WO2019210160A1 - Antibacterial compounds, compositions and uses thereof - Google Patents

Antibacterial compounds, compositions and uses thereof Download PDF

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Publication number
WO2019210160A1
WO2019210160A1 PCT/US2019/029338 US2019029338W WO2019210160A1 WO 2019210160 A1 WO2019210160 A1 WO 2019210160A1 US 2019029338 W US2019029338 W US 2019029338W WO 2019210160 A1 WO2019210160 A1 WO 2019210160A1
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compounds
mmol
compound
2xch
antibacterial activity
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PCT/US2019/029338
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French (fr)
Inventor
Michael J. PEPI
Lizbeth K. Hedstrom
Deviprasad R. Gollapalli
Shibin CHACKO
Gyan MODI
Suresh Kumar Gorla
Gregory D. Cuny
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Brandeis University
University Of Houston System
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Publication of WO2019210160A1 publication Critical patent/WO2019210160A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D263/00—Heterocyclic compounds containing 1,3-oxazole or hydrogenated 1,3-oxazole rings
    • C07D263/52—Heterocyclic compounds containing 1,3-oxazole or hydrogenated 1,3-oxazole rings condensed with carbocyclic rings or ring systems
    • C07D263/54—Benzoxazoles; Hydrogenated benzoxazoles
    • C07D263/56—Benzoxazoles; Hydrogenated benzoxazoles with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached in position 2
    • C07D263/57—Aryl or substituted aryl radicals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D209/00—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D209/02—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom condensed with one carbocyclic ring
    • C07D209/04—Indoles; Hydrogenated indoles
    • C07D209/08—Indoles; Hydrogenated indoles with only hydrogen atoms or radicals containing only hydrogen and carbon atoms, directly attached to carbon atoms of the hetero ring
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
    • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
    • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D213/24—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with substituted hydrocarbon radicals attached to ring carbon atoms
    • C07D213/36—Radicals substituted by singly-bound nitrogen atoms
    • C07D213/40—Acylated substituent nitrogen atom
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D231/00—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings
    • C07D231/02—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings not condensed with other rings
    • C07D231/10—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
    • C07D231/12—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached to ring carbon atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D277/00—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings
    • C07D277/02—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings
    • C07D277/20—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
    • C07D277/22—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached to ring carbon atoms
    • C07D277/28—Radicals substituted by nitrogen atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D277/00—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings
    • C07D277/02—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings
    • C07D277/20—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
    • C07D277/32—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D277/56—Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D295/00—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms
    • C07D295/16—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms acylated on ring nitrogen atoms
    • C07D295/18—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms acylated on ring nitrogen atoms by radicals derived from carboxylic acids, or sulfur or nitrogen analogues thereof
    • C07D295/182—Radicals derived from carboxylic acids
    • C07D295/192—Radicals derived from carboxylic acids from aromatic carboxylic acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D413/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms
    • C07D413/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings
    • C07D413/04—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings directly linked by a ring-member-to-ring-member bond
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D413/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms
    • C07D413/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings
    • C07D413/10—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings linked by a carbon chain containing aromatic rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D413/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms
    • C07D413/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings
    • C07D413/12—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings linked by a chain containing hetero atoms as chain links

