WO2011055142A2 - 1,4-quinones and their sulfur analogues useful as ligands of n-acetyltransferases - Google Patents

1,4-quinones and their sulfur analogues useful as ligands of n-acetyltransferases Download PDF

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WO2011055142A2
WO2011055142A2 PCT/GB2010/051840 GB2010051840W WO2011055142A2 WO 2011055142 A2 WO2011055142 A2 WO 2011055142A2 GB 2010051840 W GB2010051840 W GB 2010051840W WO 2011055142 A2 WO2011055142 A2 WO 2011055142A2
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compound
compounds
substituted
hydroxy
carbon atoms
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WO2011055142A3 (en
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Angela Jane Russell
Edith Sim
Steven Graham Davies
Isaac Mark Westwood
Akane Kawamura
Matthew Howard James Crawford
Nicola Laurieri
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Isis Innovation Limited
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Priority claimed from GBGB1002708.4A external-priority patent/GB201002708D0/en
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Publication of WO2011055142A2 publication Critical patent/WO2011055142A2/en
Publication of WO2011055142A3 publication Critical patent/WO2011055142A3/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D333/00—Heterocyclic compounds containing five-membered rings having one sulfur atom as the only ring hetero atom
    • C07D333/02—Heterocyclic compounds containing five-membered rings having one sulfur atom as the only ring hetero atom not condensed with other rings
    • C07D333/04—Heterocyclic compounds containing five-membered rings having one sulfur atom as the only ring hetero atom not condensed with other rings not substituted on the ring sulphur atom
    • C07D333/26—Heterocyclic compounds containing five-membered rings having one sulfur atom as the only ring hetero atom not condensed with other rings not substituted on the ring sulphur 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
    • C07D333/30—Hetero atoms other than halogen
    • C07D333/34—Sulfur atoms
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C205/00—Compounds containing nitro groups bound to a carbon skeleton
    • C07C205/27—Compounds containing nitro groups bound to a carbon skeleton the carbon skeleton being further substituted by etherified hydroxy groups
    • C07C205/35—Compounds containing nitro groups bound to a carbon skeleton the carbon skeleton being further substituted by etherified hydroxy groups having nitro groups and etherified hydroxy groups bound to carbon atoms of six-membered aromatic rings of the carbon skeleton
    • C07C205/36—Compounds containing nitro groups bound to a carbon skeleton the carbon skeleton being further substituted by etherified hydroxy groups having nitro groups and etherified hydroxy groups bound to carbon atoms of six-membered aromatic rings of the carbon skeleton to carbon atoms of the same non-condensed six-membered aromatic ring or to carbon atoms of six-membered aromatic rings being part of the same condensed ring system
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C225/00—Compounds containing amino groups and doubly—bound oxygen atoms bound to the same carbon skeleton, at least one of the doubly—bound oxygen atoms not being part of a —CHO group, e.g. amino ketones
    • C07C225/24—Compounds containing amino groups and doubly—bound oxygen atoms bound to the same carbon skeleton, at least one of the doubly—bound oxygen atoms not being part of a —CHO group, e.g. amino ketones the carbon skeleton containing carbon atoms of quinone rings
    • C07C225/26—Compounds containing amino groups and doubly—bound oxygen atoms bound to the same carbon skeleton, at least one of the doubly—bound oxygen atoms not being part of a —CHO group, e.g. amino ketones the carbon skeleton containing carbon atoms of quinone rings having amino groups bound to carbon atoms of quinone rings or of condensed ring systems containing quinone rings
    • C07C225/30—Compounds containing amino groups and doubly—bound oxygen atoms bound to the same carbon skeleton, at least one of the doubly—bound oxygen atoms not being part of a —CHO group, e.g. amino ketones the carbon skeleton containing carbon atoms of quinone rings having amino groups bound to carbon atoms of quinone rings or of condensed ring systems containing quinone rings of condensed quinone ring systems formed by two rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C255/00—Carboxylic acid nitriles
    • C07C255/49—Carboxylic acid nitriles having cyano groups bound to carbon atoms of six-membered aromatic rings of a carbon skeleton
    • C07C255/54—Carboxylic acid nitriles having cyano groups bound to carbon atoms of six-membered aromatic rings of a carbon skeleton containing cyano groups and etherified hydroxy groups bound to the carbon skeleton
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C311/00—Amides of sulfonic acids, i.e. compounds having singly-bound oxygen atoms of sulfo groups replaced by nitrogen atoms, not being part of nitro or nitroso groups
    • C07C311/15—Sulfonamides having sulfur atoms of sulfonamide groups bound to carbon atoms of six-membered aromatic rings
    • C07C311/20—Sulfonamides having sulfur atoms of sulfonamide groups bound to carbon atoms of six-membered aromatic rings having the nitrogen atom of at least one of the sulfonamide groups bound to a carbon atom of a ring other than a six-membered aromatic ring
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C50/00—Quinones
    • C07C50/26—Quinones containing groups having oxygen atoms singly bound to carbon atoms
    • C07C50/32—Quinones containing groups having oxygen atoms singly bound to carbon atoms the quinoid structure being part of a condensed ring system having two rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C50/00—Quinones
    • C07C50/38—Quinones containing —CHO or non—quinoid keto groups
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D295/00—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms
    • C07D295/04—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms
    • C07D295/12—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by singly or doubly bound nitrogen atoms
    • C07D295/135—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by singly or doubly bound nitrogen atoms with the ring nitrogen atoms and the substituent nitrogen atoms separated by carbocyclic rings or by carbon chains interrupted by carbocyclic rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D307/00—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom
    • C07D307/02—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings
    • C07D307/34—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
    • C07D307/38—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members with substituted hydrocarbon radicals attached to ring carbon atoms
    • C07D307/52—Radicals substituted by nitrogen atoms not forming part of a nitro radical
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D333/00—Heterocyclic compounds containing five-membered rings having one sulfur atom as the only ring hetero atom
    • C07D333/50—Heterocyclic compounds containing five-membered rings having one sulfur atom as the only ring hetero atom condensed with carbocyclic rings or ring systems
    • C07D333/52—Benzo[b]thiophenes; Hydrogenated benzo[b]thiophenes
    • C07D333/54—Benzo[b]thiophenes; Hydrogenated benzo[b]thiophenes with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached to carbon atoms of the hetero ring
    • C07D333/58—Radicals substituted by nitrogen atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C2601/00—Systems containing only non-condensed rings
    • C07C2601/06—Systems containing only non-condensed rings with a five-membered ring
    • C07C2601/08—Systems containing only non-condensed rings with a five-membered ring the ring being saturated
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C2602/00—Systems containing two condensed rings
    • C07C2602/02—Systems containing two condensed rings the rings having only two atoms in common
    • C07C2602/04—One of the condensed rings being a six-membered aromatic ring
    • C07C2602/10—One of the condensed rings being a six-membered aromatic ring the other ring being six-membered, e.g. tetraline

Definitions

  • This invention relates to ligands for arylamine /V-acetyltransferases (NATs). More particularly, it relates to compounds useful as probes for NATs and compounds useful as antagonists for NATs, as well as the use thereof. The invention further relates to preparing the described products, compositions comprising them and other subject matter.
  • NATs arylamine /V-acetyltransferases
  • Arylamine /V-acetyltransferases are drug metabolising enzymes which are found in a wide range of prokaryotic and eukaryotic species.
  • NATs catalyse the transfer of an acetyl group from acetyl coenzyme A (AcCoA) to the amine of a variety of arylamines.
  • Human NAT1 hNAT1
  • ER+ oestrogen receptor- positive
  • hNAT1 is upregulated in some ER+ tumours compared with ER-negative tumours, and hence is identified as a putative ER-responsive gene.
  • NAT1 plays a biological role in breast cancer progression and it has therefore emerged as a diagnostic marker and drug target for breast cancer.
  • hNAT1 has been generated in a pure form and its substrate specificity profile has been demonstrated to be very similar to that of mouse NAT2 (Kawamura A D. Phil. Thesis, University of Oxford 2005; Wang H et al. Protein J. 2005, 24, 55; Kawamura A et al. Biochem. Pharmacol. 2008, 75, 1550).
  • Russell A J et al. Bioorg. Med. Chem. 2009, 77, 905-918 is included herein by reference and describes selective small molecule inhibitors of hNAT1 and its murine homologue, mouse arylamine /V-acetyltransferase 2 (mNAT 2).
  • the inhibitors are 5- (benzylidene)-2-thioxothiazolidinones and analogues thereof, in particular analogues in which the benzene ring of the benzylidene moiety is replaced by naphthyl or another hydrocarbyl moiety containing 6 or more carbon atoms.
  • the present invention provides in one aspect compounds useful as probes to detect NATs, as inhibitors of NATs, or as both probes and inhibitors of NATs.
  • the invention therefore provides in one aspect the use of 1 ,4-quinones, in particular 1 ,4-naphthoquinones, and their sulfur analogues in which one or both dione oxo groups are replaced by thioxo groups, as probes and/or inhibitors of hNATL
  • Some of said 1 ,4-quinones and their mono-and di-thioxo analogues are chromogenic in the presence of hNAT1 , in that they change colour (or potentially become coloured/decoloured) when contacted with hNATL
  • the invention includes methods of detecting for and/or measuring hlMATI comprising contacting a sample with a said chromogenic 1 ,4-quinone or a thioxo analogue thereof.
  • the invention also includes methods of inhibiting hlMATI in the treatment of disease, for example in delaying the progression of disease, comprising administering to a subject a therapeutically effective amount of a said 1 ,4-quinone or a thioxo analogue thereof.
  • compounds selected from those having a structure disclosed herein and salts and prodrugs thereof, for use in inhibiting hlMATI in the treatment of disease may be substituted at least at the 2- and 3- positions; for example the substituents at the 2- and 3- positions may be the same or different and selected from residues comprising a functional group capable of forming hydrogen bonds or ionic bonds and, spaced from the (thioxo)quinone ring, a ring.
  • the invention provides, amongst other things, chromogenic compounds which bind selectively to hlMATI and change colour, or become coloured/decoloured, on binding thereto.
  • a compound is contacted with a sample, in particular one containing one or more intact or lysed human cells, to form a mixture, and the mixture is observed in order to detect development/alteration of any colour characteristic of the compound when bound to hNATL
  • the intensity of the light absorbed or emitted at a wavelength characteristic of the hNATI -bound compound is used to measure the amount of hNAT1 .
  • the invention provides compounds having the structure of Formula I and salts and physiological precursors thereof:
  • X is O or S
  • Y is O or S;
  • R 1 is R 5 , J, halogen, trifluoromethyl, cyano, nitro, -O-L-R 7 , -S-L-R 7 or -OC(0)-L-R 7 ;
  • L is a bond, alkylene optionally substituted by hydroxy and having 1 , 2, 3, 4 or 5 carbon atoms and unsubstituted or substituted by hydroxy, alkylene optionally substituted by hydroxy and having 1 , 2, 3 or 4 carbon atoms and interrupted and/or terminated adjacent R 7 by -0-, -S-, or -C(O)-;
  • R 7 is H or a 3-7 membered monocyclic ring or an 8-13 membered bicyclic ring, which rings are unsubstituted or are substituted by one or more R 8 , wherein:
  • R 8 is selected from halogen, hydroxy, alkyl optionally substituted by hydroxy and having 1 , 2, 3, 4 or 5 carbon atoms, alkyl optionally substituted by hydroxy and having 1 , 2, 3 or 4 carbon atoms and interrupted by -0-, -S-, or -C(O)-, a 3 to 6- membered ring bonded to the remainder of the molecule either directly or through a Ci -4 alkylene group, trifluoromethyl, trifluoromethoxy, cyano, nitro, oxo, amidino, -OR 9 , -C(0)R 9 , -C(0)OR 9 , -OC(0)R 9 , -N(R 9 )R 10 , -C(0)N(R 9 )R 10 , - S(0) a R 9 and -S(0) a OR 9 , wherein R 9 and R 10 are the same or different and are each hydrogen or are selected from alkyl optionally substituted by hydroxy and having 1 , 2,
  • R 5 is H; alkyl having 1 , 2, 3, 4 or 5 carbon atoms and unsubstituted or substituted by hydroxy; alkyl having 1 , 2, 3 or 4 carbon atoms, unsubstituted or substituted by hydroxy and interrupted by -0-, -S-, or -C(O)-; -L-R 7 ; -S(0) b -L-R 7 ; -C(0)-N(R 19 )-[S(0) b ] r L-R 7 , -C(0)-L-R 7 ; -C(NR 19 )-L-R 7 ; -C(NR 19 )0-L-R 7 ; -C(NR 19 )NR 30 -L-R 7 ; -C(0)S-L-R 7 ; -C(NR 19 )S- L-R 7 -C(0)0-L-R 7 ; 2-pyridyl or 4-pyridyl, wherein 2-pyridyl and 4-pyr
  • L-R and R are independently selected from the possibilities for L-R 7 , L and R 7 are as defined previously, and b is 0, 1 or 2;
  • R 5 is as defined previously;
  • R 6 is selected from the possibilities for L-R 7 , e.g. is H or alkyl having 1 , 2, 3, 4 or 5 carbon atoms;
  • R 2 is J, wherein if both R 1 and R 2 are J they may be the same or different, -(CH 2 ) e -0-M-R 16 or -(CH 2 ) e -S-M-R 16 , wherein e is 0, 1 , 2, 3 or 4, M is selected from the list of possibilities for L and R 16 is selected from the list of possibilities for R 7 ;
  • R 3 and R 4 are each independently H or R 8 ;
  • each -L-R 7 may be the same as or different from any other -L-R 7
  • each R 8 may be the same as or different from any other R 8 .
  • the identities of R 6 , R 19 and R 30 are chosen independently of each other and independently of the identity of the or each -L-R 7
  • the invention provides compounds having the structure of Formula l(i) and salts and physiological precursors thereof:
  • X is O or S
  • Y is O or S
  • R 1 is R 5 , J, halogen, trifluoromethyl, cyano, nitro, -O-L-R 7 , -S-L-R 7 or -OC(O)
  • L is a bond, alkylene optionally substituted by hydroxy and having 1 , 2, 3, 4 or 5 carbon atoms and unsubstituted or substituted by hydroxy, alkylene optionally substituted by hydroxy and having 1 , 2, 3 or 4 carbon atoms and interrupted and/or terminated adjacent R 7 by -0-, -S-, or -C(O)-;
  • R 7 is H or a 3-7 membered monocyclic ring or an 8-13 membered bicyclic ring, which rings are unsubstituted or are substituted by one or more R 8 , wherein:
  • R 8 is selected from halogen, hydroxy, alkyl optionally substituted by hydroxy and having 1 , 2, 3, 4 or 5 carbon atoms, alkyl optionally substituted by hydroxy and having 1 , 2, 3 or 4 carbon atoms and interrupted by -0-, -S-, or -C(O)-, a 3 to 6- membered ring bonded to the remainder of the molecule either directly or through a Ci -4 alkylene group, trifluoromethyl, cyano, nitro, oxo, amidino, -OR 9 , -C(0)R 9 , -C(0)OR 9 , -OC(0)R 9 , -N(R 9 )R 10 , -C(0)N(R 9 )R 10 , -S(0) a R 9 and - S(0) a OR 9 , wherein R 9 and R 10 are the same or different and are each hydrogen or are selected from alkyl optionally substituted by hydroxy and having 1 , 2, 3, 4 or 5 carbon atom
  • R 5 is H; alkyl having 1 , 2, 3, 4 or 5 carbon atoms and unsubstituted or substituted by hydroxy; alkyl having 1 , 2, 3 or 4 carbon atoms, unsubstituted or substituted by hydroxy and interrupted by -0-, -S-, or -C(O)-; -L-R 7 ; -S(0) b -L-R 7 ; -C(0)-N(R 19 )-[S(0) b ] r L-R 7 , -C(0)-L-R 7 ; -C(NR 19 )-L-R 7 ; -C(NR 19 )0-L-R 7 ; -C(NR 19 )NR 30 -L-R 7 ; -C(0)S-L-R 7 ; -C(NR 19 )S- L-R 7 -C(0)0-L-R 7 ; 2-pyridyl or 4-pyridyl, wherein 2-pyridyl and 4-pyr
  • L-R and R are independently selected from the possibilities for L-R 7 , L and R 7 are as defined previously, and b is 0, 1 or 2;
  • R 5 is as defined previously;
  • R 6 is selected from the possibilities for L-R 7 , e.g. is H or alkyl having 1 , 2, 3, 4 or 5 carbon atoms;
  • R 2 is J, wherein if both R 1 and R 2 are J they may be the same or different, -(CH 2 ) e -0-M-R 16 or -(CH 2 ) e -S-M-R 16 , wherein e is 0, 1 , 2, 3 or 4, M is selected from the list of possibilities for L and R 16 is selected from the list of possibilities for R 7 ;
  • R 3 and R 4 are each independently H or R 8 ;
  • each -L-R 7 may be the same as or different from any other -L-R 7
  • each R 8 may be the same as or different from any other R 8 .
  • the identities of R 6 , R 19 and R 30 are chosen independently of each other and independently of the identity of each -L-R 7 .
  • the compounds additionally include structures wherein R 1 is -L-R 7 or O-L-R 7 , L is a bond and R 7 is a substituted or unsubstituted ring selected from 3-7 membered monocyclic rings and 8-13 membered bicyclic rings optionally substituted with one or more R 8 selected from halogen, hydroxy, alkyl optionally substituted by hydroxy and having 1 , 2, 3, 4 or 5 carbon atoms, alkyl optionally substituted by hydroxy and having 1 , 2, 3 or 4 carbon atoms and interrupted by -0-, -S-, or -C(O)-, a 3 to 6- membered ring bonded to the remainder of the molecule either directly or through a Ci -4 alkylene group, trifluoromethyl, trifluoromethoxy, cyano, nitro, oxo, amidino, -OR 9 , -C(0)R 9 , -C(0)OR 9 , -
  • the invention includes a sub-class of the above compounds comprising chromogenic compounds.
  • This sub-class is compounds selected from the structure of Formula I and salts and physiological precursors thereof wherein the symbols have the following meanings:
  • X is O or S
  • Y is O or S;
  • R 1 is R 5 , J, halogen, trifluoromethyl, cyano, nitro, -O-L-R 7 , -S-L-R 7 or -OC(0)-L-R 7 ;
  • L is a bond, alkylene optionally substituted by hydroxy and having 1 , 2, 3, 4 or 5 carbon atoms and unsubstituted or substituted by hydroxy, alkylene optionally substituted by hydroxy and having 1 , 2, 3 or 4 carbon atoms and interrupted and/or terminated adjacent R 7 by -0-, -S-, or -C(O)-;
  • R 7 is H or a 3-7 membered monocyclic ring or an 8-13 membered bicyclic ring, which rings are unsubstituted or are substituted by one or more R 8 , or is a dye, wherein:
  • R 8 is selected from halogen, hydroxy, alkyl optionally substituted by hydroxy and having 1 , 2, 3, 4 or 5 carbon atoms, alkyl optionally substituted by hydroxy and having 1 , 2, 3 or 4 carbon atoms and interrupted by -0-, -S-, or -C(O)-, a 3 to 6- membered ring bonded to the remainder of the molecule either directly or through a Ci -4 alkylene group, trifluoromethyl, trifluoromethoxy, cyano, nitro, oxo, amidino, -OR 9 , -C(0)R 9 , -C(0)OR 9 , -OC(0)R 9 , -N(R 9 )R 10 , -C(0)N(R 9 )R 10 , and -S(0) a R 9 , wherein R 9 and R 10 are the same or different and are each hydrogen or are selected from alkyl optionally substituted by hydroxy and having 1 , 2, 3, 4 or 5 carbon atoms
  • R 5 is H; alkyl having 1 , 2, 3, 4 or 5 carbon atoms and unsubstituted or substituted by hydroxy; alkyl having 1 , 2, 3 or 4 carbon atoms, unsubstituted or substituted by hydroxy and interrupted by -0-, -S-, or -C(O)-; -L-R 7 ; -S(0) b -L-R 7 ; -C(0)-N(R 19 )-[S(0) b ] r L-R 7 , -C(0)-L-R 7 ; -C(NR 19 )-L-R 7 ; -C(NR 19 )0-L-R 7 ; -C(NR 19 )NR 30 -L-R 7 ; -C(0)S-L-R 7 ; -C(NR 19 )S- L-R 7 -C(0)0-L-R 7 ; 2-pyridyl or 4-pyridyl, wherein 2-pyridyl and 4-pyr
  • R 5 is as defined previously;
  • R 6 is selected from the possibilities for L-R 7 , e.g. is H or alkyl having 1 , 2, 3, 4 or 5 carbon atoms;
  • R 2 is of Formula (Ila): wherein R 5a is -S(0) b -L-R 7 ; -C(0)-N(R 19 )-[S(0) b ] r L-R 7 , -C(0)-L-R 7 ; -C(NR 19 )-L-R 7 ; - C(NR 19 )0-L-R 7 ; -C(NR 19 )NR 30 -L-R 7 ; -C(0)S-L-R 7 ; -C(NR 19 )S-L-R 7 , -C(0)0-L-R 7 , ; 2- pyridyl or 4-pyridyl, wherein 2-pyridyl and 4-pyridyl are unsubstituted or are substituted by one or more substituents independently selected from L-R 7 and R 8 ,
  • L, R 7 , R 8 , R 19 , b and f are each as previously defined, independently of the identity of R 1 ;
  • R 3 and R 4 are each independently H or R 8 ; or R 3 and R 4 taken together form a benzene ring which is unsubstituted or substituted by one or more R 8 ,
  • each -L-R 7 may be the same as or different from any other -L-R 7
  • each R 8 may be the same as or different from any other R 8 .
  • the compounds of the disclosure include chemically permissible structures falling within the described possibilities.
  • the compounds of the disclosure can exist in different forms, such as free acids, free bases, esters and other prodrugs, salts and tautomers, for example, and the disclosure includes all variant forms of the compounds, including those with isotopic substitution by non-normal or radioactive isotopes.
  • the extent of protection includes counterfeit or fraudulent products which contain or purport to contain a compound of the invention irrespective of whether they do in fact contain such a compound and irrespective of whether any such compound is contained in a therapeutically effective amount.
  • packages which include a description or instructions which indicate that the package contains a species or pharmaceutical formulation of the invention and a product which is or comprises, or purports to be or comprise, such a formulation or species. Such packages may be, but are not necessarily, counterfeit or fraudulent.
  • a structural formula herein comprising a chiral centre does not indicate chirality (e.g. where all bonds are shown as lines and there is no "wedge" bond)
  • the structure refers to all corresponding compounds or moieties irrespective of chirality and includes reference to individual compounds or moieties in which the chiral centre is of (R)-configuration, individual compounds or moieties in which the chiral centre is of (S)-configuration and mixtures of (R)- and (S)- isomers as, for example, in the case of racemic mixtures, amongst others.
  • Figure 1 is a pH titration curve for compound 1 .
  • Figure 2 relates to determination of the mode of inhibition of 1 with mouse Nat2. Inhibitory activity was determined by monitoring for rate of AcCoA hydrolysis.
  • Figure 2(a) is Lineweaver-Burk plots showing mNat2 rates of hydrolysis with pABA as arylamine substrate, at inhibitor concentrations of 3 ⁇ (crosses), 1 ⁇ (triangles), 0.5 ⁇ (squares) and ⁇ (diamonds).
  • Figure 2(b) shows slope values from Lineweaver-Burk graphs plotted against concentration of 1 .
  • Figure 3 is a barchart showing percentage inhibition of NAT preparations by compounds of the invention.
  • alkyl in this specification includes linear and branched alkyl groups containing, unless otherwise stated, 1 , 2, 3, 4 or 5 carbon atoms, for example methyl, ethyl, n- propyl, iso-propyl, tert-butyl and n-pentyl.
  • Alkylene groups may likewise be linear or branched and may, for example, correspond to one of those alkyl groups listed in the preceding sentence.
  • the alkyl groups may be substituted by inert substituents, notably halogen.
  • physiological precursors denotes compounds which, when administered to a living cell or organism, are converted to a compound of the disclosure.
  • the term therefore includes prodrugs and compounds which, whilst falling outside the pharmaceutical field, have prodrug structures. Prodrugs are further described later in this specification.
  • phrases "pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings or animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • halogen herein includes reference to F, CI, Br and I, particularly F, CI and Br. In some instances halogen is CI. In one class of compounds, halogen is F.
  • heterocyclic ring refers to a ring system which has at least one (e.g. 1 , 2 or 3) in-ring heteroatoms.
  • heteroatom includes oxygen, sulfur and nitrogen, of which sulfur is sometimes less preferred. Heterocycles may be aromatic or non-aromatic. Examples are thiophene, pyrrole, pyrazole, furan, pyrrolidine, pyrazolidine, imidazoline, pyridine, piperidine, pyrimidine and triazine.
  • heteroaryl refers to a ring system which has at least one (e.g. 1 , 2 or 3) in- ring heteroatoms and has a conjugated in-ring double bond system.
  • heteroatom includes oxygen, sulfur and nitrogen, of which sulfur is sometimes less preferred.
  • Treating" a disease with the compounds discussed herein is defined as administering one or more of the compounds discussed herein, with or without additional therapeutic agents, in order to palliate, ameliorate, stabilize, reverse, slow, delay, reduce, or eliminate either the disease or one or more symptoms of the disease, or to retard or stop the progression of the disease or of one or more symptoms of the disease, when a compound or composition is administered to treat a disease, disorder or occurrence, it does not mean that the treatment will be completely successful, since curative treatments may fail in any individual patient. It should be noted that the use of the compounds and/or methods for treatment and the use of the compounds and/or methods for prevention need not be mutually exclusive.
  • “Therapeutic use” of the compounds discussed herein is defined as using one or more of the compounds discussed herein to treat or prevent a disease, as defined above.
  • a “therapeutically effective amount” of a compound is an amount of the compound, which, when administered to a subject, is sufficient to treat, prevent, reduce, or eliminate either the disease or one or more symptoms of the disease, or to retard the progression of the disease or of one or more symptoms of the disease, or to reduce the severity of the disease or of one or more symptoms of the disease.
  • a “therapeutically effective amount” can be given in one or more administrations.
  • the compounds of the disclosure include those having the structure of Formula I and salts and physiological precursors thereof:
  • X is O or S and Y is O or S.
  • X and Y may be the same or different.
  • X is O and Y is S.
  • X is S and Y is O.
  • X and Y are both S.
  • X and Y are both O.
  • R 1 is R 5 , J, halogen, trifluoromethyl, cyano, nitro, -O-L-R 7 , -S-L-R 7 or -OC(0)-L-R 7 .
  • R 2 is J, -(CH 2 ) e -0-M-R 16 or -(CH 2 ) e -S-M-R 16 , wherein e is 0, 1 , 2, 3 or 4, M is selected from the list of possibilities for L and R 16 is selected from the list of possibilities for R 7 (see below).
  • R 16 is H in one embodiment; in a sub-class -M-R 16 is H. In one embodiment, e is 0; in another embodiment e is not 0, e.g. is 1 or 2.
  • both R 1 and R 2 are J they may be the same or different. In one embodiment, both R 1 and R 2 are J. In one embodiment, R 1 is a moiety not falling within the definition of J whilst R 2 is a J moiety. In another embodiment, both of R 1 and R 2 are moieties not falling within the definition of J.
  • the disclosure includes compounds in which R 2 is -(CH 2 ) e -0-M-R 16 or -(CH 2 ) e -S-M-R 16 , in particular the former, e.g. -(CH 2 ) e OH or -(CH 2 ) e OH, especially -(CH 2 ) e OH, and R 1 is a J moiety or, in other embodiments, is a moiety not falling within the definition of J.
  • R 1 is R 5 , halogen, trifluoromethyl, cyano, nitro, -O-L-R 7 or -OC(O)- L-R 7 .
  • R 1 is therefore halogen in one embodiment, in particular F, CI or Br, e.g. it is F in one class of compounds and CI in another class of compounds.
  • Compounds in which R 1 is CI or Br may be used as intermediates for making other compounds of the disclosure.
  • R 1 is -O-L-R 7 or -OC(0)-L-R 7 , in particular -O-L-R 7 .
  • R 1 is R 5 , in particular H, alkyl having 1 , 2, 3, 4 or 5 carbon atoms and unsubstituted or substituted by hydroxy; or alkyl having 1 , 2, 3 or 4 carbon atoms, unsubstituted or substituted by hydroxy and interrupted by -0-, -S-, or -C(O)-; for example R 1 may be H or Ci -5 alkyl, particularly H.
  • R 1 is an R 5 selected from -S(0) b -L-R 7 ; -C(0)-L-R 7 ; -C(0)0-L-R 7 , or -L-R 7 , wherein R 7 is H or alkyl having 1 , 2, 3, 4 or 5 carbon atoms; for example it is an R 5 selected from - S(0) b -L-R 7 ; -C(0)-L-R 7 ; -C(0)0-L-R 7 .
  • -L-R 7 is in particular H or alkyl optionally substituted by hydroxy and having 1 , 2, 3, 4 or 5 carbon atoms, e.g.
  • -L-R 7 is, for example, a said alkyl group substituted by a 3-7 membered monocyclic ring or by an 8-13 membered bicyclic ring, in particular by a 3-7 membered monocyclic ring. These rings may be unsubstituted or, in another embodiment, substituted as described herein.
  • L is a bond in one class of compounds.
  • L is alkylene which has 1 , 2, 3, 4 or 5 carbon atoms and which is unsubstituted or substituted by hydroxy.
  • L is alkylene having 1 , 2, 3 or 4 carbon atoms and interrupted by, and/or terminated adjacent R 7 by, -0-, -S-, or -C(O)-, in particular -0-.
  • L is alkylene or alkylene interrupted and/or terminated as just described, it may be unsubstituted or it may be substituted by a hydroxy group.
  • L is a said alkylene.
  • L is hydroxyalkylene having 1 , 2, 3, 4 or 5 carbon atoms. Alkylene moieties mentioned in this paragraph may be linear or branched and in particular are linear.
  • R 7 is H, a dye or a 3-7 membered monocyclic ring or an 8-13 membered bicyclic ring, which rings are unsubstituted or are substituted by one or more R 8 .
  • R 7 is H.
  • R 7 is a monocyclic ring having 3, 4, 5, 6 or 7 ring-forming atoms, for example a carbocyclic ring, such as a cycloalkyi ring or a benzene ring, or a heterocyclic ring, in particular a said carbocyclic ring.
  • R 7 is an 8-13 membered bicyclic ring, for example a carbocyclic ring (e.g.
  • R 7 is a ring, it may be unsubstituted or substituted by one or more R 8 , in particular by 1 , 2 or 3 R 8 moieties.
  • R 7 is, independently wherever it occurs, a ring substituted with at least one R 8 .
  • R 7 is, independently wherever it occurs, a benzene ring substituted with at least one R 8 .
  • R 8 is selected from halogen, hydroxy, alkyl optionally substituted by hydroxy and having 1 , 2, 3, 4 or 5 carbon atoms, alkyl optionally substituted by hydroxy and having 1 , 2, 3 or 4 carbon atoms and interrupted by -0-, -S-, or -C(O)-, C 3- 6 carbocyclyl bonded to the remainder of the molecule either directly or through a Ci -4 alkylene group, trifluoromethyl, trifluoromethoxy, cyano, nitro, oxo, amidino, -OR 9 , -C(0)R 9 , -C(0)OR 9 , -OC(0)R 9 , -N(R 9 )R 10 , - C(0)N(R 9 )R 10 , -S(0) a R 9 and -S(0) a OR 9 , wherein R 9 and R 10 are the same or different and are each hydrogen or are selected from alkyl optionally substituted by hydroxy and having 1 ,
  • R 8 does not include trifluoromethoxy.
  • exemplary R 8 moieties are F, CI, Br, methyl, ethyl, n-propyl, iso-propyl, n-butyl, tert-butyl and other Ci -4 alkyl, trifluoromethyl, trifluoromethoxy, methoxy, ethoxy and other Ci -4 alkoxy, hydroxy, hydroxymethyl, hydroxyethyl, nitro, cyano, amino, amino mono- or disubstituted by Ci_ 4 alkyl such as methyl or ethyl, carboxy or carboxy esterified by Ci -5 alkyl.
  • R 8 moieties comprises halogen and moieties containing 1 , 2 or 3 (e.g. 1 or 2) multivalent atoms, for example F, CI, Br, methyl, ethyl, trifluoromethyl, trifluoromethoxy, methoxy, hydroxy, hydroxymethyl, nitro, cyano, amino or methylamino, particularly halogen (e.g. F, CI or Br), methyl or trifluoromethyl.
  • R 8 moieties are selected from halogen (e.g. F, CI or Br), hydroxy, alkyl optionally substituted by hydroxy and having 1 , 2, 3, 4 or 5 carbon atoms (e.g.
  • R 7 is a said ring, it may therefore be substituted by an R 8 moiety mentioned in this paragraph; any one or more ring-forming atoms which is available for substitution by an R 8 moiety may be substituted, e.g. 1 , 2 or 3 such ring-forming atoms.
  • each R 8 may be chosen independently, i.e. any one R 8 may be the same as or different from any other R 8 .
  • R 7 is a dye, for example a fluorescent dye.
  • Fluorescent dyes useful as detectable labels are well known to those skilled in the art and numerous examples can be found in the Handbook of Fluorescent Probes and Research Chemicals 6th Edition, Richard Haugland, Molecular Probes, Inc., 1996 (ISBN 0-9652240-0-7).
  • fluorophores include, but are not limited to, coumarin, acridine, furan, indole, quinoline, cyanine, benzofuran, quinazolinone, benzazole, borapolyazaindacene and xanthenes, with the latter including fluorescein, rhodamine, rhodol, and rosamine.
  • fluorescent dyes incorporated in compounds of the disclosure are environmentally sensitive and undergo a change in absorption/emission properties when in the environment of a protein, e.g. when a compound of the disclosure binds to hNAT1 .
  • a particular class of fluorescent dyes consists of pH-sensitive dyes, for example for example naphthalene dyes.
  • Exemplary fluorescent dyes are dansyl amide, 5- (dimethylamino)naphthalene-l -sulfonamide, and other naphthalene-1 -sulfonamides.
  • R 5 is H; alkyl having 1 , 2, 3, 4 or 5 carbon atoms and unsubstituted or substituted by hydroxy; alkyl having 1 , 2, 3 or 4 carbon atoms, unsubstituted or substituted by hydroxy and interrupted by -0-, -S-, or -C(O)-; -L-R 7 ; -S(0) b -L-R 7 ; -C(0)-N(R 19 )-[S(0) b ] r L-R 7 , -C(0)-L-R 7 ; - C(NR 19 )-L-R 7 ; -C(NR 19 )0-L-R 7 ; -C(NR 19 )NR 30 -L-R 7 ; -C(0)S-L-R 7 ; -C(NR 19 )S-L-R 7 -C(0)0-L-R 7 ; 2-pyridyl or 4-pyridyl, wherein 2-pyridyl
  • substituents independently selected from L-R and R wherein R and R are independently selected from amongst the possibilities for L-R 7 , L and R 7 are as defined previously and R 7 is in particular a said ring (whether unsubstituted or substituted), each R 7 being the same as or different from the other R 7 where there are two R 7 moieties in the compound, and b is 0, 1 or 2. In one embodiment b is 1 . In one embodiment b is 2. In one embodiment, the number of L-R 7 substituents on 2-pyridyl and 4-pyridyl is 0. In one embodiment, the number of L-R 7 substituents on 2-pyridyl and 4-pyridyl is 1 .
  • any R 8 substituents on 2-pyridyl and 4-pyridyl are selected from exemplary R 8 moieties described previously herein.
  • R 5 is H or a said alkyl group. In some compounds, R 5 is H. In some compounds, R 5 is not H. In some compounds, R 5 is -L-R 7 . In some compounds, R 5 is S(0) b -L-R 7 ; -C(0)-L-R 7 ; or -C(0)0-L-R 7 . As examples of -L-R 7 , there may be mentioned groups in which L is a bond and R 7 is as described in the preceding paragraph.
  • -L-R 7 are groups in which L is alkylene having 1 , 2, 3, 4 or 5 carbon atoms and R 7 is as described in the preceding paragraph and is in particular a said ring, as well as groups in which L is a said Ci -5 alkylene substituted by a hydroxy group and R 7 is as described in the preceding paragraph, in particular a said ring.
  • J is a group of Formula (II): wherein
  • R 18 is H or alkyl having 1 , 2, 3, 4 or 5 carbon atoms, in particular H;
  • R 5 is as defined previously;
  • R 6 is selected from the possibilities for L-R 7 , e.g. is H or alkyl having 1 , 2, 3, 4 or 5 carbon atoms, and in particular is H.
  • R 5 may be, for example, -S(0) b -L-R 7 , -C(0)-L-R 7 or -C(0)0-L-R 7 , and particularly - S(0) b -L-R 7 , and b is 1 or 2 and normally 2.
  • Z is -S(0) c - where c is in particular 1 or 2 (e.g.
  • R 5 may be, for example, -L-R 7 , -C(0)-L-R 7 , -C(0)0-L-R 7 or -C(0)-N(R 19 )-S(0) b -L-R 7 , and particularly -L-R 7 , or another moiety described herein as a possibility for R 5 .
  • Z is a bond in one class of compounds.
  • R 1 is a J group.
  • Z is a bond and R 5 is as defined herein and in particular is -L-R 7 .
