Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D519/00—Heterocyclic compounds containing more than one system of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring system not provided for in groups C07D453/00 or C07D455/00
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/04—Antibacterial agents
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
  • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
  • C07D487/04—Ortho-condensed systems
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
  • C07D487/12—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains three hetero rings
  • C07D487/16—Peri-condensed systems
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
  • C07D487/22—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains four or more hetero rings
• • 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
  • C07D498/22—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 four or more hetero rings
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D515/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen, oxygen, and sulfur atoms as the only ring hetero atoms, not provided for in groups C07D463/00, C07D477/00 or C07D499/00 - C07D507/00
  • C07D515/22—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen, oxygen, and sulfur atoms as the only ring hetero atoms, not provided for in groups C07D463/00, C07D477/00 or C07D499/00 - C07D507/00 in which the condensed system contains four or more hetero rings

Definitions

• the present disclosure relates to the field of medicinal chemistry and in particular to compounds, and pharmaceutical compositions thereof, that are useful as antibiotics.
• tricyclic gyrase compounds inhibit DNA Gyrase B (GyrB) and Topoisomerase IV (ParE) enzymes.
• Related methods of treating bacterial infections and methods of making the compounds using novel intermediates are also contemplated.
• Gyrase B DNA Gyrase B
• ParentE Topoisomerase IV enzymes necessary for DNA replication.
• Gyrase inhibitors have been disclosed in RE40,245, which is hereby incorporated by reference in its entirety.
• the GyrB enzymatic pocket has been characterized in detail in Wigley, D.B. et al, Nature, 351(6328), 624-629, 1991. See also, Tsai FT, et al, The high-resolution crystal structure of a 24-kDa gyrase B fragment from E. coli complexed with one of the most potent coumarin inhibitors, clorobiocin, Proteins. 1997 May; 28(l):41-52.
• ParE enzymatic pocket has been characterized in detail in Bellon, S., et al. Crystal structures of Escherichia coli topoisomerase IV ParE subunit (24 and 43 kilodaltons) : a single residue dictates differences in novobiocin potency against topoisomerase IV and DNA gyrase, Antimicrob. Agents Chemother. 48: 1856- 1864 (2004). These references are hereby incorporated by reference in their entirety.
• PCT/US2012/029104 filed by the same assignees in the present application, discloses tricyclic gyrase inhibitors and is incorporated herein by reference in its entirety.
• Tricyclic gyrase compounds of Formula I include compounds herein, inhibit DNA Gyrase B (GyrB) and Topoisomerase IV (ParE) enzymes.
• the claims do not include the compounds disclosed in PCT/US2012/029104, with the exception of unexpectedly advantageous species of compounds, which have not been previously disclosed but may fall within the genus of compounds disclosed in PCT/US2012/029104.
• the present claims do not include: A compound having the structure of Formula ⁇
• L is O or S
• R 8 is H or an interacting substituent having a length of about 1 A to about 5 A from the carbon attachment point on the A Ring to the terminal atom in R and a width of about 3.3 A or less;
• X, Y and Z are independently selected from the group consisting of N, CR X , CR Y , and CR Z , provided that no more than two of X, Y and Z are N,
• R x is H or an interacting substituent having a length of about 1 A to about 2 A from the carbon in CR X to the terminal atom in R x ;
• R Y is H or an interacting substituent having a length of about 1 A to about 3 A from the carbon in CR Y to the terminal atom in R Y ;
• R z is H or an interacting substituent having a length of about 1 A to about 2 A from the carbon in CR Z to the terminal atom in R z ;
• R is a 6-membered aryl or heteroaryl ring containing 0-3 O, S, or N heteroatoms, optionally substituted with 0-3 noninterfering substituents, wherein 2 adjacent noninterfering substituents on R 2 may form one or more fused rings with the 6-membered aryl or heteroaryl ring;
• R 4 is:
• the optional substituent is 0-3 noninterfering substituents; wherein R a is a 5-6 membered aryl or heteroaryl containing 0-3 O, S, or N heteroatoms optionally substituted with 0-3 noninterfering substituents; wherein the R 4 substituent does not project greater than about 3 A below the plane of the A, B and C Rings toward the GyrB/ParE binding pocket floor in the bound conformation; and wherein R does not sterically interfere with R or Z when the compound is in the bound conformation.
• R 8 is:
• R a is H or an interacting substituent having a length of about 1 A to about 3.3 A from the adjacent nitrogen to the terminal atom in R 8a and a width of about 3.3 A or less;
• R 8b and R 8c are independently H or C1-C6 alkyl
• R 8d is or a pharmaceutically acceptable salt thereof
• Q is CH or N
• R 8e is (CR 8g 2) n -basic amine, wherein each R 8g may be independently H or C1-C3 alkyl;
• n 0-2;
• R 8f is hydrogen or C1-C6 alkyl optionally substituted with OH or NH 2 ;
• R 8e and R 8f may join to form a C3-C12 hydrocarbyl ring containing 0-3 heteroatoms selected from O, N and S optionally substituted with 0-3 noninterfering substituents;
• R 8j and R 8k are independently H or C1-C8 hydrocarbyl residue
• R ya is H or CI -4 alkyl
• R 9 is:
• the 5-membered heteroaryl ring of R 2 has O, S, N, NH, CH, CF, or CC1, independently at each of the positions immediately adjacent the position where R 2 attaches to L, if L is O, S, NH, CH 2 , CHF, or CF 2 ;
• 6-membered or 5-membered non-aryl or non-heteroaryl ring of R has O, S, N, NH, CH, CF, or CH 2 , independently at each position immediately adjacent the position where R 2 attaches to L, if L is O, S, NH, CH 2 , CHF, or CF 2 ;
• 2 adjacent noninterfering substituents of R in a)-f) may form one or more fused rings with the 6-membered aryl or heteroaryl ring, the 5- membered heteroaryl ring, or the 6-membered or 5-membered non-aryl or non-heteroaryl ring;
• R 2a contains an oxygen residue derived from an R 2 as in a)-f), wherein R 2 has an OH group, wherein the R 2 OH is replaced with an oxygen residue in R 2a , and wherein the oxygen residue is linked to P; h) joined with R 4 to form a fused ring, wherein R 2 is a ring as recited in a)-f) attached to R 4 ; wherein if a 6-membered ring of R 2 is attached to L and R 4 , R 4 is attached through the meta or para position of the 6-membered ring with respect to the point of attachment to L;
• R 2b , R 2c , R 2d , R 2f and R 2g independently may be each N or CR 2e wherein R C 1 -C4 alkyl optionally substituted with a noninterfering substituent; or
• Ra is a 5-6 membered aryl or heteroaryl containing 0-3 O, S, or N heteroatoms optionally substituted with 0-3 noninterfering substituents;
• the optional substituent is 0-3 noninterfering substituents; wherein the R 4 substituent of a)-d) does not project greater than about 3 A below the plane of the A, B and C Rings toward the GyrB/ParE binding pocket floor in the bound conformation; and
• R 4 does not sterically interfere with R 2 or Z when the compound is in the bound conformation
• R 4a contains an oxygen residue derived from a non-prodrug R 4 as in b)-d) or g)-i), wherein the non-prodrug R 4 has an OH group, wherein the R 4 OH is replaced with an oxygen residue in R 4a , and wherein the oxygen residue is linked to P;
• R 4b and R 4c are independently H or C1-C6 alkyl
• Q is CH or N
• R 4e is (CR 4g 2) n -basic amine, wherein each R 4g may be independently H or C1-C3 alkyl;
• n 0-2;
• R 4f is hydrogen or C1-C6 alkyl optionally substituted with OH or
• R 4e and R 4f may join to form a ring
• R 4 ⁇ and R 4k are independently H or C1-C8 hydrocarbyl residue; f) a prodrug-containing substituent, wherein the compound has the structure of Formula ⁇ "
• R 4 is a 5- to 15-member hydrocarbyl linker containing 0-6 O, S or N atoms in the backbone of the linker that attaches to the R group wherein atoms in the 5- to 15-member hydrocarbyl linker are optionally substituted with a noninterfering substituent;
• X is N or CR , wherein R is H or an interacting substituent having a length of about 1 A to about 2 A from the carbon in CR X to the terminal atom in R x ;
• Y is N or CR Y , wherein R Y is H or an interacting substituent having a length of about 1 A to about 3 A from the carbon in CR Y to the terminal atom in R Y ;
• R 41 is CR 10 , CR 10 CR n , NR 12 , O or S;
• R 4m is CR 10 , CR 10 CR n , or NR 12 ;
• R 4n is CR 10 , CR 10 CR n , NR 12 , O or S
• each of R 10 , R 11 and R 12 is independently H or a noninterfering substituent; wherein 2 adjacent noninterfering substituents on either R 41 and R 4m or R 4m and R 4n may form one or more fused rings;
• the dashed lines indicate an optional double bond when two adjacent R 41 , R 4m , and R 4n are CR 10 and R 4 ° is CH or N;
• R 4 ° is:
• R 4n may be CH, CH 2 , S, NH, O, CHF, or CF 2 ;
• D Ring contains at least one N in the backbone or wherein a substituent on the D Ring contains at least one N;
• the D ring does not project greater than about 3 A below the plane of the A, B and C Rings toward the GyrB/ParE binding pocket floor in the bound conformation; and wherein the D ring does not sterically interfere with R when the compound is in the bound conformation;
• R 8 is: a. a prodrug-containing substituent, wherein the compound has the structure of Formula II:
• R a is H or an interacting substituent having a length of about 1 A to about 3.3 A from the adjacent nitrogen to the terminal atom in R 8a and a width of about 3.3 A or less;
• R 8b and R 8c are independently H or C 1 -C6 alkyl; wherein R 8d is
• R 8 , OR 8k or a pharmaceutically acceptable salt thereof
• Q is CH or N
• R 8e is (CR 8g 2 ) n -basic amine, wherein each R 8g may be independently H or C1-C3 alkyl;
• n 0-2;
• R is hydrogen or C1-C6 alkyl optionally substituted with OH or NH 2 ;
• R 8e and R 8f may join to form a C3-C12 hydrocarbyl ring containing 0-3 heteroatoms selected from O, N and S optionally substituted with 0-3 noninterfering substituents; wherein R 8 ⁇ and R 8k are independently H or C1-C8 hydrocarbyl residue; or
• R 9a is H or CI -4 alkyl
• R 9 is linked to an N in R 8 to form a prodrug-containing substituent, wherein the compound has the structure of Formula III;
• the 5-membered heteroaryl ring of R has O, S, N, NH, CH, CF, or CC1, independently at each of the positions immediately adjacent the position where R attaches to L, if L is O, S, NH, CH 2 , CHF, or CF 2 ;
• 6-membered or 5-membered non-aryl or non-heteroaryl ring of R 2 has O, S, N, NH, CH, CF, or CH 2 , independently at each position immediately adjacent the position where R attaches to L, if L is O, S, NH, CH 2 , CHF, or CF 2 ;
• a 6-membered aryl or heteroaryl ring containing 0-3 O, S, or N heteroatoms, optionally substituted with 0-3 noninterfering substituents ii. a 5-membered heteroaryl ring containing 1-4 O, S, or N heteroatoms, optionally substituted with 0-2 noninterfering substituents, or iii. a 6-membered or 5-membered non-aryl or non-heteroaryl ring containing 0-3 O, S, or N heteroatoms, optionally with 0-3 noninterfering substituents,
• L contains two or more members in the backbone thereof in any of e.i. to e.iii.;
• 2 adjacent noninterfering substituents of R in a.-e. may form one or more fused rings with the 6-membered aryl or heteroaryl ring, the 5- membered heteroaryl ring, or the 6-membered or 5-membered non-aryl or non-heteroaryl ring;
• R 2a contains an oxygen residue derived from an R 2 as in a)-f), wherein R 2 has an OH group, wherein the R 2 OH is replaced with an oxygen residue in R 2a , and wherein the oxygen residue is linked to P, which is discussed below in more detail;
• R 2 is a ring as recited in a)-f) attached to R 4 ; wherein if a 6-membered ring of R 2 is attached to L and R 4 , R 4 is attached through the meta or para position of the 6-membered ring with respect to the point of attachment to L
• R 2b , R 2c , R 2d , R 2f and R 2g independently may be each N or CR 2e wherein R 2e is H or C1-C4 alkyl optionally substituted with a noninterfering substituent, such as, R may be N, or such as R m aayy bbee NN aanndd RR 2c0 aanndd RR 2d mmaayy eeaacchh bbee CCRR 2 e , for example, CH; or
• R 4a contains an oxygen residue derived from a non- prodrug R 4 as in b)-d) or g)-i), wherein the non-prodrug R 4 has an OH group, wherein the R 4 OH is replaced with an oxygen residue in R 4a , and wherein the oxygen residue is linked to P;
• R 4b and R 4c are independently H or C 1 -C6 alkyl
• Q is CH or N
• R 4e is (CR 4g 2) n -basic amine, wherein each R 4g may be independently H or C1-C3 alkyl;
• n 0-2;
• R 4f is hydrogen or C1-C6 alkyl optionally substituted with OH or NH 2 ;
• R 4e and R 4f may join to form a ring
• R 4j and R 4k are independently H or C1-C8 hydrocarbyl residue; or a prodrug-containing substituent, wherein the compound has the structure of Formula ⁇ "
• R is a 5- to 15- member hydrocarbyl linker containing 0-6 O, S or N atoms in the backbone of the linker that attaches to the R 2 group wherein atoms in the 5- to 15-member hydrocarbyl linker are optionally substituted with a noninterfering substituent; or
• R 41 is CR 10 , CR 10 CR n , NR 12 , O or S;
• R 4m is CR 10 , CR 10 CR n , or NR 12 ;
• R 4n is CR 10 , CR 10 CR n , NR 12 , O or S wherein each of R 10 , R 11 and R 12 is independently H or a noninterfering substituent;
• R 41 and R 4m or R 4n andR 4m may form one or more fused rings;
• the dashed lines indicate an optional double bond when two adjacent R 41 , R 4m , and R 4n are CR 10 and R 4 ° is CH or N;
• R 4 ° is:
• R 4n may be CH, CH 2 , S, NH, O, CHF, or CF 2 ;
• D Ring contains at least one N in the backbone or wherein a substituent on the D Ring contains at least one N;
• the D ring does not project greater than about 3 A below the plane of the A, B and C Rings toward the GyrB/ParE binding pocket floor in the bound conformation; and wherein the D ring does not sterically interfere with R 2 when the compound is in the bound conformation.
