L CROSS-REFENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. §1 19(e) of U.S. Provisional Patent Application No. 61/777,547, filed March 12, 2013. The foregoing application is incorporated herein by reference in its entirety.

II. STATEMENT OF GOVERNMENT INTEREST

This invention was made with government support under Contract HDTRA1 -07- C-0079 awarded by the United States Department of Defense. The government has certain rights in this invention.

Ill BACKGROUND

A. Field

This invention pertains generally to treating infections caused by gram-negative bacteria. More specifically, the invention described herein pertains to treating gram-negative infections by inhibiting activity of UDP-3-0-(R-3-hydroxydecanoyi)- -acetylgiucosamine deacetylase (LpxC). The present invention provides small molecule inhibitors of LpxC, pharmaceutical formulations containing such inhibitors, methods of treating patients with such pharmaceutical formulations, and methods of preparing such pharmaceutical formulations and inhibitors. The invention described herein pertains to treating gram-negative infections by administering compounds capable of inhibiting activity of UDP-3-G-(j -3-hydroxydecanoyl)-/V- acetyiglucosamine deacetylase (LpxC), either alone or in combination with administering a second antibacterial compound.

B, Introduction

Over the past several decades, the frequency of antimicrobial resistance and its association with serious infectious diseases have increased at alarming rates. The problem of antibacterial resistance is compounded by the existence of bacterial strains resistant to multiple antibacterials. Thus there is a need for new antibacterials, particularly antibacterials with novel mechanisms of action. A previously unexpioited but highly conserved target, LpxC, provides a new opportunity for developing broad-spectrum antibacterial small molecules that comprise a new class of active bactericidal chemical entities that should encounter little, if any, naturally- occurring, target-related resistance. LpxC (the enzyme uridyldiphospho-3-0-(R- hydroxydecanoyl)-N-acetylglucosamine deacetylase) is present across all Gram-negative bacterial species of interest and is involved in the first committed step in outer membrane biosynthesis. Thus LpxC Is essential for survival and presents an ideal target for antibiotic activity in Gram-negative bacterial species.

Researchers have identified some compounds with antibacterial activity that target lipid A biosynthesis. For example, Jackman et ai. (J. Biol. Chem., 2000, 275(15), 1 1002- 1 1009); Wyckoff et al. (Trends in Microbiology, 1998, 6(4), 154-159); U.S. Patent Application Publication No. 2001/0053555 (published 20 December 2001 , corresponding to International PCT Publication No. WO 98/18754, published 7 May 1998); International PCT Publication No. VVO 00/61 134 (published 19 October 2000); U.S. Patent Application Publication No.

2004/0229955 (published 18 November 2004); International PCT Publication No. VVO

2008/027466 (published 6 March 2008); International PCT Publication No, VVO 2008/105515 (published 4 September 2008); international PCT Publication No. WO 2008/154642 (published 18 December 2008); International PCT Publication No. WO 2009/158369 (published 30 December 2009); international PCT Publication No. WO 2010/017060 (published 1 1 February 2010); International PCT Publication No. WO 2010/024356 (published 4 March 2010);

International PCT Publication No. WO 2010/031750 (published 25 March 2010); international PCT Publication No. WO 2010/032147 (published 25 March 2010); International PCT

Publication No. WO 2010/100475 (published 10 September 2010); International PCT

Publication No. WO 2011/045703 (published 21 April 201 1 ); International PCT Publication No. WO 201 1/073845 (published 23 June 201 1 ); and International PCT Publication No. WO 201 1/132712 (published 27 October 2011 ) ail disclose compounds having antibacterial anti- LpxC activity. The commercial development of these LpxC inhibitors has been complicated by toxicity of these compounds in mammalian animals at concentrations at or near those required for antibacterial activity. The compounds presented herein are significantly better tolerated, more active and/or less protein-bound than other closely related compounds having anti-LpxC activity.

Although there have been advances in the field, there remains a need for LpxC inhibitors that have activity as bactericidal agents against gram-negative bacteria and have an acceptable efficacy and toxicity/tolerance profile. It is, accordingly, an object of this invention to provide compounds and combinations of such compounds for use in the preparation of nontoxic antibacteriais and other pharmaceuticals capable of inhibiting gram-negative bacterial infections.

IV, BRIEF SUGA Y OF THE INVENTION

The present invention provides novel compounds, pharmaceutical formulations including the compounds, methods of inhibiting UDP-3-0-( ?-3-hydroxydecanoyl)-/V- acetylglucosamine deacetylase (LpxC), and methods of treating gram-negative bacterial infections.

In one I:

and stereoisomers, pharmaceutically acceptable salts and esters thereof, wherein

A is azetidinyl or pyrrolidiny!, wherein said azetidinyl or pyrrolidinyi is unsubstituted or substituted with 1 or 2 groups selected from substituted or unsubstituted Ci-C ₃ alkyl, halogen, cyano, -C{0)R ₄ , oxo, hydroxyl, -8(G) ₂ R ₄ , substituted or unsubstituted C ₃ -C ₆ heterocyclyl and substituted or unsubstituted C ₄ -C ₇ heterocyclylalkyl;

B is absent, a Ci-C ₂ aikylene or C ₃ -C ₆ cycloalkyi wherein said Ci-C ₂ alky!ene or C ₃ -C ₆ cycioaikyl is unsubstituted or substituted with 1 or 2 groups selected from substituted or unsubstituted C C ₃ alkyl, halogen, cyano, -C(0)R ₄ , oxo, hydroxyl, --3(0) ₂ ί¾, substituted or unsubstituted C ₃ -C ₆ heterocyc!y! and substituted or unsubstituted C4-C7 heterocyclyla!ky!;

or A and B together form a fused 5-9 membered bicyclic ring system, wherein A is azetidinyl or pyrrolidinyi and B is a C ₃ -C ₆ cyc!oa!kyi and wherein each of A and B is unsubstituted or substituted with 1 or 2 groups selected from substituted or unsubstituted C C ₃ alkyl, ha!ogen, cyano, -C(0)R ₄ , oxo, hydroxy!, -S(0) ₂ R4, substituted or unsubstituted C ₃ -C ₆ heterocyciyi and substituted or unsubstituted C4-C7 heterocyclylaiky!;

G is selected from the group consisting of -C≡C-, -CH=CH-C≡C~ -C≡C-CH=CH-, and ~ 0 ~ — o~o— _>

Q is O or NR, wherein R is hydrogen or an unsubstituted C _r C ₃ aikyl;

R, and R ₂ independently are selected from the group consisting of hydrogen, substituted or unsubstituted C C ₃ alkyl, substituted or unsubstituted C ₃ -C ₆ cycloalkyl, substituted or unsubstituted 4-8 membered heterocyciyi, and substituted or unsubstituted heteroaryl, or R-, and R ₂ , together with the carbon atom to which they are attached, form an unsubstituted C ₃ -C ₆ cycloalkyl group or an unsubstituted 4-6 membered heterocyclic group:

R ₃ is selected from the group consisting of hydrogen, substituted or unsubstituted C C ₆ alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted cyc!oaiky!alkyl, substituted or unsubstituted heterocyciyi, substituted or unsubstituted heterocyclylalky!, substituted or unsubstituted heteroaryl, and substituted or unsubstituted heteroarylalkyi;

each R ₄ is independently hydrogen, substituted or unsubstituted C C ₃ alkyl, substituted or unsubstituted C ₃ -C ₆ cycloalkyl or NR ₅ R ₆ ; and

each R ₅ and R ₆ is independently hydrogen or C ₁ -C3 alkyl.

In another aspect, the present invention provides a pharmaceutical composition comprising a compound of Formula I, or or a stereoisomer, pharmaceutically acceptable salt, or ester thereof, and a pharmaceutically acceptable carrier or diluent. In another aspect, the present invention provides a pharmaceutical composition or formulation comprising an effective amount of an antibacterial compound of Formula I, or a stereoisomer, pharmaceutically acceptable salt, or ester thereof, and a pharmaceutically acceptable carrier or diluent.

In another aspect, the present invention provides a method of inhibiting a deacetylase enzyme in gram-negative bacteria, thereby affecting bacterial growth, comprising administering to a patient in need of such inhibition an LpxC-inhibitory compound of Formula I or a stereoisomer, pharmaceutically acceptable salt, or ester thereof.

In another aspect, the present invention provides a method of inhibiting LpxC, thereby modulating the virulence of a bacterial infection, comprising administering to a patient in need of such inhibition an LpxC-inhibitory compound of Formula 1 or a stereoisomer, pharmaceutically acceptable salt, or ester thereof.

In another aspect, the present invention provides a method for treating a patient having a bacterial infection comprising administering to the patient in need thereof an antibacterialiy effective amount of a compound of Formula I, or a stereoisomer,

pharmaceutically acceptable salt, or ester thereof, in a more specific embodiment of the method of treatment, the bacterial infection is a gram-negative bacterial infection, in a further specific embodiment the patient is a mammal and in certain embodiments, a human.

In another aspect, the present invention provides a method of administering an antibacterialiy effective amount of a compound of Formula I or a stereoisomer, pharmaceutically acceptable salt, or ester thereof, to a patient infected with a fermentative or non-fermentative gram-negative bacteria. Examples of such bacteria include Pseudomonas aeruginosa, Stenotrophomonas maltophila, Burkhoideria cepacia, Aicaiigenes xyiosoxidans,

Enterobacteriaceae, Haemophilus, Franciscellaceae (e.g., Franciscella tuiarensis) and

Neisseria species.

In another aspect, the present invention provides a method of administering an antibacterialiy effective amount of a compound of Formula I, or a stereoisomer,

pharmaceutically acceptable salt, or ester thereof, to a patient infected with gram-negative bacteria. Examples of such bacteria include Enterobacteriaceae, such as Serratia, Proteus, Klebsiella, Enterobacter, Citrobacter, Salmonella, Providencia, Yersinia (e.g., Yersinia pestis), Morganeila, Cedecea, Edwardsielia species and Escherichia coii.

These and other aspects of the invention will be evident upon reference to the following detailed description.

V. DETAILED DESCRIPTION OF THE INVENTION

The present invention provides novel compounds, methods for inhibiting LpxC in gram-negative bacteria, and novel methods for treating bacterial infections. The compounds provided herein can be formulated into pharmaceutical formulations and medicaments that are useful in the methods of the invention. The invention also provides for the use of the

compounds in preparing medicaments and pharmaceutical formulations, for use of the compounds in inhibiting LpxC, and for use of the compounds in treating bacterial infections in a patient. The invention further provides compositions and methods for treating gram-negative infections by administering compounds capable of inhibiting activity of UDP~3~G-(R~3~ hydroxydecanoyl)-/V-acetyiglucosamine deacetyiase (LpxC), either alone or in combination with administering a second antibacterial compound.

A. Definitions

The following abbreviations and definitions are used throughout this application:

"LpxC" is an abbreviation that stands for UDP-3-0-(R-3-hydroxydecanoyl)-W- acetylgiucosamine deacetyiase.

As used herein, the following definitions shall apply unless otherwise indicated.

"Alkyi" refers to monovalent saturated aliphatic hydrocarbyl groups having from 1 to 10 carbon atoms and preferably 1 to 6 carbon atoms. This term includes, by way of example, linear and branched hydrocarbyl groups such as methyl (CH ₃ -), ethyl (CH ₃ CH ₂ -), n-propyl

(CH ₃ CH ₂ CH ₂ -), isopropyl ((CH ₃ ) ₂ CH-), n-butyl (CH ₃ CH ₂ CH ₂ CH ₂ -), isobutyl ((CH ₃ ) ₂ CHCH ₂ -), sec- butyl ((CH ₃ )(CH ₃ CH ₂ )CH-), i-but l ((CH ₃ ) ₃ C-), n-pentyl (CH ₃ CH ₂ CH ₂ CH ₂ CH ₂ -), and neopentyl ((CH ₃ ) ₃ CCH _r ). "Alkoxy" refers to the group -O-a!ky!, wherein alky! is as defined herein. Alkoxy includes methoxy, etboxy, n-propoxy, isopropoxy, π-butoxy, i-butoxy, sec-butoxy, n-pentoxy, and the like.

"Amino" refers to the group -NH ₂ .

"Alkenyl" refers to straight chain or branched hydrocarby! groups having from 2 to 6 carbon atoms and preferably 2 to 4 carbon atoms and having at least 1 and preferably from 1 to 2 sites of vinyl (>C=C<) unsaturation. Such groups are exemplified by viny!, ally!, and but-3-en-1-yl. Included within this term are the c/s and trans isomers or mixtures of these isomers.

"Alkynyi" refers to straight or branched monovalent hydrocarby! groups having from 2 to 6 carbon atoms and preferably 2 to 3 carbon atoms and having at least 1 and preferably from 1 to 2 sites of acetylenic ~C≡C- unsaturation. Examples of such a!kynyl groups include acetylenyl (-C≡CH), and propargyl (~CH ₂ C≡CH).

"Carboxyl" or "carboxy" refers to -CQOH or salts thereof.

"Cvano" or "nitri!e" refers to the group -CN.

"Cvcioalkyl" refers to a monocyclic or po!ycyclic alky! group consisting , which may include fused or bridged ring systems, having from 3 to 13 carbon atoms. Examples of cycloa!kyl groups include cyclopropy!, cyciobutyl, cyclopentyl, cyclooctyl, and the like.

"Halo" or "halogen" refers to fiuoro, chioro, bromo, and iodo and is typically fiuoro or chioro.

"Hydroxy" or "hydroxyl" refers to the group -OH,

"Heterocycle," "heterocyclic," and "heterocyciyi" refer to a saturated or unsaturated monocyclic or polycyc!ic group, which may include fused or bridged ring systems, having from 3 to 15 ring atoms, including 1 to 4 hetero atoms. These ring atoms are selected from the group consisting of nitrogen, sulfur, or oxygen. In one implementation, the nitrogen and/or sulfur atom(s) of the heterocyclic group are optionally oxidized to provide for the N-oxide, -S(O)-, or -S0 ₂ - moieties.

"Nitro" refers to the group -NQ ₂ .

"Oxo" refers to the atom (=0). "Substituted" refers to a group having one or more hydrogens replaced with substituents selected from the group consisting of alkoxy, acyi, acylamino, acyloxy, amino, aminocarbonyl, aminocarbonylamino, aminothiocarbonylamino, aminocarbonyioxy, amidino, carboxyl ester, (carboxyl ester)amino, (carboxyi ester)oxy, cyano, halo, hydroxy, nitro, suifony!, thioacyl, and alkylthio, wherein said substituents are as defined herein, in certain substituted cyclic groups, "substituted" also refers to a group having one or more hydrogens replaced with an alkyl group or "substituted" refers a group having two hydrogens replaced with a single double bonded oxygen atom (an oxo group) or a single double bonded sulfur atom (thioxo). in some implementations, the substituted group has 1 to 3 of the aforementioned substituents. in other implementations, the substituted group has 1 to 2 of the aforementioned substituents

"Suifony!" refers to the group -S0 ₂ -alkyi, -S0 ₂ -substituted alkyl, ~S0 ₂ -alkenyl, -S0 ₂ -substituted alkenyl, wherein alkyl, substituted alkyl, aikenyi, substituted aikenyi, alkynyl, and substituted alkynyl are as defined herein. Sulfonyl includes groups such as methyi-S0 ₂ -.

"Thioacyl" refers to the groups H-C(S)-, alkyl-C(S)-, substituted a!ky!-C(S)-, a!keny!-C(S)-, substituted aikenyi-C(S)-, a!kyny!-C(S)-, and substituted alkynyl-C(S)-, wherein alkyl, substituted alkyl, aikenyi, substituted alkenyl, alkynyl, and substituted alkynyl are as defined herein.

"Thioxo" refers to the atom (=S).

"Alkylthio" refers to the group -S-a!ky!, wherein alkyl is as defined herein. In other implementations, sulfur may be oxidized to -S(O)-. The sulfoxide may exist as one or more stereoisomers.

Unless indicated otherwise, the nomenclature of substituents that are not explicitly defined herein are arrived at by naming the terminal portion of the functionality followed by the adjacent functionality toward the point of attachment. For example, the substituent "arylalkyloxycarbony!" refers to the group (ary!)-(alkyl)-0-C(0)-.

Generally, reference to a certain element such as hydrogen or H is meant to include all isotopes of that element. For example, if a substituent group is defined to include hydrogen or H, if also includes deuterium and tritium. The subject invention also includes isotopicaliy-labeied compounds of the present invention, that are structurally identical to those disclosed herein, but for the fact that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes that can be incorporated into compounds of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorous, sulfur, fluorine and chlorine, such as ² H, ³ H, ¹³ C, ¹⁴ C, ¹⁵ N, ⁸ 0, ¹⁷ 0, ^{3 '} P, ³ P, ³⁵ S, ⁸ F and ^j6 Ci, respectively. Compounds of the present invention, prodrugs thereof, and pharmaceutically acceptable salts of said compounds and of said prodrugs that contain the aforementioned isotopes and/or other isotopes of other atoms are within the scope of this invention. Certain isotopicaliy labeled compounds of the present invention, for example those into which radioactive isotopes such as ³ H and ^'4 C are incorporated, are useful in drug and/or substrate tissue distribution assays. Tritiated, i.e., ³ H, and carbon-14, i.e., ¹⁴ C, isotopes are particularly preferred for their ease of preparation and detectability. Further, substitution with heavier isotopes such as deuterium, i.e., ² H, may afford certain therapeutic advantages resulting from greater metabolic stability, for example increased in vivo half-life or reduced dosage requirements and, hence, may be preferred in some circumstances, isotopicaliy labeled compounds of this invention and prodrugs thereof can generally be prepared by carrying out known or referenced procedures and by substituting a readily available isotopicaliy labeled reagent for a non-isotopicaily labeled reagent.

"Stereoisomer" and "stereoisomers" refer to compounds that have same atomic connectivity but different atomic arrangement in space. Stereoisomers include cis-trans isomers, E and Z isomers, enantiomers, and diastereomers.

"Tautomer" refers to alternate forms of a molecule that differ in the position of a proton, such as enol-keto and imine-enamine tautomers, or the tautomeric forms of neteroary! groups containing a -N=C(H)-NH- ring atom arrangement, such as pyrazoies, imidazoles, benzimidazoles, triazoies, and tetrazoies. A person of ordinary skill in the art would recognize that other tautomeric ring atom arrangements are possible.

"Patient" refers to human and non-human animals, especially mammals. "Pharmaceutically acceptable salt" refers to pharmaceutically acceptable salts of a compound, which salts are derived from a variety of organic and inorganic counter ions well known in the art and include, by way of example only, sodium, potassium, calcium, magnesium, ammonium, tetraa!kyiammonium, and the like: and when the molecule contains a basic functionality, salts of organic or inorganic acids, such as hydrochloride, hydrobromide, tartrate, mesylate, acetate, maleate, oxalate, phosphate, sulfate and the like.

" Pharmaceutically effective amount" and "therapeuticaliy effective amount" refer to an amount of a compound sufficient to treat a specified disorder or disease or one or more of its symptoms and/or to prevent the occurrence of the disease or disorder.

The term "antibacterial agent" refers to agents that have either bactericidal or bacteriostatic activity. The term "inhibiting the growth" indicates that the rate of increase in the numbers of a population of a particular bacterium is reduced. Thus, the term includes situations in which the bacterial population increases but at a reduced rate, as well as situations where the growth of the population is stopped, as well as situations where the numbers of the bacteria in the population are reduced or the population even eliminated, if an enzyme activity assay is used to screen for inhibitors, one can make modifications in uptake/efflux, solubility, half-life, etc. to compounds in order to correlate enzyme inhibition with growth inhibition. The activity of antibacterial agents is not necessarily limited to bacteria but may also encompass activity against parasites, virus, and fungi.

Unless the context requires otherwise, throughout the specification and claims which follow, the word "comprise" and variations thereof, such as, "comprises" and "comprising" are to be construed in an open, inclusive sense, that is, as "including, but not limited to".

Reference throughout this specification to "one embodiment" or "an embodiment" means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, the appearances of the phrases "in one embodiment" or "in an embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment.

Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments. 8, Compounds, Compositions and Use Thereof

in one I:

stereoisomer, pharmaceutically acceptable salt or ester thereof, wherein

A is azetidinyi or pyrroiidinyi, wherein said azetidinyi or pyrroiidinyl is unsubstituted or substituted with 1 or 2 groups selected from substituted or unsubstituted Ci-C ₃ alkyl, halogen, cyano, -C(0)R ₄ , oxo, hydroxyl, -S(0) ₂ R4, substituted or unsubstituted C ₃ -C ₆ heterocyclyl and substituted or unsubstituted C ₄ -C ₇ heterocyclylalkyl;

B is absent, a CrC ₂ aiky!ene or C ₃ ~C ₆ cycloalkyl wherein said Ci-C ₂ aikyiene or C ₃ -C ₆ cycioalkyl is unsubstituted or substituted with 1 or 2 groups seiected from substituted or unsubstituted C C ₃ alkyi, halogen, cyano, -C(0)R ₄ , oxo, hydroxy!, -S(0) ₂ R4, substituted or unsubstituted C ₃ -C ₆ heterocyclyl and substituted or unsubstituted C4-C7 heterocyciyiaikyl;

or A and B together form a fused 5-9 membered bicyclic ring system, wherein A is azetidinyi or pyrroiidinyi and B is a C ₃ -C ₃ cycloalkyl and wherein each of A and B is unsubstituted or substituted with 1 or 2 groups selected from substituted or unsubstituted Ci-C ₃ alkyi, halogen, cyano, -C(0)R ₄ , oxo, hydroxy!, ---S(0) ₂ R ₄ , substituted or unsubstituted C ₃ -C ₆ heterocyc!y! and substituted or unsubstituted C4-C7 heterocyciyiaikyl;

G is selected from the group consisting of -C≡C-, -CH=CH-C≡C--, -C≡C-CH=CH-, and " 0 ~ ~~C -~ """ .

Q is O or NR, wherein R is hydrogen or an unsubstituted d-C ₃ alkyl;

Ri and R ₂ independently are seiected from the group consisting of hydrogen, substituted or unsubstituted Ci-C ₃ alkyl, substituted or unsubstituted C ₃ -C ₆ cycioalkyl, substituted or unsubstituted 4-6 membered heterocyclyl, and substituted or unsubstituted heteroaryl, or R-. and R ₂ , together with the carbon atom to which they are attached, form an unsubstituted C ₃ -C ₆ cycloaikyi group or an unsubstituted 4-8 membered heterocyclic group;

R ₃ is selected from the group consisting of hydrogen, substituted or unsubstituted Ci-C ₆ alkyi, substituted or unsubstituted cycloaikyi, substituted or unsubstituted cycloaikyla!ky!, substituted or unsubstituted heterocyclyl, substituted or unsubstituted heterocyclylalkyi, substituted or unsubstituted heteroaryl, and substituted or unsubstituted heteroaryla!ky!;

each R ₄ is independently hydrogen, substituted or unsubstituted C C ₃ alkyi, substituted or unsubstituted C ₃ -C ₆ cycloaikyi or NR _S R ₆ ; and

each R ₅ and R ₆ is independently hydrogen or C C ₃ alkyi.

in certain embodiments, R ³ is hydrogen or a substituted or unsubstituted C,-C ₆ aikyl .

In some embodiments G is - ≡C-C≡C-.

In some embodiments, Q is NR, wherein R is hydrogen or unsubstituted Ci-C ₃ aikyl. In certain embodiments where Q is NR, R is hydrogen. In certain embodiments, Q is O and R ₃ is H. in certain embodiments both R ¹ and R ² are methyl.

In certain embodiments B is absent, in other embdiments B is Ci~C ₂ alky!ene. In still other embodiments B is C ₃ -C ₆ cycloaikyi.

In some embodiments A is unsubstituted azetidinyl. In other embodiments A is azetidinyl substituted with 1 or 2 groups selected from substituted or unsubstituted C C ₃ aikyi, -C(0)R ₄ , C ₃ -C ₆ heterocyclyl, hydroxy! and -S(0) ₂ R ₄ . In some embodiments A is azetidinyl substituted with substituted or unsubstituted Ci~C ₃ aikyl. In other embodiments A is azetidinyl substituted with -C(0)R ₄ . In certain embodiments where A is azetidinyl substituted with

-C(0)R ₄ , R is hydrogen, in other embodiments where A is azetidinyl substituted with -C(0)R ₄ , R is methyl. In certain embodiments where A is azetidinyl substituted with -C{0)R ₄ , R is Ci-C ₃ aikyl substituted with hydroxy!, in certain embodiments where A is azetidinyl substituted with -C(0)R ₄ , R ₄ is Ci-C ₃ aikyl substituted with amino. In certain embodiments where A is azetidinyl substituted with ~C(0)R _4l R ₄ is C;~C ₃ a!ky! substituted with hydroxy! and amino, in certain embodiments where A is azetidinyi substituted with -C(0)R ₄ , R ₄ is C ₃ -C ₆ cycioa!ky!. in certain embodiments A is azetidinyi substituted with -C(0)R ₄ and hydroxy!, !n certain embodiments A is azetidinyi substituted with C ₃ -C ₆ heterocycly!. In certain embodiments A is azetidinyi substituted with hydroxyl. In certain embodiments A is azetidinyi substituted with -S(0) ₂ R . In certain embodiments where A is azetidinyi substituted with -S(0) ₂ R ₄ , R is methyl, in certain other embodiments where A is azetidinyi substituted with -8(G) ₂ R ₄ , R ₄ is NR ₅ R ₆ . In certain

embodiments where A is azetidinyi substituted with -S(0) ₂ R ₄ , where R ₄ is NR ₅ R ₆ , R ₅ and R ₆ are both hydrogen. In certain embodiments where A is azetidinyi substituted with -S(0) ₂ R, _: , where R ₄ is NR ₅ R _6> R ₅ is Ci~C ₃ alky! and R ₆ is hydrogen. In certain embodiments where A is azetidinyi substituted with ~S(0) ₂ R , where R ₄ is NR ₅ R ₆ , R ₅ is methyl and R ₆ is hydrogen.

In some embodiments A is unsubstituted pyrrolidinyi. in other embodiments A is pyrrolidinyi substituted with 1 or 2 groups selected from substituted or unsubstituted C,-C ₃ alkyl, -C(0)R ₄ , C ₃ -C ₆ heterocyclyl, hydroxyl and ~8(G) ₂ R ₄ . in some embodiments A is pyrrolidinyi substituted with substituted or unsubstituted Ci~C ₃ aiky!. In some embodiments A is pyrrolidinyi substituted with substituted or unsubstituted C ₃ -C ₃ heterocyclyl. In some embodiments A is pyrrolidinyi substituted with oxo. In some embodiments A is pyrrolidinyi substituted with hydroxyl.

In certain embodiments G is -CH=CH--C≡C- and A is azetidinyi substituted with 1 or 2 groups selected from substituted or unsubstituted C;~C ₃ alkyl, -C(G)R ₄ , C ₃ -C ₆

heterocyclyl, hydroxyl and -S(0) ₂ R ₄ . In certain embodiments where G is -CH=CH-C≡C- A is azetidinyi substituted with -C(0)R ₄ or -S{0) ₂ R . In certain embodiments where G is -CH=CB- C≡C- and A is azetidinyi substituted with -C(0)R ₄ or -S(0) ₂ R ₄ , R ₄ is hydrogen or methyl. In certain embodiments A is pyrrolidinyi substituted with 1 or 2 groups selected from substituted or unsubstituted C _r C ₃ alkyl, oxo, C ₃ -C ₆ heterocyclyl, hydroxy! and -S(0) ₂ R ₄ .

