WO2012051708A1 - Anti-bacterial pyruvate kinase modulator compounds, compositions, uses, and methods - Google Patents

Anti-bacterial pyruvate kinase modulator compounds, compositions, uses, and methods Download PDF

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Publication number
WO2012051708A1
WO2012051708A1 PCT/CA2011/001175 CA2011001175W WO2012051708A1 WO 2012051708 A1 WO2012051708 A1 WO 2012051708A1 CA 2011001175 W CA2011001175 W CA 2011001175W WO 2012051708 A1 WO2012051708 A1 WO 2012051708A1
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WIPO (PCT)
Prior art keywords
phenyl
compound
och
nmr
con
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PCT/CA2011/001175
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French (fr)
Inventor
Neil E. Reiner
Barton B. Finlay
Robert C. Brunham
Artem Tcherkassov
Leonard J. Foster
William R. Mcmaster
Raymond H. See
Roya Zoraghi
Michael M. K. Hsing
Kendall G. Byler
Peter J. Axerio-Cilies
Fuqiang Ban
Nag Kumar
Anne Moreau
Christophe Labriere
Robert N. Young
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The University Of British Columbia
Simon Fraser University
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Publication of WO2012051708A1 publication Critical patent/WO2012051708A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
    • A61K31/404—Indoles, e.g. pindolol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/4164—1,3-Diazoles
    • A61K31/4184—1,3-Diazoles condensed with carbocyclic rings, e.g. benzimidazoles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • 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
    • C07D209/10—Indoles; Hydrogenated indoles with substituted hydrocarbon radicals attached to carbon atoms of the hetero ring
    • C07D209/14—Radicals substituted by nitrogen atoms, not forming part of a nitro radical
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D235/00—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, condensed with other rings
    • C07D235/02—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, condensed with other rings condensed with carbocyclic rings or ring systems
    • C07D235/04—Benzimidazoles; Hydrogenated benzimidazoles
    • C07D235/06—Benzimidazoles; Hydrogenated benzimidazoles with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached in position 2
    • C07D235/14—Radicals substituted by nitrogen atoms
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D277/00—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings
    • C07D277/60—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings condensed with carbocyclic rings or ring systems
    • C07D277/62—Benzothiazoles
    • C07D277/64—Benzothiazoles with only hydrocarbon or substituted hydrocarbon radicals attached in position 2

Definitions

  • This invention relates to therapeutics, their uses and methods for the treatment of various indications, including infections.
  • therapeutic compositions and methods of treatment of bacteria that have developed resistance to other antibiotics are particularly preferred.
  • MRSA methicillin-resistant Staphylococcus aureus
  • Staphylococcus aureus is a facultatively anaerobic, gram-positive coccus and is the most common cause of staph infections.
  • Escherichia coli are gram-negative rod-shaped bacterium found in the digestive systems of warm blooded organisms. Many E. coli strains are harmless. However, other strains are capable of causing serious food poisoning in humans. Resistance to beta-lactam antibiotics is problematic as resistant bacteria are able to produce extended-spectrum beta- lactamases, which result in resistance to most of the penicillins and cephalosporins.
  • Bacillus anthracis is also a gram-positive bacterium, but is also spore-forming and is capable of surviving in both aerobic and anaerobic conditions. Bacillus anthracis causes anthrax, which is an acute disease of humans and other animals. Anthrax is often lethal, but sometimes responds well to antibiotic treatment. Vaccines against anthrax are also available.
  • MRSA also known as oxacillin-resistant Staphylococcus aureus (ORSA)
  • RSA oxacillin-resistant Staphylococcus aureus
  • MRSA is defined as any strain of Staphylococcus aureus bacteria with resistance to beta-lactam antibiotics, such as the penicillins: methicillin; dicloxacillin; nafcillin; oxacillin; etc. and the cephalosporins.
  • MRSA infection is made when a biological sample (for example, a skin sample, pus from a wound, blood, urine, or other biopsy material) is cultured for S. aureus. If a positive culture is found, the bacteria are then tested for resistance to antibiotics, such as beta-lactam antibiotics including methicillin. S. aureus that grows in the presence of methicillin is identified as MRSA.
  • a biological sample for example, a skin sample, pus from a wound, blood, urine, or other biopsy material
  • antibiotics such as beta-lactam antibiotics including methicillin.
  • S. aureus that grows in the presence of methicillin is identified as MRSA.
  • Mild infections of MRSA may be initially treated by draining the infected area and with topical disinfection.
  • antibiotic treatment should be guided by the susceptibility profile of the cultured S. aureus.
  • the testing for antibiotic resistance when a bacteria is cultured often defines a susceptibility profile for a cultured bacteria and guides the subsequent treatments.
  • glycopeptide antibiotics for example, vancomycin and teicoplanin.
  • strains of MRSA have also shown antibiotic resistance even to glycopeptide antibiotics and have been called vancomycin intermediate- resistant Staphylococcus aureus (VISA) or vancomycin-resistant Staphylococcus aureus (VRSA) or multi-drug resistant Staphylococcus aureus (MDRSA).
  • treatments for MRSA are or oxazolidinone class compounds (for example, linezolid), quinupristin/dalfopristin, daptomycin, tigecycline, trimethoprim-sulfamethoxazole, doxycycline, and clindamycin.
  • oxazolidinone class compounds for example, linezolid
  • quinupristin/dalfopristin for example, daptomycin
  • tigecycline for example, trimethoprim-sulfamethoxazole, doxycycline, and clindamycin.
  • trimethoprim-sulfamethoxazole doxycycline
  • clindamycin a number of alternative antibiotics against MRSA are in clinical trials (for example, ceftobiprole, ceftaroline, dalbavancin, telavancin, aurograb, torezolid, iclaprim, and nemonoxaci
  • This invention is based, in part, on the discovery that compounds described herein selectively inhibit microbial pyruvate kinase (PK) and do not inhibit mammalian pyruvate kinases. Furthermore, as disclosed herein, the selective inhibition of microbial pyruvate kinase is based on selective binding to the monomer interface of microbial PK to interfere with dimer/tertramer formation.
  • PK microbial pyruvate kinase
  • the compounds described herein may be used for in vivo or in vitro research uses (i.e. non-clinical) to investigate alternative treatments for microbial infection. Furthermore, these compounds may be used individually or as part of a kit for in vivo or in vitro research to investigate mechanisms of microbial resistance or microbial infection using recombinant proteins, cells maintained in culture, and/or animal models.
  • R 1 may be N-R 5 , S, or O;
  • R 2 may be C-H, or N;
  • R 3 may be C-H, C-OR 5 , N, C-OCH 2 C ⁇ CH, C-OCH 2 OCH 3 , C-OCH 2 OCH 2 CH 3 , C- OC(0)CH 3 , or C-OCH 2 (OCH 2 CH 2 ) n OR 5 ;
  • R 4 may be C
  • R 5 may be H, or a C1-C6 branched or unbranched, saturated or unsaturated, alkyl or acyl group;
  • n may be 1-5;
  • a 1 may be R 5 , OR 5 , F, CI, Br, I, C ⁇ CH, S0 2 R 5 , NHCOR 5 , NHS0 2 R 5 , N0 2 , CON(R 5 ) 2 , CF 3 , CBr 3 , CC1 3 , CI 3 , phenyl or a C6-C10 aryl group;
  • a 2 may be H, F, CI, Br, I, C ⁇ CH, OR 5 , CF 3 , CBr 3 , CC1 3 , CI 3 , phenyl or a C6-C10 aryl group;
  • a 3 may be F, CI, Br, I, C ⁇ CH, OR 5 , R 5 , S0 2 R 5 , NHCOR 5 , NHS0 2 R 5 , N0 2 , CON(R 5 ) 2 , CF 3 , CBr 3 , CCI3, CI 3 , phenyl or a C6-C10 aryl group;
  • a 4 may be F, CI, Br, I, C ⁇ CH, OR 5 , R 5 , S0 2 R 5 , NHCOR 5 , NHS0 2 R 5 , N0 2 , CON(R 5 ) 2 ,
  • Q 1 may be R 5 , OR 5 , F, CI, Br, I, N 3 , C ⁇ CH, phenyl or a C6-C10 aryl group;
  • Q 2 may be R 5 , OR 5 , F, CI, Br, I, N 3 , C ⁇ CH, phenyl or a C6-C10 aryl group;
  • Q 3 may be R 5 , OR 5 , F, CI, Br, I, N 3 , C ⁇ CH, phenyl or a C6-C10 aryl group;
  • Q 4 may be H or OH
  • Q 1 and Q 2 may optionally form a benzene ring optionally substituted with R 5 , OR 5 , F, CI, Br, I, N 3 , C ⁇ CH, phenyl or a C6-C10 aryl group; or Q 2 and Q 3 may optionally form a benzene ring optionally substituted with R 5 , OR 5 , F, CI, Br, I, N 3 , C ⁇ CH, phenyl or a C6-C10 aryl group;
  • Q 3 and Q 4 may optionally form a benzene ring, optionally substituted with R 5 , OR 5 , F, CI, Br, I, N 3 , C ⁇ CH, or phenyl;
  • D 1 may be R 5 ;
  • E 1 may be R 5 , phenyl or a C6-C10 aryl group
  • E may be R , phenyl or a C6-C10 aryl group
  • E may be R , phenyl or a C6-C10 aryl group
  • R 3 may be C-H, C-OR 5 , N, C-OCH 2 C ⁇ CH, C-OCH 2 OCH 3 , C- OC3 ⁇ 4OCH 2 CH 3 , or C-OC(0)CH 3 .
  • R 3 may be C-H, C-OH, C-OMe, C-OEt, N, C- OCH 2 C ⁇ CH, C-OCH 2 OCH 3 , C-OCH 2 OCH 2 CH 3 , or C-OC(0)CH 3 .
  • R 3 may be C-H, C-OR 5 , or N.
  • R 3 may be C-H, C-OH, C-OMe, C-OEt, or N.
  • R 1 may be N-H, N-Me, N-Et, S, or O.
  • R 1 may be N-R 5 .
  • R 1 may be N-H, N-Me, or N-Et.
  • R 1 may be S, or O.
  • R 2 may be N.
  • R 2 may be C-H.
  • R 5 may be a CI -C6 branched or unbranched, saturated or unsaturated, alkyl.
  • a 1 may be R 5 , OR 5 , F, CI, Br, I, C ⁇ CH, or phenyl.
  • a 1 may be H, O-Me, F, CI, Br, I, C ⁇ CH, or phenyl.
  • a 1 may be H, O-Me, F, CI, Br, or I.
  • a 2 may be H, F, CI, Br, I, C ⁇ CH, or phenyl.
  • a 2 may be H, O-Me, F, CI, Br, I, C ⁇ CH, or phenyl.
  • a 2 may be H, O-Me, F, CI, Br, or I.
  • a 3 may be F, CI, Br, I, C ⁇ CH, OR 5 , R 5 , or phenyl.
  • a 3 may be F, CI, Br, I, C ⁇ CH, H, or O-Me.
  • a 3 may be H, O-Me, F, CI, Br, or I.
  • a 3 may be F, CI, Br, I, OR 5 , R 5 , or phenyl.
  • a 4 may be F, CI, Br, I, C ⁇ CH, OR 5 , R 5 , or phenyl.
  • a 4 may be F, CI, Br, I, C ⁇ CH, OR 5 , R 5 , N0 2 , CON(R 5 ) 2 or phenyl.
  • a 4 may be F, CI, Br, I, OR 5 , R 5 , or N0 2 .
  • a 4 may be H, O-Me, F, CI, Br, or I.
  • Q 1"3 may be independently selected from R 5 , OR 5 , F, CI, Br, I, and N 3 .
  • Q 1"3 may be independently
  • Q 4 may be H.
  • L may be .
  • D 1 may be H or
  • E 1"3 may be independently selected from H, Me, Et, phenyl, and tert-butyl. E 1"3 may be independently selected from H, Me, and Et. E 1 may be selected from H, Me, Et, phenyl, and tert-butyl. E 1 may be selected from phenyl, Me, and Et. E 1"3 may be independently selected from Me and Et. E 1"3 may be Me. E 1 may be Et, Me, or phenyl. E 1 may be Me.
  • R 1 is N-R 5 ;
  • R 2 may be C-H;
  • R 3 is C-OH;
  • R 5 may be H or Me or Et;
  • a 1 may be R 5 , F, CI, Br, or I;
  • a 2 may be H, F, CI, Br, or I;
  • a 3 may be OMe, F, CI, Br, I, or R 5 ;
  • a 4 may be F, CI, Br, I, or R 5 ;
  • Q 1 may be R 5 , F, CI, Br, or I;
  • Q 2 may be R 5 , F, CI, Br, or I;
  • Q 3 may be R 5 ,
  • R 1 may be N-H or N-Me; R 3 may be C-OH; A 1 may be H; A 2 may be H, or F; A 3 may be OMe, F, CI, Br, or H; A 4 may be H, or F; Q 1 may be H; Q 2 may be H; Q 3 may be Br, or I; Q 4 may be H; D 1 may be H; and E 1 may be Me, Et, or phenyl.
  • a compound or a salt thereof selected from TABLE 2A.
  • a compound or a salt thereof selected from TABLE 2B.
  • G 1 may be G 5 , OG 5 , F, CI, Br, I, C ⁇ CH, S0 2 G 5 , NHCOG 5 , NHS0 2 G 5 , N0 2 , CON(G 5 ) 2 or phenyl;
  • G 2 may be G 5 , OG 5 , F, CI, Br, I, C ⁇ CH, S0 2 G 5 , NHCOG 5 , NHS0 2 G 5 , N0 , CON(G 5 ) 2 or phenyl;
  • G 3 may be G 5 , OG 5 , F, CI, Br, I, C ⁇ CH, S0 2 G 5 , NHCOG 5 , NHS0 2 G 5 , N0 2 , CON(G 5 ) 2 or phenyl;
  • G 4 may be G 5 , OG 5 , F, CI, Br, I, C ⁇ CH, S0 2 G 5 , NHCOG 5 , NHS0 2 G 5 , N0 2 , CON(G 5 ) 2 or phenyl;
  • G 5 may be H, or a C1-C6 branched or unbranched, saturated or unsaturated, alkyl; J 1 maybe N-G 5 , S, or O;
  • M 1 may be G 5 , OG 5 , F, CI, Br, I, N 3 , C ⁇ CH, or phenyl;
  • M 2 may be G 5 , OG 5 , F, CI, Br, I, N 3 , C ⁇ CH, or phenyl;
  • M 3 may be G 5 , OG 5 , F, CI, Br, I, N 3 , C ⁇ CH, or phenyl;
  • M 4 may be G 5 , OG 5 , F, CI, Br, I, N 3 , C ⁇ CH, or phenyl;
  • M 5 may be G 5 , OG 5 , F, CI, Br, I, N 3 , C ⁇ CH, or phenyl;
  • M 6 may be G 5 , OG 5 , F, CI, Br, I, N 3 , C ⁇ CH, or phenyl; M 7 may be H or OH;
  • a method of treating a microbial infection including administering a compound of Formula (A) or Formula (B):
  • R 1 may be -R 5 , S, or O;
  • R 2 may be C-H, or N;
  • R 3 may be C-H, C-OR 5 , N, C-OCH 2 C ⁇ CH, C-OCH 2 OCH 3 , C-OCH 2 OCH 2 CH 3 , C- OC(0)CH 3 , or C-OCH 2 (OCH 2 CH 2 ) n OR 5 ;
  • R 4 may be C
  • R 5 may be H, or a C1-C6 branched or unbranched, saturated or unsaturated, alkyl or acyl group;
  • n may be 1-5;
  • a 1 may be R 5 , OR 5 , F, CI, Br, I, C ⁇ CH, S0 2 R 5 , NHCOR 5 , NHS0 2 R 5 , N0 2 , CON(R 5 ) 2 , CF 3 , CBr 3 , CC1 3 , CI 3 , or phenyl;
  • a 2 may be H, F, CI, Br, I, C ⁇ CH, OR 5 , CF 3 , CBr 3 , CC1 3 , CI 3 , or phenyl;
  • a 3 may be F, CI, Br, I, C ⁇ CH, OR 5 , R 5 , S0 2 R 5 , NHCOR 5 , NHS0 2 R 5 , N0 2 , CON(R 5 ) 2 ,
  • a 4 may be F, CI, Br, I, C ⁇ CH, OR 5 , R 5 , S0 2 R 5 , NHCOR 5 , NHS0 2 R 5 , N0 2 , CF 3 , CBr 3 ,
  • Q 1 may be R 5 , OR 5 , F, CI, Br, I, N 3 , C ⁇ CH, or phenyl;
  • Q 2 may be R 5 , OR 5 , F, CI, Br, I, N 3 , C ⁇ CH, or phenyl;
  • Q 3 may be R 5 , OR 5 , F, CI, Br, I, N 3 , C ⁇ CH, or phenyl;
  • Q 4 may be H or OH
  • Q and Q may optionally form a benzene ring optionally substituted with R , OR , F, CI, Br, I, N 3 , C ⁇ CH, phenyl or a C6-C10 aryl group;
  • Q and Q may optionally form a benzene ring optionally substituted with R , OR , F, CI, Br, I, N 3 , C ⁇ CH, or phenyl;
  • Q 3 and Q 4 may optionally form a benzene ring optionally substituted with R 5 , OR 5 , F, CI, Br, I, N 3 , C ⁇ CH, phenyl or a C6-C10 aryl group;
  • R 3 may be C-H, C-OR 5 , N, C-OCH 2 C ⁇ CH, C-OCH 2 OCH 3 , C- OCH 2 OCH 2 CH 3 , or C-OC(0)CH 3 .
  • R 3 may be C-H, C-OH, C-OMe, C-OEt, N, C- OCH 2 C ⁇ CH, C-OCH 2 OCH 3 , C-OCH 2 OCH 2 CH 3 , or C-OC(0)CH 3 .
  • R 3 may be C-H, C-OR 5 , or N.
  • R 3 may be C-H, C-OH, C-OMe, C-OEt, or N.
  • R 1 may be N-H, N-Me, N-Et, S, or O.
  • R' may be N-R 5 .
  • R 1 may be N-H, N-Me, or N-Et.
  • R 1 may be S, or O.
  • R 2 may be N.
  • R 2 may be C-H.
  • R 5 may be a C1-C6 branched or unbranched, saturated or unsaturated, alkyl.
  • a 1 may be R 5 , OR 5 , F, CI, Br, I, C ⁇ CH, or phenyl.
  • a 1 may be H, O-Me, F, CI, Br, I, C ⁇ CH, or phenyl.
  • a 1 may be H, O-Me, F, CI, Br, or I.
  • a 2 may be H, F, CI, Br, I, C ⁇ CH, or phenyl.
  • a 2 may be H, O-Me, F, CI, Br, I, C ⁇ CH, or phenyl.
  • a 2 may be H, O-Me, F, CI, Br, or I.
  • a 3 may be F, CI, Br, I, C ⁇ CH, OR 5 , R 5 , or phenyl.
  • a 3 may be F, CI, Br, I, C ⁇ CH, H, or O-Me.
  • a 3 may be H, O-Me, F, CI, Br, or I.
  • a 3 may be F, CI, Br, I, OR 5 , R 5 , or phenyl.
  • a 4 may be F, CI, Br, I, C ⁇ CH, OR 5 , R 5 , or phenyl.
  • a 4 may be F, CI, Br, I, C ⁇ CH, OR 5 , R 5 , N0 2 , CON(R 5 ) 2 or phenyl.
  • a 4 may be F, CI, Br, I, OR 5 , R 5 , or N0 2 .
  • a 4 may be H, O-Me, F, CI, Br, or I.
  • Q 1"3 may be independently selected from R 5 , OR 5 , F, CI, Br, I, and N 3 . 1"3 may be independently
  • Q 4 may be H.
  • L may be .
  • D 1 may be H or
  • E 1"3 may be independently selected from H, Me, Et, phenyl, and tert-butyl. E 1"3 may be independently selected from H, Me, and Et. E 1 may be selected from H, Me, Et, phenyl, and tert-butyl. E may be selected from phenyl, Me, and Et. E “ may be independently selected from Me and Et. E " may be Me. E may be Et, Me, or phenyl. E may be Me.
  • a method of treating a microbial infection includin administering a compound of Formula (A):
  • R may be N-R J ; R may be C-H; R may be C-OH; R may be H or Me or Et; A 1 may be R 5 , F, CI, Br, or I; A 2 may be H, F, CI, Br, or I; A 3 may be OMe, F, CI, Br, I, or R 5 ; A 4 may be F, CI, Br, I, or R 5 ; Q 1 may be R 5 , F, CI, Br, or I; Q 2 may be R 5 , F, CI, Br, or I; Q 3 may be R 5 , F, CI, Br, or I; Q 4 may be H or OH;
  • L may be ; wherein, D 1 may be R 5 ; and E 1 may be R 5 or phenyl.
  • a method of treating microbial infection including administering a compound selected from TABLE 2A r
  • a method treating microbial infection comprising administering a compound selected from TABLE 2B
  • a method treating microbial infection comprising administering a compound selected from TABLE 2C
  • a method treating a microbial infection comprising administering a compound selected from TABLE 2D or
  • a method of treating a microbial infection including administering a compound of the Formula (C):
  • G 1 may be G 5 , OG 5 , F, CI, Br, I, C ⁇ CH, S0 2 G 5 , NHCOG 5 , NHS0 2 G 5 , N0 2 , CON(G 5 ) 2 or phenyl;
  • G 2 may be G 5 , OG 5 , F, CI, Br, I, C ⁇ CH, S0 2 G 5 , NHCOG 5 , NHS0 2 G 5 , N0 2 , CON(G 5 ) 2 or phenyl;
  • G 3 may be G 5 , OG 5 , F, CI, Br, I, C ⁇ CH, S0 2 G 5 , NHCOG 5 , NHS0 2 G 5 , N0 2 , CON(G 5 ) 2 or phenyl;
  • G 4 may be G 5 , OG 5 , F, CI, Br, I, C ⁇ CH, S0 2 G 5 , NHCOG 5 , NHS0 2 G 5 , N0 2 , CON(G 5 ) 2 or phenyl;
  • M 1 may be G 5 , OG 5 , F, CI, Br, I, N 3 , C ⁇ CH, or phenyl;
  • M 2 may be G 5 , OG 5 , F, CI, Br, I, N 3 , C ⁇ CH, or phenyl;
  • M 3 may be G 5 , OG 5 , F, CI, Br, I, N 3 , C ⁇ CH, or phenyl;
  • M 4 may be G 5 , OG 5 , F, CI, Br, I, N 3 , C ⁇ CH, or phenyl;
  • M 5 may be G 5 , OG 5 , F, CI, Br, I, N 3 , C ⁇ CH, or phenyl;
  • M 6 may be G 5 , OG 5 , F, CI, Br, I, N 3 , C ⁇ CH, or phenyl;
  • M 7 may be H or OH.
  • a method of treating a microbial infection including administering a compound having the
  • R 1 may be N-R 5 , S, or O;
  • R 2 may be C-H, or N;
  • R 3 may be C-H, C-OR 5 , N, C-OCH 2 C ⁇ CH, C-OCH 2 OCH 3 , C-OCH 2 OCH 2 CH 3 , C- OC(0)CH 3 , or C-OCH 2 (OCH 2 CH 2 ) n OR 5 ;
  • R 4 may be C
  • R 5 may be H, or a C1-C6 branched or unbranched, saturated or unsaturated, alkyl or acyl group;
  • n may be 1-5;
  • a 1 may be R 5 , OR 5 , F, CI, Br, I, C ⁇ CH, S0 2 R 5 , NHCOR 5 , NHS0 2 R 5 , N0 2 , CF 3 , CBr 3 , CC1 3 , CI 3 , CON(R 5 ) 2 , or phenyl;
  • a 2 may be H, F, CI, Br, I, C ⁇ CH, OR 5 , CF 3 , CBr 3 , CC1 3 , CI 3 , or phenyl;
  • a 3 may be F, CI, Br, I, C ⁇ CH, OR 5 , R 5 , S0 2 R 5 , NHCOR 5 , NHS0 2 R 5 , N0 2 , CF 3 , CBr 3 ,
  • a 4 may be F, CI, Br, I, C ⁇ CH, OR 5 , R 5 , S0 2 R 5 , NHCOR 5 , NHS0 2 R 5 , N0 2 , CF 3 , CBr 3 ,
  • Q 1 may be R 5 , OR 5 , F, CI, Br, I, N 3 , C ⁇ CH, or phenyl;
  • Q 2 may be R 5 , OR 5 , F, CI, Br, I, N 3 , C ⁇ CH, or phenyl;
  • Q 3 may be R 5 , OR 5 , F, CI, Br, I, N 3 , C ⁇ CH, or phenyl;
  • Q 4 may be H or OH
  • Q and Q" may optionally form a benzene ring optionally substituted with R , OR , F, CI, Br, I, N 3 , C ⁇ CH, phenyl or a C6-C10 aryl group;
  • Q and Q may optionally form a benzene ring optionally substituted with R , OR , F, CI, Br, I, N 3 , C ⁇ CH, or phenyl;
  • Q 3 and Q 4 may optionally form a benzene ring optionally substituted with R 5 , OR 5 , F, CI, Br, I, N 3 , C ⁇ CH, phenyl or a C6-C10 aryl group;
  • D 1 may be R 5 ;
  • E 1 may be R 5 or phenyl
  • E may be R or phenyl
  • E may be R or phenyl
  • R 1 may be N-R 5 , S, or O;
  • R 2 may be C-H, or N;
  • R 3 may be C-H, C-OR 5 , N, C-OCH 2 C ⁇ CH, C-OCH 2 OCH 3 , C-OCH 2 OCH 2 CH 3 , C-
  • R 4 may be C
  • R 5 may be H, or a C1-C6 branched or unbranched, saturated or unsaturated, alkyl or acyl group;
  • n may be 1-5;
  • a 1 may be R 5 , OR 5 , F, CI, Br, I, C ⁇ CH, S0 2 R 5 , NHCOR 5 , NHS0 2 R 5 , N0 2 , CF 3 , CBr 3 , CC1 3 , CI 3 , CON(R 5 ) 2 , or phenyl;
  • a 2 may be H, F, CI, Br, I, C ⁇ CH, OR 5 , CF 3 , CBr 3 , CC1 3 , CI 3 , or phenyl;
  • a 3 may be F, CI, Br, I, C ⁇ CH, OR 5 , R 5 , S0 2 R 5 , NHCOR 5 , NHS0 2 R 5 , N0 2 , CF 3 , CBr 3 ,
  • a 4 may be F, CI, Br, I, C ⁇ CH, OR 5 , R 5 , S0 2 R 5 , NHCOR 5 , NHS0 2 R 5 , N0 2 , CF 3 , CBr 3 ,
  • Q 1 may be R 5 , OR 5 , F, CI, Br, I, N 3 , C ⁇ CH, or phenyl;
  • Q 2 may be R 5 , OR 5 , F, CI, Br, I, N 3 , C ⁇ CH, or phenyl;
  • Q 3 may be R 5 , OR 5 , F, CI, Br, I, N 3 , C ⁇ CH, or phenyl;
  • Q 4 may be H or OH
  • Q 1 and Q 2 may optionally form a benzene ring optionally substituted with R 5 , OR 5 , F, CI, Br, I, N 3 , C ⁇ CH, phenyl or a C6-C10 aryl group;
  • Q 2 and Q 3 may optionally form a benzene ring optionally substituted with R 5 , OR 5 , F, CI, Br, I, N 3 , C ⁇ CH, or phenyl; or Q 3 and Q 4 may optionally form a benzene ring optionally substituted with R , OR , F, CI, Br, I, N 3 , C ⁇ CH, phenyl or a C6-C10 aryl group;
  • D 1 may be R 5 ;
  • E 1 may be R 5 or phenyl
  • E may be R or phenyl
  • E may be R or phenyl
  • the compound may be for the manufacture of a medicament for treating a microbial infection.
  • R 3 may be C-H, C-OR 5 , N, C-OCH 2 C ⁇ CH, C-OCH 2 OCH 3 , C- OCH 2 OCH 2 CH 3 , or C-OC(0)CH 3 .
  • R 3 may be C-H, C-OH, C-OMe, C-OEt, N, C- OCH 2 C ⁇ CH, C-OCH 2 OCH 3 , C-OCH 2 OCH 2 CH 3 , or C-OC(0)CH 3 .
  • R 3 may be C-H, C-OR 5 , or N.
  • R 3 may be C-H, C-OH, C-OMe, C-OEt, or N.
  • R 1 may be N-H, N-Me, N-Et, S, or O.
  • R' may be N-R 5 .
  • R 1 may be N-H, N-Me, or N-Et.
  • R 1 may be S, or O.
  • R 2 may be N.
  • R 2 may be C-H.
  • R 5 may be a C1-C6 branched or unbranched, saturated or unsaturated, alkyl.
  • a 1 may be R s , OR 5 , F, CI, Br, I, C ⁇ CH, or phenyl.
  • a 1 may be H, O-Me, F, CI, Br, I, C ⁇ CH, or phenyl.
  • a 1 may be H, O-Me, F, CI, Br, or I.
  • a 2 may be H, F, CI, Br, I, C ⁇ CH, or phenyl.
  • a 2 may be H, O-Me, F, CI, Br, I, C ⁇ CH, or phenyl.
  • a 2 may be H, O-Me, F, CI, Br, or I.
  • a 3 may be F, CI, Br, I, C ⁇ CH, OR 5 , R 5 , or phenyl.
  • a 3 may be F, CI, Br, I, C ⁇ CH, H, or O-Me.
  • a 3 may be H, O-Me, F, CI, Br, or I.
  • a 3 may be F, CI, Br, I, OR 5 , R 5 , or phenyl.
  • a 4 may be F, CI, Br, I, C ⁇ CH, OR 5 , R 5 , or phenyl.
  • a 4 may be F, CI, Br, I, C ⁇ CH, OR 5 , R 5 , N0 2 , CON(R 5 ) 2 or phenyl.
  • a 4 may be F, CI, Br, I, OR 5 , R 5 , or N0 2 .
  • a 4 may be H, O-Me, F, CI, Br, or I.
  • Q 1"3 may be independently selected from R 5 , OR 5 , F, CI, Br, I, and N 3 .
  • Q 1 3 may be independently selected from H, OMe, or OH.
  • Q 4 may be H.
  • L may be .
  • D 1 may be H or
  • E 1 "3 may be independently selected from H, Me, Et, phenyl, and tert-butyl.
  • E 1"3 may be independently selected from H, Me, and Et.
  • E 1 may be selected from H, Me, Et, phenyl, and tert-butyl.
  • E may be selected from phenyl, Me, and Et.
  • E " may be independently selected from Me and Et.
  • E " may be Me.
  • E may be Et, Me, or phenyl.
  • E may be Me.
  • R may be N-R 5 ;
  • R 2 may be C-H;
  • R may be C-OH;
  • R may be H or Me or Et;
  • a 1 may be R 5 , F, CI, Br, or I;
  • a 2 may be H, F, CI, Br, or I;
  • a 3 may be OMe, F, CI, Br, I, or R 5 ;
  • a 4 may be F, CI, Br, I, or R 5 ;
  • Q 1 may be R 5 , F, CI, Br, or I;
  • Q 2 may be R 5 , F, CI, Br, or I;
  • Q 3 may be R 5 , F, CI, Br, or I;
  • Q 4 may be H or OH;
  • R 1 may be N-H or N-Me; R 3 may be C-OH; A 1 may be H; A 2 may be H, or F; A 3 may be OMe, F, CI, Br, or H; A 4 may be H, or F; Q 1 may be H; Q 2 may be H; Q 3 may be Br, or I; Q 4 may be H; D 1 may be H; and E 1 may be Me, Et, or phenyl.
  • G 1 may be G 5 , OG 5 , F, CI, Br, I, C ⁇ CH, S0 2 G 5 , NHCOG 5 , NHS0 2 G 5 , N0 2 , CON(G 5 ) 2 or phenyl;
  • G 2 may be G 5 , OG 5 , F, CI, Br, I, C ⁇ CH, S0 2 G 5 , NHCOG 5 , NHS0 2 G 5 , N0 2 , CON(G 5 ) 2 or phenyl;
  • G 3 may be G 5 , OG 5 , F, CI, Br, I, C ⁇ CH, S0 2 G 5 , NHCOG 5 , NHS0 2 G 5 , N0 2 , CON(G 5 ) 2 or phenyl;
  • G 4 may be G 5 , OG 5 , F, CI, Br, I, C ⁇ CH, S0 2 G 5 , NHCOG 5 , NHS0 2 G 5 , N0 2 , CON(G 5 ) 2 or phenyl;
  • M 1 maybe G 5 , OG 5 , F, CI, Br, I, N 3 , C ⁇ CH, or phenyl;
  • M 2 may be G 5 , OG 5 , F, CI, Br, I, N 3 , C ⁇ CH, or phenyl;
  • M 3 may be G 5 , OG 5 , F, CI, Br, I, N 3 , C ⁇ CH, or phenyl;
  • M 4 may be G 5 , OG 5 , F, CI, Br, I, N 3 , C ⁇ CH, or phenyl;
  • M 5 may be G 5 , OG 5 , F, CI, Br, I, N 3 , C ⁇ CH, or phenyl;
  • M 6 maybe G 5 , OG 5 , F, CI, Br, I, N 3 , C ⁇ CH, or phenyl;
  • M 7 may be H or OH
  • the compound may be any organic compound.
  • G 1 may be G 5 , OG 5 , F, CI, Br, I, C ⁇ CH, S0 2 G 5 , NHCOG 5 , NHS0 2 G 5 , N0 2 , CON(G 5 ) 2 or phenyl;
  • G 2 may be G 5 , OG 5 , F, CI, Br, I, C ⁇ CH, S0 2 G 5 , NHCOG 5 , NHS0 2 G 5 , N0 2 , CON(G s ) 2 or phenyl;
  • G 3 may be G 5 , OG 5 , F, CI, Br, I, C ⁇ CH, S0 2 G 5 , NHCOG 5 , NHS0 2 G 5 , N0 2 , CON(G 5 ) 2 or phenyl;
  • G 4 may be G 5 , OG 5 , F, CI, Br, I, C ⁇ CH, S0 2 G 5 , NHCOG 5 , NHS0 2 G 5 , N0 2 , CON(G 5 ) 2 or phenyl;
  • M 1 may be G 5 , OG 5 , F, CI, Br, I, N 3 , C ⁇ CH, or phenyl;
  • M 2 may be G 5 , OG 5 , F, CI, Br, I, N 3 , C ⁇ CH, or phenyl;
  • M 3 may be G 5 , OG 5 , F, CI, Br, I, N 3 , C ⁇ CH, or phenyl;
  • M 4 may be G 5 , OG 5 , F, CI, Br, I, N 3 , C ⁇ CH, or phenyl;
  • M 5 may be G 5 , OG 5 , F, CI, Br, I, N 3 , C ⁇ CH, or phenyl;
  • M 6 may be G 5 , OG 5 , F, CI, Br, I, N 3 , C ⁇ CH, or phenyl;
  • M 7 may be H or OH
  • the compound may be for the manufacture of a medicament for treating a microbial infection.
  • the compound may be
  • a pharmaceutical composition for treating a microbial infection may include a compound described herein and a pharmaceutically acceptable carrier.
  • a commercial package comprising (a) a compound described herein; and (b) instructions for the use thereof for treating a microbial infection.
  • commercial package comprising (a) a pharmaceutical composition comprising a compound described herein and a pharmaceutically acceptable carrier; and (b) instructions for the use thereof for treating a microbial infection.
  • method for testing a candidate compound for selective binding to a pathogen pyruvate kinase including: (a) combining the candidate compound with a pathogen pyruvate kinase monomelic subunits; (b) combining the candidate compound with human pyruvate kinase monomelic subunits; and (c) assaying for pyruvate kinase tetramer/dimer formation in both (a) and (b).
  • the pathogen may be MRSA.
  • FIGURE 1 shows a inhibition studies with NSK4-77 and NSK5-15.
  • NSK4-77 was used at 0 nM (O), 100 nM ( ⁇ ), 200 nM (V) and 400 nM (O).
  • NSK5-15 was used at 0 nM (O), 50 nM ( ⁇ ), 100 nM (V), and 200 nM (O).
  • PK enzymatic activity was assayed as described in Materials and Methods. The data shown are derived from one of two experiments performed in triplicate Error bars indicate range of values within the single experiment shown.
  • FIGURE 2 shows a cytotoxicity assessment of PK lead compounds against HeLa cells (A) compared to that of the MDRSA S. aureus (B).
  • Compounds NSK4-77 (O), NSK5- 15 ( ⁇ ) and AM-165(V) were used at 0-500 ⁇ to determine CC 5 o and MIC as described in Material and Methods.
  • the data presented are representative of three experiments performed in triplicate.
  • FIGURE 3 shows a time-kill curve of S. aureus incubated with AM- 165.
  • S. aureus ATCC 25923 was incubated with vehicle alone (e.g., DMSO) (O) as growth control, or AM- 165 at 1 x MIC (A), 2 x MIC (V), 4 x MIC (O) and 8 x MIC ( ⁇ ) or vancomycin at 2 x MIC ( ⁇ ) and 4 x MIC ( ⁇ ) and sampled at the indicated time points.
  • vehicle alone e.g., DMSO
  • O DMSO
  • AM- 165 at 1 x MIC (A), 2 x MIC (V), 4 x MIC (O) and 8 x MIC ( ⁇ ) or vancomycin at 2 x MIC ( ⁇ ) and 4 x MIC ( ⁇ ) and sampled at the indicated time points.
  • the logio values of cfu/ml were plotted versus time as described in Material and Methods. The data shown are
  • FIGURE 4. shows a serial passage of S. aureus RN4220 in either NSK5-15 ( ⁇ ) or fusidic acid ( ⁇ ).
  • the highest sublethal concentration of compound (denoted as fold-MIC) is plotted versus the number of days of serial passage for each compound as describes in Material and Methods.
  • FIGURE 5 shows a pyruvate concentrations in S. aureus cells challenged with PK lead compound AM- 165. Pyruvate concentrations were measured in S. aureus RN4220 cells incubated in the absence (Control) or presence of the highest sublethal concentration of AM- 165 (e.g, 1.25 as described in Material and Methods. The data presented are mean + SD of three independent experiments each performed in triplicates.
  • FIGURE 6. shows a series of plots (Top) NSK-460 and 465 (10 ⁇ ) selectively inhibit MRSA PK enzymatic activity; (Middle) Effects of NSK-460 and 465 on S. aureus growth; (Bottom) Toxicity evaluation of NSK-460 and 465 for human HeLa cells.
  • FIGURE 7 shows a resolved structure of MRSA252 pyruvate kinase tetramer showing the domain boundaries and tetramer architecture, wherein each monomer primarily fills one quadrant of the image to facilitate the identification of the small and large interface shown as lines, and the binding site are shown located at the small interface sits in between two alpha helices formed two PK monomers shown as circles.
  • This Figure was generated by MOE version 2007.09 [Larkin MA et al. Clustal W and Clustal X version 2.0. Bioinformatics
  • FIGURE 8A shows a sequence alignment with the interface region (highlighted residues) for pyruvate kinase (PK) from Staphylococcus aureus and Homo sapiens. The poorly conserved residues between MRSA and human PK are highlighted dark. The small interface also encompasses an insertion region in human PK (residues SD) and the corresponding deletion area in MRSA PK.
  • This Figure was generated by ClustalX version 2.0 [Tulloch BL et al. Sulphate removal induces a major conformational change in Leishmania mexicana Pyruvate kinase in the crystalline state. Journal of Molecular Biology
  • FIGURES 8B and C show a structural modelS of the interface-binding site for MRSA and human PK, respectfully.
  • the spheres show the interface cavity in MRSA and human PK, which demonstrates an accessible binding pocket located at the interface of two PK monomers in MRSA FIGURE 8B, as compared to the pocket in human PK which is partially obstructed by five amino acid residues (Glu418-B, Arg399-A, B and Arg400-A, FIGURE 8C.
  • This figure was generated using the MOE molecular package [Larkin MA et al. Clustal W and Clustal X version 2.0. Bioinformatics 2007, 23(21):2947-2948].
  • FIGURES 9A and B show a binding model of NSK-465 at the interface binding site.
  • FIGURE 9A shows a two-dimensional map of the binding interactions between NSK-465 and the interface site based on its co-crystallization with MRSA PK, wherein the arrows depict hydrogen-accepting interactions between NSK-465 and MRSA PK residues from the interface.
  • FIGURE 9B shows the binding orientation of NSK-465 within the interface- binding pocket based on the protein-ligand crystal structure.
  • C ]-6 alkyl or "Q-Cg alkyl” is used as it is normally understood to a person of skill in the art and often refers to a chemical entity that has a carbon skeleton or main carbon chain comprising a number from 1 to 6 (with all individual integers within the range included, including integers 1 and 6) of carbon atoms.
  • a "Ci-6 alkyl” is a chemical entity that has 1, 2, 3, 4, 5, or 6 carbon atom(s) in its carbon skeleton or main chain.
  • branched is used as it is normally understood to a person of skill in the art and often refers to a chemical entity that comprises a skeleton or main chain that splits off into more than one contiguous chain.
  • the portions of the skeleton or main chain that split off in more than one direction may be linear, cyclic or any combination thereof.
  • Non-limiting examples of a branched alkyl are tert-butyl and isopropyl.