Definitions

  • Tuberculosis is a global health threat. Each year there are more than 10 million new cases of tuberculosis TB which have led to more than 1 million deaths. Approximately one-third of the world’s population is infected with the causative agent Mycobacterium tuberculosis ( Mtb ), many with latent infections that can recrudesce if the patient becomes immunosuppressed.
  • Mtb Mycobacterium tuberculosis
  • Mtb can be free living or intracellular in vivo, and bacteria also occupy diverse environments that vary in pH, nutrient and oxygen availability as well as drug accessibility. Different metabolic pathways are vulnerable in each growth state. In addition, a population of bacteria exists in a quiescent state that is phenotypically resistant to many antibiotics. Several different antibiotics are required to eradicate bacteria from these diverse
  • Treatment typically requires administration with isoniazid, rifampicin pyrazinamide and ethambutol, but almost 0.5 million cases involve Mtb strains that are resistant to isoniazid and rifampicin. Extensively drug resistant strains have also emerged that are resistant to the second line drugs fluoroquinolones and at least one of capreomycin, kanamycin and amikacin.
  • One aspect of the present invention relates to compounds and pharmaceutically acceptable salts thereof, which are useful as inhibitors of IMPDH. Further, the invention provides pharmaceutical compositions comprising one or more compounds of the invention. The invention also relates to methods of treating various bacterial infections in a subject.
  • Fig. 1A and Fig. IB show the correlation between antibacterial activity and enzyme inhibition for Q series compounds. Values from Table 6 and Makowska-Grzyska et ah, PLoS ONE 10, e0l38976. Mtb H37Rv cultured in GAST medium (Fig. 1A) and 7H9 medium
  • Figs. 2A-2D are plots showing that knockdown of MtbIMPDH2 hypersensitizes Mtb to Q compounds. Regulated expression of guaB2 is achieved in a TET-OFF system.
  • ATc anhydrotetracy cline
  • Fig. 3 is a plot showing the cytotoxicity of select Q compounds in HepG2 cells after
  • Fig. 4 is a plot showing the depletion of MtbIMPDH2 had no effect on the antibacterial activity of Ql 12. Regulated expression of guaB2 is achieved in a TET-OFF system as described in Fig. 2. Addition of anhydrotetracycline (ATc) represses expression of guaB2, decreasing the level of MtbIMPDH2 within the bacteria. ATc concentrations (ng/mL) are 0 (dark orange), 0.08 (dark blue), 0.15 (green), 0.31 (sky blue), 0.62 (yellow), 1.25 (gray), 2.5 (orange) and 5 (royal blue).
  • Fig. 5 is a bar graph showing the antibacterial activity of Ql 12 in macrophages.
  • Fig. 6 depicts certain A and P compounds with antibacterial activity against S.
  • Fig. 7 is a plot of K ⁇ data for select A and P compounds showing that the structure activity relationship for these compounds in 7GIMPDH and V/IMPDH are similar.
  • the black line denotes equal values of /ri.app for both enzymes.
  • the dotted lines denote 2-fold differences.
  • Fig. 8A and Fig. 8B are plots showing physicochemical properties of enzyme inhibition and antibacterial activity. P compounds are denoted with circles, A compounds with squares.
  • Fig. 9A and Fig. 9B are plots showing the cytotoxicity of select A and P compounds in HepG2 cells after 24 h.
  • the invention provides substituted ureas (P series compounds) and substituted benzoxazoles (Q series compounds), and pharmaceutical compositions thereof.
  • P series compounds substituted ureas
  • Q series compounds substituted benzoxazoles
  • such compounds disclosed herein are useful as inhibitors of inosine 5'- monophosphate dehydrogenase (IMPDH).
  • IMPDH inosine 5'- monophosphate dehydrogenase
  • IMPDH catalyzes the NAD + -dependent conversion of inosine 5 '-monophosphate (IMP) to xanthosine 5 '-monophosphate (XMP). This reaction is the first and the rate limiting step in the de novo biosynthesis of guanine nucleotides and therefore controls the size of the guanine nucleotide pool.
  • IMPDH is an attractive target for the development of new antibiotics due to the essential role of guanine nucleotides in DNA and RNA synthesis, signal transduction, energy transfer, glycoprotein biosynthesis and many other processes involved in cell proliferation.
  • Mtb has three genes, designated as guaBJ guaB2 and guaB3 , which encode IMPDH homologs.
  • guaB2 Only guaB2 is essential, and only guaB2 encodes an active IMPDH ( / MPDH2).
  • the proteins encoded by guaBl and guaB3 are each missing key catalytic residues and neither display IMPDH activity. The functions of these proteins are currently unknown.
  • the compounds disclosed herein exploit the highly diverged cofactor binding site, which explains their selectivity for bacterial over eukaryotic orthologs of IMPDH, including / MPDH2.
  • the compounds disclosed herein can be used as antibacterial agents, particular with respect to treating Mycobacterium tuberculosis bacterial infections.
  • Exemplary compounds disclosed herein are depicted in Tables 1 and 2.
  • the compounds of Table 1 and 2 may be depicted as the free base or the conjugate acid.
  • compounds of the invention may be prodrugs of the compounds of Table 1 and 2, e.g., wherein a hydroxyl in the parent compound is presented as an ester or a carbonate, or carboxylic acid present in the parent compound is presented as an ester.
  • the prodrug is metabolized to the active parent compound in vivo (e.g., the ester is hydrolyzed to the corresponding hydroxyl, or carboxylic acid).
  • compounds of the invention may be racemic. In certain embodiments, compounds of the invention may be enriched in one enantiomer. For example, a compound of the invention may have greater than 30% ee, 40% ee, 50% ee, 60% ee, 70% ee, 80% ee, 90% ee, or even 95% or greater ee. The compounds of the invention have more than one stereocenter. Consequently, compounds of the invention may be enriched in one or more diastereomer. For example, a compound of the invention may have greater than 30% de, 40% de, 50% de, 60% de, 70% de, 80% de, 90% de, or even 95% or greater de.
  • the compound is not P177, P188, P221, P226, or P227.
  • the invention is a method of killing or inhibiting the growth of a microbe, comprising the step of contacting said microbe with an effective amount of a compound disclosed herein (e.g., a compound of Table 1 or Table 2), or a pharmaceutically acceptable salt thereof.
  • a compound disclosed herein e.g., a compound of Table 1 or Table 2
  • the method may be a method of treating or preventing a microbial infection in a mammal, comprising the step of administering to a mammal in need thereof a therapeutically effective amount of a compound disclosed herein (e.g., a compound of Table 1 or Table 2), or a pharmaceutically acceptable salt thereof.
  • the method may be a method of treating or preventing a parasitic infection in a mammal, comprising the step of
  • a compound disclosed herein e.g., a compound of Table 1 or Table 2
  • a pharmaceutically acceptable salt thereof e.g., a compound of Table 1 or Table 2
  • provided herein are methods for treating a bacterial infection in a subject by administering to the subject an effective amount of a compound disclosed herein (e.g., compounds of Table 1 and 2) to thereby treat the infection. Also provided herein are methods for ameliorating the signs or symptoms of an infection in a subject by bacterial cells, the method comprising administering to the subject an effective amount of a compound disclosed herein (e.g., compounds of Table 1 and 2) to thereby ameliorate the signs and symptoms of the infection.
  • a compound disclosed herein e.g., compounds of Table 1 and 2
  • provided herein are methods for preventing or reducing the likelihood of a productive bacterial infection in a subject by administering to a subject an effective amount of a compound disclosed herein (e.g., compounds of Table 1 and 2) to thereby prevent or reduce the likelihood of a productive bacterial infection in the subject.
  • a compound disclosed herein e.g., compounds of Table 1 and 2
  • the bacteria or bacterial cells are Mycobacterium.
  • the Mycobacterium may be Mycobacterium tuberculosis.
  • the Mycobacterium may be any Mycobacterium species that causes tuberculosis or belongs to the Mycobacterium
  • tuberculosis complex Species in this complex include M. africanum M. bovis, M. canetti ,
  • M. caprae M. microti , M. mungi , M. orygis , M. pinnipedii , M. suricattae and M.
  • the Mycobacterium may be any other species of Mycobacterium (e.g., any species disclosed herein), such as nontuberculous mycobacteria.
  • Nontuberculous mycobacteria are commonly present in soil and water and are usually much less virulent in humans than is Mycobacterium tuberculosis. Infections with these organisms have been called atypical, environmental, and nontuberculous mycobacterial infections. Exposures and infections by these organisms often require a defect in local or systemic host defenses; the frail elderly and immunocompromised people are at the highest risk.
  • M. avium complex (MAC) including the closely related species of M. avium and M. intracellulare , accounts for many of nontuberculosis mycobacterial diseases.
  • Other causative species include M kansasii, M. xenopi, M. marinum, M. ulcer ans, M. fortuitum, M. abscessus , andM chelonae.
  • Mycobacertium include M. asiaticum, M. gordonae, M. gastri,
  • M. kansasii M. hiberniae, M. icosiumassiliensis, M. nonchromogenicum, M. terrae, M. triviale, M. ulcerans , M. pseudoshottsii, M. shottsii, M. florentinum, M. genavense, M.
  • aquaticum M. bacteremicum, M. bohemicum, M. botniense, M. branderi, M. celatum, M. chimaera, M. conspicuum, M. cookii, M. doricum, M. farcinogenes, M. haemophilum, M. homeeshornense, M. intracellulare, M. lacus, M. leprae , M lepraemurium, M. lepromatosis , M liflandii, M. llatzerense, M. malmoense, M. marinum , causes a rare disease
  • brisbanense M. cosmeticum, M. fortuitum, M. fortuitum subsp. acetamidolyticum, M.
  • chubuense M. conceptionense, M. confluentis, M. duvalii, M. elephantis, M. flavescens, M. gadium, M. gilvum, M. hassiacum, M. holsaticum, M. iranicum, M. komossense, M.
  • madagascariense M. massilipolynesiensis, M. moriokaense, M. obuense, M. phlei, M.
  • M. psychrotolerans M. pulveris, M. pyrenivorans, M. smegmatis, M. goodii, M. wolinskyi, M. sphagni, M. thermoresistibile, M. vanbaalenii, M. arosiense, M. aubagnense, M.
  • chlorophenolicum M. fluoroanthenivorans, M. kumamotonense, M. novocastrense,
  • the bacteria or bacterial cells are from the genera
  • the invention relates to any one of the aforementioned methods, wherein said microbial infection is caused by a protozoan or bacterium.
  • the invention relates to any one of the aforementioned methods, wherein said microbial infection is caused by a protozoan or a bacterium selected from the group consisting of the genera Cryptosporidium, Entamoeba, Leishmania ,
  • Burkholderia Burkholderia, Brachyspira, Campylobacter, Clostridia , Coxiella , Enterococcus ,
  • provided herein are method of treating tuberculosis in a subject, the method comprising administering to the subject an effective amount of a compound disclosed herein (e.g., compounds of Table 1 and 2) to thereby treat the tuberculosis.
  • a compound disclosed herein e.g., compounds of Table 1 and 2
  • the subject is a mammal, e.g., a human.
  • the invention relates to a method of treating or preventing a bacterial infection in a mammal, comprising the step of co-administering to a mammal in need thereof: (a) a first amount of a compound of Table 1 or Table 2, or a pharmaceutically acceptable salt thereof; and (b) a second amount of an antibacterial agent, wherein, taken together, the first amount and the second amount are effective.
  • the invention relates to a method of treating a recurrent or persistent bacterial infection in a mammal, comprising the step of co-administering to a mammal in need thereof: (a) a first amount of a compound of Table 1 or Table 2, or a pharmaceutically acceptable salt thereof; and (b) a second amount of an antibacterial agent, wherein, taken together, the first amount and the second amount are therapeutically effective.
  • the invention relates to a method of preventing the recurrence of a bacterial infection in a mammal, comprising the step of co-administering to a mammal in need thereof: (a) a first amount of a compound of Table 1 or Table 2, or a
  • the antibacterial agent is an anti-translation agent, for example, an agent that interferes with bacterial protein synthesis, for example, by interacting with prokaryotic ribosomes.
  • the antibacterial agent inhibits DNA gyrase or a topoisomerase in a bacterium.
  • the antibacterial agent interferes with the transpeptidation reaction, thereby inhibits synthesis of peptidoglycan, a component of bacterial cell walls.
  • the antibacterial agent is a penicillin.
  • the antibacterial agent is a tetracycline. In certain embodiments, the antibacterial agent is a cephalosporin. In certain embodiments, the antibacterial agent is a quinolone, such as a fluoroquinolone. In certain embodiments, the antibacterial agent is a linomycin. In certain embodiments, the antibacterial agent is a macrolide. In certain embodiments, the antibacterial agent is a sulfonamide. In certain embodiments, the antibacterial agent is a glycopeptide. In certain embodiments, the antibacterial agent is an aminoglycoside. In certain embodiments, the antibacterial agent is a carbapenem.
  • the invention relates to any one of the compositions described herein, further comprising an antibacterial agent selected from the group consisting of amikacin, amoxicillin, azithromycin, cefdinir, ceftriaxone, cefuroxime, cephalexin, ciprofloxacin, clarithromycin, clindamycin, co-trimoxazole, dalbavancin, doripenem, doxycycline, ertapenem, erythromycin, gentamicin, imipenem/cilastatin, kanamycin, levofloxacin, lincomycin, meropenem, metronidazole, minocycline,
  • an antibacterial agent selected from the group consisting of amikacin, amoxicillin, azithromycin, cefdinir, ceftriaxone, cefuroxime, cephalexin, ciprofloxacin, clarithromycin, clindamycin, co-trimoxazole,
  • moxifloxacin moxifloxacin, ofloxacin, oritavancin, oxacillin, penicillin, sulfamethoxazole, sulfasalazine, sulfisoxazole, telavancin, tetracycline, tobramycin, trimethoprim, and vancomycin.
  • the mammal prior to co-administration of (a) and (b), the mammal had been administered a first antibacterial agent.
  • the first antibacterial agent may be selected from the group consisting of amikacin, amoxicillin, azithromycin, cefdinir, ceftriaxone, cefuroxime, cephalexin, ciprofloxacin, clarithromycin, clindamycin, co- trimoxazole, dalbavancin, doripenem, doxycycline, ertapenem, erythromycin, gentamicin, imipenem/cilastatin, kanamycin, levofloxacin, lincomycin, meropenem, metronidazole, minocycline, moxifloxacin, ofloxacin, oritavancin, oxacillin, penicillin, sulfamethoxazole, sulfasalazine, sulfisoxazole
  • the present invention provides pharmaceutical compositions comprising a compound of Table 1 or 2 and a pharmaceutically acceptable carrier.
  • the present invention provides pharmaceutical compositions comprising a compound of Table 1 or 2, an antibacterial agent, and a pharmaceutically acceptable carrier.
  • the antibacterial agent is an anti-translation agent, for example, an agent that interferes with bacterial protein synthesis, for example, by interacting with prokaryotic ribosomes.
  • the antibacterial agent inhibits DNA gyrase or a topoisomerase in a bacterium.
  • the antibacterial agent interferes with the transpeptidation reaction, thereby inhibits synthesis of peptidoglycan, a component of bacterial cell walls.
  • the antibacterial agent is a penicillin.
  • the antibacterial agent is a tetracycline. In certain embodiments, the antibacterial agent is a cephalosporin. In certain embodiments, the antibacterial agent is a quinolone, such as a fluoroquinolone. In certain embodiments, the antibacterial agent is a linomycin. In certain embodiments, the antibacterial agent is a macrolide. In certain embodiments, the antibacterial agent is a sulfonamide. In certain embodiments, the antibacterial agent is a glycopeptide. In certain embodiments, the antibacterial agent is an aminoglycoside. In certain embodiments, the antibacterial agent is a carbapenem.
  • the invention relates to any one of the compositions described herein, further comprising an antibacterial agent selected from the group consisting of amikacin, amoxicillin, azithromycin, cefdinir, ceftriaxone, cefuroxime, cephalexin, ciprofloxacin, clarithromycin, clindamycin, co-trimoxazole, dalbavancin, doripenem, doxycycline, ertapenem, erythromycin, gentamicin, imipenem/cilastatin, kanamycin, levofloxacin, lincomycin, meropenem, metronidazole, minocycline,
  • an antibacterial agent selected from the group consisting of amikacin, amoxicillin, azithromycin, cefdinir, ceftriaxone, cefuroxime, cephalexin, ciprofloxacin, clarithromycin, clindamycin, co-trimoxazole,
  • moxifloxacin moxifloxacin, ofloxacin, oritavancin, oxacillin, penicillin, sulfamethoxazole, sulfasalazine, sulfisoxazole, telavancin, tetracycline, tobramycin, trimethoprim, and vancomycin.
  • compositions and methods of the present invention may be utilized to treat an individual in need thereof.
  • the individual is a mammal such as a human, or a non-human mammal.
  • the composition or the compound is preferably administered as a pharmaceutical composition comprising, for example, a compound of the invention and a pharmaceutically acceptable carrier.
  • compositions include, for example, aqueous solutions such as water or physiologically buffered saline or other solvents or vehicles such as glycols, glycerol, oils such as olive oil, or injectable organic esters.
  • aqueous solutions such as water or physiologically buffered saline or other solvents or vehicles such as glycols, glycerol, oils such as olive oil, or injectable organic esters.
  • the aqueous solution is pyrogen-free, or substantially pyrogen-free.
  • the excipients can be chosen, for example, to effect delayed release of an agent or to selectively target one or more cells, tissues or organs.
  • the pharmaceutical composition can be in dosage unit form such as tablet, capsule (including sprinkle capsule and gelatin capsule), granule, lyophile for reconstitution, powder, solution, syrup, suppository, injection or the like.
  • the composition can also be present in a transdermal delivery system, e.g., a skin patch.
  • the composition can also be present in a solution suitable for topical administration, such as an eye drop.
  • a pharmaceutically acceptable carrier can contain physiologically acceptable agents that act, for example, to stabilize, increase solubility or to increase the absorption of a compound such as a compound of the invention.
  • physiologically acceptable agents include, for example, carbohydrates, such as glucose, sucrose or dextrans, antioxidants, such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins or other stabilizers or excipients.
  • the choice of a pharmaceutically acceptable carrier, including a physiologically acceptable agent depends, for example, on the route of administration of the composition.
  • the preparation or pharmaceutical composition can be a self-emulsifying drug delivery system or a self-microemulsifying drug delivery system.
  • the pharmaceutical composition also can be a liposome or other polymer matrix, which can have incorporated therein, for example, a compound of the invention.
  • Liposomes for example, which comprise phospholipids or other lipids, are nontoxic, physiologically acceptable and metabolizable carriers that are relatively simple to make and administer.
  • phrases "pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • pharmaceutically acceptable carrier means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient.
  • materials which can serve as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide;
  • a pharmaceutical composition can be administered to a subject by any of a number of routes of administration including, for example, orally (for example, drenches as in aqueous or non-aqueous solutions or suspensions, tablets, capsules (including sprinkle capsules and gelatin capsules), boluses, powders, granules, pastes for application to the tongue); absorption through the oral mucosa (e.g., sublingually); anally, rectally or vaginally (for example, as a pessary, cream or foam); parenterally (including intramuscularly, intravenously, subcutaneously or intrathecally as, for example, a sterile solution or suspension); nasally; intraperitoneally; subcutaneously; transdermally (for example as a patch applied to the skin); and topically (for example, as a cream, ointment or spray applied to the skin, or as an eye drop).
  • routes of administration including, for example, orally (for example, drenches as in aqueous or
  • the compound may also be formulated for inhalation.
  • a compound may be simply dissolved or suspended in sterile water. Details of appropriate routes of administration and compositions suitable for same can be found in, for example, U.S. Pat. Nos. 6,110,973, 5,731,000, 5,541,231, 5,427,798, 5,358,970 and 4,172,896, as well as in patents cited therein.
  • the formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy.
  • the amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated, the particular mode of administration.
  • the amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect. Generally, out of one hundred percent, this amount will range from about 1 percent to about ninety-nine percent of active ingredient, preferably from about 5 percent to about 70 percent, most preferably from about 10 percent to about 30 percent.
  • Methods of preparing these formulations or compositions include the step of bringing into association an active compound, such as a compound of the invention, with the carrier and, optionally, one or more accessory ingredients.
  • an active compound such as a compound of the invention
  • the formulations are prepared by uniformly and intimately bringing into association a compound of the present invention with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.
  • Formulations of the invention suitable for oral administration may be in the form of capsules (including sprinkle capsules and gelatin capsules), cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), lyophile, powders, granules, or as a solution or a suspension in an aqueous or non-aqueous liquid, or as an oil- in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and/or as mouth washes and the like, each containing a predetermined amount of a compound of the present invention as an active ingredient.
  • Compositions or compounds may also be administered as a bolus, electuary or paste.
  • the active ingredient is mixed with one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate, and/or any of the following: (1) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents,
  • pharmaceutically acceptable carriers such as sodium citrate or dicalcium phosphate, and/or any of the following: (1) fillers or extenders, such as starches, lactose
  • compositions may also comprise buffering agents.
  • Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like.
  • a tablet may be made by compression or molding, optionally with one or more accessory ingredients.
  • Compressed tablets may be prepared using binder (for example, gelatin or hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agent.
  • Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.
  • the tablets, and other solid dosage forms of the pharmaceutical compositions may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical-formulating art. They may also be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile, other polymer matrices, liposomes and/or microspheres.
  • compositions may be sterilized by, for example, filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions that can be dissolved in sterile water, or some other sterile injectable medium immediately before use.
  • These compositions may also optionally contain opacifying agents and may be of a composition that they release the active ingredient(s) only, or preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner.
  • embedding compositions that can be used include polymeric substances and waxes.
  • the active ingredient can also be in micro- encapsulated form, if appropriate, with one or more of the above-described excipients.
  • Liquid dosage forms useful for oral administration include pharmaceutically acceptable emulsions, lyophiles for reconstitution, microemulsions, solutions, suspensions, syrups and elixirs.
  • the liquid dosage forms may contain inert diluents commonly used in the art, such as, for example, water or other solvents, cyclodextrins and derivatives thereof, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1, 3-butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.
  • inert diluents commonly used in the art, such as
  • the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.
  • adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.
  • Suspensions in addition to the active compounds, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.
  • suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.
  • Formulations of the pharmaceutical compositions for rectal, vaginal, or urethral administration may be presented as a suppository, which may be prepared by mixing one or more active compounds with one or more suitable nonirritating excipients or carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax or a salicylate, and which is solid at room temperature, but liquid at body temperature and, therefore, will melt in the rectum or vaginal cavity and release the active compound.
  • suitable nonirritating excipients or carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax or a salicylate, and which is solid at room temperature, but liquid at body temperature and, therefore, will melt in the rectum or vaginal cavity and release the active compound.
  • Formulations of the pharmaceutical compositions for administration to the mouth may be presented as a mouthwash, or an oral spray, or an oral ointment.
  • compositions can be formulated for delivery via a catheter, stent, wire, or other intraluminal device. Delivery via such devices may be especially useful for delivery to the bladder, urethra, ureter, rectum, or intestine.
  • Formulations which are suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams or spray formulations containing such carriers as are known in the art to be appropriate.
  • Dosage forms for the topical or transdermal administration include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants.
  • the active compound may be mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants that may be required.
  • the ointments, pastes, creams and gels may contain, in addition to an active compound, excipients, such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.
  • Powders and sprays can contain, in addition to an active compound, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances.
  • Sprays can additionally contain customary propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane.
  • Transdermal patches have the added advantage of providing controlled delivery of a compound of the present invention to the body.
  • dosage forms can be made by dissolving or dispersing the active compound in the proper medium.
  • Absorption enhancers can also be used to increase the flux of the compound across the skin. The rate of such flux can be controlled by either providing a rate controlling membrane or dispersing the compound in a polymer matrix or gel.
  • Ophthalmic formulations eye ointments, powders, solutions and the like, are also contemplated as being within the scope of this invention.
  • Exemplary ophthalmic materials are also contemplated as being within the scope of this invention.
  • liquid ophthalmic formulations have properties similar to that of lacrimal fluids, aqueous humor or vitreous humor or are compatible with such fluids.
  • a preferred route of administration is local administration (e.g ., topical administration, such as eye drops, or administration via an implant).
  • parenteral administration and “administered parenterally” as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion.
  • compositions suitable for parenteral administration comprise one or more active compounds in combination with one or more pharmaceutically acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.
  • aqueous and nonaqueous carriers examples include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate.
  • polyols such as glycerol, propylene glycol, polyethylene glycol, and the like
  • vegetable oils such as olive oil
  • injectable organic esters such as ethyl oleate.
  • Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.
  • compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents that delay absorption such as aluminum monostearate and gelatin.
  • a liquid suspension of crystalline or amorphous material having poor water solubility The rate of absorption of the drug then depends upon its rate of dissolution, which, in turn, may depend upon crystal size and crystalline form.
  • delayed absorption of a parenterally administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle.
  • Injectable depot forms are made by forming microencapsulated matrices of the subject compounds in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of drug to polymer, and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly (anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions that are compatible with body tissue.
  • active compounds can be given per se or as a pharmaceutical composition containing, for example, 0.1 to 99.5% (more preferably, 0.5 to 90%) of active ingredient in combination with a pharmaceutically acceptable carrier.
  • Methods of introduction may also be provided by rechargeable or biodegradable devices.
  • Various slow release polymeric devices have been developed and tested in vivo in recent years for the controlled delivery of drugs, including proteinacious biopharmaceuticals.
  • a variety of biocompatible polymers including hydrogels, including both biodegradable and non-degradable polymers, can be used to form an implant for the sustained release of a compound at a particular target site.
  • Actual dosage levels of the active ingredients in the pharmaceutical compositions may be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.
  • the selected dosage level will depend upon a variety of factors including the activity of the particular compound or combination of compounds employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound(s) being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compound(s) employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.
  • a physician or veterinarian having ordinary skill in the art can readily determine and prescribe the therapeutically effective amount of the pharmaceutical composition required.
  • the physician or veterinarian could start doses of the pharmaceutical composition or compound at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.
  • therapeutically effective amount is meant the concentration of a compound that is sufficient to elicit the desired therapeutic effect. It is generally understood that the effective amount of the compound will vary according to the weight, sex, age, and medical history of the subject. Other factors which influence the effective amount may include, but are not limited to, the severity of the patient's condition, the disorder being treated, the stability of the compound, and, if desired, another type of therapeutic agent being administered with the compound of the invention.
  • a larger total dose can be delivered by multiple administrations of the agent.
  • Methods to determine efficacy and dosage are known to those skilled in the art (Isselbacher et al. (1996) Harrison’s Principles of Internal Medicine 13 ed., 1814-1882, herein incorporated by reference).
  • a suitable daily dose of an active compound used in the compositions and methods of the invention will be that amount of the compound that is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above.
  • the effective daily dose of the active compound may be administered as one, two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms.
  • the active compound may be administered two or three times daily. In preferred embodiments, the active compound will be administered once daily.
  • the patient receiving this treatment is any animal in need, including primates, in particular humans, and other mammals such as equines, cattle, swine and sheep; and poultry and pets in general.
  • compounds of the invention may be used alone or conjointly administered with another type of therapeutic agent.
  • the phrase“conjoint administration” refers to any form of administration of two or more different therapeutic compounds such that the second compound is administered while the previously
  • the administered therapeutic compound is still effective in the body (e.g ., the two compounds are simultaneously effective in the patient, which may include synergistic effects of the two compounds).
  • the different therapeutic compounds can be administered either in the same formulation or in a separate formulation, either concomitantly or sequentially.
  • the different therapeutic compounds can be administered within one hour, 12 hours, 24 hours, 36 hours, 48 hours, 72 hours, or a week of one another.
  • an individual who receives such treatment can benefit from a combined effect of different therapeutic compounds.
  • conjoint administration of compounds of the invention with one or more additional therapeutic agent(s) provides improved efficacy relative to each individual administration of the compound of the invention (e.g., compounds of Table 1 and 2) or the one or more additional therapeutic agent(s).
  • the conjoint administration provides an additive effect, wherein an additive effect refers to the sum of each of the effects of individual administration of the compound of the invention and the one or more additional therapeutic agent(s).
  • This invention includes the use of pharmaceutically acceptable salts of compounds of the invention in the compositions and methods of the present invention.
  • “pharmaceutically acceptable salt” as used herein includes salts derived from inorganic or organic acids including, for example, hydrochloric, hydrobromic, sulfuric, nitric, perchloric, phosphoric, formic, acetic, lactic, maleic, fumaric, succinic, tartaric, glycolic, salicylic, citric, methanesulfonic, benzenesulfonic, benzoic, malonic, trifluoroacetic, trichloroacetic, naphthalene-2-sulfonic, and other acids.
  • Pharmaceutically acceptable salt forms can include forms wherein the ratio of molecules comprising the salt is not 1 :1.
  • the salt may comprise more than one inorganic or organic acid molecule per molecule of base, such as two hydrochloric acid molecules per molecule of compounds of Table 1 and 2.
  • the salt may comprise less than one inorganic or organic acid molecule per molecule of base, such as two molecules of compounds of Table 1 and 2 per molecule of tartaric acid.
  • contemplated salts of the invention include, but are not limited to, alkyl, dialkyl, trialkyl or tetra-alkyl ammonium salts.
  • contemplated salts of the invention include, but are not limited to, L-arginine, benenthamine, benzathine, betaine, calcium hydroxide, choline, deanol, diethanolamine, diethylamine, 2- (diethylamino)ethanol, ethanolamine, ethylenediamine, N-methylglucamine, hydrabamine, lH-imidazole, lithium, L-lysine, magnesium, 4-(2-hydroxyethyl)morpholine, piperazine, potassium, 1 -(2-hydroxy ethyl)pyrrolidine, sodium, triethanolamine, tromethamine, and zinc salts.
  • contemplated salts of the invention include, but are not limited to, Na, Ca, K, Mg, Zn or other metal salts
  • the pharmaceutically acceptable acid addition salts can also exist as various solvates, such as with water, methanol, ethanol, dimethylformamide, and the like. Mixtures of such solvates can also be prepared.
  • the source of such solvate can be from the solvent of crystallization, inherent in the solvent of preparation or crystallization, or adventitious to such solvent.
  • wetting agents such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.
  • antioxidants examples include: (1) water-soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabi sulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal-chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.
  • water-soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabi sulfite, sodium sulfite and the like
  • oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (
  • a reference to“A and/or B”, when used in conjunction with open-ended language such as“comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.
  • “or” should be understood to have the same meaning as“and/or” as defined above.
  • “or” or“and/or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as“only one of’ or“exactly one of,” or, when used in the claims,“consisting of,” will refer to the inclusion of exactly one element of a number or list of elements.
  • the phrase“at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements.
  • This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase“at least one” refers, whether related or unrelated to those elements specifically identified.
  • “at least one of A and B” or, equivalently,“at least one of A or B,” or,
  • equivalently“at least one of A and/or B”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.
  • compositions of the present invention may exist in particular geometric or stereoisomeric forms.
  • the present invention contemplates all such compounds, including cis- and trans-isomers, R- and //-enantiomers, diastereomers, (D)- isomers, (L)-isomers, the racemic mixtures thereof, and other mixtures thereof, as falling within the scope of the invention. All such isomers, as well as mixtures thereof, are intended to be included in this invention.
  • a particular enantiomer of compound of the present invention may be prepared by asymmetric synthesis, or by derivation with a chiral auxiliary, where the resulting diastereomeric mixture is separated and the auxiliary group cleaved to provide the pure desired enantiomers.
  • the molecule contains a basic functional group, such as amino, or an acidic functional group, such as carboxyl,
  • diastereomeric salts are formed with an appropriate optically-active acid or base, followed by resolution of the diastereomers thus formed by fractional crystallization or chromatographic means well known in the art, and subsequent recovery of the pure enantiomers.
  • a therapeutic that“prevents” a disorder or condition refers to a compound that, in a statistical sample, reduces the occurrence of the disorder or condition in the treated sample relative to an untreated control sample, or delays the onset or reduces the severity of one or more symptoms of the disorder or condition relative to the untreated control sample.
  • prophylactic and/or therapeutic treatments includes prophylactic and/or therapeutic treatments.
  • prophylactic or therapeutic treatment is art-recognized and includes administration to the host of one or more of the subject compositions. If it is administered prior to clinical manifestation of the unwanted condition (e.g., disease or other unwanted state of the host animal) then the treatment is prophylactic (i.e., it protects the host against developing the unwanted condition), whereas if it is administered after manifestation of the unwanted condition, the treatment is therapeutic, (i.e., it is intended to diminish, ameliorate, or stabilize the existing unwanted condition or side effects thereof).
  • the unwanted condition e.g., disease or other unwanted state of the host animal
  • A“recurrent” or“persistent” bacterial infection results from certain bacterial pathogens that are able to evade the host immune system and persist within the human host.
  • the consequences of persistent bacterial infections potentially include increased morbidity and mortality from the infection itself as well as an increased risk of dissemination of disease. Eradication of persistent infections is difficult, often requiring prolonged or repeated courses of antibiotics.
  • a population or subpopulation of bacteria exists that is refractory to traditional antibiotics, possibly in a non-replicating or
  • metabolically altered (“quiescent”) state As used herein,“preventing the recurrence of’ an infection describes eradication of the agent that caused the original infection. The efficacy of preventing the recurrence of the infection is determined by a successful clinical outcome and does not require 100% elimination of the microorganisms involved in the infection.
  • prodrug is intended to encompass compounds which, under physiologic conditions, are converted into the therapeutically active agents of the present invention (e.g., compounds of Table 1 and 2).
  • a common method for making a prodrug is to include one or more selected moieties which are hydrolyzed under physiologic conditions to reveal the desired molecule.
  • the prodrug is converted by an enzymatic activity of the host animal.
  • esters or carbonates e.g., esters or carbonates of alcohols or carboxylic acids
  • some or all of the compounds of Table 1 and 2 in a formulation represented above can be replaced with the corresponding suitable prodrug, e.g., wherein a hydroxyl in the parent compound is presented as an ester or a carbonate or carboxylic acid present in the parent compound is presented as an ester.
  • Arylbenzoxazoles 4 were directly synthesized by oxidative cyclization methods using 2-amino-4-nitrophenol and aldehydes in the presence of activated carbon (Darco KB) under an oxygen atmosphere. The nitro group was reduced using Pd/C under 1 atm hydrogen to give 5-amine-2-arylbenzoxazoles 5.
  • Enantiomerically pure phenyl ethers 6 were synthesized from (+)-methyl D-lactate and the corresponding phenol using Mitsunobu reaction conditions.
  • Reagents and conditions (a) ArCHO, DarcoKB, O2 (1 atm), xylene 140 °C, 6-8 h, 70-80%; (b) 10% Pd/C, H 2 (1 atm), EtOAc:MeOH, 3 h, 85-94%; (c) PPh 3 , DEAD, (+)-methyl D- lactate, THF, 0 °C to rt, 4 h, 85-90%, (d) Li OH, THF :MeOH, 4 h, 86-91%; (e) 5, EDC HC1, DMF, 12 h, 62-79%.
  • Scheme 2 depicts the functional group transformations to various substituents attached to the benzoxazole aryl group.
  • Compounds 8i and 8j were subjected to alkylation reactions with ethyl 2-bromoacetate in the presence of K2CO3 and DMF to give 18a and 18b, respectively. These compounds were hydrolyzed using LiOH in THF:MeOH (1 :3) to yield 19a and 19b, respectively.
  • Carboxylic acid 19a was also converted to ester 20 using propargyl bromide and K2CO3 in dry DMF.
  • Ester 8h was also converted to an array of derivatives. For example, it was treated with hydroxylamine hydrochloride to provide the corresponding hydroxamic acid 21.
  • Reagents and conditions (a) BrCH 2 COOEt, K2CO3, DMF, rt, 6 h, 80-83%; (b) LiOH, THF:MeOH, rt, 86-88%; (c) NH2OH ⁇ O, KOH, MeOH, rt, 12 h, 65%; (d) NaOH,
  • Scheme 3 illustrates the synthesis of an array of derivatives via functional group transformation of 8f.
  • 8f was treated with hydroxylamine hydrochloride in absolute ethanol in the presence of triethylamine to afford hydroxamidine 25, which upon treatment with CDI and DIPEA gave l,2,4-oxadiazolone 26.
  • the tetrazole derivative 27 was prepared by treatment of 8f with sodium azide in the presence of ammonium chloride in dry DMF.
  • Primary amide 28 was generated by treating 8f with /-BuOK in /-BuOH.
  • Reagents and conditions (a) NH2OH HCI, Et 3 N, EtOH, reflux, 4 h, 82%; (b) CDI, DIPEA, DMF, rt, 2 h, 61%; (c) NaN 3 , NFECl, DMF, 100 °C, 6 h, 62%; (d) /-BuOK, /-BuOH, 12 h, 54%; (e) 1. NiCh 6H2O, NaBH 4 , B0C2O, MeOH:THF, rt, 2 h, 2. TFA, DCM, rt, 4 h, 67%.
  • the synthesis of 2-pyridone derivative 30 was carried out by treating 8b with LiCl and p-toluenesulfonic acid in anhydrous methanol (Scheme 4).
  • the pyridyl of 8a was oxidized using w-CPBA in DCM to give the corresponding pyridine N-oxide 31.
  • the nitrile in 811 was reduced using NiCh ⁇ EEO and NaBFE then protected as the corresponding t-butyl carbamate. Removal of the protecting group using trifluoroacetic acid in DCM provided amine 32.
  • the benzyl ether of 8v was removed by hydrogenolysis in presence of Pd/C under a hydrogen atmosphere to yield 33.
  • Reagents and conditions (a) LiCl, p-TSA, DMF, 120 °C, 2 h, 77%; (b) w-CPBA, DCM, rt, 12 h, 74%; (c) NiCh H 2 0, NaBH 4 , and then B0C2O, THF, MeOH, rt, 4 h; (d) TFA, DCM, 68%; (e) 10% Pd/C, H2, MeOH:EtOAc, rt, 3 h, 90%.
  • reaction was quenched with water and extracted with ethyl acetate (20 mL) and organic layer was washed with brine. The organic layer was dried using MgS0 4 , filtered and concentrated under reduced pressure. The crude product was purified through column chromatography.
  • hydroxylamine hydrochloride (2 mmol) was added at 0 °C and stirred well.
  • KOH (3 mmol) was added to the cold solution and stirred overnight at room temperature. After completion, the solvent was removed under reduced pressure and the residue was dissolved in water and extracted with ethyl acetate. The organic layer was washed with brine and dried using anhydrous MgSCri. Solvent was removed under reduced pressure and the product was purified using column chromatography.
  • reaction mixture was stirred for 2 h at room temperature.
  • the reaction mixture was diluted with water and extracted with ethyl acetate.
  • the combined extract was washed with brine, dried using anhydrous MgSCri, filtered and concentrated.
  • the residue was purified through column chromatography (2-8% MeOH gradient in CH2CI2) to give the title compound.
  • reaction mixture was filtered through celite and the filtrate was concentrated under reduced pressure.
  • the crude residue was poured into 1N HC1 and extracted with ethyl acetate.
  • the organic layer was washed with brine and dried using anhydrous MgSCri.
  • Solvent was removed under reduced pressure and crude reaction mixture was purified through column chromatography.
  • Trifluoroacetic acid (1 mL) was added to the stirred solution of Boc protected amine (1 mmol) in dry DCM (5 mL) at 0 °C. After 4 h, the reaction was quenched with a saturated NaHCCh solution and extracted with DCM. The organic layer was washed with brine and dried using anhydrous MgSCh.
  • the cofactor binding sites are widely diverged in bacterial and eukaryotic IMPDHs, and the / MPDE12 inhibitors exploit this divergence. Only four compounds, 8f, 8p, 17a and 24, modestly inhibited human IMPDH2, and in these cases selectivity ranged from a factor of 200 to 1000 (Table 5). No inhibition of human IMPDH2 was observed for the other compounds (maximum concentration tested was 5 mM).
  • GMP reductase (GMPR) is closely related to IMPDH, and catalyzes a similar reaction, the reduction of GMP by NADPH to produce IMP, NADP + and ammonia.
  • the adenosine site of human GMPR2 contains the Ala- Tyr motif that characterizes the inhibitor binding site of / MPDH2.
  • none of the compounds inhibited human GMPR2 (maximum concentration tested was 5 mM).
  • Antibacterial activity was determined for A///4MPDH2ACBS inhibitors with /ri.app ⁇ 40 nM and selected additional compounds by monitoring the growth oiMtb H37Rv (ATCC 27294) after one week (Table 6). Since in vitro antibiotic efficacy can vary unpredictably with growth conditions, antibacterial activity was assessed in both GAST/Fe and
  • FIG. 1 shows the dependence of antibacterial activity on inhibition of
  • the failure of guanine to decrease the antibacterial activity of most Q compounds would usually suggest that antibacterial activity derives from the engagement of another target.
  • guanine salvage may not be sufficient to support growth in the presence of potent / MPDH2 inhibition.
  • the Q inhibitors stabilize/disrupt a protein complex, thereby perturbing a moonlighting activity of / MPDH2 in addition to enzyme activity.
  • SRMV2.6 This strain expresses the mutant / MPDH2/Y487C, which is resistant to an isoquinoline sulfonamide inhibitor.
  • Tyr487 interacts with the benzoxazole group, so the substitution of Cys is expected to disrupt the binding of all the Q inhibitors.
  • SRMV2.6 was resistant to 1 and 17b (Table 7), further confirming that the antibacterial activity of these compounds resulted from on-target inhibition of A///4MPDH2 Interestingly, however, SRMV2.6 remained sensitive to 18a and 22.
  • H37Rv is the wild-type strain b.
  • SRMV2.6 contains / MPDH2/Y487C, which confers resistance to the isoquinoline sulfonamide inhibitor VCC234718.
  • strain SRMV2.6 should also be resistant to 1 and 18a, yet it remained sensitive to these compounds. Perhaps 1 and 18a are concentrated or metabolized in the bacteria, or perhaps these compounds interfere with a moonlighting function.
  • these compounds may engage an additional target(s). Irrespective of the ambiguous mechanism of action of 1 and 18a, 17b demonstrates that on-target inhibition of / MPDH2 can be impervious to guanine rescue, suggesting that it is a vulnerable target.
  • Bacteria were cultured in 7H9/Glycerol/OADC/Tween and growth was measured with AlamarBlue as previously described, +ATc, 100 ng/mL. d. 20% inhibition at 50 mM. e. 5-10% inhibition at 15 pM. f. 80% inhibition at 50 pM.
  • MfMMPDH Inhibition of MfMMPDH.
  • the /ri.app values were determined by measuring the initial velocities at varying concentrations of the inhibitors (1-10,000 nM) with fixed concentrations of IMP (0.5 mM) and NAD + (1.5 mM) and A / lb I M P D H 2 (20-50 nM).
  • Inhibition of human IMPDH2 was assayed IMP using (0.25 mM) and NAD + (0.060 mM) and MMPDH2 (250 nM). Inhibition of human GMPR2 was assayed using GMP (0.050 mM), NADPH (0.045 mM) and enzyme (100 nM).
  • the assay buffer contained 50 mM TrisCl, pH 8.0, 100 mM KC1 and 1 mM dithiothreitol.
  • MIC determinations were determined as previously described. MIC values were determined in at least triplicate according to the broth microdilution methods using compounds from DMSO stock solutions. Isoniazid was used as a positive control and DMSO was utilized as a negative control. Isolated Mtb cells (ATCC 27294) were cultured to an OD 0.2-0.3 in the required medium, then diluted to deliver approximately 1 x 10 4 bacteria per well of a 96 well clear round-bottom plate. Plates were read after 1 week with an inverted enlarging mirror plate reader and graded as either growth or no growth.
  • GAST/Fe medium (per liter) consisted of 0.3 g of Bacto Casitone (Difco), 4.0 g of dibasic potassium phosphate, 2.0 g of citric acid, 1.0 g of L-alanine, 1.2 g of magnesium chloride hexahydrate, 0.6 g of potassium sulfate, 2.0 g of ammonium chloride, 1.80 ml of 10 N sodium hydroxide, and 10.0 ml of glycerol, 0.05% Tween 80 and 0.05 g of ferric ammonium citrate adjusted to pH 6.6.
  • 7H9/glycerol/glucose/BSA/Tween medium consisted of Middlebrook 7H9 broth base supplemented per liter with 0.2% glucose, 0.2% glycerol, 0.5% BSA fraction V, 0.08% NaCl and 0.05% Tween 80. Cultures were supplemented with 200 mM guanine as noted.
  • Mtb strains guaB2 cKD guaB2-B3 Tet-OFF attB::guaB3
  • SRMV2.6 which carries gtiaB2 Y4 1(: were cultured in Middlebrook 7H9 media (Difco) supplemented with 0.2% glycerol, Middlebrook oleic acid-albumin-dextrose-catalase (OADC) enrichment (Difco) and 0.05% Tween 80 (7H9/Glycerol/OADC/Tween).
  • Middlebrook 7H9 media Difco
  • OADC Middlebrook oleic acid-albumin-dextrose-catalase
  • Hygromycin Hyg
  • kanamycin Km
  • gentamycin Gm
  • ATc Sigma
  • concentrations up to 100 ng/mL For pairwise combination (checkerboard) assays, a two-dimensional array of serial dilutions of test compound and ATc was prepared in 96-well plates, as previously described. MIC testing was carried out by broth microdilution using the AlamarBlue (AB, Invitrogen) assay.
  • Hep G2 cells (ATCC, purchased February 2017) were cultivated in EMEM supplemented with 10% heat inactivated FBS and IX penicillin/streptomycin under standard conditions (37 °C in a 5% CO2 humidified atmosphere).
  • HEK293T, MCF7 and HELA cells were cultured in DMEM with 10% heat inactivated FBS and IX penicillin/streptomycin.
  • Active cell cultures routinely tested for presence of Mycoplasma (MycoAlertTM detection kit, Lonza) and confirmed to be
  • Example 13 - 0112 has antibacterial activity against Mtb and Mma.
  • the antibacterial activity of Q112 against Mtb varies less than 2-fold with different carbon sources (glucose, glycerol, cholesterol, dipalmitoylphosphatidyl-choline, butyrate) and is also active in macrophages (Fig. 5).
  • Mma is a ubiquitous fish pathogen that can also infect humans, commonly causing skin infections, and invasive infections can occur when the patient is immune compromised.
  • Q112 does not display antibacterial activity against S.
  • Q112 is not cytotoxic against HepG2 cells or in the NCI 60 panel of cancer cell lines (LDso> 10 mM). These observations indicate that anti -Mtb activity does not arise from a nonspecific process. Moreover, neither Q151 nor Q121 display cytotoxicity against HepG2 cells, and a single 20 mg/kg does of Q151 is well tolerated in mice, which suggest that the Q scaffold does not have toxicity liabilities even though it engages multiple bacterial targets. Table 8. Antibacterial activity of selected Q compounds.
  • WT is Mtb strain H37Rv
  • SRMV2.6 contains MtbIMPDH2/Y487C, which confers resistance to MtbIMPDH2 inhibitors
  • MtbIMPDH2 Inhibition of purified MtbIMPDH2/Y487C.
  • GAST/Fe glycerol
  • 7H9 glycerol/glucose
  • P146 and P150 The polarity of P146 and P150 was first addressed with the addition of a secondary amine that will be charged at physiologic pH (P148), sulfonamides and sulfoxides (P161, P162 and P163), as well as small heterocycles (P181, P182) in the 3-position (Table 9). Most of these substitutions had little impact on enzyme inhibition, with the exception of the piperazine amides P148 and P162, which were deleterious and therefore not tested for antibacterial activity. P161 and P163 displayed markedly reduced antibacterial activity relative to P146 and P150. The trifluoromethylpyrazole P181 and trifluoromethylthiazole P182 maintained comparable antibacterial activity. Curiously, the ketone analogs of P181 and P182 (P185 and P184) displayed little antibacterial activity despite similar affinities for &IMPDH.
  • oxime in ring A was replaced with hydroxyamidine to further increase topological polar surface area (P200, P202, P265, P208, P219, P269, P209, P210, P211, P212, P263 and P239).
  • the effect of the hydroxamidine substitution on antibacterial activity was variable, improving by more than lO-fold for P200, but with no effect for P211, and decreasing the antibacterial activity of P208 relative to their oxime analogs.
  • the addition of small heterocycles to the B ring in the context of the oxime substituted A ring increased antibacterial activity by 40-60-fold even though they had a deleterious effect on enzyme affinity.
  • the addition of the small heterocycles had little effect on enzyme affinity in the context of the hydroxyamidine substituted A ring and only a 4-fold increase in antibacterial activity (compare P209 and P211 to P200).
  • Three compounds, P219, P209 and P211 displayed MIC ⁇ l mM.
  • Example 15 Structures of enzyme-inhibitor complexes.
  • the x-ray crystal structures of the complexes of Tk/IMPDH and IMP with five inhibitors: P182, P200, P176, P178 and P221 was solved to determine how the thiazole, ribose, arabinose and hydroxyamidine groups interact with the enzyme.
  • the unit cell of the P182 complex contained one tetramer, while two tetramers where found in the unit cells of the other four complexes.
  • the overall structures are very similar, although the loop comprised of residues 390-418, designated the“flap”, displays varying amounts of disorder in different complexes as well as in different subunits of the same complex.
  • the thiazole ring of P182 binds in the site occupied by the adenosine ribose of NAD + , where it interacts with Ser257 in two active sites.
  • the oxime has rotated from its position in P32, so that it now forms a hydrogen bond with the side chain of Glu4l6 in all four active sites. This hydrogen bond is not observed in the P32 complex.
  • An additional hydrogen bond with the side chain of Thr310 is found in the chain B-C active site, and two additional hydrogen bonds, one to Thr3 l0 and the second to the hydroxyl of Tyr445’, are observed in the chain D-B active site.
  • the hydroxyamidine forms different hydrogen bonding patterns in different active sites.
  • amidine NEb binds in the same location as the oxime OH of P182, and makes a hydrogen bond to the carboxylate of Glu4l6 in six active sites. In the two active sites, an additional water mediated hydrogen bond is observed between the hydroxyamidine OH and carboxylate of Asp25l.
  • P176, P178 and P221 bind in the nicotinamide ribose site as expected, but make different interactions in each inhibitor.
  • P176, which links the ribose to the B ring via NH makes a hydrogen bond to Thr252 with the 3’ OH and an intramolecular hydrogen bond between the 5’-OH and the urea carbonyl.
  • P178 which links the ribose to the B ring via an oxygen, forms two hydrogen bonds to the enzyme, one between the 2’-OH and the backbone NH of His254 and the second between the 5’-OH and the OH of Ser257.
  • the arabinose containing compound P221 makes two hydrogen bonds via the T - OH, one to the Thr252 OH and the other to the main chain carbonyl of His254.
  • Example 16 - Antibacterial activity is due to inhibition of N/IMPDH.
  • Figures 8A and 8B show the dependence of antibacterial activity on inhibition of N/IMPDH for the P and A compounds described above. Greater antibacterial activity is generally associated with more potent enzyme inhibition, as expected if antibacterial activity derived from inhibition of N/IMPDH.
  • Table 9 the values of MIC in the presence of guanosine (Table 9), which provides an alternative source of guanine nucleotides that allows cells to grow in the absence of IMPDH activity, was determined. None of the inhibitors displayed antibacterial activity in the presence of guanosine (MIC >30 mM, Tables 8 and 9), with the exception of P200, where the value of MIC increased by a factor of 7 to 28 pM.
  • Example 17 The physicochemical properties of antibacterial activity.
  • cytotoxicity was tested for cytotoxicity in HepG2 cells by monitoring release of lactate dehydrogenase (Figs. 9A and 9B). Significant cytotoxicity (>10% release) was only observed in cells treated with 25 mM P211 and P221. Approximately 6% LDH release was observed when cells were treated with P219 and P251. No cytotoxicity ( ⁇ 2% release) was observed in HepG2 cells treated with P146, P171, P182, and P221.
  • P219 The pharmacokinetics of P219 were evaluated to further assess the antibiotic potential of the P scaffold.
  • P219 75 mg/kg i.v.
  • the value of CO greatly exceeds MIC (1.2 pM).
  • Plasma protein binding was also estimated by determining the increase in Ai app when enzyme inhibition was measured in the presence of 50% plasma. P219 displays high plasma protein binding, with a free fraction of 0.0043, which suggests the value of Co for free drug would be 0.8 pM.
  • the expression vector was acquired/constructed according to methods known in the art.
  • the vector was transformed in BL21 (DE3) guaB.
  • the cells were grown in LB media with 50 pg/ml of kanamycin at 37 ° C to an OD 6 oo 0.5-0.8.
  • Protein expression was initiated by 0.25 mM isopropyl b- ⁇ - thiogalactoside (IPTG) and the culture was maintained at 30 ° C overnight.
  • IPTG isopropyl b- ⁇ - thiogalactoside
  • N/IMPDH was purified according to a standard protocol for His tagged protein.
  • Protease inhibitor cocktail (Roche, Indianapolis, IN; 50 mL/g of wet cells) were added to the thawed cell suspension. The cells lysed on ice by sonication. The lysate was clarified by centrifugation at 18000 for 1 h and filtered through a 0.44 pm membrane.
  • Clarified lysate was applied to a 5 mL Ni-NTA column (McLab, San Francisco CA). The column was washed with 10 column volume (CV) lysis buffer, 10 CV of lysis buffer containing 30 mM imidazole, and the protein was eluted with the same buffer containing 250 mM imidazole.
  • riaIMPDH was dialyzed against 20 mM Hepes (pH 8.0), 150 mM KC1, 3 mM EDTA and 2 mM DTT, concentrated, flash-frozen, and stored in liquid nitrogen.
  • Enzyme assays were performed in 50 mM Tris-HCl, pH 8.0, 100 mM
  • Vi Vo/( 1 + [I]/ arr) (1)
  • v is initial velocity in the presence of inhibitor (I)
  • vo is the initial velocity in the absence of inhibitor.
  • the Morrison equation (Equation 2) was used to evaluate tight-binding inhibitors.
  • Vi/vo ⁇ Eo - Jo - K* + [(£b - /o - K ⁇ ,arr ) 2 + 4 ⁇ Eo ⁇ K, arr ⁇ 5 ⁇ / 2 ⁇ Eo (2)
  • v is the initial velocity in the presence of inhibitor
  • vo is the initial velocity in the absence of inhibitor
  • Eo is the total concentration of enzyme
  • Io is the total concentration of inhibitor
  • Ki app is the apparent inhibition constant.

