Lactivicin compounds, their preparation and use as antibacterial agents

Abstract

Provided are lactivicin compounds of Formula (I) and pharmaceutically acceptable salts of the compounds of Formula (I), wherein the compounds include antibiotics suitable for use alone or in combination with beta-lactamase inhibitors and / or other antibiotics, including beta-lactam and non-beta-lactam antibiotics, for the treatment or prevention of bacterial infections,

Description

Technical Field

[0001] This invention relates to novel lactivicin compounds having a unique bicyclic dipeptide structure, namely 2-[(4S)-4-acetamido-3-oxo-1,2-oxazolidin-2-yl]-5-oxooxolane-2-carboxylicacid, pharmaceutically acceptable salts thereof, uses, and methods for preparing these compounds. More specifically, novel lactivicin compounds with enhanced antibacterial activity and combinations of these compounds with β-lactamase inhibitors are provided, said combinations being active against a wide range of drug-resistant pathogenic microorganisms. Background Technology

[0002] Public health experts and officials consider the emergence and spread of antibiotic-resistant bacteria to be one of the major public health problems of the 21st century. While most resistant strains continue to emerge in hospital settings, physicians and epidemiologists are encountering increasing numbers of resistant bacteria in the community, even among individuals with no prior medical exposure. Infections caused by multidrug-resistant Gram-negative pathogens, including the bacteria that make up ESKAPE organisms, typically involve six pathogens: Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter, for which treatments are particularly limited.

[0003] Historically, highly successful and well-tolerated β-lactam antibiotics have been the primary treatments for infections caused by Gram-negative pathogens. However, the vast array of over 1,000 different β-lactamases, generated by bacterial and other bacterial resistance mechanisms, seriously threatens the current medium-term availability of β-lactam antibiotics. Extended-spectrum β-lactamases (ESBLs) and carbapenemases are particularly important drivers of resistance, inactivation of β-lactam antibiotics, and their ineffectiveness in treating infections. Consequently, morbidity and mortality from bacterial infections continue to rise in hospital and community settings, and bacterial resistance has become a major public health problem.

[0004] There is an urgent need for new agents with anti-destructive properties to fill the gap. Therefore, there is a need to develop new classes of antibiotics that exhibit more effective antibacterial activity and are particularly effective against a variety of β-lactamases that produce Gram-negative bacteria.

[0005] Lactivicin is the first non-β-lactam antibiotic with a bicyclic dipeptide backbone described in the literature. It was isolated from soil samples collected in Japan by the Takeda Group. Lactivicin possesses a unique bicyclic dipeptide structure and exhibits bacterial penicillin-binding protein (PBP) affinity similar to that of β-lactam antibiotics. However, it has low sensitivity to β-lactamases. [Nature, 1987, Vol. 325, pp. 179-180; J. Chem. Soc. Chem. Commun., 1987, (2), pp. 62-63; Tetrahedron Lett.,1986,Vol.27,No.51,pp.6229-6232; Tetrahedron,1988,Vol.44,No.11,pp.3231-3240; Tetrahedron,1988,Vol.44, No.21, pp.6589-6606; J. Antibiot., 1989, Vol. 42, No. 1, pp. 84-93; Chem. Pharm. Bull., 1990, Vol. 38. No. 1, pp. 116-122; US 4,851,422(1989); EP 0191989A1; WO 87 / 00527; EP 0219923 A1].

[0006] As specific examples of lactivicin, US 4,851,422 and EP0219923B1 disclose 2-(3-oxo-2-isoxazolyl)-5-oxo-2-tetrahydrofuran carboxylate of the following formula, which is an exemplary structure of lactivicin:

[0007]

[0008] Where R 5 R 6 R 7 and R 8 Independently hydrogen, C 1-6 Alkyl, phenyl, or benzyl groups, which can be C 1-6 Acylamino or C 1-6 Alkoxy substitution, which can be achieved through bonding with oxygen or sulfur atoms, or R 5 and / or R 6 With R 7 Or R 8 Forming chemical bonds to form double bonds, or R5 Or R 6 With R 7 Or R 8 A benzene ring is formed.

[0009] In search of different classes of antibiotics with novel nuclei, the Takeda Group prepared several derivatives of lactivicin with various amide moieties at the C-4 position [J. Chem. Soc. Chem. Commun., 1987, (2), pp. 62-63]. Compounds with a 2-aminothiazol-4-yl-(Z)-2-methoxyiminoacetyl side chain showed improved and enhanced antibacterial activity compared to natural lactivicin. Furthermore, some compounds showed protective effects in experimentally infected mice [Chem. Pharm. Bull., 1990, Vol. 38, No. 1, pp. 116-122; Tetrahedron, 1988, Vol. 44, No. 21, pp. 6589-6606].

[0010] Attempts to enhance cellular uptake of β-lactams through the use of iron-siderophore uptake systems in microorganisms are a concept explored in the field of monocyclic β-lactams (Basilea (WO2007 / 065288), NAEJA Pharmaceuticals (WO2002 / 022613), and Squibb & Sons (US5290929, EP531976, EP484881)). Furthermore, BMS-180680 is a catechol-containing monocyclic β-lactam that appears to enhance bacterial outer membrane uptake using Cir and Fiu iron-regulated outer membrane receptor proteins and a TonB-dependent iron transport system. Therefore, BMS-180680 exhibits excellent activity against many Gram-negative bacteria [Antimicrob. Agents & Chemother., 1997, Vol. 41, No. 5, pp. 1010-1016]. Furthermore, Shionogi & Co. Ltd. discovered a novel cephalosporin containing catechol [Antimicrob. Agents & Chemother., 2016, Vol. 60, No. 12, pp. 7396-7401] and introduced it clinically as cefidil. Recently, Pfizer re-examined the siderophore receptor-mediated uptake of catechol-containing lactivicin in Gram-negative bacteria [J. Med. Chem. 2014, 57, 3845-3855]. Another group [Antimicrob. Agents & Chemother., 2016, Vol. 60, No. 7, pp. 4170-4175] showed that iron-containing modification of lactivicin significantly increased its potency against clinical isolates of extensively drug-resistant Stenotrophomonas maltophilia. In summary, iron-containing conjugates of lactivicin represent a clearly unexplored class with a novel bicyclic dipeptide core, warranting further investigation through the preparation of additional novel analogs to identify new and effective antimicrobial agents. This invention aims to achieve these and other important objectives. Summary of the Invention

[0011] Given the increasing resistance of pathogenic bacteria (including those exhibiting multidrug resistance) to known classes of antimicrobial agents, there remains a need to find new antimicrobial substances, especially compounds with structural motifs different from those of traditional antimicrobial molecules.

[0012] Although research on lactivicin has shown promising results, there is still a need for novel lactivicin compounds with enhanced antibiotic efficacy, particularly in highly resistant Gram-negative bacteria, which have structural features significantly different from those described in the aforementioned patents and publications.

[0013] Therefore, the present invention relates to and includes compounds of formula (I).

[0014]

[0015] Where A is defined by equation (Ia).

[0016]

[0017] Where X is N or CR 3 R 3 This indicates hydrogen or halogen. Suitable halogens include chlorine and fluorine.

[0018] In equation (I), R 1 and R 2 Together with the carbon atoms bonded to them, they can form (C3-C8) cycloalkyl groups, wherein (i) the cycloalkyl group may contain a heteroatom selected from O, N, and S; and / or

[0019] (ii) The cycloalkyl group may be substituted with one, two, three, or four substituents, which are independent of each other and are selected from (C1-C3) alkyl groups and halogens. Suitable halogens include chlorine and fluorine.

[0020] Alternatively, in equation (I), R 1 and R 2 They can be independent of each other, representing hydrogen or (C1-C3)alkyl, wherein the (C1-C3)alkyl can be substituted with substituents selected from hydroxyl and chlorine.

[0021] In formula (I), B is a fragment containing a bicyclic catechol or a hydroxypyridinone moiety shown in formula (Ia′), which has the ability to enhance the intracellular uptake of compound (I) by means of bacterial iron uptake processes in Gram-negative bacteria.

[0022]

[0023] In formula (Ia′), the P ring is an unsaturated 5-membered or 6-membered ring, which may optionally contain one carbonyl (CO) group or two carbonyl groups, or one sulfone (SO2) group, or a combination of one carbonyl (CO) group and one sulfone (SO2) group, and may further contain two additional N atoms. In a particular embodiment, the P ring is an unsaturated 5-membered ring.

[0024] In formula (Ia′), the Q ring can contain a maximum of two N atoms, where the substituent R 4 Selected from hydrogen, (C1-C3)alkyl, carbonyl, trifluoromethyl, cyano, and halogens. Specific examples of the Q ring are benzene and pyridine. Suitable halogens include fluorides and chlorides.

[0025] The present invention also includes salts of compounds of formula (I), solvates of compounds of formula (I), and solvates of salts of compounds of formula (I).

[0026] Based on their structures, the compounds of the present invention can exist in stereoisomer forms (e.g., enantiomers, diastereomers). Therefore, the present invention also includes enantiomers and diastereomers of the compounds of formula (I) as defined herein, as well as mixtures thereof. Stereoisomeric homogeneous components, which are also compounds of the present invention, can be isolated from such mixtures of enantiomers and / or diastereomers in a known manner.

[0027] Regarding the compounds of formula (I), it is understood that these compounds include Z-isomers, E-isomers, and mixtures thereof. As used herein, the term "isomer" refers to different compounds having the same molecular formula but different atomic arrangements and configurations, such as geometric isomers and optical isomers.

[0028] If the compounds of the present invention may appear in tautomer forms, then the present invention includes all tautomer forms of the compounds of formula (I).

[0029] Preferred salts for the purposes of this invention are “pharmaceutically acceptable salts” of the compounds of this invention, i.e., compounds of formula (I). Also included are salts that are not inherently suitable for pharmaceutical applications (“non-pharmaceutically acceptable salts”), but can be used, for example, for the separation or purification of the compounds of this invention.

[0030] Examples of pharmaceutically acceptable salts of compounds of formula (I) include inorganic base salts, such as ammonium salts, alkali metal salts (especially sodium or potassium salts), and alkaline earth metal salts (especially magnesium or calcium salts); organic base salts derived from n-propylamine, n-butylamine, cyclohexylamine, benzylamine, octylamine, ethanolamine, diethanolamine, diethylamine, triethylamine, dicyclohexylamine, procaine, choline, methylpyridine, N,N-dibenzylethylenediamine, meglumine, morpholine, pyrrolidine, pyridine, piperidine, N-ethylpiperidine, and N-methylmorpholine. Basic amino acids that can form basic amino acid salts include lysine, arginine, ornithine, and histidine. As will be understood by those skilled in the art, compounds of formula (I) containing a basic nitrogen atom are capable of forming acid addition salts. This document includes salts containing pharmaceutically acceptable acids. Examples of these acids are hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, citric acid, oxalic acid, maleic acid, fumaric acid, glycolic acid, mandelic acid, tartaric acid, aspartic acid, succinic acid, malic acid, formic acid, acetic acid, trifluoroacetic acid, methanesulfonic acid, ethanesulfonic acid, trifluoromethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, etc.

[0031] Furthermore, some compounds of formula (I), when containing basic groups such as NH, NH2, or pyridine, piperazine, etc., may form internal zwitterionic salts with COOH groups. These internal salts are considered and included in this document.

[0032] The solvates used for the purposes of this invention refer to those forms in which the compounds of this invention form complexes by coordination with solvent molecules in the solid or liquid state. A hydrate is a specific form of solvate in which coordination with water occurs. Molecular complexes of the compound or a portion thereof with a solvent can be stabilized by non-covalent intramolecular forces, such as electrostatic forces, van der Waals forces, or hydrogen bonds. The compounds of this invention can form molecular complexes with one or more solvent molecules in stoichiometric or non-stoichiometric amounts.

[0033] In the context of this invention, the substituents defined in formula (I) have the following definitions, unless otherwise specified.

[0034] The term "cycloalkyl" refers to aliphatic C3-C8 rings, preferably C3-C6 rings, particularly rings such as cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl, but also includes C3-C4, C3-C5, and C3-C7 rings. The term "cycloalkyl" includes aliphatic C3, C4, C5, C6, C7, and C8 rings.

[0035] The term "halogen" refers to fluorine and chlorine.

[0036] The term "alkyl" refers to a straight-chain or branched (C1-C6) alkyl group, preferably (C1-C4) alkyl, particularly methyl, ethyl, propyl, butyl, isopropyl, isobutyl, and tert-butyl, and also includes (C1-C2) alkyl, (C1-C3) alkyl, (C1-C5) alkyl, and (C1-C6) alkyl. The term alkyl includes straight-chain or branched C3 alkyl, straight-chain or branched C4 alkyl, straight-chain or branched C5 alkyl, and straight-chain or branched C6 alkyl.

[0037] The term "5-membered ring" refers to a 5-membered heterocycle containing at least one to at most three N atoms and optionally one S atom.

[0038] The term "6-membered ring" refers to a 6-membered heterocycle containing at least one nitrogen atom.

[0039] Therefore, in some embodiments of the present invention, lactivicin compounds and pharmaceutically acceptable salts thereof are provided, namely compounds of formula (I) and pharmaceutically acceptable salts thereof. In some respects, these compounds may exhibit activity against pathogenic microorganisms and thus can be used alone or in combination with one or more β-lactam antibiotics, other non-β-lactam antibiotics, and β-lactamase inhibitors for the treatment and / or prevention of bacterial infections in humans or animals.

[0040] In other embodiments of the invention, pharmaceutical compositions comprising one or more compounds of formula (I) or pharmaceutically acceptable salts and pharmaceutically acceptable carriers or diluents are provided. In some respects, these compositions may exhibit activity against pathogenic microorganisms.

[0041] In other embodiments of the invention, methods for preparing novel lactivicin compounds and their salts are provided, namely compounds of formula (I) and their pharmaceutically acceptable salts.

[0042] In other embodiments of the invention, pharmaceutical compositions are provided comprising (i) one or more compounds of formula (I) or pharmaceutically acceptable salts thereof, (ii) one or more β-lactamase inhibitors, and (iii) a pharmaceutically acceptable carrier or diluent. In some respects, these compositions may exhibit activity against pathogenic microorganisms.

[0043] In other embodiments of the invention, the following methods for treating or preventing bacterial infection in a subject are provided, including administering medication to the subject as needed:

[0044] (i) an effective therapeutic amount of one or more of the (I) compound or a pharmaceutically acceptable salt thereof;

[0045] (ii) An effective therapeutic amount of a pharmaceutical composition comprising one or more compounds of formula (I) or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or diluent;

[0046] (iii) A therapeutically effective amount of the composition, comprising (a) one or more compounds of formula (I) or a pharmaceutically acceptable salt thereof, and (b) one or more β-lactamase inhibitors; administered in any order or at any time;

[0047] (iv) An effective therapeutic amount of a pharmaceutical composition comprising (a) one or more compounds of formula (I) or a pharmaceutically acceptable salt thereof, (b) one or more β-lactamase inhibitors, and (c) a pharmaceutically acceptable carrier or diluent.

[0048] In other embodiments of the invention, the use of the compound of formula (I) for treating or preventing bacterial infections in a subject is provided. In some aspects, the compound is included in a composition, which may further comprise a β-lactamase inhibitor and may relate to the preparation of an effective therapeutic medicament.

[0049] According to embodiments of the present invention, the subject may be a human or an animal, wherein the compound and composition may provide beneficial antibacterial effects. Detailed Implementation

[0050] According to embodiments herein, pharmaceutically acceptable salts of formula (I) and formula (I) compounds are provided, wherein said compounds comprise antibiotics suitable for use alone or in combination with β-lactamase inhibitors and / or other antibiotics (including β-lactam and non-β-lactam antibiotics) for the treatment or prevention of bacterial infections. The term "antibiotic" refers to a compound or composition that reduces the viability of microorganisms or inhibits their growth or proliferation, and is further intended to include antimicrobial agents, bacteriostatic agents, or bactericidal agents.

[0051] The compounds of the present invention

[0052] In particular, the present invention relates to compounds of formula (I).

[0053]

[0054] Where A is defined by formula (Ia)

[0055]

[0056] Where X is N or CR 3 R 3 Indicates hydrogen or halogen;

[0057] R 1 and R 2 Together with the carbon atoms they are bonded to, they can form (C3-C8) cycloalkyl groups, in which...

[0058] (i) The cycloalkyl group may contain a heteroatom selected from O, N and S and / or

[0059] (ii) The cycloalkyl group may be substituted with one, two, three, or four substituents, which are independent of each other and selected from (C1-C3) alkyl groups and halogens; or

[0060] R 1 and R 2 They can be independent of each other, representing hydrogen or (C1-C3)alkyl, wherein the (C1-C3)alkyl can be substituted by a substituent selected from hydroxyl and chlorine;

[0061] B is a fragment containing a bicyclic catechol or hydroxypyridinone moiety, as defined by formula (Ia′).

[0062]

[0063] The p-ring is an unsaturated 5- or 6-membered ring that may optionally contain one or two carbonyl (CO) groups, or one sulfone (SO2) group, or a combination of one carbonyl (CO) and one sulfone (SO2) group, and may further contain two additional nitrogen atoms; and

[0064] Q can contain up to two N atoms, and the substituent R 4 Selected from hydrogen, (C1-C3)alkyl, carbonyl, trifluoromethyl, cyano and halogen; and their salts, their solvates and their solvates.

[0065] In formula (I), specific examples of halogens include fluorine and chlorine.

[0066] In formula (Ia′), a specific instance of the P-ring is an unsaturated 5-membered ring.

[0067] In formula (Ia′), specific instances of the Q ring are benzene and pyridine.

[0068] In formula (Ia′), a specific bicyclic catechol or fragment containing a hydroxypyridinone moiety has the ability to enhance intracellular uptake of compound (I) by using bacterial iron uptake in Gram-negative bacteria.

[0069] In equation (I), Examples include, but are not limited to, fragments:

[0070]

[0071] In equation (I), instances of “A” include, but are not limited to, the following:

[0072]

[0073] In equation (I), instances of “B” include, but are not limited to, the following:

[0074]

[0075] R 4 It is selected from hydrogen, (C1-C3)alkyl, trifluoromethyl, cyano or halogen, wherein the halogen is preferably chlorine or fluorine.

[0076] Table 1 below provides examples of compounds of formula (I) described in this invention, but is not limited to any particular compound.

[0077] Table 1.

[0078]

[0079]

[0080]

[0081]

[0082]

[0083]

[0084]

[0085]

[0086]

[0087]

[0088]

[0089]

[0090]

[0091]

[0092]

[0093]

[0094]

[0095]

[0096]

[0097]

[0098] As used herein, references to compounds of formula (I) specifically exclude the following compounds I', I” and I”'.

[0099]

[0100] The compounds of the present invention are therefore compounds of formula (I) and their salts, solvates thereof and solvates thereof, including the compounds described in Table 1, but excluding compounds I', I” and I”'.

[0101] The compounds of formula (I) and Table 1 of this invention include enantiomers or diastereomers and mixtures thereof.

[0102] The compounds of formula (I) and Table 1 of this invention include Z-isomers, E-isomers and mixtures thereof.

[0103] The compounds of formula (I) and Table 1 of this invention may appear in tautomer forms, and this invention includes all tautomer forms.

[0104] The salts of the compounds of this invention can be "pharmaceutically acceptable salts", such as those defined above.

[0105] The compounds of the present invention can be internal salts of formula (I), such as zwitterionic salts formed by COOH groups and basic groups (e.g., NH, NH2, pyridine or piperazine).

[0106] The composition of the present invention

[0107] The compounds of the present invention exhibit broad-spectrum antibacterial effects. In some aspects, the compounds may exhibit activity against pathogenic microorganisms and thus may be used alone or in combination with one or more: β-lactam antibiotics, other non-β-lactam antibiotics, and β-lactamase inhibitors for the treatment and / or prevention of bacterial infections in humans or animals. Therefore, the compounds of the present invention are suitable for pharmaceutical compositions for the treatment, prevention, and / or prevention of diseases in humans and animals, and as medicaments for the treatment, prevention, and / or prevention of diseases in humans and animals.

[0108] Accordingly, the present invention also relates to pharmaceutical compositions comprising (i) one or more compounds of formula (I), or pharmaceutically acceptable salts thereof, and (ii) pharmaceutically acceptable carriers or diluents. In some respects, these compositions may exhibit antipathogenic activity. As used herein, the terms “pharmaceutical composition” and “pharmaceutical” are synonyms.

[0109] In other embodiments, the present invention relates to pharmaceutical compositions comprising (i) one or more compounds of formula (I) or pharmaceutically acceptable salts thereof, (ii) one or more β-lactamase inhibitors, and (iii) a pharmaceutically acceptable carrier or diluent. In some aspects, these compositions may exhibit activity against pathogenic microorganisms. When used for the treatment or prevention of bacterial infections, such combinations may exhibit synergistic effects.

[0110] The present invention further relates to pharmaceutical compositions comprising (i) one or more compounds of formula (I) or pharmaceutically acceptable salts thereof, and (ii) one or more β-lactamase inhibitors, for example, wherein the β-lactamase inhibitor is selected from formula (I). a ) to (1 z′ (i) and (iii) pharmaceutically acceptable carriers or diluents. It should be understood that compounds of formula (i) may be used in combination with other β-lactamase inhibitors. Such combinations may exhibit synergistic effects when used to treat or prevent bacterial infections.

[0111]

[0112]

[0113]

[0114] The term "β-lactamase inhibitor" refers to a compound that can inhibit β-lactamase activity, such as formula (1). a ) to (1 Z′ Compounds containing β-lactamase activity, wherein inhibition of β-lactamase activity means inhibition of the activity of class A, B, C, and / or D lactamases. The term "β-lactamase" refers to an enzyme capable of inactivating antibiotic β-lactams.

[0115] In this invention, it has been found that the effectiveness of the lactivicin compound of formula (I) against Gram-negative bacteria can be enhanced by co-use with a β-lactamase inhibitor selected from the above formula (I). a ) to (1 Z′ Any of the above-mentioned β-lactamase inhibitors may be used, but the present invention should not be construed as being limited to the use of the above-mentioned β-lactamase inhibitors.

[0116] The compounds and compositions of the present invention can act systemically and / or locally. For this purpose, they can be administered in suitable manner, for example, parenterally, pulmonaryly, nasally, sublingually, lingually, buccally, rectally, dermis, percutaneously, conjunctiva, ocularly, or as implants or scaffolds.

[0117] For these routes of administration, pharmaceutical compositions comprising the compounds of the present invention can be administered in a suitable form.

[0118] Parenteral administration can be performed without absorption steps (such as intravenous, intra-arterial, intracardiac, intraspinal, or intralumbar) or with absorption steps (such as intramuscular, subcutaneous, intradermal, percutaneous, or intraperitoneal). Suitable parenteral administration formulations include injectable and infusion formulations in the form of solutions, suspensions, emulsions, lyophilized forms, or sterile powders.

[0119] Other routes of administration include, for example, inhaled drug forms (including powders, nebulizers), nasal drops, solutions, sprays, tablets, films / sheets or capsules for tongue, sublingual or oral administration, suppositories, ear or eye preparations, aqueous suspensions (washes, shaken mixtures), lipophilic suspensions, ointments, creams, transdermal therapy systems (such as patches), pastes, foams, powders, implants or stents.

[0120] The compounds of the present invention can be converted into the aforementioned dosage form, i.e., the pharmaceutical compositions of the present invention. This can be achieved by mixing with inert, non-toxic, pharmaceutically acceptable carriers or diluents, such as starch, glucose, lactose, sucrose, gelatin, gum arabic, malt, rice, flour, calcium carbonate, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skim milk powder, glycerin, propylene glycol, water, water for injection, saline, buffered saline, glucose, glycerin, ethanol, propylene glycol, and polysorbate 80 (Tween-80). TM ), polyethylene glycol (PEG) 300 and 400, polyethylene glycolated castor oil (such as polyoxyethylene castor oil (CremophorEL)), poloxamer 407 and 188, hydrophilic and hydrophobic carriers, and combinations thereof. Hydrophobic carriers include, for example, fat emulsions, lipids, polyethylene glycolated phospholipids, polymer matrices, biocompatible polymers, lipospheres, vesicles, particles, and liposomes. The terminology specifically excludes cell culture media.

[0121] If desired, the pharmaceutical compositions of the present invention may also contain small amounts of wetting agents (e.g., sodium lauryl sulfate, polyethylene oxide sorbitan monooleate), dispersants or emulsifiers, pH buffers, and preservatives. Additionally, they may include adjuvants, stabilizers, thickeners, lubricants, and colorants. The pharmaceutical compositions can be formulated in a conventional manner. The correct formulation depends on the chosen route of administration.

[0122] In pharmaceutical compositions containing compounds of formula (I), the weight ratio of the active ingredient to the carrier is typically in the range of 1:20 to 20:1.

[0123] When administered in combination with a β-lactamase inhibitor, this compound and the β-lactamase inhibitor may provide a synergistic effect. The term "synergistic effect" refers to an effect produced when two or more agents are administered simultaneously, which is greater than the additive effect produced when the agents are administered alone. Optionally, during treatment with a β-lactamase inhibitor, the compound of formula (I) or a salt thereof may be administered as a single agent.

[0124] "Effective therapeutic dose" is a dose of a compound that, when administered to a subject to treat a disease or condition, is sufficient to affect the treatment of the disease or condition or to improve the symptoms of the disease or condition. Effective therapeutic doses may vary, for example, depending on the compound, the disease, condition and / or symptoms of the disease, the severity of the disease or condition, and the age, weight, and / or health status of the patient being treated.

[0125] Typically, the effective therapeutic dose of the compounds of this invention for adults is about 50 mg to about 3000 mg of the compound of formula (I). In another embodiment, the effective therapeutic dose is about 100 mg to about 2000 mg. In another embodiment, the effective therapeutic dose is about 500 mg to about 1200 mg. Typically, the dose (the amount) is administered 1 to 4 times daily. In one embodiment, the dose is administered 3 times daily. In some cases, doses exceeding these limits may be required.

[0126] The terms “dose,” “unit dose,” “unit dosage,” or “effective dose” refer to a physically discrete unit containing a predetermined amount of active ingredient, calculated to produce the desired therapeutic effect.

[0127] The present invention also relates to pharmaceuticals comprising at least one compound of the present invention, typically used with one or more inert, non-toxic, pharmaceutically acceptable carriers or diluents, and their use for the purposes described above. The compositions may be in the form of injectable formulations, suspensions, emulsions, coated tablets, granules, gelatin capsules, liquid-containing capsules, powders, granules, sustained-release formulations, suppositories, aerosols, sprays, ointments, creams, or any other suitable form.

[0128] Treatment

[0129] The present invention also relates to the use of the compounds of the invention for the treatment, prevention and / or prophylaxis of diseases caused by bacteria, particularly Gram-negative bacteria.

[0130] The present invention also relates to the use of the compounds of the present invention in the manufacture of medicaments for the treatment, prevention and / or prophylactic treatment of diseases, particularly bacterial infections.

[0131] Accordingly, the present invention includes methods for treating, preventing, and / or preventing bacterial infections by administering the compounds of the present invention to subjects in need.

[0132] In one embodiment, the method of the present invention includes administering an effective therapeutic amount of one or more compounds of formula (I) to a subject suffering from or at risk of bacterial infection.

[0133] In another embodiment, the method of the present invention includes administering an effective therapeutic amount of a pharmaceutical composition to a subject suffering from or at risk of developing a bacterial infection, said pharmaceutical composition comprising one or more compounds of formula (I) as defined herein, and a pharmaceutically acceptable carrier or diluent.

[0134] In a further embodiment, the method of the present invention includes administering an effective therapeutic amount of a pharmaceutical composition to a subject suffering from or at risk of bacterial infection, said pharmaceutical composition comprising (i) one or more compounds of formula (I) as defined herein, (ii) a pharmaceutically acceptable carrier or diluent, and (iii) one or more β-lactamase inhibitors. In subsequent embodiments, for example, the β-lactamase inhibitor may be selected from formula (I) a ) to (1 z′ ).

[0135] The compounds of this invention exhibit an excellent antibacterial spectrum against Gram-negative bacteria. The compounds of this invention are particularly suitable for human treatment and veterinary medicine for prophylactic treatment, prevention and / or treatment of local and systemic infections. Therefore, in each embodiment and aspect of this invention, the subject is a human, a non-human primate, a bird, a horse, a cattle, a goat, a sheep, a companion animal (such as a dog, cat, or rodent), or other mammal.

[0136] The compounds described in this invention include examples of bacteria with antibacterial activity, but are not limited to, Enterobacterales, Escherichia coli, Enterobacter spp., Klebsiella spp., Serratia spp., Pseudomonass spp., Stenotrophomonas spp., Citrobacter spp., Acinetobacter spp., Campylobacter spp., Helicobacter spp., Vibrio spp., Bordetella spp., Salmonella spp., Shigella spp., and Francisella spp. spp.), Burkholderia spp., Clostridia spp., Alcaligenes spp., Moraxella spp., Proteus spp., Neisseria spp., Haemophilus spp., Achromobacter spp., and Erwinia spp.

[0137] Manufacturing method

[0138] The present invention also relates to a method for preparing compounds of formula (I). The following scheme 1 illustrates a general method for preparation, which is not intended to be limited to any particular compound described herein.

[0139] Option 1

[0140]

[0141] In formulas (II), (III), and (IV), Pg′ and Pg″ represent carboxyl protecting groups that are frequently used in β-lactam chemistry to protect the carboxyl group.

[0142] The carboxyl protecting groups Pg′ and Pg″ may be residues that form aliphatic or aryl alcohols in ester formation.

[0143] Examples of carboxyl protecting groups include isopropyl, tert-butyl, methoxymethyl, ethoxymethyl, isobutoxymethyl, acetoxymethyl, propionyloxymethyl, butyryloxymethyl, valeratemethyl, 1-methoxycarbonyloxyethyl, 1-ethoxycarbonyloxyethyl, p-methoxybenzyl, benzyl, o-nitrophenyl, p-nitrophenyl, 2,4-dinitrophenylmethyl, diphenylmethyl, phthaloyl, and allyl.

[0144] In any intermediate of formula (I), when an amino group is present in the molecule, it should be understood that it must be protected by a suitable amino protecting group as is commonly used in β-lactam chemistry.

[0145] Examples of amino protecting groups include triphenylmethyl, p-nitrobenzyloxycarbonyl (PNZ), and tert-butyloxycarbonyl.

[0146] Similarly, it can be understood that the hydroxyl groups of the catechols or hydroxypyridinones present in formulas (III) and (IV) must be protected with appropriate hydroxyl protecting groups.

[0147] Examples of suitable hydroxyl protecting groups include, but are not limited to, benzyl ethers, diphenylmethyl ethers, and diphenylmethylene ketals.

[0148] The protecting group can be removed by any convenient method suitable for removing the protecting group, as described in the literature or known to a professional chemist, such as the method chosen so that the removal of the protecting group causes minimal interference to other groups in the molecule.

[0149] The removal of the carboxyl, amino, and hydroxyl protecting groups from the catechol or hydroxypyridinone in intermediate (IV) can be carried out through conventional procedures, such as treatment with acids or reducing agents. Examples of acids include trifluoroacetic acid, formic acid, acetic acid, and hydrochloric acid. Examples of Lewis acids used are boron trifluoride ether, zinc chloride, tin tetrachloride, aluminum chloride, titanium tetrachloride, or boron trichloride.

[0150] When removal is carried out by catalytic hydrogenolysis, any procedure using palladium or platinum catalysts can be employed.

[0151] The removal of carboxyl protecting groups, hydroxyl protecting groups of catechol and hydroxypyridinone moieties, and amino protecting groups can preferably be carried out simultaneously or through a stepwise method.

[0152] The present invention also includes methods for preparing specific fragments, such as compounds (IIIa′) and (IIIa″) of formula (III) in Scheme 1, which are described in Schemes 2 and 3 below, respectively, illustrating only general methods of preparation and not intended to be limited to any specific compound described herein.

[0153]

[0154]

[0155] The compounds of the present invention can be prepared by removing the protecting group from the compound of formula (IV) under acidic conditions and catalytic hydrogenolysis.

[0156] Acidic conditions may involve treating compound (IV) with boron trichloride, formic acid, acetic acid, trifluoroacetic acid, or hydrochloric acid for 10 minutes to 16 hours in a temperature range of -78°C to 100°C. Boron trichloride is preferably used for 1 to 3 hours in a temperature range of -78°C to -20°C.

[0157] Compounds of formula (II) may be synthesized according to the following references, all of which are incorporated herein by reference: Chem. Pharm. Bull., 1990, Vol. 38, No. 11, pp. 116-122; J. Chem. Soc., Chem. Commun., 1987, (2), pp. 62-63; J. Med. Chem., 2014, Vol. 57, pp. 3845-3855, or by means of adaptation known to those skilled in the art.

[0158] The substituted 2-ketoglutarate of formula (III) can be synthesized according to the following reference: J. Med. Chem., 2014, Vol. 57, pp. 3845-3855, or by adapting the reference procedure in a manner known to those skilled in the art. The preparation of specific fragments such as (IIIa′) and (IIIa″) are shown in Schemes 2 and 3, respectively.

[0159] In the presence of a coupling agent, the coupling reaction between intermediate (II) and intermediate (III) typically occurs in an inert solvent, where, where applicable, a base is added for 1-24 hours at a temperature range of -20°C to 80°C, preferably overnight at 20-30°C. Inert solvents are, for example, dichloromethane (DCM), toluene, tetrahydrofuran (THF), 1,4-dioxane, N,N-dimethylformamide (DMF), N,N-dimethylacetamide (DMA), N-methylpyrrolidone-2-one (NMP), and acetonitrile, as well as mixtures of the above solvents. Tetrahydrofuran is the preferred solvent.

[0160] Suitable coupling agents are, for example, carbodiimides, such as N,N'-diethyl-, N,N'-dipropyl-, N,N'-diisopropyl-, N,N'-dicyclohexylcarbodiimide, N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), carbonyl diimidazole (CDI), and O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethylurea hexafluorophosphate (HBTU). Or O-(7-azabenzotriazol-1-yl)-N,N,N′,N'-tetramethylurea hexafluorophosphate (HATU) or 1-hydroxybenzotriazole (HOBT) or (benzotriazol-1-yloxy)tris(dimethylamino)hexafluorophosphate (BOP), or (benzotriazol-1-yloxy)tripyrrolidine hexafluorophosphate (PyBOP), or N-hydroxysuccinimide, or mixtures thereof, with or without a base. Suitable bases are, for example, carbonates and bicarbonates, triethylamine, diisopropylethylamine, N-methylmorpholine, N-methylpiperidine, or 4-dimethylaminopyridine.

[0161] The reaction typically occurs in a single solvent or a mixture of solvents, and is carried out over a temperature range of 0°C to 100°C for 1–24 hours. Suitable protic solvents include, for example, methanol, ethanol, isopropanol, tert-butanol, and water. Solvents suitable for forming mixtures include, for example, dichloromethane, trichloromethane, tetrahydrofuran, 1,4-dioxane, acetonitrile, and N,N-dimethylformamide.

[0162] Example

[0163] As described above, the present invention also includes methods for preparing compounds of formula (I). The following examples provide specific methods for preparing certain specific compounds in Table 1.

[0164] In the following instructions, the following symbols are used to indicate specific meanings:

[0165] Ac: Acetyl group

[0166] br: Broad (spectral)

[0167] Boc: tert-Butoxycarbonyl

[0168] Bn: Benzyl

[0169] d: Bimodal (spectral)

[0170] DBU: 1,8-diazabicycloundec-7-ene

[0171] DCM: Dichloromethane

[0172] DCC: N,N′-Dicyclohexylcarbodiimide

[0173] DI: Deionized

[0174] DIC: N,N′-Diisopropylcarbodiimide

[0175] DIPEA: N,N-Diisopropylethylamine

[0176] DMA: N,N-dimethylacetamide

[0177] DMAP: 4-Dimethylaminopyridine

[0178] DMFH: N,N-Dimethylformamide

[0179] DMSO: Dimethyl sulfoxide

[0180] h: hours

[0181] HATU: 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridine-3-oxide hexafluorophosphate

[0182] m: multiple (spectral)

[0183] min: minutes

[0184] MW: Microwave

[0185] NHS: N-hydroxysuccinimide

[0186] Ph: Phenyl

[0187] s: Single peak (spectral)

[0188] t: triplet (spectral)

[0189] t-Bu: tert-butyl

[0190] TBSCl: tert-butyldimethylsilyl chloride

[0191] TEA: Triethylamine

[0192] THF: Tetrahydrofuran

[0193] TMEDA: Tetramethylethylenediamine

[0194] TMS: Trimethylsilyl

[0195] TMSCN: Trimethylsilyl cyanide

[0196] Example 1

[0197] (4S)-2-[(4S)-4-{[(2Z)-2-(2-amino-1,3-thiazolyl-4-yl)-2-{[(1-carboxycyclopropyl)oxy]imino}acetyl]amino}-3-oxo-1,2-oxazolidine-2-yl]-4-(5,6-dihydroxy-1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)-5-oxooxopentane-2-carboxylic acid (Compound 1, Table 1)

[0198]

[0199] Step 1: 1-(aminooxy)cyclopropane-1-carboxylic acid tert-butyl ester (2)

[0200]

[0201] A solution of anhydrous THF (120 mL) containing 1-hydroxycyclopropane-1-carboxylic acid tert-butyl ester (4.86 g, 30.72 mmol) was cooled to 0–5 °C, and O-diphenylphosphinohydroxylamine (9.32 g, 39.97 mmol) was added, followed by sodium tert-butoxide (3.84 g, 39.96 mmol). The reaction mixture was stirred at 0–10 °C for 2 hours, and then hexane (30 mL) and brine (75 mL) were added. The resulting suspension was stirred at 15–25 °C for 30 minutes. The precipitated solid was removed by filtration and washed with hexane containing 10% ethyl acetate. The organic phase of the filtrate was separated, and the aqueous layer was further extracted with ethyl acetate. The combined organic extracts were washed with brine, dried over sodium sulfate, filtered, and concentrated under reduced pressure. The residue was treated with hexane and filtered. The filtrate was concentrated under reduced pressure to provide 1-(aminooxy)cyclopropane-1-carboxylic acid tert-butyl ester 2 (5.02 g, 94%) as a yellow oil.

[0202] 1 HNMR (400MHz, CDCl3) δ5.78(brs,2H),1.49(s,9H),1.34-1.28(m,2H),1.22-1.16(m,2H).

[0203] Step 2: (2Z)-{2-[(tert-butoxycarbonyl)amino]-1,3-thiazolyl-4-yl}({[1-(tert-butoxycarbonyl)cyclopropyl]oxy}imino)acetic acid (3)

[0204]

[0205] To a MeOH solution containing tert-butyl 1-(aminooxy)cyclopropane-1-carboxylic acid ester 2 (5.92 g, 34.18 mmol), {2-[(tert-butyloxycarbonyl)amino]-1,3-thiazolyl}(oxo)acetic acid (8.46 g, 31.07 mmol) was added, and the mixture was stirred at room temperature for 4 hours. The reaction mixture was then concentrated to half its volume and quenched by adding water (100 mL) and 0.5 M HCl solution (100 mL). The mixture was extracted with ethyl acetate, the combined organic extracts were washed with brine, dried over sodium sulfate, filtered, and concentrated under reduced pressure. The residue was dissolved in EtOH (50 mL), and water was slowly added until no more precipitate formed. The resulting suspension was stirred at 0-10°C for 15 minutes, and the precipitated solid was collected by filtration and air drying to provide (2Z)-{2-[(tert-butoxycarbonyl)amino]-1,3-thiazolyl-4-yl}({[1-(tert-butoxycarbonyl)cyclopropyl]oxy}imino)acetic acid 3 (12.20 g, 92%), which is a grayish-white solid.

[0206] 1 HNMR (400MHz, DMSO-d6) δ13.91(brs,1H),11.78(s,1H),7.42(s,1H),1.45(s,9H),1.41-1.31(m,11H),1.27-1.19(m,2H).

[0207] MS(ESI)m / z: [M+1] + 428.1

[0208] Step 3: 1-{[(Z)-(1-{2-[(tert-butyloxycarbonyl)amino]-1,3-thiazolyl-4-yl}-2-[(2,5-dioxopyrrolidone-1-yl)oxy]-2-oxoethylidene)amino]oxy}cyclopropane-1-carboxylic acid tert-butyl ester (4)

[0209]

[0210] At 0 °C, DIC (4.59 mL, 29.64 mmol) was slowly added to anhydrous DCM (120 mL) containing a mixture of (2Z)-{2-[(tert-butoxycarbonyl)amino]-1,3-thiazolyl}({1-(tert-butoxycarbonyl)-cyclopropyl]oxy}imino)acetic acid 3 (10.93 g, 25.57 mmol) and NHS (3.53 g, 30.67 mmol). The reaction mixture was stirred at 0 °C for 15 min, then at room temperature for 3 h. The resulting suspension was filtered, and the solids were washed with DCM. The filtrate was concentrated under reduced pressure, and the residue was treated with a mixture of methanol (50 mL) and heptane (40 mL). The mixture was stirred at room temperature for 30 minutes, then stirred again at about 10°C for 30 minutes, and then filtered to produce 1-{[(Z)-(1-{2-[(tert-butyloxycarbonyl)amino]-1,3-thiazolyl-4-yl}-2-[(2,5-dioxopyrrolidine-1-yl)oxy]-2-oxoethylidene)amino]oxy}cyclopropane-1-carboxylic acid tert-butyl ester 4 (13.50 g, 100 g)), a white solid.

[0211] 1 HNMR (400MHz, CDCl3) δ8.36(s,1H),7.60(s,1H),2.99-2.83(m,4H),1.54(s,9H),1.51-1.45(m,4H),1.43(s,9H).