One aspect of the invention provides compounds selected from the group of: (S)-A-(3-amino-1-(hydroxyamino)-3-methyl-1-oxobutan-2-y!)-4- (azetidin-3-ylbuta-1 ,3- diyny!)benzamide (1 ); (S)-4-((1-acetyIazetidin-3-yI)buta-1 ,3-diyn-1-y!)-A/-(3-hydroxy-1 -(hydroxyamino)-3-methyl-1- oxobutan-2-yl)benzamide (2a);

(S)-4-((azetidin-3-y!)buta-1 ,3-diyn-1-y!)-A/-(3-hydroxy-1-(hydroxyamino)-3-methyl-1-oxob utan-2- yl)benzamide (2b);

(S)-4-((1-formylazetidin-3-yl)buta-1 ,3-diyn-1-yl)-A/-(3-hydroxy-1-(hydroxyamino)-3-methy!-1- oxobutan-2-yl)benzamide (3);

(S)-4-((1-(2-aminoacety!)azetidin-3-yl)buta-1 ,3-diyn-1-yl)-W-(3-hydroxy-1-(hydroxyamino)-3- methyi-1-oxobutan-2-yi)benzamide (4);

4-({1-(2-amino-3-hydroxypropanoyi)azetidin-3-yl)buia-1 ,3-diyn-1-yi)-A -((S)-3-hydroxy-1- (hydroxyamino)-3-methy!-1-oxobutan-2-y!)benzamide (5);

(S)-A/-(3-hydroxy-1-(hydroxyamino)-3-methy!-1-oxobutan-2-yi) -4-((1-(2-hydroxyethyl)azetidin-3- yl)buta-1 ,3-diyn-1-y!)benzamide (6);

(S)-A/-(3-hydroxy-1-(hydroxyamino)-3-meihyi-1-oxobuian-2-y!) -4-((1-methyiazetidin-3-yi)buta- 1 ,3-diyn-1-y!)benzamide (7);

(S)-A/-(3-amino-1-(hydroxyamino)-3-methyI-1-oxobutan-2-y!)-4 -((1-(oxeian-3-yi)azetidin-3- yl)buta-1 ,3-diyn-1-y!)benzamide (8);

A/-{(S)-3-hydroxy-1-(hydroxyamino)-3-methyi-1-oxobutan-2-yl) -4-(5-((2/?,3S)-3- hydroxypyrroiidin-2-y!)periia-1 ,3-diyn-1-y!)benzamide (9);

(S)-4-((1-acetyiazetidin-3-yi)buta-1 _l 3-diyny!)-A/-(3-amino-1-(hydroxyamino)-3-methy!-1- oxobutan-2-yl)benzamide 2,2,2-trif!uoroacetate (10);

(S)-3-((4-(3-amino-1-(hydroxyamino)-3-methyl-1-oxobuian-2-yl carbamoyl)phenyl)buta-1 ,3- diyny!)-A/-methylazetidine-1-carboxamide (1 1 );

(S)-3-((4-(3-amino-1-(hydroxyamino)-3-meihyl-1-oxobuian-2-yl carbamoyl)phenyl)buta-1 _l 3- diyny!)azetidine-1-carboxamide (12);

(S)-4-((1-acety!azetidin-3-y!)buta-1 ,3-diynyi)-W-(3-(dimethyiamino)-1-(hydroxyamino)-3-met 1-oxobutan-2-y!)benzamide (13);

(S)-A/-(3-amino-1-{hydroxyamino)-3-methyl-1-oxobutan-2-y!)-4 -{(1-(2-hydroxyacetyl)azetidin-3- yl)buta-1 ,3-diyn-1-y!)benzamide (14);

(S)-A/-(3-amino-1-(hydroxyamino)-3-methyl-1-oxobutan-2-y!)-4 -((1-(2-hydroxyethyi)azetidin-3- yl)buta-1 ,3-diyn-1-y!)benzamide (15);

(S)-A/-(3-amino-1-(hydroxyamino)-3-methyl-1-oxobutan-2-y!)-4 -((1-formylazetidin-3-yl)buta-1 ,3- diyn-1-y!)benzamide (16);

(S)-4-((1-formylazetidin-3-yl)buta-1 ,3-diyn-1-yl)-A -(1-(hydroxyamino)-3-methyi-3-(methyiami 1-oxobutan-2-y!)benzamide (17);

(S)-4-((1-acety!azeiidin-3-yI)buia-1 ,3-diyn-1-y!)-A/-(1-(hydroxyamino)-3-methy!-3-(methylamino)^ 1-oxobutan-2-y!)benzamide (18);

(S)-4-((1-acetyi-3-hydroxyazetidin-3-y!)buta-1 ,3-diyn-1-yi)-A/-(3-amino-1-(hydroxyamino)-3- methyi-1-oxobutan-2-y!)benzamide (19);

W-((2S,3R)-3-amino-1-(hydroxyamino)-1-oxobutan-2-y!)-4-((1-f ormy!azetidin-3-y!)buta-1 ,3-diyn- 1-y!)benzamide (20);

(S)-A/-(3-amino-1-(hydroxyamino)-3-methyl-1-oxobutan-2-y!)-4 -((1-{methy!sulfonyl)azetidin-3- yl)buta-1 ,3-diyn-1-yi)benzatnide (21 );

4-((1-formylazetidin-3-yl)buta-1 ,3-diyn-1-yl)-W-((2S,3/ )-1-(hydroxyamino)-3-(methyiamino)-1- oxobutan-2-yl)benzamide (22);

(S)-A/-(3-hydroxy-1-(hydroxyamino)-3-methyi-1-oxobutan-2-y!) -4-((1-(oxetan-3-y!)azetidin-3- yl)buta-1 ,3-diyn-1-y!)benzamide (23);

(S)-A/-(3-amino-1-(hydroxyamino)-3-methyl-1-oxobutan-2-y!)-4 -(5-(azetidin-3-yi)penta-1 ,3-diyr!- 1-y!)benzamide (24);

(S)-A/-(3-amino-1-(hydroxyamino)-3-methyl-1-oxobutan-2-y!)-4 -(5-(1-formyiazetidin-3-yl)penta- 1 ,3-diyn-1-yl)benzamide (25);

(S,E)-A/-(3-amino-1-(hydroxyamino)-3-methyi-1-oxobutan-2-yi) -4-(4-(1-(methyisulfonyi)azetidi 3-yi)but-3-en-1 -yn-1 -yl)benzamide (26);

(S _> E)-W-(3-amino-1-(hydroxyamino)-3-methyi-1-oxobutan-2-y !)-4-(4-(1-formylazetidin-3-yi)but^ en-1-yn-1-y!)benzamide (27);

(S _! £)-4-(4-(1-acety!azetidin-3-y!)but-3-en-1-yn-1-yl)-A -(3-amino-1-(hydroxyamino)-3-met^ oxobutan-2-yl)benzamide (28);

(S _! E)-4-(4-(1-acety!azetidin-3-y!)bui-3-en-1-yn-1-yl)-A/- (3-amino-1-(hydroxyamino)-3-methyl-1- oxobutan-2-yl)benzamide (29); W-((S)-3-amino-1-(hydroxyamino)-3-methyl-1-oxobutan-2-y!)-4- ((1-methy!-5-oxopyrrolidin-3- yl)buta-1 ,3-diynyi)benzamide (30);

A -((S)-3-amino-1-(hydroxyamino)-3-methy!-1-oxobutan-2-y!)-4-( (1-(2- (hydroxymethy!)cyc!opropanecarbonyl)azetidin-3-y!)ethyny!)be nzamide (31 );

A/-((S)-3-hydroxyamino)-3-methy!-1-oxobutan-2-yl)-4-((1-( oxetan-3-yl)pyrrolidiri-3-yl)buta-1 _! 3- diyn-1-yi)benzamide (32);

A/-((S)-3-hydroxy-1-(hydroxyamino)-3-methyi-1-oxobutan-2-yl) -4-(((S)-1-(2- hydroxyethyl)pyrrolidin-3-yl)buta-1 ,3-diyn-1-y!)benzamide (33);

4-((1-(1-fluoropropan-2-yl)azetidin-3-yl)buta-1 _l 3-diyn-1-yl)-N-((S)-3-hydroxy-1-(hydroxyamino)-3- methyi-1-oxobutan-2-y!)benzamide (34);

N-(3-hydroxy-1-(hydroxyamino)-3-methyl-1-oxobutan-2-yl)-4-(( 3-(3-hydroxyazetidin-1- yl)cyciobutyl)buta-1 _! 3-dsyn-1-yl)benzamide (35);

N-((S)-3-hydroxy-1-(hydroxyamino)-3-meihyi-1-oxobutan-2-y!)- 4-((3-((S)-3-hydroxypyrro!idin-1- yl)cyc!obutyl)buta-1 ,3-diyn-1-yI)benzamide (36);

N-((S)-3-hydroxy-1-(hydroxyamino)-3-methyi-1-oxobutan-2-y!)- 4-((3-((R)-3-hydroxypyrro!idin-1- yl)cyc!obutyl)buta-1 ,3-diyn-1-yl)benzamide (37);

4-((3-(3-fluoroazetidin-1-yl)cyclobutyl)buta-1 ,3-diyn-1-yi)-N-(3-hydroxy-1-(hydroxyamino)-3- methyi-1-oxobutan-2-yl)benzamide (38);

4-((1-(2-cyanoethyI)azeiidin-3-y!)buta-1 ,3-diyn-1-y!)-N-(3-hydroxy-1-(hydroxyamino)-3-methyl-1- oxobutan-2-yl)benzamide(39);

4-((1-(2 _! 2-difluoroethyl)azetidin-3-y!)buta-1 ,3-diyn-1-y!)-N-(3-hydroxy-1-(hydroxyamino)-3- methyi-1-oxobutan-2-yl)benzamide (40);

4-((1-(2-amino-2-oxoethyi)azetidin-3-yi)buta-1 ,3-diyn-1-yi)-N-(3-hydroxy-1-(hydroxyamino)-3- methy!-1-oxobutan-2-yl)benzamide (41 );

4-((3-(3-fluoropyrrolidin-1-y!)cyc!obutyl)buta-1 ,3-diyn-1-yl)-N-(3-hydroxy-1-(hydroxyamino)-3- methyi-1-oxobutan-2-yl)benzamide (42);

4-((3-(3 _! 3-difluoroazeiidin-1-yl)cyclobutyl)buta-1 ,3-diyri-1-yl)-N-(3-hydroxy-1-(hydroxyamino)-3- methyi-1-oxobutan-2-y!)benzamide (43); (S)-N-(3-hydroxy-1-(hydroxyamino)-3-methy!-1-oxobutan-2-yi)- 4-(6-(3-hydroxyazetidin-1- yl)hexa-1 ,3-diyn-1-yi)benzamide (44);

(S)-4-(6-(3-f!uoroazetidin-1-yl)hexa-1 ,3-diyn-1-yl)-N-(3-hydroxy-1-(hydroxyamino)-3-methy!-1- oxobutan-2-yl)benzamide (45);

(S)-4-(6-(3,3-dif!uoroazetidin-1-yl)hexa-1 ,3-diyn-1-yl)-N-(3-hydroxy-1-(hydroxyamino)-3-methy!- 1-oxobutan-2-y!)benzamide (48);

N-((S)-3-hydroxy-1-(hydroxyamino)-3-methyi-1-oxobutan-2-y!)- 4-(6-(3-hydroxypyrro!idin-1- yl)hexa-1 ,3-diyn-1-yl)benzamide (47);

4-(6-(3-fiuoropyrrolidin-1-y!)hexa-1 ,3-diyn-1-yl)-N-({S)-3-hydroxy-1-(hydroxyamino)-3-nriethyl-1 - oxobutan-2-yl)benzamide (48);

(S^-ie-iS.S-difluoropyrroiidin-l-ylJhexa-l ^-diyn-l-y -N-iS-hydroxy-l-ihydroxyaminoJ-S-meihy!-

1- oxobutan-2-y!)benzamide (49);

(S)-4-(azetidin-3-yibuta-1 ,3-diyn-1-yl)-N-(3-hydroxy-1-(hydroxyamino)-3-methyl-1-oxobu tari-2- yl)benzamide (50);

4-(azetidin-3-ylbuta-1 _l 3-diyn-1-y!)-N-((2S,3R)-3-hydroxy-1-(hydroxyamino)-1-o xobutan-2- yl)benzamide (51 );

4-({1-formylazetidin-3-yl)buta-1 ,3-diyn-1-yi)-N-((2S,3R)-3-hydroxy-1-(hydroxyarnino)-1- oxobutan-2-yl)benzamide (52);

4-((1-gIycyiazetidin-3-yl)buta-1 _l 3-diyn-1-y!)-N-((2S,3R)-3-hydroxy-1-(hydroxyamino)-1-o xobuian-

2- yl)benzamide (53);

N-((2S,3R)-3-hydroxy-1-(hydroxyamino)-1-oxobutan-2-yl)-4-((1 -serylazetidin-3-yl)buta-1 ,3-diyn- 1-yl)benzamide (54);

N-((2S,3R)-3-hydroxy-1-(hydroxyamino)-1-oxobutan-2-yl)-4-((1 -{2-hydroxyethy!)azetidin-3- yl)buta-1 ,3-diyn-1-y!)benzamide (55);

N-((2S,3R)-3-hydroxy-1-(hydroxyamino)-1-oxobutan-2-yl)-4-((1 -methylazetidin-3-yl)buta-1 _! 3- diyn-1-yi)benzamide (56);

N-{(2S,3R)-3-hydroxy-1-(hydroxyarnino)-1-oxobutan-2-yl)-4-(( 1-(meihyisulfonyi)azeiidin-3- y!)buta-1 ,3-diyn-1-y!)benzamide (57); N-((2S,3R)-3-hydroxy-1-(hydroxyamino)-1-oxobutan-2-yI)-4-((1 -(oxetan-3-y!)azetidin-3-y!)buta- 1 ,3-diyn-1-yl)benzamide (58);

N-((2S,3R)-3-hydroxy-1-(hydroxyamino)-1-oxobutan-2-yl)-4-(5- ((2R,3S)-3-hydroxypyrro!idin-2- yl)penta-1 ,3-diyn-1-yl)benzamide (59);

(S)-3-((4-((3-hydroxy-1-(hydroxyamino)-3-meihyl-1-oxobuian-2 -yl)carbamoyl)phenyl)buta-1 ,3- diyn-1-yi)-N-meihylazetidine-1-carboxamide (60);

3-((4-(((2S,3R)-3-hydroxy-1-(hydroxyamino)-1-oxobutan-2-yl)c arbamoy!)pheny!)buta-1 ,3-diyn-1- yl)-N-methylazetidine-1-carboxamide (61 );

(S)-3-((4-{(3-hydroxy-1-(hydroxyamino)-3-methyl-1-oxobutan-2 -yl)carbamoyl)phenyl)buta-1 ,3- diyn-1-y!)azeiidine-1-carboxamide (62);

3- ((4-(((2S,3R)-3-hydroxy-1-(hydroxyamino)-1-oxobutan-2-yl)car bamoy!)phenyi)buta-1 ,3-diyn-1- yl)azetidine-1-carboxamide (63);

(S)-N-(3-hydroxy-1-(hydroxyarnino)-3-meihyi-1-oxobutan-2-y!) -4-((1-(2-hydroxyacety!)azetidin-3- yl)buta-1 ,3-diyn-1-y!)benzamide (64);

N-((2S,3R)-3-hydroxy-1-(hydroxyamino)-1-oxobutan-2-yl)-4-((1 -(2-hydroxyaceiy!)azetidin-3- yl)buta-1 ,3-diyn-1-y!)benzamide (65);

(S)-N-(3-hydroxy-1-(hydroxyamino)-3-methyi-1-oxobutan-2-yi)- 4-({1-(2-hydroxyethyl)azetidin-3- yl)buta-1 ,3-diyn-1-y!)benzamide (66);

N-((2S,3R)-3-hydroxy-1-(hydroxyamino)-1-oxobutan-2-yI)-4-((1 -(2-hydroxyethyi)azetidin-3- yl)buta-1 ,3-diyn-1-y!)benzamide (67);

(S)-4-((1-acetyi-3-hydroxyazetidin-3-yl)buta-1 ,3-diyn-1-y!)-N-(3-hydroxy-1-(hydroxyamino)-3- methyi-1-oxobutan-2-yi)benzamide (68);

4- ((1-aceiyi-3-hydroxyazetidin-3-yl)buia-1 ,3-diyn-1-y!)-N-((2S,3R)-3-hydroxy-1-{hydroxyamino)- 1-oxobutan-2-y!)benzamide (69);

(S)-N-(3-hydroxy-1-(hydroxyamino)-3-methyi-1-oxobutan-2-yl)- 4-((1-(methylsu!fonyi)azetidin-3- yl)buta-1 ,3-diyn-1-y!)benzamide (70);

N-{(2S,3R)-3-hydroxy-1-(hydroxyamino)-1-oxobutan-2-yl)-4-((1 -(meihyisulfonyi)azeiidin-3- yl)buta-1 ,3-diyn-1-y!)benzamide (71 ); (S)-4-(5-(azetidin-3-y!)penta-1 ,3-diyn-1-yl)-N-(3-hydroxy-1-(hydroxyamino)-3-methyl-1- oxobutan-2-yl)benzamide (72);

4-(5-(azetidin-3-y!)penta-1 ,3-diyn-1-y!)-N-((2S _! 3R)-3-hydroxy-1-(hydroxyamino)-1-oxobutan-2- yl)benzamide (73);

(S)-4-(5-(1-formylazetidin-3-yl)penta-1 ,3-diyn-1-y!)-N-(3-hydroxy-1-(hydroxyamino)-3-methyl-1- oxobutan-2-yl)benzamide (74);

4-(5-(1-formylazetidin-3-y!)penta-1 _! 3-diyn-1-y!)-N-((2S,3R)-3-hydroxy-1-(hydroxyamino)-1- oxobutan-2-yl)benzamide (75);

(S _! E)-N-(3-hydroxy-1-(hydroxyamino)-3-methy!-1-oxobutan-2 -yl)-4-(4-(1- (methylsuifony!)azetidin-3-yI)but-3-en-1-yn-1-y!)benzamide (76);

N-((2S,3R)-3-hydroxy-1-(hydroxyamino)-1-oxobutan-2-y!)-4-((E )-4-(1-(methy!sulfony!)azetidin-3- yl)but-3-en-1-yn-1-yl)benzamide (77);

(S _l E)-4-(4-(1-formylazetidin-3-yl)but-3-en-1-yn-1-y!)-N-( 3-hydroxy-1-(hydroxyamino)-3-methyl-1- oxobutan-2-yl)benzamide (78);

4-((E)-4-(1-formylazetidin-3-yl)but-3-en-1-yn-1-y!)-N-((2S,3 R)-3-hydroxy-1-(hydroxyamino)-1- oxobutan-2-yl)benzamide (79);

N-{(S)-3-hydroxy-1-(hydroxyamino)-3-methyi-1-oxobutan-2-yi)- 4-((5-oxopyrrolidin-3-y!)buia-1 ,3- diyn-1-yl)benzamide (80);

N-((2S,3R)-3-hydroxy-1-(hydroxyamino)-1-oxobutan-2-yI)-4-((5 -oxopyrrolidin-3-yl)buia-1 ,3-diyn- 1-yl)benzamide (81 );

N-((S)-3-hydroxy-1-(hydroxyamino)-3-methy!-1-oxobutan-2-yi)- 4-((1-methyl-5-oxopyrro!idin-3- yl)buta-1 ,3-diyn-1-y!)benzamide (82);

N-((2S _l 3R)-3-hydroxy-1-(hydroxyamino)-1-oxobutan-2-yl)-4-((1- methy!-5-oxopyrro!idin-3-yl)buta- 1 ,3-diyn-1-yI)benzamide (83);

N-((2S,3R)-3-hydroxy-1-(hydroxyamino)-1-oxobutan-2-yl)-4-((( S)-1-(oxetan-3-yl)pyrrolidin-3- yl)buta-1 ,3-diyn-1-y!)benzamide (84);

N-{(2S,3R)-3-hydroxy-1-(hydroxyamino)-1-oxobutan-2-yl)-4-((( S)-1-(2-hydroxyethyl)pyrroiidir!-3- y!)buta-1 ,3-diyn-1-y!)benzamide (85); 4-((1-(1-fluoropropan-2-yl)azetidin-3-yi)buta-1 _> 3-diyn-1-yl)-N-((2S,3R)-3-hydroxy-1- (hydroxyamino)-1-oxobutan-2-y!)benzamide (88);

4-((1-(2 _! 2-difluoroethyl)azetidin-3-y!)buta-1 ,3-diyn-1-y!)-N-((3R)-3-hydroxy-1-(hydroxyamino)-1- oxobutan-2-yl)benzamide (87);

4-((1-(2-cyanoethyl)azeiidin-3-yi)buta-1 ,3-diyn-1-yi)-N-((3R)-3-hydroxy-1-(hydroxyamino)-1- oxobutan-2-yl)benzamide (88);

4-((1-(2-amino-2-oxoethy!)azetidin-3-yi)buta-1 ,3-diyn-1-y!)-N-((3R)-3-hydroxy-1-(hydroxyamino)- 1-oxobutan-2-y!)benzamide (89);

N-{(2S,3R)-3-hydroxy-1-(hydroxyamino)-1-oxobutan-2-yl)-4-((1 -((1-methyi-1 H-imidazol-4- yI)methyl)azetidin-3-y!)buta-1 ,3-diyn-1-yl)benzamide (90);

N-((3R)-3-hydroxy-1-(hydroxyamino)-1-oxobutan-2-y!)-4-((3 -(3-hydroxyazetidin-1- y cyciobuty buta-l ^-diyn-l-y benzamide (91 );

4-((3-(3-fluoroazetidiri-1-yl)cyclobutyl)buta-1 ,3-diyn-1-yi)-N-((3R)-3-hydroxy-1-(hydroxyamino)-1- oxobutan-2-yl)benzamide (92);

N-((3R)-3-hydroxy-1-(hydroxyamino)-1-oxobutan-2-y!)-4-((3-(3 -hydroxypyrrolidin-1- yl)cyc!obutyl)buta-1 ,3-diyn-1-yl)benzamide (93);

4-((3-(3-fluoropyrrolidin-1-y!)cyc!obutyl)buta-1 ,3-diyn-1-yl)-N-{(3R)-3-hydroxy-1-(hydroxyamino)- 1-oxobutan-2-yi)benzatnide (94);

4-((3-(3 _! 3-difluoroazetidin-1-yl)cyclobuty!)buta-1 ,3-diyn-1-yl)-N-((3R)-3-hydroxy-1- (hydroxyamino)-1-oxobutan-2-y!)benzamide (95);

N-((2S,3R)-3-hydroxy-1-(hydroxyamino)-1-oxobutan-2-yl)-4-(6- (3-hydroxyazetidin-1-yl)hexa-1 ,3- diyn-1-yi)benzamide (96);

4-(6-(3-f!uoroazeiidin-1-yl)hexa-1 ,3-diyn-1-yl)-N-({2S _! 3R)-3-hydroxy-1-(hydroxyamino)-1- oxobutan-2-yl)benzamide (97);

4-(6-(3,3-dif!uoroazetidin-1-yl)hexa-1 ,3-diyn-1-yl)-N-((2S,3R)-3-hydroxy-1-(hydroxyarnino)-1- oxobutan-2-yl)benzamide (98);

N-{(2S,3R)-3-hydroxy-1-(hydroxyamino)-1-oxobutan-2-yl)-4-(6- (3-hydroxypyrrolidin-1-y!)hexa- 1 ,3-diyn-1-yl)benzamide (99); 4-(6-(3-fiuoropyrrolidin-1-y!)hexa-1 ,3-diyn-1-yi)-N-((2S _! 3R)-3-hydroxy-1-(hydroxyamino)-1- oxobutan-2-yl)benzamide (100);

4-(6-(3,3-difiuoropyrroiidin-1-y!)hexa-1 ,3-diyn-1-yi)-N-((2S,3R)-3-hydroxy-1 -(hydroxyamino)-1- oxobutan-2-yl)benzamide (101 );

4-((1-(4 _l 5-dimeihyi-4,5-dihydrooxazo!-2-yi)azetidin-3-yi)buta- 3-diyn-1-yl)-N-((2S,3R)-3- hydroxy-1-(hydroxyamino)-1-oxobutan-2-yl)benzamide (102);

4-((1-(4 _! 5-dimethyl-4,5-dihydrooxazol-2-y!)azetidin-3-y!)buta-1 _! 3-diyn-1-y!)-N-((S)-3-hydroxy-1- (hydroxyamino)-3-methy!-1-oxobutan-2-y!)benzamide (103); and

(S)-N-(3-hydroxy-1-(hydroxyamino)-3-meihy!-1-oxobutan-2-yi)- 4-((1-((1-methyl-1 H-imidazol-4- yI)methyl)azeiidin-3-yI)buta-1 ,3-diyn-1-yl)benzamide (104).