• Figure 1 illustrates a schematic representation of the receptor constraints on the compound, particularly, the binding modes of the tricyclic inhibitors to the GyrB/ParE active-site pocket (from crystallographic data). The measurements provided for the lengths are measured from atom center of the A Ring member to the atom center of the nearest non- hydrogen atom on the active site pocket.
• the figure indicates a length of about 6 A to about 8 A from the C atom attached to R 8 to the atom on the active site pocket; about 4 A to about 5 A from the A Ring atom of X to the atom on the active site pocket; about 4 A to about 6 A from the A Ring atom of Y to the atom on the active site pocket; and about 4 A to about 6 A from the A Ring atom of Z to the atom on the active site pocket.
• the relative positions of the R 8 , R 4 , and cyclic R 2 substituents are shown.
• the approximate shape of a cross-section of a representative GyrB/ParE active-site pocket in and above the plane of the tricyclic scaffold i.e., the A, B and C Rings
• the hatched area having unbroken lines depicts regions of the inhibitor that are covered on both surfaces by the active-site pocket.
• the approximate shape of a cross-section of a representative GyrB/ParE active-site pocket below the plane of the tricyclic scaffold is shown.
• the hatched area having dashed lines depict regions of the inhibitor that make contact with the floor surface of the active-site pocket, while the plane above the tricyclic ring system is solvent exposed.
• the approximate position of the conserved substrate -binding Asp side-chain and structural water molecule are shown in Figure 1 , along with the constellation of potential hydrogen-bonds (depicted as dotted lines) observed between the tricyclic scaffold and the Asp and water.
• the solvent exposed and solvent sheltered faces of the active-site pocket are highlighted.
• the solvent refers to the in vivo surroundings of GyrB/ParE active site as part of a protein, which generally includes an aqueous environment in which the protein is situated within a cell. Also, the R 4 moiety in some aspects does not project atoms greater than about 3 A below the plane of the tricyclic ring system towards the GyrB/ParE binding pocket floor in the bound state.
• Figure 2 illustrates a schematic representation of the intramolecular constraints on the compound wherein R is a 6-membered ring. Specifically, the molecular geometry and the conformations of R-groups necessary to allow binding of tricyclic inhibitors to the GyrB/ParE active-site pockets constrain the size and composition of substituents at certain positions on the inhibitor scaffold. This figure illustrates regions of potential steric interference between the R 4 substituent and the R 2 or R z substituent in the bound conformation.
• Figure 3 illustrates an example of relative positions of a primary amine that is encompassed within R 4 when bound to GyrB/ParE. This illustration also applies to a secondary amine, which is not shown in Figure 3.
• the volume occupied by the R 4 amine with respect to the tricyclic scaffold across the amines was determined using a four point trilateration procedure based on distances between the R 4 amine and four different atoms on the tricyclic scaffold from 17 different crystal structures of complexes of E. faecalis GyrB with tricyclic inhibitors containing a diverse set of R 4 amines comprising a secondary or tertiary amine attached to the C Ring through the secondary or tertiary amine N and a primary or secondary amine that is not attached to the C Ring.
• the relative position of the primary (or secondary, not shown) amine would be above the plane of the tricyclic scaffold, to avoid impinging the floor of the active site.
• Compounds of Formula I herein include in some aspects, one or more of the variables in the Formula ⁇ , i.e., L, X, Y, Z, R 2 , R 4 , or R 8 , disclosed in PCT/US2012/029104, which has been replaced with one or more "new" variable substituents.
• Cardiovascular side effects are a major reason for drug failures.
• Inhibition of hERG human ether a go-go related gene
• hERG human ether a go-go related gene
• QTc interval prolongation of the QTc interval
• Compounds 9.1 and 9.2 were found to be unexpectedly and significantly more selective in the hERG assay than previous compounds in the series. These compounds also have excellent broad-spectrum antibacterial activity.
• L is a linker that bridges R to the C Ring.
• L may be O or S.
• An O linker provides potent compounds.
• an S linker also provides potent compounds, for example, if R 2 is a 5-membered ring.
• these linkers may impart additional flexibility between the scaffold and R 2 , which may improve dual targeting between ParE and GyrB active-sites.
• compounds having a CH 2 linker include:
• aryl refers to optionally-substituted monocyclic and fused bicyclic hydrocarbyl moiety. Any monocyclic or fused ring bicyclic system which has the characteristics of aromaticity in terms of electron distribution throughout the ring system is included in this definition. Typically, the ring systems contain 5-12 ring member atoms.
• Heteroaryl refers to optionally-substituted aromatic monocyclic and fused bicyclic heterocycles containing one or more heteroatoms selected from N, O and S. The inclusion of a heteroatom permits inclusion of 5-membered rings as well as 6-membered rings.
• alkyl include straight- and branched-chain and cyclic monovalent substituents. Examples include methyl, ethyl, propyl, isopropyl, and cyclopropyl. Where indicated, the alkyl substituents may contain 1-10C (1 to 10 carbon atoms) such as 1-3C, 1-6C, or 1-8C.
• hydrocarbyl residue refers to a residue which contains only carbon and hydrogen.
• the hydrocarbyl residue may be saturated or unsaturated, aliphatic or aromatic, straight-chain, branched-chain, or cyclic including a single ring, a fused ring system, a bridge ring system, or a spiro ring system, or a combination hydrocarbyl groups.
• the hydrocarbyl residue when so stated however, may contain heteroatoms over and above the carbon and hydrogen members of the substituent residue. Thus, when specifically noted as containing such heteroatoms, the hydrocarbyl residue may also contain heteroatoms such as O, S or N within the "backbone" of the hydrocarbyl residue.
• a hydrocarbyl group may include a combination hydrocarbyl containing moieties such as a heterocyclic group, linked to a heteroalkyl containing a combination of a straight chain alkyl and a cycloalkyl group.
• cyclic residue refers to a cyclic hydrocarbyl residue, which contains only carbon and hydrogen.
• the cyclic residue when so stated however, may contain heteroatoms over and above the carbon and hydrogen members of the substituent residue.
• the heterocyclic residue may also contain heteroatoms such as O, S or N within the "backbone" of the cyclic residue.
• the cyclic residue when so stated, is a cycloaliphatic or cycloheteroaliphatic residue.
• a saturated cycloaliphatic or saturated cycloheteroaliphatic residue refers to a ring containing saturated bonds between each ring member.
• unsaturated cyclic residue refers to an at least partially unsaturated or aromatic cyclic hydrocarbyl residue, which contains only carbon and hydrogen.
• the unsaturated cyclic residue when so stated however, may contain heteroatoms over and above the carbon and hydrogen members of the substituent residue.
• the unsaturated heterocyclic residue may also contain heteroatoms such as O, S or N within the "backbone” of the unsaturated cyclic residue.
• the term “members” or “membered” in the context of heterocyclic and heteroaryl groups refers to the total atoms, carbon and heteroatoms N, O and/or S, which form the ring.
• an example of a 6-membered saturated cycloheteroaliphatic ring is piperidine and an example of a 6-membered heteroaryl ring is pyridine.
• the bound conformation refers to the conformation (i.e., the spatial arrangement of atoms) the tricyclic gyrase compound would assume if it was bound to the GyrB/ParE active-site pocket in the enzyme's interior.
• the compound may interact with the active site pocket and inhibit the ATPase activity.
• some substituents interact with certain amino acids and thus the substituents' ability to rotate freely about a bond is constrained.
• more useful measurements may be made to determine distances relevant for determining the dimensions of proper substituents.
• measurements are based on the relative positions of substituents on the compound while hypothetically bound to the GyrB/ParE active-site pocket.
• references to the bound conformation with respect to the compound should not be interpreted as literally encompassing the GyrB/ParE active-site pocket in combination with the compound.
• the bound conformation is characterized via measurements derived from the three dimensional structure from x-ray crystallo graphic data on the inhibitor complexed with a protein construct that typically encompasses the 24 or 46 kDa ATP -binding domain of one or more representative bacterial GyrB or ParE orthologs. Given the high degree of sequence identity between GyrB and ParE enzymes in most pathogenic organisms of interest, structural information derived from a protein ortholog from any pathogen of clinical relevance should be sufficient to describe the bound conformation. Briefly, crystallographic structures are generated using the following methods: Proteins of interest (e.g., E.
• E. coli GyrB E. coli GyrB, F. tularensis ParE or E. coli ParE
• the open reading frames are cloned into an expression plasmid (e.g., pET28a), and expressed in and appropriate E. coli expression strain (e.g., BL21 (DE3)).
• E. coli expression strain e.g., BL21 (DE3)
• 24 kDa and 46 kDa ATP binding domains are cloned with a C(His) 6 tag to aid purification by metal affinity chromatography. This robust chromatography step typically yields greater than 80% pure protein.
• Polishing steps including ion exchange and size exclusion chromatography are performed as needed until satisfactory (>95%) purity is achieved.
• complexes of GyrB or ParE and the inhibitor molecule of interest are generated by mixing a stoichiometric excess of the inhibitor of interest with the recombinant protein target in solution and crystallizing the complex using established crystallization methods (typically vapor diffusion, as described in Drenth J. (1999) In Principles of protein x-ray crystallography. 2 nd ed. Springer, New York).
• x-ray diffraction data are collected on single crystals of the protein-inhibitor complexes using monochromatic x-rays generated by a rotating anode or synchrotron radiation source.
