitrogen, and sulfur. In some embodiments, heteroaliphatic groups contain 1-4 carbon atoms, wherein 1-2 carbon atoms are optionally and independently replaced with heteroatoms selected from oxygen, nitrogen, and sulfur. In yet other embodiments, heteroaliphatic groups contain 1-3 carbon atoms, wherein 1 carbon atom is optionally and independently replaced with a heteroatom selected from oxygen, nitrogen, and sulfur. Suitable heteroaliphatic groups include, but are not limited to, linear or branched, heteroalkyl, heteroalkenyl, and heteroalkynyl groups. For example, a 1- to 10 atom heteroaliphatic group includes the following exemplary groups: -O-CH₃, -CH₂-O-CH₃, -O-CH₂- CH₂-O-CH₂-CH₂-O-CH₃, and the like.

[0035] Heteroaryl: The terms “heteroaryl” and “heteroar-”, used alone or as part of a larger moiety, e.g., “heteroaralkyl”, or “heteroaralkoxy”, refer to monocyclic or bicyclic ring groups having 5 to 10 ring atoms (e.g., 5- to 6-membered monocyclic heteroaryl or 9- to 10-membered bicyclic heteroaryl); having 6, 10, or 14 π -electrons shared in a cyclic array; and having, in addition to carbon atoms, from one to five heteroatoms. Heteroaryl groups include, without limitation, thienyl, furanyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, indolizinyl, purinyl, naphthyridinyl, pteridinyl, imidazo[l ,2-a]pyrimidinyl, imidazo[l ,2-a]pyridyl, imidazo[4,5-b]pyridyl, imidazo[4,5-c]pyridyl, pyn’olopyridyl, pyrrolopyrazinyl, thienopyrimidinyl, triazolopyridyl, and benzoisoxazolyl. The terms “heteroaryl” and “heteroar-”, as used herein, also include groups in which a heteroaromatic ring is fused to one or more aryl, cycloaliphatic, or heterocyclyl rings, where the radical or point of attachment is on the heteroaromatic ring (i.e., a bicyclic heteroaryl ring having 1 to 3 heteroatoms). Nonlimiting examples include indolyl, isoindolyl, benzothienyl, benzofuranyl, dibenzofuranyl, indazolyl, benzimidazolyl, benzotriazolyl, benzothiazolyl, benzothiadiazolyl, benzoxazolyl, quinolyl, isoquinolyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, 4//-quinolizinyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, pyrido[2,3-b]-l,4-oxazin-3(4H)-one, 4H-thieno[3,2-b]pyrrole, and benzoisoxazolyl. The term “heteroaryl” may be used interchangeably with the terms “heteroaryl ring”, “heteroaryl group”, or “heteroaromatic”, any of which terms include rings that are optionally substituted.

[0036] Heteroatom: The term “heteroatom” as used herein refers to nitrogen, oxygen, or sulfur, and includes any oxidized form of nitrogen or sulfur, and any quatemized form of a basic nitrogen.

[0037] Heterocycle: As used herein, the terms “heterocycle”, “heterocyclyl”, “heterocyclic radical”, and “heterocyclic ring” are used interchangeably and refer to a stable 3- to 8-membered monocyclic, a 6- to 10-membered bicyclic, or a 10- to 16-membered polycyclic heterocyclic moiety that is either saturated or partially unsaturated, and having, in addition to carbon atoms, one or more, such as one to four, heteroatoms, as defined above. When used in reference to a ring atom of a heterocycle, the term "nitrogen" includes a substituted nitrogen. As an example, in a saturated or partially unsaturated ring having 0-3 heteroatoms selected from oxygen, sulfur or nitrogen, the nitrogen may be N (as in 3,4-dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl), or NR⁺ (as in N-substituted pyrrolidinyl). A heterocyclic ring can be attached to its pendant group at any heteroatom or carbon atom that results in a stable structure and any of the ring atoms can be optionally substituted. Examples of such saturated or partially unsaturated heterocyclic radicals include, without limitation, azetidinyl, oxetanyl, tetrahydrofuranyl, tetrahydrothienyl, pyrrolidinyl, piperidinyl, decahydroquinolinyl, oxazolidinyl, piperazinyl, dioxanyl, dioxolanyl, diazepinyl, oxazepinyl, thiazepinyl, morpholinyl, and thiamorpholinyl. A heterocyclyl group may be mono-, bi-, tri-, or polycyclic, preferably mono-, bi-, or tricyclic, more preferably mono- or bicyclic. A bicyclic heterocyclic ring also includes groups in which the heterocyclic ring is fused to one or more aryl rings. Exemplary bicyclic heterocyclic groups include indolinyl, isoindolinyl, benzodioxo lyl, 1,3-dihydroisobenzofuranyl, 2,3-dihydrobenzofuranyl, and tetrahydroquinolinyl. A bicyclic heterocyclic ring can also be a spirocyclic ring system (e.g., 7- to 11-membered spirocyclic fused heterocyclic ring having, in addition to carbon atoms, one or more heteroatoms as defined above (e.g., one, two, three or four heteroatoms)). A bicyclic heterocyclic ring can also be a bridged ring system (e.g., 7- to 11-membered bridged heterocyclic ring having one, two, or three bridging atoms).

[0038] Oral: The phrases “oral administration” and “administered orally” as used herein have their art-understood meaning referring to administration by mouth of a compound or composition. [0039] Parenteral: The phrases “parenteral administration” and “administered parenterally” as used herein have their art-understood meaning referring to modes of administration other than enteral and topical administration, usually by injection, and include, without limitation, intravenous, intramuscular, intra-arterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticulare, subcapsular, subarachnoid, intraspinal, and intrasternal injection and infusion.

[0040] Partially unsaturated: As used herein, the term “partially unsaturated” refers to a ring moiety that includes at least one double or triple bond between ring atoms. The term “partially unsaturated” is intended to encompass rings having multiple sites of unsaturation, but is not intended to include aromatic (e.g., aryl or heteroaryl) moieties, as herein defined.

[0041] Patient or subject: As used herein, the term “patient” or “subject” refers to any organism to which a provided composition is or may be administered, e.g., for experimental, diagnostic, prophylactic, cosmetic, and/or therapeutic purposes. Typical patients or subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and/or humans). In some embodiments, a patient is a human. In some embodiments, a patient or a subject is suffering from or susceptible to one or more disorders or conditions. In some embodiments, a patient or subject displays one or more symptoms of a disorder or condition. In some embodiments, a patient or subject has been diagnosed with one or more disorders or conditions. In some embodiments, a patient or a subject is receiving or has received certain therapy to diagnose and/or to treat a disease, disorder, or condition. [0042] Pharmaceutical composition: As used herein, the term “pharmaceutical composition” refers to an active agent, formulated together with one or more pharmaceutically acceptable carriers. In some embodiments, the active agent is present in unit dose amount appropriate for administration in a therapeutic or dosing regimen that shows a statistically significant probability of achieving a predetermined therapeutic effect when administered to a relevant population. In some embodiments, pharmaceutical compositions may be specially formulated for administration in solid or liquid form, including those adapted for the following: oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, e.g., those targeted for buccal, sublingual, and systemic absorption, boluses, powders, granules, pastes for application to the tongue; parenteral administration, for example, by subcutaneous, intramuscular, intravenous or epidural injection as, for example, a sterile solution or suspension, or sustained-release formulation; topical application, for example, as a cream, ointment, or a controlled-release patch or spray applied to the skin, lungs, or oral cavity; intravaginally or intrarectally, for example, as a pessary, cream, or foam; sublingually; ocularly; transdermally; or nasally, pulmonary, and to other mucosal surfaces.

[0043] Pharmaceutically acceptable: As used herein, the phrase “pharmaceutically acceptable” refers to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

[0044] Pharmaceutically acceptable salt: The term “pharmaceutically acceptable salt”, as used herein, refers to salts of such compounds that are appropriate for use in pharmaceutical contexts, i.e., salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, S. M. Berge, et al. describes pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 66: 1-19 (1977).

[0045] Prevent or prevention: As used herein, the terms “prevent” or “prevention”, when used in connection with the occurrence of a disease, disorder, and/or condition, refer to reducing the risk of developing the disease, disorder and/or condition and/or to delaying onset of one or more characteristics or symptoms of the disease, disorder or condition. Prevention may be considered complete when onset of a disease, disorder or condition has been delayed for a predefined period of time.

[0046] Substituted or optionally substituted: As described herein, compounds of the invention may contain “optionally substituted” moieties. In general, the term “substituted,” whether preceded by the term “optionally” or not, means that one or more hydrogens of the designated moiety are replaced with a suitable substituent. “Substituted” applies to one or more hydrogens that are either explicit or implicit from the structure (e.g.,

refers to at least

Unless otherwise indicated, an “optionally substituted” group may have a suitable

substituent at each substitutable position of the group, and when more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituent may be either the same or different at every position. Combinations of substituents envisioned by this invention are preferably those that result in the formation of stable or chemically feasible compounds. The term “stable,” as used herein, refers to compounds that are not substantially altered when subjected to conditions to allow for their production, detection, and, in certain embodiments, their recovery, purification, and use for one or more of the purposes provided herein. Groups described as being “substituted” preferably have between 1 and 4 substituents, more preferably 1 or 2 substituents. Groups described as being “optionally substituted” may be unsubstituted or be “substituted” as described above.

[0047] Suitable monovalent substituents on a substitutable carbon atom of an “optionally substituted” group are independently halogen; -(CH₂)_{0- 4}R°; -(CH₂)_0-4OR°; -O(CH₂)_0-4R°, -O- (CH₂)_0-4C(O)OR°; -(CH₂)_0-4CH(OR°)₂; -(CH₂)_0-4SR°; -(CH₂)_0-4Ph. which may be substituted with R°; -(CH₂)_0-4O(CH₂)_0-1Ph which may be substituted with R°; -CH=CHPh, which may be substituted with R°; -(CH₂)_0-4O(CH₂)_0-1 -pyridyl which may be substituted with R°; -NO₂; -CN; -N₃; -(CH₂)_0-4N(R°)₂; -(CH₂)_0-4N(R°)C(0)R°; -N(R°)C(S)R°; -(CH₂)O- ₄N(R°)C(O)NR°₂; -N(R°)C(S)NR°₂; -(CH₂)_0-4N(R°)C(O)OR°;

N(R°)N(R°)C(O)R°; -N(R°)N(R°)C(O)NR°₂; -N(R°)N(R°)C(O)OR°; -(CH₂)_0-4C(O)R°; C(S)R°; -(CH₂)_0-4C(O)OR°; -(CH₂)_0-4C(O)SR°; -(CH₂)(wC(O)OSiR°₃; -(CH₂)_{0 4}OC(O)R°; -

OC(0)(CH₂)OMSR°; -(CH₂)_0-4SC(O)R°; -(CH₂)_0-4C(O)NR°₂; -C(S)NR°₂; -C(S)SR°; - SC(S)SR°, -(CH₂)Q ₄OC(O)NR°₂; -C(O)N(OR°)R°; -C(O)C(O)R°; -C(O)CH₂C(O)R° ; - C(NOR°)R°; -(CH₂)_0-4SSR°; -(CH₂)OMS(0)₂R°; -(CH₂)_0-4S(O)₂OR°: -(CH₂)_0-4OS(O)₂R°; - S(O)₂NR°₂; -(CH₂)_{O 4}S(O)R°; -N(R°)S(O)₂NR°₂; -N(R°)S(O)₂R°; -N(OR°)R°; -C(NH)NR°₂; - P(O)₂R°; -P(O)R°₂; -OP(O)R°₂; -OP(O)(OR°)₂; SiR°₃; - (CIM straight or branched alkylene)O- N(R°)₂; or -(C_1-4 straight or branched alkylene)C(O)O-N(R°)₂, wherein each R° may be substituted as defined below and is independently hydrogen, Ci-6 aliphatic, -CH₂Ph, -0(CH₂)o~ ₁Ph, -CH₂-(5- to 6-membered heteroaryl ring), or a 3- to 6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or, notwithstanding the definition above, two independent occurrences of R°, taken together with their intervening atom(s), form a 3- to 12-membered saturated, partially unsaturated, or aryl mono- or bicyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, which may be substituted as defined below.

[0048] Suitable monovalent substituents on R° (or the ring formed by taking two independent occurrences of R° together with their intervening atoms), are independently halogen, -(CH₂)o-₂R^●, -(haloR^●), -(CH₂)O ₂OH, -(CH₂)O-₂OR^●, -(CH₂)O ₂CH(OR^●)₂, -O(haloR’), -CN, -N₃, -(CH₂)o- ₂C(O)R^●, -(CH₂)_{O 2}C(O)OH, -(CH₂)_{O 2}C(O)OR^●, -(CH₂)O_₂SR\ -(CH₂)O- ₂SH. -(CH₂)O ₂NH₂, - (CH₂)O ₂NHR^● -(CH₂)_0-2 NR^●2, -NO₂, -SiR^● ₃, -OSiR^● ₃, -C(O)SR^●. -(C_1-4 straight or branched alkylene)C(O)OR“, or -SSR^● wherein each R^● is unsubstituted or where preceded by “halo” is substituted only with one or more halogens, and is independently selected from CIM aliphatic, - CH₂Ph, -0(CH₂)_0-1Ph, or a 3- to 6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. Suitable divalent substituents on a saturated carbon atom of R° include =0 and =S.

[0049] Suitable divalent substituents on a saturated carbon atom of an “optionally substituted” group include the following: =0 (“oxo”), =S, =NNR*₂, =NNHC(O)R* =NNHC(O)OR*, =NNHS(O)₂R*, =NR*, =NOR*, -O(C(R*₂))_2-3O-, or -S(C(R*₂))_2-3S-, wherein each independent occurrence of R is selected from hydrogen, C_1-6 aliphatic which may be substituted as defined below, or an unsubstituted 5- to 6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. Suitable divalent substituents that are bound to vicinal substitutable carbons of an “optionally substituted” group include: -O(CR ₂)_{2 3}O-, wherein each independent occurrence of R is selected from hydrogen, C_1-6 aliphatic which may be substituted as defined below, or an unsubstituted 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0050] Suitable substituents on the aliphatic group of R* include halogen, -R^●, -(haloR^●), -OH, -OR^●, -O(haloR’), -CN, -C(O)OH, -C(O)OR^●, -NH₂, -NHR^●, -NR^● ₂, or -NO₂, wherein each R^● is unsubstituted or where preceded by “halo” is substituted only with one or more halogens, and is independently CIM aliphatic, -CH₂Ph, -0(CH₂)o iPh, or a 3- to 6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0051] Suitable substituents on a substitutable nitrogen of an “optionally substituted” group include -R', -NR'₂, -C(O)R^f, -C(O)OR^f, -C(O)C(O)R^f,

C(O)CH₂C(O)R^t, -S(O)₂R^t, -S(O)₂NR^t ₂, -C(S)NR^f ₂, -C(NH)NR^f ₂, or -N(R⁺)S(O)₂R⁺; wherein each R¹ is independently hydrogen, C_1-6 aliphatic which may be substituted as defined below, unsubstituted -OPh, or an unsubstituted 3- to 6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or, notwithstanding the definition above, two independent occurrences of R^f, taken together with their intervening atom(s) form an unsubstituted 3- to 12-membered saturated, partially unsaturated, or aryl mono- or bicyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0052] Suitable substituents on the aliphatic group of R are independently halogen, - R^●, -(haloR^●), -OH, -OR^●, -O(haloR^●), -CN, -C(O)OH, -C(O)OR^●, -NH₂, -NHR^●, -NR^● ₂, or -NO₂, wherein each R^● is unsubstituted or where preceded by “halo” is substituted only with one or more halogens, and is independently C_1-4 aliphatic, -CH₂Ph, -0(CH₂)o-iPh, or a 3- to 6- membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0053] Small molecule: As used herein, the term “small molecule” means a low molecular weight organic and/or inorganic compound. In general, a “small molecule” is a molecule that is less than about 5 kilodaltons (kD) in size. In some embodiments, a small molecule is less than about 4 kD, 3 kD, about 2 kD, or about 1 kD. In some embodiments, the small molecule is less than about 800 daltons (D), about 600 D, about 500 D, about 400 D, about 300 D, about 200 D, or about 100 D. In some embodiments, a small molecule is less than about 2000 g/mol, less than about 1500 g/mol, less than about 1000 g/mol, less than about 800 g/mol, or less than about 500 g/mol. In some embodiments, a small molecule is not a polymer.

[0054] In some embodiments, a small molecule does not include a polymeric moiety. In some embodiments, a small molecule is not and/or does not comprise a protein or polypeptide (e.g., is not an oligopeptide or peptide). In some embodiments, a small molecule is not and/or does not comprise a polynucleotide (e.g., is not an oligonucleotide). In some embodiments, a small molecule is not and/or does not comprise a polysaccharide; for example, in some embodiments, a small molecule is not a glycoprotein, proteoglycan, glycolipid, etc.). In some embodiments, a small molecule is not a lipid.

[0055] In some embodiments, a small molecule is a modulating agent (e.g., is an inhibiting agent or an activating agent). In some embodiments, a small molecule is biologically active. In some embodiments, a small molecule is detectable (e.g., comprises at least one detectable moiety). In some embodiments, a small molecule is a therapeutic agent.

[0056] Those of ordinary skill in the art, reading the present disclosure, will appreciate that certain small molecule compounds described herein may be provided and/or utilized in any of a variety of forms such as, for example, crystal forms (e.g., polymorphs, solvates, etc), salt forms, protected forms, pro-drug forms, ester forms, isomeric forms (e.g., optical and/or structural isomers), isotopic forms, etc.

[0057] Those of ordinary skill in the art will appreciate that certain small molecule compounds have structures that can exist in one or more steroisomeric forms. In some embodiments, such a small molecule may be utilized in accordance with the present disclosure in the form of an individual enantiomer, diastereomer or geometric isomer, or may be in the form of a mixture of stereoisomers; in some embodiments, such a small molecule may be utilized in accordance with the present disclosure in a racemic mixture form.

[0058] Those of skill in the art will appreciate that certain small molecule compounds have structures that can exist in one or more tautomeric forms. In some embodiments, such a small molecule may be utilized in accordance with the present disclosure in the form of an individual tautomer, or in a form that intcrconvcrts between tautomeric forms.

[0059] Those of skill in the art will appreciate that certain small molecule compounds have structures that permit isotopic substitution (e.g., ²H or ³H for H; ¹¹C, ¹³C or ¹⁴C for ¹²C; ¹³N or ¹⁵N for ¹⁴N; ¹⁷O or ¹⁸O for ¹⁶O; ³⁶C1 for ³⁵C1 or ³⁷C1; ¹⁸F for ¹⁹F; ¹³¹I for ¹²⁷I; etc.). In some embodiments, such a small molecule may be utilized in accordance with the present disclosure in one or more isotopically modified forms, or mixtures thereof.

[0060] In some embodiments, reference to a particular small molecule compound may relate to a specific form of that compound. In some embodiments, a particular small molecule compound may be provided and/or utilized in a salt form (e.g., in an acid-addition or base-addition salt form, depending on the compound); in some such embodiments, the salt form may be a pharmaceutically acceptable salt form.

[0061] In some embodiments, where a small molecule compound is one that exists or is found in nature, that compound may be provided and/or utilized in accordance in the present disclosure in a form different from that in which it exists or is found in nature. Those of ordinary skill in the ail will appreciate that, in some embodiments, a preparation of a particular small molecule compound that contains an absolute or relative amount of the compound, or of a particular form thereof, that is different from the absolute or relative (with respect to another component of the preparation including, for example, another form of the compound) amount of the compound or form that is present in a reference preparation of interest (e.g., in a primary sample from a source of interest such as a biological or environmental source) is distinct from the compound as it exists in the reference preparation or source. Thus, in some embodiments, for example, a preparation of a single stereoisomer of a small molecule compound may be considered to be a different form of the compound than a racemic mixture of the compound; a particular salt of a small molecule compound may be considered to be a different form from another salt form of the compound; a preparation that contains only a form of the compound that contains one conformational isomer ((Z) or (E)) of a double bond may be considered to be a different form of the compound from one that contains the other conformational isomer ((E) or (Z)) of the double bond; a preparation in which one or more atoms is a different isotope than is present in a reference preparation may be considered to be a different form; etc. [0062] Those skilled in the art will further appreciate that, in small molecule structures, the symbol as used herein, refers to a point of attachment between two atoms. Additionally or alternatively, the symbol

refers to a point of attachment ring in a spirocyclic manner.

[0063] Those skilled in the art will appreciate that, in small molecule structures, the symbol as used herein, refers to a bond that is either a single bond, e.g., — , or a double bond, e.g., For example, when used in a cyclic structure, such as:

it is understood to encompass any of the following chemically feasible structures:

[0064] Therapeutic agent: As used herein, the term “therapeutic agent” in general refers to any agent that elicits a desired effect e.g., a desired biological, clinical, or pharmacological effect) when administered to a subject. In some embodiments, an agent is considered to be a therapeutic agent if it demonstrates a statistically significant effect across an appropriate population. In some embodiments, an appropriate population is a population of subjects suffering from and/or susceptible to a disease, disorder or condition. In some embodiments, an appropriate population is a population of model organisms. In some embodiments, an appropriate population may be defined by one or more criterion such as age group, gender, genetic background, preexisting clinical conditions, prior exposure to therapy. In some embodiments, a therapeutic agent is a substance that alleviates, ameliorates, relieves, inhibits, prevents, delays onset of, reduces severity of, and/or reduces incidence of one or more symptoms or features of a disease, disorder, and/or condition in a subject when administered to the subject in an effective amount. In some embodiments, a “therapeutic agent” is an agent that has been or is required to be approved by a government agency before it can be marketed for administration to humans. In some embodiments, a “therapeutic agent” is an agent for which a medical prescription is required for administration to humans. In some embodiments, therapeutic agents may be KAT inhibitors, for example, KAT-5 inhibitors, as described herein.

[0065] Therapeutically effective amount: As used herein, the term “therapeutically effective amount” refers to an amount that produces a desired effect (e.g., a desired biological, clinical, or pharmacological effect) in a subject or population to which it is administered. In some embodiments, the term refers to an amount statistically likely to achieve the desired effect when administered to a subject in accordance with a particular dosing regimen (e.g., a therapeutic dosing regimen). In some embodiments, the term refers to an amount sufficient to produce the effect in at least a significant percentage (e.g., at least about 25%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or more) of a population that is suffering from and/or susceptible to a disease, disorder, and/or condition. In some embodiments, a therapeutically effective amount is one that reduces the incidence and/or severity of, and/or delays onset of, one or more symptoms of the disease, disorder, and/or condition. Those of ordinary skill in the ail will appreciate that the term “therapeutically effective amount” does not in fact require successful treatment be achieved in a particular individual. Rather, a therapeutically effective amount may be an amount that provides a particular desired response in a significant number of subjects when administered to patients in need of such treatment, e.g., in at least about 25%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or more patients within a treated patient population. In some embodiments, reference to a therapeutically effective amount may be a reference to an amount sufficient to induce a desired effect as measured in one or more specific tissues (e.g., a tissue affected by the disease, disorder or condition) or fluids (e.g., blood, saliva, serum, sweat, tears, urine). Those of ordinary skill in the art will appreciate that, in some embodiments, a therapeutically effective amount of a particular agent or therapy may be formulated and/or administered in a single dose. In some embodiments, a therapeutically effective agent may be formulated and/or administered in a plurality of doses, for example, as part of a dosing regimen.

[0066] Treat: As used herein, the terms “treat,” “treatment,” or “treating” refer to any method used to partially or completely alleviate, ameliorate, relieve, inhibit, prevent, delay onset of, reduce severity of, and/or reduce incidence of one or more symptoms or features of a disease, disorder, and/or condition. Treatment may be administered to a subject who does not exhibit signs of a disease, disorder, and/or condition. In some embodiments, treatment may be administered to a subject who exhibits only early signs of the disease, disorder, and/or condition, for example, for the purpose of decreasing the risk of developing pathology associated with the disease, disorder, and/or condition. Bifunctional Binders of FIP200

[0067] The present disclosure provides, among other things, bifunctional compounds that arc binders of FIP200. The present disclosure encompasses, among other things, the insight that bifunctional compounds, such as those described herein, can bind to multiple FIP200 proteins, thereby promoting formation of the autophagosome, and autophagy generally.

[0068] In some embodiments, the present disclosure provides a compound represented by formula I:

or a pharmaceutically acceptable salt thereof, wherein:

A¹ and A² are each moieties that bind to or associate with FIP200; and

B is a linker moiety.

[0069] In some embodiments, a moiety that binds or associates with FIP200 is one that covalently binds directly to FIP200 or one that undergoes one or more of hydrogen bonding, electrostatic interactions, pi stacking, van der Waals interactions, or dipole-dipole interactions with FIP200.

[0070] In some embodiments of formula I, A¹ and A² are each independently selected from a moiety of formula II- 1, II-2, II-3, or II-4:

or a pharmaceutically acceptable salt thereof, wherein: each G¹ is independently an optionally substituted C₆-C₁₂ aryl or an optionally substituted 5- to 6-mcmbcrcd heteroaryl; each X¹ is independently S, N(R³), O, optionally substituted C₁-C₆ aliphatic, or optionally substituted C₃-C₆ cycloaliphatic; each X² is independently C(R³) or N, provided that, when X² is N, then X¹ is optionally substituted C₁-C₆ aliphatic, or optionally substituted C₃-C₆ cycloaliphatic; when a bond between X³ and X⁴ is a single bond, then X³ is N(R^2a), and X⁴ is C(O) when a bond between X³ and X⁴ is a double bond, then X³ is C(R^2b) and X⁴ is C(R³), or N; each R¹ is independently selected from halogen, optionally substituted C₁-C₆ aliphatic, and optionally substituted -O-C₁-C₆ aliphatic; or two instances of R¹ come together with the atoms to which they are attached to form a n optionally substituted C₆-C₁₂ aryl ring, an optionally substituted C₄-C₆ cycloaliphatic ring, a 5- to 6- membered heteroaryl ring comprising 1 to 3 heteroatoms selected from N, O, and S, or an optionally substituted 5- to 6-membered heterocyclic ring comprising 1 to 3 heteroatoms selected from N, O, and S; each R^{2 1} is independently optionally substituted C₁-C₆ aliphatic,; each R^2b is independently hydrogen, optionally substituted C₁-C₆ aliphatic, optionally substituted -N(R³)-C₁-C₆ aliphatic, or optionally substituted -O-C₁-C₆ aliphatic; each R³ is independently selected from hydrogen, halogen, and optionally substituted Ci- C₆ aliphatic; each R⁴ is independently selected from optionally substituted C₆-C₁₂ aryl, optionally substituted 5- to 12- membered heteroaryl ring comprising 1 to 3 heteroatoms selected from N, O, and S, optionally substituted 4- to 12-membered heterocyclyl comprising 1 to 3 heteroatoms selected from N, O, and S, and optionally substituted C₁-C₆ aliphatic; each R⁵ is independently an optionally substituted 4- to 6-membered heterocyclic ring, an optionally substituted 5- to 6-membered heteroaryl ring, an optionally C₃-C₆ cycloaliphatic ring, an optionally substituted C₆-C₁₂ aryl, or an optionally substituted C₁-C₆ aliphatic; each L¹ is independently a bond, -C(O)-, -S(O)-, -S(O)₂-, or -NR³-; each n is independently 0, 1 , 2, 3, 4, 5, or 6; each X⁵, X⁶, and X⁷ arc independently selected from the group consisting of N and CH; each R⁷ is independently hydrogen, an optionally substituted -O-C₁-C₆ aliphatic, - S(O)₂R³, optionally substituted C₁-C₆ aliphatic, an optionally substituted 4- to 12- membered heterocycle ring comprising 1 to 4 heteroatoms selected from N, O, and S, an optionally substituted C₆-C₁₂ aryl, or an optionally substituted 5- to 12-membered heteroaryl comprising 1 to 4 heteroatoms selected from N, O, and S each G² is independently an optionally substituted C₆-C₁₂ aryl, optionally substituted 4- to 12-membered heterocycle comprising 1 to 4 heteroatoms selected from N, O, and S, optionally substituted 5- to 6-membered heteroaryl comprising 1 to 4 heteroatoms selected from N, O, and S, or optionally substituted C₃-C₆ cycloaliphatic; each G³ is independently an optionally substituted 4- to 12- membered heterocycle ring comprising 1 to 4 heteroatoms selected from N, O, and S, an optionally substituted 5- to 6-membered heteroaryl ring comprising 1 to 4 heteroatoms selected from N, O, and S, an optionally substituted C₃-C₆ cycloaliphatic ring or optionally substituted C₆-C₁₂ aryl; each L² is independently a bond, -NR³-C(O)-, -C(O)-NR³-, optionally substituted C₁-C₆ aliphatic, optionally substituted 4- to 6-membered heterocyclic, or optionally substituted C₃-C₆ cycloaliphatic; each L³ is independently -NR³-, -O-, -C(O)-, -NR³-C(O)-, -NR³-S(O)₂-, -C(O)-NR³-, - S(O)₂ NR³-, -NR³-C(O)-NR³-, optionally substituted C₁-C₆ aliphatic, or an optionally substituted C₃-C₆ cycloaliphatic ring;

* represents a point of attachment to moiety B ; and wherein: when A¹ or A² is a moiety of formula II- 3 then R⁶ is a bond, optionally substituted C₁-C₆ aliphatic, optionally substituted 2- to 6-membered heteroaliphatic, optionally substituted 4- to 6-membered heterocycle comprising one 1 to 3 heteroatoms selected from N, O, and S, optionally substituted C₆-C₁₂ aryl, or optionally substituted C₃-C₆ cycloaliphatic; and R⁸ is independently halogen, -OR³, -C(O)N(R³)₂, -C(O)OR³, optionally substituted C₁-C₆ aliphatic, optionally substituted C₃-C₆ cycloaliphatic, optionally substituted C₆-C₁₂ aryl; and when A¹ or A² is of formula II-4, then R⁶ is H, halogen, optionally substituted C₁-C₆ aliphatic, optionally substituted 2- to 6-mcmbcrcd heteroaliphatic, optionally substituted 4- to 6-membered heterocycle comprising one 1 to 3 heteroatoms selected from N, O, and S, optionally substituted C₆-C₁₂ aryl, or optionally substituted C₃-C₆ cycloaliphatic; and R⁸ is a bond, -O-, -C(O)NR³-, -C(O)OR³, optionally substituted C₁-C₆ aliphatic, optionally substituted C₃-C₆ cycloaliphatic, or optionally substituted C₆-C₁₂ aryl.

[0071] In some embodiments, A¹ and A² are the same. In some embodiments A¹ and A² are different.

[0072] In some embodiments, A¹ and A² are each a moiety of formula II- 1. In some embodiments, A¹ and A² are each a moiety of formula II-2. In some embodiments, A¹ and A² are each a moiety of formula II-3. In some embodiments, A¹ and A² are each a moiety of formula II-

4. In some embodiments, A¹ is a moiety of formula II-l, and A² is a moiety of formula II-2, II-3, or II-4. In some embodiments, A¹ is a moiety of formula II-2, and A² is a moiety of formula II- 1, II-3, or II-4. In some embodiments, A¹ is a moiety of formula II-3, and A² is a moiety of formula II- 1, II-2 , or II-4. In some embodiments, A¹ is a moiety of formula II-4, and A² is a moiety of formula II- 1, II-2, or II-3. In some embodiments, A¹ is a moiety of formula II- 1, II-2, or II-3 and A² is a moiety of formula II-4. In some embodiments, A¹ is a moiety of formula II- 1, II-2, or II-4 and A² is a moiety of formula II-3. In some embodiments, A¹ is a moiety of formula II- 1 , II-3, or II-4 and A² is a moiety of formula II-2. In some embodiments, A¹ is a moiety of formula II-2, II-

3, or II-4 and A² is a moiety of formula II- 1.

[0073] In some embodiments, A¹ is a moiety of formula II- 1, and A² is a moiety of formula II-

2. In some embodiments, A¹ is a moiety of formula II- 1, and A² is a moiety of formula II-3. In some embodiments, A¹ is a moiety of formula II- 1, and A² is a moiety of formula II-4. In some embodiments, A¹ is a moiety of formula II-2, and A² is a moiety of formula II- 1. In some embodiments, A¹ is a moiety of formula II- 2, and A² is a moiety of formula II-3. In some embodiments, A¹ is a moiety of formula II-2, and A² is a moiety of formula II-4. In some embodiments, A¹ is a moiety of formula II- 3, and A² is a moiety of formula II- 1. In some embodiments, A¹ is a moiety of formula II-3, and A² is a moiety of formula II-2. In some embodiments, A¹ is a moiety of formula II-3, and A² is a moiety of formula II-4. In some embodiments, A¹ is a moiety of formula II-4, and A² is a moiety of formula II- 1. In some embodiments, A¹ is a moiety of formula II-4, and A² is a moiety of formula TT-2. In some embodiments, A¹ is a moiety of formula II-4, and A² is a moiety of formula II-3.

[0074] In some embodiments of formula I, A¹ and A² are each independently selected from a moiety of formula II- 1 or II-2:

or a pharmaceutically acceptable salt thereof, wherein: each G¹ is independently an optionally substituted C₆-C₁₂ aryl or an optionally substituted

5- to 6-membered heteroaryl; each X¹ is independently -S-, -N(R³)-, -O-, optionally substituted C₁-C₆ aliphatic, or optionally substituted C₃-C₆ cycloaliphatic; each X² is independently C(R³) or N, provided that, when X² is N, then X¹ is optionally substituted C₁-C₆ aliphatic, or optionally substituted C₃-C₆ cycloaliphatic; when a bond between X³ and X⁴ is a single bond, then X³ is N(R^2a), and X⁴ is C(O) when a bond between X³ and X⁴ is a double bond, then X³ is C(R^2b) and X⁴ is C(R³), or

N; each R¹ is independently selected from halogen, optionally substituted C₁-C₆ aliphatic, and optionally substituted -O-C₁-C₆ aliphatic; or two instances of R¹ come together with the atoms to which they are attached to form a n optionally substituted C₆-C₁₂ aryl ring, an optionally substituted C₄-C₆ cycloaliphatic ring, a 5- to 6- membered heteroaryl ring comprising 1 to 3 heteroatoms selected from N, O, and S, or an optionally substituted 5- to 6-membered heterocyclic ring comprising 1 to 3 heteroatoms selected from N, O, and S; each R^2a is independently optionally substituted C₁-C₆ aliphatic,; each R^2b is independently hydrogen, optionally substituted C₁-C₆ aliphatic, optionally substituted -N(R³)-C₁-C₆ aliphatic, or optionally substituted -O-C₁-C₆ aliphatic; each R³ is independently selected from hydrogen, halogen, and optionally substituted Ci- C₆ aliphatic; each R⁴ is independently selected from optionally substituted C₆-C₁₂ aryl; optionally substituted 5- to 12- membered heteroaryl ring comprising 1 to 3 heteroatoms selected from N, O, and S; and optionally substituted C₁-C₆ aliphatic; each R⁵ is independently an optionally substituted 4- to 6-membered heterocyclic ring, an optionally substituted 5- to 6-membered heteroaryl ring, an optionally C₃-C₆ cycloaliphatic ring, an optionally substituted C₆-C₁₂ aryl, or an optionally substituted C₁-C₆ aliphatic; each L¹ is independently a bond, -C(O)-, -S(O)-, -S(O)₂-, or -NR³-; each n is independently 0, 1, 2, 3, 4, 5, or 6; and

* represents a point of attachment to moiety B .

[0075] In some embodiments of formula I, one or both of A¹ and A² are a moiety of formula II- 1:

II I or a pharmaceutically acceptable salt thereof, wherein: each G¹ is independently an optionally substituted C₆-C₁₂ aryl or an optionally substituted 5- to 6-membered heteroaryl; each X¹ is independently -S-, -N(R³)-, -O-, optionally substituted C₁-C₆ aliphatic, or optionally substituted C₃-C₆ cycloaliphatic; each X² is independently C(R³) or N, provided that, when X² is N, then X¹ is optionally substituted C₁-C₆ aliphatic, or optionally substituted C₃-C₆ cycloaliphatic; when a bond between X³ and X⁴ is a single bond, then X³ is N(R^2a), and X⁴ is C(O) when a bond between X³ and X⁴ is a double bond, then X³ is C(R^2b) and X⁴ is C(R³), or N; each R¹ is independently selected from halogen, optionally substituted C₁-C₆ aliphatic, and optionally substituted -O-C₁-C₆ aliphatic; or two instances of R¹ come together with the atoms to which they are attached to form a n optionally substituted C₆-C₁₂ aryl ring, an optionally substituted C₄-C₆ cycloaliphatic ring, a 5- to 6- membered heteroaryl ring comprising 1 to 3 heteroatoms selected from N, O, and S, or an optionally substituted 5- to 6-membered heterocyclic ring comprising 1 to 3 heteroatoms selected from N, O, and S; each R^2a is independently optionally substituted C₁-C₆ aliphatic,; each R^2b is independently hydrogen, optionally substituted C₁-C₆ aliphatic, optionally substituted -N(R³)-C₁-C₆ aliphatic, or optionally substituted -O-C₁-C₆ aliphatic; each R³ is independently selected from hydrogen, halogen, and optionally substituted Ci- C₆ aliphatic; each R⁴ is independently selected from optionally substituted C₆-C₁₂ aryl; optionally substituted 5- to 12- membered heteroaryl ring comprising 1 to 3 heteroatoms selected from N, O, and S; and optionally substituted C₁-C₆ aliphatic; each R⁵ is independently an optionally substituted 4- to 6-membered heterocyclic ring, an optionally substituted 5- to 6-membered heteroaryl ring, an optionally C₃-C₆ cycloaliphatic ring, an optionally substituted C₆-C₁₂ aryl, or an optionally substituted C₁-C₆ aliphatic; each L¹ is independently a bond, -C(O)-, -S(O)-, -S(O)₂-, or -NR³-; each n is independently 0, 1, 2, 3, 4, 5, or 6; and

* represents a point of attachment to moiety B.

[0076] In some embodiments of formula I, one or both of A¹ and A² is a moiety of formula II-

2:

or a pharmaceutically acceptable salt thereof, wherein: each G¹ is independently an optionally substituted C₆-C₁₂ aryl or an optionally substituted 5- to 6-mcmbcrcd heteroaryl; each X¹ is independently -S-, -N(R³)-, -O-, optionally substituted C₁-C₆ aliphatic, or optionally substituted C₃-C₆ cycloaliphatic; each X² is independently C(R³) or N, provided that, when X² is N, then X¹ is optionally substituted C₁-C₆ aliphatic, or optionally substituted C₃-C₆ cycloaliphatic; when a bond between X³ and X⁴ is a single bond, then X³ is N(R^2a), and X⁴ is C(O) when a bond between X³ and X⁴ is a double bond, then X³ is C(R^2b) and X⁴ is C(R³), or N; each R¹ is independently selected from halogen, optionally substituted C₁-C₆ aliphatic, and optionally substituted -O-C₁-C₆ aliphatic; or two instances of R¹ come together with the atoms to which they are attached to form a n optionally substituted C₆-C₁₂ aryl ring, an optionally substituted C₄-C₆ cycloaliphatic ring, a 5- to 6- membered heteroaryl ring comprising 1 to 3 heteroatoms selected from N, O, and S, or an optionally substituted 5- to 6-membered heterocyclic ring comprising 1 to 3 heteroatoms selected from N, O, and S; each R^2a is independently optionally substituted C₁-C₆ aliphatic,; each R^2b is independently hydrogen, optionally substituted C₁-C₆ aliphatic, optionally substituted -N(R³)-C₁-C₆ aliphatic, or optionally substituted -O-C₁-C₆ aliphatic; each R³ is independently selected from hydrogen, halogen, and optionally substituted Ci- C₆ aliphatic; each R⁴ is independently selected from optionally substituted C₆-C₁₂ aryl; optionally substituted 5- to 12- membered heteroaryl ring comprising 1 to 3 heteroatoms selected from N, O, and S; and optionally substituted C₁-C₆ aliphatic; each R⁵ is independently an optionally substituted 4- to 6-membered heterocyclic ring, an optionally substituted 5- to 6-membered heteroaryl ring, an optionally C₃-C₆ cycloaliphatic ring, an optionally substituted C₆-C₁₂ aryl, or an optionally substituted C₁-C₆ aliphatic; each L¹ is independently a bond, -C(O)-, -S(O)-, -S(O)₂-, or -NR³-; each n is independently 0, 1, 2, 3, 4, 5, or 6; and

* represents a point of attachment to moiety B . [0077] As described herein, each X¹ is independently S, N(R³), O, optionally substituted Ci- C₆ aliphatic, or optionally substituted C₃-C₆ cycloaliphatic. In some embodiments, each X¹ is independently S, optionally substituted C₁-C₆ aliphatic or optionally substituted C₃-C₆ cycloaliphatic. In some embodiments, each X¹ is independently S or optionally substituted C₁-C₆ aliphatic. In some embodiments, X¹ is S. In some embodiments, X¹ is N(R³). In some embodiments, X¹ is N(H). In some embodiments, X¹ is O. In some embodiments, X¹ is optionally substituted C₁-C₆ aliphatic. In some embodiments, X¹ is C₁-C₆ alkylene. In some embodiments, X¹ is (CH₂). In some embodiments, X¹ is optionally substituted C₃-C₆ cycloaliphatic. In some embodiments X¹ is cyclopropyl.

[0078] As described herein, each X² is independently, C(R³) or N, provided that, when X² is N, then X¹ is optionally substituted C₁-C₆ aliphatic, or optionally substituted C₃-C₆ cycloaliphatic. In some embodiments, X²is C(R³). In some embodiments, X² is C(H). In some embodiments, X² is N.

[0079] In some embodiments, X¹ is S, N(R³), or O and X² is C(R³). In some embodiments, X¹ is optionally substituted C₁-C₆ aliphatic, or optionally substituted C₃-C₆ cycloaliphatic, and X² is N. In some embodiments, X¹ is S, and X² is C(R³). In some embodiments, X¹ is S, and X² is C(H).

[0080] In some embodiments, a moiety:

is:

[0081] As described herein, when a bond between X³ and X⁴ is a single bond, then X³ is N(R^2a), and X⁴ is C(O). In some embodiments, a bond between X³ and X⁴ is a single bond.

[0082] As described herein, when a bond between X³ and X⁴ is a double bond, then X³ is C(R^2b) and X⁴ is C(R³) or N. In some embodiments, a bond between X³ and X⁴ is a double bond. In some embodiments, when a bond between X³ and X⁴ is a double bond, when X³ is C(R^2b) and X⁴ is N. [0083] As described herein, each G¹ is independently an optionally substituted C₆-C₁₂ aryl or an optionally substituted 5- to 6-mcmbcrcd heteroaryl. In some embodiments, G¹ is optionally substituted monocyclic or bicyclic C₆-C₁₂ aryl. In some embodiments, G¹ is optionally substituted phenyl.

[0084] In some embodiments, G¹ is optionally substituted naphthyl. In some embodiments, G¹ is unsubstituted naphthyl.

[0085] In some embodiments, G¹ is phenyl or naphthyl.

[0086] As described herein, each R¹ is independently selected from halogen, optionally substituted C₁-C₆ aliphatic, and optionally substituted -O-C₁-C₆ aliphatic. In some embodiments, R¹ is halogen. In some embodiments, R¹ is chloride. In some embodiments, n is 2 and R¹ is chloride. In some embodiments, n is 2, and one instance of R¹ is halogen, and another instance of R¹ is C₁-C₆ aliphatic or -O-C₁-C₆ aliphatic. In some embodiments, R¹ is optionally substituted Ci- C₆ aliphatic. In some embodiments, R¹ is C₁-C₆ alkyl. In some embodiments, R¹ is methyl. In some embodiments, R¹ is optionally substituted O-C₁-C₆ aliphatic. In some embodiments, R¹ is optionally substituted -O-C₁-C₆ alkyl. In some embodiments, R¹ is -O-CH₃.

[0087] In some embodiments, two instances of R¹ come together with the atoms to which they are attached to form an optionally substituted C₆-C₁₂ aryl ring, an optionally substituted C₄-C₆ cycloaliphatic ring, a 5- to 6- membered heteroaryl ring comprising 1 to 3 heteroatoms selected from N, O, and S, or an optionally substituted 5- to 6-membered heterocyclic ring comprising 1 to 3 heteroatoms selected from N, O, and S.

[0088] In some embodiments, two instances of R¹ come together with the atoms to which they are attached to form an optionally substituted C₆-C₁₂ aryl ring.

[0089] In some embodiments, two instances of R¹ come together with the atoms to which they are attached to form an optionally substituted C₄-C₆ cycloaliphatic ring. In some embodiments, two instances of R¹ come together to form a cyclopentyl or cyclohexyl ring.

[0090] In some embodiments, two instances of R¹ come together with the atoms to which they are attached to form a 5- to 6- membered heteroaryl ring comprising 1 to 3 heteroatoms selected from N, O, and S. In some embodiments, two instances of R¹ come together with the atoms to which they are attached to form an optionally substituted 5- to 6-membered heterocyclic ring comprising 1 to 3 heteroatoms selected from N, O, and S.

[0091] In some embodiments, a moiety:

[0092] In some embodiments, a moiety:

[0093] As described herein, each R^2a is independently optionally substituted C₁-C₆ aliphatic, optionally substituted -N(R³)-C₁-C₆ aliphatic, or optionally substituted -O-C₁-C₆ aliphatic. In some embodiments, R^2a is optionally substituted C₁-C₆ aliphatic. In some embodiments, R^2a is Ci- C₆ alkyl. In some embodiments is R^2a is methyl, ethyl, propyl, butyl, pentyl, or hexyl. In some embodiments, R^2a is methyl or ethyl. In some embodiments, R^2a is ethyl. In some embodiments, R^2a is Ci-C₆ optionally substituted with halogen. In some embodiments, R^2a is -CH₂-CHF2.

[0094] As described herein, each R^2b is independently hydrogen, optionally substituted C₁-C₆ aliphatic, optionally substituted -N(R³)- C₁-C₆ aliphatic, or optionally substituted -O-C₁-C₆, aliphatic. In some embodiments, R^2b is hydrogen. In some embodiments, R^2b is optionally substituted C₁-C₆ aliphatic. In some embodiments, R^2b is In some embodiments, R^2b is optionally substituted C₁-C₆ aliphatic. In some embodiments, R^2b is C₁-C₆ alkyl. In some embodiments is R^2b is methyl, ethyl, propyl, butyl, pentyl, or hexyl. In some embodiments, R^2a is methyl or ethyl. In some embodiments, R^2a is ethyl.

[0095] In some embodiments, R^2b is optionally substituted -N(R³)-C₁-C₆ aliphatic. In some embodiments, R^2b is -N(H)-C₁-C₆ aliphatic. In some embodiments, R^2b is -N(H)-CH₂-CH₃.

[0096] In some embodiments, R^2b is optionally substituted -O-C₁-C₆ aliphatic. In some embodiments, R^2b is -O- C₁-C₆, alkyl. In some embodiments, R^2b is -O-CH₂-CH₃.

[0097] In some embodiments of formula II- 1 or II-2, a moiety:

[0098] In some embodiments of formula II- 1 or II-2, a moiety:

[0099] As described herein, each R³ is independently selected from hydrogen, halogen, and optionally substituted C₁-C₆ aliphatic. In some embodiments, R³ is hydrogen. In some embodiments, R³ is halogen. In some embodiments, R³ is optionally substituted C₁-C₆ aliphatic. In some embodiments, R³ is optionally substituted C₁-C₆ alkyl. In some embodiments, R³ is methyl.

