WO2005085189A2 - Analogues of congo red and evaluation of their anti-prion activity - Google Patents

Analogues of congo red and evaluation of their anti-prion activity Download PDF

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WO2005085189A2
WO2005085189A2 PCT/GB2005/000808 GB2005000808W WO2005085189A2 WO 2005085189 A2 WO2005085189 A2 WO 2005085189A2 GB 2005000808 W GB2005000808 W GB 2005000808W WO 2005085189 A2 WO2005085189 A2 WO 2005085189A2
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conh
compound according
cor
alkyl
compound
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WO2005085189A3 (en
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Ian Gilbert
Shane Sellarajah
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Medical Research Council
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C39/00—Compounds having at least one hydroxy or O-metal group bound to a carbon atom of a six-membered aromatic ring
    • C07C39/205—Compounds having at least one hydroxy or O-metal group bound to a carbon atom of a six-membered aromatic ring polycyclic, containing only six-membered aromatic rings as cyclic parts with unsaturation outside the rings
    • C07C39/21—Compounds having at least one hydroxy or O-metal group bound to a carbon atom of a six-membered aromatic ring polycyclic, containing only six-membered aromatic rings as cyclic parts with unsaturation outside the rings with at least one hydroxy group on a non-condensed ring
    • C07C39/215—Compounds having at least one hydroxy or O-metal group bound to a carbon atom of a six-membered aromatic ring polycyclic, containing only six-membered aromatic rings as cyclic parts with unsaturation outside the rings with at least one hydroxy group on a non-condensed ring containing, e.g. diethylstilbestrol
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C215/00—Compounds containing amino and hydroxy groups bound to the same carbon skeleton
    • C07C215/74—Compounds containing amino and hydroxy groups bound to the same carbon skeleton having hydroxy groups and amino groups bound to carbon atoms of six-membered aromatic rings of the same carbon skeleton
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C229/00—Compounds containing amino and carboxyl groups bound to the same carbon skeleton
    • C07C229/52—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to carbon atoms of six-membered aromatic rings of the same carbon skeleton
    • C07C229/54—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to carbon atoms of six-membered aromatic rings of the same carbon skeleton with amino and carboxyl groups bound to carbon atoms of the same non-condensed six-membered aromatic ring
    • C07C229/60—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to carbon atoms of six-membered aromatic rings of the same carbon skeleton with amino and carboxyl groups bound to carbon atoms of the same non-condensed six-membered aromatic ring with amino and carboxyl groups bound in meta- or para- positions
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C233/00—Carboxylic acid amides
    • C07C233/64—Carboxylic acid amides having carbon atoms of carboxamide groups bound to carbon atoms of six-membered aromatic rings
    • C07C233/67—Carboxylic acid amides having carbon atoms of carboxamide groups bound to carbon atoms of six-membered aromatic rings having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by singly-bound oxygen atoms
    • C07C233/75—Carboxylic acid amides having carbon atoms of carboxamide groups bound to carbon atoms of six-membered aromatic rings having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by singly-bound oxygen atoms with the substituted hydrocarbon radical bound to the nitrogen atom of the carboxamide group by a carbon atom of a six-membered aromatic ring
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C251/00—Compounds containing nitrogen atoms doubly-bound to a carbon skeleton
    • C07C251/02—Compounds containing nitrogen atoms doubly-bound to a carbon skeleton containing imino groups
    • C07C251/24—Compounds containing nitrogen atoms doubly-bound to a carbon skeleton containing imino groups having carbon atoms of imino groups bound to carbon atoms of six-membered aromatic rings
    • 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
    • C07C311/21—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 six-membered aromatic ring
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C65/00—Compounds having carboxyl groups bound to carbon atoms of six—membered aromatic rings and containing any of the groups OH, O—metal, —CHO, keto, ether, groups, groups, or groups
    • C07C65/01—Compounds having carboxyl groups bound to carbon atoms of six—membered aromatic rings and containing any of the groups OH, O—metal, —CHO, keto, ether, groups, groups, or groups containing hydroxy or O-metal groups
    • C07C65/19—Compounds having carboxyl groups bound to carbon atoms of six—membered aromatic rings and containing any of the groups OH, O—metal, —CHO, keto, ether, groups, groups, or groups containing hydroxy or O-metal groups having unsaturation outside the aromatic ring

Definitions

  • the present invention relates to chemical analogues of the diazo compound known as Congo Red. More specifically, the invention relates to compounds that are useful in the treatment or prevention of transmissible spongiform encephalopathies.
  • TSE Transmissible spongiform encephalopathies
  • GSS Gerstmann- Staussler-Scheinker Syndrome
  • FFI Familial Fatal Insomnia
  • BSE Bovine Spongiform Encephalopathy
  • PrP-res misfolded prion protein form
  • PrP-res normal cellular prion protein form
  • Compounds shown to have anti-prion activity include sulphated polysaccharides such as pentosan polysulfate, Congo Red and other azo dyes, amphotericin B and analogues, anthracyclines, phthalocyanines and porphyrins, inorganic ions, branched polyamines, and antagonists of the N-methyl-D-aspartate (NMD A) receptor such as memantine. 6 Recently there has been interest in quinacrine and other acridine derivatives.
  • the azo-dye Congo Red has been shown to have in vivo anti-prion activity against various models of TSEs, including a cell free polymerisation assay, 11 cellular assays 12 ' 13 and in vivo activity against golden Syrian hamsters. 14
  • the molecule itself has a number of shortfalls. 15 Firstly, it has a lack of specificity. In addition it does not have significant permeability through the blood brain barrier 16 ' 17 presumably due to its charged nature, primarily caused by the presence of two sulfonate groups. Toxicity is also a problem as the diazo bonds in Congo Red can be cleaved by enzymes present in the mammalian gut and intestines. ' The most common side product of this cleavage is benzidine, a highly carcinogenic compound strongly associated with urinary bladder cancer. 20 However, unlike many of the other compounds reported to have activity in various models of TSEs, Congo Red is a small molecule, which is amenable to chemical synthesis and structure activity relationship studies, with the aim of producing a low molecular weight therapeutic agent.
  • the present invention seeks to provide small molecule analogues of Congo Red, paricularly those which may have therapeutic applications in the treatment of TSEs. Ideally, the invention seeks to provide low molecular weight compounds which are specific in the interaction with PrP Sc , have good cellular uptake, good penetration of the blood brain barrier, and which exhibit low toxicity.
  • a first aspect of the invention relates to a compound of formula I, or a pharmaceutically acceptable salt thereof,
  • R 1"5 are each independently selected from H, OH, OR 6 , CO 2 H, CO 2 R 7 , NO 2 , CN CONR 8 R 9 , CONHR 10 , CO 2 (CH 2 ) n CO 2 R ⁇ , CO 2 (CH 2 ) m O(CO)R 12 , CO 2 CHR 13 COR 14 , CONH(CH 2 ) p CO 2 R 15 , CONH(CH 2 ) q O(CO)R 16 , CONHCHR 17 COR 18 ;
  • the replacement of the diazo group of Congo Red with sulphonamide, amide, imine, aminomethylene and alkene bonds prevents the molecule being broken down to benzidine.
  • a second aspect of the invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a compound of formula I as defined above admixed with a pharmaceutically acceptable diluent, excipient or carrier.
  • a third aspect of the invention relates to use of a compound of formula II, or a pharmaceutically acceptable salt thereof,
  • X is selected from
  • R 1"5 are each independently selected from H, OH, OR 6 , CO 2 H, CO 2 R 7 , NO 2 , CN CONR 8 R 9 , CONHR 10 , CO 2 (CH 2 ) ⁇ CO 2 R ⁇ , CO 2 (CH 2 ) m O(CO)R 12 , CO 2 CHR 13 COR 14 , CONH(CH 2 ) p CO 2 R 15 , CONH(CH 2 ) q O(CO)R 16 , CONHCHR 17 COR 18 ; ⁇ R 6"18 are each independently hydrocarbyl; n, m, p and q are each independently 1, 2, 3 or 4. in the preparation of a medicament for treating transmissible spongiform encephalopathy (TSE).
  • TSE transmissible spongiform encephalopathy
  • a fourth aspect of the invention relates to a method of treating, or preventing the onset of, transmissible spongiform encephalopathy (TSE) in a subject, said method comprising administering to a the subject a compound of formula II as defined above.
  • TSE transmissible spongiform encephalopathy
  • a fifth aspect of the invention relates to the use of a compound of formula II, or a pharmaceutically acceptable salt thereof, in an assay for identifying further candidate compounds capable of inhibiting the polymerisation of PrP by PrP-res.
  • a sixth aspect of the invention relates to the use of a compound of formula LT, or a pharmaceutically acceptable salt thereof, in an assay for identifying further candidate compounds capable of inl ibiting the infectivity of the abnormal form of prion protein (PrP Sc ).
  • hydrocarbyl refers to a group comprising at least C and H that may Optionally comprise one or more other suitable substituents.
  • substituents may include OH, COOH, halo-, alkoxy-, nitro-, an alkyl group, or a cyclic group.
  • a combination of substituents may form a cyclic group. If the hydrocarbyl group comprises more than one C then those carbons need not necessarily be linked to each other. For example, at least two of the carbons may be linked via a suitable element or group. Thus, the hydrocarbyl group may contain heteroatoms.
  • heteroatoms will be apparent to those skilled in the art and include, for instance, sulphur, nitrogen, oxygen, phosphorus and silicon.
  • the hydrocarbyl group is an aryl, heteroaryl, alkyl, cycloalkyl, aralkyl or alkenyl group, more preferably, an aryl or alkyl group, more preferably still, an alkyl group.
  • alkyl includes both saturated straight chain and branched alkyl groups which may be substituted (mono- or poly-) or unsubstituted.
  • the alkyl group is a C ⁇ - 2 o alkyl group, more preferably a C MS , more preferably still a C ⁇ - ⁇ 2 alkyl group, more preferably still, a C ⁇ _ 6 alkyl group, more preferably a C ⁇ - 3 alkyl group.
  • Particularly preferred alkyl groups include, for example, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl and hexyl.
  • Suitable substituents include, for example, one or more halo-, OH, alkoxy-, nitro-, COOH, CF 3 , or alkyl groups.
  • cycloalkyl refers to a cyclic alkyl group which may be substituted (mono- or poly-) or unsubstituted.
  • the cycloalkyl group is a C 3 _ ⁇ 2 cycloalkyl group.
  • Suitable substituents include those listed above for alkyl.
  • alkenyl refers to a group containing one or more carbon-carbon double bonds, which may be branched or unbranched, substituted (mono- or poly-) or unsubstituted.
  • the alkenyl group is a C - 20 alkenyl group, more preferably a C 2 . ⁇ 5 alkenyl group, more preferably still a C 2 . ⁇ 2 alkenyl group, or preferably a C 2 . 6 alkenyl group, more preferably a C 2 . 3 alkenyl group.
  • Suitable substituents include those listed for alkyl above.
  • aryl refers to a C 6 _ ⁇ 2 aromatic group which may be substituted (mono- or poly-) or unsubstituted. Typical examples include phenyl and naphthyl etc. Suitable substituents include those listed for alkyl above.
  • heteroaryl refers to a C . ⁇ 2 aromatic, substituted (mono- or poly- ) or unsubstituted group, which comprises one or more heteroatoms.
  • Preferred heteroaryl groups include pyrrole, pyrazole, pyrimidine, pyrazine, pyridine, quinoline, thiophene and furan. Again, suitable substituents include those listed for alkyl above.
  • aralkyl includes, but is not limited to, a group having both aryl and alkyl functionalities.
  • the term includes groups in which one of the hydrogen atoms of the alkyl group is replaced by an aryl group, e.g. a phenyl group optionally having one or more substituents such as halo, alkyl, alkoxy, hydroxy, and the like.
  • Typical aralkyl groups include benzyl, phenethyl and the like.
  • R " are each independently alkyl or aryl, more preferably alkyl.
  • one of R 1"5 is selected from H, OH and OR 6 ; one of R 1"5 is selected from OH, CO 2 H, CN, CO 2 -alkyl and CONH-alkyl; and the remainder of R 1"5 are H.
  • R 3 is CN, OH or CO 2 H, and R 1 , R 2 , R 4 and R 5 are all H;
  • R 1 is OH, R 3 is CO 2 H, CO 2 R 7 or H, and R 2 , R 4 and R 5 are all H;
  • R 1 is OR 6 , R 4 is CO 2 R 7 or CO 2 H, and R 2 , R 3 and R 5 are all H;
  • R 2 and R 4 are CO 2 R 7 , and R 1 , R 3 and R 5 are all H;
  • R 3 is CO 2 R 7 , R 4 is OH or OR 6 , and R 1 , R 2 and R 5 are all H; or
  • R 3 is OH
  • R 4 is CO 2 R 7 or CO 2 H
  • R 1 , and R 2 and R 5 are all H.
  • R 3 is CN, OH or CO 2 H, and R 1 , R 2 , R 4 and R 5 are all H;
  • R 1 is OH, R 3 is CO 2 H, CO 2 Me or H, and R 2 , R 4 and R 5 are all H;
  • R 1 is OMe, R 4 is CO 2 Me or CO 2 H, and R 2 , R 3 and R 5 are all H; (iv) R 2 and R 4 are CO 2 Me, and R 1 , R 3 and R 5 are all H;
  • R 3 is CO 2 Me or CO 2 Et, R 4 is OH or OMe, and R 1 , R 2 and R 5 are all H; or
  • R 3 is OH
  • R 4 is CO 2 Me, CO 2 Et or CO 2 H
  • R 1 , and R 2 and R 5 are all H.
  • said compound is of formula la, or a pharmaceutically acceptable salt thereof
  • R 1 is OH
  • R 3 is CO 2 R 7 , CONR 8 R 9 , CONHR 10 , CO 2 (CH 2 ) classroomCO 2 R ⁇ , CO 2 (CH 2 ) m O(CO)R 12 , CO 2 CHR 13 COR 14 , CONH(CH 2 ) p CO 2 R 15 , CONH(CH 2 ) q O(CO)R 16 or CONHCHR 17 COR 18 , and R 2 , R 4 and R 5 are all H; or (ii) R 1 is OR 6 , R 5 is CO 2 H, CO 2 R 7 CONR 8 R 9 , CONHR 10 , CO 2 (CH 2 ) n CO 2 R n , CO 2 (CH 2 ) m O(CO)R 12 , CO 2 CHR 13 COR 14 , CONH(CH 2 ) p CO 2 R 15 , CONH(CH 2 ) q O(CO)R 16 or CONHCHR 17 COR 18 , and R 2"4 are all H.
  • R 1 is OH
  • R 3 is CO 2 R 7
  • R 2 , R 4 and R 5 are all H
  • R 1 is OR 6
  • R 5 is CO 2 H or CO 2 R 7
  • R 2"4 are all H.
  • R 1 is OH, R 3 is CO 2 alkyl, and R 2 , R 4 and R 5 are all H; or (ii) R 1 is O-alkyl, R 5 is CO 2 H or CO 2 -alkyl, and R 2"4 are all H.
  • R 1 is OH, R 3 is CO 2 Me, and R 2 , R 4 and R 5 are all H; or (ii) R 1 is OMe, R 5 is CO 2 H or CO 2 Me, and R 2"4 are all H.
  • the compound is of formula lb, or a pharmaceutically acceptable salt thereof
  • R 1 is OH
  • R 3 is CO 2 R 7 , CONR 8 R 9 , CONHR 10 , CO 2 (CH 2 ) n CO 2 R ⁇ , CO 2 (CH 2 ) m O(CO)R 12 , CO 2 CHR 13 COR 14 , CONH(CH 2 ) p CO 2 R 15 , CONH(CH 2 ) q O(CO)R 16 or CONHCHR 17 COR 18 , and R 2 , R 4 and R 5 are H;
  • R 2 is OR 6
  • R 3 is CO 2 R 7 , CONR 8 R 9 , CONHR 10 , CO 2 (CH 2 ) n CO 2 R 11 , CO 2 (CH 2 ) m O(CO)R 12 , CO 2 CHR 13 COR 14 , CONH(CH 2 ) p CO 2 R 15 , CONH(CH 2 ) q O(CO)R 16 or CONHCHR 17 COR 18 , and R 1 , R 4 and R 5 are H;
  • R 1 is OH, R 3 is CO 2 R 7 , and R 2 , R 4 and R 5 are H;
  • R 2 is OR 6 , R 3 is CO 2 R 7 , and R 1 , R 4 and R 5 are H;
  • R 2 is CO 2 R 7 , R 3 is OH, and R 1 , R 4 and R 5 are H.
  • R 1 is OH, R 3 is CO 2 -alkyl, and R 2 , R 4 and R 5 are H;
  • R 2 is O-alkyl, R 3 is CO 2 -alkyl, and R 1 , R 4 and R 5 are H;
  • R 2 is CO 2 -alkyl, R 3 is OH, and R 1 , R 4 and R 5 are H.
  • R 1 is OH, R 3 is CO 2 Me, and R 2 , R 4 and R 5 are H;
  • R 2 is OMe, R 3 is CO 2 Me, and R 1 , R 4 and R 5 are H;
  • R 2 is CO 2 Me, R 3 is OH, and R 1 , R 4 and R 5 are H.
  • the compound according to claim 1 is of formula Ic, or a pharmaceutically acceptable salt thereof
  • R 1"5 are as defined above.
  • R 3 is CN, and R 1 , R 2 , R 4 and R 5 are H;
  • R 2 and R 4 are CO 2 R 7 , CONR 8 R 9 , CONHR 10 , CO 2 (CH 2 ) n CO 2 R ⁇ , CO 2 (CH 2 ) m O(CO)R 12 , CO 2 CHR 13 COR 14 , CONH(CH 2 ) p CO 2 R 15 , CONH(CH 2 ) q O(CO)R 16 or CONHCHR 17 COR 18 , and R 1 , R 3 and R 5 are H; or (iv) R 1 is OH, R 3 is COR 7 and R 2 , R 4 and R 5 are all H.
  • R and R are CO 2 R , and R , R and R are H.
  • R 2 and R 4 are CO 2 -alkyl, and R 1 , R 3 and R 5 are H, or R 1 is OH, R 3 is CO 2 -alkyl and R 2 , R 4 and R 5 are all H.
  • R 2 and R 4 are CO Me, and R 1 , R 3 and R 5 are H, or R 1 is OH, R 3 is CO 2 Me and R 2 , R 4 and R 5 are all H.
  • the compound is of formula Id, or a pharmaceutically acceptable salt thereof Id
  • R 1"5 are as defined above.
  • R 3 is CO 2 H, CO 2 R 7 , CONR 8 R 9 , CONHR 10 , CO 2 (CH 2 ) n CO 2 R n , CO 2 (CH 2 ) m O(CO)R 12 , CO 2 CHR 13 COR 14 , CONH(CH 2 ) p CO 2 R 15 , CONH(CH 2 ) q O(CO)R 16 or CONHCHR 17 COR 18 , R 1 is OH, and R 2 , R 4 and R 5 are H; or
  • R 3 is OH, and R 1 , R 2 , R 4 and R 5 are H;
  • R 3 is CO 2 H, or CO 2 R 7 , R 1 is OH, and R 2 , R 4 and R 5 are H.
  • R 3 is CO 2 H, or CO 2 -alkyl, R 1 is OH, and R 2 , R 4 and R 5 are H.
  • R 3 is CO 2 H, or CO 2 Me, R 1 is OH, and R 2 , R 4 and R 5 are H.
  • the compound is of formula le, or a pharmaceutically acceptable salt thereof
  • R 1 is OR 6 , R 5 is CO 2 H, CO 2 R 7 , CONR 8 R 9 , CONHR 10 , CO 2 (CH 2 ) n CO 2 R 11 , CO 2 (CH 2 ) m O(CO)R 12 , CO 2 CHR 13 COR 14 , CONH(CH 2 ) p CO 2 R 15 , CONH(CH 2 ) q O(CO)R 16 or CONHCHR 17 COR 18 , and R 2 , R 3 and R 4 are H; (ii) R 1 is OH, R 3 is CO 2 H or H, and R 2 , R 4 and R 5 are H; (iii) R 3 is CN or CO 2 H, and R 1 , R 2 , R 4 and R 5 are H;
  • R 2 is CO 2 H, CO 2 R 7 , CONR 8 R 9 , CONHR 10 , CO 2 (CH 2 ) n CO 2 R 11 , CO 2 (CH 2 ) m O(CO)R 12 , CO 2 CHR 13 COR 14 , CONH(CH 2 ) p CO 2 R 15 , CONH(CH 2 ) q O(CO)R 16 or CONHCHR 17 COR 18 , R 3 is OH, R 1 , R 4 and R 5 are H; or (v) R 2 is OH, R 3 is CO 2 R 7 , CONR 8 R 9 , CONHR 10 , CO 2 (CH 2 ) n CO 2 R ⁇ , CO 2 (CH 2 ) ra O(CO)R 12 , CO 2 CHR 13 COR 14 , CONH(CH 2 ) p CO 2 R 15 , CONH(CH 2 ) q O(CO)R 16 or CONHCHR 17 COR 18 , and R 1 , R 4 and R 5 are
  • R 1 is OR 6 , R 5 is CO 2 H or CO 2 R 7 , and R 2 , R 3 and R 4 are H; (ii) R 2 is CO 2 H or CO 2 R 7 , R 3 is OH, R 1 , R 4 and R 5 are H; or (iii) R 2 is OH, R 3 is CO 2 R 7 , and R 1 , R 4 and R 5 are H.
  • R 1 is O-alkyl
  • R 5 is CO 2 H or CO 2 -alkyl
  • R 2 , R 3 and R 4 are H
  • R 2 is CO 2 H or CO 2 alkyl
  • R 3 is OH
  • R 1 , R 4 and R 5 are H
  • R 2 is OH
  • R 3 is CO 2 -alkyl
  • R 1 , R 4 and R 5 are H.
  • R 1 is OMe, R 5 is CO 2 H or CO 2 Me, and R 2 , R 3 and R 4 are H;
  • R 2 is CO 2 H or CO 2 Et, R 3 is OH, R 1 , R 4 and R 5 are H; or
  • R 2 is OH, R 3 is CO 2 Et, and R 1 , R 4 and R 5 are H.
  • the compound is of formula If, or a pharmaceutically acceptable salt thereof
  • R 1 is OH
  • R 3 is H, CO 2 H, CO 2 R 7 , CONR 8 R 9 , CONHR 10 , CO 2 (CH 2 ) n CO 2 R ⁇ , CO 2 (CH 2 ) m O(CO)R 12 , CO 2 CHR 13 COR 14 , CONH(CH 2 ) p CO 2 R 15 , CONH(CH 2 ) q O(CO)R 16 or CONHCHR 17 COR 18 , and R 2 , R 4 and R 5 are H; or
  • R 1 is OR 6
  • R 4 is CO 2 R 7 , CONR 8 R 9 , CONHR 10 , CO 2 (CH2) n CO 2 R 11 , CO 2 (CH 2 ) m O(CO)R 12 , CO 2 CHR 13 COR 14 , CONH(CH 2 ) p CO 2 R 15 , CONH(CH 2 ) q O(CO)R 16 or CONHCHR 17 COR 18
  • R 2 , R 3 and R 5 are H.
  • R 1 is OH, R 3 is H, CO 2 H or CO 2 -alkyl, and R 2 , R 4 and R 5 are H; or (ii) R 1 is O-alkyl, R 4 is CO 2 -alkyl, and R 2 , R 3 and R 5 are H.
  • R 1 is OH, R 3 is H, CO 2 H or CO 2 Me, and R 2 , R 4 and R 5 are H; or (iii) R 1 is OMe, R 4 is CO 2 Me, and R 2 , R 3 and R 5 are H.
  • the compound is of formula Ig, or a pharmaceutically acceptable salt thereof
  • R is OH, and R 1 , ⁇ R»2 , ⁇ R>4 and R are H.
  • the compound is selected from those set forth in Tables 1-8.
  • sulphonamide analogues of the invention can be synthesised from the readily available 4-4'-biphenyldisulfonyl chloride 8 and the appropriate amine utilising DMAP as a catalyst 15. and anhydrous pyridine as the solvent.
  • biphenyl amide analogues of the invention can be synthesised either by the reaction of the corresponding acid chloride with the appropriate aniline derivative in the presence of triethylamine or using TBTU, a uronium based peptide coupling reagent (Scheme 3).
  • Scheme 3 a uronium based peptide coupling reagent
  • the amount of triethylamine used in the coupling reaction is varied, specifically if the hydrochloride salt of the amine is to be used for coupling, a further equivalent of base (triethylamine) isbe needed to optimise the reaction.
  • the imine analogues of the invention can be made by condensation of the corresponding aldehyde with the appropriate amine.
  • the products (imines) are generally insoluble in ethanol and precipitate from solution: filtration and washing generally provides the compounds in good yield.
  • the biphenyl analogues prove less soluble but also the reaction time is longer in these cases.
  • the mono-phenyl imines (series 5) can be synthesised from the commerically available terephthalaldehyde, whereas the biphenyl analogues (series 4) require 4-4 '-biphenyl dialdehyde to be made first.
  • the amines of the invention can be synthesised by reduction of the imines with sodium borohydride.
  • the only limiting factor is the initial solubility of the imines, many of which prove insoluble in any solvent once formed.
  • the reduction is favourable in a methanol / dichloromethane mixture.
  • Transition metal chemistry plays a part in the synthesis of the alkenes of the invention (Scheme 7); the Heck reaction is preferred, so as to achieve only one of the stereoisomers, the tr ⁇ r ⁇ -stereoisomer.
  • the reaction is sensitive to the choice of solvent.
  • a 17:3 DMF:water mixture is optimal, the water being a crucial element.
  • 1,4-divinylbenzene can be purchased commercially but only as a mixture with 1,2-divinylbenzene, therefore it is preferable to synthesise this compound from terephthaldehyde using a Wittig reaction.
