Therapeutic compounds and methods

Abstract

The disclosure provides compound of formula (I): or a salt thereof, wherein R'-R10, W, X, Y, and Z have any of the values described in the specification, as well as compositions comprising such compounds or salts, methods of making such compounds and salts, and methods of using such compounds and salts, e.g., as inhibitors of bacterial RNA polymerase and as antibacterial agents.

Description

[0001] THERAPEUTIC COMPOUNDS AND METHODS

[0002] CROSS-REFERENCE TO RELATED APPLICATION

[0003] This application claims priority to United States Provisional Application Number 63 / 614,741 that was filed on 26 December 2023. The entire content of the application referenced above is hereby incorporated by reference herein.

[0004] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0005] This invention was made with government support under AI109713 and AI142731 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0006] BACKGROUND OF THE INVENTION

[0007] Bacterial infectious diseases kill 100,000 persons each year in the US and 11 million persons each year worldwide, representing nearly a fifth of deaths each year worldwide (Heron et al., Final Data for 2006. National Vital Statistics Reports, Vol. 57 (Centers for Disease Control and Prevention, Atlanta GA) and World Health Organization (2008) The Global Burden of Disease: 2004 Update (World Health Organization, Geneva)). In the US, hospital-acquired bacterial infections strike 2 million persons each year, resulting in 90,000 deaths and an estimated $30 billion in medical costs (Klevins et al., (2007) Estimating health care-associated infections and deaths in U.S. hospitals. Public Health Reports, 122, 160-166; Scott, R. (2009) The direct medical costs of healthcare-associated infections in U.S. hospitals and benefits of prevention (Centers for Disease Control and Prevention, Atlanta GA)). Worldwide, the bacterial infectious disease tuberculosis kills nearly 2 million persons each year. One third of the world's population currently is infected with tuberculosis, and the World Health Organization projects that there will be nearly 1 billion new infections by 2020, 200 million of which will result in serious illness, and 35 million of which will result in death. Bacterial infectious diseases also are potential instruments of biowarfare and bioterrorism.

[0008] For six decades, antibiotics have been a bulwark against bacterial infectious diseases. This bulwark is failing due to the appearance of resistant bacterial strains. For all major bacterial pathogens, strains resistant to at least one current antibiotic have arisen. For several bacterial pathogens, including tuberculosis, strains resistant to all current antibiotics have arisen.

[0009] Bacterial RNA polymerase (RNAP) is a proven target for antibacterial therapy (Darst, S. (2004) Trends Biochem. Sci. 29, 159-162; Chopra, I. (2007) Curr. Opin. Investig. Drugs 8, 600-607; Villain-Guillot, P., Bastide, L., Gualtieri, M. & Leonetti, J. (2007) Drug Discov. Today 12, 200-208; Ho, M., Hudson, B., Das, K., Arnold, E., Ebright, R. (2009) Curr. Opin. Struct. Biol. 19, 715-723; and Srivastava et al. (2011) Curr. Opin. Microbiol. 14, 532-543). The suitability of bacterial RNAP as a target for antibacterial therapy follows from the fact that bacterial RNAP is an essential enzyme (permitting efficacy), the fact that bacterial RNAP subunit sequences are highly conserved (permitting broad-spectrum activity), and the fact that bacterial RNAP-subunit sequences are highly conserved in human RNAP I, RNAP II, and RNAP III (permitting therapeutic selectivity).

[0010] The rifamycin antibacterial agents function by binding to and inhibiting bacterial RNAP (Darst, S. (2004) Trends Biochem. Sci. 29, 159-162; Chopra, I. (2007) Curr. Opin. Investig. Drugs 8, 600-607; Villain-Guillot, P., Bastide, L., Gualtieri, M. & Leonetti, J. (2007) Drug Discov. Today 12, 200-208; and Ho, M., Hudson, B., Das, K., Arnold, E., Ebright, R. (2009) Curr. Opin. Struct. Biol. 19, 715-723). The rifamycins bind to a site on bacterial RNAP adjacent to the RNAP active center and prevent extension of RNA chains beyond a length of 2-3 nt. The rifamycins are in current clinical use in treatment of both Gram-positive and Gram-negative bacterial infections. The rifamycins are of particular importance in treatment of tuberculosis; the rifamycins are first-line anti-tuberculosis agents and are among the few antituberculosis agents able to kill non-replicating tuberculosis bacteria.

[0011] The clinical utility of the rifamycin antibacterial agents is threatened by the existence of bacterial strains resistant to rifamycins (Darst, S. (2004) Trends Biochem. Sci. 29, 159-162; Chopra, I. (2007) Curr. Opin. Investig. Drugs 8, 600-607; Villain-Guillot, P., Bastide, L., Gualtieri, M. & Leonetti, J. (2007) Drug Discov. Today 12, 200-208; and Ho, M., Hudson, B., Das, K., Arnold, E., Ebright, R. (2009) Curr. Opin. Struct. Biol. 19, 715-723). Resistance to rifamycins typically involves substitution of residues in or immediately adjacent to the rifamycin binding site on bacterial RNAP— i.e., substitutions that directly decrease binding of rifamycins.

[0012] In view of the public-health threat posed by rifamycin-resistant and multidrugresistant bacterial infections, there is an urgent need for new antibacterial agents that (i) inhibit bacterial RNAP (and thus have the same biochemical effects as rifamycins), but that (ii) inhibit bacterial RNAP through binding sites that do not overlap the rifamycin binding site (and thus do not share cross-resistance with rifamycins.

[0013] A new drug target— the "switch region"— within the structure of bacterial RNAP has been identified (W02007 / 094799; Mukhopadhyay, J. et al. (2008) Cell. 135, 295-307; see also Belogurov, G. et al. (2009) Nature. 45, 332-335; Ho et al. (2009) Curr. Opin. Struct. Biol. 19, 715-723; Srivastava et al. (2011) Curr. Opin. Microbiol. 14, 532-543). The switch region is a structural element that mediates conformational changes required for RNAP to bind and retain the DNA template in transcription. The switch region is located at the base of the RNAP active-center cleft and serves as the hinge that mediates opening of the active-center cleft to permit DNA binding and that mediates closing of the active-center cleft to permit DNA retention. The switch region can serve as a binding site for compounds that inhibit bacterial gene expression and kill bacteria. Since the switch region is highly conserved in bacterial species, compounds that bind to the switch region are active against a broad spectrum of bacterial species. Since the switch region does not overlap the rifamycin binding site, compounds that bind to the switch region are not cross-resistant with rifamycins.

[0014] It has been shown that the α-pyrone antibiotic myxopyronin (Myx) functions through interactions with the bacterial RNAP switch region (W02007 / 094799; Mukhopadhyay, J. et al. (2008) Cell. 135, 295-307; see also Belogurov, G. et al. (2009) Nature. 45, 332-335; Ho et al. (2009) Curr. Opin. Struct. Biol. 19, 715-723; Srivastava et al. (2011) Curr. Opin. Microbiol. 14, 532-543). Myx binds to the RNAP switch region, traps the RNAP switch region in a single conformational state, and interferes with formation of a catalytically competent transcription initiation complex. Amino acid substitutions within RNAP that confer resistance to Myx occur only within the RNAP switch region. There is no overlap between amino acid substitutions that confer resistance to Myx and amino acid substitutions that confer resistance to rifamycins and, accordingly, there is no cross-resistance between Myx and rifamycins.

[0015] A crystal structure of a non-pathogenic bacterial RNAP, Thermus thermophilus RNAP, in complex with Myx has been determined, and homology models of pathogenic bacterial RNAP, including Mycobacterium tuberculosis RNAP and Staphylococcus aureus RNAP, in complex with Myx have been constructed (W02007 / 094799; Mukhopadhyay, J. et al. (2008) Cell. 135, 295-307; see also Belogurov, G. et al. (2009) Nature. 45, 332-335; Ho et al. (2009) Curr. Opin. Struct. Biol. 19, 715-723; Srivastava et al. (2011) Curr. Opin. Microbiol. 14, 532-543). The crystal structure and homology models define interactions between RNAP and Myx and can be used to understand the roles of the "west" and "east" Myx sidechains as well as the Myx α-pyrone core.

[0016] United States Patent Numbers 9,133,155, 9,187,446, 9,315,495 and 9,592,221 relate to pyronin compounds that are reported to possess antibacterial activity. Additionally, International Patent Application Publication Number WO / 2019 / 160873 relates to pyronin compounds that are reported to possess antibacterial activity. In spite of these disclosures, there remains a need for pyronin antibacterial compounds that possess improved metabolic stability, improved in vivo pharmacokinetics, improved in vitro antibacterial activity, and / or improved in vivo antibacterial efficacy.

[0017] SUMMARY OF THE INVENTION

[0018] The disclosure provides new compositions of matter that inhibit bacterial RNA polymerase and inhibit bacterial growth. The compounds have applications in analysis of RNA polymerase structure and function, control of bacterial gene expression, control of bacterial growth, antibacterial prophylaxis, antibacterial therapy, and / or drug discovery.

[0019] The disclosure provides new compositions of matter that inhibit bacterial RNA polymerase and inhibit bacterial growth.

[0020] Compounds of this disclosure differ from previously disclosed pyronins with RNA- polymerase-inhibitory and antibacterial activities by halogenation on a specific heterocyclic ring.

[0021] Applicant has discovered— surprisingly— that halogenation on a specific heterocyclic ring improves in vitro RNA polymerase inhibitory activity, in vitro antibacterial activity, in vivo bioavailability, and / or in vivo antibacterial efficacy.

[0022] Certain compounds exhibit higher in vitro RNA polymerase inhibitory activity.

[0023] Certain compounds exhibit superior in vitro antibacterial activity.

[0024] Certain compounds exhibit superior in vivo pharmacokinetics.

[0025] Certain compounds exhibit superior in vivo antibacterial efficacy.

[0026] An object of this disclosure is to provide antibacterial compounds that possess one or more of the following: 1) improved metabolic stability, 2) in vivo pharmacokinetics, 3) improved in vitro antibacterial efficacy, and / or 4) improved in vivo antibacterial efficacy.

[0027] The compounds and salts have utility as inhibitors of bacterial RNAP.

[0028] The salts of the invention also have utility as inhibitors of bacterial growth.

[0029] A particular object of this disclosure is to provide compounds and pharmaceutical compositions that have utility in the treatment of bacterial infections in a mammal. In one embodiment the invention provides a compound of formula (I): or a salt thereof, wherein:

[0030] W, X, Y, and Z are individually carbon, sulfur, oxygen, selenium, or nitrogen, wherein at least two of W, X, Y, and Z are carbon; one of R1and R2is C1-C10alkyl, C2-C10alkenyl, C1-C10alkoxy, aryloxy, heteroaryloxy, or NRaRb, wherein any C1-C10alkyl, C2-C10alkenyl, or C1-C10alkoxy, is optionally substituted by at least one of halogen, hydroxy, C1-C5alkoxy, tetrahydrofuranyl, or furanyl, and wherein any aryloxy or heteroaryloxy is optionally substituted by at least one of halogen, hydroxy, C1-C5alkyl, C3-C6cycloalkyl, C1-C5alkoxy, aryl, or heteroaryl, wherein any C1-C5alkyl, C3-C6cycloalkyl, C1-C5alkoxy, aryl, and heteroaryl is optionally substituted by at least one of halogen, hydroxy, C1-C5alkyl, C3-C6cycloalkyl, or C1-C5alkoxy; or one of R1and R2is a 5-6-membered saturated, partially unsaturated, or aromatic heterocycle that is optionally substituted by at least one of halogen, hydroxy, C1-C10alkyl, C2-C10alkenyl, or C1-C10alkoxy; and the other of R1and R2is absent or is one of H, halogen, C1-C10alkyl, C2- C10 alkenyl, or C1-C10alkoxy, wherein any C1-C10alkyl, C2-C10alkenyl, or C1-C10alkoxy is optionally substituted by at least one of halogen, hydroxy, or C1-C5alkoxy;

[0031] R3is absent or is one of H, C1-C2alkyl, or halogen- substituted C1-C2alkyl;

[0032] R4is halo;

[0033] R5is H or M+, where M+is a pharmaceutically acceptable cation;

[0034] R6is H, halogen, or methyl that is optionally substituted with halogen;

[0035] R9is C1-C10alkyl or C2-C10alkenyl, wherein any C1-C10alkyl or C2-C10alkenyl is optionally substituted by at least one of halogen, hydroxy, alkoxy, or NRaRb; and

[0036] R10is C1-C5alkyl that is optionally substituted with one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11) deuterium atoms.

[0037] The invention also provides a compound of formula (I) or a pharmaceutically acceptable salt thereof for use in medical treatment. The invention also provides a compound of formula (I) or a pharmaceutically acceptable salt thereof for use in the prophylaxis or treatment of a bacterial infection.

[0038] The invention also provides a composition comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0039] The invention also provides a composition comprising a salt of formula (I) and a pharmaceutically acceptable carrier. In one embodiment the composition is suitable for intravenous administration. In one embodiment the pharmaceutically acceptable carrier is water. In one embodiment the composition is substantially free of organic co-solvents. In one embodiment the composition is substantially free of surfactants.

[0040] The invention also provides the use of a compound as an inhibitor of a bacterial RNA polymerase.

[0041] The invention also provides the use of a compound as an antibacterial agent.

[0042] The invention also provides the use of a compound as a disinfectant, a sterilant, an antispoilant, an antiseptic, or an anti-infective.

[0043] The invention also provides the use of a compound of formula (I) or a pharmaceutically acceptable salt thereof for the preparation of a medicament for prophylaxis or treatment of a bacterial infection in a mammal.

[0044] The invention also provides a method of inhibiting a bacterial RNA polymerase, comprising contacting a bacterial RNA polymerase with a compound.

[0045] The invention also provides a method of treating a bacterial infection in a mammal, comprising administering to the mammal a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof.

[0046] DETAILED DESCRIPTION OF THE INVENTION Definitions

[0047] The following definitions are used, unless otherwise indicated.

[0048] The term “halo” means fluoro, chloro, bromo, or iodo.

[0049] The term "alkyl" used alone or as part of a larger moiety, includes both straight and branched chains. For example, C1-C10alkyl includes both straight and branched chained alkyl groups having from one to ten carbon atoms. The term alkyl also includes cycloalkyl groups (e.g. cyclopropyl, cyclobutyl, cyclopently, cyclohexyl, cycloheptyl, and cyclooctyl), as well as (cycloalkyl)alkyl groups (e.g. 3 -cyclohexylpropyl, cyclopentylmethyl, 2- cyclohexylethyl, and 2-cyclopropylethyl). The term "alkenyl" used alone or as part of a larger moiety, includes an alkyl that has one or more double bonds. For example, C2-C10alkenyl includes both straight and branched chained groups having from two to ten carbon atoms and one or more (e.g. 1, 2, or 3) double bonds, as well as (cycloalkyl)alkyl groups having one or more double bonds in the cycloalkyl portion or in the alkyl portion of the (cycloalkyl)alkyl.

[0050] The term “alkoxy” used alone or as part of a larger moiety is a group alkyl-O-, wherein alkyl has any of the values defined herein.

[0051] The term “aryl” denotes a phenyl radical or an ortho-fused bicyclic carbocyclic radical having about nine to ten ring atoms in which at least one ring is aromatic. For example, aryl can be phenyl, indenyl, or naphthyl.

[0052] The term “heteroaryl” encompasses a radical of a monocyclic aromatic ring containing five or six ring atoms consisting of carbon and one to four heteroatoms each selected from the group consisting of non-peroxide oxygen, sulfur, and N(X) wherein X is absent or is H, O, (Ci-C4)alkyl, phenyl or benzyl, as well as a radical of an ortho-fused bicyclic heterocycle of about eight to ten ring atoms comprising one to four heteroatoms each selected from the group consisting of non-peroxide oxygen, sulfur, and N(X). For example heteroaryl can be furyl, imidazolyl, triazolyl, triazinyl, oxazoyl, isoxazoyl, thiazolyl, isothiazoyl, pyrazolyl, pyrrolyl, pyrazinyl, tetrazolyl, pyridyl, (or its N-oxide), thienyl, pyrimidinyl (or its N-oxide), indolyl, isoquinolyl (or its N-oxide) or quinolyl (or its N- oxide).

[0053] The term “heterocycle” or “heterocyclyl” ring as used herein refers to a ring that has at least one atom other than carbon in the ring, wherein the atom is selected from the group consisting of oxygen, nitrogen and sulfur. The ring can be saturated, partially unsaturated, or aromatic. The term includes single (e.g., monocyclic) saturated, partially unsaturated, and aromatic rings (e.g., 3, 4, 5, 6 or 7-membered rings) from about 1 to 6 carbon atoms and from about 1 to 4 heteroatoms selected from the group consisting of oxygen, nitrogen and sulfur in the ring. In one embodiment the term includes 5-6 membered saturated, partially unsaturated, and aromatic heterocycles that include 1-5 carbon atoms and 1-4 heteroatoms.

[0054] A bond designated herein represents a double bond that can optionally be cis, trans, or a mixture thereof.

[0055] A combination of substituents or variables is permissible only if such a combination results in a stable or chemically feasible compound. The term “stable compounds,” as used herein, refers to compounds which possess stability sufficient to allow for their manufacture and which maintain the integrity of the compound for a sufficient period of time to be useful for the purposes detailed herein (e.g., formulation into therapeutic products, intermediates for use in production of therapeutic compounds, isolatable or storable intermediate compounds, treating a disease or condition responsive to therapeutic agents.

[0056] Unless otherwise stated, structures depicted herein are also meant to include all stereochemical forms of the structure (i.e., the R and S configurations for each asymmetric center). Therefore, single stereochemical isomers, as well as enantiomeric and diastereomeric mixtures, of the present compounds are within the scope of the invention. Similarly, E- and Z-isomers, or mixtures thereof, of olefins within the structures also are within the scope of the invention.

[0057] It is understood by one skilled in the art that this invention also includes any compound claimed that may be enriched at any or all atoms above naturally occurring isotopic ratios with one or more isotopes such as, but not limited to, deuterium (2H or D). As a non-limiting example, a -CH3 group may be substituted with -CD3. When a compound is shown or named as containing a specific isotope, it is understood that the compound is enriched in that isotope above the natural abundance of that isotope. In one embodiment the compound may be enriched by at least 2-times the natural abundance of that isotope. In one embodiment the compound may be enriched by at least 10-times the natural abundance of that isotope. In one embodiment the compound may be enriched by at least 100-times the natural abundance of that isotope. In one embodiment the compound may be enriched by at least 1000-times the natural abundance of that isotope.

[0058] The term “substituted with one or more deuterium atoms” means that one or more hydrogen atoms in a group (e.g., a C1-C5alkyl group) the has been replaced with a deuterium, such that the deuterium is present above the natural abundance of deuterium. In one embodiment the deuterium is enriched by at least 2-times its natural abundance. In one embodiment the deuterium is enriched by at least 10-times its natural abundance. In one embodiment the deuterium is enriched by at least 100-times its natural abundance. In one embodiment the deuterium is enriched by at least 1000-times its natural abundance. When R6is a C1-C5alkyl that is substituted with one or more deuterium atoms, it is understood that one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11) of the hydrogens on the C1-C5alkyl group has been replaced with a deuterium as described.

[0059] Compounds of this invention may exist in tautomeric forms, such as keto-enol tautomers. The depiction of a single tautomer is understood to represent the compound in all of its tautomeric forms. The term “pharmaceutically acceptable,” as used herein, refers to a component that is, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and other mammals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit / risk ratio. A “pharmaceutically acceptable salt” means any non-toxic salt that, upon administration to a recipient, is capable of providing, either directly or indirectly, a compound of this invention.

[0060] The term “pharmaceutically acceptable cation” includes sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum and the like. Particularly acceptable cations are the monovalent catins, including sodium, potassium, lithium, and ammonium, and the like. The term pharmaceutically acceptable cation” also includes cations formed by protonation or alkylation of a pharmaceutically acceptable organic nontoxic bases such as primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins. For example, the term “pharmaceutically acceptable cation” includes cations formed from unsubstituted or hydroxyl-substituted mono-, di-, or tri-alkylamines, dicyclohexylamine; tributyl amine; pyridine; N-methyl, N-ethylamine; diethylamine; triethylamine; mono-, bis-, or tris-(2-OH-(C1-C6)-alkylamine), such as N,N-dimethyl-N-(2- hydroxyethyl)amine or tri-(2-hydroxyethyl)amine; N-methyl-D-glucamine; morpholine; thiomorpholine; piperidine; pyrrolidine; and amino acids such as arginine, lysine, and the like.

[0061] Antibacterial Agents

[0062] The invention provides new compositions of matter that highly potently inhibit bacterial RNA polymerase and inhibit bacterial growth. Certain compounds of this invention exhibit potencies higher than the potencies of the natural products myxopyronin A and B and of other known analogs of myxopyronin A and B.

[0063] Certain embodiments of the invention also provide methods for preparation of a compound according to general structural formula (I).

[0064] Certain embodiments of the invention also provide an assay for inhibition of a RNA polymerase comprising contacting a bacterial RNA polymerase with a compound according to general structural formula (I).

[0065] Certain embodiments of the invention also provide an assay for antibacterial activity comprising contacting a bacterial RNA polymerase with a compound according to general structural formula (I). Certain embodiments of the invention also provide the use of a compound according to general structural formula (I), as an inhibitor of a bacterial RNA polymerase.

[0066] Certain embodiments of the invention also provide the use of a compound according to general structural formula (I) as an antibacterial agent.

[0067] Certain embodiments of the invention also provide the use of a compound according to general structural formula (I) as one of a disinfectant, a sterilant, an antispoilant, an antiseptic, or an anti-infective.

[0068] Embodiments

[0069] In one embodiment: W, X, Y, and Z are individually carbon, sulfur, oxygen, selenium, or nitrogen, wherein at least two of W, X, Y, and Z are carbon; one of R1and R2is C1-C10alkyl, C2-C10alkenyl, C1-C10alkoxy, aryloxy, heteroaryloxy, or NRaRb, wherein any C1-C10alkyl, C2-C10alkenyl, or C1-C10alkoxy, is optionally substituted by at least one of halogen, hydroxy, C1-C5alkoxy, tetrahydrofuranyl, or furanyl, and wherein any aryloxy or heteroaryloxy is optionally substituted by at least one of halogen, hydroxy, C1-C5alkyl, C1-C5alkoxy, aryl, or heteroaryl, wherein any C1-C5alkyl, C1-C5alkoxy, aryl, and heteroaryl is optionally substituted by at least one of halogen, hydroxy, C1-C5alkyl, or C1-C5alkoxy; or one of R1and R2is a 5-6-membered saturated, partially unsaturated, or aromatic heterocycle that is optionally substituted by at least one of halogen, hydroxy, C1-C10alkyl, C2-C10alkenyl, or C1-C10alkoxy; and the other of R1and R2is absent or is one of H, halogen, C1-C10alkyl, C2-C10alkenyl, or C1-C10alkoxy, wherein any C1-C10alkyl, C2-C10alkenyl, or C1-C10alkoxy is optionally substituted by at least one of halogen, hydroxy, or C1-C5alkoxy;

[0070] R3is absent or is one of H, C1-C2alkyl, or halogen- substituted C1-C2alkyl;

[0071] R4is halo;

[0072] R5is H or M+, where M+is a pharmaceutically acceptable cation;

[0073] R6is H, halogen, or methyl that is optionally substituted with halogen;

[0074] R9is C1-C10alkyl or C2-C10alkenyl, wherein any C1-C10alkyl or C2-C10alkenyl is optionally substituted by at least one of halogen, hydroxy, alkoxy, or NRaRb; and

[0075] R10is C1-C5alkyl that is optionally substituted with one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11) deuterium atoms.

[0076] In one embodiment: W is sulfur, oxygen, or nitrogen; and X, Y, and Z are individually carbon, sulfur, oxygen, or nitrogen, wherein at least two of X, Y, and Z are carbon. In one embodiment, the compound of formula (I) is a compound of formula (la): or a salt thereof, wherein:

[0077] Y is N or C-Ra;

[0078] R2is aryloxy that is optionally substituted with one or more groups independently selected from the group consisting of halogen, hydroxy, C1-C5alkyl, C3-C6cycloalkyl, C1-C5alkoxy, aryl, and heteroaryl, wherein any C1-C5alkyl, C3-C6cycloalkyl, C1-C5alkoxy, aryl, and heteroaryl is optionally substituted with one or more groups independently selected from the group consisting of halogen, hydroxy, C1-C5alkyl, C3-C6cycloalkyl, and C1-C5alkoxy;

[0079] R4is halo;

[0080] R5is H or M+, where M+is a pharmaceutically acceptable cation;

[0081] R6is H, halogen, or methyl that is optionally substituted with one or more halogen;

[0082] R9is C1-C5alkyl;

[0083] R10is C1-C5alkyl that is optionally substituted with one or more (e.g., 1, 2, 3, 4, 5, 6,

[0084] 7, 8, 9, 10, or 11) deuterium atoms; and

[0085] Rais H or C1-C5alkyl.

[0086] In one embodiment, the compound of formula (I) is a compound of formula (la): or a salt thereof, wherein:

[0087] Y is N or C-Ra;

[0088] R2is aryloxy that is optionally substituted with one or more groups independently selected from the group consisting of halogen, hydroxy, C1-C5alkyl, C1-C5alkoxy, aryl, and heteroaryl, wherein any C1-C5alkyl, C1-C5alkoxy, aryl, and heteroaryl is optionally substituted with one or more groups independently selected from the group consisting of halogen, hydroxy, C1-C5alkyl, and C1-C5alkoxy;

[0089] R4is halo;

[0090] R5is H or M+, where M+is a pharmaceutically acceptable cation;

[0091] R6is H, halogen, or methyl that is optionally substituted with one or more halogen;

[0092] R9is C1-C5alkyl;

[0093] R10is C1-C5alkyl that is optionally substituted with one or more (e.g., 1, 2, 3, 4, 5, 6,

[0094] 7, 8, 9, 10, or 11) deuterium atoms; and

[0095] Rais H or C1-C5alkyl.

[0096] In one embodiment, the compound of formula (I) is a compound of formula (lb):

[0097] (Ic).

[0098] In one embodiment, R2is phenyloxy that is optionally substituted with one or more groups independently selected from the group consisting of halogen, hydroxy, C1-C5alkyl, C3-C6cycloalkyl, C1-C5alkoxy, aryl, and heteroaryl, wherein any C1-C5alkyl, C3- C6cycloalkyl, C1-C5alkoxy, aryl, and heteroaryl is optionally substituted with one or more groups independently selected from the group consisting of halogen, hydroxy, C1-C5alkyl, C3-C6cycloalkyl, and C1-C5alkoxy.