  • R 6 of R 1 is a J group is in particular H.
  • R 1 may in particular be -N(R 6 )-L- R 7 and especially -NH-L-R 7 .
  • R 1 is an R 5 group which is -L-R 7 .
  • R 1 is an R 5 group which is -L-R 7 , L is a bond and R 7 is a substituted or unsubstituted ring selected from 3-7 membered monocyclic rings and 8-13 membered bicyclic rings optionally substituted with one or more R 8 .
  • R 7 is a benzene ring optionally substituted with one or more R 8 .
  • R 7 is a 5- or 6- membered heteroaromatic ring substituted with one or more R 8 or a bicyclic ring in which a 5- or 6- membered heteroaromatic ring is fused with a benzene ring substituted with one or more R 8 .
  • R 7 is selected from 3- pyridyl, 2-furyl, 3-furyl, 2-benzothiophenyl.
  • R 1 is O-L-R 7 , L is a bond and R 7 is a substituted or unsubstituted ring selected from 3-7 membered monocyclic rings and 8-13 membered bicyclic rings optionally substituted with one or more R 8 .
  • R 7 is a benzene ring optionally substituted with one or more R 8 .
  • R 8 may be selected from, for example, cyano, chloro, trifluoromethoxy, nitro and - C(0)H.
  • R 2 is of Formula (Ila): wherein R 5a is -S(0) b -L-R 7 ; -C(0)-N(R 19 )-[S(0) b ] r L-R 7 , -C(0)-L-R 7 ; -C(NR 19 )-L-R 7 ; -C(NR 19 )0-L- R 7 ; -C(NR 19 )NR 30 -L-R 7 ; -C(0)S-L-R 7 ; -C(NR 19 )S-L-R 7 -C(0)0-L-R 7 ; 2-pyridyl or 4-pyridyl, wherein 2-pyridyl and 4-pyridyl are unsubstituted or are substituted by one or more substituents independently selected from L-R 7 and R 8 .
  • R 5a is -S(0) b -L-R 7 ; -C(0)-N(R 19 )-[S(0) b ] r L
  • any R 8 substituents on 2-pyridyl and 4-pyridyl are selected from exemplary R 8 moieties described in [0038].
  • the compounds in which R 2 is of Formula (I la) are chromogenic, in that they change colour (or potentially become coloured/decoloured) when contacted with hNAT1 .
  • the invention includes an embodiment in which R 2 is of Formula (lla) and in which f is 0.
  • the invention includes an embodiment in which R 2 is of Formula (lla) and in which f is 1 .
  • Index b is for example 1 or 2, and in particular is 2.
  • R 5a is -S(0) b -L-R 7 and in particular is -S(0) 2 -L-R 7 , for example -S(0) 2 -R 7 .
  • R 5a is -C(0)-N(R 19 )-[S(0) b ] r L-R 7 , -C(0)-L-R 7 ; -C(0)S-L-R 7 , or - C(0)0-L-R 7 ; in particular compounds of this sub-class, f is 1 and S(0) b is S(0) 2 .
  • R 5a is -C(NR 19 )-L-R 7 , -C(NR 19 )0-L- R 7 , -C(NR 19 )NR 30 -L-R 7 or -C(NR 19 )S-L-R 7 .
  • R 5a is 2-pyridyl or 4-pyridyl, wherein 2-pyridyl and 4-pyridyl are unsubstituted or are substituted by one or more substituents independently selected from L-R 7 and R 8 .
  • L is a bond in one exemplary and non-limiting class of compounds.
  • R 7 of R 2 is as described herein and is in particular a substituted or unsubstituted ring selected from 3-7 membered monocyclic rings and 8-13 membered bicyclic rings or a dye, for example is phenyl or a 5- or 6- membered heteroaromatic ring (e.g. thiophenyl, particularly 2-thiophenyl) or a fluorescent dye, wherein phenyl and heteroaromatic ring are unsubstituted or are substituted by one or more R 8 , e.g. 1 , 2 or 3 R 8 ; R 8 is as described herein, e.g. selected independently from halogen, methyl and trifluoromethyl.
  • R 7 of R 2 is a substituted or unsubstituted ring selected from 3-7 membered monocyclic rings and 8-13 membered bicyclic rings, for example as mentioned in the preceding paragraph.
  • R 1 is of Formula (lla) as described with reference to R 2 and R 2 is of Formula (lla). In one embodiment, R 1 is not of Formula (lla) and R 2 is of Formula (lla). In one class of compounds, R 2 is of Formula (lla) and R 1 is a J group, in particular a J group in which R 5 is -L-R 7 and in which R 6 is as described herein, e.g. is H; L of -L-R 7 in the J group may in particular be a bond. In one class of compounds, R 2 is of Formula (lla) and R 1 is halogen (e.g.
  • R 2 is of Formula (lla) and R 1 is an R 5 group, e.g. -L-R 7 .
  • R 1 is halogen, e.g. CI or Br, are useful inter alia as intermediates for making other compounds of the disclosure.
  • R 1 and R 2 includes an R 7 which is a substituted or unsubstituted ring selected from 3-7 membered monocyclic rings and 8-13 membered bicyclic rings. Particular rings to mention are benzene and 5- and 6- membered heteroaromatic rings, for example 2-thiophenyl; these rings are unsubstituted or are substituted as previously described in relation to R 7 .
  • R 2 includes a said R 7 and is of Formula (lla).
  • both of R 1 and R 2 include an R 7 which is a substituted or unsubstituted ring selected from 3-7 membered monocyclic rings and 8-13 membered bicyclic rings. Particular rings to mention are benzene and 5- and 6- membered heteroaromatic rings, for example 2-thiophenyl; these rings are unsubstituted or are substituted as previously described in relation to R 7 .
  • R 2 may be of Formula (lla), for example.
  • L is in particular a bond
  • Ci -5 linear alkylene for example methylene or ethylene
  • Ci -4 linear alkylene for example methylene or ethylene
  • said ring may be a 5- or 6- membered carbocyclic or heterocyclic ring and is in particular a benzene ring or a 5- or 6- membered heteroaromatic ring, for example furan, pyrrole, thiophene, pyridine, pyridazine, pyrimidine and pyrazine.
  • L is a bond; in another embodiment, L is not a bond.
  • L is a bond and R 7 is a said ring, whether substituted or unsubstituted, e.g. a benzene ring or a 5- or 6- membered heteroaromatic ring.
  • the -L-R 7 moieties described in this paragraph may, for example, form the ring-containing -L-R 7 moieties mentioned in the following paragraphs [0055] and [0057] and in Tables 1 and 2 below.
  • R 1 and R 2 are both selected from the J groups mentioned in this paragraph, wherein -L-R 7 of R 1 and R 2 may be the same as or different from each other.
  • one of R 1 and R 2 is -S(0) c -N(R 6 )-L-R 7 ; in a first sub-embodiment a single one of R 1 and R 2 is -S(0) c -N(R 6 )-L-R 7 and in a second sub-embodiment both of R 1 and R 2 are - S(0)c-N(R 6 )-L-R 7 .
  • one of R 1 and R 2 is -(CH 2 ) d -N(R 6 )-S(0) b -L-R 7 , in particular -N(R 6 )-S(0) b -L-R 7 ; in a first sub-embodiment a single one of R 1 and R 2 is -(CH 2 ) d - N(R 6 )-S(0) b -L-R 7 and in a second sub-embodiment both of R 1 and R 2 are -(CH 2 ) d -N(R 6 )-S(0) b - L-R 7 .
  • Moiety -L-R 7 may be an embodiment previously described, and it will be recalled here that the invention includes embodiments in which a single one of, or both of, R 1 and R 2 include an R 7 which is a substituted or unsubstituted 3-7 membered monocyclic ring or 8-13 membered bicyclic ring.
  • R 6 is in particular H.
  • the disclosure includes J groups of this paragraph which have one or more of (e.g.
  • R and R are H or R , R and R are H.
  • the disclosure includes J groups of this paragraph in which d is 0, 1 or 2, in particular 0.
  • R 6 , R 19 and R 30 are selected independently of each other and each may be any moiety which falls within the definition of L-R 7 , but R 6 is in particular H for all embodiments of the disclosure.
  • R 6 , R 19 and R 30 may be selected from H, C1-C5 alkyl, phenyl which is unsubstituted or is substituted by one or more R 8 and 5- or 6- membered heteroaryl which is unsubstituted or is substituted by one or more R 8 ,
  • R 6 , R 19 and R 30 are selected from H and alkyl having 1 , 2, 3, 4 or 5 carbon atoms, and in particular are H.
  • R 8 moieties for R 6 , R 19 and R 30 groups which include an R 8 are F, CI, Br, methyl, ethyl, n-propyl, iso-propyl, n-butyl, tert-butyl and other Ci -4 alkyl, trifluoromethyl, trifluoromethoxy, methoxy, ethoxy and other Ci -4 alkoxy, hydroxy, hydroxymethyl, hydroxyethyl, nitro, cyano, amino, amino mono- or disubstituted by Ci -4 alkyl such as methyl or ethyl, carboxy or carboxy esterified by Ci -5 alkyl.
  • R 8 moieties comprises halogen and moieties containing 1 , 2 or 3 (e.g. 1 or 2) multivalent atoms, for example F, CI, Br, methyl, ethyl, trifluoromethyl, trifluoromethoxy, methoxy, hydroxy, hydroxymethyl, nitro, cyano, amino or methylamino, particularly halogen (e.g. F, CI or Br), methyl or trifluoromethyl.
  • 1 , 2 or 3 e.g. 1 or 2
  • multivalent atoms for example F, CI, Br, methyl, ethyl, trifluoromethyl, trifluoromethoxy, methoxy, hydroxy, hydroxymethyl, nitro, cyano, amino or methylamino, particularly halogen (e.g. F, CI or Br), methyl or trifluoromethyl.
  • R 1 is-(CH 2 ) e -0-M-R 16 or -N(R 6 )-L-R 7 , particularly -N(R 6 )-L-R 7
  • R 2 is -S(0) c -N(R 6 )-L-R 7 .
  • R 2 is-(CH 2 ) d -N(R 6 )-S(0) b -L-R 7 , in particular -N(R 6 )-S(0) b -L-R 7 .
  • Moiety - L-R 7 may be an embodiment previously described.
  • Moieties -L-R 7 of R 1 and R 2 may be the same as or different from each other, and it will be recalled here that the invention includes embodiments in which a single one of, or both of, R 1 and R 2 include an R 7 which is a substituted or unsubstituted 3-7 membered monocyclic ring or 8-13 membered bicyclic ring.
  • R 6 is in particular H.
  • the disclosure includes J groups of this paragraph which have one or more of (e.g.
  • R , R and R are H.
  • the disclosure includes J groups of this paragraph in which d is 0, 1 or 2, in particular 0.
  • integer b is in particular 2.
  • Compounds containing a fragment -(SO) b - wherein b is 2 therefore form one embodiment of the invention.
  • integer c is in particular 2.
  • Compounds containing a fragment -(SO) c - wherein c is 2 therefore form an embodiment of the invention also.
  • the invention includes compounds in which R 1 is -N(R 6 )-L-R 7 and R 2 is -NH-S(0) b -L-R 7 , wherein b is 2 and each R 7 may be the same as or different from the other and is in particular phenyl or a 5- or 6-membered heteroaromatic ring which is unsubstituted or substituted by 1 , 2 or 3 R 8 as previously described, in particular where R 8 is halogen, Ci -5 alkyl or trifluoromethyl.
  • L is in particular linear Ci -5 alkylene (e.g. methylene or ethylene) or a bond, and most particularly a bond.
  • structures of Formulae (VII) and (VIII)-(XI) discussed later in this specification may have such R 1 and R 2 groups.
  • R 3 and R 4 are each independently H or R 8 ; or R 3 and R 4 taken together form a benzene ring which is unsubstituted or substituted by one or more R 8 .
  • Exemplary R 8 moieties as described herein in paragraph [0038] are applicable to R 3 and R 4 .
  • each -L-R 7 may be the same as or different from any other -L-R 7 .
  • each R 8 may be the same as or different from any other R 8 .
  • Compounds of the invention therefore include those having the structures of Formulae (lll)-(VI) and the salts and physiological precursors thereof:
  • R 3 and R 4 are the same or different and selected from H and R 8 (e.g. R 3 and R 4 are H, R 3 is H and R 4 is an R 8 , or R 3 is an R 8 and R 4 is H), in particular H, F, CI, hydroxy, alkyl having
  • R 1 and R 2 are as previously defined and, more particularly, may be as defined in Table 1 or Table 2 below, in embodiments of which either a single one of R 1 and R 2 (in one embodiment R 1 ; in another embodiment R 2 ) or both R 1 and R 2 have an R 7 which is an unsubstituted ring or substituted ring as previously described:
  • moiety -(CH 2 ) d -N(R 6 )-S(0) b -L-R 7 on each occasion in Table 1 or Table 2 is -N(R 6 )-S(0) b -L-R 7 , in an embodiment of which R 6 is H.
  • Integer b is 2 in one class of the combinations of Table 1 and Table 2. It will be recalled that integers b and c are 2 in particular classes of the R 1 and R 2 groups in Table 1 and Table 2. It will also be recalled that R 6 , R 11 , R 12 , R 13 , R 14 , R 18 and R 19 are H in some embodiments.
  • L is in particular a bond and R 7 is in particular phenyl or a 5- or 6- membered heteroaromatic ring, which rings may be substituted as described herein or unsubstituted.
  • R 7 is in particular phenyl or a 5- or 6- membered heteroaromatic ring, which rings may be substituted as described herein or unsubstituted.
  • -L-R 7 is H in one class of compounds.
  • Compounds of Table 2 in which R 2 is -NH-2-pyridyl or - NH-4-pyridyl may comprise 1 , 2, 3 or 4 substituents on the pyridyl moiety as previously described in this specification.
  • R -R are each the same as or different from each other and selected from H and R .
  • all of R -R are H.
  • one or two of R -R are R and the remainder are H.
  • the invention also includes structures of Formulae (VIII)-(XI):
  • R u , R I , and " are the same or different and selected from H and R°, in particular H, F, CI, hydroxy, alkyl having 1 , 2, 3, 4 or 5 carbon atoms, hydroxyalkyi having 1 , 2, 3, 4 or 5 carbon atoms, alkyl having 1 , 2, 3 or 4 carbon atoms and interrupted by -0-, -S-, or - C(O)-, hydroxyalkyi having 1 , 2, 3 or 4 carbon atoms and interrupted by -0-, -S-, or -C(O)-, trifluoromethyl, trifluoromethoxy, cyano, nitro, oxo, amidino, -OR 9 , -C(0)R 9 , -C(0)OR 9 , - OC(0)R 9 , -N(R 9 )R 10 , -C(0)N(R 9 )R 10 , -S(0) a R 9 and -S(0) a OR 9 , wherein R
  • R 1 and R 2 are as defined or described in, or with reference to, Table 1 or Table 2 above.
  • R 23 is a hydrogen bond donor, for example -NHR 9 or OH. In an embodiment R 23 is -NH 2 . In certain embodiments R 5a is S0 2 7 [0074] In one embodiment of compounds of Formula (VII)-(XI), and in particular of Formula (VIII), in which R 2 is of Formula (lla), R 23 is a hydrogen bond donor, for example -NHR 9 or OH. In an embodiment R 23 is -NH 2 . In certain embodiments R 5a is S0 2 7 [0074] In one embodiment of compounds of Formula (VII)-(XI), and in particular of Formula
  • R in which R is of Formula (lla), R is not a hydrogen bond acceptor, for example cyano, nitro, -C(0)R 9 , -C(0)OR 9 , -S(0) a OR 9 .
  • R 23 is not nitro.
  • R 5a is S0 2 R 7
  • the disclosure includes compounds in which all of R -R are H or in which one, two, three or four of R -R are R , e.g. one, two or three of them are R as in the case of compounds in which one or two R -R of are R ,and the remainder are H. In some compounds, a single one of R -R is R .
  • Exemplary R moieties as described herein in paragraph [0038] are applicable to Formulae (VI l)-(XI) and embodiments described herein in relation to those Formulae, including by way of example Formulae (XII) and (XIII)-(XVIII) below.
  • R 2 is -NH-S(0) c -L-R 7 ;
  • R 1 may be an R 1 group of Table 1 or another of the possibilities described herein for R 1 .
  • Integer c may be 2.
  • R 2 may therefore be -NH-S(0) 2 -L-R 7 , where L is in particular a bond and/or R 7 is in particular phenyl or a 5- or 6- membered heteroaromatic ring, which rings may be substituted as described herein or unsubstituted.
  • R 2 is -NHS0 2 R 7 and R 1 is -NHR 7 , wherein the R 7 moieties of R 1 and R 2 are the same or different.
  • R 2 is -NHS0 2 R 25 , where R 25 is phenyl or a 5- or 6-membered heteroaromatic ring, for example is phenyl or 2-thiophenyl, in either case unsubstituted or substituted by one or more R 8 (e.g.
  • R 1 is -NHR 26 , where R 26 is phenyl which is unsubstituted or substituted by one or more R 8 (e.g. selected from halogen, alkyl and trifluoromethyl).
  • R 25 groups are unsubstituted phenyl and unsubstituted thiophenyl.
  • Exemplary R 26 groups are unsubstituted phenyl, 4-halophenyl (e.g.
  • R 21 groups comprises 4-methylphenyl and 3,5-dihalophenyl, e.g. where halo is Br.
  • the invention therefore includes compounds of Formula (XII) in which the symbols are as previously defined (so that the two R 7 moieties may be the same or different)
  • the compounds of Formula (XII) are of Formula (XIII) below: wherein R -R are as previously described (i.e. are most broadly defined as each being the same or different and H or R 8 ), and may for example be selected from H, halogen, methyl and trifluoromethyl, and more particularly are all H.
  • R 25 is phenyl or thiophenyl (particularly 2- thiophenyl), and is unsubstituted or substituted by one or more R 8 , e.g. 1 , 2 or 3 R 8 . In one embodiment phenyl and thiophenyl are unsubstituted.
  • phenyl and thiophenyl are substituted by one or more R 8 , in which case R 8 may, for example, be halogen (e.g. F, CI or Br), methyl or trifluoromethyl.
  • R 26 is phenyl which is unsubstituted or substituted by 1 , 2 or 3 R 8 ; in one embodiment phenyl is unsubstituted and, when substituted, R 8 may, for example, be halogen, hydroxy, alkyl optionally substituted by hydroxy and having 1 , 2, 3, 4 or 5 carbon atoms, alkyl optionally substituted by hydroxy and having 1 , 2, 3 or 4 carbon atoms and interrupted by -0-, -S-, or -C(O)-, trifluoromethyl, trifluoromethoxy, cyano or nitro, and in particular R 8 is in this case halogen (e.g. F, CI or Br), methyl or trifluoromethyl.
  • R 8 is in this case
  • the invention also includes compounds of Formula (XIX) in which the symbols are as previously defined (so that the two R 7 moieties ma be the same or different)
  • R -R are as previously described (i.e. are most broadly defined as each being the same or different and H or R 8 ), and may for example be selected from H, halogen, methyl and trifluoromethyl, and more particularly are all H.
  • R 25 is phenyl or thiophenyl (particularly 2- thiophenyl), and is unsubstituted or substituted by one or more R 8 , e.g. 1 , 2 or 3 R 8 . In one embodiment phenyl and thiophenyl are unsubstituted. In another embodiment phenyl and thiophenyl are substituted by one or more R 8 , in which case R 8 may, for example, be halogen (e.g.
  • R 27 is phenyl which is unsubstituted or substituted by 1 , 2 or 3 R ; in one embodiment phenyl is unsubstituted and, when substituted, R may, for example, be halogen, hydroxy, alkyl optionally substituted by hydroxy and having 1 , 2, 3, 4 or 5 carbon atoms, alkyl optionally substituted by hydroxy and having 1 , 2, 3 or 4 carbon atoms and interrupted by -0-, -S-, or -C(O)-, trifluoromethyl, trifluoromethoxy, cyano or nitro, and in particular R 8 is in this case halogen (e.g. F, CI or Br), methyl or trifluoromethyl.
  • a single one of R 25 and R 27 , particularly R 25 is a dye as previously described in this specification.
  • the invention also includes compounds of Formula (XXI) in which the symbols are as previously defined (so that the two R 7 moieties may be the same or different)
  • R -R are as previously described (i.e. are most broadly defined as each being the same or different and H or R 8 ), and may for example be selected from H, halogen, methyl and trifluoromethyl, and more particularly are all H.
  • R 25 is phenyl or thiophenyl (particularly 2- thiophenyl), and is unsubstituted or substituted by one or more R 8 , e.g. 1 , 2 or 3 R 8 . In one embodiment phenyl and thiophenyl are unsubstituted. In another embodiment phenyl and thiophenyl are substituted by one or more R 8 , in which case R 8 may, for example, be halogen (e.g.
  • R is phenyl which is unsubstituted or substituted by 1 , 2 or 3 R 8 ; in one embodiment phenyl is unsubstituted and, when substituted, R 8 may, for example, be halogen, hydroxy, alkyl optionally substituted by hydroxy and having 1 , 2, 3, 4 or 5 carbon atoms, alkyl optionally substituted by hydroxy and having 1 , 2, 3 or 4 carbon atoms and interrupted by -0-, -S-, or -C(O)-, trifluoromethyl, trifluoromethoxy, cyano or nitro, and in particular R 8 is in this case halogen (e.g.
  • a single one of R 25 and R 26 , particularly R 25 is a dye as previously described in this specification. [0085] In some structures (XII), (XIII), (XIV), (XV), (XVI), (XVII), (XVIII), (XIX), (XX), (XXI) and
  • R -R all of R -R are H.
  • Ci-C 4 alkoxy trifluoromethyl, trifluoromethoxy, cyano and nitro, and in particular the or each R 8 is in this case selected from halogen (e.g. F, CI or Br), methyl and trifluoromethyl.
  • halogen e.g. F, CI or Br
  • methyl and trifluoromethyl Compounds having structures (XII), (XIII), (XIV), (XV), (XVI), (XVII) and (XVIII), (XIX), (XX), (XXI) and (XXII) are chromogenic, and these compounds and their salts are therefore included in the compounds of the disclosure which may be used as chromogenic probes to detect the presence or absence of hlMATI .
  • the compounds of the invention bind to hlMATI and are useful to label hlMATI , or to inhibit hlMATI for medical or investigative purposes.
  • Included in the invention are chromogenic compounds in which R 3 and R 4 form a benzene ring, for example naphthoquinones in which X and Y are both O (Formula (VIII)).
  • the chromogenic compounds exhibit an optically detectable response after binding to hlMATI by changing the wavelength of absorbed or emitted light.
  • Compounds of formula (VII) in which R 1 is a halogen may be used as intermediates in the formation of chromogenic compounds of the invention. Such compounds are not themselves choromogenic. Such compounds may, however, exhibit inhibition of hNAT1 .
  • All exemplified compounds exhibited a % inhibition of hNAT1 of greater than 60% within experimental variation, except for those selected from the group comprising: 25, 40, 41 , 42, 43, 44, 46, 47, 48, 70.
  • the observed % inhibition in the case of compound 70 may have been depressed by use of an aged enzyme sample and the true inhibitory activity of compound 70 may therefore be higher than 60%.
  • the group of compounds comprising: 25, 40, 41 , 42, 43, 44, 46, 47, 48, 70 exhibited a exhibited a % inhibition of hlMATI of below 60%, within experimental variation
  • the invention comprises chromogenic compounds in which in which R 2 is of Formula (I la), i.e. is -NHR 5a .
  • the invention provides a method for detecting hlMATI , comprising contacting a sample with a chromogenic compound of the invention to form a labelling composition, incubating the labelling composition for a sufficient amount of time for the compound of the invention to bind to any hlMATI in the sample, illuminating the composition with an appropriate wavelength for the compound and observing for absorbance/emission of light having a wavelength characteristic of the compound when bound to hlMATI .
  • the sample is one which has been taken from, or is derived from, a human subject, particularly a female. Samples may contain one or more human cells or may be a lysate of one or more human cells.
  • the sample may be a lysed biopsy of breast tissue.
  • the invention also provides the ex vivo (e.g. in vitro) use of the chromogenic compounds of the disclosure as chromogenic probes to detect or measure hNAT1 in samples derived from human subjects, in particular breast cancer cells.
  • the samples may be lysates of breast tissue biopsies.
  • the samples may be, or may be derived from, tissue removed from a patient during surgery.
  • a particular benefit of using the chromogenic compounds as chromogenic probes is that the colour change (whether changing from one colour to another, colouration or decolouration) takes place virtually instantaneously (e.g. within a second) upon the compound contacting hNAT1 . This makes the compounds particularly useful for detecting cancerous hNAT1 -bearing cells during surgery.
  • a surgeon may remove a breast tumour and apply a chromogenic compound to the margins of the tumour to establish if the marginal tissue contains cancerous cells or is free of them; if the marginal tissue is free of cancerous cells (no colour change indicative of this), then the surgeon need remove no further tissue but, if the compound undergoes a colour change indicative of cancer cells in the marginal tissue, then further tissue will be removed with a view to ensuring removal of all cancerous cells.
  • the colour change may be determined by eye or using a spectrophotometer.
  • a small sample may be taken of the tissue for the purposes of the chromogenic test.
  • a similar procedure may be applied to auxiliary node tissue removed during surgery.
  • a chromogenic compound of the disclosure may be applied to an area of tissue within the body to determine whether cancerous hNAT1 -bearing cells are present in that area.
  • the invention therefore provides a method of surgical treatment of breast cancer of a patient, comprising:
  • light having a wavelength characteristic of the compound when bound to hNATI is not observed and no further tissue is removed from the patient.
  • light having a wavelength characteristic of the compound when bound to hNATI is observed further tissue is removed from the patient, the further removed tissue has applied to at least a marginal portion thereof a compound of any of claims 18 to 21 , and said at least marginal portion is observed for emission of light having a wavelength characteristic of the compound when bound to hNATI .
  • Also included in the invention is a method of detecting cancer cells during breast cancer surgery, comprising applying to an area of tissue at risk of presenting a cancer cell a chromogenic compound of the disclosure, and observed said area for emission of light having a wavelength characteristic of the compound when bound to hNATI .
  • a chromogenic compound of the disclosure for use in diagnosing tissue during surgery for the presence of cancer cells.
  • the cancer may in particular be ER+ breast cancer or an invasive ductal and/or lobular breast carcinoma.
  • a sample can be illuminated with a wavelength of light selected to give a detectable optical response, and observed with a means for detecting the optical response.
  • Equipment that is useful for illuminating the present compounds and compositions of the invention includes, but is not limited to, hand-held ultraviolet lamps, mercury arc lamps, xenon lamps, lasers and laser diodes. These illumination sources are typically optically integrated into laser scanners, fluorescence microplate readers or standard or microfluorometers.
  • the chromogenic compounds may, at any time after or during an assay, be illuminated with a wavelength of light that results in a detectable optical response, and observed with a means for detecting the optical response.
  • This optical emission is optionally detected by visual inspection, or by use of any of the following devices: CCD cameras, video cameras, photographic film, laser scanning devices, photodiodes, quantum counters, scanning microscopes, flow cytometers, or by means for amplifying the signal such as photomultiplier tubes.
  • the compounds of the disclosure are useful in inhibiting hNATI in the treatment or prevention of disease.
  • the compounds are useful in treating oestrogen receptor positive (ER+) breast cancer.
  • the compounds are useful in treating cancer having upregulated hNAT1 , in particular ER+ breast cancer having upregulated hNATL
  • the compounds are useful in treating invasive ductal and lobular breast carcinomas.
  • the invention therefore includes a method of treating cancer in a human subject, comprising administering a therapeutically effective amount of a compound.
  • the cancer may be breast cancer, in particular ER+ breast cancer or an invasive ductal and/or lobular breast carcinoma.
  • the invention further includes compounds of the disclosure for therapeutic use, particularly for use in methods of treating cancer.
  • the compounds of the disclosure may be administered in the form of pharmaceutically acceptable salts.
  • the pharmaceutically acceptable salts of the present disclosure can be synthesized from the parent compound which contains a basic or acidic moiety by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two; generally, nonaqueous media like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred. Lists of suitable salts are found in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., US, 1985, p. 1418, the disclosure of which is hereby incorporated by reference; see also Stahl et al, Eds, "Handbook of Pharmaceutical Salts Properties Selection and Use", Verlag Helvetica Chimica Acta and Wiley-VCH, 2002.
  • the disclosure thus includes pharmaceutically acceptable salts of the disclosed compounds wherein the parent compound is modified by making acid or base salts thereof, for example the conventional non-toxic salts or the quaternary ammonium salts which are formed, e.g., from inorganic or organic acids or bases.
  • acid addition salts include acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, pamoate, pectinate, persulfate, 3-phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, tos
  • Base salts include ammonium salts, alkali metal salts such as sodium and potassium salts, alkaline earth metal salts such as calcium and magnesium salts, salts with organic bases such as dicyclohexylamine salts, N-methyl-D-glucamine, and salts with amino acids such as arginine, lysine, and so forth.
  • the basic nitrogen-containing groups may be quaternized with such agents as lower alkyl halides, such as methyl, ethyl, propyl, and butyl chloride, bromides and iodides; dialkyl sulfates like dimethyl, diethyl, dibutyl; and diamyl sulfates, long chain halides such as decyl, lauryl, myristyl and stearyl chlorides, bromides and iodides, aralkyl halides like benzyl and phenethyl bromides and others.
  • lower alkyl halides such as methyl, ethyl, propyl, and butyl chloride, bromides and iodides
  • dialkyl sulfates like dimethyl, diethyl, dibutyl
  • diamyl sulfates long chain halides
  • the invention includes prodrugs for the active pharmaceutical species of the invention, for example in which one or more functional groups are protected or derivatised but can be converted in vivo to the functional group, as in the case of esters of carboxylic acids convertible in vivo to the free acid, or in the case of protected amines, to the free amino group.
  • prodrug represents in particular compounds which are rapidly transformed in vivo to the parent compound, for example, by hydrolysis in blood.
  • Prodrugs therefore include drugs having a functional group which has been transformed into a reversible derivative thereof. Typically, such prodrugs are transformed to the active drug by hydrolysis. As examples may be mentioned the following:
  • Carboxylic acid Esters including e.g. acyloxyalkyl esters, amides
  • Alcohol Esters including e.g. sulfates and phosphates as
  • Amine Amides carbamates, imines, enamines,
  • Prodrugs also include compounds convertible to the active drug by an oxidative or reductive reaction. As examples may be mentioned:
  • Groups referred to or featured herein may exist in tautomeric forms and all these tautomers are included in the scope of the disclosure. More generally, many species may exist in equilibrium, as for example in the case of organic acids and their counterpart anions; a reference herein to a species accordingly includes reference to all equilibrium forms thereof.
  • the compounds of the disclosure may also contain one or more asymmetric carbon atoms and may therefore exhibit optical and/or diastereoisomerism. All diastereoisomers may be separated using conventional techniques, e.g. chromatography or fractional crystallisation. The various stereoisomers may be isolated by separation of a racemic or other mixture of the compounds using conventional, e.g. fractional crystallisation or HPLC, techniques. Alternatively the desired optical isomers may be made by reaction of the appropriate optically active starting materials under conditions which will not cause racemisation or epimerisation, or by derivatisation, for example with a homochiral acid followed by separation of the diastereomeric derivatives by conventional means (e.g. HPLC, chromatography over silica).
  • HPLC chromatography over silica
  • Geometric isomers may also exist in the compounds of the present disclosure.
  • the present disclosure contemplates the various geometric isomers and mixtures thereof resulting from the arrangement of substituents around a carbon-carbon double bond and designates such isomers as of the Z or E configuration, wherein the term "Z" represents substituents on the same side of the carbon-carbon double bond and the term "E” represents substituents on opposite sides of the carbon-carbon double bond.
  • Compounds containing one or more asymmetric carbon atoms can exist as two or more stereoisomers. Where a compound contains an alkenyl or alkenylene group, geometric cis/trans (or Z/E) isomers are possible. Where structural isomers are interconvertible via a low energy barrier, tautomeric isomerism ('tautomerism') can occur. This can take the form of proton tautomerism in compounds containing, for example, an imino, keto, or oxime group, or so-called valence tautomerism in compounds which contain an aromatic moiety. It follows that a single compound may exhibit more than one type of isomerism.
  • Cis/trans isomers may be separated by conventional techniques well known to those skilled in the art, for example, chromatography and fractional crystallisation.
  • the racemate (or a racemic precursor) may be reacted with a suitable optically active compound, for example, an alcohol, or, in the case where the compound of formula (I) contains an acidic or basic moiety, a base or acid such as 1 -phenylethylamine or tartaric acid.
  • a suitable optically active compound for example, an alcohol, or, in the case where the compound of formula (I) contains an acidic or basic moiety, a base or acid such as 1 -phenylethylamine or tartaric acid.
  • the resulting diastereomeric mixture may be separated by chromatography and/or fractional crystallization and one or both of the diastereoisomers converted to the corresponding pure enantiomer(s) by means well known to a skilled person.
  • racemic compounds such as the racemic compound (true racemate) referred to above wherein one homogeneous form of crystal is produced containing both enantiomers in equimolar amounts.
  • the second type is the racemic mixture or conglomerate wherein two forms of crystal are produced in equimolar amounts each comprising a single enantiomer.
  • both of the crystal forms present in a racemic mixture have identical physical properties, they may have different physical properties compared to the true racemate. Racemic mixtures may be separated by conventional techniques known to those skilled in the art - see, for example, "Stereochemistry of Organic Compounds" by E. L. Eliel and S. H. Wilen (Wiley, 1994).
  • the present disclosure includes all pharmaceutically acceptable isotopically-labelled compounds) 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 which predominates in nature.
  • isotopes suitable for inclusion in the compounds of the disclosure include
  • hydrogen such as H and H
  • carbon such as C, C and C
  • chlorine such as 36 CI
  • fluorine such as 18 F
  • iodine such as 123 l and 125 l
  • nitrogen such as 13 N and 15 N
  • oxygen such as 15 0, 17 0 and 18 0, phosphorus, such as 32 P
  • sulphur such as 35 S.
  • the disclosure therefore includes all variant forms of the defined compounds, for example any tautomer or any pharmaceutically acceptable salt, ester, acid or other variant of the defined compounds and their tautomers as well as substances which, upon administration, are capable of providing directly or indirectly a compound as defined above or providing a species which is capable of existing in equilibrium with such a compound.
  • the compounds may be administered alone or as compositions in combination with pharmaceutically acceptable diluents, excipients or carriers.
  • the compounds of the invention will normally be administered orally, 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 prodrug or active 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 orally or parenterally ("parenterally” as used herein, refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion.) to a host to obtain an protease- inhibitory effect.
  • parenterally refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion.
  • the compounds of the invention may be used in monotherapy or combined or coadministered with another active pharmaceutical ingredient, particular one useful in treating cancer patients.
  • the compounds of the invention may be combined or coadministered with another active agent for use in treating cancer, e.g.
  • breast cancer in particular treating cancer having upregulated hlMATI , in particular ER+ breast cancer such as, for example, ER+ breast cancer having upregulated hlMATI .
  • the other active agent is useful in treating invasive ductal and lobular breast carcinomas.
  • 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 for to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved.
  • Envisaged suitable daily doses of the compounds of the invention in therapeutic treatment of humans are about 0.001 -100 mg/kg body weight at peroral administration and 0.001 -50 mg/kg body weight at parenteral administration.
  • a peroral dose of from 0.02 to 15 mg/Kg of body weight is envisaged.
  • the active compound may be given as a single dose, in multiple doses or as a sustained release formulation.
  • composition including a compound of the invention, in admixture with a pharmaceutically acceptable adjuvant, diluent or carrier.
  • compositions of this invention for parenteral injection suitably comprise pharmaceutically acceptable sterile aqueous or nonaqueous 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 nonaqueous 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.
  • Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants.
  • compositions may also contain adjuvants such as preservative, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol 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 aluminum monostearate and gelatin) which delay absorption.
  • adjuvants such as preservative, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol 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
  • oral formulations contain a dissolution aid.
  • the dissolution aid is not limited as to its identity so long as it is pharmaceutically acceptable. Examples include nonionic surface active agents; bile acid and salts thereof; ionic surface active agents; triethanolamine soap, and alkyl quaternary ammonium salts; and amphoteric surface active agents.
  • 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.
  • the active compounds may also be in micro-encapsulated 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 micronised.
  • 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.
  • the oral compositions may also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring 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, aluminum metahydroxide, bentonite, agar-agar, and tragacanth and mixtures thereof.
  • suspending agents such as ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar, and tragacanth and mixtures thereof.
  • compositions for rectal or vaginal administration are preferably 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.
  • liposomes are generally derived from phospholipids or other lipid substances. Liposomes are formed by mono- or multi-lamellar hydrated liquid crystals which are dispersed in an aqueous medium. Any non-toxic, physiologically acceptable and metabolisable lipid capable of forming liposomes can be used.
  • the present compositions in liposome form can contain, in addition to a compound of the present invention, stabilisers, preservatives, excipients and the like. Particular lipids are the phospholipids and the phosphatidyl cholines (lecithins), both natural and synthetic. Methods to form liposomes are known in the art, for example, Prescott, Ed., Methods in Cell Biology, Volume XIV, Academic Press, New York, N.Y. (1976), p 33 et seq.
