KR20140023869A - Hydroxamic acid derivatives and their use in the treatment of bacterial infections - Google Patents

Abstract

The present invention provides an antimicrobial compound of formula (I) as well as stereoisomers or pharmaceutically acceptable salts thereof; Pharmaceutical compositions comprising such compounds; Administering such compounds to treat bacterial infections; The use of such compounds in the treatment of bacterial infections; And methods of making such compounds:

Description

Hydrroxamic acid derivatives and their use in the treatment of bacterial infections

Cross-reference to related application

This application claims the benefit under 35 USC §119 (e) of US Provisional Application No. 61 / 412,311, filed November 10, 2010, which is incorporated herein by reference in its entirety.

A declaration of government interest

The present invention was made with government support under arrangement HDTRA1-07-C-0079 awarded by the US Department of Defense. The US government has certain rights in the invention.

The present invention generally relates to treating infections caused by Gram-negative bacteria. More specifically, the invention disclosed herein treats Gram-negative infections by inhibiting the activity of UDP-3-O- (R-3-hydroxydecanoyl) -N-acetylglucosamine deacetylase (LpxC). It's about things. The present invention provides small molecule inhibitors of LpxC, pharmaceutical formulations containing such inhibitors, methods of treating patients using such pharmaceutical formulations, and methods of preparing such pharmaceutical formulations and inhibitors. The invention disclosed herein is a compound that can inhibit the activity of UDP-3-O- (R-3-hydroxydecanoyl) -N-acetylglucosamine deacetylase (LpxC) alone or as a second antibacterial agent. And to treat Gram-negative infections by administration in combination with the agent.

Over the past decades, the association between antimicrobial resistance and its serious infectious disease has increased at an alarming rate. The problem of antibacterial resistance is exacerbated by the presence of bacterial strains resistant to many antibacterial agents. Thus, there is a need for new antibacterial agents, in particular antibacterial agents with new mechanisms of action. A previously conserved target, LpxC, which has not been used previously, is a new class of active bactericidal chemicals that should suffer from target-related tolerance and should be naturally-occurring as needed. It provides new opportunities for the development of broad-spectrum antibacterial small molecules, including beings. LpxC (enzyme uridildiphospho-3-O- (R-hydroxydecanoyl) -N-acetylglucosamine deacetylase) is present across all gram-negative bacterial species of interest and is the first in outer membrane biosynthesis. It is involved in the committed phase of Thus, LpxC is essential for survival and provides an ideal target for antibiotic activity of Gram-negative bacterial species.

The researchers identified several compounds with antibacterial activity that target lipid A biosynthesis. For example, Jackman et al. (J. Biol. Chem., 2000, 275 (15), 11002-11009); Wyckoff et al. Trends in Microbiology, 1998, 6 (4), 154-159; US Patent Publication No. 2001/0053555 (published 20 December 2001, corresponding to PCT International Publication No. WO 98/18754, published May 7, 1998); PCT International Publication No. WO 00/61134 (published 19 October 2000); US Patent Publication No. 2004/0229955 (published November 18, 2004); And PCT WO 2008/154642 (published December 18, 2008) all disclose compounds with anti-LpxC activity of antibacterial. Commercial development of these LpxC inhibitors has been difficult due to the toxicity of these compounds in mammals at their concentrations or near their concentrations required for antibacterial activity. Compounds provided herein have significantly better resistance than other closely related compounds having anti-LpxC activity.

Although progress has been made in the art, there remains a need for LpxC inhibitors that have activity as bactericides against Gram-negative bacteria and have an acceptable toxicity / tolerance profile. It is therefore an object of the present invention to provide compounds for use in the preparation of non-toxic antibacterial agents and combinations of these compounds with other agents capable of inhibiting Gram-negative bacterial infection. Another object of the present invention is to provide a synergistic combination of an antibacterial agent and an LpxC inhibitor, which has the inherent antibacterial properties as well as the ability to improve the permeability of the outer membrane of Gram-negative bacteria to other antibacterial agents. The use of synergistic combinations of drugs may result in reduced side effects of the drug due to the lower dosage or shorter duration of treatment used, faster treatment to shorten hospitalization, increased range of controlled pathogens, and resistance to antibiotics. It may have many advantages over conventional single compound chemotherapy, including reduced incidence of the development of.

The present invention provides a novel compound, a pharmaceutical formulation comprising said compound, a method of inhibiting UDP-3-O- (R-3-hydroxydecanoyl) -N-acetylglucosamine deacetylase (LpxC), and Gram- Provided are methods for treating negative bacterial infections.

In one aspect, the present invention provides a compound of formula (I) and its stereoisomers and pharmaceutically acceptable salts thereof:

Wherein A is substituted C ₃ -C ₆ cycloalkyl, wherein at least one substituent is C ₁ -C ₃ primary alcohol; B is absent or is -CH = CH-, -C≡C- or unsubstituted phenyl; C is -CH = CH-, -C≡C- or unsubstituted phenyl, wherein if B is -CH = CH-, then C is not -CH = CH-; R ¹ , R ² and R ³ are independently selected from hydrogen and substituted or unsubstituted C ₁ -C ₃ alkyl, or R ¹ and R ² are unsubstituted C ₃ -C with the carbon atom to which they are attached Form a ₆ cycloalkyl group, or R ² and R ³ together with the carbon atom and Q to which they are attached form a substituted or unsubstituted heterocyclic ring having 5 to 8 ring atoms, wherein hetero 1-2 ring atoms of the cyclic ring are selected from N, O and S; Q is O or NR, where R is hydrogen or unsubstituted C ₁ -C ₃ alkyl.

In another aspect, the present invention provides a pharmaceutical composition comprising a compound of formula (I), a stereoisomer or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or diluent.

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

In another aspect, the invention provides a method for affecting bacterial growth comprising administering an LpxC-inhibitory compound of Formula I, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof to a patient in need of such inhibition. Provided are methods for inhibiting deacetylase enzymes in negative bacteria.

In another aspect, the invention provides Gram to control the toxicity of a bacterial infection comprising administering an LpxC-inhibiting compound of Formula I, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof to a patient in need of such inhibition. Provides a method of inhibiting LpxC in negative bacteria.

In another aspect, the invention provides a method of treating a subject with a bacterial infection comprising administering to a subject in need thereof an antibacterial effective amount of a compound of Formula (I), a stereoisomer thereof, or a pharmaceutically acceptable salt thereof. Provide a way to. In a more specific embodiment of the method of treatment, the bacterial infection is a Gram-negative bacterial infection. In one such embodiment, the bacteria is a Pseudomonas rugi Ah industrial (Pseudomonas aeruginosa), Burke holde Liao (Burkholderia) (e.g., Burg holde Ria Sepharose cyano (Burkholderia cepacia)), Enterococcus when the bacteria years old child (Enterobacteriaceae), Francis Cellar years old child in (Franciscellaceae) (for example, Francis Cellar Tula alkylene sheath (Franciscella tularensis)), Serratia marcescens (Serratia), Proteus (Proteus), keulrep Ella ( Klebsiella), Enterobacter bakteo (Enterobacter), a sheet bakteo (Citrobacter), Salmonella (Salmonella), Providencia (Providencia), Yersinia (Yersinia) (e.g., Yersinia pestiviruses switch (Yersinia pestis)), a know is Nella (Morganella) or Escherichia coli (Escherichia coli). In one particular embodiment, the bacteria is a rugi labor, Burke holde Liao, Francis Cellar Seah Ah Pseudomonas, Enterobacter, Yersinia, or Escherichia coli. In one particular embodiment, the bacterium is Pseudomonas aeruginosa . In another such embodiment, the bacterium is Escherichia coli . In another embodiment, the bacterium is Stenotrophomonas maltophila), when in crab alkali to Ness xylene thiooxidans (Alcaligenes xylosoxidans ), Haemophilus , Neisseria species , Cedecea species or Edwardsiella species ). In another specific embodiment, the subject is a mammal, and in certain embodiments, a human.

One aspect of the invention provides a pharmaceutical composition comprising an inhibitor of LpxC and a second antibacterial agent. In one embodiment, the second antibacterial agent is a group consisting of vancomycin, linezolid, azithromycin, imipenem, teicoplanin, daptomycin, clindamycin, rifampin, cefotaxime, gentamycin, novobiocin, and telavancin Is selected from. In one such implementation, the second antibacterial agent is vancomycin or rifampin. In another embodiment, the LpxC inhibitor is a compound of Formula I: or a stereoisomer thereof or a pharmaceutically acceptable salt thereof:

Wherein A is substituted C ₃ -C ₆ cycloalkyl, wherein at least one substituent is C ₁ -C ₃ primary alcohol; B is absent or is -CH = CH-, -C≡C- or unsubstituted phenyl; C is -CH = CH-, -C≡C- or unsubstituted phenyl, wherein if B is -CH = CH-, then C is not -CH = CH-; R ¹ , R ² and R ³ are independently selected from hydrogen and substituted or unsubstituted C ₁ -C ₃ alkyl, or R ¹ and R ² are unsubstituted C ₃ -C with the carbon atom to which they are attached Form a ₆ cycloalkyl group, or R ² and R ³ together with the carbon atom and Q to which they are attached form a substituted or unsubstituted heterocyclic ring having 5 to 8 ring atoms, wherein hetero 1-2 ring atoms of the cyclic ring are selected from N, O and S; Q is O or NR, where R is hydrogen or unsubstituted C ₁ -C ₃ alkyl.

Another aspect of the invention provides a method of treating a patient with a Gram-negative bacterial infection comprising co-administering a synergistic amount of an antibacterial agent and an LpxC inhibitor of Formula I, eg, a synergistic amount in vivo. Provide a method. In one embodiment, the antibacterial agent is selected from the group consisting of vancomycin, lineezolide, azithromycin, imipenem, teicoplanin, daptomycin, clindamycin, rifampin, cytotaxime, gentamycin, novobiocin, and telavancin do. In one such implementation, the antibacterial agent is vancomycin or rifampin.

These and other aspects of the invention will be apparent with reference to the following detailed description.

1 shows in vivo synergy of compound I-1 and vancomycin in bacterial strain ATCC 43816.

The present invention provides new compounds, methods of inhibiting LpxC in Gram-negative bacteria, and new methods of treating bacterial infections. The compounds provided herein can be formulated into pharmaceutical formulations and medicaments useful in the methods of the invention. The present invention also provides the use of the compound in the manufacture of pharmaceuticals and pharmaceutical formulations, the use of the compound in inhibiting LpxC, and the use of the compound in treating a bacterial infection in a subject. In addition, the present invention is to provide a compound which can inhibit the activity of UDP-3-O- (R-3-hydroxydecanoyl) -N-acetylglucosamine deacetylase (LpxC) alone or claim 2 Provided are compositions and methods for treating Gram-negative infections in combination with administering bacterial compounds.

A. Definitions

The following abbreviations and definitions are used throughout this application:

"LpxC" is an abbreviation for UDP-3-O- (R-3-hydroxydecanoyl) -N-acetylglucosamine deacetylase.

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

"Alkyl" refers to a monovalent saturated aliphatic hydrocarbon group having 1 to 10 carbon atoms, preferably 1 to 6 carbon atoms. Such terms include, by way of example, linear and branched hydrocarbon 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 -), iso-butyl _{((CH 3) 2 CHCH 2} -), sec-butyl _{((CH 3) (CH 3} CH 2) CH-) , t -butyl ((CH ₃ ) ₃ C-), n -pentyl (CH ₃ CH ₂ CH ₂ CH ₂ CH _2- ), and neopentyl ((CH ₃ ) ₃ CCH _2- ).

"Alkoxy" refers to an -O-alkyl group, where alkyl is as defined herein. Alkoxy includes methoxy, ethoxy, n -propoxy, isopropoxy, n -butoxy, t -butoxy, sec -butoxy, n -pentoxy and the like.

"Amino" refers to the group -NH ₂ .

"Primary alcohol" refers to the group -alkyl-OH, where the hydroxyl radical is linked to the primary carbon. Examples include -CH ₂ OH (hydroxymethyl), -CH ₂ CH ₂ OH (hydroxymethyl) and -CH (CH ₃ ) CH ₂ OH (1-hydroxypropan-2-yl).

"Alkenyl" has 2 to 6 carbon atoms, preferably 2 to 4 carbon atoms, and has at least 1, preferably 1 to 2 vinyl (> C = C <) unsaturated sites Mention is made of linear or branched hydrocarbon groups. Examples of such groups are vinyl, allyl, and but-3-en-1-yl. Within these terms, cis and trans isomers or mixtures of these isomers are included.

"Alkynyl" is a straight or branched chain having 2 to 6 carbon atoms, preferably 2 to 3 carbon atoms, and having at least 1, preferably 1 to 2 acetylenic -C≡C- unsaturated sites Mention is made of monovalent hydrocarbon groups. Examples of such alkynyl groups include acetylenyl (-C≡CH), and propargyl (-CH ₂ C≡CH).

"Carboxyl" or "carboxy" refers to -COOH or a salt thereof.

"Cyano" or "nitrile" refers to the -CN group.

"Cycloalkyl" refers to a cyclic alkyl group of 3 to 13 carbon atoms having a single bond. Examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl, and the like.

"Guanidino" refers to the -NHC (= NH) NH ₂ group.

"Halo" or "halogen" refers to fluoro, chloro, bromo, and iodo, typically fluoro or chloro.

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

"Heterocycle", "heterocyclic" and "heterocyclyl" refer to a saturated or unsaturated group having a single ring, having 3 to 15 ring atoms, and containing 1 to 4 hetero atoms. These ring atoms are selected from the group consisting of nitrogen, sulfur or oxygen. In one embodiment, the nitrogen and / or sulfur atom (s) of the heterocyclic group are optionally oxidized to provide an N-oxide, -S (O)-, or -SO ₂ -moiety.

"Nitro" refers to the group -NO ₂ .

"Nitroso" refers to the group -NO.

"Oxo" refers to the atom (= O).

"Substituted" is alkoxy, acyl, acylamino, acyloxy, amino, aminocarbonyl, aminothiocarbonyl, aminocarbonylamino, aminothiocarbonylamino, aminocarbonyloxy, amidino, carboxyl, carboxyl Esters, (carboxy ester) amino, (carboxy ester) oxy, cyano, guanidino, halo, hydroxy, nitro, SO ₃ H, sulfonyl, sulfonyloxy, thioacyl, thiol, and alkylthio Reference is made to groups having at least one hydrogen replaced with a substituent selected from the group consisting of, wherein the substituents are as defined herein. In certain substituted cyclic groups, "substituted" also refers to a group having two hydrogens replaced with a single double bonded oxygen atom (oxo group) or a single double bonded sulfur atom (thioxo). In some implementations, the substituted group has 1 to 3 aforementioned substituents. In other implementations, the substituted group has 1 to 2 aforementioned substituents.

"Sulfonyl" refers to -SO ₂ -alkyl, -SO ₂ -substituted alkyl, -SO ₂ -alkenyl, -SO ₂ -substituted alkenyl groups, wherein alkyl, substituted alkyl, alkenyl, substituted Alkenyl, alkynyl, and substituted alkynyl are as defined herein. Sulfonyl includes groups such as methyl-SO _2- .

"Sulfonyloxy" is -OSO ₂ - alkyl, -OSO ₂ - substituted alkyl, -OSO ₂ - alkenyl, -OSO ₂ - substituted alkenyl, -OSO ₂ - alkynyl, -OSO ₂ - substituted alkynyl Nyl groups are mentioned, wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, and substituted alkynyl are as defined herein.

"Thioacyl" refers to HC (S)-, alkyl-C (S)-, substituted alkyl-C (S)-, alkenyl-C (S)-, substituted alkenyl-C (S)-, alky Nyl-C (S)-, and substituted alkynyl-C (S)-groups, wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, and substituted alkynyl are described herein. As defined in

"Thiol" refers to the group -SH.

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

"Alkylthio" refers to an -S-alkyl group, where alkyl is as defined herein. In another implementation, sulfur can be oxidized to -S (O)-. Sulfoxides may exist as one or more stereoisomers.

Unless otherwise stated, the nomenclature of substituents not explicitly defined herein is performed by naming the terminal portion of the functional group followed by contiguous functional groups for the point of attachment. For example, the substituent "arylalkyloxycarbonyl" refers to the (aryl)-(alkyl) -O-C (O)-group.

In general, reference to a specific element, such as hydrogen or H, is meant to include all isotopes of that element. For example, if a substituent is defined to include hydrogen or H, it also includes deuterium and tritium.

The present invention also encompasses isotopically-labelled compounds of the present invention, which, in addition to the fact that one or more atoms have been replaced by atoms having an atomic weight or atomic mass number different from the atomic or atomic mass numbers generally found in nature, Structurally identical to the compounds disclosed in the specification. Examples of isotopes that may be included in the compounds of the present invention are isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine and chlorine, such as ² H, ³ H, ¹³ C, ¹⁴ C, ¹⁵ N, ¹⁸ O, ¹⁷ O, ³¹ P, ³² P, ³⁵ S, ¹⁸ F and ³⁶ Cl, respectively. Compounds of the invention, prodrugs thereof, and pharmaceutically acceptable salts of the compounds and the prodrugs that contain the aforementioned isotopes and / or other isotopes of other atoms are within the scope of the present invention. Certain isotopically labeled compounds of the invention, such as those bound to radioisotopes such as ³ H and ¹⁴ C, are useful for drug and / or substrate tissue distribution assays. Tritium, ie ³ H, and carbon-14, ie ¹⁴ C isotopes are particularly preferred because of their ease of preparation and detectability. In addition, substitution with heavier isotopes such as deuterium, i.e. ² H, may provide certain therapeutic benefits resulting from greater metabolic stability, such as, for example, increased in vivo half-life or reduced dosage requirements. Therefore, it may be desirable in some situations. Isotopically labeled compounds and prodrugs thereof of the present invention are generally subjected to known or referenced procedures and replaced by readily available isotopically labeled reagents in place of isotopically unlabeled reagents. It can manufacture.

"Stereoisomers" and "stereoisomers" refer to compounds having the same atomic connectivity but different atomic arrangements in space. Stereoisomers include cis-trans isomers, E and Z isomers, enantiomers, and diastereomers.

A "tautomer" is a form of alternating molecules with different proton positions, such as enol-keto and imine-enamine tautomers, or heteroaryls containing a -N = C (H) -NH- ring atomic arrangement. Tautomeric forms of groups such as pyrazole, imidazole, benzimidazole, triazole, and tetrazole. Those skilled in the art will appreciate that arrangements of ring atoms of other tautomers are possible.

"Patient" refers to humans and non-human animals, especially mammals.

"Pharmaceutically acceptable salts" include those salts derived from a variety of organic and inorganic counter ions that are well known in the art and include by way of example only sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium, and the like; Pharmaceutically acceptable salts of compounds wherein the molecules contain basic functional groups, salts of organic or inorganic acids, such as hydrochloride, hydrobromide, tartrate, mesylate, acetate, maleate, oxalate, phosphate, sulfate, and the like. To mention.

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

The term "synergy" or "synergistic" as used herein means that the binding effect of the compound when used in combination is greater than the effect of addition of the compound when used alone. "Synergism" can be defined quantitatively as a Split Inhibitory Concentration Index (FICI) of ≤ 0.5, where FICI is the sum of the Split Inhibitory Concentrations (FIC) of individual components in a combination of two compounds FIC is defined as the ratio of the MIC of the compounds of the combination divided by the minimum inhibitory concentration (MIC) of the compound alone:

Alternatively, the "synergistic effect", more particularly the "in vivo synergy" is at least a 2-fold reduction in the static dose of the agent used in combination compared to the LpxC inhibitor or the second antibacterial agent alone. It can be defined quantitatively. In certain cases, one agent alone may never reach a static dosage. In this case, the combination may be stopped by the combined administration of two compounds in which bacterial growth alone cannot reach stasis (the same 24 hours as measured at 0 hours after infection). CFU load is synergistic.

“Combination administration” combines a single dosage form containing active agents (eg, a pharmaceutically acceptable excipient with a compound of the invention and a second antibacterial agent, wherein the two active ingredients are each optionally In separate excipient mixtures designed to independently control the rate and duration of release) or by separate administration of individual formulations containing the active agents. "Concomitant administration" also refers to simultaneous administration (as compound of the invention and second antibacterial agent, as long as both the compound of the invention and the second antibacterial agent are present in the body at a therapeutically effective concentration for at least partially overlapping time). Simultaneous administration) and time modified administration (administration of a compound of the present invention at a different time than the second antibacterial agent).

The term "antibacterial agent" refers to an agent having either bactericidal or bacteriostatic activity. The term "growth inhibition" indicates that the rate of increase of the population of a particular bacterium is reduced. Thus, the term includes not only situations in which the bacterial population increases but increases at a reduced rate, as well as situations in which the growth of the population is stopped, as well as situations in which the number of bacteria in the population is reduced or even the population is removed. When enzyme activity assays are used for screening for inhibitors, modifications can be made to the compound, such as intake / spill, solubility, half-life, etc., to correlate enzyme inhibition with growth inhibition. The activity of the antibacterial agent is not necessarily limited to bacteria, and may also include activity against parasites, viruses and fungi.

Unless the context otherwise requires, throughout the specification and the claims that follow, the words "comprises" and variations thereof, such as "comprises" and "comprising", are in an open, comprehensive sense, ie, "including, Is not limited thereto. "

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 phrases “in one embodiment” or “in one embodiment” appearing in various places throughout this specification are not necessarily all referring to the same embodiment. In addition, the particular feature, structure, or characteristic may be combined in any suitable manner in one or more embodiments.

B. Compounds, Compositions and Uses thereof

In one aspect, the present invention provides a compound of formula (I) and its stereoisomers and pharmaceutically acceptable salts thereof:

Wherein A is substituted C ₃ -C ₆ cycloalkyl, wherein at least one substituent is C ₁ -C ₃ primary alcohol; B is absent or is -CH = CH-, -C≡C- or unsubstituted phenyl; C is -CH = CH-, -C≡C- or unsubstituted phenyl, wherein if B is -CH = CH-, then C is not -CH = CH-; R ¹ , R ² and R ³ are independently selected from hydrogen and substituted or unsubstituted C ₁ -C ₃ alkyl, or R ¹ and R ² are unsubstituted C ₃ -C with the carbon atom to which they are attached Form a ₆ cycloalkyl group, or R ² and R ³ together with the carbon atom and Q to which they are attached form a substituted or unsubstituted heterocyclic ring having 5 to 8 ring atoms, wherein hetero 1-2 ring atoms of the cyclic ring are selected from N, O and S; Q is O or NR, where R is hydrogen or unsubstituted C ₁ -C ₃ alkyl.

In certain embodiments, Q is NR and in some such embodiments, Q is NH or NCH ₃ . In certain embodiments, R ¹ , R ² , and R ³ are independently selected from hydrogen and substituted or unsubstituted C ₁ -C ₃ alkyl, and in some such embodiments, hydrogen and unsubstituted C _1- C ₃ alkyl. In certain embodiments, R ¹ and R ² are independently unsubstituted C ₁ -C ₃ alkyl. In certain embodiments, A is C ₃ -C ₆ cycloalkyl substituted with hydroxymethyl. In other embodiments, B and C are both -C≡C-.

One aspect of the present invention provides a compound of formula (I-A) and its stereoisomers and pharmaceutically acceptable salts thereof:

Wherein A is substituted C ₃ -C ₆ cycloalkyl, wherein at least one substituent is C ₁ -C ₃ primary alcohol; R ¹ , R ² and R ³ are independently selected from hydrogen and substituted or unsubstituted C ₁ -C ₃ alkyl, or R ¹ and R ² together with the carbon atom to which they are attached a C ₃ -C ₆ cycloalkyl group Or R ² and R ³ together with the carbon atom and nitrogen to which they are attached form a substituted or unsubstituted heterocyclic ring having 5 to 8 ring atoms, wherein the heterocyclic ring 1-2 ring atoms of are selected from N, O and S; R is hydrogen or unsubstituted C ₁ -C ₃ alkyl. In certain embodiments, R ¹ , R ² and R ³ are independently selected from hydrogen and substituted or unsubstituted C ₁ -C ₃ alkyl, and in some such embodiments, hydrogen and unsubstituted C ₁ -C ₃ alkyl. In one embodiment, A is C ₃ -C ₆ cycloalkyl monosubstituted with a C ₁ -C ₃ primary alcohol.

Compounds of the present invention include those listed in Table 1 below. Certain compounds shown in Table 1 represent mixtures of two diastereomers. In this case, the "*" indicates that the two parts attached to the cyclic part are trans with respect to each other. "# *" Indicates that the two parts attached to the cyclic part are cis relative to each other.