[0099] In one embodiment, R2is phenyloxy that is optionally substituted with one or more groups independently selected from the group consisting of halogen, hydroxy, C1-C5alkyl, C1-C5alkoxy, aryl, and heteroaryl, wherein any C1-C5alkyl, C1-C5alkoxy, aryl, and heteroaryl is optionally substituted with one or more groups independently selected from the group consisting of halogen, hydroxy, C1-C5alkyl, and C1-C5alkoxy.

[0100] In one embodiment, R2is phenyloxy that is optionally substituted with one or more groups independently selected from the group consisting of halogen.

[0101] In one embodiment, R2is phenyloxy that is optionally substituted with one or more fluoro groups.

[0102] In one embodiment, R2is 4-fluorophenyloxy, 3,4-difluorophenyloxy, 3-chloro-4- fluorophenyloxy, or 3, 4, 5-trifluorophenyloxy.

[0103] In one embodiment, R4is fluoro.

[0104] In one embodiment, R4is chloro.

[0105] In one embodiment, R4is bromo.

[0106] In one embodiment, R4is iodo.

[0107] In one embodiment, R5is H.

[0108] In one embodiment, R5is M+and M+is a pharmaceutically acceptable cation.

[0109] In one embodiment, R6is H or methyl.

[0110] In one embodiment, R9is methyl.

[0111] In one embodiment, R10is C1-C5alkyl.

[0112] In one embodiment, R10is methyl.

[0113] In one embodiment, R10is C1-C5alkyl that is substituted with one or more deuterium atoms.

[0114] In one embodiment, R10is -CD3.

[0115] Compound Synthesis

[0116] Compounds of this invention can be synthesized according to the following general scheme:

[0117] Compounds of this invention also can be synthesized according to the following general scheme:

[0118] Certain compounds of this invention also can be synthesized according to the following general scheme:

[0119] Certain compounds of this invention also can be synthesized according to the following general scheme:

[0120]

[0121] Administration of Pharmaceutical Compositions

[0122] The compounds of Formul (I) and the pharmaceutically acceptable salts thereof may be formulated as pharmaceutical compositions and administered to a mammalian host, such as a human patient in a variety of forms adapted to the chosen route of administration (i.e., orally or parenterally, by intravenous, intramuscular, topical or subcutaneous routes).

[0123] Thus, the present compounds and salts may be systemically administered, e.g., orally, in combination with a pharmaceutically acceptable vehicle such as an inert diluent or an assimilable edible carrier. They may be enclosed in hard or soft shell gelatin capsules, may be compressed into tablets, or may be incorporated directly with the food of the patient's diet. For oral therapeutic administration, the active compound or salt may be combined with one or more excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations should contain at least 0.1% of active compound or salt. The percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 2 to about 60% of the weight of a given unit dosage form. The amount of active compound or salt in such therapeutically useful compositions is such that an effective dosage level will be obtained.

[0124] The tablets, troches, pills, capsules, and the like may also contain the following: binders such as gum tragacanth, acacia, com starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, fructose, lactose or aspartame or a flavoring agent such as peppermint, oil of wintergreen, or cherry flavoring may be added. When the unit dosage form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier, such as a vegetable oil or a polyethylene glycol. Various other materials may be present as coatings or to otherwise modify the physical form of the solid unit dosage form. For instance, tablets, pills, or capsules may be coated with gelatin, wax, shellac or sugar and the like. A syrup or elixir may contain the active compound or salt, sucrose or fructose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavoring such as cherry or orange flavor. Of course, any material used in preparing any unit dosage form should be pharmaceutically acceptable and substantially non-toxic in the amounts employed. In addition, the active compound or salt may be incorporated into sustained-release preparations and devices.

[0125] The active compound and salts may also be administered intravenously or intraperitoneally by infusion or injection. Solutions of the active compound or its salt can be prepared in water, optionally mixed with a nontoxic surfactant. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, triacetin, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

[0126] The pharmaceutical dosage forms suitable for injection or infusion can include sterile aqueous solutions or dispersions or sterile powders comprising the active ingredient which are adapted for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions, optionally encapsulated in liposomes. In all cases, the ultimate dosage form should be sterile, fluid and stable under the conditions of manufacture and storage. The liquid carrier or vehicle can be a solvent or liquid dispersion medium comprising, for example, water, ethanol, a polyol (for example, glycerol, propylene glycol, liquid polyethylene glycols, and the like), vegetable oils, nontoxic glyceryl esters, and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the formation of liposomes, by the maintenance of the required particle size in the case of dispersions or by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, buffers or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0127] Sterile injectable solutions are prepared by incorporating the active compound or salt in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filter sterilization. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze-drying techniques, which yield a powder of the active ingredient plus any additional desired ingredient present in the previously sterile-filtered solutions.

[0128] For topical administration, the present compounds or salts may be applied in pure form, i.e., when they are liquids. However, it will generally be desirable to administer them to the skin as compositions or formulations, in combination with a dermatologically acceptable carrier, which may be a solid or a liquid.

[0129] Useful solid carriers include finely divided solids such as talc, clay, microcrystalline cellulose, silica, alumina and the like. Useful liquid carriers include water, alcohols or glycols or water-alcohol / glycol blends, in which the present compounds or salts can be dissolved or dispersed at effective levels, optionally with the aid of non-toxic surfactants. Adjuvants such as fragrances and additional antimicrobial agents can be added to optimize the properties for a given use. The resultant liquid compositions can be applied from absorbent pads, used to impregnate bandages and other dressings, or sprayed onto the affected area using pump-type or aerosol sprayers.

[0130] Thickeners such as synthetic polymers, fatty acids, fatty acid salts and esters, fatty alcohols, modified celluloses or modified mineral materials can also be employed with liquid carriers to form spreadable pastes, gels, ointments, soaps, and the like, for application directly to the skin of the user.

[0131] Examples of useful dermatological compositions which can be used to deliver the compounds or salts to the skin are known to the art; for example, see Jacquet et al. (U.S. Pat. No. 4,608,392), Geria (U.S. Pat. No. 4,992,478), Smith et al. (U.S. Pat. No. 4,559,157) and Wortzman (U.S. Pat. No. 4,820,508).

[0132] Useful dosages of the compounds or salts can be determined by comparing their in vitro activity, and in vivo activity in animal models. Methods for the extrapolation of effective dosages in mice, and other animals, to humans are known to the art; for example, see U.S. Pat. No. 4,938,949. The amount of the compound or salt, or an active salt or derivative thereof, required for use in treatment will vary not only with the particular salt selected but also with the route of administration, the nature of the condition being treated and the age and condition of the patient and will be ultimately at the discretion of the attendant physician or clinician.

[0133] The compound or salt is conveniently formulated in unit dosage form; for example, containing 5 to 1000 mg, conveniently 10 to 750 mg, most conveniently, 50 to 500 mg of active ingredient per unit dosage form. In one embodiment, the invention provides a composition comprising a compound or salt of the invention formulated in such a unit dosage form.

[0134] The desired dose may conveniently be presented in a single dose or as divided doses administered at appropriate intervals, for example, as two, three, four or more sub-doses per day. The sub-dose itself may be further divided, e.g., into a number of discrete loosely spaced administrations; such as multiple inhalations from an insufflator or by application of a plurality of drops into the eye.

[0135] The following illustrate representative preferred pharmaceutical dosage forms, containing a compound of formula (I) or a pharmaceutically acceptable salt thereof, for therapeutic or prophylactic use in humans: a) A formulation comprising from about 0.25 mg / ml to about 10 mg / ml of said compound or salt, about 0% to about 10% dimethylacetamide, and about 0% to about 10% Cremophor EL; b) A formulation comprising from about 0.25 mg / ml to about 10 mg / ml of said compound or salt, about 2% to about 5% dimethylacetamide, and about 0% to about 5% Cremophor EL; c) A formulation comprising from about 0.25 mg / ml to about 10 mg / ml of a pharmaceutically acceptable salt of said compound or salt and about 5% dextrose in about 10 mM sodium phosphate at about pH 7.4; and d) A formulation comprising from about 0.25 mg / ml to about 10 mg / ml of a pharmaceutically acceptable salt of said compound or salt in phosphate-buffered saline at about pH 7.4; and e) A formulation comprising from about 0.25 mg / ml to about 10 mg / ml of a pharmaceutically acceptable salt of said compound or salt in about 0 to about 1% carboxymethylcellulose and about 0 to about 1% Tween 80.

[0136] The invention will now be illustrated by the following non-limiting Examples. EXAMPLES

[0137] Example 1. Preparation of Comparison Compound

[0138] The compound of Example 1 was prepared as described [Ebright R, Ebright Y (2023) Antibacterial agents: O-alkyl-deuterated pyronins US11685723],

[0139] Example 2. Preparation of:

[0140] The compound of Example 2 was prepared as described in a-e below. a.

[0141] To a solution of 2,4-dichlorothiazole-5-carbaldehyde (2.00 g, 11.0 mmol, 1.00 eq) in dimethylformamide (20.0 mL), was added potassium carbonate (3.04 g, 22.0 mmol, 2.00 eq) and 3,4-difluorophenol (1.72 g, 13.2 mmol, 1.20 eq). The mixture was stirred at 20°C for 1 h. The mixture was poured into water (300 mL) and extracted with ethyl acetate (3 x200 mL). The combined organic layer was dried over anhydrous sodium sulfate, filtered and concentrated under vacuum to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 20 / 1) to give 4-chloro-2-(3,4- difluorophenoxy)thiazole-5-carbaldehyde (2.30 g, 8.34 mmol, 75.9% yield) as a white solid.1H NMR (400 MHz, chloroform -d) δ= 9.96 (s, 1H), 7.27 (s, 2H), 7.13 - 7.07 (m, 1H). b.

[0142] To a solution of 4-chloro-2-(3,4-difluorophenoxy)thiazole-5-carbaldehyde (2.20 g, 7.98 mmol, 1.00 eq., Example 2a) in isopropanol (44.0 mL), was added piperidine (680 mg, 7.98 mmol, 788 pL, 1.00 eq) and methyl-(E)-6-(4-hydroxy-2-oxo-3-propionyl-2H-pyran-6- yl)hex-2-enoate (2.35 g, 7.98 mmol, 1.00 eq [Ebright R, Ebright Y (2023) Antibacterial agents: O-alkyl-deuterated pyronins US11685723]). The mixture was stirred at 70°C for 6 h. The mixture was concentrated under vacuum to give a yellow oil. The yellow oil was purified by reversed-phase HPLC (0.1% FA condition) and extracted with ethyl acetate (3 x 200 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum to give methyl-(E)-6-(3-((E)-3-(4-chloro-2-(3,4- difluorophenoxy)thiazol-5-yl)-2-methylacryloyl)-4-hydroxy-2-oxo-2H-pyran-6-yl)hex-2- enoate (96.0 mg, 164 μmol, 2.05% yield, 94.0% purity) as a yellow solid.1H NMR (400 MHz, chloroform -d) δ= 7.26 - 7.17 (m, 2H), 7.13 - 7.03 (m, 2H), 6.98 - 6.90 (m, 1H), 6.01 (s, 1H), 5.88 (d, J= 16.0 Hz, 1H), 3.75 (s, 3H), 2.56 (t, J= 7.6 Hz, 2H), 2.32 (q, J= 6.8 Hz, 2H), 2.20 - 2.10 (m, 3H), 1.90 (q, J= 7.6 Hz, 2H). To a solution of methyl-(E)-6-(3-((E)-3-(4-chloro-2-(3,4-difluorophenoxy)thiazol-5- yl)-2-methylacryloyl)-4-hydroxy-2-oxo-2H-pyran-6-yl)hex-2-enoate (100 mg, 181 μmol, 1.00 eq Example 2b) in tetrahydrofuran (1.00 mL), was added lithium hydroxide monohydrate (15.2 mg, 362 μmol, 2.00 eq and water (1.00 mL). The mixture was stirred at 20°C for 12 h. The pH of mixture was adjusted to around 3 by adding hydrochloric acid (1 M) and was extracted with ethyl acetate (3 / 30 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered and concentrated under vacuum to give (E)-6- (3-((E)-3-(4-chloro-2-(3,4-difluorophenoxy)thiazol-5-yl)-2-methylacryloyl)-4-hydroxy-2- oxo-2H-pyran-6-yl)hex-2-enoic acid (100 mg, crude) as a yellow solid. 1H NMR (400 MHz chloroform -d) δ= 7.26 - 7.17 (m, 2H), 7.14 - 6.94 (m, 3H), 6.08 - 5.97 (m, 1H), 5.89 (d, J= 16.0 Hz, 1H), 2.57 (t, J= 7.6 Hz, 2H), 2.35 (q, J= 7.2 Hz, 2H), 2.20 - 2.08 (m, 3H), 1.92 (td, J= 7.6, 15.2 Hz, 2H).

[0143] To (E)-6-(3-((E)-3-(4-chloro-2-(3,4-difluorophenoxy)thiazol-5-yl)-2-methylacryloyl)- 4-hydroxy-2-oxo-2H-pyran-6-yl)hex-2-enoic acid (100 mg, 186 μmol, 1.00 eq, Example 2c) in acetone (1.00 mL), was added diisopropylethylamine (120 mg, 930 μmol, 162 pL, 5.00 eq) and isobutyl carb onochlori date (50.8 mg, 372 μmol, 48.6 pL, 2.00 eq) at 0°C under a nitrogen atmosphere, and the mixture was stirred at 0°C for 1.5 h. Sodium azide (96.7 mg, 1.49 mmol, 8.00 eq) in water (1.00 mL) was added, and the mixture was stirred at 20°C for 40 min under nitrogen atmosphere. The reaction was queched by adding ice-water (1.00 mL). The pH of the mixture was adjusted to around 4 by adding hydrochloric acid (1 M). Organics were extracted with ethyl acetate (2 x 20.0 mL), and the combined extracts were washed with brine (20.0 mL), dried over anhydrous sodium sulfate, evaporated to an oil, and trace water was removed by azeotropic evaporation of added anhydrous stoluene azeotrope (3 x 20.0 mL) to give (E)-6-(3-((E)-3-(4-chloro-2-(3,4-difluorophenoxy)thiazol-5-yl)-2-methylacryloyl)-4- hydroxy-2-oxo-2H-pyran-6-yl)hex-2-enoyl azide (100 mg, crude) as a yellow solid. e.

[0144] A solution of (E)-6-(3-((E)-3-(4-chloro-2-(3,4-difluorophenoxy)thiazol-5-yl)-2- methylacryloyl)-4-hydroxy-2-oxo-2H-pyran-6-yl)hex-2-enoyl azide (100 mg, 178 μmol, 1.00 eq, Example 2d) in toluene (1.50 mL) was stirred at 110°C for 2 h, the mixture was cooled to room temperature, and methanol -d4 (1.50 mL) was added. The resulting mixture was stirred at 70°C for 12 h. The mixture was concentrated under vacuum to give a residue. The residue was purified by prep-HPLC (column: Phenomenex Luna C18 150 mm x 25 mm x 10 μm; mobile phase: [water (FA)-ACN]; gradient: 63%-93% B over 10 min) and was lyophilized to give m ethyl -d3 ((E)-5-(3-((E)-3-(4-chloro-2-(3,4-difluorophenoxy)thiazol-5-yl)-2- methylacryloyl)-4-hydroxy-2-oxo-2H-pyran-6-yl)pent-l-en-l-yl)carbamate (20.28 mg, 35.22 μmol, 19.8% yield, 98.9% purity) as a yellow solid.1H NMR (400 MHz, chloroform-d) δ = 7.27 - 7.18 (m, 2H), 7.14 - 7.02 (m, 2H), 6.58 - 6.41 (m, 1H), 6.25 (d, J= 8.8 Hz, 1H), 6.05 - 5.96 (m, 1H), 5.02 - 4.84 (m, 1H), 2.52 (t, J= 7.6 Hz, 2H), 2.28 - 1.98 (m, 5H), 1.77 (q, J= 7.6 Hz, 2H).

[0145] Alternatively, the compound of Example 2 was prepared as described in f-o below. f. To a solution of 2,4-dichlorothiazole-5-carbaldehyde (15.0 g, 82.4 mmol, 1.00 eq) in dimethylformamide (150 mL), was added potassium carbonate (22.8 g, 165 mmol, 2.00 eq) and 3,4-difluorophenol (10.7 g, 82.4 mmol, 1.00 eq). The mixture was stirred at 20°C for 12 h. The mixture was poured into water (500 mL) and extracted with ethyl acetate (3 x 300 mL). The combined organic layer was dried over anhydrous sodium sulfate, filtered and concentrated under vacuum to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 20 / 1) to give 4-chloro-2-(3,4- difluorophenoxy)thiazole-5-carbaldehyde (20.5 g, 74.1 mmol, 89.9% yield, 99.6% purity) as a white solid.1H NMR (400 MHz, chloroform -d) δ= 9.95 (s, 1H), 7.32 - 7.27 (m, 1H), 7.25 - 7.18 (m, 1H), 7.13 - 7.05 (m, 1H).

[0146] A solution of 4-hydroxy-6-methyl-2Z7-pyran-2-one (50.0 g, 396 mmol, 1.00 eq) and A-[bis(dimethylamino)phosphoryl]-N-methylmethanamine (204 g, 1.14 mol, 199 mL, 2.87 eq) in 2-methyloxolane (1000 mL) was degassed and purged 3x with nitrogen, butyllithium (2.50 M, 357 mL, 2.25 eq) was added dropwise at -78°C over 1.5 h, and the mixture was stirred at -78°C for 1 h under a nitrogen atmosphere. To this mixture, 4-bromobut-l-ene (107 g, 793 mmol, 80.5 mL, 2.00 eq) then was added dropwise at -78 °C over 30 min, and the mixture was stirred at 25°C for 12 h in a 3000 mL three-necked bottle under a nitrogen atmosphere. The reaction mixture was cooled to 0 °C by ice-water, the pH was adjusted to 1~2 with hydrochloric acid (6 M) at 0°C, and the mixture was stirred for 30 min at 0°C. The mixture was extracted with ethyl acetate (3 x 3000 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 1 / 1) to give 4-hydroxy-6-(pent-4-en- I -y 1 )-2H-py ran -2-one (85.0 g, 472 mmol, 59.5% yield) as a yellow oil.1H NMR (400 MHz, chloroform-d) δ= 11.53 - 10.38 (m, 1H), 6.00 (s, 1H), 5.77 (tdd, J= 6.8, 10.4, 17.2 Hz, 1H), 5.60 (d, J= 1.6 Hz, 1H), 5.09 - 4.94 (m, 2H), 2.51 (t, J= 7.6 Hz, 2H), 2.12 (q, J= 7.2 Hz, 2H), 1.82 - 1.70 (m, 2H). h.

[0147] To a solution of 4-hydroxy-6-(pent-4-en-l-yl)-2H-pyran-2-one (75.0 g, 416 mmol, 1.00 eq,- Example 2g) in toluene (1500 mL), was added propionic acid (30.8 g, 416 mmol, 31.1 mL, 1.00 eq), N,N-dimethylpyridin-4-amine (10.2 g, 83.2 mmol, 0.20 eq , and / ' / , / ' / '- di(propan-2-yl)methanediimine (52.5 g, 416 mmol, 64.5 mL, 1.00 eq at 0°C, and the mixture was stirred at 0°C for 3 h, and then heated at 100°C for 8 h in a 3000 mL three-necked bottle. The mixture was filtered, the filtrate was concentrated to dry to give a crude residue. The crude residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 20 / 1) to give 4-hydroxy-6-(pent-4-en-l-yl)-3-propionyl-2H-pyran-2-one (79.1 g, 335 mmol, 80.4% yield) as a yellow solid.1H NMR (400 MHz, chloroform-d) δ= 5.93 (s, 1H), 5.78 (tdd, J= 6.8, 10.2, 17.2 Hz, 1H), 5.13 - 4.95 (m, 2H), 3.12 (q, J= 7.2 Hz, 2H), 2.50 (t, J= 7.6 Hz, 2H), 2.13 (q, J= 7.2 Hz, 2H), 1.79 (q, J= 7.6 Hz, 2H), 1.17 (t, J= 7.2 Hz, 3H). i.

[0148] To a solution of 4-hydroxy-6-(pent-4-en-l-yl)-3-propionyl-2H-pyran-2-one (86.6 g, 367 mmol, 1.00 eq,- Example 2h) in 2-methoxy-2-methylpropane (430 mL), was added methyl-(E)-but-2-enoate (240 g, 2.40 mol, 255 mL, 6.55 eq and second-generation Hoveyda- Grubbs catalyst (7.35 g, 11.7 mmol, 0.032 eq), and the mixture was stirred at 50°C for 12 h. The mixture was concentrated under vacuum. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 5 / 1) to give methyl-(E)-6-(4-hydroxy- 2-oxo-3-propionyl-2H-pyran-6-yl)hex-2-enoate (75.0 g, 255 mmol, 69.5% yield) as a yellow solid.1H NMR (400 MHz, chloroform -d) δ= 6.93 (td, J= 6.8, 15.6 Hz, 1H), 5.94 (s, 1H), 5.87 (d, J= 15.6 Hz, 1H), 3.74 (s, 3H), 3.12 (q, J= 7.2 Hz, 2H), 2.52 (t, J= 7.6 Hz, 2H), 2.36 - 2.24 (m, 2H), 1.88 (q, J = 7.6 Hz, 2H), 1.17 (t, J = 7.2 Hz, 3H). j-

[0149] To a solution of 4-chloro-2-(3,4-difluorophenoxy)thiazole-5-carbaldehyde (12.0 g, 43.5 mmol, 1.00 eq Example 2i) in toluene (120 mL), was added 2, 2,6,6- tetramethylpiperidine (24.6 g, 174 mmol, 29.6 mL, 4.00 eq) and methyl-(E)-6-(4-hydroxy-2- oxo-3-propionyl-2H-pyran-6-yl)hex-2-enoate (12.8 g, 43.5 mmol, 1.00 eq: Example 2.9). The mixture was stirred at 40°C for 16 h. The mixture was concentrated under vacuum to give a residue. The residue was purified by reversed-phase HPLC (0.1% FA condition) and extracted with ethyl acetate (3 x 1200 mL). The combined organic layer was dried over anhydrous sodium sulfate, filtered and concentrated under vacuum to give methyl-(E)-6-(3- (3-(4-chloro-2-(3,4-difluorophenoxy)thiazol-5-yl)-3-hydroxy-2-methylpropanoyl)-4- hydroxy-2-oxo-2H-pyran-6-yl)hex-2-enoate (13.6 g, 23.9 mmol, 54.8% yield) as a yellow solid.1H NMR (400 MHz, chloroform-d) δ= 7.25 - 7.15 (m, 2H), 7.10 - 7.03 (m, 1H), 6.99 - 6.85 (m, 1H), 6.00 (s, 1H), 5.88 (d, J= 15.6 Hz, 1H), 5.29 (d, J= 92 Hz, 1H), 4.35 - 4.22 (m, 1H), 3.74 (s, 3H), 3.36 - 3.06 (m, 1H), 2.55 (t, J= 7.6 Hz, 2H), 2.31 (q, J= 7.2 Hz, 2H), 1.89

[0150] (q, J= 7.6 Hz, 2H), 1.17 (d, J = 6.8 Hz, 3H). To a solution of methyl-(E)-6-(3-(3-(4-chloro-2-(3,4-difluorophenoxy)thiazol-5-yl)-3- hydroxy-2-methylpropanoyl)-4-hydroxy-2-oxo-2H-pyran-6-yl)hex-2-enoate (13.6 g, 23.9 mmol, 1.00 eq Example 2j) in dichloromethane (270 mL), was added triethylamine (7.24 g, 71.6 mmol, 9.96 mL, 3.00 eq and methanesulfonyl chloride (5.47 g, 47.7 mmol, 3.69 mL, 2.00 eq) at 0°C. The resulting mixture was stirred at 25 °C for 0.5 h under nitrogen atmosphere. The reaction was quenched with ice water (100 mL). The pH adjusted to 4-5 with hydrochloric acid (1 M). The mixture was poured into water (1000 mL), extracted with di chloromethane (3 x 1000 mL), dried with anhydrous sodium sulfate, filtered and concentrated to give methyl-(E)-6-(3-(3-(4-chloro-2-(3,4-difluorophenoxy)thiazol-5-yl)-2- methyl-3-((methylsulfonyl)oxy)propanoyl)-4-hydroxy-2-oxo-2H-pyran-6-yl)hex-2-enoate (15.5 g, crude) as a yellow oil.

[0151] 1.

[0152] To a solution of methyl-(E)-6-(3-(3-(4-chloro-2-(3,4-difluorophenoxy)thiazol-5-yl)-2- methyl-3-((methylsulfonyl)oxy)propanoyl)-4-hydroxy-2-oxo-2H-pyran-6-yl)hex-2-enoate (15.5 g, 23.9 mmol, 1.00 eq Example 2.k) in tetrahydrofuran (270 mL), was added 2,3,4,6,7,8,9,10-octahydropyrimido[l,2-a]azepine (10.9 g, 71.6 mmol, 10.8 mL, 3.00 eq at 0 °C. The mixture was stirred at 25 °C for 1 h. The pH of mixture was adjusted to ~3 by adding hydrochloric acid (1 M). The mixture was poured into water (1000 mL) and extracted with ethyl acetate (3 x 1000 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum to give a yellow oil. The yellow oil was purified by reversed-phase HPLC (0.1% FA condition) and extracted with ethyl acetate (3 x 1000 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered and concentrated under vacuum to give methyl-(E)-6-(3-((E)-3-(4-chloro-2-(3,4- difluorophenoxy)thiazol-5-yl)-2-methylacryloyl)-4-hydroxy-2-oxo-2H-pyran-6-yl)hex-2- enoate (7.00 g, 12.7 mmol, 53.2% yield) as a yellow solid.1H NMR (400 MHz, chloroform- d) δ= 7.26 - 7.19 (m, 2H), 7.12 - 7.06 (m, 2H), 6.94 (td, J= 7.2, 15.6 Hz, 1H), 6.01 (s, 1H), 5.92 - 5.82 (m, 1H), 3.75 (s, 3H), 2.55 (t, J= 7.6 Hz, 2H), 2.38 - 2.28 (m, 2H), 2.14 (d, J= 1.2 Hz, 3H), 1.96 - 1.85 (m, 2H).