Abstract

Disclosed are compounds and pharmaceutically acceptable salts thereof, which are useful as antibacterial agents. Also disclosed are pharmaceutical compositions comprising one or more compounds of the invention. Related methods of treating various infections in mammals, such as bacterial infections, are disclosed. Moreover, the compounds may be used alone or in combination with other therapeutic or prophylactic agents, such as anti-virals, anti-inflammatory agents, antimicrobials and immunosuppressants.

Description

ANTIBACTERIAL COMPOUNDS. COMPOSITIONS AND USES THEREOF
RELATED APPLICATIONS
This application claims the benefit of priority to U.S. Provisional Patent Application serial number 62/662,922, filed April 26, 2018, which is hereby incorporated by reference in its entirety.
GOVERNMENT SUPPORT
This invention was made with government support under Grant Nos. R01 AI093459 and R01 AI125362 awarded by the National Institutes of Health. The government has certain rights in the invention.
BACKGROUND
Tuberculosis (TB) is a global health threat. Each year there are more than 10 million new cases of tuberculosis TB which have led to more than 1 million deaths. Approximately one-third of the world’s population is infected with the causative agent Mycobacterium tuberculosis ( Mtb ), many with latent infections that can recrudesce if the patient becomes immunosuppressed.
Mtb can be free living or intracellular in vivo, and bacteria also occupy diverse environments that vary in pH, nutrient and oxygen availability as well as drug accessibility. Different metabolic pathways are vulnerable in each growth state. In addition, a population of bacteria exists in a quiescent state that is phenotypically resistant to many antibiotics. Several different antibiotics are required to eradicate bacteria from these diverse
microenvironments, as well as to guard against the development of resistance. Treatment typically requires administration with isoniazid, rifampicin pyrazinamide and ethambutol, but almost 0.5 million cases involve Mtb strains that are resistant to isoniazid and rifampicin. Extensively drug resistant strains have also emerged that are resistant to the second line drugs fluoroquinolones and at least one of capreomycin, kanamycin and amikacin.
Frustratingly, resistance has already emerged against the newly approved tuberculosis drugs bedaquiline and delamanid, creating a very real threat that these agents will soon be rendered obsolete.
Therefore, there is a continuing need to discover and develop new compounds to address the emergence and spread of drug-resistant tuberculosis. SUMMARY
One aspect of the present invention relates to compounds and pharmaceutically acceptable salts thereof, which are useful as inhibitors of IMPDH. Further, the invention provides pharmaceutical compositions comprising one or more compounds of the invention. The invention also relates to methods of treating various bacterial infections in a subject.
BRIEF DESCRIPTION OF THU DRAWINGS
Fig. 1A and Fig. IB show the correlation between antibacterial activity and enzyme inhibition for Q series compounds. Values from Table 6 and Makowska-Grzyska et ah, PLoS ONE 10, e0l38976. Mtb H37Rv cultured in GAST medium (Fig. 1A) and 7H9 medium
(Fig. IB)
Figs. 2A-2D are plots showing that knockdown of MtbIMPDH2 hypersensitizes Mtb to Q compounds. Regulated expression of guaB2 is achieved in a TET-OFF system.
Addition of anhydrotetracy cline (ATc) represses expression of guaB2, decreasing the level of MtbIMPDH2 within the bacteria. ATc concentrations (ng/mL) are 0 (dark orange), 0.08 (dark blue), 0.15 (green), 0.31 (sky blue), 0.62 (yellow), 1.25 (gray), 2.5 (orange) and 5 (royal blue).
Fig. 3 is a plot showing the cytotoxicity of select Q compounds in HepG2 cells after
24 h.
Fig. 4 is a plot showing the depletion of MtbIMPDH2 had no effect on the antibacterial activity of Ql 12. Regulated expression of guaB2 is achieved in a TET-OFF system as described in Fig. 2. Addition of anhydrotetracycline (ATc) represses expression of guaB2, decreasing the level of MtbIMPDH2 within the bacteria. ATc concentrations (ng/mL) are 0 (dark orange), 0.08 (dark blue), 0.15 (green), 0.31 (sky blue), 0.62 (yellow), 1.25 (gray), 2.5 (orange) and 5 (royal blue).
Fig. 5 is a bar graph showing the antibacterial activity of Ql 12 in macrophages. Rif,
1 mM rifampicin; Q112, 10 mM, DMSO, 5 mL/mL.
Fig. 6 depicts certain A and P compounds with antibacterial activity against S.
aureus.
Fig. 7 is a plot of K\ data for select A and P compounds showing that the structure activity relationship for these compounds in 7GIMPDH and V/IMPDH are similar. The black line denotes equal values of /ri.app for both enzymes. The dotted lines denote 2-fold differences. Fig. 8A and Fig. 8B are plots showing physicochemical properties of enzyme inhibition and antibacterial activity. P compounds are denoted with circles, A compounds with squares. The dotted line denotes MIC = 1 mM. Symbols are colored according to cLogP and t-PSA as calculated by ¥ fk¥ DrugsA website
Fig. 9A and Fig. 9B are plots showing the cytotoxicity of select A and P compounds in HepG2 cells after 24 h.
DFTATUFD DESCRIPTION OF THF INVENTION
In certain aspects, the invention provides substituted ureas (P series compounds) and substituted benzoxazoles (Q series compounds), and pharmaceutical compositions thereof. In particular, such compounds disclosed herein are useful as inhibitors of inosine 5'- monophosphate dehydrogenase (IMPDH).
IMPDH catalyzes the NAD+-dependent conversion of inosine 5 '-monophosphate (IMP) to xanthosine 5 '-monophosphate (XMP). This reaction is the first and the rate limiting step in the de novo biosynthesis of guanine nucleotides and therefore controls the size of the guanine nucleotide pool. IMPDH is an attractive target for the development of new antibiotics due to the essential role of guanine nucleotides in DNA and RNA synthesis, signal transduction, energy transfer, glycoprotein biosynthesis and many other processes involved in cell proliferation. Mtb has three genes, designated as guaBJ guaB2 and guaB3 , which encode IMPDH homologs. Only guaB2 is essential, and only guaB2 encodes an active IMPDH ( / MPDH2). The proteins encoded by guaBl and guaB3 are each missing key catalytic residues and neither display IMPDH activity. The functions of these proteins are currently unknown. Not wishing to be bound by theory, the compounds disclosed herein exploit the highly diverged cofactor binding site, which explains their selectivity for bacterial over eukaryotic orthologs of IMPDH, including / MPDH2.
Thus, the compounds disclosed herein can be used as antibacterial agents, particular with respect to treating Mycobacterium tuberculosis bacterial infections.
COMPOUNDS
Exemplary compounds disclosed herein are depicted in Tables 1 and 2. The compounds of Table 1 and 2 may be depicted as the free base or the conjugate acid.
Compounds may be isolated in either the free base form, free acid form, or as a salt (e.g., a chloride salt or a sodium salt) or in both forms. In the chemical structures shown below, standard chemical abbreviations are sometimes used. Table 1. Exemplary Q series Compounds
Figure imgf000006_0001
Figure imgf000007_0001
Figure imgf000008_0001
Figure imgf000009_0001
Figure imgf000010_0001
Figure imgf000011_0001
Figure imgf000012_0001
Figure imgf000013_0001
11
Figure imgf000014_0001
Table 2 . P-series Compounds
Figure imgf000014_0002
Figure imgf000015_0001

