WO2025213185A1

WO2025213185A1 - Peptide conjugates

Info

Publication number: WO2025213185A1
Application number: PCT/US2025/023503
Authority: WO
Inventors: Mark TYE; G. Leslie BURNETT; Elsa QUINTANA; Nataliya Tovbis SHIFRIN
Original assignee: Revolution Medicines Inc
Current assignee: Revolution Medicines Inc
Priority date: 2024-04-05
Filing date: 2025-04-07
Publication date: 2025-10-09
Anticipated expiration: 2026-10-05

Abstract

Disclosed herein are peptide conjugates formed by covalently linking a peptide and a RAS tri-complex inhibitor, methods of making the peptide conjugates, and their uses for selectively labeling cells and in the treatment of cancers.

Description

PEPTIDE CONJUGATES

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated in its entirety. Said XML copy, created on April 4, 2025, is named “51432-055WO2_Sequence_Listing_4_4_25” and is 22,127 bytes in size.

BACKGROUND

The immune system plays a critical role in surveilling intracellular proteins through the mechanism of antigen presentation on major histocompatibility complex (MHC). Short peptides, typically consisting of 8-11 amino acids, are derived from proteasomal degradation of proteins and loaded onto MHC complexes. These peptide-MHC complexes (pMHCs) serve as vital indicators of intracellular health, allowing T-cells to scan for foreign antigens.

Importantly, many small molecules, such as toxins, drugs, and hormones, do not elicit an immune response when injected directly into animals. However, these molecules are capable of binding to specific antibodies, without possessing inherent antigenicity, and are therefore termed haptens. Recent findings have shown that covalent small molecule inhibitors can induce the presentation of drug-modified neoantigens by class I MHC. These neoantigens, resulting from the covalent binding to the target peptide, hold potential in effectively labeling cancer cells, notably even in cases where the cells exhibit resistance to direct inhibition by the covalent inhibitor.

There exists an ongoing and unmet need for compositions and methods useful for generating agents capable of binding to targets, including drugs covalently bound to proteins or peptides, which are useful for selectively labeling cells. The disclosure provided herein addresses these pressing needs.

BRIEF SUMMARY

Provided herein are isolated peptide conjugates which comprise a peptide covalently linked to a RAS tri-complex inhibitor, methods of making the peptide conjugates, and their uses in the treatment of cancers.

In one aspect, the disclosure provides a peptide conjugate which is formed by covalently linking a peptide and a RAS tri-complex inhibitor, wherein the peptide is derived from RAS and wherein the peptide conjugate is isolated, or a pharmaceutically acceptable salt thereof. In some embodiments, the RAS is KRAS, HRAS, or NRAS. In some embodiments, the RAS comprises a mutation. In some embodiments, the peptide comprises a segment of KRAS^G12D, HRAS^G12D, or NRAS^G12D.

In some embodiments, the RAS inhibitor selectively inhibits a RAS mutant protein over a wildtype RAS protein. In some embodiments, the RAS inhibitor is an HRAS inhibitor, a KRAS inhibitor and/or an NRAS inhibitor. In some embodiments, the RAS inhibitor is a KRAS inhibitor. In some embodiments, the RAS mutation is a KRAS mutation. In some embodiments, the KRAS mutation comprises a KRAS^G12D mutation.

In some embodiments, the peptide comprises an aspartic acid residue.

In some embodiments, the peptide conjugate is formed by covalently linking the RAS inhibitor to an Asp residue of the peptide. In some embodiments, the RAS inhibitor is a KRAS inhibitor having formula (IA), (HA), (I HA), (III A-), (IVA) or (IVA-1):

(111 A)

In some embodiments, the peptide comprises an amino acid sequence of DGVGKSALTI (SEQ ID NO: 1), ADGVGKSALT (SEQ ID NO: 2), GADGVGKSAL (SEQ ID NO: 3), VGADGVGKSA (SEQ ID NO: 4), VVGADGVGK (SEQ ID NO: 5), VVGADGVGKS (SEQ ID NO: 6), VVVGADGVGK (SEQ ID NO: 7), KLWVGADGV (SEQ ID NO: 8), YKLVVVGADG (SEQ ID NO: 9), or EYKLVVVGAD (SEQ ID NO: 10), or an isotopically labeled analog thereof. In some embodiments, the peptide has between about 7 to about 30 amino acids in length. In some embodiments, the amino acid sequence is at least 80% identical to DGVGKSALTI (SEQ ID NO: 1), ADGVGKSALT (SEQ ID NO: 2), GADGVGKSAL (SEQ ID NO: 3), VGADGVGKSA (SEQ ID NO: 4), VVGADGVGK (SEQ ID NO: 5), VVGADGVGKS (SEQ ID NO: 6), VVVGADGVGK (SEQ ID NO: 7), KLVWGADGV (SEQ ID NO: 8), YKLVVVGADG (SEQ ID NO: 9), or EYKLVVVGAD (SEQ ID NO: 10), or an isotopically labeled analog thereof. In some embodiments, the peptide comprises an amino acid sequence DGVGKSALTI (SEQ ID NO: 1), or an isotopically labeled analog thereof. In some embodiments, the peptide comprises an amino acid sequence ADGVGKSALT (SEQ ID NO: 2) or an isotopically labeled analog thereof. In some embodiments, the peptide comprises an amino acid sequence GADGVGKSAL (SEQ ID NO: 3) or an isotopically labeled analog thereof. In some embodiments, the peptide comprises an amino acid sequence VGADGVGKSA (SEQ ID NO: 4) or an isotopically labeled analog thereof. In some embodiments, the peptide comprises an amino acid sequence VVGADGVGK (SEQ ID NO: 5) or an isotopically labeled analog thereof. In some embodiments, the peptide comprises an amino acid sequence WGADGVGKS (SEQ ID NO: 6) or an isotopically labeled analog thereof. In some embodiments, the peptide comprises an amino acid sequence VWGADGVGK (SEQ ID NO: 7) or an isotopically labeled analog thereof. In some embodiments, the peptide comprises an amino acid sequence KLVVVGADGV (SEQ ID NO: 8) or an isotopically labeled analog thereof. In some embodiments, the peptide comprises an amino acid sequence YKLWVGADG (SEQ ID NO: 9) or an isotopically labeled analog thereof. In some embodiments, the peptide comprises an amino acid sequence EYKLVVVGAD (SEQ ID NO: 10) or an isotopically labeled analog thereof.

In some embodiments, the peptide conjugate is selected from Table 1 or Table 2. In some embodiments, the peptide conjugate has a structure of formula (III): wherein:

X1 is hydrogen or a peptide comprising from 1 to 50 amino acids;

X2 is OH or a peptide comprising from 1 to 50 amino acids; and

R¹ is hydrogen or cyclopropyl.

In another aspect, the disclosure provides a cell-free peptide conjugate/MHC complex comprising a peptide conjugate as described herein and a major histocompatibility complex (MHO). In some embodiments, the MHO is a human leukocyte antigen (HLA).

In another aspect, the disclosure provides a method for identifying a peptide conjugate- or peptide conjugate/MHC complex-specific antibody, the method comprising:

(a) providing (i) a peptide conjugate or (ii) a peptide conjugate/MHC complex;

(b) contacting the peptide conjugate or peptide conjugate/MHC complex with a library comprising a plurality of antibodies under conditions suitable for binding at least one antibody of the plurality of antibodies to the peptide conjugate or peptide conjugate/MHC complex; recovering the at least one antibody bound to the peptide conjugate or peptide conjugate/MHC complex. DETAILED DESCRIPTION

Definitions

In this application, unless otherwise clear from context, (i) the term “a” means “one or more”; (ii) the term "or" is used to mean "and/or" unless explicitly indicated to refer to alternatives only or the alternative are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and "and/or”; (iii) the terms “comprising” and “including” are understood to encompass itemized components or steps whether presented by themselves or together with one or more additional components or steps; and (iv) where ranges are provided, endpoints are included.

As used herein, the term “about” is used to indicate that a value includes the standard deviation of error for the device or method being employed to determine the value. In certain embodiments, the term “about” refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12% , 11%, 10% , 9% , 8% , 7% , 6% , 5% , 4% , 3% , 2% , 1 % , or less in either direction (greater than or less than) of a stated value, unless otherwise stated or otherwise evident from the context (e.g., where such number would exceed 100% of a possible value). In one embodiment, the term “about” means ±10% of a stated value. In another embodiment, the term “about” means ±15% or ± 20% of a stated value.

As used herein, the term “administration” refers to the administration of a composition (e.g., a compound, or a preparation that includes a compound as described herein) to a subject or system. Administration to an animal subject (e.g., to a human) may be by any appropriate route. For example, in some embodiments, administration may be bronchial (including by bronchial instillation), buccal, enteral, intradermal, intra-arterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, mucosal, nasal, oral, rectal, subcutaneous, sublingual, topical, tracheal (including by intratracheal instillation), transdermal, vaginal or vitreal.

The term “amino acid,” as described herein, refers to a molecule having a side chain, an amino group, and an acid group (e.g., -CO2H or -SO3H), wherein the amino acid is attached to the parent molecular group by the side chain, amino group, or acid group (e.g., the side chain). As used herein, the term “amino acid” in its broadest sense, refers to any compound or substance that can be incorporated into a polypeptide chain, e.g., through formation of one or more peptide bonds. In some embodiments, an amino acid has the general structure H2N-C(H)(R)-COOH. In some embodiments, an amino acid is a naturally-occurring amino acid. In some embodiments, an amino acid is a synthetic amino acid; in some embodiments, an amino acid is a D-amino acid; in some embodiments, an amino acid is an L-amino acid. “Standard amino acid” refers to any of the twenty standard L-amino acids commonly found in naturally occurring peptides. Exemplary amino acids include alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, optionally substituted hydroxylnorvaline, isoleucine, leucine, lysine, methionine, norvaline, ornithine, phenylalanine, proline, pyrrolysine, selenocysteine, serine, taurine, threonine, tryptophan, tyrosine, and valine.

Antibodies and antigen-binding domains or fragments thereof are collectively “binding partners” and each individually a “binding partner”. The term “antibody” includes each binding partner format herein. The antibody can comprise a polypeptide with an antigen-binding domain or fragment thereof. The binding partners bind with specificity to a protein or fragment thereof, or a peptide provided in peptide form, that comprises a covalently attached molecule. The covalently attached molecule forms a peptide conjugate. The term “cell” refers to a cell that is in vitro, ex vivo or in vivo. In some embodiments, an ex vivo cell can be part of a tissue sample excised from an organism such as a mammal. In some embodiments, an in vitro cell can be a cell in a cell culture. In some embodiments, an in vivo cell is a cell living in an organism such as a mammal.

The term “compound” as used herein is meant to include all stereoisomers, geometric isomers, tautomers and isotopes of the structures depicted. The term is also meant to refer to compounds of the inventions, regardless of how they are prepared, e.g., synthetically, through biological process (e.g., metabolism or enzyme conversion), or a combination thereof.

Throughout this specification, the word “comprise” or variations such as “comprises” or “comprising” will be understood to imply the inclusion of a stated integer (or components) or group of integers (or components), but not the exclusion of any other integer (or components) or group of integers (or components).

As used herein, the term “conjugate” refers to a compound formed by the joining (e.g., via a covalent bond forming reaction) of two or more chemical compounds (e.g., a compound including a crosslinking group and a peptide or a protein such as a target protein or a presenter protein).

The term “contacting” refers to the bringing together of indicated moieties in an in vitro system or an in vivo system. For example, “contacting” KRAS with a compound described herein includes the administration of a compound described herein to an individual or patient, such as a human, having KRAS, as well as, for example, introducing a compound described herein into a sample containing a cellular or purified preparation containing KRAS.

As used herein, the term “cross-linking group” refers to a group comprising a reactive functional group capable of chemically attaching to specific functional groups (e.g., primary amines, sulfhydryls) on proteins or other molecules. A “moiety capable of a chemoselective reaction with an amino acid,” as used herein refers to a moiety comprising a reactive functional group capable of chemically attaching to a functional group of a natural or non-natural amino acid (e.g., primary and secondary amines, sulfhydryls, alcohols, carboxyl groups, carbonyls, or triazole forming functional groups such as azides or alkynes). Examples of cross-linking groups include sulfhydryl-reactive cross-linking groups (e.g., groups comprising maleimides, haloacetyls, pyridyldisulfides, thiosulfonates, or vinylsulfones), amine-reactive cross-linking groups (e.g., groups comprising esters such as NHS esters, imidoesters, and pentafluorophenyl esters, or hydroxymethylphosphine), carboxyl-reactive cross-linking groups (e.g., groups comprising primary or secondary amines, alcohols, or thiols), carbonyl-reactive cross-linking groups (e.g., groups comprising hydrazides or alkoxyamines), and triazole-forming cross-linking groups (e.g., groups comprising azides or alkynes).

As used herein, the term “dosage form” refers to a physically discrete unit of a compound (e.g., a compound of the present invention) for administration to a subject. Each unit contains a predetermined quantity of compound. 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). Those of ordinary skill in the art appreciate that the total amount of a therapeutic composition or compound administered to a particular subject is determined by one or more attending physicians and may involve administration of multiple dosage forms. As used herein, the term “dosing regimen” refers 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 compound (e.g., a compound of the present invention) 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 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).

The term “including” is used to mean “including but not limited to.” “Including” and “including but not limited to” are used interchangeably.

As used herein, the term “inhibitor” refers to a compound that i) inhibits, decreases or reduces the effects of a target protein (e.g., a eukaryotic target protein such as a mammalian target protein or a fungal target protein or a prokaryotic target protein such as a bacterial target protein); and/or ii) inhibits, decreases, reduces, or delays one or more biological events. An inhibitor may be direct (in which case it exerts its influence directly upon its target) or indirect (in which case it exerts its influence by other than binding to its target; e.g., by interacting with a regulator of the target protein (e.g., a eukaryotic target protein such as a mammalian target protein or a fungal target protein or a prokaryotic target protein such as a bacterial target protein), for example so that level or activity of the target protein is altered).

Unless otherwise stated, structures depicted herein are also meant to include compounds or amino acids that differ only in the presence of one or more isotopically enriched atoms. Exemplary isotopes that can be incorporated into compounds of the present disclosure include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, chlorine, and iodine, such as ²H, ³H, ¹¹C, ¹³C, ¹⁴C, ¹³N, ¹⁵N, ¹⁵O, ¹⁷O, ¹⁸0, ³²P, ³³P, ³⁵S, ¹⁸F, ³⁶CI, ¹²³l and ¹²⁵l. Isotopically-labeled compounds (e.g., those labeled with ³H and ¹⁴C) can be useful in compound or substrate tissue distribution assays. Tritiated (i.e., ³H) and carbon-14 (i.e., ¹⁴C) isotopes can be useful for their ease of preparation and detectability. Further, substitution with heavier isotopes such as deuterium (i.e., ²H) may afford certain therapeutic advantages resulting from greater metabolic stability (e.g., increased in vivo half-life or reduced dosage requirements). In some embodiments, one or more hydrogen atoms are replaced by ²H or ³H, or one or more carbon atoms are replaced by ¹³C- or ¹⁴C-enriched carbon. Positron emitting isotopes such as ¹⁵O, ¹³N, ¹¹C, and ¹⁸F are useful for positron emission tomography (PET) studies to examine substrate receptor occupancy. Preparations of isotopically labelled compounds are known to those of skill in the art. For example, isotopically labeled compounds can generally be prepared by following procedures analogous to those disclosed for compounds of the present disclosure described herein, by substituting an isotopically labeled reagent for a non-isotopically labeled reagent. In some embodiments, one or more carbon atoms in the amino acids of the peptide conjugates as disclosed herein can be replaced by ¹³C atoms and/or one or more nitrogen atoms in the amino acids of the peptide conjugates as disclosed herein can be replaced by ¹⁵N atoms.

The term “isolated” refers to an object (e.g., peptide) that is removed from its natural environment (e.g., separated). “Isolated” objects are at least 50% free, preferably 75% free, more preferably at least 90% free, and most preferably at least 95% (e.g., 95%, 96%, 97%, 98%, or 99%) free from other components with which they are naturally associated.

The term “complex” refers to a peptide conjugate comprising: a peptide and a chemical fragment of a selective covalent inhibitor, presented by a major histocompatibility complex (MHC). For example, the peptide conjugate can be formed by the covalent reaction of a selective covalent inhibitor with a residue (e.g., a cysteine residue) in a peptide. In some embodiments, the peptide conjugate is formed by the covalent reaction of a RAS inhibitor with a KRAS^G12C peptide. In some embodiments, the peptide conjugate is formed by the covalent reaction of a RAS inhibitor with a KRAS^G12D peptide.

As used herein the term “KRAS^G12C” refers to the KRAS protein with a G12C mutation, i.e., a cysteine at amino acid position 12.

As used herein the term “KRAS^G12D” refers to the KRAS protein with a G12D mutation, i.e., an aspartic acid at amino acid position 12.

As used herein, the term “linker” refers to a divalent organic moiety connecting a first moiety (e.g., one portion of a macrocycle) to a second moiety (e.g., an amino acid residue of a peptide).

The term "mutation" as used herein indicates any modification of a nucleic acid or polypeptide which results in an altered nucleic acid or polypeptide. The term "mutation" may include, for example, point mutations, deletions or insertions of single or multiple residues in a polynucleotide, which includes alterations arising within a protein-encoding region of a gene as well as alterations in regions outside of a protein-encoding sequence, such as, but not limited to, regulatory or promoter sequences, as well as amplifications or chromosomal breaks or translocations. In particular embodiments, the mutation results in an amino acid substitution in the encoded protein.

As used herein, the term “mutant RAS protein” means a RAS protein (e.g., KRAS, NRAS, HRAS) that comprises at least one mutation in which a non-aspartic acid amino acid in the corresponding wildtype RAS protein is mutated to an aspartic acid.

The term “peptide,” as used herein, refers broadly to a sequence of two or more amino acids joined together by peptide bonds. It should be understood that this term does not connote a specific length of a polymer of amino acids, nor is it intended to imply or distinguish whether the polypeptide is produced using recombinant techniques, chemical or enzymatic synthesis, or is naturally occurring.

A “peptide conjugate” as used herein means any protein or peptide that has been modified so that it is covalently conjugated to another molecule.

As used herein, the term “peptide conjugate-MHC complex” or “peptide conjugate/MHC complex” refers to a peptide conjugate comprising: a peptide and a chemical fragment of a targeted covalent inhibitor, presented by a major histocompatibility complex (MHC). For example, the peptide conjugate can be formed by the covalent reaction of a targeted covalent inhibitor with a residue (e.g., a cysteine residue) in a peptide. In some embodiments, the peptide conjugate is formed by the covalent reaction of covalent tri-complex RAS inhibitor with a KRAS^G12D peptide. As used herein, the term “pharmaceutical composition” refers to an active compound, formulated together with one or more pharmaceutically acceptable carriers. In some embodiments, active compound is present in unit dose amount appropriate for administration in a therapeutic 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.

A “pharmaceutically acceptable excipient,” as used herein, refers to any inactive ingredient (for example, a vehicle capable of suspending or dissolving the active compound) having the properties of being nontoxic and non-inflammatory in a subject. Typical excipients include, for example: antiadherents, antioxidants, binders, coatings, compression aids, disintegrants, dyes (colors), emollients, emulsifiers, fillers (diluents), film formers or coatings, flavors, fragrances, glidants (flow enhancers), lubricants, preservatives, printing inks, sorbents, suspensing or dispersing agents, sweeteners, or waters of hydration. Excipients include, but are not limited to: butylated optionally substituted hydroxyltoluene (BHT), calcium carbonate, calcium phosphate (dibasic), calcium stearate, croscarmellose, crosslinked polyvinyl pyrrolidone, citric acid, crospovidone, cysteine, ethylcellulose, gelatin, optionally substituted hydroxylpropyl cellulose, optionally substituted hydroxylpropyl methylcellulose, lactose, magnesium stearate, maltitol, mannitol, methionine, methylcellulose, methyl paraben, microcrystalline cellulose, polyethylene glycol, polyvinyl pyrrolidone, povidone, pregelatinized starch, propyl paraben, retinyl palmitate, shellac, silicon dioxide, sodium carboxymethyl cellulose, sodium citrate, sodium starch glycolate, sorbitol, starch (corn), stearic acid, stearic acid, sucrose, talc, titanium dioxide, vitamin A, vitamin E, vitamin C, and xylitol. Those of ordinary skill in the art are familiar with a variety of agents and materials useful as excipients. See, e.g., Ansel, et al., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems. Philadelphia: Lippincott, Williams & Wilkins, 2004; Gennaro, et al., Remington: The Science and Practice of Pharmacy. Philadelphia: Lippincott, Williams & Wilkins, 2000; and Rowe, Handbook of Pharmaceutical Excipients. Chicago, Pharmaceutical Press, 2005. In some embodiments, a composition includes at least two different pharmaceutically acceptable excipients.

The term “pharmaceutically acceptable salt,” as use herein, refers to those salts of the compounds described here that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and 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, pharmaceutically acceptable salts are described in: Berge et al., J. Pharmaceutical Sciences 66:1-19, 1977 and in Pharmaceutical Salts: Properties, Selection, and Use, (Eds. P.H. Stahl and C.G. Wermuth), Wiley-VCH, 2008. The salts can be prepared in situ during the final isolation and purification of the compounds described herein or separately by reacting the free base group with a suitable organic acid.

The term “RAS protein” means a protein from the RAS family of related GTPase proteins including KRAS, HRAS, and NRAS. A RAS protein may be a wild-type protein or a mutant protein. In some embodiments, a RAS protein is not a wild-type protein.

KRAS is encoded by the KRAS gene. The term “KRAS” also refers to natural variants of the wildtype KRAS protein, such as proteins having at least 85% identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9% identity, or more) to the amino acid sequence of wild-type KRAS, which is set forth in SEQ ID NO: 11 .

SEQ ID NO: 11

MTEYKLVWG AGGVGKSALT IQLIQNHFVD EYDPTIEDSY RKQVVIDGET

CLLDILDTAG QEEYSAMRDQ YMRTGEGFLC VFAINNTKSF EDIHHYREQI

KRVKDSEDVP MVLVGNKCDL PSRTVDTKQA QDLARSYGIP FIETSAKTRQ

RVEDAFYTLV REIRQYRLKK ISKEEKTPGC VKIKKCIIM

HRAS is encoded by the HRAS gene. The term “HRAS” also refers to natural variants of the wildtype HRAS protein, such as proteins having at least 85% identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9% identity, or more) to the amino acid sequence of wild-type HRAS, which is set forth in SEQ ID NO: 12.