Compounds of the present invention can be readily synthesized using the methods described herein, or other methods well known in the art. For example, the synthesis of hydroxamic acid or similar scaffolds with a wide variety of substituents is described by Kline, T., et al., “Potent, novel in vitro inhibitors of the Pseudomonas aeruginosa deacetylase LpxC " J. Med Chem . 2002, 45 (14), 3112-29; US Pat. No. 5,925,659; Pirrung, MC, et al.," A Convenient Procedure for the Preparation of Amino Acid Hydroxamate from Esters " J. Org Chem 1995, 60, 8084-8085; ... Nhu, K., et al, "A New and Efficient Solid Phase Synthesis of Hydroxamic Acids" J. Org Chem 1997, 62, 7088-7089;.. PCT International Publication WO98 / 18754; Mellor, SL, et al., "N-Fmoc-aminoxy-2-chlortrityl Polystyrene Resin: A Facile Solid-phase Methodology for the Synthesis of Hydroxamic Acids" Tetrahedron Lett . 1997 , 38, 3311-3314; Khan, SI, et al., "A Facile and Convenient Solid-phase Procedure for Synthesizing Nucleoside Hydroxamic Acids" Tetrahedron . Lett . 1998 , 39, 8031-8034; Zhang, Y., et al., "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 al., “Synthetic Reactions by Complex Catalysts XXXV” Syn . Commun . 1974 , 4, 97-103; Witte, H., et al., "Cyclische Imidsaurester aus Nitrilen 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, RJ, et al., "Total Synthesis of Thiangazole, A Novel Naturally Occurring HIV-1 Inhibitor from Polyangium sp." Tetrahedron 1995 , 51, 7321-7330; Galeotti, N., et al., "Synthesis of Peptidyl Aldehydes from Thiazolines" Tetrahedron . Lett . 1997 , 38, 2459-2462; Charette, AB, et al., "Mild Method for the Synthesis of Thiazolines from Secondary and Tertiary Amides" J. Org . Chem . 1998 , 63, 908-909; Bergeron, RJ, et al., "Effects of C-4 Stereochemistry and C-4 'Hydroxylation on the Iron Clearing Efficiency and Toxicity of Desferrithiocin Analogues" J. Med . Chem. 1999 , 42, 2432-2440; Raman, P., et al., "Titanium (IV) -mediated Tandem Deprotection-cyclodehydration of Protected Cysteine N-Amides: Biomimetic Synthesis of Thiazoline- and Thiazole-containing Heterocycles" Org . Lett . 2000 , 2, 3289-3292; Fernandez, X., et al., "Novel Synthesis of 2-Thioazolines" Tetrahedron Lett . 2000 , 41, 3381-3384; And Wipf, P., et al., "C. Thiolysis of Oxazolinenes: A New, Selective Method for the Direct Conversion of Peptide Oxazolines into Thiazolines" Tetrahedron Lett . Extensively reviewed in 1995 , 36, 6395-6398, which are incorporated herein by reference.

In another aspect, the present invention provides a pharmaceutical composition comprising a compound of formula (I) or a stereoisomer or pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or diluent.

In another aspect, the invention provides Gram-negative bacteria for affecting bacterial growth comprising administering a compound of Formula I, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof to a patient in need of such inhibition. Provided are methods for inhibiting deacetylase enzymes in a stomach.

In another aspect, the invention provides a bacterial infection by inhibiting LpxC in Gram-negative bacteria comprising administering to a patient in need thereof such a compound of formula (I), a stereoisomer thereof or a pharmaceutically acceptable salt thereof Provides a way to control toxicity. In certain embodiments of the method of inhibiting LpxC using a compound of the present invention, the IC ₅₀ value of the compound is 10 μM or less for LpxC. In other embodiments, the IC ₅₀ value is 1 μM or less, 0.1 μM or less, 0.050 μM or less, 0.030 μM or less, 0.025 μM or less, 0.010 μM or less for LpxC.

In another aspect, the present invention is directed to a gram-negative bacterial infection comprising administering an antibacterial effective amount of a compound of formula (I) or a stereoisomer thereof or a pharmaceutically acceptable salt thereof to a subject in need thereof. Provide a method of treating a subject. In one such embodiment, the bacterium is Pseudomonas aeruginosa , Berkholderia (eg, Berkholdereria Sephacia ), Enterobacteriaceae , Franciscellacea (eg, Franciscella Tula alkylene cis), Serratia, Proteus, keulrep when Ella, Enterobacter bakteo, sheet by bakteo, Salmonella, Providencia, Yersinia (e.g. Yersinia pestiviruses's), know the Nella or Escherichia coli to be. In one particular embodiment, the bacterium is Pseudomonas aeruginosa , Berkholdereria , Francis Celacea , Enterobacter , Yersinia or Escherichia coli . In one such embodiment, the bacterium is Pseudomonas aeruginosa . In another such embodiment, the bacterium is Escherichia coli S. Kane. In another embodiment, the bacterium is Stenotropomonas maltophila, Alkaligenes xyloxyxidans, Haemophilus, Neisseria spp., Cedesea spp. Or Edward siela spp.

In certain embodiments, the subject can be a mammal and in some embodiments can be a human.

Bacterial infections susceptible to treatment in accordance with the invention include primary and co-infection caused by bacterial species and, for example, one or more additional infectious agents such as bacteria, viruses, parasites and fungi. It includes.

The compounds of the invention can be used to treat conditions caused by bacteria using LpxC in bacterial production of endotoxins, in particular Gram-negative bacteria, and biosynthesis of lipopolysaccharide (LPS) or endotoxins.

The compounds of the present invention are also acute in conditions caused or exacerbated by bacterial production of lipid A and LPS or endotoxins such as sepsis, septic shock, systemic inflammation, local inflammation, chronic obstructive pulmonary disease (COPD) and chronic bronchitis Useful for treating aggravation (AECB). For these conditions, treatment comprises administering a compound of the invention, or a combination of compounds of the invention, optionally in combination with a second agent, wherein the second agent is a second antibacterial or non-antibacterial agent. Jay.

For sepsis, septic shock, systemic inflammation, local inflammation, chronic obstructive pulmonary disease (COPD) and acute exacerbation of chronic bronchitis (AECB), representative non-antibacterial agents are endotoxin receptor-binding antibodies, endotoxin-binding antibodies Anti-toxins, including anti-CD14-binding protein antibodies, anti-lipopolysaccharide-binding protein antibodies, and tyrosine kinase inhibitors.

In the treatment of severe or chronic airway infections, the compounds of the present invention may also be used with non-antibacterial agents administered by inhalation. Representative non-antibacterial agents used in such treatments include anti-inflammatory steroids, non-steroidal anti-inflammatory agents, bronchodilators, mucolytics, anti-asthmatic agents, and lung fluid surfactants. In particular, the non-antibacterial agents include albuterol, salbuterol, budesonide, beclomethasone, dexamethasone, nidochromyl, beclomethasone, fluticasone, flunisolide, triamcinolone, ibuprofine, lofecoxib, Naproxen, celecoxib, nidochromyl, ipratropium, metaproterenol, pybuterol, salmeterol, formoterol, indacaterol, bronchodilators, expectorants, calfactants, belacants ( beractant, poractant alfa, sulfaxin or pulmozyme (also called domase alfa).

The compounds of the invention alone or in, or Enterococcus bakteo Oh Eroge Ness (Enterobacter aerogenes), Enterobacter in bakteo claw ACCA (Enterobacter cloacae), Escherichia coli, keulrep when Ella pneumoniae in (Klebsiella pneumoniae ), Klebsiella Oxy cytokine (Klebsiella oxytoca), Proteus Mira Billy's (Proteus mirabilis), Serratia Mar Seth sense (Serratia marcescens), Pomona to Stephen's Notes Maltophilia ( Stenotrophomonas maltophilia ), Pseudomonas aeruginosa , Berkholderia Sepharose cyano, when in a Alcaligenes xylene thiooxidans, flat Labo tumefaciens menin gosep tikum (Flavobacterium meningosepticum), Providencia Stuttgart are CHANTILLY (Providencia stuartii) and a sheet bakteo Serious lung and nosocomial infections such as those caused by the presence undi (Citrobacter freundi), by the writing brush Russ influenza (Haemophilus Influenzae), Legionella species (Legionella species), morak Cellar Qatar Harleys (Moraxella catarrhalis), Blanc and melanoma Qatar Harleys (Branhamella catarrhalis), Enterobacter species, keulrep when Ella species, and group (community) such as those caused by Proteus species lung infections, and Nei serie O species (Neisseria species), Shigella species (Shigella species), Salmonella species, Helicobacter Pylori ( Helicobacter pylori), Vibrio or years old child in (Vibrionaceae) and Bor detel La species (Bordetella species) during Brucella species (Brucella species) as well as the infection caused by other bacterial species, such as a cellar Francisco Tularensis and / or Yersinia The may be used in combination with a second antibacterial agent for the treatment of serious or chronic respiratory tract infections, including infections caused by the pace tooth.

When used to treat a subject infected with a Gram-negative bacterial infection, the compounds of the present invention can be used to sensitize Gram-negative bacteria to the effect of the second antibacterial agent.

The present invention provides methods for treating subjects infected with Gram-negative bacteria, as well as new combinations of compounds comprising a compound of Formula (I) or a stereoisomer thereof or a pharmaceutically acceptable salt thereof. The novel combinations provided herein can be formulated into pharmaceutical formulations and medicaments useful in the methods of the invention. The present invention also provides the use of the new combination in the manufacture of pharmaceuticals and pharmaceutical formulations for use of the combination in treating a bacterial infection in a patient.

One traditional method for assessing synergy, referred to as checkerboard assay, is used to predict the efficacy of antibacterial agents, which is described in Scribner et. al., (1982, Antimicrobial Agents and Chemotherapy 21 (6): 939-943) and Goodman & Gilman (1980, The Pharmacological Basis of Therapeutics, Sixth Edition, pp. 1097-1098). The checkerboard assay involves making a series of 2-fold dilutions of individual antibiotics and combinations thereof in broth, and then inoculating the microorganisms tested therein. After incubation, the minimum inhibitory concentration (MIC) of each drug used separately and the drug used in combination is measured (Note, MIC is the lowest concentration of drug that inhibits growth in the medium). Synergistic effects indicate a decrease in the MIC of each drug when used in combination. Antagonism refers to an increase in the MIC of either or both drugs when used in combination. Alternative methods of assessing synergy are described in Greco, et al., Pharmacological Reviews 47 (2): 331-285 (1995), which is incorporated herein by reference in its entirety.

However, positive results in a checkerboard assay, i.e., results that exhibit synergy below the MIC, do not necessarily lead to synergistic behavior in vivo. U.S. Patent Application Publication No. 2004-229955A1 discloses : Erythromycin and LpxC inhibitors against E. coli strain ATCC 25922 N-[(1S) -1- (aminomethyl) -2- (hydroxyamino) -2oxoethyl] -4- (4- {4-[({ Strong synergy between [(3methylphenyl) methyl] amino} acetyl) amino] phenyl} buta-1,3diynyl) benzamide has been reported. PCT International Application No. PCT / US2010 / 33910 demonstrated that the combination of erythromycin and various LpxC inhibitors showed no synergy in vivo.

LpxC, an essential gene of Gram-negative bacteria, encodes the uridildiphospho-3-O- (R-hydroxydecanoyl) -N-acetylglucosamine deacetylase enzyme. These enzymes catalyze an early devoted stage in the biosynthesis of lipid A, the lipid portion of lipopolysaccharide, an essential component of all Gram-negative bacteria. Above MIC, LpxC inhibitors are expected to destroy the outer membrane, thus allowing other antibacterial compounds to penetrate the outer membrane. Once these agents have penetrated the outer membrane, they can affect the periplasmic target, such as with vancomycin, or then diffuse across the inner membrane to allow ribosomes (erythromycin) or RNA polymerase (rifampin) May interact with a target in the cell. In the absence of LpxC inhibitors, the ability of agents such as vancomycin to access these targets is greatly diminished by the outer membrane. Thus, without wishing to be bound by theory, it is believed that the biochemical mechanisms that may be the basis for the observed synergy are the enhanced permeability of the outer membrane to agents such as vancomycin when combined with LpxC inhibitors.

In one embodiment, the second antibacterial agent used in combination with a compound of formula (I) or a stereoisomer thereof or a pharmaceutically acceptable salt thereof is vancomycin, linezolide, azithromycin, imipenem, teicoplanin, dapto Mycin, clindamycin, rifampin, cefotaxime, gentamycin, novobiocin, or telavancin. In one such embodiment, the second antibacterial agent is vancomycin, teicoplanin, rifampin, azithromycin, telavancin or novobiocin. In one such embodiment, the second antibacterial agent is vancomycin or rifampin. In some embodiments of the invention, the second antibacterial agent and / or the compound of formula (I) or stereoisomer or pharmaceutically acceptable salt thereof is administered at a dosage of up to a therapeutic dose, wherein Dosages are insufficient doses to treat bacterial infections when administered alone.

The pharmaceutical composition of the present invention comprises a compound of formula (I) or a stereoisomer or pharmaceutically acceptable salt thereof, formulated with one or more pharmaceutically acceptable carriers or diluents. As used herein, the term “pharmaceutically acceptable carrier” means a nontoxic inert solid, semisolid or liquid filler, diluent, encapsulating material or any type of formulation aid. Some examples of materials that can serve as pharmaceutically acceptable carriers include 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 wax; Oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; Glycols such as propylene glycol; Esters such as ethyl oleate and ethyl laurate; Agar; Buffers such as magnesium hydroxide and aluminum hydroxide; Alginic acid; Number of exothermic substances removed; Isotonic saline; Ringer's solution; ethyl alcohol; And phosphate buffers as well as other nontoxic miscible lubricants such as sodium lauryl sulfate and magnesium stearate, as well as colorants, release agents, coatings, sweeteners, flavors and flavorings, preservatives and antioxidants, as well as the designer's judgment. Depending on the composition. The pharmaceutical compositions of the present invention can be administered orally, rectally, parenterally (such as by intravenous, intramuscular or subcutaneous injection), intactisternally, vaginally, intraperitoneally, locally to humans and other animals. It can be administered as (by powder, ointment, or mucus), buccally, or as an oral or nasal spray, or as 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 can be, for example, water or other solvents, solvating and emulsifying agents such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1, Fatty acid esters of 3-butylene glycol, dimethylformamide, oils (especially cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil, and sesame oil), glycerol, tetrahydroperferyl alcohol, polyethylene glycol and sorbitan Inert diluents commonly used in the art, such as, and mixtures thereof. In addition to inert diluents, oral compositions may also include auxiliaries such as wetting agents, emulsifying and suspending agents, sweetening agents, flavoring agents, and flavoring 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. Sterile injectable preparations may also be sterile injectable solutions, suspensions or emulsions in nontoxic parenterally acceptable diluents or solvents, such as, for example, solutions in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, 1% lidocaine, 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 may be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid are used in the preparation of injectables.

Injectable formulations can be filtered, for example, through a bacterial-retaining filter, or in the form of sterile solid compositions that can be dissolved or dispersed in sterile water or other sterile injectable media prior to use. Can be sterilized by binding.

In order to prolong the effect of the drug, slow absorption of the drug from subcutaneous or intramuscular injection is often desirable. This can be achieved by using a liquid suspension of crystalline or amorphous material with low water solubility. The rate of absorption of the drug then depends upon its rate of dissolution which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of the parenterally administered drug form can be achieved 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-polyglycolide. Depending on the ratio of drug to polymer and the nature of the particular polymer used, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly (orthoesters) and poly (anhydrides). Depot injectable formulations may also be prepared by entrapping the drug in liposomes or microemulsions that are compatible with body tissues.

Compositions for rectal or vaginal administration are preferably suitable non-irritating excipients or carriers, such as cocoa butter, polyethylene glycol or the like, which are solid at ambient temperature or liquid at body temperature and therefore melt in the rectum or vaginal cavity to release the active compound. Suppositories that can be prepared by mixing suppository waxes and the compounds of the present invention.

Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active compound comprises at least one inert, pharmaceutically acceptable excipient or carrier, such as sodium citrate or dicalcium phosphate, and / or a) filler or extender such as starch, lactose, sucrose, glucose , Mannitol, and silicic acid, b) binders such as carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and acacia, c) humectants such as glycerol, d) disintegrants, 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 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 lauryl sulfate, and mixtures thereof. In the case of capsules, tablets and pills, the dosage form may also comprise a buffer.

Similar types of solid compositions may also be used using such excipients as lactose or lactose as well as high molecular weight polyethylene glycols and the like as fillers in soft and hard filled gelatin capsules.

Solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared using coatings and shells such as enteric coatings, and other coatings well known in the pharmaceutical formulation art. They may optionally contain an opacifying agent and may also be a composition which releases only the active ingredient (s), or preferentially, in a specific part of the intestine, or in an optionally delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes.

Similar types of solid compositions may also be used using such excipients as lactose or lactose as well as high molecular weight polyethylene glycols and the like as fillers in soft and hard filled gelatin capsules.

The antibacterial compound may also be in micro-encapsulated form using one or more excipients as indicated above. Solid dosage forms of tablets, dragees, capsules, pills and granules can be prepared using coatings and shells such as enteric coatings, release controlling coatings and other coatings well known in the pharmaceutical formulation art. In such solid dosage forms, the active compound may be mixed with at least one inert diluent such as sucrose, lactose or starch. In addition, such dosage forms, as usual practice, may include additional materials other than inert diluents, such as tableting lubricants and other tableting aids such as magnesium stearate and microcrystalline cellulose. In the case of capsules, tablets and pills, the dosage form may also comprise a buffer. They may optionally contain an opacifying agent and may also be a composition which releases only the active ingredient (s), or preferentially, in a specific part of the intestine, or in an optionally 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 compound is mixed under sterile conditions with a pharmaceutically acceptable carrier and any necessary preservatives or buffers that may be required. Ophthalmic formulations, ear drops, and the like are also contemplated as being within the scope of the present invention.

Ointments, pastes, creams and gels, in addition to the active compounds of the invention, include animal and vegetable fats, oils, waxes, paraffins, starches, tragacanths, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acids, talc and zinc oxide, Or excipients such as mixtures thereof.

Compositions of the present invention may also be formulated for delivery as a liquid aerosol or inhalable dry powder. Liquid aerosol formulations can be sprayed mostly to particle sizes that can be delivered to the bronchioles of the terminal and respiratory tracts where bacteria reside in patients with bronchial infections, such as chronic bronchitis and pneumonia. Pathogenic bacteria are often present throughout the airways down the bronchus, bronchioles and lung parenchema, especially below the bronchi of the terminal and respiratory tracts. During the exacerbation of the infection, bacteria may also be present in the alveoli. The liquid aerosol and inhalable dry powder formulations are preferably delivered to the terminal bronchioles and finally to parenchymal tissue across the bronchial tree.

The aerosolized formulations of the invention are preferably aerosol-forming devices, such as jets, vibrating porous plates or ultrasonics, preferably selected to allow the formation of aerosol particles having a mass mean diameter of mostly between 1 and 5 μm. It can be delivered using a nebulizer. In addition, the formulation preferably balances osmolarity, ionic strength and chloride concentration, with a minimum aerosolizable volume capable of delivering an effective dosage of a compound of the invention to the site of infection. In addition, aerosolized formulations preferably do not negatively impair airway function and cause undesirable side effects.

Suitable aerosolization devices for the administration of the aerosol formulations of the invention include, for example, jets, vibrating porous plates, ultrasonic nebulizers and energized dry powder inhalers, most of which are aerosols in the range of 1-5 pm in size. It is possible to spray the formulations of the invention in particle size. “Most” in this application means that at least 70% of all generated aerosol particles, but preferably at least 90%, range from 1 to 5 μm. The jet nebulizer is operated by air pressure which collapses the liquid solution into aerosol droplets. The vibrating porous plate nebulizer works by using a sonic vacuum created by a rapid vibrating porous plate that extrudes a droplet of solvent through the porous plate. Ultrasonic nebulizers work by piezoelectric crystals that shear liquid into small aerosol droplets. 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 Star7 jet nebulizers ( Pari Respiratory Equipment, Inc., Richmond, Va.), And Aerosonic (DeVilbiss Medizinische Produkte (Deutschland) GmbH, Heiden, Germany) and UltraAire7 (Omron Healthcare, Inc., Vernon Hills, Ill.) Ultrasonic nebulizers, Various suitable devices are available.

The compounds of the present invention may also be used in addition to the compounds of the present invention as topical powders and sprays which may contain excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicate and polyamide powders, or mixtures of these materials. It may be formulated for. Sprays may additionally contain common propellants, such as chlorofluorohydrocarbons.

Percutaneous patches have the additional advantage of providing controlled delivery of the 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 influx of compounds through the skin. The rate can be controlled by providing a rate controlling membrane or by dispersing the compound in a polymer matrix or gel.

According to the treatment method of the present invention, a bacterial infection is administered to a patient by administering to the patient a therapeutically effective amount of a compound of formula (I), or a stereoisomer or a pharmaceutically acceptable salt thereof, in an amount necessary for this to achieve the desired result. It is treated or prevented in patients such as humans or lower mammals. By "therapeutically effective amount" of a compound of the invention is meant a sufficient amount of the compound for treating a bacterial infection at a reasonable benefit / risk ratio applicable to any medical treatment. However, it will be understood that the total daily usage of the compounds and compositions of the present invention will be determined by the attending physician within the scope of sound medical judgment. Certain therapeutically effective dosage levels for any particular patient may include the disease to be treated and the severity of the disease; The activity of the specific compound used; The particular composition used; Age, weight, overall health, sex and diet of the patient; The time of administration, route of administration, and rate of excretion of the specific compound employed; Treatment period; Drugs used in combination or coincidental with the specific compound employed; And similar factors well known in the medical arts.

The total daily dose of a compound of the invention administered to a human or other mammal in single or divided doses may be, for example, an amount of 0.01 to 200 mg / kg body weight, or more generally 0.1 to 50 mg / kg body weight. . In certain embodiments, the total daily dose administered to a human or other mammal is 1.0 to 100 mg / kg body weight or 5.0 to 25 mg / kg body weight. Single dose compositions may contain amounts or submultiples thereof that make up the daily dose. In general, therapeutic regimens according to the present invention may be administered in single or multiple doses, from about 10 mg to about 15 g, more generally from 100 mg to 5 g, even more generally in a single or multiple doses per day. Or administering from 250 mg to 1 g per day in multiple doses to a patient in need of such treatment.

Formulation methods are well known in the art and are described, for example, in Remington: The Science and Practice of Pharmacy, Mack Publishing Company, Easton, Pa., 19th Edition (1995). Pharmaceutical compositions for use in the present invention may 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 present invention includes a container for containing a pharmaceutical composition and may also include a divided container, such as a divided bottle or a divided foil packet. The container may be any conventional shape or form as known in the art made of pharmaceutically acceptable materials, such as paper or cardboard boxes, glass or plastic bottles or jars, resealable bags ( For example, it may be a "refill" of tablets for placement in another container, or a blister pack in which individual doses are pressed from the pack according to a treatment schedule. The container used may depend on the exact dosage form involved, for example a conventional cardboard box will generally not be used to hold a liquid suspension. It is feasible to use more than one container together in a single package for marketing in a single dosage form. For example, tablets can be bottled and eventually put into boxes.

An example of such a kit is a so-called blister pack. Blister packs are well known in the packaging industry and are widely used for packaging in pharmaceutical unit dosage forms (tablets, capsules, etc.). The blister pack is generally made of a sheet of relatively rigid material, preferably covered with a foil of transparent plastic material. During the packaging process recesses are formed in the plastic foil. The recess may have the size and shape of an individual tablet or capsule to be packaged or may have a size and shape to accommodate a plurality of tablets and / or capsules to be packaged. A tablet or capsule is then placed in the recess and a sheet of relatively rigid material seals the plastic foil on the foil side in the opposite direction in which the recess is formed. As a result, the tablets or capsules are individually sealed or collectively sealed in recesses between the plastic foil and the sheet, as desired. Preferably, the strength of the sheet is such that the tablet or capsule can be removed from the blister pack by hand pressing on the recess, thereby forming an opening in the sheet at the position of the recess. The tablet or capsule can then be removed through the opening.

It may be desirable for written memory aids to be provided, the written memory aids providing information and / or instructions, for example, for a physician, pharmacist or other healthcare provider, or subject. It is a type that includes the number corresponding to the prescription date on which the tablet or capsule is to be taken in the form of a number next to the tablet or capsule, or in the form of a card containing the same type of information. Another example of such a memory aid is a calendar printed on a card, such as, for example, "first week, monday, tuesday," ... etc ... "second week, monday, tuesday, ...", and the like. . Another variant of the memory aid will be readily identifiable. A "daily dose" can be a single tablet or capsule or several tablets or capsules taken on a given day. If the kit comprises individual compositions, the daily dosage of one or more compositions of the kit may consist of one tablet or capsule, while the daily dosage of another one or more compositions of the kit may consist of several tablets or capsules.