  • the compounds of the disclosure may be made by applying standard synthetic procedures to commercially available quinones or thioxoquinones, for example 2,3,5,6- tetrachloro-1 ,4-benzoquinone, 2-amino-3-chloro-1 ,4-naphthoquinone, methyl-p-benzoquinone, sodium 3-hydroxy-1 ,4-naphthoquinone-2-sulfonate, 2, 3-dihydoroxy-1 ,4-naphthoquinone, 2- alkylamino-3-alkylthio-1 ,4-naphthoquinones, 3-alkyl-2-hydroxy-1 ,4-naphthoquinones, disodium-1 ,4-dioxo-1 ,2,3,4-tetrahydro-2,3-naphthalenedisulfonate, 2-bromo-1 ,4- benzoquinone, 2, 3-dichloro-6-nitro-1
  • 2, 3-dichloronaphthalene-1 ,4-dione is used as a starting material for successive steps of nucleophilic aromatic substitution of the quinone ring.
  • a first step 2, 3-dichloronaphthalene-1 ,4-dione is contacted with a sulphonamide R 31 S0 2 NH 2 in the presence of a base to carry out a nucleophilic substitution reaction, thereby replacing a chlorine substituent with the sulphonamide to obtain a 2-sulphonamido-3-chloronaphthalene- 1 ,4-dione.
  • the reaction is suitably performed in an aprotic solvent, e.g.
  • a non-polar organic solvent for example toluene.
  • an aprotic polar solvent may be used.
  • the reaction is suitably carried out at elevated temperature, e.g. under reflux.
  • a further nucleophilic aromatic substitution is then carried out to replace the second chlorine, for example by contacting the 2-sulphonamido-3-chloronaphthalene-1 ,4-dione with an amine, e.g. a primary amine, in the presence of a Lewis acid (e.g. CeCI 3 ).
  • the reaction is suitably performed in a polar solvent and particularly an aqueous solvent, for example water.
  • the reaction is suitably carried out at elevated temperature, e.g. under reflux.
  • R is a fragment of a sulphonamide substituent R 1 as disclosed herein such that the product compound is a compound of the disclosure, e.g. it may be a moiety R 25 as defined elsewhere in this specification.
  • R 32 is a fragment of an R 2 substituent as disclosed herein such that the product compound is a compound of the disclosure, e.g. it may be a moiety R 26 as defined elsewhere in this specification.
  • the reaction is illustrated by the following reaction scheme (A):
  • reaction scheme B the 2-sulphonamido-3-chloronaphthalene-1 ,4-dione is coupled with a coupling agent (e.g. an aryl boronic acid) in the presence of a transition metal or transition metal containing compound (e.g. Pd or a palladium-containing compound such as Pd(PPh 3 ) 2 CI 2 ).
  • a coupling agent e.g. an aryl boronic acid
  • a transition metal or transition metal containing compound e.g. Pd or a palladium-containing compound such as Pd(PPh 3 ) 2 CI 2
  • reaction scheme B The reaction is suitably performed in the presence of a base.
  • the reaction may be performed in a polar aprotic solvent (eg. THF) or a polar protic solvent (eg. H 2 0).
  • the reaction is suitably carried out at elevated temperature, e.g. under reflux.
  • the reaction is illustrated by the following reaction scheme (B):
  • 3-dichloronaphthalene-1 ,4-dione is used as a starting material for successive steps of double chloride displacement with phenol and the displacement of one equivalent of phenol with a sulphonamide.
  • a double substitution reaction occurs in which 2, 3-dichloronaphthalene-1 ,4-dione is contacted with phenol in the presence of a base in an addition-elimination reaction thereby replacing the two chlorine substituents with two equivalents of a phenol.
  • the reaction is suitable carried out in an aprotic polar organic solvent, such as THF.
  • the reaction is suitably carried out at elevated temperature, e.g. under reflux.
  • the diphenol substituted naphalene is contacted with a sulphonamide in the presence of a base to carry out a nucleophilic
  • R 1 ,R 2 , R 3 and R 4 of reaction schemes A, B, and C are fragments such that the compound is a compound of the invention.
  • RP-HPLC Reverse-phase High Performance Liquid Chromatography
  • NMR spectra were recorded on Bruker Avance spectrometers in the deuterated solvent stated. Chemical shifts ( ⁇ ) are reported in parts per million (p. p.m.) and are referenced to the residual solvent peak. Coupling constant (J) are measured in Hertz and are calculated using a first-order approximation. Low resolution mass spectra (m/z) were recorded on either VG MassLab 20-250 or Micromass platform 1 spectrometer.
  • Compound 1 changes color from red to blue when it binds to mNat2 or hlMATI . Some other compounds do the same. This property is useful to provide information about the presence and/or amount of human NAT1 in a sample, for example a cell lysate mixture, and the a diagnostic kit for ER+ breast cancer.
  • Example 14 yV-(3-([1 ,1 '-Biphenyl]-4-yl)-1 ,4-dioxo-1 ,4-dihydronaphthalen-2-yl)benzenesulfonamide 14 [00169] Following General Procedure 3, /V-(3-chloro-1 ,4-dioxo-1 ,4- dihydronaphthalen-2-yl)benzenesulfonamide 3 (200 mg, 0.58
  • silica gel (eluent pet etheracetone 75:25) and subsequent
  • silica gel (eluent pet ethenacetone 75:25) and subsequent
  • DMSO-de 1 16.7, 1 18.0, 126.9, 129.7, 131 .4, 132.0, 133.0, 133.3, 133.5,
  • mouse Nat2 (mNat2) has 80% identity in amino acid sequence compared to the hlMATI isoenzyme and is easily expressed and purified as recombinant protein, the
  • mNat2 was expressed in E. coli RosettaBlue(DE3)pLysS transfected with pET28b(+) plasmid containing the mNat2 gene.
  • the expressed mNat2 with an N-terminal hexa-histidine tag was purified by ion-metal affinity chromatography. Pure recombinant mNat2 was purified with a yield of 57 mg per litre of culture according to the method previously described (Kawamura, A. et al. Biochem Pharmacol, 2008. 75(7), 1550-1560).
  • a 2mM stock solution of each of compounds 1 , 5-8 and 1 1 was prepared in DMSO.
  • the colour change test of compounds 1 , 5-8 was performed with a 2 mM inhibitor solution in DMSO (50 l_) with 10 ⁇ _ of buffer (20mM Tris.HCI, pH 8.0), as control or with 10 ⁇ _ of 4 M NaOH solution to observe the colour change.
  • Absorption spectra were recorded with a U-2001 spectrophotometer (Hitachi) from 800 to 250 nm.
  • a dose-response function was used as a model of regression for the data and the curves were estimated by the method of the least squares analysis. All the statistical tests were performed with Kyplot® software. 95% was chosen as confidence limit for these tests.
  • the quoted rates of reaction are initial rates determined from the linear initial section of graphs of absorbance versus time, assays were conducted in triplicate, and the rates and kinetic parameters quoted in this study are mean values ⁇ standard deviation. Since different sources of enzymes were used for activity assays, different protein concentrations and incubation times were selected to give a linear initial rate.
  • DTNB Immediately prior to use, pure mNAT2 solution were diluted in assay buffer (20 mM Tris.HCI, pH8.0). Samples of pure mNAT2 (5 ⁇ _ containing 50 ng protein) were preincubated with pABA (50 ⁇ _ of 1 mM stock solution, 500 ⁇ final concentration) in assay buffer for 5 min at 37 °C in a 96-well plate (Greiner). Pre-warmed (37 °C) AcCoA (40 ⁇ _ of 1 mM stock solution, 400 ⁇ final concentration) in assay buffer was added to start the reaction (final volume of 100 ⁇ _), which was allowed to proceed at 37 °C.
  • assay buffer 20 mM Tris.HCI, pH8.0.
  • DMAB Samples of enzyme (7.25 ⁇ _ containing 18 ng recombinant mNAT2 or 7.25 ⁇ _ containing 54.4 ng recombinant hlMATI ) and 4-aminobenzoic acid (pABA) (15 ⁇ _ of 450 ⁇ stock solution, 150 ⁇ final concentration) were pre-incubated at 37 °C in assay buffer (20 mM Tris.HCI, pH 8.0) for 5 min in a 96-well plate (Greiner).
  • assay buffer (20 mM Tris.HCI, pH 8.0
  • Pre-warmed AcCoA (20 ⁇ _ of 900 ⁇ stock solution, 400 ⁇ final concentration, 37 °C) in assay buffer was added to start the reaction (final volume of 45 ⁇ _), which was allowed to proceed at 37 °C.
  • the reaction was quenched with 45 ⁇ _ of 20 % (w/v) aqueous trichloroacetic acid (TCA) solution at 0 °C.
  • 150 ⁇ _ of DMAB (5% (w/v) in 90% aqueous CH 3 CN solution) was added and the absorbance was measured immediately at 450 nm in the 96-well micro plate using a plate reader (Tecan Sunrise), and the concentration of arylamine was calculated by reference to a standard curve.
  • Inhibitor assays The assays to determine NAT inhibition were carried out using the 'DTNB' method for pure recombinant mNAT2 and the 'DMAB' method for both pure recombinant mNAT2 and hlMATI . All the tested compounds were dissolved in DMSO and the final percentage of DMSO in the assay was 5 % (v/v). Inhibition percentages were determined as ratio of specific activity without the requisite inhibitor over specific activity with the inhibitor. The above described 'DTNB' assay was carried out with 30 ⁇ final inhibitor concentration and the 'DMAB' assays were conducted with 5 ⁇ final inhibitor concentration.
  • IC 50 s were estimated graphically from plots of absorbance or specific activities versus inhibitor concentrations (30, 10, 5, 2.5, 1 .25, 0.625, 0.3125, 0.15625 ⁇ ) by using GraphPad® software.
  • a dose-response function was used as a model of regression for the data and the curves were estimated by the method of the least squares analysis.
  • the activity was determined by the AcCoA hydrolysis assay.
  • the substrate 5- aminosalysilic acid 500 ⁇ was incubated with the appropriate NAT enzyme (100 ng), at 37 ° C for 5 minutes, with or without the inhibitor 7 (10 ⁇ ).
  • Pre-warmed AcCoA 400 ⁇ was added to start the reaction (final volume 100 ⁇ _).
  • Simultaneous quenching and colour development was achieved by addition of the stop reagent: 25 ⁇ _ DTNB solution (5mM DTNB in 100 mM Tris.HCI, 6.4 M guanidine.HCI, pH 7.3).
  • the absorbance was read at 405 nm immediately afterwards. Assays were conducted in triplicate, and the linear activity rates are mean values ⁇ standard deviation.
  • STNAT NAT from Salmonella typhimurium
  • MMNAT NAT from Mycobacterium marinum
  • MSNAT NAT from Mycobacterium smegmatis
  • PANAT NAT from Pseudomonas aeruginosa, were used as NAT controls
  • Percentage of inhibition was determined as the ratio of specific activity without the inhibitor over specific activity with inhibitor (100%). IC 50 s were estimated graphically from plots of specific activities versus inhibitor concentration by using Kyplot® software. A dose-response function was used as a model of regression for the data and the curves were estimated by the method of the least squares analysis. All the statistical tests were performed with Kyplot® software. 95% was chosen as confidence limit for these tests.
  • This example compares the amino acid sequences and substrate specificities in human and mouse NAT isoenzymes. Percentage inhibition at 30 ⁇ and color change of compound 1 versus NAT isoenzymes.
  • CoA Coenzyme A
  • DTNB colorimetric agent 5,5'-dithio-bis(2-nitrobenzoic acid)
  • This assay was used only with pure recombinant NATs and the appropriate arylamine substrate was chosen according to Westwood et al. (Westwood, I. M.; Kawamura, A.; Fullam, E.; Russell, A. J.; Davies, S. G.; Sim, E. Curr. Med. Chem. 2006, 1641 .)
  • mNat2 (5 ng) was preincubated with pABA (500 ⁇ ) in assay buffer (see above) for 5 min at 37 °C in a 96-well plate (Corning).
  • Pre-warmed (37 °C) AcCoA (400 ⁇ ) in assay buffer was added to start the reaction (final volume of 100 ⁇ _), which was allowed to proceed at 37 °C.
  • Simultaneous quenching and colour development was achieved by addition of the stop reagent: 25 ⁇ _ DTNB solution (5mM DTNB in 100 mM Tris.HCI, 6.4 M guanidine.HCI, pH 7.3).
  • the absorbance was read immediately after addition of the stop reagent at 405 nm using a plate reader (Tecan Sunrise). The rate of reaction was determined by reference to a standard curve.
  • FIG. 3 shows the results of screening of compounds 5-8 (5 ⁇ ) against pure mNat2 (black columns), hNAT1 in Escherichia coli cell lysate (grey columns), and hNAT1 in ZR-75-1 breast cancer cell lysate (1 and 7 only, white columns).
  • NAT activity was determined as acetylation of pABA in the presence of AcCoA.

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Abstract

The invention provides 1,4-quinones, 1,4-naphthoquinones and their sulphur analogues as inhibitors of hNAT1, an enzyme which is both a diagnostic marker and drug target for breast cancer. Some of the compounds of the invention are also chromogenic in the presence of hNAT1. The compounds of the invention are used for detecting for and/or measuring hNAT1 and for inhibiting hNAT1 in the treatment of disease.

Description

1 ,4 Quinones and their Sulfur Analogues useful as Ligands of /V-Acetyltransferases
[0001] This invention relates to ligands for arylamine /V-acetyltransferases (NATs). More particularly, it relates to compounds useful as probes for NATs and compounds useful as antagonists for NATs, as well as the use thereof. The invention further relates to preparing the described products, compositions comprising them and other subject matter.
BACKGROUND
[0002] Arylamine /V-acetyltransferases (NATs) are drug metabolising enzymes which are found in a wide range of prokaryotic and eukaryotic species. NATs catalyse the transfer of an acetyl group from acetyl coenzyme A (AcCoA) to the amine of a variety of arylamines. Human NAT1 (hNAT1 ) is one of the ten most highly overexpressed genes in oestrogen receptor- positive (ER+) breast tumours. hNAT1 is upregulated in some ER+ tumours compared with ER-negative tumours, and hence is identified as a putative ER-responsive gene. This suggests that NAT1 plays a biological role in breast cancer progression and it has therefore emerged as a diagnostic marker and drug target for breast cancer. hNAT1 has been generated in a pure form and its substrate specificity profile has been demonstrated to be very similar to that of mouse NAT2 (Kawamura A D. Phil. Thesis, University of Oxford 2005; Wang H et al. Protein J. 2005, 24, 55; Kawamura A et al. Biochem. Pharmacol. 2008, 75, 1550).
[0003] Russell A J et al. Bioorg. Med. Chem. 2009, 77, 905-918 is included herein by reference and describes selective small molecule inhibitors of hNAT1 and its murine homologue, mouse arylamine /V-acetyltransferase 2 (mNAT 2). The inhibitors are 5- (benzylidene)-2-thioxothiazolidinones and analogues thereof, in particular analogues in which the benzene ring of the benzylidene moiety is replaced by naphthyl or another hydrocarbyl moiety containing 6 or more carbon atoms.
[0004] 1 ,4- Quinones and 1 ,4-naphthoquinones have the following respective ring skeletons optionally substituted one or more times
Figure imgf000002_0001
BRIEF SUMMARY OF THE DISCLOSURE
[0005] The present invention provides in one aspect compounds useful as probes to detect NATs, as inhibitors of NATs, or as both probes and inhibitors of NATs. [0006] The invention therefore provides in one aspect the use of 1 ,4-quinones, in particular 1 ,4-naphthoquinones, and their sulfur analogues in which one or both dione oxo groups are replaced by thioxo groups, as probes and/or inhibitors of hNATL Some of said 1 ,4-quinones and their mono-and di-thioxo analogues are chromogenic in the presence of hNAT1 , in that they change colour (or potentially become coloured/decoloured) when contacted with hNATL The invention includes methods of detecting for and/or measuring hlMATI comprising contacting a sample with a said chromogenic 1 ,4-quinone or a thioxo analogue thereof. The invention also includes methods of inhibiting hlMATI in the treatment of disease, for example in delaying the progression of disease, comprising administering to a subject a therapeutically effective amount of a said 1 ,4-quinone or a thioxo analogue thereof. Further included in the invention are compounds selected from those having a structure disclosed herein and salts and prodrugs thereof, for use in inhibiting hlMATI in the treatment of disease. Said compounds may be substituted at least at the 2- and 3- positions; for example the substituents at the 2- and 3- positions may be the same or different and selected from residues comprising a functional group capable of forming hydrogen bonds or ionic bonds and, spaced from the (thioxo)quinone ring, a ring.
[0007] In particular, the invention provides, amongst other things, chromogenic compounds which bind selectively to hlMATI and change colour, or become coloured/decoloured, on binding thereto. In methods of the invention, such a compound is contacted with a sample, in particular one containing one or more intact or lysed human cells, to form a mixture, and the mixture is observed in order to detect development/alteration of any colour characteristic of the compound when bound to hNATL In some methods, the intensity of the light absorbed or emitted at a wavelength characteristic of the hNATI -bound compound is used to measure the amount of hNAT1 .
[0008] In an aspect, the invention provides compounds having the structure of Formula I and salts and physiological precursors thereof:
Figure imgf000003_0001
wherein:
X is O or S;
Y is O or S; R1 is R5, J, halogen, trifluoromethyl, cyano, nitro, -O-L-R7, -S-L-R7 or -OC(0)-L-R7; wherein:
L is a bond, alkylene optionally substituted by hydroxy and having 1 , 2, 3, 4 or 5 carbon atoms and unsubstituted or substituted by hydroxy, alkylene optionally substituted by hydroxy and having 1 , 2, 3 or 4 carbon atoms and interrupted and/or terminated adjacent R7 by -0-, -S-, or -C(O)-;
R7 is H or a 3-7 membered monocyclic ring or an 8-13 membered bicyclic ring, which rings are unsubstituted or are substituted by one or more R8, wherein:
R8 is selected from halogen, hydroxy, alkyl optionally substituted by hydroxy and having 1 , 2, 3, 4 or 5 carbon atoms, alkyl optionally substituted by hydroxy and having 1 , 2, 3 or 4 carbon atoms and interrupted by -0-, -S-, or -C(O)-, a 3 to 6- membered ring bonded to the remainder of the molecule either directly or through a Ci-4 alkylene group, trifluoromethyl, trifluoromethoxy, cyano, nitro, oxo, amidino, -OR9, -C(0)R9, -C(0)OR9, -OC(0)R9, -N(R9)R10, -C(0)N(R9)R10, - S(0)aR9 and -S(0)aOR9, wherein R9 and R10 are the same or different and are each hydrogen or are selected from alkyl optionally substituted by hydroxy and having 1 , 2, 3, 4 or 5 carbon atoms and a 3 to 6- membered ring bonded to the remainder of the molecule either directly or through a Ci-4 alkylene group, and a is 0, 1 or 2;
R5 is H; alkyl having 1 , 2, 3, 4 or 5 carbon atoms and unsubstituted or substituted by hydroxy; alkyl having 1 , 2, 3 or 4 carbon atoms, unsubstituted or substituted by hydroxy and interrupted by -0-, -S-, or -C(O)-; -L-R7; -S(0)b-L-R7; -C(0)-N(R19)-[S(0)b]rL-R7, -C(0)-L-R7; -C(NR19)-L-R7; -C(NR19)0-L-R7; -C(NR19)NR30-L-R7; -C(0)S-L-R7; -C(NR19)S- L-R7 -C(0)0-L-R7 ; 2-pyridyl or 4-pyridyl, wherein 2-pyridyl and 4-pyridyl are unsubstituted or are substituted by one or more substituents independently selected
7 8 1 Q
from L-R and R , wherein R and R are independently selected from the possibilities for L-R7 , L and R7 are as defined previously, and b is 0, 1 or 2;
J is a group of Formula (II):
Figure imgf000004_0001
wherein
Z is a bond, -S(0)c-, -S(0)c-NR18-C(0)-, -C(O)-, -(CH2)d-, -N=C(NR11R12)- or -NR11CR13R14-, wherein c is 0, 1 or 2; d is 1 , 2, 3 or 4; R11 and R12 are the same or different and are each H or alkyl having 1 , 2, 3 or 4 carbon atoms; and R and R are the same or different and are each H or alkyl having 1 , 2, 3 or 4 carbon atoms, or R13 and R14 taken together form =NR15, =0 or =S, wherein R15 is hydrogen, hydroxy or alkyl optionally substituted by hydroxy and having 1 , 2, 3, 4 or 5 carbon atoms, and R18 is H or alkyl having 1 , 2, 3, 4 or 5 carbon atoms;
R5 is as defined previously; and
R6 is selected from the possibilities for L-R7, e.g. is H or alkyl having 1 , 2, 3, 4 or 5 carbon atoms; and
R2 is J, wherein if both R1 and R2 are J they may be the same or different, -(CH2)e-0-M-R16 or -(CH2)e-S-M-R16, wherein e is 0, 1 , 2, 3 or 4, M is selected from the list of possibilities for L and R16 is selected from the list of possibilities for R7;
R3 and R4 are each independently H or R8;
or R3 and R4 taken together form a benzene ring which is unsubstituted or substituted by one or more R8,
wherein where there is there is more than one -L-R7, each -L-R7 may be the same as or different from any other -L-R7, and where there is more than one R8, each R8 may be the same as or different from any other R8. The identities of R6, R19 and R30 are chosen independently of each other and independently of the identity of the or each -L-R7
[0009] In an aspect, the invention provides compounds having the structure of Formula l(i) and salts and physiological precursors thereof:
Figure imgf000005_0001
wherein:
X is O or S;
Y is O or S;
R1 is R5, J, halogen, trifluoromethyl, cyano, nitro, -O-L-R7, -S-L-R7 or -OC(O)
wherein: L is a bond, alkylene optionally substituted by hydroxy and having 1 , 2, 3, 4 or 5 carbon atoms and unsubstituted or substituted by hydroxy, alkylene optionally substituted by hydroxy and having 1 , 2, 3 or 4 carbon atoms and interrupted and/or terminated adjacent R7 by -0-, -S-, or -C(O)-;
R7 is H or a 3-7 membered monocyclic ring or an 8-13 membered bicyclic ring, which rings are unsubstituted or are substituted by one or more R8, wherein:
R8 is selected from halogen, hydroxy, alkyl optionally substituted by hydroxy and having 1 , 2, 3, 4 or 5 carbon atoms, alkyl optionally substituted by hydroxy and having 1 , 2, 3 or 4 carbon atoms and interrupted by -0-, -S-, or -C(O)-, a 3 to 6- membered ring bonded to the remainder of the molecule either directly or through a Ci-4 alkylene group, trifluoromethyl, cyano, nitro, oxo, amidino, -OR9, -C(0)R9, -C(0)OR9, -OC(0)R9, -N(R9)R10, -C(0)N(R9)R10, -S(0)aR9 and - S(0)aOR9, wherein R9 and R10 are the same or different and are each hydrogen or are selected from alkyl optionally substituted by hydroxy and having 1 , 2, 3, 4 or 5 carbon atoms and a 3 to 6- membered ring bonded to the remainder of the molecule either directly or through a Ci-4 alkylene group, and a is 0, 1 or 2;
R5 is H; alkyl having 1 , 2, 3, 4 or 5 carbon atoms and unsubstituted or substituted by hydroxy; alkyl having 1 , 2, 3 or 4 carbon atoms, unsubstituted or substituted by hydroxy and interrupted by -0-, -S-, or -C(O)-; -L-R7; -S(0)b-L-R7; -C(0)-N(R19)-[S(0)b]rL-R7, -C(0)-L-R7; -C(NR19)-L-R7; -C(NR19)0-L-R7; -C(NR19)NR30-L-R7; -C(0)S-L-R7; -C(NR19)S- L-R7 -C(0)0-L-R7 ; 2-pyridyl or 4-pyridyl, wherein 2-pyridyl and 4-pyridyl, are unsubstituted or are substituted by one or more substituents independently selected
7 8 1 Q
from L-R and R , wherein R and R are independently selected from the possibilities for L-R7 , L and R7 are as defined previously, and b is 0, 1 or 2;
J is a group of Formula (II):
Figure imgf000006_0001
wherein
Z is a bond, -S(0)c-, -S(0)c-NR18-C(0)-, -C(O)-, -(CH2)d-, -N=C(NR11R12)- or -NR11CR13R14-,
wherein c is 0, 1 or 2; d is 1 , 2, 3 or 4; R11 and R12 are the same or different and are each H or alkyl having 1 , 2, 3 or 4 carbon atoms; and R and R are the same or different and are each H or alkyl having 1 , 2, 3 or 4 carbon atoms, or R13 and R14 taken together form =NR15, =0 or =S, wherein R15 is hydrogen, hydroxy or alkyl optionally substituted by hydroxy and having 1 , 2, 3, 4 or 5 carbon atoms, and R18 is H or alkyl having 1 , 2, 3, 4 or 5 carbon atoms;
R5 is as defined previously; and
R6 is selected from the possibilities for L-R7, e.g. is H or alkyl having 1 , 2, 3, 4 or 5 carbon atoms; and
R2 is J, wherein if both R1 and R2 are J they may be the same or different, -(CH2)e-0-M-R16 or -(CH2)e-S-M-R16, wherein e is 0, 1 , 2, 3 or 4, M is selected from the list of possibilities for L and R16 is selected from the list of possibilities for R7;
R3 and R4 are each independently H or R8;
or R3 and R4 taken together form a benzene ring which is unsubstituted or substituted by one or more R8,
wherein where there is there is more than one -L-R7, each -L-R7 may be the same as or different from any other -L-R7, and where there is more than one R8, each R8 may be the same as or different from any other R8. The identities of R6, R19 and R30 are chosen independently of each other and independently of the identity of each -L-R7. In one variant of the compounds of formula l(i), the compounds additionally include structures wherein R1 is -L-R7 or O-L-R7, L is a bond and R7 is a substituted or unsubstituted ring selected from 3-7 membered monocyclic rings and 8-13 membered bicyclic rings optionally substituted with one or more R8 selected from halogen, hydroxy, alkyl optionally substituted by hydroxy and having 1 , 2, 3, 4 or 5 carbon atoms, alkyl optionally substituted by hydroxy and having 1 , 2, 3 or 4 carbon atoms and interrupted by -0-, -S-, or -C(O)-, a 3 to 6- membered ring bonded to the remainder of the molecule either directly or through a Ci-4 alkylene group, trifluoromethyl, trifluoromethoxy, cyano, nitro, oxo, amidino, -OR9, -C(0)R9, -C(0)OR9, -OC(0)R9, -N(R9)R10, -C(0)N(R9)R10, - S(0)aR9 and -S(0)aOR9, wherein R9 and R10 are the same or different and are each hydrogen or are selected from alkyl optionally substituted by hydroxy and having 1 , 2, 3, 4 or 5 carbon atoms and a 3 to 6- membered ring bonded to the remainder of the molecule either directly or through a Ci-4 alkylene group, and a is 0, 1 or 2.
[0010] The invention includes a sub-class of the above compounds comprising chromogenic compounds. This sub-class is compounds selected from the structure of Formula I and salts and physiological precursors thereof wherein the symbols have the following meanings:
X is O or S;
Y is O or S; R1 is R5, J, halogen, trifluoromethyl, cyano, nitro, -O-L-R7, -S-L-R7 or -OC(0)-L-R7; wherein:
L is a bond, alkylene optionally substituted by hydroxy and having 1 , 2, 3, 4 or 5 carbon atoms and unsubstituted or substituted by hydroxy, alkylene optionally substituted by hydroxy and having 1 , 2, 3 or 4 carbon atoms and interrupted and/or terminated adjacent R7 by -0-, -S-, or -C(O)-;
R7 is H or a 3-7 membered monocyclic ring or an 8-13 membered bicyclic ring, which rings are unsubstituted or are substituted by one or more R8, or is a dye, wherein:
R8 is selected from halogen, hydroxy, alkyl optionally substituted by hydroxy and having 1 , 2, 3, 4 or 5 carbon atoms, alkyl optionally substituted by hydroxy and having 1 , 2, 3 or 4 carbon atoms and interrupted by -0-, -S-, or -C(O)-, a 3 to 6- membered ring bonded to the remainder of the molecule either directly or through a Ci-4 alkylene group, trifluoromethyl, trifluoromethoxy, cyano, nitro, oxo, amidino, -OR9, -C(0)R9, -C(0)OR9, -OC(0)R9, -N(R9)R10, -C(0)N(R9)R10, and -S(0)aR9, wherein R9 and R10 are the same or different and are each hydrogen or are selected from alkyl optionally substituted by hydroxy and having 1 , 2, 3, 4 or 5 carbon atoms and a 3 to 6- membered ring bonded to the remainder of the molecule either directly or through a Ci-4 alkylene group, and a is 0, 1 or 2;
R5 is H; alkyl having 1 , 2, 3, 4 or 5 carbon atoms and unsubstituted or substituted by hydroxy; alkyl having 1 , 2, 3 or 4 carbon atoms, unsubstituted or substituted by hydroxy and interrupted by -0-, -S-, or -C(O)-; -L-R7; -S(0)b-L-R7; -C(0)-N(R19)-[S(0)b]rL-R7, -C(0)-L-R7; -C(NR19)-L-R7; -C(NR19)0-L-R7; -C(NR19)NR30-L-R7; -C(0)S-L-R7; -C(NR19)S- L-R7 -C(0)0-L-R7 ; 2-pyridyl or 4-pyridyl, wherein 2-pyridyl and 4-pyridyl are unsubstituted or are substituted by one or more substituents independently selected from L-R7 and R8, wherein L and R7 are as defined previously, R19 and R30 are independently selected from the possibilities for L-R7 , and b is 0, 1 or 2;
J is a group of Formula (II):
Figure imgf000008_0001
wherein
Z is a bond, -S(0)c-, -S(0)c-NR18-C(0)-, -C(O)-, -(CH2)d-, -N=C(NR11R12)- or -NR11CR13R14-, wherein c is 0, 1 or 2; d is 1 , 2, 3 or 4; R11 and R12 are the same or different and are each H or alkyl having 1 , 2, 3 or 4 carbon atoms; and R and R are the same or different and are each H or alkyl having 1 , 2, 3 or 4 carbon atoms, or R13 and R14 taken together form =NR15, =0 or =S, wherein R15 is hydrogen, hydroxy or alkyl optionally substituted by hydroxy and having 1 , 2, 3, 4 or 5 carbon atoms, and R18 is H or alkyl having 1 , 2, 3, 4 or 5 carbon atoms;
R5 is as defined previously; and
R6 is selected from the possibilities for L-R7, e.g. is H or alkyl having 1 , 2, 3, 4 or 5 carbon atoms; and
R2 is of Formula (Ila):
Figure imgf000009_0001
wherein R5a is -S(0)b-L-R7; -C(0)-N(R19)-[S(0)b]rL-R7, -C(0)-L-R7; -C(NR19)-L-R7; - C(NR19)0-L-R7; -C(NR19)NR30-L-R7; -C(0)S-L-R7; -C(NR19)S-L-R7, -C(0)0-L-R7, ; 2- pyridyl or 4-pyridyl, wherein 2-pyridyl and 4-pyridyl are unsubstituted or are substituted by one or more substituents independently selected from L-R7 and R8,
wherein:
L, R7, R8, R19, b and f are each as previously defined, independently of the identity of R1;
R3 and R4 are each independently H or R8; or R3 and R4 taken together form a benzene ring which is unsubstituted or substituted by one or more R8,
wherein where there is there is more than one -L-R7, each -L-R7 may be the same as or different from any other -L-R7, and where there is there is more than one R8, each R8 may be the same as or different from any other R8.
[0011] The compounds of the disclosure include chemically permissible structures falling within the described possibilities. The compounds of the disclosure can exist in different forms, such as free acids, free bases, esters and other prodrugs, salts and tautomers, for example, and the disclosure includes all variant forms of the compounds, including those with isotopic substitution by non-normal or radioactive isotopes.
[0012] The extent of protection includes counterfeit or fraudulent products which contain or purport to contain a compound of the invention irrespective of whether they do in fact contain such a compound and irrespective of whether any such compound is contained in a therapeutically effective amount. [0013] Included in the scope of protection are packages which include a description or instructions which indicate that the package contains a species or pharmaceutical formulation of the invention and a product which is or comprises, or purports to be or comprise, such a formulation or species. Such packages may be, but are not necessarily, counterfeit or fraudulent.
[0014] Where a structural formula herein comprising a chiral centre does not indicate chirality (e.g. where all bonds are shown as lines and there is no "wedge" bond), then, unless the context otherwise requires, the structure refers to all corresponding compounds or moieties irrespective of chirality and includes reference to individual compounds or moieties in which the chiral centre is of (R)-configuration, individual compounds or moieties in which the chiral centre is of (S)-configuration and mixtures of (R)- and (S)- isomers as, for example, in the case of racemic mixtures, amongst others.
[0015] Further aspects and embodiments of the disclosure are set forth in the following description and claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] Embodiments of the invention are further described hereinafter with reference to the accompanying drawings, in which:
Figure 1 is a pH titration curve for compound 1 .
Figure 2 relates to determination of the mode of inhibition of 1 with mouse Nat2. Inhibitory activity was determined by monitoring for rate of AcCoA hydrolysis. Figure 2(a) is Lineweaver-Burk plots showing mNat2 rates of hydrolysis with pABA as arylamine substrate, at inhibitor concentrations of 3μΜ (crosses), 1 μΜ (triangles), 0.5μΜ (squares) and ΟμΜ (diamonds). Figure 2(b) shows slope values from Lineweaver-Burk graphs plotted against concentration of 1 .
Figure 3 is a barchart showing percentage inhibition of NAT preparations by compounds of the invention.
DETAILED DESCRIPTION
[0017] Throughout the description and claims of this specification, the words "comprise" and "contain" and variations of them mean "including but not limited to", and they are not intended to (and do not) exclude other moieties, additives, components, integers or steps. Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise. [0018] Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith. All of the features disclosed in this specification
(including any accompanying claims, abstract and drawings), and/or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of such features and/or steps are mutually exclusive. The invention is not restricted to the details of any foregoing embodiments. The invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including any accompanying claims, abstract and drawings), or to any novel one, or any novel combination, of the steps of any method or process so disclosed.
[0019] The reader's attention is directed to all papers and documents which are filed concurrently with or previous to this specification in connection with this application and which are open to public inspection with this specification, and the contents of all such papers and documents are incorporated herein by reference.
[0020] The following terms and abbreviations are used in this specification:
[0021] The term "alkyl" in this specification includes linear and branched alkyl groups containing, unless otherwise stated, 1 , 2, 3, 4 or 5 carbon atoms, for example methyl, ethyl, n- propyl, iso-propyl, tert-butyl and n-pentyl. Alkylene groups may likewise be linear or branched and may, for example, correspond to one of those alkyl groups listed in the preceding sentence. The alkyl groups may be substituted by inert substituents, notably halogen.
[0022] The term "optionally substituted" means "unsubstituted or substituted".
[0023] The term "physiological precursors" denotes compounds which, when administered to a living cell or organism, are converted to a compound of the disclosure. The term therefore includes prodrugs and compounds which, whilst falling outside the pharmaceutical field, have prodrug structures. Prodrugs are further described later in this specification.
[0024] The phrase "pharmaceutically acceptable" is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings or animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
[0025] The term "halogen" herein includes reference to F, CI, Br and I, particularly F, CI and Br. In some instances halogen is CI. In one class of compounds, halogen is F. [0026] The term "heterocyclic ring" refers to a ring system which has at least one (e.g. 1 , 2 or 3) in-ring heteroatoms. The term "heteroatom" includes oxygen, sulfur and nitrogen, of which sulfur is sometimes less preferred. Heterocycles may be aromatic or non-aromatic. Examples are thiophene, pyrrole, pyrazole, furan, pyrrolidine, pyrazolidine, imidazoline, pyridine, piperidine, pyrimidine and triazine.
[0027] The term "heteroaryl" refers to a ring system which has at least one (e.g. 1 , 2 or 3) in- ring heteroatoms and has a conjugated in-ring double bond system. The term "heteroatom" includes oxygen, sulfur and nitrogen, of which sulfur is sometimes less preferred.
[0028] "Treating" a disease with the compounds discussed herein is defined as administering one or more of the compounds discussed herein, with or without additional therapeutic agents, in order to palliate, ameliorate, stabilize, reverse, slow, delay, reduce, or eliminate either the disease or one or more symptoms of the disease, or to retard or stop the progression of the disease or of one or more symptoms of the disease, when a compound or composition is administered to treat a disease, disorder or occurrence, it does not mean that the treatment will be completely successful, since curative treatments may fail in any individual patient. It should be noted that the use of the compounds and/or methods for treatment and the use of the compounds and/or methods for prevention need not be mutually exclusive.
[0029] "Therapeutic use" of the compounds discussed herein is defined as using one or more of the compounds discussed herein to treat or prevent a disease, as defined above. A "therapeutically effective amount" of a compound is an amount of the compound, which, when administered to a subject, is sufficient to treat, prevent, reduce, or eliminate either the disease or one or more symptoms of the disease, or to retard the progression of the disease or of one or more symptoms of the disease, or to reduce the severity of the disease or of one or more symptoms of the disease. A "therapeutically effective amount" can be given in one or more administrations.
Compounds
[0030] The compounds of the disclosure are described in this section of the specification.