  • unbranched is used as it is normally understood to a person of skill in the art and often refers to a chemical entity that comprises a skeleton or main chain that does not split off into more that one contiguous chain.
  • unbranched alkyls are methyl, ethyl, n-propyl, and n-butyl.
  • substituted is used as it is normally understood to a person of skill in the art and often refers to a chemical entity that has one chemical group replaced with a different chemical group that contains one or more heteroatoms.
  • a substituted alkyl is an alkyl in which one or more hydrogen atom(s) is/are replaced with one or more atom(s) that is/are not hydrogen(s).
  • chloromethyl is a non-limiting example of a substituted alkyl, more particularly an example of a substituted methyl.
  • Aminoethyl is another non-limiting example of a substituted alkyl, more particularly it is a substituted ethyl.
  • the functional groups described herein may be substituted with, for example, and without limitation, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 substituents.
  • unsubstituted is used as it is normally understood to a person of skill in the art and often refers to a chemical entity that is a hydrocarbon and/or does not contain a heteroatom.
  • unsubstituted alkyls include methyl, ethyl, tert-butyl, and pentyl.
  • saturated when referring to a chemical entity is used as it is normally understood to a person of skill in the art and often refers to a chemical entity that comprises only single bonds.
  • saturated chemical entities include ethane, tert-butyl, and N + 3 ⁇ 4.
  • halogenated is used as it would normally be understood to a person of skill in the art and refers to a moiety or chemical entity in which a hydrogen atom is replaced with a halogen atom such as chlorine, fluorine, iodine or bromine.
  • a chlorinated side chain of a naturally occurring amino acid refers to a side chain of a naturally occurring amino acid wherein one or more hydrogen atoms occurring in the side chain of the naturally occurring amino acid is replaced with one or more chlorine atoms.
  • Non-limiting examples of saturated Ci-C 6 alkyl may include methyl, ethyl, n-propyl, i- propyl, sec-propyl, n-butyl, i-butyl, sec-butyl, t-butyl, n-pentyl, i-pentyl, sec-pentyl, t-pentyl, n-hexyl, i-hexyl, 1 ,2-dimethylpropyl, 2-ethylpropyl, l-methyl-2-ethylpropyl, l-ethyl-2- methylpropyl, 1,1 ,2-trimethylpropyl, 1 , 1 ,2-triethylpropyl, 1,1-dimethylbutyl, 2,2- dimethylbutyl, 2-ethylbutyl, 1 ,3-dimethylbutyl, 2-methylpentyl, 3-methylpentyl, sec-hexyl,
  • Non-limiting examples of C 2 -C 6 alkenyl may include vinyl, allyl, isopropenyl, 1- propene-2-yl, 1-butene-l -yl, 1 -butene-2-yl, l -butene-3-yl, 2-butene-l-yl, and 2-butene-2-yl.
  • Non-limiting examples of C 2 -C 6 alkynyl may include ethynyl, propynyl, butynyl, pentynyl, and hexynyl.
  • Saturated Ci-C 6 alkyl, C 2 -C 6 alkenyl or C 2 -C 6 alkynyl may be, for example, and without limitation, interrupted by one or more heteroatoms which are independently nitrogen, sulfur or oxygen.
  • Ci-C 6 substituted or unsubstituted acyl include acetyl, propionyl, butanoyl and pentanoyl.
  • alkoxy include methoxy, ethoxy, propoxy and butoxy.
  • Non-limiting examples of the C 6 -Cio aryl group may include phenyl (Ph), benzyl, tolyl, o-xylyl, pentalenyl, indenyl, naphthyl, and azulenyl.
  • MRSA methicillin-resistant Staphylococcus aureus
  • RSA oxacillin-resistant Staphylococcus aureus
  • MRSA multi-drug resistant Staphylococcus aureus
  • the compounds described herein may be in isolation, or may be linked to or in combination with liposomes, carbohydrate carriers, polymeric carriers or other agents or excipients as will be apparent to one of skill in the art.
  • such compounds may comprise a medicament, including other antibiotics, wherein such compounds may be present in a pharmacologically effective amount.
  • compositions or compounds according to some embodiments may be administered in any of a variety of known routes.
  • methods that may be suitable for the administration of a compound include orally, intravenous, inhalation, intramuscular, subcutaneous, topical, intraperitoneal, intra-rectal or intra-vaginal suppository, sublingual, and the like.
  • the compounds of the present invention may be administered as a sterile aqueous solution, or may be administered in a fat-soluble excipient, or in another solution, suspension, patch, tablet or paste format as is appropriate.
  • a composition comprising the compounds of the invention may be formulated for administration by inhalation. For instance, a compound may be combined with an excipient to allow dispersion in an aerosol.
  • inhalation formulations will be known to those skilled in the art.
  • Other agents may be included in combination with the compounds of the present invention to aid uptake or metabolism, or delay dispersion within the host, such as in a controlled-release formulation.
  • controlled release formulations will be known to those of skill in the art, and may include microencapsulation, embolism within a carbohydrate or polymer matrix, and the like.
  • Other methods known in the art for making formulations are found in, for example, "Remington's Pharmaceutical Sciences", (19th edition), ed. A. Gennaro, 1995, Mack Publishing Company, Easton, Pa.
  • the dosage of the compositions or compounds may vary depending on the route of administration (for example, oral, intravenous, inhalation, or the like) and the form in which the composition or compound is administered (for example, solution, controlled release or the like). Determination of appropriate dosages is within the ability of one of skill in the art.
  • an 'effective amount', a 'therapeutically effective amount', or a 'pharmacologically effective amount' of a medicament refers to an amount of a medicament present in such a concentration to result in a therapeutic level of drug delivered over the term that the drug is used. This may be dependent on mode of delivery, time period of the dosage, age, weight, general health, sex and diet of the subject receiving the medicament. Methods of determining effective amounts are known in the art.
  • PK Pyruvate kinase
  • PEP phosphoenolpyruvate
  • ADP phosphoenolpyruvate
  • ATP pyruvate and ATP
  • the PK L isozyme is major isozyme in the liver
  • the R isozyme is found in red blood cells
  • the Ml isozyme is the main form in muscle, heart and brain
  • M2 is found in early fetal tissues.
  • Pyruvate kinase isozymes M1/M2 are encoded by the PKM2 gene (alternative references include M23725 mRNA (AAA36449.1); M26252 mRNA (AAA36672.1); X56494 Genomic DNA (CAA39849.1); AK092369 mRNA (BAG52542.1); AK222927 mRNA (BAD96647.1); AK294315 mRNA (BAG57589.1 note different initiation); AK312253 mRNA (BAG35185.1); AY352517 Genomic DNA (AAQ 15274.1); AC020779 Genomic DNA; CH471082 Genomic DNA (EAW77884.1); CH471082 Genomic DNA (EAW77888.1); BC000481 mRNA (AAH00481.3); BC007640 mRNA (AAH07640.1); BC007952 mRNA (AAH07952.3); BC012811 mRNA (AAH12811.3); BC035
  • the primary difference in the Ml and M2 isozymes is at the c-terminus.
  • the pyruvate kinase isozymes R/L are encoded by the PKLR gene (alternative references include AB015983 mRNA (BAA31706.1); M15465 mRNA (AAA60104.1.); AY316591 Genomic DNA (AAP69527.1); BC025737 mRNA (AAH25737.1); S60712 mRNA (AAB26262.1); and reference sequences NP 000289.1.; NP_870986.1).
  • the pyruvate kinase isozymes R, L, Ml, and M2 form a homotetramer.
  • Pyruvate kinases from pathogenic species are also known in the art (for example, Leishmania mexicana (X74944 Genomic DNA (CAA52898.2)); Chlamydia pneumoniae (AE001363 Genomic DNA (AAD18250.1) and ref seq. NP_224305.1); Mycoplasma genitalium (L43967 Genomic DNA (AAC71435.1) U01798 Genomic DNA (AAD12324.1) and ref seq. NP_072881.1); Mycobacterium tuberculosis (BX842577 Genomic DNA (CAB08894.1) ref seq.
  • NP_216133.1 Candida albicans (S65775 mRNA); Escherichia coli 0157:H7 (AE005174 Genomic DNA (AAG56663.1) and ref seq. NP_288110.1.); Salmonella typhi (AL627271 Genomic DNA (CADOl 987.1) and ref seq. NP_456147.1); Trypanosoma brucei brucei (X57950 Genomic DNA (CAA41018.1)); Staphylococcus aureus (strain MRSA252) BX571856 Genomic DNA (CAG40767.1) and ref seq. YP_041163.1) etc.).
  • the method may include a test to determine whether a candidate compound selectively binds to a pathogen pyruvate kinase (for example, MRSA), wherein a candidate compound is combined with pathogen pyruvate kinase monomelic subunits and the candidate compound is also combined with one or more of the human pyruvate kinase monomelic subunits (i.e.
  • a pathogen pyruvate kinase for example, MRSA
  • assaying for pyruvate kinase tetramer and/or dimer formation may be accomplished using monomer- specific monoclonal antibodies may be used to quantify monomer by immunocytochemistry (see for example, Ashizawa et al. JBC (1991) 266 16842-1 846).
  • dimmer and tetramer formation may be assayed via pyruvate kinase activity assays (for example, abeamTM Pyruvate-Kinase-PK-Assay-Kit (catalog # ab83432); Sigma AldrichTM Enzymatic Assay of pyruvate kinase substrate Phospho(enol)pyruvic acid tri(cyclohexylammonium) salt Fluka (catalog # 79430); Bio VisionTM Pyruvate Kinase Assay Kit (catalog# K709-100), Gel filtration and immunodetection (see for example, Adachi et al.
  • pyruvate kinase activity assays for example, abeamTM Pyruvate-Kinase-PK-Assay-Kit (catalog # ab83432); Sigma AldrichTM Enzymatic Assay of pyruvate kinase substrate Phospho(en
  • Mass spectrometry coupled with the soft ionization processes of either matrix-assisted laser desorption (MALDI) or electrospray (ES) ionization (for example, Hernandez and Robinson (2007) Nature Protocols 2: 715 - 726), and assays for tetramer formation (for example, Ashizawa et al (1991) Biochemistry 30:7105-7111 ; and Desmaret et al. (2005) Biological Chemistry. 386:1137-1147).
  • MALDI matrix-assisted laser desorption
  • ES electrospray
  • MRSA methicillin resistant S. aureus
  • S. aureus RN4220 S. aureus RN4220 (NCTC8325 NRS144), Hyper- virulent community-acquired MSSA sequenced at the Sanger Centre (MSSA476, NRS72), MRSA strain sequenced at TIGR, (COL, NRS100) and community- acquired MRSA strain sequenced at the National Institute of Technology and Evaluation, Tokyo (USA400, MW2, NRS123) were obtained from NARSA (Network on Antimicrobial Resistance in S. aureus).
  • Methicilin sensitive & aureus ATCC 25923
  • Streptococcus Pneumoniae ATCC 49619
  • Streptococcus Pyogenes ATCC 700294
  • Listeria monocytogenes ATCC19115
  • Enterococcus faecium ATCC35667
  • Enterococcus faecalis ATCC29212
  • Enterococcus faecium ATCC700221) (VRE) were from ATCC, The Global Bioresourse Center.
  • Multi drug resistant (MDR) MRSA, Staphylococcus saprophyticus, Staphylococcus haemolyticus, Staphylococcus epidermidis, Acinetobacter baumannii and ESBL-producing Klebsiella pneumoniae were clinical isolates obtained from the Vancouver General Hospital (Vancouver, Canada).
  • Escherichia coli DYE330, Pseudomonas aeruginosa PAO-1 were obtained from the laboratory of Dr B.B. Finlay at the University of British Columbia (Vancouver, Canada).
  • IS-63 is commercially available from EnamineTM (Product Code: T5435622).
  • IS-130 is commercially available from Vitas-MTM (Product Code: STKO 15507). Analogs of IS-63 and IS-130 were synthesized as described herein. 10 mM compound stocks were prepared in DMSO and stored at -20 °C.
  • PK constructs Genomic DNA of MRSA strain Sanger 252 extracted using Dneasy Tissue KitTM (QiagenTM) was used as a template to generate the His-tagged MRSA PK. Human cDNA from MCF-7 breast cancer cell line (courtesy of Dr. J Wong, BC Cancer Research Center (Vancouver, Canada) was used as a template to generate the full- length human M2 PK enzyme. The following primer sets were used creating appropriate restriction sites (Ndel and Xhol sites underlined): For cloning of MRSA PK: M27F 5'- CTACATATGAGAAAAACTAAAATTGTATG-3 ' and M27R 5'-
  • MRSA and human constructs in pET-28a(+) were used to express relevant recombinant PK proteins in E. coli BL-21 (DE3).
  • the proteins were expressed and purified using Ni-NTA agarose (QiagenTM) according to the manufacturer's protocol. Briefly, cells were grown to an absorbance of 0.4-0.5 at 600 nm in 2xYT medium, then induced with 0.1 mM IPTG for 3 h at 20°C.
  • Cells were lysed by sonication on ice (3 x 10-s bursts with a 30-s recovery between bursts) in lysis buffer (0.2 mg/ml lysozyme, 50 mM Tris pH 7.5, 10 mM MgCl 2 , 200 mM NaCl, 100 mM KC1, 10% glycerol, 10 mM imidazole, 0.5% NP-40 and 1 mM DTT containing CompleteTM protease inhibitor). Cell lysates were cleared by centrifugation (18,000 x g in a BeckmanTM JA-20 rotor) for 20 min at 4 °C and PK isoforms were purified by batch binding to Ni-NTA resin.
  • lysis buffer 0.2 mg/ml lysozyme, 50 mM Tris pH 7.5, 10 mM MgCl 2 , 200 mM NaCl, 100 mM KC1, 10% glycerol, 10 mM imidazole
  • the resins were then packed in columns (1 x 2 cm) and washed with 400 column volumes lysis buffer containing 30 mM imidazole. His-tagged PK isoforms were eluted with the same buffer containing 300 mM imidazole.
  • the proteins were dialyzed overnight at 4 °C against 2000 volumes of ice-cold 30 mM Tris pH 7.5, 25 mM KC1, 5 mM MgCl 2 , 10% glycerol and 1 mM DTT to remove imidazole. All purification steps were done at 4°C; enzymes were flash-frozen and stored at -70 °C. Enzymatic activity of frozen protein preparations was stable for at least 10 months and up to 5 freeze/thaw cycles.
  • Protein concentration was estimated by Bradford assay (Bio-Rad Protein AssayTM) using bovine serum albumin as a standard.
  • PK activity was determined using a continuous assay coupled to lactate dehydrogenase (LDH) in which the change in absorbance at 340 nm owing to oxidation of NADH was measured using a Benchmark PlusTM microplate spectrophotometer (Bio-RadTM) ().
  • LDH lactate dehydrogenase
  • the reaction contained 60 mM Na + -HEPES, pH 7.5, 5% glycerol, 67 mM KC1, 6.7 mM MgCl 2 , 0.24 mM NADH, 5.5 units L-LDH from rabbit muscle (Sigma- AldrichTM), 2 mM ADP and 10 mM PEP (i.e. close to the K m of MRSA PK, so that the IC 50 values should approximate the Ki) in a total volume of 200 ⁇ . Reactions were initiated by the addition of 15 nM of one of the PK enzymes.
  • PK activity proportional to the rate of change at 340 nm was expressed as specific activity ( ⁇ ⁇ ⁇ /min/mg), which is defined as the amount of PK that catalyzes the formation of one micromole of either product per minute.
  • Inhibitors were dissolved in DMSO with the final concentration of the solvent never exceeding 1% of the assay volume.
  • IC 50 values were calculated by curve fitting on a four-parameter dose-response model with variable slope using Graphpad Prism 5.0TM (GraphPadTM Software Inc., La Jolla, CA). In all studies, less than 10% of total PEP was exhausted during the reaction. Reactions were performed at 30 °C for 5 min. All values determined represent three measurements, each in triplicate unless mentioned otherwise.
  • Mode-of-inhibition and K t values were determined by simultaneously changing the inhibitor concentration (0-400 nM) and substrate PEP concentration (2-20 mM) while keeping the level of the ADP substrate fixed at 2 mM.
  • the resulting curve at each inhibitor concentration was fitted by nonlinear regression to the allosteric sigmoidal kinetic model using Graphpad PrismTM. 3 ⁇ 4 values were obtained by nonlinear regression curve-fitting using the following equation ():
  • V max V max / (l + [I] / K t ) (1 )
  • PK inhibitor candidates were determined using the 96-well microtiter standard 2-fold serial broth microdilution method as described by CLSI (formerly NCCLS) () with the various gram-positive and gram- negative bacteria species mentioned above. Bacteria from a single colony were grown, overnight in BHI Broth (DifcoTM), harvested by centrifugation, and then washed twice with sterile distilled water. Each stock solution of compounds in DMSO was diluted with BHI to prepare serial two-fold dilutions in the range of 500 to 0.06 ⁇ .
  • MIC minimal inhibitory concentration
  • Freshly split cells were seeded into microtiter wells (2 x 10 4 /well) and grown for 24 hours. The original media was then removed and replaced with media containing the desired concentration of compound or solvent control ⁇ i.e., DMSO). Plates were incubated for 24 h at 37 °C in a humidified incubator with a 5% C0 2 atmosphere. At the end of the growth period, cells were lysed by the addition of 20 ⁇ of Cell Titer 96 Aqueous OneTM solution, and the incubation was continued for another 3 h at 37°C. Production of formazan was determined at 490 nm on Benchmark PlusTM microplate spectrophotometer (Bio-RadTM).
  • Soluble cell extracts were used to determine pyruvate levels by colorimetric measurement of pyruvate oxidation using Pyruvate Determination AssayTM kit (Bio VisionTM, Mountain View, CA, USA) with reference to a pyruvate standard curve, prepared as per manufacturer's instructions.
  • Time-kill studies were performed by the method of the CLSI M26-A.20 ().
  • S. aureus ATCC 25923 was cultured overnight at 37 °C in BHI.
  • Cells were diluted in medium to an initial OD 60 o of 0.1 (equal to concentration of approximately 10 cfu/ml) and incubated with shaking for 2 h at 37 °C to achieve logarithmic growth.
  • the culture was then diluted in medium to adjust the cell density to approximately 10 cfu/ml.
  • Either compound NS 5-15 to final concentrations of 1, 2, 4 and 8 times the MIC or vancomycin to final concentrations of 2 and 4 times the MIC were then added.
  • Bactericidal activity is defined as a >3 log reduction in initial cfu count within 24 h.
  • the compound was considered to be bacteriostatic at the concentration that reduced the original inoculum by 0-3 logio cfu/ml within 24 h.
  • Binding site identification The crystal structure of PK from Bacillus Stearothermophilis (PDB code: 2E28) [Edelsbrunner H et al. Measuring proteins and voids in proteins. In: Proceedings of the 28th Hawaii International Conference on Systems Science: 1995; Washington, DC: IEEE Computer Society; 1995: 256-264] with 62% sequence identity to the MRSA homologue was identified as a suitable template for the homology modelling [Rizzo RC and Jorgensen WL: OPLS All-atom model for amines: resolution of the amine hydration problem. J Am Chem 1999, 121 :4827-4836].
  • the homology-based model of the MRSA252 PK was accepted with a backbone RMSD of 0.46 A compared to its template.
  • the sequence alignment was refined using a global sequence alignment program, GGSEARCH (version 35.03 [Dombrauckas JD et al. Structural basis for tumor pyruvate kinase M2 allosteric regulation and catalysis. Biochemistry 2005, 44(27):9417-9429.]).
  • a homology model of the MRSA252 PK was built using Modeller9v4 [Friesner RA et al. Glide: a new approach for rapid, accurate docking and scoring. 1. Method and assessment of docking accuracy.
  • the 'Site Finder' functionality of the MOE package was used to identify suitable binding area around the small interface of the MRSA tetramer.
  • the site_finder is a generalization of the convex hulls method for calculating possible binding sites in receptors from 3D atomic coordinates. Alpha spheres were consequently generated to identify the binding site ( Figures 9B and C), which was subsequently used for molecular docking [Edelsbrunner H et al. 1995].
  • the interface-binding pocket of MRS A PK was optimized by the OPLS-AA force field energy minimization implemented by MOE [Rizzo RC and Jorgensen 1999].
  • the crystal structure (PDB code: 1T5A) for human PK was obtained from PDB [Dombrauckas JD et al. 2005] and used for a subsequent structural superimposition with the MRSA PK structure.
  • BBr 3 (0.8 mL, 1M in DCM, 0.8 mmol) was added to a stirred solution on l-(5-methoxy-lH- indol-2-yl)ethanone (80 mg, 0.42 mmol) in DCM (4 mL) at -78°C under Ar. The resulting mixture was stirred at 0°C for 3h. The mixture was partitioned between EtOAc (100 mL) and H 2 0 (50 mL). The organic phase was washed with brine (50 mL), dried over anhydrous Na 2 S0 4 and concentrated.
  • BBr 3 (0.65 mL, 1M in DCM, 0.65 mmol) was added to a stirred solution on l-(5-methoxy-l- methyl-lH-indol-2-yl)ethanone D2 (65 mg, 0.32 mmol) in DCM (3 mL) at -78°C under Ar. The resulting mixture was stirred at 0°C for 3h. The mixture was partitioned between EtOAc (100 mL) and H 2 0 (50 mL). The organic phase was washed with brine (50 mL), dried over anhydrous Na 2 S0 4 and concentrated.
  • Procedure A A mixture of the appropriate ketone C, D or H (0.25 mmol) and hydrazide E (0.25 mmol) in propan-l-ol (3 mL) was refluxed until completion (or good conversion) of the reaction (monitored by TLC). If the product precipitated, it was collected by filtration and the solid was washed with hot propan-l -ol (10 mL). If no precipitation was observed, the solvent was evaporated in vacuo and the compound was purified by flash column chromatography.
  • Procedure B A mixture of the appropriate ketone C, D or H (0.35 mmol) and hydrazide E (0.35 mmol) in EtOH (5 mL) was refluxed until completion (or good conversion) of the reaction (monitored by TLC). If the product precipitated, it was collected by filtration and the solid was washed with hot EtOH (2x2 mL). If no precipitation was observed, the solvent was evaporated in vacuo and the compound was purified by flash column chromatography.
  • Procedure C A mixture of the appropriate ketone C, D or H (0.41 mmol), hydrazide E (0.41 mmol) and AcOH (1 drop) in propan-l-ol (4 mL) was refluxed until completion (or good conversion) of the reaction (monitored by TLC). If the product precipitated, it was collected by filtration and the solid was washed with hot propan-l-ol (2x2 mL). If no precipitation was observed, the solvent was evaporated in vacuo and the compound was purified by flash column chromatography.
  • Procedure D A mixture of the appropriate ketone C, D or H (0.41 mmol), hydrazide E (0.41 mmol) and AcOH (1 drop) in ethanol (4 mL) was refluxed until completion (or good conversion) of the reaction (monitored by TLC). If the product precipitated, it was collected by filtration and the solid was washed with hot ethanol (2x2 mL). If no precipitation was observed, the solvent was evaporated in vacuo and the compound was purified by flash column chromatography.
  • Procedure E A mixture of the appropriate ester (1.0 eq.) and hydrazine hydrate (> 3.0 eq.) in ethanol was irradiated with microwaves for 60 minutes at 100°C. If the hydrazide E precipitated, it was collected by filtration. If no precipitation was observed, the mixture was partitioned between ethyl acetate and water, the organic layer dried over Na 2 S0 4 and evaporated in vacuo. To the hydrazide E taken up in ethanol were added the appropriate ketone C, D or H and AcOH (1 drop). The mixture was refluxed - classical heating- until completion (or good conversion) of the reaction (monitored by TLC). If the product precipitated, it was collected by filtration and the solid was washed with hot ethanol (2x2 mL). If no precipitation was observed, the solvent was evaporated in vacuo and the compound was purified by flash column chromatography.
  • Fl was prepared from lH-indole-2-carbaldehyde (obtained by reduction and oxidation of ⁇ H- indole-2-carboxylic acid, cf. scheme) and 5-bromo-2-hydroxybenzohydrazide E using general Procedure A.
  • F2 was prepared from l-(lH-indol-2-yl)propan-l-one C (obtained by addition of ethylmagnesium bromide to the Weinreb amide B, cf. scheme) and 5-bromo-2- hydroxybenzohydrazide E using general Procedure A.
  • F3 was prepared from l-(lH-indol-2-yl)-2,2-dimethylpropan-l-one C (obtained by addition of t-BuLi to the Weinreb amide B, cf. scheme) and 5-bromo-2-hydroxybenzohydrazide E using general Procedure A.
  • F5 was prepared from l-(5-fluoro-lH-indol-2-yl)ethanone CI and 5-bromo-2- hydroxybenzohydrazide E using general Procedure A.
  • F6 was prepared from l-(7-fluoro-lH-indol-2-yl)ethanone C2 and 5-bromo-2- hydroxybenzohydrazide E using general Procedure A.
  • F7 was prepared from l-(5-methoxy-lH-indol-2-yl)ethanone C and 5-bromo-2- hydroxybenzohydrazide E using general Procedure A.
  • F8 was prepared from l-(5,6-difluoro-lH-indol-2-yl)ethanone C3 and 5-bromo-2- hydroxybenzohydrazide E using general Procedure A.
  • F9 was prepared from l-(4,5-difluoro-lH-indol-2-yl)ethanone C4 and 5-bromo-2- hydroxybenzohydrazide E using general Procedure A.
  • F10 was prepared from l-(4,5,6-trifluoro-lH-indol-2-yl)ethanone C5 and 5-bromo-2- hydroxybenzohydrazide E using general Procedure A.
  • Fll was prepared from l-(5-phenyl-lH-indol-2-yl)ethanone C6 and 5-bromo-2- hydroxybenzohydrazide E using general Procedure A.
  • F12 was prepared from l-(5-bromo-lH-indol-2-yl)ethanone C and 5-bromo-2- hydroxybenzohydrazide E using general Procedure A.
  • F14 was prepared from l-(lH-indol-2-yl)ethanone C and 3,5-dibromo-2- hydroxybenzohydrazide E using general Procedure A.
  • F15 was prepared from l-(lH-indol-2-yl)ethanone C and 5-bromo-2-hydroxybenzohydrazide E using general Procedure B.
  • F16 was prepared from l-(lH-indol-2-yl)ethanone C and 5-bromobenzohydrazide E using general Procedure C.
  • F17 was prepared from l-(lH-indol-2-yl)ethanone C and 2-hydroxybenzohydrazide E using general Procedure C.
  • Br F18 was prepared from l-(lH-indol-2-yl)ethanone C and 2-methoxybenzohydrazide E using general Procedure C.
  • F19 was prepared from l-(lH-indol-2-yl)ethanone C and 2-hydroxy-5-iodobenzohydrazide E using general Procedure C.
  • F21 was prepared from l-(lH-indol-2-yl)ethanone C and 4-bromo-2-hydroxy-benzohydrazide E using general Procedure C.
  • F22 was prepared from l-(lH-indol-2-yl)ethanone C and 5-bromo-2-hydroxy-4- methoxybenzohydrazide E using general Procedure C.
  • F23 was prepared from l-(lH-indol-2-yl)ethanone C and 3-hydroxy-2-naphthohydrazide E using general Procedure C.
  • Gl was prepared from l-methyl-lH-indole-2-carbaldehyde D and 5-bromo-2- hydroxybenzohydrazide E using general Procedure A.
  • G2 was prepared from 1-(1 -methyl- lH-indol-2-yl)propan-l -one D and 5-bromo-2- hydroxybenzohydrazide E using general Procedure A.
  • G4 was prepared from ((1 -methyl- lH-indol-2-yl)(phenyl)methanone D and 5-bromo-2- hydroxybenzohydrazide E using general Procedure A.
  • G5 was prepared from l-(5-fluoro-l-methyl-lH-indol-2-yl)ethanone Dl and 5-bromo-2- hydroxybenzohydrazide E using general Procedure A.
  • G6 was prepared from l-(5-methoxy-l -methyl- lH-indol-2-yl)ethanone D2 and 5-bromo-2- hydroxybenzohydrazide E using general Procedure A.
  • G7 was prepared from l-(5,6-difluoro-l -methyl- lH-indol-2-yl)ethanone D3 and 5-bromo-2- hydroxybenzohydrazide E using general Procedure A.
  • G8 was prepared from l-(4,5-difluoro-l -methyl- lH-indol-2-yl)ethanone D4 and 5-bromo-2- hydroxybenzohydrazide E using general Procedure A.
  • G9 was prepared from l-(4,5,6-trifluoro-l-met yl-lH-indol-2-yl)ethanone D5 and 5-bromo- 2-hydroxybenzohydrazide E using general Procedure A.
  • G10 was prepared from l-(l-methyl-lH-indol-2-yl)ethanone D and 5-bromo-2- hydroxybenzohydrazide E using general Procedure B.
  • Gil was prepared from l-(5-bromo-l -methyl- lH-indol-2-yl)ethanone D6 and 2- hydroxybenzohydrazide E using general Procedure C.
  • G12 was prepared from 1-(1 -methyl- lH-indol-2-yl)ethanone D and 2-hydroxy-5- iodobenzohydrazide E using general Procedure C.
  • G13 was prepared from l-(5-chloro-l -methyl- lH-indol-2-yl)ethanone D7 and 5-bromo-2- hydroxybenzohydrazide E using general Procedure C.
  • G14 was prepared from 1-(1 -methyl- lH-indol-2-yl)ethanone D and 4-bromo-2- hydroxybenzohydrazide E using general Procedure C.
  • G15 was prepared from l-(l-methyl-lH-indol-2-yl)ethanone D and 5-bromo-2-hydroxy-4- methoxybenzohydrazide E using general Procedure C.
  • F29 was prepared from l-(6-bromo-lH-indol-2-yl)ethanone CIO and 5-bromo-2- hydroxybenzohydrazide E using general Procedure C.
  • F30 was prepared from l-(5-hydroxy-lH-indol-2-yl)ethanone Cll and 5-bromo-2- hydroxybenzohydrazide E using general Procedure C.
  • F31 was prepared from l-(5-bromo-lH-indol-2-yl)propan-l-one C12 and 3-hydroxy-2- naphthohydrazide E using general Procedure C.
  • F32 was prepared from l-(5-bromo-lH-indol-2-yl)ethanone C and 3-hydroxy-2- naphthohydrazide E using general Procedure C.
  • F33 was prepared from l-(5-bromo-lH-indol-2-yl)propan-l-one C12 and 5-bromo-2- hydroxybenzohydrazide E using general Procedure C.
  • F34 was prepared from l-(7H-indol-2-yl)ethanone C and 2-hydroxy-l-naphthohydrazide E using general Procedure D.
  • F35 was prepared from l-[5-(trifluoromethyl)-lH-indol-2-yl]ethanone C8 and 5-bromo-2- hydroxybenzohydrazide E using general Procedure D.
  • F36 was prepared from l-(5-iodo-lH-indol-2-yl)ethanone C9 and 5-bromo-2- hydroxybenzohydrazide E using general Procedure D.
  • F37 was prepared from l-(7H-indol-2-yl)ethanone C and hydrazide E of methyl 4-hydroxy- l,l'-biphenyl-3-carboxylate using general Procedure E.
  • F38 was prepared from l-(iH-indol-2-yl)ethanone C and hydrazide E of methyl 5-bromo-2- ethoxybenzoate using general Procedure E.
  • F39 was prepared from 1 -(7H-indol-2-yl)ethanone C and hydrazide E of ethyl 2-hydroxy-3,5- diisopropylbenzoate using general Procedure E.
  • G16 was prepared from l-(6-bromo-l -methyl- lH-indol-2-yl)ethanone D8 and 5-bromo-2- hydroxybenzohydrazide E using general Procedure C.
  • G18 was prepared from l-(5-bromo-l-methyl-lH-indol-2-yl)ethanone D9 and 3-hydroxy-2- naphthohydrazide E using general Procedure C.
  • G20 was prepared from l -(5-bromo-l -methyl- lH-indol-2-yl)ethanone D9 and 5-bromo-2- hydroxybenzohydrazide E using general Procedure C.
  • EXAMPLE 1 Expression and Purification of PK Proteins.
  • Recombinant His-tagged PK proteins including MRSA PK and human PK isoforms were expressed, purified to near homogeneity (>98%), and detected by SDS-PAGE [(12% (w/v) gel] as described herein.
  • the estimated molecular mass of each of the respective bands on SDS-PAGE correlated well with that predicted based on the amino acid composition for each PK protein (data not shown), indicating that all PK constructs were expressed as full-length proteins. Structural integrity of each protein was verified by the pattern of migration on SDS-PAGE and allosteric properties for each construct (data not shown). All PKs demonstrated enzyme activity comparable to those reported previously (data not shown) ().
  • MRSA and human PK proteins were used to characterize the biochemical properties of candidate MRSA PK inhibitors.
  • EXAMPLE 2 In Vitro Screening of Putative MRSA PK Inhibitors.
  • IS- 130 (NSK4-65) and IS-63 (NSK4-60) were used at a concentration of 50 ⁇ in a recombinant MRSA PK assay, with a substrate concentration of 10 mM PEP, which is close to MRSA PK K m (e.g., 6.6 mM) so that the IC 50 values should approximate the K ⁇ .
  • These compounds were also screened against human PK isoforms (Ml, M2, R and L) to test for selectively inhibit bacterial PK.
  • One compound, IS-63 (NSK4-60) was inhibitory in the nanomolar range and demonstrated selectivity for MRSA PK (FIGURE 6). However, in cell-based assays to assess activity against intact S.
  • IS-63 (NSK4-60) showed less than 10% inhibition of growth at concentrations in the 100 ⁇ range where concomitant toxicity for human cells was observed.
  • IS-130 (NSK4-65) was found to have an IC 50 of 0.10 ⁇ compared with 0.91 ⁇ for parent compound NSK4-60.
  • NSK4-65 showed complete selectivity for bacterial PK ( Figure 7, top panel).
  • IS-63 demonstrated less antibacterial activity, possibly due to lack of cellular penetration. No further inhibition of growth was achieved beyond 25 ⁇ , which was possibly due to stability of the compound over the time course of the experiment (e.g., 24 hr) or limited membrane pemieability. Subsequently, the toxic effects of IS-130 against human HeLa229 cells were evaluated. Results indicated that IS-130 had no significant growth inhibitory effects on HeLa cells (TABLE 1) up to 400 ⁇ . Despite the fact that IS-130 exhibited only modest antibacterial potency, it was a small and efficient ligand with selective on-target activity with apparently limited ability to penetrate the bacterial cell. Accordingly, IS- 130 was a good starting point for optimization through structure activity relationships (SAR) programs. Furthermore, novel compounds as shown in TABLE 2A were synthesized for testing as described herein.
  • SAR structure activity relationships
  • the preference for a compound or compounds as set out in TABLES 2B, 2C, and 2D is based on the particular circumstances under which the compounds were tested (for example, the bacteria or strain of bacteria being tested, clinical testing etc.). Accordingly, as further testing is performed the preference for a compound or compounds as set out in TABLES 2A, 2B, 2C, and 2D may change as a result of the further testing, whereby a compounds preference may be increased or be diminished or stay essentially the same depending on the particular circumstances under which the compounds are tested. Furthermore, the compound or compounds may be preferred in one circumstance and less preferred in another circumstance (for example, depending on the bacterial target).
  • EXAMPLE 3 Analog Preparation. Preparation and evaluation of IS- 130 analogs, revealed the structure dependencies of inhibition and structural modifications were found that increase both PK inhibitory activity and antibacterial potency of the inhibitors. The potency and selectivity of each derivative toward MRSA PK (IC 50 ) as well as antibacterial activities (MIC) were measured to direct the iterative rounds of synthetic chemistry. Parameters derived from these experiments are summarized in TABLE 3. The impact of different substitution patterns on the indole and benzoyl hydrazone moieties on compound function fell into three categories: (1) those important for binding affinity, (2) those crucial for cell penetration and (3) those having complex effects on binding affinity and cell penetration.
  • halogen for example, CI or F
  • MIC Minimum inhibitory concentration
  • EXAMPLE 4 PK Lead Compounds Selectively Inhibit MRSA PK.
  • PK compounds with antibacterial activity were tested against human Ml , M2, R and L PK isoforms in single-enzyme catalytic assays (TABLE 4).
  • PK compounds with potent antibacterial activity displayed submicromolar (0.16- 0.38 ⁇ ) IC 50 s toward bacterial PK with a marked 180 to 940-fold selectivity over the human isoenzymes (TABLE 4). Therefore, PK lead compounds appeared to be suitable starting point to develop highly specific antimicrobial agents.
  • EXAMPLE 5 PK Lead Compounds are Non-competitive Inhibitors.
  • PK lead compounds are non-competitive inhibitors with respect to PEP, with inhibition constant (Ki) values of 269 ⁇ 63 nM and 276 ⁇ 82 nM respectively for NSK4-77 and NSK5-15.
  • EXAMPLE 6 PK Lead Compounds Exhibit Gram-Positive Specific Antibacterial Activity.
  • PK lead compounds showed potent in vitro antibacterial activity against all strains and species of staphylococci that were tested (MIC 1.4-19.2 ⁇ g/ml) including methicillin-susceptible (e.g., RN4220, ATCC25923), methicillin-resistant S. aureus (e.g., MRSA252, COL and MW2) and a multidrug-resistant S. aureus (MDRSA) isolate that was resistant to many of the major classes of antibiotics.
  • methicillin-susceptible e.g., RN4220, ATCC25923
  • methicillin-resistant S. aureus e.g., MRSA252, COL and MW2
  • MDRSA multidrug-resistant S. aureus
  • PK compounds were inactive in vitro (MIC range, >73 to >93 pg/ml) against a range of Gram-negative human pathogens tested such as acinetobacter and pseudomonas (TABLE 5). However, as shown in TABLE 5, the PK compounds were found to also be active against a range of Gram-positive bacterial species, including several antibiotic-resistant strains. These compounds demonstrated superior antibacterial activities against Enterococcus.
  • PK compounds demonstrated potent antibacterial activities towards staphylococci, enterococci and streptococci.
  • Staphylococcus aureus CA-MRSA (USA400) 9.6 2.9 5.0 a 0.75, b l
  • Staphylococcus aureus HA-MRSA252 9.6 2.9 5.0 a >10, b 0.5
  • MIC were determined in BHI for all bacterial strains as described in Material and Methods, unless for Enterococcal species ( v ) and Streptococcal species ( A ), which MIC were determined in CAMHB (cation-adjusted Mueller Hinton broth, containing 20 mg of Ca 2+ /L, and 10 mg of Mg 2+ /L) and CAMHB containing 2 - 5 % laked horse blood, respectively.
  • CAMHB cation-adjusted Mueller Hinton broth, containing 20 mg of Ca 2+ /L, and 10 mg of Mg 2+ /L
  • CAMHB containing 2 - 5 % laked horse blood
  • the cytotoxicity of NSK4-77, NS 5-15 and AM- 165 compounds was determined using HeLa cells in a 1-day incubation assay as described herein.
  • the results shown in FIGURE 2A show that PK lead compounds exhibited little cytotoxicity toward mammalian cells (i.e., less than 30% of cell death at 500 ⁇ equals to 193, 232 and 202 ⁇ of NSK4- 77, NSK5-15 and AM- 165, respectively) with CC 50 values of > 200 ⁇ for all three compounds tested.
  • EXAMPLE 8 Bactericidal Activity of PK Compounds.
  • FIGURE 3 shows representative time-kill curves for compound NSK5-15 against a methicillin-sensitive S. aureus (MSSA) ATCC25923. Vancomycin was also used as a comparator drug. As shown in FIGURE 3, with NSK5-15 maximum killing was observed at concentrations of 4 x MIC, with a 3-log drop in the numbers of cfu/ml occurring by 24 h after compound addition, consistent with a bactericidal mode of action.
  • MSSA methicillin-sensitive S. aureus
  • mutants able to grow in concentrations up to 32 to 128 x appeared within 5 to 10 passages, indicating the emergence of resistant mutants.
  • 10 generation of the bacteria that had developed resistance to fusidic acid was tested against NSK5-15, it was found to be susceptible, with MIC similar to initial exposure (e.g., 1.4 ⁇ g/ml).
  • MIC similar to initial exposure e.g., 1.4 ⁇ g/ml
  • EXAMPLE 10 PK Compounds Reduced Pyruvate Production.
  • the target selectivity of AM- 165 was determined by measuring pyruvate concentrations in S. aureus RN4220 strains incubated either with vehicle (e.g, DMSO) as control or sub-lethal concentration of compound AM- 165. As shown in FIGURE 5, pyruvate concentration was significantly reduced (by 4.3-fold) in AM- 165 treated cells (0.86 ⁇ 0.31 per 5 x 10 7 cell) compared to the control cells (0.19 ⁇ 0.12 per 5 x 10 7 cell) indicating that essential PK () is the target of lead compounds for the inhibition of bacterial growth. Together with the demonstration that PK compounds inhibited PK enzymatic activity directly (FIGURE 1 and TABLE 4) provide direct validation that the compounds are targeting PK. This mechanism of action was further confirmed with recent availability of X-ray structure of the MRSA PK in complex with PK lead inhibitors (Axerio-Cilies, in preparation).
  • EXAMPLE 11 Crystallization and Binding Site Analysis for MRS A252 PK and NSK-465.
  • NSK-465 is shown herein to be a potent inhibitor of the MRSA252 PK.