[0153] To a solution of methyl-(E)-6-(3-((E)-3-(4-chloro-2-(3,4-difluorophenoxy)thiazol-5- yl)-2-methylacryloyl)-4-hydroxy-2-oxo-2H-pyran-6-yl)hex-2-enoate (7.00 g, 12.7 mmol, 1.00 eq., Example 2.1) in tetrahydrofuran (70.0 mL), was added lithium hydroxide monohydrate (1.06 g, 25.4 mmol, 2.00 eq) and water (70.0 mL). The mixture was stirred at 20°C for 12 h. The pH of mixture was adjusted to ~3 by adding hydrochloric acid (I M) and extracted with ethyl acetate (3 x 700 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum to give (E)-6-(3-((E)-3- (4-chloro-2-(3,4-difluorophenoxy)thiazol-5-yl)-2-methylacryloyl)-4-hydroxy-2-oxo-2H- pyran-6-yl)hex-2-enoic acid (6.90 g, crude) as a yellow solid.1H NMR (400 MHz, chloroform -d) δ= 7.26 - 7.25 (m, 1H), 7.27 - 7.19 (m, 1H), 7.12 - 6.97 (m, 3H), 6.02 (s, 1H), 5.89 (d, J= 16.0 Hz, 1H), 2.57 (t, J= 7.6 Hz, 2H), 2.40 - 2.30 (m, 2H), 2.16 - 2.12 (m, 3H), 1.96 - 1.90 (m, 2H).

[0154] To a solution of (E)-6-(3-((E)-3-(4-chloro-2-(3,4-difluorophenoxy)thiazol-5-yl)-2- methylacryloyl)-4-hydroxy-2-oxo-2H-pyran-6-yl)hex-2-enoic acid (6.90 g, 12.8 mmol, 1.00 eq., Example 2m) in acetone (70.0 mL), was added diisopropylethylamine (8.29 g, 64.1 mmol, 11.2 mL, 5.00 eq) and isobutyl carb onochlori date (3.50 g, 25.7 mmol, 3.36 mL, 2.00 eq) at 0°C under a nitrogen atmosphere, and the mixture was stirred at 0°C for 1.5 h. Sodium azide (6.67 g, 103 mmol, 8.00 eq) in water (70.0 mL) was added, and the mixture was stirred at 25 °C for 40 min under a nitrogen atmosphere. The reaction was quenched by adding icewater (100.0 mL). The pH of the mixture was adjusted to ~5 by adding hydrochloric acid (1 M). Organics were extracted with ethyl acetate (2 x 700 mL), and the combined extracts were washed with brine (700 mL), dried over anhydrous sodium sulfate, and evaporated to an oil. Trace water was removed by azeotropic evaporation of added anhydrous toluene azeotrope (3 x 50.0 mL) to give (E)-6-(3-((E)-3-(4-chloro-2-(3,4-difluorophenoxy)thiazol-5-yl)-2- methylacryloyl)-4-hydroxy-2-oxo-2H-pyran-6-yl)hex-2-enoyl azide (7.22 g, crude) as a yellow oil.

[0155] 0.

[0156] A solution of (E)-6-(3-((E)-3-(4-chloro-2-(3,4-difluorophenoxy)thiazol-5-yl)-2- methylacryloyl)-4-hydroxy-2-oxo-2H-pyran-6-yl)hex-2-enoyl azide (7.22 g, 12.8 mmol, 1.00 eq., Example 2n) in toluene (35.0 mL) was stirred at 110°C for 2 h, the mixture was cooled to room temperature, and methanol -d4 (35.0 mL) was added. The resulting mixture was stirred at 70°C for 3 h. The mixture was concentrated under vacuum to give a residue. The residue was purified by prep-HPLC (column: Phenomenex Luna C18 (250 mm x 70 mm x 10 μm); mobile phase: [water(FA)-ACN]; B%:35%, isocratic elution mode) and was lyophilized to give m ethyl -d ((E)-5-(3-((E)-3-(4-chloro-2-(3,4-difluorophenoxy)thiazol-5-yl)-2- methylacryloyl)-4-hydroxy-2-oxo-2H-pyran-6-yl)pent-l-en-l-yl)carbamate (3.60 g, 6.32 mmol, 49.3% yield) as a yellow solid.1H NMR (400 MHz, chi oroform-d) d = 7.26 - 7.18 (m, 2H), 7.14 - 7.01 (m, 2H), 6.60 - 6.40 (m, 1H), 6.34 - 6.15 (m, 1H), 6.00 (s, 1H), 5.09 - 4.83 (m, 1H), 2.61 - 2.46 (m, 2H), 2.26 - 2.04 (m, 5H), 1.77 (td, J= 7.6, 14.8 Hz, 2H).

[0157] Example 3. Preparation of:

[0158] The compound of Example 3 was prepared as follows:

[0159] A mixture of methyl -d3 ((E)-5-(3-((E)-3-(4-chloro-2-(3,4-difluorophenoxy)thiazol-5- yl)-2-methylacryloyl)-4-hydroxy-2-oxo-2H-pyran-6-yl)pent-l-en-l-yl)carbamate (3.50 g, 6.14 mmol, 1.00 eq, Example 2) in sodium carbonate (0.01 M, 1.84 L, 3.00 eq) was stirred at 37°C for 2 h. The mixture was poured into water (300 mL) and extracted with ethyl acetate (3 x500 mL). The combined organic layers were dried over anhydrous sodium sulfate, were evaporated to an oil, and trace water was removed by azeotropic evaporation of added ethanol azeotrope (3 x 30.0 mL) to give a yellow solid with residual ethanol. Then ethanol was removed by evaporation of added anhydrous acetonitrile (3 x 30.0 mL) to give sodium- 3-((E)-3-(4-chloro-2-(3,4-difluorophenoxy)thiazol-5-yl)-2-methylacryloyl)-6-((E)-5- (((methoxy-d3)carbonyl)amino)pent-4-en-l-yl)-2-oxo-2H-pyran-4-olate (3.44 g, 5.74 mmol, 93.5% yield, 98.9% purity) as a yellow solid.1H NMR (400 MHz, methanol-d4) δ= 7.54 - 7.36 (m, 3H), 7.28 - 7.18 (m, 1H), 6.44 (d, J= 14.0 Hz, 1H), 5.77 (s, 1H), 5.20 - 5.01 (m, 1H), 2.44 (t, J= 7.6 Hz, 2H), 2.17 - 2.02 (m, 5H), 1.73 (q, J= 7.2 Hz, 2H).

[0160] Example 4. Preparation of:

[0161] The compound of Example 4 was prepared as described in a-f below. a.

[0162] To a solution of 3-chlorothiophene-2-carbaldehyde (10.0 g, 68.2 mmol, 1.00 eq) in dichloromethane (100 mL), was added bromine (27.3 g, 171 mmol, 8.79 mL, 2.50 eq . The mixture was stirred at 20 °C for 12 h. The mixture was poured into water (1000 mL) and extracted with di chloromethane (3 x 1000 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 1 / 0) to give 5-bromo-3-chlorothiophene-2-carbaldehyde (12.4 g, 55.0 mmol, 80.6% yield) as a yellow solid.1H NMR (400 MHz, DMSO-d6) δ= 9.85 (s, 1H), 7.66 (s, 1H). b.

[0163] To a solution of 5-bromo-3-chlorothiophene-2-carbaldehyde (12.4 g, 55.0 mmol, 1.00 eq., Example 4a) in dimethylformamide (120 mL), was added potassium carbonate (15.2 g, 110 mmol, 2.00 eq and 3,4-difluorophenol (7.15 g, 55.0 mmol, 1.00 eq). The reaction mixture was stirred at 20°C for 12 h, poured into water (2000 mL), and extracted with ethyl acetate (3 x 1000 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 20 / 1) to give a yellow oil. The yellow oil was purified by prep-HPLC (column: Welch Ultimate XB-CN 250 μm x 70 μm x 10 μm; mobile phase: [Hexane-EtOH]; B%: 3%, isocratic elution mode) and was concentrated under vacuum to give 3-chloro-5-(3,4-difluorophenoxy)thiophene-2- carbaldehyde (5.80 g, 21.1 mmol, 38.4% yield) as a yellow solid.1H NMR (400 MHz, chloroform-d) δ= 9.94 (s, 1H), 7.26 - 7.19 (m, 1H), 7.07 (ddd, J= 3.2, 6.8, 10.0 Hz, 1H), 6.99 - 6.94 (m, 1H), 6.40 (s, 1H). c.

[0164] To a solution of 3-chloro-5-(3,4-difluorophenoxy)thiophene-2-carbaldehyde (1.00 g, 3.64 mmol, 1.00 eq,- Example 4b) in isopropanol (20.0 mL), was added piperidine (310 mg, 3.64 mmol, 360 pL, 1.00 eq) and methyl-(E)-6-(4-hydroxy-2-oxo-3-propionyl-2H-pyran-6- yl)hex-2-enoate (1.07 g, 3.64 mmol, 1.00 eq,- Example 2i). The reaction mixture was stirred at 70°C for 8 h and then concentrated under vacuum to give a residue. The residue was purified by prep-HPLC (column: Phenomenex Luna C18 150 mm x 25 mm x 10 μm; mobile phase: [water (FA)-ACN]; gradient: 69%-99% B over 10 min) and extracted with ethyl acetate (3 x 100 mL). The combined organic layer was dried over anhydrous sodium sulfate, filtered and was concentrated under vacuum to give methyl-(E)-6-(3-((E)-3-(3-chloro-5-(3,4- difluorophenoxy)thiophen-2-yl)-2-methylacryloyl)-4-hydroxy-2-oxo-2H-pyran-6-yl)hex-2- enoate (740 mg, 1.34 mmol, 36.9% yield) as a yellow oil.1H NMR (400 MHz, chloroform- d) δ= 7.28 (s, 1H), 7.19 (q, J= 9.2 Hz, 1H), 7.06 - 6.99 (m, 1H), 6.98 - 6.86 (m, 2H), 6.49 (s, 1H), 6.00 (s, 1H), 5.88 (d, J= 15.6 Hz, 1H), 3.75 (s, 3H), 2.60 - 2.49 (m, 2H), 2.31 (q, J = 6.8 Hz, 2H), 2.15 (s, 3H), 1.90 (q, J= 7.6 Hz, 2H). d.

[0165] To a solution of methyl-(E)-6-(3-((E)-3-(3-chloro-5-(3,4-difluorophenoxy)thiophen-2- yl)-2-methylacryloyl)-4-hydroxy-2-oxo-2H-pyran-6-yl)hex-2-enoate (740 mg, 1.34 mmol, 1.00 eq., Example 4c) in tetrahydrofuran (7.50 mL), was added lithium hydroxide monohydrate (113 mg, 2.69 mmol, 2.00 eq) and water (7.50 mL). The reaction mixture was stirred at 20°C for 12 h. The pH was adjusted to ~3 by adding hydrochloric acid (1 M), and the reaction mixture extracted with ethyl acetate (3 x 70.0 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered and concentrated under vacuum to give (E)-6-(3-((E)-3-(3-chloro-5-(3,4-difluorophenoxy)thiophen-2-yl)-2-methylacryloyl)-4- hydroxy-2-oxo-2H-pyran-6-yl)hex-2-enoic acid (720 mg, crude) as a yellow solid.1H NMR (400 MHz, chloroform-d) δ= 7.28 (d, J= 1.2 Hz, 1H), 7.19 (q, J = 9.2 Hz, 1H), 7.09 - 6.97 (m, 2H), 6.96 - 6.86 (m, 1H), 6.49 (s, 1H), 6.01 (s, 1H), 5.89 (d, J= 15.6 Hz, 1H), 2.56 (t, J= 7.6 Hz, 2H), 2.35 (q, J= 6.8 Hz, 2H), 2.15 (d, J= 1.2 Hz, 3H), 1.92 (q, J= 7.6 Hz, 2H). e.

[0166] To a solution of (E)-6-(3-((E)-3-(3-chloro-5-(3,4-difluorophenoxy)thiophen-2-yl)-2- methylacryloyl)-4-hydroxy-2-oxo-2H-pyran-6-yl)hex-2-enoic acid (720 mg, 1.34 mmol, 1.00 eq., Example 4d) in acetone (7.00 mL), was added diisopropylethylamine (867 mg, 6.70 mmol, 1.17 mL, 5.00 eq and isobutyl carbonochloridate (366 mg, 2.68 mmol, 351 pL, 2.00 eq) atO °C under a nitrogen atmosphere, and the reaction mixture was stirred at 0°C for 1.5 h Sodium azide (697 mg, 10.7 mmol, 8.00 eq in water (7.00 mL) was added, and the mixture was stirred at 25 °C for 40 min under a nitrogen atmosphere. The reaction was quenched by adding ice-water (7.00 mL). The pH of the mixture was adjusted to ~5 by adding hydrochloric acid (1 M). Organics were extracted with ethyl acetate (2 x 70.0 mL), and the combined extracts were washed with brine (70.0 mL), dried over anhydrous sodium sulfate, and evaporated to an oil. Trace water was removed by azeotropic evaporation of added anhydrous toluene azeotrope (3 x 10 mL) to give (E)-6-(3-((E)-3-(3-chloro-5-(3,4- difluorophenoxy)thiophen-2-yl)-2-methylacryloyl)-4-hydroxy-2-oxo-2H-pyran-6-yl)hex-2- enoyl azide (750 mg, crude) as a yellow oil. f.

[0167] A solution of (E)-6-(3-((E)-3-(3-chloro-5-(3,4-difluorophenoxy)thiophen-2-yl)-2- methylacryloyl)-4-hydroxy-2-oxo-2H-pyran-6-yl)hex-2-enoyl azide (750 mg, 1.33 mmol, 1.00 eq: Example 4e) in toluene (11.0 mL) was stirred at 110°C for 2 h, the mixture was cooled to room temperature, and methanol -d4 (11.0 mL) was added. The resulting reaction mixture was stirred at 70°C for 12 h, and was concentrated under vacuum to give a yellow oil. The yellow oil was purified by prep-HPLC (column: Phenomenex Luna C18 150 mm x 25 mm x 10 μm; mobile phase: [water(FA)-ACN]; gradient: 63%-93% B over 10 min) and was lyophilized to give methyl-d3-((E)-5-(3-((E)-3-(3-chloro-5-(3,4-difluorophenoxy)- thiophen-2-yl)-2-methylacryloyl)-4-hydroxy-2-oxo-2H-pyran-6-yl)pent-l-en-l-yl)carbamate (230 mg, 404 μmol, 30.3% yield) as a yellow solid.1H NMR (400 MHz, methanol-d4) δ= 7.46 (d, .7= 1.2 Hz, 1H), 7.35 (q, J= 9.2 Hz, 1H), 7.24 (ddd, J = 3.2, 6.2, 11.2 Hz, 1H), 7.09 - 7.01 (m, 1H), 6.66 (s, 1H), 6.47 - 6.37 (m, 1H), 6.16 (s, 1H), 5.14 - 5.00 (m, 1H), 2.57 (t, J= 7.6 Hz, 2H), 2.16 - 2.00 (m, 5H), 1.75 (q, J= 7.2 Hz, 2H). Example 5. Preparation of:

[0168] The compound of Example 5 was prepared as follows:

[0169] A solution of methyl -d ((E)-5-(3-((E)-3-(3-chloro-5-(3,4-difluorophenoxy)thiophen- 2-yl)-2-methylacryloyl)-4-hydroxy-2-oxo-2H-pyran-6-yl)pent- 1 -en- 1 -yl)carbamate (230 mg, 404 μmol, 1.00 eq Example 4) in sodium carbonate (1.00 M, 808 pL, 2.00 eq) was stirred at 37°C for 16 h. The reaction solution was diluted with methanol (2.00 mL) to give a residue. The residue was purified by prep-HPLC (column: Phenomenex C18 150 mm x 25 mm x 10 μm; mobile phase: [Water-MeOH]; gradient: 28%-58% B over 11 min) and was lyophilized to give sodium-3-((E)-3-(3-chloro-5-(3,4-difluorophenoxy)thiophen-2-yl)-2-methylacryloyl)- 6-((E)-5-(((methoxy-d3)carbonyl)amino)pent-4-en-l-yl)-2-oxo-2H-pyran-4-olate (133.04 mg, 225.12 μmol, 55.7% yield, 100% purity) as a yellow solid.1H NMR (400 MHz, methanol-d4) 5 = 7.67 (s, 1H), 7.39 - 7.26 (m, 1H), 7.21 (ddd, J= 3.2, 6.8, 11.2 Hz, 1H), 7.08 - 6.95 (m, 1H), 6.62 (s, 1H), 6.49 - 6.35 (m, 1H), 5.80 (s, 1H), 5.18 - 4.98 (m, 1H), 2.45 (t, J= 7.6 Hz, 2H), 2.17 - 1.94 (m, 5H), 1.72 (q, J= 7.2 Hz, 2H). Example 6. Preparation of:

[0170] The compound of Example 6 was prepared as described in a-f below.

[0171] To a solution of 2,4-dichlorothiazole-5-carbaldehyde (2.00 g, 11.0 mmol, 1.00 eq) in dimethylformamide (20.0 mL), was added potassium carbonate (3.04 g, 22.0 mmol, 2.00 eq) and 3, 4, 5 -trifluorophenol (1.63 g, 11.0 mmol, 1.00 eq). The reaction mixture was stirred at 20°C for 2 h. The reaction mixture was poured into water (200 mL) and extracted with ethyl acetate (3 x200 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 20 / 1) to give 4-chloro-2- (3,4,5-trifluorophenoxy)thiazole-5-carbaldehyde (3.20 g, 10.9 mmol, 99.2% yield) as a solid.1H NMR (400 MHz, chloroform-d) δ= 9.97 (s, 1H), 7.16 - 6.97 (m, 2H). b. To a solution of 4-chloro-2-(3,4,5-trifluorophenoxy)thiazole-5-carbaldehyde (1.50 g, 5.11 mmol, 1.00 eq Example 6a) in toluene (15.0 mL), was added 2, 2,6,6- tetramethylpiperidine (2.89 g, 20.4 mmol, 3.47 mL, 4.00 eq and methyl-(E)-6-(4-hydroxy-2- oxo-3-propionyl-2H-pyran-6-yl)hex-2-enoate (1.50 g, 5.11 mmol, 1.00 eq: Example 2i). The reaction mixture was stirred at 40°C for 16 h, and concentrated under vacuum to give a residue. The residue was purified by reversed-phase HPLC (0.1% FA condition) and extracted with ethyl acetate (3 x 500 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum to give methyl-(E)-6-(3- (3-(4-chl oro-2-(3, 4, 5-tri fluorophenoxy )thiazol-5-yl)-3-hydroxy-2-methylpropanoyl)-4- hydroxy-2-oxo-2H-pyran-6-yl)hex-2-enoate (1.40 g, 2.38 mmol, 46.6% yield) as a yellow oil.1H NMR (400 MHz, chloroform-d) δ= 7.10 - 7.00 (m, 2H), 6.98 - 6.86 (m, 1H), 6.00 (s, 1H), 5.88 (d, J= 15.6 Hz, 1H), 5.30 (dd, J= 6.0, 8.8 Hz, 1H), 4.30 (dd, J= 7.2, 8.8 Hz, 1H), 3.75 (s, 3H), 3.23 (d, .7= 6.0 Hz, 1H), 2.55 (t, = 7.6 Hz, 2H), 2.31 (q, = 7.2 Hz, 2H), 1.96 - 1.85 (m, 2H), 1.18 (d, J = 7.2 Hz, 3H). c.

[0172] To a solution of methyl-(E)-6-(3-(3-(4-chloro-2-(3,4,5-trifluorophenoxy)thiazol-5-yl)- 3-hydroxy-2-methylpropanoyl)-4-hydroxy-2-oxo-2H-pyran-6-yl)hex-2-enoate (1.10 g, 1.87 mmol, 1.00 eq: Example 6b) in dichloromethane (22.0 mL), was added triethylamine (568 mg, 5.61 mmol, 781 pL, 3.00 eq) and methanesulfonyl chloride (429 mg, 3.74 mmol, 290 pL, 2.00 eq) at 0°C, the reaction mixture was stirred at 25°C for 0.5 h under a nitrogen atmosphere. The reaction was quenched with ice water (20.0 mL), and the pH was adjusted to to 4-5 with hydrochloric acid (1 M). The reaction mixture was poured into water (80.0 mL), extracted with di chloromethane (3 x 80.0 mL), dried with magnesium sulfate, filtered and concentrated to give methyl-(E)-6-(3-(3-(4-chloro-2-(3,4,5-trifluorophenoxy)thiazol-5-yl)-2- methyl-3-((methylsulfonyl)oxy)propanoyl)-4-hydroxy-2-oxo-2H-pyran-6-yl)hex-2-enoate (1.25 g, crude) as a yellow oil.

[0173] To a solution of methyl-(E)-6-(3-(3-(4-chloro-2-(3,4,5-trifluorophenoxy)thiazol-5-yl)- 2-methyl-3-((methyl sulfonyl )oxy)propanoyl)-4-hydroxy-2-oxo-2H-pyran-6-yl)hex-2-enoate (1.25 g, 1.88 mmol, 1.00 eq., Example 6c) in tetrahydrofuran (22.0 mL), was added 2,3,4,6,7,8,9,10-octahydropyrimido[l,2-a]azepine (857 mg, 5.63 mmol, 849 pL, 3.00 eq) at 0°C, and the reaction mixture was stirred at 25°C for 1 h. The pH of the reaction mixture was adjusted to ~3 by adding hydrochloric acid (1 M). The mixture was poured into water (100 mL) and was extracted with ethyl acetate (3 x 100 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum to give a yellow oil. The yellow oil was purified by reversed-phase HPLC (0.1% FA condition) and extracted with ethyl acetate (3 x 100 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum to give m ethyl -(E)-6-(3- ((E)-3-(4-chloro-2-(3, 4, 5-tri fluorophenoxy )thiazol-5-yl)-2-methylacryloyl)-4-hydroxy-2-oxo- 2H-pyran-6-yl)hex-2-enoate (620 mg, 1.09 mmol, 58.0% yield) as a yellow solid.1H NMR (400 MHz, choloroform-d) δ= 7.27 - 7.27 (m, 1H), 7.11 - 7.03 (m, 2H), 6.99 - 6.88 (m, 1H), 6.01 (s, 1H), 5.88 (td, J= 1.6, 15.6 Hz, 1H), 3.75 (s, 3H), 2.56 (t, J= 7.6 Hz, 2H), 2.38 - 2.28 (m, 2H), 2.19 - 2.12 (m, 3H), 1.90 (q, J= 7.6 Hz, 2H). e.

[0174] To a solution of methyl-(E)-6-(3-((E)-3-(4-chloro-2-(3,4,5-trifluorophenoxy)thiazol- 5-yl)-2-methylacryloyl)-4-hydroxy-2-oxo-2H-pyran-6-yl)hex-2-enoate (670 mg, 1.18 mmol, 1.00 eq-. Example 6d) in tetrahydrofuran (7.00 mL), was added lithium hydroxide monohydrate (98.7 mg, 2.35 mmol, 2.00 eq) and water (7.00 mL). The reaction mixture was stirred at 25°C for 12 h. The pH of the reaction mixture was adjusted to ~3 by adding hydrochloric acid (1 M), and the reaction mixture was extracted with ethyl acetate (3 x 100 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum to give a yellow solid. The yellow solid was purified by reversed- phase HPLC (0.1% FA condition) and was extracted with ethyl acetate (3 x 100 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum to give (E)-6-(3-((E)-3-(4-chloro-2-(3,4,5-trifluorophenoxy)thiazol-5-yl)-2- methylacryloyl)-4-hydroxy-2-oxo-2H-pyran-6-yl)hex-2-enoic acid (440 mg, 792 μmol, 67.3% yield) as a yellow solid. 1H NMR (400 MHz, chloroform-d) 5 = 7.27 - 7.26 (m, 1H), 7.08 - 7.00 (m, 3H), 6.02 (s, 1H), 5.89 (d, J= 15.6 Hz, 1H), 2.57 (t, J= 7.6 Hz, 2H), 2.35 (q, J= 7.2 Hz, 2H), 2.16 (s, 3H), 1.95 - 1.89 (m, 2H).

[0175] To a (E)-6-(3-((E)-3-(4-chloro-2-(3,4,5-trifluorophenoxy)thiazol-5-yl)-2- methylacryloyl)-4-hydroxy-2-oxo-2H-pyran-6-yl)hex-2-enoic acid (440 mg, 792 μmol, 1.00 eq., Example 6e) in acetone (4.40 mL), was added diisopropyl ethylamine (511 mg, 3.96 mmol, 689 pL, 5.00 eq and isobutyl carbonochloridate (216 mg, 1.58 mmol, 207 pL, 2.00 eq) at 0 °C under a nitrogen atmosphere, and the reaction mixture was stirred at 0°C for 1.5 h. Sodium azide (412 mg, 6.33 mmol, 8.00 eq in water (4.40 mL) was added, and the reaction mixture was stirred at 25°C for 40 min under a nitrogen atmosphere. The reaction was quenched by adding ice-water (5 mL). The pH of the reaction mixture was adjusted to ~5 by adding hydrochloric acid (1 M). Organics were extracted with ethyl acetate (2 x 50.0 mL), and the combined extracts were washed with brine (50.0 mL), dried over anhydrous sodium sulfate, and evaporated to an oil. Trace water was removed by azeotropic evaporation of added anhydrous stoluene azeotrope (3 x 10.0 mL) to give (E)-6-(3-((E)-3-(4-chloro-2-(3,4,5- tri fluorophenoxy )thi azol-5-yl)-2-methylacryloyl)-4-hy droxy -2-oxo-2H-pyran-6-yl)hex-2- enoyl azide (460 mg, crude) as a yellow oil. A solution of (E)-6-(3-((E)-3-(4-chloro-2-(3,4,5-trifluorophenoxy)thiazol-5-yl)-2- methylacryloyl)-4-hydroxy-2-oxo-2H-pyran-6-yl)hex-2-enoyl azide (460 mg, 792 μmol, 1.00 eq., Example 6f) in toluene (6.50 mL) was stirred at 110°C for 2 h, and was cooled to room temperature. Methanol -d4 (6.50 mL) was added at 70°C, and the reaction mixture was stirred at 70°C for 6 h. The reaction mixture was concentrated under vacuum to give a residue. The residue was purified by prep-HPLC (column: CD05-Phenomenex Luna C18 150 mm x 40 mm x 10 μm; mobile phase: [water(FA)-ACN]; gradient: 57%-87% B over 11 min) and was lyophilized to give methyl-d3-((E)-5-(3-((E)-3-(4-chloro-2-(3,4,5-trifluorophenoxy)thiazol-5- yl)-2-methylacryloyl)-4-hydroxy-2-oxo-2H-pyran-6-yl)pent-l-en-l-yl)carbamate (160 mg, 272 μmol, 34.4% yield) as a yellow solid.1H NMR (400 MHz, methanol-d4) δ= 7.42 - 7.28 (m, 2H), 7.23 (s, 1H), 6.51 - 6.38 (m, 1H), 6.18 (s, 1H), 5.13 - 5.01 (m, 1H), 2.58 (t, J= 6.0 Hz, 2H), 2.20 - 2.02 (m, 5H), 1.75 (dd, J= 5.2, 6.8 Hz, 2H).