Another specific embodiment of the kit is a dispenser designed to dispense the daily dosage one at a time in the intended order of use. Preferably, the dispenser is provided with memory-assisting means to further facilitate prescription compliance. An example of such a memory-assistance means is a mechanical counter indicating the number of daily doses dispensed. Another example of such a memory aid is an audible liquid crystal readout, or, for example, reading the date on which the last daily dose was taken and / or reminding the date on which the next dose should be taken. audible) Battery-powered microchip memory with reminder signals.

Kits of the invention may also comprise one or more additional pharmaceutically active compounds, in addition to the compounds of the invention. For example, the additional compound is a second antibacterial agent. The additional compound may be administered in the same dosage form as the compound of the invention or in a different dosage form. Likewise, the additional compound may be administered simultaneously or at another time with the compound of the present invention.

Compositions of the compounds of the present invention may also be useful in the preparation of (1) a number of organisms that may require other agents in a spectrum other than these compounds to enhance the treatment of severe Gram-negative infections covered by the spectrum of such compounds. It can be used in combination with other known antibacterial agents of a similar spectrum to add the coverage of this suspected severe infection. Potential agents include members of aminoglycosides, penicillins, cephalosporins, fluoroquinolones, macrolides, glycopeptides, lipopeptides and oxazolidinones. The treatment may involve the administration of a composition having both a compound of the invention and a second antibacterial compound, or administration of a compound of the invention, and subsequent or prior administration of a second antibacterial agent.

The foregoing may be better understood with reference to the following examples, which are provided for illustration and are not intended to limit the scope of the inventive concept.

VII . Example

In connection with the following examples, the compounds of the present invention were characterized by high performance liquid chromatography (HPLC) using a Waters Millenium chromatography system with the 2690 Separation Module (Milford, Mass.). The analytical column was Alltima C-18 reverse phase, 4.6 × 250 mm from Alltech (Deerfield, Ill.). Gradient elution was used, typically starting with 5% acetonitrile / 95% water and progressing over a period of 40 minutes with 100% acetonitrile. All solvents contained 0.1% trifluoroacetic acid (TFA). Compounds were detected by ultraviolet (UV) absorption at 220 or 254 nm. In some cases, purity was assessed by thin layer chromatography (TLC) using glass or plastic backed silica gel plates such as Baker-Flex Silica Gel 1 B2-F flexible sheets. TLC results can be detected visually easily under ultraviolet light, or using well known iodine vapors and various other dyeing techniques.

Mass spectroscopic analysis was performed on one of two LCMS instruments: Waters System. (Alliance HT HPLC and a Micromass ZQ Mass Spectrometer; Column: Eclipse XDB-C-18, 2.1 × 50 mm; Solvent System: 5-95% (or 35-95%, in water containing 0.05% TFA) Or 65-95% or 95-95%) acetonitrile; flow rate 0.8 mL / min; molecular weight range 500-1500; cone voltage 20 V; column temperature 40 ° C.) or Hewlett Packard System (Series 1100 HPLC; Column: Eclipse XDB-C18, 2.1 × 50 mm; solvent system: 1-95% acetonitrile in water containing 0.05% TFA; flow rate 0.4 mL / min; molecular weight range 150-850; cone voltage 50 V; column temperature 30 ° C.). All masses were recorded as the mass of protonated mother ions.

GCMS analysis was performed on a Hewlet Packard instrument (HP6890 Series gas chromatograph with Mass Selective Detector 5973; injector volume: 1 μl; 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 m × 25 m × 0.25 m).

Nuclear magnetic resonance (NMR) analysis was performed using a Varian 300 MHz NMR (Palo Alto, Calif.). The reference for the spectrum was chemical shift of TMS or known solvents. Some compound samples were run at elevated temperatures (eg, 75 ° C.) to promote increased sample solubility.

The purity of some of the compounds of the present invention was evaluated by an elemental analyzer (Desert Analytics, Tucson, Ariz.).

Melting points were measured on a Laboratory Devices Mel-Temp apparatus (Holliston, Mass.).

Preparative separation can be performed using a Flash 40 chromatography system and KP-Sil, 60A (Biotage, Charlottesville, Va.), Or flash column chromatography using silica gel (230-400 mesh) packing material. Or by HPLC using a C-18 reversed phase column. Typical solvents used in the Flash 40 Biotage system and flash column chromatography were dichloromethane, methanol, ethyl acetate, hexane, acetone, aqueous hydroxylamine solution and triethyl amine. Typical solvents used for reverse phase HPLC were acetonitrile and water at various concentrations containing 0.1% trifluoroacetic acid.

A. N-((S) -3-amino-1- (hydroxy Amino ) -3-methyl-1- Oxobutane -2- Work ) -4-(((1,2-trans) -2- ( Hydroxymethyl ) Cyclopropyl ) Buta -1,3- Diinil ) Benzamide  Synthesis of Compound (I-1)

methyl  2- Ethynylcyclopropanecarboxylate (2)

Racemic ethyl 2-formylcyclopropanecarboxylate 1 (10 g, 70.3 mmol) and Bestmann Ohira reagent (16.4 g, 85 mmol) were dissolved in anhydrous methanol (100 ml) under N ₂ . Then potassium carbonate (19.4 g, 141 mmol) was added slowly in portions and the solution was stirred for 18 hours. The solvent is removed at 20 ° C. under reduced pressure, water (100 ml) is added and the product is extracted with dichloromethane (2 × 200 ml), then dried over sodium sulfate and concentrated slowly to give racemic methyl 2-ethynylcyclopropanecarboxylate ( 4.16 g) was confirmed by NMR.

(2- Ethynylcyclopropyl Methanol (3)

Then lithium borohydride (175 mg, 8.06 mmol) was added slowly to racemic methyl 2-ethynylcyclopropanecarboxylate (1 g, 20.6 mmol) in dry THF (20 ml) under nitrogen and stirring continued for 2 hours. It was. The reaction mixture was quenched with several drops of acetic acid and the solvent was removed. The crude product was extracted with ethyl acetate (2x50ml), dried over sodium sulfate and then slowly concentrated to give racemic (2-trans-ethynylcyclopropyl) methanol, 3 as a yellow liquid (735mg) which was obtained in the next step. It was used as it is.

(2S) - methyl  3- ( Rat - Butoxycarbonyl Amino) -2- (4-((2- ( Hydroxymethyl ) Cyclopropyl ) Buta -1,3- Diinil Benzamido) -3- Methylbutanoate  (5)

CuCl (42 mg, 0.416 mmol) in racemic (2-trans-ethynylcyclopropyl) methanol (400 mg, 4.16 mmol), methyl 2 in a mixture of THF (20 ml), methanol (10 ml) and butyl amine (10 ml) under nitrogen. To a stirred solution of-(S)-(4- (bromoethynyl) benzamido) -3- (tert-butoxycarbonylamino) -3-methylbutanoate (1.9 g, 4.16 mmol) and Several crystals of hydroxylamine hydrochloride (30 mgs) were added. Stirring was continued for 4 hours. The solvent was removed under reduced pressure, water (100 ml) was added followed by extraction with ethyl acetate (2x150 ml), then dried over sodium sulfate, concentrated and purified over ISCO to give 432 mg of (2-S) -methyl 3- (tert -Butoxycarbonylamino) -2- (4-((2- (hydroxymethyl) cyclopropyl) buta-1,3-diynyl) benzamido) -3-methylbutanoate was obtained.

I-1 : N-((S) -3-amino-1- ( Hydroxyamino ) -3- methyl -One- Oxobutane -2- Work ) -4-(((1,2 trans) -2- ( Hydroxymethyl ) Cyclopropyl ) Buta -1,3- Diinil ) Benzamide

(2-S) -methyl 3- (tert-butoxycarbonylamino) -2- (4-((2- (hydroxymethyl) cyclopropyl) buta-1,3-diynyl) benzamido)- 3-methylbutanoate 5 (0.370 g, 0.814 mmol) was added to dichloromethane (25 ml), treated with TFA (2 ml) and stirred for 20 minutes. Excess solvent and TFA were removed under reduced pressure to obtain deprotected material which was redissolved in IPA (10 mL), treated with aqueous hydroxylamine solution (50%, 2 mL, excess) and placed in the refrigerator for 2 days. Excess solvent was removed under reduced pressure and the crude product was purified by reverse phase HPLC to afford 42 mgs of I-1 as trifluoroacetic acid salt. LC-MS (M + 1) 370, Formula: C ₂₀ H ₂₃ N ₃ O ₄ , Exact Mass: 369.17. ¹ HNMR (DMSO-d6), TFA salt: δ 0.9 (m, 2H) 1.24 (s, 3H), 1.29 (s, 3H), 1.40 (m, 1H), 3.24 (m, 1H), 3.4 (m, 1H), 4.64 (d, 1H), 4.69 (d, 1H), 7.61 (d, 2H), 7.88 (d, 2H), 7.90 (br.d, 1NH), 8.65 (d, 1NH), 9.1 (br) .S, OH).

B. N-((S) -3-amino-1- (hydroxy Amino ) -3-methyl-1- Oxobutane -2- Work ) -4-(((1R, 2R) -2- (hydroxymethyl) cyclopropyl) buta-1,3-diynyl) benzamide (I-2) Synthesis of

(E) -4-hydroxybut-2- Enil  Acetate (2)

Sodium hydride in a solution of (E) -but-2-ene-1,4-diol 1 (264 g, 3.0 mol) in 1.5 L of THF (120 g, 3.0 mol) was added in portions under -20 ° C. After addition, the mixture was stirred at -20 ° C for 30 minutes. Acetyl chloride (235.5 g, 3 mol) was then added dropwise and the mixture was allowed to warm to room temperature and then stirred at room temperature for an additional 3 hours. The mixture was filtered and the residue was washed with THF. The combined organic layers were dried and concentrated to give crude product 2 , which was then purified by silica gel column (PE: EA = 5: 1-2: 1) to give 210 g of 2 as a colorless oil. Yield: 54%. 1 HNMR: CP-0005065-043 (CDCl 3, 400 M HZ) δ: 5.85 (m, 1H), 5.62 (m, 1H), 4.67 (t, J = 6.2 Hz, 2H), 4.26 (t, J = 6.0 Hz , 2H), 2.10 (s, 1H), 2.06 (s, 3H).

(E) -4-oxo Boot -2- Enil  Acetate (3)

To a suspension of manganese dioxide (active, 1305 g, 15 mol) in 2.5 L dichloromethane (E) -4-hydroxybut- 2 -enyl acetate 2 (195 g) was added in portions. The mixture was stirred at room temperature for 48 hours. The mixture was filtered and the residue was washed with dichloromethane. The combined organic layers were dried and concentrated to give crude product 3 , which was then purified by silica gel column (PE: EA = 10: 1-5: 1) to give 130 g of 3 as a colorless oil. Yield 64%. 1 HNMR: CP-0005065-044 (CDCl 3, 400 M HZ) δ: 10.01 (d, J = 6.4 Hz, 1H), 6.52 (m, 1H), 6.10 (m, 1H), 5.08 (m, 2H), 2.10 (s, 3 H).

(E) -4,4- Diethoxy boot -2- Enil  Acetate (4)

Ammonium nitrate in a solution of (E) -4-oxobut-2-enyl acetate 3 (96.0 g, 0.75 mol) and triethoxymethane (133.2 g, 0.9 mol) in 500 ml ethanol (3.0 g, 0.038 mol) was added and the mixture was stirred at room temperature for 15 hours. The mixture was diluted with 800 ml EtOAc and washed with saturated sodium bicarbonate. The aqueous layer was back extracted with EtOAc (300ml x 2). The combined organic layers were dried and concentrated to afford 140 g of crude 4 as red oil, which was used for the next step without further purification.

(4R, 5R, E)- Diiso Propyl-2- (3-acetoxyprop-1- Enil ) -1,3- Dioxolane -4,5- Dicarboxylate  (5)

(E) -4,4-diethoxybut-2-enyl acetate 4 (60.6 g, 0.3 mol) and (2R, 3R) -diisopropyl 2,3-dihydroxysuccinate (77.2 g in 500 ml benzene , 0.33 mol) PPTS (3.8 g, 15 mmol) was added to the solution, and the mixture was heated to 90 ° C. to distill ethanol for 15 hours. The mixture was cooled to room temperature and then concentrated in vacuo. Purification by silica gel (PE: EA = 50: 1-30: 1) gave 38.5 g of 5 as a colorless oil. Yield: 37.3% .GCMS: CP-0005065-070-2 (85% purity).

(4R, 5R)- Diisopropyl -2-((1S, 2R) -2- (acetoxy methyl Cyclo profile ) -1,3-dioxolane-4,5-diocarboxylate (6)

(4R, 5R, E) -Diisopropyl-2- (3-acetoxyprop-1-enyl) -1,3-dioxolane-4,5-dicarboxylate 5 (34.4 in hexane (1.5 L) g, 0.1 mol) diethyl zinc in solution (1M in hexane, 500 mL) was added in portions under argon at −20 ° C. After addition, diiodoethane was added dropwise at −20 ° C. or lower with vigorous stirring. The mixture was allowed to warm to room temperature and stirred for an additional 8 hours. The reaction mixture was quenched with 800 ml cold aqueous ammonium chloride solution and then extracted with ether (800 ml × 5). The combined organic layers were washed with aqueous sodium thiosulfate solution, water and brine, dried and concentrated to give crude product 6 , which was then purified by silica gel column (PE: EA = 30: 1-10: 1) to give 16 as colorless oil. 6 of g was obtained. Yield 44.7%. 1 HNMR: PO 5 (CDCl 3, 400 MHZ) δ: 5.13 (m, 2H), 4.95 (d, J = 5.6 Hz, 1H), 4.67 (d, J = 3.6 Hz, 1H), 4.57 (d, J = 4.0 Hz , 1H), 4.07 (m, 1H), 3.88 (m, 1H), 2.06 (s, 3H), 1.38 (s, 12H), 1.23 (m, 1H), 0.83 (m, 1H), 0.66 (m, 1H).

((1R, 2R) -2-formyl Cyclopropyl Methyl Acetate (7)

(4R, 5R) -Diisopropyl-2-((1S, 2R) -2- (acetoxymethyl) cyclopropyl) -1,3-dioxolane-4,5-dicarboxylate in 140 ml 80% acetic acid A mixture of 6 (14.3 g, 40 mmol) was heated to 80 ° C. and stirred at this temperature for 2 hours. When TLC showed little 6 remaining, the mixture was added dropwise to 300 ml saturated sodium bicarbonate and then extracted with dichloromethane (200 ml x 3). The combined organic layers were washed with water and brine, dried and concentrated to give crude product 7 which was purified by silica gel column (PE: EA = 10: 1-5: 1) to give 3.5 g of 7 as a colorless oil. . Yield 62%. 1 HNMR: CP-0005065-072 (CDCl 3, 400 M HZ) δ: 9.15 (s, 1H), 4.11 (m, 1H), 3.91 (m, 1H), 2.08 (s, 3H), 2.10 (s, 3H) , 1.88 (m, 2H), 1.39 (m, 1H), 1.12 (m, 1H).

((1R, 2R) -2- (2,2- Dibromovinyl Cyclo profile Methyl Acetate (8)

To a solution of carbon tetrabromide (13.9 g, 42 mmol) in 30 mL of dichloromethane was added dropwise a solution of triphenylphosphine (22.0 g, 84 mmol) in 50 mL dichloromethane at -20 ° C under argon. The mixture was stirred at this temperature for 30 minutes and then cooled to -78 ° C. A solution of ((1R, 2R) -2-formylcyclopropyl) methyl acetate 7 (3.00 g, 21 mmol) in 40 ml dichloromethane was added dropwise and the temperature was maintained for an additional 30 minutes. The mixture was allowed to warm to room temperature over 30 minutes. The solvent was removed and the residue was purified by a silica gel column (PE: EA = 100: 1 to 50: 1) to give 4.3 g of 8 as a colorless oil. Yield 69%. 1 HNMR: CP-0005065-075 (CDCl 3, 400 M HZ) δ: 5.85 (d, J = 8.8 Hz, 1H), 3.98 (m, 2H), 2.09 (m, 3H), 1.61 (m, 1H), 1.35 (m, 1 H), 0.88 (m, 2 H).

Methyl 4-(((1R, 2R) -2- (acetoxy methyl Cyclo profile ) Buta -1,3-diynyl) benzoate (10)

DMF ((1R, 2R) -2- (2,2-dibromovinyl) cyclopropyl) methyl acetate 8 (4.3 g, 14.5 mmol), Pd ₂ dba ₂ (86.3 mg, 0.15 mmol), tri ( To a solution of 4-methylphenyl) phosphine (204 mg, 0.58 mmol), triethylamine (4.35 g, 43.5 mmol) was treated with methyl 4-ethynylbenzoate 9 (2.64 g, 11 mmol) under argon. The mixture was stirred at room temperature for 5 hours. When TLC showed little compound 8 remaining, the reaction was diluted with EtOAc (300 mL) and washed with water (3x100 mL), then the organic layer was dried and concentrated to give crude product 10 , which was then silica gel. Purified by column chromatography PE: EA = 50: 1 to 30: 1 to obtain 2.0 g of 10 as a yellow solid. Yield: 46.5%, LCMS: CP-0005065-085-2 (ESI) m / z = 297 (M + 1) Purity: 92.4% (214 nm).

4-(((1R, 2R) -2- (hydroxy methyl Cyclo profile ) Buta -1,3- Diinil Benzoic acid (11)

Methyl 4-(((1R, 2R) -2- (acetoxymethyl) cyclopropyl) buta-1,3-diynyl) benzoate 10 (1.92 g, 6.5 mmol) was dissolved in THF (40 mL) , A solution of sodium hydroxide (2.60 g, 65 mmol) in 10 mL water was added. The mixture was stirred at room temperature for 8 hours. When LCMS showed little compound 10 remaining, the solvent was removed under reduced pressure, the residue was diluted with water (50 mL), the pH was adjusted to 4.0 and then extracted with ethyl acetate (4x50 mL) The organic layer was dried and concentrated to give 1.4 g of crude product 11 as a yellow solid which was used for the next step without further purification. LCMS: CP-0005065-088-3 (ESI) m / z = 241 (M + l) Purity: 89% (214 nm). Yield: 89%.

(S) -methyl 2- (4-(((1R, 2R) -2- (acetoxy) methyl Cyclo profile ) Buta -1,3-diynyl) benzamido) -3- (tert-butoxycarbonylamino) -3-methylbutanoate (12)

4-(((1R, 2R) -2- (hydroxymethyl) cyclopropyl) buta-1,3-diynyl) benzoic acid 11 (1.20 g, 5.0 mmol) in 50 mL DMF, HATU (2.34 g, 6 mmol) in solution (S) -methyl 2-amino-3- (tert-butoxycarbonylamino) -3-methylbutanoate (1.48g, 6.0mmoL) and DIPEA (3.58g, 20mmoL ). The mixture was stirred at room temperature for 5 hours. When LCMS showed little compound 11 remaining, the reaction was diluted with EtOAc (100 mL), washed with 5% lithium chloride (3x50 mL), and then the organic layer was dried and concentrated to give 12 as a yellow oil. This was purified by silica gel column chromatography PE: EA = 2: 1 to obtain 2.0 g of 12 as a colorless oil. Yield: 70%, LCMS: CP-0005065-091-3 (ESI) m / z = 469 (M + l) Purity: 95% (214 nm).

(S) -methyl 3-amino-2- (4-(((1R, 2R) -2- (hydroxy methyl Cyclo profile ) Buta -1,3-diynyl) benzamido) -3-methylbutanoate hydrochloride (13)

(S) -methyl2- (4-(((1R, 2R) -2- (acetoxymethyl) cyclopropyl) buta-1,3-diynyl) benzamido) -3- (tert-butoxycarbo Nylamino) -3-methylbutanoate 12 (1.87 g, 4.0 mmol) was dissolved in methanol (50 mL) and treated with dry HCl _g for 10 minutes. When LCMS showed little compound 12 remaining, the HCl flow was stopped. The solvent was removed under reduced pressure to yield 1.45 g of 13 as a yellow solid. Yield: 91%, LCMS: CP-0005065-096-4-LCMSA019 (ESI) m / z = 369 (M + 1) Purity: 98% (214 nm). 1 H NMR: CP-0005065-096-4 (DMSO- d ₆ , 400 MHz) δ: 9.04 (d, J = 6.8 Hz, 1H), 8.36 (s, 3H), 7.98 (d, J = 6.4 Hz, 2H ), 7.64 (d, J = 6.8 Hz, 2H), 4.89 (d, J = 6.8 Hz, 1H), 3.73 (s, 3H), 3.44 (m, 1H), 3.22 (m, 1H), 1.48 (m , 2H), 1.40 (s, 6H), 0.94 (m, 2H).

(S) -methyl 3-amino-2- (4-(((1R, 2R) -2- (hydroxymethyl) cyclopropyl) buta-1,3-diynyl) benzamido) -3-methylbuta Noate hydrochloride 13 (1.25 g, 3.1 mmol) was treated with isopropanol (10 mL) and 50% aqueous hydroxylamine solution (4.1 mL, 61.7 mmol, 20 equiv) until the majority appeared complete by LCMS.

The crude material was purified by reverse phase HPLC (slope of 0-30% acetonitrile in water, containing 0.1% TFA each, over 120 minutes) and the desired fractions were collected and lyophilized to yield N-(( S) -3-amino-1- (hydroxyamino) -3-methyl-1-oxobutan-2-yl) -4-(((1R, 2R) -2- (hydroxymethyl) cyclopropyl) buta -1,3-diynyl) benzamide was obtained (white solid, 647 mg, 1.3 mmol, 43%). Mass spectral data: predicted (M + 1): 370.4, found: 370.2. Proton NMR (400 MHz, dmso-d6): 11.20 (s, 1H), 9.43 (br s, 1H), 8.55 (d, 1H, J = 9.6 Hz), 8.00 (br s, 3H), 7.89 (dd, 2H , J = 1.8, 6.6 Hz), 7.60 (dd, 2H, J = 2.0, 6.8 Hz), 8.66 (d, 1H, J = 9.2 Hz), 3.39 (dd, 1H, J = 4.8, 11.6 Hz), 3.22 (dd, 1H, J = 5.8, 11.4 Hz), 1.39-1.46 (m, 2H), 1.30 (s, 3H), 1.25 (s, 3H), 0.84-0.93 (m, 2H)

C. N-((S) -3-amino-1- (hydroxy Amino ) -3-methyl-1- Oxobutane -2- Work Synthesis of) -4-(((1S, 2S) -2- (hydroxymethyl) cyclopropyl) buta-1,3-diynyl) benzamide (I-3)

(4S, 5S, E)- Diisopropyl -2- (3-acetoxyprop-1- Enil ) -1,3- Dioxolane -4,5-dicarboxylate (5)

(E) -4,4-diethoxybut-2-enyl acetate 4 (75 g, 0.37 mol) and diisopropyl (-)-(S, S) -tartrate (86.8 g, 0.3 mol) in 800 ml benzene To the solution was added PPTS (4.66 g, 0.0185 mol) and the mixture was heated to 9 ° C. to distill ethanol for 15 hours. The mixture was cooled to room temperature and concentrated in vacuo. This was distilled and purified to obtain compound 5 (50.0 g, 39%) as a colorless oil. 1 H NMR: (CDCl 3, 400 MHz) δ: 6.04-6.01 (m, 1H), 5.81-5.80 (m, 1H), 5.57 (d, J = 8 Hz, 1H), 5.07-5.01 (m, 2H), 4.65 (d, J = 4 Hz, 1H), 4.57 (d, J = 4 Hz, 1H), 4.54-4.53 (m, 2H), 1.99 (s, 3H), 1.23-1.19 (m, 12H).

(4S, 5S)- Diisopropyl -2-((1R, 2S) -2- (acetoxy methyl Cyclo profile ) -1,3-dioxolane-4,5-diocarboxylate (6)

(4S, 5S, E) -Diisopropyl-2- (3-acetoxyprop-1-enyl) -1,3-dioxolane-4,5-dicarboxylate 5 (40 g) in hexane (1.0 L) , 0.12mol) diethyl zinc in solution (1M in hexanes, 1.16 L) was added in portions under argon at −20 ° C. After addition, diiodomethane (623 g, 2.40 mol) was added dropwise at −20 ° C. or lower with vigorous stirring. The mixture was allowed to warm to room temperature and then stirred for an additional 8 hours. The reaction mixture was quenched with 800 ml cold ammonium chloride aqueous solution and then extracted with ether (800 mL x 5). The combined organic layers were washed with aqueous sodium thiosulfate solution, water, brine, dried and concentrated to give crude compound 6, and then purified by silica gel column using ethyl acetate (3% -10% v / v) in petroleum ether. Purification gave compound 6 (20.0 g, 50%) as a colorless oil. ¹ H NMR: (CDCl ₃ , 400 MHz) δ: 5.15-5.08 (m, 2H), 4.95 (d, J = 5.6 Hz, 1H), 4.67 (d, J = 3.6 Hz, 1H), 4.57 (d, J = 4.0 Hz, 1H), 4.07-4.04 (m, 1H), 3.91-3.86 (m, 1H), 2.06 (s, 3H), 1.40-1.37 (m, 1H), 1.31-1.28 (m, 12H) , 1.24-1.22 (m, 1 H), 0.85-0.82 (m, 1 H), 0.68-0.63 (m, 1 H).