[0212] MS(ESI)m / z: [M+1] + 525.0

[0213] Step 4: 1-{[(Z)-(1-{2-[(tert-butyloxycarbonyl)amino]-1,3-thiazolyl}-2-oxo-2-{[(4S)-3-oxo-1,2-oxazolidine-4-yl]amino}ethylene)amino]oxy}cyclopropane-1-carboxylic acid tert-butyl ester (5)

[0214]

[0215] At room temperature, DIPEA (5.34 mL, 30.72 mmol) was added to an anhydrous DMF solution containing 1-{[(Z)-(1-{2-[(tert-butyloxycarbonyl)amino]-1,3-thiazolyl}-2-[(2,5-dioxopyrrolidone-1-yl)oxy]-2-oxoethylidene)amino]oxy}cyclopropane-1-carboxylic acid tert-butyl ester 4 (13.50 g, 25.57 mmol) and L-cycloserine (3.13 g, 30.66 mmol). The reaction mixture was stirred at 45 °C for 18 hours, then concentrated under reduced pressure and dried. The residue was purified by silica gel column chromatography using a gradient DCM containing 0-2.5% methanol, followed by grinding with DCM and hexane to produce 5 (5.56 g, 43%) of 1-{[(Z)-(1-{2-[(tert-butyloxycarbonyl)amino]-1,3-thiazolyl}-2-oxo-2-{[(4S)-3-oxo-1,2-oxazolidine-4-yl]amino}ethylene)amino]oxy}cyclopropane-1-carboxylic acid tert-butyl ester, a white solid.

[0216] 1 HNMR(400MHz,DMSO-d6)δ11.81(s,1H),11.59(s,1H),9.00(d,J=8.0Hz,1H),7.45(s,1H),4.90(d,J =7.4Hz,1H),4.58(t,J=8.5Hz,1H),4.08-3.96(m,1H),1.45(s,9H),1.37(s,9H),1.34-1.18(m,4H).

[0217] MS(ESI)m / z: [m+Na] + 534.1

[0218] Step 5: (4S)-2-[(4S)-4-{[(2Z)-2-{2-[(tert-butoxycarbonyl)amino]-1,3-thiazolyl-4-yl}-2-({[1-(tert-butoxycarbonyl)cyclopropyl]oxy}imino)acetyl]amino}-3-oxo-1,2-oxazolidine-2-yl]-4-(5,7-dioxo-2,2-diphenyl-5,7-dihydro-2H,6H-[1,3]dioxono[4,5-f]isoindol-6-yl)-5-oxooxopentane-2-carboxylic acid tert-butyl ester (7)

[0219]

[0220] To 5 (1.04 g, 2.03 mmol) of 1-{[(Z)-(1-{2-[(tert-butoxycarbonyl)amino]-1,3-thiazolyl}-2-oxo-2-{[(4S)-3-oxo-1,2-oxazolidine-4-yl]amino}ethylene)amino]oxy}cyclopropane-1-carboxylic acid tert-butyl ester and (2S)-5-tert-butoxy-2-(5,7-dioxo-2,2-diphenyl-5,7-dihydro-2H, DMAP (50 mg, 0.41 mmol) was added to an anhydrous THF (60 mL) solution of 6H-[1,3]dioxacyclo[4,5-f]isoindol-6-yl)-4,5-dioxovaleric acid 6 (1.10 g, 2.02 mmol, prepared as described in J. Med. Chem., 2014, Vol. 57, pp. 3845-3855), followed by DCC (585 mg, 2.84 mmol). The reaction mixture was stirred at room temperature for 18 hours, followed by concentration under reduced pressure. The residue was treated with hexane containing 30% DCM, and the precipitated solids were removed by filtration. The filtrate was concentrated under vacuum, and the residue was purified by silica gel column chromatography using a gradient of hexane containing 10-35% ethyl acetate to give (4S)-2-[(4S)-4-{[(2Z)-2-{2-[(tert-butoxycarbonyl)amino]-1,3-thiazolyl-4-yl}-2-({[1-(tert-butoxycarbonyl)cyclopropyl]oxy}imino)acetyl]amino}-3-oxo-1,2-oxazolidine-2-yl]-4-(5,7-dioxo-2,2-diphenyl-5,7-dihydro-2H,6H-[1,3]dioxono[4,5-f]isoindol-6-yl)-5-oxooxopentane-2-carboxylic acid tert-butyl ester 7 (720 mg, 34%), as a brown foam.

[0221] 1 HNMR (400MHz, CDCl3) δ8.68-8.56(m,1H),8.56-8.26(m,1H),7.58-7.49(m,4H),7.46-7.35(m,7H),7.30(d,J=1.1Hz,2H),5.3 8(m,1H),5.21-4.81(m,2H),4.40-4.19(m,1H),3.70-3.28(m,1H),2.93-2.70(m,1H),1.66-1.46(m,21H),1.46-1.30(m,10H).

[0222] MS(ESI)m / z: [m+Na] + 1059.4

[0223] Step 6: (4S)-2-[(4S)-4-{[(2Z)-2-(2-amino-1,3-thiazolyl-4-yl)-2-{[(1-carboxycyclopropyl)oxy]imino}acetyl]amino}-3-oxo-1,2-oxazolidine-2-yl]-4-(5,6-dihydroxy-1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)-5-oxooxopentane-2-carboxylic acid (Compound 1, Table 1)

[0224]

[0225] At -50°C, BCl3 solution (1.0 M in DCM, 4.34 mL, 4.34 mmol) was added dropwise to an anhydrous DCM (20 mL) solution containing (4S)-2-[(4S)-4-{[(2Z)-2-{2-[(tert-butyloxycarbonyl)amino]-1,3-thiazolyl}-2-({[1-(tert-butyloxycarbonyl)cyclopropyl]oxy}imino)acetyl]amino}-3-oxo-1,2-oxazolidine-2-yl]-4-(5,7-dioxo-2,2-diphenyl-5,7-dihydro-2H,6H-[1,3]dioxacyclo[4,5-f]isoindol-6-yl)-5-oxooxohexane-2-carboxylic acid tert-butyl ester 7 (562 mg, 0.54 mmol). The reaction mixture was stirred for 2.5 hours at a temperature of -50°C to -35°C. Then, a mixture of NaHCO3 (873 mg) and Na2HPO4 (273.5 mg) dissolved in water (47 mL) was added to the reaction mixture at -50°C. The resulting heterogeneous mixture was stirred at 0 to 5°C using an ice-water bath instead of a cold bath until the aqueous phase thawed (approximately 40 minutes), and the phases were carefully separated. The aqueous layer was filtered using a 1.0 μm pinhole filter and immediately subjected to C18 reversed-phase column chromatography using a Biotage system and a gradient of acetonitrile containing 0.1% formic acid and an aqueous solution containing 0.1% formic acid (0-30%). The products containing fractions were combined and lyophilized to provide compound 1 (182 mg, 51%) as a grayish-white solid.

[0226] 1 ¹H NMR (400 MHz, mixture of D₂O and CD₃CN) δ 7.25 (s, 2H), 7.15–7.10 (m, 1H), 5.45–5.18 (m, 1H), 5.18–5.02 (m, 1H), 4.77–4.66 (m, 1H), 4.31–4.19 (m, 1H), 3.60–3.24 (m, 1H), 2.92–2.65 (m, 1H), 1.55–1.45 (m, 2H), 1.45–1.33 (m, 2H). No interchangeable protons were observed in D₂O.

[0227] MS(ESI)m / z: [M+1] + 661.1

[0228] Example 2

[0229] (4S)-2-[(4S)-4-{[(2Z)-2-(2-amino-1,3-thiazolyl-4-yl)-2-{[(1-carboxycyclobutyl)oxy]imino}acetyl]amino}-3-oxo-1,2-oxazolidine-2-yl]-4-(5,6-dihydroxy-1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)-5-oxooxopentane-2-carboxylic acid (Compound 4, Table 1)

[0230]

[0231] Step 1: 1-{[(Z)-(1-{2-[(tert-butyloxycarbonyl)amino]-1,3-thiazolyl}-2-oxo-2-{[(4S)-3-oxo-1,2-oxazolidine-4-yl]amino}ethylene)amino]oxy}cyclobutane-1-carboxylic acid tert-butyl ester (9)

[0232]

[0233] DIPEA (4.23 mL, 24.51 mmol) was added to a mixed solution of anhydrous DMF (110 mL) containing 1-{(Z)-(1-{2-[(tert-butoxycarbonyl)amino]-1,3-thiazolyl-4-yl}-2-[(2,5-dioxopyrrolidone-1-yl)oxy]-2-oxoethylidene)amino]oxy}cyclobutane-1-carboxylic acid tert-butyl ester 8 (11.0 g, 20.42 mmol, prepared as described in WO2012 / 073138) and L-cycloserine (2.5 g, 20.42 mmol). The reaction mixture was heated overnight at 45 °C and then concentrated under reduced pressure and dried. The residue was analyzed by silica gel column chromatography using a gradient DCM containing 0-3% MeOH to obtain 9 (4.5 g, 42 g) of 1-{[(Z)-(1-{2-[(tert-butyloxycarbonyl)amino]-1,3-thiazolyl}-2-oxo-2-{[(4S)-3-oxo-1,2-oxazolidine-4-yl]amino}ethylene)amino]oxy}cyclobutane-1-carboxylic acid tert-butyl ester, a grayish-white solid.

[0234] 1HNMR(400MHz,DMSO-d6)δ11.83(s,1H),11.59(s,1H),9.10(d,J=7.9Hz,1H),7.43(s,1H),4.92(brs,1H),4.60(t,J =8.4Hz,1H),4.12-4.03(m,1H),2.46-2.33(m,2H),2.32-2.14(m,2H),1.94-1.70(m,2H),1.45(s,9H),1.40(s,9H).

[0235] Step 2: (4S)-2-[(4S)-4-{[(2Z)-2-{2-[(tert-butoxycarbonyl)amino]-1,3-thiazolyl-4-yl}-2-({[1-(tert-butoxycarbonyl)cyclobutyl]oxy}imino)acetyl]amino}-3-oxo-1,2-oxazolidine-2-yl]-4-(5,7-dioxo-2,2-diphenyl-5,7-dihydro-2H,6H-[1,3]dioxono[4,5-f]isoindol-6-yl)-5-oxooxopentane-2-carboxylic acid tert-butyl ester (10)

[0236]

[0237] At room temperature, 9 (1.0 g, 1.84 mmol) of 1-{[(Z)-(1-{2-[(tert-butoxycarbonyl)amino]-1,3-thiazolyl}-2-oxo-2-{[(4S)-3-oxo-1,2-oxazolidine-4-yl]amino}ethylene)amino]oxy}cyclobutane-1-carboxylic acid tert-butyl ester was prepared. Anhydrous DCM (30 mL) of 2-diphenyl-5,7-dihydro-2H,6H-[1,3]dioxane[4,5-f]isoindol-6-yl)-4,5-dioxovaleric acid 6 (1.06 g, 2.02 mmol, prepared as described in J. Med. Chem., 2014, Vol. 57, pp. 3845-3855) was added to DCC (0.53 g, 2.58 mmol). The reaction mixture was stirred overnight at room temperature and then filtered to remove the precipitated solids. The filtrate was concentrated under reduced pressure and dried. The residue was purified by silica gel column chromatography using a gradient of hexane containing 0-35% ethyl acetate to give 10 (1.0 g, 57%) of (4S)-2-[(4S)-4-{[(2Z)-2-{2-[(tert-butoxycarbonyl)amino]-1,3-thiazolyl}-2-({[1-(tert-butoxycarbonyl)cyclobutyl]oxy}imino)acetyl]amino}-3-oxo-1,2-oxazolidine-2-yl]-4-(5,7-dioxo-2,2-diphenyl-5,7-dihydro-2H,6H-[1,3]dioxacyclo[4,5-f]isoindol-6-yl)-5-oxooxopentane-2-carboxylic acid tert-butyl ester, as a gray solid.

[0238] 1 HNMR(400MHz, CDCl3)δ8.33-8.25(m,1H),7.57-7.49(m,4H),7.45-7.36(m,6H),7.30(s,1H),7.26(s,2H),5.44-5.31(m,1H),5.19-4.95(m,1H) ,4.94-4.81(m,1H),4.23(m,1H),3.49-3.24(m,1H),2.94-2.74(m,1H), 2.65-2.43(m,4H),2.14-1.90(m,2H),1.58-1.50(m,18H),1.48(s,9H).

[0239] MS(ESI)m / z: [M+1] + 1073.4

[0240] Step 3: (4S)-2-[(4S)-4-{[(2Z)-2-(2-amino-1,3-thiazolyl-4-yl)-2-{[(1-carboxycyclobutyl)oxy]imino}acetyl]amino}-3-oxo-1,2-oxazolidine-2-yl]-4-(5,6-dihydroxy-1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)-5-oxooxopentane-2-carboxylic acid (Compound 4, Table 1)

[0241]

[0242] A mixture of 10 mL of anhydrous DCM containing 10 (300 mg, 0.28 mmol) of (4S)-2-[(4S)-4-{[(2Z)-2-{2-[(tert-butyloxycarbonyl)amino]-1,3-thiazolyl}-2-({[1-(tert-butyloxycarbonyl)cyclobutyl]oxy}imino)acetyl]amino}-3-oxo-1,2-oxazolidine-2-yl]-4-(5,7-dioxo-2,2-diphenyl-5,7-dihydro-2H,6H-[1,3]dioxacyclo[4,5-f]isoindol-6-yl)-5-oxooxopentane-2-carboxylic acid tert-butyl ester was cooled to -50 °C under nitrogen. After 10 minutes, DCM containing BCl3 (1.0 M, 2.28 mL, 2.28 mmol) was added dropwise over a period of more than 10 minutes, maintaining the external temperature at -50 °C. The reaction mixture was stirred at -50 °C to -40 °C for 2.5 hours, and then 25.2 mL of buffer solution (prepared by dissolving 776 mg NaHCO3 and 243 mg Na2HPO4 in 42 mL of DI water) was added at -50 °C. The resulting heterogeneous mixture was stirred at 0 to 5 °C using an ice-water bath instead of a cold bath until the aqueous phase thawed (approximately 30 minutes), and the phases were carefully separated. The aqueous layer was filtered using a 1.0 μm pinhole filter and immediately subjected to C18 reversed-phase column chromatography using a Biotage system and a gradient of acetonitrile containing 0.1% formic acid and an aqueous solution containing 0.1% formic acid at 0-30%. The products containing fractions were combined and lyophilized to give compound 4 (105 mg, 55%) as a pale yellow solid.

[0243] 1 ¹H NMR (400 MHz, mixture of D₂O and CD₃CN) δ 7.43 (s, 2H), 7.29 (s, 1H), 5.62–5.52 (m, 1H), 5.33 (m, 1H), 4.92 (m, 1H), 4.53–4.40 (m, 1H), 3.51 (m, 1H), 2.92 (m, 1H), 2.79–2.63 (m, 2H), 2.58–2.41 (m, 2H), 2.21–1.96 (m, 2H). No interchangeable proton O was observed in D₂O.

[0244] MS(ESI)m / z: [M+1] + 675

[0245] Example 3

[0246] (4S)-2-[(4S)-4-{[(2Z)-2-(2-amino-1,3-thiazolyl-4-yl)-2-{[(3-carboxyoxobutane-3-yl)oxy]imino}acetyl]amino}-3-oxo-1,2-oxazolidine-2-yl]-4-(5,6-dihydroxy-1,3-dioxo-2,3-dihydro-2H-isoindol-2-yl)-5-oxooxopentane-2-carboxylic acid (compound 5, Table 1) and (4 S)-2-[(4S)-4-{[(2Z)-2-(2-amino-1,3-thiazolyl-4-yl)-2-{[(2-carboxy-1-chloro-3-hydroxypropane-2-yl)oxy]imino}acetyl]amino}-3-oxo-1,2-oxazolidine-2-yl]-4-(5,6-dihydroxy-1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)-5-oxooxopentane-2-carboxylic acid (Compound 6, Table 1)

[0247]

[0248] Step 1: 3-[(trimethylsilyl)oxy]oxetane-3-nitrile (12)

[0249]

[0250] 3-oxetane 11 (8.10 mL, 138.18 mmol) was added to LiClO4 (14.70 g, 138.18 mmol), followed by TMSCN (22.0 mL, 175.85 mmol). The resulting mixture was stirred at room temperature for 2 hours, then diluted with DCM and filtered. The filtrate was washed with water, dried over sodium sulfate, filtered, and concentrated under reduced pressure. Crude 3-[(trimethylsilyl)oxy]oxetane-3-nitrile 12 (22.05 g, 93%) was obtained as a brown oil for the next step without further purification.

[0251] 1 HNMR (400MHz, CDCl3) δ4.95-4.84(m,2H), 4.78-4.64(m,2H), 0.43-0.20(s,9H).

[0252] Step 2: 3-hydroxyoxetane-3-nitrile (13)

[0253]

[0254] 2M hydrochloric acid solution (130 mL) was slowly added to a mixture of 22.05 g (128.74 mmol) of 3-[(trimethylsilyl)oxy]oxetane-3-nitrile 12 and 130 mL of THF at room temperature. The reaction mixture was stirred at room temperature for 2 hours, then saturated with solid NaCl and extracted with ethyl acetate. The combined organic extracts were dried over sodium sulfate, filtered, and concentrated under reduced pressure. The residue was ground with DCM and hexane to give 3-hydroxyoxetane-3-nitrile 13 (11.12 g, 87%) as a grayish-white product.

[0255] 1 HNMR(400MHz,DMSO-d6)δ7.48(s,1H),4.96-4.71(m,2H),4.68-4.38(m,2H).

[0256] Step 3: 3-(benzyloxy)oxetane-3-nitrile (14)

[0257]

[0258] To a THF (200 mL) solution containing 3-hydroxyoxetane-3-nitrile 13 (6.69 g, 67.51 mmol), NaOH (powder, 8.10 g, 202.5 mmol) was added. The resulting mixture was stirred at room temperature for 10 min, then cooled to 5–10 °C (cold water bath), and benzyl bromide (12.05 mL, 101.31 mmol) was added, followed by n-Bu4NI (2.50 g, 6.77 mmol). The reaction mixture was allowed to be slowly heated to room temperature and stirred for 19 h. The mixture was then quenched with water and extracted with diethyl ether. The combined organic extracts were washed with brine, dried over sodium sulfate, filtered, and concentrated under reduced pressure. The residues were purified by silica gel column chromatography using a gradient of hexane containing 0–8% ethyl acetate. The resulting product was ground with hexane to give pure 3-(benzyloxy)oxetane-3-nitrile 14 (2.61 g, 20%) as a white crystalline solid.

[0259] 1 HNMR (400MHz, CDCl3) δ7.51-7.32 (m, 5H), 4.85 (d, J = 8.1Hz, 2H), 4.74-4.61 (m, 4H).

[0260] Step 4: 3-(benzyloxy)oxetane-3-carboxylic acid (15)

[0261]

[0262] An aqueous NaOH solution (2M, 29.2 mL) containing 5.51 g (29.12 mmol) of 3-(benzyloxy)oxetane-3-nitrile 14 was added. The reaction mixture was heated at reflux for 6 hours, then cooled to room temperature and concentrated under reduced pressure to remove the MeOH. The aqueous solution was cooled to 0 °C and acidified to pH approximately 2 using 1M hydrochloric acid. The precipitate was collected as a white solid by filtration and then dissolved in DCM containing 10% MeOH. The resulting solution was dried over sodium sulfate, filtered, and concentrated under reduced pressure to give 5.30 g (87%) of 3-(benzyloxy)oxetane-3-carboxylic acid 15 as a white solid.

[0263] 1 HNMR (400MHz, DMSO-d6) δ13.42 (brs, 1H), 7.46-7.19 (m, 5H), 4.76 (d, J = 7.2Hz, 2H), 4.59 (d, J = 7.1Hz, 2H), 4.47 (s, 2H).

[0264] MS(ESI)m / z: [m-1] - 207.0

[0265] Step 5: 3-(benzyloxy)oxetane-3-carboxylic acid tert-butyl ester (16)

[0266]

[0267] At 0 °C, tert-butyl N,N'-diisopropylcarbamate (15 mL, 57.6 mmol) was slowly added to a mixture of 5-(benzyloxy)oxetane-3-carboxylic acid 15 (5.30 g, 25.46 mmol) and 80 mL of anhydrous THF. The reaction mixture was stirred at 0 °C for 10 min, then at room temperature for 5 h. The reaction mixture was concentrated under reduced pressure, and the residue was treated with hexane containing 25% DCM and filtered. The filtrate was concentrated under vacuum, and the crude mixture was purified by silica gel column chromatography using a gradient of hexane containing 0–8% ethyl acetate to yield tert-butyl 3-(benzyloxy)oxetane-3-carboxylic acid 16 (5.65 g, 84%) as a white solid.

[0268] 1 HNMR (500MHz, CDCl3) δ7.45-7.30(m,5H),4.92-4.87(m,2H),4.76-4.71(m,2H),4.51(s,2H),1.58(s,9H).

[0269] Step 6: 3-Hydroxybutane-3-carboxylic acid tert-butyl ester (17)

[0270]

[0271] Pd(OH)₂ (20 wt%, 1.30 g) was added to a mixed solution of ethyl acetate and MeOH (1:1, 120 mL) containing 5.65 g (21.38 mmol) of 3-(benzyloxy)oxetane-3-carboxylic acid tert-butyl ester 16. The reaction mixture was purged three times with hydrogen, then shaken at room temperature under hydrogen pressure (50 psi) for 5 hours and filtered through a diatomaceous earth mat. The filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography using a gradient of hexane containing 10–30% ethyl acetate to give 3-hydroxyoxetane-3-carboxylic acid tert-butyl ester 17 (3.31 g, 89%) as a white solid.

[0272] 1 HNMR (500MHz, CDCl3) δ4.88 (d, J = 6.9 Hz, 2H), 4.75 (d, J = 7.3 Hz, 2H), 3.75 (s, 1H), 1.60 (s, 9H).

[0273] Step 7: 3-(aminooxy)oxetane-3-carboxylic acid tert-butyl ester (18)

[0274]

[0275] Sodium tert-butoxide (2.19 g, 22.79 mmol) was added to 90 mL of anhydrous THF containing 3-hydroxyoxetane-3-carboxylic acid tert-butyl ester 17 (3.31 g, 19.0 mmol) and O-diphenylphosphinohydroxylamine (5.32 g, 22.81 mmol) at 0–5 °C. The reaction mixture was stirred at 0–5 °C for 2 hours, then brine (50 mL) and hexane (30 mL) were added, and the resulting mixture was stirred at 15–25 °C for 30 minutes. The precipitated solid was removed by filtration and washed with hexane containing 30% ethyl acetate. The two layers of the two-phase filtrate were separated, and the aqueous layer was further extracted with ethyl acetate. The combined organic extracts were washed with brine, dried over sodium sulfate, filtered, and concentrated under reduced pressure. The residue was treated with hexane, the resulting mixture was filtered, and the filtrate was concentrated under vacuum to give 3-(aminooxy)oxetane-3-carboxylic acid tert-butyl ester 18 (3.41 g, 95%) as a yellow solid.

[0276] 1 HNMR (500MHz, CDCl3) δ4.88 (dd, J = 7.2, 0.8Hz, 2H), 4.76-4.64 (m, 2H), 1.62-1.55 (m, 9H).

[0277] Step 8: (2z)-{2-[(tert-butoxycarbonyl)amino]-1,3-thiazolyl-4-yl}({3-(tert-butoxycarbonyl)oxetane-3-yl]oxy}imino)acetic acid (19)

[0278]

[0279] To a MeOH (50 mL) solution containing tert-butyl 3-(aminooxy)oxetane-3-carboxylic acid ester 18 (3.41 g, 18.02 mmol), {2-[(tert-butyloxycarbonyl)amino]-1,3-thiazol-4-yl}(oxo)acetic acid was added, and the reaction mixture was stirred at room temperature for 4 hours. Water (100 mL) and an aqueous hydrochloric acid solution (0.1 M, 100 mL) were then added, and the mixture was extracted with ethyl acetate. The combined organic extracts were washed with brine, dried over sodium sulfate, filtered, and concentrated under reduced pressure. The residue was treated with hexane containing 5% diethyl ether, and the resulting suspension was stirred for 1 hour and then filtered to give (2z)-{2-[(tert-butyloxycarbonyl)amino]-1,3-thiazol-4-yl}({3-(tert-butyloxycarbonyl)oxetane-3-yl]oxy}imino)acetic acid 19 (6.96 g, 96%) as a white solid.

[0280] 1 HNMR (400MHz, DMSO-d6) δ11.81(s,1H),7.43(s,1H),4.95-4.78(m,2H),4.76-4.54(m,2H),1.60-1.43(m,9H),1.41(s,9H).

[0281] MS(ESI)m / z: [M+1] + 443.8

[0282] Step 9: 3-{[(Z)-(1-{2-[(tert-butyloxycarbonyl)amino]-1,3-thiazolyl-4-yl}-2-[(2,5-dioxopyrrolidone-1-yl)oxy]-2-oxoethylidene)amino]oxy}oxetane-3-carboxylic acid tert-butyl ester (20)

[0283]

[0284] DIC (2.82 mL, 18.21 mmol) was slowly added to an anhydrous DCM (80 mL) solution containing (2z)-{2-[(tert-butoxycarbonyl)amino]-1,3-thiazolyl}({3-(tert-butoxycarbonyl)oxetane-3-yl]oxy}imino)acetic acid 19 (6.96 g, 15.69 mmol) and NHS (2.17 g, 18.85 mmol). The reaction mixture was stirred at 0 °C for 15 min, then at room temperature for 3 h. The suspension was filtered and the solids were washed with DCM. The filtrate was concentrated under reduced pressure, and the residue was treated with a mixture of methanol (20 mL) and n-heptane (20 mL). The resulting mixture was stirred at room temperature for 30 minutes, then stirred at about 10°C for 30 minutes, and then filtered to obtain 20 (7.69 g, 91%) of 3-{[(Z)-(1-{2-[(tert-butyloxycarbonyl)amino]-1,3-thiazolyl-4-yl}-2-[(2,5-dioxopyrrolidine-1-yl)oxy]-2-oxoethylidene)amino]oxy}oxetane-3-carboxylic acid tert-butyl ester, a white solid.

[0285] 1 HNMR (400MHz, CDCl3) δ8.51(brs,1H),7.61(s,1H),5.09-5.03(m,2H),4.94-4.88(m,2H),3.05-2.77(m,4H),1.65-1.53(m,9H),1.53-1.47(m,9H).

[0286] MS(ESI)m / z: [M+1] + 541.1

[0287] Step 10: 3-{[(Z)-(1-{2-[(tert-butyloxycarbonyl)amino]-1,3-thiazolyl}-2-oxo-2-{[(4S)-3-oxo-1,2-oxazolidine-4-yl]amino}ethylene)amino]oxy}oxetane-3-carboxylic acid tert-butyl ester (21)

[0288]

[0289] At room temperature, DIPEA (2.97 mL, 17.05 mmol) was added to anhydrous DMF (80 mL) containing a mixture of 20 tert-butyl 3-carboxylic acid ester (7.69 g, 14.23 mmol) and L-cycloserine (1.74 g, 17.05 mmol). The reaction mixture was stirred at 45 °C for 18 hours, then cooled to room temperature and concentrated under reduced pressure until dry. The crude mixture was purified by silica gel column chromatography using a gradient DCM of 0–2.5% methanol. The fractions of the product were combined and concentrated under vacuum, and the residue was further purified by grinding with diethyl ether and hexane to give 21 (2.37 g, 32%) of 3-{[(Z)-(1-{2-[(tert-butoxycarbonyl)amino]-1,3-thiazolyl}-2-oxo-2-{[(4S)-3-oxo-1,2-oxazolidine-4-yl]amino}ethylene)amino]oxy}oxetane-3-carboxylic acid tert-butyl ester, a white solid.

[0290] 1 HNMR(400MHzDMSO-d6)δ11.85(s,1H),11.63(s,1H),9.26(d,J=7.8Hz,1H),7.49(s,1H),4.95(brs,1H) ,4.81(d,J=7.3Hz,2H),4.62(dd,J=12.3,7.4Hz,3H),4.10(t,J=9.1Hz,1H),1.45(s,9H),1.43(s,9H).

[0291] MS(ESI)m / z: [M+1] + 528.0

[0292] Step 11: (4S)-2-[(4S)-4-{[(2Z)-2-{2-[(tert-butoxycarbonyl)amino]-1,3-thiazolyl-4-yl}-2-({[3-(tert-butoxycarbonyl)oxetane-3-yl]oxy}imino)acetyl]amino}-3-oxo-1,2-oxazolidine-2-yl]-4-(5,7-dioxo-2,2-diphenyl-5,7-dihydro-2H,6H-[1,3]dioxetane[4,5-f]isoindol-6-yl)-5-oxooxopentane-2-carboxylic acid tert-butyl ester (22)

[0293]

[0294] At 0 °C, tert-butyl ester 21 (444 mg, 0.84 mmol) containing 3-{[(Z)-(1-{2-[(tert-butoxycarbonyl)amino]-1,3-thiazolyl}-2-oxo-2-{[(4S)-3-oxo-1,2-oxazolidine-4-yl]amino}ethylene)amino]oxy}oxetane-3-carboxylic acid tert-butyl ester 21 (444 mg, 0.84 mmol) and (2S)-5-tert-butoxy-2-(5,7-dioxo-2,2-diphenyl-5,7-dioxane ... A mixture of dihydro-2H,6H-[1,3]dioxane-[4,5-f]isoindol-6-yl)-4,5-dioxovaleric acid 6 (457 mg, 0.84 mmol, prepared as described in J. Med. Chem., 2014, Vol. 57, pp. 3845-3855) was added to anhydrous THF with DMAP (21 mg, 0.17 mmol), followed by DCC (243 mg, 1.18 mmol). The reaction mixture was allowed to be slowly heated to room temperature and stirred for 18 hours. The mixture was then concentrated under reduced pressure, and the residue was treated with hexane containing 30% DCM. The solids were removed by filtration, and the filtrate was concentrated. The residue was purified by silica gel column chromatography using a gradient of hexane containing 10-40% ethyl acetate to obtain (4S)-2-[(4S)-4-{[(2Z)-2-{2-[(tert-butoxycarbonyl)amino]-1,3-thiazolyl-4-yl}-2-({[3-(tert-butoxycarbonyl)oxetane-3-yl]oxy}imino)acetyl]amino}-3-oxo-1,2-oxazolidine-2-yl]-4-(5,7-dioxo-2,2-diphenyl-5,7-dihydro-2H,6H-[1,3]dioxetane[4,5-f]isoindol-6-yl)-5-oxooxopentane-2-carboxylic acid tert-butyl ester 22 (550 mg, 62%), as a white foam.

[0295] 1 HNMR(400MHz, CDCl3)δ8.46(brs,1H),8.13-7.91(m,1H),7.63-7.52(m,4H),7.47-7.38(m,7H),7.33(d,J=1.6Hz, 2H),5.38(m,1H),5.25-4.86(m,6H),4.39-4.20(m,1H),3.75-3.24(m,1H),2.93-2.72(m,1H),1.62-1.49(m,27H).

[0296] MS(ESI)m / z: [m+Na] + 1075.4

[0297] Step 12: (4S)-2-[(4S)-4-{[(2Z)-2-(2-amino-1,3-thiazolyl-4-yl)-2-{[(3-carboxyoxobutane-3-yl)oxy]imino}acetyl]amino}-3-oxo-1,2-oxazolidine-2-yl]-4-(5,6-dihydroxy-1,3-dioxo-2,3-dihydro-2H-isoindol-2-yl)-5-oxooxopentane-2-carboxylic acid (compound 5, Table 1) and (4S)-2-[(4S)-4-{[(2Z)-2-(2-amino-1,3-thiazolyl-4-yl)-2-{[(2-carboxy-1-chloro-3-hydroxypropane-2-yl)oxy]imino}acetyl]amino}-3-oxo-1,2-oxazolidine-2-yl]-4-(5,6-dihydroxy-1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)-5-oxooxopentane-2-carboxylic acid (Compound 6, Table 1)

[0298]

[0299] Add BCl3 solution (in 1.0 M in DCM, 1.14 mL, 1.14 mmol) dropwise to an anhydrous DCM solution (7 mL) containing (4S)-2-[(4S)-4-{[(2Z)-2-{2-[(tert-butyloxycarbonyl)amino]-1,3-thiazolyl}-2-({[3-(tert-butyloxycarbonyl)oxetane-3-yl]oxy}imino)acetyl]amino}-3-oxo-1,2-oxazolidine-2-yl]-4-(5,7-dioxo-2,2-diphenyl-5,7-dihydro-2H,6H-[1,3]dioxetane[4,5-f]isoindol-6-yl)-5-oxooxopentane-2-carboxylic acid tert-butyl ester 22 (150 mg, 0.14 mmol). The reaction mixture was stirred at -78°C for 2.5 hours, and then an aqueous solution (12.6 mL) containing NaHCO3 (233 mg) and Na2HPO4 (73 mg) was added at -78°C. The resulting heterogeneous mixture was stirred at 0–5°C using an ice-water bath instead of a cold bath until the aqueous phase thawed (approximately 30 minutes), and the phases were carefully separated. The aqueous layer was filtered using a 1.0 μm pinhole filter and immediately subjected to C18 reversed-phase column chromatography using a Biotage system with a gradient of acetonitrile containing 0–30% 0.1% formic acid and an aqueous solution containing 0.1% formic acid. The fractions containing the purified products were lyophilized to give compound 5 (8.5 mg, 9%) and compound 6 (38 mg, 19%) as off-white solids.

[0300] For compound 5:

[0301] 1¹H NMR (400 MHz, mixture of D₂O and CD₃CN) δ 10.05 (s, 2H), 10.01–9.95 (m, 1H), 8.23–8.05 (m, 1H), 8.02–7.88 (m, 1H), 7.80 (d, J = 7.8 Hz, 2H), 7.65–7.49 (m, 3H), 7.14–7.05 (m, 1H), 6.38–6.10 (m, 1H), 5.76–5.52 (m, 1H). No interchangeable protons were observed in D₂O.

[0302] MS(ESI)m / z: [M+1] + 677.1

[0303] For compound 6:

[0304] 1 ¹H NMR (400 MHz, mixture of D₂O and CD₃CN) δ 10.05 (s, 2H), 9.98 (s, 1H), 8.28–8.03 (m, 1H), 8.03–7.83 (m, 1H), 7.60–7.46 (m, 1H), 7.14–7.07 (m, 1H), 6.89–6.72 (m, 5H), 6.38–6.07 (m, 1H), 5.75–5.49 (m, 1H). No interchangeable protons were observed in D₂O.

[0305] MS(ESI)m / z: [M+1] + 713.0

[0306] Example 4

[0307] 3-({(Z)-[1-(2-amino-1,3-thiazolyl-4-yl)-2-({(4S)-2-[(4S)-2-carboxy-4-(5,6-dihydroxy-1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)-5-oxooxopentane-2-yl]-3-oxo-1,2-oxazolidine-4-yl}amino)-2-oxoethylidene]amino}oxy)azacyclobutane-3-carboxylic acid (Compound 7, Table 1)

[0308]

[0309] Step 1: 3-Hydroxyazine-1,3-dicarboxylic acid ditert-butyl ester (24)

[0310]

[0311] To a solution of 100 mL of anhydrous THF containing 1-(tert-butoxycarbonyl)-3-hydroxyazacyclobutane-3-carboxylic acid 23 (4.33 g, 19.93 mmol, prepared as described in WO2013 / 96771A1), N,N′-diisopropylcarbamate tert-butyl ester (7.4 mL, about 39.86 mmol) was added, and the reaction mixture was stirred at room temperature for 14 hours. The precipitated solid was removed by filtration and washed with THF. The filtrate was concentrated under reduced pressure, and the residue was treated with a mixture of DCM and hexane (1:1, 100 mL). The resulting suspension was stirred at about 5 °C for 10 minutes, the solid was filtered off, and washed with a mixture of DCM and hexane (1:1, 20 mL). The filtrate was collected and concentrated under vacuum. The residue was purified by silica gel column chromatography using a gradient of hexane containing 10-20% ethyl acetate to give 24 di-tert-butyl 3-hydroxyazacyclobutane-1,3-dicarboxylic acid, as a grayish-white solid (4.35 g, 80% yield).

[0312] 1 HNMR (400MHz, CDCl3): δ4.21 (d, J = 13.7Hz, 2H), 3.97 (d, J = 13.7Hz, 2H), 1.53 (s, 9H), 1.44 (s, 9H).

[0313] Step 2: 3-(aminooxy)azacyclobutane-1,3-dicarboxylic acid ditert-butyl ester (25)

[0314]

[0315] O-diphenylphosphonohydroxylamine (4.43 g, 19.0 mmol) was added to an anhydrous THF (100 mL) solution containing 4.33 g (15.84 mmol) of di-tert-butyl 3-hydroxyazacyclobutane-1,3-dicarboxylic acid 24. The resulting heterogeneous mixture was cooled to 0 °C, and sodium tert-butoxide (1.83 g, 19.0 mmol) was added. The reaction mixture was stirred at 0 °C for 2 h, and then 200 mL of 5% aqueous NaCl solution was added. The mixture was stirred at 10–15 °C for 30 min. Ethyl acetate (200 mL) was then added, and the mixture was stirred for 15 min. The organic phase was separated, and the aqueous layer was further extracted with 100 mL of ethyl acetate. The combined organic extracts were washed with brine, dried over sodium sulfate, filtered, and concentrated under reduced pressure to give 25 of 3-(aminooxy)azacyclobutane-1,3-dicarboxylic acid di-tert-butyl 25 as a deep yellow oil (4.08 g, 89% yield).

[0316] 1HNMR (400MHz, CDCl3): δ4.15 (d, J = 9.5 Hz, 2H), 3.94 (d, J = 9.54 Hz, 2H), 1.52 (s, 9H), 1.45 (s, 9H).

[0317] Step 3: (2z)-({[1,3-bis(tert-butoxycarbonyl)azacyclobutane-3-yl]oxy}imino){2-[(tert-butoxycarbonyl)amino]-1,3-thiazolyl-4-yl}acetic acid (26)

[0318]

[0319] To a solution of di-tert-butyl 3-(aminooxy)azacyclobutane-1,3-dicarboxylic acid 25 (3.97 g, 13.77 mmol) in anhydrous methanol (40 mL), 2-(2-(tert-butoxycarbonylamino)thiazol-4-yl)-2-oxoacetic acid (3.41 g, 12.51 mmol) was added, and the reaction mixture was stirred at room temperature for 3 hours. Most of the methanol was removed under reduced pressure, and the residue was dissolved in 200 mL of ethyl acetate. The organic phase was washed with diluted hydrochloric acid solution (100 mL of water and 13 mL of 1M hydrochloric acid), dried over sodium sulfate (100 mL), filtered, and concentrated under vacuum to give (2z)-({[1,3-bis(tert-butoxycarbonyl)azacyclobutane-3-yl]oxy}imino){2-[(tert-butoxycarbonyl)amino]-1,3-thiazol-4-yl}acetic acid 26, as a grayish-white foamy solid (7.19 g, crude product).

[0320] 1 HNMR (400MHz, DMSO-d6): δ7.45 (s, 1H), 4.21 (d, J = 9.6Hz, 2H), 3.90 (d, J = 9.6Hz, 2H), 1.45 (s, 9H), 1.41 (s, 9H), 1.38 (s, 9H).

[0321] Step 4: 3-{[(Z)-(1-{2-[(tert-butoxycarbonyl)amino]-1,3-thiazolyl-4-yl}-2-[(2,5-dioxopyrrolidone-1-yl)oxy]-2-oxoethylidene)amino]oxy}azacyclobutane-1,3-dicarboxylic acid ditert-butyl ester (27)

[0322]

[0323] A solution of anhydrous DCM (140 mL) containing (2z)-({[1,3-bis(tert-butoxycarbonyl)azacyclobutane-3-yl]oxy}imino){2-[(tert-butoxycarbonyl)amino]-1,3-thiazolyl-4-yl}acetic acid 26 (7.19 g crude product, approx. 13.25 mmol) and NHS (1.83 g, 15.90 mmol) was cooled to 0 °C. N,N'-diisopropylcarbodiimide (2.00 g, 15.90 mmol) was added dropwise, and the reaction mixture was stirred at 0 °C for 15 min, then at room temperature for 3 h. The precipitated solid was filtered off, and the filtrate was concentrated under reduced pressure. The residue was dissolved in 20 mL of methanol, and the mixture was concentrated to approximately 5 mL. 20 mL of n-heptane was added. The mixture was stirred at room temperature for 30 min, then cooled to approximately 10 °C and stirred for another 15 min. The precipitate was collected by filtration, washed with heptane, and dried under vacuum to give 27 di-tert-butyl 3-{[(Z)-(1-{2-[(tert-butoxycarbonyl)amino]-1,3-thiazolyl-4-yl}-2-[(2,5-dioxopyrrolidine-1-yl)oxy]-2-oxoethylidene)amino]oxy}azacyclobutane-1,3-dicarboxylic acid, as a grayish-white solid (8.41 g, 99% yield).

[0324] 1 HNMR (400MHz, CDCl3): δ8.24(brs,1H),7.57(s,1H),4.36(d,J=9.8Hz,2H),4.20(d,J=9.8Hz,2H),2.91(s,4H),1.53(s,9H),1.45(s,9H),1.44(s,9H).