[0176] Example 7. Preparation of:

[0177] The compound of Example 7 was prepared as follows:

[0178] A solution of methyl-d3-((E)-5-(3-((E)-3-(4-chloro-2-(3,4,5-trifluorophenoxy)thiazol- 5-yl)-2-methylacryloyl)-4-hydroxy-2-oxo-2H-pyran-6-yl)pent- 1 -en- 1 -yl)carbamate (140 mg, 238 μmol, 1.00 eq Example 6) in sodium carbonate (0.10 M, 7.14 mL, 3.00 eq) was stirred at 37°C for 2 h. The reaction mixture was poured into water (50.0 mL) and was extracted with ethyl acetate (3 x50.0 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum to give a yellow oil. The yellow oil was lyophilized to give sodium-3-((E)-3-(4-chloro-2-(3,4,5-trifluorophenoxy)thiazol-5-yl)-2- methylacryloyl)-6-((E)-5-(((methoxy-d3)carbonyl)amino)pent-4-en-l-yl)-2-oxo-2H-pyran-4- olate (133.27 mg, 214.76 μmol, 90.2% yield, 98.3% purity) as a yellow solid.1H NMR (400 MHz, methanol-A) δ= 7.40 (s, 1H), 7.33 (dd, J= 6.0, 8.0 Hz, 2H), 6.42 (d, J= 14.0 Hz, 1H), 5.76 (s, 1H), 5.16 - 5.02 (m, 1H), 2.42 (t, J= 7.6 Hz, 2H), 2.19 - 1.96 (m, 5H), 1.76 - 1.65 (m, 2H).

[0179] Example 8. Preparation of:

[0180] The compound of Example 8 was prepared as described in a-f below. a.

[0181] To a solution of 3-chlorothiophene-2-carbaldehyde (10.0 g, 68.2 mmol, 1.00 eq) in dichloromethane (100 mL), was added bromine (27.3 g, 171 mmol, 8.79 mL, 2.50 eq). The mixture was stirred at 25°C for 16 h. The mixture was poured into water (500 mL) and extracted with di chloromethane (3 x500 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 1 / 0) to give 5-bromo-3-chloro-thiophene-2-carbaldehyde (14.0 g, 62.1 mmol, 91.0% yield) as yellow solid.1H NMR (400 MHz, DMSO-d6) δ= 9.85 (s, 1H), 7.65 (s, 1H). To a solution of 5-bromo-3-chloro-thiophene-2-carbaldehyde (13.0 g, 57.7 mmol, 1.00 eq,- Example 8a) in dimethylformamide (130 mL), was added potassium carbonate (15.9 g, 115 mmol, 2.00 eq and 3,4,5-trifluorophenol (8.54 g, 57.7 mmol, 1.00 eq), and the mixture was stirred at 25°C for 12 h. The mixture was poured into water (1000 mL) and extracted with ethyl acetate (3 x 1000 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 20 / 1) to give a yellow oil. The yellow oil was purified by prep-HPLC (column: Welch Ultimate XB-CN 250 mm x 70 mm x 10 μm; mobile phase: [Hexane-EtOH]; B%: 3%, isocratic elution mode) and was concentrated under vacuum to give 3-chloro-5-(3,4,5- trifluorophenoxy)thiophene-2-carbaldehyde (7.70 g, 26.3 mmol, 45.6% yield) as a white solid. 'H NMR (400 MHz, chloroform-d) δ= 9.96 (s, 1H), 6.96 - 6.82 (m, 2H), 6.46 (s, 1H). c.

[0182] To a solution of 3-chloro-5-(3,4,5-trifluorophenoxy)thiophene-2-carbaldehyde (1.00 g, 3.42 mmol, 1.00 eq,- Example 8b) in isopropanol (20.0 mL), was added piperidine (291 mg, 3.42 mmol, 337 pL, 1.00 eq) and methyl-(E)-6-(4-hydroxy-2-oxo-3-propionyl-2H-pyran-6- yl)hex-2-enoate (1.01 g, 3.42 mmol, 1.00 eq,- Example 2i), and the mixture was stirred at 70°C for 8 h. The mixture was concentrated under vacuum to give a residue. The residue was purified by reversed-phase HPLC (0.1% FA condition) and was extracted with ethyl acetate (3 x 200 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum to give methyl-(E)-6-(3-((E)-3-(3-chloro-5-(3,4,5- tri fluorophenoxy )thi ophen-2 -yl)-2-methylacryloyl)-4-hydroxy-2-oxo-2H-pyran-6-yl)hex-2- enoate (940 mg, 1.65 mmol, 48.4% yield) as a yellow solid.1H NMR (400 MHz, chloroform-d) δ= 7.26 (d, J= 1.2 Hz, 1H), 7.00 - 6.89 (m, 1H), 6.81 (dd, J= 5.6, 8.0 Hz, 2H), 6.55 (s, 1H), 6.00 (s, 1H), 5.88 (td, J= 1.6, 15.6 Hz, 1H), 3.75 (s, 3H), 2.55 (t, J= 7.6 Hz, 2H), 2.38 - 2.28 (m, 2H), 2.24 - 2.13 (m, 3H), 1.96 - 1.84 (m, 2H). d.

[0183] To a solution of methyl-(E)-6-(3-((E)-3-(3-chloro-5-(3,4,5-trifluorophenoxy)thiophen- 2-yl)-2-methylacryloyl)-4-hydroxy-2-oxo-2H-pyran-6-yl)hex-2-enoate (940 mg, 1.65 mmol, 1.00 eq., Example 8c) in tetrahydrofuran (9.40 mL). was added lithium hydroxide monohydrate (139 mg, 3.30 mmol, 2.00 eq) and water (9.40 mL),a nd the mixture was stirred at 20°C for 12 h. The pH of the mixture was adjusted to ~3 by adding hydrochloric acid (1 M), and the mixture was extracted with ethyl acetate (3 x 100 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum to give (E)-6-(3-((E)-3-(3-chloro-5-(3,4,5-trifluorophenoxy)thiophen-2-yl)-2-methylacryloyl)-4- hydroxy-2-oxo-2H-pyran-6-yl)hex-2-enoic acid (950 mg, crude) as a yellow solid.1H NMR (400 MHz, chloroform-d) δ= 7.26 (s, 1H), 7.08 - 6.98 (m, 1H), 6.81 (dd, J= 6.0, 8.0 Hz, 2H), 6.55 (s, 1H), 6.01 (s, 1H), 5.89 (d, J= 15.6 Hz, 1H), 2.57 (t, J= 7.6 Hz, 2H), 2.40 - 2.30 (m, 2H), 2.17 (s, 3H), 1.95 - 1.88 (m, 2H).

[0184] To a solution of (E)-6-(3-((E)-3-(3-chloro-5-(3,4,5-trifluorophenoxy)thiophen-2-yl)-2- methylacryloyl)-4-hydroxy-2-oxo-2H-pyran-6-yl)hex-2-enoic acid (950 mg, 1.71 mmol, 1.00 eq., Example 8d) in acetone (9.50 mL), was added diisopropylethylamine (1.11 g, 8.56 mmol, 1.49 mL, 5.00 eq and isobutyl carbonochloridate (468 mg, 3.42 mmol, 448 pL, 2.00 eq) at 0°C under a nitrogen atmosphere, and the mixture was stirred at 0°C for 1.5 h. Sodium azide (890 mg, 13.7 mmol, 8.00 eq) in water (9.50 mL) was added, and the mixture was stirred at 25°C for 40 min under a nitrogen atmosphere. The reaction was quenched by adding icewater (10.0 mL). The pH of the mixture was adjusted to ~5 by adding hydrochloric acid (1 M). Organics were extracted with ethyl acetate (2 x 90.0 mL), and the combined extracts were washed with brine (90.0 mL), dried over anhydrous sodium sulfate, and evaporated to an oil. Trace water was removed by azeotropic evaporation of added anhydrous toluene azeotrope (3 x 10.0 mL) to give (E)-6-(3-((E)-3-(3-chloro-5-(3,4,5-trifluorophenoxy)- thiophen-2-yl)-2-methylacryloyl)-4-hydroxy-2-oxo-2H-pyran-6-yl)hex-2-enoyl azide (990 mg, crude) as a yellow oil. f.

[0185] A solution of (E)-6-(3-((E)-3-(3-chloro-5-(3,4,5-trifluorophenoxy)thiophen-2-yl)-2- methylacryloyl)-4-hydroxy-2-oxo-2H-pyran-6-yl)hex-2-enoyl azide (990 mg, 1.71 mmol, 1.00 eq., Example 8e) in toluene (15.0 mL) was stirred at 110°C for 2 h, was cooled to room temperature. Methanol -d4 (15.0 mL) was added at 70°C, and the mixture was stirred at 70°C for 12 h. The mixture was concentrated under vacuum to give a residue. The residue was purified by prep-HPLC (column: CD05-Phenomenex LC18 150 mm x 40 mm x 10 μm; mobile phase: [water(FA)-ACN]; gradient: 62%-92% B over 11 min) and was lyophilized to give methyl-d3-((E)-5-(3-((E)-3-(3-chl oro-5-(3, 4, 5 -trifluorophenoxy )thi ophen-2 -yl)-2- methylacryloyl)-4-hydroxy-2-oxo-2H-pyran-6-yl)pent-l-en-l-yl)carbamate (570 mg, 971 μmol, 56.9% yield) as a yellow solid.1H NMR (400 MHz, methanol -d4) δ= 7.45 (d, J= 1.2 Hz, 1H), 7.08 (dd, J= 6.0, 8.4 Hz, 2H), 6.78 - 6.69 (m, 1H), 6.51 - 6.36 (m, 1H), 6.16 (s, 1H), 5.06 (dd, J= 6.8, 14.0 Hz, 1H), 2.63 - 2.51 (m, 2H), 2.21 - 2.00 (m, 5H), 1.83 - 1.63 (m, 2H).

[0186] Example 9. Preparation of:

[0187] A solution of methyl-d3-((E)-5-(3-((E)-3-(3-chloro-5-(3,4,5-trifluorophenoxy)- thiophen-2-yl)-2-methylacryloyl)-4-hydroxy-2-oxo-2H-pyran-6-yl)pent-l-en-l-yl)carbamate (520 mg, 886 μmol, 1.00 eq Example 8) in sodium carbonate (0.10 M, 31.0 mL, 3.50 eq) was stirred at 37°C for 16 h. The mixture was poured into water (100 mL) and extracted with ethyl acetate (3 x 100 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum to give a yellow oil. The yellow oil was lyophilized to give sodium-3-((E)-3-(3-chloro-5-(3,4,5-trifluorophenoxy)thiophen-2-yl)-2- methylacryloyl)-6-((E)-5-(((methoxy-d3)carbonyl)amino)pent-4-en-l-yl)-2-oxo-2H-pyran-4- olate (515.71 mg, 836.28 μmol, 94.4% yield, 98.8% purity) as a yellow solid.1H NMR (400 MHz, methanol-A) δ= 7.61 (s, 1H), 7.05 (dd, J= 6.0, 8.4 Hz, 2H), 6.72 (s, 1H), 6.47 - 6.39 (m, 1H), 5.80 (s, 1H), 5.15 - 4.98 (m, 1H), 2.44 (t, J= 7.6 Hz, 2H), 2.14 - 2.03 (m, 5H), 1.71 (q, J = 7.2 Hz, 2H).

[0188] Example 10. Preparation of:

[0189] The compound of Example 10 was prepared as described in a-g below. a.

[0190] To a solution of 2,4-dibromothiazole-5-carbaldehyde (3.00 g, 11.1 mmol, 1.00 eq) in dimethylformamide (30.0 mL), was added potassium carbonate (3.06 g, 22.2 mmol, 2.00 eq) and 3,4-difluorophenol (1.58 g, 12.2 mmol, 1.10 eq), and the mixture was stirred at 40°C for 8 h. The mixture was poured into water (500 mL) and extracted with ethyl acetate (3 / 300 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 20 / 1) to give 4-bromo-2-(3,4- difluorophenoxy)thiazole-5-carbaldehyde (2.53 g, 7.90 mmol, 71.4% yield) as a white solid.1H NMR (400 MHz, chloroform-d) δ= 9.87 (s, 1H), 7.32 - 7.27 (m, 1H), 7.24 - 7.20 (m, 1H), 7.13 - 7.04 (m, 1H). b.

[0191] To a solution of 4-bromo-2-(3,4-difluorophenoxy)thiazole-5-carbaldehyde (2.40 g, 7.50 mmol, 1.00 e , Example 10a) in toluene (24.0 mL), was added 2, 2,6,6- tetramethylpiperidine (4.24 g, 30.0 mmol, 5.09 mL, 4.00 eq) and methyl-(E)-6-(4-hydroxy-2- oxo-3-propionyl-2H-pyran-6-yl)hex-2-enoate (2.21 g, 7.50 mmol, 1.00 eq,' Example 2i), and the mixture was stirred at 40°C for 8 h. The mixture was concentrated in vacuum to give a residue. The residue was purified by reversed-phase HPLC (0.1% FA condition) and extracted with ethyl acetate (3 x 200 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered and concentrated under vacuum to give methyl-(E)-6-(3- (3-(4-bromo-2-(3,4-difluorophenoxy)thiazol-5-yl)-3-hydroxy-2-methylpropanoyl)-4- hydroxy-2-oxo-2H-pyran-6-yl)hex-2-enoate (2.12 g, 3.45 mmol, 46.0% yield) as a yellow solid.1H NMR (400 MHz, chloroform-d) δ= 7.25 - 7.15 (m, 2H), 7.07 (d, J= 8.0 Hz, 1H), 6.98 - 6.84 (m, 1H), 6.00 (s, 1H), 5.88 (d, J= 15.6 Hz, 1H), 5.26 (dd, J= 5.2, 8.0 Hz, 1H), 4.29 (s, 1H), 3.75 (s, 3H), 3.19 (d, J = 5.2 Hz, 1H), 2.55 (t, J = 7.6 Hz, 2H), 2.31 (q, J= 6.8 Hz, 2H), 1.89 (q, .7= 7.6 Hz, 2H), 1.18 (d, J= 6.8 Hz, 3H).

[0192] To a solution of methyl-(E)-6-(3-(3-(4-bromo-2-(3,4-difluorophenoxy)thiazol-5-yl)-3- hydroxy-2-methylpropanoyl)-4-hydroxy-2-oxo-2H-pyran-6-yl)hex-2-enoate (1.76 g, 2.86 mmol, 1.00 eq,' Example 10b) in di chloromethane (35.0 mL). was added triethylamine (870 mg, 8.59 mmol, 1.20 mL, 3.00 eq), and then methanesulfonyl chloride (656 mg, 5.73 mmol, 443 pL, 2.00 eq) was added dropwise at 0°C. The resulting mixture was stirred at 25°C for 0.5 h. The reaction was quenched with ice water (50.0 mL). The pH was adjusted to 4-5 with hydrochloric acid (1 M). Organics were extracted with di chloromethane (3 x 100 mL), dried with magnesium sulfate, filtered, and concentrated to give methyl -(E)-6-(3 -(3 -(4-bromo-2- (3,4-difluorophenoxy)thiazol-5-yl)-2-methyl-3-((methylsulfonyl)oxy)propanoyl)-4-hydroxy- 2-oxo-2H-pyran-6-yl)hex-2-enoate (1.98 g, crude) as a yellow oil. d.

[0193] To a solution of methyl-(E)-6-(3-(3-(4-bromo-2-(3,4-difluorophenoxy)thiazol-5-yl)-2- methyl-3-((methylsulfonyl)oxy)propanoyl)-4-hydroxy-2-oxo-2H-pyran-6-yl)hex-2-enoate (1.98 g, 2.86 mmol, 1.00 eq Example 10c) in tetrahydrofuran (35.0 mL), was added 2,3,4,6,7,8,9,10-octahydropyrimido[l,2-a]azepine (1.31 g, 8.58 mmol, 1.29 mL, 3.00 eq) at 0 °C, and the mixture was stirred at 25°C for 1 h. The pH of mixture was adjusted to ~3 by adding hydrochloric acid (1 M). The mixture was poured into water (200 mL) and was extracted with ethyl acetate (3 x 200 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum to give a yellow oil. The yellow oil was purified by reversed-phase HPLC (0.1% FA condition) and extracted with ethyl acetate (3 x 200 mL). The combined organic layer was dried over anhydrous sodium sulfate, filtered and concentrated under vacuum to give methyl-(E)-6-(3-((E)-3-(4-bromo-2- (3,4-difluorophenoxy)thiazol-5-yl)-2-methylacryloyl)-4-hydroxy-2-oxo-2H-pyran-6-yl)hex- 2-enoate (350 mg, 587 μmol, 20.5% yield) as a yellow soild.1H NMR (400 MHz, chloroform-d) δ=7.25 - 7.18 (m, 2H), 7.12 - 7.06 (m, 1H), 7.04 (d, J= 1.6 Hz, 1H), 6.94 (td, J= 7.2, 15.6 Hz, 1H), 6.01 (s, 1H), 5.92 - 5.84 (m, 1H), 3.75 (s, 3H), 2.55 (t, J= 7.6 Hz, 2H), 2.37 - 2.27 (m, 2H), 2.14 (d, J= 1.2 Hz, 3H), 1.90 (q, J= 7.6 Hz, 2H). e.

[0194] To a solution of methyl-(E)-6-(3-((E)-3-(4-bromo-2-(3,4-difluorophenoxy)thiazol-5- yl)-2-methylacryloyl)-4-hydroxy-2-oxo-2H-pyran-6-yl)hex-2-enoate (550 mg, 922 μmol, 1.00 eq., Example lOd) in tetrahydrofuran (5.50 mL), was added lithium hydroxide monohydrate (77.4 mg, 1.84 mmol, 2.00 eq) and water (5.50 mL), and the mixture was stirred at 20°C for 12 h. The pH of mixture was adjusted to ~3 by adding hydrochloric acid (1 M), and the mixture was extracted with ethyl acetate (3 x 50.0 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum to give (E)-6-(3-((E)-3-(4-bromo-2-(3,4-difluorophenoxy)thiazol-5-yl)-2-methylacryloyl)-4- hydroxy-2-oxo-2H-pyran-6-yl)hex-2-enoic acid (550 mg, crude) as a yellow solid.1H NMR (400 MHz, chloroform-d) δ= 7.27 (s, 3H), 7.12 - 6.97 (m, 2H), 6.02 (s, 1H), 5.89 (d, J= 15.6 Hz, 1H), 2.57 (t, J= 7.6 Hz, 2H), 2.35 (q, J= 6.8 Hz, 2H), 2.16 - 2.12 (m, 3H), 1.92 (q, J = 7.6 Hz, 2H).

[0195] To a solution of (E)-6-(3-((E)-3-(4-bromo-2-(3,4-difluorophenoxy)thiazol-5-yl)-2- methylacryloyl)-4-hydroxy-2-oxo-2H-pyran-6-yl)hex-2-enoic acid (550 mg, 944 μmol, 1.00 eq., Example lOe) in acetone (5.50 mL), was added diisopropylethylamine (610 mg, 4.72 mmol, 822 pL, 5.00 eq) and isobutyl carbonochloridate (258 mg, 1.89 mmol, 247 pL, 2.00 eq) at 0 °C under a nitrogen atmosphere, and the mixture was stirred at 0°C for 1.5 h. Sodium azide (491 mg, 7.56 mmol, 8.00 eq) in water (5.50 mL) was added, and the mixture was stirred at 25°C for 40 min under a nitrogen atmosphere. The reaction was quenched by adding ice-water (10 mL). The pH of the mixture was adjusted to ~5 by adding hydrochloric acid (1 M). Organics were extracted with ethyl acetate (2 x 50 mL), and the combined extracts were washed with brine (50 mL), dried over anhydrous sodium sulfate, and evaporated to an oil. Trace water was removed by azeotropic evaporation of added anhydrous toluene azeotrope (3 x 10 mL) to give (E)-6-(3-((E)-3-(4-bromo-2-(3,4-difluorophenoxy)thiazol-5-yl)-2- methylacryloyl)-4-hydroxy-2-oxo-2H-pyran-6-yl)hex-2-enoyl azide (574 mg, crude) as a yellow oil. g-

[0196] A solution of (E)-6-(3-((E)-3-(4-bromo-2-(3,4-difluorophenoxy)thiazol-5-yl)-2- methylacryloyl)-4-hydroxy-2-oxo-2H-pyran-6-yl)hex-2-enoyl azide (570 mg, 938 μmol, 1.00 eq,- Example 1 Of) in toluene (8.50 mL) was stirred at 110°C for 2 h, and the mixture was cooled to room temperature. Methanol -d4 (8.50 mL) was added at 70°C, and the resulting mixture was stirred at 70 °C for 4 h. The mixture was concentrated under vacuum to give a residue. The residue was purified by prep-HPLC (column: CD05-Phenomenex Luna Cl 8 150 mm x 40 mm x 10 μm; mobile phase: [water(FA)-ACN]; gradient: 60%-80% B over 11 min) and was lyophilized to give m ethyl -d3 ((E)-5-(3-((E)-3-(4-bromo-2-(3,4-difluorophenoxy)- thiazol-5-yl)-2-methylacryloyl)-4-hydroxy-2-oxo-2H-pyran-6-yl)pent-l-en-l-yl)carbamate (140 mg, 228 μmol, 24.3% yield) as a yellow solid.1H NMR (400 MHz, methanol-d4) δ= 7.52 - 7.36 (m, 2H), 7.28 - 7.18 (m, 2H), 6.51 - 6.34 (m, 1H), 6.17 (s, 1H), 5.16 - 5.00 (m, 1H), 2.58 (t, J= 7.6 Hz, 2H), 2.17 - 1.99 (m, 5H), 1.84 - 1.68 (m, 2H). Example 11. Preparation of:

[0197] The compound of Example 11 was prepared as follows:

[0198] A solution of methyl -d ((E)-5-(3-((E)-3-(4-bromo-2-(3,4-difluorophenoxy)thiazol-5- yl)-2-methylacryloyl)-4-hydroxy-2-oxo-2H-pyran-6-yl)pent-l-en-l-yl)carbamate (130 mg, 212 μmol, 1.00 eq: Example 10) in sodium carbonate (0.10 M, 6.35 mL, 3.00 eq) was stirred at 37°C for 1 h. The mixture was poured into water (50.0 mL) and extracted with ethyl acetate (3 x50 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum to give a yellow oil. The yellow oil was purified by prep-HPLC (column: CD02-Waters Xbidge BEH Cl 8 150 mm x 25 mm x 10 μm; mobile phase: [Water-MeOH]; gradient: 26%-56% B over 10 min) and was lyophilized to give a yellow solid, and trace water was removed by azeotropic evaporation of added ethanol azeotrope (3 x 10.0 mL) to give a yellow solid with residual ethanol. Residual ethanol was removed by evaporation of added anhydrous acetonitrile (3 x 10.0 mL) to give sodium-3- ((E)-3-(4-bromo-2-(3,4-difluorophenoxy)thiazol-5-yl)-2-rnethylacryloyl)-6-((E)-5- (((methoxy-d3)carbonyl)amino)pent-4-en-l-yl)-2-oxo-2H-pyran-4-olate (48.38 mg, 73.73 μmol, 34.9% yield, 97.0% purity) as a yellow solid.1H NMR (400 MHz, methanol-d4) δ= 7.51 - 7.34 (m, 3H), 7.26 - 7.16 (m, 1H), 6.42 (d, J= 14.0 Hz, 1H), 5.73 (s, 1H), 5.09 (td, J= 7.2, 14.4 Hz, 1H), 2.42 (t, J= 7.6 Hz, 2H), 2.14 - 2.04 (m, 5H), 1.76 - 1.67 (m, 2H), Example 12. Preparation of:

[0199] The compound of Example 12 was prepared as described in a-g below. a.

[0200] To a solution of 3,5-dibromothiophene-2-carboxylic acid (4.90 g, 17.1 mmol, 1.00 eq) and N,O-dimethylhydroxylamine;hydrochloride (2.01 g, 20.6 mmol, 1.20 eq in dichloromethane (100 mL), was added 1 -hydroxybenzotriazole (3.47 g, 25.7 mmol, 1.50 eq), 3-(ethyliminomethylideneamino)propyl-dimethylazanium;chloride (4.93 g, 25.7 mmol, 1.50 eq and triethylamine (8.67 g, 85.7 mmol, 11.9 mL, 5.00 eq at 0°C, and the mixture was stirred at 20 °C for 16 h. The mixture was poured into water (500 mL) and extracted with ethyl acetate (3 x500 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 20 / 1) to give 3,5-dibromo- N-methoxy-N-methylthiophene-2-carboxamide (5.50 g, 16.7 mmol, 97.6% yield) as a yellow oil.1H NMR (400 MHz, chloroform-d) δ= 7.09 (s, 1H), 3.73 (s, 3H), 3.35 (s, 3H). b.

[0201] To a solution of 3,5-dibromo-N-methoxy-N-methylthiophene-2-carboxamide (5.50 g, 16.7 mmol, 1.00 eq Example 12a) in tetrahydrofuran (110 mL), was added dropwise diisobutylaluminum hydride (1.00 M, 20.1 mL, 1.20 eq at -78°C, and the mixture was stirred at -78°C for 3 h under a nitrogen atmosphere. Ethyl acetate (50.0 mL) was added, the mixture was gradually warmed to 0°C, 10% aqueous hydrochloric acid (20.0 mL) was added, and the mixture was poured into water (200 mL). The mixture was extracted with ethyl acetate (200 mL) and the combined organic layers were washed with saturated sodium bicarbonate aqueous solution and brine, dried over anhydrous sodium sulfate, filtered and evaporated. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 20 / 1) to give 3,5-dibromothiophene-2-carbaldehyde (3.70 g, 13.7 mmol, 82.0% yield) as a yellow solid.1H NMR (400 MHz, DMSO-d6) 5 = 9.76 (s, 1H), 7.69 (s, 1H).