Figure imgf000016_0001

Figure imgf000017_0001

Figure imgf000018_0001

Figure imgf000019_0001

Figure imgf000020_0001
Figure imgf000021_0001
In certain embodiments, compounds of the invention may be prodrugs of the compounds of Table 1 and 2, e.g., wherein a hydroxyl in the parent compound is presented as an ester or a carbonate, or carboxylic acid present in the parent compound is presented as an ester. In certain such embodiments, the prodrug is metabolized to the active parent compound in vivo (e.g., the ester is hydrolyzed to the corresponding hydroxyl, or carboxylic acid).
In certain embodiments, compounds of the invention may be racemic. In certain embodiments, compounds of the invention may be enriched in one enantiomer. For example, a compound of the invention may have greater than 30% ee, 40% ee, 50% ee, 60% ee, 70% ee, 80% ee, 90% ee, or even 95% or greater ee. The compounds of the invention have more than one stereocenter. Consequently, compounds of the invention may be enriched in one or more diastereomer. For example, a compound of the invention may have greater than 30% de, 40% de, 50% de, 60% de, 70% de, 80% de, 90% de, or even 95% or greater de.
In certain embodiments, the compound is not P177, P188, P221, P226, or P227.
II. METHODS
In certain embodiments, the invention is a method of killing or inhibiting the growth of a microbe, comprising the step of contacting said microbe with an effective amount of a compound disclosed herein (e.g., a compound of Table 1 or Table 2), or a pharmaceutically acceptable salt thereof.
In some aspects, provided herein are methods for treating infections. For example, the method may be a method of treating or preventing a microbial infection in a mammal, comprising the step of administering to a mammal in need thereof a therapeutically effective amount of a compound disclosed herein (e.g., a compound of Table 1 or Table 2), or a pharmaceutically acceptable salt thereof. For example, the method may be a method of treating or preventing a parasitic infection in a mammal, comprising the step of
administering to a mammal in need thereof a therapeutically effective amount of a compound disclosed herein (e.g., a compound of Table 1 or Table 2), or a pharmaceutically acceptable salt thereof.
In some aspects, provided herein are methods for treating a bacterial infection in a subject by administering to the subject an effective amount of a compound disclosed herein (e.g., compounds of Table 1 and 2) to thereby treat the infection. Also provided herein are methods for ameliorating the signs or symptoms of an infection in a subject by bacterial cells, the method comprising administering to the subject an effective amount of a compound disclosed herein (e.g., compounds of Table 1 and 2) to thereby ameliorate the signs and symptoms of the infection.
In some aspects, provided herein are methods for preventing or reducing the likelihood of a productive bacterial infection in a subject by administering to a subject an effective amount of a compound disclosed herein (e.g., compounds of Table 1 and 2) to thereby prevent or reduce the likelihood of a productive bacterial infection in the subject.
In some embodiments, the bacteria or bacterial cells are Mycobacterium. The Mycobacterium may be Mycobacterium tuberculosis. The Mycobacterium may be any Mycobacterium species that causes tuberculosis or belongs to the Mycobacterium
tuberculosis complex. Species in this complex include M. africanum M. bovis, M. canetti ,
M. caprae , M. microti , M. mungi , M. orygis , M. pinnipedii , M. suricattae and M.
tuberculosis.
The Mycobacterium may be any other species of Mycobacterium (e.g., any species disclosed herein), such as nontuberculous mycobacteria. Nontuberculous mycobacteria are commonly present in soil and water and are usually much less virulent in humans than is Mycobacterium tuberculosis. Infections with these organisms have been called atypical, environmental, and nontuberculous mycobacterial infections. Exposures and infections by these organisms often require a defect in local or systemic host defenses; the frail elderly and immunocompromised people are at the highest risk. M. avium complex (MAC), including the closely related species of M. avium and M. intracellulare , accounts for many of nontuberculosis mycobacterial diseases. Other causative species include M kansasii, M. xenopi, M. marinum, M. ulcer ans, M. fortuitum, M. abscessus , andM chelonae.
Other species of Mycobacertium include M. asiaticum, M. gordonae, M. gastri,
M. kansasii, M. hiberniae, M. icosiumassiliensis, M. nonchromogenicum, M. terrae, M. triviale, M. ulcerans , M. pseudoshottsii, M. shottsii, M. florentinum, M. genavense, M.
heidelbergense, M. interjectum, M. kubicae, M. lentiflavum, M. montefwrense, M. palustre, M. parascrofulaceum, M. simiae, M. triplex, M. arabiense, M. aromaticivorans, M.
aquaticum, M. bacteremicum, M. bohemicum, M. botniense, M. branderi, M. celatum, M. chimaera, M. conspicuum, M. cookii, M. doricum, M. farcinogenes, M. haemophilum, M. heckeshornense, M. intracellulare, M. lacus, M. leprae , M lepraemurium, M. lepromatosis , M liflandii, M. llatzerense, M. malmoense, M. marinum , causes a rare disease
called Aquarium granuloma, M neoaurum, M. monacense, M. murale, M. nebraskense, M. saskatchewanense, M. sediminis, M. scrofulaceum, M. shimoidei, M. szulgai, Mycobacterium talmoniae, M. tusciae, M. xenopi, M. yongonense, M. intermedium, M. abscessus, M. bolletii, M. massiliense, M. chelonae, M. immunogenum, M. stephanolepidis, M. boenickei, M.
brisbanense, M. cosmeticum, M. fortuitum, M. fortuitum subsp. acetamidolyticum, M.
houstonense, M. mageritense, M. neworleansense, M. peregrinum, M. porcinum, M.
senegalense, M. septicum, Mycobacterium aubagnese, M. mucogenicum, Mycobacterium phocaicum, M. austroafricanum, M. diernhoferi, M. frederiksbergense, M. hodleri, M.
neoaurum, M. parafortuitum, M. aurum, M. vaccae, M. chitae, M. fallax, M. agri, M.
aichiense, M. alvei, M. arupense, M. barrassiae, M. brumae, M. canariasense, M.
chubuense, M. conceptionense, M. confluentis, M. duvalii, M. elephantis, M. flavescens, M. gadium, M. gilvum, M. hassiacum, M. holsaticum, M. iranicum, M. komossense, M.
madagascariense, M. massilipolynesiensis, M. moriokaense, M. obuense, M. phlei, M.
psychrotolerans, M. pulveris, M. pyrenivorans, M. smegmatis, M. goodii, M. wolinskyi, M. sphagni, M. thermoresistibile, M. vanbaalenii, M. arosiense, M. aubagnense, M.
chlorophenolicum, M. fluoroanthenivorans, M. kumamotonense, M. novocastrense,
M. parmense, M. poriferae, M. rhodesiae, M. seoulense or M. tokaiense. In certain embodiments, the bacteria or bacterial cells are from the genera
Acinetobacter, Arcobacter , Bacillus, Bacteroides, Borrelia, Brucella , Burkholderia,
Campylobacter, Clostridia , Coxiella , Enterococcus , Erysipelothrix , Francisella ,
Fusobacterium, Helicobacter , Lactobacillus , Listeria , Neisseria , Pseudomonas ,
Staphylococcus , Streptococcus , and Trypanosoma.
In certain embodiments, the invention relates to any one of the aforementioned methods, wherein said microbial infection is caused by a protozoan or bacterium.
In certain embodiments, the invention relates to any one of the aforementioned methods, wherein said microbial infection is caused by a protozoan or a bacterium selected from the group consisting of the genera Cryptosporidium, Entamoeba, Leishmania ,
Trypanosoma, Acinetobacter, Arcobacter , Bacillus, Bacteroides, Borrelia , Brucella ,
Burkholderia, Brachyspira, Campylobacter, Clostridia , Coxiella , Enterococcus ,
Erysipelothrix , Francisella , Fusobacterium, Helicobacter , Lactobacillus , Listeria ,
Mycobacterium, Neisseria , Pseudomonas , Staphylococcus and Streptococcus.
In some aspects, provided herein are method of treating tuberculosis in a subject, the method comprising administering to the subject an effective amount of a compound disclosed herein (e.g., compounds of Table 1 and 2) to thereby treat the tuberculosis.
In certain embodiments, the subject is a mammal, e.g., a human.
In some embodiments, the invention relates to a method of treating or preventing a bacterial infection in a mammal, comprising the step of co-administering to a mammal in need thereof: (a) a first amount of a compound of Table 1 or Table 2, or a pharmaceutically acceptable salt thereof; and (b) a second amount of an antibacterial agent, wherein, taken together, the first amount and the second amount are effective.
In certain embodiments, the invention relates to a method of treating a recurrent or persistent bacterial infection in a mammal, comprising the step of co-administering to a mammal in need thereof: (a) a first amount of a compound of Table 1 or Table 2, or a pharmaceutically acceptable salt thereof; and (b) a second amount of an antibacterial agent, wherein, taken together, the first amount and the second amount are therapeutically effective.
In some embodiments, the invention relates to a method of preventing the recurrence of a bacterial infection in a mammal, comprising the step of co-administering to a mammal in need thereof: (a) a first amount of a compound of Table 1 or Table 2, or a
pharmaceutically acceptable salt thereof; and (b) a second amount of an antibacterial agent, wherein, taken together, the first amount and the second amount are effective to prevent the recurrence of the bacterial infection in the mammal.
In certain embodiments, the antibacterial agent is an anti-translation agent, for example, an agent that interferes with bacterial protein synthesis, for example, by interacting with prokaryotic ribosomes. In certain embodiments, the antibacterial agent inhibits DNA gyrase or a topoisomerase in a bacterium. In certain embodiments, the antibacterial agent interferes with the transpeptidation reaction, thereby inhibits synthesis of peptidoglycan, a component of bacterial cell walls.
In certain embodiments, the antibacterial agent is a penicillin. In certain
embodiments, the antibacterial agent is a tetracycline. In certain embodiments, the antibacterial agent is a cephalosporin. In certain embodiments, the antibacterial agent is a quinolone, such as a fluoroquinolone. In certain embodiments, the antibacterial agent is a linomycin. In certain embodiments, the antibacterial agent is a macrolide. In certain embodiments, the antibacterial agent is a sulfonamide. In certain embodiments, the antibacterial agent is a glycopeptide. In certain embodiments, the antibacterial agent is an aminoglycoside. In certain embodiments, the antibacterial agent is a carbapenem.
In certain embodiments, the invention relates to any one of the compositions described herein, further comprising an antibacterial agent selected from the group consisting of amikacin, amoxicillin, azithromycin, cefdinir, ceftriaxone, cefuroxime, cephalexin, ciprofloxacin, clarithromycin, clindamycin, co-trimoxazole, dalbavancin, doripenem, doxycycline, ertapenem, erythromycin, gentamicin, imipenem/cilastatin, kanamycin, levofloxacin, lincomycin, meropenem, metronidazole, minocycline,
moxifloxacin, ofloxacin, oritavancin, oxacillin, penicillin, sulfamethoxazole, sulfasalazine, sulfisoxazole, telavancin, tetracycline, tobramycin, trimethoprim, and vancomycin.
In certain embodiments, prior to co-administration of (a) and (b), the mammal had been administered a first antibacterial agent. For example, the first antibacterial agent may be selected from the group consisting of amikacin, amoxicillin, azithromycin, cefdinir, ceftriaxone, cefuroxime, cephalexin, ciprofloxacin, clarithromycin, clindamycin, co- trimoxazole, dalbavancin, doripenem, doxycycline, ertapenem, erythromycin, gentamicin, imipenem/cilastatin, kanamycin, levofloxacin, lincomycin, meropenem, metronidazole, minocycline, moxifloxacin, ofloxacin, oritavancin, oxacillin, penicillin, sulfamethoxazole, sulfasalazine, sulfisoxazole, telavancin, tetracycline, tobramycin, trimethoprim, and vancomycin. In some embodiments, the first antibacterial agent and the antibacterial agent are the same. In certain embodiments, the first antibacterial agent and the antibacterial agent are not the same.
Ill PHARMACEUTICAL COMPOSITIONS
In certain embodiments, the present invention provides pharmaceutical compositions comprising a compound of Table 1 or 2 and a pharmaceutically acceptable carrier.
In certain embodiments, the present invention provides pharmaceutical compositions comprising a compound of Table 1 or 2, an antibacterial agent, and a pharmaceutically acceptable carrier. In certain embodiments, the antibacterial agent is an anti-translation agent, for example, an agent that interferes with bacterial protein synthesis, for example, by interacting with prokaryotic ribosomes. In certain embodiments, the antibacterial agent inhibits DNA gyrase or a topoisomerase in a bacterium. In certain embodiments, the antibacterial agent interferes with the transpeptidation reaction, thereby inhibits synthesis of peptidoglycan, a component of bacterial cell walls.
In certain embodiments, the antibacterial agent is a penicillin. In certain
embodiments, the antibacterial agent is a tetracycline. In certain embodiments, the antibacterial agent is a cephalosporin. In certain embodiments, the antibacterial agent is a quinolone, such as a fluoroquinolone. In certain embodiments, the antibacterial agent is a linomycin. In certain embodiments, the antibacterial agent is a macrolide. In certain embodiments, the antibacterial agent is a sulfonamide. In certain embodiments, the antibacterial agent is a glycopeptide. In certain embodiments, the antibacterial agent is an aminoglycoside. In certain embodiments, the antibacterial agent is a carbapenem.
In certain embodiments, the invention relates to any one of the compositions described herein, further comprising an antibacterial agent selected from the group consisting of amikacin, amoxicillin, azithromycin, cefdinir, ceftriaxone, cefuroxime, cephalexin, ciprofloxacin, clarithromycin, clindamycin, co-trimoxazole, dalbavancin, doripenem, doxycycline, ertapenem, erythromycin, gentamicin, imipenem/cilastatin, kanamycin, levofloxacin, lincomycin, meropenem, metronidazole, minocycline,
moxifloxacin, ofloxacin, oritavancin, oxacillin, penicillin, sulfamethoxazole, sulfasalazine, sulfisoxazole, telavancin, tetracycline, tobramycin, trimethoprim, and vancomycin.
The compositions and methods of the present invention may be utilized to treat an individual in need thereof. In certain embodiments, the individual is a mammal such as a human, or a non-human mammal. When administered to an animal, such as a human, the composition or the compound is preferably administered as a pharmaceutical composition comprising, for example, a compound of the invention and a pharmaceutically acceptable carrier.
Pharmaceutically acceptable carriers are well known in the art and include, for example, aqueous solutions such as water or physiologically buffered saline or other solvents or vehicles such as glycols, glycerol, oils such as olive oil, or injectable organic esters. In a preferred embodiment, when such pharmaceutical compositions are for human
administration, particularly for invasive routes of administration (i.e., routes, such as injection or implantation, that circumvent transport or diffusion through an epithelial barrier), the aqueous solution is pyrogen-free, or substantially pyrogen-free. The excipients can be chosen, for example, to effect delayed release of an agent or to selectively target one or more cells, tissues or organs. The pharmaceutical composition can be in dosage unit form such as tablet, capsule (including sprinkle capsule and gelatin capsule), granule, lyophile for reconstitution, powder, solution, syrup, suppository, injection or the like. The composition can also be present in a transdermal delivery system, e.g., a skin patch. The composition can also be present in a solution suitable for topical administration, such as an eye drop.
A pharmaceutically acceptable carrier can contain physiologically acceptable agents that act, for example, to stabilize, increase solubility or to increase the absorption of a compound such as a compound of the invention. Such physiologically acceptable agents include, for example, carbohydrates, such as glucose, sucrose or dextrans, antioxidants, such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins or other stabilizers or excipients. The choice of a pharmaceutically acceptable carrier, including a physiologically acceptable agent, depends, for example, on the route of administration of the composition. The preparation or pharmaceutical composition can be a self-emulsifying drug delivery system or a self-microemulsifying drug delivery system. The pharmaceutical composition (preparation) also can be a liposome or other polymer matrix, which can have incorporated therein, for example, a compound of the invention. Liposomes, for example, which comprise phospholipids or other lipids, are nontoxic, physiologically acceptable and metabolizable carriers that are relatively simple to make and administer.
The phrase "pharmaceutically acceptable" is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. The phrase "pharmaceutically acceptable carrier" as used herein means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxic compatible substances employed in pharmaceutical formulations.
A pharmaceutical composition (preparation) can be administered to a subject by any of a number of routes of administration including, for example, orally (for example, drenches as in aqueous or non-aqueous solutions or suspensions, tablets, capsules (including sprinkle capsules and gelatin capsules), boluses, powders, granules, pastes for application to the tongue); absorption through the oral mucosa (e.g., sublingually); anally, rectally or vaginally (for example, as a pessary, cream or foam); parenterally (including intramuscularly, intravenously, subcutaneously or intrathecally as, for example, a sterile solution or suspension); nasally; intraperitoneally; subcutaneously; transdermally (for example as a patch applied to the skin); and topically (for example, as a cream, ointment or spray applied to the skin, or as an eye drop). The compound may also be formulated for inhalation. In certain embodiments, a compound may be simply dissolved or suspended in sterile water. Details of appropriate routes of administration and compositions suitable for same can be found in, for example, U.S. Pat. Nos. 6,110,973, 5,731,000, 5,541,231, 5,427,798, 5,358,970 and 4,172,896, as well as in patents cited therein.
The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated, the particular mode of administration. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect. Generally, out of one hundred percent, this amount will range from about 1 percent to about ninety-nine percent of active ingredient, preferably from about 5 percent to about 70 percent, most preferably from about 10 percent to about 30 percent.
Methods of preparing these formulations or compositions include the step of bringing into association an active compound, such as a compound of the invention, with the carrier and, optionally, one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association a compound of the present invention with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.
Formulations of the invention suitable for oral administration may be in the form of capsules (including sprinkle capsules and gelatin capsules), cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), lyophile, powders, granules, or as a solution or a suspension in an aqueous or non-aqueous liquid, or as an oil- in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and/or as mouth washes and the like, each containing a predetermined amount of a compound of the present invention as an active ingredient. Compositions or compounds may also be administered as a bolus, electuary or paste.
To prepare solid dosage forms for oral administration (capsules (including sprinkle capsules and gelatin capsules), tablets, pills, dragees, powders, granules and the like), the active ingredient is mixed with one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate, and/or any of the following: (1) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as, for example, cetyl alcohol and glycerol monostearate; (8) absorbents, such as kaolin and bentonite clay; (9) lubricants, such a talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof; (10) complexing agents, such as, modified and unmodified cyclodextrins; and (11) coloring agents. In the case of capsules (including sprinkle capsules and gelatin capsules), tablets and pills, the pharmaceutical compositions may also comprise buffering agents. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like.
A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared using binder (for example, gelatin or hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agent. Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.
The tablets, and other solid dosage forms of the pharmaceutical compositions, such as dragees, capsules (including sprinkle capsules and gelatin capsules), pills and granules, may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical-formulating art. They may also be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile, other polymer matrices, liposomes and/or microspheres. They may be sterilized by, for example, filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions that can be dissolved in sterile water, or some other sterile injectable medium immediately before use. These compositions may also optionally contain opacifying agents and may be of a composition that they release the active ingredient(s) only, or preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes. The active ingredient can also be in micro- encapsulated form, if appropriate, with one or more of the above-described excipients.
Liquid dosage forms useful for oral administration include pharmaceutically acceptable emulsions, lyophiles for reconstitution, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredient, the liquid dosage forms may contain inert diluents commonly used in the art, such as, for example, water or other solvents, cyclodextrins and derivatives thereof, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1, 3-butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.
Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.
Suspensions, in addition to the active compounds, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.
Formulations of the pharmaceutical compositions for rectal, vaginal, or urethral administration may be presented as a suppository, which may be prepared by mixing one or more active compounds with one or more suitable nonirritating excipients or carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax or a salicylate, and which is solid at room temperature, but liquid at body temperature and, therefore, will melt in the rectum or vaginal cavity and release the active compound.
Formulations of the pharmaceutical compositions for administration to the mouth may be presented as a mouthwash, or an oral spray, or an oral ointment.
Alternatively or additionally, compositions can be formulated for delivery via a catheter, stent, wire, or other intraluminal device. Delivery via such devices may be especially useful for delivery to the bladder, urethra, ureter, rectum, or intestine.
Formulations which are suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams or spray formulations containing such carriers as are known in the art to be appropriate.
Dosage forms for the topical or transdermal administration include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. The active compound may be mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants that may be required.
The ointments, pastes, creams and gels may contain, in addition to an active compound, excipients, such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof. Powders and sprays can contain, in addition to an active compound, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances. Sprays can additionally contain customary propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane.
Transdermal patches have the added advantage of providing controlled delivery of a compound of the present invention to the body. Such dosage forms can be made by dissolving or dispersing the active compound in the proper medium. Absorption enhancers can also be used to increase the flux of the compound across the skin. The rate of such flux can be controlled by either providing a rate controlling membrane or dispersing the compound in a polymer matrix or gel.
Ophthalmic formulations, eye ointments, powders, solutions and the like, are also contemplated as being within the scope of this invention. Exemplary ophthalmic
formulations are described in U.S. Publication Nos. 2005/0080056, 2005/0059744,
2005/0031697 and 2005/004074 and U.S. Patent No. 6,583,124, the contents of which are incorporated herein by reference. If desired, liquid ophthalmic formulations have properties similar to that of lacrimal fluids, aqueous humor or vitreous humor or are compatible with such fluids. A preferred route of administration is local administration ( e.g ., topical administration, such as eye drops, or administration via an implant).
The phrases "parenteral administration" and "administered parenterally" as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion. Pharmaceutical compositions suitable for parenteral administration comprise one or more active compounds in combination with one or more pharmaceutically acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.
Examples of suitable aqueous and nonaqueous carriers that may be employed in the pharmaceutical compositions of the invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.
These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents that delay absorption such as aluminum monostearate and gelatin.
In some cases, in order to prolong the effect of a drug, it is desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This may be
accomplished by the use of a liquid suspension of crystalline or amorphous material having poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution, which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle.
Injectable depot forms are made by forming microencapsulated matrices of the subject compounds in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of drug to polymer, and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly (anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions that are compatible with body tissue.
For use in the methods of this invention, active compounds can be given per se or as a pharmaceutical composition containing, for example, 0.1 to 99.5% (more preferably, 0.5 to 90%) of active ingredient in combination with a pharmaceutically acceptable carrier.
Methods of introduction may also be provided by rechargeable or biodegradable devices. Various slow release polymeric devices have been developed and tested in vivo in recent years for the controlled delivery of drugs, including proteinacious biopharmaceuticals. A variety of biocompatible polymers (including hydrogels), including both biodegradable and non-degradable polymers, can be used to form an implant for the sustained release of a compound at a particular target site.
Actual dosage levels of the active ingredients in the pharmaceutical compositions may be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.
The selected dosage level will depend upon a variety of factors including the activity of the particular compound or combination of compounds employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound(s) being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compound(s) employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.
A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the therapeutically effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could start doses of the pharmaceutical composition or compound at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. By “therapeutically effective amount” is meant the concentration of a compound that is sufficient to elicit the desired therapeutic effect. It is generally understood that the effective amount of the compound will vary according to the weight, sex, age, and medical history of the subject. Other factors which influence the effective amount may include, but are not limited to, the severity of the patient's condition, the disorder being treated, the stability of the compound, and, if desired, another type of therapeutic agent being administered with the compound of the invention. A larger total dose can be delivered by multiple administrations of the agent. Methods to determine efficacy and dosage are known to those skilled in the art (Isselbacher et al. (1996) Harrison’s Principles of Internal Medicine 13 ed., 1814-1882, herein incorporated by reference).
In general, a suitable daily dose of an active compound used in the compositions and methods of the invention will be that amount of the compound that is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above. If desired, the effective daily dose of the active compound may be administered as one, two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms. In certain embodiments of the present invention, the active compound may be administered two or three times daily. In preferred embodiments, the active compound will be administered once daily.
The patient receiving this treatment is any animal in need, including primates, in particular humans, and other mammals such as equines, cattle, swine and sheep; and poultry and pets in general.
In certain embodiments, compounds of the invention may be used alone or conjointly administered with another type of therapeutic agent. As used herein, the phrase“conjoint administration” refers to any form of administration of two or more different therapeutic compounds such that the second compound is administered while the previously
administered therapeutic compound is still effective in the body ( e.g ., the two compounds are simultaneously effective in the patient, which may include synergistic effects of the two compounds). For example, the different therapeutic compounds can be administered either in the same formulation or in a separate formulation, either concomitantly or sequentially. In certain embodiments, the different therapeutic compounds can be administered within one hour, 12 hours, 24 hours, 36 hours, 48 hours, 72 hours, or a week of one another. Thus, an individual who receives such treatment can benefit from a combined effect of different therapeutic compounds.
In certain embodiments, conjoint administration of compounds of the invention with one or more additional therapeutic agent(s) (e.g., one or more additional chemotherapeutic agent(s)) provides improved efficacy relative to each individual administration of the compound of the invention (e.g., compounds of Table 1 and 2) or the one or more additional therapeutic agent(s). In certain such embodiments, the conjoint administration provides an additive effect, wherein an additive effect refers to the sum of each of the effects of individual administration of the compound of the invention and the one or more additional therapeutic agent(s).
This invention includes the use of pharmaceutically acceptable salts of compounds of the invention in the compositions and methods of the present invention. The term