[0325] Step 5: 3-{[(Z)-(1-{2-[(tert-butoxycarbonyl)amino]-1,3-thiazolyl}-2-oxo-2-{[(4S)-3-oxo-1,2-oxazolidine-4-yl]amino}ethylene)amino]oxy}azacyclobutane-1,3-dicarboxylic acid ditert-butyl ester (28)

[0326]

[0327] At room temperature, DIPEA (2.7 mL, 15.7 mmol) was added to 100 mL of anhydrous DMF containing a mixture of 3-{[(Z)-(1-{2-[(tert-butoxycarbonyl)amino]-1,3-thiazo-4-yl}-2-[(2,5-dioxopyrrolidone-1-yl)oxy]-2-oxoethylidene)amino]oxy}azacyclobutane-1,3-dicarboxylic acid ditert-butyl ester 27 (8.4 g, 13.1 mmol) and L-cycloserine (1.60 g, 15.7 mmol). The heterogeneous reaction mixture was stirred at 45 °C for 18 hours under nitrogen, then cooled to room temperature and concentrated under reduced pressure for drying. The residue was purified by silica gel column chromatography using 30% acetonitrile, followed by a 30% acetonitrile-DCM mixture containing 2% MeOH as the eluent, to give 3-{[(Z)-(1-{2-[(tert-butoxycarbonyl)amino]-1,3-thiazo-4-yl}-2-oxo-2-{[(4S)-3-oxo-1,2-oxazolidine-4-yl]amino}ethylene)amino]oxy}azacyclobutane-1,3-dicarboxylic acid ditert-butyl ester 28, as a white solid (3.1 g, 37% yield).

[0328] 1 HNMR (400MHz, DMSO-d6): δ9.23(d,J=8.0Hz,1H),7.46(s,1H),5.45(d,J=7.4Hz,1H),4.92(brs,1H ),4.63-4.60(m,1H),4.21-4.17(m,2H),4.01-3.85(m,2H),1.45(s,9H),1.42(s,9H),1.38(s,9H).

[0329] MS(ESI)m / z: [M+1] + 627.0

[0330] Step 6: 3-({(Z)-[1-{2-[(tert-butoxycarbonyl)amino]-1,3-thiazolyl-4-yl}-2-({(4S)-2-[(4S)-2-(tert-butoxycarbonyl)-4-(5,7-dioxo-2,2-diphenyl-5,7-dihydro-2H,6H-[1,3]dioxacyclo[4,5-f]isoindol-6-yl)-5-oxooxopentane-2-yl]-3-oxo-1,2-oxazolidine-4-yl}amino)-2-oxoethylidene]amino}oxy)azacyclobutane-1,3-dicarboxylic acid ditert-butyl ester (29)

[0331]

[0332] Anhydrous THF (30 mL) containing a mixture of (2S)-5-tert-butoxy-2-(5,7-dioxo-2,2-diphenyl-5,7-dihydro2H,6H-[1,3]dioxo-[4,5-f]isoindol-6-yl)-4,5-dioxovaleric acid 6 (1.09 g, 2.0 mmol, prepared as described in J. Med. Chem., 2014, Vol. 57, pp. 3845-3855) and 3-{(Z)-(1-{2-[(tert-butoxycarbonyl)amino]-1,3-thiazo-4-yl}-2-oxo-2-{[(4S)-3-oxo-1,2-oxazolidine-4-yl]amino}ethylene)amino]oxy}azacyclobutane-1,3-dicarboxylic acid ditert-butyl ester 28 was cooled to 0 °C. DMAP (50 mg, 0.40 mmol) was added, followed by DCC (0.58 g, 2.80 mmol). The reaction mixture was stirred at 5–10 °C for 1 hour, then allowed to stand overnight at room temperature. The mixture was concentrated under reduced pressure at 25 °C, and the residue was extracted with hexane (25 mL) containing 40% DCM. The precipitate was filtered off, washed with hexane (25 mL) containing 30% DCM, and then washed with hexane. The filtrate was concentrated under vacuum, and the crude product was purified by silica gel column chromatography using a gradient of hexane containing 10-35% ethyl acetate to give 29 tert-butyl 3-({(Z)-[1-{2-[(tert-butoxycarbonyl)amino]-1,3-thiazolyl-4-yl}-2-({(4S)-2-[(4S)-2-(tert-butoxycarbonyl)-4-(5,7-dioxo-2,2-diphenyl-5,7-dihydro-2H,6H-[1,3]dioxono[4,5-f]isoindol-6-yl)-5-oxooxopentane-2-yl]-3-oxo-1,2-oxazolidine-4-yl}amino)-2-oxoethylidene]amino}oxy)azacyclobutane-1,3-dicarboxylic acid ditert-butyl ester, a grayish-white foamy solid (1.44 g, 62% yield).

[0333] 1 H NMR (400MHz, CDCl3): δ8.03(d,J=6.0Hz,1H),7.58-7.53(m,4H),7.47-7.40(m,6H),7.35-7.31(m,2H),5.42-5.34(m,1H),5.05-4.85(m,1H),4.4 2-4.19(m,5H),3.55-3.25(m,1H),2.93-2.81(m,1H),2.00-1.91(m,1H) ,1.77-1.68(m,1H),1.59(m,9H),1.56(m,9H),1.53(m,9H),1.48(m,9H).

[0334] Step 7: 3-({(Z)-[1-(2-amino-1,3-thiazolyl-4-yl)-2-({(4S)-2-[(4S)-2-carboxy-4-(5,6-dihydroxy-1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)-5-oxooxopentane-2-yl]-3-oxo-1,2-oxazolidine-4-yl}amino)-2-oxoethylidene]amino}oxy)azacyclobutane-3-carboxylic acid (Compound 7, Table 1)

[0335]

[0336] At -50°C, boron trichloride solution (1.0 M DCM solution, 1.7 mL, 1.7 mmol) was added dropwise to an anhydrous DCM solution containing 29 (240 mg, 0.21 mmol) of ditert-butyl 3-({(Z)-[1-{2-[(tert-butyloxycarbonyl)amino]-1,3-thiazolyl}-2-({(4S)-2-[(4S)-2-(tert-butyloxycarbonyl)-4-(5,7-dioxo-2,2-diphenyl-5,7-dihydro-2H,6H-[1,3]dioxacyclo[4,5-f]isoindol-6-yl)-5-oxooxopentane-2-yl]-3-oxo-1,2-oxazolidine-4-yl}amino)-2-oxoethylidene]amino}oxy)azacyclobutane-1,3-dicarboxylic acid ditert-butyl ester 29 (240 mg, 0.21 mmol). The reaction mixture was stirred at -50 to -35 °C for 2.5 h, then cooled to -50 °C and quenched by adding 18.3 mL of buffer solution (prepared by dissolving 776 mg NaHCO3 and 243 mg Na2HPO4 in 42 mL of water). The resulting heterogeneous mixture was stirred at 0 to 5 °C using an ice-water bath instead of a cold bath until the aqueous phase thawed (approximately 30 minutes), and the phases were carefully separated. The aqueous layer was filtered using a 1.0 μm pinhole filter and immediately subjected to C18 reversed-phase column chromatography using a Biotage system with a gradient of acetonitrile containing 0–30% 0.1% formic acid and water containing 0.1% formic acid. The fraction containing the purified product was lyophilized to give compound 7 (40.5 mg, 29%) as a pale yellow, foamy solid.

[0337] 1 A mixture of ¹H NMR (400 MHz, D₂O and CD₃CN) values: δ 7.53 (s, 2H), 7.40 (s, 1H), 5.73–5.58 (m, 1H), 5.51–5.37 (m, 1H), 5.02 (m, 1H), 4.84–4.73 (m, 3H), 4.55–4.45 (m, 2H), 3.68–3.53 (m, 1H), 3.07–2.94 (m, 1H). No interchangeable protons were observed in D₂O.

[0338] MS(ESI)m / z: [M+1] + 676.1

[0339] Example 5

[0340] 4-({(Z)-[1-(2-amino-1,3-thiazolyl-4-yl)-2-({(4S)-2-[(4S)-2-carboxy-4-(5,6-dihydroxy-1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)-5-oxooxopentane-2-yl]-3-oxo-1,2-oxazolidine-4-yl}amino)-2-oxoethylidene]amino}oxy)oxohexane-4-carboxylic acid (Compound 8, Table 1)

[0341]

[0342] Step 1: 4-[(trimethylsilyl)oxy]oxohexyl-4-nitrile (31)

[0343]

[0344] Tetrahydropyran-4-one 30 (10.0 mL, 108.75 mmol) was added to LiClO4 (11.57 g, 108.75 mmol), followed by TMSCN (17.20 mL, 137.48 mmol), and the mixture was stirred at room temperature for 3.5 hours. The reaction mixture was diluted with DCM and filtered. The filtrate was washed with water, dried over sodium sulfate, filtered, and concentrated under reduced pressure. Crude 4-[(trimethylsilyl)oxy]oxohexylcyclo-4-onitrile 31 (20.69 g, 96%) was given as a colorless oil and used in the next step without further purification.

[0345] 1 H NMR (400MHz, CDCl3) δ3.95-3.85(m,2H), 3.66-3.61(m,2H), 2.13-2.02(m,2H), 1.90-1.81(m,2H), 0.48-0.08(m,9H).

[0346] MS(ESI)m / z: [M+1] + 200.1

[0347] Step 2: 4-Hydroxyhexyl-4-carboxylic acid (32)

[0348]

[0349] A stirred mixture of 4-[(trimethylsilyl)oxy]oxohexylcyclo-4-nitrile 31 (20.69 g, 103.80 mmol) and ice-cold AcOH (45 mL) was cooled to 0 °C. Concentrated HCl (37%, 45 mL) was added dropwise at 0 °C, the cold bath was removed, and the resulting mixture was heated at 90 °C for 4 hours. The reaction mixture was then cooled to room temperature and concentrated under reduced pressure. The residue was diluted with water (80 mL), and the aqueous phase was extracted with ethyl acetate (4 × 120 mL). The aqueous layer was then saturated with solid NaCl and further extracted with ethyl acetate (4 × 120 mL). The combined organic extracts were dried over sodium sulfate, filtered, and concentrated under vacuum. Trace amounts of solvent were removed by co-evaporation with toluene to give 4-hydroxyoxohexylcyclo-4-carboxylic acid 32 (15.05 g, 99%) as a light brown solid.

[0350] 1 H NMR (400MHz, DMSO-d6) δ5.26(br s,1H),3.68-3.56(m,4H),1.96-1.79(m,2H),1.55-1.42(m,2H).

[0351] MS(ESI)m / z: [m-1] - 145.0

[0352] Step 3: 4-Hydroxyhexyl-4-carboxylic acid tert-butyl ester (33)

[0353]

[0354] N,N'-diisopropylcarbamate tert-butyl ester (32 mL, 122.9 mmol) was slowly added to anhydrous THF (150 mL) containing 32 (8.08 g, 55.29 mmol) of 4-hydroxyoxohexane-4-carboxylic acid at 0 °C. The reaction mixture was stirred at 0 °C for 10 min, then the cold bath was removed, and stirring was continued at room temperature for 5 h. The residue was treated with hexane containing 30% DCM, and urea byproducts were filtered off. The filtrate was concentrated under vacuum, and the residue was purified by silica gel column chromatography using a gradient of hexane containing 0-20% ethyl acetate to give 33 (9.42 g, 84%) of 4-hydroxyoxohexane-4-carboxylic acid as a white solid.

[0355] 1 HNMR (400MHz, CDCl3) δ3.88-3.74(m,4H),3.16(s,1H),2.16-2.03(m,2H),1.53-1.43(m,11H).

[0356] MS(ESI)m / z: [m+Na] + 225.1

[0357] Step 4: 4-(aminooxy)oxohexyl-4-carboxylic acid tert-butyl ester (34)

[0358]

[0359] Sodium tert-butoxide (3.56 g, 37.08 mmol) was added to 120 mL of anhydrous THF containing a mixture of 4-hydroxyoxohexylcyclo-4-carboxylic acid tert-butyl ester 33 (5.00 g, 24.72 mmol) and O-diphenylphosphinohydroxylamine (8.65 g, 37.09 mmol). The reaction mixture was stirred at 0–10 °C for 6 hours. Brine (65 mL) was added, followed by hexane (40 mL), and the resulting mixture was stirred at 15–25 °C for 30 minutes. The two layers were separated, and the aqueous phase was extracted with ethyl acetate. The combined organic extracts were washed with brine, dried over sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography using a gradient of hexane containing 0–40% ethyl acetate to give 4-(aminooxy)oxohexylcyclo-4-carboxylic acid tert-butyl ester 34 (4.14 g, 77%) as a grayish-white solid.

[0360] 1 H NMR (400MHz, CDCl3) δ5.57(br s,2H),3.79-3.62(m,4H),2.10-1.97(m,2H),1.94-1.83(m,2H),1.49(s,9H).

[0361] Step 5: (2Z)-{2-[(tert-butoxycarbonyl)amino]-1,3-thiazolyl-4-yl}({4-(tert-butoxycarbonyl)oxohexylcyclo-4-yl]oxy}imino)acetic acid (35)

[0362]

[0363] 2-(2-(tert-Butoxycarbonylamino)thiazol-4-yl)-2-oxoacetic acid (4.70 g, 17.26 mmol) was added to a MeOH solution (50 mL) containing 4-(aminooxy)oxohexylcyclo-4-carboxylic acid tert-butyl ester 34 (4.12 g, 18.96 mmol) at room temperature. The reaction mixture was stirred at room temperature for 4 hours, and then water (75 mL) and an aqueous hydrochloric acid solution (0.1 M, 80 mL) were added. The resulting mixture was extracted with ethyl acetate. The combined organic extracts were washed with brine, dried over sodium sulfate, filtered, and concentrated under reduced pressure. The residue was ground with hexane containing 5% diethyl ether to give (2Z)-{2-[(tert-Butoxycarbonyl)amino]-1,3-thiazol-4-yl}({4-(tert-Butoxycarbonyl)oxohexylcyclo-4-yl]oxy}imino)acetic acid 35 (7.94 g, 98%) as a white solid.

[0364] 1 H NMR (400MHz, DMSO-d6) δ11.76(s,1H),7.37(s,1H),3.69-3.48(m,4H),2.03-1.89(m,2H),1.83(m,2H),1.45(s,9H),1.39(s,9H).

[0365] MS(ESI)m / z: [M+1] + 472.1

[0366] Step 6: 4-{[(Z)-(1-{2-[(tert-butyloxycarbonyl)amino]-1,3-thiazolyl-4-yl}-2-[(2,5-dioxopyrrolidone-1-yl)oxy]-2-oxoethylidene)amino]oxy}oxohexane-4-carboxylic acid tert-butyl ester (36)

[0367]

[0368] At 0 °C, DIC (3.03 mL, 19.57 mmol) was slowly added to anhydrous DCM (90 mL) of a mixture of (2Z)-{2-[(tert-butoxycarbonyl)amino]-1,3-thiazolyl}({4-(tert-butoxycarbonyl)oxohexylcyclo-4-yl]oxy}imino)acetic acid 35 (7.94 g, 16.84 mmol) and NHS (2.33 g, 20.24 mmol). The reaction mixture was stirred at 0 °C for 15 min, the cold bath was removed, and stirring was continued at room temperature for 3 h. The precipitated solids were removed by filtration and washed with DCM. The filtrate was concentrated under reduced pressure, and the residue was extracted by a mixture of methanol (20 mL) and n-heptane (20 mL). The resulting suspension was stirred at room temperature for 30 min, and then stirred again at about 10 °C for 30 min. After filtration, 36 (8.83 g, 92%) of 4-{[(Z)-(1-{2-[(tert-butyloxycarbonyl)amino]-1,3-thiazolyl-4-yl}-2-[(2,5-dioxopyrrolidine-1-yl)oxy]-2-oxoethylidene)amino]oxy}oxohexane-4-carboxylic acid tert-butyl ester was obtained as a white solid.

[0369] 1 H NMR (400MHz, CDCl3) δ8.07(br s,1H),7.50(s,1H),3.92-3.67(m,4H),3.06-2.77(m,4H),2.29-2.06(m,4H),1.53(s,9H),1.43(s,9H).

[0370] MS(ESI)m / z: [M+1] + 569.0

[0371] Step 7: 4-{[(Z)-(1-{2-[(tert-butyloxycarbonyl)amino]-1,3-thiazolyl}-2-oxo-2-{[(4S)-3-oxo-1,2-oxazolidine-4-yl]amino}ethylene)amino]oxy}oxohexane-4-carboxylic acid tert-butyl ester (37)

[0372]

[0373] At room temperature, DIPEA (3.25 mL, 18.66 mmol) was added to anhydrous DMF containing a mixture of 36 tert-butyl 4-[(Z)-(1-{2-[(tert-butyloxycarbonyl)amino]-1,3-thiazolyl-4-yl}-2-[(2,5-dioxopyrrolidone-1-yl)oxy]-2-oxoethylidene)amino]oxy}oxohexyl-4-carboxylic acid tert-butyl ester 36 (8.83 g, 15.53 mmol) and L-cycloserine (1.91 g, 18.67 mmol). The reaction mixture was stirred at 45 °C for 18 hours and then concentrated under reduced pressure. The residue was purified by silica gel column chromatography using a gradient DCM containing 0-30% acetonitrile, followed by a DCM mixture containing 0-2% methanol and a gradient of 30% acetonitrile, to give 37 (3.16 g, 37%) of 4-{[(Z)-(1-{2-[(tert-butyloxycarbonyl)amino]-1,3-thiazolyl}-2-oxo-2-{[(4S)-3-oxo-1,2-oxazolidine-4-yl]amino}ethylene)amino]oxy}oxohexane-4-carboxylic acid tert-butyl ester, as a white foamy solid.

[0374] 1 HNMR(400MHz,DMSO-d6)δ11.69(s,1H),11.48(s,1H),9.05(d,J=7.8Hz,1H),7.29(s,1H),4.82(br s,1H),4.49(t,J=8.4Hz,1H),3.97(t,J=9.0Hz,1H),3.66-3.38(m,4H),1.91-1.66(m,4H),1.35(s,9H),1.28(s,9H).

[0375] MS(ESI)m / z: [M+1] + 556.0

[0376] Step 8: 4-({(Z)-[1-{2-[(tert-butyloxycarbonyl)amino]-1,3-thiazolyl-4-yl}-2-({(4S)-2-[(4S)-2-(tert-butyloxycarbonyl)-4-(5,7-dioxo-2,2-diphenyl-5,7-dihydro-2H,6H-[1,3]dioxono[4,5-f]isoindol-6-yl)-5-oxooxopentane-2-yl]-3-oxo-1,2-oxazolidine-4-yl}amino)-2-oxoethylidene]amino}oxy)oxohexane-4-carboxylic acid tert-butyl ester (38)

[0377]

[0378] At 0 °C, DCC (289 mg, 1.40 mmol) was added to an anhydrous THF (20 mL) solution containing 37 tert-butyl 4-[(Z)-(1-{2-[(tert-butoxycarbonyl)amino]-1,3-thiazo-4-yl}-2-[(2,5-dioxopyrrolidone-1-yl)oxy]-2-oxoethylidene)amino]oxy}oxohexane-4-carboxylic acid tert-butyl ester 37 (556 mg, 1.00 mmol) and (2S)-5-tert-butoxy-2-(5,7-dioxo-2,2-diphenyl-5,7-dihydro2H,6H-[1,3]dioxo-[4,5-f]isoindol-6-yl)-4,5-dioxovaleric acid 6 (571 mg, 1.05 mmol). The reaction mixture was allowed to be gradually heated to room temperature and stirred for 18 hours. The mixture was then concentrated under reduced pressure, the residue was treated with hexane containing 30% DCM, and then filtered. The filtrate was concentrated under vacuum, and the crude product was purified by silica gel column chromatography using a gradient of hexane containing 10-40% ethyl acetate to give 38 (756 mg, 70%) of 4-({(Z)-[1-{2-[(tert-butyloxycarbonyl)amino]-1,3-thiazolyl-4-yl}-2-({(4S)-2-[(4S)-2-(tert-butyloxycarbonyl)-4-(5,7-dioxo-2,2-diphenyl-5,7-dihydro-2H,6H-[1,3]dioxono[4,5-f]isoindol-6-yl)-5-oxooxopentane-2-yl]-3-oxo-1,2-oxazolidine-4-yl}amino)-2-oxoethylidene]amino}oxy)oxohexane-4-carboxylic acid tert-butyl ester, as a grayish-white foam.

[0379] 1H NMR (400MHz, CDCl3) δ8.23-7.96(m,2H),7.58-7.49(m,4H),7.45-7.36(m,6H),7.35-7.31(m,1H),7.30(s,2H),5.42-5.31(m,1H),5.22-4.83( m,2H),4.38-4.16(m,1H),3.92-3.65(m,4H),3.63-3.24(m,1H),2.91- 2.69(m,1H),2.31-2.07(m,4H),1.63-1.48(m,18H),1.48-1.34(m,9H).

[0380] MS(ESI)m / z: [m+Na] + 1103.4

[0381] Step 9: 4-({(Z)-[1-(2-amino-1,3-thiazolyl-4-yl)-2-({(4S)-2-[(4S)-2-carboxy-4-(5,6-dihydroxy-1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)-5-oxooxopentane-2-yl]-3-oxo-1,2-oxazolidine-4-yl}amino)-2-oxoethylidene]amino}oxy)oxohexane-4-carboxylic acid (Compound 8, Table 1)

[0382]

[0383] At -50°C, boron trichloride solution (1.0 M in DCM, 0.74 mL, 0.74 mmol) was added dropwise to anhydrous DCM containing 38 (100 mg, 0.092 mmol) of 4-({(Z)-[1-{2-[(tert-butyloxycarbonyl)amino]-1,3-thiazolyl}-2-({(4S)-2-[(4S)-2-(tert-butyloxycarbonyl)-4-(5,7-dioxo-2,2-diphenyl-5,7-dihydro-2H,6H-[1,3]dioxane[4,5-f]isoindol-6-yl)-5-oxooxopentane-2-yl]-3-oxo-1,2-oxazolidine-4-yl}amino)-2-oxoethylidene]amino}oxy)oxohexane-4-carboxylic acid tert-butyl ester 38. The reaction mixture was stirred at -50°C to -35°C for 2.5 hours, then cooled to -50°C, and an aqueous solution (8.2 mL) of NaHCO3 (152 mg) and Na2HPO4 (48 mg) was added at -50°C. The resulting heterogeneous mixture was stirred at 0 to 5°C using an ice-water bath instead of a cold bath until the aqueous phase thawed (approximately 20 minutes), and the phases were carefully separated. The aqueous layer was filtered using a 1.0 μm pinhole filter and immediately subjected to C18 reversed-phase column chromatography using a Biotage system with a gradient of acetonitrile containing 0–30% formic acid and water containing 0.1% formic acid. The fraction containing the purified product was lyophilized to give compound 8 (46 mg, 71%) as a grayish-white solid.

[0384] 1 ¹H NMR (400 MHz, mixture of D₂O and CD₃CN) δ 7.25 (s, 2H), 7.20–7.09 (m, 1H), 5.46–5.24 (m, 1H), 5.21–5.08 (m, 1H), 4.75 (m, 1H), 4.35–4.26 (m, 1H), 3.83–3.73 (m, 2H), 3.64–3.26 (m, 3H), 2.88–2.70 (m, 1H), 2.18–1.99 (m, 4H). No interchangeable protons were observed in D₂O.

[0385] MS(ESI)m / z: [M+1] + 705.1

[0386] Example 6

[0387] 4-({(Z)-[1-(2-amino-1,3-thiazolyl-4-yl)-2-({(4S)-2-[(4S)-2-carboxy-4-(5,6-dihydroxy-1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)-5-oxooxopentane-2-yl]-3-oxo-1,2-oxazolidine-4-yl}amino)-2-oxoethylidene]amino}oxy)-1-methylpiperidine-4-carboxylic acid (Compound 9, Table 1)

[0388]

[0389] Step 1: 1-Methyl-4-[(trimethylsilyl)oxy]piperidine-4-nitrile (40)

[0390]

[0391] Trimethylcyanosilane (12.4 mL, 99.0 mmol) was added to a mixture of 1-methylpiperidin-4-one (39) (11.0 mL, 90.0 mmol) and 60 mL of anhydrous THF at room temperature. The reaction mixture was heated at 65 °C under nitrogen for 5 hours, then allowed to cool to room temperature and stirred overnight. Volatile substances were removed under reduced pressure, and the residue was further dried under high vacuum to give 1-methyl-4-[(trimethylsilyl)oxy]piperidin-4-onitrile 40, as a brown oil (20.64 g, quantitative yield), which was used directly for the next step.

[0392] 1 H-NMR (400MHz; CDCl3): δ2.76-2.54(m,2H), 2.41-2.28(m,2H), 2.31(s,3H), 2.10-1.99(m,2H), 1.93-1.79(m,2H), 0.22(s,9H).

[0393] Step 2: 4-Hydroxy-1-methylpiperidine-4-carboxylic acid hydrochloride (41)

[0394]

[0395] A mixture of 1-methyl-4-((trimethylsilyl)oxy)piperidine-4-onitrile 40 (20.64 g, about 90 mmol) and glacial acetic acid (50 mL) was cooled to 0 °C, and concentrated hydrochloric acid solution (50 mL) was added dropwise over 15 minutes. The resulting reaction mixture was stirred at 0 °C for 15 minutes, then at room temperature for 15 minutes. Subsequently, the reaction mixture was heated at 110 °C and stirred for 4 hours. The mixture was allowed to cool to room temperature, volatile substances were removed under reduced pressure, and the residue was further dried under high vacuum. The crude product was ground with ethyl acetate (3 x 75 mL), filtered, and dried under high vacuum to give 4-hydroxy-1-methylpiperidine-4-carboxylic acid hydrochloride (41) as a light brown solid (24.1 g, crude product), which was used for the next step without further purification.

[0396] 1 H-NMR (400MHz; D2O): δ3.39-3.36(m,2H), 3.21-3.14(m,2H), 2.79(s,3H), 2.22-2.14(m,2H), 1.97-1.85(m,2H).

[0397] MS(ESI)m / z: [M+1] + 160.2

[0398] Step 3: 4-Hydroxy-1-methylpiperidine-4-carboxylic acid tert-butyl ester (42)

[0399]

[0400] Triethylamine (12.5 mL, 90 mmol) was added to a mixed suspension of anhydrous THF (200 mL) containing 4-hydroxy-1-methylpiperidine-4-carboxylic acid hydrochloride 41 (12.01 g, crude product, about 45 mmol). The resulting mixture was stirred at room temperature for 15 min, and then tert-butyl N,N′-diisopropylcarbamate (prepared as described in EP2471792A1, 27 mL, about 135 mmol) was added. The reaction mixture was stirred at room temperature for 14 h, and an additional tert-butyl N,N′-diisopropylcarbamate (20 mL) was added, and stirring was continued for 24 h. The precipitated solid was removed by filtration, washed with THF, and the combined filtrates were concentrated under reduced pressure. The residue was extracted with a mixture of DCM and hexane (1:1200 mL), and the resulting suspension was cooled using an ice-water bath for 10 min. The solid was filtered and washed with a mixture of DCM and hexane (1:1, 50 mL). The filtrates were combined and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography using a gradient DCM containing 0-6% methanol, followed by a DCM containing 6% methanol-ammonia solution as the eluent, to give 42 tert-butyl 4-hydroxy-1-methylpiperidine-4-carboxylic acid (5.41 g, 56% yield) as a light brown solid.

[0401] 1 H-NMR (400MHz; CDCl3): δ3.18-3.06(m,1H),2.71-2.69(m,2H),2.37-2.33(m, 5H), 2.10 (td, J=12.9, 4.4Hz, 2H), 1.58 (dd, J=13.6, 2.6Hz, 2H), 1.47 (s, 9H).

[0402] MS(ESI)m / z: [M+1] + 216.2

[0403] Step 4: 4-(aminooxy)-1-methylpiperidine-4-carboxylic acid tert-butyl ester (43)

[0404]

[0405] Sodium tert-butoxide (3.27 g, 34.0 mmol) was added to an anhydrous THF (300 mL) solution containing 42 g (4.89 g, 22.7 mmol) of tert-butyl 4-hydroxy-1-methylpiperidin-4-carboxylic acid. The mixture was stirred at room temperature until a clear solution was obtained. O-Diphenylphosphinohydroxylamine (6.35 g, 27.2 mmol) was added, and the heterogeneous mixture was stirred at room temperature for 4 hours. Then, additional sodium tert-butoxide (1.63 g, 17.0 mmol) and O-Diphenylphosphinohydroxylamine (3.17 g, 13.6 mmol) were added, and the mixture was stirred overnight at room temperature. The addition of sodium tert-butoxide (1.63 g, 17.0 mmol) and O-Diphenylphosphinohydroxylamine (3.17 g, 13.6 mmol) was repeated, and the mixture was stirred at room temperature for 14 hours to complete the reaction. Most of the THF was removed under reduced pressure, and the residue was obtained from DCM (300 mL). Add 20 mL of saturated ammonium chloride aqueous solution, separate the organic phase and concentrate under reduced pressure. Elute the residue with a silica gel pad using DCM containing 6% ammonium methoxide solution (7N) to give 43 (2.32 g, crude product) of 4-(aminooxy)-1-methylpiperidine-4-carboxylic acid tert-butyl ester, a brown liquid.

[0406] 1 H-NMR (400MHz; CDCl3): δ5.28(bs,2H),2.60-2.55(m,2H),2.27(s,3H),2.25-2.16(m,2H),2.02-1.96(m,4H),1.46(s,9H).

[0407] MS(ESI)m / z: [M+1] + 231.2

[0408] Step 5: (2Z)-{2-[(tert-butoxycarbonyl)amino]-1,3-thiazolyl-4-yl}({4-(tert-butoxycarbonyl)-1-methylpiperidin-4-yl]oxy}imino)acetic acid (44)

[0409]

[0410] To anhydrous MeOH (40 mL) solution containing 43 g (2.33 g, 10.14 mmol) of 4-(aminooxy)-1-methylpiperidin-4-carboxylic acid tert-butyl ester 4-, {2-[(tert-butyloxycarbonyl)amino]-1,3-thiazo-4-yl}(oxo)acetic acid (1.38 g, 5.07 mmol), glacial acetic acid (0.3 mL, 5.2 mmol) was added at room temperature. The reaction mixture was stirred at room temperature for 14 hours, and then concentrated under reduced pressure. The crude product was purified by C18 reversed-phase column chromatography using an aqueous solution containing 0.1% formic acid and acetonitrile containing 0.1% formic acid as eluents. The fractions were combined and freeze-dried to give (2Z)-{2-[(tert-butoxycarbonyl)amino]-1,3-thiazolyl-4-yl}({4-(tert-butoxycarbonyl)-1-methylpiperidin-4-yl]oxy}imino)acetic acid 44 (1.27 g, 52%), a white solid.

[0411] 1 H-NMR (400MHz; DMSO-d6): δ11.66(s,1H),7.18(s,1H),3.14-2.91(m,2H),2.67(s,3H),2.13-1.99(m,4H),1.44(s,9H),1.41(s,9H).

[0412] MS(ESI)m / z: [M+1] + 485.6 and [m-1] - 483.4

[0413] Step 6: 4-{[(Z)-(1-{2-[(tert-butyloxycarbonyl)amino]-1,3-thiazolyl}-2-oxo-2-{[(4S)-3-oxo-1,2-oxazolidine-4-yl]amino}ethylene)amino]oxy}-1-methylpiperidine-4-carboxylic acid tert-butyl ester (45)

[0414]

[0415] N,N-diisopropylethylamine (0.39 mL, 2.26 mmol) was added to a suspension of (2Z)-{2-[(tert-butyloxycarbonyl)amino]-1,3-thiazolyl-4-yl}({4-(tert-butyloxycarbonyl)-1-methylpiperidin-4-yl]oxy}imino)acetic acid 44 (0.73 g, 1.51 mmol), and the mixture was stirred at room temperature for 10 min. Then HATU (0.57 g, 1.51 mmol) was added, and the mixture was stirred at room temperature for 14 h. Subsequently, anhydrous DMF (15 mL) and N,N-diisopropylethylamine (1.05 mL, 6.02 mmol) were added, and the mixture was stirred at room temperature for 10 min before adding L-cycloserine (0.23 g, 2.26 mmol). The reaction mixture was stirred at room temperature for 1 h, and then concentrated under reduced pressure. The crude mixture was purified by C18 reversed-phase column chromatography using an aqueous solution containing 0.1% formic acid and acetonitrile containing 0.1% formic acid as eluents. The fractions were combined and lyophilized to give 45 (702 mg, 76%) of 4-{[(Z)-(1-{2-[(tert-butyloxycarbonyl)amino]-1,3-thiazolyl}-2-oxo-2-{[(4S)-3-oxo-1,2-oxazolidine-4-yl]amino}ethylene)amino]oxy}-1-methylpiperidine-4-carboxylic acid tert-butyl ester as a white solid.

[0416] 1 H-NMR (400MHz; DMSO-d6): δ11.77-11.66(m,2H),9.28(d,J=8.0Hz,1H),7.42(s,1H),4.97(q,J=8.5Hz,1H),4.61(t ,J=8.5Hz,1H),4.08(t,J=9.0Hz,1H),3.25-2.99(m,4H),2.76(s,3H),2.17-2.07(m,4H),1.45(s,9H),1.40(s,9H).

[0417] MS(ESI)m / z: [M+1] + 569.2

[0418] Step 7: 4-({(Z)-[1-{2-[(tert-butyloxycarbonyl)amino]-1,3-thiazolyl-4-yl}-2-({(4S)-2-[(4S)-2-(tert-butyloxycarbonyl)-4-(5,7-dioxo-2,2-diphenyl-5,7-dihydro-2H,6H-[1,3]dioxono[4,5-f]isoindol-6-yl)-5-oxooxopentane-2-yl]-3-oxo-1,2-oxazolidine-4-yl}amino)-2-oxoethylidene]amino}oxy)-1-methylpiperidine-4-carboxylic acid tert-butyl ester (46)

[0419]

[0420] At 0 °C, DMAP (50 mg, 0.40 mmol) was added to anhydrous THF (20 mL) containing a mixture of 4-{[(Z)-(1-{2-[(tert-butoxycarbonyl)amino]-1,3-thiazo-4-yl}-2-oxo-2-{[(4S)-3-oxo-1,2-oxazolidine-4-yl]amino}ethylene)amino]oxy}-1-methylpiperidin-4-carboxylic acid tert-butyl ester 45 (455 mg, 0.80 mmol) and (2S)-5-tert-butoxy-2-(5,7-dioxo-2,2-diphenyl-5,7-dihydro2H,6H-[1,3]dioxo-[4,5-f]isoindol-6-yl)-4,5-dioxovaleric acid 6 (435 mg, 0.80 mmol). Then DCC (231 mg, 1.12 mmol) was added. The reaction mixture was stirred at 0°C for 1 hour, then left overnight at room temperature. The mixture was concentrated under reduced pressure at 25°C, and the residue was dissolved in DCM (15 mL). Hexane (15 mL) was then added, the precipitate was removed by filtration, and the mixture was washed sequentially with hexane (25 mL) containing 50% DCM and hexane. The filtrates were combined and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography using a gradient DCM containing 0-5% MeOH to obtain 46 (435 mg, 50%) of 4-({(Z)-[1-{2-[(tert-butyloxycarbonyl)amino]-1,3-thiazolyl-4-yl}-2-({(4S)-2-[(4S)-2-(tert-butyloxycarbonyl)-4-(5,7-dioxo-2,2-diphenyl-5,7-dihydro-2H,6H-[1,3]dioxono[4,5-f]isoindol-6-yl)-5-oxooxopentane-2-yl]-3-oxo-1,2-oxazolidine-4-yl}amino)-2-oxoethylidene]amino}oxy)-1-methylpiperidin-4-carboxylic acid tert-butyl ester, as a beige solid.

[0421] 1 H-NMR (400MHz; CDCl3): δ7.53 (dd, J=5.5, 2.7Hz, 4H), 7.40 (t, J=3.1Hz, 6H), 7.33-7.29 (m, 3H), 5.39 (td, J=10.0, 4.0Hz, 1H), 5.25-5. 03(m,1H),4.86-4.73(m,1H),4.41-4.24(m,1H),3.32-3.29(m,2H),2.89-2.75(m,5H),2.52-2.42(m,4H),1.54(m,18H),1.44(s,9H).

[0422] Step 8: 4-({(Z)-[1-(2-amino-1,3-thiazolyl-4-yl)-2-({(4S)-2-[(4S)-2-carboxy-4-(5,6-dihydroxy-1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)-5-oxooxopentane-2-yl]-3-oxo-1,2-oxazolidine-4-yl}amino)-2-oxoethylidene]amino}oxy)-1-methylpiperidine-4-carboxylic acid (Compound 9, Table 1)

[0423]

[0424] At -50°C, boron trichloride (in 1.0 M DCM, 3.1 mL, 3.09 mmol) was added dropwise to anhydrous DCM containing 4-({(Z)-[1-{2-[(tert-butyloxycarbonyl)amino]-1,3-thiazolyl}-2-({(4S)-2-[(4S)-2-(tert-butyloxycarbonyl)-4-(5,7-dioxo-2,2-diphenyl-5,7-dihydro-2H,6H-[1,3]dioxane[4,5-f]isoindol-6-yl)-5-oxooxopentane-2-yl]-3-oxo-1,2-oxazolidine-4-yl}amino)-2-oxoethylene]amino}oxy)-1-methylpiperidin-4-carboxylic acid tert-butyl ester 46 (422 mg, 0.386 mmol). The reaction mixture was stirred at -45 to -35°C for 3 hours, then cooled to -50°C, and a buffer solution (34 mL, prepared by dissolving 776 mg NaHCO3 and 243 mg Na2HPO4 in 42 mL of water) was added. The heterogeneous mixture was stirred in an ice-water bath instead of a cold bath until the aqueous phase thawed. The phase was carefully separated, and the aqueous layer was immediately subjected to C18 reversed-phase chromatography using acetonitrile containing 0.1% formic acid and water containing 0.1% formic acid as eluents to give compound 9 (32 mg, 12%), a pale yellow solid, using acetonitrile containing 0.1% formic acid and water containing 0.1% formic acid as eluents.

[0425] 1¹H-NMR (400 MHz, mixture of D₂O and CD₃CN): δ 7.25 (s, 2H), 6.99 (s, 1H), 5.41 (t, J = 10.0 Hz, 1H), 5.20–5.08 (m, 1H), 4.74 (t, J = 8.6 Hz, 1H), 3.49–3.26 (m, 3H), 3.17–3.01 (m, 2H), 2.85–2.70 (m, 4H), 2.45–2.27 (m, 2H), 2.27–2.07 (m, 2H). No interchangeable protons were observed in D₂O.

[0426] MS(ESI)m / z: [M+1] + 718.1

[0427] Example 7

[0428] (4S)-2-[(4S)-4-{[(2Z)-2-(2-amino-1,3-thiazolyl-4-yl)-2-{[(1-carboxy-4,4-difluorocyclohexyl)oxy]imino}acetyl]amino}-3-oxo-1,2-oxazolidine-2-yl]-4-(5,6-dihydroxy-1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)-5-oxooxopentane-2-carboxylic acid (Compound 10, Table 1),

[0429] (4S)-2-[(4S)-4-{[(2Z)-2-(2-amino-1,3-thiazolyl-4-yl)-2-{[(1-carboxy-4-chloro-4-fluorocyclohexyl)oxy]imino}acetyl]amino}-3-oxo-1,2-oxazolidine-2-yl]-4-(5,6-dihydroxy-1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)-5-oxooxopentane-2-carboxylic acid (Compound 11, Table 1)

[0430] And (4S)-2-[(4S)-4-{[(2Z)-2-(2-amino-1,3-thiazolyl-4-yl)-2-{[(1-carboxy-4,4-dichlorocyclohexyl)oxy]imino}acetyl]amino}-3-oxo-1,2-oxazolidine-2-yl]-4-(5,6-dihydroxy-1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)-5-oxooxopentane-2-carboxylic acid (compound 12, Table 1).

[0431]

[0432] Step 1. 4,4-Difluoro-1-hydroxycyclohexane-1-carboxylic acid tert-butyl ester (48)

[0433]

[0434] At 0 °C, N,N'-diisopropylcarbamate tert-butyl ester (23 mL, about 90 mmol) was slowly added to an anhydrous THF (80 mL) solution of 4,4-difluoro-1-hydroxycyclohexane-1-carboxylic acid 47 (5.19 g, 28.8 mmol). The reaction mixture was stirred at 0 °C for 10 min, then stirred at room temperature for 5 h, and then concentrated under reduced pressure. The residue was treated with hexane containing 30% DCM, and the precipitated solid was removed by filtration. The filtrate was concentrated, and the crude product was purified by silica gel column chromatography using a gradient of hexane containing 0% to 8% ethyl acetate to give 4,4-difluoro-1-hydroxycyclohexane-1-carboxylic acid tert-butyl ester 48 (5.63 g, 83%) as a grayish-white solid.

[0435] 1 H NMR (400MHz, CDCl3) δ3.13(s,1H),2.24-1.97(m,6H),1.75-1.67(m,2H),1.49(s,9H).

[0436] Step 2: 1-(aminooxy)-4,4-difluorocyclohexane-1-carboxylic acid tert-butyl ester (49)

[0437]

[0438] At 0–5 °C, sodium tert-butoxide (1.48 g, 15.4 mmol) was added to a stirred mixture of anhydrous THF (120 mL) containing 4,4-difluoro-1-hydroxycyclohexane-1-carboxylic acid tert-butyl ester 48 (3.03 g, 12.8 mmol) and O-diphenylphosphinohydroxylamine (3.59 g, 15.4 mmol). The reaction mixture was stirred at 0–10 °C for 1 hour, then additional O-diphenylphosphinohydroxylamine (2.39 g, 10.3 mmol) was added at 0–10 °C, followed by sodium tert-butoxide (990 mg, 10.3 mmol). Stirring was continued at 0–10 °C for 1 hour, then additional O-diphenylphosphinohydroxylamine (1.50 g, 6.43 mmol) and sodium tert-butoxide (620 mg, 6.45 mmol) were added at 0–10 °C, and the reaction mixture was stirred for 1 hour. Then, brine (40 mL) and hexane (30 mL) were added, and the mixture was stirred at 15–25 °C for 30 minutes. The two layers were separated, and the aqueous layer was extracted with ethyl acetate. The combined organic extracts were washed with brine, dried over sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography using a gradient of hexane containing 0–15% ethyl acetate to give 49 g (2.99 g, 93%) of 1-(aminooxy)-4,4-difluorocyclohexane-1-carboxylic acid tert-butyl ester as a white solid.

