WO2017189866A1 - Polymyxin analogs 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 A, 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 to a patient infected with the bacteria, simultaneously or sequentially, a therapeutically effective amount of the antibacterial agent and a compound of formula (I).

Description

POLYMYXIN ANALOGS USEFUL AS ANTIBIOTIC POTENTIATORS

CROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims priority from U.S. Provisional Application No. 62/328, 146, filed April 27, 2016, which is hereby incorporated by reference in its entirety.

TECI-!NICAL FIELD

[0002] The present disclosure provides polymyxin analogues useful for treating bacterial infections and/or useful for sensitizing bacteria, including Gram negative bactena, to the effects of other antibacterial agents and thereby increasing the efficacy of the other antibacterial agents.

BACKGROUND

[0003] Grain-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 die 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 polymyxa and related organisms. These cationtc drugs are rela tively simple peptides with molecular weights of about 1000. Polymyxins, such as polymyxin B, are decapeptide antibiotics, i.e., they are made often (10) aminoacyl residues. They are bactericidal and especially effective against Gram- negative bacteria such as Escherichia coli and other species oi Enterobacteriaceae, Pse domonas.

Acinetobacter baurtiannii, 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 Gram negative bacteria. Polymyxins have maintained their effectiveness against these emergent multiresistant strains of Gram-negative bacteria. Accordingly, polymyxins have been recalled to die therapeutic arsenal, although, due to their toxicity, they are considered a therapeutic of last resort. Their systemic (i.e., non-topical) use is, however, largely restricted to the therapy of life- threatening infections caused by multiply resistant strains of Pseudomon s aeruginosa and A. baiirnanrdi as well as by carbapenem-resistant Enterobacteriaceae .

[0006] Numerous efforts have been made to reduce the toxicity of polymyxin s, 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 derivatives 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 Gram negative bacteria or capable of potentiating the effects of other antibacterial agents against Gram negative bacteria without the toxicity of early polymyxin deri vatives. This disclosure fulfills this need and provides additional advantages described herein.

SUMMARY

[000 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 Ci- C4 alkyl.

[0010] R² is selected from -C(0)-heterocyclyl, -heterocyclyl, and -C(0)-aryl, each of which is optionally substituted; or R² is selected from -C(0)NH-N(R⁴)-C(0)R⁶ and -W-X-Y.

[0011] Or, R¹ and R² are taken together to form an optionally substituted oxo-substituted heterocyclyl.

[0012] W is selected from C(O), CH₂, P(0)(OH), and S(0)₂.

[0013] X is selected from CH(R³) and optionally substituted phen-l,2-diyl, wherein R³ is selected from -CH(OH)CH₃, -CH(CH₃)₂ and -heterocyclyl.

[0014] Y is selected from -C(0)N(R⁴)(R⁵), -N(R⁴)C(0)R⁶, -N(R⁴)(R⁵), -N(R⁴)S(0)₂-R⁶, - C(R⁴)(R⁷)-R⁶, -N(R⁴)-C(R⁴)=N-CN, and optionally substituted N-linked heterocyclyl. [0015] R⁴ is selected independently selected at each occurrence from hydrogen and optionally substituted C1-C4 alkyl and R⁵ is selected from C1-C4 alkyl, heterocyclyl and cycloalkyl, wherein R⁵ is optionally substituted; or R⁴ and R⁵ bound to the same nitrogen atom may be taken together to form an optionally substituted heterocyclyl group.

[0016] R⁶ is selected from C1-C4 alkyl, C₂-C₄alkenyl, C₂-C₄alkynyl, -NH(Ci-C₄ alkyl), N(Ci-C₄ alkyl)2, heterocyclyl and cycloalkyl, wherein R⁶ is optionally substituted.

[0017] R⁷ is selected from hydrogen, -OH, and optionally substituted C1-C4 alkyl.

The dashed line (— ) in formula I represents a stereospecific bond selected from (R) and (S).

[0018] The disclosure provides that when R¹ is hydrogen, W is C(O), X is -CH(OH)CH₃, and Y is -NHC(0)R⁶, then R⁶ is other than methyl, 2-aminocyclopentyl, cyclohexylhydroxymethyl, 1- cyclohexyl-l-aminoethan-2-yl, or 5-(sec-butyl)-piperidin-3-yl.

[0019] The disclosure further includes pharmaceutical compositions comprising a compound of formula I together with a pharmaceutically acceptable carrier.

[0020] 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. The compound of formula I may be administered as the only active agent or administered in combination with one or more additional active agents.

[0021] 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

[0022] 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.

[0023] The term "formula I" encompasses all compounds that satisfy formula I, including any enantiomers, racemates, stereoisomers, tautomers, as well as all pharmaceutically acceptable salts, of such compounds. "Formula I" includes all subgeneric groups of formula I unless clearly contraindicated by the context in which this phrase is used.

[0024] 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.

[0025] 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. The disclosure includes embodiments in which any one or more hydrogen atoms are replaced with deuterium atoms.

[0026] 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 and II 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.

[0027] 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.

[0028] 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.

[0029] "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.

[0030] "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.

[0031] "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.

[0032] "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.

[0033] "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.

[0034] "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.

[0035] "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.

[0036] "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.

[0037] 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. [0038] "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.

[0039] "Halo" or "halogen" indicates any of fluoro, chloro, bromo, and iodo.

[0040] "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.

[0041] "Haloalkoxy" indicates a haloalkyl group as defined herein attached through an oxygen bridge (oxygen of an alcohol radical).

[0042] 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, morpholinyl, piperazinyl, piperidinyl, and pyrrolidinyl. In certain embodiments a heterocycle is chosen from pyridinyl, pyrimidinyl, furanyl, thienyl, and pyrrolyl.

[0043] 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.

[0044] "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.

[0045] 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).

[0046] 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.

[0047] 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.

[0048] 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, moφholinyl, 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, 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.

[0049] 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.

[0050] "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.

[0051] "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.

[0052] 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.

[0053] 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.

[0054] A "patient" is a human or non-human animal in need of medical treatment.. In some embodiments the patient is a human patient.

[0055] "Providing" means giving, administering, selling, distributing, transferring (for profit or not), manufacturing, compounding, or dispensing.

[0056] "Treatment," as used herein includes providing a compound of this disclosure such as a compound of any of formula I, 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.

[0057] 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

[005

(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, and R⁷ 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.

[0059] (1) R¹ is selected from hydrogen and methyl.

[0060] (2) R² is -W-X-Y.

[0061] (3) R² is W-X-Y and is selected from: -C(0)NH-N(R⁴)-C(0)R⁶;

-C(0)-CH(R )-N(R⁴)-C(R⁴)=N-CN; -C(0)-CH(R )-N(R⁴)C(0)R⁶; -C(0)-CH(R )-N(R⁴)(R⁵);

-C(0)-CH(R )-C(R⁴)(R⁷)-R⁶; -C(0)-CH(R )-S(0)₂-R⁶; -C(0)-CH(R )-C(0)N(R⁴)(R⁵);

-P(0)(OH)-CH(R )-N(R⁴)C(0)R⁶, -S 0₂)-CH(R )-N(R⁴)C(0)R⁶;

-C(0)-CH(R )-N(R⁴)S(0)₂-R⁶; and

, wherein the phenyl moiety is optionally substituted. [0062] (4) R³ is an optionally substituted 4-, 5- or 6-membered heterocyclyl group having 1, 2, 3, or 4 heteroatoms independently chosen from N, O, and S .

[0063] (5) R³ is a heterocyclyl selected from oxazol-2-yl, l,2,4-oxadiazol-3-yl, pyridin-l-yl, azetidin- l-yl, and pyrrolidin-l-yl, and wherein R³ is optionally substituted.

[0064] (6) R³ is 5-methyl-oxazol-2-yl, 5 -methyl- l,2,4-oxadiazol-3-yl, 2-oxo-pyridin-l-yl, 2-oxo- azetidin-l -yl, and 2-oxo-pyrrolidin-l -yl.

[0065] (7) R³ is-CH(OH)CH₃ or -CH(CH₃)₂.

[0066] (8) R⁴ is independently selected at each occurrence from hydrogen and methyl.

[0067] (9) R⁵ is selected from methyl, -CH(CH₃)-CF₃, and -CH(CH₃)(CF₃)-CF₃.

[0068] (10) R⁶ is selected from -CH₃, -NHCH₃, -CH₂CF₃, tetrahydrofuranyl, furanyl, and cyclopropyl.

[0069] (1 1) R² is optionally substituted -C(0)heterocyclyl or optionally substituted -C(0)phenyl.

[0070] (12) R² is optionally substituted -C(0)heterocyclyl or optionally substituted -C(0)phenyl and the heterocyclyl of the -C(0)heterocyclyl is an optionally substituted 5- or 6-membered heterocyclyl group having group having 1, 2, 3, or 4 heteroatoms independently chosen from N, O, and S.

[0071] (13) R² is optionally substituted -C(0)heterocyclyl and heterocyclyl of the

-C(0)heterocyclyl is chosen from pyrimidinyl, oxazolyl, morpholinyl, piperidinyl, piperazinyl, thiomorpholinyl, isothiazolyl, pyrrolidinyl, oxadiazolyl, oxadiazolyl, oxadiazolyl substituted with benzyl, pyrazolyl, pyrazinyl, oxazolidinyl, isothiazolidinyl, imidiazolyl, pyridazinyl, pyridinyl, pyrrolyl, thiazolyl, thienyl, and furanyl, each of which is optionally substituted with one or more substituents independently chosen from 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.

[0072] (14) R² is selected from -C(0)heterocyclyl, -heterocyclyl, and -C(0)-phenyl, wherein each heterocyclyl is selected from pyrimidin-2-yl, oxazol-4-yl, morpholin-3-yl, isothiazol-3-yl, pyrrolidin-2-yl, l,2,4-oxadiazol-3-yl, pyrazol-4-yl, pyrazin-2-yl, oxazolidin-4-yl, isothiazolidin-3-yl, pyridazin-3-yl, pyridin-2-yl, and furan-2-yl; and wherein each heterocyclyl or phenyl is optionally substituted.

[0073] (15) R² is selected from -C(0)heterocyclyl, -heterocyclyl, and -C(0)-phenyl, wherein each heterocyclyl is selected from pyrimidin-2-yl, 2-acetylamino-pyrimidin-4-yl, 2-methyl-oxazol-4-yl, 2,5-dimethyl-oxazol-4-yl, 5-oxo-mo holin-3-yl, 5-methyl-isothiazol-3-yl, 5-oxo-pyrrolidin-2-yl, 4,4- dimethyl-5 -oxo-pyrrolidin-2-yl, 4-hydroxy-5 -oxo-pyrrolidin-2-yl, 2-hydroxymethyl-5 -oxo-pyrrolidin-2- yl, 3 -hydroxymethyl-5 -oxo-pyrrolidin-2-yl, 5 -methyl- 1 ,2,4-oxadiazol-3 -yl, 5 -methyl-2-oxo-oxazolidin-4- yl, 2-oxo-pyrrolidin-l-yl, 2-oxo-3 -benzyl -oxazolidin-4-yl, 3 -benzyl oxy-5-oxo-pyrrolidin-2-yl, 1,3,5- trimethyl-pyrazol-4-yl, 6-methyl-pyrazin-2-yl, 2-oxo-oxazolidin-4-yl, l, l-dioxo-isothiazolidin-3-yl, 1, 1- dioxo-4-hydroxy-isothiazolidin-3-yl, l, l-dioxo-4-hydroxymethyl-isothiazolidin-3-yl, 6-methyl-pyridazin- 3-yl, 6-acetylamino-pyridazin-3-yl, 6-methyl-pyridin-2-yl, 6-acetylamino-pyridin-2-yl, 2,5- dimethylfuran-3-yl, 4,5-dimethylfuran-2-yl, and the phenyl is 2-acetylamino-5-fluoro-phenyl.

[0074] (16) R¹ and R² are taken together to form a ring selected from pyrrolidinyl and pyridinyl, wherein the ring is optionally substituted.

[0075] (17) R¹ and R² are taken together to form a ring selected from 2-oxo-3-aminoacetyl-4- hydroxymethyl-pyrrolidinyl, 2-oxo-3-aminoacetyl-pyrrolidinyl and 2-oxo-3-acetylamino-pyridinyl.

[0076] (18) The disclosure also includes compounds in which, when R² is W-X-Y and W is - C(O), X is CH(OH)CH₃ and Y is -NR⁴C(0)R⁶, the R⁶ is not methyl, 2-aminocyclopentyl,

cyclohexylhydroxymethyl, l-cyclohexyl-laminoethan-2-yl, or 5-(sec-butyl)piperidin-3-yl.

[0077] (19) The disclosure also includes compounds in which when R² is W-X-Y and W is - C(O), X is CH(OH)CH₃ and Y is -NR⁴C(0)R⁶, the R⁶ is not methyl, ethyl, or a group containing a cyclopentyl, cyclohexyl, or piperidinyl moiety.

[0078] (20) R² is selected from optionally substituted -C(0)-heterocyclyl, optionally

substituted -heterocyclyl, and substituted -C(0)-aryl; or R² is selected from -C(0)NH-N(R⁴)-C(0)R⁶ and - W-X-Y. Wherein the substituted aryl is substituted with one or more substituents independently chosen from 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 and at least one substituent is other than methyl.

[0079] (21) R² is selected from -C(0)-heterocyclyl, -heterocyclyl, and -C(0)-aryl; or R² is selected from -C(0)NH-N(R⁴)-C(0)R⁶ and -W-X-Y and when R² is W-X-Y, and W is C(O), X is - CH(OH)CH₃ or -CH(CH₃)₂ and Y is-N(R⁴)(R⁵), N(R⁴)C(0)R⁶, or -N(R⁴)-S(0)₂-R⁶, R⁵ is not alkyl and R⁶ is not alkyl or -NH(alkyl).

[0080] (22) R² is -W-X-Y, wherein

[0081] W is C(O);

[0082] X is CH(R³), wherein R³ is selected from -CH(OH)CH₃, and

[0083] Y is -N(R⁴)C(0)R⁶, wherein R⁴ at each occurrence is hydrogen, and R⁶ 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, or -N(Qi)(Q2), wherein Qi, and Q2 are each independently selected from hydrogen, a C1-C10 alkyl group, and a Ce-Cn aryl group.

[0084] (23) R² carries the definition given in definition (22) wherein R⁶ is represented by one of the following formulae:

[0085] (24). The compound of Formula 1, wherein "-heterocyclyl" in any of the embodiments listed herein which recite a heterocyclyl group, 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.

[0086] (25) The compound of claim 1, wherein R² is selected from -C(0)-heterocyclyl, wherein a "-heterocycl l" is selected from one of the following formulae:

PHARMACEUTICAL COMPOSITIONS

[0087] The disclosure includes a pharmaceutical composition containing at least one compound of formula I as the active agent together with a pharmaceutically acceptable carrier.

[0088] 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.

[0089] 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

[0090] 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.

[0091] 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.

[0092] In some embodiments, the bacterial infection or antibiotic-tolerant or antibiotic-resistant infection is caused by a Gram-negative bacterium.

[0093] 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.

[0094] In some embodiments, the Gram-negative bacterium is a. Pseudomonas, e.g., P.

aeruginosa.

[0095] In some embodiments, the Gram-negative bacterium is Burkholderia species.

[0096] In some embodiments, the Gram-negative bacterium is Acinetobacter, e.g., A. baumannii.

[0097] 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 marcescens, e.g., Salmonella typhimurium, e.g., Shigella dysenteriae, e.g., Proteus mirabilis, e.g., Citrobacter freundii, e.g., Yersinia pestis.

[0098] 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.

[0099] 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.

[0100] 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.

[0101] 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.

[0102] 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.

[0103] In some embodiments, the Gram-negative infection is caused by Pseudomonas aeruginosa.

[0104] In other embodiments the disclosure includes treating an infection caused by Gram- positive bacteria, such as Staphylococcus epidermidis and Staphylococcus aureus. [0105] In some embodiments, the subject is a trauma patient or a burn patient suffering from a burn or skin wound.

[0106] 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.

[0107] In some embodiments, the method further includes identifying said subject suffering from an infection with bacteria resistant to antimicrobial therapy.

[0108] The disclosure includes methods of treatment in which a compound or composition of the disclosure is administered orally, topically, intravenously, parenterally, or inhaled.

[0109] 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

[0110] LC Method 1. The retention times (Rt) were obtained on an Acquity SDS system with an Agilent Ultimate XB-C18 4.6 x 50 nm, 5μιη column. A gradient of H₂0 (H₂0 : CFLCN : 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.

[0111] LC Method 2. The retention times (Rt) were obtained on an Acquity SDS system with an Agilent Ultimate XB-C18 4.6 x 50 nm, Sum column. A gradient of H?0 (¾G : CH₃CN : formic acid = 90 : 10 : 0.05)/C¾CN (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 min and held for 0.5 min (1.0 mL/min. as solvent flow) at an oven temperature of 35 °C.

[0112] LC Method 3. The retention times (Rt) were obtained on an Acquity SDS system with an Welch Ultimate XB-C18 4.6 x 50 nm, Sum column. A gradient of H∑0 (I : CHsC 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 rain (1 .0 mL/min. as solvent flow) at an oven tempera ture of 35 °C. [0113] NMR spectra were obtained on a 400 MHz high-resolution speciroineter using the indicated solvent.

ABBREVIATIONS

HOBt 1 -Hydroxybenzotriazole TBAF tetrabutylammonium fluoride

Hz hertz TBDPSC1 tert-butyl diphenylsilyl chloride

i-PrOH Isopropyl alcohol TBDPSO tert- butyldiphenylsilyloxy

J dipole dipole coupling value TBTU 0-(benzotriazol- 1 -yl)- Ν,Ν,Ν',Ν'- tetramethyunronium tetrafluoroborate

KOH potassium hydroxide TEA Trimethylamine

K₂C0₃ potassium carbonate TFA trifluoroacetic acid

LC liquid chromatography TsCl 4-toluenesulfonyl chloride

LCMS liquid chromatography mass μιη Micrometers

spectrometry

LiHMDS lithium hexamethyldisilazide Zn Zinc

LiOH lithium hydroxide

[01 14] Synthesis of key synthetic Intermediate A- di-fert-butyl (((2S,5R$S,\ \S,\4S,nS,22S)- 22-amino-5 -benzyl- 1 1 -(2-((teri-butoxycarbonyl)amino)ethyl)- 17-((R)- 1 -hydroxyethyl)-8-isobutyl- 3,6,9, 12, 15, 18,23-heptaoxo-l,4,7, 10, 13, 16, 19-heptaazacyclotricosane-2, 14-diyl)bis(ethane-2, l- diyl))dicarbamate. Intermediate A was prepared in a two-step process, from commercially available polymyxin B according to the procedure described in Synthesis (2015) 47: 2088-2092.

[0115] LCMS: rt = 1.03min (LC method 3). m/z = 1062 (M+l). ¾ NMR (400 MHz, D₂0) δ 8.68 (d, J= 8.0 Hz, 1H), 8.37 (d, J= 8.0 Hz, 1H), 8.30 (d, J= 4.0 Hz, 1H), 8.05 (s, 1H), 7.72 (s, 1H), 7.25 - 7.15 (m, 5H), 6.82 (s, 1H), 6.63 - 6.57 (m, 2H), 4.86 (d, J= 5.2 Hz, 1H), 4.43 - 4.42 (m, 1H), 4.35 (q, J = 8.0 Hz, 1H), 4.15 - 4.06 (m, 2H), 3.98 - 3.90 (m, 2H), 3.15 - 2.78 (m, 12H), 1.99 - 1.37 (m, 40H), 1.02 (d, J= 8 Hz, 4H), 0.73 (d, J= 8.0 Hz, 3H), 0.66 (d, J= 8.0 Hz, 3H).

[0116] Compound 1 is not compound of Formula I. The synthesis of this compound is provided to exemplify a synthetic method by which many compounds of Formula I can be prepared.

[0117] 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), Intermediate A (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 the title compound. (40 mg, 4.5 % yield), LCMS: rt =1.96 minutes (LC method 1), 1 185 (M+l).

[0118] 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) .

[0119] 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) in DCM (5 mL) 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 vacuum and the solid was freeze-dried to give the title compound (30 mg, 95 % yield). LCMS: rt = 0 26 and 0.62 minutes (double peak, LC method 2). m!z --- 885 (M+ 1). 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, l lH), 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).

[0120] Synthesis of (3^,6^9^12^15i?,18^21¾-21-amino-6,9,18-tris(2-aminoethyl)-15-benzyl- 3-((i?)-l-hydroxyethyl)-12-isobutyl-l,4,7,10, 13,16, 19-heptaazacyclotricosane-2,5,8, l l, 14,17,20-heptaone (Compound 2). To a solution of intermediate A (80 mg, 0.08 mmol) in DCM (3 mL) was added TFA (0.75 mL), dropwise. The resulting mixture was stirred at room temperature. After the reaction was complete, the crude mixture was concentrated to dryness, and the residue was re-dissolved in 10% aq. formic acid solution (5 mL). The solvent was removed in vacuo and the solid was freeze-dried to give the title compound (60 mg, 93 % yield). LCMS: rt = 2.084 min (LC method 3). m/z = 962 (M+l). 1H NMR (400 MHz, D₂0) δ 8.16 (si 1H), 7.29 - 7.00 (m, 3H), 7.23 - 7.20 (m, 2H), 4.60 (t, J= 8.0 Hz, 1H), 4.64 (dd, J= 8.0, 5.8 Hz, 1H), 4.26 - 4.11 (m, 5H), 3.96 (t, J= 6.8 Hz, 1H), 3.31 - 3.16 (m, 2H), 3.10 - 2.94 (m, 6H), 2.79 - 2.63 (m, 2H), 2.24 -2.09 (m, 4H), 2.02 - 1.82 (m, 4H), 1.42 (dd, J= 9.8, 4.8 Hz, 2H), 1.11 (d, J= 6.4 Hz, 3H), 0.96 (m, 1H), 0.70 (dd, J= 8.0, 6.4 Hz, 6H).

D 3

[0121] ( ^-amino-S-hydroxy-N-^S^^S, \2S, 15R, 18S,2 lS)-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 3) was prepared according to the coupling then deprotection sequence described for Example 2, Compound 1. LCMS: rt = 0.284 min (LC method 2). m/z = 849.6 (M+l).

[0122] 'H NMR (400 MHz, D₂0) δ 7.27 - 7.23 (m, 3H), 7.20 - 7.12 (m, 2H), 4.44 (t, J= 8.2 Hz, 1H), 4.35 (dd, J= 8.8, 5.6 Hz, 1H), 4.24 - 4.17 (m, 1H), 4.16 -4.01 (m, 6H), 3.86 (qd, J= 12.4, 4.8 Hz, 2H), 3.33 - 3.23 (m, 1H), 3.06 - 2.85 (m, 7H), 2.81 - 2.72 (m, 1H), 2.69 - 2.61 (m, 1H), 2.17 - 1.96 (m, 4H), 1.90 - 1.71 (m, 4H), 1.36 - 1.23 (m, 2H), 1.04 (d, J= 6.2 Hz, 3H), 0.72 - 0.50 (m, 7H). 4

[0123] (2S,3i?)-2-amino-3-hydroxy-N-((i?)-3-hydroxy- 1 -oxo- 1 -(((3S,6S,9S,12S, 15R, 18^,21.S)- 6,9J8 ris(2-aminoethyl)-15-benzyl-3 (i?)-l-hydroxyemyl)-12-isobutyl-2,5,841J447,20-heptaoxo- l,4,7,10, 13,16, 19-heptaazacyclotricosan-21-yl)amino)propan-2-yl)butanamide was prepared according to the coupling then deprotection sequence described for Example 2, Compound 1. LCMS: rt = 2.71 min (LC method 3). m/z = 951 (M+l). ¾ NMR (400 MHz, D₂0) δ 7.39 - 7.09 (m, 5H), 4.41 (s, 1H), 4.12 (s, 5H), 3.98 - 3.72 (m, 2H), 2.97 (d, J= 31.8 Hz, 8H), 1.26 (d, J= 6.2 Hz, 6H), 1.12 (d, J= 6.4 Hz, 3H), 0.87 (t, J= 73 Hz, 3H), 0.72 (s, 3H), 0.63 (d, J= 6.7 Hz, 3H).

EXAMPLE 6. SYNTHESIS OF COMPOUND 5

[0124] (2,S',3i?)-2-acetamido-3 -hydroxy-N-((5)-3 -hydroxy- 1 -oxo- 1 - (((3S,6S,9S, US, 15R, 185,215)-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)amino)propan-2- yl)butanamide (Compound 5) was prepared according to the coupling then deprotection sequence described for compound 15, Example 7, below. EXAMPLE 7. SYNTHESIS OF COMPOUND 15

[0125] N-((2S,3R)-3-hydroxy-l-((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-ylamino)- 1 -oxobutan-2-yl) furan-2- carboxamide (Compound 15) is prepared according to the following procedure.

[0126] Step 1. Synthesis of (R)-methyl 2-((2S,3R)-2-(tert-butoxycarbonylamino)-3-hydroxy butanamido)-3-(tert-butyldiphenylsilyloxy)propanoate (Intermediate Compound 8). To a solution of (2S,3R)-2-(tert-butoxycarbonylamino)-3-hydroxybutanoic acid 6 (3 g, 13.68 mmol) and (R)-methyl 2- amino-3-(tert-butyldiphenylsilyloxy) propanoate 7 (4.89 g, 13.68 mmol) in anhydrous DMF (40 mL) was added DIPEA (3.54 g, 27.37 mmol), HOBt (2.22 g, 16.42 mmol) and HBTU (6.23 g, 16.42 mmol). The reaction mixture was stirred at room temperature for 2 h. The crude mixture was poured into water (100 mL) and extracted with ethyl acetate (500 mL). The organic layer was dried over anhydrous Na2SC>4, filtered, and then concentrated to dryness. The residue was purified by silica gel column (PE : EA = 5 : 2) to give title compound 8 (3.5 g, 45% yield). LC-MS (LC method 1): m/z 559 (M+l)⁺.

[0127] Step 2. Synthesis of (R)-methyl 2-((2S,3R)-2-amino-3-hydroxybutanamido)-3-(tert- butyldiphenylsilyloxy)propanoate (Intermediate compound 9). To an ice water cooled solution of (R)- methyl 2-((2S,3R)-2-(tert-butoxycarbonylamino)-3-hydroxybutanamido)-3-(tert-butyldiphenylsilyloxy) propanoate 8 (500 mg, 0.89 mmol) in DCM (10 mL) at room temperature was added TFA (2 mL, 1.79 mmol). The resulting mixture was stirred at this temperature for 3h. The crude mixture was basified with aq. NaHCC to adjust the pH to 7-8. The resulting mixture was extracted with DCM (100 mL). The organic layer was dried over anhydrous Na2SC>4, filtered, and then concentrated to dryness. The residue was purified by silica gel column (PE : EA = 5:2) to give title compound 9 (350 mg, 85.2% yield).

LC-MS (LC method 1): m/z 459 (M+l)⁺.

[0128] Step 3. Synthesis of (R)-methyl 3-(tert-butyldiphenylsilyloxy)-2-((2S,3R)-2-(furan-2- carboxamido)-3-hydroxybutanamido)propanoate (Intermediate Compound 11). To a solution of (R)- methyl 2-((2S,3R)-2-amino-3-hydroxybutanamido)-3-(tert-butyldiphenylsilyloxy)propanoate 9 (200 mg, 0.44 mmol) and furan-2-carboxylic acid 10 (200 mg, 0.44 mmol) in anhydrous DCM (5 mL) was added DIPEA (112.5 mg, 0.87 mmol) and HBTU (247.9 mg, 0.65 mmol). The reaction mixture was stirred at room temperature for 2 h. The crude mixture was poured into water (100 mL) and extracted with ethyl acetate (50 mL). The organic layer was dried over anhydrous Na2SC>4, filtered, and then concentrated to dryness. The residue was purified by silica gel column (PE : EA = 5 : 2) to give title compound 11 (205 mg, 46% yield). LC-MS (LC method 1): m/z 553 (M+l)⁺.

[0129] Step 4. Synthesis of (R)-3-(tert-butyldiphenylsilyloxy)-2-((2S,3R)-2-(furan-2-carbox amido)-3-hydroxybutanamido)propanoic acid (Intermediate Compound 12). To a solution of (R)-methyl 3-(tert-butyldiphenylsilyloxy)-2-((2S,3R)-2-(furan-2-carboxamido)-3-hydroxybutanamido)propanoate 11 (205 mg, 0.37 mmol) in THF (4 mL) was added aq. LiOH (31.16 mg, 0.74 mmol in 1 mL H₂0) at 0 °C. The reaction mixture was stirred at room temperature for 1 h. The crude mixture was then acidified with IN HC1 to adjusted pH to 3-4. The resulting mixture was extracted with ethyl acetate (20 mL). The organic layer was washed with brine, dried over anhydrous Na2SC>4, filtered, and then concentrated to give Compound 12 (180 mg, 90% yield). LC-MS (LC method 1): m/z 539 (M+l)⁺.

