WO2013036196A1 - Novel thiophene compounds and method for in vivo imaging - Google Patents

Novel thiophene compounds and method for in vivo imaging Download PDF

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WO2013036196A1
WO2013036196A1 PCT/SE2012/050946 SE2012050946W WO2013036196A1 WO 2013036196 A1 WO2013036196 A1 WO 2013036196A1 SE 2012050946 W SE2012050946 W SE 2012050946W WO 2013036196 A1 WO2013036196 A1 WO 2013036196A1
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mmol
added
thiophen
mixture
mecn
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PCT/SE2012/050946
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French (fr)
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Peter ÅSBERG
Kristin Hammer
Johan Olsson
Martin Henriksson
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Biochromix Ab
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D333/00—Heterocyclic compounds containing five-membered rings having one sulfur atom as the only ring hetero atom
    • C07D333/02—Heterocyclic compounds containing five-membered rings having one sulfur atom as the only ring hetero atom not condensed with other rings
    • C07D333/04—Heterocyclic compounds containing five-membered rings having one sulfur atom as the only ring hetero atom not condensed with other rings not substituted on the ring sulphur atom
    • C07D333/06—Heterocyclic compounds containing five-membered rings having one sulfur atom as the only ring hetero atom not condensed with other rings not substituted on the ring sulphur atom with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached to the ring carbon atoms
    • C07D333/24—Radicals substituted by carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/041—Heterocyclic compounds
    • A61K51/0429—Heterocyclic compounds having sulfur as a ring hetero atom
    • A61K51/0431—Heterocyclic compounds having sulfur as a ring hetero atom having five-membered rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D285/00—Heterocyclic compounds containing rings having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by groups C07D275/00 - C07D283/00
    • C07D285/01—Five-membered rings
    • C07D285/02—Thiadiazoles; Hydrogenated thiadiazoles
    • C07D285/04—Thiadiazoles; Hydrogenated thiadiazoles not condensed with other rings
    • C07D285/12—1,3,4-Thiadiazoles; Hydrogenated 1,3,4-thiadiazoles
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
    • C07D403/10—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings linked by a carbon chain containing aromatic rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D409/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms
    • C07D409/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing two hetero rings
    • C07D409/04—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing two hetero rings directly linked by a ring-member-to-ring-member bond
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D409/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms
    • C07D409/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing two hetero rings
    • C07D409/10—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing two hetero rings linked by a carbon chain containing aromatic rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D409/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms
    • C07D409/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing three or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D413/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms
    • C07D413/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings
    • C07D413/10—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings linked by a carbon chain containing aromatic rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
    • C07D417/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings
    • C07D417/10—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings linked by a carbon chain containing aromatic rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
    • C07D417/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing three or more hetero rings

Definitions

  • Novel thiophene compounds and method for in vivo imaging are novel thiophene compounds and method for in vivo imaging
  • the present invention relates to novel labelled compounds for use in imaging amyloid deposits and aggregated protein in living patients. It further relates to imaging methods using labelled or unlabelled compounds and the use of unlabelled molecules in such methods.
  • Imaging that allows non-invasive assessment of biological and biochemical processes in living subjects is of great importance. Noninvasive identification and observation of the molecular events underlying neurodegenerative diseases is especially beneficial since it is difficult to sample information from the brain in other ways, like autopsies. Therefore imaging technologies are important when it comes to increase our understanding of diseases, diagnosing diseases, for drug activity assessment, during preclinical development, during clinical drug development and to monitor treatment of diseases.
  • Natural biopolymers such as proteins, frequently have ordered conformations, such as alpha- helix and beta-sheets, which contribute to the three-dimensional ordered structure and the specific function of the biopolymer.
  • ordered conformations such as alpha- helix and beta-sheets
  • the structure of a protein is essential for the protein's function; it has been shown by many scientists that an unfolded protein may not be functional. More important, in the last few years there is increasing awareness of the danger of protein misfolding and misassembly into for example amyloid and other pathological forms.
  • Misfolding can change a protein from something that is useful into nonfunctional, harmful or even toxic.
  • Human health relies on properly folded protein, and in vivo deposition of amyloid fibrils is associated with many diseases related to protein conformation, including
  • AD Alzheimer's disease
  • HD Huntington's disease
  • ALS Amyotrophic Lateral Sclerosis
  • PD Parkinson's disease
  • Tauopathies multiple
  • systemic amyloidoses and the prion diseases.
  • the prion diseases i.e. transmissible spongiform encephalopathy (TSE), in animals [e.g.
  • PrP Sc normal cellular prion protein
  • PrP Sc an infectious pathogenic disease-associated isoform
  • proteins frequently alter their conformation due to different external stimuli and the importance of conformational changes of proteins leading to pathogenic states has been well documented.
  • proteins can aggregate into characteristic fibrillar assemblies, known as amyloid fibrils.
  • beta-sheet rich protein assemblies have distinctively different conformations compared to that of the native state.
  • the misfolded prion protein is even self-propagating (infectious), a property which is entirely encoded within the misfolded conformation.
  • ethylenedioxythiophene EDOT
  • benzothiadizole fluorene
  • fluorene fluorene
  • phenyl in homo and hetero oligomers and polymers with ionic or polar sidechains
  • EDOT ethylenedioxythiophene
  • benzothiadizole fluorene
  • fluorene fluorene
  • phenyl in homo and hetero oligomers and polymers with ionic or polar sidechains
  • the interaction between amyloid like fibrils of insulin and anionic, zwitterionic and cationic poly- and oligo thiophenes was shown by WO 2005/109005.
  • Several of the mentioned oligomers and polymers have been shown to bind to amyloid, aP and PrP deposits in histological sections, as disclosed in WO 2007/091973. Imaging using thiophene derivatives is known from WO 2010/044744.
  • polymer of EDOT showed high affinity for amyloid like fibrils [Hamedi, M. et al.;. Nano Lett.; (2008); 8, 1736-1740]. Moreover a substituted polyfluorene and an alternating polyfluorene with a polyethylene oxide were demonstrated to strongly associate with amyloid like fibrils in vitro [Tanaka, H. et al.; Nano Lett.; (2008) 8, 2858- 2861].
  • the present invention relates to novel compounds of formula (I) (C) n -B-(A) m -B-(C) n (I) wherein m is 0 or 1, and n is independently 0, 1, 2 or 3,
  • each B and each C are independently selected from phenylene and five- and six-membered heteroaromatic rings, and for a terminal ring B or C also from bicyclic heteroaromatic fused rings having seven to ten ring members, wherein the bond between at least two of the rings A to C may be replaced by a carbonyl group ( -CO- ), wherein at least two of the rings A to C are substituted with one or two groups R, and wherein each ring A to C further optionally is substituted with one or two groups R 1 , wherein each group R is independently selected from hydroxy, hydroxyalkyl, hydroxyalkoxy, hydroxyalkoxyalkyl, hydroxypolyoxyalkylene, alkoxy, alkoxyalkyl, polyoxyalkylene, carboxy, carboxyalkyl, carboxyalkoxy, carboxyalkoxyalkyl, carboxypolyoxyalkylene, alkoxycarbonyl, alkoxycarbonylalkyl, alkoxycarbonylalkoxy
  • alkylamino)(carboxy)alkoxy (dialkylamino)(carboxy)alkoxy, (amino)(carboxy)alkoxyalkyl, (alkylamino)(carboxy)alkoxyalkyl, (dialkylamino)(carboxy)alkoxyalkyl,
  • alkoxycarbonyl)(dialkylamino)alkoxy (alkoxycarbonyl)(dialkylamino)alkoxy, (alkoxycarbonyl)(amino)alkoxyalkyl, (alkoxycarbonyl)(alkylamino)alkoxyalkyl, (alkoxycarbonyl)(dialkylamino)alkoxyalkyl, (alkoxycarbonyl)(amino)polyoxyalkylene, (alkoxycarbonyl)(alkylamino)polyoxyalkylene, (alkoxycarbonyl)(dialkylamino)polyoxyalkylene, acylamino, acylaminoalkyl,
  • acylaminoalkoxy acylaminoalkoxyalkyl, acylaminopolyoxyalkylene, acylalkylamino, acylalkylaminoalkyl, acylalkylaminoalkoxy, acylalkylaminoalkoxyalkyl,
  • hydrazinocarbonylpolyoxyalkylene nitro, nitroalkyl, nitroalkoxy, nitroalkoxyalkyl, nitropolyoxyalkylene, cyano, cyanoalkyl, cyanoalkoxy, cyanoalkoxyalkyl,
  • sulfopolyoxyalkylene alkylsulphonyl, alkylsulphinyl, alkylaminosulphonyl, acyloxyalkyl, carboxyalkenyl, pyrrolidincarbonyl, morpholinoalkyl, cycloalkylcarbamoyl, trifluoroxy, carbamoyl, hydroxyalkylaminoalkyl, carboxyalkylaminoalkyl, aminoalkylaminoalkyl, piperidinealkyl, piperazinealkyl, or any two groups R attached to the same ring taken together are alkylenedioxy, optionally substituted with sulfoalkyl, sulfoalkoxy, sulfoalkoxyalkyl or sulfopolyoxyalkylene; and wherein each group R 1 is independently selected from halogen, alkyl, alkenyl, alkynyl, trifluoro, with the provisos that:
  • any H2 group may optionally be protected as a tert-butyl carbamate, benzyl carbamate or 9-fluorenylmethyl carbamate or substituted with a biotinyl moiety; wherein any alkyl or alkylene moiety is Ci-6-alkyl or Ci-6-alkylene, and any alkenyl and alkynyl moiety is C 2 -6-alkenyl and C 2 -6-alkynyl, respectively; or a pharmaceutically acceptable salt thereof, wherein the compounds are labelled with a label detectable with an in vivo imaging method.
  • the invention relates
  • the invention relates to a method for imaging of misfolded or aggregated forms of proteins in a sample or subject, comprising the steps of:
  • the invention relates to a method for diagnosis of a disease involving misfolded or aggregated forms of proteins in a mammalian subject, comprising the steps of
  • the invention relates to the use of a compound of formula I as defined above in a method according to the third or fourth aspect above.
  • the invention relates to the use of a labelled or unlabeled compound of formula I as defined above in imaging ⁇ in vivo or ex vivo).
  • Figure 1 is a plot of normalized fluorescence vs time showing fluorescence dynamics in plaques in in vivo imaging with a compound of Example 1.
  • Figure 2 is a plot of fluorescence vs time showing fluorescence dynamics in plaques in in vivo imaging with a compound of Example 21. Detailed description of the invention
  • the present invention relates to a compound of formula (I), optionally labeled with a label detectable with an in vivo imaging method, or a
  • in vivo imaging refers to any method which permits the detection of a compound in vivo as described herein.
  • a method for in vivo imaging is e.g. imaging of compounds with near infrared (NIR) spectra.
  • NIR imaging can be non-invasive imaging using NIR light and methods of NIR imaging are known in the art [Hintersteiner, M. et al, Nat. Biotechnol. 23, 577-583 (2005)]. Further methods include PET, SPECT, MRS, MRI, CAT, TPM and MPM.
  • NIR Near Infra-red imaging.
  • PET Positron Emission Tomography.
  • SPECT Single Photon Emission Computed Tomography.
  • MRS Magnetic Resonance Spectroscopy
  • MRI Magnetic Resonance Imaging
  • CAT means Computer Assisted Tomography.
  • TPM Two Photon Fluorescence Microscopy Imaging.
  • MPM Multi Photon Microscopy Imaging
  • alkyl or alkylene moiety is a C1-C6 alkyl or alkylene moiety, e.g. a C1-C4 alkyl or alkylene moiety and is intended to encompass also the alkyl or alkylene portion of any functional group, e.g. an alkoxy, alkylamino or carboxypolyoxyalkylene group.
  • any alkyl in an alkoxy or alkylamino group according to the invention is a C1-C6 alkyl group, e.g. a C1-C4 alkyl group.
  • alkenyl is a C2-C 6 alkenyl moiety, e.g. a C1-C4 alkenyl moiety, and is intended to encompass also the alkenyl portion of any functional group,
  • alkynyl is a C2-C6 alkynyl moiety, e.g. a C2-C4 alkynyl moiety, and is intended to encompass also the alkynyl portion of any functional group,
  • any alkyl, or alkylene alkenyl or alkynyl group according to the invention may be branched or unbranched.
  • alkyl includes the monoradical derived from a branched or unbranched Ci-C 6 alkane, or C1-C4 alkane.
  • alkyl group examples include methyl (CH3-), ethyl (CH3CH2-), propyl (-CH 2 CH 2 CH 2 -) and isopropyl ((CH 3 ) 2 CH-).
  • alkylene includes the diradical derived from a branched or unbranched Ci-C 6 alkane, or C1-C4 alkane.
  • alkylene group examples include methylene (-CH 2 -), ethylene
  • phenylene include diradicals derived from benzene, pyridine, pyrazine, pyridazine, pyrimidine, thiphene, thiazole, thiadiazole, oxazole, furane, triazole, pyrazole, imidazole, pyrrolylene, benzo[c]thiophene, benzodiazole
  • hydroxypolyoxyalkylene include an alkyl, alkoxy, alkoxyalkyl and polyoxyalkylene radical, respectively, carrying a hydroxy function.
  • alkoxy includes a group R-0-, wherein R is alkyl.
  • alkoxyalkyl includes an alkyl radical carrying an alkoxy function.
  • polyoxyalkylene includes a group of the general formula
  • n is an integer from 1 to 6, e.g. from 1 to 4, or 1 or 2; R is an alkyl radical and each R ' is an independently selected alkylene radical.
  • R is an alkyl radical and each R ' is an independently selected alkylene radical.
  • Carboxypolyoxyalkylene include an alkyl, alkoxy, alkoxyalkyl and polyoxyalkylene radical, respectively, carrying a carboxy function.
  • alkoxycarbonyl includes a radical -COOR, viz. an alkyl ester of a carboxylic acid function.
  • alkoxycarbonylalkyl include an alkyl, alkoxy, alkoxyalkyl and polyoxyalkylene radical, respectively, carrying an alkoxycarbonyl function.
  • alkylamino includes - HR wherein R is alkyl.
  • dialkylamino includes -NRR ' wherein R and R ' are independently selected alkyl groups.
  • aminoalkyl include an alkyl radical carrying an amino, alkylamino or dialkylamino function, respectively.
  • aminoalkoxy includes an alkoxy radical carrying an amino, alkylamino or dialkylamino function, respectively.
  • aminoalkoxyalkyl includes an alkoxyalkyl radical carrying an amino, alkylamino or dialkylamino function, respectively.
  • aminopolyoxyalkylene "alkylaminopolyoxyalkylene”
  • dialkylaminopolyoxyalkylene include a polyoxyalkylene radical carrying an amino, alkylamino or dialkylamino function, respectively.
  • acylamino includes a moiety - H-C(0)-alkyl.
  • acylaminoalkyl acylaminoalkoxy
  • acylaminoalkoxyalkyl acylaminoalkoxyalkyl
  • acylaminopolyoxyalkylene include an alkyl, alkoxy, alkoxyalkyl and polyoxyalkylene radical, respectively, carrying an acylamino function.
  • acylalkylamino includes a moiety - R-C(0)-alkyl wherein R is alkyl.
  • acylalkylaminoalkyl include an alkyl, alkoxy, alkoxyalkyl and polyoxyalkylene radical, respectively, carrying an acylalkylamino function.
  • hydrazinocarbonyl includes a moiety a -C(0) H- H 2 .
  • hydrazinocarbonylalkoxyalkyl and “hydrazinocarbonylpolyoxyalkylene”, include an alkyl, alkoxy, alkoxyalkyl and polyoxyalkylene radical, respectively, carrying a hydrazinocarbonyl function.
  • amino(carboxy)alkoxyalkyl and “(amino)(carboxy)polyoxyalkylene” include an alkyl, alkoxy, alkoxyalkyl and polyoxyalkylene radical, respectively, carrying a carboxy and an amino function, preferably attached to the same carbon atom.
  • alkylamino)(carboxy)alkoxyalkyl and "(alkylamino)(carboxy)polyoxyalkylene”, include an alkyl, alkoxy, alkoxyalkyl and polyoxyalkylene radical, respectively, carrying a carboxy and an alkylamino function, preferably attached to the same carbon atom.
  • dialkylamino)(carboxy)alkoxyalkyl and "(dialkylamino)(carboxy)polyoxyalkylene" include an alkyl, alkoxy, alkoxyalkyl and polyoxyalkylene radical, respectively, carrying a carboxy and a dialkylamino function, preferably attached to the same carbon atom.
  • alkoxycarbonyl)(amino)alkoxyalkyl and "(alkoxycarbonyl)(amino)polyoxyalkylene”, include an alkyl, alkoxy, alkoxyalkyl and polyoxyalkylene radical, respectively, carrying an alkoxycarbonyl and an amino function, preferably attached to the same carbon atom.
  • (alkoxycarbonyl)(alkylamino)alkyl include an alkyl, alkoxy, alkoxyalkyl and polyoxyalkylene radical, respectively, carrying an alkoxycarbonyl and an alkylamino function, preferably attached to the same carbon atom.
  • (alkoxycarbonyl)(dialkylamino)alkyl include an alkyl, alkoxy, alkoxyalkyl and polyoxyalkylene radical, respectively, carrying an alkoxycarbonyl and a dialkylamino function, preferably attached to the same carbon atom.
  • nitroalkyl include an alkyl, alkoxy, alkoxyalkyl and polyoxyalkylene radical, respectively, carrying a nitro function.
  • cyanoalkyl include an alkyl, alkoxy, alkoxyalkyl and polyoxyalkylene radical, respectively, carrying a cyano function.
  • sulfoalkyl include an alkyl, alkoxy, alkoxyalkyl and polyoxyalkylene radical, respectively, carrying a sulfo function.
  • alkylsulphonyl and “alkylaminosulphonyl” include an alkyl and alkylamino radical, respectively, carrying a sulphonyl function.
