WO2024112201A1 - Inhibiteurs d'adn gyrase - Google Patents

Inhibiteurs d'adn gyrase Download PDF

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Publication number
WO2024112201A1
WO2024112201A1 PCT/NL2023/050618 NL2023050618W WO2024112201A1 WO 2024112201 A1 WO2024112201 A1 WO 2024112201A1 NL 2023050618 W NL2023050618 W NL 2023050618W WO 2024112201 A1 WO2024112201 A1 WO 2024112201A1
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compound
mmol
mhz
nmr
alkyl
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PCT/NL2023/050618
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Alexander T. BAKKER
Ioli KOTSOGIANNI
Mario Van Der Stelt
Nathaniel I. MARTIN
Dmitry GHILAROV
Mariana AVALOS
Jeroen M. PUNT
Bing Liu
Diana PIERMARINI
Constant A.A. Van Boeckel
Gilles P. Van Wezel
Richard J.H.B.N VAN DEN BERG
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Universiteit Leiden
John Innes Centre
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Publication of WO2024112201A1 publication Critical patent/WO2024112201A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/472—Non-condensed isoquinolines, e.g. papaverine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/472—Non-condensed isoquinolines, e.g. papaverine
    • A61K31/4725—Non-condensed isoquinolines, e.g. papaverine containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/12—Cyclic peptides, e.g. bacitracins; Polymyxins; Gramicidins S, C; Tyrocidins A, B or C
    • 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
    • C07D215/00—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems
    • C07D215/02—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen atoms or carbon atoms directly attached to the ring nitrogen atom
    • C07D215/16—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen atoms or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D215/36—Sulfur atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
    • C07D401/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings linked by a chain containing hetero atoms as chain links
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing three or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D409/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms
    • C07D409/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing two hetero rings
    • C07D409/12—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing two hetero rings linked by a chain containing hetero atoms as chain links

Definitions

  • This invention relates to compounds useful for treating bacterial infections.
  • the compounds may be capable of binding a horseshoe-like hydrophobic pocket on the DNA-binding surface GyrA subunit of DNA gyrase.
  • formulations comprising such compounds, as well uses of such compounds or formulations, e.g. for the treatment of bacterial infection, and/or as a DNA gyrase inhibitor.
  • Target-based screening is an effective approach to identify novel small molecules as hits in a drug discovery program, but it is less successful in the search for novel Gram-negative-specific antibiotics.
  • 67 Lack of target validation and the additional outer membrane (OM) of Gram-negative bacteria constitute major bottlenecks for target-based drug discovery.
  • Rational modification of existing drugs 89 and phenotypic screening have instead emerged as promising strategies to overcome these challenges.
  • 10 11 A major advantage of phenotypic screening is the opportunity to discover compounds with an unprecedented MoA, and historically the mining of libraries of natural product extracts presented a fruitful strategy to search for new target-drug combinations.
  • DNA gyrase has proven to be an excellent target for antibiotics with a number of clinically used anti-infectives operating by inhibiting its activity. 14 Given that DNA gyrase lacks a direct human homolog, has multiple target sites, and is essential to bacterial DNA function, inhibitors have the potential to selectively target bacterial cells vs host cells. Notably, the DNA negative supercoiling activity of DNA gyrase is a multi-step process that can be interrupted at multiple stages through pharmacological intervention. 15 Key examples are the classical fluoroquinolone antibiotics 16 and the more recent NBTIs 17 that intercalate in DNA, the aminocoumarins 18 that compete with ATP, and SD8 19 which prevents DNA binding with the gyrase complex.
  • An aim of certain embodiments of the present invention is to provide compounds with antibiotic activity against clinically relevant bacteria, such as Gram-negative bacteria, e.g. E. coli and K. pneumoniae.
  • An aim of certain embodiments of the present invention is to provide compounds that inhibit bacterial DNA gyrase.
  • An aim of certain embodiments of the present invention is to provide compounds that have activity against bacterial strains that have developed a resistance to prior art compounds, e.g. fluoroquinolones.
  • a compound of formula I wherein: a single one of X 1 , X 2 and X 3 is N and the remaining two of X 1 , X 2 and X 3 are CR 7 ;
  • R 1 is selected from H, halo, C1-C4 alkyl and C1-C4 haloalkyl;
  • R 2 is selected from H, halo, C1-C4 alkyl and C1-C4 haloalkyl; or R 1 and R 2 together form a 1 , 2 or 3 membered alkylene chain, optionally substituted, where chemically possible, with one, two or three groups independently selected from halo, C1-C4 alkyl, C1-C4 haloalkyl, CN; each R 3a and R 3b is independently selected from halo, C1-C4 alkyl, C1-C4 haloalkyl, CN, OR a and NR b R b , wherein R a is selected from H, C1-C4 alkyl and C1-C4 haloalkyl and each R b is independently selected from H and C1-C4 alkyl;
  • R 4 is selected from H, C1-C4 alkyl, C1-C4 haloalkyl
  • R 5 is selected from phenyl and a 5- or 6-membered heteroaryl group, optionally substituted with 1 or 2 R 3b groups; each R 6 is independently selected from halo, C1-C4 alkyl, C1-C4 haloalkyl, CN, -O-C1-C4 alkyl and -O-C1-C4 haloalkyl; each R 7 is independently selected from H, halo, C1-C4 alkyl, C1-C4 haloalkyl, CN, -O-C1-C4 alkyl and -O-C1-C4 haloalkyl; n is 0, 1 , 2 or 3; and p is 0, 1 , 2 or 3; or a pharmaceutically acceptable salt, stereoisomer, solvate, or prodrug thereof.
  • the compound of formula I is not a compound selected from
  • R 1 , R 2 , R 3a , R 3b , R 4 and p are as defined above for compounds of formula I; and q is 0, 1 or 2;
  • X 4 is N, CH, or CR 10 ;
  • X 5 is N, CH, or CR 11 ;
  • R 10 is selected from halo, C1-C4 alkyl, C1-C4 haloalkyl and CN;
  • R 11 is selected from halo, C1-C4 alkyl, C1-C4 haloalkyl and CN, with the proviso that when X 4 is N, X 5 is CH or CR 11 , and when X 5 is N, X 4 is CH or CR 10 , or a pharmaceutically acceptable salt, stereoisomer, solvate, or prodrug thereof.
  • the compound of formula I is a compound of formula III: wherein R 1 , R 2 , R 3a , R 4 , R 5 and p are as defined above for compounds of formula I, or a pharmaceutically acceptable salt, stereoisomer, solvate, or prodrug thereof.
  • X 1 , X 2 , X 3 , R 1 , R 2 , R 3a , R 3b , R 4 , R 6 , R 7 , n and p are as defined above for compounds of formula I; and q is 0, 1 or 2;
  • X 4 is N, CH, or CR 10 ;
  • X 5 is N, CH, or CR 11 ;
  • R 10 is selected from halo, C1-C4 alkyl, C1-C4 haloalkyl and CN;
  • R 11 is selected from halo, C1-C4 alkyl, C1-C4 haloalkyl and CN, with the proviso that when X 4 is N, X 5 is CH or CR 11 , and when X 5 is N, X 4 is CH or CR 10 , or a pharmaceutically acceptable salt, stereoisomer, solvate, or prodrug thereof.
  • the compound of formula I is a compound of formula V:
  • X 1 , X 2 , X 3 , R 3a , R 4 , R 5 , R 6 , R 7 , n and p are as defined above for compounds of formula I; and each R 8 and R 9 is independently selected from H, halo, C1-C4 alkyl, C1-C4 haloalkyl and CN; and m is 0, 1 or 2, or a pharmaceutically acceptable salt, stereoisomer, solvate, or prodrug thereof.
  • the compound of formula I is a compound of formula VI: wherein R 3a , R 3b , R 4 and p are as defined above for compounds of formula I; and q is 0, 1 or 2; X 4 is N, CH, or CR 10 ; X 5 is N, CH, or CR 11 ; R 10 is selected from halo, C1-C4 alkyl, C1-C4 haloalkyl and CN; R 11 is selected from halo, C1-C4 alkyl, C1-C4 haloalkyl and CN, with the proviso that when X 4 is N, X 5 is CH or CR 11 , and when X 5 is N, X 4 is CH or CR 10 ; each R 8 and R 9 is independently selected from H, halo, C1-C4 alkyl, C1-C4 haloalkyl and CN; and m is 0, 1 or 2, or a pharmaceutically acceptable salt, stereoisomer,
  • the compound of formula (I) is a compound of formula VII: wherein R 3a , R 4 , R 5 and p are as defined above for compounds of formula I; and each R 8 and R 9 is independently selected from H, halo, C1-C4 alkyl, C1-C4 haloalkyl and CN; and m is 0, 1 or 2, or a pharmaceutically acceptable salt, stereoisomer, solvate, or prodrug thereof.
  • the compound of formula I is a compound of formula VIII: or 2; X 4 is N, CH, or CR 10 ; X 5 is N, CH, or CR 11 ; R 10 is selected from halo, C1-C4 alkyl, C1-C4 haloalkyl and CN; and R 11 is selected from halo, C1-C4 alkyl, C1-C4 haloalkyl and CN, with the proviso that when X 4 is N, X 5 is CH or CR 11 , and when X 5 is N, X 4 is CH or CR 10 ; each R 8 and R 9 is independently selected from H, halo, C1-C4 alkyl, C1-C4 haloalkyl and CN; and m is 0, 1 or 2, or a pharmaceutically acceptable salt, stereoisomer, solvate, or prodrug thereof.
  • the compound of formula I is a compound of formula IX: wherein X 1 , X 2 , X 3 , R 3a , R 4 , R 5 , R 6 , R 7 , n and p are as defined above for compounds of formula I; and each R 8 and R 9 is independently selected from H, halo, C1-C4 alkyl, C1-C4 haloalkyl and CN; and m is 0, 1 or 2, or a pharmaceutically acceptable salt, stereoisomer, solvate, or prodrug thereof.
  • the compound of formula I is a compound of formula X: wherein R 3a , R 3b , R 4 and p are as defined above for compounds of formula I; q is 0, 1 or 2; X 4 is N, CH, or CR 10 ; X 5 is N, CH, or CR 11 ; R 10 is selected from halo, C1-C4 alkyl, C1-C4 haloalkyl and CN; and R 11 is selected from halo, C1-C4 alkyl, C1-C4 haloalkyl and CN, with the proviso that when X 4 is N, X 5 is CH or CR 11 , and when X 5 is N, X 4 is CH or CR 10 ; each R 8 and R 9 is independently selected from H, halo, C1-C4 alkyl, C1-C4 haloalkyl and CN; and m is 0, 1 or 2, or a pharmaceutically acceptable salt, stereoisomer
  • the compound of formula I is a compound of formula XI:
  • R 3a , R 4 , R 5 and p are as defined above for compounds of formula I; each R 8 and R 9 is independently selected from H, halo, C1-C4 alkyl, C1-C4 haloalkyl and CN; and m is 0, 1 or 2, or a pharmaceutically acceptable salt, stereoisomer, solvate, or prodrug thereof.
  • the compound of formula I is a compound of formula XII: wherein X 1 , X 2 , X 3 , R 3a , R 3b , R 4 , R 6 , R 7 , n and p are as defined above for compounds of formula I; q is 0, 1 or 2; X 4 is N, CH, or CR 10 ; X 5 is N, CH, or CR 11 ; R 10 is selected from halo, C1-C4 alkyl, C1-C4 haloalkyl and CN; and R 11 is selected from halo, C1-C4 alkyl, C1-C4 haloalkyl and CN, with the proviso that when X 4 is N, X 5 is CH or CR 11 , and when X 5 is N, X 4 is CH or CR 10 ; each R 8 and R 9 is independently selected from H, halo, C1-C4 alkyl, C1-C4 ;
  • the compound of formula I is a compound of formula XIII: wherein X 1 , X 2 , X 3 , R 3a , R 4 , R 5 , R 6 , R 7 , n and p are as defined above for compounds of formula I; and each R 8 and R 9 is independently selected from H, halo, C1-C4 alkyl, C1-C4 haloalkyl and CN; and m is 0, 1 or 2, or a pharmaceutically acceptable salt, stereoisomer, solvate, or prodrug thereof.
  • the compound of formula I is a compound of formula XIV:
  • X 5 is N, CH, or CR 11 ;
  • R 10 is selected from halo, C1-C4 alkyl, C1-C4 haloalkyl and CN; and
  • R 11 is selected from halo, C1-C4 alkyl, C1-C4 haloalkyl and CN, with the proviso that when X 4 is N, X 5 is CH or CR 11 , and when X 5 is N, X 4 is CH or CR 10 ;
  • each R 8 and R 9 is independently selected from H, halo, C1-C4 alkyl, C1-C4 haloalkyl and CN; and
  • m is 0, 1 or 2, or a pharmaceutically acceptable salt, stereoisomer, solvate, or prodrug thereof.