  • X is selected from
  • R 1"5 are each independently selected from H, OH, OR 6 , CO 2 H, CO 2 R 7 , NO 2 , CN CONR 8 R 9 , CONHR 10 , CO 2 (CH 2 ) conflictCO 2 R ⁇ , CO 2 (CH 2 ) m O(CO)R 12 , CO 2 CHR 13 COR 14 ,
  • R 6"18 are each independently hydrocarbyl; n, m, p and q are each independently 1, 2, 3 or 4. in the preparation of a medicament for treating transmissible spongiform encephalopathy (TSE).
  • TSE transmissible spongiform encephalopathy
  • said compound of formula II is as defined above for compounds of formula I.
  • said compound is as defined above for compounds of formula Ig and wherein:
  • R 3 is OH, and R 1 , R 2 , R 4 and R 5 are H; or
  • R J is OH, R >2 4 i ⁇ s CO 2 H, and R 1 1 , r R>4 4 and R D are H
  • preparation of a medicament includes the use of compounds of formula II directly as the medicament in addition to their use in a screening programme for identifying further therapeutic agents or in any stage of the manufacture of such a medicament.
  • Another aspect of the invention relates to a method of treating, or preventing the onset of, transmissible spongiform encephalopathy (TSE) in a subject, said method comprising administering to the subject a compound of formula II as defined above.
  • TSE transmissible spongiform encephalopathy
  • the TSE is selected from Creutzfeldt Jakob Disease (CJD), Gerstmann- Staussler-Scheinker Syndrome (GSS), Familial Fatal Insomnia (FFI), Kuru, variant CJD, Bovine Spongiform Encephalopathy (BSE) and scrapie.
  • CJD Creutzfeldt Jakob Disease
  • GSS Gerstmann- Staussler-Scheinker Syndrome
  • FCI Familial Fatal Insomnia
  • Kuru variant CJD
  • Bovine Spongiform Encephalopathy BSE
  • the compound of formula II is administered in an amount sufficient to inhibit the infectivity of the abnormal form of prion protein (PrP Sc ).
  • the compound of formula II is administered in an amount sufficient to inhibit the polymerisation of PrP by PrP-res.
  • the compounds of the invention are active in both the cellular and polymerisation assay described in the accompanying Examples section. Without wishing to be bound by theory, it is believed that these compounds probably prevent PrP-res formation in cells by preventing polymerisation of PrP c .
  • Preferred compounds falling into this category include, for example, WSP677, 905, 1008, 795, 1011 718 and 798.
  • the compounds of the invention are active in the polymerisation assay, but not in the cellular assay. Without wishing to be bound by theory, it is possible that these compounds are too weak at inhibition of polymerisation to be active in cellular assays, or they are inactivated in a cellular system, or they are unable to access the molecular target.
  • Preferred compounds falling into this category include, for example, WSP675, 676, 678, 917, 1012, 794, 738, 741, 742, 760, 980 and 759.
  • the compounds of the invention are active in the cellular assay, but not the polymerisation assay. Without wishing to be bound by theory, it is possible that these compounds preventPrP-res formation in the SMB cells by a mode of action other than inhibition of polymerisation. Preferred compounds falling into this category include, for example, WSP 1009, 906, 915, 740 and 911.
  • Both the cellular and polymerisation assay provide useful information in the context of a drug discovery programme.
  • the polymerisation assay can identify most, but not all molecules which reduce PrP-res formation in cellular assays. Thus, on its own it is not a useful screen to select out compounds for cellular assays.
  • the cellular assay employed on its own also has limitations. The assay does not identify all compounds that prevent polymerisation. These compounds may be a useful source of information for future rounds of drug design.
  • the polymerisation assay also provides information on the possible modes of action of compounds.
  • the biphenyl amides showed the most promise; WSP677 is of particular interest.
  • WSP677 is of particular interest.
  • the biphenyl amides without wishing to be bound by theory, there seems to be some correlation between prevention of PrP-res formation in cell culture and prevention of polymerisation of PrP c . This suggests that the anti-TSE activity of at least some of the compounds may be related to their ability to prevent polymerisation.
  • the sulfonamides appeared less active than the biphenyl amides (Series 2).
  • the sulfonamide group is a bioisostere of the amide group.
  • the NH of the sulfonamides is ionisable and may well be ionised at physiological pH. Whilst ionisation may well increase the solubility of these compounds, interaction with the prion protein may be also be prevented, hi spite of this, some of the sulfonamides still showed weak activity in a cellular model and inhibition of polymerisation.
  • Imines are normally readily subject to hydrolysis.
  • the imines of the present invention are highly conjugated, which stablises them to hydrolysis.
  • any hydrolysis should not give rise to benzidene or its analogues.
  • the biphenyl imines (Series 4) showed higher activity than the monophenyl imines (Series 5) in the cellular assay, although several of the monophenyl imines showed potent inhibition of polymerisation.
  • these compounds tended to be those with carboxylates on the terminator, e.g. WSP738, WSP741, WSP742, WSP760.
  • the aminomethylene compounds (Series 6) show very promising activity, particularly in the cellular assay, but also in the polymerisation assay.
  • Structure activity relationships indicate that the most active compounds in cellular assays preferably have a methyl carboxylate attached to the terminator and either a hydroxy or methoxy group.
  • the terminator found in compound found in WSP677 is active in both the cellular and polymerisation assays. However other regio-isomers are also active (WSP905, 1009). Some compounds containing a carboxylic acid on the terminator are active in inhibition of polymerisation, but not in the cellular assay. Conversely, compounds which are active in the cellular assay and contain a methyl ester are not be active in the polymerisation assay (WSP1009, 906, 740).
  • Another aspect of the invention relates to the use of compounds of formula I, or pharmaceutically acceptable salts thereof, in the preparation of a medicament for treating Alzheimer's disease.
  • a fourth aspect of the invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a compound of formula I as defined for said first aspect admixed with one or more pharmaceutically acceptable diluents, excipients or carriers.
  • the compounds of the present invention can be administered alone, they will generally be administered in admixture with a pharmaceutical carrier, excipient or diluent, particularly for human therapy.
  • the pharmaceutical compositions may be for human or animal usage in human and veterinary medicine.
  • Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985).
  • suitable carriers include lactose, starch, glucose, methyl cellulose, magnesium stearate, mannitol, sorbitol and the like.
  • suitable diluents include ethanol, glycerol and water.
  • the choice of pharmaceutical carrier, excipient or diluent can be selected with regard to the intended route of administration and standard pharmaceutical practice.
  • the pharmaceutical compositions may comprise as, or in addition to, the carrier, excipient or diluent any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s), solubilising agent(s).
  • Suitable binders include starch, gelatin, natural sugars such as glucose, anhydrous lactose, free-flow lactose, beta-lactose, corn sweeteners, natural and synthetic gums, such as acacia, tragacanth or sodium alginate, carboxymethyl cellulose and- polyethylene glycol.
  • Suitable lubricants include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride and the like.
  • Preservatives, stabilizers, dyes and even flavoring agents may be provided in the pharmaceutical composition.
  • preservatives include sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid.
  • Antioxidants and suspending agents may be also used.
  • the compounds of formula I or II can be present as salts or esters, in particular pharmaceutically acceptable salts or esters.
  • salts of the compounds of the invention include suitable acid addition or base salts thereof.
  • suitable pharmaceutical salts may be found in Berge et al, J Pharm Sci, 66, 1-19 (1977). Salts are formed, for example with strong inorganic acids such as mineral acids, e.g.
  • sulphuric acid, phosphoric acid or hydrohalic acids with strong organic carboxylic acids, such as alkanecarboxylic acids of 1 to 4 carbon atoms which are unsubstituted or substituted (e.g., by halogen), such as acetic acid; with saturated or unsaturated dicarboxylic acids, for example oxalic, malonic, succinic, maleic, fumaric, phthalic or tetraphthalic; with hydroxycarboxylic acids, for example ascorbic, glycolic, lactic, malic, tartaric or citric acid; with aminoacids, for example aspartic or glutamic acid; with benzoic acid; or with organic sulfonic acids, such as (C ⁇ -C 4 )-alkyl- or aryl-sulfonic acids which are unsubstituted or substituted (for example, by a halogen) such as methane- or p-toluene sulfonic acid.
  • Esters are formed either using organic acids or alcohols/hydroxides, depending on the functional group being esterified.
  • Organic acids include carboxylic acids, such as alkanecarboxylic acids of 1 to 12 carbon atoms which are unsubstituted or substituted (e.g., by halogen), such as acetic acid; with saturated or unsaturated dicarboxylic acid, for example oxalic, malonic, succinic, maleic, fumaric, phthalic or tetraphthalic; with hydroxycarboxylic acids, for example ascorbic, glycolic, lactic, malic, tartaric or citric acid; with aminoacids, for example aspartic or glutamic acid; with benzoic acid; or with organic sulfonic acids, such as (C ⁇ -C 4 )-alkyl- or aryl-sulfonic acids which are unsubstituted or substituted (for example, by a halogen) such as methane- or p-tol
  • Suitable hydroxides include inorganic hydroxides, such as sodium hydroxide, potassium hydroxide, calcium hydroxide, aluminium hydroxide.
  • Alcohols include alkanealcohols of 1-12 carbon atoms which may be unsubstituted or substituted, e.g. by a halogen).
  • the invention includes, where appropriate all enantiomers and tautomers of compounds of formula I or II.
  • the man skilled in the art will recognise compounds that possess an optical properties (one or more chiral carbon atoms) or tautomeric characteristics.
  • the corresponding enantiomers and/or tautomers may be isolated/prepared by methods known in the art. STEREO AND GEOMETRIC ISOMERS
  • the present invention contemplates the use of all the individual stereoisomers and geometric isomers of those inhibitor agents, and mixtures thereof.
  • the tenns used in the claims encompass these forms, provided said forms retain the appropriate functional activity (though not necessarily to the same degree).
  • the present invention also includes all suitable isotopic variations of the agents or pharmaceutically acceptable salts thereof.
  • An isotopic variation of an agent of the present invention or a pharmaceutically acceptable salt thereof is defined as one in which at least one atom is replaced by an atom having the same atomic number but an atomic mass different from the atomic mass usually found in nature.
  • isotopes that can be incorporated into the agent and pharmaceutically acceptable salts thereof include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulphur, fluorine and chlorine such as 2 H, 3 H, 13 C, 14 C, 15 N, 17 O, 18 O, 31 P, 32 P, 35 S, 18 F and 36 C1, respectively.
  • isotopic variations of the agent and pharmaceutically acceptable salts thereof are useful in drug and/or substrate tissue distribution studies. Tritiated, i.e., H, and carbon-14, i.e., 14 C, isotopes are particularly preferred for their ease of preparation and detectability. Further, substitution with isotopes such as deuterium, i.e., 2 H, may afford certain therapeutic advantages resulting from greater metabolic stability, for example, increased in vivo half-life or reduced dosage requirements and hence may be preferred in some circumstances. Isotopic variations of the agent of the present invention and pharmaceutically acceptable salts thereof of this invention can generally be prepared by conventional procedures using appropriate isotopic variations of suitable reagents. SOLVATES
  • the present invention also includes the use of solvate forms of the compounds of the present invention.
  • the terms used in the claims encompass these forms.
  • the invention furthermore relates to the compounds of the present invention in their various crystalline forms, polymorphic forms and (an)hydrous forms. It is well established within the pharmaceutical industry that chemical compounds may be isolated in any of such forms by slightly varying the method of purification and or isolation form the solvents used in the synthetic preparation of such compounds.
  • the invention further includes the compounds of the present invention in prodrug form.
  • Such prodrugs are generally compounds of formula I or II wherein one or more appropriate groups have been modified such that the modification may be reversed upon administration to a human or mammalian subject.
  • Such reversion is usually performed by an enzyme naturally present in such subject, though it is possible for a second agent to be administered together with such a prodrug in order to perform the reversion in vivo.
  • compositions of the present invention may be adapted for oral, rectal, vaginal, parenteral, intramuscular, intraperitoneal, intraarterial, intrathecal, intrabronchial, subcutaneous, intradermal, intravenous, nasal, buccal or sublingual routes of administration.
  • parenteral intramuscular, intraperitoneal, intraarterial, intrathecal, intrabronchial, subcutaneous, intradermal, intravenous, nasal, buccal or sublingual routes of administration.
  • intramuscular intraperitoneal
  • intraarterial intrathecal
  • intrabronchial subcutaneous, intradermal, intravenous, nasal, buccal or sublingual routes of administration.
  • these compositions contain from 1 to 250 mg and more preferably from 10-100 mg, of active ingredient per dose.
  • compositions of the present invention may also be in form of suppositories, pessaries, suspensions, emulsions, lotions, ointments, creams, gels, sprays, solutions or dusting powders.
  • the active ingredient can be incorporated into a cream consisting of an aqueous emulsion of polyethylene glycols or liquid paraffin.
  • the active ingredient can also be incorporated, at a concentration of between 1 and 10% by weight, into an ointment consisting of a white wax or white soft paraffin base together with such stabilisers and preservatives as may be required.
  • Injectable forms may contain between 10 - 1000 mg, preferably between 10 - 250 mg, of active ingredient per dose.
  • compositions may be formulated in unit dosage form, i.e., in the form of discrete portions containing a unit dose, or a multiple or sub-unit of a unit dose.
  • a person of ordinary skill in the art can easily determine an appropriate dose of one of the instant compositions to administer to a subject without undue experimentation.
  • a physician will determine the actual dosage which will be most suitable for an individual patient and it will depend on a variety of factors including the activity of the specific compound employed, the metabolic stability and length of action of that compound, the age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular condition, and the individual undergoing therapy.
  • the dosages disclosed herein are exemplary of the average case. There can of course be individual instances where higher or lower dosage ranges are merited, and such are within the scope of this invention.
  • the agent may be administered at a dose of from 0.01 to 30 mg/kg body weight, such as from 0.1 to 10 mg/kg, more preferably from 0.1 to 1 mg/kg body weight.
  • one or more doses of 10 to 150 mg/day will be administered to the patient for the treatment of TSE.
  • the one or more compounds of the invention are administered in combination with one or more other agents, for example, existing drugs available on the market.
  • the compounds of the invention may be administered consecutively, simultaneously or sequentially with the one or more other active agents.
  • Drugs in general are often more effective when used in combination.
  • combination therapy is desirable in order to avoid an overlap of major toxicities, mechanism of action and resistance mechanism(s).
  • the major advantages of combining drugs are that it may promote additive or possible synergistic effects through biochemical interactions and also may decrease the emergence of resistance in cells which would have been otherwise responsive to initial therapy with a single agent.
  • Beneficial combinations may be suggested by studying the activity of the test compounds with agents known or suspected of being valuable in the treatment of a particular disorder.
  • This procedure can also be used to determine the order of administration of the agents, i.e. before, simultaneously, or after delivery. Such scheduling may be a feature of all the agents identified herein.
  • Another aspect of the invention relates to the use of a compound of formula I or II, or a pharmaceutically acceptable salt thereof, as defined above, in an assay for identifying candidate compounds capable of inhibiting the polymerisation of PrP c by PrP-res.
  • Yet another aspect relates to the use of a compound of formula I or II, or a pharmaceutically acceptable salt thereof, as defined above, in an assay for identifying candidate compounds capable of inhibiting the infectivity of the abnormal form of prion protein (PrP Sc ).
  • the assay is a competitive binding assay.
  • One aspect of the invention relates to a process comprising the steps of: (a) performing an assay method described hereinabove;
  • Another aspect of the invention provides a process comprising the steps of: (a) performing an assay method described hereinabove; (b) identifying one or more candidate compounds capable of inhibiting the polymerisation of PrP c by PrP-res or inhibiting the infectivity of the abnormal form of prion protein (PrP Sc ); and
  • Another aspect of the invention provides a process comprising the steps of:
  • the invention also relates to a candidate compound identified by the method described hereinabove.
  • Yet another aspect of the invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a candidate compound identified by the method described hereinabove.
  • Another aspect of the invention relates to the use of a candidate compound identified by the method described hereinabove in the preparation of a pharmaceutical composition for use in the treatment or prevention of a TSE.
  • said candidate compound is generated by conventional SAR modification of a compound of the invention.
  • conventional SAR modification refers to standard methods known in the art for varying a given compound by way of chemical derivatisation.
  • Figure 1 shows the cell viability of cerebellar neurones isolated from mice when incubated with WSP677 and WSP1012.
  • WSP676 (0.1 OOg, 0.156mmol) was placed in a 10ml round bottomed flask, NaOH
  • WSP675 (0.086g, 0.14mmol) was reacted with aqueous NaOH (0.023g, 0.56mmol) in water (4ml) and the product WSP679 was isolated as a light brown solid.
  • WSP906 (0.250g, 0.456mmo ⁇ ) was reacted with aqueous NaOH (0.291g, 0.729mmol) in water (4ml) and the product WSP907 was isolated as a light grey solid.
  • WSP908 was isolated as a white solid.
  • This compound was made via an identical preparation to the preparation of WSP677 (alternative procedure). 2-aminophenol (0.234g, 2.15mmol) and triethylamine (0.45ml, 3.21mmol) was reacted with 4-4'-biphenyldicarboxyl chloride (0.300g, 1.07mmol) in anhydrous THF (10ml). An identical work-up procedure was employed, the pure product WSP912 was isolated as an off-white solid.
  • WSP905 (0.1 OOg, 0.176mmol) was reacted with aqueous NaOH (0.070g, 1.76mmol) in water (1ml) and THF (5ml).
  • the product WSP971 was isolated as a brown solid.
  • This compound was made via an identical preparation to the preparation of WSP677 (alternative procedure), with the exception of adding DMAP as a catalyst (0.009g, 0.689mmol). 2-amino-5-nitrophenol (1.062g, 6.89mmol) and triethylamine (0.45ml, 3.21mmol) was reacted with terephthaloyl chloride (0.700g, 3.44mmol) in anhydrous THF (15ml). An identical work-up procedure was employed, the pure product WSP974 was isolated as an orange solid.
  • terephthalaldehyde (0.200g, 1.49mmol) and methyl-4-amino-3-hydroxy-benzoate (0.498g, 2.98mmol).
  • Ethanol (15ml) was added and the reaction mixture heated to 55°C for 5 hours under a nitrogen atmosphere.
  • the precipitate which forms was filtered tlirough a sinter funnel and washed with ethanol (40ml).
  • the precipitate, the product WSP714 was isolated as a yellow powder and dried under high vacuum. No further purification was necessary.
  • WSP714 (0.300g, 0.693mmol) was dissolved in dichloromethane (10ml) and reacted with sodium borohydride (0.158g, 4.16mmol) in methanol (5ml). In this case water (20ml) was added to precipitate the product, which was washed further with water (20ml) and dichloromethane (20ml).
  • the product WSP740 was isolated as a light brown solid.
  • WSP740 Into a 25ml round bottomed flask was placed WSP740 (0.075g, 0.171mmol). A solution of NaOH (0.070g, 1.71mmol) in water (6ml) was added dropwise and the reaction mixture was left stirring at room temperature for 45 minutes. 2N HCl (8ml) was added dropwise to the reaction mixture till a neutral pH was achieved. A solid precipitated which was filtered and washed with water (20ml). The product WSP798 was obtained as a white solid without further need for purification.
  • Drug treatment Drugs were resuspended in DMSO at a stock concentration of lOmM.
  • SMB cells were plated at 50% confluency per well in 6-well plates. Cells were left for 24h to allow for attachment. Media was then replaced with fresh media containing the appropriate dilution of the lOmM drug stock. 4 days after the addition of drug the media was removed and the protein extracted.
  • the MTT formazan product was released from cells by addition of dimethylsulphoxide (Sigma) and measured at 570 mn in a spectrophotometer (Bio50, Cary). Relative survival in comparison to control treated with the DMSO vehicle could then be determined.
  • SMB cells are persistently scrapie-infected mouse cells, cloned from a scrapie-infected mouse brain, but of non-neuronal origin. ' ' ' These cells are highly phagocytic and may mimic cells involved in the initial uptake and replication of the agent in peripheral infection. 12 In addition these cells show stable persistent scrapie-infection over many passages, suggesting that they are suitable for drug screening. 23 hideed, this cell line has been 10 1 ⁇ successfully used to screen many derivatives of Congo Red.
  • Tables 1-7 The results from these assays are shown in Tables 1-7, for the seven series of compounds: sulfonamides (Table 1); amides with a biphenyl linker (Table 2); amides with a phenyl linker (Table 3); imines with a biphenyl linker (Table 4); imines with a phenyl linker (Table 5); amines with a phenyl linker (Table 6); and alkenes with a phenyl linker (Table 7).
  • Compound WSP979 which is a regioisomer of WSP677 and WSP1009 was inactive. This suggests that the relative orientation of the ester and they hydroxyl group are important in the terminator.
  • Compound WSP971 which is the analogue of WSP905 with the free carboxylate did not show activity, indicating that the ester has a role, perhaps as a prodrug.
  • Other compounds with the methyl carboxylate removed or replaced (WSP912, 916, 971, 978, 1013) were inactive in the cell culture assay. This suggests that the ester may have a role in the activity of the molecule; by altering the structure of the ester it may be possible to modify the potency and physicochemical properties of the compounds.
  • the compounds of series 3 were the amides with a phenyl core (Table 3).
  • Compound 27 showed activity and following a fuller dose-response experiment, the ED 50 was found to be 250nM.
  • WSP759 is X- 34, a compound used by Klunk for diagnosis of Alzheimer's disease. 24 ' 25 This compound showed no activity at lO ⁇ M, but weak activity at 50 ⁇ M.
  • WSP762 showed activity at 2 ⁇ M, the mechanism of action is unknown, but may well differ from that of the other molecules described herein. It is possible that this molecule could be acting through an antioxidant mode of action.
  • WSP794 and WSP795 show some inhibition of polymerisation.
  • the two compounds which inhibit polymerisation are the two compounds which also have some effect in cell culture assays, indicating that the mode of action of these compounds, may include inhibition of polymerisation.
  • the terminator group on WSP794 is the same as that on WSP677 and that on WSP795 differs only in the carboxylate ester being replaced by a free carboxylate group.
  • the compounds of Series 5 are those with a phenyl linker and an imine bond (Table 5). A number of these show inliibition of PrP c polymerisation; WSP716, 738, 741, 742, 760, 980.
  • the terminator group found in WSP677 is not active in this series of compounds (compound WSP714).
  • the presence of a carboxylate group (either as the free acid or as an ester) seems to be important for activity.
  • Compounds not containing these functionalities are not active in this assay.
  • Compound WSP739 is a direct homologue of WSP738 (one of the most active compounds) except it has an extra methylene between the terminator and the imine. WSP739 is inactive, suggesting that conjugation with the terminator is an important feature.
  • WSP759 (Table 7) shows some inhibition of polymerisation.
  • the efficacy of the compounds of the invention against Alzheimer's disease was investigated by performing a beta-amyloid assay to measure amyloid aggregation in accordance with the methodology set forth in Fernanda G. De Felice et al, FASEB Journal, 2001, vol 15, 1297-1299.
  • the effect of the compounds on Alzheimer beta- amyloid peptide (Abeta) polymerization and the growth of amyloid fibrils was investigated using the methodology disclosed in Kuner et al, Journal of Biological Chemistry, 2000, vol 275, 1673-1687.
  • Prusiner S. B. Prions. Proc. Natl. Acad. Sci. U. S. A. 1998, 95, 13363-13383.
  • WSP number Entry R MW Yield Inhibition Polymerof PrP-res isation formation (% of control) in SMB cells at lO ⁇ M WSP905 2b Me0 2 C— ⁇ X ⁇ - 568.57 46 +ve 70 MeO WSP912 2c 424.45 14 -ve 430 OH precipitated WSP916 2d VJ 392.45 48 -ve 171 WSP971 2e 540.52 32 -ve 472
  • Phenyl Imines Synthetic Yield, Inhibition of PrP-res formation in SMB cells at lO ⁇ M and Inhibition of Polymerisation at lOO ⁇ M. J ⁇ ⁇ - N-R
  • Phenyl amines Synthetic Yield, Inhibition of PrP-res formation in SMB cells at lO ⁇ M and Inhibition of Polymerisation at lOO ⁇ M.
  • Phenyl alkenes Synthetic Yield, Inhibition of PrP-res formation in SMB cells at lO ⁇ M and inhibition of Polymerisation at lOO ⁇ M.
  • WSP Entry R MW Yield hihibition of PrP- Polymer- number res formation in isation SMB cells at (% of control) lO ⁇ M WSP762 7b HO 314.4 (+ve 2 ⁇ M)
  • Table 8 ED50 values of compounds in SMB cells showing activity ⁇ 10 ⁇ M. WSP number Entry Structure Polymerisation (% of control) nM

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Abstract

The present invention relates to compounds of formula (I), or pharmaceutically acceptable salts thereof, wherein X is selected from (II) and (III), Y is selected from SO2NH, CONH, CH=N, CH2NH and CH=CH; R1-5 are each independently selected from H, OH, OR6, CO2H, C02R7, NO2, CN CONR8R9, CONHR10, CO2(CH2)nCO2R11, CO2(CH2)mO(CO)R12, CO2CHR13COR14, CONH(CH2)pCO2R15, CONH(CH2)qO(CO)R16, CONHCHRI7COR18; R6-18 are each independently hydrocarbyl; n, m, p and q are each independently 1, 2, 3 or 4; with the proviso that when X is phenyl, Y is CH=CH, R3 is OH and R1, R4 and R5 are H, R2 is other than COON. Further aspects of the invention relate to the use of such compounds in the preparation of a medicament for the treatment or prevention of transmissible spongiform encephalopathies (TSEs), and pharmaceutical compositions comprising the same.

Description

COMPOUNDS
The present invention relates to chemical analogues of the diazo compound known as Congo Red. More specifically, the invention relates to compounds that are useful in the treatment or prevention of transmissible spongiform encephalopathies.