[0031] It will be understood that the disclosure specifically includes variants of individual or exemplary compounds or compound classes in which one or more moieties have been replaced by alternatives described in this application.
[0032] The compounds of the disclosure include those having the structure of Formula I and salts and physiological precursors thereof:
Figure imgf000013_0001
[0033] The symbols in Formula (I) will be described next.
[0034] X is O or S and Y is O or S. X and Y may be the same or different. In one embodiment, X is O and Y is S. In another embodiment. X is S and Y is O. In a further embodiment, X and Y are both S. In a particular embodiment, X and Y are both O.
[0035] R1 is R5, J, halogen, trifluoromethyl, cyano, nitro, -O-L-R7, -S-L-R7 or -OC(0)-L-R7. R2 is J, -(CH2)e-0-M-R16 or -(CH2)e-S-M-R16, wherein e is 0, 1 , 2, 3 or 4, M is selected from the list of possibilities for L and R16 is selected from the list of possibilities for R7 (see below). R16 is H in one embodiment; in a sub-class -M-R16 is H. In one embodiment, e is 0; in another embodiment e is not 0, e.g. is 1 or 2. If both R1 and R2 are J they may be the same or different. In one embodiment, both R1 and R2 are J. In one embodiment, R1 is a moiety not falling within the definition of J whilst R2 is a J moiety. In another embodiment, both of R1 and R2 are moieties not falling within the definition of J.
[0036] The disclosure includes compounds in which R2 is -(CH2)e-0-M-R16 or -(CH2)e-S-M-R16, in particular the former, e.g. -(CH2)eOH or -(CH2)eOH, especially -(CH2)eOH, and R1 is a J moiety or, in other embodiments, is a moiety not falling within the definition of J.
[0037] In one embodiment, R1 is R5, halogen, trifluoromethyl, cyano, nitro, -O-L-R7 or -OC(O)- L-R7. R1 is therefore halogen in one embodiment, in particular F, CI or Br, e.g. it is F in one class of compounds and CI in another class of compounds. Compounds in which R1 is CI or Br may be used as intermediates for making other compounds of the disclosure. In some compounds, R1 is -O-L-R7 or -OC(0)-L-R7, in particular -O-L-R7. In one embodiment, R1 is R5, in particular H, alkyl having 1 , 2, 3, 4 or 5 carbon atoms and unsubstituted or substituted by hydroxy; or alkyl having 1 , 2, 3 or 4 carbon atoms, unsubstituted or substituted by hydroxy and interrupted by -0-, -S-, or -C(O)-; for example R1 may be H or Ci-5 alkyl, particularly H. In one embodiment, R1 is an R5 selected from -S(0)b-L-R7; -C(0)-L-R7; -C(0)0-L-R7, or -L-R7, wherein R7 is H or alkyl having 1 , 2, 3, 4 or 5 carbon atoms; for example it is an R5 selected from - S(0)b-L-R7; -C(0)-L-R7; -C(0)0-L-R7. In embodiments mentioned in this paragraph, -L-R7 is in particular H or alkyl optionally substituted by hydroxy and having 1 , 2, 3, 4 or 5 carbon atoms, e.g. is H in one class of compounds and a said alkyl in another class of compounds; in alternative embodiments mentioned in this paragraph, -L-R7 is, for example, a said alkyl group substituted by a 3-7 membered monocyclic ring or by an 8-13 membered bicyclic ring, in particular by a 3-7 membered monocyclic ring. These rings may be unsubstituted or, in another embodiment, substituted as described herein.
[0038] L is a bond in one class of compounds. In another class of compounds, L is alkylene which has 1 , 2, 3, 4 or 5 carbon atoms and which is unsubstituted or substituted by hydroxy. In a third class of compounds, L is alkylene having 1 , 2, 3 or 4 carbon atoms and interrupted by, and/or terminated adjacent R7 by, -0-, -S-, or -C(O)-, in particular -0-. Where L is alkylene or alkylene interrupted and/or terminated as just described, it may be unsubstituted or it may be substituted by a hydroxy group. In some embodiments, L is a said alkylene. In other embodiments, L is hydroxyalkylene having 1 , 2, 3, 4 or 5 carbon atoms. Alkylene moieties mentioned in this paragraph may be linear or branched and in particular are linear.
[0039] R7 is H, a dye or a 3-7 membered monocyclic ring or an 8-13 membered bicyclic ring, which rings are unsubstituted or are substituted by one or more R8. In one embodiment, R7 is H. In another embodiment, R7 is a monocyclic ring having 3, 4, 5, 6 or 7 ring-forming atoms, for example a carbocyclic ring, such as a cycloalkyi ring or a benzene ring, or a heterocyclic ring, in particular a said carbocyclic ring. In a further embodiment, R7 is an 8-13 membered bicyclic ring, for example a carbocyclic ring (e.g. a benzene ring or a naphthalene ring) or a heteroaromatic moiety in which one or both of the rings contain at least one heteroatom selected from O, N and S. In those embodiments where R7 is a ring, it may be unsubstituted or substituted by one or more R8, in particular by 1 , 2 or 3 R8 moieties.
[0040] In one embodiment R7 is, independently wherever it occurs, a ring substituted with at least one R8. In another embodiment R7 is, independently wherever it occurs, a benzene ring substituted with at least one R8.
[0041] R8 is selected from halogen, hydroxy, alkyl optionally substituted by hydroxy and having 1 , 2, 3, 4 or 5 carbon atoms, alkyl optionally substituted by hydroxy and having 1 , 2, 3 or 4 carbon atoms and interrupted by -0-, -S-, or -C(O)-, C3-6 carbocyclyl bonded to the remainder of the molecule either directly or through a Ci-4 alkylene group, trifluoromethyl, trifluoromethoxy, cyano, nitro, oxo, amidino, -OR9, -C(0)R9, -C(0)OR9, -OC(0)R9, -N(R9)R10, - C(0)N(R9)R10, -S(0)aR9 and -S(0)aOR9 , wherein R9 and R10 are the same or different and are each hydrogen or are selected from alkyl optionally substituted by hydroxy and having 1 , 2, 3, 4 or 5 carbon atoms and C3-6 carbocyclyl bonded to the remainder of the molecule either directly or through a Ci-4 alkylene group, and a is 0, 1 or 2. For all embodiments of the invention, there are disclosed also structures in which R8 does not include trifluoromethoxy. Exemplary R8 moieties are F, CI, Br, methyl, ethyl, n-propyl, iso-propyl, n-butyl, tert-butyl and other Ci-4 alkyl, trifluoromethyl, trifluoromethoxy, methoxy, ethoxy and other Ci-4 alkoxy, hydroxy, hydroxymethyl, hydroxyethyl, nitro, cyano, amino, amino mono- or disubstituted by Ci_ 4 alkyl such as methyl or ethyl, carboxy or carboxy esterified by Ci-5 alkyl. One class of said exemplary R8 moieties comprises halogen and moieties containing 1 , 2 or 3 (e.g. 1 or 2) multivalent atoms, for example F, CI, Br, methyl, ethyl, trifluoromethyl, trifluoromethoxy, methoxy, hydroxy, hydroxymethyl, nitro, cyano, amino or methylamino, particularly halogen (e.g. F, CI or Br), methyl or trifluoromethyl. In one class of compounds, R8 moieties are selected from halogen (e.g. F, CI or Br), hydroxy, alkyl optionally substituted by hydroxy and having 1 , 2, 3, 4 or 5 carbon atoms (e.g. CrC4 alkyl), alkyl optionally substituted by hydroxy and having 1 , 2, 3 or 4 carbon atoms and interrupted by -0-, -S-, or -C(O)- (e.g. Ci-C4 alkoxy), trifluoromethyl, trifluoromethoxy, cyano and nitro, and in particular the or each R8 is in this case selected from halogen (e.g. F, CI or Br), Ci-4 alkyl and especially methyl, and trifluoromethyl. Other exemplary R8 moieties to be mentioned are S03H and Ci-C5 alkyl esters thereof. Where R7 is a said ring, it may therefore be substituted by an R8 moiety mentioned in this paragraph; any one or more ring-forming atoms which is available for substitution by an R8 moiety may be substituted, e.g. 1 , 2 or 3 such ring-forming atoms. In the case of compounds having two or more R8 moieties, each R8 may be chosen independently, i.e. any one R8 may be the same as or different from any other R8.
[0042] In embodiments, R7 is a dye, for example a fluorescent dye. Fluorescent dyes useful as detectable labels are well known to those skilled in the art and numerous examples can be found in the Handbook of Fluorescent Probes and Research Chemicals 6th Edition, Richard Haugland, Molecular Probes, Inc., 1996 (ISBN 0-9652240-0-7). Numerous fluorophores are therefore known to those skilled in the art and include, but are not limited to, coumarin, acridine, furan, indole, quinoline, cyanine, benzofuran, quinazolinone, benzazole, borapolyazaindacene and xanthenes, with the latter including fluorescein, rhodamine, rhodol, and rosamine. Advantageously, fluorescent dyes incorporated in compounds of the disclosure are environmentally sensitive and undergo a change in absorption/emission properties when in the environment of a protein, e.g. when a compound of the disclosure binds to hNAT1 . A particular class of fluorescent dyes consists of pH-sensitive dyes, for example for example naphthalene dyes. Exemplary fluorescent dyes are dansyl amide, 5- (dimethylamino)naphthalene-l -sulfonamide, and other naphthalene-1 -sulfonamides.
[0043] R5 is H; alkyl having 1 , 2, 3, 4 or 5 carbon atoms and unsubstituted or substituted by hydroxy; alkyl having 1 , 2, 3 or 4 carbon atoms, unsubstituted or substituted by hydroxy and interrupted by -0-, -S-, or -C(O)-; -L-R7; -S(0)b-L-R7; -C(0)-N(R19)-[S(0)b]rL-R7, -C(0)-L-R7; - C(NR19)-L-R7; -C(NR19)0-L-R7; -C(NR19)NR30-L-R7; -C(0)S-L-R7; -C(NR19)S-L-R7 -C(0)0-L-R7 ; 2-pyridyl or 4-pyridyl, wherein 2-pyridyl and 4-pyridyl are unsubstituted or are substituted by
7 8 1 Q
one or more substituents independently selected from L-R and R , wherein R and R are independently selected from amongst the possibilities for L-R7, L and R7 are as defined previously and R7 is in particular a said ring (whether unsubstituted or substituted), each R7 being the same as or different from the other R7 where there are two R7 moieties in the compound, and b is 0, 1 or 2. In one embodiment b is 1 . In one embodiment b is 2. In one embodiment, the number of L-R7 substituents on 2-pyridyl and 4-pyridyl is 0. In one embodiment, the number of L-R7 substituents on 2-pyridyl and 4-pyridyl is 1 . In particular compounds, any R8 substituents on 2-pyridyl and 4-pyridyl are selected from exemplary R8 moieties described previously herein. In some compounds, R5 is H or a said alkyl group. In some compounds, R5 is H. In some compounds, R5 is not H. In some compounds, R5 is -L-R7. In some compounds, R5 is S(0)b-L-R7; -C(0)-L-R7; or -C(0)0-L-R7. As examples of -L-R7, there may be mentioned groups in which L is a bond and R7 is as described in the preceding paragraph. Also to be mentioned as examples of -L-R7 are groups in which L is alkylene having 1 , 2, 3, 4 or 5 carbon atoms and R7 is as described in the preceding paragraph and is in particular a said ring, as well as groups in which L is a said Ci-5 alkylene substituted by a hydroxy group and R7 is as described in the preceding paragraph, in particular a said ring.
[0044] J is a group of Formula (II):
Figure imgf000016_0001
wherein
Z is a bond, -S(0)c-, -S(0)c-NR18-C(0)-, -C(O)-, -(CH2)d-, -N=C(NR11R12)- or -NR11CR13R14-,
wherein c is 0, 1 or 2; d is 1 , 2, 3 or 4 and in particular 1 or 2; R11 and R12 are the same or different and are each H or alkyl having 1 , 2, 3 or 4 carbon atoms, and in particular
11 1? 1 ^ 14
one or both of R and R is H; and R and R are the same or different and are each H or alkyl having 1 , 2, 3 or 4 carbon atoms, or R13 and R14 taken together form =NR15, =0 or =S, wherein R15 is hydrogen, hydroxy or alkyl optionally substituted by hydroxy and having 1 , 2, 3, 4 or 5 carbon atoms, and in particular one or both of R and R are
H, and R18 is H or alkyl having 1 , 2, 3, 4 or 5 carbon atoms, in particular H;
R5 is as defined previously; and
R6 is selected from the possibilities for L-R7, e.g. is H or alkyl having 1 , 2, 3, 4 or 5 carbon atoms, and in particular is H.
[0045] R5 may be, for example, -S(0)b-L-R7, -C(0)-L-R7 or -C(0)0-L-R7, and particularly - S(0)b-L-R7, and b is 1 or 2 and normally 2. [0046] In one class of compounds Z is -S(0)c- where c is in particular 1 or 2 (e.g. 2), and R5 may be, for example, -L-R7, -C(0)-L-R7, -C(0)0-L-R7 or -C(0)-N(R19)-S(0)b-L-R7, and particularly -L-R7, or another moiety described herein as a possibility for R5.
[0047] Z is a bond in one class of compounds.
[0048] In one embodiment, applicable to all R2 moieties disclosed herein, R1 is a J group. In a sub-embodiment Z is a bond and R5 is as defined herein and in particular is -L-R7. In compounds in which R1 is a J group, R6 of R1 is a J group is in particular H. For all embodiments of the invention (and all R2 moieties), therefore, R1 may in particular be -N(R6)-L- R7 and especially -NH-L-R7. In another embodiment, applicable to all R2 moieties disclosed herein, R1 is an R5 group which is -L-R7.
[0049] In an embodiment, applicable to all R2 moieties disclosed herein, R1 is an R5 group which is -L-R7, L is a bond and R7 is a substituted or unsubstituted ring selected from 3-7 membered monocyclic rings and 8-13 membered bicyclic rings optionally substituted with one or more R8. In an embodiment R7 is a benzene ring optionally substituted with one or more R8. In another embodiment R7 is a 5- or 6- membered heteroaromatic ring substituted with one or more R8 or a bicyclic ring in which a 5- or 6- membered heteroaromatic ring is fused with a benzene ring substituted with one or more R8. In another embodiment R7 is selected from 3- pyridyl, 2-furyl, 3-furyl, 2-benzothiophenyl.
[0050] In another embodiment, applicable to all R2 moieties disclosed herein, R1 is O-L-R7, L is a bond and R7 is a substituted or unsubstituted ring selected from 3-7 membered monocyclic rings and 8-13 membered bicyclic rings optionally substituted with one or more R8. In an embodiment R7 is a benzene ring optionally substituted with one or more R8. In this embodiment R8 may be selected from, for example, cyano, chloro, trifluoromethoxy, nitro and - C(0)H.
[0051] A particular embodiment of the invention resides in compounds in which R2 is of Formula (Ila):
Figure imgf000017_0001
wherein R5a is -S(0)b-L-R7; -C(0)-N(R19)-[S(0)b]rL-R7, -C(0)-L-R7; -C(NR19)-L-R7; -C(NR19)0-L- R7; -C(NR19)NR30-L-R7; -C(0)S-L-R7; -C(NR19)S-L-R7 -C(0)0-L-R7; 2-pyridyl or 4-pyridyl, wherein 2-pyridyl and 4-pyridyl are unsubstituted or are substituted by one or more substituents independently selected from L-R7 and R8. The meanings of the symbols used in this paragraph are as described previously herein and the specific option chosen for any one symbol is independent of the specific option chosen - if necessary - for that symbol in relation to R1. In one embodiment, the number of L-R7 substituents on 2-pyridyl and 4-pyridyl is 0. In one embodiment, the number of L-R7 substituents on 2-pyridyl and 4-pyridyl is 1 . In particular compounds, any R8 substituents on 2-pyridyl and 4-pyridyl are selected from exemplary R8 moieties described in [0038]. The compounds in which R2 is of Formula (I la) are chromogenic, in that they change colour (or potentially become coloured/decoloured) when contacted with hNAT1 . The invention includes an embodiment in which R2 is of Formula (lla) and in which f is 0. The invention includes an embodiment in which R2 is of Formula (lla) and in which f is 1 . Index b is for example 1 or 2, and in particular is 2.
[0052] In one sub-class of the compounds in which R2 is of Formula (lla), R5a is -S(0)b-L-R7 and in particular is -S(0)2-L-R7, for example -S(0)2-R7. In another sub-class of the compounds in which R2 is of Formula (lla), R5a is -C(0)-N(R19)-[S(0)b]rL-R7, -C(0)-L-R7; -C(0)S-L-R7, or - C(0)0-L-R7; in particular compounds of this sub-class, f is 1 and S(0)b is S(0)2. In another sub-class of the compounds in which R2 is of Formula (lla), R5a is -C(NR19)-L-R7, -C(NR19)0-L- R7, -C(NR19)NR30-L-R7 or -C(NR19)S-L-R7. In another sub-class of the compounds in which R2 is of Formula (lla), R5a is 2-pyridyl or 4-pyridyl, wherein 2-pyridyl and 4-pyridyl are unsubstituted or are substituted by one or more substituents independently selected from L-R7 and R8. For all compounds mentioned in this paragraph, L is a bond in one exemplary and non-limiting class of compounds.
[0053] Continuing to discuss the compounds in which R2 is of Formula (lla), L of R2 is as previously described, for example in one sub-class is a bond. R7 of R2 is as described herein and is in particular a substituted or unsubstituted ring selected from 3-7 membered monocyclic rings and 8-13 membered bicyclic rings or a dye, for example is phenyl or a 5- or 6- membered heteroaromatic ring (e.g. thiophenyl, particularly 2-thiophenyl) or a fluorescent dye, wherein phenyl and heteroaromatic ring are unsubstituted or are substituted by one or more R8, e.g. 1 , 2 or 3 R8; R8 is as described herein, e.g. selected independently from halogen, methyl and trifluoromethyl.
[0054] In an embodiment of compounds in which R2 is of Formula (lla), R7 of R2 is a substituted or unsubstituted ring selected from 3-7 membered monocyclic rings and 8-13 membered bicyclic rings, for example as mentioned in the preceding paragraph.
[0055] In one embodiment, R1 is of Formula (lla) as described with reference to R2 and R2 is of Formula (lla). In one embodiment, R1 is not of Formula (lla) and R2 is of Formula (lla). In one class of compounds, R2 is of Formula (lla) and R1 is a J group, in particular a J group in which R5 is -L-R7 and in which R6 is as described herein, e.g. is H; L of -L-R7 in the J group may in particular be a bond. In one class of compounds, R2 is of Formula (lla) and R1 is halogen (e.g. F or CI), trifluoromethyl, cyano, nitro, -O-L-R7, -S-L-R7 or -OC(0)-L-R7, wherein L may for example be a bond. In one class of compounds, R2 is of Formula (lla) and R1 is an R5 group, e.g. -L-R7.
[0056] Compounds in which R1 is halogen, e.g. CI or Br, are useful inter alia as intermediates for making other compounds of the disclosure.
[0057] In one embodiment, exactly one of R1 and R2 includes an R7 which is a substituted or unsubstituted ring selected from 3-7 membered monocyclic rings and 8-13 membered bicyclic rings. Particular rings to mention are benzene and 5- and 6- membered heteroaromatic rings, for example 2-thiophenyl; these rings are unsubstituted or are substituted as previously described in relation to R7. In certain compounds of this paragraph, R2 includes a said R7 and is of Formula (lla).
[0058] In one embodiment, both of R1 and R2 include an R7 which is a substituted or unsubstituted ring selected from 3-7 membered monocyclic rings and 8-13 membered bicyclic rings. Particular rings to mention are benzene and 5- and 6- membered heteroaromatic rings, for example 2-thiophenyl; these rings are unsubstituted or are substituted as previously described in relation to R7. In the compounds of this paragraph, R2 may be of Formula (lla), for example.
[0059] In those -L-R7 moieties in which R7 is a said ring, whether substituted or unsubstituted, L is in particular a bond, Ci-5 linear alkylene for example methylene or ethylene, or Ci-4 linear alkylene (for example methylene or ethylene) joined to R7 through an ether linkage, and said ring may be a 5- or 6- membered carbocyclic or heterocyclic ring and is in particular a benzene ring or a 5- or 6- membered heteroaromatic ring, for example furan, pyrrole, thiophene, pyridine, pyridazine, pyrimidine and pyrazine. In one embodiment of the -L-R7 moieties mentioned in this paragraph, L is a bond; in another embodiment, L is not a bond. In some compounds, L is a bond and R7 is a said ring, whether substituted or unsubstituted, e.g. a benzene ring or a 5- or 6- membered heteroaromatic ring. The -L-R7 moieties described in this paragraph may, for example, form the ring-containing -L-R7 moieties mentioned in the following paragraphs [0055] and [0057] and in Tables 1 and 2 below.
[0060] As particular J groups may be mentioned: -N(R6)-L-R7, -S(0)c-N(R6)-L-R7, -S(0)c-NR18- C(0)-N(R6)-L-R7, -C(0)-N(R6)-L-R7, -(CH2)d-N(R6)-S(0)b-L-R7; -(CH2)d-N(R6)-C(0)-N(R19)-S(0)b- L-R7; -C(0)-L-R7; -C(0)0-L-R7, -N=C(NR11R12)-N(R6)-S(0)b-L-R7; and -N(R11)CR13R14-N(R6)- S(0)b-L-R7. In one embodiment, R1 and R2 are both selected from the J groups mentioned in this paragraph, wherein -L-R7 of R1 and R2 may be the same as or different from each other. In one embodiment one of R1 and R2 is -S(0)c-N(R6)-L-R7; in a first sub-embodiment a single one of R1 and R2 is -S(0)c-N(R6)-L-R7 and in a second sub-embodiment both of R1 and R2 are - S(0)c-N(R6)-L-R7. In another embodiment, one of R1 and R2 is -(CH2)d-N(R6)-S(0)b-L-R7, in particular -N(R6)-S(0)b-L-R7; in a first sub-embodiment a single one of R1 and R2 is -(CH2)d- N(R6)-S(0)b-L-R7 and in a second sub-embodiment both of R1 and R2 are -(CH2)d-N(R6)-S(0)b- L-R7. Moiety -L-R7 may be an embodiment previously described, and it will be recalled here that the invention includes embodiments in which a single one of, or both of, R1 and R2 include an R7 which is a substituted or unsubstituted 3-7 membered monocyclic ring or 8-13 membered bicyclic ring. R6 is in particular H. The disclosure includes J groups of this paragraph which have one or more of (e.g. all of) the following features: b is 2, c is 2, R11 is H, R12 is H, R13 is H, R14 is H; and R6 is H; some J groups have the following combinations of features: b or c is 2 and R6 is H; b or c is 2, R6 is H and R18 or R19 is H; b is 2, R6 is H and R11
11 1^ 14
and R are H or R , R and R are H. The disclosure includes J groups of this paragraph in which d is 0, 1 or 2, in particular 0.
[0061] As previously mentioned, R6, R19 and R30 are selected independently of each other and each may be any moiety which falls within the definition of L-R7, but R6 is in particular H for all embodiments of the disclosure. For example R6, R19 and R30 may be selected from H, C1-C5 alkyl, phenyl which is unsubstituted or is substituted by one or more R8 and 5- or 6- membered heteroaryl which is unsubstituted or is substituted by one or more R8, In one embodiment, R6, R19 and R30 are selected from H and alkyl having 1 , 2, 3, 4 or 5 carbon atoms, and in particular are H. Exemplary R8 moieties for R6, R19 and R30 groups which include an R8 are F, CI, Br, methyl, ethyl, n-propyl, iso-propyl, n-butyl, tert-butyl and other Ci-4 alkyl, trifluoromethyl, trifluoromethoxy, methoxy, ethoxy and other Ci-4 alkoxy, hydroxy, hydroxymethyl, hydroxyethyl, nitro, cyano, amino, amino mono- or disubstituted by Ci-4 alkyl such as methyl or ethyl, carboxy or carboxy esterified by Ci-5 alkyl. One class of said exemplary R8 moieties comprises halogen and moieties containing 1 , 2 or 3 (e.g. 1 or 2) multivalent atoms, for example F, CI, Br, methyl, ethyl, trifluoromethyl, trifluoromethoxy, methoxy, hydroxy, hydroxymethyl, nitro, cyano, amino or methylamino, particularly halogen (e.g. F, CI or Br), methyl or trifluoromethyl.
[0062] In one embodiment, R1 is-(CH2)e-0-M-R16 or -N(R6)-L-R7, particularly -N(R6)-L-R7,and R2 is -S(0)c-N(R6)-L-R7, -S(0)c-NR18-C(0)-N(R6)-L-R7, -C(0)-N(R6)-L-R7, -(CH2)d-N(R6)-S(0)b-L- R7; -(CH2)d-N(R6)-C(0)-N(R19)-S(0)b-L-R7; -C(0)-L-R7; -C(0)0-L-R7, -N=C(NR11R12)-N(R6)- S(0)b-L-R7; or -N(R11)CR13R14-N(R6)-S(0)b-L-R7. In one embodiment, R2 is -S(0)c-N(R6)-L-R7. In another embodiment, R2 is-(CH2)d-N(R6)-S(0)b-L-R7, in particular -N(R6)-S(0)b-L-R7. Moiety - L-R7 may be an embodiment previously described. Moieties -L-R7 of R1 and R2 may be the same as or different from each other, and it will be recalled here that the invention includes embodiments in which a single one of, or both of, R1 and R2 include an R7 which is a substituted or unsubstituted 3-7 membered monocyclic ring or 8-13 membered bicyclic ring. R6 is in particular H. The disclosure includes J groups of this paragraph which have one or more of (e.g. all available combinations of) the following features: b is 2, c is 2, R11 is H, R12 is H, R13 is H, R14 is H; and R6 is H; some J groups have the following combinations of features: b or c is 2 and R6 is H; b or c is 2, R6 is H and R18 or R19 is H; b is 2, R6 is H and R11 and R12 are H or
11 1 14
R , R and R are H. The disclosure includes J groups of this paragraph in which d is 0, 1 or 2, in particular 0.
[0063] For all embodiments of the invention containing a fragment S(0)b, integer b is in particular 2. Compounds containing a fragment -(SO)b- wherein b is 2 therefore form one embodiment of the invention.
[0064] For all embodiments of the invention containing a fragment S(0)c, integer c is in particular 2. Compounds containing a fragment -(SO)c- wherein c is 2 therefore form an embodiment of the invention also.
[0065] It will be understood from the aforegoing that the invention includes compounds in which R1 is -N(R6)-L-R7 and R2 is -NH-S(0)b-L-R7, wherein b is 2 and each R7 may be the same as or different from the other and is in particular phenyl or a 5- or 6-membered heteroaromatic ring which is unsubstituted or substituted by 1 , 2 or 3 R8 as previously described, in particular where R8 is halogen, Ci-5 alkyl or trifluoromethyl. L is in particular linear Ci-5 alkylene (e.g. methylene or ethylene) or a bond, and most particularly a bond. For example, structures of Formulae (VII) and (VIII)-(XI) discussed later in this specification may have such R1 and R2 groups.
[0066] R3 and R4 are each independently H or R8; or R3 and R4 taken together form a benzene ring which is unsubstituted or substituted by one or more R8. Exemplary R8 moieties as described herein in paragraph [0038] are applicable to R3 and R4.
[0067] In compounds in which there is more than one -L-R7, each -L-R7 may be the same as or different from any other -L-R7. In compounds in which there is more than one R8, each R8 may be the same as or different from any other R8.
[0068] Compounds of the invention therefore include those having the structures of Formulae (lll)-(VI) and the salts and physiological precursors thereof:
Figure imgf000021_0001
Figure imgf000022_0001
[0069] Included in the disclosure are the structures of Formula (III), Formula (IV), Formula (V) and Formula (VI) in which:
R3 and R4 are the same or different and selected from H and R8 (e.g. R3 and R4 are H, R3 is H and R4 is an R8, or R3 is an R8 and R4 is H), in particular H, F, CI, hydroxy, alkyl having
1 , 2, 3, 4 or 5 carbon atoms, hydroxyalkyi having 1 , 2, 3, 4 or 5 carbon atoms, alkyl having 1 ,
2, 3 or 4 carbon atoms and interrupted by -0-, -S-, or -C(O)-, hydroxyalkyi having 1 , 2, 3 or 4 carbon atoms and interrupted by -0-, -S-, or -C(O)-, trifluoromethyl, trifluoromethoxy, cyano, nitro, oxo, amidino, -OR9, -C(0)R9, -C(0)OR9, -OC(0)R9, -N(R9)R10, -C(0)N(R9)R10, -S(0)aR9 and -S(0)aOR9 , wherein R9 and R10 are the same or different and are each hydrogen or are selected from alkyl optionally substituted by hydroxy and having 1 , 2, 3, 4 or 5 carbon atoms, the aforesaid alkyl moieties more particularly being methyl or ethyl; and
R1 and R2 are as previously defined and, more particularly, may be as defined in Table 1 or Table 2 below, in embodiments of which either a single one of R1 and R2 (in one embodiment R1; in another embodiment R2) or both R1 and R2 have an R7 which is an unsubstituted ring or substituted ring as previously described:
Table 1 : Selected Combinations of R1 and R2 groups
R1 R2 R1 R2
1 -O-L-R7 -O-L-R7 -(CH2)d-N(R6)-S(0)b- -O-L-R7
L-R7
2 -O-L-R7 -S-L-R7 -(CH2)d-N(R6)-S(0)b- -S-L-R7
L-R7
3 -O-L-R7 -N(R6)-L-R7 -(CH2)d-N(R6)-S(0)b- -N(R6)-L-R7
L-R7
4 -O-L-R7 -S(0)c-N(R6)-L-R7 -(CH2)d-N(R6)-S(0)b- -S(0)c-N(R6)-L-R7
L-R7
5 -O-L-R7 -S(0)c-NR18-C(0)- -(CH2)d-N(R6)-S(0)b- -S(0)c-NR18-C(0)- N(R6)-L-R7 L-R7 N(R6)-L-R7
6 -O-L-R7 -C(0)-N(R6)-L-R7 -(CH2)d-N(R6)-S(0)b- -C(0)-N(R6)-L-R7
L-R7
7 -O-L-R7 -(CH2)d-N(R6)-S(0)b- -(CH2)d-N(R6)-S(0)b- -(CH2)d-N(R6)-S(0)b- L-R7 L-R7 L-R7
8 -O-L-R7 -(CH2)d-N(R6)-C(0)- -(CH2)d-N(R6)-S(0)b- -(CH2)d-N(R6)-C(0)- N(R19)-S(0)b-L-R7 L-R7 N(R19)-S(0)b-L-R7 -O-L-R7 -C(0)-L-R7 -(CH2)d-N(R6)-S(0)b- -C(0)-L-R7
L-R7
-O-L-R7 -C(0)0-L-R7 -(CH2)d-N(R6)-S(0)b- -C(0)0-L-R7
L-R7
-O-L-R7 -N=C(NR11R12)- -(CH2)d-N(R6)-S(0)b- -N=C(NR11R12)- N(R6)-S(0)b-L-R7 L-R7 N(R6)-S(0)b-L-R7
-O-L-R7 -N(R11)CR13R14- -(CH2)d-N(R6)-S(0)b- -N(R11)CR13R14- N(R6)-S(0)b-L-R7 L-R7 N(R6)-S(0)b-L-R7
-S-L-R7 -O-L-R7 -(CH2)d-N(R6)-C(0)- -O-L-R7
N(R19)-S(0)b-L-R7
-S-L-R7 -S-L-R7 -(CH2)d-N(R6)-C(0)- -S-L-R7
N(R19)-S(0)b-L-R7
-S-L-R7 -N(R6)-L-R7 -(CH2)d-N(R6)-C(0)- -N(R6)-L-R7
N(R19)-S(0)b-L-R7
-S-L-R7 -S(0)c-N(R6)-L-R7 -(CH2)d-N(R6)-C(0)- -S(0)c-N(R6)-L-R7
N(R19)-S(0)b-L-R7
-S-L-R7 -S(0)c-NR18-C(0)- -(CH2)d-N(R6)-C(0)- -S(0)c-NR18-C(0)- N(R6)-L-R7 N(R19)-S(0)b-L-R7 N(R6)-L-R7
-S-L-R7 -C(0)-N(R6)-L-R7 -(CH2)d-N(R6)-C(0)- -C(0)-N(R6)-L-R7
N(R19)-S(0)b-L-R7
-S-L-R7 -(CH2)d-N(R6)-S(0)b- -(CH2)d-N(R6)-C(0)- -(CH2)d-N(R6)-S(0)b- L-R7 N(R19)-S(0)b-L-R7 L-R7
-S-L-R7 -(CH2)d-N(R6)-C(0)- -(CH2)d-N(R6)-C(0)- -(CH2)d-N(R6)-C(0)- N(R19)-S(0)b-L-R7 N(R19)-S(0)b-L-R7 N(R19)-S(0)b-L-R7
-S-L-R7 -C(0)-L-R7 -(CH2)d-N(R6)-C(0)- -C(0)-L-R7
N(R19)-S(0)b-L-R7
-S-L-R7 -C(0)0-L-R7 -(CH2)d-N(R6)-C(0)- -C(0)0-L-R7
N(R19)-S(0)b-L-R7
-S-L-R7 -N=C(NR11R12)- -(CH2)d-N(R6)-C(0)- -N=C(NR11R12)- N(R6)-S(0)b-L-R7 N(R19)-S(0)b-L-R7 N(R6)-S(0)b-L-R7
-S-L-R7 -N(R11)CR13R14- -(CH2)d-N(R6)-C(0)- -N(R11)CR13R14- N(R6)-S(0)b-L-R7 N(R19)-S(0)b-L-R7 N(R6)-S(0)b-L-R7
-N(R6)-L-R7 -O-L-R7 -C(0)-L-R7 -O-L-R7
-N(R6)-L-R7 -S-L-R7 -C(0)-L-R7 -S-L-R7
-N(R6)-L-R7 -N(R6)-L-R7 -C(0)-L-R7 -N(R6)-L-R7
-N(R6)-L-R7 -S(0)c-N(R6)-L-R7 -C(0)-L-R7 -S(0)c-N(R6)-L-R7
-N(R6)-L-R7 -S(0)c-NR18-C(0)- -C(0)-L-R7 -S(0)c-NR18-C(0)- N(R6)-L-R7 N(R6)-L-R7
-N(R6)-L-R7 -C(0)-N(R6)-L-R7 -C(0)-L-R7 -C(0)-N(R6)-L-R7
-N(R6)-L-R7 -(CH2)d-N(R6)-S(0)b- -C(0)-L-R7 -(CH2)d-N(R6)-S(0)b- L-R7 L-R7
-N(R6)-L-R7 -(CH2)d-N(R6)-C(0)- -C(0)-L-R7 -(CH2)d-N(R6)-C(0)- N(R19)-S(0)b-L-R7 N(R19)-S(0)b-L-R7
-N(R6)-L-R7 -C(0)-L-R7 -C(0)-L-R7 -C(0)-L-R7
-N(R6)-L-R7 -C(0)0-L-R7 -C(0)-L-R7 -C(0)0-L-R7
Figure imgf000024_0001
61 -C(0)-N(R6)-L-R7 -O-L-R7 -N(R11)CR13R14- -O-L-R7
N(R6)-S(0)b-L-R7
62 -C(0)-N(R6)-L-R7 -S-L-R7 -N(R11)CR13R14- -S-L-R7
N(R6)-S(0)b-L-R7
63 -C(0)-N(R6)-L-R7 -N(R6)-L-R7 -N(R11)CR13R14- -N(R6)-L-R7
N(R6)-S(0)b-L-R7
64 -C(0)-N(R6)-L-R7 -S(0)c-N(R6)-L-R7 -N(R11)CR13R14- -S(0)c-N(R6)-L-R7
N(R6)-S(0)b-L-R7
65 -C(0)-N(R6)-L-R7 -S(0)c-NR18-C(0)- -N(R11)CR13R14- -S(0)c-NR18-C(0)- N(R6)-L-R7 N(R6)-S(0)b-L-R7 N(R6)-L-R7
66 -C(0)-N(R6)-L-R7 -C(0)-N(R6)-L-R7 -N(R11)CR13R14- -C(0)-N(R6)-L-R7
N(R6)-S(0)b-L-R7
67 -C(0)-N(R6)-L-R7 -(CH2)d-N(R6)-S(0)b- -N(R11)CR13R14- -(CH2)d-N(R6)-S(0)b- L-R7 N(R6)-S(0)b-L-R7 L-R7
68 -C(0)-N(R6)-L-R7 -(CH2)d-N(R6)-C(0)- -N(R11)CR13R14- -(CH2)d-N(R6)-C(0)- N(R19)-S(0)b-L-R7 N(R6)-S(0)b-L-R7 N(R19)-S(0)b-L-R7
69 -C(0)-N(R6)-L-R7 -C(0)-L-R7 -N(R11)CR13R14- -C(0)-L-R7
N(R6)-S(0)b-L-R7
70 -C(0)-N(R6)-L-R7 -C(0)0-L-R7 -N(R11)CR13R14- -C(0)0-L-R7
N(R6)-S(0)b-L-R7
71 -C(0)-N(R6)-L-R7 -N=C(NR11R12)- -N(R11)CR13R14- -N=C(NR11R12)- N(R6)-S(0)b-L-R7 N(R6)-S(0)b-L-R7 N(R6)-S(0)b-L-R7
72 -C(0)-N(R6)-L-R7 -N(R11)CR13R14- -N(R11)CR13R14- -N(R11)CR13R14- N(R6)-S(0)b-L-R7 N(R6)-S(0)b-L-R7 N(R6)-S(0)b-L-R7
Table 2: Selected Combinations of R1 and R2 groups in chromogenic compounds
Figure imgf000025_0001
Figure imgf000026_0001
Figure imgf000027_0001
Figure imgf000028_0001
[0070] In embodiments, moiety -(CH2)d-N(R6)-S(0)b-L-R7 on each occasion in Table 1 or Table 2 is -N(R6)-S(0)b-L-R7, in an embodiment of which R6 is H. Integer b is 2 in one class of the combinations of Table 1 and Table 2. It will be recalled that integers b and c are 2 in particular classes of the R1 and R2 groups in Table 1 and Table 2. It will also be recalled that R6, R11, R12, R13, R14, R18 and R19 are H in some embodiments. For all R1 and R2 groups in Table 1 and Table 2, and for all moieties described in this paragraph, L is in particular a bond and R7 is in particular phenyl or a 5- or 6- membered heteroaromatic ring, which rings may be substituted as described herein or unsubstituted. However, in the case of -O-L-R7 and -S-L-R7, -L-R7 is H in one class of compounds. Compounds of Table 2 in which R2 is -NH-2-pyridyl or - NH-4-pyridyl may comprise 1 , 2, 3 or 4 substituents on the pyridyl moiety as previously described in this specification.