SEQ ID NO: 12

MTEYKLVWG AGGVGKSALT IQLIQNHFVD EYDPTIEDSY RKQVVIDGET

CLLDILDTAG QEEYSAMRDQ YMRTGEGFLC VFAINNTKSF EDIHQYREQI

KRVKDSDDVP MVLVGNKCDL AARTVESRQA QDLARSYGIP YIETSAKTRQ

GVEDAFYTLV REIRQHKLRK LNPPDESGPG CMSCKCVLS

NRAS is encoded by the NRAS gene. The term “NRAS” also refers to natural variants of the wildtype NRAS protein, such as proteins having at least 85% identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9% identity, or more) to the amino acid sequence of wild-type NRAS, which is set forth in SEQ ID NO: 13.

SEQ ID NO: 13

MTEYKLVWG AGGVGKSALT IQLIQNHFVD EYDPTIEDSY RKQVVIDGET

CLLDILDTAG QEEYSAMRDQ YMRTGEGFLC VFAINNSKSF ADINLYREQI

KRVKDSDDVP MVLVGNKCDL PTRTVDTKQA HELAKSYGIP FIETSAKTRQ

GVEDAFYTLV REIRQYRMKK LNSSDDGTQG CMGLPCVVM

The terms “sequence identity,” “percent identity,” “percent homology,” and, for example, comprising a “sequence 80% identical to,” as used herein, refer to the extent that sequences are identical on a nucleotide-by-nucleotide basis or an amino acid-by-amino acid basis over a window of comparison. Thus, a “percentage of sequence identity” may be calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, I) or the identical amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Vai, Leu, He, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gin, Cys and Met) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity.

The term “presenter protein” refers to a protein that binds to a small molecule to form a complex that binds to and modulates the activity of a target protein (e.g., a eukaryotic target protein such as a mammalian target protein or a fungal target protein or a prokaryotic target protein such as a bacterial target protein). In some embodiments, the presenter protein is a relatively abundant protein (e.g., the presenter protein is sufficiently abundant that participation in a ternary complex does not substantially impact the biological role of the presenter protein in a cell and/or viability or other attributes of the cell). In certain embodiments, the presenter protein is a protein that has chaperone activity within a cell. In some embodiments, the presenter protein is a protein that has multiple natural interaction partners within a cell. In certain embodiments, the presenter protein is one which is known to bind a small molecule to form a binary complex that is known to or suspected of binding to and modulating the biological activity of a target protein. In some embodiments, the presenter protein is cyclophilin A (CYPA).

The terms “prevent” and “preventing” with regard to a subject refer to keeping a disease or disorder from afflicting the subject. Preventing includes prophylactic treatment. For instance, preventing can include administering to the subject a compound disclosed herein before a subject is afflicted with a disease and the administration will keep the subject from being afflicted with the disease.

The terms "RAS inhibitor" and "inhibitor of [a] RAS" are used interchangeably to refer to any inhibitor that targets, that is, selectively binds to or inhibits a RAS protein. In some embodiments, these terms include RAS(OFF) and RAS(ON) inhibitors. In some embodiments, RAS inhibitor and RAS tricomplex inhibitor are used interchangeably.

As used herein, the term “RAS(ON) inhibitor” refers to an inhibitor that targets, that is, selectively binds to or inhibits, the GTP-bound, active state of RAS (e.g., selective over the GDP-bound, inactive state of RAS). Inhibition of the GTP-bound, active state of RAS includes, for example, the inhibition of oncogenic signaling from the GTP-bound, active state of RAS. In some embodiments, the RAS(ON) inhibitor is an inhibitor that selectively binds to and inhibits the GTP-bound, active state of RAS. In certain embodiments, RAS(ON) inhibitors may also bind to or inhibit the GDP-bound, inactive state of RAS (e.g., with a lower affinity or inhibition constant than for the GTP-bound, active state of RAS).

As used herein, the term “RAS(OFF) inhibitor” refers to an inhibitor that targets, that is, selectively binds to or inhibits the GDP-bound, inactive state of RAS (e.g., selective over the GTP-bound, active state of RAS). Inhibition of the GDP-bound, inactive state of RAS includes, for example, sequestering the inactive state by inhibiting the exchange of GDP for GTP, thereby inhibiting RAS from adopting the active conformation. In certain embodiments, RAS(OFF) inhibitors may also bind to or inhibit the GTP-bound, active state of RAS (e.g., with a lower affinity or inhibition constant than for the GDP-bound, inactive state of RAS).

As used herein, the terms “react” and “reacting” refer to a process in which atoms of the same or different elements rearrange themselves to form a new substance. For example, the formation of a covalent bond between two atoms such as the reaction between a reactive amino acid on a protein and a cross-linking group to form a covalent bond. A reaction may be measured by any method known in the art, for example, formation of a reaction product can be determined by LC-MS or NMR. As used herein, the term “subject,” patient,” and “individual” may be used interchangeably and refer to any member of the animal kingdom. In some embodiments, “subject” refers to humans, at any stage of development. In some embodiments, “subject” refers to a human patient. In some embodiments, “subject” refers to non-human animals. In some embodiments, the non-human animal is a mammal (e.g., a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a sheep, cattle, a primate, or a pig). In some embodiments, subjects include, but are not limited to, mammals, birds, reptiles, amphibians, fish, or worms. In some embodiments, a subject may be a transgenic animal, genetically-engineered animal, or a clone.

The term “target protein” refers to any protein that participates in a biological pathway associated with a disease, disorder or condition. In some embodiments, the target protein is not mTOR or calcineurin. In some embodiments, the target protein is capable of forming a tripartite complex with a presenter protein and a small molecule. In some embodiments, a target protein is a naturally-occurring protein; in some such embodiments, a target protein is naturally found in certain mammalian cells (e.g., a mammalian target protein), fungal cells (e.g., a fungal target protein), bacterial cells (e.g., a bacterial target protein) or plant cells (e.g., a plant target protein). In some embodiments, a target protein is characterized by natural interaction with one or more natural presenter protein/natural small molecule complexes. In some embodiments, a target protein is characterized by natural interactions with a plurality of different natural presenter protein/natural small molecule complexes; in some such embodiments some or all of the complexes utilize the same presenter protein (and different small molecules). In some embodiments, a target protein does not substantially bind to a complex of cyclosporin, rapamycin, or FK506 and a presenter protein (e.g., FKBP). Target proteins can be naturally occurring, e.g., wild-type. Alternatively, the target protein can vary from the wild-type protein but still retain biological function, e.g., as an allelic variant, a splice mutant or a biologically active fragment. Exemplary mammalian target proteins are GTPases, GTPase activating protein, Guanine nucleotide-exchange factor, heat shock proteins, ion channels, coiled-coil proteins, kinases, phosphatases, ubiquitin ligases, transcription factors, chromatin modifier/remodelers, proteins with classical protein-protein interaction domains and motifs, or any other proteins that participate in a biological pathway associated with a disease, disorder or condition.

The term “tri-complex” as used herein means a high affinity three-component complex, or conjugate, between a synthetic ligand and two intracellular proteins which do not interact under normal physiological conditions: the target protein of interest (e.g., RAS), and a widely expressed cytosolic chaperone (presenter protein) in the cell (e.g., cyclophilin A). More specifically, in some embodiments, the inhibitors of RAS described herein induce a new binding pocket in RAS by driving formation of a high affinity tri-complex, or conjugate, between the RAS protein and the widely expressed cytosolic chaperone, cyclophilin A (CYPA). Without being bound by theory, it is postulated that non-covalent interactions of a RAS inhibitor as disclosed herein with RAS and the chaperone protein (e.g., cyclophilin A) may contribute to the inhibition of RAS activity. For example, van der Waals, hydrophobic, hydrophilic and hydrogen bond interactions, and combinations thereof, may contribute to the ability of the RAS inhibitor disclosed herein to form complexes and act as RAS inhibitors.

A “therapeutic regimen” refers to a dosing regimen whose administration across a relevant population is correlated with a desired or beneficial therapeutic outcome. The term “therapeutically effective amount” means an amount that is sufficient, when administered to a population suffering from or susceptible to a disease, disorder, and/or condition in accordance with a therapeutic dosing regimen, to treat the 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 art 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 that amount that provides a particular desired pharmacological response in a significant number of subjects when administered to patients in need of such treatment. It is specifically understood that particular subjects may, in fact, be “refractory” to a “therapeutically effective amount.” To give but one example, a refractory subject may have a low bioavailability such that clinical efficacy is not obtainable. In some embodiments, reference to a therapeutically effective amount may be a reference to an amount 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, etc.). Those of ordinary skill in the art will appreciate that, in some embodiments, a therapeutically effective amount may be formulated and/or administered in a single dose. In some embodiments, a therapeutically effective amount may be formulated and/or administered in a plurality of doses, for example, as part of a dosing regimen.

The terms “treatment,” “treat,” and “treating,” in their broadest sense, refer to any administration of a substance (e.g., provided compositions) that partially or completely alleviates, ameliorates, relives, inhibits, delays onset of, reduces severity of, and/or reduces incidence of one or more symptoms, features, and/or causes of a particular disease, disorder, and/or condition. In some embodiments, such treatment may be administered to a subject who does not exhibit signs of the relevant disease, disorder and/or condition and/or of a subject who exhibits only early signs of the disease, disorder, and/or condition. Alternatively, or additionally, in some embodiments, treatment may be administered to a subject who exhibits one or more established signs of the relevant disease, disorder and/or condition. In some embodiments, treatment may be of a subject who has been diagnosed as suffering from the relevant disease, disorder, and/or condition. In some embodiments, treatment may be of a subject known to have one or more susceptibility factors that are statistically correlated with increased risk of development of the relevant disease, disorder, and/or condition. In some embodiments, treating cancer comprises delating growth of a tumor. In other embodiments, treating cancer comprises shrinking the size of a tumor or otherwise reducing viable cancer cell numbers.

The term “variant” refers to an entity that shows significant structural identity with a reference entity but differs structurally from the reference entity in the presence or level of one or more chemical moieties as compared with the reference entity. In many embodiments, a variant also differs functionally from its reference entity. In general, whether a particular entity is properly considered to be a “variant” of a reference entity is based on its degree of structural identity with the reference entity. As will be appreciated by those skilled in the art, any biological or chemical reference entity has certain characteristic structural elements. A variant, by definition, is a distinct chemical entity that shares one or more such characteristic structural elements. To give but a few examples, a small molecule may have a characteristic core structural element (e.g., a hexahydropyridazine core) and/or one or more characteristic pendent moieties so that a variant of the small molecule is one that shares the core structural element and the characteristic pendent moieties but differs in other pendent moieties and/or in types of bonds present (single vs double, E vs Z, etc.) within the core, a polypeptide may have a characteristic sequence element comprised of a plurality of amino acids having designated positions relative to one another in linear or three-dimensional space and/or contributing to a particular biological function, a nucleic acid may have a characteristic sequence element comprised of a plurality of nucleotide residues having designated positions relative to one another in linear or three-dimensional space. For example, a variant polypeptide may differ from a reference polypeptide as a result of one or more differences in amino acid sequence and/or one or more differences in chemical moieties (e.g., carbohydrates, lipids, etc.) covalently attached to the polypeptide backbone. In some embodiments, a variant polypeptide shows an overall sequence identity with a reference polypeptide that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, or 99%. Alternatively or additionally, in some embodiments, a variant polypeptide does not share at least one characteristic sequence element with a reference polypeptide. In some embodiments, the reference polypeptide has one or more biological activities. In some embodiments, a variant polypeptide shares one or more of the biological activities of the reference polypeptide. In some embodiments, a variant polypeptide lacks one or more of the biological activities of the reference polypeptide. In some embodiments, a variant polypeptide shows a reduced level of one or more biological activities as compared with the reference polypeptide. In many embodiments, a polypeptide of interest is considered to be a “variant” of a parent or reference polypeptide if the polypeptide of interest has an amino acid sequence that is identical to that of the parent but for a small number of sequence alterations at particular positions. Typically, fewer than 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% of the residues in the variant are substituted as compared with the parent. In some embodiments, a variant has 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 substituted residue as compared with a parent. Often, a variant has a very small number (e.g., fewer than 5, 4, 3, 2, or 1) number of substituted functional residues (i.e., residues that participate in a particular biological activity). Furthermore, a variant typically has not more than 5, 4, 3, 2, or 1 additions or deletions, and often has no additions or deletions, as compared with the parent. Moreover, any additions or deletions are typically fewer than about 25, about 20, about 19, about 18, about 17, about 16, about 15, about 14, about 13, about 10, about 9, about 8, about 7, about 6, and commonly are fewer than about 5, about 4, about 3, or about 2 residues. In some embodiments, the parent or reference polypeptide is one found in nature. As will be understood by those of ordinary skill in the art, a plurality of variants of a particular polypeptide of interest may commonly be found in nature.

The term “wild-type” refers to an entity having a structure and/or activity as found in nature in a “normal” (as contrasted with mutant, diseased, altered, etc.) state or context. Those of ordinary skill in the art will appreciate that wild-type genes and polypeptides often exist in multiple different forms (e.g., alleles).

Peptide Conjugates

In one aspect, the disclosure provides an isolated peptide conjugate, which is formed by covalently linking a peptide and a RAS tri-complex inhibitor, also known as a RAS inhibitor as described herein. In some embodiments, the peptide conjugate is formed by reacting a peptide and a RAS tri- complex inhibitor, wherein the peptide is derived from RAS. In one embodiment, the peptide conjugated is formed by directly linking a peptide to a RAS inhibitor. In another embodiment, the peptide conjugated is formed by covalently linking a peptide to a RAS inhibitor through a linker.

In some embodiments, the disclosure provides a peptide conjugate, wherein the RAS protein is KRAS, HRAS, NRAS, R-RAS or M-RAS. In other embodiments, the RAS is KRAS, HRAS or NRAS. In one embodiment, RAS is KRAS. In another embodiment, RAS is HRAS. In one embodiment, RAS is KRAS. In another embodiment, RAS is NRAS.

In some embodiments, the RAS has a mutation. In one embodiment, the RAS is KRAS having a mutation. In another embodiment, RAS is HRAS having a mutation. In another embodiment, RAS is NRAS having a mutation. In some embodiments, the RAS has a G12D mutation.

In some embodiments, the RAS is KRAS with a G12D mutation.

In other embodiments, the RAS is HRAS with a G12D mutation.

In other embodiments, the RAS is NRAS with a G12D mutation.

In some embodiments, the RAS inhibitor selectively inhibits a RAS mutant protein over a wildtype RAS protein. In some embodiments, the RAS inhibitor as described herein exhibits increased inhibitory activity over a RAS mutant over a wild-type RAS protein. In some embodiments, the activity is an in vitro or an in vivo activity, e.g., an in vivo or an in vitro activity described herein. In certain embodiments, the RAS inhibitor exhibits about 1 , about 1 .5, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11 , about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 120, about 140, about 160, about 180, about 200, about 300, about 400, about 500, about 600, about 700, about 800, about 800, or about 1000-fold greater inhibitory activity over a wild-type RAS protein. In some embodiments, the RAS inhibitor as described herein exhibits at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99% or greater than 99%, 100%, 200% 300%, 400%, 500%, 700%, or 1000% greater inhibitor activity over a wild-type RAS protein.

In some embodiments, the peptide comprises an aspartic acid residue.

In some embodiments, the peptide is a segment of a RAS protein. In some embodiments, the peptide comprises a segment of KRAS^G12D, HRAS^G12D, or NRAS^G12D.

In some embodiments, the peptide conjugates are formed by covalently linking a RAS inhibitor (e.g., a targeted covalent RAS^G12D inhibitor) as described herein to an Asp residue of a peptide.

In some embodiments, the peptide conjugate has formula (IV): where X2 is hydrogen or a peptide segment, X1 is a peptide segment or OH, and the RAS inhibitor moiety is covalently linked to the carboxy group of the Asp residue either directly or through a linker, wherein L¹ is a bond or a linker. In some embodiments of formula (I), L¹ is a bond, and the RAS inhibitor moiety is covalently linked to the carboxy group of the Asp residue to form an ester bond.

In some embodiments of formula (I), L¹ is a linker, and the RAS inhibitor moiety is covalently linked to the carboxy group of the Asp residue through a linker to form an amide bond. In one embodiment, the linker i , wherein the single wavy line is connected to the RAS inhibitor moiety and the double wavy line is connected to the carboxy group of the Asp residue of the peptide.

In some embodiments, the peptide conjugate has formula (III): wherein X1 is hydrogen or a peptide comprising from 1 to 50 amino acids; X2 is OH or a peptide comprising from 1 to 50 amino acids; and R¹ is hydrogen or cyclopropyl.

In some embodiments, the RAS inhibitor moiety in formula (IV) has formula (V): where R¹ is hydrogen or C3-6 cycloalkyl. In some embodiments, R¹ is hydrogen or cyclopropyl.

In some embodiments, the RAS inhibitor moiety has formula (V), wherein R¹ is hydrogen. In other embodiments, the RAS inhibitor moiety has formula (V), wherein R¹ is cyclopropyl.

In some embodiments of formula (IV), X2 is hydrogen and X1 is a peptide (e.g., a peptide derived from RAS) having from 1 to 30 amino acids in length or an isotopically labeled analog thereof. In other embodiments, X2 is a peptide having from 1 to 30 amino acids in length or an isotopically labeled analog thereof, and X1 is OH. In other embodiments, X2 is a peptide (e.g., a peptide derived from RAS) having from 1 to 30 amino acids in length or an isotopically labeled analog thereof, and X1 is a peptide (e.g., a peptide derived from RAS) having from 1 to 30 amino acids in length or an isotopically labeled analog thereof. In some embodiments, neither X1 nor X2 contains an Asp residue.

In some embodiments, when X2 is hydrogen, then X1 is not OH. In other embodiments, when X1 is OH, then X2 is not hydrogen.

In some embodiments of formula (IV), X2 is hydrogen and X1 is a peptide comprising amino acid sequence GVGKSALTI (SEQ ID NO: 14), or an isotopically labeled analog thereof.

In some embodiments of formula (IV), X2 is Ala or an isotopically labeled analog thereof and X1 is a peptide comprising amino acid sequence GVGKSALT (SEQ ID NO: 15), or an isotopically labeled analog thereof.

In some embodiments of formula (IV), X2 is Gly-Ala, or an isotopically labeled analog thereof and X1 is a peptide comprising amino acid sequence GVGKSAL (SEQ ID NO: 16), or an isotopically labeled analog thereof.

In some embodiments of formula (IV), X2 is VGA or an isotopically labeled analog thereof, and X1 is a peptide comprising amino acid sequence GVGKSA (SEQ ID NO: 17) or an isotopically labeled analog thereof.

In some embodiments of formula (IV), X2 is VVGA (SEQ ID NO: 18) or an isotopically labeled analog thereof, and X1 is a peptide comprising amino acid sequence GVGK (SEQ ID NO: 19) or an isotopically labeled analog thereof.

In some embodiments of formula (IV), X2 is VVGA (SEQ ID NO: 18) or an isotopically labeled analog thereof, and X1 is a peptide comprising amino acid sequence GVGKS (SEQ ID NO: 20) or an isotopically labeled analog thereof.

In some embodiments of formula (IV), X2 is VVVGA (SEQ ID NO: 21) or an isotopically labeled analog thereof, and X1 is a peptide comprising amino acid sequence GVGK (SEQ ID NO: 19) or an isotopically labeled analog thereof.

In some embodiments of formula (IV), X2 is KLVVVGA (SEQ ID NO: 22) or an isotopically labeled analog thereof, and X1 is a peptide comprising amino acid sequence GV or an isotopically labeled analog thereof.

In some embodiments of formula (IV), X2 is YKLVVVGA (SEQ ID NO: 23) or an isotopically labeled analog thereof, and X1 is a peptide comprising amino acid residue Gly or an isotopically labeled analog thereof.

In some embodiments of formula (IV), X2 is EYKLVWGA (SEQ ID NO: 24) or an isotopically labeled analog thereof, and X1 is OH.

In some embodiments, the peptide of the peptide conjugates comprises a segment of KRAS^G12D, HRAS^G12D, or NRAS^G12D. In one embodiment, the peptide comprises a segment of KRAS^G12D. In some embodiments, the peptide comprises a segment of KRAS^G12D which has an amino acid sequence comprising 1 to 50 amino acids, 2 to 50 amino acids, 1 to 30 amino acids, 7 to 30 amino acids or 10 to 30 amino acids. In one embodiment, the peptide comprises a segment of KRAS^G12D which has an amino acid sequence comprising 10 amino acids. In some embodiments, the peptide comprises a segment of KRAS^G12D which has an amino acid sequence comprising 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids. In some embodiments, the peptide comprises a segment of HRAS^G12D which has an amino acid sequence comprising 1 to 50 amino acids, 2 to 50 amino acids, 1 to 30 amino acids, 7 to 30 amino acids or 10 to 30 amino acids. In one embodiment, the peptide comprises a segment of HRAS^G12D which has an amino acid sequence comprising 10 amino acids. In some embodiments, the peptide comprises a segment of HRAS^G12D which has an amino acid sequence comprising 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids.

In some embodiments, the peptide comprises a segment of NRAS^G12D which has an amino acid sequence comprising 1 to 50 amino acids, 2 to 50 amino acids, 1 to 30 amino acids, 7 to 30 amino acids or 10 to 30 amino acids. In one embodiment, the peptide comprises a segment of NRAS^G12D which has an amino acid sequence comprising 10 amino acids. In some embodiments, the peptide comprises a segment of NRAS^G12D which has an amino acid sequence comprising 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids.

In some embodiments, the RAS inhibitor is a HRAS inhibitor, a KRAS inhibitor and/or a NRAS inhibitor. In one embodiment, the RAS inhibitor is a KRAS inhibitor. In another embodiment, the RAS inhibitor is an HRAS inhibitor. In another embodiment, the RAS inhibitor is a NRAS inhibitor. In another embodiment, the RAS inhibitor is an inhibitor of KRAS, HRAS and NRAS.

In some embodiments, the disclosure provides a peptide conjugate, wherein the RAS inhibitor is a KRAS inhibitor having formulas (IA), (HA), (IIIA), (IIIA-1), (IVA) or (IVA-1):

In some embodiments, the disclosure provides a peptide conjugate, wherein the peptide comprises an amino acid sequence of DGVGKSALTI (SEQ ID NO: 1), ADGVGKSALT (SEQ ID NO: 2), GADGVGKSAL (SEQ ID NO: 3), VGADGVGKSA (SEQ ID NO: 4), WGADGVGK (SEQ ID NO: 5), VVGADGVGKS (SEQ ID NO: 6), VWGADGVGK (SEQ ID NO: 7), KLVVVGADGV (SEQ ID NO: 8), YKLVVVGADG (SEQ ID NO: 9), or EYKLVVVGAD (SEQ ID NO: 10), or an isotopically labeled analog thereof. In some embodiments, the peptide has between about 7 to about 30 amino acids in length.