  • alkyl sulphinyl includes an alkyl radical carrying a sulphinyl function.
  • the compounds of formula I can be regarded as oligomers of conjugated monomers, typically trimers, tetramers, pentamers, hexamers, heptamers, octamers, and nonamers, wherein the backbone chain is formed from monomers selected from benzene and five or six-membered heteroaromatic rings, and wherein the backbone of monomers support at least two side-chains or groups of ionic or polar character (the group R in formula I above).
  • one or more of the monomers may be substituted with one or more, e.g. one or two, other groups, such as, for example, halogen, e.g.
  • the backbone may include one or more monomers in the form of a bicyclic fused heteroaromatic rings having 7 to 10 ring members, typically in end position of the backbone.
  • heteroaromatic rings include, without limitation thereto, pyridine, pyrazine, pyridazine, pyrimidine, thiophene, thiazole, isothiazole, thiadiazole, oxazole, isoxazole, furane, triazole, triazine, oxadiazole, pyrazole, imidazole and pyrrole.
  • bicyclic fused heteroaromatic rings include, without limitation thereto, benzothiophene, benzodiazole, indole, benzoxazole, benzothiazole, benzimidazole, benzoisothiazole, and benzofurane.
  • the oligomer backbone may consist of a single monomer, such as thiophenes, or of mixed monomers, and may be symmetric or asymmetric.
  • the backbone may include one or more carbonyl groups replacing the bond between two adjacent rings.
  • the backbone comprises a central thiophene ring, i.e. A in formula I is thienylene.
  • the backbone comprises a central benzene ring, i.e, A in formula I is phenylene.
  • the backbone comprises a central phenylene ring surrounded by thiophene rings.
  • the backbone comprises a number of conjugated thiophene rings and benzene rings as end monomers.
  • Exemplary trimeric backbones include (without limitation thereto):
  • Exemplary tetrameric backbones include (without limitation thereto): thieny 1 ene-thi eny 1 ene-thi eny 1 ene-thi eny 1 ene
  • Exemplary pentameric backbones include (without limitation thereto): thieny 1 ene-thi eny 1 ene-thi eny 1 ene-thi eny 1 ene-thieny 1 ene
  • Exemplary hexameric backbones include (without limitation thereto):
  • Exemplary heptameric backbones include (without limitation thereto):
  • A is thienylene, it is preferably a 2,5-diradical:
  • A is another five-membered heteroaromat having a single heteroatom.
  • A is phenylene, it is preferably a 1,4-diradical:
  • ring A is unsubstituted.
  • the group R in formula I is a group R 2 for ring A, a group R 3 for ring B, and a group R 4 for ring C, wherein each R 2 , R 3 and R 4 are independently selected from carboxy, carboxyalkyl, aminoalkyl, acylaminoalkyl, acyloxyalkyl, hydroxyalkyl,
  • alkylsulphonyl alkylsulphinyl, alkylaminosulphonyl, alkoxycarbonylalkyl, mo holinoalkyl, carboxyalkenyl, hydroxy, alkoxycarbonyl, pyrrolidinecarbonyl, morpholino,
  • each R 2 , R 3 and R 4 are independently selected from carboxy, carboxyalkyl, aminoalkyl, acylaminoalkyl, acyloxyalkyl, hydroxyalkyl, alkylsulphonyl, alkylsulphinyl, alkylaminosulphonyl, alkoxycarbonylalkyl, morpholinoalkyl, carboxyalkenyl, hydroxy, alkoxycarbonyl, pyrrolidinecarbonyl, morpholino, cycloalkylcarbamoyl, cyano, trifluoroxy, and carbamoyl.
  • each R 2 , R 3 and R 4 are independently selected from carboxy, carboxyalkyl, aminoalkyl, acylaminoalkyl, acyloxyalkyl, hydroxyalkyl, alkylsulphonyl, alkylsulphinyl, and alkylaminosulphonyl,
  • each R 2 and R 3 are independently selected from carboxy, carboxyalkyl, alkoxycarbonylalkyl, morpholinoalkyl, aminoalkyl, acylaminoalkyl, hydroxyalkoxyalkyl, carbamoylalkyl, hydroxyalkylaminoalkyl, cyanoalkyl, aminoalkylaminoalkyl,
  • each group R 3 is independently selected from carboxy-Ci-4-alkyl, hydroxy-Ci-4-alkyl, amino-Ci-4-alkyl, Ci-6-acylamino-Ci-4-alkyl, Ci-4-alkoxycarbonylamino- Ci-4-alkyl, and morpholino-Ci-4-alkyl.
  • each R 4 is independently selected from carboxy, alkyl sulphonyl, alkylaminosulphonyl, hydroxyalkyl, carboxyalkenyl, hydroxy, alkoxycarbonyl, pyrrolidinoyl, morpholino, cycloalkylcarbamoyl (preferably cyclopropylcarbamoyl), cyano, trifluoroxy, and carbamoyl.
  • ring A is unsubstituted.
  • each R 4 is independently selected from carboxy, carboxy-Ci-4-alkyl, Ci-4-alkyl sulphonyl, carboxy-Ci-4-alkylene, hydroxy, Ci-4-alkyl, Ci-4-alkoxycarbonyl, pyrrolidinoyl, morpholino, C3-6-cycloalkylcarbamoyl, cyano, trifluoroxy, carbamoyl and amino-Ci-4-alkyl.
  • each R 2 , R 3 and R 4 may be independently selected
  • R 3 is preferably other than carboxymethyl, and preferably other than carboxyalkyl.
  • R 2 is preferably other than carboxyethyl, and preferably other than carboxyalkyl.
  • R 4 on the non-terminal rings C is preferably other than carboxymethyl, preferably other than carboxyalkyl.
  • a substituent or substituents R 4 on a terminal ring C is independently selected from carboxy, alkyl sulphonyl (e.g. methyl sulphonyl), alkylaminosulphonyl (e.g. methylaminosulphonyl), alkyl sulphinyl (e.g. methyl sulphinyl), carboxyalkenyl (e.g.
  • carboxyvinyl hydroxy, alkoxy (e.g. methoxy), cyano, alkoxycarbonyl (e.g.
  • the compound of formula I is a heptamer. In some embodiments thereof, the compound is a heptathiophene of formula (II)
  • each o is independently selected from 0-2; e.g. 0 or 1, and in particular is 0;
  • each p is independently selected from 0-2; e.g. 0 or 1, and in particular is 1;
  • each u is independently selected from 0-2; e.g. 0 or 1, and in particular is 0;
  • each v is independently selected from 0-3; e.g. 0-2, and in particular is 0 or 1,
  • each R 5 , R 6 , R 7 and R 8 are independently selected from carboxy, carboxyalkyl, aminoalkyl, acylaminoalkyl, acyloxyalkyl, hydroxyalkyl, alkylsulphonyl, alkylsulphinyl,
  • alkylaminosulphonyl alkoxycarbonylalkyl, morpholinoalkyl, carboxyalkenyl, hydroxy, alkoxycarbonyl, pyrrolidinecarbonyl, morpholino, cycloalkylcarbamoyl, cyano, trifluoroxy, carbamoyl, hydroxyalkoxyalkyl, carbamoylalkyl, hydroxyalkylaminoalkyl, cyanoalkyl, alkylaminoalkyl, aminoalkylaminoalkyl, carboxyalkylaminoalkyl, piperidinealkyl, and piperazinealkyl, and each R 8 may additionally be independently selected from halogen, alkyl and trifluoro, or a pharmaceutically acceptable salt thereof.
  • R 6 and R 8 are independently selected from carboxy, carboxy-Ci-4-alkyl, and amino-Ci-4-alkyl.
  • the group R 8 is preferably in 3- or 5-position on the thiophene ring.
  • the compounds of the present invention may be prepared by the person of ordinary skill in the art, in the light of the general description herein and the specific illustrating examples.
  • ring structures i.e. benzene, thiophene and other heteroaromates serve as basic monomer units in the inventive compounds. Substitutions of mentioned ring structures can be obtained through conventional chemistry, well known to one skilled in the art of organic synthesis and described in text books of organic synthesis, and exemplified in the synthesis examples below.
  • Stille coupling utilizes the coupling of an organotin compound with an sp 2 -hybridized organic halide catalyzed by a palladium, exemplified by the schematic reaction from three ring units to a trimer-block:
  • Suzuki coupling utilizes a reaction between an aryl- or vinyl-boronic acid or borate ester with a vinyl- or aryl-halide catalyzed by a palladium complex, exemplified by the schematic reaction:
  • PiB is a borate ester
  • the reaction may also be carried out with pseudo-halides such as triflates .
  • asymmetric compounds e.g. a stoichiometric approach may be used, where the amount of reagent added is equimolar to the compound to be derivatized, if this compound has several reaction sites.
  • the individual compounds may be separated by methods of chemical separation. Non-limiting examples of separation include flash column chromatography, preparative HPLC and distillation.
  • the compounds may be used in form of "pharmaceutically acceptable salts", referring to derivatives of the disclosed compounds, where the described compounds are modified by making acid and base salts thereof.
  • pharmaceutically acceptable salts include mineral or organic salts of basic derivatives of the mentioned It- groups such as amines and organic or inorganic, e.g. alkali salts of acidic derivatives of the mentioned R-groups such as carboxylic acids.
  • Conventional non-toxic salts and quaternary ammonium salts are included in pharmaceutically acceptable salts.
  • compositions disclosed in the present invention may be prepared from the inventive compounds described herein that contain a basic or acidic entity by conventional chemical methods.
  • proteopathies Diseases related to misfolded and aggregated proteins have also been termed proteopathies, (Walker and Levine, Curr Opin Investig Drugs. 2002 May; 3(5):782-7). Diseases featuring amyloid proteins are relevant examples for the description of diseases related to misfolded and aggregated proteins, where amyloidosis is known as a disease and may be inherited or acquired. Note that amyloidosis by default usually refers to AA amyloidosis, but any disease related to amyloid proteins, which presents amyloid deposition, is an amyloidosis. For example CJD, vCJD, Alzheimer's Disease, HD, ALS and diabetes are almost never referred to as amyloidoses. A list of proteopaties is given below.
  • Alzheimer's disease Amyloid ⁇ peptide ( ⁇ ); Tau protein
  • Parkinson's disease PD
  • a-Synuclein a-Synuclein
  • ALS Amyotrophic lateral sclerosis
  • GFAP Glial fibrillary acidic protein
  • IAPP Type II diabetes Islet amyloid polypeptide
  • SP-C Pulmonary alveolar proteinosis Surfactant protein C
  • Primary amyloidosis includes mutations in lysozyme, transthyretin, apolipoprotein B, fibrinogen and AL amyloidosis (immunoglobulin light chains, as seen with multiple myeloma).
  • Secondary amyloidosis includes AA amyloidosis (serum amyloid A protein, an acute-phase protein due to chronic inflammation) and Gelsolin amyloidosis (plasma gelsolin fragments).
  • Familial, or hereditary amyloidosis are most commonly caused by mutations in the transthyretin protein, but in rare occurrences can also be caused by apolipoprotein Al, gelsolin, fibrinogen, and lysozyme mutations, primarily caused by genetics, believed to be autosomal dominant, high probability of passage to offspring, Appalachian type amyloidosis and Shar Pei fever for amyloidosis in Shar Peis.
  • organ-specific amyloidosis examples include Diabetes mellitus type 2 (amylin, also known as IAPP), Alzheimer's disease ( ⁇ 39-42), Parkinson's disease (alpha-synuclein), Huntington's disease (huntingtin), Transmissible spongiform encephalopathies (prion protein, PrP), some examples are Creutzfeldt- Jakob disease (PrP in cerebrum), Kuru (diffuse PrP deposits in brain), Fatal Familial Insomnia (PrP in thalamus), Inclusion body myositis and Bovine spongiform encephalopathy (PrP in cerebrum of cows), Congophilic angiopathy (Amyloid beta).
  • Cardiac amyloidosis includes congestive heart failure; some instances (PrP or transthyretin in heart). Another important example is the Iatrogenic conditions like insulin amyloidosis, believed to be caused by injection-administered insulin.
  • Islet amyloid is aggregated fibrils composed primarily of the hormone peptide, islet amyloid polypeptide (IAPP, or amylin), are commonly found in the islets of Langerhans of the pancreas of patients suffering diabetes.
  • IAPP islet amyloid polypeptide
  • Non-disease amyloids are native amyloids in organisms, Curli E. coli Protein (curlin), Yeast Prions [Sup35], Podospora Anserina Prion Het-s, Malarial coat protein, spider silk,
  • Mammalian melanosomes (pMel), Tissue-type plasminogen activator (tPA) (a hemodynamic factor), Calcitonin and proteins and peptides engineered to make amyloid.
  • tPA Tissue-type plasminogen activator
  • the prion diseases e.g. bovine spongiform encephalopathy (BSE), and Creutzfeldt- Jakob disease (CJD)
  • BSE bovine spongiform encephalopathy
  • CJD Creutzfeldt- Jakob disease
  • PrP Sc normal cellular prion protein
  • PrP Sc infectious disease-associated isoform
  • the misfolded infectious form of the protein, PrP sc is the cause of a group of rare, fatal brain diseases, called prion diseases that affect humans and mammals.
  • the prion diseases are also known as transmissible spongiform encephalopathies (TSE), and they include bovine spongiform encephalopathy (BSE, or "mad cow” disease) in cattle; scrapie in sheep; chronic wasting disease in deer and elk; and in humans [Creutzfeldt Jakob disease (CJD), Gerstmann- Straussler-Scheinker disease (GSS), Kuru].
  • TSE transmissible spongiform encephalopathies
  • BSE bovine spongiform encephalopathy
  • CJD Crstmann- Straussler-Scheinker disease
  • the compounds of the present invention are intended to be used for methods for imaging of misfolded and aggregated proteins associated with these diseases and therapy of the above diseases. USE OF COMPOUNDS IN IMAGING
  • MRI magnetic resonance spectroscopy
  • NMR Nuclear magnetic resonance
  • the fluorine nucleus offers a superior signal magnitude (high gyromagnetic ratio) and hardly any imagable fluorine exists naturally in the human body. This results in a very low background signal and the detectable signal comes from the 19 F-substance administered.
  • Another option is Two Photon Fluorescence Microscopy (TPM) imaging a technique very useful when studying for example animals.
  • the amyloid binding compounds could be modified for fluorescence in the near infrared (NIR) region.
  • NIR near infrared
  • Magnetic Resonance Imaging (MRI) based on for example 19 F
  • MRI Magnetic Resonance Imaging
  • 19 F an isotope such as 19 F does not naturally occur in the body
  • 1H it eliminates the need for a so-called, pre-contrast scan.
  • This makes the diagnostic imaging procedure significantly easier.
  • the quantitative molecular imaging, 1H MRI as well as 19 F MRI, of fibrin, using a 19 F contrast agent was described by Morawski et al, Magn. Reson. Med. 52, 1255 (2004).
  • nuclei include hydrogen, helium-3, carbon-13, fluorine-19, oxygen-17, sodium-23, phosphorus-31 and xenon-129. 23 Na and 31 P are naturally abundant in the body, so they can be imaged directly. Hydrogen is the most frequently imaged nucleus in MRI due to its high gyromagnetic ratio gives a strong signal.
  • Isotopes that contain an intrinsic magnetic moment and angular momentum are those with an odd number of protons and/or of neutrons, a nonzero spin.
  • Preferred isotopes 2 H, 13 C and 19 F.
  • Two-photon imaging Two-photon imaging
  • TPM Two Photon Fluorescence Microscopy
  • TPM in vivo imaging for example multiphoton excitation laser scanning microscopy, is based on the effect of simultaneous absorption of two photons, such as in the infra-red spectrum, by the compounds of the present invention.
  • Non-linear summation of the two photons' energy results in excitation of the compounds of the present invention, which emits a detectable photon, for example in the visible spectrum.
  • Emitted photons are then collected by a suitable detector, such as sensitive photomultipliers, while the photon emission source, such as a pulsed infra-red laser, scans the plane of interest.
  • a suitable detector such as sensitive photomultipliers
  • the photon emission source such as a pulsed infra-red laser
  • Suitable computer software combines the data into a sharply focused 2D image and a 3D volumetric image can be reconstructed.
  • Suitable wavelengths include, but not limited to, the range from about 100 nm to about 2000 nm. In one embodiment, radiation in the visible range is used. It is also possible to use multiple-photon excitation, such that instead of excitation radiation of x nm, a radiation of 2x or 3x (two-photon and three-photon excitation, respectively) is used.
  • Positron emission tomography [Bailey, D.L; D.W. Townsend, P.E. Valk, M.N. Maisey (2005). Positron Emission Tomography: Basic Sciences. Secaucus, NJ: Springer- Verlag. ISBN 1-85233-798-2] is based on the positron emission from isotopes such as carbon, fluorine, iodine, nitrogen, and oxygen. It is a nuclear medicine imaging technique that produces a three-dimensional image or picture of the position of certain elements or functional processes in the body.
  • Radionuclides used in PET imaging are typically isotopes with short half-lives such as U C (about 20 min half-life), 13 N (about 10 min), 15 0 (about 2 min), and 18 F (about 110 min).
  • a compound containing a positron-emitting radionuclide (tracer) is introduced into the body and detected by a PET scanner.
  • These isotopes can replace their non-radioactive counterparts in compounds of the present invention to produce tracers that can detect the biologically relevant element in vivo.
  • 18 F is one of the most convenient labelling isotopes due to its relatively long half life of about 110 min which permits the preparation of diagnostic tracers or the study biochemical processes. 18 F-labelled
  • radiopharmaceuticals can be prepared relatively quickly by for example the nucleophilic aromatic and aliphatic fluoro-fluorination reaction.
  • novel compounds according to the present invention are labelled with a label detectable with an imaging method suitable for in vivo imaging.
  • the label may be a positron- emitting radionuclide, gamma ray emitting atoms or materials, detectable single or multiple photon emitters or magnetically active materials or any other label suitable for the imaging method of choice.
