WO2017189868A1 - Serine replacement polymyxin analogues useful as antibiotic potentiators - Google Patents

Abstract

The disclosure provides compounds of the formula I or a tautomer thereof, or a pharmaceutically acceptable salt of either of the foregoing. The variables R1 and R2 are defined in the disclosure. The disclosure further includes pharmaceutical compositions comprising a compound of formula I together with at least one pharmaceutically acceptable carrier. The disclosure also includes a method of sensitizing bacteria to an antibacterial agent, comprising administering a therapeutically effective amount of the antibacterial agent and a compound of formula I to a patient infected with the bacteria, simultaneously or sequentially.

Description

SERINE REPLACEMENT POLYMYXIN ANALOGUES USEFUL AS ANTIBIOTIC

POTENTIATORS

CROSS REFERENCE TO RELATED APPLICATION

[0001 ] This application claims priority of U.S. Provisional Application No. 62/328, 168, filed April 27, 2016, which is hereby incorporated by reference in its entirety.

TECI-INICAL FIELD

[0002] The present disclosure provides polymyxin analogues in which the amino acid in the tail portion directly attached to the polymyxin ring is other than serine. These compounds are useful for treating bacterial infections and/or useful for sensitizing bacteria, including Gram negative bacteria, to the effects of other antibacterial agents and thereby increasing the efficacy of the other antibacterial agents.

BACKGROUND

[0003] Gram-negative bacteria cause more than 40% of all septicemic infections and many of the Gram-negative bacteria are resistant to multiple antibiotics. Gram-negative bacteria possess iipopolysaceharide as a component of the outer membrane, which inhibits the diffusion of many antibacterial agents deeper into the cell, where their ultimate targets are located. Many antibacterial agents effective against Gram-positive bacteria lack activity against Gram-negative bacteria.

[0004] Polymyxins are a group of closely related antibiotic substances produced by strains of Paenibacillus polym xa and related organisms. These cationic drugs are relatively simple peptides with molecular weights of about 1000. Polymyxins, such as polymyxin B, are decapeptide antibiotics, i.e., they are made of ten (10) aminoacyl residues. They are bactericidal and especially effective against Gram- negative bacteria such as Escherichia coli and other species of Enterohacteriaceae. Pseiidomonas.

Acinetobacter baumannii, and others. However, polymyxins have severe adverse effects, including nephrotoxicity and neurotoxicity. These drugs thus have limited use as therapeutic agents because of high systemic toxicity.

[0005] Polymyxins were widely used in the therapy of serious infections caused by those bacteria since their discovery in the 1950's, but because of the toxicity, their use was largely abandoned in the 1970 s when newer, better tolerated antibiotics were developed. The recent emergence of multiresistant strains of Gram-negative bacteria has resulted in many of the less toxic antibiotics losing their effectiveness against said bacteria. Polymyxins have maintained their effectiveness against these emergent multiresistant strains of Gram-negative bacteria. Accordingly, polymyxins have been recalled to the therapeutic arsenal, although, due to their toxicity, they are considered a therapeutic of last resort. l Their systemic (i.e., non-topical) use is, however, largely restricted to the therapy of life-threatening infections caused by multiply resistant strains of Pseudomonas aeruginosa and baumannii as well as by carbapenem-res istarst Enterobacteriaeeae .

[0006] Numerous efforts have been made to reduce the toxicity of polymyxins, however these efforts have not yet proved successful. Certain polymyxin derivatives, which lack direct antibiotic activity, potentiate the effects of direct acting antibacterial agents. These polymyxin derivates sensitize bacteria to additional antibacterial agents such as hydrophobic antibiotics and large antibiotics that do not readily enter bacterial cells. The need exists for additional polymyxin derivatives with direct activity against Grain negative bacteria or capable of potentiating the effects of other antibacterial agents against Gram negati ve bacteria withoui the toxicity of early polymyxin deri vatives. This disclosure fulfills this need and provides additional advantages described herein.

SUMMARY

[0007] rmula I:

and tautomers thereof, and pharmaceutically acceptable salts of either of the foregoing.

[0008] Within formula I, R¹ and R² carry the following definitions:

[0009] R¹ is selected from hydrogen and optionally substituted C1-C4 alkyl.

[0010] R² is selected from heteroaryl substituted with -N(R⁷)(R⁸)

or -C(0)-CH(CH(OH)CH₃)-N(R )-R⁴, each of which is optionally further substituted, or R² is -C(O)- CH(CH₂OH)-X; and -C(0)-Y.

[0011] Or, R¹ and R² are taken together to form an oxo-substituted aryl or an oxo-substituted heterocyclyl, wherein the oxo-substituted aryl or oxo-substituted heterocyclyl is further substituted with -N(R⁷)-R⁸ or -(C(0))i-₂-CH(CH(OH)CH₃)-N(R )-R⁴, and is optionally further substituted. [0012] X is selected from -heterocyclyl, -aryl, -Ci-C₈ alkyl, -C2-C8 alkenyl, -C2-C8 alkynyl, -0-(Ci-C₈ alkyl), -0-C(0)-(Ci-C₈alkyl), -CH₂-C(0)-(Ci-C₈ alkyl), and -C(0)-N(R )-Ci-C₈alkyl, wherein X is optionally substituted.

[0013] Y is selected from -N(R )-N(R )-C(0)-Ci-C₈ alkyl; -N(R )-(CH₂)o-i-C(0)-Ci-C₈ alkyl; -0-N(R )-C(0)-Ci-C₈ alkyl; -(CEbVi-heteroaryl; -(CEbVi-heteroaryl-heteroaryl; -(CfbVi-aryl; -(CH2)o-i-heterocyclyl; -(CEbVi-carbocyclyl; wherein Y is optionally substituted.

[0014] Each R³ is independently selected from hydrogen, optionally substituted C1-C4 alkyl, optionally substituted C3-C6 cycloalkyl.

[0015] R⁴ is selected from -C(0)-R⁵, C1-C4 alkyl, heterocyclyl, cycloalkyl, -S(0)₂-R⁵, and -CH(R⁶)-R⁵.

[0016] R⁵ is selected from -C 1-C4 alkyl, NH(Ci-C₄ alkyl), N(Ci-C₄ alkyl)₂, heterocyclyl and cycloalkyl, wherein each R⁵ is optionally substituted.

[0017] R⁶ is selected from hydrogen, -OH, and optionally substituted C1-C4 alkyl.

[0018] R⁷ is selected from hydrogen and optionally substituted Ci- C₄ alkyl.

[0019] R⁸ is selected from -C(0)-heterocyclyl, -heterocyclyl, -C(0)-aryl,

-C(0)NH-N(R )-C(0)R⁵, and -W-Q-Z, wherein: W is selected from C(O), CH₂, P(0)(OH), and S(0)₂; Q is selected from CH(R⁹) and optionally substituted phen-l,2-diyl, wherein R⁹ is selected from

Ci-C₄alkyl, -CH(OH)CH3, -CH(CH3)2, -carbocyclyl, and -heterocyclyl; and Z is selected

from -C(0)N(R )(R¹⁰), -N(R )C(0)R⁵, -N(R )(R¹⁰), -N(R )S(0)₂-R⁵, -C(R )(R⁶)-R⁵, -N(R )-C(R )(R⁶)-R⁵, or -N(R )-C(R )=N-CN wherein, R¹⁰ is selected from C1-C4 alkyl, heterocyclyl and cycloalkyl, wherein R¹⁰ is optionally substituted.

[0020] The disclosure further includes pharmaceutical compositions comprising a compound of formula I together with a pharmaceutically acceptable carrier.

[0021] The disclosure includes a method for treating a bacterial infection in a patient, comprising administering a therapeutically effective amount of compound of formula I to the patient.

[0022] The disclosure also includes a method of sensitizing bacteria to an antibacterial agent, comprising administering to the patient, simultaneously or sequentially, a therapeutically effective amount of the antibacterial agent and a compound of formula I.

DETAILED DESCRIPTION

TERMINOLOGY

[0023] Compounds are described using standard nomenclature. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which this disclosure belongs. Unless clearly contraindicated by the context, each compound name includes the free acid or free base form of the compound as well as all pharmaceutically acceptable salts, solvates, and hydrates of the compound.

[0024] The term "formula I" encompasses all compounds that satisfy formula I, including any enantiomers, racemates and stereoisomers, as well as all pharmaceutically acceptable salts, solvates, and hydrates of such compounds. "Formula I" includes all subgeneric groups of formula I unless clearly contraindicated by the context in which this phrase is used.

[0025] The terms "a" and "an" do not denote a limitation of quantity, but rather denote the presence of at least one of the referenced item. The term "or" means "and/or". The open-ended transitional phrase "comprising" encompasses the intermediate transitional phrase "consisting essentially of and the close-ended phrase "consisting of." Claims reciting one of these three transitional phrases, or with an alternate transitional phrase such as "containing" or "including" can be written with any other transitional phrase unless clearly precluded by the context or art. Recitation of ranges of values are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. The endpoints of all ranges are included within the range and independently combinable. All methods described herein can be performed in a suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as"), is intended merely to for illustration and does not pose a limitation on the scope of the disclosure unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention as used herein. Unless defined otherwise, technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which this disclosure belongs.

[0026] Compounds of formula I include all compounds of formula I having isotopic substitutions at any position. Isotopes include those atoms having the same atomic number but different mass numbers. By way of general example, and without limitation, isotopes of hydrogen include tritium and deuterium and isotopes of carbon include ⁿC, ¹ C, and ¹⁴C. In some embodiments, any one or more hydrogen atoms are replaced with deuterium atoms.

[0027] An "active agent" means a compound (including a compound disclosed herein), element, or mixture that when administered to a patient, alone or in combination with another compound, element, or mixture, confers, directly or indirectly, a physiological effect on the subject. The indirect physiological effect may occur via a metabolite or other indirect mechanism. The "active agent" may also potentiate, or make more active another active agent. For example the compounds of formula I potentiate the activity of other antibacterial compounds when given in combination with another antibacterial compound, for example by lowering the MIC of the other antibacterial compound. [0028] A dash ("-") that is not between two letters or symbols is used to indicate a point of attachment for a substituent. For example, -C(0)NH2 is attached through carbon of the keto C(O) group.

[0029] An "aliphatic group" is a hydrocarbon group having the indicated number of carbon atoms in which the carbon atoms are covalently bound in single, double or triple covalent bonds in straight chains, branched chains, or non-aromatic rings. Aliphatic groups may be substituted.

[0030] "Alkyl" is a branched or straight chain saturated aliphatic hydrocarbon group, having the specified number of carbon atoms, generally from 1 to about 8 carbon atoms. The term Ci-Ce-alkyl as used herein indicates an alkyl group having from 1, 2, 3, 4, 5, or 6 carbon atoms. Other embodiments include alkyl groups having from 1 to 6 carbon atoms, 1 to 4 carbon atoms or 1 or 2 carbon atoms, e.g. Ci-Cs-alkyl, Ci-C/ralkyl, and Ci-C2-alkyl. Examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, 3-methylbutyl, t-butyl, n-pentyl, and sec-pentyl.

[0031] "Alkenyl" is a branched or straight chain aliphatic hydrocarbon group having one or more double carbon-carbon bonds that may occur at any stable point along the chain, having the specified number of carbon atoms. Examples of alkenyl include, but are not limited to, ethenyl and propenyl.

[0032] "Alkynyl" is a branched or straight chain aliphatic hydrocarbon group having one or more triple carbon-carbon bonds that may occur at any stable point along the chain, having the specified number of carbon atoms. Examples of alkynyl include, but are not limited to, ethynyl and propynyl.

[0033] "Alkoxy" is an alkyl group as defined above with the indicated number of carbon atoms covalently bound to the group it substitutes by an oxygen bridge (-0-). Examples of alkoxy include, but are not limited to, methoxy, ethoxy, n-propoxy, i-propoxy, n-butoxy, 2-butoxy, t-butoxy, n-pentoxy, 2-pentoxy, 3- pentoxy, isopentoxy, neopentoxy, n-hexoxy, 2-hexoxy, 3-hexoxy, and 3- methylpentoxy.

[0034] "Alkylthio" indicates an alkyl group as defined above attached through a sulfur linkage, i.e. a group of the formula alkyl-S-. Examples include ethylthio and pentylthio.

[0035] "Alkanoyl" is an alkyl group as defined above with the indicated number of carbon atoms covalently bound to the group it substitutes through a carbonyl C(O) bridge. The carbonyl carbon is included in the number of carbons, that is C2alkanoyl is a C]¾C(C))- group.

[0036] "Alkylester" is an alkyl group as defined herein covalently bound to the group it substitutes by an ester linkage. The ester linkage may be in either orientation, e.g., a group of the formula -OC(0)-alkyl or a group of the formula -C(0)0-alkyl.

[0037] "Aryl" indicates aromatic groups containing only carbon in the aromatic ring or rings. Typical aryl groups contain 1 to 3 separate, fused, or pendant rings and from 6 to about 18 ring atoms, without heteroatoms as ring members. When indicated, such aryl groups may be further substituted with carbon or non-carbon atoms or groups. Aryl groups include, for example, phenyl, naphthyl, including 1- naphthyl, 2-naphthyl, and bi-phenyl. [0038] A "carbocyclyl" is a monocyclic or bicyclic saturated, partially unsaturated, or aromatic ring system in which all ring atoms are carbon. Usually each ring of the carbocyclyl group contains from 3-6 ring atoms and a bicyclic carbocyclyl group contains from 7 to 10 ring atoms, but some other number of ring atoms may be specified. Unless otherwise indicated, the carbocycle may be attached to the group it substitutes at any carbon atom that results in a stable structure. When indicated the carbocyclic rings described herein may be substituted at any carbon atom if the resulting compound is stable. Examples of carbocyclyl groups include phenyl, naphthyl, tetrahydronaphthyl, cyclopropyl, cyclohexyl, and cyclohexenyl.

[0039] "Cycloalkyl" is a saturated hydrocarbon ring group, having the specified number of carbon atoms. Monocyclic cycloalkyl groups typically have from 3 to about 8 carbon ring atoms or from 3 to 6 (3, 4, 5, or 6) carbon ring atoms. Cycloalkyl substituents may be pendant from a substituted nitrogen, oxygen, or carbon atom, or a substituted carbon atom that may have two substituents may have a cycloalkyl group, which is attached as a spiro group. Examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

[0040] "Halo" or "halogen" indicates any of fluoro, chloro, bromo, and iodo.

[0041] "Haloalkyl" indicates both branched and straight-chain alkyl groups having the specified number of carbon atoms, substituted with 1 or more halogen atoms, up to the maximum allowable number of halogen atoms. Examples of haloalkyl include, but are not limited to, trifluoromethyl, difluoromethyl, 2-fluoroethyl, 2,2,2-trifluoroethyl, and penta-fluoroethyl.

[0042] "Haloalkoxy" indicates a haloalkyl group as defined herein attached through an oxygen bridge (oxygen of an alcohol radical).

[0043] The term "heterocyclyl" indicates a monocyclic saturated, partially unsaturated, or aromatic ring containing from 1 to 4 heteroatoms chosen from N, O, and S, with remaining ring atoms being carbon, or a bicyclic saturated, partially unsaturated, or aromatic heterocycle containing at least 1 heteroatom chosen from N, O, and S in one of the two rings of the two ring system and containing up to about 4 heteroatoms independently chosen from N, O, and S in each ring of the two ring system. Usually each ring of the heterocycle contains from 4-6 ring atoms but some other number of ring atoms may be specified. Unless otherwise indicated, the heterocycle may be attached to its pendant group at any heteroatom or carbon atom that results in a stable structure. When indicated the heterocycles described herein may be substituted on carbon, sulfur, or nitrogen atom if the resulting compound is stable. It is preferred that the total number of heteroatoms in a heterocycle is not more than 4 and that the total number of S and O atoms in a heterocycle is not more than 2, more preferably not more than 1. Examples of heterocyclyl groups include, pyridyl, indolyl, pyrimidinyl, pyridazinyl, pyrazinyl, imidazolyl, oxazolyl, furanyl, thiophenyl, thiazolyl, triazolyl, tetrazolyl, isoxazolyl, quinolinyl, pyrrolyl, pyrazolyl, benz[b]thiophenyl, isoquinolinyl, quinazolinyl, quinoxalinyl, thienyl, isoindolyl, dihydroisoindolyl, 5,6,7,8-tetrahydroisoquinoline, pyrazolyl, pyrrolidinyl, mo holinyl, piperazinyl, piperidinyl, and pyrrolidinyl. In certain embodiments a heterocycle is chosen from pyridinyl, pyrimidinyl, furanyl, thienyl, and pyrrolyl.

[0044] Additional examples of heterocyclyl groups include, but are not limited to, phthalazinyl, indolizinyl, indazolyl, benzothiazolyl, benzimidazolyl, benzofuranyl, benzoisoxolyl,

dihydro-benzodioxinyl, oxadiazolyl, thiadiazolyl, triazolyl, tetrazolyl, oxazolopyridinyl,

imidazopyridinyl, isothiazolyl, naphthyridinyl, cinnolinyl, carbazolyl, beta-carbolinyl, isochromanyl, chromanonyl, chromanyl, tetrahydroisoquinolinyl, isoindolinyl, isobenzotetrahydrofuranyl,

isobenzotetrahydrothienyl, isobenzothienyl, benzoxazolyl, pyridopyridinyl, benzotetrahydrofuranyl, benzotetrahydrothienyl, purinyl, benzodioxolyl, triazinyl, phenoxazinyl, phenothiazinyl, pteridinyl, benzothiazolyl, imidazopyridinyl, imidazothiazolyl, dihydrobenzisoxazinyl, benzisoxazinyl, benzoxazinyl, dihydrobenzisothiazinyl, benzopyranyl, benzothiopyranyl, coumarinyl, isocoumarinyl, chromanyl, tetrahydroquinolinyl, dihydroquinolinyl, dihydroquinolinonyl, dihydroisoquinolinonyl, dihydrocoumarinyl, dihydroisocoumarinyl, isoindolinonyl, benzodioxanyl, benzoxazolinonyl, pyrrolyl N-oxide, pyrimidinyl N-oxide, pyridazinyl N-oxide, pyrazinyl N-oxide, quinolinyl N-oxide, indolyl N-oxide, indolinyl N oxide, isoquinolyl N-oxide, quinazolinyl N-oxide, quinoxalinyl N-oxide, phthalazinyl N-oxide, imidazolyl N-oxide, isoxazolyl N-oxide, oxazolyl N- oxide, thiazolyl N-oxide, indolizinyl N oxide, indazolyl N-oxide, benzothiazolyl N-oxide, benzimidazolyl N-oxide, pyrrolyl N-oxide, oxadiazolyl N-oxide, thiadiazolyl N-oxide, tetrazolyl N- oxide, benzothiopyranyl S-oxide, and benzothiopyranyl S,S-dioxide. In certain embodiments the heterocyclyl group is a pyrimidinyl, oxazolyl, morpholinyl, piperidinyl, piperazinyl, thiomorpholinyl, isothiazolyl, pyrrolidinyl, oxadiazolyl, oxadiazolyl, oxadiazolyl substituted with benzyl, pyrazolyl, pyrazinyl, oxazolidinyl, isothiazolidinyl, imidiazolyl, pyridazinyl, pyridinyl, pyrrolyl, thiazolyl, thienyl, or furanyl group.

[0045] "Heteroaryl" is a stable monocyclic aromatic ring having the indicated number of ring atoms which contains from 1 to 4, or in some embodiments from 1 to 2, heteroatoms chosen from N, O, and S, with remaining ring atoms being carbon, or a stable bicyclic or tricyclic system containing at least one 5- to 7-membered aromatic ring which contains from 1 to 4, or in some embodiments from 1 to 2, heteroatoms chosen from N, O, and S, with remaining ring atoms being carbon. Monocyclic heteroaryl groups typically have from 5 to 7 ring atoms. In certain embodiments there heteroaryl group is a 5- or 6- membered heteroaryl group having 1, 2, 3, or 4 heteroatoms chosen from N, O, and S, with no more than 2 O atoms and 1 S atom. [0046] A "hydrocarbyl" group is hydrocarbon chain having the specified number of carbon atoms in which carbon atoms are joined by single, double or triple bonds, and any one carbon atom can be replaced by O, NH, or N(Ci-C₄alkyl).

[0047] The term "mono- and/ or di-alkylamino" indicates secondary or tertiary alkyl amino groups, wherein the alkyl groups are independently chosen alkyl groups, as defined herein, having the indicated number of carbon atoms. The point of attachment of the alkylamino group is on the nitrogen. Examples of mono- and di-alkylamino groups include ethylamino, dimethylamino, and

methyl-propyl-amino.

[0048] The term "substituted", as used herein, means that any one or more hydrogens on the designated atom or group is replaced with a selection from the indicated group, provided that the designated atom's normal valence is not exceeded. When the substituent is oxo (i.e., =0) then 2 hydrogens on the atom are replaced. When an oxo group substitutes a heteroaromatic moiety, the resulting molecule can sometimes adopt tautomeric forms. For example a pyridyl group substituted by oxo at the 2- or 4-position can sometimes be written as a pyridine or hydroxypyridine. Combinations of substituents and/or variables are permissible only if such combinations result in stable compounds or useful synthetic intermediates. A stable compound or stable structure is meant to imply a compound that is sufficiently robust to survive isolation from a reaction mixture and subsequent formulation into an effective therapeutic agent. Unless otherwise specified, substituents are named into the core structure. For example, it is to be understood that aminoalkyl means the point of attachment of this substituent to the core structure is in the alkyl portion and alkylamino means the point of attachment is a bond to the nitrogen of the amino group.

[0049] Suitable groups that may be present on a "substituted" or "optionally substituted" position include, but are not limited to, e.g., halogen; cyano; -OH; oxo; -NH2; nitro; azido; alkanoyl (such as a C2-C6 alkanoyl group); C(0)N¾; alkyl groups (including cycloalkyl and (cycloalkyl)alkyl groups) having 1 to about 8 carbon atoms, or 1 to about 6 carbon atoms; alkenyl and alkynyl groups including groups having one or more unsaturated linkages and from 2 to about 8, or 2 to about 6 carbon atoms; alkoxy groups having one or more oxygen linkages and from 1 to about 8, or from 1 to about 6 carbon atoms; aryloxy such as phenoxy; alkylthio groups including those having one or more thioether linkages and from 1 to about 8 carbon atoms, or from 1 to about 6 carbon atoms; alkylsulfinyl groups including those having one or more sulfinyl linkages and from 1 to about 8 carbon atoms, or from 1 to about 6 carbon atoms; alkylsulfonyl groups including those having one or more sulfonyl linkages and from 1 to about 8 carbon atoms, or from 1 to about 6 carbon atoms; aminoalkyl groups including groups having one or more N atoms and from 1 to about 8, or from 1 to about 6 carbon atoms; mono- or dialkylamino groups including groups having alkyl groups from 1 to about 6 carbon atoms; mono- or dialkylcarboxamido groups (i.e. alkylNHC(O)-, (alkyli)(alkyl₂)NC(0)-, alkylC(0)NH-, or alkyliC(0)N(alkyl₂)-) having alkyl groups from about 1 to about 6 carbon atoms; carbocyclyl such as aryl having 6 or more carbons and one or more rings, (e.g., phenyl, biphenyl, naphthyl, or the like, each ring either substituted or unsubstituted aromatic); or a saturated, unsaturated, or aromatic heterocycle having 1 to 3 separate or fused rings with 3 to about 8 members per ring and one or more N, O or S atoms, e.g. coumarinyl, quinolinyl, isoquinolinyl, quinazolinyl, pyridyl, pyrazinyl, pyrimidinyl, furanyl, pyrrolyl, thienyl, thiazolyl, triazinyl, oxazolyl, isoxazolyl, imidazolyl, indolyl, benzofuranyl, benzothiazolyl, tetrahydrofuranyl, tetrahydropyranyl, piperidinyl, morpholinyl, piperazinyl, and pyrrolidinyl. Such heterocycles may be further substituted, e.g. with hydroxy, alkyl, alkoxy, halogen and amino. In certain embodiments "optionally substituted" includes one or more substituents independently chosen from halogen, hydroxyl, oxo, amino, cyano, -CHO, -CO2H, -C(0)NH2, Ci-Ce-alkyl, C2-Ce-alkenyl, Ci-Ce-alkoxy, C2-Ce-alkanoyl, Ci-Ce- alkylester, (mono- and di-Ci-C6-alkylamino)Co-C2-alkyl, (mono- and di-Ci-C6-alkylamino)(CO)Co-C2- alkyl, Ci-C2-haloalkyl, Ci-C2haloalkoxy, and heterocyclic substituents of 5-6 members and 1 to 3 N, O or S atoms, i.e. pyridyl, pyrazinyl, pyrimidinyl, furanyl, pyrrolyl, thienyl, thiazolyl, triazinyl, oxazolyl, isoxazolyl, imidazolyl, tetrahydrofuranyl, tetrahydropyranyl, piperidinyl, morpholinyl, piperazinyl, and pyrrolidinyl, each of which heterocycle can be substituted by amino, Ci-Ce-alkyl, Ci-Ce-alkoxy,.or - CONH2. In certain embodiments "optionally substituted" includes halogen, hydroxyl, cyano, nitro, oxo, -CONH2, amino, ono- or di-Ci-C4alkylcarboxamide, and Ci-Cehydrocarbyl , which Ci-Cehydrocarbyl group, a hydrocarbon chain in which carbon atoms are joined by single, double or triple bonds, and any one carbon atom can be replaced by O, NH, or N(Ci-C4alkyl) and which hydrocarbyl group is optionally substituted with one or more substituents independently chosen from hydroxyl, halogen, and amino.

[0050] A "dosage form" means a unit of administration of an active agent. Examples of dosage forms include tablets, capsules, injections, suspensions, liquids, emulsions, creams, ointments, suppositories, inhalable forms, transdermal forms, and the like.

[0051] "Pharmaceutical compositions" are compositions comprising at least one active agent, such as a compound or salt, solvate, or hydrate of Formula (I) or a prodrug thereof, and at least one other substance, such as a carrier. Pharmaceutical compositions optionally contain one or more additional active agents. When specified, pharmaceutical compositions meet the U.S. FDA's GMP (good manufacturing practice) standards for human or non-human drugs. "Pharmaceutical combinations" are combinations of at least two active agents which may be combined in a single dosage form or provided together in separate dosage forms with instructions that the active agents are to be used together to treat a disorder, such as a Gram-negative bacterial infection.

[0052] "Pharmaceutically acceptable salts" includes derivatives of the disclosed compounds in which the parent compound is modified by making inorganic and organic, non-toxic, acid or base addition salts thereof. The salts of the present compounds can be synthesized from a parent compound that contains a basic or acidic moiety by conventional chemical methods. Generally, such salts can be prepared by reacting free acid forms of these compounds with a stoichiometric amount of the appropriate base (such as Na, Ca, Mg, or K hydroxide, carbonate, bicarbonate, or the like), or by reacting free base forms of these compounds with a stoichiometric amount of the appropriate acid. Such reactions are typically carried out in water or in an organic solvent, or in a mixture of the two. Salts of the present compounds further include solvates of the compounds and of the compound salts.

[0053] Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines or nitrogen-containing heteroaryl rings (e.g. pyridine, quinoline, isoquinoline); alkali or organic salts of acidic residues such as carboxylic acids; and the like. The pharmaceutically acceptable salts include the conventional non-toxic salts and the quaternary ammonium salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. For example, conventional non-toxic acid salts include those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, phosphoric, nitric and the like; and the salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, mesylic, esylic, besylic, sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isethionic, H02C-(CH2)_n-C02H where n is 0-4, and the like. Lists of additional suitable salts may be found, e.g., in G. Steffen Paulekuhn, et al, Journal of Medicinal Chemistry 2007, 50, 6665 and Handbook of

Pharmaceutical Salts: Properties, Selection and Use, P. Heinrich Stahl and Camille G. Wermuth Editors, Wiley-VCH, 2002.

[0054] The term "carrier" applied to pharmaceutical compositions/combinations of the disclosure refers to a diluent, excipient, or vehicle with which an active compound is provided.

[0055] A "patient" is a human or non-human animal in need of medical treatment. In some embodiments the patient is a human patient.

[0056] "Providing" means giving, administering, selling, distributing, transferring (for profit or not), manufacturing, compounding, or dispensing.

[0057] "Treatment," as used herein includes providing a compound of this disclosure such as a compound of any o, either as the only active agent or together with at least one additional active agent sufficient to: (a) inhibiting the disease, i.e. arresting its development; and (b) relieving the disease, i.e., causing regression of the disease and in the case of a bacterial infection to eliminate or reduce the virulence of the infection in the subject. "Treating" and "treatment" also means providing a

therapeutically effective amount of a compound of the disclosure as the only active agent or together with at least one additional active agent to a subject having or susceptible to a bacterial infection. "Prophylactic treatment" includes administering an amount of a compound of the disclosure sufficient to significantly reduce the likelihood of a disease from occurring in a subject who may be predisposed to the disease but who does not have it.

[0058] A "therapeutically effective amount" of a pharmaceutical composition/ combination is an amount effective, when administered to a subject, to provide a therapeutic benefit, such as to decrease the morbidity and mortality associated with bacterial infection and/ or effect a cure. In certain circumstances a subject suffering from a microbial infection may not present symptoms of being infected. Thus a therapeutically effective amount of a compound is also an amount sufficient to significantly reduce the detectable level of microorganism in the subject's blood, serum, other bodily fluids, or tissues. The disclosure also includes, in certain embodiments, using compounds of the disclosure in prophylactic treatment and therapeutic treatment. In the context of prophylactic or preventative treatment, a

"therapeutically effective amount" is an amount sufficient to significantly decrease the incidence of or morbidity and mortality associated with bacterial infection. For example, prophylactic treatment may be administered when a subject is known to be at enhanced risk of bacterial infection, such cystic fibrosis or ventilator patients. A significant reduction is any detectable negative change that is statistically significant in a standard parametric test of statistical significance such as Student's T-test, where p < 0.05.

CHEMICAL DESCRIPTION

[0059] I,

tautomers thereof, and pharmaceutically acceptable salts of either the disclosure includes embodiments in which the variables, R¹ and R² ( and variables such as R³, R⁴, R⁵, R⁶, R⁷, R⁸, X and Y that appear within the definitions of R¹ and R²) carry any of the following definitions. Definitions for the variables may be combined in any matter that results in a stable compound encompassed by the scope of formula I as defined in the SUMMARY section.

[0060] (1) R¹ is selected from hydrogen and methyl.

[0061] (2) R² is a heteroaryl substituted with -[N(R )]₀-i-C(O)-CH(CHOHCH₃)-N(R )-R⁴; and R² is optionally further substituted. [0062] (3) R² is -C(0)-Y.

[0063] (4) R² is -C(0)-Y , and Y is -heteroaryl; -CH₂-heteroaryl; -heteroaryl-heteroaryl; -(C(CH₂CH₂))heteroaryl; -CH₂-heteroaryl-heteroaryl; aryl, -CH₂-aryl; or -(C(CH₂CH₂))aryl; wherein the heteroaryl or aryl portion of Y is optionally substituted.

[0064] (5) R² is -C(0)-Y , and Y is -heteroaryl; -Clrb-heteroaryl; -heteroaryl-heteroaryl; -(C(CH₂CH₂))heteroaryl; -CH₂-heteroaryl-heteroaryl; aryl, -Cfh-aryl; or -(C(CH₂CH₂))aryl; wherein the aryl is phenyl and each heteroaryl is selected from pyridyl, triazolyl, and oxazolyl, each of which is optionally substituted with one or more substituents chosen from halogen, hydroxyl, Ci-C4alkyl, and - NHC(0)CH₃.

2 is selected from:

[0066] (7) R⁵ is methyl.

[0067] (8) R² is -C(0)-CH(CH₂OH)-X; and X is -Ci-C₆alkyl, -C₂-C₆alkenyl, -C₂- Cealkynyl, -0-C(0)-(Ci-Csalkyl), -C(0)-N(R )-Ci-Csalkyl, a 5- or 6-membered heteroaryl group; each of which X is optionally substituted with 1 or more substituents independently selected from halogen, hydroxyl, -CN, oxo, amino, Ci-Cealkyl, Ci-Cealkoxy, mono- and di-(Ci-C4alkyl)amino, mono- and di-Ci- C4alkylcarboxamide, Ci-C2haloalkyl; and Ci-C2haloalkoxy; and R³ is hydrogen or methyl.

[0068] (9) R² is -C(0)-CH(CH₂OH)-X; and X is selected from 4-acetylamino-oxazol-2-yl, 6-acetylamino-pyridin-2-yl, 2-fluoro-3-acetylamino-phenyl, (Z)-CH=CH-CH(CH(OH)CH₃)-NH-C(0)- CH₃, (E)-CH=CH-CH(CH(OH)CH₃)-NH-C(0)-CH₃„ -CH₂-CH(OH)-CH(CH(OH)CH₃)-NH-C(0)-CH₃, -CH₂-C(0)-CH(CH(OH)CH₃)-NH-C(0)-CH₃, , -C(0)-CH₂-CH(CH(OH)CH₃)-NH-C(0)-CH₃,

-0-C(0)-CH(CH(OH)CH₃)-NH-C(0)-CH₃, , -C(0)-0-CH(CH(OH)CH₃)-NH-C(0)-CH₃,

-C(0)-NH-CH(CH(OH)CH₃)-NH-C(0)-CH₃, , -NH-C(0)-CH(CH(OH)CH₃)-NH-C(0)-CH₃,

-C≡C-CH(CH(OH)CH₃)-NH-C(0)-CH₃, , -CH₂-CH₂-CH(CH(OH)CH₃)-NH-C(0)-CH₃,

-0-CH₂-CH(CH(OH)CH₃)-NH-C(0)-CH₃, -CH₂-0-CH(CH(OH)CH₃)-NH-C(0)-CH₃,

-CH(OH)-CH₂-CH(CH(OH)CH₃)-NH-C(0)-CH₃, and -CH(OH)-CH(OH)-CH(CH(OH)CH₃)-NH-C(0)- CH₃. [0069] (10) R² is -C(0)-Y, and Y is selected from: l-(4-chlorophenyl)cycloprop-l-yl, 4- ethylphenylmethyl, 2-(3 -methyl- 1 ,2,4-triazol- 1 -yl)pyridin-4-yl, 3 -methylpyrazol- 1 -ylmethyl, 2-( 1 ,2,4- triazol-l-yl)pyridin-4-yl,-N(CH2CH₂OH)-NH-C(0)-CH(CH(OH)CH₃)-NH-C(0)-CH₃,

-0-NH-C(0)-CH(CH(OH)CH₃)-NH-C(0)-CH₃, and -N(cyclopropyl)-CH₂-C(0)-CH(CH(OH)CH₃)-NH- C(0)-CH₃.

1 and R² are taken together to form an oxo-substituted ring selected from:

[0071] (12) The disclosure includes compounds o formula I or IA in which "-heteroaryl" is selected from a pyrrolyl group, an imidazolyl group, a pyrazolyl group, a pyridinyl group, a pyrazinyl group, a pyrimidinyl group, a pyridazinyl group, an isoindolyl group, an indolyl group, an indazolyl group, a purinyl group, a quinolinyl group, an isoquinolinyl group, a benzoquinolinyl group, a phthalazinyl group, a naphthyridinyl group, a quinoxalinyl group, a quinazolinyl group, a cinnolinyl group, a carbazolyl group, a phenanthridinyl group, an acridinyl group, a phenanthrolinyl group, a phenazinyl group, a benzoxazolyl group, a benzoimidazolyl group, a furanyl group, a benzoiuranyl group, a thiophenyl group, a benzothiophenyl group, a thiazolyl group, an isothiazolyl group, a benzothiazolyl group, an isoxazolyl group, an oxazolyl group, a triazolyl group, a tetrazolyl group, an oxadiazolyl group, a triazinyl group, a dibenzofuranyl group, a dibenzothiophenyl group, a benzocarbazolyl group, a dibenzocarbazolyl group, an imidazopyrimidinyl group, an imidazopyridinyl group, a pyridoindolyl group, a benzofuropyridinyl group, a benzothienopyridinyl group, a pyrimidoindolyl group, a

benzopyrimidinyl group, a benzothienopyrimidinyl group, a phenoxazinyl group, a pyridobenzoxazinyl group, and a pyridobenzothiazinyl group, each optionally substituted with at least one selected from deuterium, -F, -CI, -Br, -I, a hydroxy group, a cyano group, a nitro group, an amino group, an amidino group, a hydrazine group, a hydrazone group, a carboxylic acid group or a salt thereof, a sulfonic acid group or a salt thereof, a phosphoric acid group or a salt thereof, a Ci-Cio alkyl group, a C2-C10 alkenyl group, a C2-C10 alkynyl group, a C1-C10 alkoxy group, a phenyl group, a biphenyl group, a terphenyl group, a naphthyl group, an anthracenyl group, a pyrenyl group, a phenanthrenyl group, a fluorenyl group, a carbazolyl group, a benzocarbazolyl group, a dibenzocarbazolyl group, a pyridinyl group, a pyrimidinyl group, a pyrazinyl group, a pyridazinyl group, a triazinyl group, a quinolinyl group, an isoquinolinyl group, a phthalazinyl group, a quinoxalinyl group, a cinnolinyl group, a quinazolinyl group, and -N(Qi)(Q2), wherein Qi, and Q2 are each independently selected from hydrogen, a C1-C10 alkyl group, or a C6-C12 aryl group.

[0072] ( 13) R² is -C(0)-Y and Y is a heterocyclyl as listed in embodiment ( 12)

[0073] (4) R² is:

[0074] In certain embodiments compounds in which R² is

-C(0)-CH(CH₂OH)-X, and X is -0-(Ci-C₈ alkyl) or -0-C(0)-(Ci-C₈ alkyl) are excluded.

[0075] In addition to compounds of Formula, the disclosure further provides compounds of Formula IA

(IA),

or a tautomer thereof, or a pharmaceutically acceptable salt of either of the foregoing.

[0076] Within Formula IA, A is phenyl or a 5 - or 6-membered heteroaryl group, each of which is optionally substituted; and— represents a stereospecific bond selected from (R) and (S) .

[0077] The disclosure includes the following embodiments of Formula IA:

[0078] (1) A is phenyl or pyridyl; each of which is optionally substituted with one or more groups independently chosen from one or more substituents independently chosen from halogen, hydroxyl, cyano, nitro, oxo, -CONH2, amino, mono- or di-Ci-C4alkylcarboxamide, and Ci- Cehydrocarbyl, which Ci-Cehydrocarbyl group, a hydrocarbon chain in which carbon atoms are joined by single, double or triple bonds, and any one carbon atom can be replaced by O, NH, or N(Ci-C4alkyl) and which hydrocarbyl group is optionally substituted with one or more substituents independently chosen from hydroxyl, halogen, and amino.

[0079] (2) A is phenyl substituted with one mono- or di-Ci-C4alkylcarboxamide group and optionally substituted with one or more substituents independently chosen from hydroxyl, halogen, amino, cyano, nitro, Ci-C4alkyl, Ci-C4alkoxy, Ci-C2haloalkyl, and Ci-C2haloalkoxy.

[0080] (3) A is a group of the formula

these variables have in formula I, or in certain embodiments R⁴ is halogen and R⁶ is Ci-C4alkylNH-. PHARMACEUTICAL COMPOSITIONS

[0081] The disclosure includes a pharmaceutical composition containing at least one compound of formula I as the active agent together with a pharmaceutically acceptable carrier.

[0082] The pharmaceutical compositions of the disclosure include ocular, oral, nasal, transdermal, topical with or without occlusion, intravenous (both bolus and infusion), inhalable, and injection (intraperitoneally, subcutaneously, intramuscularly or parenterally) formulations. The composition may be in a dosage unit such as a tablet, pill, capsule, powder, granule, liposome, sterile ocular solution, parenteral solution or suspension, metered aerosol or liquid spray, drop, ampoule, auto- injector device, or suppository; for administration ocularly, orally, intranasally, sublingually, parenterally, or rectally, or by inhalation or insufflation.

[0083] The dosage form containing the composition of the disclosure contains an effective amount of the active agent necessary to provide a therapeutic effect by the chosen route of administration. The composition may contain from about 5,000 mg to about 0.5 mg (preferably, from about 1,000 mg to about 0.5 mg) of a compound of the disclosure or salt form thereof and may be constituted into any form suitable for the selected mode of administration. The dosage form may be formulated for immediate release or controlled release, including delayed release or sustained release. The pharmaceutical composition may include a compound for formula I as the only active agent or may be combined with one or more additional active agents. In certain embodiment the pharmaceutical composition includes a compound of formula I and at least one direct acting antibiotic (a compound efficacious for killing pathogenic bacteria in vivo).

METHODS OF TREATMENT

[0084] The disclosure includes a method of treating a bacterial infection in a subject by administering an effective amount of one or more compounds of the disclosure to a subject at risk for a bacterial infection or suffering from a bacterial infection. The disclosure includes a method of treatment in which a compound of formula I is used to sensitize bacteria to an antibacterial agent. In this embodiment a compound of formula I is administered to a patient having a bacterial infection, simultaneously or sequentially, with a therapeutically effective amount of the antibacterial agent. The compound of formula I increases the efficacy, often by lowering the MIC, of the other antibacterial agent.

[0085] Treatment of human patients is particularly contemplated. However, treatment of non-human subjects is within the scope of the disclosure. The disclosure includes treatment or prevention of microbial infections in fish, amphibians, reptiles or birds, but a preferred embodiment of the disclosure includes treating mammals.

[0086] In some embodiments, the bacterial infection or antibiotic-tolerant or antibiotic-resistant infection is caused by a Gram-negative bacterium.

[0087] In an embodiment of any of the methods of this disclosure, the microbial infection is the result of a pathogenic bacterial infection. Examples of pathogenic bacteria include, without limitation, bacteria within the genera Aerobacter, Aeromonas, Acinetobacter, Agrobacterium, Bacillus, Bacteroides, Bartonella, Bordetella, Brucella, Burkholderia, Calymmatobacterium, Campylobacter, Citrobacter, Clostridium, Corynebacterium, Enter obacter, Enterococcus, Escherichia, Francisella, Haemophilus, Hafnia, Helicobacter, Klebsiella, Legionella, Listeria, Morganella, Moraxella, Proteus, Providencia, Pseudomonas, Salmonella, Serratia, Shigella, Staphylococcus, Streptococcus, Treponema, Xanthomonas, Vibrio, and Yersinia. Specific examples of such bacteria include Vibrio harveyi, Vibrio cholerae, Vibrio parahemolyticus, Vibrio alginolyticus, Pseudomonas phosphoreum, Pseudomonas aeruginosa, Yersinia enterocolitica, Escherichia coli, Salmonella typhimurium, Haemophilus influenzae, Helicobacter pylori, Bacillus sub ti lis, Borrelia burgdorferi, Neisseria meningitidis, Neisseria gonorrhoeae, Yersinia pestis, Campylobacter jejuni, Mycobacterium tuberculosis, Enterococcus faecalis, Streptococcus pneumoniae, Streptococcus pyogenes, Klebsiella pneumoniae, Burkholderia cepacia, Acinetobacter baumannii, Staphylococcus epidermidis, and Staphylococcus aureus.

[0088] In some embodiments, the Gram-negative bacterium is a. Pseudomonas, e.g., P.

aeruginosa.

[0089] In some embodiments, the Gram-negative bacterium is Burkholderia species.

[0090] In some embodiments, the Gram-negative bacterium is Acinetobacter, e.g., A. baumannii.

[0091] In some embodiments, the Gram-negative bacterium is an Enterobacteriaceae, e.g.,

Klebsiella pneumonia, e.g., Escherichia coli, e.g., Enterobacter cloacae, e.g., Serratia mar cescens, e.g., Salmonella typhimurium, e.g., Shigella dysenteriae, e.g., Proteus mirabilis, e.g., Citrobacter freundii, e.g., Yersinia pestis.