((1S, 2S) -2-formyl Cyclopropyl Methyl Acetate (7)

(4S, 5S) -Diisopropyl-2-((1R, 2S) -2- (acetoxymethyl) cyclopropyl) -1,3-dioxolane-4,5-dicarboxylate 6 in 200 ml 80% acetic acid (20.0 g, 55.8 mmol) was heated to 80 ° C. and stirred at this temperature for 2 hours. When TLC showed little to 6 , the mixture was diluted with water (150 mL) and extracted with ethyl acetate (200 ml x 3). The combined organic layers were washed with saturated sodium bicarbonate, water, brine, dried and concentrated to give crude compound 7 and purified by silica gel column using ethyl acetate (8% -20% v / v) in petroleum ether. Compound 7 (3.0, 38%) was obtained as a colorless oil. ¹ H NMR: (CDCl ₃ , 400 MHz) δ: 9.15 (d, J = 4.8, 1H), 4.14-4.09 (m, 1H), 3.95-3.90 (m, 1H), 2.08 (s, 3H), 1.92 -1.88 (m, 2H), 1.38-1.36 (m, 1H), 1.12-1.09 (m, 1H).

((1S, 2S) -2- (2,2- Dibromovinyl Cyclo profile Methyl Acetate (8)

To a solution of carbon tetrabromide (13.9 g, 42.0 mmol) in dichloromethane (30 mL) was added dropwise a solution of triphenylphosphine (22.0 g, 84.0 mmol) in 50 mL dichloromethane at -20 ° C under argon. The mixture was stirred at this temperature for 30 minutes and then cooled to -78 ° C. A solution of ((1S, 2S) -2-formylcyclopropyl) methyl acetate 7 (3.0 g, 21.0 mmol) in 40 mL dichloromethane was added dropwise and maintained at this temperature for an additional 30 minutes. The mixture was allowed to warm to room temperature over 30 minutes. The solvent was removed in vacuo and the residue was purified by silica gel column with petroleum ether to give compound 8 (3.20 g, 51%) as colorless oil. This was confirmed by GC-MS.

methyl  4-(((1S, 2S) -2- (acetoxy methyl Cyclo profile ) Buta -1,3- Diinil ) Benzoate (10)

DMF ((1S, 2S) -2- (2,2-dibromovinyl) cyclopropyl) methyl acetate 8 (3.2 g, 10.85 mmol) in (50 mL), Pd ₂ dba ₂ (62.4 mg, 0.11 mmol), tri To a solution of (4-methylphenyl) phosphine (153 mg, 0.43 mmol), triethylamine (1.44 g, 14.25 mmol) was treated with methyl 4-ethynylbenzoate 9 (2.43 g, 15.2 mmol) under argon. The mixture was stirred at room temperature overnight. The reaction was diluted with EtOAc (300 mL) and washed with water (3 x 100 mL), then the combined organic layers were dried and concentrated to afford crude compound 10 , ethyl acetate in petroleum ether (3% -10% v / v). Purification by silica gel column chromatography using) afforded compound 10 (1.4 g, 46.5%) as a yellow solid. LC-MS: 297 [M + H] ⁺ .

4-(((1S, 2S) -2- (hydroxy methyl Cyclo profile ) Buta -1,3- Diinil Benzoic acid (11)

Water (10 mL) in a solution of methyl 4-(((1S, 2S) -2- (acetoxymethyl) cyclopropyl) buta-1,3-diynyl) benzoate 10 (1.4 g) in THF (30 mL) A solution of sodium hydroxide (75 mg, 18.9 mmol) in water was added. The mixture was stirred at room temperature overnight. After this time, the solvent is removed under reduced pressure, the residue is diluted with water (50 mL), adjusted to pH 4.0 with 1M HCl, extracted with EtOAc (4 x 50 mL), and then the organic layer is dried and concentrated. To give compound 11 (1.05 g, 93%) as a yellow solid, which was used for the next step without further purification. LC-MS: 241 [M + H] ⁺ .

(S) -methyl 3- ( Rat - Butoxycarbonylamino ) -2- (4-(((1S, 2S) -2- (hydroxymethyl) cyclopropyl) buta-1,3-diynyl) benzamido) -3-methylbutanoate (12)

4-(((1S, 2S) -2- (hydroxymethyl) cyclopropyl) buta-1,3-diynyl) benzoic acid 11 (1.05 g, 4.38 mmol) in DMF (25 mL), HATU (S) -methyl 2-amino-3- (tert-butoxycarbonylamino) -3-methylbutanoate (1.076 g, 4.38 mmol) and DIPEA (1.69 g, 13.1) in a (1.83 g, 4.81 mmol) solution mmoL) was added. The mixture was stirred at room temperature for 5 hours. After this time, the reaction was diluted with water (20 mL) and then extracted with ethyl acetate (60 mL x 3). The combined organic layers were washed with water and brine, then dried and concentrated to give compound 12 as a yellow oil. (1.35 g, 65%) LC-MS: 469 [M + H] ⁺ .

(S) -methyl 3-amino-2- (4-(((1S, 2S) -2- (hydroxy methyl Cyclo profile ) Buta -1,3-diynyl) benzamido) -3-methylbutanoate hydrochloride (13)

(S) -methyl 3- (tert-butoxycarbonylamino) -2- (4-(((1S, 2S) -2- (hydroxymethyl) cyclopropyl) buta-1,3 in methanol (20 mL) -Diynyl) benzamido) -3-methylbutanoate 12 (1.3 g, 2.78 mmol) solution was attached to the HCl apparatus. The reaction was then stirred at room temperature until TLC indicated total consumption of starting material. The solution was then concentrated under reduced pressure to give 13 as a yellow solid (1.1 g, 98%). LC-MS: 369 [M + H] ⁺ . 1 H-NMR: (DMSO- d ₆ , 400 MHz) δ: 9.04 (d, J = 6.8 Hz, 1H), 8.38 (s, 3H), 7.98 (d, J = 6.8 Hz, 2H), 7.64 (d, J = 6.4 Hz, 2H), 4.88 (d, J = 6.8 Hz, 1H), 3.72 (s, 3H), 3.45-3.42 (m, 1H), 3.28-3.24 (m, 1H), 1.49-1.46 (m , 2H), 1.40 (s, 6H), 0.95-0.90 (m, 2H).

(S) -methyl 3-amino-2- (4-(((1S, 2S) -2- (hydroxymethyl) cyclopropyl) buta-1,3-diynyl) benzamido) -3-methylbuta Noate hydrochloride 13 (1.15 g, 2.8 mmol) was treated with isopropanol (10 mL) and 50% aqueous hydroxylamine solution (3.8 mL, 56.8 mmol, 20 equiv) until the majority appeared complete by LCMS.

The crude product was purified by reverse phase HPLC (slope of 0-30% acetonitrile in water, containing 0.1% TFA each, over 120 minutes), and the desired fractions were combined and lyophilized to yield N- as a trifluoroacetate salt. ((S) -3-amino-1- (hydroxyamino) -3-methyl-1-oxobutan-2-yl) -4-(((1S, 2S) -2- (hydroxymethyl) cyclopropyl ) Buta-1,3-diynyl) benzamide was obtained (white solid, 446 mg, 0.92 mmol, 33%). Mass spectral data: predicted (M + 1): 370.4, found: 370.2. Proton NMR (400 MHz, dmso-d6): 11.20 (s, 1H), 9.22 (br s, 1H), 8.55 (d, 1H, J = 9.6 Hz), 7.99 (br s, 3H), 7.89 (dd, 2H , J = 2.0, 6.8 Hz), 7.61 (dd, 2H, J = 1.6, 6.8 Hz), 4.66 (d, 1H, J = 9.6 Hz), 3.40 (dd, 1H, J = 5.0, 11.4 Hz), 3.22 (dd, 1H, J = 5.8, 11.8 Hz), 1.38-1.46 (m, 2H), 1.30 (s, 3H), 1.25 (s, 1H), 0.84 -0.93 (m, 2H).

D. N-((S) -3-amino-1- (hydroxyamino) -3- methyl -One- Oxobutane -2- Work ) -4 '-(((1,2-trans) -2- (hydroxy methyl ) Cyclopropyl ) Ethynyl) biphenyl-4-carboxamide (I-5) Synthesis of

Methyl 4'-bromobiphenyl-4-carboxylate (2.0 g, 6.87 mmol, 1.0 equiv) was added to a round bottom flask, followed by racemic 2-trans-ethynylcyclopropyl) methanol dissolved in THF (20 mL). 0.991 g, 10.30 mmol, 1.5 equiv) was added thereto. To this was added palladium dichloride bis-triphenylphosphine (241 mg, 0.343 mmol, 0.05 equiv), copper (I) iodide (131 mg, 0.687 mmol, 0.1 equiv) and then triethylamine (6.87 mL , 49.3 mmol, 7.18 equiv) was added. The reaction was stirred at 75 ° C. for ˜ 2 hours. The reaction was cooled to room temperature, the solid was filtered off and rinsed with THF. The solution was placed in the freezer for 5 days. It was concentrated to dryness. DCM was added to form a precipitate. The precipitate was filtered off and rinsed with minimal DCM. TLC showed that the precipitate (ppt) mainly contained the product (˜1.5 g solids). After grinding with 10% EtOAc in hexane (5 mL) and pipetting off the solvent, TLC showed that the solid contained the product and the removed liquid contained the starting material-bromide and some baseline impurities. It was. Additional 10% EtOAc in hexane (5 mL) was added and stirred for 30 minutes, then filtered, rinsed with 10% EtOAc in hexane (5 mL) and dried to give a solid. 900 mg of crude methyl 4 ′-(((1,2-trans) -2- (hydroxymethyl) cyclopropyl) ethynyl) biphenyl-4-carboxylate were obtained. 1 H NMR (DMSO-d 6): δ 0.82-0.85 (m, 2H), 1.35-1.43 (m, 2H), 3.23-3.29 (m, 1H), 3.40-3.42 (m, 1H), 3.84 (s, 3H), 4.65-4.67 (t, 1H), 7.43-7.45 (d, 2H), 7.67-7.70 (d, 2H), 7.80-7.82 (d, 2H), 7.99-8.01 (d, 2H).

Methyl 4 '-(((1,2-trans) -2- (hydroxymethyl) cyclopropyl) ethynyl) biphenyl-4-carboxylate (900 mgs, 2.94 mmol, 1.0 equiv) in methanol (5 mL) , DMF (2 mL), and THF (5 mL). 1.0M NaOH (4.41 mL, 4.407 mmol, 1.5 equiv) was added at room temperature. The reaction was stirred for 4 days. The reaction was concentrated to remove MeOH and THF and acidified to pH ˜3 with 6N HCl (˜5 mL). Extract with EtOAc (3x50 mL), combine organic layers, wash with saturated NaCl, dry (MgSO ₄ ), filter and concentrate. 890 mg of 4 '-(((1,2-trans) -2- (hydroxymethyl) cyclopropyl) ethynyl) biphenyl-4-carboxylic acid were obtained. 1 H NMR (DMSO-d 6): δ 0.79-0.88 (m, 2H), 1.34-1.44 (m, 2H), 3.22-3.26 (m, 1H), 3.40-3.44 (m, 1H), 7.42-7.44 (d , 2H), 7.66-7.68 (d, 2H), 7.76-7.78 (d, 2H), 7.97-7.99 (d, 2H), 8.10 (s, 1H).

(S) -methyl 2-amino-3- (tert-butoxycarbonylamino) -3-methylbutanoate (150 mg, 0.609 mmol, 1.0 equiv) and 4 '-(((1,2-trans) 2- (hydroxymethyl) cyclopropyl) ethynyl) biphenyl-4-carboxylic acid (178 mg, 0.609 mmol, 1.0 equiv) was dissolved in DMF (2 mL). To this was added DIPEA (0.266 mL, 1.523 mmol, 2.5 equiv) followed by HATU (278 mg, 0.731 mmol, 1.2 equiv). It was stirred at room temperature for ˜48 hours. The reaction was partitioned between 1M 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 and then evaporated to dryness. 370 mg crude (S) -methyl 3- (tert-butoxycarbonylamino) -2- (4 '-(((1S, 2S) -2- (hydroxymethyl) cyclopropyl) ethynyl) ratio Phenyl-4-ylcarboxamido) -3-methylbutanoate was obtained. LCMS M + 1 predicted = 521.3, found = 521.3.

(S) -methyl-3- (tert-butoxycarbonylamino) -2- (4 '-(((1,2-trans) -2- (hydroxymethyl) cyclopropyl) ethynyl) biphenyl- 4-ylcarboxamido) -3-methylbutanoate (crude compound) was dissolved in methanol (1 mL). At room temperature was added 4.0M HCl in dioxane (1.283 mL, 5.13 mmol, 8.43 equiv). The reaction was complete after 90 minutes (HPLC). The reaction was concentrated on rotovap at 0 ° C. To this was added IPA (1 mL) followed by hydroxylamine solution (0.804 mL, 12.18 mmol, 20 equiv). The flask was placed at 4 ° C. for ˜ 120 hours. The reaction was concentrated to a gummy mass (store reaction at 0 ° C.). To this was added water (3 mL) and ACN (0.5 mL). Acidified with TFA (3 mL) at 0 ° C. Additional water (1 mL) and ACN (1 mL) were added. Purified by RP HPLC (1 "column, 25 mL / min, 0.1% TFA in water / ACN, equilibrated at 10% B). Loaded using a syringe filter on 1" column (10 mL / min, 5% B). (2X6.5 mL). Ramped to 25 mL / min over 1 minute. The product was eluted at 41-48 minutes with 10% B for 15 minutes and then 10-70% B over 80 minutes. Desired fractions were combined, frozen and placed in lyo. 105 mg N-((S) -3-amino-1- (hydroxyamino) -3-methyl-1-oxobutan-2-yl) -4 '-(((1,2-trans) -2 -(Hydroxymethyl) cyclopropyl) ethynyl) biphenyl-4-carboxamide (I-5), TFA was obtained. LCMS M + 1 Found = 422.2, Found = 422.2. 1 H NMR (DMSO-d 6): δ 0.80-0.88 (m, 2H), 1.27 (s, 3H), 1.31 (s, 3H), 1.36-1.44 (m, 2H), 3.21-3.29 (m, 1H), 3.40-3.45 (m, 1H), 4.65-4.71 (m, 2H), 7.44-7.46 (d, 2H), 7.68-7.71 (d, 2H), 7.79-7.81 (d, 2H), 7.97-7.99 (d , 2H), 8.46-8.48 (d, 1H), 9.22 (s, 1H), 11.21 (br, 1H).

E. N-((S) -1- (hydroxy Amino ) -3-methyl-3- (methyl Amino )-One- Oxobutane -2- Work ) -4-(((1,2-trans) -2- (hydroxymethyl) cyclopropyl) buta-1,3-diynyl) benzamide (I-6) And N-((S) -1- (hydroxy Amino ) -3- (2-hydroxyethyl Amino ) -3-methyl-1- Oxobutane -2- Work ) -4-(((1,2-trans) -2- (hydroxymethyl) cyclopropyl) buta-1,3-diynyl) benzamide (I-7) Synthesis of

In a 250 mL round-bottom flask, N-((S) -3-amino-1- (hydroxyamino) -3-methyl-1-oxobutan-2-yl) -4-((( 1,2-trans) -2- (hydroxymethyl) cyclopropyl) buta - 1,3 - diynyl) benzamide ( I-1 ) (1.2 g, 3.25 mmol), triethylamine (2 mL, 14.35 mmol ) And formaldehyde (0.2 g, 6.66 mmol) were added to obtain a yellow solution. The reaction mixture was stirred for 3 hours. Excess formaldehyde was quenched with n-butylamine (2 ml). Sodium cyanoborohydride (570 mg, 14.3 mmol) and acetic acid (4 ml) were added at room temperature and 0 ° C. The reaction mixture was checked by LCMS. After removing the solvent, the product was purified by HPLC to give 440 mg of N-((S) -1- (hydroxyamino) -3-methyl-3- (methylamino) -1-oxobutan-2-yl) -4 -(((1,2-trans) -2- (hydroxymethyl) cyclopropyl) buta-1,3-diynyl) benzamide and I-6 were obtained. LC-MS (M + 1) 384; Chemical formula: C ₂₁ H ₂₅ N ₃ O ₄ , Exact mass: 383.18. 1 HNMR (DMSO-d 6), TFA salt: δ 0.88 (m, 2H), 1.30 (s, 3H), 1.35 (s, 3H), 1.44 (m, 1H), 2.51 (s, H), 3.39 (m, 1H), 3.41 (m, 1H), 4.7 (br.d, 1H), 4.84 (d, 1H), 7.58 (d, 2H), 7.91 (d, 2H), 8.51 (br.m, 1NH), 8.64 (d, 1 NH), 9.23 (S, NH), 11.17 (s, OH).

In a 250 mL round-bottom flask, N-((S) -3-amino-1- (hydroxyamino) -3-methyl-1-oxobutan-2-yl) -4-((( 1,2-trans) -2- (hydroxymethyl) cyclopropyl) buta - 1,3 - diynyl) benzamide ( I-1 ) (1 g, 2.71 mmol), triethylamine (0.377 ml, 2.71 mmol) , And acetaldehyde (0.119 g, 2.71 mmol) were added to give a yellow solution. The reaction mixture was stirred for 3 hours. Sodium cyanoborohydride (570 mg, 14.3 mmol) and acetic acid (2 ml) were added at room temperature. The reaction mixture was checked by LCMS. The reaction mixture was concentrated and the product was purified by HPLC to give 642 mg of N-((S) -1- (hydroxyamino) -3- (2-hydroxyethylamino) -3-methyl-1-oxobutane-2- Yl) -4-(((1,2-trans) -2- (hydroxymethyl) cyclopropyl) buta-1,3-diynyl) benzamide and I-7 were obtained. LC-MS (M + 1) 398; Chemical formula: C ₂₂ H ₂₇ N ₃ O ₄ , Exact mass: 397.20. 1 HNMR (DMSO-d 6), TFA salt: δ 0.85 (m, 2H), 1.14 (t, 3H), 1.29 (s, 3H), 1.39 (s, 3H), 1.44 (m, 1H), 2.96 (br. m, 2H) 3.24 (m, 2H) 3.39 (m, 2H), 4.64 (br.d, 1H), 4.85 (d, 1H), 7.60 (d, 2H), 7.92 (d, 2H), 8.59 (br m, 1 NH) 8.45 (brt, 1 NH), 9.23 (S, NH), 11.15 (s, OH).

F. N-((S) -1- (hydroxy Amino ) -3- (2-hydroxyethyl Amino ) -3-methyl-1- Oxobutane -2-yl) -4-(((1,2-trans) -2- (hydroxymethyl) cyclopropyl) buta-1,3-diynyl) benzamide (I-8) Synthesis of

In a 250 mL round-bottom flask (t = g) N-((S) -3-amino-1- (hydroxyamino) -3-methyl-1-oxobutan-2-yl)-in DMF (20 ml)- 4-(((1,2-trans) -2- (hydroxymethyl) cyclopropyl) buta-1,3-diynyl) benzamide (1 g, 2.71 mmol), and triethylamine (0.377 ml, 2.71 mmol ) To give a yellow solution. 2- (tert-butyldimethylsilyloxy) acetaldehyde (0.494 g, 2.83 mmol) was added. The reaction mixture was stirred for 18 hours and concentrated to dryness. The residue was dissolved in THF (15 ml) and acetic acid (2 ml) and sodium cyanoborohydride (600 mg, 15 mmol) were added at room temperature. The reaction mixture was checked by LCMS. After stirring at room temperature for 2 hours, TFA (5 ml) was added and stirred for an additional 3 hours. After completion of the reaction was confirmed by LCMS and the reaction was concentrated, the product was purified on HPLC to give 370 mg of N-((S) -1- (hydroxyamino) -3- (2-hydroxyethylamino) -3- Methyl-1-oxobutan-2-yl) -4-(((1,2-trans) -2- (hydroxymethyl) cyclopropyl) buta-1,3-diynyl) benzamide, I-8 Got it. LC-MS (M + 1) 414; Chemical formula: C ₂₂ H ₂₇ N ₃ O ₅ , Exact mass: 413.20; 1 HNMR (DMSO-d 6), TFA salt: δ 0.88 (m, 2H) 1.30 (s, 3H), 1.35 (s, 3H), 1.44 (m, 1H), 3.22 (m, 2H), 3.39 (m, 2H ), 4.64 (br.d, 1H), 4.90 (d, 1H), 5.25 (br.S, 1H), 7.60 (d, 2H), 7.91 (d, 2H), 8.59 (br.m, 1NH), 8.71 (d, 1 NH), 9.26 (S, NH), 11.19 (s, OH).

G. N-((S) -3- ( Dimethylamino ) -1- (hydroxy Amino ) -3-methyl-1- Oxobutane -2- Work ) -4-(((1,2-trans) -2- (hydroxymethyl) cyclopropyl) buta-1,3-diynyl) benzamide (I-9) Synthesis of

N-((S) -3-amino-1- (hydroxyamino) -3-methyl-1-oxobutan-2-yl) -4-((2- (hydroxymethyl) cyclopropyl) buta-1 , 3-diynyl) benzamide (300 mg, 0.81 mmol) was dissolved in N, N -dimethylformamide (10 mL) and paraformaldehyde (732 mg, 8.1 mmol, 10eq) and N, N -di at room temperature for 16 hours. Treated with isopropylethylamine (0.56 mL, 3.3 mmol, 4eq). Trifluoroacetic acid (1.3 mL, 16.2 mmol, 20 eq) and sodium cyanoborohydride (101 mg, 1.6 mmol, 2 eq) were added.

The crude product was purified by reverse phase HPLC and the desired fractions were collected and lyophilized to yield N-((S) -3- (dimethylamino) -1- (hydroxyamino) -3-methyl-1- as trifluoroacetate salt. Oxobutan-2-yl) -4-(((1,2-trans) -2- (hydroxymethyl) cyclopropyl) buta-1,3-diynyl) benzamide was obtained (white solid, 2.9 mg, 5.3 μmol, 0.7%). Mass spectral data: predicted (M + 1): 398.5, found: 398.2. Proton NMR (400 MHz, dmso-d6): 11.16 (s, 1H), 9.25 (br s, 1H), 9.05 (br s, 1H), 8.68 (d, 1H, J = 10.0 Hz), 7.94 (dd, 2H , J = 1.6, 8.0HZ), 7.59 (dd, 2H, J = 1.8, 8.2Hz), 5.06 (d, 1H, J = 9.6Hz), 3.4 (peak unclear by water), 3.21 (dd, 1H , J = 5.2, 11.2 Hz), 2.75 (d, 3H, J = 5.6 Hz), 2.73 (d, 3H, J = 5.2 Hz), 1.48 (s, 3H), 1.39-1.45 (m, 2H), 1.28 (s, 3H), 0.85-0.91 (m, 2H).

H. N-((S) -3-hydroxy-1- (hydroxy Amino ) -3-methyl-1- Oxobutane -2- Work ) -4-(((1,2-trans) -2- (hydroxymethyl) cyclopropyl) buta-1,3-diynyl) benzamide (I-10) Synthesis of

4-(((1,2-trans) -2- (hydroxymethyl) cyclopropyl) buta-1,3-diynyl) benzoic acid (750 mg, 3.12 mmol), HATU (in room temperature DMF (30 ml) To a stirred solution of 570 mg, 1.5 mmol) and DIEA (3.4 ml excess) was added (S) -methyl 2-amino-3-hydroxy-3-methylbutanoate hydrochloride (402 mg, 1.5 mmol). The reaction mixture was stirred for 1 h at ambient temperature and then diluted with water (50 ml). The solution was extracted with ethyl acetate (100 ml x 3) and brine (20 ml). The organic layer was dried over MgSO ₄ and evaporated. The resulting product (1.3 g) was dissolved in IPA (20 mL), treated with NH ₂ OH (10 mL, excess) and stirred at room temperature for 3 days. Excess solvent was removed and the crude product was purified on reverse phase HPLC to give 178 mg of N-((S) -3-hydroxy-1- (hydroxyamino) -3-methyl-1-oxobutan-2-yl) -4-(((1,2-trans) -2- (hydroxymethyl) cyclopropyl) buta-1,3-diynyl) benzamide I-10 was obtained; LC-MS (M + 1) 371; Chemical formula: C ₂₀ H ₂₂ N ₂ O ₅ Exact mass: 370.15. 1 HNMR (DMSO-d6): δ 0.88 (m, 2H), 1.11 (s, 3H), 1.16 (s, 3H), 1.44 (m, 1H), 3.20 (m, 1H), 3.40 (m, 2H), 4.34 (d, 1H), 4.84 (d, 1H), 7.58 (d, 2H), 7.84 (d, 2H), 7.6 (d, 1NH), 8.88 (br.s, NH), 10.57 (s, OH) .