BACKGROUND TO THE INVENTION
Transmissible spongiform encephalopathies (TSE) are a group of rare neurological degenerative disorders.1 The diseases can arise spontaneously, have a genetic origin or can occur by infection, hi humans the TSEs are Creutzfeldt Jakob Disease, Gerstmann- Staussler-Scheinker Syndrome (GSS), Familial Fatal Insomnia (FFI), Kuru and more recently a new form of the disease called variant CJD.2,3 TSEs affect other organisms, those of note include Bovine Spongiform Encephalopathy (BSE) in cattle and scrapie in sheep. There is no cure currently available for these diseases, which are fatal.
The causative agent of these diseases is thought to be or to contain an abnormally folded protein, the prion protein. During the course of the disease, there is an increase in the concentration of the misfolded prion protein form (PrP-res) over the normal cellular prion protein form (PrP ). PrP is a glycohpid-anchored membrane protein and is easily degraded by protein kinases, whereas PrP-res, while having a lower molecular mass, is highly resistant to degradation under many denaturing conditions.
A number of compounds have shown anti-prion activity in numerous assays using various r n different strains of scrapie infectivity. " Compounds shown to have anti-prion activity include sulphated polysaccharides such as pentosan polysulfate, Congo Red and other azo dyes, amphotericin B and analogues, anthracyclines, phthalocyanines and porphyrins, inorganic ions, branched polyamines, and antagonists of the N-methyl-D-aspartate (NMD A) receptor such as memantine.6 Recently there has been interest in quinacrine and other acridine derivatives.9,10 The azo-dye Congo Red has been shown to have in vivo anti-prion activity against various models of TSEs, including a cell free polymerisation assay,11 cellular assays12'13 and in vivo activity against golden Syrian hamsters.14
Congo Red Benzidine
Figure imgf000003_0001
terminator connector linker
The molecule itself has a number of shortfalls.15 Firstly, it has a lack of specificity. In addition it does not have significant permeability through the blood brain barrier16'17 presumably due to its charged nature, primarily caused by the presence of two sulfonate groups. Toxicity is also a problem as the diazo bonds in Congo Red can be cleaved by enzymes present in the mammalian gut and intestines. ' The most common side product of this cleavage is benzidine, a highly carcinogenic compound strongly associated with urinary bladder cancer.20 However, unlike many of the other compounds reported to have activity in various models of TSEs, Congo Red is a small molecule, which is amenable to chemical synthesis and structure activity relationship studies, with the aim of producing a low molecular weight therapeutic agent.
The present invention seeks to provide small molecule analogues of Congo Red, paricularly those which may have therapeutic applications in the treatment of TSEs. Ideally, the invention seeks to provide low molecular weight compounds which are specific in the interaction with PrPSc, have good cellular uptake, good penetration of the blood brain barrier, and which exhibit low toxicity. STATEMENT OF INVENTION
A first aspect of the invention relates to a compound of formula I, or a pharmaceutically acceptable salt thereof,
Figure imgf000004_0001
wherein
X is selected from
Figure imgf000004_0002
Y is selected from SO2NH, CONH, CH=N, CH2NH and CH=CH;
R1"5 are each independently selected from H, OH, OR6, CO2H, CO2R7, NO2, CN CONR8R9, CONHR10, CO2(CH2)nCO2Rπ, CO2(CH2)mO(CO)R12, CO2CHR13COR14, CONH(CH2)pCO2R15, CONH(CH2)qO(CO)R16, CONHCHR17COR18; R " are each independently hydrocarbyl; n, m, p and q are each independently 1, 2, 3 or 4; with the proviso that when X is phenyl, Y is CH=CH, R3 is OH and R1, R4 and R5 are H, R2 is other than COOH.
Advantageously, the replacement of the diazo group of Congo Red with sulphonamide, amide, imine, aminomethylene and alkene bonds (bio-isoteres of the diazo group) prevents the molecule being broken down to benzidine.
A second aspect of the invention relates to a pharmaceutical composition comprising a compound of formula I as defined above admixed with a pharmaceutically acceptable diluent, excipient or carrier. A third aspect of the invention relates to use of a compound of formula II, or a pharmaceutically acceptable salt thereof,
Figure imgf000005_0001
II wherein
X is selected from
Figure imgf000005_0002
Y is selected from SO2NH, CONH, CH=N, CH2NH and CH=CH;
R1"5 are each independently selected from H, OH, OR6, CO2H, CO2R7, NO2, CN CONR8R9, CONHR10, CO2(CH2)πCO2Rπ, CO2(CH2)mO(CO)R12, CO2CHR13COR14, CONH(CH2)pCO2R15, CONH(CH2)qO(CO)R16, CONHCHR17COR18; ■ R6"18 are each independently hydrocarbyl; n, m, p and q are each independently 1, 2, 3 or 4. in the preparation of a medicament for treating transmissible spongiform encephalopathy (TSE).
A fourth aspect of the invention relates to a method of treating, or preventing the onset of, transmissible spongiform encephalopathy (TSE) in a subject, said method comprising administering to a the subject a compound of formula II as defined above.
A fifth aspect of the invention relates to the use of a compound of formula II, or a pharmaceutically acceptable salt thereof, in an assay for identifying further candidate compounds capable of inhibiting the polymerisation of PrP by PrP-res. A sixth aspect of the invention relates to the use of a compound of formula LT, or a pharmaceutically acceptable salt thereof, in an assay for identifying further candidate compounds capable of inl ibiting the infectivity of the abnormal form of prion protein (PrPSc).
DETAILED DESCRIPTION
As used herein, the term "hydrocarbyl" refers to a group comprising at least C and H that may Optionally comprise one or more other suitable substituents. Examples of such substituents may include OH, COOH, halo-, alkoxy-, nitro-, an alkyl group, or a cyclic group. In addition to the possibility of the substituents being a cyclic group, a combination of substituents may form a cyclic group. If the hydrocarbyl group comprises more than one C then those carbons need not necessarily be linked to each other. For example, at least two of the carbons may be linked via a suitable element or group. Thus, the hydrocarbyl group may contain heteroatoms. Suitable heteroatoms will be apparent to those skilled in the art and include, for instance, sulphur, nitrogen, oxygen, phosphorus and silicon. Preferably, the hydrocarbyl group is an aryl, heteroaryl, alkyl, cycloalkyl, aralkyl or alkenyl group, more preferably, an aryl or alkyl group, more preferably still, an alkyl group.
As used herein, the term "alkyl" includes both saturated straight chain and branched alkyl groups which may be substituted (mono- or poly-) or unsubstituted. Preferably, the alkyl group is a Cι-2o alkyl group, more preferably a CMS, more preferably still a Cι-ι2 alkyl group, more preferably still, a Cι_6 alkyl group, more preferably a Cι-3 alkyl group. Particularly preferred alkyl groups include, for example, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl and hexyl. Suitable substituents include, for example, one or more halo-, OH, alkoxy-, nitro-, COOH, CF3, or alkyl groups. As used herein, the term "cycloalkyl" refers to a cyclic alkyl group which may be substituted (mono- or poly-) or unsubstituted. Preferably, the cycloalkyl group is a C3_ι2 cycloalkyl group. Suitable substituents include those listed above for alkyl.
As used herein, the term "alkenyl" refers to a group containing one or more carbon-carbon double bonds, which may be branched or unbranched, substituted (mono- or poly-) or unsubstituted. Preferably the alkenyl group is a C -20 alkenyl group, more preferably a C2. ι5 alkenyl group, more preferably still a C2.ι2 alkenyl group, or preferably a C2.6 alkenyl group, more preferably a C2.3 alkenyl group. Suitable substituents include those listed for alkyl above.
As used herein, the term "aryl" refers to a C6_ι2 aromatic group which may be substituted (mono- or poly-) or unsubstituted. Typical examples include phenyl and naphthyl etc. Suitable substituents include those listed for alkyl above.
As used herein, the term "heteroaryl" refers to a C .ι2 aromatic, substituted (mono- or poly- ) or unsubstituted group, which comprises one or more heteroatoms. Preferred heteroaryl groups include pyrrole, pyrazole, pyrimidine, pyrazine, pyridine, quinoline, thiophene and furan. Again, suitable substituents include those listed for alkyl above.
As used herein, the term "aralkyl" includes, but is not limited to, a group having both aryl and alkyl functionalities. By way of example, the term includes groups in which one of the hydrogen atoms of the alkyl group is replaced by an aryl group, e.g. a phenyl group optionally having one or more substituents such as halo, alkyl, alkoxy, hydroxy, and the like. Typical aralkyl groups include benzyl, phenethyl and the like.
By way of clarification, where Y is asymmetrical (i.e. other than CH=CH), each Y may be attached via either end to X; in other words, Y is selected from SO2NH, NHSO2, CONH, NHCO, CH=N, N=CH, CH2NH and NHCH2. In a preferred embodiment, R " are each independently alkyl or aryl, more preferably alkyl.
Preferably, for compounds of formula I, one of R1"5 is selected from H, OH and OR6; one of R1"5 is selected from OH, CO2H, CN, CO2-alkyl and CONH-alkyl; and the remainder of R1"5 are H.
Even more preferably, for compounds of formula I,
(i) R3 is CN, OH or CO2H, and R1 , R2, R4 and R5 are all H; (ii) R1 is OH, R3 is CO2H, CO2R7 or H, and R2, R4 and R5 are all H;
(iii) R1 is OR6, R4 is CO2R7 or CO2H, and R2, R3 and R5 are all H;
(iv) R2 and R4 are CO2R7, and R1, R3 and R5 are all H;
(v) R3 is CO2R7, R4 is OH or OR6, and R1, R2 and R5 are all H; or
(vi) R3 is OH, R4 is CO2R7 or CO2H, and R1, and R2 and R5 are all H.
More preferably still, for compounds of formula I,
(i) R3 is CN, OH or CO2H, and R1, R2, R4 and R5 are all H;
(ii) R1 is OH, R3 is CO2H, CO2Me or H, and R2, R4 and R5 are all H;
(iii) R1 is OMe, R4 is CO2Me or CO2H, and R2, R3 and R5 are all H; (iv) R2 and R4 are CO2Me, and R1 , R3 and R5 are all H;
(vi) R3 is CO2Me or CO2Et, R4 is OH or OMe, and R1, R2 and R5 are all H; or
(vi) R3 is OH, R4 is CO2Me, CO2Et or CO2H, and R1, and R2 and R5 are all H.
In one preferred embodiment of the invention, said compound is of formula la, or a pharmaceutically acceptable salt thereof
Figure imgf000008_0001
la wherein R . 1-"5 are as defined above.
Preferably, for compounds of formula la,
(i) R1 is OH, R3 is CO2R7, CONR8R9, CONHR10, CO2(CH2)„CO2Rπ, CO2(CH2)mO(CO)R12, CO2CHR13COR14, CONH(CH2)pCO2R15, CONH(CH2)q O(CO)R16 or CONHCHR17COR18, and R2, R4 and R5 are all H; or (ii) R1 is OR6, R5 is CO2H, CO2R7 CONR8R9, CONHR10, CO2(CH2)nCO2Rn, CO2(CH2)mO(CO)R12, CO2CHR13COR14, CONH(CH2)pCO2R15, CONH(CH2)q O(CO)R16 or CONHCHR17COR18, and R2"4 are all H.
More preferably, for compounds of formula la,
(i) R1 is OH, R3 is CO2R7, and R2, R4 and R5 are all H; or
(ii) R1 is OR6, R5 is CO2H or CO2R7, and R2"4 are all H.
Even more preferably, for compounds of formula la,
(i) R1 is OH, R3 is CO2alkyl, and R2, R4 and R5 are all H; or (ii) R1 is O-alkyl, R5 is CO2H or CO2-alkyl, and R2"4 are all H.
More preferably still, for compounds of formula la, (i) R1 is OH, R3 is CO2Me, and R2, R4 and R5 are all H; or (ii) R1 is OMe, R5 is CO2H or CO2Me, and R2"4 are all H.
In another preferred embodiment of the invention, the compound is of formula lb, or a pharmaceutically acceptable salt thereof
Figure imgf000009_0001
lb wherein R , 1-"5 are as defined above. Preferably, for compounds of formula lb,
(i) R1 is OH, R3 is CO2R7, CONR8R9, CONHR10, CO2(CH2)nCO2Rπ, CO2(CH2)mO(CO)R12, CO2CHR13COR14, CONH(CH2)pCO2R15, CONH(CH2)q O(CO)R16 or CONHCHR17COR18, and R2, R4 and R5 are H; (ii) R2 is OR6, R3 is CO2R7, CONR8R9, CONHR10, CO2(CH2)nCO2R11, CO2(CH2)mO(CO)R12, CO2CHR13COR14, CONH(CH2)pCO2R15, CONH(CH2)q O(CO)R16 or CONHCHR17COR18, and R1, R4 and R5 are H; (iii) R2 is CO2R7, CONR8R9, CONHR10, CO2(CH2)nCO2Rπ, CO2(CH2)mO(CO)R12, CO2CHR13COR14, CONH(CH2)pCO2R15, CONH(CH2)q O(CO)R16 or CONHCHR17COR18, R3 is OH, and R1, R4 and R5 are H.
More preferably, for compounds of formula lb, (i) R1 is OH, R3 is CO2R7, and R2, R4 and R5 are H; (ii) R2 is OR6, R3 is CO2R7, and R1, R4 and R5 are H; (iii) R2 is CO2R7, R3 is OH, and R1, R4 and R5 are H.
Even more preferably, for compounds of formula lb, (i) R1 is OH, R3 is CO2-alkyl, and R2, R4 and R5 are H; (ii) R2 is O-alkyl, R3 is CO2-alkyl, and R1, R4 and R5 are H; (iii) R2 is CO2-alkyl, R3 is OH, and R1, R4 and R5 are H.
More preferably still, for compounds of formula lb, (i) R1 is OH, R3 is CO2Me, and R2, R4 and R5 are H; (ii) R2 is OMe, R3 is CO2Me, and R1, R4 and R5 are H; (iii) R2 is CO2Me, R3 is OH, and R1, R4 and R5 are H.
In another preferred embodiment of the invention, the compound according to claim 1 is of formula Ic, or a pharmaceutically acceptable salt thereof
Figure imgf000011_0001
wherein R1"5 are as defined above.
Preferably, for compounds of formula Ic, (i) R^ are H;
(ii) R3 is CN, and R1 , R2, R4 and R5 are H;
(iii) R2 and R4 are CO2R7, CONR8R9, CONHR10, CO2(CH2)nCO2Rπ, CO2(CH2)mO(CO)R12, CO2CHR13COR14, CONH(CH2)pCO2R15, CONH(CH2)q O(CO)R16 or CONHCHR17COR18, and R1, R3 and R5 are H; or (iv) R1 is OH, R3 is COR7 and R2, R4 and R5 are all H.
More preferably, for compounds of formula Ic, R and R are CO2R , and R , R and R are H.
In one preferred embodiment, for compounds of formula Ic, R2 and R4 are CO2-alkyl, and R1, R3 and R5 are H, or R1 is OH, R3 is CO2-alkyl and R2, R4 and R5 are all H.
More preferably still, for compounds of formula Ic, R2 and R4 are CO Me, and R1, R3 and R5 are H, or R1 is OH, R3 is CO2Me and R2, R4 and R5 are all H.
In another preferred embodiment of the invention, the compound is of formula Id, or a pharmaceutically acceptable salt thereof
Figure imgf000012_0001
Id
wherein R1"5 are as defined above.
Preferably, for compounds of formula Id,
(i) R3 is CO2H, CO2R7, CONR8R9, CONHR10, CO2(CH2)nCO2Rn, CO2(CH2)mO(CO)R12, CO2CHR13COR14, CONH(CH2)pCO2R15, CONH(CH2)q O(CO)R16 or CONHCHR17COR18, R1 is OH, and R2, R4 and R5 are H; or
(ii) R3 is OH, and R1, R2, R4 and R5 are H;
More preferably, for compounds of formula Id,
R3 is CO2H, or CO2R7, R1 is OH, and R2, R4 and R5 are H.
Even more preferably, for compounds of formula Id,
R3 is CO2H, or CO2-alkyl, R1 is OH, and R2, R4 and R5 are H.
More preferably still, for compounds of formula Id,
R3 is CO2H, or CO2Me, R1 is OH, and R2, R4 and R5 are H.
In another preferred embodiment of the invention, the compound is of formula le, or a pharmaceutically acceptable salt thereof
Figure imgf000012_0002
le wherein R " are as defined above.
Preferably, for compounds of formula le,
(i) R1 is OR6, R5 is CO2H, CO2R7, CONR8R9, CONHR10, CO2(CH2)nCO2R11, CO2(CH2)mO(CO)R12, CO2CHR13COR14, CONH(CH2)pCO2R15, CONH(CH2)q O(CO)R16 or CONHCHR17COR18, and R2, R3 and R4 are H; (ii) R1 is OH, R3 is CO2H or H, and R2, R4 and R5 are H; (iii) R3 is CN or CO2H, and R1, R2, R4 and R5 are H;
(iv) R2 is CO2H, CO2R7, CONR8R9, CONHR10, CO2(CH2)nCO2R11, CO2(CH2)mO(CO)R12, CO2CHR13COR14, CONH(CH2)pCO2R15, CONH(CH2)q O(CO)R16 or CONHCHR17COR18, R3 is OH, R1, R4 and R5 are H; or (v) R2 is OH, R3 is CO2R7, CONR8R9, CONHR10, CO2(CH2)nCO2Rπ, CO2(CH2)raO(CO)R12, CO2CHR13COR14, CONH(CH2)pCO2R15, CONH(CH2)q O(CO)R16 or CONHCHR17COR18, and R1, R4 and R5 are H.
More preferably, for compounds of formula le, (i) R1 is OR6, R5 is CO2H or CO2R7, and R2, R3 and R4 are H; (ii) R2 is CO2H or CO2R7, R3 is OH, R1, R4 and R5 are H; or (iii) R2 is OH, R3 is CO2R7, and R1, R4 and R5 are H.
Even more preferably, for compounds of formula le, (i) R1 is O-alkyl, R5 is CO2H or CO2-alkyl, and R2, R3 and R4 are H; (ii) R2 is CO2H or CO2alkyl, R3 is OH, R1, R4 and R5 are H; or (iii) R2 is OH, R3 is CO2-alkyl, and R1, R4 and R5 are H.
More preferably still, for compounds of formula le, (i) R1 is OMe, R5 is CO2H or CO2Me, and R2, R3 and R4 are H; (ii) R2 is CO2H or CO2Et, R3 is OH, R1, R4 and R5 are H; or (iii) R2 is OH, R3 is CO2Et, and R1, R4 and R5 are H. In yet another preferred embodiment of the invention, the compound is of formula If, or a pharmaceutically acceptable salt thereof
Figure imgf000014_0001
If wherein R1"5 are as defined above.
Preferably, for compounds of formula If,
(i) R1 is OH, R3 is H, CO2H, CO2R7, CONR8R9, CONHR10, CO2(CH2)nCO2Rπ, CO2(CH2)mO(CO)R12, CO2CHR13COR14, CONH(CH2)pCO2R15, CONH(CH2)q O(CO)R16 or CONHCHR17COR18, and R2, R4 and R5 are H; or
(ii) R1 is OR6, R4 is CO2R7, CONR8R9, CONHR10, CO2(CH2)nCO2R11, CO2(CH2)mO(CO)R12, CO2CHR13COR14, CONH(CH2)pCO2R15, CONH(CH2)q O(CO)R16 or CONHCHR17COR18, and R2, R3 and R5 are H.
More preferably, for compounds of formula If, ((ii)) RR11 iiss OOHH,, RR33 iiss HH,, CCOO22HH oorr CCOO22RR77,, aanncd R2 R4 and R5 are H; or (ii) R1 is OR6, R4 is CO2R7, and R2, R3 and R5 are H
Even more preferably, for compounds of formula If, (i) R1 is OH, R3 is H, CO2H or CO2-alkyl, and R2, R4 and R5 are H; or (ii) R1 is O-alkyl, R4 is CO2-alkyl, and R2, R3 and R5 are H.
More preferably still, for compounds of formula If, (ii) R1 is OH, R3 is H, CO2H or CO2Me, and R2, R4 and R5 are H; or (iii) R1 is OMe, R4 is CO2Me, and R2, R3 and R5 are H. In yet another preferred embodiment of the invention, the compound is of formula Ig, or a pharmaceutically acceptable salt thereof
5 Ig wherein R1"5 are as defined above.
Preferably, for compounds of formula Ig, R is OH, and R 1 , τ R»2 , τR>4 and R are H.
10 In one especially preferred embodiment of the invention, the compound is selected from those set forth in Tables 1-8.
CHEMICAL SYNTHESIS The sulphonamide analogues of the invention can be synthesised from the readily available 4-4'-biphenyldisulfonyl chloride 8 and the appropriate amine utilising DMAP as a catalyst 15. and anhydrous pyridine as the solvent.
Figure imgf000015_0002
Scheme 1: Sulphonamide coupling The amines utilised in the reactions are either readily commercially available or straightforwardly synthesised via simple one step esterifications (for example see Scheme 2).
Figure imgf000016_0001
Scheme 2: Synthesis of amines
The majority of biphenyl amide analogues of the invention can be synthesised either by the reaction of the corresponding acid chloride with the appropriate aniline derivative in the presence of triethylamine or using TBTU, a uronium based peptide coupling reagent (Scheme 3). Depending upon the nature of the amine utilised in the reaction, the amount of triethylamine used in the coupling reaction is varied, specifically if the hydrochloride salt of the amine is to be used for coupling, a further equivalent of base (triethylamine) isbe needed to optimise the reaction.
Figure imgf000016_0002
Scheme 3: Amide coupling The mono-phenyl analogues of the invention can be synthesised using terephthaloyl chloride and the appropriate aniline.
The imine analogues of the invention can be made by condensation of the corresponding aldehyde with the appropriate amine. The products (imines) are generally insoluble in ethanol and precipitate from solution: filtration and washing generally provides the compounds in good yield. The biphenyl analogues prove less soluble but also the reaction time is longer in these cases. The mono-phenyl imines (series 5) can be synthesised from the commerically available terephthalaldehyde, whereas the biphenyl analogues (series 4) require 4-4 '-biphenyl dialdehyde to be made first.
Ethanol, 55°C OH
Figure imgf000017_0002
Figure imgf000017_0001
Scheme 4: Imine Formation
Hθ2C CH0
Figure imgf000017_0003
Scheme 5: Synthesis of biphenyl dialdehyde. (i) BH SMe , THF; (ii) Pyridinium Dichromate, Dichloromethane
The amines of the invention can be synthesised by reduction of the imines with sodium borohydride. The only limiting factor is the initial solubility of the imines, many of which prove insoluble in any solvent once formed. Generally the reduction is favourable in a methanol / dichloromethane mixture.
Figure imgf000018_0001
Scheme 6: Reduction of Imines
Transition metal chemistry plays a part in the synthesis of the alkenes of the invention (Scheme 7); the Heck reaction is preferred, so as to achieve only one of the stereoisomers, the trαrø-stereoisomer. The reaction is sensitive to the choice of solvent. A 17:3 DMF:water mixture is optimal, the water being a crucial element. 1,4-divinylbenzene can be purchased commercially but only as a mixture with 1,2-divinylbenzene, therefore it is preferable to synthesise this compound from terephthaldehyde using a Wittig reaction.
Figure imgf000018_0002
Scheme 7: Alkene coupling
THERAPEUTIC USE
Initial studies indicate that certain compounds of the invention may have potential therapeutic applications in the treatment or prevention of TSE. Thus another aspect of the invention relates to the use of a compound of formula II, or a pharmaceutically acceptable salt thereof,
Figure imgf000019_0001
II wherein
X is selected from
Figure imgf000019_0002
Y is selected from SO2NH, CONH, CH=N, CH2NH and CH=CH;
R1"5 are each independently selected from H, OH, OR6, CO2H, CO2R7, NO2, CN CONR8R9, CONHR10, CO2(CH2)„CO2Rπ, CO2(CH2)mO(CO)R12, CO2CHR13COR14,
CONH(CH2)pCO2R15, CONH(CH2)qO(CO)R16, CONHCHR17COR18;
R6"18 are each independently hydrocarbyl; n, m, p and q are each independently 1, 2, 3 or 4. in the preparation of a medicament for treating transmissible spongiform encephalopathy (TSE).
Preferably, said compound of formula II is as defined above for compounds of formula I.
In one preferred embodiment, said compound is as defined above for compounds of formula Ig and wherein:
R3 is OH, and R1, R2, R4 and R5 are H; or
RJ is OH, R >24 i ■s CO2H, and R 11, r R>44 and RD are H
As used herein the phrase "preparation of a medicament" includes the use of compounds of formula II directly as the medicament in addition to their use in a screening programme for identifying further therapeutic agents or in any stage of the manufacture of such a medicament.
Another aspect of the invention relates to a method of treating, or preventing the onset of, transmissible spongiform encephalopathy (TSE) in a subject, said method comprising administering to the subject a compound of formula II as defined above.
Preferably, the TSE is selected from Creutzfeldt Jakob Disease (CJD), Gerstmann- Staussler-Scheinker Syndrome (GSS), Familial Fatal Insomnia (FFI), Kuru, variant CJD, Bovine Spongiform Encephalopathy (BSE) and scrapie.
Preferably, the compound of formula II is administered in an amount sufficient to inhibit the infectivity of the abnormal form of prion protein (PrPSc).
Preferably, the compound of formula II is administered in an amount sufficient to inhibit the polymerisation of PrP by PrP-res.
hi one preferred embodiment, the compounds of the invention are active in both the cellular and polymerisation assay described in the accompanying Examples section. Without wishing to be bound by theory, it is believed that these compounds probably prevent PrP-res formation in cells by preventing polymerisation of PrPc. Preferred compounds falling into this category include, for example, WSP677, 905, 1008, 795, 1011 718 and 798.
hi another preferred embodiment, the compounds of the invention are active in the polymerisation assay, but not in the cellular assay. Without wishing to be bound by theory, it is possible that these compounds are too weak at inhibition of polymerisation to be active in cellular assays, or they are inactivated in a cellular system, or they are unable to access the molecular target. Preferred compounds falling into this category include, for example, WSP675, 676, 678, 917, 1012, 794, 738, 741, 742, 760, 980 and 759.
h yet another preferred embodiment, the compounds of the invention are active in the cellular assay, but not the polymerisation assay. Without wishing to be bound by theory, it is possible that these compounds preventPrP-res formation in the SMB cells by a mode of action other than inhibition of polymerisation. Preferred compounds falling into this category include, for example, WSP 1009, 906, 915, 740 and 911.