[0100] As described herein, each R⁴ is independently selected from optionally substituted Cf>- C12 aryl, optionally substituted 5- to 12- membered heteroaryl ring comprising 1 to 3 heteroatoms selected from N, O, and S; and optionally substituted C₁-C₆ aliphatic. [0101] In some embodiments, R⁴ is optionally substituted C₆-C₁₂ aryl. In some embodiments, R⁴ is optionally substituted phenyl. In some embodiments, R⁴ is phenyl optionally substituted with halogen, -(CH₂)_0-4R°. -(CH₂)_0-4OR°. or -(CH₂)_0-4N(R°)₂. In some embodiments, R⁴ is phenyl optionally substituted with halogen. In some embodiments, R⁴ is phenyl substituted with bromide or chloride. In some embodiments, R⁴ is phenyl substituted with bromide. In some embodiments,

R⁴ is unsubstituted phenyl. In some embodiments, R⁴ is optionally substituted napthyl.

[0102] In some embodiments, of formula II- 1, R⁴ is:

[0103] In some embodiments of formula II-2, R⁴ is:

where * represents a point of attachment to moiety B of formula I.

[0104] In some embodiments, R⁴ is optionally substituted 5- to 12- membered heteroaryl ring comprising 1 to 3 heteroatoms selected from N, O, and S. In some embodiments, R⁴ is optionally substituted monocyclic 5- to 6- membered heteroaryl comprising 1 to 3 heteroatoms selected from N, O, and S. In some embodiments, R⁴ is pyrrolidine, pyrazole, imidazole, pyridine, pyrimidine, or pyrazine. In some embodiments of formula II- 1, R⁴ is:

[0105] In some embodiments, R⁴ is optionally substituted 4- to 12-membered heterocyclyl comprising 1 to 3 heteroatoms selected from N, O, and S. In some embodiments, R⁴ is optionally substituted monocyclic 4- to 6-membered heterocyclyl comprising 1 to 3 heteroatoms selected from N, O, and S. In some embodiments, R⁴ is optionally substituted tetrahydropyran. In some embodiments of formula II- 1, R⁴ is:

[0106] In some embodiments, R⁴ is optionally substituted C₁-C₆ aliphatic. In some embodiments, R⁴ is optionally substituted C₁-C₆ alkyl. In some embodiments, R⁴ is methyl, ethyl, propyl, butyl, pentyl, or hexyl. In some embodiments, R⁴ is methyl.

In some embodiments of formula II- 1, R⁴ is -CH₃,

[0107] As described herein, each R⁵ is independently an optionally substituted 4- to 6- membered heterocyclic ring, an optionally substituted 5- to 6-membered heteroaryl ring, an optionally C₃-C₆ cycloaliphatic ring, an optionally substituted C₆-C₁₂ aryl, or an optionally substituted C₁-C₆ aliphatic.

[0108] In some embodiments, R⁵ is optionally substituted 4- to 6-membered heterocyclic ring comprising 1 to 3 heteroatoms selected from N, O, and S. In some embodiments, R⁵ is optionally substituted 5-membered heterocyclyl. In some embodiments, R⁵ is optionally substituted 6- membered heterocyclyl. In some embodiments, R⁵ is azetidine, pyrrolidine, piperdine, or piperazine. In some embodiments of formula II- 1, R⁵ is:

where * represents a point of attachment to moiety B of formula I.

[0109] In some embodiments of formula II- 1 , R⁵ is:

[0110] As described herein, each L¹ is independently a bond, -C(O)-, -S(O)-, -S(O)₂-, or -NR³- . In some embodiments, L¹ is a bond. In some embodiments, L¹ is -C(O)-. In some embodiments, L¹ is -S(O)-. In some embodiments, L¹ is -S(O)₂-. In some embodiments, L¹ is -NR³-. In some embodiments L¹ is -NH-. [0111] As described herein, each n is independently 0, 1 , 2, 3, 4, 5, or 6. In some embodiments, n is 0. In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 3.

In some embodiments, n is 4. In some embodiments, n is 5. In some embodiments, n is 6.

[0112] In some embodiments of formula I, A¹ and A² are each independently selected from a moiety of formula II- 3 or II-4:

or a pharmaceutically acceptable salt thereof, wherein: each X⁵, X⁶, and X⁷ arc independently selected from the group consisting of N and CH; each R⁷ is independently hydrogen, an optionally substituted -O-C₁-C₆ aliphatic, - S(O)₂ ³, optionally substituted C₁-C₆ aliphatic, an optionally substituted 4- to 12- membered heterocycle ring comprising 1 to 4 heteroatoms selected from N, O, and S, an optionally substituted C₆-C₁₂ aryl, or an optionally substituted 5- to 12-membered heteroaryl comprising 1 to 4 heteroatoms selected from N, O, and S each G² is independently an optionally substituted C₆-C₁₂ aryl, optionally substituted 4- to 12-membered heterocycle comprising 1 to 4 heteroatoms selected from N, O, and S, optionally substituted 5- to 6-membered heteroaryl comprising 1 to 4 heteroatoms selected from N, O, and S, or optionally substituted C₃-C₆ cycloaliphatic; each G³ is independently an optionally substituted 4- to 12- membered heterocycle ring comprising 1 to 4 heteroatoms selected from N, O, and S, an optionally substituted 5- to 6-membered heteroaryl ring comprising 1 to 4 heteroatoms selected from N, O, and S, an optionally substituted C₃-C₆ cycloaliphatic ring or optionally substituted C₆-C₁₂ aryl; each L² is independently a bond, -NR³-C(O)-, -C(O)-NR³-, optionally substituted C₁-C₆ aliphatic, optionally substituted 4- to 6-membered heterocyclic, or optionally substituted C₃-C₆ cycloaliphatic; each L³ is independently -NR³-, -0-, -C(0)-, -NR³-C(O)-, -NR³-S(O)₂-, -C(O)-NR³-, - S(O)₂ NR³-, -NR³-C(O)-NR³-, optionally substituted C₁-C₆ aliphatic, or an optionally substituted C₃-C₆ cycloaliphatic ring;

* represents a point of attachment to moiety B ; and wherein: when A¹ or A² is a moiety of formula II- 3 then R⁶ is a bond, optionally substituted C₁-C₆ aliphatic, optionally substituted 2- to 6-membered heteroaliphatic, optionally substituted 4- to 6-membered heterocycle comprising one 1 to 3 heteroatoms selected from N, O, and S, optionally substituted C₆-C₁₂ aryl, or optionally substituted C₃-C₆ cycloaliphatic; and R⁸ is independently halogen, -OR³, -C(O)N(R³)₂, -C(O)OR³, optionally substituted C₁-C₆ aliphatic, optionally substituted C₃-C₆ cycloaliphatic, optionally substituted C₆-Ci₂ aryl; and when A¹ or A² is of formula II-4, then R⁶ is H, halogen, optionally substituted C₁-C₆ aliphatic, optionally substituted 2- to 6-membered heteroaliphatic, optionally substituted 4- to 6-membered heterocycle comprising one 1 to 3 heteroatoms selected from N, O, and S, optionally substituted C6-Ci₂ aryl, or optionally substituted C₃-C₆ cycloaliphatic; and R⁸ is a bond, -O-, -C(O)NR³-, -C(O)OR³, optionally substituted C₁-C₆ aliphatic, optionally substituted C₃-C₆ cycloaliphatic, or optionally substituted C6-Ci₂ aryl.

[0113] In some embodiments of formula I, one or both of A¹ and A² is a moiety of formula II-

3:

II-3 or a pharmaceutically acceptable salt thereof, wherein: each X⁵, X⁶, and X⁷ are independently selected from the group consisting of N and CH; each R⁷ is independently hydrogen, an optionally substituted -O-C₁-C₆ aliphatic, -

S(O)₂R³, optionally substituted C₁-C₆ aliphatic, an optionally substituted 4- to 12- membered heterocycle ring comprising 1 to 4 heteroatoms selected from N, O, and S, an optionally substituted C₆-C₁₂ aryl, or an optionally substituted 5- to 12-mcmbcrcd heteroaryl comprising 1 to 4 heteroatoms selected from N, O, and S each G² is independently an optionally substituted C₆-C₁₂ aryl, optionally substituted 4- to 12-membered heterocycle comprising 1 to 4 heteroatoms selected from N, O, and S, optionally substituted 5- to 6-membered heteroaryl comprising 1 to 4 heteroatoms selected from N, O, and S, or optionally substituted C₃-C₆ cycloaliphatic; each G³ is independently an optionally substituted 4- to 12- membered heterocycle ring comprising 1 to 4 heteroatoms selected from N, O, and S, an optionally substituted 5- to 6-membered heteroaryl ring comprising 1 to 4 heteroatoms selected from N, O, and S, an optionally substituted C₃-C₆ cycloaliphatic ring or optionally substituted C₆-C₁₂ aryl; each L² is independently a bond, -NR³-C(O)-, -C(O)-NR³-, optionally substituted C₁-C₆ aliphatic, optionally substituted 4- to 6-membered heterocyclic, or optionally substituted C₃-C₆ cycloaliphatic; each L³ is independently -NR³-, -O-, -C(O)-, -NR³-C(O)-, -NR³-S(O)₂-, -C(O)-NR³-, - S(O)₂ NR³-, -NR³-C(O)-NR³-, optionally substituted C₁-C₆ aliphatic, or an optionally substituted C₃-C₆ cycloaliphatic ring;

* represents a point of attachment to moiety B ; and

R⁶ is a bond, optionally substituted C₁-C₆ aliphatic, optionally substituted 2- to 6- membered heteroaliphatic, optionally substituted 4- to 6-membered heterocycle comprising one 1 to 3 heteroatoms selected from N, O, and S, optionally substituted C₆-C₁₂ aryl, or optionally substituted C₃-C₆ cycloaliphatic; and

R⁸ is independently halogen, -OR³, -C(O)N(R³)₂, -C(O)OR³, optionally substituted C₁-C₆ aliphatic, optionally substituted C₃-C₆ cycloaliphatic, optionally substituted C₆-C₁₂ aryl.

[0114] In some embodiments of formula I, one or both of A¹ and A² is a moiety of formula II- 4:

or a pharmaceutically acceptable salt thereof, wherein: each X⁵, X⁶, and X⁷ are independently selected from the group consisting of N and CH; each R⁷ is independently hydrogen, an optionally substituted -O-C₁-C₆ aliphatic, - S(O)₂ ³, optionally substituted C₁-C₆ aliphatic, an optionally substituted 4- to 12- membered heterocycle ring comprising 1 to 4 heteroatoms selected from N, O, and S, an optionally substituted C₆-C₁₂ aryl, or an optionally substituted 5- to 12-membered heteroaryl comprising 1 to 4 heteroatoms selected from N, O, and S each G² is independently an optionally substituted C₆-C₁₂ aryl, optionally substituted 4- to 12-membered heterocycle comprising 1 to 4 heteroatoms selected from N, O, and S, optionally substituted 5- to 6-membered heteroaryl comprising 1 to 4 heteroatoms selected from N, O, and S, or optionally substituted C₃-C₆ cycloaliphatic; each G³ is independently an optionally substituted 4- to 12- membered heterocycle ring comprising 1 to 4 heteroatoms selected from N, O, and S, an optionally substituted 5- to 6-membered heteroaryl ring comprising 1 to 4 heteroatoms selected from N, O, and S, an optionally substituted C₃-C₆ cycloaliphatic ring or optionally substituted C₆-C₁₂ aryl; each L² is independently a bond, -NR³-C(O)-, -C(O)-NR³-, optionally substituted C₁-C₆ aliphatic, optionally substituted 4- to 6-membered heterocyclic, or optionally substituted C₃-C₆ cycloaliphatic; each L³ is independently -NR³-, -O-, -C(O)-, -NR³-C(O)-, -NR³-S(O)₂-, -C(O)-NR³-, - S(O)₂ NR³-, -NR³-C(O)-NR³-, optionally substituted C₁-C₆ aliphatic, or an optionally substituted C₃-C₆ cycloaliphatic ring;

* represents a point of attachment to moiety B ;

R⁶ is H, halogen, optionally substituted C₁-C₆ aliphatic, optionally substituted 2- to 6- membered heteroaliphatic, optionally substituted 4- to 6-membered heterocycle comprising one 1 to 3 heteroatoms selected from N, O, and S, optionally substituted C₆-C₁₂ aryl, or optionally substituted C₃-C₆ cycloaliphatic; and

R⁸ is a bond, -O-, -C(O)NR³-, -C(O)OR³, optionally substituted C₁-C₆ aliphatic, optionally substituted C₃-C₆ cycloaliphatic, or optionally substituted C₆-C₁₂ aryl.

[0115] As described herein, each X⁵, X⁶, and X⁷ are independently selected from the group consisting of N and CH. In some embodiments, each of X⁵, X⁶, and X⁷ are N. In some embodiments, each of X⁵, X⁶, and X⁷ are CH. In some embodiments, X⁵ is N and X⁶ and X⁷ are CH. In some embodiments, X⁶ is N and X⁵ and X⁷ are CH. In some embodiments, X⁷ is N, and X⁵ and X⁶ are CH. In some embodiments, X⁵ and X⁶ are N and X⁷ is CH. In some embodiments, X⁵ and X⁷ are N and X⁶ is CH. In some embodiments, X⁶ and X⁷ are N and X⁵ is CH.

[0116] As described herein, each R⁷ is independently hydrogen, an optionally substituted -O- C₁-C₆ aliphatic, -S(O)₂R³, optionally substituted C₁-C₆ aliphatic, an optionally substituted 4- to 12- membered heterocycle ring comprising 1 to 4 heteroatoms selected from N, O, and S, an optionally substituted C₆-C₁₂ aryl, or an optionally substituted 5- to 12-membered heteroaryl comprising 1 to 4 heteroatoms selected from N, O, and S.

[0117] In some embodiments, R⁷ is hydrogen.

[0118] In some embodiments, each R⁷ is independently an optionally substituted -O-C₁-C₆ aliphatic, -S(O)₂R³, optionally substituted C₁-C₆ aliphatic, an optionally substituted 4- to 12- membered heterocycle ring comprising 1 to 4 heteroatoms selected from N, O, and S, an optionally substituted C₆-C₁₂ aryl, or an optionally substituted 5- to 12-membered heteroaryl comprising 1 to 4 heteroatoms selected from N, O, and S.

[0119] In some embodiments, R⁷ is optionally substituted -O-C₁-C₆ aliphatic. In some embodiments, R⁷ is optionally substituted -O-C₁-C₆ alkyl. In some embodiments, R⁷ is -O-CH₃.

[0120] In some embodiments, R⁷ is -S(O)₂R³. In some embodiments, R⁷ is optionally substituted -S(O)₂-Ci-C6 aliphatic.

[0121] In some embodiments, R⁷ is optionally substituted C₁-C₆ aliphatic.

[0122] In some embodiments, R⁷ is an optionally substituted 4- to 12-membered heterocycle ring comprising 1 to 4 heteroatoms selected from N, O, and S. In some embodiments, R⁷ is an optionally substituted monocyclic 4- to 6-membered heterocycle ring comprising 1 to 3 heteroatoms selected from N, O, and S. In some embodiments, R⁷ is optionally substituted tetrahydropyran or morpholine. In some embodiments, R⁷ is a bicyclic or spirocyclic 6- to 12- membered heterocycle ring comprising 1 to 4 heteroatoms selected from N, O, and S. In some embodiments, R⁷ is:

[0123] In some embodiments, R⁷ is optionally substituted C₆-C₁₂ aryl. In some embodiments, R⁷ is optionally substituted phenyl. In some embodiments, R⁷ is phenyl optionally substituted with -(CH₂)_0-4R° or -(CH₂)_0-4N(R°)₂. In some embodiments, R⁷ is phenyl optionally substituted with -NH₂. In some embodiments, R⁷ is optionally substituted naphthyl. In some embodiments, R⁷ is:

[0124] In some embodiments, R⁷ is optionally substituted 5- to 12-membered heteroaryl comprising 1 to 4 heteroatoms selected from N, O, and S. In some embodiments, R⁷ is optionally substituted monocyclic 5- to 6-membered heteroaryl comprising 1 to 3 heteroatoms selected from N, O, and S. In some embodiments, R⁷ is monocyclic 5- to 6-membered heteroaryl comprising 1 to 3 heteroatoms selected from N, O, and S optionally substituted with -(CH₂)_0-4R° or -(CH₂)o 4N(R°)₂. In some embodiments, R⁷ is monocyclic 5- to 6-membered heteroaryl comprising 1 to 3 heteroatoms selected from N, O, and S optionally substituted with -CH₃ or -NH₂. In some embodiments, R⁷ is optionally substituted pyridine, pyrazine, pyrimidine, pyrazolc, pyrrole, or imidazole. In some embodiments, R⁷ is:

[0126] As described herein, each G² is independently an optionally substituted C₆-C₁₂ aryl, optionally substituted 4- to 12-mcmbcrcd heterocycle comprising 1 to heteroatoms selected from N, O, and S, optionally substituted 5- to 6-membered heteroaryl comprising 1 to 4 heteroatoms selected from N, O, and S, or optionally substituted C₃-C₆ cycloaliphatic.

[0127] In some embodiments, G² is optionally substituted C₆-C₁₂ aryl. In some embodiments, G² is optionally substituted phenyl. In some embodiments, G² is unsubstituted phenyl. In some embodiments, G² is naphthyl.

[0128] In some embodiments, G² is optionally substituted 4- to 12-membered heterocycle comprising 1 to 4 heteroatoms selected from N, O, and S. In some embodiments, G² is optionally substituted monocyclic 4- to 6- membered heterocycle comprising 1 to 3 heteroatoms selected from N, O, and S. In some embodiments, G² is optionally substituted bicyclic 7- to 12-membered heterocycle comprising 1 to 4 heteroatoms selected from N, O, and S.

[0129] In some embodiments, G² is optionally substituted 5- to 6-membered heteroaryl comprising 1 to 4 heteroatoms selected from N, O, and S.

[0130] In some embodiments, G² is optionally substituted C₃-C₆ cycloaliphatic. In some embodiments, G² is optionally substituted partially unsaturated C₆ cycloaliphatic.

[0131] In some embodiments of formula II- 3 or II-4, a moiety:

[0132] As described herein, each G³ is independently an optionally substituted 4- to 12- membered heterocycle ring comprising 1 to 4 heteroatoms selected from N, O, and S, an optionally substituted 5- to 6-membered heteroaryl ring comprising 1 to 4 heteroatoms selected from N, O, and S, an optionally substituted C₃-C₆ cycloaliphatic ring, or optionally substituted C₆-C₁₂ aryl.

[0133] In some embodiments, G³ is optionally substituted 4- to 12-membered heterocycle ring comprising 1 to 4 heteroatoms selected from N, O, and S. In some embodiments, G³ is optionally substituted monocyclic 4- to 6-mcmbcrcd heterocycle ring comprising 1 to 3 heteroatoms selected from N, O, and S. In some embodiments, G³ is optionally substituted pyrrolidine or piperdine. In some embodiments, G³ is pyrrolidine or piperdine optionally substituted with -(CH₂)_0-4R° or -

(CH₂)_0-4Ph, which may be substituted with R°.

[0134] In some embodiments, G³ is optionally substituted bicyclic or spirocyclic 6- to 12- mcmbcrcd heterocyclyl comprising 1 to 4 heteroatoms selected from N, O, and S. In some embodiments, G³ is bicyclic or spirocyclic 6- to 12-membered heterocyclyl comprising 1 to 4 heteroatoms selected from N, O, and S, optionally substituted with -(CH₂)_0-4R°.

[0135] In some embodiments, G³ is an optionally substituted 5- to 6-membered heteroaryl ring comprising 1 to 4 heteroatoms selected from N, O, and S.

[0136] In some embodiments, G³ is an optionally substituted C₃-C₆ cycloaliphatic ring

[0137] In some embodiments, G³ is optionally substituted C₆-C₁₂ aryl.

[0138] As described herein, each L² is independently a bond, -NR³-C(O)-, -C(O)-NR³-, optionally substituted C₁-C₆ aliphatic, optionally substituted 4- to 6-membered heterocyclic, or optionally substituted C₃-C₆ cycloaliphatic.

[0139] In some embodiments, L² is a bond.

[0140] In some embodiments, L² is optionally substituted C₁-C₆ aliphatic. In some embodiments, L² is C₁-C₆ alkylene. In some embodiments, L² is -C(H)(CH₃)-, -C(CH₃)₂-, or - CH₂-. In some embodiments, L² is:

In some embodiments, L² is:

[0141] In some embodiments, L² is -NR³-C(O)-. In some embodiments, L² is -NH-C(O)-.

[0142] In some embodiments, L² is -C(O)-NR³-. In some embodiments, L² is -C(O)-NH-.

[0143] In some embodiments, L² is optionally substituted 4- to 6-membered heterocyclic. In some embodiments, L² is optionally substituted azetidine or oxetane. In some embodiments, L² is:

[0144] In some embodiments, L² is optionally substituted C₃-C₆ cycloaliphatic. In some embodiments, L² is cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl. In some embodiments, L² is:

[0145] As described herein, each L³ is independently -NR³-, -O-, -C(O)-, -NR³-C(O)-, -NR³- S(O)₂-, -C(O)-NR³-, -S(O)₂ NR³-, -NR³-C(O)-NR³-, optionally substituted Ci-C₆ aliphatic, or an optionally substituted C₃-C₆ cycloaliphatic ring. In some embodiments, L³ is -NR³-. In some embodiments, L³ is -NH-. In some embodiments, L³ is -O-. In some embodiments, L³ is -C(O)-. In some embodiments, L³ is -NR³-C(O)-. In some embodiments, L³ is -NH-C(O)-. In some embodiments, L³ is -NR³-S(O)₂-. In some embodiments, L³ is -NH-S(O)₂-. In some embodiments, L³ is -C(O)-NR³-. In some embodiments, L³ is -C(O)-NH-. In some embodiments, L³ is -S(O)₂ NR³-. In some embodiments, L³ is-S(O)₂-NH-. In some embodiments, L³ is -NR³-C(O)-NR³-. In some embodiments, L³ is -NH-C(O)-NH-.

[0146] In some embodiments, L³ is optionally substituted C₁-C₆ aliphatic. In some embodiments, L³ is optionally substituted C₁-C₆ alkylene. In some embodiments, L³ is -(CH₂)I-6- . In some embodiments, L³ is -CH₂-.

[0147] In some embodiments, L³ is an optionally substituted C₃-C₆ cycloaliphatic ring. In some embodiments, L³ is cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl. In some embodiments, L³ is cyclopropyl. [0148] As described herein with respect to formula IT-3, R⁶ is a bond, optionally substituted C₁-C₆ aliphatic, optionally substituted 2- to 6-mcmbcrcd heteroaliphatic, optionally substituted 4- to 6-membered heterocycle comprising one 1 to 3 heteroatoms selected from N, O, and S, optionally substituted C₆-C₁₂ aryl, or optionally substituted C₃-C₆ cycloaliphatic. In some embodiments of formula II-3, R⁶ is a bond or optionally substituted C₁-C₆ aliphatic.

[0149] In some embodiments of formula II-3, R⁶ is a bond.

[0150] In some embodiments of formula II-3, R⁶ is optionally substituted C₁-C₆ aliphatic. In some embodiments, R⁶ is C₁-C₆ alkylene. In some embodiments, R⁶ is -CH₂-. In some embodiments, R⁶ is C2-C6 alkynlene.

[0151] In some embodiments of formula II-3, R⁶ is optionally substituted 2- to 6-membered heteroaliphatic.

[0152] In some embodiments of formula II-3, R⁶ is optionally substituted 4- to 6-membered heterocycle comprising one 1 to 3 heteroatoms selected from N, O, and S.

[0153] In some embodiments of formula II-3, R⁶ is optionally substituted C₆-C₁₂ aryl.

[0154] In some embodiments of formula II-3, R⁶ is optionally substituted C₃-C₆ cycloaliphatic. [0155] As described herein with respect to formula II-3, each R⁸ is independently halogen, - OR³, -C(O)N(R³)₂, -C(O)OR³, optionally substituted C₁-C₆ aliphatic, optionally substituted C₃-C₆ cycloaliphatic, optionally substituted C₆-C₁₂ aryl.

[0156] In some embodiments of formula II-3, R⁸ is halogen.

[0157] In some embodiments of formula II-3, R⁸ is -OR³. In some embodiments of formula II-

3, R⁸ is -OH. In some embodiments of formula II-3, R⁸ is -O-CH₃.

[0158] In some embodiments of formula II-3, R⁸ is -C(O)N(R³)₂. In some embodiments of formula II-3, R⁸ is -C(O)NH₂. In some embodiments of formula II-3, R⁸ is -C(O)N(H)(C₁-C₆ aliphatic). In some embodiments of formula II-3, R⁸ is -C(O)N(H)(CH₃). In some embodiments of formula II-3, R⁸ is -C(O)N(H)-CH₂-CH₂-O-CH₃.

[0159] In some embodiments of formula II-3, R⁸ is -C(O)OR³. In some embodiments, R⁸ is - C(O)OH. In some embodiments, R⁸ is -C(O)OCH₃.

[0160] In some embodiments of formula II-3, R⁸ is optionally substituted C₁-C₆ aliphatic. In some embodiments, R⁸ is methyl, ethyl, or propyl. In some embodiments, R⁸ is -CH₃ or - C(H)(CH₃)₂. [0161] In some embodiments of formula II-3, R⁸ is optionally substituted C₃-C₆ cycloaliphatic.

In some embodiments, R⁸ is optionally substituted cyclopropyl.

[0162] In some embodiments of formula II- 3 , R⁸ is optionally substituted C₆-C₁₂ aryl. In some embodiments, R⁸ is optionally substituted phenyl.

[0163] In some embodiments, with respect to formula II-3, a moiety;

is:

[0164] As described herein with respect to formula II-4, R⁶ is H, halogen, optionally substituted C₁-C₆ aliphatic, optionally substituted 2- to 6-membered heteroaliphatic, optionally substituted 4- to 6-membered heterocycle comprising one 1 to 3 heteroatoms selected from N, O, and S, optionally substituted C₆-C₁₂ aryl, or optionally substituted C₃-C₆ cycloaliphatic.

[0165] In some embodiments of formula II-4, R⁶ is H.

[0166] In some embodiments of formula II-4, R⁶ is halogen.

[0167] In some embodiments of formula II-4, R⁶ is optionally substituted C₁-C₆ aliphatic.

[0168] In some embodiments of formula II-4, R⁶ is optionally substituted 2- to 6-membered heteroaliphatic.

[0169] In some embodiments of formula II-4, R⁶ is optionally substituted 4- to 6-membered heterocycle comprising one 1 to 3 heteroatoms selected from N, O, and S.

[0170] In some embodiments of formula II-4, R⁶ is optionally substituted C₆-C₁₂ aryl.

[0171] In some embodiments of formula II-4, R⁶ is optionally substituted C₃-C₆ cycloaliphatic. [0172] As described herein with respect to formula II-4, R⁸ is a bond, -O-, -C(O)NR³-, - C(O)OR³, optionally substituted C₁-C₆ aliphatic, optionally substituted C₃-C₆ cycloaliphatic, or optionally substituted C₆-C₁₂ aryl.

[0173] In some embodiments of formula II-4, R^s is a bond.

[0174] In some embodiments of formula II-4, R⁸ is -O-.

[0175] In some embodiments of formula II-4, R⁸ is -C(O)NR³-. In some embodiments, R⁸ is

-C(O)NH-. In some embodiments, R⁸ is -C(O)N(C₁-C₆ aliphatic)-.

[0176] In some embodiments of formula II-3, R⁸ is -C(O)OR³-. In some embodiments, R⁸ is -C(O)OCH₂-.

[0177] In some embodiments of formula II-4, R⁸ is optionally substituted C₁-C₆ aliphatic. In some embodiments, R⁸ is methyl, ethyl, or propyl. In some embodiments, R⁸ is -CH₃ or - C(H)(CH₃)₂.

[0178] In some embodiments of formula II-4, R⁸ is optionally substituted C₃-C₆ cycloaliphatic. In some embodiments, R⁸ is optionally substituted cyclopropyl.

[0179] In some embodiments of formula II-4, R⁸ is optionally substituted C₆-C₁₂ aryl.

[0180] In some embodiments, with respect to formula II-4, a moiety;

or

where * indicates a point of attachment to moiety B.

[0181] In some embodiments, one or both of A¹ and A² are each independently selected from:

wherein R¹, R^2a, R^2b, R⁴, R⁵, L¹, G¹, n, G², G³, L³, R⁶, R⁷, and R⁸ are as described in classes and subclasses herein, and * represents a point of attachment to moiety B.

[0182] In some embodiments of formula I, one or both of A¹ and A² are each independently selected from:

[0183] In some embodiments, the present disclosure provides a compound of formula I:

A¹-B-A²

I or a pharmaceutically acceptable salt thereof, wherein:

A¹ and A² are each a moiety of formula II- 1:

II- 1 wherein: each G¹ is independently an optionally substituted C₆-C₁₂ aryl;

X¹ is -S-;

X² is -C(R³)-; a bond between X³ and X⁴ is a single bond each X³ is independently -C(H)(R^2b)- or -N(R^2a)-;

X⁴ is -C(O)-; each R¹ is independently selected from halogen, optionally substituted C₁-C₆ aliphatic, and optionally substituted -O-C₁-C₆ aliphatic; or two instances of R¹ come together with the atoms to which they are attached to form a n optionally substituted C₆-C₁₂ aryl ring, an optionally substituted C₄-C₆ cycloaliphatic ring, a 5- to 6- membered heteroaryl ring comprising 1 to 3 heteroatoms selected from N, O, and S, or an optionally substituted 5- to 6-membered heterocyclic ring comprising 1 to 3 heteroatoms selected from N, O, and S; each R^2a is independently optionally substituted C₁-C₆ aliphatic; each R^2b is independently hydrogen, optionally substituted C₁-C₆ aliphatic, optionally substituted -N(R³)-C₁-C₆ aliphatic, or optionally substituted -O-C₁-C₆ aliphatic; each R³ is independently selected from hydrogen, halogen, and optionally substituted Ci- C& aliphatic; each R⁴ is independently selected from optionally substituted C₆-C₁₂ aryl, optionally substituted 5- to 12- membered heteroaryl ring comprising 1 to 3 heteroatoms selected from N, O, and S; each R⁵ is independently an optionally substituted 4- to 6-membered heterocyclic ring, an optionally substituted 5- to 6-membered heteroaryl ring, or an optionally C₃-C₆ cycloaliphatic ring; each L¹ is independently a bond, -C(O)-; each n is independently 0, 1, 2, 3, 4, 5, or 6;

* represents a point of attachment to moiety B ; and

B is a linker moiety that is an optionally substituted C_2-30 aliphatic group wherein one or more carbons are optionally and independently replaced by -Cy-, -NR^Z-, -N(R^Z)C(O)- , -C(O)N(R^Z)-, -N(R^Z)C(O)O-, -OC(O)N(R^Z)-, -N(R^Z)C(O)N(R^Z) -, -OC(O)O-, -O-, - C(O)-, -OC(O)-, -C(O)O-, -SO-, -SO₂-, wherein each -Cy- is independently an optionally substituted 3-12 membered heterocyclyl ring having 1-3 heteroatoms selected from N, O, and S, an optionally substituted 3-8 membered heteroaryl ring having 1-4 heteroatoms selected from N, O, and S, an optionally substituted C₃-C₆ cycloalkyl, or an optionally substituted C₆-C₁₂ aryl, and each R^z is independently H or an optionally substituted group selected from C1-C20 aliphatic, or C3-C12 cycloaliphatic. [0184] In some embodiments, the present disclosure provides a compound of formula I:

A³-B-A²

I or a pharmaceutically acceptable salt thereof, wherein:

A¹ and A² arc each a moiety of formula II-2:

wherein: each G¹ is independently an optionally substituted C₆-C₁₂ aryl;

X¹ is -S-;

X⁴ is -C(O)-; each R¹ is independently selected from halogen, optionally substituted C₁-C₆ aliphatic, and optionally substituted -O-C₁-C₆ aliphatic; or two instances of R¹ come together with the atoms to which they are attached to form a n optionally substituted C₆-C₁₂ aryl ring, an optionally substituted C₄-C₆ cycloaliphatic ring, a 5- to 6- membered heteroaryl ring comprising 1 to 3 heteroatoms selected from N, O, and S, or an optionally substituted 5- to 6-membered heterocyclic ring comprising 1 to 3 heteroatoms selected from N, O, and S; each R^2a is independently optionally substituted C₁-C₆ aliphatic; each R^2b is independently hydrogen, optionally substituted C₁-C₆ aliphatic, optionally substituted -N(R³)-C₁-C₆ aliphatic, or optionally substituted -O-C₁-C₆ aliphatic; each R³ is independently selected from hydrogen, halogen, and optionally substituted Ci- C& aliphatic; each R⁴ is independently selected from optionally substituted C₆-C₁₂ aryl, optionally substituted 5- to 12- membered heteroaryl ring comprising 1 to 3 heteroatoms selected from N, O, and S; each R⁵ is independently an optionally substituted 4- to 6-membered heterocyclic ring, an optionally substituted 5- to 6-mcmbcrcd heteroaryl ring, or an optionally C₃-C₆ cycloaliphatic ring; each L¹ is independently a bond, -C(O)-; each n is independently 0, 1, 2, 3, 4, 5, or 6;

* represents a point of attachment to moiety B; and

B is a linker moiety that is an optionally substituted C2-30 aliphatic group wherein one or more carbons are optionally and independently replaced by -Cy-, -NR^Z-, -N(R^Z)C(O)- , -C(O)N(R^Z)-, -N(R^Z)C(O)O-, -OC(O)N(R^Z)-, -N(R^Z)C(O)N(R^Z) -, -OC(O)O-, -O-, - C(O)-, -OC(O)-, -C(O)O-, -SO-, -SO₂-, wherein each -Cy- is independently an optionally substituted 3-12 membered heterocyclyl ring having 1-3 heteroatoms selected from N, O, and S, an optionally substituted 3-8 membered heteroaryl ring having 1-4 heteroatoms selected from N, O, and S, an optionally substituted C₃-C₆ cycloalkyl, or an optionally substituted C₆-C₁₂ aryl, and each R^z is independently H or an optionally substituted group selected from C1-C20 aliphatic, or C₃-C₁₂ cycloaliphatic. [0185] In some embodiments, the present disclosure provides a compound of formula I:

A¹-B-A²

I or a pharmaceutically acceptable salt thereof, wherein:

A¹ and A² are each a moiety of formula II-3:

each X⁵, X⁶, and X⁷ are independently selected from the group consisting of N and CH; each R⁷ is independently an optionally substituted 4- to 12- membered heterocycle ring comprising 1 to 4 heteroatoms selected from N, O, and S, an optionally substituted C₆-C₁₂ aryl, or an optionally substituted 5- to 12-membered heteroaryl comprising 1 to 4 heteroatoms selected from N, O, and S each G² is independently an optionally substituted C₆-C₁₂ aryl, or optionally substituted 5- to 6-mcmbcrcd heteroaryl comprising 1 to 4 heteroatoms selected from N, O, and S; each G³ is independently an optionally substituted 4- to 12- membered heterocycle ring comprising 1 to 4 heteroatoms selected from N, O, and S; each L² is independently optionally substituted C₁-C₆ aliphatic, or optionally substituted C₃-C₆ cycloaliphatic; each L³ is independently -NR³-, -O-, -C(O)-, -NR³-C(O)-, -NR³-S(O)₂-, -C(O)-NR³-, - S(O)₂ NR³-, or -NR³-C(O)-NR³-;

* represents a point of attachment to moiety B ;

R⁶ is a bond; and

[0186] In some embodiments, the present disclosure provides a compound of formula I:

A¹-B-A²

I or a pharmaceutically acceptable salt thereof, wherein:

A¹ and A² are each a moiety of formula II-4:

each X⁵, X⁶, and X⁷ are independently selected from the group consisting of N and CH; each R⁷ is independently an optionally substituted 4- to 12-membered heterocycle ring comprising 1 to 4 heteroatoms selected from N, O, and S, an optionally substituted C₆-C₁₂ aryl, or an optionally substituted 5- to 12-membered heteroaryl comprising 1 to 4 heteroatoms selected from N, O, and S each G² is independently an optionally substituted C₆-C₁₂ aryl, or optionally substituted 5- to 6-mcmbcrcd heteroaryl comprising 1 to 4 heteroatoms selected from N, O, and S; each G³ is independently an optionally substituted 4- to 12- membered heterocycle ring comprising 1 to 4 heteroatoms selected from N, O, and S; each L² is independently optionally substituted C₁-C₆ aliphatic, or optionally substituted C₃-C₆ cycloaliphatic; each L³ is independently -NR³-, -O-, -C(O)-, -NR³-C(O)-, -NR³-S(O)₂-, -C(O)-NR³-, - S(O)₂ NR³-, or -NR³-C(O)-NR³-;

* represents a point of attachment to moiety B ;

R⁸ is a bond.

[0187] In some embodiments, one or both of A¹ and A² is represented by:

wherein R¹, n, G¹, R^2a, R⁴, R⁵, and L¹ are as described in classes and subclasses herein, both singly and in combination.

[0188] In some embodiments, one or both of A¹ and A² is represented by:

[0189] In some embodiments, one or both of A¹ and A² is represented by:

wherein R¹, n, G¹, R^2b, R⁴, R⁵, and L¹ are as described in classes and subclasses herein, both singly and in combination.

[0190] In some embodiments, one or both of A¹ and A² is represented by:

[0191] In some embodiments, one or both of A¹ and A² is represented by:

wherein R⁶, G², G³, L², R⁷, and L³ are as described in classes and subclasses herein, both singly and in combination.

[0192] In some embodiments, one or both of A¹ and A² is represented by:

[0193] In some embodiments, one or both of A¹ and A² is represented by:

wherein R⁶, G³, L², R⁷, and L³ are as described in classes and subclasses herein, both singly and in combination.

[0194] In some embodiments, one or both of A¹ and A² is represented by:

wherein R⁶, G , L², R⁷, and L³ are as described in classes and subclasses herein, both singly and in combination.

[0195] In some embodiments, one or both of A¹ and A² is represented by:

wherein R⁶, G², L², R⁷, and L³ are as described in classes and subclasses herein, both singly and in combination.

[0196] In some embodiments, one or both of A¹ and A² is represented by:

[0197] In some embodiments, a compound of formula I is represented by formula III- 1 :

or a pharmaceutically acceptable salt thereof, wherein R¹, n, G¹, R^2a, R⁴, L¹, R⁵, and B are as defined in classes and subclasses herein, both singly and in combination.

[0198] In some embodiments, a compound of formula I is represented by formula III-2:

[0199] In some embodiments, a compound of formula I is represented by formula III-3 :

or a pharmaceutically acceptable salt thereof, wherein R¹, n, G¹, R^2a, R⁴, L¹, R⁵, B, R⁶, L³, G³, R⁸, L³, G², and R⁷ are as defined in classes and subclasses herein, both singly and in combination.

[0200] In some embodiments, a compound of formula I is represented by formula III-4:

or a pharmaceutically acceptable salt thereof, wherein B, R⁶, L³, G³, R⁸, L³, G², and R⁷ are as defined in classes and subclasses herein, both singly and in combination.

[0201] In some embodiments, a compound of formula I is represented by formula III-5 :

[0202] In some embodiments, a compound of formula I is a compound of formula IV- 1:

or a pharmaceutically acceptable salt thereof, wherein R^2a, R⁴, and B are as described in classes and subclasses herein, both singly and in combination.

[0203] In some embodiments, a compound of formula I is a compound of formula IV-2:

IV-2 or a pharmaceutically acceptable salt thereof, wherein R^2a, R⁵, and B are as described in classes and subclasses herein, both singly and in combination.

[0204] In some embodiments, a compound of formula I is a compound of formula IV-3:

IV-3 or a pharmaceutically acceptable salt thereof, wherein R⁸, L², G², B, and R⁷ are as described in classes and subclasses herein, both singly and in combination.

[0205] In some embodiments, a compound of formula I is a compound of formula IV-4:

or a pharmaceutically acceptable salt thereof, wherein R⁷, L², G², B, and R⁷ are as described in classes and subclasses herein, both singly and in combination. [0206] As described herein, B is a linker moiety.

[0207] In some embodiments, B is a linker moiety that is an optionally substituted C2-30 aliphatic group wherein one or more carbons are optionally and independently replaced by -Cy-, - NR^Z-, -N(R^Z)C(O)-, -C(O)N(R^Z)-, -N(R^Z)C(O)O-, -OC(O)N(R^Z)-, -N(R^Z)C(O)N(R^Z) -, -OC(O)O- , -O-, -C(O)-, -OC(O)-, -C(O)O-, -SO-, -SO₂-, wherein each -Cy- is independently an optionally substituted 3-12 membered heterocyclyl ring having 1-3 heteroatoms selected from N, O, and S, an optionally substituted 3-8 membered heteroaryl ring having 1-4 heteroatoms selected from N, O, and S, an optionally substituted C₃-C₆ cycloalkyl, or an optionally substituted C₆-C₁₂ aryl, and each R^z is independently H or an optionally substituted group selected from C1-C20 aliphatic, or C3-C12 cycloaliphatic.

[0208] In some embodiments, B is selected from Table B:

Table B

[0209] In some embodiments, a compound of formula I is selected from Table 1:

Table 1

[0210] In some embodiments, a compound of formula I is selected from Table 2:

Table 2

[0211] In some embodiments, provided compounds are provided and/or utilized in a salt form (e.g., a pharmaceutically acceptable salt form). Reference to a compound provided herein is understood to include reference to salts thereof, unless otherwise indicated.

Uses, Formulation, and Administration

Pharmaceutically Acceptable Compositions

[0212] According to another embodiment, the present disclosure provides a composition comprising a compound described herein or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier, adjuvant, or vehicle. In certain embodiments, a composition described herein is formulated for administration to a patient in need of such composition. In some embodiments, a composition described herein is formulated for oral administration to a patient.

[0213] Compounds and compositions, according to method of the present disclosure, are administered using any amount and any route of administration effective for treating or lessening the severity of a disorder provided herein. The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the infection, the particular agent, its mode of administration, and the like. Compounds described herein are preferably formulated in unit dosage form for ease of administration and uniformity of dosage.

[0214] Compositions of the present disclosure may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally, intraperitoneally, intracistemallyor via an implanted reservoir. In some embodiments, the compositions are administered orally, intraperitoneally or intravenously.

[0215] Sterile injectable forms of the compositions described herein may be aqueous or oleaginous suspension. These suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed arc water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium.

[0216] For this purpose, any bland fixed oil may be employed including synthetic mono- or diglycerides. Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically-acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions. These oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant, such as carboxymethyl cellulose or similar dispersing agents that are commonly used in the formulation of pharmaceutically acceptable dosage forms including emulsions and suspensions. Other commonly used surfactants, such as Tweens, Spans and other emulsifying agents or bioavailability enhancers which are commonly used in the manufacture of pharmaceutically acceptable solid, liquid, or other dosage forms may also be used for the purposes of formulation.

[0217] Injectable formulations can be sterilized, for example, by filtration through a bacterial- retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use. [0218] In order to prolong the effect of a compound of the present disclosure, it is often desirable to slow the absorption of the compound from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material with poor water solubility. The rate of absorption of the compound then depends upon its rate of dissolution that, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered compound form is accomplished by dissolving or suspending the compound in an oil vehicle. Injectable depot forms are made by forming microencapsule matrices of the compound in biodegradable polymers such as polylactide-polyglycolide. Depending upon the ratio of compound to polymer and the nature of the particular polymer employed, the rate of compound release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the compound in liposomes or microemulsions that are compatible with body tissues.

[0219] In some embodiments, provided pharmaceutically acceptable compositions are formulated for oral administration. Such formulations may be administered with or without food. In some embodiments, pharmaceutically acceptable compositions described herein are administered without food. In other embodiments, pharmaceutically acceptable compositions described herein arc administered with food. Pharmaceutically acceptable compositions described herein may be orally administered in any orally acceptable dosage form including, but not limited to, capsules, tablets, aqueous suspensions or solutions. In the case of tablets for oral use, carriers commonly used include lactose and corn starch. Lubricating agents, such as magnesium stearate, are also typically added. For oral administration in a capsule form, useful diluents include lactose and dried cornstarch. When aqueous suspensions are required for oral use, the active ingredient is combined with emulsifying and suspending agents. If desired, certain sweetening, flavoring or coloring agents may also be added.

[0220] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active compound is mixed with at least one inert, pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and/or a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol, and silicic acid, b) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia, c) humectants such as glycerol, d) disintegrating agents such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, e) solution retarding agents such as paraffin, f) absorption accelerators such as quaternary ammonium compounds, g) wetting agents such as, for example, cetyl alcohol and glycerol monostearate, h) absorbents such as kaolin and bentonite clay, and/or i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets and pills, the dosage form may also comprise buffering agents.

[0221] Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular’ weight polyethylene glycols and the like. The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical formulating art. They may optionally contain opacifying agents and can also be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polethylene glycols and the like.

[0222] The active compounds can also be in micro-encapsulated form with one or more excipients as noted above. The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings, release controlling coatings and other coatings well known in the pharmaceutical formulating art. In such solid dosage forms the active compound may be admixed with at least one inert diluent such as sucrose, lactose or starch. Such dosage forms may also comprise, as is normal practice, additional substances other than inert diluents, e.g., tableting lubricants and other tableting aids such a magnesium stearate and microcrystalline cellulose. In the case of capsules, tablets and pills, the dosage forms may also comprise buffering agents. They may optionally contain opacifying agents and can also be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes.

[0223] Liquid dosage forms for oral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active compounds, the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

[0224] Alternatively, pharmaceutically acceptable compositions described herein may be administered in the form of suppositories for rectal administration. These can be prepared by mixing the agent with a suitable non-irritating excipient that is solid at room temperature but liquid at rectal temperature and therefore will melt in the rectum to release the drug. Such materials include cocoa butter, beeswax and polyethylene glycols.

[0225] Compositions for rectal or vaginal administration are preferably suppositories which can be prepared by mixing the compounds described herein with suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol or a suppository wax which are solid at ambient temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active compound.

[0226] Pharmaceutically acceptable compositions described herein may also be administered topically, especially when the target of treatment includes areas or organs readily accessible by topical application, including diseases of the eye, the skin, or the lower intestinal tract. Suitable topical formulations are readily prepared for each of these areas or organs.

[0227] Topical application for the lower intestinal tract can be effected in a rectal suppository formulation (see above) or in a suitable enema formulation. Topically-transdermal patches may also be used.

[0228] For topical applications, provided pharmaceutically acceptable compositions may be formulated in a suitable ointment containing the active component suspended or dissolved in one or more carriers. Carriers for topical administration of compounds described herein include, but are not limited to, mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compound, emulsifying wax and water. Alternatively, provided pharmaceutically acceptable compositions can be formulated in a suitable lotion or cream containing the active components suspended or dissolved in one or more pharmaceutically acceptable carriers. Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.

[0229] For ophthalmic use, provided pharmaceutically acceptable compositions may be formulated as micronized suspensions in isotonic, pH adjusted sterile saline, or, preferably, as solutions in isotonic, pH adjusted sterile saline, either with or without a preservative such as benzylalkonium chloride. Alternatively, for ophthalmic uses, the pharmaceutically acceptable compositions may be formulated in an ointment such as petrolatum.