I. N-((S) -3-amino-1- (hydroxy Amino ) -3-methyl-1- Oxobutane -2- Work Synthesis of) -4-(((1S, 2R) -2- (hydroxymethyl) -2-methylcyclopropyl) buta-1,3-diynyl) benzamide (I-11)

(S, E) -ethyl 3- (2,2- dimethyl -1,3- Dioxolane -4- Work ) -2-methylacrylate (2006.01)

To a stirred solution of D-mannitol diacetonide 1 (13.0 g, 49.6 mmol) in sodium bicarbonate (5% aq, 60 ml) was added dropwise a saturated solution of sodium periodate (12.74 g, 59.5 mmol) at room temperature. After addition, the mixture was stirred at room temperature for 2 hours. Then ethyl 2- (diethoxyphosphoryl) propanoate 2 (23.0 g, 99.2 mmol) is added followed by potassium carbonate (14.3 g, 103.6 mmol) and the mixture is stirred at room temperature for an additional 120 hours. It was. The mixture was added to 500 ml water and extracted with EtOAc (300 mL × 3). The combined organic layers were dried over anhydrous sodium sulfate, concentrated and chromatographed with PE: EA (30/1) to afford 5.5 g of the desired compound 3 as a colorless oil. Yield: 51.8%.

(S, E) -3- (2,2-dimethyl-1,3- Dioxolane Yl) -2- Methyl propyl 2-en-1-ol (4)

DIBAL-H in a solution of (S, E) -ethyl 3- (2,2-dimethyl-1,3-dioxolan-4-yl) -2-methylacrylate 3 (5.35 g, 5 mmol) in 150 ml THF The solution (50 mL, 50 mmol) was added dropwise at 0 ° C. under argon. After addition, the mixture was stirred at 0 ° C. for 3 hours. When TLC showed little compound 3 remaining, the reaction was quenched with 30 ml ammonia water and stirred with 300 ml dichloromethane for an additional 1 hour. The mixture was filtered, the residue was washed with THF, the combined organic layers were dried and concentrated to give crude compound 4, and then purified by silica gel column using PE: EA = 10: 1-5: 1. 3.0 g of compound 4 was obtained as a colorless oil. Yield: 70%.

(S, E) -3- (2,2- dimethyl -1,3- Dioxolane -4- Work ) -2-methylallyl acetate (5)

(S, E) -3- (2,2-dimethyl-1,3-dioxolan-4-yl) -2-methylprop-2-en-1-ol 4 (2.92 g, 17 in 100 ml THF mmol) was added triethylamine (4.3 g, 42.5 mmol). Acetyl chloride (2.7 g, 34 mmol) was then added dropwise at 0 ° C. under argon. After addition, the mixture was stirred at 0 ° C. for 3 hours. When TLC showed little compound 4 remaining, the reaction was poured into 150 ml water. Extracted with EtOAc (50mLx3). The combined organic layers were dried over anhydrous sodium sulfate, concentrated and chromatographed with PE: EA (10/1) to afford 3.4 g of the desired compound 5 as a colorless oil. Yield 89%.

((Trans) -2-((S) -2,2- dimethyl -1,3- Dioxolane -4- Work ) -1-methylcyclo profile Methyl Acetate (6)

Diethyl zinc in a solution of (S, E) -3- (2,2-dimethyl-1,3-dioxolan-4-yl) -2-methylallyl acetate 5 (3.2 g, 15 mmol) in 200 mL of hexane (1M in hexane, 75 mL) was added in portions at -20 ° C. under argon. After addition, diiodomethane (40 g, 150 mmol) was added dropwise at −20 ° C. or lower with vigorous stirring. The mixture was stirred for an additional 8 hours at -20 ° C or less and then warmed to room temperature and then stirred for an additional 8 hours. The reaction mixture was quenched with 100 ml cold NH ₄ Cl aqueous solution and then extracted with ether (100 ml × 5). The combined organic layers were washed with aqueous sodium thiosulfate solution, water and brine, dried and concentrated to give crude compound 6 , which was purified by a silica gel column (PE: EA = 30: 1-20: 1) to give a colorless oil. 3.0 g of 6 was obtained. Yield: 88%.

((Trans) -2-((S) -1,2- Dihydroxyethyl ) -1-methylcyclo profile Methyl Acetate (7)

((Trans) -2-((S) -2,2-dimethyl-1,3-dioxolan-4-yl) -1-methylcyclopropyl) methyl acetate 6 (2.97 g, 13 in 30 ml 80% acetic acid mmol) was stirred at room temperature for 15 hours. When TLC showed little 6 remaining, the mixture was added dropwise to 300 ml saturated aqueous sodium bicarbonate solution. Then extracted with dichloromethane (200 ml x 3). The combined organic layers were washed with water, brine, dried and concentrated to give crude compound 7 which was then purified by silica gel column (PE: EA = 3: 1-1: 1) to give 1.5 g of compound 7 as colorless oil. Got. Yield 62%.

((Trans) -2-formyl-1-methylcyclo profile Methyl Acetate (8)

Sodium bicarbonate To a stirred solution of ((trans) -2-((S) -1,2-dihydroxyethyl) -1-methylcyclopropyl) methyl acetate 7 (1.41 g, 7.5 mmol) in 20 ml (5% aqueous solution) A saturated solution of sodium periodate (1.93 g, 9 mmol) was added dropwise at room temperature. After addition, the mixture was stirred at room temperature for 2 hours. When TLC showed little 7 remaining, the mixture was added to 50 ml water and extracted with EtOAc (100 mL × 3). The combined organic layers were dried over anhydrous sodium sulfate, concentrated and chromatographed with PE: EA (20/1) to afford 1.0 g of the desired compound 8 as a colorless oil. Yield: 85%.

((Trans) -2-ethynyl-1-methylcyclo profile Methanol (9)

Bestman's reagent (1.44 g, 7.5 mmol) and potassium carbonate in a solution of ((trans) -2-formyl-1-methylcyclopropyl) methyl acetate 8 (780 mg, 5 mmol) in 15 mL methanol (2.07 g, 15 mmol) was added and the mixture was stirred at room temperature for 5 hours. When TLC showed little 8 remained, the reaction mixture was diluted with water (20 mL). After extraction with ether (20mLx3), the combined organic layers were dried and concentrated to give crude compound 9 , which was then purified by silica gel column (PE: Et ₂ O = 10: 1-5: 1) to give 400 mg of colorless oil. 9 was obtained. Yield: 73%.

methyl  4-(((trans) -2- ( Hydroxymethyl )-2- Methylcyclopropyl ) Buta -1,3- Diinil ) Benzoate  (11)

THF ((Trans) -2-ethynyl-1-methylcyclopropyl) methanol 9 (400 mg, 3.6 mmol), Pd (PPh ₃ ) ₂ Cl ₂ (126 mg, 0.18 mmol) in 20 mL, CuI (68.4 mg, 0.36 mmol), DIPEA (1.09 g, 10.8 mmol) solution was treated with methyl 4- (bromoethynyl) benzoate 10 (870 mg, 3.6 mmol) under argon. The mixture was stirred at room temperature for 5 hours. When TLC showed little compound 9 remained, the solvent was removed under reduced pressure, the residue was diluted with water (50 mL), extracted with dichloromethane (3x50 mL), the organic layer was dried and concentrated to Crude compound 11 was obtained as a red oil, and then purified by silica gel column chromatography PE: EA = 20: 1 to 10: 1 to obtain 430 mg of 11 as a yellow solid. Yield 40%.

4-(((1S, 2R) -2- (hydroxy methyl ) -2-methylcyclo profile ) Buta -1,3- Diinil Benzoic acid ( ACHL02 -112-intermediate-A)

To a stirred solution of methyl 4-(((trans) -2- (hydroxymethyl) -2-methylcyclopropyl) buta-1,3-diynyl) benzoate 11 (400 mg, 1.5 mmol) in 16 ml of THF. A solution of lithium hydroxide mono-hydrate (240 mg, 6 mmol) in 4 mL water was added. After addition, the mixture was stirred at room temperature for 15 hours. When LCMS showed little Compound 11 remaining, the mixture was added to 50 ml water. The pH was adjusted to 3.0 with 1M HCl at 0 ° C. and extracted with EtOAc (100 mL × 3). The combined organic layers were dried over anhydrous sodium sulfate and concentrated to yield 370 mg of the desired compound as a yellow powder. Yield: 92%. LCMS: CP-0004344-006-3-03665-LCMSA035 (ESI) m / z = 255 (M + l). 1 HNMR: CP-0004344-006-3 (DMSO- d ₆ , 400 MHz) δ: 13.1 (s, 1H), 7.91 (d, J = 8.4 Hz, 2H), 7.62 (d, J = 8.4 Hz, 2H) , 4.75 (s, 1H), 3.22 (m, 2H), 1.59 (m, 1H), 1.18 (s, 3H), 1.08 (m, 1H), 0.66 (m, 1H).

(S) -methyl 2-amino-3- (tert-butoxycarbonylamino) -3-methylbutanoate (347 mg, 1.408 mmol, 1.0 equiv) and 4-(((1S, 2R) -2- (Hydroxymethyl) -2-methylcyclopropyl) buta-1,3-diynyl) benzoic acid (358 mg, 1.408 mmol, 1.0 equiv) was dissolved in DMF (3 mL). To this was added DIPEA (0.615 mL, 3.52 mmol, 2.5 equiv) followed by HATU (642 mg, 1.689 mmol, 1.2 equiv). It was stirred at room temperature for ˜ 16 hours. The reaction was partitioned between 1M 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 and then evaporated to dryness. 912 mg of crude compound (S) -methyl 3- (tert-butoxycarbonylamino) -2- (4-(((1S, 2R) -2- (hydroxymethyl) -2-methylcyclopropyl) buta -1,3-diynyl) benzamido) -3-methylbutanoate was obtained. LCMS M + l predicted = 483.2, found = 483.2.

(S) -methyl-3- (tert-butoxycarbonylamino) -2- (4-(((1S, 2R) -2- (hydroxymethyl) -2-methylcyclopropyl) buta-1,3 -Diynyl) benzamido) -3-methylbutanoate (crude compound) was dissolved in methanol (1 mL). At room temperature was added 4.0M HCl in dioxane (2.97 mL, 11.86 mmol, 8.43 equiv). The reaction was complete after 60 minutes (HPLC). The reaction was concentrated on rotovap at 0 ° C. To this was added IPA (2 mL) followed by hydroxylamine solution (1.859 mL, 28.1 mmol, 20 equiv). The flask was placed at 4 ° C. for ˜ 40 hours. The reaction was concentrated to a gummy mass (the reaction was stored at 0 ° C.). To this was added water (3 mL) and ACN (0.5 mL). Acidified at 0 ° C. with TFA (3 mL). Additional water (1 mL) and ACN (1 mL) were added. Purified by RP HPLC (2 "column, 50 mL / min, 0.1% TFA in water / ACN, equilibrated at 5% B). Loaded using a syringe filter on 2" column (10 mL / min, 5% B). (2X6.5 mL). Gradient was 50 mL / min over 1 minute. The reaction was carried out at 5-30% B over 66 minutes, and the product (prod) eluted at 25% B. Desired fractions were combined, frozen and placed in a lyo. 410 mg of N-((S) -3-amino-1- (hydroxyamino) -3-methyl-1-oxobutan-2-yl) -4-(((1S, 2R) -2- (hydroxy Oxymethyl) -2-methylcyclopropyl) buta-1,3-diynyl) benzamide ( I-11 ), TFA was obtained. LCMS M + l predicted = 384.2, found = 384.2. 1 H NMR (DMSO-d6): δ 0.61-0.64 (t, 1H), 1.02-1.06 (m, 1H), 1.16 (s, 3H), 1.25 (s, 3H), 1.30 (s, 3H), 1.55- 1.60 (m, 1H), 3.17-3.29 (m, 2H), 4.65-4.67 (d, 1H), 7.61-7.63 (d, 2H), 7.88-7.90 (d, 2H), 7.98 (s, 2H), 8.53-8.56 (d, 1 H), 9.21 (br, 1 H), 11.19 (s, 1 H).

J. N-((S) -3-amino-1- ( Hydroxyamino ) -3- methyl -One- Oxobutane -2-yl) -4-(((1,3-cis) -3- ( Hydroxymethyl ) Cyclobutyl ) Buta -1,3- Diinil ) Benzamide  (I-12)  And N-((S) -3-amino-1- ( Hydroxyamino ) -3- methyl -One- Oxobutane -2-yl) -4-(((1,3- Tran Synthesis of S) -3- (hydroxymethyl) cyclobutyl) buta-1,3-diynyl) benzamide (I-13)

3- Methylenecyclobutanecarboxylic acid  (2)

A solution of 3-methylenecyclobutanecarbonitrile 1 (27.9 g, 0.3 mol) in ethanol (200 mL) and water (200 mL) was treated with potassium hydroxide (84 g, 1.5 mol). The resulting mixture was stirred and heated to 105 ° C. for 4 hours. Ethanol was removed under reduced pressure. The residue was cooled to 0 ° C., the pH adjusted to 1-2 and then extracted with ethyl acetate, dried over sodium sulfate, filtered and the solvent removed. (30.2 g, yield = 90%).

Methyl 3-methylene Cyclobutane carboxylate  (3)

3-methylenecyclobutanecarboxylic acid 2 (29.12 g, 0.26 mol), Potassium carbonate (70.2 g, 0.52 mol), acetone (500 mL) and dimethyl sulfate (39.312 g, 0.312 mol) were heated at reflux for 2 hours. The reaction mixture was cooled to room temperature and filtered. The solvent was removed under reduced pressure. The residue was purified by silica gel chromatography to give the desired product as a colorless oil. (27.5 g, yield = 84%).

methyl  3- ( Hydroxymethyl Cyclobutane Carboxylate  (4)

Methyl 3-methylenecyclobutanecarboxylate 3 (24.46 g, 0.21 mol) and dry THF (130 ml) were added to a dry three-necked flask, cooled to -10 ° C, and the borane-THF complex (70.0 mL) was syringed. Dropping through The resulting mixture was stirred at room temperature for 4 hours, and then cooled to -20 ° C to -10 ° C; Methanol was added and stirred for 15 minutes. Sodium hydroxide (3M 30 mL) and hydrogen peroxide (34.0 g, 0.210 mol) were added sequentially. The mixture was stirred for 2 hours and saturated sodium sulfite solution (100 mL) was added. The reaction mixture was diluted with water, then extracted with ethyl acetate, washed with water and brine, dried over sodium sulfate, filtered, the solvent was removed and the residue was purified by silica gel chromatography. (28.73g, 95%)

(Trans) -methyl 3-formyl Cyclobutane carboxylate  (5a) and (cis) -methyl 3-formylcyclobutanecarboxylate (5b)

To a solution of oxalyl chloride (17.8 g, 139 mmol, 2.0 eq) in CH ₂ Cl ₂ (450 mL) was slowly added DMSO (21.7 g, 278 mmol, 4.0 eq) in dichloromethane (50 mL) at −78 ° C. After 30 minutes, methyl 3- (hydroxymethyl) cyclobutanecarboxylate 4 (10 g, 69.4 mmol, 1.0 eq) in dichloromethane (150 mL) was added dropwise at -78 ° C. The mixture was stirred at -78 ° C for an additional 2 hours, then triethylamine (70 g, 700 mmol, 10.0eq) was added at -78 ° C. After 20 minutes, the mixture was warmed to room temperature and saturated aqueous NH ₄ Cl solution was added. The layers were separated and the aqueous layer was extracted with dichloromethane (3 × 200 mL). The combined organic layers were washed with brine, dried (sodium sulfate), filtered and concentrated to afford the crude product as a mixture of cis and trans isomers. It was purified by silica column to give 5a (trans, 3.1 g) and 5b (cis, 2.9 g).

(Cis) -methyl 3- (2,2- Dibromovinyl Cyclobutane Carboxylate  (6b)

Triphenylphosphine in 80 mL dichloromethane in a solution of carbon tetrabromide (13.56 g, 40.89 mmol, 2.0 eq) in 40 mL dichloromethane (21.41 g, 81.80 mmol, 4.0 eq) solution was added dropwise under -20 ° C under argon. The mixture was stirred for 30 min at -20 ° C and then cooled to -78 ° C. (Cis) -methyl 3-formylcyclobutanecarboxylate 5b (2.9 g, 20.40 mmol, 1.0 eq) in 80 mL dichloromethane was added dropwise and stirred at −78 ° C. for a further 1 hour. The mixture was allowed to warm to room temperature. The reaction mixture was added dropwise to 300 mL PE under stirring. The mixture was filtered and the residue washed with PE. The combined organic layers were dried over sodium sulfate and concentrated to afford the desired compound and purified by silica gel column. (2.3g, yield = 37%)

((Cis) -3- (2,2- Dibromovinyl Cyclobutyl) Methanol (7b)

To compound 6b (2.5 g, 8.4 mmol, 1.0 eq) dissolved in THF (10 mL) was added lithium borohydride (203 mg) at 0 ° C. The reaction mixture was stirred at room temperature for 3 hours. Water (5 mL) was added and extracted with ethyl acetate. The combined ethyl acetate layer was dried and concentrated under reduced pressure. The crude product was purified by silica gel column using PE: EA = 5: 1. (1.88 g yield = 83%).

Methyl 4-(((cis) -3- (hydroxy methyl Cyclobutyl) Buta -1,3- Diinil ) Benzoate (9b)

((Cis) -3- (2,2-dibromovinyl) cyclobutyl) methanol 7b (1.88 g, 6.9 mmol, 1.0 eq) in dry DMF (30 mL), Methyl 4-ethynylbenzoate 8 (1.56g, 9.7mmol, 1.4eq), tri (4-methylphenyl) phosphine (98mg, 0.28mmol, 0.04eq), Pd (dba) ₄ (64mg, 0.069mmol, 0.01eq To the mixture was added triethylamine (2.11 g, 20.9 mmol, 3.0 eq) under a nitrogen atmosphere. The reaction mixture was stirred at 80 ° C. for 15 hours. The reaction was monitored by LCMS. The reaction mixture was diluted with NH ₄ Cl _aq solution and extracted with ethyl acetate. The combined ethyl acetate layers were washed with water, brine and dried. The solvent was removed under reduced pressure to give crude product which was purified by silica gel column using PE: EA = 5: 1. (450 mg, yield = 24%).

4-(((cis) -3- (hydroxy methyl Cyclobutyl) Buta -1,3- Diinil Benzoic acid ( ACHL02 -146-cis)

Lithium hydroxide mono- in a solution of methyl 4-(((cis) -3- (hydroxymethyl) cyclobutyl) buta-1,3-diynyl) benzoate 9b (450 mg, 1.0 equiv) in THF (200 mL) Hydrate (282 mg, 4.0 equiv) was added and the mixture was then reacted at 25 ° C. for 5 h. The reaction solvent was then removed and neutralized with 3 N HCl to PH 5-6 and extracted with ethyl acetate. The combined ethyl acetate layer was washed with brine and dried. The solvent was evaporated to give the desired compound. (400 mg, yield = 94%).

(Trans) -methyl 3- (2,2- Dibromovinyl Cyclobutane Carboxylate  (6a)

Triphenylphosphine in 80 mL dichloromethane in a solution of carbon tetrabromide (11.2 g, 33.80 mmol, 2.0 eq) in 40 mL dichloromethane (17.7 g, 67.60 mmol, 4.0 eq) solution was added dropwise under -20 ° C under argon. The mixture was stirred for 30 min at -20 ° C and then cooled to -78 ° C. (Trans) -methyl 3-formylcyclobutanecarboxylate 5a (2.4 g, 16.90 mmol, 1.0 eq) in 80 mL dichloromethane was added dropwise and stirred at -78 ° C for a further 1 hour. The mixture was allowed to warm to room temperature. The reaction mixture was added dropwise to 300 mL PE under stirring. Filtration and the residue washed with PE. The combined organic layers were dried over sodium sulfate and concentrated to afford the desired compound and purified by silica gel column. (2.5 g, yield = 50%).

((Trans) -3- (2,2- Dibromovinyl Cyclobutyl) Methanol (7a)

Lithium borohydride (203 mg) was dissolved in (trans) -methyl 3- (2,2-dibromovinyl) cyclobutanecarboxylate 6a (2.5 g, 8.4 mmol, 1.0 eq) dissolved in THF (10 mL) at 0 ° C. Was added. The reaction mixture was stirred at room temperature for 3 hours. Water (5 mL) was added and extracted with ethyl acetate. The combined ethyl acetate layer was dried and concentrated under reduced pressure. The residue was purified by silica gel column using PE: EA = 5: 1. (1.70 g, yield = 75%).

Methyl 4-(((trans) -3- (hydroxy methyl Cyclobutyl) Buta -1,3- Diinil ) Benzoate (9a)

((Trans) -3- (2,2-dibromovinyl) cyclobutyl) methanol 7a (1.70 g, 6.3 mmol, 1.0 eq), compound 8 (1.41 g, 8.8 mmol, 1.4 in dried DMF (30 mL) eq), tri (4-methylphenyl) phosphine (89 mg, 0.25 mmol, 0.04 eq), Pd (dba) ₄ (58 mg, 0.063 mmol, 0.01 eq) in a mixture of triethylamine (1.91 g, 18.9 mmol, 3.0 eq) ) Was added under nitrogen. The reaction mixture was stirred at 80 ° C. for 15 hours. The reaction was monitored by LCMS. The reaction mixture was diluted with NH ₄ Cl _aq solution and extracted with ethyl acetate. The combined ethyl acetate layers were washed with water, brine and dried. The solvent was evaporated under reduced pressure to give crude product which was purified by silica gel column using PE: EA = 5: 1. (550 mg, yield = 33%).

4-(((trans) -3- (hydroxy methyl Cyclobutyl) Buta -1,3- Diinil Benzoic acid ( ACHL02 -146-trans)

Lithium hydroxide mono- in a solution of methyl 4-(((trans) -3- (hydroxymethyl) cyclobutyl) buta-1,3-diynyl) benzoate 9a (550 mg, 1.0 equiv) in THF (20 mL) Hydrate (345 mg, 4.0 equiv) was added and the mixture was then reacted at 25 ° C. for 5 h. The reaction solvent was then removed and neutralized to pH 5-6 with 3 N HCl and extracted with ethyl acetate. The combined ethyl acetate layer was washed with brine and dried. The solvent was evaporated to give the desired compound. (340 mg, yield = 65%).

(S) -methyl 2-amino-3- (tert-butoxycarbonylamino) -3-methylbutanoate (387 mg, 1.573 mmol, 1.0 equiv) and 4-(((1,3- cis )- 3- (hydroxymethyl) cyclobutyl) buta-1,3-diynyl) benzoic acid (400 mg, 1.573 mmol, 1.0 equiv) was dissolved in DMF (3 mL). To this was added DIPEA (0.687 mL, 3.93 mmol, 2.5 equiv) followed by HATU (718 mg, 1.888 mmol, 1.2 equiv). It was stirred at room temperature for ˜ 16 hours. The reaction was partitioned between 1M 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 and then evaporated to dryness. 1.035 g of crude compound (S) -methyl 3- (tert-butoxycarbonylamino) -2- (4-(((1,3- cis ) -3- (hydroxymethyl) cyclobutyl) buta-1 , 3-diynyl) benzamido) -3-methylbutanoate was obtained. LCMS M + l predicted = 483.2, found = 483.2.