[0130] Step 5. Synthesis of tert-butyl 2,2',2"-((2S,5R,8S, l lS, 14S, 17S,22S)-5-benzyl-22-((R)-3- (tert-butyldiphenylsilyloxy)-2-((2S,3R)-2-(furan-2-carboxamido)-3-hydroxy butanamido)propanamido)- 17-((R)-l-hydroxyethyl)-8-isobutyl-3,6,9, 12,15, 18,23-heptaoxo-l,4,7, 10, 13,16, 19- heptaazacyclotricosane-2, l l,14-triyl)tris (ethane-2, l-diyl)tricarbamate (Intermediate Compound 13). To a solution of (R)-3 -(tert-butyldiphenylsilyloxy)-2-((2S,3R)-2-(furan-2-carboxamido)-3 - hydroxybutanamido)propanoic acid 12 (180 mg, 0.33 mmol) and Intermediate A (350 mg, 0.33 mmol) in anhydrous DMF (4 mL) was added DIPEA (86.21 mg, 0.67 mmol), HOBt (54.17 mg, 0.4 mmol) and HBTU (151.98 mg, 0.4 mmol). The reaction mixture was stirred at room temperature overnight for 5h. The crude mixture was poured into brine (10 mL). The resulting mixture was extracted with ethyl acetate (50 mL). The organic layer was dried over anhydrous Na2SC>4, filtered, and then concentrated to dryness to give crude title compound 13 (310 mg). LC-MS (LC method 1): m/z 1582.8 (M+l)⁺.

[0131] Step 6. Synthesis of tert-butyl 2,2',2"-((2S,5R,8S,l lS,14S, 17S,22S)-5-benzyl-22-((R)-2- ((2S,3R)-2-(furan-2-carboxamido)-3-hydroxybutanamido)-3-hydroxypro panamido)-17-((R)-l- hydroxyethyl)-8-isobutyl-3,6,9, 12,15, 18,23-heptaoxo-l,4,7,10, 13, 16, 19-heptaazacyclotricosane-2, l l,14- triyl)tris(ethane-2, l-diyl) tricarbamate (Intermediate Compound 14). To a solution of tert-butyl 2,2',2"- ((2S,5R,8S, 1 IS, 14S, 17S,22S)-5-benzyl-22-((R)-3-(tert-butyldiphenylsilyloxy)-2-((2S,3R)-2-(furan-2- carboxamido)-3 -hydroxy butanamido)propanamido)- 17-((R)- 1 -hydroxyethyl)-8-isobutyl- 3,6,9, 12, 15,18,23-heptaoxo-l,4,7, 10, 13, 16, 19-heptaazacyclotricosane-2, 11, 14-triyl)tris(ethane-2, l- diyl)tricarbamate 13 (290 mg, 0.18 mmol) in THF (4 mL) was added TBAF (0.37 mL, 1 M THF solution). The resulting mixture was stirred at room temperature overnight. The crude mixture was quenched with aq. NH4CI (50 mL). The resulting mixture was extracted with ethyl acetate (50 mL). The organic layer was separated, dried over anhydrous Na2SC>4, filtered, and then concentrated to dryness. The residue was purified by prep. HPLC (eluting by CFLCN/water, additive: formic acid) to give title compound 14 (120 mg, 48.72% yield). LC-MS (LC method 1 ): m/z 1344.7 (M+l)⁺.

[0132] Step 7. Synthesis of N-((2S,3R)-3-hydroxy-l-((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-iso butyl- 2,5,8,11, 14,17,20-heptaoxo- 1, 4,7, 10,13, 16, 19-heptaazacyclotricosan-21 -ylamino)propan-2-ylamino)- 1 - .

[0133] Compound 15, was prepared according to the deprotection sequence described

Compound 1. LCMS: rt === 0.39 minutes (LC method 2). m z == 1044.5 (M+I). ¾ NMR (400 MHz, D₂0) δ 8.07 (s, 1H), 7.73 (d, J= 8.6 Hz, 1H), 7.67 (d, J= 8.6 Hz, 1H), 7.61 - 7.53 (m, 1H), 7.50 - 7.43 (m, 1H), 7.28 - 7.18 (m, 3H), 7.12 (d, J= 7.4 Hz, 2H), 4.44 (t, J= 8.2 Hz, 1H), 4.40 - 4.31 (m, 1H), 4.23 - 3.99 (m, 8H), 3.98 - 3.82 (m, 3H), 3.40 - 3.15 (m, 3H), 3.16 - 2.82 (m, 10H), 2.81 - 2.49 (m, 3H), 2.22 - 1.98 (m, 5H), 2.02 - 1.90 (m, 4H), 1.91 - 1.63 (m, 5H), 1.46 - 1.21 (m, 3H), 1.14 (ddd, J= 20.5, 13.5, 6.9 Hz, 1H), 1.03 (t, J= 5.6 Hz, 6H), 0.70 (s, 1H), 0.62 (d, J= 6.0 Hz, 3H), 0.55 (d, J= 6.0 Hz, 3H).

16

[0134] N-((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,l l, 14,17,20-heptaoxo-l,4,7, 10, 13,16, 19- heptaazacyclotricosan-21-ylamino)propan-2-yl) pyrimidine-2-carboxamide (Compound 16) was prepared according to the coupling then deprotection sequence described for Example 7, Compound 15.

[0135] LCMS: rt = 0.54/0.85 minutes (double peak) (LC method 3). m z = 956 (M+l). ¾ NMR (400 MHz, D₂0) δ 8.99 (dd, J= 14.0, 4.8 Hz, 2H), 7.70 (t, J= 4.8 Hz, 1H), 7.32 - 7.19 (m, 5H), 4.56 (d, J= 6.4 Hz, 2H), 4.49 - 4.26 (m, 5H), 4.15 - 3.97 (m, 4H), 3.82 - 3.68 (m, lH), 3.12 - 2.94 (m, 9H), 2.67 - 2.53 (m, 1H), 2.29 (dd, J= 16.0, 9.2 Hz, 1H), 2.15 (dd, J= 17.2, 11.6 Hz, 2H), 2.09 - 1.95 (m, 4H), 1.54 - 1.44 (m, 1H), 1.39 - 1.27 (m, 3H), 1.22 - 1.12 (m, 3H), 0.88 (d, J= 6.8 Hz, 1H), 0.65 (dd, J = 19.0, 12.6 Hz, 6H).

17

[0136] N-((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,l l, 14,17,20-heptaoxo-l,4,7, 10, 13,16, 19- heptaazacyclotricosan-21-ylamino)propan-2-yl)-l,3,5-trimethyl-lH-pyrazole-4-carboxamide (Compound 17) was prepared according to the coupling then deprotection sequence described for Example 7, Compound 15. LCMS: ft = 0.41 minutes (LC method 3). m/z = 986 (M+1 ). ¾ NMR (400 MHz, D₂0) δ 7.29 (m, 3H), 7.19 (m, 2H), 4.55 - 4.44 (m, 2H), 4.40 (dt, J= 11.6, 6.0 Hz, 1H), 4.35 - 4.19 (m, 3H), 4.16 - 4.07 (m, 3H), 3.88 (t, J= 6.6 Hz, 2H), 3.63 (s, 3H), 3.42 - 3.26 (m, 1H), 3.15 - 2.95 (m, 7H), 2.82-2.71 (m, 2H), 2.36-2.20 (m, 9H), 2.02 - 1.75 (m, 5H), 1.37 (dt, J= 13.6, 8.0 Hz, 2H), 1.11 (t, J= 5.2 Hz, 3H), 0.82 (s, 1H), 0.71 (dd, J= 6.4, 2.6 Hz, 3H), 0.67 - 0.59 (m, 3H).

EXAMPLE 10. SYNTHESIS OF COMPOUND 18

[0137] N-((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,ll,14,17,20-heptaoxo-l,4,7,10,13,16,19- heptaazacyclotricosan-21 -ylamino)propan-2-yl)-6-methylpyrazine-2-carboxamide was prepared according to the coupling then deprotection sequence described for Example 7, Compound 15. LCMS: rt = 1.45 minutes (LC method 2). m/z = 969.6 (M+l). ¾ NMR (400 MHz, D₂0) δ 8.80 (s, 1H), 8.56 (s, 1H), 7.25 - 7.16 (m, 3H), 7.10 - 7.08 (m, 2H), 4.53 (dt, J= 10.2, 6.8 Hz, 2H), 4.40 (dd, J= 9.2, 5.0 Hz, 1H), 4.35- 4.32 (m, 3H), 4.06 - 4.00 (m, 3H), 3.90 - 3.89 (d, J= 4.0 Hz, 1H), 3.30 - 3.28 (m, 1H), 3.10 - 2.85 (m, 8H), 2.77-2.67 (m, 2H), 2.17 - 2.02 (m, 3H), 1.88 - 1.76 (m, 3H), 1.31 - 1.24 (m, 2H), 1.03 (d, J = 6.0 Hz, 3H), 0.63-0.52 (m, 7H).

19

[0138] N-((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,ll,14,17,20-heptaoxo-l,4,7,10,13,16,19- heptaazacyclotricosan-21-ylamino)propan-2-yl)-6-methylpyridazine-3-carboxamide, Compound 19, was prepared according to the coupling then deprotection sequence described for Example 7, Compound 15. LCMS: rt = 0.44/0.61 minutes (double peak) (LC method 3). mz = 969 (M+l). ¾ NMR (400 MHz, D₂0) δ 8.07 (t, J= 8.0 Hz, 1H), 7.76 (d, J= 8.8 Hz, lH), 7.28 (dd, J= 12.8, 6.8 Hz, 3H), 7.17 (d, J= 7.2 Hz, 2H), 4.67 - 4.59 (m, 2H), 4.51 - 4.38 (m, 2H), 4.33 - 4.18 (m, 3H), 4.15 - 4.05 (m, 3H), 3.98 (d, J = 4.8 Hz, 2H), 3.45 -3.34 (m, 1H), 3.12 - 2.91 (m, 7H), 2.88 - 2.80 (m, lH), 2.79- 2.72 (m, lH), 2.69 (s, 3H), 2.29-2.10 (m, 3H), 2.03 - 1.83 (m, 4H), 1.42- 1.26 (m, 2H), 1.10 (d, J= 5.8 Hz, 3H), 0.66(td,J = 18.0, 5.8 Hz, 7H).

20

[0139] N-((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,ll,14,17,20-heptaoxo-l,4,7,10,13,16,19- heptaazacyclotricosan-21-ylamino)propan-2-yl)-6-methylpicolinamide (Compound 20) was prepared according to the coupling then deprotection sequence described for Example 7, Compound 15. LCMS: rt = 2.03 minutes (LC method 3). m/z = 968 (M+l). ¾NMR (400 MHz, D₂0) δ 8.13 (t, J= 8.0 Hz, 1H), 7.98 (d, J= 7.8 Hz, 1H), 7.69 (d, J= 8.0 Hz, 1H), 7.29 (dq, J= 14.2, 7.2 Hz, 3H), 7.18 (d, J= 6.8 Hz, 2H), 4.59 (t, J= 5.2 Hz, 1H), 4.51 - 4.38 (m, 2H), 4.33 - 4.07 (m, 6H), 3.96 (d, J= 5.2 Hz, 2H), 3.41 - 3.31 (m, 1H), 3.11 -2.92 (m, 7H), 2.87 - 2.79 (m, lH), 2.73 (dd,J= 17.8, 11.0 Hz, 1H), 2.64 (s, 3H), 2.26-2.09 (m, 3H), 2.06- 1.80 (m, 5H), 1.35 (qd,J= 13.8, 6.4 Hz, 2H), 1.11 (d,J=6.2Hz, 3H), 0.77- 0.60 (m, 7H).

21

[0140] N-((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,ll,14,17,20-heptaoxo-l,4,7,10,13,16,19- heptaazacyclotricosan-21-ylamino)propan-2-yl)-2,5-dimethylfuran-3-carboxamide (Compound 21) was prepared according to the coupling then deprotection sequence described for Example 7, Compound 15.

LCMS: rt = 0.58 minutes (LC method 3). m/z = 972 (M+l). ¾ NMR (400 MHz, D₂0) δ 7.43 - 7.33 (m, 3H), 7.26 (d, J= 7.2 Hz, 2H), 6.30 (s, 1H), 4.60 - 4.46 (m, 3H), 4.41 - 4.25 (m, 3H), 4.19 (ddd, J= 14.8, 9.8, 5.4 Hz, 3H), 3.94 (dd, J= 9.2, 5.2 Hz, 2H), 3.47 - 3.36 (m, 1H), 3.18 - 3.03 (m, 7H), 2.93 - 2.74 (m, 2H), 2.42 (d, J= 12.8 Hz, 3H), 2.34 - 2.18 (m, 6H), 2.13-1.88(m, 5H), 1.69 - 1.31 (m, 3H), 1.18 (dd, J = 6.0, 2.8 Hz, 3H), 0.84 - 0.66 (m, 7H).

22

[0141] N-((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,l l, 14,17,20-heptaoxo-l,4,7, 10, 13,16, 19- heptaazacyclotricosan-21-ylamino)propan-2-yl)-4,5-dimethylfuran-2-carboxamide (Compound 22) was prepared according to the coupling then deprotection sequence described for Example 7, Compound 15. LCMS: rt = 2.37 minutes (LC method 3). m/z = 972 (M+l). ¾ NMR (400 MHz, D₂0) δ 7.35 - 7.24 (m, 3H), 7.21 - 7.15 (m, 2H), 6.97 (d, J= 2.4 Hz, 1H), 4.47 (dt, J= 10.2, 6.8 Hz, 2H), 4.39 (dd, J= 9.2, 5.0 Hz, 1H), 4.32 - 4.17 (m, 3H), 4.14 - 4.06 (m, 3H), 3.92 - 3.84 (m, 2H), 3.41 - 3.29 (m, 1H), 3.11 - 2.91 (m, 7H), 2.86-2.68(m, 2H), 2.29 - 2.07 (m, 6H), 2.05 - 1.92 (m, 3H), 1.92 - 1.76 (m, 5H), 1.44 - 1.29 (m, 2H), 1.10 (d, J= 6.0 Hz, 3H), 0.75-0.60 (m, 7H).

23

[0142] N-((2S,3R)-3-hydroxy-l-((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-iso butyl-2,5,8, 11,14, 17,20-heptaoxo- 1, 4, 7, 10, 13, 16, 19-heptaazacyclotricosan-21 -ylamino)propan-2-ylamino)- 1 -oxobutan-2- yl)cyclopropanecarboxamide (Compound 23) was prepared according to the coupling then deprotection sequence described for Example 7, Compound 15. LCMS: rt = 0.22 minutes (LC method 2). m/z = 1018.6 (M+l). ¾ NMR (400 MHz, D₂0) δ 8.19 (s, 1H), 7.36 - 7.24 (m, 3H), 7.20 (d, J= 6.7 Hz, 2H), 4.50 - 4.00 (m, 14H), 3.80 (m, 4H), 3.36 (s, 1H), 3.16 - 2.90 (m, 8H), 2.78 (d, J= 38.2 Hz, 2H), 2.33 - 1.50 (m, 11H), 1.29 (m, 4H), 1.16 (d, J= 6.4 Hz, 3H), 1.11 (t, J= 4.8 Hz, 3H), 0.98 (s, 1H), 0.91 - 0.77 (m, 3H), 0.70 (s, 4H), 0.62 (d, J= 4.0 Hz, 2H).

24

[0143] (S)-N-((2S,3R)-3-hydroxy-l-((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, l l, 14,17,20-heptaoxo- 1, 4, 7, 10, 13, 16, 19-heptaazacyclotricosan-21 -ylamino)propan-2-ylamino)- 1 -oxobutan-2- yl)tetrahydrofuran-2-carboxamide (Compound 24) was prepared according to the coupling then deprotection sequence described for Example 7, Compound 15. LCMS: rt = 0.45 minutes (LC method 2). m/z = 1048.6 (M+l). ¾ NMR (400 MHz, D₂0) δ 8.08 (s, 2H), 7.29 - 7.16 (m, 3H), 7.12 (d, J= 6.7 Hz, 2H), 4.47 - 4.36 (m, 1H), 4.28 (dd, J= 8.8, 5.1 Hz, 3H), 4.24 - 3.98 (m, 8H), 3.89 (d, J= 6.5 Hz, 1H), 3.82 - 3.69 (m, 3H), 2.92 (d, J= 8.2 Hz, 7H), 2.78 - 2.55 (m, 2H), 2.27 - 1.68 (m, 14H), 1.36 - 1.24 (m, 2H), 1.18 (t, J= 5.6 Hz, 2H), 1.11 - 1.00 (m, 7H), 0.84 - 0.78 (m, 3H), 0.67 - 0.60 (m, 3H), 0.55 (t, J = 6.7 Hz, 3H).

25

[0144] 4S,5R)-N-((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,l l, 14, 17,20-heptaoxo- 1, 4, 7, 10, 13, 16, 19-heptaazacyclotricosan-21 -ylamino) propan-2-yl)-5 -methyl -2 -oxooxazolidine-4- carboxamide (Compound 25) was prepared according to the coupling then deprotection sequence described for Example 7, Compound 15. LCMS: rt = 0.25 minutes (LC method 2). m/z = 976.6

(M+l). ¾ NMR (400 MHz, D₂0) δ 7.19 (m, 5H), 4.49 - 3.94 (m, 10H), 3.75 (d, J= 5.4 Hz, 2H), 3.36 - 3.18 (m, 1H), 3.03 - 2.56 (m, 9H), 2.23 - 1.64 (m, 9H), 1.41 - 1.20 (m, 5H), 1.06 (dd, J= 18.3, 6.4 Hz,3H), 0.80 - 0.45 (m, 7H).

26

[0145] (2S,3R)-3-hydroxy-N-((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- l,4,7,10, 13,16, 19-heptaazacyclotricosan-21-yl amino)propan-2-yl)-2-(3,3,3- trifluoropropanamido)butanamide (Compound 26) was prepared according to the coupling then deprotection sequence described for Example 7, Compound 15. LCMS: rt = 0.41 minutes (LC method 2). m/z = 1060.6 (M+l). ¾ NMR (400 MHz, D₂0) δ 8.18 (s, 2H), 7.32 (dd, J= 14.5, 6.9 Hz, 5H), 7.22 (d, J= 6.8 Hz, 3H), 4.60 - 4.29 (m, 10H), 4.30 - 4.03 (m, 10H), 3.82 (dd, J= 18.4, 12.6 Hz, 4H), 3.37 (dd, J = 19.2, 10.1 Hz, 4H), 3.21 - 2.93 (m, 13H), 2.81 (m, 4H), 2.67 (s, lH), 2.57 (t, J= 5.6 Hz, 1H), 2.19 (s, 5H), 1.93 (m, 10H), 1.40 (s, 4H), 1.21 (m, 14H), 0.84 - 0.56 (m, 12H).

27

[0146] (2S,3R)-3-hydroxy-N-((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 -yl amino)propan-2-yl)-2-(2-oxopyrrolidin- 1 -yl)butanamide (Compound 27) was prepared according to the coupling then deprotection sequence described for Example 7, Compound 15. LCMS: rt = 0.32 minutes (LC method 2). m/z = 1018.6 (M+l). ¾ NMR (400 MHz, D₂0) δ 7.36 - 7.09 (m, 5H), 4.49 (t, J= 8.4 Hz, 1H), 4.43 - 4.02 (m, 10H), 3.82 (d, J= 5.0 Hz, 2H), 3.58 (s, 2H), 3.33 (s, 1H), 3.02 (t, J= 15.5 Hz, 7H), 2.64 (s, 3H), 2.49 - 1.67 (m, 12H), 1.39 (s, 2H), 1.14 (dd, J= 21.0, 6.0 Hz, 6H), 0.91 - 0.52 (m, 7H).

28

[0147] 2-acetamido-5-fluoro-N-((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-iso butyl-2,5,8, 11,14, 17,20-heptaoxo- 1,4,7,10, 13, 16, 19-heptaazacyclotricosan-21-ylamino)propan-2-yl)benzamide (Compound 28) was prepared according to the coupling then deprotection sequence described for Example 7, Compound 15. LCMS: rt = 0.21 minutes (LC method 2). m/z = 1028.6 (M+l). ¾ NMR (400 MHz, D₂0) δ 8.07 (s, 1H), 7.34 - 7.06 (m, 8H), 4.71 (d, J= 30.0 Hz, 3H), 4.49 - 4.16 (m, 4H), 4.16 - 3.85 (m, 5H), 3.80 (d, J= 5.4 Hz, 2H), 3.30 (ddd, J= 14.4, 9.7, 5.1 Hz, 1H), 3.20 - 2.85 (m, 7H), 2.85 - 2.53 (m, 2H), 2.31 - 1.72 (m, 11H), 1.38 - 1.24 (m, 2H), 1.03 (d, J= 6.2 Hz, 3H), 0.63 (d, J= 6.4 Hz, 3H), 0.55 (d, J= 6.3 Hz, 2H).

29

[0148] (S)-2-acetamido-N-((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-iso butyl-2,5,8, 11,14, 17,20-heptaoxo- 1, 4, 7, 10, 13, 16, 19-heptaazacyclotricosan-21 -ylamino)propan-2-yl)-3 -methylbutanamide (Compound 29) was prepared according to the coupling then deprotection sequence described for Example 7, Compound 15. LCMS: rt = 0.34 minutes (LC method 3). m/z = 990.6 (M+l). ¾ NMR (400 MHz, D₂0) δ 7.23 (m, 3H), 7.12 (m, 2H), 4.49 - 4.21 (m, 4H), 4.08 - 3.81 (m, 4H), 3.81 - 3.53 (m, 2H), 3.31 - 2.58 (m, 10H), 2.66 (s, 1H), 2.12 - 1.79 (m, 9H), 1.79 - 1.74 (m, 1H), 1.22 (d, J= 66.3 Hz, 2H), 1.10 - 0.90 (m, 3H), 0.82 (dd, J= 9.6, 5.0 Hz, 6H), 0.67 - 0.50 (m, 6H).

[0149] Step 1. Synthesis of Intermediate Compound 33. To a stirred suspension of D-serine methyl ester hydrochloride 30 (5 g, 40.7 mmol) and ethyl acetimidate hydrochloride 31 (6.18 g, 50 mmol) in CH2CI2 (100 mL) was added a solution of triethylamine (12.4 mL, 89.5 mmol) in CH2CI2 (35 mL) dropwise during 25 min, then reaction mixture was stirred at rt for 16 h. The crude mixture was filtered and the filter cake was washed with ether. The filtrate was concentrated to dryness to give the crude 4,5- dihydro-2-methyloxazole-4-carboxylic acid methyl ester 32 as yellow oil. Hexamethylenetetramine (14.3 g, 101.8 mmol) and l,8-diazabicyclo[5.4.0]undec-7-ene (DBU) (15.2 mL, 101.8 mmol) were added to a stirred suspension of CuBr2 (22.7 g, 101.8 mmol) at 0 °C. After 20 min, the above crude 4,5-dihydro-2- methyloxazole-4-carboxylic acid methyl ester 32 was added, and the reaction mixture was stirred at room temperature for 2 h. The crude mixture was concentrated to dryness, the residue was partitioned between EtOAc (150 mL) and saturated aqueous M Cl/saturated aqueous NH4OH (1 : 1, 100 mL). The aqueous layer was extracted with EtOAc (150 mL x 2), the combined organic layers were washed with saturated aqueous NH₄C1 : saturated aqueous NH₄OH (1 : 1, 100 mL), 10% citric acid (100 mL), NaHC0₃ (100 mL), and brine, dried over anhydrous Na2SC>4, filtered and concentrated to dryness. The residue was purified by silica gel column (PE : EA = 5 : 1) to give the title compound methyl 2-methyloxazole-4- carboxylate 33 (1.2 g, 20% yield). LC-MS (LC method 1): m/z 142 (M+l)⁺. ¾ NMR (CDC1₃) δ 2.54 (s, 3H), 3.93 (s, 3H), 8.16 (s, 1H).

[0150] Step 2. Synthesis of Intermediate Compound 34. To a solution of methyl 2- methyloxazole-4-carboxylate 33 (1.2 g, 8.5 mmol) in the mixture of THF (18 mL), methanol (6 mL) and water (6 mL) at room temperature was added LiOH (5 10 mg, 21.3 mmol). The reaction mixture was stirred at room temperature for 2 h. The crude mixture was acidified by 2M HC1 to adjust pH to 5. The resulting mixture was extracted with DCM/i-PrOH (100 mL, 3 : 1 v/v). The organic layer was washed with brine, dried over anhydrous Na2SC>4, filtered, and then concentrated to give the crude title compound 34, 2-methyloxazole-4-carboxylic acid, (800 mg) without further purification. LC-MS (LC method 1): m/z 128 (M+l)⁺.

Compound 40

[0152] N-((R)-3-hydroxy-l-oxo-l-((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, l l, 14, 17,20-heptaoxo- l,4,7, 10, 13, 16, 19- heptaazacyclotricosan-21-ylamino)propan-2-yl)-2-methyloxazole-4-carboxamide (Compound 40) was prepared according to the coupling, hydrolysis, the second coupling then deprotection sequence described for example 7, Compound 15. LCMS : rt = 0.36 minutes (LC method 3). m/z = 959 (M+l). ¾ NMR (400 MHz, D₂0) δ 8.54 (s, 1H), 8.38 (d, J= 10.8 Hz, 1H), 7.29 (dt, J= 18.6, 7.4 Hz, 5H), 4.59 - 4.54 (m, 1H), 4.51 - 4.15 (m, 6H), 4.14 - 3.88 (m, 4H), 3.81 - 3.64 (m, lH), 3.15-2.95 (m, 8H), 2.5 1 (d, J = 6.8Hz, 3H), 2.32 - 2.22 (m, 1H), 2.21 - 1.82 (m, 7H), 1.52 (t, J= 12.0 Hz, 1H), 1.40 - 1.27 (m, 2H), 1.18 (dt, J= 18.4, 9.2 Hz, 3H), 0.98 - 0.81 (m, lH), 0.76-0.65 (m, 6H). EXAMPLE 23. SYNTHESIS OF COMPOUND 52

[0153] Step 1. Synthesis of Intermediate Compound 43. To a solution of L-Serine 41 (2.3 g, 17.32 mmol) in 2M NaOH (50 mL) was added 4-methoxybenzaldehyde 42 (2 g, 19.05mmol). The resulting mixture was stirred at room temperature overnight. After cooling to 0 °C, Sodium borohydride (1.64 g, 26 mmol) was added portionwise such that an internal temperature of below 10 °C. After addition the reaction mixture was allowed to stir at room temperature for 1 h. The crude mixture was extracted with Et20 (100 mL) and the aqueous phase was acidified by Cone. HC1 to adjust pH to 5. The resultant white precipitate was filtered and washed with water. The product was dried under vacuum to give the title compound 43, (S)-3-hydroxy-2-(4-methoxybenzylamino)propanoic acid (3.5 g, 89.7%). LC-MS (LC method 1): m/z 226 (M+l)⁺. [0154] Step 2. Synthesis of Intermediate Compound 44. To a solution of (S)-3-hydroxy-2-(4- methoxybenzylamino)propanoic acid 43 (3.5 g, 15.56 mmol) in aq. NaOH solution (0.82 g in 40 mL) at 0°C was slowly added chloroacetyl chloride (2.55 mL, 31. 12 mmol). The reaction mixture was allowed to warm to room temperature and stirred for 30 minutes. Additional NaOH solution (30 wt %) was added to keep pH= 13. The resulting mixture was stirred at room temperature overnight. The crude mixture was acidified by 2 M HCl to adjust pH to 2-3. The reaction mixture was extracted with ethyl acetate (100 mL). The organic layer was dried over anhydrous Na2SC>4, filtered, and concentrated to dryness to give the title compound 44, (S)-4-(4-methoxybenzyl)-5-oxomorpholine-3-carboxylic acid (2.356 g) without further purification. LC-MS (LC method 1): m/z 266 (M+l)⁺.

[0155] Step 3. Synthesis of Intermediate Compound 45. To a solution of (S)-4-(4 - methoxybenzyl)-5-oxomoφholine-3-carboxylic acid 44 (2.356 g, 8.96 mmol) in DMF (20 mL), was added BnBr (3.4 g, 17.92 mmol) and K2CO3 (3.709 g, 26.88 mmol). The reaction mixtures were stirred at room temperature overnight. The crude mixture was diluted with water (200 mL), the reaction mixture was extracted with ethyl acetate (100 mL). The organic layer were dried over anhydrous Na2SC>4, filtered, and concentrated. The crude was purified using silica gel column (PE : EA = 1 : 1) to give the title compound 45, (S)-benzyl 4-(4-methoxybenzyl)-5-oxomoφholine-3-carboxylate, (1.36 g, 43%). LC-MS (LC method 1): m/z 356 (M+l)⁺.

[0156] Step 4. Synthesis of Intermediate Compound 46. To an ice water cooled solution of (S)- benzyl 4-(4-methoxybenzyl)-5-oxomorpholine-3-carboxylate 45 (886 mg, 2.5 mmol) in the mixture of CH₃CN (1 1 mL) and water (1 1 mL), was added CAN (6.84 g, 12.5 mmol). The reaction mixture was stirred at this temperature for 1 h. The crude mixture was diluted with water ( 100 mL), the reaction mixture was extracted with ethyl acetate (100 mL). The organic layer was washed with brine, dried over anhydrous Na2SC>4, filtered, and then concentrated to dryness. The residue was purified with silica gel column (PE : EA = 1 : 1) to give the intermediate, (S)-benzyl 5-oxomorpholine-3-carboxylate (517 mg). To a mixture of (S)-benzyl 5-oxomorpholine-3-carboxylate (517 mg, 2.2 mmol) in MeOH (20 mL) was added 10% Pd/C ( 16 mg). The reaction was stirred under an atmosphere of hydrogen overnight at room temperature. The crude mixture was filtered through a Celite pad and concentrated in vacuo to give the title compound the crude (S)-5-oxomorpholine-3-carboxylic acid 46 (400 mg). LC-MS (LC method 1): m/z 146 (M+l)⁺. d 52

[0158] (S)-N-((R)-3-hydroxy-l -oxo-l-((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, l l, 14, 17,20-heptaoxo-l,4,7, 10, 13, 16, 19- heptaazacyclotricosan-21-ylamino)propan-2-yl)-5-oxomoφholine-3-carboxamide (Compound 52) was prepared according to the coupling, hydrolysis, the second coupling then deprotection sequence described for example 7, Compound 15. LCMS: rt = 0.32 minutes (LC method 3). m/z = 977 (M+l). ¾ NMR (400 MHz, D₂0) δ 7.19 (m, 5H), 4.44 (t, J= 8.0 Hz, 1H), 4.35 - 4.19 (m, 3H), 4.16 - 3.82 (m, 10H), 3.81 - 3.66 (m, 2H), 3.29 (t, J= 10.4 Hz, 1H), 3.05 - 2.80 (m, 7H), 2.72 (m, 1H), 2.63 (m, lH), 2.24 - 1.95 (m, 4H), 1.97 - 1.60 (m, 5H), 1.42 - 1.15 (m, 3H), 1.03 (d, J= 6.0 Hz, 3H), 0.76 (m, 1H), 0.57 (ddd, J = 63.1, 31.8, 27.3 Hz, 6H).