  • the compound of formula I is a compound of formula XVI wherein R 3a , R 4 , R 5 and p are as defined above for compounds of formula I; each R 8 and R 9 is independently selected from H, halo, C1-C4 alkyl, C1-C4 haloalkyl and CN; and m is 0, 1 or 2, or a pharmaceutically acceptable salt, stereoisomer, solvate, or prodrug thereof.
  • the compound of formula I is a compound of formula XVI: or 2; X 4 is N, CH, or CR 10 ; X 5 is N, CH, or CR 11 ; R 10 is selected from halo, C1-C4 alkyl, C1-C4 haloalkyl and CN; and R 11 is selected from halo, C1-C4 alkyl, C1-C4 haloalkyl and CN, with the proviso that when X 4 is N, X 5 is CH or CR 11 , and when X 5 is N, X 4 is CH or CR 10 ; each R 8 and R 9 is independently selected from H, halo, C1-C4 alkyl, C1-C4 haloalkyl and CN; and m is 0, 1 or 2, or a pharmaceutically acceptable salt, stereoisomer, solvate, or prodrug thereof.
  • X 1 , X 2 and X 3 are N and the remaining two of X 1 , X 2 and X 3 are CH. It may be that X 2 is N and X 1 and X 2 are both CH.
  • Each R 7 may be independently selected from H, halo, C1-C2 alkyl, C1-C2 haloalkyl, CN, -O-C1-C2 alkyl and -O-C1-C2 haloalkyl.
  • Each R 7 may be independently selected from H, halo, CH3, Ci haloalkyl, CN, -OCH3 and -O-C1 haloalkyl.
  • Each R 7 may be independently selected from halo or H.
  • Each R 7 may be independently selected from halo.
  • each R 7 is H.
  • X 2 is N and X 1 and X 3 are independently selected from CH and CR 7a , wherein each R 7a is independently selected from F and CF3.
  • n is 0 or 1 .
  • n is 0.
  • Each R 6 may be independently selected from halo, C1-C2 alkyl, C1-C2 haloalkyl, CN, -O-C1-C2 alkyl and -O-C1-C2 haloalkyl.
  • Each R 6 may be independently selected from H, halo, CH3, Ci haloalkyl, CN, - OCH3 and -O-C1 haloalkyl. It may be that each R 6 is independently selected from halo or H, e.g. each R 6 may be independently selected from halo. It may be that n is 1 and R 6 is halo, e.g. F.
  • R 1 is selected from H, halo, C1-C2 alkyl and C1-C2 haloalkyl.
  • R 1 may be selected from H, halo, CH3 and Ci haloalkyl.
  • R 1 is selected from H and halo.
  • R 1 may be H.
  • R 2 is selected from H, halo, C1-C2 alkyl and C1-C2 haloalkyl.
  • R 2 may be selected from H, halo, CH3 and Ci haloalkyl. It may be that R 2 is selected from H and halo.
  • R 2 may be H.
  • R 1 and R 2 may be H.
  • R 1 and R 2 together may form a 1 , 2 or 3 membered alkylene chain, optionally substituted, where chemically possible, with one, two or three groups independently selected from halo, C1-C4 alkyl, C1-C4 haloalkyl and CN.
  • R 1 and R 2 together may form a 1 , 2 or 3 membered alkylene chain, optionally substituted, where chemically possible, with one, two or three groups independently selected from halo, C1-C2 alkyl, C1-C2 haloalkyl and CN.
  • R 1 and R 2 together may form a 1 , 2 or 3 membered alkylene chain, optionally substituted, where chemically possible, with one, two or three groups independently selected from halo, CH3, CF3 and CN. It may be that R 1 and R 2 together form an unsubstituted 1 , 2 or 3 membered alkylene chain.
  • R 1 and R 2 together may form a 2 or 3 membered alkylene chain, optionally substituted with one, two or three groups independently selected from halo, C1-C4 alkyl, C1-C4 haloalkyl and CN.
  • R 1 and R 2 together may form a 2 or 3 membered alkylene chain, optionally substituted with one, two or three groups independently selected from halo, C1-C2 alkyl, C1-C2 haloalkyl and CN.
  • R 1 and R 2 together may form a 2 or 3 membered alkylene chain, optionally substituted with one, two or three groups independently selected from halo, CH3, CF3 and CN. It may be that R 1 and R 2 together form an unsubstituted 2 or 3 membered alkylene chain.
  • R 1 and R 2 together may form a 2 membered alkylene chain, optionally substituted, with one, two or three groups independently selected from halo, C1-C4 alkyl, C1-C4 haloalkyl and CN.
  • R 1 and R 2 together may form a 2 membered alkylene chain, optionally substituted, with one, two or three groups independently selected from halo, C1-C2 alkyl, C1-C2 haloalkyl and CN.
  • R 1 and R 2 together may form a 2 membered alkylene chain, optionally substituted, with one, two or three groups independently selected from halo, CH3, CF3 and CN.
  • R 1 and R 2 together may form an unsubstituted 2 membered alkylene chain.
  • p is 0, 1 or 2. It may be that p is 0 or 1 . p may be 1 . p may be 0.
  • each R 3a is independently selected from halo, C1-C2 alkyl, C1-C2 haloalkyl, CN, OR a and NR b R b , wherein R a is selected from H, C1-C2 alkyl and C1-C2 haloalkyl and each R b is independently selected from H and C1-C2 alkyl.
  • each R 3a is independently selected from halo, CH3, CF3, OH, CN and NH2.
  • Each R 3a may be independently selected from halo, e.g. F. It may be that p is 1 and R 3a is halo, e.g. F.
  • R 4 may be selected from H, C1-C2 alkyl, C1-C2 haloalkyl.
  • R 4 may be selected from H, CH3 and CF3.
  • R 4 is H.
  • R 5 may be phenyl.
  • R 5 may be unsubstituted phenyl.
  • R 5 may be phenyl substituted with 1 or 2 R 3b groups.
  • R 5 may be phenyl substituted with 1 R 3b group.
  • R 5 may be a 5- or 6-membered heteroaryl group.
  • R 5 may be an unsubstituted 5- or 6-membered heteroaryl group.
  • R 5 may be a 5- or 6-membered heteroaryl group, optionally substituted with 1 or 2 R 3b groups.
  • R 5 may be a 5- or 6-membered heteroaryl group, optionally substituted with 1 R 3b group.
  • R 5 may be a 6-membered heteroaryl group, e.g. pyridinyl.
  • R 5 may be an unsubstituted 6- membered heteroaryl group, e.g. pyridinyl.
  • R 5 may be a 6-membered heteroaryl group, e.g. pyridinyl, optionally substituted with 1 or 2 R 3b groups.
  • R 5 may be a 6-membered heteroaryl group, e.g. pyridinyl, optionally substituted with 1 R 3b group.
  • R 5 may be a 5-membered heteroaryl group.
  • R 5 may be an unsubstituted 5-membered heteroaryl group.
  • R 5 may be a 5-membered heteroaryl group, optionally substituted with 1 or 2 R 3b groups.
  • R 5 may be a 5-membered heteroaryl group, optionally substituted with 1 R 3b group.
  • Exemplary five-membered heteroaryl groups include furanyl, thiophenyl, pyrrolyl, oxazolyl, triazolyl and thiazolyl. It may be that R 5 is furanyl or thiophenyl. It may be that R 5 is thiophenyl.
  • each R 3b is independently selected from halo, C1-C2 alkyl, C1-C2 haloalkyl, CN, OR a and NR b R b , wherein R a is selected from H, C1-C2 alkyl and C1-C2 haloalkyl and each R b is independently selected from H and C1-C2 alkyl. It may be that each R 3b is independently selected from halo, CH3, CF3, OH, CN and NH2. Each R 3a may be independently selected from halo, CH3 and CN.
  • R 5 may be: , wherein f indicates the point of attachment to the rest of the compound.
  • R 5 may be: , wherein: X 4 is N, CH, or CR 10 ; X 5 is N, CH, or CR 11 ; R 10 is selected from halo, C1-C4 alkyl, C1-C4 haloalkyl and CN; R 11 is selected from halo, C1-C4 alkyl, C1-C4 haloalkyl and CN; q is 0, 1 or 2; and / indicates the point of attachment to the rest of the compound, with the proviso that when X 4 is N, X 5 is CH or CR 11 , and when X 5 is N, X 4 is CH or CR 10 .
  • X 4 may be N. It may be that X 4 is N and X 5 is CH. It may be that X 4 is N and X 5 is CR 11 . X 5 may be N. It may be that X 5 is N and X 4 is CH. It may be that X 5 is CH or CR 12 . X 5 may be CH. It may be that X 4 is CH or CR 10 . X 4 may be is CH. It may be that X 4 and X 5 are each CH. It may be that X 4 is CH and X 5 is CR 11
  • R 10 may be selected from halo, CH3, CF3 and CN.
  • R 10 may be CN.
  • R 11 may be selected from halo, CH3, and CF3 and CN.
  • R 11 may be halo, e.g. F.
  • q is 0. It may be that q is 1 . It may be that q is 1 and R 3b is selected from halo, CH3, CF3 and CN It may be that q is 1 and R 3b is Me.
  • each R 8 and R 9 is independently selected from H, halo, C1-C2 alkyl, C1-C2 haloalkyl and CN.
  • each R 8 and R 9 may be independently selected from H, halo, CH3, Ci haloalkyl and CN.
  • R 8 is H.
  • each R 9 is H.
  • m is 1 or 2.
  • m is 1 .
  • R 8 and R 9 are both H.
  • the compound of formula I may be a compound selected from: or a pharmaceutically acceptable salt, stereoisomer, solvate, or prodrug thereof.
  • the compound of formula I may be a compound selected from: or a pharmaceutically acceptable salt, stereoisomer, solvate, or prodrug thereof.
  • the compound of formula (I) may be:
  • DNA gyrase is a well-validated essential bacterial topoisomerase that is targeted by ciprofloxacin (CIP), a fluoroquinolone for which widespread resistance has been observed in the clinic.
  • CIP ciprofloxacin
  • the compounds described herein did not show any cross- resista nee with CIP or vice versa.
  • the present inventors carried out extensive structural biology studies using cryo-electron microscopy and identified an unprecedented allosteric binding mode of 102 at a site distinct from CIP.
  • This novel binding site (also referred to herein as “a binding pocket”) is a horseshoe-like hydrophobic pocket on the DNA-binding surface GyrA subunit of DNA gyrase.
  • a binding pocket is a horseshoe-like hydrophobic pocket on the DNA-binding surface GyrA subunit of DNA gyrase.
  • a binding pocket is a horseshoe-like hydrophobic pocket on the DNA-binding surface GyrA subunit of DNA gyrase.
  • the compound may be a compound according to the first aspect.
  • the compound binds to both GyrA subunits of DNA gyrase. It may be that the compound is capable of binding a horseshoe-like hydrophobic pocket on the DNA-binding surface GyrA subunit of DNA gyrase thereby inhibiting DNA cleavage.
  • the compound may comprise an amine containing group A.
  • the amine may be a secondary amine.
  • the amine may be a cyclic amine.
  • the amine may be a cyclic secondary amine.
  • Illustrative cyclic secondary amines include pyrrolidine, pyrrole, 3-pyrroline, 2-pyrroline, piperidine, imidazole, 2- imidazoline, 3-imidazoline , 4-imidazoline, imidazolidine, pyrazole, 2-pyrazoline, pyrazolidine, and piperazine. It may be that the amine is pyrrolidine.
  • amine forms a hydrogen bond with amino acid residue S97 of SEQ ID NO: 1 , or a corresponding amino acid residue in a homolog thereof, or a conservative modifications thereof.
  • a, or the, oxygen atom of the sulfur containing group B forms a hydrogen bond with amino acid residue S172 of SEQ ID NO: 1 , or a corresponding amino acid residue in a homolog thereof, or a conservative modifications thereof. It may be that a, or the, oxygen atom of the sulfur containing group B forms a hydrogen bond with amino acid residue K42 of SEQ ID NO: 1 , or a corresponding amino acid residue in a homolog thereof, or a conservative modifications thereof.
  • a, or the, oxygen atom of the sulfur containing group B forms a hydrogen bond with amino acid residue S172 of SEQ ID NO: 1 , or a corresponding amino acid residue in a homolog thereof, or a conservative modifications thereof; and also forms a hydrogen bond with amino acid residue K42 of SEQ ID NO: 1 , or a corresponding amino acid residue in a homolog thereof, or a conservative modifications thereof.
  • the compound may comprise amine containing group A and sulfur containing group B. It may be that the nitrogen atom of amine containing group A and the sulfur atom of sulfur containing group B are spaced from 2 to 5 atoms apart, e.g. from 2 to 4 atoms apart. It may be that the nitrogen atom of amine containing group A and the sulfur atom of sulfur containing group B are spaced 3 atoms apart.
  • the compound may comprise a quinoline or isoquinoline.
  • the quinoline or isoquinoline may be proximate to the sulfur containing group B.