• X-ray data processing, analysis and subsequent structure solution and refinement are carried out using well established computational methods (reviewed in Drenth J. (1999) In Principles of protein x-ray crystallography. 2 nd ed. Springer, New York).
• Interacting substituents on the compound that interact with the GyrB/ParE active-site pocket include those substituents that would be located within the protein's interior when the compound is in the bound conformation. Interactions of interacting substituents generally include hydrophobic interactions (which favor the apposition of lipophilic surfaces on the inhibitor and active-site pocket), and electrostatic interactions such as Van der Waals, dipole-dipole, coulombic interactions or hydrogen-bonding between atoms on the compound and atoms in the GyrB/ParE active-site pocket. For example, R 8 , R x , R Y , and R interact with various portions of the protein's interior.
• R , R , R , or R is NH 2 or NHR (where R is, for example, a small alkyl group)
• the H atom(s) on the nitrogen may interact with electronegative atoms, such as nitrogen or oxygen, proximally located in the GyrB/ParE active-site pocket to which the compound may bind.
• R 8 , R x , R Y , and R z are non-polar (e.g., a methyl group)
• the interacting substituent may also electrostatically interact with an atom in the protein's interior via Van der Waals interactions, and desolvate complementary lipophilic surfaces in the active-site pocket to form favorable hydrophobic interactions.
• the shape and size of the active-site may place restrictions on the dimensions of compound's substituents that would be sterically compatible with the active-site pocket.
• the dimensions of a substituent may be provided and are associated with the dimensions of the pocket in which the compound would be situated if in a bound conformation.
• the length of a substituent may be given based on its distance from the atom on the tricyclic scaffold to the substituent's atom that is positioned farthest from the tricyclic scaffold, i.e., the terminal atom. The distance is measured based on the center of a first atom such as a C on the tricyclic scaffold, to the center of the terminal atom. The distance is measured from point to point in a straight line regardless of the fact that the bonds in the substituent are not linearly aligned, such as an ethyl or OH substituent.
• the R 8 substituent generally projects into the R 8 pocket along an axis that projects through the C atom on the A Ring that is attached to R 8 , and the C atom on the same ring in the meta position that shares a common C atom with the B ring when the compound is in bound conformation.
• the width of the R 8 substituent refers to the width at its widest point measured from atom center to atom center that are farthest apart approximately perpendicularly about such an axis, when the compound is in the bound conformation.
• the R substituent may be able to adopt a conformation, when the compound is in the bound conformation, having a width that does not exceed about 3.3 A.
• the NHMe moiety on R has a width of approximately 2.8 A.
• This width is derived by summing the distance of atom center of a methyl proton oriented trans to the N-H proton perpendicularly from the axis described above, with the distance of the center of the N-H proton perpendicularly from the same axis.
• the width of a cyclopropyl substituent would be approximately 3.1 A, measured as the distance between the centers of protons on adjacent carbon atoms on opposite faces of the cyclopropyl ring.
• R 8 may be H or an interacting substituent having a length of about 1 A to about 5 A from the carbon attachment point on the A Ring to the terminal atom in R 8 and a width of about 3.3 A or less.
• the length of R 8 is appropriate for the length from the tricyclic scaffold carbon to the active site pocket based on crystallographic data, which is about 6 A to about 8 A as shown in Figure 1.
• R 8 is H, CI, F, Br, NH 2 , OH, 1-3C alkyl, amino- 1 -3C alkyl, aminocyclopropyl, OCH 3 , OCH 2 CH 3 , cyclopropyl, CH 2 cyclopropyl, CH 2 C1, CH 2 F, CHF 2 , CF 3 , CH 2 CH 2 F, CH 2 CHF 2 , CH 2 CF 3 , NHNH 2 , NHOH, NHNHCH 3 , NHOCH 3 , NHCD 3 , SCH 3 , or NHCOH, where D is deuterium.
• R 8 is H, CI, F, Br, NH 2 , 1 -3C alkyl, amino- l-3C alkyl, aminocyclopropyl, OCH 3 , OCH 2 CH 3 , cyclopropyl, CH 2 cyclopropyl, CH 2 C1, CHC1 2 , CH 2 F, CHF 2 , CF 3 , CH 2 CH 2 F, CH 2 CHF 2 , CH 2 CF 3 , CHCH 2 , NHNH 2 , HOH, NHNHCH 3 , NHOCH 3 , NHCD 3 , SCH 3 , or NHCOH.
• R 8 may be H, CH 3 , CH 2 CH 3 , CI, OCH 3 , NHCD 3 , NHCH 3 , NHCH 2 CH 3 , or NH 2 , such as NHCH 3 .
• R may be a prodrug-containing substituent, wherein the prodrug is cleaved to form a compound that has dimensions appropriate for the length from the tricyclic scaffold carbon to the active site pocket based on crystallographic data as described above.
• prodrugs such as Formula II, among others, are described in more detail below.
• R 9 may be H or may be linked to R 8 to form a prodrug-containing substituent, such as a compound having the structure of Formula III described in more detail below.
• X, Y and Z may be independently selected from the group consisting of N, CR X , CR Y , and CR Z , provided that no more than two of X, Y and Z are N.
• R x may be H or is an interacting substituent having a length of about 1 A to about 2 A from the carbon in CR X to the terminal atom in R x .
• R Y may be H or an interacting substituent having a length of about 1 A to about 3 A from the carbon in CR Y to the terminal atom in R Y .
• R Y would not be a methoxy substituent because a methoxy substituent is longer than 3 A.
• R z may be H or is an interacting substituent having a length of about 1 A to about 2 A from the carbon in CR to the terminal atom in R . These lengths of CR , CR , and CR are appropriate in comparison to the lengths from the tricyclic scaffold carbon to the active site pocket based on crystallographic data shown in Figure 1.
• X, Y and Z are CR X , CR Y , and CR Z respectively.
• R x may be H, CH 3 , CI, Br, or F, such as H or F.
• R Y may be H, CH 3 , CHF 2 , CF 3 , CN, CH 2 CH 3 , CI, Br, or F, such as H, F, CI, or CF 3 .
• R z may be H, CH 3 , CN, CI, Br, or F, such as H, CH 3 or F.
• Z may be C linked to R 4 .
• the potency and/or selectivity may be increased because the conformational entropy is reduced when Z joins with R 4 to form a fused ring.
• Z may be C linked to R 4 , wherein the compound has the structure of Formula VI
• R 41 may be CR 10 , CR 10 CR n , NR 12 , O or S.
• R 4m may be CR 10 , CR 10 CR n , or NR 12 .
• R 4n may be CR 10 , CR 10 CR n , NR 12 , O or S.
• Each of R 10 , R 11 and R 12 is independently H or a noninterfering substituent.
• Noninterfering substituents include amine, CI -CIO hydrocarbyl, halogen such as F or CI, alkyl amine such as C1 -C4 alkylamine, for example, methylamine.
• optionally substituted R 41 may be O, CH, NH, or NCH 3 .
• Optionally substituted R 4n may be CH, NH, CH 2 or CHNH 2 .
• R 41 and R 4m may form one or more fused rings.
• the dashed lines indicate an optional double bond when two adjacent R 41 , R 4m , and R 4n are CR 10 and R 4 ° is CH or N.
• Formula VI may have the structure of Formula Via:
• R 41 and R 4m together with two noninterfering substituents may form a fused aromatic six membered E Ring containing 0-1 N atoms, optionally substituted with halogen, such as chloro.
• R 41 and R 4m together with two noninterfering substituents may form a fused five-membered unsaturated, partially unsaturated, or saturated E Ring comprising 0-2 heteroatoms selected from O, S and N.
• R 41 may be N and together with R 4m and two noninterfering substituents may form a fused five-membered saturated heterocyclic E Ring.
• Additional examples of R 41 and R 4m together with two noninterfering substituents include saturated 5-, 6-, 7-, 8-, or 9-membered rings that may be fused, spiro or bridged optionally containing an amine substituent.
• R 4n is NH or CH 2 , attached to R 4 ° through the C or N, -R 4n -R 4m -R 41 - may be selected from the following moieties, for example, forming an E Ring:
• R n may be replaced with CH 2 .
• R 4n may be replaced with NH.
• R 4 ° may be a) a bond, wherein a 7-membered D ring is formed, wherein R 4n may be CH, CH 2 , S, NH, O, CHF, or CF 2 ; b) a 1 member link in the backbone of the D ring wherein an 8-membered D Ring is formed, wherein the 1 member link may be CH, CH 2 , S, NH, O, CHF, or CF 2 ; c) a 2 member link in the backbone of the D ring that forms a 9- membered D Ring, wherein the member adjacent the A Ring may be CH, CH 2 , S, NH, O, CHF, or CF 2 . ,4 ⁇
• -R 4o -R 4n -R 4m -R 41 - i.e., ⁇ R 4m — R 41 may be selected from the following moieties,
• D Ring contains at least one N in the backbone, or a substituent on the D Ring contains at least one N.
• two adjacent noninterfering substituents on R 4m and R 4n may form one or more fused rings.
• the dashed lines indicate an optional double bond when two adjacent R , R m , and R n are CR and R 0 is CH or N.
• Formula VI may have the structure of Formula VIb:
• R 4 ° may not be a bond. If R 4 ° is a 1 member link, the portion of the F Ring linking R 4m and R 4n , if present, may be an unsubstituted CI residue or CI substituted with a small substituent such as F or NH 2 substituent forming an unsubstituted cyclopropyl residue with R 4m and R 4n .
• R 4 ° is a 1 member link
• the portion of the F Ring linking R 4m and R 4n may be a C2-C15 hydrocarbyl residue containing 0-5 O, S, or N heteroatoms, however, the position on the F ring immediately adjacent R 4n may be unsubstituted or substituted with a small substituent such as F or NH 2 .
• R 4 ° is a 2 member link and the F Ring is present, any substituents on the F Ring may be sufficiently distal to avoid steric hindrance and other interference with enzyme binding.
• R 4m and R 4n together with two noninterfering substituents may form a fused 6-membered unsaturated, partially unsaturated, or saturated F Ring such as an aromatic 6-membered F Ring containing 0-1 N atoms, optionally substituted with halogen, such as fluoro or NH 2 .
• R m and R n together with two noninterfering substituents may form a fused five-membered unsaturated, partially unsaturated, or saturated F Ring comprising 0-2 heteroatoms selected from O, S and N.
• R 4m and R 4n may both be CH and together with two noninterfering substituents may form a fused five- membered saturated heterocyclic F Ring.
• Additional examples of R 4m and R 4n together with two noninterfering substituents include saturated 5-, 6-, 7-, 8-, or 9-membered rings that may be fused, spiro or bridged optionally containing an amine substituent.
• the D Ring avoids steric hindrance and avoids interference with the compound's binding to the enzyme's active site.
• the D ring does not project greater than about 3 A below the plane of the A, B and C Rings toward the GyrB/ParE binding pocket floor in the bound conformation; and the D ring does not sterically interfere with R 2 when the compound is in the bound conformation.
• the compound of Formula VI may be selected from the group consisting of:
• R 2 may be useful for conferring selectivity and potency against eukaryotic ATP binding proteins, such as kinases and HSP90.
• one of the compounds' benefits includes avoiding toxicity due to off target binding, such as to a kinase, due in part to R 's selectivity as part of the compound.
• the compounds are not potent inhibitors for eukaryotic kinases.
• R is a 6-membered aryl or heteroaryl ring containing 0-3 O, S, or N heteroatoms, optionally substituted with 0-3 noninterfering substituents, wherein 2 adjacent noninterfering substituents on R 2 may form one or more fused rings with the 6-membered aryl or heteroaryl ring.
• R 2 may be an optionally substituted 6-membered aryl or heteroaryl ring containing 0-3 O, S, or N heteroatoms such as optionally substituted pyrimidinyl, phenyl, or pyridyl.
• R 2 is a heteroaryl ring such as 6-membered heteroaryl.