Both the cellular and polymerisation assay provide useful information in the context of a drug discovery programme. The polymerisation assay can identify most, but not all molecules which reduce PrP-res formation in cellular assays. Thus, on its own it is not a useful screen to select out compounds for cellular assays. In addition, the cellular assay employed on its own also has limitations. The assay does not identify all compounds that prevent polymerisation. These compounds may be a useful source of information for future rounds of drug design. The polymerisation assay also provides information on the possible modes of action of compounds.
The biphenyl amides showed the most promise; WSP677 is of particular interest. For the biphenyl amides, without wishing to be bound by theory, there seems to be some correlation between prevention of PrP-res formation in cell culture and prevention of polymerisation of PrPc. This suggests that the anti-TSE activity of at least some of the compounds may be related to their ability to prevent polymerisation.
The sulfonamides (Series 1) appeared less active than the biphenyl amides (Series 2). The sulfonamide group is a bioisostere of the amide group. However, the NH of the sulfonamides is ionisable and may well be ionised at physiological pH. Whilst ionisation may well increase the solubility of these compounds, interaction with the prion protein may be also be prevented, hi spite of this, some of the sulfonamides still showed weak activity in a cellular model and inhibition of polymerisation.
Imines are normally readily subject to hydrolysis. However, the imines of the present invention are highly conjugated, which stablises them to hydrolysis. Furthermore, by making sure that the nitrogen of the imine is attached to the terminator rather than the linker, any hydrolysis should not give rise to benzidene or its analogues. The biphenyl imines (Series 4) showed higher activity than the monophenyl imines (Series 5) in the cellular assay, although several of the monophenyl imines showed potent inhibition of polymerisation. Preferably, these compounds tended to be those with carboxylates on the terminator, e.g. WSP738, WSP741, WSP742, WSP760.
The aminomethylene compounds (Series 6) show very promising activity, particularly in the cellular assay, but also in the polymerisation assay.
Structure activity relationships indicate that the most active compounds in cellular assays preferably have a methyl carboxylate attached to the terminator and either a hydroxy or methoxy group. Clearly the terminator found in compound found in WSP677 is active in both the cellular and polymerisation assays. However other regio-isomers are also active (WSP905, 1009). Some compounds containing a carboxylic acid on the terminator are active in inhibition of polymerisation, but not in the cellular assay. Conversely, compounds which are active in the cellular assay and contain a methyl ester are not be active in the polymerisation assay (WSP1009, 906, 740). Without wishing to be bound by theory, this could imply, assuming the mechanism of action of these compounds is inhibition of polymerisation, that the methyl esters are metabolised to the active carboxylic acid form by the cells, which then inhibits polymerisation. By way of evidence, WSP798, the demethylated analogue of WSP740 is active in the polymerisation assay. The compounds which contain free carboxylic acids may not be active in the cellular assays as they cannot access the site of action. In addition there are several compounds that are active in cellular and or polymerisation assays, which do not have a carboxylate substituent, implying that this is not an essential substituent for activity.
Another aspect of the invention relates to the use of compounds of formula I, or pharmaceutically acceptable salts thereof, in the preparation of a medicament for treating Alzheimer's disease.
PHARMACEUTICAL COMPOSITIONS
A fourth aspect of the invention relates to a pharmaceutical composition comprising a compound of formula I as defined for said first aspect admixed with one or more pharmaceutically acceptable diluents, excipients or carriers. Even though the compounds of the present invention (including their pharmaceutically acceptable salts, esters and pharmaceutically acceptable solvates) can be administered alone, they will generally be administered in admixture with a pharmaceutical carrier, excipient or diluent, particularly for human therapy. The pharmaceutical compositions may be for human or animal usage in human and veterinary medicine.
Examples of such suitable excipients for the various different forms of pharmaceutical compositions described herein may be found in the "Handbook of Pharmaceutical Excipients, 2nd Edition, (1994), Edited by A Wade and PJ Weller.
Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985).
Examples of suitable carriers include lactose, starch, glucose, methyl cellulose, magnesium stearate, mannitol, sorbitol and the like. Examples of suitable diluents include ethanol, glycerol and water. The choice of pharmaceutical carrier, excipient or diluent can be selected with regard to the intended route of administration and standard pharmaceutical practice. The pharmaceutical compositions may comprise as, or in addition to, the carrier, excipient or diluent any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s), solubilising agent(s).
Examples of suitable binders include starch, gelatin, natural sugars such as glucose, anhydrous lactose, free-flow lactose, beta-lactose, corn sweeteners, natural and synthetic gums, such as acacia, tragacanth or sodium alginate, carboxymethyl cellulose and- polyethylene glycol.
Examples of suitable lubricants include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride and the like.
Preservatives, stabilizers, dyes and even flavoring agents may be provided in the pharmaceutical composition. Examples of preservatives include sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid. Antioxidants and suspending agents may be also used.
SALTS/ESTERS
The compounds of formula I or II can be present as salts or esters, in particular pharmaceutically acceptable salts or esters.
Pharmaceutically acceptable salts of the compounds of the invention include suitable acid addition or base salts thereof. A review of suitable pharmaceutical salts may be found in Berge et al, J Pharm Sci, 66, 1-19 (1977). Salts are formed, for example with strong inorganic acids such as mineral acids, e.g. sulphuric acid, phosphoric acid or hydrohalic acids; with strong organic carboxylic acids, such as alkanecarboxylic acids of 1 to 4 carbon atoms which are unsubstituted or substituted (e.g., by halogen), such as acetic acid; with saturated or unsaturated dicarboxylic acids, for example oxalic, malonic, succinic, maleic, fumaric, phthalic or tetraphthalic; with hydroxycarboxylic acids, for example ascorbic, glycolic, lactic, malic, tartaric or citric acid; with aminoacids, for example aspartic or glutamic acid; with benzoic acid; or with organic sulfonic acids, such as (Cι-C4)-alkyl- or aryl-sulfonic acids which are unsubstituted or substituted (for example, by a halogen) such as methane- or p-toluene sulfonic acid.
Esters are formed either using organic acids or alcohols/hydroxides, depending on the functional group being esterified. Organic acids include carboxylic acids, such as alkanecarboxylic acids of 1 to 12 carbon atoms which are unsubstituted or substituted (e.g., by halogen), such as acetic acid; with saturated or unsaturated dicarboxylic acid, for example oxalic, malonic, succinic, maleic, fumaric, phthalic or tetraphthalic; with hydroxycarboxylic acids, for example ascorbic, glycolic, lactic, malic, tartaric or citric acid; with aminoacids, for example aspartic or glutamic acid; with benzoic acid; or with organic sulfonic acids, such as (Cι-C4)-alkyl- or aryl-sulfonic acids which are unsubstituted or substituted (for example, by a halogen) such as methane- or p-toluene sulfonic acid. Suitable hydroxides include inorganic hydroxides, such as sodium hydroxide, potassium hydroxide, calcium hydroxide, aluminium hydroxide. Alcohols include alkanealcohols of 1-12 carbon atoms which may be unsubstituted or substituted, e.g. by a halogen).
ENANTIOMERS/TAUTOMERS
In all aspects of the present invention previously discussed, the invention includes, where appropriate all enantiomers and tautomers of compounds of formula I or II. The man skilled in the art will recognise compounds that possess an optical properties (one or more chiral carbon atoms) or tautomeric characteristics. The corresponding enantiomers and/or tautomers may be isolated/prepared by methods known in the art. STEREO AND GEOMETRIC ISOMERS
Some of the specific agents exist as stereoisomers and/or geometric isomers - e.g. they may possess one or more asymmetric and/or geometric centres and so may exist in two or more stereoisomeric and/or geometric forms. The present invention contemplates the use of all the individual stereoisomers and geometric isomers of those inhibitor agents, and mixtures thereof. The tenns used in the claims encompass these forms, provided said forms retain the appropriate functional activity (though not necessarily to the same degree).
The present invention also includes all suitable isotopic variations of the agents or pharmaceutically acceptable salts thereof. An isotopic variation of an agent of the present invention or a pharmaceutically acceptable salt thereof is defined as one in which at least one atom is replaced by an atom having the same atomic number but an atomic mass different from the atomic mass usually found in nature. Examples of isotopes that can be incorporated into the agent and pharmaceutically acceptable salts thereof include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulphur, fluorine and chlorine such as 2H, 3H, 13C, 14C, 15N, 17O, 18O, 31P, 32P, 35S, 18F and 36C1, respectively. Certain isotopic variations of the agent and pharmaceutically acceptable salts thereof, for example, those in which a radioactive isotope such as 3H or 14C is incorporated, are useful in drug and/or substrate tissue distribution studies. Tritiated, i.e., H, and carbon-14, i.e., 14C, isotopes are particularly preferred for their ease of preparation and detectability. Further, substitution with isotopes such as deuterium, i.e., 2H, may afford certain therapeutic advantages resulting from greater metabolic stability, for example, increased in vivo half-life or reduced dosage requirements and hence may be preferred in some circumstances. Isotopic variations of the agent of the present invention and pharmaceutically acceptable salts thereof of this invention can generally be prepared by conventional procedures using appropriate isotopic variations of suitable reagents. SOLVATES
The present invention also includes the use of solvate forms of the compounds of the present invention. The terms used in the claims encompass these forms.
POLYMORPHS
The invention furthermore relates to the compounds of the present invention in their various crystalline forms, polymorphic forms and (an)hydrous forms. It is well established within the pharmaceutical industry that chemical compounds may be isolated in any of such forms by slightly varying the method of purification and or isolation form the solvents used in the synthetic preparation of such compounds.
PRODRUGS
The invention further includes the compounds of the present invention in prodrug form.
Such prodrugs are generally compounds of formula I or II wherein one or more appropriate groups have been modified such that the modification may be reversed upon administration to a human or mammalian subject. Such reversion is usually performed by an enzyme naturally present in such subject, though it is possible for a second agent to be administered together with such a prodrug in order to perform the reversion in vivo.
Examples of such modifications include ester (for example, any of those described above), wherein the reversion may be carried out be an esterase etc. Other such systems will be well known to those skilled in the art.
ADMINISTRATION
The pharmaceutical compositions of the present invention may be adapted for oral, rectal, vaginal, parenteral, intramuscular, intraperitoneal, intraarterial, intrathecal, intrabronchial, subcutaneous, intradermal, intravenous, nasal, buccal or sublingual routes of administration. For oral administration, particular use is made of compressed tablets, pills, tablets, gellules, drops, and capsules. Preferably, these compositions contain from 1 to 250 mg and more preferably from 10-100 mg, of active ingredient per dose.
Other forms of administration comprise solutions or emulsions which may be injected intravenously, intraarterially, intrathecally, subcutaneously, intradermally, intraperitoneally or intramuscularly, and which are prepared from sterile or sterilisable solutions. The pharmaceutical compositions of the present invention may also be in form of suppositories, pessaries, suspensions, emulsions, lotions, ointments, creams, gels, sprays, solutions or dusting powders.
An alternative means of transdermal administration is by use of a skin patch. For example, the active ingredient can be incorporated into a cream consisting of an aqueous emulsion of polyethylene glycols or liquid paraffin. The active ingredient can also be incorporated, at a concentration of between 1 and 10% by weight, into an ointment consisting of a white wax or white soft paraffin base together with such stabilisers and preservatives as may be required.
Injectable forms may contain between 10 - 1000 mg, preferably between 10 - 250 mg, of active ingredient per dose.
Compositions may be formulated in unit dosage form, i.e., in the form of discrete portions containing a unit dose, or a multiple or sub-unit of a unit dose.
DOSAGE
A person of ordinary skill in the art can easily determine an appropriate dose of one of the instant compositions to administer to a subject without undue experimentation. Typically, a physician will determine the actual dosage which will be most suitable for an individual patient and it will depend on a variety of factors including the activity of the specific compound employed, the metabolic stability and length of action of that compound, the age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular condition, and the individual undergoing therapy. The dosages disclosed herein are exemplary of the average case. There can of course be individual instances where higher or lower dosage ranges are merited, and such are within the scope of this invention.
Depending upon the need, the agent may be administered at a dose of from 0.01 to 30 mg/kg body weight, such as from 0.1 to 10 mg/kg, more preferably from 0.1 to 1 mg/kg body weight.
In an exemplary embodiment, one or more doses of 10 to 150 mg/day will be administered to the patient for the treatment of TSE.
COMBINATIONS
In a particularly preferred embodiment, the one or more compounds of the invention are administered in combination with one or more other agents, for example, existing drugs available on the market. In such cases, the compounds of the invention may be administered consecutively, simultaneously or sequentially with the one or more other active agents.
Drugs in general are often more effective when used in combination. In particular, combination therapy is desirable in order to avoid an overlap of major toxicities, mechanism of action and resistance mechanism(s). Furthermore, it is also desirable to administer most drugs at their maximum tolerated doses with minimum time intervals between such doses. The major advantages of combining drugs are that it may promote additive or possible synergistic effects through biochemical interactions and also may decrease the emergence of resistance in cells which would have been otherwise responsive to initial therapy with a single agent. Beneficial combinations may be suggested by studying the activity of the test compounds with agents known or suspected of being valuable in the treatment of a particular disorder.
This procedure can also be used to determine the order of administration of the agents, i.e. before, simultaneously, or after delivery. Such scheduling may be a feature of all the agents identified herein.
ASSAYS
Another aspect of the invention relates to the use of a compound of formula I or II, or a pharmaceutically acceptable salt thereof, as defined above, in an assay for identifying candidate compounds capable of inhibiting the polymerisation of PrPc by PrP-res.
Yet another aspect relates to the use of a compound of formula I or II, or a pharmaceutically acceptable salt thereof, as defined above, in an assay for identifying candidate compounds capable of inhibiting the infectivity of the abnormal form of prion protein (PrPSc).
Preferably, the assay is a competitive binding assay.
One aspect of the invention relates to a process comprising the steps of: (a) performing an assay method described hereinabove;
(b) identifying one or more candidate compounds capable of inhibiting the polymerisation of PrP by PrP-res or inhibiting the infectivity of the abnormal form of prion protein (PrPSc); and
(c) preparing a quantity of said one or more candidate compounds.
Another aspect of the invention provides a process comprising the steps of: (a) performing an assay method described hereinabove; (b) identifying one or more candidate compounds capable of inhibiting the polymerisation of PrPc by PrP-res or inhibiting the infectivity of the abnormal form of prion protein (PrPSc); and
(c) preparing a pharmaceutical composition comprising said one or more candidate compounds.
Another aspect of the invention provides a process comprising the steps of:
(a) performing an assay method described hereinabove;
(b) identifying one or more candidate compounds capable of inhibiting the polymerisation of PrP by PrP-res or inhibiting the infectivity of the abnormal form of prion protein (PrPSc);
(c) modifying said one or more candidate compounds capable of inhibiting the polymerisation of PrPc by PrP-res or inhibiting the infectivity of the abnormal form of prion protein (PrPSc); and (d) performing the assay method described hereinabove;
(e) optionally preparing a pharmaceutical composition comprising said one or more candidate compounds.
The invention also relates to a candidate compound identified by the method described hereinabove.
Yet another aspect of the invention relates to a pharmaceutical composition comprising a candidate compound identified by the method described hereinabove.
Another aspect of the invention relates to the use of a candidate compound identified by the method described hereinabove in the preparation of a pharmaceutical composition for use in the treatment or prevention of a TSE. Preferably, said candidate compound is generated by conventional SAR modification of a compound of the invention.
As used herein, the term "conventional SAR modification" refers to standard methods known in the art for varying a given compound by way of chemical derivatisation.
The present invention is further described by way of example, and with reference to the following figures wherein:
Figure 1 shows the cell viability of cerebellar neurones isolated from mice when incubated with WSP677 and WSP1012.
EXAMPLES
General Procedures
1H and 13C NMR spectra were recorded as a solution (in specified solvent) on a Bruker Advance DPX300 spectrometer at 300MHz and 75MHz respectively. Chemical shifts are quoted in parts per million (ppm) downfield with respect to tetramethylsilane (δ = 0.00 ppm). Coupling constants (J) are quoted in Hertz to the nearest 0.5 Hertz. Low resolution ES mass spectrometry was performed on a Fisons VG platform II electrospray mass spectrometer at the Welsh School of Pharmacy, Cardiff university. Low resolution El, CI, FAB, and all high resolution mass spectra were recorded on a VG ZAB spectrometer at the Engineering and Physical Sciences Research Council (EPSRC) mass spectrometry centre, at the University College of Wales, Swansea. LR spectra were recorded on an Perkin Elmer 1600 series spectrophotometer using sodium chloride plates for liquid samples and potassium bromide for solid samples. Elemental Analyses were obtained from MED AC analytical and chemical consultancy services Ltd. Melting points are uncorrected values and were determined using a Gallenkamp melting point apparatus.
All reactions were performed in oven dried apparatus under an atmosphere of nitrogen. All solvents were of reagent grade. Anhydrous diethyl ether, methanol, and tetrahydrofuran were purchased from Fluka . Anhydrous NN-dimethylacetamide, NN- dimethylformamide, and 1,4-dioxane were purchased from Aldrich chemical company. Reaction temperatures of -78°C and 0°C were achieved by the use of acetone/dry ice baths and water/ice baths respectively. Thin layer chromatography (TLC) was performed on Fluka aluminium backed silica plates. Compounds were visualised under a UN lamp, with aqueous potassium permanganate solution and/or phosphomolybdic acid in ethanolic solution. Column chromatography was performed on Fluka silica gel (with the solvent mixtures specified). Synthesis of Sulphonamide Analogues
Bis-methyl 4-({[4 '-(aminosulfonyl)-l, 1 '-biphenyl-4-yl]sulfonyl}amino)-3-hydroxybenzoate (WSP675) In a 25ml flask was placed DMAP (0.012g, 0.099mmol) and methyl-4-amino-3- hydroxybenzoate (0.366g, 2.2mmol). Anhydrous pyridine (10ml) was added and the flask cooled to 0°C for 10 minutes. 4-4'-biphenyldisulphonyl chloride (0.350g, 0.99mmol) was added as a solid in small 50mg portions. The reaction was left stirring at room temperature overnight (14 hours). 2N HC1 (50ml) was added and the organic phase was extracted with ethyl acetate (2 x 25ml). The organic layer was separated, dried (MgSO4) and evaporated in vacuo to leave a crude brown product. Column chromatography in a 4:1 ethyl acetate :petroleum ether eluent mixture yielded the product WSP675 as a white solid. Yield 0.169g (0.276mmol, 28%); mp 289-290°C; 1H NMR (300MHz, -DMSO) δ 3.77 (6H, s, OCH3), 7.35 (6H, m, C4-H + C5-H + C8-H), 7.89 (8H, br, s, Cio-H + di-H), 9.97 (4H, br, s, NH + OH); 13C NMR (75MHz, -pyridine) δ 54.2 (d), 119.2 (C5), 123.9 (C8), 125.0 (C3), 129.7 (C4), 130.3 (Cio), 130.5 (Cu), 133.7 (C6), 143.4 (C9), 145.5 (Cι2), 152.4 (C7), 168.9 (C2); ES" m/z 610.9 (M"); high resolution ES" m/z found 611.0787 C28H23N2Oi0S2 (M-H) requires 611.0794. (Anal. Calcd. for C28H24N2OιoS2 : C, 54.90; H, 3.95; N, 4.57; S, 10.47%. Found: C, 55.78; H, 4.37; N, 4.59; S, 9.17%).
Bis-methyl-3-({[4'-(aminosulfonyl)-l,l '-biphenyl-4-yl]sulfonyl}amino)-4-methoxybenzoate
(WSP676)
An identical procedure to the one employed for WSP675 was used. DMAP (0.017g,
0.142mmol) and methyl-3-amino-4-methoxybenzoate (0.567g, 3.13mmol) was reacted with 4-4'-biphenyldisulphonyl chloride (0.350g, 0.99mmol) in anhydrous pyridine (10ml). The product WSP676 was isolated as an off-white solid.
Yield 0.458g (0.71mmol, 50%); mp 265-268°C; 1H NMR (300MHz, -DMSO) δ 3.57 (6H, s, C9-H), 3.80 (6H, s, Q-H), 7.01 (2H, d, J = 8.7Hz, C5-H), 7.78-7.90 (12H, m, C4-H + C8-H + Cπ-H + Cι2-H), 9.89 (2H, s, NH); 13C NMR (75MHz, -DMSO) δ 52.3 (d), 56.1 (C9), 112.0 (C5), 122.0 (C8), 125.6 (C4), 126.4 (C3), 127.7 (Cu), 127.9 (Cι2), 128.8 (C7), 140.3 (Cio), 142.7 (C13), 156.5 (C6), 165.8 (C2); ES" m/z 638.8 (M"); high resolution ES+ m/z found 658.1531 C30H32N3OιoS2 (M+NH4) requires 658.1529. (Anal. Calcd. for C3oH28N2Oi0S2 : C, 56.24; H, 4.40; N, 4.37%. Found: C, 55.89; H, 4.38; N, 4.43%)
Bis-3-({[4'-(aminosulfonyl)-l, 1 '-biphenyl-4-yl]sulfonyl}amino)-4-methoxybenzoic acid
(WSP678)
WSP676 (0.1 OOg, 0.156mmol) was placed in a 10ml round bottomed flask, NaOH
(0.025g, 0.625mmol) was dissolved in water (4ml) and added to the reaction flask dropwise. The mixture was left stirring at ambient temperature for 2 hours. 2N HCl (5ml) was added dropwise till a white precipitate crashed out. The solid was filtered through a sinter funnel and dried under high vacuum. The product WSP678 was isolated as a white solid. Yield 0.030g (0.076mmol, 49%); mp >360°C (decomp.); 1H NMR (300MHz, /-DMSO) δ 3.53 (6H, s, C8-H), 7.00 (2H, d, J = 8.6Hz, C4-H), 7.72-7.90 (12H, m, C3-H + C7-H + Cio-H +C11-H), 9.84 (2H, s, NH); 13C NMR (75MHz, /-DMSO) δ 56.1 (C8), 111.8 (C4), 123.2 (C7), 125.4 (C3), 126.7 (C2), 127.7 (Cio), 127.9 (Cu), 128.9 (C6), 140.4 (C9), 142.7 (Cι2), 156.3 (C5), 166.9 (CO; ES"m/z 610.8 (M"), 305.0 (m/2z).
Bis-4-({[4'-(anιinosulfonyl)-l, 1 '-biphenyl-4-yl]sulfonyl}amino)-3-hydroxybenzoic acid
(WSP679)
An identical procedure to the one employed for WSP678 was used. WSP675 (0.086g, 0.14mmol) was reacted with aqueous NaOH (0.023g, 0.56mmol) in water (4ml) and the product WSP679 was isolated as a light brown solid. Yield 0.045g (0.094mmol, 55%); mp >360°C (decomp.); 1H NMR (300MHz, /-DMSO) δ 7.32 (6H, br, s, C3-H + C6-H + C7-H), 7.89-8.04 (8H, br, m, C9-H + Cio-H), 9.76 (2H, s, OH), 10.14 (2H, s, NH), 12.77 (2H, br, s, CO2H); 13C NMR (75MHz, /-DMSO) δ 116.5 (C6), 118.5 (C3), 122.7 (C2), 124.1 (C7), 129.6 (C9), 129.9 (C10), 130.9 (C8), 142.2 (C5), 144.5 (Cu), 150.9 (C4), 168.9 (Ci); ES" m/z 582.5 (M"), 291.1 (m/2z); high resolution ES" m/z found 583.0494 C26Hι9N2O10S2 (M-H) requires 583.0487; (Anal. Calcd. for C26H16N2Na4θι0S2 + 2H2O: C, 44.07; H, 2.85; N, 3.95%. Found: C, 44.88; H, 2.98; N, 3.99%).
Bis-dimethyl-5-({[4 '-(aminosulfonyl)- 1, 1 '-biphenyl-4-yl]sulfonyl}amino)isophthalate (WSP684)
An identical procedure to the one employed for WSP675 was used. DMAP (0.024g, 0.199mmol) and dimethyl-5-aminoisophthalate (0.917g, 4.38mmol) was reacted with 4-4'- biphenyldisulphonyl chloride (0.700g, 1.99mmol) in anhydrous pyridine (15ml). The required product (the tetra-methyl tetra-ester) was not formed, instead it was hydrolysed to WSP684 during the work-up. The product WSP684 was isolated as an off-white solid. Yield 0.0618g (0.96mmol, 48%); mp 278-281°C; 1H NMR (300MHz, /-DMSO) δ 7.83- 7.95 (12H, m, C4-H + C7-H + C8-H), 8.12 (2H, s, C3-H), 10.89 (2H, s, NH), 13.34 (4H, br, s, CO2H); 13C NMR (75MHz, /-DMSO) δ 124.3 (C3), 125.7 (C4), 127.6 (C7), 128.6 (C8), 132.7 (C2), 138.8 (C6), 139.2 (C9), 142.9 (C5), 166.3 (Ci); ES" m/z 638.5(M"), 319.0 (m/2z); high resolution ES" m/z found 639.0373 C28Hι9N2Oι2S2 (M-H) requires 639.0379; (Anal. Calcd. for C28H20N2Oι2S2 : C, 52.50; H, 3.15; N, 4.37%. Found C, 48.95; H, 3.39; N, 4.06%).
Bis-phenyl 4-({[4'-(aminosulfonyl)-l, 1 '-biphenyl-4-yl]sulfonyl}amino)-2-hydroxybenzoate (WSP685)
An identical procedure to the one employed for WSP675 was used. DMAP (0.026g, 0.216mmol) and phenyl-4-aminosalicylate (1.09g, 4.76mmol) was reacted with 4-4'- biphenyldisulphonyl chloride (0.760g, 2.16mmol) in anhydrous pyridine (15ml). The product WSP685 was isolated as an white solid.