  • the sulfur containing group B may be attached to a quinoline or an isoquinoline at one of C5, C6, C7 or C8 by a covalent bond or linker (such as C1-C4 alkyl).
  • the compound may comprise the moiety: wherein R 1 , R 2 and R 4 are as defined elsewhere (e.g. in relation to formula
  • An oxygen atom of the sulfonamide group may form a hydrogen bond with amino acid residue S172 of SEQ ID NO: 1 , or a corresponding amino acid residue in a homolog thereof, or a conservative modifications thereof.
  • An oxygen atom of the sulfonamide group may form a hydrogen bond with amino acid residue K42 of SEQ ID NO: 1 , or a corresponding amino acid residue in a homolog thereof, or a conservative modifications thereof.
  • An oxygen atom of the sulfonamide group may form a hydrogen bond with amino acid residue S172 of SEQ ID NO: 1 , or a corresponding amino acid residue in a homolog thereof, or a conservative modifications thereof; and form a hydrogen bond with amino acid residue K42 of SEQ ID NO: 1 , or a corresponding amino acid residue in a homolog thereof, or a conservative modifications thereof.
  • the rightmost amine group i.e. positioned between R 1 and R 2 ) may form a hydrogen bond with amino acid residue S97 of SEQ ID NO: 1 , or a corresponding amino acid residue in a homolog thereof, or a conservative modifications thereof.
  • the compound may have a molecular weight of ⁇ 1000 g.mol 1 , e.g. ⁇ 750 g.mol 1 .
  • binding pocket is described in more detail hereinbelow.
  • the inventors’ identification of the isoquinoline sulfonamides as allosteric DNA gyrase inhibitors paves the way for the development of a novel class of antibiotics targeting bacteria, including those that have become resistant to fluoroquinolones. Due to the amino acids defining the binding pocket being broadly conserved across many species of bacteria, the inventors believe that the compounds described herein may have antibiotic activity against Gram-negative and/or Gram-positive bacteria. Suitably, the compounds described herein may have antibiotic activity against Gram-negative bacteria (for example Escherichia coli, as described in more detail in the Examples section of the present disclosure).
  • Gram-negative bacteria for example Escherichia coli, as described in more detail in the Examples section of the present disclosure.
  • the binding pocket may be defined by residues 92, 97, 98, 169, 172, 266, 267, 268 and 269 of SEQ ID NO: 1 (amino acid sequence of DNA gyrase subunit A of Escherichia coli), or corresponding amino acid residues in homologs thereof.
  • residues 92, 97, 98, 169, 172, 266, 267, 268 and 269 of SEQ ID NO: 1 are respectively, M92, S97, L98, N169, S172, Y266, Q267, V268 and N269 of SEQ ID NO: 1 , or conservative modifications thereof. What constitutes a conservative modification will be known to a person skilled in the art. However, examples of conservative modifications are provided herein below.
  • the binding pocket may be defined by residues 42, 92, 96, 97, 98, 169, 172, 266, 267, 268 and 269 of SEQ ID NO: 1 (amino acid sequence of DNA gyrase subunit A of Escherichia coli), or corresponding amino acid residues in homologs thereof.
  • residues 92, 96, 97, 98, 169, 172, 266, 267, 268 and 269 of SEQ ID NO: 1 are respectively, K42, M92, F96, S97, L98, N169, S172, Y266, Q267, V268 and N269 of SEQ ID NO: 1 , or conservative modifications thereof. What constitutes a conservative modification will be known to a person skilled in the art. However, examples of conservative modifications are provided herein below.
  • the reference polypeptide may be the amino acid sequence according to SEQ ID NO: 1.
  • SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25 and 26 provide non-limiting examples of homologs from other bacterial species.
  • the term “corresponding amino acid” refers to an amino acid which is present within a corresponding region and which is the counterpart of a given amino acid of SEQ ID NO: 1 in a sequence alignment (for example as shown in Figures 1 to 26).
  • the amino acid of SEQ ID NO: 2 that corresponds to amino acid residue M92 of SEQ ID NO: 1 is M96.
  • the amino acid of SEQ ID NO: 2 that corresponds to the amino acid residue S97 of SEQ ID NO: 1 is S100.
  • the amino acid of SEQ ID NO: 2 that corresponds to the amino acid residue L98 of SEQ ID NO: 1 is Q101 .
  • the amino acid of SEQ ID NO: 2 that corresponds to the amino acid residue N169 of SEQ ID NO: 1 is N172.
  • the amino acid of SEQ ID NO: 2 that corresponds to the amino acid residue S172 of SEQ ID NO: 1 is S175.
  • the amino acid of SEQ ID NO: 2 that corresponds to the amino acid residue Y266 of SEQ ID NO: 1 is Y268.
  • the amino acid of SEQ ID NO: 2 that corresponds to the amino acid residue Q267 of SEQ ID NO: 1 is Q269.
  • amino acid of SEQ ID NO: 2 that corresponds to the amino acid residue V268 of SEQ ID NO: 1 is V270.
  • amino acid of SEQ ID NO: 2 that corresponds to the amino acid residue N269 of SEQ ID NO: 1 is N271 .
  • the corresponding amino acid does not have to be the same amino acid as in the reference polypeptide.
  • the corresponding amino acid may be a similar amino acid.
  • Similar amino acid residues are grouped by chemical characteristics of the side chains into families. Said families are described below for “conservative amino acid substitutions”.
  • sequence alignment may be obtained by using bioinformatics tools such as EMBOSS Needle (pair wise alignment; available at www.ebi.ac.uk).
  • EMBOSS Needle air wise alignment; available at www.ebi.ac.uk.
  • the “percent identity” is a function of the number of matching positions divided by the number of positions compared and multiplied by 100%. For instance, if 6 out of 10 sequence positions are identical, then the identity is 60%.
  • the percent identity between two protein sequences can, e.g., be determined using the Needleman and Wunsch algorithm (NEEDLEMAN, S. B. and Wunsch, C. D. A general method applicable to the search for similarities in the amino acid sequence of two proteins. Journal of Molecular Biology 1970, vol. 48, p. 443-453) which has been incorporated into EMBOSS Needle.
  • the % identity is typically determined over the entire length of the query sequence on which the analysis is performed.
  • Two molecules having the same primary amino acid sequence are identical irrespective of any chemical and/or biological modification. For example, two antibodies having the same primary amino acid sequence but different glycosylation patterns are identical by this definition. Similar protein sequences are those which, when aligned, share similar amino acid residues and most often, but not mandatorily, identical amino acid residues at the same positions of the sequences to be compared.
  • the novel binding pocket is defined by residues M92, F96, S97, L98, N169, S172, Y266, Q267, V268 and N269 of SEQ ID NO: 1 (amino acid sequence of DNA gyrase subunit A of Escherichia col'i), or corresponding amino acid residues in homologs thereof, the binding pocket may also be defined by amino acids that are conservative modifications of these amino acids.
  • the compounds described herein may inhibit DNA gyrase by binding a horseshoe-like hydrophobic pocket on the DNA-binding surface GyrA subunit of DNA gyrase.
  • the compounds described herein may bind one or more amino acid residues selected from the group consisting 92, 96, 97, 98, 169, 172, 266, 267, 268 and 269 of SEQ ID NO: 1 (amino acid sequence of DNA gyrase subunit A of Escherichia co/i), or corresponding amino acid residues in homologs thereof.
  • the compounds described herein may bind one or more amino acid residues selected from the group consisting of M92, F96, S97, L98, N169, S172, Y266, Q267, V268 and N269 of SEQ ID NO:1 , or corresponding amino acid residues in homologs thereof, or conservative modifications thereof.
  • the compounds may bind one amino acid residue selected from the group consisting of M92, F96, S97, L98, N169, S172, Y266, Q267, V268 and N269 of SEQ ID NO:1 , or corresponding amino acid residues in homologs thereof, or conservative modifications thereof.
  • the compounds may bind two amino acid residues selected from the group consisting of M92, F96, S97, L98, N169, S172, Y266, Q267, V268 and N269 of SEQ ID NO:1 , or corresponding amino acid residues in homologs thereof, or conservative modifications thereof.
  • the compounds may bind three amino acid residues selected from the group consisting of M92, F96, S97, L98, N169, S172, Y266, Q267, V268 and N269 of SEQ ID NO:1 , or corresponding amino acid residues in homologs thereof, or conservative modifications thereof.
  • the compounds may bind four amino acid residues selected from the group consisting of M92, F96, S97, L98, N169, S172, Y266, Q267, V268 and N269 of SEQ ID NO:1 , or corresponding amino acid residues in homologs thereof, or conservative modifications thereof.
  • the compounds may bind five amino acid residues selected from the group consisting of M92, F96, S97, L98, N169, S172, Y266, Q267, V268 and N269 of SEQ ID NO:1 , or corresponding amino acid residues in homologs thereof, or conservative modifications thereof.
  • the compounds may bind six amino acid residues selected from the group consisting of M92, F96, S97, L98, N169, S172, Y266, Q267, V268 and N269 of SEQ ID NO:1 , or corresponding amino acid residues in homologs thereof, or conservative modifications thereof.
  • the compounds may bind seven amino acid residues selected from the group consisting of M92, F96, S97, L98, N169, S172, Y266, Q267, V268 and N269 of SEQ ID NO:1 , or corresponding amino acid residues in homologs thereof, or conservative modifications thereof.
  • the compounds may bind eight amino acid residues selected from the group consisting of M92, F96, S97, L98, N169, S172, Y266, Q267, V268 and N269 of SEQ ID NO:1 , or corresponding amino acid residues in homologs thereof, or conservative modifications thereof.
  • the compounds may bind all nine amino acid residues selected from the group consisting of M92, F96, S97, L98, N169, S172, Y266, Q267, V268 and N269 of SEQ ID NO:1 , or corresponding amino acid residues in homologs thereof, or conservative modifications thereof.
  • the compounds may bind to amino acid residue S97 or S172 of SEQ ID NO: 1 , or a corresponding amino acid residue in a homolog thereof, or a conservative modifications thereof.
  • the compounds may bind to amino acid residue S97 and S172 of SEQ ID NO: 1 , or a corresponding amino acid residue in a homolog thereof, or a conservative modifications thereof.
  • the binding to residue S97 and/or S172 of SEQ ID NO: 1 , or a corresponding amino acid residue in a homolog thereof, or a conservative modification thereof is by hydrogen bonding.
  • the compounds may bind to amino acid residue K42 of SEQ ID NO: 1 , or a corresponding amino acid residue in a homolog thereof, or a conservative modifications thereof.
  • the binding to residue K42 of SEQ ID NO: 1 , or a corresponding amino acid residue in a homolog thereof, or a conservative modification thereof, is by hydrogen bonding.
  • Binding of a compound of the invention to the novel binding pocket can be confirmed using suitable structural biology techniques known in art, such as cryogenic electron microscopy (Cryo EM). This can be combined with functional assay to confirm inhibitory activity.
  • a suitable functional assay to confirm inhibitory activity is a DNA gyrase supercoiling assay as disclosed herein.
  • the structural biology technique and/or the functional assay is preferably performed using an enzyme comprising a polypeptide of SEQ ID NO: 1 or a homolog (such as SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, or 26).
  • the DNA gyrase may contain a mutation that confers fluoroquinolone resistance. It may be that the mutation is a mutation to an amino acid residue in SEQ ID NO: 1 , or in or a corresponding amino acid residue in a homolog thereof. It may be that the mutation is a mutation to S83 in SEQ ID NO: 1 , or to the corresponding amino acid residue in a homolog thereof. It may be that the mutation is a mutation to D87 in SEQ ID NO: 1 , or to the corresponding amino acid residue in a homolog thereof. It may be that the mutation is a D87N mutation in SEQ ID NO: 1 . It may be that the mutation is an S83L mutation in SEQ ID NO: 1 .
  • the compounds of the present invention are typically active against such fluoroquinone resistant DNA gyrase, as the present compounds are active at a novel binding site.
  • a compound of the invention may have an IC50 of less than about 10 pM in a DNA gyrase supercoiling assay.
  • the compound may have an IC50 of less than about 1 pM in a DNA gyrase supercoiling assay.
  • the compound may have an IC50 of less than about 500 nM in a DNA gyrase supercoiling assay.
  • the compound may have an IC50 of less than about 400 nM in a DNA gyrase supercoiling assay.
  • the compound may have an IC50 of less than about 300 nM (e.g.
  • the compound may have an IC50 of less than about 100 nM (e.g. less than about 80 nm) in a DNA gyrase supercoiling assay.
  • the DNA gyrase supercoiling assay may be the DNA gyrase supercoiling assay described below in the Examples (See “DNA gyrase supercoiling assay” the the Materials and Methods part of “Antibacterial Activity”).
  • Formulations [0083] According to a third aspect of the invention there is provided a pharmaceutical formulation or composition including a compound of the invention, optionally in admixture with at least one pharmaceutically acceptable adjuvant, diluent or carrier.
  • the formulation or composition may be a parenteral formulation or an oral formulation.
