US20150018543A1 - Anti-infective compounds - Google Patents

Anti-infective compounds Download PDF

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US20150018543A1
US20150018543A1 US14/263,218 US201414263218A US2015018543A1 US 20150018543 A1 US20150018543 A1 US 20150018543A1 US 201414263218 A US201414263218 A US 201414263218A US 2015018543 A1 US2015018543 A1 US 2015018543A1
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nmr
mhz
cdcl
pyrido
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Priscille Brodin
Thierry Christophe
Zaesung No
Jaeseung Kim
Auguste Genovesio
Denis Philippe Cedric Fenistein
Heekyoung Jeon
Fanny Anne Ewann
Sunhee Kang
Saeyeon Lee
Min Jung Seo
Eunjung PARK
Monica Contreras Dominguez
Ji Youn Nam
Eun Hye Kim
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Institut National de la Sante et de la Recherche Medicale INSERM
Institut Pasteur Korea
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Institut National de la Sante et de la Recherche Medicale INSERM
Institut Pasteur Korea
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    • 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
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/18—Testing for antimicrobial activity of a material
    • 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
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • 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
    • A61P31/06—Antibacterial agents for tuberculosis
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    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C255/00—Carboxylic acid nitriles
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    • C07C255/65—Carboxylic acid nitriles containing cyano groups and nitrogen atoms further bound to other hetero atoms, other than oxygen atoms of nitro or nitroso groups, bound to the same carbon skeleton with the nitrogen atoms further bound to nitrogen atoms
    • C07C255/66—Carboxylic acid nitriles containing cyano groups and nitrogen atoms further bound to other hetero atoms, other than oxygen atoms of nitro or nitroso groups, bound to the same carbon skeleton with the nitrogen atoms further bound to nitrogen atoms having cyano groups and nitrogen atoms being part of hydrazine or hydrazone groups bound to the same carbon skeleton
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    • C07C335/00—Thioureas, i.e. compounds containing any of the groups, the nitrogen atoms not being part of nitro or nitroso groups
    • C07C335/40—Thioureas, i.e. compounds containing any of the groups, the nitrogen atoms not being part of nitro or nitroso groups having nitrogen atoms of thiourea or isothiourea groups further bound to other hetero atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D223/00—Heterocyclic compounds containing seven-membered rings having one nitrogen atom as the only ring hetero atom
    • C07D223/14—Heterocyclic compounds containing seven-membered rings having one nitrogen atom as the only ring hetero atom condensed with carbocyclic rings or ring systems
    • C07D223/18—Dibenzazepines; Hydrogenated dibenzazepines
    • C07D223/22—Dibenz [b, f] azepines; Hydrogenated dibenz [b, f] azepines
    • C07D223/24—Dibenz [b, f] azepines; Hydrogenated dibenz [b, f] azepines with hydrocarbon radicals, substituted by nitrogen atoms, attached to the ring nitrogen atom
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D233/00—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings
    • C07D233/54—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings having two double bonds between ring members or between ring members and non-ring members
    • C07D233/66—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings having two double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D233/84—Sulfur atoms
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    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D251/00—Heterocyclic compounds containing 1,3,5-triazine rings
    • C07D251/02—Heterocyclic compounds containing 1,3,5-triazine rings not condensed with other rings
    • C07D251/12—Heterocyclic compounds containing 1,3,5-triazine rings not condensed with other rings having three double bonds between ring members or between ring members and non-ring members
    • C07D251/26—Heterocyclic compounds containing 1,3,5-triazine rings not condensed with other rings having three double bonds between ring members or between ring members and non-ring members with only hetero atoms directly attached to ring carbon atoms
    • C07D251/40—Nitrogen atoms
    • C07D251/48—Two nitrogen atoms
    • C07D251/52—Two nitrogen atoms with an oxygen or sulfur atom attached to the third ring carbon atom
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    • C07D251/00—Heterocyclic compounds containing 1,3,5-triazine rings
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    • C07D251/12—Heterocyclic compounds containing 1,3,5-triazine rings not condensed with other rings having three double bonds between ring members or between ring members and non-ring members
    • C07D251/26—Heterocyclic compounds containing 1,3,5-triazine rings not condensed with other rings having three double bonds between ring members or between ring members and non-ring members with only hetero atoms directly attached to ring carbon atoms
    • C07D251/40—Nitrogen atoms
    • C07D251/54—Three nitrogen atoms
    • C07D251/66—Derivatives of melamine in which a hetero atom is directly attached to a nitrogen atom of melamine
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    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D271/00—Heterocyclic compounds containing five-membered rings having two nitrogen atoms and one oxygen atom as the only ring hetero atoms
    • C07D271/02—Heterocyclic compounds containing five-membered rings having two nitrogen atoms and one oxygen atom as the only ring hetero atoms not condensed with other rings
    • C07D271/10—1,3,4-Oxadiazoles; Hydrogenated 1,3,4-oxadiazoles
    • C07D271/113—1,3,4-Oxadiazoles; Hydrogenated 1,3,4-oxadiazoles with oxygen, sulfur or nitrogen atoms, directly attached to ring carbon atoms, the nitrogen atoms not forming part of a nitro radical
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    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
    • C07D401/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings linked by a chain containing hetero atoms as chain links
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D409/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms
    • C07D409/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing two hetero rings
    • C07D409/12—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing two hetero rings linked by a chain containing hetero atoms as chain links
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
    • C07D417/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings
    • C07D417/12—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings linked by a chain containing hetero atoms as chain links
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
    • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
    • C07D471/04—Ortho-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D498/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms
    • C07D498/02—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms in which the condensed system contains two hetero rings
    • C07D498/04—Ortho-condensed systems

Definitions

  • the present invention relates to small molecule compounds and their use in the treatment of bacterial infections, in particular Tuberculosis.
  • Tuberculosis (TB) as a disease continues to result in millions of deaths each year.
  • Inadequate use of chemotherapy has led to an increasing number of drug resistant cases. This situation is likely to worsen with the emergence of extremely resistant strains to all currently known drugs (Van Rie and Enarson, 2006).
  • the internationally recommended TB control strategy also referred to as directly observed short-course chemotherapy (DOTS), relies on a combination of five antibacterial agents to be taken for a protracted period of more than six months (http://www.who.int/tb/dots/en/).
  • the bacillus mainly localizes inside phagocytic cells, such as macrophages and dendritic cells, and it has clearly been established that the tubercle bacillus adopts a different phenotype in the host macrophage's phagosome compared to growth in extracellular conditions (Rohde et al., 2007; Schnappinger et al., 2003).
  • phagocytic cells such as macrophages and dendritic cells
  • the present invention relates to compounds having the general formula VIII:
  • n 0, 1, 2, or 3;
  • X 3 is selected from the group comprising CH 2 , O, S and NH;
  • X 4 is selected from the group comprising halide, alkyl, OR 23 , SR 24 and NR 25 R 26 ;
  • R 20 is selected from the group comprising acyl, alkoxy, alkyl, alkylamino, alkylcarboxylic acid, arylcarboxylic acid, alkylcarboxylic alkylester, alkylene, alkylether, alkylhydroxy, alkylthio, alkynyl, amido, amino, aryl, arylalkoxy, arylamino, arylthio, carboxylic acid, cyano, cycloalkyl, carboxylic acid, ester, halo, haloalkoxy, haloalkyl, haloalkylether, heteroaryl, heteroarylamino, heterocycloalkyl and hydrogen, any of which is optionally substituted;
  • R 21 and R 22 are each independently selected from the group comprising alkoxy, alkyl, alkylamino, alkylene, alkylether, alkylthio, alkynyl, amido, amino, aryl, arylether, arylalkoxy, arylamino, arylthio, carboxy, cyano, cycloalkyl, ester, halo, haloalkoxy, haloalkyl, heteroaryl, heteroarylamino, heterocycloalkyl, hydroxyl, hydrogen, nitro, thio, sulfonate, sulfonyl and sulfonylamino, any of which is optionally substituted;
  • R 23 is selected from the group comprising acyl, alkyl, alkylamino, alkylene, alkynyl, aryl, arylalkoxy, arylamino, arylthio, carboxy, cycloalkyl, ester, ether, haloalkyl, heteroaryl, heteroarylamino, heterocycloalkyl, hydrogen, thio, sulfonate, and sulfonylamino, any of which is optionally substituted;
  • R 24 is selected from the group comprising alkyl, alkylaryl, alkylene, alkynyl, aryl, cycloalkyl, ester, halo, haloalkyl, heteroaryl, heterocycloalkyl, and hydrogen, any of which is optionally substituted; and
  • R 25 and R 26 are each independently selected from the group comprising acyl, alkyl, aminoalkyl, alkylene, alkylthio, alkynyl, aryl, arylalkoxy, arylamino, arylthio, carboxy, cycloalkyl, ester, ether, halo, haloalkoxy, haloalkyl, haloalkylether, heteroaryl, heteroarylamino, heterocycloalkyl and hydrogen, any of which is optionally substituted.
  • the term “optionally substituted” as used herein is meant to indicate that a group, such as alkyl, alkylen, alkynyl, aryl, cycloalkyl, heterocycloalkyl, or heteroaryl, may be unsubstituted or substituted with one or more substituents. “Substituted” in reference to a group indicates that a hydrogen atom attached to a member atom within a group is replaced.
  • the present invention relates to compounds having the general formula VIIIa:
  • X 5 is selected from the group comprising CH 2 , C ⁇ O and C ⁇ S;
  • Z 1 and Z 2 are each independently selected from the group comprising alkoxy, alkyl, alkylamino, alkylene, alkylether, alkylthio, alkynyl, amido, amino, aryl, arylether, arylalkoxy, arylamino, arylthio, carboxy, cyano, cycloalkyl, ester, halo, haloalkoxy, haloalkyl, heteroaryl, heteroarylamino, heterocycloalkyl, hydroxyl, and hydrogen, or two groups are connected each other to make five or six membered cyclic, heterocyclic and heteroaryl rings, any of which is optionally substituted;
  • R 27 and R 28 are each independently selected from the group comprising alkoxy, alkylamino, alkylene, alkylether, alkylthio, alkynyl, amido, amino, aryl, arylether, arylalkoxy, arylamino, arylthio, carboxy, cyano, cycloalkyl, ester, halo, haloalkoxy, haloalkyl, heteroaryl, heteroarylamino, heterocycloalkyl, hydroxyl, hydrogen, nitro, thio, sulfonate, sulfonyl and sulfonylamino, any of which is optionally substituted;
  • R 29 and R 30 are each independently selected from the group comprising alkoxy, alkyl, alkylamino, alkylene, alkylether, alkylthio, alkynyl, amido, amino, aryl, arylether, arylalkoxy, arylamino, arylthio, carboxy, cyano, cycloalkyl, ester, halo, haloalkoxy, haloalkyl, heteroaryl, heteroarylamino, heterocycloalkyl, hydroxyl, hydrogen, nitro, thio, sulfonate, sulfonyl and sulfonylamino, or two groups are connected each other to make five or six membered cyclic, heterocyclic, aryl, and heteroaryl rings, any of which is optionally substituted.
  • alkyl as used herein is meant to indicate that a group, such as substituted or non-substituted C 1 -C 10 alkyl group which has the straight or branched chain.
  • cycloalkyl as used herein is meant to indicate that a group, such as substituted or non-substituted cyclic compound of C 3 -C 8 ring structure.
  • heteroaryl as used herein is meant to indicate that a group, such as substituted or non-substituted 5- to 9-membered aromatic compounds which have more than one heteroatom of N, O, and S in the ring structure itself.
  • a hydrogen atom attached to a member atom within a group is possibly replaced by group, such as C 1 -C 10 alkyl, halogen including fluorine, OH, NO 2 , OR 31 , CN, NR 31 R 32 , COR 31 , SOR 32 , SO 2 R 31 , SO 2 NR 31 , CR 31 ⁇ CR 31 R 32 , CR 31 ⁇ NR 32 , aryl, aryloxy, C 4 -C 10 heteroaryl group, or —NR 31 —COR 32 , —O—COR 31 .
  • group such as C 1 -C 10 alkyl, halogen including fluorine, OH, NO 2 , OR 31 , CN, NR 31 R 32 , COR 31 , SOR 32 , SO 2 R 31 , SO 2 NR 31 , CR 31 ⁇ CR 31 R 32 , CR 31 ⁇ NR 32 , aryl, aryloxy, C 4 -C 10 heteroaryl group,
  • R 31 and R 32 are each independently selected from the group comprising hydrogen, alkyl, alkyloxy, alkylamino, alkylcarbonyl, alkylcarbonylamino, alkylcarbonyloxy, alkylaminocarbonyl, alkyloxycarbonyl, cycloalkyl, cycloalkyloxy, cycloalkylamino, cycloalkylcarbonyl, cycloalkylcarbonylamino, cycloalkylcarbonyloxy, cycloalkylaminocarbonyl, cycloalkyloxycarbonyl, heteroaryl, heteroaryloxy, heteroaryl amino, heteroaryl carbonyl, heteroaryl carbonylamino, heteroaryl carbonyloxy, heteroaryl aminocarbonyl, heteroaryl oxycarbonyl, heteroaryl alkyl, heteroaryl alkyloxy, heteroaryl alkylamino, heteroaryl alkylcarbonyl, heteroaryl alkylcarbonyl, heteroaryl
  • the present invention relates to compounds having one of the formulas 125-301 as shown in Example 7, preferably 132-135, 137, 139-140, 147, 151-152, 160, 163, 173, 180, 184-185, 193, 195, 199-201, 204, 206-222, 224, 226, 229, 231-243, 245-278, 280-286 and 290-301 as shown in Table 3.
  • Particularly preferred compounds are compounds having one of the formulas 133, 232 and 264 as shown in Table 3.
  • the present invention relates to compounds having the general formula II:
  • R 5 and R 6 are each independently selected from the group comprising acyl, alkyl, alkylamino, alkylene, alkylthio, alkynyl, aryl, arylalkoxy, arylamino, arylthio, carboxy, cycloalkyl, ester, haloalkoxy, haloalkyl, heteroaryl, heteroarylamino, heterocycloalkyl, hydroxyl, hydrogen, sulfonate and sulfonyl, any of which is optionally substituted and
  • R 7 , R 8 and R 9 are each independently selected from the group comprising alkoxy, alkyl, alkylamino, alkylene, alkylthio, alkynyl, amido, amino, aryl, arylalkoxy, arylamino, arylthio, carboxy, cyano, cycloalkyl, ester, halo, haloalkoxy, haloalkyl, heteroaryl, heteroarylamino, heterocycloalkyl, hydroxyl, hydrogen, nitro, thio, sulfonate, sulfonyl and sulfonylamino, any of which is optionally substituted.
  • the present invention relates to compounds with the general formula II, wherein R 5 and R 6 are connected, having the general formula IIa:
  • n 0, 1, 2, or 3;
  • Y and Z are each independently selected from the group comprising CH 2 , CHOR 10 , CHNR 10 R 11 , CR 10 R 11 and NR 10 ;
  • R 10 and R 11 are each independently selected from the group comprising acyl, alkyl, alkylamino, alkylene, alkylthio, alkynyl, aryl, arylalkoxy, arylamino, arylthio, carboxy, cycloalkyl, ester, haloalkoxy, haloalkyl, heteroaryl, heteroarylamino, heterocycloalkyl, hydrogen, sulfonate and sulfonyl, any of which is optionally substituted.
  • the present invention relates to compounds having one of the formulas with the general formula/scaffold II as shown in FIGS. 9-1 to 9 - 41 , as well as one of the formulas 1-123 as shown in Example 6, preferably 1-24, 26-34, 54, 56, 58-61, 63-64, 67, 90-101, 103-105, 107-109, 112, 114-116 and 118-121 as shown in Table 3.
  • Particularly preferred compounds are compounds having one of the formulas 4 and 24 as shown in Table 3.
  • the compounds as defined above have an inhibitory activity, preferably an inhibitory activity above 65%, on bacterial growth, preferably on the growth of M tuberculosis, inside a host cell, preferably a macrophage, at a concentration between 5-20 ⁇ M, preferably less than 5 ⁇ M.
  • the present invention relates to compounds as defined above for use in the treatment of bacterial infections.
  • the present invention relates to compounds as defined above for use in the treatment of Tuberculosis.
  • the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a compound as defined above.
  • the present invention relates to a method of treatment of Tuberculosis, comprising the application of a suitable amount of a compound as defined above to a person in need thereof.
  • the present invention relates to compounds having one of the general formulas/scaffolds I, III-VII and IX-XX as shown in Table 2.
  • the present invention relates to compounds having the general formula I:
  • X 1 and X 2 are each independently selected from the group comprising CH and NH;
  • R 1 and R 2 are each independently selected from the group comprising alkoxy, alkyl, alkylamino, alkylene, alkylthio, alkynyl, amido, amino, aryl, arylalkoxy, arylamino, arylthio, carboxy, cyano, cycloalkyl, ester, halo, haloalkoxy, haloalkyl, heteroaryl, heteroarylamino, heterocycloalkyl, hydroxyl, hydrogen, nitro, thio, sulfonate, sulfonyl and sulfonylamino, any of which is optionally substituted; and
  • R 3 and R 4 are each independently selected from the group comprising alkoxy, alkyl, alkylamino, alkylene, alkylthio, alkynyl, aryl, arylalkoxy, arylamino, arylthio, cyano, cycloalkyl, haloalkoxy, haloalkyl, heteroaryl, heteroarylamino, heterocycloalkyl and hydrogen, any of which is optionally substituted.
  • the present invention relates to compounds having the general formula III:
  • R 10 and R 11 are each independently selected from the group comprising alkoxy, alkyl, alkylamino, alkylene, alkylthio, alkynyl, amido, amino, aryl, arylalkoxy, arylamino, arylthio, carboxy, cyano, cycloalkyl, ester, halo, haloalkoxy, haloalkyl, heteroaryl, heteroarylamino, heterocycloalkyl, hydroxyl, hydrogen, nitro, thio, sulfonate, sulfonyl and sulfonylamino, any of which is optionally substituted.
  • the present invention relates to compounds having the general formula IV:
  • A is an optionally substituted heteroaryl, naphthyl and phenyl and
  • R 12 is selected from the group comprising alkoxy, alkyl, alkylamino, alkylene, alkylthio, alkynyl, amido, amino, aryl, arylalkoxy, arylamino, arylthio, carboxy, cyano, cycloalkyl, ester, halo, haloalkoxy, haloalkyl, heteroaryl, heteroarylamino, heterocycloalkyl, hydroxyl, hydrogen, nitro, thio, sulfonate, sulfonyl and sulfonylamino, any of which is optionally substituted.
  • the present invention relates to compounds having the general formula V:
  • R 13 , R 14 and R 15 are each independently selected from the group comprising alkoxy, alkyl, alkylamino, alkylene, alkylthio, alkynyl, amido, amino, aryl, arylalkoxy, arylamino, arylthio, carboxy, cyano, cycloalkyl, ester, halo, haloalkoxy, haloalkyl, heteroaryl, heteroarylamino, heterocycloalkyl, hydroxyl, hydrogen, nitro, thio, sulfonate, sulfonyl and sulfonylamino, any of which is optionally substituted.
  • the present invention relates to compounds having the general formula VI:
  • R 16 is selected from the group comprising alkoxy, alkyl, alkylamino, alkylene, alkynyl, amino, aryl, arylalkoxy, arylamino, arylthio, cycloalkyl, haloalkoxy, haloalkyl, heteroaryl, heteroarylamino, heterocycloalkyl, hydroxyl and hydrogen, any of which is optionally substituted and
  • R 17 is selected from the group comprising alkoxy, alkyl, alkylamino, alkylene, alkylthio, alkynyl, amido, amino, aryl, arylalkoxy, arylamino, arylthio, carboxy, cyano, cycloalkyl, ester, halo, haloalkoxy, haloalkyl, heteroaryl, heteroarylamino, heterocycloalkyl, hydroxyl, hydrogen, nitro, thio, sulfonate, sulfonyl and sulfonylamino, any of which is optionally substituted.
  • the present invention relates to compounds having the general formula VII:
  • R 18 and R 19 are each independently selected from the group comprising alkoxy, alkyl, alkylamino, alkylene, alkylthio, alkynyl, amido, amino, aryl, arylalkoxy, arylamino, arylthio, carboxy, cyano, cycloalkyl, ester, halo, haloalkoxy, haloalkyl, heteroaryl, heteroarylamino, heterocycloalkyl and hydrogen, any of which is optionally substituted.
  • the present invention relates to compounds having one of the formulas with the general formulas I, III-VII and IX-XX as shown in FIGS. 9-1 to 9 - 41 .
  • the present invention relates to a compound listed in Table 1.
  • the present invention relates to compounds as defined above for use in the treatment of bacterial infections.
  • the present invention relates to compounds as defined above for use in the treatment of Tuberculosis.
  • the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a compound as defined above.
  • the present invention relates to a method of treatment of Tuberculosis, comprising the application of a suitable amount of a compound as defined above to a person in need thereof.
  • the present invention relates to a screening method comprising the steps of
  • the screening method according to the present invention represents a phenotypic cell-based assay enabling the search for drugs that interfere with the multiplication of M. tuberculosis within host macrophages.
  • the assay makes use of fluorescently labeled living macrophages infected with fluorescently labeled mycobacteria and uses automated confocal fluorescence microscopy to measure intracellular mycobacterial growth.
  • the assay has been set-up for the high throughput screening (HTS) of large scale chemical libraries.
  • FIG. 1 shows the monitoring of tubercle bacillus intracellular growth inside macrophages by automated confocal microscopy:
  • FIG. 2 shows the pharmacological validation and MIC (minimal inhibitory concentration) comparison of the reference drugs in the in vitro growth fluorescence assay and the phenotypic cell-based assay:
  • FIG. 3 shows assay automation validation of the phenotypic cell-based assay:
  • FIG. 4 shows primary screening results for the phenotypic cell-based assay and the in vitro growth assay for 26500 compounds: (a) Percent inhibition based on infection ratio relative to each compound and distribution. (b) Percent inhibition based on RFU relative to each compound and distribution. (c) Comparison of inhibition percentage for the phenotypic cell-based assay and the in vitro growth assay for each compound;
  • FIG. 5 shows serial dilution results from the in vitro growth fluorescence assay and the phenotypic cell-based assay: Typical curves for compounds inhibiting (a,b,c) in vitro bacterial growth (d,e,f) both in vitro and intracellular growth and (g,h,i) intracellular growth only. (a,d,g) Infection ratio relative to compound concentration. (b,e,h) Cell number relative to compound concentration. (c,f,i) Relative fluorescence intensity relative to compound concentration. Compound concentration is given in M;
  • FIG. 6 shows (a) a scheme of assay automation. (b) a 384-plate format description; (c) a 384-plate dose-response curve description.
  • a to P and a to b correspond to 2-fold serial dilution of INH and Rifampin respectively with a starting concentration of 20 mg/mL in well A or a; RIF: Rifampin 5 ⁇ g/mL, Cpd: compound, INH100 1 ⁇ g/mL, INH50 0.05 ⁇ g/mL;
  • FIG. 7 shows the anti-tuberculosis effect of compounds 4 and 24 (5 ⁇ M) on M. tuberculosis H37Rv-GFP in (a) Raw267.4 (10 4 cells), (b) mouse bone marrow-derived macrophages and (c) human primary macrophages differentiated with 50 ng/mL rhM-CSF (1.5*10 4 ) after 7 days of infection with MOI 2.5:1 (control INH at 5 ⁇ M);
  • FIG. 8 illustrates the colony forming units (CFUs) recovered from macrophages at different time points after infection with M. tuberculosis H37Rv.
  • CFUs colony forming units
  • FIGS. 9-1 to 9 - 41 list 121 compounds which demonstrated an inhibitory activity above 65% at 2 ⁇ M without any apparent cell toxicity at 20 ⁇ M and consequently were selected for further confirmation by ten 3-fold serial dilutions;
  • Table 1 lists 340 hits whose inhibitory activity was confirmed in an intracellular (QIM) assay or an in vitro (QUM) assay, wherein the abbreviation “QIM” stands for Quantification of Intracellular Mycobacteria, the abbreviation “QUM” stands for Quantification of in vitro grown Mycobacteria, and the abbreviation “CellNb” stands for cell number;
  • Table 2 summarizes the independent/general molecular scaffolds/formulas of the 121 hits listed in FIGS. 9-1 to 9 - 41 ;
  • Table 3 lists dinitrobenzamide and pyridopyrimidinone derivatives (general scaffold II and VIII, respectively, see Table 2) with their respective inhibitory activities, wherein the numbers in bold print refer to the compounds listed in Examples 6 and 7;
  • Table 4 shows the cytotoxicity and antibacterial spectrum of dinitrobenzamide compounds 4 and 24 (see Table 3);
  • Table 5 shows the cytotoxicity and antibacterial spectrum of pyridopyrimidinone compound 133 (see Table 3).
  • Table 6 shows the frequency of spontaneous resistance for representative dinitrobenzamide and pyridopyrimidinone compounds.
  • H37Rv-GFP green fluorescent protein
  • tuberculosis H37Rv-GFP were prepared from 400 mL of a 15 days old Middlebrook 7H9 culture (Difco, Sparks Md., USA) supplemented with albumin-dextrose-catalase (ADC, Difco, Sparks Md., USA), glycerol and 0.05% Tween 80.
  • Bacilli were harvested by centrifugation at 3000 g for 20 min, washed twice with H 2 O at room temperature, and resuspended in 1-2 mL of 10% glycerol at room temperature after recentrifugation. 250 ⁇ l of bacilli were mixed with green fluorescent protein encoding plasmid and electroporated using a Biorad Gene Pulser (Biorad).
  • bacilli were resuspended in medium and left one day at 37° C.
  • Transformants were selected on Middlebrook 7H11 medium (Difco, Sparks Md., USA) supplemented with oleic acid-albumin-dextrose-catalase (OADC, Difco, Sparks Md., USA) and 50 ⁇ g/mL hygromycin (Invitrogen, Carlsbad, Calif. USA). The selected hygromycin-resistant and green fluorescent colonies appeared after 3 weeks.
  • a 100 mL culture of the H37Rv-GFP strain was grown in Middlebrook 7H9-ADC medium supplemented with 0.05% Tween 80 and 50 ⁇ g/mL of hygromycin.
  • Bacteria were harvested, washed twice and suspended in 50 mM sodium phosphate buffer (pH 7.5). The bacteria were then sonicated and allowed to stand for 1 hour to allow residual aggregates to settle. The bacterial suspensions were then aliquoted and frozen at ⁇ 80° C. A single defrosted aliquot was used to quantify the CFUs (colony forming units) prior to inoculation and typical stock concentrations were about 2 to 5 ⁇ 10 8 CFU/mL.
  • CFUs colony forming units
  • the small synthetic molecules from the screening libraries were suspended in pure DMSO (Sigma, D5879-500 mL) at a concentration of 10 mM (Master plates) in Corning 96 well clear V-bottom polypropylene plates (Corning, #3956). The compounds were then reformatted in Greiner 384 well V-shape polypropylene plates (Greiner, #781280) and diluted to a final concentration of 2 mM in pure DMSO. The compounds were kept frozen until use. For screening, compound plates were incubated at room temperature until thawed.
  • the compounds were directly added into the assay plates from the DMSO stock using an EVObird liquid handler (Evotec Technologies), which transfers 250 n1 of compound twice to reach a final dilution of 1:100. This one-step dilution reduces the risk of compound precipitation in intermediate plates and allows for a low final DMSO concentration (1%).
  • Positive control antibiotics Isoniazid (Sigma, 13377-50G) and Rifampin (Euromedex, 1059-8, 5 g)
  • negative controls DMSO
  • Cells were first seeded in 50 ⁇ l at a density of 20,000 cells per well of a 384-well plate (Evotec technologies #781058) for 16 hours and then infected with bacterial suspensions at a multiplicity of infection (MOI) varying from 10:1 to 1:1 (bacteria:host cells). After 2 hours, cells were washed three times with phosphate buffered saline (PBS) and the compounds diluted in fresh culture medium were added. Cells were incubated at 37° C., 5% CO 2 for up to seven days.
  • MOI multiplicity of infection
  • Cells (1.5 ⁇ 10 8 cells) were infected with H37Rv-GFP suspension at a MOI of 1:1 in 300 mL for 2 hours at 37° C. with shaking (100 rpm). After two washes by centrifugation at 1100 rpm (Beckman SX4250, 165 g) for 5 min., the remaining extracellular bacilli from the infected cells suspension were killed by a 1 hour amykacin (20 ⁇ M, Sigma, A2324-5G) treatment. After a final centrifugation step, cells were dispensed with the Wellmate (Matrix) into 384-well Evotec plates (#781058) preplated with 10 ⁇ l of the respective compound diluted in cell medium.
  • Infected cells were then incubated in the presence of the compound for 5 days at 37° C., 5% CO 2 . After five days, macrophages were stained with SYTO 60 (Invitrogen, S11342) followed by plate sealing and image acquisition. During screening, staining of the live cells was carried out on a set of three plates every two hours to limit cell death due to prolonged incubation with cell chemical stain.
  • Infected cells are then defined as those having at least a given number of pixels (usually 3) whose intensity in the green channel is above a given intensity threshold. The ratio of infected cells to the total number of cells is the measure of interest (named infection ratio). For each well, 4 pictures were recorded and for each parameter, the mean of the four images was used.
  • IDBS ActivityBase
  • a frozen aliquot of M. tuberculosis H37Rv-GFP was diluted at 1.5 ⁇ 10 6 CFU/mL in Middlebrook 7119-ADC medium supplemented with 0.05% Tween 80.
  • Greiner ⁇ clear-black 384-well plates (Greiner, #781091) were first preplated with 0.5 ⁇ l of compound dispensed by EVOBird (Evotec) in 10 ⁇ l of Middlebrook 7H9-ADC medium supplemented with 0.05% Tween 80.
  • 40 ⁇ l of the diluted H37Rv-GFP bacterial suspension was then added on top of the diluted compound resulting in a final volume of 50 ⁇ l containing 1% DMSO. Plates were incubated at 37° C., 5% CO 2 for 10 days after which GFP-fluorescence was recorded using a Victor 3 reader (Perkin-Elmer Life Sciences).
  • Raw 264.7 (ATCC #TIB-71) (1.5*10 8 cells) were infected with H37Rv-GFP (Abadie et al., 2005, Cremer et al., 2002) in suspension at a MOI of 1:1 for 2 hours at 37° C. with shaking. After two washes by centrifugation, the remaining extracellular bacilli from the infected cell suspension were killed by a 1 hour Amikacin (20 ⁇ M. Sigma, A2324) treatment. After a final centrifugation step, cells were dispensed into 384-well Evotec plates (#781058) preplated with compounds and controls. Infected cells were then incubated for 5 days at 37° C., 5% CO 2 .
  • BMDM Murine Bone Marrow-Derived Macrophages
  • FCS heat-inactivated fetal calf serum
  • PBMC Peripheral Blood Mononuclear Cells
  • Buffy coat diluted in PBS supplemented with 1% FCS was treated with 15 ml of Ficoll-Paque Plus (Amersham Biosciences, Sweden) and centrifuged at 2500 ⁇ g for 20 min.
  • PBMC were obtained by CD14 + bead separation (Miltenyi Biotec, Germany), washed 3-times with PBS (1% FCS) and transferred to 75 cm 2 culture flasks containing RPMI 1640 media, 10% FCS and 50 ng/ml of recombinant-human macrophage colony stimulating factor (R & D systems, Minneapolis).
  • Mycobacteria-GFP were detected using a 488-nm laser coupled with a 535/50 nm detection filter and cells labeled with a 635-nm laser coupled with a 690/40 nm detection filter. Four fields were recorded for each plate well and each image was then processed using dedicated in-house image analysis software (IM) as described elsewhere (Fenistein et al., in press).
  • IM in-house image analysis software
  • Mycobacterium tuberculosis H37Rv, H37Ra and BCG Pasteur were used as reference strains. All strains were diluted at 1.5 ⁇ 10 6 CFU/mL in Middlebrook 7H9-ADC medium supplemented with 0.05% Tween 80. 384-well plates (Greiner, #781091) were first preplated with 0.5 ⁇ l of compound dispensed by EVOBird (Evotec) in 10 ⁇ l of Middlebrook 7H9-ADC medium supplemented with 0.05% Tween 80. Forty microliters of the diluted H37Rv-GFP bacterial suspension was then added to the diluted compound resulting in a final volume of 50 ⁇ l containing 1% DMSO.
  • resazurin test In order to address compound toxicity, seven cell lines originating from different body tissues were cultivated in the presence of 3-fold dilutions of compounds starting from 100 ⁇ M. After 5 days of culture, cell viability was assessed by the resazurin test. Briefly, cells were incubated with 10 ⁇ g/mL of resazurin (Sigma-Aldrich St. Louis, Mo.) for 4 h at 37° C. under 5% CO 2 . Resofurin fluorescence (RFU) was measured as indicated above.
  • the frequency of spontaneous mutations was determined on 7H10 plates containing increasing concentrations of dintirobenzamide (0.2, 0.8, 1.6 and 3.2 ⁇ g/ml) or pyridopyrimidinone (0.4, 0.8, 1.6 and 3.2 ⁇ g/ml) compounds.
  • 10 6 , 10 7 and 10 8 CFU containing bacterial suspensions were spread on compound containing agar plates. After 5-6 weeks at 37° C., colonies were counted and frequency of mutation was evaluated as the ratio of colonies relative to the original inoculum.
  • DMSO and INH were used as negative and positive controls, respectively.
  • Raw264.7 macrophages were first infected with mycobacteria that constitutively express green fluorescent protein (GFP) at different multiplicities of infection (MOI) followed by kinetic analysis.
  • GFP green fluorescent protein
  • MOI multiplicities of infection
  • the host live cells were daily labeled with the red chemical fluorescent dye Syto60, and confocal images of live samples were acquired using an automated confocal microscope. Typical images are displayed in FIG. 1 a .
  • a few discrete weakly fluorescent bacteria localized within the cells.
  • the average number of cells had increased and mycobacteria had started to spread into neighboring cells leading to zones of strongly fluorescent bacteria.
  • the localization of the green signal is always within a distance of 5 ⁇ m to that of the red cell signal and in most cases actually overlaps with the cell signal. This confirms the intracellular nature of the mycobacteria growth.
  • the cell number has significantly diminished and the bacteria have formed large, highly fluorescent aggregates, which cover almost the entire image from day 5 onwards.
  • non-infected cells grew up to confluence at day 2 and remained alive until day 7.
  • an in-house image analysis script was developed. This script enables the automated quantification of the number of cells and the percentage of infected cells, whereby an infected cell is a cell containing at least three green pixels with an intensity above a defined threshold ( FIG. 1 b ). 2 hours after infection, between 2 and 10% of Raw264.7 cells were found to harbor a low number of bacilli ( FIG. 1 c ). The percentage of infected cells, hereafter named infection ratio, continued to increase from 72 hours post-infection reaching up to 70% at seven days post infection. This increase in infection ratio correlated with an increase in cell mortality ( FIG. 1 d/e ).
  • FIG. 2 a A clear inhibition dose-response curve was obtained by image-extracted analysis ( FIG. 2 b ).
  • INH minimal inhibitory concentration
  • macrophages were infected in batch with M. tuberculosis before being dispensed onto the compounds.
  • the batch infection was carried out with macrophages in suspension at 37° C. under mild shaking. Free unbound mycobacteria were removed by washing three times with PBS and differential centrifugation, as well as by an additional one-hour incubation step with amykacin, an antibiotic known to selectively kill extracellular microbes ( FIG. 6 a ).
  • M. tuberculosis infected macrophages were then seeded in plates that had been previously dispensed with the compounds, DMSO or antibiotic controls. The day-to-day as well as plate-to plate reproducibility was first tested.
  • a 26500 small molecule compound library that was selected for its high chemical diversity and drug-like properties according to the Lipinski rules (Lipinski et al., 2001), was chosen as the first library to be screened using the validated phenotypic cell-based assay.
  • the primary screen was carried out with compounds at 20 ⁇ M in singleton.
  • the throughput was set to about 6000 compounds per working day encompassing 25 plates.
  • the screening was performed with Raw264.7 cells that had been expanded from frozen stocks for ten days before infection with M. tuberculosis H37Rv-GFP.
  • the MICs obtained from 2 serial dilutions of INH and Rifampin processed at the beginning and at the end of the screening day should show similar results compared to the values obtained during the validation (see above).
  • Each screened plate is then accepted by the quality control procedure if the window between DMSO and INH (1 ⁇ g/ml) is higher than 3 and the CV calculated for the 320 compounds present in each plate is lower than 25.
  • Such quality control criteria allow the identification of hits with an activity higher than 75%.
  • the percent inhibition for each compound was determined relative to the corresponding mean infection ratio between 1 ⁇ g/mL INH (100%) and DMSO (0%) in the same 384-well plate. The percent inhibition distribution is centered around ⁇ 20% of inhibition ( FIG. 4 a ). It was decided to select compounds that have an inhibitory effect greater than 65% which corresponds to a little less than 1.5% of the total compounds.
  • cell cytotoxicity An important parameter that can be measured during image analysis is the total cell number, also referred to as cell cytotoxicity.
  • a low cell number can be the result of two independent phenomena, the compound toxicity and M. tuberculosis growth mediated cell toxicity. Indeed, at day 5 after infection with M. tuberculosis , the cell number decreased to less than 100 cells per image compared to more than 500 cells per image for uninfected cells ( FIG. 1 e ). In contrast, a high cell number is obtained only when the compound is not toxic and prevents mycobacterial growth. This turns out to be a second relevant measurement of a compound's anti-mycobacterial activity.
  • the 657 selected hits were first confirmed at 3 different concentrations, 20 ⁇ M, 2 ⁇ M and 0.2 ⁇ M.
  • the activity was confirmed either at 20 ⁇ M or 2 ⁇ M, on the intracellular or the in vitro assay (see Table 1).
  • 121 compounds demonstrated an inhibitory activity above 65% at 2 ⁇ M without any apparent cell toxicity at 20 ⁇ M and consequently were selected for further confirmation by ten 3-fold serial dilutions (see FIGS. 9-1 to 9 - 41 ). All 121 compounds were confirmed by serial dilution with a MIC ranging between 250 nM and 20 ⁇ M. The results shown in FIG.
  • FIG. 5 are representative of the three types of behavior observed: most of the compounds exhibited a clear dose response curve when activity was measured as infection ratio ( FIG. 5 b/e/h ).
  • Compounds active on the bacilli level present a similar activity in the extracellular assay ( FIG. 5 c/f ) even if the MIC is different from one assay to the other.
  • a few compounds don't present clear activity on the in vitro bacilli ( FIG. 5 i ) and may represent drugs acting through a cellular target or on a bacilli target involved only during the infection process.
  • toxic compounds can be identified thanks to a dramatic decrease in the cell number when the compound concentration increases ( FIG.
  • FIGS. 9-1 to 9 - 41 The serial dilution results from all 121 compounds are presented in FIGS. 9-1 to 9 - 41 .
  • the 121 confirmed hits can be clustered as 20 independent/general scaffolds (Table 2).
  • the number of compounds for each scaffold varied, ranging from 1 to 69 molecules.
  • the molecules from the 69-compound scaffold share a common structure which is similar to INH thereby validating the screening results.
  • One scaffold contains molecules that were only active in the intracellular assay and its mechanism of action will be the focus of further investigation.
  • benzamide compounds (scaffold II; see Table 2) underwent derivatization according to the methods outlined below (Schemes 1-7). Formation of the amide can be performed under general conditions using EDC or DCC coupling reagents with acids instead of acyl chloride. Resulting derivatives were examined for inhibitory activity using the assay described above and the results are summarized in Table 3.
  • reaction mixture was diluted with methylene chloride (10 mL) and washed with 1 M HCl aqueous solution (30 mL), saturated Na 2 CO 3 aqueous solution (30 mL) and brine (30 mL). The organic layer was dried over anhydrous MgSO 4 and concentrated in vacuo.
  • the crude product was purified by silica gel flash column chromatography (3:1 hexanes/ethyl acetate) and recrystallized from a mixture of hexanes and ethyl acetate to give A2.
  • pyridopyrimidinone compounds (scaffold VIII; see Table 2) underwent derivatization according to the methods outlined below (Schemes 8-10). Resulting derivatives were examined for inhibitory activity using the assay described above and the results are summarized in Table 3.
  • G3 (36.6 ⁇ mol) was dissolved in 760 ⁇ l of tert-butyl alcohol and 180 ⁇ l of 2-methyl-2-butene.

Abstract

The present invention relates to 4H-pyrido[1,2-a]pyrimidin-4-one compounds and their use in the treatment of bacterial infections, in particular Tuberculosis.

Description

    CROSS REFERENCE TO A RELATED APPLICATION
  • This application is a continuation application of Ser. No. 12/999,095, filed Mar. 11, 2011, now U.S. Pat. No. 8,785,452; which is a National Stage Application of International Application Number PCT/EP2009/004379, filed Jun. 17, 2009; which claims the benefit of U.S. Provisional Application Ser. No. 61/132,285, filed Jun. 17, 2008; which are incorporated herein by reference in their entirety.
    • FIELD OF INVENTION
  • The present invention relates to small molecule compounds and their use in the treatment of bacterial infections, in particular Tuberculosis.
    • BACKGROUND OF THE INVENTION
  • Tuberculosis (TB) as a disease continues to result in millions of deaths each year. Inadequate use of chemotherapy has led to an increasing number of drug resistant cases. This situation is likely to worsen with the emergence of extremely resistant strains to all currently known drugs (Van Rie and Enarson, 2006). The internationally recommended TB control strategy, also referred to as directly observed short-course chemotherapy (DOTS), relies on a combination of five antibacterial agents to be taken for a protracted period of more than six months (http://www.who.int/tb/dots/en/). With the use of a mathematical model, taking into consideration treatment duration and TB dynamics, benefits of reduced treatment length were predicted to be substantial and likely to greatly contribute to a reduced global TB burden (Salomon et al., 2006).
  • Current chemotherapy consists of compounds that directly target Mycobacterium tuberculosis bacillus, either by neutralizing general information pathways and critical processes such as RNA polymerization and protein synthesis inhibition or by interfering with mycobacterial specific cell envelope synthesis. The most widely used dedicated anti-tubercular drugs isoniazid, ethionamide and pyrazinamide are pro-drugs that first require activation. As active forms, they demonstrate inhibitory activity on a wide range of mycobacterial targets, which have not yet been fully characterized. As for other chronic infectious diseases like human immunodeficiency virus, a multi-therapy approach, including drugs that target a wide range of critical features of M. tuberculosis, proved to be the most successful strategy to date. It is, thus, likely that a combination of current drug inhibitors, having different mechanisms of action against M. tuberculosis, will be the solution for the control of the disease.
  • The most challenging approaches for discovering new anti-TB drugs rely on screening for active compounds that target critical features essential for the survival of the bacillus. Although there is still a lack of understanding of the biological mechanisms behind tubercle bacillus persistence, i.e. the location and state of latent bacteria, in humans, M. tuberculosis is thought to reside in primary granulomas under hypoxic conditions (Lenaerts et al., 2007) as well as to hide within various types of cells (Houben et al., 2006; Neyrolles et al., 2006). The bacillus mainly localizes inside phagocytic cells, such as macrophages and dendritic cells, and it has clearly been established that the tubercle bacillus adopts a different phenotype in the host macrophage's phagosome compared to growth in extracellular conditions (Rohde et al., 2007; Schnappinger et al., 2003). Upon infection, an inflammatory response is induced, thereby initiating recruitment of T lymphocytes that release interleukins and cytokines, which in turn activate the infected macrophages to enable the destruction of the pathogen. Upon the appropriate trigger, the host macrophage is, thus, able to eliminate the invading bacillus. This is further supported by the fact that of the people that inhale M. tuberculosis, more than 95% percent do not develop the disease, suggesting that the human host response is sufficient in most cases to thwart M. tuberculosis induced pathogenesis. This gives rise to the hypothesis that small molecular compounds could mimic the immune cell response signals and induce the host cells to clear the mycobacteria.
  • Accordingly, it was an object of the present invention to develop a phenotypic cell-based assay suitable for high throughput screening that allows for the search of compounds that would prevent M. tuberculosis multiplication inside the host macrophage. Up to now, this type of investigation of the tubercle bacillus growth within host cells relied on colony forming units (CFUs) determination after host cell lysis followed by serial dilutions and a 3-week incubation period required for bacterial growth on agar plates. Luciferase-expressing mycobacteria have been shown to be efficient in reducing the experiment duration, although cell lysis and luciferin substrate addition steps are still required (Arain et al., 1996). Also, these types of experiments are not easily amenable to large scale screening.
  • It was another object of the present invention to identify compounds effective against bacterial infections, in particular compounds that would prevent M. tuberculosis multiplication inside the host macrophage.
    • DESCRIPTION OF THE INVENTION
  • In one aspect, the present invention relates to compounds having the general formula VIII:
  • Figure US20150018543A1-20150115-C00001
  • wherein
  • m is 0, 1, 2, or 3;
  • X3 is selected from the group comprising CH2, O, S and NH;
  • X4 is selected from the group comprising halide, alkyl, OR23, SR24 and NR25R26;
  • R20 is selected from the group comprising acyl, alkoxy, alkyl, alkylamino, alkylcarboxylic acid, arylcarboxylic acid, alkylcarboxylic alkylester, alkylene, alkylether, alkylhydroxy, alkylthio, alkynyl, amido, amino, aryl, arylalkoxy, arylamino, arylthio, carboxylic acid, cyano, cycloalkyl, carboxylic acid, ester, halo, haloalkoxy, haloalkyl, haloalkylether, heteroaryl, heteroarylamino, heterocycloalkyl and hydrogen, any of which is optionally substituted;
  • R21 and R22 are each independently selected from the group comprising alkoxy, alkyl, alkylamino, alkylene, alkylether, alkylthio, alkynyl, amido, amino, aryl, arylether, arylalkoxy, arylamino, arylthio, carboxy, cyano, cycloalkyl, ester, halo, haloalkoxy, haloalkyl, heteroaryl, heteroarylamino, heterocycloalkyl, hydroxyl, hydrogen, nitro, thio, sulfonate, sulfonyl and sulfonylamino, any of which is optionally substituted;
  • R23 is selected from the group comprising acyl, alkyl, alkylamino, alkylene, alkynyl, aryl, arylalkoxy, arylamino, arylthio, carboxy, cycloalkyl, ester, ether, haloalkyl, heteroaryl, heteroarylamino, heterocycloalkyl, hydrogen, thio, sulfonate, and sulfonylamino, any of which is optionally substituted;
  • R24 is selected from the group comprising alkyl, alkylaryl, alkylene, alkynyl, aryl, cycloalkyl, ester, halo, haloalkyl, heteroaryl, heterocycloalkyl, and hydrogen, any of which is optionally substituted; and
  • R25 and R26 are each independently selected from the group comprising acyl, alkyl, aminoalkyl, alkylene, alkylthio, alkynyl, aryl, arylalkoxy, arylamino, arylthio, carboxy, cycloalkyl, ester, ether, halo, haloalkoxy, haloalkyl, haloalkylether, heteroaryl, heteroarylamino, heterocycloalkyl and hydrogen, any of which is optionally substituted.
  • In general, the term “optionally substituted” as used herein is meant to indicate that a group, such as alkyl, alkylen, alkynyl, aryl, cycloalkyl, heterocycloalkyl, or heteroaryl, may be unsubstituted or substituted with one or more substituents. “Substituted” in reference to a group indicates that a hydrogen atom attached to a member atom within a group is replaced.
  • In another aspect, the present invention relates to compounds having the general formula VIIIa:
  • Figure US20150018543A1-20150115-C00002
  • wherein
  • X5 is selected from the group comprising CH2, C═O and C═S;
  • Z1 and Z2 are each independently selected from the group comprising alkoxy, alkyl, alkylamino, alkylene, alkylether, alkylthio, alkynyl, amido, amino, aryl, arylether, arylalkoxy, arylamino, arylthio, carboxy, cyano, cycloalkyl, ester, halo, haloalkoxy, haloalkyl, heteroaryl, heteroarylamino, heterocycloalkyl, hydroxyl, and hydrogen, or two groups are connected each other to make five or six membered cyclic, heterocyclic and heteroaryl rings, any of which is optionally substituted;
  • R27 and R28 are each independently selected from the group comprising alkoxy, alkylamino, alkylene, alkylether, alkylthio, alkynyl, amido, amino, aryl, arylether, arylalkoxy, arylamino, arylthio, carboxy, cyano, cycloalkyl, ester, halo, haloalkoxy, haloalkyl, heteroaryl, heteroarylamino, heterocycloalkyl, hydroxyl, hydrogen, nitro, thio, sulfonate, sulfonyl and sulfonylamino, any of which is optionally substituted;
  • R29 and R30 are each independently selected from the group comprising alkoxy, alkyl, alkylamino, alkylene, alkylether, alkylthio, alkynyl, amido, amino, aryl, arylether, arylalkoxy, arylamino, arylthio, carboxy, cyano, cycloalkyl, ester, halo, haloalkoxy, haloalkyl, heteroaryl, heteroarylamino, heterocycloalkyl, hydroxyl, hydrogen, nitro, thio, sulfonate, sulfonyl and sulfonylamino, or two groups are connected each other to make five or six membered cyclic, heterocyclic, aryl, and heteroaryl rings, any of which is optionally substituted.
  • The term “alkyl” as used herein is meant to indicate that a group, such as substituted or non-substituted C1-C10 alkyl group which has the straight or branched chain.
  • The term “cycloalkyl” as used herein is meant to indicate that a group, such as substituted or non-substituted cyclic compound of C3-C8 ring structure.
  • The term “heteroaryl” as used herein is meant to indicate that a group, such as substituted or non-substituted 5- to 9-membered aromatic compounds which have more than one heteroatom of N, O, and S in the ring structure itself.
  • The term “optionally substituted” as used herein is meant to indicates that a hydrogen atom attached to a member atom within a group is possibly replaced by group, such as C1-C10 alkyl, halogen including fluorine, OH, NO2, OR31, CN, NR31R32, COR31, SOR32, SO2R31, SO2NR31, CR31═CR31R32, CR31═NR32, aryl, aryloxy, C4-C10 heteroaryl group, or —NR31—COR32, —O—COR31.
  • R31 and R32 are each independently selected from the group comprising hydrogen, alkyl, alkyloxy, alkylamino, alkylcarbonyl, alkylcarbonylamino, alkylcarbonyloxy, alkylaminocarbonyl, alkyloxycarbonyl, cycloalkyl, cycloalkyloxy, cycloalkylamino, cycloalkylcarbonyl, cycloalkylcarbonylamino, cycloalkylcarbonyloxy, cycloalkylaminocarbonyl, cycloalkyloxycarbonyl, heteroaryl, heteroaryloxy, heteroaryl amino, heteroaryl carbonyl, heteroaryl carbonylamino, heteroaryl carbonyloxy, heteroaryl aminocarbonyl, heteroaryl oxycarbonyl, heteroaryl alkyl, heteroaryl alkyloxy, heteroaryl alkylamino, heteroaryl alkylcarbonyl, heteroaryl alkylcarbonylamino, heteroaryl alkylcarbonyloxy, heteroaryl alkylaminocarbonyl, heteroaryl alkyloxycarbonyl, phenyl, phenyloxy, phenylamino, phenylcarbonyl, phenylcarbonylamino, phenylcarbonyloxy, phenylaminocarbonyl, and phenyloxycarbonyl, any of which is optionally substituted.
  • In another aspect, the present invention relates to compounds having one of the formulas 125-301 as shown in Example 7, preferably 132-135, 137, 139-140, 147, 151-152, 160, 163, 173, 180, 184-185, 193, 195, 199-201, 204, 206-222, 224, 226, 229, 231-243, 245-278, 280-286 and 290-301 as shown in Table 3. Particularly preferred compounds are compounds having one of the formulas 133, 232 and 264 as shown in Table 3.
  • In one aspect, the present invention relates to compounds having the general formula II:
  • Figure US20150018543A1-20150115-C00003
  • wherein
  • R5 and R6 are each independently selected from the group comprising acyl, alkyl, alkylamino, alkylene, alkylthio, alkynyl, aryl, arylalkoxy, arylamino, arylthio, carboxy, cycloalkyl, ester, haloalkoxy, haloalkyl, heteroaryl, heteroarylamino, heterocycloalkyl, hydroxyl, hydrogen, sulfonate and sulfonyl, any of which is optionally substituted and
  • R7, R8 and R9 are each independently selected from the group comprising alkoxy, alkyl, alkylamino, alkylene, alkylthio, alkynyl, amido, amino, aryl, arylalkoxy, arylamino, arylthio, carboxy, cyano, cycloalkyl, ester, halo, haloalkoxy, haloalkyl, heteroaryl, heteroarylamino, heterocycloalkyl, hydroxyl, hydrogen, nitro, thio, sulfonate, sulfonyl and sulfonylamino, any of which is optionally substituted.
  • In another aspect, the present invention relates to compounds with the general formula II, wherein R5 and R6 are connected, having the general formula IIa:
  • Figure US20150018543A1-20150115-C00004
  • wherein
  • n is 0, 1, 2, or 3;
  • Y and Z are each independently selected from the group comprising CH2, CHOR10, CHNR10R11, CR10R11 and NR10; and
  • R10 and R11 are each independently selected from the group comprising acyl, alkyl, alkylamino, alkylene, alkylthio, alkynyl, aryl, arylalkoxy, arylamino, arylthio, carboxy, cycloalkyl, ester, haloalkoxy, haloalkyl, heteroaryl, heteroarylamino, heterocycloalkyl, hydrogen, sulfonate and sulfonyl, any of which is optionally substituted.
  • In another aspect, the present invention relates to compounds having one of the formulas with the general formula/scaffold II as shown in FIGS. 9-1 to 9-41, as well as one of the formulas 1-123 as shown in Example 6, preferably 1-24, 26-34, 54, 56, 58-61, 63-64, 67, 90-101, 103-105, 107-109, 112, 114-116 and 118-121 as shown in Table 3. Particularly preferred compounds are compounds having one of the formulas 4 and 24 as shown in Table 3.
  • Preferably, the compounds as defined above have an inhibitory activity, preferably an inhibitory activity above 65%, on bacterial growth, preferably on the growth of M tuberculosis, inside a host cell, preferably a macrophage, at a concentration between 5-20 μM, preferably less than 5 μM.
  • In one aspect, the present invention relates to compounds as defined above for use in the treatment of bacterial infections.
  • In one aspect, the present invention relates to compounds as defined above for use in the treatment of Tuberculosis.
  • In one aspect, the present invention relates to a pharmaceutical composition comprising a compound as defined above.
  • In one aspect, the present invention relates to a method of treatment of Tuberculosis, comprising the application of a suitable amount of a compound as defined above to a person in need thereof.
  • In another aspect, the present invention relates to compounds having one of the general formulas/scaffolds I, III-VII and IX-XX as shown in Table 2.
  • In one aspect, the present invention relates to compounds having the general formula I:
  • Figure US20150018543A1-20150115-C00005
  • wherein
  • X1 and X2 are each independently selected from the group comprising CH and NH;
  • R1 and R2 are each independently selected from the group comprising alkoxy, alkyl, alkylamino, alkylene, alkylthio, alkynyl, amido, amino, aryl, arylalkoxy, arylamino, arylthio, carboxy, cyano, cycloalkyl, ester, halo, haloalkoxy, haloalkyl, heteroaryl, heteroarylamino, heterocycloalkyl, hydroxyl, hydrogen, nitro, thio, sulfonate, sulfonyl and sulfonylamino, any of which is optionally substituted; and
  • R3 and R4 are each independently selected from the group comprising alkoxy, alkyl, alkylamino, alkylene, alkylthio, alkynyl, aryl, arylalkoxy, arylamino, arylthio, cyano, cycloalkyl, haloalkoxy, haloalkyl, heteroaryl, heteroarylamino, heterocycloalkyl and hydrogen, any of which is optionally substituted.
  • In one aspect, the present invention relates to compounds having the general formula III:
  • Figure US20150018543A1-20150115-C00006
  • wherein
  • R10 and R11 are each independently selected from the group comprising alkoxy, alkyl, alkylamino, alkylene, alkylthio, alkynyl, amido, amino, aryl, arylalkoxy, arylamino, arylthio, carboxy, cyano, cycloalkyl, ester, halo, haloalkoxy, haloalkyl, heteroaryl, heteroarylamino, heterocycloalkyl, hydroxyl, hydrogen, nitro, thio, sulfonate, sulfonyl and sulfonylamino, any of which is optionally substituted.
  • In another aspect, the present invention relates to compounds having the general formula IV:
  • Figure US20150018543A1-20150115-C00007
  • wherein
  • A is an optionally substituted heteroaryl, naphthyl and phenyl and
  • R12 is selected from the group comprising alkoxy, alkyl, alkylamino, alkylene, alkylthio, alkynyl, amido, amino, aryl, arylalkoxy, arylamino, arylthio, carboxy, cyano, cycloalkyl, ester, halo, haloalkoxy, haloalkyl, heteroaryl, heteroarylamino, heterocycloalkyl, hydroxyl, hydrogen, nitro, thio, sulfonate, sulfonyl and sulfonylamino, any of which is optionally substituted.
  • In one aspect, the present invention relates to compounds having the general formula V:
  • Figure US20150018543A1-20150115-C00008
  • wherein
  • R13, R14 and R15 are each independently selected from the group comprising alkoxy, alkyl, alkylamino, alkylene, alkylthio, alkynyl, amido, amino, aryl, arylalkoxy, arylamino, arylthio, carboxy, cyano, cycloalkyl, ester, halo, haloalkoxy, haloalkyl, heteroaryl, heteroarylamino, heterocycloalkyl, hydroxyl, hydrogen, nitro, thio, sulfonate, sulfonyl and sulfonylamino, any of which is optionally substituted.
  • In another aspect, the present invention relates to compounds having the general formula VI:
  • Figure US20150018543A1-20150115-C00009
  • wherein
  • R16 is selected from the group comprising alkoxy, alkyl, alkylamino, alkylene, alkynyl, amino, aryl, arylalkoxy, arylamino, arylthio, cycloalkyl, haloalkoxy, haloalkyl, heteroaryl, heteroarylamino, heterocycloalkyl, hydroxyl and hydrogen, any of which is optionally substituted and
  • R17 is selected from the group comprising alkoxy, alkyl, alkylamino, alkylene, alkylthio, alkynyl, amido, amino, aryl, arylalkoxy, arylamino, arylthio, carboxy, cyano, cycloalkyl, ester, halo, haloalkoxy, haloalkyl, heteroaryl, heteroarylamino, heterocycloalkyl, hydroxyl, hydrogen, nitro, thio, sulfonate, sulfonyl and sulfonylamino, any of which is optionally substituted.