  • Examples are detectable isotopes such as 18 F, 19 F, 123 I, n C 2 H, n C, 13 C, 14 C, 18 C, 13 N, 15 N, 15 0 17 0, 18 0, 18 F, 35 S, 36 C1, 75 Br, 76 Br, 77 Br, 82 Br, 120 I, 123 I, 124 I, 125 I, 131 I, 67 Ga, 81m Kr, 82 Rb, m In, 133 Xe, 201 T1, 90 Y or 99m Tc. It may also be a detectable micro- or
  • nanoparticle such as a gold particle, a magnetic, supramagnetic or ferromagnetic particle, a lanthanide particle (e.g. Gd, Eu or Nd) optionally doped with metal, or a nanocrystal (such as a quantum dot commercially available from e.g. Invitrogen and Quantum Dot Corporation). What specific label is used will vary with the used imaging method and may be chosen by the skilled person.
  • Preferred isotopes for PET 18 F, 19 F, 123 I, U C, 13 C, 14 C, 18 C, 13 N, 15 N, 15 0 ⁇ 17 0, 18 0, 120 1, 124 I,
  • one or more atoms of a compound of formula I above may be replaced by a desired isotope.
  • a desired isotope Such an atom may be a ring atom (ring A, B or C) or an atom of a group R or R 1 .
  • the isotope or isotopes may be attached directly or via a suitable linker to the compound of formula I (to a ring A, B or C, or to a group R or R 1 ).
  • the amyloid binding compounds could be modified for fluorescence in the near infrared (NIR) region.
  • the NIR spectra permit non-invasive detection of amyloid deposits using NIR light or two photon spectroscopy in combination with diffuse optical tomography.
  • Direct non-invasive imaging of aggregated proteins, e.g. amyloid deposits, in vivo is difficult.
  • Deviations in physical characteristics, e.g. density and water-content, between aggregated proteins and normal tissues is generally too small to enable imaging with magnetic resonance imaging (MRI) and computer assisted tomography (CAT).
  • MRI magnetic resonance imaging
  • CAT computer assisted tomography
  • the compounds in the invention may be used to detect, in terms of presence and/or location, and/or quantify aggregated proteins in the body, whole or in parts of the body such as the brain, of animals or humans.
  • the inventive compounds may be used in conjunction with non-invasive imaging techniques such as magnetic resonance spectroscopy (MRS) or imaging (MRI), positron emission tomography (PET), single photon emission computed tomography (SPECT) and optical methods such as near infra-red imaging (NIR) and multi photon imaging (MPM).
  • non-invasive imaging techniques such as magnetic resonance spectroscopy (MRS) or imaging (MRI), positron emission tomography (PET), single photon emission computed tomography (SPECT) and optical methods such as near infra-red imaging (NIR) and multi photon imaging (MPM).
  • the present invention has the potential to be used together with many or all of the imaging technologies described above. This is possible since the compounds in the invention have the
  • amyloid binding compounds/probes are made and described in the present invention, are useful for in vivo imaging and quantification of amyloid deposition.
  • These compounds are to be used in conjunction with non-invasive neuroimaging or body imaging techniques such as NIR, MRS, MRI, PET and SPECT.
  • the compounds may be engineered to cross the blood-brain barrier after peripheral injection, or they will be delivered into the cerebral spinal fluid directly.
  • the compounds may be used to detect aggregated proteins with NIR imaging.
  • NIR imaging includes the use of NIR light and diffuse optical tomography.
  • Inventive compounds for NIR imaging have near infrared spectra and affinity for aggregated protein, including but not limited to amyloid plaques.
  • NIR imaging included methods known to those skilled in the art and examples of NIR compounds can be found in [Hintersteiner, M. et al. Nat. Biotechnol. 23, 577-583, 2005].
  • the inventive compounds may be used for multi photon imaging.
  • the principle of MPM is that two or more low energy photons interact nearly simultaneously with a fluorescent molecule resulting in an electronic transition, an excitation, equivalent to the absorption of a photon of twice as much energy.
  • Methods of MPM include, but are not limited to, using excitation at the NIR wavelengths (700-1000 nm).
  • the use of MPM furthermore includes, but is not limited to, the use of gradient index (GRIN) lenses and/or cranial windows as well as conventional multiphoton imaging techniques known to those skilled in the art.
  • GRIN gradient index
  • Inventive compounds usable for MPM have the sufficient two-photon cross section to enable MPM.
  • MPM may be used in conjunction with the inventive compounds to image aggregated proteins within cells and/or limited parts or tissue. In other embodiments the inventive compounds may be used to image or detect aggregated proteins in tissue culture slides.
  • the compounds will contain additional detectable labels, including fluorescent labels and isotopic labels.
  • the present invention includes the use compounds that are "isotopically-labelled", “radio-labelled”, “radio-ligands”, “detectable” by the means that one or more atoms in the compounds are substituted or replaced with an atom having a different atom mass or number than the normally naturally occurring. 19 F is one non- limiting exception which is a stable isotope that can be used to label the inventive compounds.
  • the invention includes without limitations labeling of the compounds with gamma-emitters, beta-emitters, positron-emitters and x-ray.
  • Suitable radionuclides that may be used to label the inventive compounds include but are not limited to ; 2 H (also noted as D for deuterium), U C, 13 C, 14 C, 13 N, 15 N, 15 0 17 0, 18 0, 18 F, 35 S, 36 C1, 75 Br, 76 Br, 77 Br, 82 Br, 120 I, 123 I, 124 I, 125 I and 131 I, preferably U C, 18 F. It is understood that an inventive compound only need to be detectable, with or without labeling, to the extent necessary for the technical application, i.e. mode of detection.
  • Suitable labels of inventive compounds for MRI and MRS include, but are not limited to, 13 C and 19 F. Labelling of the inventive compounds with 13 C and 19 F may be done with general organic synthesis, known to one skilled in the art. Both PET and SPECT imaging positron emitters are suitable for labeling of the inventive compounds. For SPECT imaging 123 I and 13 I are suitable isotopes for labeling the inventive compounds. Labeling procedures are well known to those skilled in the art (see e.g.
  • the inventive compounds may also be done with metal radiolabels.
  • metal radiolabels For SPECT imaging 99m Tc is one suitable label.
  • the compounds may be labeled by either of the isotopes n C,
  • the choice of detection instrument for imaging will be a major factor for choosing a certain label and will guide the selection between a radionuclide, a stable isotope or R active labels.
  • the label in question must have a signal detectable by the instrument, e.g. a radionuclide must have a decay detectable by the instrument.
  • the half life of the mentioned radionuclide must be considered. The half life time must be sufficient to ensure maximum uptake by the target but short enough to minimize harmful radiation to the subject. "Subject" is referring to the human or animal studied with the imaging method.
  • Labels of the inventive compounds include gamma emitters, which enable detection of the compounds through emitted gamma radiation of suitable wavelength.
  • PET and SPECT are included in methods detecting gamma radiation.
  • Suitable for SPECT is an isotope that lacks a specific emission but emits a vast number of photons in the energy range 140-200 keV.
  • PET imaging a positron emitting label will annihilate to form two gamma rays which can be detected in the PET camera.
  • imaging technologies could be of great benefit during drug development since they could provide important information to select candidates that seem most likely to be successful or even halt the development of drugs that does not give the desired response.
  • medical imaging modalities the use of selective imaging agents to target specific molecular targets that are associated with a disease, like the compounds of the present invention for Alzheimer's disease, allows earlier diagnosis, better management and follow up of the disease as well as more efficient drug development. Therefore, targeting agents that distribute preferentially to distinct body sites, e.g. amyloid deposits or aggregated protein, are by virtue of active targeting of particular interest.
  • the targeting agent binds to amyloid deposits or aggregated protein in various tissues, at cell surfaces, in body fluids or other places at the target site and can cross the blood brain barrier if needed.
  • a central criterion for a successful molecular imaging agent for use on living humans and animals is that it is specific and that excess substance can be removed through renal and/or hepatobiliary systems in order to achieve a high contrast between the target and the surroundings.
  • the present invention is based on oligomeric aromate/heteroaromate derivatives and in such the often problematic imaging antibodies can be avoided.
  • a labeled antibody for imaging studies in humans have shown that the maximum concentration of antibody at the target site is attainable within 24 h but that several more days are required before the concentration of a labeled antibody in circulation and in non- target tissue decreases to levels low enough for successful imaging to take place. This is not ideal for nuclear probes, because these constantly produce signal by decaying. Consequently, the present invention is based on polythiophene or other aromate/heteroaromate imaging agents for amyloid deposits or aggregated proteins in vivo that has the potential to reach the desired contrast much faster.
  • novel substituted derivatives that are suitable for imaging amyloid deposits and aggregated protein and imaging methods of the invention, in part, include the determination of the presence and location of amyloid deposits in an organ or body area, preferably brain, spinal cord, and/or blood vessels of a patient.
  • the novel derivatives can also be used to discriminate between misfolded and native proteins.
  • Certain embodiments of the methods of the invention include administration of a detectable quantity of a pharmaceutical composition containing the inventive compound described herein and analogues thereof, referred to as a the compounds of the present invention or a pharmaceutically acceptable water-soluble salt thereof, to a patient.
  • a detectable compound is a radioactively labeled compound, magnetically tagged compound and in some embodiments of the invention a compound fluorescent properties are employed.
  • the invention employs detectable compounds which, in conjunction with non- invasive neuroimaging techniques such as magnetic resonance spectroscopy (MRS), imaging (MRI), or gamma imaging such as positron emission tomography (PET), single-photon emission computed tomography
  • SPECT single photon emission tomography
  • NIR near infrared
  • multiphoton imaging may be used to quantify amyloid deposition or protein aggregates in vivo.
  • One mode in which the novel compounds based in vivo imaging methods is performed is by preparing the compound with a suitable label, administered to the organism whereby the compound captures, binds to, interacts with or detects mis-assembled/aggregated forms of proteins, especially misfolded or aggregated forms of proteins, and at the same time acting as transducer reporting the capture event in detectable signals, is therefore described in the present invention.
  • the inventive compounds may be administrated by any means known to one of ordinary skill in the art.
  • the present invention includes "pharmaceutically acceptable" compositions based on an amount of the inventive compound necessary for effective administration to a patient together with one or more pharmaceutical carriers, such as additive and/or diluent.
  • the formulation of the inventive compounds may be determined by the means of administration.
  • the formulation for administration of the inventive compounds may be solid, liquid or in aerosol form.
  • Administration of the inventive compounds to an animal or human may be local or systemic accomplished parenterally, orally, by inhalation, rectally, nasally, buccally, vaginally or via an implanted reservoir.
  • parenteral refers to administration outside the alimentary tract including subcutaneous, intravenous, intramuscular, intraarterial, intraspinal, intracranial, subdermal, intradermal by injection or infusion.
  • Carrier agents may be added to the pharmaceutical thiophene derivates compositions in order to achieve the desired distribution and acceptance in the living organism.
  • Such agents include, but are not limited to, lipids, phospholipids, cellulose membranes, sugar coatings, hyaluronic acid, detergents, peptides, proteins, ions, salts, chelators and solvents.
  • the present invention relates to a method for imaging the misfolded protein species in order to detect, quantify and/or localize said misfolded protein species inside a living organism ⁇ in vivo) or where an organ or tissue has been removed from the organism ⁇ ex vivo) comprising the steps of: - providing a compound according to the invention, optionally substituted with a detectable label for MRI, PET, SPECT, NIR, infrared, two-photon or fluorescence detection;
  • any misfolded protein species can be detected, quantified and/or localized.
  • the imaging method according to this aspect may be used for diagnosis of a disease involving misfolded or aggregated forms of proteins, such as Alzheimer's disease, Creutzfeldt Jacob disease (CJD), variant Creutzfeldt Jacob disease (vCJD), Secondary amyloidosis, type 2 diabetes or transmissible spongiform encephalopathy (TSE, such as, CWD, Scrapie, GSS and Kuru, bovine spongiform encephalopathy (BSE)).
  • TSE transmissible spongiform encephalopathy
  • BSE bovine spongiform encephalopathy
  • the radiation used in the method of the present invention has wavelengths in the range from about 100 nm to about 2000 nm. In one embodiment, radiation in the visible range is used. It is also possible to use multiple-photon excitation, such that instead of excitation radiation of x nm, a radiation of 2x or 3x (two-photon and three-photon excitation, respectively) is used.
  • Unlabelled compounds may be suitable for, but not limited to, NIR, single-photon, two- photon or multiple-photon imaging according to the present invention.
  • o is independently 0, 1, 2, 3, 4, 5, 6 or 7,
  • p is independently 0, 1, 2, 3, 4, 5, 6 or 7, and
  • R L is a linker, e.g. a group R or R 1 as defined for formula I above, or an additional group selected from groups R and R 1 .
  • A, B and C may also independently be labelled with an isotope according to the present invention.
  • o is zero, the isotope is situated directly on the main compound.
  • Isotopes are suitable for, but not limited to, PET, MRI or SPECT imaging according to the present invention.
  • NBS (9.53 g, 53.6 mmol) was added portionwise to a solution of 3-thiopheneethanol 6.87 g, 53.6 mmol) in AcOH (30 ml). The temperature was raised to 40 °C and mixture was stirred for 1 h. DCM (400 ml) and water was added. The organic layer was washed with water and sat. NaHC0 3 . The organic layer evaporated and residue purified by flash chromatography using 25-33% EtOAc in iso-hexane. Yield: 5.39 g (49%); colourless oil.
  • NBS (37.5 g, 21 mmol) was added in small portions into a solution of the material from above (40.0 g, 24 mmol) in chloroform (300 ml) and acetic acid (300 ml) below 5°C and then the reaction was stirred at RT. The completion of the reaction was indicated by the disappearance of starting material by HPLC. The reaction mixture was concentrated and ice-water was added. The mixture was extracted with dichloromethane and the organic extracts were washed with water, sat. NaHC03, brine, dried with anhydrous Na 2 S0 4 , filtered and concentrated in vacuo . Yield: 52.0 g (87%); pale brown oil.
  • NIS (2.31 g, 10.3 mmol) was added portion-wise to a solution of intermediate C (4.03 g, 10.3 mmol) in AcOH (15 ml) and CHCI 3 (15 ml) at 0 °C. The mixture was allowed to slowly reach ambient temperature overnight. DCM and sat. Na 2 C03 was added. Organic phase was evaporated and residue purified by flash chromatography using 2% EtOAc in toluene as eluent.
  • Methyl iodide (232 ⁇ , 3.74 mmol) was added to a mixture of the carboxylic acid from above (705 mg, 2.50 mmol) and sodium carbonate (529 mg, 4.99 mmol) in DMF (10 ml). The mixture was stirred at ambient temperature overnight. Water and diethyl ether were added. The aqueous layer was extracted with diethyl ether, The combined organic layers were concentrated and residue purified by flash chromatography using 5-20% EtOAc in iso- hexane. Yield: 364 mg (49%); white solid.
  • PEPPSI-iPrTM (15 mg, 0.0228 mmol) was added and the mixture heated at 60 °C for 45 min under argon. Orange material precipitated during the reaction. Water and CHC1 3 were added. The organic layer was separated and silica was added to the mixture and the solvents were evaporated. The dry silica was applied on a flash column and eluted with 0-3% MeOH in DCM. Yield: 230 mg (75%); orange solid.
  • PEPPSI-iPrTM (22 mg, 0.0323 mmol) was added, and the mixture heated in a microwave reactor at 100 °C for 30 min. Solvents were evaporated and residue dissolved in 1 M Na 2 C0 3 and filtered. The product was isolated by preparative hplc (10-35% MeCN in 50 mM buffer, big XBridge). Pure fractions were combined and amount of solvents reduced and solid material precipitated by addition of 6 M HCl. Solid material was isolated by centrifugation, washed with water and dried under high vacuum for 2 d. Yield: 65 mg (36%); red shiny solid.
  • the ester from above (98 mg, 0.15 mmol) was dissolved in dioxane (2.5 ml) and 2 M NaOH (2.5 ml) was added. After 18 hrs at RT, the reaction mixture was diluted with water (15 ml) and acidified using 1 M HC1 (approx. 13 ml). The formed precipitate was isolated by centrifugation and washed three times with water. The material was dried under high vacuum for 2d. Yield: 68 mg (72%); yellow solid.
  • the ester from above (70 mg, 0.10 mmol) was added dioxane (3 ml) and 2 M NaOH (3 ml) and the mixture was heated at 80 °C for 90 min. The solution was cooled to r.t. and added 6 M HC1 (1.5 ml). The formed precipitate was isolated by centrifugation and washed three times with water. The material was added some MeOH and the solvent was removed in vacuo. The yellow solid was then dried under high vacuum for 2 days. Yield: 55 mg (82%); yellow solid.
  • the ester from above (64 mg, 0.08 mmol) was dissolved in dioxane (2 ml) and 2 M NaOH (2 ml) was added. After 2 hrs at 80 °C followed by 18 hrs at RT, the reaction mixture was diluted with water (20 ml) and acidified using 2 M HC1 (approx. 5 ml). The formed precipitate was isolated by centrifugation and washed three times with water. The material was dried under high vacuum for 2d. Yield: 50 mg (79%); Yellow solid.
  • the ester from above (42 mg, 0.05 mmol) was dissolved in dioxane (2 ml) and 2 M NaOH (2 ml) was added. After 18 hrs at RT, the reaction mixture was diluted with water (20 ml) and acidified using 2 M HC1 (approx. 5 ml). The formed precipitate was isolated by centrifugation and washed three times with water. The material was dried under high vacuum for 2d. Yield: 23 mg (59%); Green-yellow solid.
  • the crude material was dissolved in MeOH (8 ml), filtered and purified by prep-HPLC (30-70% MeCN in 0.1% TFA aq). The pure fractions were combined and freeze dried. Not pure fractions containing product were combined and concentrated to dryness. The red solid was dissolved in MeOH (2 ml) and purified on prep-HPLC (30-70% MeCN in 0.1% TFA aq). The pure fractions were combined and freeze dried. The 2 batches were combined.