  • the formulation may be a parenteral formulation, for example a formulation for intravenous injection.
  • the formulation may be an oral formulation.
  • Compounds, formulations or compositions of the invention may be administered orally, topically, intravenously, subcutaneously, buccally, rectally, dermally, nasally, tracheally, bronchially, by any other parenteral route, as an oral or nasal spray or via inhalation.
  • the compounds may be administered in the form of pharmaceutical preparations comprising the compound either as a free compound or, for example, a pharmaceutically acceptable non-toxic organic or inorganic acid or base addition salt, in a pharmaceutically acceptable dosage form.
  • the compositions may be administered at varying doses.
  • the pharmaceutical compounds of the invention may be administered parenterally (“parenterally” as used herein, refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion) or orally to a host to obtain an antibacterial effect.
  • parenterally refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion
  • the pharmaceutical compounds of the invention may be administered by intravenous injection or infusion.
  • the compounds may be administered alone or as compositions in combination with pharmaceutically acceptable diluents, excipients or carriers.
  • Actual dosage levels of active ingredients in the pharmaceutical formulations and pharmaceutical compositions of this invention may be varied so as to obtain an amount of the active compound(s) that is effective to achieve the desired therapeutic response for a particular patient, compositions and mode of administration.
  • the selected dosage level will depend upon the activity of the particular compound, the route of administration, the severity of the condition being treated and the condition and prior medical history of the patient being treated. However, it is within the skill of the art to start doses of the compound at levels lower than required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. Suitable doses are generally in the range of from 0.01 - 100 mg/kg/day, for example in the range of 0.1 to 50 mg/kg/day.
  • compositions or compositions of this invention for parenteral (e.g. intravenous) injection may comprise pharmaceutically acceptable sterile aqueous or non-aqueous solutions, dispersions, suspensions or emulsions as well as sterile powders for reconstitution into sterile injectable solutions or dispersions just prior to use.
  • suitable aqueous and non-aqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol and the like), and suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters, such as ethyl oleate.
  • Formulations or compositions for parenteral injection may represent preferred formulations or compositions of the invention. [0089] These compositions may also contain adjuvants such as preservative, wetting agents, emulsifying agents and dispersing agents. Inhibition of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol or phenol sorbic acid. It may also be desirable to include isotonic agents, such as sugars or sodium chloride, for example. Prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents (for example, aluminium monostearate and gelatine) which delay absorption.
  • agents for example, aluminium monostearate and gelatine
  • Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules.
  • the active compound is typically mixed with at least one inert, pharmaceutically acceptable excipient or carrier, such as sodium citrate or dicalcium phosphate and/or one or more: a) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol and silicic acid; b) binders, such as carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and acacia; c) humectants, such as glycerol; d) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates and sodium carbonate; e) solution retarding agents, such as paraffin; f) absorption accelerators, such as quaternary ammonium compounds; g) wetting agents, such as cetyl
  • the dosage form may also comprise buffering agents.
  • Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycol, for example.
  • Oral formulations may contain a dissolution aid.
  • dissolution aids include nonionic surface active agents, such as sucrose fatty acid esters, glycerol fatty acid esters, sorbitan fatty acid esters (e.g.
  • sorbitan trioleate polyethylene glycol, polyoxyethylene hydrogenated castor oil, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene alkyl ethers, methoxypolyoxyethylene alkyl ethers, polyoxyethylene alkylphenyl ethers, polyethylene glycol fatty acid esters, polyoxyethylene alkylamines, polyoxyethylene alkyl thioethers, polyoxyethylene polyoxypropylene copolymers, polyoxyethylene glycerol fatty acid esters, pentaerythritol fatty acid esters, propylene glycol monofatty acid esters, polyoxyethylene propylene glycol monofatty acid esters, polyoxyethylene sorbitol fatty acid esters, fatty acid alkylolamides, and alkyamine oxides; bile acid and salts thereof (e.g.,
  • ionic surface active agents such as sodium laurylsulfate, fatty acid soaps, alkylsufonates, alkylphosphates, ether phosphates, fatty acid salts of basic amino acids; triethanolamine soap, and alkyl quaternary ammonium salts; and amphoteric surface active agents, such as betaines and aminocarboxylic acid salts.
  • the solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical formulating art. They may optionally contain opacifying agents and may also be of a composition such that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, and/or in delayed fashion. Examples of embedding compositions include polymeric substances and waxes.
  • the active compounds may also be in microencapsulated form, if appropriate, with one or more of the above-mentioned excipients.
  • the active compounds may be in finely divided form, for example it may be micronized.
  • Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups and elixirs.
  • the liquid dosage forms may contain inert diluents commonly used in the art such as water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 ,3-butylene glycol, dimethyl formamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan and mixtures thereof.
  • inert diluents commonly used in the art such as water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol,
  • the oral compositions may also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavouring and perfuming agents.
  • Suspensions in addition to the active compounds, may contain suspending agents such as ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminium metahydroxide, bentonite, agar-agar, and traganacanth and mixtures thereof.
  • compositions for rectal or vaginal administration may be in the form of suppositories which can be prepared by mixing the compounds of this invention with suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol or a suppository wax which are solid at room temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active compound.
  • suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol or a suppository wax which are solid at room temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active compound.
  • Dosage forms for topical administration of a compound of this invention include powders, sprays, creams, foams, gels, ointments and inhalants.
  • the active compound is mixed under sterile conditions with a pharmaceutically acceptable carrier and any needed preservatives, buffers or propellants which may be required. Ophthalmic formulations, eye ointments, powders and solutions
  • Liquid (e.g. aqueous) formulations and compositions may comprise additional compound(s) to help prevent precipitation of the active compound.
  • Compounds of the invention are glycopeptide derivatives. Precipitation of such compounds in aqueous solution may be avoided or minimised by including a monosaccharide in the solution.
  • aqueous formulations or compositions may comprise glucose.
  • a parenteral (e.g. intravenous injection) formulation or composition may comprise a compound of the invention, water for injection and glucose.
  • the formulations or compositions according to the present subject matter may contain other active agents intended, in particular, for use in treating a bacterial infection.
  • the formulation may further comprise an additional antibiotic.
  • the antibiotic may be a fluoroquinolone, e.g. ciprofloxacin.
  • the formulation may further comprise a polymyxin, e.g. Polymyxin B nonapeptide.
  • the formulations according to the present subject matter may also contain inactive components. Suitable inactive components are well known in the art and are described in standard textbooks, such as Goodman and Gillman’s: The Pharmacological Bases of Therapeutics, 13 th Ed., Brunton et al., Eds. McGraw-Hill Education (2017), and Remington’s Pharmaceutical Sciences, 17 th Ed., Mack Publishing Co., Easton, Pa. (1990), both of which are incorporated by reference herein in their entirety.
  • the formulations may be used in combination with an additional pharmaceutical dosage form to enhance their effectiveness in treating any of the disorders described herein.
  • the present formulations may be administered as part of a regimen additionally including any other pharmaceutical and/or pharmaceutical dosage form known in the art as effective for the treatment of any of these disorders.
  • a compound or formulation of the invention for use as a medicament.
  • a compound or formulation of the invention for use in the treatment of a bacterial infection.
  • a pharmaceutical combination for use in the treatment of a bacterial infection wherein the combination comprises a compound of the invention, or formulation of the invention; and another antibiotic.
  • the another antibiotic may be a fluoroquinolone, e.g. ciprofloxacin.
  • the bacterial infection is a Gram-negative bacterial infection.
  • Illustrative Gramnegative bacterial infections include infections caused by bacteria of a genus selected from Campylobacter, Vibrio, Escherichia, Haemophilus, Klebsiella, Enterobacter, Salmonella, Shigella, Legionella, Yersinia, Pseudomonas, or Acinetobacter.
  • the Gram-negative bacterial infection is an infection caused by bacteria selected from Campylobacter jejuni, Vibrio cholerae, Escherichia coli, Haemophilus influenzae, Klebsiella pneumoniae, Enterobacter cloacae, Salmonella enterica, Shigella dysenteriae, Legionella pneumophila, Yersinia pestis, Pseudomonas aeruginosa, or Acinetobacter baumannii. It may be that the Gramnegative bacterial infection is an infection caused by bacteria selected from E. coli, K. pneumoniae, Klebsiella, Acinetobacter, P. aeruginosa, Salmonella, Helicobaceter. It may be that the Gram-negative bacterial infection is an infection caused by E. coli or K. pneumoniae. It may be that the Gram-negative bacterial infection is an infection caused by A. baumannii or P. aeruginosa.
  • the Gram-negative bacterial infection may be an infection caused by drug-resistant Gramnegative bacteria, e.g. ciprofloxacin-resistant Gram-negative bacteria.
  • the bacterial infection is a Gram-positive bacterial infection.
  • Illustrative Grampositive bacterial infections include infections caused by bacteria of a genus selected from Bacillus, Corynebacterium, Enterococcus, Erysipelothrix, Listeria, Streptococcus, Staphylococcus, Clostridioides or Mycobacterium.
  • the Gram-positive bacterial infection is an infection caused by bacteria selected from Bacillus anthracis, Corynebacterium diphtheriae, Enterococcus faecalis, Enterococcus faecium, Erysipelothrix rhusiopathiae, Listeria monocytogenes, Streptococcus pneumoniae, Streptococcus pyogenes, Staphylococcus aureus, Clostridioides difficile, Mycobacterium tuberculosis, Mycobacterium leprae, Mycobacterium avium, or Mycobacterium abscessus.
  • Exemplary bacterial (e.g. Gram-negative) infections that may be treated by compounds of the invention include skin and structure infections, lower respiratory tract infections, bacteremia, sepsis, septicemia, infective endocarditis, peritonitis (e.g. associated with continuous ambulatory peritoneal dialysis), enterocolitis (e.g. 21taphylococcal), mastitis, Clostridium difficile infection-associated diarrhoea and colitis.
  • the infection that may be treated may be selected from skin and structure infections and bacteremia.
  • Exemplary skin and structure infections include cellulitis/erysipelas, major cutaneous abscesses, and wound infections.
  • Exemplary lower respiratory tract infections include pneumonia, community-acquired pneumonia (CAP), nosocomial pneumonia, and pleural empyema.
  • a seventh aspect of the invention there is provided a method of treating a bacterial infection in a patient, comprising administering to the patient an effective amount of a compound or formulation of the invention.
  • the bacterial infection may be as described above in relation to the fifth aspect of the invention.
  • the method may further comprise administering to the patient an effective amount of a fluoroquinolone, e.g. ciprofloxacin.
  • Figure 1 shows sequence alignment of Escherichia coll (strain K12) sp
  • Figure 2 shows sequence alignment of Escherichia coll (strain K12) sp
  • Figure 3 shows sequence alignment of Escherichia coll (strain K12) sp
  • Figure 4 shows sequence alignment of Escherichia coll (strain K12) sp
  • Figure 5 shows sequence alignment of Escherichia coll (strain K12) sp
  • Figure 6 shows sequence alignment of Escherichia coli (strain K12) sp
  • Figure 7 shows sequence alignment of Escherichia coli (strain K12) sp
  • Figure 8 shows sequence alignment of Escherichia coli (strain K12) sp
  • Figure 9 shows sequence alignment of Escherichia coli (strain K12) sp
  • Figure 10 shows sequence alignment of Escherichia coli (strain K12) sp
  • Figure 11 shows sequence alignment of Escherichia coli (strain K12) sp
  • Figure 12 shows sequence alignment of Escherichia coli (strain K12) sp
  • Figure 13 shows sequence alignment of Escherichia coli (strain K12) sp
  • Figure 14 shows sequence alignment of Escherichia coli (strain K12) sp
  • GYRA_ECOLI (SEQ ID NO: 1) with Mycobacteroides abscessus SV 1tr
  • Figure 15 shows sequence alignment of Escherichia coli (strain K12) sp
  • Figure 16 shows sequence alignment of Escherichia coli (strain K12) sp
  • Figure 17 shows sequence alignment of Escherichia coli (strain K12) sp
  • Figure 18 shows sequence alignment of Escherichia coli (strain K12) sp
  • Figure 19 shows sequence alignment of Escherichia coli (strain K12) sp
  • Figure 20 shows sequence alignment of Escherichia coli (strain K12) sp
  • Figure 21 shows sequence alignment of Escherichia coli (strain K12) sp
  • Figure 22 shows sequence alignment of Escherichia coli (strain K12) sp
  • Figure 23 shows sequence alignment of Escherichia coli (strain K12) sp
  • Figure 24 shows sequence alignment of Escherichia coli (strain K12) sp
  • Figure 25 shows sequence alignment of Escherichia coli (strain K12) sp
  • Figure 26 shows the hemolytic activity of certain compounds of the invention after 20 h incubation with sheep blood cells.