[0092] In some embodiments, the infection is a polymicrobial infection, e.g., an infection comprising more than one organism. In some embodiments, the infection comprises at least one of the organisms listed above, e.g., one or more of Pseudomonas, e.g., P. aeruginosa, Klebsiella, e.g., Klebsiella pneumoniae, and/or Acinetobacter, e.g., A. baumannii.

[0093] In some embodiments, the methods further include administering an additional active agent in combination with a compound of the disclosure, such as an antibiotic selected from the group consisting of but not limited to: beta-lactams such as penicillins, cephalosporins, carbacephems, cephamycins, carbapenems, monobactams, quinolones including fluoroquinolones and similar DNA synthesis inhibitors, tetracyclines, aminoglycosides, macrolides, glycopeptides, chloramphenicols, glycylcyclines, lincosamides, lipopeptides, lipodepsipeptides, such as daptomycin, and oxazolidinones.

[0094] In some embodiments, the bacterial infection is an upper and lower respiratory tract infection, pneumonia, bacteremia, a systemic infection, sepsis and septic shock, a urinary tract infection, a gastrointestinal infection, endocarditis, a bone infection, central nervous system infections such as meningitis, or an infection of the skin and soft tissue.

[0095] In some embodiments, the subject is a mammal, e.g., a human or non-human mammal. In some embodiments, the methods include treating one or more cells, e.g., cells in a culture dish.

[0096] In one aspect, the present disclosure features a method of treating a Gram-negative infection in a subject, the method comprising administering to said subject in need of such treatment a therapeutically effective amount of a compound described herein.

[0097] In some embodiments, the Gram-negative infection is caused by Pseudomonas aeruginosa.

[0098] In other embodiments the disclosure includes treating an infection caused by Gram- positive bacteria, such as Staphylococcus epidermidis and Staphylococcus aureus.

[0099] In some embodiments, the subject is a trauma patient or a burn patient suffering from a burn or skin wound.

[0100] In a further aspect, the present disclosure features a method of reducing bacterial tolerance in a subject, the method comprising administering to said subject a therapeutically effective amount of a compound described herein.

[0101] In some embodiments, the method further includes identifying said subject suffering from an infection with bacteria resistant to antimicrobial therapy.

[0102] The disclosure includes methods of treatment in which a compound or composition of the disclosure is administered orally, topically, intravenously, parenterally, or inhaled.

[0103] A compound of the disclosure may be administered about 1 to about 5 times per day. Daily administration or post-periodic dosing may be employed. Frequency of dosage may also vary depending on the compound used, the particular disease treated and the bacteria causing the disease. It will be understood, however, that the specific dose level for any particular subject will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, route of administration, and rate of excretion, drug combination and the severity of the particular disease undergoing therapy.

EXAMPLES

General Methods

[0104] LC Method 1. The retention times (Rt) were obtained on an ACQUITY SDS system with an Agilent Ultimate XB-C18 4.6 x 50 run, 5μπι column. A gradient of H₂O (H₂O : CH₃CN : formic acid = 90 : 10 : 0.05)/CH₃CN (100% CH₃CN) 80/20 to 0/100 was applied over 2.1 min., then held for 0.5 min. 80/20 was applied at 2.7 min and held for 0.3 min (1.0 mL/min. as solvent flow) at an oven temperature of 35 °C.

[0105] LC Method 2. The retention times (Rt) were obtained on an ACQUITY SDS system with an Agilent Ultimate XB-C18 4.6 x 50 ran, 5μηι column , A gradient of H?0 (H₂0 : CH3CN : formic acid = 90 : 10 : 0.05)/CH₃CN ( 100% CH₃CN) 100/0 to 70/30 was applied over 3.5 mm., then held for 0.2 mm. 0/100 was applied at 4.50 mm and held for 0.5 mm (I .OmL/mm. as solvent flow) at an oven temperature of 35 °C.

[0106] LC Method 3- The retention times (Rt) were obtained on an ACQUITY SDS system with a Welch Ultimate XB-C18 4.6 x 50 nm, 5μιη column. A gradient of H₂0 (H₂0 : CH₃CN : formic acid = 90 : 10 : 0.05)/CH₃CN (CH3CN : H₂0 = 90 : 10 : 0.05) 95/5 to 70/30 was applied over 3.5 min., then held for 0.8 min. 100/0 was applied at 4.3 min and held for 0.6 min (1.0 mL/min. as solvent flow) at an oven temperature of 35 °C.

[0107] NMR spectra were obtained on a 400 MHz high resolution spectrometer using the indicated solvent.

ABBREVIATIONS

Dddd doublet of doublet of doublet of mL millileters doublets

DCM dichloromethane Mm millimeters

DIEA/ DIPEA N,N-diisopropylethylamine Mmol millimoles

DMAP dimethylaminopyridine m/z mass to charge ratio

DMF dimethylformamide N normal

EtOH ethanol PE: EA petroleum ether: ethyl acetate

EtOAc ethyl acetate PMBHB0C3 para-methoxy benzyl

(intermediate hydride tri- fert-butyl

A) dicarbonate

Et₂0 Diethyl ether rt room temperature

G Grams S singlet

H Hours t triplet

HBTU (2-( IH-benzotriazol- 1 -yl)- 1, 1,3,3- i-BuOH t-butyl alcohol

tetramethyluronium

hexafluorophosphate

HPLC High performance liquid TFA trifluoroacetic acid chromatography

LCMS liquid chromatography mass μιη micrometers

spectrometry -3

(1-2) (1-3)

[0108] Step 1. Synthesis of (₁S)-2,4-diamino-N-((3₁S^*,6,S^*,9₁S^*,12₁S^*,15i?, 18₁S^*,21₁S)-6,9, 18-tris(2- aminoethyl)-15-benzyl-3-((i?)-l-hydroxyethyl)-12-isobutyl-2,5,8,l l, 14,17,20-heptaoxo- l,4,7,10, 13,16, 19-heptaazacyclotricosan-21-yl)butanamide (1-2). Intermediate compound (1-2) is produced by enzymatic cleavage with savinase.

[0109] Step 2. Synthesis of Intermediate l-3,_fert-butyl (2-((2S,5R,8S,\ \S,\4S,nS,22S)-22-((S)- 2-amino-4-((teri-butoxycarbonyl)amino)butanamido)-5 -benzyl- 1 l, 14-bis(2-((tert- butoxycarbonyl)amino)ethyl)- 17-((i?)- 1 -hydroxyethyl)-8-isobutyl-3 ,6, 9, 12, 15,18,23 -heptaoxo- l,4,7,10, 13,16, 19-heptaazacyclotricosan-2-yl)ethyl)carbamate, is prepared by Boc protection of intermediate 1-2 generated by savinase cleavage in Step 1.

(1-3) (1-4)

[0110] Step 1. Synthesis of tert-butyl 2,2',2"-((2S,5R,8S, l lS,14S, 17S,22S)-5-benzyl-17-((R)-l- hydroxyethyl)-8-isobutyl-22-(2-(3-methyl-lH-pyrazol-l-yl)acetamido)-3,6, 9, 12,15,18,23-heptaoxo- 1,4,7,10, 13, 16, 19-heptaazacyclotricosane-2,l l, 14-triyl)tris(ethane-2, l-diyl)tricarbamate. To a solution of 2-(3-methylpyrazol-l-yl)acetic acid (100 mg, 0.71 mmol) in DMF (15 mL) was added HOBT (20.74 mg, 0.15 mmol), HBTU (53.38 mg, 0.14 mmol), PMBHBoc₃ (1-3) (136.01mg, 0.13 mmol), and DIEA (33.03 mg, 0.26 mmol). The reaction mixture was stirred at room temperature overnight. The crude mixture was poured into water and extracted with EtOAc, the organic phase was concentrated to dryness. The crude mixture was purified by prep-TLC (DCM : MeOH = 20 : 1) to give title compound, 1-4. (40 mg, 4.5 % yield), LCMS: rt =1.96 minutes (LC method 1), 1185 (M+l).

[0111] Step 2. Synthesis of 2-(3-methyl-lH-pyrazol-l-yl)-N-((3S,6S,9S,12S,15R,18S,21S)- 6,9, 18-tris(2-aminoethyl)-15-benzyl-3-((R)-l-hydroxyethyl)-12-isobutyl-2,5,8, 11, 14, 17,20-heptaoxo- -yl)acetamide. Compound 1.

[0112] To a solution of tert-butyl 2,2',2"-((2S,5R,8S, l lS, 14S, 17S,22S)-5-benzyl-17-((R)-l- hydroxyethyl)-8-isobutyl-22-(2-(3-methyl-lH-pyrazol-l-yl)acetamido)-3,6,9, 12, 15, 18,23-heptaoxo- 1, 4, 7, 10, 13, 16, 19-heptaazacyclotricosane-2, 11 , 14-triyl)tris(ethane-2, 1 -diyl)tricarbamate (30 mg, 0.03 mmol) (1-4) in DCM (5mL) was added TFA (1.0 mL) dropwise. The resulting mixture was stirred at room temperature. After completion of the reaction, the crude mixture was then concentrated to dryness, the residue was re-dissolved in 10% aq. formic acid solution (5 mL). The solvent was removed under vacuo and the solid was freeze-dried to give the title compound (Compound 1) (30 mg, 95 % yield). LCMS: ri = 0.26 and 0.62 mmutes (double peak, LC method 2). m/z = 885 (M÷l), ¾ NMR (400 MHz, D₂0) δ 7.63 - 7.43 (m, 1H), 7.24 (m, 5H), 6.18 (s, lH), 4.96 - 4.82 (m, 2H), 4.43 (t, J= 8.0 Hz, 2H), 4.27 - 3.98 (m, 6H), 3.35 (d, J= 4.9 Hz, 1H), 3.12 - 2.57 (m, 9H), 2.24 - 1.68 (m, 11H), 1.30 (dd, J = 23.8, 10.0 Hz, 2H), 1.09 (d, J= 6.0 Hz, 3H), 0.63 (t, J= 23.8 Hz, 7H).

2

[0113] 2-(lH-l,2,4-triazol-l-yl)-N-((3S,6S,9S,12S,15R,18S,21S)-6,9, 18-tris(2-aminoethyl)-15- benzyl-3-((R)-l-hydroxyethyl)-12-isobutyl-2,5,8,l l, 14,17,20-heptaoxo-l,4,7, 10, 13,16, 19- heptaazacyclotricosan-21-yl)isonicotinamide (Compound 2) was prepared according to the coupling, then deprotection sequence, described for example 2, compound 1. LCMS: rt = 0 33 minutes (LC method 2) m/z = 934.5 (M+l). ¾ NMR (400 MHz, D₂0) δ 9.23 (s, 1H), 8.61 (s, 1H), 8.21 (s, 1H), 8.12 (s, lH), 7.74 (s, 1H), 7.27 (m, 5H), 4.55 - 3.92 (m, 9H), 3.48 (s, lH), 3.30 - 2.61 (m, 9H), 2.09 (m, 8H), 1.37 (s, 2H), 1.13 (s, 3H), 0.69 (s, 3H), 0.62 (s, 3H).

EXAMPLE 4. SYNTHESIS OF COMPOUND 3

[0114] l-(4-chlorophenyl)-N-((3S,6S,9S,12S,15R,18S,21S)-6,9,18-tris(2-aminoethyl)-15- benzyl-3-((R)-l-hydroxyethyl)-12-isobutyl-2,5,8,l l, 14,17,20-heptaoxo-l,4,7, 10, 13,16, 19- heptaazacyclotricosan-21-yl)cyclopropane carboxamide (Compound 3) was prepared according to the coupling then deprotection sequence described for Compound 1, Example 2. LCMS: rt ^::: 0.26 minutes (LC method 2) mz = 940.5 (M+l ). ¾ NMR (400 MHz, D₂0) δ 7.48 - 7.07 (m, 9H), 4.52 - 4.31 (m, 2H), 4.14 (dddd, J = 21.7, 14.4, 12.5, 6.6 Hz, 6H), 3.31 - 3.17 (m, lH), 3.09 - 2.87 (m, 7H), 2.83 - 2.57 (m, 2H), 2.27 - 1.58 (m, 9H), 1.36 (dt, J = 23.6, 11.2 Hz, 4H), 1.24 - 1.02 (m, 5H), 0.77 - 0.52 (m, 7H).

4

[0115] 2-(4-Ethylphenyl)-N-((3S,6S,9S,12S,15R,18S,21S)-6,9,18-tris(2-aminoethyl)-15-benzyl- 3-((R)-l-hydroxyethyl)-12-isobutyl-2,5,8,ll,14,17,20-heptaoxo-l,4,7,10,13,16,19- heptaazacyclotricosan-21-yl)acetamide (Compound 4), was prepared according to the coupling then

[0116] Step 1. Preparation of intermediate tert-butyl 2,2',2"-((2S,5R,8S,l lS,14S,17S,22S)-5- benzyl-22-(2-(4-ethylphenyl)acetamido)-17-((R)-l-hydroxyethyl)-8-isobutyl-3,6,9,12,15,18,23-heptaoxo- 1, 4, 7, 10,13, 16,19-heptaazacyclotricosane-2, 11 , 14-triyl)tris(ethane-2, 1 -diyl)tricarbamate (4-2) . To a solution of 2-(4-ethylphenyl)acetic acid (129.8 mg, 0.79 mmol) and tert-butyl N-[2- [(2S,5R,8S,llS,14S,17S,22S)-22-amino-5-benzyl-ll,14-bis[2-(tert-butoxycarbonylamino)ethyl]-17- [(lR)-l-hydroxyethyl]-8-isobutyl-3,6,9,12,15,18,23-heptaoxo-l,4,7,10,13,16,19-heptazacyclotricos-2- yl]ethyl]carbamate (4-1) (800 mg, 0.75 mmol) in DMF (20 mL), was added HBTU (571 mg, 1.51 mmol), HOBT (102 mg, 0.75 mmol) and DIPEA (0.37 mL, 2.26 mmol). The resulting mixture was stirred at room temperature overnight. The crude mixture was poured into water and extracted with ethyl acetate (50 niL x 2), the combined organic phase were washed with water and brine, dried over anhydrous Na2SC>4, filtered and concentrated to dryness. The residue was purified by prep-HPLC (acidic condition, modifier: HCOOH) to give the title compound intermediate 4-2 (400 mg, 44 % yield). LCMS: rt = 2.04 minutes (LC method 1), 1209 (M+l)⁺.

[0117] Step 2. Synthesis of 2-(4-ethylphenyl)-N-((3S,6S,9S, 12S,15R,18S,21S)-6,9, 18-tris(2- aminoethyl)-15-benzyl-3-((R)-l-hydroxyethyl)-12-isobutyl-2,5,8,l l, 14, 17,20-heptaoxo- l,4,7,10, 13,16, 19-heptaazacyclotricosan-21-yl)acetamide (4). To the solution of tert-butyl N-[2- [(2S,5R,8S, 1 IS, 14S, 17S,22S)-5-benzyl-l 1, 14-bis[2-(tert-butoxycarbonylamino)ethyl]-22-[[2-(4- ethylphenyl)acetyl] amino] -17-[(1R)-1 -hydroxyethyl] -8-isobutyl-3 ,6,9, 12,15, 18,23 -heptaoxo- 1,4,7,10, 13, 16, 19-heptazacyclotricos-2-yl]ethyl]carbamate (4-2) (380 mg, 0.31 mmol) in DCM (8 mL), was added TFA (2 mL, 26.93 mmol) dropwise. The resulting mixture was stirred at room temperature. After completion of the reaction, the crude mixture was then concentrated to dryness, the residue was re- dissolved in 10% aq. formic acid solution (lOmL). The solvent was removed under vacuum and the solid was freeze-dried to give the title compounds 2-(4-ethylphenyl)-N-[(3S,6S,9S, 12S,15R, 18S,21S)-6,9, 18- tris(2-aminoethyl)-15-benzyl-3-[(lR)-l-hydroxyethyl]-12-isobutyl-2,5,8, l l,14, 17,20-heptaoxo- 1,4,7,10, 13, 16, 19-heptazacyclotricos-21-yl]acetamide; formic acid (4). LCMS: rt = 0.34 minutes (double, peak, LC method 3). m/z = 908 (M+l). ¾ NMR (400 MHz, D₂0) δ 7.29 (dq, J = 14.4, 7.0 Hz, 3H), 7.23 - 7.14 (m, 6H), 4.49 - 4.40 (m, 2H), 4.26 - 4.15 (m, 3H), 4.12 - 4.02 (m, 3H), 3.55 (d, J = 3.0 Hz, 2H), 3.38-3.31 (m, 1H), 3.10 - 2.90 (m, 7H), 2.82 - 2.74 (m, lH), 2.66 (dd, J = 13.8, 6.4 Hz, lH), 2.56 (q, J = 7.6 Hz, 2H), 2.24 - 2.09 (m, 3H), 2.07 - 1.93 (m, 2H), 1.90-1.74 (m, 3H), 1.42 - 1.26 (m, 2H), 1.11 (t, J = 7.6 Hz, 6H), 0.74 - 0.55 (m, 7H).

. 5

[0118] 2-(3 -methyl- 1H- 1 ,2,4-triazol- 1 -yl)-N-((3S,6S,9S, 12S, 15R, 18^,2 lS)-6,9, 18-tris(2- aminoethyl)-15-benzyl-3-((i?)-l-hydroxyethyl)-12-isobutyl-2,5,8,l l, 14,17,20-heptaoxo- l,4,7,10, 13,16, 19-heptaazacyclotricosan-21-yl)isonicotinamide (Compound 5), was prepared according to the coupling then deprotection sequence describe for Example 2, Compound 1. LCMS: rt ==^: 0.17 minutes (LC method 1 ). m/z - 908.6 (M-t I). ¾ NMR (400 MHz, D₂0) δ δ 9.26 (s, IH), 8.52 (d, J= 5.1 Hz, IH), 8.08 (s, IH), 8.00 (s, IH), 7.67 (d, J= 5.1 Hz, IH), 7.29 - 7.18 (m, 3H), 7.12 (m, 2H), 4.43 (t, J= 7.1 Hz, 2H), 4.33 (t, J= 7.2 Hz, IH), 4.28 - 4.13 (m, 2H), 4.13 - 3.94 (m, 4H), 3.47 - 3.38 (m, IH), 3.15 - 3.06 (m, IH), 3.01 - 2.77 (m, 8H), 2.72 (s, IH), 2.38 (s, 3H), 2.37 - 1.75 (m, 9H), 1.28 (d, J= 12.7 Hz, 2H), 1.06 (d, J= 6.0 Hz, 3H), 0.62 (s, 3H), 0.53 (d, J= 4.6 Hz, 3H).

(6-10) Compound 6

[0119] Compound 6, (2S,3R)-2-acetamido-3-hydroxy-N-((S)-2-oxo-l- ((3S,6S,9S, 12S, 15R, 18S,21S)-6,9,18-tris(2-aminoethyl)-15-^^^

2,5,8,11, 14,17,20-heptaoxo- 1,4,7, 10,13, 16, 19-heptaazacyclotricosan-21 -yl)piperidin-3 -yl)butanamide was prepared as follows.

[0120] Step 1. Intermediate (S)-2-amino-5-methoxy-5-oxopentanoic acid hydrochloride (6-2). Thionyl chloride (3.45 mL, 47.58 mmol) was added to methanol (20 niL) at -10 °C, and the resulting solution was stirred at this temperature for 45 min. Then, (2S)-2-aminopentanedioic acid (6-1) (5 g, 33.98 mmol) was added at once, the reaction solution was stirred at this temperature for 30 min and then warmed up to room temperature for 2 h. Slow addition of Et20 (80 mL) led to formation of a crystalline solid. The suspension was cooled to -10 °C and additional Et20 (80 mL) was added. The solid was collected by filtration, and then dried to give compound 6-2. (. LC-MS (LC method 1): m/z 162 (M+H)⁺.

[0121] Step 2. Intermediate (S)-2-(benzyloxycarbonylamino)-5 -methoxy-5 -oxopentanoic acid (6-3). (2S)-2-amino-5-methoxy-5-oxo-pentanoic acid hydrochloride (6-2) (5.g, 25.3 mmol) was dissolved in a solution (cooled to 0 °C) of NaOH (1.32 g, 32.89 mmol) and NaHC0₃ (4.25 g, 50.6 mmol) in H₂0 (50 mL) (pH ~ 9). After 15 min, benzyl carbonochloridate (2.14mL, 15.18 mmol) was added, and the mixture was vigorously stirred at 0 °C for 30 min and then for 2 h at r.t. (at the final pH ~ 8). After further addition of NaHCC (4.25 g, 50.6 mmol) and benzyl carbonochloridate (2.14 mL, 15.18 mmol), the reaction mixture were vigorously stirred at r.t. for an additional 3.5 h. The mixture was then extracted with ethyl acetate (200 mL). Thereafter, the aqueous phase was acidified with 6 M HC1 solution to adjust pH = 2-3. The reaction mixture was again extracted with ethyl acetate (500 mL). The organic phase was washed with brine, dried over Na2SC>4 and concentrated to give compound 6-3. LC-MS (LC method 1): m/z 296 (M+H)⁺.

[0122] Step 3. Intermediate (S)-l-tert-butyl 5 -methyl 2-(benzyloxycarbonylamino)

pentanedioate (6-4). To a solution of 2-(benzyloxycarbonylamino)-5-methoxy-5-oxo-pentanoic acid (6-3) (8 g, 27.09 mmol) in tert-butanol (lOOmL) was added (Boc)₂0 (7.1 g, 32.51 mmol) and DMAP (662 mg, 5.42 mmol). The resulting mixture was stirred at 30 °C for 3 h. The crude mixture was extracted with ethyl acetate (100 ml) and the organic phase was washed with water and brine, dried over Mg2SC>4, filtered and concentrated. The residue was purified by flash column chromatography on silica gel (PE : EA = 10 : 1) to give the compound 6-4. LC-MS (LC method 1): m/z 374 (M+Na)⁺.

[0123] Step 4. Intermediate (S)-tert-butyl-2-(benzyloxycarbonylamino)-5-hydroxypentanoate (6-

5) . To a solution of (S)-l-tert-butyl 5 -methyl 2-(benzyloxycarbonylamino) pentanedioate (760 mg, 2.16 mmol) 6-4 (760 mg, 2.16 mmol) in DCM (5 mL) was added L1BH4 (1.62 mL, 3.24 mmol) dropwise at 0 °C. After addition, the reaction mixture was stirred at this temperature for 5 h. The crude mixture was quenched with aq. NH4CI (50 mL), extracted with ethyl acetate (50 mL). The organic phase was separated and dried over anhydrous Na2SC>4, filtered and then concentrated. The residue was purified by flash column chromatography on silica gel (PE : EA = 3 : 1) to give the title compound (6-5). LC-MS (LC method 1): m/z 324 (M+H)⁺.

[0124] Step 5. Intermediate (S)-tert-butyl 2-(benzyloxycarbonylamino)-5-bromopentanoate (6-

6) . To a solution of carbontetrabromide (492.25 mg, 1.48 mmol) in DCM (10 mL) was added triphenylphosphine (389.34 mg, 1.48 mmol) and (S)-tert-butyl-2-(benzyloxycarbonylamino)-5- hydroxypentanoate (400 mg, 1.24 mmol). The mixture was stirred at 20 °C overnight. The crude mixture was diluted with DCM (10 ml), washed with water and brine, dried over anhydrous Na2SC>4, filtered and concentration. The residue was purified by flash column chromatography on silica gel (PE : EA = 5 : 1) to give the title compound 6-6 as yellow oil. LC-MS (LC method 1): m/z 330 (M+H-t-Bu)⁺.

[0125] Step 6. Intermediate (S)-2-(benzyloxycarbonylamino)-5-bromopentanoic acid (6-7). To a solution of (S)-tert-butyl 2-(benzyloxycarbonylamino)-5-bromopentanoate (6-6) (280 mg, 0.72 mmol) 6 in DCM (5 mL) was added trifluoroacetic acid (330.6 mg, 2.9 mmol) dropwise at 0 °C. The mixture was stirred at 0 °C for 3 h. The crude mixture was concentrated to give the title compound 6-7. LC-MS (LC method 1): m/z 330 (M+H)⁺. [0126] Step 7. Intermediate tert-butyl N-[2-[(2S,5R,8S,l lS,14S,17S,22S)-5-benzyl-22-[[2- (benzyloxycarbonylamino)-5 -bromo-pentanoyl] amino] -11, 14-bis[2-(tert-butoxycarbonylamino)ethyl] -17- [( 1 R)- 1 -hydroxyethyl] -8-isobutyl-3 ,6, 94^

yl] ethyl] carbamate (6-8). To a solution of (2S)-2-(benzyloxycarbonylamino)-5-bromo-pentanoic acid (6- 7) (100 mg, 0.3 mmol) and PMBHBoc₃ (321 mg, 0.30 mmol) in anhydrous DMF (4 mL) was added DIPEA (78.29 mg, 0.61 mmol), HOBt (49.1 mg, 0.36 mmol) and HBTU (137.83 mg, 0.36 mmol). The reaction mixture was stirred at room temperature overnight and then quenched with brine (10 mL), extracted with ethyl acetate (50 mL). The organic phase was separated, dried over anhydrous Na2SC>4, filtered and concentrated to give the title compound 6-8 (310 mg). LC-MS (LC method 1 ): m/z 1375.6 (M+H)⁺

[0127] Step 8. Intermediate tert-butyl N-[2-[(2S,5R,8S,l lS,14S,17S,22S)-5-benzyl-22-[(3S)-3- (benzyloxycarbonylamino)-2-oxo- 1 -piperidyl] -11, 14-bis [2-(tert-butoxycarbonylamino)ethyl] -17-[(1R)-1- hydroxyethyl]-8-isobutyl-3,6,9, 12, 15, 18,23-heptaoxo-l,4,7, 10, 13, 16, 19-heptazacyclotricos-2- yl]ethyl]carbamate (6-9). To a solution of (tert-butyl N-[2-[(2S,5R,8S, l lS, 14S,17S,22S)-5-benzyl-22- [[2-(benzyloxycarbonylamino)-5 -bromo-pentanoyl]amino] -11, 14-bis [2-(tert-butoxy

carbonylamino)ethyl] -17-[(1R)-1 -hydroxyethyl] -8-isobutyl-3 ,6,9, 12,15, 18,23 -heptaoxo- 1,4,7,10, 13, 16, 19-heptazacyclotricos-2-yl]ethyl]carbamate (6-8) (50 mg, 0.04 mmol) in anhydrous DMF (3 mL) was added K2CO3 (10.06 mg, 0.073 mmol). The reaction mixture was stirred at room temperature for 5 h. The crude mixture was quenched with brine (10 mL). The resulting mixture was extracted with ethyl acetate (50 mL). The organic phase was separated, dried over anhydrous Na2SC>4, filtered and concentrated. The residue was purified with prep. HPLC (acidic condition, modifier: HCOOH) to afford product 6-9. LC-MS (LC method 1 ): m/z 1293.7 (M+H)⁺.

[0128] Step 9. Intermediate tert-butyl N-[2-[(2S,5R,8S,l lS,14S,17S,22S)-22-[(3S)-3-amino-2- oxo- 1 -piperidyl] -5 -benzyl- 11 , 14-bis [2-(tert-butoxycarbonylamino)ethyl] -17-[(1R)-1 -hydroxyethyl] -8- isobutyl-3,6,9, 12,15, 18,23-heptaoxo-l,4,7, 10,13, 16, 19-heptazacyclotricos-2-yl]ethyl]carbamate 6-10. To a solution oftert-butyl N-[2-[(2S,5R,8S, l lS, 14S,17S,22S)-5-benzyl-22-[(3S)-3-

(benzyloxycarbonylamino)-2-oxo- 1 -piperidyl] -11, 14-bis[2-(tert-butoxy carbonylamino)ethyl] -17-[(1R)-1- hydroxyethyl]-8-isobutyl-3,6,9, 12, 15, 18,23-heptaoxo- 1,4,7, 10, 13, 16, 19-heptazacyclotricos-2- yl]ethyl]carbamate (6-9) (70 mg, 0.05 mmol) in MeOH (5 mL) was added Pd(OH)₂/C (3 mg). The reaction mixture was stirred under H2 balloon (~1 atm) for 3 h. The catalyst was filtered off through Celite, the filtrate was concentrated to give the title compound (6-10). LC-MS (LC method 1 ): m/z 1159.7 (M+H)⁺.

[0129] Step 10. Intermediate tert-butyl N-[2-[(2S,5R,8S, l lS, 14S, 17S,22S)-22-[(3S)-3- [[(2R,3R)-2-acetamido-3 -hydroxy-butanoyl] amino] -2-oxo- 1 -piperidyl] -5 -benzyl- 11 , 14-bis [2-(tert- butoxycarbonylamino)ethyl] -17-[(1R)-1 -hydroxyethyl] -8-isobutyl-3 ,6,9, 12, 15, 18,23 -heptaoxo- 1,4,7, 10, 13, 16, 19-heptazacyclotricos-2-yl]ethyl]carbamate (6-11). To a solution of tert-butyl N-[2- [(2S,5R,8S, l lS, 14S, 17S,22S)-22-[(3S)-3-amino-2-oxo- l-piperidyl]-5-benzyl-l l, 14-bis[2-(tert- butoxycarbonylamino)ethyl] -17-[(1R)-1 -hydroxyethyl] -8-isobutyl-3 ,6, 9, 12, 15, 18,23 -heptaoxo-

1.4.7.10.13.16.19- heptazacyclotricos-2-yl]ethyl]carbamate (6-10) (60 mg, 0.05 mmol) and (2S,3R)-2- acetamido-3-hydroxybutanoic acid (8.34 mg, 0.05mmol) in anhydrous DMF (4 mL) was added DIPEA (13.38 mg, 0.1 mmol), HOBt (8.39 mg, 0.06 mmol) and HBTU (36.76 mg, 0.1 mmol). The reaction mixture was stirred at room temperature overnight. The crude mixture was quenched with brine ( 10 mL), extracted with ethyl acetate (50 mL). The organic phase was separated and dried over anhydrous Na2SC>4, filtered and concentrated. The residue was purified by prep. HPLC (acidic condition, modifier:HCOOH) to give the title compound 6-11 (23 mg, 34.7% yield). LC-MS (LC method 1): m/z 1302.6 (M+H)⁺.

[0130] Synthesis of Compound 6, (2S,3R)-2-acetamido-3-hydroxy-N-[(3S)-2-oxo-l- [(3S,6S,9S, 12S, 15R, 18S,21 S)-6,9, 18-tris(2-aminoethyl)-15-benzyl-3-[(lR)- l-hydroxyethyl]-12-isobutyl-

2.5.8.1 1.14.17.20- heptaoxo- 1,4,7, 10, 13, 16, 19-heptazacyclotricos-21 -yl] -3 -piperidyl]butanamide was prepared according to the coupling then deprotection sequence describe for Example 2, Compound 1. LCMS: rt = 0.363 minutes (LC method 2). m/z = 1002.6 (M+l). ¾ NMR (400 MHz, D₂0) 5 8.16 (s, 1H), 7.37 - 7.23 (m, 3H), 7.19 (m, 2H), 4.52 (m, 2H), 4.43 (m, lH), 4.39 - 4.29 (m, lH), 4.29 - 3.98 (m, 7H), 3.79 (m, 1H), 3.72 - 3.54 (m, 1H), 3.44 - 3.22 (m, lH), 3.19 - 2.89 (m, 8H), 2.86 - 2.60 (m, 3H), 2.36 - 1.67 (m, 16H), 1.39 (m, 2H), 1.24 (m, lH), 1.14 (dd, J = 21.0, 5.8 Hz, 7H), 0.80 (s, 1H), 0.70 (d, J = 6.3 Hz, 3H), 0.63 (d, J = 6.3 Hz, 3H).

Compound 7

[0131] (2S,3R)-((R)-3-hydroxy-l-oxo-l-((3S,6S,9S,12S,15R,18S,21S)-6,9,18-tris(2- aminoethyl)- 15 -benzyl-3 -((R)- 1 -hydroxyethyl)- 12-isobutyl-2,5 , 8, 11 , 14, 17,20-heptaoxo- 1, 4, 7, 10, 13, 16, 19-heptaazacyclotricosan-21 -ylamino)propan-2-yl) 2-acetamido-3 -hydroxybutanoate (Compound 7) was prepared according to the following procedure.

[0132] Step 1. Intermediate (S)-benzyl 3-(benzyloxy)-2-hydroxypropanoate (7-2). To a solution of (2S)-3-benzyloxy-2-hydroxy-propanoic acid (150 mg, 5.35 mmol) (7-1) and CS2CO3 (2615 mg, 8.03 mmol) in DMF (20 mL), was added bromomethyl benzene (0.7 niL, 5.89 mmol) dropwise. The reaction mixture was then stirred at room temperature overnight. The crude mixture was partitioned between ethyl acetate (100 mL) and water (100 mL), the organic phase was separated and washed with 10% LiCl, brine, dried over Na2SC>4 and concentrated. The residue was purified by flash column chromatography on silica gel (PE : EA = 2 : 1) to give the title compound 7-2. LC-MS (LC method 1): m/z 287 (M+H)⁺. [0133] Step 2. Intermediate (S)-benzyl 3-(benzyloxy)-2-(methylsulfonyloxy)propanoate (7-3). To a solution of benzyl (2S)-3-benzyloxy-2-hydroxy-propanoate (7-2) (950 mg, 3.32 mmol) and triethylamine (1.38 mL, 9.95 mmol) in DCM (15 mL), was added methanesulfonyl chloride (0.3 lmL, 3.98mmol) at 0 °C. The reaction mixture was then stirred at room temperature for 2 h. The crude mixture was partitioned between DCM (50 mL) and water (50 mL), and the organic phase was washed with brine, dried over Na2SC>4 filtered and concentrated, the residue was purified by flash column chromatography on silica gel (PE : EA = 3 : 1) to give the title compound 7-3 as white solid. LC-MS (LC method 1): m/z 296 (M+H)⁺.

[0134] Step 3. Intermediate (2S,3R)-((R)-l,3-bis(benzyloxy)- l-oxopropan-2-yl) 2-acetamido-3- hydroxybutanoate (7-4). To a solution of benzyl (2S)-3-benzyloxy-2-methylsulfonyloxy-propanoate (900 mg, 2.47 mmol) 7-3 and (2S,3R)-2-acetamido-3-hydroxy-butanoic acid (398 mg, 2.47 mmol) in DMF (15 mL) was added CS2CO3 (1609 mg, 4.94 mmol). The mixture was then stirred at 80 °C overnight. The crude mixture was partitioned between EA (50 mL) and water (50 mL), the organic phase was washed with brine, dried over Na2SC>4, filtered and concentrated. The residue was purified by prep-TLC (PE : EA = 2 : 1) to give the title compound (2S,3R)-((R)-l,3-bis(benzyloxy)-l-oxopropan-2-yl) 2-acetamido-3- hydroxybutanoate 7-4 (200 mg, 23% yield) LC-MS (LC method 1): m/z 430 (M+H)⁺.

[0135] Step 4. Intermediate (R)-2-((2S,3R)-2-acetamido-3-hydroxybutanoyloxy)-3- hydroxypropanoic acid (7-5). To a solution of (2S,3R)-((R)-l,3-bis(benzyloxy)-l-oxopropan-2-yl) 2- acetamido-3 -hydroxybutanoate (7-4) (60 mg, 0.12 mmol) in EtOH (3 mL) was added 10% Pd/C (5 mg). The reaction mixture was stirred under ¾ balloon (~1 atm) overnight. The catalyst was filtered off through Celite, the filtrate was concentrated to give the title compound 7-5 (25 mg, 86 % yield). LC-MS (LC method 1): m/z 250 (M+H)⁺.

[0136] Step 5. Synthesis of (2S,3R)-((R)-3-hydroxy-l-oxo-l-((3S,6S,9S,12S,15R, 18S,21S)- 6,9, 18-tris(2-aminoethyl)-15-benzyl-3-((R)-l-hydroxyethyl)-12-isobutyl-2,5, 8, 11, 14,17,20-heptaoxo- 1, 4, 7, 10, 13, 16, 19-heptaazacyclotricosan-21 -ylamino)propan-2-yl) 2-acetamido-3 -hydroxybutanoate (Compound 7). Compound 7 was prepared according to the coupling then deprotection sequence describe for Example 2, Compound 1. LCMS: rt = 0.19 and 0.32 minutes (double peak) (LC method 2). m/z = 993.6 (M+l). ¾ NMR (400 MHz, D₂0) δ 7.26-7.17 (m, 3H), 7.14 - 7.07 (m, 2H), 4.44 - 4.25 (m, 4H), 4.21 - 3.89 (m, 7H), 3.69-3.65 (m, 1H), 3.32-3.19 (m, 1H), 3.08 - 2.85 (m, 7H), 2.82-2.66 (m, 2H), 2.25 - 1.58 (m, 11H), 1.37 - 0.96 (m, 7H), 0.66-0.47 (m, 7H).

Compound 8

[0137] (2S,3R)-2-acetamido-3-(benzyloxy)-N-((3S,6S,9S, 12S, 15R, 18S,21S)-6,9, 18-tris(2- aminoethyl)- 15 -benzyl-3 -((R)- 1 -hydroxyethyl)- 12-isobutyl-2,5 , 8, 11 , 14, 17,20-heptaoxo- l,4,7,10, 13,16, 19-heptaazacyclotricosan-21-ylcarbamoyloxy)butanamide is prepared according to the following procedure. (Compound 8)

[0138] Step 1. Intermediate (2S,3R)-2-acetamido-3-(benzyloxy)-N-hydroxybutanamide (8-2). To a solution of (2S,3R)-2-acetamido-3-benzyloxy-butanoic acid (8-1) (1000 mg, 3.98 mmol) in THF (3 mL) at -30 °C was added 4-methylmorpholine (0.53 mL, 4.78 mmol), followed by the slow addition of ethyl carbonochloridate (0.45 mL, 4.78 mmol). The resulting reaction mixture was stirred at -30 °C for 2h. Then to the reaction mixture was added freshly prepared hydroxylamine (331 mg, 4.78 mmol). The resulting mixture was stirred at room temperature for 2.5 h. The crude mixture was then concentrated under reduced pressure, the residue was purified by reverse ISCO (acidic condition, modifier: TFA) to give the title compound 8-2. LC-MS (LC method 1): m/z 267 (M+H)⁺.

[0139] Step 2. Intermediate tert-butyl N-[2-[(2S,5R,8S, l l S, 14S, 17S,22S)-22-[[[(2S,3R)-2- acetamido-3 -benzyl oxy-butanoyl] amino] oxycarbonylamino] -5 -benzyl- 1 1 , 14-bis [2-(tert- butoxycarbonylamino)ethyl] -17-[(1R)-1 -hydroxyethyl] -8-isobutyl-3 ,6,9, 12, 15, 18,23 -heptaoxo- 1,4,7, 10, 13, 16, 19-heptazacyclotricos-2-yl]ethyl]carbamate (8-3). To a solution of N-[(l S,2R)-2- benzyloxy-l-(hydroxycarbamoyl)propyl] acetamide (8-2) (200 mg, 0.75 mmol) in MeCN (5 mL) was added CDI (133.84 mg, 0.83 mmol) at 0 °C. The reaction mixture was then stirred at room temperature for 2 h, then cooled to 0°C, tert-butyl N-[2-[(2S,5R,8S, l l S, 14S, 17S,22S)-22-amino-5-benzyl-l l, 14-bis[2- (tert-butoxycarbonylamino)ethyl] -17-[(1R)-1 -hydroxyethyl] -8-isobutyl-3 ,6,9, 12, 15 , 18,23 -heptaoxo-

1.4.7.10.13.16.19- heptazacyclotricos-2-yl]ethyl]carbamate (8-3) (797.82 mg, 0.75 mmol) was added to the reaction mixture. The resulting mixture was stirred at room temperature for 2h. The precipitate was filtered off and the filtrate was concentrated, the residue was purified by prep. HPLC (acidic condition, modifier: HCOOH) to give the title compound 8-3 LC-MS (LC method 1): m/z 1354.7 (M+H)⁺.

[0140] Step 3. Synthesis of (2S,3R)-2-acetamido-3-(benzyloxy)-N- ((3S,6S,9S, 12S, 15R, 18S,21 S)-6,9, 18-tris(2-aminoethyl)-15-benzyl-3-((R)- l-hydroxyethyl)-12-isobutyl^

2.5.8.1 1.14.17.20- heptaoxo- 1,4,7, 10, 13, 16, 19-heptaazacyclotricosan-21 -ylcarbamoyloxy)butanamide. Compound 8 was prepared according to the coupling then deprotection sequence describe for Example 2, Compound 1. LCMS: rt = 0.18 and 0.32 minutes (double peak) (LC method 2). m/z = 1054.6 (M+l). ¾ NMR (400 MHz, D₂0) δ 7.40 - 7.23 (m, 8H), 7.18 (d, J = 7.0 Hz, 2H), 4.62 - 4.57 (m, 1H), 4.51-4.41 (m, 4H), 4.28-4.19 (m, 2H), 4.15-4.08 (m, 3H), 4.07 - 3.94 (m, 2H), 3.46 - 3.33 (m, 1H), 3.13 - 2.94 (m, 7H), 2.86 - 2.67 (m, 2H), 2.24 - 2.10 (m, 3H), 2.07 - 1.77 (m, 8H), 1.44 - 1.29 (m, 2H), 1.22 - 1.08 (m, 6H), 0.78 - 0.58 (m, 7H).

EXAMPLE 10. SYNTHESIS OF COMPOUNDS 9 and 10

Compound 9 Compound 10

[0141] Step 1. Synthesis of Intermediate Compound 9-2. 2-(5-bromo-2-fluoro-phenyl)acetic acid (2g, 8.58 mmol) (9-1) was added in small portions over a period of 20 min to fuming nitric acid (10 mL) chilled to -10 °C (methanol/ice). The addition was closely monitored to keep the temperature below 5 °C. After addition, the solution stirred at -10 °C for 15 min. The crude mixture was poured into ice water and extracted with ethyl acetate (100 mL). The organic phase was washed with brine, dried over Na2SC>4, filtered and concentrated to dryness to give methyl 2-(5-bromo-2-fluoro-3-nitrophenyl)acetate (9-2), which was used in the next step without purification. LCMS (LC method 1): m/z 278(M+1)⁺.

[0142] Step 2. Synthesis of Intermediate Compound 9-3. To a mixture of Intermediate Compound 9-2 (2.4g, 8.63 mmol) and NaHC0₃ (1.45g, 17.26 mmol) in DMF (10 mL), was added Mel (0.59 mL, 9.5 mmol). The resulting mixture was stirred at room temperature overnight. The crude mixture was poured into water (100 mL) and extracted with ethyl acetate (50 mL x 2). The combined organic phase were washed with brine and dried over anhydrous Na2SC>4 and concentrated to dryness. The residue was purified by silica gel column chromatography (pet ether/ethyl acetate = 10: 1 to 3 : 1) to give Intermediate Compound 9-3, methyl 2-(5-bromo-2-fluoro-3-nitrophenyl)acetate. (2.0g, 79% yield). LCMS (LC method 1): m/z 292(M+1)⁺.