[0071] It will further be recalled that the invention includes compounds of Formula (VII) and salts and physiological precursors thereof:
Figure imgf000029_0001
[0072] wherein X, Y, R and R are as previously described, and R -R are each the same as or different from each other and selected from H and R . In one embodiment, all of R -R are H. In another embodiment, one or two of R -R are R and the remainder are H.The invention also includes structures of Formulae (VIII)-(XI):
Figure imgf000029_0002
in which:
R u, R I, and " are the same or different and selected from H and R°, in particular H, F, CI, hydroxy, alkyl having 1 , 2, 3, 4 or 5 carbon atoms, hydroxyalkyi having 1 , 2, 3, 4 or 5 carbon atoms, alkyl having 1 , 2, 3 or 4 carbon atoms and interrupted by -0-, -S-, or - C(O)-, hydroxyalkyi having 1 , 2, 3 or 4 carbon atoms and interrupted by -0-, -S-, or -C(O)-, trifluoromethyl, trifluoromethoxy, cyano, nitro, oxo, amidino, -OR9, -C(0)R9, -C(0)OR9, - OC(0)R9, -N(R9)R10, -C(0)N(R9)R10, -S(0)aR9 and -S(0)aOR9, wherein R9 and R10 are the same or different and are each hydrogen or are selected from alkyl optionally substituted by hydroxy and having 1 , 2, 3, 4 or 5 carbon atoms, the aforesaid alkyl moieties more particularly being methyl or ethyl; and
R1 and R2 are as defined or described in, or with reference to, Table 1 or Table 2 above.
[0073] In one embodiment of compounds of Formula (VII)-(XI), and in particular of Formula (VIII), in which R2 is of Formula (lla), R23 is a hydrogen bond donor, for example -NHR9 or OH. In an embodiment R23 is -NH2. In certain embodiments R5a is S02 7 [0074] In one embodiment of compounds of Formula (VII)-(XI), and in particular of Formula
(VIII), in which R is of Formula (lla), R is not a hydrogen bond acceptor, for example cyano, nitro, -C(0)R9, -C(0)OR9, -S(0)aOR9. In an embodiment R23 is not nitro. In certain embodiments R5a is S02R7
[0075] The disclosure includes compounds in which all of R -R are H or in which one, two, three or four of R -R are R , e.g. one, two or three of them are R as in the case of compounds in which one or two R -R of are R ,and the remainder are H. In some compounds, a single one of R -R is R . Exemplary R moieties as described herein in paragraph [0038] are applicable to Formulae (VI l)-(XI) and embodiments described herein in relation to those Formulae, including by way of example Formulae (XII) and (XIII)-(XVIII) below.
[0076] In one class of structures of Formula (VII), Formula (VIII), Formula (IX), Formula (X) and Formula (XI), R2 is -NH-S(0)c-L-R7; R1 may be an R1 group of Table 1 or another of the possibilities described herein for R1. Integer c may be 2. R2 may therefore be -NH-S(0)2-L-R7, where L is in particular a bond and/or R7 is in particular phenyl or a 5- or 6- membered heteroaromatic ring, which rings may be substituted as described herein or unsubstituted.
[0077] Particularly to be mentioned are compounds, especially those featuring structures (VII), (VIII), (IX), (X) and (XI) above, in which R2 is -NHS02R7 and R1 is -NHR7, wherein the R7 moieties of R1 and R2 are the same or different. In particular, R2 is -NHS02R25, where R25 is phenyl or a 5- or 6-membered heteroaromatic ring, for example is phenyl or 2-thiophenyl, in either case unsubstituted or substituted by one or more R8 (e.g. selected from halogen, alkyl and trifluoromethyl); and R1 is -NHR26, where R26 is phenyl which is unsubstituted or substituted by one or more R8 (e.g. selected from halogen, alkyl and trifluoromethyl). Exemplary R25 groups are unsubstituted phenyl and unsubstituted thiophenyl. Exemplary R26 groups are unsubstituted phenyl, 4-halophenyl (e.g. 4-bromophenyl, 4-chlorophenyl or 4- fluorophenyl) and 3,5-dimethylphenyl; another class of R21 groups comprises 4-methylphenyl and 3,5-dihalophenyl, e.g. where halo is Br.
[0078] The invention therefore includes compounds of Formula (XII) in which the symbols are as previously defined (so that the two R7 moieties may be the same or different)
Figure imgf000030_0001
[0079] In a particular embodiment, the compounds of Formula (XII) are of Formula (XIII) below:
Figure imgf000031_0001
wherein R -R are as previously described (i.e. are most broadly defined as each being the same or different and H or R8), and may for example be selected from H, halogen, methyl and trifluoromethyl, and more particularly are all H. R25 is phenyl or thiophenyl (particularly 2- thiophenyl), and is unsubstituted or substituted by one or more R8, e.g. 1 , 2 or 3 R8. In one embodiment phenyl and thiophenyl are unsubstituted. In another embodiment phenyl and thiophenyl are substituted by one or more R8, in which case R8 may, for example, be halogen (e.g. F, CI or Br), methyl or trifluoromethyl. R26 is phenyl which is unsubstituted or substituted by 1 , 2 or 3 R8; in one embodiment phenyl is unsubstituted and, when substituted, R8 may, for example, be halogen, hydroxy, alkyl optionally substituted by hydroxy and having 1 , 2, 3, 4 or 5 carbon atoms, alkyl optionally substituted by hydroxy and having 1 , 2, 3 or 4 carbon atoms and interrupted by -0-, -S-, or -C(O)-, trifluoromethyl, trifluoromethoxy, cyano or nitro, and in particular R8 is in this case halogen (e.g. F, CI or Br), methyl or trifluoromethyl. In one variant embodiment of Formula (XIII), a single one of R25 and R26, particularly R25, is a dye as previously described in this specification.
[0080] Particular structures (XIII) are formulae (XIV)-(XVII) below, in which each R8 is selected inde endently of each other R8:
(XV)
Figure imgf000031_0002
Figure imgf000032_0001
[0081] The invention also includes compounds of Formula (XIX) in which the symbols are as previously defined (so that the two R7 moieties ma be the same or different)
Figure imgf000032_0002
[0082] In a particular embodiment, the compounds of Formula (XIX) are of Formula (XX) below:
Figure imgf000032_0003
wherein R -R are as previously described (i.e. are most broadly defined as each being the same or different and H or R8), and may for example be selected from H, halogen, methyl and trifluoromethyl, and more particularly are all H. R25 is phenyl or thiophenyl (particularly 2- thiophenyl), and is unsubstituted or substituted by one or more R8, e.g. 1 , 2 or 3 R8. In one embodiment phenyl and thiophenyl are unsubstituted. In another embodiment phenyl and thiophenyl are substituted by one or more R8, in which case R8 may, for example, be halogen (e.g. F, CI or Br), methyl or trifluoromethyl. R27 is phenyl which is unsubstituted or substituted by 1 , 2 or 3 R ; in one embodiment phenyl is unsubstituted and, when substituted, R may, for example, be halogen, hydroxy, alkyl optionally substituted by hydroxy and having 1 , 2, 3, 4 or 5 carbon atoms, alkyl optionally substituted by hydroxy and having 1 , 2, 3 or 4 carbon atoms and interrupted by -0-, -S-, or -C(O)-, trifluoromethyl, trifluoromethoxy, cyano or nitro, and in particular R8 is in this case halogen (e.g. F, CI or Br), methyl or trifluoromethyl. In one variant embodiment of Formula (XX), a single one of R25 and R27, particularly R25, is a dye as previously described in this specification.
[0083] The invention also includes compounds of Formula (XXI) in which the symbols are as previously defined (so that the two R7 moieties may be the same or different)
Figure imgf000033_0001
[0084] In a particular embodiment, the compounds of Formula (XXI) are of Formula (XXII) below:
Figure imgf000033_0002
wherein R -R are as previously described (i.e. are most broadly defined as each being the same or different and H or R8), and may for example be selected from H, halogen, methyl and trifluoromethyl, and more particularly are all H. R25 is phenyl or thiophenyl (particularly 2- thiophenyl), and is unsubstituted or substituted by one or more R8, e.g. 1 , 2 or 3 R8. In one embodiment phenyl and thiophenyl are unsubstituted. In another embodiment phenyl and thiophenyl are substituted by one or more R8, in which case R8 may, for example, be halogen (e.g. F, CI or Br), methyl or trifluoromethyl. R is phenyl which is unsubstituted or substituted by 1 , 2 or 3 R8; in one embodiment phenyl is unsubstituted and, when substituted, R8 may, for example, be halogen, hydroxy, alkyl optionally substituted by hydroxy and having 1 , 2, 3, 4 or 5 carbon atoms, alkyl optionally substituted by hydroxy and having 1 , 2, 3 or 4 carbon atoms and interrupted by -0-, -S-, or -C(O)-, trifluoromethyl, trifluoromethoxy, cyano or nitro, and in particular R8 is in this case halogen (e.g. F, CI or Br), methyl or trifluoromethyl. In one variant embodiment of Formula (XXII), a single one of R25 and R26, particularly R25, is a dye as previously described in this specification. [0085] In some structures (XII), (XIII), (XIV), (XV), (XVI), (XVII), (XVIII), (XIX), (XX), (XXI) and
(XXII) all of R -R are H. In other structures, one, two, three or four (e.g. one or two, as in the case of one) of R -R is/are an R moiety (the remainder being H), e.g. selected from halogen, hydroxy, alkyl optionally substituted by hydroxy and having 1 , 2, 3, 4 or 5 carbon atoms, alkyl optionally substituted by hydroxy and having 1 , 2, 3 or 4 carbon atoms and interrupted by -0-, -S-, or -C(O)- (e.g. Ci-C4 alkoxy), trifluoromethyl, trifluoromethoxy, cyano and nitro, and in particular the or each R8 is in this case selected from halogen (e.g. F, CI or Br), methyl and trifluoromethyl. Compounds having structures (XII), (XIII), (XIV), (XV), (XVI), (XVII) and (XVIII), (XIX), (XX), (XXI) and (XXII) are chromogenic, and these compounds and their salts are therefore included in the compounds of the disclosure which may be used as chromogenic probes to detect the presence or absence of hlMATI .
Uses
[0086] The compounds of the invention bind to hlMATI and are useful to label hlMATI , or to inhibit hlMATI for medical or investigative purposes. Included in the invention are chromogenic compounds in which R3 and R4 form a benzene ring, for example naphthoquinones in which X and Y are both O (Formula (VIII)). The chromogenic compounds exhibit an optically detectable response after binding to hlMATI by changing the wavelength of absorbed or emitted light.
[0087] It may readily be determined which compounds are chromogenic, for example by testing them following the procedure of Example 57.
[0088] All exemplified compounds exhibited a colour change with varying pH except for 3, 4, 20, 34, 46, 47, 53.
[0089] Compounds of formula (VII) in which R1 is a halogen may be used as intermediates in the formation of chromogenic compounds of the invention. Such compounds are not themselves choromogenic. Such compounds may, however, exhibit inhibition of hNAT1 .
[0090] Compounds of formula (VII) in which both R1 and R2 are -O-L-R7 may be used as intermediates in the formation of chromogenic compounds of the invention. The colour changes observed with varying pH exhibited by these compounds may be due to hydrolysis rather than deprotonation. All exemplified compounds of this form exhibit inhibition of hNAT1 .
[0091] All exemplified compounds exhibited a % inhibition of hNAT1 of greater than 60% within experimental variation, except for those selected from the group comprising: 25, 40, 41 , 42, 43, 44, 46, 47, 48, 70. The observed % inhibition in the case of compound 70 may have been depressed by use of an aged enzyme sample and the true inhibitory activity of compound 70 may therefore be higher than 60%. [0092] The group of compounds comprising: 25, 40, 41 , 42, 43, 44, 46, 47, 48, 70 exhibited a exhibited a % inhibition of hlMATI of below 60%, within experimental variation
[0093] It will be recalled that the invention comprises chromogenic compounds in which in which R2 is of Formula (I la), i.e. is -NHR5a. The invention provides a method for detecting hlMATI , comprising contacting a sample with a chromogenic compound of the invention to form a labelling composition, incubating the labelling composition for a sufficient amount of time for the compound of the invention to bind to any hlMATI in the sample, illuminating the composition with an appropriate wavelength for the compound and observing for absorbance/emission of light having a wavelength characteristic of the compound when bound to hlMATI . The sample is one which has been taken from, or is derived from, a human subject, particularly a female. Samples may contain one or more human cells or may be a lysate of one or more human cells. The sample may be a lysed biopsy of breast tissue.
[0094] The invention also provides the ex vivo (e.g. in vitro) use of the chromogenic compounds of the disclosure as chromogenic probes to detect or measure hNAT1 in samples derived from human subjects, in particular breast cancer cells. The samples may be lysates of breast tissue biopsies. The samples may be, or may be derived from, tissue removed from a patient during surgery.
[0095] A particular benefit of using the chromogenic compounds as chromogenic probes is that the colour change (whether changing from one colour to another, colouration or decolouration) takes place virtually instantaneously (e.g. within a second) upon the compound contacting hNAT1 . This makes the compounds particularly useful for detecting cancerous hNAT1 -bearing cells during surgery. For example, a surgeon may remove a breast tumour and apply a chromogenic compound to the margins of the tumour to establish if the marginal tissue contains cancerous cells or is free of them; if the marginal tissue is free of cancerous cells (no colour change indicative of this), then the surgeon need remove no further tissue but, if the compound undergoes a colour change indicative of cancer cells in the marginal tissue, then further tissue will be removed with a view to ensuring removal of all cancerous cells. The colour change may be determined by eye or using a spectrophotometer. Suitably, a small sample may be taken of the tissue for the purposes of the chromogenic test. A similar procedure may be applied to auxiliary node tissue removed during surgery. If desired, a chromogenic compound of the disclosure may be applied to an area of tissue within the body to determine whether cancerous hNAT1 -bearing cells are present in that area.
[0096] .The invention therefore provides a method of surgical treatment of breast cancer of a patient, comprising:
removing tissue known or to comprise, or at risk of comprising, cancer cells; applying a chromogenic compound of the disclosure to at least a marginal portion of the removed tissue; and
observing said at least marginal portion for emission of light having a wavelength characteristic of the compound when bound to hNATI .
[0097] In one embodiment, light having a wavelength characteristic of the compound when bound to hNATI is not observed and no further tissue is removed from the patient. In another embodiment, light having a wavelength characteristic of the compound when bound to hNATI is observed, further tissue is removed from the patient, the further removed tissue has applied to at least a marginal portion thereof a compound of any of claims 18 to 21 , and said at least marginal portion is observed for emission of light having a wavelength characteristic of the compound when bound to hNATI .
[0098] Also included in the invention is a method of detecting cancer cells during breast cancer surgery, comprising applying to an area of tissue at risk of presenting a cancer cell a chromogenic compound of the disclosure, and observed said area for emission of light having a wavelength characteristic of the compound when bound to hNATI . Further provided by the invention is a chromogenic compound of the disclosure for use in diagnosing tissue during surgery for the presence of cancer cells.
[0099] In those aspects of the invention where tissue is contacted with a chromogenic compound to determine if breast cancer cells are present, the cancer may in particular be ER+ breast cancer or an invasive ductal and/or lobular breast carcinoma.
[00100] A sample can be illuminated with a wavelength of light selected to give a detectable optical response, and observed with a means for detecting the optical response. Equipment that is useful for illuminating the present compounds and compositions of the invention includes, but is not limited to, hand-held ultraviolet lamps, mercury arc lamps, xenon lamps, lasers and laser diodes. These illumination sources are typically optically integrated into laser scanners, fluorescence microplate readers or standard or microfluorometers.
[00101] The chromogenic compounds may, at any time after or during an assay, be illuminated with a wavelength of light that results in a detectable optical response, and observed with a means for detecting the optical response. This optical emission is optionally detected by visual inspection, or by use of any of the following devices: CCD cameras, video cameras, photographic film, laser scanning devices, photodiodes, quantum counters, scanning microscopes, flow cytometers, or by means for amplifying the signal such as photomultiplier tubes.
[00102] The compounds of the disclosure are useful in inhibiting hNATI in the treatment or prevention of disease. In embodiments, the compounds are useful in treating oestrogen receptor positive (ER+) breast cancer. In embodiments, the compounds are useful in treating cancer having upregulated hNAT1 , in particular ER+ breast cancer having upregulated hNATL In embodiments, the compounds are useful in treating invasive ductal and lobular breast carcinomas.
[00103] The invention therefore includes a method of treating cancer in a human subject, comprising administering a therapeutically effective amount of a compound. The cancer may be breast cancer, in particular ER+ breast cancer or an invasive ductal and/or lobular breast carcinoma.
[00104] The invention further includes compounds of the disclosure for therapeutic use, particularly for use in methods of treating cancer.
[00105] The compounds of the disclosure may be administered in the form of pharmaceutically acceptable salts. The pharmaceutically acceptable salts of the present disclosure can be synthesized from the parent compound which contains a basic or acidic moiety by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two; generally, nonaqueous media like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred. Lists of suitable salts are found in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., US, 1985, p. 1418, the disclosure of which is hereby incorporated by reference; see also Stahl et al, Eds, "Handbook of Pharmaceutical Salts Properties Selection and Use", Verlag Helvetica Chimica Acta and Wiley-VCH, 2002.
[00106] The disclosure thus includes pharmaceutically acceptable salts of the disclosed compounds wherein the parent compound is modified by making acid or base salts thereof, for example the conventional non-toxic salts or the quaternary ammonium salts which are formed, e.g., from inorganic or organic acids or bases. Examples of such acid addition salts include acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, pamoate, pectinate, persulfate, 3-phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, tosylate, and undecanoate. Base salts include ammonium salts, alkali metal salts such as sodium and potassium salts, alkaline earth metal salts such as calcium and magnesium salts, salts with organic bases such as dicyclohexylamine salts, N-methyl-D-glucamine, and salts with amino acids such as arginine, lysine, and so forth. Also, the basic nitrogen-containing groups may be quaternized with such agents as lower alkyl halides, such as methyl, ethyl, propyl, and butyl chloride, bromides and iodides; dialkyl sulfates like dimethyl, diethyl, dibutyl; and diamyl sulfates, long chain halides such as decyl, lauryl, myristyl and stearyl chlorides, bromides and iodides, aralkyl halides like benzyl and phenethyl bromides and others.
[00107] The invention includes prodrugs for the active pharmaceutical species of the invention, for example in which one or more functional groups are protected or derivatised but can be converted in vivo to the functional group, as in the case of esters of carboxylic acids convertible in vivo to the free acid, or in the case of protected amines, to the free amino group. The term "prodrug," as used herein, represents in particular compounds which are rapidly transformed in vivo to the parent compound, for example, by hydrolysis in blood. A thorough discussion is provided in T. Higuchi and V. Stella, Pro-drugs as Novel Delivery Systems, Vol. 14 of the A.C.S. Symposium Series, Edward B. Roche, ed., Bioreversible Carriers in Drug Design, American Pharmaceutical Association and Pergamon Press, 1987; H Bundgaard, ed, Design of Prodrugs, Elsevier, 1985; and Judkins, et al. Synthetic Communications, 26(23), 4351 -4367 (1996), each of which is incorporated herein by reference.
[00108] Prodrugs therefore include drugs having a functional group which has been transformed into a reversible derivative thereof. Typically, such prodrugs are transformed to the active drug by hydrolysis. As examples may be mentioned the following:
Functional Group Reversible derivative
Carboxylic acid Esters, including e.g. acyloxyalkyl esters, amides
Alcohol Esters, including e.g. sulfates and phosphates as
well as carboxylic acid esters
Amidine Amidoximes, carbamateamidino
Amine Amides, carbamates, imines, enamines,
Carbonyl (aldehyde, Imines, oximes, acetals/ketals, enol esters,
ketone) oxazolidines and thiazoxolidines [00109] Prodrugs also include compounds convertible to the active drug by an oxidative or reductive reaction. As examples may be mentioned:
Oxidative activation
• N- and O- dealkylation
• Oxidative deamination
· N-oxidation • Epoxidation
Reductive activation
• Azo reduction
• Sulfoxide reduction
· Disulfide reduction
• Bioreductive alkylation
• Nitro reduction.
[00110] Also to be mentioned as metabolic activations of prodrugs are nucleotide activation, phosphorylation activation and decarboxylation activation. For additional information, see "The Organic Chemistry of Drug Design and Drug Action", R B Silverman (particularly Chapter 8, pages 497 to 546), incorporated herein by reference.
[00111] The use of protecting groups is fully described in "Protective Groups in Organic Chemistry", edited by J W F McOmie, Plenum Press (1973), and "Protective Groups in Organic Synthesis" , 2nd edition, T W Greene & P G M Wutz, Wiley-lnterscience (1991 ).
[00112] Thus, it will be appreciated by those skilled in the art that, although protected derivatives of compounds of the disclosure may not possess pharmacological activity as such, they may be administered, for example parenterally or orally, and thereafter metabolised in the body to form compounds of the disclosure which are pharmacologically active. Such derivatives are therefore examples of "prodrugs". All prodrugs and physiological precursors of the described compounds are included within the scope of the disclosure.
[00113] Groups referred to or featured herein (especially those containing heteroatoms and conjugated bonds) may exist in tautomeric forms and all these tautomers are included in the scope of the disclosure. More generally, many species may exist in equilibrium, as for example in the case of organic acids and their counterpart anions; a reference herein to a species accordingly includes reference to all equilibrium forms thereof.
[00114] The compounds of the disclosure may also contain one or more asymmetric carbon atoms and may therefore exhibit optical and/or diastereoisomerism. All diastereoisomers may be separated using conventional techniques, e.g. chromatography or fractional crystallisation. The various stereoisomers may be isolated by separation of a racemic or other mixture of the compounds using conventional, e.g. fractional crystallisation or HPLC, techniques. Alternatively the desired optical isomers may be made by reaction of the appropriate optically active starting materials under conditions which will not cause racemisation or epimerisation, or by derivatisation, for example with a homochiral acid followed by separation of the diastereomeric derivatives by conventional means (e.g. HPLC, chromatography over silica). All stereoisomers are included within the scope of the disclosure. [00115] Geometric isomers may also exist in the compounds of the present disclosure. The present disclosure contemplates the various geometric isomers and mixtures thereof resulting from the arrangement of substituents around a carbon-carbon double bond and designates such isomers as of the Z or E configuration, wherein the term "Z" represents substituents on the same side of the carbon-carbon double bond and the term "E" represents substituents on opposite sides of the carbon-carbon double bond.
[00116] Compounds containing one or more asymmetric carbon atoms can exist as two or more stereoisomers. Where a compound contains an alkenyl or alkenylene group, geometric cis/trans (or Z/E) isomers are possible. Where structural isomers are interconvertible via a low energy barrier, tautomeric isomerism ('tautomerism') can occur. This can take the form of proton tautomerism in compounds containing, for example, an imino, keto, or oxime group, or so-called valence tautomerism in compounds which contain an aromatic moiety. It follows that a single compound may exhibit more than one type of isomerism.
[00117] Included within the scope of the present invention are all stereoisomers, geometric isomers and tautomeric forms of the compounds of formula I, including compounds exhibiting more than one type of isomerism, and mixtures of one or more thereof. Also included are acid addition or base salts wherein the counter ion is optically active, for example, c/-lactate or /- lysine, or racemic, for example, c//-tartrate or c//-arginine.
[00118] Cis/trans isomers may be separated by conventional techniques well known to those skilled in the art, for example, chromatography and fractional crystallisation.
[00119] Conventional techniques for the preparation/isolation of individual enantiomers include chiral synthesis from a suitable optically pure precursor or resolution of the racemate (or the racemate of a salt or derivative) using, for example, chiral high pressure liquid chromatography (HPLC).
[00120] Alternatively, the racemate (or a racemic precursor) may be reacted with a suitable optically active compound, for example, an alcohol, or, in the case where the compound of formula (I) contains an acidic or basic moiety, a base or acid such as 1 -phenylethylamine or tartaric acid. The resulting diastereomeric mixture may be separated by chromatography and/or fractional crystallization and one or both of the diastereoisomers converted to the corresponding pure enantiomer(s) by means well known to a skilled person.
[00121] When any racemate crystallises, crystals of two different types are possible. The first type is the racemic compound (true racemate) referred to above wherein one homogeneous form of crystal is produced containing both enantiomers in equimolar amounts. The second type is the racemic mixture or conglomerate wherein two forms of crystal are produced in equimolar amounts each comprising a single enantiomer. [00122] While both of the crystal forms present in a racemic mixture have identical physical properties, they may have different physical properties compared to the true racemate. Racemic mixtures may be separated by conventional techniques known to those skilled in the art - see, for example, "Stereochemistry of Organic Compounds" by E. L. Eliel and S. H. Wilen (Wiley, 1994).
[00123] The present disclosure includes all pharmaceutically acceptable isotopically-labelled compounds) 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 which predominates in nature.
[00124] Examples of isotopes suitable for inclusion in the compounds of the disclosure include
¾ 11 1^ 14
isotopes of hydrogen, such as H and H, carbon, such as C, C and C, chlorine, such as 36CI, fluorine, such as 18F, iodine, such as 123l and 125l, nitrogen, such as 13N and 15N, oxygen, such as 150, 170 and 180, phosphorus, such as 32P, and sulphur, such as 35S.
[00125] The disclosure therefore includes all variant forms of the defined compounds, for example any tautomer or any pharmaceutically acceptable salt, ester, acid or other variant of the defined compounds and their tautomers as well as substances which, upon administration, are capable of providing directly or indirectly a compound as defined above or providing a species which is capable of existing in equilibrium with such a compound.
Administration of the Products of the Disclosure
[00126] In the context of therapeutic use, the compounds may be administered alone or as compositions in combination with pharmaceutically acceptable diluents, excipients or carriers.
[00127] The compounds of the invention will normally be administered orally, 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 prodrug or active 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. Depending upon the disorder and patient to be treated and the route of administration, the compositions may be administered at varying doses.
[00128] Typically, therefore, the pharmaceutical compounds of the invention may be administered orally or parenterally ("parenterally" as used herein, refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion.) to a host to obtain an protease- inhibitory effect. [00129] The compounds of the invention may be used in monotherapy or combined or coadministered with another active pharmaceutical ingredient, particular one useful in treating cancer patients. In particular,the compounds of the invention may be combined or coadministered with another active agent for use in treating cancer, e.g. breast cancer, in particular treating cancer having upregulated hlMATI , in particular ER+ breast cancer such as, for example, ER+ breast cancer having upregulated hlMATI . In embodiments, the other active agent is useful in treating invasive ductal and lobular breast carcinomas.
[00130] Actual dosage levels of active ingredients in the 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 for to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved.
[00131] Envisaged suitable daily doses of the compounds of the invention in therapeutic treatment of humans are about 0.001 -100 mg/kg body weight at peroral administration and 0.001 -50 mg/kg body weight at parenteral administration. A peroral dose of from 0.02 to 15 mg/Kg of body weight is envisaged.
[00132] The active compound may be given as a single dose, in multiple doses or as a sustained release formulation.
[00133] According to a further aspect of the invention there is thus provided a pharmaceutical composition including a compound of the invention, in admixture with a pharmaceutically acceptable adjuvant, diluent or carrier.
[00134] Pharmaceutical compositions of this invention for parenteral injection suitably comprise pharmaceutically acceptable sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions as well as sterile powders for reconstitution into sterile injectable solutions or dispersions just prior to use. Examples of suitable aqueous and nonaqueous 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. Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants.
[00135] These compositions may also contain adjuvants such as preservative, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol 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 aluminum monostearate and gelatin) which delay absorption.
[00136] Suitably, oral formulations contain a dissolution aid. The dissolution aid is not limited as to its identity so long as it is pharmaceutically acceptable. Examples include nonionic surface active agents; bile acid and salts thereof; ionic surface active agents; triethanolamine soap, and alkyl quaternary ammonium salts; and amphoteric surface active agents.
[00137] 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. The active compounds may also be in micro-encapsulated form, if appropriate, with one or more of the above-mentioned excipients.
[00138] The active compounds may be in finely divided form, for example it may be micronised.
[00139] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups and elixirs. In addition to the active compounds, the liquid dosage forms may contain inert diluents commonly used in the art. Besides inert diluents, the oral compositions may also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring 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, aluminum metahydroxide, bentonite, agar-agar, and tragacanth and mixtures thereof.
[00140] Compositions for rectal or vaginal administration are preferably 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.
[00141] Compounds of the present invention can also be administered in the form of liposomes. As is known in the art, liposomes are generally derived from phospholipids or other lipid substances. Liposomes are formed by mono- or multi-lamellar hydrated liquid crystals which are dispersed in an aqueous medium. Any non-toxic, physiologically acceptable and metabolisable lipid capable of forming liposomes can be used. The present compositions in liposome form can contain, in addition to a compound of the present invention, stabilisers, preservatives, excipients and the like. Particular lipids are the phospholipids and the phosphatidyl cholines (lecithins), both natural and synthetic. Methods to form liposomes are known in the art, for example, Prescott, Ed., Methods in Cell Biology, Volume XIV, Academic Press, New York, N.Y. (1976), p 33 et seq.
Synthesis
[00142] The compounds of the disclosure may be made by applying standard synthetic procedures to commercially available quinones or thioxoquinones, for example 2,3,5,6- tetrachloro-1 ,4-benzoquinone, 2-amino-3-chloro-1 ,4-naphthoquinone, methyl-p-benzoquinone, sodium 3-hydroxy-1 ,4-naphthoquinone-2-sulfonate, 2, 3-dihydoroxy-1 ,4-naphthoquinone, 2- alkylamino-3-alkylthio-1 ,4-naphthoquinones, 3-alkyl-2-hydroxy-1 ,4-naphthoquinones, disodium-1 ,4-dioxo-1 ,2,3,4-tetrahydro-2,3-naphthalenedisulfonate, 2-bromo-1 ,4- benzoquinone, 2, 3-dichloro-6-nitro-1 ,4-naphthoquinone, 2, 5-dibromo-1 ,4-benzoquinone, 2,3- dichloro-5-nitro-1 ,4-naphthoquinone, 2,3-dichloro-5,8-dihydroxy-1 ,4-naphthoquinone, 2- hydroxy-1 ,4-naphthoquinone, 5-hydroxy-1 ,4-naphthoquinone or 2, 3-dichloronaphthalene-1 ,4- dione. The (naphtha)quinone starting material may be substituted and the molecule then further elaborated as desired.
[00143] In one exemplary procedure, 2, 3-dichloronaphthalene-1 ,4-dione is used as a starting material for successive steps of nucleophilic aromatic substitution of the quinone ring. In a first step, 2, 3-dichloronaphthalene-1 ,4-dione is contacted with a sulphonamide R31S02NH2 in the presence of a base to carry out a nucleophilic substitution reaction, thereby replacing a chlorine substituent with the sulphonamide to obtain a 2-sulphonamido-3-chloronaphthalene- 1 ,4-dione. The reaction is suitably performed in an aprotic solvent, e.g. a non-polar organic solvent, for example toluene. Alternatively, an aprotic polar solvent may be used. The reaction is suitably carried out at elevated temperature, e.g. under reflux. A further nucleophilic aromatic substitution is then carried out to replace the second chlorine, for example by contacting the 2-sulphonamido-3-chloronaphthalene-1 ,4-dione with an amine, e.g. a primary amine, in the presence of a Lewis acid (e.g. CeCI3). The reaction is suitably performed in a polar solvent and particularly an aqueous solvent, for example water. The reaction is suitably carried out at elevated temperature, e.g. under reflux. R is a fragment of a sulphonamide substituent R1 as disclosed herein such that the product compound is a compound of the disclosure, e.g. it may be a moiety R25 as defined elsewhere in this specification. R32 is a fragment of an R2 substituent as disclosed herein such that the product compound is a compound of the disclosure, e.g. it may be a moiety R26 as defined elsewhere in this specification. The reaction is illustrated by the following reaction scheme (A):
Reaction scheme (A)
Figure imgf000045_0001
[00144] In a further exemplary procedure the 2-sulphonamido-3-chloronaphthalene-1 ,4-dione is coupled with a coupling agent (e.g. an aryl boronic acid) in the presence of a transition metal or transition metal containing compound (e.g. Pd or a palladium-containing compound such as Pd(PPh3)2CI2). This reaction is illustrated by reaction scheme B. The reaction is suitably performed in the presence of a base. The reaction may be performed in a polar aprotic solvent (eg. THF) or a polar protic solvent (eg. H20). The reaction is suitably carried out at elevated temperature, e.g. under reflux. The reaction is illustrated by the following reaction scheme (B):
Reaction scheme (B)
Figure imgf000045_0002
[00145] In another exemplary procedure 2, 3-dichloronaphthalene-1 ,4-dione is used as a starting material for successive steps of double chloride displacement with phenol and the displacement of one equivalent of phenol with a sulphonamide. In a first step a double substitution reaction occurs in which 2, 3-dichloronaphthalene-1 ,4-dione is contacted with phenol in the presence of a base in an addition-elimination reaction thereby replacing the two chlorine substituents with two equivalents of a phenol. The reaction is suitable carried out in an aprotic polar organic solvent, such as THF. The reaction is suitably carried out at elevated temperature, e.g. under reflux. In a second step the diphenol substituted naphalene is contacted with a sulphonamide in the presence of a base to carry out a nucleophilic
substitution reaction, thereby replacing one phenolic substituent with the sulphonamide. The procedure is exemplified by reaction scheme (C):
Reaction scheme (C)
Figure imgf000045_0003
[00146] R1,R2, R3 and R4 of reaction schemes A, B, and C are fragments such that the compound is a compound of the invention.
[00147] Compounds of the disclosure in which R1 is halogen, in particular CI, Br or I, are therefore useful as intermediates for making compounds in which R1 is another group, for example one having a nitrogen directly bonded to the quinone ring.
EXAMPLES
[00148] All reactions involving moisture-sensitive reagents were carried out under an atmosphere of dry nitrogen via standard vacuum line techniques, using flame-dried glassware allowed to cool under vacuum. Reactions which are described as being performed at temperatures over 25 °C were heated by means of a silicone oil bath. All solvents were reagent grade and used as supplied unless otherwise stated. Solvents were dried according to the proceeding outlined by Grubbs and co-workers. Pangborn, A. B.; Giardello, M. A.; Grubbs, R. H.; Rosen, R. K.; Timmers, F. J. Organometallics, 1996, 15, 1518. All reagents were used as supplied (analytical or HPLC grade), without further purification. Unless otherwise stated, all aqueous solutions were saturated and magnesium sulphate was used to dry organic layers. Column chromatography was performed using silica gel (Kieselgel 60). Thin layer chromatography was performed on Merck aluminium sheets coated with 0.2 mm silica gel 60 F254. Plates were visualised either by UV light (254 nm) or potassium permanganate 1 % (w/v) in 2% (v/v) aqueous acetic acid, containing 7% (w/v) potassium carbonate). Reverse-phase High Performance Liquid Chromatography (RP-HPLC) was performed on a Gilson instrument equipped with Gilson 306 pumps, a Gilson 81 1 C dynamic mixer, a Gilson 806 manometric module with automated sample injection on a Gilson 215 Liquid Handler, configured with a Gilson 819 valve actuator. Separations were performed on a Varian Omnisphere 5 C18 (analytical) column (5 μηι particle size, 150.0 mm x 4.6 mm). All experiments were performed under gradient elution: solvent A (H20 containing 0.1 % (v/v) TFA) and solvent B (CH3CN), starting from 95% A, 5% B to 5% A, 95% B over 8 minutes, then isocratic for 4 minutes, unless otherwise stated. The flow rate was 1 .0 mL/min. Detection was at wavelengths of 220, 254 and 290nm using a Gilson 170 Diode Array Detector. Melting points were measured on a Gallenkamp Hot Stage apparatus and are uncorrected. Infrared spectra were recorded as KBr discs using a Bruker Tensor 27 FT-IR spectrophotometer. Selected characteristics peaks are reported in cm-1 . NMR spectra were recorded on Bruker Avance spectrometers in the deuterated solvent stated. Chemical shifts (δ) are reported in parts per million (p. p.m.) and are referenced to the residual solvent peak. Coupling constant (J) are measured in Hertz and are calculated using a first-order approximation. Low resolution mass spectra (m/z) were recorded on either VG MassLab 20-250 or Micromass platform 1 spectrometer. Accurate mass measurements were run on either a Bruker MicroTOF internally calibrated with polyalanine, or a Micromass CGT instrument fitted with a Scientific Glass Instruments BPX5 column (15 m χ 0.25 mm) using amyl acetate as a lock mass, by the mass spectrometry department of the Chemistry Research Laboratory, University of Oxford, UK. Major peaks are listed with intensities quoted as percentages of the base peak.