[0202] To a solution of 3,5-dibromothiophene-2-carbaldehyde (3.50 g, 13.0 mmol, 1.00 eq Example 12b) in dimethylformamide (35.0 mL), was added potassium carbonate (3.58 g, 25.9 mmol, 2.00 eq and 3,4-difluorophenol (1.69 g, 13.0 mmol, 1.00 eq , and the mixture was stirred at 40°C for 8 h. The mixture was poured into water (500 mL) and extracted with ethyl acetate (3 x500 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 20 / 1) to give a yellow solid. The yellow solid was purified by prep-HPLC (column: Welch Ultimate XB-CN 250 mm x 50 mm x 10 μm; mobile phase: [Hexane-EtOH]; B%: 1.5%, isocratic elution mode) and was concentrated under vacuum to give 3-bromo-5-(3,4-difluorophenoxy)thiophene-2- carbaldehyde (1.20 g, 3.76 mmol, 29.0% yield) as a yellow solid.1H NMR (400 MHz, chloroform-d) δ= 9.84 (s, 1H), 7.26 - 7.20 (m, 1H), 7.11 - 7.03 (m, 1H), 7.00 - 6.93 (m, 1H), 6.47 (s, 1H). d.

[0203] To a solution of 3-bromo-5-(3,4-difluorophenoxy)thiophene-2-carbaldehyde (1.10 g, 3.45 mmol, 1.00 eq., Example 12c) in isopropanol (22.0 mL), was added piperidine (294 mg, 3.45 mmol, 340 pL, 1.00 eq and methyl-(E)-6-(4-hydroxy-2-oxo-3-propionyl-2H-pyran-6- yl)hex-2-enoate (1.01 g, 3.45 mmol, 1.00 eq Example 2i), and the mixture was stirred at 70°C for 8 h. The mixture was concentrated under vacuum to give a residue. The residue was purified by prep-HPLC (column: Phenomenex Luna Cl 8 (250 mm x 70 mm x 10 μm); mobile phase: [water (FA)-ACN]; gradient: 65%-95% B over 20 min) and was extracted with ethyl acetate (3 x 100 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum to give methyl-(E)-6-(3-((E)-3-(3-bromo-5- (3,4-difluorophenoxy)thiophen-2-yl)-2-methylacryloyl)-4-hydroxy-2-oxo-2H-pyran-6- yl)hex-2-enoate (1.20 g, 2.02 mmol, 58.5% yield) as a yellow solid.1H NMR (400 MHz, chloroform-d) δ= 7.24 - 7.22 (m, 1H), 7.20 - 7.14 (m, 1H), 7.02 (ddd, J= 3.2, 6.4, 10.8 Hz, 1H), 6.98 - 6.87 (m, 2H), 6.54 (s, 1H), 6.00 (s, 1H), 5.88 (d, J= 15.6 Hz, 1H), 4.13 (q, J= 7.2 Hz, 1H), 3.75 (s, 3H), 2.55 (t, J= 7.6 Hz, 2H), 2.31 (q, J= 7.2 Hz, 2H), 2.19 - 2.13 (m, 3H), 1.89 (q, .7= 7.6 Hz, 2H). e.

[0204] To a solution of methyl-(E)-6-(3-((E)-3-(3-bromo-5-(3,4-difluorophenoxy)thiophen- 2-yl)-2-methylacryloyl)-4-hydroxy-2-oxo-2H-pyran-6-yl)hex-2-enoate (1.20 g, 2.02 mmol, 1.00 eq Example 12d) in tetrahydrofuran (12.0 mL), was added lithium hydroxide monohydrate (169 mg, 4.03 mmol, 2.00 eq) and water (12.0 mL), and the mixture was stirred at 20°C for 12 h. The pH of mixture was adjusted to ~3 by adding hydrochloric acid (1 M), and the mixture was extracted with ethyl acetate (3 x 120 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum to give (E)-6-(3-((E)-3-(3-bromo-5-(3,4-difluorophenoxy)thiophen-2-yl)-2-methylacryloyl)-4- hydroxy-2-oxo-2H-pyran-6-yl)hex-2-enoic acid (1.20 g, crude) as a yellow solid.1H NMR (400 MHz, chloroform-d) δ= 7.24 - 7.14 (m, 2H), 7.08 - 6.97 (m, 2H), 6.94 - 6.86 (m, 1H), 6.54 (s, 1H), 6.01 (s, 1H), 5.89 (d, J= 15.6 Hz, 1H), 2.56 (t, J= 6.8 Hz, 2H), 2.35 (d, J= 7.6 Hz, 2H), 2.16 (s, 3H), 1.97 - 1.89 (m, 2H).

[0205] To a solution of (E)-6-(3-((E)-3-(3-bromo-5-(3,4-difluorophenoxy)thiophen-2-yl)-2- methylacryloyl)-4-hydroxy-2-oxo-2H-pyran-6-yl)hex-2-enoic acid (1.20 g, 2.06 mmol, 1.00 eq.:Example 12e) in acetone (12.0 mL), was added diisopropylethylamine (1.33 g, 10.3 mmol, 1.80 mL, 5.00 eq and isobutyl carbonochloridate (564 mg, 4.13 mmol, 540 pL, 2.00 eq) at 0°C under a nitrogen atmosphere, and the mixture was stirred at 0°C for 1.5 h. Sodium azide (1.07 g, 16.5 mmol, 8.00 eq in water (12.0 mL) was added, and the mixture was stirred at 25°C for 40 min under a nitrogen atmosphere. The reaction was quenched by adding icewater (50.0 mL). The pH of the mixture was adjusted to ~5 by adding hydrochloric acid (1 M). Organics were extracted with ethyl acetate (2 x 100 mL), and the combined extracts were washed with brine (100 mL), dried over anhydrous sodium sulfate, and evaporated to an oil. Trace water was removed by azeotropic evaporation of added anhydrous toluene azeotrope (3 x 20.0 mL) to give (E)-6-(3-((E)-3-(3-bromo-5-(3,4-difluorophenoxy)thiophen-2-yl)-2- methylacryloyl)-4-hydroxy-2-oxo-2H-pyran-6-yl)hex-2-enoyl azide (1.25 g, crude) as a yellow oil. g-

[0206] A solution of (E)-6-(3-((E)-3-(3-bromo-5-(3,4-difluorophenoxy)thiophen-2-yl)-2- methylacryloyl)-4-hydroxy-2-oxo-2H-pyran-6-yl)hex-2-enoyl azide (1.25 g, 2.06 mmol, 1.00 eq, Example 12f) in toluene (6.00 mL) was stirred at 110°C for 2 h, and the mixture was cooled to room temperature. Methanol -d4 (6.00 mL) was added, and the mixture was stirred at 70°C for 12 h. The mixture was concentrated under vacuum to give a residue. The residue was purified by prep-HPLC (column: CD05-Phenomenex Luna C18 150 mm x 40 mm x 10 μm; mobile phase: [water(FA)-ACN]; gradient: 58%-88% B over 11 min) and was lyophilized to give m ethyl -d3 ((E)-5-(3-((E)-3-(3-bromo-5-(3,4-difluorophenoxy)thiophen-2- yl)-2-methylacryloyl)-4-hydroxy-2-oxo-2H-pyran-6-yl)pent-l-en-l-yl)carbamate (440 mg, 717 μmol, 34.8% yield) as a yellow solid.1H NMR (400 MHz, methanol -d4) δ= 7.45 (d, J = 1.0 Hz, 1H), 7.39 - 7.30 (m, 1H), 7.24 (ddd, J= 3.2, 6.8, 11.2 Hz, 1H), 7.10 - 7.00 (m, 1H), 6.71 (s, 1H), 6.54 - 6.35 (m, 1H), 6.16 (s, 1H), 5.15 - 5.01 (m, 1H), 2.58 (t, J= 7.6 Hz, 2H), 2.23 - 2.04 (m, 5H), 1.76 (q, J= 7.2 Hz, 2H).

[0207] Example 13. Preparation of:

[0208] The compound of Example 13 was prepared as follows:

[0209] A solution of methyl -d ((E)-5-(3-((E)-3-(3-bromo-5-(3,4-difluorophenoxy)thiophen- 2-yl)-2-methylacryloyl)-4-hydroxy-2-oxo-2H-pyran-6-yl)pent- 1 -en- 1 -yl)carbamate (430 mg, 701 μmol, 1.00 eq Example 12) in sodium carbonate (0.10 M, 21.0 mL, 3.00 eq) was stirred at 37°C for 16 h. The mixture was poured into water (30.0 mL) and extracted with ethyl acetate (3 x 100 mL). The combined organic layers were dried over anhydrous sodium sulfate, evaporated to an oil, and trace water was removed by azeotropic evaporation of added ethanol azeotrope (3 x 10.0 mL), to give a yellow solid with residual ethanol. Residual ethanol was removed by evaporation of added anhydrous acetonitrile (3 x 10.0 mL) to give sodium 3-((E)-3-(3-bromo-5-(3,4-difluorophenoxy)thiophen-2-yl)-2-methylacryloyl)-6-((E)- 5-(((methoxy-d3)carbonyl)amino)pent-4-en-l-yl)-2-oxo-2H-pyran-4-olate (442.37 mg, 676.46 μmol, 96.5% yield, 97.2% purity) as a yellow solid.1H NMR (400 MHz, methanol- A) δ= 7.70 (s, 1H), 7.41 - 7.27 (m, 1H), 7.20 (ddd, J= 2.8, 6.8, 11.2 Hz, 1H), 7.08 - 6.96 (m, 1H), 6.66 (s, 1H), 6.49 - 6.32 (m, 1H), 5.82 - 5.69 (m, 1H), 5.09 (td, J= 7.2, 14.0 Hz, 1H), 2.43 (t, J= 7.6 Hz, 2H), 2.17 - 2.03 (m, 5H), 1.71 (td, J= 7.2, 14.4 Hz, 2H).

[0210] Example 14. Preparation of:

[0211] The compound of Example 14 was prepared as described in a-g below. a.

[0212] To a solution of 2,4-dichlorothiazole-5-carbaldehyde (3.00 g, 16.5 mmol, 1.00 eq) in dimethylformamide (30.0 mL), was added potassium carbonate (4.56 g, 33.0 mmol, 2.00 eq and 4-fluorophenol (1.85 g, 16.5 mmol, 1.00 eq), and the mixture was stirred at 20°C for 12 h. The mixture was poured into water (400 mL) and extracted with ethyl acetate (3 x 400 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 20 / 1) to give 4-chloro-2-(4- fluorophenoxy)thiazole-5-carbaldehyde (4.10 g, 15.9 mmol, 96.6% yield) as a yellow solid.1H NMR (400 MHz, chloroform-d) δ= 9.93 (s, 1H), 7.27 (s, 2H), 7.20 - 7.14 (m, 2H). b.

[0213] To a solution of 4-chloro-2-(4-fluorophenoxy)thiazole-5-carbaldehyde (2.00 g, 7.76 mmol, 1.00 eq Example 14a) in toluene (20.0 mL), was added 2,2,6,6-tetramethylpiperidine (4.39 g, 31.1 mmol, 5.27 mL, 4.00 eq and methyl-(E)-6-(4-hydroxy-2-oxo-3-propionyl-2H- pyran-6-yl)hex-2-enoate (2.28 g, 7.76 mmol, 1.00 eq Example 2i), and the mixture was stirred at 40°C for 16 h. The reaction was quenched with ice water (50.0 mL), and the pH was adjusted to 4-5 with hydrochloric acid (1 M). The reaction mixture was poured into water (400 mL), extracted with ethyl acetate (3 x 300 mL), dried with anhydrous sodium sulfate, filtered, and concentrated to give a yellow oil. The yellow oil was purified by re -HPLC (column: Phenomenex Luna C18 (250 mm x 70 mm, 10 μm); mobile phase: [water (FA)- ACN]; gradient: 44%-74% B over 30 min) and extracted with ethyl acetate (3 / 300 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum to give methyl-(E)-6-(3-(3-(4-chloro-2-(4-fluorophenoxy)thiazol-5-yl)-3- hydroxy-2-methylpropanoyl)-4-hydroxy-2-oxo-2H-pyran-6-yl)hex-2-enoate (2.90 g, 5.25 mmol, 67.7% yield) as a yellow oil.1H NMR (400 MHz, chloroform-d) δ= 7.27 (s, 3H), 7.18 - 7.05 (m, 2H), 6.93 (td, J= 7.2, 15.6 Hz, 1H), 6.00 (s, 1H), 5.88 (d, J= 15.6 Hz, 1H), 5.29 (d, J= 9.2 Hz, 1H), 4.32 - 4.20 (m, 1H), 3.80 - 3.70 (m, 3H), 3.27 - 3.02 (m, 1H), 2.55 (t, J = 7.6 Hz, 2H), 2.38 - 2.25 (m, 2H), 1.89 (q, J= 7.6 Hz, 2H), 1.23 - 1.10 (m, 3H).

[0214] To a solution of methyl-(E)-6-(3-(3-(4-chloro-2-(4-fluorophenoxy)thiazol-5-yl)-3- hydroxy-2-methylpropanoyl)-4-hydroxy-2-oxo-2H-pyran-6-yl)hex-2-enoate (2.90 g, 5.25 mmol, 1.00 eq Example 14b) in dichloromethane (58.0 mL), was added triethylamine (1.59 g, 15.8 mmol, 2.19 mL, 3.00 eq), and then methanesulfonyl chloride (1.20 g, 10.5 mmol, 813 pL, 2.00 eq) was added dropwise at 0°C, and the mixture was stirred at 25°C for 0.5 h. The reaction was quenched with ice water (50.0 mL), and the pH was adjusted to 4-5 with hydrochloric acid (1 M). The reaction mixture was poured into water (400 mL), extracted with dichloromethane (3 x 300 mL), dried with anhydrous sodium sulfate, filtered and concentrated to give methyl-(E)-6-(3-(3-(4-chloro-2-(4-fluorophenoxy)thiazol-5-yl)-2- methyl-3-((methylsulfonyl)oxy)propanoyl)-4-hydroxy-2-oxo-2H-pyran-6-yl)hex-2-enoate (3.30 g, crude) as a yellow oil. d

[0215] To a solution of methyl-(E)-6-(3-(3-(4-chloro-2-(4-fluorophenoxy)thiazol-5-yl)-2- methyl-3-((methylsulfonyl)oxy)propanoyl)-4-hydroxy-2-oxo-2H-pyran-6-yl)hex-2-enoate (3.30 g, 5.24 mmol, 1.00 eq Example 14c) in tetrahydrofuran (60.0 mL), was added 2,3,4,6,7,8,9,10-octahydropyrimido[l,2-a]azepine (2.39 g, 15.7 mmol, 2.37 mL, 3.00 eq) at 0°C, and the mixture was stirred at 25°C for 1 h. The pH of mixture was adjusted to ~3 by adding hydrochloric acid (1 M).The mixture was poured into water (300 mL) and was extracted with ethyl acetate (3 x 300 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum to give a yellow oil. The yellow oil was purified by prep-HPLC (column: YMC-Triart Prep C18 250 mm x 50 mm x 10 μm; mobile phase: [water (FA)-ACN]; gradient: 50%-80% B over 20 min) and was extracted with ethyl acetate (3 x 300 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered and concentrated under vacuum to give methyl-(E)-6-(3- ((E)-3-(4-chloro-2-(4-fluorophenoxy)thiazol-5-yl)-2-methylacryloyl)-4-hydroxy-2-oxo-2H- pyran-6-yl)hex-2-enoate (1.40 g, 2.62 mmol, 50.1% yield) as a yellow solid.1H NMR (400 MHz, chloroform-d) δ= 7.33 - 7.27 (m, 2H), 7.18 - 7.11 (m, 2H), 7.09 (d, J= 1.2 Hz, 1H), 6.94 (td, J= 7.2, 15.6 Hz, 1H), 6.00 (s, 1H), 5.88 (td, J= 1.6, 15.6 Hz, 1H), 3.75 (s, 3H), 2.55 (t, J = 7.6 Hz, 2H), 2.31 (dq, J= 1.2, 7.2 Hz, 2H), 2.12 (d, J = 1.2 Hz, 3H), 1.89 (q, J = 7.6 Hz, 2H). e.

[0216] To a solution of methyl-(E)-6-(3-((E)-3-(4-chloro-2-(4-fluorophenoxy)thiazol-5-yl)-2- methylacryloyl)-4-hydroxy-2-oxo-2H-pyran-6-yl)hex-2-enoate (1.30 g, 2.43 mmol, 1.00 eq Example 14d) in tetrahydrofuran (13.0 mL), was added lithium hydroxide monohydrate (245 mg, 5.84 mmol, 2.40 eq) and water (13.0 mL), and the mixture was stirred at 20°C for 12 h. The pH of the mixture was adjusted to ~3 by adding hydrochloric acid (1 M), and the mixture was extracted with ethyl acetate (3 x 200 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum to give (E)-6-(3-((E)-3- (4-chloro-2-(4-fluorophenoxy)thiazol-5-yl)-2-methylacryloyl)-4-hydroxy-2-oxo-2H-pyran-6- yl)hex-2-enoic acid (1.40 g, crude) as a yellow solid.1H NMR (400 MHz, chloroform-d) d = 7.33 - 7.27 (m, 2H), 7.19 - 7.11 (m, 2H), 7.10 - 6.99 (m, 2H), 6.06 - 5.98 (m, 1H), 5.89 (d, J= 15.6 Hz, 1H), 2.56 (t, .7= 7.6 Hz, 2H), 2.35 (q, J= 6.8 Hz, 2H), 2.11 (d, J= 1.2 Hz, 3H), 1.97 - 1.88 (m, 2H).

[0217] To a solution of (E)-6-(3-((E)-3-(4-chloro-2-(4-fluorophenoxy)thiazol-5-yl)-2- methylacryloyl)-4-hydroxy-2-oxo-2H-pyran-6-yl)hex-2-enoic acid (1.40 g, 2.69 mmol, 1.00 eq.:Example 14e) in acetone (14.0 mL), was added diisopropylethylamine (1.74 g, 13.5 mmol, 2.35 mL, 5.00 eq and isobutyl carb onochlori date (735 mg, 5.39 mmol, 704 pL, 2.00 eq) at 0°C under qa nitrogen atmosphere, and the mixture was stirred at 0°C for 1.5 h. Sodium azide (1.40 g, 21.5 mmol, 8.00 eq in water (14.0 mL) was added, and the mixture was stirred at 25°C for 40 min under a nitrogen atmosphere. The reaction was quenched by adding ice-water (10 mL). The pH of the mixture was adjusted to ~5 by adding hydrochloric acid (1 M). Organics were extracted with ethyl acetate (2 x 150 mL), and the combined extracts were washed with brine (150 mL), dried over anhydrous sodium sulfate, and evaporated to an oil. Trace water was removed by azeotropic evaporation of added anhydrous toluene azeotrope (3 x 20 mL) to give (E)-6-(3-((E)-3-(4-chloro-2-(4-fluorophenoxy)thiazol- 5-yl)-2-methylacryloyl)-4-hydroxy-2-oxo-2H-pyran-6-yl)hex-2-enoyl azide (1.50 g, crude) as a yellow oil.

[0218] A solution of (E)-6-(3-((E)-3-(4-chloro-2-(4-fluorophenoxy)thiazol-5-yl)-2- methylacryloyl)-4-hydroxy-2-oxo-2H-pyran-6-yl)hex-2-enoyl azide (1.50 g, 2.75 mmol, 1.00 eq in toluene (7.00 mL) was stirred at 110°C for 2 h, and the mixture was cooled to room temperature. Methanol -d4 (7.00 mL) was added, and the resulting mixture was stirred at 70°C for 2 h. The mixture was concentrated in vacuum to give a residue. The residue was purified by prep-HPLC (column: CD05-Phenomenex Luna C18 150 mm x 40 mm x 10 μm; mobile phase: [water (FA)-ACN]; gradient: 50%-80% B over 10 min) and was extracted with ethyl acetate (3 x 100 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum to give m ethyl -d ((E)-5-(3-((E)-3-(4-chloro-2-(4- fluorophenoxy)thiazol-5-yl)-2-methylacryloyl)-4-hydroxy-2-oxo-2H-pyran-6-yl)pent- 1 -en- 1 - yl)carbamate (610 mg, 1.11 mmol, 40.2% yield) as a yellow solid.1H NMR (400 MHz, methanol^) δ= 7.51 - 7.35 (m, 2H), 7.30 - 7.17 (m, 3H), 6.52 - 6.39 (m, 1H), 6.17 (s, 1H), 5.11 - 5.03 (m, 1H), 2.57 (t, 7= 7.6 Hz, 2H), 2.19 - 2.03 (m, 5H), 1.84 - 1.69 (m, 2H).

[0219] Example 15. Preparation of:

[0220] The compound of Example 15 was prepared as follows:

[0221] A solution of methyl -d ((E)-5-(3-((E)-3-(4-chloro-2-(4-fluorophenoxy)thiazol-5-yl)- 2-methylacryloyl)-4-hydroxy-2-oxo-2H-pyran-6-yl)pent-l-en-l-yl)carbamate (600 mg, 1.09 mmol, 1.00 eq Example 14) in sodium carbonate (0.01 M, 326 mL, 3.00 eq) was stirred at 37°C for 2 h. The mixture was poured into water (100 mL) and extracted with ethyl acetate (3 x 200 mL). The combined organic layers were dried over anhydrous sodium sulfate, evaporated to an oil, and trace water was removed by azeotropic evaporation of added ethanol azeotrope (3 x 10.0 mL), to give a yellow solid with residual ethanol. Residual ethanol was removed by evaporation of added anhydrous acetonitrile (3 x 10.0 mL) to give sodium-3-((E)-3-(4-chloro-2-(4-fluorophenoxy)thiazol-5-yl)-2-methylacryloyl)-6-((E)-5- (((methoxy -d3)carbonyl)amino)pent-4-en-l-yl)-2-oxo-2H-pyran-4-olate (521.44 mg, 891.98 μmol, 82.1% yield, 98.2% purity) as a yellow solid.1H NMR (400 MHz, methanol -d4) δ= 7.56 - 7.35 (m, 3H), 7.32 - 7.17 (m, 2H), 6.44 (d, J= 14.0 Hz, 1H), 5.76 (s, 1H), 5.17 - 5.04 (m, 1H), 2.44 (t, J= 7.6 Hz, 2H), 2.16 - 2.02 (m, 5H), 1.80 - 1.66 (m, 2H). Example 16. Preparation of:

[0222] The compound of Example 16 was prepared as described in a-g below. a.

[0223] To a solution of 2,4-dichlorothiazole-5-carbaldehyde (3.00 g, 16.5 mmol, 1.00 eq) in dimethylformamide (30.0 mL), was added potassium carbonate (4.56 g, 33.0 mmol, 2.00 eq) and 3-chloro-4-fluoro-phenol (2.42 g, 16.5 mmol, 1.00 eq), and the mixture was stirred at 20°C for 12 h. The mixture was poured into water (400 mL) and extracted with ethyl acetate (3 x400 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 20 / 1) to give 4-chloro-2-(3- chloro-4-fluorophenoxy)thiazole-5-carbaldehyde (2.70 g, 9.24 mmol, 56.1% yield) as a white solid.1H NMR (400 MHz, chloroform-d) δ= 9.95 (s, 1H), 7.41 (dd, J= 2.8, 6.0 Hz, 1H),

[0224] 7.26 - 7.19 (m, 2H). b. To a solution of 4-chloro-2-(3-chloro-4-fluorophenoxy)thiazole-5-carbaldehyde (2.00 g, 6.85 mmol, 1.00 eq Example 16a) in toluene (20.0 mL), was added 2, 2,6,6- tetramethylpiperidine (3.87 g, 27.4 mmol, 4.65 mL, 4.00 eq) and methyl-(E)-6-(4-hydroxy-2- oxo-3-propionyl-2H-pyran-6-yl)hex-2-enoate (2.01 g, 6.85 mmol, 1.00 eq: Example 2i), and the mixture was stirred at 40°C for 16 h. The reaction was quenched with ice water (50.0 mL), and the pH was adjusted to 4-5 with hydrochloric acid (1 M). The reaction mixture was poured into water (400 mL), extracted with ethyl acetate (3 x 300 mL), dried with anhydrous sodium sulfate, filtered, and concentrated to give a yellow oil. The yellow oil was purified by prep-HPLC (column: Phenomenex Luna Cl 8 (250 mm x 70 mm x 10 μm); mobile phase: [water (FA)-ACN]; gradient: 55%-85% B over 30 min) and was extracted with ethyl acetate (3 x 300 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum to give methyl-(E)-6-(3-(3-(4-chloro-2-(3-chloro-4- fluorophenoxy)thiazol-5-yl)-3-hydroxy-2-methylpropanoyl)-4-hydroxy-2-oxo-2H-pyran-6- yl)hex-2-enoate (2.90 g, 4.95 mmol, 72.2% yield) as a yellow oil.1H NMR (400 MHz, chloroform-d) δ= 7.42 - 7.35 (m, 1H), 7.23 - 7.14 (m, 2H), 6.93 (td, J= 7.2, 15.6 Hz, 1H), 6.00 (s, 1H), 5.88 (td, J= 1.6, 15.6 Hz, 1H), 5.29 (d, J= 9.2 Hz, 1H), 4.36 - 4.20 (m, 1H), 3.83 - 3.65 (m, 3H), 3.35 - 3.13 (m, 1H), 2.55 (t, J= 7.6 Hz, 2H), 2.31 (dq, J= 1.6, 7.2 Hz, 2H), 1.89 (q, .7= 7.6 Hz, 2H), 1.17 (d, J= 7.2 Hz, 3H).