In some embodiments, the disclosure provides a peptide conjugate, wherein the peptide comprises an amino acid sequence of at least 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55% or 50% identical to DGVGKSALTI (SEQ ID NO: 1), ADGVGKSALT (SEQ ID NO: 2), GADGVGKSAL (SEQ ID NO: 3), VGADGVGKSA (SEQ ID NO: 4), WGADGVGK (SEQ ID NO: 5), VVGADGVGKS (SEQ ID NO: 6), VWGADGVGK (SEQ ID NO: 7), KLVVVGADGV (SEQ ID NO: 8), YKLVVVGADG (SEQ ID NO: 9), or EYKLVVVGAD (SEQ ID NO: 10), or an isotopically labeled analog thereof.

In some embodiments, the disclosure provides a peptide conjugate, wherein the peptide comprises an amino acid sequence of at least 80% identical to DGVGKSALTI (SEQ ID NO: 1), ADGVGKSALT (SEQ ID NO: 2), GADGVGKSAL (SEQ ID NO: 3), VGADGVGKSA (SEQ ID NO: 4), WGADGVGK (SEQ ID NO: 5), VVGADGVGKS (SEQ ID NO: 6), VWGADGVGK (SEQ ID NO: 7), KLWVGADGV (SEQ ID NO: 8), YKLVVVGADG (SEQ ID NO: 9), or EYKLVVVGAD (SEQ ID NO: 10), or an isotopically labeled analog thereof.

In some embodiments, the disclosure provides a peptide conjugate, wherein the peptide comprises an amino acid sequence having at least 70% sequence identity to DGVGKSALTI (SEQ ID NO: 1), ADGVGKSALT (SEQ ID NO: 2), GADGVGKSAL (SEQ ID NO: 3), VGADGVGKSA (SEQ ID NO: 4), WGADGVGK (SEQ ID NO: 5), VVGADGVGKS (SEQ ID NO: 6), WVGADGVGK (SEQ ID NO: 7), KLVVVGADGV (SEQ ID NO: 8), YKLWVGADG (SEQ ID NO: 9), or EYKLVWGAD (SEQ ID NO: 10), or an isotopically labeled analog thereof.

In some embodiments, the disclosure provides a peptide conjugate, wherein the peptide comprises an amino acid sequence of DGVGKSALTI (SEQ ID NO: 1), or an isotopically labeled analog thereof. In one embodiment, the peptide comprises an amino acid sequence having at least 80% sequence identity to DGVGKSALTI (SEQ ID NO: 1).

In some embodiments, the disclosure provides a peptide conjugate, wherein the peptide comprises an amino acid sequence of ADGVGKSALT (SEQ ID NO: 2), or an isotopically labeled analog thereof. In one embodiment, the peptide comprises an amino acid sequence of at least 80% identical to ADGVGKSALT (SEQ ID NO: 2).

In some embodiments, the disclosure provides a peptide conjugate, wherein the peptide comprises an amino acid sequence of GADGVGKSAL (SEQ ID NO: 3), or an isotopically labeled analog thereof. In one embodiment, the peptide comprises an amino acid sequence of at least 80% identical to GADGVGKSAL (SEQ ID NO: 3).

In some embodiments, the disclosure provides a peptide conjugate, wherein the peptide comprises an amino acid sequence of VGADGVGKSA (SEQ ID NO: 4), or an isotopically labeled analog thereof. In one embodiment, the peptide comprises an amino acid sequence of at least 80% identical to VGADGVGKSA (SEQ ID NO: 4).

In some embodiments, the disclosure provides a peptide conjugate, wherein the peptide comprises an amino acid sequence of WGADGVGK (SEQ ID NO: 5), or an isotopically labeled analog thereof. In one embodiment, the peptide comprises an amino acid sequence of at least 80% identical to VVGADGVGK (SEQ ID NO: 5).

In some embodiments, the disclosure provides a peptide conjugate, wherein the peptide comprises an amino acid sequence of VVGADGVGKS (SEQ ID NO: 6), or an isotopically labeled analog thereof. In one embodiment, the peptide comprises an amino acid sequence of at least 80% identical to VVGADGVGKS (SEQ ID NO: 6).

In some embodiments, the disclosure provides a peptide conjugate, wherein the peptide comprises an amino acid sequence of VWGADGVGK (SEQ ID NO: 7), or an isotopically labeled analog thereof. In one embodiment, the peptide comprises an amino acid sequence of at least 80% identical to WVGADGVGK (SEQ ID NO: 7).

In some embodiments, the disclosure provides a peptide conjugate, wherein the peptide comprises an amino acid sequence of KLVWGADGV (SEQ ID NO: 8), or an isotopically labeled analog thereof. In one embodiment, the peptide comprises an amino acid sequence of at least 80% identical to KLWVGADGV (SEQ ID NO: 8).

In some embodiments, the disclosure provides a peptide conjugate, wherein the peptide comprises an amino acid sequence of YKLWVGADG (SEQ ID NO: 9), or an isotopically labeled analog thereof. In one embodiment, the peptide comprises an amino acid sequence of at least 80% identical to YKLWVGADG (SEQ ID NO: 9).

In some embodiments, the disclosure provides a peptide conjugate, wherein the peptide comprises an amino acid sequence of EYKLVVVGAD (SEQ ID NO: 10), or an isotopically labeled analog thereof. In one embodiment, the peptide comprises an amino acid sequence of at least 80% identical to EYKLVVVGAD (SEQ ID NO: 10).

In some embodiments, the disclosure provides peptide conjugates shown in Table 1 , or a pharmaceutically acceptable salt thereof.

Table 1 : Certain Compounds of the Present Invention

In some embodiments, the disclosure provides peptide conjugates shown in Table 2, or a pharmaceutically acceptable salt thereof.

Table 2: Certain Compounds of the Present Invention

In an aspect, provided herein is a cell-free peptide conjugate comprising: a) a chemical moiety having the formula: covalently bonded to the aspartic acid residue in a peptide comprising the amino acid sequence DGVGKSALTI (SEQ ID NO: 1), ADGVGKSALT (SEQ ID NO: 2), GADGVGKSAL (SEQ ID NO: 3), VGADGVGKSA (SEQ ID NO: 4), VVGADGVGK (SEQ ID NO: 5), WGADGVGKS (SEQ ID NO: 6), VVVGADGVGK (SEQ ID NO: 7), KLVVVGADGV (SEQ ID NO: 8), YKLVVVGADG (SEQ ID NO: 9), or EYKLWVGAD (SEQ ID NO: 10); and optionally (b) an MHO. In some embodiments, provided herein is a cell-free peptide conjugate comprising: a) a chemical moiety having the formula: , covalently bonded to the aspartic acid residue in a peptide comprising the amino acid sequence DGVGKSALTI (SEQ ID NO: 1), ADGVGKSALT (SEQ ID NO: 2), GADGVGKSAL (SEQ ID NO: 3), VGADGVGKSA (SEQ ID NO: 4), WGADGVGK (SEQ ID NO: 5), VVGADGVGKS (SEQ ID NO: 6), VVVGADGVGK (SEQ ID NO: 7), KLWVGADGV (SEQ ID NO: 8), YKLVVVGADG (SEQ ID NO: 9), or EYKLVVVGAD (SEQ ID NO: 10); and optionally (b) an MHO.

In another aspect, the disclosure provides a cell-free peptide conjugate-MHC complex. The peptide conjugate-MHC complex comprises an isolated peptide conjugate as disclosed herein and a major histocompatibility complex (MHO). In some embodiments, the MHO is a human leukocyte antigen (HLA).

In some embodiments, the MHO is a human leukocyte antigen (HLA), optionally wherein the HLA is an HLA-A, HLA-B, or HLA-C. In some embodiments, the HLA molecule is an HLA-A*02:01 , HLA- A*03:01 , HLA-A*01 :01 , HLA-A*11 :01 , HLAA* 24:02, HLA-A*26:01 , HLA-B*07:02, HLA-B*08:01 , HLA- B*27:05, HLA-B*39:01 , HLA-B*40:01 , HLA-B*58:01 , and/or HLA-B*15:01 molecule. In some embodiments, the MHC is HLA-A*02:01 , HLA-A*03:01 , and/or HLAA* 11 :01.

In embodiments, the RAS inhibitor is conjugated to a peptide that comprises, or consists of, 7, 8, 9, 10, 11 , 12, 13, 14, or 15 amino acids, and which may be presented in an MHC Class I context. In embodiments, the RAS inhibitor is conjugated to a peptide that is 9-30 amino acids, inclusive, and including all numbers and ranges of numbers there between, and which may be presented in an MHC Class II context. In embodiments, the RAS inhibitor is conjugated to a peptide that comprises at least 7 amino acids. Non-classical MHC class I molecules function to mediate inhibitory or activating stimuli in natural killer (NK) cells. Non-classical MHC class I molecules can be expressed by immune and tumor cells. For example, expression of non-classical MHC complexes on malignant cells hampers cytotoxic activity of effector cells in the immune system. Overexpression of non-classical MHC class I molecules, including but not limited to HLA-E, HLA-F, and HLA-G, can be found in cancer cells. In some embodiments, the RAS inhibitor is conjugated to a peptide that is 9-30 amino acids, inclusive, and including all numbers and ranges of numbers there between, and which may be presented in a non- classical MHC Class I context. In some embodiments, the peptide conjugate forms a complex with a non- classical MHC class I molecule. In some embodiments, the non-classical MHC class I molecule is selected from the group consisting of HLA-E, HLA-F, and HLA-G. In some embodiments, the non- classical MHC class I molecule is selected from HLA-E, HLA-F, HLA-G, or some combination thereof.

In another aspect, the present disclosure provides a method for generating a peptide conjugate- MHC complex. The method includes contacting a cell with a peptide conjugate as disclosed herein and isolating the peptide conjugate-MHC complex. In some embodiments, the method comprises identifying the peptide conjugate/MHC complex. In some embodiments, the peptide conjugate is presented on said cell in the context of an MHC molecule.

Methods of Synthesis

The compounds and peptide conjugates described herein can be made from commercially available starting materials or synthesized using known organic, inorganic, or enzymatic processes.

The peptide conjugates of the present invention can be prepared in a number of ways well known to those skilled in the art of organic synthesis. By way of example, peptide conjugates of the present invention can be synthesized using the methods described in the Schemes below, together with synthetic methods known in the art of synthetic organic chemistry, or variations thereon as appreciated by those skilled in the art. These methods include but are not limited to those methods described in the Schemes below.

Scheme 1. General synthesis of functionalized peptides

As shown in Scheme 1 , compounds of this type may be prepared by the deprotection of a protecting group (PG) from an appropriate functionalized resin (1a) to afford the carboxylic acid (2a), which is coupled to an appropriately protected peptide X1 (3a) to afford compound 4a. Deprotection of the amine affords compound 5a and subsequent coupling of an appropriately protected peptide X2 (6a) affords compound 7a. Cleavage of the functionalized peptide (7a) from the resin then affords compound 8a. PGi, PG2, PG3, PG4 and PG5 are hydroxy and/or amino protecting groups. X1 and X2 are peptide fragments. Scheme 2. General synthesis of peptide containing macrocycles

As shown in Scheme 2, compounds of this type can be prepared by the reaction of a macrocyclic amine (I) with a carboxylic acid containing peptide (8a), where X1 is OH or an appropriately protected peptide and X2 is hydrogen, when PG4 is an appropriate protecting group, or an appropriately protected peptide, when PG4 is hydrogen, in the presence an amide coupling reagent to afford compound. II. Deprotection would then afford the final compound (III), where X1 is OH or an appropriate peptide and X2 is hydrogen or an appropriate peptide.

In some embodiments of the peptide conjugates in Scheme 2, X2 is hydrogen and X1 is a peptide having from 1 to 30 amino acids in length. In other embodiments, X2 is a peptide having from 1 to 30 amino acids in length and X1 is OH. In other embodiments, X2 is a peptide having from 1 to 30 amino acids in length and X1 is a peptide having from 1 to 30 amino acids in length.

In some embodiments of the peptide conjugates in Scheme 2, X1 and X2 are each independently a peptide comprising 1 , 2, 3, 4, 5, 6, 7, 8 or 9 amino acids in length. In some embodiments, X1 is a peptide comprising 1 , 2, 3, 4, 5, 6, 7, 8 or 9 amino acids in length and X2 is a peptide comprising 1 , 2, 3, 4, 5, 6, 7, 8 or 9 amino acids in length.

In one embodiment of the peptide conjugates in Scheme 2, X2 is hydrogen and X1 is a peptide comprising 1 , 2, 3, 4, 5, 6, 7, 8 or 9 amino acids in length. In one embodiment, X2 is hydrogen and X1 is a peptide comprising 9 amino acids in length.

In another embodiment of the peptide conjugates in Scheme 2, X2 comprises a single amino acid residue and X1 is a peptide comprising 1 , 2, 3, 4, 5, 6, 7 or 8 amino acids in length. In one embodiment, X2 comprises a single amino acid and X1 is a peptide comprising 8 amino acids in length.

In another embodiment of the peptide conjugates in Scheme 2, X2 is a peptide comprising two amino acids and X1 is a peptide comprising 1 , 2, 3, 4, 5, 6, or 7 amino acids in length. In one embodiment, X2 comprises two amino acids and X1 is a peptide comprising 7 amino acids in length. In another embodiment of the peptide conjugates in Scheme 2, X2 is a peptide comprising three amino acids and X1 is a peptide comprising 1 , 2, 3, 4, 5, or 6 amino acids in length. In one embodiment, X2 comprises three amino acids and X1 is a peptide comprising 6 amino acids in length.

In another embodiment of the peptide conjugates in Scheme 2, X2 is a peptide comprising four amino acids and X1 is a peptide comprising 1 , 2, 3, 4 or 5 amino acids in length. In one embodiment, X2 comprises 4 amino acids and X1 is a peptide comprising 5 amino acids in length.

In another embodiment of the peptide conjugates in Scheme 2, X2 is a peptide comprising five amino acids and X1 is a peptide comprising 1 , 2, 3, or 4 amino acids in length. In one embodiment, X2 comprises 5 amino acids and X1 is a peptide comprising 4 amino acids in length.

In another embodiment of the peptide conjugates in Scheme 2, X2 is a peptide comprising six amino acids and X1 is a peptide comprising 1 , 2, or 3 amino acids in length. In one embodiment, X2 comprises 6 amino acids and X1 is a peptide comprising 3 amino acids in length.

In another embodiment of the peptide conjugates in Scheme 2, X2 is a peptide comprising seven amino acids and X1 is a peptide comprising 1 or 2 amino acids in length. In one embodiment, X2 comprises 7 amino acids and X1 is a peptide comprising 2 amino acids in length.

In another embodiment of the peptide conjugates in Scheme 2, X2 is a peptide comprising eight amino acids and X1 comprises a single amino acid residue.

In one embodiment of the peptide conjugates in Scheme 2, X2 is hydrogen and X1 is a peptide comprising an amino acid sequence of GVGKSALTI (SEQ ID NO: 14). In another embodiment, X2 comprises amino acid residue Ala and X1 is a peptide comprising an amino acid sequence of GVGKSALT (SEQ ID NO: 15). In another embodiment, X2 is a peptide comprising an amino acid sequence Gly-Ala and X1 is a peptide comprising an amino acid sequence of GVGKSALT (SEQ ID NO: 15). In another embodiment, X2 is a peptide comprising an amino acid sequence VGA and X1 is a peptide comprising an amino acid sequence of GVGKSA (SEQ ID NO: 17). In another embodiment, X2 is a peptide comprising an amino acid sequence VVGA (SEQ ID NO: 18) and X1 is a peptide comprising an amino acid sequence of GVGK (SEQ ID NO: 19). In another embodiment, X2 is a peptide comprising an amino acid sequence VVGA (SEQ ID NO: 18) and X1 is a peptide comprising an amino acid sequence of GVGKS (SEQ ID NO: 20). In another embodiment, X2 is a peptide comprising an amino acid sequence VVVGA (SEQ ID NO: 21) and X1 is a peptide comprising an amino acid sequence of GVGK (SEQ ID NO: 19). In another embodiment, X2 is a peptide comprising an amino acid sequence KLVVVGA (SEQ ID NO: 22) and X1 is a peptide comprising an amino acid sequence of GV. In another embodiment, X2 is a peptide comprising an amino acid sequence YKLVVVGA (SEQ ID NO: 23) and X1 is a peptide comprising an amino acid residue Gly. In another embodiment, X2 is a peptide comprising an amino acid sequence EYKLVWGA (SEQ ID NO: 24) and X1 is OH.

Those of ordinary skill in the art, reading the present disclosure, will appreciate that certain compounds described herein may be provided or utilized in any of a variety of forms such as, for example, salt forms, protected forms, pro-drug forms, ester forms, isomeric forms (e.g., optical or structural isomers), isotopic forms, etc. In some embodiments, reference to a particular compound may relate to a specific form of that compound. In some embodiments, reference to a particular compound may relate to that compound in any form. In some embodiments, for example, a preparation of a single stereoisomer of a compound may be considered to be a different form of the compound than a racemic mixture of the compound; a particular salt of a compound may be considered to be a different form from another salt form of the compound; a preparation containing one conformational isomer ((Z) or (E)) of a double bond may be considered to be a different form from one containing 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.

Compounds of Table 1 herein are prepared using methods disclosed herein or were prepared using methods described herein combined with the knowledge of one of skill in the art.

Methods of Use

In an aspect, provided herein is a method of identifying a binding partner of a peptide conjugate or peptide conjugate/MHC complex as disclosed above, the method comprising: (a) providing a peptide conjugate or peptide conjugate/MHC complex of the disclosure; (b) contacting the peptide conjugate or peptide conjugate/MHC complex with a library comprising a plurality of antibodies under conditions suitable for binding at least one antibody of the plurality of antibodies to the peptide conjugate or peptide conjugate/MHC complex; and (c) recovering the at least one antibody bound to the peptide conjugate or peptide conjugate/MHC complex to identify the peptide conjugate or peptide conjugate/MHC complex specific antibody.

In another aspect, provided herein is a method of identifying a cell containing a peptide conjugate or peptide conjugate/MHC complex provided, the method comprising contacting the cell with a binding partner specific for the peptide conjugate or peptide conjugate/MHC complex.

Various techniques have been developed for the production of binding partners and are included in the scope of this disclosure. In embodiments, the disclosure provides for screening of a binding partner. This approach comprises providing a plurality of distinct binding partners, exposing the plurality of distinct (e.g., different) binding partners to one or a diversity of peptide conjugates of the disclosure, and selecting binding partners that bind with specificity to the peptide conjugates that contain the RAS inhibitor, but do not bind to the protein or peptide that does not comprise the covalently conjugated RAS inhibitor. As described above, this approach can be performed in a manner that either does, or does not, require the amino acid sequence of the protein or peptide to be part of the antigenic determinant. The described approach can be used to screen for binding partners that are specific for presentation of a peptide conjugate as a component of any MHC complex.

In embodiments, the peptide conjugate specific binding partners are produced by host cells by way of recombinant expression vectors. The present disclosure includes all polynucleotide sequences encoding the amino acid sequences described herein, expression vectors comprising such polynucleotide sequences, and in vitro cell cultures comprising such expression vectors. In embodiments, the cell cultures include prokaryotic cells or eukaryotic cells. In embodiments, the cell cultures are mammalian cells. In embodiments, the cells are CHO cells. In embodiments, the cells are HEK293 cells and their derivatives. Kits comprising the binding partners, and/or cell cultures expressing the binding partners, are provided by this disclosure. In general, the kits comprise one or more sealed containers that contain the binding partners, or cells expressing them. Instructions for using the binding partners for therapeutic and/or diagnostic purposes can be included in the kits.

Cells that are modified to express any described binding partner include but are not necessarily limited to CD4+ T cells, CD8+ T cells, Natural Killer T cells, y6 T cells, neutrophils, mucosal-associated invariant T (MAIT) cells, and cells that are progenitors of T cells, such as hematopoietic stem cells or other lymphoid progenitor cells, such as immature thymocytes (double-negative CD4-CD8-) cells, or double-positive thymocytes (CD4+CD8+). In some embodiments, the cell is optionally a totipotent, multipotent, or pluripotent stem cell, wherein optionally the stem cell has an induced stem cell phenotype, or wherein the cell is optionally a leukocyte. In embodiments, the cell is a macrophage. In some embodiments, the progenitor cells comprise markers, such as CD34, CD117 (c-kit) and CD90 (Thy-1). In embodiments, the modified cells comprise macrophages. In some embodiments, the modified cells comprise neutrophils. In some embodiments, the modified cell is a neutrophil. The described modified cells may be used therapeutically or prophylactically.

In embodiments, peptide conjugate specific binding partners may be used in any immunological diagnostic test, including but not limited to the imaging approaches described above. In embodiments, one or more binding partners described herein can be used as a component in any form of, for example, enzyme-linked immunosorbent assay (ELISA) assay, including but not limited to a direct ELISA, a sandwich ELISA, a competitive ELISA, and a reverse ELISA. In embodiments, one or more binding partners described herein can also be incorporated into an immunodiagnostic device, such as a microfluidic device, a lateral flow device, and the like. Binding partners may also be used in, for example, Western blots and immunoprecipitation assays. The peptide conjugate or peptide conjugate/MHC complex of the disclosure may be useful in the context of the above-described assays as controls or in the use of standards.

Pharmaceutical Compositions and Methods of Administration

The compounds and peptide conjugates as described herein are useful in the treatment of cancer or for identifying suitable agents for the treatment of cancer. The disclosure provides a pharmaceutical composition containing a compound as disclosed herein or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient, as well as methods of using the compounds to prepare such compositions.

Compounds peptide conjugates described herein, whether expressly stated or not, may be provided or utilized in salt form, e.g., a pharmaceutically acceptable salt form, unless expressly stated to the contrary. The term “pharmaceutically acceptable salt,” as use herein, refers to those salts of the compounds described herein that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and other 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, pharmaceutically acceptable salts are described in: Berge et al., J. Pharmaceutical Sciences 66:1-19, 1977 and in Pharmaceutical Salts: Properties, Selection, and Use, (Eds. P.H. Stahl and C.G. Wermuth), Wiley-VCH, 2008. The salts can be prepared in situ during the final isolation and purification of the compounds described herein or separately by reacting the free base group with a suitable organic acid. The compounds and peptide conjugates of the invention may have ionizable groups so as to be capable of preparation as pharmaceutically acceptable salts. These salts may be acid addition salts involving inorganic or organic acids or the salts may, in the case of acidic forms of the compounds of the invention, be prepared from inorganic or organic bases. In some embodiments, the compounds are prepared or used as pharmaceutically acceptable salts prepared as addition products of pharmaceutically acceptable acids or bases. Suitable pharmaceutically acceptable acids and bases are well-known in the art, such as hydrochloric, sulfuric, hydrobromic, acetic, lactic, citric, or tartaric acids for forming acid addition salts, and potassium hydroxide, sodium hydroxide, ammonium hydroxide, caffeine, various amines, and the like for forming basic salts. Methods for preparation of the appropriate salts are well-established in the art.