Another aspect of the invention provides compounds selected from the group of: (S)-A/-(3-amino-1-(hydroxyamino)-3-methyl-1-oxobutan-2-yi)-4 -(azetidin-3-y!buta-1 _l 3- diyny!)benzamide (1 );

(S)-4-((1-acetylazetidin-3-yl)buta-1 ,3-diyn-1-y!)-A/-(3-hydroxy-1-(hydroxyamino)-3-methyI-1- oxobutan-2-yl)benzamide (2a);

(S)-4-((azetidin-3-yi)buta-1 ,3-diyn-1-y!)-A/-(3-hydroxy-1-{hydroxyamino)-3-methyi-1-oxob utan y!)benzamide (2b);

(S)-4-((1-formyIazetidin-3-yI)buta-1 ,3-diyn-1-yl)-W-(3-hydroxy-1-(hydroxyamino)-3-methy!-1- oxobutan-2-yl)benzamide (3);

(S)-4-((1-(2-aminoacetyi)azetidin-3-yl)buta-1 _! 3-diyn-1-yl)-W-(3-hydroxy-1-(hydroxyamino)-3- methyi-1-oxobutan-2-yi)benzamide (4);

4-((1-(2-amino-3-hydroxypropanoy!)azetidiri-3-yl)buta-1 ,3-diyri-1-y!)-W-((S)-3-hydroxy-1-

(hydroxyamino)-3-methy!-1-oxobutan-2-y!)benzamide (5);

(S)~A/~(3-hydroxy-1-(hydroxyamino)~3~m

yl)buta-1 ,3-diyn-1-y!)benzamide (6);

(S)-W-(3-hydroxy-1-(hydroxyamino)-3-methy!-1-oxob

1 ,3-diyn-1-y!)benzamide (7); (S)-A/-(3-amino-1-(hydroxyamino)-3-methyl-1-oxobutan-2-y!)-4 -((1-(oxetan-3-y!)azetidin-3- yl)buta-1 ,3-diyn-1-y!)benzamide (8);

A -((S)-3-hydroxy-1-(hydroxyamino)-3-methy!-1-oxobutan-2-yl)-4 -(5-((2/ ^: ?,3S)-3- hydroxypyrroiidin-2-yi)penta-1 ,3-diyn-1-y!)benzamide (9);

(S)-4-((1-acety!azei!din-3-y!)buia-1 ,3-diynyi)-W-(3-amino-1-(hydroxyamino)-3-methyi-1- oxobutan-2-yl)benzamide 2,2,2-trif!uoroacetate (10);

(S)-3-((4-(3-amino-1-(hydroxyamino)-3-methyl-1-oxobutan-2-yl carbamoyl)phenyl)buta-1 _! 3- diyny!)-A/-methylazetidine-1-carboxamide (1 1 );

(S)-3-((4-(3-amino-1-(hydroxyamino)-3-methyl-1-oxobuiari-2-y lcarbamoyl)phenyl)buta-1 _! 3- diyny!)azetidine-1-carboxamide (12);

(S)-4-((1-acetyiazetidin-3-yi)buta-1 ,3-diyny!)-A/-(3-(dimethy!amino)-1-(hydroxyamino)-3-meth 1-oxobutan-2-y!)benzamide (13);

(S)-A/-(3-amino-1-(hydroxyamino)-3-methyl-1-oxobuian-2-yi)-4 -((1-{2-hydroxyacetyl)azetidin-3- yl)buta-1 ,3-diyn-1-y!)benzamide (14);

(S)-A/-(3-amino-1-(hydroxyamino)-3-methyI-1-oxobutan-2-y!)-4 -((1-(2-hydroxyethy!)azeiidin-3- yl)buta-1 ,3-diyn-1-y!)benzamide (15);

(S)-A/-(3-amino-1-(hydroxyamino)-3-methyl-1-oxobutan-2-y!)-4 -((1-formylazetidin-3-yl)buta-1 ,^ diyn-1-y!)benzamide (16);

(S)-4-((1-formyIazetidin-3-yI)buta-1 ,3-diyn-1-yl)-W-(1-(hydroxyamino)-3-methyi-3-(meihyiami 1-oxobutan-2-y!)benzamide (17);

(S)-4-((1-acetyiazetidin-3-yl)buta-1 ,3-diyn-1-y!)-A/-(1-(hydroxyamino)-3-methy!-3-(methylamin 1-oxobutan-2-y!)benzamide (18);

(S)-4-((1-acetyl-3-hydroxyazetidin-3-yi)buta-1 ,3-diyn-1-yl)-A-(3-amino-1-(hydroxyamino)-3- methy!-1-oxobutan-2-yi)benzamide (19);

A/-((2S,3R)-3-amino-1-(hydroxyamino)-1-oxobutan-2-y!)-4-((1- formy!azetidin-3-y!)buta-1 ,3-diyn- 1-yl)benzamide (20);

(S)-A/-(3-amino-1-(hydroxyamino)-3-methyl-1-oxobutan-2-y!)-4 -((1-(methyisulfonyl)azetidin-3- yl)buta-1 ,3-diyn-1-y!)benzamide (21 ); 4-((1-formylazetidin-3-yl)buia-1 ,3-diyn-1-yI)-W-((2S _l 3R)-1-(hydroxyamino)-3-(methy!ami oxobutan-2-yl)benzamide (22);

(S)-A/-(3-hydroxy-1-(hydroxyamino)-3-methy!-1-oxobutan-2-yi) -4-((1-(oxetan-3-y!)azetidin-3- yl)buta-1 ,3-diyn-1-y!)benzamide (23);

(S)-A/-(3-amino-1-(hydroxyamino)-3-methyl-1-oxobutan-2-yi)-4 -(5-(azetidin-3-yl)penta-1 ,3-diyn- 1-yl)benzamide (24);

(S)-A/-(3-amino-1-(hydroxyamino)-3-methy!-1-oxobutan-2-y!)-4 -(5-(1-formy!azetidin-3-yl)penta- 1 ,3-diyn-1-yl)benzamide (25);

(S,£)-A/-(3-amino-1-(hydroxyamino)-3-methy!-1-oxobutan-2-yi )-4-(4-{1-(methy!sulfony!)azetidi

3- y!)but-3-en-1-yn-1-yI)benzamide (26);

(S,£)-A/-(3-amino-1-(hydroxyamino)-3-methy!-1-oxobutan-2-yi )-4-(4-(1-formylazetidin-3-y!)but-3- en-1-yn-1-y!)benzamide (27);

(S,£)-4-(4-(1-ace1y!azeiidin-3-y!)bui-3-en-1-yn-1-yl)-W-{3- amino-1-(hydroxyamino)-3-methyl-1- oxobutan-2-yl)benzamide (28);

(S _l E)-4-(4-(1-acety!azetidin-3-y!)but-3-en-1-yn-1-yl)-W-( 3-amino-1-(hydroxyamino)-3-methyl-1- oxobutan-2-yl)benzamide (29);

A/-{(S)-3-amino-1-(hydroxyamino)-3-methyl-1-oxobutan-2-y!)-4 -((1-methyi-5-oxopyrrolidin-3- yl)buta-1 ,3-diynyi)benzamide (30);

A/-((S)-3-amino-1-(hydroxyamino)-3-methy!-1-oxobutan-2-y!)-4 -((1-(2- (hydroxymethy!)cyc!opropanecarbonyl)azetidin-3-y!)ethyny!)be nzamide (31 );

A -((S)-3-hydroxyamino)-3-methy!-1-oxobutan-2-yl)-4-((1-(oxeta n-3-yl)pyrrolidin-3-yl)buta-1 _! 3- diyn-1-yi)benzamide (32);

A/-((S)-3-hydroxy-1-(hydroxyamino)-3-methyl-1-oxobutan-2-yl) -4-(((S)-1-(2- hydroxyethyl)pyrrolidin-3-yI)buta-1 ,3-diyn-1-y!)benzamide (33);

4- ((1-(1-fluoropropan-2-yl)azetidin-3-yl)buta-1 ,3-diyn-1-yl)-N-((S)-3-hydroxy-1-(hydroxyamino)-3- methyi-1-oxobutan-2-yi)benzamide (34);

N-{3-hydroxy-1-{hydroxyamino)-3-methyl-1-oxobutan-2-yl)-4-(( 3-(3-hydroxyazetidin-1- yI)cyc!obutyl)buta-1 ,3-diyn-1-yl)benzamide (35); N-((S)-3-hydroxy-1-(hydroxyamino)-3-methy!-1-oxobutan-2-yi)- 4-((3-((S)-3-hydroxypyrroiidin yl)cyciobutyl)buta-1 ,3-diyn-1-yi)benzamide (36);

N-((S)-3-hydroxy-1-(hydroxyamino)-3-methy!-1-oxobutan-2-yi)- 4-((3-((R)-3-hydroxypyrro!idin-1- yl)cyciobutyl)buta-1 ,3-diyn-1-yi)benzamide (37);

4-((3-(3-fluoroazetidin-1-yl)cyclobutyl)buta-1 ,3-diyn-1-yi)-N-(3-hydroxy-1-(hydroxyamino)-3- methy!-1-oxobutan-2-yl)benzamide (38); and

4-((1-(2-cyanoethyl)azetidin-3-yi)buta-1 ,3-diyn-1-y!)-N-(3-hydroxy-1-(hydroxyamino)-3-methyl-1- oxobutan-2-yl)benzamide{39).

Compounds of the present invention can be readily synthesized using the methods described herein, or other methods, that are we!i known in the art. For example, the synthesis of hydroxamic acids or similar scaffolds having a wide variety of substituents are comprehensively reviewed in Kline, T., et a!., "Potent, novel in vitro inhibitors of the

Pseudomonas aeruginosa deacetyiase LpxC" J. Med Chem. 2002, 45(14), 31 12-29; U.S. Patent No. 5,925,659; Pirrung, M. C, et a!., "A Convenient Procedure for the Preparation of Amino Acid Hydroxamates from Esters" J. Org. Chem. 1995, 60, 8084-8085; Nhu, K., et a!., "A New and Efficient Solid Phase Synthesis of Hydroxamic Acids" J. Org. Chem. 1997, 62, 7088-7089; international PCT Publication No. W098/18754; Mellor, S. L, et al., "N-Fmoc-aminoxy-2- chlortrityl Polystyrene Resin: A Facile Solid-phase Methodology for the Synthesis of Hydroxamic Acids" Tetrahedron Lett. 1997, 38, 331 1-3314; Khan, S. I., et a!., "A Facile and Convenient Solid-phase Procedure for Synthesizing Nucleoside Hydroxamic Acids" Terahedron. Lett. 1998, 39, 8031-8034; Zhang, Y., et a!., "Design, Combinatorial Chemical Synthesis, and in vitro Characterization of Novel Urea Based Gelatinase inhibitors" Bioorg. Med. Chem. Lett. 1999, 9, 2823-2826; Ito, Y., et al., "Synthetic Reactions by Complex Catalysts. XXXI, A Novel and Versatile Method of Heterocycle Synthesis" J. Am Chem. Soc. 1973, 95, 4447-4448; Ito, Y., et a!., "Synthetic Reactions by Complex Catalysts XXXV" Syn. Commun. 1974, 4, 97-103; Witte, H., et al., "Cyclische imidsaurester aus Nitriien und Aminoalkoholen" Liebigs Ann. Chem. 1974, 996-1009; Pattenden, G., et al., "Naturally Occurring Linear Fused Thiazoline-Thiazole

Containing Metabolites: Total Synthesis of (-) Didehydromirabazole A, a Cytotoxic Alkaloid from Blue-Green Algae" J. Chem. Soc. Perkin Trans 1993, 1 , 1629-1636; Boyce, R. J., et al., "Total Synthesis of Thiangazo!e, A Novel Naturally Occurring HiV-1 Inhibitor from Polyangium sp." Tetrahedron 1995, 51 , 7321-7330; Galeotti, N., et a!., "Synthesis of Peptidyl Aldehydes from Thiazoiines" Tetrahedron. Lett. 1997, 38, 2459-2462; Charette, A. B., et a!., "Mild Method for the Synthesis of Thiazoiines from Secondary and Tertiary Amides" J. Org. Chem. 1998, 83, 908- 909; Bergeron, R. J., et a!., "Effects of C-4 Stereochemistry and C-4' Hydroxyiation on the Iron Clearing Efficienc and Toxicity of Desferrithiocin Analogues" J. Med, Chem. 1999, 42, 2432- 2440; Raman, P., et a!., "Titanium (IV)-mediated Tandem Deprotection-cyc!odehydration of Protected Cysteine N-Amides: Biomimetic Synthesis of Thiazoline- and Thiazole-containing Heterocycies" Org. Lett 2000, 2, 3289-3292; Fernandez, X., et al., "Novel Synthesis of 2- Thioazo!ines" Tetrahedron Lett. 2000, 41 , 3381-3384; and Wipf, P., et a!., "C. Thiolysis of Oxazolinenes: A New, Selective Method for the Direct Conversion of Peptide Oxazolines into Thiazoiines" Tetrahedron Lett. 1995, 36, 6395-6398, which are incorporated herein by reference.

The compounds of Formula i are distinguished from previously known substances by a surprisingly low degree of binding to plasma proteins and can therefore provide a relatively high concentration of free, i.e. pharmacologically effective and available drug concentration, in order to achieve an antibacterial effect, the MIC for the target organism must be reached in vivo. Binding of the antibacterial agent to plasma proteins will decrease the available plasma concentration of the agent, making it more difficult to achieve a concentration at or above the MIC. The compounds disclosed herein demonstrate decreased protein binding as compared to previously known substances, and therefore can more easily achieve a therapeutic concentration in the patient.

in another aspect, the present invention provides a pharmaceutical composition comprising a compound of Formula I, or a stereoisomer, pharmaceutically acceptable salt, or ester thereof, and a pharmaceutically acceptable carrier or diluent.

In another aspect, the invention provides a method of inhibiting a deacetylase enzyme in a gram-negative bacteria, thereby affecting bacterial growth, comprising

administering to a patient in need of such inhibition a compound of Formula I or a stereoisomer, pharmaceutically acceptable salt, or ester thereof. In another aspect, the invention provides a method of inhibiting LpxC, thereby modulating the virulence of a bacteria! infection, comprising administering to a patient in need of such inhibition a compound of Formula or a stereoisomer, pharmaceutically acceptable salt, or ester thereof. In certain embodiments of the method of inhibiting LpxC using a compound of the present invention, the IC ₅₀ value of the compound is less than or equal to 10 μΜ with respect to LpxC. in other embodiments, the !C ₅ o value is less than or equal to 1 μΜ, is less than or equal to 0.1 μΜ, is less than or equal to 0.050 μ , is less than or equal to 0.030 μΜ, is less than or equal to 0.025 μ , or is less than or equal to 0.010 μΜ.

In another aspect, the invention provides a method for treating a patient having a gram-negative bacterial infection comprising administering to the patient in need thereof an antibacterially effective amount of a compound of Formula I or a stereoisomer, pharmaceutically acceptable salt, or ester thereof.

In another aspect, the invention provides a method of administering a

therapeutically effective amount of a compound of Formula I or a stereoisomer,

pharmaceutically acceptable salt, or ester thereof, to a patient infected with a fermentative or non-fermentative gram-negative bacteria. Examples of fermentative or non-fermentative gram- negative bacteria include Pseudomonas aeruginosa, Stenotrophomonas maltophila,

Burkholderia cepacia, Alcaiigenes xyiosoxidans, Enterobacteriaceae, Haemophilus,

Francisce!iaceae (e.g., Francisce!la tularensis) and Neisseria species.

In another aspect, the invention provides a method of administering an inhibitory amount of a compound described herein to gram-negative bacteria, such as Enterobacieriaceae which is selected from the group consisting of organisms such as Serratia, Proteus, Klebsiella, Enterobacter, Citrobacter, Salmonella, Providencia, Yersinia (e.g., Yersinia pestis), Morganella, Cedecea, Edwardsiella species and Escherichia coli.

In certain embodiments, the patient may be a mammal, and in some embodiments, a human.

Bacteria! infections susceptible to treatment according to the present invention include primary infections and co-infections caused by a species of bacteria and one or more additional infectious agents such as, for example, bacteria, virus, parasite and fungus. Compounds of the invention can be used for treating conditions caused by the bacterial production of endotoxin and, in particular, by gram-negative bacteria and bacteria that use LpxC in the biosynthesis of !ipopolysaccharide (LPS) or endotoxin.

Compounds of the invention also are useful in treating conditions that are caused or exacerbated by the bacteria! production of lipid A and LPS or endotoxin, such as sepsis, septic shock, systemic inflammation, localized inflammation, chronic obstructive pulmonary disease (COPD) and acute exacerbations of chronic bronchitis (AECB). For these conditions, treatment includes the administration of a compound of the invention, or a combination of compounds of the invention, optionally with a second agent wherein the second agent is a second antibacterial agent or a non-antibacterial agent.

For sepsis, septic shock, systemic inflammation, localized inflammation, chronic obstructive pulmonary disease (COPD) and acute exacerbations of chronic bronchitis (AECB), representative non-antibacterial agents include antiendotoxins including endotoxin receptor- binding antibodies, endotoxin-binding antibodies, anti-CD14-binding protein antibodies, antilipopo!ysaccharide-binding protein antibodies and tyrosine kinase inhibitors.

In treatment of serious or chronic respiratory tract infections, compounds of the present invention may also be used with non-antibacterial agents administered via inhalation. Representative non-antibacterial agents used in this treatment include anti-inflammatory steroids, non-steroidal anti-inflammatory agents, bronchiodiiators, mucolytics, anti-asthma therapeutics and lung fluid surfactants, in particular, the non-antibacterial agent may be albuterol, salbuterol, budesonide, bec!omethasone, dexamethasone, nedocromi!,

beclomethasone, fluticasone, fluniso!ide, triamcinolone, ibuprofin, rofecoxib, naproxen, celecoxib, nedocromil, ipratropium, metaproterenol, pirbuteroi, saimeterol, formoterol, indacatero!, bronchiodiiators, mucolytics, calfactant, beractant, poractant alfa, surfaxin or pu!mozyme (also called domase alfa).

Compounds of the invention can be used alone or in combination with a second antibacterial agent for the treatment of a serious or chronic respiratory tract infection including serious lung and nosocomial infections such as those caused by Enterobacter aerogenes, Enterobacter cloacae, Escherichia co/ , Klebsiella pneumoniae, Klebsiella oxytoca, Proteus rnirabiiis, Serratia marcescens, Stenotrophornonas ma!tophi!ia, Pseudornonas aeruginosa, Burkhoidena cepacia, Alcaligenes xylosoxidans, Fiavobacierium meningosepticum, Providencia siuartii and Citrobacter freundi, community lung infections such as those caused by

Haemophilus Influenzae, Legionella species, Moraxeila catarrhalis, Branhamella catarrhal, Enterobacter species, Klebsiella species, and Proteus species, infections caused by other bacterial species such as Neisseria species, Shigella species, Salmonella species, Helicobacter pylori, Vibrionaceae and Bordeteila species, as well as infections caused by a Brucella species, Francisetla tularensis and/or Yersinia Pestis.

When used for treating patients infected with gram-negative bacterial infections, compounds of the present invention can be used to sensitize gram-negative bacteria to the effects of a second agent.

The present invention provides novel combinations of compounds including a compound of Formula I or a stereoisomer, pharmaceutically acceptable salt, or ester thereof, as well as methods for treating patients infected with gram-negative bacteria. The novel combinations provided herein can be formulated into pharmaceutical formulations and medicaments that are useful in the methods of the invention. The invention also provides for the use of the novel combinations in preparing medicaments and pharmaceutical formulations, for use of the combinations in treating bacterial infections in a patient.

In one embodiment, a second antibacterial agent is used in combination with a compound of Formula I, or stereoisomer or pharmaceutically acceptable salt thereof. Examples of suitable second antibactierai agents include, but are not limited to, vancomycin, linezolid , azithromycin, imipenem, teicop!anin, daptomycin, clindamycin, rifampin, cefotaxime, gentamicin, novobiocin or telavancin. in one such embodiment, the antibacterial agent is vancomycin, teicoplanin, rifampin, azithromycin, telavancin or novobiocin. Most preferably, the antibacterial agent is vancomycin or rifampin, in some embodiments of the invention, the antibacterial agent and/or the compound of Formula I, or stereoisomer or pharmaceutically acceptable salt thereof, is administered at a sub-therapeutic dose, wherein a subtherapeutic dose is a dose that would be insufficient to treat bacterial infections, if administered alone. Pharmaceutical compositions of the present invention comprise a therapeutically effective amount of a compound of Formula I, or a stereoisomer or pharmaceutically acceptable salt thereof, formulated together with one or more pharmaceutically acceptable carriers or diluents. As used herein, the term "pharmaceutically acceptable carrier" means a non-toxic, inert solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. Some examples of materials that can serve as pharmaceutically acceptable carriers are sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil; saffiower oil; sesame oil; olive oil; corn oil and soybean oil; glycols; such a propylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; aiginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol, and phosphate buffer solutions, as well as other non-toxic compatible lubricants such as sodium iauryi sulfate and magnesium sfearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the composition, according to the judgment of the formuiaior. The pharmaceutical compositions of this invention can be administered to humans and other animals orally, rectally, parenteraily (as by intravenous, intramuscular or subcutaneous injection), intracistemaily, intravaginaliy, intraperitonea!!y, topically (as by powders, ointments, or drops), bucally, or as an oral or nasal spray, or a liquid aerosol or dry powder formulation for inhalation.

Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active compounds, the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubiiizing agents and emu!sifiers such as ethyl alcohol, isopropyi alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 ,3-butylene glycol, dimethyiformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution, suspension or emulsion in a nontoxic parenteraliy acceptable diluent or solvent, for example, as a solution in 1 ,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, 1 % iidocaine, U.S. P. and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono- or diglycerides. in addition, fatty acids such as oleic acid are used in the preparation of injectabies.

The injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions that can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.

In order to prolong the effect of a drug, it is often desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material with poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution that, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenteraliy administered drug form may be accomplished by dissolving or suspending the drug in an oil vehicle, injectable depot forms are made by forming microencapsule matrices of the drug in biodegradable polymers such as polylactide-poiygiycoiide. Depending upon the ratio of drug to polymer and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poiy(orthoesters) and poly(anhydrides). Depot injectable formulations may also be prepared by entrapping the drug in liposomes or microemuisions that are compatible with body tissues. Compositions for rectal or vaginal administration are preferably suppositories that can be prepared by mixing the compounds of this invention with suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol or a suppository wax which are solid at ambient temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active compound.

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

Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.

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

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

Dosage forms for topical or transdermal administration of a compound of this invention include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants or patches. The active component is admixed under sterile conditions with a pharmaceutically acceptable carrier and any needed preservatives or buffers as may be required. Ophthalmic formulations, ear drops, and the like areaiso contemplated as being within the scope of this invention.

The ointments, pastes, creams and gels may contain, in addition to an active compound of this invention, excipients such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.

Compositions of the invention may also be formulated for delivery as a liquid aerosol or inhalable dry powder. Liquid aerosol formulations may be nebulized predominantly into particle sizes that can be delivered to the terminal and respiratory bronchioles where bacteria reside in patients with bronchial infections, such as chronic bronchitis and pneumonia. Pathogenic bacteria are commonly present throughout airways down to bronchi, broncbioli and lung parenchema, particularly in terminal and respiratory bronchioles. During exacerbation of infection, bacteria can also be present in alveoli. Liquid aerosol and inhalabie dry powder formulations are preferably delivered throughout the endobronchial tree to the terminal bronchioles and eventually to the parenchymal tissue.

Aerosolized formulations of the invention may be delivered using an aerosol forming device, such as a jet, vibrating porous plate or ultrasonic nebulizer, preferably selected to allow the formation of a aerosol particles having with a mass medium average diameter predominantly between 1 to 5 μπι. Further, the formulation preferably has balanced osmolarity ionic strength and chloride concentration, and the smallest aerosoiizabie volume able to deliver effective dose of the compounds of the invention to the site of the infection. Additionally, the aerosolized formulation preferably does not impair negatively the functionality of the airways and does not cause undesirable side effects.

Aeroso!ization devices suitable for administration of aerosol formulations of the invention include, for example, jet, vibrating porous plate, ultrasonic nebulizers and energized dry powder inhalers, that are able to nebulize the formulation of the invention into aerosol particle size predominantly in the size range from 1 -5 pm. Predominantly in this application means that at least 70% but preferably more than 90% of all generated aerosol particles are 1 to 5 m range. A jet nebulizer works by air pressure to break a liquid solution into aerosol droplets. Vibrating porous plate nebulizers work by using a sonic vacuum produced b a rapidly vibrating porous plate to extrude a solvent droplet through a porous plate. An ultrasonic nebulizer works by a piezoelectric crystal that shears a liquid into small aerosol droplets. A variety of suitable devices are available, including, for example, AeroNeb and AeroDose vibrating porous plate nebulizers (AeroGen, inc., Sunnyvale, Calif.), Sidestream7 nebulizers (Medic-Aid Ltd., West Sussex, England), Pari LC7 and Pari LC Siar7 jet nebulizers (Pari Respiratory Equipment, Inc., Richmond, Va.), and Aerosonic (DeViibiss Medizinische Produkte (Deutsch!and) GmbH, Heiden, Germany) and UltraAire7 (Omron Healthcare, inc., Vernon Hills, ill.) ultrasonic nebulizers. Compounds of the invention may also be formulated for use as topical powders and sprays that can contain, in addition to the compounds of this invention, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances. Sprays can additionally contain customary propeilants such as chlorofluorohydrocarbons.

Transdermal patches have the added advantage of providing controi!ed delivery of a compound to the body. Such dosage forms can be made by dissolving or dispensing the compound in the proper medium. Absorption enhancers can also be used to increase the flux of the compound across the skin. The rate can be controlled by either providing a rate controlling membrane or by dispersing the compound in a polymer matrix or gel.

According to the methods of treatment of the present invention, bacterial infections are treated or prevented in a patient such as a human or lower mammal by administering to the patient a therapeutically effective amount of a compound of Formula I, or a stereoisomer or pharmaceutically acceptable salt thereof, in such amounts and for such time as is necessary to achieve the desired result. By a "therapeutically effective amount" of a compound of the invention is meant a sufficient amount of the compound to treat bacterial infections, at a reasonable benefit/risk ratio applicable to any medical treatment. It will be understood, however, that the total daily usage of the compounds and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the specific compound employed; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed; and like factors well known in the medical arts.

The total daily dose of the compounds of this invention administered to a human or other mammal in single or in divided doses can be in amounts, for example, from 0.01 to 200 mg/kg body weight or more usually from 0.1 to 50 mg/kg body weight, in certain embodiments, the total daily dose administered to a human or other mammal is from 1.0 to 100 mg/kg body weight or from 5.0 to 25 mg/kg body weight. Single dose compositions may contain such amounts or submultip!es thereof to make up the daily dose. In general, treatment regimens according to the present invention comprise administration to a patient in need of such treatment from about 10 mg to about 15 g of the compound(s) of this invention per day in single or multiple doses, more usually, from 100 mg to 5 g, and even more usually from 250 mg to 1 g per day in single or multiple doses.

Methods of formulation are well known in the art and are disclosed, for example, in Remington: The Science and Practice of Pharmacy, Mack Publishing Company, Easton, Pa., 19th Edition (1995). Pharmaceutical compositions for use in thepresent invention can be in the form of sterile, non-pyrogenic liquid solutions or suspensions, coated capsules, suppositories, lyophilized powders, transdermal patches or other forms known in the art.

A "kit" as used in the instant application includes a container for containing the pharmaceutical compositions and may also include divided containers such as a divided bottle or a divided foil packet. The container can be in any conventional shape or form as known in the art that is made of a pharmaceutically acceptable material, for example a paper or cardboard box, a glass or plastic bottle or jar, a resealabie bag (for example, to hold a "refill" of tablets for placement into a different container), or a blister pack with individual doses for pressing out of the pack according to a therapeutic schedule. The container employed can depend on the exact dosage form involved, for example a conventional cardboard box would not generally be used to hold a liquid suspension. It is feasible that more than one container can be used together in a single package to market a single dosage form. For example, tablets may be contained in a bottle that is in turn contained within a box.

An example of such a kit is a so-called blister pack. Blister packs are well known in the packaging industry and are being widely used for the packaging of pharmaceutical unit dosage forms (tablets, capsules, and the like). Blister packs generally consist of a sheet of relatively stiff material covered with a foil of a preferably transparent plastic material. During the packaging process, recesses are formed in the plastic foil. The recesses have the size and shape of individual tablets or capsules to be packed or may have the size and shape to accommodate multiple tablets and/or capsules to be packed. Next, the tablets or capsules are placed in the recesses accordingly and the sheet of relatively stiff materia! is sealed against the plastic foil at the face of the foil that is opposite from the direction in which the recesses were formed. As a result, the tablets or capsules are individually sealed or collectively sealed, as desired, in the recesses between the plastic foil and the sheet. Preferably the strength of the sheet is such that the tablets or capsules can be removed from the blister pack by manually applying pressure on the recesses whereby an opening is formed in the sheet at the place of the recess. The tablet or capsule can then be removed via said opening.

It maybe desirable to provide a written memory aid, where the written memory aid is of the type containing information and/or instructions for the physician, pharmacist or other health care provider, or patient, e.g., in the form of numbers next to the tablets or capsules whereby the numbers correspond with the days of the regimen that the tablets or capsules so specified should be ingested or a card that contains the same type of information. Another example of such a memory aid is a calendar printed on the card e.g., as follows "First Week, Monday, Tuesday,". . . etc . . . "Second Week, Monday, Tuesday, . . ." etc. Other variations of memor aids will be readily apparent. A "daily dose" can be a single tablet or capsule or several tablets or capsules to be taken on a given day. When the kit contains separate compositions, a daily dose of one or more compositions of the kit can consist of one tablet or capsule while a daily dose of another one or more compositions of the kit can consist of several tablets or capsules.

Another specific embodiment of a kit is a dispenser designed to dispense the daily doses one at a time in the order of their intended use. Preferably, the dispenser is equipped with a memory-aid, so as to further facilitate compliance with the regimen. An example of such a memory-aid is a mechanical counter, that indicates the number of daily doses that has been dispensed. Another example of such a memory-aid is a battery-powered micro-chip memory coupled with a liquid crystal readout, or audible reminder signal that, for example, reads out the date that the last daily dose has been taken and/or reminds one when the next dose is to be taken. The kits of the present invention may also include, in addition to a compound of the present invention, one or more additional pharmaceutically active compounds. For example, the additional compound second antibacterial. The additional compounds may be administered in the same dosage form as the compound of the present invention or in a different dosage form. Likewise, the additional compounds can be administered at the same time as the compound of the present invention or at different times.

Compositions of the present compounds may also be used in combination with other known antibacterial agents of similar spectrum to (1 ) enhance treatment of severe gram- negative infections covered by the spectrum of this compound or (2) add coverage in severe infections in which multiple organisms are suspected in which another agent of a different spectrum may be required in addition to this compound. Potential agents include members of the aminoglycosides, peniciiiins, cephalosporins, fluoroquinolones, macrolides, glycopeptides, lipopeptides and oxazolidsnones. The treatment can involve administering a composition having both a compound of the present invention and a second antibacterial compound or

administration of a compound of the present inventive compounds followed by or preceded by administration of a second antibacterial agent.

The foregoing may be better understood by reference to the following examples, that are presented for illustration and not to limit the scope of the inventive concepts.