  • Argon was bubbled through a mixture of intermediate O (174 mg, 0.244 mmol), 2- methoxycarbonylphenylboronic acid (131 mg, 0.731 mmol) and K 2 CO 3 (168 mg, 1.22 mmol) in toluene (10 ml) and methanol (10 ml).
  • PEPPSI-iPrTM (3 mg, 0.005 mmol) was added and the mixture was heated at 70 °C for 30 min. Solvents were evaporated and residue dissolved in dioxane (5 ml) and 2 M NaOH (5 ml) was added and the mixture heated at 80 °C for 1 h.
  • Argon was bubbled through a mixture of intermediate D (193 mg, 0.351 mmol), lH-Indole-5- boronic acid (141 mg, 0.0.877 mmol) and K 2 C0 3 (150 mg, 1.09 mmol) in toluene (2 ml) and methanol (2 ml).
  • PEPPSI-iPrTM (5 mg, 0.007 mmol) was added and the mixture heated in a microwave reactor at 90 °C for 10 min. Solvents were evaporated, the residue added dioxane (2 ml) and 2 M NaOH (2 ml), and the mixture heated at 80 °C for 1 h.
  • Argon was bubbled through a mixture of material from above (109 mg, 0.250 mmol), K 2 CO 3 (104 mg, 0,750 mmol) and DMSO (2 ml) for 5 min.
  • Bis(pinacolato)diboron (254 mg, 0.125 mmol) and l, l'-bis(diphenylphosphino)ferrocene-palladium(II)di chloride (9.1 mg, 0,013 mmol) were added and the reaction was stirred at 80°C in a sealed tube overnight.
  • the reaction was added water (0.60 ml) and 5 M NaOH (0.60 ml) and stirred at 80°C for 1 hour.
  • the mixture was cooled and filtered.
  • Example 50 2- ⁇ 5-[4-(Carboxymethyl)-5-[5-(4-methanesulfonylphenyl)thiophen-2- yl]thiophen-2-yl]-2-[5-(4-methanesulfonylphenyl)thiophen-2-yl]thiophen-3-yl ⁇ acetic acid (9708_053)
  • Example 51 3-(2- ⁇ 5-[3-(2-Carboxyethyl)-5-(4-methanesulfonylphenyl)thiophen-2- yl]thiophen-2-yl ⁇ -5-(4-methanesulfonylphenyl)thiophen-3-yl)propanoic acid (9708 054)
  • the intermediate C2 (201.9 mg, 0.35 mmol) and 4-(methylsulfonyl)phenylboronic acid (177.6 mg, 0.89 mmol) were dissolved in methanol (5 ml) and toluene (5 ml).
  • the formed diester was hydrolyzed in 2 M NaOH (3,5 ml) and 1,4-dioxane (3 ml) at 60 °C for 1,5 h. Ethyl acetate (2 ml) was added and the phases were separated. The desired compound precipitated upon addition of 6 M HCl. The obtained crystals were collected by centrifugation and washed with water two times. Further purification using preparative HPLC was necessary. Yield: 26.5 mg, (4 %); orange solid.
  • the intermediate C2 (199 mg, 0.34 mmol) and (5-methoxycarbonyl-2-thiophenyl) boronic acid (166 mg, 0.89 mmol) were dissolved in methanol (5 ml) and toluene (5 ml). Potassium carbonate (241 mg, 1.74 mmol) was added and argon (g) was bubbled through the reaction mixture before the PEPPSI-iPrTM (12 mg, 0.017 mmol) was added. The reaction mixture was heated for 30 minutes at 60 °C and temperature was then elevated to 80 °C for another 30 minutes. Solution went from bright yellow to brown. The solvents were removed under reduced pressure.
  • the diester was hydrolyzed using 1,4-dioxane (7 ml) and 2 M NaOH (7 ml) and the mixture was left to stir over night. The temperature was raised to 80 °C and stirred for 30 minutes and the solution was dark and transparent. H 2 0 and EtOAc were added to the reaction and the phases were separated and the acid was precipitated using 6 M HC1. The crystals were collected by centrifugation and washed twice with water and purified by preparative HPLC. The desired compound was obtained as a red solid in 23 % yield (51 mg). 1H NMR (400
  • Example 54 4- ⁇ 5-[3-(Carboxymethyl)-5- ⁇ 5-[4-(carboxymethyl)-5-[5-(4- carboxyphenyl)thiophen-2-yl]thiophen-2-yl]thiophen-2-yl ⁇ thiophen-2-yl]thiophen-2- yllbenzoic acid (9708_057)
  • Argon was bubbled through a mixture of the material from above (204 mg, 0.452 mmol), 2,5- thiophenediboronic acid (38 mg, 0.226 mol) and K 2 CO 3 (78 mg, 0.565 mmol) in toluene (8 ml) and MeOH (8 ml).
  • PEPPSI-iPrTM (6 mg, 0.009 mmol) was added and mixture heated at 60 °C under argon for 45 min. Solvents were evaporated and residue added dioxane (5 ml) and 2 M NaOH (5 ml). The mixture was heated and 80 °C for 1 h.
  • NBS 260 mg, 1.46 mmol
  • a solution of the material from above (546 mg, 1.46 mmol) in chloroform (5 ml) and AcOH (5 ml) at 0 °C and the mixture was stirred at 4 °C overnight.
  • Solvent was evaporated and residue purified by flash chromatography using 2% EtOAc in toluene as eluent. Yield: 512 mg (78%); pale yellow solid.
  • Argon was bubbled through a mixture of the material from above (174 mg, 0.386 mmol), bispinacolatodiboron (49 mg, 0.193 mg) and K 2 C0 3 (266 mg, 1.93 mmol) in DMSO (2 ml).
  • l, l '-Bis(diphenylphosphino)ferrocene dichloropalladium (II) 49 mg, 0.193 mmol
  • 2 M NaOH (2 ml) was added and heating was continued at 80 °C for 1 h. Solid material was precipitated by addition of 2 M HC1 and isolated by centrifugation.
  • Argon was bubbled through a mixture of intermediate O (118 mg, 0.165 mmol), 3-(2- methoxy-2-oxoethyl)phenylboronic acid, pinacol ester (114 mg, 0.413 mmol) and K 2 C0 3 (114 mg, 0.826 mmol) in toluene (8 ml) and MeOH (8 ml).
  • PEPPSI-iPrTM 5 mg, 0.0074 mmol was added and the mixture heated at 70 °C in a sealed tube for 45 min. Solvents were evaporated and residue added dioxane (5 ml) and 2 M NaOH (5 ml).
  • Example 58 2-(2-(5-r3-(Carboxymethvn-5-(5-r4-(carboxymethvn-5-(5-r3- (carboxymethyl)-5-chlorothiophen-2-yl]thiophen-2-yl ⁇ thiophen-2-yl]thiophen-2-yl ⁇ thiophen- 2-yllthiophen-2-yl
  • NCS (73 mg, 0.547 mmol) was added to a solution of intermediate E (258 mg, 0.547 mmol) in AcOH (5 ml). The mixture was stirred at rt for 3 days. Solvent was evaporated and residue purified by flash chromatography using 2% EtOAc in toluene. Yield: 205.8 mg (74%); yellow oil.1H MR (400 MHz, CDC1 3 ): ⁇ 3.69 (s, 2H), 3.71 (s, 2H), 3.74 (s, 6H), 6.90 (s, 1H), 7.03 (s, 1H), 7.09 (s, 2H).
  • Argon was bubbled through a mixture of the material from above (205 mg, 0.407 mmol), 2,5- thiophenediboronic acid and K 2 C0 3 (224 mg, 1.63 mmol) in toluene (4 ml) and MeOH (4 ml).
  • PEPPSI-iPrTM was added and the mixture heated at 60 °C for 30 min under argon.
  • the coupled product was dissolved in 1,4-dioxane (3 ml) and NaOH 2 M (3 ml) and heated at 60 °C for 30 minutes.
  • the acid was precipitated by adding HC1 6 M (0.5 ml) and solid material purified by preparative HPLC (Xbridge 5%-50% MeCN) was necessary.
  • the compound was precipitated using 6 M HC1 and collected 38.2 mg (18 %) pure compound.
  • Example 62 3-(2-(5-[3-(2-Carboxyethvn-5-(5-[4-(2-carboxyethvn-5-(5-[3-(2- carboxyethyl)thiophen-2-yl]thiophen-2-yl ⁇ thiophen-2-yl]thiophen-2-yl ⁇ thiophen-2- yl]thiophen-2-yl ⁇ thiophen-3-yl)propanoic acid (9708 065).
  • the intermediate B2 (373.6 mg, 0.89 mmol) was dissolved chloroform (4 ml) and acetic acid (4 ml). The solution was cooled to 0 °C and N-iodosuccinimide (199.8 mg, 0.89 mmol) was added. The reaction was slowly warmed to room temperature over night. The reaction mixture was poured onto Na 2 S 2 0 3 and extracted with DCM (x2). The organic phases were combined and washed with water and brine and dried with MgS0 4 . Concentration under reduced pressure gave a 519 mg red residue. The residue was purified by column chromatography, 2% ethyl acetate in toluene: 191.5 mg (38%) of a yellow oil.
  • the mixture was purified by prep-HPLC (20-50% MeCN, in 50 mM H3/NH 4 HCO3 buffer). The combined pure fractions were concentrated to dryness. The compound was dissolved in water (20 ml) and some 2 M HC1 was added. The solid was collected and washed several times with water and dried. Yield: 25 mg (18%) as a orange solid.
  • Example 65 3- ⁇ 5-[3-(Carboxymethyl)-5- ⁇ 5-[4-(carboxymethyl)-5-[5-(3- carboxyphenyl)thiophen-2-yl]thiophen-2-yl]thiophen-2-yl ⁇ thiophen-2-yl]thiophen-2- yllbenzoic acid (9708 068)
  • NBS (358 mg, 2.01 mmol) was added portion-wise to the material from above (749 mg, 2.01 mmol) in CHCI 3 (8 ml) and AcOH (8 ml) and the mixture was stirred at ambient temperature overnight. Solvents were evaporated and residue purified by flash chromatography using 2% EtOAc in toluene.Yield: 702 mg, pure on tic, pale yellow solid.
  • Argon was bubbled through a mixture of material from above (204 mg, 0.452 mmol), 2,5- thiophenediboronic acid (35 mg, 0.204 mmol) and K 2 C0 3 (140 mg, 1.02 mmol) in toluene (8 ml) and MeOH (8 ml).
  • PEPPSI-iPrTM was added and mixture heated at 60 °C under argon for 2h. Solvents were evaporated and residue added dioxane (5 m,l) and 2 M NaOH (5 ml) the mixture was heated at 80 °C for 1 h. Material was purified by prep. hplc. Pure fractions were combined and some solvents evaporated.
  • Example 68 2-(2- ⁇ 5-[3-(Carboxymethyl)-5-(4-hydroxy-3-methoxyphenyl)thiophen-2- yl]thiophen-2-yl ⁇ -5-(4-hydroxy-3-methoxyphenyl)thiophen-3-yl)acetic acid (9708 071) Argon was flushed through a solution of intermediate D (130 mg, 0.236 mmol) and 4- hydroxy-3-methoxyphenylboronic acid (120 mg, 0.714 mmol) in a mixture of toluene (3 ml) and MeOH (3 ml).
  • PEPPSI-iPrTM (26.1 mg, 0.036 mmol) and KF (125 mg, 2.14 mmol) were added and the reaction heated at 55 °C for 120 min.
  • the solvents were removed in vacuo and the crude was added water (3 ml), 5 M NaOH (1 ml) and dioxane (2 ml).
  • the reaction was heated to 70°C for 30 min then cooled, filtered and washed with water. 1 M NH 4 HCO3 was added and the product was collected as a solid by filtration. Water and 2 M HCl were added. The solid was once more collected by filtration and washed with water. Yield: 110 mg (77%) as a off white solid.
  • Example 70 2-(2- ⁇ 5-[3-(Carboxymethyl)-5-[5-(methoxycarbonyl)thiophen-2-yl]thiophen-2-yl]thiophen-2-yl ⁇ -5-[5-(methoxycarbonyl)thiophen-2-yl]thiophen-3-yl)acetic acid (9708 073) Argon was bubbled through a mixture of intermediate Q (90 mg, 0.172 mmol), 5- methoxycarbonyl)thiophen-2-ylboronic acid (80 mg, 0.431 mmol) and KF (50 mg, 0.862 mmol) in toluene (5 ml) and MeOH (10 ml).
  • intermediate Q 90 mg, 0.172 mmol
  • 5- methoxycarbonyl)thiophen-2-ylboronic acid 80 mg, 0.431 mmol
  • KF 50 mg, 0.862 mmol
  • the aqueous phase was diluted with some 1 M H 4 HCO 3 filtered and purified by prep. hplc. Pure fractions were combined, some solvents were removed, the compound was precipitated by addition of 1 M HCl, isolated by centrifugation, washed three times with water and freeze-dried for 2 d. Yield: 45.3 mg (45%); bright yellow powder.
  • Argon was bubbled through a mixture of intermediate D (55 mg, 0.100 mmol), l-methyl-3- trifluoromethylpyrazole-5-boronic acid (48.4 mg, 0.250 mmol) and KF (29 mg, 0.500 mmol) in toluene (1 ml) and MeOH (1 ml).
  • PEPPSI-iPrTM (2 mg, 0.003 mmol) was added and the mixture heated at 80 °C for 30 min in a sealed tube. Solvents were evaporated and dioxane (1 ml) and 2 M NaOH (1 ml) was added. The mixture was heated at 80 °C for 2 h.
  • Example 78 2-(2-(5-r3-(Carboxymethyl)-5-r3-fluoro-4-(pyrrolidine-l- carbonyl)phenyl]thiophen-2-yl]thiophen-2-yl ⁇ -5-[3-fluoro-4-(pyrrolidine-l- carbonyl)phenyl]thiophen-3-yl)acetic acid (9708 081)
  • Argon was bubbled through a mixture of intermediate D (55 mg, 0.100 mmol), 3-fluoro-4- (pyrrolidine-l-carbonyl)phenylboronic acid (59 mg, 0.250 mmol) and KF (29 mg, 0.500 mmol) in toluene (1 ml) and MeOH (1 ml).
  • PEPPSI-iPrTM (2 mg, 0.003 mmol) was added and the mixture heated at 80 °C for 30 min in a sealed tube. Solvents were evaporated and dioxane (1.5 ml) and 2 M NaOH (1.5 ml) was added. The mixture was heated at 80 °C for 1 h.
  • Example 80 2-(2- ⁇ 5-[3-(Carboxymethyl)-5-[3-(cyclopropylcarbamoyl)phenyl]thiophen-2- yl]thiophen-2-yl ⁇ -5-[3-(cyclopropylcarbamoyl)phenyl]thiophen-3-yl)acetic acid (9708 083) Argon was bubbled through a mixture of intermediate D (55 mg, 0.100 mmol), 3-
  • the ester from above (55 mg, 0.08 mmol) was dissolved in dioxane (2 ml) and 2 M NaOH (2 ml) was added. After stirring 45 min at 70 °C, the reaction was cooled to RT, diluted with water (11 ml) and acidified using 2 M HC1 (approx. 3 ml). The formed precipitate was isolated by centrifugation and washed two times with water. The material was dried under high vacuum for 48 hrs.
  • Example 84 2-(2- ⁇ 5-[3-(Carboxymethyl)-5- ⁇ 5-[3-(carboxymethyl)thiophen-2-yl]thiophen-2- yl ⁇ thiophen-2-yl]thiophen-2-yl ⁇ -5- ⁇ 5-[3-(carboxymethyl)thiophen-2-yl]thiophen-2- yl ⁇ tmophen-3-yl)acetic acid (9708 087)
  • Argon was flushed through a solution of the boronic ester from above (52.1 mg, 0.185 mmol), intermediate O (60 mg, 0.084 mmol) and KF (36.6 mg, 0.0630 mmol) in a mixture of toluene (5 ml) and MeOH (5 ml) for 10 min.
  • PEPPSI-iPrTM (3.0 mg, 0.0042 mmol) was added and the reaction heated at 60°C for 3 hours. The solvents were removed in vacuo.
  • the crude material was added dioxane (1 ml), 5 M NaOH (1 ml) and water (2 ml) and the reaction was heated to 65°C for 30 min.
  • the solution was filtered and purified by prep-HPLC (5-45% MeCN, in 50 mM NH3/NH 4 HCO3 buffer). The pure fractions were combined and concentrated to dryness. The solid was dissolved in water and 2 M HC1 (6 ml) was added. A red solid was collected, washed several times with water and dried in the rotavapor and at high vacuum. Yield: 33 mg (49%) as a red solid.
  • Example 86 2-(2- ⁇ 5-[3-(Carboxymethyn-5-ethynylthiophen-2-yllthiophen-2-yl ⁇ -5- ethynylthiophen-3-yOacetic acid (9708 089) Argon was bubbled through a stirred mixture of the intermediate G2 (50 mg, 0.078 mmol), Cul (3.0 mg, 0.016 mmol) water (0.056 ml, 3.1 mmol) and toluene (10 ml) for 10 min.
  • PdCl 2 (PPh 3 ) 2 (3.3 mg, 0.0047 mmol) and trimethylsilylacetylene (0.0215 ml, 0.155 mmol) were added and the mixture stirred in a sealed tube at r.t. over night.
  • the solvent was removed in vacuo and the residue purified by flash chromatography (1% EtOAc in toluene, 24 g silica).
  • the solid was dissolved in dioxane (2 ml) and 5 M NaOH (0,5 ml) and water (1 ml) were added. The mixture was stirred at r.t. overnight, filtered and purified by prep-HPLC (10-50% MeCN, in 50 mM H 3 /NH 4 HC0 3 buffer).