Representative acid addition salts include acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptonate, hexanoate, hydrobromide, hydrochloride, hydroiodide, 2-optionally substituted hydroxyl-eth an esulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, toluenesulfonate, undecanoate, valerate salts and the like. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium and the like, as well as nontoxic ammonium, quaternary ammonium, and amine cations, including, but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine and the like.

For use as treatment of subjects, the compounds of the invention, or a pharmaceutically acceptable salt thereof, can be formulated as pharmaceutical or veterinary compositions. Depending on the subject to be treated, the mode of administration, and the type of treatment desired, e.g., prevention, prophylaxis, or therapy, the compounds, or a pharmaceutically acceptable salt thereof, are formulated in ways consonant with these parameters. A summary of such techniques may be found in Remington: The Science and Practice of Pharmacy, 21^st Edition, Lippincott Williams & Wilkins, (2005); and Encyclopedia of Pharmaceutical Technology, eds. J. Swarbrick and J. C. Boylan, 1988-1999, Marcel Dekker, New York, each of which is incorporated herein by reference.

Compositions can be prepared according to conventional mixing, granulating or coating methods, respectively, and the present pharmaceutical compositions can contain from about 0.1% to about 99%, from about 5% to about 90%, or from about 1% to about 20% of a compound of the present invention, or pharmaceutically acceptable salt thereof, by weight or volume. In some embodiments, compounds, or a pharmaceutically acceptable salt thereof, described herein may be present in amounts totaling 1-95% by weight of the total weight of a composition, such as a pharmaceutical composition.

The composition may be provided in a dosage form that is suitable for intraarticular, oral, parenteral (e.g., intravenous, intramuscular), rectal, cutaneous, subcutaneous, topical, transdermal, sublingual, nasal, vaginal, intravesicular, intraurethral, intrathecal, epidural, aural, or ocular administration, or by injection, inhalation, or direct contact with the nasal, genitourinary, reproductive or oral mucosa. Thus, the pharmaceutical composition may be in the form of, e.g., tablets, capsules, pills, powders, granulates, suspensions, emulsions, solutions, gels including hydrogels, pastes, ointments, creams, plasters, drenches, osmotic delivery devices, suppositories, enemas, injectables, implants, sprays, preparations suitable for iontophoretic delivery, or aerosols. The compositions may be formulated according to conventional pharmaceutical practice.

Formulations may be prepared in a manner suitable for systemic administration or topical or local administration. Systemic formulations include those designed for injection (e.g., intramuscular, intravenous or subcutaneous injection) or may be prepared for transdermal, transmucosal, or oral administration. A formulation will generally include a diluent as well as, in some cases, adjuvants, buffers, preservatives and the like. Compounds, or a pharmaceutically acceptable salt thereof, can be administered also in liposomal compositions or as microemulsions.

For injection, formulations can be prepared in conventional forms as liquid solutions or suspensions or as solid forms suitable for solution or suspension in liquid prior to injection or as emulsions. Suitable excipients include, for example, water, saline, dextrose, glycerol and the like. Such compositions may also contain amounts of nontoxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents and the like, such as, for example, sodium acetate, sorbitan monolaurate, and so forth.

Various sustained release systems for drugs have also been devised. See, for example, U.S. Patent No. 5,624,677.

Systemic administration may also include relatively noninvasive methods such as the use of suppositories, transdermal patches, transmucosal delivery and intranasal administration. Oral administration is also suitable for compounds of the invention, or a pharmaceutically acceptable salt thereof. Suitable forms include syrups, capsules, and tablets, as is understood in the art.

Each compound, or a pharmaceutically acceptable salt thereof, as described herein, may be formulated in a variety of ways that are known in the art. For example, the first and second agents of the combination therapy may be formulated together or separately. Other modalities of combination therapy are described herein.

The individually or separately formulated agents can be packaged together as a kit. Non-limiting examples include, but are not limited to, kits that contain, e.g., two pills, a pill and a powder, a suppository and a liquid in a vial, two topical creams, etc. The kit can include optional components that aid in the administration of the unit dose to subjects, such as vials for reconstituting powder forms, syringes for injection, customized IV delivery systems, inhalers, etc. Additionally, the unit dose kit can contain instructions for preparation and administration of the compositions. The kit may be manufactured as a single use unit dose for one subject, multiple uses for a particular subject (at a constant dose or in which the individual compounds, or a pharmaceutically acceptable salt thereof, may vary in potency as therapy progresses); or the kit may contain multiple doses suitable for administration to multiple subjects (“bulk packaging”). The kit components may be assembled in cartons, blister packs, bottles, tubes, and the like.

Formulations for oral use include tablets containing the active ingredient(s) in a mixture with non-toxic pharmaceutically acceptable excipients. These excipients may be, for example, inert diluents or fillers (e.g., sucrose, sorbitol, sugar, mannitol, microcrystalline cellulose, starches including potato starch, calcium carbonate, sodium chloride, lactose, calcium phosphate, calcium sulfate, or sodium phosphate); granulating and disintegrating agents (e.g., cellulose derivatives including microcrystalline cellulose, starches including potato starch, croscarmellose sodium, alginates, or alginic acid); binding agents (e.g., sucrose, glucose, sorbitol, acacia, alginic acid, sodium alginate, gelatin, starch, pregelatinized starch, microcrystalline cellulose, magnesium aluminum silicate, carboxymethylcellulose sodium, methylcellulose, optionally substituted hydroxylpropyl methylcellulose, ethylcellulose, polyvinylpyrrolidone, or polyethylene glycol); and lubricating agents, glidants, and antiadhesives (e.g., magnesium stearate, zinc stearate, stearic acid, silicas, hydrogenated vegetable oils, or talc). Other pharmaceutically acceptable excipients can be colorants, flavoring agents, plasticizers, humectants, buffering agents, and the like.

Two or more compounds may be mixed together in a tablet, capsule, or other vehicle, or may be partitioned. In one example, the first compound is contained on the inside of the tablet, and the second compound is on the outside, such that a substantial portion of the second compound is released prior to the release of the first compound.

Formulations for oral use may also be provided as chewable tablets, or as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent (e.g., potato starch, lactose, microcrystalline cellulose, calcium carbonate, calcium phosphate or kaolin), or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example, peanut oil, liquid paraffin, or olive oil. Powders, granulates, and pellets may be prepared using the ingredients mentioned above under tablets and capsules in a conventional manner using, e.g., a mixer, a fluid bed apparatus or a spray drying equipment.

Dissolution or diffusion-controlled release can be achieved by appropriate coating of a tablet, capsule, pellet, or granulate formulation of compounds, or by incorporating the compound, or a pharmaceutically acceptable salt thereof, into an appropriate matrix. A controlled release coating may include one or more of the coating substances mentioned above or, e.g., shellac, beeswax, glycowax, castor wax, carnauba wax, stearyl alcohol, glyceryl monostearate, glyceryl distearate, glycerol palmitostearate, ethylcellulose, acrylic resins, dl-poly lactic acid, cellulose acetate butyrate, polyvinyl chloride, polyvinyl acetate, vinyl pyrrolidone, polyethylene, polymethacrylate, methylmethacrylate, 2-optionally substituted hydroxylmethacrylate, methacrylate hydrogels, 1 ,3 butylene glycol, ethylene glycol methacrylate, or polyethylene glycols. In a controlled release matrix formulation, the matrix material may also include, e.g., hydrated methylcellulose, carnauba wax and stearyl alcohol, carbopol 934, silicone, glyceryl tristearate, methyl acrylate-methyl methacrylate, polyvinyl chloride, polyethylene, or halogenated fluorocarbon.

The liquid forms in which the compounds, or a pharmaceutically acceptable salt thereof, and compositions of the present invention can be incorporated for administration orally include aqueous solutions, suitably flavored syrups, aqueous or oil suspensions, and flavored emulsions with edible oils such as cottonseed oil, sesame oil, coconut oil, or peanut oil, as well as elixirs and similar pharmaceutical vehicles.

Generally, when administered to a human, the oral dosage of any of the compounds of the invention, or a pharmaceutically acceptable salt thereof, will depend on the nature of the compound, and can readily be determined by one skilled in the art. A dosage may be, for example, about 0.001 mg to about 2000 mg per day, about 1 mg to about 1000 mg per day, about 5 mg to about 500 mg per day, about 100 mg to about 1500 mg per day, about 500 mg to about 1500 mg per day, about 500 mg to about 2000 mg per day, or any range derivable therein. In some embodiments, the daily dose range for oral administration, for example, may lie within the range of from about 0.001 mg to about 2000 mg per kg body weight of a human, in single or divided doses. On the other hand, it may be necessary to use dosages outside these limits in some cases.

In some embodiments, the pharmaceutical composition may further comprise an additional compound having antiproliferative activity. Depending on the mode of administration, compounds, or a pharmaceutically acceptable salt thereof, will be formulated into suitable compositions to permit facile delivery. Each compound, or a pharmaceutically acceptable salt thereof, of a combination therapy may be formulated in a variety of ways that are known in the art. For example, the first and second agents of the combination therapy may be formulated together or separately. Desirably, the first and second agents are formulated together for the simultaneous or near simultaneous administration of the agents.

It will be appreciated that the compounds and pharmaceutical compositions of the present invention can be formulated and employed in combination therapies, that is, the compounds and pharmaceutical compositions can be formulated with or administered concurrently with, prior to, or subsequent to, one or more other desired therapeutics or medical procedures. The particular combination of therapies (therapeutics or procedures) to employ in a combination regimen will take into account compatibility of the desired therapeutics or procedures and the desired therapeutic effect to be achieved. It will also be appreciated that the therapies employed may achieve a desired effect for the same disorder, or they may achieve different effects (e.g., control of any adverse effects).

Administration of each drug in a combination therapy, as described herein, can, independently, be one to four times daily for one day to one year, and may even be for the life of the subject. Chronic, long-term administration may be indicated.

Examples

The disclosure is further illustrated by the following examples and synthesis examples, which are not to be construed as limiting this disclosure in scope or spirit to the specific procedures herein described. It is to be understood that the examples are provided to illustrate certain embodiments and that no limitation to the scope of the disclosure is intended thereby. It is to be further understood that resort may be had to various other embodiments, modifications, and equivalents thereof which may suggest themselves to those skilled in the art without departing from the spirit of the present disclosure or scope of the appended claims.

Chemical Syntheses

Definitions used in the following examples and elsewhere herein are:

CH2CI2, DCM methylene chloride, dichloromethane

CH3CN, MeCN acetonitrile

Cui copper (I) iodide

DIPEA diisopropylethyl amine

DMF A/,A/-dimethylformamide

EtOAc ethyl acetate h hour H₂0 water

HCI hydrochloric acid

K3PO4 potassium phosphate (tribasic)

MeOH methanol

Na₂SO4 sodium sulfate

NMP A/-methyl pyrrolidone

Pd(dppf)CI₂ [1 ,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(ll)

Instrumentation

Mass spectrometry data collection took place with a Shimadzu LCMS-2020, an Agilent 1260LC- 6120/6125MSD, a Shimadzu LCMS-2010EV, or a Waters Acquity UPLC, with either a QDa detector or SQ Detector 2. Samples were injected in their liquid phase onto a C-18 reverse phase. The compounds were eluted from the column using an acetonitrile gradient and fed into the mass analyzer. Initial data analysis took place with either Agilent ChemStation, Shimadzu LabSolutions, or Waters MassLynx. NMR data was collected with either a Bruker AVANCE III HD 400MHz, a Bruker Ascend 500MHz instrument, or a Varian 400MHz, and the raw data was analyzed with either TopSpin or Mestrelab Mnova.

Example 1. Synthesis of Intermediates

Intermediate 1 : Synthesis of (S)-3-(5-bromo-2-(5-(4-cyclopropylpiperazin-1-yl)-2-(1- methoxyethyl)pyridin-3-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-3-yl)-2,2-dimethylpropyl acetate

Step 1'. Synthesis of (1 S)-1-(3-bromopyridin-2-yl)ethanol

To a stirred mixture of HCO₂H (66.3 g, 1 .44 mol) in EtaN (728 g, 7.2 mol) at 0 °C under an atmosphere of argon was added (4S,5S)-2-chloro-2-methyl-1-(4-methylbenzenesulfonyl)-4,5-diphenyl- 1 ,3-diaza-2-ruthenacyclopentane cymene (3.9 g, 6.0 mmol) portion-wise. The mixture was heated to 40 °C and stirred for 15 min, then cooled to room temperature and 1-(3-bromopyridin-2-yl)ethanone (120 g, 600 mmol) was added in portions. The mixture was heated to 40 °C and stirred for an additional 2 h, then the reaction was concentrated under reduced pressure. Brine (2 L) was added to the residue, the mixture was extracted with EtOAc (4 x 700 mL), dried over anhydrous Na₂SO4 and filtered. The filtrate was concentrated under reduced pressure and the residue was purified by silica gel column chromatography to give the product (100 g, 74% yield) as an oil. LCMS (ESI) m/z: [M + H] calcd for C HsBrNO: 201 .99; found: 201.9.

Step 2: Synthesis of 3-bromo-2-[(1 S)-1-methoxyethyl]pyridine

To a stirred mixture of (1 S)-1-(3-bromopyridin-2-yl)ethanol (100 g, 495 mmol) in DMF (1 L) at 0 °C was added NaH, 60% dispersion in oil (14.25 g, 594 mmol) in portions. The mixture was stirred at 0 °C for 1 h. Mel (140.5 g, 990 mmol) was added dropwise at 0 °C and the mixture was warmed to room temperature and stirred for 2 h. The mixture was cooled to 0 °C and sat. aq. NH4CI (5 L) was added. The mixture was extracted with EtOAc (3 x 1.5 L), dried over anhydrous Na2SO4 and filtered. The filtrate was concentrated under reduced pressure and the residue was purified by silica gel column chromatography to give the product (90 g, 75% yield) as an oil. LCMS (ESI) m/z: [M + H] calcd for CsH-ioBrNO: 216.00; found: 215.9.

Step 3: Synthesis of 5-[2-[(1 S)-1-methoxyethyl]pyridin-3-yl]-2,2-dimethyl-5-oxopentanoic acid

To a mixture of /-PrMgCI (2 M in in THF, 0.5 L) at -10 °C under an atmosphere of nitrogen was added n-BuLi (2.5 M in hexane, 333 mL, 833 mmol) dropwise over 15 min. The mixture was stirred for 30 min at -10 °C then 3-bromo-2-[(1 S)-1-methoxyethyl]pyridine (180 g, 833 mmol) in THF (0.5 L) added dropwise over 30 min at -10 °C. The resulting mixture was warmed to -5 °C and stirred for 1 h, then 3,3- dimethyloxane-2, 6-dione (118 g, 833 mmol) in THF (1 .2 L) was added dropwise over 30 min at -5 °C. The mixture was warmed to 0 °C and stirred for 1 .5 h, then quenched with the addition of pre-cooled 4 M HCI in 1 ,4-dioxane (0.6 L) at 0 °C to adjust pH ~5. The mixture was diluted with H2O (3 L) at 0 °C and extracted with EtOAc (3 x 2.5 L). The combined organic layers were dried over anhydrous Na2SO4, filtered, the filtrate was concentrated under reduced pressure and the residue was purified by silica gel column chromatography to give the product (87 g, 34% yield) as a solid. LCMS (ESI) m/z: [M + H] calcd for C15H21NO4: 280.15; found: 280.1.

Step 4: Synthesis of ethyl (S)-3-(5-bromo-2-(2-(1-methoxyethyl)pyridin-3-yl)-1/7-indol-3-yl)-2,2- dimethylpropanoate

To a solution of 5-[2-[(1 S)-1-methoxyethyl]pyridin-3-yl]-2,2-dimethyl-5-oxopentanoic acid (180 g, 644.39 mmol) in EtOH (1.26 L) was added (4-bromophenyl)hydrazine hydrochloride (171.38 g, 766.82 mmol). The reaction mixture was stirred for 3 h at 85 °C, was then cooled to room temperature mixture, and then HCI in 1 ,4-dioxane (161 .33 mL, 5309.7 mmol) was added. The reaction mixture was stirred for an additional 16 h at 85 °C and was then concentrated under reduced pressure. The residue was dissolved in TFA (1 .8 kg) and was stirred for 2 h at 65 °C. The resulting mixture was concentrated under reduced pressure and the residue was basified to pH 6-7 with NaOH. The aqueous layer was extracted with EtOAc and the combined organic layers were concentrated under reduced pressure to afford the crude product (300 g, 97% yield) as a red solid. Four reactions were run in parallel to afford 1100 g of crude product, which was used directly in the next step. LCMS (ESI) m/z [M + H] calcd for C23H2?BrN2O3: 459.13; found: 459.1.

Step 5: Synthesis of ethyl (S)-3-(5-bromo-2-(2-(1-methoxyethyl)pyridin-3-yl)-1 -(2,2,2- trifluoroethyl)-1/7-indol-3-yl)-2,2-dimethylpropanoate

To a solution of ethyl (S)-3-(5-bromo-2-(2-(1-methoxyethyl)pyridin-3-yl)-1/7-indol-3-yl)-2,2- dimethylpropanoate (1100 g, 2394.5 mmol) and CS2CO3 (3900.9 g, 11972.6 mmol) in MeCN (5500 mL) was added 2,2,2-trifluoroethyl trifluoromethanesulfonate (1111.5 g, 4789.0 mmol). The reaction mixture was stirred for 16 h at 50 °C and was then cooled to room temperature and filtered. The filtrate was concentrated under reduced pressure to afford the desired crude product (1380 g, 84% yield) as a red solid. LCMS (ESI) m/z: [M + H] calcd for C25H28BrF₃N2O₃: 541 .13; found: 451 .0.

Step 6: Synthesis of (S)-3-(5-bromo-2-(2-(1-methoxyethyl)pyridin-3-yl)-1-(2,2,2-trifluoroethyl)-1/7- indol-3-yl)-2,2-dimethylpropan-1-ol

To a solution of ethyl (S)-3-(5-bromo-2-(2-(1-methoxyethyl)pyridin-3-yl)-1-(2,2,2-trifluoroethyl)-1/7- indol-3-yl)-2,2-dimethylpropanoate (260 g, 480.2 mmol) in THF (1 .56 L) at 0 °C was added LiBF (26.15 g, 1200.6 mmol). The resulting mixture was stirred for 16 h at 60 °C and was cooled to 0 °C and quenched with 1 M NF CI. The aqueous layer was extracted with EtOAc, and the combined organic layers were washed with brine. The organic layer was concentrated under reduced pressure to afford the desired crude mixture. Six batches were run in parallel and were combined to afford the crude mixture (1300 g, crude) as a red solid. The crude mixture (1300 g) was dissolved in DCM (1 L) and was loaded onto silica gel and purified by normal phase chromatography. Elution with 14% EtOAc/pet. ether afforded the desired atropisomer (100 g, 80% purity) followed by elution with 20% EtOAc/pet. ether to afford additional desired atropisomer (470 g, 90% purity) as a yellow solid. Elution with 9% MeOH/EtOAc afforded the undesired atropisomer (510 g) as a brown oil. LCMS (ESI) m/z: [M + H] calcd for C2₃H26BrF₃N2O2: 499.12; found: 499.0.

Step 7: Synthesis of (S)-3-(5-bromo-2-(2-(1-methoxyethyl)pyridin-3-yl)-1-(2,2,2-trifluoroethyl)-1/7- indol-3-yl)-2,2-dimethylpropyl acetate

To a stirred solution of (S)-3-(5-bromo-2-(2-(1-methoxyethyl)pyridin-3-yl)-1-(2,2,2-trifluoroethyl)- 1/7-indol-3-yl)-2,2-dimethylpropan-1-ol (60 g, 0.12 mol) and Et₃N (24.33 g, 0.24 mol) in DCM (600 mL) were added DMAP (1.46 g, 0.012 mol) and acetic anhydride (14.7 g, 144 mmol) dropwise at 0 °C under an atmosphere of argon. The resulting mixture was stirred for 2 h at room temperature. The resulting mixture was concentrated under reduced pressure and washed with HCI (500 mL). The resulting mixture was washed with sat. aq. NaHC0₃ (500 mL). The combined organic layers were washed with brine (500 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure to afford the product (59.6 g, 92% yield) as an oil. LCMS (ESI) m/z: [M + H] calcd C25H2sBrF₃N2O₃: 541.13; found: 543.2.

Step 8: Synthesis of (S)-(5-(3-(3-acetoxy-2,2-dimethylpropyl)-5-bromo-1-(2,2,2-trifluoroethyl)-1/7- i nd o l-2-y l)-6-(1 -methoxyethyl) pyridin-3-yl)boronic acid

To a stirred mixture of (S)-3-(5-bromo-2-(2-(1-methoxyethyl)pyridin-3-yl)-1-(2,2,2-trifluoroethyl)- 1/7-indol-3-yl)-2,2-dimethylpropyl acetate (55.1 g, 101.771 mmol) and 4,4,5,5-tetramethyl-2-(tetramethyl- 1 ,3,2-dioxaborolan-2-yl)-1 ,3,2-dioxaborolane (38.77 g, 152.656 mmol) in THF (40 mL) were added dtbpy (4.10 g, 15.266 mmol) and chloro(1 ,5-cyclooctadiene)iridium(l) dimer (3.42 g, 5.089 mmol) in portions at room temperature under an atmosphere of argon. The resulting mixture was stirred for 5 h at 75 °C. The resulting mixture was concentrated under reduced pressure to afford the product (102.4 g, crude) as an oil. LCMS (ESI) m/z: [M + H] calcd C25H29BBrF₃N₂O₅: 585.14; found: 585.2.

Step 9 Synthesis of (S)-3-(5-bromo-2-(5-(4-cyclopropylpiperazin-1-yl)-2-(1-methoxyethyl)pyridin- 3-yl)-1-(2,2,2-trifluoroethyl)-1/7-indol-3-yl)-2,2-dimethylpropyl acetate

To a stirred solution of (S)-(5-(3-(3-acetoxy-2,2-dimethylpropyl)-5-bromo-1-(2,2,2-trifluoroethyl)- 1/7-indol-2-yl)-6-(1-methoxyethyl)pyridin-3-yl)boronic acid (40 g, 61.514 mmol) and Et₃N (12.45 g, 123.028 mmol) in MeCN (1000 mL) was added 4A MS (8 g) and 1 -cyclopropylpiperazine (38.82 g, 307.570 mmol) in portions under an oxygen atmosphere. The resulting mixture was stirred for 1 h at room temperature under an oxygen atmosphere. To the above mixture was added Cu(OAc)2 (22.35 g, 123.028 mmol) and then the vessel was evacuated, backfilled with oxygen, and then stirred overnight at room temperature. The resulting mixture was filtered and was concentrated under reduced pressure. The residue was diluted with EtOAc (300 mL) and the organic layer was washed with NHa'HLO (4 x 100 mL), dried over Na2SO4, and concentrated under reduce pressure. The residue was purified by column chromatography (50% EtOAc/pet. ether) to afford the desired product (23.7 g, 56% yield) as a solid. LCMS (ESI) m/z: [M + H] calcd for C₃2H4oBrF₃N₄0₃: 665.23; found: 666.0.