[0143] Step 3. Synthesis of Intermediate Compound 9-4. To a solution of Intermediate Compound 9-3 (6.34g, 21.7 mmol) in DMSO (30 mL) at room temperature was added paraformaldehyde (683.8 mg, 22.79 mmol) and NaHCC (91.2 mg, 1.09 mmol). The resulting reaction mixture was stirred at 45°C overnight and then diluted with brine (100 mL). The resulting mixture was extracted with ethyl acetate (250 mL x 2). The organic layer was separated, dried over anhydrous Na2SC>4, filtered, and then concentrated. The residue was purified by column chromatography on silica gel (pet ether/ethyl acetate= 3 : 1) to give Intermediate Compound 9-4, methyl 2-(5-bromo-2-fluoro-3-nitrophenyl)-3-hydroxy- propanoate. LCMS (LC method 1): m/z 322 (M+l)⁺.

[0144] Step 4. Synthesis of Intermediate Compound 9-5. Pd/C (10% w/w, 500 mg) was added to a solution of Intermediate Compound 9-4 (3.09g, 9.59 mmol) in methanol (15 mL) and stirred at room temperature under atmospheric pressure of hydrogen overnight. The catalyst was filtered off and washed with methanol (2 mL). The combined filtrates were concentrated under reduced pressure to yield Intermediate Compound 9-5, methyl 2-(3-amino-2-fluorophenyl)-3-hydroxypropanoate.

LCMS (LC method 1): m/z 214 (M+l)⁺.

[0145] Step 5. Synthesis of Intermediate Compound 9-6. To an ice water cooled solution of Intermediate Compound 9-5 (lg, 4.69 mmol) and DIPEA (1.64 mL, 9.38 mmol) in DCM (10 mL) was added acetyl acetate (0.44 mL, 4.69 mmol) dropwise. After addition, the reaction mixture was stirred for another 4 h. Then the solvent and excessive acetic anhydride were removed in vacuo, the residue was purified column chromatography on silica gel (pet ether/ethyl acetate = 1 : 1) to give Intermediate Compound 9-6, methyl 2-(3-acetamido-2-fluorophenyl)-3-hydroxypropanoate. LCMS (LC method 1): m/z 256 (M+l)⁺.

[0146] Step 6. Synthesis of Intermediate Compound 9-7. To a solution of compound

Intermediate Compound 9-6 (251 mg, 0.98 mmol) in a mixture of THF (4 mL) and water ( 1 mL) at 0 °C was added LiOH- H2O (41.3 mg, 0.98 mmol) . The reaction mixture was stirred at room temperature for 3 h and then the pH was adjusted to 3-4 with 2N HC1. The resulting mixture was extracted with ethyl acetate (10 mL). The organic phase was washed with brine, dried over anhydrous Na2SC>4, filtered and concentrated to give Intermediate Compound 9-7, 2-(3-acetamido-2-fluorophenyl)-3-hydroxypropanoic acid. LCMS (LC method 1): m/z 242 (M+l)⁺. [0147] Step 7. Synthesis of Compound 9. Compound 9, (S)-2-(3-acetamido-2-fluorophenyl)-3- hydroxy-N-((3S,6S,9S, 12S, 15R,18S,21S)-6,9, 18-tris(2-aminoethyl)-15-benzyl-3-((R)-l-hydroxyethyl)- 12-isobutyl-2,5 , 8, 11, 14,17,20-heptaoxo- 1, 4,7, 10,13, 16, 19-heptaazacyclotricosan-21 -yl)propanamide, was prepared according to the coupling, hydrolysis, the second coupling then deprotection sequence described for Example 2, Compound 1. LCMS: rt =0.56min (LC method 1). m/z = 985.6 (M+l)⁺. ¾ NMR (400 MHz, D₂0) δ 8.07 (s, 1H), 7.21 (m, 5H), 7.13 - 6.99 (m, 3H), 4.44 - 4.28 (m, 3H), 4.16 (dd, J= 8.3, 5.5 Hz, 3H), 4.07 - 3.68 (m, 7H), 3.21 (m, lH), 3.08 - 2.40 (m, 1 1H), 2.24 - 1.53 (m, 14H), 1.40 - 1.19 (m,

= 20.3, 5.8 Hz, 3H), 0.60 (s, 3H), 0.52 (s, 3H).

[0148] Step 7(con ). Synthesis of Compound 10. Compound 10, (S)-2-(3-acetamido-2- fluorophenyl)-3-hydroxy-N (3S,6S,9S,12S,15R,18S,21S)-6,9, 18 ris(2-aminoethyl)-15-benzyl-3-((R)-l- hydroxyethyl)- 12-isobutyl-2,5 ,8, 11 , 14, 17,20-heptaoxo- 1 ,4,7, 10, 13 , 16, 19-heptaazacyclotricosan-21 - yl)propanamide, was prepared according to the coupling, hydrolysis, the second coupling then deprotection sequence described for Example 2, Compound 1. LCMS: rt =0.26min (LC method 1). m/z = 985.6 (M+l)⁺. 1H NMR (400 MHz, D₂0) δ 8.16 (s, 2H), 7.30 (m, 5H), 7.15 (m, 3H), 4.51 - 4.34 (m, 3H), 4.57 - 4.12 (m, 10H), 3.34 (m, 1H), 3.04 - 2.98 (m, 8H), 2.83 - 2.44 (m, 2H), 2.33 - 1.63 (m, 13H), 1.47 - 1.19 (m, 3H), 1.48 - 0.99 (m, 7H), 1.13 (t, J= 7.8 Hz, 4H), 0.69 (s, 3H), 0.60 (s, 3H).

11

[0149] 2-(4-fluorophenyl)-N-((3 S,6S,9S, 12S, 15R, 18S,21 S)-6,9, 18-tris(2-aminoethyl)- 15-benzyl- 3 -((R)- 1 -hydroxyethyl)- 12-isobutyl-2,5 ,8, 11 , 14, 17,20-heptaoxo- 1 ,4,7, 10, 13 , 16, 19-heptaazacyclotricosan- 21-yl)acetamide (Compound 11), was prepared according to the coupling then deprotection sequence describe for Compound 4. LCMS: rt = 0.20 minutes (LC method 2). m/z = 898.5 (M+l)⁺. ¾ NMR (400 MHz, D₂0): δ 7.29 (m, 3H), 7.25 - 7.15 (m, 4H), 7.06 (m, 2H), 4.51 - 4.41 (m, 2H), 4.26 - 4.14 (m, 3H), 4.09 (m, 3H), 3.58 (s, 2H), 3.35 (s, 1H), 3.10 - 2.89 (m, 7H), 2.78 (s, 1H), 2.68 (s, 1H), 2.14 (m, 3H), 2.07 - 1.92 (m, 2H), 1.84 (m, 3H), 1.43 - 1.27 (m, 2H), 1.11 (d, J= 5.4 Hz, 3H), 0.67 (s, 3H), 0.59 (s, 3H).

12

[0150] 2-(4-methoxyphenyl)-N-((3S,6S,9S, 12S, 15R,18S,21S)-6,9, 18-tris(2-aminoethyl)-15- benzyl-3-((R)-l-hydroxyethyl)-12-isobutyl-2,5,8,l l, 14,17,20-heptaoxo-l,4,7, 10, 13,16, 19- heptaazacyclotricosan-21-yl)acetamide Compound 12 was prepared according to the coupling then deprotection sequence described for Compound 4. LCMS: rt = 0.18 minutes (LC method 2). m/z = 910.5 (M+l)⁺. ¾ NMR (400 MHz, D₂0): δ 7.41 - 7.24 (m, 3H), 7.18 (m, 4H), 6.92 (d, J= 8.4 Hz, 2H), 4.44 (m, 2H), 4.26 - 4.15 (m, 3H), 4.08 (m, 3H), 3.76 (s, 3H), 3.53 (s, 2H), 3.35 (m, lH), 3.00 (m, 7H), 2.78 (s, 1H), 2.68 (s, 1H), 2.14 (m, 3H), 2.00 (m, 2H), 1.84 (d, J= 9.6 Hz, 3H), 1.43 - 1.28 (m, 2H), 1.11 (d, J= 5.2 Hz, 3H), 0.67 (s, 3H), 0.59 (s, 3H).

13

[0151] (R)-2-phenyl-N-((3S,6S,9S,12S,15R,18S,21S)-6,9,18-tris(2-aminoethyl)-15-benzyl-3- ((R)- 1 -hydroxyethyl)- 12-isobutyl-2,5 , 8, 11 , 14, 17,20-heptaoxo- 1, 4,7, 10, 13, 16, 19-heptaazacyclotricosan- 21-yl)propanamide was prepared according to the coupling then deprotection sequence describe for

Compound 4. LCMS: rt = 0.18 & 0.33 minutes (double peak, LC method 2). m/z = 894.5 (M+l)⁺. ¾ NMR (400 MHz, D₂0): δ 7.39 - 7.24 (m, 8H), 7.18 (d, J = 7.6 Hz, 2H), 4.46 (t, J = 7.6 Hz, 2H), 4.21 (dd, J = 14.4, 8.0 Hz, 2H), 4.17-4.00 (m, 4H), 3.78 (m, 1H), 3.30-3.19 (m, lH), 3.07-2.92 (m, 7H), 2.86-2.69 (m, 2H), 2.23-2.10 (m, 3H), 2.03 (dd, J = 15.4, 6.8 Hz, 2H), 1.91-1.77 (m, 3H), 1.44-1.28 (m, 5H), 1.10 (t, J = 8.6 Hz, 3H), 0.70-0.55 (m, 7H).

D 14

[0152] (S)-2-phenyl-N-((3S,6S,9S,12S,15R,18S,21S)-6,9, 18-tris(2-aminoethyl)-15-benzyl-3- ((R)- 1 -hydroxyethyl)- 12-isobutyl-2,5 , 8, 11 , 14, 17,20-heptaoxo- 1, 4,7, 10, 13, 16, 19-heptaazacyclotricosan- 21-yl)propanamide Compound 14 was prepared according to the coupling then deprotection sequence describe for Compound 4. LCMS: rt = 0.24 & 0.35 minutes (double peak, LC method 2). m/z = 894.5 (M+l)⁺. ¾ NMR (400 MHz, D₂0): δ 7.39 - 7.24 (m, 8H), 7.19 (d, J = 7.2 Hz, 2H), 4.50 - 4.42 (m, 1H), 4.37 (dd, J = 9.2, 5.0 Hz, 1H), 4.31 - 4.21 (m, 2H), 4.20 - 4.13 (m, 2H), 4.09 (dt, J = 11.2, 6.0 Hz, 2H), 3.77 (q, J = 7.2 Hz, 1H), 3.42 - 3.32 (m, 1H), 3.15 - 2.89 (m, 7H), 2.66 - 2.57 (m, 1H), 2.51 - 2.41 (m, 1H), 2.29 - 2.02 (m, 4H), 1.94 - 1.76 (m, 3H), 1.71-1.62 (m, 1H), 1.48 - 1.23 (m, 5H), 1.13 (d, J = 6.4 Hz,

3H), 0.73 - 0.51 (m, 7H).

15

[0153] 2-(6-ethylpyridin-3-yl)-N-((3S,6S,9S,12S,15R,18S,21S)-6,9, 18-tris(2-aminoethyl)-15- benzyl-3-((R)-l-hydroxyethyl)-12-isobutyl-2,5,8,l l, 14,17,20-heptaoxo-l,4,7, 10, 13,16, 19- heptaazacyclotricosan-21-yl)acetamide Compound 15 was prepared according to the coupling then deprotection sequence describe for Compound 4. LCMS: rt = 0.42 minutes (LC method 2). m/z = 909.5 (M+l)⁺. ¾ NMR (400 MHz, D₂0): δ 8.47(s, 1H),8.31 (d, J = 8.0 Hz, 1H), 8.15 (s, 1H),7.84 (d, J = 8.0 Hz, 1H), 7.32 - 7.28 (m, 3H), 7.19 - 7.17 (m, 2H), 4.50 (t, J = 8.0 Hz, lH), 4.43- 4.39 (m, 1H), 4.25-4.12 (m, 4H),3.83 (s, 2H), 3.38-3.30 (m, 1H), 3.10-2.98 (m, 6H), 2.79-2.71 (m, 2H), 2.18-2.08 (m, 3H), 1.88- 1.85 (m, 3 H), 1.44-1.27 (m, 5H), 1.10 (d, J = 4.0 Hz, 3H), 0.80 (s, lH), 0.80-0.68 (m, 4H), 0.63 (d, J = 8.0 Hz, 3H).

16

[0154] 2-(4-chlorophenyl)-N-((3S,6S,9S,12S,15R, 18S,21S)-6,9,18-tris(2-aminoethyl)-15- benzyl-3-((R)-l-hydroxyethyl)-12-isobutyl-2,5,8,l l, 14,17,20-heptaoxo-l,4,7, 10, 13,16, 19- heptaazacyclotricosan-21-yl)acetamide Compound 16 was prepared according to the coupling then deprotection sequence describe for Compound 4. LCMS: rt = 0.20 & 0.30 minutes (double peak, LC method 2). m/z = 914.5 (M+l)⁺. ¾ NMR (400 MHz, D₂0): δ 8.16 (s, 1H), 7.31 (m, 5H), 7.19 (m, 4H), 4.51-4.40 (m, 2H), 4.28-4.14 (m, 3H), 4.08 (m, 3H), 3.58 (s, 2H), 3.35 (s, 1H), 3.15-2.90 (m, 7H), 2.78 (s, 1H), 2.67 (s, 1H), 2.14 (m, 3H), 1.99 (m, 2H), 1.83 (m, 3H), 1.45-1.25 (m, 2H), 1.11 (m, 4H), 0.69 (s, 1H), 0.67 (s, 3H), 0.59 (s, 3H).

17

[0155] 2-phenyl-N-((3S,6S,9S,12S,15R, 18S,21S)-6,9,18-tris(2-aminoethyl)-15-benzyl-3-((R)-l- hydroxyethyl)- 12-isobutyl-2,5 ,8, 11 , 14, 17,20-heptaoxo- 1 ,4,7, 10, 13 , 16, 19-heptaazacyclotricosan-21 - yl)acetamide Compound 17 was prepared according to the coupling then deprotection sequence describe for Compound 4. LCMS: rt = 0.25 & 0.30 minutes (double peak, LC method 2). m/z = 880.5 (M+l)⁺. ¾ NMR (400 MHz, D₂0): δ 8.08 (s, 1H), 7.30-7.21 (m, 5H), 7.17 (m, 3H), 7.10 (d, J= 7.2 Hz, 2H), 4.36 (m, 2H), 4.12 (m, 3H), 4.05-3.95 (m, 3H), 3.58-3.46 (m, 2H), 3.36-3.23 (m, lH), 3.04-2.82 (m, 7H), 2.73- 2.65 (m, 1H), 2.57 (m, 1H), 2.08 (m, 3H), 1.99-1.85 (m, 2H), 1.82-1.63 (m, 3H), 1.24 (m, 2H), 1.03 (m, 3H), 0.71-0.61 (m, 1H), 0.59 (s, 3H), 0.51 (s, 3H).

18

[0156] 2-(biphenyl-4-yl)-N-((3S,6S,9S,12S,15R,18S,21S)-6,9, 18-tris(2-aminoethyl)-15-benzyl- 3-((R)-l-hydroxyethyl)-12-isobutyl-2,5,8, l l,14, 17,20-heptaoxo-l,4,7, 10, 13,16, 19-heptaazacyclotricosan- 21-yl)acetamide Compound 18 was prepared according to the coupling then deprotection sequence describe for Compound 4. LCMS: rt = 0.30 minutes (LC method 2). m/z = 957.5 (M+l)⁺. ¾ NMR (400 MHz, D₂0): δ 8.18 (s, 1H), 7.65 (m, 4H), 7.48 (m, 2H), 7.33 (m, 6H), 7.18 (d, J= 7.2 Hz, 2H), 4.45 (m, 2H), 4.29-4.18 (m, 3H), 4.09 (d, J= 5.8 Hz, 3H), 3.72-3.60 (m, 2H), 3.38 (m, 1H), 3.11-2.95 (m, 7H), 2.79 (m, 1H), 2.67 (m, 1H), 2.30-2.11 (m, 3H), 2.08-1.77 (m, 5H), 1.42-1.30 (m, 2H), 1.10 (d, J= 5.6 Hz, 3H), 0.70 (s, 1H), 0.69 (s, 3H), 0.61 (s, 3H).

EXAMPLE 19. SYNTHESIS OF COMPOUND 19

[0157] Synthesis of 2-(4-tert-butylphenyl)-N-((3 S,6S,9S, 12S, 15R, 18S,21 S)-6,9, 18-tris(2- aminoethyl)-15-benzyl-3-((R)-l-hydroxyethyl)-12-isobutyl-2,5,8,l l, 14, 17,20-heptaoxo- 1,4,7,10, 13, 16, 19-heptaazacyclotricosan-21-yl)acetamide. Compound 19 was prepared according to the coupling then deprotection sequence describe for Compound 4. LCMS: rt = 0.29 minutes (LC method 2). m/z = 936.6 (M+l)⁺. ¾ NMR (400 MHz, D₂0): δ 8.18 (s, 1H), 7.44 (d, J= 7.5 Hz, 2H), 7.31 (m, 3H), 7.21 (m, 4H), 4.47 (m, 2H), 4.31-4.15 (m, 3H), 4.10 (s, 3H), 3.64-3.52 (m, 2H), 3.33 (m, lH), 3.17-2.91 (m, 7H), 2.81 (s, 1H), 2.70 (s, 1H), 2.28-2.10 (m, 3H), 2.09-1.95 (m, 2H), 1.94-1.74 (m, 3H), 1.45-1.32 (m, 2H), 1.24 (s, 9H), 1.12 (d, J= 4.7 Hz, 3H), 0.71 (s, 1H), 0.70 (s, 3H), 0.62 (s, 3H).

20

[0158] 2-(4-isopropylphenyl)-N-((3S,6S,9S,12S,15R,18S,21S)-6,9, 18-tris(2-aminoethyl)-15- benzyl-3-((R)-l-hydroxyethyl)-12-isobutyl-2,5,8,l l, 14,17,20-heptaoxo-l,4,7, 10, 13,16, 19- heptaazacyclotricosan-21-yl)acetamide, Compound 20, was prepared according to the coupling then deprotection sequence describe for Compound 4. LCMS: rt = 0.23 &0.29 minutes (double peak, LC method 2). m/z = 922.6 (M+l)⁺. ¾ NMR (400 MHz, D₂0): δ 8.18 (s, 1H), 7.37-7.25 (m, 5H), 7.20 (d, J = 8.0 Hz, 4H), 4.46 (m, 2H), 4.22 (m, 3H), 4.15-4.05 (m, 3H), 3.65-3.50 (m, 2H), 3.36 (m, lH), 3.12-2.92 (m, 7H), 2.90-2.76 (m, 2H), 2.69 (m, 1H), 2.29-2.1 1 (m, 3H), 2.08-1.94 (m, 2H), 1.84 (m, 3H), 1.35 (m, 2H), 1.16 (d, J= 6.9 Hz, 6H), 1.12 (d, J= 5.6 Hz, 3H), 0.72 (d, J= 8.8 Hz, 1H), 0.69 (s, 3H), 0.61 (s, 3H).

21

[0159] 2-(5-ethylpyridin-2-yl)-N-((3S,6S,9S,12S,15R,18S,21S)-6,9, 18-tris(2-aminoethyl)-15- benzyl-3-((R)-l-hydroxyethyl)-12-isobutyl-2,5,8,l l, 14,17,20-heptaoxo-l,4,7, 10, 13,16, 19- heptaazacyclotricosan-21-yl)acetamide, Compound 21, was prepared according to the coupling then deprotection sequence describe for Compound 4. LCMS: rt = 0.33 minutes (double peak, LC method 2). m/z = 909.5 (M+l). ¾ NMR (400 MHz, D₂0): δ 8.51 (s, 1H), 8.34 (d, J = 8.4 Hz, 1H), 8.17 (s, 1H), 7.78 (d, J = 8.4 Hz, 1H), 7.29 (m, 3H), 7.18 (d, J = 6.9 Hz, 2H), 4.51 (m, lH), 4.40 (m, lH), 4.30-4.09 (m, 8H), 3.32-3.23 (m, 1H), 3.12-2.97 (m, 7H), 2.81-2.75 (m, 4H), 2.18-2.13 (m, 4H), 1.22-1.18 (m, 2H), 1.12 (m, 3H), 1.11 (m, 3H), 0.80 (m, 1H), 0.71-0.67 (m, 6H).

22

[0160] 2 2-ethylphenyl)-N (3S,6S,9S, 12S, 15R, 18S,21S)-6,9, 18-tris(2-aminoethyl)-15-benzyl- 3 -((R)- 1 -hydroxyethyl)- 12-isobutyl-2,5 ,8, 11 , 14, 17,20-heptaoxo- 1 ,4,7, 10, 13 , 16, 19-heptaazacyclotricosan- 21-yl)acetamide, Compound 22 was prepared according to the coupling then deprotection sequence describe for Compound 4. LCMS: rt = 0.33 & 0.40 minutes (double peak, LC method 2). m/z = 908.6 (M+l)⁺. ¾ NMR (400 MHz, D₂0): δ 8.20 (s, 1H), 7.35-7.24 (m, 5H), 7.18 (d, J= 5.2 Hz, 4H), 4.52-4.39 (m, 2H), 4.21 (m, 3H), 4.08 (m, 3H), 3.74-3.62 (m, 2H), 3.38 (m, lH), 3.02 (m, 7H), 2.81 (m, 1H), 2.72 (m, 1H), 2.53 (m, 2H), 2.25-2.08 (m, 3H), 2.00 (m, 2H), 1.86 (m, 3H), 1.32 (m, 2H), 1.09 (m, 6H), 0.69 (s, 1H), 0.66 (s, 3H), 0.58 (s, 3H).

23

[0161] l-phenyl-N-((3S,6S,9S,12S,15R, 18S,21S)-6,9,18-tris(2-aminoethyl)-15-benzyl-3-((R)-l- hydroxyethyl)- 12-isobutyl-2,5 ,8, 11 , 14, 17,20-heptaoxo- 1 ,4,7, 10, 13 , 16, 19-heptaazacyclotricosan-21 - yl)cyclopropanecarboxamide, Compound 23, was prepared according to the coupling then deprotection sequence described for Compound 4. LCMS: rt = 0.28 minutes (LC method 2). m/z = 906.5 (M+l)⁺. ¾ NMR (400 MHz, D₂0): δ 8.09 (s, 1H), 7.35 (d, J= 7.4 Hz, 2H), 7.24 (m, 6H), 7.10 (d, J= 7.3 Hz, 2H), 4.39 (m, 1H), 4.32 (m, 1H), 4.19-4.04 (m, 4H), 4.00 (m, 2H), 3.23-3.11 (m, lH), 3.01-2.83 (m, 7H), 2.77- 2.68 (m, 1H), 2.61 (m, 1H), 2.17-1.86 (m, 5H), 1.76 (m, 2H), 1.65-1.54 (m, lH), 1.38-1.20 (m, 4H), 1.12 (m, 1H), 1.04 (d, J= 6.3 Hz, 4H), 0.66 (m, 1H), 0.60 (s, 3H), 0.52 (s, 3H).

D 24

[0162] 2-p-tolyl-N-((3S,6S,9S,12S,15R, 18S,21S)-6,9,18-tris(2-aminoethyl)-15-benzyl-3-((R)-l- hydroxyethyl)- 12-isobutyl-2,5 ,8, 11 , 14, 17,20-heptaoxo- 1 ,4,7, 10, 13 , 16, 19-heptaazacyclotricosan-21 - yl)acetamide, Compound 24, was prepared according to the coupling then deprotection sequence described for Compound 4. LCMS: rt = 0.21 &0.29 minutes (double peak, LC method 2). m/z = 894.5 (M+l)⁺. ¾ NMR (400 MHz, D₂0): δ 8.07 (s, 1H), 7.21 (m, 3H), 7.10 (d, J= 8.6 Hz, 4H), 7.05 (d, J= 7.9 Hz, 2H), 4.47-4.32 (m, 2H), 4.21-4.06 (m, 3H), 4.00 (m, 3H), 3.54-3.41 (m, 2H), 3.27 (m, lH), 2.92 (m, 7H), 2.71 (m, 1H), 2.64-2.53 (m, 1H), 2.17 (s, 3H), 2.06 (m, 3H), 1.99-1.85 (m, 2H), 1.74 (m, 3H), 1.24 (m, 2H), 1.04 (m, 3H), 0.62 (d, J= 8.4 Hz, 1H), 0.56 (s, 3H), 0.50 (s, 3H).

25

[0163] 2-(2,3-dihydrobenzo[b][l,4]dioxin-6-yl)-N-((3S,6S,9S,12S, 15R, 18S,21S)-6,9, 18-tris(2- aminoethyl)-15-benzyl-3-((R)-l-hydroxyethyl)-12-isobutyl-2,5,8,l l, 14, 17,20-heptaoxo- l,4,7,10, 13,16, 19-heptaazacyclotricosan-21-yl)acetamide, Compound 25, was prepared according to the coupling then deprotection sequence described for Compound 4. LCMS: rt = 0.24 minutes (double peak, LC method 2). m/z = 961 (M+Na)⁺. 1H NMR (400 MHz, D₂0): δ 7.39-7.27 (m, 3H), 7.19-7.17 (m, 2H), 6.85-6.83 (d, J = 8.0 Hz, 2H), 4.48-4.44 (m, 3H), 4.22-4.16 (m, 7H), 4.08 (m, 3H), 3.48 (m, 2H), 2.80 (m, 1H), 2.69 (s, 1H), 2.32-1.72 (m, 8H), 1.34 (m, 2H), 1.11-1.10 (m, 3H), 0.68-0.60 (m, 7H).

26

[0164] 2-(4-(trifluoromethyl)phenyl)-N-((3 S,6S,9S, 12S, 15R, 18S,21 S)-6,9, 18-tris(2-aminoethyl)- 15-benzyl-3 (R)-l-hydroxyethyl)-12-isobutyl-2,5,8,l l, 14,17,20-heptaoxo-l,4,7, 10, 13,16, 19- heptaazacyclotricosan-21-yl)acetamide, Compound 26, was prepared according to the coupling then deprotection sequence described for Compound 4. LCMS: rt = 0.30 minutes (LC method 2). m/z = 948.5 (M+l)⁺. ¾ NMR (400 MHz, D₂0): δ 8.18 (s, 1H), 7.66 (d, J= 8.0 Hz, 2H), 7.42 (d, J= 7.9 Hz, 2H), 7.32 (m, 3H), 7.20 (d, J= 7.0 Hz, 2H), 4.52-4.42 (m, 2H), 4.22 (m, 3H), 4.11 (m, 3H), 3.68 (m, 2H), 3.36 (m, 1H), 3.11-2.92 (m, 7H), 2.79 (m, 1H), 2.71 (m, 1H), 2.28-2.11 (m, 3H), 2.09-1.95 (m, 2H), 1.86 (m, 3H), 1.35 (m, 2H), 1.12 (d, J = 5.5 Hz, 3H), 0.71 (s, 1H), 0.69 (s, 3H), 0.62 (s, 3H).

27

[0165] 2-(3-ethylphenyl)-N-((3S,6S,9S, 12S, 15R, 18S,21S)-6,9, 18-tris(2-aminoethyl)-15-benzyl- 3-((R)-l-hydroxyethyl)-12-isobutyl-2,5,8, l l,14, 17,20-heptaoxo-l,4,7, 10, 13,16, 19-heptaazacyclotricosan- 21-yl)acetamide, Compound 27, was prepared according to the coupling then deprotection sequence described for Compound 4. LCMS: rt = 0.24 &0.30 minutes (double peak, LC method 2). m/z = 908.6 (M+l)⁺. ¾ NMR (400 MHz, D₂0): δ 8.18 (s, 1H), 7.36-7.25 (m, 4H), 7.23-7.12 (m, 4H), 7.07 (d, J= 7.3 Hz, 1H), 4.46 (m, 2H), 4.31-4.17 (m, 3H), 4.10 (m, 3H), 3.65-3.52 (m, 2H), 3.36 (m, lH), 3.01 (m, 7H), 2.78 (m, 1H), 2.68 (m, 1H), 2.58 (m, 2H), 2.15 (m, 3H), 2.08-1.94 (m, 2H), 1.92-1.74 (m, 3H), 1.46-1.28 (m, 2H), 1.13 (m, 6H), 0.72 (s, 1H), 0.69 (s, 3H), 0.61 (s, 3H).

28

[0166] 2-(4-(trifluoromethoxy)phenyl)-N-((3 S,6S,9S, 12S, 15R, 18S,21 S)-6,9, 18-tris(2- aminoethyl)- 15 -benzyl-3 -((R)- 1 -hydroxyethyl)- 12-isobutyl-2,5 , 8, 11 , 14, 17,20-heptaoxo- l,4,7,10, 13,16, 19-heptaazacyclotricosan-21-yl)acetamide, Compound 28, was prepared according to the coupling then deprotection sequence described for Compound 4. LCMS: rt = 0.38 (LC method 2). m/z = 964.5 (M+l)⁺. 1H NMR (400 MHz, D20): δ 8.17 (s, 1H), 7.32-7.17 (m, 9H), 4.43-4.41 (m, 2H), 4.25- 4.15 (m, 3H), 4.10-4.08 (m, 3H), 3.34-3.33 (s, 1H), 3.08-2.96 (m, 6H), 2.79 (m, 1H), 2.68 (m, 1H), 2.17- 1.83 (m, 8H), 1.35-1.32 (m, 2H),.1.11-1.10 (d, J=4.0 Hz, 3H), 0.68 (s, 3H), 0.60 (s, 3H).

29

[0167] 2-(2,4-dichlorophenyl)-N-((3S,6S,9S,12S,15R,18S,21S)-6,9, 18-tris(2-aminoethyl)-15- benzyl-3-((R)-l-hydroxyethyl)-12-isobutyl-2,5,8,l l, 14,17,20-heptaoxo-l,4,7, 10, 13,16, 19- heptaazacyclotricosan-21-yl)acetamide, Compound 29, was prepared according to the coupling then deprotection sequence described for Compound 4. LCMS: rt = 0.18 minutes (LC method 2). m/z = 950.5 (M+l)⁺. ¾ NMR (400 MHz, D₂0): δ 8.20 (s, 1H), 7.51 (d, J= 1.9 Hz, 1H), 7.36-7.25 (m, 5H), 7.19 (d, J = 6.9 Hz, 2H), 4.51-4.41 (m, 2H), 4.23 (m, 3H), 4.17-4.05 (m, 3H), 3.83-3.69 (m, 2H), 3.49-3.32 (m, 1H), 3.03 (m, 7H), 2.88-2.79 (m, 1H), 2.73 (m, 1H), 2.26-2.1 1 (m, 3H), 2.09-1.97 (m, 2H), 1.88 (m, 3H), 1.34 (m, 2H), 1.13 (d, J= 5.8 Hz, 3H), 0.77-0.71 (m, 1H), 0.68 (s, 3H), 0.62 (d, J= 9.3 Hz, 3H).

30

[0168] 2-(3-fluorophenyl)-N-((3S,6S,9S, 12S,15R,18S,21S)-6,9,18-tris(2-aminoethyl)-15-benzyl- 3-((R)-l-hydroxyethyl)-12-isobutyl-2,5,8, l l,14, 17,20-heptaoxo-l,4,7, 10, 13,16, 19-heptaazacyclotricosan- 21-yl)acetamide, Compound 30, was prepared according to the coupling then deprotection sequence describe for Compound 4. LCMS: rt = 0.23 minutes (double peak, LC method 2). m/z = 898.5 (M+l)⁺. 'H NMR (400 MHz, D₂0): δ 8.19 (s, 1H), 7.33 (m, 4H), 7.20 (d, J= 7.3 Hz, 2H), 7.05 (m, 3H), 4.47 (m, 2H), 4.23 (m, 3H), 4.12 (m, 3H), 3.63 (s, 2H), 3.38 (m, lH), 3.14-2.92 (m, 7H), 2.80 (m, lH), 2.72 (m, 1H), 2.26-2.11 (m, 3H), 2.09-1.95 (m, 2H), 1.94-1.77 (m, 3H), 1.35 (m, 2H), 1.13 (d, J= 5.8 Hz, 3H), 0.72 (s, 1H), 0.70 (s, 3H), 0.62 (s, 3H).

31

[0169] 2-(3,4-dichlorophenyl)-N-((3S,6S,9S,12S,15R,18S,21S)-6,9, 18-tris(2-aminoethyl)-15- benzyl-3-((R)-l-hydroxyethyl)-12-isobutyl-2,5,8,l l, 14,17,20-heptaoxo-l,4,7, 10, 13,16, 19- heptaazacyclotricosan-21-yl)acetamide, Compound 31, was prepared according to the coupling then deprotection sequence described for Compound 4. LCMS: rt = 0.19 minutes (double peak, LC method 2). m/z = 950.5 (M+l)⁺. 'H NMR (400 MHz, D₂0): δ 8.17 (s, 1H), 7.49-7.39 (m, 2H), 7.29 (m, 3H), 7.16 (m, 3H), 4.51-4.39 (m, 2H), 4.28-4.14 (m, 3H), 4.08 (m, 3H), 3.58 (s, 2H), 3.34 (m, lH), 3.12-2.92 (m, 7H), 2.79 (m, 1H), 2.69 (m, 1H), 2.16 (m, 3H), 2.07-1.93 (m, 2H), 1.82 (m, 3H), 1.44-1.27 (m, 2H), 1.11 (d, J= 5.8 Hz, 3H), 0.70 (s, 1H), 0.68 (s, 3H), 0.60 (s, 3H).

32

[0170] 2-(4-(methylamino)phenyl)-N-((3 S,6S,9S, 12S, 15R, 18S,21 S)-6,9, 18-tris(2-aminoethyl)- 15-benzyl-3-((R)-l-hydroxyethyl)-12-isobutyl-2,5,8,l l, 14,17,20-heptaoxo-l,4,7, 10, 13,16, 19- heptaazacyclotricosan-21-yl)acetamide, Compound 32, was prepared according to the coupling then deprotection sequence described for Compound 4. LCMS: rt = 0.21 minutes (LC method 2). m/z = 910 (M+l)⁺. ¾ NMR (400 MHz, D₂0): δ 7.29 (m, 5H), 7.16 (m, 4H), 4.45 (m, 2H), 4.18 (m, 6H), 3.70 - 3. (m, 2H), 3.43-3.24 (m, 1H), 3.13-2.91 (m, 9H), 2.87 (s, 3H), 2.84-2.60 (m, 3H), 2.28-1.73 (m, 9H), 1.50 1.15 (m, 3H), 1.10 (d, J = 5.7 Hz, 3H), 0.73 (m, 1H), 0.68 (s, 3H), 0.60 (d, J= 4.6 Hz, 3H).

33

[0171] 2-(4-acetamidophenyl)-N-((3S,6S,9S, 12S, 15R,18S,21S)-6,9,18-tris(2-aminoethyl)-15- benzyl-3-((R)-l-hydroxyethyl)-12-isobutyl-2,5,8,l l, 14,17,20-heptaoxo-l,4,7, 10, 13,16, 19- heptaazacyclotricosan-21-yl)acetamide, Compound 33, was prepared according to the coupling then deprotection sequence described for Compound 4. LCMS: rt = 0.17 minutes (LC method 2). m/z = 937.6 (M+l)⁺. ¾ NMR (400 MHz, D₂0): δ 8.08 (s, 1H), 7.26-7.13 (m, 8H), 7.10 (d, J= 7.1 Hz, 2H), 4.36 (dd, J= 15.4, 7.3 Hz, 2H), 4.12 (ddd, J= 17.3, 12.6, 6.5 Hz, 3H), 4.05-3.95 (m, 3H), 3.50 (s, 2H), 3.35-3.22 (m, 1H), 3.03-2.81 (m, 8H), 2.66 (s, 1H), 2.56 (s, 1H), 2.20-1.61 (m, 13H), 1.24 (dd, J= 23.3, 10.5 Hz, 2H), 1.03 (d, J= 5.7 Hz, 3H), 0.59 (s, 3H), 0.52 (d, J= 9.2 Hz, 3H).

34

[0172] l-(4-ethylphenyl)-3-((3S,6S,9S,12S,15R,18S,21S)-6,9, 18-tris(2-aminoethyl)-15-benzyl- 3-((R)-l-hydroxyethyl)-12-isobutyl-2,5,8, l l,14, 17,20-heptaoxo-l,4,7, 10, 13,16, 19-heptaazacyclotricosan- 21-yl)urea, Compound 34, was prepared according to the coupling then deprotection sequence described for Compound 4. LCMS: rt = 0.25 &0.36 minutes (double peak, LC method 2). m/z = 909.6 (M+l)⁺. ¾ NMR (400 MHz, D₂0): δ 7.29-7.03 (m, 9H), 4.44-4.33 (m, 2H), 4.22-4.12 (m, 2H), 4.10-3.96 (m, 4H), 3.41-3.32 (m, 1H), 3.06-2.86 (m, 7H), 2.83-2.76 (m, 1H), 2.72-2.65 (m, 1H), 2.47 (m, 2H), 2.15-1.91 (m, 5H), 1.88-1.78 (m, 2H), 1.77-1.69 (m, 1H), 1.36-1.24 (m, 2H), 1.03 (t, J = 7.4 Hz, 5H), 0.70-0.48 (m, 7H).

35

[0173] 2-(4-(2-oxopiperidin-l-yl)phenyl)-N-((3S,6S,9S, 12S,15R,18S,21S)-6,9,18-tris(2- aminoethyl)- 15 -benzyl-3 -((R)- 1 -hydroxyethyl)- 12-isobutyl-2,5 , 8, 11 , 14, 17,20-heptaoxo- l,4,7,10, 13,16, 19-heptaazacyclotricosan-21-yl)acetamide, Compound 35, was prepared according to the coupling then deprotection sequence described for Compound 4. LCMS: rt = 0.23 & 0.37 minutes (double peak, LC method 2). m/z = 977.6 (M+l)⁺. ¾ NMR (400 MHz, D₂0): δ 7.36-7.24 (m, 5H), 7.18 (t, J = 7.6 Hz, 4H), 4.48-4.41 (m, 2H), 4.27-4.03 (m, 6H), 3.63-3.56 (m, 4H), 3.45-3.35 (m, 1H), 3.09-2.91 (m, 8H), 2.83-2.71 (m, 1H), 2.69-2.62 (m, 1H), 2.45 (t, J= 5.8 Hz, 2H), 2.28-1.94 (m, 7H), 1.92-1.74 (m, 7H), 1.51 (s, 1H), 1.42-1.28 (m, 2H), 1.28-1.00 (m, 7H), 0.75 (t, J= 7.0 Hz, 2H), 0.70-0.58 (m, 7H).

36

[0174] 2-(4-moφholinophenyl)-N-((3S,6S,9S, 12S, 15R, 18S,21S)-6,9, 18-tris(2-aminoethyl)-15- benzyl-3-((R)-l-hydroxyethyl)-12-isobutyl-2,5,8,l l, 14,17,20-heptaoxo-l,4,7, 10, 13,16, 19- heptaazacyclotricosan-21-yl)acetamide, Compound 36, was prepared according to the coupling then deprotection sequence described for Compound 4. LCMS: rt = 0.17 minutes (LC method 2). m/z = 965.5 (M+l)⁺. ¾ NMR (400 MHz, D₂0): δ 8.17 (s, 1H), 7.51 (d, J= 8.6 Hz, 2H), 7.43 (d, J= 8.5 Hz, 2H), 7.31 (m, 3H), 7.19 (d, J= 7.5 Hz, 2H), 4.53-4.41 (m, 2H), 4.27-4.17 (m, 3H), 4.16-4.09 (m, 3H), 4.07 (m, 4H), 3.68 (d, J= 3.8 Hz, 2H), 3.64 (d, J= 4.5 Hz, 4H), 3.31 (m, 1H), 3.11-2.96 (m, 7H), 2 88-2 79 (m, IH), 2.77-2.65 (m, IH), 2.17 (m, 3H), 2.02 (m, 2H), 1.92-1.75 (m, 3H), 1 .46-1.30 (m, 2H), 1.12 (m, 3H), 0.72 (s, IH), 0.70 (s. 31 ! }. 0.62 (d,■/ 5.2 Hz, 3H).

37

[0175] 2-(4-(dimethylamino)phenyl)-N-((3S,6S,9S,12S,15R, 18S,21S)-6,9,18-tris(2-aminoethy 15-benzyl-3-((R)-l-hydroxyethyl)-12-isobutyl-2,5,8,l l, 14,17,20-heptaoxo-l,4,7, 10, 13,16, 19- heptaazacyclotricosan-21-yl)acetamide, Compound 37, was prepared according to the coupling then deprotection sequence described for Compound 4. LCMS: rt = 0.23 minutes (LC method 2). m/z = 923.6 (M+l). ¾ NMR (400 MHz, D₂0): δ 8.17 (s, IH), 7.51 (m, 2H), 7.43 (m, 2H), 7.37-7.24 (m, 3H), 7.19 (m, 2H), 4.47 (m, 11H), 4.31-3.96 (m, 9H), 3.75 (m, 3H), 3.25 (m, IH), 3.21 (m, 5H), 3.12 - 2.54 (m, 10H), 2.31-1.61 (m, 7H), 1.34 (m, 2H), 1.10 (d, J= 5.4 Hz, 4H), 0.68 (s, 4H), 0.61 (s, 3H).

38

[0176] (S)-2-amino-2-phenyl-N-((3S,6S,9S,12S,15R,18S,21S)-6,9, 18-tris(2-aminoethyl)-15- benzyl-3-((R)-l-hydroxyethyl)-12-isobutyl-2,5,8,l l, 14,17,20-heptaoxo-l,4,7, 10, 13,16, 19- heptaazacyclotricosan-21-yl)acetamide, Compound 38, was prepared according to the coupling then deprotection sequence described for Compound 4. LCMS: rt = 0.33 minutes (LC method 3). m/z = 895 (M+l). ¾ NMR (400 MHz, D₂0): δ 8.15 (s, 1H), 7.45- 7.41 (m, 5H), 7.32-7.17 (m, 5H), 5.12 (s, 1H), 4.54 (t, J= 8.0 Hz, 1H), 4.32-4.10 (m, 6H), 3.31-3.28 (m, lH), 3.03-2.97 (m, 6H), 2.65 (m, lH), 2.48 (m, 1H), 2.15-2.07 (m, 4H), 1.83-1.65 (m, 4H), 1.39 (m, 2H), 1.12 (d, J = 4.0 Hz, 3H), 0.82 (m, lH), 0.72 (d, J= 8.0 Hz, 3H), 0.62 (d, J= 8.0 Hz, 3H).

39

[0177] (R)-2-amino-2-phenyl-N-((3S,6S,9S,12S,15R,18S,21S)-6,9,18-tris(2-aminoethyl)-15- benzyl-3-((R)-l-hydroxyethyl)-12-isobutyl-2,5,8,l l, 14,17,20-heptaoxo-l,4,7, 10, 13,16, 19- heptaazacyclotricosan-21-yl)acetamide, Compound 39, was prepared according to the coupling then deprotection sequence described for Compound 4. LCMS: rt = 0.33 minutes (LC method 3). m/z = 895 (M+l). ¾ NMR (400 MHz, D₂0): δ 8.23 (s, 1H), 7.47-7.43 (m, 5H), 7.34-7.19 (m, 5H), 5.12 (s, lH), 4.55 (t, J= 8.0 Hz, 1H), 4.43 (t, J = 4.0 Hz, 1H), 4.27-4.10 (m, 5H), 3.96 (d, J= 4.0 Hz, 1H), 3.07-2.98 (m, 6H), 2.84-2.80 (m, 1H), 2.74-2.73 (m, 2H), 2.16-2.06 (m, 4H), 1.98-1.82 (m, 3H), 1.68 (m, 1H), 1.42- 1.33 (m, 2H), 1.10 (d, J= 8.0 Hz, 3H), 0.82 (m, 1H), 0.71 (d, J= 8.0 Hz, 3H), 0.65 (d, J= 8.0 Hz, 3H).