[0230] Pharmaceutically acceptable compositions described herein may also be administered by nasal aerosol or inhalation. Such compositions are prepared according to techniques well-known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other conventional solubilizing or dispersing agents. [0231] Dosage forms for topical or transdermal administration of a compound disclosed herein include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants or patches. The active component is admixed under sterile conditions with a pharmaceutically acceptable carrier and any needed preservatives or buffers as may be required. Ophthalmic formulation, ear drops, and eye drops are also contemplated as being within the scope of this disclosure. Additionally, the present disclosure contemplates the use of transdermal patches, which have the added advantage of providing controlled delivery of a compound to the body. Such dosage forms can be made by dissolving or dispensing the compound in the proper medium. Absorption enhancers can also be used to increase the flux of the compound across the skin. The rate can be controlled by either providing a rate controlling membrane or by dispersing the compound in a polymer matrix or gel.

F1P200 and Autophagy for Treatment of Diseases

[0232] As described herein, the present disclosure provides compounds for use in promoting autophagy. In particular, the present disclosure encompasses, among other things, the insight that small molecule bifunctional binders of FIP200 can aggregate FIP200, thereby promoting formation of the autophagosome, which is a critical step in the autophagy pathway.

[0233] Augmenting, enhancing, or promoting autophagy, as used herein, refers to an increased flux in the autophagy pathway, which mimic’s the body’s natural starvation or exercise response. Promoting autophagy allows for increased clearance of defective organelles, protein aggregates, cellular components, and the like. Promotion of autophagy has potential benefit in treatment of disorders associated with protein aggregopathies, such as Alzheimer’s disease, Huntington’s disease, and al-antitrypsin deficiency; lysosomal storage diseases, such as Niemann Pick C, and Gaucher’s disease; infectious diseases, such as tuberculosis; and inflammatory diseases, such as Crohn’s disease.

[0234] Without wishing to be bound by theory, it is hypothesized that accumulation of cytotoxic/dysfunctional components in cells underlie many human diseases. For example, intracellular aggregates such as Lewy bodies, neurofibrillary tangles, and TDP43; dysfunctional cellular organelles such as mitochondria, lipid droplets, ribosomes, and endoplasmic reticulum; dysregulated or mutated proteins such a oncoproteins and MYC and KRAS; and invading pathogens such as bacteria and viruses, can all contribute to various diseases. Promoting autophagy clears said cytotoxic substrates and can restore cellular homeostasis.

[0235] For example, in some embodiments, the present disclosure provides a method of treating a disease, disorder, or condition by promoting autophagy in a subject. In some embodiments, said disease, disorder, or condition is neurodegenerative diseases, inflammatory diseases, muscle disorders, metabolic disorders, infectious diseases, oncology, and liver diseases.

[0236] Autophagy functions by sequestering substrates, such as said protein aggregates or cellular components, within an autophagosome, which is then delivered to the lysosome for degradation. Autophagy is induced by nutrient starvation. See Fujioka and Noda, Curr. Opin. in Cell Bio., 69:23-29 (2021). As part of the process, ULK1 autophosphorylates and activates itself through clustering promoted by FIP200, ATG13, and ATG101, and the corresponding complex (the ULK1 complex) has been found to be critical to autophagy induction. See Ganley, et al. , Molecular Basis of Cell and Developmental Biology, 284(18):P12297-12305 (May 2009). In a specialized form of autophagy, known as selective autophagy, p62 recruits FIP200 as part of the ULK1 complex to ubiquitin-positive condensates. The recruitment through FIP200 causes the ULK1 complex to form condensates, which leads to the ultimate formation of the autophagosome. See Turco, et al., Molecular Cell, 74: 330-346 (2019).

[0237] The present disclosure encompasses, among other things, an insight that forming FIP200 dimers, trimers, or tetramers, for example through the use of small molecule binders of FIP200, promotes activation of ULK1. Multiple types of dimers of FIP200 have been identified through the use of small molecule compounds described herein, for example, as illustrated in FIG. 1.

[0238] Accordingly, compounds described herein are useful for the treatment of a variety of diseases, disorders, and conditions associated with autophagy, for example, a protein aggregopathy, a lysosomal storage disease, an infectious disease, or an inflammatory disease.

[0239] In some embodiments, a protein aggregopathy is Alzheimer’s disease, Huntington’s disease, Parkinson’s disease, amyotrophic lateral sclerosis, dementia with Lewy Bodies, cystic fibrosis, frontotemporal dementia, amyloid transthyretin cardiomyopathy, type-2 diabetes, and an al -antitrypsin deficiency.

[0240] In some embodiments, a lysosomal storage disease is Niemann-Pick disease type C, Gaucher disease, Fabry disease, cystinosis, Pompe disease, Tay Sachs disease, Sandhoff disease, metachromatic leukodystrophy, mucolipidosis, mucopolysaccharide storage disease, and Schindler disease.

[0241] In some embodiments, an infectious disease is tuberculosis, viral infections including influenza, HIV, Hepatitis C Virus and herpesvirus, salmonella, listeria, toxoplasma gondii, chlamydia, and leishmania.

[0242] In some embodiments, an inflammatory disease is Crohn’s disease, ulcerative colitis, rheumatoid arthritis, systemic lupus erythematosus, psoriasis, gout, atherosclerosis, neurodegenerative diseases, cystic fibrosis, age-related macular- degeneration, and asthma.

EXEMPLARY EMBODIMENTS

[0243] The present disclosure includes the following non-limiting enumerated embodiments.

Embodiment 1. A compound represented by formula I:

A¹-B-A² or a pharmaceutically acceptable salt thereof, wherein:

Embodiment 2. The compound of Embodiment 1, wherein A¹ and A² are each independently selected from formula 11-1, 11-2, 11-3, or 11-4:

5- to 6-membered heteroaryl; each X¹ is independently S, N(R³), O, optionally substituted C₁-C₆ aliphatic, or optionally substituted C₃-C₆ cycloaliphatic; each X² is independently C(R³) or N, provided that, when X² is N, then X¹ is optionally substituted C₁-C₆ aliphatic, or optionally substituted C₃-C₆ cycloaliphatic; when a bond between X³ and X⁴ is a single bond, then X³ is N(R^2a), and X⁴ is C(O) when a bond between X³ and X⁴ is a double bond, then X³ is C(R^2b) and X⁴ is C(R³), or N; each R¹ is independently selected from halogen, optionally substituted C₁-C₆ aliphatic, and optionally substituted -O-C₁-C₆ aliphatic; or two instances of R¹ come together with the atoms to which they are attached to form a n optionally substituted C₆-C₁₂ aryl ring, an optionally substituted C₄-C₆ cycloaliphatic ring, a 5- to 6- membered heteroaryl ring comprising 1 to 3 heteroatoms selected from N, O, and S, or an optionally substituted 5- to 6-membered heterocyclic ring comprising 1 to 3 heteroatoms selected from N, O, and S; each R^2a is independently optionally substituted C₁-C₆ aliphatic; each R^2b is independently hydrogen, optionally substituted C₁-C₆ aliphatic, optionally substituted -N(R³)-C₁-C₆ aliphatic, or optionally substituted -O-C₁-C₆ aliphatic; each R is independently selected from hydrogen, halogen, and optionally substituted Ci- C₆ aliphatic; each R⁴ is independently selected from optionally substituted C₆-C₁₂ aryl, optionally substituted 5- to 12- membered heteroaryl ring comprising 1 to 3 heteroatoms selected from N, O, and S, optionally substituted 4- to 12-membered heterocyclyl comprising 1 to 3 heteroatoms selected from N, O, and S, and optionally substituted C₁-C₆ aliphatic; each R⁵ is independently an optionally substituted 4- to 6-membered heterocyclic ring, an optionally substituted 5- to 6-membered heteroaryl ring, an optionally C₃-C₆ cycloaliphatic ring, an optionally substituted C₆-C₁₂ aryl, or an optionally substituted C₁-C₆ aliphatic; each L¹ is independently a bond, -C(O)-, -S(O)-, -S(O)₂-, or -NR³-; each n is independently 0, 1, 2, 3, 4, 5, or 6; each X⁵, X⁶, and X⁷ are independently selected from the group consisting of N and CH; each R⁷ is independently hydrogen, an optionally substituted -O- C₁-C₆, aliphatic, - S(O)₂R³, optionally substituted C₁-C₆ aliphatic, an optionally substituted 4- to 12- membered heterocycle ring comprising 1 to 4 heteroatoms selected from N, O, and S, an optionally substituted C₆-C₁₂ aryl, or an optionally substituted 5- to 12-membered heteroaryl comprising 1 to 4 heteroatoms selected from N, O, and S each G² is independently an optionally substituted C₆-C₁₂ aryl, optionally substituted 4- to 12-membered heterocycle comprising 1 to 4 heteroatoms selected from N, O, and S, optionally substituted 5- to 6-membered heteroaryl comprising 1 to 4 heteroatoms selected from N, O, and S, or optionally substituted C₃-C₆ cycloaliphatic; each G³ is independently an optionally substituted 4- to 12- membered heterocycle ring comprising 1 to 4 heteroatoms selected from N, O, and S, an optionally substituted 5- to 6-membered heteroaryl ring comprising 1 to 4 heteroatoms selected from N, O, and S, an optionally substituted C₃-C₆ cycloaliphatic ring or optionally substituted C₆-C₁₂ aryl; each L² is independently a bond, -NR³-C(O)-, -C(O)-NR³-, optionally substituted C₁-C₆ aliphatic, optionally substituted 4- to 6-membered heterocyclic, or optionally substituted C₃-C₆ cycloaliphatic; each L³ is independently -NR³-, -O-, -C(O)-, -NR³-C(O)-, -NR³-S(O)₂-, -C(O)-NR³-, - S(O)₂ NR³-, -NR³-C(O)-NR³-, optionally substituted C₁-C₆ aliphatic, or an optionally substituted C₃-C₆ cycloaliphatic ring;

* represents a point of attachment to moiety B; and wherein: when A¹ or A² is a moiety of formula IT-3 then R⁶ is a bond, optionally substituted C₁-C₆ aliphatic, optionally substituted 2- to 6-mcmbcrcd heteroaliphatic, optionally substituted 4- to 6-membered heterocycle comprising one 1 to 3 heteroatoms selected from N, O, and S, optionally substituted C₆-C₁₂ aryl, or optionally substituted C₃-C₆ cycloaliphatic; and R⁸ is independently halogen, -OR³, -C(O)N(R³)₂, -C(O)OR³, optionally substituted C₁-C₆ aliphatic, optionally substituted C₃-C₆ cycloaliphatic, optionally substituted C₆-C₁₂ aryl; and when A¹ or A² is of formula II-4, then R⁶ is H, halogen, optionally substituted C₁-C₆ aliphatic, optionally substituted 2- to 6-membered heteroaliphatic, optionally substituted 4- to 6-membered heterocycle comprising one 1 to 3 heteroatoms selected from N, O, and S, optionally substituted C₆-C₁₂ aryl, or optionally substituted C₃-C₆ cycloaliphatic; and R⁸ is a bond, -O-, -C(O)NR³-, -C(O)OR³, optionally substituted C₁-C₆ aliphatic, optionally substituted C₃-C₆ cycloaliphatic, or optionally substituted C₆-C₁₂ aryl.

Embodiment 3. The compound of Embodiments 1 or 2, wherein A¹ and A² are the same.

Embodiment 4. The compound of Embodiments 1 or 2, wherein A¹ and A² are different.

Embodiment 5. The compound of any one of Embodiments 2-4, wherein two R¹ come together, with the atoms to which they are attached, to form an optionally substituted C₆- C12 aryl ring or a 5- to 6-membered heteroaryl ring comprising 1 to 3 heteroatoms selected from N, O, and S.

Embodiment 6. The compound of any one of Embodiments 2-5, wherein n is 2 and each R¹ is halogen.

Embodiment 7. The compound of any one of Embodiments 2-5, wherein each R¹ is chloride. Embodiment 8. The compound of any one of Embodiments 2-7, wherein a bond between X⁴ and X³ is a single bond.

Embodiment 9. The compound of any one of Embodiments 2-7, wherein a bond between X⁴ and X³ is a double bond, X³ is C(R^2b) and X⁴ is N.

Embodiment 10. The compound of any one of Embodiments 2-9, wherein X¹ is S.

Embodiment 11. The compound of any one of Embodiments 2- 10, wherein R⁴ is optionally substituted phenyl or optionally substituted 5- to 6-membered heteroaryl comprising 1 to 3 heteroatoms selected from N, O, and S.

Embodiment 12. The compound of any one of Embodiments 2-11, wherein a moiety:

is:

Embodiment 13. The compound of any one of Embodiments 2-12, wherein G¹ is phenyl or naphthyl.

Embodiment 14. The compound of any one of Embodiments 2-13, wherein R⁵ is optionally substituted 5- to 6-membered heterocycle comprising 1 to 3 heteroatoms selected from N, O, and S.

Embodiment 15. The compound of any one of Embodiments 2-14, wherein L¹ is -C(O)-.

Embodiment 16. The compound of Embodiment 2, wherein A¹ and/or A² arc of formula II-

1 or II-2, X⁴ is C(O), a bond between X⁴ and X³ is a single bond, G¹ is napthyl, X¹ is S, X² is C(R³), R⁴ is optionally substituted phenyl, and R⁵ is optionally substituted 5- to 6- mcmbcrcd heterocycle.

Embodiment 17. The compound of Embodiment 2, wherein X⁵ and X⁷ are each N and X⁶ is CH.

Embodiment 18. The compound of Embodiments 2 or 17, wherein G³ is an optionally substituted 4- to 12-membered heterocycle ring comprising 1 to 4 heteroatoms selected from N, O, and S.

Embodiment 19. The compound of Embodiment 18, wherein G³ is optionally substituted 4- to 6-membered heterocycle comprising 1 to 3 heteroatoms selected from N, O, and S.

Embodiment 20. The compound of Embodiment 18, wherein G³ is optionally substituted 6- to 12-membered bicyclic heterocycle comprising 1 to 4 heteroatoms selected from N, O, and S.

Embodiment 21. The compound of any one of Embodiments 2 or 17-20, wherein L² is optionally substituted C₁-C₆ aliphatic.

Embodiment 22. The compound of any one of Embodiments 2 or 17-20, wherein L² is a bond or selected from:

-CH₂-,

Embodiment 23. The compound of Embodiment 22, wherein L² is:

Embodiment 24. The compound of any one of Embodiments 2 or 17-23, wherein G² is optionally substituted C₆-C₁₂ aryl or optionally substituted 5- to 6-mcmbcrcd heteroaryl comprising 1 to 4 heteroatoms selected from N, O, and S.

Embodiment 25. The compound of any one of Embodiments 2 or 17-24, wherein R⁷ is optionally substituted 4- to 12-membered heterocycle comprising 1 to 4 heteroatoms selected from N, O, and S.

Embodiment 26. The compound of Embodiment 2, wherein A¹ and/or A² are of formula II- 3 or II-4, X⁵ and X⁷ are each N, X⁶ is CH, G³ is optionally substituted 4- to 12-membered heterocycle comprising 1 to 4 heteroatoms selected from N, O, and S, L² is optionally substituted C₁-C₆ aliphatic, L³ is -NR³-C(O)- or -C(O)-NR³-, G² is optionally substituted phenyl, and R⁷ is optionally substituted 4- to 12-membered heterocycle comprising 1 to 4 heteroatoms selected from N, O, and S.

Embodiment 27. The compound of Embodiment 2, wherein a moiety:

Embodiment 28. The compound of Embodiment 2, wherein a moiety:

is:

Embodiment 29. The compound of Embodiment 2, wherein A¹ and A² are each independently selected from:

Embodiment 30. The compound of Embodiment 2, wherein A¹ and A² are each independently selected from:

Embodiment 31. The compound of any one of Embodiments 1-30, wherein B is a linker moiety that is an optionally substituted C2-30 aliphatic group wherein one or more carbons are optionally and independently replaced by -Cy-, -NR^Z-, -N(R^Z)C(O)-, -C(O)N(R^Z)-, - N(R^Z)C(O)O-, -OC(O)N(R^Z)-, -N(R^Z)C(O)N(R^Z) -, -OC(O)O-, -O-, -C(O)-, -OC(O)-, - C(O)O-, -SO-, -SO₂-, wherein each -Cy- is independently an optionally substituted 3-12 membered heterocyclyl ring having 1-3 heteroatoms selected from N, O, and S, an optionally substituted 3-8 membered heteroaryl ring having 1-4 heteroatoms selected from N, O, and S, an optionally substituted C₃-C₆ cycloalkyl, or an optionally substituted C₆-C₁₂ aryl, and each R^z is independently H or an optionally substituted group selected from Ci- C20 aliphatic, or C3-C12 cycloaliphatic.

Embodiment 32. The compound of any one of Embodiments 1-30, wherein B is selected from Table B. Embodiment 33. The compound of Embodiment 2, wherein the compound is represented by formula III- 1 :

III-l or a pharmaceutically acceptable salt thereof.

Embodiment 34. The compound of Embodiment 2, wherein the compound is represented by formula III-2:

III-2 or a pharmaceutically acceptable salt thereof.

Embodiment 35. The compound of Embodiment 2, wherein the compound is represented by formula III- 3:

III-3 or a pharmaceutically acceptable salt thereof. Embodiment 36. The compound of Embodiment 2, wherein the compound is represented by formula III-4:

III-4 or a pharmaceutically acceptable salt thereof.

Embodiment 37. The compound of Embodiment 2, wherein the compound is represented by formula III- 5:

III-5 or a pharmaceutically acceptable salt thereof.

Embodiment 38. The compound of Embodiment 2, wherein the compound is represented by formula IV- 1:

IV- 1 or a pharmaceutically acceptable salt thereof.

Embodiment 39. The compound of Embodiment 2, wherein the compound is represented by formula IV-2:

or a pharmaceutically acceptable salt thereof.

Embodiment 40. The compound of Embodiment 2, wherein the compound is represented by formula IV-3:

IV-3 or a pharmaceutically acceptable salt thereof.

Embodiment 41. The compound of Embodiment 2, wherein the compound is represented by formula IV-4:

IV-4 or a pharmaceutically acceptable salt thereof.

Embodiment 42. The compound of Embodiment 2, wherein the compound is selected from Table 1.

Embodiment 43. A pharmaceutical composition comprising a compound of any one of Embodiments 1-42, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, diluent, or excipient.

Embodiment 44. A method of treating a disease, disorder, or condition in a subject comprising administering to the subject a compound of any one of Embodiments 1-42 or a pharmaceutical composition of Embodiment 43.

Embodiment 45. The method of Embodiment 44, wherein the disease, disorder, or condition is selected from a protein aggregopathy, a lysosomal storage disease, an infectious disease, or an inflammatory disease.

Embodiment 46. The method of Embodiment 45, wherein the protein aggregopathy is Alzheimer’s disease, Huntington’s disease, Parkinson’s disease, amyotrophic lateral sclerosis, dementia with Lewy Bodies, cystic fibrosis, frontotemporal dementia, amyloid transthyretin cardiomyopathy, type-2 diabetes, and an al -antitrypsin deficiency. Embodiment 47. The method of Embodiment 45, wherein the lysosomal storage disease is Niemann-Pick disease type C, Gaucher disease, Fabry disease, cystinosis, Pompe disease, Tay Sachs disease, Sandhoff disease, metachromatic leukodystrophy, mucolipidosis, mucopolysaccharide storage disease, and Schindler disease.

Embodiment 48. The method of Embodiment 45, wherein the infectious disease is tuberculosis, viral infections (e.g., influenza, HIV, Hepatitis C Virus and herpesvirus), salmonella, listeria, toxoplasma gondii, chlamydia, and leishmania.

Embodiment 49. The method of Embodiment 45, wherein the inflammatory disease is Crohn’s disease, ulcerative colitis, rheumatoid arthritis, systemic lupus erythematosus, psoriasis, gout, atherosclerosis, neurodegenerative diseases, cystic fibrosis, age-related macular degeneration, and asthma.

Embodiment 50. A method of promoting autophagy in a subject comprising administering to the subject a compound of any one of Embodiments 1-42, or a pharmaceutical composition of Embodiment 43.

Embodiment 51. Use of a compound of any one of Embodiments 1-42 or a pharmaceutical composition of Embodiment 43 for the treatment of a disease, disorder, or condition.

Embodiment 52. Use of a compound of any one of Embodiments 1-42 or a pharmaceutical composition of Embodiment 43 for promoting autophagy in a subject.

EXAMPLES

[0244] As described in the Examples below, in certain exemplary embodiments, compounds are prepared according to the following general procedures. It will be appreciated that, although the general methods depict the synthesis of certain compounds of the present disclosure, the following general methods and other methods known to one of ordinary skill in the art can be applied to all compounds and subclasses and species of each of these compounds, as described herein.

Table of Abbreviations

ACN: acetonitrile

CDI: l,l'-carbonyldiimidazole

DBU : 1 ,8-diazabicyclo(5.4.0)undec-7 -ene

DCM: dichloromethane

DDQ: 2,3-dichloro-5,6-dicyano- 1 ,4-benzoquinone

DEA: diethanolamine

DIAD: diisopropyl azodicarboxylate

DIEA, DIPEA: N,N-diisopropylethylamine

DMF: dimethylformamide

DMSO: dimethyl sulfoxide dppf: l,l' -bis(diphenylphosphino)ferrocene

EDC: 1 -ethyl-3-(3-dimethylaminopropyl)carbodiimide equiv. or eq.: equivalent or equivalents h or hr: hour or hours

HATU: l-[Bis(dimethylamino)methylene]-lH-l,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate

HEP: heptane

HOBt: hydroxy benzotriazole

HPLC: high performance liquid chromatography

IPA: isopropyl alcohol

LDA: lithium diisopropylamidc

LCMS: liquid chromatography mass spectrometry

LiHDMS: lithium bis(trimethylsilyl)amide m-CPBA: meta-chloroperbenzoic acid

MTBE: methyl tert-butyl ether

NMP: N-methyl-2-pyrrolidone

NMR: nuclear magnetic resonance

PE: petroleum ether

RT or rt: room temperature TBAB: tetrabutylazanium bromide

TBAF: tctrabutylammonium fluoride

Synthesis of Certain Intermediates

INT-1. Synthesis of l-[2-(2-aminoethoxy) ethoxy] -6-chlorohexane hydrochloride, INT-1

Step-1: Synthesis of tert-butyl (2-(2-((6-chlorohexyi) ' oxy) ethoxy) ethyl) carbamate

[0245] To a solution of tcrt-butyl N-[2-(2-hydroxycthoxy)cthyl]carbamatc(10.0 g, 48.7 mmol) in THF (100 mL) was added NaH (2.33 g, 97.4 mmol, 60% in a mineral oil) at 0 °C. The mixture was stirred for 1.0 hour at 0 °C, and after was added l-chloro-6-iodohexane (12.0 g, 48.7 mmol). The mixture was stirred for 16 h at room temperature. The resulting mixture was concentrated in vacuo and then the residue was purified by silica gel chromatography and eluted with (PE:EA=1 : 1) to afford tert-butyl N-[2-(2-hydroxyethoxy)ethyl]carbamate (10.0 g, 48.7 mmol, 90% purity, 45.8% yield) as a yellow oil.

Step-2. Synthesis of l-[2-(2-aminoethoxy) ethoxy] -6-chlorohexane hydrochloride, INT-1

[0246] A solution of tert-butyl N-(2-{2-[(6-chlorohexyl)oxy]ethoxy]ethyl)carbamate (10.0 g, 30.8 mmol) in HCI/dioxane (4 mol/L 50 mL) was stirred for 2 hours at room temperature. The resulting mixture was concentrated in vacuo to afford l-[2-(2-aminoethoxy)ethoxy]-6- chlorohexane hydrochloride (7.00 g, 26.9 mmol, crude) as a brown solid, which was used for next step directly. INT-2. Synthesis of 2-((3-ethyl-4-oxo-3,4-dihydrobenzo[g]quinazolin-2-yl)thio)-2- phenylacetic acid

Step-1. Synthesis of bromo(phenyl)acetic acid

[0247] To a solution of NaNO₂ (219.1 g, 3175 mmol, 1.60 equiv) in water (600 mL) was slowly added (+/-)-a-phenylglycine (300.0 g, 1984 mmol, 1.00 equiv) in HBr in water (5L, 30%) at -15°C under nitrogen atmosphere. The reaction was allowed to proceed for 3 h and warmed from - 15°C to room temperature. The reaction mixture was extracted by diethyl ether (3x2L). The combined organic layers were washed with brine (1x800 mL), dried over anhydrous Na₂SO₄, filtered and concentrated under reduced pressure to afford product bromo(phenyl)acetic acid (310 g, 69%) as a brown oil, which was used for next step without purification.

Step-2. 3 -ethyl-2-sulfanylbenzo[ g ]quinazolin-4-one

[0248] To a mixture of 3-amino-2-naphthoic acid (250 g, 1335 mmol, 1.00 equiv) and TEA (297.3 g, 2.94 mol, 2.20 equiv) in anhydrous EtOH (8 L) was added isothiocyanatoethane (128.0 g, 1.47 mol, 1.10 equiv). The mixture was refluxed until the starling material was consumed (16 h; TLC, ethyl acetate/methanol, 99.9:0.1) and cooled to room temperature. The formed precipitate was filtered and washed with cold ethanol (2x500 mL) to give 3-ethyl-2-sulfanylbenzo[g]quinazolin- 4-one (260 g, 71 %) as a light-yellow solid.

Step-3a. Synthesis of ({3-ethyl-4-oxobenzo[g]quinazolin-2-yl}sulfanyl)(phenyl)acetic acid

[0249] To a suspension of 3-ethyl-2-sulfanylbenzo[g]quinazolin-4-one (250 g, 975 mmol, 1.00 equiv) in MeCN (5000 mL) was added NaH (58.5 g, 1463 mmol, 1.50 equiv, 60%) in portions at room temperature under N2 atmosphere. The reaction was stirred for 3.0 h at room temperature, bromo(phenyl)acetic acid (293.6 g, 1365 mmol, 1.40 equiv) in MeCN (800 mL) was added dropwise. The mixture was stirred for 5 h. The reaction was quenched by the addition of HC1 (6 M in water, 300 mL) and diluted with water (5 L). The precipitated solids were collected by filtration and washed with water (3x100 mL). The resulting solid was dried under infrared light. This resulted in ({3-ethyl-4-oxobenzo[g]quinazolin-2-yl}sulfanyl)(phenyl)acetic acid (211 g, 50%) as a yellow solid, INT-2. Yield: 211 g, 50.0%; Appearance: Yellow solid; ¹H NMR (300 MHz, DMSO-d₆) δ 13.24 (s, 1H), 8.81 (s, 1H), 8.20 (d, J= 8.3 Hz, 1H), 8.11 (d, J = 8.4 Hz, 1H), 8.08 (s, 1H), 7.67 (ddd, J = 8.3, 6.7, 1.3 Hz, 1H), 7.63-7.54 (m, 3H), 7.48-7.37 (m, 3H), 5.70 (s, 1H), 4.10 (q, J = 7.3 Hz, 2H), 1.30 (t, 7= 7.0 Hz, 3H). HPLC purity: 99.5%; LCMS Calculated for C₂₂H₁₈N₂O₃S: 390.10; Observed: 391.1 [M+H]⁺.

Step-3b. Synthesis of ethyl 2-({3-ethyl-4-oxo-3H,4H-benzo[g]quinazolin-2-yl}sulfanyl)-2- phenylacetate

[0250] To a 2-L 4-necked round-bottom flask were added 3-ethyl-2-sulfanyl-3H,4H- benzo[g]quinazolin-4-one (20.0 g, 78.0 mmol), DIEA (30.0 g, 233.0 mmol) and ethyl 2-chloro-2- phenylacetate (30.9 g, 156.0 mmol) in DMSO (500 mL) at room temperature. The reaction mixture was stirred for 16 hours at 50 °C. The resulting mixture was allowed to cool down to room temperature and diluted with H₂O (2L). The formed solid was filtered, washed with distilled water (50 mL x 3), and dried at room temperature to give ethyl 2-({3-ethyl-4-oxo-3H,4H- benzo[g]quinazolin-2-yl}sulfanyl)-2-phenylacetate (30.0 g, 71.6 mmol, 92.0% yield) as a yellow solid.

Step-4. Synthesis of 2-({3-ethyl-4-oxo-3H,4H-benzo[g]quinazolin-2-yl}sulfanyl)-2-phenylacetic acid, INT-2 [0251] To a solution of ethyl 2-({3-ethyl-4-oxo-3H,4H-benzo[g]quinazolin-2-yl}sulfanyl)-2- phcnylacctatc (30.0 g, 71.6 mmol) in EtOH:H₂O (4:1, 1 L) was added LiOH (5.7 g, 143.0 mmol) and stirred for 16 hours at room temperature. The resulting mixture was adjusted pH=3 with 2M HC1, the precipitated solids were collected by filtration and washed with water (3 x 50 mL). The resulting solid was dried under infrared light. This resulted in 2-({3-ethyl-4-oxo-3H,4H- benzo[g]quinazolin-2-yl}sulfanyl)-2-phenylacetic acid (17.0 g, 43.5 mmol, 60.9% yield) as a yellow solid.

INT-3 & INT-4. Synthesis of ((R)-2-((3-ethyl-4-oxo-3,4-dihydrobenzo[g]quinazolin-

2-yl)thio)-2-phenylacetyl)-D-proline, INT-3, & ((S)-2-((3-ethyl-4-oxo-3,4- dihydrobenzo[g]quinazolin-2-yl)thio)-2-phenylacetyl)-D-proline, INT-4,

[0252] To a solution of ({3-ethyl-4-oxobenzo[g]quinazolin-2-yl}sulfanyl)(phenyl)acetic acid (40 g, 102.443 mmol, 1 equiv), HATU (46.74 g, 122.93 mmol, 1.2 equiv) in DCM (1 L), was added DIEA (39.72 g, 307.33 mmol, 3 equiv). After stirring for 30 mins, tert-butyl (2R)- pyrrolidine-2-carboxylate (19.30 g, 112.69 mmol, 1.1 equiv) was added. The reaction was stirred at 50 °C for 16 h under N2 atmosphere. The reaction mixture was concentrated to dryness and purified by silica gel column chromatography (Ethyl acetate: Petroleum ether =30% - 90%) to give product tert-butyl (2R)- 1 - [2-( { 3-cthyl-4-oxobcnzo[g]quinazolin-2-yl} sulfanyl)-2- phenylacetyl]pyrrolidi ne-2-carboxylate (45 g, 80.80%) as a red solid.

Step-2. Synthesis of (2-((3-ethyl-4-oxo-3,4-dihydrobenzo[g]quinazolin-2-yl)thio)-2-phenylacetyl)- D-proline

[0253] A solution of tert-butyl (2R)-l-[(2R)-2-({3-ethyl-4-oxobenzo[g]quinazolin-2- yl}sulfanyl)-2-phenylacetyl]pyrrolidine-2-carboxylate (45 g, 82.769 mmol, 1 equiv) in TFA (140 mL) was stirred for 2.0 h at room temperature. The resulting mixture was concentrated in vacuo. The residue was diluted with H₂O (300 mL), extracted with EtOAc(100 mLx3), washed with brine(100 mL), dried over anhydrous sodium sulfate, filtered and concentrated in vacuo. The residue was purified by trituration with EtOAc( 100 mL) and filtered. The collected solids were dried in vacuo to afford (2R)-l-[(2R)-2-({ 3-ethyl-4-oxobenzo[g]quinazolin-2-yl}sulfanyl)-2- phenylacetyl]pyrrolidine-2-carboxylic acid (24 g, 59.47%) as a yellow solid.

Step-3. Synthesis of ((R)-2-((3-ethyl-4-oxo-3,4-dihydrobenzo[g]quinazolin-2-yl)th io)-2- phenylacetyl)-D-proline, INT-3 & ( (S)-2-( ( 3-ethyl-4-oxo-3,4-dihydrobenzo[g]quinazolin-2- yl)thio)-2-phenylacetyl)-D-proline, INT-4.

[0254] (2R)-l-[2-({3-ethyl-4-oxobenzo[g]quinazolin-2-yl]sulfanyl)-2-phenylacetyl]pyrr olidine- 2-carboxylic acid (24 g, 49.224 mmol, 1 equiv) was purified by Chiral-SFC under the following conditions: Column: CHIRAL ART Cellulose-SJ, 3*25 cm, 5 pm; Mobile Phase A: CO2, Mobile Phase B: MeOH-Preparative; Flow rate: 80 mL/min; Gradient: isocratic 45% B; Column Temperature(°C): 25; Back Pressure(bar): 100; Wave Length: 220 nm; RTl(min): 2.85; RT2 (min): 5.00; Sample Solvent: MeOH: DCM=2: 1; Injection Volume: 9 mL; Number Of Runs: 80. This resulted in (2R)-l-[(2R)-2-({3-ethyl-4-oxobenzo[g]quinazolin-2-yl}sulfanyl)-2- phenylacetyl]pyrrolidine -2-carboxylic acid, INT-3 (11.2 g, 46.67%) as a yellow solid, Yield: 10.8 g, 46.7%; Appearance: White solid; ¹H NMR (300 MHz, DMSO-d₆) δ 12.43 (s, 1H), 8.79 (d, J = 8.1 Hz, 1H), 8.27 - 8.14 (m, 2H), 8.13 - 7.94 (m, 1H), 7.75 - 7.51 (m, 4H), 7.46 - 7.28 (m, 3H), 6.12 (d, 7 = 42.0 Hz, 1H), 4.35 - 4.15 (m, 2H), 4.13 - 4.01 (m, 2H), 3.50 - 3.41 (m, 1H), 3.18 (d, J= 4.1 Hz, 1H), 2.14 - 2.04 (m, 1H), 1.99 - 1.86 (m, 2H), 1.27 (t, 7= 7.0 Hz, 3H). HPLC purity: 99.5%; LCMS Calculated for C₂₇H₂₅N₃O₄S: 487.16; Observed: 488.2 [M+H]⁺. This resulted in (S)-2-((3-ethyl-4-oxo-3,4-dihydrobenzo [g]quinazolin-2-yl)thio)-2-phenylacetyl)- D-prolinc, INT-4. Yield: 10.8 g, 46.7%; Appearance: White solid; ¹H NMR (300 MHz, DMSO- d6) 5 12.43 (s, 1H), 8.80 (d, 7 = 8.1 Hz, 1H), 8.21 - 8.18 (m, 2H), 8.12 - 7.96 (m, 1H), 7.70 - 7.53 (m, 4H), 7.44 - 7.31 (m, 3H), 6.12 - 6.08 (m, 1H), 4.35 - 4.15 (m, 2H), 4.14 - 4.04 (m, 2H), 3.48 - 3.41 (m, 1H), 3.17 (d, J = 4.1 Hz, 1H), 2.15 - 2.04 (m, 1H), 1.99 - 1.86 (m, 2H), 1.30 (t, 7= 7.0 Hz, 3H). HPLC purity: 99.5%; LCMS Calculated for C27H25N3O4S: 487.16; Observed: 488.2 [M+H]⁺.

INT-5 & INT-6. Synthesis of tert-butyl (((R)-l-((R)-2-((3-ethyl-4-oxo-3,4- dihydrobenzo[g]quinazolin-2-yl)thio)-2-phenylacetyl)pyrrolidin-2-yl)methyl)car hamate, INT-5 & tert-butyl (((R)-l-((S)-2-((3-ethyl-4-oxo-3,4- dihydrobenzo[g]quinazolin-2-yl)thio)-2-phenylacetyl)pyrrolidin-2- yl)methyl)carbamate, INT-6:

Step-1. Synthesis of tert-butyl (((2R)-l-(2-((3-ethyl-4-oxo-3,4-dihydroben zo[g]quinazolin-2- yl)thio)-2-phenylacetyl)pyrrolidin-2-yl)methyl)carbamate

To a solution of ({3-ethyl-4-oxobenzo[g]quinazolin-2-yl}sulfanyl)(phenyl)acetic acid (50 g, 128.054 mmol, 1 equiv) and HATU (58.43 g, 153.665 mmol, 1.2 equiv) in DCM (1 L) was added DIEA (49.65 g, 384.162 mmol, 3 equiv) at room temperature. After stirring for 30 mins, tert-butyl N-[(2R)-pyrrolidin-2-ylmethyl]carbamate (28.21 g, 140.859 mmol, 1.1 equiv) was added. The reaction was stirred at 50 °C for 16 h under N2 atmosphere. The reaction mixture was concentrated to dryness and purified by silica gel column chromatography ( Ethyl acetate : Petroleum ether =15% - 90%) to give product tert-butyl N-{ [(2R)-l-[2-({3-ethyl-4- oxobenzo[g]quinazolin-2-yl}sulfanyl)-2-phenylacetyl]pyrrolidin-2-yl]methyl}carbamate (25 g, 34.02%) as a red solid.

Step-2. Synthesis of tert-butyl (((R)-l-((R)-2-((3-ethyl-4-oxo-3,4-dihydrobenz.olg]quinaz.olin-2- yl)thio)-2-phenylacetyl)pyrrolidin-2-yl)methyl)carbam ate, INT-5, & tert-butyl (((R)-l-((S)-2-((3- ethyl-4-oxo-3,4-dihydr obenzo[ g ]quinazolin-2-yl )thio)-2 -phenylacetyl )pyrrolidin-2-yl )methyl) carbamate, INT-6,

[0255] tert-butyl N-{[(2R)-l-[2-({3-ethyl-4-oxobenzo[g]quinazolin-2-yl}sulfanyl)-2- phenylacetyl]pyrrolidin-2-yl]methyl}carbamate (25 g, 43.651 mmol, 1 equiv) was purified by Chiral-SFC under the following conditions: Column: CHIRALPAK IA, 5*25 cm, 5 p m; Mobile Phase A: CO2, Mobile Phase B: MEOH: DCM=2: 1(0.1% 2M NH3-MeOH); Flow rate: 150 mL/min; Gradient: isocratic 60% B; Column Temperature(°C): 35; Back Pressure(bar): 100; Wave Length: 220 nm; RTl(min): 3.32; RT2(min): 5.31; Sample Solvent: MeOH: DCM=2: 1; Injection Volume: 4.8 mL; Number Of Runs: 45. This resulted in tert-butyl N-{ [(2R)-l-[(2R)-2-({3-ethyl- 4-oxobenzo[g]quinazolin-2-yl}sulfanyl)-2-phenylacetyl]pyrrolidin-2-yl]methyl}carbamate, INT- 5,. Yield: 10.6 g, 42.4%; Appearance: Yellow solid; ¹H NMR (400 MHz, DMSO-d₆) δ 8.80 (d, J = 4.0 Hz, 1H), 8.20 (d, J = 8.3 Hz, 1H), 8.16 - 8.07 (m, 1H), 8.06 - 7.93 (m, 1H), 7.76 - 7.63 (m, 3H), 7.56 (ddd, J = 9.5, 7.2, 3.1 Hz, 1H), 7.48 - 7.29 (m, 3H), 6.78 (t, J = 5.9 Hz, 1H), 6.20 (d, J= 148.0 Hz, 1H), 4.09 (tq, J= 14.0, 7.5 Hz, 3H), 4.00 - 3.75 (m, 1H), 3.44 (t, J= 9.6 Hz, 1H), 3.31 - 2.61 (m, 1H), 2.08 (s, 1H), 2.00 - 1.73 (m, 2H), 1.40 (d, 7 = 40.0 Hz, 9H), 1.29 (t, 7 = 7.0 Hz, 3H). HPLC purity: 97.7%; LCMS Calculated for C₃₂H₃₆N₄O₄S: 572.25; Observed: 573.4 [M+H]⁺.

This resulted in tert-butyl (((R)-l-((S)-2-((3-ethyl-4-oxo-3,4-dihydroben zo[gj quinazolin-2- yl)thio)-2-phenylacetyl)pyrrolidin-2-yl)methyl)carbamate, INT-6 . Yield: 12.6 g, 50.4%; Appearance: Red solid; ¹H NMR (400 MHz, DMSO-d₆) 8 8.80 (d, 7 = 4.0 Hz, 1H), 8.20 (d, 7 = 8.3 Hz, 1H), 8.16 - 8.07 (m, 1H), 8.06 - 7.93 (m, 1H), 7.76 - 7.63 (m, 3H), 7.56 (ddd, 7 = 9.5, 7.2, 3.1 Hz, 1H), 7.48 - 7.29 (m, 3H), 6.78 (t, 7 = 5.9 Hz, 1H), 6.20 (d, 7 = 148.0 Hz, 1H), 4.09 (tq, 7 = 14.0, 7.5 Hz, 3H), 4.00 - 3.75 (m, 1H), 3.44 (t, J = 9.6 Hz, 1H), 3.31 - 2.61 (m, 1H), 2.08 (s, 1H), 2.00- 1.73 (m, 2H), 1.40 (d, J= 40.0 Hz, 9H), 1.29 (t, J= 7.0 Hz, 3H). HPLC purity: 97.5%; LCMS Calculated for C32H36N4O4S: 572.25; Observed: 573.4 [M+H]⁺.

INT-7. Synthesis of 2-(((R)-2-((R)-2-(aminomethyl)pyrrolidin-l-yl)-2-oxo-l- phenylethyI)thio)-3-ethylbenzo[g]quinazolin-4(3H)-one, INT-7:

Step-1. Synthesis of 2-(((R)-2-((R)-2-( aminomethyl )pyrrolidin-l -yl)-2-oxo-l -phenylethyl )thio )-3- ethylbenzo[g ]quinazolin-4( 3H)-one, INT- 7

[0256] A solution of tert-butyl (((R)-l-((R)-2-((3-ethyl-4-oxo-3,4-dihydrobenzo[g]quinazolin-2- yl)thio)-2-phenylacetyl)pyrrolidin-2-yl)methyl)carbamate (200 mg, 349 μmol) in TFA:DCM (1:5,

6 mL) was stirred for 1 hour at room temperature. The resulting mixture was concentrated in vacuo to afford 2-(((R)-2-((R)-2-(aminomethyl)pyrrolidin-l-yl)-2-oxo-l-phenylethyl)thio)-3-ethylben zo[g]quinazolin-4(3H)-one (155 mg, 327 μmol, 85% purity, 94% yield) as a colorless oil, which was used for next step directly.

INT-8. Synthesis of 2-(((S)-2-((R)-2-(aminomethyl)pyrrolidin-l-yl)-2-oxo-l- phenylethyl)thio)-3-ethylbenzo[g]quinazolin-4(3H)-one, INT-8:

Step-1. Synthesis of 2-(((S)-2-((R)-2-( aminomethyl )pyrrolidin- 1 -yl)-2 -oxo-1 -phenylethyl )thio )-3 - ethylbenzo [g]quinazolin-4( 3H)-one, INT-8

[0257] A solution of tert-butyl (((R)-l-((S)-2-((3-ethyl-4-oxo-3,4-dihydrobenzo[g]quinazolin-2- yl)thio)-2-phenylacetyl)pyrrolidin-2-yl)methyl)carbamate (200 mg, 349 μmol) in TFA:DCM (1:5, 12 mL) was stirred for 1 hour at room temperature. The resulting mixture was concentrated in vacuo to afford 2-(((S)-2-((R)-2-(aminomethyl)pyrrolidin-l-yl)-2-oxo-l-phenylethyl)thio)-3- ethylben zo[g]quinazolin-4(3H)-one (150 mg, 317 μmol, 95% purity, 91% yield) as a white oil, which was used for next step directly.

INT-9 & INT-10. Synthesis of ((R)-2-((6,7-dichloro-3-ethyl-4-oxo-3,4- dihydroquinazolin-2-yl)thio)-2-phenylacetyl)-D-proline, INT-9 & ((S)-2-((6,7- dichloro-3-ethyl-4-oxo-3,4-dihydroquinazolin-2-yl)thio)-2-phenylacetyl)-D-proline, INT-10:

Step-1. Synthesis of methyl 2-amino-4,5-dichlorobenzoate

[0258] To a 5 L 4-necked round-bottom flask were added methyl 2-amino-4-chlorobenzoate (400 g, 2.16 mol, 1.00 cquiv) and DMF (3 L). followed by NCS (288 g, 2.16 mol, 1.00 cquiv) at room temperature. The resulting mixture was stirred overnight at 50°C. The mixture was allowed to cool down to room temperature and then the resulting mixture was diluted with water (4 L) and extracted with ethyl acetate (2x3 L). The combined organic layers were washed with brine (2x2 L) anddricd over anhydrous Na₂SO₄. After filtration, the filtrate was concentrated under reduced pressure. The crude product was purified by Prep-HPLC with the following conditions (EA:PE=5:100) to afford methyl 2-amino-4,5-dichlorobenzoate (230 g, 1.04 mol, 95% purity, 48% yield) as a white solid.

Step-2. Synthesis of 2-amino-4,5-dichlorobenzoic acid

[0259] To a 5 L 4-necked round-bottom flask were added methyl methyl 2-amino-4,5- dichlorobenzoate (240 g, 1.09 mol, 1.00 equiv), MeOH (2000 mL), H₂O (500 mL) and LiOH (52.2 g, 2.18 mol, 2.00 equiv). The resulting mixture was stirred overnight at room temperature. The residue was acidified to pH 1 with HC1 (cone.). The resulting mixture was extracted with EA (2x2 L) and then the combined organic layers were washed with brine (2x2 L) and dried over anhydrous Na₂SO₄. After filtration, the filtrate was concentrated under reduced pressure to yield 2-amino- 4,5-dichlorobenzoic acid (220 g, 1.06 mol, 96% purity, 98% yield) as a white solid.

Step-3. Synthesis of 6, 7-dichloro-3-ethyl-2-sulfanylquinazolin-4-one

[0260] To a 10 L 4-necked round-bottom flask were added 2-amino-4,5-dichlorobenzoic acid (215 g, 1.04 mol, 1.00 equiv), isothiocyanatoethane (90.9 g, 1.04 mol, 1.00 equiv), EtOH (4000 mL) and TEA (264 g, 2.61 mol, 2.50 equiv). The resulting mixture was stirred overnight at 80°C. The mixture was allowed to cool down to room temperature. The precipitated solids were collected by filtration and washed with EtOH (2x200 mL) to yield6,7-dichloro-3-ethyl-2- sulfanylquinazolin-4-one (215 g, 781mmol, 98% purity, 75% yield) as a white solid.

Step-4. Synthesis of [(6,7-dichloro-3-ethyl-4-oxoquinazolin-2-yl)sulfanyl](phenyl) acetic acid

[0261] To a 5 L 4-necked round-bottom flask were added 6,7-dichloro-3-ethyl-2- sulfanylquinazolin-4-one (215 g, 781mmol, 1.00 equiv), 2-chloro-2-phenylacetic acid (133 g, 781 mmol, 1.00 equiv), DMSO (2000 mL) and DIEA (252.5 g, 1950 mmol, 2.50 equiv). The resulting mixture was stirred overnight at 50°C. The mixture was allowed to cool down to room temperature and then acidified to pH =3 with cone. HC1. The resulting mixture was extracted with CH₂Q2 (2x2 L). The combined organic layers were washed with brine (2x2 L) and dried over anhydrous Na₂SO₄. After filtration, the filtrate was concentrated under reduced pressure. The residue was stirred with ethyl acetate (500 mL). The precipitated solids were collected by filtration and washed with Et₂O (2x200 mL) to yield [(6,7-dichloro-3-ethyl-4-oxoquinazolin-2-yl) sulfanyl](phcnyl)acctic acid (198 g, 484 mmol, 97% purity, 62% yield) as a white solid.