Yield 0.065g (0.088mmol, 4%); mp 266-270°C; 1H NMR (300MHz, /-DMSO) δ 6.77- 6.81 (4H, m, C8-H + Cio-H), 7.21-7.46 (10H, m, Ci-H + C2-H + C3-H), 7.87 (2H, d, J = 9.3Hz, Cii-H), 7.96 (8H, s, C13-H + Cι4-H), 10.39 (2H, s, NH), 11.28 (2H, br, s, OH); 13C NMR (75MHz, /-DMSO) δ 103.9 (C8), 106.4 (Cι0), 108.1 (C6), 120.4 (C3), 124.5 (Ci), 125.9 (Cι3), 126.7 (CM), 127.9 (C2), 130.8 (Cu), 137.5 (Cι2), 141.2 (Cι5), 148.5 (C4 + C9), 159.7 (C7), 164.7 (C5); ES" m/z 734.7 (M"); high resolution ES" m/z found 735.1100 C38H27N2Oi0S2 (M-H) requires 735.1107; (Anal. Calcd. for C38H28N2Oι0S2 + 0.2H2O : C, 61.65; H, 3.87; N, 3.78%. Found: C, 61.57; H, 3.74; N, 3.56%).
Synthesis of Amide Analogues
Bis-methyl4-({[4 '-(aminocarbonyl)-! , 1 '-biphenyl-4-yl]carbonyl}amino)-3-hydroxybenzoate (WSP677) A mixture of biphenyl 4-4' dicarboxylic acid (0.300g, 1.24mmol) and TBTU (0.875g, 2.72mmol) was placed in a dry 50ml round bottomed flask. Anhydrous DMA (15ml) was added, followed by DJJPEA (0.7ml, 3.71mmol). After stirring at room temperature for 15 minutes, methyl-4-amino-2-hydroxybenzoate (0.455g, 2.72mmol) was dissolved in DMA (5ml) in a separate vial and added dropwise to the reaction flask. The reaction mixture was left stirring for 48 hours at room temperature under a nitrogen atmosphere. 2N HCl (40ml) was added and the organic product was extracted with ethyl acetate (2 x 40ml). The organic phases were first combined and dried (MgSO4), then evaporated in vacuo to leave the crude product as an off-white solid. The pure compound WSP677 was obtained after recrystallisation from ethyl acetate. Yield 0.070g (0.13mmol, 10%); mp 286-288°C (decomp.); 1H NMR (300MHz, /-DMSO) δ 3.75 (6H, br, s, OCH3), 6.08 (4H, br s, NH + OH), 6.80 (2H, br, s, C8-H), 7.58 (4H, br, s, C4-H + C5-H), 8.00 (4H, br, s, Cι2-H), 8.27 (4H, br, s, Cπ-H); 13C NMR (75MHz, /- DMSO) δ 52.2 (Ci), 115.4 (C8), 116.6 (C5), 124.8 (C4), 127.9 (C3), 129.1 (Cι2), 129.6 (Cu), 131.6 (C6), 136.1 (Cio), 144.4 (Cι3), 146.5 (C7), 164.9 (C9), 166.5 (C2); ES" m/z 538.9 (M"); high resolution ES+ m/z found 558.1878 C3oH28N3O8 (M+NH4) requires 558.1876; (Anal. Calcd. for C30H24N2O8 : C, 66.66; H, 4.48; N, 5.18%. Found: C, 66.47; H, 4.42; N, 5.15%). Bis-methyl4-({[4'-(aminocarbonyl)-l,r-biphenyl-4-yl]carbonyl}amino)-3-hydroxybenzoate ( SP677) (alternative procedure)
A mixture of the amine (0.438g, 2.15mmol) and triethylamine (0.45ml, 3.22mmol) dissolved in anhydrous THF (15ml) was cooled to 0°C in an ice bath. After 10 minutes at 0°C, the acid chloride (0.300g, 1.07mmol) was added dropwise to the reaction mixture. A precipitate was seen almost immediately. The mixture was left stirring at room temperature for 18 hours. Water (50ml) was added to the reaction mixture. The organic product was extracted with ethyl acetate (2x40ml). The organic phase was separated and washed with 2N HCl (50ml), NaHCO3 (50ml) and brine (50ml). The organic solvent phase was then dried (MgSO4) and concentrated in vacuo. Column chromatography (1:1 ethyl acetate:hexane) afforded the product WSP677 as a slightly off-white solid. Yield 0.080g (0.15mmol, 14%); Characterisation data was identical to the data obtained with the first experimental procedure.
Methyl3-[(4-{[(2-methoxy-5-(methoxycarbonyl)phenyl)amino]carbonyl}benzoyl)amino]-4- methoxybenzoate (WSP904)
This compound was made via an identical preparation to the preparation of SP677 (alternative procedure). Methyl-3-amino-4-methoxybenzoate (0.892g, 4.92mmol) and triethylamine (1.1ml, 7.38mmol) was reacted with terephthaloyl chloride (0.500g, 2.46mmol) in anhydrous THF (10ml). An identical work-up procedure was employed, the pure product WSP904 was isolated as an off-white solid.
Yield 1.006g (2.04mmol, 83%); mp 266-268°C; 1H NMR (300MHz, /-DMSO) δ 3.89 (6H, s, C7-H), 3.98 (6H, s, d-H), 7.27 (2H, d, J = 8.6Hz, C5-H), 7.88 (2H, d, J = 8.7Hz, C4-H), 8.15 (4H, s, Ci2-H), 8.48 (2H, s, C9-H), 9.85 (2H, s, NH); 13C NMR (75MHz, /- DMSO) δ 52.3 (Ci), 56.5 (C7), 111.7 (C5), 121.8 (C9), 125.7 (C8), 128.1 (Cι2), 129.9 (C4), 137.2 (Cu), 155.8 (C6), 164.9 (Cio), 166.1 (C2); (Anal. Calcd. for C26H24N2O8 + 0.2H2O: C, 62.95; H, 4.96; N, 5.65%. Found C, 62.83; H, 4.95; N, 5.55%) Bis-methyl 4-({[4'-(aminocarbonyl)-l, 1 '-biphenyl-4-yl]carbonyl}amino)-2- methoxybenzoate (WSP905)
This compound was made via an identical preparation to the preparation of WSP677 (alternative procedure). Methyl-4-amino-2-methoxybenzoate (0.260g, 1.43mmol) and triethylamine (0.2ml, 1.42mmol) was reacted with 4-4'-biphenyldicarboxyl chloride (0.200g, 0.71mmol) in anhydrous THF (10ml). An identical work-up procedure was employed, the pure product WSP905 was isolated as an off-white solid. Yield 0.186g (0.33mmol, 46%); mp 280-283°C; 1H NMR (300MHz, /-DMSO) δ 3.77 (6H, s, d-H), 3.84 (6H, s, Ci-H), 7.55 (2H, d, J = 6.0Hz, C8-H), 7.74 (4H, m, C6-H + C9- H), 7.98 (4H, d, J = 6.9Hz, Cι3-H), 8.13 (4H, d, J = 6.9Hz, Cι2-H), 10.58 (2H, s, NH); 13C NMR (75MHz, /-DMSO) δ 51.9 (Ci), 55.9 (C5), 103.9 (C6), 111.6 (C3), 114.5 (C8), 127.3 (C13), 128.9 (Cι2), 130.3 (C9), 132.4 (Cu), 134.2 (Cu), 142.5 (C7), 144.6 (C4), 159.6 (Cio), 165.7 (C2); El m/z 568.3 (M "); high resolution El m/z found 568.1842 C32H28N2O8 requires 568.1840; (Anal. Calcd. for C32H28N2O8 + 0.5H2O : C, 66.54; H, 5.06; N, 4.85%. Found: C, 66.49; H, 4.91; N, 4.67%).
Methyl3-[(4-{ f(3-(methoxycarbonyl)-5 ' (methoxy carbonyl)phenyl) amino) ' carbonyljbenzoyl) amino] -5-(methoxycarbonyl)benzoate (WSP906)
This compound was made via an identical preparation to the preparation of WSP677 (alternative procedure). Dimethyl-5-aminoisophthalate (1.030g, 4.92mmol) and triethylamine (1.1ml, 7.38mmol) was reacted with terephthaloyl chloride (0.500g,
2.46mmol) in anhydrous THF (15ml). An identical work-up procedure was employed, the pure product WSP906 was isolated as an white solid.
Yield 1.266g (2.31mmol, 94%); mp 303-306°C; 1H NMR (300MHz, CDC13) δ 3.92 (12H, s, OMe), 8.15 (4H, s, Ci-H), 8.22 (2H, s, C7-H), 8.74 (4H, s, C5-H), 10.80 (2H, s, NH); 13C
NMR could not be done due to insolubility of compound; (Anal. Calcd. for C28H24N2Oι0 + lH2O: C, 59.36; H, 4.63; N, 4.94%. Found C, 59.15; H, 4.57; N, 4.80%). 3-[(4-{[(3-carboxy-5-carboxyphenyl)amino]carbonyl}benzoyl)amino]-5-carboxybenzoic acid (WSP907)
An identical procedure to the one employed for WSP678 was used. WSP906 (0.250g, 0.456mmoι) was reacted with aqueous NaOH (0.291g, 0.729mmol) in water (4ml) and the product WSP907 was isolated as a light grey solid.
Yield 0.172g (0.35mmol, 77%); mp 320°C (decomp.); 1H NMR (300MHz, CDC13) δ 8.17 (4H, s, Ci-H), 8.24 (2H, s, C7-H), 8.68 (4H, s, C5-H), 10.79 (2H, s, NH), 13.31 (4H, br, s, CO2H); 13C NMR (75MHz, /-DMSO) δ 125.1 (C5), 125.5 (C7), 128.2 (Ci), 132.0 (C6), 137.4 (C2), 140.0 (C4), 165.4 (C8), 166.8 (C3); (Anal. Calcd. for C24Hι6N2Oι0 + 2HC1 + 2H2O: C, 47.94; H, 3.69; N, 4.66%. Found C, 47.99; H, 3.49; N, 4.41%)
Methyl4-[(4-{[(4-(methoxycarbonyl)phenyl)amino]carbonyl}benzoyl)amino]benzoate
(WSP908)
This compound was made via an identical preparation to the preparation of WSP677 (alternative procedure). Methyl-4-aminobenzoate (0.55 lg, 2.95mmol) and triethylamine
(1.1ml, 7.38mmol) was reacted with terephthaloyl chloride (0.300g, 1.48mmol) in anhydrous THF (10ml). An identical work-up procedure was employed, the pure product
WSP908 was isolated as a white solid.
Yield 0.303g (0.70mmol, 47%); mp 340-343°C; 1H NMR (300MHz, /-DMSO) δ 3.85 (3H, s, OMe), 7.99 (8H, m, C5-H + C6-H), 8.12 (4H, s, Ci-H), 10.74 (2H, s, NH); 13C
NMR (75MHz, /-DMSO) δ 52.3 (C9), 120.0 (C5), 124.8 (C7), 128. (C6), 130.5 (Ci), 137.6
(C2), 143.7 (C ), 165.6 (C3), 166.1 (C8); (Anal. Calcd. for C24H2oN2O6 : C, 66.66; H, 4.66;
N, 6.48%. Found C, 66.50; H, 4.69; N, 6.31%)
Bis-N,N'-(2-hydroxyphenyl)-l , 1 '-biphenyl-4,4'-dicarboxamide (WSP912)
This compound was made via an identical preparation to the preparation of WSP677 (alternative procedure). 2-aminophenol (0.234g, 2.15mmol) and triethylamine (0.45ml, 3.21mmol) was reacted with 4-4'-biphenyldicarboxyl chloride (0.300g, 1.07mmol) in anhydrous THF (10ml). An identical work-up procedure was employed, the pure product WSP912 was isolated as an off-white solid.
Yield 0.065g (0.153mmol, 14%); mp 260-263°C (decomp.); 1H NMR (300MHz, /- DMSO) δ 6.87 (2H, t, J = 7.5Hz, C2-H), 6.95 (2H, d, J = 7.9Hz, C3-H), 7.07 (2H, t, J = 7.5Hz, C4-H), 7.71 (2H, d, J = 7.9Hz, C5-H), 7.95 (4H, d, J = 8.2Hz, C9-H), 8.12 (4H, d, J = 8.2Hz, Cio-H), 9.64 (2H, s, NH), 9.78 (2H, br, s, OH); 13C NMR (75MHz, /-DMSO) δ 116.3 (C2), 119.4 (C4), 124.7 (C5), 126.2 (C6), 127.3 (C3), 128.7 (Cio), 134.1 (C9), 142.3 (C8), 149.9 (Cu), 159.9 (Ci), 165.1 (C7); (Anal. Calcd. for C26H2oN2O4 : C, 73.57; H, 4.75; N, 6.60%. Found C, 73.37; H, 4.73; N, 6.11%).
N,N'-diphenyl-l, 1 '-biphenyl-4, 4 '-dicarboxamide (WSP916)
To a 25ml round bottomed flask was added 4-4'-biphenyldicarboxyl chloride (0.300g, 1.07mmol) and aniline (0.2ml, 2.15mmol) in anhydrous THF (15ml). After stirring for lhr at room temperature, a solid crashed out. This was filtered and washed with water (30ml) and hexane (20ml) to leave the product SP916 as a white solid.
Yield 0.202g (0.51mmol, 48%); mp >360°C (decomp.); 1H NMR (300MHz, /-DMSO) δ 7.12 (2H, m, C9-H), 7.35 (4H, m, C8-H), 7.80 (4H, d, J = 7.7Hz, C7-H), 7.93 (4H, d, J = 8.3Hz, C2-H), 8.09 (4H, d, J = 8.3Hz, C3-H), 10.34 (2H, s, NH); 13C NMR could not be done due to insolubility; (Anal. Calcd. for C26H20N2θ2 + 0.5HC1: C, 76.04; H, 5.03; N, 6.82%. Found C, 76.02; H, 4.84; N, 6.57%).
N,N'-diphenylterephthalamide (WSP917)
An identical procedure to the one employed for WSP916 was carried out. Aniline (0.5ml, 4.92mmol) and terephthaloyl chloride (0.500g, 2.46mmoι) were reacted in anhydrous THF (20ml). The product WSP917 was isolated as a white solid.
Yield 0.719g (2.27mmol, 92%); mp 340-342°C; 1H NMR (300MHz, /-DMSO) δ 7.14 (2H, t, J = 7.4Hz, Ci-H), 7.39 (4H, t, J = 7.9Hz, C2-H), 7.81 (4H, d, J = 7.9Hz, C3-H), 8.10 (4H, s, C7-H), 10.42 (2H, s, NH); 13C NMR (75MHz, /-DMSO) δ 120.8 (C3), 124.2 (Ci), 128.1 (C7), 129.0 (C2), 137.8 (C6), 139.3 (C4), 165.2 (C5); (Anal. Calcd. for C20Hι6N2O2 : C, 75.93; H, 5.10; N, 8.85%. Found C, 75.92; H, 5.12; N, 8.86%).
Bis-4-({[4 '-(aminocarbonyl)- 1, 1 '-biphenyl-4-yl]carbonyl}amino)-2-methoxybenzoic acid (WSP971)
An identical procedure to the one employed for WSP678 was used. WSP905 (0.1 OOg, 0.176mmol) was reacted with aqueous NaOH (0.070g, 1.76mmol) in water (1ml) and THF (5ml). The product WSP971 was isolated as a brown solid.
Yield 0.030g (0.05mmol, 32%); mp 280°C (decomp.); 1H NMR (300MHz, /-DMSO) δ 3.84 (6H, s, C4-H), 7.54 (2H, d, J = 8.6Hz, C7-H), 7.86-8.16 (12H, m, C5-H + C8-H + Cii-H + Ci2-H), 10.60 (2H, s, NH), 12.70 (2H, br, s, CO2H); 13C NMR (75MHz, /- DMSO) δ 55.9 (C4), 104.0 (C5), 111.6 (C2), 115.6 (C7), 127.3 (C12), 127.5 (Cu), 130.6 (C8), 132.5 (Cio), 142.5 (C13), 144.3 (C6), 159.6 (C3), 165.7 (C9), 167.4 (Ci).
N,N'-bis-(2-hydroxy-4-nitrophenyl)terephthalamide (WSP974)
This compound was made via an identical preparation to the preparation of WSP677 (alternative procedure), with the exception of adding DMAP as a catalyst (0.009g, 0.689mmol). 2-amino-5-nitrophenol (1.062g, 6.89mmol) and triethylamine (0.45ml, 3.21mmol) was reacted with terephthaloyl chloride (0.700g, 3.44mmol) in anhydrous THF (15ml). An identical work-up procedure was employed, the pure product WSP974 was isolated as an orange solid.
Yield 1.362g (3.1mmol, 90%); mp 283-286°C; 1H NMR (300MHz, /-DMSO) δ 7.71 (2H, s, C2-H), 7.73 (2H, d, J = 9.4Hz, C5-H), 8.08 (4H, s, C9-H), 8.26 (2H, d, J = 9.4Hz, C6-H), 9.86 (2H, br, s, OH); 13C NMR (75MHz, /-DMSO) δ 109.8 (C2), 114.0 (C6), 121.4 (C5), 128.2 (C9), 133.6 (C4), 137.2 (C8), 143.9 (Ci), 150.4 (C3), 164.7 (C7); (Anal. Calcd. for C20Hι4N4O8 + 0.4H2O + 0.4Et3N: C, 55.36; H, 4.31; N, 12.68%. Found C, 55.49; H, 4.57; N, 12.37%). Bis-N,N'-(2-hydroxy-4-nitrophenyl)-l, 1 '-biphenyl-4,4'-dicarboxamide (WSP978) This compound was made via an identical preparation to the preparation of WSP677 (alternative procedure). 2-amino-5-nitrophenol (0.331g, 2.15mmol) and triethylamine (0.45ml, 3.21mmol) was reacted with 4-4'-biphenyldicarboxyl chloride (0.300g, 1.07mmol) in anhydrous THF (10ml). An identical work-up procedure was employed, the pure product WSP978 was isolated as an orange/red solid.
Yield 0.406g (0.79mmol, 74%); mp 302-304°C; 1H NMR (300MHz, /-DMSO) δ 6.81 (2H, s, C2-H), 6.87 (2H, d, J = 9.0Hz, C5-H), 7.98 (2H, d, J = 9.0Hz, C6-H), 8.05 (4H, d, J = 8.2Hz, Cio-H), 8.30 (4H, d, J = 8.2Hz, C9-H); 13C NMR (75MHz, /-DMSO) δ 119.9 (C2), 114.3 (C6), 120.2 (C5), 127.6 (Cio), 128.9 (C9), 131.4 (C4), 134.9 (C8), 135.4 (Cu), 144.1 (Ci), 148.7 (C3), 164.4 (C7); (Anal. Calcd. for C26Hι8N4O8 + 0.3H2O: C, 60.07; H, 3.61; N, 10.78%. Found C, 59.91; H, 3.63; N, 10.57%).
Bis-methyl4-({[4'-(aminocarbonyl)-l,l '-biphenyl-4-yl]carbonyl}amino)-2-hydroxybenzoate (WSP979)
This compound was made via an identical preparation to the preparation of WSP677 (alternative procedure). Methyl-4-amino-2-hydroxybenzoate (0.238g, 1.43mmol) and triethylamine (0.30ml, 2.13mmol) was reacted with 4-4'-biphenyldicarboxyl chloride (0.200g, 0.71mmol) in anhydrous THF (10ml). An identical work-up procedure was employed, the pure product WSP979 was isolated as an white solid.
Yield 0.230g (0.43mmol, 60%); mp 246-250°C; 1H NMR (300MHz, /-DMSO) δ 3.85 (6H, s, Ci-H), 7.39 (2H, d, J = 8.3Hz, C7-H), 7.87-8.35 (12H, m, C5-H + C8-H + Cn-H + C12-H); 10.75 (2H, s, NH), 10.90 (2H, br, s, OH); 13C NMR (75MHz, /-DMSO) δ 51.9 (Ci), 106.9 (C5), 111.8 (C7), 115.0 (C3), 127.3 (C]2), 127.6 (Cu), 130.3 (C8), 131.4 (Cio), 144.9 (C13), 151.3 (C6), 156.4 (C4), 163.2 (C ), 166.0 (C2); (Anal. Calcd for C30H24N2O8 + 0.4H2O: C, 65.79; H, 4.56; N, 5.11%. Found C, 66.17; H, 4.54; N, 4.67%). Bis-methyl5-({[4'-(aminocarbonyl)-l,l '-biphenyl-4-yl]carbonyl}amino)-2-h.ydroxybenzoate (WSP1009)
This compound was made via an identical preparation to the preparation of WSP677 (alternative procedure). Methyl-3-amino-5-hydroxybenzoate (0.357g, 2.15mmol) and triethylamine (0.45ml, 3.21mmol) was reacted with 4-4'-biphenyldicarboxyl chloride (0.300g, 1.07mmol) in anhydrous THF (10ml). An identical work-up procedure was employed, the pure product WSP1009 was isolated as a white solid. Yield 0.294g (0.54mmol, 51%); mp 290-292°C; 1H NMR (300MHz, /-DMSO) δ 3.93 (6H, s, Ci-H), 7.03 (2H, d, J = 8.9Hz, C5-H), 8.11 (4H, d, J = 7.4Hz, Cn-H), 7.94 (6H, m, C6-H + C12-H), 8.35 (2H, s, C8-H), 10.37 (4H, s, OH + NH); 13C NMR (75MHz, /- DMSO) δ 52.9 (Ci), 112.8 (C5), 117.8 (C3), 121.8 (C8), 127.2 (C6), 128.7 (C12), 128.9 (Cu), 131.4 (C7), 134.8 (Cio), 142.3 (C13), 156.7 (C4), 165.1 (C9), 169.4 (C2); El m/z 540.2; high resolution El m/z found 541.1605 C3oH25N2O8 (M+H) requires 541.1604; (Anal. Calcd. for C30H24N2O8 + 0.3H2O: C, 66.00; H, 4.54; N, 5.13%. Found C, 65.96; H, 4.40; N, 4.91%).
N,N'-bis-(4-cyanophenyl)terephthalamide (WSP1012)
This compound was made via an identical preparation to the preparation of SP677
(alternative procedure). 4-aminobenzonitrile (0.349g, 2.95mmol) and triethylamine (0.6ml, 4.44mmol) was reacted with terephthaloyl chloride (0.300g, 1.48mmol) in anhydrous THF (20ml). An identical work-up procedure was employed, the pure product WSP1012 was isolated as a white solid.
Yield 0.351g (0.96mmol, 65%); mp 343-346°C; 1H NMR (300MHz, /-DMSO) δ 7.86 (4H, d, J = 8.8Hz, C3-H), 8.03 (4H, d, J = 8.8Hz, C4-H), 8.13 (4H, s, C8-H), 10.82 (2H, s, NH); 13C NMR (75MHz, /-DMSO) δ 106.0 (C2), 119.4 (Ci), 120.7 (C4), 128.4 (C8), 133.6 (C3), 137.6 (C7), 143.6 (C5), 165.8 (C6); (Anal. Calcd. for C22Hι4N4O2 + 0.2Et3N + 0.1H2O: C, 71.40; H, 4.42; N, 15.07%. Found C, 71.70; H, 4.18; N, 14.71%). Bis-N,N'-(4-cyanophenyl)-l, 1 '-biphenyl-4,4'-dicarboxamide (WSP1013) This compound was made via an identical preparation to the preparation of WSP677 (alternative procedure). 4-aminobenzonitrile (0.254g, 2.15mmol) and triethylamine (0.5ml, 3.21ιnmol) was reacted with terephthaloyl chloride (0.300g, 1.07mmol) in anhydrous THF (20ml). An identical work-up procedure was employed, the pure product WSP1013 was isolated as a white solid.
Yield 0.300g (0.68mmol, 63%); mp 335-338°C; 1H NMR (300MHz, /-DMSO) δ 7.84- 8.13 (16H, m, C3-H + C4-H + C9-H + C10-H), 10.74 (2H, s, NH); 13C NMR (75MHz, /- DMSO) δ 105.8 (C2), 120.6 (Ci), 127.5 (C4), 129.0 (C9), 130.4 (C8), 133.5 (C3), 134.1 (C7), 142.7 (Cio), 143.8 (C5), 166.1 (C6); (Anal. Calcd. for C28Hι8N4O2 + 0.6H2O: C, 74.19; H, 4.27; N, 12.36%. Found C, 74.07; H, 4.20; N, 10.60%).
Synthesis of Imines
Methyl3-hydroxy-4-({(lZ)-[4-((Z)-{[2-hydroxy-4-(methoxycarbonyl)phenyl]imino}methyl) phenyl] methylene}amino)benzoate (WSP714)
To an oven dried 50ml round bottomed flask was added terephthalaldehyde (0.200g, 1.49mmol) and methyl-4-amino-3-hydroxy-benzoate (0.498g, 2.98mmol). Ethanol (15ml) was added and the reaction mixture heated to 55°C for 5 hours under a nitrogen atmosphere. The precipitate which forms was filtered tlirough a sinter funnel and washed with ethanol (40ml). The precipitate, the product WSP714, was isolated as a yellow powder and dried under high vacuum. No further purification was necessary. Yield 0.491g (1.13mmol, 76%); mp 263-266°C; 1H NMR (300MHz, /-DMSO) δ 3.84 (6H, s, Ci-H), 7.26 (2H, d, J = 8.1Hz, C7-H), 7.50 (4H, m, C4-H + C8-H), 8.16 (4H, s, Cn- H), 8.78 (2H, s, C9-H), 9.68 (2H, br, s, OH); 13C NMR (75MHz, /-DMSO) δ 52.4 (Ci), 116.9 (C4), 120.4 (C7), 121.1 (C8), 128.3 (Cu), 129.6 (C3), 138.9 (Cio), 143.1 (C6), 150.9 (C5), 161.6 (C9), 166.2 (C2); (Anal. Calcd. for C24H20N2O6 + 0.1H2O : C, 66.38; H, 4.69; N, 6.45%. Found C, 66.25; H, 4.36; N, 6.26%). Methyl 2~hydroxy-4-({(lZ)-[4-((Z)-{[3-hydroxy-4-(phenoxycarbonyl)phenyl]imino}methyl) phenyl] methylene}amino)benzoate (WSP715)
An identical procedure to the one employed for WSP714 was used. Terephthalaldehyde (0.500g, 3.72mmol) and phenyl-4-aminosaιicylate (1.708g, 7.45mmol) was reacted in ethanol (25ml). The product WSP715 was isolated as a yellow solid.