[0225] To a solution of methyl-(E)-6-(3-(3-(4-chloro-2-(3-chloro-4-fluorophenoxy)thiazol-5- yl)-3 -hydroxy -2-methylpropanoyl)-4-hydroxy-2-oxo-2H-pyran-6-yl)hex-2-enoate (2.90 g, 4.95 mmol, 1.00 eq: Example 16b) in dichloromethane (58.0 mL), was added triethylamine (1.50 g, 14.8 mmol, 2.06 mL, 3.00), and then methanesulfonyl chloride (1.13 g, 9.89 mmol, 766 pL, 2.00 eq was added dropwise at 0°C, and the mixture was stirred at 25°C for 0.5 h. The reaction was quenched with ice water (50.0 mL), and the pH was adjusted to 4-5 with hydrochloric acid (1 M). The reaction mixture was poured into water (400 mL), extracted with dichloromethane (3 x 300 mL), dried with anhydrous sodium sulfate, filtered, and concentrated to give methyl-(E)-6-(3-(3-(4-chloro-2-(3-chloro-4-fluorophenoxy)thiazol-5-yl)- 2-methyl-3-((methyl sulfonyl )oxy)propanoyl)-4-hydroxy-2-oxo-2H-pyran-6-yl)hex-2-enoate (3.30 g, crude) as a yellow oil.

[0226] To a solution of methyl-(E)-6-(3-(3-(4-chloro-2-(3-chloro-4-fluorophenoxy)thiazol-5- yl)-2-methyl-3-((methylsulfonyl)oxy)propanoyl)-4-hydroxy-2-oxo-2H-pyran-6-yl)hex-2- enoate (3.30 g, 4.97 mmol, 1.00 eq., Example 16c) in tetrahydrofuran (60.0 mL), was added 2,3,4,6,7,8,9,10-octahydropyrimido[l,2-a]azepine (2.27 g, 14.9 mmol, 2.25 mL, 3.00 eq) at 0°C, and the mixture was stirred at 25°C for 1 h. The pH of the mixture was adjusted to ~3 by adding hydrochloric acid (1 M). The mixture was poured into water (300 mL) and was extracted with ethyl acetate (3 x 300 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum to give a yellow oil. The yellow oil was purified by prep-HPLC (column: YMC-Triart Prep C18 250 mm x 50 mm x 10 μm; mobile phase: [water (FA)-ACN]; gradient: 25%-55% B over 20 min) and was extracted with ethyl acetate (3 x 300 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum to give methyl-(E)-6-(3- ((E)-3-(4-chloro-2-(3-chloro-4-fluorophenoxy)thiazol-5-yl)-2-methylacryloyl)-4-hydroxy-2- oxo-2H-pyran-6-yl)hex-2-enoate (1.40 g, 2.46 mmol, 49.6% yield) as a yellow solid. 1H NMR (400 MHz, chloroform-d) 5 = 7.44 - 7.39 (m, 1H), 7.24 - 7.20 (m, 2H), 7.08 (d, J= 1.2 Hz, 1H), 6.94 (td, J= 7.2, 15.6 Hz, 1H), 6.01 (s, 1H), 5.88 (td, J= 1.6, 15.6 Hz, 1H), 3.75 (s, 3H), 2.55 (t, J= 7.6 Hz, 2H), 2.32 (dq, J= 1.2, 7.2 Hz, 2H), 2.14 (d, J= 1.2 Hz, 3H), 1.96 - 1.84 (m, 2H). e.

[0227] To a solution of methyl-(E)-6-(3-((E)-3-(4-chloro-2-(3-chloro-4-fluorophenoxy)- thiazol-5-yl)-2-methylacryloyl)-4-hydroxy-2-oxo-2H-pyran-6-yl)hex-2-enoate (1.30 g, 2.29 mmol, 1.00 eq., Example 16d) in tetrahydrofuran (13.0 mL), was added lithium hydroxide monohydrate (230 mg, 5.49 mmol, 2.40 eq) and water (13.0 mL), and the mixture was stirred at 20°C for 12 h. The pH of the mixture was adjusted to ~3 by adding hydrochloric acid (1 M), and the mixture was extracted with ethyl acetate (3 x 200 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum to give (E)-6-(3-((E)-3-(4-chloro-2-(3-chloro-4-fluorophenoxy)thiazol-5-yl)-2-methylacryloyl)- 4-hydroxy-2-oxo-2H-pyran-6-yl)hex-2-enoic acid (1.40 g, crude) as a yellow solid.1H NMR (400 MHz, chloroform-d) δ= 7.46 - 7.37 (m, 1H), 7.26 - 7.18 (m, 2H), 7.10 - 6.99 (m, 2H), 6.02 (s, 1H), 5.94 - 5.85 (m, 1H), 2.57 (t, J = 7.6 Hz, 2H), 2.41 - 2.31 (m, 2H), 2.17 - 2.12 (m, 3H), 1.97 - 1.87 (m, 2H).

[0228]

[0229] To a solution of (E)-6-(3-((E)-3-(4-chloro-2-(3-chloro-4-fluorophenoxy)thiazol-5-yl)- 2-methylacryloyl)-4-hydroxy-2-oxo-2H-pyran-6-yl)hex-2-enoic acid (1.40 g, 2.53 mmol, 1.00 eq., Example 16e) in acetone (14.0 mL), was added diisopropylethylamine (1.63 g, 12.6 mmol, 2.20 mL, 5.00 eq and isobutyl carb onochlori date (690 mg, 5.05 mmol, 661 pL, 2.00 eq) at 0°C under a nitrogen atmosphere, and the mixture was stirred at 0°C for 1.5 h. Sodium azide (1.31 g, 20.2 mmol, 8.00 eq in water (14.0 mL) was added, and the mixture was stirred at 25°C for 40 min under a nitrogen atmosphere. The reaction was quenched by adding ice-water (10.0 mL), and the pH of the mixture was adjusted to ~5 by adding hydrochloric acid (1 M). Organics were extracted with ethyl acetate (2 x 150 mL), and the combined extracts were washed with brine (150 mL), dried over anhydrous sodium sulfate, and evaporated to an oil. Trace water was removed by azeotropic evaporation of added anhydrous toluene azeotrope (3 x 20.0 mL) to give (E)-6-(3-((E)-3-(4-chloro-2-(3-chloro-4- fluorophenoxy )thiazol-5-yl)-2-methylacryloyl)-4-hydroxy-2-oxo-2H-pyran-6-yl)hex-2-enoyl azide (1.50 g, crude) as a yellow oil. A solution of (E)-6-(3-((E)-3-(4-chloro-2-(3-chloro-4-fluorophenoxy)thiazol-5-yl)-2- methylacryloyl)-4-hydroxy-2-oxo-2H-pyran-6-yl)hex-2-enoyl azide (1.50 g, 2.59 mmol, 1.00 eq) in toluene (7.00 mL) was stirred at 110°C for 2 h, and the mixture was cooled to room temperature. Methanol -d4 (7.00 mL) was added, the mixture was stirred at 70°C for 2 h, and the mixture was concentrated in vacuum to give a residue. The residue was purified by prep-HPLC (column: CD05-Phenomenex Luna Cl 8 150 mm x 40 mm x 10 μm; mobile phase: [water (FA)-ACN]; gradient: 56%-86% B over 10 min) and extracted with ethyl acetate (3 x 100 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum to give a yellow solid. The yellow solid was triturated with methyl tert-butyl ether (5.00 mL) to give methyl-d3-((E)-5-(3-((E)-3-(4-chloro- 2-(3-chloro-4-fluorophenoxy)thiazol-5-yl)-2-methylacryloyl)-4-hydroxy-2-oxo-2JH-pyran-6- yl)pent-l-en-l-yl)carbamate (400 mg, 682 μmol, 26.4% yield) as a yellow solid. 'H NMR (400 MHz, methanol-A) δ= 7.71 - 7.61 (m, 1H), 7.46 - 7.37 (m, 2H), 7.25 (s, 1H), 6.51 - 6.42 (m, 1H), 6.19 (s, 1H), 5.18 - 5.06 (m, 1H), 2.59 (t, J= 7.6 Hz, 2H), 2.24 - 2.06 (m, 5H), 1.77 (q, J = 7.2 Hz, 2H).

[0230] Example 17. Preparation of:

[0231] The compound of Example 17 was prepared as follows:

[0232] A solution of methyl-d3-((E)-5-(3-((E)-3-(4-chloro-2-(3-chloro-4- fluorophenoxy)thiazol-5-yl)-2-methylacryloyl)-4-hydroxy-2-oxo-2H-pyran-6-yl)pent- 1 -en- 1 - yl)carbamate (390 mg, 665 μmol, 1.00 eq.:Example 16) in sodium carbonate (0.01 M, 200 mL, 3.00 eq) was stirred at 37°C for 2 h. The mixture was poured into water (100 mL) and extracted with ethyl acetate (3 x200 mL). The combined organic layers were dried over anhydrous sodium sulfate, evaporated to an oil, and trace water was removed by azeotropic evaporation of added ethanol azeotrope (3 x 10.0 mL), to give a yellow solid with residual ethanol. Residual ethanol was removed by evaporation of added anhydrous acetonitrile (3 x 10.0 mL), to give sodium-3-((E)-3-(4-chloro-2-(3-chloro-4-fluorophenoxy)thiazol-5-yl)-2- methylacryloyl)-6-((E)-5-(((methoxy-d3)carbonyl)amino)pent-4-en-l-yl)-2-oxo-2H-pyran-4- olate (362.64 mg, 589.73 μmol, 88.7% yield, 98.9% purity) as a yellow solid.1H NMR (400 MHz, methanol-A) δ= 7.67 - 7.54 (m, 1H), 7.44 (s, 1H), 7.37 (dd, J= 2.0, 6.0 Hz, 2H), 6.42 (d, J= 14.4 Hz, 1H), 5.73 (s, 1H), 5.17 - 5.03 (m, 1H), 2.42 (t, J= 7.6 Hz, 2H), 2.16 - 1.98 (m, 5H), 1.80 - 1.63 (m, 2H).

[0233] Examples 18-62.

[0234] Example 18: Preparation of: 1H NMR (400 MHz, methanol-d4) 5 = 7.46 (d, J = 1.2 Hz, 1H), 7.40 (dd, J = 3.2, 6.0 Hz, 1H), 7.37 - 7.30 (m, 1H), 7.26 - 7.17 (m, 1H), 6.65 (s, 1H), 6.55 - 6.36 (m, 1H), 6.16 (s, 1H), 5.06 (br dd, J = 7.2, 14.4 Hz, 1H), 2.68 - 2.51 (m, 2H), 2.19 - 2.02 (m, 5H), 1.84 - 1.66 (m, 2H). MS (LC / MS): calculated: m / z 584.1 found: 585.1.

[0235] Example 19. Preparation of: 1H NMR (400 MHz, methanol-d4) 5 = 7.72 (s, 1H), 7.37 (dd, J = 3.2, 6.0 Hz, 1H), 7.34 - 7.27 (m, 1H), 7.19 (td, J = 3.6, 9.2 Hz, 1H), 6.61 (s, 1H), 6.50 - 6.31 (m, 1H), 5.74 (s, 1H), 5.09 (td, J = 7.2, 14.4 Hz, 1H), 2.42 (t, J = 7.2 Hz, 2H), 2.14 - 2.00 (m, 5H), 1.79 - 1.66 (m, 2H). MS (LC / MS): calculated: m / z 606.0 (MNa+); found: 607.1. Example 20. Preparation of: 1H NMR (400 MHz, methanol-d4) 5 = 7.55 - 7.33 (m, 2H), 7.22 (d, J = 1.6 Hz, 1H), 6.50 - 6.38 (m, 1H), 6.29 - 6.11 (m, 1H), 5.15 - 5.00 (m, 1H), 2.58 (t, J = 7.6 Hz, 2H), 2.19 - 2.04 (m, 5H), 1.83 - 1.68 (m, 2H). MS (LC / MS): calculated: m / z 603.1 (M+H+); found: 603.9.

[0236] Example 21. 1H NMR (400 MHz, methanol-d4) 5 = 7.68 - 7.13 (m, 3H), 6.52 - 6.09 (m, 1H), 5.85 - 5.61 (m, 1H), 5.08 (br dd, J = 6.8, 14.0 Hz, 1H), 2.43 (br t, J = 7.2 Hz, 2H), 2.25 - 1.93 (m, 5H), 1.77 - 1.61 (m, 2H). MS (LC / MS): calculated: m / z 625.0 (M+Na+); found: 626.1.

[0237] Example 22. Preparation of: 1H NMR (400 MHz, methanol-d4) 5 = 7.74 (dd, J = 2.8, 5.6 Hz, 1H), 7.53 - 7.15 (m, 3H), 6.44 (br dd, J = 9.6, 13.6 Hz, 1H), 6.16 (s, 1H), 5.08 (br d, J = 6.8 Hz, 1H), 2.57 (t, J = 7.6 Hz, 2H), 2.22 - 1.98 (m, 5H), 1.85 - 1.66 (m, 2H). MS (LC / MS): calculated: m / z 631.0 (M+H+); found: 632.1. Example 23. Preparation of:

[0238] JHNMR (400 MHz, methanol-d4) 5 = 7.83 - 7.53 (m, 1H), 7.49 - 7.17 (m, 3H), 6.55 - 6.24 (m, 1H), 5.95 (br s, 1H), 5.15 - 5.04 (m, 1H), 2.50 (br t, J = 7.2 Hz, 2H), 2.21 - 1.94 (m, 5H), 1.80 - 1.64 (m, 2H). MS (LC / MS): calculated: m / z 653.0 (M+Na+); found: 654.0.

[0239] Example 24. Preparation of:

[0240] 1H NMR (400 MHz, methanol-d4) 5 = 7.53 (dd, J = 2.8, 5.6 Hz, 1H), 7.46 (s, 1H), 7.35 - 7.19 (m, 2H), 6.65 (s, 1H), 6.49 - 6.39 (m, 1H), 6.16 (s, 1H), 5.06 (br dd, J = 7.2, 14.0 Hz, 1H), 2.58 (t, J = 7.6 Hz, 2H), 2.18 - 2.05 (m, 5H), 1.83 - 1.69 (m, 2H). MS (LC / MS): calculated: m / z 630.0 (M+H+); found: 631.1.

[0241] Example 25. Preparation of: 1H NMR (400 MHz, methanol-d4) 5 = 7.70 (s, 1H), 7.49 (dd, J = 2.8, 5.6 Hz, 1H), 7.38 - 7.13 (m, 2H), 6.61 (s, 1H), 6.54 - 6.32 (m, 1H), 5.74 (s, 1H), 5.18 - 5.00 (m, 1H), 2.43 (br t, J = 7.2 Hz, 2H), 2.16 - 1.97 (m, 5H), 1.79 - 1.61 (m, 2H). MS (LC / MS): calculated: m / z 652.0 (M+Na+); found: 653.0. Example 26. Preparation of: 1H NMR (400 MHz, methanol-d4) 5 = 7.68 - 7.51 (m, 1H), 7.47 - 7.32 (m, 1H), 7.31 - 7.11 (m, 2H), 6.51 - 6.39 (m, 1H), 6.25 - 6.11 (m, 1H), 5.12 - 5.06 (m, 1H), 2.57 (br t, J = 7.6 Hz, 2H), 2.18 - 1.98 (m, 5H), 1.86 - 1.67 (m, 2H). MS (LC / MS): calculated: m / z 585.1 (M+H+); found: 586.2.

[0242] Example 27. Preparation of: 1H NMR (400 MHz, methanol-d4) 5 = 7.66 - 7.47 (m, 1H), 7.46 - 7.06 (m, 3H), 6.51 - 6.17 (m, 1H), 5.94 - 5.73 (m, 1H), 5.16 - 5.01 (m, 1H), 2.47 (br t, J = 7.6 Hz, 2H), 2.18 - 1.99 (m, 5H), 1.81 - 1.62 (m, 2H). MS (LC / MS): calculated: m / z 607.0 (M+Na+); found: 608.2.

[0243] Example 28. Preparation of: 1H NMR (400 MHz, methanol-d4) 5 = 7.54 (t, J = 8.8 Hz, 1H), 7.46 (d, J = 0.8 Hz, 1H), 7.19 (dd, J = 2.8, 10.0 Hz, 1H), 7.06 (td, J = 1.6, 9.2 Hz, 1H), 6.73 (s, 1H), 6.44 (br d, J = 14.0 Hz, 1H), 6.17 (s, 1H), 5.14 - 5.05 (m, 1H), 2.58 (t, J = 7.2 Hz, 2H), 2.17 - 2.01 (m, 5H), 1.75 (quin, J = 7.2 Hz, 2H). MS (LC / MS): calculated: m / z 584.1 (M+H+); found: 585.0. Example 29. Preparation of: 1H NMR (400 MHz, methanol-d4) 5 = 7.67 - 7.47 (m, 2H), 7.17 (br d, J = 9.6 Hz, 1H), 7.05 (br d, J = 8.4 Hz, 1H), 6.71 (s, 1H), 6.44 (br d, J = 14.0 Hz, 1H), 5.85 (br s, 1H), 5.15 - 5.07 (m, 1H), 2.47 (br t, J = 6.8 Hz, 2H), 2.14 - 2.01 (m, 5H), 1.78 - 1.67 (m, 2H). MS (LC / MS): calculated: m / z 606.0 (M+Na+); found: 607.1.

[0244] Example 30. Preparation of: 1H NMR (400 MHz, methanol-d4) 5 = 7.75 - 7.59 (m, 2H), 7.37 (dd, J = 2.8, 8.8 Hz, 1H), 7.24 (s, 1H), 6.54 - 6.32 (m, 1H), 6.16 (s, 1H), 5.08 (br d, J = 6.8 Hz, 1H), 2.57 (t, J = 7.2 Hz, 2H), 2.25 - 1.95 (m, 5H), 1.75 (quin, J = 7.6 Hz, 2H). MS (LC / MS): calculated: m / z 603.0 (M+H+); found: 604.0.

[0245] Example 31. Preparation of: 1H NMR (400 MHz, methanol-d4) 5 = 7.76 - 7.48 (m, 2H), 7.47 - 7.15 (m, 2H), 6.55 - 6.17 (m, 1H), 5.93 - 5.69 (m, 1H), 5.16 - 5.06 (m, 1H), 2.56 - 2.33 (m, 2H), 2.21 - 1.93 (m, 5H), 1.72 (td, J = 7.6, 15.2 Hz, 2H). MS (LC / MS): calculated: m / z 625.0 (M+Na+); found: 626.0 Example 32. Preparation of: 1H NMR (400 MHz, methanol-d4) 5 = 7.56 (t, J = 2.4 Hz, 1H), 7.47 (dd, J = 2.8, 9.6 Hz, 1H), 7.23 (s, 1H), 6.53 - 6.36 (m, 1H), 6.17 (s, 1H), 5.08 (br d, J = 6.8 Hz, 1H), 2.58 (br t, J = 7.6 Hz, 2H), 2.23 - 2.03 (m, 5H), 1.83 - 1.68 (m, 2H). MS (LC / MS): calculated: m / z 621.0 (M+H+); found: 622.0.

[0246] Example 33. Preparation of: 1H NMR (400 MHz, methanol-d4) 5 = 7.55 (t, J = 2.0 Hz, 1H), 7.50 - 7.39 (m, 1H), 7.39 - 7.26 (m, 1H), 6.52 - 6.21 (m, 1H), 5.93 (s, 1H), 5.07 (br dd, J = 7.2, 14.0 Hz, 1H), 2.53 - 2.43 (m, 2H), 2.21 - 1.92 (m, 5H), 1.81 - 1.66 (m, 2H). MS (LC / MS): calculated: m / z 643.0 (M+Na+); found: 644.3.

[0247] Example 34. Preparation of: 1H NMR (400 MHz, methanol-d4) 5 = 7.76 - 7.63 (m, 1H), 7.46 (d, J = 1.2 Hz, 1H), 7.16 (dd, J = 2.8, 9.6 Hz, 1H), 7.01 (ddd, J = 1.2, 2.8, 8.8 Hz, 1H), 6.73 (s, 1H), 6.51 - 6.38 (m, 1H), 6.16 (s, 1H), 5.09 (br s, 1H), 2.58 (t, J = 7.6 Hz, 2H), 2.20 - 2.05 (m, 5H), 1.76 (td, J = 7.2, 14.8 Hz, 2H). MS (LC / MS): calculated: m / z 630.0 (M+H+); found: 631.1. Example 35. Preparation of: 1H NMR (400 MHz, methanol-d4) 5 = 7.67 (dd, J = 7.6, 16.4 Hz, 2H), 7.13 (dd, J = 2.8, 9.6 Hz, 1H), 6.98 (ddd, J = 1.2, 2.8, 8.8 Hz, 1H), 6.69 (s, 1H), 6.49 - 6.36 (m, 1H), 5.74 (s, 1H), 5.09 (td, J = 7.2, 14.4 Hz, 1H), 2.43 (br t, J = 7.6 Hz, 2H), 2.14 - 2.02 (m, 5H), 1.79 - 1.66 (m, 2H). MS (LC / MS): calculated: m / z 652.0 (M+Na+); found: 653.1.

[0248] Example 36. Preparation of: 1H NMR (400 MHz, methanol-d4) 5 = 7.84 (dd, J = 7.6, 8.8 Hz, 1H), 7.47 (s, 1H), 7.07 (dd, J = 2.4, 8.4 Hz, 1H), 6.89 (dd, J = 2.0, 8.0 Hz, 1H), 6.73 (s, 1H), 6.44 (br d, J = 14.4 Hz, 1H), 6.13 (s, 1H), 5.06 (br dd, J = 6.8, 13.6 Hz, 1H), 2.57 (br t, J = 7.6 Hz, 2H), 2.17 - 2.04 (m, 5H), 1.83 - 1.70 (m, 2H). MS (LC / MS): calculated: m / z 676.0 (M+H+); found: 677.0.

[0249] Example 37. Preparation of: 1H NMR (400 MHz, methanol-d4) 5 = 7.83 (t, J = 8.4 Hz, 1H), 7.69 (br s, 1H), 7.04 (dd, J = 2.4, 8.8 Hz, 1H), 6.86 (br dd, J = 2.0, 8.4 Hz, 1H), 6.69 (s, 1H), 6.50 - 6.33 (m, 1H), 5.74 (s, 1H), 5.09 (td, J = 7.6, 14.8 Hz, 1H), 2.42 (br t, J = 7.6 Hz, 2H), 2.15 - 1.99 (m, 5H), 1.77 - 1.65 (m, 2H). MS (LC / MS): calculated: m / z 698.0 (M+Na+); found: 699.0. Example 38. Preparation of: 1H NMR (400 MHz, methanol-d4) 5 = 7.48 (d, J = 1.2 Hz, 1H), 7.17 - 7.03 (m, 3H), 6.54 (s, 1H), 6.48 - 6.40 (m, 1H), 6.15 (s, 1H), 5.14 - 4.99 (m, 1H), 2.73 - 2.63 (m, 2H), 2.57 (br t, J = 8.0 Hz, 2H), 2.16 - 2.05 (m, 5H), 1.81 - 1.70 (m, 2H), 1.65 - 1.55 (m, 2H), 1.45 - 1.33 (m, 2H), 1.00 - 0.89 (m, 3H). MS (LC / MS): calculated: m / z 606.0 (M+H+); found: 607.2.

[0250] Example 39. Preparation of: 1H NMR (400 MHz, methanol-d4) 5 = 7.70 (s, 1H), 7.18 - 7.00 (m, 3H), 6.50 (s, 1H), 6.43 (br d, J = 14.0 Hz, 1H), 5.76 (s, 1H), 5.17 - 5.03 (m, 1H), 2.72 - 2.60 (m, 2H), 2.43 (br t, J = 7.6 Hz, 2H), 2.13 - 2.01 (m, 5H), 1.78 - 1.66 (m, 2H), 1.60 (td, J = 7.2, 15.2 Hz, 2H), 1.41 - 1.32 (m, 2H), 0.94 (t, J = 7.2 Hz, 3H). MS (LC / MS): calculated: m / z 628.2 (M+Na+); found: 629.0.

[0251] Example 40. Preparation of: 1H NMR (400 MHz, methanol-d4) 5 = 7.32 (dd, J = 2.8, 6.4 Hz, 1H), 7.28 - 7.06 (m, 3H), 6.50 - 6.34 (m, 1H), 6.21 - 6.10 (m, 1H), 5.09 (br s, 1H), 3.29 - 3.21 (m, 1H), 2.57 (br t, J = 7.6 Hz, 2H), 2.16 - 2.01 (m, 5H), 1.82 - 1.70 (m, 2H), 1.31 - 1.22 (m, 6H). MS (LC / MS): calculated: m / z 593.1 (M+H+); found: 594.1. Example 41. Preparation of: 1H NMR (400 MHz, methanol-d4) 5 = 7.38 (br s, 1H), 7.31 (dd, J = 2.8, 6.0 Hz, 1H), 7.24 - 7.00 (m, 2H), 6.52 - 6.17 (m, 1H), 5.93 - 5.70 (m, 1H), 5.15 - 5.02 (m, 1H), 3.29 - 3.17 (m, 1H), 2.56 - 2.34 (m, 2H), 2.20 - 1.92 (m, 5H), 1.72 (td, J = 7.2, 14.8 Hz, 2H), 1.32 - 1.21 (m, 6H). MS (LC / MS): calculated: m / z 615.1 (M+Na+); found: 616.2.

[0252] Example 42. Preparation of: 1H NMR (400 MHz, methanol-d4) 5 = 7.23 (s, 1H), 7.19 - 7.02 (m, 2H), 6.98 (br d, J = 5.2 Hz, 1H), 6.56 - 6.34 (m, 1H), 6.16 (s, 1H), 5.11 - 5.06 (m, 1H), 2.57 (br t, J = 7.6 Hz, 2H), 2.24 - 1.98 (m, 6H), 1.75 (td, J = 7.2, 14.8 Hz, 2H), 1.11 - 0.97 (m, 2H), 0.83 - 0.66 (m, 2H). MS (LC / MS): calculated: m / z 591.1 (M+H+); found: 592.1.

[0253] Example 43. Preparation of: 1H NMR (400 MHz, methanol-d4) 5 = 7.39 (br s, 1H), 7.23 - 6.79 (m, 3H), 6.52 - 6.16 (m, 1H), 5.94 - 5.71 (m, 1H), 5.16 - 5.05 (m, 1H), 2.46 (br t, J = 7.6 Hz, 2H), 2.25 - 1.92 (m, 6H), 1.77 - 1.64 (m, 2H), 1.09 - 0.97 (m, 2H), 0.83 - 0.65 (m, 2H). MS (LC / MS): calculated: m / z 613.1 (M+Na+); found: 614.2. Example 44. Preparation of: 1H NMR (400 MHz, methanol-d4) 5 = 7.48 (d, J = 0.8 Hz, 1H), 7.26 - 6.94 (m, 3H), 6.54 (s, 1H), 6.44 (br dd, J = 10.4, 14.4 Hz, 1H), 6.15 (s, 1H), 5.06 (br dd, J = 7.2, 14.4 Hz, 1H), 3.29 - 3.19 (m, 1H), 2.57 (br t, J = 7.2 Hz, 2H), 2.24 - 2.03 (m, 7H), 1.96 - 1.47 (m, 8H). MS (LC / MS): calculated: m / z 618.2 (M+H+); found: 619.2.