Step-5. Synthesis of tert-butyl(2R )-l-{2-[ ( 6, 7-dichloro-3-ethyl-4-oxoquinazolin-2-yl)sulfanyl ]-2- phenyl acetyl }pyrrolidine-2-carboxylate

[0262] To a 2 L 4-necked round-bottom flask were added tert-butyl (2R)-pyrrolidine-2- carboxylate hydrochloride (22.8 g, 110 mmol, 1.00 equiv), [(6,7-dichloro-3-ethyl-4- oxoquinazolin-2-yl)sulfanyl](phenyl)acetic acid (45.0 g, 110 mmol, 1.00 equiv), THF (900 mL), DIEA (42.6 g, 330 mmol, 3.00 equiv) and HATU (54.4 g, 143 mmol, 1.30 equiv). The resulting mixture was stirred for 16 hours at 70°C, cooled to room temperature and then concentrated under reduced pressure. The resultant residue was purified by silica gel column chromatography, eluted with Petroleum ether/Ethyl acetate (1:1) to afford tert-butyl(2R)-l-{ 2-[(6,7-dichloro-3-ethyl-4- oxoquinazolin-2-yl)sulfanyl]-2-phenylacetyl}pyrrolidine-2-carboxylate (52.0 g, 92.4 mmol, 95% purity, 84% yield) as a white solid.

Step-6. Synthesis of (2R)-l-{2-[(6,7-dichloro-3-ethyl-4-oxoquin azolin-2-yl)sulfanyl]-2- phenylacetyl} pyroledine -2 -carboxylic acid

[0263] To a 1 L 4-necked round-bottom flask were added tert-butyl (2R)-l-{2-[(6,7-dichloro-3- ethyl-4-oxoquinazolin-2-yl)sulfanyl]-2-phenylacetyl]pyrrolidine-2-carboxylate (52.0 g, 92 mmol, 1 .0 equiv) and TFA (400 mL) at room temperature. The resulting mixture was stirred for 5 hours at room temperature, concentrated under reduced pressure, diluted with water (500 mL), and then extracted with ethyl acetate (2x500 mL). The combined organic layers were washed with brine (2x200 mL) and dried over anhydrous Na₂SO₄. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with Petroleum ether /ethyl acetate (1:2) to afford (2R)-l-{2-[(6, 7- dichloro-3-ethyl-4-oxoquinazolin-2-yl)sulfanyl]-2-phenylacetyl]pyrrolidine-2-carboxylic acid (35 g, 69.1 mmol, 88% purity, 75% yield) as a white solid.

Step-7. Synthesis of (2R)-l-[(2R)-2-[(6,7-dichloro-3-ethyl-4-oxoquinazolin-2-yl)sulfanyl] -2- phenylacetyl] pyrrolidine-2-carboxylic acid (1NT-9) and (2R)-1 -[(2S)-2-[(6,7-dichloro-3-ethyl-4- oxoquinazolin-2-yl)sulfanyl]-2-phenylacetyl]pyrrolidine-2-carboxylic acid (INT-10) [0264] The crude product (2R)-l-{2-[(6,7-dichloro-3-ethyl-4-oxoquin azolin-2-yl)sulfanyl]-2- phcnylacctyl}pyrrolidinc-2-carboxylic acid (30.0 g) was purified by CHIRAL-HPLC, Column: CHIRAL ART Cellulose-SJ, 3*25 cm, 5 pm; Mobile Phase A: CO₂, Mobile Phase B: IPA:ACN=1:1; Flow rate: 80 mL/min; Gradient: isocratic 30% B; Column Temperature(°C): 35; Back Pressure(bar): 100; Wave Length: 220 nm; RTl(min): 3.9; RT2(min): 5.2; Sample Solvent: IPA:ACN=1:1; Injection Volume: 2 mL; Number Of Runs: 170. This resulted in (2R)-l-[(2R)-2- [(6,7-dichloro-3-ethyl-4-oxoquinazolin-2-yl)sulfanyl]-2-phenylacetyl]pyrrolidine-2-carboxylic acid (INT-9, 25.6 mmol, 13.0 g, 99% purity, 43% yield) as a white solid and (2R)-l-[(2S)-2-[(6,7- dichloro-3-ethyl-4-oxoquinazolin-2-yl)sulfanyl]-2-phenylacetyl]pyrrolidine-2-carboxylic acid (INT-10, 12.0 g, 23.6 mmol, 99% purity, 40% yield) as a white solid.

INT-9: LC-MS Calculated for C₂₃H₂₁C₁₂N₃O₄S: 505.06; Observed: 506.06 [M+H]⁺, Peak I: Chiral-SFC: Column: IJ 100x4.6mm 3.0um; Mobile Phase A: CO₂, Mobile Phase B: ACN(0.1%TFA); Flow rate: 3.00 mL/min; Gradient: isocratic 10% B; Column Temperature(°C): 35; Back Pressure (bar): 15 MPa; Wave Length: 254 nm; RT1 (min): 2.058; RT2 (min): 2.778; Sample Solvent: ACN(0.1%TFA); Injection Volume: 1 pL; RT = 2.058 min, ee =100%. ¹H NMR: (300 MHz, DMSO-d₆₎ ppm) 5 8.17-8.08 (m, 1H), 7.97-7.89 (m, 1H), 7.72-7.60 (m, 2H),

7.46-7.36 (m, 3H), 7.36-7.28 (m, 1H), 6.05 (d, J = 2.9 Hz, 1H), 4.27-4.18 (m, 1H), 4.11 (td, J = 8.2, 7.1, 3.2 Hz, 1H), 4.04 (q, J = 7.0 Hz, 2H), 3.35 (d, J = 8.0 Hz, 1H), 2.04 (dq, J= 12.5, 8.5, 6.1 Hz, 2H), 1.95-1.83 (m, 2H), 1.24 (t, J = 7.0 Hz, 3H).

INT-10: LC-MS Calculated for C₂₃H₂₁C₁ N₃O₄S:505.06; Observed: 506.06[M+H]⁺, Peak II, Chiral-SFC: Column: IJ 100x4.6mm 3.0um; Mobile Phase A: CO₂, Mobile Phase B: ACN(0.1%TFA); Flow rate: 3.00 mL/min; Gradient: isocratic 10% B; Column Temperature(°C): 35; Back Pressure (bar): 15 MPa; Wave Length: 254 nm; RT1 (min): 2.058; RT2 (min): 2.778; Sample Solvent: ACN(0.1%TFA); Injection Volume: 1 pL; Number Of Runs: 12, solvent H, RT=2.778 min, ee = 99%.

^!HNMR: (300 MHz, DMSO-d₆, ppm) δ 8.15 (d, J = 3.9 Hz, 1H), 7.78 (s, 1H), 7.66-.56 (m, 2H),

7.46-7.31 (m, 3H), 6.03 (d, J = 50.3 Hz, 1H), 4.32 (ddd, J = 21.4, 7.8, 3.6 Hz, 1H), 4.03 (dtt, J = 6.9, 4.8, 2.5 Hz, 3H), 3.63-3.39 (m, 1H), 2.20 (dtd, J = 16.6, 9.2, 8.2, 3.5 Hz, 1H), 2.08-1.97 (m, 1H), 1.97-1.74 (m, 2H), 1.35-1.01 (m, 3H). INT-11: Synthesis of (2S,3aS,7aS)-l-(6-((S)-l-(4'-((tert-butoxycarbonyl)amino)-[l,r- biphenyl]-4-carboxamido)ethyl)-2-methylpyrimidin-4-yl)octahydro-lH-indole-2- carboxylic acid, INT-11:

Step-1. Synthesis of ethyl (4S)-4-{[(tert-butoxy)carbonyl]amino}-3-oxopentanoate

[0265] To a stirred solution of (2S)-2-{ [(ter/-butoxy)carbonyl]amino}propanoic acid (30.0 g, 158 mmol) in THF (300 mL) was added l-(lH-imidazole-l-carbonyl)-lH-imidazole (38.2 g, 236 mmol) at room temperature. The reaction was stirred for 1 hour at room temperature and then 1- ethyl 3-potassium propanedioate (40.1 g, 236 mmol) and dichloromagnesium (15.0 g, 158 mmol) were added. The reaction mixture was then quenched with saturated ammonium chloride aqueous solution (50.0 mL) and extracted with ethyl acetate (150 mL x 2). The combined organic layers were washed with brine (150 mL) and dried over anhydrous NaiSCL. After filtration, the filtrate was concentrated under reduced pressure to afford ethyl (4S)-4-{[(tert-butoxy)carbonyl] amino }- 3-oxopentanoate (40.0 g, 154 mmol, 95% purity, 97% yield)) as a red oil.

Step-2. Synthesis of tert-butyl (S )-(!-( 6-hydroxy-2-methylpyrimidin-4-yl )ethyl )carbamate [0266] To a stirred solution of ethanimidamide hydrochloride (5.45 g, 57.7 mmol) in methanol (100 mL) was added sodium mcthanolatc (30% in McOH) (10.3 g, 57.7 mmol) at room temperature. The reaction was stirred for 20 min at room temperature. Ethyl (4S)- 4-{ [(tert-butoxy)carbonyl]amino]-3-oxopentanoate (10 g, 38.5 mmol) was added and then the reaction mixture was stirred further for 16 hours at room temperature. The resulting mixture was filtered, the filter cake was washed with MeOH (20 mL), and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography and eluted with ethyl acetate (100%) to afford tert-butyl N-[( lS)-l-(6-hydroxy-2-methylpyrimidin-4- yl)ethyl]carbamate (6.50 g, crude). The crude product (6.5 g) was separated by the following condition: Column: CHIRAL ART Cellulose-SC, 3*25 cm, 5 pm; Mobile Phase A: CO₂, Mobile Phase B: IPA: HEX=1: 1(0.1% 2M NH₃-MeOH); Flow rate: 80 mL/min; Gradient: isocratic 40% B; Column Temperature(°C): 35; Back Pressure(bar): 100; Wave Length: 220 nm; RT2(min): 4.8; Sample Solvent: MeOH-- HPLC; Injection Volume: 1.5 mL; Number Of Runs: 40 to afford tert- butyl N-[(lS)-l-(6-hydroxy-2-methylpyrimidin-4-yl)ethyl]carbamate (4.50 g, 17.7 mmol, 95% purity, ee>95%, Rt = 2.648 min, 45% yield) as a white solid.

Chiral-SFC: Column Name : 1:SC 100x4.6mm 3.0um; Co Solvent : B:IPA (50%Hex); Start Cone, of Pump B : 10.0%; Total Flow : 3.0000 mL/min; BPR Pressure : 15.00 MPa; Oven Temperature : 35 C. Rt = 2.648 min.

Step-3. Synthesis of6-[( / S)-l -aminoethyl]-2-methylpyrimidin-4-ol

[0267] A solution of tert-butyl N-[(lS)-l-(6-hydroxy-2-methylpyrimidin-4- yl)ethyl]carbamate (4.00 g, 15.7 mmol) in trifluoro acetic acid (10.0 mL) and methylene chloride (40.0 mL) was stirred for 2 hours at room temperature. The resulting mixture was concentrated under reduced pressure. The crude residue was used to next reaction directly.

Step-4. Synthesis of tert-butyl N-(4'-{[(lS)-l-(6-hydroxy-2-methylpyrimidin-4- yl)ethyl]carbamoyl}-[ 1,1 '-biphenyl]-4-yl)carbamate

[0268] To a stirred solution of 6-[(lS)-l-aminoethyl]-2-methylpyrimidin-4-ol (3.90 g, crude, 15.5 mmol) and 4'-{ [(tert-butoxy)carbonyl]amino}-[l,T-biphenyl]-4-carboxylic acid (4.85 g, 15.5 mmol) in methylene chloride (40.0 mL) were added ethylbis(propan-2-yl)amine (10.0 g, 77.5 mmol) and l-[(dimethylamino)(dimethyliminiumyl)methyl]-3-oxo-lH,2H,3H-3V- [l ,2,3]triazolo[5,4-b]pyridin-3-ylium-2-ide and hexafluoro-λ.⁵-phosphanuide (7.03 g, 18.5 mmol) at room temperature. The resulting mixture was stirred for 2 hours at room temperature, the solids were collected by filtration and the fdter cake was washed with water (10 mL x 3) and DCM (10 mL x 3). The resulted solid was dried with an infrared lamp to afford tert-butyl N-(4'-{[(lS)- l-(6-hydroxy-2-methylpyrimidin-4-yl)ethyl]carbamoyl}-[l,T-biphenyl]-4-yl)cai'bamate (5.10 g, 11.3 mmol, 95% purity) as a grey solid.

Step-5. Synthesis of tert-butyl N-(4'-{[(lS)-l-[2-methyl-6-

( trifluoromethane sulfonyloxy )pyrimidin-4-yl ] ethyl ] carbamoyl} -[ 1, 1 '-biphenyl ]-4-yl )carbamate

[0269] To a stirred solution of tert-butyl N-(4'-{ [(lS)-l-(6-hydroxy-2-methylpyrimidin-4- yl)ethyl]carbamoyl}-[l,T-biphenyl]-4-yl)carbamate (6.00 g, 13.3 mmol) in pyridine (60 mL) was added trifluoromethanesulfonyl trifluoromethanesulfonate (11.2 g, 39.9 mmol) dropwise at 0 °C, and then the reaction mixture was stirred for 3 hours at the same temperature. The reaction was diluted with 50 mL of water and acidified with hydrochloric acid (1 M) to pH = 5-6. The resulting mixture was extracted with ethyl acetate (20 mL x 2) and the combined organic phase was washed with brine (2 mL). The organic phase was concentrated under reduced pressure and the residue was purified by silica gel column chromatography (ethyl acetate) to afford tert-butyl N-(4'-{[(lS)-l- [2-methyl-6-(trifluoromethanesulfonyloxy)pyrimidin-4-yl]ethyl] carbamoyl } - [ 1 , 1 '-biphenyl] -4- yl)carbamate (3.60 g, 6.20 mmol, 95% purity, 46.4% yield) as an off-white solid.

Step-6. Synthesis of (2S,3aS,7aS)-l -{6-[( 1 S)-l -[(4'-{[(tert-butoxy)carbonyl]amino}-[l , I '- biphenyl]-4-yl)formamido]ethyl]-2-methylpyrimidin-4-yl}-octahydro-lH-indole-2-carboxylic acid, INT-11

[0270] To a stirred solution of tert-butyl N-(4'-{ [(lS)-l-[2-methyl-6- (trifluoromethanesulfonyloxy)pyrimidin-4-yl]ethyl]carbamoyl}-[l,T-biphenyl]-4- yl)carbamate (2.00 g, 3.44 mmol) and ethylbis(propan-2-yl)amine (1.77 g, 13.7 mmol) in isopropyl alcohol (20 mL ) was added (2S,3aS,7aS)-octahydro-lH-indole-2-carboxylic acid (871 mg, 5.15 mmol). The reaction mixture was stirred for 0.5 hour at 80 °C and then diluted with 5 mL of water and acidified with hydrochloric acid (1 M) to Ph = 5-6. The resulting mixture was extracted with ethyl acetate (2 mL x 2), then the combined organic phase was washed with brine (2 mL) and concentrated under reduced pressure. The resultant residue was purified by silica gel column chromatography (ethyl acetate) to afford (2S,3aS,7aS)-l-{6-[(lS)-l-[(4'-{ [(tert- butoxy)carbonyl] amino }-[l , 1'-biphenyl]-4-yl)formamido]ethyl]-2-methylpyri midin-4-yl}- octahydro-lH-indolc-2-carboxylic acid, INT-11. Yield: 1.7 g, 82.5%; Appearance: White solid; ¹H NMR (400 MHz, DMSO-d₆) 59.50 (s, 1H), 8.85 (d, J= 6.9 Hz, 1H), 7.96 (d, J= 8.3 Hz, 2H), 7.77 (d, J = 8.2 Hz, 2H), 7.67 (d, J = 8.8 Hz, 2H), 7.58 (d, J = 8.8 Hz, 2H), 6.78 (s, 1H), 5.01 (s, 1H), 4.49 (t, J = 8.9 Hz, 1H), 4.03 (d, J= 6.3 Hz, 1H), 2.45 (s, 3H), 2.28 (s, 1H), 2.11 - 1.96 (m, 1H), 1.89 - 1.58 (m, 4H), 1.54 (d, J = 6.9 Hz, 3H), 1.50 (s, 9H), 1.46 - 1.10 (m, 5H). HPLC purity: 97.93%; LCMS Calculated for C34H41N5O5: 599.31; Observed: 600.4[M+H]⁺.

INT-12 & INT-13. Synthesis of (2R)-4-[(tert-butoxy)carbonyl]-l-[(2R)-2-({3-ethyl-4- oxo-3H,4H-benzo[g]quinazolin-2-yl}sulfanyl)-2-phenylacetyl]piperazine-2- carboxylic acid (peakl, INT-12) and (2R)-4-[(tert-butoxy)carbonyl]-l-[(2S)-2-({3- ethyl-4-oxo-3H,4H-benzo[g]quinazolin-2-yl}sulfanyl)-2-phenylacetyl] piperazine-2- carboxylic acid (peak2, INT-13):

Step-1. Synthesis of 1-tert-butyl 3-meth.yl (3R)-4-[2-({3-ethyl-4-oxobenzo[g]quinazolin-2- yl}sulfanyl)-2-phenylacetyl]piperazine-l,3-dicarboxylate

[0271] A mixture of ({3-ethyl-4-oxobenzo[g]quinazolin-2-yl}sulfanyl)(phenyl)acetic acid (5.0 g, 12.8 mmol), 1 -( rt-butyl) 3-methyl (R)-piperazine-l,3-dicarboxylate (3.8 g, 15.4 mmol) and DIEA (4.9 g, 38.4 mmol, 3.0 equiv) in DCE (70 mL) was stirred at room temperature for 10 min. HATU (5.8 g, 15.0 mmol, 1.2 equiv) was then added, and the reaction mixture was stirred for 16.0 hours at 80°C. The resulting mixture was cooled down to room temperature and concentrated in vacuo. The residue was purified by prep-HPLC (NH₃.H₂O buffer) to give 1 -tert-butyl 3-methyl (3R)-4-[2-({3-ethyl-4-oxobenzo[g]quinazolin-2-yl] sulf anyl)-2-phenylacetyl]piperazine- 1,3- dicarboxylate (3.6 g, 45.6% yield) as a yellow solid.

Step-2. Synthesis of ( 2R )-4-( tert-butoxy carbonyl )-l-[2-({ 3-ethyl-4-oxobenzo[g ]quinaz.olin-2- yl}sulfanyl)-2-phenylacetyl]piperazine-2-carboxylic acid

[0272] To a solution of 1-tert-butyl 3-methyl (3R)-4-[2-({3-ethyl-4-oxobenzo[g]quinazolin-2- yl}sulfanyl)-2-phenylacetyl]piperazine-l,3-dicarboxylate (3.6 g, 5.8 mmol, 1.0 equiv) in THF (30 mL) and H₂O (10 mL) was added LiOH.ELO (0.4 g, 17.0 mmol, 3.0 equiv) at room temperature. The resulting mixture was stirred for 2 hours at room temperature and adjusted to pH 3 with 2M HC1. The precipitated solids were collected by filtration, washed with water (3 x 20 mL) and then dried under infrared light to afford (2R)-4-(tert-butoxycarbonyl)-l-[2-({3-ethyl-4- oxobenzo[g]quinazolin-2-yl}sulfanyl)-2-phenylacetyl]piperazine-2-carboxylic acid (2.4 g, 64.9% yield) as a white solid.

Step-3. Synthesis of (2R)-4-[(tert-butoxy)carbonyl]-l-[(2R)-2-({3-ethyl-4-oxo-3H,4H- benzo[g]quinazolin-2-yl}sulfanyl)-2-phenylacetyl]piperazine-2-carboxylic acid (peakl, labelled as INT-4A)1NT-4A and (2R)-4-[(tert-butoxy)carbonyl]-l-[(2S)-2-({3-ethyl-4-oxo-3H,4H- benzo[g]c[uinazolin-2-yl}sulfanyl)-2-phenylacetyl]piperazine-2-carboxylic acid (peak2, labelled as INT-4B) INT-12

[0273] The (2R)-4-[(tert-butoxy)carbonyl]-l-[2-({3-ethyl-4-oxo-3H,4H-benzo[g]quinazolin-2- yl}sulfanyl)-2-phenylacetyl]piperazine-2-carboxylic acid (2.40 g, 3.98 mmol) was purified by Chiral-SFC under the following conditions: Column: CHIRALPAK IA, 5*25 cm, 5 pm; Mobile Phase A: CO₂, Mobile Phase B: MeOH: DCM=1:1 (2011M NH₃); Flow rate: 3 mL/min; Gradient: isocratic 50% B; Column Temperature (°C): 35; Back Pressure (bar): 150; Wave Length: 254 nm; RT1 (min): 1.17; RT2 (min): 1.96; Sample Solvent: MeOH: DCM=1:1; Injection Volume: 0.002 mL; Number of Runs: 100. This resulted in (2R)-4-[(tert-butoxy)carbonyl]-l-[(2R)-2-({3-ethyl-4- oxo-3H,4H-benzo[g]quinazolin-2-yl}sulfanyl)-2-phenylacetyl]piperazine-2-carboxylic acid (peakl, INT-12) (1.20 g, 1.99 mmol) as a white solid and (2R)-4-[(tert-butoxy)carbonyl]-l-[(2S)- 2-({3-ethyl-4-oxo-3H,4H-benzo[g]quinazolin-2-yl}sulfanyl)-2-phenylacetyl]piperazine-2- carboxylic acid (pcak2, labelled as INT-13) (1.20 g, 1.99 mmol) as a white solid.

INT-14 & INT-15. Synthesis of (2R)-l-[(2R)-2-({3-ethyl-4-oxo-3H,4H- benzo[g]quinazolin-2-yl}sulfanyl)-2-(pyridin-3-yl)acetyl]pyrrolidine-2-carboxylic acid (peakl, INT-14), and (2R)-l-[(2S)-2-({3-ethyl-4-oxo-3H,4H-benzo[g]quinazolin- 2-yl}sulfanyl)-2-(pyridin-3-yl)acetyl]pyrrolidine-2-carboxylic acid (peak2, INT-15):

Step-1. Synthesis of ethyl 2-bromo-2-(pyridin-3-yl) acetate [0274] A mixture of ethyl 2-(pyridin-3-yl)acetate (15 g, 90.8 mmol), NBS (19.2 g, 108 mmol) and AIBN (1.48 g, 9.08 mmol) in CCU (1.5 L) was stirred for 2 h at 80 °C under nitrogen atmosphere. The resulting mixture was cooled to room temperature, concentrated under reduced pressure, and then purified by silica gel column chromatography, (ethyl acetate/petroleum ether (0-100%, 15 min)) to afford ethyl 2-bromo-2-(pyridin-3-yl) acetate (20.0 g, 81.9 mmol, 90.4% yield) as a brown solid.

Step-2. Synthesis of ethyl 2-({3-ethyl-4-oxo-3H,4H-benzo[g]quinazolin-2-yl}sulfanyl)-2-(pyridin- 3-yl)acetate

[0275] To a 1 L 3-necked round-bottom flask was added 3-ethyl-2-sulfanyl-3H,4H-benzo[g]quin azolin-4-one (13.5 g, 52.6 mmol), DIEA (20.2 g, 157.0 mmol) and ethyl 2-bromo-2-(pyridin-3- yl)acetate (16.6 g, 68.3 mmol) in DMSO (300 mL) at room temperature. The resulting mixture was stirred for 3 hours at 50 °C, then cooled to room temperature and diluted with H₂O (IL). The formed solids were filtered, and the filter cake was washed with H₂O (3x 100 mL) to afford 3- ethy 1-2-s ulfanyl-3H,4H-benzo[g]quinazolin-4-one (30.0 g, 117.0 mmol, 88.2% yield) as a yellow solid.

Step-3. Synthesis of 2-({3-ethyl-4-oxo-3H,4H-benz.o[g]quinaz.olin-2-yl}sulfanyl)-2-(pyridin-3- yl)acetic acid

[0276] To a suspension of ethyl 2-({3-ethyl-4-oxo-3H,4H-benzo[g]quinazolin-2-yl}sulfanyl)-2- (pyridin-3-yl)acetate (7.0 g, 16.6 mmol) in EtOH/H₂ O (4:1, 70 mL) was added LiOH (1.4 g, 33.2 mmol) at room temperature. The reaction mixture was stirred for 3 hours at room temperature. The resulting mixture was adjusted to pH 3 with 2M HC1, and then the precipitated solids were collected by filtration, washed with water (3 x 50 mL)and dried under infrared light to afford 2- ({3-ethyl-4-oxo-3H,4H-benzo[g]quinazolin-2-yl}sulfanyl)-2-(pyridin-3-yl)acetic acid (4.0 g, 10.2 mmol, 61.6% yield) as a yellow solid.

Step-4. Synthesis of tert-butyl (2R)-l-[2-({3-ethyl-4-oxo-3H,4H-benzo[g]quinazolin-2- yl}sulfanyl)-2-(pyridin-3-yl)acetyl]pyrrolidine-2-carboxylate

[0277] To a 100 mL round bottom flask was added 2-({3-ethyl-4-oxo-3H,4H-benzo[g]quinazolin- 2-yl}sulfanyl)-2-(pyridin-3-yl)acetic acid (4.0 g, 10.2 mmol), tert-butyl D-prolinate hydrochloride (2.5 g, 12,2 mmol), HATU (4.6 g, 12.2 mmol), DIEA (3.9 g, 30.5 mmol) and DCM (40 mL) at room temperature. The resulting mixture was stirred overnight at 50 °C under nitrogen atmosphere. The mixture was then allowed to cool down to room temperature and concentrated in vacuo. The residue was purified by prep-HPLC (NH₃ H₂O buffer) to afford tert-butyl(2R)- l -[2-( {3-cthyl-4- oxo-3H,4H-benzo[g]quinazolin-2-yl}sulfanyl)-2-(pyridin-3-yl)acetyl]pyrrolidine-2-carboxylate (2.0 g, 3.7 mmol, 36.0% yield) as a yellow oil

Step-5. Synthesis of (2R)-l-[2-({3-ethyl-4-oxo-3H,4H-benzo[g]quinazolin-2-yl}sulfanyl)-2- (pyridin-3-yl)acetyl]pyrrolidine-2-carboxylic acid

[0278] A stirred solution of tert-butyl (2R)-l-[2-({3-ethyl-4-oxo-3H,4H-benzo[g]quinazo lin-2- yl}sulfanyl)-2-(pyridin-3-yl)acetyl]pyrrolidine-2-carboxylate (2.0 g, 3.7 mmol) in TFA (20 mL) at room temperature was cooled to 0°C and stirred for 3.0 hours. The reaction mixture was concentrated under reduced pressure to afford the crude product (2R)-l-[2-({3-ethyl-4-oxo- 3H,4H-benzo[g]quinazolin-2-yl)sulfanyl)-2-(pyridin-3-yl)acetyl]pyrrolidine-2-carboxylic acid (1.0 g, 2.0 mmol, 55.8% yield) as yellow solid, which was used to the next step directly without under purification.

Step-6. Synthesis of (2R)-l-[(2R)-2-({3-ethyl-4-oxo-3H,4H-benzo[g]quinazolin-2-yl}sulfanyl)-2- (pyridin-3-yl)acetyl]pyrrolidine-2-carboxylic acid (peakl, INT-14) and (2R)-l-[(2S)-2-({3-ethyl- 4-oxo-3H,4H-benzo[g]quinazolin-2-yl}sulfanyl)-2-(pyridin-3-yl)acetyl]pyrrolidine-2-carboxylic acid (peak2, INT-15)

[0279] tert-butyl (2R)-l-[2-({3-ethyl-4-oxo-3H,4H-benzo[g]quinazolin-2-yl]sulfanyl)-2- (pyridin-3-yl)acetyl]pyrrolidine-2-carboxylic acid (1.0 g, 2.0 mmol, 55.8% yield) was purified by Chiral-HPLC with the following conditions: Column: YMC Cellulose-SB, 50*4.6mm, 3um; Mobile Phase A: Ethanol(0.1%DEA), Mobile Phase B: DCM; Flow rate: 100 mL/min; Gradient: isocratic 20.0% B; Column Temperature (°C): 25; Back Pressure (bar): 100; Wave Length: 280 nm; RT1 (min): 1.89; RT2 (min): 1.30; Samp le Solvent: MeOH: DCM-2: 1 ; Injection Volume: 20 mL; Number of Runs: 60. This resulted in (2R)-l-[(2R)-2-({3-ethyl-4-oxo-3H,4H- benzo[g]quinazolin-2-yl}sulfanyl)-2-(pyridin-3-yl)acetyl]pyrrolidine-2-carboxylic acid (peakl, INT-14) (300 mg, 42.4%) as a white solid and (2R)-l-[(2S)-2-({3-ethyl-4-oxo-3H,4H- benzo[g]quinazolin-2-yl}sulfanyl)-2-(pyridin-3-yl)acetyl]pyrrolidine-2-carboxylic acid (400.0 mg, 42.4% yield) (peak2, INT-15) as a white solid. INT-16 & INT-17. Synthesis of 3-ethyl-2-(((R)-2-((S)-3-hydroxypyrrolidin-l-yl)-l-(3- iodophenyl)-2-oxoethyl)thio)benzo[g]quinazolin-4(3H)-one (INT-16) and 3-ethyl-2- (((S)-2-((S)-3-hydroxypyrrolidin-l-yl)-l-(3-iodophenyl)-2-oxoethyl)thio) benzo[g] quinazolin-4(3H)-one (INT - 17) :

Step-1. Synthesis of ethyl 2-(3-iodophenyl)acetate

[0280] To a solution of 2-(3-iodophenyl)acetic acid (20 g, 76.3 mmol) in EtOH(200 mL ) was added concentrated H₂SO₄ (1 mL). The resulting solution was refluxed for Ih. The reaction mixture was cooled to RT and concentrated in vacuo. The residue was diluted with PE (200 mL)/EA (200 mL) and the mixture was washed with saturated Na₂CO₃ (2 X 100 mL), H₂O (150 mL) and brine(150 mL) successively, dried over anhydrous MgSO₄ and concentrated in vacuo to yield ethyl 2-(3-iodophenyl)acetate (20.5 g, 70.9 mmol, 98% purity, 93% yield) as a yellow oil.

Step-2. Synthesis of ethyl 2-bromo-2-(3-iodophenyl)acetate

[0281] To a solution of ethyl 2-(3-iodophenyl)acetate (19 g, 62.0 mmol) in CCI4 (200 mL ) was added NBS (13.2 g, 74.3 mmol), followed by BPO(1.50 g, 6.20 mmol). The mixture was stirred at 80 °C overnight, thencooled to RT and filtered. The filter cake was washed with hexane. The combined filtrates were concentrated in vacuo. The residue was then purified by column chromatography on silica gel (eluted with PE/EA=5/1 ) to afford ethyl 2-bromo-2-(3- iodophcnyl)acctatc (9.95 g, 26.9 mmol, 95% purity, 42% yield) as yellow oil.

Step-3. Synthesis of ethyl 2-((3-ethyl-4-oxo-3,4-dihydrobenzo[ ]quinazolin-2-yl)thio)-2-(3- iodophenyl)acetate

[0282] To a solution of ethyl 2-bromo-2-(3-iodophenyl)acetate (10.7 g, 28.9 mmol) and DIPEA (13.6 mL, 78.8 mmol) in DMSO (100 mL ) was added 3-ethyl-2-sulfanyl-37 ,47 - benzo[g]quinazolin-4-one(6.71 g, 26.2 mmol). The resulting mixture was stirred at 50 °C for 4 h under N2. After being cooled to room temperature, the reaction mixture was diluted with distilled water (500 mL) and extracted with EA(500 mL X 3). The combined organic layer was concentrated in vacuo. The residue was purified by column chromatography(eluted with EA/PE=1/3~1/1) to afford ethyl 2-({3-ethyl-4-oxo-377,477-benzo[g]quinazolin-2-yl}sulfanyl)-2-(3- iodophenyl)acetate (11.0 g, 20.2 mmol, 95% purity, 73% yield) as a white solid.

Step-4. Synthesis of 2-((3-ethyl-4-oxo-3,4-dihydrobenzo[g]quinazolin-2-yl)thio)-2-(3-iodo phenyl)acetic acid

[0283] To a solution of ethyl 2-({3-ethyl-4-oxo-3H,4H-benzo[g]quinazolin-2-yl }sulfanyl)-2- phenylacetate (11 g, 20.2 mmol) in water (50 mL ) and THF (150 mL) was added LiOH (2.42 g, 101 mmol). The resulting mixture was stirred for 20 h at RT. After the reaction was complete, the reaction mixture was diluted with distilled water (100 mL) and adjusted with HC1 (1 mol/L, aq.) to pH =3. The precipitate was filtered off, washed with water (100 mL X 2) and dried under vacuum to afford 2-({3-ethyl-4-oxo-3H,4H-benzo[g]quinazolin-2-yl}sulfanyl)-2-phenylacetic acid (10.0 g, 19.37 mmol, 95% purity, 96% yield) as a yellow solid that was used for the next step without further purification.

Step-5. Synthesis of 3-ethyl-2-((2-((S)-3-hydroxypyrrolidin-l-yl)-l-(3-iodophenyl)-2- oxoethyl)thio)benzo[g]quinazolin-4(3H)-one

[0284] A solution of 2-({3-ethyl-4-oxo-377,477-benzo[g]quinazolin-2-yl sulfanyl)-2-(3- iodophenyl) acetic acid (10 g, 19.4 mmol), HATU (10.4 g, 27.6 mmol) and DIPEA (14.8 mL, 85.2 mmol) in dry DMF (50 mL) was stirred at RT for 10 min. Next, (3S)-pyrrolidin-3-ol hydrochloride (3.94 g, 31.9 mmol) was added. The resulting mixture was stirred at 50°C for 16 h under N2. The reaction mixture was concentrated to dryness and the residue was purified by silica gel column chromatography (EA/PE = 1/1 —2/1) to give 3-ethyl-2-({2-[(3S')-3-hydroxypyrrolidin-l-yl]-l-(3- iodophcnyl)-2-oxocthyl } sulfanyl )-3H,4H-bcnzo[g]quinazolin-4-onc (5.0 g, 8.54 mmol, 95% purity, 44% yield) as a red solid.

Step-6. Synthesis of 3-ethyl-2-(((R)-2-((S)-3-hydroxypyrro lidin- l-yl)-l -(3-iodophenyl)-2- oxoethyl)thio)benzo[g]quinazolin-4(3H)-one and 3-ethyl-2-(((5)-2-((5)-3-hydroxypyrrolidin-l- yl)-l-(3-iodophenyl)-2-oxoethyl)thio)benzo[g]quinazolin-4(3//)-one

[0285] 3-ethyl-2-({ 2- [(35)-3-hydroxypyiTolidin-l-yl]-l-(3-iodophenyl)-2-oxoethyl} sulfanyl) 3H,4H-benzo[ ]quinazolin-4-one (5.0 g, 8.54 mmol) was separated with SFC to afford 3-ethyl-2-

(((R )-2-((S)-3-hydroxypyrrolidin-l-yl)-l-(3-iodophenyl)-2-oxoethyl) thio)benzo[g]quinazolin- 4(3H)-one, INT-16 (2.8 g, 56%yield) as light red solid and 3-ethyl-2-(((S)-2-((5)-3- hydroxypyrrolidin-l-yl)-l-(3-iodophenyl)-2-oxoethyl)thio)benzo [g]quinazolin-4(3H)-one, INT- 17 (2.0 g, 40%yield) as light red solid.

INT-18. Synthesis of (2S,3aS,7aS)-l-(6-((S)-l-(4'-((tert-butoxycarbonyl)amino)-[l,l'- biphenyl] -4-carboxamido)ethyl)-2-methyl pyrimidin-4-yl) octahydro- lH-indole-2- carboxylic acid, INT-18:

Step-1. Synthesis of ethyl (4S)-4-{[(tert-hutoxy)carbonyl]amino}-3-oxopentanoate

[0286] To a stirred solution of (2S)-2-{ [(tert-butoxy)carbonyl]amino]propanoic acid (30.0 g, 158 mmol) in tetrahydrofuran (300 mL) were added l-(lH-imidazole-l -carbonyl)- IH-imidazole (38.2 g, 236 mmol) at room temperature. The reaction was stirred for 16.0 hours at room temperature, and thenl-ethyl 3-potassium propanedioate (40.1 g, 236 mmol) and dichloromagnesium (15.0 g, 158 mmol) were added. The reaction mixture was quenched with saturated ammonium chloride aqueous solution (50.0 mL) and the resulting mixture was extracted with ethyl acetate (150 mL x 2). The combined organic layers were washed with brine (150 mL) and dried over anhydrous Na₂SO₄. After filtration, the filtrate was concentrated under reduced pressure to afford ethyl (4S)- 4-{ [(tert-butoxy)carbonyl]amino}-3-oxopentanoate (40.0 g, 154 mmol, 95% purity, 97% yield)) as a red oil.

Step-2. Synthesis of tert-butyl (S )-(1-( 6-hydroxy-2-methylpyrimidin-4-yl )ethyl )carbamate

[0287] To a stirred solution of ethanimidamide hydrochloride (5.45 g, 57.7 mmol) in methanol (100 mL) were added sodium methanolate (30% in MeOH) (10.3 g, 57.7 mmol) at room temperature. The reaction was stirred for 20 min at room temperature. To the above mixture was then added ethyl (4S)-4-{[(tert-butoxy)carbonyl] amino }-3-oxopentanoate (10 g, 38.5 mmol) and the reaction mixture was stirred 3.0 hours at room temperature. The resulting mixture was filtered and the filter cake was washed with MeOH (20 mL). The filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with ethyl acetate (100%) to afford tert-butyl N-[(lS)-l-(6-hydroxy-2-methylpyrimidin-4-yl)ethyl]carbamate (6.50 g, crude). The crude product (6.5 g) was separated by the following condition: Column: CHIRAL ART Cellulose-SC, 3*25 cm, 5 pm; Mobile Phase A: CO₂, Mobile Phase B: IPA: HEX=1: 1(0.1% 2M NH₃-MeOH); Flow rate: 80 mL/min; Gradient: isocratic 40% B; Column Temperature(°C): 35; Back Pressure(bar): 100; Wave Length: 220 nm; RT2(min): 4.8; Sample Solvent: MeOH— HPLC; Injection Volume: 1.5 mL; Number Of Runs: 40. This resulted in tert-butyl N-[(lS)-l-(6- hydroxy-2-methylpyrimidin-4-yl)ethyl]carbamate (4.50 g, 17.7 mmol, 95% purity, ee>95%, Rt = 2.648 min, 45% yield) as a white solid.

Chiral-SFC: Column Name : 1:SC 100x4.6mm 3.0um; Co Solvent : B:IPA (50%Hex); Start Cone, of Pump B : 10.0%; Total Flow : 3.0000 mL/min; BPR Pressure : 15.00 MPa; Oven Temperature : 35 C. Rt = 2.648 min. Step-3. Synthesis of6-[( 1 S)-1 -aminoethyl]-2-methylpyrimidin-4-ol

[0288] A solution of tert-butyl N-[(lS)-l-(6-hydroxy-2-methylpyrimidin-4-yl)ethyl]cai’bamate (4.0 g, 15.7 mmol) in trifluoroacetic acid (10.0 mL) and methylene chloride (40.0 mL) was stirred for 2 hours at room temperature. The resulting mixture was concentrated under reduced pressure. The crude residue was used to next reaction directly.

[0289] To a stirred solution of 6-[(lS)-l-aminoethyl]-2-methylpyrimidin-4-ol (3.90 g, crude, 15.5 mmol) and 4'-{[(tert-butoxy)carbonyl]amino}-[l,l'-biphenyl]-4-carboxylic acid (4.85 g, 15.5 mmol) in methylene chloride (40.0 mL) were added ethylbis(propan-2-yl)amine (10.0 g, 77.5 mmol) and l-[(dimethylamino)(dimethyliminiumyl)methyl]-3-oxo- 1H,2H,3H-3 - [l,2,3]triazolo[5,4-b]pyridin-3-ylium-2-ide; hexafluoro- -phosphanuide (7.03 g, 18.5 mmol) at room temperature. The resulting mixture was stirred for 2 hours at room temperature. The solids were collected by filtration and the filter cake was washed with water (10 mL x 3) and DCM (10 mL x 3). The resulted solid was dried with an infrared lamp to afford tert-butyl N-(4'-{ [( 1 S)- 1 -(6- hydroxy-2-methylpyrimidin-4-yl)ethyl] carbamoyl] -[l,T-biphenyl]-4-yl)carbamate (5.10 g, 11.3 mmol, 95% purity) as a grey solid.

Step-5. Synthesis of tert-butyl N-(4'-{[(lS)-l-[2-methyl-6-

[0290] To a stirred solution of tert-butyl N-(4'-{ [(lS)-l-(6-hydroxy-2-methylpyrimidin-4- yl)ethyl]carbamoyl]-[l,T-biphenyl]-4-yl)carbamate (6.0 g, 13.3 mmol) in pyridine (60 mL) was added trifluoromethanesulfonyl trifluoromethanesulfonate (11.2 g, 39.9 mmol) dropwise at 0 °C and the reaction mixture was stirred for 3 hours at the same temperature. The reaction was diluted with 50 mL of water and acidified with hydrochloric acid (1 M) to pH = 5-6. The resulting mixture was extracted with ethyl acetate (20 mL x 2) and the combined organic phase was washed with brine (2 mL). The organic phase was concentrated under reduced pressure and the residue was purified by silica gel column chromatography (ethyl acetate) to afford tert-butyl N-(4'-{ [(lS)-l- [2-methyl-6-(trifluoromethanesulfonyloxy)pyrimidin-4-yl]ethyl]carbamoyl}-[1, 1'-biphenyl]-4- yl)carbamate (3.60 g, 6.20 mmol, 95% purity, 46.4% yield) as an off-white solid. Step-6. Synthesis of (2S,3aS, 7aS)-l-{6-[( 1 S)-l -[(4'-{ [(tert-butoxy)carbonyl]amino}-[1 , biphenyl]-4-yl)fonnamido]ethyl]-2-methylpyrimidin-4-yl}-octahydro-lH-indole-2-carboxylic acid, INT-18

[0291] To a stirred solution of tert-butyl N-(4'-{ [(lS)-l-[2-methyl-6- (trifluoromethanesulfonyloxy)pyrimidin-4-yl]ethyl]carbamoyl}-[l,T-biphenyl]-4-yl)carbamate (2.00 g, 3.44 mmol) and ethylbis(propan-2-yl)amine (1.77 g, 13.7 mmol) in isopropyl alcohol (20 mL) was added (2S,3aS,7aS)-octahydro-lH-indole-2-carboxylic acid (871 mg, 5.15 mmol). The reaction mixture was stirred for 0.5 hour at 80 °C. The reaction was diluted with 5 mL of water and acidified with hydrochloric acid (1 M) to pH = 5-6. The resulting mixture was extracted with ethyl acetate (2 mL x 2) and the combined organic phase was washed with brine (2 mL). The organic phase was concentrated under reduced pressure and the residue was purified by silica gel column chromatography (ethyl acetate) to afford (2S,3aS,7aS)-l-{6-[(lS)-l-[(4'-{[(tert- butoxy )carbonyl] amino } - [ 1, 1 '-biphenyl] -4-yl)formamido] ethyl] -2-methylpyri midin-4-yl } - octahydro- lH-indole-2-carboxylic acid, INT-18. Yield: 1.7 g, 82.5%; Appearance: White solid; ¹H NMR (400 MHz, DMSO-d₆) δ 9.50 (s, 1H), 8.85 (d, J= 6.9 Hz, 1H), 7.96 (d, J= 8.3 Hz, 2H), 7.77 (d, J = 8.2 Hz, 2H), 7.67 (d, J = 8.8 Hz, 2H), 7.58 (d, J = 8.8 Hz, 2H), 6.78 (s, 1H), 5.01 (s, 1H), 4.49 (t, J = 8.9 Hz, 1H), 4.03 (d, J = 6.3 Hz, 1H), 2.45 (s, 3H), 2.28 (s, 1H), 2.11 - 1.96 (m, 1H), 1.89 - 1.58 (m, 4H), 1.54 (d, J = 6.9 Hz, 3H), 1.50 (s, 9H), 1.46 - 1.10 (m, 5H). HPLC purity: 97.93%; LCMS Calculated for C₃₄H₄₁N₅O₅: 599.31 ; Observed: 600.4[M+H]⁺.

INT-19. Synthesis of (2S,3aS,7aS)-l-{2-bromo-6-[(lS)-l-{[4-(morpholin-4-yl)phenyl] formamido}ethyl]pyrimidin-4-yl}-N-methyl-octahydro-lH-indoIe-2-carboxamide, INT-19:

Step-1. Synthesis of (2S,3aS,7aS)-l-{6-[(lS)-l-aminoethyl]-2-bromopyrimidin-4-yl}-N-methyl- octahydro- lH-indole-2-carboxamide

[0292] To a solution of tert-butyl N-[( lS)-l-{6-[(2S,3aS,7aS)-2-(methylcarbamoyl)-octahydro- lH-indol-l-yl]-2-bromopyrimidin-4-yl}ethyl]carbamate (10 g, 20.7 mmol) in DCM (100 mL) was added TFA (50 mL). The reaction mixture was stirred at r.t. for 2 h and then concentrated under reduced pressure to give the crude product as a TFA salt, which was directly used in the next steps.

Step-2. Synthesis of (2S,3aS,7aS)-l-{2-bromo-6-[(lS*)-l-{ [4-(morpholin-4- yl)phenyl]formamido } ethyl]pyrimidin-4-yl } -N-methyLoctahydro- 1 H-indole-2-carboxamide

[0293] To a solution of 4-(morpholin-4-yl)benzoic acid (5.11 g, 24.7 mmol) and DIPEA (26.6 g, 206 mmol) in DMF (150 mL) was added HATU (8.97 g, 23.6 mmol). After stirring for 1 hr at r.t., the mixture was cooled to -40 °C, (2S,3aS,7aS)-l-[6-(l-aminoethyl)-2-bromopyrimidin-4-yl]-N- methyl-octahydro-lH-indole-2-carboxamide (7.9 g, 20.6 mmol) was added dropwise at -40 °C, and then the mixture was stirred further for 1 hr. Next, the mixture was concentrated under reduced pressure to give the crude residue, which was purified by column chromatography on silica gel eluting with EtOAc/PE (0- 66%) to give (2S,3aS,7aS)-l-(2-bromo-6-(l-(4- morpholinobenzamido)ethyl)pyrimidin-4-yl)-N-methyloctahydro-lH-indole-2-carboxamide (11.9 g). Next, the product was separated with SFC to give (2S,3aS,7aS)-l-{2-bromo-6-[(lS)-l- { [4-(morpholin-4-yl)phenyl]formamido]ethyl]pyrimidin-4-yl}-N-methyl-octahydro-lH-indole- 2-carboxamide (Peak-1), INT-19. Yield: 4.4 g, 34 %; Appearance: White solid; ^JH NMR (400 MHz, DMSO- d) 5 8.46 (d, 7 = 6.8 Hz, 1H), 8.14 - 7.89 (m, 1H), 7.79 (d, J = 8.8 Hz, 2H), 6.97 (d, J= 9.0 Hz, 2H), 6.64 - 5.87 (m, 1H), 4.84 (t, J = 12 Hz, 1H), 4.36 - 4.16 (m, 1H), 3.83 - 3.68 (m, 4H), 3.32 (s, 1H), 3.25 - 3.19 (m, 4H), 2.65 - 2.53 (m, 3H), 2.39 - 1.80 (m, 4H), 1.72 - 1.53 (m, 3H), 1.48 - 1.36 (m, 5H), 1.30 - 1.01 (m, 2H); HPLC purity: 91.07%; LCMS Calculated for C27H35B1N6O3: 571.52; Observed: 571.4 [M+H]⁺.