[0159] Step 1. Synthesis of Intermediate Compound 54. To an ice water cooled solution of benzyl 3-oxobutanoate 53 (1.46 g, 7.6 mmol) in glacial AcOH (5 mL) stirred at 0 °C was added a solution of NaNC (1.31 g, 19.0 mmol) in water (4 mL) dropwise over a period of 1 h. The resulting mixture was stirred at amibient temperature overnight. The reaction mixture was then partitioned between ethyl acetate (100 mL) and water (100 mL). The organic extract was separated, washed with brine, dried over anhydrous Na2SC>4 and concentrated, the residue was purified by silica gel column (PE : EA = 5 : 1) to give (E)-benzyl 2-(hydroxyimino)-3-oxobutanoate 54 (1.68 g, 99% yield). LC-MS (LC method 1): m/z 222 (M+l)⁺.

[0160] Step 2. Synthesis of Intermediate Compound 55. Zn powder (2.47 g, 38 mmol) was slowly added to a solution of (E)-benzyl 2-(hydroxyimino)-3-oxobutanoate 53 (1.68 g, 7.6 mmol) and acetic anhydride (1.9 g, 19 mmol) in glacial AcOH (4 mL), and the resulting mixture was stirred room temperature for 3 h. The crude mixture was poured into ice water, filtrated through Celite, and the solid residue washed with dichloromethane (50 mL). The organic layer was separated, and the aqueous mixture was extracted with dichloromethane (50 mL). The combined organic layers were washed with brine and dried over anhydrous Na2SC>4 and concentrated to dryness. The residue was purified by silica gel column (PE : EA = 5 : 1) to give the title compound 54, benzyl 2-acetamido-3-oxobutanoate (700 mg, 40% yield). LC-MS (LC method 1): m/z 250 (M+l)⁺.

[0161] Step 3. Synthesis of Intermediate Compound 56. Triethylamine (323 mg, 3.2 mmol) was added to a solution of triphenylphosphine (419 mg, 1.6 mmol) and iodine (406 mg, 1.6 mmol) in dry dichloromethane (5 mL) and stirred for 30 min. Then a solution of the benzyl 2-acetamido-3- oxobutanoate 55 (200 mg, 0.8 mmol) in dry dichloromethane (4 mL) was added and the reaction mixture stirred until completion of reaction (followed by TLC). The crude mixture was concentrated to dryness, the residue was purified by silica gel column (PE : EA = 5 : 1) to give the title compound 55, benzyl 2,5- dimethyloxazole-4-carboxylate (0.15 g, 81% yield) LC-MS (LC method 1): m/z 232 (M+l)⁺.

[0162] Step 4. Synthesis of Intermediate Compound 57. Pd/C (10% w/w, 150 mg) was added to a solution of benzyl 2,5-dimethyl oxazole-4-carboxylate 56 (2.1 g, 9 mmol) in dry ethyl acetate (10 mL) and stirred at room temperature under atmospheric pressure of hydrogen overnight. The catalyst was filtered off through Celite and washed with methanol (2 mL). The filtrates were concentrated to dryness to give title compound 57, 2,5-dimethyloxazole-4-carboxylic acid (1.1 g, 84% yield). LC-MS (LC method 1) m/z 142 (M+l)⁺.

Compound 63

[0164] N-((R)-3-hydroxy- l-oxo-l-((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, l l, 14, 17,20-heptaoxo- l,4,7, 10, 13, 16, 19- heptaazacyclotricosan-21 -ylamino)propan-2-yl)-2,5 -dimethyloxazole-4-carboxamide (Compound 63) was prepared according to the coupling, hydrolysis, the second coupling then deprotection sequence described for example 7, Compound 15. LCMS : rt = 0.29/0.42 minutes (double peak) (LC method 3). m/z = 973 (M+l). ¾ NMR (400 MHz, D₂0) δ 7.29 (m, 3H), 7.18 m, 2H), 4.57 - 4.36 (m, 3H), 4.33 - 4.17 (m, 3H), 4.16 - 4.01 (m, 3H), 3.97 - 3.75 (m, 2H), 3.35 (m, 1H), 3.20 - 2.92 (m, 8H), 2.86 - 2.74 (m, 2H), 2.45 (s, 3H), 2.35 (s, 3H), 2.29 - 2.10 (m, 3H), 2.07 - 1.73 (m, 5H), 1.57 (m, 1H), 1.48 - 1.24 (m, 3H), 1.19 (t, J = 7.2 Hz, 3H), 1.10 (t, J= 5.5 Hz, 1H), 0.87 (t, J= 7.4 Hz, 1H), 0.80 - 0.51 (m, 7H).

[0165] Step 1. Synthesis of Intermediate Compound 66. To the solution of furan-2-carbonitrile 64 (5 g, 53.7 mmol) and pentane-2,4-dione 65 (6.45 g, 53.7 mmol) in toluene (100 mL), was added AICI3 (7.1 g, 53.7 mmol). The resulting mixture was stirred at 100 °C for 4 h. After cooling the reaction, the crude mixture was quenched with 4 M HC1 and stirred for 4 h. The resulting mixture was extracted with ethyl acetate (3 x 100 mL) The combined organic phase were washed with brine, dried over anhydrous Na2SC>4, filtered and concentrated to dryness. The residue was purified by silica gel column (PE : EA = 4 : 1) to give title compound 66, (Z)-4-amino-4-(furan-2-yl)but-3-en-2-one (2 g, 25% yield). LC-MS (LC method 1): m/z 152 (M+l)⁺ _.

[0166] Step 2. Synthesis of Intermediate Compound 67. To a solution of compound 66 (1.5 g, 10 mmol) in THF (30 mL) was added P2S5 (1.11 g, 5 mmol). The resulting mixture was stirred at room temperature for 36 h. The crude mixture was concentrated to dryness, the residue was dissolved in MTBE (30 mL), treated with 30% H2O2 (20 mL), the reaction mixture was stirred for additional 30 h. Then the crude mixture was quenched with aq. Na2S2C>3 carefully, extracted with ethyl acetate (100 mL x 2). The combined organic phase were washed with brine, dried over anhydrous Na2SC>4, filtered and concentrated to dryness. The residue was purified by silica gel column (PE : EA = 40 : 1) to give title compound 67, 3- (furan-2-yl)-5-methylisothiazole (0.4 g, 24% yield). LC-MS (LC method 1): m/z 166 (M+l)⁺

[0167] Step 3. Synthesis of Intermediate Compound 68. To a mixture of 3-(furan-2-yl)-5- methylisothiazole 67 (0.4 g, 2.4 mmol) in acetone 10 (mL) and water (20 mL), was added KMn04 (0.77 g, 4.8 mmol). The reaction mixture was stirred for 1.5 h at room temperature. Then 2 M NaOH (25 mL) was added and heated to 50 °C for 1.5 h. The resulting mixture was acidified with 2M HCl and extracted with ethyl acetate (100 mL). The organic extract was washed with brine, dried over anhydrous Na2SC>4, filtered and concentrated to give title compound 68, 5 -methylisothiazole-3 -carboxylic acid (0.2 g).

LC-MS (LC method 1): m/z 144 (M+l)⁺.

74

[0169] N-((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,l l, 14,17,20-heptaoxo-l,4,7, 10, 13,16, 19- heptaazacyclotricosan-21 -ylamino)propan-2-yl)-5 -methylisothiazole-3 -carboxamide (Compound 74) was prepared according to the coupling, hydrolysis, the second coupling then deprotection sequence described for example 7, Compound 15. LCMS: rt = 0.4 minutes (LC method 3). m/z = 974 (M+l). ¾ ΝΜΡν (400 MHz, D₂0) δ 7.47 (s, 1H), 7.36 - 7.22 (m, 3H), 7.18 (d, J= 7.4 Hz, 2H), 4.57 - 4.01 (m, 9H), 3.92 (t, J= 12.5 Hz, 2H), 3.51 - 3.31 (m, 1H), 3.18 - 2.65 (m, 13H), 2.55 (s, 3H), 2.30 - 1.77 (m, 8H), 1.67 - 1.49 (m, 4H), 1.42 - 1.20 (m, 5H), 1.16 - 1.02 (m, 3H), 0.86 (dd, J= 12.7, 7.3 Hz, 5H), 0.74 - 0.53 (m, 7H).

[0170] Step 1. Synthesis of Intermediate Compound 76. To a solution of L-pyroglutamic acid 1 (13 g, 100 mmol) in anhydrous ethanol (50 mL) was slowly added SOC (16.7 g, 130 mmol at 0°C, and the reaction mixture was stirred at this temperature for additional 3 h. The crude mixture was concentrated to dryness, the residue was dissolved in DCM (300 mL). The organic layer was washed brine and dried over anhydrous Na2SC>4, filtered and concentrated to dryness to give title compound 76, (S)-ethyl 5-oxopyrrolidine-2-carboxylate (13 g, 82% yield). (LC method 1): m/z 158 (M+l)⁺.

[0171] Step 2. Synthesis of Intermediate Compound 77. To a solution of (S)-ethyl 5- oxopyrrolidine-2-carboxylate 76 (15 g, 95.54 mol) and dimethylaminopyridine ( 1.166 g, 9.554 mol) in dichloromethane (40 mL) was added B0C2O (20.85 g, 95.54 mol). The resulting mixture was stirred at room temperature overnight. The crude mixture was washed with saturated NH4CI solution. The organic phase was separated, dried over anhydrous Na2SC>4, filtered and concentrated. The residue was purified by silica gel column (PE : EA = 5 : 1) to give title compound 77, (S)-l-tert-butyl 2-ethyl 5-oxopyrrolidine- 1,2-dicarboxylate (22 g, 89.6% yield) . LC-MS (LC method 1): m/z 258 (M+l)⁺.

[0172] Step 3. Synthesis of Intermediate Compound 78. To a solution of (S)-l-tert-butyl 2-ethyl 5-oxopyrrolidine-l,2-dicarboxylate 77 (5.1 g, 20 mmol) in dry THF (100 mL) stirred at -78 °C was added lithium hexamethyldisilazide (44 mL, 44 mmol, 1 M in THF). After the reaction mixture was stirred at - 78 °C for 1 hour, was added a solution of iodomethane (6.2 g, 44 mmol) in dry THF (100 mL) at -78 °C and the mixture was allowed to stir for 15 minutes at this temperature and 2 hours at 25 °C. The crude mixture was quenched with saturated NH4CI solution and extracted with dichloromethane (500 mL). The organic layer was dried over anhydrous Na2SC>4, filtered, and concentrated to dryness. The residue was purified by silica gel column (PE : EA= 500 : 1 to 2: 1) to give title compound 78, (S)-l-tert-butyl 2-ethyl 4,4-dimethyl-5-oxopyrrolidine-l,2-dicarboxylate (4.1 g, 71%). 1H NMR δ: 4.38-4.34 (m, 1 H), 4.08-4.05 (m, 2 H), 2.09-2.03 (m, 1 H), 1.77-1.73 (m, 1 H), 1.35 (s, 9H), 1.12 (t, J= 8 Hz, 3H), 1.06 (s, 6H).

LC-MS (LC method 1): m/z 286 (M+l)⁺.

[0173] Step 4. Synthesis of Intermediate Compound 79. To a solution of (S)-l-tert-butyl 2-ethyl 4,4-dimethyl-5-oxopyrrolidine-l,2-dicarboxylate 78 (570 mg, 2 mmol) in DCM (30 mL), was added TFA (1.5 mL). The reaction mixtures were stirred at room temperature overnight. The crude mixture was concentrated to dryness to give title compound 79, (S)-ethyl 4,4-dimethyl-5-oxopyrrolidine-2- carboxylate (400 mg, 95% yield) without further purification. LC-MS (LC method 1): m/z 186 (M+l)⁺.

[0174] Step 5. Synthesis of Intermediate Compound 80. To a solution of (S)-ethyl 4,4- dimethyl-5-oxopyrrolidine-2-carboxylate 79 (400 mg, 2.6 mmol) in EtOH (5 mL) at 0 °C was added aq. LiOH (5 mL, 4M solution). The reaction mixture was stirred at room temperature for 2 h. The crude mixture was acidified with 2M HC1 to adjust the pH to ~5. The resulting mixture was extracted with DCM/i-PrOH (50 mL, 3 : 1 v/v). The organic layer was washed with brine, dried over anhydrous Na2SC>4, filtered, and concentrated to dryness to give title compound 80, (S)-4,4-dimethyl-5-oxopyrrolidine-2- carboxylic acid 6 (200 mg, 49% yield). LC-MS (LC method 1): m/z 158 (M+l)⁺.

86

[0176] (S)-N-((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,l l, 14, 17,20-heptaoxo-l ,4,7, 10, 13,16, 19- heptaazacyclotricosan-21 -ylamino)propan-2-yl)-4,4-dimethyl-5 -oxopyrrolidine-2-carboxamide

(Compound 86) was prepared according to the coupling, hydrolysis, the second coupling then deprotection sequence described for example 7, Compound 15. LCMS: rt = 0.33 minutes (LC method 2). m/z = 988.6 (M+l). ¾ NMR (400 MHz, D₂0) δ 7.44 - 6.92 (m, 5H), 4.44 (t, J= 8.2 Hz, 1H), 4.37 - 4.17 (m, 4H), 4.15 - 3.92 (m, 4H), 3.74 (d, J= 5.2 Hz, 2H), 3.27 (m, 1H), 2.96 (t, J= 19.5 Hz, 7H), 2.80 - 2.53 (m, 2H), 2.27 (dd, J= 13.0, 8.8 Hz, 1H), 2.20 - 2.00 (m, 3H), 1.99 - 1.57 (m, 6H), 1.50 (s, lH), 1.42 - 1.15 (m, 3H), 1.14 - 0.91 (m, 8H), 0.77 (m, 1H), 0.59 (dd, J= 30.4, 6.1 Hz, 6H).

[0177] Step 1. Synthesis of Intermediate Compound 88. To a solution of ethyl cyanoformate 87 (4 g, 40 mmol) in the mixture of ethanol (43 mL) and water (27 mL), was added hydroxylamine hydrochloride (5.6 g, 80 mmol) and sodium carbonate (3.3 g, 40 mmol). The resulting mixture was stirred at room temperature for lOh. After completion of the reaction, the crude mixture was concentrated to dryness, the residue was extracted with methylene chloride ( 100 mL x 3) The combined organic phase were washed with brine , dried anhydrous Na2SC>4, filtered and concentrated to dryness to give title compound 88, (Z)-ethyl 2-amino-2-(hydroxyimino)acetate (2 g, 38% yield) without further purification. LC-MS (LC method 1): m/z 133 (M+l)⁺.

[0178] Step 2. Synthesis of Intermediate Compound 89. A mixture of compound 88 (1 g, 7.5 mmol) and AC2O (5 mL) was stirred at room temperature for 30 min. The crude mixture was concentrated to give title compound 89, (Z)-ethyl 2-(acetoxyimino)-2-aminoacetate (700 mg), which used in the next step without purification.

[0179] Step 3 Synthesis of Intermediate Compound 90. A mixture of compound 89 (644 mg, 3.7 mmol) and HO Ac (3 mL) was heated at 130 °C under microwave for 1.5 h. The crude mixture was concentrated to dryness, the residue was purified by silica gel column (DCM : MeOH = 20 : 1) to give title compound 90, ethyl 5-methyl-l,2,4-oxadiazole-3-carboxylate (500 mg, 78% yield). LC-MS (LC method 1): m/z 157 (M+l)⁺.

[0180] Step 4. Synthesis of Intermediate Compound 91. To a solution of compound 90 (780 mg, 5 mmol) in EtOH (1 mL) was added 10 M KOH (1 mL) at room temperature. After being stirred at room temperature for 15 min, the reaction mixture was filtered to give the title compound potassium 5-methyl- l,2,4-oxadiazole-3-carboxylate 5 (500 mg, 64% yield). LC-MS (LC method 1): m z 129 (M+l)⁺.

ound 97

[0182] N-((R)-3-hydroxy-l-oxo-l-((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, l l, 14, 17,20-heptaoxo- l,4,7, 10, 13, 16, 19- heptaazacyclotricosan-21 -ylamino)propan-2-yl)-5 -methyl- 1 ,2,4-oxadiazole-3 -carboxamide (Compound 97) was prepared according to the coupling, hydrolysis, the second coupling then deprotection sequence described for example 7, Compound 15. LCMS : rt = 1.53 minutes (LC method 2). m/z = 959.6 (M+l). 'H NMR (400 MHz, D₂0) δ 7.54 - 6.93 (m, 5H), 4.49 - 4.27 (m, 3H), 4.27 - 3.95 (m, 6H), 3.86 (d, J= 5.0 Hz, 2H), 3.32 (m, 1H), 3.15 - 2.82 (m, 7H), 2.74 (m, 2H), 2.54 (s, 3H), 2.30 - 1.62 (m, 8H), 1.26 (m, 2H), 1.03 (d, J= 5.9 Hz, 3H), 0.61- 0.53 (m, 7H).

EXAMPLE 28. SYNTHESIS OF COMPOUND 104

104

[0183] (S)-N-((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,l l, 14, 17,20-heptaoxo-l,4,7, 10, 13,16, 19- heptaazacyclotricosan-21-ylamino)propan-2-yl)-5-oxopyrrolidine-2-carboxamide (Compound 104) was prepared according to the coupling, hydrolysis, the second coupling then deprotection sequence described for example 7, Compound 15. LCMS: rt = 0.28/0.44 minutes (double peak) (LC method 3). m z = 961 (M+l). ¾ NMR (400 MHz, D₂0) 57.27 (m, 5H), 4.50 (t, J= 12.0 Hz, 1H), 4.43 - 4.26 (m, 4H), 4.25 _ 4.02 (m, 5H), 3.82 (t, J= 6.9 Hz, 2H), 3.41 - 3.23 (m, 1H), 3.12 - 2.87 (m, 7H), 2.81 - 2.70 (m, 2H), 2.45 (m, 1H), 2.43 - 2.29 (m, 2H), 2.29 - 2.05 (m, 3H), 2.07 - 1.66 (m, 6H), 1.50 - 1.27 (m, 2H), 1.13 (t, J= 12.2 Hz, 3H), 0.81 (m, 1H), 0.76 - 0.48 (m, 6H) EXAMPLE 29. SYNTHESIS OF COMPOUND 116

[0184] Step 1. Synthesis of Intermediate Compound 106. To a solution of compound 105 (10 g, 43.3 mmol) DMF (150 mL), was added K₂C0₃ (12 g, 86.6 mmol) and Mel (6.1 g, 43.3 mmol). The resulting mixture was stirred at room temperature overnight. The crude mixture was diluted with water (500 mL), extracted with DCM (500 mL). The organic layer was dried over anhydrous Na2SC>4, filtered, and concentrated to dryness to give title compound 106, (2S,4R)-l-tert-butyl 2-methyl 4- hydroxypyrrolidine-l,2-dicarboxylate (10.5 g) without further purification. LC-MS (LC method 1): m/z 246 (M+l)⁺.

[0185] Step 2. Synthesis of Intermediate Compound 107. To a solution of (2S,4R)-l-tert-butyl 2-methyl 4-hydroxypyrrolidine-l,2-dicarboxylate 106 (10.5 g, 42.9 mmol) in DMF (200 mL) was added imidazole (5.8 g, 85.7 mmol) and TBDPSC1 (9.7 g, 64.3 mmol). The resulting mixture was stirred at room temperature overnight. The crude mixture was diluted with water (500 mL), extracted with DCM (500 mL). The organic layer was dried over anhydrous Na2SC>4, filtered, and concentrated to dryness. The residue was purified by silica gel column (PE : EA = 10 : 1) to provide 107, (2S,4R)-l-tert-butyl 2-methyl 4-(tert-butyldiphenyl silyloxy)pyrrolidine- 1,2-dicarboxylate (13 g, 87% yield). LC-MS (LC method 1): m/z 484 (M+l)⁺.

[0186] Step 3. Synthesis of Intermediate Compound 108. To a solution of NalC (1.5 g, 7.0 mmol) in water ( 160 mL) was added RuC .LbO (74 mg, 0.56 mmol) under the atmosphere of nitrogen. The resulting green-yellow solution was stirred at room temperature for 5 min followed by addition of (2S,4R)- l-tert-buty2-methyl4-((tert-butyldimethyl silyl)oxy)pyrrolidine - 1,2-dicarboxylate 107 (1.0 g, 2.8 mmol) in EtOAc (8 mL) in one portion. The mixture was stirred at room temperature overnight. The crude mixture was then diluted with EtOAc and filtered through a pad of Celite. The organic phase was separated and washed with saturated aq. NaHSC , which resulted in precipitation of Ru black. The organic phase was washed with brine and dried over anhydrous Na2SC>4 and concentrated to give the crude title compound 108, (2S,4R)-l-tert-butyl 2-methyl 4-(tert-butyldiphenylsilyloxy)-5-oxopyrrolidine- 1,2-dicarboxylate (1.0 g) without further purification. LC-MS (LC method 1): m/z 498 (M+l)⁺.

[0187] Step 4. Synthesis of Intermediate Compound 109. To a solution of (2S,4R)-l-tert-butyl 2-methyl 4-(tert-butyldiphenylsilyloxy)-5-oxopyrrolidine- 1,2-dicarboxylate 108 (900 mg, 2.4 mmol) in THF (10 mL) was added TBAF (4.8 mL, 1 M THF solution). The resulting mixture was stirred at room temperature overnight. The crude mixture was quenched with aq. NH4CI (10 mL). The resulting mixture was extracted with ethyl acetate (100 mL). The organic layer was separated, dried over anhydrous Na2SC>4, filtered, and then concentrated to give crude title compound 109, (2S,4R)-l-tert-butyl-2-methyl- 4-hydroxy-5-oxo pyrrolidine- 1,2-dicarboxylate (45 mg, 32% yield) without further purification. LC-MS (LC method 1): m/z 260 (M+l)⁺.

[0188] Step 5. Synthesis of Intermediate Compound 110. To a solution of (2S,4R)-l-tert-butyl 2-methyl 4-hydroxy-5-oxopyrrolidine- 1,2-dicarboxylate 109 (2 g, 7.7 mmol) in DCM (20 mL) was added TFA (2 mL). The resulting mixture was stirred at room temperature overnight and then concentrated to dryness. The residue was dissolved in THF (10 mL), to the mixture was added aq. LiOH (470 mg in 5 mL H2O). The resulting mixture was stirred for additional 30 min and then acidified by 2M HC1 to adjust pH to 2-3. The resulting mixture was extracted with ethyl acetate (50 mL). The organic layer was washed with brine, dried over anhydrous Na2SC>4, filtered, and concentrated to dryness to give title compound 110, (2S,4R)-4-hydroxy-5-oxopyrrolidine-2-carboxylic acid (400 mg) without further purification.

LC-MS (LC method 1): m/z 146 (M+l)⁺. 116

[0190] (2S,4R)-4-hydroxy-N-((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 -yl amino)propan-2-yl)-5 -oxopyrrolidine-2-carboxamide (Compound 116) was prepared according to the coupling, hydrolysis, the second coupling then deprotection sequence described for example 7, Compound 15. LCMS: rt = 0.27 minutes (LC method 2). m/z = 976.5 (M+l). ¾ NMR (400 MHz, D₂0) δ 7.19 (m, 5H), 4.44 (t, J= 8.2 Hz, 1H), 4.35 (t, J= 8.4 Hz, 1H), 4.25 (m, 4H), 4.06 (m, 5H), 3.86 - 3.66 (m, 2H), 3.77 (dd, J= 20.7, 5.6 Hz, 2H), 3.27 (m, 1H), 3.01-2.85 (m, 6H), 2.79 - 2.66 (m, 1H), 2.59 (m, lH), 2.45 - 2.31 (m, lH), 2.21 - 1.57 (m, 11H), 1.30 (m, 2H), 1.05 (t, J= 14.0 Hz, 3H), 0.71 (s, 1H), 0.68 - 0.41 (m, 6H).

117 119 120

[0191] Step 1. Synthesis of Intermediate Compound 119. To a solution of (R)-methyl 5- oxopyrrolidine-2-carboxylate 117 (4.2 g, 29.37 mmol) in dry THF (123 mL) stirred at -78 °C was added lithium hexamethyldisilazide (62.5 mL, 62.5 mmol, 1 M in THF). After addition, the reaction mixture was warmed to -30 °C over lh. Then the reaction mixture was cooled to -78 °C again, and

((chloromethoxy)methyl)benzene 118 (5.53 g, 35.24 mmol) was added. The mixture was allowed to stir at -30 °C for 1 h. The crude mixture was quenched with saturated NH4CI solution and extracted with dichloromethane (100 mL x 2). The combnined organic phase were dried over anhydrous Na2SC>4, filtered, and concentrated to dryness. The residue was purified by silica gel column (PE : EA = 2 : 1) to give title compound 119, methyl 2-(benzyloxymethyl)-5-oxopyrrolidine -2-carboxylate (2.1 g, 27%). LC-MS (LC method 1): m/z 264 (M+l)⁺.

[0192] Step 2. Synthesis of Intermediate Compound 120. To a solution of methyl 2- (benzyloxymethyl)-5-oxopyrrolidine-2-carboxylate 119 (500 mg, 1.9 mmol) in the mixture of THF ( 10 mL) and water (2 mL) at 0 °C was added Li OH (319 mg, 7.6 mmol). The reaction mixture was stirred at room temperature for 3 h. The crude mixture was acidified by 2M HCl to adjusted pH to 3-4 and then extracted with ethyl acetate (50 mL). The organic phase was washed with brine, dried over anhydrous Na2SC>4, filtered, and then concentrated to give title compound 120, 2-(benzyloxymethyl)-5- oxopyrrolidine-2-carboxylic acid (500 mg). LC-MS (LC method 1): m/z 250 (M+l)⁺.

128

[0194] (S)-N-((R)-3-hydroxy-l-oxo- l-((3S,6S,9S, 12S, 15R, 18S,21S)-6,9, 18-tri s(2-aminoethyl)- 15-benzyl-3-((R)- l-hydroxyethyl)-12-isobutyl-2,5,8, l 1, 14, 17, 20-heptaoxo- l,4,7, 10, 13, 16, 19- heptaazacyclotricosan-21-ylamino)propan-2-yl)-2-(hydroxymethyl)-5-oxopyrrolidine-2-carboxamide (Compound 128) was prepared according to the coupling, hydrolysis, the second coupling then deprotection sequence described for example 7, Compound 15. LCMS: rt = 0.35 minutes (LC method 2). m/z = 990.6 (M+l). ¾ NMR (400 MHz, D20) 57.19 (m, 5H), 4.43 (t, J= 8.3 Hz, 1H), 4.35 - 4.19 (m, 3H), 4.16 - 3.95 (m, 5H), 3.78 (t, J= 9.7 Hz, 3H), 3.54 (dd, J= 15.0, 9.9 Hz, 1H), 3.23 (d, J= 31.6 Hz, 1H), 3.06 - 2.83 (m, 7H), 2.78 - 2.53 (m, 2H), 2.32 (t, J= 6.9 Hz, 2H), 1.94 (dddd, J= 25.4, 21.9, 14.8, 9.2 Hz, 1 1H), 1.36 - 1.21 (m, 2H), 1.014 (d, J= 6.1 Hz, 3H), 0.68 - 0.51 (m, 6H). 129

[0196] (R)-N-((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, l l, 14, 17,20-heptaoxo-l,4,7, 10, 13, 16, 19- heptaazacyclotricosan-21-ylamino)propan-2-yl)-2-(hydroxymethyl)-5-oxopyrrolidine-2-carboxamide (Compound 129) was prepared according to the coupling, hydrolysis, the second coupling then deprotection sequence described for example 7, Compound 15. LCMS: rt = 0.73 minutes (LC method 3). m/z = 991 (M+l). ¾ NMR (400 MHz, D₂0) 57.19 (m, 5H), 4.43 (t, J= 8.2 Hz, 1H), 4.34 - 4.20 (m, 3H), 4.16 - 3.98 (m, 5H), 3.79 (dt, J= 7.4, 5.7 Hz, 3H), 3.58 - 3.51 (m, 1H), 3.31 - 3.16 (m, lH), 3.03 - 2.84 (m, 7H), 2.78 - 2.55 (m, 2H), 2.37 - 2.26 (m, 2H), 2.21 - 1.63 (m, 12H), 1.37 - 1.24 (m, 2H), 1.03 (d, J= 6.2 Hz, 3H), 0.60 (dd, J= 29.6, 6.4 Hz, 6H).