[0439] 1H NMR (400MHz, CDCl3) δ5.34(s,2H),2.17-2.13(m,2H),2.00-1.91(m,6H),1.49(s,9H).

[0440] Step 3: (2Z)-{2-[(tert-butoxycarbonyl)amino]-1,3-thiazolyl-4-yl}({1-(tert-butoxycarbonyl)-4,4-difluorocyclohexyl]oxy}imino)acetic acid (50)

[0441]

[0442] At room temperature, {2-[(tert-butyloxycarbonyl)amino]-1,3-thiazol-4-yl}({1-(tert-butyloxycarbonyl)-4,4-difluorocyclohexane-1-carboxylic acid tert-butyl ester 49 (2.99 g, 11.9 mmol)}(oxo)acetic acid (2.95 g, 10.83 mmol) was added to a MeOH (32 mL) solution containing 1-(aminooxy)-4,4-difluorocyclohexane-1-carboxylic acid tert-butyl ester 49. The reaction mixture was stirred at room temperature for 3 hours, and then water (60 mL) and hydrochloric acid aqueous solution (0.5 M, 40 mL) were added. The resulting mixture was extracted with ethyl acetate, the combined organic extracts were washed with brine, dried over sodium sulfate, filtered, and concentrated under reduced pressure. The residue was ground with hexane containing 5% diethyl ether to give (2Z)-{2-[(tert-butyloxycarbonyl)amino]-1,3-thiazol-4-yl}({1-(tert-butyloxycarbonyl)-4,4-difluorocyclohexyl]oxy}imino)acetic acid 50 (5.37 g, 98%) as a white solid.

[0443] 1 H NMR (400MHz, DMSO-d6) δ11.77(s,1H),7.40(s,1H),2.19-2.05(m,2H),1.98-1.87(m,6H),1.45(s,9H),1.39(s,9H).

[0444] MS(ESI)m / z: [M+1] + 506.4

[0445] Step 4: 1-{[(Z)-(1-{2-[(tert-butyloxycarbonyl)amino]-1,3-thiazolyl-4-yl}-2-[(2,5-dioxopyrrolidone-1-yl)oxy]-2-oxoethylidene)amino]oxy}-4,4-difluorocyclohexane-1-carboxylic acid tert-butyl ester (51)

[0446]

[0447] At 0 °C, DIC (1.84 mL, 11.88 mmol) was slowly added to anhydrous DCM (60 mL) containing a mixture of (2Z)-{2-[(tert-butoxycarbonyl)amino]-1,3-thiazolyl}({1-(tert-butoxycarbonyl)-4,4-difluorocyclohexyl]oxy}imino)acetic acid 50 (5.17 g, 10.23 mmol) and N-hydroxysuccinimide (1.41 g, 12.25 mmol). The reaction mixture was stirred at 0 °C for 15 min, and then stirred at room temperature for 3 h. The precipitated solids were removed by filtration and washed with DCM. The filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography using a gradient of hexane containing 0-20% ethyl acetate to obtain 51 (5.96 g, 97%) of 1-{[(Z)-(1-{2-[(tert-butyloxycarbonyl)amino]-1,3-thiazolyl-4-yl}-2-[(2,5-dioxopyrrolidine-1-yl)oxy]-2-oxoethylidene)amino]oxy}-4,4-difluorocyclohexane-1-carboxylic acid tert-butyl ester, as a white foam.

[0448] 1 H NMR (400MHz, CDCl3) δ8.13(bs,1H),7.51(s,1H),2.91(s,4H),2.38-2.36(m,2H),2.17-1.99(m,6H),1.53(s,9H),1.42(s,9H).

[0449] MS(ESI)m / z: [M+1] + 603.4

[0450] Step 5: 1-{[(Z)-(1-{2-[(tert-butyloxycarbonyl)amino]-1,3-thiazolyl}-2-oxo-2-{[(4S)-3-oxo-1,2-oxazolidine-4-yl]amino}ethylene)amino]oxy}-4,4-difluorocyclohexane-1-carboxylic acid tert-butyl ester (52)

[0451]

[0452] At room temperature, DIPEA (2.07 mL, 11.88 mmol) was added to anhydrous DMF (50 mL) containing a mixture of 1-{[(Z)-(1-{2-[(tert-butyloxycarbonyl)amino]-1,3-thiazolyl}-2-[(2,5-dioxopyrrolidone-1-yl)oxy]-2-oxoethylidene)amino]oxy}-4,4-difluorocyclohexane-1-carboxylic acid tert-butyl ester 51 (5.96 g, 9.89 mmol) and L-cycloserine (1.21 g, 11.85 mmol). The reaction mixture was stirred at 45 °C for 18 hours, and then concentrated under reduced pressure. The residue was dissolved in a mixture of diethyl ether and ethyl acetate (6:4), the organic phase was washed with water and brine, dried over sodium sulfate, filtered, and concentrated. The crude mixture was purified by silica gel column chromatography using a gradient of hexane containing 0-30% acetonitrile to obtain 52 (1.87 g, 32%) of 1-{[(Z)-(1-{2-[(tert-butyloxycarbonyl)amino]-1,3-thiazolyl}-2-oxo-2-{[(4S)-3-oxo-1,2-oxazolidine-4-yl]amino}ethylene)amino]oxy}-4,4-difluorocyclohexane-1-carboxylic acid tert-butyl ester, as a white foam.

[0453] 1 H NMR (400MHz, DMSO-d6) δ11.79(s,1H),11.59(s,1H),9.20(d,J=8.0Hz,1H),7.41(s,1H),4.96-4.88 (m,1H),4.58(t,J=8.4Hz,1H),4.04(t,J=9.0Hz,1H),2.16-1.82(m,8H),1.45(s,9H),1.38(s,9H).

[0454] MS(ESI)m / z: [M+1] + 590.2

[0455] Step 6: (4S)-2-[(4S)-4-{[(2Z)-2-{2-[(tert-butoxycarbonyl)amino]-1,3-thiazolyl-4-yl}-2-({[1-(tert-butoxycarbonyl)-4,4-difluorocyclohexyl]oxy}imino)acetyl]amino}-3-oxo-1,2-oxazolidine-2-yl]-4-(5,7-dioxo-2,2-diphenyl-5,7-dihydro-2H,6H-[1,3]dioxono[4,5-f]isoindol-6-yl)-5-oxooxopentane-2-carboxylic acid tert-butyl ester (53)

[0456]

[0457] At 0 °C, DMAP (9 mg, 0.074 mmol) was added to anhydrous THF (6 mL) containing a mixture of 1-{[(Z)-(1-{2-[(tert-butoxycarbonyl)amino]-1,3-thiazo-4-yl}-2-oxo-2-{[(4S)-3-oxo-1,2-oxazolidine-4-yl]amino}ethylene)amino]oxy}-4,4-difluorocyclohexane-1-carboxylic acid tert-butyl ester 52 (199 mg, 0.34 mmol) and (2S)-5-tert-butoxy-2-(5,7-dioxo-2,2-diphenyl-5,7-dihydro-2H,6H-[1,3]dioxo-[4,5-f]isoindol-6-yl)-4,5-dioxovaleric acid 6 (188 mg, 0.35 mmol). Then DCC (98 mg, 0.47 mmol) was added. The reaction mixture was allowed to be slowly heated to room temperature and stirred for 18 hours. The mixture was concentrated under reduced pressure, and the residue was extracted with hexane containing 25% DCM. Insoluble substances were removed by filtration, the filtrate was concentrated, and the crude product was purified by silica gel column chromatography using a gradient of hexane containing 0-30% ethyl acetate to obtain (4S)-2-[(4S)-4-{[(2Z)-2-{2-[(tert-butyloxycarbonyl)amino]-1,3-thiazolyl}-2-({[1-(tert-butyloxycarbonyl)-4,4-difluorocyclohexyl]oxy}imino)acetyl]amino}-3-oxo-1,2-oxazolidine-2-yl]-4-(5,7-dioxo-2,2-diphenyl-5,7-dihydro-2H,6H-[1,3]dioxono[4,5-f]isoindol-6-yl)-5-oxooxopentane-2-carboxylic acid tert-butyl ester (53) (215 mg, 57%), which is a light brown foam.

[0458] 1 H NMR (400MHz, CDCl3) δ8.15-8.09(m,2H),7.55-7.51(m,4H),7.42-7.39(m,6H),7. 34(d,J=3.8Hz,1H),7.30(d,J=1.2Hz,2H),5.36(td,J=10.1,3.0Hz,1H),5.23-4. 87(m,2H),4.37-4.18(m,1H),3.66-3.26(m,1H),2.88-2.71(m,1H),2.51-2.31(m ,2H),2.18-1.87(m,6H),1.60-1.58(m,9H),1.51-1.49(m,9H),1.48-1.41(m,9H).

[0459] Step 7: (4S)-2-[(4S)-4-{[(2Z)-2-(2-amino-1,3-thiazolyl-4-yl)-2-{[(1-carboxy-4,4-difluorocyclohexyl)oxy]imino}acetyl]amino}-3-oxo-1,2-oxazolidine-2-yl]-4-(5,6-dihydroxy-1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)-5-oxooxopentane-2-carboxylic acid (Compound 10, Table 1),

[0460] (4S)-2-[(4S)-4-{[(2Z)-2-(2-amino-1,3-thiazolyl-4-yl)-2-{[(1-carboxy-4-chloro-4-fluorocyclohexyl)oxy]imino}acetyl]amino}-3-oxo-1,2-oxazolidine-2-yl]-4-(5,6-dihydroxy-1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)-5-oxooxopentane-2-carboxylic acid (Compound 11, Table 1)

[0461] And (4S)-2-[(4S)-4-{[(2Z)-2-(2-amino-1,3-thiazolyl-4-yl)-2-{[(1-carboxy-4,4-dichlorocyclohexyl)oxy]imino}acetyl]amino}-3-oxo-1,2-oxazolidine-2-yl]-4-(5,6-dihydroxy-1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)-5-oxooxopentane-2-carboxylic acid (compound 12, Table 1).

[0462]

[0463]

[0464] At -50°C, boron trichloride (1 M in DCM, 1.54 mL, 1.54 mmol) was added dropwise to an anhydrous DCM solution containing (4S)-2-[(4S)-4-{[(2Z)-2-{2-[(tert-butoxycarbonyl)amino]-1,3-thiazolyl}-2-({[1-(tert-butoxycarbonyl)-4,4-difluorocyclohexyl]oxy}imino)acetyl]amino}-3-oxo-1,2-oxazolidine-2-yl]-4-(5,7-dioxo-2,2-diphenyl-5,7-dihydro-2H,6H-[1,3]dioxono[4,5-f]isoindol-6-yl)-5-oxooxopentane-2-carboxylic acid tert-butyl ester 53 (215 mg, 0.19 mmol). The reaction mixture was stirred at -50°C to -35°C for 2.5 hours, and then quenched at -50°C by adding NaHCO3 (314 mg) and Na2HPO4 (98 mg) in 17 mL of water. The mixture was then stirred at 0–5°C (ice-water bath) for 20 minutes, followed by stirring at room temperature until the aqueous phase thawed. The suspension was filtered through a 1.0 μm pinhole filter, and the different phases were carefully separated. The aqueous solution was immediately subjected to C-18 reversed-phase column chromatography using a Biotage system and acetonitrile containing 0.1% formic acid and water containing 0.1% formic acid as eluents. The fraction containing the pure product was collected and lyophilized to obtain compound 10 (32 mg, 22%) of (4S)-2-[(4S)-4-{[(2Z)-2-(2-amino-1,3-thiazo-4-yl)-2-{[(1-carboxy-4,4-difluorocyclohexyl)oxy]imino}acetyl]amino}-3-oxo-1,2-oxazolidine-2-yl]-4-(5,6-dihydroxy-1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)-5-oxooxopentane-2-carboxylic acid. Compound 11 (41 mg, 28%) of compound 12 (17 mg, 11%) of compound 11 (41 mg, 28%) of compound 12 of compound 11 (4-3-oxo-1,2-oxazolidine-2-yl)-4-(5,6-dihydroxy-1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)-5-oxooxopentane-2-carboxylic acid is a grayish-white solid.

[0465] For compound 10 (Table 1):

[0466] 1 ¹H NMR (400 MHz, mixture of D₂O and CD₃CN) δ 7.25 (s, 2H), 7.11–7.08 (m, 1H), 5.45–5.24 (m, 1H), 5.18–5.09 (m, 1H), 4.76–4.68 (m, 1H), 4.31–4.24 (m, 1H), 3.58–3.26 (m, 1H), 2.88–2.70 (m, 1H), 2.29–1.76 (m, 8H). No interchangeable protons were observed in D₂O.

[0467] MS(ESI)m / z: [M+1] + 739.1

[0468] For compound 11 (Table 1):

[0469] 1 ¹H NMR (400 MHz, mixture of D₂O and CD₃CN) δ 7.25 (s, 2H), 7.12–7.09 (m, 1H), 5.44–5.24 (m, 1H), 5.18–5.08 (m, 1H), 4.76–4.68 (m, 1H), 4.30–4.24 (m, 1H), 3.57–3.27 (m, 1H), 2.88–2.70 (m, 1H), 2.32–2.05 (m, 8H). No interchangeable protons were observed in D₂O.

[0470] MS(ESI)m / z: [M+1] + 755.0

[0471] For compound 12 (Table 1):

[0472] 1 ¹H NMR (400 MHz, mixture of D₂O and CD₃CN) δ 7.25 (s, 2H), 7.10–7.08 (m, 1H), 5.45–5.24 (m, 1H), 5.18–5.08 (m, 1H), 4.76–4.69 (m, 1H), 4.32–4.24 (m, 1H), 3.59–3.28 (m, 1H), 2.88–2.70 (m, 1H), 2.46–2.10 (m, 8H). No interchangeable protons were observed in D₂O.

[0473] MS(ESI)m / z: [M+1] + 771.0

[0474] Example 8

[0475] 3-({(Z)-[1-(2-amino-1,3-thiazolyl-4-yl)-2-({(4S)-2-[(4S)-2-carboxy-4-(5,6-dihydroxy-1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)-5-oxooxopentane-2-yl]-3-oxo-1,2-oxazolidine-4-yl}amino)-2-oxoethyl]amino}oxy)-8-oxabicyclo[3.2.1]octane-3-carboxylic acid (compound 13, Table 1)

[0476]

[0477] Step 1: 3-[(trimethylsilyl)oxy]-8-oxabicyclo[3.2.1]octane-3-nitrile (55)

[0478]

[0479] Zinc iodide (174 mg, 0.55 mmol) was placed in a dry round-bottom flask and gently heated under high vacuum for 30 minutes. An anhydrous DCM solution (60 mL) of oxadicyclo[3.2.1]octane-3-one 54 (3.44 g, 27.25 mmol) was added, and the resulting mixture was cooled to 0 °C. Trimethylcyanosyl silane (4.09 mL, 32.7 mmol) was slowly added at 0 °C, and the reaction mixture was allowed to heat to room temperature and stirred for 5 hours. The mixture was concentrated under reduced pressure, and the residue was extracted in hexane (75 mL) containing 10% DCM. The solids were removed by filtration, the filtrate was concentrated under reduced pressure, and the resulting brown oil was further dried under high vacuum to give 3-[(trimethylsilyl)oxy]-8-oxadicyclo[3.2.1]octane-3-onitrile 55 (6.41 g, quantitative yield).

[0480] 1 H-NMR (400MHz; CDCl3): δ4.47-4.43(m,2H),2.38(d,J=4.3Hz,1H),2.34(d,J=4.3Hz ,1H),2.20(s,1H),2.18(s,1H),2.05-2.02(m,2H),1.94-1.90(m,2H),0.29(s,9H).

[0481] Step 2: 3-Hydroxy-8-oxabicyclo[3.2.1]octane-3-carboxylic acid (56)

[0482]

[0483] A mixture of 3-[(trimethylsilyl)oxy]-8-oxobicyclo[3.2.1]octane-3-nitrile 55 (6.4 g crude, about 27 mmol) and glacial acetic acid (30 mL) was cooled at 0 °C and concentrated hydrochloric acid solution (30 mL) was added dropwise over 10 minutes. The reaction mixture was stirred at 0 °C for 15 minutes, then at room temperature for 15 minutes, and then stirred and heated at 110 °C for 4 hours. The reaction mixture was cooled to room temperature, and volatiles were removed under reduced pressure. The residue was further dried under high vacuum. The residue was dissolved in water (100 mL), and the aqueous phase was saturated with solid NaCl and extracted with ethyl acetate (2 × 150 mL). The extracts were combined, washed with water and salt, dried over sodium sulfate, filtered, concentrated under vacuum, and dried under high vacuum to give 3-hydroxy-8-oxabicyclo[3.2.1]octane-3-carboxylic acid 56 (2.43 g, 52% yield) as a brown solid.

[0484] 1 H-NMR (400MHz; DMSO-d6): δ12.50-12.34(m,1H),4.29-4.25(m,2H),2.18-2.13(m,2H),2 .07(d,J=4.3Hz,1H),2.03(d,J=3.4Hz,1H),1.72-1.66(m,2H),1.58(s,1H),1.54(s,1H).

[0485] MS(ESI)m / z: [m-1] - 171.0

[0486] Step 3: 3-hydroxy-8-oxabicyclo[3.2.1]octane-3-carboxylic acid tert-butyl ester (57)

[0487]

[0488] To a solution of 56 octane-3-carboxylic acid 56 (2.44 g, 14.18 mmol) in anhydrous THF (50 mL), N,N'-diisopropylcarbamate tert-butyl ester (11.4 mL, 56.72 mmol, prepared as described in EP2471792A1) was added, and the reaction mixture was stirred at room temperature for 16 hours. The precipitated solid was removed by filtration and washed with THF. The filtrates were combined and concentrated under reduced pressure. The residue was ground at 0–4 °C using a mixture of DCM and hexane (1:2, 90 mL), the precipitate was filtered, and washed with a mixture of DCM and hexane (1:2, 50 mL). The filtrate was collected and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography using a gradient of hexane containing 10-40% ethyl acetate to obtain 57 (2.31 g, 71% yield) of 3-hydroxy-8-oxabicyclo[3.2.1]octane-3-carboxylic acid tert-butyl ester, a pale yellow solid.

[0489] 1 H-NMR (400MHz; CDCl3): δ4.48-4.45 (m, 2H), 3.40 (s, 1H), 2.37 (d, J = 4.3Hz, 1H), 2.35-2. 33(m,3H),1.96-1.90(m,2H),1.59(t,J=1.1Hz,1H),1.55(t,J=1.1Hz,1H),1.49(s,9H).

[0490] MS(ESI)m / z: [m-1] - 226.8

[0491] Step 4: 3-(aminooxy)-8-oxabicyclo[3.2.1]octane-3-carboxylic acid tert-butyl ester (58)

[0492]

[0493] At 0 °C, sodium hydride (0.46 g, 11.43 mmol, in 60% mineral oil) was added in portions to anhydrous THF (75 mL) containing 57 tert-butyl 3-hydroxy-8-oxabicyclo[3.2.1]octane-3-carboxylic acid. The mixture was stirred for 15 min, then O-diphenylphosphinohydroxylamine (2.67 g, 11.43 mmol) was added, and stirring was continued at 0 °C for 30 min. The reaction mixture was allowed to heat to room temperature and stirred for 17 h. Subsequently, additional sodium hydride (0.23 g, 5.72 mmol, in 60% mineral oil) and O-diphenylphosphinohydroxylamine (1.33 g, 5.72 mmol) were added, and the resulting mixture was stirred at room temperature for 24 h. Most of the THF was then removed under reduced pressure, brine (75 mL) was added, and the mixture was extracted with ethyl acetate (2 × 150 mL). The combined organic phases were dried over sodium sulfate, filtered, concentrated under vacuum, and further dried under high vacuum. The residues were analyzed by silica gel column chromatography using a gradient of hexane containing 10-30% ethyl acetate to give 58 (1.06 g, 57% yield) of 3-(aminooxy)-8-oxabicyclo[3.2.1]octane-3-carboxylic acid tert-butyl ester as a white solid.

[0494] 1 H-NMR (400MHz; CDCl3): δ5.29 (bs, 2H), 4.42-4.39 (m, 2H), 2.23 (d, J = 4.3Hz, 1 H), 2.20 (d, J = 4.4Hz, 1H), 2.04-1.97 (m, 4H), 1.96-1.88 (m, 2H), 1.49 (s, 9H).

[0495] Step 5: (2Z)-{2-[(tert-butoxycarbonyl)amino]-1,3-thiazolyl-4-yl}({3-(tert-butoxycarbonyl)-8-oxabicyclo[3.2.1]octane-3-yl]oxy}imino)acetic acid (59)

[0496]

[0497] To an anhydrous MeOH (10 mL) solution containing tert-butyl 3-(aminooxy)-8-oxabicyclo[3.2.1]octane-3-carboxylic acid 58 (0.42 g, 1.73 mmol), {2-[(tert-butyloxycarbonyl)amino]-1,3-thiazol-4-yl}(oxo)acetic acid (0.47 g, 1.73 mmol) was added, and the reaction mixture was stirred at room temperature for 17 hours. The reaction mixture was concentrated under reduced pressure, and the crude product was further dried under high vacuum to give (2Z)-{2-[(tert-butyloxycarbonyl)amino]-1,3-thiazol-4-yl}({3-(tert-butyloxycarbonyl)-8-oxabicyclo[3.2.1]octane-3-yl]oxy}imino)acetic acid 59 (0.86 g, 100%) as a white solid.

[0498] 1 H-NMR (400MHz; CDCl3): δ7.37(s,1H),4.41-4.40(m,2H),2.33-2.23(m,4H),2.05-2.00(m,2H),1.86-1.81(m,2H),1.55(s,9H),1.44(s,9H).

[0499] MS(ESI)m / z: [M+1] + 498.1

[0500] Step 6: 3-{[(Z)-(1-{2-[(tert-butyloxycarbonyl)amino]-1,3-thiazolyl}-2-oxo-2-{[(4S)-3-oxo-1,2-oxazolidine-4-yl]amino}ethylene)amino]oxy}-8-oxabicyclo[3.2.1]octane-3-carboxylic acid tert-butyl ester (60)

[0501]

[0502] N,N-diisopropylethylamine (0.31 mL, 1.80 mmol) was added to an anhydrous DMF (8 mL) solution containing (2Z)-{2-[(tert-butyloxycarbonyl)amino]-1,3-thiazolyl}({3-(tert-butyloxycarbonyl)-8-oxabicyclo[3.2.1]octane-3-yl]oxy}imino)acetic acid 59 (0.60 g, 1.20 mmol). The resulting mixture was stirred at room temperature for 10 min. Then HATU (0.46 g, 1.20 mmol) was added, and the mixture was stirred at room temperature for 14 h. The reaction mixture was then diluted with anhydrous DMF (8 mL), and DIPEA (0.84 mL, 4.80 mmol) and L-cycloserine (184 mg, 1.80 mmol) were added. The mixture was stirred at room temperature for 1 h, and then concentrated under reduced pressure. The residue was dissolved in DCM (50 mL), and the organic phase was washed with water (20 mL) and brine (20 mL), dried over sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography using a gradient of DCM containing 0-3% MeOH to give 60 (0.69 g, 98%) of 3-{[(Z)-(1-{2-[(tert-butyloxycarbonyl)amino]-1,3-thiazolyl}-2-oxo-2-{[(4S)-3-oxo-1,2-oxazolidine-4-yl]amino}ethylene)amino]oxy}-8-oxabicyclo[3.2.1]octane-3-carboxylic acid tert-butyl ester, as a grayish-white solid.

[0503] 1 H-NMR (400MHz; DMSO-d6): δ11.84(bs,1H),11.55(bs,1H),9.09(d,J=7.8Hz,1H),7.46(s,1H),4.98-4.90(m,1H),4.59(t,J=8.5Hz,1H ),4.34-4.27(m,2H),4.13(dd,J=9.9,8.4Hz,1H),2.16-2.06(m,2H),1.94-1.89(m,4H),1.71-1.66(m,2H),1.47(s,9H),1.36(s,9H).

[0504] MS(ESI)m / z: [M+1] + 582.2

[0505] Step 7: 3-({(Z)-[1-{2-[(tert-butyloxycarbonyl)amino]-1,3-thiazolyl-4-yl}-2-({(4S)-2-[(4S)-2-(tert-butyloxycarbonyl)-4-(5,7-dioxo-2,2-diphenyl-5,7-dihydro-2H,6H-[1,3]dioxacyclo[4,5-f]isoindol-6-yl)-5-oxooxopentane-2-yl]-3-oxo-1,2-oxazolidine-4-yl}amino)-2-oxoethylidene]amino}oxy)-8-oxabicyclo[3.2.1]octane-3-carboxylic acid tert-butyl ester (61)

[0506]

[0507] At 0°C, tert-butyl octane-3-carboxylic acid ester 60 (0.38 g, 0.65 mmol) containing 3-{[(Z)-(1-{2-[(tert-butoxycarbonyl)amino]-1,3-thiazolyl}-2-oxo-2-{[(4S)-3-oxo-1,2-oxazolidine-4-yl]amino}ethylene)amino]oxy}-8-oxabicyclo[3.2.1]octane-3-carboxylic acid tert-butyl ester 60 (0.38 g, 0.65 mmol) and (2S)-5-tert-butoxy- In anhydrous THF (15 mL) of a mixture of 2-(5,7-dioxo-2,2-diphenyl-5,7-dihydro-2H,6H-[1,3]dioxo-[4,5-f]isoindol-6-yl)-4,5-dioxovaleric acid 6 (0.353 g, 0.65 mmol), DMAP (16 mg, 0.13 mmol) was added, followed by DCC (188 mg, 0.91 mmol). The reaction mixture was stirred at 0 °C for 1 hour, then allowed to stand overnight at room temperature, and concentrated under reduced pressure at 25 °C. The residue was ground with hexane (20 mL) containing 30% DCM, the precipitate was removed by filtration, and the mixture was washed with hexane (15 mL) and hexane (15 mL) containing 30% DCM. The filtrates were combined and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography using a gradient of hexane containing 10-50% ethyl acetate to obtain 3-({(Z)-[1-{2-[(tert-butyloxycarbonyl)amino]-1,3-thiazolyl-4-yl}-2-({(4S)-2-[(4S)-2-(tert-butyloxycarbonyl)-4-(5,7-dioxo-2,2-diphenyl-5,7-dihydro-2H,6H-[1,3]dioxacyclo[4,5-f]isoindol-6-yl)-5-oxooxopentane-2-yl]-3-oxo-1,2-oxazolidine-4-yl}amino)-2-oxoethylidene]amino}oxy)-8-oxabicyclo[3.2.1]octane-3-carboxylic acid tert-butyl ester 61 (355 mg, 49%), a brown solid.

[0508] 1H-NMR (400MHz; CDCl3): δ7.57-7.54(m,4H),7.45-7.42(m,6H),7.36(m,1H) ,7.33(m,2H),5.42-5.36(m,1H),5.22-4.89(m,2H),4.49-4.45(m,2H),4.2 9-4.22(m,1H),3.54-3.47(m,1H),3.43-3.31(m,1H),2.91-2.84(m,1H),2. 44-2.28(m,4H),2.03-1.88(m,4H),1.75-1.69(m,1H),1.59-1.42(m,27H).

[0509] Step 8: 3-({(Z)-[1-(2-amino-1,3-thiazolyl-4-yl)-2-({(4S)-2-[(4S)-2-carboxy-4-(5,6-dihydroxy-1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)-5-oxooxopentane-2-yl]-3-oxo-1,2-oxazolidine-4-yl}amino)-2-oxoethyl]amino}oxy)-8-oxabicyclo[3.2.1]octane-3-carboxylic acid (compound 13)

[0510]

[0511] At -50°C, an agent containing 3-({(Z)-[1-{2-[(tert-butoxycarbonyl)amino]-1,3-thiazo-4-yl}-2-({(4S)-2-[(4S)-2-(tert-butoxycarbonyl)-4-(5,7-dioxo-2,2-diphenyl-5,7-dihydro-2H,6H-[1,3]dioxohetero[4,5-f]isoindol-6-yl)-5-oxo Boron trichloride (1.0 M in 2.5 mL of DCM, 2.50 mmol) was added dropwise to an anhydrous DCM solution of tert-butyl 61 octane-3-carboxylic acid (345 mg, 0.31 mmol) in 16 mL of [3.2.1]octane-3-carboxylic acid. The reaction mixture was stirred at -45 to -35 °C for 3 hours, then cooled to -50 °C and 31 mL of buffer solution (prepared by dissolving 776 mg NaHCO3 and 243 mg Na2HPO4 in 42 mL of water) was added. An ice-water bath was used instead of a cold water bath, and the resulting mixture was stirred until the aqueous layer thawed, separating the different phases. The organic phase was carefully removed, and the Biotage system was used with acetonitrile containing 0.1% formic acid and water containing 0.1% formic acid as the mobile phase. The collected aqueous phase was immediately subjected to C18 reversed-phase column chromatography to give 3-({(Z)-[1-(2-amino-1,3-thiazolyl-4-yl)-2-({(4S)-2-[(4S)-2-carboxy-4-(5,6-dihydroxy-1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)-5-oxooxopentane-2-yl]-3-oxo-1,2-oxazolidine-4-yl}amino)-2-oxoethylene]amino}oxy)-8-oxabicyclo[3.2.1]octane-3-carboxylic acid compound 13 (100 mg, 44%) as a pale yellow foamy solid.

[0512] 1 ¹H-NMR (400 MHz; mixture of D₂O and CD₃CN): δ 7.25 (s, 2H), 7.08 (s, 1H), 5.41–5.33 (m, 1H), 5.16–5.06 (m, 1H), 4.76–4.66 (m, 1H), 4.32–4.24 (m, 2H), 3.35–3.27 (m, 1H), 2.79–2.68 (m, 1H), 2.32–2.18 (m, 2H), 2.11–2.01 (m, 2H), 1.86–1.71 (m, 4H). No interchangeable protons were observed in D₂O.

[0513] MS(ESI)m / z:[M+H] + 731.0

[0514] Example 9

[0515] (4S)-2-[(4S)-4-{[(2Z)-2-(5-amino-1,2,4-thiadiazol-3-yl)-2-{[(1-carboxycyclopropyl)oxy]imino}acetyl]amino}-3-oxo-1,2-oxazolidine-2-yl]-4-(5,6-dihydroxy-1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)-5-oxooxopentane-2-carboxylic acid (Compound 18, Table 1)

[0516]

[0517] Step 1: (2Z)-{5-[(tert-butoxycarbonyl)amino]-1,2,4-thiadiazol-3-yl}({1-(tert-butoxycarbonyl)cyclopropyl]oxy}imino)acetic acid (63)

[0518]

[0519] To an anhydrous MeOH (10 mL) solution containing tert-butyl 1-(aminooxy)cyclopropane-1-carboxylic acid ester 2 (364 mg, 2.10 mmol, prepared as described in step 1 of Example 1), {5-[(tert-butyloxycarbonyl)amino]-1,2,4-thiadiazol-3-yl}(oxo)acetic acid 62 (546 mg, 2.00 mmol, prepared as described in WO 2017 / 155765) was added. The reaction mixture was stirred at room temperature for 14 hours, and then concentrated under reduced pressure. The residue was extracted in diethyl ether (50 mL), the organic phase was washed with 0.1 M hydrochloric acid solution (33 mL) and hydrochloric acid (30 mL), dried over sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was further dried under high vacuum to obtain (2Z)-{5-[(tert-butoxycarbonyl)amino]-1,2,4-thiadiazol-3-yl}({1-(tert-butoxycarbonyl)cyclopropyl]oxy}imino)acetic acid 63 (696 mg, 81%), a pale yellow solid.

[0520] 1 H-NMR (400MHz; DMSO-d6): δ12.62(s,1H),12.51(s,1H),1.49(s,9H),1.36(m,11H),1.28(m,2H).

[0521] Step 2: 1-{[(Z)-(1-{5-[(tert-butyloxycarbonyl)amino]-1,2,4-thiadiazol-3-yl}-2-oxo-2-{[(4S)-3-oxo-1,2-oxazolidine-4-yl]amino}ethylene)amino]oxy}cyclopropane-1-carboxylic acid tert-butyl ester (64)

[0522]

[0523] N,N-diisopropylethylamine (0.31 mL, 1.80 mmol) was added to an anhydrous DMF (8 mL) solution containing (2Z)-{5-[(tert-butoxycarbonyl)amino]-1,2,4-thiadiazol-3-yl}({1-(tert-butoxycarbonyl)cyclopropyl]oxy}imino)acetic acid 63 (514 mg, 1.20 mmol), and the mixture was stirred at room temperature for 10 min. HATU (456 mg, 1.20 mmol) was then added, and the mixture was stirred at room temperature for 14 h. Subsequently, anhydrous DMF (10 mL) and N,N-diisopropylethylamine (0.84 mL, 4.8 mmol) were added, followed by L-cycloserine (184 mg, 1.80 mmol), and the reaction mixture was stirred at room temperature for 1 h. Volatile substances were then removed under reduced pressure, and the residue was extracted in DCM (30 mL). The organic phase was washed with water, dried over sodium sulfate, filtered, and concentrated. The crude product was purified by silica gel column chromatography using a gradient DCM containing 0-3% methanol to obtain 1-{[(Z)-(1-{5-[(tert-butyloxycarbonyl)amino]-1,2,4-thiadiazol-3-yl}-2-oxo-2-{[(4S)-3-oxo-1,2-oxazolidine-4-yl]amino}ethylene)amino]oxy}cyclopropane-1-carboxylic acid tert-butyl ester 64 (204 mg, 33%), as a grayish-white solid.

[0524] 1 H-NMR (400MHz; DMSO-d6): δ12.59(s,1H),11.57(s,1H),9.07(d,J=7.6Hz,1H),4.91-4.78(m,1H) ,4.59(t,J=8.3Hz,1H),3.91(t,J=8.8Hz,1H),1.49(s,9H),1.38(s,9H),1.34(m,2H),1.24(m,2H)

[0525] Step 3: (4S)-2-[(4S)-4-{[(2Z)-2-{5-[(tert-butoxycarbonyl)amino]-1,2,4-thiadiazol-3-yl}-2-({[1-(tert-butoxycarbonyl)cyclopropyl]oxy}imino)acetyl]amino}-3-oxo-1,2-oxazolidine-2-yl]-4-(5,7-dioxo-2,2-diphenyl-5,7-dihydro-2H,6H-[1,3]dioxono[4,5-f]isoindol-6-yl)-5-oxooxopentane-2-carboxylic acid tert-butyl ester (65)

[0526]

[0527] The mixture of 1-{[(Z)-(1-{5-[(tert-butoxycarbonyl)amino]-1,2,4-thiadiazol-3-yl}-2-oxo-2-{[(4S)-3-oxo-1,2-oxazolidine-4-yl]amino}ethylene)amino]oxy}cyclopropane-1-carboxylic acid tert-butyl ester 64 (196 mg, 0.38 mmol) and (2S)-5-tert-butoxy-2-(5,7-dioxo-2,2-diphenyl-5,7-dihydro2H,6H-[1,3]dioxo-[4,5-f]isoindol-6-yl)-4,5-dioxovaleric acid 6 (207 mg, 0.38 mmol) was stirred and cooled to 0 °C. DMAP (14 mg, 0.11 mmol) was added, followed by DCC (110 mg, 0.53 mmol). The reaction mixture was stirred at 0 °C for 1 hour, then left overnight at room temperature, and concentrated under reduced pressure at 25 °C. The residue was treated with hexane (25 mL) containing 40% DCM, the precipitate was removed by filtration, and the mixture was washed with hexane (25 mL) and hexane (15 mL) containing 40% DCM. The filtrates were combined and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography using a gradient of hexane containing 10-40% ethyl acetate to obtain (4S)-2-[(4S)-4-{[(2Z)-2-{5-[(tert-butoxycarbonyl)amino]-1,2,4-thiadiazol-3-yl}-2-({[1-(tert-butoxycarbonyl)cyclopropyl]oxy}imino)acetyl]amino}-3-oxo-1,2-oxazolidine-2-yl]-4-(5,7-dioxo-2,2-diphenyl-5,7-dihydro-2H,6H-[1,3]dioxono[4,5-f]isoindol-6-yl)-5-oxooxopentane-2-carboxylic acid tert-butyl ester 65 (197 mg, 50%), a white solid.

[0528] 1 H-NMR (400MHz; CDCl3): δ8.56(s,1H),8.18(dd,J=8.1,5.0Hz,1H),7.54-7.51(m,4H),7.41(q,J=3.3Hz,6H),7.30(s,2H),5.38(td,J=1 0.0,8.3Hz,1H),5.16-4.96(m,2H),4.30-4.22(m,1H),3.49-3.29(m,1H),2.88-2.82(m,1H),1.57-1.53(m,22H),1.43(d,J=2.7Hz,9H).

[0529] Step 4: (4S)-2-[(4S)-4-{[(2Z)-2-(5-amino-1,2,4-thiadiazol-3-yl)-2-{[(1-carboxycyclopropyl)oxy]imino}acetyl]amino}-3-oxo-1,2-oxazolidine-2-yl]-4-(5,6-dihydroxy-1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)-5-oxooxopentane-2-carboxylic acid (Compound 18, Table 1)

[0530]

[0531] A solution of (4S)-2-[(4S)-4-{[(2Z)-2-{5-[(tert-butyloxycarbonyl)amino]-1,2,4-thiadiazol-3-yl}-2-({[1-(tert-butyloxycarbonyl)cyclopropyl]oxy}imino)acetyl]amino}-3-oxo-1,2-oxazolidine-2-yl]-4-(5,7-dioxo-2,2-diphenyl-5,7-dihydro-2H,6H-[1,3]dioxane[4,5-f]isoindol-6-yl)-5-oxooxopentane-2-carboxylic acid tert-butyl ester 65 (190 mg, 0.183 mmol) in anhydrous DCM was cooled to -50 °C and boron trichloride (1.0 M in DCM, 1.46 mL, 1.46 mmol) was added dropwise. The reaction mixture was stirred at -45 to -35°C for 3 hours, then cooled to -50°C, and 16.5 mL of buffer solution (prepared by dissolving 776 mg NaHCO3 and 243 mg Na2HPO4 in 42 mL of water) was added. An ice-water bath was used instead of a cold water bath, and the resulting mixture was stirred until the aqueous layer thawed. The organic phase was carefully separated using a Biotage system and an aqueous solution containing 0.1% formic acid in acetonitrile and 0.1% formic acid as eluent. The aqueous phase was immediately subjected to C18 reversed-phase chromatography to give compound 18 (50 mg, 41%) as a pale yellow foamy solid.

[0532] 1¹H-NMR (400 MHz; mixture of D₂O and CD₃CN): δ 7.25 (s, 2H), 5.44–5.34 (m, 1H), 5.15–5.04 (m, 1H), 4.73 (q, J = 8.4 Hz, 1H), 4.32–4.14 (m, 1H), 3.45–3.31 (m, 1H), 2.91–2.73 (m, 1H), 1.55–1.45 (m, 2H), 1.45–1.42 (m, 2H). No interchangeable protons were observed in D₂O.

[0533] MS(ESI) m / z: [M+H]+662.1

[0534] Example 10

[0535] (4S)-2-[(4S)-4-{[(2Z)-2-(2-amino-1,3-thiazolyl-4-yl)-2-{[(2-carboxypropane-2-yl)oxy]imino}acetyl]amino}-3-oxo-1,2-oxazolidine-2-yl]-4-(2,3-dihydroxy-5,7-dioxo-5,7-dihydro-6H-pyrrolo[3,4-b]pyridin-6-yl)-5-oxooxopentane-2-carboxylic acid (Compound 36, Table 1)

[0536]

[0537] Step 1: Diethyl 2,2-diphenyl-2H-[1,3]dioxane[4,5-b]pyridine-5,6-dicarboxylic acid ester (66)

[0538]

[0539] Anhydrous DMA (60 mL) containing a stirred mixture of diethyl 5-hydroxy-6-oxo-1,6-dihydropyridine-2,3-dicarboxylic acid 66 (6.83 g, 26.76 mmol, prepared as described in US 5252538(A), 1993) and potassium carbonate (4.07 g, 29.45 mmol) was heated to 100 °C, and a DMA solution (20 mL) containing 1,1′-(dichloromethane)diphenyl (5.65 mL, 29.43 mmol) was added dropwise. The reaction mixture was then heated to 170 °C and stirred at 170 °C for 20 hours. Subsequently, the reaction mixture was cooled to room temperature and water was added. The resulting mixture was extracted with a mixture of diethyl ether and ethyl acetate (2:1). The combined organic extracts were washed with brine, dried over sodium sulfate, filtered, and concentrated under reduced pressure. The residues were purified by silica gel column chromatography using a gradient of hexane containing 0–16% ethyl acetate. Further purification of the product by recrystallization from diethyl ether and hexane yielded 67 (1.66 g, 15%) of 2,2-diphenyl-2H-[1,3]dioxane[4,5-b]pyridine-5,6-dicarboxylic acid diethyl ester, a white solid.

[0540] 1 H NMR (400MHz, CDCl3) δ7.57-7.53(m,4H),7.45(s,1H),7.41-7.38(m,6H),4.40(q, J=7.2Hz, 2H), 4.34 (q, J=7.1Hz, 2H), 1.38 (t, J=7.2Hz, 3H), 1.34 (t, J=7.1Hz, 3H).