VI. EXAMPLES

A. Compound synthesis

The following are abbreviations used in the examples:

ACN: Acetonitriie

AcOH: Acetic acid

aq: Aqueous

BOC: tert-butoxycarbonyl

DCM: Dichloromethane

DIBAL-H: Diisobutyla!uminium hydride

DIPEA: Diisopropylethylamine DMAP: 4-Dimethylaminopyridine

DMF: N,N-Dimethylformamide

DMSO: Dimethyl sulfoxide

EA: Ethyl acetate

Et ₂ 0: Diethy!ether

HATU: 2-(1 H-7-Azabenzotriazol-1-yi)-1 _! 1 ,3,3-tetramethyl uronium

hexafluorophosphate Methanaminium

HOBt: N-Hydroxybenzotriazoie

I PA: Isopropy! alcohol

-CPBA: meia-Chloroperoxybenzoic acid

MTBE: Methyl tert-butyl ether

NBS N-Bromosuccinimide

NMO: N-methylmorphoiine N-oxide

PCC: Pyridinium Chlorochromate

PE: Petroleum Ether

PE:EA: Petroleum Ether: Ethyl acetate

RP HPLC: Reverse phase high performance liquid chromatography

TBAF: Tetra-n-buty!ammonium fluoride

TEA: Triethyiamine

TFA: Trifluoroacetic acid

THF: Tetrah drofuran

TLC: Thin layer chromatography

Referring to the examples that follow, compounds of the present invention were characterized by high performance liquid chromatography (HPLC) using a Waters Millenium chromatography system with a 2690 Separation Module (Miiford, MA) or an Agilent 1 100 series chromatography system (Santa Clara, CA). The analytical columns were Phenomenex Luna C18(2) reversed phase, 10pm, 100 A, axia packed, 2, 0x50mm and the preparative columns were Phenomenex Luna C18(2) reversed phase, 10pm, 100 A, axia packed, 21.2x250 or 50x250mm. A gradient eiution was used, typically starting with 100% water and progressing to 100% acetonitriie over a varying lengths of time All solvents contained 0.1 % acetic acid (AcOH). Compounds were detected by ultraviolet light (UV) absorption at either 220 or 254 nm. in some instances, purity was assessed by thin layer chromatography (TLC) using glass or plastic backed silica gel plates, such as, for example, Baker-Flex Silica Gel 1 B2-F flexible sheets. TLC results were readily detected visually under ultraviolet light, or by employing well known iodine vapor and other various staining techniques

Mass spectrometry analysis was performed on one of three LCMS instruments: a Waters System. (Alliance HT HPLC and a icromass ZQ mass spectrometer; Column:

Eclipse XDB-C-18, 2.1 x50 mm; solvent system: 5-95% (or 35-95%, or 65-95% or 95-95%) acetonitriie in water with 0.05%TFA; flow rate 0.8 mL/min; molecular weight range 500-1500; cone Voltage 20 V; column temperature 40° C.) or a Hewlett Packard System (Series 1 100 HPLC; Column: Eclipse XDB-C18, 2.1 x50 mm; solvent system: 1-95% acetonitriie in water with 0.05% TFA; flow rate 0.4 mL/min; molecular weight range 150-850; cone Voltage 50 V; column temperature 30° C). or an Agilent System (Series 1 100 HPLC; Column: Waters Sunfire C18 reversed phase, 2.5 m, 100 A, 2.1x50mm; solvent system: 1-95% acetonitriie in water with 0.1 % TFA; flow rate 0.5 mL/min; molecular weight range 150-1500; cone Voltage 70 V; column temperature 35° C).

GCMS analysis was performed on a Hewlett Packard instrument (HP6890 Series gas chromatograph with a Mass Selective Detector 5973; injector volume: 1 μί; initial column temperature: 50° C; final column temperature: 250C; ramp time: 20 minutes; gas flow rate: 1 mL/min; column: 5% phenyl methyl siloxane, Model #HP 190915-443, dimensions: 30.0 mx25 mx0.25 m).

Nuclear magnetic resonance (NMR) analysis was performed with a Varian 300 MHz NMR (Palo Alto, Calif,), and a Varian Unity Enova 400 MHz NMR spectrometer (Palo Alto, Calif.). The spectral reference was either TMS or the known chemical shift of the solvent. Some compound samples were run at elevated temperatures (e.g. 75° C.) to promote increased sample solubility. Procedure 1 (C~C coupling reaction using CuC!-Cadiot): Hydroxylamine hydrochloride (0.235 mmo!, 0.06 eq) and CuCI (0.08, 0.02 eq) were dissolved in 23% aqueous n-butylamine (1 mL) and the resulting solution was cooled to 0°C. A solution of the alkyne (4.3 mmol, 1.1 eq) in 23% aqueous n-buty!amine (2 mL) was then added. The bromo-aikyne (3.92 mmo!) and hydroxylamine hydrochloride (0.235 mmoi, 0.06 eq) were dissolved in 23% aqueous n-butylamine (2 mL) and THF (3 mL), and they were slowly added to the reaction mixture. The reaction was stirred for 1 hr, followed by quenching with EtOAc and water. The organic layer was separated and washed with brine, dried over Na ₂ S0 ₄ , filtered and concentrated under reduced pressure to yield the desired coupled product.

Procedure 2 (Boc deprotection using TFA): To the Boc-protected compound (3.39 mmoi) at 0°C was added a TFA:DCM solution (9 mL, 2: 1 ) and the reaction was stirred for 1 hour. The reaction was concentrated under reduced pressure to yield a crude residue, which was azeotroped with I PA twice to yield the desired deprotected product.

Procedure 3 (Hydroxa ate formation): To a stirring solution of the ester (3.38 mmol) in IPA (4 mL) at 0°C was slowly added 50% aqueous hydroxylamine (40 eq), and the reaction was stirred overnight. The reaction was quenched with AcOH (0.121 mo!, 20 eq) or until the pH is 6. The volatiies were removed under reduced pressure, and the resulting solution was purified by RP HPLC.

Procedure 4A (formation of srnine in reductive a snation to NH e): To a stirred solution of the amine (2.37 g, 7.20 mmoi) in DMF (14.39 mL) was added DIPEA (1.885 mL, 10.79 mmol) followed by formaldehyde (37% in water) (1.071 mL, 14.39 mmol) and the reaction was stirred for 2 hours. The excess aidehyde was quenched with n-butylamine (30% in water) (2.63 g, 10.79 mmol) and stirred for one hour. The reaction mixture was diluted with water, and iyophiiized to yield the desired imine.

Procedure 4B (reduction to amine in reductive amination to NHMe): To a stirring solution of the imine (3.96 g, 1 1.60 mmoi) in THF (23.17 mL) and MeOH (2.439 mL) was added acetic acid (1.328 mL, 23.20 mmol) followed by sodium cyanoborohydride (10.94 g, 174 mmol) and the reaction was stirred for 1 hour. The reaction mixture was diluted with wafer (7 mL) and concentrated under reduced pressure to yield the amine. {S)-meihyl! 2-(4-{bromoethyn S)ben2:amido)-3-{(terf~butoxycarbony1)amino)- 3-methy!buianoaie {!NT-1}:

Ethyny!trimethylsilane (82.4 g, 0.84 mol, 1.2 equiv) was added dropwise over 10 min under a nitrogen atmosphere to a solution of methyl 4-bromobenzoate (150 g, 0.7 mol 1.0 equiv), PdCI ₂ (PPh ₃ ) ₂ (15 g, 0.021 mol, 0.03 equiv) and Cul (13 g, 0.068 mol, 0.1 equiv) in TEA (1.5 L). The reaction was stired at 90°C for 30 minutes, whereupon LCMS showed complete consumption of methyl 4-bromobenzoate. The reaction mixture was filtered and the filter cake was washed with EtOAc (5 x 500 mL). The filtrate was concentrated under reduced pressure to give a residue, which was distilled under reduced pressure to yield methyl 4-({trimethylsilyl) ethynyi) benzoate (!NT-1.2) as an off-white solid (156 g, 96%).

To a solution of methyl 4-((trimethylsiIyI) ethynyi) benzoate (156 g, 0.67 mol, 1.0 equiv) in methanol (800 mL) was added dropwise KOH/methanol (18 g/250 mL) keeping the temperature below 10°C, the mixture was allowed to warm to room temperature for 5 min. The reaction mixture was neutralized with 2 HCI. The reaction suspension was filtered to collect methyl 4-ethynyibenzoate (!NT-1 ,3) as a white solid (97 g, 90%). MS: m/z calcd for Ci ₀ H ₈ Q ₂ 160.0, found [M+H]* 161.

To a solution of methyl 4-ethynylbenzoate (50 g, 0.3125 mol, 1.0 equiv) in acetone (750 mL) was added AgN0 ₃ (5 g, 29.7 mmol 0.095 equiv) and the reaction mixture was stirred for 1 hr. NBS (61.2 g, 0.344 mol, 1.1 equiv) was added and the reaction mixture was stirred at room temperature for 20 hr, filtered and concentrated under reduced pressure. The residue was diluted in EA, and washed with iced 20% H ₂ S0 ₄ . The EA layer was washed with water and brine, dried (Na ₂ S0 ₄ ), and filtered. The filtrate was concentrated under reduced pressure to give a residue, which was recrystallized from MeOH (1 mL/4 g) to yield methyl 4- (bromoethynyl) benzoate (INT-1.4) as an off-yei!ow solid (67 g, 90%). ^' H NMR (400 MHz, CDCI ₃ ) δ 7.98 (d, J = 8.8 Hz, 2H), 7.51 (d, J = 8.8 Hz, 2H), 3.92 (s, 3H).

To a solution of methyl 4-(bromoethynyl) benzoate (67 g, 280 mmol, 1.0 equiv) in CH ₃ OH/THF/H ₂ 0=5/5/1 (1 100 mL) was added NaOH (44.84 g, 4.0 equiv) and the reaction mixture was stirred at 25°C for 3 hr. The volati!es were removed under reduced pressure and the resulting solution was neutralized with 1 N HCi to pH 3-5. 4~(bromoethynyl) benzoic acid (iNT-1.5) was collected by filtration. The filter cake was washed with water, and dried in an oven at 50°C for 5 hr (61g, 96%).

(S)-methyl 2-amino-3-(iert-butoxycarbonylamino)-3-methylbutanoate oxalate (9.34 g, 27.8 mmol) (synthesized as described in WO 2008/154642 at pages 240-6) was suspended in ethyl acetate (80 mL) and water (80 mL). While cooling in an ice bath, potassium carbonate (7.67 g, 55.5 mmol) was added and the reaction was stirred for 10 min. The aqueous layer was separated and extracted with ethyl acetate (2 x 75 mL) and the combined organic extracts were dried over sodium sulfate, and concentrated under reduced pressure to give a clear oil. To a stirring solution of 4-(bromoethynyl)benzoic acid (5.68 g, 25.2 mmol) in D F (45 mL) at 0°C was added a solution of (S)-methyl 2-amino-3-(fert-butoxycarbonylamino)-3- methyibutanoate in DMF (40 mL), followed by HATU (1 1.52 g, 30.3 mmol) and A -ethy!-W- isopropylpropan-2-amine (13.19 mL, 76 mmol) and the reaction was stirred for two hours with warming to room temperature. The reaction mixture was poured into water (300 mL) and was extracted with ethyl acetate (3 x 200 mL), The combined extracts were washed with saturated NaCI, dried over sodium sulfate and concentrated under reduced pressure to yield a crude, which was purified by flash chromatography (silica ge!/20-60% EtOAc/Hexanes) to yield the desired product, (S)-methyl 2-(4-(biOmoethynyl)benzamido)-3-((te/f-butoxycarbonyi)amino) -3- methylbutanoate {INT-1), (9.24g) as a white foam: TLC ethyl acetate/hexanes 1 : 1 R _f 0.5; MS: m/z calcd for CaoHzsBr^Os 452.09, found [M-Boc+H 353.1.

ΜΊ-2 was generated from !M!T-1 according to Procedure 2,

{S)-methy! 3~({ferf-butoxycarbonyI)amino)~2~(4-iodobenzam!do)-3~ methy!butanoate (f T-3):

To a stirring solution of iodobenzoic acid (8.62 g, 34.8 mmol) in acetonitrile (69.5 mL) was added HATU (26.4 g, 69.5 mmol), followed by TEA (4.84 mL, 34.8 mmol). INT-1 ,6 (1 1.69 g, 34.8 mmol) was then added and the reaction was stirred for 1 hr. Additional TEA (1 eq) was added and the reaction was stirred for 2.5 hr. The mixture was concentrated under reduced pressure to give a crude, which was purified by flash chromatography (silica gel/10- 50% EtOAc in hexanes) to yield \HJ-Z (1 1 .7 g, 70.7%).

Bestmann reagent:

To a solution of dimethyl 2-oxopropyiphosphonate (65.3 g, 331 mmol) in acetonitrile (150 mL) at 0°C was added K ₂ C0 ₃ (45.8 g, 331 mmol) followed by the dropwise addition of 4-methy!benzenesulfony! azide (50 g, 300 mmol) and the reaction mixture was stirred overnight. It was diluted with EA (500 mL), washed with water (3 x 300 mL), brine (3 x 100 mL), dried (Na ₂ S0 ₄ ), and filtered. The filtrate was concentrated under reduced pressure to a residue, which was dissolved in Et ₂ 0 (300 mL) and recrystailized with DCM and PE to remove 4-methylbenzenesulfonamide as a white solid byproduct several times. The remaining solution was concentrated under reduced pressure to give the Bestmann reagent as a yellow oil (43 g, 75%). H NMR (400 MHz, CDC! ₃ ) δ 2.28 (s, 3 H), 3.85 (d, J = 8.0 Hz, 6 H). G. Roth, B. Liepoid, S. u!!er and H. J. Bestmann, Synthesis 2004, 1 , 59-62.

1. {S}-N-(3-8mino-1-(hydroxyaminQ}~3-methyi-1-Qxobutan-2^!)-4- (azetidin-S-y!tuta-l^-diynyljbenzamide (1)

ferf-butyl 3-(hydroxymethyl)a∑etidine-1 -carboxy!ate (1.2)

To a stirring solution of BH THF (285 mL) at -78°C was added 1-(ferf- butoxycarbony!)azetidine-3-carboxy!ic acid (22.53 g, 1 12 mmol) in portions under a N ₂ atmosphere, and the reaction was stirred for 30 min. The reaction was then warmed to rt and stirred for 2 hr. The reaction mixture was poured into ice (500 mL), and extracted with EA (3 x 300 mL). The combined organic layers were washed with brine (3 x 100 mL), dried (Na ₂ S0 ₄ ), and filtered. The filtrate was concentrated under reduced pressure to yield compound 1.2 as clear oil (20.9 g, 100%). MS: rn/z calcd for C ₉ H ₁₇ N0 ₃ 187.1 , found [M-H] 186.1. ferf-butyl 3-formyl!azet!dme-1-carb0xy!ate (1.3) Reagent MW Eqiv. Mmo! Amount

Compound 1.2 187.24 1.0 1 12 20,96 g

!BX 280.02 2.0 224 62.69 g

EA 500 mL

To a stirring solution of ferf-butyi 3-(hydroxymethyl)azetidine-1-carboxylate (20.96 g, 1 12 mmoi) in EA (500 mL) was added IBX (62.69 g, 224 mmoi) and the reaction was refluxed overnight. The reaction was cooled to rt, PE (500 mL) was added and the reaction mixture was filtered. The filtrate was concentrated under reduced pressure to give compound 1.3 as yellow oii product (20.56 g, 99%). H NMR (400 MHz, CDCI ₃ ) δ 1.44 (s, 9 H), 3.32-3.40 (m, 1 H), 4.07-4.14 (m, 4 H), 9.85 (d, J = 2.0 Hz, 3 H). ierf-buty S-eihyny azetsdme-l -earboxyJaie (1.4)

To a stirring solution of aldehyde 1.3 (5 g, 27 mmoi) in MeOH (70 mL) at 0°C was added the Bestmann reagent (6 g, 31.2 mmoi), followed by potassium carbonate (14.92 g, 108 mmoi) and the reaction was stirred for 3 hr. The reaction mixture was then diluted with EtOAc (150 mL), filtered and concentrated under reduced pressure to give a crude, which was purified by flash chromatography (silica ge!/ 0-100% EtOAc in hexanes) to yield compound 1.4 (4.6 g, 94%). Ή NMR (400 MHz, DMSO-d ₆ ) δ 1.34 (s, 9 H), 3.22-3,23 (m, 1 H), 3.34-3.39 (m, 1 H), 3.68 (t, J = 6.8, 2 H), 4.04 (t, J = 8.4 Hz, 2 H).

3-{{4-(3~ammo-1 -methoxy-3-methy!-1 -oxobutan-2-y carbamoy!)pheny!)buta- :etsdine-1-carboxy§aie 2,2,2-tnfiuoroac

To a stirring solution of SNT-1 (12,5 g, 27.6 mrnol) in DCM (70 mL) was added TFA (80 mL) and the reaction was stirred for 10 min. The mixture was concentrated under reduced pressure from DCM (150 mL) twice to yield which was dissolved in THF (25 ml).

TEA (33.3 mL) was added followed by a solution of compound 1.4 (5 g, 27.6 mrnol) in THF (75 mL) and PdC! ₂ (PPh ₃ ) ₂ (0.48 g) and Cul (0,26 g), and the reaction was heated to 75°C for 20 min. The reaction mixture was cooled to 0°C, filtered and washed with THF. The filtrate was concentrated under reduced pressure to give a crude, which was purified by RP HPLC (6", 0.1 % TFA in water/ACN) to yield compound 1.5 (6.5 g, 42%). MS: m/z calcd for sHai NaQs 435.23, found [IVHHf 454.2.

(S)~methyl 3-amino~2-{4-(azet!dsn~3~y!buta~1 ,3~dsynyI)ben amido)-3-rnethyibutanoate bss{2,2,2-trif!uoroacetate) (1.6)

To a stirring solution of compound 1.5 (250 mg, 0.44 mmol) in DCM (2 mL) was added TFA (2 mL) and the reaction was stirred for 5 min. Reaction was concentrated under reduced pressure from DCM twice to yield compound 1.6 (370 mg), which was carried through to the next step without further purification. MS: rn/z calcd for C20H23 3O3 353.17, found [M+H 354.1 .

{S)-/^-(3~amsno-1 -(hydroxyamino)~3~m

diyny!)benzamide (1)

To a stirring solution of compound 1.6 (370 mg) in I PA (1 mL) was added hydroxylamine (50% aq, 1 mL) and the reaction was stirred at 0°C overnight. The reaction mixture was acidified with TFA, diluted with water, and purified by RP HPLC (1 ", 0.1 % TFA in water/ACN) to yield compound 1 (86 mg, 21 .6%). MS: rn/z calcd for C19H22N4O3 354.17, found [M+H] ⁺ 355.1. 4-{{1-(terf~butoxycarbony!)azet!dsn~3~y!)buta~1 ,3-diyn~1-y!)benzo!C acid (2.1)

To a stirring solution of compound 1.4 (10.1 1 g, 55.8 mmo!) in 30% aqueous n- butylamine (29.6 ml) at 0°C were added copper chloride (0.1 10 g, 1.1 16 mmol) and

hydroxy!amine hydrochloride (0.233 g, 3.35 mmol). In a separate flask, to a stirring solution of

4-(bromoethyny!)benzoic acid (12,55 g, 55.8 mmol) in 30% aqueous n-butylamine (63.4 ml) was added hydroxylamine hydrochloride (0.233 g, 3.35 mmol) and this solution was then added dropwise to the solution containing compoud 1.4, and the reaction was stirred for 1 hr. The reaction mixture was diluted with MeTHF (400 mL), and acidified to pH 1 with concentrated HCi.

The resulting solution was filtered through Celite, and the layers were separated. The aqueous layer was extracted with MeTHF (200 mL), and the combined organic layers were washed with 2M HCI (2 x 20 mL), water (200 mL), brine (200 mL), dried over Na2SC>4, filtered, and concentrated under reduced pressure to yield compound 2.1 as a white solid (16.6 g, 91 %), which was carried through to the next step without further purification. MS: m/z caicd for C ₁₉ H ₁₉ N0 ₄ 325.13, found [M+Na] ⁺ 348.2,

4-{a2etid!n-3-yibuta-1 ,3-d!yn-1-yi)ben2:o!C add (2,2)

To a stirring solution of compound 2.1 (3 g, 9.22 mmol) in DCM (30.7 ml) was added TFA (61.5 ml) and the reaction was stirred for 1 hr. The reaction was azeotroped with DCM (3 x 75 mL) and then concentrated under reduced pressure to yield compound 2.2 as a white solid, which was carried through to the next step without further purification. MS: m/z calcd for Ci ₄ Hii 0 ₂ 225.1 , found [M+Hf 226.0.

4~((1~acetyla∑etidin~3~yf)buta-1,3~d!yn~1~yf)benzoic acid (2,3)

To a stirring solution of compound 2.2 (2 g, 8.88 mmol) in water (22.2 mL) and dioxane (22.2 mL) were added Ac ₂ 0 (1.089 ml, 1 1.54 mmol) and NMO (1.30 mi, 10.21 mmol) and the reaction was stirred for 18 hr. The reaction was filtered and washed with water:dioxane (1 : 1 , 2 x 30 mL). The solids were then dried under reduced pressure to yield compound 2.3 a white solid, which was carried through to the next step without further purification.

{5)-methy 2-{4-C(1-aceiy!a;£etidin-^

methy!butarsoate (2.4)

To a stirring solution of methyl 2-amino-3-hydroxy-3-methylbutanoate (2.4, synthesized as described in WO 2008/154642 at page 247 ei seq) (2.065 g, 14.03 mmol) in

DMF (76 mL) was added K ₂ C0 ₃ (5.82 g, 42.1 mmol) and the reaction was stirred for 1 hr. Then compound 2.3 (2.5 g, 9.35 mmol) and DI PEA (2.45 mi, 14.03 mmol) were added, followed by

HATU (3.91 g, 10.29 mmoi) and the reaction was stirred for 3 hr. The reaction was diluted with ethyl acetate, washed with 1 M citric acid, aHC03 and brine, dried over a2S04 _< filtered, and concentrated under reduced pressure to yield compound 2.5 (2.18 g, 58.8%) as a brown oil. MS: m/z calcd for C ₂₂ H ₂₄ N ₂ O _s 396,2, found [M+H] ⁺ 397.2. (S)-4-{(1 -acetyl! a£etidin-3-y!)buta-1 ,3~dsyn-1 -y!)- /-{3-hydrox -1 -(hydroxyamsno)-3-methy!- 1 -oxobutan-2-yi}benzamide (2a) and (S)-4-((azetidin-3-yl)buta-1 ,3-diyn-1 -yl)-Af-(3-hydroxy- 1 -(hydroxyamsno)-3-methyM -oxobutan-2-yi)benz:am!de (2b)

To a stirring solution of compound 2,5 (3 g, 7.57 mmoi) in IPA (32.5 mL) was added hydroxy!amine (50% solution in water, 30.0 mL, 454 mmol) and the reaction was stirred for 18 hr. The reaction mixture was then concentrated under reduced pressure to a thick oil, which was diluted with AcOH (70 mL) and purified by RP HPLC (6", 0.1 % AcOH in water/ACN) to yield compound 2a (0.35 g, 1 1.2%) MS: m/z calcd for C21 H23 3O5 397.16, found [M+H 398.3 and 2b (0.51 g) MS: m/z calcd for C19H21 N3O4 355.15, found [M+Hf 356.3

3. (S)-4-{{1 ormy!azeiidin-3-y!)buta-1,3-diyn-1-y!)-N-{3-hydroxy-1- (hydroxya ino}-3- ethyS-1-Qxobutan-2-yi}benzamide (3)

To a stirring solution of compound 2b (536 mg, 1.14 mmol) in DMF (4 ml) at 0°C was added DIPEA (0.24 ml, 1.37 mmol), followed by 2,2,2-trifluoroethyi formate (0.1 1 ml, 114 mmol), and the reaction was stirred for 75 min. The reaction mixture was acidified with AcOH (0.098 ml, 1.71 mmol) and purified by RP HPLC (2", 0.1 % AcOH in ACN/water) to yield (S)-4- ((1-formyiazetidin-3-yi)buta-1 _! 3-diyn-1-y1)-W-(3-hydroxy-1-(hydroxyamjno)-3-methyi-1- oxobutan- 2-yi)benzamide (412.6 mg, 1.08 mmol, 94%). MS: m/z caicd for C> >-N ,O _b 383.1 , found [M+H] ⁺ 384.1.

4, {S ^l-^-aminoacety azetidin-S-y buia-l S-diyn-l-y -N-fS- hydrQxy-1-(hydroxyamino}-3-methy!-1-oxobiitan-2-y!}benzamide (4)

(S)-(9H-fluoren-9-y ^m © hyl (2-{3-{{4-({3-hydroxy-1 -{hydroxyamino)-3-meihyl-1 -oxobutan-2- yl)carbamoyl)phenyl)buta-1 ,3-d!y'"i- -yl)-2-oxoeihy!)carbamaie (4.1)

To a stirring solution of 2-((({9H-fluoren-9-yl)methoxy)carbonyl)amino)acetic acid (6.97 mg, 0.023 mrnol) and HATU (8.10 mg, 0.021 mrnol) in DMF (0.2 mL) was added DiPEA (9.30 μΙ_, 0.053 mrnol), and the resulting solution was added to compound 2b (10 mg, 0.021 mrnol), and the reaction was stirred overnight. The crude solution containing 4.1 was carried through to the next step. MS: m/z calcd for C36H34N4O7 634.24, found [M+H] ⁺ 635.4.

(S)-4-((1 -{2-aminoaeety!}azet!d!n~3~ l}buta~1 ,3-diyn-1 -yl}-A ~(3~hydroxy-1 -(hydroxyamino)- 3-rnethyI-1 -oxobutan-2-yl!)benzamide (4)

To a stirring solution of compound 4.1 (13.52 mg, 0.021 mrnol) in DMF (0.2 mL) was added piperidine (10.52 pL, 0.107 mrnol) and the reaction was stirred for 30 min. The reaction mixture was acidified with AcOH (8.54 pL, 0.149 mrnol) and purified by RPHPLC (1 ", 0.1 %TFA in water /ACN) to yield (S)-4-((1-(2-aminoacetyl)azetidin-3-yl)buta-1 ,3-diyn-1-yl)-W-(3- hydroxy-1-(hydroxyamino)-3-methyi-1-oxobutan-2-yi)benzamide (0.0046 g, 0.01 1 mrnol, 52.4%). MS: m/z calcd for C21 H24N4O5 412.2, found [M+H] ⁺ 413.3. 5. 4-{(1~{2~amino~3~hydmxypropanoy!)azetidiii~3~y!)buta-1,3~diy ii~1~y!)- N~({S}~3~hydroxy~1-(hydroxyamsno}-3-methyM~oxobutan~2^!}ben {5} teri-butyl-(3-hydroxy-1 -(3-((4-(((S)-3-hydroxy-1 -(hydroxyamino)-3-methy 1 -oxobistan-2- yi)carbamoy!)phenyI)buta-1 ,3~diyn~1 ~yf)a∑etidin~1 ~yI)-1 -oxopropan~2~yf)carbarnate (5.1)

To a stirring solution of 2-((iert-butoxycarbony!)amino)-3-hydroxypropanoic acid (17.34 mg, 0.084 mmo!) and HATU (32.1 mg, 0.084 mmoi) in DCM (0.2 mL) was added DIPEA (0.025 ml, 0.146 mmoi) and the resulting solution was added to compound 2b (19.5 mg, 0.047 mmoi) and the reaction was stirred for 45 min. Additional HATU, Boc-Ser and DIPEA (half the original amounts) in DMF (0.075 mL) were added and the reaction was stirred for another 45 min. Piperidine (0.023 ml, 0.235 mmo!) was added followed by 40% methylamine (100 μί). Solvent evaporation under reduced pressure gave compound 5.1 , which was carried through to the next step without further purification. MS: m/z calcd for C27H34 4O8 542.2, found [M+Na] ⁺ 565.3. 4~{{1-(2-ammo-3-hydrQxypropanoy^a;^

(hydroxyamino)-3-methyi-1-oxobutan-2-y!)benz:amide (5)

To a stirring solution of compound 5.1 (47 mg, 0.047 mmol) in DCM (0.2 mL) was added TFA (0.20 mL) and the reaction was stirred for 30 min. The reaction mixture was concentrated under reduced pressure, and purified by RP HPLC (1 " column, 0.1 % TFA in water/ACN) to yield 4-((1-(2-amino-3-hydroxypropanoyl)azetidin-3-y!)buta-1 ,3-diyn-1 -yl)-A -((S)- 3-hydroxy-1-(hydroxyarnino)-3-methyl-1-oxobutan-2-y!)benzami de (0.0072 g, 0.013 mmol, 27.7%). MS: m/z caicd for C ₂ 2H ₂₆ N ₄ 0 ₆ 442.2, found [M+H] ⁺ 443.3.