40

[0178] l-(4-chlorophenyl)-N-((3S,6S,9S,12S,15R, 18S,21S)-6,9,18-tris(2-aminoethyl)-15- benzyl-3-((R)-l-hydroxyethyl)-12-isobutyl-2,5,8,l l, 14,17,20-heptaoxo-l,4,7, 10, 13,16, 19- heptaazacyclotricosan-21-yl)cyclobutanecarboxamide, Compound 40, was prepared according to the coupling then deprotection sequence described for Compound 4. LCMS: rt = 0.28 & 0.33 minutes (double peak, LC method 2). m/z = 955.5 (M+l). ¾ NMR (400 MHz, D₂0): δ 8.16 (s, 1H), 7.48-7.22 (m, 7H), 7.18 (d, J= 6.8 Hz, 2H), 4.46 (m, 1H), 4.40-4.33 (m, 1H), 4.23 (m, 2H), 4.18-4.11 (m, 2H), 4.09 (m, 2H), 3.25-3.15 (m, 1H), 3.13-2.84 (m, 7H), 2.64 (m, 3H), 2.55-2.37 (m, 3H), 2.28-2.10 (m, 3H), 2.05 (m, 1H), 1.83 (m, 5H), 1.64 (m, 1H), 1.45-1.28 (m, 2H), 1.1 1 (d, J= 6.2 Hz, 3H), 0.71 (s, 1H), 0.69 (s, 3H), 0.61 (s, 3H).

41

[0179] 2-(4-(lH-pyrazol-l-yl)phenyl)-N-((3S,6S,9S, 12S, 15R,18S,21S)-6,9, 18-tris(2- aminoethyl)-15-benzyl-3-((R)-l-hydroxyethyl)-12-isobutyl-2,5,8,l l, 14, 17,20-heptaoxo- l,4,7,10, 13,16, 19-heptaazacyclotricosan-21-yl)acetamide, Compound 41, was prepared according to the coupling then deprotection sequence described for Compound 4. LCMS: it = 0.19 minutes (LC method 2). m/z = 946.5 (M+l). ¾ NMR (400 MHz, D₂0): δ 8.15 (s, 1H), 8.11 (d, J= 2.1 Hz, 1H), 7.75 (s, 1H), 7.59 (d, J= 8.4 Hz, 2H), 7.37 (d, J= 8.4 Hz, 2H), 7.28 (m, 3H), 7.17 (d, J= 7.3 Hz, 2H), 6.54 (s, 1H), 4.53-4.40 (m, 2H), 4.27-4.15 (m, 3H), 4.12-4.04 (m, 3H), 3.71-3.55 (m, 2H), 3.44-3.31 (m, 1H), 3.09- 2.91 (m, 7H), 2.81-2.72 (m, 1H), 2.65 (m, 1H), 2.24-2.09 (m, 3H), 2.08-2.00 (m, lH), 1.87 (m, 4H), 1.35 (m, 2H), 1.09 (d, J= 5.8 Hz, 3H), 0.69 (d, J= 9.2 Hz, 1H), 0.67 (s, 3H), 0.61 (d, J= 10.5 Hz, 3H).

D 42

[0180] 2-mesityl-N-((3S,6S,9S, 12S, 15R,18S,21S)-6,9, 18-tris(2-aminoethyl)-15-benzyl-3-((R)-l- hydroxyethyl)- 12-isobutyl-2,5 ,8, 11 , 14, 17,20-heptaoxo- 1 ,4,7, 10, 13 , 16, 19-heptaazacyclotricosan-21 - yl)acetamide, Compound 42, was prepared according to the coupling then deprotection sequence described for Compound 4. LCMS: rt = 0.42 minutes (LC method 2). m/z = 922.5 (M+l). ¾ NMR (400 MHz, D₂0): δ 8.16 (s, 1H), 7.31 (m, 3H), 7.18 (d, J= 6.6 Hz, 2H), 6.92 (s, 2H), 4.44 (m, 2H), 4.21 (m, 3H), 4.14-4.01 (m, 3H), 3.66 (m, 2H), 3.40 (m, 1H), 3.09-2.90 (m, 7H), 2.83 (m, lH), 2.73 (m, 1H), 2.16 (d, J= 11.5 Hz, 12H), 2.01 (m, 3H), 1.85 (m, 3H), 1.33 (m, 2H), 1.11 (m, 3H), 0.69 (s, lH), 0.65 (s, 3H), 0.58 (s, 3H).

43

[0181] 2-(3-acetamidophenyl)-N-((3S,6S,9S, 12S, 15R,18S,21S)-6,9,18-tris(2-aminoethyl)-15- benzyl-3-((R)-l-hydroxyethyl)-12-isobutyl-2,5,8,l l, 14,17,20-heptaoxo-l,4,7, 10, 13,16, 19- heptaazacyclotricosan-21-yl)acetamide, Compound 43, was prepared according to the coupling then deprotection sequence described for Compound 4. LCMS: rt = 0.22 minutes (LC method 2). m/z = 937.5 (M+l). ¾ NMR (400 MHz, D₂0): δ 8.15 (s, 1H), 7.36 - 7.25 (m, 5H), 7.19 (m, 3H), 7.06 (d, J= 7.5 Hz, 1H), 4.44 (m, 2H), 4.26-4.15 (m, 3H), 4.12-4.02 (m, 3H), 3.65-3.54 (m, 2H), 3.37 (m, lH), 2.99 (m, 7H), 2.82-2.72 (m, 1H), 2.66 (m, 1H), 2.24-2.08 (m, 6H), 2.05-1.94 (m, 2H), 1.83 (m, 3H), 1.29 (m, 3H), 1.10 (m, 3H), 0.72 (m, 1H), 0.65 (s, 3H), 0.57 (s, 3H).

D 44

[0182] (S)-4-amino-5-oxo-5-((3S,6S,9S, 12S, 15R,18S,21S)-6,9, 18-tris(2-aminoethyl)-15-benzyl- 3-((R)-l-hydroxyethyl)-12-isobutyl-2,5,8, l l,14, 17,20-heptaoxo-l,4,7, 10, 13,16, 19-heptaazacyclotricosan- 21-ylamino)pentanoic acid, Compound 44, was prepared according to the coupling then deprotection sequence described for Compound 4. LCMS: rt = 0.30 minutes (LC method 2). m/z = 891.5 (M+l). 1H NMR (400 MHz, D₂0): δ 8.16 (s, 1H), 7.29 (dd, J= 14.9, 6.9 Hz, 3H), 7.19 (d, J= 6.8 Hz, 2H), 4.58-4.36 (m, 4H), 4.34-3.95 (m, 8H), 3.30 (m, 1H), 3.03 (m, 7H), 2.77 (m, 3H), 2.47 (m, 2H), 1.99 (m, 10H), 1.37 (m, 2H), 1.12 (d, J= 5.9 Hz, 3H), 0.71 (m, 4H), 0.63 (d, J= 5.5 Hz, 2H).

45

[0183] (S)-2-acetamido-6-amino-N-((3S,6S,9S,12S,15R,18S,21S)-6,9, 18-tris(2-aminoethyl)-15- benzyl-3-((R)-l-hydroxyethyl)-12-isobutyl-2,5,8,l l, 14,17,20-heptaoxo-l,4,7, 10, 13,16, 19- heptaazacyclotricosan-21-yl)hexanamide, Compound 45, was prepared according to the coupling then deprotection sequence described for Compound 4. LCMS: rt = 0.29 minutes (LC method 3). m/z = 933 (M+l). ¾ NMR (400 MHz, D₂0): δ 7.34-7.25 (m, 3H), 7.19 (d, J= 6.8 Hz, 2H), 4.50 (t, J= 8.2 Hz, 1H), 4.41 (dd, J= 8.8, 5.2 Hz, 1H), 4.28-4.09 (m, 7H), 3.34-3.24 (m, lH), 3.1 1-2.91 (m, 10H), 2.85-2.77 (m, 1H), 2.76-2.67 (m, 1H), 2.27-2.06 (m, 4H), 1.98 (d, J= 10.4 Hz, 3H), 1.95-1.70 (m, 5H), 1.69-1.55 (m, 3H), 1.46-1.29 (m, 4H), 1.12 (d, J= 6.2 Hz, 3H), 0.80-0.73 (m, 1H), 0.70 (d, J= 6.0 Hz, 3H), 0.62 (d, J = 6.0 Hz, 3H).

EXAMPLE 46. SYNTHESIS OF COMPOUND 46

[0184] (S)-2-amino-5-guanidino-N-((3S,6S,9S, 12S,15R,18S,21S)-6,9, 18-tris(2-aminoethyl)-15- benzyl-3-((R)-l-hydroxyethyl)-12-isobutyl-2,5,8,l l, 14,17,20-heptaoxo-l,4,7, 10, 13,16, 19- heptaazacyclotricosan-21-yl)pentanamide, Compound 46, was prepared according to the coupling then deprotection sequence described for Compound 4. LCMS: rt = 0.63 minutes (LC method 3). m/z = 919 (M+l). ¾ NMR (400 MHz, D₂0): δ 8.16 (s, 2H), 7.30 (m, 3H), 7.19 (d, J= 7.0 Hz, 2H), 4.50 (m, 2H), 4.18 (m, 6H), 4.00 (m, 1H), 3.35 - 3.24 (m, 1H), 3.22 - 2.94 (m, 9H), 2.87 - 2.77 (m, lH), 2.76 - 2.63 (m, 1H), 2.14 (m, 4H), 1.98 - 1.75 (m, 6H), 1.65 (m, 2H), 1.42 (m, 2H), 1.12 (d, J= 6.2 Hz, 3H), 0.71 (s, 1H), 0.69 (s, 3H), 0.62 (d, J= 5.5 Hz, 3H).

47

[0185] (S)-2-acetamido-3-(lH-imidazol-4-yl)-N-((3 S,6S,9S, 12S, 15R, 18S,21 S)-6,9, 18-tris(2- aminoethyl)-15-benzyl-3-((R)-l-hydroxyethyl)-12-isobutyl-2,5,8,l l, 14, 17,20-heptaoxo- l,4,7,10, 13,16, 19-heptaazacyclotricosan-21-yl)propanamide, Compound 47, was prepared according to the coupling then deprotection sequence described for Compound 4. LCMS: rt = 0.36 minutes (LC method 3). m/z = 941 (M+l). ¾ NMR (400 MHz, D₂0): δ 8.57 (s, 1H), 8.20 (s, 1H), 7.36-7.24 (m, 4H), 7.19 (d, J= 7.2 Hz, 2H), 4.62-4.49 (m, 3H), 4.42 (m, 1H), 4.30-4.10 (m, 6H), 3.20 (m, 2H), 3.1 1-2.94 (m, 8H), 2.87-2.76 (m, 1H), 2.71 (m, 1H), 2.26-2.04 (m, 4H), 1.98-1.74 (m, 7H), 1.45-1.31 (m, 2H), 1.12 (d, J = 6.1 Hz, 3H), 0.75 (s, 1H), 0.70 (d, J= 6.1 Hz, 3H), 0.63 (m, 3H).

48

[0186] 4-amino-N-((3S,6S,9S,12S,15R, 18S,21S)-6,9,18-tris(2-aminoethyl)-15-benzyl-3-((R)-l- hydroxyethyl)- 12-isobutyl-2,5 ,8, 11 , 14, 17,20-heptaoxo- 1 ,4,7, 10, 13 , 16, 19-heptaazacyclotricosan-21 - yl)piperidine-4-carboxamide, Compound 48, was prepared according to the coupling then deprotection sequence described for Compound 4. LCMS: rt = 0.63 minutes (LC method 3). m/z = 888 (M+l). ¾ NMR (400 MHz, D₂0): δ 7.34-7.25 (m, 3H), 7.20 (d, J= 7.2 Hz, 2H), 4.55-4.42 (m, 2H), 4.33-4.07 (m, 6H), 3.46-3.26 (m, 5H), 3.16-2.96 (m, 7H), 2.86-2.68 (m, 2H), 2.60-2.47 (m, 2H), 2.26-2.07 (m, 6H), 2.00-1.83 (m, 4H), 1.45-1.29 (m, 2H), 1.12 (d, J= 6.2 Hz, 3H), 0.77-0.57 (m, 7H).

49

[0187] (S)-2-amino-3-(pyridin-4-yl)-N-((3 S,6S,9S, 12S, 15R, 18S,21 S)-6,9, 18-tris(2-aminoethyl)- 15-benzyl-3-((R)-l-hydroxyethyl)-12-isobutyl-2,5,8,l l, 14,17,20-heptaoxo-l,4,7, 10, 13,16, 19- heptaazacyclotricosan-21-yl)propanamide, Compound 49, was prepared according to the coupling then deprotection sequence described for Compound 4. LCMS: rt = 0.25 & 0.37 minutes (double peak, LC method 3). m/z = 910 (M+l). ¾ NMR (400 MHz, D₂0): δ 8.72 (d, J= 6.6 Hz, 2H), 7.97 (d, J= 6.6 Hz, 2H), 7.29 (dq, J= 14.4, 7.0 Hz, 3H), 7.19 (d, J= 6.8 Hz, 2H), 4.51 (t, J= 8.2 Hz, 1H), 4.42 (dd, J= 8.6, 6.0 Hz, 2H), 4.26 - 4.09 (m, 6H), 3.52 (dd, J = 14.2, 6.8 Hz, lH), 3.40 (dd, J= 14.2, 7.8 Hz, 1H), 3.30 - 3.19 (m, 1H), 3.13 - 2.96 (m, 7H), 2.88 - 2.79 (m, 1H), 2.77 - 2.67 (m, lH), 2.21 - 2.07 (m, 4H), 2.01 - 1.71 (m, 4H), 1.45 - 1.28 (m, 2H), 1.12 (d, J = 6.2 Hz, 3H), 0.74 (dd, J= 13.0, 7.0 Hz, 1H), 0.68 (d, J = 5.6 Hz, 3H), 0.62 (t, J= 6.4 Hz, 3H).

50

[0188] (S)-2-amino-3-(pyridin-2-yl)-N-((3 S,6S,9S, 12S, 15R, 18S,21 S)-6,9, 18-tris(2-aminoethyl)- 15-benzyl-3-((R)-l-hydroxyethyl)- 12-isobutyl-2,5,8,l l, 14, 17,20-heptaoxo-l,4,7, 10, 13, 16, 19- heptaazacyclotricosan-21-yl)propanamide, Compound 50, was prepared according to the coupling then deprotection sequence described for Compound 4. LCMS: rt = 0.40 minutes (LC method 3). m/z = 910 (M+l). ¾ NMR (400 MHz, D₂0): δ 8.58 (d, J= 4.8 Hz, 1H), 8.27 (m, 1H), 8.08 (s, 1H), 7.73 (d, J= 7.9 Hz, 2H), 7.27 - 7. 16 (m, 3H), 7.1 1 (d, J= 6.9 Hz, 2H), 4.43 (m, 1H), 4.34 (m, 2H), 4. 18 - 3.99 (m, 6H), 3.47 (m, 2H), 3.02 - 2.88 (m, 7H), 2.79 - 2.71 (m, 1H), 2.68 - 2.61 (m, lH), 2.59 - 2.5 1 (m, 1H), 2. 13 - 1.98 (m, 4H), 1.89 - 1.74 (m, 2H), 1.67 (m, 1H), 1.53 (m, lH), 1 .28 (m, 3H), 1 .08 (m, 5H), 0.69 (s, lH), 0.62 (d, J= 6.2 Hz, 3H), 0.55 (d, J= 6.2 Hz, 3H).

5 1

[0189] (R)-2-amino-3 pyridin-2-yl)-N (3S,6S,9S, 12S, 15R, 18S,21S)-6,9, 18-tris(2-aminoethyl)- 15-benzyl-3-((R)-l-hydroxyethyl)-12-isobutyl-2,5,8,l l, 14,17,20-heptaoxo-l,4,7, 10, 13,16, 19- heptaazacyclotricosan-21-yl)propanamide, Compound 51, was prepared according to the coupling then deprotection sequence described for Compound 4. LCMS: rt = 0.39 minutes (LC method 3). m/z = 910 (M+l). ¾ NMR (400 MHz, D₂0): δ 8.61 (d, J= 5.1 Hz, 1H), 8.23 (m, 1H), 8.16 (s, 1H), 7.72 (m, 2H), 7.30 (m, 3H), 7.20 (d, J= 6.6 Hz, 2H), 4.52 (m, 1H), 4.46 - 4.37 (m, 2H), 4.28 - 4.07 (m, 6H), 3.57 - 3.39 (m, 2H), 3.31 - 3.16 (m, 1H), 3.11 - 2.96 (m, 7H), 2.80 (m, 1H), 2.67 (m, 1H), 2.13 (m, 4H), 1.80 (m, 4H), 1.53 - 1.25 (m, 3H), 1.15 (m, 5H), 0.75 (s, 1H), 0.70 (d, J= 6.0 Hz, 3H), 0.63 (d, J= 6.1 Hz, 3H).

52

[0190] (2S,4R)-4-amino-N-((3S,6S,9S,12S,15R,18S,21S)-6,9,18-tris(2-aminoethyl)-15-benzyl- 3 -((R)- 1 -hydroxyethyl)- 12-isobutyl-2,5 ,8, 11 , 14, 17,20-heptaoxo- 1 ,4,7, 10, 13 , 16, 19-heptaazacyclotricosan- 21-yl)pyrrolidine-2-carboxamide, Compound 52, was prepared according to the coupling then deprotection sequence described for Compound 4. LCMS: rt = 0.30 minutes (LC method 3). m/z = 874 (M+l). ¾ NMR (400 MHz, D₂0): δ 8.17 (s, 1H), 7.29 (m, 3H), 7.19 (m, 2H), 4.52 (m, 2H), 4.43 (m, 1H), 4.28 (dd, J= 9.7, 4.4 Hz, 1H), 4.17 (m, 6H), 3.84 (dd, J= 12.9, 8.1 Hz, 1H), 3.50 (dt, J= 43.5, 21.8 Hz, 1H), 3.36 - 3.20 (m, 1H), 3.18 - 2.88 (m, 8H), 2.88 - 2.60 (m, 2H), 2.14 (m, 5H), 2.00 - 1.67 (m, 4H), 1.54 - 1.25 (m, 2H), 1.12 (d, J= 6.3 Hz, 3H), 0.83 - 0.65 (m, 4H), 0.62 (d, J= 6.1 Hz, 3H).

53

[0191] (R)-2-amino-3-(lH-indol-2-yl)-N-((3S,6S,9S,12S,15R, 18S,21S)-6,9, 18-tris(2- aminoethyl)-15-benzyl-3-((R)-l-hydroxyethyl)-12-isobutyl-2,5,8,l l, 14, 17,20-heptaoxo- l,4,7,10, 13,16, 19-heptaazacyclotricosan-21-yl)propanamide, Compound 53, was prepared according to the coupling then deprotection sequence described for Compound 4. LCMS: rt = 0.33 minutes (LC method 3). m/z = 934 (M+l). ¾ NMR (400 MHz, D₂0): δ 8.31 (s, 1H), 7.58 - 7.45 (m, 2H), 7.37 - 7.25 (m, 4H), 7.23 - 7.01 (m, 4H), 5.49 (m, 1H), 4.65 - 4.44 (m, 2H), 4.28 - 4.11 (m, 5H), 3.98 (m, 1H), 3.30 (m, 1H), 3.09 - 2.95 (m, 7H), 2.71 (m, 2H), 2.41 - 2.21 (m, 2H), 2.14 (m, 3H), 1.95 - 1.62 (m, 4H), 1.45 (m, 4H), 1.14 (m, 3H), 0.76 (d, J= 6.3 Hz, 3H), 0.71 - 0.67 (m, 3H), 0.63 (d, J= 5.5 Hz, 1H).

54

[0192] (S)-2-amino-3 pyridin-4-yl)-N-((3S,6S,9S, 12S, 15R, 18S,21S)-6,9, 18-tris(2-aminoethyl)- 15-benzyl-3-((R)-l-hydroxyethyl)-12-isobutyl-2,5,8,l l, 14,17,20-heptaoxo-l,4,7, 10, 13,16, 19- heptaazacyclotricosan-21-yl)propanamide, Compound 54, was prepared according to the coupling then deprotection sequence described for Compound 4. LCMS: rt = 0.34 minutes (LC method 3). m/z = 934 (M+l). ¾ NMR (400 MHz, D₂0): δ 8.29 (s, 1H), 7.53 (m, 2H), 7.37 - 7.26 (m, 4H), 7.20 (m, 4H), 5.51 (s, 1H), 4.60 (m, 1H), 4.52 - 4.30 (m, 2H), 4.30 - 4.05 (m, 5H), 4.04 - 3.93 (m, 1H), 3.24 (m, 1H), 3.10 - 2.92 (m, 7H), 2.84 (m, 1H), 2.78 - 2.66 (m, 1H), 2.63 - 2.52 (m, lH), 2.26 - 2.02 (m, 5H), 1.86 (m, 4H), 1.63 (m, 1H), 1.47 (m, 4H), 1.19 - 1.10 (m, 4H), 1.03 (m, 2H), 0.75 (m, 3H), 0.71 - 0.66 (m, 3H), 0.63 (m, 1H).

55

[0193] (S)-2-acetamido-3-(pyridin-2-yl)-N-((3 S,6S,9S, 12S, 15R, 18S,21 S)-6,9, 18-tris(2- aminoethyl)-15-benzyl-3-((R)-l-hydroxyethyl)-12-isobutyl-2,5,8,l l, 14, 17,20-heptaoxo- l,4,7,10, 13,16, 19-heptaazacyclotricosan-21-yl)propanamide, Compound 55, was prepared according to the coupling then deprotection sequence described for Compound 4. LCMS: rt = 0.35 minutes (LC method 3). m/z = 952 (M+l). ¾ NMR (400 MHz, D₂0): δ 8.15 (s, 1H), 7.45-7.41 (m, 5H), 7.32-7.17 (m,

5H), 5.12 (s, 1H), 4.54 (t, J= 8.0 Hz, 1H), 4.32-4.10 (m, 6H), 3.31-3.28 (m, 1H), 3.03 - 2.97 (m, 6H),

2.65 (m, 1H), 2.48 (m, 1H), 2.15-2.07 (m, 4H), 1.83-1.65 (m, 4H), 1.39 (m, 2H), 1.12 (d, J= 4.0 Hz, 3H),

0.82 (m, 1H), 0.72 (d, J= 8.0 Hz, 3H), 0.62 (d, J= 8.0 Hz, 3H).

EXAMPLE 56. SYNTHESIS OF COMPOUND 56

[0194] 3-(2-(aminomethyl)phenyl)-N-((3 S,6S,9S, 12S, 15R, 18S,21 S)-6,9, 18-tris(2-aminoethyl)- 15-benzyl-3-((R)-l-hydroxyethyl)-12-isobutyl-2,5,8,l l, 14,17,20-heptaoxo-l,4,7, 10, 13,16, 19- heptaazacyclotricosan-21-yl)picolinamide, Compound 56, was prepared according to the coupling then deprotection sequence described for Compound 4. LCMS: rt = 0.35 minutes (LC method 3). m/z = 972 (M+l). ¾ NMR (400 MHz, D₂0): δ 8.62 (s, 1H), 8.15 (s, 1H), 7.84 (m, 1H), 7.66 (m, 1H), 7.50-7.45 (m, 3H),7.32-7.19 (m, 5H), 4.50-4.39 (m, 4H), 4.18-4.12 (m, 6H), 3.93 (m, 2H), 3.01-2.85 (m, 6H), 2.80-2.59 (m, 3H), 2.20-2.12 (m, 3H), 1.95 -1.60 (m, 5H), 1.41-1.30 (m, 2H), 1.20-0.95 (m, 3H), 0.81 (m, 1H), 0.71 (d, J= 8.0 Hz, 3H), 0.64 (d, J= 8.0 Hz, 3H).

57

[0195] 3-(benzylamino)-2-((benzylamino)methyl)-N-((3S,6S,9S, 12S, 15R, 18S,21S)-6,9, 18- tris(2-aminoethyl)-15-benzyl-3-((R)-l-hydroxyethyl)-12-isobutyl-2,5,8,l l, 14,17,20-heptaoxo- l,4,7,10, 13,16, 19-heptaazacyclotricosan-21-yl)propanamide, Compound 57, was prepared according to the coupling then deprotection sequence described for Compound 4. LCMS: rt = 0.34 minutes (LC method 3). m/z = 1043 (M+l). ¾ NMR (400 MHz, D₂0): δ 8.18 (s, 1H), 7.47 - 7.37 (m, 10H), 7.31 (m, 3H), 7.19 (d, J= 6.8 Hz, 2H), 4.50 (m, 1H), 4.42 (m, 1H), 4.27 - 4.10 (m, 9H), 4.08 (m, lH), 3.41 - 3.34 (m, 1H), 3.24 (m, 5H), 3.11 - 2.94 (m, 7H), 2.83 - 2.73 (m, lH), 2.71 - 2.60 (m, lH), 2.15 (m, 4H), 1.85 (m, 4H), 1.41 (m, 1H), 1.32 (m, 1H), 1.10 (d, J= 6.4 Hz, 3H), 0.73 (m, 1H), 0.69 (d, J= 5.4 Hz, 3H), 0.62 (d, J= 5.8 Hz, 3H).

D 58

[0196] 3-amino-2-(aminomethyl)-N-((3S,6S,9S, 12S,15R,18S,21S)-6,9, 18-tris(2-aminoethyl)-15- benzyl-3-((R)-l-hydroxyethyl)-12-isobutyl-2,5,8,l l, 14,17,20-heptaoxo-l,4,7, 10, 13,16, 19- heptaazacyclotricosan-21-yl)propanamide, Compound 58, is prepared as follows. To a solution of 3- (benzylamino)-2-[(benzylamino)methyl]-N-[(3 S,6S,9S, 12S, 15R, 18S,21 S)-6,9, 18-tris(2-aminoethyl)-15- benzyl-3-[(lR)-l-hydroxyethyl]-12-isobutyl-2,5,8,l l, 14,17,20-heptaoxo-l,4,7, 10, 13, 16, 19- heptazacyclotricos-21-yl]propanamide; formic acid (15 mg, O.OlOOmmol) in Methanol (5mL), was added Pd(OH)2 (5 mg, 20% wt). The resulting mixture was stirred at room temperature under ¾ balloon overnight. The catalyst was filtered off through CELITE. The filtrate was concentrated to dryness and dissolved with 10% aq. formic acid solution (5 mL). The solution was then concentrated. After which, it was re-dissolved with 10% aq. formic acid solution (3 mL) and freeze-dried to afford the desired product 3-amino-2^aminomethyl)-N-[(3S,6S,9S, 12S,15R,18S,21S)-6,9, 18 ris(2-aminoethyl)-15-benzyl-3-[(lR)- l-hydroxyethyl]-12-isobutyl-2,5, 8, 11, 14, 17,20-heptaoxo- 1,4,7, 10, 13,16, 19-heptazacyclotricos-21- yl]propanamide, Compound 58 (12 mg, 95.9% yield). LCMS: rt = 0.32 minutes (LC method 3). m/z = 862 (M+l)⁺. ¾ NMR (400 MHz, D₂0): 5 8.19 (s, 5H), 7.30 (m, 3H), 7.19 (d, J = 7.1 ! !:··· . 2H), 4.48 (m, 2H), 4.30 - 4.07 (m, 6H), 3.39 - 3.27 {m, 2H), 3.22 (m, 4H), 3.05 (m, 7H), 2.89 - 2.80 {m, 1H), 2.79 - 2.68 i . IH), 2.15 (m. 4! ! . 2.00 -^■ 1.77 (m, 4H), 1 .41 id. ./ 4.6 Hz, H i ). 1 .35 - 1.17 (m, 21 I 1 . 12 id, J

6.4 Hz, 3H), 0.73 - 0.68 (m, IH), 0.67 (s, 3H), 0.61 (s, 3H).

D 59

[0197] (S)-4-amino-2-pivalamido-N-((3 S,6S,9S, 12S, 15R, 18S,21 S)-6,9, 18-tris(2-aminoethyl)- 15-benzyl-3-((R)- l-hydroxyethyl)- 12-isobutyl-2,5,8, l l, 14, 17,20-heptaoxo-l,4,7, 10, 13, 16, 19- heptaazacyclotricosan-21-yl)butanamide, Comopund 59, was prepared according to the coupling then deprotection sequence described for Compound 4. LCMS : rt = 0.45 minutes (double peak, LC method 3). m/z = 946 (M+l). ¾ NMR (400 MHz, D₂0): δ 8.16 (s, IH), 7.29 (m, 3H), 7.19 (d, J= 7.0 Hz, 2H), 4.52 (m, IH), 4.43 (m, IH), 4.32 (m, IH), 4.20 (m, 3H), 4.14 (m, 2H), 3.26 (m, IH), 3.12 - 2.95 (m, 9H), 2.84 - 2.76 (m, IH), 2.75 - 2.64 (m, IH), 2.12 (m, 6H), 1.97 - 1.72 (m, 4H), 1.33 (m, 3H), 1.12 (s, 9H), 1.10 (d, J= 6.4 Hz, 2H), 0.78 (s, IH), 0.70 (d, J= 6.4 Hz, 3H), 0.63 (d, J= 6.2 Hz, 3H).

D 60

[0198] 4-acetamido-N-((3S,6S,9S, 12S, 15R, 18S,21 S)-6,9, 18-tris(2-aminoethyl)-15-benzyl-3- ((R)- 1 -hydroxyethyl)- 12-isobutyl-2,5 , 8, 1 1 , 14, 17,20-heptaoxo- 1, 4,7, 10, 13, 16, 19-heptaazacyclotricosan- 21-yl)piperidine-4-carboxamide, Compound 60, was prepared according to the coupling then deprotection sequence described for Compound 4. LCMS: rt = 0.31 minutes (LC method 3). m/z = 930 (M+l). ¾ NMR (400 MHz, D₂0): δ 7.34-7.25 (m, 3H), 7.20 (d, J= 7.0 Hz, 2H), 4.54 (t, J= 8.2 Hz, IH), 4.34-4.29 (m, 2H), 4.22 - 4.08 (m, 5H), 3.40 - 3.10 (m, 6H), 3.08 - 2.94 (m, 7H), 2.85 - 2.75 (m, IH), 2.70 - 2.61 (m, 1H), 2.29 - 2.08 (m, 7H), 2.05 - 1.79 (m, 8H), 1.46 - 1.31 (m, 2H), 1.12 (d, J= 6.2 Hz, 3H), 0.91 - 0.82 (m, 1H), 0.72 (d, J= 6.6 Hz, 3H), 0.66 (d, J= 6.4 Hz, 3H).

D 61

[0199] (S)-N-((3S,6S,9S,12S,15R,18S,21S)-6,9, 18-tris(2-aminoethyl)-15-benzyl-3-((R)-l- hydroxyethyl)- 12-isobutyl-2,5 ,8, 11 , 14, 17,20-heptaoxo- 1 ,4,7, 10, 13 , 16, 19-heptaazacyclotricosan-21 - yl)piperazine-2-carboxamide, Compound 61, was prepared according to the coupling then deprotection sequence described for Compound 4. LCMS: rt = 0.30 minutes (LC method 3). m/z = 874 (M+l). ¾ NMR (400 MHz, D₂0): δ 8.17 (s, 1H), 7.29 (dq, J= 14.5, 7.1 Hz, 3H), 7.19 (d, J= 6.9 Hz, 2H), 4.56 - 4.38 (m, 2H), 4.34 - 4.06 (m, 7H), 3.82 (dd, J= 13.7, 3.5 Hz, 1H), 3.61 (m, 2H), 3.46 - 3.19 (m, 4H), 3.18 - 2.91 (m, 7H), 2.88 - 2.57 (m, 2H), 2.29 - 1.72 (m, 9H), 1.31 (m, 3H), 1.12 (d, J= 6.3 Hz, 3H), 0.81 - 0.66 (m, 4H), 0.62 (d, J= 4.9 Hz, 3H).

EXAMPLE 62. SYNTHESIS OF COMPOUND 62

[0200] (R)-2-amino-3-(4-carbamimidoylphenyl)-N-((3S,6S,9S, 12S, 15R,18S,21S)-6,9, 18-tris(2- aminoethyl)-15-benzyl-3-((R)-l-hydroxyethyl)-12-isobutyl-2,5,8,l l, 14, 17,20-heptaoxo- l,4,7,10, 13,16, 19-heptaazacyclotricosan-21-yl)propanamide, Compound 62, was prepared according to the coupling then deprotection sequence described for Compound 4. LCMS: rt = 0.40 minutes (LC method 3). m/z = 951 (M+l). ¾ NMR (400 MHz, D₂0): δ 8.37 (s, 2H), 7.72 (d, J= 8.0 Hz, 2H), 7.46 (d, J= 8.0 Hz, 2H), 7.39 - 7.07 (m, 5H), 4.55 - 4.38 (m, 3H), 4.33 - 4.00 (m, 7H), 3.41 - 3.24 (m, lH), 3.26 - 2.92 (m, 10H), 2.93 - 2.56 (m, 3H), 2.32 - 1.64 (m, 8H), 1.39 (m, 2H), 1.20 (m, 2H), 1.12 (d, J= 6.1 Hz, 3H), 0.79 - 0.69 (m, 2H), 0.67 (s, 3H), 0.59 (s, 3H).

EXAMPLE 63. SYNTHESIS OF COMPOUND 63

[0201] (2S,4R)-4-acetamido-N-((3S,6S,9S,12S,15R, 18S,21S)-6,9, 18-tris(2-aminoethyl)-15- benzyl-3-((R)-l-hydroxyethyl)-12-isobutyl-2,5,8,l l, 14,17,20-heptaoxo-l,4,7, 10, 13,16, 19- heptaazacyclotricosan-21-yl)pyrrolidine-2-carboxamide, Compound 63, was prepared according to the coupling then deprotection sequence described for Compound 4. LCMS: rt = 0.569 minutes (LC method 3). m/z = 916 (M+l). ¾ NMR (400 MHz, D₂0): δ 8.17 (s, 1H), 7.30 (m, 3H), 7.19 (d, J= 7.3 Hz, 2H), 4.53 (t, J= 8.2 Hz, 2H), 4.49 - 4.31 (m, 4H), 4.31 - 4.00 (m, 6H), 3.61 (m, lH), 3.43 - 3.19 (m, 2H), 3.19 - 2.89 (m, 7H), 2.73 (m, 3H), 2.33 - 1.61 (m, 13H), 1.40 (m, 2H), 1.12 (d, J= 6.2 Hz, 3H), 0.81 (m, 1H), 0.75 - 0.67 (d, J= 6.4 Hz 3H), 0.64 (d, J= 6.3 Hz, 3H).

ND 64

[0202] (R)-2-amino-2-(piperidin-4-yl)-N-((3 S,6S,9S, 12S, 15R, 18S,21 S)-6,9, 18-tris(2- aminoethyl)-15-benzyl-3-((R)-l-hydroxyethyl)-12-isobutyl-2,5,8,l l, 14, 17,20-heptaoxo- l,4,7,10, 13,16, 19-heptaazacyclotricosan-21-yl)acetamide, Compound 64, was prepared according to the coupling then deprotection sequence described for Compound 4. LCMS: rt = 0.260 minutes (LC method 3). m/z = 902 (M+l). ¾ NMR (400 MHz, D₂0): δ 7.34-7.25 (m, 3H), 7.19 (d, J= 6.8 Hz, 2H), 4.54 - 4.40 (m, 2H), 4.30 - 4.10 (m, 6H), 3.87 (d, J= 6.8 Hz, 1H), 3.55 - 3.41 (m, 2H), 3.34 - 3.24 (m, lH), 3.19 - 2.90 (m, 9H), 2.85-2.68 (m, 2H), 2.25 - 2.07 (m, 5H), 2.04 - 1.78 (m, 6H), 1.70 - 1.31 (m, 4H), 1.12 (d, J= 6.4 Hz, 3H), 0.80 - 0.58 (m, 7H).

65

[0203] (S)-2-amino-3-(lH-benzo[d]imidazol-2-yl)-N-((3S,6S,9S, 12S, 15R, 18S,21S)-6,9, 18- tris(2-aminoethyl)- 15 -benzyl-3 -((R)- 1 -hydroxyethyl)- 12-isobutyl-2,5 , 8, 11 , 14, 17,20-heptaoxo- l,4,7,10, 13,16, 19-heptaazacyclotricosan-21-yl)propanamide, Compound 65, was prepared according to the coupling then deprotection sequence described for Compound 4. LCMS: rt = 0.56 minutes (double peak, LC method 3). m/z = 949 (M+1). ¾ NMR (400 MHz, D₂0): δ 8.17 (s, 1H), 7.73 (m, 2H), 7.53 (m, 2H), 7.37 - 7.24 (m, 4H), 7.20 (d, J= 6.9 Hz, 2H), 4.53 (m, 2H), 4.42 (m, 1H), 4.34 - 4.28 (m, IH), 4.15 (m, 4H), 4.07 (m, 2H), 3.77 (m, 2H), 3.27 - 3.17 (m, IH), 3.11 - 2.91 (m, 9H), 2.79 (m, IH), 2.68 (m, IH), 2.11 (m, 5H), 2.00 - 1.71 (m, 5H), 1.53 - 1.12 (m, 7H), 1.10 (m, 3H), 0.76 (d, J= 6.6 Hz, IH), 0.72 (m, IH), 0.69 (d, J= 5.9 Hz, 3H), 0.61 (d, J= 5.9 Hz, 3H).

66

[0204] (R)-2-amino-3-(4-aminothiazol-2-yl)-N-((3 S,6S,9S, 12S, 15R, 18S,21 S)-6,9, 18-tris(2- aminoethyl)-15-benzyl-3-((R)-l-hydroxyethyl)-12-isobutyl-2,5,8,l l, 14, 17,20-heptaoxo- l,4,7,10, 13,16, 19-heptaazacyclotricosan-21-yl)propanamide, Compound 66, was prepared according to the coupling then deprotection sequence described for Compound 4. LCMS: rt = 0.33 minutes (double peak, LC method 3). m/z 931 (M+1). ¾ NMR (400 MHz, D₂0): δ 8.16 (s, IH), 7.29 (m, 3H), 7.19 (d, J= 7.3 Hz, 2H), 6.62 (s, 1H), 4.51 (m, 1H), 4.47 - 4.40 (m, 1H), 4.17 (m, 6H), 3.25 - 2.87 (m, 10H), 2.81 (m, 1H), 2.71 (m, 1H), 2.26 - 2.01 (m, 4H), 1.83 (m, 4H), 1.48 - 1.29 (m, 3H), 1.12 (d, J= 6.1 Hz, 3H), 0.77 (m, 1H), 0.69 (d, J= 5.5 Hz, 3H), 0.63 (d, J= 5.8 Hz, 3H).

67

[0205] (S)-2-amino-3-(4-aminothiazol-2-yl)-N-((3S,6S,9S, 12S, 15R,18S,21S)-6,9, 18-tris(2- aminoethyl)- 15 -benzyl-3 -((R)- 1 -hydroxyethyl)- 12-isobutyl-2,5 , 8, 11 , 14, 17,20-heptaoxo- l,4,7,10, 13,16, 19-heptaazacyclotricosan-21-yl)propanamide, Compound 67, was prepared according to the coupling then deprotection sequence described for Compound 4. LCMS: rt = 0.34 minutes (double peak, LC method 3). m/z = 931 (M+l). ¾ NMR (400 MHz, D₂0): δ 8.20 (s, 1H), 7.34 (m, 3H), 7.23 (m, 2H), 6.65 (s, 1H), 4.54 (m, 1H), 4.46 (m, 1H), 4.21 (m, 6H), 3.27 (m, 1H), 3.23 - 2.93 (m, 9H), 2.84 (m, lH), 2.74 (m, lH), 2.17 (m, 4H), 1.94 (m, 4H), 1.51 - 1.31 (m, 3H), 1.15 (d, J= 5.6 Hz, 3H), 0.77 (m, lH), 0.72 (s, 3H), 0.65 (s, 3H).

68

[0206] (S)-2-amino-Nl-((3S,6S,9S,12S,15R,18S,21S)-6,9, 18-tris(2-aminoethyl)-3-((R)-l- hydroxyethyl)-12, 15-diisobutyl-2,5,8, l l,14, 17,20-heptaoxo-l,4,7, 10, 13,16, 19-heptaazacyclotricosan-21- yl)succinamide, Compound 68 was prepared according to the coupling then deprotection sequence described for Compound 4. LCMS: rt = 0.34 minutes (LC method 3). m/z = 864 (M+Na). ¾ NMR (400 MHz, D₂0): δ 8.17 (s, 1H), 4.43 (m, 1H), 4.36 - 4.12 (m, 7H), 3.36 (m, 1H), 3.03 (m, 9H), 2.22 (m, 2H), 2.15 - 1.92 (m, 5H), 1.81 (m, 1H), 1.53 (m, 6H), 1.12 (d, J= 5.6 Hz, 3H), 0.89 - 0.78 (m, 11H). 69

[0207] 4-amino-N-((3 S,6S,9S, 12S, 15R, 18S,21 S)-6,9, 18-tris(2-aminoethyl)-3-((R)- 1 - hydroxyethyl)- 1245-diisobutyl-2,5,84 14447,20-heptaoxo-l,4,74043J649-heptaazacyclotricosan-21- yl)piperidine-4-carboxamide, Compound 69, was prepared according to the coupling then deprotection sequence described for . LCMS: rt = 0.36 minutes (LC method 3). m/z = 855 (M+l). ¾ NMR (400 MHz, D₂0): δ 4.44 (dd, J= 9.2, 5.0 Hz, 1H), 4.25 (dd, J= 14.4, 7.4 Hz, 2H), 4.19 - 4.02 (m, 5H), 3.35 - 3.18 (m, 5H), 3.08 (dd, J= 13.6, 7.0 Hz, 1H), 3.00-2.86 (m, 6H), 2.55 - 2.43 (m, 2H), 2.19 - 1.82 (m, 10H), 1.53-1.39 (m, 6H), 1.06 (d, J= 6.2 Hz, 3H), 0.85 - 0.66 (m, 12H).

ND 70

[0208] (3S,6S,9S, 12S, 15R, 18S,21 S)-6,9, 18-tris(2-aminoethyl)-21-(2,4-dioxo- l,3,8- triazaspiro[4.5]decan-3-yl)-3-((R)- l-hydroxyethyl)-12, 15-diisobutyl- l,4,7, 10, 13, 16, 19- heptaazacyclotricosane-2,5,8, l l, 14, 17,20-heptaone, Compound 70, was prepared according to the coupling then deprotection sequence described for Compound 4. LCMS: rt = 0.24 & 0.42 minutes (double peak, LC method 3). m/z = 880 (M+l), 902 (M+Na). ¾ NMR (400 MHz, D₂0): δ 4.47 (dd, J = 15.4, 10.0 Hz, 1H), 4.34 (d, J= 9.6 Hz, 1H), 4.24-4.13 (m, 5H), 3.49-3.38(m, 2H), 3.27-2.90 (m, 10H), 2.35 - 2.04 (m, 8H), 2.01-1.91 (m, 4H), 1.69 - 1.45 (m, 6H), 1.39 (dd, J= 5.6, 5.6 Hz, 3H), 1.14 (d, J= 5.6 Hz, 3H), 0.93 - 0.71 (m, 12H). EXAMPLE 71. SYNTHESIS OF COMPOUND 71

[0209] Step 1. Intermediate 71-1, tert-butyl N-[2-[(2S,5R,8S,l lS,14S,17S,22S)-5-benzyl-l l,14- bis[2-(tert-butoxycarbonylamino)ethyl]-22-[[(2R)-2-(tert-butoxycarbonylamino)-3-hydroxy- propanoyl]amino]-17-[(lR)-l-hydroxyethyl]-8-isobutyl-3,6,9,12, 15, 18,23-heptaoxo-l,4,7, 10, 13, 16, 19- , shown below, is synthesized as follows.