EXAMPLE 31. SYNTHESIS OF COMPOUND 137

[0197] Step 1. Synthesis of Intermediate Compound 131. To a solution of 1 M NaOH (45 mL, 45 mmol) was added L-serine 130 (1.58 g, 15 mmol), followed by the addition of triphosgene(4.0 g, 15 rnmol) in dioxane (30 mL). The reaction mixtures were stirred at room temperature until clear solutions were obtained and were further stirred for 2 h. The crude mixture was concentrated to give title compound 131, (S)-2-oxooxazolidine-4-carboxylic acid (0.5 g, 8.5% yield) without further purification. LC-MS (LC method 1): m/z 132 (M+l)⁺.

d 137

[0199] (S)-N-((R)-3-hydroxy-l -oxo-l-((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, l l, 14, 17,20-heptaoxo-l,4,7, 10, 13, 16, 19- heptaazacyclotricosan-21-ylamino)propan-2-yl)-2-oxooxazolidine-4-carboxamide (Compound 137) was prepared according to the coupling, hydrolysis, the second coupling then deprotection sequence described for example 7, Compound 15. LCMS: rt = 0.35 minutes (LC method 2). m/z = 962.5 (M+l). ¾ NMR (400 MHz, D₂0) δ 7.27 (m, 5H), 4.56 (m, 2H), 4.65-4.50 (m, 3H), 4.43 -4.38 (m, 3H), 4.21-4.01 (m, 4H), 3.83 (d, J= 5.3 Hz, 2H), 3.43 - 3.27 (m, 2H), 3.22 - 2.88 (m, 8H), 1.57 (s, lH), 1.34 (ddd, J = 28.0, 14.4, 8.7 Hz, 2H), 1.11 (d, J = 6.1 Hz, 2H), 0.87 (m, 2H), 0.67 (dd, J = 30.0, 6.3 Hz, 6H).

138

[0200] (5)-3 -benzyl-JV-((R)-3 -hydroxy- 1 -oxo- 1 -(((3S,6S,9S, 12S, 15R, 185,21 S)-6,9, 18-tris(2- aminoethyl)-15-benzyl-3-((i?)- l-hydroxyethyl)-12-isobutyl-2,5,8, l l, 14, 17,20-heptaoxo- 1, 4, 7, 10, 13, 16, 19-heptaazacyclotricosan-21 -yl)amino)propan-2-yl)-2-oxooxazolidine-4-carboxamide, (Compound 138) was synthesized according to the method of Example 31, starting from benzyl protected Compound 131

[0201] Step 1. Synthesis of Intermediate Compound 140. To a solution 2-hydrazinylethanol 139 (5 g, 65.7 mmol) in acetone (50 mL) was added Na2SC>4 (23 g, 887.5 mmol). The resulting mixture was stirred at 0 °C overnight. The crude mixtures was filtrated and concentrated to dryness to give title compound 139, 2-(2-(propan-2-ylidene)hydrazinyl)ethanol (5 g) without further purification. LC-MS (LC method 1): m/z 117 (M+l)⁺.

[0202] Step 2. Synthesis of Intermediate Compound 141. To an ice water cooled solution 2-(2- (propan-2-ylidene)hydrazinyl)ethanol 140 (5 g, 42.7 mmol) in DCM (20 mL) was added TEA (4.3 g, 42.7 mmol) and AC2O (4.4 g, 42.7 mmol). The resulting mixture was stirred at room temperature overnight. The crude mixture was concentrated to dryness to give title compound 141, N-(2-hydroxyethyl)-N'- (propan-2-ylidene)acetohydrazide (6.0 g) without further purification. LC-MS (LC method 1): m z 159 (M+l)⁺.

[0203] Step 3. Synthesis of Intermediate Compound 142. To a solution of N-(2-hydroxyethyl)- N'-(propan-2-ylidene)acetohydrazide 141 (6.0 g, 37.5 mmol) in THF (50 mL) was added 1 M HC1 (10 mL). The resulting mixture was stirred at room temperature overnight and then concentrated to dryness. The residue was basified by aq. NaHCC to pH = 8 and extracted with DCM (500 mL). The organic phase was washed with brine and dried over anhydrous Na2SC>4, filtrated and concentrated to dryness. The residue was purified by silica gel column (PE : EA = 1 :2) to give title compound 142, crude N-(2- hydroxyethyl) acetohydrazide (4.0 g, 89% yield). LC-MS (LC method 1): m/z 1 19 (M+l)⁺.

[0204] Step 4. Synthesis of Intermediate Compound 144. To a solution of N-(2- hydroxyethyl)acetohydrazide 142 (4 g, 33.8 mmol) and (R)-methyl 2-amino-3-(tert- butyldiphenylsilyloxy)propanoate 143 (1.2 g, 3.38 mmol) in anhydrous DMF (25 mL) was added CDI (5.48 g, 33.8 mmol). The reaction mixture was stirred at room temperature overnight. The crude mixture was quenched with brine (100 mL) and extracted with ethyl acetate (250 mL). The organic phase was dried over anhydrous Na2SC>4, filtered, and then concentrated to dryness. The residue was purified by silica gel column (PE : EA = 1 : 1) to give title compound 144, (R)-methyl 3 -acetyl- 1 -hydroxy- 1 1, 1 1- dimethyl-5-oxo-10, 10-diphenyl-9-oxa-3,4,6-triaza-10-siladodecane-7-carboxylate (600 mg, 40% yield). LC-MS: m/z 502 (M+l)⁺.

148

[0206] 2-acetyl-N-((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, 1 1, 14, 17,20-heptaoxo- 1, 4, 7, 10, 13, 16, 19-heptaazacyclotricosan-21 -ylamino)pro pan-2-yl)-2-(2- hydroxyethyl)hydrazinecarboxamide (Compound 148) was prepared according to the coupling, hydrolysis, the second coupling then deprotection sequence described for example 7, Compound 15. LCMS: rt = 0.41 minutes (LC method 3). m/z = 994 (M+l). ¾ NMR (400 MHz, D₂0) δ 7.29 (m, 3H), 7.19 (m, 2H), 4.52 - 4.04 (m, 10H), 3.80 (d, J= 5.1 Hz, 2H), 3.60 (s, 1H), 3.34 (d, J= 9.1 Hz, 1H), 3.17 - 2.89 (m, 7H), 2.88 - 2.63 (m, 2H), 2.31 - 1.74 (m, 12H), 1.35 (dd, J= 27.7, 13.0 Hz, 2H), 1.1 1 (d, J= 6.1 Hz, 3H), 0.65 (dd, J= 31.1, 5.3 Hz, 7H). EXAMPLE 34. SYNTHESIS OF COMPOUND 156

[0207] Step 1. Synthesis of Intermediate Compound 150. To an ice water cooled solution of (2S,3R)-2-amino-3-hydroxybutanoic acid 149 (2 g, 16.8 mmol) in 4 M Na2CC>3 (10 mL) was added a solution of MsCl (1.92 g, 16.8 mmol) in THF (5 mL) dropwise during 30 min. The resulting mixture was stirred at this temperature overnight. The crude mixture was acidified by 2 M HC1 to adjust pH = 3 and extracted with ethyl acetate (100 mL x 2). The combined solution was washed with brine, dried over anhydrous Na2SC>4, filtered, and then concentrated to give title Compound 150, (2S,3R)-3-hydroxy-2- (methylsulfonamido) butanoic acid ( 0.5 g, 15.1 % yield) without further purification. LC-MS (LC method 1): m/z 198 (M+l)⁺. 156

[0209] (2S,3R)-3-hydroxy-N-((R)-3-hydroxy-l-oxo-l-((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 -ylam ino)propan-2-yl)-2-(methylsulfonamido)butanamide (Compound 156) was prepared according to the coupling, hydrolysis, the second coupling then deprotection sequence described for example 7, Compound 15. LCMS: rt = 1.6 minutes (LC method 2). m/z = 1028.6 (M+l). ¾ NMR (400 MHz, D₂0) δ 7.45 - 7.05 (m, 1H), 4.57 - 3.96 (m, 10H), 3.86 (dd, J = 23.1, 5.3 Hz, 2H), 3.36 (s, 1H), 3.03 (dd, J= 21.9, 9.7 Hz, 9H), 2.77 (d, J= 48.3 Hz, 2H), 2.34 - 1.66 (m, 9H), 1.49 - 1.02 (m, 9H), 0.86 - 0.48 (m, 7H).

[0210] Step 1. Synthesis of Intermediate Compound 158. To a solution compound 1 (2.9 g, 10 mmol) in DCM (15 mL) was added TFA (3 mL). The resulting mixture was stirred at this temperature overnight. The crude mixture was concentrated to dryness to provide crude 158, (S)-2-amino-3- (benzyloxy) propanoic acid (2 g). LC-MS (LC method 1): m/z 196 (M+l)⁺.

[021 1] Step 2. Synthesis of Intermediate Compound 159. To an ice water cooled solution (S)-2- amino-3-(benzyloxy)propanoic acid 158 (2 g, 10 mmol) TFA (7 mL), was added aq. NaNC>2 solution (1.0 g in 60 mL FLO). Then resulting mixture was stirred at room temperature overnight. The crude mixture was diluted with water (100 mL) and extracted with ethyl acetate (100 mL). The organic phase was dried over anhydrous Na2SC>4, filtered, and concentrated to dryness to give crude title compound 159, (2.0 g). LC-MS (LC method 1): m/z 197 (M+l)⁺.

[0212] Step 3. Synthesis of Intermediate Compound 160. To a solution of (S)-3-(benzyloxy)-2- hydroxypropanoic acid 159 (2.0 g, 10 mmol) in MeOH (10 mL), was added CH(OMe)3 (5 mL) and FbSO/t mL). The resulting mixture was stirred at reflux overnight. The crude mixture was concentrated and the residue was dissolved with ethyl acetate (100 mL), washed with water, aq. NaHCC and brine. The organic phase was dried over anhydrous Na2SC>4 and concentrated. The residue was purified by silica gel column (PE : EA = 5 : 1) to give title compound crude 160, (S)-methyl 3-(benzyloxy)-2- hydroxypropanoate (1.0 g, 47% yield). LC-MS (LC method 1): m/z 211 (M+l)⁺

[0213] Step 4. Synthesis of Intermediate Compound 161. To an ice water cooled solution (S)- methyl 3-(benzyloxy)-2 -hydroxy propanoate 160 (500 mg, 2.4 mmol) DCM (50 mL) was added TEA (500 mg, 5.0 mmol) and MsCl (0.33 g, 2.88 mmol). Then resulting mixture was stirred at room temperature for 2 h. The crude mixture was diluted with aq. NaHCC (50 mL) and extracted with DCM (100 mL). The organic phase was dried over anhydrous Na2SC>4, filtered, and concentrated to dryness to give crude title compound 160, (S)-methyl 3-(benzyloxy)-2-(methylsulfonyloxy)propanoate (450 mg) without further purification. LC-MS (LC method 1): m/z 289 (M+l)⁺.

[0214] Step 5. Synthesis of Intermediate Compound 163. To an ice water cooled solution of 3- nitropyridin-2(lH)-one 162 (450 mg, 3.2 mmol) in anhydrous DMF (20 mL) was added 60% NaH (160 mg, 3.84 mmol). After stirring for 30 min, to the mixture was added (S)-methyl 3-(benzyloxy)-2- (methylsulfonyloxy)propanoate 161 (1.0 g, 3.0 mmol). The reaction mixture was stirred at room temperature overnight. The crude mixture was quenched with sat. NH4CI (100 mL) and extracted with ethyl acetate (100 mL). The organic phase was washed with brine, dried over anhydrous Na2S04 and concentrated to dryness. The residue was purified by prep. TLC (PE : EA = 3 : 1) to give title compound 163, (R)-methyl 3-(benzyloxy)-2-(3-nitro-2-oxopyridin-l(2H)-yl)propanoate (100 mg, 10% yield).

LC-MS (LC method 1): m/z 333 (M+l)⁺.

[0215] Step 6. Synthesis of Intermediate Compound 164. Pd/C (10% w/w, 10 mg) was added to a solution of (R)-methyl 3-(benzyloxy)-2-(3-nitro-2-oxopyridin-l(2H)-yl)propanoate 7 (100 mg, 0.3 mmol) in MeOH (10 mL) and stirred at room temperature under atmospheric pressure of hydrogen overnight. The catalyst was filtered off through Celite and washed with methanol (2 mL). The combined filtrates were concentrated to dryness to give pure title compound 164, (R)-methyl 2-(3-amino-2- oxopyridin-l (2H)-yl)-3 -(benzyl oxy)propanoate (90 mg, 90% yield). LC-MS (LC method 1): m/z 303 (M+l)⁺.

[0216] Step 7. Synthesis of Intermediate Compound 165. To a solution of (R)-methyl 2-(3- amino-2-oxopyridin-l(2H)-yl)-3-(benzyloxy) propanoate 164 (90 mg, 0.3 mmol) in DCM (10 mL) was added TEA (30 mg, 0.3 mmol) and AcCl (30.6 mg, 0.3 mmol). The resulting mixture was stirred at room temperature overnight and then concentrated to give crude title compound 165, (R)-methyl 2-(3- acetamido-2-oxopyridin-l(2H)-yl)-3-(benzyloxy)propanoate (100 mg). LC-MS (LC method 1): m/z 345 (M+l)⁺.

[0217] Step 8. Synthesis of Intermediate Compound 166. To a solution of (R)-methyl 2-(3- acetamido-2-oxopyridin-l(2H)-yl)-3-(benzyloxy)propanoate 165 (300 mg, 0.87 mmol) in the mixture of THF (5 mL) and water (5 mL) at 0 °C was added LiOH (35 mg, 0.87 mmol). The reaction mixture was stirred at room temperature for 3 h. The crude mixture was acidified by 2M HCl to adjuste pH to 3-4 and then extracted with ethyl acetate (50 mL). The organic phase was washed with brine, dried over anhydrous Na2SC>4, filtered, and then concentrated to give title compound 166, (R)-2-(3-acetamido-2- oxopyridin-l(2H)-yl)-3-(benzyloxy)propanoic acid (250 mg, 87% yield). LC-MS (LC method 1): m/z 331 (M+l)⁺.

nd 169.

[0219] (R)-2-(3-acetamido-2-oxopyridin-l(2H)-yl)-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 1, 14, 17,20-heptaoxo- l,4,7,10, 13,16, 19-heptaazacyclotricosan-21-yl)propanamide (Compound 169) was prepared according to the coupling then deprotection sequence described for example 7, Compound 15. LCMS: rt = 0.246 minutes (LC method 2). m/z = 984.5 (M+l). ¾ NMR (400 MHz, D₂0) δ 7.97 (d, J= 8 Hz, 1H), 7.54 (d, J= 8 Hz, 1H), 7.32 - 7.19 (m, 5H), 6.50 (t, J= 8 Hz, 1H), 5.23 (m, 1H), 4.75-4.39 (m, 3H), 4.33 -4.10 (m, 6H), 3.36 (m, 1H), 3.11 - 2.66 (m, 7H), 2.71-2.64 (m, 2H), 2.16 - 1.84 (m, 12H), 1.42 - 1.36 (m, 3H), 1.19 (d, J= 28 Hz, 3H), 0.93 - 0.38 (m, 7H).

[0220] Step 1. Synthesis of Intermediate Compound 172. To a solution of (2S,3R)-benzyl 2- amino-3-hydroxybutanoate 170 (209 mg, 1 mmol) in C¾CN (5 mL) was added N-methyl-lH-imidazole- 1-carboxamide 171 (125 mg, 1 mmol) and DIPEA (387 mg, 3 mmol). The resulting mixture was stirred at room temperature overnight. The crude mixture was concentrated to dryness. The residue was purified by prep-TLC (PE : EA = 3 : 1) to give title compound 172, (2S,3R)-benzyl 3-hydroxy-2-(3- methylureido)butanoate ( 70 mg, 26.3% yield). LC-MS (LC method 1): m/z 267 (M+l)⁺.

[0221] Step 2. Synthesis of Intermediate Compound 173. Pd/C (10% w/w, 30 mg) was added to a solution of (2S,3R)-benzyl 3-hydroxy-2-(3-methylureido)butanoate 172 ( 160 mg, 0.6 mol) in MeOH (10 mL) and stirred at room temperature under atmospheric pressure of hydrogen overnight. The catalyst was filtered off through Celite and washed with methanol (10 mL). The filtrates was concentrated to dryness to give title compound 173, (2S,3R)-3-hydroxy-2-(3-methylureido)butanoic acid (80 mg, 75.6 % yield). LC-MS (LC method 1): m/z 177 (M+l)⁺. 179

[0223] (2S,3R)-3-hydroxy-N-((R)-3-hydroxy-l-oxo-l-((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 -yl amino)propan-2-yl)-2-(3 -methylureido)butanamide (Compound 179) was prepared according to the coupling, hydrolysis, the second coupling then deprotection sequence described for example 7, Compound 15. LCMS: rt = 0.28/0.36 minutes (double peak) (LC method 2). m z = 1007.6 (M+l). ¾ NMR (400 MHz, D₂0) δ 8.07 (s, 1H), 7.19 (m, 5H), 4.44 _ 4.17 (m₅ 4H), 4.15 - 3.93 (m, 6H), 3.85 - 3.60 (m, 2H), 3.35 - 3.09 (m, 1H), 2.93 (m, 7H), 2.70 (m, 2H), 2.58 - 2.45 (m, 3H), 2.32 - 1.55 (m, 8H), 1.44 - 1.16 (m, 2H), 1.05 (dd, J= 15.5, 6.2 Hz, 6H), 0.74 - 0.38 (m, 7H).

[0224] Step 1. Synthesis of Intermediate Compound 182. To a solution of (2S,3R)-2-(tert- butoxycarbonylamino)-3-hydroxybutanoic acid 180 (3 g, 13.68 mmol) and (R)-methyl 2-amino-3-(tert- butyldiphenylsilyloxy) propanoate 181 (4.89 g, 13.68 mmol) in anhydrous DMF (40 mL) was added DIPEA (3.54 g, 27.37 mmol), HOBt (2.22 g, 16.42 mmol) and HBTU (6.23 g, 16.42 mmol). The reaction mixture was stirred at room temperature for 2 h. The crude mixture was quenched with water ( 100 mL) and extracted with ethyl acetate (500 mL). The organic phase was dried over anhydrous Na2SC>4, filtered, and then concentrated to dryness. The residue was purified by silica gel column (PE : EA = 5 : 2) to give title compound 182, (6R,9S)-methyl 9-((R)-l-hydroxyethyl)-2,2, 13, 13-tetramethyl-8, l l-dioxo-3,3- diphenyl-4, 12-dioxa-7, 10-diaza-3-silatetradecane-6-carboxy late (3.5 g, 45.8% yield). LC-MS (LC method 1): m/z 559 (M+l)⁺

[0225] Step 2. Synthesis of Intermediate Compound 183. To a solution of (6R, 9 S) -methyl 9- ((R)- 1 -hydroxyethyl)-2,2, 13, 13 -tetramethyl-8, 1 1 -dioxo-3 ,3 -diphenyl-4, 12-dioxa-7, 10-diaza-3 - silatetradecane-6-carboxylate 183 ( 1 g, 1.79 mmol) was in dry THF (15 mL), was added BH3.SMe2 (4.5 mL, 8.95 mmol). The reaction mixture was stirred at room temperature overnight. The crude mixture was quenched with MeOH carefully. The reaction mixture was extracted with ethyl acetate (100 mL). The organic phase was dried over Na2SC>4, filtered, and concentrated to dryness. The residue was purified by prep-TLC (PE : EA = 3 : 1) to give title compound 183, (6R,9R)-methyl-9-((R)-l-hydroxyethyl)- 2,2, 13, 13-tetramethyl-l l-oxo-3,3-diphenyl-4, 12-dioxa-7, 10-diaza-3-silatetra decane-6-carboxylate (260 g, 26.7 % yield). LC-MS (LC method 1): m/z 545 (M+l)⁺.

[0226] Step 3. Synthesis of Intermediate Compound 184. To an ice water cooled solution of (6R,9R)-methyl 9-((R) - 1 -hydroxyethyl)-2,2, 13, 13 -tetramethyl- 1 1 -oxo-3 ,3 -diphenyl-4, 12-dioxa-7, 10- diaza-3-silatetradecane -6-carboxylate 4 (90 mg, 0.17 mmol) in DCM (10 mL) at room temperature was added TFA (2 mL). The resulting mixture was stirred at 0 °C for 3 h. The crude mixture was basified by aq. NaHCC to pH = 7-8 and extracted with DCM (50 mL). The organic layer was dried over anhydrous Na2SC>4, filtered, and concentrated to dryness to give title compound 184, (R)-methyl 2-((2R,3R)-2- amino-3-hydroxybutylamino)-3-(tert-butyldiphenylsilyloxy)propanoate (60 mg, 81.7% yield). LC-MS (LC method 1): m/z 445 (M+l)⁺ _.

[0227] Step 4. Synthesis of Intermediate Compound 185. A mixture of (R)-methyl 2-((2R,3R)- 2-amino-3-hydroxybutylamino)-3-(tert-butyldiphenylsilyloxy)propanoate 184 (80 mg, 0.18 mmol) in DCM ( 3 mL), was added AC₂O (27.6 mg, 0.27 mmol) and pyridine (42.7 g, 0.54 mmol). The resulting mixture was stirred at 0 °C for 2 h. The crude mixture was quenched with aq. NaHCCh, extracted with DCM (50 mL). The organic phase was washed with brine, dried over anhydrous Na2SC>4 and concentrated to give title compound 185, (6R,9R)-methyl 9-((R)-l-hydroxyethyl)-2,2-dimethyl-l l-oxo-3,3-diphenyl-4- oxa-7, 10-diaza-3-siladodecane-6-carboxylate. LC-MS (LC method 1): m/z 487 (M+l)⁺.

[0228] Step 5. Synthesis of Intermediate Compound 186. To a solution of (6R,9R) -methyl 9- ((R)-l-hydroxyethyl)-2,2-dimethyl-l l-oxo-3,3-diphenyl-4-oxa-7, 10-diaza-3-siladodecane-6-carboxylate 185 (60 mg, 0.12 mmol) in the mixture of THF (2 mL), methanol (2 mL) and water (1 mL), was added LiOH (10.34 mg, 0.25 mmol) at 0 °C. The reaction mixture was stirred at room temperature for 1 h. The crude mixture was acidified by 2M HC1 to adjust pH to 3-4 and extracted with ethyl acetate (20 mL). The organic phase was washed with brine, dried over anhydrous Na2SC>4, filtered, and concentrated to dryness give title compound 186, (6R,9R)-9-((R)-l-hydroxyethyl)-2,2-dimethyl-l l-oxo-3,3-diphenyl-4-oxa-7,10- diaza-3-siladodecane-6-carboxylic acid (180 mg, 90% yield). LC-MS (LC method 1): m z 473 (M+l)⁺.

189

[0230] (R)-2-((2R,3R)-2-acetamido-3-hydroxybutylamino)-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 (Compound 189) was prepared according to the coupling, hydrolysis, the second coupling then deprotection sequence described for example 7, Compound 15. LCMS: rt = 0.32 minutes (LC method 2). m/z = 978.6 (M+l). ¾ NMR (400 MHz, D₂0) 5 8.15 (s, 1H), 7.63 (d, J= 1 1.5 Hz, 1H), 7.31 (dd, J= 13.9, 6.8 Hz, 3H), 7.18 (s, 2H), 6.59 (s, 1H), 4.54 - 3.99 (m, 10H), 3.84 (s, 2H), 3.33 (s, lH), 3.05 (d, J= 30.9 Hz, 6H), 2.88 - 2.62 (m, 2H), 2.16 (s, 3H), 1.95 (s, 2H), 1.83 (s, 3H), 1.57 (s, lH), 1.48 - 1.25 (m, 3H), 1.20 (d, J = 6.0 Hz, 2H), 1.09 (s, 2H), 0.86 (t, J= 7.2 Hz, 1H), 0.64 (d, J= 31.8 Hz, 6H).

[0231] Step 1. Synthesis of Intermediate Compound 192. To a solution of compound 190 (5.3 g, 21.48 mmol) and compound 191 (2.1, 19.53 mmol) in DCM (20 mL), was added DIPEA (7.56 g, 58.58 mmol). The resulting mixture was stirred at room temperature overnight. Then to the mixture was added NaBH4 (0.82 g, 21.48 mmol). The resulting mixture was stirred for 4 h. The crude mixture was quenched with aq. NH4CI (50 mL) and extracted with ethyl acetate (200 mL x 2). The combined organic phase was washed with brine, dried over anhydrous Na2SC>4, filtrated and concentrated to dryness. The residue was purified by silica gel column (PE : EA = 10 : 1) to give title compound 192, (2S,3R)-methyl 2- (benzylamino)-3-hydroxybutanoate (2.5 g, 52% yield). LC-MS (LC method 1): m/z 224 (M+l)⁺.

[0232] Step 2. Synthesis of Intermediate Compound 193. To a solution of (2S,3R)-methyl 2- (benzylamino)-3-hydroxybutanoate 192 (2.23g, 10 mmol) was in dry DMF (10 mL) was added Mel (1.4 g, 10 mmol) and NaHCC (2.5 g, 30 mmol). The reaction was stirred at room temperature overnight. The crude mixture was poured into water (200 mL) and extracted with ethyl acetate (200 mL). The organic layer was dried over anhydrous Na2SC>4, filtered, and concentrated to dryness. The residue was purified by silica gel column (PE : EA = 5 : 1) to give title compound 193, (2S,3R)-methyl 2- (benzyl(methyl)amino)-3-hydroxybutanoate (2 g, 84% yield). LC-MS (LC method 1): m/z 238 (M+l)⁺.

[0233] Step 3. Synthesis of Intermediate Compound 194. To a solution of (2S,3R)-methyl 2- (benzyl(methyl)amino)-3 -hydroxy butanoate 193 (711 mg, 3 mmol) in THF at 0 °C was added 3 M LiOH (10 mL). The reaction mixture was stirred at room temperature for 3 h. The crude mixture was acidified by 2M HC1 to adjust the pH to 3-4 and extracted with ethyl acetate (100 mL). The organic layer was washed with brine, dried over anhydrous Na2SC>4, filtered, and then concentrated to dryness to give title compound 194, (2S,3R)-2-(benzyl(methyl)amino)-3-hydroxybutanoic acid (600 mg, 90% yield). LC-MS (LC method 1): m/z 224 (M+l)⁺.

[0234] Step 4. Synthesis of Intermediate Compound 195. To a solution of (2S,3R)-2- (benzyl(methyl)amino)-3-hydroxybutanoic acid 194 (500 mg, 2.24 mmol) and (S)-methyl 2-amino-3- (tert-butyldiphenylsilyloxy)pro panoate (800 mg, 2.24 mmol) in anhydrous DMF (10 mL) was added DIPEA (866 mg, 6.72 mmol) and HBTU (848 mg, 2.24 mmol). The reaction mixture was stirred at room temperature overnight. The crude mixture was quenched with brine (50 mL) and extracted with ethyl acetate (100 mL). The organic layer was dried over anhydrous Na2SC>4, filtered, and then concentrated to dryness. The residue was purified by silica gel column (DCM : MeOH = 20 : 1) to give title compound 195, (S)-methyl 2-((2S,3R)-2-(benzyl(methyl)amino)-3-hydroxybutanamido)-3-(tert-butyldi phenylsilyloxy)propanoate (1 g). LC-MS (LC method 1): m/z 563 (M+l)⁺.

[0235] Step 5. Synthesis of Intermediate Compound 196 . Pd/C (10% w/w, 200 mg) was added to a solution of (S)-methyl 2-((2S,3R)-2-(benzyl(methyl)amino)-3-hydroxybutanamido)-3-(tert- butyldiphenylsilyloxy) propanoate 195 (1 g, 1.78 mmol) in dry ethyl acetate (10 mL) and stirred at room temperature under atmospheric pressure of hydrogen overnight. The catalyst was filtered off through Celite and washed with methanol (10 mL). The combined filtrates were concentrated to dryness to give title compound 196, (S)-methyl 3-(tert-butyldiphenylsilyloxy)-2-((2S,3R)-3-hydroxy-2- (methylamino)butan amido)propanoate (0.472 g, 56% yield). LC-MS (LC method 1): m/z 473 (M+l)⁺. [0236] Step 6. Synthesis of Intermediate Compound 197. To a solution of (S)-methyl 3-(tert- butyldiphenylsilyloxy)-2-((2S,3R)-3-hydroxy-2-(methylamino)butanamido)propanoate 196 ( 472.65 mg, 1 mmol) in DCM ( 10 mL), was added AC2O (102.09 mg, 1 mmol) and pyridine (158 mg, 2 mmol). The reaction mixture was stirred at room temperature for 1 h. The crude mixture was concentrated to dryness to give title compound 197, (S)-methyl 3-(tert-butyldiphenylsilyloxy)-2-((2S,3R)-3-hydroxy-2-(N- methylacetamido)butan amido)propanoate (380 mg, 73.8 % yield). LC-MS (LC method 1): m/z 515

(M+l)⁺.