  • the product was dissolved in dioxane (2 ml) and 5 M NaOH (0.5 ml) and water (1 ml) were added. The mixture was stirred at r.t. over night. The mixture was concentrated to dryness added water ( ⁇ 2 ml) and MeCN ( ⁇ 2 ml). The water phase was removed and new water was added ( ⁇ 2 ml). The homogen solution was filtered and purified by prep-HPLC (10- 60% MeCN, in 50 mM NH 3 /NH 4 HC0 3 buffer). The combined pure fractions were
  • the ester from above (60 mg, 0.09 mmol) was dissolved in dioxane (2 ml), followed by addition of 2 M NaOH (2 ml) and water (1 ml). After stirring 35 min at 70 °C, the reaction was cooled to RT, diluted with water (10 ml) and acidified using 1 M HCl (approx. 4 ml). The formed precipitate was isolated by centrifugation and washed two times with water. The material was dried under high vacuum for 48 hrs. The obtained solid was dissolved in 1 M Na 2 C0 3 and purified by preparative hplc (5-40% MeCN in 50 mM buffer, XBridge). Pure fractions were combined and some solvents were evaporated.
  • Example 90 2-[5-(3-Carbamoylphenyl)-2- ⁇ 5-[5-(3-carbamoylphenyl)-3- (carboxymethyl)thiophen-2-yl]thiophen-2-yl ⁇ thiophen-3-yl]acetic acid (9708 093) Argon was flushed through a solution of intermediate D (60.0 mg, 0.109 mmol), 3- aminocarbonylphenylboronic acid (41.0 mg, 0.249 mmol) and KF (47.5 mg, 0.818 mmol) in a mixture of toluene (2 ml) and MeOH (2 ml) for 10 min.
  • PEPPSI-iPrTM (4.0 mg, 0.0054 mmol) was added and the reaction heated at 60°C for 3 hours. The solvents were removed in vacuo and the crude was dissolved in dioxane (2 ml) and 5 M NaOH (0,5 ml) and water (1 ml) were added. The mixture was stirred at r.t. overnight, concentrated to dryness and dissolved in 0.1 M NaOH solution. 2 M HC1 was added and the solid was collected by centrifugation. -1/3 of the solid was dissolved in a mixture of water ( ⁇ 7 ml), MeCN ( ⁇ 1 ml) and a small amount of cone NH 3 .
  • Example 92 and 93 2-(2-(5-r3-(Carboxymethvn-5-(thiophene-2-carbonyl)thiophen-2- yllthiophen-2-yl

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Abstract

The present invention relates to novel labelled compounds of formula (I) (C)n-B-(A)m-B-(C)n (I) wherein m is 0 or 1, and n is independently 0, 1, 2 or 3, A, each B and each C are independently selected from phenylene and five- and six-membered heteroaromatic rings, and for a terminal ring B or C also from bicyclic heteroaromatic fused rings having seven to ten ring members, wherein the bond between at least two of the rings A to C may be replaced by a carbonyl group ( -CO- ), wherein at least two of the rings A to C are substituted with one or two groups R, and wherein each ring A to C further optionally is substituted with one or two groups R1, for use in imaging amyloid deposits and aggregated protein in living patients. The invention further relates to imaging methods using labelled or unlabelled compounds of formual I and the use of unlabelled compounds in such methods.

Description

Novel thiophene compounds and method for in vivo imaging
Field of the invention
The present invention relates to novel labelled compounds for use in imaging amyloid deposits and aggregated protein in living patients. It further relates to imaging methods using labelled or unlabelled compounds and the use of unlabelled molecules in such methods.
Background of the invention
Molecular imaging that allows non-invasive assessment of biological and biochemical processes in living subjects is of great importance. Noninvasive identification and observation of the molecular events underlying neurodegenerative diseases is especially beneficial since it is difficult to sample information from the brain in other ways, like autopsies. Therefore imaging technologies are important when it comes to increase our understanding of diseases, diagnosing diseases, for drug activity assessment, during preclinical development, during clinical drug development and to monitor treatment of diseases.
Natural biopolymers, such as proteins, frequently have ordered conformations, such as alpha- helix and beta-sheets, which contribute to the three-dimensional ordered structure and the specific function of the biopolymer. The structure of a protein is essential for the protein's function; it has been shown by many scientists that an unfolded protein may not be functional. More important, in the last few years there is increasing awareness of the danger of protein misfolding and misassembly into for example amyloid and other pathological forms.
Misfolding can change a protein from something that is useful into nonfunctional, harmful or even toxic. Human health relies on properly folded protein, and in vivo deposition of amyloid fibrils is associated with many diseases related to protein conformation, including
Alzheimer's disease (AD), Huntington's disease (HD), Amyotrophic Lateral Sclerosis (ALS), Parkinson's disease (PD), Tauopathies (multiple), systemic amyloidoses, and the prion diseases. The prion diseases, i.e. transmissible spongiform encephalopathy (TSE), in animals [e.g. bovine spongiform encephalopathy (BSE), Scrapie and chronic wasting disease (CWD)] and in humans [Creutzfeldt Jakob disease (CJD), Gerstmann-Straussler-Scheinker disease (GSS), Kuru] are associated with the conformational conversion of the normal cellular prion protein, (PrPc), to an infectious pathogenic disease-associated isoform denoted PrPSc. Proteins frequently alter their conformation due to different external stimuli and the importance of conformational changes of proteins leading to pathogenic states has been well documented. Especially under conditions that destabilize the native state, proteins can aggregate into characteristic fibrillar assemblies, known as amyloid fibrils. These beta-sheet rich protein assemblies have distinctively different conformations compared to that of the native state. The misfolded prion protein is even self-propagating (infectious), a property which is entirely encoded within the misfolded conformation.
Chronic human diseases seriously affect the healthcare system. It is well recognized that rapid and accurate diagnostic tools are necessary to afford early intervention and therapy. Only symptomatic therapy is available, like in Alzheimer's disease for example, and these have limited therapeutic efficacy. Presently there are no antemortem molecular diagnostic tests of Alzheimer's disease or transmissible spongiform encephalopathies (TSEs), and the clinical diagnostics that are performed require that disease progression is severe. Further, there are no efficient treatments available yet, and immunotherapy in for example Alzheimer's disease holds great promise. The lack of reliable methods to measure misfolded and/or aggregated proteins in living subjects, monitoring both treatment and disease progression is however a severe shortcoming in treatment of most protein misfolding related diseases.
The affinity between misfolded proteins in amyloid plaques, amyloid fibrils and amyloid like fibrils, and conjugated molecules compromised of repeating units of thiophene,
ethylenedioxythiophene (EDOT), benzothiadizole, fluorene, and phenyl in homo and hetero oligomers and polymers with ionic or polar sidechains has been demonstrated in several in vitro studies. The interaction between amyloid like fibrils of insulin and anionic, zwitterionic and cationic poly- and oligo thiophenes was shown by WO 2005/109005. Several of the mentioned oligomers and polymers have been shown to bind to amyloid, aP and PrP deposits in histological sections, as disclosed in WO 2007/091973. Imaging using thiophene derivatives is known from WO 2010/044744. An anionic, more specifically an
alkoxysulfonate derivative, polymer of EDOT showed high affinity for amyloid like fibrils [Hamedi, M. et al.;. Nano Lett.; (2008); 8, 1736-1740]. Moreover a substituted polyfluorene and an alternating polyfluorene with a polyethylene oxide were demonstrated to strongly associate with amyloid like fibrils in vitro [Tanaka, H. et al.; Nano Lett.; (2008) 8, 2858- 2861]. It has previously been demonstrated that the chemical class that the compounds of the present invention belongs to can be used for in vivo imaging of aggragated beta- Amyloid (AB), aggragated Tau and neurofibrillary tangles ( FTs) [WO 2010/044744; and Aslund et al., ACS Chem. Biol. (2009), Vol. 4(8), p. 673-684.]. The present invention introduces novel and innovative compounds that potentially can be derivatized with, for example:
2H, 13 C and 19F such as in MRI applications;
UC, 13N, 150 or 18F such as in PET applications;
123I or 131\ such as in SPECT applications, and used in in vivo imaging of aggregated beta- Amyloid (AB), aggregated Tau, neurofibrillary tangles (NFTs), aggregated prion protein, SOD-1 protein aggregation, huntingtin as well as other aggraging or misfolding proteins. Since there are many different kinds of diseases that are related to aggregated proteins the compounds of the present invention may be used as a powerful diagnostic tool. In addition, many of the aggregated proteins are therapeutic targets suggesting that the compounds described herein can be used during drug development or monitoring treatment. As demonstrated in the experimental part of the present invention it is clear that not every single compound described herein can be used to image every single aggregating or misfolding protein that exists. Also, as exemplified herein it is not possible to derivatize every single compound with all isotopes suggested as suitable in different imaging modalities. However, a person skilled in the art of chemistry relating to the present invention can select a suitable isotope, suitable chemical synthesis pathway and suitable compound of the present invention in order to produce a suitable imaging probe. Likewise can a person skilled in the art of protein aggregating and misfolding relating to the present invention validate suitable non- labeled or labeled compounds of the present invention for binding to the desired aggregating or misfolding protein. The description and examples presented herein describe how to, but not limited to, accomplish this.
In WO 2006/020156 various poly cyclic dye compounds for in vivo imaging and diagnosis of Alzheimer's are disclosed. These dye compounds are primarily designed to be NIR
compounds and no derivates with potential for imaging via methods such as PET, SPECT and NMR are exemplified, although mentioned in the text.
Summary of the invention
In a first aspect, the present invention relates to novel compounds of formula (I) (C)n-B-(A)m-B-(C)n (I) wherein m is 0 or 1, and n is independently 0, 1, 2 or 3,
A, each B and each C are independently selected from phenylene and five- and six-membered heteroaromatic rings, and for a terminal ring B or C also from bicyclic heteroaromatic fused rings having seven to ten ring members, wherein the bond between at least two of the rings A to C may be replaced by a carbonyl group ( -CO- ), wherein at least two of the rings A to C are substituted with one or two groups R, and wherein each ring A to C further optionally is substituted with one or two groups R1, wherein each group R is independently selected from hydroxy, hydroxyalkyl, hydroxyalkoxy, hydroxyalkoxyalkyl, hydroxypolyoxyalkylene, alkoxy, alkoxyalkyl, polyoxyalkylene, carboxy, carboxyalkyl, carboxyalkoxy, carboxyalkoxyalkyl, carboxypolyoxyalkylene, alkoxycarbonyl, alkoxycarbonylalkyl, alkoxycarbonylalkoxy, alkoxycarbonylalkoxyalkyl, alkoxycarbonylpolyoxyalkylene, amino, alkylamino, dialkylamino, aminoalkyl,
alkylaminoalkyl, dialkylaminoalkyl, aminoalkoxy, alkylaminoalkoxy, dialkylaminoalkoxy, aminopolyoxyalkylene, alkylaminopolyoxyalkylene, dialkylaminopolyoxyalkylene, aminoalkoxyalkyl, alkylaminoalkoxyalkyl, dialkylaminoalkoxyalkyl, (amino)(carboxy)alkyl, (alkylamino)(carboxy)alkyl, (dialkylamino)(carboxy)alkyl, (amino)(carboxy)alkoxy,
(alkylamino)(carboxy)alkoxy, (dialkylamino)(carboxy)alkoxy, (amino)(carboxy)alkoxyalkyl, (alkylamino)(carboxy)alkoxyalkyl, (dialkylamino)(carboxy)alkoxyalkyl,
(amino)(carboxy)polyoxyalkylene, (alkylamino)(carboxy)polyoxyalkylene,
(dialkylamino)(carboxy)polyoxyalkylene, (alkoxycarbonyl)(amino)alkyl,
(alkoxycarbonyl)(alkylamino)alkyl, (alkoxycarbonyl)(dialkylamino)alkyl,
(alkoxycarbonyl)(amino)alkoxy, (alkoxycarbonyl)(alkylamino)alkoxy,
(alkoxycarbonyl)(dialkylamino)alkoxy, (alkoxycarbonyl)(amino)alkoxyalkyl, (alkoxycarbonyl)(alkylamino)alkoxyalkyl, (alkoxycarbonyl)(dialkylamino)alkoxyalkyl, (alkoxycarbonyl)(amino)polyoxyalkylene, (alkoxycarbonyl)(alkylamino)polyoxyalkylene, (alkoxycarbonyl)(dialkylamino)polyoxyalkylene, acylamino, acylaminoalkyl,
acylaminoalkoxy, acylaminoalkoxyalkyl, acylaminopolyoxyalkylene, acylalkylamino, acylalkylaminoalkyl, acylalkylaminoalkoxy, acylalkylaminoalkoxyalkyl,
acylalkylaminopolyoxyalkylene, hydrazinocarbonyl, hydrazinocarbonylalkyl,
hydrazinocarbonylalkoxy, hydrazinocarbonylalkoxyalkyl,
hydrazinocarbonylpolyoxyalkylene, nitro, nitroalkyl, nitroalkoxy, nitroalkoxyalkyl, nitropolyoxyalkylene, cyano, cyanoalkyl, cyanoalkoxy, cyanoalkoxyalkyl,
cyanopolyoxyalkylene, sulfo, sulfoalkyl, sulfoalkoxy, sulfoalkoxyalkyl,
sulfopolyoxyalkylene, alkylsulphonyl, alkylsulphinyl, alkylaminosulphonyl, acyloxyalkyl, carboxyalkenyl, pyrrolidincarbonyl, morpholinoalkyl, cycloalkylcarbamoyl, trifluoroxy, carbamoyl, hydroxyalkylaminoalkyl, carboxyalkylaminoalkyl, aminoalkylaminoalkyl, piperidinealkyl, piperazinealkyl, or any two groups R attached to the same ring taken together are alkylenedioxy, optionally substituted with sulfoalkyl, sulfoalkoxy, sulfoalkoxyalkyl or sulfopolyoxyalkylene; and wherein each group R1 is independently selected from halogen, alkyl, alkenyl, alkynyl, trifluoro, with the provisos that:
(i) when m=0, then one n is other than 0 or both B represent a bicyclic heteroaromatic fused ring, and, preferably,
(ii) when m=l and each n=l, A, B and C are thienylene, and C is unsubstituted or mono- substituted with carboxy or iodo, then a substituent R on a ring B in ortho-position to A is other than carboxymethyl, methoxycarbonylmethyl and aminoethyl; wherein any H2 group may optionally be protected as a tert-butyl carbamate, benzyl carbamate or 9-fluorenylmethyl carbamate or substituted with a biotinyl moiety; wherein any alkyl or alkylene moiety is Ci-6-alkyl or Ci-6-alkylene, and any alkenyl and alkynyl moiety is C2-6-alkenyl and C2-6-alkynyl, respectively; or a pharmaceutically acceptable salt thereof, wherein the compounds are labelled with a label detectable with an in vivo imaging method. In a second aspect, the invention relates to a composition comprising a compound of the first aspect above, and optionally a pharmaceutically acceptable buffer, diluent, excipient and/or carrier.
In a third aspect, the invention relates to a method for imaging of misfolded or aggregated forms of proteins in a sample or subject, comprising the steps of:
- bringing the sample in contact with, or administering to the subject, a compound of formula I as defined above, optionally labelled with a label detectable with an in vivo imaging method;
- allowing said compound to bind to said misfolded or aggregated forms of proteins; and
- detecting the presence of said compound;
- optionally localizing said compound in said sample or subject;
- optionally quantifying the amount of said compound in said sample or subject. In a fourth aspect, the invention relates to a method for diagnosis of a disease involving misfolded or aggregated forms of proteins in a mammalian subject, comprising the steps of
- bringing a sample from said subject in contact with, or administering to the subject, a compound of formula I as defined above, optionally labeled with a label detectable with an in vivo imaging method;
- allowing said compound to bind to said misfolded or aggregated forms of proteins in said sample or subject;
- allowing unbound amounts of said compound to be cleared from the subject, or
washing unbound amounts of said compound from said sample; and
- detecting the presence of said compound in said subject or sample;
- optionally localizing said compound in said subject or sample;
- optionally quantifying the amount of said compound in said subject or sample;
wherein the presence of said compound in said subject or sample is indicative of a disease involving misfolded or aggregated forms of protein. In a fifth aspect, the invention relates to the use of a compound of formula I as defined above in a method according to the third or fourth aspect above. In a sixth aspect, the invention relates to the use of a labelled or unlabeled compound of formula I as defined above in imaging {in vivo or ex vivo).
Brief description of the drawings
Figure 1 is a plot of normalized fluorescence vs time showing fluorescence dynamics in plaques in in vivo imaging with a compound of Example 1.
Figure 2 is a plot of fluorescence vs time showing fluorescence dynamics in plaques in in vivo imaging with a compound of Example 21. Detailed description of the invention
As mentioned above, in a first aspect the present invention relates to a compound of formula (I), optionally labeled with a label detectable with an in vivo imaging method, or a
pharmaceutically acceptable salt thereof, for in vivo imaging of misfolded or aggregated forms of proteins in a mammal.
DEFINITIONS
All words and abbreviations used in the present application shall be construed as having the meaning usually given to them in the relevant art, unless otherwise indicated. For clarity, some terms are however specifically defined below.
The term "in vivo imaging" refers to any method which permits the detection of a compound in vivo as described herein. One illustrative example of a method for in vivo imaging is e.g. imaging of compounds with near infrared (NIR) spectra. NIR imaging can be non-invasive imaging using NIR light and methods of NIR imaging are known in the art [Hintersteiner, M. et al, Nat. Biotechnol. 23, 577-583 (2005)]. Further methods include PET, SPECT, MRS, MRI, CAT, TPM and MPM.
NIR means Near Infra-red imaging. PET means Positron Emission Tomography.
SPECT means Single Photon Emission Computed Tomography.
MRS means Magnetic Resonance Spectroscopy.
MRI means Magnetic Resonance Imaging.
CAT means Computer Assisted Tomography.
TPM means Two Photon Fluorescence Microscopy Imaging.
MPM means Multi Photon Microscopy Imaging.