• R 2 may be attached to L through a carbon atom in the 6-membered aryl or heteroaryl ring.
• solvent sheltered faces of the GyrB/ParE active-site pockets may restrict the size of substituents on the compound proximal those solvent sheltered faces.
• the 6-membered aryl or heteroaryl ring may contain a CH at the ring positions immediately adjacent the position where R attaches to L.
• FIG. 2 illustrates R as an optionally substituted 6-membered heteroaryl ring, although the positioning of the substituents also applies to a 6-membered aryl ring.
• a and E are C.
• R b and R° face the solvent in the bound conformation, and thus the substituents at this position may be varied and may include prodrugs. Cyclization between R b and R° may be permitted.
• R d is partially solvent exposed, and cyclization between R° and R d (for example, with an H-bond acceptor in the R d position) may be permitted. Large substituents such as large branched groups at R d may collide with the outer rim of the pocket.
• the optionally substituted 6-membered aryl or heteroaryl ring of R 2 in combination with the one or more fused rings formed from optional substituents may be selected from the group consisting of optionally substituted indolyl, azaindolyl, pyrimidopyridyl, quinazolinyl, quinoxalinyl, naphthyridinyl, purinyl, imidizopyridinyl, furopyridinyl, isoindolylinyl, benzodioxinyl, dihydrobenzodioxinyl, benzothiazolyl, pyrrolopyridinyl, dihydropyrrolopyridinyl, benzoimidazolyl, imidazopyridinyl, dihydroimidazopyridinyl, tetrahydroisoindolyl, chromenyl, benzthiophene, benztriazolyl, benzfur
• R 2 may be a 6-membered aryl or heteroaryl ring containing 0-3 O, S, or N heteroatoms, optionally substituted with 0-3 noninterfering substituents, wherein the 6-membered aryl or heteroaryl ring of R 2 has a CF at each positions immediately adjacent the position where R attaches to L, if L is O or S.
• R 2 does not include the 6-membered aryl or heteroaryl groups in PCT/US2012/029104.
• R 2 may be a 6-membered aryl or heteroaryl ring containing 0-3 O, S, or N heteroatoms, optionally substituted with 0-3 noninterfering substituents, wherein the 6-membered aryl or heteroaryl ring of R 2 has a CF at each positions immediately adjacent the position where R attaches to L, if L is O or S.
• R 2 may be a 6- membered aryl or heteroaryl ring containing 0-3 O, S, or N heteroatoms, optionally substituted with 0-3 noninterfering substituents, wherein the 6-membered aryl or heteroaryl ring of R has a CH or CF independently at each position immediately adjacent the position where R 2 attaches to L, if L is NH, CH 2 , CHF, or CF 2 .
• R may be a 5- membered heteroaryl ring containing 1-4 O, S, or N heteroatoms, optionally substituted with
• the 5-membered heteroaryl ring of R has O, S, N, NH, CH, CF, or CCl, independently at each of the positions immediately adjacent the position where R 2 attaches to L, if L is O, S, NH, CH 2 , CHF, or CF 2 .
• R contains O or S in the backbone of the 5-membered heteroaryl, one O or S may be present.
• R may be a 6-membered or 5-membered non-aryl or non-heteroaryl ring containing 0-3 O, S, or N heteroatoms, optionally substituted with 0-3 noninterfering substituents; wherein the 6- membered or 5-membered non-aryl or non-heteroaryl ring of R z has a O, S, N, NH, CH, CF, or CH 2 , independently at each position immediately adjacent the position where R attaches to L, if L is O, S, NH, CH 2 , CHF, or CF 2 .
• R may be a 6-membered aryl or heteroaryl ring containing 0-3 O, S, or N heteroatoms, optionally substituted with 0-3 noninterfering substituents; a 5-membered heteroaryl ring containing 1 -4 O, S, or N heteroatoms, optionally substituted with 0-2 noninterfering substituents, or a 6- membered or 5-membered non-aryl or non-heteroaryl ring containing 0-3 O, S, or N heteroatoms, optionally with 0-3 noninterfering substituents.
• Examples of 5-membered heterocycles include thiadiazole, oxadiazole, isoxazole, tetrazole, imidazole, pyrrole, pyrazole, thiazole, oxazole, isothiazole, triazole, furan and thiophen.
• Adjacent noninterfering substituents of R 2 may form one or more fused rings with the 6-membered aryl or heteroaryl ring, the 5-membered heteroaryl ring, or the 6- membered or 5-membered non-aryl or non-heteroaryl ring.
• 5/6 fused rings include indole, azaindole, purine, imidazolepyrimidine, furopyridinyl, azabenzothiazole, pyrrolopyridinyl, azabenzoimidazole, imidazopyridine, azabenzthiophene, azabenztriazole, azabenzfuran, azabenzoxazole, or azaindazole.
• R 2 may include
• R 2b , R 2c , R 2d , R 2f and R 2g independently may be each N or CR 2e wherein R 2e is H or C1-C4 alkyl optionally substituted with a noninterfering substituent, such as OH, CH 3 or CH 2 OH. In comparison to the leading compounds in PCT/US2012/029104, many compounds containing these R species are significantly more potent against certain bacteria.
• R 2b is N.
• R 2d is N.
• R 2b may N and R 2c and R 2d may each be CR 2e , for example, CH. Examples of R 2 include
• compounds wherein R may be a 5-membered heteroaryl ring containing 1 -4 O, S, or N heteroatoms, optionally substituted with 0-2 noninterfering substituents, or a 6-membered or 5-membered non-aryl or non-heteroaryl ring containing 0-3 O, S, or N heteroatoms, may have the following structures of Formula VII, Formula VIII and Formula IX:
• a , A , A A B , z , B J and B are independently optionally substituted CH, CH 2 , NH, N, O or S, wherein the optional substituents are noninterfering substituents.
• Noninterfering substituents may include CI -CI 2 hydrocarbyl group containing 0-6 hetero atoms such as O, S, or N, such as a C1-C6 alkyl or a 5-membered or a 6-membered carbocyclic, aromatic or hetero aromatic ring, which may in turn be optionally substituted with a noninterfering substituent.
• Two adjacent noninterfering substituents on R may form one or more fused rings.
• R may also include:
• 5-membered non-aromatic rings may include:
• 6-membered non-aromatic rings may include:
• R 2 groups examples include:
• R 2 a 5-membered heteroaryl ring containing 1-4 O, S, or N heteroatoms, optionally substituted with 0-2 noninterfering substituents, or a 6- membered or 5-membered non-aryl or non-heteroaryl ring containing 0-3 O, S, or N heteroatoms, optionally with 0-3 noninterfering substituents include:
• Solvent exposed faces of the GyrB/ParE active-site pockets allow portions of the compound to be exposed to a solvent environment when in use as illustrated in Figure 1.
• noninterfering substituents may be water soluble to afford compatibility with an aqueous solvent environment. Proportions of the substituents in the direction of a potential solvent environment are not critical but one skilled in the art would understand that sterically unhindered substituents are useful. Thus, proportions of the solvent-exposed substituents may be diverse.
• a "noninterfering substituent” is a substituent which leaves the ability of the compound herein such as compounds of Formula I to inhibit bacterial growth of at least one type of bacterium qualitatively intact.
• the noninterfering substituent would leave the ability of the compound to provide antibacterial efficacy based on a minimum inhibitory concentration (MIC) of less than 32 ⁇ g/ml, or based on inhibition of ATPase activity of DNA Gyrase B (GyrB) or Topoisomerase IV (ParE) of less than 10 nm.
• MIC minimum inhibitory concentration
• the substituent may alter the degree of inhibition based on MIC or ATPase activity.
• substituents that are noninterfering substituents on one R group may also be noninterfering substituents on another R group, subject to the steric hindrances, size limitations and the ability to inhibit bacteria as discussed herein.
• the noninterfering substituents on R2 may also be noninterfering substituents on R4.
• R may have 0-3 noninterfering substituents on a ring.
• R may have a noninterfering substituent selected from the group consisting of OH, C0 2 H, CN, NH 2 , Br, CI, F, S0 3 H, S0 2 NH 2 , S0 2 CH 3 , SOCH 3 , NHOH, NHOCH 3 , and N0 2 .
• the nitrogen atom may be oxidized to a pyridine N-oxide; thus, an OH substituent may be in the form of an oxide, thus for example, permitting a pyridyl having an N-oxide wherein the N is a ring heteroatom.
• the Cl -15 hydrocarbyl residue containing 0-5 O, S, or N heteroatoms may include a combination of hydrocarbyl groups such as a combination of aliphatic rings or chains and aromatic rings linked together.
• two adjacent noninterfering substituents on R 2 form one or more fused rings.
• the optional substituents may occupy all positions of the R 2 ring structure that are not adjacent L such as one position, 1 -2 positions, or 1-3 positions. In some aspects, one position is optionally substituted. These substituents may be optionally substituted with substituents similar to those listed. Of course, some substituents, such as halo, are not further substituted, as known to one skilled in the art.
• R may be pyrimidinyl or pyridinyl optionally substituted with CH(OH)CH 3 , C(OH)(CH 3 ) 2 , OCH 3 , CN, CH 3 , CH 2 CH 3 , O-cyclopropyl, SCH 3 , Br, CI, F, or NH 2 .
• the noninterfering substituents on R 's ring that may be solvent exposed in the bound conformation may include large substituents such as prodrugs.
• R 2 may be selected from the substituents in the following Chart 1.
• R 2 may be selected from the substituents in the following Chart 2. Chart 2
• L-R 2 includes: [0090] Figures 1 and 2 show that the compound is solvent exposed in the bound conformation along the R 4 bond axis and in a 0-90° counterclockwise sweep from the R 4 bond axis. Choices for prodrugs and substituents on R 4 , therefore, may be varied. In selecting the R 4 substituent, in some aspects the R 4 groups do not sterically interfere with R 2 or Z groups in the bound conformation, which is illustrated in Figure 2. A skilled artisan would understand that to avoid steric interference, atoms on R 4 should not approach atoms on R 2 or R z (in the bound conformation) such that the interatomic distances of the closest atoms are less than the sums of their Van der Waals radii.
• the R 4 substituent does not project greater than about 3 A below the plane of the A, B and C Rings toward the GyrB/ParE binding pocket in the bound conformation.
• "Toward the GyrB/ParE binding floor pocket” refers to not projecting greater than about 3 A below the plane within about 5-6 bonds from the point of attachment of R 4 to the scaffold.
• portions of R 4 that extend greater than about 5-6 bonds away from the point of attachment of R 4 to the C Ring may project greater than about 3 A below the plane of the A, B and C Rings as these portions are not constrained by the floor of the GyrB/ParE binding pocket.
• the distance is defined as the perpendicular distance from the plane aligned with atom centers of the tricyclic scaffold to the center of the most distal atom (from the plane) on the R 4 substituent in the bound conformation.
• R 4 may be H.
• R 4 may also be an optionally substituted OR a ; wherein R a is a 5-6 membered aryl or heteroaryl containing 0-3 O, S, or N heteroatoms optionally substituted with 0-3 noninterfering substituents.
• the ring positions adjacent the position where O attaches to R a may be substituted with small substituents such as those having 2 atoms in the backbone, such as OCH 3 , CH 3 , CH 2 CH 3 , OH, NH 2 , F, CI, Br, I, or NO. In the remaining positions, substituents can be larger and diverse as substituents in these positions are solvent exposed in the bound conformation.
• R a is an optionally substituted pyrimidinyl or pyridinyl, such as unsubstituted pyrimidinyl or pyrimidinyl substituted with CH 3 or NH 2 .
• OR a is one of the following substituents in Chart 3. Chart 3
• R 4 may be an optionally substituted secondary or tertiary amine attached to the C Ring through the secondary or tertiary amine N.
• Secondary amine refers to an N-containing substituent that contains one H attached to the secondary amine N when the substituent is attached to the remainder of the molecule.
• Tertiary amine refers to an N-containing substituent that contains no H attached to the tertiary amine N when the substituent is attached to the remainder of the molecule.