[0237] Step 7. Synthesis of Intermediate Compound 198. To a solution of (S)-methyl 3-(tert- butyldiphenylsilyloxy)-2-((2S,3R)-3-hydroxy-2-(N-methylacetamido)butanamido)propanoate 197 ( 350 mg, 0.68 mmol) in the mixture of THF ( 5 mL) and water ( 3 mL) at 10 °C was added LiOH ( 85.68 mg, 2.04 mmol). The reaction mixture was stirred at room temperature for 3 h. The crude mixture was acidified by 2M HC1 to adjust pH to 3-4 and extracted with ethyl acetate (50 mL). The organic phase was washed with brine, dried over anhydrous Na2SC>4, filtered, and then concentrated to give title compound 198, (S)-3-(tert-butyldiphenylsilyloxy)-2-((2S,3R)-3-hydroxy-2-(N-methylacetamido)butan

amido)propanoic acid (300 mg). LC-MS (LC method 1): m/z 501 (M+l)⁺.

201

[0239] (2S,3R)-3-hydroxy-N-((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 -yl amino)propan-2-yl)-2-(N-methylacetamido)butanamide (Compound 201) was prepared according to the coupling, hydrolysis, the second coupling then deprotection sequence described for example 7, Compound 15. LCMS: rt = 0.27/0.37 minutes (double peak) (LC method 2). m/z = 1006.6 (M+l). ¾ NMR (400 MHz, D20) δ 8.21 (s, 1H), 7.78 - 6.77 (m, 5H), 4.42 (s, 2H), 4.18 (m, 7H), 3.84 (s, 2H), 3.28 (s, 1H), 3.15 (d, J= 102.8 Hz, 8H), 2.75 (d, J= 57.1 Hz, 5H), 2.00 (dd, J= 89.1, 33.1 Hz, 11H), 1.55 - 1.23 (m, 4H), 1.12 (d, J= 4.7 Hz, 3H), 0.68 (dd, J = 29.8, 5.2 Hz, 7H). EXAMPLE 39. SYNTHESIS OF COMPOUND 216

202 203 204

Boc_?0 NHBoc NHBoc

Mel

OH OH O.

HO^' HO^' HO^'

NaHC0₃

205 206 207

NHBoc

MsCI

O.

MsO^'

TEA

208

NaHCQ₃ dioxane

208

[0240] Step 1. Synthesis of Intermediate Compound 203. To an ice water cooled solution of compound 202 (17 g, 1 10 mmol) in DMF (75 mL) was added imidazole (27 g, 38.5 mmol) and TBDPSCl (35.8 g, 130 mmol). The resulting mixture was stirred at this temperature overnight. The crude mixture was diluted with a sat. Li CI solution (500 mL). The mixture was extracted with ethyl acetate (500 mL). The organic layer was dried over anhydrous Na2SC>4, filtered, and concentrated to dryness to give title compound 203, (R)-methyl 2-(tert-butoxycarbonylamino)-3-(tert-butyldiphenylsilyloxy) propanoate (28 g) without further purification. LC-MS (LC method 1): m/z 458 (M+l)⁺.

[0241] Step 2. Synthesis of Intermediate Compound 204. To an ice water cooled solution of (R)-methyl 2-(tert-butoxycarbonylamino) -3-(tert-butyldiphenylsilyloxy) propanoate 203 (4.58 g, 10 mmol) DCM (50 mL) was added TFA (0.97 g, 10 mmol). The resulting mixture was stirred at room temperature for 2 h. The crude mixture was diluted with aq. NaHCC (100 mL) and extracted with DCM (500 mL). The organic phase was dried over anhydrous Na2SC>4, filtered, and concentrated. The crude was purified by silica gel column (PE : EA = 1 : 1) to give title compound 204, (R)-methyl 2-amino-3-(tert- butyldiphenyl silyloxy) propanoate (2.5 g, 71% yield). LC-MS (LC method 1): m/z 358 (M+l)⁺.

[0242] Step 3. Synthesis of Intermediate Compound 206. To a solution of (S)-2-amino-4- hydroxybutanoic acid 205 (5 g, 41.6 mmol) in EtOH (50 mL) at room temperature was added TEA (4.2 g, 41.6 mmol) and B0C2O (9.1 g, 41.6 mmol). The resulting mixture was stirred at room temperature overnight. The crude mixture was concentrated to dryness to give crude title compound 206, (S)-2-(tert- butoxycarbonylamino)-4-hydroxybutanoic acid (8.5 g). LC-MS (LC method 1): m/z 220 (M+l)⁺. [0243] Step 4. Synthesis of Intermediate Compound 207. To the solution (S)-2-(tert- butoxycarbonylamino)-4-hydroxybutanoic acid 206 (8.5 g, 38 mmol) DMF (50 mL), was added K2CO3 (5.24 g, 38 mmol) and Mel (5.4 g, 38 mmol). The resulting mixture was stirred at room temperature overnight. The reaction mixture was diluted with water (500 mL) and extracted with DCM (500 mL). The organic layer was dried over anhydrous Na2SC>4, filtered, and concentrated to dryness to give crude title compound 207, (S)-methyl 2-(tert-butoxycarbonylamino)-4-hydroxybutanoate (8.5 g, 90% yield).

LC-MS (LC method 1): m/z 234 (M+l)⁺.

[0244] Step 5. Synthesis of Intermediate Compound 208. To an ice water cooled solution of (S)-methyl 2-(tert-butoxycarbonyl amino)-4-hydroxybutanoate 207 (2.6 g, 1 1.15 mmol) and TEA (1.69 g, 16.72 mmol) in anhydrous DCM (60 mL) was added methanesulfonyl chloride ( 1.52 g, 13.4 mmol). The reaction mixture was stirred at room temperature overnight. The crude mixture was diluted with DCM (100 mL), washed with aq. NaHCC , brine, dried over anhydrous Na2SC>4. The organic layer was concentrated to dryness to give title compound 208, methyl (2S)-2-(tert-butoxycarbonylamino)-4- methylsulfonyloxy-butanoate (3.0 g, 86% yield). LC-MS (LC method 1): m/z 312 (M+l)⁺.

[0245] Step 6. Synthesis of Intermediate Compound 209. To a mixture of (R)-methyl 2-amino- 3-(tert-butyldiphenylsilyloxy)pro panoate 204 (57.4 mg, 0.16 mmol) and methyl (2S)-2-(tert- butoxycarbonylamino)-4-methylsulfonyloxy-butanoate 208 (50 mg , 0.16 mmol) in 1,4-dioxane (10 mL), was added NaHCC (26.9 mg, 0.32 mmol). The resulting mixture was stirred at 100 °C overnight. The crude mixture was diluted with water (50 mL) and extracted with ethyl acetate (50 mL). The organic layer was washed with brine, dried over anhydrous Na2SC>4, filtered, and then concentrated to dryness. The residue was purified by prep-TLC (PE : EA = 5 : 1) to give title compound 209, (S)-methyl 2-(tert- butoxycarbonylamino)-4-((R)-3 -(tert-butyldiphenylsilyloxy)- 1 -methoxy- 1 -oxopropan-2- ylamino)butanoate (10 mg, 1 1%). LC-MS (LC method 1): m/z 425 (M+l)⁺.

[0246] Step 7. Synthesis of Intermediate Compound 210. To a solution of (S)-methyl 2-(tert- butoxycarbonylamino)-4-((R)-3 -(tert-butyldiphenylsilyloxy)- 1 -methoxy- 1 -oxopropan-2- ylamino)butanoate 209 (50 mg, 0.087 mmol) in m-Xylene (8 mL) was added DMAP (16.0 mg, 0.13 mmol). The resulting mixture was stirred at 150 °C for 6 h. The crude mixture was concentrated to dryness. The residue was purified by prep-TLC (PE : EA = 5 : 1) to give title compound 210, (R)-methyl 2-((S)-3-(tert-butoxycarbonylamino)-2-oxopyrrolidin-l-yl)-3-(tert-butyldiphenylsilyloxy)propanoate (10 mg, 21%). LC-MS (LC method 1): m/z 393 (M+l)⁺.

[0247] Step 8. Synthesis of Intermediate Compound 211. To a solution of (R)-methyl 2-((S)-3- (tert-butoxycarbonylamino)-2-oxopyrro lidin-l-yl)-3 -(tert-butyldiphenylsilyloxy )propanoate 210 (1 10 mg, 0.2 mmol) in DCM (5 mL) at room temperature was added TFA (1 16 mg, 1 mmol). The resulting mixture was stirred at room temperature overnight and then concentrated to give crude title compound 211, (R)-methyl 2-((S)-3-amino-2-oxopyrrolidin-l-yl)-3-(tert-butyldiphenylsilyloxy) propanoate (90 mg) without further purification. LC-MS (LC method 1): m/z 293 (M+l)⁺.

[0248] Step 9. Synthesis of Intermediate Compound 212. To a solution of (R)-methyl 2-((S)-3 - amino-2-oxopyrrolidin-l-yl)-3-(tert-butyldiphenylsilyloxy)propanoate 211 (85 mg, 0.19 mmol) in DCM (3 mL) at room temperature was added pyridine (45.7 mg, 0.58 mmol) and AC2O (29.5 mg, 0.29 mmol). The resulting mixture was stirred at room temperature for 6 h and then concentrated to give crude title compound 212, (R)-methyl 2-((S)-3-acetamido-2-oxopyrrolidin-l-yl)-3-(tert-butyldiphenylsilyloxy) propanoate (92 mg). LC-MS (LC method 1): m/z 335 (M+l)⁺.

[0249] Step 10. Synthesis of Intermediate Compound 213. To a solution of (R)-methyl 2-((S)- 3-acetamido-2-oxopyrrolidin-l-yl)-3-(tert-butyl diphenylsilyloxy) propanoate 212 (90 mg, 0.19 mmol) in the mixture of THF (3 mL) and water (3 mL) at 0 °C was added LiOH (4.5 mg, 0.19 mmol). The reaction mixture was stirred at room temperature for 1 h. The crude mixture was acidified by 2M HCl to adjuste pH to 3-4 and extracted with ethyl acetate (50 mL). The organic layer was washed with brine, dried over anhydrous Na2SC>4, filtered, and concentrated to give crude title compound 213, (R)-2-((S)-3-acetamido- 2-oxopyrrolidin-l-yl)-3-(tert-butyldiphenylsilyloxy)propanoic acid (70 mg) without further purification. LC-MS (LC method 1): m/z 321 (M+l)⁺.

nd 216

[0251] (R)-2-((S)-3-acetamido-2-oxopyrrolidin-l-yl)-3-hydroxy-N-((3S,6S,

9S, 12S, 15R, 18S,21S)-6,9, 18-tris(2-aminoethyl)-15-benzyl-3-((R)-l-hydroxy ethyl)- 12-isobutyl- 2,5,8,11, 14,17,20-heptaoxo- 1, 4,7, 10,13, 16, 19-heptaaza cyclotricosan-21 -yl)propanamide (Compound 216) was prepared according to the coupling, hydrolysis, the second coupling then deprotection sequence described for example 7, Compound 15. LCMS: rt = 0.27 minutes (LC method 2). m/z = 974.6 (M+l). ¾ NMR (400 MHz, D₂0) δ 7.45 - 7.08 (m, 5H), 4.56 - 4.01 (m, 9H), 4.03 - 3.27 (m, 6H), 3.20 - 2.66 (m, 10H), 2.48 - 1.73 (m, 13H), 1.68 - 1.02 (m, 10H), 0.93 - 0.38 (m, 7H). EXAMPLE 40. SYNTHESIS OF COMPOUNDS 228 AND 229

TFA

[0252] Step 1. Synthesis of Intermediate Compound 218. To an ice water cooled solution of l,l, l-trifluoropropan-2-ol 217 (1 g, 8.77 mmol) in ether (35 mL) was added TsCl (1.84 g, 9.64 mmol) and TEA (1.77 g, 17.53 mmol). The resulting mixture was stirred at room temperature overnight. The crude mixture was filtered; the filtrate was diluted with water (15 mL) and extracted with ethyl acetate (20 mL x 2). The combined organic phase were washed with brine, dried over anhydrous Na2SC>4, filtered, and then concentrated. The residue was purified by silica gel column (PE : EA = 4 : 1) to give crude title compound 218, l, l,l-trifluoropropan-2-yl 4-methylbenzenesulfonate ( 1.885 g, 80.16 % yield).

[0253] Step 2. Synthesis of Intermediate Compound 220. To a solution of 1, 1, 1 -trifluoropropan- 2-yl 4-methylbenzenesulfonate 218 (1.24 g, 4.61 mmol) and (2S,3R)-benzyl 2-amino-3-hydroxybutanoate 219 (1.49 g, 4.61 mmol) in CH₃CN (20 mL), was added NaHC0₃ (1.94 g, 23.05 mmol). The resulting mixture was stirred at 70 °C overnight. The crude mixture was quenched with water (20 mL) and extracted with ethyl acetate (50 mL x 2). The combined organic phase was washed with brine, dried over anhydrous Na2SC>4, filtered, and then concentrated to dryness. The residue was purified by silica gel column (PE : EA = 3 : 1) to give title compound 220, (2S,3R)-benzyl 3-hydroxy-2-(l,l, l-trifluoropropan- 2-ylamino) butanoate (700 mg, 49.75% yield). LC-MS (LC method 1): m/z 306.1 (M+l)⁺.

[0254] Step 3. Synthesis of Intermediate Compound 221. Pd/C (10% w/w, 50 mg) was added to a solution of (2S,3R)-benzyl 3-hydroxy-2-(l, l, l-trifluoropropan-2-ylamino)butanoate 220 ( 700 mg, 2.29 mmol) in MeOH (10 mL) and stirred at room temperature under atmospheric pressure of hydrogen overnight. The catalyst was filtered off through Celite and washed with methanol (10 mL). The filtrates was concentrated to dryness to give title compound 221, (2S,3R)-3-hydroxy-2-(l, l, l-trifluoropropan-2- ylamino)butanoic acid ( 478 mg, 96 % yield). LC-MS (LC method 1): m/z 216.1 (M+l)⁺. 228

[0256] (2S,3R)-3-hydroxy-N-((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 -yl amino)propan-2-yl)-2-((S)- 1,1, 1 -trifluoropropan-2- ylamino)butanamide (Compound 228), was prepared according to the coupling, hydrolysis, the second coupling then deprotection sequence described for example 7, Compound 15. LCMS: rt = 0.34 minutes (LC method 2). m/z = 1046.6 (M+l). ¾ NMR (400 MHz, D20) δ 7.22 (m, 3H), 7.11 (m, 2H), 4.46 - 3.96 (m, 11H), 3.75 (dd, J= 18.7, 5.8 Hz, 2H), 3.25 (s, 1H), 3.06 - 2.80 (m, 8H), 2.65 (dd, J= 31.6, 23.6 Hz, 2H), 2.21 - 1.60 (m, 9H), 1.24 (d, J= 47.6 Hz, 2H), 1.17 - 1.07 (m, 3H), 1.03 (d, J= 5.3 Hz, 4H), 0.63 (d, J= 3.5 Hz, 3H), 0.54 (s, 3H).

[0258] (2S,3R)-3-hydroxy-N-((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 -yl amino)propan-2-yl)-2-((R)- 1,1, 1 -trifluoropropan-2- ylamino)butanamide (Compound 229) LCMS: rt = 1.06 minutes(LC method 2). m/z = 1046.6

(M+l). ¾ NMR (400 MHz, D₂0) δ 7.29 (m, 3H), 7.19 (m, 2H), 4.32 (m, 11H), 3.92 - 3.72 (m, 2H), 3.30 (s, 1H), 3.01 (s, 8H), 2.64 (s, 12H), 1.98 (dd, J= 84.1, 45.1 Hz, 8H), 1.37 (s, 2H), 1.20 (dd, J= 19.1, 6.1 Hz, 3H), 1.11 (d, J= 5.7 Hz, 3H), 0.71 (d, J= 3.3 Hz, 3H), 0.63 (d, J= 5.3 Hz, 3H). EXAMPLE 41. SYNTHESIS OF COMPOUND 246

[0259] Step 1. Synthesis of Intermediate Compound 232. To an ice water cooled solution of phenylmethanol 230 (3 g, 27.74 mmol) in THF (10 mL), was added 60% NaH (1.2 g, 50 mmol) in THF (40 mL). The resulting mixture was stirred at this temperature for 0.5 h. The crude mixture was added ethyl 2-bromoacetate 231 (4.63 g, 27.74 mmol). The resulting mixture was stirred at room temperature for 4 h. The crude mixture was quenched with ¾0 (20 mL), extracted with ethyl acetate (100 mL x 2). The combined organic phase was washed with brine, dried over anhydrous Na2SC>4, filtered, and then concentrated. The residue was purified by silica gel column (PE : EA = 10 : 1) to give title compound 232, ethyl 2-(benzyloxy)acetate (2.78 g, 52% yield). LC-MS (LC method 1): m/z 195 (M+l)⁺.

[0260] Step 2. Synthesis of Intermediate Compound 233. To the solution of ethyl 2- (benzyloxy)acetate 232 (2.78g, 14.31mmol) was in dry DCM (47 mL), was added DIBAL-H (17 mL, 17.18mmol) at -78 °C dropwise during 10 min, The reaction was stirred for 1.5 h. The crude mixture was quenched with 3 mL H2O and acidified by 1M HC1 to adjust pH = 3. The reaction mixture was extracted with ethyl acetate (100 mL). The organic phase was dried over anhydrous Na2SC>4, filtered, and concentrated to give crude title compound 233, 2-(benzyloxy)acetaldehyde (1.6 g). LC-MS (LC method 1): m/z 151 (M+l)⁺.

[0261] Step 3. Synthesis of Intermediate Compound 235. A mixture of 2- (benzyloxy)acetaldehyde 233 (1.6 g, 10.6 mmol) and compound 234 (3.7 g, 10.6) in toluene (50 mL), was stirred at reflux overnight and then concentrated to dryness. The residue was purified by silica gel column (PE : EA = 10 : 1) to give crude title compound 235, (E)-ethyl 4-(benzyloxy)but-2-enoate (0.8 g, 34% yield). LC-MS (LC method 1): m/z 221 (M+l)⁺.

[0262] Step 4. Synthesis of Intermediate Compound 236. To a solution of (E)-ethyl 4- (benzyloxy)but-2-enoate 235 (800 mg, 3.6 mmol) in the mixture of THF (10 mL) and water (10 mL) at 0 °C was added LiOH (576 mg, 14.4 mmol). The reaction mixture was stirred at room temperature for 3 h. The crude mixture was acidified by 2M HC1 TO adjust pH to 3-4 and extracted with ethyl acetate (100 mL). The organic phase was washed with brine, dried over anhydrous Na2SC>4, filtered, and then concentrated to give title compound 236, (E)-4-(benzyloxy)but-2-enoic acid (670 mg, 95% yield). LC-MS (LC method 1): m/z 193 (M+l)⁺.

[0263] Step 5. Synthesis of Intermediate Compound 238. To a solution of (E)-4- (benzyloxy)but-2-enoic acid 236 (2.5g, 13.01mmol) and triethylamine (1.45g, 14.31 mmol) in anhydrous THF (40 mL) under nitrogen at -78 °C was added trimethylacetyl chloride (1.72g, 14.3 lmmol). The reaction mixture was stirred at -78 °C for 10 minutes, 0 °C for 1 hour, then re-cooled to -78°C.

[0264] At the same time, in a seperate flask charged with a solution of (S)-4-phenyloxazolidin-2- one 237 (0.42g, 2.6mmol) in anhydrous THF (40 mL) under nitrogen at -78 °C was added dropwise a solution of LiHMDS (14.31 mL, 14.3 lmmol). The mixture was stirred at -78 °C for 20 minutes and then transferred via a cannula into the reaction flask containing the mixed anhydride at -78 °C. The reaction mixture was stirred at 0 °C for 1 hour, then warmed to room temperature and stirred for 18 hours. The crude mixture was quenched with sat. NH4CI (200 mL), and concentrated in vacuo, to about half of its original volume under reduced pressure to remove THF. The remaining mixture was extracted with ethyl acetate (150 mL x 2). The organic layer was separated, combined, dried over anhydrous Na2SC>4 and concentrated to dryness. The residue was purified by silica gel column (PE : EA = 2 : 1) to give 238, (S,E)-3-(4-(benzyloxy)but-2-enoyl)-4-phenyloxazolidin-2-one (3 g, 68% yield). LC-MS (LC method 1): m/z 338 (M+l)⁺.

[0265] Step 6. Synthesis of Intermediate Compound 240. To a solution of Nickle intermediate 239 (1.0 g, 2.2 mmol) in DMF (15 mL) was added (S,E)-3-(4-(benzyloxy)but-2-enoyl)-4- phenyloxazolidin-2-one 238 (0.68 g, 2.02 mmol). After stirring at room temperature for 20 min, 1 ,8- Diazabicyclo[5.4.0]undec-7-ene (0.46 g, 3.02 mmol) was added. The reaction was stirred at room temperature for 30 min. The crude mixture was poured into water, and the precipitate was collected by filtration. The solid was re-dissolved in MeOH (40 mL), then 3M HC1 (30 mL) was added, and the mixture stirred at 70 °C overnight. After cooling the reaction, the crude mixture was extracted with AcOEt (100 mL). The organic phase was dried over anhydrous Na2SC>4, and concentrated to give title compound 240, (2S,3S)-3-(benzyloxymethyl)-5-oxopyrrolidine-2-carboxylic acid (200 mg, 39% yield). LC-MS (LC method 1): m/z 250 (M+l)⁺.

[0267] (2S,3S)-N-((R)-3-hydroxy-l-oxo-l-((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, 1 1, 14, 17,20-heptaoxo- l,4,7, 10, 13, 16, 19-heptaazacyclotricosan-21-ylamino) propan-2-yl)-3-(hydroxymethyl)-5-oxopyrrolidine- 2-carboxamide (Compound 246) was prepared according to the coupling, hydrolysis, then deprotection sequence described for example 7, Compound 15. LCMS: rt = 0.26 minutes (LC method 2). m/z = 991.6 (M+l). 'H NMR (400 MHz, D₂0) δ 7.30 - 7.05 (m, 5H), 4.47 - 3.99 (m, 10H), 3.81 - 3.17 (m, 5H), 3.06 - 2.41 (m, 1 1H), 2.23 - 1.65 (m, 9H), 1.30 (dd, J= 14.3, 9.8 Hz, 2H), 1.04 (d, J = 6.2 Hz, 3H), 0.59 (dd, J = 31.1, 6.1 Hz, 7H).

[0268] (2₁S^*,3₁S)-3-((benzyloxy)methyl)-N-((i?)-3 -hydroxy- 1 -oxo- 1 -(((3S,6S,9S, 12S, 15R, 18^,215)- 6,9, 18-tris(2-aminoethyl)-15-benzyl-3-((i?)- l-hydroxye1hyl)-12-isobutyl-2,5,8, l l, 14, 17,20-heptaoxo- 1, 4, 7, 10, 13, 16, 19-heptaazacyclotricosan-21 -yl)amino)propan-2-yl)-5 -oxopyrrolidine-2-carboxamide (Compound 247) was prepared according to the deprotection sequence described for example 7, Compound 15. LCMS: rt =0.35min (LC method 2). m/z = 1 126.6 (M+l). ¾ NMR (400 MHz, D₂0) δ 7.47- 7.09 (m, 10H), 4.57-4.45 (m, 3H), 4.41 - 4.02 (m, 9H), 3.77-3.50 (m, 4H), 3.38 - 2.46 (m, 14H), 2.27- 1.69 (m, 10H), 1.63 -1.20 (m, 4H), 1.10 (d, J= 5.9 Hz, 3H), 0.76 (m, 8H).

EXAMPLE 43. SYNTHESIS OF COMPOUND 257

[0269] Step 1. Synthesis of Intermediate Compound 250. To a solution compound 248 (2 g, 13.42 mmol) in 1,4-dioxane (50 mL) was added compound 249 (4.8 g, 13.42 mmol) and DIPEA (5.2 g,

40.27 mmol). The resulting mixture was stirred at 100 °C overnight. The crude mixture was concentrated to dryness. The residue was purified by silica gel column (pure DCM) to give the title compound 250, (R)-methyl 3-(tert-butyldiphenylsilyloxy)-2-(2-chloropyrimidin-4-ylamino)propanoate (3.4 g, 53.88% yield). LC-MS (LC method 1): m/z 471 (M+l)⁺.

[0270] Step 2. Synthesis of Intermediate Compound 251. To a solution of (R)-methyl 3-(tert- butyldiphenylsilyloxy)-2-(2-chloro pyrimidin-4-ylamino)propanoate 250 (3.5 g, 7.45 mmol) in 1,4- dioxane (60 mL), was added PMBNH₂ (1.12 g, 8.19 mmol) and DIPEA (2.88 g, 22.34mmol). The resulting mixture was stirred at 120 °C overnight. The crude mixture was concentrated to dryness. The residue was purified by silica gel column (PE : EA = 5 : 1 to 1 : 1) to give title compound 251, (R)-methyl 3-(tert-butyldiphenylsilyloxy)-2-(2-(4-methoxybenzylamino)pyrimidin-4-ylamino) propanoate (3.2 g, 75.3% yield). LC-MS (LC method 1): m/z 571(M+1)⁺.

[0271] Step 3. Synthesis of Intermediate Compound 252. To an ice water cooled solution of (R)-methyl 3-(tert-butyldiphenyl silyloxy)-2-(2-(4-methoxybenzylamino)pyrimidin-4-ylamino)propanoate 251 (3 g, 5.26 mmol) in the mixture of CH₃CN (120 mL) and water (24 mL), was added CAN ( 14.41 g,

26.28 mmol). The resulting mixture was stirred at room temperature for 3 h. The crude mixture was poured into water (100 mL) and extracted with ethyl acetate (250 mL). The organic phase was washed with brine, dried over anhydrous Na2SC>4 and concentrated to dryness. The residue was purified by silica gel column (DCM : MeOH = 50 : 1) to give title compound 252, (R)-methyl 2-(2-aminopyrimidin-4- ylamino)-3-(tert-butyldiphenylsilyloxy)propanoate ( 1.5 g, 63.3 % yield). LC-MS (LC method 1): m/z 451 (M+l)⁺.

[0272] Step 4. Synthesis of Intermediate Compound 253. To a solution of (R)-methyl 2-(2- aminopyrimidin-4-ylamino)-3-(tert-butyldi phenylsilyloxy)propanoate 252 (800 mg, 1.78 mmol) in DCM (15 mL), was added AC₂O (181.2 mg, 1.78 mmol) and pyridine (421.3 mg, 5.33 mmol). The resulting mixture was stirred at 80 °C overnight. The crude mixture was quenched with aq. NaHCC , extracted with ethyl acetate (50 mL). The organic phase was washed with brine, dried over anhydrous Na2SC>4 and concentrated to dryness. The residue was purified by silica gel column (DCM : MeOH = 80 : 1) to give title compound 253, (R)-methyl 2-(2-acetamidopyrimidin-4-ylamino)-3-(tert-butyldiphenylsilyloxy) propanoate ( 400 mg, 45.7% yield). LC-MS (LC method 1): m/z 493 (M+l)⁺.

[0273] Step 5. Synthesis of Intermediate Compound 254. To a solution of (R)-methyl 2-(2- acetamidopyrimidin-4-ylamino)-3-(tert-butyldiphenylsilyloxy) propanoate 253 (200 mg, 0.40 mmol) in the mixture of THF (4 mL) and water (2 mL), was added LiOH (51.15 mg, 1.22 mmol) at 0 °C. The reaction mixture was stirred at room temperature for 1 h. The crude mixture was acidified by 2M HC1 to adjust pH to 6. The resulting mixture was extracted with ethyl acetate (20 mL). The organic phase was washed with brine, dried over anhydrous Na2SC>4, filtered, and then concentrated to give the title compound (R)-2-(2-acetamidopyrimidin-4-ylamino)-3-(tert-butyldiphenylsilyloxy)propanoic acid 254, (160 mg, 82.3% yield). LC-MS (LC method 1): m/z 479 (M+l)⁺.

[0275] (R)-2-(2-acetamidopyrimidin-4-ylamino)-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 1, 14, 17,20-heptaoxo- l,4,7,10, 13,16, 19-heptaazacyclotricosan-21-yl)propanamide (Compound 257) was prepared according to the coupling, hydrolysis, the second coupling then deprotection sequence described for example 7, Compound 15. LCMS: rt = 0.37 minutes (LC method 3). m/z = 985 (M+l)⁺.

'H NMR (400 MHz, D₂0) δ 8.07 (s, 1H), 7.34 - 7.06 (m, 8H), 4.71 (d, J= 30.0 Hz, 3H), 4.49 - 4.16 (m, 4H), 4.16 - 3.85 (m, 5H), 3.80 (d, J= 5.4 Hz, 2H), 3.30 (ddd, J= 14.4, 9.7, 5.1 Hz, 1H), 3.20 - 2.85 (m, 7H), 2.85 - 2.53 (m, 2H), 2.31 - 1.72 (m, 1 1H), 1.38 - 1.24 (m, 2H), 1.03 (d, J= 6.2 Hz, 3H), 0.63 (d, J = 6.4 Hz, 3H), 0.55 (d, J= 6.3 Hz, 2H).

EXAMPLE 44. SYNTHESIS OF COMPOUND 263

258 259

[0276] Step 1. Synthesis of Intermediate Compound 259. To a solution of methyl(2S)-2- [[(2S,3S)-2-amino-3-hydroxy-butanoyl]amino]-3-[tert-butyl(diphenyl)silyl]oxy-propanoate (500 mg, 1.09 mmol) (258) in DMF (10 mL) was added 3-bromopropanoyl chloride (280.33 mg, 1.64 mmol) dropwise at 0 °C. After being stirred at 20 °C for 10 min, to the mixture was added K2CO3 (452 mg, 3.27 mmol). The resulting mixture was stirred at 20 °C for 10 h. Then the reaction mixture was poured into ice water, extract with ethyl acetate (10 mLx2). The organic layer was washed with brine, dried over anhydrous Na2SC>4, filtered and concentrated to give Intermediate Compound 259, (R)-methyl 2-((2S,3R)-2-(3- bromopropanamido)-3-hydroxybutanamido)-3-(tert-butyldiphenylsilyloxy)propanoate. (350 mg, 63% yield). LCMS (LC method 1): m/z 513 (M+l)⁺.