Yield 0.291g (0.52mmol, 14%); mp 185-186°C; 1H NMR (300MHz, /-DMSO) δ 6.92 (2H, m, C8-H + Cio-H), 7.35 (3H, m, d-H + C3-H), 7.50 (2H, m, C2-H), 8.06 (1H, d, J = 8.3Hz, Cii-H), 8.14 (2H, s, Cι4-H), 8.76 (1H, s, C12-H), 10.5 (1H, br s, OH); 13C NMR (75MHz, /-DMSO) δ 109.4 (C8), 110.7 (Cio), 113.4 (C6), 122.3 (C3), 126.5 (Ci), 129.8 (C2), 129.9 (CM), 132.3 (Cu), 138.7 (C13), 150.5 (C4), 158.5 (C9), 161.7 (C7), 162.7 (C12), 167.0 (C5); (Anal. Calcd. for C34H24N2O6 : C, 73.37; H, 4.35; N, 5.03%. Found C, 73.32; H, 4.29; N, 5.07%).
Methyl4-methoxy-3-({(lZ)-[4-((Z)-{[2-methoxy-5-(methoxycarbonyl)phenyl]imino} methyl)phenyl] methylene} amino)benzoate (WSP716)
An identical procedure to the one employed for WSP714 was used. Terephthalaldehyde (0.500g, 3.72mmol) and methyl-3-amino-4-methoxybenzoate (1.350g, 7.45mmol) was reacted in ethanol (25ml). The product WSP716 was isolated as a yellow solid. Yield 0.764g (1.65mmol, 45%); mp 182-183°C; 1H NMR (300MHz, CDC13) δ 3.97 (6H, s, C7-H), 4.03 (6H, s, Ci-H), 7.03 (2H, d, J = 8.6Hz, C5-H), 7.78 (2H, d, J = 2.1Hz, C9-H), 7.99 (2H, dd, J = 2.1Hz and 8.6Hz, C4-H), 8.10 (4H, s, C12-H), 8.63 (2H, s, Cio-H); 13C could not be carried out due to solubility problems; (Anal. Calcd. for C26H24N2O6 + 0.2H2O : C, 67.29; H, 5.30; N, 6.04%. Found C, 67.20; H, 5.45; N, 6.01%).
4-{[(lZ)-(4-{(Z)-[(4-carboxyphenyl)imino]methyl}phenyl)methylene]amino}benzoic acid (WSP738)
An identical procedure to the one employed for WSP714 was used. Terephthalaldehyde (0.300g, 2.23mmol) and 4-aminobenzoic acid (0.628g, 4.58mmol) was reacted in ethanol (20ml). The product WSP738 was isolated as a yellow solid. Yield 0.647g (1.73mmol, 78%); mp >360°C; 1H NMR (300MHz, /-DMSO) δ 7.37 (4H, d, J = 8.5Hz, C4-H), 8.00 (4H, d, J = 8.5Hz, C3-H), 8.12 (4H, s, C8-H), 8.74 (2H, s, C6-H), 12.93 (2H, br, s, CO2H); 13C NMR (75MHz, /-DMSO) δ 121.5 (C4), 128.6 (C8), 129.7 (C2), 130.9 (C3), 138.8 (C7), 155.5 (C5), 162.2 (C6), 167.3 (Ci); (Anal. Calcd. for C22Hι6N2O4 + 0.1H2O : C, 70.62; H, 4.36; N, 7.49%. Found C, 70.76; H, 4.39; N, 7.20%).
4-({[(lZ)-(4-{(Z)-[(4-carboxybenzyl)imino]methyl}phenyl)methylene]amino}methyl) benzoic acid (WSP739)
An identical procedure to the one employed for WSP714 was used. Terephthalaldehyde (0.250g, 1.86mmol) and 4-(aminomethyl)benzoic acid (0.563g, 3.72mmol) was reacted in ethanol (15ml). The product WSP739 was isolated as a white solid.
Yield 0.475g (1.19mmol, 64%); mp >235°C (decomp.); 1H NMR (300MHz, /-DMSO) δ 4.88 (4H, s, C6-H), 7.46 (4H, d, J = 8.2Hz, C4-H), 7.89 (4H, s, C9-H), 7.92 (4H, d, J =
8.2Hz, C3-H), 8.59 (2H, s, C7-H), 12.95 (2H, br, s, CO2H); 13C NMR (75MHz, /-DMSO) δ 63.8 (C6), 128.2 (C2), 128.7 (C4), 129.7 (C9), 129.8 (C3), 138.2 (C8), 144.8 (C5), 162.4
(C7), 167.6 (Ci); (Anal. Calcd. for C24H2oN2O4 + 0.2H2O : C, 71.35; H, 5.09; N, 6.93%.
Found C, 71.45; H, 5.03; N, 6.81%).
3-{[(lZ)-(4-{(Z)-[(5-carboxy-2-methoxyphenyl)imino]methyl}phenyl)methylene]amino}-4- methoxybenzoic acid (WSP741)
An identical procedure to the one employed for WSP714 was used. Terephthalaldehyde (0.080g, 0.596mmol) and 3-amino-4-methoxybenzoic acid (0.192g, 1.19mmol) was reacted in ethanol (10ml). The product WSP741 was isolated as a yellow solid. Yield 0.216g (0.50mmol, 84%); mp >335°C (decomp.); 1H NMR (300MHz, /-DMSO) δ 3.90 (6H, s, C6-H), 7.20 (2H, d, J = 8.6Hz, C4-H), 7.62 (2H, d, J = 2.0Hz, C8-H), 7.85 (2H, dd, J = 2.0Hz and 8.6Hz, C3-H), 8.09 (4H, s, Cn-H), 8.69 (2H, s, C9-H), 12.76 (2H, br, s, CO2H); 13C NMR (75MHz, /-DMSO) δ 56.3 (C6), 112.0 (C4), 121.2 (C2), 123.6 (C8), 129.5 (Cu), 138.8 (C3), 141.2 (Cio), 156.0 (C7), 161.8 (C5), 167.3 (C9), 172.3 (Ci); (Anal. Calcd. for C24H20N2O6 + 1H20 : C, 63.99; H, 4.92; N, 6.22%. Found C, 63.90; H, 4.42; N, 6.10%).
5-{[(lZ)-(4-{(Z)-[(3-carboxy-4-lιydroxyplιenyl)imino]methyl}phenyl)methylene]amino}-2- hydroxybenzoic acid (WSP742)
An identical procedure to the one employed for WSP714 was used. Terephthalaldehyde (0.200g, 1.49mmol) and 5-aminosalicylic acid (0.456g, 2.98mmol) was reacted in ethanol (10ml). The product WSP742 was isolated as a grey solid. Yield 0.400g (0.99mmol, 66%); mp >360°C; 1H NMR (300MHz, /-DMSO) δ 7.03 (2H, d, J = 8.8Hz, C4-H), 7.60 (2H, dd, J = 2.7Hz and 8.8Hz, C5-H), 7.78 (2H, d, J = 2.7Hz, C7- H), 8.06 (4H, s, Cio-H), 8.78 (2H, s, C8-H); 13C NMR (75MHz, /-DMSO) δ 113.7 (C3), 118.3 (C2), 122.8 (C7), 129.1 (C5), 129.4 (Cio), 138.7 (C9), 142.6 (C6), 158.7 (C3), 160.3 (C8), 172.0 (Ci); (Anal. Calcd. for C22H16N2O6 + 0.5H2O : C, 63.92; H, 4.15; N, 6.78%. Found C, 63.93; H, 4.13; N, 6.74%).
4-{[(lZ)-(4-{(Z)-[(4-carboxy-2-hydroxyphenyl)imino]?nethyl}phenyl)methylene]amino}-3- hydroxybenzoic acid (WSP760)
An identical procedure to the one employed for WSP714 was used. Terephthalaldehyde (0.1 OOg, 0.74mmol) and 4-amino-3 -hydroxybenzoic acid (0.239g, 1.56mmol) was reacted in ethanol (10ml). The product WSP760 was isolated as a brown solid.
Yield 0.220g (0.54mmol, 74%); mp >360°C; 1H NMR (300MHz, /-DMSO) δ 7.24 (2H, d, J = 8.1Hz, C6-H), 7.44 (4H, m, C3-H + C7-H), 8.17 (4H, s, Cι0-H), 8.79 (2H, s, C8-H), 9.57 (2H, br, s, OH), 12.79 (2H, br, s, CO2H); 13C NMR (75MHz, /-DMSO) δ 117.1 (C3), 120.2 (C6), 121.2 (C7), 129.6 (Cio), 129.8 (C2), 138.9 (C9), 142.7 (C5), 150.8 (C4), 161.3 (C8), 167.4 (Ci); (Anal. Calcd. for C22Hι6N2O6 + 0.2H2O : C, 64.77; H, 4.05; N, 6.87%. Found C, 64.88; H, 4.02; N, 6.73%). Bis-methyl 3-{[(lZ)-l,l'-biphenyl-4-ylmethylene]amino}-4-methoxybetιzoate (WSP761) An identical procedure to the one employed for WSP714 was used. 4-4'-biphenyl- dialdehyde (0.1 OOg, 0.47mmol) and methyl-3-amino-4-methoxybenzoate (0.181g, 0.99mmol) was reacted in ethanol (5ml). The product WSP761 was isolated as a yellow solid.
Yield 0.035g (0.065mmol, 14%); mp 188-190°C; 1H NMR (300MHz, /-DMSO) δ 3.85 '(6H, s, C7-H), 3.91 (6H, s, Ci-H), 7.22 (2H, d, J = 8.6Hz, C5-H), 7.62 (2H, d, J - 2.0Hz, C9-H), 7.87 (2H, dd, J - 2.0Hz and 8.6Hz, C4-H), 7.96 (4H, d, J = 8.3Hz, C13-H), 8.08 (4H, d, J = 8.3Hz, C12-H), 8.67 (2H, s, Cio-H); 13C NMR (75MHz, /-DMSO) δ 52.3 (Ci), 56.3 (C7), 112.1 (C5), 120.9 (C3), 122.4 (C9), 127.6 (C13), 128.7 (C4), 129.9 (C12), 135.9 (Cu), 141.6 (C8), 142.4 (CM), 156.3 (C6), 162.1 (Cio), 166.3 (C2); (Anal. Calcd. for C32H28N2O6 : C, 71.63; H, 5.26; N, 5.22%. Found C, 71.50; H, 5.21; N, 5.15%).
Bis-methyl 4-{[(lZ)-l,l '-biphenyl-4-ylmethylene]amino}-3-hydroxybenzoate (WSP794) An identical procedure to the one employed for WSP714 was used. 4-4'-biphenyl- dialdehyde (0.044g, 0.209mmol) and methyl-4-amino-3-hydroxybenzoate (0.070g, 0.418mmol) was reacted in ethanol (10ml). The product WSP794 was isolated as a yellow solid.
Yield 0.048g (0.094mmol, 45%); mp 221°C (decomp.); 1H NMR (300MHz, /-DMSO) δ 3.85 (6H, s, Ci-H), 7.27 (2H, d, J = 7.9Hz, C7-H), 7.48-7.51 (4H, m, C4-H + C8-H), 7.98 (4H, d, J = 8.1Hz, C12-H), 8.15 (4H, d, J = 8.1Hz, Cn-H), 8.78 (2H, s, C9-H), 9.63 (2H, s, OH); 13C NMR (75MHz, /-DMSO) δ 51.6 (Ci), 112.8 (C4), 114.8 (C7), 116.5 (C8), 122.9 (Cu), 128.3 (Cu), 130.6 (Cio), 136.2 (C3), 142.7 (d3), 143.1 (C6), 144.7 (C5), 160.1 (C9), 166.8 (C2); (Anal. Calcd. for C3oH24N2O6 : C, 70.86; H, 4.76; N, 5.51%. Found C, 70.57; H, 4.62; N, 4.74%).
Bis-4-{[(lZ)-l,l'-biphenyl-4-ylmethylene]amino}-3-hydroxybenzoic acid (WSP795)
An identical procedure to the one employed for WSP714 was used. 4-4'-biphenyl- dialdehyde (0.060g, 0.285mmol) and 4-amino-3 -hydroxybenzoic acid (0.087g, 0.570mmol) was reacted in ethanol (10ml). The product SP795 was isolated as a yellow solid.
Yield 0.039g (0.081mmol, 28%); mp >360°C; 1H NMR (300MHz, /-DMSO) δ 7.25 (2H, d, J = 8.0Hz, C6-H), 7.45-7.50 (4H, m, C3-H + C7-H), 7.97 (4H, d, J = 7.9Hz, di-H), 8.16 (4H, d, J = 7.9Hz, Cio-H), 8.78 (2H, s, C8-H), 9.53 (2H, s, OH); 13C NMR (75MHz, /- DMSO) δ 112.8 (C3), 115.1 (C6), 117.8 (C7), 122.9 (Cu), 128.3 (Cio), 130.6 (C9), 136.1 (C2), 142.3 (Cι2), 143.1 (C5), 144.7 (C4), 160.1 (C8), 168.0 (Ci); (Anal. Calcd. for C28H2oN2O6 : C, 69.99; H, 4.20; N, 5.83%. Found C, 70.34; H, 4.46; N, 5.23%).
2~{[(lZ)-(4-{(Z)-[(2-hydroxyphenyl)imino]methyl}phenyl)methylene]amino}phenol (WSP909)
An identical procedure to the one employed for WSP714 was used. Terephthalaldehyde (0.300g, 2.23mmol) and 2-aminophenol (0.488g, 4.47mmol) was reacted in ethanol (15ml). The product WSP909 was isolated as a yellow solid. Yield 0.387g (1.22mmol, 55%); mp 188-190°C; 1H NMR (300MHz, /-DMSO) δ 6.84- 6.93 (4H, m, C2-H + C3-H), 7.12 (2H, t, J = 6.8Hz, C4-H), 7.27 (2H, d, J = 6.8Hz, C5-H), 8.17 (4H, s, C9-H), 8.81 (2H, s, C7-H), 9.11 (2H, s, OH); 13C NMR (75MHz, /-DMSO) δ 116.5 (C2), 119.5 (C4), 119.9 (C5), 128.1 (C3), 129.4 (C9), 137.9 (C8), 138.9 (C6), 151.8 (Ci), 160.0 (C7); (Anal. Calcd. for C20Hι6N2O2 : C, 75.93; H, 5.10; N, 8.85%. Found C, 75.61; H, 4.97; N, 8.86%).
4-{[(lZ)-(4-{(Z)-[(4-hydroxyphenyl)imino]methyl}phenyl)methylene]amino}phenol (WSP910)
An identical procedure to the one employed for SP714 was used. Terephthalaldehyde (0.300g, 2.33mmol) and 4-aminophenol (0.496g, 4.47mmol) was reacted in ethanol (15ml). The product WSP910 was isolated as a yellow solid.
Yield 0.244g (0.77mmol, 33%); mp 260-262°C; 1H NMR (300MHz, /-DMSO) δ 6.81 (4H, d, J = 8.6Hz, C2-H), 7.25 (4H, d, J = 8.6Hz, C3-H), 8.01 (4H, s, C7-H), 8.69 (2H, s, C5-H), 9.58 (2H, s, OH); 13C NMR (75MHz, /-DMSO) δ 116.1 (C2), 123.0 (C3), 128.9 (C7), 138.7 (C6), 142.7 (C4), 156.7 (Ci), 160.2 (C5); (Anal. Calcd for C20Hι6N2O2 + 0.1H2O : C, 75.50; H, 5.13; N, 8.80%. Found C, 75.20; H, 4.96; N, 8.68%).
2-{[(lZ)-(4'-{(Z)-[(2-hydroxyphenyl)imino]methyl}-l,l'-biphenyl-4-yl)methylene] amino} phenol (WSP914)
An identical procedure to the one employed for SP714 was used. 4-4'-biphenyl- dialdehyde (O.lOOg, 0.47mmol) and 2-aminophenol (0.103g, 0.95mmol) was reacted in ethanol (15ml). The product WSP914 was isolated as a yellow solid. Yield 0.129g (0.33mmol, 70%); mp 226-230°C; 1H NMR (300MHz, /-DMSO) δ 6.84- 6.94 (4H, m, C2-H + C3-H), 7.10 (2H, t, J = 7.1Hz, C4-H), 7.26 (2H, d, J = 7.8Hz, C5-H), 7.95 (4H, d, J = 8.3Hz, Cio-H), 8.16 (4H, d, J = 8.3Hz, C9-H), 8.79 (2H, s, C7-H), 9.07 (2H, br, s, OH); 13C NMR (75MHz, /-DMSO) δ 116.4 (C2), 119.4 (C4), 119.8 (C5), 127.4 (Cio), 127.9 (C3), 129.9 (C9), 136.3 (C8), 138.1 (Cu), 142.0 (C6), 151.7 (Ci), 158.9 (C7); (Anal. Calcd. for C26H20N2O2 : C, 79.57; H, 5.14; N, 7.13%. Found C, 79.57; H, 5.06; N, 7.19%)
4-{[(lZ)-(4'-{(Z)-[(4-hydroxyphenyl)imino]methyl}-l,l '-biphenyl-4-yl)methylene] amino} phenol (WSP915)
An identical procedure to the one employed for WSP714 was used. 4-4'-biphenyl- dialdehyde (O.lOOg, 0.47mmol) and 4-aminophenol (0.103g, 0.95mmol) was reacted in ethanol (15ml). The product WSP915 was isolated as a yellow solid.
Yield 0.127g (0.32mmol, 69%); mp >360°C; 1H NMR (300MHz, /-DMSO) δ 6.82 (4H, d, J = 8.6Hz, C2-H), 7.25 (4H, d, J = 8.6Hz, C3-H), 7.90 (4H, d, J = 8.2Hz, C8-H), 8.01
(4H, d, J = 8.2Hz, C7-H). 8.69 (2H, s, C5-H), 9.56 (2H, s, OH); 13C NMR (75MHz, /- DMSO) δ 116.1 (C2), 122.9 (C3), 127.4 (C8), 129.3 (C7), 136.3 (C6), 141.7 (C9), 142.9
(C4), 156.8 (Ci), 156.9 (C5); (Anal. Calcd. for C26H20N2O2 + 0.3H2O : C, 78.49; H, 5.22;
N, 7.04%. Found C, 78.58; H, 5.03; N, 7.02%). N-((lZ)-{4-[(Z)-(phenylimino)methyl]phenyl}methylene)aniline (WSP919) An identical procedure to the one employed for WSP714 was used. Terephthalaldehyde (0.200g, 1.49mmol) and aniline (0.27ml, 2.98mmol) was reacted in ethanol (15ml). The product WSP919 was isolated as a yellow solid.
Yield 0.313g (l.lmmol, 74%); mp 158-160°C; 1H NMR (300MHz, /-DMSO) δ 7.25-7.34 (6H, m, Ci-H + d-H), 7.45 (4H, t, J = 8.1Hz, C2-H), 8.09 (4H, s, C7-H), 8.72 (2H, s, C5- H); 13C NMR (75MHz, /-DMSO) δ 121.5 (C3), 126.7 (Ci), 129.4 (C7), 129.6 (C2), 138.8 (C6), 151.5 (C4), 160.4 (C5); (Anal. Calcd. for C20Hι6N2 : C, 84.48; H, 5.67; N, 9.85%. Found C, 84.41; H, 5.68; N, 9.89%).
N-((lZ)-{4'-[(Z)-(phenylimino)methyl] -1 , 1 '-biphenyl-4-yl}methylene) aniline (WSP920) An identical procedure to the one employed for WSP714 was used. 4-4'-biphenyl- dialdehyde (0.200g, 0.95mmol) and aniline (0.17ml, 1.90mmol) was reacted in ethanol (20ml). The product WSP920 was isolated as a yellow solid. Yield 0.252g (0.69mmol, 74%); mp 237-238°C; 1H NMR (300MHz, /-DMSO) δ 7.26- 7.33 (6H, m, Ci-H + C3-H), 7.45 (4H, t, J = 7.5Hz, C2-H), 7.96 (4H, d, J = 8.3Hz, C8-H), 8.08 (4H, d, J = 8.3Hz, C7-H), 8.71 (2H, s, C5-H); 13C NMR (75MHz, /-DMSO) δ 121.4 (C3), 126.0 (Ci), 127.6 (C8), 129.6 (C7), 129.7 (C2), 135.9 (C6), 142.2 (C9), 151.7 (C4), 160.5 (C5); (Anal. Calcd. for C26H20N2 : C, 86.64; H, 5.59; N, 7.77%. Found C, 86.25; H, 5.49; N, 7.82%).
4-{[(lZ)-(4-{(Z)-[(4-cyanophenyl)imino]methyl}naphthyl)methyleiie]amino}naphthonitrile (WSP972)
An identical procedure to the one employed for WSP714 was used. Terephthalaldehyde (0.050g, 0.372mmol) and 4-amino-l -naphthalene carbonitrile (0.125g, 0.745mmol) was reacted in ethanol (6ml). The product WSP972 was isolated as a yellow solid. Yield- 0.020g (0.046mmol, 12%); 1H NMR (300MHz, /-DMSO, 313K) δ 7.46 (2H, d, J = 7.8Hz, C4-H), 7.80 (2H, t, J = 7.3Hz, C8-H), 7.91 (2H, t, J = 7.3Hz, C7-H), 8.20 (2H, d, J = 8.5Hz, C6-H), 8.26 (2H, d, J = 7.6Hz, C9-H), 8.34 (4H, s, C13-H), 8.47 (2H, d, J = 7.8Hz, C3-H), 8.92 (2H, s, C12-H); 13C could not be done due to a problem with solubility.
2-{[(lZ)-(4-{(Z)-[(2-hydroxy-4-nitrophenyl)imino]methyl}phenyl)methylene]amino}-5- nitrophenol ( SP973)
An identical procedure to the one employed for WSP714 was used. Terephthalaldehyde (0.300g, 2.2mmol) and 2-amino-5-nitrophenol (0.690g, 4.4mmol) was reacted in ethanol (15ml). The product WSP973 was isolated as a dark yellow solid. Yield 0.573g (1.41mmol, 64%); mp 268-270°C; 1H NMR (300MHz, /-DMSO) δ 7.35 (2H, d, J - 8.4Hz, C5-H), 7.76 (4H, m, C2-H + C4-H), 8.18 (4H, s, C9-H), 8.78 (2H, s, C7- H), 10.32 (2H, br, s, OH); 13C NMR (75MHz, /-DMSO) δ 110.9 (C2), 115.5 (C4), 121.0 (C5), 129.9 (C9), 138.9 (C8), 145.6 (C6), 145.9 (C3), 151.0 (Ci), 163.0 (C7); (Anal. Calcd. for C20H14N4O6 : C, 59.12; H, 3.47; N, 13.78%. Found C, 59.28; H, 3.61; N, 13.29%).
Methyl 2-hydroxy-4~({(lZ)-[4-((Z)-{[3-hydroxy-4-(methoxycarbonyl)phenyl]imino}methyl) phenyl] methylene}amino)benzoate (WSP975)
An identical procedure to the one employed for WSP714 was used. Terephthalaldehyde (0.200g, 1.49mmol) and methyl-4-amino-2-hydroxybenzoate (0.49g, 2.98mmol) was reacted in ethanol (13ml). The product WSP975 was isolated as a yellow solid.
Yield 0.267g (0.62mmol, 41%); mp 198-20FC; 1H NMR (300MHz, /-DMSO) δ 3.84 (6H, br, s, Ci-H), 6.78 (4H, br, s, C5-H + C7-H), 7.77 (2H, br, s, C8-H), 8.03 (4H, br, s, Cn- H), 8.65 (2H, br, s, C9-H), 10.63 (2H, br, s, OH); 13C could not be done due to a problem with solubility; (Anal. Calcd. for C24H2oN2O6 + 0.5H2O : C, 65.30; H, 4.79; N, 6.35%. Found C, 65.33; H, 4.67; N, 6.21%).
Ethyl 2-hydroxy-4-({(lZ)-[4-((Z)-{[3-hydroxy-4-(ethoxycarbonyl)phenyl]imino}methyl) phenyl] methylene}amino)benzoate (WSP976) An identical procedure to the one employed for WSP714 was used. Terephthalaldehyde (0.060g, 0.44mmol) and ethyl-4-amino-2-hydroxybenzoate (0.161g, 0.88mmol) was reacted in ethanol (5ml). The product WSP976 was isolated as a yellow solid. Yield 0.060g (0.13mmol, 30%); mp 190-191°C; 1H NMR (300MHz, /-DMSO) δ 1.37 (6H, t, J = 7.1Hz, Ci-H), 4.39 (4H, q, J = 7.1Hz, C2-H), 6.85 (2H, s, C6-H), 6.88 (2H, d, J = 8.1Hz, C8-H), 7.86 (2H, d, J = 8.1Hz, C9-H), 8.11 (4H, s, Cι2-H), 8.73 (2H, s, Cι0-H), 10.78 (2H, s, OH); 13C could not be done due to a problem with solubility; (Anal. Calcd. for C26H24N2O6 + 0.4H2O : C, 66.77; H, 5.34; N, 5.99%. Found C, 66.73; H, 5.22; N, 5.70%).
Methyl 2-hydroxy-5-({(lZ)-[4-((Z)-{[4-hydroxy-3-(methoxycarbonyl)phenyl]imino}methyl) phenyl] methylene}amino)benzoate (WSP977)
An identical procedure to the one employed for WSP714 was used. Terephthalaldehyde (O.lOOg, 0.75mmol) and methyl-3-amino-5-hydroxybenzoate (0.250g, 1.49mmol) was reacted in ethanol (7ml). The product WSP977 was isolated as a yellow solid.