  • In one aspect, the present invention relates to compounds having the general formula VII:
  • Figure US20150018543A1-20150115-C00010
  • wherein
  • R18 and R19 are each independently selected from the group comprising alkoxy, alkyl, alkylamino, alkylene, alkylthio, alkynyl, amido, amino, aryl, arylalkoxy, arylamino, arylthio, carboxy, cyano, cycloalkyl, ester, halo, haloalkoxy, haloalkyl, heteroaryl, heteroarylamino, heterocycloalkyl and hydrogen, any of which is optionally substituted.
  • In another aspect, the present invention relates to compounds having one of the formulas with the general formulas I, III-VII and IX-XX as shown in FIGS. 9-1 to 9-41.
  • In one aspect, the present invention relates to a compound listed in Table 1.
  • In one aspect, the present invention relates to compounds as defined above for use in the treatment of bacterial infections.
  • In one aspect, the present invention relates to compounds as defined above for use in the treatment of Tuberculosis.
  • In one aspect, the present invention relates to a pharmaceutical composition comprising a compound as defined above.
  • In one aspect, the present invention relates to a method of treatment of Tuberculosis, comprising the application of a suitable amount of a compound as defined above to a person in need thereof.
  • In another aspect, the present invention relates to a screening method comprising the steps of
  • (a) batch infection of host cells with fluorescently labeled M. tuberculosis mycobacteria;
    (b) removing free unbound mycobacteria;
    (c) adding compounds that are to be tested to a multi-well plate;
    (d) seeding said host cells infected with fluorescently labeled M. tuberculosis mycobacteria into said multi-well plate containing said compounds;
    (e) incubating said multi-well plate containing host cells infected with fluorescently labeled M. tuberculosis mycobacteria and said compounds;
    (f) fluorescently labeling said host cells;
    (g) analyzing said multi-well plate using automated confocal microscopy.
  • The screening method according to the present invention represents a phenotypic cell-based assay enabling the search for drugs that interfere with the multiplication of M. tuberculosis within host macrophages. The assay makes use of fluorescently labeled living macrophages infected with fluorescently labeled mycobacteria and uses automated confocal fluorescence microscopy to measure intracellular mycobacterial growth. The assay has been set-up for the high throughput screening (HTS) of large scale chemical libraries.
    • BRIEF DESCRIPTION OF THE FIGURES AND TABLES
  • Reference is now made to the figures and tables, wherein
  • FIG. 1 shows the monitoring of tubercle bacillus intracellular growth inside macrophages by automated confocal microscopy: (a) Representative pictures of Raw264.7 cells infected with M. tuberculosis H37Rv-GFP at different time points after infection. (b) Image analysis: 1: Typical 2-color image; 2: Circled object corresponds to detected cells; 3: Circled object corresponds to bacterial aggregates; 4: Filled purple cells correspond to infected cells. (c,d,e) Image-based quantification of the percentage of infected cells and the mean number of cells from 2 hours to day 7 after infection with H37Rv-GFP at a multiplicity of infection of 0.5 (gray square), 1 (black circle) and 2 (dark gray triangle). Non-infected cells (black diamonds) were used as the negative control;
  • FIG. 2 shows the pharmacological validation and MIC (minimal inhibitory concentration) comparison of the reference drugs in the in vitro growth fluorescence assay and the phenotypic cell-based assay: (a) Representative pictures of infected cells in presence of INH at 1 μg/mL or DMSO control. (b,c,d) Dose-response of INH, rifampin and ethionamide; black square and line corresponds to growth inhibition in cell-based assay; gray circle and line correspond to in vitro growth inhibition; shown is a representative data set;
  • FIG. 3 shows assay automation validation of the phenotypic cell-based assay: (a) Percent of M. tuberculosis infected cells relative to 384-plate well-index. Black square, dark gray square, gray square and open square correspond to INH 1 μg/mL, rifampin 5 μg/mL, PBS and DMSO control respectively. (b,c) Percent of M. tuberculosis infected cells relative to INH and rifampin concentration. Experiments were performed on four different plates on two independent days;
  • FIG. 4 shows primary screening results for the phenotypic cell-based assay and the in vitro growth assay for 26500 compounds: (a) Percent inhibition based on infection ratio relative to each compound and distribution. (b) Percent inhibition based on RFU relative to each compound and distribution. (c) Comparison of inhibition percentage for the phenotypic cell-based assay and the in vitro growth assay for each compound;
  • FIG. 5 shows serial dilution results from the in vitro growth fluorescence assay and the phenotypic cell-based assay: Typical curves for compounds inhibiting (a,b,c) in vitro bacterial growth (d,e,f) both in vitro and intracellular growth and (g,h,i) intracellular growth only. (a,d,g) Infection ratio relative to compound concentration. (b,e,h) Cell number relative to compound concentration. (c,f,i) Relative fluorescence intensity relative to compound concentration. Compound concentration is given in M;
  • FIG. 6 shows (a) a scheme of assay automation. (b) a 384-plate format description; (c) a 384-plate dose-response curve description. A to P and a to b correspond to 2-fold serial dilution of INH and Rifampin respectively with a starting concentration of 20 mg/mL in well A or a; RIF: Rifampin 5 μg/mL, Cpd: compound, INH100 1 μg/mL, INH50 0.05 μg/mL;
  • FIG. 7 shows the anti-tuberculosis effect of compounds 4 and 24 (5 μM) on M. tuberculosis H37Rv-GFP in (a) Raw267.4 (104 cells), (b) mouse bone marrow-derived macrophages and (c) human primary macrophages differentiated with 50 ng/mL rhM-CSF (1.5*104) after 7 days of infection with MOI 2.5:1 (control INH at 5 μM);
  • FIG. 8 illustrates the colony forming units (CFUs) recovered from macrophages at different time points after infection with M. tuberculosis H37Rv. Either Raw264.7 cells (a) or murine BMDM (b) were infected at an MOI of 1:1 and treated with the indicated amount of pyridopyrimidione compound 232 (20 μM) with DMSO, INH (10 μM) and RIF (10 μM) as controls;
  • FIGS. 9-1 to 9-41 list 121 compounds which demonstrated an inhibitory activity above 65% at 2 μM without any apparent cell toxicity at 20 μM and consequently were selected for further confirmation by ten 3-fold serial dilutions;
    •
  • Table 1 lists 340 hits whose inhibitory activity was confirmed in an intracellular (QIM) assay or an in vitro (QUM) assay, wherein the abbreviation “QIM” stands for Quantification of Intracellular Mycobacteria, the abbreviation “QUM” stands for Quantification of in vitro grown Mycobacteria, and the abbreviation “CellNb” stands for cell number;
  • Table 2 summarizes the independent/general molecular scaffolds/formulas of the 121 hits listed in FIGS. 9-1 to 9-41;
  • Table 3 lists dinitrobenzamide and pyridopyrimidinone derivatives (general scaffold II and VIII, respectively, see Table 2) with their respective inhibitory activities, wherein the numbers in bold print refer to the compounds listed in Examples 6 and 7;
  • Table 4 shows the cytotoxicity and antibacterial spectrum of dinitrobenzamide compounds 4 and 24 (see Table 3);
  • Table 5 shows the cytotoxicity and antibacterial spectrum of pyridopyrimidinone compound 133 (see Table 3); and
  • Table 6 shows the frequency of spontaneous resistance for representative dinitrobenzamide and pyridopyrimidinone compounds.
    • EXAMPLES
  • The invention is now further described by reference to the following examples which are intended to illustrate, not to limit the scope of the invention.
    • Materials and Methods Genetic Constructs and Mycobacterial Strains
  • A recombinant strain of M. tuberculosis H37Rv expressing the green fluorescent protein (H37Rv-GFP) was obtained by transformation of an integrative plasmid (Abadie et al., 2005; Cremer et al., 2002). Within this plasmid, which is derived from the Ms6 mycobacteriophage, the gfp gene is cloned and constitutively expressed under the strong mycobacterial promoter pBlaF. Electrocompetent cells for M. tuberculosis H37Rv-GFP were prepared from 400 mL of a 15 days old Middlebrook 7H9 culture (Difco, Sparks Md., USA) supplemented with albumin-dextrose-catalase (ADC, Difco, Sparks Md., USA), glycerol and 0.05% Tween 80. Bacilli were harvested by centrifugation at 3000 g for 20 min, washed twice with H2O at room temperature, and resuspended in 1-2 mL of 10% glycerol at room temperature after recentrifugation. 250 μl of bacilli were mixed with green fluorescent protein encoding plasmid and electroporated using a Biorad Gene Pulser (Biorad). After electroporation, bacilli were resuspended in medium and left one day at 37° C. Transformants were selected on Middlebrook 7H11 medium (Difco, Sparks Md., USA) supplemented with oleic acid-albumin-dextrose-catalase (OADC, Difco, Sparks Md., USA) and 50 μg/mL hygromycin (Invitrogen, Carlsbad, Calif. USA). The selected hygromycin-resistant and green fluorescent colonies appeared after 3 weeks. A 100 mL culture of the H37Rv-GFP strain was grown in Middlebrook 7H9-ADC medium supplemented with 0.05 % Tween 80 and 50 μg/mL of hygromycin. Bacteria were harvested, washed twice and suspended in 50 mM sodium phosphate buffer (pH 7.5). The bacteria were then sonicated and allowed to stand for 1 hour to allow residual aggregates to settle. The bacterial suspensions were then aliquoted and frozen at −80° C. A single defrosted aliquot was used to quantify the CFUs (colony forming units) prior to inoculation and typical stock concentrations were about 2 to 5×108 CFU/mL.
    • Host Cells
  • Mouse macrophage cell lines Raw 264.7 (ATCC #TIB-71), J774A.1 (ATCC #TIB-67) or human monocytes (ATCC #TIB-202) differentiated with 50 ng/mL PMA (Sigma) were grown in RPMI 1640 (Gibco) with 10% heat-inactivated fetal calf serum (Gibco).
    • Chemical Compounds
  • The small synthetic molecules from the screening libraries were suspended in pure DMSO (Sigma, D5879-500 mL) at a concentration of 10 mM (Master plates) in Corning 96 well clear V-bottom polypropylene plates (Corning, #3956). The compounds were then reformatted in Greiner 384 well V-shape polypropylene plates (Greiner, #781280) and diluted to a final concentration of 2 mM in pure DMSO. The compounds were kept frozen until use. For screening, compound plates were incubated at room temperature until thawed. The compounds were directly added into the assay plates from the DMSO stock using an EVObird liquid handler (Evotec Technologies), which transfers 250 n1 of compound twice to reach a final dilution of 1:100. This one-step dilution reduces the risk of compound precipitation in intermediate plates and allows for a low final DMSO concentration (1%).
  • Positive control antibiotics (Isoniazid (Sigma, 13377-50G) and Rifampin (Euromedex, 1059-8, 5 g)) as well as negative controls (DMSO) were added manually in each plate in columns 1-2 and 23-24 (see FIG. 6 b for plate description).
  • A total of 26500 compounds were tested. These compounds came from commercial libraries from Timtec (25000 from the ActiProbe diverse library, 1000 from the Kinase inhibitors ActiTargK library and 500 from the Protease inhibitors ActitargP library). The screened compounds were selected based on high diversity and drug-like properties (using Lipinski rule-of-five (Lipinski et al., 2001)). They were first screened at one concentration (primary screen, concentration=20 μM). The “positive” compounds selected from the primary screen were then confirmed by testing at 3 concentrations (20, 2 and 0.2 μM) to identify the most active and/or by ten 3-fold ten dilutions (from 20 μM to 0.5 nM).
    • Macrophage Invasion Assay Set-Up
  • Cells were first seeded in 50 μl at a density of 20,000 cells per well of a 384-well plate (Evotec technologies #781058) for 16 hours and then infected with bacterial suspensions at a multiplicity of infection (MOI) varying from 10:1 to 1:1 (bacteria:host cells). After 2 hours, cells were washed three times with phosphate buffered saline (PBS) and the compounds diluted in fresh culture medium were added. Cells were incubated at 37° C., 5% CO2 for up to seven days.
    • Macrophage Batch Infection Assay Scale-Up
  • Cells (1.5×108 cells) were infected with H37Rv-GFP suspension at a MOI of 1:1 in 300 mL for 2 hours at 37° C. with shaking (100 rpm). After two washes by centrifugation at 1100 rpm (Beckman SX4250, 165 g) for 5 min., the remaining extracellular bacilli from the infected cells suspension were killed by a 1 hour amykacin (20 μM, Sigma, A2324-5G) treatment. After a final centrifugation step, cells were dispensed with the Wellmate (Matrix) into 384-well Evotec plates (#781058) preplated with 10 μl of the respective compound diluted in cell medium. Infected cells were then incubated in the presence of the compound for 5 days at 37° C., 5% CO2. After five days, macrophages were stained with SYTO 60 (Invitrogen, S11342) followed by plate sealing and image acquisition. During screening, staining of the live cells was carried out on a set of three plates every two hours to limit cell death due to prolonged incubation with cell chemical stain.
    • Image Acquisition and Data Analysis
  • Confocal images were recorded on an automated fluorescent confocal microscope Opera™ (Evotec Technologies) using a 20×-water objective (NA 0.70), 488-nm and 635-nm lasers and a 488/635 primary dichroic mirror. Each image was then processed using dedicated in-house image analysis software (1M). Parameters determined were the total cell number and the number of infected cells. Briefly, the algorithm first segments the cells on the red channel using a sequence of processing steps as described elsewhere (Fenistein et al., manuscript in press). It is generally based on a succession of 1) thresholding the histogram of the original image (3 classes K-means) 2) gaussian filtering the original image with a standard deviation that is set equal to the cells' average radius, 3) searching for the local maxima of the filtered image that provides cell centers as seeds for 4) region growing that defines each cell's own surface and finally 5) removing extremely small cells as potential artifacts or noise. This step provides the total number of cells in the red channel. Infected cells are then defined as those having at least a given number of pixels (usually 3) whose intensity in the green channel is above a given intensity threshold. The ratio of infected cells to the total number of cells is the measure of interest (named infection ratio). For each well, 4 pictures were recorded and for each parameter, the mean of the four images was used.
  • Data obtained from either the intracellular assay image analysis or from the conventional antibacterial assay (see below) were then processed using ActivityBase (IDBS) to calculate the statistical data (% of inhibition, Z score for each compound, Z′, CV etc. for the control plates) and to store the data in an Oracle database. Additional analyses with regards to both quality control of the screens and hit identification were performed with various software packages including Spotfire (Tibco) and Pipelinepilot (Accelrys).
    • In Vitro Aerobic Bacterial Growth Assay
  • A frozen aliquot of M. tuberculosis H37Rv-GFP was diluted at 1.5×106 CFU/mL in Middlebrook 7119-ADC medium supplemented with 0.05% Tween 80. Greiner μclear-black 384-well plates (Greiner, #781091) were first preplated with 0.5 μl of compound dispensed by EVOBird (Evotec) in 10 μl of Middlebrook 7H9-ADC medium supplemented with 0.05% Tween 80. 40 μl of the diluted H37Rv-GFP bacterial suspension was then added on top of the diluted compound resulting in a final volume of 50 μl containing 1% DMSO. Plates were incubated at 37° C., 5% CO2 for 10 days after which GFP-fluorescence was recorded using a Victor 3 reader (Perkin-Elmer Life Sciences).
    • Macrophage Infection Assay and Image Analysis
  • Raw 264.7 (ATCC #TIB-71) (1.5*108 cells) were infected with H37Rv-GFP (Abadie et al., 2005, Cremer et al., 2002) in suspension at a MOI of 1:1 for 2 hours at 37° C. with shaking. After two washes by centrifugation, the remaining extracellular bacilli from the infected cell suspension were killed by a 1 hour Amikacin (20 μM. Sigma, A2324) treatment. After a final centrifugation step, cells were dispensed into 384-well Evotec plates (#781058) preplated with compounds and controls. Infected cells were then incubated for 5 days at 37° C., 5% CO2. Murine Bone Marrow-Derived Macrophages (BMDM) were produced as described previously (Brodin et al., 2006). Briefly, cells were extracted from the femurs and tibia of 6 weeks old female mice (C57BL/6, Orientbio) and cultivated in RPMI 1640 media containing 10% heat-inactivated fetal calf serum (FCS) (both from Gibco® at Invitrogen, Carlsbad, Calif.) and 10% L-929 cell conditioned medium. Peripheral Blood Mononuclear Cells (PBMC) were isolated from Buffy coat from healthy volunteers. Buffy coat diluted in PBS supplemented with 1% FCS was treated with 15 ml of Ficoll-Paque Plus (Amersham Biosciences, Sweden) and centrifuged at 2500×g for 20 min. PBMC were obtained by CD14+ bead separation (Miltenyi Biotec, Germany), washed 3-times with PBS (1% FCS) and transferred to 75 cm2 culture flasks containing RPMI 1640 media, 10% FCS and 50 ng/ml of recombinant-human macrophage colony stimulating factor (R & D systems, Minneapolis). Six day old adherent murine BMDM and PBMC derived human macrophages were infected with H37Rv-GFP (Abadie et al., 2005) in suspension at a MOI of 1:1 for 2 hours at 37° C. and then extensively washed and finally incubated with compounds or controls. After several days, macrophages were stained with SYTO 60 (Invitrogen, S11342) and image acquisition was performed on an EVOscreen-MarkIII fully automated platform (PerkinElmer) integrated with an Opera™ (20×-water objective, NA 0.70) and located in a BSL-3 safety laboratory. Mycobacteria-GFP were detected using a 488-nm laser coupled with a 535/50 nm detection filter and cells labeled with a 635-nm laser coupled with a 690/40 nm detection filter. Four fields were recorded for each plate well and each image was then processed using dedicated in-house image analysis software (IM) as described elsewhere (Fenistein et al., in press).
    • Mycobacterial Strains and In Vitro Bacterial Growth Assay
  • Mycobacterium tuberculosis H37Rv, H37Ra and BCG Pasteur were used as reference strains. All strains were diluted at 1.5×106 CFU/mL in Middlebrook 7H9-ADC medium supplemented with 0.05% Tween 80. 384-well plates (Greiner, #781091) were first preplated with 0.5 μl of compound dispensed by EVOBird (Evotec) in 10 μl of Middlebrook 7H9-ADC medium supplemented with 0.05% Tween 80. Forty microliters of the diluted H37Rv-GFP bacterial suspension was then added to the diluted compound resulting in a final volume of 50 μl containing 1% DMSO. Plates were incubated at 37° C., 5% CO2 for 10 days. Mycobacterial growth was determined by measuring GFP-fluorescence using a Victor 3 reader (Perkin-Elmer Life Sciences) for H37Rv-GFP or with resazurin method. Isoniazid at 0.05 μg/mL and 1 μg/mL (Sigma, 13377), Rifampin at 1 μg/mL (Euromedex) and DMSO were used as controls.
    • Cytotoxicity Assay
  • In order to address compound toxicity, seven cell lines originating from different body tissues were cultivated in the presence of 3-fold dilutions of compounds starting from 100 μM. After 5 days of culture, cell viability was assessed by the resazurin test. Briefly, cells were incubated with 10 μg/mL of resazurin (Sigma-Aldrich St. Louis, Mo.) for 4 h at 37° C. under 5% CO2. Resofurin fluorescence (RFU) was measured as indicated above. Percentage of toxicity on cells was calculated as follows: Cytotoxicity (%)=(RFUDMSO−RFUBlank)−(RFUcompound−RFUblank)/(RFUDMSO−RFUBlank)×100. Percentage of cytotoxicity was plotted against compound concentration and the minimal toxic concentration (MTC50) was determined by non-linear regression analysis as the lowest compound concentration where fifty percent toxicity was observed on the corresponding cell line.
    • Frequency of Spontaneous Resistance
  • The frequency of spontaneous mutations was determined on 7H10 plates containing increasing concentrations of dintirobenzamide (0.2, 0.8, 1.6 and 3.2 μg/ml) or pyridopyrimidinone (0.4, 0.8, 1.6 and 3.2 μg/ml) compounds. 106, 107 and 108 CFU containing bacterial suspensions were spread on compound containing agar plates. After 5-6 weeks at 37° C., colonies were counted and frequency of mutation was evaluated as the ratio of colonies relative to the original inoculum. DMSO and INH were used as negative and positive controls, respectively.
    • Example 1 Phenotypic Macrophage-Based Assay Set-Up and Automated Image Quantification
  • To set-up the optimal conditions of M. tuberculosis infection, Raw264.7 macrophages were first infected with mycobacteria that constitutively express green fluorescent protein (GFP) at different multiplicities of infection (MOI) followed by kinetic analysis. Up to 7 days post bacillus infection, the host live cells were daily labeled with the red chemical fluorescent dye Syto60, and confocal images of live samples were acquired using an automated confocal microscope. Typical images are displayed in FIG. 1 a. During the first 24 hours, a few discrete weakly fluorescent bacteria localized within the cells. By day 2, the average number of cells had increased and mycobacteria had started to spread into neighboring cells leading to zones of strongly fluorescent bacteria. The localization of the green signal is always within a distance of 5 μm to that of the red cell signal and in most cases actually overlaps with the cell signal. This confirms the intracellular nature of the mycobacteria growth. By day 4, the cell number has significantly diminished and the bacteria have formed large, highly fluorescent aggregates, which cover almost the entire image from day 5 onwards. As a control, non-infected cells grew up to confluence at day 2 and remained alive until day 7.
  • In order to automatically quantify the intracellular bacterial load, an in-house image analysis script was developed. This script enables the automated quantification of the number of cells and the percentage of infected cells, whereby an infected cell is a cell containing at least three green pixels with an intensity above a defined threshold (FIG. 1 b). 2 hours after infection, between 2 and 10% of Raw264.7 cells were found to harbor a low number of bacilli (FIG. 1 c). The percentage of infected cells, hereafter named infection ratio, continued to increase from 72 hours post-infection reaching up to 70% at seven days post infection. This increase in infection ratio correlated with an increase in cell mortality (FIG. 1 d/e).
    • Example 2 Comparative Minimal Inhibitory Concentration of Known Anti-Tubercular Drugs
  • To validate the assay set-up, the effect of current anti-tuberculosis drugs on M. tuberculosis intracellular growth was investigated. 2-fold serial dilutions of isoniazid (INH), rifampin and ethionamide were performed, followed by testing on macrophages that had previously been infected with M. tuberculosis H37Rv-GFP. After 5 days of incubation, macrophages were stained, and images acquired on an automated confocal microscope as described above. A larger number of cells and a fewer number of bacteria are clearly seen on pictures corresponding to samples that were incubated with INH compared to the DMSO negative control. This shows that INH prevents both intracellular M. tuberculosis growth and bacillus mediated cytotoxicity (FIG. 2 a). A clear inhibition dose-response curve was obtained by image-extracted analysis (FIG. 2 b). In parallel, inhibition of M. tuberculosis H37Rv-GFP in vitro growth by INH was monitored by recording green fluorescence intensity under the same conditions. In both experiments, the minimal inhibitory concentration (MIC) for INH was 0.1 μg/mL, which is in accordance with the MIC reported in the literature for extracellular M. tuberculosis growth (Andries et al., 2005). Similar results were obtained with the standard anti-tuberculosis drugs ethionamide (FIG. 2 c) and ethambutol (data not shown), whereas for rifampin, there was a log-fold decrease in the MIC in the cell-based assay compared to the in vitro assay (FIG. 2 d). The diminished efficacy of rifampin in the cell-based assay is likely due to impaired cell penetration and further demonstrates that it is the intracellular antibacterial activity that is being monitored in this assay. Thus, adaptation of both the intracellular and the in vitro M. tuberculosis growth assay for high throughput screening (HTS) was performed.
    • Example 3 Assay Scale-Up and Validation
  • To simplify the protocol for FITS purposes, macrophages were infected in batch with M. tuberculosis before being dispensed onto the compounds. The batch infection was carried out with macrophages in suspension at 37° C. under mild shaking. Free unbound mycobacteria were removed by washing three times with PBS and differential centrifugation, as well as by an additional one-hour incubation step with amykacin, an antibiotic known to selectively kill extracellular microbes (FIG. 6 a). M. tuberculosis infected macrophages were then seeded in plates that had been previously dispensed with the compounds, DMSO or antibiotic controls. The day-to-day as well as plate-to plate reproducibility was first tested. To this end, either serial dilutions of INH or rifampin were dispensed into 8 plates along with the regular DMSO and positive control (INH at 1 μg/mL (MIC100) and at 0.05 μg/mL (MIC90) and rifampin at 1 μg/mL) wells that were subsequently seeded with infected cells. The same experiment was repeated over 2 consecutive days. After incubation for 5 days and macrophage staining, pictures from each plate were acquired. The mean infection ratio determined for the DMSO negative controls in each plate for the 2 days of experiments was between 50% and 70%, whereas for the INH and rifampin samples, the mean infection ratio fell to below 20% (FIG. 3 a). Despite some variation in the mean infection ratio between the two experiments, the difference between the INH-positive and DMSO-negative controls was above five-fold for both days. P values calculated for each plate using a paired t-student test also confirmed a significant difference between the positive and negative controls (p<0.000001, data not shown). In addition, the inventors performed an experiment to determine if inhibitors of M. tuberculosis intracellular growth infection dispensed in any well on the plate could be detected by performing double-blind controls (spike of INH and rifampin at 3 different concentrations). Indeed, one hundred percent of the spikes were identified (data not shown). Taken together, these results prove that the assay is sensitive enough to be able to identify inhibitors under HTS conditions. Finally, the robustness of the assay was checked by monitoring the dose-response of reference compounds. Almost identical MICs for the antibiotic positive controls were determined independent of the plate or the day of the experiment (FIG. 3 b/c). Calculated MICs from the image based quantification of the infection ratio were 0.16+/−0.05 μg/mL and 2.4+/−1.3 μg/mL for INH and rifampin, respectively, and were confirmed by CFU plating (data not shown). In parallel, the extracellular growth assay was validated with a similar approach (data not shown).
    • Example 4 Primary Screening of a Large Library of Small Synthetic Compounds Using the Phenotypic Cell-Based Assay
  • A 26500 small molecule compound library, that was selected for its high chemical diversity and drug-like properties according to the Lipinski rules (Lipinski et al., 2001), was chosen as the first library to be screened using the validated phenotypic cell-based assay. The primary screen was carried out with compounds at 20 μM in singleton. The throughput was set to about 6000 compounds per working day encompassing 25 plates. The screening was performed with Raw264.7 cells that had been expanded from frozen stocks for ten days before infection with M. tuberculosis H37Rv-GFP. To accept the screening results, the MICs obtained from 2 serial dilutions of INH and Rifampin processed at the beginning and at the end of the screening day should show similar results compared to the values obtained during the validation (see above). Each screened plate is then accepted by the quality control procedure if the window between DMSO and INH (1 μg/ml) is higher than 3 and the CV calculated for the 320 compounds present in each plate is lower than 25. Such quality control criteria allow the identification of hits with an activity higher than 75%. Subsequently, the percent inhibition for each compound was determined relative to the corresponding mean infection ratio between 1 μg/mL INH (100%) and DMSO (0%) in the same 384-well plate. The percent inhibition distribution is centered around −20% of inhibition (FIG. 4 a). It was decided to select compounds that have an inhibitory effect greater than 65% which corresponds to a little less than 1.5% of the total compounds.
  • In parallel, the same compounds were only tested for their inhibitory activity on the M. tuberculosis H37Rv-GFP bacterial growth. The results from this assay, which are based on classical fluorescence intensity, showed a higher degree of reproducibility and the criteria for plate validation was set to a Z′ value (DMSO/INH) greater than 0.35. The throughput for this fluorescence based assay was approximately 20,000 compounds per day. Compounds that prevented M. tuberculosis growth in vitro with an inhibitory effect above 65% were then selected as hits (1.4%) as they belong to a clear independent population compared to the inactive population centered to 0% (FIG. 4 b).
  • The results gathered from the two different screenings were then compiled and compared (FIG. 4 c). Four different populations could be identified: compounds that are i) active only on extracellular bacteria, ii) active only on intracellular bacteria, iii) active in both settings or iv) not active. 657 compounds (2.5%) belonged to one of the first three categories and, thus, were selected for further investigation.
  • An important parameter that can be measured during image analysis is the total cell number, also referred to as cell cytotoxicity. A low cell number can be the result of two independent phenomena, the compound toxicity and M. tuberculosis growth mediated cell toxicity. Indeed, at day 5 after infection with M. tuberculosis, the cell number decreased to less than 100 cells per image compared to more than 500 cells per image for uninfected cells (FIG. 1 e). In contrast, a high cell number is obtained only when the compound is not toxic and prevents mycobacterial growth. This turns out to be a second relevant measurement of a compound's anti-mycobacterial activity. However, this criterion was not applied for the selection of hits from the primary screen as a low cell number was found for only a few compounds and the inventors wanted to avoid failing to select highly active compounds that would later on prove to be active at much lower concentrations despite a cell toxicity at 20 μM. An additional validation criterion of a Z′ (DMSO/INH) value of the total cell number greater than 0.2 was added for the following screening steps.
    • Example 5 Confirmation of Screening Results, Dose-Response Analysis and Hit Classification
  • The 657 selected hits were first confirmed at 3 different concentrations, 20 μM, 2 μM and 0.2 μM. For 340 hits the activity was confirmed either at 20 μM or 2 μM, on the intracellular or the in vitro assay (see Table 1). From this latter list, 121 compounds demonstrated an inhibitory activity above 65% at 2 μM without any apparent cell toxicity at 20 μM and consequently were selected for further confirmation by ten 3-fold serial dilutions (see FIGS. 9-1 to 9-41). All 121 compounds were confirmed by serial dilution with a MIC ranging between 250 nM and 20 μM. The results shown in FIG. 5 are representative of the three types of behavior observed: most of the compounds exhibited a clear dose response curve when activity was measured as infection ratio (FIG. 5 b/e/h). Compounds active on the bacilli level present a similar activity in the extracellular assay (FIG. 5 c/f) even if the MIC is different from one assay to the other. A few compounds don't present clear activity on the in vitro bacilli (FIG. 5 i) and may represent drugs acting through a cellular target or on a bacilli target involved only during the infection process. Furthermore, toxic compounds can be identified thanks to a dramatic decrease in the cell number when the compound concentration increases (FIG. 5 d) and activity of non-toxic compounds are validated by a dose response protective effect on the cell number (FIG. 5 a). Consequently cell number detection represents an independent secondary assay in the same well as the primary assay. The serial dilution results from all 121 compounds are presented in FIGS. 9-1 to 9-41.
  • The 121 confirmed hits can be clustered as 20 independent/general scaffolds (Table 2). The number of compounds for each scaffold varied, ranging from 1 to 69 molecules. The molecules from the 69-compound scaffold share a common structure which is similar to INH thereby validating the screening results. One scaffold contains molecules that were only active in the intracellular assay and its mechanism of action will be the focus of further investigation.
    • Example 6 Derivatization of the Benzamide Compounds
  • The benzamide compounds (scaffold II; see Table 2) underwent derivatization according to the methods outlined below (Schemes 1-7). Formation of the amide can be performed under general conditions using EDC or DCC coupling reagents with acids instead of acyl chloride. Resulting derivatives were examined for inhibitory activity using the assay described above and the results are summarized in Table 3.
  • Figure US20150018543A1-20150115-C00011
  • Figure US20150018543A1-20150115-C00012
    • General procedure for the synthesis of 2-phenoxyethyl isoindoline-1,3-dione (A1)
  • To a solution of 2-(2-hydroxyethyl)isoindoline-1,3-dione (1.68 mmol) in methylene chloride (10 mL) was added ADDP (1.68 mmol), triphenylphosphine (1.68 mmol) and phenol (3.18 mmol) and stirred at room temperature. After stirring overnight, the reaction mixture was diluted with methylene chloride (30 mL) and washed with 1 M NaOH aqueous solution (50 mL), and brine (50 mL). The organic layer was dried over anhydrous MgSO4 and concentrated in vacuo. The crude product was purified by silica gel flash column chromatography (4:1 hexanes/ethyl acetate) and recrystallized from a mixture of hexanes and ethyl acetate to give A1.
    • General Procedure for the Synthesis of N-(2-Phenoxyethyl)-Benzamide (A2)
  • To a solution of A1 (1.14 mmol) in methanol (10 mL) was added hydrazine monohydrate (1.42 mmol) and the resulting mixture was refluxed under a nitrogen atmosphere. After 3 h, the reaction mixture was allowed to cool to room temperature and concentrated in vacuo. The residue was precipitated with methylene chloride (10 mL). The resulting precipitate was filtered through Celite and the filtrate was concentrated in vacuo to afford an amine intermediate. To a solution of the amine in methylene chloride (10 mL) was added triethylamine (0.45 mmol) and a benzoylchloride (0.45 mmol) at 0° C. and the resulting mixture was stirred at room temperature. After 3 h, the reaction mixture was diluted with methylene chloride (10 mL) and washed with 1 M HCl aqueous solution (30 mL), saturated Na2CO3 aqueous solution (30 mL) and brine (30 mL). The organic layer was dried over anhydrous MgSO4 and concentrated in vacuo. The crude product was purified by silica gel flash column chromatography (3:1 hexanes/ethyl acetate) and recrystallized from a mixture of hexanes and ethyl acetate to give A2.
    • 3,5-Dinitro-N-(2-phenoxyethyl)benzamide (1)
  • Figure US20150018543A1-20150115-C00013
  • 1H NMR (400 MHz, Acetone-d6) δ 3.88 (t, J=4.4 Hz, 2H), 4.21 (t, J=5.2 Hz, 2H), 6.89 (d, J=8.4 Hz, 3H), 7.24 (t, J=8.0 Hz, 2H), 8.78 (brs, 1H), 9.02 (d, J=2.0 Hz, 1H), 9.07 (d, J=2.0 Hz, 2H); 13C NMR (100 MHz, Acetone-d6) δ 40.1, 66.0, 114.5, 120.8, 127.6, 129.6, 137.8, 148.8, 158.9, 163.0.
    • N-(2-(2-Methoxyphenoxy)ethyl)-3,5-dinitrobenzamide (2)
  • Figure US20150018543A1-20150115-C00014
  • 1H NMR (400 MHz, CDCl3) δ 3.89 (s, 3H), 3.92 (dd, J=5.2, 10.4 Hz, 2H), 4.23 (t, J=4.8 Hz, 2H), 6.91-7.02 (m, 4H), 7.63 (brs, 1H), 9.02 (d, J=1.6 Hz, 2H), 9.14 (t, J=2.0 Hz, 1H); 13C NMR (100 MHz, CDCl3) δ40.0, 56.1, 68.8, 112.2, 115.8, 121.0, 121.5, 122.9, 127.3, 137.8, 147.5, 148.6, 149.8, 162.6.
    • N-(2-(3-Methoxyphenoxy)ethyl)-3,5-dinitrobenzamide (3)
  • Figure US20150018543A1-20150115-C00015
  • 1H NMR (400 MHz, Acetone-d6) δ 3.74 (s, 3H), 3.85 (dd, J=5.6 Hz, 4.8 Hz, 2H), 4.21 (t, J=5.2 Hz, 2H), 6.50 (m, 3H), 7.14 (t, J=8.4 Hz, 1H), 8.75 (brs, 1H), 9.04 (s, 1H), 9.08 (s, 2H); 13C NMR (100 MHz, Acetone-d6) δ 40.1, 54.8, 66.1, 100.9, 106.5, 106.8, 120.9, 127.5, 130.0, 137.9, 148.8, 160.2, 161.2, 163.0.
    • N-(2-(4-Methoxyphenoxy)ethyl)-3,5-dinitrobenzamide (4)
  • Figure US20150018543A1-20150115-C00016
  • 1H NMR (400 MHz, CDCl3) δ 3.72 (s, 3H), 3.91 (dd, J=5.2, 10.8 Hz, 2H), 4.12 (t, J=4.8 Hz, 2H), 6.74-6.80 (m, 4H), 7.21 (brs, 1H), 8.95 (d, J=2.0 Hz, 2H), 9.07 (t, J=2.0 Hz, 1H); 13C NMR (100 MHz, CDCl3) δ 40.4, 55.6, 66.8, 114.7, 115.4, 121.0, 127.2, 137.6, 148.5, 152.2, 154.3, 163.1; LC-MS (ESI, m/z): 361 [M+H]+.
    • N-(2-(2-Chlorophenoxy)ethyl)-3,5-dinitrobenzamide (5)
  • Figure US20150018543A1-20150115-C00017
  • 1H NMR (400 MHz, CDCl3) δ 3.97 (dd, J=5.2, 10.4 Hz, 2H), 4.25 (t, J=5.2 Hz, 2H), 6.93-6.95 (m, 2H), 7.19-7.24 (m, 2H), 7.35 (dd, J=1.2, 8.0 Hz, 1H), 8.98 (d, J=2.0 Hz, 2H), 9.12 (t, J=2.0 Hz, 1H); 13C NMR (100 MHz, CDCl3) δ 34.9, 63.0, 109.7, 116.2, 117.7, 118.2, 122.3, 123.1, 125.5, 132.6, 143.7, 148.7, 157.9.
    • N-(2-(3-Chlorophenoxy)ethyl)-3,5-dinitrobenzamide (6)
  • Figure US20150018543A1-20150115-C00018
  • 1H NMR (400 MHz, CDCl3) δ 3.97 (dd, J=5.6, 10.8 Hz, 2H), 4.19 (t, J=4.8 Hz, 2H), 6.80-6.98 (m, 4H), 7.24 (t, J=8.0 Hz, 1H), 8.96 (d, J=2.0 Hz, 2H), 9.17 (t, J=2.0 Hz, 1H); 13C NMR (100 MHz, CDCl3) δ 40.1, 66.4, 110.7, 115.0, 121.2, 121.7, 127.2, 130.4, 135.1, 137.6, 148.7, 158.8, 163.0.
    • N-(2-(4-Chlorophenoxy)ethyl)-3,5-dinitrobenzamide (7)
  • Figure US20150018543A1-20150115-C00019
  • 1H NMR (400 MHz, CDCl3) δ 3.96 (dd, J=5.6, 10.4 Hz, 2H), 4.17 (t, J=4.8 Hz, 2H), 6.78 (brs, 1H), 6.86 (dd, J=2.4, 6.8 Hz, 2H), 7.23 (dd, J=2.0, 6.8 Hz, 2H), 8.96 (d, J=2.4 Hz, 2H), 9.17 (t, J=2.0 Hz, 1H); 13C NMR (100 MHz, CDCl3) δ40.1, 66.5, 115.7, 121.2, 126.5, 127.2, 129.6, 137.6, 148.9, 156.8, 163.0.
    • N-(2-(2-Fluorophenoxy)ethyl)-3,5-dinitrobenzamide (8)
  • Figure US20150018543A1-20150115-C00020
  • 1H NMR (400 MHz, CDCl3) δ 3.97 (dd, J=5.2, 10.8 Hz, 2H), 4.25 (t, J=5.2 Hz, 2H), 6.91-7.06 (m, 4H), 7.39 (brs, 1H), 8.97 (d, J=2.0 Hz, 2H), 9.15 (t, J=2.0 Hz, 1H); 13C NMR (100 MHz, CDCl3) δ 40.1, 68.3, 115.7, 116.3 (d, J=20 Hz, due to F), 121.1, 122.3 (d, J=7Hz, due to F), 124.6 (d, J=5 Hz, due to F), 127.3, 137.6, 146.2, 148.6, 152.8 (d, J=250 Hz, due to F), 163.1; LC-MS (ESI, m/z): 350 [M+H]+.
    • N-(2-(4-Fluorophenoxy)ethyl)-3,5-dinitrobenzamide (9)
  • Figure US20150018543A1-20150115-C00021
  • 1H NMR (400 MHz, Acetone-d6) δ 3.88 (dd, J=5.2, 10.8 Hz, 2H), 4.23 (t, J=5.2 Hz, 2H), 6.95-7.07 (m, 4H), 8.79 (brs, 1H), 9.07 (t, J=2.4 Hz, 1H), 9.11 (d, J=2.0 Hz, 2H).
    • N-(2-(4-Hydroxyphenoxy)ethyl)-3,5-dinitrobenzamide (10)
  • Figure US20150018543A1-20150115-C00022
  • 1H NMR (400 MHz, DMSO-d6) δ 3.66 (dd, J=5.6, 11.2 Hz, 2H), 4.06 (t, J=5.2 Hz, 2H), 6.65-6.68 (m, 2H), 6.76-6.80 (m, 2H), 8.91 (brs, 1H), 8.98 (t, J=2.0 Hz, 1H), 9.08 (d, J=2.4 Hz, 2H), 9.42 (brs, 1H); 13C NMR (100 MHz DMSO-d6) δ 40.1, 66.9, 116.2, 116.4, 121.5, 128.2, 137.4, 148.8, 151.8, 152.0, 163.1.
    • N-(2-(3-(Trifluoromethoxy)phenoxy)ethyl)-3,5-dinitrobenzamide (11)
  • Figure US20150018543A1-20150115-C00023
  • 1H NMR (400 MHz, Acetone-d6) δ 3.89 (dd, J=5.6, 11.2 Hz, 2H), 4.29 (t, J=5.6 Hz, 2H), 6.88 (d, J=6.0 Hz, 2H), 6.99 (d, J=8.0 Hz, 1H), 7.38 (t, J=8.4 Hz, 1H), 8.79 (brs, 1H), 9.05 (d, J=1.2 Hz, 1H), 9.08 (d, J=1.2 Hz, 2H); 13C NMR (100 MHz, Acetone-d6) δ 39.9, 66.7, 107.8, 113.1, 113.6, 120.9, 127.6, 130.9, 137.8, 148.9, 150.1, 160.2, 163.0.
    • N-(2-(4-(Trifluoromethoxy)phenoxy)ethyl)-3,5-dinitrobenzamide (12)
  • Figure US20150018543A1-20150115-C00024
  • 1H NMR (400 MHz, Acetone-d6) δ 3.88 (dd, J=10.8 Hz, 5.2 Hz, 2H), 4.27 (t, J=5.6 Hz, 2H), 7.03 (dd, J=7.2, 2.0 Hz, 2H), 7.23 (d, J=8.8 Hz, 2H), 8.78 (brs, 1H), 9.04 (d, J=2.0 Hz, 1H), 9.08 (d, J=2.0 Hz, 2H); 13C NMR (100 MHz, Acetone-d6) δ 40.0, 66.8, 115.7, 120.9, 122.7, 127.6, 137.8, 142.7, 142.8, 148.9, 157.9, 163.1.
    • Methyl 4-(2-(3,5-dinitrobenzamido)ethoxy)benzoate (13)
  • Figure US20150018543A1-20150115-C00025
  • 1H NMR (400 MHz, Acetone-d6) δ 3.81 (s, 3H), 3.91 (t, J=5.6 Hz, 2H), 4.33 (t, J=5.6 Hz, 2H), 7.00 (t, J=2.8 Hz, 1H), 7.03 (t, J=2.8 Hz, 1H), 7.90 (t, J=2.8 Hz, 1H), 7.92 (t, J=2.8 Hz, 1H), 8.78 (brs, 1H), 9.03 (t, J=2.4 Hz, 1H), 9.07 (d, J=2.4 Hz, 2H); 13C NMR (100 MHz, Acetone-d6) δ39.9, 51.3, 66.5, 114.4, 120.9, 123.0, 127.6, 131.5, 137.8, 148.9, 162.8, 163.0, 166.1.
    • N-(2-(4-Aminophenoxy)ethyl)-3,5-dinitrobenzamidehydrochloride (14)
  • Figure US20150018543A1-20150115-C00026
  • 1H NMR (400 MHz, DMSO-d6) δ 3.67 (d, J=5.2 Hz, 2H), 4.15 (t, J=5.2 Hz, 2H), 7.03 (d, J=1.6 Hz, 2H), 7.29 (d, J=1.6 Hz, 2H), 8.91 (d, J=2.0 Hz, 1H), 9.04 (d, J=2.0 Hz, 2H), 9.52 (brs, 1H), 10.28 (brs, 3H); 13C NMR (100 MHz, DMSO-d6) δ 40.1, 66.1, 115.4, 120.8, 124.3, 124.5, 127.5, 136.7, 148.1, 157.8, 162.4.
    • N-(2-(4-(t-Butoxycarbonylamino)phenoxy)ethyl)-3,5-dinitrobenzamide (15)
  • Figure US20150018543A1-20150115-C00027
  • 1H NMR (400 MHz, Acetone-d6) δ 1.44 (s, 9H), 3.83 (m, 2H), 4.18 (m, 2H), 6.84 (dd, J=3.2, 9.2 Hz, 2H), 7.40 (d, J=7.6 Hz, 2H), 8.15 (brs, 1H), 8.73 (brs, 1H), 9.03 (t, J=2.0 Hz, 1H), 9.08 (d, J=2.0 Hz, 2H); 13C NMR (100 MHz, Acetone-d6) δ 27.8, 40.1, 66.4, 78.9, 114.8, 119.9, 120.9, 127.6, 133.3, 137.9, 148.8, 153.2, 154.4, 163.0; LC-MS (ESI, m/z): 469[M+Na]+.
    • N-(2-(4-Methoxyphenoxy)ethyl)-3-nitrobenzamide (16)
  • Figure US20150018543A1-20150115-C00028
  • 1H NMR (400 MHz, CDCl3) δ 3.69 (s, 3H), 3.81 (dd, J=5.2, 10.4 Hz, 2H), 4.06 (t, J=5.6 Hz, 2H), 6.73-6.78 (m, 4H), 7.48 (brs, 1H), 7.53 (t, J=8.0 Hz, 1H), 8.13 (d, J=7.6 Hz, 1H), 8.24 (d, J=10.4 Hz, 1H), 8.56 (t, J=2.0 Hz, 1H); 13C NMR (100 MHz, CDCl3) δ 39.8, 55.4, 66.7, 114.4, 115.2, 121.9, 125.8, 129.5, 133.1, 135.7, 147.8, 152.3, 153.9, 165.2.
    • N-(2-(2-Fluorophenoxy)ethyl)-3-nitrobenzamide (17)
  • Figure US20150018543A1-20150115-C00029
  • 1H NMR (400 MHz, CDCl3) δ 3.92 (dd, J=5.6, 10.8 Hz, 2H), 4.23 (t, J=4.8 Hz, 2H), 6.90-7.09 (m, 4H and brs, 1H), 7.62 (t, J=8.0 Hz, 1H), 8.14 (d, J=8.0 Hz, 1H), 8.33 (d, J=8.0 Hz, 1H), 8.63 (t, J=2.0 Hz, 1H); 13C NMR (100 MHz, CDCl3) δ 39.8, 68.3, 115.6, 116.6 (d, J=18.6 Hz, due to F), 122.3 (d, J=5.3 Hz, due to F), 124.7 (d, J=4.5 Hz, due to F), 126.0, 129.7, 133.0, 135.8, 146.3 (d, J=10.4 Hz, due to F), 148.1, 152.6 (d, J=245 Hz, due to F), 165.2.
    • N-(2-(4-Methoxyphenoxy)ethyl)benzamide (18)
  • Figure US20150018543A1-20150115-C00030
  • 1H NMR (400 MHz, CDCl3) δ 3.72 (s, 3H), 3.80 (dd, J=5.2, 10.8 Hz, 2H), 4.05 (t, J=5.6 Hz, 2H), 6.78-6.83 (m, 4H), 7.03 (brs, 1H), 7.35-7.45 (m, 4H), 7.74 (d, J=11.2 Hz, 1H); 13C NMR (100 MHz, CDCl3) δ9.4, 55.4, 67.1, 114.5, 115.2, 126.8, 128.3, 131.3, 134.1, 152.4, 153.9, 167.6.
    • N-(2-(4-Methoxyphenoxy)ethyl)-N-methyl-3,5-dinitrobenzamide (19)
  • Figure US20150018543A1-20150115-C00031
  • (Two rotamers, 1:1) 1H NMR (400 MHz, CDCl3) δ 3.18 (brs, 3H), 3.65 (brs, 1H), 3.75 (s, 3H), 3.94 (brs, 1H), 4.03 (brs, 1H), 4.27 (brs, 1H), 6.79-6.84 (brd, 4H), 8.55 (brs, 1H), 8.72 (brs, 1H), 9.04 (brs, 1H).
    • N-Ethyl-N-(2-(4-methoxyphenoxy)ethyl)-3,5-dinitrobenzamide (20)
  • Figure US20150018543A1-20150115-C00032
  • (Two rotamers, 1:1) 1H NMR (400 MHz, CDCl3) δ 1.22-1.30 (m, 3H), 3.42 (brs, 1H), 3.63 (brs, 2H), 3.75 (s, 3H), 3.89 (brs, 1H), 4.01 (brs, 1H), 4.26 (brs, 1H), 6.80 (br, 4H), 8.53 (brs, 1H), 8.72 (brs, 1H), 9.04 (brs, 1H).
    • N-(3-(4-Methoxyphenoxy)propyl)-3,5-dinitrobenzamide (21)
  • Figure US20150018543A1-20150115-C00033
  • 1H NMR (400 MHz, CDCl3) δ 2.04-2.20 (m, 2H), 3.76 (t, J=6.0 Hz, 2H), 3.77 (s, 3H), 4.17 (t, J=5.2 Hz, 2H), 6.85-6.91 (m, 4H), 7.24 (brs, 1H), 8.96 (d, J=2.0 Hz, 2H), 9.16 (t, J=102.0 Hz, 1H).
    • Methyl 4-(3-(3,5-dinitrobenzamido)propoxy)benzoate (22)
  • Figure US20150018543A1-20150115-C00034
  • 1H NMR (400 MHz, CDCl3) δ 2.21-2.24 (m, 2H), 3.77 (dd, J=6.0, 12.0 Hz, 2H), 3.89 (s, 3H), 4.24 (t, J=5.6 Hz, 2H), 6.95 (d, J=8.8 Hz, 2H), 7.04 (brs, 1H), 8.00 (d, J=8.8 Hz, 2H), 8.96 (d, J=2.0 Hz, 2H), 9.16 (s, 1H); 13C NMR (100 MHz, CDCl3) δ 28.4, 39.3, 52.0, 67.2, 113.9, 121.1, 123.3, 127.0, 131.8, 137.8, 148.6, 161.9, 162.5, 166.6.
    • N-(3-(2-Fluorophenoxy)propyl)-3,5-dinitrobenzamide (23)
  • Figure US20150018543A1-20150115-C00035
  • 1H NMR (400 MHz, CDCl3) δ 2.19-2.25 (m, 2H), 3.83 (dd, J=5.2, 11.2 Hz, 2H), 4.27 (t, J=5.2 Hz, 2H), 6.90-7.11 (m, 4H), 7.50 (brs, 1H), 8.99 (d, J=2.0 Hz, 2H), 9.16 (t, J=2.0 Hz, 1H); 13C NMR (100 MHz, CDCl3) δ 28.2, 40.0, 69.5, 114.0, 116.3 (d, J=18 Hz, due to F), 120.9, 121.8 (d, J=7.4 Hz, due to F), 124.7 (d, J=3.7 Hz, due to F), 127.2, 127.3, 138.1, 147.3 (d, J=245 Hz, due to F), 153.5, 162.7.
  • Figure US20150018543A1-20150115-C00036
    • General Procedure for the Synthesis of N-(2-(benzyloxy)ethyl)-dinitrobenzamide (B2)
  • To a solution of 2-(2-hydroxyethyl)isoindoline-1,3-dione (1.17 mmol) in dimethyl formamide (10 mL) was added sodium hydride (2.34 mmol) and a benzyl bromide (1.40 mmol) at 0° C. and the resulting mixture was stirred at room temperature. After stirring overnight, distilled water (50 mL) was added and the resulting precipitate was collected by filtration to afford B1.
  • To a solution of B1 (0.85 mmol) in methanol (10 mL) was added hydrazine monohydrate (0.85 mmol) and the resulting mixture was refluxed under a nitrogen atmosphere. After 3 h, the reaction mixture was allowed to cool to room temperature and concentrated in vacuo. The residue was precipitated with methylene chloride (10 mL). The resulting precipitate was filtered off through Celite, and the filtrate was concentrated in vacuo to afford an amine.
  • To a solution of the amine in methylene chloride (10 mL) was added triethylamine (113 μl, 0.81 mmol) and a benzoylchloride (0.81 mmol) at 0° C. and the resulting mixture was stirred at room temperature. After 3 h, the reaction mixture was diluted with methylene chloride (30 mL) and washed with 1 M HCl aqueous solution (50 mL), saturated Na2CO3 aqueous solution (50 mL) and brine (50 mL). The organic layer was dried over anhydrous MgSO4 and concentrated in vacuo. The crude product was purified by silica gel flash column chromatography (3:1 hexanes/ethyl acetate) and recrystallized from a mixture of hexanes and ethyl acetate to give B2.
    • N-(2-(Benzyloxy)ethyl)-3,5-dinitrobenzamide (24)
  • Figure US20150018543A1-20150115-C00037
  • 1H NMR (400 MHz, CDCl3) δ 3.68-3.72 (m, 4H), 4.55 (s, 2H), 6.75 (brs, 1H), 7.24-7.33 (m, 5H), 8.91 (d, J=2.0 Hz, 2H), 9.13 (t, J=2.0 Hz, 1H); 13C NMR (100 MHz, CDCl3) δ 40.4, 68.1, 73.4, 121.0, 127.2, 128.0, 128.2, 128.7, 137.5, 138.0, 148.6, 162.7; LC-MS (ESI, m/z): 346[M+H]+.
    • N-(2-(3-Methoxybenzyloxy)ethyl)-3,5-dinitrobenzamide (25)
  • Figure US20150018543A1-20150115-C00038
  • 1H NMR (400 MHz, CDCl3) δ 3.71-3.74 (m, 4H), 3.76 (s, 3H), 4.52 (s, 2H), 6.77-6.90 (m, 3H), 6.97 (brs, 1H), 7.23 (t, J=8.0 Hz, 1H), 8.91 (d, J=2.0 Hz, 2H), 9.12 (t, J=2.0 Hz, 1H); 13C NMR (100 MHz, CDCl3) δ 40.5, 55.2, 68.2, 73.1, 113.1, 113.6, 120.0, 120.9, 127.2, 129.6, 137.8, 139.1, 148.5, 159.7, 162.8.
    • N-(2-(4-Methoxybenzyloxy)ethyl)-3,5-dinitrobenzamide (26)
  • Figure US20150018543A1-20150115-C00039
  • 1H NMR (400 MHz, CDCl3) δ 3.65-3.71 (m, 4H), 3.75 (s, 3H), 4.47 (s, 2H), 6.71 (brs, 1H), 6.84 (dd, J=6.8, 2.0 Hz, 2H), 7.23 (d, J=8.4 Hz, 2H), 8.87 (d, J=2.4 Hz, 2H), 9.13 (t, J=2.0 Hz, 1H); 13C NMR (100 MHz, CDCl3) δ 40.5, 55.3, 67.8, 73.1, 114.0, 121.0, 127.1, 129.6, 130.0, 137.9, 148.6, 159.5, 162.7.
    • N-(2-(4-Chlorobenzyloxy)ethyl)-3,5-dinitrobenzamide (27)
  • Figure US20150018543A1-20150115-C00040
  • 1H NMR (400 MHz, CDCl3) δ 3.68-3.76 (m, 4H), 4.53 (s, 2H), 6.77 (brs, 1H), 7.25-7.32 (m, 4H), 8.91 (d, J=2.0 Hz, 2H), 9.15 (t, J=2.0 Hz, 1H); 13C NMR (100 MHz, CDCl3) δ 40.4, 68.3, 72.6, 121.1, 127.2, 128.8, 129.2, 134.0, 136.0, 137.8, 148.6, 162.7.
    • N-(2-(3-chlorobenzyloxy)ethyl)-3,5-dinitrobenzamide (28)
  • Figure US20150018543A1-20150115-C00041
  • 1H NMR (400 MHz, CDCl3) δ 3.68-3.76 (m, 4H), 4.52 (s, 2H), 6.79 (brs, 1H), 7.17-7.29 (m, 4H), 8.91 (d, J=2.0 Hz, 2H), 9.13 (t, J=2.0 Hz, 1H); 13C NMR (100 MHz, CDCl3) δ 40.4, 68.4, 72.5, 121.1, 125.8, 127.2, 127.8, 128.1, 129.2, 134.5, 137.8, 139.6, 148.6, 162.8.
    • N-(2-(4-Fluorobenzyloxy)ethyl)-3,5-dinitrobenzamide (29)
  • Figure US20150018543A1-20150115-C00042
  • 1H NMR (400 MHz, CDCl3) δ 3.68-3.76 (m, 4H), 4.53 (s, 2H), 6.74 (brs, 1H), 7.02-7.06 (m, 2H), 7.30-7.33 (m, 2H), 8.92 (d, J=2.0 Hz, 2H), 9.16 (t, J=2.0 Hz, 1H); 13C NMR (100 MHz, CDCl3) δ 40.4, 68.1, 72.6, 115.5 (d, J=22 Hz, due to F), 121.1, 127.1, 130.0 (d, J=8.2 Hz, due to F), 133.5 (d, J=3.0 Hz, due to F), 137.8, 148.6, 162.5 (d, J=245 Hz, due to F), 162.7.
    • N-(2-(2-Fluorobenzyloxy)ethyl)-3,5-dinitrobenzamide (30)
  • Figure US20150018543A1-20150115-C00043
  • 1H NMR (400 MHz, CDCl3) δ 3.75 (s, 4H), 4.64 (s, 2H), 7.07-7.17 (m, 3H), 7.29-7.39 (m, 1H and brs. 1H), 8.94 (d, J=2.0 Hz, 2H), 9.17 (t, J=2.0 Hz, 1H).
    • 3,5-Dinitro-N-(2-(4-(trifluoromethoxy)benzyloxy)ethyl)benzamide (31)
  • Figure US20150018543A1-20150115-C00044
  • 1H NMR (400 MHz, CDCl3) δ 3.72-3.76 (m, 4H), 4.54 (s, 2H), 7.13 (d, J=8.0 Hz, 2H), 7.31-7.35 (m, 2H and brs, 1H), 8.94 (d, J=2.0 Hz, 2H), 9.08 (t, J=2.0 Hz, 1H); 13C NMR (100 MHz, CDCl3) δ0.4, 68.4, 72.2, 120.9, 121.0, 127.2, 129.1, 136.3, 137.7, 148.4, 148.7, 148.9, 162.9.
    • 3,5-Dinitro-N-(2-(3-(trifluoromethyl)benzyloxy)ethyl)benzamide (32)
  • Figure US20150018543A1-20150115-C00045
  • 1H NMR (400 MHz, CDCl3) δ 3.72-3.79 (m, 4H), 4.61 (s, 2H), 7.06 (brs, 1H), 7.45-7.55 (m, 4H), 8.93 (d, J=2.0 Hz, 2H), 9.10 (t, J=2.0 Hz, 1H); 13C NMR (100 MHz, CDCl3) δ 40.4, 68.7, 72.4, 121.0, 124.1, 124.6, 124.7, 127.2, 129.0, 130.6 (q, J=32 Hz, due to F), 130.8, 137.7, 138.6, 148.6, 162.9.