• R 4 when R 4 is the optionally substituted secondary or tertiary amine attached to the C Ring through the secondary or tertiary amine N, R 4 may further comprise a primary or secondary amine, wherein the primary or secondary amine is not directly attached to the C Ring.
• Primary amine refers to an amine group that contains two H atoms attached to the primary amine N when attached to the remainder of the substituent.
• secondary amine refers to an amine group that contains one H atom attached to the secondary amine N when attached to the remainder of the substituent.
• the primary or secondary amine that is not directly attached to the C Ring may be positioned in the compound in the bound conformation wherein:
• the distance between the C or N atom of Y and the primary or secondary amine N is about 7 A to about 10.5 A;
• the distance between the C atom to which R is attached and the primary or secondary amine N is about 6 A to about 9 A;
• R 4 may be an optionally substituted tertiary amine that is an optionally substituted 4- 14 membered saturated cycloheteroaliphatic tertiary amine ring system containing 1-3 N atoms, 0-3 O atoms and 0-1 S atoms; and wherein the 4-14 membered saturated cycloheteroaliphatic ring system is a single ring, a fused ring system, a bridge ring system, or a spiro ring system.
• R 4 may be the optionally substituted tertiary amine attached to the C ring through the tertiary amine N, wherein the optionally substituted tertiary amine contains at least one additional N separated from the tertiary amine N by 2-3 atoms.
• the atoms separating the N's need not be located in the same ring. For example, one atom separating the N's may be in a ring and the second atom may be found in a substituent, or both atoms separating the N's may be in the backbone in, or a substituent on, the same or different rings.
• the optionally substituted secondary or tertiary amine of R 4 is one of the following substituents in Chart 4.
• R 4 may be a noncyclic secondary or tertiary amine substituted with 1-2 noninterfering substituents.
• R 4 may be selected from the group consisting of optionally substituted pyrazolyl, phenyl, piperazinyl, pyridinyl, and tetrahydropyridinyl.
• R 4 may be an optionally substituted 5-10 membered unsaturated cyclic or heterocyclic residue containing 0-3 N, O or S heteroatoms.
• the optional substituents may include 0-2 optional substituents selected from the group consisting of C3 ⁇ 4, NH 2 , F, CI, and CH 2 H 2 .
• the optionally substituted 5-10 membered unsaturated cyclic or heterocyclic residue containing 0-3 N, O or S heteroatoms of R 4 is one of the following substituents in Chart 5.
• the optional substituent on R 4 may include 0-3 noninterfering substituents.
• an OH substituent may be in the form of an oxide, thus for example, permitting a pyridyl having an N-oxide wherein the N is a ring heteroatom.
• the Cl-15 hydrocarbyl residue containing 0-5 O, S, or N heteroatoms may include a combination of hydrocarbyl groups such as a combination of aliphatic rings or chains and aromatic rings linked together.
• R 4 may be selected from the substituents in the following Chart 6.
• R 4 may also be a prodrug-containing substituent, wherein the compound has the structure of Formula V or Formula V:
• R 4a contains an oxygen residue derived from a non- prodrug R 4 as in b)-d) or g)-i), wherein the non-prodrug R 4 has an OH group, wherein the R 4 OH is replaced with an oxygen residue in R 4a , and wherein the oxygen residue is linked to P;
• R 4b and R 4c are independently H or C 1 -C6 alkyl
• Q is CH or N
• R 4e is (CR 4g 2) n -basic amine, wherein each R 4g may be independently H or C1-C3 alkyl;
• n 0-2;
• R 4f is hydrogen C1-C6 alkyl optionally substituted with OH or NH 2 ;
• R 4e and R 4f may join to form a ring
• R 4j and R 4k are independently H or C1-C8 hydrocarbyl residue; R 4 may also also be a prodrag-containing substituent, wherein the compound has the structure of Formula ⁇ "
• R 4 may also be joined with R 2 to form a fused ring, wherein R 4 is a 5- to 15-member hydrocarbyl linker containing 0-6 O, S or N atoms in the backbone of the linker that attaches to the R group wherein atoms in the 5- to 15-member hydrocarbyl linker are optionally substituted with a noninterfering substituent.
• R 4 may also be joined with Z to form a fused ring, discussed in more detail herein.
• the compound may be one of the compounds exemplified in the Examples.
• the compounds of Chart 7, or any compound recited in PCT/US2012/029104 may be the active moiety associated with the new prodrugs described herein.
• the compounds in Chart 8 have either unexpectedly higher activity than the most active species of compounds disclosed in PCT/US2012/029104, or were expected to have lower activity than compounds disclosed in PCT/US2012/029104 but had unexpectedly higher activity.
• the hERG IC50 values of compounds in Chart 8, or other compoudns herein are unexpectedly higher than the most active species of compounds disclosed in PCT/US2012/029104.
• R may join with R to form a fused ring with the ABC ring.
• the potency and/or selectivity may be increased because the conformational entropy is reduced when R 2 joins with R 4 to form a fused ring.
• the oversimplified structure below which does not show the details of L, R 2 and R 4 , illustrates R 2 joined with R 4 forming a ring that is fused with the ABC ring:
• R When part of the fused ring, R may be a ring as recited herein.
• L may have a single member in its backbone such as O, S, NH, CH 2 , CHF, or CF 2 .
• R 4 may be attached through the meta or para position of the 6-membered ring with respect to the point of attachment to L.
• R 4 may be attached through any position on R 2 's 5-membered ring except the position where L attaches to R 2 .
• R 2 's 6- membered or 5-membered ring may be a carbon or an appropriate heteroatom such as nitrogen in the ring.
• R 2 is a 6-membered ring as described herein, e.g., a 6- membered aryl, heteroaryl, non-aryl or non-heteroaryl ring
• R 2 may be attached to R 4 through the meta or para position of R 's 6-membered ring with respect to the point of attachment to L.
• L has a single member in its backbone and if R 2 is a 5-membered ring as described herein, e.g., a 5-membered aryl, heteroaryl, non-aryl or non-heteroaryl ring, the first position is the ring member attached to L.
• the second position is the ring member at the position in the five membered ring adjacent to the first position.
• the third position is the ring member at the position in the five membered ring adjacent to the second position.
• R may be attached to R through the third position in the five membered ring to avoid any stearic hindrance or interfere with enzyme binding.
• R may contain more than one ring. If R is a multi-ring substituent, the attachment point to R 4 may be sufficiently distal to avoid steric hindrance and other interference with enzyme binding, if the attachment point in not in a ring that is adjacent L.
• the attachment point of R 2 to R 4 may be sufficiently distal to avoid steric hindrance and other interference with enzyme binding.
• R 4 in this case may be a 5- to 15-membered, such as a 9- to 12-membered hydrocarbyl residue linker and may contain 0-6 O, S or N atoms in the backbone of the hydrocarbyl residue linker that attaches to the R 2 group wherein atoms in the 5- 15 member hydrocarbyl residue linker are optionally substituted with noninterfering substituents.
• the hydrocarbyl residue linker may be saturated, partially unsaturated, or unsaturated.
• Optional substituents include any noninterfering substituents recited herein including hydrocarbyl residues optionally containing heteroatoms in the backbone which, in turn, may be optionally substituted with noninterfering substituents.
• the R 4 linker When in the bound conformation, the R 4 linker will be open to the solvent space above the plane of the A, B and C Rings so little interference is expected with the GyrB/ParE binding pocket floor, regardless of the size of the optionally substituted linker.
• a portion of the GyrB/ParE binding pocket floor is open to solvent space, allowing the linker freedom of movement both above and below the plane of the A, B, and C Rings.
• practical considerations in drug design may limit the size of the linker, such as size of the resulting pharmaceutical, which may increase costs.
• Protecting groups are useful for chemoselectivity and are known in the art. Typical protecting groups included tert-butyloxycarbonyl (BOC) and carbobenzyloxy (Cbz). When the protecting group is BOC, an acid may be used for deprotection, protecting group is Cbz, catalytic hydrogenation may be used for deprotection.
• BOC tert-butyloxycarbonyl
• Cbz carbobenzyloxy
• the process may further comprise reacting the compound of Formula XX
• G 1 and G 2 are leaving groups independently selected from the group consisting of CI, Br, F, I, SR, SOR, S0 2 R, OS0 2 R, and O- benzotriazole (OBt); wherein R may be Cl-8 alkyl, aryl, or heteroaryl containing 0-5 O, S, or N atoms optionally substituted with CI -4 alkyl, CI -4 alkyloxy, CI, Br, F, I, or N0 2 , such as methyl, benzyl and p-methoxybenzyl, to make the compound having the structure
• the compounds wherein R 4 is an optionally substituted secondary or tertiary amine attached to the C Ring through the secondary or tertiary amine N may also be made using a process comprising treating
• R LH under basic conditions such as with the anion of phenol, thiophenol, heteroarylhydroxy or heteroarylthiol, wherein G is a leaving group selected from the group consisting of CI, Br, F, and I; and optionally further comprising, before the treating step immediately above, protecting R 8 with a protecting group, or protecting an amine in R 4 which is not the secondary or tertiary amine N, if present, with a protecting group; and deprotecting R 8 and R 4 after the treating step.
• the process may further comprise reacting the compound of Formula XX
• G is a leaving group selected from the group consisting of CI, Br, F, and I.
• a process of making the compound wherein R 4 is an optionally substituted secondary or tertiary amine attached to the C Ring through the secondary or tertiary amine N may comprise treating
• G is a leaving group derived from S0 2 halide, bis(2-oxo-3-oxazolidinyl)phosphine (BOP), or benzotriazol- 1 -yl-oxytripyrrolidinophosphonium hexafluorophosphate (pyBOP), with HR 4 to make the compounds herein.
• This process may also optionally further comprise, before the treating step immediately above, protecting R with a protecting group, or protecting an amine in R 4 which is not the secondary or tertiary amine N, if present, with a
• the process may further comprise reacting
• G'X 1 is S0 2 halide, bis(2-oxo-3-oxazolidinyl)phosphine (BOP), or benzotriazol-l-yl- oxytripyrrolidinophosphonium hexafluorophosphate (pyBOP).
• the process may further comprise coupling
• an intermediate compound has the structure of Formula
• G 1 and G 2 are leaving groups independently selected from the group consisting of SH, OH, CI, Br, F, I, SR, SOR, S0 2 R, OS0 2 R, OAr, and OBt;
• R is CI -8 alkyl, aryl, or heteroaryl;
• Ar is aryl or heteroaryl containing 0-5 O, S, or N atoms optionally substituted with Cl-4 alkyl, Cl-4 alkoxy, halo or N0 2 ;
• Bt is benzotriazole;
• R is an interacting substituent having a length of about 1 A to about 5 A from the carbon attachment point on the A Ring to the terminal atom in R and a width of about 3.3 A or less;
• X, Y and Z are independently selected from the group consisting of N, CR X , CR Y , and CR Z respectively, provided that no more than two of X, Y and Z are N, wherein R x is H or
• the intermediate compound is an amine-protected intermediate
• one or more nitrogens in the compound may be protected with carbobenzyloxy (Cbz) or BOC.
• G 1 and G 2 may be leaving groups independently selected from the group consisting tosylate, mesylate, trifilate, O-pyrimidine, O-phenyl and O-pyridine.
• a wide variety of amines and substituted amines can be introduced into the A Ring of the pyrimidoindole system as shown in Scheme 1.
• Ortho-fluoro-nitrobenzenes SI can be readily displaced by amines to yield the orthoamino analogs S2.
• a protecting group can be introduced by incorporation in the starting material (as in S 3b) or introduced after the fluoroaryl displacement reaction (as in S 3c).
• nitration may be used to introduce the nitro group ortho to the R 8 group S3d.
• chromatography may be used to isolate the desired isomer.