  • the newly identified MRSA pyruvate kinase small molecule binding surface shown in Figure 3 was further studied by superimposing the MRSA and human PK structures to examine whether the site constituted a suitable compound-binding pocket that is unique to the MRSA.
  • 10 amino acid residues were identified highlighted in Figure 8A (Thr348, Thr353, Ser354, Ala358, Ile361, Ser362, His365, Thr366, Asn369, and Leu370) in each subunit of the MRSA PK which contribute to the formation of the binding pocket.
  • Analysis of the crystal structure showed two His365 residues one from each subunit, which are positioned just above and below the interface cavity and, which are turned inwards into the cavity.

Abstract

Compounds having a structure of Formulas A-C are provided. Uses of such compounds as an antibiotic, including both gram-negative and gram-positive micro-organisms, as well as methods of treatment and uses involving such compounds are provided.

Description

W
ANTI-BACTERIAL PYRUVATE KINASE MODULATOR COMPOUNDS,
COMPOSITIONS, USES, AND METHODS
TECHNICAL FIELD
This invention relates to therapeutics, their uses and methods for the treatment of various indications, including infections. In particular, to therapeutic compositions and methods of treatment of bacteria that have developed resistance to other antibiotics.
BACKGROUND
Infectious diseases caused by bacterial and eukaryotic pathogens continue to be a threat to human health. In particular, many bacteria are developing antibiotic resistance. It is not surprising that the effectiveness of the available antimicrobial drugs against bacteria such as methicillin-resistant Staphylococcus aureus (MRSA) is diminishing at a rapid pace.
Staphylococcus aureus is a facultatively anaerobic, gram-positive coccus and is the most common cause of staph infections.
Escherichia coli are gram-negative rod-shaped bacterium found in the digestive systems of warm blooded organisms. Many E. coli strains are harmless. However, other strains are capable of causing serious food poisoning in humans. Resistance to beta-lactam antibiotics is problematic as resistant bacteria are able to produce extended-spectrum beta- lactamases, which result in resistance to most of the penicillins and cephalosporins.
Bacillus anthracis is also a gram-positive bacterium, but is also spore-forming and is capable of surviving in both aerobic and anaerobic conditions. Bacillus anthracis causes anthrax, which is an acute disease of humans and other animals. Anthrax is often lethal, but sometimes responds well to antibiotic treatment. Vaccines against anthrax are also available.
MRSA (also known as oxacillin-resistant Staphylococcus aureus (ORSA)), is defined as any strain of Staphylococcus aureus bacteria with resistance to beta-lactam antibiotics, such as the penicillins: methicillin; dicloxacillin; nafcillin; oxacillin; etc. and the cephalosporins.
A determination of MRSA infection is made when a biological sample (for example, a skin sample, pus from a wound, blood, urine, or other biopsy material) is cultured for S. aureus. If a positive culture is found, the bacteria are then tested for resistance to antibiotics, such as beta-lactam antibiotics including methicillin. S. aureus that grows in the presence of methicillin is identified as MRSA.
Mild infections of MRSA may be initially treated by draining the infected area and with topical disinfection.
If antibiotic treatment is clinically indicated, it should be guided by the susceptibility profile of the cultured S. aureus. The testing for antibiotic resistance when a bacteria is cultured often defines a susceptibility profile for a cultured bacteria and guides the subsequent treatments. Many MRSAs are treatable with glycopeptide antibiotics (for example, vancomycin and teicoplanin). However, strains of MRSA have also shown antibiotic resistance even to glycopeptide antibiotics and have been called vancomycin intermediate- resistant Staphylococcus aureus (VISA) or vancomycin-resistant Staphylococcus aureus (VRSA) or multi-drug resistant Staphylococcus aureus (MDRSA). Alternative, treatments for MRSA are or oxazolidinone class compounds (for example, linezolid), quinupristin/dalfopristin, daptomycin, tigecycline, trimethoprim-sulfamethoxazole, doxycycline, and clindamycin. Furthermore, a number of alternative antibiotics against MRSA are in clinical trials (for example, ceftobiprole, ceftaroline, dalbavancin, telavancin, aurograb, torezolid, iclaprim, and nemonoxacin).
SUMMARY
This invention is based, in part, on the discovery that compounds described herein selectively inhibit microbial pyruvate kinase (PK) and do not inhibit mammalian pyruvate kinases. Furthermore, as disclosed herein, the selective inhibition of microbial pyruvate kinase is based on selective binding to the monomer interface of microbial PK to interfere with dimer/tertramer formation.
The compounds described herein may be used for in vivo or in vitro research uses (i.e. non-clinical) to investigate alternative treatments for microbial infection. Furthermore, these compounds may be used individually or as part of a kit for in vivo or in vitro research to investigate mechanisms of microbial resistance or microbial infection using recombinant proteins, cells maintained in culture, and/or animal models.
In accordance with one embodiment, there is provided a compound of the Formula (A) or Formula (B):
Figure imgf000004_0001
or a salt thereof, wherein:
R1 may be N-R5, S, or O;
R2 may be C-H, or N;
R3 may be C-H, C-OR5, N, C-OCH2C≡CH, C-OCH2OCH3, C-OCH2OCH2CH3, C- OC(0)CH3, or C-OCH2(OCH2CH2)nOR5;
R4 may be C;
R5 may be H, or a C1-C6 branched or unbranched, saturated or unsaturated, alkyl or acyl group;
n may be 1-5;
A1 may be R5, OR5, F, CI, Br, I, C≡CH, S02R5, NHCOR5, NHS02R5, N02, CON(R5)2, CF3, CBr3, CC13, CI3, phenyl or a C6-C10 aryl group;
A2 may be H, F, CI, Br, I, C≡CH, OR5, CF3, CBr3, CC13, CI3, phenyl or a C6-C10 aryl group;
A3 may be F, CI, Br, I, C≡CH, OR5, R5, S02R5, NHCOR5, NHS02R5, N02, CON(R5)2, CF3, CBr3, CCI3, CI3, phenyl or a C6-C10 aryl group;
A4 may be F, CI, Br, I, C≡CH, OR5, R5, S02R5, NHCOR5, NHS02R5, N02, CON(R5)2,
CF3, CBr3, CCI3, CI3, phenyl or a C6-C10 aryl group;
Q1 may be R5, OR5, F, CI, Br, I, N3, C≡CH, phenyl or a C6-C10 aryl group;
Q2 may be R5, OR5, F, CI, Br, I, N3, C≡CH, phenyl or a C6-C10 aryl group;
Q3 may be R5, OR5, F, CI, Br, I, N3, C≡CH, phenyl or a C6-C10 aryl group;
Q4 may be H or OH;
or Q1 and Q2 may optionally form a benzene ring optionally substituted with R5, OR5, F, CI, Br, I, N3, C≡CH, phenyl or a C6-C10 aryl group; or Q2 and Q3 may optionally form a benzene ring optionally substituted with R5, OR5, F, CI, Br, I, N3, C≡CH, phenyl or a C6-C10 aryl group;
or Q3 and Q4 may optionally form a benzene ring, optionally substituted with R5, OR5, F, CI, Br, I, N3, C≡CH, or phenyl;
L may be
Figure imgf000005_0001
wherein,
D1 may be R5;
E1 may be R5, phenyl or a C6-C10 aryl group;
E may be R , phenyl or a C6-C10 aryl group;
E may be R , phenyl or a C6-C10 aryl group;
provided that the compound is not
Figure imgf000005_0002
Alternatively, R3 may be C-H, C-OR5, N, C-OCH2C≡CH, C-OCH2OCH3, C- OC¾OCH2CH3, or C-OC(0)CH3. R3 may be C-H, C-OH, C-OMe, C-OEt, N, C- OCH2C≡CH, C-OCH2OCH3, C-OCH2OCH2CH3, or C-OC(0)CH3. R3 may be C-H, C-OR5, or N. R3 may be C-H, C-OH, C-OMe, C-OEt, or N. R1 may be N-H, N-Me, N-Et, S, or O. R1 may be N-R5. R1 may be N-H, N-Me, or N-Et. R1 may be S, or O. R2 may be N. R2 may be C-H. R5 may be a CI -C6 branched or unbranched, saturated or unsaturated, alkyl. A1 may be R5, OR5, F, CI, Br, I, C≡CH, or phenyl. A1 may be H, O-Me, F, CI, Br, I, C≡CH, or phenyl. A1 may be H, O-Me, F, CI, Br, or I. A2 may be H, F, CI, Br, I, C≡CH, or phenyl. A2 may be H, O-Me, F, CI, Br, I, C≡CH, or phenyl. A2 may be H, O-Me, F, CI, Br, or I. A3 may be F, CI, Br, I, C≡CH, OR5, R5, or phenyl. A3 may be F, CI, Br, I, C≡CH, H, or O-Me. A3 may be H, O-Me, F, CI, Br, or I. A3 may be F, CI, Br, I, OR5, R5, or phenyl. A4 may be F, CI, Br, I, C≡CH, OR5, R5, or phenyl. A4 may be F, CI, Br, I, C≡CH, OR5, R5, N02, CON(R5)2 or phenyl. A4 may be F, CI, Br, I, OR5, R5, or N02. A4 may be H, O-Me, F, CI, Br, or I. Q1"3 may be independently selected from R5, OR5, F, CI, Br, I, and N3. Q1"3 may be independently
Figure imgf000006_0001
selected from H, OMe, or OH. Q4 may be H. L may be . D1 may be H or
Me. E1"3 may be independently selected from H, Me, Et, phenyl, and tert-butyl. E1"3 may be independently selected from H, Me, and Et. E1 may be selected from H, Me, Et, phenyl, and tert-butyl. E1 may be selected from phenyl, Me, and Et. E1"3 may be independently selected from Me and Et. E1"3 may be Me. E1 may be Et, Me, or phenyl. E1 may be Me.
In accordance with a further embodiment, there is provided a compound or a salt thereof, the compound havin the Formula (A) :
Figure imgf000006_0002
wherein: R1 is N-R5; R2 may be C-H; R3 is C-OH; R5 may be H or Me or Et; A1 may be R5, F, CI, Br, or I; A2 may be H, F, CI, Br, or I; A3 may be OMe, F, CI, Br, I, or R5; A4 may be F, CI, Br, I, or R5; Q1 may be R5, F, CI, Br, or I; Q2 may be R5, F, CI, Br, or I; Q3 may be R5,
F, CI, Br, or I; Q4 is H or OH; L may be
Figure imgf000006_0003
; wherein, D1 may be R5; and E1 may be R5 or phenyl.
Alternatively, R1 may be N-H or N-Me; R3 may be C-OH; A1 may be H; A2 may be H, or F; A3 may be OMe, F, CI, Br, or H; A4 may be H, or F; Q1 may be H; Q2 may be H; Q3 may be Br, or I; Q4 may be H; D1 may be H; and E1 may be Me, Et, or phenyl.
In accordance with a further embodiment, there is provided a compound or a salt thereof, selected from TABLE 2A. In accordance with a further embodiment, there is provided a compound or a salt thereof, selected from TABLE 2B.
In accordance with a further embodiment, there is provided a compound or a salt thereof, selected from TABLE 2C.
In accordance with a further embodiment, there is provided a compound or a salt thereof, selected from TABLE 2D.
In accordance with a further embodiment, there is provided a compound or a salt thereof, having the Formula (C):
Figure imgf000007_0001
or a salt thereof, wherein:
G1 may be G5, OG5, F, CI, Br, I, C≡CH, S02G5, NHCOG5, NHS02G5, N02, CON(G5)2 or phenyl;
G2 may be G5, OG5, F, CI, Br, I, C≡CH, S02G5, NHCOG5, NHS02G5, N0 , CON(G5)2 or phenyl;
G3 may be G5, OG5, F, CI, Br, I, C≡CH, S02G5, NHCOG5, NHS02G5, N02, CON(G5)2 or phenyl;
G4 may be G5, OG5, F, CI, Br, I, C≡CH, S02G5, NHCOG5, NHS02G5, N02, CON(G5)2 or phenyl;
G5 may be H, or a C1-C6 branched or unbranched, saturated or unsaturated, alkyl; J1 maybe N-G5, S, or O;
M1 may be G5, OG5, F, CI, Br, I, N3, C≡CH, or phenyl;
M2 may be G5, OG5, F, CI, Br, I, N3, C≡CH, or phenyl;
M3 may be G5, OG5, F, CI, Br, I, N3, C≡CH, or phenyl;
M4 may be G5, OG5, F, CI, Br, I, N3, C≡CH, or phenyl;
M5 may be G5, OG5, F, CI, Br, I, N3, C≡CH, or phenyl;
M6 may be G5, OG5, F, CI, Br, I, N3, C≡CH, or phenyl; M7 may be H or OH;
provided that the compound is
Figure imgf000008_0001
In accordance with a further embodiment, there is provided a method of treating a microbial infection including administering a compound of Formula (A) or Formula (B):
Figure imgf000008_0002
or a salt thereof, wherein:
R1 may be -R5, S, or O;
R2 may be C-H, or N;
R3 may be C-H, C-OR5, N, C-OCH2C≡CH, C-OCH2OCH3, C-OCH2OCH2CH3, C- OC(0)CH3, or C-OCH2(OCH2CH2)nOR5;
R4 may be C;
R5 may be H, or a C1-C6 branched or unbranched, saturated or unsaturated, alkyl or acyl group;
n may be 1-5;
A1 may be R5, OR5, F, CI, Br, I, C≡CH, S02R5, NHCOR5, NHS02R5, N02, CON(R5)2, CF3, CBr3, CC13, CI3, or phenyl;
A2 may be H, F, CI, Br, I, C≡CH, OR5, CF3, CBr3, CC13, CI3, or phenyl;
A3 may be F, CI, Br, I, C≡CH, OR5, R5, S02R5, NHCOR5, NHS02R5, N02, CON(R5)2,
CF3, CBr3, CC13, CI3, or phenyl; A4 may be F, CI, Br, I, C≡CH, OR5, R5, S02R5, NHCOR5, NHS02R5, N02, CF3, CBr3,
CC13, CI3, CON(R5)2, or phenyl;
Q1 may be R5, OR5, F, CI, Br, I, N3, C≡CH, or phenyl;
Q2 may be R5, OR5, F, CI, Br, I, N3, C≡CH, or phenyl;
Q3 may be R5, OR5, F, CI, Br, I, N3, C≡CH, or phenyl;
Q4 may be H or OH;
1 2 5 5 or Q and Q may optionally form a benzene ring optionally substituted with R , OR , F, CI, Br, I, N3, C≡CH, phenyl or a C6-C10 aryl group;
2 5 5 or Q and Q may optionally form a benzene ring optionally substituted with R , OR , F, CI, Br, I, N3, C≡CH, or phenyl;
or Q3 and Q4 may optionally form a benzene ring optionally substituted with R5, OR5, F, CI, Br, I, N3, C≡CH, phenyl or a C6-C10 aryl group;
L may be
Figure imgf000009_0001
1 5 1 5 2 5 3 5 wherein, D may be R ; E may be R or phenyl; E may be R or phenyl; and E may be R or phenyl.
Alternatively, R3 may be C-H, C-OR5, N, C-OCH2C≡CH, C-OCH2OCH3, C- OCH2OCH2CH3, or C-OC(0)CH3. R3 may be C-H, C-OH, C-OMe, C-OEt, N, C- OCH2C≡CH, C-OCH2OCH3, C-OCH2OCH2CH3, or C-OC(0)CH3. R3 may be C-H, C-OR5, or N. R3 may be C-H, C-OH, C-OMe, C-OEt, or N. R1 may be N-H, N-Me, N-Et, S, or O. R' may be N-R5. R1 may be N-H, N-Me, or N-Et. R1 may be S, or O. R2 may be N. R2 may be C-H. R5 may be a C1-C6 branched or unbranched, saturated or unsaturated, alkyl. A1 may be R5, OR5, F, CI, Br, I, C≡CH, or phenyl. A1 may be H, O-Me, F, CI, Br, I, C≡CH, or phenyl. A1 may be H, O-Me, F, CI, Br, or I. A2 may be H, F, CI, Br, I, C≡CH, or phenyl. A2 may be H, O-Me, F, CI, Br, I, C≡CH, or phenyl. A2 may be H, O-Me, F, CI, Br, or I. A3 may be F, CI, Br, I, C≡CH, OR5, R5, or phenyl. A3 may be F, CI, Br, I, C≡CH, H, or O-Me. A3 may be H, O-Me, F, CI, Br, or I. A3 may be F, CI, Br, I, OR5, R5, or phenyl. A4 may be F, CI, Br, I, C≡CH, OR5, R5, or phenyl. A4 may be F, CI, Br, I, C≡CH, OR5, R5, N02, CON(R5)2 or phenyl. A4 may be F, CI, Br, I, OR5, R5, or N02. A4 may be H, O-Me, F, CI, Br, or I. Q1"3 may be independently selected from R5, OR5, F, CI, Br, I, and N3. 1"3 may be independently
Figure imgf000010_0001
selected from H, OMe, or OH. Q4 may be H. L may be . D1 may be H or
Me. E1"3 may be independently selected from H, Me, Et, phenyl, and tert-butyl. E1"3 may be independently selected from H, Me, and Et. E1 may be selected from H, Me, Et, phenyl, and tert-butyl. E may be selected from phenyl, Me, and Et. E " may be independently selected from Me and Et. E " may be Me. E may be Et, Me, or phenyl. E may be Me.
In accordance with a further embodiment, there is provided a method of treating a microbial infection includin administering a compound of Formula (A):
Figure imgf000010_0002
or a salt thereof, wherein: R may be N-RJ; R may be C-H; R may be C-OH; R may be H or Me or Et; A1 may be R5, F, CI, Br, or I; A2 may be H, F, CI, Br, or I; A3 may be OMe, F, CI, Br, I, or R5; A4 may be F, CI, Br, I, or R5; Q1 may be R5, F, CI, Br, or I; Q2 may be R5, F, CI, Br, or I; Q3 may be R5, F, CI, Br, or I; Q4 may be H or OH;
Figure imgf000010_0003
L may be ; wherein, D1 may be R5; and E1 may be R5 or phenyl.
Alternatively, R1 may be N-H or N-Me; R3 may be C-OH; A1 may be H; A2 may be H, or F; A3 may be OMe, F, CI, Br, or H; A4 may be H, or F; Q1 may be H; Q2 may be H; Q3 may be Br, or I; Q4 may be H; D1 may be H; and E1 may be Me, Et, or phenyl. In accordance with a further embodiment, there is provided a method of treating microbial infection including administering a compound selected from TABLE 2A r
Figure imgf000011_0001
In accordance with a further embodiment, there is provided a method treating microbial infection comprising administering a compound selected from TABLE 2B
Figure imgf000011_0002
In accordance with a further embodiment, there is provided a method treating microbial infection comprising administering a compound selected from TABLE 2C
Figure imgf000011_0003
In accordance with a further embodiment, there is provided a method treating a microbial infection comprising administering a compound selected from TABLE 2D or
Figure imgf000011_0004
In accordance with a further embodiment, there is provided a method of treating a microbial infection including administering a compound of the Formula (C):
Figure imgf000012_0001
or a salt thereof, wherein:
G1 may be G5, OG5, F, CI, Br, I, C≡CH, S02G5, NHCOG5, NHS02G5, N02, CON(G5)2 or phenyl;
G2 may be G5, OG5, F, CI, Br, I, C≡CH, S02G5, NHCOG5, NHS02G5, N02, CON(G5)2 or phenyl;
G3 may be G5, OG5, F, CI, Br, I, C≡CH, S02G5, NHCOG5, NHS02G5, N02, CON(G5)2 or phenyl;
G4 may be G5, OG5, F, CI, Br, I, C≡CH, S02G5, NHCOG5, NHS02G5, N02, CON(G5)2 or phenyl;
G5 may be H, or a C1-C6 branched or unbranched, saturated or unsaturated, alkyl; J1 may be N-G5, S, or O;
M1 may be G5, OG5, F, CI, Br, I, N3, C≡CH, or phenyl;
M2 may be G5, OG5, F, CI, Br, I, N3, C≡CH, or phenyl;
M3 may be G5, OG5, F, CI, Br, I, N3, C≡CH, or phenyl;
M4 may be G5, OG5, F, CI, Br, I, N3, C≡CH, or phenyl;
M5 may be G5, OG5, F, CI, Br, I, N3, C≡CH, or phenyl;
M6 may be G5, OG5, F, CI, Br, I, N3, C≡CH, or phenyl; and
M7 may be H or OH.
In accordance with a further embodiment, there is provided a method of treating a microbial infection including administering a compound having the
structure
Figure imgf000013_0001
In accordance with a further embodiment, there is provided a compound of Formula r Formula (B):
Figure imgf000013_0002
or a salt thereof, wherein:
R1 may be N-R5, S, or O;
R2 may be C-H, or N;
R3 may be C-H, C-OR5, N, C-OCH2C≡CH, C-OCH2OCH3, C-OCH2OCH2CH3, C- OC(0)CH3, or C-OCH2(OCH2CH2)nOR5;
R4 may be C;
R5 may be H, or a C1-C6 branched or unbranched, saturated or unsaturated, alkyl or acyl group;
n may be 1-5;
A1 may be R5, OR5, F, CI, Br, I, C≡CH, S02R5, NHCOR5, NHS02R5, N02, CF3, CBr3, CC13, CI3, CON(R5)2, or phenyl;
A2 may be H, F, CI, Br, I, C≡CH, OR5, CF3, CBr3, CC13, CI3, or phenyl;
A3 may be F, CI, Br, I, C≡CH, OR5, R5, S02R5, NHCOR5, NHS02R5, N02, CF3, CBr3,
CCI3, CI3, CON(R5)2, or phenyl; A4 may be F, CI, Br, I, C≡CH, OR5, R5, S02R5, NHCOR5, NHS02R5, N02, CF3, CBr3,
CC13, CI3, CON(R5)2, or phenyl;
Q1 may be R5, OR5, F, CI, Br, I, N3, C≡CH, or phenyl;
Q2 may be R5, OR5, F, CI, Br, I, N3, C≡CH, or phenyl;
Q3 may be R5, OR5, F, CI, Br, I, N3, C≡CH, or phenyl;
Q4 may be H or OH;
5 5 or Q and Q" may optionally form a benzene ring optionally substituted with R , OR , F, CI, Br, I, N3, C≡CH, phenyl or a C6-C10 aryl group;
2 5 5 or Q and Q may optionally form a benzene ring optionally substituted with R , OR , F, CI, Br, I, N3, C≡CH, or phenyl;
or Q3 and Q4 may optionally form a benzene ring optionally substituted with R5, OR5, F, CI, Br, I, N3, C≡CH, phenyl or a C6-C10 aryl group;
L may be
Figure imgf000014_0001
wherein,
D1 may be R5;
E1 may be R5 or phenyl;
2 5
E may be R or phenyl; and
E may be R or phenyl;
for the treatment of a microbial infection.
In accordance with a further embodiment, there is provided the use of a compound of Formula (A) or Formula (B):
Figure imgf000015_0001
t thereof, wherein:
R1 may be N-R5, S, or O;
R2 may be C-H, or N;
R3 may be C-H, C-OR5, N, C-OCH2C≡CH, C-OCH2OCH3, C-OCH2OCH2CH3, C-
OC(0)CH3, or C-OCH2(OCH2CH2)nOR5;
R4may be C;
R5 may be H, or a C1-C6 branched or unbranched, saturated or unsaturated, alkyl or acyl group;
n may be 1-5;
A1 may be R5, OR5, F, CI, Br, I, C≡CH, S02R5, NHCOR5, NHS02R5, N02, CF3, CBr3, CC13, CI3, CON(R5)2, or phenyl;
A2 may be H, F, CI, Br, I, C≡CH, OR5, CF3, CBr3, CC13, CI3, or phenyl;
A3 may be F, CI, Br, I, C≡CH, OR5, R5, S02R5, NHCOR5, NHS02R5, N02, CF3, CBr3,
CC13, CI3, CON(R5)2 or phenyl;
A4 may be F, CI, Br, I, C≡CH, OR5, R5, S02R5, NHCOR5, NHS02R5, N02, CF3, CBr3,
CCI3, CI3, CON(R5)2, or phenyl;
Q1 may be R5, OR5, F, CI, Br, I, N3, C≡CH, or phenyl;
Q2 may be R5, OR5, F, CI, Br, I, N3, C≡CH, or phenyl;
Q3 may be R5, OR5, F, CI, Br, I, N3, C≡CH, or phenyl;
Q4 may be H or OH;
or Q1 and Q2 may optionally form a benzene ring optionally substituted with R5, OR5, F, CI, Br, I, N3, C≡CH, phenyl or a C6-C10 aryl group;
or Q2 and Q3 may optionally form a benzene ring optionally substituted with R5, OR5, F, CI, Br, I, N3, C≡CH, or phenyl; or Q3 and Q4 may optionally form a benzene ring optionally substituted with R , OR , F, CI, Br, I, N3, C≡CH, phenyl or a C6-C10 aryl group;
L ma be
Figure imgf000016_0001
wherein,
D1 may be R5;
E1 may be R5 or phenyl;
2 5
E may be R or phenyl; and
E may be R or phenyl;
for the treatment of a microbial infection.
Alternatively the compound may be for the manufacture of a medicament for treating a microbial infection.
Alternatively, R3 may be C-H, C-OR5, N, C-OCH2C≡CH, C-OCH2OCH3, C- OCH2OCH2CH3, or C-OC(0)CH3. R3 may be C-H, C-OH, C-OMe, C-OEt, N, C- OCH2C≡CH, C-OCH2OCH3, C-OCH2OCH2CH3, or C-OC(0)CH3. R3 may be C-H, C-OR5, or N. R3 may be C-H, C-OH, C-OMe, C-OEt, or N. R1 may be N-H, N-Me, N-Et, S, or O. R' may be N-R5. R1 may be N-H, N-Me, or N-Et. R1 may be S, or O. R2 may be N. R2 may be C-H. R5 may be a C1-C6 branched or unbranched, saturated or unsaturated, alkyl. A1 may be Rs, OR5, F, CI, Br, I, C≡CH, or phenyl. A1 may be H, O-Me, F, CI, Br, I, C≡CH, or phenyl. A1 may be H, O-Me, F, CI, Br, or I. A2 may be H, F, CI, Br, I, C≡CH, or phenyl. A2 may be H, O-Me, F, CI, Br, I, C≡CH, or phenyl. A2 may be H, O-Me, F, CI, Br, or I. A3 may be F, CI, Br, I, C≡CH, OR5, R5, or phenyl. A3 may be F, CI, Br, I, C≡CH, H, or O-Me. A3 may be H, O-Me, F, CI, Br, or I. A3 may be F, CI, Br, I, OR5, R5, or phenyl. A4 may be F, CI, Br, I, C≡CH, OR5, R5, or phenyl. A4 may be F, CI, Br, I, C≡CH, OR5, R5, N02, CON(R5)2 or phenyl. A4 may be F, CI, Br, I, OR5, R5, or N02. A4 may be H, O-Me, F, CI, Br, or I. Q1"3 may be independently selected from R5, OR5, F, CI, Br, I, and N3. Q1 3 may be independently selected from H, OMe, or OH. Q4 may be H. L may be
Figure imgf000017_0001
. D1 may be H or
Me. E1 "3 may be independently selected from H, Me, Et, phenyl, and tert-butyl. E1"3 may be independently selected from H, Me, and Et. E1 may be selected from H, Me, Et, phenyl, and tert-butyl. E may be selected from phenyl, Me, and Et. E " may be independently selected from Me and Et. E " may be Me. E may be Et, Me, or phenyl. E may be Me.
In accordance with a further embodiment, there is provided the use of a compound of Formula (A) :
Figure imgf000017_0002
or a salt thereof, wherein: R may be N-R5; R2 may be C-H; R may be C-OH; R may be H or Me or Et; A1 may be R5, F, CI, Br, or I; A2 may be H, F, CI, Br, or I; A3 may be OMe, F, CI, Br, I, or R5; A4 may be F, CI, Br, I, or R5; Q1 may be R5, F, CI, Br, or I; Q2 may be R5, F, CI, Br, or I; Q3 may be R5, F, CI, Br, or I; Q4may be H or OH;
Figure imgf000017_0003
Alternatively, R1 may be N-H or N-Me; R3 may be C-OH; A1 may be H; A2 may be H, or F; A3 may be OMe, F, CI, Br, or H; A4 may be H, or F; Q1 may be H; Q2 may be H; Q3 may be Br, or I; Q4may be H; D1 may be H; and E1 may be Me, Et, or phenyl.
In accordance with a further embodiment, there is provided a compound selected from
r
Figure imgf000017_0004
TABLE 2A or for the treatment of a microbial infection. In accordance with a further embodiment, there is provided a compound selected from r
TABLE 2B or
Figure imgf000018_0001
for the treatment of a microbial infection.
In accordance with a further embodiment, there is provided a compound selected from
TABLE 2C or
Figure imgf000018_0002
for the treatment of a microbial infection.
In accordance with a further embodiment, there is provided a compound selected from r
TABLE 2D or
Figure imgf000018_0003
for the treatment of a microbial infection.
In accordance with a further embodiment, there is provided a compound of Formula
(C):
Figure imgf000018_0004
or a salt thereof, wherein:
G1 may be G5, OG5, F, CI, Br, I, C≡CH, S02G5, NHCOG5, NHS02G5, N02, CON(G5)2 or phenyl;
G2 may be G5, OG5, F, CI, Br, I, C≡CH, S02G5, NHCOG5, NHS02G5, N02, CON(G5)2 or phenyl;
G3 may be G5, OG5, F, CI, Br, I, C≡CH, S02G5, NHCOG5, NHS02G5, N02, CON(G5)2 or phenyl; G4 may be G5, OG5, F, CI, Br, I, C≡CH, S02G5, NHCOG5, NHS02G5, N02, CON(G5)2 or phenyl;
G5 may be H, or a C1-C6 branched or unbranched, saturated or unsaturated, alkyl; J1 may be N-G5, S, or O;
M1 maybe G5, OG5, F, CI, Br, I, N3, C≡CH, or phenyl;
M2 may be G5, OG5, F, CI, Br, I, N3, C≡CH, or phenyl;
M3 may be G5, OG5, F, CI, Br, I, N3, C≡CH, or phenyl;
M4 may be G5, OG5, F, CI, Br, I, N3, C≡CH, or phenyl;
M5 may be G5, OG5, F, CI, Br, I, N3, C≡CH, or phenyl;
M6 maybe G5, OG5, F, CI, Br, I, N3, C≡CH, or phenyl; and
M7 may be H or OH;
for the treatment of a microbial infection.
The compound may be
Figure imgf000019_0001
In accordance with a further embodiment, there is provided the use of a compound a compound of Formula (C):
Figure imgf000019_0002
or a salt thereof, wherein:
G1 may be G5, OG5, F, CI, Br, I, C≡CH, S02G5, NHCOG5, NHS02G5, N02, CON(G5)2 or phenyl;
G2 may be G5, OG5, F, CI, Br, I, C≡CH, S02G5, NHCOG5, NHS02G5, N02, CON(Gs)2 or phenyl; G3 may be G5, OG5, F, CI, Br, I, C≡CH, S02G5, NHCOG5, NHS02G5, N02, CON(G5)2 or phenyl;
G4 may be G5, OG5, F, CI, Br, I, C≡CH, S02G5, NHCOG5, NHS02G5, N02, CON(G5)2 or phenyl;
G5 may be H, or a C1-C6 branched or unbranched, saturated or unsaturated, alkyl; J1 may be N-G5, S, or O;
M1 may be G5, OG5, F, CI, Br, I, N3, C≡CH, or phenyl;
M2 may be G5, OG5, F, CI, Br, I, N3, C≡CH, or phenyl;
M3 may be G5, OG5, F, CI, Br, I, N3, C≡CH, or phenyl;
M4 may be G5, OG5, F, CI, Br, I, N3, C≡CH, or phenyl;
M5 may be G5, OG5, F, CI, Br, I, N3, C≡CH, or phenyl;
M6 may be G5, OG5, F, CI, Br, I, N3, C≡CH, or phenyl; and
M7 may be H or OH;
for the treatment of a microbial infection.
Alternatively the compound may be for the manufacture of a medicament for treating a microbial infection. The compound may be
Figure imgf000020_0001
In accordance with a further embodiment, there is provided a pharmaceutical composition for treating a microbial infection, may include a compound described herein and a pharmaceutically acceptable carrier.
In accordance with a further embodiment, there is provided a commercial package comprising (a) a compound described herein; and (b) instructions for the use thereof for treating a microbial infection. In accordance with a further embodiment, there is provided commercial package comprising (a) a pharmaceutical composition comprising a compound described herein and a pharmaceutically acceptable carrier; and (b) instructions for the use thereof for treating a microbial infection.
In accordance with a further embodiment, there is provided method for testing a candidate compound for selective binding to a pathogen pyruvate kinase, the method including: (a) combining the candidate compound with a pathogen pyruvate kinase monomelic subunits; (b) combining the candidate compound with human pyruvate kinase monomelic subunits; and (c) assaying for pyruvate kinase tetramer/dimer formation in both (a) and (b).
The pathogen may be MRSA.
BRIEF DESCRIPTION OF THE DRAWINGS
FIGURE 1. shows a inhibition studies with NSK4-77 and NSK5-15. (A) NSK4-77 was used at 0 nM (O), 100 nM (□), 200 nM (V) and 400 nM (O). (B) NSK5-15 was used at 0 nM (O), 50 nM (□), 100 nM (V), and 200 nM (O). PK enzymatic activity was assayed as described in Materials and Methods. The data shown are derived from one of two experiments performed in triplicate Error bars indicate range of values within the single experiment shown.
FIGURE 2. shows a cytotoxicity assessment of PK lead compounds against HeLa cells (A) compared to that of the MDRSA S. aureus (B). Compounds NSK4-77 (O), NSK5- 15 (□) and AM-165(V) were used at 0-500 μΜ to determine CC5o and MIC as described in Material and Methods. The data presented are representative of three experiments performed in triplicate.
FIGURE 3. shows a time-kill curve of S. aureus incubated with AM- 165. S. aureus ATCC 25923 was incubated with vehicle alone (e.g., DMSO) (O) as growth control, or AM- 165 at 1 x MIC (A), 2 x MIC (V), 4 x MIC (O) and 8 x MIC (Δ) or vancomycin at 2 x MIC (□) and 4 x MIC (·) and sampled at the indicated time points. The logio values of cfu/ml were plotted versus time as described in Material and Methods. The data shown are from one of three independent experiments with similar results.
FIGURE 4. shows a serial passage of S. aureus RN4220 in either NSK5-15 (·) or fusidic acid (□). The highest sublethal concentration of compound (denoted as fold-MIC) is plotted versus the number of days of serial passage for each compound as describes in Material and Methods.
FIGURE 5. shows a pyruvate concentrations in S. aureus cells challenged with PK lead compound AM- 165. Pyruvate concentrations were measured in S. aureus RN4220 cells incubated in the absence (Control) or presence of the highest sublethal concentration of AM- 165 (e.g, 1.25
Figure imgf000022_0001
as described in Material and Methods. The data presented are mean + SD of three independent experiments each performed in triplicates. FIGURE 6. shows a series of plots (Top) NSK-460 and 465 (10 μΜ) selectively inhibit MRSA PK enzymatic activity; (Middle) Effects of NSK-460 and 465 on S. aureus growth; (Bottom) Toxicity evaluation of NSK-460 and 465 for human HeLa cells.
FIGURE 7. shows a resolved structure of MRSA252 pyruvate kinase tetramer showing the domain boundaries and tetramer architecture, wherein each monomer primarily fills one quadrant of the image to facilitate the identification of the small and large interface shown as lines, and the binding site are shown located at the small interface sits in between two alpha helices formed two PK monomers shown as circles. This Figure was generated by MOE version 2007.09 [Larkin MA et al. Clustal W and Clustal X version 2.0. Bioinformatics
2007, 23(21):2947-2948].
FIGURE 8A. shows a sequence alignment with the interface region (highlighted residues) for pyruvate kinase (PK) from Staphylococcus aureus and Homo sapiens. The poorly conserved residues between MRSA and human PK are highlighted dark. The small interface also encompasses an insertion region in human PK (residues SD) and the corresponding deletion area in MRSA PK. This Figure was generated by ClustalX version 2.0 [Tulloch BL et al. Sulphate removal induces a major conformational change in Leishmania mexicana Pyruvate kinase in the crystalline state. Journal of Molecular Biology
2008, 383(3):615-626].
FIGURES 8B and C. show a structural modelS of the interface-binding site for MRSA and human PK, respectfully. The spheres show the interface cavity in MRSA and human PK, which demonstrates an accessible binding pocket located at the interface of two PK monomers in MRSA FIGURE 8B, as compared to the pocket in human PK which is partially obstructed by five amino acid residues (Glu418-B, Arg399-A, B and Arg400-A, FIGURE 8C. This figure was generated using the MOE molecular package [Larkin MA et al. Clustal W and Clustal X version 2.0. Bioinformatics 2007, 23(21):2947-2948].
FIGURES 9A and B. show a binding model of NSK-465 at the interface binding site. FIGURE 9A shows a two-dimensional map of the binding interactions between NSK-465 and the interface site based on its co-crystallization with MRSA PK, wherein the arrows depict hydrogen-accepting interactions between NSK-465 and MRSA PK residues from the interface. FIGURE 9B shows the binding orientation of NSK-465 within the interface- binding pocket based on the protein-ligand crystal structure. These figures were generated using the MOE molecular package [Larkin MA et al. Clustal W and Clustal X version 2.0. Bioinformatics 2007, 23(21 ):2947-2948].
DETAILED DESCRIPTION
As used herein, the phrase "C]-6 alkyl" or "Q-Cg alkyl" is used as it is normally understood to a person of skill in the art and often refers to a chemical entity that has a carbon skeleton or main carbon chain comprising a number from 1 to 6 (with all individual integers within the range included, including integers 1 and 6) of carbon atoms. For example a "Ci-6 alkyl" is a chemical entity that has 1, 2, 3, 4, 5, or 6 carbon atom(s) in its carbon skeleton or main chain.
As used herein, the term "branched" is used as it is normally understood to a person of skill in the art and often refers to a chemical entity that comprises a skeleton or main chain that splits off into more than one contiguous chain. The portions of the skeleton or main chain that split off in more than one direction may be linear, cyclic or any combination thereof. Non-limiting examples of a branched alkyl are tert-butyl and isopropyl.
As used herein, the term "unbranched" is used as it is normally understood to a person of skill in the art and often refers to a chemical entity that comprises a skeleton or main chain that does not split off into more that one contiguous chain. Non-limiting examples of unbranched alkyls are methyl, ethyl, n-propyl, and n-butyl.
As used herein, the term "substituted" is used as it is normally understood to a person of skill in the art and often refers to a chemical entity that has one chemical group replaced with a different chemical group that contains one or more heteroatoms. Unless otherwise specified, a substituted alkyl is an alkyl in which one or more hydrogen atom(s) is/are replaced with one or more atom(s) that is/are not hydrogen(s). For example, chloromethyl is a non-limiting example of a substituted alkyl, more particularly an example of a substituted methyl. Aminoethyl is another non-limiting example of a substituted alkyl, more particularly it is a substituted ethyl. The functional groups described herein may be substituted with, for example, and without limitation, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 substituents.
As used herein, the term "unsubstituted" is used as it is normally understood to a person of skill in the art and often refers to a chemical entity that is a hydrocarbon and/or does not contain a heteroatom. Non-limiting examples of unsubstituted alkyls include methyl, ethyl, tert-butyl, and pentyl.
As used herein, the term "saturated" when referring to a chemical entity is used as it is normally understood to a person of skill in the art and often refers to a chemical entity that comprises only single bonds. Non-limiting examples of saturated chemical entities include ethane, tert-butyl, and N+¾.
As used herein the term "halogenated" is used as it would normally be understood to a person of skill in the art and refers to a moiety or chemical entity in which a hydrogen atom is replaced with a halogen atom such as chlorine, fluorine, iodine or bromine. For example, a chlorinated side chain of a naturally occurring amino acid refers to a side chain of a naturally occurring amino acid wherein one or more hydrogen atoms occurring in the side chain of the naturally occurring amino acid is replaced with one or more chlorine atoms.
Non-limiting examples of saturated Ci-C6 alkyl may include methyl, ethyl, n-propyl, i- propyl, sec-propyl, n-butyl, i-butyl, sec-butyl, t-butyl, n-pentyl, i-pentyl, sec-pentyl, t-pentyl, n-hexyl, i-hexyl, 1 ,2-dimethylpropyl, 2-ethylpropyl, l-methyl-2-ethylpropyl, l-ethyl-2- methylpropyl, 1,1 ,2-trimethylpropyl, 1 , 1 ,2-triethylpropyl, 1,1-dimethylbutyl, 2,2- dimethylbutyl, 2-ethylbutyl, 1 ,3-dimethylbutyl, 2-methylpentyl, 3-methylpentyl, sec-hexyl, and t-hexyl. Non-limiting examples of C2-C6 alkenyl may include vinyl, allyl, isopropenyl, 1- propene-2-yl, 1-butene-l -yl, 1 -butene-2-yl, l -butene-3-yl, 2-butene-l-yl, and 2-butene-2-yl. Non-limiting examples of C2-C6 alkynyl may include ethynyl, propynyl, butynyl, pentynyl, and hexynyl. Saturated Ci-C6 alkyl, C2-C6 alkenyl or C2-C6 alkynyl may be, for example, and without limitation, interrupted by one or more heteroatoms which are independently nitrogen, sulfur or oxygen.