[00149] General Procedure 1 : Substitution of 2,3-dichloronaphthalene-1 ,4-dione 2
2,3-Dichloronaphthalene-1 ,4-dione 2 (1 .0 eq), the requisite sulphonamide (1.3 eq) and Cs2C03 (1.3 eq) were refluxed in the stated solvent under N2 over the specified time.
[00150] General Procedure 2: Substitution of /V-(3-Chloro-1 ,4-dioxo-1 , 4-dihvdronaphthalen-2- vD-arylsulfonamide with amines
/V-(3-Chloro-1 ,4-dioxo-1 ,4-dihydronaphthalen-2-yl)-arylsulfonamide (1 .0 eq) was stirred with CeCI3.7H20 (0.4 eq) in toluene at RT for 90 minutes. The requisite amine (3.0 eq) was added and the mixture was refluxed for 24 h (with anilines) or 6 h (with aliphatic amines). The solution was cooled to RT and partitioned between EtOAc and sat. aq. NH4CI. The organic extract was washed with brine, before being dried, filtered and concentrated in vacuo to give the crude reaction mixture.
[00151] General Procedure 3: Coupling between /V-(3-Chloro-1 ,4-dioxo-1 ,4- dihydronaphthalen-2-yl)-arylsulfonamide and an aryl boronic acid
/V-(3-Chloro-1 ,4-dioxo-1 ,4-dihydronaphthalen-2-yl)-arylsulfonamide (1 .0 eq) and Pd(PPh3)2CI2 (0.1 eq, 42 mg, 0.06 mmol) were stirred in THF for 10 mintues. The requisite arylboronic acid and sat. aq. NaHC03 were added and reaction mixture was refluxed for 18 h. The solution was cooled to RT and partitioned between EtOAc and 1 M aq. HCI. The organic extract was washed with brine, before being dried, filtered and concentrated in vacuo to give the crude reaction mixture.
[00152] General Procedure 4: Disubstitution of 2,3-dichloronaphthalene-1 ,4-dione 2
2, 3-Dichloronaphthalene-1 ,4-dione 2 (1 .0 eq, 454 mg, 2 mmol), the requisite phenol (2.2 eq) and Cs2C03 (2.2 eq, 1 .43 g, 4.4 mmol) were refluxed in THF for 18 h. The solution was cooled to RT and partitioned between EtOAc and 0.1 M aq. NaOH. The organic extract was washed with sat. aq. NH4CI and brine, before being dried, filtered and concentrated in vacuo to give the crude reaction mixture.
[00153] General Procedure 5: Substitution of diphenols
The requisite diphenol (1 .0 eq), benzenesulfonamide (1 .2 eq) and Cs2C03 (1 .2 eq) were refluxed in THF for 1 h. The solution was cooled to RT and partitioned between EtOAc and 1 M aq. HCI. The organic extract was washed with brine, before being dried, filtered and concentrated in vacuo to give the crude reaction mixture which was purified by recrystallisation from toluene to give the required phenoxy analogue in pure form.
[00154]
Example 1
Preparation of W-(3'-chloro-1 ',4'-dioxo-1 ',4'-dihydronaphthalen-2'-yl)benzene- sulphonamide 3
Figure imgf000048_0001
[00155] 2,3-Dichloronaphthalene-1 ,4-dione (5.0 g, 22.03 mmol) was reacted with benzenesulphonamide (4.5 g, 28.64 mmol) and Cs2C03 (9.3 g, 28.64 mmol) in toluene (100 ml_). The reaction mixture was concentrated in vacuo and re-suspended in water (40 ml_). The suspension was filtered and the residue was washed with hot water to yield 3 (5.5 g, 72%) as a purple solid; mp 250 °C; δΗ (400 MHz, DMSO-d6) 7.38 - 7.48 (2H, m, Ar), 7.62 - 7.70 (2H, m, Ar), 7.74 (1 H, t, J 7.51 , Ar), 7.83 - 7.89 (2H, m, Ar), 7.92 (2H, d, J 7.17, Ar); 5C (50 MHz, DMSO-de) 125.3, 125.4, 126.2, 128.0, 128.1 , 128.4, 128.5, 129.6, 130.4, 131 .1 , 132.0, 132.6, 133.9, 148.4, 175.8 (C=0), 180.1 (C=0); m/z (ESI") 346 (M-H, 100 %); HRMS (ESI") Ci6H9CIN04S [35M-H]~ requires 345.9935, found 345.9941 .
Example 2
Preparation of thiophene-2-sulphonamide
Figure imgf000048_0002
[00156] Thiophene-2-sulfonyl chloride (0.5 g, 2.74 mmol) was added in a 25 % ammonium hydroxide solution (5 ml_, 33.45 mmol) refluxing at 50 °C, and stirred for 15 minutes. The reaction mixture was filtered while boiling and the residue was washed with boiling 25% ammonium hydroxide aqueous solution. Ammonium hydroxide solution was removed in vacuo until the ammonia smell disappeared and the target compound was re-crystallised from water (0.328 g, 77%) as a white solid; mp 145 - 146 °C; vmax (KBr) 3290 (N-H), 3224 (N-H); δΗ (400 MHz, DMSO-de) 7.15 (1 H, dd, J 5.0, 3.7, C(4)H), 7.55 (1 H, dd, J 3.7, 1 .4, C(3)H), 7.66 (2H, s, NH2), 7.85 (1 H, dd, J 5.0, 1 .4, C(5)H); 5C (100 MHz, DMSO-d6) 127.3, 130.0, 131 .1 , 145.7; m/z (ESI") 162 ([M-H] ", 100 %); HRMS (ESI") C4H4N02S2 " ([M-H] ") requires 161 .9689; found 161 .9695.
Example 3 Preparation of /V-(3'-chloro-1 ',4'-dioxo-1 ',4'-dihydronaphthalen-2'-yl)thiophene-2- sulphonamide 4
Figure imgf000049_0001
[00157] 2,3-dichloronaphthalene-l ,4-dione (56 mg, 0.24 mmol), thiophene-2-sulfonamide (49 mg, 0.31 mmol) and Cs2C03 (102 mg, 0.31 mmol) were stirred in refluxing toluene (10 mL) at 1 10 °C for 18 h. The reaction mixture was concentrated in vacuo and re-suspended in ethyl acetate (5 mL). The suspension was washed with 1 M aqueous K2C03 solution (3 x 5 mL), the organic phase was separated, dried, filtered and concentrated in vacuo, affording 4 (45 mg, 54 %) as a red solid; mp >250 °C; vmax (KBr) 3328 (N-H), 1663 (C=0), 713 (C-CI); δΗ (400 MHz, DMSO-de) 6.99 (1 H, dd, J 5.0, 3.7, C(4)H), 7.43 (1 H, dd, J 3.7, 1.4, C(3)H), 7.59 (1 H, dd, J 5.0, 1 .4, C(5)H), 7.70 (1 H, td, J 7.00, 1 .86, C(6')H or C(7')H), 7.77 (1 H, td, J 7.00, 1 .86, C(6')H or C(7')H), 7.90 (1 H, dd, J 7.00, 1 .86, C(5')H or C(8')H), 7.93 (1 H, dd, J 7.00, 1 .86, C(5')H or C(8')H); 5c (100 MHz, DMSO-d6) 124.6, 125.4, 125.5, 125.6, 126.0, 126.3, 130.9, 131 .4, 132.2, 132.4, 133.9, 134.5, 175.93 (C=0), 179.56 (C=0); m/z (ESI") 352 ([M-H]", 100 %); HRMS (ESI") Ci4H7CIN04S2" ([35M-H]") requires 351 .951 1 ; found 351 .9508.
Example 4
Preparation of Λ/-(1 ',4'-dioxo-3'-(phenylamino)-1 ',4'-dihydronaphthalen-2'-yl)benzene- sulphonamide 1
Figure imgf000049_0002
1
[00158] /V-(3'-chloro-1 ',4'-dioxo-1 ',4'-dihydronaphthalen-2'-yl)benzenesulphonamide 3 (0.22 g, 0.63 mmol) was stirred with CeCI3.7H20 (0.10 g, 0.25 mmol) in toluene (20 mL) for 1 h, then aniline (66 μί, 0.70 mmol) was added and refluxed at 1 10 °C for 2 days. The reaction mixture was cooled and filtered and the residue was re-crystallised from ethyl acetate to give 1 (0.16 g, 63 %) as a dark red solid; mp >300 °C {lit. mp >300 °C}; δΗ (400 MHz, DMSO-d6) 6.99 - 7.10 (2H, m, Ar), 7.23 (2H, t, J 7.9, Ar), 7.31 - 7.41 (3H, m, Ar), 7.45 - 7.51 (1 H, m, Ar), 7.52 - 7.58 (1 H, m, Ar), 7.72 - 7.82 (3H, m, Ar), 7.98 - 8.04 (3H, m, Ar, NH), 8.98 - 9.04 (1 H, m, NH); 5C (126 MHz, DMSO-de) 123.5, 125.6, 125.6, 126.2, 126.5, 127.5, 128.5, 128.9, 130.4, 131 .5, 131 .8, 132.1 , 133.0, 134.9, 138.6, 141 .0, 178.5 (C=0), 182.3 (C=0); m/z (ESI") 403 (M-H, 100 %); HRMS (ESI") C22H16N2Na04S [M+Na]+ requires 427.0723, found 427.0726.
[00159] Compound 1 changes color from red to blue when it binds to mNat2 or hlMATI . Some other compounds do the same. This property is useful to provide information about the presence and/or amount of human NAT1 in a sample, for example a cell lysate mixture, and the a diagnostic kit for ER+ breast cancer.
Example 5
Preparation of W-(3'-(4"-bromophenylamino)-1 ',4'-dioxo-1 ',4'-dihydronaphthalen-2'-yl) benzen
Figure imgf000050_0001
[00160] Following General Procedure 2, /V-(3'-chloro-1 ',4'-dioxo-1 ',4'-dihydronaphthalen-2'- yl)benzenesulphonamide 3 (0.2 g, 0.57 mmol) was stirred with CeCI3.7H20 (0.09 g, 0.23 mmol) in toluene (15 mL) for 1 h, then 4-bromoaniline (0.1 1 g, 0.63 mmol) was added and refluxed at 1 10 °C for 2 days. Purification of the crude product via flash column chromatography (1 :9 to 1 :1 ethyl acetate/30-40o petrol) afforded 5 (27 mg, 10 %) as a dark red solid; mp 200 °C; δΗ (400 MHz, DMSO-de) 7.35 - 7.60 (5H, m, Ar), 7.73 - 7.79 (3H, m, Ar), 7.81 - 7.86 (4H, m, Ar),7.96 - 8.02 (1 H, m, Ar) ; 5C (100 MHz, DMSO-d6) 125.6, 126.2, 127.4, 128.3, 128.7, 128.9, 129.1 , 129.6, 130.1 , 131 .3, 131 .5, 131 .7, 131 .8, 133.1 , 133.3, 144.1 , 178.5(C=0), 183.2 (C=0); m/z (ESI") 481 ([79M-H]", 100%), 483 ([81M-H]", 97%); HRMS (ESI") C22H14BrN204S" ([M- H]") requires 480.9863; found 480.9875.
Example 6
Preparation of W-(3'-(4"-tert-butylphenylamino)-1 ',4'-dioxo-1 ',4'-dihydronaphthalen-2'- yl)benzenesulphonamide 6
Figure imgf000051_0001
[00161] Following General Procedure 2, /V-(3'-chloro-1 \4'-dioxo-1 ',4'-dihydronaphthalen-2'- yl)benzenesulphonamide 3 (0.2 g, 0.57 mmol) was stirred with CeCI3.7H20 (0.09 g, 0.23 mmol) in toluene (12 ml.) for 1 h, then 4-ie f-butylaniline (100 μΙ_, 0.63 mmol) was added and refluxed at 1 10 °C for 2 days. Purification of the crude product via flash column chromatography (5:95 to 1 :2 ethyl acetate/30-40o petrol) gave 6 (0.16 g, 60 %) as a dark violet solid; mp > 300 °C; vmax (KBr) 3445 (N-H), 1683 (C=0); δΗ (400 MHz, DMSO-d6) 1.30 (9H, s, *Bu), 6.90 - 7.26 (4H, C(2")H, C(3")H, C(5")H and C(6")H), 7.33 - 7.59 (5H, m, C(2)H, C(3)H, C(4)H, C(5)H and C(6)H), 7.72 - 8.01 (4H, m, C(5')H, C(6')H, C(7')H and C(8')H); 5C (100 MHz, DMSO-d6) 31 .3 (Me), 34.1 (C(Me)3), 122.9, 124.2, 125.6, 126.1 , 126.5, 127.4, 128.5, 129.0, 129.6, 130.5, 131 .5, 132.0, 132.2, 132.9, 134.0, 134.8, 178.3(C=0), 182.3 (C=0); m/z (ESI") 459.1 ([M-H]\ 100%); HRMS (ESI+) C26H24N2Na04S+ ([M+Na]+) requires 483.1349; found 483.1338.
Example 7
Preparation of W-(3'-(3",5"-dimethylphenylamino)-1 ',4'-dioxo-1 ,4-dihydronaphthalen-2- yl)benzenesulphonamide 7
Figure imgf000051_0002
7
[00162] Following General Procedure 2, /V-(3'-chloro-1 ',4'-dioxo-1 ',4'-dihydronaphthalen-2'- yl)benzenesulphonamide 3 (0.2 g, 0.57 mmol) was stirred with CeCI3.7H20 (0.09 g, 0.23 mmol) in toluene (12 mL) for 1 h, then 3,5-dimethylaniline (80 μΙ_, 0.63 mmol) was added and refluxed at 1 10 °C for 2 days. Purification of the crude product via flash column chromatography (5:95 to 1 :4 ethyl acetate/30-40o petrol) yielded 7 (0.13 g, 53 %) as a dark red solid; mp 180 °C; vmax (KBr) 3314 (N-H), 3186 (N-H), 1670 (C=0); δΗ (400 MHz, DMSO-d6) 2.25 (6H, s, Me), 6.60 (2H, s, C(2")H and C(6")H), 6.68 (1 H, s, C(4")H), 7.32 - 7.58 (5H, m, C(2)H, C(3)H, C(4)H, C(5)H and C(6)H), 7.75 - 8.05 (4H, m, C(5')H, C(6')H, C(7')H and C(8')H), 8.78 (1 H, s, NH), 9.05 (1 H, s, NH); 5C (100 MHz, DMSO-d6) 21 .0 {Me), 120.9, 125.3, 125.6, 125.7, 126.2, 126.4, 128.4, 128.9, 130.3, 131 .6, 131 .8, 131 .9, 133.0, 134.9, 136.4, 138.1 , 178.7 (C=0), 182.3 (C=0); m/z (ESI") 431 .1 ([M-H]\ 100%); HRMS (ESI+) C24H2oN2Na04S+ ([M+Na]+) requires 455.1036; found 455.1031 .
Example 8
Preparation of Λ/-(1 4'-dioxo-3'-(phenylamino)-1 ',4'-dihydronaphthalen-2'-yl)thiophene-2- sulphonamide 8
Figure imgf000052_0001
[00163] Following General Procedure 2, /V-(3'-chloro-1 ',4'-dioxo-1 ',4'-dihydronaphthalen-2'- yl)thiophene-2-sulphonamide 4 (20 mg, 0.057 mmol) was stirred with CeCI3.7H20 (9 mg, 0.023 mmol) in toluene (5 mL) for 1 h, then aniline (6 μΙ_, 0.063 mmol) was added and refluxed at 1 10 °C for 2 days. Purification of the crude product by flash column chromatography (1 :9 to 1 :3 ethyl acetate/30-40o petrol) yielded 8 (20 mg, 88 %) as a red solid; 180 °C; δΗ (400 MHz, DMSO-de) 6.97 - 6.99 (1 H, m, C(4)H), 7.24 - 7.29 (2H, m, Ar), 7.32 - 7.35 (1 H, m, C(3)H), 7.37 - 7.41 - 1 H, m, Ar), 7.37 - 7.53 (1 H, m, Ar), 7.54 - 7.58 (1 H, m, Ar), 7.72 - 7.76 (1 H, m, C(5)H), 7.78 - 7.86 (3H, m, Ar), 8.02 - 8.06 (1 H, m, Ar), 9.1 1 (1 H, s, NH), 9.18 (1 H, s, NH); 5C (100 MHz, DMSO-de) 123.8, 125.7, 126.3, 127.2, 127.4, 127.9, 128.4, 128.5, 129.0, 129.6, 131 .6, 131 .7, 132.5, 133.0, 138.4, 142.3, 178.4 (C=0), 182.3 (C=0); m/z (ESI") 409 ([M-H]", 100 %); C20H14N2NaO4S2 + ([M+Na]+) requires 433.0287; found 433.0284.
Example 9 yV-(3-(2-Chlorophenyl)-1 ,4-dioxo-1 ,4-dihydronaphthalen-2-yl)benzenesulfonamide 9
[00164] Following General Procedure 3, /V-(3-chloro-1 ,4-dioxo-1 ,4- dihydronaphthalen-2-yl)benzenesulfonamide 3 (200 mg, 0.58 mmol) and
2-chlorophenylboronic acid (181 mg, 1 .14 mmol) gave the crude reaction
mixture. Purification via column chromatography on silica gel (eluent pet
ether:acetone 75:25) and subsequent recrystallisation from toluene gave
Figure imgf000052_0002
9 as a bright yellow solid (1 19 mg, 48%); mp 194-196 °C; umax (KBr) 3206 (N-H), 1673 (C=0), 1656 (C=0); δΗ (400 MHz, Acetone-d6) 7.21 -7.29 (1 H, m, Ar), 7.33 (3H, m, Ar) 7.44-7.53 (2H, m, Ar), 7.59 (1 H, s, Ar), 7.67-7.76 (2H, m, Ar), 7.85-7.97 (2H, m, Ar), 8.05-8.14 (2H, m, Ar), 8.61 (1 H, br. s, NH); 5C (100 MHz, Acetone-d6) 126.4, 126.8, 126.8, 126.9, 129.1 , 129.3, 129.4, 130.8, 131 .2, 131 .6, 132.4, 132.6, 132.9, 134.4, 134.8, 135.2, 140.7, 141 .8, 181 .2 (C=0), 182.5 (C=0); m/z (ESI") 422 ([M-H]\ 100%); HRMS (ESI+) C22H14CINNa04S+, ([M+Na]+) requires 446.0224; found 446.0225.
Example 10
A/-(3-(3-Chlorophenyl)-1 ,4-dioxo-1 ,4-dihydronaphthalen-2-yl)benzenesulfonamide 10
[00165] Following General Procedure 3, /V-(3-chloro-1 ,4-dioxo-1 ,4- dihydronaphthalen-2-yl)benzenesulfonamide 3 (200 mg, 0.58 mmol) and
3-chlorophenylboronic acid (181 mg, 1 .14 mmol) gave the crude reaction
mixture. Purification via column chromatography on silica gel (eluent pet
etheracetone 75:25) and subsequent recrystallisation from toluene gave
Figure imgf000053_0001
10 as a bright yellow solid (87 mg, 35%); mp 168-172°C; umax (KBr) 3229 (N-H), 1670 (C=0), 1651 (C=0); δΗ (400 MHz, Acetone-d6) 7.26-7.31 (3H, m, Ar), 7.33 (1 H, s, Ar), 7.44-7.51 (2H, m, Ar), 7.57-7.63 (1 H, m, Ar), 7.63-7.69 (2H, m, Ar), 7.84-7.94 (2H, m,
Figure imgf000053_0002
8.04-8.13 (2H, m,
Figure imgf000053_0003
8.53 (1 H, br. s, NH); 5C (100 MHz, Acetone-c/6) 126.6, 126.7, 126.9, 128.9, 129.1 , 129.6, 129.7, 130.9, 131 .2, 132.5, 133.1 , 133.3, 134.3, 134.4, 135.1 , 136.1 , 139.7, 141 .7, 181 .6 (C=0), 183.4 (C=0); m/z (ESI") 422 ([M-H]", 100%); HRMS (ESI") C22H13CIN04S", ([M-H]") requires 422.0259; found 422.0258.
Example 1 1
A/-(3-(4-Chlorophenyl)-1 ,4-dioxo-1 ,4-dihydronaphthalen-2-yl)benzenesulfonamide 11
[00166] Following General Procedure 3, /V-(3-chloro-1 ,4-dioxo-1 ,4- dihydronaphthalen-2-yl)benzenesulfonamide 3 (200 mg, 0.58 mmol) and
4-chlorophenylboronic acid (181 mg, 1 .14 mmol) gave the crude
reaction mixture. Purification via column chromatography on silica gel
Figure imgf000053_0004
(eluent pet etheracetone 80:20) and subsequent recrystallisation from toluene gave 11 as a bright yellow solid (1 15 mg, 47%); mp 222-223°C; umax (KBr) 3240 (N-H), 1627 (C=0), 1655 (C=0); δΗ (400 MHz, DMSO-d6) 7.21 -7.27 (2H, m, chlorophenyMr), 7.30-7.38 (2H, m, chlorophenyl-/Ar), 7.43-7.51 (2H, m, sulphonamide-/Ar), 7.54-7.66 (3H, m, sulphonamide-/Ar), 7.80-7.93 (2H, m, naphthoquinone-/*/-), 7.97-8.05 (2H, m, naphthoquinone-/*/-), 9.93 (1 H, br. s, N-H); 5c (100 MHz, DMSO-d6) 126.4, 126.7, 127.0, 127.2, 128.4, 129.5, 129.8, 131 .2, 131 .5, 132.4, 132.7, 133.0, 133.1 , 134.2, 135.1 , 135.5 (C-CI), 140.0 (S02-C), 142.7 (C-NHS02Ph), 182.0 (C=0), 184.0 (C=0), rotameric compound - 22 signals observed; m/z (ESI") 422 ([M-H]", 100%); HRMS (ESI") C22H13CIN04S", ([M-H]") requires 422.0259; found 422.0260. Example 12
W-(3-(3-Formylphenyl)-1 ,4-dioxo-1 ,4-dihydronaphthalen-2-yl)benzenesulfonamide 12
[00167] Following General Procedure 3, /V-(3-chloro-1 ,4-dioxo-1 ,4- dihydronaphthalen-2-yl)benzenesulfonamide 3 (200 mg, 0.58 mmol)
and 3-formylphenylboronic acid (174 mg, 1 .14 mmol) gave the crude
reaction mixture. Purification via column chromatography on silica gel
Figure imgf000054_0001
(eluent pet etheracetone 70:30) and subsequent recrystallisation
from toluene gave 12 as a bright yellow solid (74.1 mg, 31 %); mp 200-201 °C; umax (KBr) 3229 (N-H), 1698 (C=0), 1671 (C=0), 1651 (C=0); δΗ (400 MHz, Acetone-d6) 7.37-7.45 (2H, m, Ar), 7.49-7.61 (4H, m, Ar), 7.63-7.70 (1 H, m, Ar), 7.75-7.85 (2H, m, Ar), 7.87-7.97 (2H, m, Ar- naphthoquinone), 8.06-8.16 (2H, m,
Figure imgf000054_0002
8.69 (1 H, br. s, N-H), 9.89 (1 H, s, CHO); 5c (100 MHz, Acetone-d6) 126.5, 126.7, 126.9, 128.7, 129.2, 129.8, 131 .2, 132.1 , 132.5, 133.0, 133.4, 134.4, 135.2, 136.2, 136.5, 137.2, 139.6, 141 .7, 181 .7 (C=0 a- sulphonamide), 183.6 (C=0 a-aryl), 192.0 (CHO); m/z (ESI") 416 ([M-H]", 100%); HRMS (ESI") C23H14NO5S", ([M-H]") requires 416.0598; found 416.0596.
Example 13
W-(3-(4-Formylphenyl)-1 ,4-dioxo-1 ,4-dihydronaphthalen-2-yl)benzenesulfonamide 13 [00168] Following General Procedure 3, /V-(3-chloro-1 ,4-dioxo-1 ,4- dihydronaphthalen-2-yl)benzenesulfonamide 3 (200 mg, 0.58 mmol)
and 4-formylbenzeneboronic acid (174 mg, 1 .14 mmol) gave the
crude reaction mixture. Purification via column chromatography on
Figure imgf000054_0003
silica gel (eluent pet etheracetone 70:30) and subsequent recrystallization from toluene gave 13 as a bright yellow solid (59 mg, 24%); mp 200-202 °C; umax (KBr) 3234 (N-H), 1698 (C=0), 1668 (C=0), 1650 (C=0); δΗ (400 MHz, DMSO-d6) 7.37-7.50 (4H, m, formylphenyMr), 7.51 - 7.65 (3H, m, sulphonamide-/*/-), 7.76-7.84 (2H, m, sulphonamide- */-), 7.85-7.96 (2H, m, naphthoquinone-/*/-), 7.98-8.08 (2H, m, naphthoquinone-/*/-), 10.02 (1 H, s, CHO), N-H signal not observed; 5C (100 MHz, DMSO-d6) 126.7, 127.0, 127.2, 129.3, 129.5, 131 .5, 132.1 , 132.4, 133.1 , 135.1 , 135.6, 136.5, 138.7, 139.1 , 140.0, 142.5, 182.0 (C=0), 183.9 (C=0), 193.8 (CHO); m/z (ESI") 416 ([M-H]", 100%); HRMS (ESI") C23H14N05S", ([M-H]") requires 416.0598; found 416.0600.
Example 14 yV-(3-([1 ,1 '-Biphenyl]-4-yl)-1 ,4-dioxo-1 ,4-dihydronaphthalen-2-yl)benzenesulfonamide 14 [00169] Following General Procedure 3, /V-(3-chloro-1 ,4-dioxo-1 ,4- dihydronaphthalen-2-yl)benzenesulfonamide 3 (200 mg, 0.58
mmol) and 4-biphenylboronic acid (230 mg, 1 .14 mmol) gave the
crude reaction mixture. Purification via column chromatography on
silica gel (eluent pet etheracetone 75:25) and subsequent
Figure imgf000055_0001
recrystallisation from toluene gave 14 as a bright yellow solid (107 mg, 40%); mp 228-230 °C; umax (KBr) 3264 (N-H), 1671 (C=0), 1646 (C=0); δΗ (400 MHz, DMSO-d6) 7.31 -7.38 (2H, m, Ar), 7.38-7.45 (3H, m, Ar), 7.48-7.56 (3H, m, Ar), 7.58-7.67 (4H, m, Ar), 7.68-7.75 (2H, m, Ar), 7.80-7.94 (2H, m, naphthoquinone-o/f/?o-H), 7.99-8.07 (2H, m, naphthoquinone-mefa-H), 9.95 (1 H, br. s, N-H); 5C (100 MHz, DMSO-d6) 126.4, 126.6, 126.7, 127.0, 127.2, 127.6, 128.6, 129.4, 129.9, 131 .4, 131 .5, 131 .9, 132.5, 132.9, 135.1 , 135.5, 139.8, 140.5, 141 .0, 142.8 (C- NHS02Ph), 182.1 (C=0 a-sulphonamide), 184.3 (C=0 a-biphenyl); m/z (ESI") 464 ([M-H]\ 100%); HRMS (ESI") C28H18N04S", ([M-H]") requires 464.0962; found 464.0963.
Example 15 yV-(1 ,4-Dioxo-3-(4-(trifluoromethoxy)phenyl)-1 ,4-dihydronaphthalen-2- yl)benzenesulfonamide 15
[00170] Following General Procedure 3, /V-(3-chloro-1 ,4-dioxo-1 ,4- dihydronaphthalen-2-yl)benzenesulfonamide 3 (200 mg, 0.58 mmol)
and 4-trifluoromethoxybenzeneboronic acid (239 mg, 1 .14 mmol) gave
the crude reaction mixture. Purification via column chromatography on
Figure imgf000055_0002
silica gel (eluent pet etheracetone 80:20) and subsequent recrystallisation from toluene gave 15 as a bright yellow solid (103 mg, 37%); mp 178-180 °C; umax (KBr) 3245 (N-H), 1669 (C=0), 1651 (C=0); δΗ (400 MHz, Acetone-d6) 7.16-7.26 (2H, m, Ar), 7.43-7.53 (4H, m, Ar), 7.58-7.65 (1 H, m, Ar), 7.67-7.72 (2H, m, Ar), 7.85-7.96 (2H, m, Ar), 8.06-8.14 (2H, m, Ar), 8.55 (1 H, br.s, N-H); 5c (100 MHz, Acetone-d6) 120.2, 126.6, 126.8, 126.9, 129.1 , 131 .2, 132.5, 133.0, 133.1 , 134.4, 135.1 , 136.4, 139.6, 141 .8, 144.9, 149.4, 181 .6 (C=0), 183.5 (C=0), CF3 not observed; m/z (ESI") 472 ([M-H]", 100%); HRMS (ESI+) C23H14F3NNa05S+, ([M+Na]+) requires 496.0437; found 496.0439.
Example 16
W-(3-(2-Methoxyphenyl)-1 ,4-dioxo-1 ,4-dihydronaphthalen-2-yl)benzenesulfonamide 16 [00171] Following General Procedure 3, /V-(3-chloro-1 ,4-dioxo-1 ,4- o
^lL
dihydronaphthalen-2-yl)benzenesulfonamide 3 (200 mg, 0.58 mmol)
and 2-methoxybenzeneboronic acid (176 mg, 1 .14 mmol) gave the U
o
crude reaction mixture. Purification via column chromatography on silica JU gel (eluent pet etheracetone 70:30) and subsequent recrystallisation from toluene gave 16 as a bright yellow solid (97 mg, 40%); mp 223-224 °C; umax (KBr) 3241 (N-H), 1661 (C=0), 1626 (C=0); δΗ (400 MHz, DMSO-d6) 3.67 (3H, s, OMe); 6.90-6.97 (1 H, m, Ar), 7.01 (1 H, s, Ar) 7.05-7.13 (1 H, m, Ar), 7.37 (1 H, m, Ar), 7.44-7.53 (2H, m, Ar), 7.54-7.63 (1 H, m, Ar), 7.66-7.75 (2H, m, Ar), 7.80-7.92 (2H, m, Ar), 7.93-8.08 (2H, m, Ar), 9.74 (1 H, br. s, N-H); 5C (100 MHz, DMSO-de) 56.3 {OMe), 1 12.0, 120.7, 121 .1 , 126.8, 127.1 , 129.4, 129.8, 131 .2, 131 .6, 131 .8, 132.3, 132.9, 135.0, 135.5, 141 .2, 143.2, 157.7, 181 .7 (C=0), 183.6 (C=0); m/z (ESI") 418 ([M- H]", 100%); HRMS (ESI+) C23H17NNa05S+, ([M+Na]+) requires 442.0720; found 442.0719.
Example 17
W-(3-(3-Methoxyphenyl)-1 ,4-dioxo-1 ,4-dihydronaphthalen-2-yl)benzenesulfonamide 17
[00172] Following General Procedure 3, /V-(3-chloro-1 ,4-dioxo-1 ,4- o
dihydronaphthalen-2-yl)benzenesulfonamide 3 (200 mg, 0.58 mmol)
and 3-methoxybenzeneboronic acid (176 mg, 1 .14 mmol) gave the
o
crude reaction mixture. Purification via column chromatography on u silica gel (eluent pet etheracetone 70:30) and subsequent recrystallisation from toluene gave 17 as a bright yellow solid (141 mg, 58%); mp 165-169 °C; umax (KBr) 3228 (N-H), 1675 (C=0), 1649 (C=0); δΗ (400 MHz, Acetone-d6) 3.75 (3H, s, OMe), 6.82-6.95 (2H, m, Ar), 7.16-7.29 (1 H, m, Ar), 7.44-7.53 (2H, m, Ar), 7.55-7.64 (1 H, m, Ar), 7.67-7.76 (2H, m, Ar), 7.82-7.95 (2H, m,
Figure imgf000056_0001
8.35 (1 H, s, N-H); 5C (100 MHz, Acetone-de) 54.9 {OMe), 1 14.8, 1 16.2, 123.3, 126.5, 126.8, 126.8, 129.0, 129.2, 131 .4, 132.5, 132.8, 133.0, 134.2, 134.9, 137.1 , 139.8, 142.1 , 159.5, 181 .6 (C=), 183.5 (C=0); m/z (ESI") 418 ([M-H]", 100%); HRMS (ESI") C23H16N05S", ([M-H]") requires 418.0755; found 418.0735.
Example 18 Λ/-(1 ,4-Dioxo-3-(2,3,4-trimethoxyphenyl)-1 ,4-dihydronaphthalen-2-yl)benzenesulfonamide 18
[00173] Following General Procedure 3, /V-(3-chloro-1 ,4-dioxo-1 ,4- dihydronaphthalen-2-yl)benzenesulfonamide 3 (200 mg, 0.58 mmol)
Figure imgf000056_0002
and 2,3,4-trimethoxybenzeneboronic acid (246 mg, 1 .14 mmol) gave the crude reaction mixture. Purification via column chromatography on silica gel (eluent pet ethenacetone 75:25) and subsequent recrystallisation from toluene gave 32 as a bright yellow solid (63 mg, 23%); mp 158-162 °C; umax (KBr) 3251 (N-H), 1672 (C=0), 1632 (C=0); δΗ (400 MHz, DMSO-d6) 3.68 (3H, s, O-Me), 3.76 (3H, s O-Me), 3.83 (3H, s O-Me), 6.65-6.73 (1 H, m, Ar), 6.76-6.83 (1 H, m, Ar), 7.44-7.54 (2H, m, Ar), 7.56-7.63 (1 H, m, Ar), 7.66-7.74 (2H, m, Ar), 7.82-7.94 (2H, m, Ar), 7.96-8.08 (2H, m, Ar), 9.78 (1 H, br. s, N-H); 5C (100 MHz, DMSO-d6) 56.7, 61 .0, 61 .3, 107.9, 1 18.7, 126.0, 126.7, 127.1 , 129.4, 129.8, 131 .5, 132.4, 133.0, 135.0, 135.5, 138.4, 140.9, 141 .9, 143.1 , 152.4, 155.1 , 181 .8 (C=0), 183.8 (C=0); m/z (ESI") 478 ([M-H]\ 100%); HRMS (ESI+) C25H21NNa07S+, ([M+Na]+) requires 502.0931 ; found 502.0932.
Example 19 yV-(3-(3-Fluoro-4-methoxyphenyl)-1 ,4-dioxo-1 ,4-dihydronaphthalen-2- yl)benzenesulfonamide 19 [00174] Following General Procedure 3, /V-(3-chloro-1 ,4-dioxo-1 ,4- dihydronaphthalen-2-yl)benzenesulfonamide 3 (200 mg, 0.58 mmol)
and 3-fluoro-4-methoxyphenylboronic acid (197 mg, 1 .14 mmol) gave
the crude reaction mixture. Purification via column chromatography on
Figure imgf000057_0001
silica gel (eluent pet ethenacetone 75:25) and subsequent
recrystallisation from toluene gave 19 as a bright yellow solid (94 mg, 37%); mp 253-255 °C; umax (KBr) 3443 (N-H), 1679 (C=0), 1649 (C=0); δΗ (400 MHz, DMSO-d6) 3.86 (3H, s, O-Me), 6.99-7.09 (3H, m, 3-fluoro-4-methoxyphenyl-H), 7.41 -7.51 (2H, m, sulphonamide-H), 7.53-7.67 (3H, m, sulphonamide-H), 7.83-7.92 (2H, m, naphthoquinone-H) 7.97-8.06 (2H, m, naphthoquinone-H), 9.87 (1 H, br. s, N-H); 5C (100 MHz, DMSO-d6) 56.7 (O-Me), 1 13.6, 1 18.9, 1 19.1 , 124.5, 126.7, 126.9, 127.2, 128.0, 129.4, 131 .5, 132.4, 133.0, 135.0, 135.4, 139.7, 142.8 (C-NHS02Ph), 148.2 (C-OMe), 152.6 (C-F), 182.1 (C=0 a-sulphonamide), 184.2 (C=0 a-3-fluoro-4-methoxyphenyl); m/z (ESI") 436 ([M-H]", 100%); HRMS (ESI") C23H15FN05S", ([M- H]") requires 436.0660; found 436.0663.