  • Figure 28 shows Microscopy images of E. coli BW25113 from the BCP experiment. Conditions are, from top to bottom: DMSO, ampicillin (AMP), rifampicin (RIF), tetracycline (TET), ciprofloxacin (CIP), 99, 102 (LEI-800). From left to right the channels indicate: DIC, DAPI, FM4-64, and DAPI/FM4-64 combined. The bacteria treated with 99 displayed a phenotype that is consistent with the DMSO control, while incubation with 102 (LEI-800) led to a phenotype resembling the CIP and TET morphological profile. Scale bars represent 10 pm.
  • Figure 29 shows (a), Individual graphs of six exemplary features extracted from BCP microscopy after image analysis. Each dot represents the measured value for an individual bacterium, (b), PCA plot of all the morphological features extracted from three independent BCP experiments.
  • Figure 30 shows (a) Agar containing 60 (5x MIC) was inoculated with 10 7 CFU of E. coli. After one day, nine viable colonies were isolated and used for MIC testing and whole-genome sequencing (WGS). (b) Susceptibility assays confirm spontaneous resistance to 60 and WGS identified mutations located in the DNA gyrase, predominantly in subunit A. Similar mutations are similarly coloured. Crossresistance is observed for conformationally restricted compounds 101 and 102. (c) Topology of the mutations in the core of the DNA gyrase heterotetramer outline a possible binding site of isoquinoline sulfonamides, (d) All mutations are found in the cleavage-reunion domain of DNA gyrase. Domain organization of DNA gyrase, GyrB (coral) and GyrA (beige) subunits.
  • Figure 31 shows Whole-genome sequencing results.
  • Figure 32 shows (a)Schematic representation of negative supercoiling.
  • relaxed (R) DNA is transformed into supercoiled (SC) DNA by DNA gyrase.
  • SC supercoiled
  • FIG. 32 Three compounds tested for supercoiling inhibition. CIP (green) is used as positive control, 99 (blue) is used as antimicrobially inactive compound, and 102 (LEI-800) (red) is used as lead compound,
  • Dose-response curves of DNA gyrase supercoiling inhibition, based on n 3 gels. Error bars and dotted lines represent the standard deviation at each concentration and the 95% Cl respectively,
  • Figure 33 shows the effects of compound 102 (LEI-800) and compound 60 on ATP- independent relaxation of negatively supercoiled DNA by gyrase.
  • Lane 1 negatively supercoiled pBR322;
  • lane 2 relaxation by 200 nM gyrase;
  • lane 3 DNA relaxation in the presence of CIP (30 pM);
  • subsequent lanes the effect of increasing concentrations of 102 (LEI-800) (0.0005, 0.0015, 0.005, 0.015, 0.05, 0.15, 0.5, 1.5, 5, 10, 15, 20, 30 pM).
  • Lane 1 negatively supercoiled pBR322; lane 2: relaxation by 200 nM gyrase; lane 3: DNA relaxation in the presence of CIP (10 pM); subsequent lanes: the effect of increasing concentrations of compound 60 (0.0005, 0.0015, 0.005, 0.015, 0.05, 0.1 , 0.15, 0.25, 0.5, 1.5, 5, 10 pM).
  • Figure 34 shows (a) A selection of 2D classes, box size in angstroms indicated, (b) Processing scheme (see Methods for description), (c) FSC curve for the final reconstruction as output by cryoSPARC. (d) Euler angle distribution as output by cryoSPARC. (e). Local resolution map to illustrate resolution distribution from ⁇ 4 A next to the DNA and compound to >7 at the ends of the cleavagereunion domain, (f). Map-to-model curve, (g). Comparison of Gyr-Mu217-102 (LEI-800) map (grey surface) to the gepotidacin EM structure 6RKW (cartoon model). Note the significant change in the position of the CTDs and DNA.
  • Figure 35 shows (a) Overview of the model of the Gyr-Mu217-102 complex.
  • GyrA (8-524) and GyrB (402-790) are depicted as beige and coral cartoon representations.
  • the modelled central part of Mu217 DNA is shown in teal green.
  • Two molecules of 102 (LEI-800) observed in a single gyrase heteroteramer are shown as golden van der Waals spheres. Uniform colour scheme is used throughout the manuscript, (b) A close-up of the 102 (LEI-800) binding pocket on the DNA-binding surface of GyrA.
  • GyrA is shown as molecular surface, DNA as cartoon representation and 102 (LEI-800) as van der Waals spheres, (c) Molecular interactions between GyrA and 102 (LEI-800). 102 (LEI-800) is shown as stick representation. Main GyrA residues important for 102 (LEI-800) binding are labelled.
  • a quinoline ring of 102 (LEI-800) shares binding pocket with the aminocoumarin moiety of SD8, but unlike SD8, 102 (LEI-800) does not interfere with DNA binding, (e) Coulomb potential density map for 102 (LEI-800), (f) A 2D diagram of 102 (LEI-800) binding site generated by LigPlot. Key hydrogen bonds to Ser97 and Ser172 are shown in gold and distances in A are indicated. Spiked red arcs show non-bonded interactions with residues within 3.9 A distance.
  • Figure. 36 shows that CryoEM reveals a unique binding pocket of 102 (LEI-800) within DNA gyrase.
  • Low-resolution contour (white, 5o) illustrates the position of GyrA CTDs and GyrB ATPase domains.
  • High-resolution part (12o) is coloured according to the scheme: coral, GyrB; beige, GyrA; teal, DNA; golden, LEI-800,
  • GyrA (8-524) and GyrB (402-800) are depicted as beige and coral cartoon representations.
  • the modelled 26- bp central part of Mu217 DNA is shown in teal.
  • Two molecules of 102 (LEI-800) observed in a single gyrase heteroteramer are shown as golden van der Waals spheres.
  • Uniform colour scheme is used throughout the manuscript,
  • GyrA is shown as molecular surface, DNA as cartoon representation and 102 (LEI-800) as van der Waals spheres.
  • Figure 37 shows Cryo-EM data processing for Gyr-Mu217-LEI800.
  • (a) a representative motion-corrected micrograph, gyrase particles are encircled,
  • (b) a selection of 2D classes, box size in angstroms indicated,
  • Figure 38 shows that molecular interactions of 102 (LEI-800) explain the mutation data and SAR.
  • A Molecular interactions between GyrA and 102 (LEI-800).
  • 102 (LEI-800) is shown as stick representation.
  • Main GyrA residues important for 102 (LEI-800) binding are labelled.
  • a quinoline ring of 102 (LEI-800) shares binding pocket with the aminocoumarin moiety of SD8, but unlike SD8, 102 (LEI-800) does not interfere with DNA binding, c, A 2D diagram of 102 (LEI-800) binding site generated by LigPlot. Key hydrogen bonds to Ser97 and Ser172 are shown in black and distances in A are indicated. Spiked red arcs show non-bonded interactions and green arcs hydrophobic interactions with residues within 3.9 A distance.
  • Figure 39 activity of 102 (LEI-800) and 99 (LEI-801) against reconstituted EcGyrA S97A /GyrB complex, a, Plasmid supercoiling assays showing the activity of WT (GyrA2B2) gyrase and GyrA S97L and GyrA s172A variants produced in this work.
  • First lane relaxed pBR322, subsequent lanes: effect of increasing enzyme concentration (5, 10, 15 nM) on relaxed plasmid. Positions of nicked, relaxed and supercoiled DNA are indicated to the left of each gel. In GyrA s172A assay higher enzyme concentrations were used (15, 20, 25, 30, 40, 50 nM).
  • Concentrations used for 102 (LEI-800): 30, 50, 100, 200, 300, 325, 350, 375, 400; for 99 (LEI-801): 30, 40, 50, 75, 100, 150, 200, 300, 400.
  • Figure 40 Activity of 102 (LEI-800), 99 (LEI-801) and compound 60 against reconstituted EcGyrA s172A /GyrB complex, a, Plasmid supercoiling assay showing the inhibitory activity of LEI-800 and LEI-801 against GyrA s172A /GyrB complex.
  • First lane relaxed pBR322
  • second lane relaxed pBR322 with 30 nM reconstituted (A2B2) GyrA s172A /GyrB gyrase complex
  • third lane effect of 10 pM ciprofloxacin (CIP) on gyrase activity
  • subsequent lanes effect of increasing compound concentration (0.0005, 0.0015, 0.005, 0.05, 0.15, 0.5, 1 .5, 5, 10, 15, 20, 30 pM) on GyrA s172A /GyrB complex activity.
  • Positions of nicked, relaxed and supercoiled DNA are indicated to the left of each gel. When absent, compound was replaced with the corresponding amount of DMSO.
  • Dose-response curves of DNA gyrase supercoiling inhibition, based on n 3 gels. Error bars represent the standard deviation at each concentration.
  • Figure 41 Comparison of LEI800 binding pocket between Gram-negative and Gram-positive species
  • a Comparison of LEI800 binding pocket in E. coli (this paper, GyrA coloured beige, DNA coloured teal) and AlphaFold generated model of K. pneumoniae 312(UniProt: B5XNZ4_KLEP3; AF-DB: B5XNZ4) GyrA (blue). Ser97 and Leu98 are indicated. No significant differences are found in the neighbouring residues
  • b Comparison of LEI800 binding pocket in E. coli and AlphaFold generated model of A. baumanii GyrA (UniProt: GYRA_ACIBA; AF-DB: Q2FCU6) coloured purple.
  • Figure 42 Activity of 102 (LEI-800) against purified A. baumannii and P. aeuruginosa gyrases.
  • a Plasmid supercoiling assay showing the inhibitory activity of 102 (LEI-800) against A. baumannii gyrase.
  • baumannii gyrase third lane: effect of 10 pM ciprofloxacin (CIP) on WT gyrase (5 U) activity, subsequent lanes: effect of increasing 102 (LEI-800) concentration (0.0005, 0.0015, 0.005, 0.05, 0.15, 0.5, 1.5, 5, 10, 15, 20, 30 pM) on WT gyrase activity. Positions of nicked, relaxed and supercoiled DNA are indicated to the left of each gel. When absent, compound was replaced with the corresponding amount of DMSO.
  • Plasmid supercoiling assay showing the inhibitory activity of 102 (LEI-800) against P. aeruginosa gyrase.
  • First lane relaxed pBR322
  • second lane relaxed pBR322 with 5 U P. aeruginosa gyrase
  • third lane effect of 10 pM ciprofloxacin (CIP) on WT gyrase (5 U) activity
  • subsequent lanes effect of increasing 102 (LEI- 800) concentration (0.0005, 0.0015, 0.005, 0.05, 0.15, 0.5, 1 .5, 5, 10, 15, 20, 30 pM) on WT gyrase activity.
  • Positions of nicked, relaxed and supercoiled DNA are indicated to the left of each gel. When absent, compound was replaced with the corresponding amount of DMSO.
  • Dose-response curves of DNA gyrase supercoiling inhibition, based on n 3 gels. Error bars represent the standard deviation at each concentration.
  • FIG 43 102 (LEI-800) is an allosteric DNA cleavage inhibitor, a, Metal binding site in Gyr- Mu217-LEI-800 complex.
  • Catalytic tyrosine (Tyr122), scissile phosphate (+1), and neighbouring GyrB residues (within 6 A distance from the metal, shown as lime sphere) are shown as stick representations.
  • GyrA is coloured beige, GyrB - coral, DNA-teal.
  • Map density is shown as blue mesh and is contoured at 9o level. Distances in A between the catalytic tyrosine and phosphate and between the metal and the closest Glu side chains are indicated.
  • binding of 102 hinders loop movement preventing formation of a phosphotyrosine bond and DNA cleavage, inhibiting the enzyme, c, A time-course of DNA cleavage by ciprofloxacin and effect of 102 (LEI-800).
  • the reactions contained 5 pM ciprofloxacin and 5 pM 102 (LEI-800) (as indicated). After completion, the reactions were run on a gel with EtBr. The amount of cleaved DNA is reduced when 102 (LEI-800) is present, present, d, A timecourse of DNA cleavage by Ca 2+ and effect of 102 (LEI-800).
  • the reactions contained 5 mM Ca 2+ and 5 pM 102 (LEI-800) (as indicated). After completion, the reactions were run on a gel with EtBr. The amount of cleaved DNA is reduced when 102 (LEI-800) is present, e, Linear DNA was quantified and plotted. Error bars represent the SD of at least three independent experiments.
  • Figure 44 Checkerboard broth microdilution assay between 102 (LEI-800) and CIP showing the absence of any synergistic effect.
  • the invention concerns amongst other things the treatment of a disease.
  • treatment and the therapies encompassed by this invention, include the following and combinations thereof: (1) hindering, e.g. delaying initiation and/or progression of, an event, state, disorder or condition, for example arresting, reducing or delaying the development of the event, state, disorder or condition, or a relapse thereof in case of maintenance treatment or secondary prophylaxis, or of at least one clinical or subclinical symptom thereof; (2) preventing or delaying the appearance of clinical symptoms of an event, state, disorder or condition developing in an animal (e.g.
  • the benefit to a patient to be treated may be either statistically significant or at least perceptible to the patient or to the physician.
  • compositions and methods described herein are of use for therapy and/or prophylaxis of the mentioned conditions.