Intermediate 2: Synthesis of (2²S,6³S,4S)-4-amino-1²-(5-(4-cyclopropylpiperazin-1-yl)-2-((S)- 1-methoxyethyl)pyridin-3-yl)-10,10-dimethyl-1¹-(2,2,2-trifluoroethyl)-6¹,6²,6³,6⁴,6⁵,6⁶-hexahydro-1¹H- 8-oxa-2(4,2)-morpholina-1 (5, 3)-indola-6(1 ,3)-pyridazinacycloundecaphane-5, 7-dione

Step 1: Synthesis of methyl (S)-1-((S)-3-((S)-4-(3-(3-acetoxy-2,2-dimethylpropyl)-2-(5-(4- cyclopropylpi perazin-1 -yl)-2-((S)-1 -methoxyethyl) pyridin-3-yl)-1 -(2, 2, 2-trifluoroethyl)-1 /7-indol-5- yl)morpholin-2-yl)-2-((fe/Y-butoxycarbonyl)amino)propanoyl)hexahydropyridazine-3-carboxylate

To a solution of 3(S)-3-(5-bromo-2-(5-(4-cyclopropylpiperazin-1-yl)-2-(1-methoxyethyl)pyridin-3- yl)-1-(2,2,2-trifluoroethyl)-1 /7-indol-3-yl)-2,2-dimethylpropyl acetate (65.0 g, 97.66 mmol) and methyl (S)- 1-((S)-2-((te/Y-butoxycarbonyl)amino)-3-((S)-morpholin-2-yl)propanoyl)hexahydropyridazine-3-carboxylate (78.2 g, 0.195 mol) in 1 ,4-dioxane (650 mL) was added RuPhos (27.3 g, 58.60 mmol), RuPhos-G2-Pd (22.7 g, 29.30 mmol), and Cs2CO₃ (95.5 g, 0.29 mol). The resulting mixture was stirred overnight at 80 °C. The reaction mixture was then filtered, the filter cake was washed with EtOAc (3 x 300 mL), and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography to afford the desired product (63 g, 65% yield) as a solid. LCMS (ESI) m/z [M + H] calcd for CsoH/iFsNsOg: 985.54; found: 985.8. Step 2 Synthesis of (S)-1-((S)-2-((te/Y-butoxycarbonyl)amino)-3-((S)-4-(2-(5-(4- cyclopropylpi perazin-1 -yl)-2-((S)-1 -methoxyethyl) pyridin-3-yl)-3-(3-hydroxy-2,2-dimethylpropyl)-1 -(2, 2, 2- trifluoroethyl)-1 /7-indol-5-yl)morpholin-2-yl)propanoyl)hexahydropyridazine-3-carboxylic acid

To a solution of methyl (S)-1-((S)-3-((S)-4-(3-(3-acetoxy-2,2-dimethylpropyl)-2-(5-(4- cyclopropylpiperazin-1 -yl)-2-((S)-1 -methoxyethyl) py rid i n-3-y I)- 1 -(2 ,2 , 2-trifl u oroethy l)-1 H-\ nd o I-5- yl)morpholin-2-yl)-2-((te/Y-butoxycarbonyl)amino)propanoyl)hexahydropyridazine-3-carboxylate (79 g, 80.19 mmol) in THF (700 mL) was added a solution of LiOH»H2O (16.7 g, 0.398 mol) in H2O (150 mL) at 0 °C. The resulting mixture was stirred for 5 h at room temperature. The mixture was then acidified to pH 5 with 1 M HCI(aq). The aqueous layer was extracted with DCM (3 x 500 mL) and the organic layer was dried over Na2SO4, filtered, and concentrated under reduced pressure to afford the desired product (70 g, crude) as a solid. LCMS (ESI) m/z: [M + H] calcd for C47H67F3N8O8: 929.51 ; found: 929.4.

Step 3: Synthesis of tert-butyl ((2²S,6³S,4S)-1²-(5-(4-cyclopropylpiperazin-1-yl)-2-((S)-1- methoxyethyl)pyridin-3-yl)-10,10-dimethyl-5,7-dioxo-1¹-(2,2,2-trifluoroethyl)-6¹,6²,6³,6⁴,6⁵,6⁶-hexahydro- 1¹/7-8-oxa-2(4,2)-morpholina-1 (5,3)-indola-6(1 ,3)-pyridazinacycloundecaphane-4-yl)carbamate

To a solution of (S)-1-((S)-2-((fert-butoxycarbonyl)amino)-3-((S)-4-(2-(5-(4-cyclopropylpiperazin-1- yl)-2-((S)-1 -methoxyethyl) pyridin-3-yl)-3-(3-hydroxy-2, 2-dimethylpropyl)-1 -(2, 2, 2-trifluoroethyl)-1 /7-indol-5- yl)morpholin-2-yl)propanoyl)hexahydropyridazine-3-carboxylic acid (55.7 g, 59.95 mmol) and DI PEA (208.8 mL, 1 .199 mol) in DCM (6500 mL) at 0 °C was added EDCI (229.9 g, 1 .199 mol) and HOBt (40.5 g, 0.299 mol). The resulting mixture was stirred overnight at room temperature. The reaction mixture was quenched by the addition of cold H2O (500 mL) and the aqueous layer was extracted with EtOAc (3 x 800 mL). The combined organic layers were washed with brine (2 x 500 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography to afford the desired product (35.0 g, 64% yield) as a solid. LCMS (ESI) m/z: [M + H] calcd for C47H65F3N8O7: 91 1 .50; found: 91 1 .3.

Step 4: Synthesis of (2²S,6³S,4S)-4-amino-1²-(5-(4-cyclopropylpiperazin-1-yl)-2-((S)-1- methoxyethyl)pyridin-3-yl)-10,10-dimethyl-1¹-(2,2,2-trifluoroethyl)-6¹,6²,6³,6⁴,6⁵,6⁶-hexahydro-1¹ /7-8-oxa- 2(4,2)-morpholina-1 (5,3)-indola-6(1 ,3)-pyridazinacycloundecaphane-5, 7-dione

To a solution of fert-butyl ((2²S,6³S,4S)-1²-(5-(4-cyclopropylpiperazin-1-yl)-2-((S)-1- methoxyethyl)pyridin-3-yl)-10,10-dimethyl-5,7-dioxo-1¹-(2,2,2-trifluoroethyl)-6¹,6²,6³,6⁴,6⁵,6⁶-hexahydro- 1¹/7-8-oxa-2(4,2)-morpholina-1 (5,3)-indola-6(1 ,3)-pyridazinacycloundecaphane-4-yl)carbamate (33 g, 36.07 mmol) in DCM (180 mL) at 0 °C was added HCI in 1 ,4-dioxane (180 mL). The resulting mixture was stirred for 2 h at room temperature and then the mixture was concentrated under reduced pressure to afford the desired product (33 g, crude) as a solid. LCMS (ESI) m/z: [M + H] calcd for C42H57F3N8O5: 811.45; found: 811.3. Intermediate 3: Synthesis of (S)-2-((S)-7-(tert-butoxycarbonyl)-2,7-diazaspiro[4.4]nonan-2- yl)-2-cyclopentylacetic acid

Step 1 : Synthesis of benzyl (/?)-2-cyclopentyl-2-(((trifluoromethyl)sulfonyl)oxy)acetate

To a solution of benzyl (R)-2-cyclopentyl-2-hydroxyacetate (3 g, 12.8 mmol) in DCM (50 mL) was added Tf2<D (3.79 g, 13.44 mmol) and 2,6-lutidine (1 .51 g, 14.09 mmol) at 0 °C under an atmosphere of nitrogen and the mixture was stirred at 0 °C for 2 h. The resulting mixture was diluted with H2O (30 mL) and extracted with DCM (3 x 50 mL). The combined organic layers were washed with brine (2 x 50 mL), dried over Na2SO4, filtered and the filtrate was concentrated under reduced pressure to give the product, which was used directly without further purification.

Step 2'. Synthesis of te/Y-butyl 7-((S)-2-(benzyloxy)-1-cyclopentyl-2-oxoethyl)-2,7- diazaspiro[4.4]nonane-2-carboxylate

To a solution of benzyl (/?)-2-cyclopentyl-2-(((trifluoromethyl)sulfonyl)oxy)acetate (4.86 g, 13.26 mmol) in THF (20 mL) was added tert-buty I 2,7-diazaspiro[4.4]nonane-2-carboxylate (2 g, 8.84 mmol) and CS2CO3 (8.64 g, 26.51 mmol). The mixture was stirred at room temperature for 30 min. The resulting mixture was diluted with H2O (30 mL) and extracted with EtOAc (3 x 30 mL) and the combined organic layers were washed with brine (2 x 40 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (20^100% EtOAc/pet. ether) to give the product (2.6 g, 66% yield) as a solid. LCMS (ESI) m/z: [M + H] calcd for C26H38N2O4: 443.3; found: 443.2.

Step 3: Synthesis of (S)-2-((R)-7-(te/Y-butoxycarbonyl)-2,7-diazaspiro[4.4]nonan-2-yl)-2- cyclopentylacetic acid and (S)-2-((S)-7-(te/Y-butoxycarbonyl)-2,7-diazaspiro[4.4]nonan-2-yl)-2- cyclopentylacetic acid

To a solution of te/Y-butyl 7-((S)-2-(benzyloxy)-1-cyclopentyl-2-oxoethyl)-2,7- diazaspiro[4.4]nonane-2-carboxylate (2.6 g, 5.87 mmol) in MeOH (30 mL) was added Pd/C (0.5 g, 10% on carbon w/w) under an atmosphere of nitrogen. The suspension was degassed and purged with H2. The mixture was stirred under H2 (15 psi) at room temperature for 4 h. The mixture was filtered and the filtrate was concentrated under reduced pressure to give residue. The residue was dissolved in EtOAc (5 mL) and the mixture was stirred for 10 min. Then the mixture was filtered and the filter cake was dried under reduced pressure. The solid was purified by SFC-separation (CO2/MeOH (0.1 % NH4O)) to give (S)- 2-((/?)-7-(te/Y-butoxycarbonyl)-2,7-diazaspiro[4.4]nonan-2-yl)-2-cyclopentylacetic acid (450 mg, 22% yield) and (S)-2-((S)-7-(te/Y-butoxycarbonyl)-2,7-diazaspiro[4.4]nonan-2-yl)-2-cyclopentylacetic acid (450 mg, 22% yield). Intermediate 4: Synthesis of (2S)-2-cyclopentyl-N-((2²S,6³S,4S)-1²-(5-(4- cyclopropylpiperazin-1 -y I )-2-((S)-1 -methoxyethyl)pyridin-3-yl)-10,10-dimethyl-5,7-dioxo-1¹-(2,2,2- trifluoroethyl)-6¹,6²,6³,6⁴,6⁵,6⁶-hexahydro-1¹H-8-oxa-2(4,2)-morpholina-1(5,3)-indola-6(1 ,3)- pyridazinacycloundecaphane-4-yl)-2-((S)-2,7-diazaspiro[4.4]nonan-2-yl)acetamide cyclopropylpi perazin-1 -yl)-2-((S)-1 -methoxyethyl) pyridin-3-yl)-10,10-dimethyl-5,7-dioxo-1¹-(2,2,2- trifluoroethyl)-6¹,6²,6³,6⁴,6⁵,6⁶-hexahydro-1¹/7-8-oxa-2(4,2)-morpholina-1 (5,3)-indola-6(1 ,3)- pyridazinacycloundecaphane-4-yl)amino)-2-oxoethyl)-2,7-diazaspiro[4.4]nonane-2-carboxylate To a solution of (2²S,6³S,4S)-4-amino-1²-(5-(4-cyclopropylpiperazin-1-yl)-2-((S)-1- methoxyethyl)pyridin-3-yl)-10,10-dimethyl-1¹-(2,2,2-trifluoroethyl)-6¹,6²,6³,6⁴,6⁵,6⁶-hexahydro-1¹/7-8-oxa- 2(4, 2)-morpholina-1 (5, 3)-indola-6(1 ,3)-pyridazinacycloundecaphane-5, 7-dione (28 g, 34.43 mmol) and (S)-2-((S)-7-(tert-butoxycarbonyl)-2,7-diazaspiro[4.4]nonan-2-yl)-2-cyclopentylacetic acid (18.27 g, 51.76 mmol) in DMF (300 mL) at 0 °C was added DIPEA (240.4 mL, 1 .381 mol) and COMU (19.21g, 44.88 mmol). The resulting mixture was stirred for 1 h at room temperature. The reaction was quenched with H2O and the mixture was extracted with EtOAc (2 x 500 mL). The combined organic layers were washed with brine (2150 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by prep-TLC (10% MeOH/DCM) to afford the desired product (24.4 g, 62% yield) as a solid. LCMS (ESI) m/z [M + H] calcd for CeiHs FsNioOs: 1 145.68; found: 1145.5.

Step 2: Synthesis of (2S)-2-cyclopentyl-A/-((2²S,6³S,4S)-1²-(5-(4-cyclopropylpiperazin-1-yl)-2-((S)- 1 -methoxyethyl) pyridin-3-yl)-10,10-dimethyl-5,7-dioxo-1¹-(2, 2, 2-trifluoroethyl)-6¹,6²,6³,6⁴,6⁵,6⁶-hexahydro- 1¹/7-8-oxa-2(4,2)-morpholina-1 (5,3)-indola-6(1 ,3)-pyridazinacycloundecaphane-4-yl)-2-((S)-2,7- diazaspiro[4.4]nonan-2-yl)acetamide

To a solution of tert-butyl (5S)-7-((1 S)-1-cyclopentyl-2-(((2²S,6³S,4S)-1²-(5-(4- cyclopropylpi perazin-1 -yl)-2-((S)-1 -methoxyethyl) pyridin-3-yl)-10,10-dimethyl-5,7-dioxo-1¹-(2,2,2- trifluoroethyl)-6¹,6²,6³,6⁴,6⁵,6⁶-hexahydro-1¹/7-8-oxa-2(4,2)-morpholina-1 (5,3)-indola-6(1 ,3)- pyridazinacycloundecaphane-4-yl)amino)-2-oxoethyl)-2,7-diazaspiro[4.4]nonane-2-carboxylate (27.2 g, 23.76 mol) in DCM (200 mL) at 0 °C was added HCI in 1 ,4-dioxane (240 mL). The resulting mixture was stirred for 2 h at room temperature and then the mixture was concentrated under reduced pressure to afford the desired product (28 g, crude) as a solid. LCMS (ESI) m/z: [M + H] calcd for C56H79F3N10O6: 1045.62; found: 1045.5. Intermediate 5: Synthesis of benzyl (2/?,3/?)-3-cyclopropyl-1-methylaziridine-2-carboxylate

Step 1 Synthesis of (/?,E)-A/-(cyclopropylmethylene)-4-methylbenzenesulfinamide

To a solution of cyclopropanecarbaldehyde (6 g, 85.60 mmol) in THF (120 mL) was added (R)-4- methylbenzenesulfinamide (13.29 g, 85.60 mmol) and Ti(OEt)4 (39.05 g, 171.21 mmol) at room temperature under an atmosphere of nitrogen. The mixture was stirred at 75 °C for 2 h. The reaction mixture was poured into brine/H2O (1 :1 , 600 mL) at 0-15 °C. The mixture was filtered through a pad of Celite, and the pad was washed with EtOAc (6 x 200 mL). The combined filtrates were extracted with EtOAc (2 x 200 mL). The combined organic layers were washed with brine (200 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The crude product was purified by silica gel column chromatography. (0^10% EtOAc/pet. ether) to afford the product (14.6 g, 82% yield) as a solid.

Step 2: Synthesis of benzyl (2/?,3/?)-3-cyclopropyl-1-((/?)-p-tolylsulfinyl)aziridine-2-carboxylate To a solution of (/?)-A/-(cyclopropylmethylidene)-4-methylbenzenesulfinamide (100 g, 482.4 mmol) and benzyl 2-bromoacetate (143.66 g, 627.1 mmol) in THF (1 L) at -60 °C was added LiHMDS (1 M in THF, 627.1 mL, 627.1 mmol) dropwise over 30 min. The resulting mixture was stirred at -40 °C for 1 .5 h and then cold H2O (1 .5 L) was added. The aqueous layer was extracted with EtOAc (2 x 1 L), and the combined organic layers were washed with H2O (2 x 2 L) and brine (2 L), dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (5% EtOAc/pet. ether) to afford the desired product (137 g, 80% yield) as an oil.

Step 3: Synthesis of benzyl (2R,3/?)-3-cyclopropylaziridine-2-carboxylate

To a solution of benzyl (2/?,3/?)-3-cyclopropyl-1-((/?)-p-tolylsulfinyl)aziridine-2-carboxylate (60 g, 168.80 mmol) in acetone (786 mL), H2O (131 mL) and MeOH (102 mL) at 0 °C was added TFA (96.24 g, 844.0 mmol). The resulting mixture was stirred at 0 °C for 60 min and then the reaction mixture was added to NHa'^O (500 mL of 28% NHa'^O in 1 L of H2O) at 0 °C. The resulting mixture was extracted with EtOAc (3 x 700 mL) and the combined organic layers were washed with H2O (3 x 400 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (9% EtOAc/pet. ether) to afford the desired product. LCMS (ESI) m/z: [M + H] calcd for C13H15NO2: 218.12; found: 218.3.

Step 4: Synthesis of benzyl (2/?,3/?)-3-cyclopropyl-1-methylaziridine-2-carboxylate

To a mixture of benzyl (2R,3/?)-3-cyclopropylaziridine-2-carboxylate (1.0 g, 4.60 mmol) and 4A MS (1.0 g) in THF (20.5 mL) was added CsF (2.80 g, 18.41 mmol) and methylboronic acid (826.54 mg, 13.81 mmol). To the reaction mixture was added Cu(OAc)2 (835.99 mg, 4.60 mmol) and the vessel was then placed under an oxygen atmosphere. The reaction mixture was stirred for 16 h and then THF (20.7 mL), 4A MS (2.0 g), methylboronic acid (1653.07 mg, 27.62 mmol), CsF (5593.23 mg, 36.82 mmol) and Cu(OAc)2 (1671 .98 mg, 9.21 mmol) was added. The reaction mixture was placed under an oxygen atmosphere and stirred for 16h. The resulting mixture was filtered, washed with EtOAc (2 x 20 mL), and concentrated under reduced pressure. The residue was diluted with EtOAc (50 mL), and the organic layer was washed with brine (2 x 30 mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by normal phase column chromatography (17% EtOAc/pet. ether) to afford the desired product (800 mg, 75% yield) as a yellow oil. LCMS (ESI) m/z: [M + H] calcd for C14H17NO2: 232.13; found: 231.9.

Intermediate 6: Synthesis of benzyl (2S,3S)-3-cyclopropyl-1-methylaziridine-2-carboxylate

Step 1: Synthesis of (S,E)-A/-(cyclopropylmethylene)-4-methylbenzenesulfinamide

To a solution of cyclopropanecarbaldehyde (47.4 g, 676 mmol) in THF (1 L) was added (S)-4- methylbenzenesulfinamide (100 g, 644 mmol) and Ti(OEt)4 (238 g, 838 mmol) at room temperature under an atmosphere of nitrogen. The mixture was stirred at 60 °C for 3 h. The reaction mixture was diluted with H2O at 0 °C, filtered through a pad of Celite, and the pad was washed with EtOAc (3 x 1 L). The combined filtrates were washed with brine (2 x 1 L), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography. (17% EtOAc/pet. ether) to afford the product (100 g, 75% yield) as a white solid. LCMS (ESI) m/z: [M + H] calcd for C11H13NOS: 208.08; found: 208.1 .

Step 2: Synthesis of benzyl (2S,3S)-3-cyclopropyl-1-((S)-p-tolylsulfinyl)aziridine-2-carboxylate

To a solution of (S,E)-A/-(cyclopropylmethylene)-4-methylbenzenesulfinamide (100 g, 482 mmol) and benzyl 2-bromoacetate (144 g, 627 mmol) in THF (1 L) at -50 °C was added LiHMDS (1 M in THF, 627 mL, 627 mmol) dropwise over 10 min. The resulting mixture was stirred at -40 °C for 1 .5 h and then diluted with 1 M NH4CI<aq) (2 L) at 0 °C. The aqueous layer was extracted with EtOAc (2 x 1 L), and the combined organic layers were washed with H2O (2 x 2 L) and brine (2 L), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (5% EtOAc/pet. ether) to afford the desired product (100 g, 58% yield) as an oil. LCMS (ESI) m/z: [M + H] calcd for C₂oH₂iNO_eS: 356.13; found: 356.1.

Step 3: Synthesis of benzyl (2S,3S)-3-cyclopropylaziridine-2-carboxylate

To a solution of benzyl (2S,3S)-3-cyclopropyl-1-((S)-p-tolylsulfinyl)aziridine-2-carboxylate (70 g, 197 mmol) in acetone (786 mL), H2O (131 mL) and MeOH (102 mL) at 0 °C was added TFA (73 mL, 985 mmol) under an atmosphere of nitrogen. The reaction mixture was stirred at 0 °C for 1 h under an atmosphere of nitrogen. The resulting mixture was added to NHa'FTO (500 mL of 28% NHs’FW in 1 L of H2O) at 0 °C. The resulting mixture was extracted with EtOAc (3 x 500 mL) and the combined organic layers were washed with brine (3 x 400 mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (9% EtOAc/pet. ether) to afford the desired product (35 g, 82% yield) as a light-yellow oil. LCMS (ESI) m/z: [M + H] calcd for C13H15NO2: 218.12; found: 218.2.

Step 4: Synthesis of benzyl (2S,3S)-3-cyclopropyl-1-methylaziridine-2-carboxylate

To a mixture of benzyl (2S,3S)-3-cyclopropylaziridine-2-carboxylate (35 g, 161 mmol) and 4A MS (135 g) in MeCN (700 mL) was added Cu(OAc)2 (29.3 g, 161 mmol), 2,2’-bipyridine (25.2 g, 161 mmol), Na2COs (51 .2 g, 483 mmol), and methylboronic acid (28.9 g, 483 mmol) at room temperature. The resulting mixture was stirred for 24 h at 45 °C under oxygen atmosphere. The precipitated solids were collected by filtration and washed with EtOAc (3 x 40 mL). The combined organic layers were washed with 1 M NH4CI(aq) (3 x 300 mL) at 0 °C, washed with sat. aq. NaHCOs (3 x 300 mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by reverse phase chromatography (0^50% MeCN/HLO) to afford the desired product (20 g, 54% yield) as a light-yellow oil. LCMS (ESI) m/z: [M + H] calcd for C14H17NO2: 232.13; found: 232.2.