“pharmaceutically acceptable salt” as used herein includes salts derived from inorganic or organic acids including, for example, hydrochloric, hydrobromic, sulfuric, nitric, perchloric, phosphoric, formic, acetic, lactic, maleic, fumaric, succinic, tartaric, glycolic, salicylic, citric, methanesulfonic, benzenesulfonic, benzoic, malonic, trifluoroacetic, trichloroacetic, naphthalene-2-sulfonic, and other acids. Pharmaceutically acceptable salt forms can include forms wherein the ratio of molecules comprising the salt is not 1 :1. For example, the salt may comprise more than one inorganic or organic acid molecule per molecule of base, such as two hydrochloric acid molecules per molecule of compounds of Table 1 and 2. As another example, the salt may comprise less than one inorganic or organic acid molecule per molecule of base, such as two molecules of compounds of Table 1 and 2 per molecule of tartaric acid.
In further embodiments, contemplated salts of the invention include, but are not limited to, alkyl, dialkyl, trialkyl or tetra-alkyl ammonium salts. In certain embodiments, contemplated salts of the invention include, but are not limited to, L-arginine, benenthamine, benzathine, betaine, calcium hydroxide, choline, deanol, diethanolamine, diethylamine, 2- (diethylamino)ethanol, ethanolamine, ethylenediamine, N-methylglucamine, hydrabamine, lH-imidazole, lithium, L-lysine, magnesium, 4-(2-hydroxyethyl)morpholine, piperazine, potassium, 1 -(2-hydroxy ethyl)pyrrolidine, sodium, triethanolamine, tromethamine, and zinc salts. In certain embodiments, contemplated salts of the invention include, but are not limited to, Na, Ca, K, Mg, Zn or other metal salts.
The pharmaceutically acceptable acid addition salts can also exist as various solvates, such as with water, methanol, ethanol, dimethylformamide, and the like. Mixtures of such solvates can also be prepared. The source of such solvate can be from the solvent of crystallization, inherent in the solvent of preparation or crystallization, or adventitious to such solvent.
Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.
Examples of pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabi sulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal-chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like. V. DEFINITIONS
All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and/or ordinary meanings of the defined terms.
The indefinite articles“a” and“an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean“at least one.”
The phrase“and/or,” as used herein in the specification and in the claims, should be understood to mean“either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with“and/or” should be construed in the same fashion, i.e.,“one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the“and/or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to“A and/or B”, when used in conjunction with open-ended language such as“comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.
As used herein in the specification and in the claims,“or” should be understood to have the same meaning as“and/or” as defined above. For example, when separating items in a list,“or” or“and/or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as“only one of’ or“exactly one of,” or, when used in the claims,“consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term“or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e.,“one or the other but not both”) when preceded by terms of exclusivity, such as“either,”“one of,”“only one of,” or “exactly one of.”“Consisting essentially of,” when used in the claims, shall have its ordinary meaning as used in the field of patent law.
As used herein in the specification and in the claims, the phrase“at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase“at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example,“at least one of A and B” (or, equivalently,“at least one of A or B,” or,
equivalently“at least one of A and/or B”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.
It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.
In the claims, as well as in the specification above, all transitional phrases such as “comprising,”“including,”“carrying,”“having,”“containing,”“involving,”“holding,” “composed of,” and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases“consisting of’ and“consisting essentially of’ shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03.
Certain compounds contained in compositions of the present invention may exist in particular geometric or stereoisomeric forms. The present invention contemplates all such compounds, including cis- and trans-isomers, R- and //-enantiomers, diastereomers, (D)- isomers, (L)-isomers, the racemic mixtures thereof, and other mixtures thereof, as falling within the scope of the invention. All such isomers, as well as mixtures thereof, are intended to be included in this invention.
If, for instance, a particular enantiomer of compound of the present invention is desired, it may be prepared by asymmetric synthesis, or by derivation with a chiral auxiliary, where the resulting diastereomeric mixture is separated and the auxiliary group cleaved to provide the pure desired enantiomers. Alternatively, where the molecule contains a basic functional group, such as amino, or an acidic functional group, such as carboxyl,
diastereomeric salts are formed with an appropriate optically-active acid or base, followed by resolution of the diastereomers thus formed by fractional crystallization or chromatographic means well known in the art, and subsequent recovery of the pure enantiomers.
For purposes of this invention, the chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version,“Handbook of Chemistry and Physics”, 67th Ed., 1986-87, inside cover.
As used herein, a therapeutic that“prevents” a disorder or condition refers to a compound that, in a statistical sample, reduces the occurrence of the disorder or condition in the treated sample relative to an untreated control sample, or delays the onset or reduces the severity of one or more symptoms of the disorder or condition relative to the untreated control sample.
The term“treating” includes prophylactic and/or therapeutic treatments. The term “prophylactic or therapeutic” treatment is art-recognized and includes administration to the host of one or more of the subject compositions. If it is administered prior to clinical manifestation of the unwanted condition (e.g., disease or other unwanted state of the host animal) then the treatment is prophylactic (i.e., it protects the host against developing the unwanted condition), whereas if it is administered after manifestation of the unwanted condition, the treatment is therapeutic, (i.e., it is intended to diminish, ameliorate, or stabilize the existing unwanted condition or side effects thereof).
A“recurrent” or“persistent” bacterial infection results from certain bacterial pathogens that are able to evade the host immune system and persist within the human host. The consequences of persistent bacterial infections potentially include increased morbidity and mortality from the infection itself as well as an increased risk of dissemination of disease. Eradication of persistent infections is difficult, often requiring prolonged or repeated courses of antibiotics. During persistent infections, a population or subpopulation of bacteria exists that is refractory to traditional antibiotics, possibly in a non-replicating or
metabolically altered (“quiescent”) state. As used herein,“preventing the recurrence of’ an infection describes eradication of the agent that caused the original infection. The efficacy of preventing the recurrence of the infection is determined by a successful clinical outcome and does not require 100% elimination of the microorganisms involved in the infection.
Achieving a level of antimicrobial activity at the site of infection that allows the host to survive, resolve the infection, or eradicate the causative agent is sufficient. The term“prodrug” is intended to encompass compounds which, under physiologic conditions, are converted into the therapeutically active agents of the present invention (e.g., compounds of Table 1 and 2). A common method for making a prodrug is to include one or more selected moieties which are hydrolyzed under physiologic conditions to reveal the desired molecule. In other embodiments, the prodrug is converted by an enzymatic activity of the host animal. For example, esters or carbonates (e.g., esters or carbonates of alcohols or carboxylic acids) are preferred prodrugs of the present invention. In certain embodiments, some or all of the compounds of Table 1 and 2 in a formulation represented above can be replaced with the corresponding suitable prodrug, e.g., wherein a hydroxyl in the parent compound is presented as an ester or a carbonate or carboxylic acid present in the parent compound is presented as an ester.
EXAMPLES
Examples of compounds of Table 1 and 2 or pharmaceutically acceptable salts thereof having useful biological activity are described below. The preparation of these compounds can be realized by one of skilled in the art of organic synthesis using known techniques and methodology.
Example 1 Synthesis of various aryl benzoxazoles 8. thioamide 9. amine 11. imidazorE2- alpyridine 13. methylene linked derivative 15 and N-arylated benzoxazoles !7a-b.
The synthesis used to generate ether linked Q-series derivatives is illustrated in Scheme 1. Arylbenzoxazoles 4 were directly synthesized by oxidative cyclization methods using 2-amino-4-nitrophenol and aldehydes in the presence of activated carbon (Darco KB) under an oxygen atmosphere. The nitro group was reduced using Pd/C under 1 atm hydrogen to give 5-amine-2-arylbenzoxazoles 5. Enantiomerically pure phenyl ethers 6 were synthesized from (+)-methyl D-lactate and the corresponding phenol using Mitsunobu reaction conditions. The esters 6 were hydrolyzed to the corresponding acids 7, and then coupled with various 5-amine-2-arylbenzoxazoles in the presence of EDC HCl in DMF to yield 8 Furthermore, 8 (Ar = 4-OMePh, R = 2,3-diCl) was treated with Lawesson's reagent under standard conditions to generate thioamide 9 quantitatively. Ester 6a was also treated with 1M DIBAL-H in DCM at -78 °C to generate aldehyde 10, which was subjected to reductive amination using 5 (Ar = 4-OMePh) and sodium triacetoxyborohydride in DCM to provide amine 11. The synthesis of an imidazo[l,2-a]pyridine analogue started by allowing 5- nitropyridin-2-amine to react with an a-bromoketone in the presence of NaHCCb in ethanol to give 12. Nitro group reduction using SnCb in ethanol followed by coupling with carboxylic acid 7 (R = 2,3-diCl) generated imidazo[l,2-a]pyridine 13.
The synthesis of a derivative with a methylene linker between the benzoxazole and the benzene was undertaken. 2-Amino-4-nitrophenol was coupled with a 1,1- dibromoethylene derivative using DABCO in NMP at 100 °C to generate 14. The nitro group was reduced using 10% Pd/C under a hydrogen atmosphere and the resulting amine was coupled with 7 (R = 2,3-diCl) using EDCTTCl in DMF to yield 15.
Enantiopure A -aryl ate d benzoxazole derivatives were synthesized using copper catalyzed EUlman type reactions. L-alanine was treated with aryl iodides in the presence of cesium carbonate and Cul in DMF for 24 h to provide the A-arylated amino acids 16a-b. Attempts to couple these materials with 5 (Ar = 4-CNPh) using EDC HCl failed. However, using HATU as the coupling reagent in DMF resulted in formation of 17a-b.
Scheme 1
Figure imgf000042_0001
Reagents and conditions: (a) ArCHO, DarcoKB, O2 (1 atm), xylene 140 °C, 6-8 h, 70-80%; (b) 10% Pd/C, H2 (1 atm), EtOAc:MeOH, 3 h, 85-94%; (c) PPh3, DEAD, (+)-methyl D- lactate, THF, 0 °C to rt, 4 h, 85-90%, (d) Li OH, THF :MeOH, 4 h, 86-91%; (e) 5, EDC HC1, DMF, 12 h, 62-79%. (f) Lawesson's reagent, l,4-dioxane, reflux, 2 h, 71%; (g) 1. DIBAL-H, DCM, -78 °C, lh, 89%, (h) 5 (Ar = 4-OMePh), NaBH(OAc)3, DCE, 1.5 h, 81%; (i) 4- 0MePhC(0)CH2Br, NaHCOs, EtOH, reflux, 12 h, 88%; (j) SnCb, EtOH:EtOAc, reflux, 2 h; (k) 7 (R = 2,3-diCl), EDC HCl, DMF, rt, 12 h, 60%; (1) 2-amino-4-nitrophenol, DABCO, NMP, 100 °C, 24 h, 60%; (m) 10% Pd/C, H 2, MeOEkEtOAc, 2-3 h; (n) 7 (R = 2,3-diCl), EDC HCl, DMF, rt, 12 h, 67%; (o) L-alanine, Cul, CS2CO3, DMF, 90 °C, 24 h; (p) 5 (Ar = 4-CNPh), HATU, DMF, rt, 12 h, 45-54%.
Example 2 - Synthesis of derivatives !8a-b !9a-b and 20-24
Scheme 2 depicts the functional group transformations to various substituents attached to the benzoxazole aryl group. Compounds 8i and 8j were subjected to alkylation reactions with ethyl 2-bromoacetate in the presence of K2CO3 and DMF to give 18a and 18b, respectively. These compounds were hydrolyzed using LiOH in THF:MeOH (1 :3) to yield 19a and 19b, respectively. Carboxylic acid 19a was also converted to ester 20 using propargyl bromide and K2CO3 in dry DMF. Ester 8h was also converted to an array of derivatives. For example, it was treated with hydroxylamine hydrochloride to provide the corresponding hydroxamic acid 21. Base catalyzed ester hydrolysis of 8h in
MeOH:THF Ή2O (3: 1 : 1) yielded carboxylic acid 22. Finally, ester 8h was converted to hydrazide 23 using hydrazine hydrate in EtOH, followed by treatment with carbonyl diimidazole (CDI) in the presence of DIPEA in DMF yielding 1,3,4- oxadiazolone 24.
Scheme 2
Figure imgf000044_0001
Reagents and conditions: (a) BrCH2COOEt, K2CO3, DMF, rt, 6 h, 80-83%; (b) LiOH, THF:MeOH, rt, 86-88%; (c) NH2OHΉO, KOH, MeOH, rt, 12 h, 65%; (d) NaOH,
THF :MeOH:H20 (3: 1 : 1), 2 h, rt, 90%; (e) NH2NH2Ή2O, EtOH, reflux, 5 h, 67%; (f) CDI, DIPEA, DMF, rt, 2 h, 66%; (g) CHºCCH2Br, K2CO3, DMF, rt, 6 h, 81%.
Example 3 - Synthesis of derivatives 25-29 from 8f
Scheme 3 illustrates the synthesis of an array of derivatives via functional group transformation of 8f. For example, 8f was treated with hydroxylamine hydrochloride in absolute ethanol in the presence of triethylamine to afford hydroxamidine 25, which upon treatment with CDI and DIPEA gave l,2,4-oxadiazolone 26. The tetrazole derivative 27 was prepared by treatment of 8f with sodium azide in the presence of ammonium chloride in dry DMF. Primary amide 28 was generated by treating 8f with /-BuOK in /-BuOH. Finally, 8f was treated with NiCh 6IT2O and NaBFE in the presence of B0C2O in MeOH:THF to give the Boc protected primary amine, which was deprotected using trifluoroacetic acid in DCM to provide 29. Scheme 3
Figure imgf000045_0001
Reagents and conditions: (a) NH2OH HCI, Et3N, EtOH, reflux, 4 h, 82%; (b) CDI, DIPEA, DMF, rt, 2 h, 61%; (c) NaN3, NFECl, DMF, 100 °C, 6 h, 62%; (d) /-BuOK, /-BuOH, 12 h, 54%; (e) 1. NiCh 6H2O, NaBH4, B0C2O, MeOH:THF, rt, 2 h, 2. TFA, DCM, rt, 4 h, 67%.
Example 4 - Synthesis of derivatives 30-33
The synthesis of 2-pyridone derivative 30 was carried out by treating 8b with LiCl and p-toluenesulfonic acid in anhydrous methanol (Scheme 4). The pyridyl of 8a was oxidized using w-CPBA in DCM to give the corresponding pyridine N-oxide 31. The nitrile in 811 was reduced using NiCh όEEO and NaBFE then protected as the corresponding t-butyl carbamate. Removal of the protecting group using trifluoroacetic acid in DCM provided amine 32. The benzyl ether of 8v was removed by hydrogenolysis in presence of Pd/C under a hydrogen atmosphere to yield 33.
Scheme 4
Figure imgf000046_0001
Reagents and conditions: (a) LiCl, p-TSA, DMF, 120 °C, 2 h, 77%; (b) w-CPBA, DCM, rt, 12 h, 74%; (c) NiCh H20, NaBH4, and then B0C2O, THF, MeOH, rt, 4 h; (d) TFA, DCM, 68%; (e) 10% Pd/C, H2, MeOH:EtOAc, rt, 3 h, 90%.
Example 5 - Synthesis of benzoxaborole 37
The synthesis of a benzoxaborole analogue is depicted in Scheme 5. A Mitsunobu reaction between 2-bromo-3-hydroxybenzaldehyde and (+)-methyl D-lactate was carried out in the presence of PPh3 and DEAD in THF to give 34 This aldehyde was treated with NaBH4 in ethanol to generate the primary alcohol, which was protected as the corresponding methoxymethyl ether 35 using methoxymethyl chloride (MOMC1) and DIPEA in DCM. Boronylation of the aryl bromide was carried out using bis(pinacolato)diboron in the presence of KOAc and a catalytic amount of Pd(Ph3P)2Cl2 in l,4-dioxane to give 36 Ester hydrolysis followed by amine coupling with 5 (Ar = 4-CNPh) in the presence of EDC HCl in DMF and then treatment with 6N HC1 in THF gave benzoxaborole 37 Scheme 5
Figure imgf000047_0001
Reagents and conditions: (a) (+)-Methyl D-lactate, DEAD, PPh3, THF, 0 °C, 2 h, 83%;
(b) NaBH4, EtOH, rt, 1 h; (c) MOMC1, DIPEA, DCM, it, 12 h, 72%; (d) Pin2B2,
Pd(Ph3P)2Cl2, KOAc, l,4-dioxane, 95 °C, 12 h, 64%; (e) Li OH, MeOH:THF, 4 h, 0 °C; (f) 5 (Ar = 4-CNPh), EDC HCl, DMF, rt, 12 h; (g) 4N HC1, THF, 4 h, 28%.
Example 6 - Synthetic procedures and characterization of certain compounds
General Procedure for the Synthesis of 5-nitro-2-phenylbenzo[d]oxazoles (4): To a stirred solution of 2-amino-4-nitrophenol (1 mmol) and aromatic aldehydes (1 mmol) in anhydrous xylene DarcoKB (300 mg) was added. The solution was stirred under 02 atmosphere at 140 °C for 6-8 h. After completion of the reaction as observed from TLC, reaction mixture was filtered with the aid of Celite, which was washed with hot ethyl acetate (3x20 mL). The filtrate was concentrated, and the products were either used directly or recrystallized using ethyl acetate and hexane.
General Procedure for the Synthesis of 5: 5-Nitro-2-arylbenzo[d]oxazole 4 (1 mmol) was dissolved in 10 mL EtOAc:MeOH (1 : 1) and 10% Pd/C (catalytic) was added and stirred well under a H2 atmosphere for 3 h. After the successful completion, the reaction mixture was filtered through celite and the filtrate was concentrated under reduced pressure. The crude amines were used directly or recrystallized using ethyl acetate and hexane.
General Procedure for the Synthesis of Phenyl Ethers 6: Substituted phenol (1 mmol) was added to the solution of methyl (+)-methyl D-lactate (1.38 mmol) in anhydrous THF (6 mL) under a nitrogen atmosphere. The solution was cooled to 0 °C, followed by PPh3 (1.20 mmol) was added portion wise to the stirred solution. DEAD (1.50 mmol) was added dropwise to the above solution and stirred well at room temperature for 4 h. After
completion, solvent was removed under reduced pressure and the crude residue was purified by column chromatography on silica gel using ethyl acetate/n-hexane (10:90) to yield corresponding phenyl ether as a colorless liquid (85-90% Yield).
General Procedure for Ester Hydrolysis for Preparation of 7: Li OH (1.5 mmol) was added portion wise to the stirred solution of ester 6 (1 mmol) in 5 mL THF:MeOH (2:3) at 0 °C. The reaction was brought to room temperature and stirred well for 4 h. The solvents were removed under reduced pressure, 1N HC1 was added to a pH of 4 and then the mixture was extracted with ethyl acetate. The organic layer was washed with brine, dried using anhydrous MgS04, filtered, and concentrated under reduced pressure. The crude acids were used in the next step without purification.
General Procedure for Synthesis of 8b-8v: EDC HCl (1.5 mmol) was added to the stirred solution of acid 7 (1 mmol) and amine 5 (1 mmol) in dry DMF at 0 °C under nitrogen atmosphere. The reaction mixture was stirred overnight at room temperature. After completion of the reaction, excess water was added and extracted with ethyl acetate. The organic layer was washed with brine and dried using anhydrous MgS04. The solvent was removed under reduced pressure and the crude residue was purified through column chromatography using ethyl acetate/n-hexane.
(A)-2-(2,3-Dichlorophenoxy)-/V-(2-(2-methoxypyridin-4-yl)benzo[d]oxazol-5- yl)propanamide (8b): White solid (306 mg, 66%), ¾ NMR (DMSO-i/e) 10.34 (s, 1H, NH), 8.93 (d, J= 2.00 Hz, 1H, CH), 8.39-8.36 (m, 1H, CH), 8.06 (d, J= 2.00 Hz, 1H, CH), 7.69 (d, J= 8.80 Hz, 1H, CH), 7.52 (dd, J= 8.90 Hz, J= 2.00 Hz, 1H, CH), 7.30-7.26 (m, 1H, CH), 7.22 (t, J= 1.20 Hz, 1H, CH), 7.02-7.00 (m, 2H, 2xCH), 4.98 (q, J= 6.80 Hz, 1H, CH),
3.92 (s, 3H, -OCH3), 1.59 (d, j= 6.80 Hz, 3H, CH3). Purity 98% (tR= 15.13). m.p. 165-166 C.
(A)-2-(2,3-dichlorophenoxy)-/V-(2-(6-methoxypyridin-3-yl)benzo[d]oxazol-5- yl)propanamide (8c): White solid (315 mg, 69%), ¾ NMR (DMSO-i/e) 10.34 (s, 1H, NH),
8.92 (m, 1H, CH), 8.39-8.37 (m, 1H, CH), 8.06-8.05 (m, 1H, CH), 7.69-7.67 (m, 1H, CH), 7.53- 7.51 (m, 1H, CH), 7.27-7.20 (m, 2H, 2xCH), 7.01-6.98 (m, 2H, 2xCH), 5.01 (q, J = 6.80 Hz, 1H, CH), 3.91 (s, 3H, -OCH3), 1.58 (d, j= 6.80 Hz, 3H, CH3). Purity 97% (tR = 14.85). m.p. 156-157 C.
(A)-2-(2,3-Dichlorophenoxy)-/V-(2-(4-methoxyphenyl)benzo[d]oxazol-5- yl)propanamide (8d): White solid (351 mg, 77%), ¾ NMR (DMSO-i/e) 10.32 (s, 1H, NH), 8.08 (d, J= 8.80 Hz, 2H, 2xCH), 8.03 (d, J= 2.00 Hz, 1H, CH), 7.65 (d, J= 8.80 Hz , 1H, CH), 7.50-7.48 (m, 1H, CH), 7.27 (t, J= 8.20 Hz, 1H, CH), 7.21-7.19 (m, 1H, CH), 7.10 (d, J= 8.80 Hz, 2H, 2xCH), 7.01-6.99 (m, 1H, CH), 4.98 (q, J= 6.80 Hz, 1H, CH), 3.82 (s, 3H, -OCH3), 1.59 (d, j= 6.80 Hz, 3H, CH3). Purity 99% (tR= 15.30). m.p. 230 C.
(»V)-2-(2,3-Dichlorophenoxy)-/V-(2-(4-(trifluoromethoxy)phenyl)benzo[d]oxazol-5- yl)propanamide (8e): White solid (357 mg, 70%), ¾ NMR (DMSO-<7f>) 10.41 (s, 1H, NH), 8.31 (d, J= 8.80 Hz, 2H, 2xCH), 8.16 (m, 1H, CH), 7.77 (d, J= 9.00 Hz, 1H, CH), 7.61 (d, J = 8.00 Hz, 2H, 2xCH), 7.59 (m, 1H), 7.32 (t, J= 8.20 Hz, 1H, CH), 7.26-7.24 (m, 1H, CH), 7.05 (d, J= 6.40 Hz, 1H, CH), 5.03 (q, J= 6.20 Hz, 1H, CH), 1.64 (d, J= 6.40 Hz, 3H,
CHs). Purity 99% (tR= 17.15). m.p. 140-141 C.
(»V)-/V-(2-(4-Cyanophenyl)benzo[d]oxazol-5-yl)-2-(2,3-dichlorophenoxy)propanamide (8f): Yellow solid (311 mg, 69%), ¾ NMR (DMSO-^) 10.38 (s, 1H, NH), 8.28 (dd, j =
8.20 Hz, J= 1.20, 2H, 2xCH), 8.14 (m, 1H, CH), 8.02 (dd, J= 8.20 Hz, J= 1.20 Hz, 2H, 2xCH), 7.75-7.73 (m, 1H, CH), 7.58-7.56 (m, 1H, CH), 7.26 (t, J= 1.60 Hz, 1H, CH), 7.20 (m, 1H, CH), 7.00 (d, J= 8.40 Hz m, 1H, CH), 5.01 (q, 1H, J= 6.80 Hz, CH), 1.58 (d, 3H, J = 6.80 Hz, CH3). Purity 99% (tR= 14.85). m.p. 183-184 C.
(»V)-2-(2,3-Dichlorophenoxy)-N-(2-(4-fluorophenyl)benzo[d]oxazol-5-yl)propanamide (8g): White solid (351 mg, 79%), ¾ NMR (DMSCMJ) 10.42 (s, 1H, NH), 8.26-8.23 (m, 2H, 2xCH), 8.13 (m, 1H, CH), 7.76-7.74 (m, 1H, CH), 7.59-7.44 (m, 3H, 3 xCH), 7.32-7.26 (m, 2H, 2xCH), 7.06-7.04 (m, 1H, CH), 5.04 (q, J= 6.00 Hz, 1H, CH), 1.64 (d, J= 6.00 Hz, 3H, CHs). Purity 98% (tR= 15.73). m.p. 213-214 C.
Methyl (»V)-4-(5-(2-(2,3-dichlorophenoxy)propanamido)benzo[d]oxazol-2-yl)benzoate (8h): White solid (329 mg, 68%), ¾ NMR (DMSO-^e) 10.37 (s, 1H, NH), 8.29-8.26 (m,
1H, CH), 8.11 (d, J= 8.80 Hz, 2H, 2xCH), 7.86 (m, 1H, CH), 7.73 (dd, J= 8.80 Hz, J= 3.20 Hz, 1H, CH), 7.56 (d, J= 8.20 Hz, 1H, CH), 7.40 (m, 1H, CH), 7.27-7.20 (m, 2H, 2xCH), 7.00 (dd, J= 8.80 Hz, J= 2.00 Hz, 1H, CH), 5.02 ( q, 1H, J= 6.80 Hz„ 1H, CH), 3.85 (s,
3H, -COOCH3), 1.59 (d, 3H, j= 6.00 Hz, CH3). Purity 99% (tR= 15.60). m.p. 197-198 C.
(»V)-2-(2,3-Dichlorophenoxy)-/V-(2-(4-hydroxyphenyl)benzo[d]oxazol-5- yl)propanamide (8i): White solid (313 mg, 71%), ¾ NMR (DMSCMJ) 10.30 (s, 1H, NH), 8.00-7.99 (m, 1H, CH), 7.97 (d, J= 8.40 Hz, 2H, 2xCH), 7.62 (d, J= 8.80 Hz, 1H, CH), 7.48-7.46 (m, 1H, CH), 7.27 (t, J= 8.00 Hz, 1H, CH), 7.00 (d, J= 8.40 Hz, 1H, CH), 6.91 (d, j= 8.80 Hz, 2H, 2xCH), 4.47 (q, j= 6.40 Hz, 1H, CH), 1.58 (d, j= 6.80 Hz, 3H, CH3). Purity 99% (tR= 13.11). m.p. 213.5-214.5 C. (»V)-2-(2,3-Dichlorophenoxy)-/V-(2-(3-hydroxyphenyl)benzo[d]oxazol-5- yl)propanamide (8j): White solid (300 mg, 68%), ¾ NMR (DMSO-^) 10.38 (s, 1H, NH), 9.75 (s, 1H, OH), 8.12 (s, 1H, CH), 7.74 (d, J= 9.20Hz, 1H, CH), 7.61-7.55 (m, 3H, 3xCH), 7.40 (t, J= 8.00 Hz, 1H, CH), 7.32 (t, J= 8.20 Hz, 1H, CH), 7.26 (m, 1H), 7.06-7.01 (m, 2H, 2xCH), 5.03 (q, j= 6.80 Hz, 1H, CH), 1.64 (d, j= 6.40 Hz, 3H, CHs). Purity 98% (tR =
13.36). m.p. 259 C.
(»V)-2-(2,3-Dichlorophenoxy)-/V-(2-(2-hydroxyphenyl)benzo[d]oxazol-5- yl)propanamide (8k): White solid (287 mg, 65%), ¾ NMR (DMSO-^) 11.10 (s, 1H, OH), 10.41 (s, 1H, NH), 8.13 (d, J= 2.00 Hz, 1H, CH), 7.97 (dd, J= 7.60 Hz , J= 1.20 Hz, 1H, CH), 7.76 (d, J= 8.40 Hz, 1H, CH), 7.55 (dd, J= 8.40 Hz, J= 2.00 Hz, 1H, CH), 7.54-7.48 (m, 1H, CH), 7.26 (t, J= 8.00 Hz, 1H, CH), 7.19 (m, 1H, CH), 7.09-7.00 (m, 3H, 3xCH), 5.00 (q, j= 6.80 Hz, 1H, CH), 1.59 (d, j= 6.60 Hz, 3H, CHs). Purity 99% (tR= 16.99). m.p. 209-210 C.
(»V)-2-(2,3-Difluorophenoxy)-/V-(2-(pyridin-4-yl)benzo[d]oxazol-5-yl)propanamide (81): White solid (268 mg, 68%), ¾ NMR (DMSO-^) 10.40 (s, 1H, NH), 8.79 (dd, j= 4.20 Hz, J= 1.20 Hz, 2H, CH), 8.16 (m, 1H, CH), 8.04 (t, J= 2.00 Hz, 2H, 2xCH), 7.77 (d, J = 8.80 Hz, 1H, CH), 7.62 (m, 1H, CH), 7.62-7.60 (m, 1H, CH), 4.96 (q, J= 6.80 Hz, 1H, CH), 1.58 (d, J= 6.80 Hz, 3H, CHs). Purity 99% (tR= 14.45). m.p. 189-190 C.
(»V)-2-(2,3-Difluorophenoxy)-/V-(2-(4-methoxyphenyl)benzo[d]oxazol-5- yl)propanamide (8m): White solid (322 mg, 76%), ¾ NMR (DMSO-i¾) 10.32 (s, 1H, NH), 8.08 (d, J= 9.20 Hz, 2H, 2xCH), 8.02 (d, J= 1.60 Hz, 1H, CH), 7.64 (d, J= 8.40 Hz, 1H, CH), 7.50 (d, J= 6.80 Hz, 1H, CH), 7.10 (d, J= 8.80 Hz, 3H, 3xCH), 6.99 (m, 1H, CH),
6.89 (t, J= 2.00 Hz, 1H, CH), 4.95 (q, J= 6.80 Hz, 1H, CH), 1.57 (d, J= 6.40 Hz, 3H, CHs). Purity 97% (tR= 13.95). m.p. 179-180 C.
(»V)-2-(2,3-Difluorophenoxy)-/V-(2-(4-hydroxyphenyl)benzo[d]oxazol-5- yl)propanamide (8n): White solid (291 mg, 71%), ¾ NMR (DMSCMJ) 10.36 (s, 1H, OH), 10.33 (s, 1H, NH), 8.04 (d, J= 2.00 Hz, 2H, 2xCH), 8.01 (m, 1H, CH), 7.67 (d, J= 8.80 Hz, 1H, CH), 7.52 (d, J= 8.20 Hz„ 1H, CH), 7.15-6.97 (m, 3H, 3xCH), 6.96 (d, J= 8.40 Hz, 2H, 2xCH), 4.99 (q, 1H, j= 6.80 Hz, CH), 1.62 (d, 3H, j= 6.40 Hz, CH3). Purity 98% (tR =
11.36). m.p. 182-183 C.
(»V)-2-(2,3-Difluorophenoxy)-/V-(2-(6-methoxypyridin-3-yl)benzo[d]oxazol-5- yl)propanamide (8o): White solid (311 mg, 69%), ¾ NMR (DMSO-<7e) 10.49 (s, 1H, NH),
8.97 (s„ 1H, CH), 8.40 (dd, j= 8.40 Hz, j= 2.00 Hz, 1H, CH), 8.11 (m, 1H, CH), 7.73 (d, J = 8.00 Hz, 1H, CH), 7.59-7.56 (m, 1H, CH), 7.19-6.98 (m, 3H, 3xCH), 6.94 (t, J= 2.40 Hz, 1H, CH), 5.00 (q, J= 6.40 Hz, 1H, CH), 3.17 (s, 3H, -OCH3), 1.63 (d, J= 6.40 Hz, 3H,
CHs). Purity 99% (tR= 13.25). m.p. 177-178 C.
(»V)-/V-(2-(4-Cyanophenyl)benzo[d]oxazol-5-yl)-2-(2,3-difluorophenoxy)propanamide (8p): White solid (289 mg, 69%), ¾ NMR (DMSO-^) 10.44 (s, 1H, NH), 8.34 (d, J= 8.40 Hz, 2H, 2xCH), 8.19 (d, J= 1.60 Hz, 1H, CH), 8.07 (d, J= 8.8 Hz, 2H, 2xCH), 7.79 (d, J =
8.80 Hz, 1H, CH), 7.65-7.62 (m, 1H, CH), 7.17-7.12 (m, 1H, CH), 7.06-7.02 (m, 1H, CH), 6.95 (t, J= 2.00 Hz, 1H, CH), 5.01 (q„ J= 6.40 Hz, 1H, CH), 1.63 (d, J= 6.8 Hz, 3H, CH3). Purity 98% (tR= 13.24). m.p. 176-177 C.
(»V)-2-(2,3-Difluorophenoxy)-/V-(2-(4-fluorophenyl)benzo[d]oxazol-5-yl)propanamide (8q): White solid (317 mg, 77%), ¾ NMR (DMSO-^) 10.40 (s, 1H, NH), 8.24 (dd, j =
8.60 Hz, J= 5.40 Hz, 2H, 2xCH), 8.13 (d, 7= 1.60 Hz,IH, CH), 7.74 (d, J= 9.20 Hz,, 1H, CH), 7.58 (dd, J= 8.80 Hz, J= 2.00 Hz, 1H, CH), 7.46 (td, J= 8.80, J= 1.00 Hz, 2H, 2xCH), 7.17-7.12 (m, 1H, CH), 7.06-7.02 (m, 1H, CH), 6.96-6.92 (m, 1H, CH), 5.00 (q, J = 6.40 Hz, 1H, CH), 1.63 (d, J= 6.80 Hz, 3H, CH3). Purity 97% (tR= 14.29). m.p. 194-195 C.
(»V)-/V-(2-(4-Cyanophenyl)benzo [d] oxazol-5-yl)-2-(2,3,4- trifluorophenoxy)propanamide (8r): White solid (270 mg, 62%), ¾ NMR (DMSO-7e) 10.42 (s, 1H, NH), 8.34 (d, J= 6.80 Hz, 2H, 2xCH), 8.19 (s, 1H, CH), 8.08 (d, J= 7.20 Hz , 2H, 2xCH), 7.80 (d, J= 8.00 Hz, 1H, CH), 7.63 (d, J= 8.40 Hz, 1H, CH), 7.27 (d, J= 10.00 Hz 1H, CH), 6.98 (m, 1H, CH), 4.98 (q, J= 6.40 Hz, 1H, CH), 1.61 (d, J= 6.80 Hz, 3H, CH3). Purity 99% (tR= 13.60). m.p. 184-185 C.
(»V)-2-(2-Cyanophenoxy)-/V-(2-(pyridin-4-yl)benzo[d]oxazol-5-yl)propanamide (8s): White solid (261 mg, 68%), ¾ NMR (DMSO-7e) 10.49 (s, 1H, NH), 8.84 (dd, J= 4.00 Hz, J= 1.60 Hz, 2H, 2xCH), 8.21 (d, J= 2.00 Hz, 1H, CH), 8.09 (dd, J= 4.40 Hz, J= 1.60 Hz, 2H, 2xCH), 7.83-7.77 (m, 2H, 2xCH), 7.68-7.63 (m, 2H, 2xCH), 7.14-7.11 (m, 2H, 2xCH), 5.11 (q, J= 6.80 Hz, 1H, CH), 1.66 ( d, J= 6.80 Hz, 3H, CH3). Purity 98% (tR= 12.67). m.p. 229-230 C.
(»V)-2-(2-Cyano-3-fluorophenoxy)-/V-(2-(pyridin-4-yl)benzo[d]oxazol-5- yl)propanamide (8t): White solid (285 mg, 71%), ¾ NMR (DMSCMJ) 10.51 (s, 1H, NH), 8.84 (dd, J= 6.00 Hz, J= 1.00 Hz, 2H, 2xCH), 8.20 (s, 1H, CH), 8.09 (dd, J= 6.00 Hz, J = 1.00 Hz, 2H, 2xCH), 7.84-7.81 (m, 1H, CH), 7.73-7.63 (m, 2H, 2xCH), 7.10 (t, J= 8.80 Hz, 1H, CH), 6.99-6.97 (d, J= 8.80 Hz, 1H, CH), 5.16 (q, J= 6.40 Hz, 1H, CH), 1.67 (d, J =
6.80 Hz, 3H, CHs). Purity 98% (tR= 13.44). m.p. 189-dec C. (A)-2-(2-Cyanophenoxy)-/V-(2-(4-methoxyphenyl)benzo[d]oxazol-5-yl)propanamide (8u): White solid (318 mg, 77%), ¾ NMR (DMSO-^) 10.41 (s, 1H, NH), 8.13 (d, J= 8.80 Hz, 2H, 2xCH), 8.07 (m, 1H, CH), 7.78 (d, J= 8.80 Hz, 1H, CH), 7.72 (m, 2H, 3 xCH), 7.54 (d, J= 8.00 Hz, 1H, CH), 7.21-7.04 (m, 4H, 4xCH), 5.09 (q, J= 6.00 Hz, 1H, CH), 3.82 (s, 3H, OCH3), 1.65 (d, j= 6.40 Hz, 3H, CH3). Purity 99% (tR = 12.99). m.p. 162-163 C.
(A)-2-(2-(Benzyloxy)phenoxy)-/V-(2-(4-cyanophenyl)benzo[d]oxazol-5-yl)propanamide (8v): White solid (337 mg, 69%), ¾ NMR (DMSO-^) 10.23 (s, 1H, NH), 8.34 (d, j= 8.00 Hz, 2H, 2xCH), 8.21 (m, 1H, CH), 8.08 (d, J= 8.00 Hz, 2H, 2xCH), 7.76 (d, J= 8.80 Hz,
1H, CH), 7.61-7.58 (m, 1H, CH), 7.50 (d, J= 7.60 Hz, 2H, 2xCH), 7.38-7.29 (m, 3H,
3 xCH), 7.08 (m, 2H, 2xCH), 6.93(m, 2H, 2xCH), 5.17 (s, 2H, -OCH2-), 4.82 (q, J= 6.40 Hz, 1H, CH), 1.57 (d, J= 6.80 Hz, 3H, CH3). Purity 99% (tR = 15.31). m.p. 144-145 C.
(A)-2-(2,3-Dichlorophenoxy)-/V-(2-(4-methoxyphenyl)benzo[d]oxazol-5- yl)propanethioamide (9): A mixture of amide 8 (Ar = 4-OMePh, R = 2,3 -diCl, 1 mmol) and Lawesson's reagent (1 mmol) was dissolved in dry dioxane (5 mL) and refluxed well for 2 h. After cooling to room temperature, dioxane was removed under reduced pressure and the crude product was purified through column chromatography. White solid (335 mg, 71%), 'H NMR (DMSO-i&) 11.72 (s, 1H, NH), 8.10-8.08 (m, 3H, 3 xCH), 7.76-7.70 (m, 1H, CH), 7.51-7.50 (m, 1H, CH), 7.32-7.29 (m, 1H, CH), 7.22-7.20 (m, 1H, CH), 7.14-7.02 (m, 2H, 2xCH), 7.02-7.00 (m, 1H, CH), 5.20 (q, J= 6.80 Hz, 1H, CH), 1.70 (d, J= 6.80 Hz, 3H, CHs). Purity 98% (tR= 17.09). m.p. 132-139 C.
(A)-2-(2,3-Dichlorophenoxy)propanal (10): DIBAL-H (1.0 M, in cyclohexane, 2 mmol) was added dropwise to the stirred solution of ester 6a (1 mmol) in dry CH2CI2 (4 mL) under a nitrogen atmosphere at -78 °C. After completion of the addition, stirring was continued at the same temperature for 1 h. Aq. HC1 (1 N, 3 mL) was added carefully. The bath was removed and the mixture was extracted with Et20 (3 x30 mL). The combined organic layers were washed with water (10 mL) and brine (10 mL) and dried over anhydrous MgSCri. The solvent was removed under reduced pressure and the crude reaction mixture was purified through column chromatography. Colorless oil (193 mg, 89%), ¾ NMR (CDCh) 9.72 (s,
1H, CH), 7.10 (m, 2H, 2xCH), 6.74-6.71 (m, 1H, CH), 4.62-4.60 (m, 1H, CH), 1.51 (d, J = 7.20 Hz, 3H, CH3).
(A)-/V-(2-(2,3-Dichlorophenoxy)propyl)-2-(4-methoxyphenyl)benzo[d]oxazol-5-amine (11): Aldehyde 10 (1 mmol) was dissolved in DCE (25 mL) at 0 °C. Benzoxazole amine 5
(Ar = 4-OMePh, 1 mmol) and sodium triacetoxyborohydride (2 mmol) were added to the above solution and stirred well. The reaction mixture was stirred for 1.5 h and quenched with satd. NaHCCb solution (20 mL), and the product was extracted with CH2CI2 (2 c 20 mL).
The organic layer was washed with brine and dried using anhydrous MgS04. The solvent was removed under reduced pressure and crude mixture was purified through column chromatography. Colorless oil (351 mg, 81%), ¾ NMK (CDCb) 8.11 (dd, J= 9.20 Hz, J = 2.00 Hz, 2H, 2xCH), 7.29 (dd, J= 8.80 Hz, J= 2.40 Hz, 1H, CH), 7.05-7.02 (m, 2H, 2xCH), 6.96 (dd, J= 9.00 Hz, J= 2.60 Hz, 2H, 2xCH), 6.92-6.91 (m, 1H, CH), 6.81-6.78 (m, 1H, CH), 6.59 (dd, j= 8.60 Hz, j= 2.20 Hz, 1H, CH), 4.64 (m, 1H, CH), 4.21 (bs, 1H, NH),
3.84 ( s, 3H, -OCH3), 3.47-3.39 (m, 2H, -CH2-), 1.39 (d, j= 6.20 Hz, 3H, CH3). Purity 98% (tR= 17.11).