Yield 0.070g (0.16mmol, 22%); mp 207-210°C; 1H NMR (300MHz, /-DMSO) δ 3.88 (6H, s, Ci-H), 7.04 (2H, dd, J = 1.2Hz and 8.8Hz, C6-H), 7.59 (2H, d, J = 8.8Hz, C5-H), 7.72 (2H, s, C8-H), 8.02 (4H, s, Cn-H), 8.74 (2H, s, C9-H), 10.47 (2H, s, OH); 13C could not be done due to a problem with solubility; (Anal. Calcd. for C2 H2oN2O6 : C, 66.66; H, 4.66; N, 6.48%. Found C, 66.33; H, 4.54; N, 6.28%).
Ethyl 2-hydroxy-5-({(lZ)-[4-((Z)-{[4-hydroxy-3~(ethoxycarbonyl)phenyl]imino}methyl) phenyl] methylene} amino)benzoate (WSP980)
An identical procedure to the one employed for WSP714 was used. Terephthalaldehyde (0.050g, 0.37mmol) and ethyl-3-amino-5-hydroxybenzoate (0.134g, 0.74mmol) was reacted in ethanol (7ml). The product WSP980 was isolated as a yellow solid. Yield 0.117g (0.25mmol, 69%); mp 171-173°C; 1H NMR (300MHz, /-DMSO) δ 1.33 (6H, t, J = 7.1Hz, Ci-H), 4.36 (4H, q, J = 7.1Hz, C2-H), 7.04 (2H, d, J = 8.8Hz, C6-H), 7.60 (2H, d, J = 8.8Hz, C7-H), 7.72 (2H, s, C9-H), 8.03 (4H, s, C12-H), 8.74 (2H, s, C10-H), 10.55 (2H, s, OH); 13C could not be carried out to the insolubility of the compound; (Anal. Calcd. for C26H24N2O6 + 0.2H2O : C, 67.29; H, 5.30; N, 6.04%. Found C, 67.19; H, 5.24; N, 6.00%).
2-{[(lZ)-(4-{(Z)-[(2-hydroxy-4-methylphenyl)imino]methyl}phenyl)methylene]amino}-5- methylphenol (WSP1010)
An identical procedure to the one employed for WSP714 was used. Terephthalaldehyde (0.300g, 2.2mmol) and 2-amino-5-methylphenol (0.550g, 4.4mmol) was reacted in ethanol (15ml). The product WSP1010 was isolated as a yellow solid. Yield 0.649g (1.88mmol, 86%); mp 205-207°C; 1H NMR (300MHz, /-DMSO) δ 2.26 (6H, s, Ci-H), 6.68 (2H, d, J - 8.1Hz, C7-H), 6.75 (2H, s, C3-H), 7.23 (2H, d, J = 8.1Hz, C6-H), 8.15 (4H, s, Cio-H), 8.81 (2H, s, C8-H), 8.99 (2H, s, OH); 13C NMR (75MHz, /- DMSO) δ 21.3 (Ci), 117.0 (C3), 118.9 (C7), 120.6 (C6), 129.3 (Cio), 135.1 (C9), 138.0 (C5), 138.9 (C2), 151.9 (C4), 157.4 (C8); (Anal. Calcd. for C22H20N2O2 + 0.2H2O : C, 75.93; H, 5.91; N, 8.05%. Found C, 75.95; H, 5.72; N, 7.94%).
4-{[(lZ)-(4-{(Z)-[(4-cyanophenyl)imino]methyl}phenyl)methylene]amino}benzonitrile
(WSP1011)
An identical procedure to the one employed for WSP714 was used. Terephthalaldehyde (0.200g, 1.49mmol) and 4-amino-benzonitrile (0.352g, 2.98mmol) was reacted in ethanol
(10ml). The product WSP1011 was isolated as a yellow solid.
Yield 0.207g (0.62mmol, 42%); mp 209-210°C; 1H NMR (300MHz, /-DMSO) δ 7.46
(4H, d, J = 8.2Hz, C4-H), 7.92 (4H, d, J = 8.2Hz, C3-H), 8.13 (4H, s, C8-H), 8.74 (2H, s,
C6-H); 13C NMR (75MHz, /-DMSO) δ 95.9 (C2), 113.8 (Ci), 122.5 (C4), 130.4 (C8), 133.8 (C3), 133.9 (C7), 140.1 (C5), 153.4 (C6); (Anal. Calcd. for C22Hι4N4 + 0.2H2O : C,
78.18; H, 4.29; N, 16.58%. Found C, 78.17; H, 4.17; N, 16.59%). Synthesis of Amines
Bis-methyl 3-{[4-(aminomethyl)benzyl]amino}-4-methoxybenzoate (WSP718) To a 25ml round bottomed flask was added sodium borohydride (O.lOOg, 2.6mmol) Anhydrous methanol (8ml) was added slowly dropwise, effervescence occurs. In a separate flask WSP716 (0.200g, 0.42mmol) was dissolved in anhydrous dichloromethane (8ml) and then this solution was added slowly dropwise to the main reaction vessel. After stirring at room temperature for 12 hours, a precipitate forms, which was filtered and washed with 2N HCl (10ml), water (10ml), methanol (20ml) and dichloromethane (20ml). The product WSP718 was obtained as a white solid without need for further purification. Yield 0.044g (0.095mmol, 23%); mp 218-220°C; 1H NMR (300MHz, /-DMSO) δ 3.71 (6H, s, C7-H), 3.88 (6H, s, Ci-H), 4.30 (4H, d, J = 5.9Hz, Cio-H), 5.77 (2H, t, J = 5.9Hz, NH), 6.89 (4H, m, C9-H + C5-H), 7.22 (6H, m, C4-H + C12-H); 13C could not be done due to a problem with solubility; ES+ m/z 462.9 (M+); (Anal. Calcd. for C26H28N2O6 + 0.7HC1 : C, 63.73; H, 5.90; N, 5.72%. Found C, 63.85; H, 5.72; N, 5.67%).
Bis-methyl 4-{[4-(aminomethyl)benzyl]amino}-3-hydroxybenzoate (WSP740)
An identical procedure to the one employed for WSP718 was used. WSP714 (0.300g, 0.693mmol) was dissolved in dichloromethane (10ml) and reacted with sodium borohydride (0.158g, 4.16mmol) in methanol (5ml). In this case water (20ml) was added to precipitate the product, which was washed further with water (20ml) and dichloromethane (20ml). The product WSP740 was isolated as a light brown solid. Yield 0.148g (0.34mmol, 49%); mp 239-240°C; 1H NMR (300MHz, /-DMSO) δ 3.71 (6H, s, Cl-H), 4.33 (4H, d, J = 6.2Hz, C9-H), 6.15 (2H, t, J = 6.2Hz, NH), 6.36 (2H, d, J = 8.7Hz, C7-H), 7.23 (8H, m, C4-H + C8-H + Cn-H), 9.74 (2H, br, s, OH); 13C NMR (75MHz, /-DMSO) δ 45.7 (d), 51.5 (C9), 108.6 (C7), 113.5 (C4), 116.1 (C3), 123.0 (C8), 127.3 (Cu), 138.5 (C6), 142.2 (Cio), 143.5 (C5), 166.7 (C2); ES+ m/z 459.5 (100%, M+Na+); high resolution ES+ m/z found 459.1538 C24H24N2O6Na (M+Na+) requires 459.1532; (Anal. Calcd. for C24H24N2O6 + 0.7H2O : C, 64.19; H, 5.70; N, 6.24%. Found C, 64.15; H, 5.43; N, 6.17%).
4-[(4-{[(4-carboxy-2-hydroxyphenyl)amino]methyl}benzyl)amino]-3-hydroxybenzoic acid (WSP798)
Into a 25ml round bottomed flask was placed WSP740 (0.075g, 0.171mmol). A solution of NaOH (0.070g, 1.71mmol) in water (6ml) was added dropwise and the reaction mixture was left stirring at room temperature for 45 minutes. 2N HCl (8ml) was added dropwise to the reaction mixture till a neutral pH was achieved. A solid precipitated which was filtered and washed with water (20ml). The product WSP798 was obtained as a white solid without further need for purification.
Yield 0.015g (0.037mmol, 21%); mp >360°C (decomp.); 1H NMR (300MHz, /-DMSO) δ 4.34 (4H, br, s, C8-H), 6.08 (2H, br, s, NH), 6.38 (2H, br, s, C6-H), 7.21-7.30 (8H, m, C3-H + C7-H + Cio-H), 9.67 (2H, br, s, OH); 13C NMR (75MHz, /-DMSO) δ 53.6 (C8), 115.9 (C6), 118.1 (C3), 119.6 (C2), 122.4 (C7), 129.8 (Cio), 132.1 (C5), 140.6 (C9), 143.6 (C4), 169.9 (Ci); (Anal. Calcd. for C22H20N2O6 + 0.8HC1 : C, 60.39; H, 4.79; N, 6.40%. Found C, 60.43; H, 4.57; N, 6.22%).
2-[(4-{[(2-hydroxyphenyl)amino]methyl}benzyl)amino]phenol (WSP911) An identical procedure to the one employed for WSP718 was used. WSP909 (0.200g, 0.63mmol) was dissolved in dichloromethane (10ml) and reacted with sodium borohydride (0.158g, 4.16mmol) in methanol (10ml). After stirring for 12 hours, dichloromethane (20ml) was added and the solution was washed with 2N HCl (20ml) and saturated sodium bicarbonate solution (20ml). The organic phase was separated, dried (MgSO4) and evaporated in vacuo. The crude was then purified by column chromatography (1:1 ethyl acetate:hexane) to leave the product SP911 as a light orange coloured solid. Yield 0.020g (0.062mmol, 10%); 1H NMR (300MHz, /-DMSO) δ 4.25 (4H, d, J = 6.0Hz, C7-H); 5.19 (2H, t, J = 6.0Hz, NH), 6.36 (4H, m, C3-H + C5-H), 6.51 (2H, t, J = 7.3Hz, C4- H), 6.63 (2H, d, J = 7.8Hz, C2-H), 7.28 (4H, s, C9-H), 9.25 (2H, s, OH); 13C NMR (75MHz, /-DMSO) δ 46.6 (C7), 110.4 (C5), 113.7 (C2), 116.0 (C3), 119.8 (C4), 127.3 (C9), 137.5 (C6), 139.1 (C8), 144.3 (C ; (Anal. Calcd. for C2oH20N2O2 + 0.1HC1 + 0.1H2O : C, 73.72; H, 6.28; N, 8.60%. Found C, 73.68; H, 5.92; N, 7.95%).
Synthesis of Alkene Analogues
5-((E)-2-{4-[(E)-2-(3-carboxy-4-hydroxyphenyl)vinyl]phenyl}vinyl)-2-hydroxybenzoic acid
(WSP759)
To a 50ml round bottomed flask was placed 1,4-divinylbenzene (0.200g, 1.53mmol), 5- iodosalicylic acid (0.81 lg, 3.07mmol), 10 mol% palladium acetate (0.034g, 0.153mmol) and potassium carbonate (0.424g, 3.07mmol). DMF (17ml) and water (3ml) were added to the reaction mixture, which was then stirred and heated to 100°C with a condenser attached. After stirring at 100°C for 18 hours, water (75ml) was added followed by 2M HCl (10ml) to acidify mixture. Upon addition of the acid a solid precipitated which was filtered. The solid was dissolved in ethyl acetate (80ml) and left to slowly recrystallise. After filtration and washing with petroleum ether (20ml) the product WSP759 was isolated as a light green crystalline solid.
Yield 0.080g (0.19mmol, 13%); mp >300°C; 1H NMR (300MHz, /-DMSO) δ 6.98 (IH, d, J = 8.6Hz, C -H), 7.11 (IH, d, J = 16.4Hz, C9-H), 7.25 (IH, d, J = 16.4Hz, C8-H), 7.59 (2H, s, Cn-H), 7.81 (IH, dd, J = 1.9Hz and 8.7Hz, C5-H), 7.99 (IH, d, J = 1.9Hz, C7-H), 11.44 (IH, br, s, CO2H); 13C NMR (75MHz, /-DMSO) δ 113.5 (C ), 118.0 (C2), 126.8 (C9), 126.9 (C8), 127.4 (Cu), 128.8 (C6), 128.9 (C7), 133.4 (C5), 136.6 (Cio), 161.0 (C3), 172.1 (Ci); (Anal. Calcd. for C24Hι8O6 : C, 71.64; H, 4.51%. Found C, 71.44; H, 4.57%).
4-((E)-2-{4-[(E)-2-(4-hydroxyphenyl)vinyl]phenyl}vinyl)phenol (WSP762)
An identical procedure to the one employed for WSP759 was used. 1,4-divinylbenzene (0.240g, 1.84mmol), 4-iodophenol (0.81 lg, 3.68mmol), 10 mol% palladium acetate (0.042g, 0.184mmol) and potassium carbonate (0.509g, 3.68mmol) were reacted together in DMF (15ml) and water (5ml). A crude solid was obtained after ethyl acetate extraction and needed further purification. It was columned in a 1:1 ethyl acetate:hexane eluent mixture. The product WSP762 was finally isolated as a grey-green solid. Yield 0.025g (0.079mmol, 4%); 1H NMR (300MHz, -MeOD) δ 6.81 (4H, d, J = 8.6Hz, C2-H), 7.08 (2H, d, J = 16.4Hz, C6-H), 7.33 (2H, d, J = 16.4Hz, C5-H), 7.47 (4H, C8-H), 7.88 (4H, d, J - 8.6Hz, C3-H); 13C NMR (75MHz, /-DMSO) δ 117.0 (C2), 125.7 (C5), 128.0 (C6), 129.9 (C8), 130.1 (C4), 131.6 (C3), 133.9 (C7), 159.7 (Ci); ES- rn/z 312.7 (M-); high resolution ES+ m/z found 315.1382 C22Hι8O2 (M+H+) requires 315.1380.
Methyl 4-{[4-({[2-hydroxy-4-(methoxycarbonyl)phenyl]amino}carbonyl)benzoyl]amino}3- hydroxybenzoate (WSP1077)
Figure imgf000059_0001
Activity Result +ve at lOμm
This compound was made via an identical preparation to the preparation of WSP677 (alternative procedure). Methyl-4-amino-4-hydroxybenzoate hydrogen sulphate salt (0.522g, 1.97mmol) and triethylamine (1.4ml, 9.85mmol) was reacted with terephthaloyl chloride (0.200g, 0.98mmol) in anhydrous THF (3ml). An identical work-up procedure was employed, the pure product WSP1077 was isolated as an light brown solid. Yield 0.258g (0.56mmol, 56%); mp 287-290°C; 1H NMR (300MHz, /-DMSO) δ 3.82 (6H, s, Ci-H), 7.48 (2H, d, J = 8.3Hz, C7-H), 7.51 (2H, s, C4-H), 8.01 (2H, d, J = 8.3Hz, C8-H), 8.09 (4H, s, Cn-H), 9.66 (2H, s, NH), 10.45 (2H, br, s, OH); 13C NMR (75MHz, /- DMSO) δ 52.4 (Ci), 116.1 (C4), 120.8 (C7), 123.0 (C8), 126.5 (Cu), 128.2 (C3), 130.9 (C6), 137.3 (Cio), 148.9 (C5), 164.9 (C9), 166.3 (C2); (Anal. Calcd. for C24H20N2O8 + 0.5H2O: C, 60.89; H, 4.47; N, 5.92%. Found C, 60.75; H, 4.49; N, 6.22%). Methyl 4-aminobenzoate (WSP1079)
Figure imgf000060_0001
Activity Result +ve at lOμm, ED50 = lμm
To a 500ml round bottomed flask was added 4-aminobenzoic acid (lOg, 72.90mmol) and methanol (150ml). Concentrated sulfuric acid (7ml) was added dropwise slowly. The reaction mixture was heated to reflux (65°C) in an oil bath for 36 hours. The solution remaining was evaporated in vacuo to dryness. Water (100ml) was added and the pH of the solution was adjusted to 3 using a 2M solution of NaOH. The precipitate which was formed was filtered through a sinter funnel and washed with water (50ml). The product WSP 1079 was isolated pure as an off- white solid.
Yield 7.980g (52.80mmol, 72%); mp 107-110°C; 1H NMR (300MHz, /-DMSO) δ 3.77 (3H, s, Ci-H), 6.60 (2H, d, J = 8.7Hz, C5-H), 7.68 (2H, d, J = 8.7Hz, C4-H); 13C NMR (75MHz, /- DMSO) δ 51.5 (Q), 113.1 (C5), 116.2 (C3), 131.4 (C4), 153.8 (C6), 166.7 (C2); ES+ m/z 174.0 (M+Na+), 325.1 (2M+Na+).
Methyl-4-amino-3-hydroxybenzoate (WSP1017)
Figure imgf000060_0002
Activity Result +ve at 10 ,m, ED50 = 1 μm
To a 500ml round bottomed flask was added 4-amino-3-hydroxybenzoic acid (5.0g, 32.65mmol) and methanol (140ml). Concentrated sulfuric acid (3.5ml) was added dropwise slowly. The reaction mixture was heated to reflux (65°C) in an oil bath for 36 hours. The solution remaining was evaporated in vacuo to dryness. Ice water (125ml) was added and the pH of the solution was adjusted to 5 using a 2M NaOH solution. The brown precipitate which was formed was filtered through a sinter funnel and washed with water (50ml). The product WSP 1017 was isolated pure as a brown solid. Yield 3.360g (20.10mmol, 62%); mp 116-117°C; 1H NMR (300MHz, /-MeOD) δ 3.82 (3H, s, Ci-H), 6.67 (IH, d, J=8.20 Hz, C4-H), 7.32 (IH, d, J=1.89 Hz, C7-H), 7.36 (IH, d, J=1.89 Hz and 8.20 Hz, C3-H); 13C NMR (75MHz, /-MeOD) δ 52.4 (Ci), 114.9 (C4), 116.3 (C7), 119.8 (C3), 124.6 (C8), 143.7 (C6), 145.2 (C5), 169.8 (C2); ES+ m/z 167.5 (M+), 189.9 (M+Na+).
Cell Culture Assays
Cell Culture SMB cells were grown in tissue culture treated flasks in 199 medium (Gibco) supplemented with 10%(v/v) foetal calf serum, 5%(v/v) newborn calf serum (Gibco), lOOU/mL penicillin and lOOμg/mL streptomycin at 37°C, 5% CO2.
Drug treatment Drugs were resuspended in DMSO at a stock concentration of lOmM. To assess the effects of a drug, SMB cells were plated at 50% confluency per well in 6-well plates. Cells were left for 24h to allow for attachment. Media was then replaced with fresh media containing the appropriate dilution of the lOmM drug stock. 4 days after the addition of drug the media was removed and the protein extracted.
Protein Extraction
Cells were lysed in PBS containing 1% Triton-X 100, 1% Igepal CA-630 for 20 minutes at 37°C. Cell lysates were either placed on ice or treated with 80μg/mL proteinase k for lh at 37°C. Proteins were concentrated from the total cell lysate by methanol precipitation and the protein pellet resuspended and denatured by boiling for 5 minutes in 8M Urea. Western Blotting
Samples were electrophoresed on a 12% acrylamide gel and transferred electrophoretically to PNDF membrane (Immobilon-P, Millipore). PrP was detected using the primary antibody DR1 as previously described27 and a HRP conjugated secondary antibody (Dako). Specific protein bands were visualised using ECL Plus chemiluminescent reagent (Amersham Pharmacia Biotech) followed by autoradiography. Autoradiographs were analysed using Scion Image densitometric software (Scion Corporation).
In vitro aggregation assay All measurements were performed on a Cary lOOBio UN- Visible spectrophotometer at 325nm using a quartz cuvette of 5mm path length. Substrate recombinant mouse PrP (rPrP) was refolded in the absence of metal ions and the seed for aggregation was aged manganese refolded recombinant mouse PrP (MnPrP) prepared as previously described.26'28 Briefly a seed of MnPrP induces immediate aggregation of substrate rPrP, observed as an increase in solution turbidity. The resultant scattering of UV light at 325 nm results in an increased Absorbance measurement. The abilities of the potential anti- TSE compounds to prevent this turbidity increase were measured and the results expressed as a percentage of the turbidity observed with a DMSO control. 50μg rPrP and lOOμM anti-TSE compound were preincubated in 500ul H2O pH 6.5 for 30 minutes to provide a zero for the measurement. lOμg MnPrP seed from a 400μgml"1 stock was added to the drug/rPrP mixture and an initial reading obtained immediately. A second reading was measured after 5 minutes and the increase in Absorbance over 5 minutes recorded. Time in the spectrophotometer beam was minimised due to the sensitivity to UN light of some Congo red derivatives.
Toxicity Assay
It is important that the compounds are not toxic to neural cells. Therefore a toxicity assay was carried using cultures of isolated from mice with the lead compound (WSP677) (Figure 5). Cerebellar neuronal cultures were prepared from six day old mice 129SV. Cultures were prepared as described previously.29 Briefly, the cerebella were dissociated in Hanks' media (Sigma) containing 0.5% trypsin (Sigma) and plated at 1-2 x 106 cells/cm2 in 24 well trays (Falcon) coated with poly-D-lysine (50μg/ml, Sigma). Cultures were maintained in Dulbecco's minimal essential media (Sigma) supplemented with 10% fetal calf serum 2 mM glutamine and 1% antibiotics (penicillin, streptomycin) (Sigma). Cultures were maintained at 37°C with 5% CO2. Compounds were prepared in DMSO (Sigma). After 4 days treatment the survival of the cerebellar cells was determined. MTT (3, [4,5 dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide, Sigma) was diluted to 200μM in Hanks' solution and added to cultures for 30 min at 37°C. The MTT formazan product was released from cells by addition of dimethylsulphoxide (Sigma) and measured at 570 mn in a spectrophotometer (Bio50, Cary). Relative survival in comparison to control treated with the DMSO vehicle could then be determined.
Cellular Assays: Results The compounds of the invention were tested in the SMB cell line assay. The SMB cells are persistently scrapie-infected mouse cells, cloned from a scrapie-infected mouse brain, but of non-neuronal origin. ' ' ' These cells are highly phagocytic and may mimic cells involved in the initial uptake and replication of the agent in peripheral infection.12 In addition these cells show stable persistent scrapie-infection over many passages, suggesting that they are suitable for drug screening.23 hideed, this cell line has been 10 1 ~ successfully used to screen many derivatives of Congo Red. ' The results from these assays are shown in Tables 1-7, for the seven series of compounds: sulfonamides (Table 1); amides with a biphenyl linker (Table 2); amides with a phenyl linker (Table 3); imines with a biphenyl linker (Table 4); imines with a phenyl linker (Table 5); amines with a phenyl linker (Table 6); and alkenes with a phenyl linker (Table 7).
For the sulphonamides (Table 1), none of the compounds showed inhibition of PrP-res at lOμM, although some of the compounds (WSP675 and WSP678) showed some activity at 50μM. Interestingly compound WSP675, which has the same terminator as the lead compound WSP677 showed some activity at this concentration.
Several of the amides with a biphenyl core (Table 2) showed good activity. In particular compounds WSP677, WSP905 and WSP1009 showed prevention of PrP-res formation, interestingly none of the these compounds have a free carboxylic acid, but the carboxylate group is blocked as an ester. Quite possibly the ester group is removed by esterases in the cell. In WSP677 and WSP1009 there is a free hydroxyl group, and in compound WSP905 a methoxy group attached to the phenyl ring; the hydroxyl group of WSP677 and WSP1009 could act as H-bond donors and or acceptors, whilst the methoxy group of WSP905 could act as an H-bond acceptor. Compound WSP979, which is a regioisomer of WSP677 and WSP1009 was inactive. This suggests that the relative orientation of the ester and they hydroxyl group are important in the terminator. Compound WSP971, which is the analogue of WSP905 with the free carboxylate did not show activity, indicating that the ester has a role, perhaps as a prodrug. Other compounds with the methyl carboxylate removed or replaced (WSP912, 916, 971, 978, 1013) were inactive in the cell culture assay. This suggests that the ester may have a role in the activity of the molecule; by altering the structure of the ester it may be possible to modify the potency and physicochemical properties of the compounds.
The compounds of series 3 were the amides with a phenyl core (Table 3). Compound 27 showed activity and following a fuller dose-response experiment, the ED50 was found to be 250nM.
For the biphenyl series of imines (Series 4, Table 4), two of the compounds showed inhibition of PrP-res formation in the cellular assay (WSP795 and WSP915).
The data for the corresponding series of compounds with a phenyl linker and an imine (series 5) is presented in table 5. WSP1011, which has a nitrile substituent on the terminator group, showed significant activity in the cellular assay. This substituent can act as a hydrogen bond acceptor but is not charged.
The aminomethylene compounds of series 6 (Table 3) all showed activity. Compound WSP740 showed an ED50 of about 75nM. This is a very similar value to that of WSP677. Interestingly it contains the same terminator group as WSP677.
Finally a series of compounds was prepared in which the diazo bond was replaced by an alkene (Series 7, Table 7). This group should be metabolically very stable. WSP759 is X- 34, a compound used by Klunk for diagnosis of Alzheimer's disease.24'25 This compound showed no activity at lOμM, but weak activity at 50μM. The analogue, WSP762, in which the carboxylic acid functionality has been removed from the terminator, shows potent activity.
WSP762 showed activity at 2μM, the mechanism of action is unknown, but may well differ from that of the other molecules described herein. It is possible that this molecule could be acting through an antioxidant mode of action.