    • Methyl 4-((2-(3,5-dinitrobenzamido)ethoxy)methyl)benzoate (33)
  • Figure US20150018543A1-20150115-C00046
  • 1H NMR (400 MHz, CDCl3) δ 3.71-3.74 (m, 4H), 3.84 (s, 3H), 4.55 (s, 2H), 7.29 (d, J=8.0 Hz, 2H and brs, 1H), 7.85 (d, J=8.0 Hz, 2H), 8.90 (d, J=2.0 Hz, 2H), 9.01 (t, J=2.0 Hz, 1H); 13C NMR (100 MHz, CDCl3) δ 40.6, 52.2, 68.8, 72.6, 120.9, 127.3, 129.5, 129.7, 137.8, 142.9, 148.5, 163.0, 166.8.
    • 4-((2-(3,5-Dinitrobenzamido)ethoxy)methyl)benzoic acid (34)
  • Figure US20150018543A1-20150115-C00047
  • 1H NMR (400 MHz, Acetone-d6) δ 3.74 (t, J=5.2 Hz, 2H), 3.81 (t, J=5.2 Hz, 2H), 4.72 (s, 2H), 7.56 (d, J=8.4 Hz, 2H) 7.72 (brs, 1H), 8.03 (d, J=8.4 Hz, 2H), 9.02 (d, J=2.0 Hz, 2H), 9.13 (t, J=2.0 Hz, 1H).
    • N-(2-(Benzyloxy)ethyl)benzamide (35)
  • Figure US20150018543A1-20150115-C00048
  • 1H NMR (400 MHz, CDCl3) δ 3.62-3.68 (m, 4H), 4.52 (s, 2H), 6.71 (brs, 1H), 7.24-7.49 (m, 8H), 7.73-7.76 (m, 2H); 13C NMR (100 MHz, CDCl3) δ 39.7, 68.8, 73.1, 126.9, 127.8, 128.4, 131.3, 134.5, 137.8, 167.5.
    • N-(2-(3-(Trifluoromethyl)benzyloxy)ethyl)benzamide (36)
  • Figure US20150018543A1-20150115-C00049
  • 1H NMR (400 MHz, CDCl3) δ 3.63-3.70 (m, 4H), 4.56 (s, 2H), 6.72 (brs, 1H), 7.37-7.53 (m, 6H), 7.58 (s, 1H), 7.74-7.76 (m, 2H); 13C NMR (100 MHz, CDCl3) δ 39.7, 69.3, 72.3, 124.2, 124.3, 124.6, 126.9, 128.5, 128.9, 130.8, 131.5, 134.4, 139.0, 148.6, 167.6.
    • N-(2-(3-Chlorobenzyloxy)ethyl)benzamide (37)
  • Figure US20150018543A1-20150115-C00050
  • 1H NMR (400 MHz, CDCl3) δ 3.62-3.69 (m, 4H), 4.49 (s, 2H), 6.71 (brs, 1H), 7.17-7.50 (m, 7H), 7.75-7.77 (m, 2H); 13C NMR (100 MHz, CDCl3) δ 39.7, 69.0, 72.2, 125.6, 126.8, 127.6, 127.8, 128.4, 129.7, 131.3, 134.3, 139.9, 167.4.
    • N-(2-(3-Chlorobenzyloxy)ethyl)-3,5-difluorobenzamide (38)
  • Figure US20150018543A1-20150115-C00051
  • 1H NMR (400 MHz, CDCl3) δ 3.64-3.69 (m, 4H), 4.52 (s, 2H), 6.54 (brs, 1H), 6.95 (tt, J=2.4, 11.2 Hz, 1H), 7.19-7.33 (m, 6H).
    • N-(2-(3-Chlorobenzyloxy)ethyl)-3,5-dichlorobenzamide (39)
  • Figure US20150018543A1-20150115-C00052
  • 1H NMR (400 MHz, CD3OD) δ 3.13 (t, J=5.2 Hz, 2H), 3.67 (t, J=5.2 Hz, 2H), 4.55 (s, 2H), 7.27-7.29 (m, 3H), 7.42 (s, 1H), 7.46, (s, 1H), 7.81 (s, 2H); 13C NMR (100 MHz, CDCl3) δ40.6, 67.2, 73.2, 127.0, 128.7, 128.8, 128.9, 130.7, 131.0, 135.4, 135.5, 141.4, 142.7, 171.5.
    • N-(2-(3-Chlorobenzyloxy)ethyl)-3,5-bis(trifluoromethyl)benzamide (40)
  • Figure US20150018543A1-20150115-C00053
  • 1H NMR (400 MHz, CDCl3) δ 3.64-3.68 (m, 4H), 4.49 (s, 2H), 6.89 (brs, 1H), 7.15 (d, J=3.6 Hz, 1H), 7.21-7.24 (m, 2H), 7.27 (s, 1H), 7.95 (s, 1H), 8.18 (s, 2H); 13C NMR (100 MHz, CDCl3) δ40.3, 68.37, 72.5, 121.6, 125.0, 125.1, 125.7, 127.8, 128.1, 129.9, 132.0, 134.5, 136.6, 139.8, 164.8.
    • N-(2-(3-Chlorobenzyloxy)ethyl)-3-methoxybenzamide (41)
  • Figure US20150018543A1-20150115-C00054
  • 1H NMR (400 MHz, CDCl3) δ 3.63 (d, J=3.6 Hz, 2H), 3.65 (d, J=3.6 Hz, 2H), 3.81 (s, 3H), 4.49 (s, 2H), 6.51 (brs, 1H), 7.01 (dd, J=8.0 Hz, 2.4 Hz, 1H), 7.16 (d, J=4.4 Hz, 1H), 7.28 (m, 3H), 7.25-7.34 (m, 3H); 13C NMR (100 MHz, CDCl3) δ 39.9, 55.5, 69.2, 72.5, 112.4, 117.8, 118.7, 125.8, 127.8, 128.0, 129.6, 129.9, 134.5, 136.0, 140.0, 159.9, 167.5.
    • N-(2-(3-Chlorobenzyloxy)ethyl)-4-methoxybenzamide (42)
  • Figure US20150018543A1-20150115-C00055
  • 1H NMR (400 MHz, CDCl3) δ 3.62-3.66 (m, 4H), 3.82 (s, 3H), 4.49 (S, 2H), 6.48 (brs, 1H), 6.89 (d, J=8.8 hz, 2H), 7.17 (t, J=4.4 Hz, 2H), 7.24 (m, 1H), 7.32 (s, 1H), 7.71 (d, J=8.8 Hz, 2H) 13C NMR (100 MHz, CDCl3) δ 39.8, 55.5, 69.4, 72.4, 113.8, 125.7, 126.8, 127.8, 128.0, 128.8, 129.8, 134.5, 140.1, 162.2, 167.1.
    • N-(2-(3-Chlorobenzyloxy)ethyl)-3-(trifluoromethoxy)benzamide (43)
  • Figure US20150018543A1-20150115-C00056
  • 1H NMR (400 MHz, CDCl3) δ 3.62-3.68 (m, 4H), 4.49 (s, 2H), 6.62 (brs, 1H), 7.15 (dd, J=1.2, 8.8 Hz, 1H), 7.22-7.23 (m, 2H), 7.36 (t, J=1.2 Hz, 2H), 7.43 (t, J=8.4 Hz, 1H), 7.63 (dd, J=1.2, 4.4 Hz, 2H); 13C NMR (100 MHz, CDCl3) δ 40.0, 69.0, 72.4, 119.3, 120.1, 123.8, 125.1, 125.7, 127.8, 128.0, 129.9, 130.1, 134.5, 136.6, 140.0, 149.4, 166.1.
    • N-(2-(3-Chlorobenzyloxy)ethyl)-4-(trifluoromethyl)benzamide (44)
  • Figure US20150018543A1-20150115-C00057
  • 1H NMR (400 MHz, CDCl3) δ 3.62-3.68 (m, 4H), 4.49 (s, 2H), 6.71 (brs, 1H), 7.14-7.17 (m, 1H), 7.23-7.24 (m, 2H), 7.3 (s, 1H), 7.64 (d, J=8.0 Hz, 2H), 7.83 (d, 0.1=8.0 Hz, 2H); 13C NMR (100 MHz, CDCl3) δ 40.0, 68.9, 72.4, 125.6 (q, J=3.7 Hz), 125.8, 127.5, 127.8, 128.1, 129.9, 138.1, 133.4, 134.5, 137.7, 140.0, 166.4.
    • N-(2-(3-Chlorobenzyloxy)ethyl)-3-(trifluoromethyl)benzamide (45)
  • Figure US20150018543A1-20150115-C00058
  • 1H NMR (400 MHz, CDCl3) δ 3.62 (m, 4H), 4.46 (s, 2H), 6.96 (brs, 1H), 7.14-7.27 (m, 4H), 7.47 (t, J=7.2 Hz, 1H), 7.68 (d, J=3.2 Hz, 1H), 7.89 (d, J=3.2 Hz, 1H), 8.01 (s, 1H); 13C NMR (100 MHz, CDCl3) δ 40.0, 68.9, 72.3, 122.4, 124.1, 125.7, 127.7, 127.9, 128.0, 129.1, 129.8, 130.3, 130.8, 134.4, 135.2, 140.0, 166.3.
    • Methyl 3-(2-(3-chlorobenzyloxy)ethylcarbamoyl)benzoate (46)
  • Figure US20150018543A1-20150115-C00059
  • 1H NMR (400 MHz, CDCl3) δ 3.62-3.69 (m, 4H), 3.89 (s, 3H), 4.48 (s, 2H), 6.71 (brs, 1H), 7.15-7.16 (m, 1H), 7.21-7.24 (m, 2H), 7.28 (s, 1H), 7.47 (t, J=4.0 Hz, 1H), 7.97 (d, J=4.8 Hz, 1H), 8.11 (d, J=4.8 Hz, 1H), 8.35 (t, J=1.6 Hz, 1H); 13C NMR (100 MHz, CDCl3) δ 40.0, 52.4, 69.0, 72.4, 125.7, 127.7, 127.8, 128.0, 128.9, 129.8, 130.5, 131.8, 132.4, 134.4, 134.8, 140.0, 166.3, 166.6.
    • Methyl 4-(2-(3-chlorobenzyloxy)ethylcarbamoyl)benzoate (47)
  • Figure US20150018543A1-20150115-C00060
  • 1H NMR (400 MHz, CDCl3) δ 3.62-3.66 (m, 4H), 3.90 (s, 3H), 4.48 (s, 2H), 6.65 (brs, 1H), 7.14-7.17 (m, 1H), 7.22 (d, J=5.2 Hz, 2H), 7.30 (s, 1H), 7.78 (d, J=8.0 Hz, 2H), 8.04 (d, J=8.0 Hz, 2H); 13C NMR (100 MHz, CDCl3) δ 40.0, 52.4, 69.0, 72.4, 125.7, 127.1, 127.8, 128.1, 129.9, 132.7, 134.5, 138.4, 140.0, 160.3, 166.7.
    • N-(2-(3-Chlorobenzyloxy)ethyl)-3-nitrobenzamide (48)
  • Figure US20150018543A1-20150115-C00061
  • 1H NMR (400 MHz, CDCl3) δ 3.64 (m, 4H), 4.45 (s, 2H), 7.13-7.23 (m, 5H), 7.53 (m, 1H), 8.08 (d, J=6.8 Hz, 1H) 8.22 (d, J=6.8 Hz, 1H), 8.54 (s, 1H); 13C NMR (100 MHz, CDCl3) δ 40.1, 68.7, 72.2, 122.0, 125.6, 125.9, 127.5, 127.8, 129.7, 129.8, 133.1, 134.2, 136.0, 139.9, 148.0, 165.3.
    • N-(2-(3-Chlorobenzyloxy)ethyl)-4-nitrobenzamide (49)
  • Figure US20150018543A1-20150115-C00062
  • 1H NMR (400 MHz, CDCl3) δ 3.63 (m, 4H), 4.45 (s, 2H), 6.97 (brs, 1H), 7.12-7.25 (m, 4H), 7.87 (d, J=6.4 Hz, 2H), 8.15 (d, J=6.4 Hz, 2H); 13C NMR (100 MHz, CDCl3) δ 40.1, 68.7, 72.2, 123.6, 125.6, 127.5, 127.9, 128.2, 129.7, 134.3, 139.9, 140.0, 149.4, 165.6.
    • N-(2-(3-Chlorobenzyloxy)ethyl)-3-fluorobenzamide (50)
  • Figure US20150018543A1-20150115-C00063
  • 1H NMR (400 MHz, CDCl3) δ 3.56-3.61 (m, 4H), 4.43 (s, 2H), 6.66 (brs, 1H), 7.10-7.12 (m, 2H), 7.18-7.19 (m, 2H), 7.25 (s, 1H), 7.30-7.31 (m, 1H), 7.41-7.45 (m, 2H); 13C NMR (100 MHz, CDCl3) δ 39.9, 69.0, 72.4, 114.3 (d, J=23.0 Hz, due to F), 118.4 (d, J=20.8 Hz, due to F), 122.4 (d, J=3.0 Hz, due to F), 125.7, 127.7, 128.0, 129.8, 130.2 (d, J=8.2 Hz, due to F), 134.5, 136.7 (d, J=6.7 Hz, due to F), 140.0, 163.0 (d, J=245 Hz, due to F), 166.3 (d, J=3.0 Hz, due to F).
    • N-(2-(3-Chlorobenzyloxy)ethyl)-3-chlorobenzamide (51)
  • Figure US20150018543A1-20150115-C00064
  • 1H NMR (400 MHz, CDCl3) δ 3.64 (m, 4H), 4.49 (s, 2H), 6.52 (brs, 1H), 7.17 (d, J=3.2 Hz, 1H), 7.24 (s, 2H), 7.31-7.36 (m, 2H), 7.44 (d, J=3.6 Hz, 1H), 7.59 (d, J=7.6 Hz, 1H), 7.73 (s, 1H); 13C NMR (100 MHz, CDCl3) δ40.0, 69.1, 72.5, 125.1, 125.8, 127.5, 127.8, 128.1, 129.9, 130.0, 131.6, 134.6, 134.9, 136.3, 140.0, 166.3.
    • N-(2-(3-Chlorobenzyloxy)ethyl)-4-hydroxybenzamide (52)
  • Figure US20150018543A1-20150115-C00065
  • 1H NMR (400 MHz, CDCl3) δ 3.64 (s, 4H), 4.48 (s, 2H), 6.57 (brs, 1H), 6.84 (dd, J=2.0, 8.8 Hz, 2H), 7.17 (d, J=3.2 Hz, 1H), 7.23 (d, J=3.2 Hz, 2H), 7.31 (s, 1H), 7.60 (dd, J=2.0, 8.8 Hz, 2H), 8.22 (brs, 1H); 13C NMR (100 MHz, CDCl3) δ 40.0, 69.1, 72.5, 115.7, 125.4, □125.8, 127.8, 128.1, 129.0, 129.9, 134.5, 140.0, 160.2, 168.2.
    • N-(2-(3-Chlorobenzyloxy)ethyl)-3-hydroxybenzamide (53)
  • Figure US20150018543A1-20150115-C00066
  • 1H NMR (400 MHz, CDCl3) δ 3.65 (m, 4H), 4.49 (s, 2H), 6.64 (brs, 1H), 6.98 (d, J=8.0 Hz, 1H), 7.13 (d, J=8.0 Hz, 1H), 7.17-7.26 (m, 5H), 7.30 (s, 1H), 7.50 (s, 1H); 13C NMR (100 MHz, CDCl3) δ 40.0, 69.1, 72.5, 115.1, 117.8, 119.3, 125.9, 127.3, 128.1, 129.9, 130.0, 134.6, 135.4, 139.9, 157.2, 168.0.
  • Figure US20150018543A1-20150115-C00067
    • General Procedure for the Synthesis of phenoxy-pyrrolidin-1-yl-methanone (C2)
  • To a solution of (S)-3-pyrrolidinol (10 mmol) and triethylamine (11 mmol) in methylene chloride (50 mL) was added benzoyl chloride (8.67 mmol) at 0° C. The reaction temperature was brought up to room temperature. After 2 h, the reaction mixture was diluted with methylene chloride (50 mL) and then washed with 0.5 M HCl aqueous solution (100 mL) and brine (100 mL). The organic layer was dried over anhydrous MgSO4 and concentrated in vacuo. The crude product was purified by silica gel flash column chromatography (2:1 hexanes/ethyl acetate) to give C1.
  • To a solution of C1 (1.07 mmol) in methylene chloride (10 mL) was added ADDP (1.28 mmol), triphenylphosphine (1.28 mmol) and a phenol (1.28 mmol) at room temperature. After stirring overnight, the reaction mixture was diluted with methylene chloride (30 mL) and washed with 1 M HCl aqueous solution (50 mL), saturated Na2CO3 aqueous solution (50 mL) and brine (50 mL). The organic layer was dried over anhydrous MgSO4 and concentrated in vacuo. The crude was purified by silica gel flash column chromatography (2:1 hexanes/ethyl acetate) and recrystallized from a mixture of hexanes and ethyl acetate to give C2.
    • (R)-(3,5-Dinitrophenyl)(3-(4-methoxyphenoxy)pyrrolidin-1-yl)methanone (54)
  • Figure US20150018543A1-20150115-C00068
  • (Two rotamers, 1:1 ratio), m.p. 124-125° C.; 1H NMR (400 MHz, CDCl3) δ 2.11-2.19 (m, 1H), 2.30-2.34 (m, 1H), 3.54-3.64 (m, 1H), 3.72 & 3.76 (s, 3H), 3.81-3.99 (m, 3H), 4.86-4.94 (m, 1H), 6.74-6.84 (m, 4H), 8.68 & 8.75 (d, J=1.6 Hz, 2H), 9.05 & 9.08 (brs, 1H); 13C NMR (100 MHz, CDCl3) δ 30.6, 32.4, 45.2, 47.7, 52.8, 54.8, 55.8, 55.9, 75.7, 115.0, 117.1, 117.3, 120.1, 120.2, 127.7, 127.9, 139.9, 140.0, 148.6, 150.4, 150.8, 154.8, 154.8, 164.7, 165.1; LC-MS (ESI, m/z): 388 [M+H]+.
    • (R)-(3,5-Dinitrophenyl)(3-(4-fluorophenoxy)pyrrolidin-1-yl)methanone (55)
  • Figure US20150018543A1-20150115-C00069
  • (Two rotamers, 1:1 ratio, 75%), a pale yellow solid; 1H NMR (400 MHz, CDCl3) δ 2.15-2.37 (m, 2H), 3.56-3.63 (m, 1H), 3.79-3.97 (m, 3H), 4.91-4.99 (m, 1H), 6.76-7.03 (m, 4H), 8.71 & 8.76 (d, J=1.6 Hz, 2H), 9.08 & 9.10 (brs, 1H); 13C NMR (100 MHz, CDCl3) δ 29.9, 32.3, 45.1, 47.7, 52.7, 54.8, 75.5, 77.0, 116.2, 116.5, 116.9, 117.0, 117.1, 120.1, 120.2, 127.7, 127.8, 139.8, 139.9, 148.6, 152.6, 152.9, 157.9 (d, J=245 Hz, due to F), 164.7, 165.0.
    • (R)—N-(4-(1-(3,5-Dinitrobenzoyl)pyrrolidin-3-yloxy)phenyl)acetamide (56)
  • Figure US20150018543A1-20150115-C00070
  • (Two rotamers, 1:1 ratio, 63%), a yellow solid; 1H NMR (400 MHz, CDCl3+CD3OD) δ 1.96 & 1.99 (s, 3H), 2.03-2.27 (m, 2H), 3.45-3.50 (m, 1H), 3.69-3.83 (m, 3H), 4.83-4.91 (m, 1H), 6.64 & 6.74 (d, J=8.8 Hz, 2H), 7.26 & 7.33 (d, J=8.8 Hz, 2H), 8.58 & 8.65 (d, J=2.0 Hz, 2H), 8.95-8.99 (m, 1H); 13C NMR (100 MHz, CDCl3+CD3OD) δ 23.3, 23.4, 29.7, 32.0, 45.0, 47.6, 52.6, 54.6, 75.0, 76.4, 115.8, 115.9, 120.0, 121.9, 127.4, 127.5, 127.6, 127.7, 132.4, 132.5, 139.4, 148.4, 152.8, 153.1, 165.0, 165.3, 169.7.
    • (R)-(3,5-Dinitrophenyl)(3-(4-(trifluoromethoxy)phenoxy)pyrrolidin-1-yl)methanone (57)
  • Figure US20150018543A1-20150115-C00071
  • (Two rotamers, 6:4 ratio, 67%), a white solid; 1H NMR (400 MHz, CDCl3) δ 2.20-2.40 (m, 2H), 3.59-3.66 (m, 1H), 3.84-4.00 (m, 3H), 4.97-5.05 (m, 1H), 6.83 & 6.92 (d, J=8.8 Hz, 2H), 7.12 & 7.18 (d, J=8.8 Hz, 2H), 8.73 & 8.77 (d, J=2.0 Hz, 2H), 9.09 & 9.11 (d, J=2.0 Hz, 1H); 13C NMR (100 MHz, CDCl3) δ 29.8, 32.2, 45.1, 47.6, 52.6, 54.7, 75.2, 76.7, 116.4, 120.1, 122.8, 127.7, 127.8, 139.6, 139.7, 143.4, 148.5, 155.0, 155.2, 164.7, 164.9.
    • (R)-Methyl 4-(1-(3,5-dinitrobenzoyl)pyrrolidin-3-yloxy)benzoate (58)
  • Figure US20150018543A1-20150115-C00072
  • (Two rotamers 1:1 ratio), 1H NMR (400 MHz, CDCl3) δ 2.21-2.37 (m, 2H), 3.57-3.65 (m, 1H), 3.85 & 3.87 (s, 3H), 3.89-3.99 (m, 3H), 5.03-5.11 (m, 1H), 6.82 & 6.91 (d, J=7.2 Hz, 2H), 7.93 & 7.99 (d, J=7.2 Hz, 2H), 8.70 & 8.75 (s, 2H), 9.07 & 9.09 (s, 1H); 13C NMR (100 MHz, CDCl3) δ22.1, 30.0, □ 32.4, 45.2, 47.7, 52.2, 52.8, 54.8, 74.9, 76.3, 115.0, 120.36, 123.7, 123.8, 127.8, 127.9, 132.0, 139.7, 148.6, 160.2, 160.5, 164.7, 166.7.
    • (R)-(3,5-Dinitrophenyl)(3-(2-fluorophenoxy)pyrrolidin-1-yl)methanone (59)
  • Figure US20150018543A1-20150115-C00073
  • (Two rotamers 1:1 ratio), 1H NMR (400 MHz, CD3OD) δ 2.26-2.33 (m, 2H), 3.62-3.97 (m, 3H), 4.00 & 4.36 (s, 1H), 5.06 & 5.21 (s, 1H), 7.11 & 7.27 (m, 4H), 8.78 & 8.83 (d, J=2.0 Hz, 2H), 9.01 & 9.04 (d, J=2.0 Hz, 1H); 13C NMR (100 MHz, CD3OD) δ □29.9, 31.9, 44.9, 52.3, 54.4, 77.2, 78.7, 116.62, 116.67, 116.80, 116.85, 117.8 (d, J=20 Hz, due to F), 120.04 (d, J=3.7 Hz, due to F), 122.5, 122.6, 122.70, 122.77, 125.1, 125.15 (d, J=3.7 Hz, due to F), 127.80 (d, 0.1=7.4 Hz due to F), 127.9, 139.8, 153.6 (d, J=244 Hz, due to F), 165.4, 165.5.
    • (S)-Methyl-4-(1-(3,5-dinitrobenzoyl)pyrrolidin-3-yloxy)benzoate (60)
  • Figure US20150018543A1-20150115-C00074
  • (Two rotamers 1:1 ratio), 1H NMR (400 MHz, Acetone-d6) δ 2.21-2.29 (m, 2H), 3.58 & 3.61 (s, 1H), 3.69 & 3.71 (s, 3H), 3.73-4.02 (m, 3H), 4.99 & 5.06 (s, 1H), 6.77-6.94 (m, 4H), 8.73 & 8.77 (s, 2H), 8.96 & 8.99 (s, 1H); 13C NMR (100 MHz, CDCl3) δ □29.9, 31.9, 44.1, 44.7, 52.2, 54.2, 55.1, 55.2, 76.0, 77.5, 114.82, 114.88, 117.2, 119.6, 127.7, 127.8, 140.5, 148.7, 151.1, 151.3, 154.7, 164.6, 164.7.
    • (S)-(3,5-dinitrophenyl)(3-(4-methoxyphenoxy)pyrrolidin-1-yl)methanone (61)
  • Figure US20150018543A1-20150115-C00075
  • (Two rotamers 1:1 ratio), 1H NMR (400 MHz, Acetone-d6) δ 2.19-2.28 (m, 2H) 3.60-4.01 (m, 4H), 4.98 & 5.06 (s, 1H), 6.76-6.94 (m, 4H), 8.73 & 8.76 (s, 2H), 8.95 & 8.99 (s, 1H); 13C NMR (100 MHz, Acetone-d6) δ □31.9, 44.7, 52.2, 54.2, 55.0, 55.1, 65.8, 75.9, 77.5, 114.81, 114.87, 117.2, 119.6, 127.7, 127.8, 128.6, 129.8, 140.4, 148.7, 151.3, 154.7, 164.6, 164.7.
    • (S)—N-(4-(1-(3,5-Dinitrobenzoyl)pyrrolidin-3-yloxy)phenyl)acetamide (62)
  • Figure US20150018543A1-20150115-C00076
  • (Two rotamers 1:1 ratio), 1H NMR (400 MHz, Acetone-d6) δ 1.99 (s, 3H), 2.22-2.28 (m, 2H), 3.54-4.06 (m, 3H), 5.04 & 5.11 (s, 1H), 6.80 & 6.90 (d, J=8.8 Hz, 1H), 7.46-7.70 (m, 4H, brs, 1H), 8.73 & 8.76 (s, 2H), 8.95 & 8.99 (s, 1H); 13C NMR (100 MHz, Acetone-d6) δ□24.1, 24.2, 30.0, 32.2, 45.2, 47.7, 52.7, 54.7, 75.1, 76.6, 115.9, 120.0, 120.1, 127.7, 127.8, 128.7, 128.8, 131.6, 132.0, 132.4, 132.6, 132.7, 132.8, 139.7, 148.4, 153.1, 165.0, 169.1.
    • (S)-4-(1-(3,5-Dinitrobenzoyl)pyrrolidin-3-yloxy)benzoic acid (63)
  • Figure US20150018543A1-20150115-C00077
  • (Two rotamers 1:1 ratio), 1H NMR (400 MHz, Acetone-d6) δ 2.31-2.42 (m, 2H), 3.61-3.65 (m, 1H), 3.75-4.06 (m, 3H), 5.19 & 5.28 (s, 1H), 7.02 & 7.13 (d, J=8.8 Hz, 2H), 7.98 & 8.06 (d, J=8.8 Hz, 2H), 8.72 & 8.78 (d, J=2.0 Hz, 2H), 9.02 & 9.05 (s, 1H).
    • (S)-(3,5-Dinitrophenyl)(3-(2-fluorophenoxy)pyrrolidin-1-yl)methanone (64)
  • Figure US20150018543A1-20150115-C00078
  • (Two rotamers, 1:1 ratio), 1H NMR (400 MHz, DMSO-d6) δ 2.14-2.24 (m, 2H), 3.50-3.88 (m, 4H), 4.98 & 5.08 (s, 1H), 6.86-7.15 (m, 4H), 8.65 & 8.69 (s, 2H), 8.88 & 8.92 (s, 1H); 13C NMR (100 MHz, DMSO-d6) δ □029.1, 31.1, 44.1, 51.5, 53.6, 76.4, 77.9, 115.7, 115.8, 115.9, 116.0, 117.1 (d, J=22.3 Hz, due to F), 119.2 (d, J=3.7 Hz, due to F), 121.7, 121.83, 121.88, 121.9, 124.2 (d, J=3.7 Hz, due to F), 127.0, 139.0, 144.1, 144.4, 148.0, 152.8 (d, J=242.6 Hz, due to F), 164.6, 164.7.
    • (R)-(3-(2-Fluorophenoxy)pyrrolidin-1-yl)(phenyl)methanone (65)
  • Figure US20150018543A1-20150115-C00079
  • (Two rotamers 1:1 ratio), 1H NMR (400 MHz, CDCl3) δ 2.02-2.24 (m, 2H), 3.51-3.91 (m, 4H), 4.85 & 4.98 (s, 1H), 6.86-7.09 (m, 4H), 7.36-7.48 (m, 3H), 7.52 (d, J=5.2, 1H), 7.53 (d, J=5.2 Hz, 1H); 13C NMR (100 MHz, CDCl3) δ □30.3, 32.4, 44.3, 47.5, 52.1, 54.8, 78.0, 79.0, 116.8, 117.0, 117.9, 118.6, 122.6, 122.7, 122.9, 123.0, 124.6 (d, J=3.7 Hz due to F), 127.2, 127.4, 128.5, (d, J=3.7 Hz, due to F), 130.1, 130.3, 136.7, 136.9, 144.7 (d, J=20.1 Hz due to F), 153.8 (d, J=245.6 Hz, due to F), 155.2, 170.0, 170.2.
    • (R)-(3-(4-Methoxyphenoxy)pyrrolidin-1-yl)(phenyl)methanone (66)
  • Figure US20150018543A1-20150115-C00080
  • (Two rotamers, 1:1 ratio), 1H NMR (400 MHz, CDCl3) δ 1.99-2.21 (m, 2H), 3.48-3.66 (m, 2H), 3.68 & 3.73 (s, 3H), 3.79-3.89 (m, 2H), 4.74 & 4.96 (s, 1H), 6.71 (s, 2H), 6.76 (s, 2H), 7.34 & 7.36 (d, J=5.6 Hz, 3H), 7.46 & 7.52 (d, J=5.2 Hz, 2H); 13C NMR (100 MHz, CDCl3) δ 30.2, 32.3, 44.4, 47.6, 52.1, 54.8, 55.8, 55.9, 76.0, 114.9, 115.0, □ 117.1, 117.3, 127.3, 127.4, 128.50, 128.54, 130.1, 130.2, 136.8, 137.0, 150.9, 151.1, 154.5, 154.6, 169.9, 170.2; LC-MS (ESI, m/z): 298.1 [M+H]+.
    • (R)-(3,5-Dinitrophenyl)(3-hydroxypyrrolidin-1-yl)methanone (67)
  • Figure US20150018543A1-20150115-C00081
  • (Two rotamers, 1:1 ratio), 1H NMR (400 MHz, CDCl3) δ 1.98-2.11 (m, 2H), 3.23 (brs, 1H), 3.37-3.48 (m, 1H), 3.61-3.79 (m, 3H), 4.47 & 4.56 (s, 1H), 8.62 & 8.67 (s, 2H), 8.99-9.00 (m, 1H); 13C NMR (100 MHz, CDCl3) δ □33.0, 34.9, 45.1, 47.6, 55.5, 57.5, 69.4, 70.9, 120.1, 120.2, 127.8, 139.8, 139.9, 148.5, 165.1, 165.3.
    • (R)-(3-(3-Methoxyphenoxy)pyrrolidin-1-yl)(3-methoxyphenyl)methanone (68)
  • Figure US20150018543A1-20150115-C00082
  • (Two rotamers, 1:1 ratio, 85%), a pale yellow liquid; 1H NMR (400 MHz, CDCl3) δ 1.97-2.22 (m, 2H), 3.48-3.65 (m, 2H), 3.68 & 3.71 (s, 3H), 3.73 & 3.76 (s, 3H), 3.79-3.89 (m, 2H), 4.74-4.84 (m, 1H), 6.70-6.80 (m, 4H), 6.86-6.92 (m, 1H), 6.99 & 7.01 (s, 1H), 7.04 & 7.08 (s, 1H), 7.21-7.28 (m, 1H); 13C NMR (100 MHz, CDCl3) δ 30.2, 32.3, 44.5, 47.7, 52.2, 54.8, 55.6, 55.8, 76.0, 112.6, 112.8, 114.9, 115.0, 116.1, 116.6, 117.1, 117.2, 119.4, 119.6, 179.27, 129.32, 138.1, 150.9, 151.1, 154.5, 159.7, 169.8.
    • (R)-(3-(4-Methoxyphenoxy)pyrrolidin-1-yl)(3-methoxyphenyl)methanone (69)
  • Figure US20150018543A1-20150115-C00083
  • (Two rotamers, 1:1 ratio, 83%), a pale yellow liquid; 1H NMR (400 MHz, CDCl3) δ 1.97-2.22 (m, 2H), 3.48-3.65 (m, 2H), 3.68 & 3.71 (s, 3H), 3.73 & 3.76 (s, 3H), 3.79-3.89 (m, 2H), 4.72-4.84 (m, 1H), 6.70-6.80 (m, 4H), 6.86-6.92 (m, 1H), 6.99-7.08 (m, 2H), 7.21-7.28 (m, 1H); 13C NMR (100 MHz, CDCl3) δ 29.2, 32.1, 44.4, 47.6, 52.1, 54.4, 55.33, 55.62, 75.8, 113.4, 114.7, 116.9, 128.63, 128.75, 129.16, 129.32, 131.9, 150.9, 154.3, 160.9, 169.48, 169.79.
    • (R)-Methyl 3-(3-(4-methoxyphenoxy)pyrrolidine-1-carbonyl)benzoate (70)
  • Figure US20150018543A1-20150115-C00084
  • (Two rotamers, 1:1 ratio, 87%), a pale yellow liquid; 1H NMR (400 MHz, CDCl3) δ 1.99-2.24 (m, 2H), 3.45-3.65 (m, 2H), 3.67 & 3.71 (s, 3H), 3.75-3.82 (m, 2H), 3.86 & 3.87 (s, 3H), 4.74-4.86 (m, 1H), 6.72 & 6.80 (m, 4H), 7.40-7.67 (m, 1H), 7.66 & 7.71 (d, J=7.6 Hz, 1H), 8.04 (t, J=9.0 Hz, 1H), 8.13 & 8.19 (s, 1H); 13C NMR (100 MHz, CDCl3) δ 29.9, 32.0, 44.3, 47.3, 52.2, 54.5, 55.5, 55.6, 75.7, 114.7, 114.8, 116.9, 117.0, 128.1, 128.2, 128.5, 128.6, 130.9, 134.0, 131.5, 131.6, 136.8, 136.9, 150.5, 150.7, 154.33, 154.38, 166.6, 168.6, 168.9.
    • (R)-Methyl 4-(3-(4-methoxyphenoxy)pyrrolidine-1-carbonyl)benzoate (71)
  • Figure US20150018543A1-20150115-C00085
  • (Two rotamers, 1:1 ratio, 85%), a pale yellow liquid; 1H NMR (400 MHz, CDCl3) δ 1.98-2.11 (m, 1H), 2.15-2.25 (m, 1H), 3.42-3.67 (m, 2H), 3.68 & 3.71 (s, 3H), 3.77-3.81 (m, 1H), 3.83-3.88 (m, 1H), 3.86 & 3.88 (s, 3H), 4.73-4.86 (m, 1H), 6.69-6.75 (m, 2H), 6.80 (s, 2H), 7.51 (d, J=8.0 Hz, 1H), 7.57 (d, J=8.4 Hz, 1H), 8.00 (d, J=8.4 Hz, 1H), 8.03 (d, J=8.4 Hz, 1H); 13C NMR (100 MHz, CDCl3) δ 29.9, 32.0, 44.2, 47.2, 51.9, 52.2, 54.3, 55.5, 55.6, 75.6, 114.7, 114.8, 116.8, 117.0, 127.0, 127.1, 129.5, 129.6, 131.2, 131.3, 140.7, 140.8, 150.5, 150.7, 154.3, 154.4, 168.7, 168.9.
    • (R)-(3-(4-Methoxyphenoxy)pyrrolidin-1-yl)-3-(trifluoromethyl)phenyl)methanone (72)
  • Figure US20150018543A1-20150115-C00086
  • (Two rotamers, 1:1 ratio, 82%), a pale yellow liquid; 1H NMR (400 MHz, CDCl3) δ 2.04-2.15 (m, 1H), 2.21-2.30 (m, 1H), 3.48-3.67 (m, 2H), 3.72 & 3.75 (s, 3H), 3.78-3.90 (m, 2H), 4.79-4.90 (m, 1H), 6.74-6.83 (m, 4H), 7.48-7.55 (m, 1H), 7.64-7.82 (m, 3H); 13C NMR (100 MHz, CDCl3) δ 30.0, 32.1, 44.5, 47.5, 52.2, 54.6, 55.7, 55.8, 75.8, 114.8, 114.9, 117.0, 117.2, 124.2, 124.3, 129.0, 129.1, 130.4, 130.6, 137.3, 137.4, 150.6, 150.8, 154.5, 154.6, 168.3, 168.6.
    • (R)-(3-(4-Methoxyphenoxy)pyrrolidin-1-yl)(4-(trifluoromethyl)phenyl)methanone (73)
  • Figure US20150018543A1-20150115-C00087
  • (Two rotamers, 1:1 ratio, 55%), a pale yellow solid; 1H NMR (400 MHz, CDCl3) δ 2.03-2.06 (m, 1H), 2.20-2.25 (m, 1H), 3.49-3.70 (m, 2H), 3.72 & 3.75 (s, 3H), 3.81-3.88 (m, 2H), 4.72 & 8.89 (m, 1H), 6.74-6.83 (m, 4H), 7.23-7.50 (m, 4H); 13C NMR (100 MHz, CDCl3) δ 30.0, 32.2, 44.5, 47.5, 52.2, 54.6, 55.7, 55.8, 75.8, 76.8, 114.9, 117.0, 117.2, 119.8, 120.1, 122.5, 122.6, 125.6, 125.8, 130.0, 130.1, 138.5, 149.1, 150.6, 150.9, 154.5, 168.2.
    • (R)-(3-(4-Methoxyphenoxy)pyrrolidin-1-yl)-3-(trifluoromethoxy)phenyl)methanone (74)
  • Figure US20150018543A1-20150115-C00088
  • (Two rotamers, 1:1 ratio, 67%), a yellow liquid; 1H NMR (400 MHz, CDCl3) δ 2.01-2.23 (m, 2H), 3.43-3.68 (m, 2H), 3.69 & 3.72 (s, 3H), 3.72-3.83 (m, 2H), 4.75-4.88 (m, 1H), 6.72-6.82 (m, 4H), 7.58-7.66 (m, 4H); 13C NMR (100 MHz, CDCl3) δ 29.1, 30.1, 32.2, 38.9, 44.6, 47.6, 52.2, 54.7, 55.8, 75.9, 114.9, 115.0, 117.1, 117.3, 125.5, 125.6, 127.7, 128.8, 150.8, 151.0, 154.6, 154.7, 168.5, 168.6.
    • (R)-(3-(4-Methoxyphenoxy)pyrrolidin-1-yl)(3-nitrophenyl)methanone (75)
  • Figure US20150018543A1-20150115-C00089
  • (Two rotamers, 1:1 ratio, 84%), a yellow liquid; 1H NMR (400 MHz, CDCl3) δ 2.00-2.24 (m, 2H), 3.48-3.56 (m, 1H), 3.68 & 3.72 (s, 3H), 3.73-3.88 (m, 3H), 4.79-4.89 (m, 1H), 6.71-6.83 (m, 4H), 7.52-7.59 (m, 1H), 7.81 & 7.87 (d, J=7.6H, 1H), 8.22 (t, J=9.8 Hz, 1H), 8.32 & 8.38 (s, 1H); 13C NMR (100 MHz, CDCl3) δ 29.8, 32.0, 44.5, 47.4, 52.2, 54.5, 55.5, 55.6, 75.6, 77.0, 114.7, 114.8, 116.9, 117.0, 122.2, 122.3, 124.6, 124.7, 129.6, 133.1, 133.2, 138.0, 138.1, 147.8, 150.4, 150.6, 154.3, 154.4, 166.9, 167.2.
    • (R)-(3-(4-Methoxyphenoxy)pyrrolidin-1-yl)(4-nitrophenyl)methanone (76)
  • Figure US20150018543A1-20150115-C00090
  • (Two rotamers, 1:1 ratio, 73%), a yellow solid; 1H NMR (400 MHz, CDCl3) δ 2.01-2.31 (m, 2H), 3.44-3.69 (m, 2H), 3.72 & 3.75 (s, 3H), 3.80-3.90 (m, 2H), 4.79-4.90 (m, 1H), 6.72-6.82 (m, 4H), 7.63 & 7.70 (d, J=8.0 Hz, 2H), 8.22 & 8.24 (d, J=8.2 Hz, 2H); 13C NMR (100 MHz, CDCl3) δ 30.2, 31.9, 44.3, 47.2, 52.0, 55.5, 75.5, 76.7, 114.7, 114.8, 116.8, 116.9, 123.5, 128.0, 128.2, 128.6, 142.4, 142.5, 148.4, 150.3, 150.6, 154.3, 154.4, 167.3, 167.6.
    • (R)-(3-Fluorophenyl)(3-(4-methoxyphenoxy)pyrrolidin-1-yl)methanone (77)
  • Figure US20150018543A1-20150115-C00091
  • (Two rotamers, 1:1 ratio, 78%), a pale yellow liquid; 1H NMR (400 MHz, CDCl3) δ 2.01-2.11 (m, 1H), 2.12-2.42 (m, 1H), 3.48-3.69 (m, 2H), 3.71 & 3.74 (s, 3H), 3.78-3.87 (m, 2H), 4.76-4.88 (m, 1H), 6.72-6.82 (m, 4H), 7.05-7.36 (m, 4H); 13C NMR (100 MHz, CDCl3) δ29.9, 32.0, 44.3, 47.4, 52.0, 54.5, 55.6, 75.7, 114.4, 114.8, 116.9, 117.1, 122.8, 122.9, 130.1, 130.2, 138.6, 138.7, 150.6, 150.8, 154.4, 154.5, 162.4 (d, J=245 Hz, due to F), 168.3, 168.5.
    • (R)-(3-Chlorophenyl)(3-(4-methoxyphenoxy)pyrrolidin-1-yl)methanone (78)
  • Figure US20150018543A1-20150115-C00092
  • (Two rotamers, 1:1 ratio, 87%), a pale yellow liquid; 1H NMR (400 MHz, CDCl3) δ 2.01-2.24 (m, 2H), 3.47-3.69 (m, 2H), 3.71 & 3.74 (s, 3H), 3.78-3.86 (m, 2H), 4.75-4.88 (m, 1H), 6.73-6.82 (m, 4H), 7.26-7.42 (m, 3H), 7.46 & 7.52 (s, 1H); 13C NMR (100 MHz, CDCl3) δ 29.9, 32.0, 44.3, 47.4, 52.0, 54.5, 55.6, 55.7, 75.7, 76.7, 114.7, 114.8, 116.6, 117.1, 125.1, 125.3, 127.3, 127.4, 129.7, 129.8, 130.0, 130.1, 134.3, 138.2, 138.3, 150.5, 150.7, 154.4, 168.1, 168.4
    • (R)-(3-Hydroxyphenyl)(3-(4-methoxyphenoxy)pyrrolidin-1-yl)methanone (79)
  • Figure US20150018543A1-20150115-C00093
  • (Two rotamers, 1:1 ratio, 53%), a white liquid; 1H NMR (400 MHz, CDCl3) δ 1.96-2.25 (m, 2H), 3.53-3.74 (m, 2H), 3.77 & 3.81 (s, 3H), 3.83-3.94 (m, 2H), 4.73 & 4.87 (m, 1H), 6.72-6.82 (m, 4H), 6.85-6.98 (m, 2H), 7.08-7.20 (m, 2H), 8.21 (brs, 1H);
    • (R)-(4-Hydroxyphenyl)(3-(4-methoxyphenoxy)pyrrolidin-1-yl)methanone (80)
  • Figure US20150018543A1-20150115-C00094
  • (Two rotamers, 1:1 ratio, 37%), a white solid; 1H NMR (400 MHz, CDCl3) δ 2.03-2.32 (m, 2H), 3.59-3.71 (m, 2H), 3.74 & 3.76 (s, 3H), 3.79-3.93 (m, 2H), 4.80-4.91 (m, 1H), 6.75-6.84 (m, 4H), 7.21-7.24 (m, 2H), 7.56 & 7.62 (d, J=8.0 Hz, 2H), 8.01 & 8.03 (brs, 1H).
    • (R)-(4-Hydroxy-3-nitrophenyl)(3-(4-methoxyphenoxy)pyrrolidin-1-yl)methanone (81)
  • Figure US20150018543A1-20150115-C00095
  • (Two rotamers, 1:1 ratio, 63%), a yellow liquid; 1H NMR (400 MHz, CDCl3) δ 2.01-2.14 (m, 1H), 2.25-2.27 (m, 1H), 3.56-3.65 (m, 2H), 3.72 & 3.74 (s, 3H), 3.81-3.91 (m, 2H), 4.81-4.89 (m, 1H), 6.76 (m, 4H), 7.16 (t, J=9.4 Hz, 1H), 7.78 & 7.84 (d, J=8.4 Hz, 1H), 8.29 & 8.37 (s, 1H); 13C NMR (100 MHz, CDCl3) δ 29.9, 31.8, 45.0, 47.6, 52.6, 54.9, 55.9, 115.1, 117.2, 117.3, 120.4, 124.7, 125.0, 128.8, 133.1, 136.9, 137.0, 151.0, 154.7, 156.4, 166.9, 167.3.
    • (R)-(3,5-Dichlorophenyl)(3-(4-methoxyphenoxy)pyrrolidin-1-yl)methanone (82)
  • Figure US20150018543A1-20150115-C00096
  • (Two rotamers, 1:1 ratio, 85%), a pale yellow liquid; 1H NMR (400 MHz, CDCl3) δ 2.02-2.10 (m, 1H), 2.20-2.25 (m, 1H), 3.47-3.70 (m, 2H), 3.72 & 3.74 (s, 3H), 3.75-3.85 (m, 2H), 4.78-4.87 (m, 1H), 6.74-6.82 (m, 4H), 7.34-7.41 (m, 3H); 13C NMR (100 MHz, CDCl3) δ 29.9, 32.0, 44.4, 47.4, 52.1, 54.4, 55.6, 55.7, 75.5, 114.8, 116.9, 125.6, 125.7, 130.0, 135.1, 139.2, 139.3, 150.4, 150.7, 154.4, 154.5, 166.7, 167.0.
    • (R)-(3,5-Difluorophenyl)(3-(4-methoxyphenoxy)pyrrolidin-1-yl)methanone (83)
  • Figure US20150018543A1-20150115-C00097
  • (Two rotamers, 1:1 ratio, 75%), a yellow liquid; 1H NMR (400 MHz, CDCl3) δ 2.01-2.27 (m, 2H), 3.48-3.67 (m, 2H), 3.71 & 3.74 (s, 3H), 3.77-3.85 (m, 2H), 4.78-4.88 (m, 1H), 6.73-6.87 (m, 5H), 6.99 & 7.06 (d, J=5.6 Hz, 2H); 13C NMR (100 MHz, CDCl3) δ 29.8, 32.0, 44.4, 47.3, 52.1, 54.9, 55.6, 75.6, 105.3, 105.4, 110.3, 110.4, 110.5, 110.7, 114.8, 116.9, 117.1, 150.2, 154.9, 162.4 (d, J=250 Hz, due to F), 162.5 (d, J=250 Hz, due to F), 167.0,
    • (R)-(3,5-Bis(trifluoromethyl)phenyl)(3-(4-methoxyphenoxy)pyrrolidin-1-yl)methanone (84)
  • Figure US20150018543A1-20150115-C00098
  • (Two rotamers, 1:1 ratio, 65%), a yellow liquid; 1H NMR (400 MHz, CDCl3) δ 2.08-2.14 (m, 1H), 2.24-2.29 (m, 1H), 3.47-3.67 (m, 2H), 3.71 & 3.74 (s, 3H), 3.76-3.91 (m, 2H), 4.81-4.91 (m, 1H), 6.74-6.83 (m, 4H), 7.90-8.12 (m, 3H); 13C NMR (100 MHz, CDCl3) δ 29.8, 32.1, 44.6, 47.4, 52.3, 54.5, 55.6, 75.6, 114.8, 114.9, 116, 9, 117.2, 123.7, 124.3, 127.5, 127.7, 131.1, 132.1, 138.5, 138.6, 150.4, 150.7, 154.5, 154.7, 166.5, 166.8.
    • (R)-(3-(4-Methoxyphenoxy)pyrrolidin-1-yl)(pyridin-3-yl)methanone (85)
  • Figure US20150018543A1-20150115-C00099
  • (Two rotamers, 1:1 ratio, 82%), a yellow solid; 1H NMR (400 MHz, CDCl3) δ 2.00-2.10 (m, 1H), 2.16-2.24 (m, 1H), 3.48-3.58 (m, 1H), 3.64-3.73 (m, 1H), 3.67 & 3.69 (s, 3H), 3.73-3.85 (m, 2H), 4.75-4.85 (m, 1H), 6.69-6.78 (m, 4H), 7.25-7.31 (m, 1H), 7.78 & 7.83 (d, J=7.6 Hz, 1H), 8.57-8.61 (m, 1H), 8.71 & 8.77 (s, 1H); 13C NMR (100 MHz, CDCl3) δ 29.7, 31.9, 44.3, 47.2, 51.9, 54.4, 55.49, 55.53, 75.5, 114.66, 114.69, 116.8, 116.9, 123.1, 123.2, 132.3, 134.8, 134.9, 147.9, 148.1, 150.39, 150.63, 150.83, 150.89, 154.2, 154.3, 167.0, 167.3.
    • (R)-(3-(4-Methoxyphenoxy)pyrrolidin-1-yl)(pyridin-4-yl)methanone (86)
  • Figure US20150018543A1-20150115-C00100
  • (Two rotamers, 1:1 ratio, 79%), a yellow solid; 1H NMR (400 MHz, CDCl3) δ 2.04-2.23 (m, 2H), 3.46-3.67 (m, 2H), 3.70 & 3.72 (s, 3H), 3.73-3.90 (m, 2H), 4.78-4.88 (m, 1H), 6.76-6.82 (m, 4H), 7.34 (s, 1H), 7.40 (s, 1H), 8.66 (d, J=13.2 Hz, 2H); 13C NMR (100 MHz, CDCl3) δ 29.6, 31.7, 44.0, 46.8, 51.7, 53.9, 55.3, 55.4, 75.3, 114.5, 114.6, 116.7, 116.8, 120.9, 121.0, 143.6, 143.7, 149.7, 150.2, 154.1, 154.2, 166.9, 167.1.
    • (R)-4-(3-(4-Methoxyphenoxy)pyrrolidine-1-carbonyl)pyridine 1-oxide (87)
  • Figure US20150018543A1-20150115-C00101
  • (Two rotamers, 1:1 ratio, 97%), a yellow solid; 1H NMR (400 MHz, CDCl3) δ 2.03-2.11 (m, 1H), 2.21-2.26 (m, 1H), 3.50-3.68 (m, 2H), 3.70 & 3.72 (s, 3H), 3.74-3.88 (m, 2H), 4.79-4.87 (m, 1H), 6.70-6.81 (m, 4H), 7.25-7.41 (m, 2H), 8.17-8.20 (m, 1H), 8.29 & 8.35 (brs, 1H); 13C NMR (100 MHz, CDCl3) δ 29.8, 32.0, 44, 7, 47.3, 52.3, 54.4, 55.7, 75.4, 114.8, 116.9, 117.0, 124.5, 126.0, 126.1, 135.7, 135.8, 137.9, 138.1, 140.1, 150.3, 154.5, 154.5, 164.1, 164.3.
    • (R)-4-(3-(4-Methoxyphenoxy)pyrrolidine-1-carbonyl)pyridine-1-oxide (88)
  • Figure US20150018543A1-20150115-C00102
  • (Two rotamers, 1:1 ratio, 95%), a yellow solid; 1H NMR (400 MHz, CDCl3) δ 2.03-2.10 (m, 1H), 2.22-2.27 (m, 1H), 3.52-3.68 (m, 2H), 3.70 & 3.72 (s, 3H), 3.76-3.83 (m, 2H), 4.80-4.87 (m, 1H), 6.70-6.79 (m, 4H), 7.40 (d, J=6.4 Hz, 1H), 7.47 (d, J=6.8 Hz, 1H), 8.13-8.18 (m, 2H); 13C NMR (100 MHz, CDCl3) δ 29.7, 32.1, 44.7, 47.3, 52.4, 54.4, 55.6, 75.4, 114.8, 116.9, 125.0, 125.1, 132.9, 133.0, 139.1, 150.3, 150.6, 154.4, 154.6, 165.3.
    • (R)-(3-(4-Methoxyphenoxy)pyrrolidin-1-yl)(pyrimidin-5-yl)methanone (89)
  • Figure US20150018543A1-20150115-C00103
  • (Two rotamers, 1:1 ratio, 84%), a pale yellow solid; 1H NMR (400 MHz, CDCl3) δ 2.03-2.13 (m, 1H), 2.23-2.28 (m, 1H), 3.52-3.67 (m, 2H), 3.69 & 3.72 (s, 3H), 3.78-3.88 (m, 2H), 4.79-4.89 (m, 1H), 6.70-6.80 (m, 4H), 8.56 & 8.91 (s, 2H), 9.20 & 9.22 (s, 1H); 13C NMR (100 MHz, CDCl3) δ 29.8, 32.1, 44.7, 52.3, 54.4, 55.6, 55.7, 75.5, 114.8, 116.9, 117.0 130.2, 130.3, 150.3, 150.6, 154.5, 154.6, 155.5, 155.6, 159.4, 159.5, 164.5.
    • (3,5-Dinitrophenyl)(4-hydroxypiperidin-1-yl)methanone (90)
  • Figure US20150018543A1-20150115-C00104
  • 1H NMR (400 MHz, Acetone-d6) δ 1.50-1.56 (m, 2H), 1.80-1.90 (m, 2H), 3.30-3.42 (m, 2H), 3.63 (brs, 1H), 3.94-4.05 (m, 3H), 8.61 (d, J=2.0 Hz, 2H), 8.95 (d, J=2.0 Hz, 1H); 13C NMR (100 MHz, Acetone-d6) δ 33.7, 34.5, 39.5, 44.9, 66.0, 119.1, 127.4, 140.2, 148.8, 165.1.
    • Methyl 4-(1-(3,5-dinitrobenzoyl)piperidin-4-yloxy)benzoate (91)
  • Figure US20150018543A1-20150115-C00105
  • 1H NMR (400 MHz, Acetone-d6) δ 1.84 (brs, 2H), 1.96 (brs, 2H), 3.31 (brs, 1H), 3.59-3.74 (m, 2H), 3.77 (s, 3H), 3.84-3.96 (m, 1H), 4.63-4.66 (m, 1H), 6.81-6.85 (m, 2H), 7.87-7.90 (m, 2H), 8.50 (d, J=2.0 Hz, 2H), 8.97 (d, J=2.0 Hz, 1H).
    • (3,5-Dinitrophenyl)(4-(4-methoxyphenoxy)piperidin-1-yl)methanone (92)
  • Figure US20150018543A1-20150115-C00106
  • 1H NMR (400 MHz, CDCl3) δ 1.85-1.98 (m, 4H), 3.35 (brs, 1H), 3.68-3.80 (m, 2H), 3.73 (s, 3H), 3.93 (brs, 1H), 4.49 (brs, 1H), 6.79 (d, J=8.4 Hz, 2H), 6.84 (d, J=8.4 Hz, 2H), 8.57 (s, 2H), 9.03 (s, 1H); 13C NMR (100 MHz, CDCl3) δ 30.0, 31.2, 39.3, 44.6, 55.8, 71.9, 115.0, 117.9, 119.8, 127.5, 139.6, □ 148.7, 150.8, 154.6, 165.4.
    • N-(4-(1-(3,5-Dinitrobenzoyl)piperidin-4-yloxy)phenyl)acetamide (93)
  • Figure US20150018543A1-20150115-C00107
  • 1H NMR (400 MHz, DMSO-d6) δ 1.62-1.96 (m, 4H), 1.97 (s, 3H), 3.48 (m, 3H), 3.93 (brs, 1H), 4.56 (s, 1H), 6.89 (d, J=8.4 Hz, 2H), 7.44 (d, J=8.4 Hz, 2H), 8.64 (s, 2H), 8.33 (s, 1H), 9.74 (s, 1H);
    • (3,5-Dinitrophenyl)(4-(2-fluorophenoxy)piperidin-1-yl)methanone (94)
  • Figure US20150018543A1-20150115-C00108
  • 1H NMR (400 MHz, DMSO-d6) δ 1.70-2.10 (m, 4H), 3.39-4.11 (m, 4H), 4.59 (m, 1H), 6.86-6.92 (m, 1H), 7.01-7.15 (m, 3H), 8.60 (d, J=2.0 Hz, 2H), 8.89 (d, J=2.0 Hz, 1H); 13C NMR (100 MHz, DMSO-d6) δ31.1, 31.9, 45.5, 49.6, 75.0, 117.6 (d, □ J=18.6 Hz, due to F), 119.5, 120.5, 123.3 (d, J=6.7 Hz, due to F), 126.0 (d, J=3.7 Hz, due to F), 128.6, 140.6, 146.1, 149.8, 154.8 (d, J=242.6 Hz, due to F), 166.9.
    • (3,5-Dinitrophenyl)(4-(2-methoxyphenyl)piperazin-1-yl)methanone (95)
  • Figure US20150018543A1-20150115-C00109
  • 1H NMR (400 MHz, Acetone-d6) δ 3.02-3.12 (m, 4H), 3.62 (brs, 2H), 3.82 (s, 3H), 3.87 (brs, 2H), 6.85-6.95 (m, 4H), 8.68 (d, J=2.0 Hz, 2H), 8.96 (d, J=2.4 Hz, 1H); LC-MS (ESI, m/z): 387 [M+H]+.
    • (3,5-Dinitrophenyl)(4-(4-methoxyphenyl)piperazin-1-yl)methanone (96)
  • Figure US20150018543A1-20150115-C00110
  • 1H NMR (400 MHz, Acetone-d6) δ 3.08-3.17 (m, 4H), 3.68 (brs, 2H), 3.71 (s, 3H), 3.88 (brs, 2H), 6.82 (d, J=8.8 Hz, 2H), 6.93, (d, J=8.8 Hz, 2H), 8.69 (d, J=2.0 Hz, 2H), 8.98 (d, J=2.0 Hz, 1H); 13C NMR (100 MHz, Acetone-d6) δ 42.4, 47.7, 50.5, 50.9, 54.9, 114.4, 118.8, 119.3, 127.7, 139.9, □ 145.6, 148.8, 154.5, 165.2; LC-MS (ESI, m/z): 387 [M+H]+.