2-(4-Methoxyphenyl)-6-nitroimidazo[l,2-a]pyridine (12): 5-Nitropyridin-2-amine (1 mmol) and NaHCCb (2 mmol) was added to a stirred solution of 2-bromo-4'- methoxyacetophenone (1 mmol) in ethanol at room temperature and the reaction mixture was refluxed for overnight. After completion, the solvent was removed under reduced pressure and the crude material was dissolved in water and extracted with ethyl acetate. The organic layer was washed with brine and dried using anhydrous MgSCb, filtered and concentrated under reduced pressure. The product was recrystallized using ethyl acetate and hexane. Brown solid (237 mg, 88%), ¾ NMR (DMSCMJ) 9.76 (m, 1H, CH), 8.47 (s, 1H, CH), 7.90-7.88 (m, 3H, 3xCH), 7.67-7.65 (m, 1H, CH), 7.61 (d, 2H, J= 8.80 Hz, 2xCH,
3.78 (s, 3H, -OCH3).
(A)-2-(2,3-Dichlorophenoxy)-/V-(2-(4-methoxyphenyl)imidazo[l,2-a]pyridin-6- yl)propanamide (13): To the stirred solution of 2-(4-methoxyphenyl)-6-nitroimidazo[l,2- ajpyridine 12 (1 mmol) in ethanol and ethyl acetate, SnCh (2 mmol) was added and refluxed for 2h. Solvent was removed under reduced pressure and the reaction was quenched by adding a saturated solution of NaHCCb solution and extracted with ethyl acetate. The organic phase was washed with brine, dried over anhydrous MgSCb, filtered and
concentrated under reduced pressure. The crude amine was used in the next step without any purification. EDC HCl (1.5 mmol) was added to the stirred solution of acid 7 (R = 2,3-diCl,
1 mmol) and amine (1 mmol) in dry DMF at 0 °C under a nitrogen atmosphere. The reaction mixture was stirred overnight at room temperature. After completion of the reaction, water was added and extracted with ethyl acetate. The organic layer was washed with brine and dried using anhydrous MgSCb. The solvent was removed under reduced pressure. The crude residue was purified through column chromatography to give the desired product. Brown solid (273 mg, 60%), ¾ NMR (DMSO-^) 10.35 (s, 1H, NH), 9.13 (s, 1H, CH), 8.33 (s, 1H, CH), 7.84 (d, J= 8.40 Hz, 2H, 2xCH), 7.53 (d, J= 8.00 Hz, 1H, CH), 7.24-6.92 (m, 6H, 6xCH), 5.02 (q, j= 6.40 Hz, 1H, CH), 3.79 (s, 3H, -OCH3), 1.62 (d, j= 6.40 Hz, 3H, CH3). Purity 97% (tR= 12.00). m.p. 146-147 C.
2-(4-Methoxybenzyl)-5-nitrobenzo[d]oxazole (14): DABCO (2 mmol) was added to the stirred solution of l-(2,2-dibromovinyl)-4-methoxybenzene (1 mmol) and 2-amino-4- nitrophenol (1 mmol) in NMP (5 mL) under nitrogen atmosphere. The solution was stirred well for 24 h at 100 °C. After completion, reaction was quenched by the addition of water and extracted with ethyl acetate. The organic layer was washed with brine and dried using MgSCri, filtered and concentrated under reduced pressure. The crude product was purified using column chromatography. Yellow solid (171 mg, 60%), ¾ NMR (CDCh) 8.57-8.56 (m, 1H, CH), 8.28-8.25 (m, 1H, CH), 7.57-7.55 (m, 1H, CH), 7.31-7.29 (m, 2H, 2xCH), 6.91-6.89 (m, 2H, 2xCH), 4.66 (s, 2H, -CH2-), 3.80 (s, 3H, -OCH3).
(A)-2-(2,3-dichlorophenoxy)-/V-(2-(4-methoxybenzyl)benzo[d]oxazol-5-yl)prop- anamide (15): 5-Nitro-2-arylbenzo[d]oxazoles (1 mmol) was dissolved in 10 mL
EtOAc:MeOH (1 : 1) and 10% Pd/C (catalytic) was added and stirred well under a H2 atmosphere for 2-3 h. After the successful completion, reaction mixture was filtered through celite and the filtrate was concentrated under reduced pressure. The crude amine was recrystallized using ethyl acetate and hexane. The amine was proceeded to next step without any chromatographic purification. EDC HCl (1.5 mmol) was added to the stirred solution of acid 7 (R = 2,3 -diCl, 1 mmol), amine (1 mmol) in dry DMF at 0 °C under nitrogen atmosphere. Reaction mixture was stirred well for overnight at room temperature. After completion of the reaction, excess water was added and extracted with ethyl acetate. The organic layer was washed with brine and dried using MgSCri. The solvent was removed under reduced pressure and crude residue was purified through column chromatography using ethyl acetate/n-hexane. White solid (315 mg, 67%), 1H NMR (CDCh) 8.71 (s, 1H, NH), 7.97 (bs, 1H, CH), 7.46 (d, j= 8.00 Hz, 1H, CH), 7.39 (d, j= 8.20 Hz, 1H, CH), 7.28 (d, J= 8.00 Hz, 2H, 2xCH), 7.18-7.16 (m, 2H, 2xCH), 6.91-6.86 (m, 3H, 3 xCH), 4.86 (q, J = 6.80 Hz, 1H, CH), 4.19 (s, 2H, -CH2-), 3.78 (s, 3H, -OCH3), 1.73 (d, j= 7.20 Hz, 3H, CHs). Purity 97% (tR= 14.78). m.p. 143-147 C.
Synthesis of 17: A mixture of L-alanine (163 mg, 1.82 mmol), CS2CO3 (1.19 g, 3.65 mmol), and Cul (69.7 mg, 0.36 mmol) was added to the stirred solution of l,2-dichloro-3- iodobenzene or l,2-difluoro-3-iodobenzene (1.83 mmol) in dry DMF (3 mL) under a nitrogen atmosphere. The reaction mixture was heated at 90 °C for 24 h. After completion, the reaction mixture was allowed to cool and water was added. The pH was adjusted to 3-5 by the addition of 2 N HC1 and extracted with ethyl acetate. The organic layer was washed with brine, dried over anhydrous MgS04, filtered and concentrated under reduced pressure. The crude acid 16 was used in the next step without further purification. Acid 16 (1 mmol) was added to the stirred solution of amine 5 (Ar = 4-CNPh, 1 mmol) in dry DMF at 0 °C followed by HATU (1.2 mmol) and stirred well for overnight at room temperature. After completion, excess water was added to the reaction and extracted with ethyl acetate. The organic layer was washed with brine and dried over anhydrous MgSCri, filtered, and concentrated. The residue was purified by column chromatography using ethyl acetate/n- hexane.
(A)-/V-(2-(4-Cyanophenyl)benzo [d] oxazol-5-yl)-2-((2,3- dichlorophenyl)amino)propanamide (17a): Light brown powder (202 mg, 45%), ¾NMR (DMSO rfe) 10.39 (s, 1H, NH), 8.34 (d, J= 8.00 Hz, 2H, 2xCH), 8.20 (s, 1H, CH), 8.07 (d, J = 8.40 Hz, 2H, 2xCH), 7.81 (d, J= 8.40 Hz, 1H, CH), 7.60-7.58 (m, 1H, CH), 7.18 (t, J = 8.20 Hz, 1H, CH), 6.88 (d, J= 7.60 Hz, 1H, CH), 6.65 (d, J= 8.40 Hz, 1H, CH), 5.61 (d,J = 8.00 Hz, 1H, NH), 4.26 (m, 1H, CH), 1.54 (d, J= 6.8Hz, 3H, CHs). Purity 99% (tR= 14.95). m.p. 201-202 C.
(A)-/V-(2-(4-Cyanophenyl)benzo [d] oxazol-5-yl)-2-((2,3- difluorophenyl)amino)propanamide (17b): Yellow powder (225 mg, 54%) ¾ NMR (DMSO rfe) 10.29 ( s, 1H, NH), 8.33 (d, j= 8.40 Hz, 2H, 2xCH), 8.20 (m, 1H, CH), 8.07 (d, J= 8.40 Hz, 1H, CH), 7.77 (d, J= 8.80 Hz, 1H, CH), 7.60 (d, J= 8.80 Hz, 1H, CH), 6.96 (q, J= 7.60 Hz, 1H, CH), 6.59 (m, 1H, CH), 6.51 (t, J= 7.60 Hz, 1H, CH), 5.92 (d, = 7.60 Hz, 1H, NH), 4.18 (q, j= 6.80 Hz, 1H, CH), 1.51 (d, j= 6.40 Hz, 3H, CHs). Purity 99% (tR = 12.95). m.p. 197-198 C.
Synthesis of 18: Arylbenzoxazole 8i or 8j (1 mmol) was dissolved in dry DMF (5 mL) in an oven dried round bottom flask and cooled to 0 °C. K2CO3 (2 mmol) was added to the cold solution and stirred well for ten minutes, then ethyl bromoacetate (1.5 mmol) was added and stirred well at room temperature for 6 h. After completion, the reaction was quenched with water and extracted with ethyl acetate (20 mL) and the organic layer was washed with brine. The organic layer was dried using anhydrous MgSCri, filtered and concentrated under reduced pressure. The crude residue was purified through column chromatography using hexane: ethyl acetate. Ethyl (A)-2-(4-(5-(2-(2,3-dichlorophenoxy)propanamido)benzo [d] oxazol-2- yl)phenoxy)acetate (18a): White powder (438 mg, 83%), ¾ NMR (DMSO-rfi) 10.31 (s,
1H, NH), 8.08-8.03 (m, 3H, 3 xCH), 7.66-7.64 (m, 1H, CH), 7.50-7.48 (m, 1H, CH), 7.29- 6.98 (m, 5H, 5 xCH), 4.97 (q, J= 6.40 Hz, 1H, CH), 4.87 (s, 2H, -OCH2-), 4.13 (q, J= 7.20 Hz, 2H, -OCH2-), 1.58 (d, J= 6.80 Hz, 3H, CHs), 1.17 (t, J= 7.20 Hz, 3H, CH3). Purity 99% (tR= 15.02). m.p. 144-145 C.
Ethyl(A)-2-(3-(5-(2-(2,3-dichlorophenoxy)propanamido)benzo[d]oxazol-2- yl)phenoxy)acetate (18b): White powder (422 mg, 80%), ¾ NMR (DMSO-<7e) 10.35 (s,
1H, NH), 8.09 (m, 1H, CH), 7.76 (d, J= 8.00 Hz, 1H, CH), 7.70 (d, J= 8.00 Hz, 1H, CH), 7.62-7.61 (m, 1H, CH), 7.55-7.47 (m, 2H, 2xCH), 7.27 (t, J= 6.20 Hz, 1H, CH), 7.21-7.18 (m, 2H, 2xCH), 7.02-7.00 (m, 1H, CH), 4.98 (q, J= 6.80 Hz, 1H, CH), 4.89 (s, 2H, -OCH2-), 4.15 (q, J= 7.20 Hz, 2H, -OCH2-), 1.59 (d, J= 6.40 Hz, 3H, CH3), 1.18 (t, J= 6.80 Hz, 3H, CHs). Purity 99% (tR= 13.28). m.p. 175-176 C.
Synthesis of 19: The ester 18 was subjected to ester hydrolysis using the general procedure described for 7.
(A)-2-(4-(5-(2-(2,3-Dichlorophenoxy)propanamido)benzo[d]oxazol-2- yl)phenoxy)acetic acid (19a): White powder (440 mg, 88%), ¾ NMR (DMSO-<7e) 10.36 (s, 1H, NH), 8.12 (d, J= 8.80 Hz, 2H, 2xCH), 8.08 (m, 1H, CH), 7.70 (d, J= 8.80 Hz, 1H, CH), 7.50 (m, 1H, CH), 7.32 (t, J= 8.40 Hz, 1H, CH), 7.25 (m, 1H), 7.13 (d, J= 8.80 Hz, 2H, 2xCH), 7.05 (d, j= 8.00 Hz, 1H, CH), 5.02 (q, j= 6.40 Hz, 1H, CH), 4.82 (s, 2H, -OCH2-), 1.63 (d, J= 6.40 Hz, 3H, CH3). Purity 98% (tR = 12.64). m.p. 238-239 C.
(A)-2-(3-(5-(2-(2,3-Dichlorophenoxy)propanamido)benzo[d]oxazol-2- yl)phenoxy)acetic acid (19b): White powder (430 mg, 86%), ¾ NMR (DMSO-<7e) 13.07 (bs, 1H, COOH), 10.35 (s, 1H, NH), 8.09 (m, 1H, CH), 7.76-7.70 (m, 2H, 2xCH), 7.59-7.46 (m, 3H, 3 xCH), 7.26 (t, J= 8.00 Hz, 1H, CH), 7.21-7.14 (m, 2H, 2xCH), 7.01 (d, J= 8.00 Hz, 1H, CH), 4.98 (q, j= 6.40 Hz, 1H, CH), 4.79 (s, 2H, -OCH2-), 1.59 (d, j= 6.40 Hz, 3H, CHs). Purity 97% (tR= 12.77). m.p. l9l-dec C.
Prop-2-yn-l-yl (A)-2-(4-(5-(2-(2,3-dichlorophenoxy)propanamido)benzo [d] oxa-zol-2- yl)phenoxy)acetate (20): Arylbenzoxazole 19a (1 mmol) was dissolved in dry DMF (5 mL) in an oven dried round bottom flask and cooled to 0 °C. K2CO3 (2 mmol) was added to the cold solution and stirred well for ten minutes, followed by propargyl bromide 80% solution in toluene (1.5 mmol) was added and stirred well at room temperature for 6 h. After completion, reaction was quenched with water and extracted with ethyl acetate (20 mL) and organic layer was washed with brine. The organic layer was dried using MgS04, filtered and concentrated under reduced pressure. The crude product was purified through column chromatography. White solid (435 mg, 81%), ¾ NMR (DMSCMJ) 10.37 (s, 1H, NH), 8.12 (d, J= 8.80 Hz, CH, 2xCH), 8.08 (m, 1H, CH), 7.72-7.70 (d, J= 8.80 Hz, 1H, CH), 7.55 (d, J= 8.00 Hz, 1H, CH), 7.34-7.26 (m, 2H, 2xCH), 7.17 (d, J= 8.40 Hz, 2H, 2xCH), 7.05 (d, J = 8.00 Hz, 1H, CH), 5.03-5.00 (m and s, 3H, CH, -OCH2-), 4.83 (s, 2H, -OCH2-), 3.64 (bs, 1H, CH), 1.63 (d, J= 6.00 Hz, 3H, CH3). Purity 98% (tR = 14.69). m.p. 132-133 C.
(A)-4-(5-(2-(2,3-Dichlorophenoxy)propanamido)benzo[d]oxazol-2-yl)-/V- hydroxybenzamide (21): The ester 8h (1 mmol) was dissolved in methanol and
hydroxylamine hydrochloride (2 mmol) was added at 0 °C and stirred well. KOH (3 mmol) was added to the cold solution and stirred overnight at room temperature. After completion, the solvent was removed under reduced pressure and the residue was dissolved in water and extracted with ethyl acetate. The organic layer was washed with brine and dried using anhydrous MgSCri. Solvent was removed under reduced pressure and the product was purified using column chromatography. White powder (310 mg, 65%), ¾ NMR (DMSO-ίL;) 13.29 (bs, 1H, -OH), 10.37 (s, 1H, NH), 8.26 (d, 7= 8.00 Hz, 2H, 2xCH), 8.11 (t, j= 8.40 Hz, 3H, 3 xCH), 7.74(d, J= 9.20 Hz, 1H, CH), 7.56 (d, J= 8.80 Hz, 1H, CH), 7.26 (t, J =
8.00 Hz, 1H, CH), 7.20 (m, 1H, CH), 7.00 (d, J= 8.00 Hz, 1H, CH), 4.99 (q, J= 6.80 Hz,
1H, CH), 1.59 (d, J= 6.80 Hz, 3H, CH3). Purity 98% (tR = 13.37). m.p. 253-254 C.
(A)-4-(5-(2-(2,3-Dichlorophenoxy)propanamido)benzo[d]oxazol-2-yl)benzoic acid (22): Ester 8h (1 mmol) was dissolved in THF :MeOH:H20 (3 : 1 : 1) at room temperature and NaOH (1 mmol) was added and stirred well for 2 h. After completion, the solvents were removed under reduced pressure. The crude residue was dissolved in water and pH was adjusted to 3-5 by the addition of 2N HC1 and extracted with ethyl acetate. The organic layer was washed with brine and dried using anhydrous MgS04. The crude acid was recrystallized using ethyl acetate and hexane. White powder (423 mg, 90%), 'H NMR (DMSO-r/^) 10.43 (s, 1H, NH), 8.30 (d, j= 8.40 Hz, 2H, 2xCH), 8.15 (t, j= 9.00, 3H, 3 xCH), 7.79 (d, j= 8.80 Hz, 1H, CH), 7.61 (d, J= 8.80 Hz, 1H, CH), 7.32 (t, J= 8.20 Hz, 1H, l xCH), 7.26-7.24 (m, 1H, CH), 7.05 (d, J= 8.00 Hz, 1H, CH), 5.04 (q, J= 6.80 Hz, 1H, CH), 1.64 (d, J= 6.40 Hz, 3H, CHs). Purity 98% (tR = 13.33). m.p. 244 C.
(A)-2-(2,3-Dichlorophenoxy)-/V-(2-(4-(hydrazinecarbonyl)phenyl)benzo[d]oxazol-5- yl)propanamide (23): Hydrazine hydrate (3 mmol) was added to the stirred solution of 8h
(1 mmol) in ethanol (10 mL) at room temperature and refluxed for 5 h. After completion, the solvent was removed under reduced pressure, water was added and extracted with ethyl acetate. The organic layer was washed with brine and dried using anhydrous MgSC The solvent was removed under reduced pressure and the crude mixture was purified through column chromatography. White powder (324 mg, 67%), ¾ NMR (DMSO-i/e) 10.41 (s, 1H, NH), 9.97 (s, 1H, NH), 8.25 (d, J= 6.80 Hz, 2H, 2xCH ), 8.16 (m, 1H, CH), 8.03 (d, J =
6.80 Hz, 2H, 2xCH), 7.77 (d, J= 9.2 Hz, 1H, CH), 7.60 (d, J= 8.40 Hz, 1H, CH), 7.32 (t, J = 7.60, 1H, CH), 7.25 (m, 1H), 7.05 (d, J= 8.40 Hz, 1H, CH), 5.03 (q, J= 6.80 Hz, 1H, CH), 4.60 (bs, 2H, ML·), 1.58 (d, j= 6.80 Hz, 3H, Cft). Purity 97% (tR= 10.37). m.p. 247-248 C.
(»V)-2-(2,3-Dichlorophenoxy)-/V-(2-(4-(5-oxo-4,5-dihydro-l,3,4-oxadiazol-2- yl)phenyl)benzo[d]oxazol-5-yl)propanamide (24): To the stirred solution of 23 (1 mmol) in anhydrous DMF (2 mL), carbonyldiimidazole (1.2 mmol) and diisopropylethylamine (1.2 mmol) was added. The reaction mixture was stirred for 2 h at room temperature. The reaction mixture was diluted with water and extracted with ethyl acetate. The combined extract was washed with brine, dried using anhydrous MgSCri, filtered and concentrated. The residue was purified through column chromatography (2-8% MeOH gradient in CH2CI2) to give the title compound. White solid (336 mg, 66%), ¾ NMR (DMSO-i/e) 12.78 (s, 1H, NH), 10.44 (s, 1H, NH), 8.33 (d, j= 7.60 Hz, 2H, 2xCH), 8. l7(m, 1H, CH), 8.01 (d, j =
8.00 Hz, 2H, 2xCH), 7.78 (d, J= 5.20 Hz, 1H, CH), 7.61 (d, J= 8.80 Hz, 1H, CH), 7.32 (t, J = 8.40 Hz, 1H, CH), 7.25 (m, 1H, CH), 7.06 (d, J= 8.00 Hz, 1H, CH)„ 5.04 (q, J= 6.80 Hz, 1H, CH), 1.64 (d, j= 6.00 Hz, 3H, CH3). Purity 99% (tR = 13.05). m.p. 288-dec C.
(,V,Z)-2-(2,3-Dichlorophenoxy)-/V-(2-(4-(/V- hydroxycarbamimidoyl)phenyl)benzo[d]oxazol-5-yl)propanamide (25): (S)-N-( 2-(4- cyanophenyl)benzo[d]oxazol-5-yl)-2-(2,3-dichlorophenox-y)propanamide 8f (1 mmol) was dissolved in anhydrous ethanol (5 mL) followed by hydroxylamine hydrochloride (3 mmol) and triethylamine (6 mmol) was added. The reaction mixture was refluxed for 4 h and the solvent was removed under reduced pressure. The residue was suspended in water and extracted with DCM (20 mL). Organic layer was washed with brine and dried using anhydrous MgS04. The solvent was removed under reduced pressure and the crude product was purified through column chromatography (2-8% MeOH gradient in CH2CI2) to give the title compound. White solid (396 mg, 82%), ¾ NMR (DMSO-rL) 10.34 (s, 1H, NH), 9.85 (s, 1H, OH), 8.13-8.08 (m, 2H, 2xCH), 7.86-7.84 (m, 2H, 2xCH), 7.71-7.68 (m, 1H, CH), 7.53-7.51 (m, 1H, CH), 7.28-7.18 (m, 3H, 3 xCH), 7.00-6.98 (m, 1H, CH), 5.90 (s, 2H, ML·),
4.97 (m, 1H, CH), 1.57 (m, 3H, CH3). Purity 97% (tR = 14.97). m.p. 196-197 C. (A)-2-(2,3-Dichlorophenoxy)-/V-(2-(4-(5-oxo-4,5-dihydro-l,2,4-oxadiazol-3- yl)phenyl)benzo-[d]oxazol-5-yl)propanamide (26): To the stirred solution of 25 (1 mmol) in anhydrous DMF (2 mL), carbonyldiimidazole (1.2 mmol) and diisopropylethylamine (1.2 mmol) was added. The reaction mixture was stirred for 2 h at room temperature. The reaction mixture was diluted with water and extracted with ethyl acetate. The combined extract was washed with brine, dried using anhydrous MgSCri, filtered and concentrated. The residue was purified through column chromatography (2-8% MeOH gradient in CH2CI2) to give the title compound. White solid (321 mg, 61%), ¾ NMR (DMSO-i/e) 10.49 (s, 1H, NH), 9.60 (bs, 1H, NH), 8.32 (d, J= 8.40 Hz, 2H, 2xCH), 8.15 (m, 1H, CH), 7.99 (d, J = 8.40 Hz, 2H, 2xCH), 7.72 (m, 1H, CH), 7.58 (m, 1H, CH), 7.27 (t, J= 7.60 Hz, 1H, CH), 7.21 (m, 1H, CH), 7.02 (d, j= 8.40 Hz, 1H, CH), 5.03 (q, j= 6.80 Hz, 1H, CH), 1.59 (d, j = 6.80 Hz, 3H, CHs). Purity 98% (tR= 13.04). m.p. 260 C.
(A)-/V-(2-(4-(lH-Tetrazol-5-yl)phenyl)benzo[d]oxazol-5-yl)-2-(2,3- dichlorophenoxy)propanamide (27): A mixture of (S)-N-( 2-(4- cyanophenyl)benzo[d]oxazol-5-yl)-2-(2,3-dichlorophenoxy)propanamide 8f (1 mmol), NaN3 (2 mmol), and NH4Cl (2 mmol) in DMF (1.5 mL) was heated at 100 °C for 6 h. Solvent was removed under reduced pressure, water was added and extracted with ethyl acetate. The organic layer was washed with brine, dried using anhydrous MgSCri, filtered and
concentrated under reduced pressure. The crude residue was purified through column chromatography. White solid (306 mg, 62%), ¾ NMR (DMSCMJ) 1 1.1 1 (S, 1H, NH), 10.38 (s, 1H, NH), 8.36-8.34 (m, 1H, CH), 8.22 (d, J= 5.80 Hz, 2H, 2xCH), 8.13 (m, 1H, CH), 7.90 (m, 1H, CH), 7.76-7.70 (m, 1H, CH), 7.59-7.50 (m, 1H, CH), 7.30-7.21 (m, 2H, 2xCH), 7.00 (m, 1H, CH), 5.01 (m, 1H, CH), 1.59 (m, 3H, CH3). Purity 99% (tR= 12.25). m.p. 257 C.
(A)-4-(5-(2-(2,3-Dichlorophenoxy)propanamido)benzo[d]oxazol-2-yl)benzamide (28): To the stirred solution of fV)-A-(2-(4-cyanophenyl)benzo[d]oxazol-5-yl)-2-(2,3- dichlorophenoxy)prop-anamide 8f (1 mmol) in dry tert-butyl alcohol (4 mL/mmol), KOtBu (3 mmol), was added. The reaction mixture was stirred at room temperature for 12 h under a nitrogen atmosphere, and progress of the reaction was monitored by TLC. Upon completion, the reaction mixture was quenched with water and extracted with ethyl acetate. The organic layer was washed with brine, dried using anhydrous MgSCri and solvent was removed under reduced pressure. The crude mixture was purified through column chromatography. White solid (253 mg, 54%), ¾ NMR (DMSO-^) 10.36 (s, 1H, NH), 8.21 (d, j= 8.00 Hz, 2H, 2xCH), 8.12 (s, 2H, NH2), 8.03 (d, J= 8.00 Hz, 2H, 2xCH), 7.73 (d, J= 8.40 Hz, 1H, CH), 7.55 (d, J= 8.40 Hz, 1H, CH), 7.51 (m, 1H, CH), 7.26 (t, J= 8.00 Hz, 1H, CH), 7.20 (m,
1H), 7.00 (d, J= 8.40 Hz, 1H, CH) 4.98 (q, J= 6.80 Hz, 1H, CH), 1.59 (d, J= 6.80 Hz, 3H, CHs). Purity 98% (tR= 11.56). m.p. 232 C.
(A)-/V-(2-(4-(Aminomethyl)phenyl)benzo[d]oxazol-5-yl)-2-(2,3- dichlorophenoxy)propanamide (29): To a stirred solution of (S)-N-( 2-(4- cyanophenyl)benzo[d]oxazol-5-yl)-2-(2,3-dichloroph-enoxy)propanamide 8f ( 2.0 mmol) in THF: MeOH (15 mL), B0C2O (873 mg, 4.0 mmol) and NiCl2-6H20 (48 mg, 0.2 mmol) were added at 0 °C and stirred for five minutes. After five minutes, NaBH4 (530 mg, 14.0 mmol) was added in small portions over 30 min followed by stirring at room temperature for 2h.
The reaction mixture was filtered through celite and the filtrate was concentrated under reduced pressure. The crude residue was poured into 1N HC1 and extracted with ethyl acetate. The organic layer was washed with brine and dried using anhydrous MgSCri. Solvent was removed under reduced pressure and crude reaction mixture was purified through column chromatography. Trifluoroacetic acid (1 mL) was added to the stirred solution of Boc protected amine (1 mmol) in dry DCM (5 mL) at 0 °C. After 4 h, the reaction was quenched with a saturated NaHCCh solution and extracted with DCM. The organic layer was washed with brine and dried using anhydrous MgSCh. The solvent was removed under reduced pressure and the product was recrystallized using ethyl acetate and hexane. White solid (305 mg, 67%), ¾ NMR (DMSO-^e) 10.40 (s, 1H, NH), 8.14-8.10 (m, 3H, 3 xCH),
7.73 (d, J= 9.20 Hz , 1H, CH), 7.58-7.56 (m, 3H, 3 xCH), 7.32 (t, J= 8.20 Hz, 1H, CH), 7.26-7.24 (m, 1H, CH), 7.05 (d, J= 8.00 Hz, 1H, CH), 5.03 (q, J= 6.80 Hz, 1H, CH), 3.82 (s, 3H, -OCH3), 1.64-1.63 (d, j= 6.00 Hz, 3H, CH3). Purity 97% (tR= 14.63). m.p. l89-dec C.
(A)-2-(2,3-Dichlorophenoxy)-/V-(2-(2-oxo-l,2-dihydropyridin-4-yl)benzo[d]oxazol-5- yl)propanamide (30): To the stirred solution of 8b (1 mmol) in DMF, LiCl (5 mmol) and p- TSA (5 mmol) was added and stirred well for 2 h at 120 °C. After completion, reaction was quenched with saturated NaHCCh solution and extracted with ethyl acetate. The organic layer was washed with brine and dried using MgSCh. Solvent was removed under reduced pressure and crude residue was purified through column chromatography. White solid (341 mg, 77%), ¾ NMR (DMSO-^) 12.20 (bs, 1H, NH), 10.30 (s, 1H, NH), 8.19 (s, 1H, CH), 8.02 (dd, J= 9.80 Hz, J= 2.20 Hz, 1H, CH), 7.98 (m, 1H, CH), 7.61 (d, J= 9.20 Hz, 1H,
CH), 7.46 (m, 1H, CH), 7.27 (t, J= 8.00 Hz, 1H, CH), 7.19 (m, 1H), 6.99 (d, J= 8.80 Hz, 1H, CH), 6.47 (d, J= 9.60 Hz, 1H, CH), 4.97 (q, J= 6.40 Hz, 1H, CH), 1.58 (d, J= 6.80 Hz, 3H, CH3). Purity 97% (tR = 10.25). m.p. 240-241 C.
(A)-4-(5-(2-(2,3-Dichlorophenoxy)propanamido)benzo[d]oxazol-2-yl)pyridinel-oxide (31): w-Chloroperoxybenzoic acid (2 mmol) was added to the stirred solution of 8a (1 mmol) in dry DCM at rt and stirred well for overnight. After completion, reaction was quenched with saturated NaHCCb solution and extracted with DCM. The organic layer was washed with brine, dried using MgSCri, filtered and concentrated under reduced pressure.
The crude product was purified through column chromatography. White solid (327 mg,
74%), ¾ NMR (DMSO-i&) 10.43 (s, 1H, NH), 8.38 (d, J= 6.80 Hz, 2H, 2xCH), 8.17 (m,
1H, CH), 8.09 (d, J= 6.80 Hz, 2H, 2xCH), 7.78-7.76 (m, 1H, CH), Ί.63-Ί.60 (m, 1H, CH), 7.16-7.12 (m, 1H, CH), 7.06-7.02 (m, 1H, CH), 6.95-6.92 (m, 1H, CH), 5.00 (q, J= 6.80 Hz, 1H, CH), 1.62 (d, j= 6.80 Hz, 3H, CH3). Purity 99% (tR = 10.80). m.p. l25-dec C.
(A)-2-(2-(Aminomethyl)phenoxy)-/V-(2-(4-methoxyphenyl)benzo[d]oxazol-5- yl)propanamide (32): The reaction was proceeded with 8u using the procedure, which used for the synthesis of 29. White solid (283 mg, 68%), ¾ NMR (DMSO-r/^) 11.10 (bs, 1H,
NH), 8.13-8.11 (d, J= 9.2 Hz, 2H, 2xCH), 8.02-8.01 (m, 1H, CH), 7.67 (d, J= 8.2 Hz, 1H, CH), 7.54-7.51 (m, 1H, CH), 7.29-7.14 (m, 3H, 3 xCH), 7.04-7.02 (m, 1H, CH), 6.93-6.91 (m, 1H, CH), 5.06 (q, j= 6.80 Hz, 1H, CH), 3.71 (s, 3H, -OCH3), 1.62 (d, j= 6.80 Hz, 3H, CH3). Purity 97% (tR= 14.31). m.p. 160-161 C.
(A)-/V-(2-(4-Cyanophenyl)benzo[d]oxazol-5-yl)-2-(2-hydroxyphenoxy)propanamide (33): Compound 8v (1 mmol) was dissolved in 10 mL EtOAc: MeOH (1 : 1) and 10% Pd/C (catalytic) was added and stirred well under a H2 atmosphere for 3 h. After the successful completion, reaction mixture was filtered through celite and the filtrate was concentrated under reduced pressure. The crude product was purified through column chromatography. White solid (359 mg, 90%), ¾ NMR (DMSO-i/e) 10.26 (s, 1H, NH), 9.29 (s, 1H, OH), 8.34 (d, J= 8.4 Hz, 2H, 2xCH), 8.22-8.21 (m, 1H, CH), 8.08 (d, J= 8.80 Hz, 2H, 2xCH), 7.81 (d, J= 8.80 Hz, 1H, CH), 7.68-7.66 (m, 1H, CH), 7.05-7.03 (d, J= 7.6 Hz, 1H, CH), 6.88-6.83 (m, 2H, 2xCH), 6.71-6.73 (m, 1H, CH), 4.87 (q, J= 6.80 Hz, 1H, CH), 1.57 (d, J= 6.80 Hz, 3H, CH3). Purity 99% (tR = 11.10). m.p. 185-186 C.
Methyl (A)-2-(2-bromo-3-formylphenoxy)propanoate (34): 2-Bromo-3- hydroxybenzaldehyde (1 mmol) was added to the solution of (+)-methyl-D-lactate (1.38 mmol) in anhydrous THF (6 mL) under a nitrogen atmosphere and the solution was cooled to
0 °C. PPh3 (1.20 mmol) was added portion wise and stirred for 10 min followed by the dropwise addition of DEAD (1.50 mmol) over 20 min. The reaction mixture was stirred for 2 h at room temperature. After completion, the solvent was removed under reduced pressure and the crude mixture was purified through column chromatography using ethyl acetate/n- hexane (10:90) to yield methyl ((S)-2-(2-bromo-3-formylphenoxy)propanoate (236 mg,
83%) as a white solid.
Methyl (A)-2-(2-bromo-3-((methoxymethoxy)methyl)phenoxy)propanoate (35): Under a nitrogen atmosphere, NaBEE (1.5 mmol) was added to the stirred solution of ((S)-2-(2- bromo-3-formylphenoxy)propanoate 34 (1 mmol) in ethanol at 0 °C. After 1 h at room temperature, the reaction was quenched with a saturated NaHCCh solution and extracted with ethyl acetate. The organic layer was washed with brine and dried using anhydrous MgSCri, filtered and concentrated under reduced pressure. The crude product was dissolved in dry DCM and cooled to 0 °C. DIPEA (5 mmol) was added, followed by dropwise addition of MOMC1 (3 mmol) and stirred for overnight at room temperature. After completion, the reaction was quenched with saturated NEECl and extracted with DCM. The organic layer was dried using anhydrous MgSCE and the solvent was removed under reduced pressure. The crude residue was purified through column chromatography. White solid (239 mg, 72%), 'H NMR (DMSO-i&) 7.32-7.28 (m, 1H, CH), 7.12-7.09 (m, 1H, CH), 6.90-6.88 (m, 1H, CH), 5.06 (m, 1H, CH), 4.70 (bs, 3H, -OCH3), 4.61-4.51 (m, 4H, 2x-OCH2-), 3.68 (s, 3H, - COOCH3), 1.55 (m, 3H, CH3).
Methyl (A)-2-(3-((methoxymethoxy)methyl)-2-(4,4,5,5-tetramethyl-l,3,2-dioxa- borolan-2-yl)phenoxy)propanoate (36): To a solution of methyl (S)-2-(2-bromo-3- ((methoxymethoxy)methyl)phenoxy)propanoate 35 (400 mg, 1.145 mmol) in l,4-dioxane (6 mL) was added KOAc (483 mg, 4.923 mmol), Pin2B2 (349 mg, 1.37 mmol) and
Pd(Ph3P)2Cl2 (80 mg, 0.114 mmol) under an argon atmosphere. The reaction flask was placed under vacuum and then backfilled with argon (two times). Then the reaction was stirred at 95 °C for overnight. The solvent was removed under reduced pressure and the residue was suspended in water and extracted with ethyl acetate. The organic layer was washed with brine and dried over anhydrous MgSCri, filtered and concentrated. The crude product was purified through column chromatography using hexane/ethyl acetate. White solid (243 mg, 64%), ¾ NMR (DMSO-^e) 7.28-7.24 (m, 1H, CH), 6.93-6.88 (m, 1H, CH), 6.68-6.66 (m, 1H, CH), 4.90-4.86 (m, 1H, CH), 4.56 (bs, 4H, 2x-OCH2-), 4.47 (bs, 3H, - OCH3), 3.67 (s, 3H, -COOCH3), 1.45 (m, 3H, CH3), 1.29 (bs, 12H, 4xCH3). (A)-/V-(2-(4-Cyanophenyl)benzo[d]oxazol-5-yl)-2-((l-hydroxy-l,3-dihydrobenzo- [c][l,2]oxaborol-7-yl)oxy)propanamide (37): Ester 36 (1 mmol) was dissolved in 5 mL THF: MeOH (2:3) and stirred at 0 °C. LiOH (1.5 mmol) was added portion wise to the solution and stirred well at room temperature for 4 h. The solvents were removed under reduced pressure, crude residue was dissolved in ethyl acetate and 1N HC1 was added until a pH of 4 was reached. The product was extracted with ethyl acetate and the organic extracts were combined, washed with brine, dried over anhydrous MgS04. The solvent was removed under reduced pressure and crude acid was used in the next step without chromatographic purification. EDC HCl (1.5 mmol) was added to the stirred solution of acid (1 mmol), amine 5 (Ar = 4-CNPh, 1 mmol) in dry DMF at 0 °C under nitrogen atmosphere. Reaction mixture was stirred well for overnight at room temperature. After completion of the reaction, excess water was added and extracted with ethyl acetate. The organic layer was washed with brine and dried using MgSCri. The solvent was removed under reduced pressure. The crude residue was purified through column chromatography give desired coupled product. To a solution of this MOM protected material (140 mg, 0.363 mmol) in THF (0.9 mL) 4 N HC1 (0.43 mL,
18.1 mmol) was added. The reaction was stirred at room temperature for 4 h, upon completion ethyl acetate was added and extracted. The organic phase was washed with brine, dried over anhydrous MgS04, filtered and concentrated under reduced pressure. The crude product was purified through column chromatography using hexane/ethyl acetate. Brown solid (122 mg, 28%).1H NMR (DMSO-^) 10.13 (s, 1H, NH), 9.20 (s, 1H, OH), 8.34 (d, J = 8.4 Hz, 2H, 2xCH), 8.22 (m, 1H, CH), 8.08 (d, J= 8.40 Hz, 2H, 2xCH), 7.80 (d, J= 9.2 Hz, 1H, CH), 7.70 (m, 1H, CH), 7.45 (m, 1H, CH), 7.04 (d, J= 7.60 Hz, 1H, CH), 6.93 (d, J = 7.60 Hz, 1H, CH), 5.03 (q, J= 6.80 Hz, 1H, CH), 4.98 (s, 2H, -OCH2-), 1.62 (d, J= 6.8 Hz, 3H, CH3). Purity 97% (tR = 11.27). m.p. hygroscopic.
Example 7 - Evaluation of MtbIMPDH2 inhibition.
Initially, modifications of the pyridyl of 2 were evaluated (Table 3) for MtbIMPDH2 inhibition. Pyridine A -oxide 31 demonstrated comparable activity to the parent compound 2.
Addition of a methoxy to the pyridyl 8b and 8c resulted in 5-fold more inhibition than 2.
However, changing pyridyl to 2-pyridone (30) reduced inhibitory activity by 3-fold.
Replacing pyridyl with phenyl having both electron donating groups (EDG) and electron withdrawing groups (EWG) was evaluated. Compounds 8d (4-OMe), 8e (4-OCF3) and 8f (4-
CN) resulted in 7-fold increase in inhibitory activity compared to 2. Fluorine (8g) and ester (8h) substituents were also tolerated. Replacing the pyridyl with a 4-hydroxy phenyl (8i) resulted in a 2-fold increase in inhibitory activity, whereas the 3-hydroxy (8j) and 2-hydroxy (8k) derivatives showed slightly decreased potency. Given the improved activity of 8d, the effect of other ethers was examined. For example, 18a and propargyl ester 20 shows a -12- fold increase in activity compared to 2, while the corresponding carboxylic acid (19a) and tetrazole (27) were moderately less potent. Translocating the alkoxy from the para to me la position resulted in a 4-fold loss in activity (18b and 19b). Compounds containing hydrophilic substituents at the 4-position, such as hydroxamic acid 21, carboxylic acid 22 and aminomethyl 29, were found to be much less active. Replacing the carboxylate with hydrazide 23, l,3,4-oxadiazolone 24, hydroxamidine 25, l,2,3-oxadiazolone 26 and primary amide 28 showed 2 to 7-fold increase in activity.
Table 3. f MPDFLZACBS Inhibition of Benzoxazole Derivatives Containing Various Aryl
Substituents.
Figure imgf000064_0001
Figure imgf000064_0002
Figure imgf000065_0002
a. Values are the average and range of at least two independent determinations. * Single determination.
Several 2,3-dichlorophenyl compounds (2, 8c, 8d, 8f, 8g and 8i) were compared to the corresponding 2,3-difluorophenyl analogues (81, 8o, 8m, 8p, 8p and 8n). Three of these compounds (81, 8p and 8q) displayed similar activity whereas the others were 2 to 4-fold less active. Adding another substitution on the 4-position (8r) resulted in slightly reduced activity. Replacement of the 2-chlorophenyl with a 2-cyanophenyl (8s and 8t) was well tolerated. In addition, compounds with 2-cyano (8u), 2-aminomethyl (32), and 2-hydroxy (33), and 2- benzyloxy (8v) substituents on the phenyl ether demonstrated
Mth I M P D H2 AC B S AVpp values < 45 nM. However, the benzoxaborole (37) was not as well tolerated.
Table 4. Mth I M P D H2 AC B S Inhibition of Benzoxazole Derivatives Containing Various
Phenyl Ether Substituents.
Figure imgf000065_0001
Figure imgf000066_0001
a. Values are the average and range of at least two independent determinations. * Single determination, error of the fit is listed.
Several other areas of the Q-series were examined (Table 5). For example, the amide was replaced with a thioamide (9), which demonstrated a modest loss of activity. This decrease in potency is likely due a loss of a hydrogen bond to water, while retaining the ionic-dipole interaction between the thioamide NH and the carboxylate of Glu458.
Furthermore, replacement of the amide with an amine (11) was not tolerated. In addition, activity was significantly decreased when the benzoxazole was replaced with an
imidazo[l,2-a]pyridine (13). Adding a methylene between the phenyl and benzoxazole (15) also reduced activity by a factor of 6 compared to 8d. However, replacing the aryl ether with aniline (17a and 17b) resulted in increased inhibitory activity. Table 5. Mth I M P D H2 AC B S Inhibition of Other Benzoxazole Derivatives.
Figure imgf000067_0002
a. Values are the average and range of at least two i
Figure imgf000067_0001
determinations. * Single determination.