[0294]

INT-20. Synthesis of (2S,3aS,7aS)-l-{2-iodo-6-[(lS)-l-{[4-(morpholin-4- yl)phenyl]formamido}ethyl]pyrimidin-4-yl}-N-methyl-octahydro-lH-indole-2- carboxamide, INT-20:

Step-1. Synthesis of (2S,3aS,7aS)-l-{2-iodo-6-[(lS)-l-{[4-(morpholin-4- yl)phenyl]formamido}ethyl]pyrimidin-4-yl}-N-methyl-octahydro-lH-indole-2-carboxamide,

INT-20

[0295] A mixture of (2S,3aS,7aS)-l-{2-bromo-6-[(lS)-l-{ [4-(morpholin-4- yl)phenyl]formamido]ethyl]pyrimidin-4-yl]-N-methyl-octahydro-lH-indole-2-cai’boxamide, INT-19 (1.0 g, 1.7 mmol) was dissolved in 1,4-dioxane (10 mL), then to the solution, 1, 1,1, 2,2,2- hexamethyldistannane (681.0 mg, 2.1 mmol) and Pd(PPh3)4 (200.0 mg, 174.0 μmol) were added, and the mixture was stirred at 100°C for 3 hours under nitrogen atmosphere. The reaction solution was cooled to room temperature and I2 (484.0 mg, 1 .9 mmol) was added. The solution was stirred for 1 hour at room temperature. The resulting mixture was concentrated under reduced pressure, quenched with H₂O (100 mL) and extracted with ethyl acetate (3x100 mL). The combined organic layers were washed with brine (1x100 mL) and dried over anhydrous Na₂SO₄. After filtration, the filtrate was concentrated under reduced pressure. The residue was applied onto a silica gel column and eluted with ethyl acetate/petroleum ether=l:l to give (2S,3aS,7aS)-l-{2-iodo-6-[(lS)-l-{ [4- (morpholin-4-yl)phenyl]formamido) ethyl] pyrimidin-4-yl)-N-methyl-octahydro-lH-indole-2- carboxamide, INT-20 (800.0 mg, 1.3 mmol, 90.0% purity, 74.7% yield) as yellow solid.

Example 1. Synthesis of (2R)-l-[(2R)-2-({3-ethyl-4-oxo-3H,4H-benzo[g]quinazolin-2- yl}sulfanyl)-2-phenylacetyl]-N-(l 7-{[ ( 2R )-l-[(2R )-2-( {3-ethyl-4-oxo-3H,4H- benzo[g]quinazolin2-yl}sulfanyl)-2-phenylacetyl]pyrrolidin-2-yl]formamido}-3,6,9,12,15- pentaoxaheptadecan-1 -yl)pyrrolidine-2-carboxamide, 1-1:

Step-1. Synthesis of 3,6,9, 12, 15-pentaoxaheptadecane-l , 17-diamine hydrochloride

[0296] A mixture of tert-butyl N-(17-amino-3,6,9,12,15-pentaoxaheptadecan-l-yl)carbamate (150 mg, 394 μmol) in HCl/dioxane (4M, 5 mL)was stirred for 1.0 hour at room temperature. The mixtures were concentrated under reduced pressure to afford the crude product 3,6,9,12,15- pentaoxaheptadecane-l,17-diamine as HC1 salt (140 mg, 441 μmol, 80% purity, 95% yield) as a yellow oil, which was used for next step directly.

Step-2. Synthesis of (2R)-l-[(2R)-2-({3-ethyl-4-oxo-3H,4H-benzo[g]quinazolin-2-yl}sulfanyl)-2- phenylacetyl]-N-(17-{[(2R)-l-l(2R)-2-({3-ethyl-4-oxo-3H,4H-benzo[g] quinazolin2-yl}sulfanyl)- 2-phenylacetyl]pyrrolidin-2-yl]formamiclo}-3,6,9,12,15-pent aoxaheptadecan-l-yl)pyrrolidine-2- carboxamide, 1-1

[0297] A mixture of 3,6,9,12,15-pentaoxaheptadecane-l,17-diamine (35.0 mg, 110 μmol), (2R)-

1-[(2R)-2-({3-ethyl-4-oxo3H,4H-benzo[g]quinazolin-2-yl]sulfanyl)-2-phenylac etyl]pyrrolidine-

2-carboxylic acid (53.6 mg, 110 μmol) and K₂CO₃ (45.5 mg, 330 μmol) in DMF (2 mL) was stirred at 0°C for 30 mins. HATU (62.7 mg, 165 μmol) was then added, and the reaction mixture was stirred for 1.0 hour at 0°C. The resulting mixture was concentrated in vacuo andthe residue purified by prep-HPLC (NH₃/H₂O buffer) to give (2R)-l-[(2R)-2-({3-ethyl-4-oxo-3H,4H- benzo[g] quinazolin-2-y 1 } sulf any l)-2-phenylacety 1] -N-( 17- { [(2R)- 1 - [(2R)-2-( { 3-ethyl-4-oxo- 3H,4H-benzo[g]quinazolin2-yl}sulfanyl)-2-phenylacetyl]pyrrolidin-2-yl]formamido}-

3,6,9, 12, 15 -pentaoxaheptade can-l-yl)pyrrolidine-2-carboxamide, 1-1. Yield: 30.0 mg, 22.3%; Appearance: White solid; ¹H NMR (300 MHz, DMSO-d₆) 5 8.80 - 8.78 (m, 2H), 8.24 - 8.08 (m, 4H), 8.06 - 7.98 (m, 2H), 7.80 - 7.62 (m, 7H), 7.60 - 7.50 (m, 2H), 7.44 - 7.33 (m, 6H), 6.10 - 5.93 (m, 2H), 4.44 - 4.28 (m, 2H), 4.19 - 4.00 (m, 5H), 3.53 - 3.47 (m, 13H), 3.39 - 3.33 (m, 6H), 3.29 - 3.01 (m, 8H), 2.18 - 1.80 (m, 8H), 1.34 - 1.19 (m, 6H). HPLC purity: 99.5%; LCMS Calculated for C66H74N8O11S₂: 1218.49; Observed: 1219.5 [M+H]⁺.

Example 2. Synthesis of (2R,2'R)-N,N'-(3,6,9,12-tetraoxatetradecane-l,14-diyl)bis(l-((R)-2- (f3-ethyl-4-oxo-3,4-dihydrobenzo[g]quinazolin-2-yl)thio)-2-phenylacetyl)pyrrolidine-2- carboxamide), 1-2:

Step-1. Synthesis of (2R,2'R)-N,N'-(3,6,9,12-tetraoxatetradecane-l ,14-diyl)bis(l -((R)-2-((3-ethyl- 4-oxo-3,4-dihydrobenzo[g]quinazolin-2-yl)thio)-2-phenylacetyl)pyrrolidin e-2-carboxamide), I- 2,

[0298] To a 8 mL vial were added ((R)-2-((3-ethyl-4-oxo-3,4-dihydrobenzo[g]quinazolin-2- yl)thio)-2-phenylacetyl)-D-proline (100 mg, 205 μmol), 3,6,9,12-tetraoxatetradecane-l,14- diamine (24.1 mg, 102 μmol), DIEA (39.6 mg, 307 μmol) and HATU (38.7 mg, 102 μmol) in DMF (2 mL) at room temperature. The mixture was stirred for 1 hour and then purified by prep- HPLC (NH₃ H₂O buffer) to afford (2R,2'R)-N,N'-(3,6,9,12-tetraoxatetradecane-l,14-diyl)bis(l- ((R)-2-((3-ethyl-4-oxo-3,4-dihydrobenzo[g]quinazolin-2-yl)thio)-2-phenylacetyl)pyrrolidine-2- carboxamide), 1-2. Yield: 25.0 mg, 21 %; Appearance: White solid; ¹H NMR (300 MHz, DMSO- d₆) 5 8.80 - 8.74 (m, 2H), 8.18 - 7.97 (m, 6H), 7.79 - 7.51 (m, 10H), 7.45 - 7.29 (m, 6H), 6.10 - 5.93 (m, 2H), 4.35 - 4.32 (m, 2H), 4.09 - 3.96 (m, 5H), 3.49 - 3.42 (m, 10H), 3.36 -3.33 (m, 4H), 3.21 - 3.18 (m, 5H), 3.16 - 3.03 (m, 3H), 2.12 - 2.08 (m, 4H), 2.02 - 1.76 (m, 5H), 1.25 - 1.19 (m, 6H). HPLC purity: 96%; LCMS Calculated for C₆₄H₇₀N₈O₁₀S₂: 1174.47; Observed: 1175.5 [M+H]⁺.

[0299] The following examples were prepared using standard chemical manipulations and procedures similar to those used for the preparation of the previous examples. Analytical data is given in the table below:

Example 3. Synthesis of 2-( {3-ethyl-4-oxo-3H,4H-benzo[g]quinazolin-2-yl}sulfanyl)-N-{20-[2- ({3-ethyl-4-oxo-3H,4H-benzo[g]quinazolin-2-yl}sulfanyl)-2-phenylacetamido]-3,6,9,12,15,18- hexaoxaicosan-l-yl}-2-phenylacetamide, Isomer 1 (peakl, 1-12), isomer 2 (peak2, 1-13) and Isomer 3 (peak3, 1-14):

Step-1. Synthesis of 2-({3-ethyl-4-oxo-3H,4H-benzo[g]quinazolin-2-yl}sulfanyl)-N-{20-[2-( {3- ethyl-4-oxo-3H,4H-benzo[g]quinazolin-2-yl}sulfanyl)-2-phenylacetamido]-3,6,9,12,15,18- hexaoxaicosan-l-yl}-2-phenylacetamide [0300] A mixture of 2-({3-ethyl-4-oxo-3H,4H-benzo[g]quinazolin-2-yl}sulfanyl)-2-phenylacetic acid (300.0 mg, 768.0 μmol), 3,6,9,12,15,18-hcxaoxaicosanc-l,20-diaminc (124.0 mg, 384.0 μmol), and DIEA (296.0 mg, 2.3 mmol) in DMF (3 mL) was stirred at room temperature for 10 min. HATU (349.0 mg, 921.0 μmol) was then added, and stirred at room temperature for 3.0 hours. The resulting mixture was concentrated in vacuo and the residue was purified by prep-HPLC (NH₃ H₂O buffer) to give 2-({3-ethyl-4-oxo-3H,4H-benzo[g]quinazolin-2-yl}sulfanyl)-N-{20-[2- ({3-ethyl-4-oxo-3H,4H-benzo[g]quinazolin-2-yl}sulfanyl)-2-phenylacetamido]-3,6,9,12,15,18- hexaoxaicosan-l-yl}-2-phenylacetamide (150.0 mg, 140.0 μmol, 18.2% yield) as a white solid.

Step-2. Synthesis of 2-({3-ethyl-4-oxo-3H,4H-benzo[g]quinazolin-2-yl}sulfanyl)-N-{20-[2-( {3- ethyl-4-oxo-3H,4H-benzo[g]quinazolin-2-yl}sulfanyl)-2-phenylacetamido]-3,6,9,12,15,18- hexaoxaicosan-l-yl}-2-phenylacetamide, Isomer 1 (peakl, 1-12), Isomer 2(peak2, 1-13) and isomer 3 (peak3, 1-14)

[0301] 2-({3-ethyl-4-oxo-3H,4H-benzo[g]quinazolin-2-yl]sulfanyl)-N-{20-[2-({3-ethyl-4-oxo- 3H,4H-benzo[g]quinazolin-2-yl}sulfanyl)-2-phenylacetamido]-3,6,9,12,15,18-hexao xaicosan-1- yl}-2-phenylacetamide (150.0 mg, 140.0 μmol) was purified by Chiral-HPLC with the following conditions: Column: CH1RALPAK1F-3, 50*4.6mm, 3um; Mobile Phase A: n-Hexane/DCM=l/l, Mobile Phase B: Ethanol; Flow rate: 100 mE/min; Gradient: isocratic 50.0% B; Column Temperature (°C): 25; Back Pressure (bar): 100; Wave Length: 254 nm; RT1 (min): 1.33; RT2 (min): 1.69; RT3(min): 2.13; Sample Solvent: MeOH: DCM=2:1 ; Injection Volume: 50 mL; Number of Runs: 80.

1-12: (peakl, RT1 (min): 1.33), Yield: 25.0 mg, 20.4%; Appearance: White solid; ¹H NMR (300 MHz, DMSO-d₆) 8 8.77 (s, 2H), 8.73 - 8.64 (m, 2H), 8.17 (d, J = 8.3 Hz, 2H), 8.10 - 8.03 (m, 4H), 7.70 - 7.58 (m, 4H), 7.59 - 7.49 (m, 2H), 7.44 - 7.28 (m, 6H), 5.81 (s, 2H), 4.13 - 4.04 (m, 4H), 3.53 - 3.35 (m, 26H), 3.25 - 3.14 (m, 4H), 1.28 (t, J = 7.4 Hz, 6H). HPLC purity: 98.1%;

LCMS Calculated for C₅₈H₆₄N₆O₁₀S₂: 1068.41; Observed: 1069.2 [M+H]⁺.

INT-13: (peak2, RT2 (min): 1.69), Yield: 50.0 mg, 42.4%; Appearance: White solid; ¹H NMR (300 MHz, DMSO- d₆) 8 8.78 - 8.77 (m, 2H), 8.71 - 8.68 (m, 2H), 8.11 - 8.02 (m, 2H), 8.08 - 8.06 (m, 4H), 7.70 - 7.59 (m, 6H), 7.56 - 7.54 (m, 2H), 7.44 - 7.25 (m, 6H), 5.81 (s, 2H), 4.12 - 4.07 (m, 4H), 3.41 (t, J = 2.2 Hz, 26H), 3.27 - 3.12 (m, 2H), 1.28 (t, J = 7.0 Hz, 6H). HPLC purity: 96.7%; LCMS Calculated for C₅₈H₆₄N₆O₁₀S₂: 1068.41; Observed: 1069.3 [M+H]⁺. INT-14: (peak3, RT3(min): 2.13), Yield: 28.0 mg, 23.8%; Appearance: White solid; ¹H NMR ( 300 MHz, DMSO-d6) δ 8.79 - 8.77 (m, 2H), 8.73 - 8.65 (m, 2H), 8.17 (d, J = 8.3 Hz, 2H), 8.11

- 8.02 (m, 4H), 7.70 - 7.59 (m, 6H), 7.60 - 7.49 (m, 2H), 7.44 - 7.28 (m, 6H), 5.81 (s, 2H), 4.15

- 4.02 (m, 4H), 3.45 - 3.35 (m, 24H), 3.27 - 3.13 (m, 4H), 1.33 - 1.21 (m, 6H). HPLC purity: 9 7.8%; LCMS Calculated for C₅₈H₆₄N₆O₁₀S₂: 1068.41; Observed: 1069.3 [M+H]⁺.

[0302] The following examples were prepared using standard chemical manipulations and procedures similar to those used for the preparation of the previous examples. Analytical data is given in the table below.

Example 4. Synthesis of N1,N14-bis(((R)-l-((S)-2-((3-ethyl-4-oxo-3,4- dihydrobenw[g]quinazolin-2-yl)thio)-2-phenylacetyl)pyrrolidin-2-yl)methyl)-3,6,9,12- tetraoxatetradecanediamide, 1-17:

Step-1. Synthesis of Nl,N14-bis(((R)-l-((S)-2-((3-ethyl-4-oxo-3,4-dihydroben zo[g]quinazolin-2- yl)thio)-2-phenylacetyl)pyrrolidin-2-yl)methyl)-3,6,9,12-tetraoxate tradecanediamide

[0303] To a 8 mL vial were added 2-(((S)-2-((R)-2-(aminomethyl)pyrrolidin-l-yl)-2-oxo-l- phenylethyl)thio)-3-ethylbenzo[g]quinazolin-4(3H)-one (150 mg, 262 μmol), 3,6,9,12- tetraoxatetradecanedioic acid (34.8 mg, 131 μmol), DIEA (50.6 mg, 393 μmol) and HATU (59.6 mg, 157 μmol) in DMF (3 mL) at room temperature. The mixture was stirred for 1 hour and thenthe reaction was purified by prep-HPLC (NH₃ H₂O buffer) to afford Nl,N14-bis(((R)-l-((S)-2-((3- ethyl-4-oxo-3,4-dihydrobenzo[g]quinazolin-2-yl)thio)-2-phenylacetyl)pyrrolidin-2-yl)methyl)- 3,6,9, 12-tetraoxatetradecanediamide, 1-17. Yield: 50.0 mg, 32%; Appearance: White solid; ¹H NMR (400 MHz, DMSO-d₆) 5 8.79 (t, J= 3.1 Hz, 2H), 8.18 - 8.07 (m, 4H), 7.92 - 7.84 (m, 2H), 7.74 (t, J - 5.7 Hz, 2H), 7.68 - 7.62 (m, 6H), 7.54 (t, J - 7.6 Hz, 2H), 7.46 - 7.32 (m, 6H), 6.39 - 6.00 (m, 2H), 4.19 - 4.05 (m, 8H), 3.96 (d, J = 7.4 Hz, 2H), 3.67 - 3.62 (m, 2H), 3.63 - 3.57 (m, 2H), 3.58 - 3.56 (m, 2H), 3.54 - 3.52 (m, 2H), 3.44 - 3.39 (m, 6H), 3.37 -3.32 (m, 2H), 3.24 - 2.87 (m, 4H), 2.19 -2.16 (m, 1H), 2.09 -2.07 (m, 1H), 1.87 -1.85 (m, 3H), 1.74 -1.69 (m, 3H), 1.27 (t, J = 7.1 Hz, 6H). HPLC purity: 99%; LCMS Calculated for C₆₄H₇₀N₈O₁₀S₂: 1174.47; Observed: 1175.5 [M+H]⁺.

Example 5. Synthesis of N-(((R)-l-((R)-2-((3-ethyl-4-oxo-3,4-dihydrobenzo[g]quinazolin-2- yl)thio )-2-phenylacetyl)pyrrolidin-2-yl)methyl)-3-(4-( 2-(( ((R)-l-((R)-2-( (3-ethyl-4-oxo-3,4- dihydrobenzo[g]quinazolin-2-yl)thio)-2-phenylacetyl)pyrrolidin-2-yl)methyl)amino)-2- oxoethyl)piperazin-l-yl)propanamide, INT-18:

Step-1. Synthesis of tert-butyl 4-(2-methoxy-2-oxoethyl)piperazine- 1 -carboxylate

[0304] A stirred solution of tert-butyl piperazine- 1 -carboxylate (2.0 g, 10.7 mmol) and methyl 2- bromoacetate (1.95 g, 12.8 mmol), K₂CO₃ (4.41 g, 32.0 mmol) in MeCN (40 mL) was stirred for 2 hours at 80 °C. The mixture was allowed to cool down to room temperature and thenthe residue was purified by reverse phase flash with the following conditions (column, Cl 8 silica gel; mobile phase, H₂O/0.1 % NH₄OH in MeCN, 10% to 50% gradient in 10 min; detector, UV 254 nm.) to afford tert-butyl 4-(2-mcthoxy-2-oxocthyl)pipcrazinc-l -carboxylate (2.70 g, 10.4 mmol, 96% purity, 97% yield) as a yellow solid.

Step-2. Synthesis of methyl 2 -(piperazin- 1 -yl)acet ate

[0305] A solution of tert-butyl 4-(2-methoxy-2-oxoethyl)piperazine-l -carboxylate (1.3 g, 5.03 mmol) in TFA:DCM (1:5, 18 mL) was stirred for 1 hour at room temperature. The resulting mixture was concentrated in vacuo to afford methyl 2-(piperazin-l-yl)acetate (780 mg, 4.93 mmol, 93% purity, 98% yield) as a white oil, which was used for next step directly.

Step-3. Synthesis of methyl 3-(4-(2-methoxy-2-oxoethyl)piperazin-l -yl)propanoate

[0306] A stirred solution of methyl 2-(piperazin-l-yl)acetate (800 mg, 5.05 mmol) and methyl 3- bromopropanoate (843 mg, 5.05 mmol), K₂CO₃(696 mg, 5.05 mmol) in MeCN (12 mL) was stirred for 2 hours at 80 °C. The mixture was allowed to cool down to room temperature. The residue was purified by reverse phase flash under the following conditions (column, C18 silica gel; mobile phase, H₂O/0.1% NH₄OH in MeCN, 30% to 60% gradient in 10 min; detector, UV 254 nm.) to afford methyl 3-[4-(2-methoxy-2-oxoethyl)piperazin-l-yl]propanoate (1.20 g, 4.91 mmol, 90% purity, 97% yield) as a yellow solid.

Step-4. Synthesis of 3-(4-(carboxymethyl)piperazin-l-yl)propanoic acid

[0307] A solution of methyl 3-[4-(2-mcthoxy-2-oxocthyl)pipcrazin-l-yl]propanoatc (200 mg, 818 μmol) in THF:H₂O (2:1, 3 mL) were added LiOH (102 mg, 2.45 mmol) at room temperature. The resulting mixture was stirred for 12 hours at room temperature. The mixture was acidified to pH= 2-3 with 1 M HC1. The precipitated solids were collected by filtration and washed with water (5 mL)to afford 3-[4-(carboxymethyl)piperazin-l-yl]propanoic acid (150 mg, 693 μmol, 93% purity, 85% yield) as a white solid.

Step-5. Synthesis of N-(((R)-l-((R)-2-((3-ethyl-4-oxo-3,4-dihydrobenzo[g]quinazolin-2-yl)thio)-2- phenylacetyl)pyrrolidin-2-yl)methyl)-3-(4-(2-((((R)-l-((R)-2-((3-ethyl-4-oxo-3,4- dihydrobenzo[g ]quinazolin-2-yl )thio)-2-phenylacetyl )pyrrolidin-2-yl )methyl ) amino )-2- oxoethyl )piperazin-l -yl )propanamide

[0308] To a 8 mL vial were added 3-[4-(carboxymethyl)piperazin-l-yl]propanoic acid (18.9 mg, 87.5 μmol), 2- { [(lR)-2-[(2R)-2-(aminomethyl)pyrrolidin-l-yl] -2-oxo- 1 -phenylethyl] sulfanyl] -3- ethyl-3H,4H-benzo[g]quinazolin-4-one (100 mg, 175 μmol), DIEA(33.7 mg, 262 μmol) and HATU(39.9 mg, 105 p mol) in DMF (2 mL) at room temperature. The mixture was stirred for 1 hour and then the reaction was purified by prep-HPLC (NH₃ H₂O buffer) to afford N-{ [(2R)-1- [(2R)-2-({3-ethyl-4-oxo-3H,4H-benzo[g]quinazolin-2-yl}sulfanyl)-2-phenylacetyl]pyrrolidin-2- yl]methyl } -3- { 4- [( { [(2R)- 1 - [(2R)-2-( { 3-ethyl-4-oxo-3H,4H-benzo[g]quinazolin-2-yl } sulfanyl)- 2-phenylacetyl]pyrrolidin-2-yl]methyl}carbamoyl)methyl]piperazin-l-yl}propanamide, 1-18. Yield: 10.0 mg, 10%; Appearance: White solid; ¹H NMR (400 MHz, DMSO-d₆) 8 8.90 - 8.76 (m, 2H), 8.37 - 8.35 (m, 1H), 8.18 (t, J = 6.2 Hz, 2H), 8.11 - 8.05 (m, 2H), 8.01 - 7.98 (m, 2H), 7.67 - 7.65 (m, 6H), 7.58 - 7.51 (m, 2H), 7.43 - 7.33 (m, 5H), 6.46 - 6.44 (m, 1H), 6.04 - 6.02 (m, 1H), 4.14 - 4.09 (m, 8H), 4.05 - 4.03 (m, 4H), 3.14 - 3.11 (m, 6H), 2.99 - 2.96 (m, 2H), 2.82 - 2.80 (m, 2H), 2.68 - 2.66 (m, 2H), 2.55 - 2.51 (m, 7H), 2.44 - 2.36 (m, 9H), 2.13 - 2.09 (m, 4H), 1.96 - 1.92 (m, 3H), 1.41 - 1.39 (m, 3H), 1.28 (t, J = 7.1 Hz, 6H). HPLC purity: 98%;

LCMS Calculated for C₆₃H₆₈N₁₀O₆S₂: 1124.48; Observed: 1125.5 [M+H]⁺.

[0309] The following examples were prepared using standard chemical manipulations and procedures similar to those used for the preparation of the previous examples. Analytical data is given in the table below.

Example 6. Synthesis of (2R)-l-[(2R)-2-({3-ethyl-4-oxo-3H,4H-benzo[g]quinazo lin-2- yl}sulfanyl)-2-phenylacetyl]-N-(14-{[ ( 2R )-l -[(2R )-2-( {3-ethyl-4-oxo-3H,4H-ben zo[g]quinazolin-2-yl}sulfanyl)-2-phenylacetyl]pyrrolidin-2-yl]formamido}tet radecyl)pyrrolidine-2-carboxamide, 1-25:

Step-1. Synthesis of (2R)-l-[(2R)-2-({3-ethyl-4-oxo-3H,4H-benzo[g]quinazolin-2-yl] sulfanyl)-2- phenylacetyl]-N-(14-{[(2R)-l-[(2R)-2-({3-ethyl-4-oxo-3H,4H-benzo[g]qui nazolin-2- yl}sulfanyl)-2-phenylacetyl]pyrrolidin-2-yl]formamido}tetradecyl)pyrrolidi ne-2-carboxamide

[0310] A mixture of tetradecane- 1,14-diamine (20 mg, 87.5 μmol), (2R)-l-[(2R)-2-({3-ethyl-4- oxo-3H,4H-benzo[g]quinazolin-2-yl}sulfanyl)-2-phenylacetyl]pyrrolidine-2-carboxylic acid (85.3 mg, 175 μmol) and DIEA (11.2 mg, 87.5 μmol) in DMF (1 mL) was stirred at room temperature for 10 min. HATU (33.2 mg, 87.5 μmol) was then added, and the reaction mixture was stirred for 2.0 hours at room temperature. The resulting mixture was purified by prep-HPLC (NH₃.H₂O buffer) to give (2R)-l-[(2R)-2-({3-ethyl-4-oxo-3H,4H-benzo[g]quinazolin-2- yl}sulfanyl)-2- phenylacetyl] -N-(l 4- { [(2R)-l-[(2R)-2-({3-ethyl-4-oxo-3H,4H- benzo[g]quinazolin-2-yl}sulfanyl)-2-phenylacetyl] pyrrolidin-2- yl]formamido}tetradecyl)pyrrolidine-2-carboxamide (30.0 mg, 25.6 μmol, yield 29.4 %), 1-25. Yield: 30.0 mg, 29.4 %; Appearance: White solid; ¹H NMR (300 MHz, DMSO-d₆) 88.79 - 8.77 (m, 2H), 8.17 (d, J= 7.6 Hz, 2H), 8.08 (d, J= 8.4 Hz, 2H), 7.99 (s, 2H), 7.73 -7.65 (m, 6H), 7.56 - 7.28 (m, 10H), 6.17 - 6.02 (m, 2H), 4.38 - 4.35 (m, 2H), 4.14 - 4.09 (m, 5H), 3.50 - 3.44 (m, 3H), 3.07 - 3.02 (m, 4H), 2.10 - 1.91 (m, 8H), 1.33 - 0.88 (m, 30H). HPLC purity: 97%; LCMS Calculated for: C₆₈H₇₈N₈O₆S₂ : 1166.55; Observed: 1167.5 [M+H]⁺.

Example 7. Synthesis of (2R)-l-[(2R)-2-({3-ethyl-4-oxo-3H,4H-benzo[g]quinazolin-2- yl}sulfanyl)-2-phenylacetyl]-N-(2-{[8-(2-{[(2R)-l-[(2R)-2-({3-ethyl-4-oxo-3H,4H- benzo[g]quinazolin-2-yl}sulfanyl)-2-phenylacetyl]pyrroli din-2-yl]formamido}ethoxy)oct-4-yn- l-yl]oxy}ethyl)pyrrolidine-2-carboxamide, 1-26:

Step-1. Synthesis of 1 ,4-dibromobut-2-yne [0311] To a stirred solution of but-2-yne- 1 ,4-diol (10.0 g, 1 16 mmol) and PPh₃ (75.9 g, 290 mmol) in DCM (150 mL ) was added DBTCE (79.1 g, 243 mmol) dropwisc at 0 °C under nitrogen atmosphere. The resulting mixture was stirred for 12 hours at room temperature and then quenched with sat. NaHCO₃ solution (500 mL) at 0°C. and extracted with ethyl acetate (3 x200 mL). The combined organic layers were washed with brine (2x200 mL) and dried over anhydrous Na₂SO₄. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with Ethyl acetate / Petroleum ether (0 to 30% ethyl acetate) to afford 1 ,4-dibromobut-2-yne (5.00 g, 23.5 mmol, 21% yield) as a yellow oil.

Step-2. Synthesis of 1,8-di-tert-butyl oct-4-ynedioate

[0312] To a 250 mL 3-necked round-bottom was added tert-butyl acetate (5.41 g, 46.6 mmol) in THF (100 mL). LDA(50 ml, 2M) was added to the solution at -78°C. The resulting mixture was stirred for 0.5 hour at -78°C under nitrogen atmosphere, l,4-dibromobut2-yne (4.50 g, 21.2 mmol) was added at -78°C, and the mixture was stirred for 3 hours at -78°C. The mixture was quenched with sat. NH4CI solution (200 mL) and extracted with ethyl acetate (3x300 mL). The combined organic phase was washed with brine (200 mL), dried over anhydrous sodium sulfate, filtered and concentrated in vacuo. The residue was purified by silica gel column chromatography, eluted with Petroleum ether / Ethyl acetate (0% to 30% ethyl acetate) to afford 1,8 -di- tert-butyl oct-4- ynedioate (3.50 g, 12.3 mmol, 58% yield) as a yellow oil.

Step-3. Synthesis of oct-4-yne-l ,8-diol

[0313] To a 250 mL flask was added 1,8-di-tert-butyl oct-4-ynedioate (3.40 g, 12.0 mmol) in THF (100 mL). The flask was placed in an ice-water bath and LiAlH4 (1.36 g, 36.0 mmol) was added. The mixture was stirred for 3 hours at 60 °C. After the reaction was complete, the reaction was allowed to cool to 0°C. The mixture was quenched with water (2 ml) at 0°C, after to above mixture was added NaOH solution (20% w/wt, 10ml), the mixture was stirred for 20 min at 0°C. The mixture was filtered, dried over Na₂SO₄ and then concentrated in vacuo. The residue was purified by silica gel column chromatography and eluted with Petroleum ether / Ethyl acetate (0% to 100% ethyl acetate) to afford oct-4-yne-l,8-diol (1.30 g, 9.14 mmol, 76% yield) as a yellow oil.

Step-4. Synthesis of diethyl 2,2'-(oct-4-yne-l,8-cliylbis(oxy))diacetate [0314] To a solution of oct-4-yne-l ,8-diol (200 mg, 1.40 mmol) in DCM (10 mL ) was added Rh(OAc)₂ (61.8 mg, 280 μmol) at 0°C. The mixture was stirred for 12 hours at room temperature, quenched with water (1 ml) and then concentrated in vacuo. The residue was purified by silica gel chromatography andeluted with Petroleum ether / Ethyl acetate (0% to 50% ethyl acetate) to afford diethyl 2,2'-(oct-4-yne-l,8-diylbis(oxy))diacetate (200 mg, 636 μmol, 45% yield) as a yellow oil. Step-5. Synthesis of 2,2' -( oct-4-yne-l ,8-diylbis( oxy))bis( ethan-1 -ol )

[0315] To a solution of diethyl 2,2'-(oct-4-yne-l,8-diylbis(oxy))diacetate (200 mg, 636 μmol) in THF (10 mL ) was added Li AIH4 (72.2 mg, 1.90 mmol) at O°C. The mixture was stirred for 3 hours at 0°C to room temperature, quenched with water (0.2 mL) and NaOH solution (20% w/wt, 1 mL), and filtered. The filtered solution was concentrated in vacuo and thethe residue was purified by silica gel chromatography and eluted with Petroleum ether / Ethyl acetate (0% to 100% ethyl acetate) to afford 2,2'-(oct-4-yne-l,8-diylbis(oxy))bis(ethan-l-ol) (130 mg, 564 μmol, 89% yield) as a yellow oil.

Step-6. Synthesis of2-({8-[2- (methanesulfonyloxy )ethoxy]oct-4-yn- 1 -yl}oxy)ethyl methanesulfon ate

[0316] To a stirred solution of 2-{ [8-(2-hydroxyethoxy)oct-4-yn-l-yl]oxy]ethan-l-ol (120 mg, 521 μmol) and TEA (157 mg, 1.56 mmol) in DCM (10 mL) was added methanesulfonyl chloride (130 mg, 1.14 mmol) at 0°C. The resulting mixture was stirred for 3.0 hours at 0°C, the reaction was quenched with water (20 mL) at 0°C, and theresulting mixture was extracted with DCM (2x50 mL). The combined organic layers were washed with H₂O (100 mL)and dried over anhydrous Na₂SO₄ . After filtration, the filtrate was concentrated under reduced pressureto afford 2-({ 8-[2- (methanesulfonyloxy)ethoxy]oct-4-yn-l-yl}oxy)ethyl methanesulfonate (200 mg, 517 μmol, 95 % yield) as a yellow oil.

Step-7. Synthesis of2-[2-({8-[2-(l,3-dioxo-2,3-dihydro-lH-isoindol-2-yl)ethoxy]oct-4-yn-l-yl}ox y)ethyl]-2,3-dihydro-lH-isoindole-l,3-dione

[0317] To a 50 mL flask was added 2-({8-[2-(methanesulfonyloxy)ethoxy]oct-4-yn-l- yl}oxy)ethyl methanesulfonate (200 mg, 517 μmol) and potassium l,3-dioxo-2,3-dihydro-lH- isoindol-2-ide (381 mg, 2.06 mmol) in DMF (10 mL). The mixture was stirred for 48 hours at 85°C and then cooled to room temperature. The mixture was concentrated in vacuo and thenthe residue was purified by silica gel chromatography, (Petroleum ether I Ethyl acetate (0% to 100% ethyl acetate)) to afford 2-[2-({8-[2-(l,3-dioxo-2,3-dihydro-lH-isoindol-2-yl)cthoxy]oct-4-yn-l- yl}oxy)ethyl]-2,3-dihydro-lH-isoindole-l, 3-dione (150 mg, 307 μmol, 59% yield) as a yellow solid.

Step-8. Synthesis of2-/[8-( 2-aminoethoxy )oct-4-yn-l -yl ]oxy}ethan-l -amine

[0318] To a 40 mL vial was added 2-[2-({8-[2-(l,3-dioxo-2,3-dihydro-lH-isoindol-2- yl)ethoxy]oct-4-yn-l-yl}oxy)ethyl]-2,3-dihydro-lHisoindole-l, 3-dione (140 mg, 286μmol) and diazene hydrate hydrogen hydride (143 mg, 2.86 mmol) in EtOH (8.00 mL). The resulting mixture was stirred for 3.0 hours at 80°Cand then cooled to room temperature. The mixture was filtered and the filter was concentrated in vacuoto obtain the crude product 2-{ [8-(2-aminoethoxy)oct-4- yn-l-yl]oxy}ethan-l-amine (80.0 mg, 350 μmol) as a yellow oil, which was used for next step directly.

Step-9. Synthesis of (2R)-l-[(2R)-2-({3-ethyl-4-oxo-3H,4H-benzo[g]quinazolin-2- yl}sulfanyl)-2- phenylacetyl]-N-(2-{[8-(2-{[(2R)-l-[(2R)-2-({3-ethyl-4-oxo-3H,4H-benzo[g]quinazolin-2-yl}sulf anyl )-2-phenylacetyl ]pyrrolidin-2- yl ]formamido}ethoxy)oct-4-yn-l -yl ]oxy}ethyl)pyrrolidine-2-c arboxamide:

[0319] To a mixture of 2-{ [8-(2-aminoethoxy)oct-4-yn-l-yl]oxy}ethan-l-amine (75.0 mg, 328 μmol) and (2R)-l -[(2R)-2-({3-ethyl-4-oxo-3H,4Hbenzo[g]quinazolin-2-yl}sulfanyl)-2- phenylacetyl]pyrrolidine-2-carboxylic acid (335 mg, 688 μmol), DIEA (168 mg, 1.31 mmol) in DMF (5 mL) at room temperature was added HATU (311 mg, 820 μmol). The reaction mixture was stirred for 1 hour at room temperature, and then concentrated in vacuo. The resultant residue was purified by prep-HPLC (NH₃.H₂O buffer) to give (2R)-l-[(2R)-2-({3-ethyl-4-oxo-3H,4H- benzo[g]quinazolin-2- yl}sulfanyl)-2-phenylacetyl]-N-(2-{ [8-(2-{ [(2R)-l-[(2R)-2-({3-ethyl-4- oxo-3H,4H-benzo[g]quinazolin-2-yl}sulfanyl)-2-phenylacetyl]pyrrolidin-2- yl]formamido}ethoxy)oct-4-yn-l-yl]oxy}ethyl)pyrrolidine-2-carboxamide, 1-26. Yield: 25.0 mg, 6.5%; Appearance: white solid; ¹H NMR (400 MHz, DMSO-d₆) 6 8.80 - 8.74 (m, 2H), 8.51 - 8.15 (m, 3H), 8.12 - 8.01 (m, 2H), 7.98 - 7.96 (m, 2H), 7.79 - 7.70 (m, 2H), 7.68 - 7.63 (m, 5H), 7.59 - 7.52 (m, 2H), 7.47 - 7.35 (m, 6H), 6.16 -5.86 (m, 2H), 4.43 - 4.31 (m, 2H), 4.18 - 3.62 (m, 6H), 3.51 - 3.37 (m, 5H), 3.31 - 3.27 (m, 3H), 3.25 - 3.16 (m, 3H), 3.15 - 3.00 (m, 3H), 2.22 - 2.00 (m, 7H), 2.04 - 1.79 (m, 5H), 1.64 - 1.52 (m, 3H), 1.45 - 1.31 (m, 1H),1.32 - 1.20 (m, 6H). HPLC purity: 96.6%; LCMS Calculated for C₆₆H₇₀N₈O₈S₂: 1 166.48; Observed: 1167.5 [M+H]⁺.

Example 8. Synthesis of (2R,2'R)-N,N'-((butane-l,4-diylbis(oxy))bis(butane-4,l-diyl))bis(l- ((R)-2-((3-ethyl-4-oxo-3,4-dihydrobenzo[g]quinazolin-2-yl)thio)-2-phenylacetyl)pyrrolidine-2- carboxamide), 1-27:

Step-1. Synthesis of butane -1,4 -diyl bis(4-methylbenzenesulfonate)

[0320] To a solution of butane- 1 ,4-diol (1.0 g, 11.0 mmol) and TEA (3.33 g, 33.0 mmol) in DCM (50 mL) was added TsCI (4.20 g, 22.0 mmol) at 0 °C. The resulting mixture was stirred for 2.0 hours at 0 °C. The mixture was diluted with water (100 mL) and extracted with DCM (100 mLx3). The combined organic phase was washed with brine (100 mL), dried over anhydrous sodium sulfate, filtered and concentrated in vacuo to afford butane- 1 ,4-diyl bis(4-methylbenzenesulfonate) (1.50 g, 3.76 mmol, 96% purity, 34% yield) as a yellow oil.

Step-2. Synthesis of di-tert-butyl ( (butane- 1, 4-diylbis(oxy))bis(butane-4, 1 -diyl))dicarbamate

[0321] To a mixture of tert-butyl N-(4-hydroxybutyl)carbamate (790 mg, 3.75 mmol) in THF (10 mL) was added NaH (72.0 mg, 3.00 mmol, 60% in mineral oil) at 0 °C for 30 minutes. Butane- 1,4- diyl bis(4-methylbenzenesulfonate) (500 mg, 1.25 mmol) was then added and the reaction mixture was stirred for 12 hours at room temperature. Next, the reaction mixture was diluted with water (100 mL) and extracted with DCM (30 mLx3). The combined organic phase was washed with brine (50 mL), dried over Na₂SO₄, filtered and concentrated in vacuo. The residue was purified by silica gel charomatography (Petroleum ether / ethyl acetate =1:1) to give di-tert-butyl ((butane- l,4-diylbis(oxy))bis(butane-4,l-diyl))dicarbamate (120 mg, 277 μmol, 91% purity, 22% yield) as a colorless oil.

Step-3. Synthesis of4,4’-(butane-l,4-diylbis(oxy))bis(butan-l-amine)

[0322] A solution of di-tert-butyl ((butane- l,4-diylbis(oxy))bis(butane-4,l-diyl))dicarbamate (50 mg, 115 μmol) in TFA:DCM (1:3, 4 mL) was stirred for 1 hour at room temperature. The resulting mixture was concentrated in vacuo to afford 4,4'-(butane-l,4-diylbis(oxy))bis(butan-l-amine) (26.0 mg, 111 μmol, 85% purity, 97% yield) as a colorless oil, which was used for next step directly.

Step-4. Synthesis of (2R,2'R)-N,N'-((butane-l ,4-diylbis(oxy))bis(butane-4,l -diyl)) bis(l-((R)-2- ((3-ethyl-4-oxo-3,4-dihydrobenzo[g]quinazolin-2-yl)thio)-2-phenyl acetyl)pyrrolidine-2- carboxamide)

[0323] To a 8 mL vial were added 4,4'-(butane-l,4-diylbis(oxy))bis(butan-l-amine), ((R)-2-((3- ethyl-4-oxo-3,4-dihydrobenzo[g]quinazolin-2-yl)thio)-2-phenylacetyl)-D-proline (100 mg, 205 μmol), DIEA (39.6 mg, 307 μmol) and HATU (46.3 mg, 122 μmol) in DMF (3 mL) at room temperature. The mixture was stirred for 1 hour and then purified by prep-HPLC (NH₃.H₂O buffer) to afford (2R,2'R)-N,N'-((butane-l,4-diylbis(oxy))bis(butane-4,l-diyl))bis(l-((R)-2-((3-ethyL4- oxo-3, 4-dihydrobenzo[g]quinazolin-2-yl)thio)-2-phenylacetyl)pyrrolidine-2-carboxamide), 1-27. Yield: 25.0 mg, 21%; Appearance: White solid; ¹H NMR (400 MHz, DMSO-d₆) δ 8.81 - 8.72 (m, 2H), 8.21 - 8.13 (m, 3H), 8.06 - 8.03 (m, 2H), 7.98 - 7.96 (m, 1H), 7.74 - 7.61 (m, 8H), 7.59 - 7.53 (m, 2H), 7.39 - 7.36 (m, 6H), 6.02 - 5.96 (m, 2H), 4.37 - 4.29 (m, 2H), 4.19 - 4.01 (m, 6H), 3.62 - 3.58 (m, 2H), 3.31 - 3.20 (m, 4H), 3.00 - 2.90 (m, 6H), 2.19 - 1.78 (m, 6H), 1.52 - 1.48 (m, 2H), 1.44 (t, J = 7.0 Hz, 2H), 1.35 - 1.33 (m, 7H), 1.32 - 1.20 (m, 11H). HPLC purity: 96%; LCMS Calculated for C₆₆H₇₄N₈O₈S₂: 1170.51; Observed: 1171.7 [M+H]⁺.

Example 9: Synthesis of (2R)-l-[(2R)-2-({3-ethyl-4-oxo-3H,4H-benzo[g] quinazolin-2- yl}sulfanyl)-2-phenylacetyl]-N-(14-{[ ( 2R )-l-[(2R )-2-( {3-ethyl-4-oxo-3H,4H- benzo[g]quinazolin-2-yl}sulfanyl)-2-phenylacetyl]piperazin-2-yl] formamido}-3,6,9,12- tetraoxatetradecan -l-yl)piperazine-2-carboxamide, 1-28:

Step-1. Synthesis of tert-butyl (3R)-3-[(14-{[(2R)-4-[(tert-butoxy)carbonyl]-l-[(2 R) -2-({3-ethyl-

4-oxo-3H,4H-benzo[g]quinazolin-2-yl}sulfanyl)-2-phenylacetyl]piperazI n-2-yl]formamido}- 3,6,9, 12-tetraoxatetradecan-l-yl)carbamoyl]-4-[(2R)-2-({3-ethyl-4-oxo-3H,4H- benz,o[g]quinazolin-2-yl}sulfanyl)-2-phenylacetyl]piperazine-l -carboxy late

[0324] A mixture of (2R)-4-[(tert-butoxy)carbonyl]-l-[(2R)-2-({3-ethyl-4-oxo-3H,4H- benzo[g]quinazolin-2-yl} sulfanyl)-2-phenylacetyl]piperazine-2-carboxylic acid (peakl, labelled as INT-12) (100.0 mg, 165.0 μmol), 3,6,9,12-tetraoxatetradecane-l,14-diamine (19.4 mg, 82.5 μmol) and DIEA (63.8 mg, 495.0 μmol) in DMF (1 mL) was stirred at 0°C for 10 min. HATU (75.2 mg, 198.0 μmol) was added, and then the mixture was stirred at 0 °C for 4.0 hours. The reaction mixture was concentrated in vacuo and the residue was purified by prep-HPLC (NH1 H₂O buffer) to afford tert-butyl (3R)-3-[(14-{[(2R)-4-[(tert-butoxy)carbonyl]-l-[(2R)-2-({3-ethyl-4- oxo-3H,4H-benzo[g]quinazolin-2-yl}sulfanyl)-2-phenylacetyl]piperazin-2-yl]formamido}-

3,6,9, 12-tetraoxatetradecan-l-yl)carbamoyl]-4-[(2R)-2-({3-ethyl-4-oxo-3H,4H- benzo[g]quinazolin-2-yl}sulfanyl)-2-phenylacetyl]piperazine-l-carboxylate (30.0 mg, 17.7 μmol, 11.9% yield) as white solid.