    • (4-(2-chlorophenyl)piperazin-1-yl)(3,5-dinitrophenyl)methanone (97)
  • Figure US20150018543A1-20150115-C00111
  • 1H NMR (400 MHz, Acetone-d6) δ 3.09-3.17 (m, 4H), 3.70 (brs, 2H), 3.94 (brs, 2H), 7.07 (t, J=7.6 Hz, 1H), 7.18 (d, J=8 Hz, 1H), 7.30 (t, J=8 Hz, 1H), 7.41 (d, J=8 Hz, 1H), 8.72 (s, 1H), 9.00 (s, 1H); 13C NMR (100 MHz, Acetone-d6) δ 43.3, 48.7, 51.6, 52.1, 120.0, 122.0, □ 125.3, 128.5, 128.9, 129.4, 131.4, 140.6, 149.6, 149.8, 166.1; LC-MS (ESI, m/z): 391 [M+H]+.
  • Figure US20150018543A1-20150115-C00112
    • General Procedure for the Synthesis of t-butyl-benzyloxypyrrolidine-1-carboxylate (D1)
  • To a solution of (R)-tert-butyl 3-hydroxypyrrolidine-1-carboxylate (3.2 mmol) in dimethyl formamide (10 mL) was added sodium hydride (3.2 mmol) and benzyl bromide (3.2 mmol) at 0° C. and the resulting mixture was stirred at room temperature. After stirring overnight, distilled water (50 mL) was added and the resulting precipitate was collected by filtration to afford D1.
    • General Procedure for the Synthesis of benzyloxy-pyrrolidinyl-phenylmethanone (D2)
  • D1 (0.43 mmol) was dissolved in trifluoro acetic acid (5 mL) and stirred at room temperature. After 1 h, the reaction mixture was concentrated in vacuo to afford an amine. To a solution of the amine in methylene chloride (5 mL) was added triethylamine (0.51 mmol) and a benzoylchloride (0.51 mmol) at 0° C. and the resulting mixture was stirred at room temperature. After 3 h, the reaction mixture was diluted with methylene chloride (30 mL) and washed with 1 M HCl aqueous solution (30 mL), saturated Na2CO3 aqueous solution (30 mL) and brine (30 mL). The organic layer was dried over anhydrous MgSO4 and concentrated in vacuo. The crude product was purified by silica gel flash column chromatography (3:1 hexanes/ethyl acetate) and recrystallized from a mixture of hexanes and ethyl acetate to give D2.
    • (R)-(3-(Benzyloxy)pyrrolidin-1-yl)(3,5-dinitrophenyl)methanone (98)
  • Figure US20150018543A1-20150115-C00113
  • (Two rotamers, 1:1 ratio, 23%), a white solid; 1H NMR (400 MHz, CDCl3) δ 2.18-2.29 (m, 2H), 3.53-3.58 (m, 1H), 3.76-3.93 (m, 3H), 5.12-5.37 (m, 3H), 7.34-7.44 (m, 5H), 8.67 & 8.73 (d, J=1.6 Hz, 2H), 9.08 & 9.09 (d, J=1.6 Hz, 1H).
    • ((R)-3-(3-Chlorobenzyloxy)pyrrolidin-1-yl)(3,5-dinitrophenyl)methanone (99)
  • Figure US20150018543A1-20150115-C00114
  • (Two rotamers 3:1 ratio, 75%); 1H NMR (400 MHz, CDCl3) δ 1.93-2.21 (m, 2H), 3.38-3.83 (m, 4H), 4.13-4.47 (m, 1H), 4.99 & 5.07 (s, 1H), 5.17 & 5.29 (s, 1H), 7.07-7.29 (m, 4H), 8.64 & 8.69 (s, 2H), 8.98 (s, 1H); 13C NMR (100 MHz, CDCl3) δ 29.8, 32.2, 45.1, 47.6, 52.3, 54.8, 70.3, 70.4, 76.4, 78.0, 120.0, 120.1, 125.5, 125.6, 127.5, 127.7, 127.8, 127.9, 128.1, 129.9, 134.5, 134.6, 139.7, 139.8, 139.9, 148.5, 164.7, 164.8.
    • ((R)-3-(2-Fluorobenzyloxy)pyrrolidin-1-yl)(3,5-dinitrophenyl)methanone (100)
  • Figure US20150018543A1-20150115-C00115
  • (Two rotamers 1:1 ratio), 1H NMR (400 MHz, CDCl3) δ 2.02-2.30 (m, 2H), 3.50 & 3.52 (s, 1H), 3.63-3.94 (m, 3H), 4.24 & 4.33 (s, 1H), 4.48 & 4.56 (d, J=12.0 Hz, 1H), 4.65 (s, 1H), 6.99-7.44 (m, 4H), 8.69 & 8.75 (s, 2H), 9.10 (s, 1H).
    • ((R)-3-(3-(Trifluoromethyl)benzyloxy)pyrrolidin-1-yl)(3,5-dinitrophenyl)methanone (101)
  • Figure US20150018543A1-20150115-C00116
  • (Two rotamers 2:1 ratio), 1H NMR (400 MHz, CDCl3) δ 2.06-2.29 (m, 2H), 3.53 & 3.55 (s, 1H), 3.78-3.96 (m, 3H), 4.27 & 4.35 (s, 1H), 4.51 & 4.62 (d, J=12.4 Hz, 1H), 4.65 (s, 1H), 7.47-7.62 (m, 4H), 8.69 & 8.74 (s, 2H), 9.07 (s, 1H); 13C NMR (100 MHz, CDCl3) δ 29.7, 32.1, 45.1, 47.7, 52.4, 54.9, 70.4, 70.5, 76.7, 78.2, 120.1, 124.1, 124.3, 124.83, 124.87, 127.7, 127.8, 129.2, 130.8, 130.9, 138.7, 138.8, 139.7, 139.8, 148.5, 165.0.
    • (R)-(3,5-Dinitrophenyl)(3-(pyridin-4-ylmethoxy)pyrrolidin-1-yl)methanone (102)
  • Figure US20150018543A1-20150115-C00117
  • (Two rotamers, 1:1 ratio, 75%), a brown oil; 1H NMR (400 MHz, CDCl3) δ 1.99-2.24 (m, 2H), 3.49-3.92 (m, 4H), 4.20-4.28 (m, 1H), 4.41-4.61 (m, 2H), 7.14-7.24 (m, 2H), 8.49-8.56 (m, 2H), 8.67 & 8.70 (d, J=1.6 Hz, 2H), 9.04 (d, J=1.6 Hz, 1H); 13C NMR (100 MHz, CDCl3) δ 29.7, 32.3, 45.1, 47.6, 52.3, 54.8, 69.4, 69.5, 76.9, 78.5, 120.1, 121.6, 121.7, 121.8, 127.7, 127.8, 139.8, 139.9, 146.6, 146.8, 148.5, 150.1, 150.2, 164.7.
  • Figure US20150018543A1-20150115-C00118
    • General Procedure for the Synthesis of aminopyrrolidinyl-phenyl-methanone (E3)
  • To a solution of (S)-(+)-N-Boc-3-pyrrolidinol (2.67 mmol) and triethylamine (4.01 mmol) in methylene chloride (50 mL) was added methansulfonyl chloride (4.01 mmol) under ice-bath and the resulting mixture was further stirred at 4° C. After 2 h, the residue was diluted with methylene chloride (50 mL) and washed with water (100 mL) and brine (100 mL). The organic layer was dried over anhydrous MgSO4 and concentrated in vacuo. The crude product was purified by silica gel flash column chromatography (2:1 hexanes/ethyl acetate) to give E1.
  • A solution of E1 (0.75 mmol) and an amine (3.75 mmol) was stirred at 100° C. After stirring overnight, the residue was dissolved in methylene chloride (30 mL) and washed with water (30 mL) and brine (30 mL). The organic layer was dried over anhydrous MgSO4 and concentrated in vacuo. The crude product was purified by silica gel flash column chromatography (1:1 hexanes/ethyl acetate) to give E2.
  • To a solution of E2 (0.96 mmol) in methylene chloride (20 mL) was added trifluoroacetic acid (0.5 mL). After 3 h, the solvent was removed in vacuo. The reaction mixture was dissolved in methylene chloride (20 mL) and cooled to 0° C. Triethylamine (4.83 mmol) and a benzoyl chloride (1.05 mmol) was added. After 2 h, the residue was diluted with methylene chloride (20 mL) and washed with water (40 mL) and brine (40 mL). The organic layer was dried over anhydrous MgSO4 and concentrated in vacuo. The crude was purified by silica gel flash column chromatography (1:1 hexanes/ethyl acetate) to give E3.
    • (R)-(3,5-Dinitrophenyl)(3-(4-methoxyphenylamino)pyrrolidin-1-yl)methanone (103)
  • Figure US20150018543A1-20150115-C00119
  • (Two rotamers, 1:1 ratio, 63%), a brown solid; 1H NMR (400 MHz, CDCl3+CD3OD) δ 1.93-2.01 (m, 1H), 2.14-2.30 (m, 1H), 3.26-3.30 & 3.44-3.50 (m, 1H), 3.54-3.72 (m, 2H), 3.61 & 3.68 (s, 3H), 3.80-3.91 (m, 1H), 3.95-4.05 (m, 1H), 6.43 & 6.55 (d, J=8.8 Hz, 2H), 6.62 & 6.70 (d, J=8.8 Hz, 2H), 8.58 & 8.67 (d, J=2.4 Hz, 2H), 8.95-8.99 (m, 1H); 13C NMR (100 MHz, CDCl3+CD3OD) δ 30.4, 32.4, 45.2, 47.9, 52.6, 53.0, 54.4, 55.0, 55.8, 55.9, 115.0, 115.1, 115.2, 115.3, 120.1, 127.6, 127.7, 139.6, 140.5, 140.7, 148.5, 148.6, 152.8, 152.9, 165.2, 165.4.
    • (R)-(3-(4-Butoxyphenylamino)pyrrolidin-1-yl)(3,5-dinitrophenyl)methanone (104)
  • Figure US20150018543A1-20150115-C00120
  • (Two rotamers, 1:1 ratio, 54%), a brown solid; m.p. 118-120° C.; 1H NMR (400 MHz, CDCl3) δ 0.83-0.98 (m, 3H), 1.39-1.52 (m, 2H), 1.61-1.76 (m, 2H), 2.02-2.05 (m, 1H), 2.24-2.41 (m, 1H), 3.33-3.37 & 3.50-3.63 (m, 2H), 3.66-4.13 (m, 6H), 6.47 & 6.60 (d, J=8.4 Hz, 2H), 6.70 & 6.78 (d, J=8.4 Hz, 2H), 8.66 & 8.74 (s, 2H), 9.05 & 9.08 (s, 1H); 13C NMR (100 MHz, CDCl3) δ 14.0, 14.1, 19.4, 19.5, 30.9, 31.6, 31.7, 32.9, 45.3, 47.9, 52.8, 53.4, 54.6, 55.2, 68.5, 68.6, 115.0, 115.2, 116.0, 116.2, 120.2, 127.8, 127.9, 139.9, 140.1, 140.4, 148.6, 152.7, 164.9, 165.1.
    • (R)-(3,5-Dinitrophenyl)(3-(4-phenoxyphenylamino)pyrrolidin-1-yl)methanone (105)
  • Figure US20150018543A1-20150115-C00121
  • (Two rotamers, 1:1 ratio, 60%), a brown solid; 1H NMR (400 MHz, CDCl3+CD3OD) δ 2.00-2.06 (m, 1H), 2.18-2.35 (m, 1H), 3.32-3.35 & 3.48-3.54 (m, 1H), 3.61-3.78 (m, 2H), 3.82-4.12 (m, 2H), 6.47 & 6.60 (d, J=8.8 Hz, 2H), 6.77-6.97 (m, 5H), 7.17, 7.24 (m, 2H), 8.63 & 8.69 (d, J=1.6 Hz, 2H), 9.01 & 9.04 (s, 1H); 13C NMR (100 MHz, CDCl3+CD3OD) δ 30.6, 32.5, 45.3, 47.9, 52.3, 53.0, 54.0, 55.1, 114.5, 114.8, 117.4, 117.5, 120.2, 121.3, 121.4, 122.4, 122.5, 127.7, 127.8, 129.7, 139.6, 142.8, 143.0, 148.6, 148.8, 165.2, 165.3.
    • (R)-(3,5-Dinitrophenyl(3-(4-hydroxyphenylamino)pyrrolidin-1-yl)methanone (106)
  • Figure US20150018543A1-20150115-C00122
  • (Two rotamers, 1:1 ratio, 83%), a yellow solid; 1H NMR (400 MHz, DMSO-d6) δ 1.78-1.89 (m, 1H), 2.03-2.15 (m, 1H), 3.12-3.17 (m, 1H), 3.37-3.45 (m, 1H), 3.52-3.95 (m, 3H), 5.15-5.23 (m, 1H), 6.36-6.56 (m, 4H), 8.38 & 8.44 (brs, 1H), 8.64 & 8.67 (s, 2H), 8.81 & 8.84 (s, 1H); 13C NMR (100 MHz, DMSO-d6) δ 29.6, 31.3, 44.6, 46.9, 51.6, 51.9, 53.3, 54.1, 113.8, 114.2, 115.6, 115.7, 119.4, 127.4, 127.5, 139.6, 139.7, 140.3, 140.4, 148.0, 148.1, 148.5, 148.7, 164.2.
    • (R)-(3,5-Dinitrophenyl)-3-(phenylamino)pyrrolidin-1-yl)methanone (107)
  • Figure US20150018543A1-20150115-C00123
  • (Two rotamers, 1:1 ratio, 80%), a red solid; 1H NMR (400 MHz, CDCl13+CD3OD) δ 1.99-2.04 (m, 1H), 2.17-2.33 (m, 1H), 3.28-3.31 & 3.57-3.95 (m, 4H), 4.04-4.11 (m, 1H), 6.46-6.48 (m, 1H), 6.59-6.70 (m, 2H), 7.02-7.14 (m, 2H), 8.60 & 8.67 (s, 2H), 8.98 & 9.01 (s, 1H); 13C NMR (100 MHz, CDCl3+CD3OD) δ 30.3, 32.2, 45.1, 47.8, 51.7, 52.8, 53.3, 54.9, 113.2, 113.5, 118.3, 118.4, 120.0, 127.6, 127.7, 129.4, 139.5, 146.3, 146.4, 148.4, 148.5, 165.1, 165.3.
    • (R)-(3,5-Dinitrophenyl)(3-(pyridin-2-ylamino)pyrrolidin-1-yl)methanone (108)
  • Figure US20150018543A1-20150115-C00124
  • (Two rotamers, 1:1 ratio, 70%), a yellow solid; 1H NMR (400 MHz, CDCl3) δ 2.00-2.44 (m, 2H), 3.38-4.11 (m, 4H), 4.38 & 4.50 (m, 1H), 6.36 & 6.44 (d, J=8.4 Hz, 1H), 6.57 & 6.64 (t, J=6.0 Hz, 1H), 7.37 & 7.44 (t, J=7.8 Hz, 1H), 7.98 & 8.11 (d, J=5.2 Hz, 1H), 8.67 & 8.73 (s, 2H), 9.05 & 9.09 (s, 1H); 13C NMR (100 MHz, CDCl3) δ 30.4, 32.6, 45.1, 47.7, 51.7, 52.9, 55.3, 76.7, 101.8, 108.3, 113.7, 119.9, 127.7, 137.5, 137.7, 147.8, 147.9, 148.3, 148.4, 157.2, 157.4, 164.8, 164.9.
    • (R)-(3-(Cyclohexylamino)pyrrolidin-1-yl)(3,5-dinitrophenyl)methanone (109)
  • Figure US20150018543A1-20150115-C00125
  • (Two rotamers, 1:1 ratio, 69%), a pale yellow solid; 1H NMR (400 MHz, CDCl3+CD3OD) δ 0.99-1.35 (m, 6H), 1.60-1.98 (m, 5H), 2.15-2.32 (m, 1H), 2.39-2.57 (m, 1H), 3.24-3.60 (m, 2H), 3.63-3.73 (m, 2H), 3.81-3.91 (m, 1H), 8.73 & 8.78 (s, 2H), 9.10 (s, 1H); 13C NMR (100 MHz, CDCl3+CD3OD) δ 24.7, 24.8, 24.9, 25.6, 25.7, 30.6, 32.4, 33.3, 33.4, 45.2, 47.7, 52.6, 54.5, 54.8, 54.9, 55.1, 119.7, 127.4, 127.5, 139.5, 148.3, 164.9, 165.0.
    • (R)—N-Cyclohexyl-N-(1-(3,5-dinitrobenzoyl)pyrrolidin-3-yl)-3,5-dinitrobenzamide (110)
  • Figure US20150018543A1-20150115-C00126
  • (Two rotamers, 1:1 ratio, 15%), a white solid; 1H NMR (400 MHz, CDCl3) δ 1.01-1.22 (m, 3H), 1.62-1.86 (m, 6H), 2.18-2.26 (m, 1H), 2.74-2.89 (m, 1H), 3.30-3.35 (m, 1H), 3.50-3.78 (m, 2H), 3.97-4.19 (m, 4H), 8.51 & 8.56 (s, 2H), 8.74 (s, 2H), 9.09-9.10 (m, 2H); 13C NMR (100 MHz, CDCl3) δ 24.8, 24.9, 25.3, 25.5, 27.3, 30.0, 31.8, 45.6, 48.7, 48.9, 50.0, 53.6, 54.8, 60.5, 119.8, 120.0, 126.7, 127.8, 139.8, 140.1, 140.2, 140.4, 148.6, 148.2, 164.4, 164.7, 166.6, 166.7.
    • (R)-(3-(4-Methoxyphenylamino)pyrrolidin-1-yl)(phenyl)methanone (111)
  • Figure US20150018543A1-20150115-C00127
  • (Two rotamers, 1:1 ratio, 75%), a pale yellow solid; 1H NMR (400 MHz, CDCl3) δ 1.84-1.88 (m, 1H), 2.08-2.32 (m, 1H), 3.26-3.34 & 3.49-4.03 (m, 5H), 3.69 & 3.72 (s, 3H), 6.48 & 6.50 (d, J=6.4 Hz, 2H), 6.71 & 6.76 (d, J=6.4 Hz, 2H), 7.36-7.51 (m, 5H); 13C NMR (100 MHz, CDCl3) δ 30.7, 32.5, 44.5, 47.7, 52.6, 52.7, 54.2, 55.2, 55.8, 55.9, 114.7, 114.9, 115.0, 127.2, 128.3, 130.1, 136.7, 140.8, 141.0, 152.6, 170.0.
    • (R)-(3-(3-Chlorobenzylamino)pyrrolidin-1-yl)(3,5-dinitrophenyl)methanone (112)
  • Figure US20150018543A1-20150115-C00128
  • (Two rotamers, 1:1 ratio, 32%) as a pale yellow solid; 1H NMR (400 MHz, CDCl3+CD3OD) δ 1.83-1.89 (m, 1H), 2.01-2.08 & 2.14-2.19 (m, 1H), 2.75 (brs, 1H), 3.15-3.19 & 3.35-3.83 (m, 7H), 7.05-7.23 (m, 4H), 8.58 & 8.67 (d, J=2.0 Hz, 2H), 8.97-8.99 (m, 1H); 13C NMR (100 MHz, CDCl3+CD3OD) δ 30.4, 32.1, 45.2, 47.7, 51.2, 51.4, 52.4, 54.9, 55.2, 57.5, 119.8, 126.0, 126.2, 127.2, 127.3, 127.5, 127.6, 127.8, 128.0, 129.7, 129.8, 134.1, 134.2, 139.5, 139.6, 141.3, 141.7, 148.2, 148.3, 164.7, 164.8.
    • (R)—N-(3-Chlorobenzyl)-N-(1-(3,5-dinitrobenzoyl)pyrrolidin-3-yl)-3,5-dinitrobenzamide (113)
  • Figure US20150018543A1-20150115-C00129
  • (Two rotamers, 1:1 ratio, 44%), a white solid; 1H NMR (400 MHz, CDCl3) δ 2.26-2.35 (m, 2H), 3.56-4.05 (m, 4H), 4.57-4.65 (m, 3H), 7.06-7.15 (m, 2H), 7.24-7.35 (m, 2H), 8.50-8.62 (m, 4H), 8.97-9.02 (m, 2H); LC-MS (ESI, m/z): 599 [M+H]+.
    • (R)-(3-(Benzylamino)pyrrolidin-1-yl)(3,5-dinitrophenyl)methanone (114)
  • Figure US20150018543A1-20150115-C00130
  • 1H NMR (400 MHz, CDCl3) δ 1.59 (brs, 1H), 1.87-1.94 (m, 1H), 2.06-2.24 (m, 1H), 3.20 (dd, J=4.8, 10.4 Hz, 0.5H), 3.46-3.89 (m, 6.5H), 7.15-7.36 (m, 5H), 8.63 (d, J=2.0 Hz, 1H), 8.71 (d, J=2.0 Hz, 111), 9.03 (t, J=2.0 Hz, 0.5H), 9.06 (t, J=2.0 Hz, 0.5H).
    • (R)-(3,5-Dinitrophenyl)(3-(3-(trifluoromethyl)benzylamino)pyrrolidin-1-yl)methanone (115)
  • Figure US20150018543A1-20150115-C00131
  • 1H NMR (400 MHz, CDCl3) δ 1.51 (brs, 1H), 1.89-1.94 (m, 1H), 2.10-2.28 (m, 1H), 3.24 (dd, J=5.2, 10.0 Hz, 0.5H), 3.45-3.92 (m, 6.5H), 7.40-7.61 (m, 4H), 8.65 (d, J=2.0 Hz, 1H), 8.72 (d, J=2.0 Hz, 1H), 9.06 (t, J=2.0 Hz, 0.5H), 9.08 (t, J=2.0 Hz, 0.5H).
    • (R)-(3,5-Dinitrophenyl)-3-(2-fluorobenzylamino)pyrrolidin-1-yl)methanone (116)
  • Figure US20150018543A1-20150115-C00132
  • (Two rotamers, 1:1 ratio, 75%), a yellow solid; 1H NMR (400 MHz, CDCl3) δ 1.89-1.94 (m, 1H), 2.11-2.25 (m, 1H), 3.22-3.89 (m, 7H), 6.93 & 7.02 (t, J=8.6 Hz, 2H), 7.20 & 7.33 (m, 2H), 8.66 & 8.72 (d, J=2.0 Hz, 2H), 9.06 (s, 1H); 13C NMR (100 MHz, CDCl3) δ 30.7, 32.5, 45.3, 47.8, 51.3, 51.5, 52.7, 55.1, 55.4, 57.7, 115.1, 115.3, 119.8, 119.9, 127.6, 127.7, 129.4, 129.6, 135.4, 135.5, 139.8, 148.3, 148.4, 162.0 (d, J=245 Hz, due to F), 162.1 (d, J=245 Hz, due to F), 164.5, 164.6.
    • (R)-(3,5-Dinitrophenyl)(3-(2-fluorobenzylamino)pyrrolidin-1-yl)methanone hydrochloride (117)
  • Figure US20150018543A1-20150115-C00133
  • (Two rotamers, 1:1 ratio, 92%), a white solid; 1H NMR (400 MHz, CD3OD+D2O) δ 2.24-2.35 (m, 1H), 2.48-2.63 (m, 1H), 3.48-4.34 (m, 7H), 7.13 & 7.24 (t, J=8.6 Hz, 2H), 7.47 & 7.58 (q, J=7.0 Hz, 2H), 8.73 & 8.8 (d, J=2.0 Hz, 2H), 9.16 (brs, 1H); 13C NMR (100 MHz, CD3OD+D2O) δ 28.1, 29.7, 45.5, 50.6, 50.7, 51.9, 56.6, 57.7, 81.1, 117.0, 117.1, 127.7, 128.6, 128.7, 133.2, 133.3, 139.0, 147.1, 149.7, 167.5, 167.6.
    • (R)-(3,5-Dinitrophenyl)(3-(pyridin-4-ylmethylamino)pyrrolidin-1-yl)methanone (118)
  • Figure US20150018543A1-20150115-C00134
  • (Two rotamers, 1:1 ratio, 69%), a yellow solid; 1H NMR (400 MHz, CDCl3) δ 1.80 (br, 1H), 1.88-2.23 (m, 2H), 3.23-3.89 (m, 7H), 7.17 & 7.26 (d, J=5.2 Hz, 2H), 8.45 & 8.52 (d, J=5.6 Hz, 2H), 8.65 & 8.69 (d, J=2.0 Hz, 2H), 9.04 (t, J=2.0 Hz, 1H); 13C NMR (100 MHz, CDCl3) δ 30.2, 32.4, 44.7, 47.7, 52.4, 54.8, 55.9, 76.0, 115.0, 115.1, 117.2, 117.4, 124.4, 124.5, 127.0, 129.1, 130.6, 130.8, 137.5, 137.7, 150.8, 151.0, 154.7, 154.8, 168.5, 168.8.
  • Figure US20150018543A1-20150115-C00135
    • General Procedure for the Synthesis of (R)—N-benzoylpyrrolidinyl-benzamide (F5)
  • To a solution of F1 (3.77 mmol) in DMF (15 mL) was added sodium azide (11.00 mmol) and the resulting mixture was warmed to 70° C. After 3 h, the solvent was removed in vacuo, dissolved in ethylacetate (50 mL) and washed with water (50 mL) and brine (50 mL). The organic layer was dried over anhydrous MgSO4 and concentrated in vacuo. The crude product was purified by silical gel flash column chromatography (1:1 hexanes/ethyl acetate) to give F2.
  • To a solution of F2 (2.68 mmol) was added 10% palladium on activated carbon and stirred overnight under hydrogen atmosphere. The reaction mixture was filtered using cellite 545 and the resulting filtrate was concentrated in vacuo to give F3.
  • To a solution of F3 (0.77 mmol) and triethylamine (1.16 mmol) in methylene chloride (10 mL) was added benzoyl chloride (1.00 mmol) under ice bath. The reaction mixture was brought up to room temperature. After 2 h, the reaction mixture was diluted with methylene chloride (20 mL) and washed with water (30 mL) and brine (30 mL). The organic layer was dried over anhydrous MgSO4 and concentrated in vacuo. The crude product was purified by silica gel flash column chromatography (2:1 hexanes/ethyl acetate) to give F4.
  • To a solution of F4 (0.59 mmol) in methylene chloride (10 mL) was added trifluoroacetic acid (0.5 mL) and stirred at room temperature. After 3 h, the solvent was removed in vacuo. The crude product was dissolved in methylene chloride (10 mL) and triethylamine (0.41 mL, 2.96 mmol) was added. The reaction mixture was cooled to 0° C. and then 3,5-dichlorobenzoyl chloride (0.65 mmol) was added. The resulting mixture was brought up to room temperature. After 2 h, the solvent was removed in vacuo and the crude residue was purified by silica gel flash column chromatography (1:1 hexanes/ethyl acetate) to give F5.
    • (R)—N-(1-(3,5-Dinitrobenzol)pyrrolidin-3-yl)-3-(trifluoromethoxy)benzamide (119)
  • Figure US20150018543A1-20150115-C00136
  • (Two rotamers, 1:1 ratio, 67%), a pale yellow solid; 1H NMR (400 MHz, CDCl3) δ 2.07-2.18 (m, 1H), 2.29-2.40 (m, 1H), 3.49-3.60 (m, 1H), 3.68-3.76 (m, 1H), 3.87-3.98 (m, 2H), 4.60-4.74 (m, 1H), 7.19-7.60 (m, 5H), 8.51 & 8.59 (s, 2H), 8.91 & 8.96 (s, 1H); 13C NMR (100 MHz, CDCl3) δ 29.9, 32.5, 45.3, 48.0, 49.2, 50.8, 51.9, 54.8, 119.9, 120.0, 120.2, 124.3, 125.7, 127.6, 130.2, 135.7, 136.0, 139.4, 148.4, 148.5, 149.2, 164.9, 165.0, 166.5, 166.6.
    • (R)—N-(1-(3,5-Dinitrobenzoyl)pyrrolidin-3-yl)-4-methoxybenzamide (120)
  • Figure US20150018543A1-20150115-C00137
  • (Two rotamers, 1:1 ratio, 0.19 g, 76%), a white solid; 1H NMR (400 MHz, CDCl3) δ 2.08-2.15 (m, 1H), 2.35-2.47 (m, 1H), 3.47-4.08 (m, 4H), 3.81 & 3.84 (s, 3H), 4.62-4.64 & 4.77-4.78 (m, 1H), 6.45 & 6.50 (brs, 1H), 6.82 & 6.88 (d, J=8.4 Hz, 2H), 7.62 & 7.72 (d, J=8.4 Hz, 2H), 8.62 & 8.71 (s, 2H), 9.04 & 9.08 (s, 1H); 13C NMR (100 MHz, CDCl3) δ□ 30.0, 32.8, 45.2, 48.0, 48.9, 50.5, 52.2, 55.2, 55.6, 60.6, 113.9, 120.2, 125.6, 126.1, 127.7, 127.8, 129.0, 139.5, 148.5, 162.7, 164.9, 165.0, 167.4.
    • (R)-3-Chloro-N-(1-(3,5-dinitrobenzoyl)pyrrolidin-3-yl)benzamide (121)
  • Figure US20150018543A1-20150115-C00138
  • (Two rotamers, 1:1 ratio, 66%), a pale yellow solid; 1H NMR (400 MHz, DMSO-d6) δ 1.94-2.20 (m, 2H), 3.33-3.83 (m, 4H), 4.42-4.55 (m, 1H), 7.43-7.60 (m, 2H), 7.71-7.90 (m, 2H), 8.66 & 8.69 (d, J=2.0 Hz, 2H, brs, 1H), 8.83-8.86 (m, 1H); 13C NMR (100 MHz, DMSO-d6) δ 29.3, 31.5, 44.6, 47.0, 48.4, 49.9, 51.1, 53.3, 119.4, 119.5, 126.2, 126.3, 127.0, 127.1, 127.5, 130.2, 130.3, 131.1, 133.0, 133.1, 136.1, 136.3, 139.5, 139.6, 148.0, 164.0, 164.1, 165.0, 165.1.
    • (S)-1-(3,5-Dinitrobenzoyl)pyrrolidin-3-yl methanesulfonate (122)
  • Figure US20150018543A1-20150115-C00139
  • (Two rotamers, 1:1 ratio, 92%), a white solid; m.p. 138-140° C.; 1H NMR (400 MHz, CDCl3) δ 2.25-2.46 (m, 2H), 3.03 & 3.10 (s, 3H), 3.59-3.67 & 3.75-4.03 (m, 4H), 5.28-5.40 (m, 1H), 8.68 & 8.73 (s, 2H), 9.08 (s, 1H); 13C NMR (100 MHz, CDCl3) δ 31.1, 33.6, 38.9, 39.0, 44.7, 47.2, 53.3, 55.3, 78.2, 78.6, 120.5, 127.8, 127.9, 139.3, 148.7, 164.8, 165.0; LC-MS (ESI, m/z): 360 [M+H]+.
    • (R)-1-(3,5-Dinitrobenzoyl)pyrrolidin-3-yl methanesulfonate (123)
  • Figure US20150018543A1-20150115-C00140
  • (Two rotamers, 1:1 ratio, 89%), a white solid; 1H NMR (400 MHz, CDCl3+CD3OD) δ 2.16-2.32 (m, 2H), 2.94 & 3.02 (s, 3H), 3.50-3.91 (m, 4H), 5.19-5.30 (m, 1H), 8.58 & 8.63 (s, 2H), 8.97 (s, 1H); 13C NMR (100 MHz, CDCl3+CD3OD) δ 30.7, 33.1, 38.3, 38.4, 44.5, 46.9, 53.0, 55.0, 78.5, 79.0, 120.1, 127.6, 139.0, 148.4, 164.9, 165.0.
    • Example 7 Derivatization of the Pyridopyrimidinone Compounds
  • The pyridopyrimidinone compounds (scaffold VIII; see Table 2) underwent derivatization according to the methods outlined below (Schemes 8-10). Resulting derivatives were examined for inhibitory activity using the assay described above and the results are summarized in Table 3.
  • Figure US20150018543A1-20150115-C00141
    • General Procedure for the Synthesis of G1
  • 2-Amino-3-picoline (1.0 mmol) was dissolved in diethyl malonate (1.0 mmol). The solution was heated to 1701′ for 12 h. After cooling, the dark residue was triturated with CH2Cl2 (10 mL). The residual pale solid was collected by filtration and washed with CH2Cl2 to give G1.
    • General Procedure for the Synthesis of G2
  • To a DMF (2.0 mL) was added POCl3 (3.0 mmol) at 0° C. After the mixture was stirred at 0 for 40 min, a solution of G1 (1.0 mmol) in DMF (2.0 mL) was added and stirred at 80° C. for 1 h. The mixture was cooled and concentrated in vacuo. The residue was diluted with water and extracted with CH2Cl2 (10 mL×3). The combined organic layers were washed with brine, dried over MgSO4 and concentrated. The residue was purified by flash column chromatography to give G2.
    • General Procedure for the Synthesis of G3
  • To a stirred solution of G2 (1.0 mmol) in THF (2.0 mL) was added Et3N (2.0 mmol). The mixture was cooled to 0° C. After 5 min, an amine (1.0 mmol) was added dropwise and the mixture was stirred at room temperature overnight. The reaction mixture was diluted with CH2Cl2 (10 mL) and washed with brine (10 mL). The organic layer was dried over anhydrous MgSO4 and concentrated in vacuo. The crude product was purified by flash column chromatography to give G3.
    • General Procedure for the Synthesis of G4
  • G2 (0.5 mmol) was dissolved in 10.4 mL of tert-butyl alcohol and 2.5 mL of 2-methyl-2-butene. A solution of sodium chlorite (4.59 mmol) and sodium dihydrogenphosphate (3.46 mmol) in 4.2 mL of water was added dropwise. The reaction mixture was stirred at room temperature overnight. Volatile components were then removed under vacuum, and the residue was dissolved in 10 ml of water and extracted with two 10 ml portions of hexane. The aqueous layer was acidified to pH=3 with HCl(aq) and extracted with 10 mL portions of methylene chloride. The combined organic layers were washed with 20 mL of cold water, dried and concentrated to give G4.
    • General Procedure for the Synthesis of G5 from G3
  • G3 (36.6 μmol) was dissolved in 760 μl of tert-butyl alcohol and 180 μl of 2-methyl-2-butene. A solution of sodium chlorite (335 μmol) and sodium dihydrogenphosphate (253 μmol) in 300 μl of water was added dropwise. The reaction mixture was stirred at room temperature overnight. Volatile components were then removed under vacuum and the residue was dissolved in 10 ml of water and extracted with two 10 ml portions of hexane. The aqueous layer was acidified to pH=3 with HCl(aq) and extracted with 10 ml portions of methylene chloride. The combined organic layers were washed with 20 ml of cold water, dried and concentrated to give G5.
    • General Procedure for the Synthesis of G5 from G4
  • To a stirred solution of G4 (1.0 mmol) in DMF (2.0 mL) was added Et3N (2.0 mmol) and amine (1.5 mmol) and the mixture was stirred at 60° C. overnight. The reaction mixture was diluted with CH2Cl2 (10 mL) and washed with brine (10 ml). The organic layer was dried over anhydrous MgSO4 and concentrated in vacuo. The crude product was purified by recrystallization from a mixture of hexanes and methylene chloride to give G5.
    • General Procedure for the Synthesis of G6
  • The solution of 2-amino-3-picoline (4.0 mmol) in a solution of CH2Cl2 (3 mL) and dried pyridine (1 mL) was added dropwise at room temperature to a stirred solution of ethyl 3-chloro-3-oxo-propionate (5.3 mmol) in CH2Cl2 (3 mL) (an exothermic reaction with emission of white fume occurred during the addition). The resulting warm mixture was stirred at room temperature for 30 min and then poured into 30 mL of cold water; an excess of sodium carbonate was carefully added with stirring and the mixture was further stirred at room temperature for 1 h. The organic layer was then collected and the aqueous phase was extracted several times with CH2Cl2. The combined organic layers were washed with water, dried over anhydrous Na2SO4, and concentrated in vacuo. The crude product was purified by flash column chromatography to give G6.
    • General Procedure for the Synthesis of G7
  • A mixture of G6 (1.83 mmol), POCl3 (0.5 mL) and polyphosphoric acid (137 mg) was heated with stirring at 130° C. for 3 h. After cooling, anhydrous ethanol was added and the mixture was refluxed for 30 min, then allowed to cool. The mixture was treated with aqueous sodium carbonate and exhaustively extracted with CH2Cl2 (10 mL×3). The combined layers were washed with water (10 mL), brine (10 mL), dried over MgSO4, filtered and concentrated in vacuo. The crude product was purified by flash column chromatography to give G7.
    • General Procedure for the Synthesis of G8
  • To a solution of G6 (1 mmol) in DMF (0.96 mL) was added potassium carbonate (5.0 mmol) followed by phenol (1.94 mmol). After 12 h at 100° C., the solution was allowed to cool to 23° C. The reaction mixture was washed with H2O (50 mL), and the aqueous layer was extracted with CH2Cl2 (20 mL×3). The combined organic layers were washed with 1N HCl (20 mL×2), filtered, and concentrated in vacuo. The crude product was purified by flash column chromatography to give G8.
    • General Procedure for the Synthesis of G9
  • To DMF (2.0 mL) was added POCl3 (3.0 mmol) at 0° C. After the mixture was stirred at 0° C. for 40 min, a solution of G8 (1.0 mmol) in DMF (2.0 mL) was added and stirred at 80° C. for 1 h. The mixture was cooled and concentrated in vacuo. The residue was diluted with water and extracted with CH2Cl2 (10 mL×3). The combined organic layers were washed with brine, dried over MgSO4 and concentrated. The residue was purified by flash column chromatography to give G9.
    • Ethyl 3-(3-methylpyridin-2-ylamino)-3-oxopropanoate (124)
  • Figure US20150018543A1-20150115-C00142
  • 1H NMR (400 MHz, CDCl3) δ 1.25 (t, J=7.0 Hz, 3H), 2.25 (s, 3H), 3.45 (s, 2H), 4.20 (q, J=7.2 Hz, 2H), 7.47 (d, J=8.4 Hz, 1H), 8.03 (d, J=8.4 Hz, 1H), 8.07 (s, 1H), 9.67 (brs, 1H); 13C NMR (100 MHz, CDCl3) δ 13.9, 17.7, 42.6, 61.7, 113.8, 129.3, 138.8, 147.6, 148.8, 163.5, 168.4.
    • 2-Hydroxy-9-methyl-4H-pyrido[1,2-a]pyrimidin-4-one (125)
  • Figure US20150018543A1-20150115-C00143
  • 1H NMR (400 MHz, DMSO-d6) δ 2.48 (s, 3H), 5.44 (s, 1H), 7.20 (t, J=7.0 Hz, 1H), 7.87 (d, J=6.8 Hz, 1H), 8.84 (d, J=6.8 Hz, 1H), 11.52 (brs, 1H).
    • 2-Hydroxy-8-methyl-4H-pyrido[1,2-a]pyrimidin-4-one (126)
  • Figure US20150018543A1-20150115-C00144
  • 1H NMR (400 MHz, DMSO-d6) δ 2.50 (s, 3H), 4.88 (s, 1H), 7.20-7.24 (m, 2H), 8.85 (d, J=6.8 Hz, 1H), 11.98 (br s, 1H); 13C NMR (100 MHz, DMSO-d6) δ 20.6, 80.3, 114.4, 117.1, 127.7, 146.7, 153.5, 155.3, 162.3.
    • 2-Chloro-9-methyl-4H-pyrido[1,2-a]pyrimidin-4-one (127)
  • Figure US20150018543A1-20150115-C00145
  • 1H NMR (400 MHz, CDCl3) δ 2.57 (s, 3H), 6.45 (s, 1H), 7.12 (t, J=7.0 Hz, 1H), 7.68 (d, J=6.8 Hz, 1H), 8.93 (d, J=6.8 Hz, 1H); 13C NMR (100 MHz, CDCl3) δ 18.0, 102.3, 115.8, 125.7, 134.7, 136.9, 150.0, 157.6, 157.9.
    • 2-Chloro-9-methyl-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carbaldehyde (128)
  • Figure US20150018543A1-20150115-C00146
  • 1H NMR (400 MHz, CDCl3) δ 2.64 (s, 3H), 7.30 (t, J=7.0 Hz, 1H), 7.92 (d, J=7.2 Hz, 1H), 9.10 (d, J=6.4 Hz, 1H), 10.42 (s, 1H); 13C NMR (100 MHz, CDCl3) δ 17.7, 107.3, 117.7, 127.0, 135.6, 140.6, 150.0, 156.4, 160.2, 187.1.
    • 2-Chloro-8-methyl-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carbaldehyde (129)
  • Figure US20150018543A1-20150115-C00147
  • 1H NMR (400 MHz, CDCl3) δ 2.59 (s, 3H), 7.24 (d, J=7.2 Hz, 1H), 7.52 (s, 1H), 9.09 (d, J=7.2 Hz, 1H), 10.40 (s, 1H).
    • 2-Chloro-7-methyl-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carbaldehyde (130)
  • Figure US20150018543A1-20150115-C00148
  • 1H NMR (400 MHz, DMSO-d6) δ 2.32 (s, 3H), 7.49 (d, J=8.8 Hz, 1H), 7.78 (d, J=8.8 Hz, 1H), 8.79 (s, 1H), 10.16 (s, 1H).
    • 2-Chloro-6-methyl-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carbaldehyde (131)
  • Figure US20150018543A1-20150115-C00149
  • 1H NMR (400 MHz, CDCl3) δ 3.11 (s, 3H), 6.98 (d, J=7.2 Hz, 1H), 7.51 (d, J=8.8 Hz, 1H), 7.79 (t, J=8.0 Hz, 1H), 10.29 (s, 1H).
    • 9-Methyl-4-oxo-2-(phenylamino)-4H-pyrido[1,2-a]pyrimidine-3-carbaldehyde (132)
  • Figure US20150018543A1-20150115-C00150
  • 1H NMR (400 MHz, CDCl3) δ 2.44 (s, 3H), 6.89 (t, J=6.8 Hz, 1H), 7.11 (t, J=7.2 Hz, 1H), 7.34 (t, J=7.6 Hz, 2H), 7.62 (d, J=6.4 Hz, 1H), 7.76 (d, J=8.0 Hz, 2H), 8.80 (d, J=6.8 Hz, 1H), 10.27 (s, 1H), 11.67 (brs, 1H); 13C NMR (100 MHz, CDCl3) δ 18.1, 94.6, 113.6, 121.8, 124.2, 125.9, 128.7, 133.6, 138.1, 138.9, 152.5, 153.8, 160.2, 190.2.
    • 2-(3-Chlorophenylamino)-9-methyl-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carbaldehyde (133)
  • Figure US20150018543A1-20150115-C00151
  • 1H NMR (400 MHz, CDCl3) δ 2.50 (s, 3H), 6.97 (t, J=6.8 Hz, 1H), 7.08 (d, J=8.0 Hz, 1H), 7.25 (t, J=8.0 Hz, 1H), 7.42 (d, J=8.0H, 1H), 7.69 (d, J=6.8 Hz, 1H), 8.18 (s, 1H), 8.84 (d, J=6.8 Hz, 1H), 10.27 (s, 1H), 11.72 (brs, 1H).
    • 9-Methyl-4-oxo-2-(3-(trifluoromethoxy)phenylamino)-4H-pyrido[1,2-a]pyrimidine-3-carbaldehyde (134)
  • Figure US20150018543A1-20150115-C00152
  • 1H NMR (400 MHz, CDCl3) δ 2.50 (s, 3H), 6.99 (t, J=7.0 Hz, 1H), 7.36 (t, J=8.0 Hz, 1H), 7.42 (d, J=8.0 Hz, 1H), 7.70 (d, J=6.8 Hz, 1H), 8.16 (s, 1H), 8.88 (d, J=8.0 Hz, 1H), 10.32 (s, 1H), 11.86 (brs, 1H); 13C NMR (100 MHz, CDCl3) δ 18.0, 94.7, 114.2, 114.7, 116.5, 119.7, 126.1, 129.7, 133.8, 139.4, 139.7, 149.4, 152.6, 157.0, 160.1, 190.4.
    • 9-Methyl-4-oxo-2-(3-(trifluoromethyl)phenylamino)-4H-pyrido[1,2-a]pyrimidine-3-carbaldehyde (135)
  • Figure US20150018543A1-20150115-C00153
  • 1H NMR (400 MHz, CDCl3) δ 2.49 (s, 1H), 6.98 (t, J=6.8 Hz, 1H), 7.37 (d, J=7.6 Hz, 1H), 7.45 (d, J=7.6 Hz, 1H), 7.61 (d, J=8.0 Hz, 1H), 7.70 (d, J=6.0 Hz, 1H), 8.61 (s, 1H), 8.87 (d, J=6.8 Hz, 1H), 10.30 (s, 1H), 11.85 (brs, 1H).
    • 2-(4-tert-Butylphenylamino)-9-methyl-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carbaldehyde (136)
  • Figure US20150018543A1-20150115-C00154
  • 1H NMR (400 MHz, CDCl3) δ 1.32 (s, 9H), 2.48 (s, 3H), 6.89 (t, J=7.0 Hz, 1H), 7.37 (d, J=8.4 Hz, 1H), 7.62 (d, J=6.8 Hz, 1H), 7.73 (d, J=8.8 Hz, 1H), 8.81 (d, J=7.2 Hz, 1H), 10.30 (s, 1H), 11.68 (br s, 1H); 13C NMR (100 MHz, CDCl3) δ 18.2, 31.3, 34.3, 94.6, 113.5, 121.4, 125.6, □ 125.9, 133.6, 135.6, 138.8, 147.2, 152.6, 156.7, 160.4, 190.2.
    • 2-(3-Chlorobenzylamino)-9-methyl-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carbaldehyde (137)
  • Figure US20150018543A1-20150115-C00155
  • 1H NMR (400 MHz, CDCl3) δ 2.40 (s, 3H), 4.80 (d, J=6.0 Hz, 2H), 6.87 (t, J=7.0 Hz, 1H), 7.24-7.26 (m, 3H), 7.37 (s, 1H), 7.59 (d, J=6.8 Hz, 1H), 8.79 (d, J=7.2 Hz, 1H), 10.34 (brs, 1H), 10.30 (s, 1H).
    • 9-Methyl-2-morpholino-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carbaldehyde (138)
  • Figure US20150018543A1-20150115-C00156
  • 1H NMR (400 MHz, CDCl3) δ 2.30 (s, 3H), 3.65 (d, J=2.4 Hz, 4H), 3.72 (d, J=3.2 Hz, 4H), 6.74-6.77 (m, 1H), 7.49 (d, J=6.8 Hz, 1H), 8.62 (d, J=7.2 Hz, 1H), 10.01 (s, 1H); 13C NMR (100 MHz, CDCl3) δ 17.6, 49.5, 67.0, 95.9, 112.9, 125.7, 133.0, 138.1, 150.5, 158.4, 162.3, 186.2
    • 2-(4-(2-Chlorophenyl)piperazin-1-yl)-9-methyl-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carbaldehyde (139)
  • Figure US20150018543A1-20150115-C00157
  • 1H NMR (400 MHz, CDCl3) δ 2.41 (s, 3H), 3.19 (t, J=4.8 Hz, 4H), 3.92 (t, J=4.6 Hz, 4H), 6.82 (t, J=7.0 Hz, 1H), 6.98 (t, J=7.6 Hz, 1H), 7.04 (d, J=7.2 Hz, 1H), 7.21 (t, J=7.6 Hz, 1H), 7.36 (d, J=7.6 Hz, 1H), 7.55 (d, J=6.4 Hz, 1H), 8.73 (d, J=6.8 Hz, 1H), 10.15 (s, 1H); 13C NMR (100 MHz, CDCl3) δ 17.6, 49.3, 51.4, 96.1, 112.7, 120.5, 124.0, 125.8, 127.6, 128.8, 130.6, 133.0, 137.8, 148.7, 150.5, 158.6, 162.5, 186.4.
    • 2-(3,4-Dihydroisoquinolin-2(1H)-yl)-9-methyl-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carbaldehyde (140)
  • Figure US20150018543A1-20150115-C00158
  • 1H NMR (400 MHz, CDCl3) δ 2.43 (s, 3H), 3.05 (t, J=5.8 Hz, 2H), 4.03 (t, J=5.8 Hz, 2H), 4.73 (s, 2H), 6.78 (t, J=7.0 Hz, 1H), 7.06-7.17 (m, 4H), 7.52 (d, J=6.8 Hz, 1H), 8.70 (d, J=7.6 Hz, 1H), 10.21 (s, 1H); 13C NMR (100 MHz, CDCl3) δ 17.6, 28.7, 46.3, 52.0, 96.1, 112.5, 125.8, 126.2, 126.6, 128.4, 133.0, 133.9, 134.6, 137.5, 150.3, 158.6, 162.3, 186.7.
    • 2-(Isobutylamino)-9-methyl-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carbaldehyde (141)
  • Figure US20150018543A1-20150115-C00159
  • 1H NMR (400 MHz, CDCl3) δ 0.95 (d, J=4 Hz, 6H), 1.90 (m, 1H), 2.37 (s, 3H), 3.41 (t, J=6.8 Hz, 2H), 6.76 (t, J=6.8 Hz, 1H), 7.24-7.52 (m, 1H), 8.69 (dd, J=0.8, 7.2 Hz, 1H), 9.67 (brs, 1H), 10.22 (s, 1H); 13C NMR (100 MHz, CDCl3) δ 17.9, 20.4, 28.7, 48.1, 94.4, 112.5, 125.9, 133.2, 138.1, 152.8, 159.5, 160.7, 190.2.
    • 2-(Diethylamino)-9-methyl-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carbaldehyde (142)
  • Figure US20150018543A1-20150115-C00160
  • 1H NMR (400 MHz, CDCl3) δ 1.25 (t, J=6.8 Hz, 6H), 2.36 (s, 3H), 3.65 (q, J=6.8 Hz, 4H), 6.72 (t, J=6.8 Hz, 1H), 7.47 (d, J=6.8 Hz, 1H), 8.65 (d, J=6.4 Hz, 1H), 10.12 (s, 1H); 13C NMR (100 MHz, CDCl3) δ 13.2, 17.7, 45.3, 96.2, 112.2, 125.8, 133.0, 137.3, 150.2, 158.5, 162.6, 186.9.
    • 2-(Cyclohexylmethylamino)-9-methyl-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carbaldehyde (143)
  • Figure US20150018543A1-20150115-C00161
  • 1H NMR (400 MHz, CDCl3) δ 0.93-1.02 (m, 2H), 1.11-1.25 (m, 3H), 1.57-1.77 (m, 6H), 2.36 (s, 3H), 3.43 (t, J=6.0 Hz, 2H), 6.75 (t, J=7.2 Hz, 1H), 7.50 (d, J=7.2 Hz, 1H), 8.67 (d, J=6.8 Hz, 1H), 9.65 (brs, 1H), 10.21 (s, 1H); 13C NMR (100 MHz, CDCl3) δ 17.9, 26.0, 26.5, 31.1, 38.2, 47.0, 94.4, 112.5, 125.8, 133.2, 138.0, 152.8, 159.4, 160.6, 190.2
    • 2-Chloro-9-methyl-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carboxylic acid (144)
  • Figure US20150018543A1-20150115-C00162
  • 1H NMR (400 MHz, DMSO-d6) δ 2.58 (s, 3H), 7.53 (t, J=7.0 Hz, 1H), 8.14 (d, J=7.2 Hz, 1H), 8.97 (d. J=6.8 Hz, 1H), 13.53 (brs, 1H); 13C NMR (100 MHz, DMSO-d) δ 16.7, 108.1, 117.1, 125.6, 133.3, 138.7, 148.2, 152.0, 154.6, 163.9.
    • 2-Chloro-7-methyl-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carboxylic acid (145)
  • Figure US20150018543A1-20150115-C00163
  • 1H NMR (400 MHz, DMSO-d6) δ 2.49 (s, 3H), 7.76 (d, J=8.8 Hz, 1H), 8.11 (d, J=8.8 Hz, 1H), 8.89 (s, 1H), 13.46 (br s, 1H).
    • 2-Chloro-6-methyl-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carboxylic acid (146)
  • Figure US20150018543A1-20150115-C00164
  • 1H NMR (400 MHz, DMSO-d6) δ 3.00 (s, 3H), 7.19 (d, J=7.6 Hz, 1H), 7.52 (d, J=8.0 Hz, 1H), 7.92 (t, J=8.0 Hz, 1H), 13.35 (br s, 1H).
    • 9-Methyl-4-oxo-2-(phenylamino)-4H-pyrido[1,2-a]pyrimidine-3-carboxylic acid (147)
  • Figure US20150018543A1-20150115-C00165
  • 1H NMR (400 MHz, CDCl3) δ 2.50 (s, 3H), 6.70 (dd, J=6.8, 7.2 Hz, 1H), 7.15 (dd, J=7.2, 7.2 Hz, 1H), 7.37 (dd, J=7.2, 7.6 Hz, 2H), 7.65 (d, J=6.8 Hz, 1H), 7.76 (d, J=8.4 Hz, 2H), 8.76 (d, J=7.2 Hz, 1H), 11.70 (brs, 1H), 14.31 (s, 1H).
    • 2-(3-Chlorophenylamino)-9-methyl-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carboxylic acid (148)
  • Figure US20150018543A1-20150115-C00166
  • 1H NMR (400 MHz, DMSO-d6) δ 2.55 (s, 3H), 7.04 (t, J=7.0 Hz, 1H), 7.12 (d, J=8.0 Hz, 1H), 7.28 (J=8.0 Hz, 1H), 7.71 (d, J=8.0 Hz, 1H), 8.17 (s, 1H), 8.79 (d, J=7.6 Hz, 1H), 11.78 (brs, 1H).
    • 2-(3-Chlorophenylamino)-8-methyl-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carboxylic acid (149)
  • Figure US20150018543A1-20150115-C00167
  • 1H NMR (400 MHz, CDCl3) δ 2.49 (s, 3H), 6.93 (d, J=7.6 Hz, 1H), 7.12 (d, J=7.6 Hz, 1H), 7.25-7.29 (m, 2H), 7.46 (d, J=7.2 Hz, 1H), 7.96 (s, 1H), 8.76 (d, J=7.2 Hz, 1H), 11.72 (br s, 1H), 14.19 (s, 1H).
    • 2-(3-Chlorophenylamino)-7-methyl-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carboxylic acid (150)
  • Figure US20150018543A1-20150115-C00168
  • 1H NMR (400 MHz, CDCl3) δ 2.41 (s, 3H), 7.12 (d, J=8.0 Hz, 1H), 7.27 (t, J=8.6 Hz, 1H), 7.41 (d, J=8.8 Hz, 1H), 7.47 (d, J=7.6 Hz, 1H), 7.96 (s, 1H), 8.68 (s, 1H), 11.70 (br s, 1H), 14.28 (s, 1H).
    • 2-(3-Chlorophenylamino)-6-methyl-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carboxylic acid (151)
  • Figure US20150018543A1-20150115-C00169
  • 1H NMR (400 MHz, CDCl3) δ 3.03 (s, 3H), 6.70 (d, J=6.8 Hz, 1H), 7.10 (d, J=8.0 Hz, 1H), 7.23-7.27 (m, 2H), 7.44 (d, J=8.0 Hz, 1H), 7.56 (t, J=8.0 Hz, 1H), 7.91 (s, 1H), 11.76 (br s, 1H), 14.37 (s, 1H).
    • 2-(3-Fluorophenylamino)-9-methyl-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carboxylic acid (152)
  • Figure US20150018543A1-20150115-C00170
  • 1H NMR (400 MHz, CDCl3) δ 2.54 (s, 3H), 6.81-6.87 (m, 1H), 7.03 (t, J=7.2 Hz, 1H), 7.28-7.31 (m, 2H), 7.71 (d, J=6.8 Hz, 1H), 7.89 (d, J=10.4 Hz, 1H), 8.79 (d, J=7.2 Hz 1H), 11.83 (b s, 1H), 14.26 (br s, 1H).
    • 9-Methyl-4-oxo-2-(3-(trifluoromethyl)phenylamino)-4H-pyrido[1,2-a]pyrimidine-3-carboxylic acid (153)
  • Figure US20150018543A1-20150115-C00171
  • 1H NMR (400 MHz, CDCl3) δ 2.54 (s, 3H), 7.05 (t, J=7.0 Hz, 1H), 7.40 (d, J=7.6 Hz, 1H), 7.47 (t, J=8.0 Hz, 1H), 7.61 (d, J=8.0 Hz, 1H), 7.73 (d, J=6.8 Hz, 1H), 8.58 (s 1H), 8.81 (d, J=6.8 Hz, 1H), 11.91 (br s, 1H).
    • 9-Methyl-4-oxo-2-(3-(trifluoromethoxy)phenylamino)-4,1-pyrido[1,2-a]pyrimidine-3-carboxylic acid (154)
  • Figure US20150018543A1-20150115-C00172
  • 1H NMR (400 MHz, CDCl3) δ 2.58 (s, 3H), 7.00 (d, J=8.0 Hz, 1H), 7.05 (t, J=7.0 Hz, 1H), 7.36 (t, J=8.0 Hz, 1H), 7.42 (d, J=8.0 Hz, 1H), 7.72 (d, J=6.8 Hz, 1H), 8.09 (s, 1H), 8.81 (d, J=7.2 Hz, 1H), 11.89 (br s, 1H), 14.26 (br s, 1H).
    • 9-Methyl-2-(3-nitrophenylamino)-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carboxylic acid (155)
  • Figure US20150018543A1-20150115-C00173
  • 1H NMR (400 MHz, DMSO-d6) δ 2.60 (s, 3H), 7.40 (t, J=7.0 Hz, 1H), 7.73 (t, J=8.2 Hz, 1H), 7.96 (d, J=7.6 Hz, 1H), 8.02 (d, J=7.6 Hz, 1H), 8.13 (d, J=6.8 Hz, 1H), 8.90 (d, J=7.2 Hz, 1H), 9.33 (s, 1H), 11.84 (br s, 1H), 14.43 (br s, 1H).
    • 2-(3-(Methoxycarbonyl)phenylamino)-9-methyl-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carboxylic acid (156)
  • Figure US20150018543A1-20150115-C00174
  • 1H NMR (400 MHz, CDCl3) δ 2.57 (s, 3H), 3.92 (s, 3H), 7.052 (t, J=6.8 Hz, 1H), 7.43 (t, J=8.0 Hz, 1H), 7.71 (t, J=7.0 Hz, 2H), 7.82 (d, J=8.0 Hz, 1H), 8.79 (d, J=6.8 Hz, 1H), 8.83 (s, 1H), 11.83 (br s, 1H), 14.28 (br s, 1H).