6. {S)~N~{3~hydroxy~1~(hydroxyamino)~3~methyi-1-oxobutan-2~yl)~ 4~{{1~ {2~hydroxyethy!}azetidsn~3~yl}buta~1,3~diyn~1~yl}benza sde (6)

To a stirring solution of compound 2b (27.7 mg, 0.067 mmo!) in THF (0.2 mL) was added DIPEA (10.48 pL, 0.060 mmol) foiiowed by methanol (0.1 mL). Next, 2-({tert~ butyldimethylsilyl)oxy)acetaldehyde (0.038 ml, 0.200 mmol) was added foiiowed by sodium triacetoxyborohydride and the reaction was stirred for 20 min. Methanol (0.3 mL) was added followed by water (0.3 mL) and TFA (0.3 mL, 3.89 mmol) and the reaction was stirred for 90 min. The reaction mixture was concentrated under reduced pressure, diluted with water and DMF (0.1 mL) and purified by RP HPLC (0.1 % TFA in water/ACN) to yield (S)-/V-(3~hydroxy-1- (hydroxyamino)-3-methyl-1-oxobutan-2-yl)-4-((1-(2-hydroxyeth yl)azetidin-3-yi)buta- yhbenzamide (1 1.6 mg, 0.023 mmol, 33.8%). MS: m/z caicd for C ₂₁ H _{25 3} 0 ₅ 399.2, found [M+H] ⁺ 400.2.

7. {S)~N~(3~hydmxy-1-(hydroxya !no)~3~ ethyl-1'Oxobutan'2^!)~4~{{1- methyiazet!din-3-y!)buta- 1,3-diyn- 1 -y!)benza ide {7}

To a stirring solution of compound 2b (22.7 mg, 0.055 mmol) in THF (0.2 mL) was added DIPEA (8.59 μΙ_, 0.049 mmol) followed by MeOH (0.1 ml), formaldehyde (0.012 ml, 0.164 mmol) and molecular sieves. After 5 min sodium triacetoxyborohydride (57.9 mg, 0.273 mmol) was added and the reaction was stirred for 25 min. The reaction mixture was concentrated under reduced pressure to give a residue, which was dissolved in water, acidified with AcOH and purified by RP HPLC (1 ", 0.1 % TFA in ACN/water) to yield (S)-A/-(3-hydroxy-1- (hydroxyam!no)-3-methyl-1-oxobutarv2-yl)-4-((1-methyiazetidi n-3-yl)buta-1 _l 3-diyn-1- yl)benzamide (6, 1 mg, 0.013 mmol, 23.1 %). MS: m z calcd for C20H23N3O4 369,2, found [M+H] ⁺ 370.2.

8, {S)-N-{3-amsno-1-{hydroxyamino}-3-methyl-1-Qxobutan-2-yi}-4- ((1- (oxetan~3~yl}azetidin~3~yl}biita~1,3~diyn~1~y!}benzamide (8)

{S)-methy! 2-{4-{a∞iidin-3-y!buia-1 ,3-diyn-1 -y )ben^amido)~3~{(ferf- butoxycarbony am5no)-3-methy!butanoaie (8.2)

To a stirring solution of compound 1.4 (2 g, 11.04 mmol) in DCM (20 mL) at 0°C was added TFA (20 mL) and the reaction was warmed to rt and stirred for 1 hr. The reaction mixture was concentrated under reduced pressure to yield compound 8.1 , which was carried through to the next step. Coupling of compound 8.1 to !NT-1 was carried out according to procedure 1 to yield compound 8,2, which was carried through to the next step. MS: m/z calcd for C ₂₅ H3iN ₃ 05 453.2, found [M+Hf 454.3.

(5)-i¥-(3-amino-1-(hydr0xyam!no)-3-me^^

yi)buia~1 ,3-diyn-l -yi)benzamide (8)

To a stirring solution of (S)-methyl 2-{4-(azetidin-3-yibuta-1 _! 3-diyn-1- yI)benzamido)-3-((tert-butoxycarbony!)amino)-3-methyIbutanoa te (3 g, 6.61 mmol) in MeOH (20 mL) was added oxetan-3-one (0.6 g, 8,33 mmol) followed by sodium cyanoborohydrate (0.523 g, 8.33 mmol) and the reaction was stirred. Additional NaCNBH ₃ (200 mg), and oxetan-3-one (100 g) were added and the reaction was stirred for 18 hr. Excess MeOH was removed under reduced pressure, water (20 mL) was added and the reaction was extracted with EA (2 x 200 mL). The combined organic layers were dried over Na ₂ S0 ₄ and concentrated under reduced pressure to yield compound 8.3 (3.8 g) (MS: m/z calcd for C ₂₈ H _{35 3} 0 ₆ 509.2, found [M+Hf 510.3), which was dissolved in DCM (20 mL) and TFA (6.2 mL) and stirred for 2 hr. Solvent removal under reduced pressure gave compound 8.4 as a TFA salt, which was neutralized with aqueous saturated K ₂ C0 ₃ (pH 9) and extracted with EA (3 x 100 mL), dried over Na ₂ S0 , filtered and concentrated under reduced pressure to yield compound 8.4 (3.2 g) MS: m/z calcd for C23H27 3O4 409.2, found [M+Hj ⁺ 410.2). To a stirring solution of compound 8.4 in I PA (10 mL) was added hydroxylamine (50% aq, 17 mL) and the reaction was stirred until deemed complete by LCMS. IPA was removed under reduced pressure, and the mixture was acidified with AcOH and purified by RP HPLC (2" column, 0.1 % AcOH in water/ACN) to yield (S)-/V-(3- amino-1-(hydroxyamino)-3-methyl-1-oxobutan-2-yi)-4-((1-(oxet an-3-yl)azetidin-3-yi)buta-1 ,3- diyn-1-yl)benzamide (70.2 mg, 0.136 mmoi, 12.8 %). MS: m/z calcd for C ₂ 2H _{26 4} 0 ₄ 410.2, found [M+H] ⁺ 41 1.3.

9. N-((S)-3-hydroxy-1-(hydroxya ino)-3- ethyl-1-oxohutan-2-y!)-4-(5-

{ ( 2R, 3S}-3-hydrQxypyrrQiidin-2-y!)penta- 1, 3-d iyn- 1 -y!jbenzamide ( 9}

(S)~methyl 2-(4~{bromoethynyI)ben amido)-3-hydroxy~3~methylbutanoate (9.1)

To a stirring solution of (f?)-methyi 2-amino-3-hydroxy-3-methylbutanoate, HCi

(2.441 g , 13.29 mmol) in ACN (25 mL) was added INT-1.5 (3 g, 13.33 mmol) followed by TEA

(5.56 mL, 39.9 mmol). HATU (6.06 g, 15.95 mmol) was added portionwise over 20 minutes, and the reaction was stirred for 3 hours. The reaction was concentrated under reduced pressure to a residue, which was diluted with ethyl acetate (30 mL), and solids were removed by filtration. The filtrate was washed with 1 M citric acid (2 x 30 mL), saturated aqueous θΗΟΟ

(4 x 30 mL), 1 M NaOH (30 mL), and brine (30 mL). The organic layer was concentrated under reduced pressure to yield compound 9,1 (4.49 g, 95%) as a brown solid, which was carried through to the next step without further purification. MS: m/z calcd for C ₁₅ H ₁₆ BrN0 353.03 & 355.02, found [M+H] ⁺ 354.0 & 356.0. eth S (3S)-3-hydroxy-L-pro!inate hydrochloride (9.3)

To a stirring solution of acetyl chloride (7.6 mL, 1 10 mmol) in methanol (70 mL) at 0°C was added (3S)-3-hydroxy-L-pro!ine (9.2, 10.0 g, 76.3 mmol) and the reaction was warmed to ambient temperature and was then heated at 65 °C for 6 hr. The reaction was cooled to ambient temperature and was diluted with ether (150 mL) and a white precipitate formed. The white precipitate was collected by filtration and the solids were washed with Et ₂ 0 (2 x 25 mL) and dried overnight under reduced pressure to afford compound 9.3 (12.6 g, 91 %) as a white soiid. ¹ H NMR (400 MHz, DMSO-d ₆ ) δϋ 0.87 (br. s., 2 H), 5.93 (br. s., 1 H), 4.50 (br. s. , 1 H), 4.18 (d, J = 2.07 Hz, 1 H), 3.77 (s, 3 H), 3.35 (m, 2 H), 1 .94 (m, 2 H).

1 ~terf~buiyi 2~methy! (2$, SSJ-S-hydroxypyrrolidine-i ^-dicarboxyiate (9.4) _r -'\ !siaHCO, r-

^~~~ HCI Bo¾0 HCr ^"" {

9.3 8.4

To a stirring solution of methyl (3S)-3-hydroxy-L-pro!inate hydrochloride (9.3, 12.62 g, 69.4 mmoi) in tetrahydrofu an (500 mL) and water (20 mL) at 0°C was added sodium bicarbonate (14.6 g, 0.174 mol) followed by the dropwise addition of a solution of di-feri- buty!dicarbonate (22.7 g, 0.104 rno!) in tetrahydrofuran (75 mL), and the reaction was stirred at ambient temperature overnight. The reaction was concentrated under reduced pressure and the resulting aqueous layer was extracted with ethyl acetate (3 x 50 mL), The combined organic layers were washed with H ₂ 0 and brine, dried over MgS0 ₄ , filtered and concentrated under reduced pressure to afford a crude product, which was purified by flash chromatography (silica gel/ 0 to 30% ethyl acetate in DC ) to yield compound 9.4 (16.6 g, 97%). H NMR (400 MHz, CDCIs) 50 4.48 {m, 1 H), 4.31 (s _s 0,40 H, CH), 4.19 (s, 0.60 H,CH), 3.76 (s, 3 H), 3.64 (m, 2 H), 2.16 (m, 1 H), 2.12 (m, 1 H), 1.93 (m, 1 H), 1.49 (s, 4 H), 1.43 (s, 5 H).

1 -terf-Butyl 2-methy! {2S,3S)~3~{[ferf-butyl(d!methyf)siIyi]oxy}pyrro dine-1 ,2~dicarboxy!ate (9.5)

HCT ~i imidazois TBSO* ^

^O e DMF ^^~QUe

9.4 9.5

To a stirring solution of 1 -tert-Butyl 2-methy! (2S,3S)-3-hydroxypyrroiidine-1 ,2~ dicarboxylate (9.4, 16.6 g, 67.7 mmoi) in DMF (100 mL) was added 1 H-lmidazoie (6.91 g, 102 mmoi) followed by ferf-buty!dimethylsilyl chloride (12.2 g, 81.2 mmoi) and the reaction was stirred for 2.5 hr. Additional 1 H-imidazoie (1.4 g, 20.0 mmoi) and ferf-butyldimethyisilyl chloride (1.5 g, 10.0 mmoi) were added and the reaction was stirred for an additional hour and then quenched by the addition of water. The organic product was extracted with diethyl ether (2 x 200 mL) and the combined organic layers were washed with water (2 x 100 mL), saturated sodium bicarbonate (1 x 100 rnL), and brine (1 x 100 mL) and then dried over gS0 ₄ . The filtered product was concentrated under reduced pressure to afford a clear oil, which was purified by flash chromatography (silica gel/ 0-10% ethyl acetate in hexanes) to yield 9.5 (23.4 g, 96%). ¹ H NMR (400 MHz, CDCI ₃ ) δ 4.25 (m, 1 H), 4.08 (s, 0.4 H), 3.94 (d, J = 2.28 Hz, 0.6 H), 3.64 (s, 1 H, CH ₃ ), 3.63 (s, 2 H, CH ₃ ), 3.46 (m, 2 H), 1 .91 (m, 1 H), 1 .71 (m, 1 H), 1 .37 (s, 4 H), 1.31 (s, 5 H, t-Bu rotamer), 0.78 (s, 9 H), -0.01 (s, 3 H), -0.02 (s, 3 H).

carboxylaie (9.6)

stirring solution of terf-butyl 2-methyl (2S,ZS)-3-{[tert butyl(dimethyi)siiyl3oxy}pyrrolidine-1 ,2-dicarboxylate (9.5, 23.4 g, 65.1 mmoi) in tetrahydrofuran (400 rnL) at 0°C was added in portions lithium tetrahydroborate (2.13 g, 97.6 mmoi) and the reaction was stirred with warming to rt for 18 nr. The reaction was concentrated under reduced pressure to give a residue, which was partitioned between 0.1 M aqueous HCI (150 mL) and ice/water (300 mL). 1 M HCI was added until the pH was slightly acidic and the two layers separated. The aqueous layer was extracted with chloroform and the combined organic layers were washed with water (200 mL), brine (200 mL), dried over MgS0 , filtered and concentrated under reduced pressure to give an oil, which was purified by flash chromatography (silica gei/0- 30% ethyl acetate in hexanes) to yield alcohol 9.6 (20.7 g, 96%). ^' H NMR (400 MHz, CDCI ₃ ) ϋδϋ 4.24 (m, 1 H) _« 3.92 <m _« 1 H), 3.85 (m, 2 H) _: 3,53 (br. s. _: 1 H). 3.46 (i J = 8.60 Hz, 1 H), 3.27 (m, 1 H), 1.85 (m, 1 H), 1 .69 (m, 1 H), 1.39 (s, 9 H), 0.80 (s, 9 H), -0.00 (s, 3 H), -0.01 (s, 3

H). teri-Buty! (2S,3S}-3-{[teri-butyKdsmeihyi)sHy oxy}-2-formyipyrro!idine-1 -carboxySate (9.7) NBoc ^Swarn . NBoc

TBSO TBSO ΐ

A 500-mL round bottom flask equipped with a thermometer and addition funnel under an atmosphere of nitrogen was charged with CH ₂ Ci ₂ (930 mL) and cooled to -78 °C . Oxa!y! chloride (10.6 mL, 0.125 mol) was added followed by the dropwise addition of dimethyl sulfoxide (17.7 mL, 0.250 moi) and the reaction was stirred for 10 minutes. A solution of tert- Butyi (2 3S)-3-{[lerf-butyi(dimethy!)s!iyi]oxy}-2-(hydroxymethyi)pyrr olidine-1-carboxyiate (9.6, 20.7 g, 62.5 mmol) in CH CI ₂ (200 mL) was added dropwise and the reaction was stirred for 20 minutes. Triethylamine (34.8 mL, 0.250 mol) was added and the reaction mixture was stirred for 1 nr. The reaction was quenched with wafer and the product was extracted with CH ₂ CI ₂ (3 x 200 mL). The combined organic layers were washed with water (2 x 50 mL) and brine (2 x 50 mL), dried over MgS0 ₄ , filtered and concentrated under reduced pressure to afford a crude, which was purified by flash chromatography (silica gel/ 0-30% ethyl acetate in hexanes) to yield 9.7 (17.7 g, 86%). Ή NMR (400 MHz, CDCI ₃ ) Οδ 9.49 (s, 0.4 H), 9.39 (d, J = 2.70 Hz, 0.6 H), 4.29 (m, 1 H), 4.05 (s, 0.4 H), 3.84 (t, J = 2.70 Hz, 0.6 H), 3.50 (m, 2 H), 1.80 (m, 2 H), 1.39 (m, 4 H), 1.34 (s, 5 H), 0.80 (s, 9 H), 0.01 (s, 3 H), -0.00 (s, 3 H). ierf-Buty! (2/?,3S)-3-hydroxy-2-(2-oxoeihy pyrro!id!ne-1 -carboxylate (9.9)

To a stirring solution of (methoxymethyl)triphenylphos-phonium chloride (46.0 g, 0.134 moi) in tetrahydrofuran (400 mL) at 0 °C was added in portions potassium ferf-butoxide (13.9 g, 0.124 mol) and the reaction was warmed to ambient temperature and stirred for 2 hr. The reaction was cooled to 0°C and a solution of terf-butyl (2S,3S)-3-{[ierf- butyl(dimethyl)siiyl3oxy}-2-formylpyrroiidine-1-carboxylate (9.7, 17.7 g, 53.7 mmol) in tetrahydrofuran (200 mL) was added dropwise over 20 min and the reaction mixture was stirred overnight . The reaction was quenched by pouring into ice-water and the organic product was extracted with ethyl acetate (2 x 150 mL). The combined organic layers were washed with brine, dried over MgS0 , filtered and concentrated under reduced pressure to afford a crude, which was purified by flash chromatography (silica gel/ 0-15% ethyl acetate in hexanes) to yield 9.8. To a stirring solution of enol ether 9.8 in acetonitriie (200 mL) was added 5% aqueous TFA (100 mL) and the reaction was stirred for 2 hours. The reaction was quenched by the addition of saturated aqueous sodium bicarbonate (200 mL). The volatsles were removed under reduced pressure and the product was extracted with ethyl acetate (3 x 10GmL). The combined organic layers were washed with wafer (150 ml) and brine (150 mL), dried over MgS0 ₄ , filtered and concentrated under reduced pressure to afford a crude, which was purified by flash chromatography (silica gei/0-30% ethyl acetate in hexanes) to yield compound 9.9 (7.1 1 g, 58%) ¹ H NMR (400 MHz, CDCi ₃ )G δ 9.72 is, 1 H) _« 4.06 (m, 1 H), 4.01 (m, 1 H , 3.52 (m, 1 H), 3.33 (m, 1 H), 3.01 (d, J = 18.45 Hz, 0.5 H), 2.79 (d, J = 18.87 Hz, 0.5 H), 2.37 (m, 1 H), 2.10 (br. s. , 1 H), 1 .96 (m, 1 H), 183 (m, 1 H), 1 .38 (s, 9 H). ferf-Butyl (2 3S)-3-hydroxy-2-prop-2-yn-1 -y pyrrolidine-1 -carboxyiaie (9.10)

To a stirring solution of dimethyl 2-oxopropylphosphon-ate (6.0 mL, 43 mmol) in acetonitriie (30 mL) at 0°C was added potassium carbonate (12 g, 86 mmo!) and the reaction was stirred for 5 minutes. A solution of 4-methyibenzenesuifony! azide (8.5 g, 43 mmol) in acetonitriie (20 mL) was added dropwise over 10 min and the reaction was warmed to room temperature and allowed to stir for 2 hr. The reaction was cooled in a water bath and a solution of ferf-butyl (2R, 3S)-3-hydroxy-2-(2-oxoethyl)pyrrolidine-1 -carboxylate (9.9, 6.60 g, 28.8 mmol) in CH ₃ OH (500 mL) was added dropwise over 20 min, and the reaction was stirred overnight. The reaction was quenched by the addition of water, voiatiies were removed under reduced pressure and the reaction mixture was partitioned between ethyl acetate (150 mL) and water (100 mL). The product was extracted with ethyl acetate (2 x 100 mL) and the combined organic layers were washed with water and brine, dried over MgS0 ₄ , filtered and concentrated under reduced pressure to afford a crude, which was purified by flash chromatography (0-20% ethyl acetate in CH ₂ CI ₂ ) to yield compound 9,10 (5.6 g, 86%). H N R (400 MHz, CDCi ₃ ) δ 4,40 (br. s„ 1 H), 3.76 (m, 1 H), 3.57 (m, 1 H), 3.44 (m, 1 H), 2.75 (d, J = 16.59 Hz, 1 H), 2.64 (d, J = 17.00 Hz, 1 H), 2.24 (m, 1 H), 2.15 (m, 1 H), 2.02 (m, 1 H), 1 ,88 (m, 1 H), 1 .75 (m, 1 H), 1 .47 (s, 9 H).

{2 ,3S)~terf~butyi 3~hydroxy~2~fS-{4-(((S)-3-hydroxy~1 ~meihoxy-3-rnethyl-1 -oxobutan-2- y^carbamoy!lpheny^penta^^-diyn-l -yllpyrroydine-l -carboxySate (9.11)

To a stirring solution of hydroxylamine hydrochloride (2.68 mg, 0.039 mmol) and copper chloride (1.27 mg, 0.013 mmol) in 23% n-buty!amine/water (0.6 mL) at 0°C was added a solution of (2f?,3SHerf-butyl 3-hydroxy-2-(prop-2-yn-1-y!)pyrro!idine-1-carboxy!ate (0.15 g, 0.675 mmol) in 23% n-butylamine/water (1.2 mL), followed by a solution containing (S)-methyl 2-(4-(bromoethynyi)benzamido)-3-hydroxy-3-methy!butanoate (0.2 g, 0.565 mmol) and hydroxylamine hydrochloride (2.68 mg, 0.039 mmol) in MeOH (1 mL) and 23% n- butylamine/water (0.6 mL), and the reaction was stirred for 1.5 hr at 0°C. The reaction mixture was partioned between EtOAc and water/brine (1 : 1 ). The organic layer was dried over sodium sulfate, filtered and concentrated under reduced pressure to yield compound 9.11 (313 mg, 98%) as a green foam, which was carried through to the next step without further purification. MS: m/z calcd for C ₂₇ H ₃ 4N ₂ 0 ₇ 498.2, found [M-s-H] ⁴ 499.3.

(21?, 3 SHeri-b uiy 3 -h yd roxy-2 -{5 -( ^

oxobutan-2-y!)carbamoyI)phenyf)penta-2,4~diyn~1 ~yI)pyrro!idine-1 -carboxy!ate (9.12)

To a stirring solution of compound 9.11 (313 mg, 0.628 mmol) in I PA (2 mL) at 0°C was added 50% hydroxylamine in water (2.074 ml, 31.4 mmol) and the reaction was stirred at 5-10°C overnight. The IPA was removed under reduced pressure and the product was extracted with MeTHF. The organic layer was washed with 1 M citric acid (2 x) and brine, then dried over sodium sulfate, filtered and concentrated under reduced pressure to yield compound 9.12 (494 mg) as a yellow oil, which was carried through to the next step without further purification. MS: m/z calcd for C ₂₆ H ₃₃ N ₃ G ₇ 499.2, found [M-Boc+H]* 400.2.

M-{{S}-3*ydroxy-1 hydroxyamino} ^» 3 ^» methyi-1 -oxobuian-2-y!} ^» 4-(5-{(2 ? _s 3S)-3- hydroxypyrroHdin-2-yl)penta-1,3~diyn-1-y!)benzamide (9)

To a stirring solution of compound 9.12 (494 mg, 0,628 mmol) in DCM (1 mL) was added TFA (1 mL) and the reaction was stirred for 1 hr. The reaction was concentrated under reduced pressure to give a crude, which was dissolved in water and purified by RP HPLC (2", 0,1%TFA in water/ACN) to yield A/-((S)-3-hydroxy-1-(hydroxyamino)-3-methyi-1-oxobutan-2- yI)-4-(5-((2 ?,3S)-3-hydroxypyrro!idin-2-y!)penta-1 _> 3-diyn-1-yl)benzamide (42.5 rng, 0.082 mmoi, 13,0%). MS: m/z calcd for C21H25N3O5399.2, found [M+Hf 400.3,

10. {S)-4-((1-aGety!azetidsn-3-y!)buta-1,3-dsyny!)-N-{3-amino-1- (hydmxyairssno}-3- ethyl~1~oxobutan~2~yl}benza ide ^,Ζ-ί ίίυοίθθθθίθίθ (10)

{S)-?eff -but l 3^(4-{3-{{(9H-f!uoren-9-y!)methoxy}carbonyiamino)-1-rnethoxy -3-rneihyl-1- oxobutan-2-yicarbamoy!)phenyS)buta-1,3-diyny!)a.:et!d!ne-1-c arboxy!ate (10,1)

Reactant W Eq Moles Sample Mass Vol d

(mmol) (rug) (mL) (g/ml)

D!PEA 129,24 3.3 1.249 161 0.218 0.74

To a stirring solution of compound 1.6 (220 mg, 0.38 mmol) in MeOH (2 mL) was added D!PEA (0.22 mL) and the reaction was cooled in an Ice/acetone bath. Boc-ONB (95 mg, 0.34 mmol) was added and the reaction was stirred for 2 hr. Additional Boc-ONB (7 mg) was added and the reaction was stirred for 15 min. Fmoc-OSu (166 mg, 0.49 mmol) was added and the reaction was stirred overnight. Additional Fmoc-OSu (17 mg) was added and the reaction was stirred for 2 days. N,N-dimeihylaminopropyiamine (40 μί) was added and the reaction was stirred for 5 min at rt. The reaction mixture was partitioned between EtOAc and 1 M citric acid, and the organic layer was washed with 1 M citric acid, saturated sodium bicarbonate, brine, dried over magnesium suifate, and concentrated under reduced pressure to yield compound 10.1 (340 mg), which was carried through to the next step without further purification. MS: m/z calcd for C40H41 N3O7 675.29, found [M+H] ^4' 676.3.

CS)-methy!-3-CCC9H-fluoren-9-y!}meth^

d5ynyi)benzam!do)-3-methy§buianoate 2,2,2-irifSuoroacetate (10,2}

To a stirring solution of compound 10.1 (340 mg, 0.50 mmol) in DCM (4 mL) was added TFA (4 mL) and the reaction was stirred at rt for 10 min. The reaction was concentrated under reduced pressure from DCM twice to give a residue, which was lyophilized from water/ACN to yield compound 10.2 (305 mg, 88%), which was carried through to the next step without further purification. CS)-methy 2~C4~{f1 -aceiy!azet! !^

methy!butanoate 2,2,2-tnf!uoroaeetate (10.3)

To a stirring solution of compound 10.2 (280 mg, 0.41 mmo!) in pyridine (0.7 mL) was added acetic anhydride (0.1 mL) and the reaction was stirred for 30 min. eOH (5 mL) was added and the reaction was stirred for 30 min. Pyrrolidine (1 mL) was added and the reaction was stirred for 3.5 hr. The reaction mixture was acidified with TFA and purified by RP HPLC (2" column, 0.1 % TFA in water/ACN) to yield compound 10.3 (190 mg). MS: m/z calcd for C22H25N3O4 395.18, found [M+H]' 396.2.

(§)-4-{{1 -acetyl! as3iidin-3-y!)buta-1 ,3-dsyny!)- f-f3-arriino-1 -(hydroxyamino)-3-methy1-1- oxobutan-2-yi)benzamide 2,2,2-trifiuoroacetate (10)

To a stirring solution of compound 10.3 (3.5 g, 6.87 mmol) in IPA (10 mL) at 0°C was added hydroxylamine (50% aq., 2 mL) and the reaction was stirred at 5-10°C overnight. The reaction mixture was acidified with TFA, diluted with water and purified by RP HPLC (2", 0.1 % TFA in water/ACN) to yield compound 10. MS: m/z calcd for C21 H24N4O4 396.18, found [M+H] ⁺ 397.2.