(S) -methyl 3- (tert-butoxycarbonylamino) -2- (4-(((1,3- cis ) -3- (hydroxymethyl) cyclobutyl) buta-1,3-diynyl Benzamido) -3-methylbutanoate (crude compound) was dissolved in methanol (1 mL). At room temperature was added 4.0M HCl in dioxane (3.3 mL, 13.26 mmol, 8.43 equiv). The reaction was complete after 60 minutes (HPLC). The reaction was concentrated at 0 ° C. on a rotary evaporator. To this was added IPA (2 mL) followed by hydroxylamine solution (2 mL, 31.5 mmol, 20 equiv). The flask was placed at 4 ° C. for 24 hours. The reaction was concentrated to a gummy mass (the reaction was stored at 0 ° C.). To this was added water (3 mL) and ACN (0.5 mL). Acidified with TFA (3 mL) at 0 ° C. Additional water (1 mL) and ACN (1 mL) were added. Purified by RP HPLC (2 "column, 50 mL / min, 0.1% TFA in water / ACN, equilibrated at 5% B). Loaded using a syringe filter on 2" column (10 mL / min, 5% B). (2X6 mL). Gradient was 50 mL / min over 1 minute. The reaction was carried out at 5-35% B over 55 minutes and the product eluted at 24.5-26% B. Desired fractions were combined, frozen and placed in a lyo. 460 mg of N-((S) -3-amino-1- (hydroxyamino) -3-methyl-1-oxobutan-2-yl) -4-(((1,3- cis ) -3- (Hydroxymethyl) cyclobutyl) buta-1,3-diynyl) benzamide, TFA was obtained. LCMS M + l predicted = 384.2, found = 384.2. 1 H NMR (DMSO-d 6): δ 1.25 (s, 3H), 1.30 (s, 3H), 1.81-1.88 (m, 2H), 2.23-2.34 (m, 3H), 3.08-3.15 (m, 1H), 3.30-3.31 (d, 1H), 4.54 (br, 1H), 4.65-4.67 (d, 1H), 7.61-7.64 (d, 2H), 7.89-7.91 (d, 2H), 7.99 (s, 2H), 8.54-8.56 (d, 1 H), 9.22 (br, 1 H), 11.20 (s, 1 H).

(S) -methyl 2-amino-3- (tert-butoxycarbonylamino) -3-methylbutanoate (329 mg, 1.377 mmol, 1.0 equiv) and 4-(((1,3-trans)- 3- (hydroxymethyl) cyclobutyl) buta-1,3-diynyl) benzoic acid (340 mg, 1.377 mmol, 1.0 equiv) was dissolved in DMF (3 mL). To this was added DIPEA (0.584 mL, 3.34 mmol, 2.5 equiv) followed by HATU (610 mg, 1.605 mmol, 1.2 equiv). It was stirred at room temperature for ˜ 16 hours. The reaction was partitioned between 1M 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 and then evaporated to dryness. 990 mg of crude compound (S) -methyl 3- (tert-butoxycarbonylamino) -2- (4-(((1,3-trans) -3- (hydroxymethyl) cyclobutyl) buta-1 , 3-diynyl) benzamido) -3-methylbutanoate was obtained. LCMS M + l predicted = 483.2, found = 483.2.

(S) -methyl-3- (tert-butoxycarbonylamino) -2- (4-(((1,3-trans) -3- (hydroxymethyl) cyclobutyl) buta-1,3-diyne Yl) benzamido) -3-methylbutanoate (crude compound) was dissolved in methanol (1 mL). At room temperature was added 4.0M HCl in dioxane (2.82 mL, 11.27 mmol, 8.43 equiv). The reaction was complete after 60 minutes (HPLC). The reaction was concentrated at 0 ° C. on a rotary evaporator. To this was added IPA (2 mL) followed by hydroxylamine solution (1.77 mL, 26.7 mmol, 20 equiv). The flask was placed at 4 ° C. for 96 hours. The reaction was then concentrated to a gum mass (the reaction was stored at 0 ° C.). To this was added water (3 mL) and ACN (0.5 mL). The solution was acidified with TFA (3 mL) at 0 ° C. Additional water (1 mL) and ACN (1 mL) were added. The compound was purified by RP HPLC (2 "column, 50 mL / min, 0.1% TFA in water / ACN, equilibrated at 5% B); using a syringe filter on a 2" column (10 mL / min, 5% B) After loading (2X7 mL); Gradient to 50 mL / min over 1 minute and 5-35% B over 55 minutes. The product eluted at 22.9-25.5% B. Desired fractions were combined, frozen and placed in lyophilizer. This procedure results in 410 mg of N-((S) -3-amino-1- (hydroxyamino) -3-methyl-1-oxobutan-2-yl) -4-(((1,3-trans) 3- (hydroxymethyl) cyclobutyl) buta-1,3-diynyl) benzamide, TFA was obtained. LCMS M + l predicted = 384.2, found = 384.2. 1 H NMR (DMSO-d 6): δ 1.25 (s, 3H), 1.30 (s, 3H), 2.07-2.11 (t, 4H), 2.40-2.43 (m, 1H), 3.22-3.26 (m, 1H), 3.37-3.38 (d, 1H), 4.57 (br, 1H), 4.65-4.67 (d, 1H), 7.62-7.64 (d, 2H), 7.89-7.91 (d, 2H), 7.97 (s, 2H), 8.54-8.56 (d, 1 H), 9.21 (br, 1 H), 11.19 (s, 1 H).

K. N-((S) -3-amino-1- (hydroxy Amino ) -3-methyl-1- Oxobutane -2- Work Synthesis of) -4-(((1,3-trans) -3- (hydroxymethyl) cyclopentyl) buta-1,3-diynyl) benzamide (I-14)

(Cis) -cyclopentane-1,3- Dicarboxylic acid (2)

Bicyclo [2.2.1] hept-2 in acetone (15 mL) to a stirred suspension of potassium permanganate (47 g, 0.297 mol) and magnesium sulfate (4.3 g, 35.4 mmol) in water (500 mL) cooled to 5 ° C. -En 1 (10 g, 0.106 mol) solution was added in one portion (5 ° C.-40 ° C.). The ice bath was removed and the reaction stirred for an additional 2 hours. After this time, the mixture was filtered and the filtrate was treated with sodium hydrogensulfite and acidified to pH = 2 with 1N HCl. Then extracted with ethyl acetate (800 mL x 3) and the combined organic layers were washed with water (1 L), brine (1 L), dried and concentrated to afford the desired compound 2 as a white solid without purification for the next step. (7.5 g, 46%) was obtained.

(Cis)- dimethyl  Cyclopentane-1,3- Dicarboxylate (3)

To a stirred solution of (cis) -cyclopentane-1,3-dicarboxylic acid 2 (7.5 g, 47.5 mmol) in methanol (200 mL) was added thionyl chloride (22.6 g, 0.19 mol) in an ice bath. The reaction mixture was then heated at reflux overnight. After this time, the mixture was concentrated to give compound 3 (8.3 g, 94%) as a yellow oil without purification for the next step. Confirmed by GC-MS.

Cyclopentane -1,3- Diyl dimethanol (4)

To a stirred solution of LAH (3.4 g, 89.2 mmol) in THF (100 mL) was added a solution of (cis) -dimethyl cyclopentane-1,3-dicarboxylate 3 (8.3 g, 44.6 mmol) in THF (50 mL) on ice. Added in bath. The reaction mixture was then stirred at room temperature overnight. After this time, the mixture is diluted with water (6.5 mL), then filtered, the filtrate is concentrated and the residue is purified by silica-gel chromatography (PE: EA = 2: 1) to give the compound as a yellow oil. 4 (4.0 g, 69%) was obtained. HNMR (400 MHz, DMSO-d6) 0.53-0.50 (m, 1H), 0.96-1.00 (m, 2H), 1.29-1.35 (m, 2H), 1.47-1.54 (m, 1H), 1.66-1.73 (m , 2H), 2.98-3.01 (m, 4H), 4.29 (s, 1H).

3- (hydroxy methyl Cyclopentyl) methyl acetate (5)

To a stirred solution of cyclopentane-1,3-diyldimethanol 4 (4.0 g, 31 mmol) in THF (60 mL) was added NaH (60%) (1.1 g, 28 mol) in an ice bath. The reaction mixture was then stirred for 0.5 h in an ice bath, then acetyl chloride (2.7 g, 34 mmol) was added to the reaction mixture. The mixture was then stirred at room temperature overnight. After this time, the mixture was diluted with water (1.0 mL), filtered and the filtrate was concentrated and the residue was purified by silica-gel chromatography to give compound 5 (1.7 g, 25%) as a yellow oil. . HNMR (400 MHz, DMSO-d6) 0.84-0.87 (m, 1H), 1.28-1.31 (m, 2H), 1.62-1.65 (m, 2H), 1.80-1.83 (m, 1H), 1.99 (s, 1H ), 2.10-2.18 (m, 1H), 3.26-3.29 (m, 2H), 3.89 (d, J = 8, 2H), 4.43 (s, 1H).

3-formyl Cyclopentyl Methyl Acetate (6)

To a stirred solution of 3- (hydroxymethyl) cyclopentyl) methyl acetate 5 (1.7 g, 9.9 mmol) in CH ₂ Cl ₂ (50 mL) was added in portions PCC (6.4 g, 29.7 mmol). The reaction mixture was then stirred at room temperature overnight. After this time, the mixture was concentrated and the residue was purified by silica-gel chromatography (PE: EA = 6: 1) to give compound 6 (0.7 g, 44%) as a yellow oil.

3-ethynyl Cyclopentyl ) Methanol (7)

Potassium carbonate in a stirred solution of 3-formylcyclopentyl) methyl acetate 6 (0.7 g, 4.1 mmol) in MeOH (50 mL) (0.85 g, 6.15 mmol) and Bestman's reagent (1.18 g, 6.15 mmol) were added. The reaction mixture was stirred at room temperature overnight. After this time, the mixture is diluted with water (50 mL), extracted with ether (80 mL x 3), the combined organic layers are dried, concentrated and the residue is subjected to silica-gel chromatography (PE: EA = 2: 1) to give compound 7 (0.31 g, 60%) as a yellow oil.

Methyl 4-((3- (hydroxy methyl Cyclopentyl) Buta -1,3- Diinil ) Benzoate ( ACHL02 -149)

Under nitrogen, DIPEA (936 mg, 7.26 mmol) was added 3-ethynylcyclopentyl) methanol 7 (0.3 g, 2.42 mmol) in THF (60 mL), methyl 4- (bromoethynyl) benzoate 8 (0.7 g, 2.9 mmol), PdCl ₂ (PPh ₃ ) ₂ (70 mg, 0.1 mmol) and CuI (46 mg, 0.242 mmol) were added dropwise at room temperature. The reaction mixture was stirred at room temperature overnight. The reaction was monitored by LCMS. The reaction mixture was diluted with water (60 mL) and extracted with ethyl acetate (80 mL x 3). The ethyl acetate layer was washed with saturated aqueous NH ₄ Cl solution, water and brine. After drying, the ethyl acetate solution was concentrated under reduced pressure. The residue was purified by silica-gel column (PE: EA = 5: 1) to give compound ACHL02-149 (as a mixture of cis and trans isomers) (140 mg, 25%). H NMR (400 MHz, DMSO-d6): 7.95 (d, J = 8.4 Hz, 2H), 7.51 (d, J = 8 Hz, 2H), 3.91 (s, 1H), 3.61 (d, J = 4 Hz, 1H) , 3.53 (d, J = 8 Hz, 1H), 2.89-2.87 (m, 1H), 2.43-2.40 (m, 1H), 2.21-2.17 (m, 1H), 2.02-1.99 (m, 1H), 1.82- 1.76 (m, 1 H), 1.54-1.45 (m, 1 H), 1.36-1.31 (m, 1 H).

Methyl 4-((3- (hydroxymethyl) cyclopentyl) buta-1,3-diynyl) benzoate (115 mg, 0.407 mmol, 1.0 equiv) was dissolved in MeOH (5 mL) and lithium hydroxide ( 29.3 mg, 1.222 mmol, 3.0 equiv) and water (5 mL) were added. It was stirred at rt for 72 h. The reaction was concentrated to remove MeOH and acidified to pH ˜3 with 6N HCl (˜5 mL). Extract with EtOAc (3x50 mL), combine organic layers, wash with saturated NaCl, then dry (MgSO ₄ ), filter and concentrate. 0.14 g of crude compound 4-((3- (hydroxymethyl) cyclopentyl) buta-1,3-diynyl) benzoic acid was obtained. LCMS M + 1 Found = 269.1, Found = 269.1.

(S) -methyl 2-amino-3- (tert-butoxycarbonylamino) -3-methylbutanoate (100 mg, 0.406 mmol, 1.0 equiv) and 4-((3- (hydroxymethyl) cyclo Pentyl) buta-1,3-diynyl) benzoic acid (crude compound) (109 mg of theory, 0.406 mmol, 1.0 equiv) was dissolved in DMF (1 mL). To this was added DIPEA (0.177 mL, 1.016 mmol, 2.5 equiv) followed by HATU (185 mg, 0.488 mmol, 1.2 equiv). It was stirred at room temperature for ˜ 16 hours. The reaction was partitioned between 1M 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 and then evaporated to dryness. 500 mg of crude compound (S) -methyl 3- (tert-butoxycarbonylamino) -2- (4-((3- (hydroxymethyl) cyclopentyl) buta-1,3-diynyl) benzami Fig.-3-Methylbutanoate was obtained. LCMS M + 1 Found = 497.3, Found = 497.2.

(2S) -methyl-3- (tert-butoxycarbonylamino) -2- (4-((3- (hydroxymethyl) cyclopentyl) buta-1,3-diynyl) benzamido) -3 Methylbutanoate (crude compound) was dissolved in methanol (1 mL). At room temperature was added 4.0M HCl in dioxane (0.857 mL, 3.43 mmol, 8.43 equiv). The reaction was complete after 3 hours (HPLC). The reaction was concentrated at 0 ° C. on a rotary evaporator. To this was added IPA (1 mL) followed by hydroxylamine solution (0.537 mL, 8.14 mmol, 20 equiv). The flask was placed at 4 ° C. for 168 hours. The reaction was concentrated to a gummy mass (the reaction was stored at 0 ° C.). To this was added water (3 mL) and ACN (0.5 mL). Acidified with TFA (3 mL) at 0 ° C. Additional water (1 mL) and ACN (1 mL) were added. Purified by RP HPLC (1 "column, 20 mL / min, 0.1% TFA in water / ACN, equilibrated at 5% B). Loaded using a syringe filter on 1" column (10 mL / min, 5% B). (2X8 mL). Gradient to 20 mL / min over 1 minute. Performed at 5-19% B over 80 minutes and the product eluted at 19% B. Desired fractions were combined, frozen and placed in lyophilizer. 145 mg of N-((S) -3-amino-1- (hydroxyamino) -3-methyl-1-oxobutan-2-yl) -4-((3- (hydroxymethyl) cyclopentyl) Buta-1,3-diynyl) benzamide, I-14 , TFA was obtained. LCMS M + l predicted = 398.2, found = 398.2. 1 H NMR (DMSO-d 6): δ 1.25 (s, 3H), 1.29 (s, 3H), 1.38-1.45 (m, 1H), 1.53-1.77 (m, 2H), 1.88-2.17 (m, 3H), 2.83-2.91 (m, 1H), 3.21-3.38 (m, 4H), 4.52 (br, 1H), 4.65-4.67 (d, 1H), 7.61-7.63 (d, 2H), 7.88-7.90 (d, 2H ), 7.97 (s, 2H), 8.53-8.56 (d, 1H), 9.21 (br, 1H), 11.19 (s, 1H).

L. N-((S) -3-amino-1- (hydroxy Amino ) -3-methyl-1- Oxobutane -2- Work ) -4-(((1,4-cis) -4- (hydroxymethyl) cyclohexyl) buta-1,3-diynyl) benzamide (I-15)  And N-((S) -3-amino-1- (hydroxy Amino ) -3-methyl-1- Oxobutane -2- Work Synthesis of) -4-(((1,4-trans) -4- (hydroxymethyl) cyclohexyl) buta-1,3-diynyl) benzamide (I-16)

Step 1: dimethyl Cyclohexane -1,4- Dicarboxylate (2)

Methanol (500 mL) was cooled in an ice-salt bath and thionyl chloride (138 g, 4.0eq) was added dropwise. To the solution of HCl in methanol was added cyclohexane-1,4-dicarboxylic acid 1 (50 g, 1.0 equiv) and the reaction mixture was refluxed for 1 h. The reaction was monitored by TLC. The solvent was evaporated and the residue was diluted with water and extracted with ethyl acetate. The ethyl acetate layer was washed with NaHCO ₃ solution, brine and dried over Na ₂ SO ₄ . Evaporation of the solvent gave the desired compound (56.4 g, 97%).

Step 2: Cyclohexane -1,4- Diyl dimethanol (3)

To an ice-cooled suspension of LiAlH ₄ in THF was added dropwise a solution of dimethyl cyclohexane-1,4-dicarboxylate 2 at 0 ° C. The reaction mixture was stirred for 30 min at ambient temperature. The reaction was monitored by TLC. The reaction was quenched by the addition of 12eq water (32.4 mL). The reaction mixture was filtered and the filtrate was concentrated. The residue was diluted with Et ₂ O and dried over Na ₂ SO ₄ . Et ₂ O was removed to collect the desired compound and used for the next step without further purification (19.9 g, 92%).

Step 3:  (4-( Hydroxymethyl ) Cyclohexyl ) methyl  acetate (4)

AcCl was added dropwise at 0 ° C. to an ice-cooled suspension of NaH and cyclohexane-1,4-diyldimethanol 3 in THF. The reaction was stirred at 15 ° C. for 3 hours. The reaction was monitored by TLC. The reaction was quenched by the addition of 1 eq H ₂ O (2.5 mL). The reaction mixture was filtered and the filtrate was diluted with ethyl acetate. The ethyl acetate layer was washed with brine (5 mL) and dried over Na ₂ S0 ₄ . The solvent was removed under reduced pressure. The residue was purified by silica-gel column (DCM: MeOH = 50: 1 to 10: 1) to afford the desired compound (8.6 g, 33%).

Step 4:  (4- Formylcyclohexyl ) methyl  acetate (5)

To a solution of oxalyl chloride in DCM (60 mL) was added dropwise DMSO in DCM (60 mL) at -78 ° C. After 30 minutes, (4- (hydroxymethyl) cyclohexyl) methyl acetate 4 in DCM (80 mL) was added dropwise at -78 ° C. The reaction mixture was stirred at −78 ° C. for a further 2 hours, then the reaction was quenched by dropwise addition of TEA at −78 ° C. The reaction mixture was allowed to warm to room temperature. The reaction mixture was diluted with DCM and washed with saturated aqueous ammonium chloride solution and brine. The DCM layer was dried and concentrated. The residue was purified by silica-gel column (DCM) to afford the desired compound (9.0 g, still wet with dichloromethane).

Step 5:  (4- Ethynylcyclohexyl ) Methanol (6)

Bestman reagent (11.56 g, 1.3 eq, 80) in a mixture of (4-formylcyclohexyl) methyl acetate 5 (8.5 g, 1.0 eq), K ₂ CO ₃ (19.04 g, 3.0 eq) in CH ₃ OH (100 mL) %) Was added at 0 ° C. The mixture was stirred at room temperature for 15 hours. The reaction was monitored by TLC. The mixture was diluted with water, extracted with ethyl acetate, washed with brine, dried over Na ₂ S0 ₄ and concentrated under reduced pressure. The crude product was purified by silica-gel column (PE: EA = 3: 1) to afford the desired compound (2.6 g, 40%).

Step 6: methyl  4-((4- ( Hydroxymethyl ) Cyclohexyl ) Buta -1,3- Diinil ) Benzoate (8)

Under nitrogen, DIPEA (3.92 g, 3.0 equiv) was added (4-ethynylcyclohexyl) methanol 6 (1.4 g, 1.0 equiv) in THF (150 mL), methyl 4- (bromoethynyl) benzoate 7 (2.9 g, 1.2 equiv), PdCl ₂ (PPh ₃ ) ₂ (213 mg, 0.03 equiv) and CuI (194 mg, 0.1 equiv) were added dropwise at room temperature. The reaction mixture was stirred at room temperature overnight. The reaction was monitored by LCMS. The reaction mixture was diluted with water (100 mL) and extracted with ethyl acetate (300 mL). The ethyl acetate layer was washed with saturated aqueous NH ₄ Cl solution, water and brine. After drying, the ethyl acetate solution was concentrated under reduced pressure. The residue was purified by silica-gel column (PE: EA = 5: 1 to 3: 1) to afford the desired compound (1.2 g, 40% as a mixture of cis and trans isomers). ¹ H NMR (400 MHz, DMSO-d ₆ ): 7.959-7.937 (d, J = 8.8 Hz, 2H), 7.682-7.660 (d, J = 8.8 Hz, 2H), 4.421-4.395 (t, J = 5.2 Hz , 1H), 3.860 (s. 3H), 3.207-3.179 (t, J = 5.6 Hz, 2H), 2.085 (s, 1H), 1.980-1.941 (m, 2H), 1.747-1.708 (m, 2H), 1.366-1.328 (m, 3H), 0.935-0.897 (m, 2H).

4-((4- (hydroxy methyl Cyclohexyl) Buta -1,3- Diinil ) Benzoic acid (2)

Lithium hydroxide in water (5 mL) in a solution of methyl 4-((4- (hydroxymethyl) cyclohexyl) buta-1,3-diynyl) benzoate 1 (1.2 g, 4.1 mmol) in THF (20 mL) Mono-hydrate (517 mg, 12.3 mmol) solution was added. The mixture was stirred at room temperature overnight. After this time, the solvent was removed under reduced pressure and the residue was diluted with water (40 mL), then the pH was adjusted to 4.0 using 1M HCl and then extracted with ethyl acetate (3 x 60 mL). The organic layer was dried and concentrated to afford 4-((4- (hydroxymethyl) cyclohexyl) buta-1,3-diynyl) benzoic acid 2 (1.0 g, 93%) as a yellow solid, which was obtained without further purification. It was used for. LC-MS: 283 [M + H] ⁺ .

(S) - methyl  3- ( Rat - Butoxycarbonylamino ) -2- (4-((4- ( Hydroxymethyl ) Cyclohexyl ) Buta -1,3- Diinil ) Benzamido ) -3- Methylbutanoate  (3)

4-((4- (hydroxymethyl) cyclohexyl) buta-1,3-diynyl) benzoic acid 2 (1.05 g, 3.72 mmol) in DMF (25 mL), HATU (1.55 g, 4.1 mmol) in solution (S) -methyl 2-amino-3- (tert-butoxycarbonylamino) -3-methylbutanoate (915 mg, 3.72 mmol) and DIPEA (1.44 g, 11.2 mmoL) was added. The mixture was stirred at room temperature for 5 hours. After this time, the reaction was diluted with water (20 mL) and then extracted with ethyl acetate (60 mL x 3). The combined organic layers were washed with water and then brine, dried and concentrated, and then the residue was purified by silica-gel chromatography (PE: EA 2: 1) to give (S) -methyl 3- (as a yellow solid). Tert-butoxycarbonylamino) -2- (4-((4- (hydroxymethyl) cyclohexyl) buta-1,3-diynyl) benzamido) -3-methylbutanoate 3 was obtained. (1.6 g, 85%). LC-MS: 511 [M + H] ⁺ . 455 [M-56]

(S) -methyl 3-amino-2- (4-((4- (hydroxy methyl Cyclohexyl) Buta -1,3-diynyl) benzamido) -3-methylbutanoate hydrochloride (4)

(S) -methyl 3- (tert-butoxycarbonylamino) -2- (4-((4- (hydroxymethyl) cyclohexyl) buta-1,3-diynyl) benzami in methanol (20 mL) FIG. 3-3-Methylbutanoate 3 (1.6 g, 3.14 mmol) solution was attached to the HCl apparatus. The reaction was then stirred at room temperature until TLC showed total consumption of starting material. After that time the solution was concentrated under reduced pressure to afford (S) -methyl 3-amino-2- (4-((4- (hydroxymethyl) cyclohexyl) buta-1,3-diynyl) benzamido as a yellow solid. ) -3-methylbutanoate hydrochloride 4 was obtained (1.4 g, 99%). LC-MS: 411 [M + H] ⁺ . The cis and trans isomers were separated by chiral HPLC to give (S) -methyl 3-amino-2- (4-(((1,4- cis ) -4- (hydroxymethyl) cyclohexyl) buta-1 as peak 1 , 3-diynyl) benzamido) -3-methylbutanoate (ACHL02-148-coupled, 90 mg) and (S) -methyl 3-amino-2- (4- ( ((1,4-trans) -4- (hydroxymethyl) cyclohexyl) buta-1,3-diynyl) benzamido) -3-methylbutanoate (ACHL02-147-coupled, 750 mg).