Example 8 - Evaluation of Selectivity of inhibition
The cofactor binding sites are widely diverged in bacterial and eukaryotic IMPDHs, and the / MPDE12 inhibitors exploit this divergence. Only four compounds, 8f, 8p, 17a and 24, modestly inhibited human IMPDH2, and in these cases selectivity ranged from a factor of 200 to 1000 (Table 5). No inhibition of human IMPDH2 was observed for the other compounds (maximum concentration tested was 5 mM). GMP reductase (GMPR) is closely related to IMPDH, and catalyzes a similar reaction, the reduction of GMP by NADPH to produce IMP, NADP+ and ammonia. The adenosine site of human GMPR2 contains the Ala- Tyr motif that characterizes the inhibitor binding site of / MPDH2. However, none of the compounds inhibited human GMPR2 (maximum concentration tested was 5 mM). These experiments demonstrate that compounds selectively inhibit bacterial IMPDHs and do not affect related host enzymes.
Example 9 - Evaluation of antibacterial activity
Antibacterial activity was determined for A///4MPDH2ACBS inhibitors with /ri.app < 40 nM and selected additional compounds by monitoring the growth oiMtb H37Rv (ATCC 27294) after one week (Table 6). Since in vitro antibiotic efficacy can vary unpredictably with growth conditions, antibacterial activity was assessed in both GAST/Fe and
7H9/ ADC/Tween media, both of which lack purines (e.g. -Gua). Two compounds, 18a and 20, displayed MICs less than 1 mM in both media. An additional 8 compounds, 8f, 81, 8m,
8p, 8u, 17a, 17b and 18b, displayed MIC < 5 mM in both media. Six of these compounds retained antibacterial activity over two weeks: 81, 8u, 17a, 17b, 18a and 20. Two compounds (21 and 22) were active in GAST/Fe medium (MIC < 5 mM) but considerably less effective in 7H9/ ADC/Tween medium. These compounds also retained activity over two weeks. Table 6. Antitubercular activity of / MPDH2 inhibitors against Mth H37Rv.
Figure imgf000068_0001
The number of independent determinations is shown in parentheses a. MICs determined after 1 week in culture b. Values from Makowska-Grzyska et al 9. +Gua, 200 mM guanine.
Figure 2 shows the dependence of antibacterial activity on inhibition of
Mth I M P D H2 AC B S for the Q compounds described above as well as those reported previously. Uptake and metabolism also play important roles in antibacterial efficacy, so it is not surprising that some potent / MPDH2ACBS inhibitors fail to display antibacterial activity. Nonetheless, more potent enzyme inhibition is generally associated with greater antibacterial activity, as expected if antibacterial activity derived from inhibition of
MtbIMPDH2.
Many bacteria have the ability to salvage guanine or guanosine, and thus overcome inhibition of IMPDH. Mtb can salvage guanine, but not guanosine, and previously reported IMPDH inhibitors were much less effective in the presence of high guanine concentrations. For example, the MIC values of indazole sulfonamide inhibitors of / MPDF12 increased more than 25-fold in 100-125 mM guanine. However, the MIC of only one of the new Q compounds, 8n, increased substantially in the presence of guanine in both media.
Intriguingly, this compound was one of the weaker inhibitors of / MPDH2 (/riapp = 121 nM). The MIC of one additional compound, 8c (Ki, app = 20 nM), increased in the presence of guanine in 7H9/ ADC/Tween medium but not in GAST/Fe. The failure of guanine to decrease the antibacterial activity of most Q compounds would usually suggest that antibacterial activity derives from the engagement of another target. Alternatively, guanine salvage may not be sufficient to support growth in the presence of potent / MPDH2 inhibition. It is also possible that the Q inhibitors stabilize/disrupt a protein complex, thereby perturbing a moonlighting activity of / MPDH2 in addition to enzyme activity.
Two experiments were performed to further address whether antibacterial activity derived from on-target inhibition of / MPDH2. First, the effect of / MPDH2 depletion on the antibacterial activity of four / MPDH2 inhibitors was evaluated, including one compound with guanine-dependent antibacterial activity (1), and three compounds with guanine-independent antibacterial activity (17b, 18a and 22; note that 22 is only active in GAST/Fe). The downregulation of / MPDH2 was achieved using strain guaB2 cKD, in which guaB2 expression is suppressed by anhydrotetracycline (ATc). The antibacterial activities of 1, 17b, 18a, and 22 against the wild-type strain were not affected by the addition of ATc (Table 6). In contrast, treatment with ATc hypersensitized the guaB2 cKD strain to all four compounds (Figs. 2A-2D), suggesting that antibacterial activity derives primarily from the inhibition of / MPDH2.
The antibacterial activity of 1, 17b, 18a, and 22 was also assessed against Mtb strain
SRMV2.6. This strain expresses the mutant / MPDH2/Y487C, which is resistant to an isoquinoline sulfonamide inhibitor. As noted above, Tyr487 interacts with the benzoxazole group, so the substitution of Cys is expected to disrupt the binding of all the Q inhibitors.
SRMV2.6 was resistant to 1 and 17b (Table 7), further confirming that the antibacterial activity of these compounds resulted from on-target inhibition of A///4MPDH2 Interestingly, however, SRMV2.6 remained sensitive to 18a and 22. H37Rv is the wild-type strain b. SRMV2.6 contains / MPDH2/Y487C, which confers resistance to the isoquinoline sulfonamide inhibitor VCC234718.
The inhibition of recombinant / MPDH2/Y487C was measured to determine if the Y487C mutation decreased the affinity of all the Q compounds as expected (Table 7). The values of /ri.app for 1 and 17b were increased by more than 300-fold and 60-fold,
respectively, which can account for the resistance of strain SRMV2.6. These observations further confirm that the antibacterial activity of 1 and 17b derive from inhibition of
/ MPDH2. The values of Ki, app for 18a and 22 were similarly increased in
/ MPDH2/Y487C, by at least 2000-fold and approximately 40-fold, respectively. These observations suggest that strain SRMV2.6 should also be resistant to 1 and 18a, yet it remained sensitive to these compounds. Perhaps 1 and 18a are concentrated or metabolized in the bacteria, or perhaps these compounds interfere with a moonlighting function.
Alternatively, these compounds may engage an additional target(s). Irrespective of the ambiguous mechanism of action of 1 and 18a, 17b demonstrates that on-target inhibition of / MPDH2 can be impervious to guanine rescue, suggesting that it is a vulnerable target.
Table 7. Antibacterial activity against SRMV2.6.
Figure imgf000070_0001
a. Inhibition of purified wild-type / MPDH2. b. Inhibition of purified
/ MPDH2/Y487C c. Bacteria were cultured in 7H9/Glycerol/OADC/Tween and growth was measured with AlamarBlue as previously described, +ATc, 100 ng/mL. d. 20% inhibition at 50 mM. e. 5-10% inhibition at 15 pM. f. 80% inhibition at 50 pM.
Example 10 - Evaluation of Human Cytotoxicity Activity
Compounds displaying potent antibacterial activity (81, 8p, 8u, 17b, 18a and 20) were evaluated for cytotoxicity against HepG2 cells using a LDH release assay. Only compounds 8p, 8u and 17b displayed measurable cytotoxicity (9-13% lactate dehydrogenase release) at 25 mM (LDso > 25 mM, Fig. 3). Since 18a and 20 are esters of 19a, the
cytotoxicity of this compound was also examined, and again no cytotoxicity was observed. The cytotoxicity of 17b (25 pM) was also less than 10% in Hela, HEK293T and MCF7 cells. These experiments demonstrate that all of the compounds display a greater than lO-fold selectivity for antibacterial activity versus cytotoxicity (the recommended criteria for A ///?), with the selectivity of some exceeding lOO-fold.
Example 11 - Preliminary pharmacokinetic evaluation of !7b.
The stability of 17b and 18a in mouse liver microsomes was tested. 18a was rapidly metabolized (ti/2 = 1.3 min) in an NADPH-independent process. Compound 19a, the ester of 18a was also metabolized in an NADPH-independent process, although with a much longer half-life (ti/2 = 23 min). These observations suggest that both the ester and amide bonds of 18a may be liabilities. Compound 17b was metabolized in an NADPH-dependent process with 11/2 = 26 min. No decomposition of 17b was observed when it was incubated in mouse plasma at 37 °C for 2 h. Based on these results 17b was selected for further analysis. This compound displayed promising pharmacokinetics in mice, with a single 20 mg/kg oral dose [formulated using Tween 80 (1%) and 0.5% (w/v) methylcellulose in water (99%)] producing a maximum plasma concentration level comparable to MIC (Cmax = 3 mM) in 0.5 h (Tmax) with a plasma elimination half-life of 5 h. However, 17b also displays high serum protein binding (>99%), which suggests that the free drug concentration is insufficient to achieve in vivo efficacy.
Example 12 - General Biological Protocols for O compounds
Inhibition of MfMMPDH. The /ri.app values were determined by measuring the initial velocities at varying concentrations of the inhibitors (1-10,000 nM) with fixed concentrations of IMP (0.5 mM) and NAD+ (1.5 mM) and A / lb I M P D H 2 (20-50 nM).
Inhibition of human IMPDH2 was assayed IMP using (0.25 mM) and NAD+ (0.060 mM) and MMPDH2 (250 nM). Inhibition of human GMPR2 was assayed using GMP (0.050 mM), NADPH (0.045 mM) and enzyme (100 nM). The assay buffer contained 50 mM TrisCl, pH 8.0, 100 mM KC1 and 1 mM dithiothreitol.
The values of /ri.app were obtained using the equations (3) and (4)
Vi = vo / (1+ [I]/ IC50) (3)
Ai,app = IC50— [E]/2 (4) where Vi is the initial velocity in the presence of inhibitor and vo is the initial velocity in the absence of the inhibitor. If the IC50 value is comparable to the enzyme concentration, the Morrison tight binding equation was used to determine ATapp (5)
Vi/vo = l-(([E] + [I] + a.app) - (([E] + [I] + Lapp)2 - 4[E][I])° 5)/(2[E]) (5) where [E] is the concentration of the enzyme. All the initial velocity measurements were performed in triplicates. The Ai,app values reported are the average of at least two
independent experiments unless otherwise noted.
MIC determinations. MICs were determined as previously described. MIC values were determined in at least triplicate according to the broth microdilution methods using compounds from DMSO stock solutions. Isoniazid was used as a positive control and DMSO was utilized as a negative control. Isolated Mtb cells (ATCC 27294) were cultured to an OD 0.2-0.3 in the required medium, then diluted to deliver approximately 1 x 104 bacteria per well of a 96 well clear round-bottom plate. Plates were read after 1 week with an inverted enlarging mirror plate reader and graded as either growth or no growth. GAST/Fe medium (per liter) consisted of 0.3 g of Bacto Casitone (Difco), 4.0 g of dibasic potassium phosphate, 2.0 g of citric acid, 1.0 g of L-alanine, 1.2 g of magnesium chloride hexahydrate, 0.6 g of potassium sulfate, 2.0 g of ammonium chloride, 1.80 ml of 10 N sodium hydroxide, and 10.0 ml of glycerol, 0.05% Tween 80 and 0.05 g of ferric ammonium citrate adjusted to pH 6.6. 7H9/glycerol/glucose/BSA/Tween medium consisted of Middlebrook 7H9 broth base supplemented per liter with 0.2% glucose, 0.2% glycerol, 0.5% BSA fraction V, 0.08% NaCl and 0.05% Tween 80. Cultures were supplemented with 200 mM guanine as noted.
fMMPDH2 downregulation and susceptibility of SRMV2.6. Mtb strains guaB2 cKD ( guaB2-B3 Tet-OFF attB::guaB3 ) and SRMV2.6, which carries gtiaB2Y4 1(: were cultured in Middlebrook 7H9 media (Difco) supplemented with 0.2% glycerol, Middlebrook oleic acid-albumin-dextrose-catalase (OADC) enrichment (Difco) and 0.05% Tween 80 (7H9/Glycerol/OADC/Tween). Hygromycin (Hyg), kanamycin (Km) and gentamycin (Gm) were used in guaB2 cKD culture at final concentrations of 50, 25 and 2.5 pg/mL, respectively. ATc (Sigma) was used at concentrations up to 100 ng/mL. For pairwise combination (checkerboard) assays, a two-dimensional array of serial dilutions of test compound and ATc was prepared in 96-well plates, as previously described. MIC testing was carried out by broth microdilution using the AlamarBlue (AB, Invitrogen) assay.
Mammalian Cell Culture. Hep G2 cells (ATCC, purchased February 2017) were cultivated in EMEM supplemented with 10% heat inactivated FBS and IX penicillin/streptomycin under standard conditions (37 °C in a 5% CO2 humidified atmosphere). HEK293T, MCF7 and HELA cells were cultured in DMEM with 10% heat inactivated FBS and IX penicillin/streptomycin. Active cell cultures routinely tested for presence of Mycoplasma (MycoAlert™ detection kit, Lonza) and confirmed to be
Mycoplasma free.
LDH Cytotoxicity Assay. All compounds were dissolved in DMSO and further diluted with culture medium before use in tissue culture assays (final DMSO concentrations were < 0.1%). To determine cytotoxicity, LDH release was measured with the LDH
Cytotoxicity Assay Kit (Pierce) according to manufacturer’s protocol. Briefly, 96 well plates were seeded with 13,000 Hep G2 cells (all other cell lines seeded at 6,000 to 8,000 cells per well) and the cells were cultured for 24 h prior to drug treatment. The cells were incubated in 110 pL of EMEM containing compound or DMSO (vehicle only, control) for 24 h at 37°C. At least four wells from each plate were used as either‘spontaneous’ LDH controls or as ‘maximum’ LDH controls per manufacturer’s instructions. Cytotoxicity was determined by measuring absorbance on a microplate reader. Data represent two independent experiments each performed in quadruplicate (n =8).
Example 13 - 0112 has antibacterial activity against Mtb and Mma.
The antibacterial activity of Q112 against Mtb varies less than 2-fold with different carbon sources (glucose, glycerol, cholesterol, dipalmitoylphosphatidyl-choline, butyrate) and is also active in macrophages (Fig. 5). Q112 also displays potent activity against M. marinum {Mma, MIC = 0.78 mM), but is less effective against M smegmatis (MIC = 25 mM) andM avium (MIC = 6.2 mM). Mma is a ubiquitous fish pathogen that can also infect humans, commonly causing skin infections, and invasive infections can occur when the patient is immune compromised. Q112 does not display antibacterial activity against S. aureus, E.faecalis, S. pneumoniae, P. aeruginosa, E. coli or K. pneumoniae. Most importantly, Q112 is not cytotoxic against HepG2 cells or in the NCI 60 panel of cancer cell lines (LDso> 10 mM). These observations indicate that anti -Mtb activity does not arise from a nonspecific process. Moreover, neither Q151 nor Q121 display cytotoxicity against HepG2 cells, and a single 20 mg/kg does of Q151 is well tolerated in mice, which suggest that the Q scaffold does not have toxicity liabilities even though it engages multiple bacterial targets. Table 8. Antibacterial activity of selected Q compounds.
Figure imgf000074_0001
Parentheses denote whether antibacterial activity derives from on-target inhibition of MtbIMPDH2. WT is Mtb strain H37Rv, SRMV2.6 contains MtbIMPDH2/Y487C, which confers resistance to MtbIMPDH2 inhibitors (a) Inhibition of purified wild-type
MtbIMPDH2. (b) Inhibition of purified MtbIMPDH2/Y487C. (c) GAST/Fe (glycerol) medium for 1 week (d) 7H9 (glycerol/glucose) media for 1 week (e)
7H9/Glycerol/OADC/Tween and growth was measured (f) 80% inhibition at 50 mM. All MICs were performed with rifampicin as the positive control. Values are the average of at least two independent determinations n.a., not applicable.
Example 14 - Design of P-Series Compounds to increase antibacterial activity
The polarity of P146 and P150 was first addressed with the addition of a secondary amine that will be charged at physiologic pH (P148), sulfonamides and sulfoxides (P161, P162 and P163), as well as small heterocycles (P181, P182) in the 3-position (Table 9). Most of these substitutions had little impact on enzyme inhibition, with the exception of the piperazine amides P148 and P162, which were deleterious and therefore not tested for antibacterial activity. P161 and P163 displayed markedly reduced antibacterial activity relative to P146 and P150. The trifluoromethylpyrazole P181 and trifluoromethylthiazole P182 maintained comparable antibacterial activity. Curiously, the ketone analogs of P181 and P182 (P185 and P184) displayed little antibacterial activity despite similar affinities for &IMPDH.
Recent x-ray crystal structures of cofactor complexes of M. tuberculosis IMPDH2 ( / MPDH2) and Vibrio cholera IMPDH reveal that the bacterial IMPDH inhibitors bind in the cofactor binding site (Makowska-Grzyska, 2015 #3290;Makowska-Grzyska, 2015 #3289}. Overlay of the structures of the /A/IMPDH*P32 complex an d Mth I M P D H2· cofactor complex indicates that the B-ring of the P compounds binds in the adenosine subsite, in the location occupied by the adenine base, but does not extend into the ribose binding subsite. Further inspection suggested that modifications of the 3 -position of the B-ring would extend into the ribose binding site, providing another attractive strategy for increasing potency and polarity. Next, Pll was used as the starting scaffold because it contains a vinyl group on the A ring that is more easily synthesized than the oximes. Although Pll was a potent inhibitor of N/IMPDH, it displayed little antibacterial activity. The oxime analog P37 was likewise a potent enzyme inhibitor with little antibacterial activity (Table 9). 3-NH2 (P171) and 3-OH (P177) substitutions were first added to enable further elaboration of the 3-position. These compounds were potent inhibitors of N/IMPDH, equivalent to Pll (Table 9). Both P171 and P177 displayed improved antibacterial activity (MIC = 3 and 1.7 mM, respectively).
Addition of the dimethylamine to the 3’ position (P220) decreased enzyme affinity by a factor of 6, yet improved antibacterial activity by a factor of 4. A similar decrease in enzyme affmity/increase in antibacterial potency was observed when the B ring was replaced with a 5-substituted indole (P173). However, replacement of the B ring with 6-indole decreased enzyme affinity by 50-fold (P172).
Next, added hydrogen bonding moieties were added to the 3 -position of the B ring with the intent of introducing an interaction with Ser257 (P225, P224, P226, P227 and
P256). Addition of an acetic acid group (P225) increased enzyme affinity, but eliminated antibacterial activity. Such substitutions have been similarly debilitating in the antibacterial activity of topoisomerase inhibitors. The corresponding ethyl ester derivative (P224) had a deleterious effect on enzyme affinity, and also failed to display antibacterial activity. In contrast, the amide (P226) and hydroxamic acid (P227) derivatives maintained enzyme affinity and displayed superior antibacterial activity. Extension of the amide from the phenyl ring by one methylene group (P256) decreased both enzyme affinity and antibacterial activity. Since the oxime substituted A ring usually displayed improved antibacterial activity, we synthesized the oxime versions of P224 (P251) and P226 (P359). P251 displayed similar enzyme inhibition as P224. Nonetheless, antibacterial activity improved by more than a factor of 30. In contrast, no improvement in antibacterial activity was observed for P359 relative to P226, although enzyme inhibition improved by a factor of approximately 2.5.
Further inspection of the crystal structure of /i c / 1 M P D H · I M P · P 32 suggested that a sugar attached to the 3 -position of the B-ring would bind in the site occupied by the nicotinamide ribose of the cofactor. Therefore, ribose (P176 and P178) and arabinose (P221) were appended to this position. These substitutions decreased the potency of enzyme inhibition by 2 to 4-fold relative to P171 and P177. The antibacterial activity of P176 was similarly decreased, while P178 and P221 maintained antibacterial activity.
Lastly, the oxime in ring A was replaced with hydroxyamidine to further increase topological polar surface area (P200, P202, P265, P208, P219, P269, P209, P210, P211, P212, P263 and P239). Three were potent antibacterials (MIC < 2 mM, Table 9). This substitution had little effect on enzyme affinity (e.g., P181 vs. P211, P163 vs. P208 and P209 vs. P182), with the exception of P200, where affinity increased 3-fold relative to P37. The effect of the hydroxamidine substitution on antibacterial activity was variable, improving by more than lO-fold for P200, but with no effect for P211, and decreasing the antibacterial activity of P208 relative to their oxime analogs. As noted above, the addition of small heterocycles to the B ring in the context of the oxime substituted A ring increased antibacterial activity by 40-60-fold even though they had a deleterious effect on enzyme affinity. Disappointingly, the addition of the small heterocycles had little effect on enzyme affinity in the context of the hydroxyamidine substituted A ring and only a 4-fold increase in antibacterial activity (compare P209 and P211 to P200). Three compounds, P219, P209 and P211 displayed MIC ~l mM.
Table 9 - Biological data for certain P-Series compounds
Figure imgf000076_0001
Figure imgf000077_0001
Figure imgf000078_0001
Example 15 - Structures of enzyme-inhibitor complexes.
The x-ray crystal structures of the complexes of Tk/IMPDH and IMP with five inhibitors: P182, P200, P176, P178 and P221 was solved to determine how the thiazole, ribose, arabinose and hydroxyamidine groups interact with the enzyme. The unit cell of the P182 complex contained one tetramer, while two tetramers where found in the unit cells of the other four complexes. The overall structures are very similar, although the loop comprised of residues 390-418, designated the“flap”, displays varying amounts of disorder in different complexes as well as in different subunits of the same complex. In four complexes, the last visible residues of the flap are observed in two (P178, P200 and P221) or three (P176) distinct locations in different subunits, suggesting that the flap has multiple “open” conformations. All five inhibitors bind in the same site as P32. The A ring interacts with IMP and the B ring interacts with Tyr445’ in the adjacent subunit as expected. All of the inhibitors also interact with residues Ala253, His254, Glu4l6 and Pro27’, and all form a halogen bond between the 4-C1 on the B ring and the carbonyl oxygen of Gly444.
The thiazole ring of P182 binds in the site occupied by the adenosine ribose of NAD+, where it interacts with Ser257 in two active sites. The oxime has rotated from its position in P32, so that it now forms a hydrogen bond with the side chain of Glu4l6 in all four active sites. This hydrogen bond is not observed in the P32 complex. An additional hydrogen bond with the side chain of Thr310 is found in the chain B-C active site, and two additional hydrogen bonds, one to Thr3 l0 and the second to the hydroxyl of Tyr445’, are observed in the chain D-B active site. In the P200 complex, the hydroxyamidine forms different hydrogen bonding patterns in different active sites. The amidine NEb binds in the same location as the oxime OH of P182, and makes a hydrogen bond to the carboxylate of Glu4l6 in six active sites. In the two active sites, an additional water mediated hydrogen bond is observed between the hydroxyamidine OH and carboxylate of Asp25l.
The sugar moieties of P176, P178 and P221 bind in the nicotinamide ribose site as expected, but make different interactions in each inhibitor. P176, which links the ribose to the B ring via NH, makes a hydrogen bond to Thr252 with the 3’ OH and an intramolecular hydrogen bond between the 5’-OH and the urea carbonyl. In contrast, P178, which links the ribose to the B ring via an oxygen, forms two hydrogen bonds to the enzyme, one between the 2’-OH and the backbone NH of His254 and the second between the 5’-OH and the OH of Ser257. The arabinose containing compound P221 makes two hydrogen bonds via the T - OH, one to the Thr252 OH and the other to the main chain carbonyl of His254.
Example 16 - Antibacterial activity is due to inhibition of N/IMPDH.
Figures 8A and 8B show the dependence of antibacterial activity on inhibition of N/IMPDH for the P and A compounds described above. Greater antibacterial activity is generally associated with more potent enzyme inhibition, as expected if antibacterial activity derived from inhibition of N/IMPDH. To further confirm on-target antibacterial activity, the values of MIC in the presence of guanosine (Table 9), which provides an alternative source of guanine nucleotides that allows cells to grow in the absence of IMPDH activity, was determined. None of the inhibitors displayed antibacterial activity in the presence of guanosine (MIC >30 mM, Tables 8 and 9), with the exception of P200, where the value of MIC increased by a factor of 7 to 28 pM. The failure of some potent inhibitors to display antibacterial activity is possibly due to lack of uptake or chemical transformation to an inactive metabolite. Although a tenfold improvement in inhibitor potency was achieved relative to the starting compound P146, only a twofold improvement in antibacterial activity was observed.
Example 17 - The physicochemical properties of antibacterial activity.
Although some studies suggested that antibiotics are smaller and more polar than Lipinski/Veber guidelines, little correlation exists between antibacterial activity and hydrophobicity (cLogP) or topological polar surface area (t-PSA)(Figs. 8A and 8B). The average values of cLogP and t-PSA are 3.2 and 103 A2, respectively, for the 11 P
compounds with values of MIC less than 2 pM. The best antibacterial compounds, P209, P219 and P359, vary in cLogP from 1.87 to 4.15, while P186 and P265 do not display antibacterial activity despite being potent inhibitors (Ai,app = 4 and 13 nM, respectively) with low lipophilicity (cLogP = 2.33 and 1.21, respectively). All of the A compounds have comparable antibacterial activity despite large differences in hydrophobicity (cLogP varies from 2.96 to 6.19) and polar surface area (t-PSA varies from 49.55 to 84.29 A2). These observations suggest that decreasing lipophilicity may not be a general strategy for antibiotic design. Example 18 - Cytotoxicity
Compounds were tested for cytotoxicity in HepG2 cells by monitoring release of lactate dehydrogenase (Figs. 9A and 9B). Significant cytotoxicity (>10% release) was only observed in cells treated with 25 mM P211 and P221. Approximately 6% LDH release was observed when cells were treated with P219 and P251. No cytotoxicity (<2% release) was observed in HepG2 cells treated with P146, P171, P182, and P221.
Example 19 - Preliminary pharmacokinetic evaluation.
The pharmacokinetics of P219 were evaluated to further assess the antibiotic potential of the P scaffold. For P219 (75 mg/kg i.v.) the average value of Co = 195 pM and ti/2 = 1.1 h. The value of CO greatly exceeds MIC (1.2 pM). Plasma protein binding was also estimated by determining the increase in Aiapp when enzyme inhibition was measured in the presence of 50% plasma. P219 displays high plasma protein binding, with a free fraction of 0.0043, which suggests the value of Co for free drug would be 0.8 pM.
Example 20 - General Biological Protocols for A and P compounds
Expression and purification of SaIMPDH. Briefly, the expression vector was acquired/constructed according to methods known in the art. The vector was transformed in BL21 (DE3) guaB. The cells were grown in LB media with 50 pg/ml of kanamycin at 37 °C to an OD6oo 0.5-0.8. Protein expression was initiated by 0.25 mM isopropyl b-Ό- thiogalactoside (IPTG) and the culture was maintained at 30 °C overnight. Cells were harvested, resuspended in lysis buffer [50 mM phosphate (pH 8.0), 500 mM KC1, 10 mM imidazole, 0.5 mM (tris(2-carboxyethyl)phosphine) (TCEP) and 10% glycerol], and stored at -80 °C. N/IMPDH was purified according to a standard protocol for His tagged protein. Protease inhibitor cocktail (Roche, Indianapolis, IN; 50 mL/g of wet cells) were added to the thawed cell suspension. The cells lysed on ice by sonication. The lysate was clarified by centrifugation at 18000 for 1 h and filtered through a 0.44 pm membrane. Clarified lysate was applied to a 5 mL Ni-NTA column (McLab, San Francisco CA). The column was washed with 10 column volume (CV) lysis buffer, 10 CV of lysis buffer containing 30 mM imidazole, and the protein was eluted with the same buffer containing 250 mM imidazole. riaIMPDH was dialyzed against 20 mM Hepes (pH 8.0), 150 mM KC1, 3 mM EDTA and 2 mM DTT, concentrated, flash-frozen, and stored in liquid nitrogen.
Enzyme assays. IMPDH assays were performed in 50 mM Tris-HCl, pH 8.0, 100 mM
KC1, 3 mM EDTA and 1 mM DTT. Activity was routinely assayed in the presence of 20-50 nM IMPDH at 25 °C. NADH production was monitored either by following absorbance change at wavelength 340 nm using a Hitachi U-2000 spectrophotometer (e = 6.2 mM 1 cm- ') or by following fluorescence on a Biotek plate reader (excitation wavelength = 340 nm, emission wavelength = 460 nm). Each determination of Akapp was derived from duplicate measurements of enzyme activity in the presence of twelve different inhibitor
concentrations. Reported values are averages of at least two independent determinations. Enzyme was incubated with inhibitor (50 pM - 100 mM) for 10 min at room temperature prior to addition of substrates. The concentrations of IMP and NAD+ were fixed at 1.0 mM (saturating) and 80 mM (2.4 x Km), respectively /ri.app values were calculated for each inhibitor according to Equation 1 (when Ki, app » [E]) or Equation 2 (when Ki, app ~ [E]) using the SigmaPlot program (SPSS, Inc.):
Vi = Vo/( 1 + [I]/ arr) (1) where v; is initial velocity in the presence of inhibitor (I) and vo is the initial velocity in the absence of inhibitor. The Morrison equation (Equation 2) was used to evaluate tight-binding inhibitors.
Vi/vo = {Eo - Jo - K* + [(£b - /o - Kί,arr)2 + 4·Eo· K,arr†·5 } / 2·Eo (2) where v; is the initial velocity in the presence of inhibitor, vo is the initial velocity in the absence of inhibitor, Eo is the total concentration of enzyme, Io is the total concentration of inhibitor, and Ki app is the apparent inhibition constant.
Cytotoxicity. All compounds were dissolved in DMSO and further diluted with culture medium before use in tissue culture assays (final DMSO concentrations were <
0.1%). To determine cytotoxicity, LDH release was measured with the LDH Cytotoxicity Assay Kit (Pierce) according to manufacturer’s protocol. Briefly, 96 well plates were seeded with 13,000 Hep G2 cells (all other cell lines seeded at 6,000 to 8,000 cells per well) and the cells were cultured for 24 h prior to drug treatment. The cells were incubated in 110 mL of EMEM containing compound or DMSO (vehicle only, control) for 24 h at 37°C. At least four wells from each plate were used as either‘spontaneous’ LDH controls or as‘maximum’ LDH controls per manufacturer’s instructions. Cytotoxicity was determined by measuring absorbance on a microplate reader. Data represent two independent experiments each performed in quadruplicate (n =8).
Incorporation by Reference
All publications and patents mentioned herein are hereby incorporated by reference in their entirety as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control.
Equivalents
While specific embodiments of the subject invention have been discussed, the above specification is illustrative and not restrictive. Many variations of the invention will become apparent to those skilled in the art upon review of this specification and the claims below. The full scope of the invention should be determined by reference to the claims, along with their full scope of equivalents, and the specification, along with such variations.