[0541] Step 2: 2,2-Diphenyl-2H-[1,3]dioxane[4,5-b]pyridine-5,6-dicarboxylic acid (68)

[0542]

[0543] Sodium hydroxide solution (835 mg, 20.88 mmol in 10 mL of water) was added to a stirred suspension of 2,2-diphenyl-2H-[1,3]dioxon[4,5-b]pyridine-5,6-dicarboxylic acid diethyl ester 67 (1.75 g, 4.17 mmol) in MeOH, THF, and water (1:1:1, 30 mL). The reaction mixture was heated at 80 °C for 5 hours, then cooled to room temperature and concentrated under reduced pressure to remove THF and MeOH. The aqueous phase was washed twice with diethyl ether, cooled to 0 °C, and acidified to pH 1. The resulting mixture was extracted with ethyl acetate, and the combined organic extracts were dried over sodium sulfate, filtered, and concentrated under reduced pressure to give crude 2,2-diphenyl-2H-[1,3]dioxon[4,5-b]pyridine-5,6-dicarboxylic acid 68 (1.46 g, 96%) as a white solid, which was used directly for the next step without further purification.

[0544] 1 H NMR (400MHz, DMSO-d6) δ7.78 (s, 1H), 7.56-7.52 (m, 4H), 7.49-7.45 (m, 6H).

[0545] Step 3: 2,2-Diphenyl-2H-[1,3]dioxane[4,5-b]furano[3,4-e]pyridine-5,7-dione (69)

[0546]

[0547] Acetic anhydride (16 mL) was added to 2,2-diphenyl-2H-[1,3]dioxono[4,5-b]pyridine-5,6-dicarboxylic acid 68 (1.46 g, 4.02 mmol) at room temperature. The resulting mixture was heated to 100 °C and stirred for 3 hours. The reaction mixture was then cooled to room temperature and concentrated under reduced pressure. Trace amounts of volatiles were removed by co-evaporation with toluene (3×), and the residue was further dried under high vacuum to give 2,2-diphenyl-2H-[1,3]dioxono[4,5-b]furano[3,4-e]pyridine-5,7-dione 69 (1.27 g, 92%) as a grayish-white foam, which was used directly for the next step.

[0548] 1 H NMR (400MHz, DMSO-d6) δ8.08(s,1H),7.58-7.55(m,4H),7.52-7.49(m,6H).

[0549] Step 4: (2S)-5-tert-butoxy-2-(5,7-dioxo-2,2-diphenyl-5,6-dihydro-2H,6H-[1,3]dioxane[4,5-b]pyrrolo[3,4-e]pyridin-6-yl)-4,5-dioxovalerate (71)

[0550]

[0551] At room temperature, an anhydrous pyridine solution (25 mL) of 2,2-diphenyl-2H-[1,3]dioxane[4,5-b]furano[3,4-e]pyridine-5,7-dione 69 (crude product, 1.27 g, 3.68 mmol) was added to (2S)-2-amino-5-tert-butoxy-4,5-dioxane 70 (2.03 g, 7.68 mmol, prepared as described in J. Med. Chem. 2014, Vol. 57, pp. 3845-3855). The reaction mixture was stirred and heated at 90 °C for 2.5 h, then cooled to room temperature and concentrated under reduced pressure. The residue was dissolved in DCM (10 mL) and water (120 mL) was added. The mixture was extracted with ethyl acetate, the combined organic extracts were washed with brine, dried over sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography using a gradient DCM containing 0-16% acetonitrile to obtain (2S)-5-tert-butoxy-2-(5,7-dioxo-2,2-diphenyl-5,6-dihydro-2H,6H-[1,3]dioxane[4,5-b]pyrrolo[3,4-e]pyridin-6-yl)-4,5-dioxovalerate 71 (390 mg, 19%), which was a brown foam.

[0552] 1 H NMR (400MHz, DMSO-d6) δ7.93(s,1H),7.57-7.53(m,4H),7.51-7.47(m,6H),5.19-5.16(m,1H),3.71-3.62(m,1H),3.37-3.34(m,1H),1.44(s,9H).

[0553] MS(ESI)m / z: [M+1] + 545.2

[0554] Step 5: (4S)-2-[(4S)-4-{[(2Z)-2-{2-[(tert-butoxycarbonyl)amino]-1,3-thiazolyl-4-yl}-2-{[(1-tert-butoxy-2-methyl-1-oxopropane-2-yl)oxy]imino}acetyl]amino}-3-oxo-1,2-oxazolidine-2-yl]-4-(5,7-dioxo-2,2-diphenyl)-5,7-dihydro-2H,6H-[1,3]dioxane[4,5-b]pyrrolo[3,4-e]pyridin-6-yl)-5-oxooxopentane-2-carboxylic acid tert-butyl ester (73)

[0555]

[0556] At 0 °C, (390 mg, 0.72 mmol) of (2S)-5-tert-butoxy-2-(5,7-dioxo-2,2-diphenyl-5,6-dihydro-2H,6H-[1,3]dioxane[4,5-b]pyrrolo[3,4-e]pyridin-6-yl)-4,5-dioxovaleric acid 71 (390 mg, 0.72 mmol) and 2-{[(Z)-(1-{2-[(tert-butoxycarbonyl)amino]-1,3-thiazo-4-yl}-2-oxo-2-{[( A mixture of tert-butyl 4S)-3-oxo-1,2-oxazolidine-4-yl]amino}ethylene)amino]oxy}-2-methylpropionate 72 (368 mg, 0.72 mmol, prepared as described in J. Med. Chem., 2014, Vol. 57, pp. 3845-3855) was mixed with anhydrous THF (20 mL) and DMAP (18 mg, 0.15 mmol) was added, followed by DCC (207 mg, 1.0 mmol). The reaction mixture was allowed to be slowly heated to room temperature and stirred for 18 hours. The mixture was then concentrated under reduced pressure, and the residue was ground with hexane containing 30% DCM. The precipitated solids were removed by filtration, and the filtrate was concentrated under vacuum. The residue was purified by silica gel column chromatography using a gradient of hexane containing 10-35% ethyl acetate to obtain (4S)-2-[(4S)-4-{[(2Z)-2-{2-[(tert-butoxycarbonyl)amino]-1,3-thiazolyl-4-yl}-2-{[(1-tert-butoxy-2-methyl-1-oxopropane-2-yl)oxy]imino}acetyl]amino}-3-oxo-1,2-oxazolidine-2-yl]-4-(5,7-dioxo-2,2-diphenyl)-5,7-dihydro-2H,6H-[1,3]dioxono[4,5-b]pyrrolo[3,4-e]pyridin-6-yl)-5-oxooxopentane-2-carboxylic acid tert-butyl ester 73 (375 mg, 50%), as a brown foam.

[0557] 1H NMR (400MHz, CDCl3) δ8.44-8.39(m,1H),7.57-7.54(m,4H),7.44-7.40(m,8H),5.45-5.39(m,1H),5.18-4.85(m,2H),4.38-4. 21(m,1H),3.68-3.33(m,1H),2.89-2.77(m,1H),1.66-1.65(m,6H),1.57-1.55(m,9H),1.54-1.52(m,9H),1.45-1.44(m,9H).

[0558] Step 6: (4S)-2-[(4S)-4-{[(2Z)-2-(2-amino-1,3-thiazolyl-4-yl)-2-{[(2-carboxypropane-2-yl)oxy]imino}acetyl]amino}-3-oxo-1,2-oxazolidine-2-yl]-4-(2,3-dihydroxy-5,7-dioxo-5,7-dihydro-6H-pyrrolo[3,4-b]pyridin-6-yl)-5-oxooxopentane-2-carboxylic acid (Compound 36, Table 1)

[0559]

[0560] At -50°C, boron trichloride (at 1.0 M in DCM, 0.77 mM) was added dropwise to an anhydrous DCM (5 mL) solution containing ((4S)-2-[(4S)-4-{[(2Z)-2-{2-[(tert-butoxycarbonyl)amino]-1,3-thiazolyl}-2-{[(1-tert-butoxy-2-methyl-1-oxopropane-2-yl)oxy]imino}acetyl]amino}-3-oxo-1,2-oxazolidine-2-yl]-4-(5,7-dioxo-2,2-diphenyl)-5,7-dihydro-2H,6H-[1,3]dioxane[4,5-b]pyrrolo[3,4-e]pyridin-6-yl)-5-oxooxopentane-2-carboxylic acid tert-butyl ester 73 (100 mg, 0.096 mmol). The reaction mixture was stirred at -50°C to -35°C for 2.5 hours, and then an aqueous solution (8.5 mL) of NaHCO3 (157 mg) and Na2HPO4 (49 mg) was added at -50°C. The mixture was stirred for 20 minutes using an ice-water bath instead of a cold bath. Subsequently, the heterogeneous mixture was stirred at room temperature until the aqueous phase was completely thawed. The resulting mixture was filtered through a 1 μm pinhole filter to carefully remove the organic layer. The aqueous phase was immediately passed through a C-18 reversed-phase chromatograph using a Biotage system and acetonitrile containing 0.1% formic acid and an aqueous solution containing 0.1% formic acid as eluents. The fractions were combined and lyophilized to give (4S)-2-[(4S)-4-{[(2Z)-2-(2-amino-1,3-thiazolyl-4-yl)-2-{[(2-carboxypropane-2-yl)oxy]imino}acetyl]amino}-3-oxo-1,2-oxazolidine-2-yl]-4-(2,3-dihydroxy-5,7-dioxo-5,7-dihydro-6H-pyrrolo[3,4-b]pyridin-6-yl)-5-oxooxopentane-2-carboxylic acid compound 36 (23 mg, 36%), as a yellow solid.

[0561] 1 ¹H NMR (400 MHz, mixture of D₂O and CD₃CN) δ 7.09–7.08 (m, 1H), 7.08–7.06 (m, 1H), 5.42–5.22 (m, 1H), 5.17–5.08 (m, 1H), 4.76–4.68 (m, 1H), 4.34–4.23 (m, 1H), 3.53–3.29 (m, 1H), 2.88–2.64 (m, 1H), 1.53–1.45 (m, 6H). No interchangeable protons were observed in D₂O.

[0562] MS(ESI)m / z: [M+1] + 664.1

[0563] Example 11

[0564] (4S)-2-[(4S)-4-{[(2Z)-2-(2-amino-1,3-thiazolyl-4-yl)-2-{[(1-carboxycyclopropyl)oxy]imino}acetyl]amino}-3-oxo-1,2-oxazolidine-2-yl]-4-(2,3-dihydroxy-5,7-dioxo-5,7-dihydro-6H-pyrrolo[3,4-b]pyridin-6-yl)-5-oxooxopentane-2-carboxylic acid (Compound 3, Table 1)

[0565]

[0566] Step 1: (4S)-2-[(4S)-4-{[(2Z)-2-{2-[(tert-butoxycarbonyl)amino]-1,3-thiazolyl-4-yl}-2-({[1-(tert-butoxycarbonyl)cyclopropyl]oxy}imino)acetyl]amino}-3-oxo-1,2-oxazolidine-2-yl]-4-(5,7-dioxo-2,2-diphenyl-5,7-dihydro-2H,6H-[1,3]dioxane[4,5-b]pyrrolo[3,4-e]pyridin-6-yl)-5-oxooxopentane-2-carboxylic acid tert-butyl ester (74)

[0567]

[0568] At 0°C, (105 mg, 0.2 mmol, prepared as described in step 4 of Example 10) 71 containing (2S)-5-tert-butoxy-2-(5,7-dioxo-2,2-diphenyl-5,6-dihydro-2H,6H-[1,3]dioxane[4,5-b]pyrrolo[3,4-e]pyridin-6-yl)-4,5-dioxovalerate acid 71 (prepared as described in step 4 of Example 10) and 1-{[(Z)-(1-{2-[(tert-butoxycarbonyl)amino]-1,3-thiazo-4-yl)-4-dioxovalerate acid 71 (prepared as described in step 4 of Example 10) and 1-{[(Z)-(1-{2-[(tert-butoxycarbonyl)amino]-1,3-thiazo-4-yl)-2-dioxane-4-yl)-2-dioxane-2-(2S)-5-tert-butoxy-2-(5,7-dioxo-2,2-diphenyl-5,6-dihydro-2H,6H-[1,3]dioxane[4,5-b]pyrrolo[3,4-e]pyridin-6-yl)-4,5-dioxovalerate acid 71 (prepared as described in step 4 of Example 10) and 1-{[(Z)-(1-{2-[(tert-butoxycarbonyl)amino]-1,3-thiazo-4-yl)-2-[2S)-5-tert-butoxycarbonyl)amino]-1,3-thiazo-4 ... The mixture of 5 mg (5 mg, 0.04 mmol) of tert-butyl cyclopropane-1-carboxylic acid ester (100 mg, 0.2 mmol, prepared as described in step 4 of Example 1) was added to anhydrous THF (2 mL) with DMAP (5 mg, 0.04 mmol), followed by DCC (56 mg, 0.27 mmol). The reaction mixture was allowed to gradually heat to room temperature and stirred continuously for 18 hours. The reaction mixture was concentrated under reduced pressure, and the residue was ground with hexane containing 25% DCM. Insoluble substances were filtered off, and the filtrate was concentrated. The crude product was purified by silica gel column chromatography using a gradient of hexane containing 10-40% ethyl acetate to obtain (4S)-2-[(4S)-4-{[(2Z)-2-{2-[(tert-butoxycarbonyl)amino]-1,3-thiazolyl-4-yl}-2-({[1-(tert-butoxycarbonyl)cyclopropyl]oxy}imino)acetyl]amino}-3-oxo-1,2-oxazolidine-2-yl]-4-(5,7-dioxo-2,2-diphenyl-5,7-dihydro-2H,6H-[1,3]dioxane[4,5-b]pyrrolo[3,4-e]pyridin-6-yl)-5-oxooxopentane-2-carboxylic acid tert-butyl ester 74 (50 mg, 25%), as a white foam.

[0569] 1 H-NMR (599MHz; CDCl3) δ8.59 (t, J = 7.5Hz, 1H), 8.20-8.05 (bs, 1H) 7.58-7.52 (m ,4H),7.43-7.40(m,7H),5.43(dt,J=19.8,10.0Hz,1H),5.15-5.12(m,1H),4.9 0(dt,J=26.1,8.4Hz,1H),4.30-4.21(m,1H),3.50-3.32(m,J=9.8Hz,1H),2.86 (dd,J=13.3,11.3Hz,1H),1.59-1.57(m,9H),1.55-1.51(m,13H),1.44(s,9H).

[0570] Step 2: (4S)-2-[(4S)-4-{[(2Z)-2-(2-amino-1,3-thiazolyl-4-yl)-2-{[(1-carboxycyclopropyl)oxy]imino}acetyl]amino}-3-oxo-1,2-oxazolidine-2-yl]-4-(2,3-dihydroxy-5,7-dioxo-5,7-dihydro-6H-pyrrolo[3,4-b]pyridin-6-yl)-5-oxooxopentane-2-carboxylic acid (Compound 3)

[0571]

[0572] A solution of 74 tert-butyl ester of (4S)-2-[(4S)-4-{[(2Z)-2-{2-[(tert-butyloxycarbonyl)amino]-1,3-thiazolyl-4-yl}-2-({[1-(tert-butyloxycarbonyl)cyclopropyl]oxy}imino)acetyl]amino}-3-oxo-1,2-oxazolidine-2-yl]-4-(5,7-dioxo-2,2-diphenyl-5,7-dihydro-2H,6H-[1,3]dioxane[4,5-b]pyrrolo[3,4-e]pyridin-6-yl)-5-oxooxopentane-2-carboxylic acid tert-butyl ester 74 (50 mg, 0.05 mmol) was cooled to -50 °C and boron trichloride solution (1.0 M in DCM, 0.39 mL, 0.39 mmol) was added dropwise. The reaction mixture was stirred at -30°C to -25°C for 2.5 hours, then cooled to -50°C, and a solution of NaHCO3 (75 mg) and Na2HPO4 (24 mg) in H2O (4 mL) was added. The resulting heterogeneous mixture was stirred at 0–5°C (ice-water bath) for 20 minutes, then stirred at room temperature until the aqueous phase thawed. The different layers were separated, and the organic layer was carefully removed. Purification was then performed by C-18 reversed-phase column chromatography using a Biotage system and a mixture of acetonitrile containing 0.1% formic acid and an aqueous solution containing 0.1% formic acid as the mobile phase. The fraction containing the pure product was collected and lyophilized to give (4S)-2-[(4S)-4-{[(2Z)-2-(2-amino-1,3-thiazolyl-4-yl)-2-{[(1-carboxycyclopropyl)oxy]imino}acetyl]amino}-3-oxo-1,2-oxazolidine-2-yl]-4-(2,3-dihydroxy-5,7-dioxo-5,7-dihydro-6H-pyrrolo[3,4-b]pyridin-6-yl)-5-oxooxopentane-2-carboxylic acid compound 3 (8 mg, 25%), as a yellow solid.

[0573] 1¹H-NMR (599 MHz; mixture of D₂O and CD₃CN) δ 7.06 (s, ¹H), 7.04 (s, ¹H), 5.40–5.16 (m, ¹H), 5.14–5.03 (m, ¹H), 4.69 (dt, J = 17.4, 8.7 Hz, ¹H), 4.29–4.18 (m, ¹H), 3.52–3.26 (m, ¹H), 2.87–2.63 (m, ¹H), 1.42 (s, 2H), 1.34 (s, 2H). No interchangeable protons were observed in D₂O.

[0574] MS(ESI)m / z:[M+H] + 662.0

[0575] Example 12

[0576] 4-({(Z)-[1-(2-amino-1,3-thiazolyl-4-yl)-2-({(4S)-2-[(4S)-2-carboxy-4-(2,3-dihydroxy-5,7-dioxo-5,7-dihydro-6H-pyrrolo[3,4-b]pyridin-6-yl)-5-oxooxopentane-2-yl]-3-oxo-1,2-oxozolidine-4-yl}amino)-2-oxoethylidene]amino}oxy)oxohexane-4-carboxylic acid (Compound 26, Table 1)

[0577]

[0578] Step 1: 4-({(Z)-[1-{2-[(tert-butyloxycarbonyl)amino]-1,3-thiazolyl-4-yl}-2-({(4S)-2-[(4S)-2-(tert-butyloxycarbonyl)-4-(5,7-dioxo-2,2-diphenyl-5,7-dihydro-2H,6H-[1,3]dioxacyclo[4,5-b]pyrrolo[3,4-e]pyridin-6-yl)-5-oxooxopentane-2-yl]-3-oxo-1,2-oxazolidine-4-yl}amino)-2-oxoethylidene]amino}oxy)oxohexane-4-carboxylic acid tert-butyl ester (75)

[0579]

[0580] At 0°C, tert-butyl 4-({(Z)-[1-{2-[(tert-butoxycarbonyl)amino]-1,3-thiazolyl}-2-oxo-2-{(4S)-2-[(4S)-3-oxo-1,2-oxazolidine-4-yl]amino}ethylene)amino]oxy)-oxohexane-4-carboxylic acid tert-butyl ester 37 (293 mg, 0.53 mmol, as in step 7 of Example 5) and (2S)-5-tert-butoxy-2-(5,7-dioxane)carboxylic acid tert-butyl ester was added to the mixture. A mixture of 2,2-diphenyl-5,7-dihydro-2H,6H-[1,3]dioxane[4,5-b]pyrrolo[3,4-e]pyridin-6-yl)-4,5-dioxovaleric acid 71 (316 mg, 0.58 mmol, prepared as described in step 4 of Example 10) was added to anhydrous THF (9 mL) with DMAP (14 mg, 0.11 mmol), followed by DCC (153 mg, 0.74 mmol). The reaction mixture was allowed to be slowly heated to room temperature and stirred for 18 hours. The mixture was then concentrated, the residue was treated with hexane containing 30% DCM, and the precipitated solids were removed by filtration. The filtrate was concentrated under reduced pressure, and the crude product was purified by silica gel column chromatography using a gradient of hexane containing 10-40% ethyl acetate to obtain 75 (238 mg, 42%) of 4-({(Z)-[1-{2-[(tert-butyloxycarbonyl)amino]-1,3-thiazolyl-4-yl}-2-({(4S)-2-[(4S)-2-(tert-butyloxycarbonyl)-4-(5,7-dioxo-2,2-diphenyl-5,7-dihydro-2H,6H-[1,3]dioxacyclo[4,5-b]pyrrolo[3,4-e]pyridin-6-yl)-5-oxooxopentane-2-yl]-3-oxo-1,2-oxazolidine-4-yl}amino)-2-oxoethylidene]amino}oxy)oxohexane-4-carboxylic acid tert-butyl ester, which was a light brown foam.

[0581] 1 H NMR (400MHz, CDCl3) δ8.26-8.01(m,2H),7.59-7.56(m,4H),7.46-7.42(m,7H),7.37-7.34(m,1H),5.46-4.88(m,3H),4.39-4. 21(m,1H),3.89-3.33(m,5H),2.91-2.77(m,1H),2.26-2.12(m,4H),1.60-1.57(m,9H),1.56-1.51(m,9H),1.49-1.42(m,9H).

[0582] Step 2: 4-({(Z)-[1-(2-amino-1,3-thiazolyl-4-yl)-2-({(4S)-2-[(4S)-2-carboxy-4-(2,3-dihydroxy-5,7-dioxo-5,7-dihydro-6H-pyrrolo[3,4-b]pyridin-6-yl)-5-oxooxopentane-2-yl]-3-oxo-1,2-oxozolidine-4-yl}amino)-2-oxoethylene]amino}oxy)oxohexyl-4-carboxylic acid (Compound 26)

[0583]

[0584] At -50°C, a solution containing 4-({(Z)-[1-{2-[(tert-butoxycarbonyl)amino]-1,3-thiazolyl-4-yl}-2-({(4S)-2-[(4S)-2-(tert-butoxycarbonyl)-4-(5,7-dioxo-2,2-diphenyl-5,7-dihydro-2H,6H-[1,3]dioxane-[4,5-b]pyrrolo[3,4-e]pyridine) was prepared. [-6-yl]-5-oxooxopentane-2-yl]-3-oxo-1,2-oxazolidine-4-yl]amino]-2-oxoethylidene]amino]oxy)oxohexane-4-carboxylic acid tert-butyl ester 75 (238 mg, 0.22 mmol) in anhydrous DCM (17 mL), with boron trichloride solution (1.0 M in DCM, 1.76 mL, 1.76 mmol) added dropwise. The reaction mixture was stirred at -50 °C to -25 °C for 2.5 h, and then an aqueous solution of NaHCO3 (362 mg) and Na2HPO4 (114.5 mg) (19.6 mL) was added at -50 °C. The resulting heterogeneous mixture was stirred at 0–5 °C (ice-water bath) for 20 min, and then stirred at room temperature until the aqueous phase thawed. The mixture was filtered through a pinhole filter, and the organic layer was carefully separated. The aqueous layer was then purified by C-18 reversed-phase chromatography using a Biotage system and a mixture of acetonitrile containing 0.1% formic acid and an aqueous solution containing 0.1% formic acid as the mobile phase. The product containing fractions was collected and lyophilized to give 26 (47 mg, 30%) of 4-({(Z)-[1-(2-amino-1,3-thiazolyl-4-yl)-2-({(4S)-2-[(4S)-2-carboxy-4-(2,3-dihydroxy-5,7-dioxo-5,7-dihydro-6H-pyrrolo[3,4-b]pyridin-6-yl)-5-oxooxopentane-2-yl]-3-oxo-1,2-oxozolidine-4-yl}amino)-2-oxoethylene]amino}oxy)oxohexane-4-carboxylic acid as a yellow solid.

[0585] 1¹H NMR (400 MHz, mixture of D₂O and CD₃CN) δ 7.07–7.06 (m, 2H), 5.39–5.07 (m, 2H), 4.74–4.67 (m, 1H), 4.30–4.23 (m, 1H), 3.78–3.72 (m, 2H), 3.60–3.28 (m, 3H), 2.86–2.64 (m, 1H), 2.12–1.96 (m, 4H). No interchangeable protons were observed in D₂O.

[0586] MS(ESI)m / z: [M+1] + 706.0

[0587] Example 13

[0588] 3-({(Z)-[1-(2-amino-1,3-thiazo-4-yl)-2-({(4S)-2-[(4S)-2-carboxy-4-(2,3-dihydroxy-5,7-dioxo-5,7-dihydro-6H-pyrrolo[3,4-b]pyridin-6-yl)-5-oxooxopentane-2-yl]-3-oxo-1,2-oxazolidine-4-yl}amino)-2-oxoethylidene]amino}oxy)-8-oxoheterocyclic[3.2.1]octane-3-carboxylic acid (compound 27, Table 1)

[0589]

[0590] Step 1: 3-({(Z)-[1-{2-[(tert-butyloxycarbonyl)amino]-1,3-thiazolyl-4-yl}-2-({(4S)-2-[(4S)-2-(tert-butyloxycarbonyl)-4-(5,7-dioxo-2,2-diphenyl-5,7-dihydro-2H,6H-[1,3]dioxacyclo[4,5-b]pyrrolo[3,4-e]pyridin-6-yl)-5-oxooxopentane-2-yl]-3-oxo-1,2-oxazolidine-4-yl}amino)-2-oxoethylidene]amino}oxy)-8-oxabicyclo[3.2.1]octane-3-carboxylic acid tert-butyl ester (76)

[0591]

[0592] At 0°C, tert-butyl octane-3-carboxylic acid 60 (337 mg, 0.62 mmol, prepared as described in step 6 of Example 8) containing 3-{[(Z)-(1-{2-[(tert-butoxycarbonyl)amino]-1,3-thiazolyl}-2-oxo-2-{[(4S)-3-oxo-1,2-oxazolidine-4-yl]amino}ethylene)amino]oxy}-8-oxabicyclo[3.2.1]octane-3-carboxylic acid tert-butyl ester 60 (337 mg, 0.62 mmol, prepared as described in step 6 of Example 8) and (2S)-5-tert-butoxy-2- (5,7-dioxo-2,2-diphenyl-5,6-dihydro-2H,6H-[1,3]dioxane[4,5-b]pyrrolo[3,4-e]pyridin-6-yl)-4,5-dioxovaleric acid 71 (343 mg, 0.59 mmol, prepared as described in step 4 of Example 1) was added to THF (9 mL) with DMAP (15 mg, 0.12 mmol), followed by DCC (171 mg, 0.83 mmol). The reaction mixture was allowed to be gradually heated and stirred at room temperature for 18 hours. Subsequently, the mixture was concentrated, and the residue was ground with hexane containing 30% DCM and filtered. The filtrate was concentrated under reduced pressure, and the crude product was purified by silica gel column chromatography using a gradient of hexane containing 10-40% ethyl acetate to obtain 76 (274 mg, 42%) of 3-({(Z)-[1-{2-[(tert-butyloxycarbonyl)amino]-1,3-thiazolyl-4-yl}-2-({(4S)-2-[(4S)-2-(tert-butyloxycarbonyl)-4-(5,7-dioxo-2,2-diphenyl-5,7-dihydro-2H,6H-[1,3]dioxacyclo[4,5-b]pyrrolo[3,4-e]pyridin-6-yl)-5-oxooxopentane-2-yl]-3-oxo-1,2-oxazolidine-4-yl}amino)-2-oxoethylidene]amino}oxy)-8-oxabicyclo[3.2.1]octane-3-carboxylic acid tert-butyl ester, a light brown foam.

[0593] 1 H NMR (400MHz, CDCl3) δ8.19-8.01(m,2H),7.60-7.54(m,4H),7.47-7.42(m,7H),7.36(d,J=4.2Hz,1H),5.47-4.89(m,3H),4.50-4 .22(m,3H),3.71-3.34(m,1H),2.92-2.79(m,1H),2.44-2.19(m,4H),2.05-1.87(m,4H),1.61-1.53(m,18H),1.46-1.44(m,9H).

[0594] Step 2: 3-({(Z)-[1-(2-amino-1,3-thiazolyl-4-yl)-2-({(4S)-2-[(4S)-2-carboxy-4-(2,3-dihydroxy-5,7-dioxo-5,7-dihydro-6H-pyrrolo[3,4-b]pyridin-6-yl)-5-oxooxopentane-2-yl]-3-oxo-1,2-oxazolidine-4-yl}amino)-2-oxoethylidene]amino}oxy)-8-oxoheterocyclic[3.2.1]octane-3-carboxylic acid (compound 27)

[0595]

[0596] At -50°C, an agent containing 3-({(Z)-[1-{2-[(tert-butoxycarbonyl)amino]-1,3-thiazolyl-4-yl}-2-({(4S)-2-[(4S)-2-(tert-butoxycarbonyl)-4-(5,7-dioxo-2,2-diphenyl-5,7-dihydro-2H,6H-[1,3]dioxono[4,5-b]pyrrolo[3,4-e]pyridin-6-yl)) [-5-oxooxopentane-2-yl]-3-oxo-1,2-oxazolidine-4-yl]amino)-2-oxoethylidene]amino)oxy)-8-oxabicyclo[3.2.1]octane-3-carboxylic acid tert-butyl ester 76 (265 mg, 0.24 mmol) was added dropwise to anhydrous DCM (17 mL), with boron trichloride solution (1.0 M in DCM, 1.91 mL, 1.91 mmol). The reaction mixture was stirred at -50 °C to -25 °C for 2.5 h, and then a solution of NaHCO3 (393 mg) and Na2HPO4 (124 mg) in water (21.3 mL) was added at -50 °C. The resulting heterogeneous mixture was stirred at 5–10 °C for 20 min using an ice-water bath instead of a cold water bath, and then stirred at room temperature until the aqueous phase was completely thawed. The mixture was filtered through a pinhole filter, and the organic layer was carefully separated. Using a Biotage system and acetonitrile containing 0.1% formic acid and an aqueous solution containing 0.1% formic acid as eluents, the aqueous solution was immediately subjected to C-18 reversed-phase column chromatography. Fractions containing the pure product were combined and lyophilized to give 27 (95 mg, 54%) of 3-({(Z)-[1-(2-amino-1,3-thiazo-4-yl)-2-({(4S)-2-[(4S)-2-carboxy-4-(2,3-dihydroxy-5,7-dioxo-5,7-dihydro-6H-pyrrolo[3,4-b]pyridin-6-yl)-5-oxooxopentane-2-yl]-3-oxo-1,2-oxazolidine-4-yl}amino)-2-oxoethylidene]amino}oxy)-8-oxoheterocyclic[3.2.1]octane-3-carboxylic acid, as a yellow solid.

[0597] 1¹H NMR (400 MHz, mixture of D₂O and CD₃CN) δ 7.13–7.11 (m, 1H), 7.07–7.06 (m, 1H), 5.38–5.24 (m, 1H), 5.14–5.07 (m, 1H), 4.74–4.68 (m, 1H), 4.42–4.39 (m, 2H), 4.34–4.24 (m, 1H), 3.48–3.28 (m, 1H), 2.87–2.66 (m, 1H), 2.30–2.23 (m, 2H), 2.09–2.04 (m, 2H), 1.83–1.70 (m, 4H). No interchangeable protons were observed in D₂O.

[0598] MS(ESI)m / z: [M+1] + 732.0

[0599] Example 14

[0600] 4S)-2-[(4S)-4-{[(2Z)-2-(2-amino-1,3-thiazolyl-4-yl)-2-{[(2-carboxypropane-2-yl)oxy]imino}acetyl]amino}-3-oxo-1,2-oxazolidine-2-yl]-4-(5,6-dihydroxy-1-oxo-1,3-dihydro-2H-isoindol-2-yl)-5-oxooxopentane-2-carboxylic acid (Compound 35, Table 1)

[0601]

[0602] Step 1: Methyl 6-methyl-2,2-diphenyl-2H-1,3-benzodioxazole-5-carboxylate (78)

[0603]

[0604] A mixture of methyl 4,5-dihydroxy-2-methylbenzoate 77 (7.45 g, 40.90 mmol) and 1,1′-(dichloromethane)diphenyl (7.86 mL, 40.94 mmol) was heated to 170 °C and stirred at 170 °C for 30 min. The reaction mixture was cooled and then concentrated under reduced pressure. The crude product was purified by silica gel column chromatography using a gradient of hexane containing 0–6% ethyl acetate, followed by grinding with hexane to give methyl 6-methyl-2,2-diphenyl-2H-1,3-benzodioxazole-5-carboxylic acid 78 (12.37 g, 87%) as a white solid.

[0605] 1H NMR (400MHz, CDCl3) δ7.54-7.50(m,4H),7.45(s,1H),7.36-7.32(m,6H),6.72(s,1H),3.81(s,3H),2.50(s,3H).

[0606] MS(ESI)m / z: [M+1] + 347.1

[0607] Step 2: Methyl 6-(bromomethyl)-2,2-diphenyl-2H-1,3-benzodioxazole-5-carboxylic acid ester (79)

[0608]

[0609] At room temperature, AIBN (587 mg, 3.57 mmol) was added to 120 mL of carbon tetrachloride containing a mixture of methyl 6-methyl-2,2-diphenyl-2H-1,3-benzodioxazole-5-carboxylic acid 78 (12.37 g, 35.71 mmol) and NBS (7.0 g, 39.33 mmol). The reaction mixture was heated to 70 °C and stirred overnight at 70 °C, then cooled and concentrated under reduced pressure. The residue was ground with hexane and the insoluble matter was filtered off. The filtrate was concentrated, and the crude product was purified by silica gel column chromatography using a gradient of hexane containing 0–4% ethyl acetate to give methyl 6-(bromomethyl)-2,2-diphenyl-2H-1,3-benzodioxazole-5-carboxylic acid 79 (9.83 g, 65%) as a colorless, viscous oil.

[0610] 1 H NMR (400MHz, CDCl3) δ7.56-7.53(m,4H),7.51(s,1H),7.40-7.36(m,6H),6.96(s,1H),4.93(s,2H),3.89(s,3H).

[0611] Step 3: 4-tert-butyl 1-methyl(2S)-2-(5-oxo-2,2-diphenyl-5,7-dihydro-2H,6H-[1,3]dioxane[4,5-f]isoindol-6-yl)succinic acid (81)

[0612]

[0613] DIPEA (4.76 mL, 27.33 mmol) was added dropwise to anhydrous acetonitrile (60 mL) containing a mixture of methyl 6-(bromomethyl)-2,2-diphenyl-2H-1,3-benzodioxazole-5-carboxylic acid 79 (5.28 g, 12.42 mmol) and 1-methyl-L-aspartic acid 4-tert-butyl hydrochloride 80 (3.28 g, 13.68 mmol). The reaction mixture was stirred at 0 °C for 10 min, then at room temperature for 1 h. Subsequently, the reaction mixture was heated to reflux and stirred at reflux temperature for 20 h, then cooled and concentrated. The residue was treated with ethyl acetate, the solids were removed by filtration, and the filtrate was concentrated. The crude product was purified by silica gel column chromatography using a gradient of hexane containing 0-4% ethyl acetate to obtain 4-tert-butyl 1-methyl(2S)-2-(5-oxo-2,2-diphenyl-5,7-dihydro-2H,6H-[1,3]dioxane[4,5-f]isoindol-6-yl)succinate 81 (5.08 g, 79%) as a white foam.

[0614] 1 H NMR (400MHz, CDCl3) δ7.59-7.54(m,4H),7.42-7.35(m,6H),7.30(s,1H),6.91(d,J=0.3Hz,1H) ,5.30(dd,J=8.5,5.7Hz,1H),4.45-4.28(m,2H),3.71(s,3H),3.01-2.84(m,2H),1.39(s,9H).

[0615] MS(ESI)m / z: [M+1] + 516.2

[0616] Step 4: (3S)-4-methoxy-4-oxo-3-(5-oxo-2,2-diphenyl-5,7-dihydro-2H,6H-[1,3]dioxane[4,5-f]isoindol-6-yl)butyric acid (82)

[0617]

[0618] At -78 °C, boron trichloride solution (0.86 mL, 1.0 M in DCM, 0.86 mmol) was added dropwise to an anhydrous DCM solution (12 mL) containing 1-methyl(2S)-2-(5-oxo-2,2-diphenyl-5,7-dihydro-2H,6H-[1,3]dioxono[4,5-f]isoindol-6-yl)succinic acid tert-butyl 81 (245 mg, 0.475 mmol). The reaction mixture was stirred at -78 °C for 30 min, then gradually heated to -45 °C. Subsequently, the reaction mixture was diluted with ethyl acetate, and water was added at -50 °C. The resulting mixture was stirred until the aqueous phase thawed, then the organic layer was separated, dried over sodium sulfate, filtered, and concentrated under reduced pressure. The residue was ground with a mixture of ethyl acetate and hexane, the precipitated solid was collected by filtration and further dried under high vacuum to give (3S)-4-methoxy-4-oxo-3-(5-oxo-2,2-diphenyl-5,7-dihydro-2H,6H-[1,3]dioxane[4,5-f]isoindol-6-yl)butyric acid 82 (154 mg, 71%) as a white solid.

[0619] 1 H NMR(400MHz,DMSO-d6)δ12.52(bs,1H),7.54-7.50(m,4H),7.47-7.40(m,6H),7.26(s, 1H), 7.25 (s, 1H), 5.08 (t, J = 7.0Hz, 1H), 4.33 (s, 2H), 3.60 (s, 3H), 2.99-2.83 (m, 2H).

[0620] MS(ESI)m / z: [M+1] + 460.1

[0621] Step 5: (2S)-5-cyano-4-oxo-2-(5-oxo-2,2-diphenyl-5,7-dihydro-2H,6H-[1,3]dioxane[4,5-f]isoindol-6-yl)-5-(1λ 4 methyl thiophene-1-yl)valerate (83)

[0622]

[0623] HATU (172 mg, 0.45 mmol) was added to an anhydrous DMF (4 mL) solution containing (3S)-4-methoxy-4-oxo-3-(5-oxo-2,2-diphenyl-5,7-dihydro-2H,6H-[1,3]dioxono[4,5-f]isoindol-6-yl)butyric acid 82 (189 mg, 0.41 mmol). The resulting mixture was cooled to 0 °C, and DIPEA (0.22 mL, 1.26 mmol) was added, followed by 1-(cyanomethyl)thiophene-1-ammonium bromide (112 mg, 0.54 mmol). The reaction mixture was stirred at 0 °C for 30 min, then allowed to slowly heat and stir overnight at room temperature. Subsequently, the mixture was diluted with water and extracted with ethyl acetate by quenching the reaction with a saturated ammonium chloride solution at 0 °C. The combined organic extracts were washed with brine, dried over sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography using a gradient of acetone containing 0-40% ethyl acetate to give (2S)-5-cyano-4-oxo-2-(5-oxo-2,2-diphenyl-5,7-dihydro-2H,6H-[1,3]dioxane[4,5-f]isoindol-6-yl)-5-(1λ) 4 Methyl thiophene-1-yl)valerate 83 (247 mg, quantitative), was a white foam.

[0624] 1 H NMR (400MHz, CDCl3) δ7.59-7.53(m,4H),7.42-7.36(m,6H),7.27(s,1H),6.95-6.91(m,1H),5.55-5.50 (m,1H),4.51-4.36(m,2H),3.77-3.71(m,3H),3.55-3.12(m,6H),2.53-2.40(m,2H),2.12-2.00(m,2H).

[0625] Step 6: (4S)-5-methoxy-2,5-dioxo-4-(5-oxo-2,2-diphenyl-5,7-dihydro-2H,6H-[1,3]dioxane[4,5-f]isoindol-6-yl)valerate (84)

[0626]

[0627] To contain (2S)-5-cyano-4-oxo-2-(5-oxo-2,2-diphenyl-5,7-dihydro-2H,6H-[1,3]dioxane[4,5-f]isoindol-6-yl)-5-(1λ 4To a mixture of THF and water (1:1, 9 mL) of methyl thiophene-1-yl)valerate 83 (244 mg, 0.41 mmol), OXONE (potassium persulfate complex salt) (264 mg, 0.86 mmol) and the reaction mixture were added, followed by the addition of additional OXONE (132 mg, 0.43 mmol) and water (2 mL), and stirring was continued at room temperature for 1 hour. The reaction mixture was concentrated to remove THF, and the aqueous phase was extracted with ethyl acetate. The combined organic extracts were dried over sodium sulfate, filtered, and concentrated under reduced pressure to give (4S)-5-methoxy-2,5-dioxo-4-(5-oxo-2,2-diphenyl-5,7-dihydro-2H,6H-[1,3]dioxono[4,5-f]isoindol-6-yl)valerate 84 (220 mg), as a white foam, for the next step.

[0628] 1 H NMR (400MHz, CDCl3) δ7.56-7.53(m,4H),7.39-7.36(m,6H),7.28(s,1H),6.88(s,1H),5.38-5.34( m,1H),4.43-4.27(m,2H),3.74(dd,J=17.8,6.3Hz,1H),3.70(s,3H),3.46(dd,J=17.8,7.7Hz,1H).

[0629] Step 7: 1-tert-butyl-5-methyl(4S)-2-oxo-4-(5-oxo-2,2-diphenyl-5,7-dihydro-2H,6H-[1,3]dioxane[4,5-f]isoindole-6-yl)glutarate (85)

[0630]

[0631] At 0 °C, tert-butyl N,N′-di(propane-2-yl)carbamate (0.4 mL, 1.54 mmol) was slowly added to an anhydrous THF solution containing (4S)-5-methoxy-2,5-dioxo-4-(5-oxo-2,2-diphenyl-5,7-dihydro-2H,6H-[1,3]dioxono[4,5-f]isoindol-6-yl)valerate 84 (220 mg, 0.41 mmol). The reaction mixture was stirred at 0 °C for 10 min, then stirred at room temperature for 3 h. The mixture was concentrated under reduced pressure, and the residue was ground with hexane containing 25% DCM. The insoluble substances were removed by filtration, the filtrate was concentrated, and the crude product was purified by silica gel column chromatography using a gradient of hexane containing 10-30% ethyl acetate to obtain 1-tert-butyl-5-methyl(4S)-2-oxo-4-(5-oxo-2,2-diphenyl-5,7-dihydro-2H,6H-[1,3]dioxane[4,5-f]isoindol-6-yl)glutarate 85 (181 mg, less than 81%) as a white solid.