[0210] To a solution oftert-butyl N-[2-[(2S,5R,8S, l lS, 14S,17S,22S)-22-amino-5-benzyl-l l,14- bis[2-(tert-butoxycarbonylamino)ethyl]-17-[(lR)-l-hydroxyethyl]-8-isobutyl-3,6,9, 12, 15, 18,23-heptaoxo- 1,4,7,10, 13, 16, 19-heptazacyclotricos-2-yl]ethyl]carbamate (500 mg, 0.47 mmol) and (2R)-2-(tert- butoxycarbonylamino)-3-hydroxy-propanoic acid (96.59 mg, 0.47 mmol) in DMF (2 mL) was added Ν,Ν-Diisopropylethylamine (DIPEA) (121.44 mg, 0.94 mmol) and (2-(lH-benzotriazol-l-yl)- 1, 1,3,3- tetramethyluronium hexafluorophosphate) (HBTU) (178.39 mg, 0.47 mmol). The resulting mixture was stirred at room temperature for 2 hrs (confirmed by LCMS). The reaction mixture was diluted with ethyl acetate (50 mL); the organic phase was washed with water and brine. The organic layer was concentrated and purified by preparative HPLC (acidic condition, modifier: HCOOH) to afford the compound

intermediate 71-1 (300 mg, 0.24 mmol, 51% yield). LCMS:rt = 1.39 minutes (LC method 1), 1249 (M+l).

[0211] Synthesis of example (2R)-2-amino-3-hydroxy-N-[(3S,6S,9S, 12S, 15R, 18S,21S)-6,9, 18- tris(2-aminoethyl)-15-benzyl-3-[(lR)-l-hydroxyethyl]-12-isobutyl-2,5,8, l l,14, 17,20-heptaoxo- 1, 4, 7, 10, 13, 16, 19-heptazacyclotric -21 -yl]propanamide, Compound 71.

[0212] To a solution of intermediate 71-1 (110 mg, 0.09 mmol) in DCM (5 mL) was added TFA (0.5 mL) dropwise. After the addition, the mixture was stirred at room temperature until the reaction was complete, monitored by LCMS. The mixture was then concentrated under reduced pressure, and the residue was dissolved with 10% aq. formic acid solution (10 mL). The solution was concentrated, after which, it was re-dissolved with 10% aq. formic acid solution (10 mL) and freeze-dried to afford the desired product (2R)-2-amino-3-hydroxy-N-[(3S,6S,9S,12S,15R,18S,21S)-6,9, 18-tris(2-aminoethyl)-15- benzyl-3-[(lR)-l-hydroxyethyl]-12-isobutyl-2,5,8,l l, 14,17,20-heptaoxo-l,4,7, 10, 13, 16, 19- heptazacyclotricos-21-yl]propanamide, Compound 71 (75 mg, 0.08 mmol, 90.4 % yield). LCMS: rt = 0.27 minutes ( LC method 2). m/z == 849.5 (M+l ). ¾ NMR (400 MHz, D₂0) δ 8.16 (s, 1H), 7.29 (m, 3H), 7.19 (d, J= 7.1 Hz, 2H), 4.52 (t, J= 8.2 Hz, 1H), 4.43 (dd, J= 8.8, 5.4 Hz, 1H), 4.30 - 4.24 (m, 1H), 4.19 - 4.10 (m, 6H), 3.97 - 3.91 (m, 2H), 3.38-3.34 (m, lH), 3.15 - 2.95 (m, 6H), 2.83-2.80 (m, lH), 2.73-2.72 (m, 1H),2.29 - 2.06 (m, 4H), 1.95 - 1.84 (m, 4H), 1.41 - 1.34 (m, 2H), 1.12 (d, J= 6.1 Hz, 3H), 0.75 (m, 1H), 0.70 (t, J= 6.7 Hz, 3H), 0.63 (d, J= 6.3 Hz, 3H).

EXAMPLE 72. SYNTHESIS OF COMPOUND 72

[0213] Compound 72, 2-amino-N-[(3S,6S,9S,12S,15R, 18S,21S)-6,9, 18-tris(2-aminoethyl)-15- benzyl-3-[(lR)-l-hydroxyethyl]-12-isobutyl-2,5,8,l l, 14,17,20-heptaoxo-l,4,7, 10, 13, 16, 19- heptazacyclotricos-21-yl]acetamide, was prepared according to the coupling then deprotection sequence described for Compound 71. LCMS: rt = 0.19 & 0.32 minutes (double peak, LC method 2). m/z = 819.5 (M+l) ¾ NMR (400 MHz, D₂0) δ 7.34-7.25 (m, 3H), 7.19 (d, J= 7.4 Hz, 2H), 4.53 (t, J= 8.2 Hz, 1H), 4.42 (dd, v = 8.6, 5.4 Hz, 1H), 4.31 - 4.08 (m, 6H), 3.81 (s, 2H), 3.37-3.27 (m, lH), 3.14 - 2.95 (m, 7H), 2.85-2.78 (m, 1H), 2.74-2.66 (m, 1H), 2.26 - 2.03 (m, 4H), 1.99 - 1.76 (m, 4H), 1.45 - 1.31 (m, 2H), 1.12 (d, J= 6.0 Hz, 3H), 0.85 - 0.77 (m, 1H), 0.74 - 0.58 (m, 6H). EXAMPLE 73. SYNTHESIS OF COMPOUND 73

[0214] Compound 73, (2S)-2-amino-3-hydroxy-N-[(3S,6S,9S,12S,15R, 18S,21S)-6,9,18-tris(2- aminoethyl)- 15 -benzyl-3 -[( 1 R)- 1 -hydroxyethyl] - 12-isobutyl-2,5 , 8, 11 , 14, 17,20-heptaoxo- l,4,7,10, 13,16, 19-heptazacyclotricos-21-yl]propanamide was prepared according to the coupling then deprotection sequence described for Compound 71. LCMS: rt = 0.22 &0.37 minutes (double peak, LC method 2). m/z == 849.5 (M+l ). ¾ NMR (400 MHz, D₂0) δ 7.35 - 7.24 (m, 3H), 7.19 (d, J= 7.0 Hz, 2H), 4.52 (d, J= 8.4 Hz, 1H), 4.44 - 4.37 (m, 1H), 4.31 - 4.09 (m, 7H), 3.93 (s, 2H), 3.37-3.30 (m, lH), 3.12 - 2.93 (m, 7H), 2.84 - 2.62 (m, 3H), 2.25 - 2.03 (m, 4H), 1.96-1.78 (m, 4H), 1.46 - 1.31 (m, 2H), 1.12 (d, J= 5.8 Hz, 3H), 0.86-0.78 (m, 1H), 0.67 (dd, J= 6.0, 8.0 Hz, 6H).

EXAMPLE 74. SYNTHESIS OF COMPOUND 74

[0215] Compound 74, (2S)-2-amino-N-[(3S,6S,9S,12S, 15R,18S,21S)-6,9, 18-tris(2- aminoethyl)- 15 -benzyl-3 - [( 1 R)- 1 -hydroxyethyl] - 12-isobutyl-2,5 , 8, 11 , 14, 17,20 -heptaoxo- 1,4,7,10, 13, 16, 19-heptazacyclotricos-21-yl]butanamide, was prepared according to the coupling then deprotection sequence described for Compound 71. LCMS: ft = 0.18 &0.25 minutes (double peak, LC method 2). m/z == 847.5 (M+l ). ¾ NMR (400 MHz, D₂0) δ 7.42-7.33 (m, 3H), 7.28 (d, J= 7.4 Hz, 2H), 4.63-4.51 (m, 2H), 4.38 - 4.13 (m, 6H), 4.02 (t, J= 6.4 Hz, 1H), 3.44-3.32 (m, 1H), 3.22 - 3.04 (m, 7H), 2.94 - 2.86 (m, 1H), 2.81-2.74 (m, 1H), 2.35 - 2.17 (m, 4H), 2.10 - 2.01 (m, lH), 1.99 - 1.88 (m, 4H), 1.56 - 1.39 (m, 2H), 1.31 (t, J = 7.0 Hz, 1H), 1.21 (d, J = 6.0 Hz, 3H), 1.10 (t, J= 7.4 Hz, 1H), 0.99 (t, J = 7.4 Hz, 3H), 0.86-0.70 (m, 7H). COMPOUND 75

[0216] Compound 75, (2R)-N-[(3S,6S,9S, 12S, 15R, 18S,21S)-6,9, 18-tris(2-aminoethyl)-15- benzyl-3-[(lR)-l-hydroxyethyl]-12-isobutyl-2,5,8,l l, 14,17,20-heptaoxo-l,4,7, 10, 13, 16, 19- heptazacyclotricos-21-yl]pyrrolidine-2-carboxamide, was prepared according to the coupling then deprotection sequence described for Compound 71. LCMS: rt = 0.16 &0.32 minutes (double peak, LC method 2). m/z == 859.5 (M M). ¾ NMR (400 MHz, D₂0) δ 7.34-7.25 (m, 3H), 7.20 (d, J= 7.2 Hz, 2H), 4.53 (t, JJ = 8.2 Hz, 1H), 4.46 - 4.25 (m, 3H), 4.24 - 4.07 (m, 5H), 3.43 - 3.28 (m, 3H), 3.10-2.94 (m, 7H), 2.87 - 2.78 (m, 1H), 2.76 - 2.67 (m, 1H), 2.48 - 2.38 (m, 1H), 2.25-2.12 (m, 3H), 2.09-1.80 (m, 8H), 1.47 - 1.29 (m, 2H), 1.12 (d, J= 6.0 Hz, 3H), 0.83 - 0.58 (m, 7H).

COMPOUND 76

[0217] Compound 76, (2S)-N-[(3S,6S,9S, 12S, 15R, 18S,21S)-6,9, 18-tris(2-aminoethyl)-15- benzyl-3-[(lR)-l-hydroxyethyl]-12-isobutyl-2,5,8,l l, 14,17,20-heptaoxo-l,4,7, 10, 13, 16, 19- heptazacyclotricos-21-yl]pyrrolidine-2-carboxamide. was prepared according to the coupling then deprotection sequence described for Compound 71. LCMS: rt = 0.19 &0.36 minutes (double peak, LC method 2). m/z == 859.5 (M M). ¾ NMR (400 MHz, D₂0) δ 7.34-7.24 (m, 3H), 7.19 (d, J= 7.2 Hz, 2H), 4.51 (t, J= 8.2 Hz, 1H), 4.44 (dd, J= 8.6, 5.6 Hz, 1H), 4.40 - 4.32 (m, 1H), 4.29 - 4.08 (m, 6H), 3.39 - 3.27 (m, 3H), 3.15 - 2.95 (m, 7H), 2.85 - 2.77 (m, 1H), 2.73-2.64(m, lH), 2.47 - 2.35 (m, lH), 2.25 - 2.06 (m, 4H), 2.04 - 1.78 (m, 7H), 1.46 - 1.29 (m, 2H), 1.12 (d, J= 6.2 Hz, 3H), 0.83 - 0.58 (m, 7H). COMPOUND 77

[0218] Compound 77, (2S)-2-amino-N-[(3S,6S,9S,12S,15R,18S,21S)-6,9,18-tris(2- aminoethyl)- 15 -benzyl-3 - [( 1 R)- 1 -hydroxyethyl] - 12-isobutyl-2,5 , 8, 11 , 14, 17,20-heptaoxo- l,4,7,10,13,16,19-heptazacyclotricos-21-yl]pentanediamide, was prepared according to the coupling then deprotection sequence described for Compound 71. LCMS: ft ^::: 2.22 minutes (LC method 3). ra/z ^::: 890 (M+l). ¾NMR (400 MHz, D₂0) δ 8.18 (s, 1H), 7.29 (m, 3H), 7.19 id../ 7.2 Hz, 2H), 4.57 - 4.40 (m, 2H), 4.35 - 3.96 (m, 6H), 3.47 - 3.19 (m, 1H), 3.19 - 2.58 (m, 9H), 2.41 (dq, J= 16.4, 8.4 Hz, 2.H), 2.2.7 - 1.73 (m, 9H), 1.46 - 1.15 (m, 4H), 1.12 (d, J= 5.6 Hz, 3H), 0.77 - 0.65 (m, 3H), 0.65 - 0.53 (m, 3H).

D 78

[0219] Compound 78, (2R)-2-amino-3-(lH-indol-3-yl)-N-[(3S,6S,9S,12S,15R,18S,21S)-6,9,18- tris(2-aminoethyl)-15-benzyl-3-[(lR)-l-hydroxyethyl]-12-isobutyl-2,5,8,ll,14,17,20-heptaoxo- l,4,7,10,13,16,19-heptazacyclotricos-21-yl]propanamide, was prepared according to the coupling then deprotection sequence described for Compound 71. LCMS: ft ^::: 0.33 minutes (LC method 3). m/z ^::: 949 (M+l). ¾NMR (400 MHz, D₂0) δ 8.18 (s, 1H), 7.53 id../ 8.0 Hz, 1H), 7.45 (d, J 8.0 Hz, 1H), 7.36 - 7.24 (m, 4H), 7.23 - 7.16 (m, 3H), 7.09 (m, 1H), 4.55 (m, 1H), 4.35 - 4.22 (m, 3H), 4.21 - 4.04 (m, 5H), 3.41 (m, 1H), 3.24 (m, 2H), 3.10 - 2.89 im 111).2.79 - 2.69 im. ill) 2.55 (m, I Hi.2.09 im 4H), 1.94 - 1.70 (m, 4H).1.39 (m, 2H), 1.12 (m.31!}.0.88 (s, Hi).0.71 (d, ./ 6.4 Hz, 3H), 0.62 (m, 3H). 79

[0220] Compound 79, 2-(lH-indol-3-yl)-N-[(3S,6S,9S, 12S, 15R, 18S,21S)-6,9,18-tris(2- aminoethyl)- 15 -benzyl-3 - [( 1 R)- 1 -hydroxyethyl] - 12-isobutyl-2,5 , 8, 11 , 14, 17,20-heptaoxo- 1,4,7,10, 13, 16, 19-heptazacyclotricos-21-yl]acetamide, was prepared according to the coupling then deprotection sequence described for Compound 71. LCMS: rt ^::: 0.21 minutes (LC method 2). m/z ^:== 919.5 CM+1). ¾ NMR (400 MHz, D₂0) 5 8.16 (s, I B), 7.54 (d, J= 7.9 Hz, 1H), 7.46 (d, J = 8.1 Hz, 1H), 7.29 (m, 4H), 7.19 (ra, 3H), 7.10 (m, IH), 4.47 - 4.35 (m, 2H), 4.22 (ra, 3H), 4.07 (m, 3H), 3.74 (s, 2H), 3.35 (m, I H), 2.99 (m, 7H), 2.65 (m, 1 H), 2.55 (m, 1H), 2.14 (m, 3H), 2.04 - 1.97 (m, 1 H), 1.85 (m, 3H), 1.73 - 1.61 (m, IH), 1.42 - 1.28 (m, 2H), 1.09 (d, .1 5.7 Hz, 3H), 0.70 (s, IH), 0.66 (s, 3H), 0.58 (s, 3H). EXAMPLE 80. SYNTHESIS OF COMPOUND 80

[0221] Compound 80, (2S)-2-amino-N-[(3S,6S,9S,12S,15R,18S,21S)-6,9, 18-tris(2- aminoethyl)- 15 -benzyl-3 -[( 1 R)- 1 -hydroxyethyl] - 12-isobutyl-2,5 , 8, 11 , 14, 17,20-heptaoxo- l,4,7,10, 13,16, 19-heptazacyclotricos-21-yl]butanediamide, was prepared according to the coupling then deprotection sequence described for Compound 71. LCMS: rt = 0.27 & 0.39 minutes (double peak, LC method 3). m/z = 876 (M+l). ¾ NMR (400 MHz, D₂0) δ 7.34-7.25 (m, 3H), 7.19 (d, J= 7.0 Hz, 2H), 4.53 (t, J= 8.2 Hz, IH), 4.40 - 4.23 (m, 4H), 4.21 - 4.08 (m, 4H), 3.41 - 3.32 (m, IH), 3.12 - 2.99 (m, 6H), 2.97 - 2.92 (m, 2H), 2.85 - 2.75 (m, IH), 2.67 (dd, J= 17.4, 10.4 Hz, IH), 2.27 - 1.73 (m, 9H), 1.44 - 1.30 (m, 2H), 1.11 (d, J= 5.8 Hz, 3H), 0.87 - 0.78 (m, IH), 0.67 (dd, J= 6.8, 6.4 Hz, 6H). EXAMPLE 81. SYNTHESIS OF COMPOUND 81

[0222] Compound 81, (2S)-2,6-diamino-N-[(3S,6S,9S,12S,15R, 18S,21S)-6,9,18-tris(2- aminoethyl)- 15 -benzyl-3 - [( 1 R)- 1 -hydroxyethyl] - 12-isobutyl-2,5 , 8, 11 , 14, 17,20-heptaoxo- l,4,7,10, 13,16, 19-heptazacyclotricos-21-yl]hexanamide, was prepared according to the coupling then deprotection sequence described for Compound 71. LCMS: rt ^::: 0.31 &0.39 minutes (double peak, LC method 3). mlz = 891 (M+l). 1H NMR (400 MHz, D₂0) δ 7.35-7.25 (m, 3H), 7.20 (d, J= 6.8 Hz, 2H), 4.55 - 4.40 (m, 2H), 4.28 - 4.09 (m, 6H), 3.98 (t, J= 6.6 Hz, 1H), 3.34 - 3.24 (m, 1H), 3.14 - 2.92 (m, 9H), 2.86 - 2.78 (m, 1H), 2.76 - 2.67 (m, 1H), 2.1520-2.09 (m, 4H), 2.00 - 1.78 (m, 6H), 1.66 (dt, J = 15.2, 7.6 Hz, 2H), 1.48 - 1.29 (m, 4H), 1.13 (d, J= 6.4 Hz, 3H), 0.75 (dd, J= 12.2, 7.4 Hz, 1H), 0.69 (d, J = 5.4 Hz, 3H), 0.62 (d, J= 5.8 Hz, 3H).

D 82

[0223] Compound 82, (2R)-2-acetamido-3-(lH-indol-2-yl)-N-[(3S,6S,9S, 12S,15R,18S,21S)- 6,9, 18-tris(2-aminoethyl)-15-benzyl-3-[(lR)-l-hydroxyethyl]-12-isobutyl-2,5,8,l l,14, 17,20-heptaoxo- l,4,7,10, 13,16, 19-heptazacyclotricos-21-yl]propanamide, was prepared according to the coupling then deprotection sequence described for Compound 71. LCMS: rt ^::: 0.37 minutes (LC method 3). m/z ^::: 991 (M+l). ¾ NMR (400 MHz, D₂0) δ 8.19 (s, 1H), 7.56 id. ./ 7.8 Hz, IH), 7.41 (d, J= 8.2 Hz, 1H), 7.30 (m . 3H), 7.17 (m, 4H), 7.09 (m M i ). 4.51 (m, 2H), 4.34 ( , I B), 4.18 - 4.07 (m 3H), 4.03 (m, 2H), 3.90 (m, Mb.3.18 id. 7.1 Hz, 111).3.05 - 2.88 (m, 7H), 2.77 (m, 1H), 2.63 (m.3H), 2.19 - 1.97 (m, 5H), 1.91 (m, 4H), 1.76 (m, 2H), 1.56 (m, 1H), 1.41 - 1.34 (m, 2H), 1.18 (m, ill). 1.10 id../ 5.9 Hz, 3H), 0.75 (m, 1H), 0.69 (d, = 6.3 Hz, 3H), 0.61 (d, J= 6.2 H .3H).

COMPOUND 83

[0224] Compound 83, (2S)-2-amino-3-(lH-imidazol-4-yl)-N-[(3S,6S,9S,12S,15R,18S,21S)- 6,9,18 ris(2-aminoethyl)-15-benzyl-3-[(lR)-l-hydroxy^

l,4,7,10,13,16,19-heptazacyclotricos-21-yl]propanamide, was prepared according to the coupling then deprotection sequence described for Compound 71. LCMS: rt = 0.34 minutes (LC method 3} m/z = 899 (M+l). ¾NMR (400 MHz, D₂0) δ 8.66 (s, IH), 8.19 (s, 1H), 7.39 (s, 1H), 7.35 - 7.25 (m, 3H), 7.20 (d, ./ 7.3 Hz, 2H), 4.52 (m, IH), 4.46 (m, 1H), 4.29 (m, 111).4.23 - 4.11 (m, 5H), 3.33 id../ 6.8 Hz, 111). 3.26 (m, IH), 3.14 - 2.97 (m, 7H), 2.81 (m, IH), 2.77 - 2.67 (m, IH), 2.14 (m, 4H), 1.88 (m, 4H), 1.38 (ro, 2H), 1.12 (d, ./= 6.2 Hz, 3H), 0.73 (m, H), 0.69 (d, J = 6.0 Hz, 3H), 0.62 (d, J= 6.0 Hz, 3H).

Example 84 Scheme 0

[0225] Synthesis of Compound 84. (S)-3-amino-4-oxo-4-((3S,6S,9S, 12S,15R, 18S,21S)-6,9, 18- tris(2-aminoethyl)-15-benzyl-3-((R)-l-hydroxyethyl)-12-isobutyl-2,5,8,l l, 14,17,20-heptaoxo-

1.4.7.10.13.16.19- heptaazacyclotricosan-21-ylamino)butanoic acid, was performed as shown in Example 84 Scheme, described in more detail below.

[0226] Step 1. Synthesis of Intermediate 84-2, benzyl (3S)-4-[[(3S,6S,9S, 12S, 15R, 18S,21S)-15- benzyl-6,9, 18-tris [2-(tert-butoxycarbonylamino)ethyl] -3 -[( 1 R)- 1 -hydroxyethyl] - 12-isobutyl-

2.5.8.11.14.17.20- heptaoxo-l,4,7, 10,13, 16,19-heptazacyclotricos-21-yl]amino]-3-(tert- butoxycarbonylamino)-4-oxo-butanoate.

[0227] To a solution of (2S)-4-benzyloxy-2-(tert-butoxycarbonylamino)-4-oxo-butanoic acid (60.88 mg, 0.19 mmol) in anhydrous DMF (3 mL) was added HBTU (53.5 mg, 0.14 mmol) and DIPEA (0.05 mL, 0.28 mmol). Then tert-butyl N-[2-[(2S,5R,8S,l lS, 14S, 17S,22S)-22-amino-5-benzyl-l 1,14- bis [2-(tert-butoxycarbonyl amino)ethyl] -17-[(1R)-1 -hydroxyethyl] -8-isobutyl-3 ,6,9, 12,15, 18,23- heptaoxo-l,4,7, 10,13, 16,19-heptazacyclotricos-2-yl]ethyl]carbamate (100 mg, 0.09 mmol) was added to the mixture. The reaction mixture was stirred at room temperature for 2 hrs. The crude mixture was quenched with water (10 mL). The resulting mixture was extracted with ethyl acetate (15 mL x 2). The combined organic layer were washed with water and brine, dried over anhydrous Na2SC>4, filtered and concentrated, the residue was purified by Prep-HPLC (eluting by CLLCN/Water, modifier: formic acid) to afford intermediate 84-2, benzyl (3S)-4-[[(3S,6S,9S,12S,15R,18S,21S)-15-benzyl-6,9,18-tris[2-(tert- butoxycarbonylamino)ethyl] -3 - [( 1 R)- 1 -hydroxyethyl] - 12-isobutyl-2,5 , 8, 11,14,17,20-heptaoxo-

1.4.7.10.13.16.19- heptazacyclotricos-21 -yl]amino] -3 -(tert-butoxycarbonylamino)-4-oxo-butanoate (85 mg, 0.06 mmol, 66 % yield). LC-MS (LC method 1): m/z 1367.7 (M+H)⁺.

[0228] Step 2. Synthesis of Intermediate 84-3, (3S)-4-[[(3S,6S,9S,12S,15R,18S,21S)-15-benzyl- 6,9,18-tris [2-(tert-butoxycarbonylamino)ethyl] -3 - [( 1 R)- 1 -hydroxyethyl] - 12-isobutyl-2,5 , 8, 11,14,17,20- heptaoxo- 1,4,7,10,13,16, 19-heptazacyclotricos-21 -yl]amino] -3 -(tert-butoxycarbonylamino)-4-oxo- butanoic acid 84-3. To a solution of Intermediate 84-2, benzyl (3S)-4-[[(3S,6S,9S,12S,15R,18S,21S)-15- benzyl-6,9, 18-tris [2-(tert-butoxycarbonylamino)ethyl] -3 -[( 1 R)- 1 -hydroxyethyl] - 12-isobutyl-

2.5.8.11.14.17.20- heptaoxo-l,4,7,10,13,16,19-heptazacyclotricos-21-yl]amino]-3-(tert- butoxycarbonylamino)-4-oxo-butanoate (85 mg, 0.06 mmol), in Methanol (5 niL) that was degassed with N2 three times, Pd/C (8.5 mg, 10% wt) was added. The resulting mixture was degassed again and stirred at room temperature under a ¾ balloon for 2 hrs. The crude mixture was filtered through CELITE. The filtrate was concentrated under reduced pressure to give Intermediate 84-3, (3S)-4-[[(3S,6S,9S,12S,15R,18S,21S) - 15 -benzyl-6,9, 18-tris [2-(tert-butoxycarbonylamino)ethyl] -3 - [( 1 R)- 1 -hydroxyethyl] - 12-isobutyl- 2,5,8,ll,14,17,20-heptaoxo-l,4,7,10,13,16,19-heptazacyclotricos-21-yl]amino]-3-(tert- butoxycarbonylamino)-4-oxo-butanoic acid 84-3 (35 mg, 0.027 mmol, 44.1 % yield). LC-MS (LC method 1): m/z 1277 (M+H)⁺.

[0229] Step 3. Synthesis of Compound 84, (3S)-3-amino-4-oxo-4

[[(3S,6S,9S,12S,15R,18S,21S)-6,9,18-tris(2-aminoethyl)-15-benzyl-3-[(lR)-l-hydroxyethyl]-12-isobutyl- 2,5,8,11,14,17,20-heptaoxo- 1,4,7,10,13,16, 19-heptazacyclotricos-21 -yl] amino] butanoic acid. Compound 84 was prepared from Intermediate 84-3 according to the deprotection sequence described for Compound 71. LCMS: rt = 2.22&2.24 (double peak, LC method 3). m/z = 877 (M+l). ¾ NMR (400 MHz, D₂0) δ 8.17 (s, 1H), 7.30 (m, 3H), 7.19 (d, j = 7.0 Hz, 2H), 4.49 (ra, 1H), 4.35 (m, 3H), 4.23-4.11 (m, 5H), 3.38 (m, 1H), 3.00 (m, 8H), 2.82 (m, 4H), 2.22 (m, 2H), 2.03 (m, 6H), 1.79 (m₅1 IT), 1.44 - 1.26 (m, 2H), 1.11 (d, J 5.8 Hz, 3H), 0.72 (s, 1H), 0.69 (s, 3H), 0.61 (d, J - 5.0 Hz.3H).

EXAMPLE 85. SYNTHESIS OF COMPOUND 85

[0230] Compound 85, (2S)-2,4-diamino-N-[(3S,6S,9S,12S,15R, 18S,21S)-6,9,18-tris(2- aminoethyl)- 15 -benzyl-3 - [( 1 R)- 1 -hydroxyethyl] - 12-isobutyl-2,5 , 8, 11 , 14, 17,20-heptaoxo- 1,4,7,10, 13, 16, 19-heptazacyclotricos-21-yl]butanamide, was prepared according to the coupling then deprotection sequence described for Compound 71. I .( MS rt = 0.20 minutes (LC method 1 ). m/z = 862.5 (M+l ). ¾ NMR (400 MHz, D₂0) δ 8.22 (s, 1H), 7.30 (m, 3H), 7.20 (m, 2H), 4.58 - 4.36 (m, 2H), 4.33 - 3.93 (m, 7H), 3.40 - 3.21 (m, 1H), 3.19 - 2.91 (m, 9H), 2.90 - 2.59 (m, 2H), 2.31 - 2.04 (m, 6H), 2.04 - 1.73 (m, 4H), 1.51 - 1.18 (m, 3H), 1.13 (d, J= 6.4 Hz, 3H), 0.78 - 0.65 (m, 4H), 0.62 (d, J= 5.5 Hz, 3H).

Example 86 Scheme [0231] Synthesis of Compound 86, (S)-2-acetamido-4-amino-N-((3S,6S,9S,12S,15R,18S,21S)- 6,9,18-tris(2-aminoethyl)-15-benzyl-3-((R)-l-hydroxyethyl)-12-isobutyl-2,5,8,ll,14,17,20-heptaoxo- l,4,7,10,13,16,19-heptaazacyclotricosan-21-yl)butanamide, was performed as shown in Example 86Scheme, described in more detail below.

[0232] Step 1. Synthesis of Intermediate 86-2, tert-butyl N-[2-[(2S,5R,8S,l lS,14S,17S,22S)-5- benzyl-22-[[(2S)-2-(benzyloxycarbonylamino)-4-(tert-butoxycarbonylamino)butanoyl]amino]-ll,14- bis[2-(tert-butoxycarbonylamino)ethyl]-17-[(lR)-l-hydroxyethyl]-8-isobutyl-3,6,9,12,15,18,23-heptaoxo- 1,4,7,10,13, 16,19-heptazacyclotricos-2-yl]ethyl]carbamate 86-2.To a solution of (2S)-2- (benzyloxycarbonylamino)-4-(tert-butoxycarbonylamino)butanoic acid (100 mg, 0.28 mmol), hydroxybenzotriazole (HOBT) (76.6 mg, 0.57 mmol), and tert-butyl N-[2-[(2S,5R,8S,l 1S,14S,17S,22S)- 22-amino-5 -benzyl- 11 , 14-bis [2-(tert-butoxycarbonylamino)ethyl] -17-[(1R)-1 -hydroxyethyl] -8-isobutyl - 3,6,9,12,15, 18,23-heptaoxo-l,4,7,10,13,16,19-heptazacyclotricos-2-yl]ethyl] carbamate (301.5 mg, 0.28 mmol) in DMF (5 mL) was added l-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI) (163.2 mg, 0.85 mmol). The mixture was stirred at 20 °C for 2 hrs. The reaction was diluted with water (25 mL), extracted with ethyl acetate (10 mL x2). The combined organic phases were washed with water and brine, dried over Na2SC>4, filtered, and concentrated. The residue was purified by flash column chromatography (DCM : MeOH = 10 : 1) to give Intermediate 86-2, tert-butyl N-[2-[(2S,5R,8S,l lS,14S,17S,22S)-5- benzyl-22-[[(2S)-2-(benzyloxycarbonylamino)-4-(tert-butoxycarbonylamino)butanoyl]amino]-ll,14- bis[2-(tert-butoxycarbonylamino)ethyl]-17-[(lR)-l-hydroxyethyl]-8-isobutyl-3,6,9,12,15,18,23-heptaoxo- 1,4,7,10,13, 16,19-heptazacyclotricos-2-yl]ethyl]carbamate (270 mg, 0.19 mmol, 68.1 % yield). LC-MS (LC method 1): m/z 1396 (M+H)⁺.

[0233] Step 2. Synthesis of Intermediate 86-3, tert-butyl N-[(3S)-3-amino-4- [[(3S,6S,9S,12S,15R,18S,21S)-15-benzyl-6,9,18-tris[2-(tert-butoxycarbonylamino)ethyl]-3-[(lR)-l- hydroxyethyl]-12-isobutyl-2,5,8, 11,14, 17,20-heptaoxo- 1,4,7, 10, 13,16, 19-heptazacyclotricos-21- yl]amino]-4-oxo-butyl]carbamate 86-3. To a solution of isopropyl N-[2-[(2S,5R,8S,l lS,14S,17S,22S)-5- benzyl-22-[[(2S)-2-(benzyloxycarbonylamino)-4-(tert-butoxycarbonylamino)butanoyl]amino] -11,14- bis[2-(tert-butoxycarbonylamino)ethyl]-17-[(lR)-l-hydroxyethyl]-8-isobutyl-3,6,9,12,15,18,23-heptaoxo- 1,4,7,10,13, 16,19-heptazacyclotricos-2-yl]ethyl]carbamate (86-2) (270 mg, 0.20 mmol) in methanol (10 mL) was added Pd(OH)2 (27 mg, 0.20 mmol). The mixture was stirred under an ¾ balloon at 20°C overnight. The catalyst was filtered off through CELITE. The filtrate was concentrated to give the crude product, which was purified by preparative-HPLC to give Intermediate 86-3, tert-butyl N-[(3S)-3-amino- 4-[[(3S,6S,9S,12S,15R,18S,21S)-15-benzyl-6,9,18-tris[2-(tert-butoxycarbonylamino)ethyl]-3-[(lR)-l- hydroxyethyl]-12-isobutyl-2,5,8,l 1,14, 17,20-heptaoxo- 1,4,7, 10, 13,16, 19-heptazacyclotricos-21- yl]amino]-4-oxo-butyl]carbamate (170 mg, 0.13 mmol, 68.9 % yield). LC-MS (LC method 1): m/z 1263 (M+H)⁺.

[0234] Step 4. Synthesis of Intermediate 86-4, tert-butyl N-[(3 S)-3-acetamido-4- [[(3S,6S,9S,12S,15R, 18S,21S)-15-benzyl-6,9,18-tris[2-(tert-butoxycarbonylamino)ethyl]-3-[(lR)-l- hydroxyethyl]-12-isobutyl-2,5,8, 11,14, 17,20-heptaoxo- 1,4,7, 10, 13, 16, 19-heptazacyclotricos-21- yl]amino]-4-oxo-butyl]carbamate. To a solution of tert-butyl N-[(3S)-3-amino-4- [[(3S,6S,9S,12S,15R, 18S,21S)-15-benzyl-6,9,18-tris[2-(tert-butoxycarbonylamino)ethyl]-3-[(lR)-l- hydroxyethyl]-12-isobutyl-2,5,8, l 1,14, 17,20-heptaoxo- 1,4,7, 10, 13, 16, 19-heptazacyclotricos-21- yl]amino]-4-oxo-butyl]carbamate (120 mg, 0.10 mmol) and acetic anhydride (10.7 mg, 0.10 mmol) in DCM (5 mL), was added pyridine (15 mg, 0.19 mmol). The reaction mixture was stirred at 0 °C for 2 hrs. The crude mixture was diluted with DCM (10 ml) and the organic phase was washed with water and brine, dried over Na2SC>4, filtered, and concentrated. The residue was purified by preparative HPLC to get Intermediate 86-4, tert-butyl N-[(3S)-3-acetamido-4-[[(3S,6S,9S,12S,15R,18S,21S)-15-benzyl-6,9,18- tris[2-(tert-butoxycarbonylamino)ethyl]-3-[(lR)-l-hydroxyethyl]-12-isobutyl-2,5,8, 11, 14, 17,20- heptaoxo-l,4,7, 10,13, 16,19-heptazacyclotricos-21-yl]amino]-4-oxo-butyl]carbamate (41 mg, 0.031 mmol, 33.1 % yield). LC-MS (LC method 1): m/z 1304 (M+H)⁺.

[0235] Step 5. Compound 86, (2S)-2-acetamido-4-amino-N-[(3S,6S,9S,12S, 15R,18S,21S)- 6,9, 18-tris(2-aminoethyl)-15-benzyl-3-[(lR)-l-hydroxyethyl]-12-isobutyl-2,5,8,l l,14, 17,20-heptaoxo- l,4,7,10, 13,16, 19-heptazacyclotricos-21-yl]butanamide, was prepared from Intermediate 86-4 according to the deprotection sequence described for Compound 71. LCMS: rt = 0.20 (LC method 2). m/z = 904.6 (M+l). lH NMR (400 MHz, D₂0) δ 8.16 (s, 3H), 7.29 (m, 3H), 7.19 (m, 2H), 4.50 (t, J= 8.4 Hz, 1H), 4.41 (dd, J = 8.8, 5.2 Hz, 1H), 4.33 - 4.04 (m, 7H), 3.40 - 3.18 (m, 1H), 3.20 - 2.94 (m, 9H), 2.88 - 2.55 (m, 2H), 2.11 (m, 6H), 1.98 (s, 3H), 1.94 - 1.71 (m, 4H), 1.48 - 1.15 (m, 3H), 1.12 (d, J = 6.2 Hz, 3H), 0.73 (m, 1H), 0.69 (d, J= 5.8 Hz, 3H), 0.61 (d, J= 6.0 Hz, 3H).

COMPOUND 87

[0236] Compound 87, (2S)-2-acetamido-5-amino-N-[(3S,6S,9S,12S,15R,18S,21S)-6,9, 18- tris(2-aminoethyl)-15-benzyl-3-[(lR)-l-hydroxyethyl]-12-isobutyl-2,5,8, 11,14, 17,20-heptaoxo- l,4,7,10, 13,16, 19-heptazacyclotricos-21-yl]pentanamid, was prepared according to the coupling then deprotection sequence described for Compound 71. LCMS: rt ^:== 0.27 minutes (LC method 3). m/z ^:== 918 (M+1) ¾ NMR (400 MHz, D₂0) δ 8.24 (s, 1H), 7.32- 7.27 (m, 3H), 7.20-7.18 (m, 2H), 4.53 (q, J= 8.0 Hz, 1H), 4.42-4.13 (m, 6H), 3.31-3.25 (m, 1H), 3.14-3.12 (m, 2H), 3.02-2.96 (m, 7H), 2.81-2.79 (m, 1H), 2.72-2.65 (m, 1H), 2.16-2.15 (m, 4H), 1.97-1.94 (s, 3H), 1.85-1.61 (m, 5H), 1.44-1.25 (m, 2H), 1.13 (d, J = 8.0 Hz, 3H), 0.78-0.75 (m, 1H), 0.70 (t, J= 4.0 Hz, 3H), 0.64 (t, J= 8.0 Hz, 3H).

Example 88 Scheme

[0237] Synthesis of Compound 88, (S)-2-acetamido-5-guanidino-N-((3S,6S,9S,12S,15R, 18S,21S)-6,9,18-tris(2-aminoethyl)-15-benzyl-3-((R)-l-hydroxyethyl)-12-isobutyl-2,5,8,l 1, 14,17,20- heptaoxo- 1,4,7, 10,13, 16, 19-heptaazacyclotricosan-21 -yl)pentanamide, was performed as shown in Scheme , described in more detail below.

[0238] Step. 1. Synthesis of Intermediate 88-2, (2S)-2-acetamido-5- (benzyloxycarbonylamino)pentanoic acid 88-2. To a stirred solution of (2S)-2-amino-5- (benzyloxycarbonylamino)pentanoic acid (1 g, 3.76 mmol) in water (lOmL) was added saturated

NaHCC (10 mL, 3.76 mmol) and acetyl acetate (421.7 mg, 4.13 mmol). The reaction mixture was stirred at room temperature (r.t.) for 3hrs. The crude mixture was extracted with ethyl acetate (100 mL x 3), the combined organic phases were washed with water and brine, dried over Na2SO*, filtered, and

concentrated to give the crude intermediate (2S)-2-acetamido-5-(benzyloxycarbonylamino)pentanoic acid 88-2 (470 mg, 1.52 mmol, 40.6 % yield) LC-MS (LC method 3): m/z 309 (M+H)⁺.

[0239] Step 2. Intermediate 88-3, tert-butyl N-[2-[(2S,5R,8S, l lS, 14S, 17S,22S)-22-[[(2S)-2- acetamido-5 -(benzyl oxycarbonylamino)pentanoyl]amino] -5 -benzyl- 11 , 14-bis [2-(tert- butoxycarbonylamino)ethyl] -17-[(1R)-1 -hydroxyethyl] -8-isobutyl-3 ,6,9, 12, 15, 18,23 -heptaoxo- 1, 4, 7, 10, 13, 16, 19-heptazacyclotricos-2-yl]ethyl]carbamate 88-3.

[0240] To a solution of tert-butyl N-[2-[(2S,5R,8S, l lS, 14S,17S,22S)-22-amino-5-benzyl-l 1,14- bis[2-(tert-butoxycarbonylamino)ethyl]-17-[(lR)-l-hydroxyethyl]-8-isobutyl-3,6,9, 12, 15, 18,23-heptaoxo- 1,4,7,10, 13, 16, 19-heptazacyclotricos-2-yl]ethyl]carbamate (400 mg, 0.38 mmol) and (2S)-2-acetamido-5- (benzyloxy carbonylamino)pentanoic acid (116.1 mg, 0.38 mmol) in DMF (5 mL), was added DIPEA (48.58 mg, 0.38 mmol) and l-[Bis(dimethylamino)methylene]-lH-l,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate (HATU) (142.71 mg, 0.38 mmol). The resulting mixture was stirred at room temperature overnight. Then the crude mixture was quenched with water (50 mL), and extracted with ethyl acetate (50 mL). The organic layer was washed with water and brine, dried over azSC , filtered, and concentrated. The residue was purified by preparative TLC (DCM : MeOH = 10 : 1) to afford intermediate tert-butyl N-[2-[(2S,5R,8S,l lS,14S,17S,22S)-22-[[(2S)-2-acetamido-5-(benzyloxycarbonyl amino)pentanoyl] amino] -5 -benzyl- 11 , 14-bis [2-(tert-butoxycarbonylamino)ethyl] -17-[(1R)-1- hydroxyethyl]-8-isobutyl-3,6,9, 12, 15, 18,23-heptaoxo-l,4,7, 10, 13, 16, 19-heptazacyclotricos-2- yl]ethyl]carbamate 88-3 (300 mg, 0.22 mmol, 58.9 % yield). LC-MS (LC method 3): m/z 1353 (M+H)⁺.

[0241] Step 3. Synthesis of Intermediate 88-4, tert-butyl N-[2-[(2S,5R,8S,l lS,14S,17S,22S)-22- [[(2S)-2-acetamido-5-amino-pentanoyl]amino]-5-benzyl-l l, 14-bis[2-(tert-butoxy carbonylamino)ethyl]- 17-[(lR)-l-hydroxyethyl]-8-isobutyl-3,6,9, 12J

yl]ethyl]carbamate. To a solution of tert-butyl N-[2-[(2S,5R,8S, l lS,14S,17S,22S)-22-[[(2S)-2- acetamido-5 -(benzyl oxycarbonylamino)pentanoyl]amino] -5 -benzyl- 1 1 , 14-bis [2-(tert- butoxycarbonylamino)ethyl] -17-[(1R)-1 -hydroxyethyl] -8-isobutyl-3 ,6,9, 12, 15, 18,23 -heptaoxo- 1,4,7, 10, 13, 16, 19-heptazacyclotricos-2-yl]ethyl] carbamate (300 mg, 0.22 mmol) in ethanol (15 niL), was added Pd/C (30 mg, 0.22 mmol). The resulting mixture was stirred at room temperature under a hydrogen balloon overnight. The catalyst was filtered off through celite and the filtrate was concentrated. The residue was purified by preparative HPLC (eluting with CHsCN/Water, modifier : formic acid) to afford intermediate tert-butyl N-[2-[(2S,5R,8S, l l S, 14S, 17S,22S)-22-[[(2S)-2-acetamido-5-amino- pentanoyl] amino] -5 -benzyl- 1 1 , 14-bis [2-(tert-butoxycarbonylamino)ethyl] -17-[(1R)-1 -hydroxyethyl] -8- isobutyl-3,6,9, 12, 15, 18,23-heptaoxo-l,4,7, 10, 13, 16, 19-heptazacyclotricos-2-yl]ethyl]carbamate, 88-4 (120 mg, 0.09 mmol, 42.2 % yield). LC-MS (LC method 3): m/z 1219 (M+H)⁺.