[0277] Step 2. Synthesis of Intermediate Compound 260. To a solution of Intermediate Compound 259 (350 mg, 0.68 mmol) in the mixture of THF (4 mL) and Water ( 1 niL) at 0 °C was added LiOH H₂0 (57.2 mg, 1.40 mmol). The reaction mixture was stirred at room temperature for 3 h. The crude mixture was acidified by 2M HCl to adjust the pH to 3-4. The resulting mixture was extracted with ethyl acetate (10 mL). The organic layer was washed with brine, dried over anhydrous Na2SC>4, filtered and then concentrated to afford crude Intermediate Compound 260, (R)-2-((2S,3R)-2-acrylamido-3- hydroxybutanamido)-3-(tert-butyldiphenylsilyloxy)propanoic acid. (350 mg, 99% yield).

LCMS (LC method 1): m/z 499 (M+l)⁺.

[0279] 2S,3R)-2-acrylamido-3-hydroxy-N-((R)-3-hydroxy- 1 -oxo- 1 - ((3 S,6S,9S, 12S, 15R, 18S,21 S)-6,9, 18-tris(2-aminoe^

2,5,8, 1 1, 14, 17,20-heptaoxo- 1, 4,7, 10, 13, 16, 19-heptaazacyclotricosan-21 -ylamino)propan-2-yl)butanamide (Compound 263) was prepared according to the coupling and deprotection sequence described for example 7, Compound 15. LCMS : rt =0.33min (LC method 3). m/z = 1004.6 (M+l)⁺. ¾ NMR (400 MHz, D₂0) δ 8.16 (s, 1H), 7.41 - 7.08 (m, 5H), 6.43 - 6.07 (m, 2H), 5.76 (m, lH), 4.54 - 4.05 (m, 13H), 3.84 (m, 2H), 3.32 (s, 1H), 3.20 - 2.89 (m, 10H), 2.79 (m, 2H), 2.64 (s, 2H), 2.34 - 1.72 (m, 9H), 1.57 (m, 3H), 1.31 (m, 5H), 1.22 - 1.03 (m, 6H), 0.87 (t, J = 7.2 Hz, 4H), 0.70 (s, 3H), 0.62 (s, 3H). EXAMPLE 45. SYNTHESIS OF COMPOUND 269

269

Compound 269 (2S,3R)-3-hydroxy-N-((R)-3-hydroxy-l-oxo-l-((3S,6S,9S, 12S,15R,18S,21S)- 6,9J8 ris(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)propan-2-yl)-2-(N- methylsulfamoylamino)butanamide was prepared as follows.

[0280] Step 1. Intermediate benzyl (2S,3R)-benzyl 3-(benzyloxy)-2-(N- methylsulfamoylamino)butanoate 265. To a solution of (2S,3R)-benzyl 2-amino-3 -(benzyl oxy)butanoate 1 (1.5 g, 5.01 mmol) in DCM (15 mL), was added pyridine (793 mg, 10 mmol) dropwise at 0 °C, then N- methylsulfamoyl chloride (650 mg, 5 mmol) was added at to the mixture, the resulting mixture was stirred at 0 °C for 2 h. The crude mixture was then poured into ice-water, extracted with DCM (50 mL x 2) and the combined organic phase were washed with water and brine, dried over anhydrous Na2SC>4, filtered and concentrated. The residue was purified by flash column chromatography (PE : EA = 10 : 1) to give the title compound benzyl (2S,3R)-3-benzyloxy-2-(methylsulfamoylamino)butanoate 2 (1 g, 51% yield). LC-MS (LC method 1 ): m/z 393 (M+H)⁺

[0281] Step 2. Intermediate (2S,3R)-3-hydroxy-2-(N-methylsulfamoylamino)butanoic acid 3. To a solution of benzyl (2S,3R)-3-benzyloxy-2-(methylsulfamoylamino) butanoate 265 (900 mg, 2.29 mmol) in Methanol (30 mL), was added Pd/C (100 mg, 2.29mmol). The resulting mixture was stirred under ¾ balloon at room temperature for 10 h. The catalyst was filtered off through Celite, the filtrate was concentrated to give the title compound (2S,3R)-3-hydroxy-2-(methylsulfamoylamino)butanoic acid 266 (450 mg, 92.5% yield). LC-MS (LC method 1): m/z 213 (M+H)⁺. [0282] Synthesis of Compound 269, (2S,3R)-3-hydroxy-N-((R)-3-hydroxy-l-oxo-l- ((3S,6S,9S42S45R48S,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-(N- methylsulfamoylamino)butanamide was prepared according to the coupling then deprotection sequence described for Compound 15, Example 7. LCMS: rt = 0.84 minutes (LC method 3). m/z = 1043 (M+l) ⁺. ¾ NMR (400 MHz, D₂0) 5 8.16 (s, 1H), 7.30 (m, 3H), 7.19 (m, 2H), 4.52 - 4.25 (m, 5H), 4.24 - 4.05 (m, 5H), 4.05 - 3.94 (m, 1H), 3.83 (m, 2H), 3.72 (d, J = 5.0 Hz, lH), 3.38 (m, lH), 3.15 - 2.67 (m, 10H), 2.60 (s, 3H), 2.38 - 1.73 (m, 9H), 1.31 (m, 2H), 1.20 (d, J = 6.2 Hz, 3H), 1.1 1 (d, J = 5.8 Hz, 3H), 0.69 (m, 4H), 0.61 (s, 3H).

270

[0283] (2S,3R)-3-(benzyloxy)-N-((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,l l, 14,17,20-heptaoxo-

I, 4, 7, 10, 13, 16, 19-heptaazacyclotricosan-21 -ylamino)propan-2-yl)-2-(N- methylsulfamoylamino)butanamide Compound 270 was prepared according to the coupling then deprotection sequence describe for Example 7, Compound 15. LCMS: rt = 1.01 minutes (LC method 3). m/z = 1133 (M+l). ¾ NMR (400 MHz, D₂0) δ 7.43 - 7.24 (m, 9H), 7.19 (d, J = 7.5 Hz, 2H), 4.59 (d, J =

I I .6 Hz, 1H), 4.42 (ddd, J = 14.1, 10.5, 4.6 Hz, 3H), 4.36 - 4.26 (m, lH), 4.21 - 4.04 (m, 6H), 3.92 - 3.81 (m, 2H), 3.73 (ddd, J = 29.2, 11.5, 4.9 Hz, 2H), 3.38 (t, J = 9.9 Hz, lH), 3.18 - 2.89 (m, 8H), 2.78 (dd, J = 22.7, 15.0 Hz, 2H), 2.55 (s, 3H), 2.31 - 2.06 (m, 3H), 2.06 - 1.73 (m, 5H), 1.33 (dd, J = 26.5, 10.1 Hz, 2H), 1.21 (t, J = 8.5 Hz, 4H), 1.10 (d, J = 5.8 Hz, 3H), 0.69 (s, 4H), 0.61 (d, J = 4.8 Hz, 3H). 271

[0284] N-[(lR)-l-(hydroxymethyl)-2-oxo-2-[[(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] amino] ethyl] -1, 1 -dioxo- 1 ,2-thiazolidine-3 -carboxamide

Compound 271 was prepared according to the coupling then deprotection sequence describe for

Example 7, compound 15. LCMS: rt = 0.33 minutes (LC method 2). m/z = 996.5 (M+l). ¾ NMR (400 MHz, D₂0) δ 8.18 (s, 1H), 7.30 (m, 3H), 7.19 (m, 2H), 4.50 (m, 1H), 4.34 (m, 4H), 4.24 - 4.03 (m, 5H), 3.84 (m, 2H), 3.30 (m, 2H), 3.23 - 2.91 (m, 8H), 2.89 - 2.63 (m, 3H), 2.41 - 1.64 (m, 9H), 1.36 (m, 2H), 1.30 - 1.15 (m, 1H), 1.11 (d, J = 6.1 Hz, 3H), 0.75 (d, J = 7.5 Hz, IH), 0.71 (d, J = 6.1 Hz, 3H), 0.65 - 0.58 (m, 3H).

[0285] N-((2S,3R)-3-hydroxy-l-((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,l l, 14,17,20-heptaoxo- 1 ,4,7, 10, 13, 16, 19-heptaazacyclotricosan-21 -ylamino)propan-2-ylamino)- 1 -oxobutan-2-yl)- 1 -methyl- 1H- imidazole-2 -carboxamide Compound 272 was prepared according to the coupling then deprotection sequence describe for Example 7, compound 15. LCMS: rt = 0.32 minutes (LC method 2). m/z = 1058.6 (M+l)⁺. ¾ NMR (400 MHz, D₂0) δ 8.15 (s, IH), 7.53 - 7.24 (m, 5H), 7.19 (d, J = 7.4 Hz, 2H), 4.55 - 4.33 (m, 3H), 4.33 - 4.23 (m, 2H), 4.19 (m, 2H), 4.11 (m, 3H), 4.00 - 3.90 (m, 3H), 3.90 - 3.77 (m, 2H), 3.31 (s, 1H), 3.18 - 2.88 (m, 7H), 2.90 - 2.50 (m, 2H), 2.01 (m, 8H), 1.45 - 1.31 (m, 2H), 1.30 - 1.25 (m, 1H), 1.21 (d, J = 10.1 Hz, 2H), 1.10 (t, J = 6.9 Hz, 3H), 0.73 (s, lH), 0.70 (d, J = 5.7 Hz, 3H), 0.62 (d, J = 5.8 Hz, 3H).

273

[0286] N-((2S,3R)-3-hydroxy-l-((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-ylamino)- 1 -oxobutan-2-yl)- lH-imidazole- 2-carboxamide Compound 273 was prepared according to the coupling then deprotection seq. LCMS : rt = 0.36 minutes (LC method 2). m/z = 1044.6 (M+l)⁺. ¾ NMR (400 MHz, D₂0) δ 8.05 (d, J = 20.4 Hz, 1H), 7.41 (m, 2H), 7.30 - 7.16 (m, 3H), 7.11 (d, J = 7.2 Hz, 2H), 4.52 - 3.92 (m, 10H), 3.92 - 3.67 (m, 2H), 3.21 (s, 1H), 3.10 - 2.81 (m, 7H), 2.69 (m, 2H), 2.27 - 1.58 (m, 8H), 1.38 - 1.23 (m, 2H), 1.16 (dd, J = 20.5, 10.4 Hz, 3H), 1.03 (t, J = 7.2 Hz, 3H), 0.65 (m, 1H), 0.62 (d, J = 3.2 Hz, 3H), 0.54 (d, J = 5.4 Hz, 3H).

274

[0287] (2S,3R)-3-hydroxy-N-((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,l l, 14,17,20-heptaoxo- 1, 4, 7, 10, 13, 16, 19-heptaazacyclotricosan-21 -ylamino)propan-2-yl)-2-(2-oxopyridin- 1 (2H)-yl)butanamide Compound 274 was prepared according to the coupling then deprotection sequence describe for example 7, Compound 15. LCMS: rt = 0.29 minutes (LC method 3). m/z = 1029 (M+l) ⁺. ¾ NMR (400 MHz, D₂0) δ 7.91 (d, J= 5.1 Hz, 1H), 7.73 - 7.57 (m, 1H), 7.19 (m, 5H), 6.99 - 6.79 (m, 2H), 5.07 (dt, J= 13.7, 6.7 Hz, 1H), 4.45 - 3.93 (m, 10H), 3.78 - 3.67 (m, 2H), 3.22 (s, lH), 3.03 - 2.50 (m, 10H), 2.24 - 1.57 (m, 8H), 1.36 - 0.95 (m, 8H), 0.62 (m, 7H).

[0288] N-((2S,3R)-3-hydroxy-l-((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-ylamino)- 1 -oxobutan-2-yl)-4,5 - dimethylfuran-2-carboxamide Compound 275 was prepared according to the coupling then deprotection sequence describe for example 7, Compound 15. LCMS: rt = 0.19 & 0.35 minutes (double peak, LC method 2). m/z = 1072.6 (M+l)⁺. ¹HNMR:(400 MHz, D₂0) δ 7.34-7.27 (m, 3H), 7.17 (d, J = 7.2 Hz, 2H), 7.00 (s, 1H), 4.49 - 4.28 (m, 5H), 4.26 - 4.03 (m, 6H), 3.85 (dd, J = 9.8, 4.8 Hz, 2H), 3.30 (dd, J = 17.6, 7.6 Hz, 1H), 3.10 - 2.90 (m, 7H), 2.85 - 2.71 (m, 2H), 2.30 - 2.06 (m, 6H), 2.05 - 1.74 (m, 8H), 1.36 (d, J = 6.6 Hz, 1H), 1.26 (dd, J = 14.8, 8.4 Hz, 2H), 1.19 (d, J = 6.4 Hz, 3H), 1.09 (d, J = 6.2 Hz, 3H), 0.76 - 0.53 (m, 7H).

[0289] N-((2S,3R)-3-hydroxy-l-((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,l l, 14,17,20-heptaoxo- 1, 4, 7, 10, 13, 16, 19-heptaazacyclotricosan-21 -ylamino)propan-2-ylamino)- 1 -oxobutan-2-yl)furan-3 - carboxamide Compound 276 was prepared according to the coupling then deprotection sequence describe for example 7, Compound 15. LCMS: rt = 0.17 minutes (LC method 2). m/z = 1044.5 (M+l)⁺. 'H NMR (400 MHz, D₂0) δ 8.29 (s, 1H), 8.08 (d, J = 17.5 Hz, 1H), 7.55 (s, 1H), 7.32-7.20 (m, 5H), 6.76 (d, J = 17.1 Hz, 1H), 4.54 - 4.25 (m, 4H), 4.23 - 3.98 (m, 6H), 3.85 (m, lH), 3.33 (m, 1H), 3.02 (m, 6H), 2.74 (m, 1H), 2.16 (m, 2H), 1.89 (m, 4H), 1.43 - 1.01 (m, 6H), 0.66 (dd, J = 30.2, 7.2 Hz, 5H).

[0290] (2S,3R)-2-(2-(furan-2-yl)acetamido)-3-hydroxy-N-((R)-3-hydroxy- 1 -oxo- 1 - ((3S,6S,9S, 12S, 15R, 18S,21S)-6,9,18-tris(2-aminoethyl)-15-benzyl-3-((R)-l-hydroxyethyl)-12-isobu^ 2,5,8,11, 14,17,20-heptaoxo- 1,4,7, 10,13, 16, 19-heptaazacyclotricosan-21 -ylamino)propan-2-yl)butanamide Compound 277 was prepared according to the coupling then deprotection sequence describe for example 7, Compound 15. LCMS: rt = 0.28 minutes (LC method 2). m/z = 1058.6 (M+l)⁺. ¾ NMR (400 MHz, D₂0) δ 7.60 - 7.05 (m, 6H), 6.32 (s, 1H), 6.25(s, 1H), 4.53 - 4.01 (m, 12H), 3.83 (m, 2H), 3.64 (m, 2H), 3.29 (m, 1H), 3.05 (m, 8H), 2.76 (m, 2H), 2.15 (m, 3H), 1.93 (m, 5H), 1.47 - 0.99 (m, 13H), 0.67 (dd, J = 29.4, 6.1 Hz, 7H).

[0291] 5-chloro-N-((2S,3R)-3-hydroxy-l-((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-ylamino)- 1 - oxobutan-2-yl)furan-2 -carboxamide Compound 278 was prepared according to the coupling then deprotection sequence describe for example 7, Compound 15. LCMS: rt = 0.33 minutes (LC method 2). m/z = 1078.5 (M+l)⁺. ¾ NMR (400 MHz, D₂0) δ 7.33 - 7.15 (m, 3H), 7.16 - 7.02 (m, 3H), 6.39 (d, J = 4.3 Hz, 1H), 4.42 (m, 3H), 4.35 - 3.92 (m, 12H), 3.77 (d, J = 5.4 Hz, 3H), 3.53 (m, lH), 3.26 (s, lH), 3.08 - 2.57 (m, 10H), 2.43 (s, 2H), 2.25 - 1.99 (m, 3H), 1.99 - 1.62 (m, 5H), 1.22 (m, 2H), 1.12 (d, J = 6.3 Hz, 3H), 1.10 - 1.03 (m, 1H), 1.01 (d, J = 6.3 Hz, 3H), 0.59 (s, 4H), 0.52 (s, 3H).

279

[0292] N-((2S,3R)-3-hydroxy-l-((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,l l, 14,17,20-heptaoxo- 1, 4, 7, 10, 13, 16, 19-heptaazacyclotricosan-21 -ylamino)propan-2-ylamino)- 1 -oxobutan-2-yl)oxazole-5 - carboxamide Compound 279 was prepared according to the coupling then deprotection sequence describe for example 7, Compound 15. LCMS: rt = 0.34 minutes (LC method 2). m/z = 1045.6 (M+l) ⁺. ¾ NMR (400 MHz, D₂0) δ 8.27 (d, J = 8.0 Hz, 1H), 7.79 (s, 1H), 7.31 (td, J = 14.0, 5.6 Hz, 3H), 7.24 - 7.17 (m, 2H), 4.28-4.07 (d, J = 5.2 Hz, 1H), 4.46 - 4.28 (m, 4H), 4.28-4.07 (m, 6H), 3.85 (dd, J = 7.2, 4.4 Hz, 2H), 3.41 - 3.28 (m, 1H), 3.13 - 2.90 (m, 7H), 2.84-2.73 (m, 2H), 2.33 - 2.12 (m, 3H), 2.04-2.0 (m, 1H), 1.97 - 1.72 (m, 4H), 1.44 - 1.26 (m, 2H), 1.21 (t, J = 6.0 Hz, 2H), 1.18 - 1.07 (m, 4H), 0.73-0.61 (m, 7H).

[0293] N-((2S,3R)-3-hydroxy-l-((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-ylamino)- 1 -oxobutan-2-yl)nicotinamide Compound 280 was prepared according to the coupling then deprotection sequence describe for example 7, Compound 15. LCMS: rt = 0.18 minutes (LC method 2). m/z = 1055.6 (M+l) ⁺. ¾ NMR (400 MHz, D₂0) δ 9.00 (s, IH), 8.77 (s, IH), 8.48 (s, IH), 8.17 (s, 2H), 7.77 (s, IH), 7.32 (d, J= 7.3 Hz, 3H), 7.18 (d, J= 6.4 Hz, 2H), 4.50 (d, J= 5.2 Hz, 2H), 4.35 (d, J= 23.8 Hz, 5H), 4.24 (d, J= 5.2 Hz, 3H), 4.16 - 4.03 (m, 4H), 3.91 (d, J= 35.2 Hz, 3H), 3.33 (s, IH), 3.12 - 2.90 (m, 9H), 2.74 (s, 2H), 2.23 (d, J= 41.0 Hz, 4H), 1.93 (s, 3H), 1.82 (s, 3H), 1.32 (d, J= 12.8 Hz, 2H), 1.25 (d, J= 6.4 Hz, 3H), 1.08 (d, J= 6.4 Hz, 3H), 0.65 (d, J= 31.4 Hz, 7H).

[0294] N-((2S,3R)-3-hydroxy-l-((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,l l, 14,17,20-heptaoxo- 1, 4, 7, 10, 13, 16, 19-heptaazacyclotricosan-21 -ylamino)propan-2-ylamino)- 1 -oxobutan-2-yl)thiophene-2- carboxamide Compound 281 was prepared according to the coupling then deprotection sequence describe for example 7, Compound 15. LCMS: rt = 0.19 & 0.34 minutes (double peak, LC method 2). m/z = 1062.5 (M+l)⁺. ¾ NMR (400 MHz, D₂0) δ 8.26 (s, IH), 7.75 (m, 2H), 7.33 (m, 3H), 7.21 (m, 2H), 7.18 - 7.13 (m, IH), 4.57 - 4.04 (m, 13H), 3.88 (s, 2H), 3.35 (s, IH), 3.21 - 2.90 (m, 8H), 2.75 (m, 2H), 2.34 - 1.67 (m, 9H), 1.25 (m, 12H), 0.70 (s, 3H), 0.63 (s, 3H).

EXAMPLE 58. SYNTHESIS OF COMPOUND 282

[0295] N-((2S,3R)-3-hydroxy-l-((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-ylamino)- 1 -oxobutan-2-yl)picolinamide Compound 282 was prepared according to the coupling then deprotection sequence describe for example 7, Compound 15. LCMS: rt = 0.19 minutes (LC method 2). m/z = 1055.6 (M+l)⁺. ¾ NMR (400 MHz, D₂0) δ 8.52 (d, J = 3.9 Hz, 1H), 8.07 (s, 1H), 7.97 (d, J = 4.2 Hz, 2H), 7.56 (m, lH), 7.31 - 7.15 (m, 3H), 7.09 (d, J = 6.8 Hz, 2H), 4.48 (t, J = 7.1 Hz, 1H), 4.23 (m, 5H), 4.16 - 3.86 (m, 5H), 3.73 (d, J = 37.0, 5.8 Hz, 2H), 3.41 - 3.12 (m, 1H), 3.05 - 2.78 (m, 7H), 2.78 - 2.54 (m, 2H), 2.26 - 1.51 (m, 8H), 1.24 (m, 2H), 1.15 (d, J = 6.4 Hz, 3H), 1.11 - 1.03 (m, 1H), 1.00 (d, J = 6.2 Hz, 3H), 0.61 (s, 4H), 0.53 (d, J = 5.4 Hz, 3H).

283

[0296] N-((2S,3R)-3-hydroxy-l-((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,l l, 14,17,20-heptaoxo- 1, 4, 7, 10, 13, 16, 19-heptaazacyclotricosan-21 -ylamino)propan-2-ylamino)- 1 -oxobutan-2-yl)benzamide Compound 283 was prepared according to the coupling then deprotection sequence describe for example 7, Compound 15. LCMS: rt = 0.34 minutes (LC method 2). m/z = 1054.6 (M+l)⁺. ¾ NMR (400 MHz, D₂0) δ 8.08 (s, 2H), 7.77 - 7.64 (m, 2H), 7.60 - 7.44 (m, 2H), 7.40 (t, J = 7.6 Hz, 2H), 7.22 (m, 3H), 7.10 (d, J = 7.3 Hz, 2H), 4.42 (d, J = 8.0 Hz, 1H), 4.34 - 4.15 (m, 5H), 4.10 (m, 2H), 4.06 - 3.90 (m, 3H), 3.86 - 3.70 (m, 2H), 3.23 (m, 1H), 3.09 - 2.77 (m, 7H), 2.76 - 2.51 (m, 2H), 2.31 - 1.59 (m, 8H), 1.26 (m, 2H), 1.15 (d, J = 6.0 Hz, 3H), 1.13 - 1.03 (m, 1H), 1.01 (d, J = 8.0 Hz, 2H), 0.60 (s, 4H), 0.52 (s, 3H).

[0297] N-((2S,3R)-3-hydroxy-l-((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,l l, 14,17,20-heptaoxo- 1, 4, 7, 10, 13, 16, 19-heptaazacyclotricosan-21 -ylamino)propan-2-ylamino)- 1 -oxobutan-2-yl)-5 -methylfuran- 2-carboxamide Compound 284 was prepared according to the coupling then deprotection sequence describe for example 7, Compound 15. LCMS: rt = 0.20 minutes (LC method 2). m/z = 1058.5 (M+l)⁺. 'H NMR (400 MHz, D₂0): 8.27 (s, 1H), 7.39-7.42 (m, 3H), 7.27-7.30 (t, 2H), 7.14-7.18 (m, lH), 6.29-

6.33 (m, 1H), 4.16-4.79 (m, 11H), 3.891-3.982 (m, 2H), 3.38-3.46 (m, lH), 2.86-3.14 (m, 8H), 2.38-2.38 (d, J = 3.2 Hz, 3H), 2.22-2.33 (m, 3H), 2.01-2.08 (m, 2H), 1.87-1.94 (m, 2H), 1.42-1.49 (m, lH), 1.28-

1.34 (m, 3H), 1.17-1.21 (m, 3H).

[0298] 5-acetamido-N-((2S,3R)-3-hydroxy-l-((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-ylamino)- 1 - oxobutan-2-yl)furan-2-carboxamide Compound 285 was prepared according to the coupling then deprotection sequence describe for example 7, Compound 15. LCMS: rt = 0.22 & 0.31 minutes (double peak, LC method 2). m/z = 1101.6 (M+l)⁺. 'H NMR (400 MHz, D₂0) δ 8.17 (s, 1H), 7.26 (m, 6H), 6.36 (d, J= 3.5 Hz, 1H), 4.58 - 4.32 (m, 5H), 4.17 (m, 8H), 3.86 (s, 2H), 3.34 (s, 1H), 3.22-2.91 (m, 8H), 2.90- 2.56 (m, 3H), 2.32-2.07 (m, 6H), 1.97 (m, 3H), 1.83 (m, 3H), 1.47 - 1.34 (m, 2H), 1.28 (m, 1H), 1.21 (m, 3H), 1.10 (m, 3H), 0.73 (m, 1H), 0.68 (m, 3H), 0.62 (s, 3H).

EXAMP

[0299] N-((2S,3R)-3-hydroxy-l-((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-ylamino)- 1 -oxobutan-2-yl)-2,5 - dimethylfuran-3-carboxamide, Compound 286, was prepared according to the coupling then deprotection sequence describe for example 7, Compound 15. LCMS: rt = 0.21 & 0.34 minutes (double peak, LC method 2). m/z = 1072.6 (M+l)⁺. ¾ NMR (400 MHz, D₂0) δ 7.43-7.35 (m, 3H), 7.27 (d, J = 7.2 Hz, 2H), 6.35 (s, 1H), 4.59 - 4.45 (m, 3H), 4.40 (dd, J = 10.0, 5.0Hz, 2H), 4.34 - 4.24 (m, 3H), 4.23 - 4.13 (m, 3H), 3.98 - 3.85 (m, 2H), 3.43-3.36 (m, lH), 3.15-3.01 (m, 7H), 2.91-2.78 (m, 2H), 2.47 (s, 3H), 2.37 - 2.20 (m, 6H), 2.13 - 1.95 (m, 3H), 1.94 - 1.83 (m, 2H), 1.52 - 1.35 (m, 2H), 1.28 (d, J = 6.4 Hz, 3H), 1.18 (d, J = 6.2 Hz, 3H), 0.82-0.69 (m, 7H).

[0300] N-((2S,3R)-3-hydroxy-l-((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,l l, 14,17,20-heptaoxo- 1, 4, 7, 10, 13, 16, 19-heptaazacyclotricosan-21 -ylamino)propan-2-ylamino)- 1 -oxobutan-2-yl)-6- methylpicolinamide, Compound 287 was prepared according to the coupling then deprotection sequence describe for example 7, Compound 15.

[0301] LCMS: rt = 0.19 & 0.34 minutes (double peak, LC method 2). m/z = 1069.6 (M+l)⁺. ¾ NMR (400 MHz, D₂0) δ 7.99 (t, J = 7.8 Hz, 1H), 7.90 (d, J = 7.6 Hz, 1H), 7.57 (d, J = 7.8 Hz, lH), 7.30 (dq, J = 14.2, 7.0 Hz, 3H), 7.17 (d, J = 6.8 Hz, 2H), 4.54 (d, J = 4.4 Hz, lH), 4.39 - 4.25 (m, 5H), 4.21 - 4.04 (m, 5H), 3.85 (dt, J = 8.4, 4.2 Hz, 2H), 3.31 (dd, J = 16.6, 7.0 Hz, lH), 3.08 - 2.89 (m, 7H), 2.84 - 2.69 (m, 2H), 2.58 (s, 3H), 2.27-2.10 (m, 3H), 2.01-1.90 (m, 2H), 1.83-1.76 (m, 3H), 1.42 - 1.27 (m, 2H), 1.23 (t, J = 7.6 Hz, 3H), 1.08 (d, J = 6.2 Hz, 3H), 0.78 - 0.66 (m, 4H), 0.61 (d, J = 5.6 Hz, 3H).

288

[0302] N-((2S,3R)-3-hydroxy-l-((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,l l, 14,17,20-heptaoxo- 1, 4, 7, 10, 13, 16, 19-heptaazacyclotricosan-21 -ylamino)propan-2-ylamino)- 1 -oxobutan-2-yl)isonicotinamide Compound 288 was prepared according to the coupling then deprotection sequence describe for example 7, Compound 15. LCMS: rt = 0.19 minutes (double peak, LC method 2). m/z = 1055.6 (M+l)⁺. ¾ NMR (400 MHz, D₂0) δ 8.70 (d, J = 5.2 Hz, 2H), 7.81 (d, J = 6.0 Hz, 2H), 7.30 (m, 4H), 7.18 (d, J = 7.2 Hz, 2H), 4.51 (d, J = 5.2 Hz, 1H), 4.45 - 4.00 (m, 10H), 3.86 (m, 2H), 3.30 (m, lH), 2.99 (m, 8H), 2.87 - 2.57 (m, 3H), 2.37 - 1.70 (m, 9H), 1.32 (m, 2H), 1.24 (d, J = 6.4 Hz, 3H), 1.10 (s, 1H), 1.08 (d, J = 6.0 Hz, 3H), 0.87 (t, J = 7.2 Hz, 1H), 0.69 (s, 3H), 0.61 (d, J = 5.2 Hz, 3H).