[0632] 1 H NMR (400MHz, CDCl3) δ7.57-7.54(m,4H),7.40-7.36(m,6H),7.28(s,1H),6.90(s,1H),5.24(dd,J=7.5,5.6Hz,1H),4.45(d,J =16.5Hz,1H),4.27(d,J=16.5Hz,1H),3.71(s,3H),3.63(dd,J=18.5,5.6Hz,1H),3.49(dd,J=18.5,7.5Hz,1H),1.53(s,9H).

[0633] MS(ESI)m / z: [M+1] + 544.2

[0634] Step 8: (2S)-5-tert-butoxy-4,5-dioxo-2-(5-oxo-2,2-diphenyl-5,7-dihydro-2H,6H-[1,3]dioxane[4,5-f]isoindol-6-yl)valerate (86)

[0635]

[0636] A mixture of THF and water (1:1, 4 mL) containing 85 (181 mg, 0.33 mmol) of 1-tert-butyl glutarate of 5-methyl(4S)-2-oxo-4-(5-oxo-2,2-diphenyl-5,7-dihydro-2H,6H-[1,3]dioxono[4,5-f]isoindol-6-yl)glutarate was cooled to 0 °C, and LiOH·H₂O (14 mg, 0.33 mmol) was added. The reaction mixture was stirred at 0 °C for 40 min, then at room temperature for 10 min. The reaction mixture was then cooled to 0 °C, and 1 M HCl (0.33 mL) was added. The resulting mixture was diluted with water and extracted with ethyl acetate. The combined organic extracts were dried over sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography using hexane containing 30% ethyl acetate and then by a gradient DCM containing 0-5% MeOH to give (2S)-5-tert-butoxy-4,5-dioxo-2-(5-oxo-2,2-diphenyl-5,7-dihydro-2H,6H-[1,3]dioxono[4,5-f]isoindol-6-yl)valerate 86 (115 mg, 65%) as a white foam.

[0637] 1 H NMR(400MHz,DMSO-d6)δ7.58-7.54(m,4H),7.42-7.36(m,6H),7.32-7.27(m,1H),6.93-6.87(m,1H ),5.63-5.51(m,1H),4.39-4.23(m,2H),3.01-2.85(m,1H),2.63-2.35(m,1H),1.57-1.52(m,9H).

[0638] MS(ESI)m / z: [M+1] + 530.2

[0639] Step 9: (4S)-2-[(4S)-4-{[(2Z)-2-{2-[(tert-butoxycarbonyl)amino]-1,3-thiazolyl-4-yl}-2-{[(1-tert-butoxy-2-methyl-1-oxopropane-2-yl)oxy]imino}acetyl]amino}-3-oxo-1,2-oxazolidine-2-yl]-5-oxo-4-(5-oxo-2,2-diphenyl)5,7-dihydro-2H,6H-[1,3]dioxacyclo[4,5-f]isoindol-6-yl)oxacyclopentane-2-carboxylic acid tert-butyl ester (87)

[0640]

[0641] At 0 °C, DMAP (6 mg, 0.49 mmol) was added to anhydrous THF (4 mL) containing a mixture of (2S)-5-tert-butoxy-4,5-dioxo-2-(5-oxo-2,2-diphenyl-5,7-dihydro-2H,6H-[1,3]dioxane-[4,5-f]isoindol-6-yl)valeric acid 86 (112 mg, 0.21 mmol) and 2-{[(Z)-(1-{2-[(tert-butoxycarbonyl)amino]-1,3-thiazo-4-yl}-2-oxo-2-{[(4S)-3-oxo-1,2-oxazolidine-4-yl]amino}ethylene)amino]oxy}-2-methylpropionate tert-butyl ester 72 (109 mg, 0.21 mmol). The reaction mixture was allowed to be gradually heated to room temperature and stirred for 18 hours. The mixture was then concentrated, the residue was treated with hexane containing 25% DCM, and filtered. The filtrate was concentrated under reduced pressure, and the crude product was purified by silica gel column chromatography using a gradient of hexane containing 10-40% ethyl acetate to obtain (4S)-2-[(4S)-4-{[(2Z)-2-{2-[(tert-butoxycarbonyl)amino]-1,3-thiazolyl-4-yl}-2-{[(1-tert-butoxy-2-methyl-1-oxopropane-2-yl)oxy]imino}acetyl]amino}-3-oxo-1,2-oxazolidine-2-yl]-5-oxo-4-(5-oxo-2,2-diphenyl)5,7-dihydro-2H,6H-[1,3]dioxacyclo[4,5-f]isoindol-6-yl)oxacyclopentane-2-carboxylic acid tert-butyl ester 87 (132 mg, 61%), as a white foam.

[0642] 1 H NMR (400MHz, CDCl3) δ8.21-8.18(m,1H),7.57-7.52(m,4H),7.41-7.34(m,7H),7.30-7.29(m,1H),6.92-6.90(m,1H),5.47-5.39(m,1 H),5.20-4.85(m,2H),4.34-4.21(m,3H),3.51-3.31(m,1H),2.93-2.49(m,1H),1.61-1.58(m,6H),1.55-1.52(m,18H),1.43(s,9H).

[0643] Step 10: 4S)-2-[(4S)-4-{[(2Z)-2-(2-amino-1,3-thiazolyl-4-yl)-2-{[(2-carboxypropane-2-yl)oxy]imino}acetyl]amino}-3-oxo-1,2-oxazolidine-2-yl]-4-(5,6-dihydroxy-1-oxo-1,3-dihydro-2H-isoindol-2-yl)-5-oxooxopentane-2-carboxylic acid (Compound 35)

[0644]

[0645] A stirred solution of anhydrous DCM (7 mL) containing (4S)-2-[(4S)-4-{[(2Z)-2-{2-[(tert-butoxycarbonyl)amino]-1,3-thiazolyl-4-yl}-2-{[(1-tert-butoxy-2-methyl-1-oxopropane-2-yl)oxy]imino}acetyl]amino}-3-oxo-1,2-oxazolidine-2-yl]-5-oxo-4-(5-oxo-2,2-diphenyl)5,7-dihydro-2H,6H-[1,3]dioxacyclo[4,5-f]isoindol-6-yl)oxacyclopentane-2-carboxylic acid tert-butyl ester 87 (132 mg, 0.13 mmol) was cooled to -50 °C, and boron trichloride solution (1.03 mL, 1.03 mmol, 1.0 M in DCM) was added dropwise. The reaction mixture was stirred at -50°C to 35°C for 2.5 hours, then cooled to -50°C, and an aqueous solution (11.4 mL) of NaHCO3 (212 mg) and Na2HPO4 (67 mg) was added. Using an ice-water bath instead of a cold water bath, the heterogeneous mixture was stirred at 5–10°C for 10 minutes, then stirred at ambient temperature until the aqueous layer thawed. The mixture was filtered through a pinhole filter, and the organic layer was carefully removed. The aqueous solution was then immediately subjected to C18 reversed-phase chromatography using a Biotage system and a mixture of acetonitrile containing 0.1% formic acid and an aqueous solution containing 0.1% formic acid as the mobile phase. The pure fraction was collected and freeze-dried to give compound 35 (27 mg, 32%), a white solid, consisting of 4S)-2-[(4S)-4-{[(2Z)-2-(2-amino-1,3-thiazolyl-4-yl)-2-{[(2-carboxypropane-2-yl)oxy]imino}acetyl]amino}-3-oxo-1,2-oxazolidine-2-yl]-4-(5,6-dihydroxy-1-oxo-1,3-dihydro-2H-isoindol-2-yl)-5-oxooxopentane-2-carboxylic acid.

[0646] 1¹H NMR (400 MHz, mixture of D₂O and CD₃CN) δ 7.15–7.13 (m, 1H), 7.10–7.06 (m, 1H), 7.00–6.98 (m, 1H), 5.46–5.08 (m, 2H), 4.77–4.68 (m, 1H), 4.42–4.23 (m, 3H), 3.46–3.32 (m, 1H), 2.91–2.62 (m, 1H), 1.53–1.47 (m, 6H). No interchangeable protons were observed in D₂O.

[0647] MS(ESI)m / z: [M+1] + 649.1

[0648] Example 15

[0649] (4S)-2-[(4S)-4-{[(2Z)-2-(2-amino-1,3-thiazolyl-4-yl)-2-{[(1-carboxycyclopropyl)oxy]imino}acetyl]amino}-3-oxo-1,2-oxazolidine-2-yl]-4-(5,6-dihydroxy-1-oxo-1,3-dihydro-2H-isoindol-2-yl)-5-oxooxopentane-2-carboxylic acid (Compound 28, Table 1)

[0650]

[0651] Step 1: (4S)-2-[(4S)-4-{[(2Z)-2-{2-[(tert-butyloxycarbonyl)amino]-1,3-thiazolyl-4-yl}-2-({[1-(tert-butyloxycarbonyl)cyclopropyl]oxy}imino)acetyl]amino}-3-oxo-1,2-oxazolidine-2-yl]-5-oxo-4-(5-oxo-2,2-diphenyl-5,7-dihydro-2H,6H-[1,3]dioxacyclo[4,5-f]isoindol-6-yl)oxacyclopentane-2-carboxylic acid tert-butyl ester (88)

[0652]

[0653] The mixture of 1-{[(Z)-(1-{2-[(tert-butoxycarbonyl)amino]-1,3-thiazolyl}-2-oxo-2-{[(4S)-3-oxo-1,2-oxazolidine-4-yl]amino}ethylene)amino]oxy}cyclopropane-1-carboxylic acid tert-butyl ester 5 (358 mg, 0.70 mmol, prepared as described in step 4 of Example 1) and (2S)-5-tert-butoxy-4,5-dioxo-2-(5-oxo-2,2-diphenyl-5,7-dihydro-2H,6H-[1,3]dioxane-[4,5-f]isoindol-6-yl)valerate 86 (370 mg, 0.70 mmol, prepared as described in step 8 of Example 14) was cooled to 0°C in anhydrous THF (20 mL). DMAP (17 mg, 0.14 mmol) was added, followed by DCC (202 mg, 0.98 mmol). The reaction mixture was stirred at 0 °C for 1 hour, then incubated overnight at room temperature. The solvent was evaporated at 25 °C, and the residue was ground with hexane (25 mL) containing 25% DCM. The precipitated solid was filtered and washed with hexane (15 mL) containing 25% DCM and hexane. The combined filtrates were concentrated under reduced pressure, and the crude product was purified by silica gel column chromatography using a gradient of hexane containing 10-50% ethyl acetate as eluent to obtain (4S)-2-[(4S)-4-{[(2Z)-2-{2-[(tert-butyloxycarbonyl)amino]-1,3-thiazolyl-4-yl}-2-({[1-(tert-butyloxycarbonyl)cyclopropyl]oxy}imino)acetyl]amino}-3-oxo-1,2-oxazolidine-2-yl]-5-oxo-4-(5-oxo-2,2-diphenyl-5,7-dihydro-2H,6H-[1,3]dioxacyclo[4,5-f]isoindol-6-yl)oxacyclopentane-2-carboxylic acid tert-butyl ester 88 (460 mg, 64% yield), as a grayish-white solid.

[0654] 1 H-NMR (400MHz; CDCl3) δ8.59(d,J=6.1Hz,1H),8.17(bs,1H),7.60-7.55(m,4H),7.42-7.39(m,6H),7.32(s,1H),6.93(s,1H),5.49-5.43(m,1H), 5.23-5.17(m,1H),4.94(t,J=8.3Hz,1H),4.37-4.25(m,3H),3.45-3.35 (m,1H),2.60(dd,J=14.0,10.4Hz,1H),1.58-1.51(m,22H),1.45(s,9H).

[0655] Step 2: (4S)-2-[(4S)-4-{[(2Z)-2-(2-amino-1,3-thiazolyl-4-yl)-2-{[(1-carboxycyclopropyl)oxy]imino}acetyl]amino}-3-oxo-1,2-oxazolidine-2-yl]-4-(5,6-dihydroxy-1-oxo-1,3-dihydro-2H-isoindol-2-yl)-5-oxooxopentane-2-carboxylic acid (Compound 28)

[0656]

[0657] A 22 mL anhydrous DCM solution containing (4S)-2-[(4S)-4-{[(2Z)-2-{2-[(tert-butyloxycarbonyl)amino]-1,3-thiazolyl}-2-({[1-(tert-butyloxycarbonyl)cyclopropyl]oxy}imino)acetyl]amino}-3-oxo-1,2-oxazolidine-2-yl]-5-oxo-4-(5-oxo-2,2-diphenyl-5,7-dihydro-2H,6H-[1,3]dioxacyclo[4,5-f]isoindol-6-yl)oxacyclopentane-2-carboxylic acid tert-butyl ester 88 (450 mg, 0.44 mmol) was cooled to -50 °C. A boron trichloride solution (3.52 mL, 3.52 mmol, 1.0 M in DCM) was added dropwise at -50 °C, and the reaction mixture was stirred at -45 °C to -30 °C for 2.5 h. Subsequently, the reaction mixture was cooled to -50 °C, and 40 mL of buffer solution (prepared by dissolving 776 mg NaHCO3 and 243 mg Na2HPO4 in 42 mL of water) was added. An ice-water bath was used instead of a cold water bath to allow the frozen heterogeneous mixture to thaw and separate into two layers. The organic phase was carefully removed, and the aqueous phase was collected and immediately purified by C18 reversed-phase chromatography using a Biotage system and a mixture of acetonitrile containing 0.1% formic acid and an aqueous solution containing 0.1% formic acid as the mobile phase. The fractions were combined and lyophilized to obtain (4S)-2-[(4S)-4-{[(2Z)-2-(2-amino-1,3-thiazolyl-4-yl)-2-{[(1-carboxycyclopropyl)oxy]imino}acetyl]amino}-3-oxo-1,2-oxazolidine-2-yl]-4-(5,6-dihydroxy-1-oxo-1,3-dihydro-2H-isoindol-2-yl)-5-oxooxopentane-2-carboxylic acid 28 (70 mg, 24%), which was a grayish-white foam.

[0658] 1¹H-NMR (400 MHz; mixture of D₂O and CD₃CN) δ 7.12 (s, ¹H), 7.09 (s, ¹H), 6.97 (s, ¹H), 5.40 (t, J = 10.1 Hz, ¹H), 5.15–5.06 (m, ¹H), 4.73–4.64 (m, ¹H), 4.35–4.21 (m, ³H), 3.37–3.30 (m, ¹H), 2.64 (dd, J = 14.2, 11.1 Hz, ¹H), 1.49–1.44 (m, ²H), 1.42–1.37 (m, ²H). No interchangeable protons were observed in D₂O.

[0659] MS(ESI)m / z: [M+1] + 647.0

[0660] Example 16

[0661] 4-({(Z)-[1-(2-amino-1,3-thiazolyl-4-yl)-2-({(4S)-2-[(4S)-2-carboxy-4-(5,6-dihydroxy-1-oxo-1,3-dihydro-2H-isoindol-2-yl)-5-oxooxopentane-2-yl]-3-oxo-1,2-oxazolidine-4-yl}amino)-2-oxoethylidene}amino}oxy)oxohexane-4-carboxylic acid (Compound 29, Table 1)

[0662]

[0663] Step 1: 4-({(Z)-[1-{2-[(tert-butyloxycarbonyl)amino]-1,3-thiazolyl-4-yl}-2-({(4S)-2-[(4S)-2-(tert-butyloxycarbonyl)-5-oxo-4-(5-oxo-2,2-diphenyl-5,7-dihydro-2H,6H-[1,3]dioxacyclo[4,5-f]isoindol-6-yl)oxacyclopentane-2-yl]]-3-oxo-1,2-oxazolidine-4-yl}amino)-2-oxoethylidene]amino}oxy)oxohexane-4-carboxylic acid tert-butyl ester (89)

[0664]

[0665] At 0°C, 86 (400 mg, 0.76 mmol, prepared as described in step 8 of Example 14) containing (2S)-5-tert-butoxy-4,5-dioxo-2-(5-oxo-2,2-diphenyl-5,7-dihydro-2H,6H-[1,3]dioxane[4,5-f]isoindol-6-yl)valeric acid 86 (400 mg, 0.76 mmol, prepared as described in step 8 of Example 14) and 4-{[(Z)-(1-{2-[(tert-butoxycarbonyl)amino]-1,3-... The mixture of 37 tert-butyl thiazole-4-yl}-2-oxo-2-{[(4S)-3-oxo-1,2-oxazolidine-4-yl]amino}ethylene)amino]oxy}oxohexane-4-carboxylic acid tert-butyl ester 37 (420 mg, 0.76 mmol) in anhydrous THF (10 mL) was stirred, and DMAP (19 mg, 0.16 mmol) was added, followed by DCC (219 mg, 1.06 mmol). The reaction mixture was allowed to be gradually heated to room temperature and stirred for 18 hours. The mixture was concentrated, and the residue was treated with hexane containing 50% DCM, and the precipitated solid was filtered off. The filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography using a gradient of hexane containing 20-45% ethyl acetate to obtain 89 (545 mg, 68%) of 4-({(Z)-[1-{2-[(tert-butyloxycarbonyl)amino]-1,3-thiazolyl-4-yl}-2-({(4S)-2-[(4S)-2-(tert-butyloxycarbonyl)-5-oxo-4-(5-oxo-2,2-diphenyl-5,7-dihydro-2H,6H-[1,3]dioxacyclo[4,5-f]isoindol-6-yl)oxacyclopentane-2-yl]]-3-oxo-1,2-oxazolidine-4-yl}amino)-2-oxoethylidene]amino}oxy)oxohexane-4-carboxylic acid tert-butyl ester, as a white foam.

[0666] 1 H NMR (400MHz, CDCl3) δ8.14-8.03(m,2H),7.57-7.52(m,4H),7.41-7.36(m,6H) ),7.34-7.32(m,1H),7.30-7.28(m,1H),6.92-6.89(m,1H),5.49-5.37(m,1H) ,5.23-4.84(m,2H),4.36-4.18(m,3H),3.85-3.66(m,4H),3.49-3.29(m,1H), 2.92-2.48(m,1H),2.24-2.09(m,4H),1.56-1.52(m,18H),1.44-1.40(m,9H).

[0667] Step 2: 4-({(Z)-[1-(2-amino-1,3-thiazolyl-4-yl)-2-({(4S)-2-[(4S)-2-carboxy-4-(5,6-dihydroxy-1-oxo-1,3-dihydro-2H-isoindol-2-yl)-5-oxooxopentane-2-yl]-3-oxo-1,2-oxazolidine-4-yl}amino)-2-oxoethylidene}amino}oxy)oxohexyl-4-carboxylic acid (Compound 29)

[0668]

[0669] At -50°C, boron trichloride solution (1.0 M in DCM, 4.06 mL, 4.06 mmol) was added dropwise to an anhydrous DCM solution containing 89 (541 mg, 0.51 mmol) of 4-({(Z)-[1-{2-[(tert-butyloxycarbonyl)amino]-1,3-thiazolyl}-2-({(4S)-2-[(4S)-2-(tert-butyloxycarbonyl)-5-oxo-4-(5-oxo-2,2-diphenyl-5,7-dihydro-2H,6H-[1,3]dioxacyclo[4,5-f]isoindol-6-yl)oxacyclopentane-2-yl]]-3-oxo-1,2-oxazolidine-4-yl}amino)-2-oxoethylidene]amino}oxy)oxohexane-4-carboxylic acid tert-butyl ester 89 (541 mg, 0.51 mmol). The reaction mixture was stirred at -50°C to 25°C for 2.5 hours, then cooled to -50°C, and a solution of NaHCO3 (835 mg) and Na2HPO4 (264 mg) in water (45.2 mL) was added. The resulting heterogeneous mixture was stirred at 5–10°C (ice-water bath) for 10 minutes, then stirred at ambient temperature until the aqueous phase thawed and the two layers separated. The mixture was filtered through a 1 μm pinhole filter to carefully remove the organic layer. The aqueous solution was then purified by C18 reversed-phase chromatography using a Biotage system and a mixture of acetonitrile containing 0.1% formic acid and an aqueous solution containing 0.1% formic acid as the eluent. The pure fraction was collected and freeze-dried to give 29 (161 mg, 46%) of 4-({(Z)-[1-(2-amino-1,3-thiazolyl-4-yl)-2-({(4S)-2-[(4S)-2-carboxy-4-(5,6-dihydroxy-1-oxo-1,3-dihydro-2H-isoindol-2-yl)-5-oxooxopentane-2-yl]-3-oxo-1,2-oxazolidine-4-yl}amino)-2-oxoethylene}amino}oxy)oxohexane-4-carboxylic acid, as a white solid.

[0670] 1¹H NMR (400 MHz, mixture of D₂O and CD₃CN) δ 7.13–7.06 (m, 2H), 6.97–6.96 (m, 1H), 5.44–5.06 (m, 2H), 4.76–4.67 (m, 1H), 4.40–4.22 (m, 3H), 3.78–3.72 (m, 2H), 3.60–3.52 (m, 2H), 3.44–3.29 (m, 1H), 2.90–2.61 (m, 1H), 2.14–1.96 (m, 4H). No interchangeable protons were observed in D₂O.

[0671] MS(ESI)m / z: [M+1] + 691.0

[0672] Example 17

[0673] 3-({(Z)-[1-(2-amino-1,3-thiazolyl-4-yl)-2-({(4S)-2-[(4S)-2-carboxy-4-(5,6-dihydroxy-1-oxo-1,3-dihydro-2H-isoindol-2-yl)-5-oxooxopentane-2-yl]-3-oxo-1,2-oxazolidine-4-yl}amino)-2-oxoethylidene}amino}oxy)8-oxabicyclo[3.2.1]octane-3-carboxylic acid (compound 30, Table 1)

[0674]

[0675] Step 1: 3-({(Z)-[1-{2-[(tert-butyloxycarbonyl)amino]-1,3-thiazolyl-4-yl}-2-({(4S)-2-[(4S)-2-(tert-butyloxycarbonyl)-5-oxo-4-(5-oxo-2,2-diphenyl-5,7-dihydro-2H,6H-[1,3]dioxacyclo[4,5-f]isoindol-6-yl)oxacyclopentane-2-yl]]-3-oxo-1,2-oxazolidine-4-yl}amino)-2-oxoethylidene]amino}oxy)-8-oxobicyclo[3.2.1]octane-3-carboxylic acid tert-butyl ester (90)

[0676]

[0677] At 0°C, 86 (461 mg, 0.87 mmol, prepared as described in step 8 of Example 14) of (2S)-5-tert-butoxy-4,5-dioxo-2-(5-oxo-2,2-diphenyl-5,7-dihydro-2H,6H-[1,3]dioxono[4,5-f]isoindol-6-yl)valeric acid 86 (461 mg, 0.87 mmol, prepared as described in step 8 of Example 14) and 3-{[(Z)-(1-{2-[(tert-butoxycarbonyl)amino)-1,3-thiazolyl-4-yl}-2- A mixture of oxo-2-{[(4S)-3-oxo-1,2-oxazolidine-4-yl]amino}ethylene)amino]oxy}-8-oxabicyclo[3.2.1]octane-3-carboxylic acid tert-butyl ester 60 (507 mg, 0.87 mmol, prepared as described in step 6 of Example 8) was added to anhydrous THF (4 mL) with DMAP (21 mg, 0.17 mmol), followed by DCC (252 mg, 1.2 mmol). The reaction mixture was allowed to be slowly heated to room temperature and stirred for 18 hours. The mixture was then concentrated, and the residue was treated with hexane containing 25% DCM. The solids were filtered off, and the filtrate was concentrated under reduced pressure. The crude product was purified by silica gel column chromatography using a gradient of hexane containing 10-40% ethyl acetate to obtain 90 (460 mg, 48%) of 3-({(Z)-[1-{2-[(tert-butyloxycarbonyl)amino]-1,3-thiazolyl-4-yl}-2-({(4S)-2-[(4S)-2-(tert-butyloxycarbonyl)-5-oxo-4-(5-oxo-2,2-diphenyl-5,7-dihydro-2H,6H-[1,3]dioxacyclo[4,5-f]isoindol-6-yl)oxacyclopentane-2-yl]]-3-oxo-1,2-oxazolidine-4-yl}amino)-2-oxoethylidene]amino}oxy)-8-oxobicyclo[3.2.1]octane-3-carboxylic acid tert-butyl ester, as a white foam.

[0678] 1 H-NMR (400MHz; CDCl3) δ8.27-8.25(m,1H),8.06(d,J=6.3Hz,1H),7.64-7.51(m,4H),7.45-7.37( m,6H),7.35(s,1H),7.32(s,1H),6.93(s,1H),5.51-5.43(m,1H),5.27-5.15(m,J=6.3Hz,1H),4. 97(t,J=8.3Hz,1H),4.52-4.42(m,2H),4.36-4.22(m,3H),3.44-3.34(m,1H),2.58(dd,J=14.0,1 0.4Hz,1H),2.43-2.25(m,4H),2.03-1.88(m,4H),1.69(s,2H),1.58-1.55(m,18H),1.44(s,9H).

[0679] Step 2: 3-({(Z)-[1-(2-amino-1,3-thiazolyl-4-yl)-2-({(4S)-2-[(4S)-2-carboxy-4-(5,6-dihydroxy-1-oxo-1,3-dihydro-2H-isoindol-2-yl)-5-oxooxopentane-2-yl]-3-oxo-1,2-oxazolidine-4-yl}amino)-2-oxoethylidene}amino}oxy)8-oxabicyclo[3.2.1]octane-3-carboxylic acid (compound 30)

[0680]

[0681] At -50°C, an oxacyclopentanol containing 3-({(Z)-[1-{2-[(tert-butoxycarbonyl)amino]-1,3-thiazo-4-yl}-2-({(4S)-2-[(4S)-2-(tert-butoxycarbonyl)-5-oxo-4-(5-oxo-2,2-diphenyl-5,7-dihydro-2H,6H-[1,3]dioxacyclo[4,5-f]isoindol-6-yl)oxacyclopentanol A solution of boron trichloride (1.0 M in DCM, 2.48 mL, 2.48 mmol) was added dropwise to an anhydrous DCM solution of 90 (338 mg, 0.31 mmol) of tert-butyl octane-3-carboxylic acid (tert-butyl octane-3-carboxylic acid) in 25 mL of anhydrous DCM. The reaction mixture was stirred at -30°C to 25°C for 2.5 h, then cooled to -50°C and a buffer solution (prepared by dissolving NaHCO3 (514 mg) and Na2HPO4 (163 mg) in 28 mL of water) was added. The reaction flask was transferred to an ice-water bath and the heterogeneous mixture was stirred at 0–5°C for 20 min, then stirred at room temperature until the aqueous phase was completely thawed, and the two layers were separated. The organic layer was carefully removed, and the aqueous solution was immediately purified by C18 reversed-phase chromatography using a Biotage system and a mixture of acetonitrile containing 0.1% formic acid and an aqueous solution containing 0.1% formic acid as the eluent. The purified fractions were combined and lyophilized to give 30 (95 mg, 43%) of 3-({(Z)-[1-(2-amino-1,3-thiazolyl-4-yl)-2-({(4S)-2-[(4S)-2-carboxy-4-(5,6-dihydroxy-1-oxo-1,3-dihydro-2H-isoindol-2-yl)-5-oxooxopentane-2-yl]-3-oxo-1,2-oxazolidine-4-yl}amino)-2-oxoethylene}amino}oxy)8-oxabicyclo[3.2.1]octane-3-carboxylic acid compound as a green solid.

[0682] ¹H-NMR (599 MHz; mixture of D₂O and CD₃CN) δ 7.12 (s, ¹H), 7.02 (s, ¹H), 6.97 (s, ¹H), 5.44–5.41 (m, ¹H), 5.15–5.08 (m, ¹H), 4.75–4.68 (m, ¹H), 4.43–4.34 (m, 5H), 4.34–4.23 (m, 3H), 3.45–3.28 (m, ¹H), 2.89–2.59 (m, ¹H), 2.26–2.20 (m, 2H), 2.07–1.98 (m, 2H), 1.79–1.72 (m, 4H). No interchangeable protons were observed in D₂O.

[0683] MS(ESI)m / z: [M+1] + 717.0

[0684] Example 18

[0685] (4S)-2-[(4S)-4-{[(2Z)-2-(2-amino-1,3-thiazolyl-4-yl)-2-{[(2-carboxypropane-2-yl)oxy]imino}acetyl]amino}-3-oxo-1,2-oxazolidine-2-yl]-4-(5,6-dihydroxy-1,1,3-trioxo-1,3-dihydro-2H-1λ) 6 ,2-Benzothiazol-2-yl)-5-oxooxopentane-2-carboxylic acid (Compound 37, Table 1)

[0686]

[0687] Step 1: 1-(prop-2-en-1-yl)N-{[(9H-fluorene-9-yl)methoxy]carbonyl}-L-aspartic acid 4-tert-butyl ester (92)

[0688]

[0689] To an anhydrous acetonitrile (75 mL) solution containing allyl bromide (16 mL, 184.9 mmol) and DIPEA (12.7 mL, 72.9 mmol), 91 g of (2S)-4-tert-butoxy-2-([(9H-fluorene-9-yl)methoxy]carbonyl}amino)-4-oxobutyric acid 91 (15.0 g, 36.5 mmol) was added. The reaction mixture was heated at 40 °C and stirred at 40 °C for 4 hours, then cooled and concentrated under vacuum. The residue was extracted with EtOAc, and the organic phase was subsequently washed with 0.5 M hydrochloric acid, saturated sodium bicarbonate aqueous solution, and brine. The organic layer was then dried over sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography using a gradient of hexane containing 5% to 20% ethyl acetate to give 92 (15.19 g, 92%) of 1-(prop-2-en-1-yl)N-{[(9H-fluorene-9-yl)methoxy]carbonyl}-L-aspartic acid 4-tert-butyl ester, a white solid.

[0690] 1 H NMR (400MHz, CDCl3) δ7.80-7.78(m,2H),7.64-7.61(m,2H),7.45-7.40(m,2H), 7.33(td,J=7.5,1.2Hz,2H),5.94-5.92(m,1H),5.85-5.82(m,1H),5.38-5.34(m ,1H),5.29-5.26(m,1H),4.71-4.64(m,3H),4.46-4.43(m,1H),4.39-4.34(m,1 H),4.29-4.26(m,1H),2.98-2.97(m,1H),2.83-2.82(m,1H),1.48-1.46(m,9H).

[0691] Step 2: 1-(prop-2-en-1-yl)-L-aspartic acid 4-tert-butyl ester (93)

[0692] Diethylamine (80 mL) was added to an anhydrous DCM (80 mL) solution containing 1-(prop-2-en-1-yl)N-{[(9H-fluoren-9-yl)methoxy]carbonyl}-L-aspartic acid 4-tert-butyl ester 92 (15.19 g, 33.64 mmol). The reaction mixture was stirred at room temperature for 3 hours, and then concentrated under reduced pressure. The residue was purified by silica gel column chromatography using a gradient of hexane containing 5% to 100% ethyl acetate to give 1-(prop-2-en-1-yl)-L-aspartic acid 4-tert-butyl ester 93 (7.84 g, quantitative) as a pale yellow oil.

[0693] 1H NMR (400MHz, CDCl3) δ5.95-5.86(m,1H),5.35-5.29(m,1H),5.26-5.23(m,1H),4.67-4. 58(m,2H),3.77(dd,J=6.8,4.8Hz,1H),2.69(qd,J=14.3,5.8Hz,2H),1.71-1.67(m,3H).

[0694] Step 3: Methyl 6-bromo-2,2-diphenyl-2H-1,3-benzodioxazole-5-carboxylic acid ester (95)

[0695]

[0696] A mixture of methyl 2-bromo-4,5-dihydroxybenzoate 94 (9.53 g, 38.58 mmol) and 1,1′-(dichloromethane)diphenyl (7.41 mL, 38.59 mmol) was heated to 170 °C and stirred at 170 °C for 50 min. The reaction mixture was then cooled to room temperature and concentrated under reduced pressure. The residue was purified by silica gel column chromatography using a gradient of hexane containing 0% to 10% ethyl acetate to give methyl 6-bromo-2,2-diphenyl-2H-1,3-benzodioxazole-5-carboxylic acid 95 (15.01 g, 95%) as a white solid.

[0697] 1 H NMR (400MHz, CDCl3) δ7.57-7.53(m,4H),7.43-7.39(m,7H),7.18(s,1H),3.90(s,3H).

[0698] Step 4: Methyl 6-(benzylsulfonyl)-2,2-diphenyl-2H-1,3-benzodioxazole-5-carboxylate (96)

[0699]

[0700] At 0 °C, dropwise addition of phenylethyl mercaptan (4.30 mL, 36.63 mmol) was added to a suspension of anhydrous THF (120 mL) containing NaH (60% in mineral oil, 1.67 g, 41.75 mmol), and the resulting solution was stirred at 0 °C for 10 min. Then, 95g of methyl 6-bromo-2,2-diphenyl-2H-1,3-benzodioxazole-5-carboxylic acid (14.30 g, 34.77 mmol) was added, and the reaction mixture was heated to 45 °C and stirred at 45 °C for 13 h. The reaction mixture was cooled to room temperature, diluted with a mixture of ethyl acetate and hexane (7:3), and then water was added. The organic phase was separated, and the aqueous layer was further extracted with a mixture of ethyl acetate and hexane (7:3). The combined organic extracts were washed with brine, dried over sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography using a gradient of hexane containing 0% to 8% ethyl acetate to give methyl 6-(benzylsulfonyl)-2,2-diphenyl-2H-1,3-benzodioxazole-5-carboxylic acid 96 (7.88 g, 50%) as a white foam.

[0701] 1 H NMR (400MHz, CDCl3) δ7.57-7.53(m,5H),7.42-7.39(m,8H),7.35-7.25(m,3H),6.92(s,1H),4.12(d,J=2.4Hz,2H),3.87(s,3H).

[0702] Step 5: Methyl 6-(chlorosulfonyl)-2,2-diphenyl-2H-1,3-benzodioxazole-5-carboxylate (97)

[0703]

[0704] At 0 °C, a small amount of 1,3-dichloro-5,5-dimethylimidazolium-2,4-dione (6.84 g, 34.72 mmol) was added to a stirred suspension containing methyl 6-(benzylsulfonyl)-2,2-diphenyl-2H-1,3-benzodioxazole-5-carboxylic acid 96 (7.88 g, 17.34 mmol), acetonitrile (170 mL), acetic acid (6.3 mL), and water (4.25 mL). The reaction mixture was stirred at 0 to 5 °C for 2 hours and then concentrated under vacuum. The residue was extracted in DCM (110 mL), the resulting solution was cooled to 0 °C, and a saturated aqueous solution of sodium bicarbonate (110 mL) was slowly added while maintaining the temperature below 10 °C. The resulting mixture was stirred between 0 °C and 5 °C for 15 minutes, then the aqueous phase was separated, and the organic layer was washed with pre-cooled brine (<10 °C). The organic layer was dried over sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography using a gradient of hexane containing 0% to 10% ethyl acetate to give methyl 6-(chlorosulfonyl)-2,2-diphenyl-2H-1,3-benzodioxazole-5-carboxylic acid 97 (7.40 g, 99%) as white foam.

[0705] 1 H NMR (400MHz, CDCl3) δ7.64(s,1H),7.56-7.53(m,4H),7.46-7.43(m,6H),7.20(s,1H),3.97(s,3H).

[0706] Step 6: 1-(prop-2-en-1-yl)-N-[6-(methoxycarbonyl)-2,2-diphenyl-2H-1,3-benzodioxazole-5-sulfonyl]-L-aspartic acid 4-tert-butyl ester (98)

[0707]

[0708] A solution of anhydrous DCM (150 mL) containing methyl 6-(chlorosulfonyl)-2,2-diphenyl-2H-1,3-benzodioxazole-5-carboxylic acid 97 (7.40 g, 17.17 mmol) was cooled to 0 °C. Triethylamine (2.90 mL, 20.81 mmol), a solution of DCM (25 mL) containing 4-tert-butyl 1-(prop-2-en-1-yl)-L-aspartic acid 93 (4.33 g, 18.89 mmol), and DMAP (210 mg, 1 mL) were added sequentially at 0 °C. The reaction mixture was stirred at 0 °C for 10 min. The reaction mixture was then stirred at room temperature for 4 h and concentrated under reduced pressure. The residue was dissolved in ethyl acetate, the organic phase was washed with water and salt, dried over sodium sulfate, filtered, and concentrated under vacuum. The residue was purified by silica gel column chromatography using a gradient of hexane containing 0% to 20% ethyl acetate to give 98 (8.43 g, 79%) of 1-(prop-2-en-1-yl)-N-[6-(methoxycarbonyl)-2,2-diphenyl-2H-1,3-benzodioxazole-5-sulfonyl]-L-aspartic acid 4-tert-butyl ester, as a white foam.

[0709] 1 H NMR (400MHz, CDCl3) δ7.60 (s, 1H), 7.58-7.53 (m, 4H), 7.44-7.41 (m, 7H), 7.27 (d, J = 8.6Hz, 1H), 5.64-5.54 (m, 1H), 5.10 -5.00(m,2H),4.39-4.31(m,3H),3.96(s,3H),2.96(dd,J=16.9,4.7Hz,1H),2.82(dd,J=16.9,5.0Hz,1H),1.44(s,9H).

[0710] Step 7: 1-(prop-2-en-1-yl)(2S)-2-(1,1,3-trioxo-6,6-diphenyl-1,3-dihydro-2H,6H-1λ) 6 -[1,3]dioxane[4,5-f][1,2]benzothiazol-2-yl) 4-tert-butyl succinate (99)

[0711]

[0712] At room temperature, DMAP (544 mg, 4.45 mmol) and triethylamine (5.80 mL, 41.61 mmol) were added to anhydrous toluene (400 mL) containing 98 g (9.25 g, 14.83 mmol) of 1-(prop-2-en-1-yl)-N-[6-(methoxycarbonyl)-2,2-diphenyl-2H-1,3-benzodioxazole-5-sulfonyl]-L-aspartic acid 4-tert-butyl ester. The resulting mixture was stirred at reflux for 120 hours, then cooled and concentrated under vacuum. The residue was extracted with ethyl acetate, the organic phase was washed with water and brine, dried over sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography using a gradient of hexanes containing 0% to 15% ethyl acetate to give 1-(prop-2-en-1-yl)(2S)-2-(1,1,3-trioxo-6,6-diphenyl-1,3-dihydro-2H,6H-1λ). 6 -[1,3]dioxane[4,5-f][1,2]benzothiazolyl-2-yl) 4-tert-butyl succinate 99 (6.26 g, 71%), is a grayish-white foam.

[0713] 1 H NMR (400MHz, CDCl3) δ7.53-7.51(m,4H),7.43-7.41(m,7H),7.30(d,J=0.4Hz,1H),5.88-5.78(m,1H),5.30-5.24(m, 2H),5.20-5.16(m,1H),4.69-4.59(m,2H),3.35(dd,J=16.8,7.6Hz,1H),2.98(dd,J=16.8,7.1Hz,1H),1.45(s,9H).

[0714] Step 8: (3S)-4-oxo-4-[(prop-2-en-1-yl)oxy]-3-(1,1,3-trioxo-6,6-diphenyl-1,3-dihydro-2H,6H-1λ) 6 -[1,3]dioxane[4,5-f][1,2]benzothiazol-2-yl)butyric acid (100)

[0715]

[0716] At -78°C, an atmosphere containing 1-(prop-2-en-1-yl)(2S)-2-(1,1,3-trioxo-6,6-diphenyl-1,3-dihydro-2H,6H-1λ) was prepared. 6Trichloroboron solution (1.08 g, 1.83 mmol) was added dropwise to an anhydrous DCM (42 mL) solution of 99 g (1.08 g, 1.83 mmol) of 4-tert-butyl succinate-[1,3]dioxane-[4,5-f][1,2]benzothiazol-2-yl)succinate-4-tert-butyl ester (1.0 M in DCM, 3.29 mL, 3.29 mmol). The reaction mixture was stirred at -78 °C for 1 h, then stirred at -78 °C to -45 °C for 30 min. Subsequently, the reaction mixture was diluted with ethyl acetate and water was added at -50 °C. The heterogeneous mixture was stirred at room temperature until the different layers separated, the organic phase was dried over sodium sulfate, filtered, and concentrated under reduced pressure. The residue was initially purified by C18 reversed-phase chromatography using a mixture of acetonitrile containing 0.1% formic acid and water containing 0.1% formic acid as the eluent. The products containing fractions were combined and concentrated under reduced pressure. The product was further purified by silica gel column chromatography using a gradient DCM containing 0% to 4% methanol to give (3S)-4-oxo-4-[(prop-2-en-1-yl)oxy]-3-(1,1,3-trioxo-6,6-diphenyl-1,3-dihydro-2H,6H-1λ). 6 -[1,3]dioxane[4,5-f][1,2]benzothiazol-2-yl)butyric acid 100 (395 mg, 40%), is a white foam.

[0717] 1 H NMR (400MHz, CDCl3) δ7.57-7.52(m,4H),7.48-7.41(m,7H),7.33(s,1H),5.90-5.80(m,1H),5.32-5.27( m,2H),5.23-5.20(m,1H),4.72-4.63(m,2H),3.53(dd,J=17.1,7.8Hz,1H),3.11(dd,J=17.1,6.7Hz,1H).