11. (S 3-({4~(3~amino~1~{hydroKyamino)~3~methyi-1~oxobutan~2~ ylGarbamoyl)phenyi)buta-1,3~d!ynyl)~N-methyiamtid!ne-1-GarbO Kamide (11) (S)-methy1-3-{{(9H-f!uoren-9-y!)meihoxy)carbony!aniino)~3~ni ethy!-2-f4-{{1 - (meihy!carbamoyS)azeiidin-3-y!)buia-1 ,3-d!yny!)benz:amido)buianoaie (11.1 )

To a stirring solution of compound 10.2 (10 mg, 0.014 mmol) in pyridine (100 pL was added methylcarbamic chloride (13.6 mg, 0.145 mmol) and the reaction was stirred for 1 hr. MeOH (500 pL) was added and the reaction was stirred overnight. The reaction mixture was partitioned between 1 M citric acid and ethyl acetate. The organics were washed with saturated sodium bicarbonate, brine, dried over magnesium sulfate and concentrated under reduced pressure to yield compound 11.1 , which was carried through to the next step without further purification. MS: m/z calcd for C37H38 4G6 632.26, found [M+H 633.3.

(S)-3-((4-(3-amino-1 ^hydroxyam5no)-3-methy 1 -oxobuian-2-y!carbamoy!)pheny buta-1 _s 3- dsynyS)-iV-!Tiethy^a2eisdsne-1 -carboxa!Ti!de (11)

To a stirring solution of compound 11.1 (10 mg, 0.016 mmol) in I PA (300 μ!_) was added hydroxy!amine (50% aqueous, 300 pL) and the reaction was stirred at 0°C for 4 hr. Pyrrolidine (50 μϋ was added and the reaction was stirred for 3 hr at 0°C and at 5-10°C overnight. The reaction mixture was acidifed with TFA, diluted with MeOH and purified by RP HPLC (1 " column, 0.1 % TFA in water/ACN) to yield compound 11. MS: m/z calcd for C21 H25N5O4 41 1.2, found [M+Hf 412.2.

12. (8)-3-({4-(3~amino-1-(hydrQxyarrsinQ)~3~rrsethy!-1~oxobutan~ 2~ y!carbamoy!)pheny1)buta-1,3~diyny!)azetidine~1-carboxa ide (12)

(S)-methy! 3-({{9H-fiuoren-9-yi)meihoxy)carbonyiam!no}-2-(4-{{1 -carbamoyla-eetidin-3- y buta-1 ,3~diynyl)ben∑amido)-3-methylbutanoate (12.1)

To a stirring solution of compound 10.2 (10 mg, 0.014 mmol) in THF (100 μ[_) was added DIPEA (10 μ[_, 0.058 mmol) followed by isocyanatotrimethy!si!ane (19.27 μΐ_, 0.145 mmol) and the reaction was stirred for 1 hr. MeOH (500 pL) was added and the reaction was stirred overnight. The reaction mixture was partitioned between 1 M citric acid and ethyl acetate. The organics were washed with saturated sodium bicarbonate, brine, dried over magnesium sulfate and concentrated under reduced pressure to yield compound 12.1 , which was carried through to the next step without further purification. MS: rn/z calcd for C ₃₆ H ₃ 4 ₄ 0 ₆ 618.25, found [M+H]* 619.3. (S)-3-{(4~{3-aniino~1 ~{hydroxya mo)-3-meth !-1 -oxobutan-2- diyny a etidine-l-carboxamsde (12)

To a stirring solution of compound 12.1 (10 mg, 0.016 mmol) in IPA (300 μ!_) was added hydroxylamine (50% aqueous, 300 μ[_) and the reaction was stirred at 0°C for 4 hr. Pyrrolidine (50 pL) was added and the reaction was stirred for 3 hr at 0°C and at 5-10°C overnight. The reaction was acidified with TFA, diluted with MeOH and purified by RP HPLC (1 " column, 0.1 % TFA in water/ACN) to yield compound 12. MS: m/z calcd for C20H23N5O4 397.2, found [M+H] ⁺ 398.2.

13. (S)-4~({1-acetyiazet!dsn~3^l)buta-1,3-dsynyl)~N-(3-(dime {hydrQxyaminQ)-3-methyi-1-QXQbiitan-2-yl}benzamide (13)

To a stirring solution of compound 10 (300 mg, 0.59 mmol) in D F (3 mL) was added paraformaldehyde (176 mg, 5.88 mmol) followed by DIPEA (0.20 mL, 1.17 mmol) and the reaction was stirred for 6 hr. Sodium cyanoborohydride (1 1 1 mg, 1.76 mmol) was added, followed by methanol (3 mL) and then acetic acid (0.1 mL) and the reaction was stirred overnight. Additional sodium cyanoborohydride (1 10 mg) and TFA (91 L) were added and the reaction was stirred for 3 days. Additional sodium cyanoborohydride and TFA were added and the reaction was stirred for 2 days. The mixture was purified by RP HPLC (2" column, 0.1 % TFA in water/ACN) to yield compound 13 (262 mg, 81 %). MS: m/z calcd for C ₂ 3H _{28 4} 04 424.2, found [M+H] ^4' 425.2.

14. (S)'N-(3-amsnO'1-(hydroxyam ao}~3~methy!~1~oxobutan~2^!}-4~{(1^ hydroxyacety!)azetidin-3-y!)but8--1 _! 3-c!iyn-1-yl)benzamidle {14}

Reactant MW Eq Moles Sample Mass Vol Density

(rnfTioi) (mg) (ul) (g/rrsl)

Compound 1 354.403 1 ,000 1.806 640

DIPEA 129.243 .2 0.361 46.7 63.1 0.74

Acetoxyacetyl 136.534 .7 1 ,264 173 136 1.27 chloride

25 wt%Sodium 31.034 2.5 4,52 976 1027 .95 methoxide

AcOH 60.052 5 9.03 542 517 1.049

To a stirring solution of compound 1 (640 mg, 1.81 mmol) in DMF (4.5 mL) and water ( 100 pL) at 0°C was added DIPEA (63 μ!_) followed by acetoxyacetyl chloride (136 μΙ_, 1.26 mmol) and the reaction was stirred for 45 min. Sodium methoxide (25% in MeOH, 1 mL) was added and the reaction was stirred for 2 days. The reaction mixture was acidified with AcOH (517 pL), diluted with water and purified by RP HPLC (2", 0.1 % TFA in water/ACN) to yield compound 14 (185 mg, 25%). MS: m/z calcd for C 1 H24 4O5 412.2, found [M+H] ⁺ 413.0.

15. (S)-N-(3-ammo-1-(hydroxyammo)-3-methyl-1-oxohutan-2^ hydroxyethyi)azetidin~3~y!)buta- 1, 3~diyn~1~y!)benzamide (15)

1 -{2-{(ierf-butyid5methyisi!y!)oxy)ethyi)-3-eihyny!a etidine (15.1)

To a stirring solution of compound 8.1 (800 mg, 9.86 mmol) in DMF (49 mL) was added 2-((trimethylsilyl)oxy)acetaldehyde (1.3 g, 9.86 mmol) and the reaction was stirred for 2 hr. Sodium cyano borohydride (620 mg, 9.86 mmol) was added and the progress of the reaction was monitored by LCMS. The reaction was partitioned between water and EtOAc, the organic layer was washed with water (3 x ) and brine (2 x), concentrated under reduced pressure to give a crude, which was purified by RP HPLC (0.1 % AcOH water and ACN) to yield compound 15,1 (61 mg). MS: m/z calcd for C ₁₀ H ₁₉ NOSi 239.2, found [M+H] ⁺ 240.1.

{S)-methy! 3-{(teri-butoxycarbony1)am!no)-2-{4-({1-(2-{{ferf- buiy!dimethySsi!yi)oxy)eihy!)azetidin-3-y!)buia-1 ,3-diyn-1-y!)benzamido)-3- meihylbutanoate (15.2)

To a stirring solution of CuCI (16 mg) in 30% n-butylamine (1 : 1 THF:water, 240 μΙ_) was added hydroxylamine (50% aqueous, 18 μ!_) and the solution was cooled in an ice/MeOH bath. Next a solution of 3-ethynyl-1-(2-((trimethy!si!y!)oxy)ethyl)azetidine (57 mg, 0.24 mmol) in 30% n-buty!amine (1 : 1 THF:water, 540 μί) was added, followed by the dropwise addition (10 min) of a cold solution of INT-1 (108 mg, 0.24 mmol) and hydroxylamine (50% aqueous, 9 μ[_) in 30% n-butylamine (1 :1 THF: water, 595 μ!_), and the reacison was stirred overnight. The reaction mixture was diluted with EtOAc (20 mL), washed with 1 M citric acid (20 mL), saturated aqueous bicarbonate (20 mL), brine (2 x 10 mL), dried over Na ₂ S0 ₄ and concentrated under reduced pressure to yield compound 15.2 (130 mg), which was carried through to the next step without further purification. MS: m/z calcd for C ₃₃ H _{49 3} O ₆ S! 61 1.3, found [M+H] ⁺ 612.5.

(S)-methyl 3-amino-2-{4-((1~{2~hydroxyethy ^

methyibutanoate (15.3)

To a stirring solution of compound 15.2 (130 mg, 0.21 mmo!) in DCM (660 μΙ_) was added TFA (2 mL) followed by water (383 pL) and the reaction was stirred for 2 hr. The reaction mixture was concentrated under reduced pressure, azeotroped with IPA (2 x 10 mL) to give an oil, which was diluted with saturated aqueous NaHC0 ₃ (1 mL) and water (200 L) and extracted with eTHF, The organic layer was dried over sodium sulfate and concentrated under reduced pressure to yield compound 15,3 as an oil. MS: m/z caicd for C22H27N3O4 397.2, found [M+H] ⁺ 398.3.

{S)-^(3~am!no-1-(hydroxyamino)~3~methyl~1~oxobutan~2-yi)~ 4~(f1-{2- hydroxyeihyS)a^etsdin-3-yS)buta-1,3-diyn-1-yS)ben2:amide (15)

To a stirring solution of compound 15,3 (80 mg, 0.20 mmol) in IPA (750 μί) cooled in an ice/MeOH bath, was added cold hydroxylamine (50% aqueous, 247 pL) and the reaction was stirred overnight. Additional hydroxylamine (248 pL) was added and the reaction was stirred for 4 days. The reaction mixture was neutralized with AcOH (461 pL), IPA was removed under reduced pressure to give a crude, which was purified b RP HPLC (1 ", 0.1 % AcOH in water/ACN) to yield compound 15 (15 mg, 17.5%). MS: m/z caicd for C21 H26N4O4 398.2, found [M+H] ⁺ 399.3.

16. (S)~N~{3~amino~1~{hydrQxyaminQ)-3-methy!-1"QXQbutari"2-y!)-4 -((1~ formyiazetidin~3-yi)biita~1,3-diyn-1-yl)benzairiiide (18)

To a stirring solution of compound 1 (1.41 g, 3.40 mmol) in DMF (6.8 mL) and THF (6.8 mL) at 0°C was added 2,2,2-trifluoroethyl formate (536 mg, 4.19 mmol), followed by DIPEA (297 L, 1.70 mmol) and the reaction progress was followed by LCMS. Excess formate was quenched by N,N-Dimethy!-1 ,3-propanediamine (0.2 eq). After solvent evaporation under reduced pressure, the mixture was diluted with water (10 mL) and purified by RP HPLC (2", 0.1 % AcOH in water/ACN) to yield compound 16 (830 mg, 53.5%). MS: m/z calcd for C ₂ oH _{22 4} 0 ₄ 382.2, found [M+H] ⁺ 383.1.

17. {S)~4~{(14ormy!azetidin-3-y!)buta-1,3~d!yn-1^!)~N-(1^yd 3-methy!'3~{methy!a ino}-1-Qxobutan~2-y!)benza ide {17}

{S)"4-({1 -formy!a2etid!n~3~yl!)buta^

{meihyleneam5no)-1 -oxobutan-2-yi}benzamide (17.1)

A stirring soiution of compound 16 (1.3g, 3.12 mmol) in DMF (1 1 mL) and DiPEA (544 pL, 3.12 mmol) at 0°C was treated with 2,2,2-trifluoroetbyl formate (379 mg, 2.96 mmol). After 30 min the reaction was treated with formaldehyde (30% in water, 736 pL, 9.35mmol) and allowed to stir overnight at rt. The reaction mixture was quenched with n-butyiamine and the imine was purified by RP HPLC (2", 0.25 mM NH ₄ OH in water/ACN) to yield compound 17.1 (500 mg, 41 %). MS: m/z. calcd for C2<H ₂₂ N ₄ 0 ₄ 394.2, found [M+H] ⁺ 395.1.

(S)-4-({1 -formylazetidin-3-y§)kuta-1 ,3-diyn-l -yi)-W-(1 -{hydroxyamsno)-3-methy -3- {methylamino)~1 ~oxobutan~2~yl)benzamsde (1?)

A stirring slurry of 17.1 (500 mg, 1.27 mmol) in DMF (9 mL) was treated with NaCNBH.3 (380 mg, 6.07 mmol) at room temperature for 2hr. The reaction was then heated at 30°C for 14 hr with the progress of the reaction monitored by LCMS. The reaction solution was purified by RP HPLC (0.1 % TFA in water/ACN) to yield compound 17 (397 mg, 79%). MS: m/z calcd for Ο^Η,, ,Ο. 396.2, found [M+H] ⁺ 397.1

18. (S)-4-({1-acetyiazet!din-3-y!)buta-1 ,3-diyn-l -y!)-N-(1-{hydroxyamino)- 3-methy!-3-{methyiamino}-1-oxotutan-2-yi)benza !de (18)

Reactant MW Eq Moles Sample Vol Molarity Density %

(mmo!) Mass (ul) (molar) (g/rni) Wt

(mg) (%)

(S)-4-((1 -acety!azetidin-3- 396.440 1 .000 2.510 995

yl)buta-1 ,3-diyn-1 -yl)-/V-(3- amino-1 -(hydroxyamino)-3- methy!-1 -oxobutan-2- yl)benzamide

Formaldehyde 30.026 24.62 61 .8 5014 4600 1 .09 37

Sodium cyanoborohydride 39.853 40 100 6309

To a stirring solution of compund 16 (995 mg, 2.51 mmol) in THF (4.6 mL) was added formaldehyde (37% aqueous, 4.6 mL) and the reaction was stirred for 90 min. Cone. NH OH (20 mL) was added and the reaction was concentrated under reduced pressure to give a crude, which was dissolved in DMF (1 1 mL), acidified with AcOH (1.5 mL), and treated with NaCNBH ₃ (6.3 g) and the reaction was stirred for 1 nr. The reaction mixture was purified by RP HPLC (2", 0.1 % AcOH in water/ ACN) to yield compound 18 (295 mg). MS: m/z caicd for C ₂₂ H ₂₆ N ₄ 0 ₄ 410.2, found [M+Hf 41 1.2.

19. (S}~4~{{1-acetyl·3-hydroxyazetϊdin-3-yl)buta-1,3~dίyn-1-y l)~N-(3-amίno~ 1 ~{hydmxya snQ)~3~ ethy!-1-Qxobuian-2-y!)benza sde (19)

teri-buty^ 3-hydroxy-3-{{tnmethy!s^y eihynyl}a-eetidine-1-carboxyiaie (19.2)

To a stirring solution of etbynyltrimethylsslane (3.22 mL, 22.8 mmol) in THF (12 rnL) cooled in a dry ice/acetone bath under a nitrogen atmosphere was added r?~BuLi (3 min) (2.5 M , 9 mL) and the reaction was stirred for 5 min. A solution of lerf-butyl 3-oxoazetidine-1- carboxy!ate (0.9 g, 5.26 mmol) in THF (5 mL) was then added and the reaction was allowed to warm to rt over 2 hr. The reaction was quenched with water (10 mL) and saturated aqueous NH ₄ CI (75 ml) and diluted with Et ₂ 0 (50 mL). The organic layer was washed with saturated aqueous NH ₄ CI (2 x), dried over Na ₂ S0 and concentrated under reduced pressure to give a crude, which was purified by flash chromatography (silica gel/0-100% EtOAc/hexanes) to yield compound 19,2 (500 mg, 35.3%). MS: m/z calcd for C ₁₃ H ₂₃ N0 ₃ Si 269.1 , found [M+Na] ⁺ 292.1. terf-buty 3-eihyny ~3~hydroxya^etsdme-1 -carboxy1ate (19.3)

To a stirring solution of compound 19.2 (500 mg, 1 .86 mmoi) in MeOH (14 mL) was added K ₂ C0 ₃ (20 mg) and the reaction was stirred for 5 hr. Solvent evaporation under reduced pressure gave a crude, which was purified by flash chromatography (silica gel) to yield compound 19.3 (318 mg, 87%). MS: m/z calcd for C, ₀ H _{! 5} NO ₃ 197.1 , found [M+Na] ⁺ 220.0.

{S)-ieri-butyi 3- (4-({3-((ieri-bisioxycarbony!)amino)-1 -met hoxy-3-meihyl-1 -oxobutan-2- y^)carbamoyi)pheny!)buta-1 ,3-diyn-1 -yS)-3-hydroxyazeisdine-1 -carboxy ate (19.4)

To a stirring solution of CuCI (10 mg) in 30% r?-butylamine/water (1 : 1 waterTHF, 3 mL) and hydroxylamine (50% aqueous, 18 μί), cooled in an ice/MeOH bath was added a cold solution of compound 19.3 (300 mg, 1.52 mmoi) in 30% n-buty!amine/water (1 : 1 water/THF, 7.5 mL). Next a solution of INT-1 (1 .54 g, 2.28 mmoi) and hydroxylamine (50% aqueous, 18 pL) in 30% n-buty!amine (1 : 1 water: DMF, 7.5 mL) was added dropwise and the reaction was stirred for 15 min, and its progress was monitored by LCMS. The reaction mixture was diluted with EtOAc (100 mL), washed with 1 M citric acid (200 mL), saturated aqueous NaHC0 ₃ (100 mL), brine (100 mL), dried over sodium sulfate, filtered and concentrated under reduced pressure to yield compound 19.4 (500 mg), which was carried through to the next step without further purification. MS: m/z calcd for C ₃ oH39 ₃ 0 ₈ 569.3, found [M-s-Naf 592.4.

(S)-methy! 3-amsno-2-{4-C(3 iydroxya£etsdm-3-y!)buta-1 ,3-dsyn-1 -y!)benzamsdo)-3- methy!butarsoate dsacetaie (19,5)

To a stirring solution of compound 19.4 (600 mg, 1.05 mmol) in DCM (1 mL) at 0°C was added TFA (3 mL), and the reaction was stirred for 30 min. The mixture was concentrated under reduced pressure to give a crude, which was purified by RP HPLC to yieid compound 19.5 (145 mg, 56%). MS: m/z calcd for C20H23N3O4 369.2, found [M+Hf 370.1.

(S)-AH3~am!no-1 -(hydroxyam!nQ)~3-met^

yH)buta-1 ,3-diyn-l -yS)benzamide (19,6)

To a stirring solution of compound 19.5 (275 mg, 0.74 mmol) in IPA (3.7 mL) was added hydroxy!amine (50% aq, 931 L) and the reaction was stirred overnight. The reaction mixture was acidified with AcOH and concentrated under reduced pressure to yield compound 19.6. MS: m/z calcd for C ₁₉ H ₂₂ N ₄ 0 ₄ 370.2, found [M+Hf 371.2.

{S)-4-({1 -acety 3-hydroxyaz:et!dm-3-y!)buia-1,3-dsyn-1 -yi)-i¥-{3-amino-1 -{hydroxyam!no}-3- methyi~1 ~oxobutan~2~y!)benzamsde (19)

To a stirring solution of compound 19.6 (25 mg, 0.058 mmol) in D F (800 μί) was added DIPEA (30 μΙ_), followed by AcOH (0.66 pL) and PyBOP (36.3 mg, 0.07 mmol) and the reaction was stirred for 1 hr. The reaction mixture was diluted with water and purified by RP HPLC to yield compound 19 (1 1 mg, 45.9%). MS: m/z calcd for C21 H24N4O5 412.17, found [M+H] ^4' 413.2.

20. N-((2S _s 3R)-3-amsno~1^ydrQxyamino)-1-oxobutan-2-y!)-4-((1- fonny!azetidin~3-y!)bs ta~1,3"diyn-1-y!)benza ide (20)

{2S,ZR)- et yl 2~(4~(bromoethynyl)benzam!do)-3-((feri~butoxycarbonyi)am!no) butanoate (20.2)

To a stirring solution of 4-(bromoethynyl)benzoic acid (21 .7 g, 96 mmol) in acetonitri!e (321 mL) was added compound 20.1 (synthesized as described in WO 2008/154642 at page 236 ei seq) (25.8g, 96 mmol) followed by TEA (33.6 mL, 241 mmol) and the reaction was cooled to 0°C. HATU (40.3 g, 106 mmol) was added and the reaction was stirred while warming to rt for 1 hr. The reaction was concentrated under reduced pressure to approximately 15 mL and was diluted with EtOAc (75 mL) and washed with 1 M citric acid (25 mL). The organic layer was washed with saturated aqueous NaHC0 ₃ /water (45 mL/20 mL), brine (40 mL), and concentrated under reduced pressure to yield a crude, which was purified by flash chromatography (silica gel/10-40% EtOAc in hexanes) to yield compound 20.2 (35 g, 83%). MS: m/z calcd for C ₁₉ H ₂₃ BrN ₂ 0 ₅ 438.07 & 440.07, found [M+H] ⁺ 439.0 & 441 .0.

ierf-butyS 3-C(4-{({2S,3i?)-3-({fer^butoxycarbony ammG)-1 -meihoxy-1 -oxobutan-2- y^)carbamoyi)phenyl!)buta-1 ,3-diyn-1 -yH)azeiidine-1 -carboxy^ate {20,3}

To a stirring solution of CuC! (15mg) in 30% n-buiylamine (1 : 1 H ₂ 0 THF, 2.6 mL) and hydroxyiamine (50% aqueous, 18 pL) cooled in an ice/MeOH bath, was added a cold solution of ferf-butyl 3-ethyny!azetidine-1 -carboxylate (0.54 g, 2.98 mmol) in 30% n-butylamine (1 :1 H ₂ 0/THF, 2.6 mL). A solution of (2S,3R)~rnefhyl 2-(4-(bromoethynyl)benzamido)-3-((ierf- butoxycarbonyi)amino)butanoate (131 g, 2.98 mmo!) in 30% n-butylamine (1 : 1 H ₂ 0/THF, 10.5 mL) and hydroxylamine (50% aqueous, 18 μί) was then added dropwise over 30 min, and the reaction was stirred for 15 min. The reaction mixture was diluted with EtOAc (100 mL), and the organic layer was washed with 1 M citric acid (96 mL), saturated aqueous NaHC0 ₃ , brine, dried over sodium sulfate, filtered and concentrated under reduced pressure to yield compound 20.3 (1.7 g), which was carried through to the next step without further purification. MS: m/z caicd for C29H37N3O7 539.3, found [M+Hf 540.3.

{2S,3f?)~methy! 3-amino~2~(4-(a∑et!din~3~yf uta-1 ,3~diyn~1 ~y!)benzamido)butanoate (20.4)

To a stirring solution of compound 20.3 (1.7 g) in DCM (6 mL) at 0°C was added TFA (6 mL) and the reaction was stirred for 1 hr. The reaction was concentrated under reduced pressure from DCM twice to yieid compound 20.4, which was carried through to the next step without further purification. MS: m/z calcd for C ₁ 9H21 3O3 339.2, found [M+Hf 340.2.

N-{{2S,3 )-3-aminG-Hhy rGxyamino}-1-oxo

y benzamsde (20.5)

To a stirring solution of compound 20.4 (1.7 g) in I PA (5 mL) at 0°C was added hydroxylamine (50%, 3.13 mL) and the reaction was stirred at 5-10°C overnight. The reaction mixture was acidified with AcOH and purified by RP HPLC (2") to yield compound 20.5 (654 mg, 72,5%). MS: m/z calcd for C ₁₈ H ₂ o ₄ 0 ₃ 340.15, found [M+Hf 341.2. -C^SjSRI-S-amino-l-Ch ro yamsno)

dsyn-1-y!)ben;zarniide (20)

To a stirring solution of 20.5 (800 mg, 199 mmo!) in NMP (9.9 mL) 0°C was added DIPEA (698 L) followed by 2,2,2-trifluoroeihyl formate (2 x 40 L) and the reaction progress was monitored by LCMS. The reaction mixture was acidified with AcOH (1 mL), diluted with water (10 mL) and purified by RP HPLC (2") to yieid compound 20 (421 mg, 57.2%). MS: m/z calcd for C ₁₉ H ₂ oN ₄ 0 ₄ 368,15, found [M+Hf 369.1. 21. {S)~N~{3~a ino~1~(hydmxya ino}-3' ethyM-oxobutan-2^!}'4'((1- { ethy!su!fony!)azetidin~3~y!}buta-1^-diyn~1~y!}benza ide (21)

To a stirring solution of compound 1 (147 mg, 0.41 mmol) in DMF (1.45 mL), cooled in an ice bath, was added DIPEA (0.18 mL, 1.04 mmol) followed by methanesulfony! chloride (32 μί, 0.41 mmol) and the reaction was stirred at -15°C overnight. The reaction mixture was diluted with water (1.5 mL), acidified with AcOH (65 μί) and purified by RP HPLC (2", 0.1 % AcOH in water/ACN) to yield compound 21 (34 mg, 16.6%). MS: m/z calcd for C20H24N4O5S 432.1 , found [M+H] ⁺ 433.0,

22. 4-{(1-formylazeiidin-3-yl}buta- 1, 3-diyn- 1 -yl}-N-{{2S, 3R}-1 - {hydroxyaniino}~3~(methylamino)-1-QXQbutan~2~yl)benzaniide (22)

Reactant MW Eq Moles Sample Vol Molarity Density %

(mmol) Mass (ul) (molar) (g/ml) vt

(mg) (%)

A/-{(2S,3f?)-3-amino-1- 368.387 1.000 0.543 200

(hydroxyamino)-l- oxobutan-2-yl)-4-((1- formy!azetidin-3-y!)buta- 1 ,3-diyn-1-y!)benzamide

TEA 101.190 2 1.086 1 10 151 0.726

Formaldehyde 30,026 1.000 0.543 44.1 40.4 1.09 37

TFA 1 14.023 6 3.26 371 251 1.48

Sodium cyanoborohydride 39.853 3 1.629 102

n-Butylamine 73.137 3 1.629 1 19 161 .74

Ammonium hydroxide 18.038 10 5.43 367 14.8

To a stirring solution of compound 20 (200 mg _; 0.54 mmol) in water (3 mL) and DMF (1 mL) and MeOH (1 mL) was added TEA (151 pL) followed by formaldehyde (37% aqueous, 40.4 pL, 0.54 mmol) and the reaction progress was monitored by LCMS. Additional formaldehyde (0.5 eq) was added and again the reaction progress was monitored by LCMS. n- Butyiamine (161 pL) was added and the reaction was stirred for 3 hr. Sodium cyanoborohydride (102 mg _; 1.63 mmol) was then added, followed by TFA (251 pL, 3.26 mmol) and the reaction was stirred for 30 min. The reaction was quenched with cone. NH ₄ OH (367 pL, 5.43 mmol) and the mixture was purified by RP HPLC to yield compound 22 (16 mg, 6%). MS: m/z calcd for C ₂ oH _{22 4} 0 ₄ 382,2, found [M+H] ⁺ 383, 1.

23. {S)~N~{3~hydmxy-1^ydroxyam!no}~3~methyl-1'Oxobutan'2-y!)~4~{ {1- {Qxetan-3-y!}azetidin-3-y!}buta-1,3-d!yn-1-y!)benzamide (23)

To a stirring solution of compound 2b (502 mg, 1.21 mmol) in THF (10 mL) was added oxetan-3-one (87 mg, 1.21 mmol) and AcOH (5 mL) and the reaction was stirred for 30 min. MeOH (10 mL) was added followed by sodium cyanoborohydride (152 mg, 2.41 mmol) and the reaction was stirred for 18 hr. The mixture was concentrated under reduced pressure to give a crude, which was purified by RP HPLC (2", 0.1 % AcOH in water/ACN) to yield compound 23 (10 mg). MS: m/z caicd for C22H25N3O5 41 1.2, found [ +H] ⁺ 412.3.