289

[0303] N-((2S,3R)-3-hydroxy-l-((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-ylamino)- 1 -oxobutan-2-yl)-3 -methylfuran- 2-carboxamide Compound 289 was prepared according to the coupling then deprotection sequence describe for example 7, Compound 15. LCMS: rt = 0.20 minutes (LC method 2). m/z = 1058.6 (M+l)⁺. 'H NMR (400 MHz, D₂0) δ 8.16 (s, 1H), 7.46 (s, 1H), 7.31 (m, 3H), 7.19 (d, J = 7.1 Hz, 2H), 6.45 (s, 1H), 4.47 (d, J = 4.5 Hz, 1H), 4.42 - 3.99 (m, 10H), 3.85 (d, J = 5.0 Hz, 2H), 3.32 (m, lH), 3.13 - 2.86 (m, 7H), 2.85 - 2.61 (m, 2H), 2.24 (s, 3H), 2.19 - 1.65 (m, 7H), 1.47 - 1.24 (m, 2H), 1.20 (d, J = 6.3 Hz, 3H), 1.09 (d, J = 6.1 Hz, 3H), 0.67 (d, J = 16.8 Hz, 4H), 0.61 (d, J = 5.4 Hz, 3H).

[0304] N-((2S,3R)-3-hydroxy-l-((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,l l, 14,17,20-heptaoxo- 1, 4, 7, 10, 13, 16, 19-heptaazacyclotricosan-21 -ylamino)propan-2-ylamino)- 1 -oxobutan-2-yl)-5 - (methoxymethyl)furan-2-carboxamide Compound 290 was prepared according to the coupling then deprotection sequence describe for example 7, Compound 15. LCMS: rt = 0.18 minutes (LC method 2). m/z = 1088.6 (M+l)⁺. ¾ NMR (400 MHz, D₂0) δ 8.18 (s, 1H), 7.33 (m, 3H), 7.20 (m, 2H), 7.13 (s, 1H), 6.58 (s, 1H), 4.47 (s, 3H), 4.36 (s, 3H), 4.27 (m, 2H), 4.21 - 4.05 (m, 5H), 3.85 (s, 2H), 3.34 (s, 4H), 3.01 (m, 7H), 2.78 (m, 2H), 2.16 (m, 3H), 1.97 (m, 2H), 1.81 (m, 3H), 1.32 (m, 2H), 1.21 (m, 3H), 1.09 (m, 3H), 0.69 (s, 3H), 0.61 (s, 3H).

EXAMPLE 67. SYNTHESIS OF COMPOUND 291

[0305] 5-acetyl-N-((2S,3R)-3-hydroxy-l-((R)-3-hydroxy-l-oxo-l- ((3S,6S,9S, 12S, 15R, 18S,21S)-6,9,18-tris(2-aminoethylH^

2,5,8,11, 14,17,20-heptaoxo- 1,4,7, 10,13, 16, 19-heptaazacyclotricosan-21 -ylamino)propan-2-ylamino)- 1 - oxobutan-2-yl)furan-2-carboxamide Compound 291 was prepared according to the coupling then deprotection sequence describe for example 7, Compound 15. LCMS: rt = 0.38 minutes (LC method 2). m/z = 1086.6 (M+l)⁺. ¾ NMR (400 MHz, D₂0): 8.26 (s, lH), 7.57-7.58 (d, J = 4.0 Hz, 1H), 7.36-7.42 (m, 4H),7.26-7.28 (d, J = 6.8 Hz, 2H), 4.60-4.61 (d, J=4.8 Hz, lH), 4.16-4.48 (m, 10H), 3.95-3.96 (m, 2H), 3.39-3.47 (m, 1H), 2.83-3.15 (m, 9H), 1.87-2.38 (m, 8H), 1.37-1.49 (m, 2H), 1.30-1.32 (d, J=6.4 Hz, 3H), 1.18-1.19 (d, J=6.0 Hz, 3H),0.71-0.82 (m, 7H).

[0306] N-((2S,3R)-3-hydroxy-l-((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,l l, 14,17,20-heptaoxo- 1, 4, 7, 10, 13, 16, 19-heptaazacyclotricosan-21 -ylamino)propan-2-ylamino)- 1 -oxobutan-2-yl)-5 - isopropylfuran-2-carboxamide Compound 292 was prepared according to the coupling then deprotection sequence describe for example 7, Compound 15. LCMS: rt = 0.34 minutes (LC method 2). m/z = 1086.6 (M+l)⁺. 1H-NMR (400 MHz, D₂0): 7.35-7.44 (m, 3H), 7.27-7.28 (m, 2H), 7.20 (d, J = 3.2 Hz, lH), 6.35 (d, J = 3.2 Hz, 1H), 4.57 (d, J = 4.8 Hz, 1H), 4.15-4.49 (m, 1 1H), 3.94-3.95 (m, 2H), 3.35-3.44 (m, 1H), 3.02-3.13 (m, 9H), 2.80-2.94 (m, 2H), 2.18-2.38 (m, 3H), 1.87-2.07(m, 4H), 1.33-1.47 (m, 1H), 1.24-1.33 (m, 10H), 1.17-1.18 (d, J = 6.0 Hz, 2H), 0.69-0.79 (m, 6H).

EXAMPLE 69. SYNTHESIS OF COMPOUND 293 S

[0307] N-((2S,3R)-3-hydroxy-l-((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-ylamino)- 1 -oxobutan-2-yl)pyrazine-2- carboxamide Compound 293 was prepared according to the coupling then deprotection sequence describe for example 7, Compound 15. LCMS: rt = 0.43 minutes (LC method 2). m/z = 1056.6 (M+l)⁺. ¾ NMR (400 MHz, D₂0) δ 9.22 (d, J= 1.2 Hz, 1H), 8.86 (t, J= 2.8 Hz, 1H), 8.78 (dd, J= 6.0, 4.4 Hz, 1H), 8.27 (s, 1H), 7.41 (td, J= 14.4, 7.2 Hz, 3H), 7.29 (d, J= 6.8 Hz, 2H), 4.60 (d, J= 4.8 Hz, lH), 4.49 - 4.14 (m, 9H), 3.96 (d, J= 5.2 Hz, 2H), 3.48 (t, J= 10.8 Hz, lH), 3.25 - 2.96 (m, 7H), 2.88 - 2.82 (m, 2H), 2.74 (s, 1H), 2.45 - 2.23 (m, 3H), 2.13 - 2.01 (m, 2H), 1.90 (dd, J= 15.2, 10.8 Hz, 1H), 1.80 - 1.66 (m, 1H), 1.48 (d, J = 18.4 Hz, 1H), 1.39 - 1.23 (m, 5H), 1.18 (d, J= 6.0 Hz, 2H), 0.78 (s, 3H), 0.70 (s, 3H).

[0308] N-((2S,3R)-3-hydroxy-l-((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,l l, 14,17,20-heptaoxo- 1, 4, 7, 10, 13, 16, 19-heptaazacyclotricosan-21 -ylamino)propan-2-ylamino)- 1 -oxobutan-2-yl)-6- methylpyridazine-3-carboxamide Compound 294 was prepared according to the coupling then deprotection sequence describe for Example 7, Compound 15. LCMS: rt = 0.18 & 0.33 minutes (double peak, LC method 2). m/z = 1070.6 (M+l)⁺. ¾ NMR (400 MHz, D₂0) δ 8.15 (d, J = 7.8 Hz, 1H), 7.81 (d, J = 8.6 Hz, 1H), 7.35 - 7.22 (m, 3H), 7.19-7.16 (m, 2H), 4.62 (d, J = 4.2 Hz, lH), 4.46 - 4.24 (m, 5H), 4.22 - 4.02 (m, 5H), 3.86 (d, J = 5.4 Hz, 2H), 3.40 - 3.28 (m, lH), 3.15 - 2.80 (m, 8H), 2.79 - 2.60 (m, 4H), 2.30 - 2.08 (m, 3H), 2.01 - 1.92 (m, 2H), 1.88-1.76 (m, 3H), 1.39-1.36(m, 1H), 1.31 - 1.19 (m, 4H), 1.09 (t, J = 9.0 Hz, 3H), 0.70-0.59(m, 7H).

295

[0309] N-((2S,3R)-3-hydroxy-l-((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-ylamino)- 1 -oxobutan-2-yl)-2- methyloxazole-4-carboxamide Compound 295 was prepared according to the coupling then deprotection sequence describe for Example 7, Compound 15. LCMS: rt = 0.19 & 0.33 minutes (double peak, LC method 2). m/z = 1059.6 (M+l)⁺. ¾ NMR (400 MHz, D₂0) δ 8.36 (s, 1H), 8.25 (s, 1H), 7.43-7.36 (m, 3H), 7.28 (d, J = 7.2 Hz, 2H), 4.58 - 4.38 (m, 5H), 4.35 - 4.14 (m, 6H), 3.94 (d, J = 2.2 Hz, 2H), 3.45- 3.36 (m, 1H), 3.20 - 3.00 (m, 7H), 2.92-2.84 (m, 2H), 2.53 (s, 3H), 2.39 - 2.20 (m, 3H), 2.13 - 1.85 (m, 5H), 1.51 - 1.35 (m, 2H), 1.29 (d, J = 6.2 Hz, 3H), 1.18 (d, J = 6.0 Hz, 3H), 0.79-0.69(m, 7H).

[0310] N-((2S,3R)-3-hydroxy-l-((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,l l, 14,17,20-heptaoxo- 1, 4, 7, 10, 13, 16, 19-heptaazacyclotricosan-21 -ylamino)propan-2-ylamino)- 1 -oxobutan-2-yl)benzofuran-2- carboxamide Compound 296 was prepared according to the coupling then deprotection sequence described for Example 7, Compound 15. LCMS: rt = 0.24 & 0.33 minutes (double peak, LC method 2). m/z = 1094.6 (M+l)⁺. ¾ NMR (400 MHz, D₂0) δ 7.78 (d, J = 7.6 Hz, 1H), 7.65 - 7.58 (m, 2H), 7.51 (t, J = 7.6 Hz, 1H), 7.36 - 7.23 (m, 4H), 7.08 (d, J = 7.2 Hz, 2H), 4.60 (d, J = 4.8 Hz, lH), 4.34-4.25 (m, 4H), 4.22-4.13 (m, 2H), 4.10 - 3.99 (m, 4H), 3.87 (d, J = 5.4 Hz, 2H), 3.38-3.12 (m, 1H), 3.09 - 2.93 (m, 5H), 2.90 - 2.78 (m, 2H), 2.73 (d, J = 8.4 Hz, 2H), 2.29 - 2.07 (m, 3H), 2.01 - 1.76 (m, 5H), 1.38 - 1.30 (m, 1H), 1.27 - 1.15 (m, 4H), 1.05 (d, J = 6.2 Hz, 3H), 0.70 - 0.50 (m, 7H).

EXAMP

[0311] N-((2S,3R)-3-hydroxy-l-((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-ylamino)- 1 -oxobutan-2-yl)-5 -phenylfuran- 2-carboxamide Compound 297 was prepared according to the coupling then deprotection sequence describe for Example 7, Compound 15. LCMS: rt = 0.24 minutes (double peak, LC method 2). m/z = 1121.6 (M+l)⁺. ¾ NMR (400MHz, D₂0): 8.25 (s, 1H), 7.90-7.92 (d, J = 7.6 Hz, 2H), 7.06-7.57 (m, 10H), 4.66-4.79 (m, 1H), 3.95-4.40 (m, 12H), 3.39-3.46 (m, lH), 2.90-3.12 (m, 9H), 1.85-2.33 (m, 7H), 1.31-1.85 (m, 10H), 0.66-0.80 (m, 6H).

298

[0312] N-((2S,3R)-3-hydroxy-l-((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,l l, 14,17,20-heptaoxo- 1, 4, 7, 10, 13, 16, 19-heptaazacyclotricosan-21 -ylamino)propan-2-ylamino)- 1 -oxobutan-2-yl)oxazole-4- carboxamide Compound 298 was prepared according to the coupling then deprotection sequence describe for example 7, Compound 15. LCMS: rt = 0.19 & 0.32 minutes (double peak, LC method 2). m/z = 1044.6 (M+l)⁺. ¾ NMR (400 MHz, D₂0) δ 8.43 (s, 1H), 8.14 (s, 1H), 7.35-7.26 (m, 3H), 7.20 (d, J = 6.8 Hz, 2H), 4.48 (d, J = 4.6 Hz, 1H), 4.41 - 4.22 (m, 5H), 4.20 - 4.04 (m, 5H), 3.85 (d, J = 4.4 Hz, 2H), 3.42 - 3.28 (m, 1H), 3.10 - 2.91 (m, 7H), 2.83-2.74 (m, 2H), 2.31 - 2.10 (m, 3H), 1.97 (d, J = 12.8 Hz, 2H), 1.90 - 1.72 (m, 3H), 1.42 - 1.27 (m, 2H), 1.21 (d, J = 6.0 Hz, 3H), 1.09 (d, J = 6.0 Hz, 3H), 0.71-0.60 (m, 7H).

[0313] 5-fluoro-N-((2S,3R)-3-hydroxy-l-((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-isobu^ 2,5,8,11, 14,17,20-heptaoxo- 1,4,7, 10,13, 16, 19-heptaazacyclotricosan-21 -ylamino)propan-2-ylamino)- 1 - oxobutan-2-yl)picolinamide Compound 299 was prepared according to the coupling then deprotection sequence describe for example 7, Compound 15. LCMS: rt = 0.21 &0.33 minutes (double peak, LC method 2). m/z = 1073.6 (M+l)⁺. ¾ NMR (400 MHz, D₂0) δ 8.51 (d, J = 2.4 Hz, 1H), 8.09 (dd, J = 8.8, 4.4 Hz, 1H), 7.78 (td, J = 8.4, 2.8 Hz, 1H), 7.36-7.28 (m, 3H), 7.19 (d, J = 6.8 Hz, 2H), 4.54 (d, J = 4.4 Hz, 1H), 4.46 - 4.28 (m, 5H), 4.24 - 4.05 (m, 5H), 3.88 (dd, J = 5.2, 1.8 Hz, 2H), 3.40 - 3.29 (m, lH), 3.12 - 2.91 (m, 7H), 2.85-2.72 (m, 2H), 2.32 - 2.10 (m, 3H), 2.05-1.93 (m, 2H), 1.89 - 1.72 (m, 3H), 1.45 - 1.29 (m, 2H), 1.24 (d, J = 6.4 Hz, 3H), 1.11 (d, J = 6.4 Hz, 3H), 0.79 - 0.57 (m, 7H).

EXAMP

[0314] N-((2S,3R)-3-hydroxy-l-((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,l l, 14,17,20-heptaoxo- 1, 4, 7, 10, 13, 16, 19-heptaazacyclotricosan-21 -ylamino)propan-2-ylamino)- 1 -oxobutan-2-yl)-2,5 - dimethyloxazole-4-carboxamide Compound 300 was prepared according to the coupling then deprotection sequence describe for example 7, Compound 15. LCMS: rt = 0.21 & 0.33 minutes (double peak, LC method 2). m/z = 1073.6 (M+l)⁺. ¾ NMR (400 MHz, D20): δ 7.39 - 7.23 (m, 3H), 7.19 (d, J = 7.6 Hz, 2H), 4.48-4.35 (m, 3H), 4.31 (m, 2H), 4.28-4.03 (m, 6H), 3.86 (m, 2H), 3.32 (m, lH), 2.99 (m, 7H), 2.86-2.66 (m, 2H), 2.45 (s, 3H), 2.36 (s, 3H), 2.32 - 1.72 (m, 9H), 1.47 - 1.24 (m, 2H), 1.19 (d, J = 6.4 Hz, 3H), 1.10 (d, J = 6.0 Hz, 3H), 0.73 (d, J = 5.6 Hz, 1H), 0.73 - 0.66 (m, 4H), 0.61 (d, J = 5.6 Hz,

3H).

301

[0315] (2S,3R)-3-hydroxy-N-((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,l l, 14,17,20-heptaoxo- 1, 4, 7, 10, 13, 16, 19-heptaazacyclotricosan-21 -ylamino)propan-2-yl)-2-(2-oxoazetidin- 1 -yl)butanamide Compound 301 was prepared according to the coupling then deprotection sequence describe for

Example 7, Compound 15. LCMS: rt = 0.20 & 0.30 minutes (double peak, LC method 2). m/z = 1105.6 (M+l)⁺. ¾ NMR (400 MHz, D₂0): δ 8.18 (s, 1H), 7.40 - 7.22 (m, 3H), 7.19 (d, J = 7.4 Hz, 2H), 4.30 (m, 12H), 3.83 (m, 2H), 3.68 (m, 1H), 3.50 (m, 1H), 3.24 (m, 2H), 3.16-2.56 (m, 1 1H), 2.37-1.51 (m, 9H), 1.52-1.30 (m, 3H), 1.29-1.02 (m, 9H), 0.71 (d, J = 9.4 Hz, 3H), 0.62 (s, 3H).

[0316] 5-(difluoromethyl)-N-((2S,3R)-3-hydroxy- 1 -((R) -3 -hydroxy- 1 -oxo- 1 - ((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-ylamino)- 1 - oxobutan-2-yl)furan-2 -carboxamide Compound 302 was prepared according to the coupling then deprotection sequence describe for Example 7, Compound 15. LCMS: rt = 0.20 minutes (LC method 2). m/z = 1094.6 (M+l)⁺. ¾ NMR (400 MHz, D₂0): 7.27-7.44 (m, 6H), 6.79-7.05 (m, 2H), 4.60 (d, J = 4.4 Hz, 1H), 4.17-4.48 (m, 10H), 3.94-3.95 (d, J=5.2 Hz, 2H), 3.36-3.48 (m, lH), 2.83-3.13 (m, 9H), 1.85- 2.37 (m, 8H), 1.17-1.49 (m, 8H), 0.70-0.79 (m, 6H).

303

[0317] N-((2S,3R)-3-hydroxy-l-((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,l l, 14,17,20-heptaoxo- 1, 4, 7, 10, 13, 16, 19-heptaazacyclotricosan-21 -ylamino)propan-2-ylamino)- 1 -oxobutan-2-yl)isoxazole-5 - carboxamide Compound 303 was prepared according to the coupling then deprotection sequence describe for example 7, Compound 15. LCMS: rt = 0.19 minutes (LC method 2). m/z = 1045.5 (M+l)⁺. ¾ NMR (400 MHz, D₂0): δ 8.18 (s, 1H), 7.34 (m, 3H), 7.22 (d, J = 7.2 Hz, 2H), 7.06 (s, 1H), 4.53 (m, 1H), 4.46 - 4.33 (m, 3H), 4.32 - 4.24 (m, 2H), 4.20 - 4.07 (m, 5H), 3.88 (d, J = 5.2 Hz, 2H), 3.39 (m, 1H), 3.03 (m, 5H), 2.98 - 2.90 (m, 2H), 2.83 (m, 1H), 2.77 (m, lH), 2.66 (s, lH), 2.31 - 2.13 (m, 3H), 1.97 (d, J = 9.2 Hz, 2H), 1.91 - 1.75 (m, 3H), 1.34 (m, 2H), 1.24 (d, J= 6.4 Hz, 3H), 1.11 (d, J= 6.0 Hz, 3H), 0.73 (s, 1H), 0.71 (s, 3H), 0.63 (d, J= 5.6 Hz, 3H).

[0318] N-((2S,3R)-3-hydroxy-l-((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-ylamino)- 1 -oxobutan-2-yl)-5 - methyloxazole-4-carboxamide Compound 304 was prepared according to the coupling then deprotection sequence describe for example 7, Compound 15. LCMS: rt = 0.31 minutes (LC method 2). m/z = 1075.6 (M+l)⁺. ¾ NMR (400 MHz, D₂0): δ 8.68 (s, 1H), 7.34 (m, 3H), 7.22 (m, 2H), 4.52 (s, 3H), 4.35 (m, 5H), 4.21 - 4.09 (m, 5H), 3.88 (s, 2H), 3.37 (s, 1H), 3.02 (m, 7H), 2.77 (m, 2H), 2.67 (s, 3H), 2.19 (m, 3H), 1.97 (s, 2H), 1.82 (s, 3H), 1.37 (m, 2H), 1.23 (m, 3H), 1.1 1 (m, 3H), 0.77 (m, 1H), 0.72 (s, 3H), 0.65 (s, 3H).

305

[0319] N-((2S,3R)-3-hydroxy-l-((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,l l, 14,17,20-heptaoxo- 1, 4, 7, 10, 13, 16, 19-heptaazacyclotricosan-21 -ylamino)propan-2-ylamino)- 1 -oxobutan-2-yl)-4- methylthiazole-2-carboxamide Compound 305 was prepared according to the coupling then deprotection sequence describe for example 7, Compound 15. LCMS: rt = 0.22 & 0.37 minutes (double peak, LC method 2). m/z = 1075.6 (M+l)⁺. ¾ NMR (400 MHz, D₂0): δ 8.94 (s, 1H), 7.34-7.25 (m, 3H), 7.19 (t, J = 7.2 Hz, 2H), 4.56-4.30 (m, 5H), 4.22-4.05 (m, 6H), 3.87-3.83 (m, 2H), 3.38-3.26 (m, lH), 3.10 - 2.94 (m, 7H), 2.86 - 2.65 (m, 3H), 2.57 (s, 2H), 2.23-2.09 (m, 3H), 2.02-1.78 (m, 5H), 1.46-1.33 (m, 3H), 1.26-1.20 (m, 2H), 1.10 (dd, J = 11.6, 6.0 Hz, 3H), 0.76-0.55 (m, 7H).

EXAMPLE 82. SYNTHESIS OF COMPOUND 306

[0320] N-((2S,3R)-3-hydroxy-l-((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-ylamino)- 1 -oxobutan-2-yl)- lH-pyrrole-2- carboxamide Compound 306 was prepared according to the coupling then deprotection sequence describe for Example 7, Compound 15. LCMS: rt = 0.22 &0.33 minutes (double peak, LC method 2). m/z = 1043.5 (M+l)⁺. ¾ NMR (400 MHz, D₂0): δ 7.44-7.35 (m, 3H), 7.28 (d, J = 6.8 Hz, 2H), 7.13 (s, 1H), 7.02 (d, J = 4.4 Hz, 1H), 6.34 (s, 1H), 4.61 - 4.13 (m, 1 1H), 4.00 - 3.86 (m, 2H), 3.44-3.34 (m, 1H), 3.20 - 2.98 (m, 7H), 2.86-2.73 (m, 2H), 2.39 - 2.18 (m, 3H), 2.13 - 1.98 (m, 2H), 1.96 - 1.78 (m, 3H), 1.51 - 1.36 (m, 2H), 1.29 (d, J = 6.0 Hz, 3H), 1.18 (d, J = 6.0 Hz, 3H), 0.87 - 0.65 (m, 7H).

EXAMP

[0321] (2S,3R)-2-(2-(4-ethylphenyl)acetamido)-3-hydroxy-N-((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)butanamide Compound 307 was prepared according to the coupling then deprotection sequence describe for

Example 7, Compound 15. LCMS: rt = 0.18 & 0.33 minutes (double peak, LC method 2). m/z = 1096.6 (M+l). ¾ NMR (400 MHz, D₂0): δ 7.34-7.24 (m, 3H), 7.23-7.14 (m, 6H), 4.54-4.45 (m, lH), 4.39-4.27 (m, 3H), 4.23 (d, J = 4.6 Hz, 1H), 4.21-4.05 (m, 6H), 3.88-3.76 (m, 2H), 3.66-3.50 (m, 2H), 3.25-3.14 (m, 1H), 3.06-2.93 (m, 6H), 2.91-2.62 (m, 4H), 2.56 (q, J = 7.2 Hz, 2H), 2.30-2.07 (m, 3H), 2.02-1.77 (m, 4H), 1.74-1.62 (m, 1H), 1.45-1.30 (m, 2H), 1.13 (dd, J = 14.8, 7.6 Hz, 9H), 0.82-0.56 (m, 7H).

[0322] N-((2S,3R)-3-hydroxy-l-((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-ylamino)- 1 -oxobutan-2-yl)- lH-pyrrole-3 - carboxamide Compound 308 was prepared according to the coupling then deprotection sequence describe for example 7, Compound 15. LCMS: rt = 0.19 & 0.34 minutes (double peak, LC method 2). m/z = 1043.6 (M+l)⁺. ¾ NMR (400 MHz, D₂0): δ 7.44 (s, 1H), 7.35-7.26 (m, 3H), 7.19 (d, J = 7.2 Hz, 2H), 6.85 (s, 1H), 6.56 (s, 1H), 4.50-4.28 (m, 5H), 4.26-4.02 (m, 6H), 3.90-3.79 (m, 2H), 3.35-3.26 (m, 1H), 3.11-2.88 (m, 7H), 2.78-2.65 (m, 2H), 2.31-2.10 (m, 3H), 2.03-1.76(m, 6H), 1.42-1.27 (m, 2H), 1.20 (d, J = 6.2 Hz, 3H), 1.09 (d, J = 6.0 Hz, 3H), 0.73-0.58 (m, 7H).

[0323] (2S,3R)-2-(2-(5-ethylfuran-2-yl)acetamido)-3-hydroxy-N-((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)butanamide Compound 309 was prepared according to the coupling then deprotection sequence describe for example 7, Compound 15. LCMS: rt = 0.27 minutes (LC method 2). m/z = 898.5 (M+l)⁺. ¾ NMR (400 MHz, D₂0,): 8.35 (s, 1H), 7.26-7.43 (m, 5H), 6.21-6.22 (d, J = 2.8 Hz, 1H), 6.07-6.08 (d, J = 2.8 Hz, lH), 4.52- 4.58 (m, 1H), 4.15-4.36 (m, 6H), 3.67-3.69 (m, lH), 3.40-3.48 (m, lH), 3.02-3.22 (m, 9H), 2.76-2.94 (m, 2H), 2.58-2.64 (q, 1H), 1.87-2.35 (m, 10H), 1.38-1.51(m, 2H), 1.17-1.21(m, 6H), 0.69-0.80 (m, 7H).

310

[0324] 5-((2S,3R)-3-hydroxy-l-((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-ylamino)- 1 -oxobutan-2- ylcarbamoyl)furan-2-carboxylic acid Compound 310 was prepared according to the coupling then deprotection sequence describe for example 7, Compound 15. LCMS: rt = 0.17 minutes (LC method 2). m/z = 1088.6 (M+l)⁺. ¾ NMR (400 MHz, D₂0): δ 8.17 (s, 1H), 7.40-7.28 (m, 3H), 7.22 (m, 3H), 7.17 (m, 1H), 4.53 (m, 2H), 4.37 (m, 3H), 4.25 (m, 4H), 4.14 (m, 5H), 3.88 (d, J= 5.0 Hz, 2H), 3.43 (m, 1H), 3.02 (m, 7H), 2.92-2.76 (m, 2H), 2.18 (m, 4H), 1.97 (m, 2H), 1.81 (m, 3H), 1.32 (m, 2H), 1.23 (d, J= 6.0 Hz, 3H), 1.10 (d, J= 6.0 Hz, 3H), 0.68 (s, 3H), 0.60 (s, 3H)

EXAMPLE 87. SYNTHESIS OF COMPOUND 311

[0325] N2-((2S,3R)-3-hydroxy-l-((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,l l, 14,17,20-heptaoxo- 1, 4, 7, 10, 13, 16, 19-heptaazacyclotricosan-21 -ylamino)propan-2-ylamino)- 1 -oxobutan-2-yl)furan-2,5 - dicarboxamide Compound 311 was prepared according to the coupling then deprotection sequence describe for example 7, Compound 15. LCMS: rt = 0.23 &0.30 minutes (double peak, LC method 2). m/z = 1087.6 (M+l)⁺. ¾ NMR (400 MHz, D₂0): δ 8.20 (s, 1H), 7.32 (m, 3H), 7.26 (s, 2H), 7.20 (d, J= 73 Hz, 2H), 4.52 (d, J= 5.0 Hz, 1H), 4.40-4.23 (m, 5H), 4.18 (m, 2H), 4.10 (m, 3H), 3.88 (m, 2H), 3.34 (m, 1H), 3.13-2.92 (m, 7H), 2.83 (m, 2H), 2.19 (m, 3H), 1.97 (m, 2H), 1.83 (m, 3H), 1.44-1.29 (m, 2H), 1.23 (d, J= 6.3 Hz, 3H), 1.10 (d, J= 6.0 Hz, 3H), 0.72 (s, 1H), 0.70 (s, 3H), 0.62 (s, 3H).

312

[0326] 5-fluoro-N-((2S,3R)-3-hydroxy-l-((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-isobu^ 2,5,8,11, 14,17,20-heptaoxo- 1,4,7, 10,13, 16, 19-heptaazacyclotricosan-21 -ylamino)propan-2-ylamino)- 1 - oxobutan-2-yl)furan-2-carboxamide Compound 312 was prepared according to the coupling then deprotection sequence describe for example 7, Compound 15. LCMS: rt = 0.21 & 0.30 minutes (double peak, LC method 2). m/z = 1062.5 (M+l)⁺. ¾ NMR (400 MHz, D₂0): δ 8.30 (s, 1H), 7.34 (m, 3H), 7.22 (d, J= 7.3 Hz, 2H), 7.19 (m, 1H), 5.82 (m, 1H), 4.52-4.30 (m, 5H), 4.29-4.18 (m, 3H), 4.17-4.05 (m, 3H), 3.88 (s, 2H), 3.36 (m, 1H), 3.16-2.94 (m, 7H), 2.81 (m, 2H), 2.21 (m, 3H), 2.00 (m, 3H), 1.84 (m, 2H), 1.41 (m, 1H), 1.36-1.28 (m, 1H), 1.23 (d, J= 6.2 Hz, 3H), 1.13 (d, J= 6.0 Hz, 3H), 0.74 (s, 1H), 0.72 (s, 3H), 0.65 (s, 3H).