The term alkyl or alkylene moiety, as used herein, is a C1-C6 alkyl or alkylene moiety, e.g. a C1-C4 alkyl or alkylene moiety and is intended to encompass also the alkyl or alkylene portion of any functional group, e.g. an alkoxy, alkylamino or carboxypolyoxyalkylene group. Thus, for example, any alkyl in an alkoxy or alkylamino group according to the invention is a C1-C6 alkyl group, e.g. a C1-C4 alkyl group. The term alkenyl, as used herein, is a C2-C6 alkenyl moiety, e.g. a C1-C4 alkenyl moiety, and is intended to encompass also the alkenyl portion of any functional group,
The term alkynyl, as used herein, is a C2-C6 alkynyl moiety, e.g. a C2-C4 alkynyl moiety, and is intended to encompass also the alkynyl portion of any functional group,
Also, any alkyl, or alkylene alkenyl or alkynyl group according to the invention may be branched or unbranched.
The term "alkyl" includes the monoradical derived from a branched or unbranched Ci-C6 alkane, or C1-C4 alkane. Examples of an alkyl group are methyl (CH3-), ethyl (CH3CH2-), propyl (-CH2CH2CH2-) and isopropyl ((CH3)2CH-).
The term "alkylene" includes the diradical derived from a branched or unbranched Ci-C6 alkane, or C1-C4 alkane. Examples of an alkylene group are methylene (-CH2-), ethylene
(-CH2CH2-), propylene (-CH2CH2CH2-) and isopropylene (-CH(CH3)CH2-). The terms "phenylene", "pyridinylene", "pyrazinylene", "pyridazinylene", "pyrimidinylene", "thienylene", "thiazolylene", "thiadiazolylene", "oxazolylene", "furanylene", "triazolylene", "pyrazolylene", "imidazolylene", "pyrrolylene", "benzothienylene", "benzodiazolylene" and "indolylene" include diradicals derived from benzene, pyridine, pyrazine, pyridazine, pyrimidine, thiphene, thiazole, thiadiazole, oxazole, furane, triazole, pyrazole, imidazole, pyrrolylene, benzo[c]thiophene, benzodiazole and indole, respectively.
The terms "hydroxy alkyl", "hydroxy alkoxy" "hydroxyalkoxy alkyl" and
"hydroxypolyoxyalkylene", include an alkyl, alkoxy, alkoxyalkyl and polyoxyalkylene radical, respectively, carrying a hydroxy function.
The term "alkoxy" includes a group R-0-, wherein R is alkyl.
The term "alkoxyalkyl" includes an alkyl radical carrying an alkoxy function.
The term "polyoxyalkylene" includes a group of the general formula
RO-(R'0)n- wherein n is an integer from 1 to 6, e.g. from 1 to 4, or 1 or 2; R is an alkyl radical and each R' is an independently selected alkylene radical. The terms "carboxyalkyl", "carboxyalkoxy", "carboxyalkoxyalkyl" and
"carboxypolyoxyalkylene" include an alkyl, alkoxy, alkoxyalkyl and polyoxyalkylene radical, respectively, carrying a carboxy function.
The term "alkoxycarbonyl" includes a radical -COOR, viz. an alkyl ester of a carboxylic acid function.
The terms "alkoxycarbonylalkyl", "alkoxycarbonylalkoxy", "alkoxycarbonylalkoxyalkyl", "alkoxycarbonylpolyoxyalkylene" include an alkyl, alkoxy, alkoxyalkyl and polyoxyalkylene radical, respectively, carrying an alkoxycarbonyl function.
The term "alkylamino" includes - HR wherein R is alkyl. The term "dialkylamino" includes -NRR' wherein R and R'are independently selected alkyl groups.
The terms "aminoalkyl", "alkylaminoalkyl", and "dialkylaminoalkyl" include an alkyl radical carrying an amino, alkylamino or dialkylamino function, respectively.
The terms "aminoalkoxy", "alkylaminoalkoxy", and "dialkylaminoalkoxy" include an alkoxy radical carrying an amino, alkylamino or dialkylamino function, respectively.
The terms "aminoalkoxyalkyl", "alkylaminoalkoxyalkyl", and "dialkylaminoalkoxyalkyl" include an alkoxyalkyl radical carrying an amino, alkylamino or dialkylamino function, respectively.
The terms "aminopolyoxyalkylene", "alkylaminopolyoxyalkylene", and
"dialkylaminopolyoxyalkylene", include a polyoxyalkylene radical carrying an amino, alkylamino or dialkylamino function, respectively.
The term "acylamino" includes a moiety - H-C(0)-alkyl.
The terms "acylaminoalkyl", "acylaminoalkoxy", "acylaminoalkoxyalkyl" and
"acylaminopolyoxyalkylene" include an alkyl, alkoxy, alkoxyalkyl and polyoxyalkylene radical, respectively, carrying an acylamino function.
The term "acylalkylamino" includes a moiety - R-C(0)-alkyl wherein R is alkyl.
The terms "acylalkylaminoalkyl", "acylalkylaminoalkoxy", "acylalkylaminoalkoxyalkyl" and "acylalkylaminopolyoxyalkylene" include an alkyl, alkoxy, alkoxyalkyl and polyoxyalkylene radical, respectively, carrying an acylalkylamino function.
The term "hydrazinocarbonyl" includes a moiety a -C(0) H- H2.
The terms "hydrazinocarbonylalkyl", "hydrazinocarbonylalkoxy",
"hydrazinocarbonylalkoxyalkyl" and "hydrazinocarbonylpolyoxyalkylene", include an alkyl, alkoxy, alkoxyalkyl and polyoxyalkylene radical, respectively, carrying a hydrazinocarbonyl function.
The terms "(amino)(carboxy)alkyl", "(amino)(carboxy)alkoxy",
"(amino)(carboxy)alkoxyalkyl" and "(amino)(carboxy)polyoxyalkylene" include an alkyl, alkoxy, alkoxyalkyl and polyoxyalkylene radical, respectively, carrying a carboxy and an amino function, preferably attached to the same carbon atom.
The terms "(alkylamino)(carboxy)alkyl","(alkylamino)(carboxy)alkoxy",
"(alkylamino)(carboxy)alkoxyalkyl" and "(alkylamino)(carboxy)polyoxyalkylene", include an alkyl, alkoxy, alkoxyalkyl and polyoxyalkylene radical, respectively, carrying a carboxy and an alkylamino function, preferably attached to the same carbon atom.
The terms "(dialkylamino)(carboxy)alkyl", "(dialkylamino)(carboxy)alkoxy",
"(dialkylamino)(carboxy)alkoxyalkyl" and "(dialkylamino)(carboxy)polyoxyalkylene", include an alkyl, alkoxy, alkoxyalkyl and polyoxyalkylene radical, respectively, carrying a carboxy and a dialkylamino function, preferably attached to the same carbon atom.
The terms "(alkoxycarbonyl)(amino)alkyl", "(alkoxycarbonyl)(amino)alkoxy",
"(alkoxycarbonyl)(amino)alkoxyalkyl" and "(alkoxycarbonyl)(amino)polyoxyalkylene", include an alkyl, alkoxy, alkoxyalkyl and polyoxyalkylene radical, respectively, carrying an alkoxycarbonyl and an amino function, preferably attached to the same carbon atom.
The terms "(alkoxycarbonyl)(alkylamino)alkyl", "(alkoxycarbonyl)(alkylamino)alkoxy", "(alkoxycarbonyl)(alkylamino)alkoxyalkyl" and "(alkoxycarbonyl)(alkylamino)- polyoxyalkylene", include an alkyl, alkoxy, alkoxyalkyl and polyoxyalkylene radical, respectively, carrying an alkoxycarbonyl and an alkylamino function, preferably attached to the same carbon atom.
The terms "(alkoxycarbonyl)(dialkylamino)alkyl", "(alkoxycarbonyl)(dialkylamino)alkoxy", "alkoxycarbonyl)(dialkylamino)alkoxyalkyl" and "(alkoxycarbonyl)(dialkylamino)- polyoxyalkylene", include an alkyl, alkoxy, alkoxyalkyl and polyoxyalkylene radical, respectively, carrying an alkoxycarbonyl and a dialkylamino function, preferably attached to the same carbon atom.
The terms "nitroalkyl", "nitroalkoxy", "nitroalkoxyalkyl", "nitropolyoxyalkylene" include an alkyl, alkoxy, alkoxyalkyl and polyoxyalkylene radical, respectively, carrying a nitro function.
The terms "cyanoalkyl", "cyanoalkoxy", "cyanoalkoxyalkyl", "cyanopolyoxyalkylene" include an alkyl, alkoxy, alkoxyalkyl and polyoxyalkylene radical, respectively, carrying a cyano function.
The terms "sulfoalkyl", "sulfoalkoxy", "sulfoalkoxyalkyl", "sulfopolyoxyalkylene" include an alkyl, alkoxy, alkoxyalkyl and polyoxyalkylene radical, respectively, carrying a sulfo function.
The terms "alkylsulphonyl" and "alkylaminosulphonyl" include an alkyl and alkylamino radical, respectively, carrying a sulphonyl function.
The term "alkyl sulphinyl" includes an alkyl radical carrying a sulphinyl function.
It should be noted that the invention includes the compounds described herein in all possible geometric or stereomeric forms. Within the scope of the invention are cis- and trans-isomers, R and S enantiomers, diastereomers, and racemic mixtures of the mentioned compounds. NOVEL COMPOUNDS
The compounds of formula I can be regarded as oligomers of conjugated monomers, typically trimers, tetramers, pentamers, hexamers, heptamers, octamers, and nonamers, wherein the backbone chain is formed from monomers selected from benzene and five or six-membered heteroaromatic rings, and wherein the backbone of monomers support at least two side-chains or groups of ionic or polar character (the group R in formula I above). Optionally, one or more of the monomers may be substituted with one or more, e.g. one or two, other groups, such as, for example, halogen, e.g. chloro, iodo or bromo, trifluoro, alkyl, alkenyl or alkynyl (e.g. ethynyl) (the group R1 in formula I above). Optionally, the backbone may include one or more monomers in the form of a bicyclic fused heteroaromatic rings having 7 to 10 ring members, typically in end position of the backbone.
Examples of heteroaromatic rings include, without limitation thereto, pyridine, pyrazine, pyridazine, pyrimidine, thiophene, thiazole, isothiazole, thiadiazole, oxazole, isoxazole, furane, triazole, triazine, oxadiazole, pyrazole, imidazole and pyrrole.
Examples of bicyclic fused heteroaromatic rings include, without limitation thereto, benzothiophene, benzodiazole, indole, benzoxazole, benzothiazole, benzimidazole, benzoisothiazole, and benzofurane.
The oligomer backbone may consist of a single monomer, such as thiophenes, or of mixed monomers, and may be symmetric or asymmetric. Optionally, the backbone may include one or more carbonyl groups replacing the bond between two adjacent rings. In some embodiments, the backbone comprises a central thiophene ring, i.e. A in formula I is thienylene. In other embodiments, the backbone comprises a central benzene ring, i.e, A in formula I is phenylene.
In some embodiments the backbone comprises a central phenylene ring surrounded by thiophene rings.
In some embodiments the backbone comprises a number of conjugated thiophene rings and benzene rings as end monomers. Exemplary trimeric backbones include (without limitation thereto):
thieny 1 ene-thi eny lene-thi eny 1 ene
phenyl ene-thi eny 1 ene-pheny 1 ene
phenyl ene-thi eny 1 ene-thi eny 1 ene phenyl ene-thi adi azolyl ene-pheny 1 ene
b enzothi eny 1 ene-thieny 1 ene-b enzothi eny 1 ene
b enzodi azolyl ene-thieny 1 ene-b enzodiazolyl ene
b enzothi eny 1 ene-pheny 1 ene-b enzothi eny 1 ene
benzodiazolylene-thiadiazolylene-benzodiazolylene
b enzodi azolyl ene-pheny 1 ene-b enzodi azolyl ene .
Exemplary tetrameric backbones include (without limitation thereto): thieny 1 ene-thi eny 1 ene-thi eny 1 ene-thi eny 1 ene
thieny 1 ene-thi eny 1 ene-thi eny 1 ene-CO-thi eny 1 ene
phenyl ene-thi eny 1 ene-thi eny 1 ene-pheny 1 ene
thieny 1 ene-thi eny 1 ene-thi eny 1 ene-CO-pheny 1 ene .
Exemplary pentameric backbones include (without limitation thereto): thieny 1 ene-thi eny 1 ene-thi eny 1 ene-thi eny 1 ene-thieny 1 ene
phenyl ene-thi eny 1 ene-thi eny 1 ene-thi eny 1 ene-pheny 1 ene
thieny 1 ene-thi eny 1 ene-thi eny 1 ene-thi eny 1 ene-pyridiny 1 ene
thieny 1 ene-thi eny 1 ene-thi eny 1 ene-thi eny 1 ene-pheny 1 ene
pyridiny 1 ene-thieny 1 ene-thieny 1 ene-thi eny 1 ene-py ri diny 1 ene thieny 1 ene-thi eny 1 ene-pheny 1 ene-thi eny 1 ene-thi eny 1 ene
phenyl ene-thi eny lene-pheny 1 ene-thi eny 1 ene-pheny 1 ene
b enzothi eny 1 ene-thieny 1 ene-thi eny 1 ene-thi eny 1 ene-b enzothi eny 1 ene indolyl ene-thi enylene-thienylene-thienylene-indolyl ene.
thienylene-CO-thienylene-thienylene-thienylene-CO-thienylene phenyl ene-C O-thieny 1 ene-thi eny 1 ene-thi eny 1 ene-C O-phenyl ene thieny 1 ene-pheny 1 ene-thi eny 1 ene-pheny 1 ene-thi eny 1 ene
thieny 1 ene-py razolyl ene-thi eny 1 ene-pyrazoly 1 ene-thieny 1 ene phenyl ene-py razolyl ene-pheny 1 ene-py razolyl ene-pheny 1 ene phenyl ene-oxazolylene-phenylene-oxazolylene-phenyl ene
phenyl ene-thi adi azolyl ene-pheny 1 ene-thiadi azolyl ene-pheny 1 ene thieny 1 ene-py rrolyl ene-thi eny 1 ene-pyrroly 1 ene-thi eny 1 ene
thienylene-imidazolyl ene-thi enylene-imidazolylene-thienylene phenyl ene-imidazolylene-phenylene-imidazolylene-phenyl ene thieny 1 ene-thi eny 1 ene-thi adi azolyl ene-thi eny 1 ene-thi eny 1 ene
phenyl ene-thi eny 1 ene-thi adi azolyl ene-thi eny 1 ene-pheny 1 ene
pyrimi diny 1 ene-thi eny 1 ene-thi eny 1 ene-thi eny 1 ene-py rimi diny 1 ene
thiazolyl ene-thi azolyl ene-thi eny 1 ene-thi azolyl ene-thi azolyl ene
thiazolyl ene-thi eny 1 ene-thi eny lene-thi eny 1 ene-thi azolyl ene
thieny 1 ene-thi eny 1 ene-thi azolyl ene-thi eny 1 ene-thi eny 1 ene .
Exemplary hexameric backbones include (without limitation thereto):
thieny 1 ene-thi eny 1 ene-thi eny 1 ene-thi eny 1 ene-thieny 1 ene-thi eny 1 ene
phenyl ene-thi eny 1 ene-thi eny 1 ene-thi eny 1 ene-thi eny 1 ene-pheny 1 ene .
Exemplary heptameric backbones include (without limitation thereto):
thieny 1 ene-thi eny 1 ene-thi eny 1 ene-thi eny 1 ene-thieny 1 ene-thi eny 1 ene-thi eny 1 ene
phenyl ene-thi eny 1 ene-thi eny 1 ene-thi eny 1 ene-thi eny 1 ene-thi eny 1 ene-pheny 1 ene
thieny 1 ene-thi eny 1 ene-thi eny 1 ene-pheny 1 ene-thi eny 1 ene-thi eny 1 ene-thi eny 1 ene
tetrazole-thi eny 1 ene-thi eny 1 ene-thieny 1 ene-thieny 1 ene-thi eny 1 ene-tetrazole
thieny 1 ene-pheny 1 ene-thi eny 1 ene-pheny 1 ene-thi eny 1 ene-pheny 1 ene-thi eny 1 ene
thieny 1 ene-pheny 1 ene-thi eny lene-thi eny 1 ene-thi eny 1 ene-pheny 1 ene-thi eny 1 ene
pyridinylene-thienylene-thienylene-thienylene-thienylene-thienylene-pyridinylene
imidazolyl ene-thi enyl ene-thi enyl ene-thi eny 1 ene-thi enyl ene-thi enylene-imidazolyl ene pyrazolyl ene-thi enyl ene-thi enyl ene-thi eny lene-thieny 1 ene-thi enyl ene-py razolyl ene oxazoly 1 ene-thieny 1 ene-thi enyl ene-thi enyl ene-thi enyl ene-thi enyl ene-oxazolyl ene
pyrimi diny 1 ene-thi enyl ene-thi enyl ene-thi enyl ene-thi enyl ene-thi enyl ene-pyrimi diny 1 ene .
When A is thienylene, it is preferably a 2,5-diradical:
Figure imgf000016_0001
i.e. it is attached to the adjacent rings at the carbon atoms adjacent to its ring sulfur atom. This also applies when A is another five-membered heteroaromat having a single heteroatom.
When A is phenylene, it is preferably a 1,4-diradical:
Figure imgf000017_0001
i.e. it is attached to the adjacent rings in para-position, but it may also be a 1,3-diradical
Figure imgf000017_0002
i.e. it is attached to the adjacent rings in meta-position.
In some embodiments of the compounds, ring A is unsubstituted.
In some embodiments the group R in formula I is a group R2 for ring A, a group R3 for ring B, and a group R4 for ring C, wherein each R2, R3 and R4 are independently selected from carboxy, carboxyalkyl, aminoalkyl, acylaminoalkyl, acyloxyalkyl, hydroxyalkyl,
alkylsulphonyl, alkylsulphinyl, alkylaminosulphonyl, alkoxycarbonylalkyl, mo holinoalkyl, carboxyalkenyl, hydroxy, alkoxycarbonyl, pyrrolidinecarbonyl, morpholino,
cycloalkylcarbamoyl, cyano, trifluoroxy, carbamoyl, hydroxyalkoxyalkyl, carbamoylalkyl, hydroxyalkylaminoalkyl, cyanoalkyl, alkylaminoalkyl, aminoalkylaminoalkyl,
carboxyalkylaminoalkyl, piperidinealkyl, and piperazinealkyl.