[0242] Step 4. Synthesis of Intermediate 88-5, tert-butyl N-[N'-[(4S)-4-acetamido-5- [[(3S,6S,9S, 12S, 15R, 18S,21S)-15-benzyl-6,9, 18-tris[2-(tert-butoxycarbonylamino)ethyl]-3-[(lR)-l- hydroxyethyl]-12-isobutyl-2,5,8, 1 1, 14, 17,20-heptaoxo- 1,4,7, 10, 13, 16, 19-heptazacyclotricos-21- yl]amino]-5-oxo-pentyl]-N-tert-butoxycarbonyl-carbamimidoyl]carbamate. To a solution of tert-butyl N- [2-[(2S,5R,8S, l l S, 14S, 17S,22S)-22-[[(2S)-2-acetamido-5-amino-pentanoyl]amino]-5-benzyl-l l, 14- bis [2-(tert-butoxycarbonyl amino)ethyl] -17-[(1R)-1 -hydroxyethyl] -8-isobutyl-3 ,6,9, 12, 15, 18,23- heptaoxo-l,4,7, 10, 13, 16, 19-heptazacyclotricos-2-yl]ethyl] carbamate ( 150.mg, 0.1200mmol) and tert- butyl (NE)-N-[(tert-butoxycarbonylamino)-pyrazol-l-yl-methylene]carbamate (38.21 mg, 0.12 mmol) in DCM (5 mL), was added TEA (0.03 mL, 0.25 mmol). The resulting mixture was stirred at room temperature overnight. The crude mixture was concentrated to dryness, and the residue was purified with preparative HPLC (eluting with G¾CN/Water, modifier formic acid) to afford the intermediate tert-butyl N-[N'-[(4S)-4-acetamido-5-[[(3 S,6S,9S, 12S, 15R, 18S,21 S)-15-benzyl-6,9, 18-tris[2-(tert- butoxycarbonylamino)ethyl] -3 - [( 1 R)- 1 -hydroxyethyl] - 12-isobutyl-2,5 , 8, 1 1, 14, 17,20-heptaoxo- 1, 4, 7, 10, 13, 16, 19-heptazacyclotricos-21 -yl]amino] -5 -oxo-pentyl] -N-tert-butoxycarbonyl- carbamimidoyl]carbamate 88-5 (50 mg, 0.03mmol, 26.4 % yield) . LC-MS (LC method 1): m/z 1461 (M+H)⁺.

[0243] Step 5. Compound 88, (2S)-2-acetamido-5-guanidino-N-[(3S,6S,9S, 12S, 15R, 18S,21S)- 6,9, 18-tris(2-aminoethyl)-15-benzyl-3-[(lR)- l-hydroxyethyl]-12-isobutyl-2,5,8, l l, 14, 17,20-heptaoxo- l,4,7, 10, 13, 16, 19-heptazacyclotricos-21-yl]pentanamide, was prepared from Intermediate 88-5 according to the deprotection sequence described for Compound 71. LCMS: rt = 0.56 (LC method 3). m/z = 961 (M+l). 'H NMR (400 MHz, D₂0) δ 8.25 (s, 1H), 7.34- 7.25 (m, 3H), 7.20-7.18 (m, 2H), 4.54 (q, J= 8.0 Hz, 1H), 4.42-4.33 (m, 1H), 4.27-4.09 (m, 5H), 3.31-3.25 (m, lH), 3.15-3.13 (m, 2H), 3.07-2.99 (m, 5H), 2.81-2.79 (m, 1H), 2.72-2.65 (m, 1H), 2.28-2.07 (m, 4H), 1.97-1.94 (s, 3H), 1.89-1.61 (m, 5H), 1.44-1.25 (m, 2H), 1.13 (d, J= 8.0 Hz, 3H), 0.78-0.75 (m, 1H), 0.71 (t, J= 4.0 Hz, 3H), 0.65 (t, J= 8.0 Hz, 3H).

Example 89 Scheme

[0244] Step 1. Synthesis of ilntermediate tert-butyl 2,2',2"-((2S,5R,8S, l lS,14S,17S,22S)-5- benzyl-22-((R)-2-hydroxy-2-phenylacetamido)-17-((R)-l-hydroxyethyl)-8-isobutyl-3,6,9,12, 15, 18,23- heptaoxo- 1,4,7, 10,13, 16, 19-heptaazacyclotricosane-2, 1 1 , 14-triyl)tris(ethane-2, 1 -diyl)tricarbamate (89-2) . To a solution of (2S)-2-hydroxy-2-phenyl-acetic acid (23 mg, 0.15 mmol) in anhydrous DMF (3 mL) was added HBTU (37 mg, 0.10 mmol) and DIPEA (0.04 mL, 0.23 mmol). Then tert-butyl N-[2- [(2S,5R,8S, 1 IS, 14S, 17S,22S)-22-amino-5-benzyl-l 1, 14-bis[2-(tert-butoxycarbonylamino)ethyl]-17- [(lR)-l-hydroxyethyl]-8-isobutyl-3,6,9,12, 15,18,23-heptaoxo-l,4,7, 10, 13, 16, 19-heptazacyclotricos-2- yl] ethyl] carbamate (80 mg, 0.08 mmol) was added. The resulting mixture was stirred at room temperature for 2 h. The crude mixture was then quenched with water (10 mL), and extracted with ethyl acetate (10 mL x 2). The organic phases were combined and washed with brine (10 mL), dried over anhydrous Na2SC>4, filtered, and concentrated. The residue was purified by preparative HPLC (acidic condition, modifier: HCOOH) to give the title compound intermediate 89-2 (23 mg, 25.5 % yield). LCMS: rt = 2.03 minutes (LC method 1), 1197.7 (M+l)⁺.

[0245] Step 2. Synthesis of Compound 89, (R)-2-hydroxy-2-phenyl-N- ((3S,6S,9S, 12S, 15R, 18S,21S)-6,9,18-tris(2-aminoethyl)-15-benzyl-3-((R)-l-hydroxyethyl)-12-isobutyl- 2,5,8,11, 14,17,20-heptaoxo- 1,4,7, 10,13, 16, 19-heptaazacyclotricosan-21 -yl)acetamide ) .

[0246] To a solution of Intermediate 89-2, tert-butyl N-[2-[(2S,5R,8S,l lS, 14S, 17S,22S)-5- benzyl- 11 , 14-bis [2-(tert-butoxycarbonylamino)ethyl] -17-[(1R)-1 -hydroxyethyl] -22-[ [(2R)-2-hydroxy-2- phenyl-acetyl]amino]-8-isobutyl-3,6,9, 12,15, 18,23-heptaoxo-l,4,7, 10, 13, 16, 19-heptazacyclotricos-2- yl] ethyl] carbamate (23 mg, 0.02 mmol), in DCM (2 mL), was added TFA (0.4 mL, 5.39 mmol) dropwise at 0 °C. The resulting mixture was stirred at room temperature for 2 h. The crude mixture was then concentrated to dryness and re-dissolved in 10% aq. formic acid solution (5 mL). The solvent was removed under vacuum and the solid was freeze-dried to give Compound 89, (2R)-2-hydroxy-2-phenyl- N (3S,6S,9S,12S,15R, 18S,21S)-6,9,18-tris(2-aminoethyl)-15-benzyl-3-[(lR)-l-hydroxyethyl]-12- isobutyl-2,5,8, l l,14, 17,20-heptaoxo-l,4,7, 10,13, 16,19-heptazacyclotricos-21-yl]acetamide, formic acid (9 mg, 48.7% yield), m/z = 896.5 (M+l)⁺. ¾ NMR (400 MHz, D₂0) 5 8.19 (s, 1H), 7.38 (s, 5H), 7.35 - 7.27 (m, 3H), 7.18 (d, J= 7.2 Hz, 2H), 5.16 (s, 1H), 4.47 - 4.41 (m, 2H), 4.27 - 4.16 (m, 3H), 4.09 (m, 3H), 3.29 (m, 1H), 3.00 (m, 7H), 2.74 (s, 1H), 2.64 (s, lH), 2.14 (s, 3H), 2.05 (m, lH), 1.90 (s, 3H), 1.76 (m, 1H), 1.34 (m, 3H), 1.17 (s, 1H), 1.11 (d, J= 5.3 Hz, 4H), 0.69 (s, 1H), 0.68 (s, 3H), 0.59 (s, 3H).

90

[0247] (S)-2-hydroxy-2-phenyl-N-((3S,6S,9S, 12S, 15R, 18S,21S)-6,9, 18-tris(2-aminoethyl)-15- benzyl-3-((R)-l-hydroxyethyl)-12-isobutyl-2,5,8,l l, 14,17,20-heptaoxo-l,4,7, 10, 13,16, 19- heptaazacyclotricosan-21-yl)acetamide, Compound 90, was prepared according to the coupling then deprotection sequence described for Compound 89. LCMS: it = 0.19 (LC method 2). m/z = 896.5 (M+l). 'H NMR (400 MHz, D₂0) δ 8.17 (s, 1H), 7.40 (s, 5H), 7.34 (m, 2H), 7.30 - 7.27 (m, 1H), 7.20 (d, J= 73 Hz, 2H), 5.18 (s, 1H), 4.51 - 4.41 (m, 2H), 4.27 (m, lH), 4.22 (m, 2H), 4.13 - 4.02 (m, 3H), 3.39 (m, 1H), 3.11 - 3.05 (m, 2H), 3.05 - 2.99 (m, 3H), 2.96 (m, 2H), 2.79 - 2.70 (m, lH), 2.63 - 2.56 (m, 1H), 2.25 (m, 1H), 2.17 (m, 2H), 2.05 (m, 1H), 1.95 (m, 3H), 1.77 (m, 1H), 1.37 (m, 2H), 1.1 1 (d, J= 5.8 Hz, 3H), 0.72 - 0.66 (m, 3H), 0.62 (m, 1H), 0.59 (d, J= 5.4 Hz, 3H).

91

[0248] (S)-2-amino-2-(piperidin-4-yl)-N-((3S,6S,9S, 12S, 15R, 18S,21S)-6,9,18-tris(2- aminoethyl)-15-benzyl-3-((R)-l-hydroxyethyl)-12-isobutyl-2,5,8,l l, 14, 17,20-heptaoxo- l,4,7,10, 13,16, 19-heptaazacyclotricosan-21-yl)acetamide, Compound 91, was prepared according to the coupling then deprotection sequence described for Compound 89. LCMS: rt = 0.27 (LC method 3). m/z = 902 (M+l). ¾ NMR (400 MHz, D₂0) δ 7.34-7.5 (m, 3H), 7.19 (d, J= 6.8 Hz, 2H), 4.54 - 4.41 (m, 2H), 4.29 - 4.07 (m, 6H), 3.87 (d, J= 6.8 Hz, 1H), 3.52 - 3.43 (m, 2H), 3.41 - 3.31 (m, 1H), 3.14 - 2.92 (m, 9H), 2.89 - 2.81 (m, 1H), 2.79 - 2.70 (m, 1H), 2.27 - 2.07 (m, 5H), 2.04 - 1.81 (m, 6H), 1.68 - 1.49 (m, 2H), 1.46 - 1.26 (m, 2H), 1.13 (d, J = 6.2 Hz, 3H), 0.75 - 0.56 (m, 7H).

92

[0249] 2-amino-2-(piperidin-4-ylidene)-N-((3 S,6S,9S, 12S, 15R, 18S,21 S)-6,9, 18-tris(2- aminoethyl)-15-benzyl-3-((R)-l-hydroxyethyl)-12-isobutyl-2,5,8,l l, 14, 17,20-heptaoxo- l,4,7,10, 13,16, 19-heptaazacyclotricosan-21-yl)acetamide, Compoud 92, was prepared according to the coupling then deprotection sequence described for Compound 89. LCMS: rt = 0.26 (LC method 3). m/z = 901 (M+l). 'H NMR (400 MHz, D₂0) δ 7.33-7.25 (m, 3H), 7.19 (d, J= 6.8 Hz, 2H), 4.50-4.40 (m, 2H), 4.28 - 4.04 (m, 6H), 3.50 - 3.28 (m, 3H), 3.12 - 2.93 (m, 8H), 2.92 - 2.63 (m, 3H), 2.23-2.04 (m, 5H), 2.02 - 1.73 (m, 6H), 1.60-1.49 (m, 1H), 1.45 - 1.28 (m, 2H), 1.12 (dd, J= 5.8, 4.0 Hz, 3H), 0.82 - 0.55 (m, 7H).

93

[0250] (2S,4R)-4-acetamido-N-((3S,6S,9S,12S,15R, 18S,21S)-6,9, 18-tris(2-aminoethyl)-15- benzyl-3-((R)-l-hydroxyethyl)-12-isobutyl-2,5,8,l l, 14,17,20-heptaoxo-l,4,7, 10, 13,16, 19- heptaazacyclotricosan-21-yl)pyrrolidine-2-carboxamide, Compound 93, was prepared according to the coupling then deprotection sequence described for Compound 89. LCMS: rt = 0.33/0.51 (LC method 3, double peak), m z = 952 (M+l). ¾ NMR (400 MHz, D₂0) δ 8.72 (d, J= 6.6 Hz, 2H), 7.98 (d, J= 6.6 Hz, 2H), 7.38 (dq, J= 14.4, 7.0 Hz, 3H), 7.28 (d, J= 7.0 Hz, 2H), 4.84 (d, J= 4.8Hz, 1H), 4.61 (t, J= 8.2 Hz, 1H), 4.51 (dd, J= 8.8, 5.4 Hz, 1H), 4.37 - 4.17 (m, 6H), 3.54 (dd, J= 14.4, 4.8 Hz, 1H), 3.37-3.25(m, 2H), 3.20 - 3.05 (m, 7H), 2.95 - 2.87 (m, 1H), 2.86 - 2.76 (m, lH), 2.32 - 2.12 (m, 4H), 2.07 - 1.87 (m, 7H), 1.55 - 1.39 (m, 2H), 1.21 (d, J= 6.2 Hz, 3H), 0.92 - 0.83 (m, 1H), 0.80 (t, J= 7.2 Hz, 3H), 0.72 (t, J = 5.8 Hz, 3H).

94

[0251] (S)-5-oxo-N-((3S,6S,9S,12S,15R,18S,21S)-6,9, 18-tris(2-aminoethyl)-15-benzyl-3-((R)- 1 -hydroxyethyl)- 12-isobutyl-2,5 , 8, 11 , 14, 17,20-heptaoxo- 1 ,4,7, 10, 13 , 16, 19-heptaazacyclotricosan-21 - yl)pyrrolidine-2-carboxamide, Compound 94, was prepared according to the coupling then deprotection sequence described for Compound 89. LCMS: rt = 0.46/2.84 (LC method 3, double peak), m z = 873 (M+l). ¾ NMR (400 MHz, D₂0) δ 8.17 (s, 1H), 7.31 (m, 3H), 7.19 (d, J= 7.1 Hz, 2H), 4.47 (m, 2H), 4.32 (m, 1H), 4.25 (m, 2H), 4.18 - 4.03 (m, 4H), 3.44 - 3.29 (m, lH), 3.1 1 - 2.93 (m, 7H), 2.84 (m, 1H), 2.74 (m, 1H), 2.49 (m, 1H), 2.36 (m, 2H), 2.16 (m, 4H), 2.02 - 1.93 (m, 2H), 1.91 - 1.78 (m, 3H), 1.35 (m, 2H), 1.12 (d, J= 6.1 Hz, 3H), 0.70 (d, J= 8.1 Hz, 1H), 0.68 (s, 3H), 0.60 (s, 3H).

95

[0252] (R)-5-oxo-N-((3S,6S,9S,12S,15R,18S,21S)-6,9,18-tris(2-aminoethyl)-15-benzyl-3-((R)- 1 -hydroxyethyl)- 12-isobutyl-2,5 , 8, 11 , 14, 17,20-heptaoxo- 1 ,4,7, 10, 13 , 16, 19-heptaazacyclotricosan-21 - yl)pyrrolidine-2-carboxamide, Compound 95, was prepared according to the coupling then deprotection sequence described for Compound 89. LCMS: rt = 0.35 (LC method 3). m z = 873 (M+l). ¾ NMR (400 MHz, D₂0) δ 8.15 (s, 1H), 7.31 (m, 3H), 7.19 (d, J= 7.2 Hz, 2H), 4.48 (m, 2H), 4.29 (m, 2H), 4.22 (m, 1H), 4.10 (m, 4H), 3.39 (m, 1H), 3.11 - 2.93 (m, 7H), 2.89 - 2.79 (m, lH), 2.74 (m, lH), 2.53 - 2.43 (m, 1H), 2.36 (m, 2H), 2.26 - 2.10 (m, 3H), 2.02 (m, 3H), 1.85 (m, 3H), 1.35 (m, 2H), 1.12 (d, J= 5.9 Hz, 3H), 0.71 (d, J= 3.2 Hz, 1H), 0.68 (s, 3H), 0.61 (s, 3H).

96

[0253] (S)-4-amino-2-(3,3,3-trifluoropropanamido)-N-((3S,6S,9S, 12S, 15R, 18S,21S)-6,9, 18- tris(2-aminoethyl)-15-benzyl-3-((R)-l-hydroxyethyl)-12-isobutyl-2,5,8,l l, 14,17,20-heptaoxo- l,4,7,10, 13,16, 19-heptaazacyclotricosan-21-yl)butanamide, Compound 96, was prepared according to the coupling then deprotection sequence described for Compound 89. LCMS: rt = 0.34 (LC method 3). m/z = 972 (M+l). ¾ NMR (400 MHz, D₂0) δ 8.31 (s, 2H), 7.31 (m, 3H), 7.19 (d, J= 6.9 Hz, 2H), 4.51 (m, 1H), 4.42 (m, 1H), 4.37 (m, 1H), 4.13 (m, 7H), 3.30 (m, 3H), 3.09 - 3.01 (m, 7H), 2.99 (m, 5H), 2.79 (m, 1H), 2.75 - 2.66 (m, 1H), 2.64 (s, 2H), 2.15 (m, 7H), 1.96 - 1.79 (m, 4H), 1.38 (m, 2H), 1.19 (m, 5H), 1.12 (d, J= 6.1 Hz, 3H), 0.83 - 0.73 (m, 1H), 0.70 (d, J= 6.3 Hz, 3H), 0.62 (d, J= 6.2 Hz, 3H).

97

[0254] N-((S)-4-amino-l-oxo-l-((3S,6S,9S, 12S,15R,18S,21S)-6,9,18-tris(2-aminoethyl)-15- benzyl-3-((R)-l-hydroxyethyl)-12-isobutyl-2,5,8,l l, 14,17,20-heptaoxo-l,4,7, 10, 13,16, 19- heptaazacyclotricosan-21-ylamino)butan-2-yl)benzamide, Compound 97, was prepared according to the coupling then deprotection sequence described for Compound 59. LCMS: rt = 2.35 (LC method 3). m/z = 966 (M+l). 'H NMR (400 MHz, D₂0) δ 8.15 (s, 1H), 7.74 (d, J= 7.5 Hz, 2H), 7.58 (d, J= 7.1 Hz, 1H), 7.48 (m, 2H), 7.39 - 7.09 (m, 5H), 4.52 (m, 3H), 4.41 (m, lH), 4.15 (m, 6H), 3.37 - 3.25 (m, lH), 3.21 - 2.85 (m, 12H), 2.86 - 2.54 (m, 2H), 2.33 - 1.62 (m, 11H), 1.51 - 1.29 (m, 2H), 1.20 (t, J= 7.3 Hz, 3H), 1.10 (d, J= 6.0 Hz, 3H), 0.78 (s, 1H), 0.70 (d, J= 6.0 Hz, 3H), 0.63 (d, J= 5.9 Hz, 3H). 98

[0255] N-((S)-4-amino-l-oxo-l-((3S,6S,9S,12S,15R,18S,21S)-6,9,18-tris(2-aminoethyl)-15- benzyl-3-((R)-l-hydroxyethyl)-12-isobutyl-2,5,8,ll,14,17,20-heptaoxo-l,4,7,10,13,16,19- heptaazacyclotricosan-21-ylamino)butan-2-yl)isonicotinamide, Compound 98, was prepared according to the coupling then deprotection sequence described for Compound 89. LCMS: rt = 3.38 (LC method 3). m/z = 968 (M+l). ¾ NMR (400 MHz, D₂0) δ 9.04 (s, 1H), 8.81 (d, J= 5.4 Hz, 1H), 8.61 (d, J= 8.3 Hz, 1H), 8.17 (s, 1H), 7.89 (dd, J= 8.0, 5.6 Hz, 1H), 7.29 (m, 3H), 7.19 (m, 2H), 4.59 (m, lH), 4.52 (t, J= 8.2 Hz, 1H), 4.43 (m, 1H), 4.32 - 4.01 (m, 6H), 3.36 - 3.21 (m, lH), 3.18 - 2.90 (m, 9H), 2.87- 2.59 (m, 2H), 2.33 - 1.66 (m, 10H), 1.49- 1.25 (m, 2H), 1.11 (d,J=6.1 Hz, 3H), 0.83 -0.73 (m, 1H), 0.70 (d,J= 6.3 Hz, 3H), 0.63 (d, J= 6.1 Hz, 3H).

99

[0256] (2S,4S)-4-amino-N-((S)-4-amino-l-oxo-l-((3S,6S,9S,12S,15R,18S,21S)-6,9,18-tris(2- aminoethyl)-15-benzyl-3-((R)-l-hydroxyethyl)-12-isobutyl-2,5,8,ll,14,17,20-heptaoxo- 1, 4, 7, 10,13, 16,19-heptaazacyclotricosan-21 -ylamino)butan-2-yl)-5 -oxopyrrolidine-2-carboxamide,

Compound 99, was prepared according to the coupling then deprotection sequence described for

Compound 89. LCMS: rt = 0.28/0.43 (LC method 3, double peak), mz = 888 (M+l). ¾ NMR (400 MHz, D₂0) δ 7.29 (m, 3H), 7.19 (d, J= 6.8 Hz, 2H), 4.55 - 4.43 (m, 2H), 4.36 (dd, J= 8.6, 7.0 Hz, 1H), 4.28 - 4.07 (m, 6H), 3.95 (dd, J= 10.8, 4.0 Hz, 1H), 3.40 - 3.28 (m, 1H), 3.16 - 2.90 (m, 8H), 2.88 - 2.69 (m, 2H), 2.25 - 2.05 (m, 4H), 2.03 - 1.83 (m, 4H), 1.46 - 1.24 (m, 3H), 1.12 (d, J= 6.2 Hz, 3H), 0.79 - 0.51 (m, 7H).

100

[0257] (S)-2-amino-4-(methylsulfonyl)-N-((3S,6S,9S, 12S, 15R, 18S,21S)-6,9, 18-tris(2- aminoethyl)- 15 -benzyl-3 -((R)- 1 -hydroxyethyl)- 12-isobutyl-2,5 , 8, 11 , 14, 17,20-heptaoxo- l,4,7,10, 13,16, 19-heptaazacyclotricosan-21-yl)butanamide, Compound 100, was prepared according to the coupling then deprotection sequence described for Compound 89. LCMS: rt = 0.35 (LC method 3). m/z = 925 (M+l). ¾ NMR (400 MHz, D₂0) δ 8.17 (s, 1H), 7.34- 7.27 (m, 3H), 7.20-7.19 (m, 2H), 5.12 (s, 1H), 4.54 (t, J = 8.0 Hz, 1H), 4.45 (t, J = 4.0 Hz, 1H),4.31 (t, J = 8.0 Hz, lH), 4.21-4.08 (m, 5H), 3.41- 3.37 (m, 3H), 3.08-2.93 (m, 10H), 2.86-2.64 (m, 3H), 2.41 (m, 2H), 2.22-2.05 (m, 4H), 1.95-1.80 (m, 4H), 1.41-1.31(m, 2H), 1.22-1.19 (m, 1H), 1.13 (d, J = 4.0 Hz, 3H), 0.81-0.75 (m, lH), 0.71 (d, J = 8.0 Hz, 3H), 0.64 (d, J = 8.0 Hz, 3H).

ND 101

[0258] N-((S)-4-amino-l-oxo-l-((3S,6S,9S, 12S,15R,18S,21S)-6,9,18-tris(2-aminoethyl)-15- benzyl-3-((R)-l-hydroxyethyl)-12-isobutyl-2,5,8,l l, 14,17,20-heptaoxo-l,4,7, 10, 13,16, 19- heptaazacyclotricosan-21-ylamino)butan-2-yl)cyclopropanecarboxamide, Compound 101, was prepared according to the coupling then deprotection sequence described for Compound 89. LCMS: rt = 0.404 (LC method 3). m/z = 930 (M+l). ¾ NMR (400 MHz, D₂0) 5 8.15 (s, 1H), 7.29 (m, 3H), 7.19 (d, J = 6.9 Hz, 2H), 4.51 (m, 1H), 4.41 (m, 1H), 4.32 (m, 1H), 4.28 - 4.04 (m, 6H),.27 (m, 1H), 3.1 1 - 2.93 (m, 9H), 2.79 (m, 1H), 2.70 (m, 1H), 2.27 - 1.72 (m, 10H), 1.62 (m, 1H), 1.46 - 1.29 (m, 2H), 1.12 (d, J = 6.1 Hz, 3H), 0.79 (m, 5H), 0.70 (d, J = 6.3 Hz, 3H), 0.62 (d, J = 6.2 Hz, 3H).

102

[0259] (S)-2-acetamido-3-(lH-benzo[d]imidazol-2-yl)-N-((3S,6S,9S,12S,15R,18S,21S)-6,9, 18- tris(2-aminoethyl)- 15 -benzyl-3 -((R)- 1 -hydroxyethyl)- 12-isobutyl-2,5 , 8, 11 , 14, 17,20-heptaoxo- l,4,7,10, 13,16, 19-heptaazacyclotricosan-21-yl)propanamide, Compound 102, was prepared according to the coupling then deprotection sequence described for Compound 89. LCMS: rt = 0.40 (LC method 3). m/z = 991 (M+l). ¾ NMR (400 MHz, D₂0) δ 8.16 (s, 1H), 7.70 (m, 2H), 7.53 (m, 2H), 7.31 (m, 3H), 7.19 (d, J = 6.8 Hz, 2H), 4.87 (m, 1H), 4.52 (m, 1H), 4.40 (m, lH), 4.25 - 4.05 (m, 6H), 3.64 (m, lH), 3.54 (m, 1H), 3.21 (m, 1H), 3.07 - 2.93 (m, 7H), 2.78 (m, lH), 2.68 (m, lH), 2.14 (m, 4H), 1.95 - 1.73 (m, 7H), 1.39 (m, 2H), 1.10 (d, J = 6.2 Hz, 3H), 0.80 (s, lH), 0.70 (m, 3H), 0.63 (m, 3H).

COMPOUND 103

[0260] 2-amino-2-methyl-N-((3 S,6S,9S, 12S, 15R, 18S,21 S)-6,9, 18-tris(2-aminoethyl)- 15-benzyl- 3-((R)-l-hydroxyethyl)-12-isobutyl-2,5,8, l l,14, 17,20-heptaoxo-l,4,7, 10, 13,16, 19-heptaazacyclotricosan- 21-yl)propanamide, Compound 103, was prepared according to the coupling then deprotection sequence described for Compound 89. LCMS: rt = 0.40/0.66 (LC method 3). m z = 847 (M+l). ¾ NMR (400 MHz, D₂0) δ 7.29 (m, 3H), 7.19 (d, J = 7.8 Hz, 2H), 4.55 - 4.42 (m, 2H), 4.29 - 4.08 (m, 6H), 3.38 - 3.26 (m, 1H), 3.14 - 2.93 (m, 7H), 2.88 - 2.77 (m, 1H), 2.76 - 2.64 (m, lH), 2.23 - 2.05 (m, 4H), 1.99 - 1.78 (m, 4H), 1.57 (d, J = 9.6 Hz, 6H), 1.37 (m, 2H), 1.12 (d, J = 6.2 Hz, 3H), 0.82 - 0.55 (m, 7H). D 104

[0261] (R)-2-acetamido-2-(piperidin-4-yl)-N-((3 S,6S,9S, 12S, 15R, 18S,21 S)-6,9, 18-tris(2- aminoethyl)- 15 -benzyl-3 -((R)- 1 -hydroxyethyl)- 12-isobutyl-2,5 , 8, 11 , 14, 17,20-heptaoxo- l,4,7,10, 13,16, 19-heptaazacyclotricosan-21-yl)acetamide, Compound 104, was prepared according to the coupling then deprotection sequence described for Compound 71. LCMS: rt = 0.29/0.45 (LC method 3). m z = 944(M+1). ¾ NMR (400 MHz, D₂0) δ 7.29 (m, 3H), 7.19 (d, J = 6.8 Hz, 2H), 4.51 (t, J = 8.2 Hz, 1H), 4.41 (dd, J = 8.8, 5.4 Hz, 1H), 4.27 - 4.08 (m, 7H), 3.42 (d, J = 13.2 Hz, 2H), 3.34 - 3.22 (m, 1H), 3.13 - 2.89 (m, 9H), 2.86 - 2.77 (m, 1H), 2.71 (dt, J = 19.2, 9.8 Hz, lH), 2.23 - 1.80 (m, 14H), 1.57 - 1.30 (m, 5H), 1.12 (d, J = 6.0 Hz, 3H), 0.79 - 0.59 (m, 7H).

ND 105

[0262] (S)-2-acetamido-2-(piperidin-4-yl)-N-((3 S,6S,9S, 12S, 15R, 18S,21 S)-6,9, 18-tris(2- aminoethyl)-15-benzyl-3-((R)-l-hydroxyethyl)-12-isobutyl-2,5,8,l l, 14, 17,20-heptaoxo- l,4,7,10, 13,16, 19-heptaazacyclotricosan-21-yl)acetamide, Compound 105, was prepared according to the coupling then deprotection sequence described for Compound 89. LCMS: rt = 0.29/0.47 (LC method 3). m z = 944 (M+l). ¾ NMR (400 MHz, D₂0) δ 7.21 (m, 3H), 7.11 (d, J = 6.8 Hz, 2H), 4.45 (t, J = 8.2 Hz, 1H), 4.24 (m, 2H), 4.16 - 3.96 (m, 6H), 3.28 (m, 3H), 3.02 - 2.81 (m, 9H), 2.78 - 2.69 (m, 1H), 2.65 - 2.56 (m, 1H), 2.19 - 2.00 (m, 3H), 1.99 - 1.66 (m, 11H), 1.52 - 1.18 (m, 5H), 1.04 (d, J = 6.2 Hz, 3H), 0.81 - 0.69 (m, 1H), 0.60 (m, 6H).

Example 106 Scheme

[0263] Step 1. Intermediate tert-butyl 4-((S)-4-((3S,6S,9S, 12S,15R, 18S,21 S)-15-benzyl-6,9, 18- tris(2-(tert-butoxycarbonylamino)ethyl)-3 -(®- 1 -hydroxy ethyl)- 12-isobutyl-2,5 , 8, 1 1, 14, 17,20-heptaoxo-

1.4.7.10.13.16.19- heptaazacyclotricosan-21 -ylamino)-3 -(tert-butoxycarbonylamino)-4- oxobutanoyl)piperazine-l-carboxylate (106-2). To a solution of (3S)-4-[[(3S,6S,9S, 12S, 15R, 18S,21 S)- 15 -benzyl-6,9, 18-tris [2-(tert-butoxycarbonylamino)ethyl] -3 -[( 1 R)- 1 -hydroxyethyl] - 12-isobutyl-

2.5.8.1 1.14.17.20- heptaoxo-l,4,7, 10, 13, 16, 19-heptazacyclotricos-21-yl]amino]-3-(tert- butoxycarbonylamino)-4-oxo-butanoic acid (80 mg, 0.06 mmol) in anhydrous DMF (lOmL) was added HBTU (36 mg, 0.09 mmol) and DIPEA (0.03 mL, 0. 1900mmol). Then tert-butyl piperazine-1- carboxylate (23 mg, 0.13 mmol) was added. The resulting mixture was stirred at room temperature for 5 h. The crude mixture was quenched with water (50 mL x 2), extracted with ethyl acetate (80 mL x 2), the combined organic phases were washed with brine (50 mL), dried over anhydrous Na2S04, filtered and concentrated. The residue was purified by preparative HPLC to give the compound intermediate 106-2. LCMS: rt = 2.54 minutes (LC method 1), 1446 (M+l)⁺. [0264] Step 2. Synthesis of Compound 106, (S)-2-amino-4-oxo-4-(piperazin-l-yl)-N- ((3S,6S,9S, 12S, 15R, 18S,21S)-6,9,18-tris(2-aminoethyl)-^

2,5,8,11, 14,17,20-heptaoxo- 1,4,7, 10,13, 16, 19-heptaazacyclotricosan-21 -yl)butanamide (106-3)

Compound 106 was prepared from Intermediate 106-2 according to the deprotection sequence described for Compound 89. LCMS: rt = 0.53 (LC method 3). m/z = 945 (M+l). ¾ NMR (400 MHz, D₂0) 5 8.17 (s, 1H), 7.29 (m, 3H), 7.19 (d, J = 6.9 Hz, 2H), 4.52 (m, lH), 4.42 (m, lH), 4.38 - 4.33 (m, lH), 4.27 - 4.10 (m, 6H), 3.81 - 3.68 (m, 4H), 3.28 (m, 5H), 3.19 - 3.13 (m, 2H), 3.02 (m, 7H), 2.86 - 2.77 (m, lH), 2.76 - 2.63 (m, 1H), 2.24 - 2.03 (m, 4H), 1.89 (m, 4H), 1.36 (m, 2H), 1.28 - 1.25 (m, lH), 1.12 (d, J = 6.2 Hz, 3H), 0.76 (m, 1H), 0.71 (m, 3H), 0.63 (m, 3H).

107

[0265] 2-(6-oxopiperazin-2-yl)-N-((3 S,6S,9S, 12S, 15R, 18S,21 S)-6,9, 18-tris(2-aminoethyl)- 15- benzyl-3-((R)-l-hydroxyethyl)-12-isobutyl-2,5,8,l l, 14,17,20-heptaoxo-l,4,7, 10, 13,16, 19- heptaazacyclotricosan-21-yl)acetamide, Compound 107, was prepared according to the coupling then deprotection sequence described for Compound 89. LCMS: rt = 0.27 (LC method 3). m/z = 903 (M+l). ¾ NMR (400 MHz, D₂0) δ 8.19 (s, 1H), 7.37 - 7.23 (m, 3H), 7.19 (m, 2H), 4.54 (q, J = 8.1 Hz, lH), 4.42 - 4.23 (m, 3H), 4.23 - 4.00 (m, 5H), 3.69 - 3.48 (m, 3H), 3.43 (m, lH), 3.37 - 3.26 (m, lH), 3.17 - 2.90 (m, 9H), 2.73 (m, 2H), 2.30 - 1.62 (m, 8H), 1.49 - 1.23 (m, 2H), 1.19 (t, J = 7.3 Hz, lH), 1.11 (d, J = 6.2 Hz, 3H), 0.80 (d, J = 43.1 Hz, 1H), 0.71 (dd, J = 6.5, 2.2 Hz, 3H), 0.64 (dd, J = 6.4, 2.7 Hz, 3H).

COMPOUND 108

[0266] (R)-N-((3S,6S,9S,12S,15R,18S,21S)-6,9, 18-tris(2-aminoethyl)-15-benzyl-3-((R)-l- hydroxyethyl)- 12-isobutyl-2,5 ,8, 11 , 14, 17,20-heptaoxo- 1 ,4,7, 10, 13 , 16, 19-heptaazacyclotricosan-21 - yl)piperidine-2-carboxamide, Compound 108, was prepared according to the coupling then deprotection sequence described for Compound 89. LCMS: rt = 0.27/0.41 (LC method 3). m z = 873 (M+l). ¾ NMR (400 MHz, D₂0) δ 7.29 (dq, J = 14.4, 7.0 Hz, 3H), 7.19 (d, J = 7.0 Hz, 2H), 4.52 (t, J = 8.2 Hz, lH), 4.43 (dd, J = 8.8, 5.6 Hz, 1H), 4.27 - 4.08 (m, 6H), 3.88 (dd, J = 1 1.6, 2.8 Hz, lH), 3.41 (d, J = 12.8 Hz, 1H), 3.35 - 3.24 (m, 1H), 3.15 - 2.94 (m, 8H), 2.85 - 2.76 (m, lH), 2.68 (dd, J = 17.8, 11.0 Hz, 1H), 2.23 - 2.05 (m, 5H), 1.99 - 1.76 (m, 6H), 1.67 - 1.51 (m, 3H), 1.46 - 1.31 (m, 2H), 1.12 (d, J = 6.2 Hz, 3H), 0.85 - 0.76 (m, 1H), 0.70 (d, J = 6.4 Hz, 3H), 0.63 (d, J = 6.2 Hz, 3H).

COMPOUND 109

[0267] (S)-N-((3S,6S,9S,12S,15R,18S,21S)-6,9, 18-tris(2-aminoethyl)-15-benzyl-3-((R)-l- hydroxyethyl)- 12-isobutyl-2,5 ,8, 11 , 14, 17,20-heptaoxo- 1 ,4,7, 10, 13 , 16, 19-heptaazacyclotricosan-21 - yl)piperidine-2-carboxamide, Compound 109, was prepared according to the coupling then deprotection sequence described for Compound 89. LCMS: rt = 0.26/0.40 (LC method 3). m/z = 873 (M+l). ¾ NMR (400 MHz, D₂0) δ 7.29 (dq, J = 14.4, 7.0 Hz, 3H), 7.19 (d, J = 7.0 Hz, 2H), 4.53 (t, J = 8.2 Hz, lH), 4.41 (dd, J = 8.8, 5.4 Hz, 1H), 4.32 - 4.25 (m, 1H), 4.23 - 4.06 (m, 5H), 3.87 (dd, J = 12.2, 3.2 Hz, 1H), 3.46 - 3.29 (m, 2H), 3.12 - 2.93 (m, 8H), 2.85 - 2.77 (m, lH), 2.69 (dd, J = 18.0, 1 1.0 Hz, lH), 2.24 - 2.01 (m, 5H), 1.94-1.78 (m, 6H), 1.71-1.53 (m, 3H), 1.45-1.32 (m, 2H), 1.12 (d, J = 6.0 Hz, 3H), 0.87 - 0.76 (m, 1H), 0.71 (d, J = 6.4 Hz, 3H), 0.64 (d, J = 6.2 Hz, 3H). 110

[0268] (S)-2-amino-3 pyridin-3-yl)-N (3S,6S,9S42S,15R,18S,21S)-6,9,18-tris(2-aminoethyl)- 15-benzyl-3-((R)-l-hydroxyethyl)-12-isobutyl-2,5,8,ll,14,17,20-heptaoxo-l,4,7,10,13,16,19- heptaazacyclotricosan-21-yl)propanamide, Compound 110, was prepared according to the coupling then deprotection sequence described for Compound 89. LCMS: rt = 0.332 (LC method 3). m/z = 910 (M+l). 'HNMR (400 MHz, D₂0) δ 8.77 - 8.68 (m, 2H), 8.52 (d, J = 8.2 Hz, 1H), 8.15 (s, 1H), 8.07 - 7.99 (m, 1H), 7.36 - 7.23 (m, 3H), 7.19 (d, J = 6.9 Hz, 2H), 4.51 (m, lH), 4.43 (m, lH), 4.33 (m, lH), 4.26-4.06 (m, 6H), 3.50-3.32 (m, 2H), 3.21 (m, 1H), 3.04 (m, 7H), 2.88 -2.80 (m, lH), 2.78-2.68 (m, lH), 2.14 (m, 4H), 1.95 (m, 1H), 1.90- 1.78 (m, 2H), 1.74 (m, lH), 1.41 (m, lH), 1.33 (m, 1H), 1.11 (d, J = 6.3 Hz, 3H), 0.72 (m, 1H), 0.68 (d, J = 5.0 Hz, 3H), 0.61 (d, J = 5.6 Hz, 3H).

111

[0269] (8)-2^ίηο-3-(4^οφηο1ίηορηεη 1)-Ν-((3 S,6S,9S, 12S, 15R, 18S,21 S)-6,9, 18-tris(2- aminoethyl)-15-benzyl-3-((R)-l-hydroxyethyl)-12-isobutyl-2,5,8,ll,14,17,20-heptaoxo- l,4,7,10,13,16,19-heptaazacyclotricosan-21-yl)propanamide, Compound 111, was prepared according to the coupling then deprotection sequence described for Compound 89. LCMS: rt = 0.335 (LC method 3). m/z = 995 (M+l). 'HNMR (400 MHz, D₂0) δ 8.15 (s, 1H), 7.47-7.35 (m, 4H), 7.32- 7.27 (m, 3H), 7.20- 7.18 (m, 2H), 5.12 (s, 1H), 4.55 (t, J = 8.0 Hz, 1H), 4.45 (t, J = 4.0 Hz, lH), 4.27-4.10 (m, 8H), 4.07-3.87 (m, 5H), 3.57-3.50 (m, 4H), 3.32-3.20 (m, 2H), 3.08-2.93 (m, 10H), 2.83-2.73 (m, 2H), 2.30-2.15 (m, 4H), 1.95-1.80 (m, 4H), 1.41-1.31 (m, 2H), 1.13 (d, J

Hz, 3H), 0.61 (d, J = 8.0 Hz, 3H).

COMPOUND 112

[0270] (S)-6-oxo-N-((3S,6S,9S,12S,15R,18S,21S)-6,9, 18-tris(2-aminoethyl)-15-benzyl-3-((R)- 1 -hydroxyethyl)- 12-isobutyl-2,5 , 8, 11 , 14, 17,20-heptaoxo- 1 ,4,7, 10, 13 , 16, 19-heptaazacyclotricosan-21 - yl)piperazine-2-carboxamide, Comopound 112, was prepared according to the coupling then

deprotection sequence described for Compound 71. LCMS: rt = 3.521 (LC method 3). m/z = 888 (M+l). 'H NMR (400 MHz, D₂0) δ 8.17 (s, 1H), 7.22 (m, 3H), 7.11 (m, 2H), 4.49 - 4.21 (m, 3H), 4.25 - 3.96 (m, 6H), 3.78 (s, 2H), 3.60 (m, 1H), 3.39 (m, 1H), 3.26 (m, lH), 3.10 - 2.83 (m, 8H), 2.81 - 2.49 (m, 2H), 1.98 (m, 6.0 Hz, 8H), 1.30 (m, 2H), 1.04 (d, J = 6.3 Hz, 3H), 0.64 (m, lH), 0.61 (d, J = 5.9 Hz, 3H), 0.54 (d, J = 6.0 Hz, 3H).

113

[0271] N-((S)-4-amino-l-oxo-l-((3S,6S,9S, 12S,15R,18S,21S)-6,9,18-tris(2-aminoethyl)-15- benzyl-3-((R)-l-hydroxyethyl)-12-isobutyl-2,5,8,l l, 14,17,20-heptaoxo-l,4,7, 10, 13,16, 19- heptaazacyclotricosan-21-ylamino)butan-2-yl)furan-2-carboxamide, Compound 113, was prepared according to the coupling then deprotection sequence described for Compound 89. LCMS: rt = 2.84 (LC method 3). m/z = 956 (M+l). 'H NMR (400 MHz, D₂0) δ 8.16 (s, 1H), 7.64 (d, J = 0.9 Hz, 1H), 7.29 (m, 3H), 7.18 (m, 3H), 6.58 (dd, J = 3.5, 1.7 Hz, 1H), 4.58 - 4.46 (m, 2H), 4.40 (m, lH), 4.31 - 4.02 (m, 6H), 3.39 - 3.22 (m, 1H), 3.15 - 2.87 (m, 9H), 2.85 - 2.59 (m, 2H), 2.31 - 1.62 (m, 1 1H), 1.50 - 1.27 (m, 2H), 1.11 (d, J = 6.2 Hz, 3H), 0.75 (d, J = 21.6 Hz, lH), 0.70 (d, J = 6.3 Hz, 3H), 0.62 (d, J = 6.1 Hz, 3H).