Non-limiting examples of Ci-C6 substituted or unsubstituted acyl include acetyl, propionyl, butanoyl and pentanoyl. Non-limiting examples of alkoxy include methoxy, ethoxy, propoxy and butoxy. Non-limiting examples of the C6-Cio aryl group may include phenyl (Ph), benzyl, tolyl, o-xylyl, pentalenyl, indenyl, naphthyl, and azulenyl.
Figure imgf000025_0001
As used herein, the symbol denotes the bond at a point of attachment between two chemical entities, one of which is depicted and the other of which is typically not
Figure imgf000026_0001
depicted. For example, indicates that the chemical entity "XY" is bonded to another chemical entity via the point of attachment bond. Furthermore, the specific point of attachment to the non-depicted chemical entity may be specified by inference. For example,
Figure imgf000026_0002
the compound CH3-R3, wherein R3 is H or " " infers that when R3 is "XY", the point of attachment bond is the same bond as the bond by which R3 is depicted as being bonded to CH3.
As used herein, "MRSA" is methicillin-resistant Staphylococcus aureus (also known as oxacillin-resistant Staphylococcus aureus (ORSA)), is defined as any strain of Staphylococcus aureus bacteria with resistance to beta-lactam antibiotics, such as the penicillins: methicillin; dicloxacillin; nafcillin; oxacillin; etc. and the cephalosporins and not just methicillin. Some strains of MRSA have also shown antibiotic resistance to glycopeptide antibiotics and have been called vancomycin intermediate-resistant Staphylococcus aureus (VISA) or vancomycin-resistant Staphylococcus aureus (VRSA) or multi-drug resistant Staphylococcus aureus (MDRSA). MRSA is often responsible for difficult-to-treat infections in humans. MRSA can be especially troublesome in hospital-associated (nosocomial) infections, where patients often have open wounds, invasive devices, and weakened immune systems making them at greater risk for infection than the general public.
The compounds described herein may be in isolation, or may be linked to or in combination with liposomes, carbohydrate carriers, polymeric carriers or other agents or excipients as will be apparent to one of skill in the art. In an alternate embodiment, such compounds may comprise a medicament, including other antibiotics, wherein such compounds may be present in a pharmacologically effective amount.
Compositions or compounds according to some embodiments may be administered in any of a variety of known routes. Examples of methods that may be suitable for the administration of a compound include orally, intravenous, inhalation, intramuscular, subcutaneous, topical, intraperitoneal, intra-rectal or intra-vaginal suppository, sublingual, and the like. The compounds of the present invention may be administered as a sterile aqueous solution, or may be administered in a fat-soluble excipient, or in another solution, suspension, patch, tablet or paste format as is appropriate. A composition comprising the compounds of the invention may be formulated for administration by inhalation. For instance, a compound may be combined with an excipient to allow dispersion in an aerosol. Examples of inhalation formulations will be known to those skilled in the art. Other agents may be included in combination with the compounds of the present invention to aid uptake or metabolism, or delay dispersion within the host, such as in a controlled-release formulation. Examples of controlled release formulations will be known to those of skill in the art, and may include microencapsulation, embolism within a carbohydrate or polymer matrix, and the like. Other methods known in the art for making formulations are found in, for example, "Remington's Pharmaceutical Sciences", (19th edition), ed. A. Gennaro, 1995, Mack Publishing Company, Easton, Pa.
The dosage of the compositions or compounds may vary depending on the route of administration (for example, oral, intravenous, inhalation, or the like) and the form in which the composition or compound is administered (for example, solution, controlled release or the like). Determination of appropriate dosages is within the ability of one of skill in the art. As used herein, an 'effective amount', a 'therapeutically effective amount', or a 'pharmacologically effective amount' of a medicament refers to an amount of a medicament present in such a concentration to result in a therapeutic level of drug delivered over the term that the drug is used. This may be dependent on mode of delivery, time period of the dosage, age, weight, general health, sex and diet of the subject receiving the medicament. Methods of determining effective amounts are known in the art.
Pyruvate kinase (PK) is known to be a significant protein and is responsible for catalyzing the final step of glycolysis, which involves the transfer of the phosphoryl group of phosphoenolpyruvate (PEP) to ADP to produce pyruvate and ATP (Suzuki K, et al. J Biochem (2008) 144(3):305-312). PK is a tetrameric molecule that forms from subunits. For example, there are two major forms of PKM2 in cells, a highly active tetramer and a nearly inactive dimer. The ratio between the highly active tetramer and dimer determines whether glucose carbons are channeled to biosynthetic processes or used for glycolytic ATP production. The transition between the 2 forms contributes to the control of glycolysis.
There are 4 isozymes of pyruvate kinase in mammals: L, R, Ml and M2. The PK L isozyme is major isozyme in the liver, the R isozyme is found in red blood cells, the Ml isozyme is the main form in muscle, heart and brain, and M2 is found in early fetal tissues. Pyruvate kinase isozymes M1/M2 are encoded by the PKM2 gene (alternative references include M23725 mRNA (AAA36449.1); M26252 mRNA (AAA36672.1); X56494 Genomic DNA (CAA39849.1); AK092369 mRNA (BAG52542.1); AK222927 mRNA (BAD96647.1); AK294315 mRNA (BAG57589.1 note different initiation); AK312253 mRNA (BAG35185.1); AY352517 Genomic DNA (AAQ 15274.1); AC020779 Genomic DNA; CH471082 Genomic DNA (EAW77884.1); CH471082 Genomic DNA (EAW77888.1); BC000481 mRNA (AAH00481.3); BC007640 mRNA (AAH07640.1); BC007952 mRNA (AAH07952.3); BC012811 mRNA (AAH12811.3); BC035198 mRNA (AAH35198.1); AF025439 mRNA (AAC39559.1); and reference sequences NP_002645.3.; NP_872270.1.; NP_872271.1), and are alternative splicing variants. The primary difference in the Ml and M2 isozymes is at the c-terminus. The pyruvate kinase isozymes R/L are encoded by the PKLR gene (alternative references include AB015983 mRNA (BAA31706.1); M15465 mRNA (AAA60104.1.); AY316591 Genomic DNA (AAP69527.1); BC025737 mRNA (AAH25737.1); S60712 mRNA (AAB26262.1); and reference sequences NP 000289.1.; NP_870986.1). The pyruvate kinase isozymes R, L, Ml, and M2 form a homotetramer. Pyruvate kinases from pathogenic species are also known in the art (for example, Leishmania mexicana (X74944 Genomic DNA (CAA52898.2)); Chlamydia pneumoniae (AE001363 Genomic DNA (AAD18250.1) and ref seq. NP_224305.1); Mycoplasma genitalium (L43967 Genomic DNA (AAC71435.1) U01798 Genomic DNA (AAD12324.1) and ref seq. NP_072881.1); Mycobacterium tuberculosis (BX842577 Genomic DNA (CAB08894.1) ref seq. NP_216133.1); Candida albicans (S65775 mRNA); Escherichia coli 0157:H7 (AE005174 Genomic DNA (AAG56663.1) and ref seq. NP_288110.1.); Salmonella typhi (AL627271 Genomic DNA (CADOl 987.1) and ref seq. NP_456147.1); Trypanosoma brucei brucei (X57950 Genomic DNA (CAA41018.1)); Staphylococcus aureus (strain MRSA252) BX571856 Genomic DNA (CAG40767.1) and ref seq. YP_041163.1) etc.).
Furthermore, assay methods are described herein for the identification of additional compounds which bind to the PK monomer interface and inhibit dimmer and tetramer formation. The method may include a test to determine whether a candidate compound selectively binds to a pathogen pyruvate kinase (for example, MRSA), wherein a candidate compound is combined with pathogen pyruvate kinase monomelic subunits and the candidate compound is also combined with one or more of the human pyruvate kinase monomelic subunits (i.e. the human isoenzymes monomers for Ml, M2, L and R), followed by an assay to determine pyruvate kinase tetramer and/or dimer formation by pathogen and human pyruvate kinases in the presence of the candidate compound. For example, assaying for pyruvate kinase tetramer and/or dimer formation may be accomplished using monomer- specific monoclonal antibodies may be used to quantify monomer by immunocytochemistry (see for example, Ashizawa et al. JBC (1991) 266 16842-1 846). Alternatively, dimmer and tetramer formation may be assayed via pyruvate kinase activity assays (for example, abeam™ Pyruvate-Kinase-PK-Assay-Kit (catalog # ab83432); Sigma Aldrich™ Enzymatic Assay of pyruvate kinase substrate Phospho(enol)pyruvic acid tri(cyclohexylammonium) salt Fluka (catalog # 79430); Bio Vision™ Pyruvate Kinase Assay Kit (catalog# K709-100), Gel filtration and immunodetection (see for example, Adachi et al. (1977) Proc Natl Acad Sci U S A., 74: 501-504; Zwerschke et al. (1999) Proc Natl Acad Sci U S A., 96(4): 1291-1296; and Gupta et al. (2010) J Biol Chem., 285(22): 16864-73), MALDI-TOF (for example, Farmer and Caprioli (1991) Biological Mass Spectrometry 20: 796-800; an Moniatte et al. (1997) International Journal of Mass Spectrometry and Ion Processes, 169-170: 179-199), Mass spectrometry (MS) coupled with the soft ionization processes of either matrix-assisted laser desorption (MALDI) or electrospray (ES) ionization (for example, Hernandez and Robinson (2007) Nature Protocols 2: 715 - 726), and assays for tetramer formation (for example, Ashizawa et al (1991) Biochemistry 30:7105-7111 ; and Desmaret et al. (2005) Biological Chemistry. 386:1137-1147).
GENERAL METHODOLOGIES
Bacterial strains and compounds. Epidemic methicillin resistant S. aureus (MRSA) strain sequenced at the Sanger Centre (MRSA252, NRS71), S. aureus RN4220 (NCTC8325 NRS144), Hyper- virulent community-acquired MSSA sequenced at the Sanger Centre (MSSA476, NRS72), MRSA strain sequenced at TIGR, (COL, NRS100) and community- acquired MRSA strain sequenced at the National Institute of Technology and Evaluation, Tokyo (USA400, MW2, NRS123) were obtained from NARSA (Network on Antimicrobial Resistance in S. aureus). Methicilin sensitive & aureus (ATCC 25923), Streptococcus Pneumoniae (ATCC 49619), Streptococcus Pyogenes (ATCC 700294), Listeria monocytogenes (ATCC19115), Enterococcus faecium (ATCC35667), Enterococcus faecalis (ATCC29212) and Enterococcus faecium (ATCC700221) (VRE) were from ATCC, The Global Bioresourse Center. Multi drug resistant (MDR) MRSA, Staphylococcus saprophyticus, Staphylococcus haemolyticus, Staphylococcus epidermidis, Acinetobacter baumannii and ESBL-producing Klebsiella pneumoniae were clinical isolates obtained from the Vancouver General Hospital (Vancouver, Canada). Escherichia coli DYE330, Pseudomonas aeruginosa PAO-1 were obtained from the laboratory of Dr B.B. Finlay at the University of British Columbia (Vancouver, Canada). IS-63 is commercially available from Enamine™ (Product Code: T5435622). IS-130 is commercially available from Vitas-M™ (Product Code: STKO 15507). Analogs of IS-63 and IS-130 were synthesized as described herein. 10 mM compound stocks were prepared in DMSO and stored at -20 °C.
Generation of PK constructs. Genomic DNA of MRSA strain Sanger 252 extracted using Dneasy Tissue Kit™ (Qiagen™) was used as a template to generate the His-tagged MRSA PK. Human cDNA from MCF-7 breast cancer cell line (courtesy of Dr. J Wong, BC Cancer Research Center (Vancouver, Canada) was used as a template to generate the full- length human M2 PK enzyme. The following primer sets were used creating appropriate restriction sites (Ndel and Xhol sites underlined): For cloning of MRSA PK: M27F 5'- CTACATATGAGAAAAACTAAAATTGTATG-3 ' and M27R 5'-
GTTCTCGAGTTATAGTACGTTTGCATATCCTTC-3', for cloning of human M2 PK isoform: hM2F 5 '-GATC AT ATGATGTCG AAGCCCC ATAGTGAAGCC-3 ' and hM2R 5'- GTTCTCGAGTCACGGCACAGGAACAACACGCATG-3 '. The resulting PCR fragments for each construct were cloned into the Ndel and Xhol unique sites of the bacterial expression vector pET-28a (+) (Novagen™). This step resulted in plasmids pET-28a-MRSA and pET- 28M2, which generated N-terminally His-tagged recombinant MRSA and human M2 PKs. The sequence and the correct reading frame of all constructs were verified by sequencing. Human Ml, R and L PK constructs in pET-28-a(+) vectors (courtesy of Dr. L. Cantley, Harvard Medical, School (Boston, USA.) were used to generate relevant recombinant His- tagged human PK isoforms ( and ).
Expression and purification of recombinant His-tagged MRSA and human PKs. MRSA and human constructs in pET-28a(+) were used to express relevant recombinant PK proteins in E. coli BL-21 (DE3). The proteins were expressed and purified using Ni-NTA agarose (Qiagen™) according to the manufacturer's protocol. Briefly, cells were grown to an absorbance of 0.4-0.5 at 600 nm in 2xYT medium, then induced with 0.1 mM IPTG for 3 h at 20°C. Cells were lysed by sonication on ice (3 x 10-s bursts with a 30-s recovery between bursts) in lysis buffer (0.2 mg/ml lysozyme, 50 mM Tris pH 7.5, 10 mM MgCl2, 200 mM NaCl, 100 mM KC1, 10% glycerol, 10 mM imidazole, 0.5% NP-40 and 1 mM DTT containing Complete™ protease inhibitor). Cell lysates were cleared by centrifugation (18,000 x g in a Beckman™ JA-20 rotor) for 20 min at 4 °C and PK isoforms were purified by batch binding to Ni-NTA resin. The resins were then packed in columns (1 x 2 cm) and washed with 400 column volumes lysis buffer containing 30 mM imidazole. His-tagged PK isoforms were eluted with the same buffer containing 300 mM imidazole. The proteins were dialyzed overnight at 4 °C against 2000 volumes of ice-cold 30 mM Tris pH 7.5, 25 mM KC1, 5 mM MgCl2, 10% glycerol and 1 mM DTT to remove imidazole. All purification steps were done at 4°C; enzymes were flash-frozen and stored at -70 °C. Enzymatic activity of frozen protein preparations was stable for at least 10 months and up to 5 freeze/thaw cycles. Purity and physical integrity of proteins were assessed using SDS- polyacrylamide gel electrophoresis (SDS-PAGE) followed by coomassie blue staining. Protein concentration was estimated by Bradford assay (Bio-Rad Protein Assay™) using bovine serum albumin as a standard.
Measurement of pyruvate kinase activity. Candidate MRSA PK inhibitors were assayed for their ability to inhibit enzymatic activities of MRSA and human PKs. PK activity was determined using a continuous assay coupled to lactate dehydrogenase (LDH) in which the change in absorbance at 340 nm owing to oxidation of NADH was measured using a Benchmark Plus™ microplate spectrophotometer (Bio-Rad™) (). The reaction contained 60 mM Na+-HEPES, pH 7.5, 5% glycerol, 67 mM KC1, 6.7 mM MgCl2, 0.24 mM NADH, 5.5 units L-LDH from rabbit muscle (Sigma- Aldrich™), 2 mM ADP and 10 mM PEP (i.e. close to the Km of MRSA PK, so that the IC50 values should approximate the Ki) in a total volume of 200 μΐ. Reactions were initiated by the addition of 15 nM of one of the PK enzymes. PK activity proportional to the rate of change at 340 nm was expressed as specific activity (μιηοΐ/min/mg), which is defined as the amount of PK that catalyzes the formation of one micromole of either product per minute. Inhibitors were dissolved in DMSO with the final concentration of the solvent never exceeding 1% of the assay volume. IC50 values were calculated by curve fitting on a four-parameter dose-response model with variable slope using Graphpad Prism 5.0™ (GraphPad™ Software Inc., La Jolla, CA). In all studies, less than 10% of total PEP was exhausted during the reaction. Reactions were performed at 30 °C for 5 min. All values determined represent three measurements, each in triplicate unless mentioned otherwise. Mode-of-inhibition and Kt values were determined by simultaneously changing the inhibitor concentration (0-400 nM) and substrate PEP concentration (2-20 mM) while keeping the level of the ADP substrate fixed at 2 mM. The resulting curve at each inhibitor concentration was fitted by nonlinear regression to the allosteric sigmoidal kinetic model using Graphpad Prism™. ¾ values were obtained by nonlinear regression curve-fitting using the following equation ():
Apparent Vmax = Vmax / (l + [I] / Kt) (1 )
In vitro susceptibility testing. The antimicrobial activities of PK inhibitor candidates were determined using the 96-well microtiter standard 2-fold serial broth microdilution method as described by CLSI (formerly NCCLS) () with the various gram-positive and gram- negative bacteria species mentioned above. Bacteria from a single colony were grown, overnight in BHI Broth (Difco™), harvested by centrifugation, and then washed twice with sterile distilled water. Each stock solution of compounds in DMSO was diluted with BHI to prepare serial two-fold dilutions in the range of 500 to 0.06 μΜ. One hundred microliters of the broth containing about 5x l05 colony- forming units (cfu)/ml of bacteria was used to inoculate each well of a 96-well plate containing 100 μΐ of the same medium with the indicated concentrations of candidate inhibitors. Culture plates were incubated for 24 h at 37 °C, and optical density at 600 nm (OD60o) was measured using a Benchmark Plus™ microplate spectrophotometer (Bio-Rad™). To control for intrinsic absorbance by the inhibitors, control series containing inhibitor dilutions but no cells were run for every experiment, and the resulting absorbance values were subtracted as background from the experimental readings. Minimal inhibitory concentration (MIC) was defined as the lowest concentration of test compound leading to complete inhibition of cell growth in relation to compound-free control wells as determined by optical density. Erythromycin, vancomycin and ciprofloxicin were used as reference compounds. All assays were run in triplicate. Experiments were replicated at least three times to verify reproducibility using the above conditions. Determination of mammalian cytotoxicity. The cytotoxic activities of compounds were determined for HeLa cells 229 (ATCC:CCL-2-.l) in microtiter cultures by measuring dehydrogenase activity using CellTiter 96® AQue0us One Solution Cell Proliferation Assay™ (Promega™, Madison, WI, USA), according to the manufacturer's protocol. Freshly split cells were seeded into microtiter wells (2 x 104 /well) and grown for 24 hours. The original media was then removed and replaced with media containing the desired concentration of compound or solvent control {i.e., DMSO). Plates were incubated for 24 h at 37 °C in a humidified incubator with a 5% C02 atmosphere. At the end of the growth period, cells were lysed by the addition of 20 μΐ of Cell Titer 96 Aqueous One™ solution, and the incubation was continued for another 3 h at 37°C. Production of formazan was determined at 490 nm on Benchmark Plus™ microplate spectrophotometer (Bio-Rad™). To control for intrinsic absorbance, control series containing inhibitor dilutions but no cells were run for every experiment and the resulting absorbance values were subtracted as background from the experimental readings. Growth in compound-free control wells was considered as 100% and percentage of growth inhibition was calculated for each compound concentration. Cytotoxicity was quantified as the CC50, the concentration of compound that inhibited 50% of conversion of MTS to formazan (). The "selectivity index" is defined as the ratio of the mammalian cell cytotoxicity to the MIC against S. aureus (i.e., CC50/MIC). Positive control measurements were performed with xanthohumol (HeLa cells: CC50 ~ 9 μg/ml). All assays were performed three times in triplicate.
Determination of Pyruvate levels. Cultures of S. aureus RN4220 with initial suspensions of approximately lx lO6 cfu/ml in BHI Broth™ supplemented with either 1% DMSO (control) or the highest sub-lethal concentration (i.e., 1.25 μg/ml) of compound AM- 165 (test) were incubated at 37 °C for 3 h. Before cell harvesting, turbidity of cultures was measured at OD 0o and samples were taken to determine the number of cells (cfu/ml). Cell pellets were washed twice with PBS and lysed for 30 min at 37 °C in lysis buffer (25 mM Tris-HCl pH7.5, 120 μ^ιηΐ lysozyme, 120 μg /ml lysostaphin, 0.05% Triton X-100™, 20 μg/ml DNasel, 2 mM MgCl), followed by vortexing using a FastPrep FP120 Cell Disrupte™r (Qbiogene™, Inc., Carlsbad, CA). Insoluble cell debris was removed by centrifugation at 25,000 x g for 10 min. Soluble cell extracts were used to determine pyruvate levels by colorimetric measurement of pyruvate oxidation using Pyruvate Determination Assay™ kit (Bio Vision™, Mountain View, CA, USA) with reference to a pyruvate standard curve, prepared as per manufacturer's instructions.
Time-kill studies. The time-kill analyses were performed by the method of the CLSI M26-A.20 (). S. aureus ATCC 25923 was cultured overnight at 37 °C in BHI. Cells were diluted in medium to an initial OD60o of 0.1 (equal to concentration of approximately 10 cfu/ml) and incubated with shaking for 2 h at 37 °C to achieve logarithmic growth. The culture was then diluted in medium to adjust the cell density to approximately 10 cfu/ml. Either compound NS 5-15 to final concentrations of 1, 2, 4 and 8 times the MIC or vancomycin to final concentrations of 2 and 4 times the MIC were then added. Aliquots (0.1 ml) of the cultures were removed at 0, 2 h, 4 h, 6 h and 24 h of incubation and serial 10-fold dilutions were prepared in saline as needed. The number of viable cells was determined on drug-free BHI plates after 24 h of incubation. Rates of killing were determined by measuring the reduction in viable bacteria (logio cfu/ml) at 0, 1 h, 2 h, 4 h, 6 h, and 24 hr with fixed concentrations of compound. Experiments were performed in duplicate. If plates contained less than 10 cfu/ml, the number of colonies was considered to be below the limit of quantitation. Samples of cultures containing compound were diluted at least 10-fold to minimize drug carryover to the BHI plates. Bactericidal activity is defined as a >3 log reduction in initial cfu count within 24 h. The compound was considered to be bacteriostatic at the concentration that reduced the original inoculum by 0-3 logio cfu/ml within 24 h.
Selection for resistant mutants. Changes in the susceptibilities of bacteria to NS 5- 15 were monitored during serial passages of S. aureus RN4220 in BHI broth containing the highest sub-lethal compound and results are displayed as the highest sublethal compound concentration for each culture for each day. Two independent cultures of S. aureus RN4220 were grown in 96-well assay plates in the presence of several concentrations of compound (0.125x to 32xMIC). Cultures were recovered from the well with highest compound concentration that exhibited growth (e.g., >15% of untreated control). For the subsequent passages, the cells were grown in the liquid culture with the highest sub-lethal inhibitor concentration of the last passage. This process was repeated for 25 passages and results are displayed as the highest sublethal compound concentration for each culture for each passage. After passage 5, 10, 15, 20 and 25, colonies were isolated from cultures before determination of the MIC. Changes in the susceptibilities of RN4220 to fusidic acid were monitored for 10 passages as a positive control.
Binding site identification. The crystal structure of PK from Bacillus Stearothermophilis (PDB code: 2E28) [Edelsbrunner H et al. Measuring proteins and voids in proteins. In: Proceedings of the 28th Hawaii International Conference on Systems Science: 1995; Washington, DC: IEEE Computer Society; 1995: 256-264] with 62% sequence identity to the MRSA homologue was identified as a suitable template for the homology modelling [Rizzo RC and Jorgensen WL: OPLS All-atom model for amines: resolution of the amine hydration problem. J Am Chem 1999, 121 :4827-4836]. The homology-based model of the MRSA252 PK was accepted with a backbone RMSD of 0.46 A compared to its template. The sequence alignment was refined using a global sequence alignment program, GGSEARCH (version 35.03 [Dombrauckas JD et al. Structural basis for tumor pyruvate kinase M2 allosteric regulation and catalysis. Biochemistry 2005, 44(27):9417-9429.]). A homology model of the MRSA252 PK was built using Modeller9v4 [Friesner RA et al. Glide: a new approach for rapid, accurate docking and scoring. 1. Method and assessment of docking accuracy. J Med Chem 2004, 47(7):1739-1749] and optimized by the MMFF94x force field [Schrodinger: GLIDE™] energy minimization implemented by the Molecular Operating Environment™ (MOE™), version 2007.09 [Cherkasov A. Can 'Bacterial-Metabolite- Likeness' model improve odds of 'in silico' antibiotic discovery? J Chem Inf Model 2006, 46(3):1214-1222]. The crystal structure of human PK M2 (PDB code: 1T5A) was directly obtained from the PDB [Zsoldos Z et al. eHiTS: a new fast, exhaustive flexible ligand docking system. J Mol Graph Model 2007, 26(1): 198-212] and used for the subsequent structural superimposition with the MRSA PK model. Whereas, the crystal structure of pyruvate kinase tetramer from Staphylococcus aureus was later obtained with 3.0 A resolution. The sequence alignment between MRSA and human PK was aligned using a ClustalX with a sequence identity of 44%.
The 'Site Finder' functionality of the MOE package was used to identify suitable binding area around the small interface of the MRSA tetramer. The site_finder is a generalization of the convex hulls method for calculating possible binding sites in receptors from 3D atomic coordinates. Alpha spheres were consequently generated to identify the binding site (Figures 9B and C), which was subsequently used for molecular docking [Edelsbrunner H et al. 1995]. The interface-binding pocket of MRS A PK was optimized by the OPLS-AA force field energy minimization implemented by MOE [Rizzo RC and Jorgensen 1999]. The crystal structure (PDB code: 1T5A) for human PK was obtained from PDB [Dombrauckas JD et al. 2005] and used for a subsequent structural superimposition with the MRSA PK structure.
General synthesis methods
1H and 13C NMR spectra were recorded with either Bruker Avance II™ 600 MHz, Bruker
Avance III™ 500 MHz or Bruker Avance III™ 400 MHz. Processing of the spectra was performed with MestRec™ software. The high-resolution mass spectra were recorded in positive ion-mode with an ESI ion source on an Agilent™ Time-of-Flight LC/MS mass spectrometer. Analytical thin-layer chromatography (TLC) was performed on aluminum plates pre-coated with silica gel 60F-254 as the absorbent. The developed plates were air- dried, exposed to UV light and/or dipped in KMn04 solution and heated. Column chromatography was performed with silica gel 60 (230-400 mesh). All HPLC purifications were carried out using an Agilent™ CI 8 reverse-phase preparatory column (21.2 χ 250 mm).
General procedure for the synthesis of lH-indole-2-carboxylic acid A
A mixture of the appropriate ort/zo-iodoaniline (5 mmol), pyruvic acid (1.04 mL, 15 mmol), DABCO™ (1.683 g, 15 mmol) and Pd(OAc)2 (56 mg, 0.25 mmol) in dry DMF (15 mL) was degassed 3 times and then was stirred at 105°C for 16 h. The mixture was cooled to room temperature, neutralized with 10% aqueous hydrochloric acid and extracted with EtOAc (3x15 mL). The combined organic phases were washed with brine, dried over anhydrous Na2S04, filtered and concentrated in vacuo. The crude product was purified by flash column chromatography using 95/5 EtOAc/MeOH and recrystallized from EtOH to yield the IH- indole-2-carboxylic acid.
5,6-difluoro-l//-indoIe-2-carboxylic acid Al
Figure imgf000036_0001
Yield = 43%, pale grey powder. Mp = 292-293°C. Ή NMR (600 MHz, DMSO-c/6): δ 13.11 (bs, 1H), 11.97 (bs, 1H), 7.66 (dd, J = 10.8 Hz, 8.4 Hz, 1H), 7.33 (dd, J = 10.8 Hz, 6.6 Hz, 1H), 7.09 (d, J= 1.2 Hz, 1H). 13C NMR (150 MHz, CDC13 + DMSO-^): δ 162.5, 148.9 (dd, J = 242 Hz, 16 Hz), 146.2 (dd, J = 237 Hz, 16 Hz), 132.3 (d, J - 11 Hz), 129.5 (d, J = 4 Hz), 121.8 (d, J= 8 Hz), 107.4, 107.3 (d, J= 19 Hz), 99.5 (d, J= 22 Hz).
4,5-difluoro-lH-indole-2-carboxylic acid A2
Figure imgf000037_0001
Yield = 68%, pale brown crystals. 1H NMR (400 MHz, DMSO-J6): δ 13.30 (bs, 1H), 12.19 (bs, 1H), 7.33-7.22 (m, 2H), 7.15 (dd, J= 2.4 Hz, 1.6 Hz, 1H).
4,5,6-trifluoro-lH-indole-2-carboxylic acid A3
Figure imgf000037_0002
Yield = 66%, pale beige powder. Mp = 242-243°C. 1H NMR (400 MHz, DMSO-i¾): δ 13.33 (bs, 1H), 12.30 (s, 1H), 7.23 (dd, J = 10.2 Hz, 5.8 Hz, 1H), 7.17 (dd, J = 2.0 Hz, 0.8 Hz, 1H). 13C NMR (100 MHz, DMSO-i 6): δ 161.8, 148.9 (ddd, J= 241 Hz, 13 Hz, 2 Hz), 143.2 (ddd, J = 249 Hz, 11 Hz, 5 Hz), 134.1 (ddd, J= 237 Hz, 18 Hz, 14 Hz), 132.1 (dd, J= 13 Hz, 12 Hz), 130.6 (d, J= 3 Hz), 112.8 (d, J= 18 Hz), 103.1 (d, J= 6 Hz), 95.8 (dd, J= 22 Hz, 4 Hz).
General procedure for the synthesis of substituted l-(lH-indol-2-yl)ethanone C
To a solution of the appropriate lH-indole-2-carboxylic acid A (5 mmol) in Et20 (20 mL) at 0°C was added MeLi (10.9 mL, 1.6M in Et20, 17.4 mmol) dropwise. The mixture reaction was refluxed for 2 h, cooled to room temperature and neutralized with 10% aqueous hydrochloric acid. The organic layer was separated and the aqueous layer was extracted with EtOAc (3x15 mL). The organic phases were combined, washed with brine, dried over anhydrous Na2S04, filtered and concentrated in vacuo. The crude product was purified by flash column chromatography using 80/20 hexanes/EtOAc to yield the l-(lH-indol-2- yl)ethanone C. l-(5-fluoro-lH-indol-2-yl)ethanone CI
Figure imgf000038_0001
Yield = 73%, white powder. Mp = 183-184°C. 1H NMR (400 MHz, CDC13): δ 9.08 (bs, IH), 7.38-7.32 (m, 2H), 7.16 (dd, J = 2.4 Hz, 0.8 Hz, IH), 7.12 (td, J = 9.0 Hz, 2.4 Hz), 2.60 (s, 3H). l-(7-fluoro-lH-indol-2-yl)ethanone C2
Figure imgf000038_0002
Yield = 45%, yellow crystals. Mp = 93-94°C. Ή NMR (400 MHz, CDC13): δ 9.17 (bs, 7.50-7.45 (m, IH), 7.22 (dd, J= 3.2 Hz, 2.4 Hz, IH), 7.10-7.02 (m, 2H), 2.61 (s, 3H). l-(5,6-difluoro-lH-indol-2-yl)ethanone C3
Figure imgf000038_0003
Yield = 74%, yellow powder. Mp = 195-196°C. Ή NMR (600 MHz, CDC13 + DMSO-<¾: δ 1 1.07 (bs, IH), 7.21 (dd, J = 10.2 Hz, 7.8 Hz, IH), 7.08 (dd, J = 10.5 Hz, 6.9 Hz, IH), 6.93 (d, J= 1.2 Hz, IH), 2.36 (s, 3H). l-(4,5-difluoro-lH-indol-2-yl)ethanone C4
Figure imgf000038_0004
Yield = 72%, pale yellow cotton. Mp = 166-168°C. Ή NMR (400 MHz, CDC13): δ 9.28 (bs, IH), 7.29 (dd, J = 4.0 Hz, 0.8 Hz, IH), 7.23-7.1 1 (m, 2H), 2.62 (s, 3H). l-(4,5,6-trifluoro-l-methyl-lH-indol-2-yl)ethanone C5
Figure imgf000039_0001
Yield = 69%, grey powder. Mp = 225-226°C. 1H NMR (400 MHz, DMSO- 6): δ 12.22 (s, 1H), 7.53 (s, 1H), 7.21 (dd, J = 10.2 Hz, 5.8 Hz, 1H), 2.56 (s, 3H). 13C NMR (100 MHz, DMSO-d6): δ 189.7, 149.4 (ddd, J = 242 Hz, 13 Hz, 2 Hz), 143.6 (ddd, J = 249 Hz, 1 1 Hz, 5 Hz), 137.4 (d, J = 3 Hz), 134.1 (ddd, J= 237 Hz, 18 Hz, 14 Hz), 132.6 (dd, J= 13 Hz, 12 Hz), 112.9 (d, J= 18 Hz), 105.3 (d, J= 6 Hz), 95.9 (dd, J= 22 Hz, 4 Hz), 26.0.
Specific procedures for the synthesis of substituted l-(lH-indol-2-yI)ethanone C
l-(5-phenyl-lH-indol-2-yl)ethano
Figure imgf000039_0002
To a solution of phenylboronic acid (123 mg, 1.01 mmol) in anhydrous and degassed DME (5 mL) were added under nitrogen atmosphere, l-(5-bromo-lH-indol-2-yl)ethanone (200 mg, 0.84 mmol), Na2C03 aqueous solution (2.58 mmol in 1 mL H20) and Pd(Ph3)4 (97 mg, 0.08 mmol). The reaction was refluxed overnight. After cooling to rt, ethyl acetate and water were added to the mixture reaction and the aqueous layer was extracted with ethyl acetate. The combined organic phases were washed with brine, dried over anhydrous Na2S04, filtered and concentrated in vacuo. The crude product was purified by flash column chromatography using
20/80 EtOAc/hexanes to yield a pale yellow solid (128 mg, 65%). Mp = 181-182°C. ]H NMR (400 MHz, CDC13): δ 9.03 (bs, 1H), 7.91 (t, J = 0.6 Hz, 1H), 7.65-7.61 (m, 3H), 7.48 (d, J = 9.0 Hz, 1H), 7.46 (t, J = 7.5 Hz, 1H), 7.34 (tt, J = 7.2 Hz, 1.2 Hz, 1H), 7.25 (dd, J = 1.8 Hz, 0.6 Hz, 1H), 2.62 (s, 3H). l-(5-Azido-lH-indol-2-yl)ethanone C7
Figure imgf000040_0001
A mixture of l-(5-bromo-lH-indol-2-yl)ethanone (50 mg, 0.21 mmol), Cul (40 mg, 0.21 mmol), NaN3 (40 mg, 0.6 mmol), D/L proline (30 mg, 0.26 mmol), NaOH (10 mg, 0.25 mmol) in EtOH/H20 (7/3, 3 mL) in a sealed tube was stirred at 95 °C for 3d. The mixture was partitioned between EtOAc (100 mL) and H20 (50 mL). The organic phase was washed with brine (50 mL), dried over anhydrous Na2S04 and concentrated. The crude product was purified by flash chromatography (hexanes/EtOAc; 5/1) to give white solid (15 mg, 35%). Mp = 162-164°C. Ή NMR (CDC13): δ 9.13 (s, 1H), 7.41 (bd, J = 8.8 Hz, 1H), 7.36 (bd, J = 2.1 Hz, 1H), 7.13 (dd, J= 0.8 Hz, 2.1 Hz, 1H), 7.04 (dd, J = 2.2 Hz, 8.9 Hz, 1H), 2.60 (s, 3H). 13C NMR (CDCI3): δ 190.6, 136.7, 135.1, 133.6, 128.5, 119.1, 113.7, 112.1 , 109.1, 26.1. HRMS calcd for (Ci0H9N4O+H)+ 201.0771, found 201.0771. l-[5-(Trifluoromethyl)-lH-indol-2-yl]ethanone C8
Figure imgf000040_0002
Yield: 10%, brown powder. 1H NMR (500 MHz, DMSO-< 6): δ 12.17 (s, 1H), 8.14 (s, 1H), 7.63 (d, J= 8.8 Hz, 1H), 7.56 (dd, J= 8.8 Hz, J= 1.8 Hz, 1H), 7.51 (d, J= 1.8 Hz, 1H), 2.59 (s, 3H). 13C NMR (100 MHz, DMSO- 6): δ 190.9, 139.4, 137.9, 126.6, 121.8, 121.1, 114.2, 110.6, 26.7. HRMS calcd for (CnH8F3NO-H)- 226.0485, found 226.0482.
Specific procedures for the synthesis of substituted l-(lH-indol-2-yl)ethanone C
l-(5-Iodo-lH-indol-2-yI)ethanone
Figure imgf000040_0003
To a stirred solution of 5-iodo-lH-indole-2-carboxylic acid (1.2 g, 4.29 mmol, 1.0 eq.) and HBTU (1.8 g, 4.72 mmol, 1.1 eq.) in NMP (25 mL) at 0°C was added Ν,Ο- dimethylhydroxylamine hydrochloride (0.4 g, 4.29 mmol, 1.0 eq.) and N-methyl morpholine (1.4 mL, 12.87 mmol, 3.0 eq.). The reaction mixture was stirred at rt for 24 h, and then diluted with methyl tert-butyl ether. The organic layer was washed with H20, saturated aqueous NH4C1 and NaHC03 solutions, dried over a2S04, filtered through a silica gel pad and then concentrated under reduced pressure. The residue was taken up in anhydrous THF (30 mL) and a solution of MeMgBr in Et20 (6.3 mL, 3.0 M, 18.80 mmol, 5.0 eq.) was added under Ar at 0°C. The mixture was stirred at rt for 24 h and then more MeMgBr solution (6.3 mL, 3.0 M, 18.80 mmol, 5.0 eq.) was added. The reaction was further stirred for 48 h, cooled to 0°C, and then quenched with a saturated aqueous NH4C1 solution. The mixture was extracted with EtOAc and the organic layer was dried over Na2S04, filtered over a silica gel pad and concentrated to afford C9 (0.85 g). Yield: 69%, yellow meringue. 1H NMR (400 MHz, DMSO- ): δ 11.90 (s, 1H), 8.09 (d, J= 1.6 Hz, 1H), 7.52 (dd, J= 8.8 Hz, J= 1.6 Hz, 1H), 7.29 (m, 2H), 2.55 (s, 3H). 13C NMR (100 MHz, DMSO-</6): δ 190.7, 137.1, 136.7, 133.7, 131.4, 130.1, 115.6, 108.7, 84.3, 26.7. HRMS calcd for (C10H8INO-Hy 283.9578, found 283.9651. l-(6-Bromo-lH-indol-2-yl)ethanon C10
Figure imgf000041_0001
LDA (1M in THF, 0.9 mL, 0.9 mmol) was added to a stirred solution of 6-bromo-l- (phenylsulfonyl)-lH-indole (200 mg, 0.6 mmol) in THF (5 mL) at -78°C under Ar. The reaction mixture was stirred at 0°C for 40 min and then re-cooled to -78°C followed by the addition of Ac20 (91 uL, 0.96 mmol). The resulting mixture was stirred at -78°C for 20 min, warmed to 0°C for lh and then further stirred at rt for 3h. The mixture was partitioned between EtOAc (100 mL) and H20 (50 mL). The organic phase was washed with brine (50 mL), dried over anhydrous Na2S04 and concentrated. The residue was partially purified by flash chromatography (hexanes/EtOAc; 9/1 -> 7:13) and then subjected to TBAF (1M in THF, 1.8 mL, 1.8 mmol) in THF (5mL). The reaction mixture was stirred at rt for 3 h and then partitioned between EtOAc (100 mL) and H20 (50 mL). The organic phase was washed with brine (50 mL), dried over anhydrous Na2S04 and concentrated. The residue was purified by flash chromatography (hexanes/EtOAc; 1 1/1 - 4:1) to give pale yellow solid (64 mg, 45%). 1H NMR (CDCh): δ 8.95 (bs, 1H), 7.59 - 7.57 (m, 2H), 7.28 - 7.26 (m, 1H), 7.16 (bs, lH), 2.59 (s, 3H). l-(5-Hydroxy-l#-indol-2-yl)ethanone Cll
Figure imgf000042_0001
BBr3 (0.8 mL, 1M in DCM, 0.8 mmol) was added to a stirred solution on l-(5-methoxy-lH- indol-2-yl)ethanone (80 mg, 0.42 mmol) in DCM (4 mL) at -78°C under Ar. The resulting mixture was stirred at 0°C for 3h. The mixture was partitioned between EtOAc (100 mL) and H20 (50 mL). The organic phase was washed with brine (50 mL), dried over anhydrous Na2S04 and concentrated. The crude product was purified by flash chromatography (hexanes/EtOAc; 9/1 -» 7:3) to give pale yellow solid (52 mg, 70%). 1H NMR (CDC13): δ 8.88 (bs, 1H), 7.30 (d, J = 8.9 Hz, 1H), 7.08 (bs, 1H), 6.96 (dd, J = 2.3 Hz, 8.8 Hz, 1H), 2.58 (s, 3H).