    • 2-(3-Hydroxyphenylamino)-9-methyl-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carboxylic acid
  • Figure US20150018543A1-20150115-C00175
  • 1H NMR (400 MHz, CD3OD) δ 2.55 (s, 3H), 6.61 (d, J=8.0 Hz, 1H), 7.15-7.24 (m, 3H), 7.34 (s, 1H), 7.88 (d, J=6.8 Hz, 1H), 8.82 (d, J=7.2 Hz, 1H).
    • 2-(4-Hydroxyphenylamino)-9-methyl-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carboxylic acid (158)
  • Figure US20150018543A1-20150115-C00176
  • 1H NMR (400 MHz, CD3OD) δ 2.45 (s, 3H), 6.81 (d, J=8.8 Hz, 2H), 7.10 (t, J=7.0 Hz, 1H), 7.57 (d, J=8.8 Hz, 1H), 7.81 (d, J=6.8 Hz, 1H), 8.78 (d, J=7.2 Hz, 1H), 11.26 (br s, 1H).
    • 2-(4-tert-Butylphenylamino)-9-methyl-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carboxylic acid (159)
  • Figure US20150018543A1-20150115-C00177
  • 1H NMR (400 MHz, CDCl3) δ 1.33 (s, 9H), 2.49 (s, 3H), 6.95 (t, J=7.0 Hz, 1H), 7.37 (d, J=7.2 Hz, 2H), 7.63 (d, J=5.6 Hz, 1H), 7.69 (d, J=6.8 Hz, 2H), 8.71 (d, J=6.8 Hz, 1H), 11.64 (br s, 1H) 14.31 (br s, 1H); 13C NMR (100 MHz, CDCl3) δ 18.2, 31.3, 34.4, 85.3, 114.1, 121.3, 125.5, 125.7, 133.6, 135.4, 138.2, 147.4, 150.2, 157.0, 161.8, 169.7.
    • 2-(3-Chlorobenzylamino)-9-methyl-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carboxylic acid (160)
  • Figure US20150018543A1-20150115-C00178
  • 1H NMR (400 MHz, CDCl3) δ 2.38 (s, 3H), 4.83 (d, J=6.0 Hz, 2H), 7.17 (t, 0.1=7.0 Hz, 1H), 7.32-7.40 (m, 3H), 7.50 (s, 1H), 7.89 (d, J=6.8 Hz, 1H), 8.68 (d, J=7.2 Hz, 1H), 9.82 (d, J=6.2 Hz, 1H), 14.25 (br s, 1H).
    • 2-(Diethylamino)-9-methyl-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carboxylic acid (161)
  • Figure US20150018543A1-20150115-C00179
  • 1H NMR (400 MHz, CDCl3) δ 1.32 (t, J=6.8 Hz, 6H), 2.41 (s, 3H), 3.68 (q, J=6.8 Hz, 4H), 6.67 (t, J=7.2 Hz, 1H), 7.38 (d, J=6.8 Hz, 1H), 8.71 (d, J=7.2 Hz, 1H), 14.08 (s, 1H); 13C NMR (100 MHz, CDCl3) δ 13.8, 17.8, 45.4, 96.2, 112.2, 125.8, 133.0, 137.3, 150.2, 158.5, 162.6, 171.6.
    • 2-(Isobutylamino)-9-methyl-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carboxylic acid (162)
  • Figure US20150018543A1-20150115-C00180
  • 1H NMR (400 MHz, CDCl3) δ 0.97 (d, J=6.8 Hz, 6H), 1.93-1.99 (m, 1H), 2.40 (s, 3H), 3.43 (t, J=6.4 Hz, 2H), 6.84 (t, J=7.2 Hz, 1H), 7.53 (d, J=6.4 Hz, 1H), 8.62 (d, J=7.6 Hz, 1H), 9.52 (brs, 1H), 14.12 (s, 1H); 13C NMR (100 MHz, CDCl3) δ 17.9, 20.4, 28.7, 48.6, 84.8, 113.2, 125.7, 133.2, 137.5, 150.5, 159.7, 162.0, 169.9.
    • 2-(Cyclohexylmethylamino)-9-methyl-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carboxylic acid (163)
  • Figure US20150018543A1-20150115-C00181
  • 1H NMR (400 MHz, CDCl3) δ 0.98-1.05 (m, 2H), 1.13-1.24 (m, 3H), 1.60-1.79 (m, 6H), 2.42 (s, 3H), 3.45 (t, J=6.4 Hz, 2H), 6.83 (t, J=7.2 Hz, 1H), 7.54 (d, J=6.8 Hz, 1H), 8.62 (d, J=7.2 Hz, 1H), 9.57 (brs, 1H), 14.13 (s, 1H); 13C NMR (100 MHz, CDCl3) δ 18.0, 26.0, 26.2, 31.2, 38.2, 47.4, 84.8, 113.2, 125.7, 133.2, 137.5, 150.5, 159.6, 162.0, 170.0.
    • 2-(Cyclohexylamino)-9-methyl-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carboxylic acid (164)
  • Figure US20150018543A1-20150115-C00182
  • 1H NMR (400 MHz, CDCl3) δ 1.19-1.42 (m, 5H), 1.56-1.60 (m, 2H), 1.70-1.76 (m, 2H), 1.94-1.98 (m, 2H), 2.38 (s, 3H), 6.79 (t, J=6.8 Hz, 1H), 7.51 (d, J=6.8 Hz, 1H), 8.56 (d, J=6.8 Hz, 1H), 9.42 (d, J=6.8 Hz, 1H), 14.14 (s, 1H); 13C NMR (100 MHz, CDCl3) δ 17.8, 24.7, 25.7, 32.6, 50.0, 84.7, 113.1, 125.6, 133.1, 137.4, 150.5, 158.5, 162.0, 169.9.
    • 2-(Cyclopentylamino)-9-methyl-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carboxylic acid (165)
  • Figure US20150018543A1-20150115-C00183
  • 1H NMR (400 MHz, CDCl3) δ 1.54-1.67 (m, 4H), 1.73-1.78 (m, 2H), 2.04-2.10 (m, 2H), 2.42 (s, 3H), 4.51 (q, J=6.8 Hz, 1H), 6.83 (t, J=6.8 Hz, 1H), 7.53 (d, J=6.8 Hz, 1H), 8.59 (d, J=6.8 Hz, 1H), 9.47 (d, J=6.8 Hz, 1H), 14.15 (s, 1H); 13C NMR (100 MHz, CDCl3) δ 18.0, 24.1, 33.3, 53.0, 84.8, 113.3, 125.7, 133.3, 137.5, 150.5, 158.9, 162.0, 169.9.
    • 2-(Cycloheptylamino)-9-methyl-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carboxylic acid (166)
  • Figure US20150018543A1-20150115-C00184
  • 1H NMR (400 MHz, CDCl3) δ 1.23-1.57 (m, 4H), 1.59-1.68 (m, 4H), 1.69-1.74 (m, 2H), 1.98-2.04 (m, 2H), 2.43 (s, 3H), 4.30-4.36 (m, 1H), 6.83 (t, J=6.8 Hz, 1H), 7.53 (d, J=6.8 Hz, 1H), 8.64 (d, J=6.8 Hz, 1H), 9.53 (d, J=6.8 Hz, 1H), 14.19 (s, 1H); 13C NMR (100 MHz, CDCl3) δ 18.0, 24.6, 28.1, 34.7, 52.3, 84.8, 113.1, 125.8, 133.2, 137.4, 150.4, 158.3, 162.1, 170.0.
    • 2-(1-(tert-Butoxycarbonyl)piperidin-4-ylamino)-9-methyl-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carboxylic acid (167)
  • Figure US20150018543A1-20150115-C00185
  • 1H NMR (400 MHz, CDCl3) δ 1.51 (s, 9H), 1.61-1.65 (m, 2H), 2.01-2.03 (m, 2H), 2.42 (s, 3H), 2.99-3.05 (m, 2H), 3.98-4.00 (m, 2H), 4.26-4.33 (m, 1H), 6.88 (t, J=7.2 Hz, 1H), 7.58 (d, J=6.8 Hz, 1H), 8.67 (d, J=7.2 Hz, 1H), 9.56 (d, J=6.8 Hz), 14.12 (s, 1H); 13C NMR (100 MHz, CDCl3) δ 17.9, 28.6, 31.6, 48.5, 66.4, 79.9, 85.0, 113.5, 125.9, 133.2, 137.8, 150.6, 154.9, 158.9, 162.0, 169.9.
    • 2-(2-(4-Fluorophenoxy)ethylamino)-9-methyl-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carboxylic acid (168)
  • Figure US20150018543A1-20150115-C00186
  • 1H NMR (400 MHz, CDCl3) δ 2.44 (s, 3H), 4.01 (t, J=5.6 Hz, 2H), 4.15 (t, J=5.6 Hz, 2H), 6.83-6.96 (m, 5H), 7.59 (d, J=6.8 Hz, 1H), 8.68 (d, J=7.2 Hz, 1H), 9.81 (brs, 1H), 14.01 (s, 1H); 13C NMR (100 MHz, CDCl3) δ 18.0, 40.5, 67.1, 85.3, 113.6, 115.8, 115.9, 116.0, 116.1, 125.9, 133.2, 137.9, 150.6, 154.8, 159.8, 161.9, 169.7.
    • 9-Methyl-4-oxo-2-(2-(4-(trifluoromethoxy)phenoxy)ethylamino)-4H-pyrido[1,2-a]pyrimidine-3-carboxylic acid (169)
  • Figure US20150018543A1-20150115-C00187
  • 1H NMR (400 MHz, CDCl3) δ 2.44 (s, 3H), 4.03 (t, J=5.6 Hz, 2H), 4.18 (t, J=5.6 Hz, 2H), 6.90 (d, J=9.2 Hz, 2H), 6.91 (t, J=6.8 Hz, 1H), 7.11 (d, J=9.2 Hz, 2H), 7.60 (d, J=6.8 Hz, 1H), 9.70 (d, J=7.2 Hz, 1H), 9.82 (brs, 1H), 14.08 (s, 1H); 13C NMR (100 MHz, CDCl3) δ 18.0, 40.5, 66.9, 77.4, 85.4, 113.7, 115.7, 122.6, 126.0, 133.2, 138.0, 155.8, 157.6, 159.9, 162.0, 169.0, 170.4.
    • 9-Methyl-2-morpholino-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carboxylic acid (170)
  • Figure US20150018543A1-20150115-C00188
  • 1H NMR (400 MHz, CDCl3) δ 2.42 (s, 3H), 3.65 (t, J=4.8 Hz, 4H), 3.74 (t, J=4.8 Hz, 4H), 6.86 (t, J=6.8 Hz, 1H), 7.51 (d, J=6.8 Hz, 1H), 8.67 (d, J=6.8 Hz, 1H), 13.98 (s, 1H); 13C NMR (100 MHz, CDCl3) δ 18.1, 58.4, 64.8, 97.5, 113.6, 124.6, 132.6, 136.0, 148.1, 160.5, 161.7, 171.3.
    • 2-(3,4-Dihydroisoquinolin-2(1H)-yl)-9-methyl-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carboxylic acid (171)
  • Figure US20150018543A1-20150115-C00189
  • 1H NMR (400 MHz, CDCl3) δ 2.45 (s, 3H), 3.03 (t, J=5.8 Hz, 2H), 4.08 (m, 2H), 4.73 (m, 2H), 6.83 (t, J=7.0 Hz, 1H), 7.06-7.18 (m, 4H), 7.52 (d, J=6.8 Hz, 1H), 8.60 (d, J=7.2 Hz, 1H), 13.73 (br s, 1H); 13C NMR (100 MHz, CDCl3) δ 17.6, 28.5, 46.1, 52.4, 86.4, 113.0, 125.5, 126.1, 126.2, 126.6, 128.4, 132.9, 133.7, 134.4, 136.8, 148.1, 159.9, 163.2, 165.3.
    • 2-(4-(2-Chlorophenyl)piperazin-1-yl)-9-methyl-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carboxylic acid (172)
  • Figure US20150018543A1-20150115-C00190
  • 1H NMR (400 MHz, CDCl3) δ 2.44 (s, 3H), 3.19 (t, J=4.8 Hz, 4H), 3.96 (m, 4H), 6.87 (t, J=7.0 Hz, 1H), 6.98 (t, J=7.6 Hz, 1H), 7.02 (d, J=8.4 Hz, 1H), 7.20 (t, J=7.8 Hz, 1H), 7.36 (d, J=8.0 Hz, 1H), 7.55 (d, J=6.8 Hz, 1H), 8.66 (d, J=7.2 Hz, 1H), 13.74 (br s, 1H).
    • 2-(3-Chlorophenylamino)-8-(4-methylpiperazin-1-yl)-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carboxylic acid (173)
  • Figure US20150018543A1-20150115-C00191
  • 1H NMR (400 MHz, CDCl3) δ2.34 (s, 3H), 2.53 (t, J=4.8 Hz, 4H), 3.54 (t, J=4.8 Hz, 4H), 6.34 (d, J=2.8 Hz, 1H), 6.55 (dd, J=2.8, 8.4 Hz, 1H), 7.04 (d, J=7.2 Hz, 1H), 7.22 (t, J=8.0 Hz, 1H), 7.49 (dd, J=1.6, 8.0 Hz, 1H), 7.86 (t, J=2.0 Hz, 1H), 8.53 (d, J=8.4 Hz, 1H), 11.5 (s, 1H), 14.18 (s, 1H); 13C NMR (100 MHz, CDCl3) δ 46.1, 46.4, 54.4, 83.6, 98.8, 105.1, 120.0, 121.9, 124.0, 128.8, 129.9, 134.4, 139.9, 151.4, 155.6, 158.2, 161.8, 170.2.
  • Figure US20150018543A1-20150115-C00192
    Figure US20150018543A1-20150115-C00193
    • General Procedure for the Synthesis of H2
  • 2-Amino-3-picoline (1.0 mmol) was dissolved in diethyl ethoxymethylenemalonate (1.0 mmol). The solution was heated to 170° C. for 12 h. After cooling, the dark residue was triturated with EtOAc (10 mL). The residual pale solid was collected by filtration and washed with EtOAc to give II.
    • General Procedure for the Synthesis of H2
  • To a stirred solution of H1 (0.43 mmol) in H2O (3.0 mL) and EtOH (1.0 mL) was added LiOH (0.86 mmol). The mixture was stirred at room temperature for 3 h. The reaction mixture was diluted with CH2Cl2 (10 mL) and washed with 1 N HCl (10 ml). The organic layer was dried over anhydrous MgSO4 and concentrated in vacuo. The crude product was purified by flash column chromatography to give H2.
    • General Procedure for the Synthesis of H3
  • To a stirred solution of H1 (0.38 mmol) in THF (2.0 mL) was added LiAlH4 (0.57 mmol) at 0° C. The reaction mixture was stirred at 0° C. for 3 h. After reaction was completed, 1N NaOH (2 mL) was added dropwise. The mixture was diluted with CH2Cl2 (10 mL) and washed with H2O (10 ml). The organic layer was dried over anhydrous MgSO4 and concentrated in vacuo. The crude product was purified by flash column chromatography to give H3.
    • General Procedure for the Synthesis of H4
  • To a stirred solution of H3 (95 μmol) in CH2Cl2 (1.0 mL) was added NaHCO3 (285 μmol) and Dess-Martin Periodinane (114 μmol) at 0° C. The mixture was stirred at 0° C. for 1 h. The reaction mixture was filtered off and concentrated in vacuo. The crude product was purified by flash column chromatography to give H4.
    • General Procedure for the Synthesis of H5
  • To a stirred solution of 2-Amino-pyridine (10.6 mmol) in xylene (10.0 mL) was added diethyl ethoxymethylenemalonate (21.2 mmol). The mixture was stirred at 140 for 3 hr. After reaction was completed, the residual pale solid was collected by filtration and washed with diethyl ether to give H5.
    • General Procedure for the Synthesis of H6
  • To a stirred solution of H5 (0.42 mmol) in THF (5.0 mL) was added triethylamine (0.63 mmol) and p-toluenesulfonylchloride (0.46 mmol) at 0° C. The reaction mixture was stirred at room temperature for overnight. After reaction was completed, the mixture was diluted with CH2Cl2 (40 mL) and washed with 1N HCl (50 ml), saturated NaHCO3 (50 ml) and brine (50 ml). The organic layer was dried over anhydrous MgSO4 and concentrated in vacuo. The crude product was purified by flash column chromatography to give H6.
    • General Procedure for the Synthesis of 117
  • To a stirred solution of H6 (0.25 mmol) in THF (1.2 mL) was added triethylamine (0.5 mmol) and an amine (0.26 mmol) at 0° C. The reaction mixture was stirred at room temperature for overnight. After reaction was completed, the mixture was diluted with CH2Cl2 (10 mL) and washed with 1N HCl (10 ml), saturated NaHCO3 (10 ml) and brine (10 ml). The organic layer was dried over anhydrous MgSO4 and concentrated in vacuo. The crude product was purified by flash column chromatography to give 117.
    • General Procedure for the Synthesis of H8
  • To a stirred solution of H7 (0.27 mmol) in ethylene glycol (3.0 mL) was added methylamine (2 N solution in THF 1.3 mL). The mixture was stirred at 150° C. for 3 hr. The reaction mixture was added with ethylacetate (10 mL) and the residual pale solid was collected by filtration and washed with EtOAc. The crude product was purified by flash column chromatography to give H8.
    • General Procedure for the Synthesis of H9
  • To a stirred solution of H5 (2.13 mmol) in MeOH (8.0 mL) was added Pd/C (113 mg). The mixture was stirred at room temperature under H2 for 3 h. After reaction was completed, the reaction mixture was filtered off and concentrated in vacuo. The crude product was recrystallized with EtOAc and hexane (1:4) to give H9.
    • General Procedure for the Synthesis of H10
  • To a stirred solution of H9 (0.42 mmol) in CH2Cl2 (5.0 mL) was added triethylamine (0.63 mmol) and p-toluenesulfonylchloride (0.46 mmol) at 0° C. The reaction mixture was stirred at room temperature for overnight. After reaction was completed, the mixture was diluted with CH2Cl2 (40 mL) and washed with 1N HCl (50 ml), saturated NaHCO3 (50 ml) and brine (50 ml). The organic layer was dried over anhydrous MgSO4 and concentrated in vacuo. The crude product was purified by flash column chromatography (Hexane:EtOAc=1:2) to give H10.
    • General Procedure for the Synthesis of H11
  • To a stirred solution of H10 (0.25 mmol) in THF (2.0 mL) was added triethylamine (0.5 mmol) and an amine (0.37 mmol) at 0° C. The reaction mixture was stirred at room temperature for overnight. After reaction was completed, the mixture was diluted with CH2Cl2 (10 mL) and washed with 1N HCl (10 ml), saturated NaHCO3 (10 ml) and brine (10 ml). The organic layer was dried over anhydrous MgSO4 and concentrated in vacuo. The crude product was purified by flash column chromatography (Hexane:EtOAc=1:1) to give H11.
    • General Procedure for the Synthesis of H12
  • A solution of G3 (1.0 mmol), an amine (1.1 mmol) and triethylamine (2.0 mmol) in THF (2 mL) was refluxed for 1 h and cooled to room temperature. The solvent was evaporated to dryness, which was extracted with CH2Cl2 (20 mL×3). The reaction mixture was washed with 5% sodium bicarbonate. The organic layer was dried (MgSO4), filtered, and concentrated in vacuo. The crude product was purified by flash column chromatography to give H12.
    • General Procedure for the Synthesis of H13
  • To a solution of G3 (1.1 mmol), an amine (1.0 mmol) in CH2Cl2 (5 mL) were added sodium triacetoxyborohydride (2.0 mmol) and glacial acetic acid (2.0 mmol) at room temperature for 20 h. The reaction mixture was added saturated ammonium chloride solution and stirred for 10 min. The reaction mixture was extracted with CH2Cl2 (20 mL). The organic layer was dried (MgSO4), filtered, and concentrated in vacuo. The crude product was purified by flash column chromatography to give H13.
    • Ethyl 9-methyl-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carboxylate (174)
  • Figure US20150018543A1-20150115-C00194
  • 1H NMR (400 MHz, CDCl3) δ 1.39 (t, J=7.2 Hz, 3H), 2.62 (s, 3H), 4.39 (q, J=7.2 Hz, 2H), 7.20 (t, J=7.2 Hz, 1H), 7.77 (d, J=7.2 Hz, 1H), 9.05 (s, 1H), 9.16 (d, J=7.2 Hz, 1H); 13C NMR (100 MHz, CDCl3) 14.6, 18.2, 61.2, 105.3, 116.8, 127.0, 135.9, 138.2, 155.3, 158.4, 165.0, 189.1.
    • 9-Methyl-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carboxylic acid (175)
  • Figure US20150018543A1-20150115-C00195
  • 1H NMR (400 MHz, CDCl3) δ 2.56 (s, 3H), 7.12 (t, J=6.8 Hz, 1H), 7.79 (d, J=6.8 Hz, 1H), 8.87 (s, 1H), 9.21 (d, J=7.2 Hz), 14.13 (s, 1H); 13C NMR (100 MHz, CDCl3) δ18.3, 110.9, 117.1, 128.1, 137.6, 141.1, 155.0, 157.1, 158.3, 171.3.
    • 3-(Hydroxymethyl)-9-methyl-4H-pyrido[1,2-a]pyrimidin-4-one (176)
  • Figure US20150018543A1-20150115-C00196
  • 1H NMR (400 MHz, CDCl3) δ 2.51 (s, 3H), 3.27 (brs, 1H), 4.66 (s, 2H), 7.01 (t, J=6.8 Hz, 1H), 7.51 (d, J=6.8 Hz, 1H), 8.32 (s, 1H), 8.87 (s, 1H); 13C NMR (100 MHz, CDCl3) 18.2, 44.1, 111.2, 117.9, 127.1, 135.7, 139.8, 153.9, 155.6, 158.2.
    • 9-Methyl-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carbaldehyde (177)
  • Figure US20150018543A1-20150115-C00197
  • 1H NMR (400 MHz, CDCl3) δ 2.63 (s, 3H), 7.29 (t, J=7.2 Hz, 1H), 7.86 (d, J=7.2 Hz, 1H), 8.85 (s, 1H), 9.14 (d, J=7.2 Hz, 1H), 10.33 (s, 1H); 13C NMR (100 MHz, CDCl3) δ 18.2, 110.9, 117.5, 126.7, 136.5, 139.5, 153.1, 155.6, 158.1, 188.5.
    • Ethyl 2-hydroxy-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carboxylate (178)
  • Figure US20150018543A1-20150115-C00198
  • 1H NMR (400 MHz, CDCl3) δ 1.42 (t, J=7.2 Hz 3H), 4.45 (q, J=7.2 Hz, 2H), 7.13 (ddd, J=1.2, 6.8, 7.2 Hz, 1H), 7.49 (d, J=8.8 Hz, 1H), 7.82-7.86 (m, 1H), 9.00 (d, J=7.2 Hz, 1H), 13.64 (brs, 1H, NH); 13C NMR (100 MHz, CDCl3) δ 14.2, 62.3, 87.1, 115.3, 125.1, 128.7, 140.3, 148.4, 152.6, 155.5, 171.7.
    • 2-Hydroxy-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carboxylic acid (179)
  • Figure US20150018543A1-20150115-C00199
  • 1H NMR (400 MHz, CDCl3) δ 2.50 (s, 3H), 6.70 (dd, J=6.8, 7.2 Hz, 1H), 7.15 (dd, J=7.2, 7.2 Hz, 1H), 7.37, (dd, J=7.2, 7.6 Hz, 1H), 7.65 (d, J=6.8 Hz, 1H), 7.76 (d, J=8.4 Hz, 1H), 8.76 (d, J=7.2 Hz, 1H), 11.70 (brs, 1H), 14.31 (s, 1H).
    • Ethyl 4-oxo-2-(phenylamino)-4H-pyrido[1,2-a]pyrimidine-3-carboxylate (180)
  • Figure US20150018543A1-20150115-C00200
  • 1H NMR (400 MHz, CDCl3) δ 1.45 (t, J=7.2 Hz, 3H), 4.44 (q, J=7.2 Hz, 2H), 6.93 (dd, J=6.8, 6.8 Hz, 1H), 7.29-7.36 (m, 3H), 7.65-7.68 (m, 3H), 8.97 (d, J=7.2 Hz, 1H), 11.39 (brs, 1H); 13C NMR (100 MHz, CDCl3) δ 14.4, 61.0, 85.5, 113.6, 122.5, 124.2, 124.5, 128.4, 128.6, 138.4, 139.0, 151.6, 155.9, 159.5, 169.6.
    • Ethyl 2-(3-hydroxyphenylamino)-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carboxylate (181)
  • Figure US20150018543A1-20150115-C00201
  • 1H NMR (400 MHz, CDCl3+CD3OD) δ 1.38 (t, J=7.0 Hz, 3H), 4.37 (q, J=7.2 Hz, 2H), 6.56-6.58 (m, 1H), 6.92 (dd, J=6.8, 7.2 Hz, 1H0, 7.05 (d, J=8.4 Hz, 1h0, 7.12 (dd, J=8.0, 8.0 Hz, 1H), 7.26 (m, 1H), 7.31 (d, J=8.8 Hz, 1H), 7.66 (dd, J=7.2, 7.6 Hz, 1H), 8.90 (d, J=7.2 Hz, 1H), 11.22 (brs, 1H).
    • Ethyl 2-(2-hydroxyphenylamino)-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carboxylate (182)
  • Figure US20150018543A1-20150115-C00202
  • 1H NMR (400 MHz, CDCl3) δ 1.45 (t, J=7.2 Hz, 3H), 4.45 (q, J=6.8 Hz, 2H), 6.90 (dd, J=7.2, 8.0 Hz, 1H), 7.05-7.08 (m, 2H), 7.13 (dd, J=7.6, 8.4 Hz, 2H), 7.37 (d, J=8.4 Hz, 1H), 7.81 (dd, J=7.6, 8.0 Hz, 1H), 9.03 (d, J=6.8 Hz, 1H), 11.52 (brs, 1H); 13C NMR (100 MHz, CDCl3) 14.4, 61.3, 114.7, 120.1, 120.5, 122.9, 124.4, 127.0, 127.1, 129.0, 140.8, 149.3, 151.1, 158.6, 169.5.
    • Ethyl 2-(3-nitrophenylamino)-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carboxylate (183)
  • Figure US20150018543A1-20150115-C00203
  • 1H NMR (400 MHz, CDCl3) δ 1.46 (t, J=6.4 Hz, 3H), 4.45 (q, J=7.2 Hz, 2H), 7.05 (ddd, J=1.2, 6.8, 6.8 Hz, 1H), 7.43 (d, J=8.8 Hz, 1H), 7.47 (dd, J=8.0, 8.4 Hz, 2H), 7.77-7.82 (m, 2H), 7.93-7.96 (m, 1H), 8.97-8.98 (m, 1H), 9.04 (dd, J=0.8, 7.2 Hz, 1H), 11.74 (brs, 1H); 13C NMR (100 MHz, CDCl3) 14.4, 61.3, 86.1, 114.5, 116.9, 118.4, 124.7, 127.4, 128.6, 129.2, 139.8, 148.5, 151.5, 155.7, 159.5, 169.6.
    • Ethyl 4-oxo-2-phenoxy-4H-pyrido[1,2-a]pyrimidine-3-carboxylate (184)
  • Figure US20150018543A1-20150115-C00204
  • 1H NMR (400 MHz, CDCl3) δ 1.38 (t, J=7.2 Hz, 3H), 4.42 (q, J=7.2 Hz, 2H), 7.15-7.17 (m, 3H), 7.24 (d, J=6.4 Hz, 1H), 7.36-7.41 (m, 3H), 7.77 (ddd, J=1.6, 6.8, 6.8 Hz, 1H), 9.10 (dd, J=0.8, 6.8 Hz, 1H); ); 13C NMR (100 MHz, CDCl3) δ 14.2, 61.3, 115.7, 121.8, 125.3, 128.5, 129.2, 128.7, 150.3, 152.5, 156.7, 164.1, 165.0.
    • Ethyl 2-(3-fluorophenoxy)-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carboxylate (185)
  • Figure US20150018543A1-20150115-C00205
  • 1H NMR (400 MHz, CDCl3) δ 1.37 (t, J=7.0 Hz, 3H), 4.40 (q, J=6.8 Hz, 2H), 6.91-6.98 m, 3H), 7.19 (ddd, J=1.2, 7.2, 7.2 Hz, 1H), 7.32-7.36 (m, 1H), 7.39 (d, J=8.8 Hz, 1H), 7.78-7.82 (m, 1H), 9.10 (d, J=6.8 Hz, 1H); 13C NMR (100 MHz, CDCl3) δ 14.2, 61.4, 94.6, 109.8, 110.0, 112.2, 112.4, 115.9, 117.5, 117.6, 125.3, 128.5, 129.8, 129.9, 139.9, 150.3, 153.3, 156.6, 161.6, 163.8, 164.0, 164.5.
    • Ethyl 4-oxo-2-(3-(trifluoromethyl)phenoxy)-4H-pyrido[1,2-a]pyrimidine-3-carboxylate (186)
  • Figure US20150018543A1-20150115-C00206
  • 1H NMR (400 MHz, CDCl3) δ 1.39 (t, J=7.2 Hz, 3H), 4.43 (q, J=7.0 Hz 2H), 7.21 (dd, J=6.8, 6.8 Hz, 1H), 7.38 (d, J=8.0 Hz, 2H), 7.47-7.52 (m, 2H), 7.81 (dd, J=7.2, 8.4 Hz, 1H), 9.12 (d, J=6.8 Hz, 1H).
    • Methyl 2-chloro-9-methyl-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carboxylate (187)
  • Figure US20150018543A1-20150115-C00207
  • 1H NMR (400 MHz, CDCl3) δ 2.56 (s, 3H), 3.93 (s, 3H), 7.19 (t, J=7.2 Hz, 1H), 7.75 (d, J=6.8 Hz, 1H), 8.91 (d, J=7.2 Hz, 1H); 13C NMR (100 MHz, CDCl3) δ 17.1, 52.8, 108.0, 116.7, 126.1, 134.9, 138:3, 149.1, 155.1, 155.2, 164.2.
    • Methyl 2-(3-chlorophenylamino)-9-methyl-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carboxylate
  • Figure US20150018543A1-20150115-C00208
  • 1H NMR (400 MHz, CDCl3) δ 2.51 (s, 3H), 3.99 (s, 3H), 6.94 (t, J=7.0 Hz, 1H), 7.09 (d, J=7.6 Hz, 1H), 7.27 (d, J=8.4 Hz, 1H), 7.41 (d, J=8.0 Hz, 1H), 7.64 (d, J=6.8 Hz, 1H), 8.18 (s, 1H), 8.91 (d, J=7.2 Hz, 1H), 11.52 (br s, 1H); 13C NMR (100 MHz, CDCl3) δ 18.0, 52.1, 85.3, 113.7, 119.6, 121.9, 123.5, 126.4, 129.4, 133.2, 134.1, 138.4, 139.9, 151.0, 156.2, 158.6, 170.1.
    • Methyl 2-(3-chlorobenzylamino)-9-methyl-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carboxylate (189)
  • Figure US20150018543A1-20150115-C00209
  • 1H NMR (400 MHz, CDCl3) δ 2.35 (s, 3H), 3.92 (s, 3H), 4.77 (d, J=6.0 Hz, 2H), 6.80 (t, J=6.8 Hz, 1H), 7.20-7.24 (m, 3H), 7.34 (s, 3H), 7.50 (d, J=6.8 Hz, 1H), 8.82 (d, J=7.2 Hz, 1H), 9.69 (br s, 1H); 13C NMR (100 MHz, CDCl3) δ 17.8, 44.4, 51.8, 84.6, 112.6, 125.5, 126.4, 127.2, 127.7, 129.7, 132.7, 134.3, 137.6, 141.1, 151.3, 156.4, 160.8, 170.1.
    • Ethyl 2-hydroxy-4-oxo-6,7,8,9-tetrahydro-4H-pyrido[1,2-a]pyrimidine-3-carboxylate (190)
  • Figure US20150018543A1-20150115-C00210
  • 1H NMR (400 MHz, CDCl3) δ 1.36 (t, J=7.2 Hz, 3H), 1.82-1.93 (m, 4H), 2.86 (t, J=6.8 Hz, 2H), 3.84 (t, J=6.0 Hz, 2H), 4.39 (q, J=7.2 Hz, 2H); 13C NMR (100 MHz, CDCl3) δ 14.4, 18.9, 21.9, 32.2, 43.0, 62.4, 90.9, 159.8, 165.1, 171.7, 173.5.
    • Ethyl 4-oxo-2-(tosyloxy)-6,7,8,9-tetrahydro-4H-pyrido[1,2-a]pyrimidine-3-carboxylate (191)
  • Figure US20150018543A1-20150115-C00211
  • 1H NMR (400 MHz, CDCl3) δ 1.25 (t, J=7.2 Hz, 3H), 1.79-1.91 (m, 4H), 2.41 (s, 3H), 2.79 (t, J=6.4 Hz, 2H), 3.84 (t, J=6.4 Hz, 2H), 4.25 (q, J=7.2 Hz, 2H), 7.31 (d, J=8.0 Hz, 2H), 7.89 (d, J=8.0 Hz, 2H); 13C NMR (100 MHz, CDCl3) δ 14.2, 18.8, 21.6, 21.9, 31.8, 43.6, 61.9, 104.2, 129.1, 129.7, 134.2, 145.8, 159.4, 160.8, 162.0, 162.2.
    • Ethyl 4-oxo-2-(phenylamino)-6,7,8,9-tetrahydro-4H-pyrido[1,2-a]pyrimidine-3-carboxylate (192)
  • Figure US20150018543A1-20150115-C00212
  • 1H NMR (400 MHz, CDCl3) δ 1.40 (t, J=7.2 Hz, 3H), 1.80-1.92 (m, 4H), 2.80 (t, J=6.8 Hz, 2H), 3.87 (t, J=6.0 Hz, 2H), 4.36 (q, J=7.2 Hz, 2H), 7.08 (t, J=7.2 Hz, 1H), 7.29 (t, J=7.2 Hz, 2H), 7.53 (d, J=7.6 Hz, 2H), 11.2 (s, 1H); 13C NMR (100 MHz, CDCl3) δ 14.6, 19.2, 22.2, 32.2, 42.4, 61.0, 88.4, 122.9, 124.4, 128.8, 138.4, 160.5, 160.8, 162.2, 169.8.
    • Ethyl 2-(3-chlorophenylamino)-4-oxo-6,7,8,9-tetrahydro-4H-pyrido[1,2-a]pyrimidine-3-carboxylate (193)
  • Figure US20150018543A1-20150115-C00213
  • 1H NMR (400 MHz, CDCl3) δ 1.32 (t, J=7.2 Hz, 3H), 1.76-1.88 (m, 4H), 2.76 (t, J=6.8 Hz, 2H), 3.78 (t, J=6.0 Hz, 2H), 4.29 (q, J=7.06 (dd, J=7.2 Hz, 2H), J=1.2, 8.0 Hz, 1H), 7.27 (t, J=8.0 Hz, 1H), 7.51 (dd, J=1.2, 8.0 Hz, 1H), 7.58 (d, J=2.0 Hz, 1H); 13C NMR (100 MHz, CDCl3) δ 14.3, 18.6, 22.1, 32.1, 42.6, 61.1, 81.4, 111.2, 111.7, 113.0, 128.4, 140.4, 149.6, 158.7, 161.12, 163.2, 170.4.
    • Ethyl 4-oxo-2-(3-(trifluoromethyl)phenylamino)-6,7,8,9-tetrahydro-4H-pyrido[1,2-a]pyrimidine-3-carboxylate (194)
  • Figure US20150018543A1-20150115-C00214
  • 1H NMR (400 MHz, CDCl3) δ 1.45 (t, J=7.2 Hz, 3H), 1.88-1.97 (m, 4H), 2.87 (t, J=6.4 Hz, 2H), 3.93 (t, J=5.6 Hz, 2H), 4.41 (q, J=7.2 Hz, 2H), 7.35 (t, J=7.2 Hz, 1H), 7.35 (d, J=7.6 Hz, 1H), 7.67 (d, J=7.6 Hz, 1H), 8.05 (s, 1H), 11.2 (s, 1H);
    • Ethyl 2-(2-hydroxyphenylamino)-4-oxo-6,7,8,9-tetrahydro-4H-pyrido[1,2-a]pyrimidine-3-carboxylate 195)
  • Figure US20150018543A1-20150115-C00215
  • 1H NMR (400 MHz, CDCl3) δ 1.40 (t, J=7.2 Hz, 3H), 1.81-1.94 (m, 4H), 2.65 (t, J=6.8 Hz, 2H), 3.65 (t, J=6.0 Hz, 2H), 4.18 (q, J=6.8 Hz, 2H), 6.85 (t, J=7.2 Hz, 1H), 7.00 (d, J=7.2 Hz, 1H), 7.06-7.12 (m, 2H), 9.98 (s, 1H), 11.3 (s, 1H); 13C NMR (100 MHz, CDCl3) δ 14.6, 18.8, 21.9, 31.6, 42.6, 61.3, 88.4, 120.2, 120.7, 124.5, 127.1, 127.2, 149.1, 159.4, 159.5, 163.0, 169.6.
    • Ethyl 2-(3-hydroxyphenylamino)-4-oxo-6,7,8,9-tetrahydro-4H-pyrido[1,2-a]pyrimidine-3-carboxylate (196)
  • Figure US20150018543A1-20150115-C00216
  • 1H NMR (400 MHz, CDCl3+MeOD-d4) δ 1.26 (t, J=7.2 Hz, 3H), 1.71-1.81 (m, 4H), 2.72 (t, J=6.4 Hz, 2H), 3.74 (t, J=6.4 Hz, 2H), 4.23 (q, J=7.2 Hz, 2H), 6.47 (d, J=7.6 Hz, 1H), 6.88 (d, J=8.0 Hz, 1H), 6.99 (d, J=8.0 Hz, 1H), 7.02 (t, J=2.0 Hz, 1H); 13C NMR (100 MHz, CDCl3+MeOD-d4) δ 14.2, 18.8, 21.9, 31.8, 42.4, 60.9, 79.8, 109.8, 111.6, 114.0, 129.4, 139.4, 149.7, 159.3, 160.2, 163.1, 169.6.
    • Ethyl 2-(4-hydroxyphenylamino)-4-oxo-6,7,8,9-tetrahydro-4H-pyrido[1,2-a]pyrimidine-3-carboxylate (197)
  • Figure US20150018543A1-20150115-C00217
  • 1H NMR (400 MHz, DMSO-d6) δ 1.21 (t, J=7.2 Hz, 3H), 1.67-1.80 (m, 4H), 2.65 (t, J=6.8 Hz, 2H), 3.65 (t, J=6.0 Hz, 2H), 4.18 (q, J=7.2 Hz, 2H), 6.68 (d, J=8.8 Hz, 2H), 7.25 (d, J=8.8 Hz, 2H), 9.29 (s, 1H), 10.7 (s, 1H); 13C NMR (100 MHz, CDCl3) δ 14.9, 18.9, 21.9, 32.1, 42.3, 60.4, 87.2, 115.7, 125.0, 130.1, 154.9, 159.4, 160.6, 163.3, 169.6.
    • 2-(3-Chloro-4-fluorophenylamino)-9-methyl-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carboxamide (198)
  • Figure US20150018543A1-20150115-C00218
  • mp=218° C. (decomp.); 1H NMR (400 MHz, CDCl3) δ 2.97 (d, J=4.8 Hz, 3H), 4.41 (s, 3H), 6.89 (dd, J=7.2 Hz, 7.2 Hz, 1H), 6.97 (dd, J=1.2 Hz, 8.0 Hz, 1H), 7.05 (dd, J=8.8 Hz, 8.8 Hz, 1H), 7.40-7.44 (m, 1H), 8.46-8.51 (m, 2H), 8.82 (d, J=2.0 Hz, 1H), 12.98 (s, 1H);
    • (E)-2-(3-Chlorophenylamino)-3-((cyclohexylimino)methyl)-4H-pyrido[1,2-a]pyrimidin-4-one (199)
  • Figure US20150018543A1-20150115-C00219
  • 1H NMR (400 MHz, CDCl3) δ 1.23-1.37 (m, 3H), 1.41-1.50 (m, 2H), 1.56-1.59 (m, 1H), 1.73-1.76 (m, 4H), 3.16-3.22 (m, 1H), 6.85 (ddd, J=1.2, 6.8, 6.8 Hz, 1H), 6.94 (ddd, J=0.8, 1.2, 8.0 Hz, 1H), 7.14 (dd, J=8.0, 8.0 Hz, 1H), 7.38 (ddd, J=0.8, 1.2, 8.0 Hz, 1H), 7.54-7.58 (m, 1H), 7.90-7.91 (m, 1H), 8.83 (s, 1H), 8.85 (dd, J=0.8, 1.2 Hz, 1H), 13.40 (brs, 1H); 13C NMR (100 MHz, CDCl3) δ 24.4, 25.6, 34.9, 68.4, 91.6, 113.4, 119.2, 121.2, 123.0, 124.7, 127.6, 129.5, 134.2, 137.6, 140.8, 150.6, 156.3, 157.0, 158.3.
    • (E)-2-(3-Chlorophenylamino)-3-((3-chlorophenylimino)methyl)-4H-pyrido[1,2-a]pyrimidin-4-one (200)
  • Figure US20150018543A1-20150115-C00220
  • 1H NMR (400 MHz, CDCl3) δ 7.01 (dd, J=0.8, 1.2, 8.0 Hz, 1H), 7.28 (d, J=8.4 Hz, 1H), 7.29 (dd, J=2.0, 4.0 Hz, 1H), 7.33 (d, J=8.0 Hz, 1H), 7.44 (d, J=8.8 Hz, 1H), 7.52 (ddd, J=0.8, 1.2, 8.0 Hz, 1H), 7.17-7.76 (m, 1H), 8.02-8.04 (m, II), 8.98 (dd, J=0.8, 6.8 Hz, 1H), 9.17 (s, 1H), 12.94 (brs, 1H); 13C NMR (100 MHz, CDCl3) δ 92.6, 114.0, 119.5, 119.8, 121.8, 123.9, 125.0, 125.7, 128.0, 129.7, 130.2, 134.4, 134.8, 138.7, 140.1, 151.3, 151.8, 157.0, 158.0, 158.9.
    • 2-(3-Chlorophenylamino)-3-((cyclopentylamino)methyl)-4H-pyrido[1,2-a]pyrimidin-4-one (201)
  • Figure US20150018543A1-20150115-C00221
  • 1H NMR (400 MHz, CDCl3) δ 1.54-1.57 (m, 2H), 1.74-1.83 (m, 4H), 2.05-2.08 (m, 2H), 3.23-3.24 (m, 1H), 4.19 (s, 2H), 6.93-6.98 (m, 2H), 7.11-7.15 (m, 1H), 7.32 (d, J=8.4 Hz, 1H), 7.51 (dd, J=2.0, 8.4 Hz, 1H), 7.61-7.65 (m, 1H), 7.74-7.75 (m, 1H), 8.73 (d, J=7.2 Hz, 1H).
    • 2-(3-Chloroophenylamino)-3-((cyclohexylamino)methyl)-4H-pyrido[1,2-a]pyrimidin-4-one (202)
  • Figure US20150018543A1-20150115-C00222
  • 1H NMR (400 MHz, CDCl3) δ 1.20-1.35 (m, 4H), 1.66-1.72 (m, 2H), 1.86-1.89 (m, 2H), 2.23-2.39 (m, 2H), 3.12-3.18 (m, 1H), 6.93 (ddd, J=1.2, 6.8, 7.2 Hz, 1H), 6.99 (ddd, J=0.8, 1.2, 7.6 Hz, 1H), 7.20 (dd, J=8.0, 8.0 Hz, 1H), 7.25 (d, J=8.8 Hz, 1H), 7.52-7.57 (m, 1H), 7.61 (dd, J=1.2, 8.0 Hz, 1H), 7.84-7.85 (m, 1H), 8.76 (d, J=6.4 Hz, 1H), 9.77 (brs, 1H); 13C NMR (100 MHz, CDCl3) δ 24.6, 25.0, 41.2, 57.9, 88.9, 114.6, 119.2, 121.1, 122.8, 124.6, 127.3, 129.4, 133.7, 137.3, 140.8, 149.6, 157.2, 158.8.
    • 2-(3-Chlorophenylamino)-3-((cycloheptylamino)methyl)-4H-pyrido[1,2-a]pyrimidin-4-one (203)
  • Figure US20150018543A1-20150115-C00223
  • 1H NMR (400 MHz, CDCl3) δ 1.40-1.59 (m, 6H), 1.72-1.81 (m, 4H), 2.18-2.23 (m, 2H), 3.07-3.12 (m, 1H), 4.05 (m, 2H), 6.82 (ddd, J=1.2, 6.8, 6.8 Hz, 1H), 6.91 (dd, J=1.2, 8.0 Hz, 1H), 7.14 (dd, J=8.0, 8.0 Hz, 1H), 7.44-7.49 (m, 2H), 7.78-7.80 (m, 1H), 8.70 (d, J=6.8 Hz, 1H), 10.00 (brs, 1H); 13C NMR (100 MHz, CDCl3) δ 23.8, 32.3, 41.5, 59.7, 89.7, 114.2, 118.7, 120.6, 122.4, 124.4, 127.2, 129.3, 133.7, 136.8, 140.9, 149.4, 157.2, 158.2.
    • 2-(3-Chlorophenylamino)-3-((isopropylamino)methyl)-4H-pyrido[1,2-a]pyrimidin-4-one (204)
  • Figure US20150018543A1-20150115-C00224
  • 1H NMR (400 MHz, CDCl3) δ 1.25 (s, 3H), 1.26 (s, 3H), 2.30-3.06 (m, 1H), 4.05 (s, 2H), 6.87 (dd, J=6.4, 7.2 Hz, 1H), 6.95 (d, J=7.2 Hz, 1H), 7.17 (dd, J=8.0, 8.0 Hz, 1H), 7.32 (d, J=8.8 Hz, 1H), 7.41 (d, J=8.0 Hz, 1H), 7.54 (dd, J=7.2, 7.2 Hz, 1H), 7.81 (s, 1H), 8.83 (d, J=6.8 Hz, 1H); 13C NMR (100 MHz, CDCl3) δ 22.1, 41.7, 48.9, 91.5, 113.7, 118.2, 120.1, 122.2, 124.6, 127.5, 129.5, 134.1, 136.2, 141.2, 149.5, 157.4, 157.8.
    • 2-(3-Chlorophenylamino)-3-((cyclohexylamino)methyl)-8-(4-methylpiperazin-1-yl)-4H-pyrido[1,2-a]pyrimidin-4-one (205)
  • Figure US20150018543A1-20150115-C00225
  • 1H NMR (400 MHz, CDCl3) δ 1.20-1.34 (m, 3H), 1.71-1.91 (m, 3H), 1.92-2.04 (m, 2H), 2.20 (s, 3H), 2.23-2.36 (m, 6H), 3.04-3.10 (m, 5H), 4.01 (s, 2H), 5.87 (s, 1H), 6.55 (s, J=8.0 hz, 1H), 6.90 (d, J=8.0 Hz, 1H), 7.14 (t, J=8.0 Hz, 1H), 7.62 (d, J=7.6 Hz, 1H), 7.84 (s, 1H), 8.46 (d, J=7.6 Hz, 1H), 9.59 (s, 1H); 13C NMR (100 MHz, CDCl3) δ 24.9, 25.3, 30.2, 41.2, 46.1, 46.3, 54.2, 58.4, 86.2, 98.9, 106.5, 119.3, 121.0, 122.3, 128.3, 129.5, 133.9, 141.9, 150.8, 154.8, 157.7, 158.9.
  • Figure US20150018543A1-20150115-C00226
    • General Procedure for the Synthesis of J1
  • To a solution of an aldehyde (0.9 mmol) in methanol (0.5 mL) was added NaBH4 (1.35 mmol) at room temperature. After stirring 1 h, the reaction mixture was diluted with methylene chloride (10 mL) and washed with brine (10 ml). The organic layer was dried over MgSO4 and concentrated in vacuo. The crude product was purified by recrystallization from a mixture of hexanes and ethyl acetate to give J1.
    • General Procedure for the Synthesis of J2
  • To a stirred solution of an ester (0.06 mmol) in THF (1.0 mL) was added LiAlH4 (0.09 mmol). The reaction mixture was stirred at room temperature for 1 hr. After reaction was completed, H2O (0.1 mL) was added dropwise. The reaction mixture was filtered off and concentrated in vacuo. The crude product was purified by flash column chromatography to give J2.
    • General Procedure for the Synthesis of J3
  • To a stirred solution of J1 or J2 (0.19 mmol) in CH2Cl2 (0.6 mL) was added triethylamine (0.38 mmol) and a benzoyl chloride (0.28 mmol) at 0° C. The reaction mixture was stirred at room temperature for 1 h. After reaction was completed, the mixture was diluted with CH2Cl2 (10 mL) and washed with brine (10 ml). The organic layer was dried over anhydrous MgSO4 and concentrated in vacuo. The crude product was purified by flash column chromatography (Hexane:EtOAc=2:1) to give J3.
    • 3-(Hydroxymethyl)-2-(phenylamino)-4H-pyrido[1,2-a]pyrimidin-4-one (206)
  • Figure US20150018543A1-20150115-C00227
  • 1H NMR (400 MHz, CDCl3+CD3OD) δ 4.80 (s, 2H), 6.87-6.90 (m, 1H), 8.03 (dd, J=7.2, 7.6 Hz, 1H), 7.27 (dd, J=7.6, 8.0 Hz, 2H), 7.53-7.58 (m, 3H), 8.36 (brs, 1H), 8.82 (d, J=6.8 Hz, 1H); 13C NMR (100 MHz, CDCl3+CD3OD) δ 56.0, 94.80, 94.85, 113.8, 121.1, 121.2, 123.2, 123.3, 124.5, 127.5, 128.6, 136.4, 138.9, 139.0, 149.7, 157.1, 158.0, 158.1.
    • 2-(3-Chlorophenylamino)-3-(hydroxy methyl)-4H-pyrido-[1,2-a]pyrimidin-4-one (207)
  • Figure US20150018543A1-20150115-C00228
  • 1H NMR (400 MHz, CDCl3) δ 4.95 (d, J=6.4 Hz, 2H), 6.93 (t, J=6.8 Hz, 1H), 7.05 (d, J=8.0 Hz, 1H), 7.38 (t, J=4.4 Hz, 2H), 7.42 (s, 1H), 7.63 (t, J=6.8 Hz, 1H), 7.81 (t, J=1.6 Hz, 1H), 8.20 (s, 1H), 8.92 (d, J=7.2 Hz, 1H).
    • 2-(3-Fluorophenylamino)-3-(hydroxymethyl)-4H-pyrido[1,2-a]pyrimidine-3-carbaldehyde (208)
  • Figure US20150018543A1-20150115-C00229
  • 1H NMR (400 MHz, CDCl3) δ 4.94 (s, 2H), 6.94 (t, J=6.0 Hz, 2H), 7.17 (d, J=8.0 Hz, 1H), 7.43 (d, J=8.8 Hz, 2H), 7.63 (t, J=7.2 Hz, 2H), 7.70 (d, J=9.2 Hz, 1H), 8.26 (s, 1H), 8.93 (d, J=7.2 Hz, 1H).
    • 3-(Hydroxymethyl)-2-(3-(trifluoromethyl)phenylamino)-4H-pyrido[1,2-a]pyrimidin-4-one (209)
  • Figure US20150018543A1-20150115-C00230
  • 1H NMR (400 MHz, CDCl3) δ 4.99 (s, 2H), 6.99 (d, J=6.0 Hz, 2H), 7.32 (d, J=8.0 Hz, 1H), 7.43 (d, J=7.6 Hz, 2H), 7.69 (brs, 2H), 8.06 (s, 1H), 8.27 (s, 1H), 8.96 (d, J=7.6 Hz, 1H).
    • 3-(Hydroxymethyl)-2-(3-(trifluoromethoxy)phenylamino)-4H-pyrido[1,2-a]pyrimidin-4-one (210)
  • Figure US20150018543A1-20150115-C00231
  • 1H NMR (400 MHz, CDCl3) δ 4.95 (d, J=6.4 Hz, 2H), 6.84 (t, J=6.8 Hz, 1H), 6.92 (d, J=6.8 Hz, 1H), 7.30-7.34 (m, 3H), 7.59 (t, J=7.2 Hz, 1H), 7.86 (s, 1H), 8.36 (s, 1H), 8.87 (d, J=6.4 Hz, 1H),
    • Methyl 3-(3-(hydroxymethyl)-4-oxo-4H-pyrido[1,2-a]pyrimidin-2-ylamino)benzoate (211)
  • Figure US20150018543A1-20150115-C00232
  • 1H NMR (400 MHz, CDCl3) δ 3.92 (s, 3H), 4.99 (d, J=6.4 Hz, 2H), 6.96 (t, J=7.2 Hz, 1H), 7.38-7.42 (m, 2H), 7.63 (t, J=7.8 Hz, 1H), 7.75 (d, J=7.6 Hz, 1H), 7.88 (d, J=8.0 Hz, 1H), 8.21 (s, 1H), 8.25 (brs, 1H), 8.96 (d, J=7.6 Hz, 1H).
    • 3-(3-(hydroxymethyl)-4-oxo-4H-pyrido[1,2-a]pyrimidin-2-ylamino)benzoic acid (212)
  • Figure US20150018543A1-20150115-C00233
  • 1H NMR (400 MHz, CDCl3) δ 4.73 (s, 1H), 5.74 (s, 2H), 7.19 (t, J=7.2 Hz, 1H), 7.38-7.42 (m, 2H), 7.45 (d, J=7.6 Hz, 1H), 7.86 (t, J=8.4 Hz, 1H), 8.00 (d, J=8.0 Hz, 1H), 8.19 (s, 1H), 8.82 (s, 1H), 8.89 (d, J=6.8 Hz, 1H).
    • 2-(4-Chlorophenylamino)-3-(hydroxymethyl)-4H-pyrido[1,2-a]pyrimidin-4-one (213)
  • Figure US20150018543A1-20150115-C00234
  • 1H NMR (400 MHz, DMSO) δ 4.05 (d, J=7.2 Hz, 2H), 7.37 (d, J=8.8Hz, 2H), 7.44 (d, J=8.8 Hz, 1H), 7.75 (d, J=6.8 Hz, 2H), 7.88 (t, J=8.8 Hz, 1H), 8.81 (s, 1H), 8.88 (d, J=6.4 Hz, 1H).
    • 2-(2-Chlorophenylamino)-3-(hydroxymethyl)-4H-pyrido[1,2-a]pyrimidin-4-one (214)
  • Figure US20150018543A1-20150115-C00235
  • 1H NMR (400 MHz, CDCl3) δ 5.01 (d, J=5.6 Hz, 2H), 6.97-7.01 (m, 3H), 7.26-7.29 (m, 1H), 7.42 (t, J=8.8 Hz, 2H), 7.66 (t, J=7.2 Hz, 1H), 8.41 (t, J=5.2 Hz, 1H), 8.53 (s, 1H), 8.99 (d, J=6.8 Hz, 1H).
    • 3-(Hydroxymethyl)-2-(3-hydroxyphenylamino)-4H-pyrido[1,2-a]pyrimidin-4-one (215)
  • Figure US20150018543A1-20150115-C00236
  • 1H NMR (400 MHz, CDCl3+CD3OD) δ 4.81 (s, 2H), 6.53 (d, J=8.0 Hz, 1H), 6.99 (dd, J=6.8, 6.8 Hz, 1H), 7.04 (d, J=8.0 Hz, 1H), 7.12 (dd, J=6.8, 6.8 Hz, 1H), 7.18 (s, 1H), 7.42 (d, J=9.6 Hz, 1H), 7.64 (dd, J=6.8, 8.8 Hz, 1H), 8.88 (d, J=7.2 Hz, 1H).
    • 3-(Hydroxymethyl)-2-(4-hydroxyphenylamino)-4H-pyrido[1,2-a]pyrimidin-4-one (216)
  • Figure US20150018543A1-20150115-C00237
  • 1H NMR (400 MHz, CD3OD) δ 4.83 (s, 2H), 6.77 (dd, J=2.0, 8.8 Hz, 2H), 7.04 (dd, J=6.8, 6.8 Hz, 1H), 7.32 (d, J=8.8 Hz, 1H), 7.34-7.67 (m, 2H), 7.67-7.73 (m, 1H), 8.84 (d, J=6.8 Hz, 1H).
    • 3-(Hydroxymethyl)-2-(2-hydroxyphenylamino)-4H-pyrido[1,2-a]pyrimidin-4-one (217)
  • Figure US20150018543A1-20150115-C00238
  • 1H NMR (400 MHz, CDCl3+CD3OD) δ 3.71 (s, 1H), 4.86 (s, 2H), 6.88 (ddd, J=1.6, 7.6, 8.0 Hz, 1H), 6.93 (dd, J=1.6, 8.0 Hz, 1H), 6.98 (ddd, J=1.6, 7.2, 8.0 Hz, 1H(, 7.05 (ddd, J=1.2, 6.8, 6.8 Hz, 1H), 7.43 (d, J=8.8 Hz, 1H), 7.69-7.73 (m, 2H), 8.91 (dd, J=0.8, 6.8 Hz, 1H).
    • 2-(2,6-Dichlorophenylamino)-3-(hydroxymethyl)-4H-pyrido[1,2-a]pyrimidin-4-one (218)
  • Figure US20150018543A1-20150115-C00239
  • 1H NMR (400 MHz, CDCl3) δ 5.03 (d, J=6.0 Hz, 2H), 6.96 (t, J=7.2 Hz, 1H), 7.16 (t, J=7.6 Hz, 2H), 7.2 (s, 1H), 7.39 (d, J=8.0 Hz, 2H), 7.56 (t, J=7.6 Hz, 1H), 7.77 (s, 1H), 8.96 (d, J=7.2 Hz, 1H).
    • 2-(3,5-Dichlorophenylamino)-3-(hydroxymethyl)-4H-pyrido[1,2-a]pyrimidin-4-one (219)
  • Figure US20150018543A1-20150115-C00240
  • 1H NMR (400 MHz, CDCl3) δ 4.97 (d, J=6.0 Hz, 2H), 7.01-7.04 (m, 2H), 7.50 (t, J=6.8 Hz, 1H), 7.60 (s, 2H), 7.71 (t, J=8.4 Hz, 2H), 8.24 (s, 1H), 8.98 (d, J=7.2 Hz, 1H).