For compounds showing activity at lOμM, a full dose response study was undertaken to get a more accurate indication of the potency (Table 8). Thus compound WSP677 showed an ED50 of 25-50nM; WSP740 of 75nM; WSP906 an ED50 of 250nM; WSP1009 an ED50 of 5μM; WSP718 ED50
Polymerisation Assays: Results In order to try and understand the mode of action of the these compounds, investigations were carried out to see if these compounds could inhibit polymerisation of PrPc by PrP-res. In this assay, the seed for polymerisation was recombinant mouse PrP which was refolded and aggregated using manganese. This was then used to seed aggregation of recombinant mouse PrP (which was refolded in the absence of metal ions).26 The aggregation was monitored by looking at scattering of UV light. The effect of compounds on inhibiting aggregation was detennined. It was discovered that some of the compounds degraded when kept in the UV beam, so UV beam was not left on continually.
Some of the compounds caused precipitation prior to addition of the seed. Accordingly, this means that interpretation of the data related to these compounds is unreliable. Where this is the case it is marked in the tables.
For the sulfonamides (Table 1), several compounds showed inhibition of polymerisation. In particular, compounds WSP675 and WSP678, which showed weak activity in the cellular assays, also showed inhibition of polymerisation. Compound WSP676, which was not active in the cellular assay also showed some activity in the polymerisation assay. Compound WSP675, which showed the greatest inhibition of polymerisation contains the same terminator groups as WSP677.
For the amides with a biphenyl core (Table 2), compounds WSP677 and WSP905 showed inhibition of polymerisation. These compounds also which showed activity in the cellular assays. However, compound WSP1009, which showed inhibition of PrP-res formation in the cellular assays, appeared to stimulate aggregation in the polymerisation assay.
hi the third series of compounds investigated, the amides with a phenyl core (Table 3), compounds WSP917 and WSP1012 appeared to cause prevention of polymerisation.
Compounds of Series 4 with the biphenyl linker and an imine bond (Table 4), WSP794 and WSP795 show some inhibition of polymerisation. The two compounds which inhibit polymerisation are the two compounds which also have some effect in cell culture assays, indicating that the mode of action of these compounds, may include inhibition of polymerisation. The terminator group on WSP794 is the same as that on WSP677 and that on WSP795 differs only in the carboxylate ester being replaced by a free carboxylate group.
The compounds of Series 5 are those with a phenyl linker and an imine bond (Table 5). A number of these show inliibition of PrPc polymerisation; WSP716, 738, 741, 742, 760, 980. The only compound of this series which is active in the cellular assay, shows a very small effect on polymerisation in this assay (WSP1011). Also the terminator group found in WSP677, is not active in this series of compounds (compound WSP714). The presence of a carboxylate group (either as the free acid or as an ester) seems to be important for activity. Compounds not containing these functionalities are not active in this assay. Compound WSP739 is a direct homologue of WSP738 (one of the most active compounds) except it has an extra methylene between the terminator and the imine. WSP739 is inactive, suggesting that conjugation with the terminator is an important feature.
For the amine-linked compounds (Series 6, Table 6), several of the compounds show inhibition of PrP-res formation, WSP718, WSP798.
Finally, WSP759 (Table 7) shows some inhibition of polymerisation.
Toxicity Assays: Results
The compound was found to non-toxic at 0.1 μM. There was a very low level of toxicity at 0.5μM and an IC50 of around 30-40μM. This indicates that WSP677 causes selective action against TSE-infected cells. For comparison the toxicity of compound WSP1012 was investigated. This compound had no effect in the cell culture assays. It showed a higher toxicity to neurons compared to WSP677. Detachment Assays
Another possible mode of action of the compounds would be detaching of PrPc from the surface of the cells. Therefore, the supernatant was examined to see if there were raised levels of PrP in treated cells compared to control levels. However none was detected, suggesting that the primary mode of action of WSP677 is not removal of the PrPc from the surface of the cell.
Therapeutic Activity against Alzheimer's Disease The efficacy of the compounds of the invention against Alzheimer's disease was investigated by performing a beta-amyloid assay to measure amyloid aggregation in accordance with the methodology set forth in Fernanda G. De Felice et al, FASEB Journal, 2001, vol 15, 1297-1299. In addition, the effect of the compounds on Alzheimer beta- amyloid peptide (Abeta) polymerization and the growth of amyloid fibrils was investigated using the methodology disclosed in Kuner et al, Journal of Biological Chemistry, 2000, vol 275, 1673-1687.
Various modifications and variations of the described aspects of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes of carrying out the invention which are obvious to those skilled in the relevant fields are intended to be within the scope of the following claims. REFERENCES
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Table 1: Sulphonamides: Synthetic Yield, inhibition of PrP-res formation in SMB cells at lOμM and hihibition of Polymerisation at lOOμM. H o II 0 H R- -N- -s II- a II- -N- -R o "V. _ ~v J~ - 0 WSP Entry R MW Yield Inhibition of PrP- Polymerisation number res formation in (% of control) SMB cells at lOμM
Figure imgf000073_0001
WSP676 lb Me02C 640.69 50 -ve 77 (-ve 50μM) OMe
Figure imgf000073_0002
WSP679 Id 584.58 67 -ve 12 (-ve 50μM) precipitation WSP684 le 640.61 48 -ve 2 (-ve 50μM) after pre-
Figure imgf000073_0003
centrifugation
Figure imgf000073_0004
Table 2: Amides: Synthetic Yield, Inhibition of PrP-res formation in SMB cells at lOμM and Inhibition of Polymerisation at lOOμM Inhibition of Polymerisation at lOOμM.
Figure imgf000074_0001
WSP number Entry R MW Yield Inhibition Polymerof PrP-res isation formation (% of control) in SMB cells at lOμM
Figure imgf000074_0002
WSP905 2b Me02C— ^ X\- 568.57 46 +ve 70 MeO WSP912 2c 424.45 14 -ve 430 OH precipitated WSP916 2d VJ 392.45 48 -ve 171 WSP971 2e 540.52 32 -ve 472
WSP978 2f 514.44 74 -ve 160
Figure imgf000074_0003
WSP979 2g Me02C- w // 540.52 60 -ve NR HO precipitated WSP1009 2i πo-< >-i- 540.52 51 +ve 249 MeQ2C WSP1013 2h 442.47 63 -ve 196
Figure imgf000074_0004
Table 3: Phenyl Amides: Synthetic Yield, Inhibition of PrP-res formation in SMB cells at lOμM and Inhibition of Polymerisation at lOOμM. H H R-N- ■N-R
WSP Entry R MW Yield Inhibition Polymer- number of PrP-res isation formation (% of in SMB control) cells at lOμM
WSP904 3a Meθ2C 492.48 83 -ve 70 precipitated OMe
WSP906 3b Meθ2C 548.50 94 +ve 147 Me02C
WSP907 3c 492.39 77 -ve 1 precipitated
Figure imgf000075_0001
WSP908 3d Me02C— ^ >- - 432.13 47 -ve • 100
WSP917 3e o+ 316.35 92 -ve 54
WSP974 3f O2N-QH- 438.08 90 -ve 15 OH precipitated
WSP 1077 3h Me02C— < X\- 56 +ve 62 OH
WSP1012 3g NC_ "^ 366.37 65 -ve 68 Table 4: Biphenyl Imines: Synthetic Yield, Inhibition of PrP-res formation in SMB cells at lOμM and Inhibition of Polymerisation at lOOμM.
R-N ^^ ^— v N-R
WSP Entry Terminator MW Yield Inliibition of PrP- Polymer-isation number res formation in (% of control) SMB cells at lOμM
WSP761 Meo2c 536.6 14 -ve
OOMMe
WSP794 Meθ2c— λ-|- 508.52 45 -ve > // 49 OH (+ve 50μM) WSP795 Hθ2c- >-|- 480.47 28 +ve 62 OH
WSP914 392.45 70 -ve 281 OH
WSP915 HO 392.45 69 +ve 104
Figure imgf000076_0001
Table 5: Phenyl Imines: Synthetic Yield, Inhibition of PrP-res formation in SMB cells at lOμM and Inhibition of Polymerisation at lOOμM. J~\- N-R
WSP Entry Linker Terminator MW Yield hihibition Polymernumber of PrP-] res isation formation in (% of SMB cells control) at lOμM
WSP714 5a Phenyl Meθ2C— < X\- 432.44 76 -ve 140 OH (-ve at 50μM)
WSP715 5b Phenyl 113
Figure imgf000077_0001
50μM)
WSP716 5c Phenyl 47
Figure imgf000077_0002
WSP738 5d Phenyl H02C- >-i- 372.38 78 -ve 12
WSP739 5e Phenyl Hθ2c^T}- 400.44 64 -ve 117
WSP741 5f Phenyl 432.44 84 -ve 11
WSP742 5g Phenyl 404.38 66 -ve 53
Figure imgf000077_0003
WSP760 5h Phenyl H02C- Q- - 404.38 74 -ve 35 OH (-ve 50μM)
WSP909 5i Phenyl + 316.35 55 -ve 85 OH
WSP910 5j Phenyl 316.35 33 -ve 240 WSP919 5k Phenyl o+ 284.35 74 -ve ?
WSP972 51 Phenyl 434.15 12 -ve 148
WSP973 5m Phenyl 406.09 64 -ve 109
Figure imgf000078_0001
WSP975 5n Phenyl e02C— < -\- 432.13 41 -ve 113 HU
WSP976 5o Phenyl 460.48 30 -ve 92
Figure imgf000078_0002
WSP977 5p Phenyl 432.13 22 -ve ? Me02C
WSP980 5q Phenyl 460.48 69 -ve 60 Et02C
WSPIOIO 5r Phenyl 344.41 86 -ve 230
Figure imgf000078_0003
WSPlOll 5s Phenyl 334.37 42 +ve 81
Table 6: Phenyl amines: Synthetic Yield, Inhibition of PrP-res formation in SMB cells at lOμM and Inhibition of Polymerisation at lOOμM. V
R-NH HN-R
WSP Entry R MW Yield Inhibition of Polymer- number PrP-res isation (% formation in of control) SMB cells at lOμM
WSP718 6a Meo2c 464.52 23 +ve 20
OOMMe
WSP740 6b Me02C- +ve 166 //-|- 436-47 49 OH
WSP798 6c Ho2cX ) 408.40 21 -ve 79 OH (+ve lOμM)
WSP911 6d 320.39 10 +ve 240
Figure imgf000079_0001
Table 7: Phenyl alkenes: Synthetic Yield, Inhibition of PrP-res formation in SMB cells at lOμM and inhibition of Polymerisation at lOOμM.
WSP Entry R MW Yield hihibition of PrP- Polymer- number res formation in isation SMB cells at (% of control) lOμM
Figure imgf000080_0001
WSP762 7b HO 314.4 (+ve 2μM)
Table 8: ED50 values of compounds in SMB cells showing activity <10μM. WSP number Entry Structure Polymerisation (% of control) nM
Figure imgf000081_0001
915 250
OH OH
Figure imgf000082_0001

Claims

1. A compound of formula I, or a pharmaceutically acceptable salt thereof,
Figure imgf000083_0001
wherein
X is selected from
Figure imgf000083_0002
Y is selected from SO2NH, CONH, CH=N, CH2NH and CH=CH; R1"5 are each independently selected from H, OH, OR6, CO2H, CO2R7, NO2, CN CONR8R9, CONHR10, CC^O^CO^11, CO2(CH2)mO(CO)R12, CO2CHR13COR14, CONH(CH2)pCO2R15, CONH(CH2)qO(CO)R16, CONHCHR17COR18;
R " are each independently hydrocarbyl; n, m, p and q are each independently 1, 2, 3 or 4; with the proviso that when X is phenyl, Y is CH=CH, R3 is OH and R1, R4 and R5 are
H, R2 is other than COOH.
2. A compound according to claim 1 wherein R "18 are each independently alkyl.
3. A compound according to claim 1 or claim 2 wherein: one of R1"5 is selected from H, OH and OR6; one of R1"5 is selected from OH, CO2H, CN, CO2alkyl and CONH-alkyl; and the remainder of R1"5 are H.
4. A compound according to any preceding claim wherein: (i) R3 is CN, OH or CO2H, and R1, R2, R4 and R5 are all H;
(ii) R1 is OH, R3 is CO2H, CO2R7 or H, and R2, R4 and R5 are all H;
(iii) R1 is OR6, R4 is CO2R7 or CO2H, and R2, R3 and R5 are all H;
(iv) R2 and R4 are CO2R7, and R1, R3 and R5 are all H;
(vii) R3 is CO2R7, R4 is OH or OR6, and R1, R2 and R5 are all H; or
(vi) R3 is OH, R4 is CO2R7 or CO2H, and R1, and R2 and R5 are all H.
5. A compound according to any preceding claim wherein: (i) R3 is CN, OH or CO2H, and R1, R2, R4 and R5 are all H;
(ii) R1 is OH, R3 is CO2H, CO2Me or H, and R2, R4 and R5 are all H;
(iii) R1 is OMe, R4 is CO2Me or CO2H, and R2, R3 and R5 are all H;
(iv) R2 and R4 are CO2Me, and R1, R3 and R5 are all H;
(viii) R3 is CO2Me or CO2Et, R4 is OH or OMe, and R1, R2 and R5 are all H; or
(vi) R3 is OH, R4 is CO2Me, CO2Et or CO2H, and R1, and R2 and R5 are all H.
6. A compound according to claim 1 of formula la, or a pharmaceutically acceptable salt thereof
Figure imgf000084_0001
la
wherein R1"5 are as defined above in claim 1.
7. A compound according to claim 6 wherein:
(i) R1 is OH, R3 is CO2R7, CONR8R9, CONHR10, CO2(CH2)nCO2Rπ, CO2(CH2)mO(CO)R12, CO2CHR13COR14, CONH(CH2)pCO2R15, CONH(CH2)q O(CO)R16 or CONHCHR17COR18, and R2, R4 and R5 are all H; or (ii) R1 is OR6, R5 is CO2H, CO2R7 CONR8R9, CONHR10, CO2(CH2)nCO2Rn, CO2(CH2)mO(CO)R12, CO2CHR13COR14, CONH(CH2)pCO2R15, CONH(CH2)q O(CO)R16 or CONHCHR17COR18, and R2"4 are all H.
8. A compound according to claim 7 wherein:
(i) R1 is OH, R3 is CO2R7, and R2, R4 and R5 are all H; or (ii) R1 is OR6, R5 is CO2H or CO2R7, and R2"4 are all H.
9. A compound according to claim 8 wherein:
(i) R1 is OH, R3 is CO2-alkyl, and R2, R4 and R5 are all H; or (ii) R1 is O-alkyl, R5 is CO2H or CO2alkyl, and R2"4 are all H.
10. A compound according to claim 9 wherein:
(i) R1 is OH, R3 is CO2Me, and R2, R4 and R5 are all H; or (ii) R1 is OMe, R5 is CO2H or CO2Me, and R2"4 are all H.
11. A compound according to claim 1 of formula lb, or a pharmaceutically acceptable salt thereof
Figure imgf000085_0001
lb
wherein R , 1-"5 are as defined above in claim 1.
12. A compound according to claim 11 wherein:
(i) R1 is OH, R3 is CO2R7, CONR8R9, CONHR10, CO2(CH2)nCO2Rn, CO2(CH2)mO(CO)R12, CO2CHR13COR14, CONH(CH2)pCO2R15, CONH(CH2)q O(CO)R16 or CONHCHR17COR18, and R2, R4 and R5 are H; (ii) R2 is OR6, R3 is CO2R7, CONR8R9, CONHR10, CO2(CH2)nCθ2Rπ, CO2(CH2)mO(CO)R12, CO2CHR13COR14, CONH(CH2)pCO2R15, CONH(CH2)q O(CO)R16 or CONHCHR17COR18, and R1, R4 and R5 are H; (iii) R2 is CO2R7, CONR8R9, CONHR10, CO2(CH2)nCθ2Rπ, CO2(CH2)mO(CO)R12, CO2CHR13COR14, CONH(CH2)pCO2R15, CONH(CH2)q O(CO)R16 or CONHCHR17COR18, R3 is OH, and R1, R4 and R5 are H.
13. A compound according to claim 12 wherein:
(i) R1 is OH, R3 is CO2R7, and R2, R4 and R5 are H; . (ii) R2 is OR6, R3 is CO2R7, and R1, R4 and R5 are H; (iii) R2 is CO2R7, R3 is OH, and R1, R4 and R5 are H.
14. A compound according to claim 13 wherein:
(i) R1 is OH, R3 is CO2-alkyl, and R2, R4 and R5 are H; (ii) R2 is O-alkyl, R3 is CO2-alkyl, and R1, R4 and R5 are H; (iii) R2 is CO2-alkyl, R3 is OH, and R1, R4 and R5 are H.
15. A compound according to claim 14 wherein:
(i) R1 is OH, R3 is CO2Me, and R2, R4 and R5 are H; (ii) R2 is OMe, R3 is CO2Me, and R1, R4 and R5 are H; (iii) R2 is CO2Me, R3 is OH, and R1 , R4 and R5 are H.
16. A compound according to claim 1 of formula Ic, or a pharmaceutically acceptable salt thereof
Figure imgf000086_0001
Ic
wherein R , 1-"5 are as defined above in claim 1.
17. A compound according to claim 16 wherein: (i) R1_5 ae H;
(ii) R3 is CN, and R1, R2, R4 and R5 are H; (iii) R2 and R4 are CO2R7, CONR8R9, CONHR10, CO2(CH2)„CO2R11, CO2(CH2)mO(CO)R12, CO2CHR13COR14, CONH(CH2)pCO2R15, CONH(CH2)q O(CO)R16 or CONHCHR17COR18, and R1, R3 and R5 are H; or (iv) R1 is OH, R3 is COR7 and R2, R4 and R5 are all H.
18. A compound according to claim 17 wherein R2 and R4 are CO2R7, and R1, R3 and R5 are H.
19. A compound according to claim 17 wherein R2 and R4 are CO2-alkyl, and R1, R3 and R5 are H, or R1 is OH, R3 is CO2-alkyl and R2, R4 and R5 are all H.
20. A compound according to claim 19 wherein R2 and R4 are CO2Me, and R1, R3 and R5 are H, or R1 is OH, R3 is CO2Me and R2, R4 and R5 are all H.
21. A compound according to claim 1 of formula Id, or a pharmaceutically acceptable salt thereof
Figure imgf000087_0001
Id
wherein R , 1-"5 are as defined above in claim 1.
22. A compound according to claim 21 wherein:
(i) R3 is CO2H, CO2R7, CONR8R9, CONHR10, Cθ2(CH2)nCθ2Rπ, CO2(CH2)mO(CO)R12, CO2CHR13COR14, CONH(CH2)pCO2R15, CONH(CH2)q O(CO)R16 or CONHCHR17COR18, R1 is OH, and R2, R4 and R5 are H; or
(ii) R3 is OH, and R1, R2, R4 and R5 are H;
23. A compound according to claim 22 wherein:
R3 is CO2H, or CO2R7, R1 is OH, and R2, R4 and R5 are H.
24. A compound according to claim 23 wherein:
R3 is CO2H, or CO2-alkyl, R1 is OH, and R2, R4 and R5 are H.
25. A compound according to claim 24 wherein:
R3 is CO2H, or CO2Me, R1 is OH, and R2, R4 and R5 are H.
26. A compound according to claim 1 of formula le, or a pharmaceutically acceptable salt thereof
Figure imgf000088_0001
le
wherein R1"5 are as defined above in claim 1.
27. A compound according to claim 26 wherein:
(i) R1 is OR6, R5 is CO2H, CO2R7, CONR8R9, CONHR10, CO2(CH2)nCO2Rn, CO2(CH2)mO(CO)R12, CO2CHR13COR14, CONH(CH2)pCO2R15, CONH(CH2)q O(CO)R16 or CONHCHR17COR18, and R2, R3 and R4 are H;
(ii) R1 is OH, R3 is CO2H or H, and R2, R4 and R5 are H;
(iii) R3 is CN or CO2H, and R1, R2, R4 and R5 are H;
(iv) R2 is CO2H, CO2R7, CONR8R9, CONHR10, CO2(CH2)nCO2Rn, CO2(CH2)mO(CO)R12, CO2CHR13COR14, CONH(CH2)pCO2R15, CONH(CH2)q O(CO)R16 or CONHCHR17COR18, R3 is OH, R1, R4 and R5 are H; or
(v) R2 is OH, R3 is CO2R7, CONR8R9, CONHR10, CO2(CH2)nCO2Ru, CO2(CH2)mO(CO)R12, CO2CHR13COR14, CONH(CH2)pCO2R15, CONH(CH2)q O(CO)R16 or CONHCHR17COR18, and R1, R4 and R5 are H.
28. A compound according to claim 27 wherein:
(i) R1 is OR6, R5 is CO2H or CO2R7, and R2, R3 and R4 are H;
(ii) R2 is CO2H or CO2R7, R3 is OH, R1 , R4 and R5 are H; or
(iii) R2 is OH, R3 is CO2R7, and R1, R4 and R5 are H.
29. A compound according to claim 28 wherein:
(i) R1 is O-alkyl, R5 is CO2H or CO2-alkyl, and R2, R3 and R4 are H;
(ii) R2 is CO2H or CO2alkyl, R3 is OH, R1, R4 and R5 are H; or
(iii) R2 is OH, R3 is CO2-alkyl, and R1, R4 and R5 are H.
30. A compound according to claim 29 wherein:
(i) R1 is OMe, R5 is CO2H or CO2Me, and R2, R3 and R4 are H;
(ii) R2 is CO2H or CO2Et, R3 is OH, R1, R4 and R5 are H; or
(iii) R2 is OH, R3 is CO2Et, and R1, R4 and R5 are H.
31. A compound according to claim 1 of formula If, or a pharmaceutically acceptable salt thereof
Figure imgf000089_0001
If
wherein R 1 1-"5 are as defined above in claim 1.
32. A compound according to claim 31 wherein:
(i) R1 is OH, R3 is H, CO2H, CO2R7, CONR8R9, CONHR10, CO2(CH2)nCO2R1 CO2(CH2)mO(CO)R12, CO2CHR13COR14, CONH(CH2)pCO2R15, CONH(CH2)q O(CO)R16 or CONHCHR17COR18, and R2, R4 and R5 are H; or (ii) R1 is OR6, R4 is CO2R7, CONR8R9, CONHR10, CO2(CH2)nCO2Rn, CO2(CH2)mO(CO)R12, CO2CHR13COR14, CONH(CH2)pCO2R15, CONH(CH2)q O(CO)R16 or CONHCHR17COR18, and R2, R3 and R5 are H.
33. A compound according to claim 32 wherein:
(i) R1 is OH, R3 is H, CO2H or CO2R7, and R2, R4 and R5 are H; or (ii) R1 is OR6, R4 is CO2R7, and R2, R3 and R5 are H.
34. A compound according to claim 33 wherein:
(i) R1 is OH, R3 is H, CO2H or CO2-alkyl, and R2, R4 and R5 are H; or (ii) R1 is O-alkyl, R4 is CO2-alkyl, and R2, R3 and R5 are H.
35. A compound according to claim 34 wherein:
(i) R1 is OH, R3 is H, CO2H or CO2Me, and R2, R4 and R5 are H; or (ii) R1 is OMe, R4 is CO2Me, and R2, R3 and R5 are H.
36. A compound according to claim 1 of formula Ig, or a pharmaceutically acceptable salt thereof
Figure imgf000090_0001
ig
wherein R1"5 are as defined above in claim 1.
37. A compound according to claim 36 wherein R3 is OH, and R1, R2, R4 and R5 are H.
38. A pharmaceutical composition comprising a compound according to any one of claims 1 to 37 admixed with a pharmaceutically acceptable diluent, excipient or carrier.
39. Use of a compound according to any preceding claim in the preparation of a medicament for treating Alzheimer's disease.
40. Use of a compound of formula I, or a pharmaceutically acceptable salt thereof,
Figure imgf000091_0001
wherein
X is selected from I
Figure imgf000091_0002
Y is selected from SO2NH, CONH, CH=N, CH2NH and CH=CH;
R1"5 are each independently selected from H, OH, OR6, CO2H, CO2R7, NO2, CN
CONR8R9, CONHR10, CO2(CH2)nCO2R11, CO2(CH2)mO(CO)R12, CO2CHR13COR14,
CONH(CH2)pCO2R15, CONH(CH2)qO(CO)R16, CONHCHR17COR18;
R6"18 are each independently hydrocarbyl; n, m, p and q are each independently 1, 2, 3 or 4. in the preparation of a medicament for treating transmissible spongiform encephalopathy (TSE).
41. Use according to claim 40 wherein said compound is as defined in any one of claims 1 to 37.
42. Use according to claim 40 wherein said compound is as defined in claim 36 and wherein: R3 is OH, and R1, R2, R4 and R5 are H; or R3 is OH, R2 is CO2H, and R1, R4 and R5 are H.
43. A method of treating, or preventing the onset of, transmissible spongiform encephalopathy (TSE) in a subject, said method comprising administering to the subject a compound of formula II as defined in any one of claims 40 to 42.
44. A method according to claim 42 or use according to any one of claims 40 to 42 wherein the TSE is selected from Creutzfeldt Jakob Disease (CJD), Gerstmann- Staussler-Scheinker Syndrome (GSS), Familial Fatal Insomnia (FFI), Kuru, variant CJD, Bovine Spongiform Encephalopathy (BSE) and scrapie.
45. A method according to claim 43 or 44, or use according to any one of claims 40 to 42 wherein the compound of formula II is administered in an amount sufficient to inhibit the infectivity of the abnormal form of prion protein (PrPSc)
46. A method according to claim 43 or 44, or use according to any one of claims 40 to 42, wherein the compound of formula II is administered in an amount sufficient to inhibit the polymerisation of PrPc by PrP-res.
47. Use of a compound of formula II, or a pharmaceutically acceptable salt thereof, as defined in any one of claims 40 to 42, in an assay for identifying further candidate compounds capable of inhibiting the polymerisation of PrP by PrP-res.
48. Use of a compound of formula II, or a pharmaceutically acceptable salt thereof, as defined in any one of claims 40 to 42, in an assay for identifying further candidate compounds capable of inhibiting the infectivity of the abnormal form of prion protein (PrPSc).
49. Use according to claim 47 or 48 wherein the assay is a competitive binding assay.
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