In some embodiments each R2, R3 and R4 are independently selected from carboxy, carboxyalkyl, aminoalkyl, acylaminoalkyl, acyloxyalkyl, hydroxyalkyl, alkylsulphonyl, alkylsulphinyl, alkylaminosulphonyl, alkoxycarbonylalkyl, morpholinoalkyl, carboxyalkenyl, hydroxy, alkoxycarbonyl, pyrrolidinecarbonyl, morpholino, cycloalkylcarbamoyl, cyano, trifluoroxy, and carbamoyl.
In some embodiments each R2, R3 and R4 are independently selected from carboxy, carboxyalkyl, aminoalkyl, acylaminoalkyl, acyloxyalkyl, hydroxyalkyl, alkylsulphonyl, alkylsulphinyl, and alkylaminosulphonyl,
In some embodiments each R2 and R3 are independently selected from carboxy, carboxyalkyl, alkoxycarbonylalkyl, morpholinoalkyl, aminoalkyl, acylaminoalkyl, hydroxyalkoxyalkyl, carbamoylalkyl, hydroxyalkylaminoalkyl, cyanoalkyl, aminoalkylaminoalkyl,
carboxyalkylaminoalkyl, piperidinealkyl and piperazinealkyl. In some embodiments each group R3 is independently selected from carboxy-Ci-4-alkyl, hydroxy-Ci-4-alkyl, amino-Ci-4-alkyl, Ci-6-acylamino-Ci-4-alkyl, Ci-4-alkoxycarbonylamino- Ci-4-alkyl, and morpholino-Ci-4-alkyl.
In some embodiments each R4 is independently selected from carboxy, alkyl sulphonyl, alkylaminosulphonyl, hydroxyalkyl, carboxyalkenyl, hydroxy, alkoxycarbonyl, pyrrolidinoyl, morpholino, cycloalkylcarbamoyl (preferably cyclopropylcarbamoyl), cyano, trifluoroxy, and carbamoyl.
In some embodiments of the compounds, ring A is unsubstituted.
In some embodiments each R4 is independently selected from carboxy, carboxy-Ci-4-alkyl, Ci-4-alkyl sulphonyl, carboxy-Ci-4-alkylene, hydroxy, Ci-4-alkyl, Ci-4-alkoxycarbonyl, pyrrolidinoyl, morpholino, C3-6-cycloalkylcarbamoyl, cyano, trifluoroxy, carbamoyl and amino-Ci-4-alkyl.
In some embodiments each R2, R3 and R4 may be independently selected
from -COOH, -CH2COOH, -C2 H4cOOH, -CH2COOCH3, -CH2 H2, -C2H4 H2, - OCH2CH( H2)(COOH), and -CH2CH( H2)(COOH).
When the compound of formula I is a thienyl-tetramer, wherein m=0, n=l, rings B are unsubstituted thienyl, and rings C are substituted with a group R3, as defined above, especially in ortho-position to B, then R3 is preferably other than carboxymethyl, and preferably other than carboxyalkyl.
When the compound of formula I is a thienyl-pentamer, wherein m=l, n=l and ring A and rings C are unsubstituted thienyl, and rings B are disubstituted with R2, as defined above, then R2 is preferably other than carboxyethyl, and preferably other than carboxyalkyl.
When the compound of formula I is a thienyl-hexamer, wherein m=0, n=2, rings B are unsubstituted thienyl, both non-terminal rings C are substituted with a group R4, as defined above, especially in ortho-position to ring B, and both terminal rings C are either unsubstituted or substituted with a group R4, then R4 on the non-terminal rings C is preferably other than carboxymethyl, preferably other than carboxyalkyl.
In some embodiments, a substituent or substituents R4 on a terminal ring C is independently selected from carboxy, alkyl sulphonyl (e.g. methyl sulphonyl), alkylaminosulphonyl (e.g. methylaminosulphonyl), alkyl sulphinyl (e.g. methyl sulphinyl), carboxyalkenyl (e.g.
carboxyvinyl), hydroxy, alkoxy (e.g. methoxy), cyano, alkoxycarbonyl (e.g.
methoxycarbonyl), morpholino, pyrrolidinecarbonyl, trifluoroxy and carbamoyl.
In currently preferred embodiments, the compound of formula I is a heptamer. In some embodiments thereof, the compound is a heptathiophene of formula (II)
Figure imgf000019_0001
(R5)o (R7)u
( I I)
wherein
each o is independently selected from 0-2; e.g. 0 or 1, and in particular is 0;
each p is independently selected from 0-2; e.g. 0 or 1, and in particular is 1;
each u is independently selected from 0-2; e.g. 0 or 1, and in particular is 0;
each v is independently selected from 0-3; e.g. 0-2, and in particular is 0 or 1,
with the proviso that at least two of o, p, u and v are > 1; and each R5, R6, R7 and R8 are independently selected from carboxy, carboxyalkyl, aminoalkyl, acylaminoalkyl, acyloxyalkyl, hydroxyalkyl, alkylsulphonyl, alkylsulphinyl,
alkylaminosulphonyl, alkoxycarbonylalkyl, morpholinoalkyl, carboxyalkenyl, hydroxy, alkoxycarbonyl, pyrrolidinecarbonyl, morpholino, cycloalkylcarbamoyl, cyano, trifluoroxy, carbamoyl, hydroxyalkoxyalkyl, carbamoylalkyl, hydroxyalkylaminoalkyl, cyanoalkyl, alkylaminoalkyl, aminoalkylaminoalkyl, carboxyalkylaminoalkyl, piperidinealkyl, and piperazinealkyl, and each R8 may additionally be independently selected from halogen, alkyl and trifluoro, or a pharmaceutically acceptable salt thereof.
In one embodiment, o=0, u=0, p=l and v=l, and R6 and R8 are independently selected from carboxy, carboxy-Ci-4-alkyl, and amino-Ci-4-alkyl.
The group R8 is preferably in 3- or 5-position on the thiophene ring.
METHODS OF PREPARATION
The compounds of the present invention may be prepared by the person of ordinary skill in the art, in the light of the general description herein and the specific illustrating examples.
Generally, ring structures, i.e. benzene, thiophene and other heteroaromates serve as basic monomer units in the inventive compounds. Substitutions of mentioned ring structures can be obtained through conventional chemistry, well known to one skilled in the art of organic synthesis and described in text books of organic synthesis, and exemplified in the synthesis examples below.
To generate dimer, trimer, tetramer, pentamer, hexamer, heptamer, octamer, nonamer, etc. structures of mentioned ring structures several methods are known to those skilled in the art; the non-limiting examples of Suzuki and Stille coupling are mentioned here. Another well- known method to generate polymers and oligomers of conjugated systems and some of the inventive compounds described herein is the so called Grignard Metathesis reaction, well described by McCullough. [Loewe, R. S.; Khersonsky, S. M.; McCullough, R. D. Adv. Mater. 1999, 1 1, 250-253.]
Stille coupling utilizes the coupling of an organotin compound with an sp2-hybridized organic halide catalyzed by a palladium, exemplified by the schematic reaction from three ring units to a trimer-block:
M - X + Bu3Sn -M* - SnBu3 pd PPI ) > M -M* -M (Stille reaction)
where Bu3Sn is tributylstannyl, M and M* symbolize arbitrary ring structure. Suzuki coupling utilizes a reaction between an aryl- or vinyl-boronic acid or borate ester with a vinyl- or aryl-halide catalyzed by a palladium complex, exemplified by the schematic reaction:
M - X + PiB -M* - PiB Pd-→st > -M* -M (Suzuki reaction)
wherein PiB is a borate ester.
The reaction may also be carried out with pseudo-halides such as triflates .
A representative exemplary reaction scheme is given below.
Figure imgf000021_0001
Scheme 1
The above mentioned examples describe the preparation of symmetric compounds. To prepare asymmetric compounds, e.g. a stoichiometric approach may be used, where the amount of reagent added is equimolar to the compound to be derivatized, if this compound has several reaction sites. As is well-known to the skilled person, from any mixture of symmetrical and/or asymmetrical compounds, the individual compounds may be separated by methods of chemical separation. Non-limiting examples of separation include flash column chromatography, preparative HPLC and distillation.
In certain aspects the compounds may be used in form of "pharmaceutically acceptable salts", referring to derivatives of the disclosed compounds, where the described compounds are modified by making acid and base salts thereof. Non-limiting examples of pharmaceutically acceptable salts include mineral or organic salts of basic derivatives of the mentioned It- groups such as amines and organic or inorganic, e.g. alkali salts of acidic derivatives of the mentioned R-groups such as carboxylic acids. Conventional non-toxic salts and quaternary ammonium salts are included in pharmaceutically acceptable salts.
Pharmaceutically acceptable salts disclosed in the present invention may be prepared from the inventive compounds described herein that contain a basic or acidic entity by conventional chemical methods.
DISEASES
Diseases related to misfolded and aggregated proteins have also been termed proteopathies, (Walker and Levine, Curr Opin Investig Drugs. 2002 May; 3(5):782-7). Diseases featuring amyloid proteins are relevant examples for the description of diseases related to misfolded and aggregated proteins, where amyloidosis is known as a disease and may be inherited or acquired. Note that amyloidosis by default usually refers to AA amyloidosis, but any disease related to amyloid proteins, which presents amyloid deposition, is an amyloidosis. For example CJD, vCJD, Alzheimer's Disease, HD, ALS and diabetes are almost never referred to as amyloidoses. A list of proteopaties is given below.
Proteopathy Major aggregating protein
Alzheimer's disease Amyloid β peptide (Αβ); Tau protein
Cerebral β-amyloid angiopathy Amyloid β peptide (Αβ)
Retinal ganglion cell degeneration in
glaucoma Amyloid β peptide (Αβ)
Prion diseases (multiple) Prion protein
Parkinson's disease (PD) a-Synuclein
Microtubule-associated protein tau (Tau
Tauopathies (multiple) protein)
Frontotemporal lobar degeneration (FTLD) TDP-43
FTLD-FUS Fused in sarcoma (FUS) protein
Amyotrophic lateral sclerosis (ALS) Superoxide dismutase, TDP-43, FUS
Huntington's disease (HD) Proteins with tandem glutamine expansions
Familial British dementia ABri
Familial Danish dementia A Dan
CADASIL Notch3
Alexander disease Glial fibrillary acidic protein (GFAP)
Seipinopathies Seipin
Familial amyloidotic neuropathy Transthyretin Serpinopathies (multiple) Serpins
AL (light chain) amyloidosis Monoclonal immunoglobulin light chains
AH (heavy chain) amyloidosis Immunoglobulin heavy chains
AA (secondary) amyloidosis Amyloid A protein
Type II diabetes Islet amyloid polypeptide (IAPP; amylin)
Aortic medial amyloidosis Medin (lactadherin)
ApoAI amyloidosis Apolipoprotein AI
ApoAII amyloidosis Apolipoprotein All
ApoAIV amyloidosis Apolipoprotein AIV
Familial amyloidosis of the Finnish type
(FAF) Gelsolin
Lysozyme amyloidosis Lysozyme
Fibrinogen amyloidosis Fibrinogen
Dialysis amyloidosis Beta-2 microglobulin
Inclusion body myositis/myopathy Amyloid β peptide (Αβ)
Cataracts Crystalline
Retinitis pigmentosa rhodopsin
Medullary thyroid carcinoma Calcitonin
Cardiac atrial amyloidosis Atrial natriuretic factor
Pituitary prolactinoma Prolactin
Hereditary lattice corneal dystrophy Keratoepithelin
Cutaneous lichen amyloidosis Keratins
Mallory bodies Keratin intermediate filament proteins
Corneal lactoferrin amyloidosis Lactoferrin
Pulmonary alveolar proteinosis Surfactant protein C (SP-C)
Odontogenic (Pindborg) tumor amyloid Odontogenic ameloblast-associated protein
Seminal vesicle amyloid Semenogelin I
Cystic Fibrosis CFTR protein
Sickle cell disease Hemoglobin
Hyperproteolytic state of myosin
Critical illness myopathy (CIM) ubiquitination
In this paragraph some examples of amyloidosis with relevance to the present invention are named. Primary amyloidosis includes mutations in lysozyme, transthyretin, apolipoprotein B, fibrinogen and AL amyloidosis (immunoglobulin light chains, as seen with multiple myeloma). Secondary amyloidosis includes AA amyloidosis (serum amyloid A protein, an acute-phase protein due to chronic inflammation) and Gelsolin amyloidosis (plasma gelsolin fragments). Familial, or hereditary amyloidosis, are most commonly caused by mutations in the transthyretin protein, but in rare occurrences can also be caused by apolipoprotein Al, gelsolin, fibrinogen, and lysozyme mutations, primarily caused by genetics, believed to be autosomal dominant, high probability of passage to offspring, Appalachian type amyloidosis and Shar Pei fever for amyloidosis in Shar Peis. Examples of organ-specific amyloidosis are Diabetes mellitus type 2 (amylin, also known as IAPP), Alzheimer's disease (Αβ 39-42), Parkinson's disease (alpha-synuclein), Huntington's disease (huntingtin), Transmissible spongiform encephalopathies (prion protein, PrP), some examples are Creutzfeldt- Jakob disease (PrP in cerebrum), Kuru (diffuse PrP deposits in brain), Fatal Familial Insomnia (PrP in thalamus), Inclusion body myositis and Bovine spongiform encephalopathy (PrP in cerebrum of cows), Congophilic angiopathy (Amyloid beta). Cardiac amyloidosis includes congestive heart failure; some instances (PrP or transthyretin in heart). Another important example is the Iatrogenic conditions like insulin amyloidosis, believed to be caused by injection-administered insulin. Islet amyloid is aggregated fibrils composed primarily of the hormone peptide, islet amyloid polypeptide (IAPP, or amylin), are commonly found in the islets of Langerhans of the pancreas of patients suffering diabetes.
Some non-disease amyloids are native amyloids in organisms, Curli E. coli Protein (curlin), Yeast Prions [Sup35], Podospora Anserina Prion Het-s, Malarial coat protein, spider silk,
Mammalian melanosomes (pMel), Tissue-type plasminogen activator (tPA) (a hemodynamic factor), Calcitonin and proteins and peptides engineered to make amyloid.
The prion diseases [e.g. bovine spongiform encephalopathy (BSE), and Creutzfeldt- Jakob disease (CJD)], are associated with the conformational conversion of the normal cellular prion protein, (PrPc), to an infectious disease-associated isoform denoted PrPSc. The misfolded infectious form of the protein, PrPsc is the cause of a group of rare, fatal brain diseases, called prion diseases that affect humans and mammals. The prion diseases are also known as transmissible spongiform encephalopathies (TSE), and they include bovine spongiform encephalopathy (BSE, or "mad cow" disease) in cattle; scrapie in sheep; chronic wasting disease in deer and elk; and in humans [Creutzfeldt Jakob disease (CJD), Gerstmann- Straussler-Scheinker disease (GSS), Kuru].
The compounds of the present invention are intended to be used for methods for imaging of misfolded and aggregated proteins associated with these diseases and therapy of the above diseases. USE OF COMPOUNDS IN IMAGING
Molecular substances specifically targeting deposition of intra- or extra-cellular protein deposits or the characteristic amyloid plaques have been greatly desired for many years. For instance, a radiolabeled molecular reagent could be used for imaging of the amyloid plaques with positron emission tomography (PET) or single-photon emission computerized tomography (SPECT). A substance exhibiting active magnetic properties can be used for magnetic resonance spectroscopy (MRS) or imaging (MRI). MRI, sometimes referred as Nuclear magnetic resonance (NMR) imaging, techniques are finding increasing use in medical diagnostics. MRI is believed to be safer compared to imaging techniques employing ionizing radiation. Many MRI techniques developed have been based on imaging of hydrogen nuclei. However, other nuclei, like fluorine and 19F in particular [WO/1991/012824], is of interest and offers potential advantages. The fluorine nucleus offers a superior signal magnitude (high gyromagnetic ratio) and hardly any imagable fluorine exists naturally in the human body. This results in a very low background signal and the detectable signal comes from the 19F-substance administered. Another option is Two Photon Fluorescence Microscopy (TPM) imaging a technique very useful when studying for example animals. The amyloid binding compounds could be modified for fluorescence in the near infrared (NIR) region. The NIR spectra permits non-invasive detection of amyloid deposits using NIR light or two photon spectroscopy in combination with diffuse optical tomography. MRI, two-photon imaging and PET imaging will be described in more detail below.
MRI
Magnetic Resonance Imaging (MRI) based on for example 19F, other suitable isotopes being described elsewhere in this document, instead of 1H opens up new diagnostic possibilities. Since an isotope such as 19F does not naturally occur in the body, whereas 1H does, it eliminates the need for a so-called, pre-contrast scan. Generally this makes the diagnostic imaging procedure significantly easier. The quantitative molecular imaging, 1H MRI as well as 19F MRI, of fibrin, using a 19F contrast agent was described by Morawski et al, Magn. Reson. Med. 52, 1255 (2004).
In MRI it is possible to use any nucleus with a net nuclear spin. Such nuclei include hydrogen, helium-3, carbon-13, fluorine-19, oxygen-17, sodium-23, phosphorus-31 and xenon-129. 23Na and 31P are naturally abundant in the body, so they can be imaged directly. Hydrogen is the most frequently imaged nucleus in MRI due to its high gyromagnetic ratio gives a strong signal.
Isotopes that contain an intrinsic magnetic moment and angular momentum are those with an odd number of protons and/or of neutrons, a nonzero spin.
The most commonly studied nuclei: 1H, 13C and 19F.
Isotopes of other elements:
¾ 6Li, 10B, UB, 14N, 15N, 170, 19F, 23Na, 29Si, 31P, 35C1, 77Se, 113Cd, 129Xe, 195Pt.