  • prophylaxis includes reference to treatment therapies for the purpose of preserving health or inhibiting or delaying the initiation and/or progression of an event, state, disorder or condition, for example for the purpose of reducing the chance of an event, state, disorder or condition occurring.
  • the outcome of the prophylaxis may be, for example, preservation of health or delaying the initiation and/or progression of an event, state, disorder or condition. It will be recalled that, in any individual patient or even in a particular patient population, a treatment may fail, and this paragraph is to be understood accordingly.
  • antibiotic refers to a compound that inhibits the growth of or destroys microorganisms, such as bacteria (e.g. Gram-positive bacteria, or Gram-negative bacteria).
  • bacteria e.g. Gram-positive bacteria, or Gram-negative bacteria.
  • An “antibacterial” is an antibiotic that is active against bacteria.
  • Compounds of the invention are antibacterial, in particular with activity against bacteria (such as Gram-negative bacteria).
  • C1-C4 alkyl used herein covers any saturated straight or branched chain alkyl moiety having from 1 to 4 (e.g. 1 , 2, 3 or 4) carbon atoms.
  • the term includes, e.g., methyl, ethyl, propyl (n-propyl or isopropyl) and butyl (n-butyl, sec-butyl or tert-butyl).
  • alkylene chain by itself or as part of another substituent means a divalent radical derived from a saturated straight chain alkyl with the general formula -C n H2n-, wherein n is an integer.
  • a 1 , 2 or 3 membered alkylene chain is intended to cover any saturated straight chain alkylene moiety having 1 , 2 or 3 carbon atoms.
  • alkylene is synonymous with the term “alkanediyl”.
  • halo or halogen as used herein includes reference to F, Cl, Br or I, for example F, Cl or Br. In a particular class of embodiments, halogen is F or Cl, of which F is more common.
  • haloalkyl refers to an alkyl group (alkyl being defined as above) where one or more hydrogen atoms are substituted by a corresponding number of halogens.
  • C1-C4 haloalkyl is intended to cover any saturated straight or branched chain alkyl moiety having from 1 to 4 (e.g. 1 , 2, 3 or 4) carbon atoms where one or more hydrogen atoms are substituted by a corresponding number of halogen atoms.
  • C1-C4 haloalkyl covers, but is not limited to, trifluoromethyl (- CF3), 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, and the like.
  • 5- or 6-membered heteroaryl group may be any aromatic 5 or 6 membered ring system comprising from 1 to 4 heteroatoms independently selected from O, S and N (in other words from 1 to 4 of the atoms forming the ring system are selected from O, S and N, the rest being carbon).
  • 5- or e- membered heteroaryl groups will be monocyclic.
  • a heteroaryl group can be attached to the remainder of the molecule through a carbon or heteroatom.
  • Non-limiting examples of 5- or 6-membered heteroaryl groups include pyrrolyl, e.g. 1 -pyrrolyl, 2- pyrrolyl or 3-pyrrolyl, furanyl, e.g.
  • substituents are only at positions where they are chemically possible, the person skilled in the art being able to decide (either experimentally or theoretically) without inappropriate effort whether a particular substitution is possible.
  • amino or hydroxy groups with free hydrogen may be unstable if bound to carbon atoms with unsaturated (e.g. olefinic) bonds.
  • substituents described herein may themselves be substituted by any substituent, subject to the aforementioned restriction to appropriate substitutions as recognised by the skilled person.
  • the isomer having the lowest conformational energy may be preferred.
  • pharmaceutically acceptable includes reference to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings or animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. This term includes acceptability for both human and veterinary purposes.
  • salts are meant to include salts of the active compounds which are prepared with relatively nontoxic acids or bases, depending on the particular substituents found on the compounds described herein.
  • base addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired base, either neat or in a suitable inert solvent.
  • pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amino, or magnesium salt, or a similar salt.
  • acid addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired acid, either neat or in a suitable inert solvent.
  • Examples of pharmaceutically acceptable acid addition salts include those derived from inorganic acids like hydrochloric, hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric, monohydrogenphosphoric, dihydrogenphosphoric, sulfuric, monohydrogensulfuric, hydriodic, or phosphorous acids and the like, as well as the salts derived from relatively nontoxic organic acids like acetic, propionic, isobutyric, maleic, malonic, benzoic, succinic, suberic, fumaric, lactic, mandelic, phthalic, benzenesulfonic, p-tolylsulfonic, citric, tartaric, methanesulfonic, and the like.
  • inorganic acids like hydrochloric, hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric, monohydrogenphosphoric, dihydrogenphosphoric, sulfuric, monohydrogensulfuric, hydriodic, or phosphorous acids and
  • salts of amino acids such as arginate and the like, and salts of organic acids like glucuronic or galacturonic acids and the like (see, for example, Berge et al., “Pharmaceutical Salts”, Journal of Pharmaceutical Science, 1977, 66, 1-19).
  • Certain specific compounds of the present invention contain both basic and acidic functionalities that allow the compounds to be converted into either base or acid addition salts.
  • the neutral forms of the compounds are preferably regenerated by contacting the salt with a base or acid and isolating the parent compound in the conventional manner.
  • the parent form of the compound differs from the various salt forms in certain physical properties, such as solubility in polar solvents.
  • Certain compounds of the present invention can exist in unsolvated forms as well as solvated forms, including hydrated forms. In general, the solvated forms are equivalent to unsolvated forms and are encompassed within the scope of the present invention. Certain compounds of the present invention may exist in multiple crystalline or amorphous forms. In general, all physical forms are equivalent for the uses contemplated by the present invention and are intended to be within the scope of the present invention.
  • Certain compounds of the present invention possess asymmetric carbon atoms (optical centres) or double bonds; the racemates, diastereomers, tautomers, geometric isomers and individual isomers are encompassed within the scope of the present invention.
  • the compounds of the present invention do not include those which are known in the art to be too unstable to synthesize and/or isolate.
  • prodrug represents compounds which are transformed in vivo to the parent compound or other active compound, for example, by hydrolysis in blood.
  • An example of such a prodrug is a pharmaceutically acceptable ester of a carboxylic acid.
  • pharmaceutical formulation includes reference to a formulation comprising at least one active compound and optionally one or more additional pharmaceutically acceptable ingredients, for example a pharmaceutically acceptable carrier. Where a pharmaceutical formulation comprises two or more active compounds, or comprises at least one active compound and one or more additional pharmaceutically acceptable ingredients, the pharmaceutical formulation is also a pharmaceutical composition. Unless the context indicates otherwise, all references to a “formulation” herein are references to a pharmaceutical formulation.
  • product or “product of the invention” as used herein includes reference to any product containing a compound of the present invention.
  • product relates to compositions and formulations containing a compound of the present invention, such as a pharmaceutical composition, for example.
  • terapéuticaally effective amount refers to an amount of a drug, or pharmaceutical agent that, within the scope of sound pharmacological judgment, is calculated to (or will) provide a desired therapeutic response in a mammal (animal or human).
  • the therapeutic response may for example serve to cure, delay the progression of or prevent a disease, disorder or condition.
  • the present invention also includes all pharmaceutically acceptable isotopically-labelled compounds of formulae (I) to (XVI), wherein one or more atoms are replaced by atoms having the same atomic number, but an atomic mass or mass number different from the atomic mass or mass number most commonly found in nature.
  • stable isotopes suitable for inclusion in the compounds of the invention include isotopes of hydrogen, such as 2 H, carbon, such as 11 C and 13 C, nitrogen, such as 15 N and oxygen, such as 17 O and 18 O.
  • Isotopically-labelled compounds can generally be prepared by conventional techniques known to those skilled in the art or by processes analogous to those described using an appropriate isotopically- labelled reagent in place of the non-labelled reagent previously employed.
  • the compounds of the invention are occupied with deuterium ( 2 H) instead of hydrogen at a certain position or positions at an isotopic abundance greater than deuterium’s natural isotopic abundance (e.g. greater than about 0.015%).
  • the position or positions may be occupied with deuterium at an isotopic abundance of greater than 50%.
  • the position or positions may be occupied with deuterium at an isotopic abundance of greater than 90%, e.g. greater than 95%.
  • the position or positions may be occupied with deuterium at an isotopic abundance of greater than 99%, e.g. greater than 99.5%.
  • Isotopic abundance can be determined using conventional analytical methods known to a person skilled in the art, such as mass spectrometry and nuclear magnetic resonance spectroscopy.
  • homolog refers a polypeptide, including polypeptides from the same or different bacterial species, having greater than about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more sequence identity to a reference polypeptide, and/or having the same or substantially the same properties, and/or performing the same or substantially the same function as the reference polypeptide.
  • conservative modifications refers to modifications that are physically, biologically, chemically or functionally similar to the corresponding reference amino acid (such as a corresponding amino acid in SEQ ID NO: 1)_, e.g., has a similar size, shape, electric charge, chemical properties, including the ability to form covalent or hydrogen bonds, or the like.
  • conservative modifications include, but are not limited to, one or more amino acid substitutions, additions and deletions.
  • conservative amino acid substitutions include those in which the amino acid residue is replaced with an amino acid residue having a similar side chain.
  • Amino acid residues are usually divided into families based on common, similar side-chain properties, such as:
  • nonpolar side chains e.g., glycine, alanine, valine, leucine, isoleucine, methionine
  • uncharged polar side chains e.g., asparagine, glutamine, serine, threonine, tyrosine, proline, cysteine, tryptophan
  • acidic side chains e.g., aspartic acid, glutamic acid
  • beta-branched side chains e.g. , threonine, valine, isoleucine
  • aromatic side chains e.g., tyrosine, phenylalanine, tryptophan, histidine.
  • a conservative substitution may also involve the use of a non-natural amino acid.
  • Non-conservative substitutions i.e. exchanging members of one family against members of another family, may lead to substantial changes, e.g., with respect to the charge, dipole moment, size, hydrophilicity, hydrophobicity or conformation of the binding member, which may lead to a significant drop in the binding activity, in particular if amino acids are affected that are essential for binding to the target molecule.
  • a non- conservative substitution may also involve the use of a non-natural amino acid.
  • Conservative and non-conservative modifications can occur naturally (due to a DNA mutation) or may be introduced by a variety of standard techniques known in the art, such as combinatorial chemistry, site-directed DNA mutagenesis, PCR-mediated and/or cassette mutagenesis, peptide/protein chemical synthesis, chemical reaction specifically modifying reactive groups in the parental binding member.
  • Sensitive functional groups may need to be protected and deprotected during synthesis of a compound of the invention. This may be achieved by conventional methods, for example as described in P. G. M. Wuts, Greene's Protective Groups in Organic Synthesis, John Wiley & Sons, Inc., Michigan, 2014.
  • LC-MS measurements were performed on a Thermo Finnigan LCQ Advantage MAX ion-trap mass spectrometer (ESI+) coupled to a Surveyor HPLC system (Thermo Finnigan) equipped with a standard C18 (Gemini, 4.6 mm D x 50 mm L, 5 pm particle size, Phenomenex) analytical column and buffers A: H2O, B: ACN, C: 0.1% aq. TFA.
  • Preparative HPLC was performed on a Waters Acquity Ultra Performance LC with a C18 column (Gemini, 150 x 21 .2 mm, Phenomenex) using a ACN in H2O (+0.2% TFA) gradient. All final compounds were determined to be > 95% pure by LC-UV analysis.
  • the prolinol was co-evaporated in vacuo twice with toluene.
  • the prolinol (1 equiv.), imidazole (1 .5 equiv.) and DMAP (0.05 equiv.) were dissolved in dry DCM (0.5 M).
  • TIPS-CI was added dropwise at 0°C after which the reaction mixture was allowed to warm to RT and was stirred for 16 h. The mixture was then poured onto sat. aq. NH4CI and extracted with DCM (4x). The combined organic layers were washed with brine (2x), dried with MgSC , filtered and concentrated in vacuo. Purification of the crude material by column chromatography (80% 100% Et2O in pentane with 1% triethylamine) afforded the pure product.
  • TIPS protected starting material (1 equiv.) was dissolved in ACN (0.09 M). To this was added TBAF (5 equiv., 1 M in THF) and the mixture was steered at room temperature. The mixture was subsequently concentrated in vacuo on silica. Purification of the crude material by column chromatography (5% 40% EtOAc in pentane) afforded the pure product.
  • TrtHN ⁇ N N H M 2 Ethylenediamine (103) (267 mL, 4.00 mol) and K2CO3 (66.3 g, 440 mmol) were suspended in DCM (700 mL) after which a solution of trityl chloride (112 g, 400 mmol) in DCM (700 mL) was added dropwise over 40 min.
  • the reaction-mixture was stirred overnight at RT, filtered, concentrated under reduced pressure and co-evaporated with toluene to yield the product (123 g, quant.) which was used without further purification.