1 -(5-bromo- l/7-indol-2-yl)propan-l -one C 12
Figure imgf000042_0002
A mixture of 5-bromo- lH-indole-2-carboxylic acid (2.0 g, 8.3 mmol) and HBTU (3.5g, 9.3 mmol) in DMF (10 mL) was stirred at 0°C for 10 min followed by the addition of Ν,Ο- dimethylhydroxyamine hydrochloride (0.82g, 8.4 mmol) in DMF (6 mL) and N- methylmorpholine (2.8 mL, 25.6 mmol), respectively. The mixture was stirred at rt for 18 h and then was partitioned between EtOAc (250 mL) and H20 (100 mL). The organic phase was washed with brine (100 mL), dried over anhydrous Na2S04 and concentrated. The Weinreb amide was purified by flash chromatography (hexanes/EtOAc; 11/1 - 7:3) to give pale yellow solid (1.95g, 83%). The solid (1.35g, 4.8 mmol) was dissolved in THF (30 mL) followed by the addition of EtMgBr (1M in THF, 24 mL, 24 mmol) dropwise. The reaction mixture was stirred at 30°C for 16 h and then was partitioned between EtOAc (200 mL) and H20 (100 mL). The organic phase was washed with brine (100 mL), dried over anhydrous Na2S04 and concentrated. The crude product was purified by flash chromatography (hexanes/EtOAc; 19/1 6:1) to give pale yellow solid (l .Olg, 84%). Ή NMR (CDC13): δ 9.07 (bs, 1H), 7.85 - 7.84 (m, 1H), 7.43 (dd, J = 1.9 Hz, 8.8 Hz, 1H), 7.32 (bd, J = 8.8 Hz, 1H), 7.12 (dd, J= 0.8 Hz, 2.1 Hz, 1H), 2.99 (q, J = 7.4 Hz, 2H), 1.27 (t, J= 7.4 Hz, 3H).
General procedure for the synthesis of substituted l-(l-methyl-l//-indol-2-yl)ketone D
A mixture of l-(lH-indol-2-yl)ketone C (0.5 mmol), K2C03 (104 mg, 0.75 mmol) and Mel (47 μί, 0.75 mmol) in dry DMF (3 mL) was heated at 60°C for 2 d. The reaction was quenched with H20 (10 mL) and then extracted with EtOAc (3x10 mL). The organic phases were combined, washed with brine, dried over anhydrous Na2S04, filtered and concentrated in vacuo. The compound was purified by flash column chromatography using 90/10 hexanes/EtOAc to yield 1-(1 -methyl- lH-indol-2-yl)ketone D. l-(5-fluoro-l-methyl-lj3-indol-2-yl)ethanone Dl
Figure imgf000043_0001
Yield = 75%, white powder. Mp = 81-82°C. 1H NMR (400 MHz, CDC13): δ 7.33-7.28 (m, 2H), 7.22 (s, 1H), 7.14 (td, J = 9.2 Hz, 2.4 Hz), 4.05 (s, 3H), 2.60 (s, 3H). 13C NMR (100 MHz, CDC13): 5 191.5, 158.1 (d, J= 236 Hz), 136.7, 136.0, 125.7 (d, J = 10 Hz), 115.0 (d, J= 26 Hz), 1 1 1.4 (d, J= 4 Hz), 1 1 1.3, 106.8 (d, J= 23 Hz), 32.3, 28.0. l-(5-methoxy-l-methyI-lH-indol-2- l)ethanone D2
Figure imgf000043_0002
Yield = 83%, white powder. Mp = 128-129°C. 1H NMR (600 MHz, CDC13): δ 7.28 (d, J Hz, 1H), 7.19 (s, 1H), 7.07-7.05 (m, 2H), 4.04 (s, 3H), 3.85 (s, 3H), 2.59 (s, 3H). l-(5,6-difluoro-l-methyl-lH-indol-2-yl)ethanone D3
Figure imgf000044_0001
Yield = 97%, white powder. Mp = 123-124°C. 1H NMR (400 MHz, CDC13): δ 7.42 (dd, J = 10.0 Hz, 8.0 Hz, 1H), 7.22 (s, 1H), 7.14 (dd, J = 10.6 Hz, 6.6 Hz, 1H), 4.02 (s, 3H), 2.59 (s, 3H). 13C NMR (150 MHz, CDC13): 5190.9, 150.6 (dd, J = 246 Hz, 16 Hz), 147.2 (dd, J= 240 Hz, 16 Hz), 136.1 (d, J= 4 Hz), 135.7 (d, J= 10 Hz), 120.6 (dd, J= 8 Hz, 1 Hz), 111.8 (d, J= 5 Hz, 2 Hz), 108.8 (dd, J= 19 Hz, 1 Hz), 98.1 (d, J= 22 Hz), 32.6, 27.9. l-(4,5-difluoro-l-methyI-lH-indol-2-yl)ethanone D4
Figure imgf000044_0002
Yield = 72%, white powder. Mp = 107-108°C. 1H NMR (400 MHz, CDC13): δ 7.35 (s, 1H), 7.22 (ddd, J = 16.4 Hz, 9.2 Hz, 7.6 Hz, 1H), 7.07 (dd, J = 9.2 Hz, 3.2 Hz, 1H), 4.05 (s, 3H), 2.63 (s, 3H). l-(4,5,6-trifluoro-l-methyl-lH-indol-2-yl)ethanone D5
Figure imgf000044_0003
Yield = 85%, white cotton form. Mp = 169-170°C. Ή NMR (400 MHz, CDC13): δ 7.34 (s, 1H), 6.95 (ddd, J = 10.2 Hz, 5.4 Hz, 0.8 Hz, 1H), 4.01 (s, 3H), 2.61 (s, 3H). 13C NMR (100 MHz, CDC13): δ 190.7, 151.1 (ddd, J = 246 Hz, 13 Hz, 3 Hz), 144.4 (ddd, J= 252 Hz, 1 1 Hz, 5 Hz), 135.9 (d, J= 3 Hz), 135.2 (ddd, J= 241 Hz, 18 Hz, 14 Hz), 134.9 (t, J= 12 Hz), 112.0 (d, J= 19 Hz), 107.8 (d, J= 5 Hz), 93.4 (dd, J= 23 Hz, 5 Hz), 32.7, 27.8. l-(5-bromo-l-methyI-lH-indol-2-yl)ethanone D6
Figure imgf000044_0004
Yield = 91%, white solid. Mp = 142-144°C. 1H NMR (600 MHz, CDC13): δ 7.82 (d, J = 1.9 Hz, 1H), 7.45 (dd, J = 1.9 Hz, 8.9 Hz, 1H), 7.27 (d, J = 8.9 Hz, 1H), 7.20 (s, 1H), 4.05 (s, 3H), 2.61 (s, 3H). ,3C NMR (150 MHz, CDC13): δ 191.8, 138.8, 135.8, 129.0, 127.4, 125.3, 114.0, 1 12.2, 1 11.1, 32.6, 28.3. HRMS calcd for (CnHi0 79BrNO+H)+ 252.0019, found 252.0019. l-(5-chloro-l-methyl-lH-indol-2-yl)ethanone D7
Figure imgf000045_0001
Yield = 96%, pale yellow solid. Mp = 130-134°C. 1H NMR (600 MHz, CDC13): δ 7.67 - 7.6 (m, 1H), 7.32 - 7.31 (m, 2H), 7.21 (s, 1H), 4.06 (s, 3H), 2.61 (s, 3H). 13C NMR (150 MHz, CDC13): δ 191.8, 138.5, 136.0, 126.7, 126.5, 122.1, 111.8, 111.2, 32.6, 28.3. HRMS calcd for (CnHi0ClNO+H)+ 208.0524, found 208.0524. l-(6-bromo-l-methyl-lH-indol-2-yl)ethanone D8
Figure imgf000045_0002
Yield = 91%, white solid. !H NMR (400 MHz, CDC13): δ 7.56 - 7.54 (m, 2H), 7.26 - 7.25 (m, 2H), 4.04 (s, 3H), 2.61 (s, 3H). l-(5-bromo-l-methyl-lH-indol-2-yl)propan-l-one D9
Figure imgf000045_0003
Yield = 78%, white solid. 1H NMR (400 MHz, CDC13): δ 7.82 (d, J = 1.9 Hz, 1H), 7.44 (dd, J = 1.9 Hz, 8.9 Hz, 1H), 7.28 - 7.26 (m, 1H), 7.21 (bs, 1H), 4.06 (s, 3H), 3.01 (q, J = 7.4 Hz, 2H), 1.24 (t, J = 7.3 Hz, 3H). l-(5-Hydroxy-l-methyl-lH-indol-2-yI)ethanone D10
Figure imgf000046_0001
BBr3 (0.65 mL, 1M in DCM, 0.65 mmol) was added to a stirred solution on l-(5-methoxy-l- methyl-lH-indol-2-yl)ethanone D2 (65 mg, 0.32 mmol) in DCM (3 mL) at -78°C under Ar. The resulting mixture was stirred at 0°C for 3h. The mixture was partitioned between EtOAc (100 mL) and H20 (50 mL). The organic phase was washed with brine (50 mL), dried over anhydrous Na2S04 and concentrated. The crude product was purified by flash chromatography (hexanes/EtOAc; 9/1 - 7:3) to give pale yellow solid (48 mg, 79%). Ή NMR (CDC13): δ 7.28 - 7.26 (m, 1H), 7.16 (s, 1H), 7.06 (d, J = 2.3 Hz, 1H), 7.00 (dd, J= 2.3 Hz, 8.8 Hz, 1H), 4.04 (s, 3H), 2.60 (s, 3H).
Synthesis of substituted hvdrazides E
5-bromo-2-(prop-2-ynyloxy)benzohydrazide E 1
Figure imgf000046_0002
A mixture of prop-2-ynyl-5-bromo-2-(prop-2-ynyloxy)benzoate (900 mg, 3.1 mmol) and hydrazine hydrate (0.5 mL, 10.2 mmol) in EtOH (10 mL) was refluxed for 16 h and then concentrated. The crude product was purified by flash chromatography (49/1 : MeOH/DCM) to give white solid (750 mg, 91%). The solid was recrystallized with EtOAc and hexanes to give white crystals (410 mg, 50%). Mp - 123-126°C. 1H NMR (600 MHz, DMSO- 6): δ 9.31 (bs, 1H), 7.70 (d, J= 2.6 Hz, 1H), 7.64 (dd, J= 2.6 Hz, 8.8 Hz, 1H), 7.16 (d, J = 8.9 Hz, 1H), 4.93 (d, J= 2.4 Hz, 1H), 4.55 (bs, 2H), 3.65 (t, J= 2.4 Hz, 1H). 13C NMR (150 MHz, DMSO- d6): δ 163.6, 154.3, 134.4, 132.6, 126.1, 1 16.3, 1 13.1, 79.6, 79.0, 56.8. HRMS calcd for (CioH979BrN202+H)+ 268.9920, found 268.9924.
5-Azido-2-(prop-2-ynyIoxy)benzohydrazide E2
Figure imgf000047_0001
1) Synthesis of prop-2-ynyI 5-azido-2-(prop-2-ynyloxy)benzoate
Figure imgf000047_0002
A mixture of 5-azidosalicylic acid (600 mg, 3.4 mmol), propargyl bromide (1.0 mL, 80% in toluene, 11.2 mmol) and K2C03 (1.3 g, 9.4 mmol) in DMF (15 mL) was stirred at 65°C for 16 h and then partitioned between EtOAc (150 mL) and H20 (50 mL). The organic phase was washed with H20 (50 mL), brine (50 mL), dried over anhydrous Na2S04 and concentrated. The crude product was purified by flash chromatography (100% DCM) to give pale yellow solid (510 mg, 60%). Mp = 54-56°C. 1H NMR (CDC13): δ 7.53 (t, J= 1.8 Hz, 1H), 7.16 (d, J = 1.8 Hz, 2H), 4.90 (d, J= 2.5 Hz, 1H), 4.78 (d, J= 2.4 Hz, 1H), 2.54 (t, 7= 2.4 Hz, 1H), 2.52 (t, 7= 2.5 Hz, 1H). 13C NMR (CDC13): δ 164.2, 154.8, 133.8, 124.3, 122.4, 121.8, 116.9, 78.1, 77.7, 76.6, 75.4, 57.8, 52.9. HRMS calcd for (Co^NsOa+H^ 256.0717, found 256.0710.
2) Synthesis of 5-azido-2-(prop-2-ynyloxy)benzohydrazide E2
A mixture of prop-2-ynyl 5-azido-2-(prop-2-ynyloxy)benzoate (250 mg, 1.0 mmol) and hydrazine hydrate (250 μί, 5.1 mmol) in EtOH (6 mL) was refluxed for 90 min and then concentrated. The residue was recrystallized from EtOAc and Hexanes to give off-white solid (180 mg, 80%). Mp = 132-134°C. 1H NMR (600 MHz, DMSO-76): δ 9.30 (bs, 1H), 7.33 (t, 7 = 1.7 Hz, 1H), 7.23 (d, 7 = 1.7 Hz, 2H), 4.93 (d, 7 = 2.4 Hz, 1H), 4.56 (bs, 2H), 3.64 (t, 7 = 2.4 Hz, 1H). 13C NMR (150 MHz, DMSO-76): δ 163.5, 151.9, 132.3, 124.6, 122.0, 120.3, 115.3, 79.0, 56.5. HRMS calcd for (C10H9N5O2+H)+ 232.0829, found 232.0832.
General procedures for the synthesis of the hydrazides F, G and I
Procedure A: A mixture of the appropriate ketone C, D or H (0.25 mmol) and hydrazide E (0.25 mmol) in propan-l-ol (3 mL) was refluxed until completion (or good conversion) of the reaction (monitored by TLC). If the product precipitated, it was collected by filtration and the solid was washed with hot propan-l -ol (10 mL). If no precipitation was observed, the solvent was evaporated in vacuo and the compound was purified by flash column chromatography.
Procedure B: A mixture of the appropriate ketone C, D or H (0.35 mmol) and hydrazide E (0.35 mmol) in EtOH (5 mL) was refluxed until completion (or good conversion) of the reaction (monitored by TLC). If the product precipitated, it was collected by filtration and the solid was washed with hot EtOH (2x2 mL). If no precipitation was observed, the solvent was evaporated in vacuo and the compound was purified by flash column chromatography.
Procedure C: A mixture of the appropriate ketone C, D or H (0.41 mmol), hydrazide E (0.41 mmol) and AcOH (1 drop) in propan-l-ol (4 mL) was refluxed until completion (or good conversion) of the reaction (monitored by TLC). If the product precipitated, it was collected by filtration and the solid was washed with hot propan-l-ol (2x2 mL). If no precipitation was observed, the solvent was evaporated in vacuo and the compound was purified by flash column chromatography.
Procedure D: A mixture of the appropriate ketone C, D or H (0.41 mmol), hydrazide E (0.41 mmol) and AcOH (1 drop) in ethanol (4 mL) was refluxed until completion (or good conversion) of the reaction (monitored by TLC). If the product precipitated, it was collected by filtration and the solid was washed with hot ethanol (2x2 mL). If no precipitation was observed, the solvent was evaporated in vacuo and the compound was purified by flash column chromatography.
Procedure E: A mixture of the appropriate ester (1.0 eq.) and hydrazine hydrate (> 3.0 eq.) in ethanol was irradiated with microwaves for 60 minutes at 100°C. If the hydrazide E precipitated, it was collected by filtration. If no precipitation was observed, the mixture was partitioned between ethyl acetate and water, the organic layer dried over Na2S04 and evaporated in vacuo. To the hydrazide E taken up in ethanol were added the appropriate ketone C, D or H and AcOH (1 drop). The mixture was refluxed - classical heating- until completion (or good conversion) of the reaction (monitored by TLC). If the product precipitated, it was collected by filtration and the solid was washed with hot ethanol (2x2 mL). If no precipitation was observed, the solvent was evaporated in vacuo and the compound was purified by flash column chromatography.
(£)-N -((lH-indol-2-yI)methylene -5-bromo-2-hydroxybenzohydrazide Fl
Figure imgf000049_0001
Fl was prepared from lH-indole-2-carbaldehyde (obtained by reduction and oxidation of \H- indole-2-carboxylic acid, cf. scheme) and 5-bromo-2-hydroxybenzohydrazide E using general Procedure A.
Yield = 78% (filtration), pale yellow powder. Mp = 257-258°C. !H NMR (400 MHz, DMSO- d6): δ 11.95 (bs, 1H), 1 1.84 (s, 1H), 1 1.62 (s, 1H), 8.48 (s, 1H), 8.05 (d, J = 2.8 Hz, 1H), 7.59 (dd, J= 8.8 Hz, 2.4 Hz, 1H), 7.57 (d, J= 8.0 Hz, 1H), 7.17 (t, J= 7.4 Hz, 1H), 7.02 (t, J= 7.4 Hz, 1H), 6.97 (d, J = 8.8 Hz, 1H), 6.89 (d, J = 1.2 Hz, 1H). 13C NMR (100 MHz, DMSO-^): δ 163.0, 157.9, 141.8, 138.0, 136.0, 132.8, 130.8, 127.6, 123.5, 120.8, 119.6 (2CH), 112.1, 1 10.0, 107.6. HRMS calcd for (C16Hi2 79BrN302+H)+ 358.0186, found 358.0200.
(E)-N -(l-(lH-indol-2-yl)prop lidene)-5-bromo-2-hydroxybenzohydrazide F2
Figure imgf000049_0002
F2 was prepared from l-(lH-indol-2-yl)propan-l-one C (obtained by addition of ethylmagnesium bromide to the Weinreb amide B, cf. scheme) and 5-bromo-2- hydroxybenzohydrazide E using general Procedure A.
Yield = 73% (filtration), white powder. Mp = 255-256°C. 1H NMR (400 MHz, DMSO-<¾: δ 12.16 (bs, 1H), 11.48 (br. d, J= 0.8 Hz, 1H), 11.28 (s, 1H), 8.09 (d, J= 2.4 Hz, 1H), 7.59 (dd, J = 8.6 Hz, 2.6 Hz, 1H), 7.56 (d, J = 8.0 Hz, 1H), 7.49 (d, J = 8.0 Hz, 1H), 7.14 (td, J = 7.8 Hz, 0.6 Hz, 1H), 7.04-6.97 (m, 3H), 2.82 (q, J = 7.6 Hz, 2H), 1.24 (t, J = 7.6 Hz, 3H). 13C NMR (100 MHz, DMSO-<¾: δ 160.0, 155.4, 151.3, 137.8, 135.6, 134.8, 132.7, 127.6, 123.1, 120.6, 120.5, 1 19.3 (2CH), 1 12.1, 1 10.9, 104.4, 20.6, 10.8. HRMS calcd for (Ci8Hi6 79BrN302+H)+ 386.0499, found 386.0505.
(Z)- V-(l-(lH-indol-2-yl)-2,2-dimethylpropylidene)-5-bromo-2-hydroxybenzohydrazide F3
Figure imgf000050_0001
F3 was prepared from l-(lH-indol-2-yl)-2,2-dimethylpropan-l-one C (obtained by addition of t-BuLi to the Weinreb amide B, cf. scheme) and 5-bromo-2-hydroxybenzohydrazide E using general Procedure A.
Yield = 58% (after column 90/10 hexanes/EtOAc), white powder. Mp = 217-218°C. 1H NMR (600 MHz, DMSO- 6): δ 11.35 (s, 1H), 11.01 (s, 2H), 7.98 (d, J = 1.8 Hz, 1H), 7.63 (d, J = 8.4 Hz, 1H), 7.45-7.41 (m, 2H), 7.16 (t, J= 7.8 Hz, 1H), 7.06 (t, J= 7.5 Hz, 1H), 6.77 (d, J= 8.4 Hz, 1H), 6.52 (s, 1H), 1.21 (s, 9H). 13C NMR (150 MHz, DMSC /6): δ 159.8, 158.0, 155.0, 136.6, 135.6, 133.0, 127.7, 127.4, 122.1, 120.7, 120.0, 119.5, 119.1, 111.7, 1 10.8, 102.4, 38.5, 28.1. HRMS calcd for (C20H20 79BrN3O2 Na)+ 436.0637, found 436.0631.
{E and Z)-N -((lH-indol-2-yl)(phenyI)methylene)-5-bromo-2-hydroxybenzohydrazide F4
Figure imgf000050_0002
F4 was prepared from (lH-indol-2-yl)(phenyl)methanone C (obtained by addition of PhLi to the Weinreb amide B, cf. scheme) and 5-bromo-2-hydroxybenzohydrazide E using general Procedure A. Yield = 43% (after column 70/30 hexanes/EtOAc), pale yellow powder. NMR analysis indicated that F4 is present in two isomeric forms (11 :89).
Major isomer 1H NMR (600 MHz, DMSO-<¾: δ 11.60 (s, 1H), 11.35 (s, 1H), 11.27 (s, 1H), 8.07 (s, 1H), 7.72-7.46 (m, 8H), 7.16 (t, J- 7.2 Hz, 1H), 6.97 (t, J= 6.9 Hz, 1H), 6.85 (d, J = 8.4 Hz, 1H), 6.12 (s, 1H). 13C NMR (150 MHz, DMSO-i 6): δ 159.6, 155.0, 148.4, 137.9, 135.7, 135.5, 133.0, 132.0, 130.1, 129.4 (2CH), 128.3 (2CH), 127.5, 123.5, 120.8, 119.9, 119.5, 119.1, 112.2, 111.0, 107.4.
Minor isomer
13C NMR (150 MHz, DMSO-<¾: δ 160.4, 155.7, 146.8, 137.1, 136.9, 135.8, 133.1 , 129.9, 128.5 (2CH), 127.9 (2CH), 127.3, 127.2, 123.0, 121.2, 120.0, 119.9, 119.2, 112.1, 110.9, 105.2.
HRMS (mixture) calcd for (C22H16 79BrN302+Na)+ 456.0324, found 456.0309. (JE)-5-bromo-N -(l-(5-fluoro-lfir-indol-2- l)ethylidene)-2-hydroxybenzohydrazide F5
Figure imgf000051_0001
F5 was prepared from l-(5-fluoro-lH-indol-2-yl)ethanone CI and 5-bromo-2- hydroxybenzohydrazide E using general Procedure A.
Yield = 46% (filtration), white powder. Mp = 262-263°C. Ή NMR (400 MHz, OMSO-d6): δ 12.11 (bs, 1H), 11.42 (s, 1H), 11.32 (s, 1H), 8.09 (d, J= 2.4 Hz, 1H), 7.59 (dd, J= 8.6 Hz, 2.6 Hz, 1H), 7.48 (dd, J= 8.8 Hz, 4.8 Hz, 1H), 7.32 (dd, J- 10.0 Hz, 2.4 Hz, 1H), 7.03-6.96 (m, 3H), 2.37 (s, 3H). 13C NMR (100 MHz, DMSO-d6): δ 160.3, 157.0 (d, J = 230 Hz), 155.7, 147.0, 137.5, 135.7, 134.4, 132.5, 127.6 (d, J = 10 Hz), 120.3, 1 19.3, 1 13.2 (d, J = 9 Hz), 1 1 1.5 (d, J = 26 Hz), 1 10.8, 104.9 (d, J = 23 Hz), 104.6 (d, J = 5 Hz), 13.7. HRMS calcd for (C17H13 79BrFN302+H)+ 390.0248, found 390.0247.
(£)-5-bromo-N'-(l-(7-fluoro-lH-indol-2-yl)ethylidene)-2-hydroxybenzohydrazide F6
Figure imgf000052_0001
F6 was prepared from l-(7-fluoro-lH-indol-2-yl)ethanone C2 and 5-bromo-2- hydroxybenzohydrazide E using general Procedure A.
Yield = 86% (filtration), beige powder. Mp = 263-264°C.1H NMR (400 MHz, DMSO- 6): δ 12.11 (bs, IH), 11.75 (s, IH), 11.34 (s, IH), 8.08 (d,J= 1.6 Hz, IH), 7.60 (dd,J= 8.8 Hz, 2.4 Hz, IH), 7.42-7.38 (m, IH), 7.08 (bs, IH), 7.03-6.97 (m, 3H), 2.40 (s, 3H).13C NMR (100 MHz, DMSO- fi): δ 160.5, 155.7, 149.0 (d, J= 244 Hz), 147.1, 137.4, 135.7 (d, J = 4 Hz), 132.5 (d,J=3 Hz), 131.5 (d,J= 6 Hz), 125.5 (d,J= 13 Hz), 120.2, 119.7, 119.3 (d,J= 8 Hz), 116.9 (d,J= 8 Hz), 110.8, 108.0 (d,J= 16 Hz), 105.3 (d,J= 8 Hz), 14.0. HRMS calcd for (C17H13 79BrFN302+H)+ 390.0248, found 390.0250
(£)-5-bromo-2-hydroxy-N-l-(5-methoxy-lH-indol-2-yl)ethylidene)benzohydrazide F7
Figure imgf000052_0002
F7 was prepared from l-(5-methoxy-lH-indol-2-yl)ethanone C and 5-bromo-2- hydroxybenzohydrazide E using general Procedure A.
Yield - 79% (filtration), pale yellow cotton. Mp = 250-251°C.1H NMR (600 MHz, DMSO- d6): δ 12.14 (bs, IH), 11.30 (s, IH), 11.20 (s, IH), 8.09 (d, J= 2.4 Hz, IH), 7.59 (dd, J= 8.7 Hz, 2.7 Hz, IH), 7.37 (d, J= 9.0 Hz, IH), 7.03 (d, J= 1.8 Hz, IH), 7.01 (d, J= 8.4 Hz, IH), 6.88 (d, J= 0.8 Hz, IH), 6.80 (dd, J= 9.0 Hz, 2.4 Hz, IH), 3.75 (s, 3H), 2.36 (s, 3H).13C NMR (100 MHz, DMSO-<¾: δ 160.2, 155.7, 153.5, 147.4, 136.0, 135.6, 133.0, 132.6, 127.9, 120.3, 119.3, 113.9, 112.9, 110.9, 104.6, 101.6, 55.2, 13.8. HRMS calcd for (C,8H16 79BrN303+H)+ 402.0448, found 402.0450.
(£)-5-bromo-iV-(l-(5,6-difluoro-lH-indoI-2-yl)ethylidene)-2-hydroxybenzohydrazide F8
Figure imgf000053_0001
F8 was prepared from l-(5,6-difluoro-lH-indol-2-yl)ethanone C3 and 5-bromo-2- hydroxybenzohydrazide E using general Procedure A.
Yield = 55% (filtration), pale yellow powder. Mp = 271-272°C.1H NMR (400 MHz, DMSO- d6): δ 12.11 (bs, 1H), 11.48 (s, 1H), 11.34 (bs, 1H), 8.08 (d, J= 2.4 Hz, 1H), 7.61-7.54 (m, 2H), 7.39 (dd, J= 11.0 Hz, 7.0 Hz, 1H), 7.01 (d, J= 8.8 Hz, 1H), 6.98 (d, J= 1.6 Hz, 1H), 2.36 (s, 3H).13C NMR (150 MHz, DMSO-d6): δ 160.3, 155.7, 147.7 (dd, J= 238 Hz, 16 Hz), 146.5, 145.4 (dd,J=234 Hz, 15 Hz), 137.6 (d,J=4Hz), 135.7, 133.0 (d,J = 11 Hz), 132.6, 122.9 (d, J- 9 Hz), 120.2, 119.3, 110.8, 107.1 (d, J= 19 Hz), 104.8, 99.7 (d, J= 22 Hz), 13.6. HRMS calcd for (C17H12 79BrF2N302+H)+ 408.0159, found 408.0129.
(E)-5-bromo-N'-(l-(4,5-difluoro-lJBT-indoI-2- l)ethylidene)-2-hydroxybenzohydrazide F9
Figure imgf000053_0002
F9 was prepared from l-(4,5-difluoro-lH-indol-2-yl)ethanone C4 and 5-bromo-2- hydroxybenzohydrazide E using general Procedure A.
Yield = 75% (filtration), beige powder. Mp = 275-276°C. Ή NMR (400 MHz, DMSO-c¾: δ 12.11 (bs, 1H), 11.71 (s, 1H), 11.35 (s, 1H), 8.08 (d,J=2.8Hz, 1H), 7.60 (dd,J=8.8Hz, 2.8 Hz, 1H), 7.29 (dd,J=9.0 Hz, 3.4 Hz, 1H), 7.18 (td,J= 11.6 Hz, 8.0 Hz, 1H), 7.12 (d,J= 1.6 Hz, 1H), 7.02 (d, J= 8.4 Hz, 1H), 2.39 (s, 3H).13C NMR (150 MHz, OMSO-d6) δ 160.3, 155.6, 146.4, 143.1 (dd,J=231 Hz, 11 Hz), 141.9 (dd, J = 245 Hz, 14 Hz), 137.9, 135.8 (d, J = 9 Hz), 135.7, 132.6, 120.2, 119.3, 117.5 (d,J= 18 Hz), 112.7 (d,J=21 Hz), 110.8, 108.4 (dd, J= 7 Hz, 4 Hz), 100.3 (d, J = 5 Hz), 13.6. HRMS calcd for (Ci7Hi2 79BrF2N302+H)+ 408.0154, found 408.0161.
(£)-5-bromo-2-hydroxy-N-(l-(4,5,6-trifluoro-lH-indol-2-yl)ethylidene)benzohydrazide F10
Figure imgf000054_0001
F10 was prepared from l-(4,5,6-trifluoro-lH-indol-2-yl)ethanone C5 and 5-bromo-2- hydroxybenzohydrazide E using general Procedure A.
Yield = 46% (after column 50/50 hexanes/EtOAc then 90/10 DCM/MeOH), beige powder. 1H NMR (400 MHz, DMSO-t¾): δ 12.10 (bs, 1H), 11.78 (bs, 1H), 11.34 (s, 1H), 8.08 (d, J = 2.4 Hz, 1H), 7.60 (dd, J= 8.8 Hz, 2.4 Hz, 1H), 7.28 (dd, J= 10.2 Hz, 6.2 Hz, 1H), 7.15 (d, J= 2.0 Hz, 1H), 7.02 (d, J = 8.8 Hz, 1H), 2.38 (s, 3H). 13C NMR (150 MHz, DMSO-< 6): δ 160.3, 155.6, 148.0 (dd, J= 239 Hz, 13 Hz), 146.0, 142.7 (ddd, J= 247 Hz, 1 1 Hz, 5 Hz), 137.8 (d, J = 3 Hz), 135.6, 133.7 (ddd, J = 235 Hz, 18 Hz, 14 Hz), 132.7 (t, J = 12 Hz), 132.5, 120.2, 119.2, 113.2 (d, J = 18 Hz), 1 10.8, 100.3, 95.5 (dd, J = 22 Hz, 3 Hz), 13.5. HRMS calcd for (C17Hn 79BrF3N302+H)+ 426.0060, found 426.0070.
(£)-5-bromo-2-hydroxy-N - l-(5-phenyl-lH-indol-2-yl)ethylidene)benzohydrazide Fll
Figure imgf000054_0002
Fll was prepared from l-(5-phenyl-lH-indol-2-yl)ethanone C6 and 5-bromo-2- hydroxybenzohydrazide E using general Procedure A.
Yield = 92% (filtration), beige solid. Mp = 275-276°C. 1H NMR (600 MHz, OMSO-d6): δ 12.14 (bs, 1H), 1 1.42 (s, 1H), 1 1.33 (s, 1H), 8.10 (d, J = 2.4 Hz, 1H), 7.83 (s, 1H), 7.67 (d, J = 7.2 Hz, 2H), 7.60 (dd, J = 8.7 Hz, 2.7 Hz, 1H), 7.57 (d, J = 9.0 Hz, 1H), 7.48 (dd, 7 = 8.4 Hz, 1.2 Hz, 1H), 7.45 (t, J = 7.5 Hz, 1H), 7.31 (t, J = 7.2 Hz, 1H), 7.05 (s, 1H), 7.02 (d, J = 9.0 Hz, 1H), 2.40 (s, 3H). 13C NMR (150 MHz, DMSO-i 6): δ 160.3, 155.6, 147.2, 141.6, 137.4,
136.5, 135.7, 132.6, 131.9, 128.8 (2CH), 128.2, 126.7 (2CH), 126.3, 122.7, 120.3, 119.3,
118.6, 1 12.6, 1 10.9, 105.2, 13.8. HRMS calcd for (C23H18 79BrN302+Na)+ 470.0480, found 470.0455.
(E)-5-bromo-N'-(l-(5-bromo-li¾r-indol-2-yl)ethylidene)-2-hydroxybenzohydrazide F12
Figure imgf000055_0001
F12 was prepared from l-(5-bromo-lH-indol-2-yl)ethanone C and 5-bromo-2- hydroxybenzohydrazide E using general Procedure A.
Yield = 61% (filtration), pale yellow solid. Mp = 295-299°C. 1H NMR (600 MHz, DMSO- d6): δ 12.12 (bs, 1H), 11.53 (s, 1H), 1 1.35 (bs, 1H), 8.08 (d, J = 2.6 Hz, 1H), 7.75 (d, J = 1.8 Hz, 1H), 7.59 (dd, J= 2.6 Hz, 8.7 Hz, 1H), 7.45 (d, J= 8.7 Hz, 1H), 7.26 (dd, J= 1.9 Hz, 8.6 Hz, 1H), 7.02 (d, J= 8.7 Hz, 1H), 6.96 (d, J= 1.5 Hz, 1H), 2.37 (s, 3H). 13C NMR (150 MHz, DMSO- ): δ 161.2, 156.6, 147.6, 138.1 , 137.3, 136.6, 133.5, 130.3, 126.5, 123.7, 121.2, 120.3, 115.1, 112.7, 111.7, 105.0, 14.7. HRMS calcd for (C17H13 79Br2N302+H)+ 451.9428, found 451.9428.
(E)-N'-(l-(lH-indol-2-yl)ethylidene)-5-bromo-2-(prop-2-ynyloxy)benzohydrazide F13
Figure imgf000055_0002
F13 was prepared from l-(lH-indol-2-yl)ethanone C and El using general Procedure A. Yield = 54% (filtration), off-white solid. Mp = 246-251°C. Ή NMR (600 MHz, DMSO- ): δ 11.30 (s, 1H), 10.78 (s, 1H), 7.93 (d, J = 2.6 Hz, 1H), 7.75 (dd, J= 2.6 Hz, 8.9 Hz, 1H), 7.56 (d, J= 7.9 Hz, 1H), 7.49 (d, J= 8.1 Hz, 1H), 7.26 (d, J= 8.9 Hz, 1H), 7.14 (dt, J= 0.9 Hz, 8.0 Hz, 1H), 7.00 (dt, J = 0.7 Hz, 7.8 Hz, 1H), 6.97 (d, J = 1.5 Hz, 1H), 5.04 (d, J = 2.3 Hz, 1H), 3.75 (t, J= 2.3 Hz, 1H), 2.39 (s, 3H). 13C NMR (150 MHz, DMSO-<¾): δ 160.8, 155.2, 148.7, 138.7, 136.7, 135.7, 133.6, 128.5, 126.2, 124.1, 121.6, 120.2, 117.0, 114.0, 113.1, 105.7, 80.5, 79.3, 58.2, 15.0. HRMS calcd for (C20Hi6 81BrN3O2+H)+ 412.0481, found 412.0467.
(E)-N'-(l-(lH-indol-2-yl)ethylidene)-3,5-dibromo-2-hydroxybenzohydrazide F14
Figure imgf000056_0001
F14 was prepared from l-(lH-indol-2-yl)ethanone C and 3,5-dibromo-2- hydroxybenzohydrazide E using general Procedure A.
Yield = 55% (filtration), pale yellow solid. Mp = 146-150°C. 1H NMR (600 MHz, DMSO- d6): δ 11.64 (bs, IH), 11.39 (s, IH), 8.16 (d, J= 1.6 Hz, IH), 7.99 (d, J= 1.8 Hz, IH), 7.58 (d, J= 7.9 Hz, IH), 7.48 (d, J= 8.2 Hz, IH), 7.17 (t, J= 7.9 Hz, IH), 7.05 (s, IH), 7.00 (t, J- 7.8 Hz, IH), 2.44 (s, 3H). I3C NMR (150 MHz, DMSO- 6): δ 163.8, 156.6, 153.5, 138.6, 138.2, 135.8, 130.7, 128.0, 124.0, 121.4, 119.9, 119.2, 113.3, 112.7, 1 10.1, 106.2, 15.2. HRMS calcd for (C17H13 79Br2N302+H)+ 451.9428, found 451.9431.
(E)-iV-(l-(lH-indol-2-yl)ethylidene -5-bromo-2-hydroxybenzohydrazide F15
Figure imgf000056_0002
F15 was prepared from l-(lH-indol-2-yl)ethanone C and 5-bromo-2-hydroxybenzohydrazide E using general Procedure B.
Yield = 53% (after flash chromatography 20/1 ; DCM/MeOH), pale yellow solid. Mp = 276- 279°C. 1H NMR (600 MHz, DMSO- ): δ 12.12 (s, IH), 1 1.31 (s, 2H), 8.09 (s, IH), 7.59 (d, J = 8.7 Hz, 1H), 7.56 (d, J= 7.9 Hz, IH), 7.49 (d, J = 8.2 Hz, IH), 7.14 (t, J= 7.9 Hz, 1H), 7.01 (d, J = 7.8 Hz, IH), 7.00 (t, J = 7.4 Hz, IH), 6.98 (s, IH), 2.38 (s, 3H). 13C NMR (150 MHz, OMSO-de): δ 160.3, 155.7, 147.3, 137.7, 135.74, 135.65, 132.5, 127.6, 123.2, 120.7, 120.3, 1 19.33, 1 19.31, 1 12.1 , 110.8, 104.9, 13.8. HRMS calcd for (C] 7Hi4 79BrN302+H)+ 372.0342, found 372.0333.
(£)-N'-(l-(lH-indol-2-yl)ethylidene)-3-bromobenzohydrazide Π6
Figure imgf000056_0003
F16 was prepared from l-(lH-indol-2-yl)ethanone C and 5-bromobenzohydrazide E using general Procedure C.
Yield = 70% (after flash chromatography 3/1 ; hexanes/EtOAc), pale yellow solid. Mp = 219- 224°C.
1H NMR (600 MHz, DMSO- ): δ 11.31 (s, 1H), 10.89 (s, 1H), 8.09 (bs, 1H), 7.92 (d, J = 7.6 Hz, 1H), 7.81 (d, J = 8.0 Ηζ,ΙΗ), 7.57 (d, J = 7.9 Hz, 1H), 7.51 (t, J = 7.9 Hz, 1H), 7.49 (d, J = 7.3 Hz, 1H), 7.16 (dt, J = 0.7 Hz, 7.2 Hz, 1H), 7.01 (dt, J = 0.9 Hz, 7.5 Hz, 1H), 6.99 (bs, 1H), 2.43 (s, 3H). 13C NMR (150 MHz, DMSO-i¾): δ 163.1, 151.9, 138.5, 137.2, 136.8, 135.1, 131.5, 131.3, 128.5, 128.0, 124.1, 122.5, 121.6, 120.3, 113.1, 105.8, 15.4. HRMS calcd for (Ci7H14 79BrN30+H)+ 356.0393, found 356.0394.
(£)-N'-(l-(lH-indol-2-yI)ethylidene -2-hydroxybenzohydrazide F17
Figure imgf000057_0001
F17 was prepared from l-(lH-indol-2-yl)ethanone C and 2-hydroxybenzohydrazide E using general Procedure C.
Yield = 54% (after flash chromatography 40/1 ; DCM/MeOH), pale yellow solid. Mp = 246- 248°C. 1H NMR (600 MHz, DMSO-c/6): δ 11.86 (bs, 1H), 11.38 (bs, 1H), 11.31 (s, 1H), 8.03 (dd, J = 1.4 Hz, 7.8 Hz, 1H), 7.56 (d, J = 7.9 Hz, 1H), 7.50 (d, J = 8.1 Hz, 1H), 7.43 (dt, J =
1.8 Hz, 7.2 Hz, 1H), 7.14 (dt, J = 1.1 Hz, 8.1 Hz, 1H), 7.04 (d, J = 7.6 Hz, 1H), 7.002 (t, J =
7.9 Hz, 1H), 7.001 (t, J = 7.0 Hz, 1H), 6.97 (d, J = 1.5 Hz, 1H), 2.39 (s, 3H). 13C NMR (150 MHz, DMSC 6): δ 162.3, 157.1 , 147.2, 138.2, 136.4, 133.8, 131.0, 128.1, 123.6, 121.1, 120.1, 119.8, 118.4, 1 17.4, 1 12.6, 105.1 , 14.3. HRMS calcd for (Ci7Hi5N302+H)+ 294.1237, found 294.1237.
(J5)-iV-(l-(lH-indol-2-yl)ethylidene)-5-bromo-2-methoxybenzohydrazide F18
Figure imgf000057_0002
Br F18 was prepared from l-(lH-indol-2-yl)ethanone C and 2-methoxybenzohydrazide E using general Procedure C.
Yield = 41% (filtration), pale yellow solid. Mp = 241-244°C. !H NMR (600 MHz, DMSO- d6): δ 11.31 (s, 1H), 10.90 (s, 1H), 7.96 (d, J = 2.6 Hz, 1H), 7.73 (dd, J = 2.7 Hz, 8.8 Hz, 1H), 7.56 (d, J = 8.1 Hz, 1H), 7.49 (d, J = 8.1 Hz, 1H), 7.23 (d, J = 8.9 Hz, 1H), 7.14 (dt, J = 1.0 Hz, 7.6 Hz, 1H), 7.00 (dt, J = 0.8 Hz, 7.5 Hz, 1H), 7.23 (d, J = 8.9 Hz, 1H), 6.98 (d, J = 1.5 Hz, 1H), 3.99 (s, 3H), 2.38 (s, 3H). 13C NMR (150 MHz, DMSO- 6): δ 160.3, 156.84, 148.0, 138.2, 136.3, 135.5, 133.1, 128.1, 124.7, 123.6, 121.1, 119.8, 1 15.4, 112.8, 1 12.6, 105.3, 57.3, 14.2. HRMS calcd for (C18H,6 79BrN302+H)+ 386.0499, found 386.0515.