Intermediates 7 and 8: Synthesis of benzyl (2/?,3S)-3-(benzyloxy)-3-cyclopropyl-2- (methylamino)propanoate and benzyl (2/?,3/?)-3-(benzyloxy)-3-cyclopropyl-2- (methylamino)propanoate

Step 1: Synthesis of benzyl (2R,3S)-3-(benzyloxy)-3-cyclopropyl-2-(methylamino)propanoate and benzyl (2R,3R)-3-(benzyloxy)-3-cyclopropyl-2-(methylamino)propanoate

To a stirred solution of benzyl (2R,3R)-3-cyclopropyl-1-methylaziridine-2-carboxylate (150 g, 648 mmol) in benzyl alcohol (1.2 L) was added to BF3«Et2O (197 mL, 1.56 mol) at 0 °C under an atmosphere of nitrogen. The reaction mixture was heated to 50 °C and then stirred for 2 h at 50 °C under an atmosphere of nitrogen. The resulting mixture was diluted with sat. aq. NaHCOs (3 L) at 0 °C, extracted with EtOAc (3 x 1 L). The combined organic layers were washed with brine (3 L), dried over anhydrous Na2SO4, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by reversed phase chromatography (10^90% MeCN/FTO, 0.1% NH4HCO3). The product fractions were extracted with EtOAc (3 x 100 mL), dried over anhydrous Na2SO4, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (1 :1 EtOAc/pet. ether) to afford benzyl (2R,3S)-3-(benzyloxy)-3-cyclopropyl-2-(methylamino)propanoate (115 g, 50% yield, Rf = 0.6) as a light-yellow solid and benzyl (2R,3R)-3-(benzyloxy)-3-cyclopropyl-2- (methylamino)propanoate (63 g, 27% yield, Rf = 0.3) as a light-yellow oil. benzyl (2R,3S)-3-(benzyloxy)-3-cyclopropyl-2-(methylamino)propanoate: LCMS (ESI) m/z: [M + H] calcd for C21H25NO3: 340.19; found: 340.1. benzyl (2R,3R)-3-(benzyloxy)-3-cyclopropyl-2-(methylamino)propanoate: LCMS (ESI) m/z: [M + H] calcd for C21 H25NO3: 340.19; found: 340.1.

Intermediates 9 and 10: Synthesis of benzyl (2S,3/?)-3-(benzyloxy)-3-cyclopropyl-2-

(methylamino)propanoate and benzyl (2S,3S)-3-(benzyloxy)-3-cyclopropyl-2-

(methylamino)propanoate

Step 1 : Synthesis of benzyl (2S,3R)-3-(benzyloxy)-3-cyclopropyl-2-(methylamino)propanoate and benzyl (2S,3S)-3-(benzyloxy)-3-cyclopropyl-2-(methylamino)propanoate

To a stirred solution of benzyl (2S,3S)-3-cyclopropyl-1-methylaziridine-2-carboxylate (20 g, 86.5 mmol) in benzyl alcohol (160 mL) was added to BF3«Et2O (26.3 mL, 208 mmol) at 0 °C under an atmosphere of nitrogen. The reaction mixture was heated to 50 °C and then stirred for 2 h at 50 °C under an atmosphere of nitrogen. The resulting mixture was diluted with sat. aq. NaHCOs (500 mL) at 0 °C, extracted with EtOAc (3 x 150 mL). The combined organic layers were washed with brine (500 mL), dried over anhydrous Na2SO4, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by reversed phase chromatography (10^90% MeCN/FTO, 0.1 % NH4HCO3). The product fractions were extracted with EtOAc (3 x 100 mL), dried over anhydrous Na2SO4, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (1 :1 EtOAc/pet. ether) to afford benzyl (2S,3R)-3-(benzyloxy)-3-cyclopropyl-2-(methylamino)propanoate (10 g, 32% yield, Rf = 0.6) as a light-yellow oil and benzyl (2S,3S)-3-(benzyloxy)-3-cyclopropyl-2- (methylamino)propanoate (6 g, 19% yield, Rf = 0.3) as a light-yellow oil. benzyl (2S,3R)-3-(benzyloxy)-3-cyclopropyl-2-(methylamino)propanoate: LCMS (ESI) m/z: [M + H] calcd for C21 H25NO3: 340.19; found: 340.2. benzyl (2S,3S)-3-(benzyloxy)-3-cyclopropyl-2-(methylamino)propanoate: LCMS (ESI) m/z: [M + H] calcd for C21 H25NO3: 340.19; found: 340.1.

Intermediate 11 : Synthesis of (2/?,3S)-2-((tert-butoxycarbonyl)(methyl)amino)-3- cyclopropyl-3-hydroxypropanoic acid

Step 1'. Synthesis of benzyl (2R,3S)-3-(benzyloxy)-2-((te/Y-butoxycarbonyl)(methyl)amino)-3- cyclopropylpropanoate

To a stirred mixture of benzyl (2R,3S)-3-(benzyloxy)-3-cyclopropyl-2-(methylamino)propanoate (101 g, 298 mmol) in THF (800 mL) and H2O (800 mL) were added NaHCOs (50 g, 595 mmol) and di-fe/Y- butyl dicarbonate (97.4 g, 446 mmol) at 0 °C under an atmosphere of nitrogen. The reaction mixture was warmed to room temperature and stirred for 16 h under an atmosphere of nitrogen. The resulting mixture was diluted with H2O (2 L) and extracted with EtOAc (3 x 1.5 L). The combined organic layers were washed with brine (3 L), dried over anhydrous Na2SO4, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (14% EtOAc/pet. ether) to afford the desired product (130 g, 94% yield) as a light-yellow oil. LCMS (ESI) m/z: [M + H] calcd for C26H33NO5: 440.24; found: 440.2.

Step 2: Synthesis of (2/?,3S)-2-((te/Y-butoxycarbonyl)(methyl)amino)-3-cyclopropyl-3- hydroxypropanoic acid

To a stirred mixture of benzyl (2/?,3S)-3-(benzyloxy)-2-((te/Y-butoxycarbonyl)(methyl)amino)-3- cyclopropylpropanoate (130 g, 296 mmol) in MeOH (1 .3 L) was added Pd/C (130 g, 10 wt%) at room temperature under an atmosphere of nitrogen. The reaction mixture was stirred at room temperature for 16 h under a hydrogen atmosphere. The resulting mixture was filtered, and the filter cake was washed with MeOH (3 x 500 mL). The combined filtrates were concentrated under reduced pressure. The residue was purified by reversed phase chromatography (10^70% MeCN/H2O, 0.1 % HCO2H) to afford the desired product (57.7 g, 72% yield) as a solid. LCMS (ESI) m/z: [M + H] calcd for C12H21NO5: 260.15; found: 260.1 .

Intermediate 12: Synthesis of (2/?,3/?)-2-((tert-butoxycarbonyl)(methyl)amino)-3- cyclopropyl-3-hydroxypropanoic acid

Step 1'. Synthesis of benzyl (2/?,3/?)-3-(benzyloxy)-2-((te/Y-butoxycarbonyl)(methyl)amino)-3- cyclopropylpropanoate

To a stirred mixture of benzyl (2/?,3/?)-3-(benzyloxy)-3-cyclopropyl-2-(methylamino)propanoate (63 g, 185 mmol) in THF (500 mL) and H2O (500 mL) were added NaHCOs (31.2 g, 371 mmol) and di- tert-buty I dicarbonate (60.8 g, 278 mmol) at 0 °C under an atmosphere of nitrogen. The reaction mixture was warmed to room temperature and stirred for 16 h under an atmosphere of nitrogen. The resulting mixture was diluted with H2O (2 L) and extracted with EtOAc (3 x 1 L). The combined organic layers were washed with brine (3 L), dried over anhydrous Na2SO4, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (14% EtOAc/pet. ether) to afford the desired product (80 g, 93% yield) as a light-yellow oil. LCMS (ESI) m/z: [M + H] calcd for C26H33NO5: 440.24; found: 440.2.

Step 2: Synthesis of (2/?,3/?)-2-((te/Y-butoxycarbonyl)(methyl)amino)-3-cyclopropyl-3- hydroxypropanoic acid

To a stirred mixture of benzyl (2/?,3/?)-3-(benzyloxy)-2-((te/Y-butoxycarbonyl)(methyl)amino)-3- cyclopropylpropanoate (80 g, 752 mmol) in MeOH (800 mL) was added Pd/C (80 g, 10 wt%) at room temperature under an atmosphere of nitrogen. The reaction mixture was stirred at room temperature for 16 h under a hydrogen atmosphere. The resulting mixture was filtered, and the filter cake was washed with MeOH (3 x 500 mL). The combined filtrates were concentrated under reduced pressure. The residue was purified by reversed phase chromatography (10→70% MeCN/FW, 0.1 % HCO2H) to afford the desired product (38.3 g, 77% yield) as a solid. LCMS (ESI) m/z: [M + H] calcd for C12H21NO5: 260.15; found: 260.1 .

Intermediate 13: Synthesis of (2S,3/?)-2-((tert-butoxycarbonyl)(methyl)amino)-3- cyclopropyl-3-hydroxypropanoic acid

Step 1: Synthesis of benzyl (2S,3/?)-3-(benzyloxy)-2-((te/Y-butoxycarbonyl)(methyl)amino)-3- cyclopropylpropanoate

To a stirred mixture of benzyl (2S,3/?)-3-(benzyloxy)-3-cyclopropyl-2-(methylamino)propanoate (3.5 g, 10.3 mmol) in THF (35 mL) and H2O (35 mL) were added NaHCOs (1.73 g, 20.6 mmol) and di-te/Y- butyl dicarbonate (3.38 g, 15.5 mmol) at 0 °C under an atmosphere of nitrogen. The reaction mixture was warmed to room temperature and stirred for 16 h under an atmosphere of nitrogen. The resulting mixture was diluted with H2O (100 mL) and extracted with EtOAc (3 x 100 mL). The combined organic layers were washed with brine (300 mL), dried over anhydrous Na2SO4, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (14% EtOAc/pet. ether) to afford the desired product (4.5 g, 94% yield) as a light-yellow oil. LCMS (ESI) m/z: [M + H] calcd for C26H33NO5: 440.24; found: 440.2.

Step 2: Synthesis of (2S,3/?)-2-((te/Y-butoxycarbonyl)(methyl)amino)-3-cyclopropyl-3- hydroxypropanoic acid

To a stirred mixture of benzyl (2S,3/?)-3-(benzyloxy)-2-((te/Y-butoxycarbonyl)(methyl)amino)-3- cyclopropylpropanoate (4.7 g, 10.7 mmol) in MeOH (60 mL) was added Pd/C (4.7 g, 10 wt%) at room temperature under an atmosphere of nitrogen. The reaction mixture was stirred at room temperature for 5 h under a hydrogen atmosphere. The resulting mixture was filtered, and the filter cake was washed with MeOH (3 x 100 mL). The combined filtrates were concentrated under reduced pressure. The residue was purified by reversed phase chromatography (10^50% MeCN/H2O, 0.1 % HCO2H) to afford the desired product (1.8 g, 65% yield) as a solid. LCMS (ESI) m/z: [M + H] calcd for C12H21NO5: 260.15; found: 260.1.

Intermediate 14: Synthesis of (2S,3S)-2-((tert-butoxycarbonyl)(methyl)amino)-3- cyclopropyl-3-hydroxypropanoic acid

Step 1: Synthesis of benzyl (2S,3S)-3-(benzyloxy)-2-((te/Y-butoxycarbonyl)(methyl)amino)-3- cyclopropylpropanoate

To a stirred mixture of benzyl (2S,3S)-3-(benzyloxy)-3-cyclopropyl-2-(methylamino)propanoate (3 g, 10.3 mmol) in THF (30 mL) and H2O (30 mL) were added NaHCOs (1.48 g, 17.7 mmol) and di-te/Y-butyl dicarbonate (2.89 g, 13.3 mmol) at 0 °C under an atmosphere of nitrogen. The reaction mixture was warmed to room temperature and stirred for 16 h under an atmosphere of nitrogen. The resulting mixture was diluted with H2O (100 mL) and extracted with EtOAc (3 x 100 mL). The combined organic layers were washed with brine (300 mL), dried over anhydrous Na2SO4, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (14% EtOAc/pet. ether) to afford the desired product (3.8 g, 92% yield) as a light-yellow oil. LCMS (ESI) m/z: [M + H] calcd for C26H33NO5: 440.24; found: 440.2.

Step 2: Synthesis of (2S,3S)-2-((te/Y-butoxycarbonyl)(methyl)amino)-3-cyclopropyl-3- hydroxypropanoic acid

To a stirred mixture of benzyl (2S,3S)-3-(benzyloxy)-2-((te/Y-butoxycarbonyl)(methyl)amino)-3- cyclopropylpropanoate (3.7 g, 8.42 mmol) in MeOH (60 mL) was added Pd/C (3.7 g, 10 wt%) at room temperature under an atmosphere of nitrogen. The reaction mixture was stirred at room temperature for 5 h under a hydrogen atmosphere. The resulting mixture was filtered, and the filter cake was washed with MeOH (3 x 100 mL). The combined filtrates were concentrated under reduced pressure. The residue was purified by reversed phase chromatography (10^50% MeCN/H2O, 0.1 % HCO2H) to afford the desired product (1.28 g, 56% yield) as a solid. LCMS (ESI) m/z: [M + H] calcd for C12H21NO5: 260.15; found: 260.2.

Intermediate 15: Synthesis of (2R,3S)-3-(((S)-3-((((9H-fluoren-9- yl)methoxy)carbonyl)amino)-4-(allyloxy)-4-oxobutanoyl)oxy)-2-((tert- butoxycarbonyl)(methyl)amino)-3-cyclopropylpropanoic acid

Step 1'. Synthesis of 2-oxo-2-phenylethyl (2/?,3S)-2-((te/Y-butoxycarbonyl)(methyl)amino)-3- cyclopropyl-3-hydroxypropanoate

To a solution of (2/?,3S)-2-((fe/Y-butoxycarbonyl)(methyl)amino)-3-cyclopropyl-3- hydroxypropanoic acid (12.7 g, 49.1 mmol, 1.0 equiv) in DMF (100 mL) was added 2-bromo-1- phenylethan-1-one (19.5 g, 98.2 mmol, 2.0 equiv) and Na2COs (10.4 g, 98.2 mmol, 2.0 equiv). The mixture was stirred at room temperature for 1 h and was then concentrated under reduced pressure. The residue was diluted with H2O (1 L) and extracted with EtOAc (3 x 150 mL). The combined organic layers were washed with brine (2 x 150 mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to afford a residue. The residue was purified by silica gel column chromatography (2^50% EtOAc/pet. ether) to afford the desired product (18.4 g, 94% yield) as a yellow oil. LCMS (ESI) m/z: [M + Na] calcd for C20H27NO6: 400.17; found: 400.1.

Step 2: Synthesis of 1 -allyl 4-((1 S,2/?)-2-((te/Y-butoxycarbonyl)(methyl)amino)-1-cyclopropyl-3- oxo-3-(2-oxo-2-phenylethoxy)propyl) (((9/7-fluoren-9-yl)methoxy)carbonyl)-L-aspartate

To a solution of 2-oxo-2-phenylethyl (2/?,3S)-2-((te/Y-butoxycarbonyl)(methyl)amino)-3- cyclopropyl-3-hydroxypropanoate (18.4 g, 48.9 mmol, 1.0 equiv) in DCM (200 mL) was added DCC (14.1 g, 68.5 mmol, 1 .4 equiv), DMAP (598 mg, 4.89 mmol, 0.1 equiv), and (S)-3-((((9/7-fluoren-9- yl)methoxy)carbonyl)amino)-4-(allyloxy)-4-oxobutanoic acid (20.3 g, 51.4 mmol, 1.1 equiv). The mixture was stirred at room temperature for 3 h and was then diluted with H2O (150 mL) and extracted with EtOAc (3 x 150 mL). The combined organic layers were washed with brine (2 x 150 mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (2^50% EtOAc/pet. ether) to afford the desired product (25.6 g) as a white solid. LCMS (ESI) m/z: [M + Na] calcd for C42H46N2O11: 777.30; found: 777.3.

Step 3: Synthesis of (2/?,3S)-3-(((S)-3-((((9/7-fluoren-9-yl)methoxy)carbonyl)amino)-4-(allyloxy)-4- oxobutanoyl)oxy)-2-((te/Y-butoxycarbonyl)(methyl)amino)-3-cyclopropylpropanoic acid

To a solution of 1 -allyl 4-((1 S,2/?)-2-((fe/Y-butoxycarbonyl)(methyl)amino)-1-cyclopropyl-3-oxo-3- (2-oxo-2-phenylethoxy)propyl) (((9/7-fluoren-9-yl)methoxy)carbonyl)-L-aspartate (25.6 g, 33.9 mmol, 1.0 equiv) in 10% AcOH(aq ) (275 mL) was added Zn (55.8 g, 853 mmol, 25 equiv). The mixture was stirred at 10 °C for 1 h and then warmed to room temperature and stirred for 12 h. The reaction mixture was filtered, the residue was washed with DMF (2 x 50 mL), and the combined filtrates were concentrated under reduced pressure to afford a residue. The residue was purified by prep-HPLC to afford the desired product (15.3 g, 69% yield) as a white solid. LCMS (ESI) m/z [M + Na] calcd for C34H40N2O10: 659.23; found: 659.3.

Intermediate 16: Synthesis of (ferf-butoxycarbonyl)-L-valyl-L-valylglycyl-L-alanine

Step 1:

To a suspension of 2-CTC resin (15.0 mmol, 1.0 equiv, Sub: 1.07 mmol/g) in DCM (200 mL) was added Fmoc-Ala-OH (15.0 mmol, 1 .0 equiv) and DIPEA (60.0 mmol, 4.0 equiv). The suspension was agitated for 2 h at room temperature under an atmosphere of nitrogen. To the suspension was added MeOH (40 mL) and the suspension was agitated for an additional 30 min. The resin was then washed with DMF (5 x 300 mL).

Step 2

To the resin was added a solution of 20% piperidine in DMF (200 mL) and the suspension was agitated for 15 min at room temperature under an atmosphere of nitrogen. The resin was washed with DMF (5 x 300 mL) and filtered to afford the deprotected peptide resin.

Step 3:

To the resin was added a solution of Fmoc-Gly-OH (45.0 mmol, 3.0 equiv), HBTU (42.8 mmol, 2.85 equiv), and DIPEA (90.0 mmol, 6.0 equiv) in DMF (200 mL) and the suspension was agitated for 1 h at room temperature under an atmosphere of nitrogen. The resin was then washed with DMF (5 x 300 mL).

Step 4:

Repeat Steps 2-3 for the coupling of Fmoc-Val-OH (3.0 equiv) followed by Boc-Val-OH (3.0 equiv).

Step 5: Synthesis of (fert-butoxycarbonyl)-L-valyl-L-valylglycyl-L-alanine

The resin was washed with MeOH (5 x 300 mL) and dried under reduced pressure to afford the peptide resin. The peptide resin was treated with 20% HFIP in DCM (3 x 300 mL), filtered and concentrated under reduced pressure to afford the crude peptide (5.0 g) as a white solid. LCMS (ESI) m/z: [M + H] calcd for C20H36N4O7: 445.27; found: 445.3.

Intermediate 17: Synthesis of tert-butyl ^-(tert-butoxycarbonyO-A^-glycyl-L-valylglycyl-L- lysinate

Step 1‘.

To a suspension of 2-CTC resin (30.0 mmol, 1.0 equiv, Sub: 1.07 mmol/g) in DCM (300 mL) was added Fmoc-Gly-OH (30.0 mmol, 1 .0 equiv) and DIPEA (120 mmol, 4.0 equiv). The suspension was stirred for 2 h at room temperature under an atmosphere of nitrogen. Then, MeOH (70 mL) was added and the suspension was agitated for an additional 30 min. The resin was then washed with DMF (5 x 300 mL). Step 2:

To the resin was added a solution of 20% piperidine in DMF (300 mL) and the resin was agitated for 15 min at room temperature under an atmosphere of nitrogen. The resin was washed with DMF (5 x 300 mL) and filtered to afford the deprotected peptide resin.

Step 3:

A solution of Fmoc-Val-OH (90.0 mmol, 3.0 equiv), HBTU (85.5 mmol, 2.85 equiv), and DIPEA (180.0 mmol, 6.0 equiv) in DMF (300 mL) was added to the resin and the suspension was agitated for 1 h at room temperature under an atmosphere of nitrogen. The resin was then washed with DMF (5 x 300 mL).

Step 4:

Repeat Steps 2-3 for the coupling of Fmoc-Gly-OH (3.0 equiv) followed Cbz-OSu (3.0 equiv).

Step 5: Synthesis of ((benzyloxy)carbonyl)glycyl-L-valylglycine

The resin was washed with MeOH (5 x 300 mL) and dried under reduced pressure to afford the peptide resin. The peptide resin was treated with 20% HFIP/DCM (3 x 300 mL), filtered, and concentrated under reduced pressure to afford the crude peptide (7.5 g) as a white solid. LCMS (ESI) m/z: [M + H] calcd for C17H23N3O6: 366.17; found: 366.2.

Step 6: Synthesis of tert-butyl A/²-((benzyloxy)carbonyl)glycyl-L-valylglycyl-A/⁶-(fert- butoxycarbonyl)-L-lysinate

To a solution of ((benzyloxy)carbonyl)glycyl-L-valylglycine (7.50 g, 20.7 mmol, 1 .0 equiv) in DCM (70 mL) at 0 °C was added HOBt (8.41 g, 62.2 mmol, 3.0 equiv). The reaction mixture was stirred for 30 min and then EDCI (11.9 g, 62.2 mmol, 3.0 equiv), DIPEA (10.8 mL, 62.2 mmol, 3.0 equiv) and fert-butyl A/⁶-(tert-butoxycarbonyl)-L-lysinate hydrochloride (7.73 g, 22.8 mmol, 1.10 equiv) were added. The reaction mixture was stirred for 12 h at room temperature and then H2O (300 mL) was added, and the aqueous layer was extracted with EtOAc (3 x 200 mL). The combined organic layers were washed with brine (200 mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to afford a residue. The residue was purified by prep-HPLC to afford the desired product (10.0 g, 74% yield) as a white solid. LCMS (ESI) m/z: [M + H] calcd for C32H51 N5O9: 650.38; found: 650.4.