  • Ethylene diamine (15.1 mL, 227 mmol) was added dropwise to a cooled (0°C) and stirred solution of the crude sulfonyl chloride (10.0 g, 37.7 mmol) in DCM (600 mL). The mixture was then stirred at room temperature for 2 h. The reaction mixture was diluted with sat. aq. Na2COs (10 mL), washed with brine (50 mL) and extracted with DCM (3x). The organic layers were combined, dried over MgSC , filtered and concentrated. The residue was then co-evaporated with toluene to remove the remaining ethylene diamine giving 111 (10.3 g, 35.0 mmol, 93%) as a dark yellow solid that was used without further purification.
  • TrtHN ⁇ OH TO a solution of trityl chloride (1.4 g, 5.0 mmol) and K2CO3 (0.76 g, 5.5 mmol) in DCM (17 mL) at 0 °C was added dropwise ethanolamine (115) (1 .5 mL, 25 mmol). The reaction was allowed to warm to room temperature and stirred for 3 h before sat. aq. NaHCCh (15 mL) and H2O (15 mL) were added. The organic layer was collected and the aqueous layer extracted with DCM (3x 30 mL). The combined organic layers were dried over Na2SC>4, filtered and concentrated under reduced pressure. The residue was purified by column chromatography (20% EtOAc in pentane) to yield 116 (1 .5 g, 4.95 mmol, 99%).
  • N-(2-((4-Bromobenzyl)(methyl)amino)ethyl)isoquinoline-5-sulfonamide (120) 1.4 mmol), formaldehyde (46 mg, 1.5 mmol) and sodium triacetoxyborohydride (0.59 g, 2.8 mmol) were suspended in THF (15 mL) and MeOH (2.5 mL). The reaction mixture was stirred at room temperature overnight, after which it was diluted with sat. aq. NaHCCh (10 mL) and extracted with DCM (3x). The organic layers were combined, dried over MgSCU, filtered and concentrated. The crude product was purified by column chromatography (1 % 10% MeOH
  • TIPSO X-J Following general procedure F, 122a (1 .57 g, 6.09 mmol) was reacted with n-BuLi (1.6 M in hexanes, 3.8 mL, 6.1 mmol), benzophenone (1.33 g, 7.31 mmol) and bromobenzenelithium (9.13 mmol, prepared according to general procedure G) to afford the title compound as a crude mixture with benzhydrol (1 .5 g crude, 35% purity, 1 .6 mmol product, 26%).
  • n-BuLi 1.6 M in hexanes, 3.8 mL, 6.1 mmol
  • benzophenone (1.33 g, 7.31 mmol
  • bromobenzenelithium 9.13 mmol, prepared according to general procedure G
  • N-(2-((4-(Pyridin-3-yl)benzyl)amino)ethyl)quinazoline-6-sulfonamide (52) mg, 29 pmol, 19% over two steps) was synthesized from 108 (50 mg, 0.15 mmol) and 5-bromoquinazoline (25 mg, 0.12 mmol) according to general procedure C followed by general procedure A.
  • N-(2-((4-(Pyridin-3-yl)benzyl)amino)ethyl)-1 H-indazole-4-sulfonamide (49 mg, 20 pmol, 13% over two steps) was synthesized from 108 (50 mg, 0.15 mmol) and 4-bromo-1 /7-indazole (25 mg, 0.12 mmol) according to general procedure C followed by general procedure A.
  • N-(2-((4-(Pyridin-3-yl)benzyl)amino)ethyl)-1 H-indazole-6-sulfonamide (50) mg, 13 pmol, 9% over two steps) was synthesized from 108 (50 mg, 0.15 mmol) and 6-bromo-1 /7-indazole (47 mg, 0.24 mmol) according to general procedure C followed by general procedure A.
  • A/-Methyl-5-(A/-(2-((4-(pyridin-3-yl)benzyl)amino)ethyl)sulfamoyl)picolinamide (51) mg, 9.0 pmol, 6% over two steps) was synthesized from 108 (50 mg, 0.15 mmol) and 5-bromo-/V-methylpicolinamide (52 mg, 0.24 mmol) according to general procedure C followed by general procedure A.
  • Phenylboronic acid (27 mg, 0.23 mmol) was subjected to general procedure D with 113b (0.10 g, 0.21 mmol) followed by general procedure A to provide 80 (24 mg, 57 pmol, 27% over two steps).
  • Fluoropyridin-3-yl)boronic acid (30 mg, 0.23 mmol) was subjected to general procedure D with 113b (0.10 g, 0.21 mmol) followed by general procedure A to provide 81 (20 mg, 46 pmol, 22% over two steps).
  • Fluorophenyl)boronic acid (30 mg, 0.23 mmol) was subjected to general procedure D with 113b (0.10 g, 0.21 mmol) followed by general procedure A to provide 82 (25 mg, 57 pmol, 27% over two steps).
  • Fluoropyridin-3-yl)boronic acid (30 mg, 0.23 mmol) was subjected to general procedure D with 113c (0.10 g, 0.23 mmol) followed by general procedure A to provide 85 (16 mg, 34 pmol, 15% over two steps).
  • Fluoropyridin-3-yl)boronic acid (30 mg, 0.23 mmol) was subjected to general procedure D with 113d (0.10 g, 0.20 mmol) followed by general procedure A to provide 89 (18 mg, 40 pmol, 20% over two steps).
  • Buffers and salts were of ACS reagent grade or higher and were purchased commercially, from Carl Roth GmbH (Karlsruhe, Germany) and Sigma-Aldrich (Darmstadt, Germany), biological materials and growth media were purchased from Sigma-Aldrich, Scharlab S.L. (Barcelona, Spain) and Fischer Scientific (Landsmeer, Netherlands).
  • Antibiotics trimethoprim Sigma- Aldrich
  • ceftazidime ceftazidime pentahydrate, Thermo Scientific, Landsmeer, Netherlands
  • kanamycin kanamycin monosulfate, MP biomedicals, I llkirch, France
  • All test compounds were used from 10 mM DMSO stock solutions made from the freeze dried powder and stored at -20°C.
  • Klebsiella pneumoniae ATCC 29665 (NCTC 11228), Escherichia coll ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Acinetobacter baumannii ATCC BAA747 and Staphylococcus aureus USA300 (ATCC BAA1717) belong to the American Type Culture Collection (ATCC).
  • E. coli NCTC 13463 and 13846 belong to the National Collection of Type Cultures (UK Health Security Agency).
  • E. coli J ⁇ N5503, JW3600, JW3602, JW3605 JW3594, JW3596 belong to the Keio Collection 24 of single-gene knockouts.
  • E. coli strains 552059.1 and 552060.1 were isolated from urine 25 and acquired from the clinical Medical Microbiology department at the University Medical Center Utrecht.
  • E. coli mcr-1 (EQAS 2016 412016126, mcr-1 positive, recovered during international antimicrobial resistance programs), W3110, BW25113, belong to the laboratory collection of N.I. M. Fluoroquinolone resistant E. coli strains 965, 991 , 1022, 1075, 1104,1146, 1175, 1192, 1201 , 1233 were isolated from blood cultures, positive ciprofloxacin resistance, during July-August 2021 and were acquired from the Department of Medical Microbiology and Infection Prevention, Amsterdam University Medical Centre. The following reagents were obtained through BEI Resources, NIAID, NIH: E. coli, Strain MVAST0072, NR- 51488.
  • the bacterial suspensions were diluted 200-fold in cation adjusted Mueller-Hinton broth (CAMHB) and 99 pL were added in a library of test compounds (1 pL DMSO stock solution, per well in technical duplicates) in polypropylene 96-well microtiter plates to reach a volume of 100 pL and a final concentration of 100 pM for each test compound and a maximum of 1% DMSO.
  • the plates were sealed with breathable membranes and incubated at 37°C overnight with constant shaking (600 rpm). Screening hits were selected from the wells where no visible bacterial growth was observed, as compared to the inoculum controls, containing 1 % DMSO.
  • MIC was determined as the lowest concentration at which no visible bacterial growth was observed, as compared to the inoculum controls, from the median of a minimum of triplicates.
  • Mammalian cell culture HepG2 and HEK293T cell lines (ATCC) were cultured at 37°C and 7% CO2 in DMEM (Sigma Aldrich, D6546) with GlutaMax, penicillin (100 pg/mL), streptomycin (100 pg/mL) and 10% Fetal Calf serum. Cells were passaged twice a week by first detaching using 0.05% trypsin in PBS, and then diluting to appropriate confluence.
  • Cytotoxicity assay (MTT). Compound cytotoxicity was evaluated against HepG2 and HEK293T human cell lines based on a standard (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay protocol 27 .
  • HepG2 and HEK293T cells were seeded at a density of 1.5 x 10 4 cells per well in a clear 96-well tissue culture treated plate in a final volume of 100 pL in Dulbecco’s Modified Eagle Medium (DMEM), supplemented with Fetal Bovine Serum (1%), Glutamax and Pen/Strep.
  • DMEM Modified Eagle Medium
  • DAPI 2 pg/mL
  • FM4-64 2 pg/mL
  • SYTOX Green 0.5 pM
  • the samples were then centrifuged at 3300 x g for 50 s and the pellets resuspended in half the original volume. From this suspension, 5 pL was spotted onto a 2.0% agarose pad for microscopy.
  • Microscopy was performed on a Zeiss Axio Observer Z1/7 inverted microscope. For differential interference contrast the transmitted light source was an Aquilla TL halogen lamp (3.0 V).
  • the Zeiss Colibri.2 LED lamps were configured as follows: DAPI (365 nm, 150 ms, 25.42%), FM4-64 (590 nm, 1000 ms, 100%) and SYTOX Green (470 nm, 150 ms, 25.42%).
  • the recorded cell outlines produced by MicrobeJ were exported and used as a guide to characterize the DNA shape within each bacterium using a custom- made Imaged macro (Appendix II).
  • DAPI and SYTOX Green intensity were measured on raw images in a similar manner. Then by hand polygons were drawn that excluded bacteria to determine background intensity. After background correction the intensity was standardized to the DMSO control within the same biological replicate.
  • the BCP assay was executed for three independently biological replicates. During each experiment, images were acquired until 24 or more bacteria in total had been observed. Following feature extraction for each replicate, principal component analysis was performed using GraphPad Prism (v 9.0.0) using multiple variable analysis with standardized data. PCs were selected based on the percent of total explained variance (minimum 80%).
  • DNA was re-suspended in nuclease-free water. Genome sequencing was performed using Illumina Novaseq 6000 PE150 at Novogene Co. Ltd. (Tianjin, China). Paired-end sequence reads were generated and mapped against the reference genome of E. coli W3110. The alignment to the reference genome was performed using the Burrow-Wheeler Alignment tool (BWA vO.7.8 29 ). SNP/lnDel calling, annotation and statistics was performed using SAMtools 30 (vO.1.19) and ANNOVAR 31 (v2015MAR22). The structural variant calling annotation and statistics was performed with BreakDancer v1.4.4 and ANNOVAR 31 (v2015MAR22). The wild type E. coli W3110 was also sequenced and compared to the reference genome to confirm that the mutations found in the 60-resistant colonies were unique and related to the antibiotic resistance. Sequencing data is available at NCBI through the BioProject accession number PRJNA855320.
  • gyrA gyrA recombinant mutants in E. coli ⁇ N3110. Mutants of the DNA gyrase subunit A encoding gene (gyrA) were constructed following the protocol for gene editing via the CRISPR- Cas9 system. 32 Strains and plasmids used are listed in Table 1 , and primers used can be found in Table 2.
  • coli W3110 genomic DNA by PCR using primer pairs gyrA_P05 and gyrA_P013, and gyrA_P08 and gyrA_P14.
  • the three PCR fragments generated were cloned into the Spel and Bglll digested pTargetF via Gibson assembly.
  • desired point mutation for gyrA[S97L] four other silent point mutations were also included in HDR template. Consequently, the amino acid sequence of gyrA will remain the same in the generated mutant except for the desired mutation.
  • constructs psgRNA-gyrA-S83L and psgRNA-gyrA-D87N were created to introduce mutations S83L and D87N in gyrA, respectively.
  • spacer sequence was introduced using primers gyrA_P12 and gyrA_P17
  • HDR template was amplified using primer pairs gyrA_P05 and gyrA_P18, and gyrA_P08 and gyrA_P19.
  • spacer sequence was introduced using primers gyrA_P12 and gyrA_P20, HDR template was amplified using primer pairs gyrA_P05 and gyrA_P21 , and gyrA_P08 and gyrA_P22.
  • the verified constructs for gyrA engineering were transformed into E. coli W3110 carrying pCas9, and plated in LB containing kanamycin (50 pg/mL) for the selection of the pCas9 plasmid and spectinomycin (100 pg/mL) for the selection of psgRNA-gyrA-X.
  • Plasmids were cured by growing the strains with 0.5 mM IPTG with no antibiotics to lose first the psgRNA-gyrA plasmids. After losing the spectinomycin resistance, the strain was grown in LB with no antibiotics at 42°C to lose the pCas plasmid. Colony PCR was performed on plasmid-free colonies using primers gyrA_P9 and gyrA_P10, and PCR products were sequenced to confirm the desired mutation.