(£)-N'-(l-(lH-indol-2-yl)ethylidene -2-hydroxy-5-iodobenzohydrazide F19
Figure imgf000058_0001
F19 was prepared from l-(lH-indol-2-yl)ethanone C and 2-hydroxy-5-iodobenzohydrazide E using general Procedure C.
Yield = 32% (after flash chromatography 4/1 - 1 :1 ; hexanes/EtOAc), pale yellow solid. Mp = 251-255°C. Ή NMR (600 MHz, DMSO- 6): δ 12.10 (bs, 1H), 1 1.32 (s, 2H), 8.25 (d, J = 2.2 Hz, 1H), 7.72 (dd, J = 2.3 Hz, 8.5 Hz, 1H), 7.56 (d, J = 7.9 Hz, 1H), 7.49 (d, J = 8.0 Hz, 1H), 7.14 (dt, J= 1.0 Hz, 7.1 Hz, 1H), 7.00 (dt, J= 0.9 Hz, 7.4 Hz, 1H), 6.98 (s, 1H), 6.89 (d, J = 8.6 Hz, 1H), 2.38 (s, 3H). ,3C NMR (150 MHz, DMSO-< 6): δ 161.2, 157.3, 148.1, 142.2, 139.4, 138.7, 136.7, 128.5, 124.1, 121.6, 120.6, 120.3, 1 13.1 , 105.7, 82.6, 14.7. HRMS calcd for (Ci7H14iN302+H)+ 420.0203, found 420.0201.
(E)-5-bromo-N'-(l-(5-chloro-lH-indoI-2- l)ethylidene)-2-hydroxybenzohydrazide F20
Figure imgf000058_0002
F20 was prepared from l-(5-chloro-lH-indol-2-yl)ethanone C and 5-bromo-2- hydroxybenzohydrazide E using general Procedure C. Yield = 77% (filtration), pale yellow solid. Mp = 282-285°C. 1H NMR (600 MHz, DMSO- ): 5 12.11 (s, 1H), 11.52 (s, 1H), 1 1.34 (s, 1H), 8.09 (d, J = 2.6 Hz, 1H), 7.61 (d, J = 2.2 Hz, 1H), 7.60 (dd, J= 2.7 Hz, 8.7 Hz, 1H), 7.49 (d, J = 8.7 Hz, 1H), 7.15 (dd, J= 2.1 Hz, 8.7 Hz, 1H), 6.98 (d, J = 8.7 Hz, 1H), 6.96 (d, J = 1.5 Hz, 1H), 2.37 (s, 3H). 13C NMR (150 MHz, DMSO- ): δ 160.8, 156.1, 147.3, 137.8, 136.6, 136.2, 133.1, 129.1, 124.3, 123.6, 120.8, 120.2, 1 19.8, 114.2, 111.3, 104.7, 14.2. HRMS calcd for (Ci7Hi3 81BrClN302+H)+ 407.9931 , found 407.9931.
(^-iV-il-il -indol-l-y^ethylidene ^-bromo-l-hydroxybenzohydrazide F l
Figure imgf000059_0001
F21 was prepared from l-(lH-indol-2-yl)ethanone C and 4-bromo-2-hydroxy-benzohydrazide E using general Procedure C.
Yield = 62% (filtration), yellow solid. Mp = 281-284°C. Ή NMR (600 MHz, DMSO-< 6): δ 12.27 (bs, 1H), 11.31 (s, 1H), 1 1.25 (s, 1H), 7.94 (d, J= 8.4 Hz, 1H), 7.55 (d, J= 7.9 Hz, 1H), 7.49 (d, J= 8.2 Hz, 1H), 7.22 (d, J= 1.7 Hz, 1H), 7.20 (dd, 7= 1.7 Hz, 8.4 Hz, 1H), 7.14 (dt J = 0.9 Hz, 7.6 Hz, 1H), 7.00 (dt, J= 0.9 Hz, 7.5 Hz, 8.7 Hz, 1H), 6.98 (d, J= 1.2 Hz, 1H), 2.38 (s, 3H). 1 C NMR (150 MHz, DMSO-i/6): δ 161.8, 158.1, 148.0, 138.7, 136.7, 133.4, 128.5, 126.8, 124.1, 123.6, 121.6, 120.3, 120.2, 1 18.7, 1 13.1, 105.7, 14.7. HRMS calcd for (C17H]4 79BrN302+H)+ 372.0342, found 372.0347.
(E)-7Y'-(l-(lH-indol-2-yI)ethyIidene -5-broino-2-hydroxy-4-methoxybenzohydrazide F22
Figure imgf000059_0002
F22 was prepared from l-(lH-indol-2-yl)ethanone C and 5-bromo-2-hydroxy-4- methoxybenzohydrazide E using general Procedure C.
Yield = 82% (filtration), pale yellow solid. Mp = 242-244°C. 1H NMR (600 MHz, DMSO- ί¾): δ 12.40 (bs, 1H), 11.31 (s, 1 H), 1 1.17 (bs, 1H), 8.17 (s, 1H), 7.56 (d, J= 7.9 Hz, 1H), 7.48 (d, J = 8.2 Hz, 1H), 7.16 (dt, J = 0.9 Hz, 7.6 Hz, 1H), 6.99 (t, J = 7.1 Hz, 1H), 6.97 (s, 1H), 6.69 (s, 1H), 3.88 (s, 3H), 2.38 (s, 3H). 13C NMR (150 MHz, DMSO-d6): δ 161.1, 158.8, 158.4, 147.4, 137.7, 135.8, 133.7, 127.6, 123.1, 120.6, 119.3, 112.1, 111.4, 104.7, 100.9, 100.6, 56.5, 13.8. HRMS calcd for (Ci8H16 79BrN303+H)+ 402.0448, found 402.0454.
(EJ-N'- l-ilH-indol-l-ylJethylidene -S-hydroxy-l-naphthohydrazide FlS
Figure imgf000060_0001
F23 was prepared from l-(lH-indol-2-yl)ethanone C and 3-hydroxy-2-naphthohydrazide E using general Procedure C.
Yield = 58% (filtration), yellow solid. Mp = 252-255°C. 1H NMR (600 MHz, DMSO- ): δ 1 1.81 (bs, 1H), 11.61 (bs, 1H), 11.35 (s, 1H), 8.68 (s, 1H), 8.01 (d, J= 8.2 Hz, 1H), 7.78 (d, J - 8.2 Hz, 1H), 7.57 (d, J= 7.9 Hz, 1H), 7.53-7.50 (m, 2H), 7.38-7.36 (m, 2H), 7.15 (t, J= 7.2 Hz, 1H), 7.01-6.99 (m, 2H), 2.43 (s, 3H). 13C NMR (150 MHz, OMSO-d6) δ 161.7, 153.3, 147.2, 138.2, 136.4, 136.2, 132.7, 129.4, 128.8, 128.1 , 127.7, 126.2, 124.4, 123.6, 121.3, 121.1 , 119.8, 112.6, 11 1.2, 105.2, 14.3. HRMS calcd for (C2]H17N302+H)+ 344.1394, found 344.1392.
(E and Z)-5-bromo-2-hydroxy-N -((l-methyl-lH-indol-2-yl)methylene) benzohydrazide Gl
Figure imgf000060_0002
Gl was prepared from l-methyl-lH-indole-2-carbaldehyde D and 5-bromo-2- hydroxybenzohydrazide E using general Procedure A.
Yield = 84% (filtration), pale yellow powder. NMR analysis indicated that Gl is present in two isomeric forms (12:88). 1H NMR (400 MHz, DMSO-<¾): δ 1 1.96 (bs, 1H), 1 1.86 (s, 1H), 8.59 (s, 1H), 8.05 (d, J = 2.0 Hz, 1H), 7.63-7.57 (m, 2H), 7.51 (d, J= 8.4 Hz, 1H), 7.27 (t, J = 7.6 Hz, 1H), 7.09 (t, J = 7.4 Hz, 1H), 6.98-6.95 (m, 2H). 13C NMR (100 MHz, DMSO-i¾): δ 163.1, 158.0, 142.3, 139.5, 136.1, 133.1, 130.7, 126.8, 123.7, 121.1, 120.0, 119.6, 118.2, 110.1, 1 10.0, 108.0, 31.7. HRMS calcd for (C17Hi4 79BrN302+H)+ 372.0348, found 372.0346.
(jE' «< Z)-5-bromo-2-hydroxy-N -(l-(l-methyl-lH-indol-2-yl)propylidene)
benzohydrazide G2
Figure imgf000061_0001
G2 was prepared from 1-(1 -methyl- lH-indol-2-yl)propan-l -one D and 5-bromo-2- hydroxybenzohydrazide E using general Procedure A.
Yield = 75% (filtration), white powder. NMR analysis indicated that G2 is present in two isomeric forms (14:86).
Major isomer
1H NMR (600 MHz, DMSO-d6): δ 12.16 (bs, 1H), 11.49 (s, 1H), 8.08 (d, J = 3.0 Hz, 1H), 7.62-7.59 (m, 2H), 7.51 (d, J = 8.4 Hz, 1H), 7.26 (t, J = 7.5 Hz, 1H), 7.10-7.03 (m, 3H), 4.14 (s, 3H), 2.85 (q, J= 7.6 Hz, 2H), 1.25 (t, J = 7.6 Hz, 3H). 13C NMR (150 MHz, DMSO-<¾: δ 160.3, 155.5, 151.8, 139.6, 135.7, 135.3, 132.8, 126.4, 123.3, 120.9, 120.4, 119.8, 1 19.3, 11 1.0, 110.2, 106.0, 33.0, 21.9, 10.8.
Minor isomer
13C NMR (150 MHz, DMSO-i 6): δ 159.9, 155.1, 151.4, 137.8, 135.65, 133.0, 132.0, 127.3, 122.6, 121.0, 120.0, 1 19.8, 119.1, 1 10.9, 1 10.6, 102.1, 31.4, 31.0, 11.1.
HRMS calcd for (Ci9Hi8 79BrN302+H)+ 400.0655, found 400.0662
(Z)-5-bromo-N'-(2,2-dimethyl-l-(l-inethyl-lH-indol-2-yl)propylidene)-2- hydroxybenzohydrazide G3
Figure imgf000061_0002
G3 was prepared from 2,2-dimethyl-l-(l -methyl- l -indol-2-yl)propan-l -one D and 5- bromo-2-hydroxybenzohydrazide E using general Procedure A. Yield = 28% (after column 80/20 hexanes/EtOAc), white powder. Mp = 234-235°C. 1H NMR (600 MHz, DMSO-^): δ 10.94 (bs, 1H), 10.71 (s, 1H), 7.97 (d, J = 2.4 Hz, 1H), 7.65 (d, J = 7.8 Hz, 1H), 7.52 (d, J = 8.4 Hz, 1H), 7.43 (dd, J = 8.4 Hz, 2.4 Hz, 1H), 7.25 (t, J = 7.8 Hz, 1H), 7.12 (t, J = 7.5 Hz, 1H), 6.75 (d, J = 8.4 Hz, 1H), 6.55 (s, 1H), 3.52 (s, 3H), 1.22 (s, 9H). 13C NMR (150 MHz, DMSO-d6): δ 159.9, 156.9, 155.0, 137.4, 135.6, 133.0, 130.1, 127.1, 122.2, 120.8, 119.9, 1 19.8, 119.1, 110.8, 110.4, 101.7, 38.8, 30.6, 27.9. HRMS calcd for (C2]H22BrN302+Na)+ 450.0793, found 450.0797.
(E and Z)-5-bromo-2-hydroxy-N -((l-methyl-l/T-indol-2-yl)(phenyl)methylene) benzohydrazide G4
Figure imgf000062_0001
G4 was prepared from ((1 -methyl- lH-indol-2-yl)(phenyl)methanone D and 5-bromo-2- hydroxybenzohydrazide E using general Procedure A.
Yield = 29% (after column 80/20 hexanes/EtOAc), pale yellow solid. NMR analysis indicated that G4 is present in two isomeric forms (34:66).
Major isomer
1H NMR (600 MHz, DMSO-<¾: δ 1 1.57 (s, 1H), 11.33 (s, 1H), 8.04 (s, 1H), 7.73-6.82 (m, 12H), 3.47 (s, 3H). 13C NMR (150 MHz, DMSO-< 6): δ 160.3, 155.2, 146.1, 137.8, 136.7, 135.9, 133.0, 130.1, 129.5, 128.8 (2CH), 127.2, 127.0 (2CH), 122.8, 121.3, 120.1, 1 19.8, 1 19.2, 111.0, 110.6, 103.8, 31.0.
Minor isomer
,3C NMR (150 MHz, DMSO-c/6): δ 159.7, 155.0, 149.1 , 139.8, 135.8, 135.7, 133.0, 132.7, 130.0, 129.4 (2CH), 128.3 (2CH), 126.2, 123.7, 121.1, 1 19.9, 1 19.8, 1 19.1, 110.9, 110.2, 109.4, 33.0. HRMS calcd for (C23Hi8 79BrN302+H)+ 448.0661, found 448.0675.
(^ a«i Z)-5-bromo-/V'-(l-(5-fluoro-l-methyl-lH-indol-2-yI)ethylidene)-2- hydroxybenzohydrazide G5
Figure imgf000063_0001
G5 was prepared from l-(5-fluoro-l-methyl-lH-indol-2-yl)ethanone Dl and 5-bromo-2- hydroxybenzohydrazide E using general Procedure A.
Yield = 91% (filtration), pale yellow powder. NMR analysis indicated that G5 is present in two isomeric forms (9:91). 1H NMR (400 MHz, OMSO-d6): δ 12.10 (bs, 1H), 11.35 (s, 1H), 8.07 (d, J = 2.4 Hz, 1H), 7.60 (dd, J = 8.4 Hz, 2.4 Hz, 1H), 7.53 (dd, J= 8.8 Hz, 4.4 Hz, 1H), 7.36 (dd, J = 9.4 Hz, 2.2 Hz, 1H), 7.10 (td, J = 9.2 Hz, 2.4 Hz, 1H), 7.03-7.00 (m, 2H), 4.12 (s, 3H), 2.37 (s, 3H). 13C NMR (100 MHz, DMSO-d6): δ 160.6, 157.2 (d, J = 231 Hz), 155.8, 147.3, 137.9, 136.3, 135.7, 132.6, 126.4 (d, J = 10 Hz), 120.1, 119.3, 111.53 (d, J = 26 Hz), 1 11.47 (d, J = 10 Hz), 110.8, 106.0 (d, J = 5 Hz), 105.1 (d, J = 23 Hz), 33.2, 15.2. HRMS calcd for (Ci8H15 79BrFN302+H)+ 404.0404, found 404.0406.
(£ a«iZ)-5-bromo-2-hydroxy-N -(l-(5-methoxy-l-methyl-lH-indoI-2-yI)ethylidene) benzohydrazide G6
Figure imgf000063_0002
G6 was prepared from l-(5-methoxy-l -methyl- lH-indol-2-yl)ethanone D2 and 5-bromo-2- hydroxybenzohydrazide E using general Procedure A.
Yield = 64% (filtration), yellow powder. NMR analysis indicated that G6 is present in two isomeric forms (12:88). ]H NMR (600 MHz, DMSO-</6): δ 12.11 (bs, 1H), 1 1.31 (s, 1H), 8.09 (d, J = 2.4 Hz, 1H), 7.59 (dd, J = 8.4 Hz, 2.4 Hz, 1H), 7.41 (d, J = 9.0 Hz, 1H), 7.07 (d, J = 2.4 Hz, 1H), 7.01 (d, J= 9.0 Hz, 1H), 6.94 (s, 1H), 6.90 (dd, J = 8.4 Hz, 2.4 Hz, 1H), 4.09 (s, 3H), 3.77 (s, 3H), 2.39 (s, 3H). 13C NMR (150 MHz, DMSO-i¾): δ 160.5, 155.7, 153.8, 147.7, 136.5, 135.7, 135.0, 132.6, 126.6, 120.2, 1 19.3, 1 13.9, 111.1, 1 10.9, 106.0, 101.8, 55.3, 33.1, 15.2. HRMS calcd for (C19Hi8 79BrN303+H)+ 416.0610, found 416.0614. (£ fl«i Z)-5-bromo-N'-(l-(5,6-difluoro-l-methyl-lH-indol-2-yl)ethylidene)-2- hydroxybenzohydrazide G7
Figure imgf000064_0001
G7 was prepared from l-(5,6-difluoro-l -methyl- lH-indol-2-yl)ethanone D3 and 5-bromo-2- hydroxybenzohydrazide E using general Procedure A.
Yield = 79% (filtration), yellow powder. NMR analysis indicated that G7 is present in two isomeric forms (10:90). 1H NMR (400 MHz, DMSO-<¾: δ 12.09 (bs, IH), 1 1.33 (s, IH), 8.07 (d, J = 2.4 Hz, IH), 7.69-7.57 (m, 3H), 7.03 (s, IH), 7.01 (d, J = 8.8 Hz, IH), 4.10 (s, 3H), 2.39 (s, 3H). 13C NMR (150 MHz, DMSO-d6): δ 160.6, 155.7, 147.9 (dd, J= 239 Hz, 16 Hz), 147.0, 145.6 (dd, J = 235 Hz, 15 Hz), 138.0 (d, J= 4 Hz), 135.7, 135.1 (d, J = 10 Hz), 132.6, 121.5 (d, J= 8 Hz), 120.1, 119.3, 1 10.8, 107.2 (d, J= 19 Hz), 106.3 (d, J= 4 Hz), 98.7 (d, J = 22 Hz), 33.6, 15.0. HRMS calcd for (Ci8Hi4 79BrF2N302+H)+ 422.0310, found 422.0290.
(E a«i/Z)-5-bromo-7V'-(l-(4,5-difluoro-l-methyl-lH-indol-2-yl)ethylidene)-2- hydroxybenzohydrazide G8
Figure imgf000064_0002
G8 was prepared from l-(4,5-difluoro-l -methyl- lH-indol-2-yl)ethanone D4 and 5-bromo-2- hydroxybenzohydrazide E using general Procedure A.
Yield = 83%) (filtration), pale yellow powder. NMR analysis indicated that G8 is present in two isomeric forms (1 1 :89). 1H NMR (400 MHz, DMSO-i¾): δ 12.08 (bs, IH), 11.37 (s, IH), 8.07 (d, J - 2.8 Hz, IH), 7.60 (dd, J = 8.6 Hz, 2.6 Hz, IH), 7.37 (dd, J = 8.8 Hz, 3.2 Hz, IH), 7.33-7.25 (m, IH), 7.17 (s, IH), 7.02 (d, J = 8.8 Hz, IH), 4.13 (s, 3H), 2.43 (s, 3H). 13C NMR (150 MHz, DMSO-<¾: δ 160.6, 155.7, 146.8, 143.3 (dd, J = 232 Hz, 11 Hz), 141.7 (dd, J = 245 Hz, 15 Hz), 138.2, 137.5 (d, J = 9 Hz), 135.8, 132.6, 120.1, 119.3, 1 16.2 (d, J = 18 Hz), 1 12.7 (d, J = 21 Hz), 1 10.8, 106.8 (dd, J = 7 Hz, 4 Hz), 101.6 (d, J= 6 Hz), 33.5, 15.1. HRMS calcd for (Ci8Hi4 79BrF2N302+H)+ 422.0310, found 422.0321. (£ «rf )-5-bromo-2-hydroxy-N'-(l-(4,5,6-trifluoro-l-methyI-lH-indol-2-yl)ethylidene) benzohydrazide G9
Figure imgf000065_0001
G9 was prepared from l-(4,5,6-trifluoro-l-met yl-lH-indol-2-yl)ethanone D5 and 5-bromo- 2-hydroxybenzohydrazide E using general Procedure A.
Yield = 86% (filtration), white powder. NMR analysis indicated that G9 is present in two isomeric forms (6:94). Ή NMR (400 MHz, DMSO-<¾: δ 12.08 (bs, IH), 11.37 (s, IH), 8.06 (d, J = 2.8 Hz, IH), 7.63-7.58 (m, 2H), 7.20 (s, IH), 7.01 (d, J = 8.4 Hz, IH), 4.11 (s, 3H), 2.41 (s, 3H). 13C NMR (150 MHz, OMSO-d6) δ 160.7, 155.9, 148.2 (ddd, J= 238 Hz, 13 Hz, 2 Hz), 146.5, 142.5 (ddd, J = 247 Hz, 11 Hz, 4 Hz), 138.2 (d, J = 3 Hz), 135.8, 134.6 (t, J = 12 Hz), 133.9 (ddd, J = 235 Hz, 18 Hz, 14 Hz), 132.6, 120.1, 1 19.4, 1 11.8 (d, J - 18 Hz), 1 10.8, 101.8 (d, J = 4 Hz), 94.7 (dd, J = 23 Hz, 3 Hz), 33.8, 15.0. HRMS calcd for (C]8H13 79BrF3N302+H)+ 440.0216, found 440.0195.
(E and Z)-5-bromo-2-hydroxy-iV-(l-(l-methyHH-indol-2-yl)ethylidene)
benzohydrazide G10
Figure imgf000065_0002
Br
G10 was prepared from l-(l-methyl-lH-indol-2-yl)ethanone D and 5-bromo-2- hydroxybenzohydrazide E using general Procedure B.
Yield = 53% (filtration), pale yellow solid. NMR analysis indicated that G10 is present in two isomeric forms (9:1). Ή NMR (600 MHz, OMSO-d6): δ 12.09 (s, IH), 1 1.34 (s, IH), 8.08 (d, J = 2.6 Hz, IH), 7.61 - 7.59 (m, 2H), 7.51 (d, J = 8.3 Ηζ,ΙΗ), 7.26 (dt, J = 0.9 Hz, 7.6 Hz, IH), 7.08 (t, J = 7.4 Hz, IH), 7.05 (s, IH), 7.02 (d, J= 8.7 Hz, IH), 4.15 (s, 3H), 2.42 (s, 3H). 13C NMR (150 MHz, DMSO-c/6): δ 161.5, 159.7, 148.6, 140.5, 137.2, 136.6, 133.5, 127.3, 124.3, 121.8, 121.1, 120.7, 120.3, 111.7, 1 11.1, 107.4, 33.9, 16.1. HRMS calcd for (Ci8H16 79BrN302+H)+ 386.0499, found 386.0506.
(E n</Z)-5-bromo-iV-(l-(5-bromo-l-methyl-lH-indol-2-yl)ethylidene)-2- hydroxybenzohydrazide Gil
Figure imgf000066_0001
Gil was prepared from l-(5-bromo-l -methyl- lH-indol-2-yl)ethanone D6 and 2- hydroxybenzohydrazide E using general Procedure C.
Yield = 39% (filtration), off-white solid. NMR analysis indicated that Gil is present in two isomeric forms (9:1). 1H NMR (600 MHz, DMSO- 6): δ 12.12 (bs, IH), 11.49 (bs, IH), 8.06 (d, J = 2.6 Hz, IH), 7.79 (d, J = 1.8 Hz, IH), 7.58 (dd, J = 2.6 Hz, 8.7 Hz, IH), 7.51 (d, J = 8.8 Hz, IH), 7.36 (dd, J= 1.9 Hz, 8.8 Hz, IH), 7.02 - 6.99 (m, 2H), 4.12 (s, 3H), 2.40 (s, 3H). 13C NMR (150 MHz, DMSO- ): δ 161.6, 157.1, 147.9, 139.1, 138.6, 136.7, 133.5, 129.0, 126.5, 123.8, 121.4, 120.4, 113.3, 1 13.1, 11 1.5, 106.5, 34.2, 16.1. HRMS calcd for (Ci8H15 79Br2N302+H)+ 465.9584, found 465.9580.
(E and Z)-2-hydroxy-5-iodo-N'-(l-(l-methyl-lH-indol-2-yl)ethylidene) benzohydrazide G12
Figure imgf000066_0002
G12 was prepared from 1-(1 -methyl- lH-indol-2-yl)ethanone D and 2-hydroxy-5- iodobenzohydrazide E using general Procedure C.
Yield = 43%) (filtration), pale yellow solid. NMR analysis indicated that G12 is present in two isomeric forms (10: 1). 1H NMR (600 MHz, DMSO-c/6): δ 12.07 (bs, IH), 11.36 (s, IH), 8.24 (d, J = 2.3 Hz, IH), 7.72 (dd, J = 2.2 Hz, 8.6 Hz, IH), 7.60 (d, J = 7.8 Hz, IH), 7.51 (d, J = 8.3 Hz, IH), 7.25 (dt, J= 0.8 Hz, 7.6 Hz, IH), 7.08 (t, J= 7.5 Hz, IH), 7.04 (s, IH), 6.88 (d, J = 8.6 Hz, IH), 4.13 (s, 3H), 2.41 (s, 3H). 13C NMR (150 MHz, DMSO- 6): δ 161.1, 156.9, 148.0, 141.9, 140.1, 139.0, 136.8, 126.9, 123.8, 121.4, 121.0, 120.3, 120.2, 110.7, 106.9, 82.1, 33.5, 15.7. HRMS calcd for (C18H16IN302+H)+ 434.0360, found 434.0373.
(^ nrf j-S-bromo-iV-il-iS-chloro-l-methyl-lH-indol-l-y ethyUdeiie)-!- hydroxybenzohydrazide G13
Figure imgf000067_0001
G13 was prepared from l-(5-chloro-l -methyl- lH-indol-2-yl)ethanone D7 and 5-bromo-2- hydroxybenzohydrazide E using general Procedure C.
Yield = 73% (filtration), pale yellow solid. NMR analysis indicated that G13 is present in two isomeric forms (9:1). 1H NMR (600 MHz, DMSO- ): δ 12.10 (bs, 1H), 11.37 (bs, 1H), 8.07 (d, J = 2.4 Hz, 1H), 7.65 (d, J = 1.3 Hz, 1H), 7.60 (dd, J = 2.4 Hz, 8.6 Hz, 1H), 7.55 (d, J = 8.8 Hz, 1H), 7.24 (dd, J= 1.6 Hz, 8.7 Hz, 1H), 7.02 (s, 1H), 7.01 (d, J= 8.4 Hz, 1H), 4.12 (s, 3H), 2.41 (s, 3H). 13C NMR (150 MHz, DMSO-^6): δ 161.6, 156.7, 148.0, 138.9, 138.7, 136.7, 133.5, 128.3, 125.2, 124.1, 121.1, 120.8, 120.3, 112.9, 111.7, 106.6, 34.2, 16.1. HRMS calcd for (C,8H15 81BrClN302+H)+ 422.0088, found 422.0083.
(E and ^^-bromo-l-hydroxy-N'-il-il-methyl-lH-indol- -y^ethylidene)
benzohydrazide G14
Figure imgf000067_0002
G14 was prepared from 1-(1 -methyl- lH-indol-2-yl)ethanone D and 4-bromo-2- hydroxybenzohydrazide E using general Procedure C.
Yield = 36% (filtration), pale yellow solid. NMR analysis indicated that G14 is present in two isomeric forms (12: 1). 1H NMR (600 MHz, DMSO- 6): δ 12.24 (bs, 1H), 11.25 (s, 1H), 7.93 (d, J= 8.3 Hz, 1H), 7.60 (d, J= 7.9 Hz, 1H), 7.50 (d, J= 8.3 Hz, 1H), 7.25 (t, J= 7.8 Hz, 1H), 7.22 - 7.20 (m, 2H), 7.08 (t, J = 7.5 Hz, 1H), 7.04 (s, 1H), 4.13 (s, 3H), 2.41 (s, 3H). 13C NMR (150 MHz, DMSO-< 6): δ 162.0, 158.1, 148.3, 140.5, 137.3, 133.4, 127.3, 126.9, 124.2, 123.7, 121.8, 120.7, 120.3, 1 18.6, 1 1 1.1 , 107.3, 33.9, 16.1. HUMS calcd (C18H16 79BrN302+H)+ 386.0499, found 386.0499.
(E and Z)-5-bromo-2-hydroxy-4-methoxy-N'-(l-(l-methyl-lH-indol-2-yl)ethylidene) benzohydrazide G15
Figure imgf000068_0001
G15 was prepared from l-(l-methyl-lH-indol-2-yl)ethanone D and 5-bromo-2-hydroxy-4- methoxybenzohydrazide E using general Procedure C.
Yield = 80% (filtered), off-white solid. NMR analysis indicated that G15 is present in two isomeric forms (25:1). 1H NMR (600 MHz, DMSO-<¾): δ 12.24 (bs, 1H), 11.19 (s, 1H), 8.16 (s, 1H), 7.60 (d, J= 7.8 Hz, 1H), 7.50 (d, J= 8.4 Hz, 1H), 7.25 (t, J= 7.4 Hz, 1H), 7.07 (t, J = 7.4 Hz, 1H), 7.03 (s, 1H), 6.69 (s, 1H), 4.13 (s, 3H), 3.88 (s, 3H), 2.42 (s, 3H). 13C NMR (150 MHz, DMSO- ): δ 161.9, 159.4, 159.1 , 148.1, 140.0, 136.9, 134.2, 126.9, 123.8, 121.3, 120.2, 111.8, 1 10.7, 106.8, 101.4, 101.1, 57.0, 33.5, 15.8. HRMS calcd for (C19Hi8 79BrN303+H)+ 416.0604, found 416.0599.
{E and Z)-N -(l-(benzo[</]oxazol-2- I)ethylidene)-5-bromo-2-hydroxybenzohydrazide II
Figure imgf000068_0002
II was prepared from l-(benzo[<i]oxazol-2-yl)ethanone H and 5-bromo-2- hydroxybenzohydrazide E using general Procedure A.
Yield = 89% (filtration), white cotton. NMR analysis indicated that II is present in two isomeric forms (26:74).
Major isomer
1H NMR (400 MHz, DMSO- ¾: 512.18 (bs, 1H), 1 1.63 (s, 1H), 8.06 (s, 1H), 7.86-7.82 (m, 2H), 7.62 (dd, J = 8.8 Hz, 2.0 Hz, 1H), 7.50 (t, J = 7.4 Hz, 1H), 7.44 (t, J = 7.6 Hz, 1H), 7.03 (d, J = 8.8 Hz, 1H), 2.49 (s, 3H). 13C NMR (150 MHz, DMSO-<¾: δ 160.8, 160.7, 155.7,
150.4, 141.3, 140.9, 136.2, 132.9, 126.6, 125.0, 120.3, 120.0, 119.4, 111.2, 110.9, 13.3.
Minor isomer
13C NMR (150 MHz, DMSO-d6): δ 161.2, 155.8, 155.7, 148.1, 139.9, 136.2, 134.3, 133.3,
127.5, 125.6, 120.7, 120.0, 1 19.3, 11 1.7, 110.7, 20.2. HRMS calcd for (Ci6H12 79BrN303+H)+ 374.0135, found 374.0142.
(E and Z)-N '-(l-(benzo[< ]thiazol-2- I)ethylidene)-5-broino-2-hydroxybenzohydrazide 12
Figure imgf000069_0001
12 was prepared from l-(benzo[c/]thiazol-2-yl)ethanone H and 5-bromo-2- hydroxybenzohydrazide E using general Procedure A.
Yield = 89% (filtration), white powder. NMR analysis indicated that 12 is present in two isomeric forms (6:94). 1H NMR (600 MHz, DMSC-c^): 512.13 (bs, 1H), 11.59 (s, 1H), 8.11 (d, J = 7.8 Hz, 1H), 8.06-8.03 (m, 2H), 7.61 (dd, J = 8.4 Hz, 2.4 Hz, 1H), 7.54 (t, J = 7.5 Hz, 1H), 7.49 (t, J = 7.2 Hz, 1H), 7.02 (d, J = 9.0 Hz, 1H), 2.53 (s, 3H). 13C NMR (150 MHz, DMSO-i/6): δ 167.8, 160.5, 155.6, 152.9, 148.4, 136.1, 135.1, 132.8, 126.5, 126.4, 123.3, 122.4, 120.1, 119.4, 110.9, 12.9. HRMS calcd for (C] 6H12 79BrN302S+H)+ 389.9912, found 389.9908.
(E)-A^-(l-(benzo[b]thiophen-2-yl)ethylidene)-5-bromo-2-hydroxybenzohydrazide 13
Figure imgf000069_0002
13 was prepared from 2-acetylbenzothiophene H and 5~bromo-2-hydroxybenzohydrazide E using general Procedure A.
Yield = 65% (filtration), white solid. Mp = 301-302°C. 1H NMR (600 MHz, DMSO- ): δ 12.06 (bs, 1H), 1 1.34 (s, 1H), 8.04 (d, J = 2.6 Hz, 1H), 7.94 (d, J = 7.2 Hz, 1H), 7.91 (s, 1H), 7.86 (dd, J= 1.6 Hz, 6.9 Hz, 1H), 7.59 (dd, J= 2.6 Hz, 8.7 Hz, 1H), 7.41 - 7.37 (m, 2H), 7.01 (d, J = 8.7 Hz, 1H), 2.44 (s, 3H). 1JC NMR (150 MHz, DMSO-d6): δ 161.3, 156.6, 150.2, 144.2, 140.8, 140.4, 136.7, 133.5, 126.8, 126.3, 125.6, 125.2, 123.4, 121.1, 120.2, 111.8, 14.8. HRMS calcd for (Ci7Hi3 79BrN202S+H)+ 388.9954, found 388.9955.
(^ flMi/ZJ-S-bromo^-hydroxy-A^-il-il-methyl-lH^enzoIdJimidazol- -y^ethylidene) benzohydrazide 14
Figure imgf000070_0001
14 was prepared from l-(l-methyl-lH-benzo[d]imidazol-2-yl)ethanone H and 5-bromo-2- hydroxybenzohydrazide E using general Procedure B.
Yield = 47% (filtration), pale yellow solid. NMR analysis indicated that 14 is present in two isomeric forms (9:1). 1H NMR (600 MHz, DMSO-J6): δ 11.83 (bs, 1H), 8.06 (d, J = 2.5 Hz, 1H), 7.71 (d, J = 8.0 Hz, 1H), 7.62 (d, J = 8.1 Ηζ,ΙΗ), 7.58 (dd, J= 8.7 Hz, 2.3 Hz, 1H), 7.35 (t, J = 7.5 Hz, 1H), 7.26 (d, J = 7.3 Hz, 1H), 7.00 (d, J = 8.7 Hz, 1H), 4.1 1 (s, 3H), 2.55 (s, 3H). 13C NMR (150 MHz, DMSO- 6): δ 161.9, 157.6, 149.7, 147.3, 142.6, 138.3, 136.8, 133.6, 124.6, 123.2, 121.0, 120.6, 120.4, 11 1.6, 34.1, 15.1. HRMS calcd for (CnHi5 79BrN402+H)+ 387.0451, found 387.0467.
Specific procedures for the synthesis of the hydrazides F
(E)-N,-(l-(lH-indol-2-yI)ethyIidene)-3,5-dibromo-2-methoxybenzohydrazide F24
Figure imgf000070_0002
A mixture of l-(lH-indol-2-yl)ethanone C (50 mg, 0.31 mmol) and 3,5-dibromo-2- methoxybenzohydrazide E (101 mg, 0.31 mmol) in EtOH (2.5 mL) was irradiated with microwaves at 180°C for 3 h. The precipitate was filtered and was washed with hot EtOH (2x2mL) to give pale yellow solid (75 mg, 51%). NMR analysis indicated that F24 is present in two isomeric forms (15:1). 1H NMR (600 MHz, DMSO-i 6): δ 11.30 (bs, 1H), 10.97 (s, 1H), 8.06 (d, J = 2.4 Hz, 1H), 7.79 (d, J = 7.4 Hz, 1H), 7.56 (d, J = 7.9 Hz, 1H), 7.49 (d, J = 8.2 Hz, 1H), 7.14 (t, J = 7.9 Hz, 1H), 7.00 (t, J = 7.7 Hz, 1H), 6.97 (d, J = 1.9 Hz, 1H), 3.86 (s, 3H), 2.36 (s, 3H). 13C NMR (150 MHz, DMSO-c¾): δ 160.9, 154.1, 149.5, 138.2, 137.2, 136.2, 133.3, 132.1, 128.0, 123.7, 121.2, 119.8, 118.8, 117.0, 112.7, 105.4, 62.6, 14.7. HRMS calcd for (Ci8H15 79Br2N302+H)+ 465.9584, found 465.9597.
(jE)-N,-(l-(lH-indol-2-yl)ethylidene)-5-azido-2-( ro -2- n loxy)benzohydrazide F25
Figure imgf000071_0001
To a stirred mixture of l-(lH-indol-2-yl)ethanone C (40 mg, 0.25 mmol) and E2 (40 mg, 0.17 mg) in THF (6 mL) was added concentrated hydrochloric acid (5 μL). The resulting mixture was stirred at ambient temperature for 3 days, filtered and the filtrate was concentrated. The residue was recrystallized from hot MeOH to give a yellow solid (39 mg, 61%). Mp = decomposes > 160°C. ]H NMR (600 MHz, DMSO-<¼): δ 1 1.30 (s, 1H), 10.81 (s, 1H), 7.56 (s, 1H), 7.56 (d, J = 8.6 Hz, 1H), 7.49 (d, J = 8.2 Hz, 1H), 7.35 (s, 1H), 7.35 (d, J = 2.1 Hz, 1H), 7.14 (dt, J= 1.0 Hz, 7.1 Hz, 1H), 7.00 (dt, J = 0.9 Hz, 7.1 Hz, 1H), 6.98 (d, J = 1.6 Hz, 1H), 5.05 (d, J = 2.4 Hz, 1H), 3.75 (t, J = 2.4 Hz, 1H), 2.40 (s, 3H). 13C NMR (150 MHz, DMSO- d6) δ 160.6, 152.8, 148.1, 138.2, 136.3, 133.5, 128.1 , 124.7, 123.6, 123.5, 121.4, 121.1, 119.8, 116.1, 112.7, 105.3, 80.0, 79.0, 57.9, 14.5. HRMS calcd for (C20Hi6N6O2+H)+ 373.1408, found 373.1414.
(£)-7Y,-(l-(5-azido-lH-indoI-2-yl)ethylidene)-5-bromo-2-(prop-2-ynyIoxy)
benzohydrazide F26
Figure imgf000071_0002
A mixture of C7 (50 mg, 0.25 mmol), hydrazine hydrate (40 iL, 0.81 mmol) and AcOH (2 drops) in EtOH (3 mL) was refluxed for 3 h and then concentrated. The crude intermediate was purified by flash chromatography (DCM/MeOH; 20/1) to give the corresponding hydrazide. In a separate flask, 5-bromo-2-(prop-2-ynyloxy)benzoic acid (70 mg, 0.27 mmol) was stirred in a mixture of SOCl2 (2 mL) and DMF (1 drop) for 16 h and then concentrated. The residue was dissolved in DCM (2 mL) followed by the addition of the above hydrazide (40 mg, 0.19 mmol) in DCM (4 mL) at 0°C. The reaction mixture was stirred at 0°C to rt for 2 h and concentrated. The crude product was purified by flash chromatography (DCM/MeOH; 40/1) to give a yellow solid (32 mg, 38%). Mp = decomposes > 160°C. 1H NMR (600 MHz, DMSO- ): δ 11.46 (s, 1H), 10.80 (s, 1H), 7.92 (d, J = 2.6 Hz, 1H), 7.75 (dd, J = 2.6 Hz, 8.9 Hz, 1H), 7.52 (d, J= 8.7 Hz, 1H), 7.31 (d, J= 2.0 Hz, 1H), 7.26 (d, J= 8.9 Hz, 1H), 6.95 (d J = 1.4 Hz, 1H), 6.91 (dd, J = 2.2 Hz, 8.7 Hz, 1H), 5.03 (d, J= 2.4 Hz, 1H), 3.75 (t, J = 2.4 Hz, 1H), 2.38 (s, 3H). 13C NMR (150 MHz, DMSO- ): δ 160.4, 154.8, 147.9, 137.8, 136.0, 135.3, 133.2, 131.3, 128.8, 125.7, 1 16.6, 1 15.8, 1 14.1, 1 13.6, 1 10.4, 104.7, 80.0, 78.8, 57.7, 14.5. HRMS calcd for (C2oHi5 81BrN602+H)+ 453.0495, found 453.0497.