114

[0272] (S)-2-amino-4-(4-methylpiperazin-l-yl)-4-oxo-N-((3S,6S,9S, 12S,15R, 18S,21S)-6,9, 18- tris(2-aminoethyl)- 15 -benzyl-3 -((R)- 1 -hydroxyethyl)- 12-isobutyl-2,5 , 8, 11 , 14, 17,20-heptaoxo- l,4,7,10, 13,16, 19-heptaazacyclotricosan-21-yl)butanamide, Compound 114, was prepared according to the coupling then deprotection sequence described for Compound 71. LCMS: rt = 0.50 (LC method 3). m/z = 960 (M+l). ¾ NMR (400 MHz, D₂0) δ 8.17 (s, 1H), 7.36 - 7.24 (m, 3H), 7.19 (d, J = 6.9 Hz, 2H), 4.52 (m, 3H), 4.35 (m, 1H), 4.17 (m, 6H), 4.06 (d, J = 15.2 Hz, lH), 3.51 (m, 3H), 3.36 - 3.27 (m, lH), 3.06 (m, 12H), 2.88 (s, 3H), 2.80 (m, 1H), 2.71 (m, lH), 2.14 (m, 4H), 1.99 - 1.76 (m, 4H), 1.46 - 1.29 (m, 2H), 1.12 (d, J = 6.2 Hz, 3H), 0.76 (m, 1H), 0.70 (d, J = 6.3 Hz, 3H), 0.63 (d, J = 6.2 Hz, 3H).

115

[0273] (3S)-2-oxo-N-((3S,6S,9S,12S,15R,18S,21S)-6,9, 18-tris(2-aminoethyl)-15-benzyl-3-((R)- 1 -hydroxyethyl)- 12-isobutyl-2,5 , 8, 11 , 14, 17,20-heptaoxo- 1 ,4,7, 10, 13 , 16, 19-heptaazacyclotricosan-21 - yl)spiro[indoline-3,3'-pyrrolidine]-5'-carboxamide, Compound 115, was prepared according to the deprotection sequence described for Compound 89. LCMS: rt = 0.40 (LC method 3). m/z = 977 (M+l). 'H NMR (400 MHz, D₂0) δ 8.10 (s, 1H), 7.23 (m, 5H), 7.11 (d, J = 7.0 Hz, 2H), 7.04 (m, lH), 6.91 (d, J = 7.8 Hz, 1H), 4.83 (m, 1H), 4.46 (m, 1H), 4.22 (m, 3H), 4.12 - 4.02 (m, 2H), 3.97 (m, 2H), 3.67 (m, 2H), 3.38 - 3.25 (m, 1H), 2.94 (m, 7H), 2.70 (m, 2H), 2.39 (m, 2H), 2.16 - 1.91 (m, 4H), 1.82 (m, 4H), 1.29 (m, 2H), 1.05 (d, J = 6.2 Hz, 3H), 0.80 (m, 1H), 0.62 (d, J = 6.5 Hz, 3H), 0.53 (d, J = 6.3 Hz, 3H).

D 116

[0274] (3R)-2-oxo-N-((3S,6S,9S,12S,15R,18S,21S)-6,9, 18-tris(2-aminoethyl)-15-benzyl-3-((R)- 1 -hydroxyethyl)- 12-isobutyl-2,5 , 8, 11 , 14, 17,20-heptaoxo- 1 ,4,7, 10, 13 , 16, 19-heptaazacyclotricosan-21 - yl)spiro[indoline-3,3'-pyrrolidine]-5'-carboxamide, Compound 116, was prepared according to the deprotection sequence described for Compound 89. LCMS: rt = 0.39 (LC method 3). m/z = 977 (M+l). 'H NMR (400 MHz, D₂0) δ 8.10 (s, 1H), 7.23 (m, 5H), 7.11 (d, J = 7.0 Hz, 2H), 7.04 (m, lH), 6.91 (d, J = 7.8 Hz, 1H), 4.83 (m, 1H), 4.46 (m, 1H), 4.22 (m, 3H), 4.12 - 4.02 (m, 2H), 3.97 (m, 2H), 3.67 (m, 2H), 3.38 - 3.25 (m, 1H), 2.94 (m, 7H), 2.70 (m, 2H), 2.39 (m, 2H), 2.16 - 1.91 (m, 4H), 1.82 (m, 4H), 1.29 (m, 2H), 1.05 (d, J = 6.2 Hz, 3H), 0.80 (m, 1H), 0.62 (d, J = 6.5 Hz, 3H), 0.53 (d, J = 6.3 Hz, 3H).

EXAMPLE 117. SYNTHESIS OF COMPOUND 117

[0275] (R)-6-amino-2-(3-methylbutanamido)-N-((3 S,6S,9S, 12S, 15R, 18S,21 S)-6,9, 18-tris(2- aminoethyl)-15-benzyl-3-((R)-l-hydroxyethyl)-12-isobutyl-2,5,8,l l, 14, 17,20-heptaoxo- l,4,7,10, 13,16, 19-heptaazacyclotricosan-21-yl)hexanamide, Compound 117, was prepared according to the coupling then deprotection sequence described for Compound 89. LCMS: rt = 0.38/0.53 (LC method 3). m z = 975 (M+l). ¾ NMR (400 MHz, D₂0) δ 7.33 - 7.20 (m, 3H), 7.15 (d, J = 7.0 Hz, 2H), 4.47 (t, J = 8.2 Hz, 1H), 4.39 (dd, J = 8.6, 5.2 Hz, 1H), 4.20-4.07 (m, 7H), 3.29 - 3.18 (m, lH), 3.12 - 2.83 (m, 10H), 2.80 - 2.60 (m, 2H), 2.23 - 1.98 (m, 6H), 1.95 - 1.68 (m, 6H), 1.65 - 1.50 (m, 3H), 1.36 (dt, J = 11.8, 7.6 Hz, 4H), 1.17 (t, J = 7.4 Hz, 1H), 1.08 (d, J = 5.8 Hz, 3H), 0.81 (dd, J = 6.2, 4.6 Hz, 6H), 0.75 - 0.53 (m, 7H).

118

[0276] ®-6-amino-2-propionamido-N-((3 S,6S,9S, 12S, 15R, 18S,21 S)-6,9, 18-tris(2-aminoethyl)- 15-benzyl-3-(®-l-hydroxyethyl)-12-isobutyl-2,5,8,l l, 14, 17,20-heptaoxo-l,4,7, 10, 13, 16, 19- heptaazacyclotricosan-21-yl)hexanamide, Comopund 118, was prepared according to the coupling then deprotection sequence described for Compound 89. LCMS: rt = 0.44/0.64 (LC method 3). m z = 947 (M+l). ¾ NMR (400 MHz, D₂0) δ 7.21 (m, 3H), 7. 1 1 (d, J = 7.0 Hz, 2H), 4.42 (t, J = 8.2 Hz, 1H), 4.34 (dd, J = 8.8, 5.2 Hz, 1H), 4.20 - 4.01 (m, 7H), 3.25 - 3.15 (m, lH), 3.04 - 2.81 (m, 9H), 2.76 - 2.69 (m, 1H), 2.67 - 2.57 (m, 1H), 2.20 - 1.95 (m, 6H), 1 .93 - 1.65 (m, 5H), 1.62 - 1.49 (m, 3H), 1.40 - 1.23 (m, 4H), 1.04 (d, J = 6.0 Hz, 3H), 0.94 (t, J = 7.6 Hz, 3H), 0.72 - 0.59 (m, 4H), 0.54 (d, J = 6.0 Hz, 3H).

1 19

[0277] (S)-2-amino-3-( lH-pyrazol-5-yl)-N-((3 S,6S,9S, 12S, 15R, 18S,21 S)-6,9, 18-tris(2- aminoethyl)-15-benzyl-3-(®-l-hydroxyethyl)-12-isobutyl-2,5,8,l l, 14, 17,20-heptaoxo-l,4,7, 10, 13, i heptaazacyclotricosan-21-yl)propanamide, Compound 119, was prepared according to the coupling then deprotection sequence described for Compound 89. LCMS: rt = 0.28/0.42 (LC method 3). m/z = 899 (M+l). ¾ NMR (400 MHz, D20) δ 7.58 (d, J = 2.3 Hz, 1H), 7.22 (dq, J = 14.2, 7.0 Hz, 3H), 7.12 (d, J = 6.8 Hz, 2H), 6.21 (d, J = 2.2 Hz, 1H), 4.46 (t, J = 8.2 Hz, lH), 4.34 (dd, J = 8.6, 5.8 Hz, lH), 4.24-4.03 (m, 7H), 3.28 - 3.09 (m, 3H), 3.07 - 2.85 (m, 7H), 2.70 (dd, J = 14.0, 8.8 Hz, lH), 2.63 - 2.54 (m, lH), 2.19 - 1.95 (m, 4H), 1.93 - 1.67 (m, 4H), 1.32 (dt, J = 15.4, 10.2 Hz, 2H), 1.04 (d, J = 6.2 Hz, 3H), 0.76- 0.68 (m, 1H), 0.62 (d, J = 6.4 Hz, 3H), 0.55 (d, J = 6.2 Hz, 3H).

120

[0278] (R)-2-amino-3-(lH-pyrazol-5-yl)-N-((3S,6S,9S, 12S, 15R, 18S,21S)-6,9, 18-tris(2- aminoethyl)-15-benzyl-3-((R)-l-hydroxyethyl)-12-isobutyl-2,5,8,l l, 14, 17,20-heptaoxo- l,4,7,10, 13,16, 19-heptaazacyclotricosan-21-yl)propanamide, Compound 120, was prepared according to the coupling then deprotection sequence described for Compound 89. LCMS: rt = 0.28/0.40 (LC method 3). m z = 899 (M+l). ¾ NMR (400 MHz, D₂0) δ 7.56 (d, J = 2.2 Hz, 1H), 7.21 (dq, J = 14.4, 7.0 Hz, 3H), 7.11 (d, J = 6.8 Hz, 2H), 6.20 (d, J = 2.2 Hz, 1H), 4.44 (t, J = 8.2 Hz, 1H), 4.35 (dd, J = 8.8, 5.2 Hz, 1H), 4.17-4.02 (m, 7H), 3.13 (dd, J = 7.2, 4.2 Hz, 2H), 3.07 - 2.85 (m, 7H), 2.78 - 2.56 (m, 3H), 2.12- 1.98 (m, 4H), 1.91 - 1.70 (m, 3H), 1.58 (d, J = 9.6 Hz, lH), 1.39 - 1.22 (m, 2H), 1.05 (d, J = 6.2 Hz, 3H), 0.76 - 0.67 (m, 1H), 0.62 (d, J = 6.4 Hz, 3H), 0.55 (d, J = 6.2 Hz, 3H).

121

[0279] (3R,4S)-4-phenyl-N-((3S,6S,9S,12S,15R,18S,21S)-6,9, 18-tris(2-aminoethyl)-15-benzyl- 3 -((R)- 1 -hydroxyethyl)- 12-isobutyl-2,5 ,8, 11 , 14, 17,20-heptaoxo- 1 ,4,7, 10, 13 , 16, 19-heptaazacyclotricosan- 21-yl)pyrrolidine-3-carboxamide, Compound 121, was prepared according to the coupling then deprotection sequence described for Compound 89. LCMS: rt = 0.34 (LC method 3). m/z = 936 (M+l). 'H NMR (400 MHz, D₂0) δ 8.10 (s, 1H), 7.34 - 7.20 (m, 8H), 7.13 (d, J = 7.5 Hz, 2H), 4.44 (m, lH), 4.24 (m, 1H), 4.19 - 4.11 (m, 2H), 4.05 (m, 4H), 3.73 (s, 2H), 3.54 (m, lH), 3.48 - 3.39 (m, 1H), 3.30 (m, 2H), 3.08 (m, 1H), 2.93 (m, 7H), 2.69 - 2.59 (m, 1H), 2.47 (m, lH), 2.18 - 1.94 (m, 4H), 1.84 - 1.58 (m, 4H), 1.34 (m, 2H), 1.06 (d, J = 6.2 Hz, 3H), 0.73 (m, lH), 0.67 - 0.61 (m, 3H), 0.58 (m, 3H).

122

[0280] (3S,4R)-4-phenyl-N-((3S,6S,9S,12S,15R,18S,21S)-6,9, 18-tris(2-aminoethyl)-15-benzyl- 3-((R)-l-hydroxyethyl)-12-isobutyl-2,5,8, l l,14, 17,20-heptaoxo-l,4,7, 10, 13,16, 19-heptaazacyclotricosan- 21-yl)pyrrolidine-3-carboxamide, Compound 122, was prepared according to the coupling then deprotection sequence described for Compound 89. LCMS: rt = 0.34 (LC method 3). m/z = 936 (M+l). ¾ NMR (400 MHz, D₂0) δ 8.15 (s, 1H), 7.40 - 7.22 (m, 8H), 7.17 (d, J = 6.9 Hz, 2H), 4.51 - 4.31 (m, 2H), 4.27 - 4.00 (m, 6H), 3.82 - 3.71 (m, 2H), 3.46 (m, 3H), 3.27 (m, 1H), 3.13 - 2.87 (m, 6H), 2.71 (m, 3H), 2.25 - 1.98 (m, 5H), 1.92 (m, 1H), 1.83 (m, 1H), 1.67 - 1.55 (m, 1H), 1.50 - 1.26 (m, 3H), 1.18 - 1.08 (m, 3H), 0.72 (s, 1H), 0.69 (m, 3H), 0.61 (d, J = 5.8 Hz, 3H).

123

[0281] (3S,4R)-l-benzyl-4-phenyl-N (3S,6S,9S42S45R, 18S,21S)-6,948-tris(2-aminoethyl)- 15-benzyl-3 (R)-l-hydroxyethyl)-12-isobutyl-2,5,8,l l, 14,17,20-heptaoxo-l,4,7, 10, 13,16, 19- heptaazacyclotricosan-21-yl)pyrrolidine-3-carboxamide, Compound 123, was prepared according to the coupling then deprotection sequence described for Compound 89. LCMS: rt = 0.34 (LC method 3). m/z = 1026 (M+l). 'H NMR (400 MHz, D₂0) δ 8.19 (s, 1H), 7.46 (s, 5H), 7.39 - 7.23 (m, 8H), 7.18 (d, J = 6.9 Hz, 2H), 4.55 - 4.41 (m, 3H), 4.32 - 4.26 (m, 1H), 4.24 - 4.00 (m, 6H), 3.81 (s, 2H), 3.48 (s, 2H), 3.13 (m, 1H), 2.97 (m, 7H), 2.66 (m, 1H), 2.53 (m, 1H), 2.23 - 1.99 (m, 4H), 1.74 (m, 4H), 1.45 - 1.32 (m, 2H), 1.22 - 1.07 (m, 5H), 0.83 (m, 1H), 0.71 (d, J = 6.3 Hz, 3H), 0.63 (d, J = 6.2 Hz, 3H).

124

[0282] (3R,4S)- 1 -benzyl-4-phenyl-N-((3 S,6S,9S, 12S, 15R, 18S,21 S)-6,9, 18-tris(2-aminoethyl)- 15-benzyl-3-((R)-l-hydroxyethyl)-12-isobutyl-2,5,8,l l, 14,17,20-heptaoxo-l,4,7, 10, 13,16, 19- heptaazacyclotricosan-21-yl)pyrrolidine-3-carboxamide, Comound 124, was prepared according to the coupling then deprotection sequence described for Compound 89. LCMS: rt = 0.34 (LC method 3). m/z = 1026 (M+l). ¾ NMR (400 MHz, D₂0) δ 8.11 (s, lH), 7.38 (s, 5H), 7.30 - 7.17 (m, 8H), 7.10 (d, J = 7.2 Hz, 2H), 4.38 (d, J = 8.5 Hz, 3H), 4.29 (s, 1H), 4.07 (m, 5H), 3.94 (s, lH), 3.70 (d, J = 22.8 Hz, 3H), 3.39 (s, 3H), 3.01 - 2.84 (m, 7H), 2.72 (m, 2H), 2.56 (m, 1H), 2.14 - 1.95 (m, 5H), 1.82 (m, 1H), 1.72 (m, 1H), 1.54 (m, 1H), 1.41 - 1.23 (m, 4H), 1.16 - 0.99 (m, 7H), 0.71 (s, 1H), 0.62 (d, J = 6.4 Hz, 3H), 0.55 (d, J = 6.2 Hz, 3H).

125

[0283] (S)-2-amino-3-(thiazol-4-yl)-N-((3 S,6S,9S, 12S, 15R, 18S,21 S)-6,9, 18-tris(2-aminoethyl)- 15-benzyl-3-((R)-l-hydroxyethyl)-12-isobutyl-2,5,8,l l, 14,17,20-heptaoxo-l,4,7, 10, 13,16, 19- heptaazacyclotricosan-21-yl)propanamide, Compound 125, was prepared according to the coupling then deprotection sequence described for Compound 89. LCMS: rt = 0.352 (LC method 3). m/z = 916 (M+l). 'H NMR (400 MHz, D₂0) δ 8.88 (s, 1H), 8.07 (s, 1H), 7.36 (s, 1H), 7.21 (m, 3H), 7.11 (d, J = 7.2 Hz, 2H), 4.44 (m, 1H), 4.35 (m, 1H), 4.24 (m, 1H), 4.05 (m, 6H), 3.29 (d, J = 7.1 Hz, 2H), 3.08 - 2.88 (m, 7H), 2.78 - 2.59 (m, 3H), 2.15 - 1.96 (m, 4H), 1.89 - 1.66 (m, 3H), 1.58 (m, 1H), 1.36 - 1.23 (m, 2H), 1.05 (d, J = 6.2 Hz, 3H), 0.72 (m, 1H), 0.62 (d, J = 6.3 Hz, 3H), 0.56 (d, J = 6.2 Hz, 3H).

126

[0284] (R)-2-amino-3-(thiazol-4-yl)-N-((3 S,6S,9S, 12S, 15R, 18S,21 S)-6,9, 18-tris(2-aminoethyl)- 15-benzyl-3-((R)-l-hydroxyethyl)-12-isobutyl-2,5,8,l l, 14,17,20-heptaoxo-l,4,7, 10, 13,16, 19- heptaazacyclotricosan-21-yl)propanamide, Compound 126, was prepared according to the coupling then deprotection sequence described for Compound 89. LCMS: rt = 0.35/0.45 (LC method 3). m z = 916 (M+l). 'H NMR (400 MHz, D₂0) δ 8.84 (d, J = 1.5 Hz, 1H), 8.07 (s, 1H), 7.35 (d, J = 1.3 Hz, lH), 7.28 - 7.17 (m, 3H), 7.12 (d, J = 7.2 Hz, 2H), 4.47 (m, 1H), 4.30 (m, 2H), 4.19 (m, 2H), 4.14 - 4.01 (m, 4H), 3.26 (m, 3H), 3.05 - 2.87 (m, 7H), 2.70 (m, 1H), 2.64 - 2.51 (m, 1H), 2.19 - 1.94 (m, 4H), 1.70 (m, 4H), 1.31 (m, 2H), 1.03 (d, J = 6.2 Hz, 3H), 0.75 (m, lH), 0.63 (d, J = 6.4 Hz, 3H), 0.56 (d, J = 6.3 Hz, 3H).

127

[0285] (S)-l-benzyl-N-((3S,6S,9S, 12S,15R,18S,21S)-6,9,18-tris(2-aminoethyl)-15-benzyl-3- ((R)- 1 -hydroxyethyl)- 12-isobutyl-2,5 , 8, 11 , 14, 17,20-heptaoxo- 1, 4,7, 10, 13, 16, 19-heptaazacyclotricosan- 21-yl)piperazine-2-carboxamide, Compound 127, was prepared according to the coupling then deprotection sequence described for Compound 89. LCMS: rt = 0.44 (LC method 3). m/z = 964 (M+l). 'H NMR (400 MHz, D₂0) δ 8.15 (s, 1H), 7.42 - 7.23 (m, 8H), 7.18 (m, 2H), 4.58 - 4.34 (m, 2H), 4.31 - 4.02 (m, 6H), 3.91 (d, J= 12.7 Hz, 1H), 3.50 (m, 3H), 3.35 (m, 2H), 3.23 - 2.89 (m, 1 1H), 2.71 (m, 3H), 2.31 - 1.67 (m, 9H), 1.53 - 1.22 (m, 3H), 1.10 (d, J= 6.1 Hz, 3H), 0.72 (m, lH), 0.69 (d, J = 6.0 Hz, 3H), 0.62 (d, J = 6.0 Hz, 3H).

COMPOUND 128

[0286] (S)-3-amino-2-benzyl-N-((3S,6S,9S, 12S,15R,18S,21S)-6,9,18-tris(2-aminoethyl)-15- benzyl-3-((R)-l-hydroxyethyl)-12-isobutyl-2,5,8,l l, 14,17,20-heptaoxo-l,4,7, 10, 13,16, 19- heptaazacyclotricosan-21-yl)propanamide, Compound 128, was prepared according to the coupling then deprotection sequence described for Compound 89. LCMS: rt = 0.40 (LC method 3). m/z = 923 (M+l). 'H NMR (400 MHz, D₂0) δ 7.35 - 7.24 (m, 5H), 7.23 - 7.13 (m, 5H), 4.49 (t, J = 8.2 Hz, lH), 4.42 (dd, J = 8.6, 5.4 Hz, 1H), 4.24 - 4.07 (m, 5H), 3.96 (dd, J = 10.6, 4.2 Hz, lH), 3.25 (dd, J = 13.2, 7.4 Hz, 1H), 3.15 (dd, J = 13.2, 4.8 Hz, 1H), 3.09 - 2.91 (m, 8H), 2.82 - 2.63 (m, 4H), 2.20 - 2.02 (m, 5H), 1.96 - 1.79 (m, 2H), 1.63 - 1.51 (m, 1H), 1.46 - 1.29 (m, 3H), 1.14 (d, J = 6.0 Hz, 3H), 0.81 (td, J = 10.2, 5.6 Hz, 1H), 0.71 (t, J = 8.0 Hz, 3H), 0.63 (d, J = 6.4 Hz, 3H).

129

[0287] (2S,4R)-4-acetamido-N-((3S,6S,9S,12S,15R, 18S,21S)-6,9, 18-tris(2-aminoethyl)-15- benzyl-3-((R)-l-hydroxyethyl)-12-isobutyl-2,5,8,l l, 14,17,20-heptaoxo-l,4,7, 10, 13,16, 19- heptaazacyclotricosan-21-yl)pyrrolidine-2-carboxamide, Compound 129, was prepared according to the coupling then deprotection sequence described for Compound 89. LCMS: rt = 0.35 (LC method 3). m/z = 923 (M+l). ¾ NMR (400 MHz, D₂0) δ 7.37 - 7.22 (m, 6H), 7.22 - 7.13 (m, 4H), 4.53 (t, J = 8.2 Hz, 1H), 4.30-4.23 (m, 2H), 4.22 - 4.06 (m, 5H), 3.30 - 3.16 (m, 2H), 3.13 - 2.94 (m, 9H), 2.87 (t, J = 11.2 Hz, 2H), 2.80 - 2.70 (m, 1H), 2.62 - 2.52 (m, lH), 2.24 - 2.00 (m, 4H), 1.91 - 1.68 (m, 4H), 1.45 - 1.31 (m, 2H), 1.11 (d, J = 6.4 Hz, 3H), 0.89 - 0.78 (m, 1H), 0.71 (d, J = 6.4 Hz, 3H), 0.64 (d, J = 6.4 Hz, 3H).

COMPOUND 130

[0288] (3S,4R)-l-ethyl-4-phenyl-N-((3S,6S,9S, 12S, 15R,18S,21S)-6,9, 18-tris(2-aminoethyl)-15- benzyl-3-((R)-l-hydroxyethyl)-12-isobutyl-2,5,8,l l, 14,17,20-heptaoxo-l,4,7, 10, 13,16, 19- heptaazacyclotricosan-21-yl)pyrrolidine-3-carboxamide, Compound 130, was prepared according to the coupling then deprotection sequence described for Compound 89. LCMS: rt =0.36 (LC method 3). m/z = 964 (M+l). ¾ NMR (400 MHz, D₂0) δ 8.08 (s, 1H), 7.31 - 7.18 (m, 8H), 7.10 (d, J = 6.9 Hz, 2H), 4.40 (m, 1H), 4.36 - 4.29 (m, 1H), 4.14 - 4.01 (m, 5H), 3.99 - 3.85 (m, 2H), 3.80 (m, lH), 3.70 - 3.46 (m, 2H), 3.42 - 3.12 (m, 5H), 3.06 - 2.83 (m, 7H), 2.71 (m, 2H), 2.62 (m, lH), 2.23 - 1.91 (m, 6H), 1.88 - 1.71 (m, 2H), 1.63 - 1.49 (m, 1H), 1.21 (m, 6H), 1.06 (d, J = 6.3 Hz, 3H), 0.65 (m, lH), 0.61 (d, J = 6.2 Hz, 3H), 0.54 (d, J = 6.2 Hz, 3H).

D 131

[0289] (3R,4S)-l-ethyl-4-phenyl-N-((3S,6S,9S, 12S, 15R,18S,21S)-6,9, 18-tris(2-aminoethyl)-15- benzyl-3-((R)-l-hydroxyethyl)-12-isobutyl-2,5,8,l l, 14,17,20-heptaoxo-l,4,7, 10, 13,16, 19- heptaazacyclotricosan-21-yl)pyrrolidine-3-carboxamide, Compound 131, was prepared according to the coupling then deprotection sequence described for Compound 89. LCMS: rt = 0.35 (LC method 3). m/z = 963 (M+l). ¾ NMR (400 MHz, D₂0) δ 8.07 (s, 1H), 7.35 - 7.16 (m, 8H), 7.10 (d, J = 6.8 Hz, 2H), 4.43 (m, 1H), 4.23 (m, 1H), 4.05 (m, 5H), 3.96 - 3.86 (m, lH), 3.75 (m, lH), 3.59 (m, 2H), 3.40 (m, 1H), 3.26 (m, 3H), 3.12 - 3.03 (m, 1H), 3.01 - 2.79 (m, 7H), 2.68 - 2.58 (m, lH), 2.46 (m, lH), 2.14 - 1.92 (m, 4H), 1.82 - 1.55 (m, 4H), 1.38 - 1.18 (m, 5H), 1.03 (d, J = 6.3 Hz, 3H), 0.75 (m, lH), 0.63 (m, 3H), 0.55 (m, 3H).

D 132

[0290] (S)-2-amino-3-phenyl-N-((3S,6S,9S,12S,15R,18S,21S)-6,9, 18-tris(2-aminoethyl)-15- benzyl-3-((R)-l-hydroxyethyl)-12-isobutyl-2,5,8,l l, 14,17,20-heptaoxo-l,4,7, 10, 13,16, 19- heptaazacyclotricosan-21-yl)propanamide, Compound 132, was prepared according to the coupling then deprotection sequence described for Compound 89. LCMS: rt = 0.36 (LC method 3). m/z = 909 (M+l). 'H NMR (400 MHz, D₂0) δ 8.15 (s, 1H), 7.41 - 7.12 (m, 10H), 4.53 (m, 1H), 4.38 (m, 1H), 4.29 - 4.05 (m, 7H), 3.31 - 3.17 (m, 2H), 3.03 (m, 8H), 2.81 (m, 1H), 2.68 (m, 1H), 2.13 (m, 4H), 1.99 - 1.73 (m, 4H), 1.46 - 1.30 (m, 2H), 1.11 (d, J = 6.2 Hz, 3H), 0.80 (s, lH), 0.73 - 0.66 (m, 3H), 0.63 (m, 3H).

COMPOUND 133

[0291] (2S,3R)-2-amino-3-hydroxy-N-((3S,6S,9S,12S,15R,18S,21S)-6,9, 18-tris(2-aminoethyl)- 15-benzyl-3-((R)-l-hydroxyethyl)-12-isobutyl-2,5,8,l l, 14,17,20-heptaoxo-l,4,7, 10, 13,16, 19- heptaazacyclotricosan-21-yl)butanamide, Compound 133, was prepared according to the coupling then deprotection sequence described for Compound 89. LCMS: rt = 0.27/0.38 (LC method 3). m z = 877 (M+l). 'H NMR (400 MHz, D₂0) δ 8.14 (s, 1H), 7.28 (dq, J = 14.6, 7.2 Hz, 3H), 7.18 (d, J = 7.2 Hz, 2H), 4.53 - 4.38 (m, 2H), 4.29 - 4.05 (m, 6H), 3.88 (d, J = 6.6 Hz, lH), 3.70 (p, J = 6.2 Hz, lH), 3.41 - 3.22 (m, 4H), 3.16 - 2.91 (m, 7H), 2.87 - 2.75 (m, 1H), 2.75 - 2.63 (m, lH), 2.24 - 2.03 (m, 4H), 2.02 - 1.77 (m, 4H), 1.48 - 1.28 (m, 2H), 1.21 (d, J = 6.4 Hz, 3H), 1.13 (t, J = 10.2 Hz, 3H), 0.78 - 0.54 (m, 7H).

ND 134

[0292] (S)-2-amino-3-(diethylamino)-N-((3S,6S,9S, 12S, 15R,18S,21S)-6,9, 18-tris(2- aminoethyl)-15-benzyl-3-((R)-l-hydroxyethyl)-12-isobutyl-2,5,8,l l, 14, 17,20-heptaoxo- l,4,7,10, 13,16, 19-heptaazacyclotricosan-21-yl)propanamide, Compound 134, was prepared according to the coupling then deprotection sequence described for Compound 89. LCMS: rt = 0.26/0.41 (LC method

no 3). m/z = 904 (M+l). ¾ NMR (400 MHz, D₂0) δ 7.29 (m, 3H), 7.19 (d, J = 6.8 Hz, 2H), 4.56 - 4.39 (m, 2H), 4.29 - 4.01 (m, 7H), 3.69 - 3.55 (m, 2H), 3.48 (d, J = 14.0 Hz, lH), 3.36 - 3.21 (m, 5H), 3.15 - 2.94 (m, 7H), 2.86 - 2.64 (m, 2H), 2.27 - 2.06 (m, 4H), 1.91 (dd, J = 17.6, 1 1.6 Hz, 4H), 1.39 (ddd, J = 18.4, 12.4, 7.2 Hz, 2H), 1.29 - 1.18 (m, 6H), 1.12 (d, J = 6.2 Hz, 3H), 0.80-0.61 (m, 7H).

COMPOUND 135

[0293] (S)-l-isobutyl-N-((3S,6S,9S, 12S, 15R, 18S,21S)-6,9, 18-tris(2-aminoethyl)-15-benzyl-3- ((R)- 1 -hydroxyethyl)- 12-isobutyl-2,5 , 8, 11 , 14, 17,20-heptaoxo- 1, 4,7, 10, 13, 16, 19-heptaazacyclotricosan- 21-yl)piperazine-2-carboxamide, Compound 135, was prepared according to the coupling then deprotection sequence described for Compound 89. LCMS: rt = 0.20 (LC method 3). m/z = 930 (M+l). ¾ NMR (400 MHz, D₂0) δ 8.15 (s, 1H), 7.29 (m, 3H), 7.19 (m, 2H), 4.52 (t, J = 8.1 Hz, 1H), 4.48 - 4.39 (m, 1H), 4.33 - 4.05 (m, 6H), 3.84 (m, 1H), 3.61 (m, 3H), 3.42 - 3.20 (m, 3H), 3.18 - 2.90 (m, 8H), 2.87 - 2.51 (m, 4H), 2.26 - 1.71 (m, 9H), 1.52 - 1.25 (m, 2H), 1.12 (d, J = 6.0 Hz, 3H), 0.88 (dd, J = 1 1.4, 7.5 Hz, 6H), 0.70 (d, J = 6.0 Hz, 3H), 0.63 (d, J = 6.0 Hz, 3H).

136

[0294] (S)-2-amino-5-oxo-5-((3S,6S,9S, 12S, 15R,18S,21S)-6,9, 18-tris(2-aminoethyl)-15-benzyl- 3-((R)-l-hydroxyethyl)-12-isobutyl-2,5,8, l l,14, 17,20-heptaoxo-l,4,7, 10, 13,16, 19-heptaazacyclotricosan- 21-ylamino)pentanoic acid, Compound 136, was prepared according to the coupling then deprotection sequence describe for Compound 89. LCMS: rt = 0.31 minutes (LC method 3). m/z = 891 (M+l). 1H NMR (400 MHz, D₂0) δ 8.15 (s, 1H), 7.42 - 7.23 (m, 8H), 7.18 (m, 2H), 4.58 - 4.34 (m, 2H), 4.31 - 4.02 (m, 6H), 3.91 (d, J= 12.7 Hz, 1H), 3.50 (m, 3H), 3.35 (m, 2H), 3.23 - 2.89 (m, 1 1H), 2.71 (m, 3H), 2.31 - 1.67 (m, 9H), 1.53 - 1.22 (m, 3H), 1.10 (d, J= 6.1 Hz, 3H), 0.72 (m, lH), 0.69 (d, J= 6.0 Hz, 3H), 0.62 (d, J= 6.0 Hz, 3H).

137

[0295] (S)-3-(4-( lH-pyrazol-4-yl)phenyl)-2-amino-N-((3 S,6S,9S, 12S, 15R, 18S,21 S)-6,9, 18- tris(2-aminoethyl)- 15 -benzyl-3 -((R)- 1 -hydroxyethyl)- 12-isobutyl-2,5 , 8, 11 , 14, 17,20-heptaoxo- l,4,7,10, 13,16, 19-heptaazacyclotricosan-21-yl)propanamide, Compoound 137, was prepared according to the coupling then deprotection sequence described for Compound 89. LCMS: rt = =0.35 (LC method 3). m/z = 975 (M+l). ¾ NMR (400 MHz, D₂0) δ 8.16 (d, J = 11.0 Hz, 2H), 7.57 (m, 2H), 7.25 (m, 7H), 4.54 (m, 1H), 4.39 - 4.04 (m, 8H), 3.11 (m, 10H), 2.73 (m, 1H), 2.60 (m, lH), 2.14 (m, 4H), 1.83 (m, 4H), 1.38 (m, 2H), 1.18 - 1.04 (m, 3H), 0.81 (m, 1H), 0.78 - 0.47 (m, 6H).

EXAMPLE 138. SYNTHESIS OF COMPOUND 138

[0296] (R)-2-amino-3-phenyl-N-((3S,6S,9S,12S,15R,18S,21S)-6,9,18-tris(2-aminoethyl)-15- benzyl-3-((R)-l-hydroxyethyl)-12-isobutyl-2,5,8,l l, 14,17,20-heptaoxo-l,4,7, 10, 13,16, 19- heptaazacyclotricosan-21-yl)propanamide, Compound 138, was prepared according to the coupling then deprotection sequence described for Compound 89. LCMS: rt = 0.34 (LC method 3). m/z = 909 (M+l). 'H NMR (400 MHz, D₂0) δ 8.16 (s, 1H), 7.51 - 7.06 (m, 10H), 4.51 (m, 1H), 4.43 (m, 1H), 4.31 - 3.99 (m, 7H), 3.24 - 3.16 (m, 1H), 2.99 (m, 8H), 2.80 (m, 1H), 2.73 (m, 1H), 2.33 (m, 1H), 2.25 - 2.03 (m, 4H), 1.88 (m, 2H), 1.67 (m, 1H), 1.41 (m, 3H), 1.14 (d, J = 5.8 Hz, 3H), 0.80 (m, 1H), 0.70 (d, J = 6.2 Hz, 3H), 0.63 (d, J = 5.8 Hz, 3H).

D 139

[0297] (2S,3 S)-2-amino-3-methyl-N-((3 S,6S,9S, 12S, 15R, 18S,21 S)-6,9, 18-tris(2-aminoethyl)- 15-benzyl-3-((R)-l-hydroxyethyl)-12-isobutyl-2,5,8,l l, 14,17,20-heptaoxo-l,4,7, 10, 13,16, 19- heptaazacyclotricosan-21-yl)pentanamide, Compound 139, was prepared according to the coupling then deprotection sequence described for Compound 89. LCMS: rt = 0.47 (LC method 3). m/z = 875 (M+l). 'H NMR (400 MHz, D₂0) δ 7.27 - 7.18 (m, 3H), 7.14 - 7.09 (m, 2H), 4.46 - 4.30 (m, 2H), 4.19 - 4.01 (m, 6H), 3.95 (d, J = 14.2 Hz, 1H), 3.55 (ddd, J = 16.8, 13.6, 8.2 Hz, 3H), 3.28 - 3.19 (m, lH), 3.08 - 2.89 (m, 7H), 2.79 - 2.71 (m, 1H), 2.66 - 2.56 (m, 2H), 2.48 - 2.39 (m, lH), 2.14 - 1.99 (m, 4H), 1.88 - 1.74 (m, 4H), 1.35 - 1.22 (m, 2H), 1.05 (d, J = 6.4 Hz, 3H), 0.63 (dd, J = 17.6, 6.2 Hz, 4H), 0.54 (d, J = 5.8 Hz, 3H).

COMPOUND 140

[0298] (R)-3-amino-N-((3S,6S,9S,12S,15R,18S,21S)-6,9, 18-tris(2-aminoethyl)-15-benzyl-3- ((R)- 1 -hydroxyethyl)- 12-isobutyl-2,5 , 8, 11 , 14, 17,20-heptaoxo- 1, 4,7, 10, 13, 16, 19-heptaazacyclotricosan- 21-yl)pyrrolidine-3-carboxamide, Compound 140, was prepared according to the coupling then deprotection sequence described for Compound 89. LCMS: rt = 0.29/0.43 (LC method 3). m/z = 874 (M+l). ¾ NMR (400 MHz, D₂0) 57.27 - 7.16 (m, 3H), 7.15 - 7.09 (m, 2H), 4.46 - 4.28 (m, 2H), 4.00- 4.19 (m 5H), 3.93 (d, J = 14.1 Hz, 1H), 3.66 - 3.49 (m, 3H), 3.28 - 3.19 (m, 1H),3.13-2.88 (m, 7H), 2.74 (ddd, J = 12.8, 10.0, 5.4 Hz, 1H), 2.62 (ddd, J = 17.4, 12.4, 7.2 Hz, 2H), 2.46 - 2.37 (m, 1H), 2.18 - 1.95 (m, 4H), 1.93 - 1.71 (m, 4H), 1.34 (ddd, J = 13.8, 9.4, 4.2 Hz, lH), 1.27 (dd, J = 11.0, 3.6 Hz, lH), 1.23 - 1.12 (m, 1H), 1.05 (d, J = 6.4 Hz, 3H), 0.63 (dd, J = 16.6, 6.8 Hz, 3H), 0.55 (t, J = 6.8 Hz, 3H).

COMPOUND 141

[0299] (S)-3-amino-N-((3S,6S,9S,12S,15R,18S,21S)-6,9, 18-tris(2-aminoethyl)-15-benzyl-3- ((R)- 1 -hydroxyethyl)- 12-isobutyl-2,5 , 8, 11 , 14, 17,20-heptaoxo- 1, 4,7, 10, 13, 16, 19-heptaazacyclotricosan- 21-yl)pyrrolidine-3-carboxamide, Compound 141, was prepared according to the coupling then deprotection sequence described for Compound 89. LCMS: rt = 0.27/0.40 (LC method 3). m z = 874 (M+l). ¾ NMR (400 MHz, D₂0) δ 7.22 (dq, J = 14.6, 7.2 Hz, 3H), 7.14 - 7.09 (m, 2H), 4.46 - 4.32 (m, 2H), 4.21 - 4.01 (m, 6H), 3.95 (d, J = 14.2 Hz, 1H), 3.63 - 3.49 (m, 3H), 3.29 - 3.19 (m, lH), 3.10 - 2.89 (m, 7H), 2.80 - 2.70 (m, 1H), 2.62 (dt, J = 18.4, 9.6 Hz, 2H), 2.48 - 2.40 (m, lH), 2.14 - 1.98 (m, 4H), 1.92 - 1.74 (m, 4H), 1.38 - 1.12 (m, 3H), 1.05 (d, J = 6.4 Hz, 3H), 0.63 (dd, J = 17.8, 6.2 Hz, 4H), 0.54 (d, J = 5.8 Hz, 3H).

EXAMP 142

[0300] (S)-2-amino-3,3-dimethyl-N-((3S,6S,9S,12S,15R,18S,21S)-6,9, 18-tris(2-aminoethyl)-15- benzyl-3-((R)-l-hydroxyethyl)-12-isobutyl-2,5,8,l l, 14,17,20-heptaoxo-l,4,7, 10, 13,16, 19- heptaazacyclotricosan-21-yl)butanamide, Compound 142, was prepared according to the coupling then deprotection sequence described for Compound 89. LCMS: rt = 0.35 (LC method 3). m/z = 875 (M+l). ¾ NMR (400 MHz, D20) δ 8.17 (s, 1H), 7.34-7.18 (m, 5H), 4.54 (t, J = 8.0 Hz, 1H), 4.45-4.41 (dd, J = 8.0 Hz, 4.0 Hz, 1H), 4.29-4.09 (m, 6H), 3.68 (brs, 4H), 3.32-3.28 (m, 1H), 3.12-2.99 (m, 7H), 2.83-2.64 (m, 2H), 2.21-2.08 (m, 4H), 1.95-1.83 (m, 4H), 1.42-1.35 (m, 2H), 1.13 (d, J = 8.0 Hz, 3H), 1.00 (s, 9H),0.77 (m, 1H), 0.73 (d, J = 8.0 Hz, 3H), 0.61 (d, J = 8.0 Hz, 3H).

D 143

[0301] (S)-2-amino-4-oxo-4-((3S,6S,9S, 12S, 15R,18S,21S)-6,9, 18-tris(2-aminoethyl)-15-benzyl- 3-((R)-l-hydroxyethyl)-12-isobutyl-2,5,8, l l,14, 17,20-heptaoxo-l,4,7, 10, 13,16, 19-heptaazacyclotricosan- 21-ylamino)butanoic acid, Compound 143, was prepared according to the coupling then deprotection sequence described for Compound 89. LCMS:rt = 0.335 minutes (LC method 3). m/z = 877 (M+l). 1H NMR (400 MHz, D₂0) δ 8.09 (s, 2H), 7.38-7.10 (m, 5H), 4.47-4.45 (m, 1H), 4.41-4.37 (dd, J = 8.0 Hz, 4.0 Hz, 1H), 4.24-4.20 (dd, J = 8.0 Hz, 4.0 Hz, 1H), 3.29 (m, lH), 3.10-2.85 (m, 6H), 2.77-2.57 (m, 4H),2.15- 1.69 (m, 10H), 1.32-1.22 (m, 3H), 1.13 (m, lH), 1.04 (m, 3H), 0.69 (brs, 3H), 0.61 (brs, 3H).