(S) -methyl-3-amino-2- (4-(((1,4-trans) -4- (hydroxymethyl) cyclohexyl) buta-1,3-diynyl) benzamido) -3 Methylbutanoate (750 mg, 1.827 mmol, 1.0 equiv) was placed in a round bottom flask. To this was added IPA (6 mL), the suspension was sonicated for 5 minutes, then THF (2 mL) was added to form a solution. Hydroxylamine solution (2.4 mL, 36.5 mmol, 20 equiv) was added and the flask was placed at 4 ° C. for ˜ 96 h. The reaction was concentrated (storage at 0 ° C.) to remove IPA and THF. Acidified with TFA (4 mL) at 0 ° C. Additional ACN (2 mL) was added. Purified by RP HPLC (2 "column, 50 mL / min, 0.1% TFA in water / ACN, equilibrated at 10% B). Loaded with syringe filter on 2" column (10 mL / min, 10% B). (3X8 mL). Gradient was 50 mL / min over 1 minute. 10-40% B over 60 minutes and the product eluted at 30.5-35% B. Desired fractions were combined, frozen and placed in lyophilizer. 706 mg of N-((S) -3-amino-1- (hydroxyamino) -3-methyl-1-oxobutan-2-yl) -4-(((1,4-trans) -4- (Hydroxymethyl) cyclohexyl) buta-1,3-diynyl) benzamide, I-16 , TFA was obtained. LCMS M + 1 Found = 412.2, Found = 412.2. 1 H NMR (DMSO-d 6): δ 0.84-0.94 (m, 2H), 1.25 (s, 3H), 1.30 (s, 3H), 1.32-1.36 (m, 1H), 1.68-1.72 (m, 2H), 1.90-1.95 (m, 2H), 2.39-2.42 (m, 1H), 3.15-3.18 (m, 2H), 4.38-4.40 (t, 1H), 4.65-4.67 (d, 1H), 7.62-7.64 (d , 2H), 7.89-7.91 (d, 2H), 7.98 (br, 2H), 8.54-8.56 (d, 1H), 9.22 (br, 1H), 11.21 (br, 1H).

(S) -methyl-3-amino-2- (4-(((1,4- cis ) -4- (hydroxymethyl) cyclohexyl) buta-1,3-diynyl) benzamido) -3 Methylbutanoate (90 mg, 0.219 mmol, 1.0 equiv) was placed in a round bottom flask. IPA (0.5 mL) was added thereto, the solution was vortexed for 2 minutes, then hydroxylamine solution (0.29 mL, 4.38 mmol, 20 equiv) was added and the flask was placed at 4 ° C. for ˜72 h. . The reaction was concentrated (storing the reaction at 0 ° C.) to remove IPA. Acidified with TFA (3 mL) at 0 ° C. Acetonitrile (0.5 mL) was added. Purified by RP HPLC (1 "column, 25 mL / min, 0.1% TFA in water / ACN, equilibrated at 10% B). Loaded using a syringe filter on 1" column (10 mL / min, 10% B). (7 mL). Gradient to 25 mL / min over 1 minute. 10-40% B over 90 minutes, the product eluted at 25.5-28% B. Desired fractions were combined, frozen and placed in lyophilizer. 53 mg N-((S) -3-amino-1- (hydroxyamino) -3-methyl-1-oxobutan-2-yl) -4-(((1,4- cis ) -4- (Hydroxymethyl) cyclohexyl) buta-1,3-diynyl) benzamide, I-15 , TFA was obtained. LCMS M + 1 Found = 412.2, Found = 412.2. 1 H NMR (DMSO-d6): δ 1.20-1.35 (m, 2H), 1.26 (s, 3H), 1.30 (s, 3H), 1.47-1.59 (m, 4H), 1.71-1.75 (m, 2H), 2.97-2.99 (m, 1H), 3.21-3.22 (d, 2H), 4.30 (br, 1H), 4.65-4.68 (d, 1H), 7.64-7.66 (d, 2H), 7.89-7.91 (d, 2H ), 7.98 (br, 2H), 8.54-8.56 (d, 1H), 9.22 (br, 1H), 11.20 (s, 1H).

M. N-((S) -1- (1-aminocyclobutyl) -2- (hydroxy Amino ) -2-oxo ethyl ) -4-(((trans) -2- ( Hydroxymethyl ) Cyclopropyl ) Buta -1,3- Diinil ) Benzamide Trifluoroacetate  (I-17)  And N-((S) -1- (1- Aminocyclobutyl )-2-( Hydroxyamino )-2- Oxoethyl )-4-((( Sheath )-2-( Hydroxymethyl ) Cyclopropyl ) Buta -1,3- Diinil ) Benzamide Trifluoroacetate  Synthesis of (I-18)

4-(((trans) -2- (hydroxymethyl) cyclopropyl) buta-1,3-diynyl) benzoic acid (205 mg, 0.85 mmol, 1 eq) and (S) -methyl-2-amino-2 -(1- (tert-butoxycarbonylamino) cyclobutyl) acetate (220 mg, 0.85 mmol, 1 eq) was dissolved in N, N -dimethylformamide (3 mL). N, N -diisopropylethylamine (0.45 mL, 2.6 mmol, 3 eq) was added followed by HATU (356 mg, 0.94 mmol, 1.1 eq). The mixture was kept at room temperature for 2.5 hours and then partitioned between 1M citric acid and ethyl acetate. The organics were washed with 1M citric acid, saturated sodium bicarbonate, then saturated sodium chloride, dried over magnesium sulfate and evaporated to dryness (S) -methyl 2- (1- (tert-butoxycarbonylamino) cyclobutyl) 2- (4-(((trans) -2- (hydroxymethyl) cyclopropyl) buta-1,3-diynyl) benzamido) acetate was obtained (550 mg crude compound, 134%, used without further purification) ).

(S) -methyl 2- (1- (tert-butoxycarbonylamino) cyclobutyl) -2- (4-(((trans) -2- (hydroxymethyl) cyclopropyl) buta-1,3- Diynyl) benzamido) acetate (550 mg crude compound, 1.1 mmol) was dissolved in dichloromethane (6 mL) and treated with 6 mL trifluoroacetic acid at room temperature for 10 minutes. The volatiles were removed and the residue re-evaporated from dichloromethane to give (S) -methyl 2- (1-aminocyclobutyl) -2- (4-(((trans) -2- ()) as a trifluoroacetate salt. Hydroxymethyl) cyclopropyl) buta-1,3-diynyl) benzamido) acetate was obtained, which was used without further purification.

Crude compound (S) -methyl 2- (1-aminocyclobutyl) -2- (4-(((trans) -2- (hydroxymethyl) cyclopropyl) buta-1,3-diynyl obtained in the above reaction ) Benzamido) acetate Trifluoroacetate was dissolved in isopropanol (2 mL) and treated with 50% aqueous hydroxylamine solution (2 mL) at 4 ° C. for 20 hours. The volatiles were removed and the residue was dissolved in water / acetonitrile acidified with trifluoroacetic acid. The material was purified by reverse-phase HPLC (2 "column, 50 mL / min, 0.1% TFA in water / ACN, equilibration at 2%) using the following gradient:

2% 10 minutes

2-15 5 minutes

15-45 300 minutes

The product eluted between 58 and 78 minutes and the desired fractions were lyophilized to yield N-((S) -1- (1-aminocyclobutyl) -2- (hydroxyamino) -2 as the trifluoroacetate salt. -Oxoethyl) -4-(((trans) -2- (hydroxymethyl) cyclopropyl) buta-1,3-diynyl) benzamide was obtained (white solid, 120 mg, 0.24 mmol, 4-(((( Trans) -2- (hydroxymethyl) cyclopropyl) buta-1,3-diynyl) benzoic acid). Mass spectral data: predicted (M + 1): 382.4, found: 382.1. Proton NMR (400 MHz, dmso-d6): 11.3 (br s, 1H), 9.24 (s, 1H), 8.62 (d, 1H, J = 8.8 Hz), 8.17 (br s, 3H), 7.86 (d, 2H , J = 8.4 Hz), 7.63 (d, 2H, J = 8.4 Hz), 4.92 (d, 1H, J = 9.2 Hz), 4.69 (t, 1H, J = 5.8 Hz), 3.40 (m, 1H), 3.26 (m, 1H), 2.12-2.21 (m, 4H), 1.89 (m, 1H), 1.80 (m, 1H), 1.39-1.46 (m, 2H), 0.84-0.93 (m, 2H). An additional 80 mg was obtained under re-purification of the crude fraction.

Mainly N-((S) -1- (1-aminocyclobutyl) -2- (hydroxyamino) -2-oxoethyl) -4-(((cis) -2- (hydroxymethyl) cyclopropyl) Fractions containing buta-1,3-diynyl) benzamide (an impurity in the major isomers) were individually lyophilized and reconstituted under the same gradient. The desired fractions were combined and lyophilized to give N-((S) -1- (1-aminocyclobutyl) -2- (hydroxyamino) -2-oxoethyl) -4-(((cis as trifluoroacetate salt). ) -2- (hydroxymethyl) cyclopropyl) buta-1,3-diynyl) benzamide was obtained (3.2 mg, 8.4umol, 4-(((trans) -2- (hydroxymethyl) cyclopropyl) 1% from buta-1,3-diynyl) benzoic acid). Mass spectral data: predicted (M + 1): 382.4, found: 382.1. Proton NMR (400 MHz, dmso-d6): 11.30 (bs s, 1H), 9.24 (s, 1H), 8.62 (d, 1H, J = 9.2 Hz), 8.16 (br s, 3H), 7.86 (d, 2H , J = 7.6 Hz), 7.65 (d, 2H, J = 7.6 Hz), 4.92 (d, 1H, J = 9.2 Hz), 4.70 (t, 1H, J = 5.2 Hz), 3.49 (m, 1H), 3.40 (m, 1H), 2.12-2.22 (m, 4H), 1.82-1.95 (m, 1H), 1.70-1.80 (m, 2H), 1.36 (m, 1H), 1.08 (m, 1H), 0.58 ( m, 1 H).

N. N-((S) -1- (1- ( Dimethylamino ) Cyclobutyl) -2- (hydroxy Amino ) -2-oxo ethyl Synthesis of) -4-(((1,2-trans) -2- (hydroxymethyl) cyclopropyl) buta-1,3-diynyl) benzamide (I-19)

N-((S) -1- (1-aminocyclobutyl) -2- (hydroxyamino) -2-oxoethyl) -4-(((trans) -2- (hydroxymethyl) cyclopropyl) buta -1,3-diynyl) benzamide trifluoroacetate I-17 (80 mg, 0.16 mmol, 1 eq) is dissolved in N, N -dimethylformamide (800 μl) and paraformaldehyde (48.5 mg, 1.1 mmol, 10eq) and N, N -diisopropylethylamine (56 μl, 0.32 mmol, 2eq) were treated at room temperature for 21 hours. Sodium cyanoborohydride (30 mg, 0.48 mmol, 3 eq) was added followed by methanol (800 μl) and acetic acid (28 μl, 0.48 mmol, 3eq). The mixture was kept at room temperature for 3 days, then additional sodium cyanoborohydride and acetic acid (28 μl) were added. After an additional day of maintenance at room temperature, another 28 μl of acetic acid was added again. After another day at room temperature, the material was purified using reverse phase HPLC (1 "column, 20 mL / min, 0.1% TFA in water / acetonitrile, equilibrated at 2%) using the following gradient:

2% 5 minutes

2-18% 5 minutes

18-98% 80 minutes

The desired fractions were combined and lyophilized to yield N-((S) -1- (1- (dimethylamino) cyclobutyl) -2- (hydroxyamino) -2-oxoethyl) -4- (as trifluoroacetate salt. ((1,2-trans) -2- (hydroxymethyl) cyclopropyl) buta-1,3-diynyl) benzamide was obtained (63 mg, 0.12 mmol, 74%). Mass spectral data: predicted (M + 1): 410.5, found: 410.2. Proton NMR (400 MHz, dmso-d6): 11.29 (s, 1H), 9.72 (br s, 1H), 9.30 (s, 1H), 8.95 (d, 1H, J = 9.2 Hz), 7.88 (d, 2H, J = 8.4 Hz), 7.61 (d, 2H, J = 8.4 Hz), 5.05 (d, 1H, J = 9.2 Hz), 4.7 (br s, 1H), 3.40 (dd, 1H, J = 5.2, 11.6 Hz ), 3.22 (dd, 1H, J = 6.4, 11.2 Hz), 2.76 (d, 3H, J = 4.4 Hz), 2.68 (d, 3H, J = 4.4 Hz), 2.33-2.42 (m, 3H), 1.62 -1.78 (m, 2H), 1.37-1.46 (m, 2H), 0.84-0.92 (m, 2H).

O. N-((S) -2- (hydroxy Amino ) -1-((S) -morpholine-3- Work ) -2-oxo ethyl Synthesis of) -4-(((1,2-trans) -2- (hydroxymethyl) cyclopropyl) buta-1,3-diynyl) benzamide (I-20)

To a pre-cooled suspension of (R) -4- (tert-butoxycarbonyl) morpholine-3-carboxylic acid 1 (5 g, 0.02162 mol) in dry ether (160 mL) triethylamine (3.91 mL, 0.0281 mol) was added dropwise at −16 ° C., and after 5 minutes, the resulting mixture was treated slowly with isobutyl chloroformate (3.37 mL, 0.02600 mol) at −15 ° C. for 30 minutes, and then the reaction mixture was 16 Stir at -15 ° C to 6 ° C for hours (overnight). The reaction progress was monitored by LCMS, filtered and the solid was washed with ether (30 mL x 2), then the filtrate was used for the next step. The previous filtrate was treated with a solution of diacetylmethane (0.24 mol) in ether (480 mL) for 16 h (overnight) at 0-7 ° C., then the reaction was glacial acetic acid (5 mL), water (50 mL) After quenching with, the organic layer was washed with sodium bicarbonate (50 mL x 2), brine (50 mL), the aqueous layer was reextracted with ether (200 mL x 2), and the combined organic layers were dried over anhydrous sodium sulfate , Filtered and concentrated to afford crude residue, which was then purified by silica-gel chromatography (using 20% ethyl acetate in petroleum as eluent) and (R) -tert-butyl-3- (2- as a yellow solid. Diazoacetyl) morpholine-4-carboxylate 2 was obtained 4.56 g (87% purity, LCMS, 254 nm), yield: 77%, 2 steps.

Of (R) -tert-butyl-3- (2-diazoacetyl) morpholine-4-carboxylate 2 (4.55 g, 17.824 mmol) and silver benzoate (25 mg, 0.11 mmol) in methanol (30 mL) Triethylamine (250 μl, 1.78 mmol) was added dropwise to the precooled suspension and the suspension was stirred in a dark room at 25 ° C. for 16 hours (over night, oil bath), filtered and the filtrate was evaporated to dryness. The residue was purified by silica-gel chromatography (using 10% ethyl acetate in petroleum as eluent) to afford (S) -tert-butyl-3- (2-methoxy-2-oxoethyl) morpholine- as a colorless solid. 4-carboxylate 3 was obtained. 3.34 g, yield: 78%.

To a pre-cooled solution of (S) -tert-butyl-3- (2-methoxy-2-oxoethyl) morpholine-4-carboxylate 3 (2.737 g, 0.0106 mol) in THF (100 mL) NaHMDS ( 8.15 mL, 0.0159 mol) was added slowly at -100 ° C, the resulting suspension was stirred at -100 ° C for 1 hour, and then the solution of trisylazide (5.99 g, 0.0170 mol) in THF (10 mL) was slowly Add (keep internal temperature below -100 ° C) and continue stirring at -100 ° C for an additional 1 hour. The reaction was quenched by the rapid addition of glacial acetic acid (3.18 g, 0.0530 mol), the reaction mixture was allowed to warm to room temperature, stirred for 3 hours, finally diluted with DCM (200 mL x 2) and saturated NaHCO ₃ (100 mL), brine (100 mL) and water (100 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered and concentrated to give crude residue, then reverse phase chromatography (gradient: 10% acetonitrile in water for 5 minutes, 30% acetonitrile in water within 3 minutes). Increase, 30% acetonitrile in water for 3 minutes, 30-45% acetonitrile in water for 3 minutes, 45% acetonitrile in water for 5 minutes; flow rate: 30 mL / min) as a white solid (S) -Tert-butyl-3- (1-azido-2-methoxy-2-oxoethyl) morpholine- 4 -carboxylate 4 (mixture of two diastereomers) was obtained. 1.22 g, yield: 38%

(S) -tert-butyl-3- (1-azido-2-methoxy-2-oxoethyl) morpholine-4-carboxylate 4 (1.2 g, 4 mmol) in EtOAc / MeOH (20 mL / 3 mL), A mixture of Pd (OH) 2 / C (200 mg, 15% wt), 2M HCl (2 mL, 4 mmol) was degassed and 1 atm. Treated with hydrogen gas at 30 ° C. The mixture was filtered and the solid was washed with EtOAc (20 mL). The combined organics were washed with water (20 mL), saturated NaHCO ₃ (20 mL, to adjust pH = 8), dried over anhydrous sodium sulfate, filtered and concentrated to give crude residue followed by reversed phase chromatography ( Gradient: 10% acetonitrile in water for 5 minutes, increased to 30% acetonitrile in water within 3 minutes, 30% acetonitrile in water for 3 minutes, 30-45% acetonitrile in water for 3 minutes, for 5 minutes Purified at 45% acetonitrile in water; flow rate: 30 mL / min). The purified residue was then chiral separated to give (S) -tert-butyl-3-((S) -1-amino-2-methoxy-2-oxoethyl) morpholine-4-carboxylate (SM68-A , 170 mg, yellow oil); (S) -tert-butyl-3-((R) -1-amino-2-methoxy-2-oxoethyl) morpholine-4-carboxylate (SM68-B, 230 mg, yellow solid); And SM68-C, 30 mg, yellow oil. Total yield: 41%.

4-(((trans) -2- (hydroxymethyl) cyclopropyl) buta-1,3-diynyl) benzoic acid (140 mg, 0.58 mmol) and (S) -tert-butyl-3-((S) -1-amino-2-methoxy-2-oxoethyl) morpholine-4-carboxylate (160 mg, 0.58 mmol) was dissolved in DMF (2 mL). N, N -diisopropylethylamine (255 μl, 1.46 mmol) was added followed by HATU (266 mg, 0.70 mmol). The reaction was kept at room temperature for 30 minutes and then partitioned between 1M citric acid and ethyl acetate. The organics were washed with 1M citric acid, saturated sodium bicarbonate, then saturated sodium chloride, dried over magnesium sulfate and evaporated to dryness. 380 mg of crude compound (S) -tert-butyl-3-((S) -1- (4-(((1,2-trans) -2- (hydroxymethyl) cyclopropyl) buta-1,3- Diynyl) benzamido) -2-methoxy-2-oxoethyl) morpholine-4-carboxylate was obtained.

(S) -tert-butyl-3-((S) -1- (4-(((1,2-trans) -2- (hydroxymethyl) cyclopropyl) buta-1,3-diynyl) benz Amido) -2-methoxy-2-oxoethyl) morpholine-4-carboxylate (crude compound, 380 mg) was dissolved in dichloromethane (4 mL) and trifluoroacetic acid (4 mL) was added. . After 5 minutes at room temperature, the mixture was diluted with 10 mL dichloromethane and evaporated. (S) -methyl-2- (4-(((1,2-trans) -2- (hydroxymethyl) cyclopropyl) buta-1,3-diynyl) benzamido thus obtained) -2- ((S) -morpholin-3-yl) acetate 2,2,2-trifluoroacetate was used as crude compound in the next step.

(S) -methyl-2- (4-(((1,2-trans) -2- (hydroxymethyl) cyclopropyl) buta-1,3-diynyl) benzamido) -2-((S ) -Morpholin-3-yl) acetate 2,2,2-trifluoroacetate (crude compound from the above reaction) is dissolved in isopropanol (1 mL) and an aqueous solution of hydroxylamine (50%, 1 mL) Added. The solution was kept at 4 ° C. for 18 hours and then evaporated. Water was added and the mixture acidified with TFA. The crude product was purified by RP HPLC (2 "column, 50 mL / min, 0.1% TFA in water / ACN, equilibration at 2%):

2% 10 minutes

2-11% 5 minutes

11-41% 300 minutes

The desired compound eluted at 88-99 minutes. The desired fractions were lyophilized to yield N-((S) -2- (hydroxyamino) -1-((S) -morpholin-3-yl) -2-oxoethyl) -4- as trifluoroacetate salt. (((1,2-trans) -2- (hydroxymethyl) cyclopropyl) buta-1,3-diynyl) benzamide I-20 was obtained (white solid, 118 mg, 0.30 mmol, 4-(( (Trans) -2- (hydroxymethyl) cyclopropyl) buta-1,3-diynyl) benzoic acid). Mass spectral data: predicted (M + 1): 398.2, found: 398.1. Proton NMR (400 MHz, dmso-d6): 11.17 (s, 1H), 9.14 (s, 1H), 9.08 (br s, 1H), 8.93 (br s, 1H), 8.83 (d, 1H, J = 8.4 Hz ), 7.89 (d, 2H, J = 8.0 Hz), 7.62 (d, 2H, J = 8.0 Hz), 4.69 (br s, 1H), 4.63 (t, 1H, J = 8.8 HZ), 3.83-3.86 ( m, 2H), 3.60-3.66 (m, 2H), 3.50 (t, 1H, J = 11.0 Hz), 3.39 (m, 1H), 3.20-3.23 (m, 2H), 3.05 (m, 1H), 1.39 -1.45 (m, 2H), 0.87-0.92 (m, 2H).

P. N-((S) -2- (hydroxy Amino ) -1-((S) -4-methylmorpholine-3- Work ) -2-oxo ethyl Synthesis of) -4-(((1,2-trans) -2- (hydroxymethyl) cyclopropyl) buta-1,3-diynyl) benzamide (I-21)

N-((S) -2- (hydroxyamino) -1-((S) -morpholin-3-yl) -2-oxoethyl) -4-(((1,2-trans) -2- (Hydroxymethyl) cyclopropyl) buta-1,3-diynyl) benzamide trifluoroacetate I-20 (10 mg, 20 μmol) was dissolved in DMF (100 μl). Paraformaldehyde (5.9 mg, 200 μmol) was added followed by N, N -diisopropylethylamine (6.8 μl, 39 μmol). The mixture was stirred at room temperature for 19 hours. Sodium cyanoborohydride (3.7 mg, 59 μmol), methanol (100 μl) and acetic acid (4.5 μl, 78 μmol) were added and the mixture was stirred at room temperature for an additional 3 days. The mixture was purified by RP HPLC (2 "column, 50 mL / min, 0.1% TFA in water / ACN, equilibrated at 2%):

2% 10 minutes

2-15% 5 minutes

15-95% 80 minutes

The product eluted between 23 and 24 minutes. The desired fraction was lyophilized to give N-((S) -2- (hydroxyamino) -1-((S) -4-methylmorpholin-3-yl) -2-oxoethyl) as the trifluoroacetate salt. 4-(((1,2-trans) -2- (hydroxymethyl) cyclopropyl) buta-1,3-diynyl) benzamide was obtained (7.0 mg, 13 μmol, 68%). Mass spectral data: predicted (M + 1): 411.2, found: 412.2.

Q. N-((S) -3-amino-1- Hydroxyamino ) -3- methyl -One- Oxobutane -2-yl) -4-((E) -4- (1,2-trans) -2- ( Hydroxymethyl ) Cyclopropyl ) Boot -1-En-3-personal) Benzamide  Synthesis of (I-22)

(S) -methyl 3-amino-2- (4- (E) -4- (1S, 2S) -2- (hydroxymethyl) cyclopropyl) but-1-en-3-ynyl) benzamido) 3-Methylbutanoate (400 mg, 1.08 mmol, 1.0 equiv) was placed in a round bottom flask. To this isopropyl alcohol (2.0 mL) was added, the solution was vortexed for 2 minutes, the flask was cooled in an ice bath, then hydroxylamine solution (1.4 mL, 21.60 mmol, 20 equiv) was added and the flask Was placed at 4 ° C. for approximately 48 hours. The reaction was concentrated (at 0 ° C.) to remove isopropyl alcohol and then acidified with TFA (3 mL) at 0 ° C. Additional water (15 mL) and ACN (3 mL) were added. The product was purified by reverse phase HPLC (2 "column, 50 mL / min, 0.1% TFA in water / ACN, equilibrated at 2% B). Using a syringe filter (24 mL) at 10 mL / min, 2% B on the column Load was then ramped to 50 mL / min over 1 minute, performed over 73 minutes with gradient from 2% B to 95% B. Desired fractions were combined, frozen and placed in lyophilizer. mg of N-((S) -3-amino-1- (hydroxyamino) -3-methyl-1-oxobutan-2-yl) -4-((E) -4-((1,2- Trans) 2- (hydroxymethyl) cyclopropyl) but-1-en-3-ynyl) benzamide, I-22 , TFA were obtained LCMS M + 1 predicted = 372.2, found = 372.2. 1 H NMR (DMSO -d6): δ 0.77-0.81 (m, 2H), 1.25 (s, 3H), 1.30 (s, 3H), 1.34-1.37 (m, 1H), 3.20-3.24 (dd, 1H), 3.37-3.41 ( dd, 1H), 4.65-4.67 (d, 1H), 6.43-6.47 (d, 1H), 6.87-6.91 (d, 1H), 7.55-7.57 (d, 2H), 7.84-7.86 (d, 2H), 7.99 (br, 2H), 8.38-8.41 (d, 1H), 9.22 (br, 1H), 11.20 (s, 1H).