• R H, F, CI, Me, CF 3 S 3b
• R H, F, CI, Me, CF 3
• Scheme 2 outlines the general methods for preparing a wide variety of pyridine and pyrimidine starting materials. Nitration of 4,6-dihydroxypyrimidine followed by conversion of the hydroxyl groups to a chloro group with POCI 3 affords intermediate S4c. The chloro is readily displaced by amines and alcohols to provide the desired intermediate S3e. In a similar fashion, commercially available pyridine S4d is readily substituted with amines and alcohols to form intermediate S3f.
• the orthofluoro-nitroaromatics S3 are converted (Scheme 2) to indoles, and nitrogen substituted indoles S6a and S6b (pyrrolopyrimidines and pyrrolopyridines) by treatment with cyano ethyl acetate or cyanomalonate followed by reduction with zinc in acetic acid alternatively the nitro group can be reduced with many alternative reduction agents such as sodium bisulfite.
• indole intermediates are converted to tricyclic intermediates as shown in Scheme 4.
• Reaction of an amino ester indole S6a with an acylisothiocynate followed by treatment with base provides the tricycle S8a with an SH at the 2 position and an OH in the 4 position.
• treatment with an acylisocynate followed by base provides S8b with an OH substituent at both the 2 and 4 positions of the tricycle.
• S8a can be converted to a bis-sulfone by first alkylation at the 2-position sulfur, followed by activation of the 4-position with a reagent such as BOP or mesyl chloride followed by displacement with a sulfide then oxidation to the bis-sulfone S8f with a reagent such as sulfone.
• a reagent such as BOP or mesyl chloride
• the dihydroxy core S8b can be converted to the dichloro- tricycle S8g.
• Amino nitrile indole intermediates S6b may be converted to the bissulfone by treatment with carbon disulfide and an alkoide to provide the anion of the 2,4 dithiol tricylcle. This intermediate can be alkylated in situ and then oxidized to provide the bissulfone S8f.
• Scheme 4 Preparation of tricyclic intermediates (cont.)
• either intermediate S8f or S8g may be converted to the bis- aryloxy compound 9.
• the Aryloxy group in the 4 position can be displaced by amines or alcohols to provide the desired Formula I compound when R 4 is either an amine of an alkoxide.
• protection groups on the S8 intermediates and/or the R 4 group In those cases, an additional step may be required to remove the protecting group.
• R4 is OAryl or O Heteroaryl where R4 is an amine
• the dichloro tricyclic intermediate S8g may be treated with the R 4 group first, then followed by displacement of at the 2 position with an alkoxide of R 2 OH (Scheme 6).
• this method requires protecting groups especially when a diamine is used as the R 4 group. In these cases, removal of the protecting groups provides Formula I compound. This method is particularly useful when a costly R 2 OH group is used or the R 2 group is electron rich.
• the Formula I compounds can be prepared directly from S8a by the method in Scheme 7.
• the sulfide is coupled to an aryl halide (preferably an iodo or bromo aromatic).
• a sulfonylhalide or a coupling reagent such as BOP followed by displacement with an amine provides the desired Formula I compound.
• R 4 is an amine
• Formula I compounds where R 4 is an aryl or heteroaryl may be made as shown in Scheme 8.
• the dichloro intermediate S8g is coupled to a boronic acid using Suzuki coupling conditions.
• the resulting product is then treated with an alkoxide to provide the Formula I compound.
• R4 is aryl or heteroaryl
• the cyclic amine in Scheme 9 below may be treated with bromomalonaldehyde to generate a 6/5 bi eye lie compound. Palladium or base catalyzed hydrolysis of the bromobicyclic compound may be used to lead to the alcohol final product.
• the halide starting material may undergo Stille Coupling, for example, with (l-ethoxyvinyl)triethylstannane by catalyzed palladium, followed by acid hydrolysis to form a ketone intermediate. Then the ketone intermediate may be reduced to alcohol, for example, by sodium borohydride.
• the Boc protected methylamine-indole carboxylic ester and the nitrile was dissolved into HC1 (g) in dioxane, and stirred for overnight.
• the resulting solution may be concentrated and the resulting residue may be redissolved with the mixture solution of 1 : 1 5% NaOH in H20 and MeOH, then the solution may be heated to reflux, and LCMS may be used to monitor the progress of the reaction. After completing the reaction, the solution may be neutralized, such as with HC1, and concentrated.
• the residue may be purified, e.g., by reverse phase column to provide the triclyco pyrimidine core.
• the triclyco core may be dissolved in NMP, and coupling reagents BOP and base such as K 2 CO 3 may be added.
• the resulting intermediate may be treated with 1 to 2 equivlent mono-protected diamine, the resulting solution may be stirred for overnight, TFA may be added to the mixture to remove the protection groups.
• the resulting mixture may be concentrated under the reduced pressure and pre-HPLC separation to afford the target compounds.
• R4 is Aryl or Heteroaryl
• the bis sulfone is deprotected for example with an acid.
• the deprotected compound is then treated with HLR and a base, followed by HR 4 .
• This compound is treated with a chloroalkylchloroformate such as
• the protecting group in R4 may be removed under acidic condition such as trifluoroacetic acid.
• R 4 -NH in the scheme below represents a non-pro drug R 4 group having an NH attached to the prodrug.
• the hydroxyl group in R 4 is converted to the corresponding dialkylphosphate in the presence of alkyl phosphorochloridate and a base.
• the protecting groups in R 8 , R 4 and phosphate protecting group in R 4 may be removed under acidic conditions.
• the bis-sulfone intermediate in Scheme 18a below can be used as starting material to make compounds of Formula I where Z is joined to R 4 .
• the bis-sulfone can be first treated with arylthio or alkylthio and potassium carbonate. The reaction is carried out at room temperature with an equivalent amount of the thio reagent to regioselectively substitute the R4 sulfone. In-situ, HLR 2 group is then added and heat is applied to help the displacement of the R 2 sulfone with LR 2 . The sulfide group of the product is then oxidized by mCPBA to the resulting sulfone which is in turn hydrolyzed by lithium hydroxy.
• POCI 3 is used to transform the hydroxy functional group to CI.
• R 41 -R 4n is attached to the core via R 41 . It can take place by a metal-complex catalyzed coupling or by an amine substitution.
• the D ring can be formed by coupling the R 4n to the A ring via R 4 °.
• R 4n is an amine, it can be treated with formaldehyde and catalytic amount of TFA to form the resulting imine which is spontaneously cyclized to A ring.
• an OR 2 can replace the R 2 methylsulfonyl of compound 18.6 by heating the reaction at 90 °C in the presence of base.
• R 2 was different from R 4 2-methylpyrimidin-5-ol, more than 3 equivalences of R 2 OH were used to minmize the te-methylpyrimidine product.
• the product 18.7 of the subsitution was then treated with amine 18.8 to convert into 18.9.
• Temperature of the reaction varied from room temperature to 80 °C depended on the reactivity of amine 18.8.
• Cyclic compound 18.12 was prepared from 18.9 via C-H carbene insertion through in situ three-step sequence of imination of 18.9, formation of diazonium 18.11, and the insertion of 18.11 to form 18.12.
• the Boc protecting group of coumpound 18.12 was then removed by TFA at room temperature to afford Formula Via compound.
• 4 a is a solubilizing group such as -HNCH 3 , -NH 2 , -NHCH 2 CH 3 , -
• R groups additional compounds may also be made with various R 2 groups.
• Intermediate 18.14 in Scheme 18d below can be used to prepare the core of 18.22.
• the R 4 group of 18.14 can be selectively hydrolyzed to form the hydroxy 18.18, which upon treated with POCI 3 turns into CI 18.19.
• Suzuki coupling of 18.19 with vinyl boronic acid or Stille coupling of 18.19 with tributyl(vinyl)tin provides the vinyl 18.20.
• the compound is then subjected to ring-closing metathesis to form the advance intermediate 18.21.
• 18.22 compounds can arise from 18.21 via variety of reaction of alkene such as hydroamination, Diels-Alder reaction, cyclopropanation, etc.
• the dichloro intermediate can be used to prepare Formula VIb. It is first reacted with the F ring via a double substitution or double metal-complex catalyzed coupling or via a combination of the substitution and the coupling. HLR2 group is then attached to the core in a known manner of the substitution of CI.
• R 2 may bear a functional group, which can be optionally protected.
• This functional group could be, for example, a double bond, a carboxylic acid, a thiol, an amine or another functional group.
• the thiol at the 4-position can be converted to a leaving group, for example, by oxidation to a sulfone using MCPBA.
• This leaving group can now be displaced by HR 4 , with R 4 bearing a functional group (for example a double bond, a carboxylic acid, a thiol, an amine or another functional group).
• the functional groups at R 2 and R 4 can now be further modified or extended to be ready for the macrocyclization.
• the macrocycle can be formed by using methods available to those skilled in the art, for example ring-closing-metathesis or macro lactamization.
• Prodrugs may also be prepared from the compounds of Formula ⁇ or I.
• the term "prodrug,” as used herein, represents compounds which can be transformed in vivo to the active parent compounds defined herein.
• prodrugs may have increased oral bioavailability compared to the parent drug. Although the benefits of prodrugs are widely recognized, often prodrugs fail to achieve these advantages. Thus, significant effort and research are needed to develop an effective prodrug.
• the prodrugs herein have significantly less antibacterial activity than the parent antibacterial agent and, consequently, less disruptive to the digestive tract. Because these prodrugs are converted in blood to the active antibacterial agent, they are active systemically. Thus, the prodrug may maintain the beneficial effects of curing the bacterial infection while avoiding the significant side effects of the parent antibacterial agent on the gastrointestinal tract.
• the prodrug may have increased the water solubility compared to the parent antibacterial agent, thereby enabling a better formulation for intravenous administration.
• a prodrug may have the structure of Formula II or Formula V:
• R 2 , R 4 , and R 9 are described herein.
• the drug of Formula II or Formula V maybe cleaved by an esterase in the blood and converted to the active antibacterial agent having the following Formula Ila or Formula Ila' respectively:
• R 8a has dimensions such that it binds to the enzymatic pocket when in the bound conformation, once the prodrug is cleaved in vivo.
• R 8a may be H or an interacting substituent having a length of about 1 A to about 3.3 A from the adjacent nitrogen to the terminal atom in R 8a and a width of about 3.3 A or less.
• R 8a is H, methyl, ethyl, or cyclopropyl, such as methyl.
• R 8b or R 8c may be each independently H or C1-C6 alkyl, for example, Cl- C4 alkyl, such as methyl, ethyl, or tertiary butyl.
• R 8b may be methyl
• R 8c may be H
• R 8c may be tertiary butyl and R 8c may be H.
• one of R 8b or R 8c is H, or both are H.
• R 8d is 0 r a pharmaceutically acceptable salt thereof.
• Pharmaceutically acceptable salts are known in the art and include metal cations, for example a sodium, magnesium, calcium or potassium salt, and also include amine cations such as H 4 + or alkylated amines.
• Q may be CH or N, such as CH.
• R 8e may be (CR 8g 2 ) n -basic amine, wherein n is 0-2, such as 1 , and wherein each R 8g may be independently H or C1-C3 alkyl, such as H 2 , HCH 2 or CH 2 CH 2 .
• a basic amine is a solubilizing group that increases the solubility of the prodrug in aqueous environments such as blood upon administration to the subject.
• the basic amine may be NR 8h R 8i wherein R 8h and R 8i are independently selected from the group consisting of H, optionally substituted C1-C4 alkyl, wherein optional
• substituents may be OH, NH 2 , or NHCH 3 wherein R and R may join to form a fused ring containing 1-3 N, or 0-3 O or S heteroatoms.
• basic amines may include piperzinyl, morpholinyl, C1-C2 alkyl amine such as methyl amine, C1-C2 dialkyl amine such as dimethylamine, or NH 2 .
• R 8d may be
• R 8f is hydrogen or C1-C6 alkyl, such as methyl, ethyl, propyl, or iso-propyl, or a C1-C6 alkyl, such as methyl, ethyl, propyl, or iso-propyl, optionally substituted with OH or NH 2 .