[0718] Step 9: Propylene-2-en-1-yl(2S)-5-cyano-4-oxo-5-(1λ) 4 -thiophene-1-yl)-2-(1,1,3-trioxo-6,6-diphenyl-1,3-dihydro-2H,6H-1λ) 6 -[1,3]dioxane[4,5-f][1,2]benzothiazol-2-yl)valerate (101)

[0719]

[0720] At room temperature, to (3S)-4-oxo-4-[(prop-2-en-1-yl)oxy]-3-(1,1,3-trioxo-6,6-diphenyl-1,3-dihydro-2H,6H-1λ 6HATU (612 mg, 1.61 mmol) was added to an anhydrous DCM (14 mL) solution of 100 (783 mg, 1.46 mmol) of [1,3]dioxane-[4,5-f][1,2]benzothiazol-2-yl)butyric acid. The resulting mixture was cooled to 0 °C, and DIPEA (0.76 mL, 4.36 mmol) was added, followed by 1-(cyanomethyl)thiol-1-ammonium bromide (396 mg, 1.90 mmol). The reaction mixture was stirred at 0 °C for 5 min, then at room temperature for 1 h. The mixture was cooled to 0 °C and an aqueous saturated ammonium chloride solution was added. The mixture was then diluted with water and extracted with ethyl acetate. The combined organic extracts were washed with brine, dried over sodium sulfate, filtered, and concentrated under reduced pressure. The residues were purified by silica gel column chromatography using a gradient of hexane containing 50% to 100% ethyl acetate to give propen-2-en-1-yl(2S)-5-cyano-4-oxo-5-(1λ) 4 -thiophene-1-yl)-2-(1,1,3-trioxo-6,6-diphenyl-1,3-dihydro-2H,6H-1λ) 6 -[1,3]dioxane[4,5-f][1,2]benzothiazol-2-yl)valerate 101 (970 mg, crude product), is a white foam.

[0721] 1 H NMR (400MHz, CDCl3) δ7.57-7.52(m,4H),7.47-7.42(m,7H),7.30(s,1H),5.91-5.81(m,1H),5.36(dd,J=8.1,5.9Hz,1 H),5.32-5.27(m,1H),5.21-5.18(m,1H),4.71-4.61(m,2H),3.55-3.30(m,6H),2.65-2.56(m,2H),2.10-2.00(m,2H).

[0722] Step 10: (4S)-2,5-dioxo-5-[(prop-2-en-1-yl)oxy]-4-(1,1,3-trioxo-6,6-diphenyl-1,3-dihydro-2H,6H-1λ) 6 -[1,3]dioxane[4,5-f][1,2]benzothiazol-2-yl)valeric acid (102)

[0723]

[0724] To contain prop-2-en-1-yl(2S)-5-cyano-4-oxo-5-(1λ) 4 -thiophene-1-yl)-2-(1,1,3-trioxo-6,6-diphenyl-1,3-dihydro-2H,6H-1λ)6 A solution of 101 (970 mg, approx. 1.46 mmol) of 1,3-dioxane-[4,5-f][1,2]benzothiazol-2-yl)valerate 11% THF (11 mL) was added to water (11 mL), followed by the addition of OXONE (898 mg, 2.92 mmol). The reaction mixture was stirred at room temperature for 1 hour, with additional OXONE (898 mg, 2.92 mmol) added in two portions over a period of more than an hour, and stirring was continued at room temperature for another 2 hours. Most of the THF was removed under reduced pressure, and the aqueous solution was extracted with ethyl acetate. The combined organic extracts were dried over sodium sulfate, filtered, and concentrated to give crude (4S)-2,5-dioxo-5-[(prop-2-en-1-yl)oxy]-4-(1,1,3-trioxo-6,6-diphenyl-1,3-dihydro-2H,6H-1λ). 6 -[1,3]dioxane[4,5-f][1,2]benzothiazo-2-yl)valeric acid 102 (900 mg, crude product), as white foam, is used directly in the next step.

[0725] 1 H NMR (400MHz, CDCl3) δ7.56-7.53(m,4H),7.47-7.43(m,7H),7.33(s,1H),5.87-5.79(m,1H),5 .43-5.39(m,1H),5.32-5.22(m,2H),4.72-4.62(m,2H),4.14-4.08(m,1H),3.59-3.52(m,1H).

[0726] Step 11: 5-(prop-2-en-1-yl)(4S)-2-oxo-4-(1,1,3-trioxo-6,6-diphenyl-1,3-dihydro-2H,6H-1λ) 6 -[1,3]dioxane[4,5-f][1,2]benzothiazol-2-yl)1-tert-butyl glutarate (103)

[0727]

[0728] At 0 °C, tert-butyl isopropylcarbamate (0.38 mL, 1.52 mmol) was slowly added to an anhydrous THF solution containing 102 (900 mg, crude) of (4S)-2,5-dioxo-5-[(prop-2-en-1-yl)oxy]-4-(1,1,3-trioxo-6,6-diphenyl-1,3-dihydro-2H,6H-1λ6-[1,3]dioxo[4,5-f][1,2]benzothiazol-2-yl)valerate. The reaction mixture was stirred at 0 °C for 5 minutes, the cooling bath was removed, and stirring was continued at room temperature for 1 hour. Additional tert-butyl N,N'-diisopropylcarbamate (0.42 mL, 1.68 mmol) was added in small amounts over a period of more than 2 hours, and stirring was continued at room temperature for 1 hour. Most of the THF was removed under reduced pressure, and the mixture was treated with hexane containing 30% DCM. The precipitate was filtered off, and the filtrate was concentrated under vacuum. The residue was purified by silica gel column chromatography using a gradient of hexane containing 0% to 20% ethyl acetate to give 5-(prop-2-en-1-yl)(4S)-2-oxo-4-(1,1,3-trioxo-6,6-diphenyl-1,3-dihydro-2H,6H-1λ). 6 -[1,3]dioxane[4,5-f][1,2]benzothiazolyl-2-yl) 1-tert-butyl glutarate 103 (341 mg, 3 steps over 38%), is a white foam.

[0729] 1 H NMR (400MHz, CDCl3) δ7.53-7.50(m,4H),7.44-7.40(m,7H),7.29(s,1H),5.86-5.77(m,1H),5.41-5.37(m,1H), 5.29-5.17(m,2H),4.68-4.59(m,2H),3.93(dd,J=18.4,7.6Hz,1H),3.46(dd,J=18.4,6.8Hz,1H),1.54(s,9H).

[0730] Step 12: (2S)-5-tert-butoxy-4,5-dioxo-2-(1,1,3-trioxo-6,6-diphenyl-1,3-dihydro-2H,6H-1λ) 6 -[1,3]dioxane[4,5-f][1,2]benzothiazol-2-yl)valeric acid (104)

[0731]

[0732] At -5°C, an atmosphere containing 5-(prop-2-en-1-yl)(4S)-2-oxo-4-(1,1,3-trioxo-6,6-diphenyl-1,3-dihydro-2H,6H-1λ) was prepared. 6Morpholine (0.10 mL, 1.14 mmol) was added to an anhydrous THF (7 mL) solution of 1-tert-butyl glutarate (103) of [1,3]dioxane-[4,5-f][1,2]benzothiazo-2-yl)glutarate. The mixture was purged with nitrogen for 10 min, and Pd(PPh3)4 (23 mg, 0.02 mmol) was added. The reaction mixture was stirred under nitrogen at -5 °C to 0 °C for 2 h. The reaction mixture was diluted with diethyl ether, the organic phase was washed with 1 M HCl and brine, dried over sodium sulfate, filtered and concentrated to give (2S)-5-tert-butoxy-4,5-dioxo-2-(1,1,3-trioxo-6,6-diphenyl-1,3-dihydro-2H,6H-1λ). 6 -[1,3]dioxane[4,5-f][1,2]benzothiazo-2-yl)valeric acid 104 (231 mg, crude product), as yellow foam, is used directly in the next step.

[0733] 1 H NMR (400MHz, DMSO-d6) δ8.11(s,1H),7.74(s,1H),7.54-7.45(m,10H),5.12(t,J=7.3Hz,1H),3.75-3.68(m,1H),3.44-3.38(m,1H),1.45(s,9H).

[0734] Step 13: (4S)-2-[(4S)-4-{[(2Z)-2-{2-[(tert-butoxycarbonyl)amino]-1,3-thiazolyl-4-yl}-2-{[(1-tert-butoxy-2-methyl-1-oxopropane-2-yl)oxy]imino}acetyl]amino}-3-oxo-1,2-oxazolidine-2-yl]-5-oxo-4-(1,2,3-trioxo-6,6-diphenyl-1,3-dihydro-2H,6H-1λ) 6 -[1,3]dioxane[4,5-f][1,2]benzothiazol-2-yl)oxooxopentane-2-carboxylic acid tert-butyl ester (105)

[0735]

[0736] To contain (2S)-5-tert-butoxy-4,5-dioxo-2-(1,1,3-trioxo-6,6-diphenyl-1,3-dihydro-2H,6H-1λ 6DMAP (9 mg, 0.074 mmol) was added to anhydrous THF (7 mL) of a mixture of [1,3]dioxane-[4,5-f][1,2]benzothiazol-2-yl)valeric acid 104 (231 mg, crude product) and tert-butyl-2-{[(Z)-(1-{2-[(tert-butyloxycarbonyl)amino]-1}, followed by DCC (99 mg, 0.48 mmol) at 0 °C. The reaction mixture was allowed to be slowly heated to room temperature and stirred for 18 hours. The mixture was then concentrated under reduced pressure, and the residue was treated with hexane containing 25% DCM. The precipitate was removed by filtration. The solid was concentrated under vacuum. The residue was purified by silica gel column chromatography using a gradient of hexane containing 10% to 30% ethyl acetate to give (4S)-2-[(4S)-4-{[(2Z)-2-{2-[(tert-butoxycarbonyl)amino]-1,3-thiazo-4-yl}-2-{[(1-tert-butoxy-2-methyl-1-oxopropane-2-yl)oxy]imino}acetyl]amino}-3-oxo-1,2-oxazolidine-2-yl]-5-oxo-4-(1,2,3-trioxo-6,6-diphenyl-1,3-dihydro-2H,6H-1λ) 6 -[1,3]dioxane[4,5-f][1,2]benzothiazo-2-yl)oxooxopentane-2-carboxylic acid tert-butyl ester 105 (209 mg, 53%), is a pale yellow foam.

[0737] 1 H NMR (400MHz, CDCl3) δ8.23-8.04(m,2H),7.56-7.51(m,4H),7.47-7.43(m,7H),7.35(m,2H),5.34-4.82(m,3H),4.38-4. 21(m,1H),3.78-3.42(m,1H),3.00-2.88(m,1H),1.63-1.60(m,6H),1.58-1.54(m,9H),1.55(s,9H),1.47-1.44(m,9H).

[0738] Step 14: (4S)-2-[(4S)-4-{[(2Z)-2-(2-amino-1,3-thiazolyl-4-yl)-2-{[(2-carboxypropane-2-yl)oxy]imino}acetyl]amino}-3-oxo-1,2-oxazolidine-2-yl]-4-(5,6-dihydroxy-1,1,3-trioxo-1,3-dihydro-2H-1λ) 6 ,2-Benzothiazol-2-yl)-5-oxooxopentane-2-carboxylic acid (compound 37)

[0739]

[0740] At -50°C, an agent containing (4S)-2-[(4S)-4-{[(2Z)-2-{2-[(tert-butoxycarbonyl)amino]-1,3-thiazo-4-yl}-2-{[(1-tert-butoxy-2-methyl-1-oxopropane-2-yl)oxy]imino}acetyl]amino}-3-oxo-1,2-oxazolidine-2-yl]-5-oxo-4-(1,2,3-trioxo-6,6-diphenyl-1,3-dihydro-2H,6H-1λ] was applied. 6 -[1,3]dioxane[4,5-f][1,2]benzothiazol-2-yl)oxooxopentane-2-carboxylic acid tert-butyl ester 105 (209 mg, 0.19 mmol) in anhydrous DCM (7 mL) was added dropwise with boron trichloride solution (1.56 mL, 1.56 mmol, 1.0 M in DCM). The reaction mixture was stirred at -50 °C to 25 °C for 2.5 h, and then a solution of NaHCO3 (320 mg) and Na2HPO4 (100 mg) in water (17.3 mL) was added at -50 °C. The mixture was stirred at 0 °C to 5 °C (ice-water bath) for 10 min, followed by stirring at room temperature until the aqueous phase thawed. The mixture was filtered through a 1 μm pin filter, and the organic layer was carefully separated. The aqueous solution was then purified by C18 reversed-phase column chromatography using a Biotage system and a mixture of acetonitrile containing 0.1% formic acid and water containing 0.1% formic acid as the eluent. The fractions were combined and freeze-dried to obtain (4S)-2-[(4S)-4-{[(2Z)-2-(2-amino-1,3-thiazolyl-4-yl)-2-{[(2-carboxypropane-2-yl)oxy]imino}acetyl]amino}-3-oxo-1,2-oxazolidine-2-yl]-4-(5,6-dihydroxy-1,1,3-trioxo-1,3-dihydro-2H-1λ) 6 2-Benzothiazol-2-yl)-5-oxooxopentane-2-carboxylic acid compound 37 (45 mg, 33%) is a grayish-white solid.

[0741] 1 ¹H NMR (400 MHz, mixture of D₂O and CD₃CN) δ 7.43 (s, ¹H), 7.36–7.34 (m, ¹H), 7.08–7.06 (m, ¹H), 5.27–5.07 (m, 2H), 4.74–4.67 (m, ¹H), 4.32–4.22 (m, ¹H), 3.67–3.35 (m, ¹H), 2.98–2.78 (m, ¹H), 1.48 (s, 6H). No interchangeable protons were observed in D₂O.

[0742] MS(ESI)m / z: [M+1] + 699.0

[0743] Example 19

[0744] (4S)-2-[(4S)-4-{[(2Z)-2-(2-amino-5-chloro-1,3-thiazolyl-4-yl)-2-{[(1-carboxycyclopropyl)oxy]imino}acetyl]amino}-3-oxo-1,2-oxazolidine-2-yl]-4-(5,6-dihydroxy-1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)-5-oxooxopentane-2-carboxylic acid (Compound 24, Table 1)

[0745]

[0746] Step 1: {2-[(tert-Butoxycarbonyl)amino]-5-chloro-1,3-thiazolyl}(oxo)acetic acid (107)

[0747]

[0748] At room temperature, NCS (2.7 g, 20.2 mmol) was added to a solution of anhydrous 1,4-dioxane (35 mL) containing {2-[(tert-butoxycarbonyl)amino]-1,3-thiazol-4-yl}(oxo)acetic acid 106 (5.0 g, 18.4 mmol). The reaction mixture was heated and stirred at 40 °C for 6 hours, then cooled and concentrated under reduced pressure. The crude mixture was ground using a mixture of diethyl ether (40 mL) and hexane (20 mL). The resulting precipitate was removed by filtration, and the filtrate was concentrated under reduced pressure. The residue was further dried under high vacuum and then ground with hexane. The precipitate was collected by filtration as a grayish-white powder to give {2-[(tert-butoxycarbonyl)amino]-5-chloro-1,3-thiazol-4-yl}(oxo)acetic acid 107 (5.7 g, quantitative yield).

[0749] 1 H-NMR (400MHz; DMSO-d6) δ12.21(s,1H),1.46(s,9H).

[0750] Step 2: (2Z)-{2-[(tert-butoxycarbonyl)amino]-5-chloro-1,3-thiazolyl-4-yl}({1-(tert-butoxycarbonyl)cyclopropyl]oxy}imino)acetic acid (108)

[0751]

[0752] To a methanol (150 mL) solution containing {2-[(tert-butoxycarbonyl)amino]-5-chloro-1,3-thiazol-4-yl}(oxo)acetic acid 107 (5.6 g, 18.4 mmol), tert-butyl 1-(aminooxy)cyclopropane-1-carboxylic acid 2 (3.5 g, 20.2 mmol) was added. The reaction mixture was stirred at room temperature for 3 hours, then concentrated under reduced pressure. A trace amount of methanol was co-evaporated with hexane (3 x 50 mL), and the residue was further dried under high vacuum. The crude product was treated with hexane containing 5% diethyl ether, and the resulting suspension was stirred overnight. The precipitated solid was collected by filtration to give (2Z)-{2-[(tert-butoxycarbonyl)amino]-5-chloro-1,3-thiazol-4-yl}({1-(tert-butoxycarbonyl)cyclopropyl]oxy}imino)acetic acid 108 (8.3 g, 98%) as a grayish-white powder.

[0753] 1 H-NMR (400MHz; CDCl3) δ2.81 (s, 1H), 1.64-1.61 (m, 2H), 1.60-1.51 (m, 11H), 1.48 (s, 9H).

[0754] Step 3: 1-{[(Z)-(1-{2-[(tert-butyloxycarbonyl)amino]-5-chloro-1,3-thiazolyl}-2-oxo-2-{[(4S)-3-oxo-1,2-oxazolidine-4-yl]amino}ethylene)amino]oxy}cyclopropane-1-carboxylic acid tert-butyl ester (109)

[0755]

[0756] DIPEA (4.7 mL, 26.9 mmol) was added to an anhydrous DMF (100 mL) solution containing (2Z)-{2-[(tert-butoxycarbonyl)amino]-5-chloro-1,3-thiazolyl}({1-(tert-butoxycarbonyl)cyclopropyl]oxy}imino)acetic acid 108 (8.3 g, 18.0 mmol). The resulting mixture was stirred at room temperature for 5 minutes, then HATU was added, and stirring was continued at room temperature for 2 hours. The reaction mixture was diluted with anhydrous DMF (100 mL), then DIPEA (4.7 mL, 26.9 mmol) was added, followed by L-cycloserine (2.2 g, 21.6 mmol). The reaction mixture was stirred overnight at room temperature, then concentrated under reduced pressure to dryness. The residue was purified by silica gel chromatography using a gradient DCM containing 0-4% methanol to give 109 (7.5 g, 76 g) of 1-{[(Z)-(1-{2-[(tert-butoxycarbonyl)amino]-5-chloro-1,3-thiazo-4-yl}-2-oxo-2-{[(4S)-3-oxo-1,2-oxazolidine-4-yl]amino}ethylene)amino]oxy}cyclopropane-1-carboxylic acid tert-butyl ester, an amorphous white solid.

[0757] 1 H-NMR (400MHz; DMSO-d6) δ12.04(s,1H),11.58(s,1H),9.02(m,1H),3.95(t,J=8.8Hz,1H),1.45(s,9H),1.39(s,9H),1.34-1.26(m,4H).

[0758] Step 4: (4S)-2-[(4S)-4-{[(2Z)-2-{2-[(tert-butoxycarbonyl)amino]-5-chloro-1,3-thiazolyl-4-yl}-2-({[1-(tert-butoxycarbonyl)cyclopropyl]oxy}imino)acetyl]amino}-3-oxo-1,2-oxazolidine-2-yl]-4-(5,7-dioxo-2,2-diphenyl-5,7-dihydro-2H,6H-[1,3]dioxono[4,5-f]isoindol-6-yl)-5-oxooxopentane-2-carboxylic acid tert-butyl ester (110)

[0759]

[0760] At 0 °C, DCC (212 mg, 1.0 mmol) was added to anhydrous THF (10 mL) containing a mixture of 1-{[(Z)-(1-{2-[(tert-butoxycarbonyl)amino]-5-chloro-1,3-thiazo-4-yl}-2-oxo-2-{[(4S)-3-oxo-1,2-oxazolidine-4-yl]amino}ethylene)amino]oxy}cyclopropane-1-carboxylic acid tert-butyl ester 109 (402 mg, 0.74 mmol) and (2S)-5-tert-butoxy-2-(5,7-dioxo-2,2-diphenyl-5,7-dihydro2H,6H-[1,3]dioxane-[4,5-f]isoindol-6-yl)-4,5-dioxovaleric acid 6 (400 mg, 0.74 mmol). The resulting mixture was stirred for 5 minutes, and then DCC (212 mg, 1.0 mmol) was added at 0 °C. The reaction mixture was allowed to gradually warm to room temperature and stirred overnight. Subsequently, the reaction mixture was concentrated under reduced pressure, and the residue was ground in hexane (20 mL) containing 30% DCM. The precipitated solids were removed by filtration, and the filtrate was concentrated under vacuum. The crude mixture was purified by silica gel column chromatography using a gradient of hexane containing 0-30% ethyl acetate, and the resulting product was treated with hexane (20 mL) containing 25% ethyl acetate. The separated solids were removed by filtration, and the filtrate was concentrated to obtain (4S)-2-[(4S)-4-{[(2Z)-2-{2-[(tert-butyloxycarbonyl)amino]-5-chloro-1,3-thiazolyl-4-yl}-2-({[1-(tert-butyloxycarbonyl)cyclopropyl]oxy}imino)acetyl]amino}-3-oxo-1,2-oxazolidine-2-yl]-4-(5,7-dioxo-2,2-diphenyl-5,7-dihydro-2H,6H-[1,3]dioxono[4,5-f]isoindol-6-yl)-5-oxooxopentane-2-carboxylic acid tert-butyl ester 110 (450 mg, 57%), which was a grayish-white foam.

[0761] 1 H-NMR (400MHz; CDCl3) δ8.39-8.30(m,1H),7.97-7.93(m,1H),7.57-7.54(m,4H),7.43(t,J=3.2Hz,6H),7.33(s,2H),5.41(q,J=10.0Hz,1H),5. 11-4.93(m,2H),4.28-4.23(m,1H),3.49-3.29(m,1H),2.88(dd,J=13.7 ,10.4Hz,1H),1.63-1.58(m,9H),1.58-1.50(m,13H),1.49-1.44(m,9H).

[0762] Step 5: (4S)-2-[(4S)-4-{[(2Z)-2-(2-amino-5-chloro-1,3-thiazolyl-4-yl)-2-{[(1-carboxycyclopropyl)oxy]imino}acetyl]amino}-3-oxo-1,2-oxazolidine-2-yl]-4-(5,6-dihydroxy-1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)-5-oxooxopentane-2-carboxylic acid (Compound 24)

[0763]

[0764] At -50°C, boron trichloride solution (1.49 mL, 1.0 M in DCM) was added dropwise to an anhydrous DCM solution containing (4S)-2-[(4S)-4-{[(2Z)-2-{2-[(tert-butyloxycarbonyl)amino]-5-chloro-1,3-thiazolyl}-2-({[1-(tert-butyloxycarbonyl)cyclopropyl]oxy}imino)acetyl]amino}-3-oxo-1,2-oxazolidine-2-yl]-4-(5,7-dioxo-2,2-diphenyl-5,7-dihydro-2H,6H-[1,3]dioxono[4,5-f]isoindol-6-yl)-5-oxooxopentane-2-carboxylic acid tert-butyl ester 110 (200 mg, 0.19 mmol). The reaction mixture was stirred at -35°C to -30°C for 2.5 hours, then cooled to -50°C, and a 16 mL solution of NaHCO3 (300 mg) and Na2HPO4 (100 mg) in water was added. Using an ice-water bath instead of a cold water bath, the heterogeneous mixture was stirred at 5°C to -10°C for 20 minutes, then stirred at room temperature until the aqueous layer thawed and the different layers separated. The organic layer was carefully removed using Biotage and a mixture of acetonitrile containing 0.1% formic acid and water containing 0.1% formic acid as eluent, and the aqueous solution was purified by C18 reversed-phase column chromatography. The pure fraction was collected and freeze-dried to give compound 24 (70 mg, 54%), a grayish-white solid, which is (4S)-2-[(4S)-4-{[(2Z)-2-(2-amino-5-chloro-1,3-thiazolyl-4-yl)-2-{[(1-carboxycyclopropyl)oxy]imino}acetyl]amino}-3-oxo-1,2-oxazolidine-2-yl]-4-(5,6-dihydroxy-1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)-5-oxooxopentane-2-carboxylic acid.

[0765] 1¹H-NMR (400 MHz; CD₃CN and D₂O as cosolvents) δ 7.23 (s, 2H), 5.45–5.30 (m, 1H), 5.19–5.01 (m, 1H), 4.72–4.65 (m, 1H), 4.24–4.17 (m, 1H), 3.59–3.27 (m, 1H), 2.86–2.61 (m, 1H), 1.47 (s, 2H), 1.43 (s, 2H). No interchangeable protons were observed in D₂O.

[0766] MS(ESI)m / z: [M+1] + 695.0

[0767] Example 20

[0768] (4S)-2-[(4S)-4-{[(2Z)-2-(2-amino-1,3-thiazolyl-4-yl)-2-{[(1-carboxy-4,4-dimethylcyclohexyl)oxy]imino}acetyl]amino}-3-oxo-1,2-oxazolidine-2-yl]-4-(5,6-dihydroxy-1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)-5-oxooxopentane-2-carboxylic acid (Compound 38, Table 1)

[0769]

[0770] Step 1: 4,4-Dimethyl-1-[(trimethylsilyl)oxy]cyclohexane-1-nitrile (112)

[0771]

[0772] Zinc iodide (160 mg, 0.5 mmol) was placed in a dry round-bottom flask and dried under high vacuum (gentle heating) for 30 minutes. Anhydrous DCM (60 mL) containing 4,4-dimethylcyclohexane-1-one 111 (3.16 g, 25.0 mmol) was added, and the reaction mixture was cooled to 0 °C. Trimethylsilyl cyanide (3.75 mL, 30.0 mmol) was slowly added at 0 °C, and the ice-water bath was removed, with stirring continued at room temperature for 1 hour. The reaction mixture was concentrated under reduced pressure, and the residue was ground in hexane (100 mL) containing 10% DCM. The precipitate was removed by filtration, and the filtrate was collected and concentrated under vacuum to give 4,4-dimethyl-1-[(trimethylsilyl)oxy]cyclohexane-1-onitrile 112 (5.18 g, 92%) as a pale yellow oil.

[0773] 1H-NMR (400MHz; CDCl3) δ2.00-1.94(m,2H),1.83-1.76(m,2H),1.47(t,J=6.2Hz,4H),0.98(s,3H),0.95(s,3H),0.26(s,9H).

[0774] Step 2: 1-Hydroxy-4,4-dimethylcyclohexane-1-carboxylic acid (113)

[0775]

[0776] A solution of glacial acetic acid (25 mL) containing 4,4-dimethyl-1-[(trimethylsilyl)oxy]cyclohexane-1-nitrile 112 (5.1 g, 22.6 mmol) was cooled to 0 °C. Concentrated hydrochloric acid (25 mL) was added dropwise over 10 minutes, and the reaction mixture was stirred at 0 °C for 15 minutes, then at room temperature for 15 minutes. Subsequently, the reaction mixture was heated to 100 °C and stirred at 100 °C for 4 hours. The resulting mixture was allowed to cool to room temperature, and volatile substances were removed under reduced pressure. The residue was further dried under high vacuum and then extracted in water (100 mL). The aqueous phase was saturated with solid sodium chloride and extracted with ethyl acetate (2 × 150 mL). The combined organic extracts were washed with water and brine, dried over sodium sulfate, filtered, and concentrated under reduced pressure to give 1-hydroxy-4,4-dimethylcyclohexane-1-carboxylic acid 113 (3.79 g, 97%) as a yellow solid.

[0777] 1 H-NMR (400MHz; DMSO-d6) δ12.25(s,1H),4.79(s,1H),1.80-1.72(m,2H),1.48-1.40(m,4H),1.13-1.08(m,2H),0.87(s,3H),0.85(s,3H).

[0778] Step 3: 1-Hydroxy-4,4-dimethylcyclohexane-1-carboxylic acid tert-butyl ester (114)

[0779]

[0780] To an anhydrous THF (100 mL) solution containing 1-hydroxy-4,4-dimethylcyclohexane-1-carboxylic acid 113 (3.71 g, 21.54 mmol), N,N′-diisopropylcarbamate tert-butyl ester (17.3 mL, 86.16 mmol, prepared as described in EP2471792A1) was added, and the reaction mixture was stirred at room temperature for 14 hours. The separated solids were removed by filtration and washed with THF. The combined filtrates were collected and concentrated under vacuum. The residue was extracted with a mixture of DCM and hexane (1:3, 100 mL), and the resulting suspension was cooled in an ice-water bath for 10 min. The precipitate was removed by filtration, and the mixture was washed with DCM and hexane (1:3, 300 mL). The filtrates were combined and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography using a gradient of hexane containing 0-5% ethyl acetate to give 114 (4.12 g, 84%) of 1-hydroxy-4,4-dimethylcyclohexane-1-carboxylic acid tert-butyl ester, a yellow solid.

[0781] 1 H-NMR (400MHz; CDCl3) δ2.96 (s, 1H), 1.92 (td, J = 13.1, 4.2Hz, 2H), 1.63-1. 56(m,2H),1.54-1.49(m,11H),1.31-1.26(m,2H),0.96(s,3H),0.96(s,3H).

[0782] Step 4: 1-(aminooxy)-4,4-dimethylcyclohexane-1-carboxylic acid tert-butyl ester (115)

[0783]

[0784] A solution of anhydrous THF (50 mL) containing 1.57 g (6.88 mmol) of tert-butyl 1-hydroxy-4,4-dimethylcyclohexane-1-carboxylic acid was cooled to 0 °C under nitrogen. A small amount of sodium hydride (0.41 g, 10.32 mmol, 60% in mineral oil) was added, and the mixture was stirred at 0 °C for 15 min. Then, O-diphenylphosphinohydroxylamine (2.40 g, 10.32 mmol) was added at 0 °C, and stirring continued for 30 min. The reaction mixture was allowed to gradually heat to room temperature and stirred for 4 h. Additional O-diphenylphosphinohydroxylamine (0.5 g, 2.14 mmol) was added, and stirring continued for 14 h. Most of the THF was removed under reduced pressure, and the residue was removed by extraction in a saturated sodium chloride solution (75 mL). The aqueous phase was extracted with ethyl acetate (2 × 150 mL), and the combined organic extracts were dried over sodium sulfate, filtered, concentrated, and dried under high vacuum. The crude product was purified by silica gel column chromatography using a gradient of hexane containing 5% to 15% ethyl acetate to give 1-(aminooxy)-4,4-dimethylcyclohexane-1-carboxylic acid tert-butyl ester 115, as a grayish-white solid (1.11 g, 66% yield).

[0785] 1 H-NMR (400MHz; CDCl3): δ5.26(s,2H),1.92-1.80(m,4H),1.51(s,9H),1.46-1.39(m,2H),1.26-1.20(m,2H),0.95(s,3H),0.94(s,3H).

[0786] Step 5: (2Z)-{2-[(tert-butoxycarbonyl)amino]-1,3-thiazolyl-4-yl}({1-(tert-butoxycarbonyl)-4,4-dimethylcyclohexyl]oxy}imino)acetic acid (116)

[0787]

[0788] To an anhydrous MeOH (25 mL) solution containing 1-(aminooxy)-4,4-dimethylcyclohexane-1-carboxylic acid tert-butyl ester 115 (1.06 g, 4.35 mmol), 2-(2-(tert-butoxycarbonylamino)thiazol-4-yl)-2-oxoacetic acid (1.06 g, 3.92 mmol) was added, and the reaction mixture was stirred at room temperature for 16 hours. The reaction mixture was concentrated under reduced pressure and the residue was further dried under high vacuum to give (2Z)-{2-[(tert-butoxycarbonyl)amino]-1,3-thiazol-4-yl}({1-(tert-butoxycarbonyl)-4,4-dimethylcyclohexyl]oxy}imino)acetic acid 116, a white solid (2.07 g, crude product).

[0789] 1H-NMR(400MHz; DMSO-d6)δ11.75(s,1H),7.33(s,1H),1.83-1.80(m,4H),1. 45(s,9H),1.40-1.35(m,11H),1.17-1.13(m,2H),0.88(s,3H),0.86(s,3H).

[0790] Step 6: 1-{[(Z)-(1-{2-[(tert-butyloxycarbonyl)amino]-1,3-thiazolyl}-2-oxo-2-{[(4S)-3-oxo-1,2-oxazolidine-4-yl]amino}ethylene)amino]oxy}-4,4-dimethylcyclohexane-1-carboxylic acid tert-butyl ester (117)

[0791]

[0792] DIPEA (0.52 mL, 3.00 mmol) was added to a solution of anhydrous DMF (10 mL) containing (2Z)-{2-[(tert-butoxycarbonyl)amino]-1,3-thiazolyl-4-yl}({1-(tert-butoxycarbonyl)-4,4-dimethylcyclohexyl]oxy}imino)acetic acid 116 (1.00 g, 2.00 mmol), and the mixture was stirred at room temperature for 10 min. HATU (0.76 g, 2.00 mmol) was then added, and the mixture was stirred at room temperature for 14 h. An additional 15 mL of anhydrous DMF was added, followed by DIPEA (1.4 mL, 8.00 mmol), and the mixture was stirred at room temperature for 10 min. L-cycloserine (0.30 g, 3.00 mmol) was added to this solution, and the reaction mixture was stirred for 2 h, followed by concentration under reduced pressure. Trace amounts of DMF were removed by co-evaporation with toluene (3 x 10 mL), and the residue was dissolved in DCM (100 mL). The organic phase was washed with water (50 mL) and brine (30 mL), dried over sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography using a gradient DCM containing 0–3% MeOH to give 1-{[(Z)-(1-{2-[(tert-butyloxycarbonyl)amino]-1,3-thiazolyl}-2-oxo-2-{[(4S)-3-oxo-1,2-oxazolidine-4-yl]amino}ethylene)amino]oxy}-4,4-dimethylcyclohexane-1-carboxylic acid tert-butyl ester 117, a white solid (1.02 g, 98%).

[0793] 1H-NMR (400MHz; DMSO-d6) δ11.78(s,1H),11.57(s,1H),9.04(d,J=7.8Hz,1H),7.37(s,1H),4.92-4.86(m,1H),4.57(t,J =8.5Hz,1H),4.07(t,J=9.0Hz,1H),1.90-1.71(m,4H),1.45(s,9H),1.39-1.33(m,11H),1.18-1.06(m,2H),0.87(s,6H).

[0794] Step 7: (4S)-2-[(4S)-4-{[(2Z)-2-{2-[(tert-butoxycarbonyl)amino]-1,3-thiazolyl-4-yl}-2-({[1-(tert-butoxycarbonyl)-4,4-dimethylcyclohexyl]oxy}imino]amino}-3-oxo-1,2-oxazolidine-2-yl]-4-(5,7-dioxo-2,2-diphenyl)-5,7-dihydro-2H,6H-[1,3]dioxane[4,5-f]isoindol-6-yl)-5-oxooxopentane-2-carboxylic acid tert-butyl ester (118)

[0795]

[0796] Anhydrous THF (20 mL) containing a mixture of 1-{[(Z)-(1-{2-[(tert-butoxycarbonyl)amino]-1,3-thiazo-4-yl}-2-oxo-2-{[(4S)-3-oxo-1,2-oxazolidine-4-yl]amino}ethylene)amino]oxy}-4,4-dimethylcyclohexane-1-carboxylic acid tert-butyl ester 117 (0.38 g, 0.70 mmol) and (2S)-5-tert-butoxy-2-(5,7-dioxo-2,2-diphenyl-5,7-dihydro2H,6H-[1,3]dioxane-[4,5-f]isoindol-6-yl)-4,5-dioxovalerate 6 (0.407 g, 0.70 mmol) was cooled to 0 °C in nitrogen. DMAP (17 mg, 0.14 mmol) was added, followed by DCC (0.202 g, 0.98 mmol). The reaction mixture was stirred at 0 °C for 1 hour. The mixture was allowed to gradually warm to room temperature and stirred overnight. The mixture was then concentrated to 25 °C, and the residue was ground in hexane (40 mL) containing 30% DCM. The precipitated solid was filtered off and washed with hexane (15 mL) and hexane (15 mL) containing 30% DCM. The filtrates were combined and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography using a gradient of hexane containing 0-30% ethyl acetate to give (4S)-2-[(4S)-4-{[(2Z)-2-{2-[(tert-butoxycarbonyl)amino]-1,3-thiazolyl-4-yl}-2-({[1-(tert-butoxycarbonyl)-4,4-dimethylcyclohexyl]oxy}imino]amino}-3-oxo-1,2-oxazolidine-2-yl]-4-(5,7-dioxo-2,2-diphenyl)-5,7-dihydro-2H,6H-[1,3]dioxacyclo[4,5-f]isoindol-6-yl)-5-oxooxopentane-2-carboxylic acid tert-butyl ester 118, a grayish-white solid (0.363 g, 47%).

[0797] 1 H-NMR(400MHz; CDCl3)δ8.11-8.00(m,2H),7.58-7.53(m,4H),7.45-7.41(m,6H),7 .35-7.32(m,3H),5.42-5.32(m,1H),5.17-4.93(m,2H),4.27-4.21(m,1H),3.44-3 .31(m,1H),2.90-2.84(m,1H),2.20-1.90(m,4H),1.61-1.57(s,9H),1.57-1.53(s ,9H),1.49-1.43(s,9H),1.33-1.24(m,4H),0.99-0.96(s,3H),0.96-0.92(s,3H).

[0798] Step 8: (4S)-2-[(4S)-4-{[(2Z)-2-(2-amino-1,3-thiazolyl-4-yl)-2-{[(1-carboxy-4,4-dimethylcyclohexyl)oxy]imino}acetyl]amino}-3-oxo-1,2-oxazolidine-2-yl]-4-(5,6-dihydroxy-1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)-5-oxooxopentane-2-carboxylic acid (Compound 38)

[0799]

[0800] At -50°C, boron trichloride solution (1.0 M in DCM, 2.5 mL, 2.5 mmol) was added dropwise to an anhydrous DCM solution (16 mL, 346 mg, 0.312 mmol) containing (4S)-2-[(4S)-4-{[(2Z)-2-{2-[(tert-butyloxycarbonyl)amino]-1,3-thiazolyl}-2-({[1-(tert-butyloxycarbonyl)-4,4-dimethylcyclohexyl]oxy}imino]amino}-3-oxo-1,2-oxazolidine-2-yl]-4-(5,7-dioxo-2,2-diphenyl)-5,7-dihydro-2H,6H-[1,3]dioxane-[4,5-f]isoindol-6-yl)-5-oxooxopentane-2-carboxylic acid tert-butyl ester 118 (346 mg, 0.312 mmol) The reaction mixture was stirred at -45 to -30°C for 2.5 hours, then cooled to -50°C and quenched using 30 mL of buffer solution (prepared by dissolving 776 mg NaHCO3 and 243 mg Na2HPO4 in 42 mL of water). The mixture was stirred in an ice-water bath instead of a cold bath until the aqueous layer thawed and the two layers separated. The DCM layer was carefully removed, and the aqueous phase was collected, filtered, and immediately purified by C18 reversed-phase chromatography using a Biotage system and a mixture of acetonitrile containing 0.1% formic acid and an aqueous solution containing 0.1% formic acid as eluent. Compound 38 was obtained as a pale yellow foamy solid, with a yield of 35% (80 mg).

[0801] 1¹H-NMR (400 MHz; mixture of D₂O and CD₃CN) δ 7.23 (s, 2H), 7.05 (s, 1H), 5.38–5.29 (m, 1H), 5.14–5.04 (m, 1H), 4.74–4.66 (m, 1H), 4.31–4.27 (m, 1H), 3.35–3.26 (m, 1H), 2.80–2.68 (m, 1H), 1.98–1.88 (m, 4H), 1.35–1.24 (m, 2H), 1.24–1.15 (m, 2H), 0.88 (s, 3H), 0.84 (s, 3H). No interchangeable protons were observed in D₂O.

[0802] MS(ESI)m / z: [M+1] + 731.2

[0803] Example 21

[0804] (4S)-2-[(4S)-4-{[(2Z)-2-(2-amino-1,3-thiazolyl-4-yl)-2-{[(1-carboxy-3,3-dimethylcyclobutyl)oxy]imino}acetyl]amino}-3-oxo-1,2-oxazolidine-2-yl]-4-(5,6-dihydroxy-1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)-5-oxooxopentane-2-carboxylic acid (Compound 14, Table 1)

[0805]

[0806] Step 1: 1-Bromo-3,3-dimethylcyclobutane-1-carboxylic acid (120)

[0807]

[0808] A mixture of 3,3-dimethylcyclobutane-1-carboxylic acid 119 (5.0 g, 39.0 mmol) and bromine (3 mL, 58.5 mmol) was placed in a round-bottom pressure flask, and PBr3 (0.6 mL, 6.5 mmol) was carefully added at room temperature. An ice-water bath was used to control the temperature of the reaction mixture during the addition. The flask was sealed, and the reaction mixture was heated at 100 °C and stirred overnight. The reaction mixture was cooled to room temperature, and the flask was carefully opened to release some of the internal pressure. The mixture was diluted with ethyl acetate (100 mL) and washed with 5% NaHSO3 solution (2 × 30 mL), water (2 × 30 mL), and brine (30 mL). The organic phase was dried over sodium sulfate, filtered, and concentrated to give 1-bromo-3,3-dimethylcyclobutane-1-carboxylic acid 120 as a grayish-white solid (8.0 g, quantitative yield).

[0809] 1H-NMR (599MHz; CDCl3) δ2.89-2.86(m,2H),2.56-2.53(m,2H),1.38(s,3H),1.08(s,3H).