5-chloro-2-hydroxy-iV'-((l^)-l- -indoI-2-yI)ethylideiie)benzohydrazide F27
Figure imgf000072_0001
To a mixture of methyl 5-chloro-2-hydroxybenzoate (162 mg, 0.87 mmol) in isobutanol (0.5 mL) was added hydrazine hydrate (42 \iL, 0.87 mmol). The reaction mixture was irradiated with microwaves for 15 min at 1 15°C. Isobutanol was added and the supernatant was removed. The solid hydrazide E was taken up in isobutanol (0.5 mL) and l-(7H-indol-2- yl)ethanone C (138 mg, 0.87 mmol) was added. The reaction mixture was again irradiated with microwaves for 10 min at 1 10°C. The solid product was filtered and then, triturated with EtOH and Et20 to afford F27 as a yellow solid (90 mg, 33%). Mp = 198-200°C. !H NMR (400 MHz, OMSO-de): δ 12.46 (s, 1H), 1 1.39 (s, 1H), 10.07 (s, 1H), 7.86 (d, J= 2.4 Hz, 1H), 7.60 (d, J = 7.6 Hz, 1H), 7.47 (d, J - 7.6 Hz, 1H), 7.41 (dd, J = 8.8 Hz, 2.4 Hz, 1H), 7.19 (tm, J = 7.6 Hz), 7.03 (m, 2H), 6.93 (d, J = 8.8 Hz, 1H), 2.48 (s, 3H). 13C NMR (100 MHz, OMSO-de): δ 158.0, 153.8, 137.5, 136.5, 132.8, 127.7, 126.8, 123.4, 122.3, 120.9, 119.3, 119.1, 111.8, 104.9, 14.5. HRMS calcd for (Ci7H14 35ClN302+Na)+ 350.0663, found 350.0667. 5-bromo-2-(ethoxymethoxy)-iV-((lE)-l-(liy-indol-2-yl)ethylidene)benzohydrazide F28
Figure imgf000073_0001
To a solution of F15 (73 mg, 0.20 mmol) in anhydrous THF (30 mL) maintained at 0°C were added, under an argon atmosphere, NaH (8.1 mg, 60%, 0.24 mmol) and then, after 10 minutes stirring still at 0°C, chloromethyl ethyl ether (45 μΐ,, 0.49 mmol). The mixture was stirred at room temperature for 4 h and then, quenched with a saturated aqueous ammonium chloride solution. The aqueous layer was extracted with EtOAc and the organic layer was dried over anhydrous Na2SC>4, filtered through a silica gel pad and evaporated under reduced pressure. The residue was triturated with Et20 to afford F28 as a pale yellow solid (65 mg, 77%). Mp - 128-130°C. Ή NMR (400 MHz, DMSO- ): δ 11.32 (s, 1H), 10.90 (s, 1H), 7.94 (d, J = 2.5 Hz, 1H), 7.71 (dd, J = 9.0 Hz, 2.5 Hz, 1H), 7.57 (d, J = 7.6 Hz, 1H), 7.50 (d, J = 7.6 Hz, 1H), 7.29 (d, J = 9.0 Hz, 1H), 7.15 (t, J = 7.6 Hz, 1H), 7.00 (t, J = 7.6 Hz, 1H), 6.98 (s, 1H), 5.46 (s, 2H), 3.75 (q, J= 7.0 Hz, 2H), 2.39 (s, 3H), 1.16 (t, J= 7.0 Hz, 3H). 13C NMR (100 MHz, DMSO-c¾): 5160.0, 153.9, 147.6, 137.7, 135.8, 134.7, 132.5, 127.6, 125.6, 123.1, 120.6, 119.3, 117.6, 113.2, 112.1, 104.8, 93.9, 4.5, 14.9, 13.8. HRMS calcd for (C2oH2o79BrN303+Na)+ 453.0562, found 453.0561.
(£)-5-bromo-A^-(l-(6-bromo- -indol-2-yI)ethylidene)-2-hydroxybenzohydrazide F29
Figure imgf000073_0002
F29 was prepared from l-(6-bromo-lH-indol-2-yl)ethanone CIO and 5-bromo-2- hydroxybenzohydrazide E using general Procedure C.
Yield = 50% (after reverse-phase HPLC (ACN/H2O-0.1 % TFA, 80 - 100% for 15 min, 20 mL/min, 254 nm detection for 18 min), pale yellow solid. Mp = 338-340°C. 1H NMR (600 MHz, DMSO-</6): 6 12.17 (bs, 1H), 1 1.58 (bs, 1H), 1 1.44 (s, 1H), 8.07 (d, J = 2.3 Hz, 1H), 7.66 (s, 1H), 7.57 (dd, J = 2.4 Hz, 8.6 Hz, 1H), 7.52 (d, J= 8.4 Hz, 1H), 7.13 (dd, J= 1.8 Hz, 8.4 Hz, 1H), 7.00 (s, 1H), 6.99 (d, J = 9.3 Hz, 1H), 2.37 (s, 3H). 13C NMR (150 MHz, DMSO- ): δ 160.5, 156.3, 146.5, 138.5, 136.7, 135.6, 132.5, 126.6, 122.4, 122.2, 120.2, 1 19.5, 115.8, 114.5, 110.3, 104.8, 13.7. HRMS calcd for (C17H13 79Br2N302+H)+ 451.9428, found 451.9410.
(£)-5-bromo-2-hydroxy-N,-(l-(5-hydroxy-l£T-indol-2-yl)ethylidene)benzohydrazide F30
Figure imgf000074_0001
F30 was prepared from l-(5-hydroxy-lH-indol-2-yl)ethanone Cll and 5-bromo-2- hydroxybenzohydrazide E using general Procedure C.
Yield = 65% (filtration), pale yellow solid. Mp = 190-192°C. 1H NMR (600 MHz, DMSO- <¾): δ 12.10 (bs, 1H), 1 1.27 (s, 1H), 1 1.01 (s, 1H), 8.71 (s, 1H), 8.09 (d, .7 = 2.3 Hz, 1H), 7.59 (dd, J = 2.5 Hz, 8.7 Hz, 1H), 7.28 (d, J - 8.7 Hz, 1H), 7.01 (d, J = 8.7 Hz, 1H), 6.85 (d, J = 1.6 Hz, lH), 6.79 (s, 1H), 6.68 (dd, J = 1.9 Hz, 8.7 Hz, 1H), 2.34 (s, 3H). 13C NMR (150 MHz, DMSO-< 6): δ 160.2, 155.7, 150.8, 147.6, 135.8, 135.6, 132.5, 132.4, 128.2, 120.3, 119.3, 114.0, 112.6, 1 10.8, 104.1, 103.9, 13.8. HRMS calcd for (Ci7Hi4 7 BrN303+H)+ 388.0291 , found 388.0284.
(JE)-A,-(l-(5-bromo-lH-ind -2-yl)propylidene)-3-hydroxy-2-naphthohydrazide F31
Figure imgf000074_0002
F31 was prepared from l-(5-bromo-lH-indol-2-yl)propan-l-one C12 and 3-hydroxy-2- naphthohydrazide E using general Procedure C.
Yield = 51 % (filtration), pale brown solid. Mp = 258-260°C. Ή NMR (600 MHz, DMSO-i/6): δ 11.84 (s, 1H), 1 1.74 (s, 1H), 1 1.50 (s, 1H), 8.68 (s, 1H), 8.02 (d, J= 8.2 Hz, 1H), 7.79 (d, J = 8.2 Hz, 1H), 7.76 (d, J= 1.1 Hz, 1H), 7.53 (t, J = 7.2 Hz, 1H), 7.48 (d, J= 8.6 Hz, 1H), 7.39 (s, 1H), 7.38 (t, J = 7.8 Hz, 1H), 7.27 (dd, J = 1.7 Hz, 8.6 Hz, 1H), 6.97 (s, 1H), 2.68 (q, J = 7.5 Hz, 2H), 1.27 (t, J= 7.6 Hz,31H). 13C NMR (150 MHz, DMSO-< 6): δ 161.0, 152.4, 150.4, 136.4, 136.3, 135.7, 132.5, 129.5, 128.9, 128.3, 127.3, 125.7, 125.5, 123.9, 122.7, 120.8, 114.1, 111.7, 1 10.7, 103.5, 20.6, 10.8. HRMS calcd for (C22Hig 79BrN302+H)+ 438.0637, found 438.0645.
(£)-iV-(l-(5-bromo-lH-mdol-2-yl)ethylidene)-3-hydroxy-2-naphthohydrazide F32
Figure imgf000075_0001
F32 was prepared from l-(5-bromo-lH-indol-2-yl)ethanone C and 3-hydroxy-2- naphthohydrazide E using general Procedure C.
Yield = 83% (filtration), pale brown solid. Mp = 286-288°C. 1H NMR (600 MHz, DMSO-i 6): δ 1 1.79 (bs, 1H), 11.61 (s, 1H), 11.55 (s, 1H), 8.67 (s, 1H), 8.01 (d, J = 8.2 Hz, 1H), 7.79 (d, J = 8.3 Hz, 1H), 7.76 (s, 1 H), 7.53 (t, J= 7.3 Hz, 1H), 7.47 (d, J = 8.6 Hz, 1H), 7.38 - 7.36 (m, 2H), 7.27 (dd, J = 1.4 Hz, 8.6 Hz, 1H), 6.97 (s, 1H), 2.42 (s, 3H). 13C NMR (150 MHz, DMSO- ): δ 161.2, 152.6, 146.2, 137.2, 136.3, 135.7, 132.3, 129.4, 128.9, 128.3, 127.2, 125.7, 125.5, 123.9, 122.7, 120.7, 1 14.1, 1 1 1.7, 1 10.7, 103.9, 13.8. HRMS calcd for (C2!H16 79BrN302+H)+ 424.0481, found 424.0495.
(E)-5-bromo-N,-(l-(5-bromo- -indol-2-yI)propylidene)-2-hydroxybenzohydrazide F33
Figure imgf000075_0002
F33 was prepared from l-(5-bromo-lH-indol-2-yl)propan-l-one C12 and 5-bromo-2- hydroxybenzohydrazide E using general Procedure C.
Yield = 41% (after flash chromatography 20/1 ; DCM/MeOH), pale yellow solid. Mp = 252- 254°C. Ή NMR (600 MHz, DMSO- 6): δ 12.17 (bs, 1H), 11.60 (bs, 1H), 11.48 (s, 1H), 8.08 (d, J = 2.5 Hz, 1H), 7.75 (d, J = 1.4 Hz, 1H), 7.58 (dd, J = 2.6 Hz, 8.7 Hz, 1H), 7.46 (d, J = 8.6 Hz, 1H), 7.26 (dd, J = 1.9 Hz, 8.6 Hz, 1H), 7.02 (d, J= 8.7 Hz, 1H), 6.95 (s, 1H), 2.81 (q, J= 7.7 Hz, 2H), 1.22 (t, J= 7.6 Hz, 3H).13C NMR (150 MHz, DMSO-<6): δ 160.1, 155.7, 150.7, 136.4, 136.2, 135.6, 132.7, 129.5, 125.5, 122.7, 120.5, 119.4, 114.1, 111.7, 110.7, 103.6, 20.6, 10.7. HRMS calcd for (ClgH15 79Br2N302+H)+ 465.9584, found 465.9588.
(£)-N'-[l-(lH-indol-2-yl)ethylidene]-2-hydroxy-l-naphthohydrazide F34
Figure imgf000076_0001
F34 was prepared from l-(7H-indol-2-yl)ethanone C and 2-hydroxy-l-naphthohydrazide E using general Procedure D.
Yield: 77%, white solid. Mp = 278-280 °C.1H NMR (400 MHz, DMSO- ): δ 11.36 (s, 1H), 10.93 (s, 1H), 10.23 (s, 1H), 7.86 (d, J= 7.6 Hz, 1H), 7.83 (d, J= 7.4 Hz, 1H), 7.58 (d, J= 7.4 Hz, 1H), 7.53 (d, J= 7.4 Hz, 1H), 7.49 (tm, J= 7.4 Hz, 1H), 7.34 (tm, J= 7.4 Hz, 1H), 7.26 (d, J= 9.0 Hz, 1H), 7.16 (tm, J= 7.4 Hz, 1H), 7.02 (tm, J= 7.4 Hz, 1H), 6.94 (s, 1H), 2.34 (s, 3H). J = 7.6 Hz, 1H).13C NMR (100 MHz, DMSC /6): δ 163.7, 153.1, 148.2, 138.1, 136.7, 132.6, 130.9, 128.4, 128.1, 128.0, 127.4, 124.1, 123.5, 123.4, 121.1, 119.7, 118.9, 117.0, 112.6, 104.8, 14.8. HRMS calcd for (C2iH17N302+H)+ 344.1394, found 344.1407.
5-bromo-2-hydroxy-7Vr-{(l£)-l-[5-(trifluoromethyl)-lH-indol-2- yl]ethylidene}benzohydrazide F35
Figure imgf000076_0002
F35 was prepared from l-[5-(trifluoromethyl)-lH-indol-2-yl]ethanone C8 and 5-bromo-2- hydroxybenzohydrazide E using general Procedure D.
Yield: 90%, beige solid. Mp = 268-270 °C. Ή NMR (500 MHz, DMSO-<¾): δ 12.14 (s, IH), 11.79 (s, IH), 11.42 (s, IH), 8.09 (d, J= 2.7 Hz, s), 7.97 (d, J= 1.4 Hz, IH), 7.67 (d, J = 8.7 Hz, IH), 7.60 (dd, J= 8.7 Hz, J= 2.7 Hz, IH), 7.44 (dd, J = 8.7 Hz, J= 1.4 Hz, IH), 7.14 (m, IH), 7.02 (d, J = 8.7 Hz, IH), 2.41 (s, 3H). 13C NMR (100 MHz, DMSO-</6): δ 160.9, 156.3, 147.0, 139.6, 138.4, 136.2, 133.1, 127.4, 125.9 (q, J= 271 Hz, 1C), 120.6 (m, 1C), 119.8 (m, 1C), 118.8 (m, 1C), 113.3, 111.2, 105.8, 14.2. HRMS calcd for (C18H13 79BrF3N30-H)" 438.0070, found 438.0089.
5-bromo-2-hydroxy-N -[(lE)- -(5-iodo-lH-indoI-2-yl)ethylidene]benzohydrazide F36
Figure imgf000077_0001
F36 was prepared from l-(5-iodo-lH-indol-2-yl)ethanone C9 and 5-bromo-2- hydroxybenzohydrazide E using general Procedure D.
Yield: 64%, light brown solid. Mp = 272-274 °C. 1H NMR (500 MHz, DMSO- ): δ 12.12 (s, IH), 11.52 (s, IH), 11.37 (s, IH), 8.09 (d, J= 2.5 Hz, IH), 7.94 (m, IH), 7.60 (dd, J= 8.8 Hz, J= 2.5 Hz, IH), 7.41 (dd, J= 8.6 Hz, J= 1.4 Hz, IH), 7.34 (d, J= 8.6 Hz, IH), 7.02 (d, J= 8.8 Hz, IH), 6.94 (s, IH), 2.38 (s, 3H). 13C NMR (100 MHz, DMSO- 6): δ 160.8, 156.2, 147.2, 137.2, 137.1, 136.2, 133.0, 131.5, 130.8, 129.4, 120.6, 1 19.8, 1 15.1, 11 1.3, 104.2, 83.4, 14.3. HRMS calcd for (C17Hi3 79BriN302-Hy 495.9163, found 495.9143.
4-hydroxy-N'-[(lJE)-l-(lH-in yl-3-carbohydrazide F37
Figure imgf000077_0002
F37 was prepared from l-(7H-indol-2-yl)ethanone C and hydrazide E of methyl 4-hydroxy- l,l'-biphenyl-3-carboxylate using general Procedure E.
Yield: 22%, off-white solid. Mp = 274-276 °C. 1H NMR (400 MHz, DMSO- ): δ 12.02 (bs, 1H), 11.48 (s, 1H), 11.36 (s, 1H), 8.28 (d, J= 2.3 Hz, 1H), 7.76 (dd, J- 8.6 Hz, J = 2.3 Hz, 1H), 7.66 (d, J= 7.3 Hz, 2H), 7.57 (d, J= 7.6 Hz, 1H), 7.50 (d, J= 7.6 Hz, 1H), 7.48 (d, J= 7.3 Hz, 2H), 7.35 (t, J= 7.3 Hz, 1H), 7.16 (t, J= 7.6 Hz, 1H), 7.14 (d, J= 8.6 Hz, 1H), 7.00 (t, J= 7.6 Hz, 1H), 6.90 (bs, 1H), 2.42 (s, 3H). 13C NMR (100 MHz, DMSO- ): δ 162.3, 156.8, 147.9, 139.8, 138.2, 136.3, 132.2, 132.0, 129.4, 128.8, 128.1, 127.5, 126.7, 123.6, 121.1, 119.8, 118.7, 118.1, 112.6, 105.2, 14.4. HRMS calcd for (C23H19N302+H)+ 370.1550, found 370.1416.
(£)-N'-[l-(lH-indol-2-yl)ethylidene]-5-bromo-2-ethoxybenzohydrazide F38
Figure imgf000078_0001
F38 was prepared from l-(iH-indol-2-yl)ethanone C and hydrazide E of methyl 5-bromo-2- ethoxybenzoate using general Procedure E.
Yield: 84%, beige solid. Mp = 260-262 °C. 1H NMR (400 MHz, DMSO- ): δ 11.32 (bs, 1H), 10.81 (s, 1H), 8.01 (d, J= 2.3 Hz, 1H), 7.71 (dd, J= 8.9 Hz, J= 2.3 Hz, 1H), 7.56 (d, J= 7.6 Hz, 1H), 7.49 (d, J= 7.6 Hz, 1H), 7.22 (d, J= 8.9 Hz, 1H), 7.14 (d, J= 7.6 Hz, 1H), 6.99 (t, J= 7.6 Hz, 1H), 6.98 (s, 1H), 4.26 (q, J= 7.0 Hz, 2H), 2.39 (s, 3H), 1.47 (t, J- 7.0 Hz, 3H). 13C NMR (100 MHz, DMSO-<¾): δ 159.8, 155.7, 147.1, 137.7, 135.8, 135.2, 133.0, 127.5, 123.8, 123.1, 120.6, 119.3, 1 15.5, 112.2, 112.1, 104.8, 65.2, 14.6, 13.9. HRMS calcd for (Ci9Hi8 79BrN30 +Na)+ 422.0475, found 422.0464.
(£)-N -[l-(lH-indol-2-yI)ethylidene]-2-hydroxy-3,5-diisopropyIbenzohydrazide F39
Figure imgf000079_0001
F39 was prepared from 1 -(7H-indol-2-yl)ethanone C and hydrazide E of ethyl 2-hydroxy-3,5- diisopropylbenzoate using general Procedure E.
Yield: 80%, yellow solid. Mp = 106-108 °C. 1H NMR (400 MHz, DMSO- ): δ 12.35 (s, IH), 11.40 (s, IH), 1 1.12 (s, IH), 7.67 (d, J- 2.3 Hz, IH), 7.59 (d, J= 7.6 Hz, IH), 7.49 (d, J= 7.6 Hz, IH), 7.29 (IH, J= 2.3 Hz, IH), 7.17 (t, J= 7.6 Hz, IH), 7.04 (bs, IH), 7.02 (t, J = 7.6 Hz, IH), 3.30 (sept., J= 7.0 Hz, IH), 2.89 (spet, J= 7.0 Hz, IH), 1.24 (d, J= 7.0 Hz, 6H), 1.22 (d, J= 7.0 Hz, 6H). 13C NMR (100 MHz, DMSO- 6): δ 166.9, 156.4, 144.2, 138.8, 138.1, 136.6, 136.0, 129.1, 128.0, 123.9, 123.0, 121.3, 119.9, 114.1, 112.6, 105.8, 33.5, 26.7, 22.9, 19.0, 15.2. HRMS calcd for (Ci9H18 79BrN302+Na)+ 400.1995, found 400.1999.
Specific procedures for the synthesis of the hydrazides F
(£)- N -[(lH-indoI-2-yI)methylene]-5-bromo-2-methoxybenzohydrazide F40
Figure imgf000079_0002
To a mixture of methyl 5-bromo-2-methoxybenzoate (1.35 g, 5.50 mmol, 1.0 eq.) in ethanol (0.6 ml) was added hydrazine hydrate (535 μΐ,, 11.00 mmol, 2.0 eq.). The reaction mixture was irradiated with microwaves for 40 minutes at 100°C. Ethanol was added and the supernatant was removed. The solid hydrazide E was taken up in ethanol (5 mL) and 1H- indole-2-carbaldehyde (0.80 g, 5.50 mmol) was added. The reaction mixture was again irradiated with microwaves for 50 minutes at 100°C. The solid product was filtered and then, triturated with ethanol and diethyl ether to afford F35 (1.49 g). Yield: 73%, pale yellow solid. Mp = 108-1 10 °C. !H NMR (400 MHz, DMSO-c¾): δ 1 1.59 (s, 1H), 1 1.55 (s, 1H), 8.36 (s, 1H), 7.74 (d, J= 2.3 Hz, 1H), 7.68 (dd, J= 8.9 Hz, J= 2.3 Hz, 1H), 7.56 (d, J= 7.6 Hz, 1H), 7.44 (d, J= 7.6 Hz, 1H), 7.16 (m, 1H), 7.16 (d, J= 8.9 Hz, 1H), 7.01 (t, J= 7.6 Hz, 1H), 6.83 (bs, 1H), 3.89 (s, 3H). 1 C NMR (100 MHz, DMSO-i/6): δ 160.6, 156.0, 140.5, 137.9, 134.4, 133.0, 131.9, 127.6, 125.7, 123.3, 120.7, 119.5, 114.4, 112.0, 111.8, 107.0, 56.2. HRMS calcd for (C17H14 79BrN302+Na)+ 394.0162, found 394.0171.
(^ awi Zi-S-bromo- V-il-ie-bromo-l-methyl-lH-indol-l- eth lidene)-!- hydroxybenzohydrazide
Figure imgf000080_0001
G16 was prepared from l-(6-bromo-l -methyl- lH-indol-2-yl)ethanone D8 and 5-bromo-2- hydroxybenzohydrazide E using general Procedure C.
Yield = 74% (after reverse-phase HPLC (ACN/H2O-0.1% TFA, 80 - 100% for 15 min, 20 mL/min, 254 nm detection for 18 min)), yellow solid. NMR analysis indicated that G16 is present in two isomeric forms (11 :1). Ή NMR (600 MHz, DMSO- 6): δ 12.14 (s, 1H), 8.02 (d, J = 2.3 Hz, 1H), 7.77 (s, 1H), 7.55 (d, J = 8.4 Hz, 1H), 7.51 (dd, J = 2.1 Hz, 8.6 Hz, 1H), 7.19 (dd, J = 1.3 Hz, 8.2 Hz, 1H), 7.04 (s, 1H), 6.93 (d, J= 8.7 Hz, 1H), 4.12 (s, 3H), 2.39 (s, 3H). 13C NMR (150 MHz, DMSO-<¾): δ 161.2, 158.1, 146.4, 140.3, 137.4, 135.4, 132.3, 125.4, 122.6, 122.5, 120.1, 120.0, 116.0, 113.0, 109.0, 106.1, 33.3, 15.2. HRMS calcd for (C18Hi5 79Br2N302+H)+ 463.9604, found 463.9594.
(E and Z)-5-bromo-2-hydroxy-Nf-(l-(5-hydroxy-l-methyI-lH-indol-2- yl)ethylidene)benzohydrazi
Figure imgf000080_0002
G17 was prepared from l-(5-hydroxy-l-methyl-lH-indol-2-yl)ethanone D10 and 5-bromo-2- hydroxybenzohydrazide E using general Procedure C. Yield = 75% (after reverse-phase HPLC (ACN/H2O-0.1% TFA, 80 - 100% for 15 min, 20 mL/min, 254 ran detection for 18 min)), yellow solid. NMR analysis indicated that G17 is present in two isomeric forms (9:1). 1H NMR (600 MHz, DMSO- 6): δ 12.13 (bs, 1H), 11.60 (bs, 1H), 8.83 (s, 1H), 8.05 (d, J = 2.4 Hz, 1H), 7.55 (dd, J= 1.8 Hz, 8.4 Hz, 1H), 7.29 (d, J= 8.8 Hz, 1H), 6.97 (d, J - 8.8 Hz, 1H), 6.88 (d, J - 2.1 Hz, 1H), 6.84 (s, 1H), 6.78 (dd, J - 2.2 Hz, 8.9 Hz, 1H), 4.06 (s, 3H), 2.37 (s, 3H). i3C NMR (150 MHz, DMSO-i 6): δ 160.7, 156.0, 151.2, 147.5, 136.4, 135.5, 134.5, 132.4, 127.0, 120.1, 1 19.6, 1 14.5, 1 14.0, 1 10.6, 105.4, 104.1, 40.4, 33.0, 15.2. HRMS calcd for (Ci8H,679BrN303+H)+ 402.0448, found 402.0434.
(E and Z)-iV-(l-(5-bromo-l-methyl-lH-indol-2-yl)propylidene)-3-hydroxy-2- naphthohydrazide G18
Figure imgf000081_0001
G18 was prepared from l-(5-bromo-l-methyl-lH-indol-2-yl)ethanone D9 and 3-hydroxy-2- naphthohydrazide E using general Procedure C.
Yield = 63% (filtration), brown solid. NMR analysis indicated that G18 is present in two isomeric forms (6:1). Ή NMR (600 MHz, DMSO-c/6): δ 1 1.80 (bs, 1H), 1 1.74 (s, 1H), 8.65 (s, 1H), 7.99 (d, J = 8.1 Hz, 1H), 7.80 - 7.78 (m, 2H), 7.54 - 7.51 (m, 2H), 7.39 - 7.35 (m, 3H), 7.04 (s, 1H), 4.15 (s, 3H), 2.87 (q, J = 7.8 Hz, 2H), 1.27 (t, J = 7.6 Hz, 3H). 13C NMR (150 MHz, DMSO- ): δ 161.3, 152.5, 150.9, 138.2, 136.8, 135.8, 132.5, 128.9, 128.3, 128.1, 127.2, 125.7, 125.5, 123.9, 122.8, 120.8, 1 12.4, 112.2, 110.7, 104.9, 33.3, 22.0, 10.7. HRMS calcd for (C23H20 79BrN3O2+H)+ 452.0794, found 452.0807.
(E and Z)-N,-(l-(5-bromo-l-methyl-lH-indol-2-yl)ethylidene)-3-hydroxy-2- naphthohydrazide G19
Figure imgf000081_0002
G19 was prepared from l-(6-bromo-l -methyl- lH-indol-2-yl)ethanone D8 and 3-hydroxy-2- naphthohydrazide E using general Procedure C.
Yield = 72% (filtration), brown solid. NMR analysis indicated that G19 is present in two isomeric forms (7:1). 1H NMR (600 MHz, DMSO- ): δ 11.74 (bs, 1H), 11.59 (s, 1H), 8.65 (s, 1H), 7.99 (d, J - 8.1 Hz, 1H), 7.80 - 7.78 (m, 2H), 7.54 - 7.51 (m, 2H), 7.39 - 7.35 (m, 3H), 7.02 (s, 1H), 4.16 (s, 3H), 2.45 (s, 3H). 13C NMR (150 MHz, DMSO-i¾: δ 160.5, 151.7,
145.6, 137.1 , 136.7, 134.8, 131.3, 127.9, 127.3, 127.1, 126.2, 124.7, 124.6, 122.9, 121.8,
119.7, 111.4, 111.2, 109.7, 104.4, 32.2, 14.2. HRMS calcd for (C22H18 79BrN302+H)+ 438.0637, found 438.0643.
(E and Z)-5-bromo-iV-(l-(5-bromo-l-methyl-lH-indol-2-yl)propylidene)-2- hydroxybenzohydrazide G20
Figure imgf000082_0001
G20 was prepared from l -(5-bromo-l -methyl- lH-indol-2-yl)ethanone D9 and 5-bromo-2- hydroxybenzohydrazide E using general Procedure C.
Yield = 72% (flash chromatography 20/1 ; DCM/MeOH), white solid. NMR analysis indicated that G20 is present in two isomeric forms (5: 1). 1H NMR (600 MHz, DMSO- ): δ 12.17 (bs, 1H), 1 1.50 (s, 1H), 8.06 (d, .7= 2.5 Hz, 1H), 7.80 (d, 7= 1.5 Hz, 1H), 7.60 (dd, J= 2.6 Hz, 8.7 Hz, 1H), 7.51 (d, J = 8.8 Hz, 1H), 7.36 (dd, J = 1.7 Hz, 8.7 Hz, 1H), 7.03 - 7.02 (m, 2H), 4.11 (s, 3H), 2.84 (q, J = 7.5 Hz, 2H), 1.23 (t, J = 7.6 Hz, 3H). 13C NMR (150 MHz, DMSO- ): δ 160.4, 155.5, 151.2, 138.2, 136.6, 135.7, 132.7, 128.1, 125.6, 122.9, 120.4, 119.3, 112.4, 112.2, 110.9, 105.1, 33.3, 21.9, 10.6. HRMS calcd for (C19Hi7 79Br2N302+H)+ 479.9741 , found 479.9741.
(E)-NT-(l-(lH-indoI-2-yI)ethylidene)-5-bromo-2-methoxy-7V-methylbenzohydrazide Ol 1) Synthesis of perfluorophenyl 5-bromo-2-methoxybenzoate
Figure imgf000083_0001
A mixture of pentafluorophenol (500 mg, 2.8 mmol), DCC (550 mg, 2.4 mmol) and 5-bromo- 2-methoxybenzoic acid (400 mg, 1.7 mmol) in DMF (15 mL) was stirred at rt overnight, filtered, and the filtrate was concentrated. The crude product was purified by flash chromatography (hexanes/EtOAc; 10/1) to give white solid (653 mg, 95%). Mp = 83-86°C. 1H NMR (CDC13): δ 8.17 (d, J = 2.6 Hz, 1H), 7.71 (dd, J - 2.6 Hz, 9.0 Hz, 1H), 6.96 (d, J = 9.0 Hz, 1H), 3.94 (s, 3H). 13C NMR (CDC13): δ 160.1, 159.9, 142.4 (m, 1C), 140.7 (m, 2C), 138.9 (m, 2C), 138.5, 137.3 (m, 1C), 135.4, 117.9, 1 14.4, 1 12.5, 56.6. HRMS calcd for (Ci4H6 79BrF503+H)+ 396.9493, found 396.9499.
2) Synthesis of (^-/ -Cl-Cl^-indoI-Z- ethylideneJ-S-bromo-Z-methox -N- methylbenzohydrazide Ol
Figure imgf000083_0002
A mixture of l-(lH-indol-2-yl)ethanone C (100 mg, 0.63 mmol), methylhydrazine sulphate (200 mg, 1.34 mmol) and DIPEA (300 μΐ,, 1.72 mmol) in EtOH (2 mL) was irradiated with microwaves for 2 h at 140°C and then partitioned between EtOAc (100 mL) and brine (50 mL). The organic layer was dried over anhydrous Na2S04 and concentrated. The residue was dissolved in DCM (2 mL) followed by the addition of perfluorophenyl 5-bromo-2- methoxybenzoate (150 mg, 0.4 mmol) and pyridine (100 μΕ, 1.24 mmol). The mixture was irradiated with microwaves at 1 10°C for 3 h and then concentrated. The crude product was purified by flash chromatography (hexanes/EtOAc; 15/1 to 3/1) to give yellow solid (74 mg, 49%). NMR analysis indicated that Ol is present in two isomeric forms (2: 1).
Major isomer 1H NMR (600 MHz, DMSO- ): δ 11.62 (s, 1H), 7.65 (dd, J = 2.4 Hz, 8.8 Hz, 1H), 7.63 (d, J = 7.9 Hz, 1H), 7.50 (d, J = 2.5 Hz, 1H), 7.45 (d, J = 8.6 Hz, 1H), 7.21 (t, J = 8.1 Hz, 1H), 7.16-7.13 (m, 2H), 7.01 (t, J= 7.1 Hz, 1H), 3.88 (s, 3H), 3.1 1 (s, 3H), 2.32 (s, 3H).13C NMR (150 MHz, DMSO- ): δ 165.3, 160.7, 155.0, 138.1, 135.0, 133.7, 130.6, 128.4, 127.7, 124.4, 121.7, 120.0, 114.6, 112.59, 112.56, 107.1, 56.6, 38.7, 16.5.
Minor isomer
1H NMR (600 MHz, DMSO-cfc): δ 11.11 (s, 1H), 7.57 (d, J= 8.8 Hz, 1H), 7.45 (d, J= 8.6 Hz, 1H), 7.38 (d, J= 2.5 Hz, 1H), 7.35 (d, J= 8.2 Hz, 1H), 7.14-7.13 (m, 1H), 7.08 (d, J- 1.5 Hz, 1H), 6.99 (t, J= 7.8 Hz, 1H), 6.87 (d, J= 8.9 Hz, 1H), 3.61 (s, 3H), 3.28 (s, 3H), 2.42 (s, 3H). 13C NMR (150 MHz, DMSO-rf6): δ 166.4, 165.2, 155.2, 138.0, 134.9, 133.5, 131.1, 129.1, 127.6, 124.3, 121.6, 120.0, 114.0, 112.7, 1 11.8, 107.0, 56.1, 36.3, 16.7.
HRMS calcd for (C19H) 8 79BrN302+H)+ 400.0655, found 400.0657.
Figure imgf000085_0001
Figure imgf000086_0001
EXAMPLES
EXAMPLE 1 : Expression and Purification of PK Proteins.
Recombinant His-tagged PK proteins including MRSA PK and human PK isoforms (Ml, M2, L and R) were expressed, purified to near homogeneity (>98%), and detected by SDS-PAGE [(12% (w/v) gel] as described herein. The estimated molecular mass of each of the respective bands on SDS-PAGE correlated well with that predicted based on the amino acid composition for each PK protein (data not shown), indicating that all PK constructs were expressed as full-length proteins. Structural integrity of each protein was verified by the pattern of migration on SDS-PAGE and allosteric properties for each construct (data not shown). All PKs demonstrated enzyme activity comparable to those reported previously (data not shown) (). MRSA and human PK proteins were used to characterize the biochemical properties of candidate MRSA PK inhibitors.
EXAMPLE 2: In Vitro Screening of Putative MRSA PK Inhibitors.
IS- 130 (NSK4-65) and IS-63 (NSK4-60) were used at a concentration of 50 μΜ in a recombinant MRSA PK assay, with a substrate concentration of 10 mM PEP, which is close to MRSA PK Km (e.g., 6.6 mM) so that the IC50 values should approximate the K{. These compounds were also screened against human PK isoforms (Ml, M2, R and L) to test for selectively inhibit bacterial PK. IS-63 (Mw = 359.4) showed potent (IC50 of 0.85 μΜ) and selective MRSA PK inhibitor (with more than 45-fold selectivity over the human PKs) and IS-130 (Mw = 373.2) which showed potency (IC50 of 0.091 μΜ), selectivity (with more than 1370-fold selectivity over the human PKs) and antibacterial activity (35% growth inhibition at 25 μΜ) (see TABLE 1). One compound, IS-63 (NSK4-60) was inhibitory in the nanomolar range and demonstrated selectivity for MRSA PK (FIGURE 6). However, in cell-based assays to assess activity against intact S. aureus, IS-63 (NSK4-60) showed less than 10% inhibition of growth at concentrations in the 100 μΜ range where concomitant toxicity for human cells was observed. We hypothesized that the lack of activity of IS-63 (NSK4-60) against S. aureus might be due to poor penetration of the cell wall. IS-130 (NSK4-65) was found to have an IC50 of 0.10 μΜ compared with 0.91 μΜ for parent compound NSK4-60. Also, at 10 μΜ NSK4-65 showed complete selectivity for bacterial PK (Figure 7, top panel). TABLE 1: Two acyl hydrazone-based compounds with potent selective inhibitory activity towards MRSA PK.
Figure imgf000088_0001
All values represent the means of three independent experiments each performed in triplicate. Antibacterial and human cell toxicity activities of compounds were evaluated as described in "Experimental Procedures" using S. aureus strain RN4220 and HeLa293 cells, respectively, at compound concentration of 100 μΜ.
IS-63 demonstrated less antibacterial activity, possibly due to lack of cellular penetration. No further inhibition of growth was achieved beyond 25 μΜ, which was possibly due to stability of the compound over the time course of the experiment (e.g., 24 hr) or limited membrane pemieability. Subsequently, the toxic effects of IS-130 against human HeLa229 cells were evaluated. Results indicated that IS-130 had no significant growth inhibitory effects on HeLa cells (TABLE 1) up to 400 μΜ. Despite the fact that IS-130 exhibited only modest antibacterial potency, it was a small and efficient ligand with selective on-target activity with apparently limited ability to penetrate the bacterial cell. Accordingly, IS- 130 was a good starting point for optimization through structure activity relationships (SAR) programs. Furthermore, novel compounds as shown in TABLE 2A were synthesized for testing as described herein.
TABLE 2A: Further compounds synthesized as described herein.
Figure imgf000089_0001
Figure imgf000090_0001
Figure imgf000091_0001
Figure imgf000092_0001
Figure imgf000093_0001
Figure imgf000094_0001
Figure imgf000095_0001
Figure imgf000096_0001
Figure imgf000097_0001
(E and Z)-5-bromo-N'-(l -(6-bromo-l - methyl- 1 H-indol-2-yl)ethylidene)-2- hydroxybenzohydrazide (G16)
Figure imgf000098_0001
j HO (E and Z)-5-bromo-2-hydroxy-N'-(l-(5- hydroxy- 1 -methyl- 1 H-indol-2-
Br yl)ethylidene)benzohydrazide (G 17)
(E and Z)-N'-(l -(5 -bromo-1 -methyl- 1H- indol-2-yl)propylidene)-3-hydroxy-2-
Figure imgf000098_0002
naphthohydrazide (G18)
/ HO (E and Z)-N*-(l-(5-bromo-l -methyl- 1H- indol-2-yl)ethylidene)-3-hydroxy-2- naphthohydrazide (G19)
; V HO (E and Z)-5-bromo-N'-(l-(5-bromo-l- methyl-lH-indol-2-yl)propylidene)-2- hydroxybenzohydrazide (G20)
Br
TABLE 2B: Subset of Preferred Compounds
Figure imgf000098_0003
Figure imgf000099_0001
Figure imgf000100_0001
Figure imgf000101_0001
Figure imgf000102_0001
Figure imgf000103_0001
Figure imgf000104_0001
TABLE 2C: Subset of More Preferred Compounds
Figure imgf000104_0002
Figure imgf000105_0001
Figure imgf000106_0001
TABLE 2D: Subset of Most Preferred Compounds
Figure imgf000106_0002
Figure imgf000107_0001
It will be appreciated by a person of skill in the art that the preference for a compound or compounds as set out in TABLES 2B, 2C, and 2D is based on the particular circumstances under which the compounds were tested (for example, the bacteria or strain of bacteria being tested, clinical testing etc.). Accordingly, as further testing is performed the preference for a compound or compounds as set out in TABLES 2A, 2B, 2C, and 2D may change as a result of the further testing, whereby a compounds preference may be increased or be diminished or stay essentially the same depending on the particular circumstances under which the compounds are tested. Furthermore, the compound or compounds may be preferred in one circumstance and less preferred in another circumstance (for example, depending on the bacterial target).
EXAMPLE 3: Analog Preparation. Preparation and evaluation of IS- 130 analogs, revealed the structure dependencies of inhibition and structural modifications were found that increase both PK inhibitory activity and antibacterial potency of the inhibitors. The potency and selectivity of each derivative toward MRSA PK (IC50) as well as antibacterial activities (MIC) were measured to direct the iterative rounds of synthetic chemistry. Parameters derived from these experiments are summarized in TABLE 3. The impact of different substitution patterns on the indole and benzoyl hydrazone moieties on compound function fell into three categories: (1) those important for binding affinity, (2) those crucial for cell penetration and (3) those having complex effects on binding affinity and cell penetration. Replacement of the benzimidazole sp2 hybridized nitrogen of IS-130 (at position R2) with a CH (analog NSK4-66) resulted in a slight increase in inhibitory activity possibly due to the increased hydrophobicity of the indole ring relative to the benzimidazole ring. The presence of both the p-bromine group at Q3 (substituted in NSK4-82 with 88-fold increased IC50) and the p-hydroxyl or alkoxyl group at R3 (substituted in NSK4-93 with 2-fold increased IC50) in benzoyl moiety was also significant for PK-inhibitory activity. Furthermore, a halogen (for example, CI or F) substituent on both A3 and A2 (see NSK5-25, NSK5-69, AM-160, AM-213 and AM-215) in the compound's indole moiety resulted in improved enzyme IC50 values.
The results shown in TABLE 3 indicate that the presence of the (N)-methyl group at R1 position in the benzoheterocycle moiety (i.e. an N-methylindole moiety) (e.g., NSK4-77, NSK5-15, NSK5-68, NSK5-77, AM-179 and AM-235) conferred a remarkable increase in antibacterial activity (e.g., MIC values at the low micromolar range).
TABLE 3. Inhibition of MRSA PK and antibacterial activities of synthesized derivatives of IS-130 with notable structural- activit relationshi s.
Figure imgf000108_0001
Figure imgf000109_0001
Figure imgf000110_0001
MIC (Minimum inhibitory concentration) was determined against S. aureus RN4220, as determined by the Broth-Microdilution Method; *IS-130(NSK4-65) is the hit compound, direction of the link flipped and bF at position 15 instead of 17.
EXAMPLE 4: PK Lead Compounds Selectively Inhibit MRSA PK.
To determine if the compounds were capable of acting selectively against MRSA PK, the inhibitory effects of three compounds with antibacterial activity (e.g., NSK4-77, NSK5-15 and AM-165) were tested against human Ml , M2, R and L PK isoforms in single-enzyme catalytic assays (TABLE 4). PK compounds with potent antibacterial activity displayed submicromolar (0.16- 0.38 μΜ) IC50s toward bacterial PK with a marked 180 to 940-fold selectivity over the human isoenzymes (TABLE 4). Therefore, PK lead compounds appeared to be suitable starting point to develop highly specific antimicrobial agents.