Example 20 Λ/-(1 ,4-Dioxo-3-(pyridin-3-yl)-1 ,4-dihydronaphthalen-2-yl)benzenesulfonamide 20
[00175] Following General Procedure 3, /V-(3-chloro-1 ,4-dioxo-1 ,4- dihydronaphthalen-2-yl)benzenesulfonamide 3 (200 mg, 0.58 mmol) and
pyridine-3-boronic acid (143 mg, 1 .14 mmol) gave the crude reaction
Figure imgf000057_0002
mixture. Purification via column chromatography on silica gel (eluent pet ethenacetone 65:35) and subsequent recrystallisation from toluene gave 20 as a bright yellow solid (56 mg, 22%); mp 237-238 °C; umax (KBr) 3210 (N-H), 1671 (C=0), 1649 (C=0); δΗ (400 MHz, DMSO-d6) 7.39- 7.51 (3H, m, Ar), 7.53-7.60 (1 H, m, Ar), 7.62-7.69 (2H, m, Ar), 7.75-7.81 (1 H, m, Ar), 7.82-7.93 (2H, m, Ar), 7.96-8.06 (2H, m, Ar), 8.52 (1 H, s, pyridine-/*/-), 8.53 (1 H, s, pyridine-/*/-), N-H not observed; 5C (100 MHz, DMSO-d6) 123.8, 126.7, 127.0, 127.1 , 129.1 , 129.5, 129.8, 131 .7, 132.5, 133.0, 134.9, 135.5, 140.1 , 143.1 , 148.4, 148.5, 150.4, 181 .9 (C=0), 183.6 (C=0); m/z (ESI") 389 ([M-H]\ 100%); HRMS (ESI+) C2i H14N2Na04S+, ([M+Na]+) requires 413.0566; found 413.0565.
Example 21
W-(3-(Furan-2-yl)-1 ,4-dioxo-1 ,4-dihydronaphthalen-2-yl)benzenesulfonamide 21
[00176] Following General Procedure 3, /V-(3-chloro-1 ,4-dioxo-1 ,4- dihydronaphthalen-2-yl)benzenesulfonamide 3 (200 mg, 0.58 mmol) and
2-furanboronic acid (130 mg, 1 .14 mmol) gave the crude reaction
Figure imgf000058_0001
mixture. Purification via column chromatography on silica gel (eluent pet
ethenacetone 70:30) and subsequent recrystallisation from toluene gave 21 as a deep purple solid (100 mg, 45%); mp 144-146 °C; umax (KBr) 3242 (N-H), 1668 (C=0), 1648 (C=0); δΗ (400 MHz, Acetone-de) 6.64-6.72 (1 H, m, Ar), 7.40-7.48 (1 H, m, Ar), 7.52-7.61 (2H, m, Ar), 7.61 - 7.68 (1 H, m, Ar), 7.72 (1 H, s, Ar), 7.78-7.92 (2H, m, Ar), 7.93-7.99 (3H, m, Ar), 8.05-8.13 (1 H, m, Ar), 8.96 (1 H, s, N-H); 5C (100 MHz, Acetone-d6) 1 13.0, 1 19.5, 124.3, 126.4, 126.9, 127.3, 129.1 , 131 .2, 132.5, 133.0, 134.4, 134.8, 136.3, 142.4, 145.1 , 146.3, 180.8 (C=0), 182.7 (C=0); m/z (ESI") 378 ([M-H]", 100%); HRMS (ESI+) C2oH13NNa05S+, ([M+Na]+) requires 402.0407; found 402.0407.
Example 22
W-(3-(Furan-3-yl)-1 ,4-dioxo-1 ,4-dihydronaphthalen-2-yl)benzenesulfonamide 22
[00177] Following General Procedure 3, /V-(3-chloro-1 ,4-dioxo-1 ,4- dihydronaphthalen-2-yl)benzenesulfonamide 3 (200 mg, 0.58 mmol) and
3-furanboronic acid (130 mg, 1 .14 mmol) gave the crude reaction
mixture. Purification via column chromatography on silica gel (eluent pet
Figure imgf000058_0002
ethenacetone 75:25) and subsequent recrystallisation from toluene gave 22 as a bright yellow solid (51 mg, 23%); mp 21 1 -214 °C; umax (KBr) 3231 (N-H), 1664 (C=0), 1651 (C=0); δΗ (400 MHz, DMSO-de) 6.95 (1 H, s, Ar), 7.46-7.57 (2H, m, Ar), 7.58-7.66 (1 H, m, Ar), 7.74 (1 H, s, Ar), 7.80 (2H, s, Ar), 7.82-7.93 (3H, m, Ar), 7.99-8.09 (1 H, m, Ar), 8.14-8.22 (1 H, m, Ar), 10.03 (1 H, br. s, N-H); 5C (100 MHz, DMSO-d6) 1 12.5, 1 16.8, 126.8, 127.2, 127.3, 129.6, 131 .3, 132.6, 133.3, 134.5, 135.1 , 135.3, 137.8, 142.7, 143.5, 146.8, 181 .5 (C=0), 184.3 (C=0); m/z (ESI") 378 ([M-H]", 100%); HRMS (ESI+) C2oH13NNa05S+, ([M+Na]+) requires 402.0407; found 402.0403.
Example 23
A/-(3-(Benzo[b]thiophen-2-yl)-1 ,4-dioxo-1 ,4-dihydronaphthalen-2-yl)benzenesulfonamide 23
[00178] Following General Procedure 3, /V-(3-chloro-1 ,4-dioxo-1 ,4- dihydronaphthalen-2-yl)benzenesulfonamide 3 (200 mg, 0.58 mmol)
and benzo[b]thiophene-2-boronic acid (207 mg, 1 .14 mmol) gave the
crude reaction mixture. Purification via column chromatography on silica
Figure imgf000059_0001
gel (eluent pet ether:acetone 60:40) and subsequent recrystallisation
from toluene gave 23 as a dark red solid (90 mg, 35%); mp 245-247 °C; umax (KBr) 3210 (N-H), 1671 (C=0), 1649 (C=0); δΗ (400 MHz, DMSO-d6) 7.32-7.40 (2H, m, Ar), 7.41 -7.48 (2H, m, Ar), 7.49-7.55 (1 H, m, Ar), 7.72-7.79 (3H, m, Ar), 7.80-7.90 (2H, m, Ar), 7.90-7.95 (2H, m, Ar), 7.96-8.00 (1 H, m, Ar), 8.04-8.09 (1 H, m, Ar), 9.80 (1 H, br. s, N-H); 5C (100 MHz, DMSO-d6) 122.6, 124.8, 125.0, 125.7, 125.8, 126.8, 126.9, 127.2, 129.1 , 129.3, 129.9, 131 .5, 132.7, 132.9, 133.6, 134.0, 134.7, 135.4, 139.5, 141 .8, 181 .4 (C=0), 183.3 (C=0); m/z (ESI") 444 ([M-H]", 100%); HRMS (ESI+) C24H15NNa04S2+, ([M+Na]+) requires 468.0335; found 468.0338. Example 24
2-Amino-3-(2,3,4-trimethoxyphenyl)naphthalene-1 ,4-dione 24
[00179] 3-chloro-1 ,4-dioxo-1 ,4-dihydronaphthalen-2-ylamine (200 ' ' mg, 0.58 mmol) was treated with 2,3,4-trimethoxyphenylboronic acid
(244 mg, 1 .15 mmol), sodium carbonate (184 mg, 1 .74 mmol) and
Pd(PPh3)2CI2 (42 mg, 0.06 mmol) in 1 :1 DME:EtOH (20 mL) at 90 °C
Figure imgf000059_0002
for 18 h to give the crude reaction mixture. The residue was
partitioned between EtOAc and sat. aq. NH4CI. The organic extract was washed with brine, before being dried, filtered, and concentrated in vacuo to give the crude reaction mixture, which was then partially purified by column chromatography (eluent pet ethenEtOAc 70:30) before being recrystallised from toluene to give 24 as a bright red solid (41 mg, 21 %); umax (KBr) 3265 (N-H), 1674 (C=0), 1615 (C=0); δΗ (400 MHz, DMSO-d6) 3.63 (3H, s, OMe), 3.78 (3H, s, OMe), 3.83 (3H, s, OMe), 6.43 (2H, br. s, NH2), 6.75-6.91 (2H, m, 2,3,4- trimethoxyphenyMr), 7.70-7.87 (2H, m, naphthoquinone-H), 7.91 -8.05 (2H, m, naphthoquinone-H); 5C (100 MHz, DMSO-d6) 56.7 (OMe), 61 .1 {OMe), 61 .4 (OMe), 108.8,
1 13.3, 120.4, 126.4, 126.5, 126.7, 131 .1 , 133.1 , 133.9, 135.6, 142.9, 147.7, 152.8, 154.1 , 181 .0 (C=0), 182.6 (C=0); m/z (ESI") 338 ([M-H]", 100%); HRMS (ESI+) Ci9H17NNa05 +, ([M+Na]+) requires 362.0999; found 362.0990. Example 25
2,3-Diphenoxynaphthalene-1 ,4-dione 25
[00180] Following General Procedure 4, phenol (414 mg, 4.40 mmol)
gave 25 as a pale yellow solid (396 mg, 58%); δΗ (400 MHz, CDCI3)
6.82 (4H, m, Ar), 6.96 (2H, m, Ar), 7.15 (4H, m, Ar), 7..67-7 '.72 (2H, m,
Ar), 8.02-8.07 (2H, m, Ar); 5C (100 MHz, CDCI3) 1 16.6, 123.6, 126.8,
Figure imgf000060_0001
129.4, 130.8, 134.3, 146.1 , 156.5, 180.4 (C=0).
Example 26
Λ/-(1 ,4-Dioxo-3-phenoxy-1 ,4-dihydronaphthalen-2-yl)benzenesulfonamide 26
[00181] Following General Procedure 5, 25 (205 mg, 0.60 mmol) gave
26 as a yellow solid (74 mg, 30%); mp 227-228 °C; umax (KBr) 3237 (N- H), 1669 (C=0), 1656 (C=0); δΗ (400 MHz, DMSO-d6) 6.79-6.87 (2H, m,
Ar), 6.96-7.05 (1 H, m, Ar), 7.13-7.28 (2H, m, Ar), 7.43-7.61 (3H, m, Ar),
7.78-7.92 (5H, m, Ar), 8.00-8.07 (1 H, m, Ar), 10.29 (1 H, br. s, N-H); 5C
Figure imgf000060_0002
(100 MHz, DMSO-de) 1 17.1 , 123.6, 126.4, 126.9, 127.0, 127.2, 129.6, 130.0, 131 .4, 131 .5, 133.2, 135.3, 135.4, 142.6, 146.0, 157.3, 179.9 (C=0), 181 .5 (C=0); m/z (ESI") 405 ([M-H]", 100%); HRMS (ESI+) C22H15NNa05S+, ([M+Na]+) requires 428.0563; found 428.0565.
Example 27 2, 3-Bis(2,4,5-trichlorophenoxy)naphthalene-1 ,4-dione 27
[00182] Following General Procedure 4, 2,4,5-trichlorophenol
(869 mg, 4.40 mmol) gave 27 as a yellow solid (431 mg, 39%); δΗ
(400 MHz, CDCIs) 7.04 (2H, s, Ar), 7.46 (2H, s, >Ar-phenol), 7.78- 7.87 (2H, m, 8.10-8.17 (2H, m, Ar- naphthoquinone);
Figure imgf000060_0003
5C (100 MHz, CDCI3) 1 19.6, 122.8, 127.1 ,
128.5, 130.2, 132.2, 131 .5, 134.8, 144.1 , 150.6, 170.0 (C=0).
Example 28 2,3-Bis(4-nitrophenoxy)naphthalene-1 ,4-dione 28
[00183] Following General Procedure 4, 4-nitrophenol (612 mg,
4.40 mmol) gave 28 as a yellow solid (648 mg, 75%); δΗ (400
MHz, acetone-d6) 7.44 (4H, m, Ar), 7.92-7.97 (2H, m, Ar), 8.09- 8.14 (2H, m,), 8.21 (4H, Ar); 5C (100 MHz, acetone-d6) 1 17.5,
Figure imgf000061_0001
126.1 , 126.8, 131 .6, 135.0, 144.0, 146.1 , 161 .8, 179.9 (C=0).
Example 29
3,3'-((1 ,4-Dioxo-1 ,4-dihydronaphthalene-2,3-diyl)bis(oxy))dibenzaldehyde 29
[00184] Following General Procedure 4, 3-hydroxybenzaldehyde
(537 mg, 4.40 mmol) gave 29 as a yellow solid (219 mg, 27%); δΗ
(400 MHz, DMSO-de) 7.19-7.25 (2H, m, Ar), 7.36 (2H, s, Ar), 7.40- 7.48 (2H, m, Ar), 7.54-7.59 (2H, m, Ar), 7.76-7.87 (2H, m, Ar- naphthoquinone), 8.06-8.19 (2H, m,
Figure imgf000061_0002
9.89 (2H,
s, CHO); 5c (100 MHz, DMSO-d6) 1 15.7, 123.0, 126.0, 127.0, 130.4, 130.6, 134.7, 145.7, 156.9, 179.9 (C=0), 191 .1 (CHO).
Example 30
4,4'-((1 ,4-Dioxo-1 ,4-dihydronaphthalene-2,3-diyl)bis(oxy))dibenzaldehyde 30
[00185] Following General Procedure 4, 4- hydroxybenzaldehyde (537 mg, 4.40 mmol) gave 30 as a yellow
solid (270 mg, 34%); 1H δΗ (400 MHz, CDCI3) 7.00-7.12 (4H, m,
Ar), 7.74-7.90 (6H, m, Ar), 8.08-8.21 (2H, m, Ar), 9.91 (2H, s,
Figure imgf000061_0003
CHO); 5c (100 MHz, CDCI3) 1 16.9, 127.1 , 130.5, 131 .8, 132.4, 134.8, 145.6, 160.7, 179.5
(C=0 naphthoquinone), 190.4 (CHO).
Example 31
2,2'-((1 ,4-Dioxo-1 ,4-dihydronaphthalene-2,3-diyl)bis(oxy))dibenzonitrile 31 [00186] Following General Procedure 4, 2-cyanophenol (524 mg, 4.40
mmol) gave 31 as a yellow solid (471 mg, 59%); δΗ (400 MHz, CDCI3)
6.99 (2H, s, Ar), 7.12 (2H, m, Ar), 7.42-7.52 (4H, m, Ar), 7.72-7.77 (2H,
m, Ar), 8.02-8.08 (2H, m, Ar); 5C (400 MHz, CDCI3) 103.2, 1 15.1 , 1 17.2,
Figure imgf000061_0004
124.4, 127.1 , 130.3, 133.7, 134.4, 134.8, 144.7, 157.5, 179.0.
Example 32
2,3-Bis(4-(trifluoromethoxy)phenoxy)naphthalene-1 ,4-dione 32 [00187] Following General Procedure 4, 4- trifluoromethoxyphenol (784 mg, 4.40 mmol) gave 32 as a
pale yellow solid (735 mg, 72%); δΗ (400 MHz, CDCI3) 6.76- 6.82 (4H, m, >Ar-phenol), 7.00-7.04 (4H, m, >Ar-phenol), 7.70-
Figure imgf000062_0001
7.77 (2H, m,
Figure imgf000062_0002
8.03 - 8.09 (2H, m, ^/--naphthoquinone); 5C (100 MHz, CDCIs) 1 16.1 , 1 17.7, 1 19.1 , 122.4, 127.0, 130.6, 134.7, 145.3, 154.3, 180.2 (C=0).
Example 33 yV-(1 ,4-Dioxo-3-(2,3,4-trichlorophenoxy)-1 ,4-dihydronaphthalen-2-yl)benzenesulfonamide 33 [00188] Following General Procedure 5, 27 (176 mg, 0.32 mmol) gave
33 as a yellow solid (90 mg, 55%); mp 278-279 °C; umax (KBr) 3309 (N-H),
1672 (C=0), 1656 (C=0); δΗ (400 MHz, DMSO-d6) 7.40-7.54 (4H , m, Ar),
7.77-7.93 (6H, m, Ar), 8.01 -8.08 (1 H, m, Ar), 10.43 (1 H, br. s, N-H); 5C
(100 MHz, DMSO-de) 120.5, 122.5, 126.2, 126.8 (2 x C), 127.1 , 129.6,
Figure imgf000062_0003
129.8, 130.8, 131 .2, 131 .4, 131 .6, 133.2, 135.1 , 135.4, 142.3, 143.4,
152.4, 179.3 (C=0), 181 .2 (C=0); m/z (ESI") 506 ([M-H]\ 100%); HRMS (ESI+) C22H12CI3NNa05S+, ([M+Na]+) requires 529.9394; found 529.9396.
Example 34
A/-(3-(4-Nitrophenoxy)-1 ,4-dioxo-1 ,4-dihydronaphthalen-2-yl)benzenesulfonamide 34
[00189] Following General Procedure 5, 28 (259 mg, 0.60 mmol) gave
34 as a yellow solid (194 mg, 72%); mp 208-213 °C; umax (KBr) 3294 (N- H), 1659 (C=0), 1638 (C=0); δΗ (400 MHz, DMSO-d6) 7.05-7.12 (2H, m,
Ar), 7.43-7.62 (3H, m, Ar), 7.77-7.84 (2H, m, Ar), 7.85-7.94 (3H, m, Ar),
8.01 -8.16 (3H, m, Ar), 10.48 (1 H, br. s, N-H); 5C (100 MHz, DMSO-d6)
Figure imgf000062_0004
1 18.2, 126.0, 126.2, 126.8, 127.3, 129.1 , 129.8, 131 .4, 131 .5, 133.4,
135.3, 135.5, 142.4, 143.3, 143.8, 162.1 , 179.3 (C=0), 181 .3 (C=0); m/z (EST) 449 ([M-H]\ 100%); HRMS (ESI+) C22H14N2Na07S+, ([M+Na]+) requires 473.0414; found 473.0412. Example 35
W-(3-(3-Formylphenoxy)-1 ,4-dioxo-1 ,4-dihydronaphthalen-2-yl)benzenesulfonamide 35 [00190] Following General Procedure 5, 29 (146 mg, 0.36 mmol) gave
35 as a yellow solid (35 mg, 37%); mp 245-250 °C; umax (KBr) 3342 (N- H), 1697 (C=0), 1656 (C=0); δΗ (400 MHz, DMSO-d6) 7.17-7.22 (1 H, m,
Ar), Ί.21 -122 (1 H, m, Ar), 7.40-7.47 (3H, m, Ar), 7.47-7.53 (1 H, m, Ar),
7.54-7.61 (1 H, m, Ar), 1.11-1.82 (2H, m, Ar), 7.84-7.92 (3H, m, Ar),
Figure imgf000063_0001
8.02-8.10 (1 H, m, Ar), 9.91 (1 H, s, CHO), 10.38 (1 H, br. s, N -/-/); 5C (100 MHz, DMSO-d6) 1 17.5, 123.5, 124.9, 126.9, 127.2, 129.1 , 129.6, 129.8, 130.8, 131 .5, 133.2, 133.4, 135.3, 135.5, 138.1 , 142.5, 144.83, 157.7, 179.8 (C=0), 181 .4 (C=0), 193.4 (CH=0); m/z (EST) 432 ([M-H]", 100%); HRMS (ESI+) C23H15NNa06S+, ([M+Na]+) requires 456.0512; found 456.0512.
Example 36 yV-(3-(4-Formylphenoxy)-1 ,4-dioxo-1 ,4-dihydronaphthalen-2-yl)benzenesulfonamide 36
[00191] Following General Procedure 5, 30 (199 mg, 0.50 mmol) gave
36 as a yellow solid (89 mg, 41 %); mp 173-175 °C; umax (KBr) 3308 (N-H),
1671 (C=0), 1652 (C=0); 1633 (C=0); δΗ (400 MHz, DMSO-d6) 7.05 (2H,
m, >Ar-phenol), 7.43-7.60 (3H, m, Ar), 7.73-7.95 (7H, m, Ar), 8.01 -8.1 1
(1 H, m, Ar), 9.89 (1 H, s, CHO), 10.42 (1 H, br. s, N-H); 5C (100 MHz,
Figure imgf000063_0002
DMSO-de) 1 16.7, 1 18.0, 126.9, 129.7, 131 .4, 132.0, 133.0, 133.3, 133.5,
135.3, 135.5, 142.5, 144.5, 161 .9, 164.2, 179.5 (C=0), 181 .4 (C=0), 192.4 (CH=0); m/z (EST) 432 ([M-H]", 100%); HRMS (ESI") C23H15N06S", ([M-H]") requires 432.0547; found 432.0551 . Example 37
W-(3-(2-Cyanophenoxy)-1 ,4-dioxo-1 ,4-dihydronaphthalen-2-yl)benzenesulfonamide 37
[00192] Following General Procedure 5, 31 (235 mg, 0.60 mmol) gave 37
as a yellow solid (199 mg, 77%); mp 234-238 °C; umax (KBr) 3272 (N-H),
1658 (C=0); δΗ (400 MHz, DMSO-d6) 7.12-7.21 (2H, m, Ar), 7.40-7.62
(4H, m, Ar), 7.70-7.76 (1 H, m, Ar), 7.77-7.83 (2H, m, Ar), 7.84-7.92 (3H,
Figure imgf000063_0003
m, Ar), 8.01 -8.1 1 (1 H, m, Ar), 10.53 (1 H, br. s, N-H); 5C (100 MHz, DMSO-de) 102.3, 1 16.6, 1 18.2, 124.3, 126.4, 126.7, 126.9, 127.3, 129.7, 131 .3, 131 .5, 133.3, 134.1 , 135.2, 135.3, 135.5, 142.3, 143.6, 158.7, 179.4 (C=0), 181 .2 (C=0); m/z (EST) 429 ([M- H]", 100%); HRMS (ESI+) C23H14N2Na05S+, ([M+Na]+) requires 453.0516; found 453.0516.
Example 38 Λ/-(1 ,4-Dioxo-3-(4-(trifluoromethoxy)phenoxy)-1 ,4-dihydronaphthalen-2- yl)benzenesulfonamide 38
Figure imgf000064_0001
1 18.7, 122.9, 126.4, 126.9, 127.2, 129.6, 129.8, 131 .5, 132.7, 133.2, 133.4, 135.4, 142.5, 144.1 , 145.0, 155.9, 179.8 (C=0), 181 .4 (C=0), CF3 not observed; m/z (ESI") 488 ([M-H]", 100%); HRMS (ESI+) C23H14F3NNa06S+, ([M+Na]+) requires 512.0386; found 512.0386.
Example 39
A/-(3-(3-Hydroxyphenoxy)-1 ,4-dioxo-1 ,4-dihydronaphthalen-2-yl)benzenesulfonamide 39 [00194] Following General Procedure 5, 3,4-Di-(3-Hydroxyphenoxy)- 1 ,4-dioxo-1 ,4-dihydronaphthalene (224 mg, 0.60 mmol) gave 39 as a
brown solid (109 mg, 43%); mp 219-235 °C; umax (KBr) 3357 (O-H), 3263
(N-H), 1670 (C=0); δΗ (400 MHz, DMSO-d6) 7.36 (3H, m, Ar), 7.48-7.65
(5H, m, Ar), 7.75-7.96 (5H, m, Ar), 9.14 (1 H, s, NH), 10.52 (1 H, s, OH);
Figure imgf000064_0002
5c (100 MHz, DMSO-d6) 99.3, 103.3, 107.0, 1 17.4, 124.6, 126.4, 127.0, 129.5, 129.8, 130.4, 130.6, 132.7, 135.0, 145.0, 158.5, 159.3, 161 .0, 179.7 (C=0), 182.2 (C=0); m/z (EST) 420 ([M- H]", 100%); HRMS (ESI+) C22H15NNa06S+, ([M+Na]+) requires 444.0512; found 444.0514.
Example 40 yV-(3-((3-Methoxypropyl)amino)-1 ,4-dioxo-1 ,4-dihydronaphthalen-2- yl)benzenesulphonamide 40
[00195] Following General Procedure 2, 3 (200 mg, 0.58 mmol), CeCI3.7H20 (86 mg, 0.23 mmol) and 3-methoxypropylamine (178 μΙ_, 1 .74 mmol) gave the crude reaction mixture. Purification via column chromatography (eluent pet etheracetone 70:30) gave 40 as a bright orange solid (190 mg, 82%); mp 179-181 °C; umax (KBr) 3294 (N-H),
3216 (N-H), 1673 (C=0), 1615 (C=0); δΗ (400 MHz, CDCI3) 1 .90
(2H, quint., J 5.9, C(2)H2), 3.35 (3H, s, OMe), 3.52 (2H, t, J 5.9,
Figure imgf000065_0001
C(3)H2), 4.00 (2 H, q, J 5.9, C(1 )H2), 6.52 (1 H, br. s, NH- sulphonamide), 7.04 (1 H, br. t, J 5.9, NH-CH2CH2CH2OMe), 7.23-7.33 (2H, m, Ar), 7.36-7.43 (1 H, m, Ar), 7.46-7.56 (2H, m, Ar), 7.58-7.63 (1 H, m, Ar), 7.68-7.76 (2H, m, Ar), 7.90-7.98 (1 H, m, Ar); 5C (100 MHz, CDCI3) 29.3 (C(2)), 43.9 (C(1 )), 59.0 (OMe), 71 .6 (C(3)), 108.8, 125.9, 126.7, 127.8, 128.6, 130.1 , 132.0, 132.3, 133.0, 134.8, 138.4, 144.4, 178.5 (C=0), 181 .9 (C=0); m/z (ESI") 399 ([M-H]\ 100%); HRMS (ESI+) C20H20N2NaO5S+, ([M+Na]+) requires 423.0985; found 423.0987.
Example 41 yV-(3-((2-Methoxyethyl)amino)-1 ,4-dioxo-1 ,4-dihydronaphthalen-2- yl)benzenesulphonamide 41
[00196] Following General Procedure 2, 3 (200 mg, 0.58 mmol),
CeCI3.7H20 (86 mg, 0.23 mmol) and 3-methoxyethylamine (150 μΙ_,
1 .74 mmol) gave the crude reaction mixture. Purification via column
Figure imgf000065_0002
chromatography (eluent pet ether:acetone 70:30) gave 41 as a bright
orange solid (195 mg, 87%); mp 167-169 °C; umax (KBr) 3312 (N-H), 3235 (N-H), 1676 (C=0), 1610 (C=0); δΗ (400 MHz, CDCI3) 3.36 (3H, s, OMe), 3.60 (2H, t, J 5.2, CH2-OMe), 4.08 (2H, q, J 5.2, CH2-CH2OMe), 6.56 (1 H, br. s, NH-sulphonamide), 6.65 (1 H, br. t, J 5.2, NH- CH2CH2OMe), 7.24-7.32 (2H, m, Ar), 7.37-7.44 (1 H, m, Ar), 7.47-7.57 (2H, m, Ar), 7.58-7.64 (1 H, m, Ar), 7.68-7.75 (2H, m, Ar), 7.91 -7.99 (1 H, m, Ar); 5C (100 MHz, CDCI3) 44.2 (CH2- OMe), 58.8 (OAfe), 70.6 (CH2-CH2OMe), 109.3, 125.9, 126.8, 127.8, 128.6, 130.1 , 131 .8, 132.4, 133.1 , 134.9, 138.2, 143.9, 178.6 (C=0), 181 .7 (C=0); m/z (ESI") 385 ([M-H]", 100%); HRMS (ESI+) Ci9H18N2Na05S+, ([M+Na]+) requires 409.0829; found 409.0829.
Example 42
W-(3-((2-(Dimethylamino)ethyl)amino)-1 ,4-dioxo-1 ,4-dihydronaphthalen-2- yl)benzenesulphonamide 42
[00197] Following General Procedure 2, 3 (200 mg, 0.58 mmol),
NHS02Ph CeCI3.7H20 (86 mg, 0.23 mmol) and 2-dimethylaminoethylamine (190
μΙ_, 1 .74 mmol) gave the crude reaction mixture. Purification via uy 11
o
column chromatography (eluent pet ethenacetone 60:40) gave 42 as a dark red solid (167 mg, 72%); mp 140-143 °C; umax (KBr) 3345 (N-H), 1674 (C=0), 1608 (C=0); δΗ (400 MHz, CDCIs) 2.25 (6H, s, NMe2), 2.55 (2H, t, J 5.8, CH2- NMe2), 3.91 (2H, q, J 5.8, CH2- CH2NMe2), 7.07 (1 H, br. t, J 5.8, NH-CH2CH2NMe2), 7.24-7.33 (2H , m, Ar), 7.37-7.45 (1 H, m, Ar), 7.47-7.57 (2H, m, Ar), 7.59-7.64 (1 H, m, Ar), 7.70-7.78 (2H, m, Ar), 7.91 -7.98 (1 H, m, Ar); 5C (100 MHz, CDCI3) 41 .6 (CH2-CH2NMe2), 45.04 (NMe2), 57.5 (CH2NMe2), 109.1 , 125.9, 126.6, 127.8, 128.6, 132.0, 132.3, 133.0, 134.8, 138.6, 144.3, 178.5 (C=0), 181 .8 (C=0); m/z (ESI") 398 ([M-H]\ 100%); HRMS (ESI+) C2oH21 N3Na04S+, ([M+Na]+) requires 422.1 145; found 422.1 143.
Example 43 A/-(3-(Cyclopentylamino)-1 ,4-dioxo-1 ,4-dihydronaphthalen-2-yl)benzenesulphonamide 43
[00198] Following General Procedure 2, 3 (200 mg, 0.58 mmol),
CeCI3.7H20 (86 mg, 0.23 mmol) and cyclopentylamine (172 μΙ_, 1 .74
mmol) gave the crude reaction mixture. Purification via column
chromatography (eluent pet ether:acetone 70:30) gave 43 as a dark red
solid (204 mg, 89%); mp 199-201 °C; umax (KBr) 3318 (N-H), 3243 (N-H),
Figure imgf000066_0001
1673 (C=0), 1612 (C=0); δΗ (400 MHz, CDCI3) 1 .55 (2H , q , J 6.5, H(2)), 1 .73 (4H, quin, J 6.5, H(3)), 2.14 (2H, q, J 6.5, H(2)), 5.02 (1 H, sxt, J 6.5, H(1 )), 6.41 (1 H, d, J 6.5, NH- cyclopentylamine), 6.68 (1 H, s, NH-sulphonamide), 7.29-7.41 (2H, m, Ar), 7.43-7.52 (1 H, m, Ar), 7.54-7.65 (2H, m, Ar), 7.66-7.73 (1 H, m, Ar), 7.75-7.84 (2H, m, Ar), 7.96-8.09 (1 H, m, Ar); 5c (100 MHz, CDCI3) 24.1 (C(3)), 34.5 (C(2)), 55.0 (C(1 )), 109.1 , 125.9, 126.7, 127.8, 128.6, 130.1 , 131 .9, 132.3, 133.1 , 134.9, 138.3, 143.5, 178.5 (C=0), 182.0 (C=0); m/z (EST) 395 ([M- H]\ 100%); HRMS (ESI+) C2i H20N2NaO4S+, ([M+Na]+) requires 419.1036; found 419.1036.
Example 44 A/-(3-(4-Benzylpiperazin-1 -yl)-1 ,4-dioxo-1 ,4-dihydronaphthalen-2-yl)benzenesulphonamide 44
[00199] Following General Procedure 2, 3 (200 mg, 0.58 mmol),
CeCI3.7H20 (86 mg, 0.23 mmol) and 1 -benzylpiperazine (303 μΙ_,
1 .74 mmol) gave the crude reaction mixture. Purification via column
chromatography (eluent pet ethenacetone 70:30) gave 44 as a dark
Figure imgf000066_0002
red solid (266 mg, 72%); mp 172-174 °C; umax (KBr) 3240 (N-H),
1675 (C=0), 1640 (C=0); δΗ (400 MHz, CDCI3) 2.59 (4H, t, J 4.7, CH2-N-benzyl), 3.51 (2H, s, CH2Ph), 3.68 (4H, t, J 4.7, CH2-CH2-N-benzyl), 6.78 (1 H, br. s, NH-sulphonamide), 7.16-7.33 (7H, m, Ar), 7.33-7.40 (1 H , m, Ar), 7.44-7.55 (2H , m, Ar), 7.56-7.61 (1 H , m, Ar), 7.64-7.70 (2H , m, Ar), 7.86-7.95 (1 H, m, Ar); 5C (100 MHz, CDCI3) 51 .4 (CH2-N-benzyl), 53.3 (CH2-CH2-N- benzyl), 62.9 (CH2Ph), 1 18.4, 125.5, 126.9, 127.2, 127.5, 128.3, 128.7, 129.1 , 130.3, 132.0, 133.0, 133.1 , 133.8, 138.0, 138.6, 147.4, 179.9 (C=0), 183.1 (C=0); m/z (ESI") 386 ([M-H]\ 100%); HRMS (ESI+) C27H26N304S+, ([M+Na]+) requires 488.1639; found 488.1639.
Example 45 yV-(3-((Furan-2 -yl methyl )amino)-1 ,4-dioxo-1 ,4-dihydronaphthalen-2- yl)benzenesulphonamide 45
[00200] Following General Procedure 2, 3 (200 mg, 0.58 mmol),
CeCI3.7H20 (86 mg, 0.23 mmol) and furfurylamine (161 μΙ_, 1 .74 mmol)
gave the crude reaction mixture. Purification via column
chromatography (eluent pet ether:acetone 70:30) gave 45 as a dark
Figure imgf000067_0001
red solid (173 mg, 73%); mp 163-166 °C; umax (KBr) 3334 (N-H), 3240 (N-H), 1674 (C=0), 161 1 (C=0); δΗ (400 MHz, CDCI3) 5.06 (2H, d, J 6.1 , CH2-furyl), 6.23-6.32 (2H, m, Ar), 6.50 (1 H, br. t, J 6.1 , NH-CH2-furyl), 6.59 (1 H, br. s, NH-sulphonamide), 7.24-7.34 (3H, m, Ar), 7.36-7.46 (1 H, m, Ar), 7.49-7.58 (2H , m, Ar), 7.58-7.66 (1 H , m, Ar), 7.69-7.78 (2H , m, Ar), 7.90-7.98 (1 H, m, Ar); 5C (100 MHz, CDCI3) 41 .6 (CH2-furyl), 108.5, 1 10.0, 1 10.5, 126.0, 126.8, 127.8, 128.7, 130.0, 131 .6, 132.6, 133.2, 134.9, 138.1 , 142.8, 143.3, 150.7, 178.9 (C=0), 181 .7 (C=0); m/z (ESI") 407 ([M-H]", 100%); HRMS (ESI+) C2i H16N2Na05S+, ([M+Na]+) requires 431 .0672; found 431 .0674.
Example 46
A/-(3-Chloro-5-nitro-1 ,4-dioxo-1 ,4-dihydronaphthalen-2-yl)benzenesulphonamide 46
[00201 ] Following General Procedure 7, 2,3-dichloro-5-nitronaphthalene- 1 ,4-dione (1 .08 g, 2 mmol), benzenesulphonamide (818 mg, 5.2 mmol)
and Cs2C03 (1 .70 g, 5.2 mmol) gave the crude reaction mixture.
Purification and separation of regioisomers via column chromatography
Figure imgf000067_0002
on silica gel (eluent pet etherAcetone 50:50) gave 46 as a yellow solid (375 mg, 24%). mp 225-229 °C; umax (KBr) 3223 (N-H), 1694 (C=0), 1675 (C=0); δΗ (400 MHz, DMSO-d6) 7.57- 7.64 (2H, m, sulphonamide-^r); 7.64-7.71 (1 H, m, sulphonamide-^r); 7.86-7.93 (2H, m, sulphonamide-/Ar); 8.00-8.10 (1 H, m, naphthoquinone-/*/-); 8.14-8.21 (1 H, m, naphthoquinone- Ar); 8.22-8.29 (1 H, m, naphthoquinone-^); 5C (100 MHz, DMSO-d6) 122.8, 127.4, 128.6, 129.8, 129.9, 133.2, 133.7, 136.5, 142.7, 148.8, 176.7 (C=0), 177.0 (C=0); m/z (ESI") 391 ([M- H]\ 100%); HRMS (ESI") Ci6H8CIN206S", ([M-H]") requires 390.9797; found 390.9796.
Example 47
A/-(3-Chloro-8-nitro-1 ,4-dioxo-1 ,4-dihydronaphthalen-2-yl)benzenesulphonamide 47
[00202] Following General Procedure 1, 2,3-dichloro-5-nitronaphthalene-
1 ,4-dione (1.08 g, 2.00 mmol), benzenesulphonamide (818 mg, 5.20
mmol) and Cs2C03 (1 .70 g, 5.20 mmol) gave the crude reaction mixture.
Purification and separation of regioisomers via column chromatography
Figure imgf000068_0001
on silica gel (eluent pet etheracetone 50:50) gave 47 as a yellow solid (594 mg, 38%); mp 225-231 °C; umax (KBr) 3201 (N-H), 1687 (C=0); δΗ (400 MHz, DMSO-d6) 7.57-7.65 (2H, m, sulphonamide-/\r), 7.65-7.72 (1 H, m, sulphonamide-/\r), 7.92-7.99 (2H, m, sulphonamide-/\r), 8.00-8.06 (1 H, m,
Figure imgf000068_0002
8C (400 MHz, DMSO-de) 122.6, 127.4, 128.7, 129.8, 129.9, 132.6, 133.6, 136.4, 142.9, 148.5, 175.5 (C=0), 178.0 (C=0); m/z (ESI") 391 ([M-H]", 100%); HRMS (ESI") Ci6H8CIN206S", ([M-H]") requires 390.9797; found 390.9797.