Claims

Claims:
1 A compound selected from the group consisting of:
Figure imgf000083_0001
Figure imgf000084_0001
82
Figure imgf000085_0001
83
Figure imgf000086_0001
84
Figure imgf000087_0001
85
Figure imgf000088_0001
Figure imgf000089_0001
Figure imgf000090_0001
Figure imgf000091_0002
or a pharmaceutically acceptable salt thereof.
A compound selected from the group consisting of:
Figure imgf000091_0001
Figure imgf000092_0001
Figure imgf000093_0001
91
Figure imgf000094_0001
92
Figure imgf000095_0001
93
Figure imgf000096_0001
94
Figure imgf000097_0001
Figure imgf000098_0001
a pharmaceutically acceptable salt thereof.
3. A pharmaceutical composition comprising a compound of claim 1 or claim 2 and pharmaceutically acceptable carrier.
4. A method of treating a bacterial infection in a subject, the method comprising administering to the subject an effective amount of a compound of claim 1 or claim 2 or a composition of claim 3 to thereby treat the infection.
5. A method for preventing or reducing the likelihood of a productive bacterial infection in a subject, the method comprising administering to a subject an effective amount of a compound of claim 1 or claim 2 or a composition of claim 3, to thereby prevent or reduce the likelihood of a productive bacterial infection in the subject.
6. A method for ameliorating the signs or symptoms of an infection of a subject by bacterial cells, the method comprising administering to the subject an effective amount of a compound of claim 1 or claim 2 or a composition of claim 3 to thereby ameliorate the signs and symptoms of the infection.
7. The method of any one of claims 4 to 6, wherein the bacteria or bacterial cells are Mycobacterium .
8. The method of any one of claims 4 to 6, wherein the bacteria or bacterial cells are Staphylococcus.
9. A method of treating tuberculosis in a subject, the method comprising administering to the subject an effective amount of a compound of claim 1 or claim 2 or a composition of claim 3 to thereby treat the tuberculosis.
10. A compound selected from the group consisting of:
Figure imgf000099_0001
Figure imgf000100_0001
11. A compound for use in inhibiting Mycobacterium tuberculosis , wherein the compound is selected from the group consisting of:
Figure imgf000101_0001
A compound selected from the group consisting of:
Figure imgf000101_0002
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PCT/US2019/029338 2018-04-26 2019-04-26 Antibacterial compounds, compositions and uses thereof WO2019210160A1 (en)

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US7345068B2 (en) * 2002-02-07 2008-03-18 Hitoshi Endou Aromatic amino acid derivatives and medicinal compositions
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