Figure imgf000110_0002
Values are average ± deviation value from two independent experiments performed in triplicates. *Compounds with potent antibacterial activities. ND: Not determined.
EXAMPLE 5: PK Lead Compounds are Non-competitive Inhibitors.
To determine the nature of inhibition by NSK4-77, NSK5-15 and AM- 165 compounds, kinetic parameters of MRSA PK inhibition were determined in assays containing various concentrations of substrate (PEP) and different fixed concentrations of each inhibitor (for example, NSK4-77 and NSK5-15) (FIGURE 1). The maximal velocity (Fmax) and Michaelis-Menten constant (Km) were determined for each assay as described herein. The data suggested that inhibition by PK compounds could not be overcome by increasing the concentration of the substrate, indicating that inhibitor binds to a site on the enzyme distinct from the site that binds substrate (PEP). Furthermore, the results presented in FIGURE 1 show that Km values remained unchanged (i.e. 6.5 mM) in the absence and presence of several fixed concentrations of both NSK4-77 (FIGURE 1A) and NSK5-15 (FIGURE IB), whereas Vmax was significantly decreased (e.g., up to 80%). Thus, PK lead compounds are non-competitive inhibitors with respect to PEP, with inhibition constant (Ki) values of 269 ± 63 nM and 276 ± 82 nM respectively for NSK4-77 and NSK5-15.
EXAMPLE 6: PK Lead Compounds Exhibit Gram-Positive Specific Antibacterial Activity.
To investigate the antibacterial properties of PK inhibitors, the three compounds (NSK4-77, NSK5-15 and AM- 165) from the focused SRA study were tested in vitro for their antibacterial activities against a diverse panel of bacterial species and strains. Results in TABLE 5 indicate that PK lead compounds showed potent in vitro antibacterial activity against all strains and species of staphylococci that were tested (MIC 1.4-19.2 μg/ml) including methicillin-susceptible (e.g., RN4220, ATCC25923), methicillin-resistant S. aureus (e.g., MRSA252, COL and MW2) and a multidrug-resistant S. aureus (MDRSA) isolate that was resistant to many of the major classes of antibiotics. Compounds NSK5-15 and AM-165 also displayed MICs ranging from 1.4 to 5.1 μg/ml on other staphylococcal strains such as Staphylococcus epidermidis, Staphylococcus haemolyticus and Staphylococcus saprophyticus (TABLE 5). PK compounds were inactive in vitro (MIC range, >73 to >93 pg/ml) against a range of Gram-negative human pathogens tested such as acinetobacter and pseudomonas (TABLE 5). However, as shown in TABLE 5, the PK compounds were found to also be active against a range of Gram-positive bacterial species, including several antibiotic-resistant strains. These compounds demonstrated superior antibacterial activities against Enterococcus. faecalis, Enterococcus faecium and vancomycin-resistant E. faecium (MICs, 0.25 to2 μg/ml) as well as Streptococcus pneumoniae and Streptococcus pyogenes (MICs, 0.5 to 8 g/ml). Therefore, PK compounds demonstrated potent antibacterial activities towards staphylococci, enterococci and streptococci.
TABLE 5. Antibacterial activities of the three most potent PK inhibitors against selected staphylococci, non-staphylococcal Gram-positive pathogens, and Gram-negative pathogens compared to standard antibiotics.
Figure imgf000112_0001
Strain NSK4-77 NSK5-15 AM- 165 Control
(G10) (Gi l) (G5)
Gram positive bacteria
Staphylococcus aureus KN4220 4.8 1.4 2.5 a0.5, bl, c0.5
Staphylococcus aureus ATCC 25923 9.6 2.9 5.0 a0.5, bl
Staphylococcus aureus CA-MRSA (USA400) 9.6 2.9 5.0 a0.75, bl
Staphylococcus aureus HA-MRSA (COL) 9.6 5.8 ND a0.30
Staphylococcus aureus HA-MRSA252 9.6 2.9 5.0 a>10, b0.5
Staphylococcus aureus MDRSA* 9.6 1.4 2.5 a>16
Staphylococcus epidermidis 9.6 1.4 2.5 c>4, bl
Staphylococcus, haemolyticus 19.2 2.9 5.0 c0.1
Staphylococcus saprophytics 9.6 2.9 5.0 bl, c0.5
Enterococcus faecalis ATCC29212V >64 2 >64 b2-4, c<0.03
Enterococcus faecium ATCC35667V >64 1 >64 b2, c0.125
Enterococcus faecium ATCC700221 (VRE)V 8 0.25 ND b>64, c64
Listeria monocytogenes ATCC19115 >77 >93 ND c25
Streptococcus pneumoniae ATCC49619A 0.5 1 0.5 a<0.03, b0.25
Streptococcus pyogenes ATCC700294A 8 8 8 a<0.125, b2
Gram negative bacteria
Acinetobacter baumannii X270295 >77 >186 >162 c0.8
Escherichia coli DY330 >77 >93 ND c0.2
ESBL-producing Klebsiella pneumoniae NA >93 >81 c>50
Pseudomonas aeruginosa PA0- 1 >193 >233 >162 c0.25
Samonella typhimurium SL1344 >193 >233 ND c<0.1
MIC were determined in BHI for all bacterial strains as described in Material and Methods, unless for Enterococcal species (v) and Streptococcal species (A), which MIC were determined in CAMHB (cation-adjusted Mueller Hinton broth, containing 20 mg of Ca2+/L, and 10 mg of Mg2+/L) and CAMHB containing 2 - 5 % laked horse blood, respectively.
^Resistant to cefazolin, clindamycin, ciprofloxacin, erythromycin, penicillin, oxacillin
and methicillin. a erythromycin' Vancomycin' Ciprofloxacin. ND: not determined.
I l l EXAMPLE 7: PK Compounds Exhibit Favorable Selectivity Indices (CC50/MIC).
The cytotoxicity of NSK4-77, NS 5-15 and AM- 165 compounds was determined using HeLa cells in a 1-day incubation assay as described herein. The results shown in FIGURE 2A show that PK lead compounds exhibited little cytotoxicity toward mammalian cells (i.e., less than 30% of cell death at 500 μΜ equals to 193, 232 and 202 μ^ιηΐ of NSK4- 77, NSK5-15 and AM- 165, respectively) with CC50 values of > 200 μ^ιτιΐ for all three compounds tested. The selectivity indices (CC50/MIC) for NSK4-77, NSK5-15 and AM- 165 evaluated in relation to their very potent antibacterial activity (FIGURE 2B) against MDRSA, were respectively 20, 80 and 40, indicating remarkable selectivity for bacterial versus mammalian cells in the mode of action of these compounds.
EXAMPLE 8 : Bactericidal Activity of PK Compounds.
To determine if bioactive PK compounds were bactericidal, activity of NSK5-15 was assessed using S. aureus as a model organism. FIGURE 3 shows representative time-kill curves for compound NSK5-15 against a methicillin-sensitive S. aureus (MSSA) ATCC25923. Vancomycin was also used as a comparator drug. As shown in FIGURE 3, with NSK5-15 maximum killing was observed at concentrations of 4 x MIC, with a 3-log drop in the numbers of cfu/ml occurring by 24 h after compound addition, consistent with a bactericidal mode of action. Just slightly less than a 3-log drop in cfu/ml was also observed for NSK5-15 at 1 x and 2 x MIC, suggesting a dose dependent effect on staphylococcal killing up to a concentration equal to 4 x MIC. Killing was less rapid with 4 x MICs of NSK5-15 compared with vancomycin against the same strain. Similar rates of killing by NSK5-15 at 4 x MICs against a multidrug-resistant S. aureus (MDRSA) strain was observed (data not shown). These results indicate that PK compounds are bacteridal for both MSSA and MDRSA.
EXAMPLE 9: Resistance Studies.
To assess the potential for cells to become resistant to the antibacterial effects of PK compounds, we tried to generate resistant mutants, using S. aureus RN4220. Cells were cultured for up to 25 consecutive generations in the presence of sub-lethal concentrations of NSK5-15 or for 10 generations in the presence of sub-lethal concentrations of fusidic acid. As shown in FIGURE 4, after 25 subcultures of NSK5-15 in the presence of compound, the relative MIC of NS 5-15 against S. aureus remained stable and mutants with significant increase in resistance to the compounds (>4 x MIC) were not detected. In contrast, mutants able to grow in concentrations up to 32 to 128 x (the initial) MIC of fusidic acid appeared within 5 to 10 passages, indicating the emergence of resistant mutants. Moreover, when the 10 generation of the bacteria that had developed resistance to fusidic acid was tested against NSK5-15, it was found to be susceptible, with MIC similar to initial exposure (e.g., 1.4 μg/ml). Interestingly this demonstrated that bacterial resistance is less prone to develop in PK compound-treated staphylococci, whether they are sensitive or resistant to conventional antibiotics.
EXAMPLE 10: PK Compounds Reduced Pyruvate Production.
The target selectivity of AM- 165 was determined by measuring pyruvate concentrations in S. aureus RN4220 strains incubated either with vehicle (e.g, DMSO) as control or sub-lethal concentration of compound AM- 165. As shown in FIGURE 5, pyruvate concentration was significantly reduced (by 4.3-fold) in AM- 165 treated cells (0.86 ± 0.31 per 5 x 107 cell) compared to the control cells (0.19 ± 0.12 per 5 x 107 cell) indicating that essential PK () is the target of lead compounds for the inhibition of bacterial growth. Together with the demonstration that PK compounds inhibited PK enzymatic activity directly (FIGURE 1 and TABLE 4) provide direct validation that the compounds are targeting PK. This mechanism of action was further confirmed with recent availability of X-ray structure of the MRSA PK in complex with PK lead inhibitors (Axerio-Cilies, in preparation).
EXAMPLE 11 : Crystallization and Binding Site Analysis for MRS A252 PK and NSK-465.
The crystal structure of MRSA252 PK co-crsytallized with NSK4-65 (IS- 130) was resolved and showed that inhibitor was interacting with a small interface between two PK subunits (Figure 7). NSK-465 was binding to an inter-unit pocket located at the interface between PK subunits of the MRSA PK in a functional tetramer enzyme complex (Figure 7). This pocket was poorly conserved in human PK isoforms, likely explaining the basis for selective inhibition.
Notably, previous studies had indicated that conformational changes in these types of interfaces can disrupt the enzymatic activity of pyruvate kinase [Tulloch BL et al. Sulphate removal induces a major conformational change in Leishmania mexicana Pyruvate kinase in the crystalline state. Journal of Molecular Biology 2008, 383(3):615-626]. The crystal structure of MRSA pyruvate kinase also revealed unique architecture of the target site formed by two parallel alpha helices at the small interface which was distinct from human PK (Figure 7). Thus providing a novel site for selective targeting of MRSA PK.
The crystal structure of the bacterial protein co-crystallized with NSK4-65 clearly demonstrated that the compound was able to bind preferentially to the inter-interface cavity. NSK-465 is shown herein to be a potent inhibitor of the MRSA252 PK.
The newly identified MRSA pyruvate kinase small molecule binding surface shown in Figure 3, was further studied by superimposing the MRSA and human PK structures to examine whether the site constituted a suitable compound-binding pocket that is unique to the MRSA. 10 amino acid residues were identified highlighted in Figure 8A (Thr348, Thr353, Ser354, Ala358, Ile361, Ser362, His365, Thr366, Asn369, and Leu370) in each subunit of the MRSA PK which contribute to the formation of the binding pocket. Analysis of the crystal structure showed two His365 residues one from each subunit, which are positioned just above and below the interface cavity and, which are turned inwards into the cavity. Further examination of the crystal structure showed that the temperature factors of His365 are quite high leading to the prediction that the histidines might indeed be flexible. Based upon this consideration, we subjected the interface residues to energy minimization, which led to the generation of a well-defined cavity (see Figures 8B and C). As can be seen, the interface site of the MRSA PK is accessible to small molecules, but in the human version, the interface is blocked by five key amino acid residues, Glu418-B, Arg399-A, B and Arg400-A, B. This partially obstructed surface in human PK coupled with the poorly conserved sequence at this interface region between these two species predicts that the binding properties for small molecules should be quite distinct. To examine further the binding of NSK4-65 to MRSA PK, the protein was co- crystallized in the presence of compound. The resolved structure of the complex revealed key protein-ligand interactions and provided a rationale for the ability of NSK4-65 to bind selectively to bacterial PK. The binding orientation of NSK4-65 is shown in Figure 9 along with the corresponding hydrogen bonding patterns occurring within the interface-binding cavity. This shows that this IC50 100 nM MRSA PK inhibitor was anchored by 5 main hydrogen bonds. The schematic on the left (Figure 9A) illustrates that the sp2 hybridized nitrogen of the hydrazone moiety, the sp3 hybridized nitrogen from the indole moiety and the linker carbonyl functional group all interact with Ser362-A residue. This carbonyl group was also seen to be engaged with Ser362-B, which is located just adjacent to Ser362-A (A and B notations correspond to different MRSA PK subunits). These serines are of particular interest to target since they are not conserved in human PK (see Figure 9A) and thus are likely to influence the selectivity of the compound for bacterial PK. In addition, the hydroxyl group of NSK-465 appeared to hydrogen bond with His365-B, which is also a residue unique to the bacterial protein. Figure 9A also shows that strong hydrophobic interactions were observed with the p-bromine functional group towards Ile361-B, Ala358-B and Leu370-A residues, but amongst these only Leu370-A was noted to be unique to bacterial PK.
Protein-ligand modelling also indicated that the methyl substituent on the lead compound was involved in hydrophobic contacts with two His365 residues belonging to subunits A and B. Finally, hydrophobic contacts were observed between the indole rings and the hydrophobic residue of Ile361-A which is just orientated below the indole substructure. A similar effect was observed with Ile361-B, which is situated above the hydrophobic benzene moiety. Taken together, these findings show that the selectivity of NSK4-65 for bacterial PK can be attributed to poorly conserved interface residues, which in the case of MRSA PK interacted directly with the NSK4-65.
Although various embodiments of the invention are disclosed herein, many adaptations and modifications may be made within the scope of the invention in accordance with the common general knowledge of those skilled in this art. Such modifications include the substitution of known equivalents for any aspect of the invention in order to achieve the same result in substantially the same way. Numeric ranges are inclusive of the numbers defining the range. The word "comprising" is used herein as an open-ended term, substantially equivalent to the phrase "including, but not limited to", and the word "comprises" has a corresponding meaning. As used herein, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a thing" includes more than one such thing. Citation of references herein is not an admission that such references are prior art to the present invention.

Claims

What is claimed is:
1. A compound of the Formula (A) or Formula (B):
Figure imgf000118_0001
or a salt thereof, wherein:
R1 is N-R5, S, or O;
R2 is C-H, or N;
R3 is C-H, C-OR5, N, C-OCH2C≡CH, C-OCH2OCH3, C-OCH2OCH2CH3, C- OC(0)CH3, or C-OCH2(OCH2CH2)nOR5;
R4 is C;
R5 is H, or a C1-C6 branched or unbranched, saturated or unsaturated, alkyl or acyl group;
n is 1-5;
A1 is R5, OR5, F, CI, Br, I, C≡CH, S02R5, NHCOR5, NHS02R5, N02, CON(R5)2 or phenyl;
A2 is H, F, CI, Br, I, C≡CH, OR5, or phenyl;
A3 is F, CI, Br, I, C≡CH, OR5, R5, S02R5, NHCOR5, NHS02R5, N02, CON(R5)2 or phenyl; A4 is F, CI, Br, I, C≡CH, OR5, R5, S02R5, NHCOR5, NHS02R5, N02, CON(R5)2 or phenyl;
Q1 is R5, OR5, F, CI, Br, I, N3, C≡CH, or phenyl;
Q2 is R5, OR5, F, CI, Br, I, N3, C≡CH, or phenyl;
Q3 is R5, OR5, F, CI, Br, I, N3, C≡CH, or phenyl;
Q4 is H or OH;
*) 5 5 or Q and Q optionally form a benzene ring, optionally substituted with R , OR , F, CI, Br, I, N3, C≡CH, or phenyl;
L is
Figure imgf000119_0001
wherein,
D1 is R5;
E1 is R5 or phenyl;
2 5
E is R or phenyl;
E is R or phenyl;
provided that the compound is not
r
Figure imgf000119_0002
2. The compound of claim 1 , wherein, R3 is C-H, C-OR5, N, C-OCH2C≡CH, C- OCH2OCH3, C-OCH2OCH2CH3, or C-OC(0)CH3.
3. The compound of claim 1 or 2, wherein R3 is C-H, C-OH, C-OMe, C-OEt, N, C- OCH2C≡CH, C-OCH2OCH3, C-OCH2OCH2CH3, or C-OC(0)CH3.
4. The compound of any one of claims 1-3, wherein R3 is C-H, C-OR5, or N.
5. The compound of any one of claims 1 -4, wherein R3 is C-H, C-OH, C-OMe, C-OEt, or N.
6. The compound of any one of claims 1 -5, wherein R1 is N-H, N-Me, N-Et, S, or O.
7. The compound of any one of claims 1-5, wherein R1 is N-R5.
8. The compound of any one of claims 1 -5, wherein R1 is N-H, N-Me, or N-Et.
9. The compound of any one of claims 1 -5, wherein R1 is S, or O.
10. The compound of any one of claims 1 -9, wherein R is N.
11. The compound of any one of claims 1-9, wherein R2 is C-H.
12. The compound of any one of claims 1-1 1, wherein R5 is a C 1 -C6 branched or
unbranched, saturated or unsaturated, alkyl;
13. The compound of any one of claims 1-12, wherein A1 is R5, OR5, F, CI, Br, I, C≡CH, or phenyl.
14. The compound of any one of claims 1-12, wherein A1 is H, O-Me, F, CI, Br, I, C≡CH, or phenyl.
15. The compound of any one of claims 1-12, wherein A1 is H, O-Me, F, CI, Br, or I.
16. The compound of any one of claims 1-15, wherein A2 is H, F, CI, Br, I, C≡CH, or phenyl.
17. The compound of any one of claims 1-15, wherein A2 is H, O-Me, F, CI, Br, I, C≡CH, or phenyl.
18. The compound of any one of claims 1-15, wherein A2 is H, O-Me, F, CI, Br, or I.
19. The compound of any one of claims 1-18, wherein A3 is F, CI, Br, I, C≡CH, OR5, R5, or phenyl.
20. The compound of any one of claims 1-18, wherein A3 is F, CI, Br, I, C≡CH, H, or O- Me.
21. The compound of any one of claims 1-18, wherein A3 is H, O-Me, F, CI, Br, or I.
22. The compound of any one of claims 1-18, wherein A3 is F, CI, Br, I, OR5, R5, or
phenyl.
23. The compound of any one of claims 1 -22, wherein A4 is F, CI, Br, I, C≡CH, OR5, R5, or phenyl.
24. The compound of any one of claims 1-22, wherein A4 is F, CI, Br, I, C≡CH, OR5, R5, N02, CON(R5)2 or phenyl.
25. The compound of any one of claims 1 -22, wherein A4 is F, CI, Br, I, OR5, R5, or N02.
26. The compound of any one of claims 1 -22, wherein A4 is H, O-Me, F, CI, Br, or I.
27. The compound of any one of claims 1 -26, wherein Q1"3 are independently selected from R5, OR5, F, CI, Br, I, and N3.
28. The compound of any one of claims 1-26, wherein Q1"3 are independently selected
Figure imgf000121_0001
29. The compound of any one of claims 1 -28, wherein Q4 is H.
30. The compound of any one of claims 1-29, wherein L is
Figure imgf000121_0002
31. The compound of any one of claims 1-30, wherein D 1 i ·s H or Me.
32. The compound of any one of claims 1-29, wherein E are independently selected from H, Me, Et, phenyl, and tert-butyl.
33. The compound of any one of claims 1-29, wherein E 3 are independently selected from H, Me, and Et.
34. The compound of any one of claims 1-32, wherein E1 is selected from H, Me, Et, phenyl, and tert-butyl.
35. The compound of any one of claims 1-33, wherein E1 is selected from phenyl, Me, and
Et.
36. The compound of any one of claims 1-29, wherein E ' are independently selected from Me and Et.
37. The compound of any one of claims 1-29, wherein E1 ~3 are Me.
38. The compound of any one of claims 1-35, wherein E1 is Et, Me, or phenyl.
39. The compound of any one of claims 1-35, wherein E' is Me.
40. A compound of the Formula (A) :
Figure imgf000121_0003
or a salt thereof, wherein: R s N-R5;
R2 is C-H;
R3 is C-OH;
R5 is H or Me or Et;
A1 is R5, F, CI, Br, or I;
A2 is H, F, CI, Br, or I;
A3 is OMe, F, CI, Br, I, or R5;
A4 is F, CI, Br, I, or R5;
Q1 is R5, F, CI, Br, or I;
Q2 is R5, F, CI, Br, or I;
Q3 is R5, F, CI, Br, or I;
Q4 is H or OH;
L is
Figure imgf000122_0001
wherein,
D1 is R5; and
E1 is R5 or phenyl.
41. The compound of claim 40, wherein: R1 is N-H or N-Me; R3 is C-OH; A1 is H; A2 is H, or F; A3 is OMe, F, CI, Br, or H; A4 is H, or F; Q1 is H; Q2 is H; Q3 is Br, or I; Q4 is H; L is
Figure imgf000122_0002
42. A compound selected from TABLE 2A.
43. A compound selected from TABLE 2B.
44. A compound selected from TABLE 2C. A compound selected from TABLE 2D.
A compound of the Formula (C):
Figure imgf000123_0001
or a salt thereof, wherein:
G1 is G5, OG5, F, CI, Br, I, C≡CH, S02G5, NHCOG5, NHS02G5, N02, CON(G5)2 or phenyl;
G2 is G5, OG5, F, CI, Br, I, C≡CH, S02G5, NHCOG5, NHS02G5, N02, CON(G5)2 or phenyl;
G3 is G5, OG5, F, CI, Br, I, C≡CH, S02G5, NHCOG5, NHS02G5, N02, CON(G5)2 or phenyl;
G4 is G5, OG5, F, CI, Br, I, C≡CH, S02G5, NHCOG5, NHS02G5, N02, CON(G5)2 or phenyl;
G5 is H, or a C1-C6 branched or unbranched, saturated or unsaturated, alkyl;
J1 is N-G5, S, or O;
M1 is G5, OG5, F, CI, Br, I, N3, C≡CH, or phenyl;
M2 is G5, OG5, F, CI, Br, I, N3, C≡CH, or phenyl;
M3 is G5, OG5, F, CI, Br, I, N3, C≡CH, or phenyl;
M4 is G5, OG5, F, CI, Br, I, N3, C≡CH, or phenyl;
M5 is G5, OG5, F, CI, Br, I, N3, C≡CH, or phenyl;
M6 is G5, OG5, F, CI, Br, I, N , C≡CH, or phenyl;
M7 is H or OH;
provided that the compound is not
Figure imgf000124_0001
A method of treating a microbial infection comprising administering a compound of Formula (A) or Formula (B):
Figure imgf000124_0002
t thereof, wherein:
R1 is N-R5, S, or O;
R2 is C-H, or ;
R3 is C-H, C-OR5, N, C-OCH2C≡CH, C-OCH2OCH3, C-OCH2OCH2CH3, C- OC(0)CH3, or C-OCH2(OCH2CH2)nOR5;
R4 is C;
R5 is H, or a C1 -C6 branched or unbranched, saturated or unsaturated, alkyl or acyl group;
n is 1-5;
A1 is R5, OR5, F, CI, Br, I, C≡CH, S02R5, NHCOR5, NHS02R5, N02, CON(R5)2 or phenyl; A2 is H, F, CI, Br, I, C≡CH, OR5, or phenyl;
A3 is F, CI, Br, I, C≡CH, OR5, R5, S02R5, NHCOR5, NHS02R5, N02, CON(R5)2 phenyl;
A4 is F, CI, Br, I, C≡CH, OR5, R5, S02R5, NHCOR5, NHS02R5, N02, CON(R5)2 phenyl;
Q1 is R5, OR5, F, CI, Br, I, N3, C≡CH, or phenyl;
Q2 is R5, OR5, F, CI, Br, I, N3, C≡CH, or phenyl;
Q3 is R5, OR5, F, CI, Br, I, N3, C≡CH, or phenyl;
Q4 is H or OH;
or Q 2 and Q optionally form a benzene ring, optionally substituted with R 5 , OR 5 CI, Br, I, N3, C≡CH, or phenyl;
Figure imgf000125_0001
wherein,
D1 is R5;
E1 is R5 or phenyl;
E 2 is R 5 or phenyl; and
E is R or phenyl.
48. The method of claim 47, wherein, R3 is C-H, C-OR5, N, C-OCH2C≡CH, C- OCH2OCH3, C-OCH2OCH2CH3, or C-OC(0)CH3.
49. The method of claim 47 or 48, wherein R3 is C-H, C-OH, C-OMe, C-OEt, N, C- OCH2C≡CH, C-OCH2OCH3, C-OCH2OCH2CH3, or C-OC(0)CH3.
50. The method of any one of claims 47-48, wherein R3 is C-H, C-OR5, or N.
51. The method of any one of claims 47-50, wherein R3 is C-H, C-OH, C-OMe, C-OEt, or N.
52. The method of any one of claims 47-51 , wherein R1 is N-H, N-Me, N-Et, S, or O.
53. The method of any one of claims 47-52, wherein R1 is N-R5.
54. The method of any one of claims 47-53, wherein R1 is N-H, N-Me, or N-Et.
55. The method of any one of claims 47-54, wherein R1 is S, or O.
56. The method of any one of claims 47-55, wherein R is N.
57. The method of any one of claims 47-55, wherein R is C-H.
58. The method of any one of claims 47-57, wherein R5 is a CI -C6 branched or
unbranched, saturated or unsaturated, alkyl;
59. The method of any one of claims 47-58, wherein A1 is R5, OR5, F, CI, Br, I, C≡CH, or phenyl.
60. The method of any one of claims 47-58, wherein A1 is H, O-Me, F, CI, Br, I, C≡CH, or phenyl.
61. The method of any one of claims 47-58, wherein A1 is H, O-Me, F, CI, Br, or I.
62. The method of any one of claims 47-61 , wherein A2 is H, F, CI, Br, I, C≡CH, or
phenyl.
63. The method of any one of claims 47-61 , wherein A2 is H, O-Me, F, CI, Br, I, C≡CH, or phenyl.
64. The method of any one of claims 47-61, wherein A2 is H, O-Me, F, CI, Br, or I.
65. The method of any one of claims 47-64, wherein A3 is F, CI, Br, I, C≡CH, OR5, R5, or phenyl.
66. The method of any one of claims 47-64, wherein A3 is F, CI, Br, I, C≡CH, H, or O- Me.
67. The method of any one of claims 47-64, wherein A3 is H, O-Me, F, CI, Br, or I.
68. The method of any one of claims 47-66, wherein A3 is F, CI, Br, I, OR5, R5, or phenyl.
69. The method of any one of claims 47-68, wherein A4 is F, CI, Br, I, C≡CH, OR5, R5, or phenyl.
70. The method of any one of claims 47-68, wherein A4 is F, CI, Br, I, C≡CH, OR5, R5, N02, CON(R5)2 or phenyl.
71. The method of any one of claims 47-68, wherein A4 is F, CI, Br, I, OR5, R5, or N02.
72. The method of any one of claims 47-68, wherein A4 is H, O-Me, F, CI, Br, or I.
73. The method of any one of claims 47-72, wherein Q1"3 are independently selected from R5, OR5, F, CI, Br, I, and N3.
74. The method of any one of claims 47-72, wherein Q1"3 are independently selected from H, OMe, or OH.
75. The method of any one of claims 47-74, wherein Q4 is H.
76. The compound of any one of claims 47-75, wherein L is
Figure imgf000127_0001
77. The method of any one of claims 47-76, wherein D1 is H or Me.
78. The method of any one of claims 47-75, wherein E1"3 are independently selected from H, Me, Et, phenyl, and tert-butyl.
79. The method of any one of claims 47-75, wherein E1"3 are independently selected from H, Me, and Et.
80. The method of any one of claims 47-77, wherein E1 is selected from H, Me, Et, phenyl, and tert-butyl.
81. The method of any one of claims 47-78, wherein E1 is selected from phenyl, Me, and Et.
82. The method of any one of claims 47-75, wherein E " are independently selected from Me and Et.
83. The method of any one of claims 47-75, wherein E " are Me.
84. The method of any one of claims 47-81, wherein E1 is Et, Me, or phenyl.
85. The method of any one of claims 47-81 , wherein E1 is Me.
86. A method of treating a microbial infection comprising administering a compound of Formula (A):
Figure imgf000127_0002
or a salt thereof, wherein:
R1 is N-R5; R2 is C-H;
R3 is C-OH;
R5 is H or Me or Et;
A1 is R5, F, CI, Br, or I;
A2 is H, F, CI, Br, or I;
A3 is OMe, F, CI, Br, I, or R5;
A4 is F, CI, Br, I, or R5;
Q1 is R5, F, CI, Br, or I;
Q2 is R5, F, CI, Br, or I;
Q3 is R5, F, CI, Br, or I;
Q4 is H or OH;
L is
Figure imgf000128_0001
wherein,
D1 is R5; and
E1 is R5 or phenyl.
The method of claim 86, wherein: R1 is N-H or N-Me; R3 is C-OH; A1 is H; A2 is H, or F; A3 is OMe, F, CI, Br, or H; A4 is H, or F; Q1 is H; Q2 is H; Q3 is Br, or I; Q4 is H; L is
Figure imgf000128_0002
; wherein, D is H; and E is Me, Et, or phenyl. A method of treating a microbial infection comprising administering a compound
Figure imgf000129_0001
selected from TABLE 2A or is
A method of treating a microbial infection comprising administering a compound
Figure imgf000129_0002
selected from TABLE 2B or is
90. A method of treating a microbial infection comprising administering a compound r
Figure imgf000129_0003
selected from TABLE 2C or is
A method of treating a microbial infection comprising administering a compound r
Figure imgf000129_0004
selected from TABLE 2D or is
92. A method of treating a microbial infection comprising administering a compound of the Formula (C):
Figure imgf000129_0005
or a salt thereof, wherein: G1 is G5, OG5, F, CI, Br, I, C≡CH, S02G5, NHCOG5, NHS02G5, N02, CON(G5)2 or phenyl;
G2 is G5, OG5, F, CI, Br, I, C≡CH, S02G5, NHCOG5, NHS02G5, N02, CON(G5)2 or phenyl;
G3 is G5, OG5, F, CI, Br, I, C≡CH, S02G5, NHCOG5, NHS02G5, N02, CON(G5)2 or phenyl;
G4 is G5, OG5, F, CI, Br, I, C≡CH, S02G5, NHCOG5, NHS02G5, N02, CON(G5)2 or phenyl;
G5 is H, or a C1-C6 branched or unbranched, saturated or unsaturated, alkyl;
J1 is N-G5, S, or O;
M1 is G5, OG5, F, CI, Br, I, N3, C≡CH, or phenyl;
M2 is G5, OG5, F, CI, Br, I, N3, C≡CH, or phenyl;
M3 is G5, OG5, F, CI, Br, I, N3, C≡CH, or phenyl;
M4 is G5, OG5, F, CI, Br, I, N3, C≡CH, or phenyl;
M5 is G5, OG5, F, CI, Br, I, N3, C≡CH, or phenyl;
M6 is G5, OG5, F, CI, Br, I, N3, C≡CH, or phenyl; and
M7 is H or OH.
93. A method of treating a microbial infection comprising administering
Figure imgf000130_0001
A compound of Formula A) or Formula (B):
Figure imgf000131_0001
t thereof, wherein:
R1 is N-R5, S, or O;
R2 is C-H, or N;
R3 is C-H, C-OR5, N, C-OCH2C≡CH, C-OCH2OCH3, C-OCH2OCH2CH3, C- OC(0)CH3, or C-OCH2(OCH2CH2)nOR5;
R4 is C;
R5 is H, or a C1-C6 branched or unbranched, saturated or unsaturated, alkyl or acyl group;
n is 1-5;
A1 is R5, OR5, F, CI, Br, I, C≡CH, S02R5, NHCOR5, NHS02Rs, N02, CON(R5)2 or phenyl;
A2 is H, F, CI, Br, I, C≡CH, OR5, or phenyl;
A3 is F, CI, Br, I, C≡CH, OR5, R5, S02R5, NHCOR5, NHS02R5, N02, CON(R5)2 or phenyl;
A4 is F, CI, Br, I, C≡CH, OR5, R5, S02R5, NHCOR5, NHS02R5, N02, CON(R5)2 or phenyl;
Q1 is R5, OR5, F, CI, Br, I, N3, C≡CH, or phenyl;
Q2 is R5, OR5, F, CI, Br, I, N3, C≡CH, or phenyl;
Q3 is R5, OR5, F, CI, Br, I, N3, C≡CH, or phenyl; Q4 is H or OH;
or Q2 and Q3 optionally form a benzene ring optionally substituted with R5, OR5, F, CI,
Br, I, N3, C≡CH, or phenyl;
L is
Figure imgf000132_0001
wherein,
D1 is R5;
E1 is R5 or phenyl;
E is R or phenyl; and
E is R or phenyl;
for the treatment of a microbial infection.
95. The compound of claim 94, wherein, R3 is C-H, C-OR5, N, C-OCH2C≡CH, C- OCH2OCH3, C-OCH2OCH2CH3, or C-OC(0)CH3.
96. The compound of claim 94 or 95, wherein R3 is C-H, C-OH, C-OMe, C-OEt, N, C- OCH2C≡CH, C-OC¾OCH3, C-OCH2OCH2CH3, or C-OC(0)CH3.
97. The compound of any one of claims 94-96, wherein R3 is C-H, C-OR5, or N.
98. The compound of any one of claims 94-97, wherein R3 is C-H, C-OH, C-OMe, C-OEt, or N.
99. The compound of any one of claims 94-98, wherein R1 is N-H, N-Me, N-Et, S, or O.
100. The compound of any one of claims 94-99, wherein R1 is N-R5.
101. The compound of any one of claims 94-100, wherein R1 is N-H, N-Me, or N-Et.
102. The compound of any one of claims 94- 101, wherein R1 is S, or O.
103. The compound of any one of claims 94- 102, wherein R2 is N.
104. The compound of any one of claims 94-102, wherein R2 is C-H.
105. The compound of any one of claims 94-104, wherein R5 is a C1-C6 branched or
unbranched, saturated or unsaturated, alkyl;
106. The compound of any one of claims 94-105, wherein A1 is R5, OR5, F, CI, Br, I, C≡CH, or phenyl.
107. The compound of any one of claims 94-105, wherein A1 is H, O-Me, F, CI, Br, I, C≡CH, or phenyl.
108. The compound of any one of claims 94-105, wherein A1 is H, O-Me, F, Cl, Br, or I.
109. The compound of any one of claims 94-108, wherein A is H, F, Cl, Br, I, C≡CH, or phenyl.
110. The compound of any one of claims 94-108, wherein A is H, O-Me, F, Cl, Br, I, C≡CH, or phenyl.
111. The compound of any one of claims 94-108, wherein A is H, O-Me, F, Cl, Br, or I.
1 12. The compound of any one of claims 94- 111 , wherein A3 is F, Cl, Br, I, C≡CH, OR5, R5, or phenyl.
1 13. The compound of any one of claims 94-1 1 1 , wherein A is F, Cl, Br, I, C≡CH, H, or O-Me.
1 14. The compound of any one of claims 94-1 1 1 , wherein A3 is H, O-Me, F, Cl, Br, or I.
115. The compound of any one of claims 94- 1 13, wherein A3 is F, Cl, Br, I, OR5, R5, or phenyl.
116. The compound of any one of claims 94- 1 15, wherein A4 is F, Cl, Br, I, C≡CH, OR5, R5, or phenyl.
1 17. The compound of any one of claims 94- 1 15, wherein A4 is F, Cl, Br, I, C≡CH, OR5, R5, N02, CON(R5)2 or phenyl.
118. The compound of any one of claims 94- 115, wherein A4 is F, Cl, Br, I, OR5, R5, or N02.
119. The compound of any one of claims 94-1 15, wherein A4 is H, O-Me, F, Cl, Br, or I.
120. The compound of any one of claims 94-119, wherein Q1"3 are independently selected from R5, OR5, F, Cl, Br, I, and N3.
121. The compound of any one of claims 94-1 19, wherein Q1 "3 are independently selected from H, OMe, or OH.
122. The compound of any one of claims 94- 121 , wherein Q4 is H.
123. The compound of any one of claims 94-122, wherein L is
Figure imgf000134_0001
124. The compound of any one of claims 94-123, wherein D1 is H or Me.
125. The compound of any one of claims 94-122, wherein E " are independently selected from H, Me, Et, phenyl, and tert-butyl.
126. The compound of any one of claims 94- •122, wherein E
from H, Me, and Et.
127. The compound of any one of claims 94- •124, wherein E
phenyl, and tert-butyl.
128. The compound of any one of claims 94- ■125, wherein E
and Et.
129. The compound of any one of claims 94- ■122, wherein E
from Me and Et.
130. The compound of any one of claims 94- •122, wherein E 1 -3
131. The compound of any one of claims 94- ■128, wherein E
132. The compound of any one of claims 94- ■128, wherein E
133. A compound of Formula A) :
Figure imgf000134_0002
or a salt thereof, wherein:
R1 is N-R5;
R2 is C-H;
R3 is C-OH;
R5 is H or Me or Et;
A1 is R5, F, CI, Br, or I;
A2 is H, F, CI, Br, or I; A3 is OMe, F, CI, Br, I, or R5;
A4 is F, CI, Br, I, or R5;
Q1 is R5, F, CI, Br, or I;
Q2 is R5, F, CI, Br, or I;
Q3 is R5, F, CI, Br, or I;
Q4 is H or OH;
L is
Figure imgf000135_0001
wherein,
D1 is R5; and
E1 is R5 or phenyl;
for the treatment of a microbial infection.
134. The compound of claim 133, wherein: R1 is N-H or N-Me; R3 is C-OH; A1 is H; A2 H, or F; A3 is OMe, F, CI, Br, or H; A4 is H, or F; Q1 is H; Q2 is H; Q3 is Br, or I; H; L is
Figure imgf000135_0002
; wherein, D1 is H; and E1 is Me, Et, or phenyl.
135. A compound selected from TABLE 2A or is
Figure imgf000135_0003
for the treatment of a microbial infection. A compound selected from TABLE 2B
Figure imgf000136_0001
treatment of a microbial infection.
137. A compound selected from TABLE 2C or is
Figure imgf000136_0002
treatment of a microbial infection.
A compound selected from TABLE 2D
Figure imgf000136_0003
treatment of a microbial infection.
139. A compound of the Formula (C):
Figure imgf000136_0004
or a salt thereof, wherein:
G1 is G5, OG5, F, CI, Br, I, C≡CH, S02G5, NHCOG5, NHS02G5, N02, CON(G5)2 or phenyl;
G2 is G5, OG5, F, CI, Br, I, C≡CH, S02G5, NHCOG5, NHS02G5, N02, CON(G5)2 or phenyl;
G3 is G5, OG5, F, CI, Br, I, C≡CH, S02G5, NHCOG5, NHS02G5, N02, CON(G5)2 or phenyl; G4 is G5, OG5, F, CI, Br, I, C≡CH, S02G5, NHCOG5, NHS02G5, N02, CON(G5)2 or phenyl;
G5 is H, or a C1-C6 branched or unbranched, saturated or unsaturated, alkyl;
J1 is N-G5, S, or O;
M1 is G5, OG5, F, CI, Br, I, N3, C≡CH, or phenyl;
M2 is G5, OG5, F, CI, Br, I, N3, C≡CH, or phenyl;
M3 is G5, OG5, F, CI, Br, I, N3, C≡CH, or phenyl;
M4 is G5, OG5, F, CI, Br, I, N3, C≡CH, or phenyl;
M5 is G5, OG5, F, CI, Br, I, N3, C≡CH, or phenyl;
M6 is G5, OG5, F, CI, Br, I, N3, C≡CH, or phenyl; and
M7 is H or OH;
for the treatment of a microbial infection.
140. The compound of claim 139, having the formula
Figure imgf000137_0001
141. Use of a compound of any one of claims 94-140 for the treatment of a microbial infection.
142. Use of a compound of any one of claims 94-140 for the manufacture of a medicament for treating a microbial infection.
143. A pharmaceutical composition for treating a microbial infection, comprising a compound of any one of claims 94-140 and a pharmaceutically acceptable carrier.
144. A commercial package comprising (a) a compound of any one of claims 94-140; and (b) instructions for the use thereof for treating a microbial infection.
145. A commercial package comprising (a) a pharmaceutical composition comprising a compound of any one of claims 94-140 and a pharmaceutically acceptable carrier; and (b) instructions for the use thereof for treating a microbial infection.
146. A method for testing a candidate compound for selective binding to a pathogen pyruvate kinase, the method comprising:
(a) combining the candidate compound with a pathogen pyruvate kinase monomenc subunits;
(b) combining the candidate compound with human pyruvate kinase monomeric subunits; and
(c) assaying for pyruvate kinase tetramer/dimer formation in both (a) and (b).
147. The method of claim 146, wherein the pathogen is MRSA.
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