Step-2. Synthesis of ( 2R )-l -[( 2R)-2-({3 -ethyl-4-oxo-3H,4H-benzo[g]quinazolin-2-yl } sulfanyl )-2- phenylacetyl]-N-(14-{[(2R)-l-[(2R)-2-({3-ethyl-4-oxo-3H,4H-benzo [g]quinazolin-2- yl}sulfanyl)-2-phenylacetyl]piperaz.in-2-yl]formamido}-3,6,9,12-tet raoxatetradecan- 1 - yl)piperazine-2-carboxamide

[0325] A solution of tert-butyl (3R)-3-[(14-{ [(2R)-4-[(tert-butoxy)carbonyl]-l -[(2R)-2-({3-ethyl- 4-oxo-3H,4H-benzo[g]quinazolin-2-yl}sulfanyl)-2-phenylacetyl]piperazin-2-yl]formamido}-

3,6,9, 12-tetraoxatetradecan-l-yl)carbamoyl]-4-[(2R)-2-({3-ethyl-4-oxo3H,4H- benzo[g]quinazolin-2-yl}sulfanyl)-2-phenylacetyl]piperazine-l-carboxylate(30 mg, 21.3 μmol) in formic acid (1 mL) was stirred for 2.0 hours at room temperature. The resulting mixture was concentrated in vacuo and the residue was purified by prep-HPLC (NH₃ H₂O buffer) to afford (2R)-l-[(2R)-2-({3-ethyl-4-oxo-3H,4H-benzo[g]quinazolin-2-yl}sulfanyl)-2-phenylacetyl]-N- (14-{ [(2R)-l-[(2R)-2-({3-ethyl-4-oxo-3H,4H-benzo[g]quinazolin-2-yl]sulfanyl)-2- phenylacetyl]piperazin-2-yl]formamido]-3,6,9,12-tetraoxatetradecan-l-yl)piperazine-2- carboxamide, 1-28. Yield: 25.0 mg, 97.6%; Appearance: White solid; ¹H NMR (300 MHz, DMSO-d₆) δ 8.82 - 8.71 (m, 2H), 8.40 - 8.31 (m, 1H), 8.26 - 7.95 (m, 6H), 7.74 - 7.60 (m, 6H), 7.60 - 7.47 (m, 3H), 7.47 - 7.28 (m, 6H), 6.48 (s, 1H), 6.28 (s, 1H), 4.81 - 4.49 (m, 2H), 4.25 - 3.94 (m, 6H), 3.54 - 3.35 (m, 10H), 3.33 - 3.25 (m, 8H), 3.24 - 2.68 (m, 10H), 2.32 - 1.90 (m, 4H), 1.32 - 1.20 (m, 6H). HPLC purity: 95.8%; LCMS Calculated for C₆₄H₇₂N₁₀O₁₀S₂: 1204.49; Observed: 1205.8 [M+H]⁺.

Example 10. Synthesis of (2R)-l-[(2S)-2-({3-ethyl-4-oxo-3H,4H-benzo[g]quinazolin-2- yl}sulfanyl)-2-phenylacetyl]-N-(14-{[ ( 2R )-l-[(2S )-2-( {3-ethyl-4-oxo-3H,4H- benzo[g]quinazolin-2-yl}sulfanyl)-2-phenylacetyl]piperazin-2-yl]formamido}-3,6,9,12- tetraoxate tradecan-l-yl)piperazine-2-carboxamide, 1-29:

Step-1. Synthesis of tert-butyl (3R)-4-[(2S)-2-({3-ethyl-4-oxo-3H,4H-benzo[g]quina zolin-2- yl}sulfanyl)-2-phenylacetyl]-3-[(14-{[(2R)-l-[(2S)-2-({3-ethyl-4-oxo-3H,4H- benzo[g]quinazolin-2-yl}sulfanyl)-2-phenylacetyl]piperazin-2-yl]formamido}-3,6,9, 12- tetraoxatetradecan-1 -yl)carbamoyl ]piperazine-l -carboxylate [0326] A mixture of (2R)-4-[(tert-butoxy)carbonyl]-1 -[(2S)-2-({3-ethyl-4-oxo-3H,4H-benzo [g]quinazolin-2-yl}sulfanyl)-2-phcnylacetyl]pipcrazinc-2-carboxylic acid (pcak2, labelled as INT-13) (100.0 mg, 165 μmol), 3,6,9,12-tetraoxatetradecane-l,14-diamine (19.4 mg, 82.5 μmol) and DIEA (63.8 mg, 495.0 μmol) in DMF (1 mL) was stirred at 0°C for 10 min. HATU (75.2 mg, 198.0 μmol) was then added, and stirred at 0 °C for 4.0 hours. The reaction mixture was concentrated in vacuo and the residue was purified by prep-HPLC (NH₃· H₂O buffer) to afford tert- butyl (3R)-3-[(14-{ [(2R)-4-[(tert-butoxy)carbonyl]-l-[(2R)-2-({3-ethyl-4-oxo-3H,4H- benzo[g]quinazolin-2-yl}sulfanyl)-2-phenylacetyl]piperazin-2-yl]formamido}-3,6,9,12- tetraoxatetradecan- l-yl)carbamoyl]-4-[(2R)-2-({3-ethyl-4-oxo-3H,4H-benzo[g]quinazolin-2- yl}sulfanyl)-2-phenylacetyl]piperazine-l -carboxylate (30.0 mg, 17.7 μmol, 11.9 % yield) as white solid.

Step-2. Synthesis of (2R)-l-[(2S)-2-({3-ethyl-4-oxo-3H,4H-benw[g]quinazolin-2-yl}sulfanyl)-2- phenylacetyl]-N-(14-{[(2R)-l-[(2S)-2-({3-ethyl-4-oxo-3H,4H-benzo [g]quinazolin-2- yl}sulfanyl)-2-phenylacetyl]piperazin-2-yl]formamido}-3,6,9,12-tetraoxatetradecan-l- yl )piperazine-2-carboxamide, 1-29

[0327] A solution of tert-butyl (3R)-4-[(2S)-2-({3-ethyl-4-oxo-3H,4H-benzo[g]quinazolin-2- yl } sulfanyl)-2-phenylacetyl] -3-[( 14- { [(2R)- 1 -[(2S)-2-( { 3-ethyl-4-oxo-3H,4H-benzo [g]quinazolin-2-yl}sulfanyl)-2-phenylacetyl]piperazin-2-yl]formamido}-3,6,9,12-tetra oxatetradecan l-yl)carbamoyl]piperazine-l -carboxylate (30.0 mg, 22.9 μmol) in formic acid (1 mL) was stirred for 3.0 hours at room temperature. The resulting mixture was concentrated in vacuo and the residue was purified by prep-HPLC (NH₃ H₂O buffer) to afford (2R)-l-[(2S)-2- ({3-ethyl-4-oxo-3H,4H-benzo[g]quinazolin2-yl}sulfanyl)-2-phenylacetyl]-N-(14-{ [(2R)-l-[(2S)- 2-({3-ethyl-4-oxo-3H,4H-benzo[g]quinazolin-2-yl]sulfanyl)-2-phenylacetyl]piperazin-2- yl]formamido}-3,6,9,12-tetraoxatetradecan-l-yl)piperazine-2-carboxamide. Yield: 25.0 mg, 90.5%; Appearance: White solid; ¹H NMR (300 MHz, DMSO-d₆) δ 8.82 - 8.74 (m, 2H), 8.34 (s, 2H), 8.23 - 8.06 (m, 4H), 7.87 (t, J = 5.6 Hz, 2H), 7.75 - 7.61 (m, 4H), 7.61 - 7.49 (m, 2H), 7.48 - 7.24 (m, 8H), 6.43 (s, 2H), 4.96 - 4.77 (m, 2H), 4.20 - 3.90 (m, 4H), 3.97 - 3.87 (m, 2H), 3.65 - 3.48 (m, 2H), 3.50 - 3.36 (m, 10H), 3.31 - 3.09 (m, 8H), 3.03 - 2.79 (m, 1H), 2.63 (d, J = 5.6 Hz, 2H), 2.40 - 2.28 (m, 2H), 2.12 - 2.05 (m, 3H), 1.70 - 1.55 (m, 2H), 1.32 - 1.20 (m, 6H). HPLC purity: 97.1 %; LCMS Calculated for C₆₄H₇₂N₁₀O₁₀S₂: 1204.49; Observed: 1205.9 [M+H]⁺.

Example 11. Synthesis of (2R)-l-[(2R)-2-({3-ethyl-4-oxo-3H,4H-benzo[g]quinazolin-2- yl}sulfanyl)-2-(pyridin-3-yl)acetyl]pyrrolidine-2-carboxylic acid, 1-30:

Step-1. Synthesis of (2R)-l-[(2R)-2-({3-ethyl-4-oxo-3H,4H-benzo[g]quinazolin-2-yl}sulfanyl)-2- (pyridin-3-yl)acetyl]-N-(14-{[(2R)-l-[(2R)-2-({3-ethyl-4-oxo-3H,4H-benzo[g]quinazolin-2- yl}sulfanyl)-2-(pyridin-3-yl)acetyl]pyrrolidin-2-yl]formamido}-3,6,9,12-tetraoxatetradecan-l- yl)pyrrolidine-2-carboxamide

[0328] A mixture of (2R)-l-[(2R)-2-({3-ethyl-4-oxo-3H,4H-benzo[g]quinazolin-2-yl}sulfanyl)- 2-(pyridin-3-yl)acetyl]pyrrolidine-2-carboxylic acid (peakl, labelled as INT-14) (100 mg, 204 μmol), 3,6,9,12-tetraoxatetradecane-l,14-diamine (24.1 mg, 102 μmol) and DIEA (26.3 mg, 204 μmol) in DMF (1 mL) was stirred at 0°C for 10 min. HATU (92.7 mg, 244 μmol) was then added, and the reaction mixture was stirred for 3 hours at room temperature. The resulting mixture was concentrated in vacuo and the residue was purified by prep-HPLC (NH3· H₂O buffer) to afford (2R)-l-[(2R)-2-({3-ethyl-4-oxo-3H,4H-benzo[g]quinazolin-2-yl}sulfanyl)-2-(pyridin-3- yl)acctyl]-N-(14-{ [(2R)-l-[(2R)-2-({3-cthyl-4-oxo-3H,4H-bcnzo[g]quinazolin-2-yl]sulfanyl)-2- (pyridin-3-yl)acetyl]pyrrolidin-2-yl]formamido}-3,6,9,12-tetraoxatetradecan-l-yl)pyrrolidine-2- carboxamide, 1-30. Yield: 18.0 mg, 10.4%; Appearance: White solid; ¹H NMR (300 MHz, DMSO-d₆) 8 8.98 - 8.95 (m, 1H), 8.85 - 8.79 (m, 2H), 8.69 (d, J= 2.3 Hz, 1H), 8.56 - 8.44 (m, 2H), 8.29 - 8.14 (m, 4H), 8.12 - 7.91 (m, 4H), 7.85 - 7.78 (m, 2H), 7.73 - 7.62 (m, 2H), 7.61 - 7.50 (m, 2H), 7.48 - 7.34 (m, 2H), 6.24 (s, 1H), 5.91 (s, 1H), 5.15 - 5.05 (m, 1H), 4.36 - 4.25 (m, 1H), 4.20 - 3.98 (m, 5H), 3.53 - 3.36 (m, 16H), 3.28 - 3.19 (m, 2H), 3.13 - 3.03 (m, 5H), 2.50 - 2.40 (m, 1H), 2.09 - 1.76 (m, 7H), 1.32 - 1.20 (m, 6H). HPLC purity: 97.9%; LCMS Calculated for C₆₂H₆₈N₁₀O₁₀S₂: 1176.46; Observed: 1177.3 [M+H]⁺.

Example 12. Synthesis of (2R)-l-[(2S)-2-({3-ethyl-4-oxo-3H,4H-benzo[g] quinazolin-2- yl}sulfanyl)-2-(pyridin-3-yl)acetyl]-N-(14-{[ ( 2R )-l-[(2S )-2-( {3-ethyl-4-oxo-3H,4H- benzo[g]quinazolin-2-yl}sulfanyl)-2-(pyridin-3-yl)acetyl]pyrrolidin-2-yl]formamido}-3,6,9,12- tetraoxatetradecan-1 -yl)pyrrolidine-2-carboxamide, 1-310:

Step-1. Synthesis of ( 2R,2'R)-N,N'-( 3, 6, 9,12-tetraoxatetradecane-l, 14-diyl )bis( l-((S)-2-(( 3-elhyl- 4-oxo-3,4-dihydrobenzo[g]quinazolin-2-yl)thio)-2-(pyridin-3-yl)acetyl)pyrrolidine-2- carboxamide )

[0329] A mixture of (2R)-l-[(2S)-2-({3-ethyl-4-oxo-3H,4H-benzo[g]quinazolin-2-yl}sulfanyl)- 2-(pyridin-3-yl)acetyl]pyrrolidine-2-carboxylic acid (peak2, labelled as INT-15) (100.0 mg, 204.0 μmol), 3,6,9,12-tetraoxatetradecane-l,14-diamine (24.1 mg, 102.0 μmol) and DIEA (26.3 mg, 204.0 μmol) in DMF (1 mL) was stirred at 0°C for 10 min. HATU (92.7 mg, 244.0 μmol) was then added, and the reaction mixture was stirred for 3 hours at room temperature. The resulting mixture was concentrated in vacuo and the residue was purified by prep-HPLC (NH₃· H₂O buffer) to afford (2R,2'R)-N,N'-(3,6,9,12-tetraoxatetradecane-l,14-diyl)bis(l-((S)-2-((3-ethyl-4-oxo-3,4- dihydrobenzo[g]quinazolin-2-yl)thio)-2-(pyridin-3-yl)acetyl)pyrrolidine-2-carboxamide), I-31.

Yield: 25.0 mg, 20.0%; Appearance: White solid; ¹H NMR (300 MHz, DMSO-d₆) δ 9.04 - 8.86 (m, 2H), 8.83 - 8.71 (m, 2H), 8.64 - 8.46 (m, 2H), 8.25 - 7.96 (m, 7H), 7.86 (t, J= 5.6 Hz, 2H), 7.73 - 7.61 (m, 2H), 7.62 - 7.49 (m, 2H), 7.50 - 7.35 (m, 2H), 6.21 - 5.91 (m, 1H), 4.53 - 4.29 (m, 2H), 4.15 - 4.00 (m, 5H), 3.52 - 3.41 (m, 11H), 3.41 - 3.33 (m, 5H), 3.28 - 3.03 (m, 7H), 2.31 - 1.70 (m, 8H), 1.32 - 1.18 (m, 6H). HPLC purity: 95.2%; LCMS Calculated for C₆₂H₆₈N₁₀O₁₀S₂: 1176.46; Observed: 1177.5 [M+HJ⁺.

Example 13. Synthesis of (2R)-l-[2-({3-ethyl-4-oxo-3H,4H-benzo[g]quinazolin-2- yl}sulfanyl)pentanoyl]-N-(14-{[(2R)-l-[2-({4-oxo-3H,4H-benzo[g]quinazolin-2- yl}sulfanyl)pentanoyl]pyrrolidin-2-yl]formamido}-3,6,9,12-tetraoxatetradecan-l- yl)pyrrolidine-2-carboxamide, I-32:

Step-1. Synthesis of ethyl 2-({3-ethyl-4-oxo-3H,4H-benzo[g]quinazolin-2-yl}sulfanyl) pentanoate

[0330] To a 500 mL 3-necked round-bottom flask were added 3-ethyl-2-sulfanyl-3H,4H- benzo[g]quinazolin-4-one (10.0 g, 39.0 mmol), DIEA (14.9 g, 116.0 mmol) and ethyl 2- bromopentanoate (12.2 g, 58.4 mmol) in DMSO (300 mL) at room temperature. The resulting mixture was stirred for 4 hours at 50 °C, cooled to room temperature and then diluted with H₂O (1L). The formed solids were filtered, and the filter cake was washed with H₂O (3x 100 mL)to afford 3-cthy 1-2-s ulfanyl-3H,4H-bcnzo[g]quinazolin-4-onc (30.0 g, 117.0 mmol, 87.2% yield) as a yellow solid.

Step-2. Synthesis of2-({3-ethyl-4-oxo-3H,4H-benzo[g]quinazolin-2-yl}sulfanyl)pentanoic acid

[0331] To a solution of ethyl 2-({3-ethyl-4-oxo-3H,4H-benzo[g]quinazolin-2-yl}sulfany l)pentanoate (12.0 g, 31.2 mmol) in THF/H₂O (4:1, 300 mL) was added LiOH (1.49 g, 62.4 mmol) at room temperature. The reaction mixture was stirred for 4 hours at 50 °C and cooled down to room temperature. The resulting mixture was adjusted to pH=3 with 2M HC1, and then the precipitated solids were collected by filtration and washed with water (3 x 50 mL). The resulting solid was dried under infrared lightto afford 2-({3-ethyl-4-oxo-3H,4H-benzo[g]quinazolin-2- yl } sulfanyljpentanoic acid (9.5 g, 26.6 mmol, 85.5% yield) as a yellow solid

Step-3. Synthesis of tert-butyl (2R)-l-[2-({3-ethyl-4-oxo-3H,4H-benzo[g]quinazolin-2- yl}sulfanyl)pentanoyl]pyrrolidine-2-carboxylate

[0332] A mixture of 2-({3-ethyl-4-oxo-3H,4H-benzo[g]quinazolin-2-yl sulfanyljpentanoic acid (4.5 g, 12.6 mmol), tert-butyl (2R)-pyrrolidine-2-carboxylate hydrochloride (3.9 g, 18.9 mmol) and DIEA (4.9 g, 37.8 mmol) in DMF (500 mL ) was stirred at 0°C for 10 min. HATU (5.7 g, 15.1 mmol) was added and then solution was stirred at 0 °C for 3.0 hours. The resulting mixture was concentrated in vacuo and the residue was purified by prep-HPLC (NH₃ H₂O buffer) to afford tertbutyl (2R)-l-[2-({3-ethyl-4-oxo-3H,4H-benzo[g]quinazolin-2-yl]sulfanyl)pentanoyl]pyrrolidine- 2-carboxylate (4.0 g, 7.8 mmol, 62.3% yield) as a yellow solid.

Step-4. Synthesis of (2R)-l-[2-({3-ethyl-4-oxo-3H,4H-benzo[g]quinazolin-2-yl}sulfan I )pentanoyl ]pyrrolidine-2-carboxylic acid

[0333] A stirred solution of tert-butyl (2R)-l-[2-({3-ethyl-4-oxo-3H,4H-benzo[g]quinaz olin-2- yl}sulfanyl)pentanoyl]pyrrolidine-2-carboxylate (4.0 g, 7.84 mmol) in TFA (40 mL) was stirred for 4.0 hours at 0°C and concentrated under reduced pressure to afford the crude product (2R)-1- [2-({3-ethyl-4-oxo-3H,4H-benzo[g]quinazolin-2-yl}sulfanyl)pentanoyl]pyrrolidine-2-carboxylic acid (1.0 g, 2.2 mmol, 28.1% yield) as a yellow solid, which was used to next step without under purification. Step-5. Synthesis of (2R)-I-[2-(/3-ethyl-4-oxo-3H,4H-benzo[g]quinazolin-2- yl} sulfanyl )pentanoyl ]-N-( 14-[[(2R)-l-[2-({ 4-oxo-3H,4H-benzo[g]quinazolin-2- yl} sulfanyl )pentanoyl ]pyrrolidin-2 -yl]formamido } -3, 6, 9, 12 -tetraoxatetrade can- 1 -yl )pyrrolidine- 2-carboxamide, 1-32

[0334] A mixture of (2R)-l-[2-({4-oxo-3H,4H-benzo[g]quinazolin-2-yl}sulfanyl) pentanoyl]pyr rolidine-2-carboxylic acid (100.0 mg, 235.0 μmol), 3,6,9,12-tetraoxatetradecane-l,14-diamine (27.6 mg, 117.0 μmol) and DIEA (90.9 mg, 705.0 μmol) in DMF (3 mL) was stirred at 0°C for 10 min. HATU (107.0 mg, 282.0 μmol) was then added and the solution was stirred at 0 °C for 2.0 hours. The resulting mixture was concentrated in vacuo and the residue was purified by prep- HPLC (NH₃ H₂O buffer) to afford (2R)-l-[2-({3- ethyl-4-oxo-3H,4H-benzo[g]quinazolin-2- yl } sulfanyl)pentanoyl] -N-( 14- { [(2R)- 1 - [2-( { 4-oxo-3H,4H-benzo[g]quinazolin-2- yl} sulf anyl)pentanoyl]pyrrolidin-2-yl]formamido}-3, 6,9, 12-tetraoxatetradecan-l-yl)pyrrolidine- 2-carboxamide, 1-32. Yield: 25.0 mg, 11.8%; Appearance: White solid; ¹H NMR (300 MHz, DMSO-d₆) δ 8.76 (s, 2H), 8.26 - 7.95 (m, 5H), 7.94 - 7.84 (m, 3H), 7.70 - 7.50 (m, 4H), 5.03 - 4.86 (m, 2H), 4.33 - 4.24 (m, 3H), 4.15 - 3.99 (m, 6H), 3.80 - 3.73 (m, 2H), 3.60 - 3.44 (m, 13H), 3.32 - 3.20 (m, 3H), 3.19 - 3.10 (m, 2H), 2.98 - 2.87 (m, 1H), 2.17 - 1.72 (m, 12H), 1.66 - 1.44 (m, 4H), 1.20 - 1.40 (m, 6H), 1.05 - 0.90 (m, 6H). HPLC purity: 97.3%; LCMS Calculated for C₅₈H₇₄N₈O₁₀S₂: 1106.50; Observed: 1107.4 [M+H]⁺.

[0335] The following example was prepared using standard chemical manipulations and procedures similar to those used for the preparation of the previous examples. Analytical data is given in the table below.

Example 14. Synthesis of 2,2'-(((S,lS,rS)-(((octane-l,8-diylbis(()xy))bis(prop-l-yne-3,l- diyl))bis( 3 ,1 -phenylene ) )bis( 2-((S )-3-hydroxypyrrolidin-l-yl)-2-oxoethane-l, 1 - diyl))bis( sulfanediyl))bis( 3-ethylbenzo[g]quinazolin-4( 3H)-one), 1-34:

Step-1. Synthesis of 2,2'-(((S,1S,1 'S)-(((octane-l,8-diylbis(oxy))bis(prop-l-yne-3,l-diyl))bis(3,l- phenylene))bis(2-((S)-3-hydroxypyrrolidin- 1 -yl)-2-oxoethane- 1 , 1 -diyl))bis(sulfanediyl))bis(3- ethylbenzo[g]quinazolin-4(377)-one)

[0336] To a solution of 3-ethyl-2-{[(lS)-2-[(3S)-3-hydroxypyrrolidin-l-yl]-l-(3-iodophenyl)-2- oxoethyl ]sulfanyl }-3H,4/7-bcnzo[g ]quinazolin-4-one (100 mg, 0.17 mmol) , l,8-bis(prop-2-yn- l-yloxy)octane (18.8 mg, 0.085 mmol), Cui (6.47 mg, 0.034 mmol) and Pd(PPh₃)₂Cl₂ (23.8 mg, 0.034 mmol) in DMF (2 mL) was added TEA (1.71 g, 16.9 mmol) at RT under N2. The resulting mixture was stirred for 16 h at 60 °C under N2, quenched with water (20 mL) and extracted with EA (30 mL X 3). The combined organic layer was dried over Na₂SO₄ and concentrated under vacuum. The residue was purified by flash silica gel chromatography (EA/MeOH = 20/1) to give 2,2'-(((5,lS,l 'S)-(((octane-l,8-diylbis(oxy))bis(prop-l-yne-3,l-diyl))bis(3,l-phenylene))bis(2- ((S)-3-hydroxypyrrolidin-l-yl)-2-oxoethane-l,l-diyl))bis(sulfane diyl))bis(3- ethylbenzo[g]quinazolin-4(3//)-one). 1-34. Yield: 22.8 mg, 24%; Appearance: White solid;

NMR (400 MHz, DMSO-d₆) δ 8.77 (d, J = 4.8 Hz, 2H), 8.37 (s, 1H), 8.23 - 8.14 (m, 2H), 8.08 (d, J= 8.4 Hz, 1H), 7.96 (d, J= 7.6 Hz, 2H), 7.82 - 7.76 (m, 2H), 7.74 - 7.70 (m, 2H), 7.68 - 7.62

(m, 2H), 7.57 - 7.52 (m, 2H), 7.48 - 7.41 (m, 4H), 5.96 (d, J = 12.0 Hz, 2H), 5.25 (d, J= 2.8 Hz,

1H), 5.06 (d, J = 3.6 Hz, 1H), 4.53 (br. s, 1H), 4.37 - 4.34 (m, 5H), 4.24 - 4.17 (m, 2H), 4.13 -

4.02 (m, 4H), 3.57 - 3.49 (m, 2H), 3.48 - 3.42 (m, 6H), 2.08 - 1.90 (m, 3H), 1.89 - 1.72 (m, 1H),

1.51 - 1.43 (m, 4H), 1.29 - 1.22 (m, 16H).; HPLC purity: 95.2 %; LCMS Calculated for C₆₆H₆₈N6O₈S₂: 1136.45; Observed: 1137.1 [M+H]⁺.

Example 15. Synthesis of 3-ethyl-2-{[(lR*)-l-(3-{3-[4-(4-{4-[(3-{3-[(lR*)-l-({3-ethyl-4-oxo- 3H,4H-benzo[g]quinazolin-2-yl}sulfanyl)-2-[(3S)-3-hydroxypyrrolidin-l-yl]-2- oxoethyl]phenyl}prop-2-yn-l-yl)oxy]butyl}piperazin-l-yl)butoxy]prop-l-yn-l-yl}phenyl)-2- [(3S)-3-hydroxypyrrolidin-l-yl]-2-oxoethyl]sulfanyl}-3H,4H-benzo[g]quinazolin-4-one, 1-35:

[0337] To a solution of piperazine (4 g, 46.4 mmol) and 4-bromobutan-l-ol (21.2 g, 139 mmol) in DMF (40 mL ) was added TEA (18.7 g,185 mmol). The resulting mixture was stirred at 60 °C for 12 h. The reaction mixture was filtered and the filtrate was concentrated in vacuo. The residue was purified by silica gel chromatography (DCM/MeOH = 5/1) to give 4-[4-(4- hydroxybutyl)piperazin-l-yl]butan-l-ol (5.40 g, 23.4 mmol) as a yellow solid. 1H NM (400 MHz, Methanol-d₄) 5 3.58 (t, J= 6.0 Hz, 4H), 3.00 - 2.66 (m, 8H), 2.66 - 2.54 (m, 4H), 1 .71 - 1 .62 (m, 4H), 1 .61 - 1 .54 (m, 4H).

Step-2: l,4-bis(4-(prop-2-yn-l-yloxy)butyl)piperazine [0338] To a mixture of 4-[4-(4-hydroxybutyl)piperazin-l-yl]butan-l-ol (1 g, 4.34 mmol) and potassium tcrt-butoxidc (875 mg, 7.80 mmol) in THF (20 mL ) was added 3-bromoprop-l-ync (1.28 g, 10.8 mmol). The resulting mixture was stirred at RT for 12 h. The reaction mixture was concentrated to give residue, which was purified by flash silica gel chromatography (DCM/MeOH = 15/1) to give l,4-bis[4-(prop-2-yn-l-yloxy)butyl]piperazine (360 mg, 1.17 mmol) as a brown oil.

*H NMR (401 MHz, Methanol-d₄) δ 4.12 (d, 7= 2.4 Hz, 4H), 3.56 - 3.51 (m, 4H), 2.81 (t, 7 = 2.4 Hz, 2H), 2.70 - 2.34 (m, 12H), 1.63 - 1.55 (m, 8H).

Step-3. Synthesis of 3-ethyl-2-{ [(lR*)-l -(3-{3-[4-(4-{4-[(3-{3-[(lR*)- l-({3-ethyl-4-oxo-3H,4H- benzo[g]quinazolin-2-yl}sulfanyl)-2-[(3S)-3-hydroxypyrrolidin-l-yl]-2-oxoethyl]phenyl}prop-2- yn-l-yl)oxy]butyl}piperazin-l-yl)butoxy]prop-l-yn-l-yl}phenyl)-2-[(3S)-3-hydroxypyrrolidin- 1 -yl] -2-oxoethyl] sulfanyl } -3H,4H-benzo [g] quinazolin-4-one

[0339] To a solution of 3-ethyl-2-{ [(lR)-2-[(3S)-3-hydroxypyrrolidin-l-yl]-l-(3-iodophenyl)-2- oxoethyl]sulfanyl]-3H,4H-benzo[g]quinazolin-4-one (200 mg, 0.34 mmol) in DMF (2 mL) was added l,4-bis[4-(prop-2-yn-l-yloxy)butyl]piperazine (26.1 mg, 0.09 mmol), Pd(pph3)₂C12 (47.8 mg, 0.07 mmol), Cui (12.9 mg, 0.07 mmol) and TEA (220 mg, 1.70 mmol). The mixture was stirred at 60 °C for 4 h, concentrated and purified by reverse combi flash (Biotage, 25 g Agela C18, MeCN/H₂O(0.5% NH₄HCO₃) to give 3-ethyl-2-{ [(lR*)-l-(3-{3-[4-(4-{4-[(3-{3-[(lR*)-l- ({3-ethyl-4-oxo-3H,4H-benzo[g]quinazolin-2-yl}sulfanyl)-2-[(3S)-3-hydroxypyrrolidin-l-yl]-2- oxoethyl]phenyl }prop-2-yn- 1 -yl)oxy ]buty 1 } piperazin- 1 -yl)butoxy ]prop- 1 -y n- 1 -yl }phenyl)-2- [(3S)-3-hydroxypyrrolidin- 1-yl] -2-oxoethyl] sulfanyl]-3H,4H-benzo[g]quinazolin-4-one, 1-35, Yield: 15.0 mg, 7.05%; Appearance: White solid; ¹H NMR (400 MHz, CDCI₂) δ 8.82 - 8.75 (m, 2H), 8.17 (d, 7 = 10.8 Hz, 1H), 7.99 (dd, 7 = 12.8, 8.2 Hz, 2H), 7.90 (t, 7 = 7.2 Hz, 2H), 7.83 (d, 7 = 7.6 Hz, 1H), 7.74 (d, 7 = 10.4 Hz, 2H), 7.66 - 7.60 (m, 2H), 7.58 - 7.45 (m, 4H), 7.43 - 7.38 (m, 2H), 7.35 - 7.30 (m, 2H), 6.00 (dd, 7 = 89.6, 5.0 Hz, 2H), 4.58 (d, 7 = 33.2 Hz, 2H), 4.46 - 4.13 (m, 11H), 3.76 - 3.54 (m, 11H), 2.89 - 1.76 (m, 22H), 1.38 (t, 7= 7.2 Hz, 6H), 1.26 (s, 2H); HPLC purity: 98.5%; LCMS Calculated for C₇₀H₇₆N₈O₈S₂: 1221.55; Observed: 1221.5 [M+H]⁺.

Example 16. Synthesis of 3-ethyl-2-{[( 1S)-1 -(3-{3-[4-(4-{4-[(3-{3-[( 1 S)- l-({3-ethyl-4-oxo- 3H,4H-benzo[g]quinazolin-2-yl}sulfanyl)-2-[(3S)-3-hydroxypyrrolidin-l-yl]-2- oxoethyl]phenyl}prop-2-yn- l-yl)oxy]butyl}piperazin- l-yl)bu toxy]prop- 1-yn- 1 -yl}phenyl)-2- [(3S)-3-hydroxypyrrolidin-l-yl]-2-oxoethyl]sulfanyl}-3H,4H-benzo[g]quinazolin-4-one, 1-36:

Step-1. Synthesis of 3-ethyl-2-{[(lS)-l-(3-{3-[4-(4-{4-[(3-{3-[(lS)-l-({3-ethyl-4-oxo-3H,4H- benzo[g]quinazolin-2-yl}sulfanyl)-2-[(3S)-3-hydroxypyrrolidin-l-yl]-2-oxoethyl]phenyl}prop-2- yn-l-yl)oxy]butyl}piperazin-l-yl)butoxy]prop-l-yn-l-yl}phenyl)-2-[(3S)-3-hydroxypyrrolidin- 1 -yl] -2-oxoethyl] sulfanyl } -3H,4H-benzo [g] quinazolin-4-one

[0340] To a solution of 3-ethyl-2-{ [(lS)-2-[(2S)-2-hydroxypyrrolidin-l-yl]-l-(3-iodophenyl)-2- oxoethyl]sulfanyl}-3H,4H-benzo[g]quinazolin-4-one (200 mg, 0.3416 mmol), dichloropalladium; bis(triphenylphosphane) (23.9 mg, 0.03416 mmol) , iodocopper (6.50 mg, 0.03416 mmol) , l,4-bis[4-(prop-2-yn-l-yloxy)butyl]piperazine (52.3 mg, 0.1708 mmol) in DMF (2 mL ) was added TEA (171 mg, 1.70 mmol) . The mixture was stirred at 60 °C for 4 h. The reaction mixture was concentrated and purified by reverse combi flash (Biotage, 25 g Agela Cl 8, MeCN/H₂O(0.5% NH4HCO₃) to give 3-cthyl-2-{ [(lS)-l-(3-{ 3-[4-(4-{4-[(3-{ 3-[(lS)-l-({ 3-cthyl- 4-oxo-3H,4H-benzo[g]quinazolin-2-yl}sulfanyl)-2-[(3S)-3-hydroxypyrrolidin-l-yl]-2- oxoethyl]phenyl }prop-2-yn- 1 -yl)oxy]butyl } piperazin- 1 -yl)butoxy]prop- 1 -yn- 1 -yl }phenyl)-2- [(3S)-3-hydroxypyrrolidin-l-yl]-2-oxoethyl]sulfanyl}-3H,4H-benzo[g]quinazolin-4-one, 1-36, Yield: 20.0 mg, 8.60%; Appearance: White solid; ¹H NMR (400 MHz, CDCl₃) δ 8.81 - 8.74 (m, 2H), 8.15 (d, J = 9.2 Hz, 1H), 8.04 - 7.84 (m, 6H), 7.75 (s, 2H), 7.71 (d, 7 - 7.6 Hz, 1H), 7.60 - 7.53 (m, 2H), 7.51 - 7.47 (m, 2H), 7.44 - 7.41 (m, 2H), 7.38 - 7.33 (m, 2H), 5.92 (dd, J 7= 50.8, 4.0 Hz, 2H), 4.63 (d, J = 61.2 Hz, 2H), 4.36 - 4.12 (m, 11H), 3.78 - 3.72 (m, 2H), 3.65 - 3.52 (m, 9H), 3.41 - 2.79 (m, 12H), 2.46 - 2.01 (m, 6H), 1.86 (s, 4H), 1.40 - 1.36 (m, 6H), 1.33 - 1.25 (m, 2H); HPLC purity: 89.9%; LCMS Calculated for C₇₀H₇₆N₈O₈S₂: 1221.55; Observed: 1221.5 [M+H]⁺.

Example 17. Synthesis of 3-ethyl-2-{[(lS)-l-[3-(16-{3-[(lS)-l-({3-ethyl-4-oxo-3H,4H- benzo[g]quinazolin-2-yl}sulfanyl)-2-[(3S)-3-hydroxypyrrolidin-l-yl]-2-oxoethyl]phenyl}- 4,7,10,13-tetraoxahexadeca-l,15-diyn-l-yl)phenyl]-2-[(3S)-3-hydroxypyrrolidin-l-yl]-2- oxoethyl]sulfanyl}-3H,4H-benzo[g]quinazolin-4-one, 1-37:

Step-1. Synthesis of 3-ethyl-2-{[(lS)-l-[3-(16-{3-[(lS)-l-({3-ethyl-4-oxo-3H,4H- benzo[g]quinazolin-2-yl}sulfanyl)-2-[(3S)-3-hydroxypyrrolidin-l-yl]-2-oxoethyl] phenyl}- 4,7 , 10, 13-tetraoxahexadeca- 1,15 -diyn- 1 -yl)phenyl] -2- [(3S )-3-hydroxypyrro lidin- 1 - y 1] - 2 - oxoethyl]sulfanyl]-3H,4H-benzo[g]quinazolin-4-one

[0341] To a solution of 3-ethyl-2-{ [(lS)-2-[(3S)-3-hydroxypyrrolidin-l-yl]-l-(3-iodophenyl)-2- oxoethyl]sulfanyl}-3H,4H-benzo[g]quinazolin-4-one (150 mg, 0.26 mmol), 4,7,10,13- tetraoxahexadeca-l,15-diyne (28.9 mg, 0.13 mmol), Cui (4.86 mg, 0.03 mmol) and Pd(pph3)₂C12 (17.9 mg, 0.03 mmol) in DMF (2 mL) was added TEA (129 mg, 1.28 mmol). The mixture was stirred at 60 °C for 4 h. The reaction mixture was concentrated and purified by reverse combi flash (Biotage, 25 g Agela C18, MeCN/H2O(0.5% NH4HCO3) to give 3-ethyl-2-{[(lS)-l-[3-(16-{3- [(lS)-l-({3-ethyl-4-oxo-3H,4H-benzo[g]quinazolin-2-yl}sulfanyl)-2-[(3S)-3-hydroxypyrrolidin- 1 -yl] -2-oxoethyl]phenyl } -4,7,10, 13-tetraoxahexadeca- 1 , 15-diyn- 1 -yl)phenyl] -2- [(3S)-3- hydroxypyrrolidin-l-yl]-2-oxoethyl] sulfanyl] -3H,4H-benzo[g]quinazolin-4-one, 1-37, Yield: 35.0 mg, 23.0%; Appearance: Yellow solid; ‘H NMR (400 MHz, CDCh) 8 8.76 (dd, J= 20.4, 4.0 Hz, 2H), 8.25 (d, J= 16.0 Hz, 1H), 8.03 - 7.85 (m, 5H), 7.79 - 7.72 (m, 2H), 7.67 - 7.41 (m, 8H), 7.37 - 7.29 (m, 2H), 6.16 (dd, J= 91.2, 12.7 Hz, 2H), 4.70 - 4.47 (m, 2H), 4.42 - 4.16 (m, 10H), 3.77 - 3.43 (m, 20H), 2.23 - 2.02 (m, 4H), 1.40 - 1.35 (m, 6H); HPLC purity: 96.2%; LCMS Calculated for C₆₄H₆₄N₆O₁₀S₂: 1141.37; Observed: 1141.4 [M+H]⁺.

[0342] The following examples were prepared using standard chemical manipulations and procedures similar to those used for the preparation of the previous examples. Analytical data is given in the table below.

Example 18. Synthesis of (2R,2'R)-N,N'-(3, 6,9, 12-tetraoxatetradecane-l,14-diyl)bis(l-((R)-2- ((6, 7-dichloro-3-ethyl-4-oxo-3,4-dihydroquinazolin-2-yl)thio )-2-phenylacetyl)pyrrolidine-2- carboxamide), 1-42:

Step-1. Synthesis of ( 2R,2 'R )-N,N'-( 3, 6,9, 12-tetraoxatetradecane-l, 14-diyl)bis( 1 -( (R)-2-( ( 6, 7- dichloro-3-ethyl-4-oxo-3,4-dihydroquinazolin-2-yl)thio)-2-phenylacetyl)pyrro lidine-2- carboxamide), 1-42

[0343] To a 8 mL vial were added ((R)-2-((6,7-dichloro-3-ethyl-4-oxo-3,4-dihydroquinazolin-2- yl)thio)-2-phenylacetyl)-D-proline (150 mg, 296 μmol), 3,6,9,12-tetraoxatetradecane-l,14- diamine (34.9 mg, 148 μmol), DIEA (114 mg, 887 μmol) and HATU (112 mg, 296 μmol) in DMF (3 mL) at room temperature. The mixture was stirred for 1 hour and then purified by prep-HPLC (NH₃ H₂O buffer) to afford (2R,2'R)-N,N'-(3,6,9,12-tetraoxatetradecane-l,14-diyl)bis(l-((R)-2- ((6,7-dichloro-3-ethyl-4-oxo-3,4-dihydroquinazolin-2-yl)thio)-2-phenylacetyl)pyrrolidine-2- carboxamide), 1-42. Yield: 50.0 mg, 27%; Appearance: White solid; ¹H NMR (400 MHz, DMSO-d₆) δ 8.18 - 8.09 (m, 4H), 7.84 (t, J = 5.7 Hz, 2H), 7.68 - 7.61 (m, 3H), 7.52 - 7.45 (m, 1H), 7.43 - 7.37 (m, 3H), 7.37 - 7.28 (m, 3H), 5.95 - 5.91 (m, 2H), 4.30 (dd, J= 8.3, 2.6 Hz, 2H), 4.11 - 3.96 (m, 6H), 3.54 - 3.37 (m, 14H), 3.31 - 3.23 (m, 6H), 3.01 - 2.95 (m, 3H), 2.03 - 1.97 (m, 2H), 1.95 - 7.77 (m, 5H), 1.22 (t, J = 7.0 Hz, 6H). HPLC purity: 98%; LCMS Calculated for C₅₆H₆₂C₁₄N₈O₁₀S₂: 1210.28; Observed: 1211.3 [M+H]⁺.

Example 19. Synthesis of (2R)-N-(14-{[(2R)-l-[(2R)-2-[(6,7-dichloro-3-ethyl-4-oxo-3,4- dihydroquinazolin-2-yl )sulfanyl ]-2-phenylacetyl ]pyrrolidin-2-yl ]formamido}-3, 6,9,12- tetraoxatetradecan- 1 -yl)- 1 -[(2R)-2-( {3-ethyl-4-oxo-3H,4H-benzo[g]quinazolin-2-yl}sulfanyl)- 2-phenylacetyl]pyrrolidine-2-carboxamide, 1-43:

Step-1. Synthesis of tert-butyl N-(14-{[(2R)-1 -[(2R)-2-({3-ethyl-4-oxo-3H,4H- benzo[g]quinazolin-2-yl}sulfanyl)-2-phenylacetyl]pyrrolidin-2-yl]formamido}-3,6,9,12- tetraoxatetradecan- 1 -yl)carbamate

[0344] A mixture of (2R)-l-[(2R)-2-({3-ethyl-4-oxo-3H,4H-benzo[g]quinazolin-2-yl}sulfanyl)- 2-phenylacetyl]pyrrolidine-2-carboxylic acid (200 mg, 410 μmol), rt-butyl N-(14-amino- 3,6,9,12-tetraoxatetradecan-l-yl)carbamate (137 mg, 410 μmol) and DIEA (159 mg, 1.23 mmol) in DMF (5 mL) was stirred at0 °C for 10 min. HATU (186 mg, 491 μmol) was then added, and the reaction mixture was stirred for 1.0 hour at 0 °C. The resulting mixture was concentrated in vacuo, the residue was purified by prep-HPLC (NH₃.H₂O buffer) to give tert-butyl N-(14- { [(2R)-l-[(2R)-2-({3-ethyl-4-oxo-3H,4H-benzo[g]quinazolin-2-yl}sulfanyl)-2-phenylacetyl] pyrrolidin-2-yl]formamido}-3,6,9,12-tetraoxatetradecan-l-yl)carbamate (256 mg, 318 μmol, 98.6% purity, 77.1% yield) as a red solid.

Step-2. Synthesis of ( 2R)-N-( 14-amino-3, 6,9, 12 -tetraoxatetradecan- 1 -yl)-l-[( 2R)-2-( {3-ethyl-4- oxo-3H,4H-benzo[g]quinazolin-2-yl}sulfanyl)-2-phenylacetyl]pyrrolidine-2-carboxamide

[0345] A solution of tert-butyl N-(14-{ [(2R)-l-[(2R)-2-({3-ethyl-4-oxo-3H,4H- benzo[g]quinazolin-2-yl}sulfanyl)-2-phenylacetyl]pyrrolidin-2-yl]formamido}-3,6,9,12- tetraoxatetradecan-l-yl)carbamate (100 mg, 124 μmol) in TFA/DCM (1:4, 5 mL) was stirred for 1 hour at room temperature. The resulting mixture was concentrated in vacuo to afford (2R)-N- (14-amino-3, 6,9, 12-tetraoxatetradecan- 1-yl)- l-[(2R)-2-({ 3-ethyl-4-oxo-3H,4H- benzo[g]quinazolin-2-yl}sulfanyl)-2-phenylacetyl]pyrrolidine-2-carboxamide as TFA salt (68.4 mg, 97.0 μmol, 85.6% purity, 78.1% yield) as a brown oil, which was used for next step directly. Step-3. Synthesis of (2R)-N-(14-{[(2R)-l-[(2R)-2-[(6,7-dichloro-3-ethyl-4-oxo-3,4- dihydroquinazolin-2-yl)sulfanyl ] -2 -phenylacetyl ]pyrrolidin-2-yl Jformamido }-3, 6,9,12- tetraoxatetradecan-l-yl)-l-[(2R)-2-({3-ethyl-4-oxo-3H,4H-benzo[g]quinazolin-2-yl}sulfanyl)-2- phenylace tyl]pyrrolidine-2-carboxamide, 1-43

[0346] A mixture of (2R)-N-(14-amino-3,6,9,12-tetraoxatetradecan-l-yl)-l-[(2R)-2-({3-ethyl-4- oxo-3H,4H-benzo[g]quinazolin-2-yl}sulfanyl)-2-phenylacetyl]pyrrolidine-2-carboxamide (80.0 mg, 113 μmol), (2R)-l-[(2R)-2-[(6,7-dichloro-3-ethyl-4-oxo-3,4-dihydroquinazolin-2- yl)sulfanyl]-2-phenylacetyl]pyrrolidine-2-carboxylic acid (57.2 mg, 113 μmol) and DIEA (43.7 mg, 339 μmol) in DMF (5 mL) was stirred at 0 °C for 10 min. HATU (51 .3 mg, 135 μmol) was then added, and the reaction mixture was stirred for 1.0 hour at 0 °C. The resulting mixture was concentrated in vacuo, the residue was purified by prep-HPLC (NH₃.H₂O buffer) to give (2R)-N- (14-{ [(2R)-l-[(2R)-2-[(6,7-dichloro-3-ethyl-4-oxo-3,4-dihydroquinazolin-2-yl)sulfanyl]-2- phenylacetyl] pyrroli din-2-yl]formamido}-3,6,9,12-tetraoxatetradecan-l-yl)-l-[(2R)-2-({3-ethyl- 4-oxo-3H,4H-benzo[g]quinazolin-2-yl}sulfanyl)-2-phenylacetyl] pyrrolidine-2-carboxamide, I- 43: 55 mg, 44%; Appearance: White solid; ¹H NMR (300 MHz, DMSO-d₆) 8 8.80 - 8.78 (m, 1H), 8.24 - 8.05 (m, 4H), 7.97 (s, 1H), 7.81 - 7.60 (m, 6H), 7.56 (t, J = 7.3 Hz, 1H), 7.46 - 7.27 (m, 6H), 6.11 - 6.06 (m, 2H), 4.49 - 4.20 (m, 2H), 4.08 - 4.01 (m, 5H), 3.60 - 3.43 (m, 6H), 3.43 - 3.34 (m, 6H), 3.26 - 3.19 (m, 5H), 3.17 - 2.94 (m, 7H), 2.22 - 1.70 (m, 7H), 1.18 (t, J= 4.3 Hz, 6H). HPLC purity: 99.7%; LCMS Calculated for: C₆₀H₆₆Cl₂N₈O₁₀S₂ : 1192.37; Observed: 1193.5 [M+H]⁺.

Example 20. Synthesis of (2S,3aS,7aS)-l-(2-{4-[4-(4-{4-[(2S,3aS,7aS)-2-(methylcarbamoyl)- octahydro-lH-indol-l-yl]-6-[(lS)-l-{[4-(morpholin-4-yl)phenyl]formamido}ethyl]pyrimidin-2- yl}butyl)piperazin-l-yl]butyl}-6-[(lS)-l-{[4-(morpholin-4- yl)phenyl]formamido}ethyl]pyrimidin-4-yl)-N-methyl-octahydro-lH-indole-2-carboxamide, I- 44:

Step-1. Synthesis of 1 ,4-bis(but-3-yn-l -yl)piperazine

[0347] A mixture of 4-bromobut-l-yne (1.0 g, 7.5 mmol), piperazine (538.0 mg, 6.3 mmol) and K2CO3 (2.6 g, 18.7 mmol) in MeCN (10 mL) was stirred for overnight at 80 °Cand then cooled to room temperature and filtered. The filter cake was washed with MeCN (3x 30 mL) and the filtrate was concentrated under reduced pressure. The residue was applied onto a silica gel column and eluted with ethyl acetate/petroleum ethei-l:l to give l,4-bis(but-3-yn-l-yl)piperazine (400.0 mg, 2.1 mmol, 90.0% purity, 33.8% yield) as a white solid.