[0254] Example 45. Preparation of: 1H NMR (400 MHz, methanol-d4) 5 = 7.68 (br s, 1H), 7.22 - 6.99 (m, 3H), 6.49 (s, 1H), 6.42 (br d, J = 14.0 Hz, 1H), 5.75 (s, 1H), 5.07 (br dd, J = 6.8, 14.0 Hz, 1H), 3.26 - 3.20 (m, 1H), 2.42 (br t, J = 7.6 Hz, 2H), 2.09 - 1.99 (m, 7H), 1.83 (br dd, J = 4.4, 8.0 Hz, 2H), 1.77 - 1.66 (m, 4H), 1.62 (td, J = 2.8, 5.6 Hz, 2H). MS (LC / MS): calculated: m / z 640.2 (M+Na+); found: 641.1.

[0255] Example 46. Preparation of: 1H NMR (400 MHz, methanol-d4) 5 = 8.03 - 7.93 (m, 2H), 7.68 (d, J = 5.6 Hz, 1H), 7.45 (d, J = 5.6 Hz, 1H), 7.37 (dd, J = 2.0, 8.8 Hz, 1H), 7.25 (s, 1H), 6.48 - 6.37 (m, 1H), 6.15 (s, 1H), 5.17 - 5.04 (m, 1H), 2.56 (t, J = 7.6 Hz, 2H), 2.13 - 1.99 (m, 5H), 1.79 - 1.68 (m, 2H). MS (LC / MS): calculated: m / z 589.1 (M+H+); found: 590.0. Example 47. Preparation of: 1H NMR (400 MHz, methanol-d4) 5 = 8.04 (d, J = 8.8 Hz, 1H), 7.87 (d, J = 2.0 Hz, 1H), 7.75 (d, J = 5.6 Hz, 1H), 7.45 (d, J = 5.6 Hz, 1H), 7.35 (dd, J = 2.4, 8.8 Hz, 1H), 7.25 (s, 1H), 6.53 - 6.34 (m, 1H), 6.15 (s, 1H), 5.08 (br s, 1H), 2.56 (br t, J = 7.6 Hz, 2H), 2.13 - 1.97 (m, 5H), 1.83 - 1.67 (m, 2H). MS (LC / MS): calculated: m / z 589.1 (M+H+); found: 590.1.

[0256] Example 48. Preparation of: 1H NMR (400 MHz, methanol-d4) 5 = 7.54 - 7.31 (m, 2H), 7.28 - 7.18 (m, 2H), 6.40 (br dd, J = 8.8, 12.8 Hz, 1H), 6.17 (s, 1H), 5.05 (br d, J = 7.6 Hz, 1H), 2.78 - 2.61 (m, 1H), 2.12 - 2.10 (m, 3H), 2.03 (br s, 2H), 1.78 (br dd, J = 6.8, 13.2 Hz, 1H), 1.67 - 1.54 (m, 1H), 1.26 (d, J = 8.0 Hz, 3H). MS (LC / MS): calculated: m / z 583.1 (M+H+); found: 584.2.

[0257] Example 49. Preparation of: 1H NMR (400 MHz, methanol-d4) 5 = 7.57 - 7.02 (m, 4H), 6.51 - 6.17 (m, 1H), 5.88 - 5.70 (m, 1H), 5.05 (br dd, J = 7.2, 14.8 Hz, 1H), 2.68 - 2.44 (m, 1H), 2.15 - 1.95 (m, 5H), 1.86 - 1.67 (m, 1H), 1.60 - 1.49 (m, 1H), 1.29 - 1.19 (m, 3H). MS (LC / MS): calculated: m / z 605.1 (M+Na+); found: 606.1. Example 50. Preparation of: 1H NMR (400 MHz, methanol-d4) 5 = 7.64 (br d, J = 6.0 Hz, 1H), 7.46 - 7.34 (m, 2H), 7.26 (s, 1H), 6.49 - 6.34 (m, 1H), 6.19 (s, 1H), 5.05 (br dd, J = 7.2, 14.0 Hz, 1H), 2.78 - 2.65 (m, 1H), 2.18 - 2.10 (m, 3H), 2.09 - 1.98 (m, 2H), 1.87 - 1.72 (m, 1H), 1.62 (br dd, J = 6.4, 13.6 Hz, 1H), 1.31 - 1.23 (m, 3H). MS (LC / MS): calculated: m / z 599.1 (M+H+); found: 600.1.

[0258] Example 51. Preparation of: 1H NMR (400 MHz, methanol-d4) 5 = 7.61 (d, J = 6.0 Hz, 1H), 7.51 - 7.20 (m, 3H), 6.46 - 6.24 (m, 1H), 6.00 - 5.82 (m, 1H), 5.05 (br dd, J = 6.8, 14.0 Hz, 1H), 2.59 (td, J = 7.2, 14.0 Hz, 1H), 2.15 - 1.96 (m, 5H), 1.81 - 1.69 (m, 1H), 1.61 - 1.51 (m, 1H), 1.28 - 1.18 (m, 3H). MS (LC / MS): calculated: m / z 621.1 (M+Na+); found: 622.3.

[0259] Example 52. Preparation of: 1H NMR (400 MHz, methanol-d4) 5 = 7.57 - 7.33 (m, 2H), 7.23 (d, J = 0.8 Hz, 1H), 6.48 - 6.32 (m, 1H), 6.23 - 6.11 (m, 1H), 5.13 - 4.99 (m, 1H), 2.79 - 2.61 (m, 1H), 2.12 (s, 3H), 2.07 - 1.91 (m, 2H), 1.84 - 1.70 (m, 1H), 1.60 (br dd, J = 6.0, 12.8 Hz, 1H), 1.33 - 1.18 (m, 3H). MS (LC / MS): calculated: m / z 617.1 (M+H+); found: 618.1. Example 53. Preparation of: 1H NMR (400 MHz, methanol-d4) 5 = 7.58 - 7.23 (m, 3H), 6.47 - 6.21 (m, 1H), 6.00 - 5.80 (m, 1H), 5.05 (br dd, J = 7.6, 14.4 Hz, 1H), 2.71 - 2.46 (m, 1H), 2.22 - 1.90 (m, 5H), 1.82 - 1.69 (m, 1H), 1.56 (br dd, J = 6.5, 13.8 Hz, 1H), 1.28 - 1.18 (m, 3H). MS (LC / MS): calculated: m / z 639.1 (M+Na+); found: 640.2.

[0260] Example 54. Preparation of: 1H NMR (400 MHz, methanol-d4) 5 = 7.36 (q, J = 9.2 Hz, 1H), 7.30 - 7.19 (m, 1H), 7.10 - 7.00 (m, 1H), 6.90 (br d, J = 1.2 Hz, 1H), 6.44 (br dd, J = 9.6, 13.2 Hz, 1H), 6.19 - 6.09 (m, 1H), 5.95 - 5.83 (m, 1H), 5.12 - 5.03 (m, 1H), 2.62 - 2.46 (m, 2H), 2.23 - 2.02 (m, 5H), 1.80 - 1.65 (m, 2H). MS (LC / MS): calculated: m / z 552.1 (M+H+); found: 553.0.

[0261] Example 55. Preparation of: 1H NMR (400 MHz, methanol-d4) 5 = 7.44 - 7.31 (m, 1H), 7.30 - 7.11 (m, 2H), 7.10 - 6.98 (m, 1H), 6.45 (br d, J = 14.4 Hz, 1H), 5.95 - 5.84 (m, 1H), 5.80 - 5.69 (m, 1H), 5.18 - 5.02 (m, 1H), 2.48 - 2.25 (m, 2H), 2.22 - 1.94 (m, 5H), 1.77 - 1.58 (m, 2H). MS (LC / MS): calculated: m / z 574.1 (M+Na+); found: 575.0. Example 56. Preparation of: 1H NMR (400 MHz, methanol-d4) 5 = 7.54 - 7.35 (m, 2H), 7.29 - 7.13 (m, 2H), 6.44 (br dd, J = 10.0, 14.0 Hz, 1H), 6.14 (s, 1H), 5.13 - 5.04 (m, 1H), 2.56 (br t, J = 7.6 Hz, 2H), 2.19 - 1.99 (m, 5H), 1.82 - 1.68 (m, 2H). MS (LC / MS): calculated: m / z 553.1 (M+H+); found: 554.0.

[0262] Example 57. Preparation of: 1H NMR (400 MHz, methanol-d4) 5 = 7.72 - 7.53 (m, 2H), 7.37 (dd, J = 2.8, 9.2 Hz, 1H), 7.30 - 7.20 (m, 1H), 6.48 - 6.31 (m, 1H), 6.22 - 6.11 (m, 1H), 5.10 - 5.07 (m, 1H), 2.77 - 2.64 (m, 1H), 2.17 - 2.02 (m, 5H), 1.83 - 1.72 (m, 1H), 1.64 - 1.57 (m, 1H), 1.26 (br d, J = 4.4 Hz, 3H). MS (LC / MS): calculated: m / z 617.0 (M+H+); found: 618.1.

[0263] Example 58. Preparation of: 1H NMR (400 MHz, methanol-d4) 5 = 7.77 - 7.16 (m, 4H), 6.43 - 6.19 (m, 1H), 5.84 - 5.63 (m, 1H), 5.06 (td, J = 6.8, 14.4 Hz, 1H), 2.61 - 2.46 (m, 1H), 2.13 - 1.95 (m, 5H), 1.80 - 1.67 (m, 1H), 1.54 (br dd, J = 6.8, 14.0 Hz, 1H), 1.26 - 1.16 (m, 3H). MS (LC / MS): calculated: m / z 639.0 (M+Na+); found: 640.1. Example 59. Preparation of: 1H NMR (400 MHz, methanol-d4) 5 = 7.61 - 7.52 (m, 1H), 7.47 (dd, J = 2.8, 9.2 Hz, 1H), 7.23 (s, 1H), 6.46 - 6.31 (m, 1H), 6.16 (s, 1H), 5.09 - 5.06 (m, 1H), 2.83 - 2.60 (m, 1H), 2.19 - 2.03 (m, 5H), 1.81 - 1.73 (m, 1H), 1.63 - 1.57 (m, 1H), 1.29 - 1.26 (m, 3H). MS (LC / MS): calculated: m / z 635.0 (M+H+); found: 636.0.

[0264] Example 60. Preparation of:

[0265] JHNMR (400 MHz, methanol-d4) 5 = 7.64 - 7.24 (m, 3H), 6.50 - 6.13 (m, 1H), 5.86 - 5.63 (m, 1H), 5.06 (br dd, J = 6.8, 14.0 Hz, 1H), 2.63 - 2.43 (m, 1H), 2.20 - 1.90 (m, 5H), 1.83 - 1.65 (m, 1H), 1.55 (td, J = 7.6, 14.0 Hz, 1H), 1.29 - 1.11 (m, 3H).

[0266] MS (LC / MS): calculated: m / z 657.0 (M+Na+); found: 658.1.

[0267] Example 61. Preparation of:

[0268] 1H NMR (400 MHz, methanol-d4) 5 = 7.36 - 7.01 (m, 4H), 6.52 - 6.36 (m, 1H), 6.23 - 6.10 (m, 1H), 5.10 (br s, 1H), 2.68 - 2.48 (m, 4H), 2.20 - 2.02 (m, 5H), 1.98 - 1.84 (m, 1H), 1.83 - 1.68 (m, 2H), 0.99 - 0.90 (m, 6H). MS (LC / MS): calculated: m / z 607.2 (M+H+); found: 608.2. Example 62. Preparation of: 1H NMR (400 MHz, methanol-d4) 5 = 7.40 (br s, 1H), 7.26 - 7.03 (m, 3H), 6.55 - 6.09 (m, 1H), 5.79 (s, 1H), 5.08 (td, J = 6.8, 14.4 Hz, 1H), 2.61 - 2.39 (m, 4H), 2.20 - 1.97 (m, 5H), 1.93 (td, J = 6.8, 13.2 Hz, 1H), 1.76 - 1.63 (m, 2H), 0.96 - 0.89 (m, 6H).

[0269] MS (LC / MS): calculated: m / z 629.1 (M+Na+); found: 630.3.

[0270] The compounds of Examples 18-62 were prepared essentially as described for the compounds of Examples 2-17.

[0271] Examples 63-66.

[0272] Example 63. Preparation of: 1H NMR (400 MHz, methanol-d4) 5 = 7.53 - 7.35 (m, 2H), 7.30 - 7.18 (m, 2H), 6.40 (br dd, J = 10.0, 14.0 Hz, 1H), 6.17 (s, 1H), 5.12 - 4.99 (m, 1H), 2.78 - 2.63 (m, 1H), 2.18 - 2.01 (m, 5H), 1.84 - 1.70 (m, 1H), 1.68 - 1.52 (m, 1H), 1.31 - 1.25 (m, 3H)

[0273] MS (LC / MS): calculated: m / z 583.1 (MH+); found: 584.2.

[0274] Example 64. Preparation of: 1H NMR (400 MHz, methanol-d4) 5 = 7.56 - 7.28 (m, 3H), 7.27 - 7.04 (m, 1H), 6.47 - 6.19 (m, 1H), 5.82 (br s, 1H), 5.08 (td, J = 7.1, 14.2 Hz, 1H), 2.66 - 2.44 (m, 1H), 2.16 - 1.96 (m, 5H), 1.84 - 1.69 (m, 1H), 1.56 (qd, J = 7.0, 14.0 Hz, 1H), 1.30 - 1.16 (m, 3H) MS (LC / MS): calculated: m / z 605.1 (MH+); found: 606.3 . Example 65. Preparation of: 1H NMR (400 MHz, methanol-d4) 5 = 7.53 - 7.30 (m, 2H), 7.28 - 7.09 (m, 2H), 6.48 - 6.33 (m, 1H), 6.26 - 6.11 (m, 1H), 5.11 - 4.98 (m, 1H), 2.77 - 2.62 (m, 1H), 2.16 - 2.02 (m, 5H), 1.84 - 1.70 (m, 1H), 1.60 (br dd, J = 6.4, 14.0 Hz, 1H), 1.26 (br t, J = 4.4 Hz, 3H).

[0275] MS (LC / MS): calculated: m / z 583.1 (MH+); found: 584.2.

[0276] Example 66. Preparation of:

[0277] 1H NMR (400 MHz, methanol-d4) 5 = 7.51 - 7.27 (m, 3H), 7.25 - 7.03 (m, 1H), 6.45 - 6.15 (m, 1H), 5.79 (s, 1H), 5.06 (br dd, J = 7.2, 14.4 Hz, 1H), 2.64 - 2.42 (m, 1H), 2.12 - 1.93 (m, 5H), 1.82 - 1.66 (m, 1H), 1.60 - 1.47 (m, 1H), 1.27 - 1.13 (m, 3H)

[0278] MS (LC / MS): calculated: m / z 605.1 (MH+); found: 606.3.

[0279] The compounds of Examples 63-66 were prepared by supercritical fluid chromatography (column: SFC-AD-30-DAICEL CHIRALPAK AD (250 mm x 30 mm x 10 μm; mobile phase: [50% CO2-MeOH; 50% (0.1% NH3 in H2O)]) of the compound of Example 48 (yielding the compounds of Examples 63 and 65; retention times 1.956 min and 2.831 min), followed by conversion to sodium salts essentially as described in Example 3 (yielding the compounds of Examples 64, 66, 66, and 68). Examples 67-70.

[0280] Example 67. Preparation of: 1H NMR (400 MHz, methanol-d4) 5 = 7.62 (td, J = 1.2, 5.6 Hz, 1H), 7.43 - 7.33 (m, 2H), 7.24 (s, 1H), 6.48 - 6.33 (m, 1H), 6.17 (s, 1H), 5.06 - 5.02 (m, 1H), 2.73 - 2.67 (m, 1H), 2.14 - 2.02 (m, 5H), 1.78 (br dd, J = 6.8, 13.6 Hz, 1H), 1.64 - 1.57 (m, 1H), 1.29 - 1.25 (m, 3H) MS (LC / MS): calculated: m / z 599.1 (MH+); found: 600.0.

[0281] Example 68. Preparation of: 1H NMR (400 MHz, methanol-d4) 5 = 7.68 - 7.53 (m, 1H), 7.51 - 7.19 (m, 3H), 6.45 - 6.21 (m, 1H), 5.93 - 5.73 (m, 1H), 5.05 (br dd, J = 7.2, 14.0 Hz, 1H), 2.66 - 2.47 (m, 1H), 2.14 - 1.95 (m, 5H), 1.84 - 1.66 (m, 1H), 1.55 (qd, J = 7.2, 13.6 Hz, 1H), 1.27 - 1.16 (m, 3H) MS (LC / MS): calculated: m / z 621.1 (MH+); found: 622.3.

[0282] Example 69. Preparation of: 1H NMR (400 MHz, methanol-d4) 5 = 7.62 (br d, J = 6.4 Hz, 1H), 7.45 - 7.34 (m, 2H), 7.24 (s, 1H), 6.46 - 6.35 (m, 1H), 6.16 (s, 1H), 5.06 - 5.02 (m, 1H), 2.78 - 2.62 (m, 1H), 2.21 - 2.03 (m, 5H), 1.83 - 1.70 (m, 1H), 1.66 - 1.56 (m, 1H), 1.34 - 1.25 (m, 3H)

[0283] MS (LC / MS): calculated: m / z 599.1 (MH+); found: 600.1. Example 70. Preparation of:

[0284] ‘HNMR (400 MHz, methanol-d4) 5 = 7.69 - 7.51 (m, 1H), 7.46 - 7.22 (m, 3H), 6.48 - 6.21 (m, 1H), 5.86 (s, 1H), 5.05 (br dd, J = 7.2, 14.4 Hz, 1H), 2.69 - 2.44 (m, 1H), 2.13 - 1.95 (m, 5H), 1.75 (td, J = 7.2, 14.0 Hz, 1H), 1.55 (qd, J = 7.2, 13.6 Hz, 1H), 1.27 - 1.17 (m, 3H) MS (LC / MS): calculated: m / z 621.1 (MH+); found: 622.2.

[0285] The compounds of Examples 67-70 were prepared by supercritical fluid chromatography (column: SFC-AD-30-DAICEL CHIRALPAK AD (250 mm x 30 mm x 10 μm; mobile phase: [50% CO2-MeOH; 50% (0.1% NH3 in H2O)]) of the compound of Example 50 (yielding the compounds of Examples 67 and 69; retention times 2.419 min and 2.934 min), followed by conversion to sodium salts essentially as described in Example 3 (yielding the compounds of Examples 68 and 70).

[0286] Examples 71-76.

[0287] Example 71. Preparation of: 1H NMR (400 MHz, methanol-d4) 5= 7.47 - 7.34 (m, 2H), 7.32 - 7.10 (m, 3H), 6.51 - 6.37 (m, 1H), 6.25 - 6.13 (m, 1H), 5.07 (td, J = 7.6, 14.4 Hz, 1H), 3.66 (s, 3H), 2.57 (t, J = 7.6 Hz, 2H), 2.16 - 2.04 (m, 5H), 1.75 (quin, J = 7.2 Hz, 2H). MS (LC / MS): calculated: m / z 548.1 (MH+); found: 549.0. Example 72. Preparation of: 1H NMR (400 MHz, methanol-d4) 5= 7.45 (s, 1H), 7.43 - 7.35 (m, 2H), 7.30 - 7.11 (m, 2H),

[0288] 6.43 (br d, J = 14.4 Hz, 1H), 5.75 (s, 1H), 5.09 (td, J = 7.2, 14.4 Hz, 1H), 3.78 - 3.58 (m, 3H),

[0289] 2.43 (t, J = 7.6 Hz, 2H), 2.12 - 2.00 (m, 5H), 1.71 (quin, J = 7.6 Hz, 2H). MS (LC / MS): calculated: m / z 570.1 (MH+); found: 571.2.

[0290] Example 73. Preparation of: 1H NMR (400 MHz, methanol-d4) 5= 7.54 - 7.32 (m, 2H), 7.27 - 7.07 (m, 2H), 6.52 - 6.32 (m, 1H), 6.26 - 6.12 (m, 1H), 5.11 - 5.03 (m, 1H), 3.77 - 3.57 (m, 3H), 2.58 (t, J = 7.6 Hz, 2H), 2.22 - 2.03 (m, 5H), 1.83 - 1.69 (m, 2H). MS (LC / MS): calculated: m / z 566.1 (MH+); found: 567.0.

[0291] Example 74. Preparation of: 1H NMR (400 MHz, methanol-d4) 5 = 7.53 - 7.30 (m, 3H), 7.29 - 7.12 (m, 1H), 6.43 (br d, J = 14.4 Hz, 1H), 5.79 (s, 1H), 5.08 (td, J = 6.8, 14.0 Hz, 1H), 3.82 - 3.57 (m, 3H), 2.44 (t, J = 7.6 Hz, 2H), 2.18 - 1.98 (m, 5H), 1.71 (quin, J = 7.2 Hz, 2H). MS (LC / MS): calculated: m / z 588.1 (MH+); found: 589.2. Example 75. Preparation of: 1H NMR (400 MHz, methanol-d4) 5 = 7.69 - 7.48 (m, 1H), 7.48 - 7.17 (m, 3H), 6.48 - 6.39 (m, 1H), 6.29 - 6.13 (m, 1H), 5.12 - 5.02 (m, 1H), 3.80 - 3.60 (m, 3H), 2.58 (t, J = 7.6 Hz, 2H), 2.22 - 2.02 (m, 5H), 1.86 - 1.66 (m, 2H). MS (LC / MS): calculated: m / z 582.0 (MH+); found: 583.0.

[0292] Example 76. Preparation of: 1H NMR (400 MHz, methanol-d4) 5 = 7.67 - 7.52 (m, 1H), 7.46 (s, 1H), 7.42 - 7.24 (m, 2H), 6.43 (br d, J = 14.0 Hz, 1H), 5.73 (s, 1H), 5.09 (td, J = 7.2, 14.4 Hz, 1H), 3.77 - 3.61 (m, 3H), 2.42 (t, J = 7.6 Hz, 2H), 2.11 - 2.00 (m, 5H), 1.71 (quin, J = 7.6 Hz, 2H). MS (LC / MS): calculated: m / z 604.0 (MH+); found: 605.2.

[0293] The compounds of Examples 71, 73, and 75 were prepared essentially as described in Examples 2f-2o, but using reaction with methanol at 25°C for 1 hour in place of reaction with methanol-d4 at 70°C for 3 hours in the step corresponding to Example 2o (yielding the compounds of Examples 71, 73, and 75), followed by conversion to sodium salts essentially as described in Example 3 (yielding the compounds of Examples 72, 74, and 76).

[0294] Example 77. Assay of inhibition of bacterial RNA polymerase.

[0295] Fluorescence-detected RNA polymerase assays with E. coli RNA polymerase were performed by a modification of the procedure of Kuhlman et al., 2004 [Kuhlman, P., Duff, H. & Galant, A. (2004) A fluorescence-based assay for multisubunit DNA-dependent RNA polymerases. Anal. Biochem. 324, 183-190], Reaction mixtures contained (20 pl): 0-100 nM test salt, 75 nM E. coli RNA polymerase σ70holoenzyme, 20 nM 384 bp DNA fragment containing the bacteriophage T4 N25 promoter, 100 pM ATP, 100 pM GTP, 100 pM UTP, 100 pM CTP, 50 mM Tris-HCl, pH 8.0, 100 mM KC1, 10 mM MgCh, 1 mM DTT, 10 pg / ml bovine serum albumin, and 5.5% glycerol. Reaction components other than DNA and NTPs were pre-incubated for 10 min at 37°C. Reactions were carried out by addition of DNA and incubation for 5 min at 37°C, followed by addition of NTPs and incubation for 60 min at 37°C. DNA was removed by addition of 1 pl 5 mM CaCh and 2 U DNasel (Ambion, Inc.), followed by incubation for 90 min at 37°C. RNA was quantified by addition of 100 pl RiboGreen RNA Quantitation Reagent (Invitrogen, Inc.; 1 :500 dilution in Tris-HCl, pH 8.0, 1 mM EDTA), followed by incubation for 10 min at 25°C, followed by measurement of fluorescence intensity [excitation wavelength = 485 nm and emission wavelength = 535 nm; QuantaMaster QM1 spectrofluorometer (PTI, Inc.)]. IC50is defined as the concentration of inhibitor resulting in 50% inhibition of RNA polymerase activity.

[0296] Fluorescence-detected RNA polymerase assays with Staphylococcus aureus RNA polymerase were performed analogously, using reaction mixtures containing (20 pl): 0-100 nM test salt, 75 nM Staphylococcus aureus RNA polymerase core enzyme, 300 nM S. aureus 20 nM 384 bp DNA fragment containing the bacteriophage T4 N25 promoter, 100 pM ATP, 100 pM GTP, 100 pM UTP, 100 pM CTP, 40 mM Tris-HCl, pH 8.0, 80 mM NaCl, 5 mM MgCh, 2.5 mM DTT, and 12.7% glycerol.

[0297] Data for the compound of Example 1 and for representative compounds of this invention are shown in Table 1. Bold data indicate improvement over the compound of Example 1.

[0298] Example 78. Assay of inhibition of bacterial growth in culture: MICs.