• R 8f may be CH 2 OH, CHOHCH 3 , or (CH 2 ) 4 NH 2 .
• R 8f also may be methyl.
• R and R may join to form a ring; for example, R may be
• R 0 may be such as 0 r a pharmaceutically acceptable salt thereof as described herein.
• R 8 ⁇ and R 8k may be independently H, C1-C8 hydrocarbyl residue such as C1 -C8 alkyl, for example tertiary butyl, or benzyl.
• R 0 may be .
• R 4 has dimensions such that it binds to the enzymatic pocket when in the bound conformation, once the prodrug is cleaved in vivo.
• R 4b or R 4c may be each independently H or C1 -C6 alkyl, for example, Cl - C4 alkyl, such as methyl, ethyl, or tertiary butyl.
• R 4b may be methyl
• R 4c may be H
• R 4c may be tertiary butyl and R 4c may be H.
• one of R 4b or R 4c is
• H or both are H.
• R 4d is 0 r a pharmaceutically acceptable salt thereof.
• Pharmaceutically acceptable salts are known in the art and include metal cations, for example a sodium, magnesium, calcium or potassium salt, and also include amine cations such as ⁇ 3 ⁇ 4 + or alkylated amines.
• Q may be CH or N, such as CH.
• R 4e may be (CR 4g 2 ) folk-basic amine, wherein n is 0-2, such as 1 , and wherein each R 4g may be independently H or C1-C3 alkyl, such as H 2 , HCH 2 or CH 2 CH 2 .
• a basic amine is a solubilizing group that increases the solubility of the prodrug in aqueous environments such as blood upon administration to the subject.
• the basic amine may be NR 4h R 41 wherein R 4h and R 41 are independently selected from the group consisting of H, optionally substituted C1-C4 alkyl, wherein optional substituents may be OH, NH 2 , or NHCH 3 wherein R 4h and R 41 may join to form a fused ring containing 1-3 N, or 0-3 O or S heteroatoms.
• basic amines may include piperzinyl, morpholinyl, C1-C2 alkyl amine such as methyl amine, C1-C2 dialkyl amine such as dimethylamine, or NH 2 .
• R may be any organic radical
• R is hydrogen or C1-C6 alkyl, such as methyl, ethyl, propyl, or iso-propyl, or a C1-C6 alkyl, such as methyl, ethyl, propyl, or iso-propyl, optionally substituted with OH or NH 2 .
• R 4f may be CH 2 OH, CHOHCH 3 , or (CH 2 ) 4 NH 2 .
• R 4f also may be methyl.
• R 4e and R 4f may join to form a ring; for example, R 4d may be o o
• R 0 may be OR such as OH or a pharmaceutically acceptable salt thereof as described herein.
• R 4 ⁇ and R 4k may be independently H, C1-C8 hydrocarbyl residue such as C1 -C8 alkyl, for example tertiary butyl, or benzyl.
• R 4c may be OH 5 0 r OBu- ⁇ .
• Examples of the compound of Formula II or V include
• a prodrag may have the stracture of Formula IF, II"
• more than one prodrag substituent may be present on the compound.
• a prodrag has the stracture of Formula III:
• R 8a is the same as recited above for Formula II.
• R 9a may be H, or Cl -
• the drug of Formula III maybe cleaved by an esterase in the blood and converted to the active antibacterial agent having Formula Ila above.
• R 2a contains an oxygen residue
• R 2 groups include the following, which are shown below as attached to an O linker although other linkers may be used:
• R 4 herein comprising an OH group or substituted with an OH group may allow phosphorylation to arrive at Formula V.
• R 4a contains an oxygen residue derived from a non-prodrug R 4 .
• R 4 's OH is replaced with an oxygen residue in R 4a , upon phosphorylation, wherein the oxygen residue is linked to P in the phosphate group.
• R 2 and R 4 substituents that are disclosed in PCT/US2012/029104 maybe further substituted with -OH as known in the art.
• the prodrug of Formula IV or Formula V maybe cleaved by a phosphatase in the blood and converted to the active antibacterial agent having an R 2 or R 4 group respectively containing a hydroxy group.
• R 2a or R 4a may be derived from an active
• antibacterial compound having an hydroxy substituted R or R group respectively, wherein upon the formation of the prodrug, the hydroxy becomes the point of attachment to the phosphate.
• a prodrug formula e.g. Formula II-V or V
• a prodrug formula includes an R , R , or R 8 group
• any appropriate R 2 , R 4 , or R 8 group herein may be used.
• prodrugs for example on R 4 include NFINHCH3,
• a pharmaceutically-acceptable salt, ester, or prodrug of the compounds herein is also contemplated.
• prodrugs, salts, hydrates, solvates, and polymorphs can be produced from the compounds disclosed here, and that various isotopically-substituted variants (through, e.g., substitution
• isotopomers of deuterium for hydrogen, C for carbon, N for nitrogen, or P for phosphorus
• isotopomers can also be readily produced. All such derivatives are contemplated within the scope of this disclosure.
• Compounds herein include those structures that are set out throughout the examples, and pharmaceutically acceptable salts, esters and prodrugs thereof.
• the compound is in a pharmaceutical composition or a dosage form, wherein the pharmaceutical composition or dosage form provides an effective antibiotic amount of the compound for treating or preventing infection.
• the present disclosure relates to a pharmaceutical composition
• a pharmaceutical composition comprising one or more physiologically acceptable surface active agents, additional carriers, diluents, excipients, smoothing agents, suspension agents, film forming substances, and coating assistants, or a combination thereof; and a composition disclosed herein.
• Acceptable additional carriers or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, 18th Ed., Mack Publishing Co., Easton, PA (1990), which is incorporated herein by reference in its entirety.
• Preservatives, stabilizers, dyes, sweeteners, fragrances, flavoring agents, and the like may be provided in the pharmaceutical composition.
• sodium benzoate, ascorbic acid and esters of p-hydroxybenzoic acid may be added as preservatives.
• antioxidants and suspending agents may be used.
• alcohols, esters, sulfated aliphatic alcohols, and the like may be used as surface active agents; sucrose, glucose, lactose, starch, microcrystalline cellulose, crystallized cellulose, mannitol, light anhydrous silicate, magnesium aluminate, magnesium metasilicate aluminate, synthetic aluminum silicate, calcium carbonate, sodium acid carbonate, calcium hydrogen phosphate, calcium carboxymethyl cellulose, and the like may be used as excipients; magnesium stearate, talc, hardened oil and the like may be used as smoothing agents; coconut oil, olive oil, sesame oil, peanut oil, soya may be used as suspension agents or lubricants; cellulose acetate phthalate as a derivative of a carbohydrate such as cellulose or sugar, or methyl
• composition refers to a mixture of a compound disclosed herein with other chemical components, such as diluents or additional carriers.
• the pharmaceutical composition facilitates administration of the compound to an organism. Multiple techniques of administering a pharmaceutical composition exist in the art including, but not limited to, oral, injection, aerosol, parenteral, and topical administration.
• pharmaceutically acceptable salts of the compounds disclosed herein are provided.
• carrier refers to a chemical compound that facilitates the incorporation of a compound into cells or tissues.
• diharmonic refers to chemical compounds diluted in water that will dissolve the composition of interest as well as stabilize the biologically active form of the compound. Salts dissolved in buffered solutions are utilized as diluents in the art. One commonly used buffered solution is phosphate buffered saline because it mimics the salt conditions of human blood. Since buffer salts can control the pH of a solution at low concentrations, a buffered diluent rarely modifies the biological activity of a compound.
• an “excipient” refers to an inert substance that is added to a composition to provide, without limitation, bulk, consistency, stability, binding ability, lubrication, disintegrating ability, etc., to the composition. A “diluent” is a type of excipient.
• physiologically acceptable refers to a carrier or diluent that does not abrogate the biological activity and properties of the compound.
• a dosage form includes those forms in which the compound is admistered per se.
• a dosage form may include a pharmaceutical composition.
• the dosage form may comprise a sufficient amount of the compound to treat a bacterial infection as part of a particular administration protocol, as would be understood by those of skill in the art. Techniques for formulation and administration of the compounds of the instant application may be found in "Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, PA, 18th edition, 1990.
• Suitable routes of administration may, for example, include oral, rectal, transmucosal, topical, or intestinal administration; parenteral delivery, including intramuscular, subcutaneous, intravenous, intramedullary injections, as well as intrathecal, direct intraventricular, intraperitoneal, intranasal, or intraocular injections.
• parenteral delivery including intramuscular, subcutaneous, intravenous, intramedullary injections, as well as intrathecal, direct intraventricular, intraperitoneal, intranasal, or intraocular injections.
• the compound can also be administered in sustained or controlled release dosage forms, including depot injections, osmotic pumps, pills, transdermal (including electrotransport) patches, and the like, for prolonged and/or timed, pulsed administration at a predetermined rate.
• compositions may be manufactured in a manner that is itself known, e.g., by means of conventional mixing, dissolving, granulating, dragee- making, levigating, emulsifying, encapsulating, entrapping or tabletting processes.
• compositions may be formulated in any conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen. Any of the well-known techniques, diluents, carriers, and excipients may be used as suitable and as understood in the art; e.g. , in Remington's Pharmaceutical Sciences, above.
• injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions.
• Suitable excipients are, for example, water, saline, dextrose, mannitol, lactose, lecithin, albumin, sodium glutamate, cysteine hydrochloride, and the like.
• the injectable pharmaceutical compositions may contain minor amounts of nontoxic auxiliary substances, such as wetting agents, pH buffering agents, and the like.
• Physiologically compatible buffers include, but are not limited to, Hanks's solution, Ringer's solution, or physiological saline buffer. If desired, absorption enhancing preparations may be utilized.
• penetrants appropriate to the barrier to be permeated may be used in the formulation.
• compositions for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents that increase the solubility of the compounds to allow for the preparation of highly concentrated solutions. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative.
• compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
• the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
• the composition can be formulated readily by combining the compositions of interest with pharmaceutically acceptable carriers well known in the art.
• Such carriers which may be used in addition to the cationic polymeric carrier, enable the compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated.
• Pharmaceutical preparations for oral use can be obtained by combining the active compound with solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores.
• Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl- cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP), e.g., Povidone.
• disintegrating agents may be added, such as the cross-linked polyvinylpyrrolidone (e.g. Crospovidone), agar, or alginic acid or a salt thereof such as sodium alginate.
• Dragee cores are provided with suitable coatings.
• suitable coatings For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.
• Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.
• compositions which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol.
• the push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers.
• the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols.
• stabilizers may be added. All formulations for oral administration should be in dosages suitable for such administration.
• compositions may take the form of tablets or lozenges formulated in a conventional manner. Administration to the buccal mucosa and sublingually are contemplated.
• the composition can be conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoro methane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
• a suitable propellant e.g., dichlorodifluoro methane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
• a suitable propellant e.g., dichlorodifluoro methane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
• a suitable propellant e.g., dichlorodifluoro methane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable
• compositions well known in the pharmaceutical art for uses that include intraocular, intranasal, and intraauricular delivery.
• Suitable penetrants for these uses are generally known in the art.
• Such suitable pharmaceutical formulations are most often and preferably formulated to be sterile, isotonic and buffered for stability and comfort.
• Pharmaceutical compositions for intranasal delivery may also include drops and sprays often prepared to simulate in many respects nasal secretions to ensure maintenance of normal ciliary action.
• suitable formulations are most often and preferably isotonic, slightly buffered to maintain a pH of 5.5 to 6.5, and most often and preferably include antimicrobial preservatives and appropriate drug stabilizers.
• Pharmaceutical formulations for intraauricular delivery include suspensions and ointments for topical application in the ear. Common solvents for such aural formulations include glycerin and water.
• compositions may also be formulated in rectal compositions such as suppositories or retention enemas, e.g. , containing conventional suppository bases such as cocoa butter or other glycerides.
• compositions may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection.