Step-2. Synthesis of (2S,3aS,7aS)-l-(2-{4-[4-(4-{4-[(2S,3aS,7aS)-2-(methylcarbamoyl)- octahydro-lH-indol-1 -yl ]-6-[ 4-( morpholin-4-yl )phenyl ]formamido } ethyl ]pyrimidin-2- yl but-3-yn-l -yl)piperazin-l -yl]but-l -yn-1 -yl}-6-[( lS)-l-{[4-(morpholin-4- yl)phenyl]formamido}ethyl]pyrimidin-4-yl)-N-methyl-octahydro-lH-indole-2-carboxamide

[0348] To a solution of l,4-bis(but-3-yn-l-yl)piperazine (40.0 mg, 210.0 μmol), (2S,3aS,7aS)-l- {2-bromo-6-[(lS)-l-{ [4-(morpholin-4-yl)phenyl]formamido}ethyl]pyrimidin-4-yl}-N-methyl- octahydro-lH-indole-2-carboxamide (240.0 mg, 420.0 μmol) and TEA (63.6 mg, 630.0 μmol) in MeCN (2.4 mL) was added Cui (2.0 mg, 10.5 μmol) and Pd(PPh₃)₂Ch (1.5 mg, 2.1 μmol) at room temperature. The resulting mixture was stirred for 6 hours at 80°C under nitrogen atmosphere. The reaction mixture was cooled to room temperature and quenched by the addition of water (30.0 mL) at room temperature. The resulting mixture was extracted with ethyl acetate (3x50 mL). The combined organic layers were washed with saturated brine (100 mL)and dried over anhydrous Na₂SO₄ . After filtration, the filtrate was concentrated under vacuum to afford the crude product (2S,3aS,7aS)-l-(2-{4-[4-(4-{4-[(2S,3aS,7aS)-2-(methylcai’bamoyl)-octahydro-lH-indol-l-yl]-6- [( 1 S)- 1 - { [4-(morpholin-4-yl)phenyl]formamido }ethyl]pyrimidin-2-yl }but-3-yn- 1 -yl)piperazin- 1 - yl]but- 1 -yn- 1 -y 1 } - 6- [( 1 S ) - 1 - { [4-(morpholin-4-yl)phenyl]formamido }ethyl]pyrimidin-4-yl)-N- methyl-octahydro-lH-indole-2-carboxamide (110.0 mg, 93.8 μmol, 60.0% purity, 40.0% yield) as a yellow solid, which was used to next step without under purification.

Step-3. Synthesis of (2S,3aS,7aS)-l-(2-{4-[4-(4-{4-[(2S,3aS,7aS)-2-(methylcarbamoyl)~ octahydro- IH-indol- 1 -yl]-6-[( IS )-l-{[4-( morpholin-4-yl)phenyl]formamido jethyl ]pyrimidin-2- yl}butyl)piperazin- 1 -yl]butyl]-6-[(lS)-l -{ [4-(morpholin-4- yl)phenyl]formamido}ethyl]pyrimidin-4-yl)-N-methyl-octahydro-lH-indole-2-carboxamide, 1-44.

[0349] To a 50 mL round-bottom flask, was added (2S,3aS,7aS)-l-(2-{4-[4-(4-{4-[(2S,3aS,7aS)- 2-(methy lcarbamoyl)-octahydro- IH-indol- l-yl]-6-[(lS)- l-{ [4-(morpholin-4- yl)phenyl] formamido } ethyl]pyrimidin-2-y 1 }but-3-yn- 1 -y l)piperazin- 1 -yl] but- 1 -yn- 1 -y 1 } -6- [( 1 S )- 1 - { [4-(morpholin-4-yl)phenyl]formamido }ethyl]pyrimidin-4-yl)-N-methyl-octahydro- 1 H- indole-2-carboxamide (90.0 mg, 76.8 μmol) in MeOH ; THF (4:1, 10 mL) was added Raney-Ni (39.4 mg, 460.0 μmol) at room temperature under nitrogen atmosphere and then the reaction mixture was degassed and purged with hydrogen 3x. The resulting reaction mixture was stirred for 24.0 hours at room temperature under hydrogen atmosphere (1 atm) and filtered. The filtrate was concentrated in vacuo and then the residue was purified by C18 column (NH₃- H₂O buffer) to afford (2S,3aS,7aS)-l-(2-{4-[4-(4-{4-[(2S,3aS,7aS)-2-(methylcarbamoyl)-octahydro-lH-indol-l-yl]-6- [(1 S)- 1 -{ [4-(morpholin-4-yl)phenyl]formamido}ethyl]pyrimidin-2-yl }butyl)piperazin- 1 - yl]butyl }-6-[(lS)-l-{ [4-(morpholin-4-yl)phcnyl]fonnamido }cthyl]pyrimidin-4-yl)-N-mcthyl- octahydro-lH-indole-2-carboxamide (10.0 mg, 8.5 μmol, 89.4% purity, 11% yield) as a white solid, 1-44. Yield: 10.0 mg, 11.0%; Appearance: White solid; ¹H NMR (400 MHz, DMSO-d₆) 8 8.37 (d, J= 7.9 Hz, 2H), 7.79 - 7.77 (m, 6H), 6.97 (d, J = 8.7 Hz, 4H), 6.44 - 6.26 (m, 2H), 4.90

- 4.82 (m, 2H), 4.37 - 4.18 (m, 2H), 3.76 - 3.74 (m, 8H), 3.23 - 3.21 (m, 8H), 2.61 - 2.55 (m, 6H), 2.43 - 2.16 (m, 14H), 2.12 - 1.80 (m, 8H), 1.77 - 1.52 (m, 10H), 1.46 - 1.33 (m, 12H), 1.31

- 1.21 (m, 12H). HPLC purity: 89.4%; LCMS Calculated for C₆₆H₉4N₁₄O₆: 1178.75; Observed: 1179.8 [M+H]⁺.

Example 21. Synthesis of (2S,3aS,7aS)-l-(2-{3-[4-(3-{4-[(2S,3aS,7aS)-2-(methylcarbamoyl)- octahydro-lH-indol-l-yl]-6-[(lS)-l-{[4-(morpholin-4-yl)phenyl]formamido}ethyl]pyrimidin -2- yl}prop-2-yn-l-yl)piperazin-l-yl]prop-l-yn-l-yl}-6-[(lS)-l-{[4-(morpholin-4-yl)phenyl] formamido}ethyl]pyrimidin-4-yl)-N-methyl-octahydro-lH-indole-2-carboxamide, 1-45:

Step-1. Synthesis of l,4-bis(prop-2-yn-l-yl)piperazine

[0350] To a solution of piperazine (500 mg, 5.80 mmol) and 3-bromoprop-l-yne (1.71 g, 14.4 mmol) in DMF (20 mL) was added K₂CO₃ (2.40 g, 17.4 mmol). The mixture was stirred at 80 °C for 2 h, concentrated under reduced pressure to give the crude residue, and purified by column chromatography on silica gel using 5% MeOH in DCM to give the product l,4-bis(prop-2-yn-l- yl)piperazine (225 mg, 1.38 mmol, 98% purity, 24% yield) as a yellow solid. Step-2. Synthesis of (2S,3aS,7aS)-1 -(2-{3-[4-(3-{4-[(2S,3aS,7aS)-2-(methylcarbamoyl)-octahyd ro- IH-indol- 1 -yl] -6-[( 1 S)- 1- { [4-(morpholin-4-yl)phcnyl]formamido } cthyl]pyrimidin-2-yl }prop- 2-yn- 1 -yl)piperazin- 1 -yl]prop- 1 -yn- 1 -yl } - 6- [( 1 S)- 1- { [4-(morpholin-4-yl)phenyl]formamido } ethyl]pyrimidin-4-yl)-N-methyl-octahydro-lH-indole-2-carboxamide

[0351] To a solution of l,4-bis(prop-2-yn-l-yl)piperazine (60 mg, 369 μmol), (2S,3aS,7aS)-l-{2- bromo-6-[( 1 S)- 1 - { [4-(morpholin-4-yl)phenyl]formamido } ethyl]pyrimidin-4-yl } -N-methyl-octa hydro- lH-indole-2-carboxamide (210 mg, 369 μmol), triethylamine (372 mg, 3.68 mmol) and Bis(triphenylphoshine)palladium(II)chloride (77.2 mg, 110 μmol) in DMF (5 mL) was added Cui (20.9 mg, 110 μmol). The reaction mixture was stirred at 80 °C for 4 h and concentrated under reduced pressure to give the crude residue, which was purified by Prcp-HPLC(ACN/H₂O, with 0.1%NH₄HCO₃) to give the product (2S,3aS,7aS)-l-(2-{3-[4-(3-{4-[(2S,3aS,7aS)-2- (methylcarbamoyl)-octahydro- IH-indol- 1 -yl] -6-[( 1 S)- 1 - { [4-(morpholin-4-yl) phenyl]formamido }ethyl]pyrimidin-2-yl }prop-2-yn- 1 -yl)piperazin- 1 -yl]prop- 1 -yn- 1 -yl } -6- [( 1 S )- 1 - { [4-(morpholin-4-yl)phenyl]formamido }ethyl]pyrimidin-4-yl)-N-methyl-octahydro- 1 H-indole -2-carboxamide, 1-45. Yield: 66 mg, 15%; Appearance: White solid; ¹H NMR (400 MHz, DMSO- d₆) δ 8.44 (d, J = 7.7 Hz, 2H), 8.09 - 7.85 (m, 2H), 7.80 (d, J = 8.9 Hz, 4H), 6.97 (d, J = 9.0 Hz, 4H), 6.64 - 5.67 (m, 2H), 4.95 - 4.78 (m, 2H), 4.41 - 4.15 (m, 2H), 3.83 - 3.64 (m, 8H), 3.47 (s, 4H), 3.25 - 3.16 (m, 8H), 2.62 - 2.53 (m, 16H), 2.13 - 1.77 (m, 7H), 1.75 - 1.53 (m, 7H), 1.47 - 1.36 (m, 9H), 1.32 - 1.02 (m, 5H); HPLC purity: 93.96%; LCMS Calculated for C₆₄H₈₂N₁₄O₆: 1142.65; Observed: 1143.7 [M+H]⁺.

Example 22. Synthesis of (2S,3aS,7aS)-l-(2-{3-[4-(3-{4-[(2S,3aS,7aS)-2-(methylcarbamoyl)- octahydro-lH-indol-l-yl]-6-[(lS)-l-{[4-(morpholin-4-yl)phenyl]formamido}ethyl]pyrimidin-2- yl}propyl)piperazin-l-yl]propyl}-6-[(lS)-l-{[4-(morpholin-4-yl)phenyl]formamido}ethyl] pyrimidin-4-yl)-N-methyl-octahydro-lH-indole-2-carboxamide, 1-46:

Step-1. Synthesis of (2S,3aS,7aS)-l-(2-{3-[4-(3-{4-[(2S,3aS,7aS)-2-(methylcarbamoyl)- octahydro- 1 H-indol- 1 -yl] -6- [( 1 S )- 1 - { [4-(morpholin-4-yl)phenyl] formamido } ethyl]pyrimidin-2- yl }pro pyl)piperazin- 1 -yl]propyl } -6- [( 1 S)- 1 - { [4-(morpholin-4- yl)phenyl]formamido}ethyl]pyrimidin-4-yl)-N-methyl-octahydro-lH-indole-2-carboxamide

[0352] To a solution of (2S,3aS,7aS)-l-(2-{3-[4-(3-{4-[(2S,3aS,7aS)-2-(methylcarbamoyl)- octahydro-lH-indol-1 -yl]-6-[(l S)-l-{ [4-(morpholin-4-yl)phenyl]formamido}ethyl]pyrimidin-2- yl }prop-2-yn- 1 -yl)piperazin- 1 -yl]prop- 1 -yn- 1 -yl } -6- [( 1 S)- 1 - { [4-(morpholin-4- yl)phenyl] formamido] ethyl] pyrimidin-4-yl)-N-methyl-octahydro-lH-indole-2-carboxamide (20 mg, 17.4 μmol) in MeOH (3 mL) was added Pd/C (21.8 mg, 8.70 μmol). The reaction mixture was stirred at RT for 1 h under a H₂ atmosphere, filtered and concentrated under reduced pressure to give the crude residue which was purified by Prcp-HPLC(ACN/H₂O/0.1%NH₄HCO₃) to give the product (2S,3aS,7aS)-l-(2-{3-[4-(3-{4-[(2S,3aS,7aS)-2-(methylcarbamoyl)-octahydro-lH-indol- l-yl]-6-[(lS)-l-{ [4-(morpholin-4-y l)phenyl] formamido } ethyl]pyrimidin-2-yl }propy l)piperazin-

1 -yl]pro pyl }-6-[(lS)-l-{ [4-(morpholin-4-yl)phenyl]formamido }ethyl]pyrimidin-4-yl)-N- methyl-octahy dro-lH-indole-2-carboxamide, 1-46. Yield: 5.5 mg, 27%; Appearance: White solid; ¹H NMR (400 MHz, DMSO-J₆) 5 8.37 (d, J = 7.7 Hz, 2H), 7.98 - 7.71 (m, 6H), 6.97 (d, 7 = 9.0 Hz, 4H), 6.59 - 5.73 (m, 2H), 4.86 (t, J= 7.4 Hz, 2H), 4.60 - 4.07 (m, 2H), 3.83 - 3.63 (m, 8H), 3.25 - 3.11 (m, 8H), 2.57 (d, 7 = 4.6 Hz, 8H), 2.39 - 2.19 (m, 12H), 2.12 - 1.56 (m, 18H), 1.50 - 1.37 (m, 11H), 1.31 - 0.81 (m, 7H); HPLC purity: 98.78%; LCMS Calculated for C₆₄H₉₀N₁₄O₆: 1150.72; Observed: 1151.7 [M+H]⁺.

Example 23. Synthesis of (2S,3aS,7aS)-l-[2-(3-{2-[(3-{4-[(2S,3aS,7aS)-2-(methylcarbamoyl)- octahydro-lH-indol-l-yl]-6-[(lS)-l-{[4-(morpholin-4-yl)phenyl]formamido}ethyl]pyrimidin-2- yl}prop-2-yn-l-yl)oxy]ethoxy}prop-l-yn-l-yl)-6-[(lS)-l-{[4-(morpholin-4-yl)phenyl] formamido}ethyl]pyrimidin-4-yl]-N-methyl-octahydro-lH-indole-2-carboxamide, 1-47:

Step-1. Synthesis of (2S,3aS,7aS)-l-[2-(3-{2-[(3-{4-[(2S,3aS,7aS)-2-(methylcarbamoyl)- octahydro- 1 H-indol- 1 -y 1] -6- [( 1 S)- 1 - { [4-(morpholin-4-yl)pheny 1] formamido } ethyl] pyrimidin-2yl }prop-2-yn- 1 -yl)oxy]ethoxy }prop- 1 -yn- 1 -yl )-6- [( 1 S)-l -{ [4-(morpholin-4- yl)phcnyl]formamido]cthyl]pyrimidin-4-yl]-N-mcthyl-octahydro-lH-indolc-2-carboxamidc

[0353] To a solution of (2S,3aS,7aS)-l-{2-bromo-6-[(lS)-l-{ [4-(morpholin-4- yl)phenyl] formamido] ethyl]pyrimidin-4-yl}-N-methyl-octahydro-lH-indole-2-carboxamide (100 mg, 174 μmol), 3-[2-(prop-2-yn-l-yloxy)ethoxy]prop-l-yne (12.0 mg, 87.0 μmol), Bis(triphenylphoshine) palladium(II)chloride (12.2 mg, 17.4 μmol) and Cui (3.30 mg, 17.4 μmol) in DMF (3 mL) was added ethylbis(propan-2-yl)amine (112 mg, 870 μmol).The mixture was stirred at 80 °C under a N₂ atmosphere for 16 h. The mixture was concentrated under reduced pressure to give the crude residue, which was purified by Prep-HPLC (ACN/H₂O, with 0.1%NH₄HCO₃) to give the product (2S,3aS,7aS)-l-[2-(3-{2-[(3-{4-[(2S,3aS,7aS)-2- (methylcarbamoyl)-octahydro- IHindol- 1 -yl] - 6- [( 1 S)- 1 - { [4-(morpholin-4-yl)phenyl]formamido } ethyl]pyrimidin-2-yl }prop-2-yn- 1 -yl)oxy]ethoxy }prop- 1 -yn- 1 -y 1 ) -6- [( 1 S)- 1 - { [4-(morpholin-4- yl)phcnyl]formamido}cthyl]pyrimidin-4-yl]-N-mcthyl-octahydro-lH-indolc-2-carboxamidc, I- 47. Yield: 14 mg, 14%; Appearance: White solid; ¹H NMR (400 MHz, DMSO-d₆) δ 8.45 (d, J = 7.8 Hz, 2H), 8.09 - 7.84 (m, 2H), 7.80 (d, J = 8.9 Hz, 4H), 6.97 (d, 7= 9.0 Hz, 4H), 6.26 (m, 2H), 4.98 - 4.80 (m, 2H), 4.39 (s, 4H), 4.34 - 4.00 (m, 2H), 3.79 - 3.69 (m, 8H), 3.66 (s, 4H), 3.25 - 3.15 (m, 8H), 2.57 (d, J = 3.6 Hz, 8H), 2.15 - 1.81 (m, 7H), 1.76 - 1.52 (m, 6H), 1.51 - 1.34 (m, 10H), 1.32 - 1.01 (m, 5H); HPLC purity: 96.23%; LCMS Calculated for C₆₂H₇₈N₁₂O₈: 1118.61; Observed: 1119.6 [M+H]⁺.

[0354] The following examples were prepared using standard chemical manipulations and procedures similar to those used for the preparation of the previous examples. Analytical data is given in the table below.

Example 24. Synthesis of (2S,3aS,7aS)-N-(17-{[(2S,3aS,7aS)-l-{6-[(lS)-l-({4'-amino-[l,l'- biphenyl]-4-yl}formamido)ethyl]-2-methylpyrimidin-4-yl}- octahydro-lH-indol-2- yl]formamido}-3, 6,9, 12,15-pentaoxaheptadecan-l-yl)-l-{6-[(lS)-l -( {4'-amino-[l, 1 '-biphenyl]- 4-yl}formamido)ethyl]-2- methylpyrimidin-4-yl}-octahydro-lH-indole-2-carboxamide, 1-54:

Step-1. Synthesis of tert-butyl N-(4'-{[(lS)-l-{6-[(2S,3aS,7aS)-2-[(17-{[(2S,3aS,7aS)-l-{6-[(lS)- l-[(4'-{[( tertbutoxy)carbonyl ]amino}-[ 1, 1 '-biphenyl ]-4-yl )formamido ] ethyl] -2-methylpyrimidin- 4-yl}-octahydro-lH-indol-2-yl]formamido}-3,6,9,12,l 5- pentaoxaheptadecan- 1 -yl)carbamoyl] -o ctahydro- IH-indol- 1 -yl ]-2-methylpyrimidin-4-yl}eth.yl ]carbamoyl]-[l,l '-biphenyl ]-4-yl)carbama te

[0355] A mixture of 3,6,9,12,15-pentaoxaheptadecane-l,17-diamine (40 mg, 142 μmol), (2S ,3aS ,7aS)-l-{6-[(lS)-l - [(4'- { [(tertbutoxy )carbonyl] amino } - [ 1 , 1 '-biphenyl] -4- yl)formamido]ethyl]-2-methylpyrimidin-4-yl}-octahydro-1H-indole-2-carboxylic acid (170 mg, 284 μmol) and K₂CO₃ (58.6 mg, 425 μ mol) in DMF (4 mL) was stirred at room temperature for 30 mins. HATU (80.5 mg, 212 μmol) was then added, and the reaction mixture was stirred for 1.0 hour at room temperature. The resulting mixture was concentrated in vacuo and thenthe residue was purified by prep-HPLC (NH₃.H₂O buffer) to give tert-butyl N-(4'-{[(lS)-l-{6-[(2S,3aS,7aS)- 2-[(17-{ [(2S,3aS,7aS)-l-{ 6-[( 1 S)- 1-[(4'-{ [(tertbutoxy)carbonyl]amino}-[l,l'-biphenyl]-4- yl)formamido]ethyl]-2-methylpyrimidin-4-yl]-octahydro-lH-indol-2-yl]formamido}-

3.6.9.12.15-pentaoxaheptadecan-l-yl)carbamoyl]-octahydro-lH-indol-l-yl]-2-methylpyrimidin- 4-yl}ethyl]carbamoyl}-[l,T-biphenyl]-4-yl)carbamate (70.0 mg, 48.4 μmol, 90% purity, 34% yield) as a yellow solid.

Step-2. Synthesis of (2S,3aS,7aS)-N-( 17-{[(2S,3aS, 7aS)-l -{6- ](1S)-1-( {4'-amino-[ 1,1 ’-biphenyl] -

4-yl }formamido )ethyl ]-2 -methylpyrimidin-4-yl } - octahydro- lH-indol-2 -yl ]formamido}-3, 6, 9,12,1

5 -pentaoxaheptade can- 1 -yl)-l-{6-[(lS)-l-({4 ’-amino-] 1,1 '-biphenyl ]-4-yl jformamido )ethyl ]-2- m ethylpyrimidm-4-yl}-octahydro-lH-indole-2-carboxamide

[0356] A stirred solution of tert-butyl N-(4'-{ [(lS)-l-{6-[(2S,3aS,7aS)-2-[(17-{[(2S,3aS,7aS)-l- {6-[(lS)-l-[(4'-{ [(tert-butoxy )carbonyl] amino } - [ 1 , 1 '-biphenyl] -4-yl)formamido]ethyl] -2- methylpyrimidin-4-yl } -octahydro- 1 H-indol-2-yl]formamido } -3 ,6,9, 12, 15 -pentaoxaheptadecan- l-yl)carbamoyl]-octahydro-lH-indol-l-yl]-2-methylpyrimidin-4-yl}ethyl]carbamoyl}-[l,l'- biphenyl]-4-yl)carbamate (60 mg, 41 .5 μmol) in DCM (2 mL) and TFA (0.5 mL) was stirred for 1 hour at room temperature. The resulting mixture was concentrated under reduced pressure andthe residue was purified by reverse flash chromatography under the following conditions: column, C18 silica gel; mobile phase, MeCN in water, 10% to 80% gradient in 12min; detector, UV 220 nmto afford (2S,3aS,7aS)-N-(17-{ [(2S,3aS,7aS)-l-{6-[(lS)-l-({4’-amino-[1, 1'-biphenyl]-4- yl } formamido) ethyl] -2-methylpyrimidin-4-yl } -octahydro- 1 H-indol-2-y 1] formamido } -

3.6.9.12.15-pentaoxa heptadecan- 1-yl)- 1-{6-[(lS)-1-({4'-amino-[1, 1'-biphenyl]-4-yl}formamido) ethyl]-2-methylpyrimidin-4-yl}-octahydro-1H-indole-2-carboxamide, 1-54. Yield: 20.00 mg, 38.7%; Appearance: White solid; ¹H NMR (300 MHz, DMSO-d₆) δ 8.68 - 8.57 (m, 2H), 8.03 - 7.96 (m, 2H), 7.88 (d, J = 8.1 Hz, 4H), 7.64 (d, J = 8.1 Hz, 4H), 7.44 (d, J = 8.2 Hz, 4H), 6.66 (d, J= 8.2 Hz, 4H), 5.34 (s, 4H), 4.89 - 4.86 (m, 2H), 4.44 - 4.19 (m, 2H), 3.90 - 3.81 (m, 2H), 3.49 - 3.36 (m, 20H), 3.21 - 2.15 (m, 4H), 2.30 - 2.21 (m, 10H), 2.14 - 1.80 (m, 6H), 1.77 - 1.53 (m, 6H), 1.43 - 1.36 (m, 10H), 1.24 - 1.12 (m, 4H). HPLC purity: 95.6%; LCMS Calculated for: C70H90N12O9 : 1242.70; Observed: 1243.7 [M+H]⁺.

Example 25. Synthesis of (2S,3aS,7aS)-N-[2-(4-{4-[4-(2-{[(2S,3aS,7aS)-l-{6-[(lS)-l-({4'- amino-[l,l'-biphenyl]-4-yl}formamido)ethyl]-2-methylpyrimidin-4-yl}-octahydro-lH-indol-2- yl]formamido}ethoxy)butyl]piperazin-l-yl}butoxy)ethyl]-l-{6-[(lS)-l-({4'-amino-[l,l'- biphenyl]-4-yl}formamido)ethyl]-2-methyl pyrimidin-4-yl}-octahydro-lH-indole-2- carboxamide, 1-55:

Step-1. Synthesis of tert-butyl N-[2-(4-bromobutoxy)ethyl]carbamate [0357] A mixture of tert-butyl N-(2-hydroxyethyl)carbamate (2.0 g, 12.4 mmol), 1 ,4- dibromobutanc (4.0 g, 18.6 mmol), NaOH (992.0 mg, 24.8 mmol) and TBAB (4.0 g, 12.4 mmol) in THF (15 mL) was stirred for 2.0 hours at 80 °C. The mixture was cooled to room temperature, , quenched by the addition of H₂O (100 mL), and extracted with ethyl acetate (3x100 mL). The combined organic layers were washed with brine (1x100 mL)and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was applied onto a silica gel column and eluted with ethyl acetate/petroleum ether- 1 :2 to give tert-butyl N-[2-(4- bromobutoxy)ethyl]carbamate (1.4 g, 4.7 mmol, 90.0% purity, 38.0% yield) as yellow oil.

Step-2. Synthesis of tert-butyl N-[2-(4-{4-[4-(2-{ [(tertbutoxy )carbonyl ] amino [ethoxy [butyl ]piperazin-l -yl }butoxy[ethyl [carbamate

[0358] A mixture of tert-butyl N-[2-(4-bromobutoxy)ethyl]carbamate (400 mg, 1.35 mmol), piperazine (58.1 mg, 675.0 μmol) and K2CO3 (558.0 mg, 4.1 mmol) in MeCN (8.0 mL) was stirred for 3.0 hours at 80 °C. The mixture cooled to room temperature and purified by reverse flash chromatography under the following conditions: column, C18 silica gel; mobile phase, Aqueous phase of 0.05% ammonia in acetonitrile, 60% to 80% gradient in 20 min; detector, UV 254 nm. to afford tert-butyl N-[2-(4- { 4- [4-(2- { [(tert-butoxy)carbonyl]amino}ethoxy)butyl]piperazin- 1- yl}butoxy)ethyl]carbamate (340.0 mg, 657.0 μmol, 75% purity, 92.5% yield) as a light yellow oil. Step-3. Synthesis of 2-(4-{4-[4-(2-aminoethoxy[butyl[piperazin-l-yl[butoxy[ethan-l -amine hydrochloride

[0359] A solution of tert-butyl N-[2-(4-{4-[4-(2-{ [(tert-butoxy)carbonyl]amino} ethoxy)butyl]piperazin-l-yl}butoxy)ethyl]carbamate (100.0 mg, 193.0 μmol) in HCl/dioxane (4M, 2 mL) was stirred for 1 hour at room temperature. The resulting mixture was concentrated in vacuo to afford 2-(4-{4-[4-(2-aminoethoxy) butyl]piperazin-l-yl}butoxy)ethan-l -amine hydrochloride as HC1 salt (75.0 mg, 212.0 μmol, 70% purity, crude) as a white solid, which was used for next step directly.

Step-4. Synthesis of tert-butyl N-(4'-[[(lS[-l-{6-[(2S,3aS, 7aS)-2-[[2-(4-[4-[4-(2-[[(2S,3aS, 7aS[- 1 -{ 6-[ (1S)-l-[(4'-{[( tert-butoxy [carbonyl ]amino[-[l,l '-biphenyl [-4-yl [formamido [ethyl ]-2- methylpyrimidin-4-yl [ -octahydro- lH-indol-2-yl [formamido [ethoxy [butyl [piperazin- 1 - yl [butoxy[ethyl [carbamoyl} -octahydro- IH-indol- 1 -yl]-2-methylpyrimidin-4- yljethyl ]carbamoyl}-[ 1,1 '-biphenyl J-4-yl [carbamate [0360] A mixture of 2-(4-{4-[4-(2-aminoethoxy)butyl]piperazin-1 -yl]butoxy)ethan-l -amine hydrochloride (70.0 mg, 198.0 μmol), (2S,3aS,7aS)-l-{6-[(lS)-l-[(4'-{[(tert- butoxy )carbonyl] amino } - [ 1 , 1 '-biphenyl] -4-yl)formamido] ethyl] -2-methy lpyrimidin-4-y 1 } - octahydro- lH-indole-2-carboxylic acid (237.0 mg, 396.0 μmol) and DIEA (152.0 mg, 1.2 mmol) in DMF (2 mL) was stirred at room temperature for 30 min. HATU (75.2 mg, 198.0 μmol) was then added, and the reaction mixture was stirred for 2.0 hours at room temperature. The resulting mixture was concentrated in vacuo, and purified by reverse flash chromatography under the following conditions: column, C18 silica gel; mobile phase, Aqueous phase of 0.05% ammonia in acetonitrile, 50% to 60% gradient in 20 min; detector, UV 254 nm to afford tert-butyl N-(4'-{[(lS)- l-{6-[(2S,3aS,7aS)-2-{ [2-(4-{4-[4-(2-{ [(2S,3aS,7aS)-l-{6-[(lS)-l-[(4'-{ [(tertbutoxy )carbonyl] amino } - [ 1 , 1 '-biphenyl] -4-yl)formamido] ethyl] -2-methy lpyrimidin-4-y 1 } - octahydro- lH-indol-2-yl]formamido}ethoxy)butyl]piperazin-l-yl}butoxy)ethyl]carbamoyl}- octahydro- 1 H-indol- 1 -y 1] -2-methylpyrimidin-4-y 1 } ethyl] carbamoyl } -[ 1 , 1 '-biphenyl] -4- yl)carbamate (150.0 mg, 101.0 μmol, 90% purity, 70% yield) as a yellow solid.

Step-5. Synthesis of (2S,3aS, 7aS)-N-[2-(4-{4-[4-(2-{[(2S,3aS,7aS)-l-{6-[(lS)-l-({4'-amino-[1,1'- biphenyl ]-4-yl }formamido )ethyl ]-2-methylpyrimidin-4-yl}-octahydro-lH-indol-2- yl ]formamido } ethoxy )butyl ]piperaz.in-l -yl }butoxy)ethyl ]-l-{6-[(lS)-l-({4 '-amino- [ 1,1'- biphenyl]-4-yl}formamido)ethyl]-2-methylpyrimidin-4-yl}-octahydro-lH-indole-2-carboxamide, 1-55

[0361] A solution of tert-butyl N-(4'-{ [(lS)-l-{6-[(2S,3aS,7aS)-2-{[2-(4-{4-[4-(2- { [(2S,3aS,7aS)-l-{6-[(lS)-l-[(4'-{ [(tert-butoxy)carbonyl]amino}-[l,l'-biphenyl]-4- yl)formamido]ethyl]-2-methylpyrimidin-4-yl]-octahydro-lH-indol-2-yl]formamido} ethoxy )butyl] piperazin- 1 -yl}butoxy)ethyl]carbamoyl}-octahydro-lH-indol- l-yl]-2- methylpyrimidin-4-yl}ethyl]carbamoyl}-[l,l'-biphenyl]-4-yl)carbamate (135.0 mg, 91.2 μmol) in TFA/DCM (1:1, 1.4 mL) was stirred for 1 hour at room temperature. The resulting mixture was concentrated in vacuo and was purified by prep-HPLC (NH₃.H₂O buffer) to afford (2S,3aS,7aS)- N-[2-(4-{4-[4-(2-{[(2S,3aS,7aS)-l-{6-[(lS)-l-({4'-amino-[l,T-biphenyl]-4- yl } formamido)ethyl] -2-methy lpyrimidin-4-yl } -octahydro- 1 H-indoL2- yl]formamido}ethoxy)butyl]piperazin-l-yl}butoxy)ethyl]-l-{6-[(lS)-l-({4'-amino-[l,l'- biphenyl] -4-yl } formamido)ethyl] -2-methy lpyrimidin-4-y 1 } -octahydro- 1 H-indole-2-carboxamide, 1-55. Yield: 45.0 mg, 39.0%; Appearance: White solid; ¹H NMR (400 MHz, DMSO-d₆) δ 8.69 - 8.56 (m, 2H), 8.01 - 7.79 (m, 6H), 7.64 (dd, J = 8.5, 2.5 Hz, 4H), 7.48 - 7.41 (m, 4H), 6.69 - 6.62 (m, 4H), 5.34 (s, 4H), 4.92 - 4.83 (m, 2H), 4.40 - 4.28 (m, 2H), 3.41 - 3.32 (m, 4H), 3.25 - 2.94 (m, 4H), 2.40 - 2.14 (m, 20H), 2.10 - 1.81 (m, 5H), 1.75 - 1.55 (m, 7H), 1.52 - 1.36 (m, 18H), 1.38 - 1.22 (m, 8H), 1.20 - 0.81 (m, 2H); HPLC purity: 96.9%; LCMS Calculated for C₇₄H₉₈N₁₄O₆: 1278.78; Observed: 1279.6 [M+H]⁺.

Example 26. Synthesis of (2S,3aS/7aS)-N-(14-{[(2S,3aS,7aS)-1-{2-methyl-6-[(lS)-l-{[4- (morpholin-4-yl)phenyl]formamido}ethyl]pyrimidin-4-yl}-octahydro-lH-indol-2- yl]formamido}-3,6,9,12-tetraoxatetradecan-l-yl)-l-{2-methyl-6-[(lS)-l-{[4-(morpholin-4- yl)phenyl]formamido}ethyl]pyrimidin-4-yl}-octahydro-lH-indole-2-carboxamide, 1-56:

Step-1. Synthesis of ethyl 4-{[(benzyloxy)carbonyl]amino}-3-oxopentanoate

[0362] To a solution of 1 -ethyl 3-potassium propanedioate (2.53 g, 14.9 mmol) in THF (50 ml) was added CDI (2.41 g, 14.9 mmol) at room temperature. The reaction mixture was stirred at room temperature for 2 hours, then (2S ) -2- {[(benzyloxy)carbonyl] amino} propanoic acid (2.23 g, 9.98 mmol) and MgCl₂ (586 mg, 9.78 mmol) were added. The reaction mixture was stirred at room temperature for 48 hrs, and then concentrated, diluted with EtOAc (150 ml), washed with NaHCO₃ solution and brine, concentrated and purified by SGC (EA in PE = 0 - 20%) to afford ethyl 4- { [(bcnzyloxy)carbonyl] amino } -3-oxopcntanoatc (2.60 g, 8.86 mmol, 89.0% yield) as oil.

Step-2. Synthesis of benzyl N-[l-(6-hydroxy-2-methylpyrimidin-4-yl)ethyl]carbamate

[0363] Ethyl 4-{ [(benzyloxy)carbonyl]amino}-3-oxopentanoate (2.6 g, 8.86 mmol), ethanimidamide HC1 (837 mg, 8.86 mmol) andt-BuOK (1.47 g, 13.2 mmol) in EtOH (50 ml) were heated at 80 °C overnight. The reaction mixture was concentrated, diluted with water, and the pH adjusted to 4-5 with 2M HO aqueous solution. The resultant precipitate was filtered and washed with water, then dried to afford benzyl N-[l-(6-hydroxy-2-methylpyrimidin-4-yl)ethyl]carbamate (1.78 g, 6.19 mmol, 70.0% yield) as white solid.

Step-3. Synthesis of 6-(l-aminoethyl)-2-methylpyrimidin-4-ol

[0364] A reaction mixture of benzyl N-[l-(6-hydroxy-2-methylpyrimidin-4-yl)ethyl]carbamate (1.05 g, 3.65 mmol) and Pd/C (200 mg) in MeOH (100 ml) was stirred at room temperature under H₂ for 2 hours. The reaction mixture was filtered and concentrated to afford 6-(l-aminoethyl)-2- methylpyrimidin-4-ol (530 mg, 3.45 mmol, 94.8% yield) which was used directly in the next step. Step-4. Synthesis of N-[l-(6-hydroxy-2-methylpyrimidin-4-yl)ethyl]-4-(morpholin-4- yl)benzamide

[0365] To a solution of 4-(morpholin-4-yl)benzoic acid (486 mg, 2.35 mmol) in DMF (15 ml), was added HATU (1.15 g, 3.05 mmol) and DIPEA (757 mg, 5.87 mmol). The reaction mixture was stirred at room temperature for 2 hours and cooled to -40 °C, and then 6-(l-aminoethyl)-2- methylpyrimidin-4-ol (360 mg, 2.35 mmol) in DMF (10 mL) was added slowly. The reaction mixture was allowed to warm to room temperature and stirred further for 1 hour. The reaction mixture was concentrated and the residue was washed with EtOAc to afford N-[l-(6-hydroxy-2- methylpyrimidin-4-yl)ethyl]-4-(morpholin-4-yl)benzamide (660 mg, 1.92 mmol, 82.0% yield) as white solid.

Step-5. Synthesis of N-[(lS)-l-(6-hydroxy-2-methylpyrimidin-4-yl)ethyl]-4-(morpholin-4- yljbenzamide

[0366] N-[l-(6-hydroxy-2-methylpyrimidin-4-yl)ethyl]-4-(morpholin-4-yl)benzamide (660 mg, 1.92 mmol) was separated with SFC to afford N-[(lR)-l-(6-hydroxy-2-methylpyrimidin-4- yl)ethyl]-4-(morpholin-4-yl)benzamide, I-56-5A (300 mg) and N-[(lS)-l -(6-hydroxy-2- mcthylpyrimidin-4-yl)cthyl]-4-(morpholin-4-yl)bcnzamidc, I-56-5B (330 mg).

Step-6. Synthesis of (S)-2-methyl-6-(l-(4-morpholinobenzamido)ethyl)pyrimidin-4-yl 4- methylbenzenesulfonate

[0367] To a solution of N-[(lS)-l-(6-hydroxy-2-methylpyrimidin-4-yl)ethyl]-4-(morpholin-4- yl)benzamide, I-56-5B (100 mg, 292 μmol) in DCM (15 mL) was added TEA (3 mL) and 4- methylbenzenesulfonyl chloride (83.5 mg, 438 μmol). The reaction was stirred at 25°C for 18 h, diluted with water and extracted with EA. The organic phase was washed with brine, dried over Na₂SO₄, concentrated and purified by FC on SiO2 with (DCM/MeOH = 10:1) to afford (S)-2- methyl-6-( 1 -(4-morpholinobenzamido)ethyl)pyrimidin-4-yl 4-methylbenzenesulfonate, 1-56-6 (51.0 mg, 102 μmol, 35.1% yield) as yellow solid.

Step-7, tert-butyl (2S,3aS,7aS)-2-[(14-{ [(2S,3aS,7aS)-l-[(tert-butoxy)carbonyl]-octahydro-lH- indoL2-yl]formamido } -3 , 6,9, 12-tetraoxatetradecan- l-yl)carbamoyl] -octahydro- IH-indole- 1 - carboxylate

[0368] To a solution of (2S,3aS,7aS)-l-[(tert-butoxy)carbonyl]-octahydro-lH-indole-2- carboxylic acid (149 mg, 556 μmol) in DMF (3.6 mL) was added DIPEA (1.8 mL), HATU (211 mg, 556 μmol), and 3,6,9,12-tctraoxatctradccanc-l,14-diaminc (55 mg, 232 μmol). The reaction was stirred at 25°C for 4 h, diluted with water and extracted with EA. The organic phase was washed with brine, dried over Na₂SO₄, concentrated and purified by FC on C18 with (NH₄HCO₃) to afford tert-butyl (2S,3aS,7aS)-2-[(14-{ [(2S,3aS,7aS)-l-[(tert-butoxy)carbonyl]-octahydro-lH- indol-2-yl]formamido }-3, 6, 9, 12-tetraoxatetradecan- 1 -yl)carbamoyl] -octahydro- IH-indole- 1 - carboxylate (124 mg, 167 μmol, 72.5% yield) as yellow oil.

[0369]

Step-8. Synthesis of (2S,3aS,7aS)-N-(14-{[(2S,3aS,7aS)-octahydro-lH-indol-2-yl]formamido}- 3,6,9, 12-tetraoxatetradecan- Lyl)-octahydro-lH-indole-2-carboxamide

[0370] To a solution of tert-butyl (2S,3aS,7aS)-2-[(14-{ [(2S,3aS,7aS)-l-[(tert-butoxy)carbonyl]- octahydro- lFLindol-2-yl]formamido}-3,6,9, 12-tetraoxatetradecan- l-yl)carbamoyl]-octahydro-

1H-indole- 1 -carboxy late (124 mg, 167 μmol) in DCM (4.5 mL), was added TFA (1.5 mL). The reaction was stirred at 25 °C for 16 h and concentrated to afford (2S,3aS,7aS)-N-(14- { [(2S,3aS,7aS)-octahydro-lH-indol-2-yl]foimamido]-3,6,9,12-tctraoxatctradccan-l-yl)- octahydro-lH-indole-2-carboxamide (71.0 mg, 131 μmol, 57.3% yield) as yellow semi-solid.

Step-9 Synthesis of (2S,3aS,7aS)-N-(14-{ [(2S,3aS,7aS)-l-{2-methyl-6-[(lS)-l-{[4-(morpholin- 4-yl)phenyl]formamido}ethyl]pyrimidin-4-yl}-octahydro-lH-indol-2-yl]formamido}-3,6,9,12- tetraoxatetradecan- 1 -y 1)- 1 - { 2-methyl-6- [( 1 S )- 1 - { [4-(morpholin-4- yl)phenyl]formamido}ethyl]pyrimidin-4-yl}-octahydro-lH-indole-2-carboxamide

[0371] To a solution of (2S,3aS,7aS)-N-(14-{ [(2S,3aS,7aS)-octahydro-lH-indol-2- yl]formamido} -3,6,9, 12-tetraoxatetradecan-l-yl)-octahydro-lH-indole-2-carboxamide 1-56-8 (22 mg, 40.8 μmol), (S)-2-methyl-6-(l-(4-morpholinobenzamido) ethyl)pyrimidin-4-yl 4- methylbenzenesulfonate, 1-56-6 (44.5 mg, 89.7 μmol) in IPA (3 mL), was added DIPEA (1 mL). The reaction was stirred at 80 °C for 3 h, concentrated and purified by FC on C18 with (NH₄HCO₃) to afford (2S,3aS,7aS)-N-(14-{ [(2S,3aS,7aS)-l-{2-methyl-6-[(lS)-l-{ [4-(morpholin-4- yl)phenyl] formamido}ethyl]pyrimidin-4-yl}-octahydro-lH-indol-2-yl]formamido}-3,6,9,12- tetraoxatetradecan- 1 -yl)- 1- { 2-methyl-6- [( 1 S)- 1 - { [4-(morpholin-4-yl)phenyl] formamido}ethyl]pyrimidin-4-yl} -octahydro- 1H-indole-2-carboxamide, 1-56. Yield: 10 mg, 19.9%; Appearance: white solid; ¹H NMR (400 MHz, DMSO-d₆) δ 8.60 - 8.40 (m, 2H), 8.20 - 8.01 (m, 2H), 7.80 (d, J = 8.0 Hz, 4H), 6.98 (d, J = 7.8 Hz, 4H), 6.54 - 6.36 (m, 1H), 4.94 - 4.83 (m, 2H), 4.45 - 4.24 (m, 2H), 3.76 - 3.71 (m, 8H), 3.55 - 3.38 (m, 12H), 3.35 - 3.28 (m, 8H), 3.27 - 3.18 (m, 8H), 3.10 - 3.06 (m, 2H), 2.41 - 2.25 (m, 8H), 2.08 - 1.80 (m, 6H), 1.70 - 1.57 (m, 6H), 1.52 - 1.33 (m, 10H), 1.32 - 1.16 (m, 5H); HPLC purity: 96.6 %; LCMS Calculated for C₆₄H₉₀N₁₂O₁₀: 1187.50; Observed: 1209.9 [M+Na] ⁺.

[0372] The following examples were prepared using standard chemical manipulations and procedures similar to those used for the preparation of the previous examples. Analytical data is given in the table below.

Example 27. Synthesis of (2S,3aS,7aS)-l-{6-[(lS)-l-({4'-amino-[l,l'-biphenyl]-4- yl}formamido)ethyl]-2-methylpyrimidin-4-yl}-N-(17-{[(2R)-l-[(2R)-2-({3-ethyl-4-oxo-3H,4H- benzo[g]quinazolin-2-yl}sulfanyl)-2-phenylacetyl]pyrrolidin-2- yl]formamido}-3,6,9,12,15- pentaoxaheptadecan-l-yl)-octahydro-lH-indole-2-carboxamide, I-60:

Step-1. Synthesis of tert-butyl N-( 17-{[(2R)-l-[( 2R)-2-( { 3-ethyl-4-oxo-3H,4H-benzo[g]quinazolin -2-yl } sulfanyl) -2 -phenylacetyl ]pyrrolidin-2-yl ]formamido}-3, 6,9,12,15-pentaoxaheptadecan-l -yl )carbamate

[0373] To a stirred solution of tert-butyl N-(17-amino-3,6,9,12,15-pentaoxaheptadecan-l- yl)carbamate (80 mg, 210 μmol), K₂CO₃ (86.9 mg, 630 μmol) and HATU (119 mg, 315 μmol) in DMF (4 mL) were added (2R)-l-[(2R)-2-({3-ethyl-4-oxo-3H,4H-benzo[g]quinazolin-2- yl}sulfanyl)-2-phcnylacetyl]pyrrolidine-2-carboxylic acid, INT-3 (100 mg, 205 μmol) in portions at room temperature. The resulting mixture was stirred for 1 hour at room temperature. The residue was purified by reverse phase flash under the following conditions (column, C18 silica gel; mobile phase, H₂O/0.1% NH₄OH in MeCN, 10% to 50% gradient in 10 min; detector, UV 254 nm.) to afford tert-butyl N-(17-{ [(2R)-l-[(2R)-2-({3-ethyl-4-oxo-3H,4H-benzo[g]quinazolin-2- yl } sulfanyl)-2-phenylacetyl]pyrrolidin-2-yl]formamido } -3,6,9, 12, 15-pentaoxaheptadecan- 1 - yl)carbamate (150 mg, 176 μmol, 90% purity, 85% yield) as a yellow solid.

Step-2. Synthesis of 1 -[( 4R )-4-amino-3,3-dimethylpyrrolidin-l-yl ]ethan- 1 -one

[0374] A solution of tert-butyl N-(17-{ [(2R)-l-[(2R)-2-({3-ethyl-4-oxo-3H,4H- benzo[g]quinazolin-2-yl}sulfanyl)-2-phenylacetyl]pyrrolidin-2-yl]formamido}-3,6,9,12,15- pentaoxaheptadecan-l-yl)carbamate (140 mg, 164 μmol) in DCM (2.5 mL ) and TFA (0.5 mL) was stirred for 1 hour at room temperature. The resulting mixture was concentrated in vacuo to afford (2R)-N-(17-amino-3,6,9,12,15-pentaoxaheptadecan-l-yl)-l-[(2R)-2-({3-ethyl-4-oxo- 3H,4H-benzo[g]quinazolin-2-yl}sulfanyl)-2-phenylacetyl]pyrrolidine-2-carboxamide (130 mg, 173 μmol, 80% purity, 90% yield) as a brown solid, which was used for next step directly.