    • 2-(3,5-Difluorophenylamino)-3-(hydroxymethyl)-4H-pyrido[1,2-a]pyrimidin-4-one (220)
  • Figure US20150018543A1-20150115-C00241
  • 1H NMR (400 MHz, CDCl3) δ 4.99 (d, J=6.0 Hz, 2H), 6.52 (t, J=8.8 Hz, 1H), 7.05 (t, J=5.6 Hz, 2H), 7.29 (d, J=2.0 Hz, 2H), 7.51 (s, 1H), 7.72 (t, J=7.6 Hz, 1H), 8.30 (s, 1H), 8.99 (d, J=6.4 Hz, 1H).
    • 2-(2,6-Dimethylphenylamino)-3-(hydroxymethyl)-4H-pyrido[1,2-a]pyrimidin-4-one (221)
  • Figure US20150018543A1-20150115-C00242
  • 1H NMR (400 MHz, CDCl3) δ 2.23 (s, 6H), 5.02 (d, J=6.4 Hz, 2H), 6.92 (t, J=6.8 Hz 1H), 7.12 (s, 3H), 7.20 (d, J=8.8 Hz, 1H), 7.33 (s, 1H), 7.53 (t, J=6.8 Hz, 1H), 8.94 (d, J=6.4 Hz, 1H).
    • 3-(Hydroxymethyl)-2-phenoxy-4H-pyrido[1,2-a]pyrimidin-4-one (222)
  • Figure US20150018543A1-20150115-C00243
  • 1H NMR (400 MHz, CDCl3) δ 3.31 (brs, 1H), 4.86 (s, 2H), 7.03-7.09 (m, 3H), 7.13-7.18 (m, 1H), 7.28-7.34 (m, 3H), 7.58-7.62 (m, 1H), 8.94-8.96 (m, 1H); 13C NMR (100 MHz, CDCl3) δ6.0, 99.7, 115.2, 121.7, 125.1, 125.3, 127.4, 129.3, 136.8, 149.2, 152.8, 159.6, 164.0.
    • 2-(3-Fluorophenoxy)-3-(hydroxymethyl)-4H-pyrido[1,2-a]pyrimidin-4-one (223)
  • Figure US20150018543A1-20150115-C00244
  • 1H NMR (400 MHz, CDCl3) δ 3.62 (brs, 1H), 4.78 (s, 2H), 6.78-6.85 (m, 3H), 7.02 (ddd, J=1.2, 6.8, 7.2 Hz, 1H), 7.18-7.23 (m, 1H), 7.25 (d, J=9.2 Hz, 1H), 7.57-7.62 (m, 1H), 8.89 (d, J=6.8 Hz, 1H); 13C NMR (100 MHz, CDCl3) δ 55.3, 99.7, 109.4, 109.6, 111.7, 111.9, 115.2, 117.2, 117.3, 125.0, 127.3, 129.7, 129.8, 137.0, 149.0, 153.5, 153.6, 159.4, 161.4, 163.6, 163.8.
    • 2-(3-Chlorophenoxy)-3-(hydroxymethyl)-4-pyrido[1,2-a]pyrimidin-4-one (224)
  • Figure US20150018543A1-20150115-C00245
  • 1H NMR (400 MHz, CDCl3) δ 3.51 (t, J=6.4 Hz, 1H), 4.79 (d, J=6.4 Hz, 2H), 6.95-6.98 (m, 1H), 7.04 (dd, J=6.8, 7.2 Hz, 1H), 7.08-7.10 (m, 1H), 7.20 (dd, J=8.4, 8.8 Hz, 1H), 7.27 (d, J=8.8 Hz, 1H), 7.59-7.63 (m, 1H), 8.91 9dd, J=0.4, 7.2 Hz, 1H); 13C NMR (100 MHz, CDCl3) δ 53.3, 55.4, 99.7, 115.3, 120.1, 122.2, 125.1, 127.4, 129.8, 134.3, 137.0, 153.2, 159.2, 163.6.
    • 3-(Hydroxymethyl)-2-(phenylamino)-6,7,8,9-tetrahydro-4H-pyrido[1,2-a]pyrimidin-4-one (225)
  • Figure US20150018543A1-20150115-C00246
  • 1H NMR (400 MHz, CDCl3) δ 1.85-1.93 (m, 4H), 2.15 (s, 2H), 2.84 (t, J=6.8 Hz, 2H), 3.87 (t, J=6.2 Hz, 2H), 7.06 (t, J=7.0 Hz, 1H), 7.26 (t, J=7.0 Hz, 2H), 7.51 (d, J=7.4 Hz, 2H), 11.2 (s, 1H); 13C NMR (100 MHz, CDCl3) δ 14.6, 19.2, 22.2, 32.2, 42.4, 88.4, 122.9, 124.4, 128.8, 138.4, 160.5, 160.8, 162.2.
    • 2-(3-Chlorophenylamino)-3-(hydroxymethyl)-6,7,8,9-tetrahydro-4H-pyrido[1,2-a]pyrimidin-4-one (226)
  • Figure US20150018543A1-20150115-C00247
  • 1H NMR (400 MHz, DMSO-d6) δ 1.23-1.34 (m, 2H), 1.38-1.51 (m, 4H), 2.35-2.41 (m, 2H), 3.98-4.05 (m, 2H), 4.12 (s, 2H), 7.17-7.22 (m, 2H), 7.31 (t, J=2.0 Hz, 1H), 7.36 (t, J=8.0 Hz, 1H), 7.77 (s, 1H); 13C NMR (100 MHz, DMSO-d6) δ 15.1, 23.1, 31.4, 42.4, 59.2, 61.4, 65.7, 122.8, 123.9, 125.6, 131.6, 134.3, 139.4, 157.9, 164.3
    • 3-(Hydroxymethyl)-2-(3-(trifluoromethyl)phenylamino)-6,7,8,9-tetrahydro-4H-pyrido[1,2-a]pyrimidin-4-one (227)
  • Figure US20150018543A1-20150115-C00248
  • 1H NMR (400 MHz, DMSO-d6) δ 1.19-1.38 (m, 2H), 1.48-1.54 (m, 2H), 1.70-1.73 (m, 2H), 2.38 (t, J=12.8 Hz, 1H), 3.98-4.06 (m, 2H), 4.13 (s, 2H), 7.47 (d, J=7.6 Hz, 1H), 7.52 (d, J=8.8 Hz, 1H), 7.55-7.59 (m, 2H), 7.83 (s, 1H); 13C NMR (100 MHz, DMSO-d6) δ14.3, 22.2, 30.5, 41.5, 58.4, 77.9, 119.8, 121.2, 127.0, □ 129.8, 130.1, (d, J=26.8 due to CF3), 138.2, 146.1, 157.1, 163.6, 169.1.
    • 3-(Hydroxymethyl)-2-(2-hydroxyphenylamino)-6,7,8,9-tetrahydro-4H-pyrido[1,2-a]pyrimidin-4-one (228)
  • Figure US20150018543A1-20150115-C00249
  • 1H NMR (400 MHz, CDCl3) δ 1.78-1.94 (m, 4H), 2.13-2.23 (m, 2H), 2.61 (t, J=6.0 Hz, 1H), 3.98-4.05 (m, 2H), 4.12 (s, 2H), 6.81 (t, J=7.2 Hz, 1H), 6.89 (d, J=7.2 Hz, 1H), 6.98-7.12 (m, 2H), 10.11 (s, 1H), 11.3 (s, 1H); 13C NMR (100 MHz, CDCl3) δ 14.3, 21.4, 31.3, 42.1, 61.1, 87.7, 121.2, 126.4, 128.3, 128.6, 151.1, 161.3, 162.5, 163.7, 169.4.
    • 3-(Hydroxymethyl)-2-(3-hydroxyphenylamino)-6,7,8,9-tetrahydro-4H-pyrido[1,2-a]pyrimidin-4-one (229)
  • Figure US20150018543A1-20150115-C00250
  • 1H NMR (400 MHz, CDCl3) δ 1.41-1.61 (m, 4H), 1.62-1.77 (m, 2H), 2.72 (t, J=10.0 Hz, 1H), 3.78-3.95 (m, 2H), 4.17 (s, 2H), 6.43 (d, J=7.6 Hz, 1H), 6.81 (d, J=8.0 Hz, 1H), 6.87 (d, J=8.0 Hz, 1H), 6.98 (t, J=2.0 Hz, 1H); 13C NMR (100 MHz, CDCl3) δ 14.2, 21.8, 31.9, 42.4, 60.1, 79.8, 109.8, 111.6, 114.0, 129.4, 139.4, 149.7, 159.3, 160.2, 163.1.
    • 3-(Hydroxymethyl)-2-(4-hydroxyphenylamino)-6,7,8,9-tetrahydro-4H-pyrido[1,2-a]pyrimidin-4-one (230)
  • Figure US20150018543A1-20150115-C00251
  • 1H NMR (400 MHz, DMSO-d6) δ 1.21-1.45 (m, 4H), 1.63-1.71 (m, 2H), 2.34 (t, J=12.8 Hz, 1H), 3.98-4.05 (m, 2H), 4.19 (s, 2H), 6.75 (d, J=8.8 Hz, 2H), 7.00 (d, J=8.8 Hz, 2H); 13C NMR (100 MI-Hz, DMSO-d6) δ 14.9, 21.9, 32.1, 42.3, 60.4, 87.2, 115.7, 125.0, 130.1, 154.9, 159.4, 160.6, 163.3.
    • 3-(Hydroxymethyl)-9-methyl-2-(phenylamino)-4H-pyrido[1,2-a]pyrimidin-4-one (231)
  • Figure US20150018543A1-20150115-C00252
  • 1H NMR (400 MHz, CDCl3) δ 2.40 (s, 3H), 2.97 (brs, 1H), 4.93 (s, 2H), 6.89 (t, J=6.8 Hz, 1H), 7.11 (t, J=7.2 Hz, 1H), 7.34 (t, J=7.6 Hz, 2H), 7.62 (d, J=6.4 Hz, 1H), 8.02 (d, J=8.0 Hz, 2H), 8.73 (d, J=6.8 Hz, 1H).
    • 2-(3-Chlorophenylamino)-3-(hydroxymethyl)-9-methyl-4H-pyrido[1,2-a]pyrimidin-4-one (232)
  • Figure US20150018543A1-20150115-C00253
  • 1H NMR (400 MHz, CDCl3) δ 2.43 (s, 3H), 3.06 (t, J=6.4 Hz, 1H), 4.92 (d, 6.4 Hz, 2H), 6.69 (d, J=7.0 Hz, 1H), 7.03 (d, J=7.6 Hz, 1H), 7.23 (t, J=8.0 Hz, 1H), 7.29 (d, J=8.0 Hz, 1H), 7.44 (d, J=6.8 Hz, 1H), 8.03 (s, 1H), 8.38 (s, 1H), 8.71 (d, J=7.2 Hz, 1H).
    • 2-((3-Chlorophenyl)(methyl)amino)-3-(hydroxymethyl)-9-methyl-4H-pyrido[1,2-a]pyrimidin-4-one (233)
  • Figure US20150018543A1-20150115-C00254
  • 1H NMR (400 MHz, CDCl3) δ 2.51 (s, 3H), 4.09 (t, J=6.8 Hz, 1H), 4.12 (d, J=7.2 Hz, 2H), 6.95 (t, J=7.0 Hz, 1H), 7.04-7.06 (m, 2H), 7.20 (t, J=8.4 Hz, 1H), 7.54 (d, J=6.8 Hz, 1H), 8.84 (d, J=7.2 Hz, 1H).
    • 2-((3-Chlorophenyl)(methyl)amino)-3-(methoxymethyl)-9-methyl-4H-pyrido[1,2-a]pyrimidin-4-one (234)
  • Figure US20150018543A1-20150115-C00255
  • 1H NMR (400 MHz, CDCl3) δ 2.49 (s, 3H), 3.01 (s, 3H), 4.04 (s, 3H), 6.91 (t, J=7.0 Hz, 1H), 7.08 (d, J=8.4 Hz, 1H), 7.12 (d, J=7.2 Hz, 1H), 7.20 (s, 1H), 7.26 (t, J=8.0 Hz, 1H), 7.52 (d, J=6.8 Hz, 1H), 8.86 (d, J=7.2 Hz, 1H).
    • 3-(Hydroxymethyl)-9-methyl-2-(3-(trifluoromethoxy)phenylamino)-4H-pyrido[1,2-a]pyrimidin-4-one (235)
  • Figure US20150018543A1-20150115-C00256
  • 1H NMR (400 MHz, CDCl3) δ 2.40 (s, 3H), 3.15 (t, J=6.2 Hz, 1H), 4.93 (d, J=6.4 Hz, 1H), 6.67 (t, J=7.0 Hz, 1H), 6.91 (d, J=8.0 Hz, 1H), 7.25-7.27 (m, 1H), 7.32 (t, J=8.2 Hz, 1H), 7.43 (d, J=6.8 Hz, 1H), 7.98 (s, 1H), 8.51 (s, 1H), 8.72 (d, J=6.8 Hz, 1H).
    • 3-(Hydroxymethyl)-2-(3-hydroxyphenylamino)-9-methyl-4H-pyrido[1,2-a]pyrimidin-4-one (236)
  • Figure US20150018543A1-20150115-C00257
  • 1H NMR (400 MHz, CDCl3+CD3OD) δ 2.44 (s, 3H), 4.75 (s, 2H), 6.45 (dd, J=2.4, 8.0 Hz, 1 h), 6.84 (dd, J=6.8, 6.8 Hz, 1H), 7.06 (dd, J=8.0, 8.4 Hz, 1H), 7.11 (dd, J=2.0, 2.4 Hz, 1H), 7.17 (dd, H=2.0, 8.0 Hz, 1H), 7.45 (d, J=6.8 Hzm 1H), 8.72 (d, J=7.2 Hz, 1H).
    • 3-(Hydroxymethyl)-2-(4-hydroxyphenylamino)-9-methyl-4H-pyrido[1,2-a]pyrimidin-4-one (237)
  • Figure US20150018543A1-20150115-C00258
  • 1H NMR (400 MHz, CDCl3) δ 2.40 (s, 3H), 4.94 (d, J=4.8 Hz, 1H), 6.81-6.84 (m, 3H), 7.46 (d, J=7.2 Hz, 1H), 7.50 (d, J=8.8 Hz, 2H), 7.84 (s, 1H), 8.82 (d, J=7.2 Hz, 1H).
    • 2-(4-tert-Butylphenylamino)-3-(hydroxymethyl)-9-methyl-4H-pyrido[1,2-a]pyrimidin-4-one (238)
  • Figure US20150018543A1-20150115-C00259
  • 1H NMR (400 MHz, CDCl3) δ 1.34 (s, 9H), 2.40 (s, 3H), 3.07 (t, J=6.2 Hz, 1H), 4.91 (d, J=6.4 Hz, 2H), 6.61 (t, J=6.8 Hz, 1H), 7.34 (d, J=7.2 Hz, 2H), 7.38 (d, J=6.8 Hz, 1H), 8.21 (br s, 1H), 8.69 (d, J=7.2 Hz, H).
    • 2-(3-Chlorobenzylamino)-3-(hydroxymethyl)-9-methyl-4H-pyrido[1,2-a]pyrimidin-4-one (239)
  • Figure US20150018543A1-20150115-C00260
  • 1H NMR (400 MHz, CDCl3+CD3OD) δ 2.31 (s, 3H), 3.02 (s, 1H), 4.68 (d, J=5.6 Hz, 2H), 4.70 (s, 2H), 6.70 (dd, J=5.6, 6.0 Hz, 1H), 6.74 (dd, J=6.8, 7.2 Hz, 1H), 7.11-7.20 (m, 3H), 7.31 (s, 1H), 7.38 (d, J=6.8 Hz, 1H), 8.66 (d, J=6.8 Hz, 1H); 13C NMR (100 MHz, CDCl3+CD3OD) δ 17.7, 44.2, 44.3, 55.8, 93.1, 93.2, 112.6, 125.4, 125.5, 126.9, 127.5, 129.5, 132.6, 134.0, 134.9, 141.7, 149.45, 149.47, 157.4, 159.10, 159.16.
    • 3-(Hydroxymethyl)-2-(isobutylamino)-9-methyl-4H-pyrido[1,2-a]pyrimidin-4-one (240)
  • Figure US20150018543A1-20150115-C00261
  • 1H NMR (400 MHz, CDCl3) δ 0.96 (d, J=6.8 Hz, 6H), 1.88-1.95 (m, 1H), 2.34 (s, 3H), 3.13 (brs, 1H), 3.32 (t, J=6.0 Hz, 2H), 4.78 (d, J=6.0 Hz, 2H), 6.08 (brs, 1H), 6.72 (t, J=6.8 Hz, 1H), 7.37 (d, J=6.8 Hz, 1H), 8.66 (d, J=6.8 Hz, 1H); 13C NMR (100 MHz, CDCl3) δ 17.9, 20.5, 28.9, 48.6, 57.1, 92.5, 112.1, 126.0, 132.5, 134.6, 149.6, 157.1, 159.5.
    • 2-(Diethylamino)-3-(hydroxymethyl)-9-methyl-4H-pyrido[1,2-a]pyrimidin-4-one (241)
  • Figure US20150018543A1-20150115-C00262
  • 1H NMR (400 MHz, CDCl3) δ 1.22 (t, J=6.8 Hz, 6H), 2.35 (s, 3H), 3.41 (s, 1H), 3.63 (q, J=6.8 Hz, 4H), 4.44 (s, 2H), 6.65 (t, J=7.2 Hz, 1H), 7.31 (d, J=6.8 Hz, 1H), 8.68 (d, J=7.2 Hz, 1H) 13C NMR (100 MHz, CDCl3) δ 13.9, 17.7, 44.0, 67.0, 92.2, 111.7, 125.8, 132.5, 134.4, 148.1, 160.7, 160.8.
    • 2-(Cyclohexylmethylamino)-3-(hydroxymethyl)-9-methyl-4H-pyrido[1,2-a]pyrimidin-4-one (242)
  • Figure US20150018543A1-20150115-C00263
  • 1H NMR (400 MHz, CDCl3) δ 0.95-0.98 (m, 2H), 1.18-1.23 (m, 3H), 1.58-1.79 (m, 6H), 2.42 (s, 3H), 3.27 (t, J=6.4 Hz, 2H), 3.85 (brs, 1H), 4.74 (m, 2H), 6.21 (t, J=7.2 Hz, 1H), 6.68 (d, J=6.8 Hz, 1H), 7.33 (d, J=7.2 Hz, 1H), 8.57 (d, J=7.2 Hz, 1H); 13C NMR (100 MHz, CDCl3) δ 17.9, 26.2, 26.7, 31.3, 38.4, 47.5, 56.9, 92.8, 112.0, 126.0, 132.3, 134.5, 149.4, 156.9, 159.5.
    • 3-(Hydroxymethyl)-9-methyl-2-morpholino-4H-pyrido[1,2-a]pyrimidin-4-one (243)
  • Figure US20150018543A1-20150115-C00264
  • 1H NMR (400 MHz, CDCl3) δ 2.01 (brs, 1H), 2.43 (s, 3H), 3.62 (t, J=4.8 Hz, 4H), 3.78 (t, J=4.8 Hz, 4H), 4.62 (s, 2H), 6.85 (t, J=6.8 Hz, 1H), 7.46 (d, J=6.8 Hz, 1H), 8.76 (d, J=6.8 Hz, 1H); 13C NMR (100 MHz, CDCl3) δ 17.9, 49.7, 58.9, 67.1, 95.5, 113.3, □ 125.2, 133.4, 135.0, 148.2, 160.6, 161.7.
    • 3-(Hydroxymethyl)-9-methyl-2-morpholino-4H-pyrido[1,2-a]pyrimidin-4-one hydrochloride (244)
  • Figure US20150018543A1-20150115-C00265
  • 1H NMR (400 MHz, CDCl3) δ 2.43 (s, 3H), 3.42 (s, 1H), 3.62 (t, J=4.8 Hz, 4H), 3.78 (t, J=4.8 Hz, 4H), 4.62 (s, 2H), 6.85 (t, J=6.8 Hz, 1H), 7.46 (d, J=6.8 Hz, 1H), 8.76 (d, J=6.8 Hz, 1H); 13C NMR (100 MHz, CDCl3) δ 17.9, 49.7, 58.9, 67.1, 98.5, 113.3, 125.2, 133.4, 135.0, 148.2, 160.6, 161.7.
    • 7-Bromo-2-(3-chlorophenylamino)-3-(hydroxymethyl)-4H-pyrido[1,2-a]pyrimidin-4-one (245)
  • Figure US20150018543A1-20150115-C00266
  • 1H NMR (400 MHz, DMSO-d6) δ 4.78 (s, 2H), 5.37 (s, 1H), 7.12 (dd, J=1.6 Hz, 8.4 Hz, 1H), 7.32 (d, J=8.0 Hz 1H), 7.42 (dd, J=1.6 Hz, 8.4 Hz, 1H), 7.54 (dd, J=0.8 Hz, 8.0 Hz, 1H), 7.64 (d, J=8.0 Hz 1H), 7.91 (d, J=2.0 Hz, 1H), 8.47 (s, 1H), 8.71 (s, 1H);
    • 2-(3-Chlorophenylamino)-3-(hydroxymethyl)-7-methoxy-4H-pyrido[1,2-a]pyrimidin-4-one (246)
  • Figure US20150018543A1-20150115-C00267
  • 1H NMR (400 MHz, DMSO-d6) δ 3.86 (s, 3H), 4.70 (s, 2H), 5.22 (s, 1H), 7.02 (dd, J=0.8 Hz, 8.0 Hz, 1H), 7.28-7.32 (m, 1H), 7.41 (dd, J=1.2 Hz, 9.6 Hz, 1H), 7.58 (dd, J=0.8 Hz, 8.0 Hz, 1H), 7.64-7.68 (m, 1H), 7.87 (d, J=2.0 Hz, 1H), 8.36 (s, 1H), 8.69 (s, 1H)
    • 2-(3-Chlorophenylamino)-3-(hydroxymethyl)-8-methoxy-4H-pyrido[1,2-a]pyrimidin-4-one (247)
  • Figure US20150018543A1-20150115-C00268
  • 1H NMR (400 MHz, DMSO-d6) δ 3.92 (s, 3H), 4.62 (s, 2H), 5.07 (s, 1H), 6.71 (d, J=2.8 Hz, 1H), 6.83 (dd, J=2.8 Hz, 8.0 Hz, 1H), 7.01 (d, J=8.0 Hz, 1H), 7.28 (dd, J=8.0 Hz, J=8.0 Hz, 1H), 7.62 (d, J=8.0 Hz, 1H), 7.76 (d, J=2.0 Hz, 1H), 8.62 (s, 1H), 8.71 (d, J=8.0 Hz, 1H); 13C NMR (100 MHz, DMSO-d6) 54.8, 57.3, 93.8, 101.5, 109.3, 120.0, 120.9, 122.5, 129.5, 130.7, 133.4, 142.2, 151.9, 156.9, 157.8, 166.2.
    • 8-Chloro-2-(3-chlorophenylamino)-3-(hydroxymethyl)-4H-pyrido[1,2-a]pyrimidin-4-one (248)
  • Figure US20150018543A1-20150115-C00269
  • 1H NMR (400 MHz, CDCl3) δ 4.68 (s, 2H), 5.14 (brs, 1H), 7.03 (dd, J=1.2, 8.0 Hz, 1H), 7.19 (dd, J=2.4, 7.6 Hz, 1H), 7.28 (t, J=8.0 Hz, 1H), 7.54, (d, J=2.0 Hz, 1H), 7.58 (dd, J=1.2, 8.4 Hz, 1H), 7.57 (t, J=2.0 Hz, 1H), 8.78 (d, J=8.0 Hz, 1H).
    • 2-(3-Chlorophenylamino)-3-(hydroxymethyl)-8-(methylamino)-4H-pyrido[1,2-a]pyrimidin-4-one (249)
  • Figure US20150018543A1-20150115-C00270
  • 1H NMR (400 MHz, CDCl3) δ 2.81 (s, 3H), 3.85 (s, 2H), 6.02 (s, 1H), 6.32 (d, J=7.6 Hz, 1H), 6.93 (d, J=2 Hz, 1H), 7.12 (t, J=8.0 Hz, 1H), 7.38 (d, J=8.0 Hz, 1H), 7.81 (s, 1H), 8.42 (s, 1H), 9.93 (s, 1H).
    • 2-(3-Chlorophenylamino)-8-(diethylamino)-3-(hydroxymethyl)-4H-pyrido[1,2-a]pyrimidin-4-one (250)
  • Figure US20150018543A1-20150115-C00271
  • 1H NMR (400 MHz, CDCl3) δ 1.23 (t, J=6.8 Hz, 6H), 3.44 (q, J=6.8 Hz, 4H), 3.99 (s, 2H), 4.82 (t, J=2.1 Hz, 1H), 6.29 (d, J=2.1 Hz, 1H), 6.54 (dd, J=2.4, 8.4 Hz, 1H), 6.92 (d, J=2 Hz, 1H), 7.21 (t, J=8.0 Hz, 1H), 7.81 (d, J=2.4 Hz, 1H), 8.06 (t, J=2.0 Hz, 1H), 8.85 (d, J=8.4 Hz, 1H), 9.71 (s, 1H); 13C NMR (100 MHz, CDCl3) δ 12.7, 20.0, 44.7, 92.8, 97.1, 104.0, 118.9, 120.7, 121.9, 128.5, 129.5, 134.1, 142.8, 150.6, 151.9, 158.3, 159.2.
    • 3-(Hydroxymethyl)-8-morpholino-2-(phenylamino)-4H-pyrido[1,2-a]pyrimidin-4-one (251)
  • Figure US20150018543A1-20150115-C00272
  • 1H NMR (400 MHz, DMSO-d6) δ 3.43 (s, 4H), 3.67 (s, 4H), 4.59 (d, J=5.2 Hz, 2H), 5.05, (t, J=4.8 Hz, 1H), 6.41 (d, J=2.0 Hz, 1H), 6.95 (t, J=7.2 Hz, 1H), 7.00 (dd, J=2.8, 8.4 Hz, 1H), 7.25 (t, J=8.0 Hz, 2H), 7.64 (d, J=7.6 Hz, 2H), 8.38 (s, 1H), 8.69 (d, J=8.0 Hz, 1H); 13C NMR (100 MHz, DMSO-d6) δ 46.5, 55.1, 66.3, 91.5, 99.1, 105.4, 121.3, 122.6, 128.5, 129.1, 140.9, 151.4, 155.0, 156.7, 158.5.
    • 2-(3-Fluorophenylamino)-3-(hydroxymethyl)-8-morpholino-4H-pyrido[1,2-a]pyrimidin-4-one (252)
  • Figure US20150018543A1-20150115-C00273
  • 1H NMR (400 MHz, DMSO-d6) δ 3.46 (s, 4H), 3.68 (s, 4H), 4.59 (d, J=5.2 Hz, 2H), 5.06, (t, J=5.2 Hz, 1H), 6.47 (d, J=2.4 Hz, 1H), 6.74 (t, J=7.2 Hz, 1H), 7.03 (dd, J=2.8, 8.0 Hz, 1H), 7.26 (t, J=7.2 Hz, 1H), 7.64 (d, J=8.0 Hz, 1H), 7.79 (d, J=12.4 Hz, 1H), 8.52 (s, 1H), 8.60 (d, J=8.0 Hz, 1H); 13C NMR (100 MHz, DMSO-d6) δ 45.8, 54.2, 65.6, 91.3, 98.4, 105.0, 108.0 (d, J=20 Hz, due to F), 116.0, 128.0, 129.8 (d, J=10 Hz, due to F), 142.1 (d, J=11 Hz, due to F), 150.6, 154.4, 156.1, 157.4, 161.0, 163.3.
    • 2-(3-Chlorophenylamino)-3-(hydroxymethyl)-8-morpholino-4H-pyrido[8,2-a]pyrimidin-4-one (253)
  • Figure US20150018543A1-20150115-C00274
  • 1H NMR (400 MHz, DMSO-d6) δ 3.45 (t, J=5.6 Hz, 4H), 3.69 (t, J=5.6 Hz, 4H), 4.58 (d, J=5.2 Hz, 2H), 5.01 (t, J=5.2 Hz, 1H), 6.42 (d, J=2.8 Hz, 1H), 6.98 (d, J=8.0 Hz, 1H), 7.05 (dd, J=2.8, 8.0 Hz, 1H), 7.26 (t, J=8.0 Hz, 1H), 7.64 (d, J=8.0 Hz, 1H), 7.80 (t, J=2.0 Hz, 1H), 8.48 (s, 1H), 8.60 (d, J=8.0 Hz, 1H); 13C NMR (100 MHz, DMSO-d6) δ5.4, 53.6, 65.7, 84.7, 98.6, 105.3, 117.8, 118.7, 119.8, 127.1, 130.2, 129.2, 141.8, 149.7, 153.0, 155.3, 157.4; LC-MS (ESI, m/z): 386 [M+H]+.
    • 3-(Hydroxymethyl)-8-(4-methylpiperazin-1-yl)-2-(phenylamino)-4H-pyrido[1,2-a]pyrimidin-4-one (254)
  • Figure US20150018543A1-20150115-C00275
  • 1H NMR (400 MHz, CDCl3) δ 2.34 (s, 3H), 2.52 (t, J=5.2 Hz, 4H), 3.43 (t, J=5.2 Hz, 4H), 4.88 (s, 2H), 5.28 (s, 1H), 6.37 (s, 1H), 6.55 (d, J=8.0 Hz, 1H), 7.05 (t, J=7.2 Hz, 1H), 7.33 (t, J=7.6 Hz, 2H), 7.60 (d, J=7.6 Hz, 2H), 7.91 (s, 1H), 8.64 (d, J=8.0 Hz, 1H).
    • 2-(3-Chlorophenylamino)-3-(hydroxymethyl)-8-(4-methylpiperazin-1-yl)-4H-pyrido[1,2-a]pyrimidin-4-one (255)
  • Figure US20150018543A1-20150115-C00276
  • 1H NMR (400 MHz, CDCl3) δ 2.14 (s, 3H), 2.38 (t, J=4.4 Hz, 4H), 3.45 (t, J=4.4 Hz, 4H), 3.56 (s, 2H), 6.41 (d, J=2.4 Hz, 1H), 6.95 (dd, J=1.6, 8.0 Hz, 1H), 7.01 (dd, J=2.4, 8.0 Hz, 1H), 7.27 (t, J=8.0 Hz, 1H), 7.50 (d, J=1.6 Hz, 1H), 8.0 (d, J=8.0 Hz, 1), 10.4 (s, 1H), 14.18 (s, 1H); 13C NMR (100 MHz, CDCl3) δ 45.6, 51.6, 54.0, 55.0, 85.3, 98.3, 105.1, 117.7, 118.5, 121.0, 127.9, 130.3, 133.0, 142.1, 150.8, 154.1, 156.4, 157.8; LC-MS (ESI, m/z): 400[M+H]+.
    • 2-(3-Fluorophenylamino)-3-(hydroxymethyl)-8-(4-methylpiperazin-1-yl)-4H-pyrido[1,2-a]pyrimidin-4-one (256)
  • Figure US20150018543A1-20150115-C00277
  • 1H NMR (400 MHz, CDCl3) δ 2.35 (s, 3H), 2.54 (t, J=4.4 Hz, 4H), 3.48 (t, J=4.8 Hz, 4H), 4.87 (s, 2H), 5.23 (s, 1H), 6.42 (s, 1H), 6.60 (d, J=8.4 Hz, 1H), 6.73 (t, J=8.4 Hz, 1H), 7.12 (d, J=8.4 Hz, 1H), 7.19 (d, J=8.4 Hz, 1H), 7.71-7.75 (m, 1H), 8.04 (s, 1H), 8.71 (d, J=8.0 Hz, 1H).
    • 2-(3-Chlorophenylamino)-3-(hydroxymethyl)-8-methyl-4H-pyrido[1,2-a]pyrimidin-4-one (257)
  • Figure US20150018543A1-20150115-C00278
  • Colorless solid, mp 235° C. (decomp.); 1H NMR (400 MHz, CDCl3) δ 2.42 (s, 3H), 4.07 (q, J=7.2 Hz, 2H), 7.03 (d, J=8.8 Hz, 2H), 7.26 (t, J=8.0 Hz, 2H), 7.46 (d, J=8.4 Hz, 1H), 7.84 (t, J=2.0 Hz, 1H), 8.79 (d, J=7.2 Hz, 2H).
    • 2-(4-Chlorophenylamino)-3-(hydroxymethyl)-8-methyl-4H-pyrido[1,2-a]pyrimidin-4-one (258)
  • Figure US20150018543A1-20150115-C00279
  • Colorless solid, mp 227° C. (decomp.); 1H NMR (400 MHz, CDCl3) δ 2.42 (s, 3H), 4.10 (s, 2H), 6.85 (d, J=7.2 Hz, 1H), 7.23-7.28 (m, 4H), 7.87 (d, J=6.8 Hz, 2H), 8.94 (d, J=7.6 Hz, 1H).
    • 2-(4-Fluorophenylamino)-3-(hydroxymethyl)-8-methyl-4H-pyrido[1,2-a]pyrimidin-4-one (259)
  • Figure US20150018543A1-20150115-C00280
  • Colorless solid, mp 232° C. (decomp.); 1H NMR (400 MHz, CDCl3) δ 2.42 (s, 3H), 4.12 (s, 2H), 6.85 (d, J=6.8 Hz, 1H), 7.05 (t, J=8.4 Hz, 2H), 7.21 (s, 1H), 7.31-7.38 (m, 2H), 7.85 (q, J=4.8 Hz, 2H), 8.94 (d, J=7.2 Hz, 1H).
    • 2-(3,4-Dichlorophenylamino)-3-(hydroxymethyl)-8-methyl-4H-pyrido[1,2-a]pyrimidin-4-one (260)
  • Figure US20150018543A1-20150115-C00281
  • Colorless solid, mp 230° C. (decomp.); 1H NMR (400 MHz, CDCl3) δ 2.44 (s, 3H), 4.09 (s, 2H), 6.89 (d, J=7.2 Hz, 1H), 7.26 (s, 1H), 7.36 (d, J=8.8 Hz, 1H), 7.76 (d, J=8.4 Hz, 1H), 8.24 (d, J=2.4 Hz, 1H), 8.95 (d, J=7.2 Hz, 1H), 9.71 (s, 1H).
    • 2-(3-Chloro-4-fluorophenylamino-3-(hydroxymethyl)-8-methyl-4H-pyrido[1,2-a]pyrimidin-4-one (261)
  • Figure US20150018543A1-20150115-C00282
  • Colorless solid, mp 225° C. (decomp.); 1H NMR (400 MHz, CDCl3) δ 2.43 (s, 3H), 4.09 (s, 2H), 6.88 (d, J=7.2 Hz, 1H), 7.11 (t, J=8.8 Hz, 1H), 7.27 (s, 1H), 7.69-7.73 (m, 1H), 8.12 (d, J=6.8 Hz, 1H), 8.95 (d, J=7.2 Hz, 1H), 9.71 (s, 1H).
    • 9-Chloro-2-(3-chlorophenylamino)-3-(hydroxymethyl)-4H-pyrido[1,2-a]pyrimidin-4-one (262)
  • Figure US20150018543A1-20150115-C00283
  • Colorless solid, mp 230° C. (decomp.); 1H NMR (400 MHz, CDCl3) δ 4.95 (d, J=6.0 Hz, 2H), 6.80 (t, J=7.2 Hz, 1H), 7.06 (d, J=8.0 Hz, 1H), 7.27 (d, J=8.4 Hz, 1H), 7.46 (d, J=8.0 Hz, 1H), 7.78 (d, J=7.2 Hz, 1H), 8.18 (t, J=2.4 Hz, 1H), 8.43 (s, 1H), 8.81 (d, J=7.2 Hz, 1H).
    • 2-(3-Chlorophenylamino)-3-(hydroxymethyl)-9-(trifluoromethyl)-4H-pyrido[1,2-a]pyrimidin-4-one (263)
  • Figure US20150018543A1-20150115-C00284
  • 1H NMR (400 MHz, DMSO-d6) δ 4.77 (s, 2H), 7.11-7.13 (m, 1H), 7.32 (dd, J=7.2, 7.2 Hz, 1H), 7.35 (dd, J=8.0, 8.0 Hz, 1H), 7.48-7.50 (m, 1H), 8.13-8.14 (m, 1H), 8.41 (d, J=7.2 Hz, 1H), 9.12 (dd, J=1.2, 7.2 Hz, 1H).
    • 2-(3-Chlorophenylamino)-9-fluoro-3-(hydroxymethyl)-4H-pyrido[1,2-a]pyrimidin-4-one
  • Figure US20150018543A1-20150115-C00285
  • 1H NMR (400 MHz, DMSO-d6) δ4.76 (s, 1H), 5.31 (brs, 1H), 7.11-7.13 (m, 1H), 7.18-7.23 (m, 1H), 7.38 (dd, J=8.0, 8.0 Hz, 1H), 7.63-7.65 (m, 1H), 7.86 (dd, J=8.4, 8.8 Hz, 1H), 8.12-8.13 (m, 1H), 8.73 (d, J=7.2 Hz, 1H), 8.96 (brs, 1H).
    • 2-(4-Chlorophenylamino)-9-fluoro-3-(hydroxymethyl)-4H-pyrido[1,2-a]pyrimidin-4-one (265)
  • Figure US20150018543A1-20150115-C00286
  • 1H NMR (400 MHz, DMSO-d6) δ 4.72 (s, 2H), 5.30 (brs, 1H), 7.15-7.20 (m, 1H), 7.41-7.44 (m, 2H), 7.79-7.82 (m, 2H), 7.84-7.86 (m, 1H), 8.72 (d, J=7.2 Hz, 1H), 8.92 (brs, 1H).
    • 9-Fluoro-2-(4-fluorophenylamino)-3-(hydroxymethyl)-4H-pyrido[1,2-a]pyrimidin-4-one (266)
  • Figure US20150018543A1-20150115-C00287
  • 1H NMR (400 MHz, DMSO-d6) δ 4.75 (s, 2H), 5.25 (brs, 1H), 7.13-7.25 (m, 3H), 7.73-7.77 (m, 2H), 7.80-7.85 (m, 1H), 8.72 (d, J=7.2 Hz, 1H), 8.84 (brs, 1H). 2-(3-Chloro-4-fluorophenylamino)-9-fluoro-3-(hydroxymethyl)-4H-pyrido[1,2-a]pyrimidin-4-one (267)
  • Figure US20150018543A1-20150115-C00288
  • 1H NMR (400 MHz, DMSO-d6) δ4.74 (s, 2H), 5.24 (brs, 1H), 7.18-7.22 (m, 1H), 7.39-7.44 (m, 1H), 7.65-7.69 (m, 1H), 7.83-7.87 (m, 1H), 8.20-8.22 (m, 1H), 8.72 (d, J=7.2 Hz, 1H), 8.91 (brs, 1H).
    • 2-(3,4-Difluorophenylamino)-9-fluoro-3-(hydroxymethyl)-4H-pyrido[1,2-a]pyrimidin-4-one (268)
  • Figure US20150018543A1-20150115-C00289
  • 1H NMR (400 MHz, DMSO-d6) δ 4.75 (s, 2H), 5.26 (brs, 1H), 7.17-7.22 (m, 1H), 7.39-7.49 (m, 1H), 7.84-7.88 (m, 1H), 8.08-8.14 (m, 1H), 8.73 (m, J=7.2 Hz, 1H), 8.93 (brs, 1H).
    • 2-(3,4-Dichlorophenylamino)-9-fluoro-3-(hydroxymethyl)-4H-pyrido[1,2-a]pyrimidin-4-one (269)
  • Figure US20150018543A1-20150115-C00290
  • 1H NMR (400 MHz, DMSO-d6) δ 4.75 (s, 2H), 5.27 (brs, 1H), 7.19-7.23 (m, 1H), 7.60 (d, J=8.8 Hz, 1H), 7.7 (dd, J=2.8, 8.8 Hz, 1H), 7.85-7.89 (m, 1H), 8.83 (d, J=2.8 Hz, 1H), 8.73 (d, J=8.8 Hz, 1H), 9.00 (brs, 1H).
    • 2-(1H-Indol-5-ylamino)-9-fluoro-3-(hydroxymethyl)-4H-pyrido[12-a]pyrimidin-4-one (270)
  • Figure US20150018543A1-20150115-C00291
  • m.p=184-185° C.; 1H NMR (400 MHz, DMSO-d6) δ 4.70 (d, J=5.2 Hz, 2H), 5.18 (t, J=5.2 Hz, 1H), 6.35 (s, 1H), 7.00-7.04 (m, 1H), 7.23 (dd, 0.1=2 Hz, 8.8 Hz, 1H), 7.28-7.32 (m, 2H), 7.68 (dd, J=8 Hz, J=8 Hz, 1H), 7.82 (s, 1H), 8.61 (s, 1H), 8.64 (d, J=6 Hz, 1H), 10.98 (s, 1H); 13C NMR (100 MHz, DMSO-d6) δ5.2, 94.6, 101.7 (d, J=5.2 Hz, due to F), 111.6, 112.1 (d, J=7.4 Hz, due to F), 113.7, 118.0, 119.8 (d, J=17.1 Hz, due to F), 124.2 (d, J=4.4 Hz, due to F), 126.5, 128.2, 131.9, 133.5, 151.6, 154.1, 156.3, 157.6.
    • 3-(Hydroxymethyl)-9-methoxy-2-(phenylamino)-4H-pyrido[1,2-a]pyrimidin-4-one (271)
  • Figure US20150018543A1-20150115-C00292
  • 1H NMR (400 MHz, DMSO-d6) δ 3.93 (s, 3H), 4.71 (d, J=5.2 Hz, 2H), 5.29 (t, J=5.2 Hz, 1H), 6.97-7.01 (m, 1H), 7.06-7.10 (m, 1H), 7.27-7.32 (m, 3H), 7.83 (d, J=8.4 Hz, 2H), 8.47 (d, J=7.2 Hz, 1H), 8.68 (s, 1H).
    • 3-(Hydroxymethyl)-9-methoxy-2-(phenylamino)-4H-pyrido[1,2-a]pyrimidine-4-thione (272)
  • Figure US20150018543A1-20150115-C00293
  • 1H NMR (400 MHz, CDCl3) δ 3.98 (s, 3H), 4.11 (d, J=7.2 Hz, 2H), 6.88 (t, J=8.0 Hz, 2H), 7.04 (t, J=7.2 Hz, 1H), 7.31 (t, J=7.2 Hz, 2H), 7.82 (d, J=7.6 Hz, 2H), 7.98 (s, 1H), 8.59 (d, J=5.6 Hz, 1H); 13C NMR (100 MHz, CDCl3) 26.9, 57.1, 94.2, 111.8, 112.7, 119.9, 121.1, 123.3, 128.9, 139.8, 143.7, 151.3, 155.6, 158.6.
    • 2-(3-Chlorophenylamino)-3-(hydroxymethyl)-9-methoxy-4H-pyrido[1,2-a]pyrimidin-4-one (273)
  • Figure US20150018543A1-20150115-C00294
  • 1H NMR (400 MHz, DMSO-d6) δ 3.94 (s, 3H), 4.68 (s, 2H), 6.99 (d, J=7.6 Hz, 1H), 7.09 (dd, J=7.2 Hz, J=7.2 Hz, 1H), 7.25-7.29 (m, 2H), 7.56 (d, J=8.0 Hz, 1H), 8.42 (s, 1H), 8.45 (d, J=6.8 Hz, 1H), 8.77 (s, 1H).
    • 2-(4-Chlorophenylamino)-3-(hydroxymethyl)-9-methoxy-4H-pyrido[1,2-a]pyrimidin-4-one (274)
  • Figure US20150018543A1-20150115-C00295
  • 1H NMR (400 MHz, DMSO-d6) δ 3.90 (s, 3H), 4.65 (d, J=5.2 Hz, 2H), 5.19 (t, J=5.2 Hz, 1H), 7.03 (dd, J=7.2 Hz, 7.6 Hz, 1H), 7.23 (d, J=7.6 Hz, 1H), 7.29 (d, J=8.8 Hz, 2H), 7.85 (d, J=9.2 Hz, 2H), 8.42 (d, J=7.2 Hz, 1H), 8.72 (s, 1H).
    • 2-(4-Fluorophenylamino)-3-(hydroxymethyl)-9-methoxy-4H-pyrido[1,2-a]pyrimidin-4-one (275)
  • Figure US20150018543A1-20150115-C00296
  • 1H NMR (400 MHz, DMSO-d6) δ 3.91 (s, 3H), 4.69 (d, J=5.2 Hz, 2H), 5.19 (t, J=5.2 Hz, 1H), 7.06 (t, J=6.8 Hz, 1H), 7.13 (t, J=8.8 Hz, 1H), 7.25 (d, J=7.6 Hz, 1H), 7.83-7.86 (m, 1H), 8.45 (dd, J=1.2 Hz, 7.2 Hz, 1H), 8.66 (s, 1H).
    • 3-(Hydroxymethyl)-9-methoxy-2-(4-(trifluoromethoxy)phenylamino)-4H-pyrido[1,2-a]pyrimidin-4-one (276)
  • Figure US20150018543A1-20150115-C00297
  • 1H NMR (400 MHz, DMSO-d6) δ 3.96 (s, 3H), 4.67 (d, J=4.0 Hz, 2H), 5.20 (s, 1H), 7.07 (dd, J=7.2 Hz, J=7.2 Hz, 1H), 7.23 (s, 1H), 7.27 (d, J=8.0 Hz, 2H), 7.95 (dd, J=8.8 Hz, J=8.8 Hz, 2H), 8.45 (d, J=7.6 Hz, 1H), 8.78 (s, 1H).
    • 3-(Hydroxymethyl)-9-methoxy-2-(4-(trifluoromethyl)phenylamino)-4H-pyrido[1,2-a]pyrimidin-4-one (277)
  • Figure US20150018543A1-20150115-C00298
  • 1H NMR (400 MHz, DMSO-d6) δ 3.97 (s, 3H), 4.72 (s, 2H), 5.32 (s, 1H), 7.14, (dd, J=7.2 Hz, 7.2 Hz, 1H), 7.33 (d, J=7.6 Hz, 1H), 7.64 (d, J=8.8 Hz, 2H), 8.11 (d, J=8.8 Hz, 2H), 8.49 (d, J=7.2 Hz, 1H), 9.09 (s, 1H).
    • 2-(3-Chloro-4-fluorophenylamino)-3-(hydroxymethyl)-9-methoxy-4-pyrido[1,2-a]pyrimidin-4-one (278)
  • Figure US20150018543A1-20150115-C00299
  • 1H NMR (400 MHz, DMSO-d6) δ 3.95 (s, 3H), 4.69 (d, J=4.8 Hz, 2H), 5.16 (t, J=4.8 Hz, 1H), 7.10 (dd, J=7.2 Hz, 7.2 Hz, 1H), 7.30 (dd, J=0.8 Hz, 8.0 Hz, 1H), 7.32 (dd, J=9.2 Hz, 9.2 Hz, 1H), 7.61-7.65 (m, 1H), 8.46 (dd, J=0.8 Hz, 7.2 Hz, 1H), 8.59 (dd, J=2.8 Hz, 7.2 Hz, 1H), 8.76 (s, 1H).
    • 2-(3,4-Difluorophenylamino)-3-(hydroxymethyl)-9-methoxy-4H-pyrido[1,2-a]pyrimidin-4-one (279)
  • Figure US20150018543A1-20150115-C00300
  • m.p=231° C. (decomp.); 1H NMR (400 MHz, CDCl3) δ 3.92 (s, 3H), 4.66 (s, 2H), 5.17 (brs, 1H), 7.07 (dd, J=7.2 Hz, 7.2 Hz, 1H), 7.26-7.33 (m, 2H), 7.39-7.41 (m, 1H), 8.34-8.40 (m, 1H), 8.44 (d, J=7.2 Hz, 1H), 8.74 (s, 1H); 13C NMR (100 MHz, DMSO) δ 54.1, 56.8, 95.2, 109.1, 113.4, 116.0 (d, J=3.8 Hz, due to F), 116.8, 118.7, 137.5 (d, J=9.7 Hz, due to F), 143.2 (d, J=11.9 Hz, due to F), 145.6, 147.5 (d, J=13.4 Hz, due to F), 149.9 (d, J=13.4 Hz, due to F), 150.6, 155.5.
    • 2-(3-Chloro-4-hydroxyphenylamino)-3-(hydroxymethyl)-9-methoxy-4H-pyrido[1,2-a]pyrimidin-4-one (280)
  • Figure US20150018543A1-20150115-C00301
  • 1H NMR (400 MHz, DMSO-d6) δ 3.93 (s, 3H), 4.68 (s, 2H), 5.14 (s, 1H), 6.99 (d, J=8.4 Hz, 1H), 7.06 (dd, J=7.2 Hz, 7.2 Hz, 1H), 7.26 (dd, J=1.2 Hz, 8.0 Hz, 1H), 7.38 (dd, J=1.2 Hz, 8.0 Hz, 1H), 8.25 (d, J=2.8 Hz, 1H), 8.45 (dd, J=1.2 Hz, 7.2 Hz, 1H), 8.52 (s, 1H), 9.79 (s, 1H).
    • 2-(3,4-Dichlorophenylamino)-3-(hydroxymethyl)-9-methoxy-4H-pyrido[1,2-a]pyrimidin-4-one (281)
  • Figure US20150018543A1-20150115-C00302
  • 1H NMR (400 MHz, DMSO-d6) δ 3.93 (s, 3H), 4.66 (d, J=5.2 Hz, 2H), 5.16 (d, J=5.2 Hz, 1H), 7.09 (t, J=7.2 Hz, 1H), 7.29 (d, J=6.8 Hz, 1H), 7.48 (d, J=8.8 Hz, 1H), 7.64 (dd, J=2.8 Hz, 8.8 Hz, 1H), 8.44 (d, J=7.2 Hz, 1H), 8.67 (d, J=2.8 Hz, 1H), 8.82 (s, 1H).
    • 3-(Hydroxymethyl)-9-methoxy-2-(4-methyl-3-(trifluoromethyl)phenylamino)-4H-pyrido[1,2-a]pyrimidin-4-one (282)
  • Figure US20150018543A1-20150115-C00303
  • 1H NMR (400 MHz, DMSO-d6) δ 2.49 (t, J=2.0 Hz, 3H due to CF3), 3.93 (s, 3H), 4.70 (d, J=4.8 Hz, 2H), 5.19 (t, J=4.8 Hz, 1H), 7.10 (t, J=7.2 Hz, 1H), 7.29 (dd, J=1.2 Hz, 8.0 Hz, 1H), 7.32 (d, J=8.4 Hz, 1H), 7.74 (dd, J=1.6 Hz, 8.0 Hz, 1H), 8.46 (dd, J=1.2 Hz, 6.8 Hz, 1H), 8.81 (s, 1H), 8.85 (d, J=2.0 Hz, 1H).
    • 2-(4-Fluoro-3-(trifluoromethyl)phenylamino)-3-(hydroxymethyl)-9-methoxy-4H-pyrido[1,2-a]pyrimidin-4-one (283)
  • Figure US20150018543A1-20150115-C00304
  • 1H NMR (400 MHz, DMSO-d6) δ3.92 (s, 3H), 4.68 (d, J=5.2 Hz, 2H), 5.12 (t, J=5.2 Hz, 1H), 7.07 (dd, J=7.2 Hz, 7.2 Hz, 1H), 7.27 (d, J=7.2 Hz, 1H), 7.37-7.42 (m, 1H), 7.86-7.88 (m, 1H), 8.43 (d, J=7.2 Hz, 1H), 8.87 (s, 1H), 8.99-9.00 (m, 1H).
    • 2-(2,3-Dihydro-1H-inden-5-ylamino)-3-(hydroxymethyl)-9-methoxy-4H-pyrido[1,2-a]pyrimidin-4-one (284)
  • Figure US20150018543A1-20150115-C00305
  • 1H NMR (400 MHz, DMSO-d6) δ 1.97-2.05 (m, 2H), 2.79 (t, J=7.6 Hz, 2H), 2.85 (t, J=7.6 Hz, 2H), 3.92 (s, 3H), 4.69 (d, J=5.6 Hz, 2H), 5.26 (t, J=5.6 Hz, 1H), 7.04 (dd, J=7.2 Hz, 1H), 7.12 (d, J=8.4 Hz, 1H), 7.24 (dd, J=0.8 Hz, 7.6 Hz, 1H), 7.46 (dd, J=2.0 Hz, 8.0 Hz, 1H), 7.82 (s, 1H), 8.45 (dd, J=1.2 Hz, 7.2 Hz, 1H), 8.59 (s, 1H).
    • 2-(Benzo[d][1,3]dioxol-5-ylamino)-3-(hydroxymethyl)-9-methoxy-4H-pyrido[1,2-a]pyrimidin-4-one (285)
  • Figure US20150018543A1-20150115-C00306
  • 1H NMR (400 MHz, DMSO-d6) δ 3.91 (s, 3H), 4.68 (d, J=5.2 Hz, 2H), 5.21 (t, J=5.2 Hz, 1H), 5.98 (s, 2H), 6.84 (d, J=8.4 Hz, 1H), 7.05-7.07 (m, 1H), 7.26 (dd, J=1.2 Hz, 8.0 Hz, 1H), 7.82 (d, J=2.0 Hz, 1H), 8.46 (d, J=2.0 Hz, 1H), 8.45 (dd, J=1.2 Hz, 7.2 Hz, 1H), 8.56 (s, 1H).
    • 2-(2,3-Dihydrobenzo[b][1,4]dioxin-6-ylamino)-3-(hydroxymethyl)-9-methoxy-4H-pyrido[1,2-a]pyrimidin-4-one (286)
  • Figure US20150018543A1-20150115-C00307
  • 1H NMR (400 MHz, DMSO-d6) δ 3.92 (s, 3H), 4.19-4.24 (m, 4H), 4.67 (d, J=5.2 Hz, 2H), 5.19 (t, J=5.2 Hz, 1H), 6.77 (d, J=8.8 Hz, 1H), 7.05 (dd, J=7.2 Hz, 7.2 Hz, 1H), 7.12 (dd, J=2.4 Hz, 8.4 Hz, 1H), 7.26 (d, J=6.8 Hz, 1H), 7.64 (d, J=2.4 Hz, 1H), 8.46 (dd, J=2.0 Hz, 7.2 Hz, 1H), 8.47 (s, 1H).
    • 3-(Hydroxymethyl)-9-methoxy-2-(1-methyl-1H-indol-5-ylamino)-4H-pyrido[1,2-a]pyrimidin-4-one (287)
  • Figure US20150018543A1-20150115-C00308
  • m.p=195-197° C.; 1H NMR (400 MHz, DMSO-d6) δ 3.82 (s, 3H), 3.97 (s, 3H), 4.77 (d, J=5.2 Hz, 2H), 5.28 (t, J=5.2 Hz, 1H), 6.42 (d, J=3.0 Hz, 1H), 7.09 (dd, J=7.2, 7.6 Hz, 1H), 7.28-7.30 (m, 1H), 7.33 (d, J=3.0 Hz, 1H), 7.41 (d, J=8.8 Hz, 1H), 7.46 (dd, J=2.0, 8.8 Hz, 1H), 8.18 (d, J=2.0 Hz, 1H), 8.52 (dd, J=1.2, 6.8 Hz, 1H), 8.62 (br s, 1H).
    • 3-(Hydroxymethyl)-9-methoxy-2-(1-methyl-1H-benzo[d]imidazol-5-ylamino)-4H-pyrido[1,2-a]pyrimidin-4-one (288)
  • Figure US20150018543A1-20150115-C00309
  • m.p=186° C. (decomp.); 1H NMR (400 MHz, DMSO-d6) δ 3.87 (s, 3H), 3.98 (s, 3H), 4.79 (d, J=5.6 Hz, 2H), 5.31 (t, J=5.6 Hz, 1H), 7.08 (dd, J=7.2, 7.2 Hz, 1H), 7.28 (dd, J=0.8, 7.6 Hz, 1H), 7.50 (d, J=8.8 Hz, 1H), 7.56 (dd, J=2.0, 8.8 Hz, 1H), 8.13 (s, 1H), 8.34 (d, J=1.6 Hz, 1H), 8.53 (dd, J=0.8, 7.2 Hz, 1H), 8.73 (br s, 1H).
    • 3-(Hydroxymethyl)-9-methoxy-2-(1-methyl-1H-indazol-5-ylamino)-4H-pyrido[1,2-a]pyrimidin-4-one (289)
  • Figure US20150018543A1-20150115-C00310
  • m.p=205° C. (decomp.); 1H NMR (400 MHz, DMSO-d) δ 3.40 (s, 3H), 4.08 (s, 3H), 4.78 (d, J=4.8 Hz, 2H), 5.28 (t, J=5.0 Hz, 1H), 7.12 (dd, J=7.2, 7.6 Hz, 1H), 7.32 (1H, J=1.2, 7.6 Hz, 1H), 7.62 (d, J=9.0 Hz, 1H), 7.68 (dd, J=2.0, 9.0 Hz, 1H), 8.04 (m, 1H), 8.07 (d, J=1.2 Hz, 1H), 8.53 (dd, J=1.2, 6.8 Hz, 1H), 8.75 (br s, 1H).
    • 9-(Difluoromethoxy)-2-(4-fluorophenylamino)-3-(hydroxymethyl)-4H-pyrido[1,2-a]pyrimidin-4-one (290)
  • Figure US20150018543A1-20150115-C00311
  • 1H NMR (400 MHz, DMSO-d6) δ 4.67 (d, J=5.2 Hz, 2H), 5.14 (t, J=5.2 Hz, 1H), 7.07-7.11 (m, 3H), 7.17 (t, J=74 Hz due to F2, 1H), 7.63-7.69 (m, 3H), 8.71 (d, J=7.2 Hz, 1H), 8.75 (s, 1H).