[0327] N-((2S,3R)-3-hydroxy-l-((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,l l, 14,17,20-heptaoxo- 1, 4, 7, 10, 13, 16, 19-heptaazacyclotricosan-21 -ylamino)propan-2-ylamino)- 1 -oxobutan-2-yl)-4- methyloxazole-5 -carboxamide Compound 313 was prepared according to the coupling then deprotection sequence describe for example 7, Compound 15. LCMS: rt = 0.22 & 0.35 minutes (double peak, LC method 2). m/z = 1059.6 (M+l)⁺. ¾ NMR (400 MHz, D₂0): δ 8.14 -8.07 (m, 1H), 7.34-7.25 (m, 3H), 7.19 (d, J = 7.0 Hz, 2H), 4.48 (t, J = 6.0 Hz, 1H), 4.44-4.28 (m, 4H), 4.26-4.07 (m, 5H), 3.93-3.79 (m, 2H), 3.40-3.27 (m, 1H), 3.10-2.90 (m, 7H), 2.84-2.71 (m, 2H), 2.36 (s, 3H), 2.30-2.08 (m, 3H), 2.05-1.75 (m, 5H), 1.44-1.26 (m, 3H), 1.20 (d, J = 6.0 Hz, 3H), 1.09 (d, J = 6.2 Hz, 3H), 0.77-0.58 (m, 7H).

[0328] 5-ethyl-N-((2S,3R)-3-hydroxy-l-((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-ylamino)- 1 -oxobutan-2-yl)furan-2- carboxamide Compound 314 was prepared according to the coupling then deprotection sequence describe for example 7, Compound 15. LCMS: rt = 0.27 minutes (LC method 2). m/z = 898.5 (M+l)⁺. 1H-NMR (400 MHz, D₂0): 8.35 (s, 1H), 7.26-7.43 (m, 5H), 6.21-6.22 (d, J=2.8 Hz, lH), 6.07-6.08 (m, 1H), 4.52-4.58 (m, 1H), 4.15-4.36 (m, 6H), 3.67-3.69 (m, lH), 3.40-3.48 (m, lH), 3.02-3.22 (m, 9H), 2.76-2.94 (m, 2H), 2.58-2.64 (m, 1H), 1.87-2.35 (m, 10H), 1.38-1.51 (m, 2H), 1.17-1.21 (m, 6H), 0.69- 0.80 (m, 7H).

[0329] 3-fluoro-N-((2S,3R)-3-hydroxy-l-((R)-3-hydroxy-l-oxo-l- ((3S,6S,9S, 12S, 15R, 18S,21S)-6,9,18-tris(2-aminoethyl)-l^^^

2,5,8,11, 14,17,20-heptaoxo- 1,4,7, 10,13, 16, 19-heptaazacyclotricosan-21 -ylamino)propan-2-ylamino)- 1 - oxobutan-2-yl)picolinamide Compound 315 was prepared according to the coupling then deprotection sequence describe for example 7, Compound 15. LCMS: rt = 0.22 & 0.34 minutes (double peak, LC method 2). m/z = 1073.6 (M+l)⁺. ¾ NMR (400 MHz, D₂0): δ 8.41 (d, J = 3.4 Hz, 1H), 7.78-7.71 (m, 1H), 7.68-7.61 (m, 1H), 7.35-7.23 (m, 3H), 7.17 (d, J = 7.4 Hz, 2H), 4.52 (d, J = 4.2 Hz, lH), 4.40-4.26 (m, 5H), 4.21-4.02 (m, 5H), 3.86 (d, J = 5.2 Hz, 2H), 3.41-3.30 (m, lH), 3.09-2.90 (m, 7H), 2.83-2.71 (m, 2H), 2.28-2.07 (m, 3H), 2.04-1.77 (m, 5H), 1.41-1.27 (m, 2H), 1.22 (d, J = 5.8 Hz, 3H), 1.09 (d, J = 5.8 Hz, 3H), 0.77-0.56 (m, 7H).

[0330] 5-cyclopropyl-N-((2S,3R)-3-hydroxy- 1 -((R)-3-hydroxy- 1 -oxo- 1 - ((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-ylamino)- 1 - oxobutan-2-yl)furan-2-carboxamide Compound 316 was prepared according to the coupling then deprotection sequence describe for example 7, Compound 15. LCMS: rt = 0.21 &0.29 minutes (double peak, LC method 2). m/z = 1085.6 (M+l)⁺. ¾ NMR (400 MHz, D₂0): δ 8.20 (s, 1H), 7.34 (m, 3H), 7.21 (d, J= 7.3 Hz, 2H), 7.11 (d, J= 3.4 Hz, 1H), 6.21 (d, J= 3.4 Hz, 1H), 4.49 (m, 1H), 4.44-4.33 (m, 3H), 4.32-4.23 (m, 2H), 4.22-4.08 (m, 5H), 3.92-3.81 (m, 2H), 3.34 (m, lH), 3.01 (m, 7H), 2.81 (m, 2H), 2.30- 2.11 (m, 3H), 1.88 (m, 6H), 1.40-1.27 (m, 2H), 1.22 (d, J= 6.3 Hz, 3H), 1.1 1 (d, J= 6.0 Hz, 3H), 1.00- 0.94 (m, 2H), 0.81 (m, 2H), 0.72 (s, 1H), 0.70 (s, 3H), 0.62 (s, 3H). 317

[0331] N-((2S,3R)-3-hydroxy-l-((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-ylamino)- 1 -oxobutan-2-yl)-4- methylthiophene-2-carboxamide Compound 317 was prepared according to the coupling then deprotection sequence describe for Example 7, Compound 15. LCMS: rt = 0.18 & 0.32 minutes (double peak, LC method 2). m/z = 1074.6 (M+l)⁺. ¾ NMR (400 MHz, D₂0): δ 7.58 (s, 1H), 7.35-7.26 (m, 4H), 7.18 (d, J = 7.2Hz, 2H), 4.44 (d, J = 5.0 Hz, 1H), 4.36 (dt, J = 9.6, 6.8 Hz, 3H), 4.28 (t, J = 5.0 Hz, IH), 4.25-4.04 (m, 6H), 3.90-3.82 (m, 2H), 3.36-3.29 (m, IH), 3.10-2.89 (m, 7H), 2.83-2.67 (m, 2H), 2.30- 2.10 (m, 6H), 2.05-1.90 (m, 2H), 1.87-1.72 (m, 3H), 1.41-1.25 (m, 2H), 1.22 (d, J = 6.4 Hz, 3H), 1.08 (d, J = 6.4 Hz, 3H), 0.73-0.61 (m, 7H).

[0332] N-((2S,3R)-3-hydroxy-l-((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,l l, 14,17,20-heptaoxo- 1 ,4,7, 10, 13, 16, 19-heptaazacyclotricosan-21 -ylamino)propan-2-ylamino)- 1 -oxobutan-2-yl)- 1 -methyl- 1H- pyrazole-4-carboxamide Compound 318 was prepared according to the coupling then deprotection sequence describe for example 7, Compound 15. LCMS: rt = 0.21 & 0.35 minutes (double peak, LC method 2). m/z = 1058.6 (M+l)⁺. ¾ NMR (400 MHz, D₂0): δ 7.50 (s, IH), 7.34-7.25 (m, 3H), 7.19 (d, J = 7.4 Hz, 2H), 6.82 (s, IH), 4.46 (d, J = 4.8 Hz, IH), 4.43-4.27 (m, 4H), 4.26-4.04 (m, 6H), 3.98 (s, 3H), 3.91-3.80 (m, 2H), 3.36-3.28(m, 1H), 3.10-2.92 (m, 7H), 2.78-2.67 (m, 2H), 2.31-2.09 (m, 3H), 2.04-1.73 (m, 5H), 1.42-1.31(m, 2H), 1.21 (d, J = 6.2 Hz, 3H), 1.09 (t, J = 7.6 Hz, 3H), 0.78-0.55 (m, 7H).

EXAMPL

[0333] N-((2S,3R)-3-hydroxy-l-((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-ylamino)- 1 -oxobutan-2-yl)-5 - (hydroxymethyl)furan-2-carboxamide Compound 319 was prepared according to the coupling then deprotection sequence describe for Example 7, Compound 15. LCMS: rt = 0.21 & 0.30 minutes (double peak, LC method 2). m/z = 1074.6 (M+l)⁺. ¾ NMR (400 MHz, D₂0): δ 8.17 (s, 1H), 7.31 (m, 3H), 7.19 (d, J= 7.0 Hz, 2H), 7.13 (d, J= 3.5 Hz, 1H), 6.49 (d, J= 3.5 Hz, 1H), 4.57 (s, 2H), 4.47 (d, J= 4.8 Hz, IH), 4.41-4.32 (m, 3H), 4.26 (m, 2H), 4.21-4.14 (m, 2H), 4.13-4.04 (m, 3H), 3.87 (m, 2H), 3.34 (m, 1H), 3.09-2.88 (m, 7H), 2.86-2.69 (m, 2H), 2.32-2.09 (m, 3H), 2.02-1.91 (m, 2H), 1.87-1.68 (m, 3H), 1.40- 1.24 (m, 2H), 1.21 (d, J= 6.4 Hz, 3H), 1.09 (d, J= 6.1 Hz, 3H), 0.71 (s, IH), 0.68 (s, 3H), 0.60 (s, 3H).

[0334] N-((2S,3R)-3-hydroxy-l-((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,l l, 14,17,20-heptaoxo- 1 ,4,7, 10, 13, 16, 19-heptaazacyclotricosan-21 -ylamino)propan-2-ylamino)- 1 -oxobutan-2-yl)- 1 -methyl- 1H- pyrazole-3-carboxamide Compound 320 was prepared according to the coupling then deprotection sequence describe for example 7, Compound 15. LCMS: rt = 0.24 minutes (LC method 2). m/z = 1058.6 (M+l). ¾ NMR (400 MHz, D₂0): δ 8.09 (s, 1H), 7.52 (s, 1H), 7.22 (m, 3H), 7.10 (d, J = 7.2 Hz, 2H), 6.64 (m, 1H), 4.48 - 3.94 (m, 11H), 3.90 - 3.66 (m, 5H), 3.25 (m, lH), 3.10 - 2.80 (m, 7H), 2.68 (m, 2H), 2.31 - 1.55 (m, 8H), 1.26 (m, 4H), 1.13 (d, J = 6.3 Hz, 3H), 1.01 (d, J = 5.9 Hz, 3H), 0.61 (s, 4H), 0.53 (s, 3H).

EXAMP

[0335] N-((2S,3R)-3-hydroxy-l-((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-ylamino)- 1 -oxobutan-2-yl)-3 - methylpicolinamide Compound 321 was prepared according to the coupling then deprotection sequence describe for example 7, Compound 15. LCMS: rt = 0.22 &0.39 minutes (double peak, LC method 2). m/z = 1069.6 (M+l). ¾ NMR (400 MHz, D₂0): δ 8.35 (d, J = 3.6 Hz, 1H), 7.93 - 7.87 (m, lH), 7.55- 7.50 (m, 1H), 7.27 - 7.15 (m, 3H), 7.10 (t, J = 6.4 Hz, 2H), 4.52 - 4.45 (m, lH), 4.37 - 3.95 (m, 10H), 3.86-3.77 (m, 2H), 3.35 - 3.20 (m, 1H), 3.04 - 2.83 (m, 7H), 2.76 - 2.60 (m, 2H), 2.23-2.02 (d, J = 18.2 Hz, 3H), 2.23-2.02(m, 3H), 2.00 - 1.69 (m, 5H), 1.36 - 1.21 (m, 2H), 1.16 (d, J = 6.4 Hz, 2H), 1.02 (dd, J = 10.6, 6.2 Hz, 3H), 0.68-0.52 (m, 7H).

322

[0336] 4-ethyl-N-((2S,3R)-3-hydroxy-l-((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-ylamino)- 1 -oxobutan-2-yl)benzamide

Compound 322 was prepared according to the coupling then deprotection sequence describe for example 7, Compound 15. LCMS: rt = 0.19 &0.33 minutes (double peak, LC method 2). m/z = 1097.6 (M+l). ¾ NMR (400 MHz, D₂0): δ 7.32-7.25 (m, 3H), 7.23-7.09 (m, 6H), 4.40-4.35 (m, 2H), 4.30 (dd, J = 9.2, 5.0 Hz, 2H), 4.23-3.98 (m, 7H), 3.90-3.78 (m, 2H), 3.34-3.23 (m, lH), 3.09-2.88 (m, 7H), 2.81-2.68 (m, 2H), 2.53 (m, 2H), 2.27-2.07 (m, 3H), 2.05-1.74 (m, 5H), 1.42-1.27 (m, 2H), 1.19 (d, J = 6.0 Hz, 3H), 1.10 (t, J = 7.6 Hz, 6H), 0.74-0.61(m, 7H).

323

[0337] N-((2S,3R)-3-hydroxy-l-((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,l l, 14,17,20-heptaoxo- 1 ,4,7, 10, 13, 16, 19-heptaazacyclotricosan-21 -ylamino)propan-2-ylamino)- 1 -oxobutan-2-yl)- lH-pyrazole- 4-carboxamide Compound 323 was prepared according to the coupling then deprotection sequence describe for Example 7, Compound 15. LCMS: rt = 0.22 &0.40 minutes (double peak, LC method 2). m/z = 1044.6 (M+l). ¾ NMR (400 MHz, D₂0): δ 8.03 (s, 2H), 7.28-7.16 (m, 3H), 7.11 (d, J = 7.2 Hz, 2H), 4.38 (d, J = 5.0 Hz, 1H), 4.35-3.96 (m, 10H), 3.77 (dd, J = 9.0, 4.8 Hz, 2H), 3.28-3.18 (m, lH), 2.99- 2.85 (m, 7H), 2.73-2.59 (m, 2H), 2.22 - 2.01 (m, 3H), 1.95-1.67 (m, 5H), 1.35-1.16 (m, 3H), 1.12 (t, J = 8.6 Hz, 3H), 1.02 (dd, J = 6.2, 6.0 Hz, 3H), 0.68 - 0.46 (m, 7H).

324

[0338] 4-chloro-N-((2S,3R)-3-hydroxy-l-((R)-3-hydroxy-l-oxo-l- ((3S,6S,9S, 12S, 15R, 18S,21S)-6,9,18-tris(2-aminoethylH^

2,5,8,11, 14,17,20-heptaoxo- 1,4,7, 10,13, 16, 19-heptaazacyclotricosan-21 -ylamino)propan-2-ylamino)- 1 - oxobutan-2-yl)furan-2-carboxamide Compound 324 was prepared according to the coupling then deprotection sequence describe for example 7, Compound 15. LCMS: rt = 0.30 minutes (LC method 2). m/z = 1078.6 (M+l). 1H NMR (400 MHz, D₂0): δ 8.21 (s, 1H), 7.74 (s, 1H), 7.33-7.21 (m, 5H), 6.43 (s, 1H), 4.47 (t, J = 8.2 Hz, 1H), 4.35-4.09 (m, 7H), 3.85 (m, 2H), 3.38-3.34 (m, IH), 3.1 1 - 2.95 (m, 5H), 2.85-2.70 (m, 2H), 2.30- 2.10 (m, 4H), 1.98-1.72 (m, 4H), 1.435 (m, 2H), 1.12 (d, J = 6.1 Hz, 3H), 1.09 (d, J = 6.1 Hz, 3H), 0.68 (s, 3H), 0.60 (s, 3H).

325

[0339] N-((2S,3R)-3-hydroxy-l-((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,l l, 14,17,20-heptaoxo- 1 ,4,7, 10, 13, 16, 19-heptaazacyclotricosan-21 -ylamino)propan-2-ylamino)- 1 -oxobutan-2-yl)-4-methyl- 1H- pyrrole-3-carboxamide Compound 325 was prepared according to the coupling then deprotection sequence describe for example 7, Compound 15. LCMS: rt = 0.20 &0.35 minutes (double peak, LC method 2). m/z = 1057.6 (M+l). ¾ NMR (400 MHz, D₂0): δ 7.3-7.15 (m, 4H), 7.11 (d, J = 7.4 Hz, 2H), 6.57 (s, IH), 4.40-3.94 (m, 11H), 3.83-3.73 (m, 2H), 3.29-3.18 (m, IH), 2.98-2.85 (m, 7H), 2.73-2.56 (m, 2H), 2.25-1.98 (m, 6H), 1.97-1.66 (m, 6H), 1.34-1.21 (m, 2H), 1.03-0.99(m, 3H), 1.02 (t, J = 8.0 Hz, 3H), 0.71 - 0.47 (m, 7H).

326

[0340] 2-hydroxy-N-((2S,3R)-3-hydroxy- 1 -((R)-3-hydroxy- 1-oxo- 1 - ((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 -ylamino)propan-2-ylamino)- 1 - oxobutan-2-yl)nicotinamide Compound 326 was prepared according to the coupling then deprotection sequence describe for Example 7, Compound 15. LCMS: rt = 0.18 &0.32 minutes (double peak, LC method 2). m/z = 1071.6 (M+l). ¾ NMR (400 MHz, D₂0): δ 8.52 (d, J = 7.2 Hz, 1H), 7.82 (d, J = 6.2 Hz, 1H), 7.47-7.32 (m, 3H), 7.27 (t, J = 7.4 Hz, 2H), 6.80 (t, J = 6.4 Hz, lH), 4.59-4.51 (m, 2H), 4.47- 4.12 (m, 9H), 3.94 (d, J = 5.4Hz, 2H), 3.50-3.37 (m, lH), 3.12-3.00 (m, 7H), 2.95-2.79 (m, 2H), 2.35- 2.19(m, 3H), 2.14-1.99 (m, 3H), 1.92-1.83 (m, 2H), 1.52-1.40 (m, 2H), 1.35-1.15 (m, 7H), 0.84 (t, J = 6.8 Hz, 2H), 0.79 (s, 3H), 0.70 (d, J = 5.2 Hz, 2H).

EXAMPLE 103. RFAMPICIN POTENTIATION ASSAY

[0341] 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.

[0342] 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.

[0343] 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.

[0344] 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

MIC rifampicin* MIC

Intrinsic MIC with 8 μg/mL of Intrinsic MIC rifampicin* with

Cpd. # Cpd. #

^g/mL) potentiator g mL) 8 μg/mL of g mL) potentiator

86 128 1 298 >128 1

97 >128 0.25 299 >128 <0.016

104 >128 2 300 >128 0.063

1 16 >128 2 301 >128 0.125

128 >128 0.25 302 128 <0.016

129 >128 2 303 >128 <0.016

137 >128 2 304 >128 0.25

138 128 0.25 305 >128 0.25

148 >128 4 306 64 0.25

<0.016

63 64 1 307 8

74 128 1 308 128 0.125

156 N.D. 0.0313 309 >128 0.125

169 N.D. 4 310 >128 8

179 64 0.0156 31 1 >128 0.5

189 N.D. 2 312 >128 0.125

201 >128 4 313 128 0.063

216 N.D. 4 314 ND ND

228 N.D. 0.0625 315 >128 0.5

229 N.D. 0.0313 316 128 <0.016

246 N.D. 4 317 32 <0.016

247 N.D. 0.25 318 128 0.031

257 >128 4 319 128 0.063

263 >128 <0.0156 320 >128 4

269 64 <0.016 321 >128 1

270 32 <0.016 322 64 <0.016 Table I- In vitro Data Table for E. coli ATCC25922

MIC rifampicin* MIC

Intrinsic MIC with 8 μg/mL of Intrinsic MIC rifampicin* with

Cpd. # Cpd. #

g mL) potentiator g mL) 8 μg/mL of g mL) potentiator

271 >128 4 323 128 0.125

272 >128 0.125 324 64 <0.016

273 >128 0.063 325 16 <0.016

274 128 <0.016 326 >128 1

275 <0.016

128

*MIC of rifampicin in ATCC25922

Claims

What is claimed is:

1. A

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

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

R² is selected from -C(0)-heterocyclyl, -heterocyclyl, and -C(0)-aryl, each of which is optionally substituted; or

R² is selected from -C(0)NH-N(R⁴)-C(0)R⁶ and -W-X-Y, wherein:

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

X is selected from CH(R³) and optionally substituted phen-l,2-diyl, wherein R³ is selected from - CH(OH)CH₃, -CH(CH₃)₂ and -heterocyclyl; and

Y is selected from -C(0)N(R⁴)(R⁵), -N(R⁴)C(0)R⁶, -N(R⁴)(R⁵), -N(R⁴)S(0)₂-R⁶,

-C(R⁴)(R⁷)-R⁶, -N(R⁴)-C(R⁴)=N-CN, and optionally substituted N-linked heterocyclyl, wherein:

R⁴ is selected independently selected at each occurrence from hydrogen and optionally substituted C1-C4 alkyl;

R⁵ is selected from C1-C4 alkyl, heterocyclyl and cycloalkyl, wherein R⁵ is optionally substituted; or

R⁴ and R⁵ bound to the same nitrogen atom may be taken together to form an optionally substituted heterocyclyl group;

R⁶ is selected from C1-C4 alkyl, C₂-C4alkenyl, C₂-C4alkynyl, -NH(Ci-C4 alkyl), N(Ci-C4 alkyl)₂, heterocyclyl and cycloalkyl, wherein R⁶ is optionally substituted; and

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

R¹ and R² are taken together to form an optionally substituted oxo-substituted heterocyclyl; and

— represents a stereospecific bond selected from (R) and (<S),

provided that when R¹ is hydrogen, W is C(O), X is -CH(OH)CH₃, and Y is -NHC(0)R⁶, then R⁶ is other than methyl, 2-aminocyclopentyl, cyclohexylhydroxymethyl, l-cyclohexyl-l-aminoethan-2-yl, or 5-(sec- butyl)-piperidin-3 -yl.

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 -W-X-Y.

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

-C(0)NH-N(R⁴)-C(0)R⁶;

-C(0)-CH(R )-N(R⁴)-C(R⁴)=N-CN,

-C(0)-CH(R )-N(R⁴)C(0)R⁶, -C(0)-CH(R )-N(R⁴)(R⁵),

-C(0)-CH(R )-C(R⁴)(R⁷)-R⁶,

-C(0)-CH(R )-S(0)₂-R⁶,

-C(0)-CH(R )-C(0)N(R⁴)(R⁵),

-P(0)(OH)-CH(R )-N(R⁴)C(0)R⁶,

-S(0₂)-CH(R )-N(R⁴)C(0)R⁶,

-C(0)-CH(R )-N(R⁴)S(0)₂-R⁶, and

, wherein the phenyl moiety is optionally substituted.

5. The compound of any one of claims 1-4, wherein R³ is an optionally substituted 4-, 5- or 6- membered heterocyclyl group having 1, 2, 3, or 4 heteroatoms independently chosen from N, O, and S.

6. The compound of any one of claims 1-4, wherein R³ is a heterocyclyl selected from oxazol-2-yl, l,2,4-oxadiazol-3-yl, pyridin-l-yl, azetidin-l-yl, and pyrrolidin-l-yl, and wherein R³ is optionally substituted.

7. The compound of claim 5, wherein R³ is selected from 5-methyl-oxazol-2-yl, 5 -methyl- 1,2,4- oxadiazol-3-yl, 2-oxo-pyridin-l-yl, 2-oxo-azetidin-l-yl, and 2-oxo-pyrrolidin-l-yl.

8. The compound of any one of claims 1-4, wherein R³ is -CH(OH)C]¾ or -CH(G¾)2.

9. The compound of any one of claims 1-8, wherein each R⁴ is independently selected from hydrogen and methyl.

10. The compound of any one of claims 1-9, wherein R⁵ is selected from methyl, -CH(C]¾)-CF3, and -CH(CH₃)(CF₃)-CF₃.

11. The compound of any one of claims 1-9, wherein R⁶ is selected from -C¾, -NHCH3,

-CH2CF3, tetrahydrofuranyl, furanyl, and cyclopropyl.

12. The compound of any one of claims 1-2, wherein R² is optionally substituted -C(0)heterocyclyl or optionally substituted -C(0)phenyl.

13. The compound of claim 12, wherein the heterocyclyl of the -C(0)heterocyclyl is an optionally substituted 5- or 6-membered heterocyclyl group having group having 1, 2, 3, or 4 heteroatoms independently chosen from N, O, and S.

14. The compound of claim 13, wherein the heterocyclyl of the -C(0)heterocyclyl is chosen from pyrimidinyl, oxazolyl, morpholinyl, piperidinyl, piperazinyl, thiomorpholinyl, isothiazolyl, pyrrolidinyl, oxadiazolyl, oxadiazolyl, oxadiazolyl substituted with benzyl, pyrazolyl, pyrazinyl, oxazolidinyl, isothiazolidinyl, imidiazolyl, pyridazinyl, pyridinyl, pyrrolyl, thiazolyl, thienyl, and furanyl, each of which is optionally substituted with one or more substituents independently chosen from 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.

15. The compound of claim 1 or 2, wherein R² is selected from -C(0)heterocyclyl, -heterocyclyl, and -C(0)-phenyl, wherein each heterocyclyl is selected from pyrimidin-2-yl, oxazol-4-yl, morpholin-3-yl, isothiazol-3-yl, pyrrolidin-2-yl, l,2,4-oxadiazol-3-yl, pyrazol-4-yl, pyrazin-2-yl, oxazolidin-4-yl, isothiazolidin-3-yl, pyridazin-3-yl, pyridin-2-yl, and furan-2-yl; and wherein each heterocyclyl or phenyl is optionally substituted.

16. The compound of claim 15, wherein R² is selected from -C(0)heterocyclyl,

-heterocyclyl, and -C(0)-phenyl, wherein each heterocyclyl is selected from pyrimidin-2-yl, 2- acetylamino-pyrimidin-4-yl, 2-methyl-oxazol-4-yl, 2,5-dimethyl-oxazol-4-yl, 5-oxo-morpholin-3-yl, 5- methyl-isothiazol-3 -yl, 5 -oxo-pyrrolidin-2 -yl, 4,4-dimethyl-5 -oxo-pyrrolidin-2-yl, 4-hydroxy-5 -oxo- pyrrolidin-2-yl, 2-hydroxymethyl-5-oxo-pyrrolidin-2-yl, 3-hydroxymethyl-5-oxo-pyrrolidin-2-yl, 5- methyl-l,2,4-oxadiazol-3-yl, 5-methyl-2-oxo-oxazolidin-4-yl, 2-oxo-pyrrolidin-l-yl, 2 -oxo-3 -benzyl - oxazolidin-4-yl, 3 -benzyl oxy-5 -oxo-pyrrolidin-2-yl, 1 , 3 ,5 -trimethyl-pyrazol-4-yl, 6-methyl-pyrazin-2-yl, 2-oxo-oxazolidin-4-yl, 1 , 1 -dioxo-isothiazolidin-3 -yl, 1 , 1 -dioxo-4-hydroxy-isothiazolidin-3 -yl, 1 , 1 -dioxo- 4-hydroxymethyl-isothiazolidin-3 -yl, 6-methyl-pyridazin-3 -yl, 6-acetylamino-pyridazin-3 -yl, 6-methyl- pyridin-2-yl, 6-acetylamino-pyridin-2-yl, 2,5-dimethylfuran-3-yl, 4,5-dimethylfuran-2-yl, and the phenyl is 2-acetylamino-5-fluoro-phenyl.

17. The compound of claim 1, wherein R¹ and R² are taken together to form a ring selected from pyrrolidinyl and pyridinyl, wherein the ring is optionally substituted.

18. The compound of claim 17, wherein R¹ and R² are taken together to form a ring selected from 2- oxo-3-aminoacetyl-4-hydroxymethyl-pyrrolidinyl, 2-oxo-3-aminoacetyl -pyrrolidinyl and 2-oxo-3- acetylamino-pyridinyl.

19. The compound of claim 1, wherein R² is -W-X-Y, wherein

W is C(O),

X is CH(R³), wherein R³ is selected from -CH(OH)CH₃, and

Y is -N(R⁴)C(0)R⁶, wherein R⁴ at each occurrence is hydrogen, and R⁶ 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, or -N(Qi)(Q2), wherein Qi, and Q2 are each independently selected from hydrogen, a C1-C10 alkyl group, and a Ce-Cn aryl group.

20. The compound of claim 19, wherein R⁶ is represented by one of the following formulae:

? -¾ Z^""\ .;-ts ^'" -· ¾!H :f^{' "} ]

^:iV ° YY ^vy Y H½ ^"'¾¾S"

21.. The compound of claim 1, wherein each -heterocycly] is independently selected from

a pyrrolyl group, an imidazolyl group, a pyrazolyl group, a pyridinyl group, a pyrazinyl group, a pynmidinyl group, a pyridazinyl group, an isoindoly] 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 benzofuranyl 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-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 C i-Ci alkyl group, a C2-C1D alkenyl group, a C2-C10 alkynyl group, a C1-C10 alkoxy group, a phenyl group, a biphenyl group, a terpbenyl 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)(Qz), wherein Q], and Q2 are each independently selected from hydrogen, a C1-C10 alkyl group, or a Ce-Cii aryl group.

22. The compound of claim 1, wherein R² is selected from -C(0)-heterocyclyl, wherein a "- heteroc clyl" is selected from one of the following formulae:

23. The compound of claim 1, wherein the compound is

130

133

135

136

137

138

139

140

25. 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 25 to the patient.

26. 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 24.