Step 7: Synthesis of fert-butyl ^-(tert-butoxycarbonyQ-AP-glycyl-L-valylglycyl-L-lysinate

To a solution of fert-butyl A/²-((benzyloxy)carbonyl)glycyl-L-valylglycyl-A/⁶-(fert-butoxycarbonyl)-L- lysinate (10.0 g, 15.4 mmol, 1 .0 equiv) in MeOH (100 mL) was added Pd/C (1 .0 g, 10 wt%). The suspension was degassed under vacuum and purged with H2 several times. The mixture was stirred under H2 (15 psi) at room temperature for 1 h. The reaction mixture was filtered and concentrated to afford the desired crude product (12.5 g) as a white solid. LCMS (ESI) m/z: [M + H] calcd for C24H45N5O7: 516.34; found: 516.3. Intermediate 18: Synthesis of (10S,16S,22S,26S,27R)-27-((tert- butoxycarbonyl)(methyl)amino)-10-(((tert-butoxycarbonyl)oxy)carbonyl)-26-cyclopropyl-22- ((6S,9S,15S)-6,9-diisopropyl-2,2,15-trimethyl-4,7,10,13-tetraoxo-3-oxa-5,8,11 ,14-tetraazahexadecan- 16-amido)-16-isopropyl-2,2-dimethyl-4,12,15,18,21 ,24-hexaoxo-3,25-dioxa-5,11 ,14,17,20- pentaazaoctacosan-28-oic acid

Step 1'.

To a suspension of 2-CTC resin (5.0 mmol, 1.0 equiv, Sub: 1.07 mmol/g) in DCM (100 mL) was added (2/?,3S)-3-(((S)-3-((((9/7-fluoren-9-yl)methoxy)carbonyl)amino)-4-(allyloxy)-4-oxobutanoyl)oxy)-2- ((te/Y-butoxycarbonyl)(methyl)amino)-3-cyclopropylpropanoic acid (5.0 mmol, 1.0 equiv) and DIPEA (20.0 mmol, 4.0 equiv). The suspension was agitated for 2 h at room temperature under an atmosphere of nitrogen. Then, MeOH (10 mL) was added and the suspension was agitated for an additional 0.5 h. The resin was then washed with DMF (5 x 200 mL).

Step 2:

To the resin was added a solution of 20% piperidine in DMF (100 mL) and the suspension was agitated for 15 min at room temperature under an atmosphere of nitrogen. The resin was washed with DMF (5 x 200 mL) and filtered to afford the deprotected peptide resin.

Step 3:

To a solution of (te/Y-butoxycarbonyl)-L-valyl-L-valylglycyl-L-alanine (15.0 mmol, 3.0 equiv) in DMF (100 mL) was added HOAt (15.0 mmol, 3.0 equiv) and DIC (15.0 mmol, 3.0 equiv). The mixture was stirred at room temperature for 12 h. The resin was then washed with DMF (5 x 200 mL).

Step 4:

To the resin was added a solution of PhSiHs (50.0 mmol, 10.0 equiv) in DCM (200 mL) and Pd(PPh3)4 (0.50 mmol, 0.1 equiv). The suspension was agitated at room temperature for 15 min. The resin was washed with DCM (5 x 200 mL) and then washed DMF (5 x 200 mL).

Step 5'.

To the resin was added a solution of te/Y-butyl ^-(tert-butoxycarbonyQ-AT-glycyl-L-valylglycyl-L- lysinate (15.0 mmol, 3.0 equiv) and HOAt (15.0 mmol, 3.0 equiv) in DMF (100 mL) followed by DIC (15.0 mmol, 3.0 equiv). The mixture was agitated at room temperature 12 h. The resin was then washed with DMF (5 x 200 mL).

Step 6:Synthesis of (10S,16S,22S,26S,27/?)-27-((te/Y-butoxycarbonyl)(methyl)amino)-10-(((te/Y- butoxycarbonyl)oxy)carbonyl)-26-cyclopropyl-22-((6S,9S,15S)-6,9-diisopropyl-2,2,15-trimethyl-4,7,10,13- tetraoxo-3-oxa-5,8,1 1 ,14-tetraazahexadecan-16-amido)-16-isopropyl-2,2-dimethyl-4,12,15,18,21 ,24- hexaoxo-3,25-dioxa-5,11 ,14,17,20-pentaazaoctacosan-28-oic acid

The resin was washed with MeOH (5 x 200 mL) and dried under reduced pressure to afford the peptide resin. The peptide resin was treated with 20% HFIP/DCM (3 x 200 mL), filtered and concentrated under reduced pressure to afford the crude peptide (666 mg) as a white solid. LCMS (ESI) m/z: [M + H] calcd for C60H103N11O20: 1298.75; found: 1298.9.

Example 2: Synthesis of (S)-6-amino-2-({[(S)-2-({[(S)-2-[(S)-2-({[(S)-2-[(S)-2-amino-3- methylbutyrylamino]-3-methylbutyrylamino]methyl}carbonylamino)propionylamino]-3-[(1S,2R)-3- [(S)-7-[(S)-cyclopentyl[N-(6S,8S,14S,21 M)-21 -[5-(4-cyclopropyl-1 -piperazinyl)-2-[(S)-1 - methoxyethyl]-3-pyridyl]-18,18-dimethyl-9,15-dioxo-22-(2,2,2-trifluoroethyl)-5,16-dioxa-2,10,22,28- tetraazapentacyclo[18.5.2.1²,^s.1¹°,¹⁴.0²³, ²⁷]nonacosa-1 (26), 20, 23(27), 24-tetraen-8- ylcarbamoyl]methyl]-2,7-diaza-2-spiro[4.4]nonyl]-1 -cyclopropyl-2-(methylamino)-3- oxopropoxycarbonyl]propionylamino]methyl}carbonylamino)-3- methylbutyrylamino]methyl}carbonylamino)hexanoic acid (Compound A-5)

Step 1: Synthesis of tert-butyl A/⁶-(fert-butoxycarbonyl)-A/²-((2S)-4-((1 S,2R)-2-((fert- butoxycarbony l)(methyl)amino)-3-((5S)-7-((1 S)-1 -cyclopentyl-2-(((1²/?,2²S,6³S,4S)-1²-(5-(4- cyclopropylpi perazin-1 -yl)-2-((S)-1 -methoxyethyl) pyridin-3-yl)-10,10-dimethyl-5,7-dioxo-1¹-(2,2,2- trifluoroethyl)-6¹,6²,6³,6⁴,6⁵,6⁶-hexahydro-1¹/7-8-oxa-2(4,2)-morpholina-1 (5,3)-indola-6(1 ,3)- pyridazinacycloundecaphane-4-yl)amino)-2-oxoethyl)-2,7-diazaspiro[4.4]nonan-2-yl)-1-cyclopropyl-3- oxopropoxy)-2-((S)-2-(2-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-3- methylbutanamido)acetamido)propanamido)-4-oxobutanoyl)glycyl-L-valylglycyl-/_-lysinate

To a solution of (10S,16S,22S,26S,27/?)-27-((tert-butoxycarbonyl)(methyl)amino)-10-(((tert- butoxycarbonyl)oxy)carbonyl)-26-cyclopropyl-22-((6S,9S,15S)-6,9-diisopropyl-2,2,15-trimethyl-4,7,10,13- tetraoxo-3-oxa-5,8,11 ,14-tetraazahexadecan-16-amido)-16-isopropyl-2,2-dimethyl-4,12,15,18,21 ,24- hexaoxo-3,25-dioxa-5,11 ,14,17,20-pentaazaoctacosan-28-oic acid (666 mg, 1.0 equiv) in DMF (100 mL) was added HATU (1.10 equiv), (2S)-2-cyclopentyl-A/-((2²S,6³S,4S)-1²-(5-(4-cyclopropylpiperazin-1-yl)-2- ((S)-1 -methoxyethyl) pyridin-3-yl)-10,10-dimethyl-5,7-dioxo-1¹-(2,2,2-trifluoroethyl)-6¹,6²,6³,6⁴,6⁵,6⁶- hexahydro-1¹/7-8-oxa-2(4,2)-morpholina-1 (5,3)-indola-6(1 ,3)-pyridazinacycloundecaphane-4-yl)-2-((S)- 2,7-diazaspiro[4.4]nonan-2-yl)acetamide (1 .0 equiv) followed by DI PEA dropwise to pH 8-9. The reaction mixture was stirred at room temperature for 1 h and concentrated under reduced pressure to afford a crude residue. The residue was diluted with DCM (100 mL) and 1 M HCI(aq). The aqueous layer was extracted with DCM (3 x 100 mL) and the combined organic layers were dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to afford the crude product.

Step 2: Synthesis of (S)-6-amino-2-({[(S)-2-({[(S)-2-[(S)-2-({[(S)-2-[(S)-2-amino-3- methylbutyrylamino]-3-methylbutyrylamino]methyl}carbonylamino)propionylamino]-3-[(1 S,2/?)-3-[(S)-7- [(S)-cyclopentyl[A/-(6S,8S,14S,21 /W)-21-[5-(4-cyclopropyl-1-piperazinyl)-2-[(S)-1-methoxyethyl]-3-pyridyl]- 18,18-dimethyl-9,15-dioxo-22-(2,2,2-trifluoroethyl)-5,16-dioxa-2,10,22,28- tetraazapentacyclo[18.5.2.1²,⁶.T^lo,'^l4.0²³,²⁷]nonacosa-1 (26),20,23(27),24-tetraen-8-ylcarbamoyl]methyl]- 2,7-diaza-2-spiro[4.4]nonyl]-1-cyclopropyl-2-(methylamino)-3- oxopropoxycarbonyl]propionylamino]methyl}carbonylamino)-3- methylbutyrylamino]methyl}carbonylamino)hexanoic acid

The residue was treated with 2.5% H2O, 2.5% triisopropylsilane, 2.5% 3-mercaptopropionic acid and 92.5% TFA (100 mL) for 10 min. The crude product was precipitated with cold isopropyl ether (500 mL). The mixture was filtered, and the filter cake was washed with isopropyl ether (2 x 500 mL). The crude product was dried under reduced pressure to afford the crude product (3.2 g). The crude product was purified by prep-HPLC (0.075% TFA in FLO/MeCN) to afford the desired product (201 mg, 1 .1 % yield, TFA salt) as a white solid. LCMS (ESI) m/z: [M + 3H]/3 calcd for C97H148F3N21O19: 657.05; found: 657.4.

Intermediate 19: Synthesis of (tert-butoxycarbonyl)-L-valyl-L-valyl-L-valylglycyl-L-alanine

Step 1'.

To a suspension of 2-CTC resin (15.0 mmol, 1.0 equiv, Sub: 1.07 mmol/g) in DCM (200 mL) was added Fmoc-Ala-OH (15.0 mmol, 1 .0 equiv) and DIPEA (60.0 mmol, 4.0 equiv). The suspension was agitated for 2 h at room temperature under an atmosphere of nitrogen. Then, MeOH (40 mL) was added and the suspension was agitated for an additional 30 min. The resin was then washed with DMF (5 x 300 mL).

Step 2:

Step 3:

To the resin was added a solution of Fmoc-Gly-OH (45.0 mmol, 3.0 equiv), HBTU (42.8 mmol, 2.85 equiv), and DIPEA (90.0 mmol, 6.0 equiv) in DMF (200 mL) and the suspension was agitated for 1 h at room temperature under an atmosphere of nitrogen. The resin was then washed with DMF (5 x 300 mL). Step 4:

Successively repeat Steps 2-3 for the coupling of Fmoc-Val-OH (3.0 equiv), Fmoc-Val-OH (3.0 equiv), and Boc-Val-OH (3.0 equiv).

Step 5: Synthesis of (tert-butoxycarbonyl)-L-valyl-L-valyl-L-valylglycyl-L-alanine

The resin was washed with MeOH (5 x 300 mL) and dried under reduced pressure to afford the peptide resin. The peptide resin was treated with 20% HFIP in DCM (3 x 300 mL), filtered and concentrated under reduced pressure to afford the crude peptide (6.0 g) as a white solid. LCMS (ESI) m/z: [M + H] calcd for C25H45N5O8: 544.33; found: 544.4.

Intermediate 20: Synthesis of (6S,9S,12S,18S,21S,25S,26R)-26-((tert- butoxycarbonyl)(methyl)amino)-21 -(((10S,16S)-10-(fert-butoxycarbonyl)-16-isopropyl-2,2-dimethyl-

4,12,15,18-tetraoxo-3-oxa-5,11 ,14,17-tetraazanonadecan-19-yl)carbamoyl)-25-cyclopropyl-6,9,12- triisopropyl-2,2,18-trimethyl-4,7,10,13,16,19,23-heptaoxo-3,24-dioxa-5,8,11 ,14,17,20- hexaazaheptacosan-27-oic acid

Step 1'.

To a suspension of 2-CTC resin (5.0 mmol, 1.0 equiv, Sub: 1.07 mmol/g) in DCM (100 mL) was added (2/?,3S)-3-(((S)-3-((((9/7-fluoren-9-yl)methoxy)carbonyl)amino)-4-(allyloxy)-4-oxobutanoyl)oxy)-2- ((fe/Y-butoxycarbonyl)(methyl)amino)-3-cyclopropylpropanoic acid (6.0 mmol, 1.0 equiv) and DIPEA (20.0 mmol, 4.0 equiv). The suspension was agitated for 2 h at room temperature under an atmosphere of nitrogen. Then, MeOH (10 mL) was added and the suspension was agitated for an additional 0.5 h. The resin was then washed with DMF (5 x 200 mL).

Step 2:

To the resin was added a suspension of 20% piperidine in DMF (100 mL) and the suspension was agitated for 15 min at room temperature under an atmosphere of nitrogen. The resin was washed with DMF (5 x 200 mL) and filtered to afford the deprotected peptide resin.

Step 3:

To the resin was added a solution of (tert-butoxycarbonyl)-L-valyl-L-valyl-L-valylglycyl-L-alanine (15.0 mmol, 3.0 equiv) in DMF (100 mL) followed by HOAt (15.0 mmol, 3.0 equiv) and DIC (15.0 mmol, 3.0 equiv). The suspension was agitated at room temperature for 12 h. The resin was then washed with DMF (5 x 200 mL).

Step 4:

To the resin was added a solution of PhSiHs (50.0 mmol, 10.0 equiv) in DCM (200 mL) and Pd(PPhs)4 (0.50 mmol, 0.1 equiv). The suspension was agitated at room temperature for 15 min. The resin was washed with DCM (5 x 200 mL) and then washed with DMF (5 x 200 mL). Step 5'.

To the resin was added a solution of tert-butyl A/⁶-(tert-butoxycarbonyl)-A/²-glycyl-/--valylglycyl-/_- lysinate (15.0 mmol, 3.0 equiv) and HOAt (15.0 mmol, 3.0 equiv) in DMF (100 mL) followed by DIC (15.0 mmol, 3.0 equiv). The suspension was agitated at room temperature 12 h. The resin was then washed with DMF (5 x 200 mL).

Step 6: Synthesis of (6S,9S,12S,18S,21 S,25S,26/?)-26-((fert-butoxycarbonyl)(methyl)amino)-21- (((10S,16S)-10-(fert-butoxycarbonyl)-16-isopropyl-2,2-dimethyl-4, 12,15,18-tetraoxo-3-oxa-5,11 ,14,17- tetraazanonadecan-19-yl)carbamoyl)-25-cyclopropyl-6,9,12-triisopropyl-2,2,18-trimethyl- 4, 7,10,13,16,19, 23-heptaoxo-3,24-dioxa-5, 8,11 ,14,17, 20-hexaazaheptacosan-27-oic acid

The resin was washed with MeOH (5 x 200 mL) and dried under reduced pressure to afford the peptide resin. The peptide resin was treated with 20% HFIP/DCM (3 x 200 mL), filtered and concentrated under reduced pressure to afford the crude peptide (600 mg) as a white solid. LCMS (ESI) m/z: [M + H] calcd for C65H112N12O21: 1397.81 ; found: 1397.9.

Example 3: Synthesis of (S)-6-amino-2-({[(S)-2-({[(S)-2-[(S)-2-({[(S)-2-[(S)-2-[(S)-2-amino-3- methylbutyrylamino]-3-methylbutyrylamino]-3- methylbutyrylamino]methyl}carbonylamino)propionylamino]-3-[(1S,2/?)-3-[(S)-7-[(S)-cyclopentyl[/V-

(6S,8S,14S,21 M)-21 -[5-(4-cyclopropyl-1 -piperazinyl)-2-[(S)-1 -methoxyethyl]-3-pyridyl]-18,18- dimethyl-9,15-dioxo-22-(2,2,2-trifluoroethyl)-5,16-dioxa-2,10,22,28- tetraazapentacyclo[18.5.2.1²,^s.1¹°,¹⁴.0²³, ²⁷]nonacosa-1 (26), 20, 23(27), 24-tetraen-8- ylcarbamoyl]methyl]-2,7-diaza-2-spiro[4.4]nonyl]-1 -cyclopropyl-2-(methylamino)-3- oxopropoxycarbonyl]propionylamino]methyl}carbonylamino)-3- methylbutyrylamino]methyl}carbonylamino)hexanoic acid (Compound A-7) Step 1'. Synthesis of tert-butyl A/⁶-(fert-butoxycarbonyl)-A/²-((2S)-4-((1 S,2R)-2-((fert- butoxycarbony l)(methyl)amino)-3-((5S)-7-((1 S)-1 -cyclopentyl-2-(((1²/?,2²S,6³S,4S)-12-(5-(4- cyclopropylpi perazin-1 -yl)-2-((S)-1 -methoxyethyl) pyridin-3-yl)-10,10-dimethyl-5,7-dioxo-1¹-(2,2,2- trifluoroethyl)-6¹,6²,6³,6⁴,6⁵,6⁶-hexahydro-1¹/7-8-oxa-2(4,2)-morpholina-1 (5,3)-indola-6(1 ,3)- pyridazinacycloundecaphane-4-yl)amino)-2-oxoethyl)-2,7-diazaspiro[4.4]nonan-2-yl)-1-cyclopropyl-3- oxopropoxy)-2-((S)-2-(2-((S)-2-((S)-2-((S)-2-((fert-butoxycarbonyl)amino)-3-methylbutanamido)-3- methylbutanamido)-3-methylbutanamido)acetamido)propanamido)-4-oxobutanoyl)glycyl-L-valylglycyl-L- lysinate

To a solution of (6S,9S,12S,18S,21 S,25S,26/?)-26-((fert-butoxycarbonyl)(methyl)amino)-21- (((10S,16S)-10-(fert-butoxycarbonyl)-16-isopropyl-2,2-dimethyl-4,12,15,18-tetraoxo-3-oxa-5,1 1 ,14,17- tetraazanonadecan-19-yl)carbamoyl)-25-cyclopropyl-6,9,12-triisopropyl-2,2,18-trimethyl- 4,7,10,13,16,19,23-heptaoxo-3,24-dioxa-5,8,1 1 ,14,17,20-hexaazaheptacosan-27-oic acid (600 mg, 1.0 equiv) in DMF (100 mL) was added HATU (1.10 equiv), (2S)-2-cyclopentyl-A/-((2²S,6³S,4S)-1²-(5-(4- cyclopropylpi perazin-1 -yl)-2-((S)-1 -methoxyethyl) pyridin-3-yl)-10,10-dimethyl-5,7-dioxo-1¹-(2,2,2- trifluoroethyl)-6¹,6²,6³,6⁴,6⁵,6⁶-hexahydro-1¹/7-8-oxa-2(4,2)-morpholina-1 (5,3)-indola-6(1 ,3)- pyridazinacycloundecaphane-4-yl)-2-((S)-2,7-diazaspiro[4.4]nonan-2-yl)acetamide (1.0 equiv) followed by DIPEA dropwise to pH 8-9. The reaction mixture was stirred at room temperature for 1 h and concentrated under reduced pressure to afford a crude residue. The residue was diluted with DCM (100 mL) and 1 M HCI(aq). The aqueous layer was extracted with DCM (3 x 100 mL) and the combined layers were dried over Na2SO4, filtered, and concentrated under reduced pressure to afford the crude product.

Step 2: Synthesis of (S)-6-amino-2-({[(S)-2-({[(S)-2-[(S)-2-({[(S)-2-[(S)-2-[(S)-2-amino-3- methylbutyrylamino]-3-methylbutyrylamino]-3-methylbutyrylamino]methyl}carbonylamino)propionylamino]- 3-[(1 S,2R)-3-[(S)-7-[(S)-cyclopentyl[/V-(6S,8S, 14S,21 M)-21 -[5-(4-cyclopropyl-1 -piperazinyl)-2-[(S)-1 - methoxyethyl]-3-pyridyl]-18,18-dimethyl-9,15-dioxo-22-(2,2,2-trifluoroethyl)-5,16-dioxa-2,10,22,28- tetraazapentacyclo[18.5.2.1²,⁶.1¹⁰,'^l4.0²³,²⁷]nonacosa-1 (26),20,23(27),24-tetraen-8-ylcarbamoyl]methyl]- 2,7-diaza-2-spiro[4.4]nonyl]-1-cyclopropyl-2-(methylamino)-3- oxopropoxycarbonyl]propionylamino]methyl}carbonylamino)-3- methylbutyrylamino]methyl}carbonylamino)hexanoic acid

The residue was treated with 2.5% H2O, 2.5% triisopropylsilane, 2.5% 3-mercaptopropionic acid and 92.5% TFA (100 mL) for 10 min. The crude product was precipitated with cold isopropyl ether (500 mL). The mixture was filtered, and the filter cake was washed with isopropyl ether (2 x 500 mL). The crude product was dried under reduced pressure to afford the crude product (3.2 g). The crude product was purified by prep-HPLC (0.075% TFA in H2O/MeCN) to afford the desired product (234 mg, 2.2% yield, TFA salt) as a white solid. LCMS (ESI) m/z: [M + 2H]/2 calcd for C102H157F3N22O20: 1034.60; found: 1035.1. Intermediate 21 : Synthesis ofN²,N⁶ bisftert-butoxycarbonyQ-L-lysyl-L-leucyl-L-valyl-L- valyl-L-valylglycyl-L-alanine -

Step 1'.

Step 2:

Step 3:

Step 4:

Repeat Steps 2-3 for the coupling of Fmoc-Val-OH (3.0 equiv), Fmoc-Val-OH (3.0 equiv), Fmoc- Val-OH (3.0 equiv), Fmoc-Leu-OH (3.0 equiv) and Boc-Lys (Boc)-OH (3.0 equiv).

Step 5: Synthesis of N² ,/\/⁶-bis(tert-butoxyca rbony l)l-L-lysy l-L-leucy l-L-valy l l-L-valy ll-L-va ly Ig lycy ll-L- alanine

The resin was washed with MeOH (5 x 300 mL) and dried under reduced pressure to afford the peptide resin. The peptide resin was treated with 20% HFIP in DCM (3 x 300 mL), filtered and concentrated under reduced pressure to afford the crude peptide (10.0 g) as a white solid. LCMS (ESI) m/z: [M + H] calcd for C42H76N8O12: 885.57; found: 885.6.