    • 2-(4-Chlorophenylamino)-9-(difluoromethoxy)-3-(hydroxymethyl)-4H-pyrido[1,2-a]pyrimidin-4-one (291)
  • Figure US20150018543A1-20150115-C00312
  • 1H NMR (400 MHz, DMSO-d6) δ 4.69 (d, J=5.6 Hz, 2H), 5.23 (t, J=5.2 Hz, 1H), 7.13 (dd, J=7.2 Hz, 7.2 Hz, 1H), 7.23 (t, J=74 Hz, 1H, due to F2), 7.30-7.33 (m, 2H), 7.72-7.75 (m, 3H), 8.75 (dd, J=1.2 Hz, 7.2 Hz, 1H), 8.86 (s, 1H);
    • 9-(Difluoromethoxy)-2-(3,4-difluorophenylamino)-3-(hydroxymethyl)-4H-pyrido[1,2-a]pyrimidin-4-one (292)
  • Figure US20150018543A1-20150115-C00313
  • 1H NMR (400 MHz, DMSO-d6) δ 4.70 (d, J=5.2 Hz, 2H), 5.22 (s, 1H), 7.16 (dd, J=7.2 Hz, J=7.2 Hz, 1H), 7.26 (t, J=74 Hz, due to F2, 1H), 7.33-7.38 (m, 2H), 7.75 (d, J=7.2 Hz, 1H), 8.12 (dd, J=7.6 Hz, 12.8 Hz, 1H), 8.76 (d, J=6.8 Hz, 1H), 8.90 (s, 1H); LC-MS (ESI, m/z): 370[M+H]+.
    • 2-(3,4-Dichlorophenylamino)-9-(difluoromethoxy)-3-(hydroxymethyl)-4H-pyrido[1,2-a]pyrimidin-4-one (293)
  • Figure US20150018543A1-20150115-C00314
  • 1H NMR (400 MHz, DMSO-d6) δ 4.68 (s, 2H), 5.19 (s, 1H), 7.15 (t, J=7.2 Hz, 1H), 7.24 (t, J=74 Hz, due to F2, 1H), 7.47-7.57 (m, 2H), 7.72 (d, J=7.2 Hz, 1H), 8.32 (d, J=2.4 Hz, 1H), 8.73 (dd, J=1.6 Hz, 7.2 Hz, 1H), 8.92 (s, 1H).
    • 2-(3-Chloro-4-fluorophenylamino)-9-(difluoromethoxy)-3-(hydroxymethyl)-4H-pyrido[1,2-a]pyrimidin-4-one (294)
  • Figure US20150018543A1-20150115-C00315
  • 1H NMR (400 MHz, DMSO-d6) δ 4.68 (d, J=4.0 Hz, 2H), 5.18 (s, 1H), 7.15 (dd, J=7.2 Hz, 7.2 Hz, 1H), 7.24 (t, J=74 Hz, 1H, due to F2), 7.32 (dd, J=9.2 Hz, 9.2 Hz, 1H), 7.50-7.54 (m, 1H), 7.73 (d, J=7.6 Hz, 1H), 8.22 (dd, J=2.8 Hz, 6.8 Hz, 1H), 8.74 (dd, J=1.2 Hz, 7.2 Hz, 1H), 8.86 (s, 1H).
    • 2-(1H-Indol-5-ylamino)-9-(difluoromethoxy)-3-(hydroxymethyl)-4H-pyrido[1,2-a]pyrimidin-4-one (295)
  • Figure US20150018543A1-20150115-C00316
  • 1H NMR (400 MHz, DMSO-d6) δ 4.72 (d, J=4.8 Hz, 2H), 5.23 (t, J=4.8 Hz, 1H), 6.34 (s, 1H), 7.05-7.09 (m, 1H), 7.23 (dd, J=8.8 Hz, 8.8 Hz, 1H), 7.25 (t, J=74.4 Hz, 1H due to F2), 7.31-7.33 (m, 2H), 7.68 (d, J=7.2 Hz, 1H), 7.93 (s, 1H), 8.70 (s, 1H), 8.73 (d, J=1.2 Hz, 1H), 10.99 (s, 1H).
    • 2-(3-chlorophenylamino)-3-(hydroxymethyl)-6,8-dimethyl-4H-pyrido[1,2-a]pyrimidin-4-one (296)
  • Figure US20150018543A1-20150115-C00317
  • 1H NMR (400 MHz, CDCl3) δ 2.32 (s, 3H), 2.40 (s, 3H), 3.55 (s, 2H), 6.78 (s, 1H), 7.06 (d, J=2.0 Hz, 1H), 7.21 (dd, J=8.0 Hz, J=8.0 Hz, 1H), 7.39 (d, J=8.4 Hz, 1H), 7.69 (d, J=2.0 Hz, 1H), 7.71 (s, 1H), 9.60 (s, 1H); LC-MS (ESI, m/z): 330 [M+H]+.
    • 7,9-Dichloro-2-(3-chlorophenylamino)-3-(hydroxymethyl)-4H-pyrido[1,2-a]pyrimidin-4-one (297)
  • Figure US20150018543A1-20150115-C00318
  • 1H NMR (400 MHz, DMSO-d6) δ 4.65 (s, 2H), 5.70 (d, J=7.6 Hz, 1H), 7.29 (dd, J=8.0 Hz, J=8.0 Hz, 1H), 7.57 (dd, J=8.0 Hz, J=8.0 Hz, 1H), 8.25 (s, 1H), 8.32 (d, J=2.0 Hz, 1H), 8.76 (d, J=2.0 Hz, 1H).
    • 2-(3-Chlorophenylamino)-7,9-difluoro-3-(hydroxymethyl)-4H-pyrido[1,2-a]pyrimidin-4-one (298)
  • Figure US20150018543A1-20150115-C00319
  • 1H NMR (400 MHz, CDCl3) δ 4.69 (d, J=4.8 Hz, 2H), 5.31 (t, J=4.8 Hz, 1H), 7.06 (dd, J=1.2 Hz, 8.0 Hz, 1H), 7.32 (t, J=8.0 Hz, 1H), 7.56 (dd, J=1.2 Hz, 8.0 Hz, 1H), 8.02 (s, 1H), 8.18-8.23 (m, 1H), 8.68 (t, J=2.0 Hz, 1H), 8.90 (s, 1H).
    • (4-Oxo-2-(phenylamino)-4H-pyrido[1,2-a]pyrimidin-3-yl)methyl benzoate (299)
  • Figure US20150018543A1-20150115-C00320
  • m.p=178-179° C.; 1H NMR (400 MHz, DMSO-d6) δ 5.66 (s, 2H), 6.96 (ddd, J=1.2, 1.2, 6.8 Hz, 1H), 7.06-7.10 (m, 1H), 7.33-7.44 (m, 5H), 7.53-7.56 (m, 1H), 7.61-7.65 (m, 1H), 7.72 (m, 2H), 8.12 (dd, J=1.2, 8.4 Hz, 1H), 9.14 (brs, 1H).
    • (4-Oxo-2-(phenylamino)-4H-pyrido[1,2-a]pyrimidin-3-yl)methyl acetate (300)
  • Figure US20150018543A1-20150115-C00321
  • m.p=160-161° C.; 1H NMR (400 MHz, CDCl3) δ 2.13 (s, 3H), 6.92 (dd, J=6.8, 7.2 Hz, 1H), 7.04-7.08 (m, 1H), 7.30-7.37 (m, 3H), 7.59-7.66 (m, 3H), 8.91 (brs, 1H), 8.94 (d, J=7.2 Hz, 1H).
    • (4-Oxo-2-(phenylamino)-4H-pyrido[1,2-a]pyrimidin-3-yl)methyl isobutyrate (301)
  • Figure US20150018543A1-20150115-C00322
  • m.p=161-163° C.; 1H NMR (400 MHz, CDCl3) δ 1.17 (d, J=7.2 Hz, 6H), 2.62-2.65 (m, 1H), 6.94 (dd, J=6.8, 7.2 Hz, 1H), 7.04-7.08 (m, 1H), 7.31-7.38 (m, 3H), 7.60-7.67 (m, 3H), 8.95 (brs, 1H), 8.95 (d, J=6.8 Hz, 1H).
    • Example 8 Additional Studies on Dinitrobenzamide Compounds
  • Two representative molecules, compounds 4 and 24, were re-synthesized in-house and subjected to conventional CFU-based activity testing in primary macrophages (FIG. 7). A ten-fold decrease in the number of CFUs, similar to that seen with INH, was observed for both compounds five days after infection on three different cell lines. This confirms the potency of this series of compounds.
  • To address the issue of toxicity, compounds 4 and 24 were tested on a panel of five cell lines derived from different body tissue. Cells were incubated with increasing amounts of compound and cell viability was assessed with resazurin after 5 days of co-incubation. Percentage cytotoxicity was determined by taking as a reference the resofurin fluorescence measured by DMSO containing wells. The concentration where fifty percent of the cells died was defined as the Minimal Toxic Concentration (MTC50). Both compounds 4 and 24 showed no cytotoxicity against the panel of cell lines suggesting this series of compounds to be promising new anti-tuberculosis drugs (Table 4).
  • To gain insight into the possible specificity of activity of compounds 4 and 24, analysis of the broad antimicrobial spectrum was undertaken and showed that the effect of these dinitrobenzamide derivatives was mainly restricted to actinomycetes with the most potent activity observed against Mycobacterium (Table 4). Of particular importance, the tested DNB were also highly active against multidrug-resistant (MDR) and extensively drug-resistant (XDR) clinical isolates, suggesting that they might act on different targets than current antituberculosis compounds.
  • Mutation frequency of M. tuberculosis H37Rv was determined for compounds 4 and 24. Increasing numbers of bacteria grew on 7H10 agar medium supplemented with different concentrations of compounds. After a 6-week growth, colonies were counted in order to evaluate the proportion of spontaneous mutational frequency (Table 6). For compound 4, 1×10−6 and 1×10−8 frequencies of resistance were found at 0.2 μg/ml and 3.2 μg/ml, respectively. Spontaneous mutational rate was therefore calculated to be 1×10−7. For compound 24, at 0.2 μg/ml and 3.2 μg/ml, frequency of mutation was 7×10−7 and 1×10−8, respectively which corresponds to a mean frequency of 3.5×10−7. Overall, these values were superior to frequency of mutation observed for INH-resistant mutants (3×10−6). These results, thus, demonstrate that this class of compounds result in a low frequency of mutation.
    • Example 9 Additional Studies on Pyridopyrimidinone Compounds
  • Table 5 shows the minimal inhibitory concentration (MIC) of one representative compound, 133, on different Mycobacterial species. While it has no effect on the fast growing Mycobacterium smegmatis mc2, it was able to inhibit typical laboratory strains such as H37Rv, H37Ra and BCG Pasteur with an MIC of 2 μM. More importantly, the antimicrobial activity of 133 was also tested against clinical isolates strains of mycobacteria. The MIC values for multi-drug-resistant (MDR-TB) and extensive-drug-resistant (XDR-TB) isolates strains were within the micromolar range.
  • To address the issue of toxicity, compound 133 was tested on a panel of seven cell lines derived from different body tissue. Cells were incubated with increasing amounts of compound and cell viability was assessed with resazurin after 5 days of co-incubation. Percentage of cytotoxicity was determined by taking as a reference the resofurin fluorescence measured by DMSO containing wells. The concentration where fifty percent of the cells died was defined as the Minimal Toxic Concentration (MTC50). Compound 133 showed no cytotoxicity for all tested cell lines up to 100 μM (Table 5). The selectivity index, which consists of the ratio between antitubercular activity and cytotoxicity was therefore above 50 for both extracellular and intracellular mycobacteria suggesting this series of compounds to be promising new anti-tuberculosis drugs.
  • The effect of this series of compounds on primary macrophages was further determined. Host cells that had priory been incubated with compound 232 harbored fewer bacteria compared to DMSO control and were more abundant at day 5 after infection as shown in FIG. 8. Similar data were obtained for compound 133 (data not shown). Conventional CFU determination was then performed seven days after infection to quantify the remaining bacterial load. A ten-fold decrease in the number of CFUs, similar to that seen with INH, was observed for both compounds on both human and mouse cells (FIG. 8). This confirms the potency of this series of compounds.
  • Mutation frequency of M. tuberculosis H37Rv was determined for compound 264. Increasing numbers of bacteria grew on 7H10 agar medium supplemented with different concentrations of compound. After a 6-week growth, colonies were counted in order to evaluate the proportion of spontaneous mutational frequency (Table 6). Compound 264 gave frequencies of resistance of 3.4×10−6 and 8×10−6 at 0.4 and 0.8 μg/ml, respectively, and 2×10−8 at both 1.6 μg/ml and 3.2 μg/ml. Accordingly, spontaneous mutational rate was calculated to be 7×10−7. Overall, these values are better than the frequency of mutation observed for INH (2.9×10−6). These results, therefore, demonstrate that this class of compounds result in a low frequency of mutation.
  • One of the current challenges for TB drug discovery is the identification of compounds that are active against persistent bacteria. Although the location and state of latent bacteria remains a matter of debate, one commonly shared hypothesis for mycobacterial persistence is that M. tuberculosis bacilli are able to survive in macrophages for prolonged periods of time and, unlike other bacteria, are able to actively replicate. The intraphagosomal profile of M. tuberculosis is complex; a large variety of genes are over-expressed and timely regulated and are also dependent on environmental factors. Altogether, this makes the identification of one specific tubercle factor that could be selected as the ideal target difficult. Consequently, non-target cell-based assays are a critical tool in the search of intracellular M. tuberculosis inhibitors.
  • Investigation of bacillus growth inhibitors within macrophages has long been limited due to cumbersome CFU plating, slow bacillus growth, safety requirements and difficulties in setting-up appropriate infection conditions. As a consequence, this approach was always used as a secondary assay after the initial selection of compounds that are active on in vitro extracellular growth. With the advent of automated confocal microscopy, the above mentioned limitations could be readdressed and the inventors show the feasibility of large scale compound screening. It was decided to perform suspension macrophage batch infection in order to minimize the steps and to meet safety requirements. To this end, careful attention was paid to the removal of the extracellular non-phagocytosed mycobacteria. The centrifugation conditions used during the wash steps were set up in order to recover only the infected cells and discard most of the extracellular bacteria. By microscopy the inventors confirmed that unbound mycobacteria represented less than 10% of the total bacterial load (data not shown). Mycobacteria are able to grow independently of host cells and consequently any remaining extracellular bacilli would greatly compromise the validity of the inventors' model. To this end, an additional amikacin treatment step was added to the protocol to further eliminate any remaining mycobacteria. Thus with the optimized protocol, there is almost no non-phagocytosed mycobacteria left by the time compound is added. The obtained results also demonstrate that it is specifically the effect on the intracellular mycobacteria that is being measured with compound treatment. Indeed, the inventors observed a weak inhibition with rifampin, an antibiotic that is known to poorly penetrate cells. The 50-fold reproducible decrease in MIC for rifampin in the intracellular assay compared to the in vitro growth assay proved that the targeted bacteria are not extracellular. Otherwise no difference would have been seen in MIC between the two assays. Similarly, compounds able to inhibit mycobacterial growth in the phenotypic cell-based assay, but not the in vitro growth assay were also identified. In addition, the fact that the compounds are mixed with previously infected cells should decrease the chance for the identification of primary infection inhibitors. However, such compounds may still be identified as blockers of neighboring cell infection.
  • Compared to a conventional CFU-plating method, the microscopy based detection of fluorescent bacteria is not sufficiently sensitive to distinguish between dead and live bacilli as the GFP signal is stable for several days. Indeed, at a high concentration of INH, rifampin or active compound, there is always 10% of the cells that appear to be infected, which is similar to the initial infection ratio. Surprisingly, no CFU could be recovered after plating such samples. Owing to the fact that latent bacilli are able to recover growth (Cho et al., 2007), the microscopy-detected bacilli must be dead bacilli rather than latent bacilli. Thus, the inventors' assay detects compounds that interfere with bacilli growth within macrophages.
  • As it is well established and confirmed (FIG. 1 a), macrophages are able to support high bacterial loads which end up encompassing a large part of the cell cytoplasm and eventually lead to macrophage cell death. It is obvious when M. tuberculosis is the infectious agent compared to BCG (Bacille Calmette-Guerin), which even at high MOI fails to induce much cytotoxicity (data not shown). Taking this into account, it was decided to set the data acquisition at day 5 post-infection when the cell number in the DMSO samples had significantly decreased relative to the antibiotically protected controls. Thus, monitoring cell number was an additional parameter enabling the inventors to confirm the compound's antibacterial activity.
  • Unlike direct fluorescence based assays, analysis for image-based assays proved to be much more variable. Several parameters that are inherent to the biology of the assay partially explain the lower Z′-values that are usually accepted for HTS validation. The remaining fluorescent dead bacilli do not have much of an impact on the Z′-value, rather the variability in the infection ratio for the DMSO controls seems to account for the discrepancy. Also of importance is the fact that, upon infection, the macrophages had a tendency to migrate which in turn led to a heterogeneous set of images (FIG. 2 a). However, the aim of the primary screen was to identify compounds fully active at a concentration of 20 μM. Thus, for this purpose, a positive Z′ for the infection ratio (INH/DMSO) was considered an acceptable value. The best proof of the validity of the hit selection according to the present invention comes from the subsequent serial dilution analysis, whereby almost 100% of the hits were confirmed. For each of the hits, a nicely fitted dose-response curve for the infection ratio was obtained as well as for the non-toxic compound in terms of cell number. Again, cell number brought an additional confirmation of the results that is totally independent of green fluorescence emission and GFP expression.
  • Obviously compounds found to be active against both intracellular and in vitro M. tuberculosis growth are the most promising. The best inhibitors isolated from this library have an inhibitory activity within the same range as INH. Further structure activity relationship studies will contribute to determine if their activity could be improved. In the course of another study using this phenotypic cell-based model, MIC down to the ng/mL scale was obtained for compounds with known in vitro antibacterial efficacy showing that compounds with a lower MIC than INH can be identified by the assay according to the present invention (data not shown). Of utmost interest are the compounds that are active only in the intracellular bacteria assay as they are likely to have a new mechanism of action independent of the infecting strain suggesting that they may also be active on the non-curable multi-drug-resistant (MDR)-strains.
  • Taken together, the above results show that monitoring M. tuberculosis growth with automated fluorescence microscopy is highly robust and reliable and that this method enables fast selection of potent anti-TB compounds.
  • TABLE 1
    QIM QIM QIM QUM QUM QUM
    QIM QIM QIM Confirm Confirm Confirm Confirm Confirm Confirm
    Primary Confirm Confirm Confirm Primary % % % Primary % % %
    QIM CellNb CellNb CellNb QIM % Inhibition Inhibition Inhibition QUM % Inhibition Inhibition Inhibition
    ID Structure CellNb 20 uM 2 uM 0.2 uM Inhibition 20 uM 2 uM 0.2 uM Inhibition 20 uM 2 uM 0.2 uM
    IPK00000132
    Figure US20150018543A1-20150115-C00323
         		88.3 113.8 208.5 241.4 25.4 50.7 0.2 10.3 99.9 89.1 41.2 43.9
    IPK00000190
    Figure US20150018543A1-20150115-C00324
         		435.5 317.6 173.6 190.0 91.9 96.5 13.2 12.7 2.9 42.3 29.4 34.6
    IPK00000203
    Figure US20150018543A1-20150115-C00325
         		77.0 148.0 92.0 241.4 −28.5 −12.3 7.7 −2.0 99.7 69.7 52.4 32.5
    IPK00000217
    Figure US20150018543A1-20150115-C00326
         		235.5 249.8 541.6 472.3 26.6 24.5 70.4 54.5 98.9 49.7 67.4 56.9
    IPK00000287
    Figure US20150018543A1-20150115-C00327
         		350.3 412.9 246.1 315.9 65.9 66.0 −1.3 11.4 −13.8 36.4 36.1 45.9
    IPK00000301
    Figure US20150018543A1-20150115-C00328
         		373.5 248.3 457.3 232.6 88.2 40.4 77.1 4.7 98.7 43.0 82.8 46.4
    IPK00000389
    Figure US20150018543A1-20150115-C00329
         		72.5 103.0 200.6 265.3 27.7 84.6 86.6 20.1 100.3 67.0 76.5 44.0
    IPK00000390
    Figure US20150018543A1-20150115-C00330
         		78.0 133.4 75.6 142.3 15.7 67.9 43.2 2.2 99.7 72.7 68.8 44.0
    IPK00000391
    Figure US20150018543A1-20150115-C00331
         		63.0 128.8 148.9 220.9 31.6 76.4 36.8 2.9 99.6 76.7 41.4 46.6
    IPK00000635
    Figure US20150018543A1-20150115-C00332
         		424.3 328.8 320.9 262.8 97.6 65.2 22.8 17.7 42.0 43.4 41.9 23.7
    IPK00000731
    Figure US20150018543A1-20150115-C00333
         		61.3 166.5 308.8 393.1 −28.2 25.8 14.7 45.7 76.6 80.8 33.0 41.0
    IPK00000802
    Figure US20150018543A1-20150115-C00334
         		305.8 484.5 218.8 306.6 83.2 98.0 9.3 0.7 34.8 98.2 31.9 36.4
    IPK00000812
    Figure US20150018543A1-20150115-C00335
         		396.3 248.0 225.6 292.9 64.1 78.4 18.9 2.7 97.2 48.0 39.7 36.5
    IPK00000933
    Figure US20150018543A1-20150115-C00336
         		314.5 333.6 475.9 264.8 79.9 56.9 92.5 8.5 30.3 69.8 44.5 31.8
    IPK00000941
    Figure US20150018543A1-20150115-C00337
         		345.8 446.5 488.3 257.8 92.9 99.6 92.8 19.6 97.7 99.4 59.6 28.3
    IPK00000942
    Figure US20150018543A1-20150115-C00338
         		376.5 255.0 473.5 326.3 93.1 97.9 92.3 21.3 92.6 100.0 50.3 28.8
    IPK00000978
    Figure US20150018543A1-20150115-C00339
         		454.5 376.1 414.4 325.5 102.6 88.6 50.2 17.0 39.5 100.1 61.1 30.0
    IPK00001006
    Figure US20150018543A1-20150115-C00340
         		322.8 380.9 344.9 412.0 81.0 81.8 18.0 17.1 98.4 39.2 43.1 39.5
    IPK00001119
    Figure US20150018543A1-20150115-C00341
         		190.8 279.1 80.0 248.8 60.0 47.6 31.6 9.7 90.5 43.1 93.2 67.2
    IPK00001165
    Figure US20150018543A1-20150115-C00342
         		145.5 201.1 336.5 259.6 6.5 41.9 23.4 4.7 100.0 96.3 40.6 39.8
    IPK00001367
    Figure US20150018543A1-20150115-C00343
         		358.0 457.3 545.1 452.5 98.7 66.7 102.1 84.1 98.7 69.7 90.2 49.4
    IPK00001368
    Figure US20150018543A1-20150115-C00344
         		276.3 438.8 528.8 400.1 65.0 67.2 101.8 50.9 98.9 77.6 77.1 50.4
    IPK00001369
    Figure US20150018543A1-20150115-C00345
         		327.5 443.3 532.1 405.8 91.3 58.6 104.9 68.9 99.2 62.4 79.1 51.2
    IPK00001370
    Figure US20150018543A1-20150115-C00346
         		309.3 518.5 510.9 412.6 88.3 103.0 98.1 50.5 98.4 67.6 79.4 46.3
    IPK00001371
    Figure US20150018543A1-20150115-C00347
         		358.8 377.4 544.3 476.5 102.9 97.5 105.2 86.3 99.4 68.4 92.2 51.6
    IPK00001372
    Figure US20150018543A1-20150115-C00348
         		355.5 457.3 541.6 448.5 82.2 100.6 103.7 63.2 100.0 98.0 73.9 57.7
    IPK00001536
    Figure US20150018543A1-20150115-C00349
         		146.5 168.0 286.0 331.0 38.1 36.1 −4.8 15.8 100.3 79.5 59.4 41.9
    IPK00001600
    Figure US20150018543A1-20150115-C00350
         		93.0 300.3 265.4 278.8 −19.0 37.9 19.4 3.2 90.5 68.0 48.6 47.1
    IPK00001605
    Figure US20150018543A1-20150115-C00351
         		192.0 282.0 148.9 206.8 67.9 43.4 0.8 −2.2 98.7 70.0 42.5 36.6
    IPK00001865
    Figure US20150018543A1-20150115-C00352
         		218.0 256.4 218.3 256.6 23.1 39.2 14.0 28.0 99.9 99.0 54.0 36.4
    IPK00001866
    Figure US20150018543A1-20150115-C00353
         		63.3 130.4 296.6 258.1 −26.8 16.7 0.4 8.6 100.6 99.6 72.0 39.3
    IPK00001882
    Figure US20150018543A1-20150115-C00354
         		106.8 184.1 209.0 443.0 −57.3 4.1 17.6 42.7 99.3 67.6 41.9 41.1
    IPK00001897
    Figure US20150018543A1-20150115-C00355
         		314.0 553.9 299.8 288.5 76.7 83.8 20.0 7.3 10.3 36.1 33.9 40.5
    IPK00001984
    Figure US20150018543A1-20150115-C00356
         		402.0 610.5 329.0 287.9 88.8 94.6 2.2 7.8 −10.5 43.7 36.8 46.7
    IPK00002187
    Figure US20150018543A1-20150115-C00357
         		405.0 609.0 403.4 305.3 90.2 96.5 29.8 15.4 2.7 41.9 42.9 49.1
    IPK00002233
    Figure US20150018543A1-20150115-C00358
         		372.0 517.1 472.3 315.6 66.0 90.0 33.5 30.3 96.0 74.2 45.4 35.7
    IPK00002443
    Figure US20150018543A1-20150115-C00359
         		203.8 205.0 349.0 352.1 5.8 59.1 58.3 50.4 75.8 67.7 40.1 34.7
    IPK00002772
    Figure US20150018543A1-20150115-C00360
         		333.3 238.1 440.4 267.0 82.6 100.0 77.7 29.3 100.0 100.9 97.6 35.1
    IPK00002774
    Figure US20150018543A1-20150115-C00361
         		366.0 435.8 478.8 268.5 87.2 96.9 83.3 −2.7 98.8 100.4 97.5 43.5
    IPK00002777
    Figure US20150018543A1-20150115-C00362
         		378.0 322.3 315.3 218.1 89.1 86.3 47.7 −12.6 99.6 100.4 96.8 40.2
    IPK00002778
    Figure US20150018543A1-20150115-C00363
         		332.0 499.4 543.5 366.0 79.6 96.3 99.3 33.0 98.8 78.2 97.5 71.7
    IPK00002785
    Figure US20150018543A1-20150115-C00364
         		315.0 224.8 487.4 429.3 100.3 40.1 99.4 83.0 99.6 71.7 97.9 97.2
    IPK00002791
    Figure US20150018543A1-20150115-C00365
         		410.8 325.6 434.1 295.3 89.0 46.1 98.5 14.2 99.3 75.0 97.6 65.8
    IPK00002835
    Figure US20150018543A1-20150115-C00366
         		315.3 308.9 478.6 489.8 94.5 17.8 102.4 64.5 99.8 48.3 98.1 94.0
    IPK00003316
    Figure US20150018543A1-20150115-C00367
         		283.0 303.9 573.9 296.1 81.9 92.3 69.6 5.0 43.4 61.3 42.0 34.1
    IPK00003361
    Figure US20150018543A1-20150115-C00368
         		188.3 111.8 434.1 210.8 31.6 62.9 13.8 16.2 94.9 67.1 37.6 51.6
    IPK00003556
    Figure US20150018543A1-20150115-C00369
         		226.0 524.3 313.1 247.1 83.3 89.0 18.8 9.1 71.9 65.6 39.6 41.5
    IPK00003558
    Figure US20150018543A1-20150115-C00370
         		104.0 279.9 330.0 292.3 −51.3 2.8 34.4 5.1 87.2 65.3 46.4 45.1
    IPK00003607
    Figure US20150018543A1-20150115-C00371
         		142.3 164.4 293.9 267.4 27.7 59.5 32.0 17.2 96.4 70.6 47.9 42.3
    IPK00004014
    Figure US20150018543A1-20150115-C00372
         		95.5 330.0 262.3 321.4 −38.6 18.1 15.6 20.4 97.7 68.4 46.8 38.7
    IPK00004145
    Figure US20150018543A1-20150115-C00373
         		324.5 243.8 527.9 437.4 88.6 87.2 103.2 56.7 44.7 100.5 47.4 38.4
    IPK00004146
    Figure US20150018543A1-20150115-C00374
         		320.0 347.1 542.6 386.9 81.2 93.3 101.2 41.8 98.3 57.9 81.4 41.1
    IPK00004147
    Figure US20150018543A1-20150115-C00375
         		362.8 345.8 516.1 479.8 102.4 95.5 101.8 74.5 91.8 99.5 82.3 43.8
    IPK00004148
    Figure US20150018543A1-20150115-C00376
         		315.3 347.6 508.4 414.6 84.5 88.4 97.5 55.9 96.4 38.6 84.5 43.7
    IPK00004149
    Figure US20150018543A1-20150115-C00377
         		336.5 338.1 535.4 394.6 83.8 94.5 102.3 56.4 98.9 39.4 85.5 44.4
    IPK00004150
    Figure US20150018543A1-20150115-C00378
         		296.3 315.9 515.5 396.3 81.6 90.8 102.2 66.3 99.2 77.7 94.3 52.0
    IPK00004151
    Figure US20150018543A1-20150115-C00379
         		351.3 350.6 505.6 368.0 89.0 94.0 102.9 55.1 98.4 70.9 85.2 40.8
    IPK00004152
    Figure US20150018543A1-20150115-C00380
         		262.5 362.8 523.5 451.8 75.6 93.7 103.2 89.7 99.0 56.0 93.0 53.1
    IPK00004153
    Figure US20150018543A1-20150115-C00381
         		426.8 431.1 523.3 197.8 100.2 97.0 98.1 18.8 96.9 44.4 82.2 15.1
    IPK00004207
    Figure US20150018543A1-20150115-C00382
         		198.0 596.4 616.9 259.4 34.7 107.1 104.0 12.4 93.4 98.0 96.5 70.8
    IPK00004230
    Figure US20150018543A1-20150115-C00383
         		193.8 376.8 658.0 384.8 73.7 95.9 75.2 11.0 85.2 95.8 62.9 39.1
    IPK00004272
    Figure US20150018543A1-20150115-C00384
         		152.0 199.9 96.1 227.8 11.9 57.6 −1.9 2.5 82.5 67.7 43.7 39.6
    IPK00004277
    Figure US20150018543A1-20150115-C00385
         		367.0 425.5 255.3 352.6 104.9 48.9 7.7 22.4 99.2 67.1 51.3 33.5
    IPK00004278
    Figure US20150018543A1-20150115-C00386
         		194.5 341.9 311.5 322.6 73.9 91.7 33.0 25.8 98.7 67.1 56.4 42.9
    IPK00004293
    Figure US20150018543A1-20150115-C00387
         		321.8 451.8 532.9 387.9 108.6 105.5 105.1 49.3 96.6 98.5 76.0 44.3
    IPK00004295
    Figure US20150018543A1-20150115-C00388
         		143.5 433.9 494.6 493.0 64.7 98.8 106.0 70.4 96.6 53.1 82.0 42.2
    IPK00004296
    Figure US20150018543A1-20150115-C00389
         		216.3 477.0 472.5 491.4 84.3 105.4 101.1 77.4 94.3 97.2 83.3 43.3
    IPK00004297
    Figure US20150018543A1-20150115-C00390
         		307.8 483.0 502.4 312.9 99.7 103.4 99.2 19.2 97.0 98.8 73.9 40.1
    IPK00004298
    Figure US20150018543A1-20150115-C00391
         		350.0 554.9 494.5 279.6 102.3 105.4 95.3 19.0 30.3 82.9 48.5 31.8
    IPK00004299
    Figure US20150018543A1-20150115-C00392
         		364.0 488.3 567.3 378.4 102.7 106.3 104.1 43.4 97.9 97.1 79.3 51.8
    IPK00004300
    Figure US20150018543A1-20150115-C00393
         		333.3 413.1 537.9 371.0 104.4 101.8 101.4 37.5 98.3 98.6 70.6 51.0
    IPK00004301
    Figure US20150018543A1-20150115-C00394
         		287.8 448.5 584.3 385.1 86.6 102.6 103.6 56.3 98.6 98.8 74.2 47.4
    IPK00004302
    Figure US20150018543A1-20150115-C00395
         		229.5 422.1 483.0 476.3 81.1 99.8 98.7 71.3 98.4 98.2 82.6 48.4
    IPK00004305
    Figure US20150018543A1-20150115-C00396
         		228.8 494.4 502.8 469.0 97.1 103.8 101.6 80.2 99.4 98.8 94.4 51.4
    IPK00004306
    Figure US20150018543A1-20150115-C00397
         		200.3 436.0 520.1 213.5 81.4 98.0 98.1 −0.4 99.8 98.5 75.2 38.5
    IPK00004307
    Figure US20150018543A1-20150115-C00398
         		303.5 416.6 541.6 392.0 104.3 98.6 102.5 68.4 99.7 98.9 79.2 44.8
    IPK00004308
    Figure US20150018543A1-20150115-C00399
         		255.5 367.6 486.8 397.6 87.3 95.1 102.7 72.9 77.2 63.9 63.5 39.8
    IPK00004309
    Figure US20150018543A1-20150115-C00400
         		258.5 459.3 516.8 420.0 95.8 102.4 100.5 59.3 99.5 98.4 80.1 49.1
    IPK00004310
    Figure US20150018543A1-20150115-C00401
         		176.8 441.6 509.8 367.0 14.3 98.3 92.7 28.5 98.7 66.9 73.1 46.5
    IPK00004311
    Figure US20150018543A1-20150115-C00402
         		366.0 514.6 532.3 350.4 104.4 104.5 99.5 40.3 97.5 70.9 76.7 43.5
    IPK00004312
    Figure US20150018543A1-20150115-C00403
         		195.0 406.3 500.3 432.0 69.9 100.0 104.6 56.5 98.5 69.8 84.5 48.9
    IPK00004313
    Figure US20150018543A1-20150115-C00404
         		177.8 468.8 469.0 269.9 49.2 104.2 101.6 17.8 98.7 98.5 75.0 48.9
    IPK00004326
    Figure US20150018543A1-20150115-C00405
         		347.3 430.3 523.3 413.6 109.4 102.1 102.2 72.4 99.5 98.6 89.6 52.7
    IPK00004327
    Figure US20150018543A1-20150115-C00406
         		125.8 438.6 549.8 459.1 47.6 102.0 105.2 87.8 99.2 98.8 86.3 67.0
    IPK00004328
    Figure US20150018543A1-20150115-C00407
         		326.5 408.3 482.8 332.6 94.9 95.9 87.1 11.2 12.2 69.7 38.8 43.0
    IPK00004329
    Figure US20150018543A1-20150115-C00408
         		415.5 454.4 567.1 489.3 111.0 100.2 105.2 71.9 98.3 98.6 76.8 51.3
    IPK00004330
    Figure US20150018543A1-20150115-C00409
         		305.0 503.3 575.8 251.1 107.2 102.5 100.8 −5.6 98.0 97.9 69.7 32.8
    IPK00004331
    Figure US20150018543A1-20150115-C00410
         		334.0 442.5 526.9 321.1 94.6 100.4 101.5 43.8 98.7 52.7 69.8 24.0
    IPK00004332
    Figure US20150018543A1-20150115-C00411
         		164.0 452.4 425.4 481.6 60.5 102.5 94.5 72.0 98.9 99.1 78.1 37.0
    IPK00004333
    Figure US20150018543A1-20150115-C00412
         		270.8 522.8 515.1 362.8 83.0 104.4 94.5 31.3 98.4 98.8 59.3 34.8
    IPK00004335
    Figure US20150018543A1-20150115-C00413
         		245.5 461.5 484.3 335.4 104.0 100.9 94.4 31.6 96.1 67.1 75.7 39.9
    IPK00004362
    Figure US20150018543A1-20150115-C00414
         		393.8 523.9 561.9 502.1 98.2 104.5 98.8 63.7 99.4 98.3 71.0 42.9
    IPK00004383
    Figure US20150018543A1-20150115-C00415
         		89.0 252.3 303.8 296.4 −80.9 31.3 17.1 22.6 99.7 99.2 50.6 34.2
    IPK00004420
    Figure US20150018543A1-20150115-C00416
         		135.0 219.0 278.5 303.0 −67.5 30.7 6.3 24.5 99.6 98.5 52.4 40.3
    IPK00004441
    Figure US20150018543A1-20150115-C00417
         		126.8 307.0 377.5 260.0 −120.3 29.9 34.0 18.0 98.6 84.0 41.4 37.1
    IPK00004501
    Figure US20150018543A1-20150115-C00418
         		145.0 273.8 328.5 305.8 −9.3 30.6 24.3 19.3 99.3 66.8 57.6 41.3
    IPK00004678
    Figure US20150018543A1-20150115-C00419
         		193.3 284.4 388.9 335.3 73.3 88.9 36.5 13.9 100.8 99.1 65.2 36.5
    IPK00004680
    Figure US20150018543A1-20150115-C00420
         		238.8 389.1 277.8 353.3 76.9 64.6 3.1 38.6 99.2 73.5 41.8 44.4
    IPK00004683
    Figure US20150018543A1-20150115-C00421
         		204.0 379.6 437.5 355.0 75.8 61.7 91.7 37.2 100.1 69.6 97.1 44.3
    IPK00004686
    Figure US20150018543A1-20150115-C00422
         		129.3 285.6 305.0 300.1 26.6 40.8 15.0 17.3 100.2 70.3 41.2 38.3
    IPK00004687
    Figure US20150018543A1-20150115-C00423
         		239.0 343.3 411.1 328.6 74.4 56.3 61.3 14.4 99.2 62.5 70.4 44.2
    IPK00004692
    Figure US20150018543A1-20150115-C00424
         		303.5 309.6 325.4 304.1 90.2 93.4 8.1 31.2 17.0 53.1 38.4 42.2
    IPK00004706
    Figure US20150018543A1-20150115-C00425
         		290.8 440.0 365.8 300.0 57.3 69.9 17.6 35.0 97.4 76.4 41.9 41.1
    IPK00004715
    Figure US20150018543A1-20150115-C00426
         		281.8 432.3 482.3 298.0 69.7 85.8 61.1 13.3 99.3 97.7 36.4 36.8
    IPK00004716
    Figure US20150018543A1-20150115-C00427
         		280.5 309.1 549.8 297.5 69.4 85.1 77.9 6.0 100.4 98.3 39.0 32.0
    IPK00004717
    Figure US20150018543A1-20150115-C00428
         		152.3 196.3 536.1 328.1 57.6 88.5 69.7 46.5 99.3 98.6 36.5 37.7
    IPK00004849
    Figure US20150018543A1-20150115-C00429
         		253.8 281.3 457.3 354.8 84.5 92.9 41.7 25.8 32.6 57.7 34.3 40.3
    IPK00004871
    Figure US20150018543A1-20150115-C00430
         		56.5 98.1 331.5 255.1 62.1 70.0 22.6 4.5 85.2 72.7 44.3 39.8
    IPK00004899
    Figure US20150018543A1-20150115-C00431
         		217.8 211.4 494.5 311.4 76.8 73.5 40.3 8.6 84.7 72.0 36.4 43.6
    IPK00004900
    Figure US20150018543A1-20150115-C00432
         		108.0 212.4 503.6 349.5 32.1 74.2 72.2 24.4 86.2 73.9 38.7 48.1
    IPK00004903
    Figure US20150018543A1-20150115-C00433
         		205.3 481.3 525.4 370.8 62.6 48.6 56.0 23.4 94.5 69.1 43.7 41.3
    IPK00004920
    Figure US20150018543A1-20150115-C00434
         		155.5 157.0 405.8 296.1 71.5 88.6 54.8 36.9 28.3 27.0 31.5 36.0
    IPK00005250
    Figure US20150018543A1-20150115-C00435
         		44.0 117.6 289.6 339.1 45.0 76.4 10.8 33.8 101.0 70.4 28.8 30.9
    IPK00005275
    Figure US20150018543A1-20150115-C00436
         		48.8 290.3 265.9 256.8 −77.4 10.1 13.3 14.7 85.6 66.9 43.8 43.3
    IPK00005778
    Figure US20150018543A1-20150115-C00437
         		177.0 292.4 203.8 252.1 −10.9 35.5 12.6 4.5 93.9 86.2 39.0 43.7
    IPK00005792
    Figure US20150018543A1-20150115-C00438
         		165.3 197.4 225.8 237.3 −30.7 27.2 23.0 26.2 89.8 81.6 29.1 33.5
    IPK00005820
    Figure US20150018543A1-20150115-C00439
         		344.8 278.0 458.8 295.1 98.1 44.3 66.6 13.3 55.8 68.0 57.9 42.5
    IPK00005821
    Figure US20150018543A1-20150115-C00440
         		452.5 453.1 525.3 341.6 90.9 56.0 75.7 24.8 52.3 41.2 54.6 47.6
    IPK00005829
    Figure US20150018543A1-20150115-C00441
         		75.5 224.8 432.0 499.8 63.0 31.5 103.7 77.6 99.2 47.5 93.4 52.2
    IPK00005830
    Figure US20150018543A1-20150115-C00442
         		315.8 435.3 483.4 325.6 95.5 70.8 98.3 21.0 84.5 48.8 72.8 47.0
    IPK00006324
    Figure US20150018543A1-20150115-C00443
         		188.3 183.6 232.5 327.4 49.6 68.7 16.7 17.4 96.2 74.8 38.1 37.1
    IPK00006503
    Figure US20150018543A1-20150115-C00444
         		197.8 172.1 227.0 299.3 68.2 73.2 1.1 14.7 −16.3 38.1 35.2 29.1
    IPK00006751
    Figure US20150018543A1-20150115-C00445
         		142.3 164.5 301.5 367.1 75.2 73.2 17.9 11.0 37.4 59.5 39.9 39.0
    IPK00006760
    Figure US20150018543A1-20150115-C00446
         		164.3 278.3 179.1 326.4 63.4 46.6 25.8 17.7 99.7 65.3 46.4 40.8
    IPK00006761
    Figure US20150018543A1-20150115-C00447
         		182.0 478.4 340.5 305.0 84.3 68.5 29.2 14.3 99.3 70.6 44.5 39.6
    IPK00006887
    Figure US20150018543A1-20150115-C00448
         		156.8 183.6 221.4 295.9 73.1 71.4 −12.0 −0.6 92.9 79.3 31.0 38.1
    IPK00007311
    Figure US20150018543A1-20150115-C00449
         		147.8 224.4 168.9 256.1 49.1 56.1 −10.5 6.4 84.0 67.2 33.5 42.2
    IPK00007329
    Figure US20150018543A1-20150115-C00450
         		230.5 193.1 551.9 315.5 78.6 86.6 55.1 1.3 94.1 74.9 73.9 50.0
    IPK00007368
    Figure US20150018543A1-20150115-C00451
         		202.3 242.6 557.3 378.6 88.2 92.6 74.7 16.9 94.9 73.4 69.2 53.5
    IPK00007369
    Figure US20150018543A1-20150115-C00452
         		246.5 465.9 375.5 335.9 87.0 32.3 14.0 21.8 80.9 72.9 44.3 37.2
    IPK00007370
    Figure US20150018543A1-20150115-C00453
         		234.0 539.8 523.0 348.3 88.6 103.0 56.7 17.8 96.3 74.9 68.1 49.8
    IPK00007371
    Figure US20150018543A1-20150115-C00454
         		218.3 534.3 467.8 244.5 87.5 65.2 29.8 −4.5 8.3 45.3 59.6 44.5
    IPK00007722
    Figure US20150018543A1-20150115-C00455
         		84.5 253.9 380.5 314.1 −33.0 46.6 33.3 35.6 95.4 68.6 45.7 45.0
    IPK00007830
    Figure US20150018543A1-20150115-C00456
         		388.3 540.5 495.6 250.6 108.5 105.0 97.0 8.2 96.6 69.7 62.0 39.2
    IPK00007853
    Figure US20150018543A1-20150115-C00457
         		386.0 523.3 489.9 298.6 84.0 106.6 95.6 17.6 98.3 68.4 73.8 39.3
    IPK00007886
    Figure US20150018543A1-20150115-C00458
         		462.0 583.0 531.4 373.4 96.9 107.5 88.3 32.7 98.5 68.0 74.5 37.9
    IPK00007913
    Figure US20150018543A1-20150115-C00459
         		294.0 528.5 493.6 217.9 69.6 101.6 97.8 8.4 97.0 97.8 68.5 36.6
    IPK00007915
    Figure US20150018543A1-20150115-C00460
         		383.3 470.9 477.4 274.6 82.4 97.9 90.5 13.4 97.1 98.6 72.6 41.5
    IPK00007988
    Figure US20150018543A1-20150115-C00461
         		181.8 368.8 289.8 254.3 −69.2 28.1 7.5 0.9 92.2 88.3 34.9 39.3
    IPK00008001
    Figure US20150018543A1-20150115-C00462
         		409.8 469.1 387.1 360.5 14.3 56.6 27.4 20.1 96.1 70.4 47.4 39.5
    IPK00008024
    Figure US20150018543A1-20150115-C00463
         		188.8 321.0 492.1 323.8 0.2 23.0 67.6 13.0 99.5 73.0 62.4 45.3
    IPK00008036
    Figure US20150018543A1-20150115-C00464
         		79.8 251.4 559.6 514.4 −91.1 26.0 75.8 70.0 98.9 71.9 68.7 59.7
    IPK00008037
    Figure US20150018543A1-20150115-C00465
         		110.8 393.6 491.4 377.0 −44.0 36.0 62.1 55.4 99.6 78.6 80.6 63.1
    IPK00008038
    Figure US20150018543A1-20150115-C00466
         		307.5 371.6 337.3 264.9 21.0 3.4 32.1 13.3 98.2 73.8 58.7 52.3
    IPK00008039
    Figure US20150018543A1-20150115-C00467
         		99.0 377.1 591.3 418.3 38.3 9.2 73.0 51.8 96.3 68.2 66.6 54.7
    IPK00008069
    Figure US20150018543A1-20150115-C00468
         		123.5 328.0 358.9 289.4 −42.5 16.5 37.6 27.5 99.1 71.8 47.6 46.9
    IPK00008081
    Figure US20150018543A1-20150115-C00469
         		363.3 482.0 559.0 405.8 94.7 99.5 104.9 66.3 55.0 69.5 48.4 50.8
    IPK00008389
    Figure US20150018543A1-20150115-C00470
         		38.8 268.3 255.5 217.4 73.5 59.1 19.5 2.6 98.7 72.9 48.1 46.3
    IPK00008599
    Figure US20150018543A1-20150115-C00471
         		67.3 261.3 322.0 267.5 37.1 49.5 4.5 2.2 84.3 72.0 47.7 49.5
    IPK00009117
    Figure US20150018543A1-20150115-C00472
         		191.5 276.8 254.8 319.9 75.6 75.7 35.4 50.6 −11.9 22.4 38.8 42.6
    IPK00009149
    Figure US20150018543A1-20150115-C00473
         		110.5 228.6 265.4 388.5 90.5 95.1 9.3 30.6 94.1 65.5 33.4 37.3
    IPK00009438
    Figure US20150018543A1-20150115-C00474
         		126.3 367.8 286.9 271.6 46.0 6.0 13.6 −9.3 97.2 68.6 54.6 41.3
    IPK00009507
    Figure US20150018543A1-20150115-C00475
         		388.3 551.8 348.6 377.4 70.9 75.8 8.0 19.2 −3.7 39.6 46.1 45.0
    IPK00010207
    Figure US20150018543A1-20150115-C00476
         		116.0 124.8 516.3 476.1 94.7 84.7 99.5 79.6 99.2 100.1 84.3 77.4
    IPK00010236
    Figure US20150018543A1-20150115-C00477
         		193.3 194.8 224.0 291.8 65.6 66.5 4.4 13.2 −1.6 16.8 32.7 36.3
    IPK00010252
    Figure US20150018543A1-20150115-C00478
         		179.8 175.9 145.9 219.5 70.4 68.1 −7.0 12.6 18.3 19.1 40.8 36.7
    IPK00010328
    Figure US20150018543A1-20150115-C00479
         		134.3 164.6 419.5 263.8 71.1 71.3 49.5 11.6 5.0 48.2 47.6 39.8
    IPK00010376
    Figure US20150018543A1-20150115-C00480
         		47.8 122.4 109.5 143.1 85.7 74.5 −1.7 −13.2 45.6 50.2 50.0 39.0
    IPK00010378
    Figure US20150018543A1-20150115-C00481
         		263.5 409.1 244.8 236.5 94.6 98.0 3.5 −3.9 90.8 94.0 34.8 31.6
    IPK00010407
    Figure US20150018543A1-20150115-C00482
         		243.5 222.5 575.9 330.4 82.3 80.2 65.1 37.5 35.1 58.0 45.1 42.5
    IPK00010411
    Figure US20150018543A1-20150115-C00483
         		203.8 370.3 278.4 240.4 89.7 83.3 19.9 10.2 36.1 52.8 42.4 26.9
    IPK00010413
    Figure US20150018543A1-20150115-C00484
         		115.8 446.5 313.3 260.6 68.8 89.6 25.2 10.1 3.7 37.9 29.0 44.7
    IPK00010420
    Figure US20150018543A1-20150115-C00485
         		29.5 81.9 153.1 230.0 65.2 34.6 −2.9 5.3 67.1 90.6 56.2 40.4
    IPK00010467
    Figure US20150018543A1-20150115-C00486
         		183.0 211.4 332.9 283.1 33.8 73.7 16.1 11.9 67.7 86.2 34.2 36.4
    IPK00010519
    Figure US20150018543A1-20150115-C00487
         		374.3 425.6 566.4 521.1 98.7 105.8 104.1 88.5 98.7 66.4 93.6 70.3
    IPK00105020
    Figure US20150018543A1-20150115-C00488
         		316.5 397.9 547.6 482.3 97.1 96.4 103.8 83.8 99.6 66.0 96.0 66.6
    IPK00010547
    Figure US20150018543A1-20150115-C00489
         		125.8 172.4 468.0 270.4 60.1 50.7 68.1 13.0 75.5 95.5 54.7 47.3
    IPK00010555
    Figure US20150018543A1-20150115-C00490
         		181.3 538.8 402.4 272.8 89.3 91.5 55.5 13.0 29.2 62.2 35.1 18.8
    IPK00010556
    Figure US20150018543A1-20150115-C00491
         		225.0 169.6 442.0 371.5 65.7 86.9 79.9 28.0 36.1 68.0 39.5 36.4
    IPK00010570
    Figure US20150018543A1-20150115-C00492
         		315.5 573.6 226.4 237.8 61.2 84.6 4.7 16.8 70.7 80.7 53.8 41.8
    IPK00010630
    Figure US20150018543A1-20150115-C00493
         		146.5 171.3 409.1 233.5 65.7 67.2 28.5 9.3 19.1 61.2 36.4 17.1
    IPK00010790
    Figure US20150018543A1-20150115-C00494
         		494.3 577.5 498.0 339.3 79.3 89.6 52.8 23.7 7.0 42.6 39.6 37.4
    IPK00010827
    Figure US20150018543A1-20150115-C00495
         		171.0 369.9 332.0 321.6 54.7 44.1 19.0 14.1 95.0 71.0 47.6 49.0
    IPK00010878
    Figure US20150018543A1-20150115-C00496
         		200.3 287.6 400.0 339.8 78.8 68.8 38.3 28.2 20.9 43.1 39.2 38.4
    IPK00010900
    Figure US20150018543A1-20150115-C00497
         		87.8 171.8 348.5 312.4 −7.8 8.2 23.8 15.6 98.4 72.2 55.2 47.6
    IPK00010999
    Figure US20150018543A1-20150115-C00498
         		183.0 170.0 174.3 332.1 8.5 28.1 20.3 22.6 101.3 99.5 90.5 29.6
    IPK00011016
    Figure US20150018543A1-20150115-C00499
         		179.5 204.9 224.6 288.0 59.0 86.0 6.0 8.1 99.4 96.3 36.7 44.2
    IPK00011017
    Figure US20150018543A1-20150115-C00500
         		155.5 173.4 221.5 292.3 55.6 71.6 12.9 23.9 92.6 97.3 47.2 44.1
    IPK00011079
    Figure US20150018543A1-20150115-C00501
         		462.3 459.5 260.1 337.0 72.9 83.4 22.8 44.6 19.7 49.5 32.4 34.0
    IPK00011267
    Figure US20150018543A1-20150115-C00502
         		250.8 383.9 356.4 188.8 −36.5 32.6 27.6 8.2 91.3 68.9 59.6 43.8
    IPK00011280
    Figure US20150018543A1-20150115-C00503
         		152.0 183.0 189.0 279.9 41.1 55.6 10.7 7.7 98.3 72.1 32.5 35.9
    IPK00011305
    Figure US20150018543A1-20150115-C00504
         		162.0 107.9 309.0 264.5 55.4 56.4 31.6 10.0 74.9 67.9 45.3 41.6
    IPK00011377
    Figure US20150018543A1-20150115-C00505
         		319.8 523.4 437.4 319.8 60.9 74.3 48.4 26.6 66.7 82.9 59.5 45.0
    IPK00011401
    Figure US20150018543A1-20150115-C00506
         		163.5 236.9 198.4 233.3 3.2 −1.5 7.3 18.3 73.9 71.1 51.4 53.6
    IPK00011705
    Figure US20150018543A1-20150115-C00507
         		656.5 578.0 540.9 503.5 79.8 80.5 45.4 49.9 12.9 46.3 44.5 39.6
    IPK00011714
    Figure US20150018543A1-20150115-C00508
         		546.0 548.6 349.0 329.5 69.1 66.5 15.6 11.2 100.8 98.6 82.8 33.9
    IPK00012262
    Figure US20150018543A1-20150115-C00509
         		131.5 92.6 328.4 254.9 62.8 65.1 26.2 13.0 90.7 67.9 37.7 26.4
    IPK00012302
    Figure US20150018543A1-20150115-C00510
         		411.8 347.8 427.0 270.5 76.8 78.2 45.3 24.6 23.9 32.0 38.2 42.1
    IPK00012303
    Figure US20150018543A1-20150115-C00511
         		540.8 467.6 319.6 234.1 78.3 72.1 12.0 −8.6 21.2 34.7 35.8 36.5
    IPK00012330
    Figure US20150018543A1-20150115-C00512
         		296.8 390.9 495.6 263.1 77.2 64.1 72.5 22.6 20.9 37.0 36.6 38.0
    IPK00012390
    Figure US20150018543A1-20150115-C00513
         		205.0 226.5 374.3 230.8 73.1 87.7 26.7 −6.0 35.1 49.4 43.2 40.5
    IPK00012392
    Figure US20150018543A1-20150115-C00514
         		135.8 127.8 409.8 277.3 45.3 15.4 32.3 12.0 96.4 77.0 46.7 31.5
    IPK00012443
    Figure US20150018543A1-20150115-C00515
         		168.3 110.6 305.5 279.0 35.6 64.7 26.2 15.5 96.6 66.0 42.2 44.0
    IPK00012454
    Figure US20150018543A1-20150115-C00516
         		503.3 591.0 268.3 320.1 75.5 75.0 16.4 21.3 22.7 38.9 37.0 34.8
    IPK00012464
    Figure US20150018543A1-20150115-C00517
         		501.5 421.5 343.1 256.5 79.9 78.2 54.7 22.6 −0.6 25.1 31.1 33.9
    IPK00012465
    Figure US20150018543A1-20150115-C00518
         		490.8 577.0 416.9 267.0 77.1 94.3 45.0 7.2 5.2 38.6 38.6 36.9
    IPK00012508
    Figure US20150018543A1-20150115-C00519
         		261.5 254.5 476.3 388.9 72.8 65.1 46.9 28.2 74.4 65.1 32.8 36.9
    IPK00012515
    Figure US20150018543A1-20150115-C00520
         		233.8 153.1 186.4 237.5 87.1 88.7 4.0 4.4 95.2 55.2 40.3 40.9
    IPK00012522
    Figure US20150018543A1-20150115-C00521
         		265.5 254.5 194.1 274.0 75.4 82.0 −11.9 3.1 81.0 73.2 40.0 38.5
    IPK00012561
    Figure US20150018543A1-20150115-C00522
         		55.5 112.0 293.5 280.8 44.1 85.8 15.7 2.8 89.0 25.3 26.3 31.2
    IPK00012633
    Figure US20150018543A1-20150115-C00523
         		123.3 86.8 126.8 270.9 80.7 73.4 12.5 24.9 98.7 99.6 66.8 41.4
    IPK00012673
    Figure US20150018543A1-20150115-C00524
         		524.3 271.6 149.0 225.6 85.7 60.8 5.7 3.3 78.1 76.0 55.4 35.5
    IPK00012837
    Figure US20150018543A1-20150115-C00525
         		519.8 402.6 312.4 274.4 43.8 26.5 8.4 6.5 90.9 71.8 95.9 90.6
    IPK00012972
    Figure US20150018543A1-20150115-C00526
         		479.5 540.9 272.3 289.8 61.8 75.4 16.8 14.1 98.1 69.4 44.9 46.3
    IPK00012991
    Figure US20150018543A1-20150115-C00527
         		436.3 477.9 426.5 372.0 −36.0 38.2 25.4 25.1 69.3 74.8 51.4 52.1
    IPK00013026
    Figure US20150018543A1-20150115-C00528
         		476.8 385.9 270.4 340.1 66.3 29.9 14.5 17.8 100.0 70.1 79.7 53.0
    IPK00013054
    Figure US20150018543A1-20150115-C00529
         		539.5 507.4 515.3 384.9 66.7 65.8 45.8 25.3 34.8 45.1 56.7 46.5
    IPK00013302
    Figure US20150018543A1-20150115-C00530
         		194.8 255.4 472.4 360.5 −44.8 37.2 27.6 18.1 90.2 68.1 49.0 35.7
    IPK00013346
    Figure US20150018543A1-20150115-C00531
         		473.3 534.5 397.5 243.9 69.6 94.4 45.3 22.5 40.1 57.0 38.3 40.2
    IPK00013450
    Figure US20150018543A1-20150115-C00532
         		560.0 526.9 490.5 255.9 99.7 100.7 70.4 0.1 95.1 64.5 71.7 34.1
    IPK00013451
    Figure US20150018543A1-20150115-C00533
         		415.5 559.0 545.1 423.9 90.2 101.5 66.7 34.9 84.3 48.7 56.3 51.4
    IPK00013462
    Figure US20150018543A1-20150115-C00534
         		207.8 406.9 296.6 308.6 −56.1 25.5 11.5 13.0 89.8 68.6 44.8 46.8
    IPK00013463
    Figure US20150018543A1-20150115-C00535
         		275.3 294.1 416.9 316.6 −18.3 12.6 23.9 33.9 94.1 67.3 54.0 49.9
    IPK00013528
    Figure US20150018543A1-20150115-C00536
         		337.5 255.6 223.4 219.5 53.3 26.6 17.5 2.9 99.0 73.2 56.9 46.4
    IPK00013812
    Figure US20150018543A1-20150115-C00537
         		480.8 501.4 420.9 343.8 96.8 104.7 92.8 16.4 98.8 67.5 90.8 37.7
    IPK00013840
    Figure US20150018543A1-20150115-C00538
         		569.8 575.8 285.0 230.3 75.0 86.4 35.4 2.8 −1.0 52.4 47.7 44.6
    IPK00013843
    Figure US20150018543A1-20150115-C00539
         		514.0 521.3 361.5 276.6 76.3 78.9 25.3 6.6 16.0 47.9 22.9 48.3
    IPK00013917
    Figure US20150018543A1-20150115-C00540
         		199.8 195.3 414.0 411.5 72.9 65.8 38.1 40.8 33.8 70.4 54.4 52.3
    IPK00014081
    Figure US20150018543A1-20150115-C00541
         		208.5 384.4 460.8 276.8 94.6 91.8 51.8 11.0 12.0 37.9 35.4 35.4
    IPK00014087
    Figure US20150018543A1-20150115-C00542
         		269.5 421.0 551.0 245.0 92.2 91.1 71.8 5.9 17.0 41.7 41.3 30.4
    IPK00014108
    Figure US20150018543A1-20150115-C00543
         		529.8 625.5 270.9 200.4 67.5 84.0 11.7 4.6 13.8 34.5 47.8 41.3
    IPK00014158
    Figure US20150018543A1-20150115-C00544
         		191.5 167.0 239.1 184.0 58.2 59.2 −2.2 −14.1 92.7 95.9 45.3 42.9
    IPK00014161
    Figure US20150018543A1-20150115-C00545
         		308.3 337.6 378.3 225.5 4.2 50.8 23.0 10.3 87.4 83.4 46.7 45.5