  • DNA gyrase supercoiling assay The DNA gyrase inhibition assay was executed as instructed by the manufacturer (G1001 from Inspiralis Limited). A master mix was made including 0.5 pg relaxed plasmid (pBR322) in 35 mM Tris HCI (pH 7.5), 24 mM KCI, 4 mM MgCh, 2 mM DTT, 1.8 mM spermidine, 1 mM ATP, 6.5% (w/v) glycerol, and 0.1 mg/mL albumin per reaction.
  • pBR322 relaxed plasmid
  • Stop Dye 50% (w/v) sucrose, 100 mM Tris HCI pH 8, 10 mM EDTA, 0.5 mg/mL bromophenol Blue
  • 30 pL of a chlorofomrisoamyl alcohol solution 24:1 v/v.
  • 20 pL of the aqueous phase was loaded onto a 1% (w/v) agarose gel in TAE (Tris acetate 0.04 mM, EDTA 0.002 mM) gel.
  • the gel was run at 85 V for 2 h followed by staining (15 min) in 1 pg/mL ethidium bromide and destaining (5-10 min) in water.
  • DNA was visualized (602/50, UV Trans, auto optimal exposure) with a ChemiDoc MP (Bio-Rad Laboratories, Inc) and the percentage of supercoiled DNA relative to the total amount material per lane was determined with ImageLab 6.1 software (Bio-Rad Laboratories, Inc).
  • IC50 curves were generated in GraphPad Prism (v 9.0.0) using the nonlinear regression curve fit variable slope with four parameters and least squares regression.
  • E. coli gyrase supercoiling inhibition assays for both mutant and wild type enzymes were executed by setting up a master mix including 0.5 pg relaxed plasmid (pBR322) in 35 mM Tris HCI (pH 7.5), 24 mM KCI, 4 mM MgCI 2 , 2 mM DTT, 1 .8 mM spermidine, 1 mM ATP, 6.5% (w/v) glycerol, and 0.1 mg/mL albumin per reaction.
  • pBR322 relaxed plasmid
  • Reactions were run at 37 °C for 30 min and stopped with the addition of 30 pL STEB (40% (w/v) sucrose, 100 mM Tris HCI pH 8, 100 mM EDTA, 0.5 mg/mL Bromophenol Blue) and 30 pL of a chloroform: isoamyl alcohol solution (24:1 v/v). After brief vortexing and centrifugation at 2300 x g for 1 min, 20 pL of the aqueous phase was loaded onto 1% (w/v) agarose gels in TAE (Tris acetate 0.04 mM, EDTA 0.002 mM) gel.
  • TAE Tris acetate 0.04 mM, EDTA 0.002 mM
  • aeruginosa gyrase supercoiling inhibition assays were set up in the same way as E. coli gyrase supercoiling assays.
  • the enzyme was retrieved from Inspiralis Limited (PAG1001 and ABG1001) and tested in-house for activity with x5 u enzyme used for each reaction.
  • E. coli relaxation inhibition assays were conducted in a similar manner to supercoiling inhibition assays with the following modifications: spermidine and ATP were omitted from the assay buffer, supercoiled plasmid (pBR322) was used as DNA substrate, and 200 nM E. coli gyrase was added to each reaction. After stopping each reaction with STEB, 3 pL 2% SDS was added and tubes were vigorously vortexed before loading the aqueous phase onto the gel.
  • aqueous layers from the assays were mixed with 30 pL STEB and run on 1% agarose TAE gels with 10 pg/mL ethidium bromide at 80 V. Gels were visualized and analyzed as described for the supercoiling inhibition assays.
  • the breathable seals were removed and the plates shaken using a bench top shaker to ensure homogeneous bacterial suspensions. The plates were then transferred to a Tecan Spark plate reader and following another brief shaking (20 seconds) the GD600 was measured. The resulting GD600 values were transformed to a 2D gradient to visualize the growth/no-growth results.
  • Equation 1 Calculation of FICI.
  • the highest concentration in the dilution series was used in determing the FICI and the result reported as ⁇ the calculated value.
  • 217 bp DNA was added to the complex in a 1 :1 ratio to a final concentration of gyrase-DNA complex ⁇ 15 pM.
  • the reconstituted complex was buffer exchanged using dialysis at 4°C overnight to cryo-EM buffer (25 mM Na-HEPES pH 8, 30 mM potassium acetate, 2.5 mM magnesium acetate, 0.5 mM TCEP). After buffer exchange, the sample was concentrated to ⁇ 30 pM. Sample was supplemented with 100 pM 102 compound, 1 mM ADPNP and incubated for 30 min at 37°C. 8 mM CHAPSO was added, and sample was centrifuged (60 min at 21 ,000 x g) to remove potential aggregates.
  • Cryo-EM data were collected at the Polish national cryo-EM facility SOLARIS with a Titan Glacios microscope (Thermo Fisher Scientific) operated at 200 kV, and images (movie frames) were collected at the calibrated physical pixel size of 0.95 A per pixel with a defocus range of -3 to -0.9 pm. The images were recorded in counting mode on a Falcon IV electron direct detector (Thermo) in EER format. A dose rate and exposure time was set to generate a total dose of ⁇ 40 electrons/A 2 . Statistics for cryo-EM data collection are listed in Table 3.
  • Model building and refinement (1) The closest available structure of E. coli gyrase (PDB:6RKV 40 ) was used as a starting point for model building. 6RKV model was rigid-body fitted in ChimeraX 44 and manually adjusted in Coot 4549 . Real-space refinement was performed in phenix.refine 46 (using Ramachandran restrains and secondary structure restraints). The model and restraints for 102 were obtained using Grade server (http://grade.globalphasing.org); 102 was initially manually fitted into the density and refined in Phenix. The model was refined against the 4.23 A map described above. MolProbity 47 was used to validate the structures. Statistics for the final model is reported in Table 3.
  • CryoEM data collection and analysis (2) Aliquots of 4 pl of reconstituted complexes were applied to glow-discharged (Leica, 60 s/8 mA) Quantifoil holey carbon grids (R2/1 , 300 copper mesh). After 30 s of incubation with 95% chamber humidity at 10°C, the grids were blotted for 3.5 s and plunge- frozen in liquid ethane using a Vitrobot mark IV (FEI).
  • FEI Vitrobot mark IV
  • CryoEM data were collected at Astbury Biostructure Laboratory (ABSL) CryoEM facility (University of Leeds, UK) on Krios G2 microscope (Thermo Fisher Scientific) operated at 300 kV and nominal magnification of 120kx.
  • Movie frames were collected at the calibrated physical pixel size of 0.68 A per pixel with a defocus range of -2.8 to -0.8 pm. Movies were recorded in counting mode on a Falcon 4i direct electron detector (Thermo) in EER format. A dose rate and exposure time was set to generate a total dose of ⁇ 40 electrons/A 2 . Statistics for cryo-EM data collection are listed in Table 4.
  • cryoSPARC 4.2.1 41 17,017 movies were dose weighted and motion and CTF corrected in patch mode.
  • Particles were picked with cryoSPARC template picker and 2x2 binned particles (1 ,810,321) were subjected to several rounds of 2D classification (50 iterations, 20 full iterations, batchsize 300).
  • Cleaned particles representing holoenzyme with secondary structure elements visible (169,234) underwent 3D classification with 2 classes (ab initio, 143,883 particles retained) and was refined to 3.05 A resolution using non-uniform refinement procedure.
  • 42 Particles were re-extracted as unbinned using updated coordinates and downsampled to 1 .02 A/pix.
  • Model building and refinement (2) The closest available structure of E. coli gyrase (PDB: 7Z9C) was used as a starting point for model building. 7Z9Cmodel was rigid-body fitted in ChimeraX 44 and manually adjusted in Coot 45 and ISOLDE. 49 Real-space refinement was performed in phenix.refine 46 (using Ramachandran restrains, secondary structure restraints for protein and DNA, and NCS restraints). The model and restraints for compound 102 were obtained using Grade server (http://grade.globalphasing.org). After building protein and DNA, compound 102 was automatically fitplaced into the density by phenix. ligandfit and further refined in Phenix with the rest of the model.
  • the titer was determined by removing 100 pl of culture, serially diluted and plated (50 pL per plate, onto three plates per dilution). The plates were incubated at 37 °C, and examined for colonies every 12 h for a total of 48 h. The number of colonies on each plate was counted. Antibiotic selected colonies were randomly picked (three per plate) and tested for confirmation of resistance. Mutation frequency was calculated by dividing the number of confirmed resistant mutants obtained by the total bacteria plated.
  • PMBN Polymyxin B nonapeptide
  • CIP ciprofloxacin
  • MSC minimum synergistic concentration
  • FC fold change in MIC after addition of 4 pM PMBN
  • MCR mobile colistin resistance
  • MDR multidrug resistant
  • ESBL extended spectrum beta lactamase
  • * urinary tract infection isolates [00343] In view of this excellent profile, compound 102 was selected for further profiling. Closely related inactive compound 99 was chosen as a negative control compound.
  • BCP bacterial cytological profiling
  • AMP ampicillin
  • RAF RNA synthesis inhibitor
  • TET protein synthesis inhibitor
  • CIP DNA synthesis inhibitor
  • DAPI membrane permeable DNA dye
  • FM4-64 lipophilic membrane dye
  • SYTOX Green membrane impermeable DNA dye
  • Quantitative BCP - DIC runf'Gaussian --, "radius 1 .5"); channel Quantitative BCP - DAPI Custom made script shape descriptors Quantitative BCP - DAPI Custom made script and SYTOX Green intensity
  • Bacteria detection fit shape (rod shaped)
  • DNA gyrase is a well-validated antibiotic target, which is responsible for introducing negative supercoils in DNA ( Figure 32a), a process that is required for DNA synthesis and proliferation of bacteria.
  • DNA gyrase is a target of the fluoroquinolone antibiotics including CIP.
  • CIP CIP was taken along as a positive control and produced an IC50 value of 925 nM, which is in line with previously reported values.
  • MSC minimum synergistic concentration
  • MIC minimum inhibitory concentration
  • FICI Fractional Inhibitory Concentration Index
  • PMBN Polymyxin B nonapeptide.
  • cryogenic electron microscopy was used to determine a medium-resolution structure of E. coli gyrase holocomplex (A2B2) bound to the substrate 217-bp dsDNA, nucleotide analogue ADPNP and 102.
  • a single dataset collected on 200 kV Glacios microscope allowed us to visualise enzyme-DNA complex in the wrapped state (see Figure 34) and reconstruct a model for the cleavage-reunion domain of the enzyme (residues 8-524 of GyrA and 405-804 of GyrB) similarly to Lamour and coworkers 40 , at the resolution of 4.2 A ( Figure 35a).
  • the S97L variant was highly resistant to both 102 and LEI-801 (Figure 39), while 102 and 99 were still active on the S172A (IC50 -values of 86 and 360 nM, respectively) ( Figure 40). These biochemical results support the binding mode observed with the cryoEM study and are in line with the genetic data.
  • the 102 pocket is not exploited by any type of natural or synthetic gyrase inhibitors.
  • the only inhibitor bound in a partially overlapping site is simocyclinone D8 (SD8), a natural product preventing DNA binding to the GyrA subunit ( Figure 38c).
  • SD8 simocyclinone D8
  • Figure 38c a natural product preventing DNA binding to the GyrA subunit
  • 102 binding does not affect interaction with double-stranded DNA.
  • ciprofloxacin binding promotes double-strand cleavage by the enzyme, upon 102 binding the DNA was found to remain fully intact.
  • Results are represented as the average of three experiments with standard deviation.
  • LEI-800 is also denoted as 102.
  • the compounds of the invention exhibit potent, Gram-negative-specific antibacterial activity with little mammalian cell toxicity.
  • Cell morphology and mutation selection clearly pointed to DNA gyrase as the target for compounds of the invention after which a cryo-EM study revealed that the compounds bind to a previously unexploited site on the GyrA subunit.
  • compounds of the invention show no affinity for binding to DNA, as is common in other DNA gyrase inhibitors.

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Abstract

L'invention concerne des composés utiles pour le traitement d'infections bactériennes, par exemple une infection bactérienne à Gram négatif. Les composés peuvent être de formule (I) telle que définie dans la description. Les composés peuvent être capables de se lier à une poche hydrophobe de type en fer à cheval sur la sous-unité GyrA de surface de liaison à l'ADN de l'ADN gyrase. L'invention concerne également des formulations comprenant de tels composés, ainsi que des utilisations de tels composés ou formulations, par exemple pour le traitement d'une infection bactérienne et/ou en tant qu'inhibiteur d'ADN gyrase.
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US20040024030A1 (en) * 2000-01-18 2004-02-05 Paul Charifson Gyrase inhibitors and uses thereof
US8426426B2 (en) * 2002-06-13 2013-04-23 Vertex Pharmaceuticals Incorporated Gyrase inhibitors and uses thereof
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