[00203] The structures depicted for regioisomeric compounds 46 and 47 have been assigned based on the data currently available. This data is not sufficient for the structures to be assigned with absolute certainty. Therefore the depicted structures should be considered to be tentative until x-ray crystallographic data becomes available.
Example 48 yV-(3-((3,5-Dimethylphenyl)amino)-8-nitro-1 ,4-dioxo-1 ,4-dihydronaphthalen-2- yl)benzenesulphonamide 48 [00204] Following General Procedure 2, 46 (250 mg, 0.64 mmol) and
CeCI3.7H20 (101 mg, 0.27 mmol) and 3,5-dimethylaniline (240 μΙ_, 1 .92
mmol) gave the crude reaction mixture. Purification via column
chromatography (eluent pet etheracetone 70:30) gave 48 as a purple
solid (225 mg, 74%). mp 227-232 °C; umax (KBr) 3298 (N-H), 3219 (N-H),
Figure imgf000068_0003
1678 (C=0), 1641 (C=0); δΗ (400 MHz, CDCI3) 2.21 (6H, s, 2 χ Ar-Me), 6.61 (2H, s, aniline- ortho-H), 6.67 (1 H, s, aniline-para-H), 7.31 -7.39 (2H, m, sulphonamide-/\r), 7.46-7.55 (3H, m, sulphonamide-/\r), 7.89-7.98 (1 H, m, naphthoquinone-^/-), 8.01 -8.08 (1 H, m, naphthoquinone- Ar), 8.18-8.26 (1 H, m, naphthoquinone-/*/-), 9.06 (2H, s, aniline/sulphonamide NH); 5C (100 MHz, DMSO-de) 21 .8 (aniline-Me), 1 14.1 , 122.2, 123.0, 126.6, 127.2, 129.0, 129.3, 129.4, 132.4, 132.8, 135.0, 137.2, 138.6, 141 .8, 143.4, 148.6, 176.2 (C=0), 181 .6 (C=0); m/z (EST) 476 ([M-H]\ 100%); HRMS (ESI") C24H18N306S", ([M-H]") requires 476.0922; found 476.0920.
Example 49
W-(3-((3,5-Dimethylphenyl)amino)-5-nitro-1 ,4-dioxo-1 ,4-dihydronaphthalen-2- yl)benzenesulphonamide 49
[00205] Following General Procedure 2, 47 (392 mg, 1 .00 mmol) and
CeCI3.7H20 (149 mg, 0.40 mmol) and 3,5-dimethylaniline (375 μΙ_, 3.00
mmol) gave the crude reaction mixture. Purification via column
chromatography (eluent pet ether:acetone 70:30) gave 49 as a purple
solid (379 mg, 80%). mp 223-229 °C; umax (KBr) 3270 (N-H), 1688
Figure imgf000069_0001
(C=0); δΗ (400 MHz, DMSO-d6) 2.23 (6H, s, 2 χ Ar-Me), 6.59 (2H, s, aniline-ort/?o-H), 6.68 (1 H, s, aniline-para-H), 7.34-7.43 (2H, m, sulphonamide-/*/-), 7.47-7.55 (1 H, m, sulphonamide-/*/-), 7.58-7.66 (2H, m, sulphonamide-^r), 7.93-8.03 (2H, m, naphthoquinone-/*/-), 8.06-8.10 (1 H, m, naphthoquinone-/*/-), 9.04 (1 H, s, aniline-NH), 9.21 (1 H, s, sulphonamide-NH); 5C (100 MHz, DMSO-de) 21 .8 (aniline-Me), 1 15.5, 121 .7, 123.1 , 126.4, 127.4, 127.6, 129.1 , 129.4, 133.0, 133.3, 136.6, 137.5, 139.2, 141 .8, 144.4, 148.5, 177.5 (C=0), 180.5 (C=0); m/z (ESI") 476 ([M- H]\ 100%); HRMS (ESI") C24H18N306S", ([M-H]") requires 476.0922; found 476.0917.
[00206] The structures depicted for regioisomeric compounds 48 and 49 have been assigned based on the data currently available. This data is not sufficient for the structures to be assigned with absolute certainty. Therefore the depicted structures should be considered to be tentative until x-ray crystallographic data becomes available.
Example 50
W-(8-Amino-3-((3,5-dimethylphenyl)amino)-1 ,4-dioxo-1 ,4-dihydronaphthalen-2- yl)benzenesulphonamide 50
[00207] Treatment of 48 (100 mg, 0.21 mmol) with catalytic 10% Pd/C
(2.2 mg, 0.02 mmol) under a balloon of H2 in EtOAc at RT for 18 h gave
the crude reaction mixture. Filtration through Celite® (EtOAc) followed by
column chromatography (DCM:MeOH 95:5) gave 50 as a purple solid (87
mg, 93%); mp 220-224 °C; umax (KBr) 3337 (N-H), 3295 (N-H), 1661
Figure imgf000069_0002
(C=0), 1618 (C=0); δΗ (400 MHz, DMSO-d6) 2.20 (6H, s, 2 χ Ar-Me), 6.50 (2H, s, aniline-orf/70- H), 6.59 (1 H, s, aniline-para-H), 7.02 - 7.15 (1 H, m, Ar), 7.19-7.30 (1 H, m, Ar), 7.31 -7.43 (4H, m, Ar), 7.45-7.59 (4H, m, Ar), 8.33 (1 H, s, NH aniline), 8.94 (1 H, br. s, NH sulphonamide); 5C (100 MHz, DMSO-d6) 21 .9 (/We), 1 10.1 , 1 16.4, 1 16.5, 120.9, 125.2, 125.4, 127.2, 129.2, 132.2, 132.8, 134.1 , 137.2, 139.4, 140.8, 141 .9, 151 .3, 182.7 (C=0), 183.4 (C=0); m/z (ESI") 446 ([M- H]\ 100%); HRMS (ESI+) C24H21N3Na04S+, ([M+Na]+) requires 470.1 145; found 470.1 147.
Example 51 yV-(5-Amino-3-((3,5-dimethylphenyl)amino)-1 ,4-dioxo-1 ,4-dihydronaphthalen-2- yl)benzenesulphonamide 51
[00208] Treatment of 49 (100 mg, 0.21 mmol) with catalytic 10% Pd/C
(2.2 mg, 0.02 mmol) under a balloon of H2 in EtOAc at RT for 18 h gave
the crude reaction mixture. Filtration through Celite® (EtOAc) followed by
column chromatography (DCM:MeOH 95:5) gave 51 as a dark orange
solid (95 mg, quant.); mp 228-231 °C; umax (KBr) 3464 (N-H), 3300 (N-H),
Figure imgf000070_0001
1573 (C=0); δΗ (400 MHz, DMSO-d6) 2.21 (6H, s, 2 χ Ar-Me), 6.57 (2H, s, aniline-H), 6.63 (1 H, s, aniline-H), 6.97-7.1 1 (2H, m, sulphonamide-/Ar), 7.29-7.43 (3H, m, sulphonamide-/Ar), 7.44- 7.54 (3H, m,
Figure imgf000070_0002
7.84 (2H, br. s, NH2), 8.58 (1 H, s, aniline-H), 8.84 (1 H, br. s, sulphonamide-H); 5C (100 MHz, DMSO-d6) 21 .9 (Ar-/We), 109.0, 1 13.3, 1 15.9, 121 .7, 123.0, 126.0, 127.2, 129.2, 132.6, 133.3. 136.0, 137.2, 138.8, 142.2, 143.1 , 152.5 (C-NH2), 179.6 (C=0), 182.9 (C=0); m/z (ESI") 446 ([M-H]", 100%); HRMS (ESI+) C24H21N3Na04S+, ([M+Na]+) requires 448.1326; found 448.1326.
[00209] The structures depicted for regioisomeric compounds 50 and 51 have been assigned based on the data currently available. This data is not sufficient for the structures to be assigned with absolute certainty. Therefore the depicted structures should be considered to be tentative until x-ray crystallographic data becomes available.
Example 52 15A/-Benzenesulphonamide 52
[00210] Benzenesulphonyl chloride (256 μΙ_) was added dropwise to a 3M aq.
NaOH solution containing 15/V-labelled ammonium chloride and the solution
was heated at 90 °C over 0.5 h then allowed to cool to RT. Filtration of the
Figure imgf000070_0003
reaction mixture gave 52 as a white, crystalline solid (231 mg, 73%). mp 152-154 °C; umax (KBr) 3257 (N-H); δΗ (400 MHz, Methanol-d4) 7.51 -7.66 (3H, m), 7.87-7.96 (2H, m), NH2 not observed; 5C (100 MHz, DMSO-d6) 126.1 , 129.0, 132.2, 144.0; m/z (ESI") 157 ([M-H]", 100%); HRMS (ESI+) C6H7 15NNa02S+, ([M+Na]+) requires 181 .0060; found 181 .0059.
Example 53 W-(3-((3,5-Dimethylphenyl)amino)-1 ,4-dioxo-1 ,4-dihydronaphthalen-2-yl)15yV- benzenesulphonamide 53
[00211] Following General Procedure 1, 2,3-dichloronaphthalen-1 ,4- dione 2 (221 mg, 0.97 mmol), 15/V-labelled benzenesulfonamide 52 (200
mg, 1 .26 mmol) and Cs2C03 (41 1 mg, 1 .26 mmol) were subjected to
Figure imgf000071_0001
microwave irradiation in a sealed vessel at 170 °C in toluene over 40 min.. The reaction mixture was partitioned between DCM and 1 M aq. HCI, and the organic extract was washed with brine, dried, and concentrated in vacuo to give a crude 15/V-labelled sample of 54; m/z (EST) 347 ([M-H]", 100%); other spectroscopic data as previously reported.
Example 54 yV-(3-(3,5-dimethylphenylamino)-1 ,4-dioxo-1 ,4-dihydronaphthalen-2- yl)benzenesulfonamide 54 [00212] Following General Procedure 2, crude 53 (200 mg, 0.57 mmol)
and CeCI3.7H20 (87 mg, 0.23 mmol) and 3,5-dimethylaniline (214 μΙ_,
1 .71 mmol) gave the crude reaction mixture. Partial purification via
column chromatography (eluent pet ether:acetone 75:25) gave 54 as a
purple solid (179 mg, 72%), which was recrystallised from toluene to
Figure imgf000071_0002
give 55 (77 mg, 7% w.r.t. 15NH4CI); mp 220-221 °C; umax (KBr) 3304 (N-H), 3188 (N-H), 1664 (C=0), 1630 (C=0); δΗ (500 MHz, DMSO-d6) 2.23 (6H, s, 2 χ Ar-Me) 6.60 (2H, s, aniline-H), 6.67 (1 H, s, aniline-H), 7.32-7.41 (2H, m, Ar), 7.47-7.58 (3H, m, Ar), 7.74-7.86 (3H, m, Ar), 8.00-8.05 (1 H, m, Ar), 8.77 (1 H, s, aniline-NH), 9.04 (1 H, d, J 86.0, S02 15NH); 5C (100 MHz, CDCI3) 21 .4 (Me), 1 13.3, 121 .1 , 126.4, 126.9, 127.0, 128.7, 129.1 , 130.5, 131 .4, 132.9, 133.0, 134.8, 137.3, 138.0, 139.0, 179.1 (C=0), 181 .9 (C=0); m/z (ESI") 432 ([M-H]", 100%); HRMS (ESI+) C24H20N15NNaO4S+, ([M+Na]+) requires 456.1007; found 456.1014.
Example 55 yV-(3-((3,5-Dimethylphenyl)amino)-1 ,4-dioxo-1 ,4-dihydronaphthalen-2-yl)-yV- methylbenzenesulphonamide 55
[00213] Treatment of 7 (108 mg, 0.25 mmol) with 2M
trimethylsilyldiazomethane hexane solution (175 μΙ_, 13.5 mmol) and
anhydrous methanol (173 μΙ_, 4.25 mmol) gave the crude reaction
Figure imgf000071_0003
mixture, which was then partitioned between DCM and sat. aq. NH4CI. The organic phase was washed with brine before being dried, filtered and concentrated in vacuo. Purification by column chromatography (eluent pet ether:acetone 80:20) gave 55 as an orange solid (70 mg, 63%); mp 188-192 °C; umax (KBr) 3303 (N-H), 1678 (C=0); δΗ (400 MHz, DMSO-d6) 2.26 (6H, s, 2 x Ar-Afe), 2.82 (3H, s, N-Afe), 6.84 (3H, s, anWme-Ar), 7.37-7.64 (5H, m, S02-Ar), 7.68-7.88 (3H, m, naphthoquinone-/*/-), 7.95-8.13 (1 H, m, naphthoquinone-/*/-), 9.16 (1 H, s, NH); 5C (100 MHz, DMSO-de) 21 .6 (Ar-Afe), 37.2 (N-Afe), 1 17.2, 123.7, 126.5, 127.0, 127.8, 128.1 , 129.5, 130.8, 133.1 , 133.3, 133.6, 136.0, 137.6, 139.7, 139.8, 146.7, 179.4 (C=0), 183.4 (C=0); m/z (ESI") 488 ([M-H]\ 100%); HRMS (ESI+) C25H22N2Na04S+, ([M+Na]+) requires 469.1 192; found 469.1 191 .
Example 56
Expression of mouse Nat2
[00214] Since mouse Nat2 (mNat2) has 80% identity in amino acid sequence compared to the hlMATI isoenzyme and is easily expressed and purified as recombinant protein, the
compounds were mainly tested against mNat2.
[00215] Thus, mNat2 was expressed in E. coli RosettaBlue(DE3)pLysS transfected with pET28b(+) plasmid containing the mNat2 gene. The expressed mNat2 with an N-terminal hexa-histidine tag was purified by ion-metal affinity chromatography. Pure recombinant mNat2 was purified with a yield of 57 mg per litre of culture according to the method previously described (Kawamura, A. et al. Biochem Pharmacol, 2008. 75(7), 1550-1560).
Example 57
Colour change
[00216] A 2mM stock solution of each of compounds 1 , 5-8 and 1 1 was prepared in DMSO. The colour change test of compounds 1 , 5-8 was performed with a 2 mM inhibitor solution in DMSO (50 l_) with 10 μΙ_ of buffer (20mM Tris.HCI, pH 8.0), as control or with 10 μΙ_ of 4 M NaOH solution to observe the colour change. Absorption spectra were recorded with a U-2001 spectrophotometer (Hitachi) from 800 to 250 nm.
Table 3: Colour change of compounds 1 , 5-8
Compound No. (10 μΜ) 1 5 6 7 8
Amax (nm) in assay buffer 489 487 484 498 490
AAmax with NaOH (nm) + 72 + 49 0 + 74 + 69
AAmax with MNAT2 (nm) + 121 + 123 0 + 127 + 125 Example 58
Inhibitory Activity
[00217] The activity of pure recombinant mNat2 (5ng) was determined as hydrolysis of AcCoA (400 μΜ) in the presence of para-amino benzoic acid (500 μΜ) as arylamine substrate (Kawamura, A. et al. Biochem Pharmacol, 2008. 75(7), 1550-1560). Percentage inhibition was determined as the ratio of specific activity without the inhibitor related to specific activity with inhibitor (100%). The following (Table 4) IC50 values were estimated graphically from plots of specific activities versus inhibitor concentration by using Kyplot® software.
[00218] A dose-response function was used as a model of regression for the data and the curves were estimated by the method of the least squares analysis. All the statistical tests were performed with Kyplot® software. 95% was chosen as confidence limit for these tests.
Table 4: IC5p values
Figure imgf000073_0001
[00219] The quoted rates of reaction are initial rates determined from the linear initial section of graphs of absorbance versus time, assays were conducted in triplicate, and the rates and kinetic parameters quoted in this study are mean values ± standard deviation. Since different sources of enzymes were used for activity assays, different protein concentrations and incubation times were selected to give a linear initial rate.
[00220] DTNB: Immediately prior to use, pure mNAT2 solution were diluted in assay buffer (20 mM Tris.HCI, pH8.0). Samples of pure mNAT2 (5 μΙ_ containing 50 ng protein) were preincubated with pABA (50 μΙ_ of 1 mM stock solution, 500 μΜ final concentration) in assay buffer for 5 min at 37 °C in a 96-well plate (Greiner). Pre-warmed (37 °C) AcCoA (40 μΙ_ of 1 mM stock solution, 400 μΜ final concentration) in assay buffer was added to start the reaction (final volume of 100 μΙ_), which was allowed to proceed at 37 °C. Simultaneous quenching and colour development was achieved by addition of the stop reagent: 25 μΙ_ DTNB solution (5mM DTNB in 100 mM Tris.HCI, 6.4 M guanidine.HCI, pH 7.3). The absorbance was read immediately after addition of the stop reagent at the wavelength closest to 412 nm, which is available using a plate reader (Tecan Sunrise), i.e. at 405 nm. The rate of reaction was determined by reference to a standard curve. The standard curve for the detection of CoA was determined by addition of 25 μΙ_ of DTNB solution (see above) to 100 μΙ_ of CoA (0 - 500 μΜ) in assay buffer (as above).
[00221] DMAB: Samples of enzyme (7.25 μΙ_ containing 18 ng recombinant mNAT2 or 7.25 μΙ_ containing 54.4 ng recombinant hlMATI ) and 4-aminobenzoic acid (pABA) (15 μΙ_ of 450 μΜ stock solution, 150 μΜ final concentration) were pre-incubated at 37 °C in assay buffer (20 mM Tris.HCI, pH 8.0) for 5 min in a 96-well plate (Greiner). Pre-warmed AcCoA (20 μΙ_ of 900 μΜ stock solution, 400 μΜ final concentration, 37 °C) in assay buffer was added to start the reaction (final volume of 45 μΙ_), which was allowed to proceed at 37 °C. The reaction was quenched with 45 μΙ_ of 20 % (w/v) aqueous trichloroacetic acid (TCA) solution at 0 °C. 150 μΙ_ of DMAB (5% (w/v) in 90% aqueous CH3CN solution) was added and the absorbance was measured immediately at 450 nm in the 96-well micro plate using a plate reader (Tecan Sunrise), and the concentration of arylamine was calculated by reference to a standard curve. For the generation of the standard curve, 45 μΙ_ of 20% (w/v) aqueous TCA solution was added to 45 μΙ_ of pABA (0 - 500 μΜ) in assay buffer (as above), and then 150 μΙ_ of DMAB (as above). The absorbance at 450 nm was measured, and the triplicate results were plotted as a graph of absorbance versus arylamine concentration.
[00222] Inhibitor assays: The assays to determine NAT inhibition were carried out using the 'DTNB' method for pure recombinant mNAT2 and the 'DMAB' method for both pure recombinant mNAT2 and hlMATI . All the tested compounds were dissolved in DMSO and the final percentage of DMSO in the assay was 5 % (v/v). Inhibition percentages were determined as ratio of specific activity without the requisite inhibitor over specific activity with the inhibitor. The above described 'DTNB' assay was carried out with 30 μΜ final inhibitor concentration and the 'DMAB' assays were conducted with 5 μΜ final inhibitor concentration.
[00223] The following (Table 5) IC50s were estimated graphically from plots of absorbance or specific activities versus inhibitor concentrations (30, 10, 5, 2.5, 1 .25, 0.625, 0.3125, 0.15625 μΜ) by using GraphPad® software. A dose-response function was used as a model of regression for the data and the curves were estimated by the method of the least squares analysis.
Table 4: IC50 values (mNAT2, DTNB)
Compound IC50 (μΜ)
7 <30
9 <30
10 <30
11 <30
12 <30
14 <30 17 <30
19 <30
21 <30
22 <30
23 <30
26 <30
43 <30
45 <30
49 <30
50 <30
51 <30
55 >30
Example 59
Effect of Reducing Agents and base
[00224] The susceptibility of the naphthoquinone function towards reduction led to an investigation as to whether any reducing agents may give rise to the colour change of compound 7, especially DTT which is present in the recombinant protein solution in a concentration of 34 μΜ. DTT at 34 μΜ does not reduce compound 7 and does not affect the colour of the inhibitor, but at high concentration (100 mM) DTT and dithionite led to a decolorisation of solution of compound 7 which was reversed upon exposure to air. Other reducing agents such as NADH and NADPH gave no visible change, and only treatment with base gave the characteristic blue shift in the visible spectra of compound 7. Wavelength scan of compound 7 (10 μΜ) were recorded with a U-2001 spectrophotometer (Hitachi) from 800 to 250 nm in the presence of different reducing agents (100mM).
[00225] The effect of base is illustrated by Figure 1 , which is pH titration curve for compound 1 . Example 60
Effect of Compound 7 on activity of different NATs and shift in absorbance maxima of compound 7 in their presence
[00226] The activity was determined by the AcCoA hydrolysis assay. The substrate 5- aminosalysilic acid (500 μΜ) was incubated with the appropriate NAT enzyme (100 ng), at 37°C for 5 minutes, with or without the inhibitor 7 (10 μΜ). Pre-warmed AcCoA (400 μΜ) was added to start the reaction (final volume 100 μΙ_). Simultaneous quenching and colour development was achieved by addition of the stop reagent: 25 μΙ_ DTNB solution (5mM DTNB in 100 mM Tris.HCI, 6.4 M guanidine.HCI, pH 7.3). The absorbance was read at 405 nm immediately afterwards. Assays were conducted in triplicate, and the linear activity rates are mean values ± standard deviation.
Table 5:
Enzyme mNat2 hNatl STNAT PANAT MM NAT MS NAT
Inhibition
92.8±0.4 55.9±6.4 < 5 19.6±3.0 < 5 < 5
(%)
Amax (nm) 585 625 498 498 498 498
STNAT = NAT from Salmonella typhimurium, MMNAT = NAT from Mycobacterium marinum, MSNAT = NAT from Mycobacterium smegmatis, PANAT = NAT from Pseudomonas aeruginosa, were used as NAT controls
[00227] Although partial cross-reactivity was observed within NAT1 the colour change was observed only with mNAT2 and hNAT1 .
Example 61
Characterisation of inhibition
[00228] Percentage of inhibition was determined as the ratio of specific activity without the inhibitor over specific activity with inhibitor (100%). IC50s were estimated graphically from plots of specific activities versus inhibitor concentration by using Kyplot® software. A dose-response function was used as a model of regression for the data and the curves were estimated by the method of the least squares analysis. All the statistical tests were performed with Kyplot® software. 95% was chosen as confidence limit for these tests.
[00229] Determination of the mode of inhibition of mNat2 by 1 was performed over a range of concentrations of 1 from 0 to 3 μΜ and pABA as arylamine substrate from 40 to 300 μΜ. The results are shown in Figure 2.
Example 62
[00230] This example compares the amino acid sequences and substrate specificities in human and mouse NAT isoenzymes. Percentage inhibition at 30 μΜ and color change of compound 1 versus NAT isoenzymes.
T le 6:
Figure imgf000076_0001
hNAT1 100 - - - + > 95 + mNat2 82 100 - - + ≥95 + hNAT2 81 74 100 - + 0 - mNatl 74 81 72 100 + 0 — * = para-aminobenzoyl-L-glutamate
Example 63
Detection of arylamine acetylation
[00231] The rates of enzymic reactions with arylamine substrates were determined using the colorimetric agent 4-dimethylaminobenzaldehyde (DMAB), using a modified literature method. [ Andres, H. H.; Klem, A. J.; Szabo, S. M.; Weber. W. W. Anal. Biochem. 1985, 145, 367].
[00232] Samples of enzyme (9 ng pure recombinant mNat2, a 300-times diluted E. coli lysate containing hNAT1 , or a 2000-times diluted ZR-75-1 breast cancer cell lysate containing hNAT1 14) and para-aminobenzoic acid (pABA) (150 μΜ) were pre-incubated at 37 °C in assay buffer (20 mM Tris.HCI, pH 8.0, 1 mM DTT) for 5 min in a 96-well plate (Corning).
[00233] Pre-warmed AcCoA (400 μΜ final concentration, 37 °C) in assay buffer was added to start the reaction (final volume of 45 μΙ_), which was allowed to proceed at 37 °C. The reaction was quenched with 45 μΙ_ of 20 % (w/v) aqueous trichloroacetic acid (TCA) solution at 0 °C. 150 μΙ_ of DMAB (5% (w/v) in 90% aqueous CH3CN solution) was added and the mixture was incubated at room temperature for 15 min. The absorbance was measured at 450 nm in the 96-well micro plate using a plate reader (Tecan Sunrise), and the concentration of arylamine was calculated by reference to a standard curve. This assay was used always for hlMATI in cell lysate and for all comparable experiments.
[00234] For detection of inhibition, minor modifications were introduced in the enzymic assays containing the appropriate inhibitor. All compounds tested were dissolved in DMSO and the final percentage of DMSO in the assay was 5 % (v/v).
Detection of NAT activity by acetylCoA hydrolysis
[00235] The rate of formation of Coenzyme A (CoA) was determined spectrophotometrically using the colorimetric agent 5,5'-dithio-bis(2-nitrobenzoic acid) (Ellman's reagent, DTNB) as previously described, with some minor modifications.12 This assay was used only with pure recombinant NATs and the appropriate arylamine substrate was chosen according to Westwood et al. (Westwood, I. M.; Kawamura, A.; Fullam, E.; Russell, A. J.; Davies, S. G.; Sim, E. Curr. Med. Chem. 2006, 1641 .)
[00236] mNat2 (5 ng) was preincubated with pABA (500 μΜ) in assay buffer (see above) for 5 min at 37 °C in a 96-well plate (Corning). Pre-warmed (37 °C) AcCoA (400 μΜ) in assay buffer was added to start the reaction (final volume of 100 μΙ_), which was allowed to proceed at 37 °C. Simultaneous quenching and colour development was achieved by addition of the stop reagent: 25 μΙ_ DTNB solution (5mM DTNB in 100 mM Tris.HCI, 6.4 M guanidine.HCI, pH 7.3). The absorbance was read immediately after addition of the stop reagent at 405 nm using a plate reader (Tecan Sunrise). The rate of reaction was determined by reference to a standard curve.
[00237] In the screening of different NATs in the presence of compound 7, the same procedure was followed with 100 ng of the appropriate NAT and 5-amino salicylic acid as arylamine substrate.
[00238] For detection of inhibition, minor modifications were introduced in the enzymic assays containing the appropriate inhibitor. All compounds tested were dissolved in DMSO and the final percentage of DMSO in the assay was 5 % (v/v).
[00239] The results are shown in Figure 3, which shows the results of screening of compounds 5-8 (5 μΜ) against pure mNat2 (black columns), hNAT1 in Escherichia coli cell lysate (grey columns), and hNAT1 in ZR-75-1 breast cancer cell lysate (1 and 7 only, white columns). NAT activity was determined as acetylation of pABA in the presence of AcCoA.

Claims

P127398W O As filed WO 2011/055142 PCT/GB2010/051840 78 CLAIMS
tructure of Formula I and salts and physiological
Figure imgf000079_0001
Figure imgf000079_0002
wherein:
X is O or S;
Y is O or S;
R1 is R5, R8 or J;
wherein:
R5 is H; alkyl having 1 , 2, 3, 4 or 5 carbon atoms and unsubstituted or substituted by hydroxy; alkyl having 1 , 2, 3 or 4 carbon atoms, unsubstituted or substituted by hydroxy and interrupted by -0-, -S-, or -C(O)-; -L-R7; -S(0)b-L-R7; -C(0)-N(R19)-[S(0)b]rL-R7, -C(0)-L-R7; -C(NR19)-L-R7; -C(NR19)0-L-R7; -C(NR19)NR30-L-R7; -C(0)S-L-R7; -C(NR19)S- L-R7 -C(0)0-L-R7 ; 2-pyridyl or 4-pyridyl, wherein 2-pyridyl and 4-pyridyl are unsubstituted or are substituted by one or more substituents independently selected from L-R7 and R8,
wherein:
L is a bond, alkylene optionally substituted by hydroxy and having 1 , 2, 3, 4 or 5 carbon atoms and unsubstituted or substituted by hydroxy, alkylene optionally substituted by hydroxy and having 1 , 2, 3 or 4 carbon atoms and interrupted and/or terminated adjacent R7 by -0-, -S-, or -C(O)-;
R7 is H or a 3-7 membered monocyclic ring or an 8-13 membered bicyclic ring, which rings are unsubstituted or are substituted by one or more R8, or is a dye;
R19 is selected from the possibilities for L-R7;
b is 1 or 2; and
f is 0 or 1 ;
R is selected from halogen, hydroxy, alkyl optionally substituted by hydroxy and having 1 , 2, 3, 4 or 5 carbon atoms, alkyl optionally substituted by hydroxy and having 1 , 2, 3 P127398W O As filed
WO 2011/055142 PCT/GB2010/051840
79
or 4 carbon atoms and interrupted by -0-, -S-, or -C(O)-, a 3 to 6- membered ring bonded to the remainder of the molecule either (i) directly or (ii) through a Ci-4 alkylene group or (iii) through a Ci-4 alkylene group interrupted and/or terminated by -0-, -S-, or -C(O)-, trifluoromethyl, trifluoromethoxy, cyano, nitro, oxo, amidino, -OR9, -C(0)R9, - C(0)OR9, -OC(0)R9, -N(R9)R10, -C(0)N(R9)R10, -S(0)aR9 and -S(0)aOR9 wherein R9 and R10 are the same or different and are each hydrogen or are selected from alkyl optionally substituted by hydroxy and having 1 , 2, 3, 4 or 5 carbon atoms and a 3 to 6- membered ring bonded to the remainder of the molecule either directly or through a Ci_ 4 alkylene group, and a is 0, 1 or 2;
J is a group of Formula (II):
Figure imgf000080_0001
wherein
Z is a bond, -S(0)c-, -S(0)c-NR18-C(0)-, -C(O)-, -(CH2)d-, -N=C(NR11R12)- or -NR11CR13R14-,
wherein c is 0, 1 or 2; d is 1 , 2, 3 or 4; R11 and R12 are the same or different and are each H or alkyl having 1 , 2, 3 or 4 carbon atoms; and R and R are the same or different and are each H or alkyl having 1 , 2, 3 or 4 carbon atoms, or R13 and R14 taken together form =NR15, =0 or =S, wherein R15 is hydrogen, hydroxy or alkyl optionally substituted by hydroxy and having 1 , 2, 3, 4 or 5 carbon atoms, and R18 is H or alkyl having 1 , 2, 3, 4 or 5 carbon atoms;
R5 is as defined previously; and
R6 is selected from the possibilities for L-R7; and
R2 is H, R8 or J, provided that at least one of R1 and R2 is J and wherein if both R1 and R2 are J they may be the same or different;
R3 and R4 are each independently H or R8; or R3 and R4 taken together form a benzene ring which is unsubstituted or substituted by one or more R8,
wherein where there is there is more than one -L-R7, each -L-R7 may be the same as or different from any other -L-R7, and where there is there is more than one R8, each R8 may be the same as or different from any other R8.
2. A compound of claim 1 wherein R1 and R2 are J, R1 and R2 being the same as or different from each other. P127398W O As filed
WO 2011/055142 PCT/GB2010/051840
80
3. A compound of claim 1 or claim 2 wherein R6 is H.
4. A compound of any preceding claim wherein Z is a bond.
5. A compound of any of claims 1 to 3 wherein Z is -S(0)2-.
6. A compound of any preceding claim wherein X and Y are both O.
7. A compound of any preceding claim wherein L is a bond.
8. A compound of any of claims 1 to 6 wherein L is Ci-5 alkylene optionally substituted by hydroxy and optionally interrupted by and/or terminated adjacent R7 by -0-, -S- or -C(O)-.
9. A compound of any preceding claim wherein R7 is phenyl, naphthyl, a 5- or 6- membered heteroaromatic ring, in any of these instances optionally substituted by 1 , 2 or 3 R8 moieties, or is a dye, .
10. A compound of any preceding claim wherein b and c are each 2.
1 1 . A compound of any preceding claim wherein R8 is F, CI, Br, methyl, ethyl, trifluoromethyl, trifluoromethoxy, methoxy, hydroxy, hydroxymethyl, nitro, cyano, amino, methylamino or dimethylamino.
12. A compound of any preceding claim wherein J is selected from -N(R6)-L-R7, -S(0)c- N(R6)-L-R7, -S(0)c-NR18-C(0)-N(R6)-L-R7, -C(0)-N(R6)-L-R7, -(CH2)d-N(R6)-S(0)b-L-R7; -(CH2)d- N(R6)-C(0)-N(R19)-S(0)b-L-R7; -C(0)-L-R7; -C(0)0-L-R7, -N=C(NR11R12)-N(R6)-S(0)b-L-R7; and -N(R11)CR13R14-N(R6)-S(0)b-L-R7.
13. A compound of any preceding claim wherein R19 and R30 are each independently H or C1-C5 alkyl.
14. A compound of any preceding claim wherein R1 and R2 are a combination of groups selected from the combinations of Table 1 or Table 2.
15. A compound of any preceding claim wherein R1 is:
(i)-N(R6)-L-R7, e.g. -NH-L-R7; or
(ii) -L-R7 or O-L-R7, wherein L is a bond and R7 is a substituted or unsubstituted ring selected from 3-7 membered monocyclic rings and 8-13 membered bicyclic rings optionally substituted with one or more R8 .
16. A compound of any preceding claim which is selected from the compounds of Formulae (III), (IV), (V) and (VI): P127398W O As filed
WO 2011/055142 PCT/GB2010/051840
81
Figure imgf000082_0001
wherein R3 and R4 are the same or different and selected from H and R8.
17. A compound of any of claims 1 to 15 which is selected from the compounds of Formulae (VIII), (IX), (X) and (XI):
Figure imgf000082_0002
in which:
R20, R21, R22 and R23 are independently selected from H and R8.
18. A compound of any preceding claim, wherein R2 is of Formula (lla):
,5a
(lla)
H wherein R5a is -S(0)b-L-R7; -C(0)-N(R19)-[S(0)b]rL-R7, -C(0)-L-R7; -C(NR19)-L-R7; -C(NR19)0-L- R7; -C(NR19)NR30-L-R7; -C(0)S-L-R7; -C(NR19)S-L-R7 -C(0)0-L-R7; 2-pyridyl or 4-pyridyl, wherein 2-pyridyl and 4-pyridyl are unsubstituted or are substituted by one or more substituents independently selected from L-R7 and R8.
19. A compound of claim 18, wherein R5a is -S(0)2-L-R7. P127398W O As filed
WO 2011/055142 PCT/GB2010/051840
82
20. A compound of claim 18 or claim 19 wherein R7 is phenyl, thiophenyl or a fluorescent dye, wherein phenyl and thiophenyl are unsubstituted or are substituted by one or more R8, e.g. selected from halogen, methyl and trifluoromethyl.
21 . A compound of claim 19 which is of Formula (XIII):
Figure imgf000083_0001
wherein:
R -R are each independently H, methyl, trifluoromethyl or halogen;
R25 is phenyl or thiophenyl which in either case is unsubstituted or substituted by one or more R8 selected from halogen, methyl and trifluoromethyl;
R26 is phenyl which is unsubstituted or substituted by 1 , 2, 3 or 4 R8 selected from halogen, methyl and trifluoromethyl,
or wherein one of R25 and R26 is a dye instead of one of the possibilities previously mentioned in this claim.
22. A method for detecting hlMATI , comprising contacting a sample with a chromogenic compound of any of claims 18 to 21 to form a labelling composition, incubating the labelling composition for a sufficient amount of time for the compound to bind to any hlMATI in the sample, illuminating the composition with an appropriate wavelength for the compound and observing for emission of light having a wavelength characteristic of the compound when bound to hlMATI .
23. A method of claim 22 wherein the sample comprises intact or lysed human breast cells.
24. A method of claim 22 wherein the sample is, or is derived from, tissue removed during surgical treatment of breast cancer.
25. The in vitro use of a compound of any of claims 18 to 21 as a chromogenic probe to detect or measure hlMATI in a sample.
26. A method of treating cancer in a human subject, comprising administering to the subject a therapeutically effective amount of a compound of any of claims 1 to 21 .
27. A method of surgical treatment of breast cancer of a patient, comprising: P127398W O As filed
WO 2011/055142 PCT/GB2010/051840
83
removing tissue known or to comprise, or at risk of comprising, cancer cells;
applying a compound of any of claims 18 to 21 to at least a marginal portion of the removed tissue; and
observing said at least marginal portion for emission of light having a wavelength characteristic of the compound when bound to hlMATI .
28. A method of claim 27 wherein light having a wavelength characteristic of the compound when bound to hlMATI is not observed and no further tissue is removed from the patient.
29. A method of claim 27 wherein light having a wavelength characteristic of the compound when bound to hlMATI is observed, further tissue is removed from the patient, the further removed tissue has applied to at least a marginal portion thereof a compound of any of claims 18 to 21 , and said at least marginal portion is observed for emission of light having a wavelength characteristic of the compound when bound to hlMATI .
30. A method of detecting cancer cells during breast cancer surgery, comprising applying to an area of tissue at risk of presenting a cancer cell a compound of any of claims 18 to 21 , and observed said area for emission of light having a wavelength characteristic of the compound when bound to hNAT1 .
31 . A compound of any of claims 18 to 21 for use in diagnosing tissue during surgery for the presence of cancer cells.
32. The subject matter of any of claims 24 and 26 to 31 , wherein the cancer is ER+ breast cancer or an invasive ductal and/or lobular breast carcinoma.
33. A compound of any of claims 1 to 21 for therapeutic use, particularly for use in a method of treating cancer.
84
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