24. {S)-N-{3-amsno-1 '-{hydrox -amino)-3-methyl-1 -oxobutan-2-yi}-4-(5- (azetidin-3-yl)penta~1,3-diyn~1-yl)benzarnide (24) terf-butyl! 3~({{(trifkioromethyl)su!fonyf)oxy)methyl)azetidine-1 -carboxy!ate (24.2)

To a stirring solution of the azetidine (1.32 g, 7.05 mmoi) in DCM (35 mL) was added DIPEA (2.46 mL, 14.1 mmol) and the reaction was cooled in a dry-ice/acetone bath. Trifiic anhydride (1.25 mL, 7.40 mmol) was added and the reaction was stirred for 30 rnin. The reaction was quenched with water (20 mL), the organic layer was washed with saturated aqueous NaHC0 ₃ (10 mL), brine, dried over sodium sulfate, filtered and concentrated under reduced pressure to an oil, which was purified by flash chromatography (silica gei/0-3% MeOH in DCM) to yield compound 24.2 (1.28 g, 56.9%). MS: m/z caicd for Ο ₁₀ Η ₁₆ Ρ ₃ ΝΟ ₅ 3 319.1 , found [M+Na] ⁺ 342.1. terf-buty 3-fprop-2-yn~1 -yS)az:etidine-1 -carboxy!ate {24.4}

To a stirring solution of ethynyltrimethylsiiane (1.06 g, 10.8 mmol) in THF (10 mL) at 0°C was added r?~BuLi (1.5 M, 8.6 mL, 12.9 mmol) and the reaction was stirred for 30 min. A solution of DMPU (3.1 mL) was then added, followed by a solution of compound 24.2 (0,6 g, 1.88 mmol) in THF (3 mL) and the reaction was stirred for 30 min at -20°C. cone NH ₄ OH (1 mL) and MeOH (1 mL) were then added and the reaction was warmed to rt over 10 min. The mixture was diluted with Et ₂ 0 (100 mL) and water (100 mL). The organic layer was washed with water (2 x 50 mL), and was then concentrated under reduced pressure to yield compound 24.3 as a thick oil. To compound 24,3 was added TBAF (1 M in THF, 1.88 mL, 1.88 eq) and the reaction was stirred at 5-10°C overnight. The reaction was diluted with Et ₂ 0 and washed with a solution of water/5 M NH ₄ Ci (100 mL: 50 ml). The organic layer was washed with saturated NH ₄ CI, dried over sodium sulfate, filtered and concentrated under reduced pressure to give a crude, which was purified b flash chromatography (silica gel) to yield compound 24.4 (235 mg). Ή NMR (400 MHz, CDCI ₃ ) δ 1.44 (s, 9 H), 1.97 (s, 1 H), 2.44 (d, J = 6.8, 1 H), 2.68-2,74 (m, 1 H), 3.69 (dd, J _a = 8.4 Hz, J _b - 5.2 Hz, 2 H), 4.022 (t, J = 8.4 Hz, 2 H). (S)-fert-butyl 3~C5 4~C{3-((feri~butoxycarbonyi)amino)-1 -methoxy~3~rnethyl~1-oxobutan~2- y^carbamoy!lpheny^penta^^-diyn-l -ylla^etsdine-l-carboxySate (24.5)

To a stirring solution of CuCI (16 mg) in 30% n-butyiamine (1 :1 H ₂ 0 THF, 0.7 mL) and hydroxylamine (50% aqueous, 48 pL) cooled in an ice/MeOH bath, was added a cold solution of fert-buty! 3-(prop-2-yn-1-y!)azetidine-1-carboxy!ate (150 mg, 0.77 mmol) in 30% n- butylamine (1 : 1 H ₂ 0/THF, 1.74 mL). A cold solution of (2S,3f?)-methyl 2-(4- (bromoethynyl)benzamido)-3-((ieri-butoxycarbony!)amino)butan oate (348 mg, 0.77 mmol) in 30% n-butyiamine (1 :1 H ₂ 0 THF, 0.6 mL) and hydroxylamine (50% aqueous, 16 μί) was then added dropwise over 15 min, and the reaction was stirred for 15 min. The reaction mixture was worked as described in procedure 1 to yield compound 24.5 (300 mg), which was carried through to the next step without further purification. MS: m/z calcd for C31 H41 N3O7 567.3, found [M+Na] ⁺ 590.4. (S)-methy! 3~amino~2~{4~(5~{a∑et!d!n~3~yf)penta-1 ,3~diyn~1~yI)ben arnido)-3- methy!butanoate (24.6)

To a stirring solution of compound 24.5 (300 mg, 0.53 mmo!) in DCM (1 mL) cooled in an ice/MeOH bath was added TFA (4 mL) and the reaction was stirred for 30 min. The reaction mixture was concentrated under reduced pressure, azeotroped with EtOAc, then diluted with water and purified by RP HPLC (1 ", 0.1 % AcOH in water/ACN) to yield compound 24.6 (159 mg). MS: m/z caicd for C ₂ i H _{25 3} 0 ₃ 367.2, found [M+Hf 368.1.

{S)-/ -(3-am!no-Hhydroxyamin0)-3-met^

diyn-1-y!)benzamide (24)

To a stirring solution of compound 24.6 (69 mg, 0.19 mmol) in I PA (1050 pL) at 0°C was added hydroxylamine (50% aqueous, 750 pL) and the reaction was stirred at rt for 7 hr. AcOH (645 pL) was added and the mixture was purified by RP HPLC (1 ", 0.1 % AcOH in water/ACN) to yield compound 24 (42 mg, 607%). MS: m/z caicd for C20H2 O3 368.2, found [M+H] ⁺ 369.1.

25. {S)-N-{3-amino-1-{hydroxyamino}~3~methyi-1-Qxobutan-2-yl}~4~ {5-{1- formy!azetidin-3-yi)penta-1,3-diyn-1-yi)benzam!de (25)

To a stirring solution of compound 24 (12.9 mg, 0.035 mmol) in DMF (140 μΙ_) cooled in ice/MeOH bath was added DIPEA (9.8 μΙ_, 0.056 mmol) followed by 2,2,2-trifluoroethyi formate (3.45 μ!_, 0.035 mmol) and the reaction was stirred for 5 min. The reaction was diluted with water (2 mL), neutralized with AcOH (6 μϋ and purified by RP HPLC (1 ", 0.1 % AcOH in water/ ACN) to yield compound 25 (4.9 mg). MS: m/z calcd for C21 H24N4O4 396,2, found [M+H] ⁺ 397.1.

(S,E}-N-{ 3-amin 0- 1 -{hydroxyami: 3-methyi-1-Qxobuian-2-yl)-4-{4- <zetidin-3~yl)but~3~en~1~yn~ 1 26}

{E)-teii-hutyl 3-{2-iodoviny a etidine-1 -carboxylaie (26.1) Reagent W Eq. mmo! g, mL

Compound 1.3 185, 1 1 .0 10.80 2.0 g

CrC 122.9 5.0 54.0 6.63 g

CHI ₃ 393.7 2.0 21 .6 8.51 g

THF 60 mL

To a stirring solution of CrCI ₂ (6.63 g, 54.0 mmo!) in THF (40 mL) was added CHi ₃ (8.51 g, 21.6 mmo!) in THF (15 mL) dropwise at OX. Compound 1.3 (2.0 g, 10.8 mmol) in THF (10 mL) was then added and the reaction was stirred at 0°C for 2 hr then at room temperature for 1 hr. The reaction mixture was added to ice water, and extracted with EtOAc. The organic iayer was washed with aqueous Na ₂ S ₂ 0 ₃ , brine, concentrated under reduced pressure to a residue, which was purified by flash chromatography (siiica gel/ PE/EA 20: 1 ) to yield compound 26.1 (2.41 g, 57%) as a yellow oil (2.41 g). H NMR (400 MHz, CDCI ₃ ) δ 6.71 (dd, J = 14.5, 8.5 Hz, 1 H), 6.19 (d, J =14.5 Hz, 1 H), 4.08 (i J = 8.5 Hz, 2H), 3.78 (d, J = 6.0 Hz, 1 H), 3,74 (d, J = 4.2 Hz, 1 H), 3.25-3.15 (m, 1 H), 1 .43 (s, 9H).

{S, £}-tert- buiy! 3-{4-{4-(3-(iert-but03 sycarbo nylamino) -1 -meth Dxy-3-!Tiethyi-1 -0 5 obuian-2- yicarbamc >y!)phenyl)but-1 -en~3-yny )a∑etidi ne-1~carb< axy!ate ί 26.2)

Reagent MW Eq. mmol g, mL

Compound 28.1 309 1.0 4.2 1.30 g

Compound I T-1 374.4 1.0 4.2 1.57 g

PdCI ₂ (Ph ₃ ) ₂ 701.9 0.1 0.42 0.29 g

Cui 190.5 0.1 0.42 80 mg

Et ₃ N 101 3.0 12.6 1.82 mL

THF 15 mL

To a stirring soiution of compound !NT~1 (1.57 g, 4.2 mmol), PdCI ₂ (Ph ₃ ) ₂ (0.29 g, 0.42 mmol), and Cui (80 mg, 0.42 mmol) in THF (10 mL) was added compound 26.1 (1.30 g, 4.2 mmol) in THF (5 mL), followed by Et ₃ N (1.82 mL, 12.6 mmol) and the reaction mixture was stirred at room temperature overnight. Water was added, and the resulting mixture was extracted with EtOAc. The organic layer was washed with brine, concentrated under reduced pressure to give a crude, which was purified by flash chromatography (silica gei/PE/EA 2/1 ) to give compound 28.2 (1.20 g, 52%) as a pale yellow oil. MS: m/z calcd for CsoH^ NaGy 555.3, found [M+H] ⁺ 556.

{S,£)~methyi 3-amino~2~C4-{4-Cazet!dsn~3~y!)but^

bss{2,2,2-trifSuoroacetate) (26.3)

To a stirring solution of compound 26.2 (1.1 g, 1.98 mmol) in CH ₂ CI ₂ (20 mL) at 0°C was added TFA (1.47 mL, 19.8 mmol) dropwise and the reaction mixture was stirred at 0°C for 3 hr. The solvent was evaporated under reduced pressure to give compound 28.3 (0,65 g, 93%) as a yellow solid, MS: /z caicd for C ₂ oH _{25 3} G3 355.2, found [M+H] ⁺ 356; ^' H NMR(400 MHz, DMSO-de) δ 8.90 (d, J = 8.8 Hz, 2H), 8,70 (br s, 1 H), 8.16 (s, 3H), 7.94 (d, J = 8.4 Hz, 2H), 7.57 (d, J = 8.2 Hz, 2H), 6.50 (dd, J = 15.8, 7.9 Hz, 1 H), 6.03 (d, J = 15.9 Hz, 1 H), 4.94 (d, J = 8.8 Hz, 1 H), 4.12-4.05 (m, 2H), 3.96-3.91 (m ,2H), 3.73 (s, 3H), 3.67-3.62 (m, 2H), 1.39 (s, 3H), 1.37 (s, 3H).

{S,£)- -{3-amsno-1 -{hydroxyammo)-3-m

1 -yn-1 -yl)ben£ami e (26.4)

To a stirring solution of compound 28.3 (2, 18 g, 3.74 mmol) in IPA (8.8 mL) cooled in ice bath was added hydroxy!amine (50% aqueous, 8.8 mL) and the reaction was stirred at 5-10°C overnight. AcOH (9 mL) was added, IPA was removed under reduced pressure and the resulting mixture was purified by RP HPLC (2") to yield 26.4 (1.55 g, 71 %) as the TFA salt. MS: m/z calcd for C ₁ 9H2 ₄ N ₄ O3 356.2, found [M+H] ⁺ 357.2,

{S,E -M-{3-amino-1 -(hydroxyamino)-3-methyi-1 -oxobutan-2-y!)-4-{4-{1- ( eihylsulfonyl!)az:eisdin-3-y!)but-3-en-1 -yn-1 -y!)benzamsde (26)

Reactant W Eq Moles Sample Vol Density

(mmol) Mass (ml) (g/rn!)

(mg)

yl)benzamide

ethanesulfonyl chloride 1 14.551 1 0.205 23.52 0.016 1.47

DIPEA 129.243 2 0.41 1 53.1 0.072 0.74

AcOH 60.052 2.1 0.431 25,9 0.025 1.049

To a stirring solution of compound 26,4 (120 mg, 0.20 mmol) in DMF (1 .2 mL) was added DI PEA (72 pL, 0.41 mmol) and the reaction was cooled in an ice bath. Next, methanesulfonyl chloride (4.3 pL, 0.3 equiv) was added and the reaction was stirred for 40 min. Additional methanesulfonyl chloride (1 1 pL, 0.7 eq) was added. Additional DI PEA (1 eq) was added and the reaction was stirred fo 1 .5 hr. Additional methanesulfonyl chloride (1 eq) was added and the reaction was stirred for 30 min. AcOH (70 pL) was added, followed by water (5 mL) and the mixture was purified by RP HPLC (2', 0.1 % TFA in water/ACN) to yield compound 26 (35 mg, 31.1 %). MS: m/z calcd for C ₂₀ H _{26 4} O ₅ S 434.2, found [M+H] ⁺ 435.2.

27. {S,E}-N-{3-amino-1-{hydroxyamsno}-3-methyi-1-oxobuian-2-yl)- 4-{4- (1-formylazetidin-3-y!}biit-3-en-1-yn-1-yl)tenzam!de {27}

To a stirring solution of compound 26.4 (43 mg, 0.12 mmol) in DMF (1 mL) and water (9 drops) was added DIPEA (28 pL, 0.16 mmol) and the reaction was cooled to 0°C. 2,2,2-trif!uoroethy! formate (12 μ!_, 0.12 mmol) was then added and the reaction was stirred for 10 min. The mixture was purified by RP HPLC (1 ", 0.1 % TFA in water/ACN) to yield compound 27 (7,2 mg, 15.4%). MS: m/z calcd for C^H^INL 384.2, found [M+H] ^1" 385.1.

28. (S,E)-4-(4-(1-acety!azetidin-3-yi}but-3-en-1^n-1-yi)-N-(3-am ino-1- (hydroxyamino}-3-methy!-1-oxQtuian-2-y!}benzamide (28)

To a stirring solution of compound 26.4 (0.1 14 g, 0.242 mmol) in DMF (1 mL) was added DIPEA (0.042 mL, 0.242 mmol) and the reaction was cooled to 0°C. Acetic anhydride (7 L) was added and the reaction progress was monitored by LCMS. Additional acetic anhydride (11 μί) was added and the reaction was stirred for 2 hr. The reaction was quenched with n-butylamine (15 pL). TFA (19 pL) was added and the mixture was purified by RP HPLC (1", 0.1 % TFA in water/ACN) to yield compound 28 (67 mg, 69.4%). MS: m/z calcd for C ₂₁ H ₂₆ N ₄ 0 ₄ 398.2, found [M+Hf 399.2.

29. N~((S)~3~a ino~1^ydroxyamino)~3-methyM-oxobutan~2~yi)-4-((5~ oxopyrrolidin-3-yl)buta-1,3"diiynyi)benza ide (29)

29.11

1 ,3-dibromo-2,2-dimeihoxypropane (29,2)

To a stirring solution of compound 29.1 (157g , 2.7 mol) in CH ₃ OH (1.6 L) at 0°C was added Br ₂ (950 g, 5.9 moL) dropwise, and the reaction was stirred with warming to rt for 15 hr. The precipitate was collected by filtration and was washed with cold methanol to yield compound 29.2 (450 g, 60%) as a white solid.

Nisopropy^ 3,3-dimetho) cycyciobutane-1 ,1 -dscarboxyfat 5 ^215-3^

Reagent I MW Eq. mol g, mL Compound 29.2 261.94 1 1.35 mol 360 g

Diisopropyl ma!onate 188.22 2 2.7 mol 507 g

NaH 24.20 2.2 3.0 mol 120 g

DMF 1.0 L

To a suspension of NaH (120 g , 3.0 mol) in DMF (1 L) was added dropwise diisopropyl maionate (507 g, 2.7 mo!) under an Ar atmosphere, and the reaction was stirred. Compound 29.2 (360 g, 1.35 moi) was then added in portions and the mixture was heated to reflux for 48 hr. The reaction mixture was poured into water (2 L), and extracted with PE (6 x 1 L). The combined organic layers were dried, filtered and concentrated under reduced pressure to give a yellow oil, which was purified by distillation under reduced pressure to yield compound 29,3 (150 g, 45%) as a colorless oil.

3~oxocyclobutanecarboxylic acid (29.4)

Compound 29.3 (150 g, 0.52 mol) was added to a solution of 10 M HCi (400 ml), and the reaction mixture was heated to reflux for 60 hr. The reaction mixture was cooled to rt, extracted with Et ₂ 0 (10 x 1 L) and the combined organic layers were dried, filtered and concentrated under reduced pressure to give compound 29.4 (65 g) as a yellow oil, which was carried through to the next step without further purification.. methyl S-oxocycHobutanecarboxySate (29.5) S0CI ₂ 1 18.97 65 mL

CH ₃ 0H 650 mL

To a stirring solution of 29 .4 (65g , 0.57 mo!) in CH ₃ OH { 650 mL) at 0°C was added dropwise SOCI ₂ (65 mL) and the reaction was stirred with warming to rt for 15 hr. The solvent was removed under reduced pressure, and the residue was diluted with water (200 mL), and extracted with DCM (3 x 200 mL). The combined organic layers were dried, filtered and concentrated under reduced pressure to give a crude, which was purified by flash

chromatography (silica gel/ PE/EA 20: 1 to 50: 1 ) to yield compound 29.5 (50 g, 83%) as a yellow oil.

3-(hy roxymethyi)cydobuian0i (29.6)

To a suspension of LiAIH ₄ (0.22.8 g , 0.48 mol) in Et ₂ 0 (500 mL) under an Ar atmosphere was added dropwise a solution of 29.5 (50 g , 0.4 moL) in Et ₂ 0 (300 mL) and the reaction was stirred at rt for 3 hr. The reaction was quenched with water (44 mL), and the resulting solids were removed by filtration. The filtrate was washed with Et ₂ 0, and the combined organic Iayers were dried, filtered and concentrated under reduced pressure to yield compound 29.6 (27.8 g, 69%) as a colorless oil.

3-{{tert-buiy1dimethy s!ly!oxy)methy!}cyc!obutanoS (29.7) TBSCi 150.72 1.1 0.17 mo! 25.0 g

DMAP 122.17 0.1 0.015 mol 1.8 g

NEt ₃ 20 mL

THF 500 mL

To a stirring solution of 29.6 (15.3 g , 0.15 moL) in THF (500 mL) at 0°C was added DMAP (1.80 g, 15 mmoL), followed by TBSCI (25.0g 0.17 mol) and triethy!amine (20 mL) and the mixture was allowed to warm to rt and stirred for 8 hr. The solvent was removed under reduced pressure, and the residue was diluted with water (100 mL), and extracted with DCM (3 x 100 mL). The combined organic layers were dried, filtered and concentrated under reduced pressure to yield compound 29.7 (31 g) as a red oil, which was used in the next step without further purification.

3~((tert-butyldimethyIsilyioxy)methyl)cyciobutyl benzoate (29.8)

To a stirring solution of compound 29.7 (31.0 g , 0.15 moL) in THF (500 mL) at 0°C were added benzoyl chloride (25.2 g, 180 mmoL ) and NEt ₃ (20 mL) and the reaction mixture was heated to reflux for 15 hr. The solvent was removed under reduced pressure, and the residue was diluted with water (100 mL), and extracted with DCM (3 x 100 mL). The combined organic layers were dried, filtered and concentrated under reduced pressure to give a crude, which was purified by flash chromatography (silica ge!/PE/Et ₂ G 5: 1 to 1 :1 ) to yield compound 29.8 (25.2 g, 55%). MS: m/z calcd for C ₁₈ H ₂₈ 0 ₃ Si 320.2, found [M+Hf 321. 3~{hydroxyrnethyl)cyc!obutyl benzoate (29.9)

To a stirring solution of 29.8 (12.6 g , 39 mmol) in THF (200 mL) was added Bu ₄ NF (10.7 g, 41 mmo!) and the reaction mixture was stirred for 3 hr. The solvent was removed under reduced pressure, and the residue was diluted with water (50 mL), and extracted with DCM (3 x 100 mL). The combined organic layers were dried, filtered and concentrated under reduced pressure to give a crude, which was purified by flash chromatography (silica gel/PE/EA 5: 1 to1 : 1 ) to yield compound 29.9 (7.2 g, 88%) as a colorless oil.

S-formylcyclobutyf benzoate(29.10)

To a stirring solution of oxalyl chloride (5.04 g , 40 moL) in DCM (40 mL) at -78 °C under an Ar atmosphere was added dropwise a solution of DMSO (6.24 g, 80 mmol) in DCM (20 mL), and the reaction was stirred for 30 min. A solution of compound 29.9 (4.12 g , 20 mmol) in DCM (20 mL) was added dropwise and the reaction was stirred for 30 min. A solution of NEt ₃ (20.2 g, 200mmo!) in DCM (20 mL) was then added dropwise, and the reaction mixture was allowed to warm to rt over 30 min. The mixture was poured into cold 1 HCI (200 ml), and was extracted with DCM (3 x100 mL). The combined organic layers were dried, filtered and concentrated under reduced pressure to give a crude, which was purified by flash chromatography (silica gel/PE/EA 5: 1 to1 : 1 ) to yield compound 29.10 (3.8 g, 93%) as a colorless oil.

(1s _s 3s)-3-(2,2-dibromoviny!}cyctobuty! benzoate(29.11}

To a stirring soiution of CBr,- (9.96 g, 30 moL) in DCM (15 mL) at -20°C under an Ar atmosphere was added dropwise a solution of PPh ₃ (15.7 g, 60 mmol) in DCM (50 mL) and the reaction was stirred for 30 min. The reaction was cooled to -78°C and a solution of 29.10 (3.06 g, 15 mmol) in DCM (15 mL) was added dropwise, and the reaction was stirred for 30 min. The reaction was allowed to warm to rt over 30 min. The solvent was removed under reduced pressure, and the residue was purified by flash chromatography (silica gel/PEEA 10: 1 to 5: 1 ) to yield compound 29,11 (4.2 g, 79%) as a colorless oil.

Meth l 4-({{1s,3s)-3-{benzoyloxy)cycSobutyl}buta-1 ,3-d5ynyi)benzoate(29.12}

Reagent MVY Eq. Mmol g, mL

NEta 101 .19 3 30 3.04 g

DMF 40 mL

To a stirring solution of 29,11 (3.58 g , 10 mmol), Pd ₂ dba ₃ ( 91 .6 m g , 0.1 mmo I), TMPP (142 mg ,0.4 mmol), and NEt ₃ (3.04 g , 30 mmol) in DMF (40 mL) was added INT-1 ,3 (1 .76 g , 1 1 mmol) and the reaction was stirred under an Ar atmosphere at 80 °C for 5 hr. The reaction mixture was diluted with EtOAc (100 mL), washed with water (3 x 50 mL ), dried, filtered and concentrated under reduced pressure to give a crude, which was purified by flash chromatography (silica gel/ PE/EA 50: 1 to 10: 1 ) to yield compound 29.12 (1 .0 g, 40%) as a yellow solid. MS: m/z calcd for ·0 .Η ·«Ο, 358.1 , found [M+H] ⁺ 359. methyl ^{(flSjSsl-S-h dro yc cSo uty!l uta-l^-d n-l -y!l en^oate (29.13)

To a stirring solution of compound 29.12 (4.2 g, 1 1.78 mmol) in MeOH (150 mL) was added K ₂ C0 ₃ (4 g, 29 mmol) and the reaction was stirred overnight. The solvent was removed under reduced pressure, and water and EtOAc were added. The organic layer was washed with brine, dried, filtered and concentrated under reduced pressure to give a crude, which was purified by flash chromatography (silica gel/EtOAc/PE 1 :10 to 1 :6) to yield compound 29.13 as a solid. MS: m/z calcd for C- _B H-,0 254.1 , found [M+H] ⁺ 255. methyl 4-{(3-oxocyc!obuty buta-1,3~dsyn~1~yf)benzoate (29.14)

To a stirring solution of compound 29.13 (4.7 g, 18.48 mmoi) in DCM (80 mL) was added PCC (11.98 g, 55.45 mmoi) and the reaction was stirred overnight. The solvent was removed under reduced pressure and the residue was purified by flash chromatography (silica gel/ EtOAc/PE 1 : 10) to yield compound 29.14. m/z ca!cd for C ₆ H ₂ 0 ₃ 252.1 , found [M+Hf 253. methyl ^{(S-Ch dro ysminolc cSo uiy^ uta-l^-ds n-l-y^ en^oate (29.15)

To a stirring solution of compound 29.14 (3.58 g, 14.19 mmol) in MeOH (50 ml_) was added NH ₂ OH (1.25 g, 17,03 mmoi) and the reaction was stirred overnight. The solvent was removed under reduced pressure to yieid compound 29,15. m/z caicd for C ₁₆ H ₁₃ NQ ₃ 267.1 , found [M+H] ⁺ 268. H-NMR (400 MHz, DMSO-d ₆ ) δ 10.51 (s, 1 H), 7.95-7.97 (d, 2H), 7.88-7.70 (d, 2H), 3.88 (s, 3H), 3.340-3.48 (m, 1 H), 3.22-3.28 (m, 2H), 2.87-2.99 (m, 2H) methyi 4-{{5-oxopyrrondin-3-y!)buia-1 ,3-diyn-1 -yS)benzoate (29.16)

To a stirring solution of compound 29.15 (3,74 g, 14.03 mmoi) in DCM (50 mL) was added dropwise SOCI ₂ (3.64 g, 30.87 mmol) and the reaction was stirred for 20 min. The reaction mixture was added to ice-water and the organic Iayer was washed with brine (2 x), dried, filtered and concentrated under reduced pressure to give a crude, which was purified by flash chromatography (silica gel/MeOH/DCM) to yield compound 29.16 as a soiid. m/z calcd for C ₁₆ H ₁₃ N0 ₃ 267.1 , found [M+Hf 268. Ή-NMR (400 MHz, DMSO-d ₆ ) δ 7.95-7.97 (d, 2H), 7.82 (s, 1 H), 7.68-7.70 (d, 2H), 3.86 (s, 3H), 3.52-3.58 (m, 2H), 3.34 (s, 1 H), 3.22-3.27 (m, 1 H), 2.24- 2.29 (m, 1 H).