• the compounds may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
• a suitable pharmaceutical carrier may be a cosolvent system comprising benzyl alcohol, a nonpolar surfactant, a water-miscible organic polymer, and an aqueous phase.
• a common cosolvent system used is the VPD co-solvent system, which is a solution of 3% w/v benzyl alcohol, 8% w/v of the nonpolar surfactant Polysorbate 80TM, and 65% w/v polyethylene glycol 300, made up to volume in absolute ethanol.
• VPD co-solvent system is a solution of 3% w/v benzyl alcohol, 8% w/v of the nonpolar surfactant Polysorbate 80TM, and 65% w/v polyethylene glycol 300, made up to volume in absolute ethanol.
• the proportions of a co-solvent system may be varied considerably without destroying its solubility and toxicity characteristics.
• co-solvent components may be varied: for example, other low-toxicity nonpolar surfactants may be used instead of POLYSORBATE 80TM; the fraction size of polyethylene glycol may be varied; other biocompatible polymers may replace polyethylene glycol, e.g., polyvinyl pyrrolidone; and other sugars or polysaccharides may substitute for dextrose.
• Methods for treating bacterial infections may include administering a therapeutically effective amount of the therapeutic compounds as described herein. Treating a bacterial infection may also include prophylactically administering the therapeutic compounds to prevent infection or the spread of an infection in a subject at imminent risk of infection, such as a subject receiving or about to undergo surgery, an immunocompromised subject, or subject otherwise at risk of an infection if the compound was not administered.
• the compounds show inhibitory activity against a broad spectrum of bacteria including H. influenzae, E. coli, S. aureus, E. faecalis, E. facium, K. pneumonia, A. baumannii, S. pneumoniae, and P. aeruginosa.
• the compounds show activity against most resistant strains for example methicillin resistant Staphylococcus aureus (MRS A).
• MRSA A methicillin resistant Staphylococcus aureus
• the compounds show broad-spectrum activity against all Category A, B, and C bacterial biodefense pathogens including B. anthracis, B. pseudomallei, B. mallei, F. tularensis and Y. psetis. See the Examples.
• the compounds have excellent relative antibiotic activity with a relatively low concentration.
• the compounds may exert potent antibacterial activity versus various human and animal pathogens, including Gram-positive and Gram-negative bacteria.
• the bacterial infection that may be treated or ameliorated is MRSA.
• Methods of treating bacterial infections also include intraabdominal infection, a urinary tract infection, or melioidosis.
• Intraabdominal infections include various infections such as peritonitis, appendicitis, abscesses, sepsis, and cholecystitis, which may be complicated or uncomplicated.
• the compound here in may also be used to treat urinary tract infections, which may be caused by E. coli.
• the compounds herein are useful to treat melioidosis, which may be caused by B. pseudomallei.
• compositions or pharmaceutical compositions described herein may be administered to the subject by any suitable means.
• methods of administration include, among others, (a) administration though oral pathways, which administration includes administration in capsule, tablet, granule, spray, syrup, or other such forms; (b) administration through non-oral pathways such as rectal, vaginal, intraurethral, intraocular, intranasal, or intraauricular, which administration includes administration as an aqueous suspension, an oily preparation or the like or as a drip, spray, suppository, salve, ointment or the like; (c) administration via injection, subcutaneously, intraperitoneally, intravenously, intramuscularly, intradermally, intraorbitally, intracapsularly, intraspinally, intrasternally, or the like, including infusion pump delivery; as well as (d) administration topically; as deemed appropriate by those of skill in the art for bringing the active compound into contact with living tissue.
• compositions suitable for administration include compositions where the active ingredients are contained in an amount effective to achieve its intended purpose.
• a therapeutically effective amount of a compound is an amount effective to treat a bacterial infection, for example, in a mammalian subject (e.g., a human).
• the therapeutically effective amount of the compounds disclosed herein required as a dose will depend on the route of administration, the type of animal, including human, being treated, and the physical characteristics of the specific animal under consideration. The dose can be tailored to achieve a desired effect, but will depend on such factors as weight, diet, concurrent medication and other factors which those skilled in the medical arts will recognize.
• a therapeutically effective amount means an amount of compound effective to prevent, alleviate or ameliorate symptoms of disease or prolong the survival of the subject being treated. Determination of a therapeutically effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.
• the useful in vivo dosage to be administered and the particular mode of administration will vary depending upon the age, weight and mammalian species treated, the particular compounds employed, and the specific use for which these compounds are employed.
• the determination of effective dosage levels can be accomplished by one skilled in the art using routine pharmacological methods. Typically, human clinical applications of products are commenced at lower dosage levels, with dosage level being increased until the desired effect is achieved. Alternatively, acceptable in vitro studies can be used to establish useful doses and routes of administration of the compositions identified by the present methods using established pharmacological methods.
• dosages may range broadly, depending upon the desired effects and the therapeutic indication. Typically, dosages may be about 10 microgram/kg to about 100 mg/kg body weight, preferably about 100 microgram kg to about 10 mg/kg body weight. Alternatively dosages may be based and calculated upon the surface area of the patient, as understood by those of skill in the art.
• the exact formulation, route of administration and dosage for the pharmaceutical compositions can be chosen by the individual physician in view of the patient's condition. (See e.g., Fingl et al. 1975, in "The Pharmacological Basis of Therapeutics", which is hereby incorporated herein by reference in its entirety, with particular reference to Ch. 1 , p. 1).
• the dose range of the composition administered to the patient can be from about 0.5 to about 1000 mg/kg of the patient's body weight.
• the dosage may be a single one or a series of two or more given in the course of one or more days, as is needed by the patient.
• human dosages for compounds have been established for at least some conditions, those same dosages, or dosages that are about 0.1% to about 500%, more preferably about 25% to about 250% of the established human dosage may be used.
• a suitable human dosage can be inferred from ED 5 o or ID 50 values, or other appropriate values derived from in vitro or in vivo studies, as qualified by toxicity studies and efficacy studies in animals.
• the attending physician would know how to and when to terminate, interrupt, or adjust administration due to toxicity or organ dysfunctions. Conversely, the attending physician would also know to adjust treatment to higher levels if the clinical response were not adequate (precluding toxicity).
• the magnitude of an administrated dose in the management of the disorder of interest will vary with the severity of the condition to be treated and to the route of administration. The severity of the condition may, for example, be evaluated, in part, by standard prognostic evaluation methods. Further, the dose and perhaps dose frequency will also vary according to the age, body weight, and response of the individual patient. A program comparable to that discussed above may be used in veterinary medicine.
• the daily dosage regimen for an adult human patient may be, for example, an oral dose of about 0.1 mg to 2000 mg of the active ingredient, preferably about 1 mg to about 500 mg, e.g. 5 to 200 mg.
• an intravenous, subcutaneous, or intramuscular dose of the active ingredient of about 0.01 mg to about 100 mg, preferably about 0.1 mg to about 60 mg, e.g. about 1 to about 40 mg is used.
• dosages may be calculated as the free acid.
• the composition is administered 1 to 4 times per day.
• compositions may be administered by continuous intravenous infusion, preferably at a dose of up to about 1000 mg per day.
• the compounds disclosed herein in certain situations it may be necessary to administer the compounds disclosed herein in amounts that exceed, or even far exceed, the above-stated, preferred dosage range in order to effectively and aggressively treat particularly aggressive diseases or infections.
• the compounds will be administered for a period of continuous therapy, for example for a week or more, or for months or years.
• Dosage amount and interval may be adjusted individually to provide plasma levels of the active moiety which are sufficient to maintain the antibiotic effects, or minimal effective concentration (MEC).
• MEC minimal effective concentration
• the MEC will vary for each compound but can be estimated from in vitro data. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. However, HPLC assays or bioassays can be used to determine plasma concentrations.
• Dosage intervals can also be determined using MEC value.
• Compositions should be administered using a regimen which maintains plasma levels above the MEC for 10-90% of the time, preferably between 30-90% and most preferably between 50-90%.
• the effective local concentration of the drug may not be related to plasma concentration.
• the amount of composition administered may be dependent on the subject being treated, on the subject's weight, the severity of the infection, the manner of administration and the judgment of the prescribing physician.
• compositions disclosed herein can be evaluated for efficacy and toxicity using known methods.
• the toxicology of the compound may be established by determining in vitro toxicity towards a cell line, such as a mammalian, and preferably human, cell line. The results of such studies are often predictive of toxicity in animals, such as mammals, or more specifically, humans.
• the toxicity of particular compounds in an animal model such as mice, rats, rabbits, or monkeys, may be determined using known methods.
• the efficacy of a particular compound may be established using several recognized methods, such as in vitro methods, animal models, or human clinical trials. Recognized in vitro models exist for nearly every class of condition.
• acceptable animal models may be used to establish efficacy of chemicals to treat such conditions.
• the skilled artisan can be guided by the state of the art to choose an appropriate model, dose, and route of administration, and regime.
• human clinical trials can also be used to determine the efficacy of a compound in humans.
• compositions may, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the active ingredient.
• the pack may for example comprise metal or plastic foil, such as a blister pack.
• the pack or dispenser device may be accompanied by instructions for administration.
• the pack or dispenser may also be accompanied with a notice associated with the container in form prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the drug for human or veterinary administration. Such notice, for example, may be the labeling approved by the U.S. Food and Drug Administration for prescription drugs, or the approved product insert.
• Compositions comprising a compound formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition.
• substantially pure refers to the amount of purity required for formulating pharmaceuticals, which may include, for example, a small amount of other material that will not affects the suitability for pharmaceutical use.
• the substantially pure compound contains at least about 96% of the compound by weight, such as at least about 97%, 98%, 99%, or 100% of the compound.
• the terms “approximately, “about,” and “substantially” as used herein represent an amount close to the stated amount that still performs the desired function or achieves the desired result.
• the terms “approximately,” “about” and “substantially” may refer to an amount that is within less than 10% of, within less than 5% of, within less than 1 % of, within less than 0.1% of, and within less than 0.01% of the stated amount.
• 6-(4,4,5,5-tetramethyl-l,3,2-dioxaborolan-2-yl)thiazolo[5,4-b]pyridine A round bottom flask was charged with 6-bromothiazolo[5,4-b]pyridine (430mg, 2.00 mmol), 4,4,4',4',5,5,5',5'-octamethyl-2,2'-bi(l,3,2-dioxaborolane) (660 mg, 2.6mmol), KOAc (392mg, 4.00 mmol), toluene (10 ml), vacuum flushed with nitrogen (3x), and treated with PdCl 2 dppf (82 mg, 0.100 mmol), vacuum flushed (3x), and placed in a 100 °C oil bath.
• Step 2 To a solution of ketone (1.0 g, 5.84 mmol) in methanol ( 10 ml) was added NaB3 ⁇ 4 (666 mg, 17.5 mmol). The mixture was heated at 50 C for 30 min. After this period, the mixture was concentrated and partitioned between ethyl acetate and water. The water layer was neutralized with 2N HCl and extracted with ethyl acetate. The organic layers were dried and concentrated. Silica gel chromatography of the residue gave the title compound as a pale red solid (470 mg, 48%).
• 1H-NMR (400 MHz) ⁇ 8.12 (d, 1H), 7.23 (d, 2H), 5.20 (brs, 1H), 5.09 (m, 1H), 4.16 (d, 1H), 1.45 (d, 3H).
• Boc protected compounds 5 (30mg) was dissolved into 2ml 50% TFA in DCM, the mixture was stirred for one 1 hours, then concentrated, the residue was purified by reverse HPLC to provide final compound 6( 12mg, yield 46% ).
• tert-butyl (2,4-bis(benzylsulfonyl)-6-fluoro-9H-pyrimido[4,5-3 ⁇ 4]indol-8- yl) (methyl) carbamate (365mg, 0.584mmol), phenylmethanethiol (72.6mg, 0.584mmol) and potassium carbonate (81mg, 0.584mmol) were mixed in NMP (4ml) at 0°C. The mixture was warmed to 40°C over a period of lh.

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