[0810] Step 2: 1-Bromo-3,3-dimethylcyclobutane-1-carboxylic acid tert-butyl ester (121)

[0811]

[0812] At 0 °C, triethylamine (11.9 mL, 85.0 mmol) was added to a stirred mixture containing 120 bromo-3,3-dimethylcyclobutane-1-carboxylic acid (8.0 g, 36.6 mmol), t-BuOH (4.0 g, 54.1 mmol), and DMAP (472 mg, 3.9 mmol), followed by di-tert-butyl dicarbonate (11.0 g, 50.2 mmol). The reaction mixture was allowed to be heated to room temperature and stirred for 5 days. The reaction mixture was then separated into hexane (150 mL) and water (50 mL), and the different layers were separated. The aqueous phase was further extracted with hexane (4 × 30 mL). The combined organic extracts were washed with 1 N HCl (2 × 30 mL) and water (2 × 30 mL) and then concentrated under reduced pressure. The crude mixture was purified by silica gel column chromatography using a gradient of hexane containing 0-5% ethyl acetate to give 1-bromo-3,3-dimethylcyclobutane-1-carboxylic acid tert-butyl ester 121, a colorless liquid (6.45 g, 63%).

[0813] 1 H-NMR (400MHz; CDCl3) δ2.83-2.80(m,2H),2.53-2.49(m,2H),1.52(s,9H),1.31(s,3H),1.04(s,3H).

[0814] Step 3: 1-({(Z)-[1-(2-amino-1,3-thiazolyl-4-yl)-2-ethoxy-2-oxoethylidene]amino}oxy)-3,3-dimethylcyclobutane-1-carboxylic acid tert-butyl ester (122)

[0815]

[0816] Potassium carbonate (210 mg, 1.5 mmol) was added to anhydrous DMSO (5 mL) containing (Z)-2-(2-aminothiazol-4-yl)-2-(hydroxyimino)acetic acid (200 mg, 0.91 mmol). The resulting mixture was stirred at 50 °C for 5 minutes, and tert-butyl 121-bromo-3,3-dimethylcyclobutane-1-carboxylic acid ester (200 mg, 0.76 mmol) was added. The reaction mixture was stirred at 50 °C for 2 days, then cooled, diluted with water (30 mL), and extracted with diethyl ether (8 × 20 mL). The combined organic extracts were dried over sodium sulfate and concentrated under reduced pressure. The residue was purified by silica gel column chromatography using a gradient of hexane containing 0-20% ethyl acetate to give 122 of 1-({(Z)-[1-(2-amino-1,3-thiazolyl-4-yl)-2-ethoxy-2-oxoethylidene]amino}oxy)-3,3-dimethylcyclobutane-1-carboxylic acid tert-butyl ester, a grayish-white solid (150 mg, 50%).

[0817] 1 H-NMR (400MHz; CDCl3) δ6.61 (s, 1H), 6.57-6.57 (m, 2H), 4.41 (q, J = 7.1Hz, 2H), 2.41-2.37 (m,2H),2.09-2.06(m,2H),1.45(s,9H),1.37(t,J=7.1Hz,3H),1.20(s,3H),1.15(s,3H).

[0818] Step 4: 1-{[(Z)-(1-{2-[(tert-butyloxycarbonyl)amino]-1,3-thiazolyl}-2-ethoxy-2-oxoethylidene)amino]oxy}-3,3-dimethylcyclobutane-1-carboxylic acid tert-butyl ester (123)

[0819]

[0820] A solution of 1-({(Z)-[1-(2-amino-1,3-thiazolyl-4-yl)-2-ethoxy-2-oxoethylene]amino}oxy)-3,3-dimethylcyclobutane-1-carboxylic acid tert-butyl ester 122 (1.0 g, 2.5 mmol) in anhydrous THF (10 mL) was cooled to 0 °C. TMEDA (0.02 mL, 0.13 mmol) was added at 0 °C, followed by a solution of di-tert-butyl dicarbonate (0.577 g, 2.6 mmol) in THF (5 mL). The reaction mixture was stirred at 0 °C for 2 hours, then at room temperature for 48 hours. The reaction mixture was concentrated under reduced pressure, and trace amounts of solvent were removed by co-evaporation with MeOH (4 × 30 mL). The residue was purified by silica gel column chromatography using a gradient of hexane containing 0-20% ethyl acetate to give 1-{[(Z)-(1-{2-[(tert-butyloxycarbonyl)amino]-1,3-thiazolyl}-2-ethoxy-2-oxoethylidene)amino]oxy}-3,3-dimethylcyclobutane-1-carboxylic acid tert-butyl ester 123, as white foam (1.20 g, 96%).

[0821] 1 H-NMR (400MHz; CDCl3) δ7.21(s,1H),4.42(q,J=7.1Hz,2H),2.41(d,J=14.0Hz,2H),2.11(d ,J=14.0Hz,2H),1.52(s,9H),1.45(s,9H),1.37(t,J=7.1Hz,3H),1.21(s,3H),1.16(s,3H).

[0822] Step 5: (2Z)-{2-[(tert-butoxycarbonyl)amino]-1,3-thiazolyl-4-yl}({1-(tert-butoxycarbonyl)-3,3-dimethylcyclobutyl]oxy}imino)acetic acid (124)

[0823]

[0824] At room temperature, LiOH monohydrate (0.81 g, 19.3 mmol) was added to a MeOH solution (25 mL) containing 1-{[(Z)-(1-{2-[(tert-butyloxycarbonyl)amino]-1,3-thiazolyl}-2-ethoxy-2-oxoethylidene)amino]oxy}-3,3-dimethylcyclobutane-1-carboxylic acid tert-butyl ester 123 (3.2 g, 6.4 mmol). The reaction mixture was heated to 55 °C and stirred overnight. The reaction mixture was then concentrated under reduced pressure to remove methanol, and the pH of the aqueous solution was acidified to approximately pH 4 using 1 N HCl (approximately 20 mL). The aqueous solution was then extracted with ethyl acetate (6 × 20 mL), the combined organic extracts were washed with brine (2 × 20 mL), dried over sodium sulfate, filtered, and concentrated under reduced pressure to give (2Z)-{2-[(tert-butoxycarbonyl)amino]-1,3-thiazolyl-4-yl}({1-(tert-butoxycarbonyl)-3,3-dimethylcyclobutyl]oxy}imino)acetic acid 124, as a grayish-white foam (3.0 g, 99%). The obtained product was used for the next step without further purification.

[0825] 1 H-NMR (400MHz; CDCl3) δ7.43(s,1H),2.42-2.32(s,4H),1.55(s,9H),1.51(s,9H),1.36-1.35(m,2H),1.31-1.30(m,2H),1.26(s,3H),1.22(s,3H).

[0826] Step 6: 1-{[(Z)-(1-{2-[(tert-butyloxycarbonyl)amino]-1,3-thiazolyl}-2-oxo-2-{[(4S)-3-oxo-1,2-oxazolidine-4-yl]amino}ethylene)amino]oxy}-3,3-dimethylcyclobutane-1-carboxylic acid tert-butyl ester (125)

[0827]

[0828] At room temperature, DIPEA (1.67 mL, 9.6 mmol) and HATU (2.43 g, 6.4 mmol) were added to anhydrous DMF (35 mL) containing compound (2Z)-{2-[(tert-butoxycarbonyl)amino]-1,3-thiazolyl-4-yl}({1-(tert-butoxycarbonyl)-3,3-dimethylcyclobutyl]oxy}imino)acetic acid 124 (3.0 g, 6.4 mmol). The reaction mixture was stirred at room temperature for 4 hours, then DIPEA (1.67 mL, 9.6 mmol) and DMF (35 mL) were added, followed by L-cycloserine (0.783 g, 7.7 mmol). The resulting mixture was stirred overnight at room temperature and then diluted with ethyl acetate (150 mL). The organic phase was washed with water (6 × 30 mL), the aqueous layers were combined, and extracted again with diethyl ether (5 × 30 mL). The organic extracts were combined, washed with brine (2 × 30 mL), dried over sodium sulfate, and concentrated under reduced pressure. The residues were purified by silica gel column chromatography using a gradient DCM containing 0–5% methanol to give 125 of 1-{[(Z)-(1-{2-[(tert-butyloxycarbonyl)amino]-1,3-thiazolyl}-2-oxo-2-{[(4S)-3-oxo-1,2-oxazolidine-4-yl]amino}ethylene)amino]oxy}-3,3-dimethylcyclobutane-1-carboxylic acid tert-butyl ester, as a grayish-white solid (1.97 g, 56%).

[0829] 1 H-NMR (400MHz; DMSO-d6) δ11.85(s,1H),11.60(s,1H),9.15(d,J=8.0Hz,1H),7.44(s,1H),5.04-4.87(br,J=1.1Hz,1H),4.6 9-4.56(m,1H),4.14-4.05(m,1H),2.36-2.24(m,2H),2.09-1.98(m,2H),1.47(s,9H),1.41(s,9H),1.17(s,3H),1.13(s,3H).

[0830] Step 7: (4S)-2-[(4S)-4-{[(2Z)-2-{2-[(tert-butoxycarbonyl)amino]-1,3-thiazolyl-4-yl}-2-({[1-(tert-butoxycarbonyl)-3,3-dimethylcyclobutyl]oxy}imino)acetyl]amino}-3-oxo-1,2-oxazolidine-2-yl]-4-(5,7-dioxo-2,2-diphenyl-5,7)-dihydro-2H,6H-[1,3]dioxane[4,5-f]isoindol-6-yl)-5-oxooxopentane-2-carboxylic acid tert-butyl ester (126)

[0831]

[0832] Anhydrous THF (10 mL) containing a mixture of 1-{[(Z)-(1-{2-[(tert-butoxycarbonyl)amino]-1,3-thiazo-4-yl}-2-oxo-2-{[(4S)-3-oxo-1,2-oxazolidine-4-yl]amino}ethylene)amino]oxy}-3,3-dimethylcyclobutane-1-carboxylic acid tert-butyl ester 125 (408 mg, 0.74 mmol) and (2S)-5-tert-butoxy-2-(5,7-dioxo-2,2-diphenyl-5,7-dihydro2H,6H-[1,3]dioxane-[4,5-f]isoindol-6-yl)-4,5-dioxovalerate 6 (400 mg, 0.74 mmol) was cooled to 0 °C. DMAP (18 mg, 0.15 mmol) was added at 0 °C, followed by DCC (212 mg, 1.0 mmol). The resulting mixture was allowed to gradually warm to room temperature and stirred overnight. The reaction mixture was then concentrated under reduced pressure, and the residue was ground in hexane (20 mL) containing 30% DCM. The precipitated solids were removed by filtration, and the filtrate was concentrated under vacuum. The crude mixture was purified by silica gel column chromatography using a gradient of hexane containing 0–30% ethyl acetate, and the product was ground in hexane (20 mL) containing 25% ethyl acetate. The precipitate was removed by filtration, and the filtrate was concentrated to obtain (4S)-2-[(4S)-4-{[(2Z)-2-{2-[(tert-butyloxycarbonyl)amino]-1,3-thiazolyl-4-yl}-2-({[1-(tert-butyloxycarbonyl)-3,3-dimethylcyclobutyl]oxy}imino)acetyl]amino}-3-oxo-1,2-oxazolidine-2-yl]-4-(5,7-dioxo-2,2-diphenyl-5,7)-dihydro-2H,6H-[1,3]dioxacyclo[4,5-f]isoindol-6-yl)-5-oxooxopentane-2-carboxylic acid tert-butyl ester 126, which is a grayish-white foam (520 mg, 65%).

[0833] 1H-NMR (400MHz; CDCl3) δ8.34-8.27(m,1H),8.12-8.00(m,1H),7.57-7.54(m,4H),7.47-7. 42(m,6H),7.34-7.31(m,2H),5.44-5.35(m,1H),5.22-4.99(m,1H),4.99-4.86(m,1H),4. 29-4.17(m,1H),3.48-3.28(m,J=9.8Hz,1H),2.90-2.84(m,1H),2.48-2.26(m,4H),1.60- 1.57(m,9H),1.56-1.54(s,9H),1.51-1.48(m,9H),1.27-1.24(m,3H),1.24-1.23(m,3H).

[0834] Step 8: (4S)-2-[(4S)-4-{[(2Z)-2-(2-amino-1,3-thiazolyl-4-yl)-2-{[(1-carboxy-3,3-dimethylcyclobutyl)oxy]imino}acetyl]amino}-3-oxo-1,2-oxazolidine-2-yl]-4-(5,6-dihydroxy-1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)-5-oxooxopentane-2-carboxylic acid (Compound 14)

[0835]

[0836] At -50°C, boron trichloride solution (1.48 mL, 1.48 mmol) was added dropwise to an anhydrous DCM (16 mL) solution containing (4S)-2-[(4S)-4-{[(2Z)-2-{2-[(tert-butoxycarbonyl)amino]-1,3-thiazolyl}-2-({[1-(tert-butoxycarbonyl)-3,3-dimethylcyclobutyl]oxy}imino)acetyl]amino}-3-oxo-1,2-oxazolidine-2-yl]-4-(5,7-dioxo-2,2-diphenyl-5,7)-dihydro-2H,6H-[1,3]dioxane[4,5-f]isoindol-6-yl)-5-oxooxopentane-2-carboxylic acid tert-butyl ester 126 (200 mg, 0.19 mmol). The reaction mixture was stirred at -35°C to -30°C for 2.5 hours, then cooled to -50°C, and a solution of NaHCO3 (300 mg) and Na2HPO4 (100 mg) in H2O (16 mL) was added. Instead of a cold bath, an ice-water bath was used, and the mixture was stirred for 30 minutes, then stirred at room temperature until the aqueous layer thawed (approximately 30 minutes). The different layers were separated, and the organic phase was carefully removed. The aqueous solution was then purified by C18 reversed-phase column chromatography using a Biotage system and a mixture of acetonitrile containing 0.1% formic acid and water containing 0.1% formic acid as the eluent. The product containing fractions was collected and freeze-dried to obtain (4S)-2-[(4S)-4-{[(2Z)-2-(2-amino-1,3-thiazolyl-4-yl)-2-{[(1-carboxy-3,3-dimethylcyclobutyl)oxy]imino}acetyl]amino}-3-oxo-1,2-oxazolidine-2-yl]-4-(5,6-dihydroxy-1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)-5-oxooxopentane-2-carboxylic acid 14 (55 mg, 42%), a grayish-white solid.

[0837] 1 ¹H-NMR (400 MHz; mixture of CD₃CN and D₂O) δ 7.23 (s, 2H), 7.10 (s, 1H), 5.42–5.21 (m, 1H), 5.16–5.08 (m, 1H), 4.77–4.66 (m, 1H), 4.32–4.22 (m, 1H), 3.55–3.24 (m, 1H), 2.87–2.67 (m, 1H), 2.39 (d, J = 7.2 Hz, 2H), 2.06 (d, J = 7.2 Hz, 2H), 1.11 (s, 3H), 1.09 (s, 3H). No interchangeable protons were observed in D₂O.

[0838] MS(ESI)m / z: [M+1] + 703.1

[0839] Example 22

[0840] (4R)-2-[(4S)-4-{[(2Z)-2-(2-amino-1,3-thiazolyl-4-yl)-2-{[(2-carboxypropane-2-yl)oxy]imino}acetyl]amino}-3-oxo-1,2-oxazolidine-2-yl]-4-(5,6-dihydroxy-1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)-5-oxooxopentane-2-carboxylic acid (Compound 40, Table 1)

[0841]

[0842] Step 1: (4R)-2-[(4S)-4-{[(2Z)-2-{2-[(tert-butoxycarbonyl)amino]-1,3-thiazolyl-4-yl}-2-{[(1-tert-butoxy-2-methyl-1-oxopropane-2-yl)oxy]imino}acetyl]amino}-3-oxo-1,2-oxazolidine-2-yl]-4-(5,7-dioxo-2,2-diphenyl-5,7-dihydro-2H,6H-[1,3]dioxono[4,5-f]isoindol-6-yl)-5-oxooxopentane-2-carboxylic acid tert-butyl ester (127)

[0843]

[0844] (2R)-5-tert-butoxy-2-(5,7-dioxo-2,2-diphenyl-5,7-dihydro-2H,6H-[1,3]dioxane[4,5-f]isoindol-6-yl)-4,5-dioxoglutaric acid 128 was prepared in a manner similar to that described in J. Med. Chem., 2014, Vol. 57, pp. 3845-3855. The preparation began with 5-tert-butyl-1-methyl-N-[(benzyloxy)carbonyl]-4-oxo-D-glutamic acid and 2,2-diphenyl-2H-furan[3,4-f][1,3]benzodioxazole-5,7-dione.

[0845] Add DMAP (9 mg, 0.074 mmol) and DCC (107 mg, 0.52 mmol) to anhydrous THF (4 mL) containing a mixture of (2R)-5-tert-butoxy-2-(5,7-dioxo-2,2-diphenyl-5,7-dihydro-2H,6H-[1,3]dioxane-[4,5-f]isoindol-6-yl)-4,5-dioxoglutaric acid 128 (200 mg, 0.37 mmol) and 2-{[(Z)-(1-{2-[(tert-butoxycarbonyl)amino]-1,3-thiazo-4-yl}-2oxo-2-{[(4S)-3-oxo-1,2-oxazolidine-4-yl]amino}ethylene)amino]oxy}-2-methylpropionate tert-butyl ester 72 (189 mg, 0.37 mmol). The reaction mixture was stirred overnight at room temperature and then concentrated under reduced pressure. The residue was ground in hexane (20 mL) containing 40% DCM, the separated solids were filtered off and washed with hexane. The concentrated and combined filtrates were purified by silica gel column chromatography using a gradient of hexane containing 10-35% ethyl acetate to give (4R)-2-[(4S)-4-{[(2Z)-2-{2-[(tert-butoxycarbonyl)amino]-1,3-thiazolyl-4-yl}-2-{[(1-tert-butoxy-2-methyl-1-oxopropane-2-yl)oxy]imino}acetyl]amino}-3-oxo-1,2-oxazolidine-2-yl]-4-(5,7-dioxo-2,2-diphenyl-5,7-dihydro-2H,6H-[1,3]dioxono[4,5-f]isoindol-6-yl)-5-oxooxopentane-2-carboxylic acid tert-butyl ester 127 (174 mg, 45% yield), a light brown solid.

[0846] 1 H NMR(400MHz, CDCl3)δ8.09(br.s,1H),7.52-7.48(m,4H),7.40-7.37(m,6H),7.27(s,2H),7.23(s,1H),5.41-5.28(m,1H),5.15-4.82(m,2H),4.2 4-4.15(m,1H),3.50-3.38(m,2H),2.88-2.76(m,1H),1.95-1.85(m,1H) ,1.72-1.61(m,1H),1.58(s,6H),1.54(d,9H),1.50(s,9H),1.41(s,9H).

[0847] Step 2: (4R)-2-[(4S)-4-{[(2Z)-2-(2-amino-1,3-thiazolyl-4-yl)-2-{[(2-carboxypropane-2-yl)oxy]imino}acetyl]amino}-3-oxo-1,2-oxazolidine-2-yl]-4-(5,6-dihydroxy-1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)-5-oxooxopentane-2-carboxylic acid (compound 40)

[0848]

[0849] Anhydrous DCM (10 mL) containing (4R)-2-[(4S)-4-{[(2Z)-2-{2-[(tert-butoxycarbonyl)amino]-1,3-thiazolyl-4-yl}-2-{[(1-tert-butoxy-2-methyl-1-oxopropane-2-yl)oxy]imino}acetyl]amino}-3-oxo-1,2-oxazolidine-2-yl]-4-(5,7-dioxo-2,2-diphenyl-5,7-dihydro-2H,6H-[1,3]dioxono[4,5-f]isoindol-6-yl)-5-oxooxopentane-2-carboxylic acid tert-butyl ester 127 (170 mg, 0.163 mmol) was cooled to -50°C, and then boron trichloride solution (1.3 mL, 1.3 mmol, 1.0 M in DCM) was added dropwise. The reaction mixture was stirred at -50 to -35°C for 2.5 hours, then cooled to -50°C, and 14.3 mL of buffer solution (prepared by dissolving 776 mg NaHCO3 and 243 mg Na2HPO4 in 42 mL of water) was added. The heterogeneous mixture was stirred in an ice-water bath instead of a cold bath until the aqueous phase thawed and the two layers separated. The organic layer was carefully removed, and the aqueous phase was immediately purified by C18 reversed-phase column chromatography using Biotage and a mixture of acetonitrile containing 0.1% formic acid and an aqueous solution containing 0.1% formic acid as eluent to give compound 40 (40 mg, 37%) as a pale yellow solid.

[0850] 1¹H NMR (400 MHz, mixture of D₂O and CD₃CN) δ 7.35 (s, 2H), 7.18 (s, 1H), 5.54–5.48 (m, 1H), 5.28–5.20 (m, 1H), 4.91–4.76 (m, 1H), 4.45–4.30 (m, 1H), 3.52–3.32 (m, 1H), 2.89–2.76 (m, 1H), 1.58 (s, 6H). No interchangeable protons were observed in D₂O.

[0851] MS(ESI)m / z: [M+1] + 663.1

[0852] Bioactivity

[0853] In vitro antibacterial evaluation methods

[0854] Bacterial isolates

[0855] Gram-negative and Gram-positive bacterial strains used during in vitro minimum inhibitory concentration (MIC) assays were obtained from the American Type Culture Collection (ATCC) or clinical specimens, respectively. All strains were genotyped, and individual antibiotic resistance markers were identified for each strain. Table 2 below shows the strains used and their respective resistance markers.

[0856] Table 2. Bacterial strains used for in vitro MIC detection

[0857]

[0858]

[0859] MIC detection

[0860] The isolates were subcultured on appropriate media and stored in skim milk at -80°C. Following the two subcultures in frozen storage, in vitro antimicrobial susceptibility testing was performed using the Clinical and Laboratory Standards Institute (CLSI) broth microdilution method. In short, fresh cultures were resuspended in media to provide 5 x 10⁻⁶ broth per mL. 7 The final stock inoculum was prepared at a concentration of 128 μg / mL. The test compound was prepared at a stock solution concentration equal to 128 μg / mL and then diluted to a final initial working concentration of 32 μg / mL. 100 μl of stock solution was added to the first well of a 96-well plate, followed by a 2-fold dilution to provide a range of 32–0.025 μg / mL. Then, 100 μl of bacterial suspension was added to the 96-well plate to provide 5 x 10⁻⁶ cells / well. 5The final working inoculum amount was CFU / mL. Final compound concentrations ranged from 16 to 0.0125 μg / mL. All compounds were tested in iron-free, cation-regulated Mueller-Hinton broth, and the minimum inhibitory concentration (MIC) was read as the first well showing a significant decrease in growth compared to the growth control (inhibition zone <1 mm or insufficient turbidity). The MIC of the comparator reagents was determined using a custom-designed, in-house prepared 96-well broth microdilution plate.

[0861] Synergistic activity assay

[0862] The same method used to test the activity of the compound is used to determine its synergistic activity when bound to a β-lactamase inhibitor, but a fixed concentration or ratio of the β-lactamase inhibitor is added to the 96-well plate before the addition of bacterial culture. Synergistic activity is defined as the reduction in the MIC of a given compound when tested with a β-lactamase inhibitor compared to when the compound is tested in the absence of a β-lactamase inhibitor.

[0863] Test Results

[0864] As shown in Tables 3-6, the indicator compounds exhibited activity against Gram-negative bacteria, including resistant strains. The MICs of the indicator compounds against wild-type strains of *Acinetobacter baumannii*, *Citrobacter*, *Escherichia coli*, and *Pseudomonas aeruginosa* ranged from 0.03 to >16 μg / mL. These compounds also showed activity against resistant strains of *Citrobacter freundii*, *Enterobacter cloacae*, *Escherichia coli*, *Klebsiella pneumoniae*, *Klebsiella pneumoniae*, *Klebsiella pneumoniae*, and *Pseudomonas aeruginosa*. The MICs of these strains ranged from <0.015 to >16.0 μg / mL (Tables 3-6). The MICs of the selected compounds were enhanced using avibactam. Avibactam resulted in a 3-4 fold reduction in MICs in mice due to its inhibition of β-lactamases (Tables 7 and 8).

[0865] Table 3. Antibacterial activity of representative compounds

[0866]

[0867]

[0868] Table 4. Antibacterial activity of representative compounds

[0869]

[0870]

[0871] Table 5. Antibacterial activity of representative compounds

[0872]

[0873]

[0874]

[0875] Table 6. Antibacterial activity of representative compounds

[0876]

[0877]

[0878] Table 7. In the presence of avibactam @ 4 μg / mL, MIC-enhanced cAMP and CMY-DHA-producing strains

[0879]

[0880]

[0881]

[0882] 1 CAZ ceftazidime; 2 AVI Avibatan

[0883] Table 8. In the presence of avibactam @ 4 μg / mL, MIC-resistant strains showed increased AmpC and CMY-DHA production.

[0884]

[0885]

[0886] 1 CAZ ceftazidime; 2 AVI Avibatan

Claims

1. Compound of formula (I) (I) Where A is defined by equation (Ia). (Ia) Where X is N or CR 3 R 3 Indicates hydrogen or halogen; R 1 and R 2 Together with the carbon atoms bonded to them, they can form (C3-C8) cycloalkyl groups, in which... (i) The cycloalkyl group may contain a heteroatom selected from O, N and S and / or (ii) The cycloalkyl group may be substituted with one, two, three, or four substituents, which are independent of each other and selected from (C1-C3) alkyl groups and halogens; or R 1 and R 2 They can be independent of each other, representing hydrogen or (C1-C3) alkyl, wherein the (C1-C3) alkyl can be substituted with substituents selected from hydroxyl and chlorine; B is a fragment containing a bicyclic catechol or hydroxypyridinone moiety, as defined by formula (Ia′). (Ia') Wherein P is an unsaturated 5-membered or 6-membered ring, which may optionally contain one or two carbonyl groups, or one sulfone group, or a combination of one carbonyl and one sulfone group, and may further contain two additional N atoms; and Q can contain at most two N atoms, and R 4 Selected from hydrogen, (C1-C3)alkyl, carbonyl, trifluoromethyl, cyano, and halogen; and their salts; The compound described in formula (I) is selected from: ,or 。 2. According to claim 1, the compound of formula (I) is: (4S)-2-[(4S)-4-{[(2Z)-2-(2-amino-1,3-thiazolyl-4-yl)-2-{[(1-carboxycyclopropyl)oxy]imino}acetyl]amino}-3-oxo-1,2-oxazolidine-2-yl]-4-(5,6-dihydroxy-1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)-5-oxooxopentane-2-carboxylic acid (4S)-2-((S)-4-((Z)-2-(2-aminothiazolyl-4-yl)-2-((1-carboxycyclopropoxy)imino)acetamyl)-3-oxoisooxazolidine-2-yl)-4-(2,3-dihydroxy-5,7-dioxo-5,7-dihydro-6H-pyrrolo[3,4-b]pyridin-6-yl)-5-oxooxopentane-2-carboxylic acid (4S)-2-[(4S)-4-{[(2Z)-2-(2-amino-1,3-thiazolyl-4-yl)-2-{[(1-carboxycyclobutyl)oxy]imino}acetyl]amino}-3-oxo-1,2-oxazolidine-2-yl]-4-(5,6-dihydroxy-1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)-5-oxooxopentane-2-carboxylic acid (4S)-2-[(4S)-4-{[(2Z)-2-(2-amino-1,3-thiazolyl-4-yl)-2-{[(3-carboxyoxobutane-3-yl)oxy]imino}acetyl]amino}-3-oxo-1,2-oxazolidine-2-yl]-4-(5,6-dihydroxy-1,3-dioxo-2,3-dihydro-2H-isoindol-2-yl)-5-oxooxopentane-2-carboxylic acid (4S)-2-[(4S)-4-{[(2Z)-2-(2-amino-1,3-thiazolyl-4-yl)-2-{[(2-carboxy-1-chloro-3-hydroxypropane-2-yl)oxy]imino}acetyl]amino}-3-oxo-1,2-oxazolidine-2-yl]-4-(5,6-dihydroxy-1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)-5-oxooxopentane-2-carboxylic acid 3-({(Z)-[1-(2-amino-1,3-thiazolyl-4-yl)-2-({(4S)-2-[(4S)-2-carboxy-4-(5,6-dihydroxy-1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)-5-oxooxopentane-2-yl]-3-oxo-1,2-oxazolidine-4-yl}amino)-2-oxoethylidene]amino}oxy)azacyclobutane-3-carboxylic acid 4-({(Z)-[1-(2-amino-1,3-thiazolyl-4-yl)-2-({(4S)-2-[(4S)-2-carboxy-4-(5,6-dihydroxy-1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)-5-oxooxopentane-2-yl]-3-oxo-1,2-oxazolidine-4-yl}amino)-2-oxoethylidene]amino}oxy)oxohexyl-4-carboxylic acid 4-(((Z)-1-(2-aminothiazolyl-4-yl)-2-((4S)-2-((4S)-2-carboxy-4-(5,6-dihydroxy-1,3-dioxoisoindol-2-yl)-5-oxooxopentane-2-yl)-3-oxoisooxazolidine-4-yl)amino)-2-oxoethyl)amino)oxy)-1-methylpiperidine-4-carboxylic acid (4S)-2-((S)-4-((Z)-2-(2-aminothiazolyl-4-yl)-2-((1-carboxy-4,4-difluorocyclohexyl)oxy)imino)acetamyl)-3-oxoisooxazolidine-2-yl)-4-(5,6-dihydroxy-1,3-dioxoisoindol-2-yl)-5-oxooxopentane-2-carboxylic acid (4S)-2-((S)-4-((Z)-2-(2-aminothiazolyl-4-yl)-2-((1-carboxy-4-chloro-4-fluorocyclohexyl)oxy)imino)acetamyl)-3-oxoisooxazolidine-2-yl)-4-(5,6-dihydroxy-1,3-dioxoisoindol-2-yl)-5-oxooxopentane-2-carboxylic acid (4S)-2-((S)-4-((Z)-2-(2-aminothiazolyl-4-yl)-2-((1-carboxy-4,4-dichlorocyclohexyl)oxy)imino)acetamyl)-3-oxoisooxazolidine-2-yl)-4-(5,6-dihydroxy-1,3-dioxoisoindol-2-yl)-5-oxooxopentane-2-carboxylic acid 3-({(Z)-[1-(2-amino-1,3-thiazolyl-4-yl)-2-({(4S)-2-[(4S)-2-carboxy-4-(5,6-dihydroxy-1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)-5-oxooxopentane-2-yl]-3-oxo-1,2-oxazolidine-4-yl}amino)-2-oxoethyl]amino}oxy)-8-oxabicyclo[3.2.1]octane-3-carboxylic acid (4S)-2-[(4S)-4-{[(2Z)-2-(2-amino-1,3-thiazolyl-4-yl)-2-{[(1-carboxy-3,3-dimethylcyclobutyl)oxy]imino}acetyl]amino}-3-oxo-1,2-oxazolidine-2-yl]-4-(5,6-dihydroxy-1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)-5-oxooxopentane-2-carboxylic acid (4S)-2-[(4S)-4-{[(2Z)-2-(5-amino-1,2,4-thiadiazol-3-yl)-2-{[(1-carboxycyclopropyl)oxy]imino}acetyl]amino}-3-oxo-1,2-oxazolidine-2-yl]-4-(5,6-dihydroxy-1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)-5-oxooxopentane-2-carboxylic acid (4S)-2-[(4S)-4-{[(2Z)-2-(2-amino-5-chloro-1,3-thiazolyl-4-yl)-2-{[(1-carboxycyclopropyl)oxy]imino}acetyl]amino}-3-oxo-1,2-oxazolidine-2-yl]-4-(5,6-dihydroxy-1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)-5-oxooxopentane-2-carboxylic acid 4-({(Z)-[1-(2-amino-1,3-thiazolyl-4-yl)-2-({(4S)-2-[(4S)-2-carboxy-4-(2,3-dihydroxy-5,7-dioxo-5,7-dihydro-6H-pyrrolo[3,4-b]pyridin-6-yl)-5-oxooxopentane-2-yl]-3-oxo-1,2-oxozolidine-4-yl}amino)-2-oxoethylene]amino}oxy)oxohexyl-4-carboxylic acid 3-({(Z)-[1-(2-amino-1,3-thiazolyl-4-yl)-2-({(4S)-2-[(4S)-2-carboxy-4-(2,3-dihydroxy-5,7-dioxo-5,7-dihydro-6H-pyrrolo[3,4-b]pyridin-6-yl)-5-oxooxopentane-2-yl]-3-oxo-1,2-oxazolidine-4-yl}amino)-2-oxoethylidene]amino}oxy)-8-oxoheterocyclic[3.2.1]octane-3-carboxylic acid (4S)-2-[(4S)-4-{[(2Z)-2-(2-amino-1,3-thiazolyl-4-yl)-2-{[(1-carboxycyclopropyl)oxy]imino}acetyl]amino}-3-oxo-1,2-oxazolidine-2-yl]-4-(5,6-dihydroxy-1-oxo-1,3-dihydro-2H-isoindol-2-yl)-5-oxooxopentane-2-carboxylic acid 4-({(Z)-[1-(2-amino-1,3-thiazolyl-4-yl)-2-({(4S)-2-[(4S)-2-carboxy-4-(5,6-dihydroxy-1-oxo-1,3-dihydro-2H-isoindol-2-yl)-5-oxooxopentane-2-yl]-3-oxo-1,2-oxazolidine-4-yl}amino)-2-oxoethylene}amino}oxy)oxohexyl-4-carboxylic acid 3-({(Z)-[1-(2-amino-1,3-thiazolyl-4-yl)-2-({(4S)-2-[(4S)-2-carboxy-4-(5,6-dihydroxy-1-oxo-1,3-dihydro-2H-isoindol-2-yl)-5-oxooxopentane-2-yl]-3-oxo-1,2-oxazolidine-4-yl}amino)-2-oxoethylene}amino}oxy)8-oxabicyclo[3.2.1]octane-3-carboxylic acid (4S)-2-[(4S)-4-{[(2Z)-2-(2-amino-1,3-thiazolyl-4-yl)-2-{[(2-carboxypropane-2-yl)oxy]imino}acetyl]amino}-3-oxo-1,2-oxazolidine-2-yl]-4-(5,6-dihydroxy-1-oxo-1,3-dihydro-2H-isoindol-2-yl)-5-oxooxopentane-2-carboxylic acid (4S)-2-[(4S)-4-{[(2Z)-2-(2-amino-1,3-thiazo-4-yl)-2-{[(2-carboxypropane-2-yl)oxy]imino}acetyl]amino}-3-oxo-1,2-oxazolidine-2-yl]-4-(2,3-dihydroxy-5,7-dioxo-5,7-dihydro-6H-pyrrolo[3,4-b]pyridin-6-yl)-5-oxooxopentane-2-carboxylic acid (4S)-2-[(4S)-4-{[(2Z)-2-(2-amino-1,3-thiazolyl-4-yl)-2-{[(2-carboxypropane-2-yl)oxy]imino}acetyl]amino}-3-oxo-1,2-oxazolidine-2-yl]-4-(5,6-dihydroxy-1,1,3-trioxo-1,3-dihydro-2H-1λ) 6 2-Benzothiazol-2-yl)-5-oxooxopentane-2-carboxylic acid (4S)-2-[(4S)-4-{[(2Z)-2-(2-amino-1,3-thiazolyl-4-yl)-2-{[(1-carboxy-4,4-dimethylcyclohexyl)oxy]imino}acetyl]amino}-3-oxo-1,2-oxazolidine-2-yl]-4-(5,6-dihydroxy-1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)-5-oxooxopentane-2-carboxylic acid, or 2-[(4S)-4-{[(2Z)-2-(2-amino-1,3-thiazolyl-4-yl)-2-{[(2-carboxyl-1,3-dihydroxypropane-2-yl)oxy]imino}acetyl]amino}-3-oxo-1,2-oxazolidine-2-yl]-4-(5,6-dihydroxy-1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)-5-oxooxopentane-2-carboxylic acid.

3. A pharmaceutical composition comprising compounds 3-14, 18, 24, 26-30 and 35-39 of claim 1 or pharmaceutically acceptable salts thereof, and a pharmaceutically acceptable carrier or diluent.

4. A pharmaceutical composition comprising (i) one or more compounds 3-14, 18, 24, 26-30 and 35-39 of claim 1, or pharmaceutically acceptable salts thereof, (ii) one or more β-lactamase inhibitors, and (iii) a pharmaceutically acceptable carrier or diluent.

5. The pharmaceutical composition according to claim 4, wherein the β-lactamase inhibitor is selected from formula (1) a ) to (1 Z’ ): and .

6. Use of the pharmaceutical composition of claim 3 in the preparation of a medicament for treating or preventing bacterial infection in a subject, comprising administering an effective therapeutic amount of the pharmaceutical composition of claim 3 to a subject suffering from or at risk of developing a bacterial infection.

7. The use according to claim 6, wherein the bacterial infection is composed of one or more Enterobacteriaceae, Escherichia coli, Enterobacter spp., Klebsiella spp., Serratia spp., Pseudomonas spp., Oligotrophoblast spp., Citrobacter spp., Acinetobacter spp., Campylobacter spp., Helicobacter spp., Vibrio spp., Bordetella spp., Salmonella spp., Shigella spp., Francisella spp., Burkholderia spp., Clostridium spp., Alcaligenes spp., Moraxella spp., Proteus spp., Neisseria spp., Haemophilus spp., Achromobacter spp., and Erwinia spp.

8. Use of the pharmaceutical composition of claim 4 in the preparation of a medicament for treating or preventing bacterial infection in a subject, comprising administering an effective therapeutic amount of the pharmaceutical composition of claim 4 to a subject suffering from or at risk of developing a bacterial infection.

9. The use according to claim 8, wherein the bacterial infection is composed of one or more Enterobacteriaceae, Escherichia coli, Enterobacter spp., Klebsiella spp., Serratia spp., Pseudomonas spp., Oligotrophoblast spp., Citrobacter spp., Acinetobacter spp., Campylobacter spp., Helicobacter spp., Vibrio spp., Bordetella spp., Salmonella spp., Shigella spp., Francisella spp., Burkholderia spp., Clostridium spp., Alcaligenes spp., Moraxella spp., Proteus spp., Neisseria spp., Haemophilus spp., Achromobacter spp., and Erwinia spp.

10. A method for preparing a compound according to formula (I) of claim 1, comprising performing the reaction sequence of scheme 1, Option 1 。 11. A method for preparing a compound according to formula (I) of claim 1, comprising removing protecting groups from the compound of formula (IV), wherein Pg′ and Pg″ represent protecting groups. In formula (IV), the carboxyl protecting groups Pg′ and Pg″ are residues that form aliphatic or aryl alcohols in esters.

12. The method according to claim 11, wherein the carboxyl protecting group is independently selected from isopropyl, tert-butyl, methoxymethyl, ethoxymethyl, isobutoxymethyl, acetoxymethyl, propionyloxymethyl, butyryloxymethyl, valeratemethyl, 1-methoxycarbonyloxyethyl, 1-ethoxycarbonyloxyethyl, p-methoxybenzyl, benzyl, o-nitrophenyl, p-nitrophenyl, 2,4-dinitrophenylmethyl, diphenylmethyl, phthaloyl, and allyl.

13. The method according to claim 11, characterized in that... Compound (IV) is prepared by reacting compound (II) with compound (III). Where A is defined by equation (Ia). (Ia) Where X is N or CR 3 R 3 Indicates hydrogen or halogen; R 1 and R 2 Together with the carbon atoms they are bonded to, they can form (C3-C8) cycloalkyl groups, in which... (i) The cycloalkyl group may contain a heteroatom selected from O, N and S and / or (ii) The cycloalkyl group may be substituted with one, two, three, or four substituents, which are independent of each other and selected from (C1-C3) alkyl groups and halogens; or R 1 and R 2 They can be independent of each other, representing hydrogen or (C1-C3) alkyl, wherein the (C1-C3) alkyl can be substituted by a substituent selected from hydroxyl and chlorine; B is a fragment containing a bicyclic catechol or hydroxypyridinone moiety, as defined by formula (Ia′). (Ia') Wherein P is an unsaturated 5-membered or 6-membered ring, which may optionally contain one or two carbonyl groups, or one sulfone group, or a combination of one carbonyl and one sulfone group, and may further contain two additional N atoms; and Q can contain at most two N atoms, and R 4 It is selected from hydrogen, (C1-C3)alkyl, carbonyl, trifluoromethyl, cyano and halogen.

14. The method according to claim 13, wherein the compound of formula (III) is prepared by the reaction sequence of scheme 2 to provide an intermediate of formula (IIIa′). Option 2 。 15. The method according to claim 13, wherein the compound of formula (III) is prepared by the reaction sequence of scheme 3 to provide an intermediate of formula (IIIa''), Option 3 。 16. The method according to claim 13, wherein in an inert solvent, in the presence of a coupling agent, the intermediate (II) undergoes a coupling reaction with the intermediate (III) at a temperature ranging from -20°C to 80°C for 1 to 24 hours.

17. The method according to claim 16, wherein the inert solvent is one or more selected from dichloromethane, toluene, tetrahydrofuran, 1,4-dioxane, N,N-dimethylformamide, N,N-dimethylacetamide, acetonitrile, and N-methylpyrrolidone-2-one.

18. The method according to claim 16, wherein the coupling agent is N,N'-diethylcarbodiimide, N,N'-dipropylcarbodiimide, N,N'-diisopropylcarbodiimide, N,N'-dicyclohexylcarbodiimide, N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), carbonyl diimidazole (CDI), O-(benzotriazol-1-yl)-N,N,N',N'-tetraamineethylurea hexafluorophosphate One or more of the following: acid (HBTU), O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethylurea hexafluorophosphate (HATU), 1-hydroxybenzotriazole (HOBT), (benzotriazol-1-yloxy)tris(dimethylamino)hexafluorophosphate (BOP), (benzotriazol-1-yloxy)tripyrrolidine hexafluorophosphate (PyBOP), and N-hydroxysuccinimide, with or without the addition of a base.

19. The method of claim 16, further comprising adding a base during the coupling reaction, wherein the base is one or more of a carbonate, a bicarbonate, triethylamine, diisopropylethylamine, N-methylmorpholine, N-methylpiperidine, and 4-dimethylaminopyridine.

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