4-{{5-oxopyrrondin-3-yS)buta-1 ,3-d!yny!)benz:oic acid (29.17)

To a stirring solution of compound 29.16 (508 mg, 188 mmol) in MeOH (5 mL) and water (5 mL) was added LiOH (134 mg, 5.81 mmol) and the reaction was stirred overnight. MeOH was removed under reduced pressure, and the mixture was acidified to pH 3 with HCI (6 N, 5 mL) and extracted with EtOAc (50 mL). The solids were removed by filtration. The organic layer was concentrated under reduced pressure to yield solsds that were combined to the previously isolated solids to yield compound 29.17 (800 mg), which was carried through to the next step without further purification. MS: m/z caicd for Ci ₅ H, , 0 ₃ 253.1 , found [M+H] ⁺ 254.1 ,

(2S)-methyl 3-{ieff-butoxycarbonyiam!no}-3-meihy 2-{4-({5-oxopyrroydin-3-yl)buia-1.3- synyf)benzamido)butarsoate (29.18)

To a stirring solution of (S)-methyi 2-amino-3-{ferf-butoxycarbony!amino)-3- methyibutanoate (461 mg, 1.87 mmol) and compound 29.17 (474 mg, 1.87 mmol) in DMF (4 mL) was added DIPEA (0.82 mL, 4.7 mmol) followed by HATU (854 mg, 2,25 mmol) and the reaction was stirred overnight. The reaction mixture was partitioned between 1 M citric acid and ethyl acetate. The organics were washed with semi-saturated sodium chloride, saturated sodium bicarbonate then saturated sodium chloride, dried over magnesium sulfate, filtered and concentrated under reduced pressure to yield compound 29.18, which was carried through to the next step without further purification. MS: m/z calcd for aOe 481.2, found [M- Boc+H] ⁺ 382.1. ~f(S)~3~amsno-1-(hydroxyamino)~3~^

1 ,3-diyny!)benzarnide (29)

To a stirring solution of compound 29.18 (1.87 mmoi) in MeOH (2 mL) was added HCI (4 M in dioxane, 4 mL) and the reaction was stirred for 3 hr. The reaction was concentrated under reduced pressure at 0°C to yield compound 29.19, which was dissolved in IPA (2 mL) and treated with hydroxylamine (50% aqueous, 2 mL) at 5-10°C overnight. The reaction mixture was concentrated under reduced pressure, diluted with water (4 mL) and ACN (1 mL), acidified at 0°C with TFA (4 mL), and purified by RP HPLC (2" column, 0.1 % TFA in water/ACN) to yield compound 29 (445 mg, 47.7%). MS: m/z calcd for C ₂₀ H _{22 4} O4 382.2, found [M+H] ^4' 383.1. 30. N-{(S)~3~8m!no~1~{hydmMyamino}-3~methyM-oxobutan-2^ meihy!~5~oxopyrrQ!sdin-3-y!)buta-1,3~diyny1)benza sde (30) methyf 4~{{1 ~methyf-5-oxopyrro d!n~3~yl)buta~1 ,3-dsyn~1 ~yl)benzoate (30.1)

To a suspension of NaH (18.71 mg, 0.47mmoi) in DMF (2mL) at 0°C were added a solution of compound 29.16 (50 mg, 0.188mmo!) and Me! (53.105mg , 0.376mmol) in DMF (3mL) and the reaction was stirred for 2hr at room temperature, ice-water (10 mL) was slowly added and the reaction mixture was extracted with EA, dried, filtered and concentrated under reduced pressure to give a crude, which was purified by RP HPLC to yieid compound 30.1. MS: m/z ca!cd for C ₇ H _{1 S} N0 ₃ 281 .1 , found [M+H] ⁺ 282.

4-{(1 -methy!~S~oxopyrro din-3-y!)buta-1 ,3~diyn-1 -y!)benzosc acid (30.2)

To a stirring solution of compound 30.1 (265 mg, 0.943mmol) in THF (35 mL) was added a solution of LiOH.H ₂ 0 (158.3mg, 3.772mmo!) in water (15 mL) and the reaction was stirred for 3 hr. The reaction mixture was acidified to pH 1 -2 with HCI (3 M), and was extracted with EA. The organic layer was dried, filtered and concentrated under reduced pressure to yield compound 30.2 as yellow solid. MS: m/z calcd for Ci ₆ Hi ₃ N0 ₃ 267.1 , found [M+Hf 268.

{2S)-rnethyl 3~C{tert-butoxycarbonyI)amino)~3~methyI-2-(4-((1~rnethyl-S-o xopyrrolidin-3~ yH)buta-1 ,3-diyn~1 ~yl)ben;za!T!!do)butanQate (30.3)

To a stirring solution of compound 30.2 (251 mg, 0.94 mmoi) in DMF (40 mL) were added I T-1.6 (231 mg, 0.94 mmoi), HATU (393 mg, 1.034 mmoi) and DIPEA (364 mg, 2.82 mmoi) and the reaction was stirred overnight. The reaction mixture was diluted with H ₂ 0, and extracted with EA. The organic layer was washed with brine, dried, filtered and concentrated under reduced pressure to give a crude, which was purified by flash chromatography (silica gel MeOH/DCM) to yield compound 30.3 as a yei!ow solid. MS: m/z calcd for 495.2, found [M+H] ⁺ 496.

(2S)-meihyl 3-amsno-3-meihyi-2-{4-({1-methy!-S-oxopyrro!idin-3-y buta-1,3-d!yn-1 - yl!)benzamido)buianoate (30.4)

HCI(g) was bubbled through a solution of compound 30.3 (372 mg, 0.75 mmol) in MeOH (10 mL) for 2 hr. The solvent was removed under reduced pressure to give compound 30.4 as a yellow solid. MS: m/z calcd for C ₂₂ H ₂₅ N ₃ 0 ₄ 395.2, found [M+H] ⁺ 396. ¹ H-NMR (400 MHz, CD ₃ OD) δ 7.89-7.91 (d, 2H), 7.61 -7.83 (d, 2H), 5.01 (s, 1 H), 3.84 (s, 3H), 3.73-3.79 (m, 1 H), 3.45-3.51 (m, 2H), 3.38 (s, 1 H), 2.87 (s, 3H), 2.74-2.80 (m, 1 H), 2.45-2.52 (m, 1 H), 148-149 (d, 8H)

^-C{S)-3-amino-1"(hydroxyam!no)-3-methy^

3-y!)buta-1 _s 3-diyn-1 -y^benzamide (30)

To a stirring solution of compound 30.4 (45 mg, 0.104 mmol) in IPA (1 mL) was added hydroxyiamine (50% aqueous, 1 mL) and the reaction was stirred overnight. The reaction mixture was cooled to 0°C and acidified with TFA (2 mL), diluted with water (1 mL) and ACN (0.5 mL) and purified by RP HPLC (1 " column, 0.1 % TFA in water/ACN) to yield compound 30 (22.5 mg, 42.2%). MS: m/z ca!cd for C _2! H ₂ 4 ₄ 04 396.2, found [M+H] ⁺ 397.1 ,

31. N~{(S)-3-amino-1-{hydroxyamino}-3~methyi-1-Qxobutan-2-yl}-4~ {(1-(2- (hydroxymethyi}cyciopropanecarbonyl)azetidin-3-yl}ethynyi)be nzamide (31)

(S)-methy! 2-{4-(azetidin-3-ySethynyi)ben.:am!do)-3-{(ieri-bi!ioxycarbo ny!)am!no)-3- methy!butarsoate (31.2)

The reaction was conducted according to procedure 1 to yield compound 31.2.

(2S)-methy! 3~{f ferf-butoxycarbony!)amino)~2~{4-f (1 -{2- (hydroxymeihyl}cyctopropanecarbony!)az:etidm-3-y!)eihyr!y!)b enzamido)-3- methyibutanoate (31.3)

The reaction was carried out as described for compound IMT~% to yield compound 31.3 MS: m/z ca!cd for C ₂₈ H ₃₇ N ₃ O ₇ 527.3, found [M-Boc+H] ⁺ 428.2. Compound 31.3 was Boc-deprotected according to procedure 2 to yield compound 31.4. ^-{(SJ-S-amino-l -fh drox aminol-S-meth l -o obuian^- ^^-CCI^- {hydroxyrnethy!)cydopropanecarbony1)a2:etsdsn-3-y!)ethynyS)b en2:arnide (31)

To a stirring solution of compound 31 ,4 (6.82 g, 15.95 mmoi) in IPA (20 mL) was added hydroxylamine (50% aqueous, 20 mL) and the reaction was stirred for 28 hr. The reaction was concentrated under reduced pressure and purified by RP HPLC to yield compound 31 (34 mg). MS: /z calcd for C ₂₂ H ₂₈ N ₄ O _s 428.2, found [M-Boc+H] ^1" 429.2.

N-((S)-3-hydrQxyaminQ)-3-methyi-1-QXQbutan-2-yl)-4-({1-{Q .

(32)

{S)~ferf~buty! 3-ethyny!pyrralidine-l -carboxylate (32.2)

To a stirring solution of (R)-ferf-butyl 3-formylpyrro!idine-1-carboxyiate (2.4 g, 12.05 mmol) in ACN (25 mL) was added Bestmann reagent (2.33 g, 12.05 mmol), followed by K ₂ C0 ₃ (3.33 g, 24.09 mmol) and the reaction mixture was stirred for 24 hr. Solvent evaporation under reduced pressure gave a crude, which was purified by flash chromatography (silica gel, 2- 60% EA in DC ) to yield compound 32.2 (980 mg).

(S)-f erf-butyl 3~{(4-({{S)-3-hydroxy~1 -methoxy-S-methyl!-l -oxobutan-2- yS)carbamoyl}pheny buta-1 ,3-diyn-1-y pyrro!idine-1-carboxyiaie (32.3)

To a stirring solution of hydroxyiamine hydrochloride (8,33 mg, 0, 120 mmol) and copper chloride (3.95 mg) in 30% n-butylamine (aqueous, 5 mL) was added (S)-fert-buty! 3- ethynylpyrrolidine-1-carboxylate (0.78 g, 1 ,997 mmol) and the reaction was stirred for 20 min. A solution of (S)-methyi 2-(4-(bromoethynyl)benzamido)-3-hydroxy-3-methylbutanoate (0.707 g, 1997 mmol) in 30% n-butyiarnine (aqueous, 5 mL) and THF (10 mL) was then added and the reaction was stirred for 30min. Excess solvent was removed under reduced pressure, and the reaction mixture was extracted with EA (3 x 100 mL). Solvent evaporation under reduced pressure gave compound 32,3, which was carried through to the next step without further purification. MS: m/z calcd for CseHas sOe 468.2, found [M+H 469.2.

{2S)-methy 3-hydroxy-3-methy 2-{4-{pyrrondin-3-ySbuia-1 ,3-diyn-1 - y benxamidoJbutanoate (32.4)

Boc deprotection was carried out according to procedure 2 to yield compound 32,4. MS: m/z calcd for C21 H24N2O4 368.2, found [M+Hf 369.1.

A^((S)-3 iydroxyamino)-3 nethyl-1 ^>xobutan^

1 ,3-diyn-1 -yl)benzamide (32)

To a stirring solution of compound 32.4 (360 mg, 0.977 mmol) in THF (2 mL) was added AcOH (0.2 mL), oxetan-3-one (141 mg, 1.95 mmol) and sodium cyanoborohydride and the reaction was stirred for 3 nr. The reaction was concentrated under reduced pressure to yield compound 32. S, which was dissolved in I PA (3 mL) and treated with hydroxyiamine (50% aqueous, 3 mL) for 2 days. The reaction mixture was concentrated under reduced pressure and purified by RP HPLC to yield compound 32. MS: m/z calcd for C23H27N3G5 425.2, found [!VHH] ⁴ 426.3.

33. N-((S)'3~hydrQxy"1-(hydroxya inQ)-3' ethyl~1-Qxobutan-2-y!)'4~({(S)-- 1-(2-hydroxyethy!}pyrrolidin-3-y!}buta-1,3-dsyn-1-yl}benzami de β3)

(S)-methy! 3-hydroxy-2-(4-{{{S)-1 -{2-hydroxyethy!)pyrro din-3-y buta-1 ,3-diyn-1 ^■ y ben2amido)-3-methy!buianoate (33,2)

To a stirring solution of compound 32.3 (360 mg, 0.98 rnmoi) in MeOH (5 mL) was added 2-((ienf-butyldimethylsilyl)oxy)aceta!dehyde (170 mg, 0.98 rnmoi) and sodium cyanotrihydroborate (61.4 mg, 0.98 mmol) and the reaction was stirred for 2 hr. The mixture was concentrated under reduced pressure to yield compound 33.1 , which was dissolved in TFA (4 mL) and stirred for 18 hr. The reaction was concentrated under reduced pressure to yield a residue, which was neutralized with saturated aqueous K ₂ C0 ₃ and extracted with EA (3 x 100 mL). The combined organic layers were dried, and concentrated under reduced pressure to yield compound 33,2, which was carried through to the next step without further purification.

^-{{S)-3-h droxy~1 ~{hydroxyamino)-3-rr8eih l-1 -oxobutan-2-y!)-4-{{{S)-1 -(2- hydroxyethy pyrro!idin ^« 3-yJ)buia-1 ,3-diyn-1 -y^benzamide {33}

To a stirring solution of compound 33.2 in IPA (5 mL) was added hydroxylamine (50% aqueous, 4 mL) and the reaction was stirred for 2 days. IPA was removed under reduced pressure, and the residue was acidified with AcOH (5 mL), filtered and purified by RP HPLC (0.1 % TFA in water/ACN) to yield compound 33. MS: m/z calcd for C22H27N3O5 413.2, found [M+H] ⁺ 414.2.

4-[4-{Azetidin-3-yi)buta-1 _l 3-diynyl]-N-[(1 S)-2-hydlroxy-1 -(hydroxy carbamoyi)-2- methyi-propyi]benzamide acetate (2b, 50 mg, 0.12 mmol) was dissolved in methanol (1 mL) and cooled in an ice bath. N,N"-diisopropy!ethy!amine (0.02 mL, 0.1 1 mmol) and THF (0.50 mL) were added followed by 1-fluoropropan-2-one (0.03 mL, 0.36 mmoi). Sodium triacetoxyborohydride (127.54 mg, 0.60 mmoi) was added and the reaction was stirred at ambient temperature for 45 minutes. The reaction was concentrated under reduced pressure and redissolved in D F. It was purified by RP HPLC (0.1 % TFA in water/ACN, 5-10-30%) to give 34 as a white solid as the TFA salt (31.5 mg, 0.060 mmoi, 49% yield). MS: m/z calcd for C22H26FN3O4 415.2, found [M+Hf 416.1.

4-[4-(3-Hydroxycyc!obuty!)buta-1,3~diyny!]benzo!C acid (35.1)

Methyl 4-[4-(3~hydroxycyciobutyi)buta- 3-diynyl]benzoaie (29,13, 5,09 g, 20.02 mmol) was dissolved in THF (60 mL) and 1.0 M sodium hydroxide (60.05 mL, 60.05 mmol) was added. After 4 h, the reaction was concentrated under reduced pressure to remove most of the THF. The solution was cooled in an ice bath and acidified with 6 mL of cone HCI. A purple- white solid was filtered off and rinsed with EtOAc and then dried further under vacuum to yield compound 35.1 (4.5 g, 94%). MS: m/z caicd for C ₁₅ H ₁₂ 0 ₃ 240.1 , found [M+H] ⁺ 241.1.

Methy {2S)-3-hydroxy-2-[[4-[4-(3-hydroxycyciobuty!)buia-1^-dsynyi] ben2:oy am!no]-3- methyl-butanoate (35.2)

4-[4-(3-Hydroxycyc!obuty!)buta-1 ,3-diynyl]benzoic acid (35.1 , 500 mg, 2.08 mmol) was dissolved in DMF (4 mL) to give a clear solution containing a small amount of purple solid. The solution was cooled in an ice bath and N,N"-diisopropyiethy!amine (0.8 mL, 4.58 mmol), methyl (2S)-2-amino-3-hydroxy-3-methy!-butanoate hydrochloride (382.2 mg, 2.08 mmol) and HATU (830,8 mg, 2.19 mmol) were added. The reaction was stirred at rt for 2 h and then it was diluted with EtOAc and washed with 1 M citric acid (2X), water, sat bicarb and brine, then dried with sodium sulfate, filtered and concentrated under reduced pressure to yield compound 35.2 (755 mg, 98%). MS: m/z calcd for C ₂₁ H ₂₃ N0 ₅ 369.2, found [M+Hf 370.0. Methyl (2S)-3-hydroxy~3-methy!-2-[[4-[4-{3-oxocyc!obutyS)buta-1 ,3- diynynbenzoynaminojbutanoaie (35.3)

Methyl (2S)-3-hydroxy-2-[[4-[4-(3-hydroxycyclobutyl)buta-1 ,3-diynyl3benzoyl]amino]-3- methy!-butanoate (35,2, 755 mg, 2.04 mmol) was dissolved in DCM (10 mL), triethyl amine (1 .42 mL, 10.22 mmol) and dimethy!sulfoxide (1 .6 mL, 22.48 mmol). Sulfur trioxide pyridine complex (1.3 g, 8.18 mmol) was added and the reaction was stirred at rt for 17h, The reaction was concentrated under reduced pressure and redissoived in EtOAc. The solution was washed with 1 M citric acid (2x), water, sat bicarb and brine, then dried with sodium sulfate, filtered and concentrated under reduced pressure to yield compound 35.3 (627 mg, 84%). MS: m/z calcd for C21 H21 NO5 367, 1 , found [M+Hf 368.1 .

Methy {2S)-3-hydroxy-2-[[4-[4-[3-{3-hydroxya2eisdin-1 -y!)cyciobuiy buta-1 ,3- dsynyf]benzoyi]amsno]-3-methyi~buianoate (35.4)

Azetidin-3-ol hydrochloride (89.45 mg, 0.82 mmoi) was dissolved in MeOH (2 mL) and dipotassium carbonate (0.41 mL, 0.82 mmol) was added. The reaction was stirred at rt for 10 min. The reaction was cooled in an ice bath and then acetic acid (0.12 mL, 2.18 mmol), methyi-(2S)-3-hydroxy-3-methyi-2-[[4-[4-(3-oxocyclobuty!)but a-1 ,3- diyny!]benzoy!]amino]butanoate (35.3, 100 mg, 0.27 mmol) and sodium cyanoborohydride (34.21 mg, 0.54 mmol) were added and the reaction was stirred at rt. After 0.75 h, the reaction was diluted with EtOAc and washed with sat bicarb and brine, then dried with sodium sulfate, filtered and concentrated under reduced pressure to yield methyl {2S)-3-hydroxy-2-[[4-[4-[3-(3- hydroxyazetidin-1 -yl)cyclobutyi]buta-1 ,3-diynyi]benzoy!]amino]-3-methyi-butanoate (72.5 mg, 0.17 mmol, 62.7% yield). MS: m/z calcd for C ₂ 4H _{28 2} 0 ₅ 424.2, found [M+Hf 425.1. i¥- 3-hydroxy-1 -(hydroxya!Tiino -3-methy!-1 -oxobutan-2-yS)-4-((3-(3-hydroxya-eetidin-1 - y cyc!obuiy bisia-1 ,3-diyn-1 -yl)benza!Tiide (35)

Methy!-(2S)-3-hydroxy-2-[[4-[4-[3-(3-hydroxyazetidin-1 -yl)cyc!obutyl]buta-1 _l 3- diyny!]benzoy!]amino]-3-methyi-butanoate (35.4, 72.5 mg, 0.17 mmol) was treated according to Procedure 3 to yield compound 35 (21.7 mg, 0.040 mmol, 23.2 % yield). MS: m/z calcd for Cy,H _;¾ F _{3 3} 0/ 425.2, found [M+Hf 426.1.

36. N-({S)-3-hydtQxy-1-(hydmxya !nQ)-3- ethy!-1-oxobutan-2-y!)-4-{(3- ((S)-3-hydroxypyrfoiidin-1-yi}cy obutyl}buta-1,3-diyn-1-yl)benzamide (36)

ethyl! (S)-3^ droxy~2~{4-(f3-{{S)-3^ droxypyrro dm-1-y1)cyc!obuty!)buta-1,3~diyn-1 - yS)benzamido)-3-methy!butanoate (36.1)

(3S)-Pyrroiidin-3-ol hydrochloride (67.27 mg, 0.54 mmol) and N,N"- diisopropy!ethy!amine (0.09 mL, 0.52 mmol) were combined in MeOH (2 mL). The reaction was cooled in an ice bath and then methyl (2S)-3-hydroxy-3-methyl-2-[[4-[4-(3- oxocyclobutyi)buta-1 ,3-diynyl]benzoyl3amino]butanoate (35.3, 100 mg, 0.27 mmol) and sodium triacetoxyborohydride (230.75 mg, 1.09 mmol) were added and the reaction was stirred at rt. After 0.75 h, the reaction was diluted with EtOAc and washed with sat bicarb and brine, then dried with sodium sulfate, filtered and concentrated under reduced pressure to yield 36.1 (97 mg, 0.221 mmol, 81.3 % yield) MS: m/z calcd for C ₂ sH ₃ oN ₂ G5 438.2, found [M+Hf 439.2.

N-((S}-3- ydmxy-1-(hydrQxyamh )-3-met yi-1-QXQbutan-2-yl}-4-((3-((S^

hydrQxypyrrolld -l-yljcycSobutyljbuta-l^-diyn-l-yljbenza sde (38)

Compound 36.1 (97 mg, 0.22 mmol) was treated according to Procedure 3 to yield 36 (37 mg, 30%). MS: m/z calcd for CssHaoFa aO _? 439.2, found [M+H] ⁺ 440.1. 37. N-((S)~3~hydroxy~1~(hydroxyamino)~3~methyi-1-oxobutan-2-yl)~ 4~{(3~ {{R)-3-hydroxypyrro!idin~1~y!}cyc!obuty!)bista-1,3-diyn~1^!) benzai ide (37)

Methyl (S)-3-hydr0xy~2~{4~(f3-{(/?)~3~hy^^

y ben amido)-3-methy!butanoaie (37,1)

(3 ?)-Pyrrolidin-3-ol hydrochloride (67,27 mg, 0.54 mmol) and N,N"~ diisopropy!ethyiamine (0.09 mL, 0.52 mmoi) were combined in MeOH (2 mL). The reaction was cooled in an ice bath and then methyi-(2S)-3-hydroxy-3-methyi-2-[[4-[4-(3- oxocycIobuty!)buta-1 ,3-diynyl]benzoyI]amino]butanoate (35,3, 100 mg, 0.27 rnmol) and sodium triacetoxyborohydride (230.75 mg, 1.09 mmoi) were added and the reaction was stirred at rt. After 0.75 h, the reaction was diluted with EtOAc and washed with sat bicarb and brine, then dried with sodium sulfate, fiitered and concentrated under reduced pressure to yield compound 37,1 (105 mg, 88%). MS: m/z calcd for C ₂₅ H ₃ oN ₂ 0 ₅ 438.2, found [ +Hf 439.2.

N-((S)-3-hydroxy-1-(hydroxyamino)-3-methy!-1-oxobutan-2-y i)-4^^

hydrox pyrroHdm-1-yl}cyclobuiyl)buta-1,3-dlyri-1-yf)berizamlde (37)

Compound 37.1 {105.mg, 0.24 mmol) was treated according to Procedure 3 to yield compound 37 (35 mg, 26%). MS: m/z calcd for C ₂₆ H ₃₀ F ₃ N ₃ O ₇ 439.2, found [M+Hf 440.1 . 38. 4-({3-{3-H uoroa£etidin-1 ~y!)cydobutyf)buia-1 ,3-diyn-1 -yf)-N-(3- hydroxy-1 -{hydroxyamino}-3-methy 1 -oxobutan-2-yl}benzamide (38)

Meth HS)-2-f4-({3 3-fiuGroazet!d!n-1 ^

hydroxy-3-meihyibutanoate (38.1)

3-Fiuoroazetidine hydrochloride (57.69 mg, 0.52 mmoi) and Λ/,Λ -diisopropy!ethylamirie (0.09 mL, 0.49 mmoi) were combined in MeOH (2 mL) . The reaciion was cooled in an ice bath and then methy!-(2S)-3-hydroxy-3-methy!-2-[[4-[4-(3-oxocyclobuty!)but a-1 ,3- diyny!]benzoyi]amino]butanoate (35,3, 95 mg, 0.26 mmoi) and sodium triacetoxyborohydride (219.21 mg, 1.03 mmoi) were added and the reaction was stirred at rt. After 0.75 h, the reaction was diluted with EtOAc and washed with sat bicarb and brine, then dried with sodium sulfate, filtered and concentrated under reduced pressure to yield compound 38.1 (94 mg, 85%). MS: m/z calcd for C24H27FN2O4 426,2, found [M+H] ⁺ 427.2.

4-{{3-(3-Fiuoroazetidin-1-yl)cyctobuty!)buta-1 ,3-dsyn-1 -yi)-N^3-hydroxy-1 -(hydroxyamino)- 3-methyM ~oxobutan~2~yi)benzamide (38)

Compound 38.1 (93 mg, 0.22 mmmol) was dissolved in I PA (0.45 mL) and cooled in an ice bath. Hydroxylamine (0.58 mL, 8.72 mmoi) was added and the reaction was stirred at 4° C for 4 days. The reaction was concentrated under reduced pressure to remove the I PA and then acidified with acetic acid, diluted with D F and purified by RP HPLC (1", 0.1 % TFA in water/ACN) to yield compound 38 (32 mg, 26%). MS: m/z calcd for C ₂ 5H27F4 ₃ 0 ₆ 427.2, found [M+H] ⁺ 428.1. 39. 4-f{1-{2-Cyanoethy!)a∞tidin-3-y1)buta-1 _! 3-diyn-1 -y )~N-(3-hydroxy-1 - (hydroxyamino)-3- eihyM -oxobutan-2-y!)benzamide (39)

To a stirring solution of compound 2b (35 mg, 0.08 mmoi) in ACN (0.3 mL) and water (0.12 mL) was added prop-2-enenitri!e (0.01 mL, 0.10 mmoi) followed by potassium carbonate (5 mg) and the reaction became biphasic. The reaction was stirred vigorously at rt. After an hour, another portion of potassium carbonate (5 mg) was added and the reaction was stirred for 24 hours. The reaction was acidified with acetic acid and purified by RP HPLC (1 ", 0,1 % TFA in water/ACN) to yield compound 39 (16 mg, 36%). MS: m/z. calcd for C24H25F3 4O6 408.2, found [M+H] ⁺ 409.0,

The following compounds of the invention may be prepared according to the synthetic procedures described above:

H 3-methyl-1 -oxobuiars-2-yl)berszamide

yi)buta-1,3-diyn-1-y!)berizamide

y!)benzamide

y!)benzamide

B, Antimicrobial! Activity

1. Bacteria! Screens and Cultures

Bacterial isolates were cultivated from -70° C. frozen stocks by overnight passages at 35 C in ambient air on Mue!!er-Hinton agar (Beckton Dickinson, Franklin Lakes, NJ). Clinical isolates tested were obtained from various geographically diverse hospitals in the US and abroad (Focus Diagnostics, Herndon, VA and JMl, North Liberty, IA). Quality control strains were from the American Type Culture Collection (ATCC: Rockviile, Md.).

2, Susceptibility Testing

Minimum inhibitory Concentrations (MICs) were determined by the broth microdilution method in accordance with the Clinical and Laboratory Standards Institute (CLSl) guidelines. In brief, organism suspensions were adjusted to a 0.5 McFarland standard to yield a final inoculum between 3x 10 ⁵ and 7x 10 ^b colony-forming units (CFU)/mL. Drug dilutions and inocu!a were made in sterile, cation adjusted Mue!ier-Hinton Broth (Beckton Dickinson). An inoculum volume of 100 \iL was added to wells containing 100 uL of broth with 2-fold serial dilutions of drug. All inoculated microdilution trays were incubated in ambient air at 35° C for 18-24 h. Following incubation, the lowest concentration of the drug that prevented visible growth (OD600 nm < 0.05) was recorded as the MIC. Performance of the assay was monitored by the use of laboratory quality-control strains and levofloxacin, a compound with a defined MIC spectrum, in accordance with CL8I guidelines. Typically, compounds of the present invention have MIC values of 0.03 - 16 ng/mL. To this end, data for certain representative compounds is shown in Table I below.

Table I: Minimum Inhibitory Concentrations (MICs)

MIC Key:

A = MlC's of 1.0 Mg/mL or less

B = MlC's of greaier than 1.0 g/mL to 8.0 Mg/mL

C = MlC's of greater than 8.0 Mg/mL to 16.0 Mg/mL

D = MlC's of greater than 16.0 Mg/mL

*AEC001 is E, Coii is ATCC 25922; APAE001 is Pseudomonas aeruginosa ATCC27853; It should be understood that the organic compounds according to the invention may exhibit the phenomenon of tautomerism. As the chemical structures within this specification can only represent one of the possible tautomeric forms, it should be understood that the invention encompasses any tautomeric form of the drawn structure.

Furthermore, while particular embodiments of the present invention have been shown and described herein for purposes of illustration, it will be understood, of course, that the invention is not limited thereto since modifications may be made by persons skilled in the art, particularly in light of the foregoing teachings, without deviating from the spirit and scope of the invention. Accordingly, the invention is not limited except as by the appended claims.

Ail of the U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications and non-patent publications referred to in this specification are incorporated herein by reference, in their entirety to the extent not inconsistent with the present description.