R. Antimicrobial  activation

Bacterial Screens and Cultures

Bacterial isolates were passaged overnight at 35 ° C. on an Mueller-Hinton agar (Beckton Dickinson, Franklin Lakes, NJ) from -70 ° C. frozen stock. Clinical isolates tested were obtained from various geographically different hospitals in the United States and abroad (Focus Diagnostics, Herndon, VA and JMI, North Liberty, IA). Quality control strains were obtained from the American Type Culture Collection (ATCC; Rockville, Md.).

Sensitivity test

Minimum inhibitory concentrations (MIC) were determined by broth microdilution method according to the Clinical and Laboratory Standards Institute (CLSI) guidelines. In brief, the biosuspension was adjusted to a 0.5 McFarland standard to obtain a final inoculum of 3 × 10 ⁵ to 7 × 10 ⁵ colony-forming units (CFU) / mL. Drug dilutions and inoculum were prepared with sterile cation adjusted Mueller-Hinton Broth (Beckton Dickinson). 100 mL of inoculum volume was added to the wells containing 100 mL of liquid medium in which the drug was serially diluted 2-fold. All inoculated microdilution trays were incubated in air at 35 ° C. for 18-24 hours. After incubation, the minimum concentration of drug that was visually prevented growth (OD600 nm <0.05) was recorded as MIC. The performance of the assay was monitored according to the CLSI guidelines using laboratory quality-control strains and levofloxacin, a compound having a defined MIC spectrum. Typically, compounds of the present invention have a MIC value of 0.03-16 μg / mL. To this end, data of certain representative compounds are shown in Table 2 below.

[Table 2]

Minimum inhibitory concentration (MIC)

MIC Standard:

A = MIC of 2.0 μg / mL or less

B = MIC greater than 2.0 μg / mL up to 16.0 μg / mL

C = MIC greater than 16.0 μg / mL

* AECO001 is this. E. coli ATCC25922; APAE001 is Pseudomonas aeruginosa ATCC27853; AKPN001 is Klebsiella ATCC43816 to pneumoniae; APAE002 is a clinical isolate of Pseudomonas aeruginosa that exhibits a general level of efflux activity.

S. In vivo  Tolerance

Each compound was administered to mice by subcutaneous injection. Mice were group-accepted (5 per cage) at 18-28 ° C. and ˜50% humidity and fed to standard rodent feed. Water and diet were provided as desired. Mice were injected subcutaneously with a dose of up to 20 mL per kg of body weight.

Groups of three mice were administered subcutaneously by injection of 50, 100, 200, 400 or 600 mg / kg / day as a single dose of test compound in a formulation consisting of 15% Captisol in 20 mM acetate buffer at pH 5 It was. Subcutaneous injections were performed back to the site between the scapula. A volume of 10-20 mL / kg was injected at this site. The needle was inserted parallel to the skin surface to the depth where the tip of the needle lies in a subcutaneous pocket. A gentle but strong pressure was used on the plunger to release the contents of the syringe.

Observations were performed at various time points post-dose: 30 seconds-1 minute, 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 75 minutes, 90 minutes, 105 minutes, 2 hours, after which the animals were placed at baseline ( hourly, whichever comes first, until baseline, or until 4 hours post-dose. The minimum time range for monitoring mice was 30 minutes post-dose if animals appeared to be agile, normal and responsive. If the animals exhibited a toxic effect, they showed signs of recovery to near baseline, or closely monitored them which came first up to 4 hours post-dose. Animals were maintained for 72 hours after dosing for clinical observation, including monitoring of survival and activity levels. Observations may include central nervous system distress symptoms (eg, seizures, lethargy, transverseness, motionlessness, hyperactivity), abnormalities in the nerve roots (eg, ataxia, single switching, cramps, splayed limbs, jumps, or Kicking), autonomic symptoms (e.g., salivation, tear secretion, urination, bowel movements, piloerection or squinting), difficulty breathing (e.g., rough or rapid breathing, depression, panting or choking) , Stereotypic behavior (eg, repetitive chewing, turning, pacing, grooming, sniffing, head movement, or hunched stance), and Abnormal behaviors such as escape behaviors or wet dog shakes were assessed.

A known compound, No -_ alc (that synthesis and activation of the compound are disclosed in PCT International Publication No. 2008/06676 No. Compound 91-12) when the subcutaneous injection in the mouse the mouse within the maximum allowable dose of less than 50 mg / kg eoteuna indicate the quantity, K within the contrary mice with a dosage of 30 mg / kg by subcutaneous injection. Required as a static dose for pneumoniae . By hydroxymethyl substitution of only cyclopropyl groups, compound I-1 of the present invention, which differs from these known compounds, has antimicrobial activity comparable to No _ alc but is substantially more acceptable in mammals (mouse Maximum allowable dose of approximately 200 mg / kg when injected subcutaneously).

Table 3 shows the observations of mice administered subcutaneously. Tolerance was characterized by clinical observation. Grade A is for animals that do not exhibit toxic symptoms or who have some toxic symptoms, such as occasional short-term stops when moving, slight difficulty breathing, or a slight lethargy with rapid recovery (eg, within 10 minutes). Given. Grade B was assigned to animals exhibiting some toxic symptoms, such as longer stops when moving, and some lethargy with longer recovery times (up to 1 hour) while the animals could still move around. Grade C was assigned to animals exhibiting moderate to severe toxic symptoms such as lethargy, transverse, strabismus and lethargy with severe breathing, severe single contraction (jump, kick), or escape behavior. Finally, the D rating was given when any lethal effect (including mortality requiring a relief) occurred within clinical observation time (up to 72 hours post-dose).

[Table 3]

In vivo tolerance

T. Second With antibacterial agents  Synergy

K a bacterial isolate (isolate). In pneumoniae from a stock of frozen -70 ℃ (ATCC 43816) under an atmosphere on Mueller-Hinton agar (Beckton Dickinson, Franklin Lakes, NJ ) at 35 ℃ were sub-cultured for one night. Minimum inhibitory concentrations (MIC) were determined by broth microdilution method according to the Clinical and Laboratory Standards Institute (CLSI) guidelines. In brief, the biosuspension was adjusted to a 0.5 McFarland standard to obtain a final inoculum of 3 × 10 ⁵ to 7 × 10 ⁵ colony-forming units (CFU) / mL. Drug dilutions and inoculum were prepared with sterile cation adjusted Mueller-Hinton Broth (Beckton Dickinson). 100 mL of inoculum volume was added to the wells containing 100 mL of liquid medium in which the drug was serially diluted 2-fold. All inoculated microdilution trays were incubated in air at 35 ° C. for 18-24 hours. After incubation, the minimum concentration of drug that was visually prevented growth (OD ₆₀₀ nm <0.05) was recorded as MIC.

Standard checkerboard assays include agents described and N-((S) -3-amino-1- (hydroxyamino) -3-methyl-1-oxobutan-2-yl) -4-(((1,2 -Trans) -2- (hydroxymethyl) cyclopropyl) buta-1,3-diynyl) benzamide ( I-1 ). Table 4 shows the FICI calculated according to the standard technique. Compound I-1 showed synergy in vivo with vancomycin, teicoplanin, erythromycin, azithromycin, rifampin and novobiocin.

[Table 4]

FICI

Vancomycin and N-((S) -3-amino-1- (hydroxyamino) -3-methyl-1-oxobutan-2-yl) -4-(((1,2-trans) -2- ( In vivo synergy of hydroxymethyl) cyclopropyl) buta-1,3-diynyl) benzamide ( I-1 ) was investigated in a neutropenic femoral in vivo efficacy model. The model is essentially Craig and others (see Gudmundsson et al., “Murine Thigh Infection Model,” Handbook). of Animal Models of Infection , MA Sande and O. Zak, Eds .; London: Academic Press, 1999, pp 137-144). One briefly mentioned by the cyclophosphamide in mice administered twice presented the neutropenic infection before then, Kay. Pneumo . 10 ³ (ATCC 43816) - Using 10 ⁵ CFU of inoculum were infected intramuscularly in the thigh. Antibiotics or vehicle alone were administered 2 hours post-infection and 14 hours as negative controls. In order to minimize the effect of leukocytes on infection, animals were maintained neutropenically for the duration of the experiment so that the microbiological readout measured the in vivo interaction of drugs and bacteria. Twenty four hours post infection, thighs were collected, homogenized and plated to determine the number of surviving CFUs per thigh. In addition, thighs from a subset of animals were collected 2 hours post-infection to record the CFU present immediately before the first antibiotic treatment (pre-treatment). The static dose, defined as the dose required for the result of the CFU load at 24 hours equal to the result measured at 0 hours post infection, was calculated by the standard method in Prizm (GraphPad Software) from the dose response curve. .

The purpose of these investigations was to quantitatively assess whether the combination of I-1 and the candidate synergists gave a greater reduction in counts in this in vivo efficacy model than the sum of each agent alone.

Vancomycin is Kay. Pneumo . Significant in vivo synergy with LpxC inhibitors for the treatment of infections of ATCC43816. As shown in FIG. 1, treatment of mice infected with vancomycin alone at 220 mg / kg / day did not show a significant decrease in CFU. However, when combined with Compound I-1, the static dose of LpxC inhibitors is significantly reduced (FIG. 1 and Table 5).

[Table 5]

Taken together, these data indicate that vancomycin is N-((S) -3-amino-1- (hydroxyamino) -3-methyl-1-oxobutan-2-yl) -4-(((1,2- Trans) -2- (hydroxymethyl) cyclopropyl) buta-1,3-diynyl) benzamide ( I-1 ) shows unexpected in vivo synergy.

It is to be understood that the organic compounds according to the present invention can exhibit tautomerism. Since the chemical structure within this specification may represent only one of the possible tautomeric forms, it is to be understood that the present invention includes any tautomeric form of the depicted structure.

In addition, specific embodiments of the invention have been shown and described herein for purposes of explanation, which, of course, may be modified by those skilled in the art without departing from the spirit and scope of the invention, in particular in light of the above teaching. It will be understood that the invention is not so limited. Accordingly, the invention is not limited except as by the appended claims.

All U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent publications referred to herein are hereby incorporated by reference in their entirety to the extent that they are not inconsistent with this specification. .

Claims (23)

A compound of formula (I) or a stereoisomer thereof or a pharmaceutically acceptable salt thereof:

In this formula,
A is substituted C ₃ -C ₆ cycloalkyl, wherein at least one substituent is C ₁ -C ₃ primary alcohol;
B is absent or is -CH = CH-, -C≡C- or unsubstituted phenyl;
C is -CH = CH-, -C≡C- or unsubstituted phenyl, wherein if B is -CH = CH-, then C is not -CH = CH-;
R ¹ , R ² and R ³ are independently selected from hydrogen and substituted or unsubstituted C ₁ -C ₃ alkyl, or
R ¹ and R ² together with the carbon atom to which they are attached form an unsubstituted C ₃ -C ₆ cycloalkyl group, or
R ² and R ³ together with the carbon atom and Q to which they are attached form a substituted or unsubstituted heterocyclic ring having 5 to 8 ring atoms, wherein 1-2 of the heterocyclic ring The ring atom is selected from N, O and S;
Q is O or NR, where R is hydrogen or unsubstituted C ₁ -C ₃ alkyl.
The compound of claim 1, wherein Q is NR.
The compound of claim 1 or 2, wherein R ¹ , R ² and R ³ are independently selected from hydrogen and substituted or unsubstituted C ₁ -C ₃ alkyl.
A compound according to claim ¹ , wherein R ¹ , R ² and R ³ are independently selected from hydrogen and unsubstituted C ₁ -C ₃ alkyl.
The compound of any one of claims 1-4, wherein R ¹ and R ² are independently unsubstituted C ₁ -C ₃ alkyl.
_{6. The} compound of claim 1, wherein A is C ₃ -C ₆ cycloalkyl substituted with hydroxymethyl. ₇ .
The compound of any one of claims 1-6, wherein B is -C≡C- and C is -C≡C-.
The compound according to claim 1, wherein the compound is
N-(( S ) -3-amino-1- (hydroxyamino) -3-methyl-1-oxobutan-2-yl) -4-(((1,2-trans) -2- (hydroxy Methyl) cyclopropyl) buta-1,3-diynyl) benzamide ( I-1 );
N-((S) -3-amino-1- (hydroxyamino) -3-methyl-1-oxobutane-2-y1) -4-(((1R, 2R) -2- (hydroxymethyl) Cyclopropyl) buta-1,3-diynyl) benzamide ( I-2 );
N-((S) -3-amino-1- (hydroxyamino) -3-methyl-1-oxobutan-2-yl) -4-(((1S, 2S) -2- (hydroxymethyl) Cyclopropyl) buta-1,3-diynyl) benzamide ( I-3 );
N-((S) -3-amino-1- (hydroxyamino) -3-methyl-1-oxobutan-2-yl) -4-(((1,2-cis) -2- (hydroxy Methyl) cyclopropyl) buta-1,3-diynyl) benzamide ( I-4 );
N-((S) -3-amino-1- (hydroxyamino) -3-methyl-1-oxobutan-2-yl) -4 '-(((1,2-trans) -2- (hydr Oxymethyl) cyclopropyl) ethynyl) biphenyl-4-carboxamide ( I-5 );
N-((S) -1- (hydroxyamino) -3-methyl-3- (methylamino) -1-oxobutan-2-yl) -4-(((1,2-trans) -2- (Hydroxymethyl) cyclopropyl) buta-1,3-diynyl) benzamide ( I-6 );
N-((S) -1- (hydroxyamino) -3- (2-hydroxyethylamino) -3-methyl-1-oxobutan-2-yl) -4-(((1,2-trans ) -2- (hydroxymethyl) cyclopropyl) buta-1,3-diynyl) benzamide ( I-7 );
N-((S) -1- (hydroxyamino) -3- (2-hydroxyethylamino) -3-methyl-1-oxobutan-2-yl) -4-(((1,2-trans ) -2- (hydroxymethyl) cyclopropyl) buta-1,3-diynyl) benzamide ( I-8 );
N-((S) -3- (dimethylamino) -1- (hydroxyamino) -3-methyl-1-oxobutan-2-yl) -4-(((1,2-trans) -2- (Hydroxymethyl) cyclopropyl) buta-1,3-diynyl) benzamide ( I-9 );
N-((S) -3-hydroxy-1- (hydroxyamino) -3-methyl-1-oxobutan-2-yl) -4-(((1,2-trans) -2- (hydr Oxymethyl) cyclopropyl) buta-1,3-diynyl) benzamide ( I-10 );
N-((S) -3-amino-1- (hydroxyamino) -3-methyl-1-oxobutan-2-yl) -4-(((1S, 2R) -2- (hydroxymethyl) -2-methylcyclopropyl) buta-1,3-diynyl) benzamide ( I-11 );
N-((S) -3-amino-1- (hydroxyamino) -3-methyl-1-oxobutan-2-yl) -4-(((1,3-cis) -3- (hydroxy Methyl) cyclobutyl) buta-1,3-diynyl) benzamide ( I-12 );
N-((S) -3-amino-1- (hydroxyamino) -3-methyl-1-oxobutan-2-yl) -4-(((1,3-trans) -3- (hydroxy Methyl) cyclobutyl) buta-1,3-diynyl) benzamide ( I-13 );
N-((S) -3-amino-1- (hydroxyamino) -3-methyl-1-oxobutan-2-yl) -4-(((1,3-trans) -3- (hydroxy Methyl) cyclopentyl) buta-1,3-diynyl) benzamide ( I-14 );
N-((S) -3-amino-1- (hydroxyamino) -3-methyl-1-oxobutan-2-yl) -4-(((1,4-cis) -4- (hydroxy Methyl) cyclohexyl) buta-1,3-diynyl) benzamide ( I-15 );
N-((S) -3-amino-1- (hydroxyamino) -3-methyl-1-oxobutan-2-yl) -4-(((1,4-trans) -4- (hydroxy Methyl) cyclohexyl) buta-1,3-diynyl) benzamide ( I-16 );
N-((S) -1- (1-aminocyclobutyl) -2- (hydroxyamino) -2-oxoethyl) -4-(((trans) -2- (hydroxymethyl) cyclopropyl) buta -1,3-diynyl) benzamide trifluoroacetate ( I-17 );
N-((S) -1- (1-aminocyclobutyl) -2- (hydroxyamino) -2-oxoethyl) -4-(((cis) -2- (hydroxymethyl) cyclopropyl) buta -1,3-diynyl) benzamide trifluoroacetate ( I-18 );
N-((S) -1- (1- (dimethylamino) cyclobutyl) -2- (hydroxyamino) -2-oxoethyl) -4-(((1,2-trans) -2- (hydr Oxymethyl) cyclopropyl) buta-1,3-diynyl) benzamide ( I-19 );
N-((S) -2- (hydroxyamino) -1-((S) -morpholin-3-yl) -2-oxoethyl) -4-(((1,2-trans) -2- (Hydroxymethyl) cyclopropyl) buta-1,3-diynyl) benzamide ( I-20 );
N-((S) -2- (hydroxyamino) -1-((S) -4-methylmorpholin-3-yl) -2-oxoethyl) -4-(((1,2-trans) -2- (hydroxymethyl) cyclopropyl) buta-1,3-diynyl) benzamide ( I-21 );
N-((S) -3-amino-1- (hydroxyamino) -3-methyl-1-oxobutan-2-yl) -4-((E) -4-((1,2-trans) -2- (hydroxymethyl) cyclopropyl) but-1-en-3-ynyl) benzamide ( I-22 ); or
N-((S) -3-amino-1- (hydroxyamino) -3-methyl-1-oxobutan-2-yl) -4-((E) -4-((1,2-trans) -2- (hydroxymethyl) cyclopropyl) but-1-en-3-ynyl) benzamide ( I-23 ).
9. The compound according to claim 8, wherein the compound is
N-(( S ) -3-amino-1- (hydroxyamino) -3-methyl-1-oxobutan-2-yl) -4-(((1,2-trans) -2- (hydroxy Methyl) cyclopropyl) buta-1,3-diynyl) benzamide ( I-1 );
N-((S) -3-amino-1- (hydroxyamino) -3-methyl-1-oxobutan-2-yl) -4 '-(((1,2-trans) -2- (hydr Oxymethyl) cyclopropyl) ethynyl) biphenyl-4-carboxamide ( I-5 );
N-((S) -1- (hydroxyamino) -3-methyl-3- (methylamino) -1-oxobutan-2-yl) -4-(((1,2-trans) -2- (Hydroxymethyl) cyclopropyl) buta-1,3-diynyl) benzamide ( I-6 );
N-((S) -1- (hydroxyamino) -3- (2-hydroxyethylamino) -3-methyl-1-oxobutan-2-yl) -4-(((1,2-trans ) -2- (hydroxymethyl) cyclopropyl) buta-1,3-diynyl) benzamide ( I-7 );
N-((S) -1- (hydroxyamino) -3- (2-hydroxyethylamino) -3-methyl-1-oxobutan-2-yl) -4-(((1,2-trans ) -2- (hydroxymethyl) cyclopropyl) buta-1,3-diynyl) benzamide ( I-8 );
N-((S) -3- (dimethylamino) -1- (hydroxyamino) -3-methyl-1-oxobutan-2-yl) -4-(((1,2-trans) -2- (Hydroxymethyl) cyclopropyl) buta-1,3-diynyl) benzamide ( I-9 );
N-((S) -3-amino-1- (hydroxyamino) -3-methyl-1-oxobutan-2-yl) -4-(((1,3-cis) -3- (hydroxy Methyl) cyclobutyl) buta-1,3-diynyl) benzamide ( I-12 );
N-((S) -3-amino-1- (hydroxyamino) -3-methyl-1-oxobutan-2-yl) -4-(((1,3-trans) -3- (hydroxy Methyl) cyclobutyl) buta-1,3-diynyl) benzamide ( I-13 ); or
N-((S) -3-amino-1- (hydroxyamino) -3-methyl-1-oxobutan-2-yl) -4-(((1,3-trans) -3- (hydroxy Methyl) cyclopentyl) buta-1,3-diynyl) benzamide ( I-14 ).
The compound of claim 9, wherein the compound is
N-(( S ) -3-amino-1- (hydroxyamino) -3-methyl-1-oxobutan-2-yl) -4-(((1,2-trans) -2- (hydroxy Methyl) cyclopropyl) buta-1,3-diynyl) benzamide ( I-1 );
N-((S) -3-amino-1- (hydroxyamino) -3-methyl-1-oxobutan-2-yl) -4 '-(((1,2-trans) -2- (hydr Oxymethyl) cyclopropyl) ethynyl) biphenyl-4-carboxamide ( I-5 );
N-((S) -1- (hydroxyamino) -3-methyl-3- (methylamino) -1-oxobutan-2-yl) -4-(((1,2-trans) -2- (Hydroxymethyl) cyclopropyl) buta-1,3-diynyl) benzamide ( I-6 );
N-((S) -1- (hydroxyamino) -3- (2-hydroxyethylamino) -3-methyl-1-oxobutan-2-yl) -4-(((1,2-trans ) -2- (hydroxymethyl) cyclopropyl) buta-1,3-diynyl) benzamide ( I-7 );
N-((S) -1- (hydroxyamino) -3- (2-hydroxyethylamino) -3-methyl-1-oxobutan-2-yl) -4-(((1,2-trans ) -2- (hydroxymethyl) cyclopropyl) buta-1,3-diynyl) benzamide ( I-8 ); or
N-((S) -3- (dimethylamino) -1- (hydroxyamino) -3-methyl-1-oxobutan-2-yl) -4-(((1,2-trans) -2- (Hydroxymethyl) cyclopropyl) buta-1,3-diynyl) benzamide ( I-9 ).
A pharmaceutical composition comprising a compound of any one of claims 1 to 10 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or diluent.
A method of treating a subject with a bacterial infection comprising administering a therapeutically effective amount of a compound according to any one of claims 1 to 10 or a pharmaceutical composition of claim 11 to a subject in need thereof.
The method of claim 11, wherein the bacterial infection is a Gram-negative bacterial infection.
The method of claim 13 wherein the gram-negative bacterial infection is Pseudomonas rugi Ah industrial (Pseudomonas aeruginosa), Burke holde Liao (Burkholderia), the Enterobacter bacteria years old child (Enterobacteriaceae), Francis Cellar years old child in (Franciscellaceae), Serratia marcescens (Serratia), Proteus (Proteus), keulrep when Ella (Klebsiella), Enterobacter foil emitter (Enterobacter) the method, bakteo (Citrobacter), Salmonella (Salmonella) into a sheet, Providenciales O (Providencia), Yersinia (Yersinia), do not know the Nella (Morganella) or Escherichia coli (Escherichia coli).
15. The method of claim 14 wherein the gram-negative bacterial infection rugi labor, large burrs holde Liao, Francis Cellar Asia, Enterobacter, Yersinia, or Escherichia coli which method in Pseudomonas Ah.
The method of claim 15, wherein the Gram-negative bacterial infection is Pseudomonas aeruginosa .
The method of claim 15, wherein the Gram-negative bacterial infection is Escherichia coli .
A pharmaceutical composition comprising a second antibacterial agent and a compound of formula (I) or a stereoisomer thereof or a pharmaceutically acceptable salt thereof:

In this formula,
A is substituted C ₃ -C ₆ cycloalkyl, wherein at least one substituent is C ₁ -C ₃ primary alcohol;
B is absent or is -CH = CH-, -C≡C- or unsubstituted phenyl;
C is -CH = CH-, -C≡C- or unsubstituted phenyl, wherein if B is -CH = CH-, then C is not -CH = CH-;
R ¹ , R ² and R ³ are independently selected from hydrogen and substituted or unsubstituted C ₁ -C ₃ alkyl, or
R ¹ and R ² together with the carbon atom to which they are attached form an unsubstituted C ₃ -C ₆ cycloalkyl group, or
R ² and R ³ together with the carbon atom and Q to which they are attached form a substituted or unsubstituted heterocyclic ring having 5 to 8 ring atoms, wherein 1-2 of the heterocyclic ring The ring atom is selected from N, O and S;
Q is O or NR, where R is hydrogen or unsubstituted C ₁ -C ₃ alkyl.
The pharmaceutical composition of claim 18, wherein the antibacterial agent is vancomycin or rifampin.
The pharmaceutical composition of claim 18, wherein the combination exhibits synergy in vivo.
A method of inhibiting deacetylase enzymes in Gram-negative bacteria comprising administering a compound according to any one of claims 1 to 10 or a pharmaceutical composition of claim 11 to a patient in need of such inhibition.
The method of claim 21, wherein the Gram-negative bacteria are Pseudomonas ah rugi labor, Burke hole to Ria, in the Enterobacter bacteria Asia, Francis Cellar Asia, Serratia, Proteus, keulrep Ciel La, Enterobacter bakteo, bakteo, Salmonella into a sheet , Providencia, Yersinia, do not know the Salmonella or Escherichia coli manner.
A method of inhibiting LpxC in Gram-negative bacteria, comprising administering a compound according to any one of claims 1 to 10 or a pharmaceutical composition of claim 11 to a patient in need thereof.

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