Intermediate 22: Synthesis of tert-butyl glycyl-L-valinate Step 1: Synthesis of fert-butyl ((benzyloxy)carbonyl)glycyl-L-valinate

To a solution of ((benzyloxy)carbonyl)glycine (10.0 g, 47.8 mmol, 1.0 equiv) in DCM (100 mL) at 0 °C was added HOBt (19.4 g, 143 mmol, 3.0 equiv). The reaction mixture was stirred for 30 min and then EDCI (27.5 g, 143 mmol, 3.0 equiv), DIPEA (25 mL, 143 mmol, 3.0 equiv) and fert-butyl L-valinate (11.0 g, 52.6 mmol, 1 .10 equiv) were added. The reaction mixture was stirred at room temperature for 12 h and then H2O (300 mL) was added, and the aqueous layer was extracted with DCM (3 x 200 mL). The combined organic layers were washed with brine (200 mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to afford a residue. The residue was purified by prep-HPLC to afford the desired product (15.0 g, 83% yield) as a white solid. LCMS (ESI) m/z: [M + H] calcd for C19H28N2O5: 365.21 ; found: 365.2.

Step 7: Synthesis of fert-butyl glycyl-L-valinate

To a solution fert-butyl ((benzyloxy)carbonyl)glycyl-L-valinate (15.0 g, 41.2 mmol, 1.0 equiv) in MeOH (150 mL) was added Pd/C (1.5 g, 10 wt%). The suspension was degassed under vacuum and purged with H2 several times. The mixture was stirred under H2 (15 psi) at room temperature for 1 h. The reaction mixture was filtered and concentrated to afford the desired crude product (8.0 g) as a white solid. LCMS (ESI) m/z [M + H] calcd for C11H22N2O3: 231.17; found: 231.5.

Intermediate 23: Synthesis of (10S,13S,16S,19S,22S,28S,31S,35S,36R)-31-((2-(((S)-1-(fert- butoxy)-3-methyl-1 -oxobutan-2-yl)amino)-2-oxoethyl)carbamoyl)-36-((ferf- butoxycarbonyl)(methyl)amino)-10-((ferf-butoxycarbonyl)amino)-35-cyclopropyl-13-isobutyl- 16,19,22-triisopropyl-2,2,28-trimethyl-4,11 ,14,17,20,23,26,29,33-nonaoxo-3,34-dioxa- 5,12,15,18,21 ,24,27,30-octaazaheptatriacontan-37-oic acid

Step 1:

To a suspension of 2-CTC resin (5.0 mmol, 1.0 equiv, Sub: 1.07 mmol/g) in DCM (100 mL) was added (2/?,3S)-3-(((S)-3-((((9/7-fluoren-9-yl)methoxy)carbonyl)amino)-4-(allyloxy)-4-oxobutanoyl)oxy)-2- ((fert-butoxycarbonyl)(methyl)amino)-3-cyclopropylpropanoic acid (5.0 mmol, 1.0 equiv) and DIPEA (20.0 mmol, 4.0 equiv). The suspension was agitated for 2 h at room temperature under an atmosphere of nitrogen. Then, MeOH (10 mL) was added and the suspension was agitated for an additional 0.5 h. The resin was then washed with DMF (5 x 200 mL).

Step 2:

To the resin was added a solution of 20% piperidine in DMF (100 mL) and the suspension was agitated for 15 min at room temperature under an atmosphere of nitrogen. The resin was washed with DMF (5 x 200 mL) and filtered to afford the deprotected peptide resin. Step 3:

To the resin was added a solution of A/²,A/⁶-bis(tert-butoxycarbonyl)-/--lysyl-/--leucyl-/--valyl-/_- valyl-/_-valylglycyl-/_-alanine (15.0 mmol, 3.0 equiv) in DMF (100 mL) followed by HOAt (15.0 mmol, 3.0 equiv) and DIC (15.0 mmol, 3.0 equiv). The suspension was agitated at room temperature for 12 h. The resin was then washed with DMF (5 x 200 mL).

Step 4:

To the resin was added a solution of PhSibh (50.0 mmol, 10.0 equiv) in DCM (200 mL) and Pd(PPh3)4 (0.50 mmol, 0.1 equiv). The mixture was agitated at room temperature for 15 min. The resin was washed with DCM (5 x 200 mL) and then washed with DMF (5 x 200 mL).

Step 5'.

To the resin was added a solution of te/Y-butyl glycyl-L-valinate (15.0 mmol, 3.0 equiv) and HOAt (15.0 mmol, 3.0 equiv) in DMF (100 mL) followed by DIC (15.0 mmol, 3.0 equiv). The mixture was agitated at room temperature 12 h. The resin was then washed with DMF (5 x 200 mL).

Step 6: Synthesis of (10S,13S,16S,19S,22S,28S,31 S,35S,36R)-31-((2-(((S)-1-(fert-butoxy)-3- methyl-1-oxobutan-2-yl)amino)-2-oxoethyl)carbamoyl)-36-((te/Y-butoxycarbonyl)(methyl)amino)-10-((te/Y- butoxy carbonyl)amino)-35-cyclopropyl-13-isobuty 1-16,19, 22-triisopropyl-2, 2, 28-trimethyl- 4,11 ,14,17,20,23,26,29,33-nonaoxo-3,34-dioxa-5,12,15,18,21 ,24,27,30-octaazaheptatriacontan-37-oic acid

The resin was washed with MeOH (5 x 200 mL) and dried under reduced pressure to afford the peptide resin. The peptide resin was treated with 20% HFIP/DCM (3 x 200 mL), filtered and concentrated under reduced pressure to afford the crude peptide (621 mg) as a white solid. LCMS (ESI) m/z: [M + H] calcd for C69H120N12O21 : 1453.88; found: 1453.9.

Example 4: Synthesis of (S)-2-({[(S)-3-[(1S,2R)-3-[(S)-7-[(S)-cyclopentyl[W-(6S,8S,14S,21/W)- 21 -[5-(4-cyclopropyl-1 -piperazinyl)-2-[(S)-1 -methoxyethyl]-3-pyridyl]-18,18-dimethyl-9,15-dioxo-22- (2,2,2-trifluoroethyl)-5,16-dioxa-2,10,22,28-tetraazapentacyclo[18.5.2.1²,^s.1¹°,¹⁴.0²³,²⁷]nonacosa- 1(26),20,23(27),24-tetraen-8-ylcarbamoyl]methyl]-2,7-diaza-2-spiro[4.4]nonyl]-1 -cyclopropyl-2- (methylamino)-3-oxopropoxycarbonyl]-2-[(S)-2-({[(S)-2-[(S)-2-[(S)-2-[(S)-2-[(S)-1 ,5- diaminopentylcarbonylamino]-4-methylvalerylamino]-3-methylbutyrylamino]-3- methylbutyrylamino]-3- methylbutyrylamino]methyl}carbonylamino)propionylamino]propionylamino]methyl}carbonylamin o)-3-methylbutyric acid (Compound A-8)

Step 1: Synthesis of fert-butyl (10S,13S,16S,19S,22S,28S,31 S,37S)-31-(2-((1 S,2R)-2-((tert- butoxycarbonyl)(methyl)amino)-3-((5S)-7-((1 S)-1 -cyclopentyl-2-(((1²/?,2²S,6³S,4S)-1²-(5-(4- cyclopropylpi perazin-1 -yl)-2-((S)-1 -methoxyethyl) pyridin-3-yl)-10,10-dimethyl-5,7-dioxo-1¹-(2,2,2- trifluoroethyl)-6¹,6²,6³,6⁴,6⁵,6⁶-hexahydro-1¹/7-8-oxa-2(4,2)-morpholina-1 (5,3)-indola-6(1 ,3)- pyridazinacycloundecaphane-4-yl)amino)-2-oxoethyl)-2,7-diazaspiro[4.4]nonan-2-yl)-1-cyclopropyl-3- oxopropoxy)-2-oxoethyl)-10-((tert-butoxycarbonyl)amino)-13-isobutyl-16,19,22,37-tetraisopropyl-2,2,28- trimethyl-4,1 1 ,14,17, 20, 23, 26, 29,32, 35-decaoxo-3-oxa-5,12, 15, 18, 21 ,24,27,30,33,36- decaazaoctatriacontan-38-oate

To a solution of (10S,13S,16S,19S,22S,28S,31 S,35S,36R)-31-((2-(((S)-1-(tert-butoxy)-3-methyl- 1-oxobutan-2-yl)amino)-2-oxoethyl)carbamoyl)-36-((tert-butoxycarbonyl)(methyl)amino)-10-((tert- butoxycarbonyl)amino)-35-cyclopropyl-13-isobutyl-16,19,22-triisopropyl-2,2,28-trimethyl- 4,11 ,14,17,20,23,26,29,33-nonaoxo-3,34-dioxa-5,12,15,18,21 ,24,27,30-octaazaheptatriacontan-37-oic acid (621 mg, 1.0 equiv) in DMF (100 mL) was added HATU (1.10 equiv), (2S)-2-cyclopentyl-A/- ((2²S,6³S,4S)-1²-(5-(4-cyclopropylpiperazin-1-yl)-2-((S)-1-methoxyethyl)pyridin-3-yl)-10,10-dimethyl-5,7- dioxo-1¹-(2,2,2-trifluoroethyl)-6¹,6²,6³,6⁴,6⁵,6⁶-hexahydro-1¹/7-8-oxa-2(4,2)-morpholina-1 (5,3)-indola- 6(1 ,3)-pyridazinacycloundecaphane-4-yl)-2-((S)-2,7-diazaspiro[4.4]nonan-2-yl)acetamide (1 .0 equiv) followed by DI PEA dropwise to pH 8-9. The reaction mixture was stirred at room temperature for 1 h and concentrated under reduced pressure to afford a crude residue. The residue was diluted with DCM (100 mL) and 1 M HCI(aq). The aqueous layer was extracted with DCM (3 x 100 mL) and the combined layers were dried over Na2SO4, filtered, and concentrated under reduced pressure to afford the crude product.

Step 2: Synthesis of (S)-2-({[(S)-3-[(1S,2R)-3-[(S)-7-[(S)-cyclopentyl[/V-(6S,8S,14S,21/W)-21-[5-(4- cyclopropyl-1-piperazinyl)-2-[(S)-1-methoxyethyl]-3-pyridyl]-18,18-dimethyl-9,15-dioxo-22-(2,2,2- trifluoroethyl)-5,16-dioxa-2,10,22,28-tetraazapentacyclo[18.5.2.1²,⁶.1'^l°,'^l4.0^{23 27}]nonacosa- 1 (26), 20, 23(27), 24-tetraen-8-ylcarbamoyl]methyl]-2, 7-diaza-2-spiro[4.4]nonyl]-1 -cyclopropyl-2- (methylamino)-3-oxopropoxycarbonyl]-2-[(S)-2-({[(S)-2-[(S)-2-[(S)-2-[(S)-2-[(S)-1 ,5- diaminopentylcarbonylamino]-4-methylvalerylamino]-3-methylbutyrylamino]-3-methylbutyrylamino]-3- methylbutyrylamino]methyl}carbonylamino)propionylamino]propionylamino]methyl}carbonylamino)-3- methylbutyric acid

The residue was treated with 2.5% H2O, 2.5% triisopropylsilane, 2.5% 3-mercaptopropionic acid and 92.5% TFA (100 mL) for 2 h. The crude product was precipitated with cold isopropyl ether (500 mL). The mixture was filtered, and the filter cake was washed with isopropyl ether (2 x 500 mL). The crude product was dried under reduced pressure to afford the crude product (3.2 g). The crude product was purified by prep-HPLC (0.075% TFA in H2O/MeCN) to afford the desired product (279 mg, 2.6% yield, TFA salt) as a white solid. LCMS (ESI) m/z: [M + 2H]/2 calcd for C106H165F3N22O20: 1062.63; found: 1063.1.

Table 3: Exemplary Peptide Conjugates Prepared by Methods of the Present Invention

Example 5. MHC complex assays.

Candidate peptides are assembled with the MHC alleles (A*01 :01 , A*02:01 , A*03:01 , A*11 :01 , B*07:02, B*08:01 , C*03:04, C*04:01 , C*07:01 , and C*07:02) and analyzed using the Prolmmune REVEAL® MHC-Peptide Binding Assay to determine their level of incorporation into MHC molecules. Binding to MHC molecules was compared to that of a known T cell epitope, a positive control peptide with strong binding properties. The high-throughput Prolmmune REVEAL® MHC-Peptide Binding Assay determines the ability of each candidate peptide to bind to one or more MHC Class I alleles and stabilize the MHC-peptide complex. By comparing the binding to that of a high-affinity T cell epitope, the most likely immunogenic peptides in a protein sequence can be identified. Detection is based on the presence or absence of the native conformation of the MHC-peptide complex. Unlike traditional functional assay approaches, the Prolmmune REVEAL® Binding Assay determines the MHC restriction of peptides at the outset.

Although drug-conjugate haptens have been described, it remained unknown whether a covalently attached compound (IA) or (I I A) RAS peptide would interfere with antigen processing and subsequent binding to MHC-I complexes. To answer this question a T2 stabilization assay are performed where T2 cells (ATCC, CRL-1992) were washed twice in AIMV media 0.1x10^A6 cells were combined with 2 ug/ml beta 2 microglobulin (Millipore Sigma, Cat# M4890) and 100 pM of peptide conjugates in a 96 well U-bottom plate. After 18 hours of incubation cells were stained for HLA expression with pan-HLA W6/32 antibody clone, or for HLA A*03 (clone GAP.A3) or HLA A*11 (clone A11 .M) and analyzed on the Cytek Aurora flow cytometer.

Example 6. PLA assay.

To further test whether a covalently attached compound (I IA) RAS peptide would interfere with antigen processing and subsequent binding to MHC-I complexes a PLA assay is performed. SU.86.86 cells (ATCC CRL-1837) expressing HLA A11 are cultured in RPMI-1640 media containing 10% FBS, 1X penicillin/streptomycin, and 0.75 ug/ml puromycin supplement. Cells are plated at 15,000 cells per well in 100 pL complete media into black, clear-bottom 96-well plates and incubated for 24 hours at 37 °C, 5% CO2. The media is then replaced with fresh media containing 0.1 pg/mL interferon gamma (BioLegend 570206) and compound treatment from DMSO stocks for final concentrations of 100 nM each of parent compound or alkyne-containing analog and 0.1% DMSO. Cells are incubated with compound for 5 hours at 37 °C, 5% CO2, after which the media is removed, cells are washed 1 time with warm PBS, and media replaced with 100 pL complete media containing 0.1 pg/mL interferon gamma. Cells are further incubated for 72 hours at 37 °C, 5% CO2.

After incubation cells are washed 3 times with cold PBS and fixed with 4% formaldehyde in PBS at room temperature for 20 minutes. Cells were washed and incubated with a click labeling reaction composed of 1 mM copper(ll) sulfate, 1.25 mM tris(3-hydroxypropyltriazolylmethyl)amine, 5 mM sodium ascorbate, and 10 pM azido-PEG3-biotin (Alfa Aesar J64996) or DMSO (1%) at room temperature for 45 minutes. Cells are then washed and a proximity ligation assay (Duolink, Millipore Sigma) is performed according to the manufacturer’s instructions employing anti-biotin (1 :100, Cell Signaling Technology 5597) and anti-human MHC class I antibodies (0.01 pg/mL, Bio X Cell BE0079). Images in the DAPI (nuclei) and Cy5 channels are collected on a BioTek Lionheart FX microscope (Agilent) and analyzed using BioTek Gen5 software (Agilent). The number of Cy5 spots resulting from proximity ligation reaction are counted for each cell. Experimental wells treated with the azido-PEG3-biotin probe were normalized to wells treated with DMSO. Experimental wells treated with the alkyne-containing analog were then normalized to wells treated with the parent compound to determine the fold difference in spots per cell.

Example 7. Antibody screening.

Isolation and Characterization of antibodies and binding-partners thereof that react with compound (I I A) peptide conjugates or compound (HA) peptide conjugate/MHC complex. The peptide conjugate or peptide conjugate/MHC complex are produced using methods described herein. Antibodies are isolated using a panning platform with a human, naive Fab-phage display library. The Fab-phage library can have a diversity of, for example 4x10¹⁰, and multiple rounds of panning conducted, resulting in the identification of unique Fabs that bind with high affinity to the peptide conjugates. To further screen for binding determinants, biolayer interferometry is used with a suite of peptides. BLI quantifies the on-rate and the off-rate. Together these rates are used to calculate the dissociation constant. All Fabs are then screened against unconjugated RAS peptides and those showing no detectable affinity for unconjugated peptides indicate compound (HA) as a primary binding determinant. In addition, all Fabs can be screened against and optimized to reduce affinity for free unbound compound (I I A).

While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure come within known or customary practice within the art to which the invention pertains and may be applied to the essential features set forth herein.

All publications, patents and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety.

Claims

1 . A peptide conjugate which is formed by covalently linking a peptide and a RAS tricomplex inhibitor, wherein the peptide is derived from RAS and wherein the peptide conjugate is isolated, or a pharmaceutically acceptable salt thereof.

2. The peptide conjugate of claim 1 , wherein the RAS is KRAS, HRAS, or NRAS.

3. The peptide conjugate of claim 1 or 2, wherein the RAS comprises a mutation.

4. The peptide conjugate of any one of claims 1-3, wherein the RAS inhibitor selectively inhibits a RAS mutant protein over a wild-type RAS protein.

5. The peptide conjugate of any one of claims 1-4, wherein the peptide comprises an aspartic acid residue.

6. The peptide conjugate of any one of claims 1-5, wherein the peptide conjugate is formed by covalently linking the RAS inhibitor to an Asp residue of the peptide.

7. The peptide conjugate of any one of claims 1-6, wherein the peptide comprises a segment of KRAS^G12D, HRAS^G12D, or NRAS^G12D.

8. The peptide conjugate of any one of claims 1-7, wherein the RAS inhibitor is an HRAS inhibitor, a KRAS inhibitor and/or an NRAS inhibitor.

9. The peptide conjugate of any one of claims 1-8, wherein the RAS inhibitor is a KRAS inhibitor.

10. The peptide conjugate of any one of claims 3-9, wherein the RAS mutation is a KRAS mutation.

11 . The peptide conjugate of claim 10, wherein the KRAS mutation comprises a KRAS^G12D mutation.

12. The peptide conjugate of any one of claims 1-11 , wherein the RAS inhibitor is a KRAS inhibitor having formula (

(111 A)

13. The peptide conjugate of any one of claims 1-12, wherein the peptide comprises an amino acid sequence of DGVGKSALTI (SEQ ID NO: 1), ADGVGKSALT (SEQ ID NO: 2), GADGVGKSAL (SEQ ID NO: 3), VGADGVGKSA (SEQ ID NO: 4), VVGADGVGK (SEQ ID NO: 5), WGADGVGKS (SEQ ID NO: 6), WVGADGVGK (SEQ ID NO: 7), KLVVVGADGV (SEQ ID NO: 8), YKLWVGADG (SEQ ID NO: 9), or EYKLWVGAD (SEQ ID NO: 10), or an isotopically labeled analog thereof.

14. The peptide conjugate of any one of claims 1-13, wherein the peptide has between about 7 to about 30 amino acids in length.

15. The peptide conjugate of claim 13 or 14, wherein the amino acid sequence is at least 80% identical to DGVGKSALTI (SEQ ID NO: 1), ADGVGKSALT (SEQ ID NO: 2), GADGVGKSAL (SEQ ID NO: 3), VGADGVGKSA (SEQ ID NO: 4), VVGADGVGK (SEQ ID NO: 5), WGADGVGKS (SEQ ID NO: 6), VVVGADGVGK (SEQ ID NO: 7), KLVVVGADGV (SEQ ID NO: 8), YKLVVVGADG (SEQ ID NO: 9), or EYKLVVVGAD (SEQ ID NO: 10), or an isotopically labeled analog thereof.

16. The peptide conjugate of any one of claims 1-15, wherein the peptide comprises an amino acid sequence DGVGKSALTI (SEQ ID NO: 1), or an isotopically labeled analog thereof.

17. The peptide conjugate of any one of claims 1-15, wherein the peptide comprises an amino acid sequence ADGVGKSALT (SEQ ID NO: 2) or an isotopically labeled analog thereof.

18. The peptide conjugate of any one of claims 1-15, wherein the peptide comprises an amino acid sequence GADGVGKSAL (SEQ ID NO: 3) or an isotopically labeled analog thereof.

19. The peptide conjugate of any one of claims 1-15, wherein the peptide comprises an amino acid sequence VGADGVGKSA (SEQ ID NO: 4) or an isotopically labeled analog thereof.

20. The peptide conjugate of any one of claims 1-15, wherein the peptide comprises an amino acid sequence VVGADGVGK (SEQ ID NO: 5) or an isotopically labeled analog thereof.

21. The peptide conjugate of any one of claims 1-15, wherein the peptide comprises an amino acid sequence WGADGVGKS (SEQ ID NO: 6) or an isotopically labeled analog thereof.

22. The peptide conjugate of any one of claims 1-15, wherein the peptide comprises an amino acid sequence VVVGADGVGK (SEQ ID NO: 7) or an isotopically labeled analog thereof.

23. The peptide conjugate of any one of claims 1-15, wherein the peptide comprises an amino acid sequence KLVWGADGV (SEQ ID NO: 8) or an isotopically labeled analog thereof.

24. The peptide conjugate of any one of claims 1-15, wherein the peptide comprises an amino acid sequence YKLWVGADG (SEQ ID NO: 9) or an isotopically labeled analog thereof.

25. The peptide conjugate of any one of claims 1-15, wherein the peptide comprises an amino acid sequence EYKLVVVGAD (SEQ ID NO: 10) or an isotopically labeled analog thereof.

26. The peptide conjugate of any one of claims 1-15, having the formula selected from:

27. The peptide conjugate of any one of claims 1-15, having the formula selected from:

28. The peptide conjugate of claim 1 , having formula (III): wherein:

X1 is hydrogen or a peptide comprising from 1 to 50 amino acids;

X2 is OH or a peptide comprising from 1 to 50 amino acids; and R¹ is hydrogen or cyclopropyl.

29. A cell-free peptide conjugate/MHC complex comprising a peptide conjugate of any one of claims 1-28 and a major histocompatibility complex (MHC).

30. The complex of claim 29, wherein the MHC is a human leukocyte antigen (HLA).

31 . A method for identifying a peptide conjugate- or peptide conjugate/MHC complex -specific antibody, the method comprising:

(a) providing (i) a peptide conjugate of any one of claims 1-28, or (ii) a peptide conjugate/MHC complex of claim 29 or claim 30;

(b) contacting the peptide conjugate or peptide conjugate/MHC complex with a library comprising a plurality of antibodies under conditions suitable for binding at least one antibody of the plurality of antibodies to the peptide conjugate or peptide conjugate/MHC complex; and

(c) recovering at least one antibody bound to the peptide conjugate or peptide conjugate/MHC complex.