    IPK00014217
    Figure US20150018543A1-20150115-C00546
         		635.8 581.1 361.8 340.4 84.3 82.3 18.5 28.7 14.6 54.3 35.1 42.9
    IPK00014218
    Figure US20150018543A1-20150115-C00547
         		167.8 132.6 372.4 234.3 73.2 79.4 20.6 0.5 −1.3 38.3 39.8 39.4
    IPK00014345
    Figure US20150018543A1-20150115-C00548
         		581.8 625.4 467.4 282.6 81.2 83.5 60.3 14.4 11.5 72.5 50.0 38.3
    IPK00014422
    Figure US20150018543A1-20150115-C00549
         		262.5 377.4 452.9 318.4 −9.8 30.0 38.5 16.6 72.1 82.4 38.7 35.9
    IPK00014691
    Figure US20150018543A1-20150115-C00550
         		167.5 132.9 201.1 229.5 57.0 70.0 57.2 4.4 99.5 100.7 40.9 40.7
    IPK00014698
    Figure US20150018543A1-20150115-C00551
         		101.3 167.8 283.5 264.0 55.8 26.5 26.2 −2.8 100.3 70.3 35.1 37.6
    IPK00014717
    Figure US20150018543A1-20150115-C00552
         		81.0 177.6 186.5 330.8 11.9 40.8 47.2 22.9 98.7 74.3 40.4 45.1
    IPK00014754
    Figure US20150018543A1-20150115-C00553
         		45.3 388.4 316.0 364.1 −10.9 10.1 13.0 26.0 92.6 66.8 48.9 42.9
    IPK00014798
    Figure US20150018543A1-20150115-C00554
         		67.5 82.4 375.9 250.9 6.3 72.3 40.7 15.4 99.3 98.1 37.2 39.9
    IPK00014804
    Figure US20150018543A1-20150115-C00555
         		48.0 63.8 313.4 317.6 21.8 59.3 26.8 17.7 98.0 67.6 46.8 48.9
    IPK00014811
    Figure US20150018543A1-20150115-C00556
         		333.0 577.0 347.5 341.8 65.4 86.6 25.3 31.9 14.0 36.6 43.7 49.4
    IPK00014844
    Figure US20150018543A1-20150115-C00557
         		58.8 364.3 268.6 264.9 −15.1 8.2 14.9 11.0 98.4 67.1 43.4 41.7
    IPK00014864
    Figure US20150018543A1-20150115-C00558
         		379.3 516.6 427.3 261.9 72.5 71.4 40.3 3.7 29.2 42.0 35.3 46.5
    IPK00014865
    Figure US20150018543A1-20150115-C00559
         		47.8 73.8 338.0 247.8 41.2 29.1 17.1 6.4 99.1 65.1 43.4 43.1
    IPK00014902
    Figure US20150018543A1-20150115-C00560
         		97.0 406.3 130.0 250.3 23.5 19.1 49.7 13.5 99.1 66.8 69.8 45.8
    IPK00014944
    Figure US20150018543A1-20150115-C00561
         		259.5 465.9 298.1 200.1 57.6 47.1 21.2 −10.4 100.0 70.4 43.0 27.1
    IPK00014978
    Figure US20150018543A1-20150115-C00562
         		36.0 328.1 331.9 289.0 15.2 3.6 12.3 9.6 99.6 74.4 56.2 49.9
    IPK00015041
    Figure US20150018543A1-20150115-C00563
         		373.8 539.6 366.1 285.3 78.3 83.3 24.2 5.2 31.8 44.6 46.3 35.9
    IPK00015048
    Figure US20150018543A1-20150115-C00564
         		61.0 333.6 209.5 349.3 −96.3 16.1 −2.3 26.4 95.6 69.8 46.4 52.3
    IPK00015085
    Figure US20150018543A1-20150115-C00565
         		416.3 397.3 299.6 230.3 72.3 72.2 33.8 5.1 23.0 93.2 58.8 40.4
    IPK00015536
    Figure US20150018543A1-20150115-C00566
         		183.8 182.1 201.4 232.0 67.8 74.0 43.7 −8.6 88.3 68.3 29.0 33.2
    IPK00015751
    Figure US20150018543A1-20150115-C00567
         		321.3 227.9 377.4 393.8 96.0 46.2 100.9 39.7 99.3 47.4 82.2 47.2
    IPK00015755
    Figure US20150018543A1-20150115-C00568
         		108.3 277.0 278.0 396.4 22.0 19.9 77.5 39.0 97.6 77.0 55.2 45.1
    IPK00015849
    Figure US20150018543A1-20150115-C00569
         		96.0 266.6 168.3 248.6 −3.0 4.8 −0.8 8.4 66.6 69.1 44.3 43.2
    IPK00016045
    Figure US20150018543A1-20150115-C00570
         		436.3 371.4 285.5 296.5 66.0 70.7 43.2 31.7 17.6 25.1 30.9 45.0
    IPK00016132
    Figure US20150018543A1-20150115-C00571
         		100.3 65.1 194.0 245.4 −37.9 17.8 25.1 9.4 99.1 63.7 95.8 52.1
    IPK00016327
    Figure US20150018543A1-20150115-C00572
         		534.3 263.4 474.9 393.4 69.5 17.9 71.8 33.5 86.4 45.6 55.6 47.7
    IPK00016351
    Figure US20150018543A1-20150115-C00573
         		447.5 377.5 491.4 335.8 89.4 50.0 82.7 40.8 92.9 68.6 57.2 52.8
    IPK00016352
    Figure US20150018543A1-20150115-C00574
         		446.5 473.3 447.6 428.4 86.2 70.7 61.4 43.7 83.0 64.6 46.4 44.3
    IPK00016362
    Figure US20150018543A1-20150115-C00575
         		246.5 599.8 485.8 451.9 81.3 107.2 59.4 49.8 95.9 68.1 54.8 47.8
    IPK00016364
    Figure US20150018543A1-20150115-C00576
         		486.3 576.1 394.9 302.8 74.9 74.2 26.7 14.7 20.8 63.3 47.4 49.9
    IPK00016367
    Figure US20150018543A1-20150115-C00577
         		517.5 481.6 520.1 340.0 68.1 99.5 70.7 27.4 76.2 65.3 44.7 47.9
    IPK00016393
    Figure US20150018543A1-20150115-C00578
         		74.0 123.6 249.4 339.0 66.7 89.9 36.3 45.5 30.4 50.1 34.2 31.3
    IPK00016452
    Figure US20150018543A1-20150115-C00579
         		202.3 171.8 204.8 286.8 64.7 80.0 −2.2 0.1 91.8 86.8 36.5 38.7
    IPK00016754
    Figure US20150018543A1-20150115-C00580
         		60.3 117.3 184.8 248.0 19.5 16.5 27.7 23.2 74.3 98.3 34.3 33.3
    IPK00016810
    Figure US20150018543A1-20150115-C00581
         		106.5 185.3 137.1 294.4 38.0 65.6 17.9 8.2 100.2 54.4 38.1 43.5
    IPK00016831
    Figure US20150018543A1-20150115-C00582
         		178.8 254.9 159.9 256.1 83.9 78.1 20.6 15.7 15.9 29.3 38.8 32.3
    IPK00016832
    Figure US20150018543A1-20150115-C00583
         		167.5 254.9 352.9 312.6 75.7 81.9 37.2 18.3 50.5 29.5 31.5 35.6
    IPK00016930
    Figure US20150018543A1-20150115-C00584
         		220.0 291.1 313.0 269.3 56.2 73.7 12.4 8.8 99.3 99.4 62.8 42.8
    IPK00016942
    Figure US20150018543A1-20150115-C00585
         		106.0 218.3 410.4 361.9 −64.0 76.9 32.9 8.5 95.2 45.0 44.0 44.8
    IPK00016968
    Figure US20150018543A1-20150115-C00586
         		97.8 114.9 183.8 289.1 −5.0 −5.3 −5.9 16.8 99.1 65.3 43.9 45.5
    IPK00016976
    Figure US20150018543A1-20150115-C00587
         		62.0 137.6 188.0 301.6 −71.9 16.7 2.1 16.4 99.6 100.3 31.4 30.7
    IPK00016986
    Figure US20150018543A1-20150115-C00588
         		119.3 174.4 191.5 359.9 0.5 13.0 14.4 31.1 100.9 100.3 43.7 34.8
    IPK00016996
    Figure US20150018543A1-20150115-C00589
         		64.8 140.4 134.5 193.4 −50.2 14.7 27.5 17.0 103.8 100.6 68.8 34.4
    IPK00017027
    Figure US20150018543A1-20150115-C00590
         		59.0 118.9 319.5 499.9 −72.4 18.9 8.1 46.9 80.7 76.3 34.0 33.5
    IPK00017033
    Figure US20150018543A1-20150115-C00591
         		143.0 353.9 525.0 501.1 49.5 96.3 102.5 92.7 103.5 100.2 97.4 75.7
    IPK00017072
    Figure US20150018543A1-20150115-C00592
         		67.0 72.1 321.0 275.8 −29.5 −21.3 27.0 17.4 76.0 82.7 30.3 5.9
    IPK00017127
    Figure US20150018543A1-20150115-C00593
         		283.3 176.9 201.4 276.8 95.2 95.2 −1.5 9.0 99.2 82.5 59.9 40.9
    IPK00017146
    Figure US20150018543A1-20150115-C00594
         		55.0 108.5 82.0 236.5 24.3 72.1 0.9 11.0 100.0 59.5 73.5 39.6
    IPK00017184
    Figure US20150018543A1-20150115-C00595
         		30.0 93.9 129.5 298.4 −79.6 13.3 29.4 16.9 99.0 98.8 46.1 31.0
    IPK00017234
    Figure US20150018543A1-20150115-C00596
         		40.8 69.5 158.4 298.4 −82.4 12.5 18.2 7.7 98.2 69.9 45.1 37.8
    IPK00017235
    Figure US20150018543A1-20150115-C00597
         		104.3 185.4 470.5 345.6 0.9 59.9 55.2 28.5 101.4 81.2 53.6 41.1
    IPK00017254
    Figure US20150018543A1-20150115-C00598
         		92.0 221.3 190.0 226.6 4.1 40.4 14.5 11.4 82.1 92.0 40.3 36.2
    IPK00017306
    Figure US20150018543A1-20150115-C00599
         		54.3 314.4 224.9 308.4 44.0 15.8 9.2 26.9 74.8 73.6 58.5 44.2
    IPK00017345
    Figure US20150018543A1-20150115-C00600
         		113.0 304.0 305.5 292.8 14.9 19.2 25.3 24.8 103.7 71.5 72.0 46.7
    IPK00017527
    Figure US20150018543A1-20150115-C00601
         		44.3 177.0 131.8 302.8 −69.7 25.8 −3.3 25.1 95.8 70.7 46.5 39.9
    IPK00017824
    Figure US20150018543A1-20150115-C00602
         		111.8 140.4 158.6 199.5 −26.4 −4.1 6.9 1.3 74.0 97.0 37.9 34.0
    IPK00017905
    Figure US20150018543A1-20150115-C00603
         		124.3 190.0 194.5 352.4 −31.7 16.6 30.0 27.5 101.0 100.6 81.6 24.9
    IPK00017949
    Figure US20150018543A1-20150115-C00604
         		187.0 257.9 163.8 300.4 65.7 78.5 −8.9 17.6 16.2 54.5 44.4 48.0
    IPK00018011
    Figure US20150018543A1-20150115-C00605
         		447.8 451.5 309.9 309.1 68.2 69.7 26.0 9.7 14.8 28.9 40.6 44.8
    IPK00018016
    Figure US20150018543A1-20150115-C00606
         		217.0 234.4 323.8 332.3 46.1 69.8 12.6 13.5 93.8 68.9 41.3 43.5
    IPK00018017
    Figure US20150018543A1-20150115-C00607
         		199.3 229.5 377.5 265.0 63.2 34.5 20.2 8.6 93.8 72.0 29.6 50.6
    IPK00018076
    Figure US20150018543A1-20150115-C00608
         		467.8 565.3 289.9 347.6 70.6 92.1 37.5 43.8 90.0 63.8 40.8 32.9
    IPK00018456
    Figure US20150018543A1-20150115-C00609
         		256.3 215.1 391.5 342.5 85.1 72.7 45.0 34.4 63.3 67.3 46.9 46.5
    IPK00019245
    Figure US20150018543A1-20150115-C00610
         		219.8 386.4 376.8 334.0 88.8 65.4 22.0 16.2 −9.0 42.7 43.3 42.7
    IPK00019259
    Figure US20150018543A1-20150115-C00611
         		470.5 551.3 344.4 246.3 65.3 81.1 30.1 10.8 −8.7 41.9 31.7 42.7
    IPK00019376
    Figure US20150018543A1-20150115-C00612
         		355.5 420.4 497.1 520.4 81.4 97.4 100.4 91.9 98.6 68.4 85.6 63.9
    IPK00019599
    Figure US20150018543A1-20150115-C00613
         		305.0 488.8 537.1 477.8 79.1 69.5 105.7 96.1 98.8 74.2 97.1 70.8
    IPK00019853
    Figure US20150018543A1-20150115-C00614
         		129.5 217.9 192.3 210.1 58.5 41.9 10.2 1.8 97.3 66.2 29.5 34.9
    IPK00019854
    Figure US20150018543A1-20150115-C00615
         		153.0 189.3 394.4 309.5 38.6 77.9 27.3 20.3 88.8 55.5 44.6 31.6
    IPK00019856
    Figure US20150018543A1-20150115-C00616
         		131.5 278.1 272.5 276.0 15.1 64.7 28.7 21.2 95.9 68.3 39.0 34.3
    IPK00019970
    Figure US20150018543A1-20150115-C00617
         		227.5 256.5 301.6 345.8 70.5 69.6 11.2 23.0 −8.2 39.8 43.5 49.3
    IPK00020016
    Figure US20150018543A1-20150115-C00618
         		265.0 430.5 238.4 235.3 73.3 76.8 2.7 1.6 −16.9 50.3 42.3 40.2
    IPK00020047
    Figure US20150018543A1-20150115-C00619
         		146.0 309.6 294.9 370.5 6.3 19.1 3.5 15.8 90.3 95.0 58.9 37.1
    IPK00020208
    Figure US20150018543A1-20150115-C00620
         		283.0 279.9 354.1 396.1 65.7 67.4 18.1 23.7 4.6 28.7 38.5 39.4
    IPK00020522
    Figure US20150018543A1-20150115-C00621
         		280.0 319.8 414.0 313.6 85.1 69.7 33.2 6.2 38.2 47.4 50.0 45.5
    IPK00020542
    Figure US20150018543A1-20150115-C00622
         		205.3 314.8 430.8 330.6 71.5 69.3 58.7 19.9 44.7 47.0 65.1 51.8
    IPK00020853
    Figure US20150018543A1-20150115-C00623
         		166.8 232.5 319.1 275.9 71.6 23.3 65.1 −4.0 46.0 56.4 57.3 44.0
    IPK00021074
    Figure US20150018543A1-20150115-C00624
         		191.8 304.9 425.6 344.3 76.4 20.2 88.9 32.6 49.9 43.9 66.8 50.0
    IPK00021079
    Figure US20150018543A1-20150115-C00625
         		273.8 255.0 350.9 353.6 77.2 28.7 71.5 25.1 43.5 48.6 58.2 49.2
    IPK00021083
    Figure US20150018543A1-20150115-C00626
         		200.8 190.5 320.4 213.9 70.4 48.2 71.4 −5.0 49.8 52.8 62.8 50.6
    IPK00021926
    Figure US20150018543A1-20150115-C00627
         		548.0 218.4 476.1 435.6 91.1 51.4 98.6 63.8 98.6 46.0 84.5 45.3
    IPK00021927
    Figure US20150018543A1-20150115-C00628
         		586.3 384.6 513.9 443.3 96.9 57.4 104.8 78.1 98.7 70.3 85.0 47.6
    IPK00021928
    Figure US20150018543A1-20150115-C00629
         		623.3 240.8 480.5 433.8 106.5 46.2 98.1 57.6 98.0 40.9 83.1 42.4
    IPK00021929
    Figure US20150018543A1-20150115-C00630
         		623.5 423.8 502.5 221.3 107.4 67.6 95.1 18.5 99.3 32.9 84.5 46.5
    IPK00021930
    Figure US20150018543A1-20150115-C00631
         		700.8 300.0 564.3 356.9 103.5 93.0 101.1 13.0 99.5 99.7 82.9 44.9
    IPK00022200
    Figure US20150018543A1-20150115-C00632
         		148.3 279.5 218.5 282.6 74.4 86.2 29.9 3.6 82.3 19.7 26.8 30.8
    IPK00022204
    Figure US20150018543A1-20150115-C00633
         		181.3 124.9 210.8 261.9 65.4 85.8 69.0 40.1 54.9 78.1 21.1 30.0
    IPK00022232
    Figure US20150018543A1-20150115-C00634
         		124.8 114.3 227.8 294.8 16.2 63.2 53.1 14.4 102.4 96.7 47.9 40.7
    IPK00022459
    Figure US20150018543A1-20150115-C00635
         		60.3 95.1 248.2 333.3 72.9 86.2 41.8 14.9 30.0 31.5 37.7 45.7
    IPK00022846
    Figure US20150018543A1-20150115-C00636
         		492.8 261.5 431.4 376.6 22.5 37.8 67.1 38.9 97.5 41.0 47.1 25.0
    IPK00022950
    Figure US20150018543A1-20150115-C00637
         		488.0 322.5 352.8 412.9 60.1 83.6 21.6 24.6 99.6 28.1 39.7 36.6
    IPK00022972
    Figure US20150018543A1-20150115-C00638
         		300.3 386.8 483.6 322.8 69.4 90.7 83.2 19.6 16.9 30.0 45.7 27.1
    IPK00023002
    Figure US20150018543A1-20150115-C00639
         		223.5 277.6 453.1 388.4 −7.7 40.5 78.8 19.0 95.7 41.9 44.6 40.5
    IPK00023461
    Figure US20150018543A1-20150115-C00640
         		156.8 207.9 209.1 292.6 −61.0 −1.6 −5.8 14.6 67.9 76.3 42.7 37.4
    IPK00023509
    Figure US20150018543A1-20150115-C00641
         		91.8 251.8 346.0 276.6 46.2 36.4 32.6 3.9 98.8 45.4 70.1 22.4
    IPK00023512
    Figure US20150018543A1-20150115-C00642
         		39.8 254.0 325.5 333.5 −67.9 26.9 31.9 34.9 99.4 71.1 48.7 40.9
    IPK00023891
    Figure US20150018543A1-20150115-C00643
         		379.0 382.4 145.6 202.6 99.3 27.8 78.4 −6.0 96.7 70.1 43.2 28.6
    IPK00024037
    Figure US20150018543A1-20150115-C00644
         		132.8 156.5 116.5 213.9 17.0 67.5 11.6 −10.9 104.1 76.2 37.9 30.6
    IPK00024172
    Figure US20150018543A1-20150115-C00645
         		175.3 102.4 487.5 421.1 −43.3 28.0 67.1 53.6 100.4 97.2 92.6 68.9
    IPK00024412
    Figure US20150018543A1-20150115-C00646
         		136.0 257.8 249.4 270.0 29.9 11.8 60.1 0.5 98.4 75.7 66.8 42.3
    IPK00024744
    Figure US20150018543A1-20150115-C00647
         		584.0 332.8 158.6 174.5 89.1 97.1 17.5 12.3 −12.2 23.8 30.8 34.7
    IPK00024871
    Figure US20150018543A1-20150115-C00648
         		150.8 256.0 486.9 334.6 79.5 79.0 73.4 21.5 99.2 41.6 70.4 40.5
    IPK00024912
    Figure US20150018543A1-20150115-C00649
         		130.8 124.3 138.9 172.9 23.6 53.2 41.8 7.8 94.3 86.2 56.8 41.7
    IPK00024914
    Figure US20150018543A1-20150115-C00650
         		404.3 211.3 407.6 332.9 45.0 68.6 41.9 21.0 96.3 37.4 40.3 43.3
    IPK00024984
    Figure US20150018543A1-20150115-C00651
         		163.3 173.8 295.4 325.3 78.2 80.9 −4.9 21.4 86.0 64.6 46.3 45.8
    IPK00025149
    Figure US20150018543A1-20150115-C00652
         		162.5 282.1 327.6 285.9 27.0 15.0 18.0 0.8 92.6 65.1 55.1 44.5
    IPK00025180
    Figure US20150018543A1-20150115-C00653
         		321.0 322.1 217.8 294.5 25.5 38.2 9.2 3.9 77.6 69.2 64.3 48.0
    IPK00025412
    Figure US20150018543A1-20150115-C00654
         		179.8 351.4 306.5 225.3 30.3 13.2 13.1 −11.6 90.9 66.4 43.8 43.0
    IPK00025425
    Figure US20150018543A1-20150115-C00655
         		243.3 367.0 356.3 356.9 32.5 18.5 20.0 20.5 100.5 75.6 53.2 49.1
    IPK00025546
    Figure US20150018543A1-20150115-C00656
         		192.3 279.6 499.5 408.3 25.0 53.1 54.7 32.6 96.3 68.7 64.7 31.8
    IPK00025761
    Figure US20150018543A1-20150115-C00657
         		285.0 226.9 381.8 314.1 71.5 79.9 42.1 15.8 31.8 60.5 28.1 33.2
    IPK00025807
    Figure US20150018543A1-20150115-C00658
         		142.3 174.8 380.6 240.9 49.9 72.7 26.2 9.4 98.4 47.9 36.8 37.0
    IPK00025935
    Figure US20150018543A1-20150115-C00659
         		64.5 78.8 262.1 335.9 66.0 68.8 12.0 5.3 10.9 28.4 42.5 40.2
    IPK00025978
    Figure US20150018543A1-20150115-C00660
         		115.8 194.3 271.1 292.8 4.9 58.8 11.0 14.9 91.4 83.6 25.0 30.2
    IPK00026207
    Figure US20150018543A1-20150115-C00661
         		242.0 309.8 461.8 383.3 80.3 7.9 89.0 40.1 99.0 34.3 61.4 45.9
    IPK00026239
    Figure US20150018543A1-20150115-C00662
         		93.3 273.3 164.5 349.1 2.1 29.5 48.7 20.4 98.5 75.4 60.0 43.8
  • TABLE 2
    Scaffold Number of
    Scaffold Name Coding Compounds Scaffold Structure
    Isonicotinohydrazides I 69
    Figure US20150018543A1-20150115-C00663
    Benzamides II 19
    Figure US20150018543A1-20150115-C00664
    Thiazolhydrazides III 6
    Figure US20150018543A1-20150115-C00665
    Hydrazinecarbothioamides IV 5
    Figure US20150018543A1-20150115-C00666
    Furancarbohydrazides V 4
    Figure US20150018543A1-20150115-C00667
    Thiophenes VI 3
    Figure US20150018543A1-20150115-C00668
    Pyrazole-pyridines VII 2
    Figure US20150018543A1-20150115-C00669
    Pyridopyrimidinone VIII 1
    Figure US20150018543A1-20150115-C00670
    One hit compound IX 1
    Figure US20150018543A1-20150115-C00671
    One hit compound X 1
    Figure US20150018543A1-20150115-C00672
    One hit compound XI 1
    Figure US20150018543A1-20150115-C00673
    One hit compound XII 1
    Figure US20150018543A1-20150115-C00674
    One hit compound XIII 1
    Figure US20150018543A1-20150115-C00675
    One hit compound XIV 1
    Figure US20150018543A1-20150115-C00676
    One hit compound XV 1
    Figure US20150018543A1-20150115-C00677
    One hit compound XVI 1
    Figure US20150018543A1-20150115-C00678
    One hit compound XVII 1
    Figure US20150018543A1-20150115-C00679
    One hit compound XVIII 1
    Figure US20150018543A1-20150115-C00680
    One hit compound XIX 1
    Figure US20150018543A1-20150115-C00681
    One hit compound XX 1
    Figure US20150018543A1-20150115-C00682
  • TABLE 3
    Compound QIM (μM) QUM (μM)
    Figure US20150018543A1-20150115-C00683
       1    		 +++ +++
    Figure US20150018543A1-20150115-C00684
       2    		 ++ +++
    Figure US20150018543A1-20150115-C00685
       3    		 ++ +++
    Figure US20150018543A1-20150115-C00686
       4    		 ++ +++
    Figure US20150018543A1-20150115-C00687
       5    		 ++ +++
    Figure US20150018543A1-20150115-C00688
       6    		 ++ +++
    Figure US20150018543A1-20150115-C00689
       7    		 ++ +++
    Figure US20150018543A1-20150115-C00690
       8    		 +++ +++
    Figure US20150018543A1-20150115-C00691
       9    		 ++ +++
    Figure US20150018543A1-20150115-C00692
       10    		 ++ +++
    Figure US20150018543A1-20150115-C00693
       11    		 +++ +++
    Figure US20150018543A1-20150115-C00694
       12    		 +++ +++
    Figure US20150018543A1-20150115-C00695
       13    		 ++ +++
    Figure US20150018543A1-20150115-C00696
       14    		 ++ ++
    Figure US20150018543A1-20150115-C00697
       15    		 ++ +++
    Figure US20150018543A1-20150115-C00698
       16    		 + +
    Figure US20150018543A1-20150115-C00699
       17    		 + +
    Figure US20150018543A1-20150115-C00700
       18    		 + +
    Figure US20150018543A1-20150115-C00701
       19    		 + ++
    Figure US20150018543A1-20150115-C00702
       20    		 + +
    Figure US20150018543A1-20150115-C00703
       21    		 ++ +++
    Figure US20150018543A1-20150115-C00704
       22    		 ++ +++
    Figure US20150018543A1-20150115-C00705
       23    		 +++ +++
    Figure US20150018543A1-20150115-C00706
       24    		 +++ +++
    Figure US20150018543A1-20150115-C00707
       26    		 ++ +++
    Figure US20150018543A1-20150115-C00708
       27    		 +++ +++
    Figure US20150018543A1-20150115-C00709
       28    		 +++ +++
    Figure US20150018543A1-20150115-C00710
       29    		 +++ +++
    Figure US20150018543A1-20150115-C00711
       30    		 +++ +++
    Figure US20150018543A1-20150115-C00712
       31    		 +++ +++
    Figure US20150018543A1-20150115-C00713
       32    		 +++ +++
    Figure US20150018543A1-20150115-C00714
       33    		 ++ +++
    Figure US20150018543A1-20150115-C00715
       34    		 + +
    Figure US20150018543A1-20150115-C00716
       54    		 ++ +++
    Figure US20150018543A1-20150115-C00717
       56    		 + ++
    Figure US20150018543A1-20150115-C00718
       58    		 + +++
    Figure US20150018543A1-20150115-C00719
       59    		 ++ +++
    Figure US20150018543A1-20150115-C00720
       60    		 + +++
    Figure US20150018543A1-20150115-C00721
       61    		 + +++
    Figure US20150018543A1-20150115-C00722
       63    		 + ++
    Figure US20150018543A1-20150115-C00723
       64    		 ++ +++
    Figure US20150018543A1-20150115-C00724
       67    		 + +
    Figure US20150018543A1-20150115-C00725
       90    		 + +
    Figure US20150018543A1-20150115-C00726
       91    		 ++ +++
    Figure US20150018543A1-20150115-C00727
       92    		 + +++
    Figure US20150018543A1-20150115-C00728
       93    		 + ++
    Figure US20150018543A1-20150115-C00729
       94    		 ++ +++
    Figure US20150018543A1-20150115-C00730
       95    		 + ++
    Figure US20150018543A1-20150115-C00731
       96    		 ++ ++
    Figure US20150018543A1-20150115-C00732
       97    		 + +++
    Figure US20150018543A1-20150115-C00733
       98    		 + ++
    Figure US20150018543A1-20150115-C00734
       99    		 + ++
    Figure US20150018543A1-20150115-C00735
       100    		 + +++
    Figure US20150018543A1-20150115-C00736
       101    		 + ++
    Figure US20150018543A1-20150115-C00737
       103    		 ++ +++
    Figure US20150018543A1-20150115-C00738
       104    		 ++ +++
    Figure US20150018543A1-20150115-C00739
       105    		 +++ ++
    Figure US20150018543A1-20150115-C00740
       107    		 ++ +++
    Figure US20150018543A1-20150115-C00741
       108    		 ++ +++
    Figure US20150018543A1-20150115-C00742
       109    		 + +
    Figure US20150018543A1-20150115-C00743
       112    		 + ++
    Figure US20150018543A1-20150115-C00744
       114    		 + ++
    Figure US20150018543A1-20150115-C00745
       115    		 + ++
    Figure US20150018543A1-20150115-C00746
       116    		 + ++
    Figure US20150018543A1-20150115-C00747
       118    		 + ++
    Figure US20150018543A1-20150115-C00748
       119    		 + +++
    Figure US20150018543A1-20150115-C00749
       120    		 + +++
    Figure US20150018543A1-20150115-C00750
       121    		 + ++
    Figure US20150018543A1-20150115-C00751
       132    		 + ++
    Figure US20150018543A1-20150115-C00752
       133    		 ++ +++
    Figure US20150018543A1-20150115-C00753
       134    		 + ++
    Figure US20150018543A1-20150115-C00754
       135    		 + ++
    Figure US20150018543A1-20150115-C00755
       137    		 + +
    Figure US20150018543A1-20150115-C00756
       139    		 + +
    Figure US20150018543A1-20150115-C00757
       140    		 + +
    Figure US20150018543A1-20150115-C00758
       147    		 + +
    Figure US20150018543A1-20150115-C00759
       151    		 ++ +
    Figure US20150018543A1-20150115-C00760
       152    		 + +
    Figure US20150018543A1-20150115-C00761
       160    		 + +
    Figure US20150018543A1-20150115-C00762
       163    		 + +
    Figure US20150018543A1-20150115-C00763
       173    		 + +
    Figure US20150018543A1-20150115-C00764
       180    		 + +
    Figure US20150018543A1-20150115-C00765
       184    		 + +
    Figure US20150018543A1-20150115-C00766
       185    		 + ++
    Figure US20150018543A1-20150115-C00767
       193    		 + +
    Figure US20150018543A1-20150115-C00768
       195    		 + +
    Figure US20150018543A1-20150115-C00769
       199    		 + ++
    Figure US20150018543A1-20150115-C00770
       200    		 + +
    Figure US20150018543A1-20150115-C00771
       201    		 + +
    Figure US20150018543A1-20150115-C00772
       204    		 + +
    Figure US20150018543A1-20150115-C00773
       206    		 +++ +++
    Figure US20150018543A1-20150115-C00774
       207    		 +++ +++
    Figure US20150018543A1-20150115-C00775
       208    		 +++ +++
    Figure US20150018543A1-20150115-C00776
       209    		 +++ +++
    Figure US20150018543A1-20150115-C00777
       210    		 +++ +++
    Figure US20150018543A1-20150115-C00778
       211    		 + +++
    Figure US20150018543A1-20150115-C00779
       212    		 + +
    Figure US20150018543A1-20150115-C00780
       213    		 + +++
    Figure US20150018543A1-20150115-C00781
       214    		 + ++
    Figure US20150018543A1-20150115-C00782
       215    		 + +
    Figure US20150018543A1-20150115-C00783
       216    		 + +
    Figure US20150018543A1-20150115-C00784
       217    		 + +++
    Figure US20150018543A1-20150115-C00785
       218    		 + +
    Figure US20150018543A1-20150115-C00786
       219    		 + +
    Figure US20150018543A1-20150115-C00787
       220    		 ++ +++
    Figure US20150018543A1-20150115-C00788
       221    		 + +
    Figure US20150018543A1-20150115-C00789
       222    		 + +
    Figure US20150018543A1-20150115-C00790
       224    		 + +
    Figure US20150018543A1-20150115-C00791
       226    		 + +
    Figure US20150018543A1-20150115-C00792
       229    		 + +
    Figure US20150018543A1-20150115-C00793
       231    		 +++ +++
    Figure US20150018543A1-20150115-C00794
       232    		 +++ ++
    Figure US20150018543A1-20150115-C00795
       233    		 + +
    Figure US20150018543A1-20150115-C00796
       234    		 + +
    Figure US20150018543A1-20150115-C00797
       235    		 +++ +++
    Figure US20150018543A1-20150115-C00798
       236    		 ++ +++
    Figure US20150018543A1-20150115-C00799
       237    		 + +++
    Figure US20150018543A1-20150115-C00800
       238    		 + +
    Figure US20150018543A1-20150115-C00801
       239    		 + +
    Figure US20150018543A1-20150115-C00802
       240    		 ++ ++
    Figure US20150018543A1-20150115-C00803
       241    		 + +
    Figure US20150018543A1-20150115-C00804
       242    		 + +
    Figure US20150018543A1-20150115-C00805
       243    		 + +
    Figure US20150018543A1-20150115-C00806
       245    		 + ++
    Figure US20150018543A1-20150115-C00807
       246    		 + +
    Figure US20150018543A1-20150115-C00808
       247    		 + +++
    Figure US20150018543A1-20150115-C00809
       248    		 + +++
    Figure US20150018543A1-20150115-C00810
       249    		 + +
    Figure US20150018543A1-20150115-C00811
       250    		 + +
    Figure US20150018543A1-20150115-C00812
       251    		 + +
    Figure US20150018543A1-20150115-C00813
       252    		 + +
    Figure US20150018543A1-20150115-C00814
       253    		 + +
    Figure US20150018543A1-20150115-C00815
       254    		 + +
    Figure US20150018543A1-20150115-C00816
       255    		 + +
    Figure US20150018543A1-20150115-C00817
       256    		 + +
    Figure US20150018543A1-20150115-C00818
       257    		 +++ +++
    Figure US20150018543A1-20150115-C00819
       258    		 +++ +++
    Figure US20150018543A1-20150115-C00820
       259    		 ++ +++
    Figure US20150018543A1-20150115-C00821
       260    		 + ++
    Figure US20150018543A1-20150115-C00822
       261    		 +++ +++
    Figure US20150018543A1-20150115-C00823
       262    		 + +
    Figure US20150018543A1-20150115-C00824
       263    		 + +
    Figure US20150018543A1-20150115-C00825
       264    		 ++ +++
    Figure US20150018543A1-20150115-C00826
       265    		 +++ +++
    Figure US20150018543A1-20150115-C00827
       266    		 + +++
    Figure US20150018543A1-20150115-C00828
       267    		 +++ +++
    Figure US20150018543A1-20150115-C00829
       268    		 + ++
    Figure US20150018543A1-20150115-C00830
       269    		 ++ ++
    Figure US20150018543A1-20150115-C00831
       270    		 +++ +++
    Figure US20150018543A1-20150115-C00832
       271    		 + +++
    Figure US20150018543A1-20150115-C00833
       272    		 + +
    Figure US20150018543A1-20150115-C00834
       273    		 +++ +++
    Figure US20150018543A1-20150115-C00835
       274    		 + +++
    Figure US20150018543A1-20150115-C00836
       275    		 + +++
    Figure US20150018543A1-20150115-C00837
       276    		 + ++
    Figure US20150018543A1-20150115-C00838
       277    		 + ++
    Figure US20150018543A1-20150115-C00839
       278    		 +++ +++
    Figure US20150018543A1-20150115-C00840
       280    		 + +++
    Figure US20150018543A1-20150115-C00841
       281    		 + +++
    Figure US20150018543A1-20150115-C00842
       282    		 + +
    Figure US20150018543A1-20150115-C00843
       283    		 + +
    Figure US20150018543A1-20150115-C00844
       284    		 + +++
    Figure US20150018543A1-20150115-C00845
       285    		 + +++
    Figure US20150018543A1-20150115-C00846
       286    		 + +++
    Figure US20150018543A1-20150115-C00847
       290    		 + +
    Figure US20150018543A1-20150115-C00848
       291    		 + +
    Figure US20150018543A1-20150115-C00849
       292    		 + +
    Figure US20150018543A1-20150115-C00850
       293    		 + +
    Figure US20150018543A1-20150115-C00851
       294    		 + +
    Figure US20150018543A1-20150115-C00852
       295    		 ++ +++
    Figure US20150018543A1-20150115-C00853
       296    		 + +
    Figure US20150018543A1-20150115-C00854
       297    		 + +
    Figure US20150018543A1-20150115-C00855
       298    		 + +
    Figure US20150018543A1-20150115-C00856
       299    		 ++ +++
    Figure US20150018543A1-20150115-C00857
       300    		 ++ +++
    Figure US20150018543A1-20150115-C00858
       301    		 ++ +++
    Activity range: +++ indicates <5 uM,
    ++ indicates between 5-20 uM,
    + indicates >20 uM
  • TABLE 4
    Compounds
    4 24
    Cytotoxicity
    Host Cells Range of MTC50 (μM)
    SK-N-SH-Brain >100 >100
    HepG2-Hepatocytes >100 >100
    MRC5-Lung >100 >100
    BJ-Skin >100 >100
    HEK293-Kidney >100 >100
    Antibacterial activity & Specificity
    Range of MICs for
    Strains/Isolates Type Origin Number multiple strains (μM)
    Mycobacterium
    M. tuberculosis Drug Sensitive Tissue 1 0.38 0.31
    clinical isolates1 RIFR Sputum 2 0.05 0.08
    Tissue 2  0.02-0.05 0.08
    INHR RIFR StrepR Sputum 1 0.1 0.08
    Tissue 3 0.05-0.1 0.04-0.08
    XDR Sputum 5  0.02-0.05 0.04-0.08
    Tissue 0 0.05-0.1 0.08
    MDR Sputum 3 0.05-0.1 0.04-0.08
    Tissue 5 0.05-0.1 0.04-0.08
    M. tuberculosis H37Rv 0.6 0.6
    laboratory strains H37Ra 1.2 1.3
    Beijing 1237 0.3 0.1
    M. bovis BCG BCG Tokyo 1.2 0.6
    BCG Pasteur 1.2 1.2
    M. smegmatis mc2 155 1.2 0.6
    Gram-negative
    Acinetobacter baumannii, Escherichia coli, Enterobacter cloacae, E. aerogenes, >250 >250
    Klebsiella oxytoca, Pseudomonas aeruginosa, Salmonella enteridis, Vibrio mimicus
    Gram-positive
    Staphylococcus aureus, S. epidermis, S. capitis, S. xylosus, Micrococcus >250 >250
    luteus, Listeria innocua, Lactobacillus gallinarum, group G
    Streptococcus, Streptococcus agalactiae, S. pyogenes, Enterococcus
    faecalis, E. faecium, E. gallinarum, Bacillus pumilus
    Corynebacterium
    C. striatum 27 27
    C. jeikeium 2.7 2.7
    Fungi
    Candida albicans, C. glabrata, C. parapsilosis >250 >250
    INH: Isoniazid,
    RIF: Rifampin,
    Strep: Streptomycin,
    Rresistant.
    1The clinical isolates were isolated either from resected lung tissue or sputum specimen, which were collected from active tuberculosis in-patients from the National Masan Tuberculosis Hospital during October 2003 to March 2007.
  • TABLE 5
    Compounds
    133
    Cytotoxicity
    Range of
    Host Cells MTC50 (μM)
    SK-N-SH-Brain >100
    HepG2-Hepatocytes >100
    MRC5-Lung >100
    BJ-Skin >100
    HEK293-Kidney >100
    Jurkat-T-cell >100
    THP-1-Monocytes >100
    Primary BMDM >100
    Primary human macrophages >100
    Antibacterial activity & Specificity
    Range of
    MICs for
    multiple
    Strains/Isolates Type Origin Number strains (μM)
    Mycobacterium
    M. tuberculosis Drug Sensitive Sputum 2 5->20
    clinical isolates1 Tissue 2 2.5-5  
    RIFR Sputum 1 2.5
    Tissue 1 1.2
    INHR RIFR StrepR Sputum 3 0.3-1.2
    Tissue 1 1.2
    XDR Sputum 4 0.6-2.5
    Tissue 5 0.3-5  
    MDR Sputum 3 0.3-1.2
    Tissue 1 1.2
    M. tuberculosis H37Rv 2
    laboratory strains H37Ra 2
    BCG Pasteur-Tokyo 2
    M. smegmatis mc2 155 >100
    Gram-negative
    Acinetobacter baumannii, Escherichia coli, Enterobacter NE
    cloacae, E. aerogenes, Klebsiella oxytoca,
    Pseudomonas aeruginosa, Salmonella enteridis, Vibrio
    mimicus
    Gram-positive
    Staphylococcus aureus, S. epidermis, S. capitis, S. xylosus, NE
    Micrococcus luteus, Listeria innocua, Lactobacillus
    gallinarum, group G Streptococcus, Streptococcus
    agalactiae, S. pyogenes, Enterococcus faecalis, E. faecium,
    E. gallinarum, Bacillus pumilus
    Corynebacterium
    C. striatum NE
    C. jeikeium
    Fungi
    Candida albicans, C. glabrata, C. parapsilosis NE
    INH: Isoniazid,
    RIF: Rifampin,
    Strep: Streptomycin,
    Rresistant.
    1The clinical isolates were isolated either from resected lung tissue or sputum specimen, which were collected from active tuberculosis in-patients from the National Masan Tuberculosis Hospital during October 2003 to March 2007.
    NE: No effect up to 100 μg/mL equivalent to 320 μM.
    The antimicrobial spectrum was performed on clinical isolates from CHU d'Angers, France.
  • TABLE 6
    Concentration Bacteria inoculum (CFU) Frequency of
    Compound (μg/ml) 105 106 107 108 resistance
    4 0.2 — — 12 >100 1 × 10−6
    0.8 — — — <100
    1.6 — — — <100
    3.2 — — — 1 1 × 10−8
    24 0.2 — — 7 >100 7 × 10−7
    0.8 — — — <100
    1.6 — — — >100
    3.2 — — — 1 1 × 10−8
    INH-control 10 ND ND 33 ND 3 × 10−6
    Concentration Bacteria inoculum (CFU) Frequency of
    Compound (μg/ml) 106 107 108 resistance
    264 0.4 — 37 306 3.4 × 10−6  
    0.8 — 5 117 8 × 10−6
    1.6 — — 22 2 × 10−8
    3.2 — — 2 2 × 10−8
    INH-control 10 — 4 18 2.9 × 10−6  
    ND: not done;
    —: no colonies
    • REFERENCES
    • Abadie, V., Badell, E., Douillard, P., Ensergueix, D., Leenen, P. J., Tanguy, M., Fiette, L., Saeland, S., Gicquel, B., and Winter, N. (2005). Neutrophils rapidly migrate via lymphatics after Mycobacterium bovis BCG intradermal vaccination and shuttle live bacilli to the draining lymph nodes. Blood 106, 1843-1850.
    • Andries, K., Verhasselt, P., Guillemont, J., Gohlmann, H. W., Neefs, J. M., Winkler, H., Van Gestel, J., Timmerman, P., Zhu, M., Lee, E., et al. (2005). A diarylquinoline drug active on the ATP synthase of Mycobacterium tuberculosis. Science 307, 223-227.
    • Arain, T. M., Resconi, A. E., Singh, D. C., and Stover, C. K. (1996). Reporter gene technology to assess activity of antimycobacterial agents in macrophages. Antimicrob Agents Chemother 40, 1542-1544.
    • Brodin, P., Majlessi, L., Marsollier, L., de Jonge, M. I., Bottai, D., Demangel, Cl., Hinds, J., Neyrolles, O., Butcher, P. D., Leclerc, C., Coles, S. T., Brosch, R., (2006). Dissection of ESAT-6 system 1 of Mycobacterium tuberculosis and impact on immunogenicity and virulence. Infect Immun 74, 88-98.
    • Cho, S. H., Warit, S., Wan, B., Hwang, C. H., Pauli, G. F., and Franzblau, S. G. (2007). Low-oxygen-recovery assay for high-throughput screening of compounds against nonreplicating Mycobacterium tuberculosis. Antimicrob Agents Chemother 51, 1380-1385.
    • Cremer, I., Dieu-Nosjean, M. C., Marechal, S., Dezutter-Dambuyant, C., Goddard, S., Adams, D., Winter, N., Menetrier-Caux, C., Sautes-Fridman, C., Fridman, W. H., and Mueller, C. G. (2002). Long-lived immature dendritic cells mediated by TRANCE-RANK interaction. Blood 100, 3646-3655.
    • Fenistein, D., Lenseigne, B., Christophe, T., Brodin, P., and Genovesio, A. (2008). A fast fully automated cell segmentation algorithm for high throughput and high content screening. Cytometry part A, in press.
    • Houben, E. N., Nguyen, L., and Pieters, J. (2006). Interaction of pathogenic mycobacteria with the host immune system. Curr Opin Microbiol 9, 76-85.
    • Lenaerts, A. J., Hoff, D., Aly, S., Ehlers, S., Andries, K., Cantarero, L., Orme, I. M., and Basaraba, R. J. (2007). Location of persisting mycobacteria in a Guinea pig model of tuberculosis revealed by r207910. Antimicrob Agents Chemother 51, 3338-3345.
    • Lipinski, C. A., Lombardo, F., Dominy, B. W., and Feeney, P. J. (2001). Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings. Advanced Drug Delivery Reviews 46, 3-26.
    • Neyrolles, O., Hernandez-Pando, R., Pietri-Rouxel, F., Formes, P., Tailleux, L., Barrios Payan, J. A., Pivert, E., Bordat, Y., Aguilar, D., Prevost, M. C., et al. (2006). Is adipose tissue a place for Mycobacterium tuberculosis persistence? PLoS ONE 1, e43.
    • Rohde, K. H., Abramovitch, R. B., and Russell, D. G. (2007). Mycobacterium tuberculosis invasion of macrophages: linking bacterial gene expression to environmental cues. Cell Host Microbe 2, 352-364.
    • Salomon, J. A., Lloyd-Smith, J. O., Getz, W. M., Resch, S., Sanchez, M. S., Porco, T. C., and Borgdorff, M. W. (2006). Prospects for advancing tuberculosis control efforts through novel therapies. PLoS Med 3, e273.
    • Schnappinger, D., Ehrt, S., Voskuil, M. I., Liu, Y., Mangan, J. A., Monahan, I. M., Dolganov, G., Efron, B., Butcher, P. D., Nathan, C., and Schoolnik, G. K. (2003). Transcriptional Adaptation of Mycobacterium tuberculosis within Macrophages: Insights into the Phagosomal Environment. J Exp Med 198, 693-704.
    • Van Rie, A., and Enarson, D. (2006). XDR tuberculosis: an indicator of public-health negligence. Lancet 368, 1554-1556.

Claims (20)

We claim:
1. A screening method comprising the steps of:
(a) batch infection of host cells with fluorescently labeled M. tuberculosis mycobacteria;
(b) removing free unbound mycobacteria;
(c) adding compounds that are to be tested to a multi-well plate;
(d) seeding said host cells infected with fluorescently labeled M. tuberculosis mycobacteria into said multi-well plate containing said compounds;
(e) incubating said multi-well plate containing host cells infected with fluorescently labeled M. tuberculosis mycobacteria and said compounds;
(f) fluorescently labeling said host cells; and
(g) analyzing said multi-well plate using automated confocal microscopy.
2. The method of claim 1, wherein the screening method searches for compounds that interfere with the multiplication of M. tuberculosis within said host cells.
3. The method of claim 1, wherein the host cells are macrophages.
4. The method of claim 3, wherein the macrophages are live macrophages.
5. The method of claim 1, wherein the automated confocal fluorescence microscopy measures intracellular mycobacterial growth.
6. The method of claim 1, wherein step (g) comprises determining for each compound
a total host cell number,
a percentage of infected host cells; and/or
a percent inhibition of infection.
7. The method of claim 6, wherein the host cells are macrophages.
8. The method of claim 6, wherein step (g) further comprises analyzing the total host cell number to determine if it is high or low; and wherein
(i) a low total cell number is indicative for the compound toxicity and/or of the unrestricted growth of M. tuberculosis inside macrophages;
and/or
(ii) a high (total) cell number is indicative that the compound is not toxic and prevents mycobacterial growth.
9. The method of claim 1, further comprising the step of using control(s) selected from at least one of the following
adding a compound with known anti-tuberculosis activity instead of the compounds in step (c) as a positive control; and
adding DMSO or antibiotic control(s) instead of the compounds in step (c) as negative control.
10. The method of claim 9, wherein the compound with known anti-tuberculosis activity is selected from isoniazid, rifampin, ethionamide, and moxifloxacin.
11. The method of claim 1, wherein the M. tuberculosis mycobacteria are labeled with GFP.
12. The method of claim 1, wherein the host cells are labeled with SYTO 60.
13. The method of claim 1, wherein after step (b) an incubation with an antibiotic is carried out.
14. The method of claim 13, wherein the antibiotic is amykacin.
15. The method of claim 1, wherein the screening method is used for the high throughput screening (HTS) of large scale chemical libraries.
16. A compound that interferes with the multiplication of M. tuberculosis within host cells identified in the method of claim 1.
17. A compound that interferes with the multiplication of M. tuberculosis within host cells identified in the method of claim 3.
18. The compound of claim 17, which is represented by formula
Figure US20150018543A1-20150115-C00859
19. A compound that interferes with the multiplication of M. tuberculosis within host cells identified in the method of claim 9.
20. A compound that interferes with the multiplication of M. tuberculosis within host cells identified in the method of claim 13.
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018183382A1 (en) * 2017-03-27 2018-10-04 The Regents Of The University Of Colorado, A Body Corporate Small molecule inhibitors of bacterial efflux pumps and methods of using same

Families Citing this family (26)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2786329A1 (en) * 2010-01-13 2011-07-21 Institut National De La Sante Et De La Recherche Medicale (Inserm) Anti - infective pyrido (1,2-a) pyrimidines
HUP1000034A2 (en) * 2010-01-21 2011-09-28 Uratim Kft Pyrido[1,2-a]pyrimidine derivatives and pharmaceutical preparations thereof having anti - mycobacterial activity
ES2572387T3 (en) 2010-03-18 2016-05-31 Pasteur Institut Korea Anti-infectious compounds
WO2011136269A1 (en) 2010-04-28 2011-11-03 アステラス製薬株式会社 Tetrahydrobenzothiophene compound
US9073941B2 (en) 2010-06-28 2015-07-07 Academia Sinica Compounds and methods for treating tuberculosis infection
WO2012057599A1 (en) * 2010-10-25 2012-05-03 Universiti Sains Malaysia Anti-mycobacterial agents
CA2825905C (en) 2011-01-26 2019-06-04 University Of Rochester Small molecule rnase inhibitors and methods of use
US8877940B2 (en) 2011-02-22 2014-11-04 Institut National De La Recherche Scientifique Antibiotic tolerance inhibitors
US20130158112A1 (en) * 2011-06-20 2013-06-20 Jonathan D. SMITH Compositions and methods for inhibiting beta amyloid secretion
CN102871992A (en) * 2011-09-30 2013-01-16 中国医学科学院医药生物技术研究所 Application of 3,5-dinitrobenzene formamide in preparation of anti-tuberculosis medicaments
WO2013123081A2 (en) * 2012-02-14 2013-08-22 Children's Hospital Medical Center Use of small molecule inhibitors targeting the interaction between rac gtpase and p67 (phox)
KR20130118612A (en) * 2012-04-20 2013-10-30 (주)네오믹스 Novel aminopyridine derivatives and use thereof
ES2784223T3 (en) 2012-06-20 2020-09-23 Univ Virginia Patent Foundation Compositions and procedures for regulating glucose homeostasis and insulin action
KR102529578B1 (en) 2014-08-29 2023-05-09 (주)아모레퍼시픽 Novel adamatan derivative compound
WO2016086261A1 (en) * 2014-12-02 2016-06-09 Prana Biotechnology Limited 4H-PYRIDO[1,2-a]PYRIMIDIN-4-ONE COMPOUNDS
CZ306245B6 (en) * 2014-12-11 2016-10-26 Univerzita Karlova v Praze Farmaceutická fakulta v Hradci Králové Substituted phenyltetrazole, its use and pharmaceutical composition containing thereof
CN105712931A (en) * 2016-03-16 2016-06-29 山东师范大学 7-phenyl-5-oxo-4-substituted-1,4,5,6,7,8-hexahydroquinoline-3-carboxylic acid ethyl ester and non-catalyzed synthesis method thereof
US10596153B2 (en) * 2016-05-27 2020-03-24 Arkansas State University-Jonesboro Antimicrobial agents and the method of synthesizing the antimicrobial agents
JP7408064B2 (en) * 2017-08-21 2024-01-05 マイクロバイオティックス， インク． Metabolically stable N-acylaminoxadiazole useful as an antibacterial agent
US10357485B1 (en) 2018-09-27 2019-07-23 King Saud University Anti-cancer compound
GB2589912A (en) * 2019-12-12 2021-06-16 Chemestmed Ltd Method of suppressing cancer by RNA m6A methyltransferase mettl16 inhibitors
CN112336719A (en) * 2020-10-19 2021-02-09 济南大学 Thiazole derivative as alpha-glucosidase inhibitor and application thereof
CN113214249B (en) * 2021-04-23 2023-09-19 成都大学 Synthesis method of pyrido [1,2-a ] pyrimidine-4-thioketone compound
KR20230113192A (en) 2022-01-21 2023-07-28 재단법인 한국파스퇴르연구소 Oxazole derivative compounds having anti-bacteria activity and their medical use
EP4345092A1 (en) * 2022-09-29 2024-04-03 Faculdade de Farmácia da Universidade de Lisboa Nitrobenzamide compounds, methods and uses thereof
EP4345091A1 (en) * 2022-09-29 2024-04-03 Faculdade de Farmácia da Universidade de Lisboa Benzoic acid derivatives, methods and uses thereof

Family Cites Families (18)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
HU178910B (en) * 1977-08-19 1982-07-28 Chinoin Gyogyszer Es Vegyeszet Process for preparing 2,3-disubstituted-4-oxo-4h-pyrido/1,2-a/-pyrimidines
AU588917B2 (en) * 1985-09-25 1989-09-28 Shionogi & Co., Ltd. 9-(substituted thio)-4h-pryido(1,2-a) pyrimidin-4-one derivatives
JPH0655740B2 (en) * 1985-09-25 1994-07-27 塩野義製薬株式会社 Pyridopyrimidine derivative
HU201551B (en) * 1988-02-03 1990-11-28 Chinoin Gyogyszer Es Vegyeszet Process for producing 4-oxo-4h-pyrido(1,2-a)pyrimidine-3-carboxylic acid amide derivatives and pharmaceutical compositions comprising same
AU4231293A (en) * 1992-05-13 1993-12-13 E.I. Du Pont De Nemours And Company Substituted pyrido(1,2-A)pyrimidinone derivatives as fungicides
FR2691460B1 (en) * 1992-05-21 1994-07-22 Rhone Poulenc Rorer Sa NEW TAXANE DERIVATIVES, THEIR PREPARATION AND THE COMPOSITIONS CONTAINING THEM.
CN1312131C (en) * 2000-02-09 2007-04-25 第一制药株式会社 Anti-acid-fast bacterial agents containing pyridonecarboxylic acids as the active ingredent
US6645505B2 (en) * 2001-03-27 2003-11-11 Council Of Scientific And Industrial Research Reporter gene based method for the screening of anti-tuberculosis drugs by using essential and regulatory genes of mycobacteria as drug target
WO2002087589A1 (en) * 2001-04-26 2002-11-07 Daiichi Pharmaceutical Co., Ltd. Medicine for inhibiting drug elimination pump
EP1554586B1 (en) * 2002-10-16 2010-03-10 Council of Scientific and Industrial Research Identification of drugs against mycobacteria
WO2004037159A2 (en) 2002-10-23 2004-05-06 Obetherapy Biotechnology Compounds, compositions and methods for modulating fat metabolism
CN1580276A (en) * 2003-07-30 2005-02-16 中国科学院大连化学物理研究所 Method for screening high-flux antituberculosis bacillus medicine
EP1670797A1 (en) * 2003-09-26 2006-06-21 Jubilant Organosys Limited Process for the preparation of risperidone
EP1738170A2 (en) * 2004-03-24 2007-01-03 Rimonyx Pharmaceuticals Ltd. Screening of anti-viral drugs and pharmaceutical compositions containing thiazolidinone derivatives
CN101008029A (en) * 2006-01-27 2007-08-01 中国医学科学院医药生物技术研究所 Antitubercular drug screening model using tubercle bacillus isocitrate lyase as target
EP1916249A1 (en) * 2006-10-10 2008-04-30 LEK Pharmaceuticals D.D. 3-(benzo[d][1,3]dioxol-5-ylmethyl)-4-(thio)oxo-2-(thio)oxo-azolidin-5-ylidene derivatives as antibacterial agents
CN1945324A (en) * 2006-10-19 2007-04-11 复旦大学 Method for screening Mycobacterium tuberculosis drug-resistant protein
JP2011132142A (en) * 2009-12-22 2011-07-07 Kowa Co Phenyloxadiazole compound having erythropoietin production-promoting action

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
CAS Registry Entry for Registry No. 329221-28-5, which entered STN on March 28th, 2001 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018183382A1 (en) * 2017-03-27 2018-10-04 The Regents Of The University Of Colorado, A Body Corporate Small molecule inhibitors of bacterial efflux pumps and methods of using same
US11110080B2 (en) 2017-03-27 2021-09-07 The Regents Of The University Of Colorado, A Body Corporate Small molecule inhibitors of bacterial efflux pumps and methods of using same
US11534429B2 (en) 2017-03-27 2022-12-27 The Regents Of The University Of Colorado Small molecule inhibitors of bacterial efflux pumps and methods of using same

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