D 144

15-benzyl-3-((R)-l-hydroxyethyl)-12-isobutyl-2,5,8,l l, 14,17,20-heptaoxo-l,4,7, 10, 13,16, 19- heptaazacyclotricosan-21-yl)propanamide, Compound 144, was prepared according to the coupling then deprotection sequence described for Compound 89. LCMS: rt = 0.452 (LC method 3). m/z = 918 (M+l). ¾ NMR (400 MHz, D₂0) δ 8.15 (s, 1H), 7.28 (m, 3H), 7.18 (d, J = 7.1 Hz, 2H), 4.77 (m, 4H), 4.64 - 4.56 (m, 3H), 4.51 (t, J = 8.2 Hz, 1H), 4.41 (dd, J = 8.7, 5.6 Hz, lH), 4.17 (m, 7H), 3.73 (s, 4H), 3.41 - 3.22 (m, 1H), 3.14 - 2.85 (m, 9H), 2.73 (m, 6H), 2.12 (m, 4H), 1.99 - 1.69 (m, 4H), 1.51 - 1.26 (m, 2H), 1.11 (d, J = 6.3 Hz, 3H), 0.78 (m, 1H), 0.69 (d, J = 6.4 Hz, 3H), 0.62 (d, J = 6.2 Hz, 3H).

EXAMPL 145

15-benzyl-3-((R)-l-hydroxyethyl)-12-isobutyl-2,5,8,l l, 14,17,20-heptaoxo-l,4,7, 10, 13,16, 19- heptaazacyclotricosan-21-yl)propanamide, Compound 145, was prepared according to the coupling then deprotection sequence described for Compound 89. LCMS: rt = 0.63 (LC method 3). m/z = 918 (M+l). ¾ NMR (400 MHz, D20) δ 8.15 (s, 2H), 7.28 (m, 3H), 7.18 (d, J = 7.3 Hz, 2H),4.76 (m, 4H), 4.63 (m, 3H), 4.50 (t, J = 8.2 Hz, 1H), 4.42 (dd, J = 8.5, 5.8 Hz, lH), 4.26 (m, 2H), 4.15 (m, 5H), 3.77 (s, 4H), 3.35 - 3.20 (m, 1H), 3.18 - 2.52 (m, 16H), 2.12 (m, 4H), 1.99 - 1.69 (m, 4H), 1.36 (m, 2H), 1.10 (d, J = 6.2 Hz, 3H), 0.72 (m, 1H), 0.67 (d, J = 5.8 Hz, 3H), 0.60 (d, J = 5.9 Hz, 3H).

D 146

[0304] (S)-2-amino-2-cyclohexyl-N-((3 S,6S,9S, 12S, 15R, 18S,21 S)-6,9, 18-tris(2-aminoethyl)- 15- benzyl-3-((R)-l-hydroxyethyl)-12-isobutyl-2,5,8,l l, 14,17,20-heptaoxo-l,4,7, 10, 13,16, 19- heptaazacyclotricosan-21-yl)acetamide, Compound 146, was prepared according to the coupling then deprotection sequence described for Compound 89. LCMS: rt = 0.332 (LC method 3). m/z = 901 (M+l). ¾ NMR (400 MHz, D₂0) δ 8.15 (s, 1H), 7.29 (m, 3H), 7.19 (d, J = 7.3 Hz, 2H), 4.51 (m, lH), 4.42 (m, 1H), 4.18 (m, 6H), 3.75 (d, J = 6.3 Hz, 1H), 3.30 (m, 1H), 3.13 - 2.95 (m, 7H), 2.81 (m, 1H), 2.69 (m, 1H), 2.15 (m, 4H), 1.94 (m, 1H), 1.88 - 1.75 (m, 4H), 1.71 (m, 2H), 1.61 (m, 3H), 1.44 - 1.30 (m, 2H), 1.18 (m, 2H), 1.12 (d, J = 6.2 Hz, 3H), 1.08 - 0.94 (m, 3H), 0.74 (s, lH), 0.70 (m, 3H), 0.63 (m, 3H). EXAMPLE 147. SYNTHESIS OF COMPOUND 147

[0305] (S)-2-amino-3-(pyrrolidin-l-yl)-N-((3S,6S,9S, 12S, 15R, 18S,21S)-6,9, 18-tris(2- aminoethyl)- 15 -benzyl-3 -((R)- 1 -hydroxyethyl)- 12-isobutyl-2,5 , 8, 11 , 14, 17,20-heptaoxo- l,4,7,10, 13,16, 19-heptaazacyclotricosan-21-yl)propanamide, Compound 147, was prepared according to the coupling then deprotection sequence described for Compound 89. LCMS: rt = =0.27/0.40 (LC method 3). m z = 902 (M+l). ¾ NMR (400 MHz, D₂0) δ 7.29 (m, 3H), 7.20 (d, J = 7.8 Hz, 2H), 4.54 (t, J = 8.4 Hz, 1H), 4.45 - 4.38 (m, 2H), 4.29 - 4.24 (m, lH), 4.10-4.20 (m, 5H), 3.74 (dd, J = 14.0, 6.0 Hz, 1H), 3.65 (dd, J = 13.8, 7.2 Hz, 1H), 3.50 - 3.26 (m, 6H), 3.15 - 2.95 (m, 7H), 2.83 - 2.77 (m, 1H), 2.69 (d, J = 6.8 Hz, 1H), 2.21 - 2.10 (m, 3H), 2.06 (s, 4H), 1.94 - 1.78 (m, 5H), 1.43 - 1.33 (m, 2H), 1.12 (d, J = 6.4 Hz, 3H), 0.81 (s, 1H), 0.70 (d, J = 6.4 Hz, 3H), 0.64 (d, J = 6.2 Hz, 3H).0

148-1 148-2 148-3

PMBHBoc-,

HBTU, DIPEA, DMF

148-4 148-5

Example 148

[0306] Step 1. Synthesis of intermediate (S)-methyl 2-(tert-butoxycarbonylamino)-3- (methylsulfonyloxy) -propanoate (148-2). To a cooled (0 °C) solution of methyl (2S)-2-(tert- butoxycarbonylamino)-3-hydroxy-propanoate (200. mg, 0.9100mmol) in DCM (3 mL) was added Et N (0.38 mL, 2.74 mmol) and MsCl (0.11 mL, 1.37 mmol). The resulting mixture was then stirred at 0 °C until the reaction was completed, as determined by LCMS analysis. The crude mixture was extracted with DCM (30 mL x 3). The combined organic phases were dried over Na₂S04, filtered and concentrated in vacuo to give the crude intermediate 148-2. (200 mg, 73.7% yield). LC-MS (LC method 1): m/z 298 (M+H)⁺.

[0307] Step 2. Synthesis of intermediate (S)-methyl 3-(benzylamino)-2-(tert- butoxycarbonylamino) propanoate (148-3). To a solution of phenylmethanamine (0.16 mL, 1.49 mmol) in DCM (3 mL) was added Et3N (0.41 mL, 2.98 mmol) and methyl (2S)-2-(tert-butoxycarbonylamino)- 3-methylsulfonyloxy-propanoate (296 mg, 0.99 mmol) under N2. The mixture was then stirred at room temperature overnight. The reaction mixture was diluted with water (25 ml), extracted with EtOAc (30mL x 3). The organic phases were combined, dried over Na2SC>4, filtered and concentrated. The residue was purified by flash column chromatography (PE : EA = 3 : 1) to give the title compound intermediate 148-3. (240 mg, 78.3% yield). LC-MS (LC method 1): m/z 309 (M+H)⁺.

[0308] Step 3. Intermediate (S)-methyl 3-(benzyl(ethyl)amino)-2-(tert- butoxycarbonylamino)propanoate (148-4). To a solution of methyl (2S)-3-(benzylamino)-2-(tert- butoxycarbonylamino) propanoate (240 mg, 0.78 mmol) in MeCN (3 mL), was added K2CO3 (215 mg, 1.56 mmol) and iodoethane (0.09 mL, 1. 17 mmol). The resulting mixture was then refluxed at 75 °C under N2 overnight until the reaction was completed, as determined by LCMS analysis. The crude mixture was then extracted with EtOAc (30 mL x 3), the combined organic phases were dried over Na2SC>4, filtered and concentrated. The residue was purified by flash column chromatography (PE : EA = 2 : 1) to give the title compound intermediate 148-4. (140 mg, 53.5% yield). LC-MS (LC method 1): m/z 337 (M+H)⁺.

[0309] Step 4. Intermediate (S)-3-(benzyl(ethyl)amino)-2-(tert-butoxycarbonylamino) propanoic acid (61148-5). To a solution of methyl (2S)-3-[benzyl(ethyl)amino]-2-(tert- butoxycarbonylamino)propanoate (170. mg, 0.5100mmol) in THF (2mL), was added a solution of LiOH (63.67mg, 1.52mmol) in water (2mL). The mixture was then stirred at room temperature until the reaction was complete, as determined by LCMS analysis. Then the reaction mixture was diluted with water (50 mL), and extracted with EtOAc (50 mL). The aqueous phase was acidified to pH 5-6. The aqueous phase was extracted with EtOAc (30 mL x 2), the combined organic phases were washed with brine, dried over Na2SC>4, filtered and concentrated to give the title compound intermediate 148-5. (150 mg, 92.1% yield). LC-MS (LC method 1): m/z 323 (M+H)⁺.

[0310] Step. 5. Synthesis of Compound 148: (S)-2-amino-4-(ethylamino)-N- ((3S,6S,9S, 12S, 15R, 18S,21 S)-6,9, 18-tris(2-aminoethyl)-15-benzyl-3-((R)-

2,5,8, 1 1, 14, 17,20-heptaoxo- 1,4,7, 10, 13, 16, 19-heptaazacyclotricosan-21 -yl)butanamide 148-8 was prepared according to the coupling then deprotection sequence described for Compound 89. LCMS: rt = 0.34 (LC method 3). m/z = 876 (M+l). ¾ NMR (400 MHz, D₂0) δ 7.30 (m, 3H), 7.20 (d, J = 7.4 Hz, 2H), 4.56 - 4.29 (m, 3H), 4.21-4.10 (m, 4H), 3.59 - 3.46 (m, 2H), 3.36 - 3.26 (m, 1H), 3.20 - 2.95 (m, 8H), 2.85 - 2.63 (m, 3H), 2.21-2.07 (m, 4H), 1.98-1.81 (m, 4H), 1.48 - 1.3 1 (m, 2H), 1.30 - 1.21 (m, 3H), 1.12 (d, J = 6.2 Hz, 3H), 0.90 - 0.81 (m, 1H), 0.75 - 0.56 (m, 6H). D 149

[0311] (S)-2-amino-N-((3S,6S,9S,12S,15R,18S,21S)-6,9, 18-tris(2-aminoethyl)-15-benzyl-3- ((R)- 1 -hydroxyethyl)- 12-isobutyl-2,5 , 8, 11 , 14, 17,20-heptaoxo- 1, 4,7, 10, 13, 16, 19-heptaazacyclotricosan- 21-yl)pent-4-ynamide, Compound 149, was prepared according to the coupling then deprotection sequence described for Compound 89. LCMS: rt = 0.34 (LC method 3). m/z = 857 (M+l). ¾ NMR (400 MHz, D₂0) δ 7.34-7.24 (m, 3H), 7.19 (d, J = 7.2 Hz, 2H), 4.53 (t, J = 8.2 Hz, lH), 4.42 (dd, J = 8.6, 5.6 Hz, 1H), 4.30 - 4.11 (m, 7H), 3.33 (dt, J = 14.2, 7.2 Hz, 1H), 3.12-2.96 (m, 7H), 2.88 - 2.76 (m, 3H), 2.67 (dd, J = 18.2, 11.0 Hz, 1H), 2.54 (t, J = 2.4 Hz, 1H), 2.23 - 2.04 (m, 4H), 1.97 - 1.78 (m, 4H), 1.38 (qd, J = 14.0, 7.0 Hz, 2H), 1.12 (d, J = 6.0 Hz, 3H), 0.88 - 0.77 (m, lH), 0.71 (d, J = 6.4 Hz, 3H), 0.64 (d, J = 6.2 Hz, 3H) 0

D 150

[0312] (R)-2-amino-3-(pyrrolidin-l-yl)-N-((3S,6S,9S, 12S, 15R, 18S,21S)-6,9, 18-tris(2- aminoethyl)-15-benzyl-3-((R)-l-hydroxyethyl)-12-isobutyl-2,5,8,l l, 14, 17,20-heptaoxo- l,4,7,10, 13,16, 19-heptaazacyclotricosan-21-yl)propanamide, Compound 150, was prepared according to the coupling then deprotection sequence described for Compound 89. LCMS: rt = 0.28/0.38 (LC method 3). m z = 902 (M+l). ¾ NMR (400 MHz, D₂0) δ 7.35 - 7.26 (m, 3H), 7.20 (d, J = 7.6 Hz, 2H), 4.54 - 4.48 (m, 1H), 4.46 - 4.40 (m, 1H), 4.12-4.29 (m, 7H), 3.63 (s, lH), 3.57 (s, lH), 3.47 - 3.26 (m, 6H), 3.16 - 2.95 (m, 7H), 2.81 (s, 1H), 2.70 (s, 1H), 2.20 - 2.01 (m, 7H), 1.84-1.96 (m, 5H), 1.43 (s, 1H), 1.34 (d, J = 10.6 Hz, 1H), 1.12 (d, J = 6.2 Hz, 3H), 0.61-0.70 (m, 7H).

151

[0313] (S)-2-amino-N5-ethyl-Nl-((3S,6S,9S, 12S, 15R,18S,21S)-6,9,18-tris(2-aminoethyl)-15- benzyl-3-(®-l-hydroxyethyl)-12-isobutyl-2,5,8, l l,14, 17,20-heptaoxo-l,4,7, 10, 13,16, 19- heptaazacyclotricosan-21-yl)pentanediamide, Compound 151, was prepared according to the coupling then deprotection sequence described for Compound 89. LCMS: rt = 0.34 (LC method 3). m/z = 918 (M+l). ¾ NMR (400 MHz, D₂0) δ 8.15 (s, 1H), 7.35 - 7.24 (m, 3H), 7.19 (d, J = 7.5 Hz, 2H), 4.55 - 4.42 (m, 2H), 4.22 (m, 3H), 4.17 - 4.07 (m, 3H), 4.01 (m, lH), 3.39 - 3.26 (m, lH), 3.16 - 2.94 (m, 9H), 2.83 (m, 1H), 2.72 (m, 1H), 2.33 (m, 2H), 2.22 - 2.02 (m, 6H), 1.90 (m, 4H), 1.47 - 1.29 (m, 2H), 1.12 (d, J = 6.1 Hz, 3H), 1.03 (m, 3H), 0.72 (m, 1H), 0.69 (d, J = 5.3 Hz, 3H), 0.61 (d, J = 5.7 Hz, 3H).

EXAMP 152

[0314] (S)-2-amino-3-(piperidin-l-yl)-N-((3S,6S,9S, 12S, 15R, 18S,21S)-6,9,18-tris(2- aminoethyl)-15-benzyl-3-(®-l-hydroxyethyl)-12-isobutyl-2,5,8,l 1, 14, 17,20-heptaoxo- 1,4,7, 10, 13, 16, 19- heptaazacyclotricosan-21-yl)propanamide, Compound 152, was prepared according to the coupling then deprotection sequence described for Compound 89. LCMS: rt = 0.69 (LC method 3). m/z = 917 (M+l). ¾ NMR (400 MHz, D₂0) δ 7.29 (dq, J = 14.2, 7.0 Hz, 3H), 7.19 (d, J = 7.2 Hz, 2H), 4.56 - 4.42 (m, 3H), 4.31 (dd, J = 9.8, 4.2 Hz, 1H), 4.24 - 4.08 (m, 5H), 3.62 (dd, J = 14.2, 5.6 Hz, lH), 3.52 (dd, J = 14.4, 7.4 Hz, 1H), 3.41 - 3.25 (m, 4H), 3.16 - 2.95 (m, 7H), 2.87 - 2.69 (m, 2H), 2.14 (dt, J = 14.8, 7.6 Hz, 4H), 2.00 - 1.78 (m, 8H), 1.62 (s, 2H), 1.50 - 1.24 (m, 3H), 1.12 (d, J = 6.2 Hz, 3H), 0.76 - 0.64 (m, 4H), 0.61 (s, 3H).

COMPOUND 153

[0315] ®-2-amino-3-^iperidin-l-yl)-N (3S,6S,9S,12S,15R,18S,21S)-6,9, 18 ris(2-aminoethyl)- 15-benzyl-3-(®-l-hydroxyethyl)-12-isobutyl-2,5,8,l l, 14,17,20-heptaoxo-l,4,7, 10, 13, 16, 19- heptaazacyclotricosan-21-yl)propanamide, Compound 153, was prepared according to the coupling then deprotection sequence described for Compound 89. LCMS: rt = 0.680 (LC method 3). m/z = 917 (M+l). 'H NMR (400 MHz, D₂0) δ 7.35 - 7.25 (m, 3H), 7.19 (d, J = 7.2 Hz, 2H), 4.53 (dd, J = 15.0, 7.8 Hz, 2H), 4.42 (dd, J = 8.8, 5.6 Hz, 1H), 4.29 - 4.24 (m, 1H), 4.22 - 4.08 (m, 5H), 3.66 (dd, J = 14.6, 4.8 Hz, 1H), 3.55 (dd, J = 14.6, 7.8 Hz, 1H), 3.41 - 3.24 (m, 3H), 2.98-3.13 (m, 8H), 2.86 - 2.76 (m, lH), 2.70 (dd, J = 18.0, 11.2 Hz, 1H), 2.27 - 1.98 (m, 5H), 1.94 - 1.72 (m, 8H), 1.60 (s, 2H), 1.42 - 1.29 (m, 2H), 1.12 (d, J = 6.2 Hz, 3H), 0.79 - 0.72 (m, 1H), 0.71 - 0.65 (m, 3H), 0.63 (d, J = 6.2 Hz, 3H).

154

[0316] 4-benzyl-5-oxo-N-((3S,6S,9S, 12S,15R,18S,21S)-6,9,18-tris(2-aminoethyl)-15-benzyl-3- (®- 1 -hydroxyethyl)- 12-isobutyl-2,5 , 8, 11 , 14, 17,20-heptaoxo- 1 ,4,7, 10, 13 , 16, 19-heptaazacyclotricosan-21 - yl)piperazine-2-carboxamide, Compound 154, was prepared according to the coupling then deprotection sequence described for Compound 89. LCMS: rt = 0.330 (LC method 3). m/z = 978 (M+l). ¾ NMR (400 MHz, D₂0) δ 7.32 - 7.15 (m, 8H), 7.11 (d, J = 7.2 Hz, 2H), 4.47 - 4.36 (m, 2H), 4.34 - 4.25 (m, 1H), 4.22 - 3.87 (m, 8H), 3.77 (dd, J = 13.8, 2.8 Hz, 1H), 3.61-3.54 (m, lH), 3.21 (dt, J = 14.0, 7.0 Hz, 1H), 3.07 - 2.77 (m, 8H), 2.73-2.53 (m, 2H), 2.12-1.93 (m, 4H), 1.90 - 1.63 (m, 4H), 1.61 - 1.45 (m, 1H), 1.37-1.25 (m, 2H), 1.03 (t, J = 5.0 Hz, 3H), 0.84 - 0.69 (m, lH), 0.63 (t, J = 6.2 Hz, 3H), 0.55 (dd, J = 9.6, 6.4 Hz, 3H).

D 155

[0317] 4-(dimethylamino)-N-((3S,6S,9S, 12S, 15R, 18S,21S)-6,9,18-tris(2-aminoethyl)-15- benzyl-3-((R)-l-hydroxyethyl)-12-isobutyl-2,5,8,l l, 14,17,20-heptaoxo-l,4,7, 10, 13,16, 19- heptaazacyclotricosan-21-yl)pyrrolidine-3-carboxamide, Compound 155, was prepared according to the coupling then deprotection sequence described for Compound 89. LCMS: rt = 0.47 (LC method 3). m/z = 902 (M+l). ¾ NMR (400 MHz, D₂0) 58.31 (s, 2H), 7.30 (m, 3H), 7.19 (d, J = 7.4 Hz, 2H), 4.66 - 4.63 (m, 1H), 4.56 - 4.37 (m, 2H), 4.20 (m, 6H), 3.85 (m, 1H), 3.68 - 3.37 (m, 3H), 3.38 - 3.20 (m, 1H), 3.02 (m, 6H), 2.83 (s, 4H), 2.73 (m, 1H), 2.64 (s, 6H), 2.26 - 2.03 (m, 3H), 2.02 - 1.71 (m, 4H), 1.48 - 1.27 (m, 2H), 1.12 (t, J = 6.3 Hz, 3H), 0.72 (s, 1H), 0.69 (d, J = 4.8 Hz, 3H), 0.62 (d, J = 5.1 Hz, 3H).

156

[0318] (R)-2-amino-3-((R)-pyrrolidin-2-yl)-N-((3 S,6S,9S, 12S, 15R, 18S,21 S)-6,9, 18-tris(2- aminoethyl)-15-benzyl-3-((R)-l-hydroxyethyl)-12-isobutyl-2,5,8,l l, 14, 17,20-heptaoxo- l,4,7,10, 13,16, 19-heptaazacyclotricosan-21-yl)propanamide, Compund 156, was prepared according to the coupling then deprotection sequence described for Compound 89. LCMS: rt = 0.29/0.49 (LC method 3). m z = 902 (M+1). ¾ NMR (400 MHz, D₂0) δ 7.38 (dq, J = 14.4, 7.2 Hz, 3H), 7.28 (d, J = 7.2 Hz, 2H), 4.64 - 4.49 (m, 2H), 4.37 - 4.12 (m, 7H), 3.73 (dd, J = 13.0, 7.8 Hz, lH), 3.46 - 3.34 (m, 3H), 3.22

- 3.02 (m, 7H), 2.97 - 2.77 (m, 2H), 2.49 - 2.41 (m, 1H), 2.38 - 2.12 (m, 7H), 2.09 - 1.89 (m, 5H), 1.84

- 1.74 (m, 1H), 1.55 - 1.36 (m, 2H), 1.21 (d, J = 6.3 Hz, 3H), 0.88 - 0.66 (m, 7H).

D 157

[0319] (S)-2-amino-3-((R)-pyrrolidin-2-yl)-N-((3S,6S,9S, 12S,15R, 18S,21S)-6,9,18-tris(2- aminoethyl)-15-benzyl-3-((R)-l-hydroxyethyl)-12-isobutyl-2,5,8,l l, 14, 17,20-heptaoxo- l,4,7,10, 13,16, 19-heptaazacyclotricosan-21-yl)propanamide, Compound 157, was prepared according to the coupling then deprotection sequence described for Compound 89. LCMS: rt = 0.30/0.52 (LC method 3). m z = 902 (M+1). ¾ NMR (400 MHz, D₂0) δ 7.38¾dq,gi&a4.4,i'.Offlz,SH],^.28¾d,gi&^.Offlz,0 2H],a.62&a.5O¾m,SH],a.4Oaa 4¾m,i'H],3.8OB37O¾m,aH],3.45B3.33¾m,3H],B.27a3.O30 (m, 'H],a.95 aa.76¾m,SH],a.45Ba 2¾m,^H],a.O9Ba.89¾m,SH],a.84aa.74¾m,aH],a.54B0 1.36¾Γη,ΗΗ]^.21¾ϋ&Κ.4Μζ,ΚΗ],2Ι).84Β2Ι).65¾Γη,1Ή].

D 158

[0320] (S)-2-amino-3-( lH-pyrrol-2-yl)-N-((3 S,6S,9S, 12S, 15R, 18S,21 S)-6,9, 18-tris(2- aminoethyl)- 15 -benzyl-3 -((R)- 1 -hydroxyethyl)- 12-isobutyl-2,5 , 8, 11 , 14, 17,20-heptaoxo- l,4,7,10, 13,16, 19-heptaazacyclotricosan-21-yl)propanamide, Compound 158, was prepared according to the coupling then deprotection sequence described for Compound 89. LCMS: rt = 0.28 (LC method 3). m/z = 898 (M+l). ¾ NMR (400 MHz, D₂0) δ 7.29 (dd, J = 15.4, 5.6 Hz, 3H), 7.20 (t, J = 6.8 Hz, 2H), 6.77 (s, 1H), 6.35 (d, J = 2.4 Hz, 1H), 6.12-5.95 (dt, J = 41.1, 14.8 Hz, lH), 4.53 (dd, J = 13.8, 5.6 Hz, 1H), 4.42 (dd, J = 14.0, 7.0 Hz, 1H), 4.32 - 4.06 (m, 7H), 3.33-3.15 (m, 2H), 3.13 - 2.92 (m, 7H), 2.85- 2.69 (m, 2H), 2.23 - 2.03 (m, 4H), 1.99 - 1.64 (m, 4H), 1.45-1.31 (m, 2H), 1.16-1.1 1 (m, 4H), 0.86 - 0.58 (m, 7H).

COMPOUND 159

[0321] (R)-N-((3S,6S,9S,12S,15R,18S,21S)-6,9, 18-tris(2-aminoethyl)-15-benzyl-3-((R)-l- hydroxyethyl)- 12-isobutyl-2,5 ,8, 11 , 14, 17,20-heptaoxo- 1 ,4,7, 10, 13 , 16, 19-heptaazacyclotricosan-21 - yl)pyrrolidine-3-carboxamide, Compound 159, was prepared according to the coupling then deprotection sequence described for Compound 89. LCMS: rt = 0.284/0.493 (LC method 3). m/z = 860 (M+l). ¾ NMR (400 MHz, D₂0) δ 7.21 (dq, J = 14.4, 7.2 Hz, 3H), 7.11 (d, J = 7.4 Hz, 2H), 4.46 - 4.32 (m, 2H), 4.21 - 3.99 (m, 6H), 3.40 (dd, J = 12.0, 8.2 Hz, 1H), 3.33 - 3.12 (m, 5H), 3.05 - 2.86 (m, 7H), 2.79 - 2.69 (m, 1H), 2.64 (dd, J = 17.8, 11.0 Hz, 1H), 2.22 (dt, J = 14.2, 7.6 Hz, lH), 2.15 - 1.85 (m, 6H), 1.83 - 1.67 (m, 3H), 1.22-1.37 (m, 2H), 1.04 (d, J = 6.2 Hz, 3H), 0.63 (dd, J = 15.0, 6.4 Hz, 4H), 0.55 (t, J = 8.2 Hz, 3H).

COMPOUND 160

[0322] (S)-N-((3S,6S,9S,12S,15R,18S,21S)-6,9, 18-tris(2-aminoethyl)-15-benzyl-3-((R)-l- hydroxyethyl)- 12-isobutyl-2,5 ,8, 11 , 14, 17,20-heptaoxo- 1 ,4,7, 10, 13 , 16, 19-heptaazacyclotricosan-21 - yl)pyrrolidine-3-carboxamide, Compund 160, was prepared according to the coupling then deprotection sequence described for Compound 89. LCMS: rt = 0.278/0.465 (LC method 3). m/z = 860 (M+l). ¾ NMR (400 MHz, D₂0) δ 7.41 - 7.31 (m, 3H), 7.26 (d, J = 7.0 Hz, 2H), 4.57 (t, J = 8.2 Hz, lH), 4.49 (dd, J = 8.6, 5.4 Hz, 1H), 4.36 - 4.15 (m, 6H), 3.53 - 3.27 (m, 6H), 3.08 (dd, J = 16.0, 9.0 Hz, 7H), 2.88 (s, 1H), 2.83 - 2.76 (m, 1H), 2.40 (dt, J = 13.8, 7.8 Hz, 1H), 2.29 - 2.08 (m, 5H), 2.05 - 1.82 (m, 4H), 1.52 - 1.35 (m, 2H), 1.20 (t, J = 10.2 Hz, 3H), 0.76 (s, 4H), 0.68 (d, J = 5.6 Hz, 3H).

161 -1 161 -2 161 -3

Example 161 Scheme

[0323] Step 1 : Intermediate (S)-2-amino-4-(lH-benzo[d]imidazol-2-yl)butanoic acid (161-2). A solution of benzene- 1,2-diamine (2 g, 18.5 mmol) and (2S)-2-aminopentanedioic acid (4.08 g, 27.7 mmol) in 5.5 M HC1 (20 mL) was stirred at 100 °C overnight. The crude mixture was filtered and the filtrate was basified by adding solid Na2CC>3 to adjust pH=l . The crude mixture was filtered and the filtrate was concentrated to give the title compound Intermediate 161-2. (1.2 g, 29.6% yield). LC-MS (LC method 1): m/z 220 (M+H)⁺.

[0324] Step 2: Intermediate (S)-4-(l-(tert-butoxycarbonyl)-lH-benzo[d]imidazol-2-yl)-2-(tert- butoxycarbonylamino)butanoic acid 161-3. To a solution of (2S)-2-amino-4-(lH-benzimidazol-2- yl)butanoic acid (600 mg, 2.74 mmol) in acetone (5 mL)/water (5 mL) was added a saturated solution of Na2CC>3 to adjust pH to pH=7, then aqueous NaOH (4 mL, 8.21 mmol) and B0C2O (1.79 g, 8.21 mmol) were added. The resulting mixture was stirred for 2 h at room temperature and keeping pH = 11-12. The crude mixture was carefully acidified with 1M HC1 to adjust the pH to 3, and then extracted with DCM (100 mL). The organic phase was washed with brine, dried over Na2SC>4, filtered and concentrated. The residue was purified by preparative TLC (PE : EA = 2 : 3) to give the title compound Intermediate 161-3. (550 mg, 47.9 % yield) as a white solid. LC-MS (LC method 1): m/z 420 (M+H)⁺.

[0325] Synthesis of Compound 161 : (S)-2-amino-4-(lH-benzo[d]imidazol-2-yl)-N- ((3S,6S,9S, 12S, 15R, 18S,21S)-6,9,18-tris(2-aminoethyl)-15-benzyl-3-((R)-l-hydroxyethyl)-12-isobutyi^ 2,5,8,11, 14,17,20-heptaoxo- 1,4,7, 10,13, 16, 19-heptaazacyclotricosan-21 -yl)butanamide (74-5) was prepared according to the coupling then deprotection sequence described for Compound 89. LCMS: rt = 0.37 (LC method 3). m/z = 963 (M+l). ¾ NMR (400 MHz, D₂0) δ 8.19 (s, 1H), 7.78 - 7.63 (m, 2H), 7.53 (m, 2H), 7.29 (m, 3H), 7.16 (d, J = 7.0 Hz, 2H), 4.50 (m, 2H), 4.41 (m, 2H), 4.30 (m, lH), 4.22 - 4.09 (m, 6H), 3.28 (m, 3H), 3.02 (m, 9H), 2.80 (m, 1H), 2.71 (m, lH), 2.45 (m, lH), 2.20 (m, 5H), 1.88 (m, 5H), 1.55 - 1.17 (m, 4H), 1.12 (d, J = 5.9 Hz, 4H), 0.76 (m, lH), 0.67 (m, 3H), 0.58 (m, 3H).

EXAMPLE 162. RIFAMPICIN POTENTIATION ASSAY

[0326] To evaluate the potentiation potency of compounds, susceptibility testing was performed using rifampicin as the partner antibiotic and in accordance with the methods described in CLSI M07-A10 "Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically; approved standard - tenth edition" with the following minor modifications.

[0327] Briefly, strains from Gram-negative pathogen Escherichia coli (ATCC25922) were streaked from frozen stocks for single colonies on tryptic soy agar plates and incubated at 37°C for 18 - 24 hours. From these plates, up to 10 individual colonies were resuspended in sterile saline or Mueller Hinton II broth (MHB-II) or 3 - 5 colonies were inoculated into 3 mL MHB-II broth and grown at 37°C until sufficiently turbid. Either of these suspensions was used as the starting inoculum after adjustment to 2 - 8 x 10⁵ CFU/mL in the assay.

[0328] Compounds were prepared by making a stock solution at -100-1000 times the highest concentration to be assayed. This stock solution was used to make two-fold serial dilutions in water or MHB-II in sterile, polystyrene 96 well microtiter assay plates for a total volume (including the bacterial inoculum) of 100 uL. Plates were incubated at 35-37°C for 16 - 20 hours and the MIC was defined as the lowest concentration of compound that completely inhibited visible growth.

[0329] Table I shows the intrinsic MIC of the example compounds, as well as the potentiation activity as a function of the MIC of rifampicin and 8 μg/mL of potentiator.

Table I- In vitro Data Table for E. coli ATCC25922

Cpd. # Intrinsic MIC MIC rifampicin* Cpd. # Intrinsic MIC MIC rifampicin* g mL) with 8 μg/mL of g mL) with 8 μg/mL of potentiator potentiator

69 >128 0.25 152 >128 0.125

70 >128 0.25 153 >128 0.125

71 >128 0.125 154 >128 4

72 >128 0.125 155 >128 0.063

73 >128 0.5 156 >128 0.063

74 8 <0.016 157 >128 >0.016

75 8 <0.016 158 2 >0.016

76 >128 0.25 159 >128 0.25

77 >128 0.125 160 >128 0.125

78 >128 0.063 161 1 >0.016

79 8 >0.016

80 128 0.25

81 >128 0.25

82 >128 8

83 >128 0.063

*MIC of rifampicin in ATC25922

Claims

CLAIMS What is claimed is:

1. A compou

(I), or a tautomer thereof, of a pharmaceutically acceptable salt of either of the foregoing, wherein:

R¹ is selected from hydrogen and optionally substituted C1-C4 alkyl; and

R² is selected from heteroaryl substituted with -N(R⁷)(R⁸) or

-C(0)-CH(CH(OH)CH₃)-N(R )-R⁴, and optionally further substituted; -C(0)-CH(CH₂OH)-X; and -C(0)-Y, or

R¹ and R² are taken together to form an oxo-substituted aryl or an oxo-substituted heterocyclyl, wherein the oxo-substituted aryl or oxo-substituted heterocyclyl is further substituted with -N(R⁷)-R⁸, -(C(0))i-₂-CH(CH(OH)CH₃)-N(R )-R⁴, and optionally further substituted, wherein:

X is selected from -heterocyclyl, -aryl, -Ci-C₈ alkyl, -C2-C8 alkenyl, -C2-C8

alkynyl, -0-(Ci-C₈ alkyl), -0-C(0)-(Ci-C₈alkyl), -CH₂-C(0)-(Ci-C₈ alkyl), and -C(0)-N(R )-Ci- C₈alkyl, wherein X is optionally substituted;

Y is selected from -N(R )-N(R )-C(0)-Ci-C₈ alkyl; -N(R )-(CH₂)o-i-C(0)-Ci-C₈ alkyl; -0-N(R )-C(0)-Ci-C₈ alkyl; -(CH₂)o-i-heteroaryl; -(CH₂)₀-i-heteroaryl-heteroaryl; -(CH₂)₀- l-aryl; -(CH₂)o-i-heterocyclyl; -(CH₂)o-i-carbocyclyl; wherein Y is optionally substituted;

each R³ is independently selected from hydrogen, optionally substituted C1-C4 alkyl, optionally substituted C3-Cecycloalkyl;

R⁴ is selected from -C(0)-R⁵, C1-C4 alkyl, heterocyclyl, cycloalkyl, -S(0)₂-R⁵, and -CH(R⁶)-R⁵;

R⁵ is selected from -C1-C4 alkyl, NH(Ci-C4 alkyl), N(Ci-C4 alkyl)₂, heterocyclyl and cycloalkyl, wherein R⁵ is optionally substituted; and

R⁶ is selected from hydrogen, -OH, and optionally substituted C1-C4 alkyl; R⁷ is selected from hydrogen and optionally substituted Ci- C4 alkyl;

R⁸ is selected from -C(0)-heterocyclyl, -heterocyclyl, -C(0)-aryl,

-C(0)NH-N(R )-C(0)R⁵, and -W-Q-Z, wherein:

W is selected from C(O), CH₂, P(0)(OH), and S(0)₂;

Q is selected from CH(R⁹) and optionally substituted phen-l,2-diyl, wherein R⁹ is selected from Ci-C4alkyl, -CH(OH)CH3, -CH(G¾)2, carbocyclyl, and -heterocyclyl; and

Z is selected from -C(0)N(R )(R¹⁰), -N(R )C(0)R⁵, -N(R )(R¹⁰), -N(R )S(0)₂-R⁵, - C(R )(R⁶)-R⁵, -N(R )-C(R )(R⁶)-R⁵, or -N(R )-C(R )=N-CN wherein, R¹⁰ is selected from C1-C4 alkyl, heterocyclyl and cycloalkyl, wherein R¹⁰ is optionally substituted.

2. The compound of claim 1, wherein R¹ is selected from hydrogen and methyl.

3. The compound of claim 1 or 2, wherein R² is a heteroaryl substituted with

-[N(R )]o-i-C(0)-CH(CHOHCH₃)-N(R )-R⁴; and R² is optionally further substituted.

4. The compound of claim 1 or 2, wherein R² is -C(0)-Y.

5. The compound of claim 4, wherein Y is -heteroaryl; -Grh-heteroaryl ;

-(C(CH₂CH₂))heteroaryl; -heteroaryl-heteroaryl; -Clrh-heteroaryl-heteroaryl; aryl, -Cfb-aryl; or -(C(CH₂CH₂))aryl; wherein the heteroaryl or aryl portion of Y is optionally substituted.

6. The compound of claim 5, wherein the aryl is phenyl and each heteroaryl is selected from pyridyl, triazolyl, and oxazolyl, each of which is optionally substituted with one or more substituents chosen from halogen, hydroxyl, Ci-C4alkyl, and -NHC(0)CH3.

7. The compound of claim 3, wherein R² is selected from:

9. The compound of claim 1 or 2, wherein

R² is -C(0)-CH(CH₂OH)-X; and X is -Ci-C₆alkyl, -C₂-C₆alkenyl, -C₂-C₆alkynyl, -0-C(0)-(Ci- Csalkyl), -C(0)-N(R )-Ci-Csalkyl, a 5- or 6-membered heteroaryl group; each of which X is optionally substituted with 1 or more substituents independently selected from halogen, hydroxyl, -CN, oxo, amino, Ci-Cealkyl, Ci-Cealkoxy, mono- and di-(Ci-C4alkyl)amino, mono- and di-Ci- C4alkylcarboxamide, Ci-C2haloalkyl; and Ci-C2haloalkoxy; and R³ is hydrogen or methyl.

10. The compound of claim 1 or 2, wherein R² is -C(0)-CH(CH20H)-X; and X is selected from 4-acetylamino-oxazol-2-yl, 6-acetylamino-pyridin-2-yl, 2-fluoro-3-acetylamino-phenyl, (Z)-CH=CH-CH(CH(OH)CH₃)-NH-C(0)-CH₃,

(E)-CH=CH-CH(CH(OH)CH₃)-NH-C(0)-CH₃,

-CH₂-CH(OH)-CH(CH(OH)CH₃)-NH-C(0)-CH₃,

-CH₂-C(0)-CH(CH(OH)CH₃)-NH-C(0)-CH₃,

-C(0)-CH₂-CH(CH(OH)CH₃)-NH-C(0)-CH₃,

-0-C(0)-CH(CH(OH)CH₃)-NH-C(0)-CH₃,

-C(0)-0-CH(CH(OH)CH₃)-NH-C(0)-CH₃,

-C(0)-NH-CH(CH(OH)CH₃)-NH-C(0)-CH₃,

-NH-C(0)-CH(CH(OH)CH₃)-NH-C(0)-CH₃,

-C≡C-CH(CH(OH)CH₃)-NH-C(0)-CH₃,

-CH₂-CH₂-CH(CH(OH)CH₃)-NH-C(0)-CH₃,

-0-CH₂-CH(CH(OH)CH₃)-NH-C(0)-CH₃, -CH₂-0-CH(CH(OH)CH₃)-NH-C(0)-CH₃,

-CH(OH)-CH₂-CH(CH(OH)CH₃)-NH-C(0)-CH₃, and

-CH(OH)-CH(OH)-CH(CH(OH)CH₃)-NH-C(0)-CH₃.

11. The compound of claim 1 or 2, wherein R² is -C(0)-Y, and Y is selected from: l-(4- chlorophenyl)cycloprop- 1 -yl, 4-ethylphenylmethyl, 2-(3-methyl- 1 ,2,4-triazol- 1 -yl)pyridin-4-yl, 3 -methylpyrazol- 1 -ylmethyl, 2-( 1 ,2,4-triazol- 1 -yl)pyridin-4-yl,

-N(CH₂CH₂OH)-NH-C(0)-CH(CH(OH)CH₃)-NH-C(0)-CH₃,

-0-NH-C(0)-CH(CH(OH)CH₃)-NH-C(0)-CH₃, and

-N(cyclopropyl)-CH₂-C(0)-CH(CH(OH)CH₃)-NH-C(0)-CH₃.

12. The compound of claim 1, wherein R¹ and R² are taken together to form an oxo-

The compound of claim 8, wherein R⁵ is methyl.

14. The compound of claim 1, wherein the compound is selected from

136

137

139

141

145

149

151

152

15. The compound of claim 4, wherein "-heteroaryl" is selected from

a pyrrolyl group, an imidazolyl group, a pyrazolyl group, a pyridinyl group, a pyrazinyl group, a pyrimidinyl group, a pyridazinyl group, an isoindolyl group, an indolyl group, an indazolyl group, a purinyl group, a quinolinyl group, an isoquinolinyl group, a benzoquinolinyl group, a phthalazinyl group, a naphthyridinyl group, a quinoxalinyl group, a quinazolinyl group, a cinnolinyl group, a carbazolyl group, a phenanthridinyl group, an acridinyl group, a

phenanthrolinyl group, a phenazinyl group, a benzoxazolyl group, a benzoimidazolyl group, a furanyl group, a benzoiuranyl group, a thiophenyl group, a benzothiophenyl group, a thiazolyl group, an isothiazolyl group, a benzothiazolyl group, an isoxazolyl group, an oxazolyl group, a triazolyl group, a tetrazolyl group, an oxadiazolyl group, a triazinyl group, a dibenzofuranyl group, a dibenzothiophenyl group, a benzocarbazolyl group, a dibenzocarbazolyl group, an imidazopyrimidinyl group, an imidazopyridinyl group, a pyridoindolyl group, a

benzofuropyridinyl group, a benzothienopyridinyl group, a pyrimidoindolyl group, a

benzopyrimidinyl group, a benzothienopyrimidinyl group, a phenoxazinyl group, a

pyridobenzoxazinyl group, and a pyridobenzothiazinyl group,

each optionally substituted with at least one selected from deuterium, -F, -CI, -Br, -I, a hydroxy group, a cyano group, a nitro group, an amino group, an amidino group, a hydrazine group, a hydrazone group, a carboxylic acid group or a salt thereof, a sulfonic acid group or a salt thereof, a phosphoric acid group or a salt thereof, a Ci-Cio alkyl group, a C2-C10 alkenyl group, a C2-C10 alkynyl group, a C1-C10 alkoxy group, a phenyl group, a biphenyl group, a terphenyl group, a naphthyl group, an anthracenyl group, a pyrenyl group, a phenanthrenyl group, a fluorenyl group, a carbazolyl group, a benzocarbazolyl group, a dibenzocarbazolyl group, a pyridinyl group, a pyrimidinyl group, a pyrazinyl group, a pyridazinyl group, a triazinyl group, a quinolinyl group, an isoquinolinyl group, a phthalazinyl group, a quinoxalinyl group, a cinnolinyl group, a quinazolinyl group, and -N(Qi)(Q2), wherein Qi, and Q2 are each independently selected from hydrogen, a C1-C10 alkyl group, or a Ce-Cn aryl group.

16. The compound of claim 1, wherein R² is:

17. A pharmaceutical composition comprising a compound or any one of claims 1 to 16, together with a pharmaceutically acceptable carrier.

18. A method for treating a bacterial infection in a patient, comprising administering a therapeutically effective amount of compound of any one of claims 1 to 17 to the patient.

19. A method of sensitizing bacteria to an antibacterial agent, comprising administering to a patient infected with the bacteria, simultaneously or sequentially, a therapeutically effective amount of the antibacterial agent and a compound of any one of claims 1 to 16.