Step-3. Synthesis of tert-butyl N-(4'-{[(lS)-l-{6-[(2S,3aS, 7aS)-2-[(17-{[(2R)-l-[(2R)-2-({3-ethyl- 4-oxo-3H,4H-benzo[g]quinazolin2-yl}sulfanyl)-2-phenylacetyl]pyrrolidin-2-yl]formamido}- 3, 6, 9,12,15-pentaoxaheptadecan-l -yl)carbamoyl ]-octahydro-lH-indol-l -yl ]-2-methylpyrimidin- 4-yl}ethyl]carbamoyl}-[l,l'-biphenyl]-4-yl)carbamate

[0375] A mixture of (2R)-N-(17-amino-3,6,9,12,15-pentaoxaheptadecan-l-yl)-l-[(2R)-2-({3- ethyl-4-oxo-3H,4H-benzo[g]quinazolin-2-yl}sulfanyl)-2-phenylacetyl]pyrrolidine-2- carboxamide (120 mg, 160 μmol), (2S,3aS,7aS)-l-{6-[(lS)-l-[(4'- { [(tertbutoxy (carbonyl] amino } - [ 1 , 1 '-biphenyl] -4-yl)formamido]ethyl] -2-methyl pyrimidin-4-yl } - octahydro-lH-indole-2-carboxylic acid, INT-18 (115 mg, 192 μmol) and K₂CO₃ (66.2 mg, 480 μmol) in DMF (3 mL) was stirred at 0°C for 30 mins. HATU (91.1 mg, 240 μmol) was then added, and the reaction mixture was stirred for 1.0 hour at 0°C. The resulting mixture was concentrated in vacuo and purified by prep-HPLC (NH₃.H₂O buffer) to afford tert-butyl N-(4'-{ [(lS)-l-{6- [(2S,3aS,7aS)-2-[(17-{ [(2R)-l-[(2R)-2-({3-ethyl-4-oxo-3H,4H-benzo[g]quinazolin2- yl } sulfanyl)-2-phenylacetyl]pyrrolidin-2-yl]formamido } -3,6,9, 12, 15-pentaoxaheptadecan- 1 - yl)carbamoyl] -octahydro- 1 H-indol- 1 -yl]-2-methy lpyrimidin-4- yl } ethyl] carbamoyl } - [ 1 , T- biphenyl]-4-yl)carbamate (75.0 mg, 56.3 μmol, 90% purity, 35% yield) as a yellow solid.

Step-4. Synthesis of ( 2S,3aS, 7aS)-l -{6-[( !S)-l-({4 '-amino-[l, 1 '-biphenyl ]-4- yl}formamido )ethyl ]-2-methylpyrimidin-4-yl }-N-( 17-{ [(2R)-1 -[( 2R )-2-( { 3-ethyl-4-oxo-3H,4H-benzo[g]quinazolin-2- yl}sulfanyl)-2-phenylacetyl]pyrrolidin-2-yl]formamido }-3, 6, 9, 12,15-pentaoxaheptadecan- l-yl)-o ctahydro -lH-indole-2- carboxamide , 1-60

[0376] A solution of tert-butyl N-(4'-{[(lS)-l-{6-[(2S,3aS,7aS)-2-[(17-{ [(2R)-l-[(2R)-2-({3- ethyl-4-oxo-3H,4H-benzo[g]quinazolin-2-yl}sulfanyl)-2-phenylacetyl] pyrrolidin-2- yl]formamido}-3,6,9,12,15-pentaoxaheptadecan-l-yl)carbamoyl]-octahydro-lH-indol-l-yl]-2- methylpyrimidin-4-yl}ethyl]carbamoyl}-[l,T-biphenyl]-4-yl)carbamate (65 mg, 48.8 μmol) in DCM (2.5 mL) and TFA (0.5 mL) was stirred for 1 hour at room temperature. The resulting mixture was concentrated under reduced pressure and purified by reverse flash chromatography under the following conditions: column, C18 silica gel; mobile phase, MeCN in water, 10% to 80% gradient in 12min; detector, UV 220 nmto afford (2S,3aS,7aS)-l-{6-[(lS)-l-({4'-amino- [1 ,1 ’-biphenyl] -4- yl }formamido)ethyl] -2-methylpyrimidin-4-yl }-N-( 17 - { [(2R)- 1 - [(2R)-2-( { 3- cthyl-4-oxo-3H,4H-bcnzo[g]quinazolin-2-yl}sulfanyl)-2-phcnylacctyl]pyrrolidin-2- yl]formamido}-3,6,9,12,15-pentaoxaheptadecan-l-yl)-octahydro-lH-indole-2-carboxamide, I- 60. Yield: 15.00 mg, 25%; Appearance: White solid; ¹H NMR (300 MHz, DMSO-d₆) δ 8.82 - 8.77(m, 1H), 8.60 - 5.58 (m, 1H), 8.22 - 8.19 (m, 1H), 8.13 - 8.10 (m, 1H), 8.02 - 7.86 (m, 3H), 7.81 - 7.51 (m, 6H), 7.48 - 7.33 (m, 4H), 6.66 - 8.64 (m, 2H), 6.12 - 5.95(m, 1H), 5.32 (s, 2H), 4.88 - 4.85 (m, 1H), 4.32 - 4.26 (m, 2H), 4.12 - 4.09 (m, 3H), 3.58 - 3.42 (m, 9H), 3.42 - 3.35 (m, 7H), 3.34 - 3.32 (m, 7H), 3.21 - 3.16 (m, 2H) 3.14 - 3.09 (m, 4H), 2.31 (s, 3H), 2.21 - 1.78 (m, 6H), 1.74 - 1.52 (m, 3H), 1.44 - 1.42 (m, 4H), 1.34 - 1.20 (m, 5H). HPLC purity: 96.7%;

LCMS Calculated for: C68H82N10O10S : 1230.59; Observed: 1231.6 [M+H]⁺.

Example 28. Synthesis of N,N'-(3,6,9,12,15,18-hexaoxaicosane-l,20-diyl)bis(2-((3-ethyl-4- oxo-3, 4-dihydrobenzo[g]quinazolin-2-yl)thio)-N-methyl-2-phenylacetamide: 1-61 (Peakl, Isomer 1), 1-62 (Peak2, Isomer 2), and 1-63 (Peak3, Isomer 3)

Step-1. Synthesis of tert-butyl N-(20-{[(tert-butoxy)carbonyl]amino}-3,6,9,12,15,18- hexaoxaicosan - 1 -yl)carbamate

[0377] To a stirred solution of 3,6,9,12,15,18-hexaoxaicosane-l,20-diamine (500 mg, 1.54 mmol) and TEA (466 mg, 4.62 mmol) in DCM (10 mL) was added BOC2O (671 mg, 3.08 mmol) at 0 C. The resulting mixture was stirred for 2.0 hours at 0 °C. The reaction was quenched with H₂O (50 mL) at 0 °C. The resulting mixture was extracted with DCM (2x50 mL). The combined organic layers were washed with sat brine (100 mL), dried over anhydrous Na₂SO₄. After filtration, the filtrate was concentrated under reduced pressure. This resulted in tert-butyl N-(20-{[(tert- butoxy)carbonyl]amino}-3,6,9,12,15,18-hcxaoxaicosan-l-yl)carbamatc (600 mg, 1.14 mmol, 95% purity, 73% yield) as a yellow oil, which was used for the next step directly without further purification.

Step-2. Synthesis of tert-butyl N-(20-{[(tert-butoxy)carbonyl](methyl)amino}-3,6,9,12,15,18- hexaoxaicosan -1 -yl )-N -methylcarbamate

[0378] A solution of tert-butyl N-(20-{ [(tert-butoxy)carbonyl]amino}-3,6,9,12,15,18- hexaoxaicosan-l-yl)carbamate (540 mg, 1.02 mmol) in DMF (10 mL) was added NaH (61.1 mg, 2.55 mmol, 60% in a mineral oil) at 0°C. The mixture was stirred for 1.0 hour at 0°C, after was added CH₃I (362 mg, 2.55 mmol). The mixture was stirred for 2 hours at room temperature. The resulting mixture was concentrated in vacuum, the residue was purified by silica gel chromatography, eluted with ethyl acetate/petroleum ether=l:l to afford tert-butyl N-(20-{ [(tert- butoxy)carbonyl](methyl)amino]-3,6,9,12,15,18-hexaoxaicosan-l-yl)-N-methylcarbamate (360 mg, 651 μmol, 80% purity, 64% yield) as a yellow oil.

Step-3. Synthesis of 5,8,11,14,17, 20-hexaoxa-2, 23 -diazatetracosane dihydrochloride

[0379] A solution of tert-butyl N-(20-{[(tert-butoxy)carbonyl](methyl)amino}- 3,6,9,12,15,18-hexaoxaicosan-l-yl)-N-methylcarbamate (340 mg, 615 μmol) in HCl/dioxane (4M, 5 mL) was stirred for 3.0 hours at room temperature. The resulting mixture was concentrated in vacuum to afford 5,8,ll,14,17,20-hexaoxa-2,23-diazatetracosane dihydrochloride as HC1 salt (150 mg, 352 μmol, crude) as a brown oil, which was used for the next step directly without further purification.

Step-4. Synthesis of N,N'-(3,6,9,12,15,18-hexaoxaicosane-l,20-diyl)bis(2-((3-ethyl-4-oxo-3,4- dih.ydrobenzo[g ]quinazolin-2-yl)thio)-N-methyl-2-phenylacetamide )

[0380] A mixture of 2-({3-ethyl-4-oxo-3H,4H-benzo[g]quinazolin-2-yl}sulfanyl)-2- phenylacetic acid (153 mg, 394 μmol), HATU (187 mg, 493 μmol) and DIEA (127 mg, 987 μmol) in DCE (3 mL) was stirred at 50 °C for 0.5 hour. To above mixture was added 5,8,11,14,17,20- hexaoxa-2,23-diazatetracosane dihydrochloride (140 mg, 329 μmol), and the reaction mixture was stirred for 2.0 hours at 50 °C. The mixture was allowed to cool down to room temperature. The resulting mixture was concentrated in vacuum, the residue was purified by prep-HPLC (NH₃.H₂O buffer) to give N,N'-(3,6,9,12,15,18-hexaoxaicosane-l,20-diyl)bis(2-((3-ethyl-4-oxo-3,4- dihydrobenzo[g]quinazolin-2-yl)thio)-N-methyl-2-phenylacetamide) (100.0 mg, 91.2 μmol) as a white solid.

Step-5. Synthesis ofSynthesis ofN,N^f-(3,6,9,12,15,18-hexaoxaicosane-l,20-diyl)bis(2-((3-ethyl-4- oxo-3, 4-dihydrobenzo[g]quinazolin-2-yl)thio)-N-methyl-2-phenylacetamide: 1-61 (Peakl, Isomer 1), 1-62 (Peak2, Isomer 2), and 1-63 (Peak3, Isomer 3).

[0381] The N,N'-(3,6,9, 12,15,18-hexaoxaicosane- 1 ,20-diyl)bis(2-((3-ethyl-4-oxo-3,4- dihydro benzo[g]quinazolin-2-yl)thio)-N-methyl-2-phenylacetamide) (100.0 mg, 91.2 μmol) was purified by Chiral-SFC with the following conditions: Column: XA-CHIRAL ART Cellulose-SB, 3*25cm 5um; Mobile Phase A: HEX: DCM=3: 1, Mobile Phase B: IPA; Flow rate: 35 mL/min; Gradient: isocratic 10; Wave Length: 254 nm; RTl(min): 5.7; RT2(min): 6.4; RT3(min): 7.1; Sample Solvent: IPA: DCM=1: 1; Injection Volume: 0.5 mL; Number Of Runs: 13. This resulted in Isomer 1, (peakl, 1-61). Yield: 20.0 mg, 20.0%; Appearance: White solid; ¹H NMR (300 MHz, DMSO-d₆) δ 8.75-8.69 (m, 2H), 8.14 - 8.12 (m, 2H), 8.09 - 8.07 (m, 2H), 8.05 - 8.03 (m, 2H), 7.71 - 7.57 (m, 6H), 7.52 - 7.46(m, 2H), 7.35 - 7.21(m, 6H), 6.20 - 6.18(m, 2H), 4.08 (q, J = 7.4 Hz, 4H), 3.85 -3.51 (m, 8H), 3.55 - 3.30 (m, 18H), 3.26 (s, 6H), 2.91 - 2.88 (m, 2H), 1.30 - 1.20 (m, 6H). HPLC purity: 97.7%; LCMS Calculated for C₆₀H₆₈N₆O₁₀S₂: 1096.44; Observed: 1097.5 [M+H]⁺.

Isomer 2 (peak2, 1-62), Yield: 40.0 mg, 40.0%; Appearance: White solid; ¹H NMR (300 MHz, DMSO-d₆) δ 8.75 - 8.69 (m, 2H), 8.20 - 8.03 (m, 6H), 7.71 - 7.57 (m, 6H), 7.52 - 7.47 (m, 2H), 7.45 - 7.24 (m, 6H), 6.20 - 6.17 (m, 2H), 4.04 - 4.39 (m, 4H), 3.80 - 3.51 (m, 8H), 3.50 - 3.33 (m, 18H), 3.26 (s, 6H), 2.91 - 2.88 (m, 2H), 1.24 - 1.17 (m, 6H). HPLC purity: 96.9%; LCMS Calculated for C₆₀H₆₈N₆O₁₀S₂: 1096.44; Observed: 1097.5 [M+H]⁺.

Isomer 3 (peak3, 1-63). Yield: 10.0 mg, 10.0%; Appearance: White solid; ¹H NMR (300 MHz,

DMSO-d₆) 5 8.75 - 8.69 (m, 2H), 8.14 - 8.12 (m, 2H), 8.09 - 8.07 (m, 2H), 8.05 - 8.03 (m, 2H), 7.64 - 7.54 (m, 6H), 7.52 - 7.47 (m, 2H), 7.45 - 7.27 (m, 6H), 6.21 - 6.17 (m, 2H), 4.05 - 4.38 (m, 4H), 3.74 - 3.51 (m, 8H), 3. 49 - 3.33 (m, 18H), 3.26 (s, 6H), 2.91 - 2.88 (m, 2H), 1.38 - 1.20 (m, 6H). HPLC purity: 95.3%; LCMS Calculated for C₆₀H₆₈N₆O₁₀S₂: 1096.44; Observed: 1097.5 [M+H]⁺.

Example 29. Synthesis of (2S,3aS,7aS)-l-(2-{3-[3-(3- {4-[(2S,3aS,7aS)-2-(methylcarbamoyl)- octahydro-lH-indol-l-yl]-6-[(lS)-l-{[4-(morpholin-4-yl) phenyl]formamido}ethyl]pyrimidin-2- yl}prop-2-yn-l-yl)-l,3- diazinan-l-yl]prop-l-yn-l-yl}-6-[(lS)-l-{[4-(morpholin-4- yl)phenyl]formamido}ethyl]pyrimidin-4-yl)-N-methyl-octahydro- 1 H-indole-2-carboxamide, I- 64:

Step-1. Synthesis of tert-butyl N-(3-{[(tert-butoxy)carbonyl]amino}propyl)carbamate

To a stirred solution of tert-butyl N-(3-aminopropyl)carbamate (10.0 g, 57.3 mmol) and TEA (17.2 g, 171 mmol) in DCM (100 mL) was added BOC₂O (14.9 g, 68.7 mmol) at 0 °C. The resulting mixture was stirred for 2.0 hours at 0 °C. The reaction was quenched with H₂O (100 mL) at 0 °C. The resulting mixture was extracted with DCM (2x50 mL). The combined organic layers were washed with brine (100 mL), dried over anhydrous Na₂SO4. After filtration, the filtrate was concentrated under reduced pressure. This resulted in tert-butyl N-(3-{ [(tertbutoxy )carbonyl] amino }propyl)carbamatc (13.0 g, 47.3 mmol, 95% purity, 83% yield) as a yellow oil.

Step-2. Synthesis of tert-butyl N-(3-{[(tert-butoxy)carbonyl](prop-2-yn-l-yl)amino}propyl)-N- (prop-2-yn-l-yl)carbamate

A solution of tert-butyl N-(3-{ [(tert-butoxy)carbonyl]amino}propyl)carbamate (8.00 g, 29.1 mmol) in THF (100 mL) was added NaH (1.74 g, 72.7 mmol) at 0 °C. The mixture was stirred for 1.0 hour at 0 °C, after was added 3-bromoprop-l-yne (7.61 g, 64.0 mmol). The mixture was stirred for 2.0 hours at room temperature. The resulting mixture was poured into H₂O (200 mL) and extracted with ethyl acetate (100 mL x 3). The combined organic layers were washed with brine (100 mL), dried over anhydrous Na₂SO₄. The resulting mixture was concentrated in vacuum, the residue was purified by silica gel chromatography, eluted with ethyl acetate/petroleum ether=l:l to afford tert-butyl N-(3-{[(tert-butoxy)carbonyl](prop-2-yn-l-yl)amino}propyl)-N-(prop-2-yn- l-yl)carbamate (4.20 g, 11.9 mmol, 95% purity, 41% yield) as a yellow oil.

Step-3. Synthesis of N1,N 3 -di(prop-2-yn-l-yl)propane- 1,3 -diamine hydrochloride

A solution of tert-butyl N-(3-{ [(tert-butoxy)carbonyl](prop-2-yn-l-yl)amino}propyl)-N-(prop-2- yn-l-yl)carbamatc (4.20 g, 11.9 mmol) in HCl/dioxanc (4M, 50 mL) was stirred for 2.0 hours at room temperature. The resulting mixture was concentrated in vacuum to afford Nl, N3-di(prop-2- yn-l-yl)propane-l,3-diamine hydrochloride (2.30 g, 15.3 mmol, 80% purity, 95% yield) as a brown oil, which was used next step directly without further purification.

Step-4. Synthesis of l,3-bis(prop-2-yn-l -yl)-l ,3-diazinane

To a solution of Nl,N3-di(prop-2-yn-l-yl)propane-l,3-diamine hydrochloride (2.30 g, 15.3 mmol) in H₂O (30 mL ). Formaldehyde (2.29 g,76.5 mmol) was added and the solution was allowed to stirred for 24 hours at room temperature. After the resulting mixture was stirred for 2 hours at 80 °C. After cooling to ambient temperature, diluted with water (100 mL) and extracted with ethyl acetate (100 mLx3). The combined organic phase was washed with brine (100 mL), dried over anhydrous sodium sulfate. The resulting mixture was concentrated in vacuum, the residue was purified by silica gel chromatography, eluted with ethyl acetate/petroleum ether-1 :2 to afford 1 ,3- bis(prop-2-yn-l-yl)-l,3-diazinanc (1.20 g, 7.39 mmol) as a yellow oil.

Step-5. Synthesis of (2S,3aS,7aS)-l-(2-{3-[3-(3- {4-[(2S,3aS,7aS)-2-(methylcarbamoyl)- octahydro-lH-indol-1 -yl ]-6-[ (lS)-l-{[ 4-( morpholin-4-yl )phenyl ]fornamido } ethyl ]pyrimidin-2- yl}prop-2-yn- 1 -yl)- 1 ,3- diazinan-J-yl]prop-l-yn-l-yl}-6-[(lS)-J-{[4-(morpholin-4-yl)phenyl] formamido }ethyl ]pyrimidin-4-yl)-N-methyl-octahydro-lH-indole-2-carboxamide, 1-64.

To a stirred solution of l,3-bis(prop-2-yn-l-yl)-l,3-diazinane (30 mg, 184 μmol), (2S,3aS,7aS)-l- { 2-iodo-6-[( 1 S)- 1 - { [4-(morpholin-4-yl)phenyl]formamido }ethyl] pyrimidin-4-yl } -N-methyl- octahydro-lH-indole-2-carboxamide (227 mg, 368 μmol) , Pd(PPh₃)₂Cl₂ (25.8 mg, 36.8 μmol), TEA (55.7 mg, 552 μmol) and Cui (7.02 mg, 36.8 μmol) in MeCN (5 mL). The resulting mixture was stirred for 4 hours at 60 °C under nitrogen atmosphere. The mixture was allowed to cool down to room temperature. The mixture was diluted with water (30.0 mL) and extracted with ethyl acetate (30 mLx2). The combined organic phase was washed with brine (100 mL), dried over anhydrous sodium sulfate, filtered and concentrated in vacuum. The residue was purified by reverse phase flash with the following conditions (column, C18 silica gel; mobile phase, H₂O/0.1% NH₄OH in MeCN, 10% to 50% gradient in 10 min; detector, UV 254 nm.) to afford (2S,3aS,7aS)- l-(2-{3-[3-(3-{4-[(2S,3aS,7aS)-2-(methylcarbamoyl)-octahydro-lH-indol-l-yl]-6-[(lS)-l-{ [4- (morpholin-4-yl)phenyl]formamido}ethyl]pyrimidin-2-yl}prop-2-yn-l -yl)- l ,3-diazinan-1 - yl]prop- 1 -yn- 1 -y 1 } - 6- [( 1 S ) - 1 - { [4-(morpholin-4-yl)phenyl]formamido }ethyl]pyrimidin-4-yl)-N- methyl-octahydro-lH-indole-2-carboxamide, 1-64. Yield: 20.0 mg, 4.76%; Appearance: Yellow solid; ¹H NMR (400 MHz, DMSO-d₆) δ 8.45 - 8.39 (m, 2H), 8.10 - 7.89 (m, 2H), 7.80 - 7.76 (m, 4H), 7.00 - 6.88 (m, 4H), 6.67 - 6.59 (m, 2H), 5.00 - 4.84 (m, 2H), 4.40 - 3.80 (m, 6H), 3.71- 3.64 (m, 10H), 3.18 - 3.12 (m, 12H), 2.55 - 2.50 (m, 6H), 2.06 -1.96 (m, 2H), 1.87 -1.69 (m, 6H), 1.64 -1.56 (m, 8H), 1.51-1.46 (m, 10H), 1.37 - 1.20 (m, 3H), 1.19 - 1.09 (m, 3H). HPLC purity: 91.9%; LCMS Calculated for C₆₄H₈₂N₁₄O₆: 1142.65; Observed: 1143.5 [M+H]⁺.

Example 30. Synthesis of (2S,2'S,3aS,3a'S, 7aS,7a'S)-l, 1'-((1, 4-phenylenebis(prop-l-yne-3, 1- diyl))bis(6-((S)-l-(4-morpholinobenzamido)ethyl)pyrimidine-2,4-diyl))bis(N-methyloctahydro- 1 H-indole-2-carboxamide, 1-65:

Step-1. Synthesis of l,4-bis(3-( trimethylsilyl)prop-2-yn-l-yl)benzene

To a solution of ethynyltrimethylsilane (2.96 g, 30.2 mmol) in THF (32 mL) at 0 °C was added b romo(propan-2-yl)magnesium (15.1 mL, 30.2 mmol). The reaction mixture was stirred at room t emperature 1.0 hour. Then Cui (575 mg, 3.02 mmol) was added and stirred for 5 min. l,4-bis(bro momethyl)benzene (1.00 g, 3.78 mmol) was added and the mixture was refluxed for overnight. T he residue was quenched with NH4CI. aq. (20 ml) and extracted with ethyl acetate (25 mLx4). Th e organic layer dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure, the residue was purified by silica gel column and eluted with ethyl acetat e/petroleum ether =1:5 to give trimethyl(3-{4-[3-(trimethylsilyl)prop-2-yn-l-yl]phenyl}prop-l-y n-l-yl)silane (1.00 g, 3.34 mmol, 90 % purity, 42% yield) as a yellow oil. Step-2. Synthesis of 1 ,4-di(prop-2-yn-l -yl)benzene

To a solution of trimethyl(3-{4-[3-(trimethylsilyl)prop-2-yn-l-yl]phenyl}prop-l-yn-l-yl)silane ( 200 mg, 669 μmol) in MeOH (4 mL) was added K₂CO₃ (183 mg, 1.33 mmol) and the mixture w as stirred at room temperature for 6 hours. The mixture was quenched by water (15 mL) and extr acted with Et₂O (8 mL x 3). The organic layer dried over anhydrous sodium sulfate. After filtrati on, the filtrate was concentrated under reduced pressure, the residue was purified by silica gel col umn and eluted with ethyl acetate/petroleum ether =1:5 to give l,4-bis(prop-2-yn-l-yl)benzene ( 85.0 mg, 551 μmol, 85% purity, 72% yield) as a yellow oil.

Step-3. Synthesis of(2S,2S,3aS,3aS,7aS,7a'S)-1,1-((l,4-phenylenebis(prop-l-yne-3,l- diyl))bis(6-((S)-l-(4-morpholinobenzamido)ethyl)pyrimidine-2,4-diyl))bis(N -methyloctahydro- lH-indole-2-carboxamide) , I-65

To a stirred solution of l,4-bis(prop-2-yn-l-yl)benzene (20 mg, 129 μmol) in DMF (4 mL) was a dded (2S,3aS ,7aS)- 1 - { 2-iodo-6- [( 1 S )- 1 - { [4-(morpholin-4-yl)pheny 1] formamido } ethy l]pyrimidin -4-yl}-N-methyl-octahydro-lH-indole-2-carboxamide (159 mg, 258 μmol), CuI (2.20 mg, 11.6 p mol), DIEA (49.9 mg, 387 μmol) and Pd(PPh3)₂Cl₂ (9.05 mg, 12.9 μmol). The mixture was heate d to 50°C for 16 hours under nitrogen atmosphere. The reaction mixture was allowed to cool dow n to room temperature, diluted with H₂O (20 mL) and extracted with ethyl acetate (3 x 20 mL). T he combined organic layers were washed with saturated brine (20 mL), dried over Na₂SO₄. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by prep- HPLC (NH₃ H₂O buffer) to afford (2S,3aS,7aS)-l-(2-{3-[4-(3-{4-[(2S,3aS,7aS)-2-(mcthylcarba moyl)-octahydro- 1 H-indol- 1 -yl] -6-[( 1 S)- 1 - { [4-(morpholin-4-yl)phenyl]formamido }ethyl]pyrimi din-2-yl }prop-2-yn- 1 -yl)phenyl]prop- 1 -yn- 1 -yl } -6-[( 1 S)- 1 - { [4-(morpholin-4- yl)phenyl]formami do}ethyl]pyrimidin-4-yl)-N-methyl-octahydro-lH-indole-2-carboxamide, 1-65,. Yield: 7.50 mg, 5.1%. Appearance: white solid; ¹H NMR (400 MHz, DMSO-d₆) 5 8.42 (d, J = 7.8 Hz, 2H), 8.0 0 -7.82 (m, 2H), 7.78 (d, J = 8.8 Hz, 4H), 7.40 - 7.34 (m, 4H), 6.95 (d, J = 8.9 Hz, 4H), 4.86 (t, J = 7.3 Hz, 2H), 4.38 - 4.20 (m, 2H), 3.90 - 3.85 (m, 4H), 3.78 - 3.65 (m, 8H), 3.25 - 3.19 (m, 8 H), 2.59 - 2.53 (m, 6H), 2.36 - 2.30 (m, 2H), 2.12 -1.96 (m, 2H), 1.95- 1.75 (m, 5H), 1.72 - 1.5 0 (m, 6H), 1.50 -1.42 (m, 8H), 1.30 - 1.20 (m, 2H), 1.18 - 0.99 (m, 5H). HPLC purity: 92.7%;

LCMS Calculated for C₆₆H₇₈N₁₂O₆: 1134.62; Observed: 1135.7 [M+H] ⁺ Example 31. Biological Evaluation

Example 31a - pCTX408/pCTX378 AlphaLISA

[0382] The main materials required for the assay arc biotin-labeled Avi-FIP200 (1279-1594) and His-tagged FIP200 (1279-1594) proteins, a bead pair consisting of 20 ug/ml AlphaScreen Nickel Donor beads (PerkinElmer, Cat. No. AS101M) and 20 ug/ml Streptavidin AlphaLISA acceptor beads (PerkinElmer, Cat. No. AL125M), an assay buffer consisting of 50 mM HEPES (pH=7.5), 225 mM NaCl, 1 mM DTT, 0.01% BSA, 0.02% BRIJ-35, and 2% DMSO, and a white 384-well OptiPlate™ (PerkinElmer, Cat. No. 6007290). First, 200 nL of the compound in DMSO is dispensed to a white 384-well OptiPlate using an Echo (Labcyte) acoustic liquid transfer machine from the DMSO stock solution with a 3-fold dilution. Then, the assay reaction is initiated by adding lOpl of 3x final concentration pCTX408 Avi-FIP200 (1279-1594) to the 384-well Optiplate and left to incubate at room temperature for 30 minutes. Next, lOμl of 3x final concentration pCTX378 His-FIP200 (1279-1594) is added to the 384-well Optiplate and left to incubate at room temperature for 30 minutes. Finally, 10 pl of 3x final concentration beads mixture is added, followed by incubation at room temperature for three hours. The data is then read using an Envision (PerkinElmer) at excitation wavelength of 680 nm and emission wavelength of 570 nm. The data are plotted and EC₅₀ values generated in CDD Vault (Collaborative Drug Discovery Inc.).

[0383] Table 3 shows average EC₅₀ of certain compounds of the present disclosure. The compound numbers correspond to the compound numbers above in Table 1. Compounds having an activity designated as “++++” provided an EC50 of <0.1 pM; compounds having an activity designated as “+++” provided an EC₅₀ of 0.1-1 μM; compounds having an activity designated as “++” provided an EC50 of 1-20 μM; and compounds having an activity designated as “+” provided an EC ₅₀ of >20 μM.

Table 3

Example 31b - HDB Tandem mCherry-GFP p62 - Flux (6 hours)

[0384] USOS p62-mCherry-GFP cellsare split and expanded the day before seeding to ensure they are not overcrowded and in log growth phase using base media (DMEM (Thermo Fisher Scientific Cat. No. 11995-065), 10 % FBS (Thermo Fisher Scientific Cat. No. A31604-02), IX Pen Strep (Thermo Fisher Scientific Cat. No. 15070-063). The following day, using base media, plate 2,500 cells/well in delivery volume of 30 μL into high content 384-well plate (Perkin Elmer Cat. No. 6057300) and spin cells down at 40 x g for 10 seconds. Incubate overnight at 37°C. The following day dispense 30 nL of compound in dose response using an Echo (Labcyte) acoustic liquid transfer machine (final DMSO concentration is 0.1 %) and incubate for six hours at 37°C. After incubation, add 10 μL per well of 16% paraformaldehyde (Electron Microscopy Sciences Cat. No. 15713-S), supplemented with Hoechst stain at 1:1250. The plate is spun down at 40 x g for 10 seconds and incubated at room temperature for 15 minutes protected from light. After the plate fixation is finished, the cells are washed using a plate washer (BioTek EL 406 Washer/Dispenser). The plate is covered with aluminum or black seal film and read on PE Operetta CLS under 40X objective on six fields of view. The data are plotted and EC50 values generated in CDD Vault (Collaborative Drug Discovery Inc.).

[0385] Table 4 shows ACso of certain compounds of the present disclosure. The compound numbers correspond to the compound numbers above in Table 1. Compounds having an activity designated as “++++” provided an AC₅₀ of <5 μM ; compounds having an activity designated as “+++” provided an AC50 of 5-10 μM; compounds having an activity designated as “++” provided an ACso of 10-25 pM; and compounds having an activity designated as “+” provided an ACso of >25 pM.

Table 4

Example 31c - pATG16Ll AlphaLisa Cell Assay - U2OS Cells

[0386] The main materials required for the assay are the AlphaLISA SureFire Ultra p- ATG16L1 (Ser278) Assay Kit (PerkinElmer, Cat. No. ALSU-PATG-A500), Halt Protease and Phosphatase Inhibitor Cocktail (100X) (ThermoFisher, Cat. No. 78441), 384-well Optiplate (PerkinElmer, Cat.No.6007290), 96-well plate (Grenier, Cat.No. 655180), and U2OS Fisl-MYC- HaloTag cell line (Pn+4). Acceptor and donor reagent mixes are prepared according to the manufacturer’s instructions. First, 50 pL of U2OS cells arc seeded at 15k cclls/wcll in a 96-well plate and incubated overnight. The following day 50 μL of compound are added to each well at a top concentration of lOuM with 3-fold dilutions and eight doses total. The final DMSO concentration is 0.5% and compounds are incubated for one hour at 37°C. The cells are then lysed using 50 μL lx Lysis Buffer (dilute 5X Lysis buffer 1 :5 in MilliQ water, then add Protease and lOOx Phosphatase Inhibitor Cocktail 1:100) and are left to agitate on a plate shaker (-350 rpm) for 10 minutes at room temperature. Next, the lysate is removed and transferred 10 μL per well to wells of a 384-well Optiplate. The SureFire Ultra Assay is performed by adding 5 μL Acceptor bead mix to wells. Seal, wrap in foil and shake 1-2 minutes on plate shaker. Then incubate for one hour at 22. This step is repeated with 5 μL Donor bead mix. Finally, the plate sealer is removed and the data is read overnight using an Envision (PerkinElmer) at excitation wavelength of 680 nm and emission wavelength of 570 nm. The data are plotted and EC50 values generated in CDD Vault (Collaborative Drug Discovery Inc.).

[0387] Table 5 shows ECso of certain compounds of the present disclosure. The compound numbers correspond to the compound numbers above in Table 1. Compounds having an activity designated as “++++” provided an EC₅₀ of <1.0 μM; compounds having an activity designated as “+++” provided an ECso of 1-5 pM; compounds having an activity designated as “++” provided an ECso of 5-10 μM; and compounds having an activity designated as “+” provided an ECso of >10 μM.

Table 5

[0388] The embodiments of the disclosure described above are intended to be merely exemplary, numerous variations and modifications will be apparent to those skilled in the art. All such variations and modifications are intended to be within the scope of the present invention as defined in any appended claims.

Claims

1. A compound represented by formula I:

A¹-B-A² or a pharmaceutically acceptable salt thereof, wherein:

2. The compound of claim 1, wherein A¹ and A² are each independently selected from formula 11-1, 11-2, 11-3, or 11-4:

each G¹ is independently an optionally substituted C₆-C₁₂ aryl or an optionally substituted

5- to 6-membered heteroaryl; each X¹ is independently S, N(R³), O, optionally substituted C₁-C₆ aliphatic, or optionally substituted C₃-C₆ cycloaliphatic; each X² is independently C(R³) or N, provided that, when X² is N, then X¹ is optionally substituted C₁-C₆ aliphatic, or optionally substituted C₃-C₆ cycloaliphatic; when a bond between X³ and X⁴ is a single bond, then X³ is N(R^2a), and X⁴ is C(O) when a bond between X³ and X⁴ is a double bond, then X³ is C(R^2b) and X⁴ is C(R³), or N; each R¹ is independently selected from halogen, optionally substituted C₁-C₆ aliphatic, and optionally substituted -O-C₁-C₆ aliphatic; or two instances of R¹ come together with the atoms to which they are attached to form a n optionally substituted C₆-C₁₂ aryl ring, an optionally substituted C₄-C₆ cycloaliphatic ring, a 5- to 6- membered heteroaryl ring comprising 1 to 3 heteroatoms selected from N, O, and S, or an optionally substituted 5- to 6-membered heterocyclic ring comprising 1 to 3 heteroatoms selected from N, O, and S; each R^2a is independently optionally substituted C₁-C₆ aliphatic; each R^2b is independently hydrogen, optionally substituted C₁-C₆ aliphatic, optionally substituted -N(R³)-C₁-C₆ aliphatic, or optionally substituted -O-C₁-C₆, aliphatic; each R³ is independently selected from hydrogen, halogen, and optionally substituted Ci- C₆ aliphatic; each R⁴ is independently selected from optionally substituted C₆-C₁₂ aryl, optionally substituted 5- to 12- membered heteroaryl ring comprising 1 to 3 heteroatoms selected from N, O, and S, optionally substituted 4- to 12-membered heterocyclyl comprising 1 to 3 heteroatoms selected from N, O, and S, and optionally substituted C₁-C₆ aliphatic; each R⁵ is independently an optionally substituted 4- to 6-membered heterocyclic ring, an optionally substituted 5- to 6-membered heteroaryl ring, an optionally C₃-C₆ cycloaliphatic ring, an optionally substituted C₆-C₁₂ aryl, or an optionally substituted C₁-C₆ aliphatic; each L¹ is independently a bond, -C(O)-, -S(O)-, -S(O)₂-, or -NR³-; each n is independently 0, 1, 2, 3, 4, 5, or 6; each X⁵, X⁶, and X⁷ are independently selected from the group consisting of N and CH; each R⁷ is independently hydrogen, an optionally substituted -O-C₁-C₆ aliphatic, - S(O)₂R³, optionally substituted C₁-C₆ aliphatic, an optionally substituted 4- to 12- membered heterocycle ring comprising 1 to 4 heteroatoms selected from N, O, and S, an optionally substituted C₆-C₁₂ aryl, or an optionally substituted 5- to 12-membered heteroaryl comprising 1 to 4 heteroatoms selected from N, O, and S each G² is independently an optionally substituted C₆-C₁₂ aryl, optionally substituted 4- to 12-mcmbcrcd heterocycle comprising 1 to 4 heteroatoms selected from N, O, and S, optionally substituted 5- to 6-membered heteroaryl comprising 1 to 4 heteroatoms selected from N, O, and S, or optionally substituted C₃-C₆ cycloaliphatic; each G³ is independently an optionally substituted 4- to 12- membered heterocycle ring comprising 1 to 4 heteroatoms selected from N, O, and S, an optionally substituted 5- to 6-membered heteroaryl ring comprising 1 to 4 heteroatoms selected from N, O, and S, an optionally substituted C₃-C₆ cycloaliphatic ring or optionally substituted C₆-C₁₂ aryl; each L² is independently a bond, -NR³-C(O)-, -C(O)-NR³-, optionally substituted C₁-C₆ aliphatic, optionally substituted 4- to 6-membered heterocyclic, or optionally substituted C₃-C₆ cycloaliphatic; each L³ is independently -NR³-, -O-, -C(O)-, -NR³-C(O)-, -NR³-S(O)₂-, -C(O)-NR³-, - S(O)₂ NR³-, -NR³-C(O)-NR³-, optionally substituted C₁-C₆ aliphatic, or an optionally substituted C₃-C₆ cycloaliphatic ring;

* represents a point of attachment to moiety B ; and wherein: when A¹ or A² is a moiety of formula II- 3 then R⁶ is a bond, optionally substituted C₁-C₆ aliphatic, optionally substituted 2- to 6-membered heteroaliphatic, optionally substituted 4- to 6-membered heterocycle comprising one 1 to 3 heteroatoms selected from N, O, and S, optionally substituted C₆-C₁₂ aryl, or optionally substituted C₃-C₆ cycloaliphatic; and R⁸ is independently halogen, -OR³, -C(O)N(R³)₂, -C(O)OR³, optionally substituted C₁-C₆ aliphatic, optionally substituted C₃-C₆ cycloaliphatic, optionally substituted C₆-C₁₂ aryl; and when A¹ or A² is of formula II-4, then R⁶ is H, halogen, optionally substituted C₁-C₆ aliphatic, optionally substituted 2- to 6-membered heteroaliphatic, optionally substituted 4- to 6-membered heterocycle comprising one 1 to 3 heteroatoms selected from N, O, and S, optionally substituted C₆-C₁₂ aryl, or optionally substituted C₃-C₆ cycloaliphatic; and R⁸ is a bond, -O-, -C(O)NR³-, -C(O)OR³, optionally substituted C₁-C₆ aliphatic, optionally substituted C₃-C₆ cycloaliphatic, or optionally substituted C₆-C₁₂ aryl.

3. The compound of claims 1 or 2, wherein A¹ and A² arc the same.

4. The compound of claims 1 or 2, wherein A¹ and A² are different.

5. The compound of any one of claims 2-4, wherein two R¹ come together, with the atoms to which they are attached, to form an optionally substituted C₆-C₁₂ aryl ring or a 5- to 6- membered heteroaryl ring comprising 1 to 3 heteroatoms selected from N, O, and S.

6. The compound of any one of claims 2-5, wherein n is 2 and each R¹ is halogen.

7. The compound of any one of claims 2-5, wherein each R¹ is chloride.

8. The compound of any one of claims 2-7, wherein a bond between X⁴ and X³ is a single bond.

9. The compound of any one of claims 2-7, wherein a bond between X⁴ and X³ is a double bond, X³ is C(R^2b) and X⁴ is N.

10. The compound of any one of claims 2-9, wherein X¹ is S.

11. The compound of any one of claims 2-10, wherein R⁴ is optionally substituted phenyl or optionally substituted 5- to 6-membered heteroaryl comprising 1 to 3 heteroatoms selected from N, O, and S.

12. The compound of any one of claims 2-11, wherein a moiety:

13. The compound of any one of claims 2-12, wherein G¹ is phenyl or naphthyl.

14. The compound of any one of claims 2-13, wherein R⁵ is optionally substituted 5- to 6- membered heterocycle comprising 1 to 3 heteroatoms selected from N, O, and S.

15. The compound of any one of claims 2-14, wherein L¹ is -C(O)-.

16. The compound of claim 2, wherein A¹ and/or A² are of formula II- 1 or II-2, X⁴ is C(O), a bond between X⁴ and X³ is a single bond, G¹ is napthyl, X¹ is S, X² is C(R³), R⁴ is optionally substituted phenyl, and R⁵ is optionally substituted 5- to 6-membered heterocycle.

17. The compound of claim 2, wherein X⁵ and X⁷ are each N and X⁶ is CH.

18. The compound of claims 2 or 17, wherein G³ is an optionally substituted 4- to 12- membered heterocycle ring comprising 1 to 4 heteroatoms selected from N, O, and S.

19. The compound of claim 18, wherein G³ is optionally substituted 4- to 6-membered heterocycle comprising 1 to 3 heteroatoms selected from N, O, and S .

20. The compound of claim 18, wherein G³ is optionally substituted 6- to 12-membered bicyclic heterocycle comprising 1 to 4 heteroatoms selected from N, O, and S.

21 . The compound of any one of claims 2 or 17-20, wherein L² is optionally substituted Ci- C₆ aliphatic.

22. The compound of any one of claims 2 or 17-20, wherein L² is a bond or selected from:

23. The compound of claim 22, wherein L² is:

24. The compound of any one of claims 2 or 17-23, wherein G² is optionally substituted C₆-

C12 aryl or optionally substituted 5- to 6-membered heteroaryl comprising 1 to 4 heteroatoms selected from N, O, and S.

25. The compound of any one of claims 2 or 17-24, wherein R⁷ is optionally substituted 4- to 12-membered heterocycle comprising 1 to 4 heteroatoms selected from N, O, and S.

26. The compound of claim 2, wherein A¹ and/or A² are of formula II-3 or II-4, X⁵ and X⁷ are each N, X⁶ is CH, G³ is optionally substituted 4- to 12-membered heterocycle comprising 1 to 4 heteroatoms selected from N, O, and S, L² is optionally substituted Ci- C& aliphatic, L³ is -NR³-C(O)- or -C(O)-NR³-, G² is optionally substituted phenyl, and R⁷ is optionally substituted 4- to 12-membered heterocycle comprising 1 to 4 heteroatoms selected from N, O, and S.

27. The compound of claim 2, wherein a moiety: is:

28. The compound of claim 2, wherein a moiety:

29. The compound of claim 2, wherein A¹ and A² are each independently selected from:

30. The compound of claim 2, wherein A¹ and A² are each independently selected from:

31. The compound of any one of claims 1-30, wherein B is a linker moiety that is an optionally substituted C_2-30 aliphatic group wherein one or more carbons are optionally and independently replaced by -Cy-, -NR^Z-, -N(R^Z)C(O)-, -C(O)N(R^Z)-, -N(R^Z)C(O)O-, - OC(O)N(R^Z)-, -N(R^Z)C(O)N(R^Z) -OC(O)O-, -O-, -C(O)-, -OC(O)-, -

C(O)O-, -SO-, -SO₂-, wherein each -Cy- is independently an optionally substituted 3-12 membered heterocyclyl ring having 1-3 heteroatoms selected from N, O, and S, an optionally substituted 3-8 membered heteroaryl ring having 1-4 heteroatoms selected from N, O, and S, an optionally substituted C₃-C₆ cycloalkyl, or an optionally substituted C₆-C₁₂ aryl, and each R^z is independently H or an optionally substituted group selected from C₁- C₂₀ aliphatic, or C₃-C₁₂ cycloaliphatic.

32. The compound of any one of claims 1-30, wherein B is selected from Table B.

33. The compound of claim 2, wherein the compound is represented by formula ITI-1 :

or a pharmaceutically acceptable salt thereof.

34. The compound of claim 2, wherein the compound is represented by formula III-2:

or a pharmaceutically acceptable salt thereof.

35. The compound of claim 2, wherein the compound is represented by formula III-3:

or a pharmaceutically acceptable salt thereof.

36. The compound of claim 2, wherein the compound is represented by formula III-4:

or a pharmaceutically acceptable salt thereof.

37. The compound of claim 2, wherein the compound is represented by formula III-5 :

or a pharmaceutically acceptable salt thereof.

38. The compound of claim 2, wherein the compound is represented by formula IV- 1:

or a pharmaceutically acceptable salt thereof.

39. The compound of claim 2, wherein the compound is represented by formula IV-2:

or a pharmaceutically acceptable salt thereof.

40. The compound of claim 2, wherein the compound is represented by formula IV-3:

or a pharmaceutically acceptable salt thereof.

41. The compound of claim 2, wherein the compound is represented by formula IV-4:

or a pharmaceutically acceptable salt thereof.

42. The compound of claim 2, wherein the compound is selected from Table 1.

43. A pharmaceutical composition comprising a compound of any one of claims 1-42, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, diluent, or excipient.

44. A method of treating a disease, disorder, or condition in a subject comprising administering to the subject a compound of any one of claims 1-42 or a pharmaceutical composition of claim 43.

45. The method of claim 44, wherein the disease, disorder, or condition is selected from a protein aggregopathy, a lysosomal storage disease, an infectious disease, or an inflammatory disease.

46. The method of claim 45, wherein the protein aggregopathy is Alzheimer’s disease, Huntington’s disease, Parkinson’s disease, amyotrophic lateral sclerosis, dementia with Lewy Bodies, cystic fibrosis, frontotemporal dementia, amyloid transthyretin cardiomyopathy, type-2 diabetes, and an αl -antitrypsin deficiency.

47. The method of claim 45, wherein the lysosomal storage disease is Niemann-Pick disease type C, Gaucher disease, Fabry disease, cystinosis, Pompe disease, Tay Sachs disease, Sandhoff disease, metachromatic leukodystrophy, mucolipidosis, mucopolysaccharide storage disease, and Schindler disease.

48. The method of claim 45, wherein the infectious disease is tuberculosis, viral infections (e.g., influenza, HIV, Hepatitis C Virus and herpesvirus), salmonella, listeria, toxoplasma gondii, chlamydia, and leishmania.

49. The method of claim 45, wherein the inflammatory disease is Crohn’s disease, ulcerative colitis, rheumatoid arthritis, systemic lupus erythematosus, psoriasis, gout, atherosclerosis, neurodegenerative diseases, cystic fibrosis, age-related macular degeneration, and asthma.

50. A method of promoting autophagy in a subject comprising administering to the subject a compound of any one of claims 1-42, or a pharmaceutical composition of claim 43.

51. Use of a compound of any one of claims 1-42 or a pharmaceutical composition of claim 43 for the treatment of a disease, disorder, or condition.

52. Use of a compound of any one of claims 1-42 or a pharmaceutical composition of claim 43 for promoting autophagy in a subject.