[0299] Minimal inhibitory concentrations (MICs) for Staphylococcus aureus, Staphylococcus epidermidis. Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus agalactiae, Enterococcus faecalis, Enterococcus faecium, Enterococcus gallinarum, Acinetobacter baumannii, Burkholderia cepacia, Burkholderia dolosa, Burkholderia mutivorans, Escherichia coli, Haemophilus influenzae, Moraxella catarrhalis, and Legionella pneumophila were quantified using broth microdilution assays and a starting cell density of 5xl05cfu / ml [Clinical and Laboratory Standards Institute (CLIS / NCCLS) (2012) Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically;

[0300] Approved Standard, Ninth Edition. CLIS Document M07-A9 (CLIS, Wayne PA); Clinical and Laboratory Standards Institute (CLIS / NCCLS) (2015) Methods for Antimicrobial Dilution and Disk Susceptibility Testing of Infrequently Isolated or Fastidious Bacteria That Grow Aerobically; Approved Standard, Second Edition. CLIS Document M45-A2 (CLIS, Wayne PA)]. MICs for Staphylococcus spp., Enterococcus spp., Acinetobacter spp., Burkholderia spp., and Escherichia coli were determined using cation-adjusted Mueller- Hinton II broth (MHII; BD Biosciences, Inc.), an air atmosphere, and incubation for 16 h at 37°C with shaking at 180 rμm). MICs for Streptococcus spp. were determined using Todd- Hewitt broth (THB; BD Biosciences, Inc.), a 5% CO2 / 95% air atmosphere, and incubation for 16 h at 37°C without shaking. MICs for Haemophilus influenzae were determined using Haemophilus Test Medium broth (HTM; BD Biosciences, Inc), an air atmosphere, and incubation for 20 h at 37°C with shaking at 180 rμm. MICs for Legionella pneumophilia were determined using buff ered-y east-extract broth (prepared by mixing 10 g yeast extract, 10 g ACES buffer, and 1 g α -ketoglutarate in 980 ml water; autoclaving; aseptically adding 0.4 g L-cysteine HC1 in 10 ml water and 0.25 g iron (III) nitrate nonahydrate in 10 ml water; and then aseptically adjusting pH to 6.9), an air atmosphere, and incubation for 48 h at 37°C with shaking at 180 rμm.

[0301] MICs for Mycoplasma pneumoniae were quantified in colorimetric broth microdilution assays as follows: Cultures of Mycoplasma pneumoniae ATCC 15531 grown in SP4 glucose broth (Thermo Fisher, Inc.) at the stage in which cultures start to turn yellow were diluted 100-fold with fresh SP4 glucose broth, were dispensed into wells of 96-well plates, were supplemented with serial dilutions of test compounds, and were incubated 10 days at 37°C. Untreated control cultures started to turn yellow between day 5 and day 7. The MIC was defined as the lowest concentration of test compound that prevented color change by day 10. SP4 glucose broth contains 10% fetal bovine serum. To correct for sequestration of test compounds by serum protein binding, the raw MIC for each test compound was divided by the serum MIC shift for the test compound (defined as the ratio of brothmicrodilution MICs against Escherichia coli D21f2tolC with and without 10% serum).

[0302] MICs for Chlamydia pneumoniae were quantified in intracellular infection microdilution assays essentially as in [Bao, X., Gylfe, A., Sturdevant, G., Gong, Z., Xu, S., Caldwell, H., Elofsson, M., Fan, H. (2014) Benzylidene acylhydrazides inhibit chlamydial growth in a type III secretion- and iron chelation-independent manner. J Bacteriol 196:2989- 3001], Monolayers of human cervical epithelial HEp2 cells grown in Dulbecco's Modified Eagle Medium (Thermo Fisher, Inc.) on 96-well plates were infected by replacing culture medium with fresh medium containing elementary bodies of Chlamydia pneumoniae ATCC 53592 (multiplicity of infection = 0.2), 1 pg / ml cycloheximide, and serial dilutions of test compounds. Plates were centrifuged 10 min at 900xg and incubated 48 h at 37°C. Cells were fixed with cold methanol, reacted with rabbit anti -Chlamydia-pneumoniae IgG, washed, reacted with fluorescein-isothiocyanate-conjugated goat anti-rabbit IgG, washed, and evaluated for inclusion formation by fluorescence microscopy using an Olympus 1X51 fluorescence microscope and the green fluorescence channel. The MIC was defined as the lowest concentration of test compound that prevented inclusion formation.

[0303] MICs for Chlamydia trachomatis were quantified in intracellular infection microdilution assays essentially as in [Lu, B., Qiao, Q., Park, E., Wang, Y., Gilleran, J., Pan, M., Pilch, D., Wu, X., Roberge, J., Fan, H. (2023) Acylpyrazoline-based third-generation selective antichlamydial compounds with enhanced potency. ACS Omega 8:6597-6607], Monolayers of human from mouse fibroblast L929 cells grown in Dulbecco's Modified Eagle Medium (Thermo Fisher, Inc.) on 96-well plates were infected by replacing culture medium with fresh medium containing elementary bodies of Chlamydia trachomatis ATCC VR- 902B / mKate, which expresses red fluorescence protein mKate2 ([Lu, B., Qiao, Q., Park, E., Wang, Y., Gilleran, J., Pan, M., Pilch, D., Wu, X., Roberge, J., Fan, H. (2023) Acylpyrazoline-based third-generation selective antichlamydial compounds with enhanced potency. ACS Omega 8:6597-6607]; multiplicity of infection = 0.2), 1 pg / ml cycloheximide, and serial dilutions of test compounds. Plates were centrifuged 10 min at 900xg and incubated 40 h at 37°C. Cells were evaluated for inclusion formation by fluorescence microscopy using an Olympus 1X51 fluorescence microscope and the red fluorescence channel. The MIC was defined as the lowest concentration of test compound that prevented inclusion formation.

[0304] Chlamydia trachomatis ATCC VR-902B / mKate was described in [Lu, B., Qiao, Q., Park, E., Wang, Y., Gilleran, J., Pan, M., Pilch, D., Wu, X., Roberge, J., Fan, H. (2023) Acylpyrazoline-based third-generation selective antichlamydial compounds with enhanced potency. ACS Omega 8:6597-6607] and was provided by Dr. H. Fan of Rutgers University. Other bacterial strains were obtained from the American Type Culture Collection (ATCC) and the CDC & FDA Antibiotic Resistance Isolate Bank (AR Isolate Bank).

[0305] Data for the compound of Example 1 and for representative compounds of this invention are shown in Tables 2-4. Bold data indicate improvement over the compound of Example 1. Example 79. Assay of inhibition of bacterial growth in culture: panel MIC50s and panel MIC90s.

[0306] Panel median MICs (MIC50s) and panel 90th-percentile MICs (MIC90s) for panels of Staphylococcus spp. strains (Staphylococcus aureus and Staphylococcus epidermidis: MSSA, MRS A, VISA, VRSA, and MDR; n = 33), panels of Streptococcus spp. strains (Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus agalactiae,' SPN, GAS, GBS, DR, and MDR; n = 22), and panels of Enterococcus spp. strains (Enterococcus faecalis, Enterococcus faecium, and Enterococcus gallinarum,' VSE, VRE, and MDR; n = 11) were determined using the procedures of in Example 78. Vancomycin-resistant strains were cultured in agar and broth containing 4 ug / ml vancomycin, prior to dilution in the absence of vancomycin and addition to assay plates.

[0307] Rifampin-resistant derivatives of Staphylococcus aureus ATCC 12600 were described in [Srivastava, A., Degen, D., Ebright, Y.W., Ebright. R.H. (2012) Frequency, spectrum, and nonzero fitness costs of resistance to myxopyronin in Staphylococcus aureus. Antimicrob Agents Chemother. 56:6250-6255], Rifampin-resistant derivatives of Streptococcus pyogenes ATCC 12344 were described in [Maffioli, S., Zhang, Y., Degen, D., Carzaniga, T., Del Gatto, G., Serina, S., Monciardini, P., Mazzetti, C., Guglierame, P., Candiani, G., Chiriac, A., Facchetti, G., Kaltofen, P., Sahl, H., Deho, G., Donadio, S., Ebright, R.H. (2017) Antibacterial nucleoside-analog inhibitor of bacterial RNA polymerase. Cell. 169: 1240- 1248], Daptomycin-resistant isolates of Staphylococcus aureus were described in [Friedman, L., Alder, J., Silverman, J. (2006) Genetic changes that correlate with reduced susceptibility to daptomycin in Staphylococcus aureus. Antimicrob Agents Chemother. 50:2137-2145; Song, Y., Rubio, A., Jayaswal, R., Silverman, J., Wilkinson, B. (2013) Additional routes to Staphylococcus aureus daptomycin resistance as revealed by comparative genome sequencing, transcriptional profiling, and phenotypic studies. PLoS One. 8:e58469] and were provided by Dr. J. Silverman of Cubist Pharmaceuticals. Other panel isolates were obtained from the American Type Culture Collection (ATCC), the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ), the Network on Antimicrobial Resistance in Staphylococcus aureus (NARSA), the CDC & FDA Antibiotic Resistance Isolate Bank (AR Isolate Bank), the Antibacterial Resistance Leadership Group (ARLG), and BEI Resources.

[0308] Cross-resistances were determined from the MICs of drug-resistant and multidrugresistant strains in panels. Data for the compound of Example 1 and for representative compounds of this invention are shown in Tables 5-7. Bold data indicate improvement over the compound of Example 1.

[0309] Example 80. Assay of pharmacokinetics in mice.

[0310] For intravenous pharmacokinetics, test compounds (5 mg / kg; 0.1 ml of 1.25 mg / ml solution in 5% dimethylacetamide / 95% (5% L-dextrose / 100 mM sodium citrate pH = 7.4) were administered to mice (CD-I; 0.024-0.26 kg; n = 3) by intravenous injection into a tail vein at t = 0 h. For oral pharmacokinetics, test compounds (25 mg / kg; 0.1 ml of 6.25 mg / ml solution in 5% dimethylacetamide / 95% (5% L-dextrose / 100 mM sodium citrate pH = 7.4) were administered to mice (CD-I; 0.024-0.26 kg; n = 3) by oral gavage at t = 0 h. Blood samples were collected by tail-vein bleed at t = 0.08, 0.25, 1, 3, 7, and 24 h, and plasma concentrations of test compounds were determined by LC-MS / MS.

[0311] Data for the compound of Example 1 and for representative compounds of this invention are shown in Tables 8-9. Bold data indicate improvement over the compound of Example 1.

[0312] Example 81. Assay of antibacterial efficacy in mouse model of Staphylococcus aureus lung infection ("neutropenic lung infection model").

[0313] Female Swiss Webster mice (0.18-0.20 kg) were rendered immunosuppressed by intraperitoneal injection of 150 mg / kg cyclophosphamide on day -4 and 100 mg / kg cyclophosphamide on day -1 and then were infected by intranasal administration of 1 x 107colony forming units of methicillin-resistant Staphylococcus aureus (MRSA) strain BAA- 1556. Test compounds in vehicle (5% dimethylacetamide / 95% (4% Cremphor-EL / 100 mM sodium phosphate, pH 7.4), positive control in vehicle (linezolid at 50 mg / kg), and negative control (vehicle only), were administered by intravenous injection into a tail vein (200 pl) 1 h post-infection to provide single intravenous doses of 6.25, 12.5, 25, 50, 100, and / or 200 mg / kg or by oral gavage (200 pl) 1 h post-infection to provide oral doses of 6.25, 12.5, 25, 50, 100, and / or 200 mg / kg. Mice were euthanized and lungs were harvested and homogenized 26 h post-infection; and viable bacteria were quantified. The effective dose (ED) was defined as the minimum test-compound dose resulting in P < 0.05 reduction in bacterial burden. Data for the compound of Example 1 and for representative compounds of this invention are shown in Table 10 (for intravenous dosing) and Table 11 (for oral dosing). Bold data indicate improvement over the compound of Example 1. Example 82. Assay of antibacterial efficacy in mouse model of Staphylococcus aureus thigh infection ("neutropenic thigh model").

[0314] Assays were performed as in Example 75, except that mice were infected by injection of 1 x 105colony forming units of methicillin-resistant Staphylococcus aureus (MRSA) strain BAA-1556 or BAA-1707 (USA-400, MW2) into thighs, thighs were harvested and homogenized 24 h post-infection; and viable bacteria were quantified.

[0315] Data for the compound of Example 1 and for representative compounds of this invention are shown in Tables 10-11. Bold data indicate improvement over the compound of Example 1. Table 1: in vitro RNAP-inhibitory activity

[0316] a fluorescence-detected transcription assays.

[0317] Table 2: in vitro antibacterial activity, Gram-positive bacterial pathogens

[0318]

[0319]

[0320]

[0321]

[0322] a broth -microdilution assays.

[0323] Table 3: in vitro antibacterial activity, fastidious Gram-negative bacterial pathogens

[0324]

[0325]

[0326] a broth-microdilution assays. b intracellular infection microdilution assays. c broth-microdilution assays in presence of 10% serum; raw data divided by measured MIC shift for 10% serum.

[0327] Table 4: in vitro antibacterial activity, non-fastidious Gram-negative bacterial pathogens

[0328]

[0329]

[0330] Ill

[0331] a broth -microdilution assays. Table 5: in vitro antibacterial activity, Gram-positive bacterial pathogens, panel

[0332] MIC50s a broth -microdilution assays. Table 6: in vitro antibacterial activity, Gram-positive bacterial pathogens, panel

[0333] MIC90s a broth -microdilution assays. Table 7: in vitro antibacterial activity, cross-resistance properties

[0334] 3broth -microdilution assays (Rutgers). Table 8: in vivo pharmacokinetics, intravenous a LC / MS-detected mouse iv PK; CD-I mice.

[0335] Table 9: in vivo pharmacokinetics, oral

[0336] a LC / MS-detected mouse po PK; CD-I mice.

[0337] Table 10: in vivo antibacterial efficacy, intravenous dosing a Balb / c mice; S. aureus MRSA BAA-1556; n = 5-8 / arm; P < 0.05. b CD-I mice; A aureus MRSA BAA-1556 or MRSA BAA-1707; n = 10 / arm; P < 0.05. Table 11: in vivo antibacterial efficacy, oral dosing a Balb / c mice; S. aureus MRSA BAA-1556; n = 5-8 / arm; P < 0.05. b CD-I mice; A aureus MRSA BAA-1556 or MRSA BAA-1707; n = 10 / arm; P < 0.05.

[0338] The data in Table 1 show that certain compounds according to general structural formula (I) are at least approximately 4 times more potent in inhibiting Gram-positive and Gram-negative bacterial RNA polymerases than the compound of Example 1, the most potent arylmyxopyronin for which data previously were disclosed.

[0339] The data in Table 2 show that certain compounds according to general structural formula (I) are at least approximately 4 times more potent in inhibiting the growth of certain clinically relevant Gram-positive bacterial pathogens than the compound of Example 1, the most potent arylmyxopyronin for which data previously were disclosed.

[0340] The data in Table 3 show that certain compounds according to general structural formula (I) are at least approximately 4 times more potent in inhibiting the growth of certain clinically relevant fastidious Gram-negative bacterial pathogens than the compound of Example 1, the most potent arylmyxopyronin for which data previously were disclosed.

[0341] The data in Table 4 show that certain compounds according to general structural formula (I) are at least approximately 4 times more potent in inhibiting the growth of certain clinically relevant non-fastidious Gram-negative bacterial pathogens than the compound of Example 1, the most potent arylmyxopyronin for which data previously were disclosed.

[0342] The data in Tables 5-7 show that certain compounds according to general structural formula (I) are at least approximately 4 times more potent in inhibiting the growth of panels of strains— including drug-resistant isolates, multi-drug-resistant isolates, and current clinical isolates— of certain clinically relevant Gram-positive bacterial pathogens than the compound of Example 1, the most potent arylmyxopyronin for which data previously were disclosed.

[0343] The data in Tables 2-7 show that certain compounds according to general structural formula (I) provide coverage of Gram-positive, fastidious Gram-negative, and non-fastidious Gram-negative pathogens relevant to lower respiratory tract infections, skin / wound infections, and bloodstream infections— including drug-resistant, multi-drug-resistance, and current clinical isolates.

[0344] The data in Table 8 show that certain compounds according to general structural formula (I) exhibit superior intravenous pharmacokinetics in mice (higher AUCinf, higher Cmax, and / or higher plasma half-life, t0.5) than the compound of Example 1, the most potent arylmyxopyronin for which data previously were disclosed.

[0345] The data in Table 9 show that certain compounds according to general structural formula (I) exhibit superior oral pharmacokinetics in mice (higher AUCinf, higher Cmax, and / or higher oral availability fraction, F) than the compound of Example 1, the most potent arylmyxopyronin for which data previously were disclosed.

[0346] The data in Table 10 show that certain compounds according to general structural formula (I) are at least approximately 2 to 4 times more efficacious in clinically relevant mouse methicillin-resistant Staphylococcus aureus (MRSA) infection assays— including both a mouse MRSA lung infection assay with intravenous dosing and a mouse MRSA thigh infection assay with intravenous dosing— than the compound of Example 1, the most potent arylmyxopyronin for which data previously were disclosed.

[0347] The data in Table 11 show that certain compounds according to general structural formula (I) are at least approximately 4 times more efficacious in clinically relevant mouse methicillin-resistant Staphylococcus aureus (MRSA) infection assays— including both a mouse MRSA lung infection assay with oral dosing and a mouse MRSA thigh infection assay with oral dosing— than the compound of Example 1, the most potent arylmyxopyronin for which data previously were disclosed.

[0348] The data in Examples 2-76 define a new genus of arylmyxopyronins, with potency and properties suggesting that it can be developed to provide a novel, first-in-class, orally available drug for treatment of lower respiratory tract infections, skin / wound infections, and bloodstream infections caused by, at minimum, Staphylococcus spp. (MSSA, MRSA, VRSA, and MDR), Streptococcus spp. (SPN, GAS, GBS, and MDR), Enterococcus spp. (VSE, VRE, and MDR), fastidious Gram negatives (Haemophilus influenzae, Moraxella catarrhalis, Legionella pneumophila, Chlamydia pneumoniae, and Mycoplasma pneumoniae), and certain non-fastidious Gram negatives (Acinetobacteria spp. and Burkholderia spp.).

Claims

CLAIMSWhat is claimed is:

1. A compound of formula (I):or a salt thereof, wherein:W, X, Y, and Z are individually carbon, sulfur, oxygen, selenium, or nitrogen, wherein at least two of W, X, Y, and Z are carbon; one of R1and R2is C1-C10alkyl, C2-C10alkenyl, C1-C10alkoxy, aryloxy, heteroaryloxy, or NRaRb, wherein any C1-C10alkyl, C2-C10alkenyl, or C1-C10alkoxy, is optionally substituted by at least one of halogen, hydroxy, C1-C5alkoxy, tetrahydrofuranyl, or furanyl, and wherein any aryloxy or heteroaryloxy is optionally substituted by at least one of halogen, hydroxy, C1-C5alkyl, C3-C6cycloalkyl, C1-C5alkoxy, aryl, or heteroaryl, wherein any C1-C5alkyl, C3-C6cycloalkyl, C1-C5alkoxy, aryl, and heteroaryl is optionally substituted by at least one of halogen, hydroxy, C1-C5alkyl, C3-C6cycloalkyl, or C1-C5alkoxy; or one of R1and R2is a 5-6-membered saturated, partially unsaturated, or aromatic heterocycle that is optionally substituted by at least one of halogen, hydroxy, C1-C10alkyl, C2-C10alkenyl, or C1-C10alkoxy; and the other of R1and R2is absent or is one of H, halogen, C1-C10alkyl, C2- C10 alkenyl, or C1-C10alkoxy, wherein any C1-C10alkyl, C2-C10alkenyl, or C1-C10alkoxy is optionally substituted by at least one of halogen, hydroxy, or C1-C5alkoxy;R3is absent or is one of H, C1-C2alkyl, or halogen- substituted C1-C2alkyl;R4is halo;R5is H or M+, where M+is a pharmaceutically acceptable cation;R6is H, halogen, or methyl that is optionally substituted with halogen;R9is C1-C10alkyl or C2-C10alkenyl, wherein any C1-C10alkyl or C2-C10alkenyl is optionally substituted by at least one of halogen, hydroxy, alkoxy, or NRaRb; andR10is C1-C5alkyl that is optionally substituted with one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11) deuterium atoms.

2. The compound or salt of claim 1, wherein:W, X, Y, and Z are individually carbon, sulfur, oxygen, selenium, or nitrogen, wherein at least two of W, X, Y, and Z are carbon; one of R1and R2is Ci-Cio alkyl, C2-C10alkenyl, C1-C10alkoxy, aryloxy, heteroaryloxy, or NRaRb, wherein any C1-C10alkyl, C2-C10alkenyl, or C1-C10alkoxy, is optionally substituted by at least one of halogen, hydroxy, C1-C5alkoxy, tetrahydrofuranyl, or furanyl, and wherein any aryloxy or heteroaryloxy is optionally substituted by at least one of halogen, hydroxy, C1-C5alkyl, C1-C5alkoxy, aryl, or heteroaryl, wherein any C1-C5alkyl, C1-C5alkoxy, aryl, and heteroaryl is optionally substituted by at least one of halogen, hydroxy, C1-C5alkyl, or C1-C5alkoxy; or one of R1and R2is a 5-6-membered saturated, partially unsaturated, or aromatic heterocycle that is optionally substituted by at least one of halogen, hydroxy, C1-C10alkyl, C2-C10alkenyl, or C1-C10alkoxy; and the other of R1and R2is absent or is one of H, halogen, C1-C10alkyl, C2-C10alkenyl, or C1-C10alkoxy, wherein any C1-C10alkyl, C2-C10alkenyl, or C1-C10alkoxy is optionally substituted by at least one of halogen, hydroxy, or C1-C5alkoxy;R3is absent or is one of H, C1-C2alkyl, or halogen- substituted C1-C2alkyl;R4is halo;R5is H or M+, where M+is a pharmaceutically acceptable cation;R6is H, halogen, or methyl that is optionally substituted with halogen;R9is C1-C10alkyl or C2-C10alkenyl, wherein any C1-C10alkyl or C2-C10alkenyl is optionally substituted by at least one of halogen, hydroxy, alkoxy, or NRaRb; andR10is C1-C5alkyl that is optionally substituted with one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11) deuterium atoms.

3. The compound or salt of claim 1 or 2, wherein:W is sulfur, oxygen, or nitrogen; andX, Y, and Z are individually carbon, sulfur, oxygen, or nitrogen, wherein at least two of X, Y, and Z are carbon.

4. The compound or salt of claim 1, which is a compound of formula (la):or a salt thereof, wherein:Y is N or C-Ra;R2is aryloxy that is optionally substituted with one or more groups independently selected from the group consisting of halogen, hydroxy, C1-C5alkyl, C3-C6cycloalkyl, C1-C5alkoxy, aryl, and heteroaryl, wherein any C1-C5alkyl, C3-C6cycloalkyl, C1-C5alkoxy, aryl, and heteroaryl is optionally substituted with one or more groups independently selected from the group consisting of halogen, hydroxy, C1-C5alkyl, C3-C6cycloalkyl, and C1-C5alkoxy;R4is halo;R5is H or M+, where M+is a pharmaceutically acceptable cation;R6is H, halogen, or methyl that is optionally substituted with one or more halogen;R9is C1-C5alkyl;R10is C1-C5alkyl that is optionally substituted with one or more (e.g., 1, 2, 3, 4, 5, 6,7, 8, 9, 10, or 11) deuterium atoms; andRais H or C1-C5alkyl.

5. The compound or salt of claim 1 wherein the compound of formula (I) is a compound of formula (lb):

6. The compound or salt of claim 1 wherein the compound of formula (I) is a compound of formula (Ic):

7. The compound or salt of any one of claims 1-6, wherein R2is phenyloxy that is optionally substituted with one or more groups independently selected from the group consisting of halogen, hydroxy, C1-C5alkyl, C3-C6cycloalkyl, C1-C5alkoxy, aryl, and heteroaryl, wherein any C1-C5alkyl, C3-C6cycloalkyl, C1-C5alkoxy, aryl, and heteroaryl is optionally substituted with one or more groups independently selected from the group consisting of halogen, hydroxy, C1-C5alkyl, C3-C6cycloalkyl, and C1-C5alkoxy.

8. The compound or salt of any one of claims 1-6, wherein R2is phenyloxy that is optionally substituted with one or more groups independently selected from the group consisting of halogen.

9. The compound or salt of any one of claims 1-6, wherein R2is phenyloxy that is optionally substituted with one or more fluoro groups.

10. The compound or salt of any one of claims 1-6, wherein R2is 4-fluorophenyloxy, 3,4- difluorophenyloxy, 3-chloro-4-fluorophenyloxy, or 3,4,5-trifluorophenyloxy.

11. The compound or salt of any one of claims 1-10, wherein R4is fluoro.

12. The compound or salt of any one of claims 1-10, wherein R4is chloro.

13. The compound or salt of any one of claims 1-10, wherein R4is bromo.

14. The compound or salt of any one of claims 1-10, wherein R4is iodo.

15. The compound or salt of any one of claims 1-13, wherein R5is H.

16. The salt of any one of claims 1-13, wherein R5is M+and M+is a pharmaceutically acceptable cation.

17. The compound or salt of any one of claims 1-15, wherein R6is H or methyl.

18. The compound or salt of any one of claims 1-16, wherein R9is methyl.

19. The compound or salt of any one of claims 1-17, wherein R10is C1-C5alkyl.

20. The compound or salt of any one of claims 1-17, wherein R10is methyl.

21. The compound or salt of any one of claims 1-17, wherein R10is C1-C5alkyl that is substituted with one or more deuterium atoms.

22. The compound or salt of any one of claims 1-17, wherein R10is -CD3.

23. A compound or salt selected from the group consisting of:

24. A pharmaceutical composition comprising a compound as described in any one of claims 1-23 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

25. The composition of claim 24, which is suitable for intravenous administration.

26. The composition of claim 24 or 25 wherein the pharmaceutically acceptable carrier is an aqueous solution.

27. The composition of any one of claims 24-26 that is substantially free of co-solvents and surfactants.

28. The composition of any one of claims 24-26 that is substantially free of organic cosolvents.

29. A method of inhibiting a bacterial RNA polymerase, comprising contacting a bacterial RNA polymerase with a compound or salt of any one of claims 1-23.

30. A method of treating a bacterial infection in a mammal, comprising administering to the mammal a therapeutically effective amount of a compound as described in any one of claims 1-23 or a pharmaceutically acceptable salt thereof.

31. A compound as described in any one of claims 1-23 or a pharmaceutically acceptable salt thereof for use in medical treatment.

32. A compound as described in any one of claims 1-23 or a pharmaceutically acceptable salt thereof for use in the prophylaxis or treatment of a bacterial infection.

33. The use of a compound as described in any one of claims 1-23 or a salt thereof as an inhibitor of a bacterial RNA polymerase.

34. The use of a compound as described in any one of claims 1-23 or a salt thereof as an antibacterial agent.

35. The use of a compound as described in any one of claims 1-23 or a salt thereof as a disinfectant, sterilant, antispoilant, antiseptic, or anti-infective.

36. The use of a compound as described in any one of claims 1-23 or a pharmaceutically acceptable salt thereof for the preparation of a medicament for prophylaxis or treatment of a bacterial infection in a mammal.

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