Preferred isotopes: 2H, 13C and 19F. Two-photon imaging
Two Photon Fluorescence Microscopy (TPM) is a fluorescence imaging technique that greatly reduces photodamage and allows imaging of living specimens up to a very high depth, that is up to about one millimeter. TPM in vivo imaging, for example multiphoton excitation laser scanning microscopy, is based on the effect of simultaneous absorption of two photons, such as in the infra-red spectrum, by the compounds of the present invention. Non-linear summation of the two photons' energy results in excitation of the compounds of the present invention, which emits a detectable photon, for example in the visible spectrum. Emitted photons are then collected by a suitable detector, such as sensitive photomultipliers, while the photon emission source, such as a pulsed infra-red laser, scans the plane of interest. Suitable computer software combines the data into a sharply focused 2D image and a 3D volumetric image can be reconstructed.
Suitable wavelengths include, but not limited to, the range from about 100 nm to about 2000 nm. In one embodiment, radiation in the visible range is used. It is also possible to use multiple-photon excitation, such that instead of excitation radiation of x nm, a radiation of 2x or 3x (two-photon and three-photon excitation, respectively) is used.
PET imaging
Positron emission tomography (PET) [Bailey, D.L; D.W. Townsend, P.E. Valk, M.N. Maisey (2005). Positron Emission Tomography: Basic Sciences. Secaucus, NJ: Springer- Verlag. ISBN 1-85233-798-2] is based on the positron emission from isotopes such as carbon, fluorine, iodine, nitrogen, and oxygen. It is a nuclear medicine imaging technique that produces a three-dimensional image or picture of the position of certain elements or functional processes in the body. Radionuclides used in PET imaging are typically isotopes with short half-lives such as UC (about 20 min half-life), 13N (about 10 min), 150 (about 2 min), and 18F (about 110 min). A compound containing a positron-emitting radionuclide (tracer) is introduced into the body and detected by a PET scanner. These isotopes can replace their non-radioactive counterparts in compounds of the present invention to produce tracers that can detect the biologically relevant element in vivo. 18F is one of the most convenient labelling isotopes due to its relatively long half life of about 110 min which permits the preparation of diagnostic tracers or the study biochemical processes. 18F-labelled
radiopharmaceuticals can be prepared relatively quickly by for example the nucleophilic aromatic and aliphatic fluoro-fluorination reaction.
Accordingly, the novel compounds according to the present invention are labelled with a label detectable with an imaging method suitable for in vivo imaging. The label may be a positron- emitting radionuclide, gamma ray emitting atoms or materials, detectable single or multiple photon emitters or magnetically active materials or any other label suitable for the imaging method of choice. Examples are detectable isotopes such as 18F, 19F, 123I, nC 2H, nC, 13C, 14C, 18C, 13N, 15N, 150 170, 180, 18F, 35S, 36C1, 75Br, 76Br, 77Br, 82Br, 120I, 123I, 124I, 125I, 131I, 67Ga, 81mKr, 82Rb, mIn, 133Xe, 201T1, 90Y or 99mTc. It may also be a detectable micro- or
nanoparticle, such as a gold particle, a magnetic, supramagnetic or ferromagnetic particle, a lanthanide particle (e.g. Gd, Eu or Nd) optionally doped with metal, or a nanocrystal (such as a quantum dot commercially available from e.g. Invitrogen and Quantum Dot Corporation). What specific label is used will vary with the used imaging method and may be chosen by the skilled person.
Preferred isotopes for PET: 18F, 19F, 123I, UC, 13C, 14C, 18C, 13N, 15N, 150< 170, 180, 1201, 124I,
125I or 131I.
As will be described in more detail below, in an isotopically labelled compound of the invention, one or more atoms of a compound of formula I above may be replaced by a desired isotope. Such an atom may be a ring atom (ring A, B or C) or an atom of a group R or R1. Alternatively, the isotope or isotopes may be attached directly or via a suitable linker to the compound of formula I (to a ring A, B or C, or to a group R or R1). The amyloid binding compounds could be modified for fluorescence in the near infrared (NIR) region. The NIR spectra permit non-invasive detection of amyloid deposits using NIR light or two photon spectroscopy in combination with diffuse optical tomography. Direct non-invasive imaging of aggregated proteins, e.g. amyloid deposits, in vivo is difficult. Deviations in physical characteristics, e.g. density and water-content, between aggregated proteins and normal tissues is generally too small to enable imaging with magnetic resonance imaging (MRI) and computer assisted tomography (CAT). The compounds in the invention may be used to detect, in terms of presence and/or location, and/or quantify aggregated proteins in the body, whole or in parts of the body such as the brain, of animals or humans. The inventive compounds may be used in conjunction with non-invasive imaging techniques such as magnetic resonance spectroscopy (MRS) or imaging (MRI), positron emission tomography (PET), single photon emission computed tomography (SPECT) and optical methods such as near infra-red imaging (NIR) and multi photon imaging (MPM).
The present invention has the potential to be used together with many or all of the imaging technologies described above. This is possible since the compounds in the invention have the
18 19 123 desired properties or can be synthesized with functional imaging groups, such as F, F, I, UC, etc, at specific groups without harming its binding capacity for aggregated or amyloid proteins. In such amyloid binding compounds/probes are made and described in the present invention, are useful for in vivo imaging and quantification of amyloid deposition. These compounds are to be used in conjunction with non-invasive neuroimaging or body imaging techniques such as NIR, MRS, MRI, PET and SPECT. The compounds may be engineered to cross the blood-brain barrier after peripheral injection, or they will be delivered into the cerebral spinal fluid directly.
In certain embodiments of the invention the compounds may be used to detect aggregated proteins with NIR imaging. NIR imaging includes the use of NIR light and diffuse optical tomography. Inventive compounds for NIR imaging have near infrared spectra and affinity for aggregated protein, including but not limited to amyloid plaques. NIR imaging included methods known to those skilled in the art and examples of NIR compounds can be found in [Hintersteiner, M. et al. Nat. Biotechnol. 23, 577-583, 2005]. In certain embodiments the inventive compounds may be used for multi photon imaging. The principle of MPM is that two or more low energy photons interact nearly simultaneously with a fluorescent molecule resulting in an electronic transition, an excitation, equivalent to the absorption of a photon of twice as much energy. Methods of MPM include, but are not limited to, using excitation at the NIR wavelengths (700-1000 nm). The use of MPM furthermore includes, but is not limited to, the use of gradient index (GRIN) lenses and/or cranial windows as well as conventional multiphoton imaging techniques known to those skilled in the art. Inventive compounds usable for MPM have the sufficient two-photon cross section to enable MPM. In certain aspects of the invention MPM may be used in conjunction with the inventive compounds to image aggregated proteins within cells and/or limited parts or tissue. In other embodiments the inventive compounds may be used to image or detect aggregated proteins in tissue culture slides.
In certain aspects of the invention the compounds will contain additional detectable labels, including fluorescent labels and isotopic labels. The present invention includes the use compounds that are "isotopically-labelled", "radio-labelled", "radio-ligands", "detectable" by the means that one or more atoms in the compounds are substituted or replaced with an atom having a different atom mass or number than the normally naturally occurring. 19F is one non- limiting exception which is a stable isotope that can be used to label the inventive compounds. The invention includes without limitations labeling of the compounds with gamma-emitters, beta-emitters, positron-emitters and x-ray. Suitable radionuclides that may be used to label the inventive compounds include but are not limited to ; 2H (also noted as D for deuterium), UC, 13C, 14C, 13N, 15N, 150 170, 180, 18F, 35S, 36C1, 75Br, 76Br, 77Br, 82Br, 120I, 123I, 124I, 125I and 131I, preferably UC,18F. It is understood that an inventive compound only need to be detectable, with or without labeling, to the extent necessary for the technical application, i.e. mode of detection. For example, if a certain compound included in the invention is labeled with UC, a fraction of the labeled compounds may constitute 12C or another isotope of carbon of the carbon site in question of labeling. Suitable labels of inventive compounds for MRI and MRS include, but are not limited to, 13C and 19F. Labelling of the inventive compounds with 13C and 19F may be done with general organic synthesis, known to one skilled in the art. Both PET and SPECT imaging positron emitters are suitable for labeling of the inventive compounds. For SPECT imaging 123I and 13 I are suitable isotopes for labeling the inventive compounds. Labeling procedures are well known to those skilled in the art (see e.g. Eersels et al, J Label Compd Radiopharm 2005; 48: 241-257). Labeling of the inventive compounds may also be done with metal radiolabels. For SPECT imaging 99mTc is one suitable label. In certain aspects of the invention the compounds may be labeled by either of the isotopes nC,
15 18 75 76 120
1JC" 10F, ,JBr, °Br and i KJl for PET imaging by techniques well known in the art. Some examples of synthesis of such derivates have been described by Adams MJ and Wilbur DS [Adams MJ and Wilbur DS, Chem. Soc. Rev. 2005, 34, 2, 153-163]. Isotopes UC and 18F are the preferred labels for PET imaging. 3H, 125I and 14C are suitable for in vitro studies of aggregated proteins. Apart from the mentioned methods any conventional method for imaging or visualizing diagnostic probes can be used in accordance with this invention.
The choice of detection instrument for imaging will be a major factor for choosing a certain label and will guide the selection between a radionuclide, a stable isotope or R active labels. The label in question must have a signal detectable by the instrument, e.g. a radionuclide must have a decay detectable by the instrument. Furthermore the half life of the mentioned radionuclide must be considered. The half life time must be sufficient to ensure maximum uptake by the target but short enough to minimize harmful radiation to the subject. "Subject" is referring to the human or animal studied with the imaging method. The choice of detection instrument will furthermore affect the resolution of the detection or imaging, giving a resolution of 1-10 mm for PET and SPECT, 0.1 - 1 mm for MRI, 0.001 mm for MPM and 1 mm for NIR imaging. Labels of the inventive compounds include gamma emitters, which enable detection of the compounds through emitted gamma radiation of suitable wavelength. Without limitations PET and SPECT are included in methods detecting gamma radiation. Suitable for SPECT is an isotope that lacks a specific emission but emits a vast number of photons in the energy range 140-200 keV. For PET imaging a positron emitting label will annihilate to form two gamma rays which can be detected in the PET camera. The use of imaging technologies could be of great benefit during drug development since they could provide important information to select candidates that seem most likely to be successful or even halt the development of drugs that does not give the desired response. In medical imaging modalities, the use of selective imaging agents to target specific molecular targets that are associated with a disease, like the compounds of the present invention for Alzheimer's disease, allows earlier diagnosis, better management and follow up of the disease as well as more efficient drug development. Therefore, targeting agents that distribute preferentially to distinct body sites, e.g. amyloid deposits or aggregated protein, are by virtue of active targeting of particular interest. The targeting agent binds to amyloid deposits or aggregated protein in various tissues, at cell surfaces, in body fluids or other places at the target site and can cross the blood brain barrier if needed.
A central criterion for a successful molecular imaging agent for use on living humans and animals is that it is specific and that excess substance can be removed through renal and/or hepatobiliary systems in order to achieve a high contrast between the target and the surroundings. The present invention is based on oligomeric aromate/heteroaromate derivatives and in such the often problematic imaging antibodies can be avoided. A labeled antibody for imaging studies in humans have shown that the maximum concentration of antibody at the target site is attainable within 24 h but that several more days are required before the concentration of a labeled antibody in circulation and in non- target tissue decreases to levels low enough for successful imaging to take place. This is not ideal for nuclear probes, because these constantly produce signal by decaying. Consequently, the present invention is based on polythiophene or other aromate/heteroaromate imaging agents for amyloid deposits or aggregated proteins in vivo that has the potential to reach the desired contrast much faster.
The novel substituted derivatives that are suitable for imaging amyloid deposits and aggregated protein and imaging methods of the invention, in part, include the determination of the presence and location of amyloid deposits in an organ or body area, preferably brain, spinal cord, and/or blood vessels of a patient. The novel derivatives can also be used to discriminate between misfolded and native proteins. Certain embodiments of the methods of the invention include administration of a detectable quantity of a pharmaceutical composition containing the inventive compound described herein and analogues thereof, referred to as a the compounds of the present invention or a pharmaceutically acceptable water-soluble salt thereof, to a patient. In some embodiments of the invention, a detectable compound is a radioactively labeled compound, magnetically tagged compound and in some embodiments of the invention a compound fluorescent properties are employed. The invention employs detectable compounds which, in conjunction with non- invasive neuroimaging techniques such as magnetic resonance spectroscopy (MRS), imaging (MRI), or gamma imaging such as positron emission tomography (PET), single-photon emission computed tomography
(SPECT), near infrared (NIR) or multiphoton imaging may be used to quantify amyloid deposition or protein aggregates in vivo.
One mode in which the novel compounds based in vivo imaging methods is performed is by preparing the compound with a suitable label, administered to the organism whereby the compound captures, binds to, interacts with or detects mis-assembled/aggregated forms of proteins, especially misfolded or aggregated forms of proteins, and at the same time acting as transducer reporting the capture event in detectable signals, is therefore described in the present invention.
The inventive compounds may be administrated by any means known to one of ordinary skill in the art. The present invention includes "pharmaceutically acceptable" compositions based on an amount of the inventive compound necessary for effective administration to a patient together with one or more pharmaceutical carriers, such as additive and/or diluent. The formulation of the inventive compounds may be determined by the means of administration. The formulation for administration of the inventive compounds may be solid, liquid or in aerosol form. Administration of the inventive compounds to an animal or human may be local or systemic accomplished parenterally, orally, by inhalation, rectally, nasally, buccally, vaginally or via an implanted reservoir. The term "parenteral" refers to administration outside the alimentary tract including subcutaneous, intravenous, intramuscular, intraarterial, intraspinal, intracranial, subdermal, intradermal by injection or infusion.
Carrier agents may be added to the pharmaceutical thiophene derivates compositions in order to achieve the desired distribution and acceptance in the living organism. Such agents include, but are not limited to, lipids, phospholipids, cellulose membranes, sugar coatings, hyaluronic acid, detergents, peptides, proteins, ions, salts, chelators and solvents.
Illustration of positions, but not limited to, on the molecular scaffold oh the inventive compounds where isotopes can be introduced
The illustrations presented below are only presented as non-limiting examples of compounds according to the invention. Suitable substitutions of mentioned ring structures can be obtained through conventional chemistry, well known to one skilled in the art of organic synthesis and described in text books of organic synthesis, and exemplified in the illustrations below, not verified, and in the synthesis examples herein.
Illustration based on the compound of synthesis Example 1 below.
Figure imgf000033_0001
Figure imgf000034_0001
Figure imgf000034_0002
Illustration based on the compound of synthesis Example 21 below.
Figure imgf000034_0003
Figure imgf000035_0001
Illustration based on the compound of synthesis Example 19 below.
Figure imgf000035_0002
Figure imgf000035_0003
Illustration based on the compound of synthesis Example 62 below.
Figure imgf000036_0001
References:
Flourinated thiophenes: "Tetrahedron Letters 42 (2001) 8797-8800"; and "Fluorinated pharmaceuticals", Basil Wakefield, Ultrafine, IPT Online, The Pharmaceutical Technology Journal, January 2000.
Deuterated benzenes: Organic Chemistry, Maitland Jones, 4th edition, Chapter 14.
Additional references can be found elsewhere in the description.
In one aspect the present invention relates to a method for imaging the misfolded protein species in order to detect, quantify and/or localize said misfolded protein species inside a living organism {in vivo) or where an organ or tissue has been removed from the organism {ex vivo) comprising the steps of: - providing a compound according to the invention, optionally substituted with a detectable label for MRI, PET, SPECT, NIR, infrared, two-photon or fluorescence detection;
- providing, or preparation of, a pharmaceutical or non-pharmaceutical composition containing the optionally substituted compound;
- administrating the compound to the subject, organism, organ, tissue or sample;
- subjecting the subject, organism, organ, tissue or sample to the appropriate imaging method;
- optionally exposing the subject, organism, organ, tissue or sample to appropriate radiation or magnetic field;
- analyzing the results using appropriate hardware and software;
whereby any misfolded protein species can be detected, quantified and/or localized.
The imaging method according to this aspect may be used for diagnosis of a disease involving misfolded or aggregated forms of proteins, such as Alzheimer's disease, Creutzfeldt Jacob disease (CJD), variant Creutzfeldt Jacob disease (vCJD), Secondary amyloidosis, type 2 diabetes or transmissible spongiform encephalopathy (TSE, such as, CWD, Scrapie, GSS and Kuru, bovine spongiform encephalopathy (BSE)). The presence of misfolded or aggregated forms of protein in a subject or a sample from the subject is indicative of the disease.
If detection of misfolded protein by a compound of the invention is performed, the radiation used in the method of the present invention has wavelengths in the range from about 100 nm to about 2000 nm. In one embodiment, radiation in the visible range is used. It is also possible to use multiple-photon excitation, such that instead of excitation radiation of x nm, a radiation of 2x or 3x (two-photon and three-photon excitation, respectively) is used.
Figure imgf000037_0001
(C)n-B-(A)m-B-(C> 'n (I)
wherein A, B, C, m and n are as defined above for formula I. Unlabelled compounds may be suitable for, but not limited to, NIR, single-photon, two- photon or multiple-photon imaging according to the present invention.
Example
Figure imgf000038_0001
Labelled
Figure imgf000038_0002
(III) wherein A, B, C, m and n are as defined above for formula I,
o is independently 0, 1, 2, 3, 4, 5, 6 or 7,
p is independently 0, 1, 2, 3, 4, 5, 6 or 7, and
RL is a linker, e.g. a group R or R1 as defined for formula I above, or an additional group selected from groups R and R1.
A, B and C may also independently be labelled with an isotope according to the present invention. When o is zero, the isotope is situated directly on the main compound.
Isotopes are suitable for, but not limited to, PET, MRI or SPECT imaging according to the present invention.
Example
Figure imgf000038_0003
o is 0 (= R is nothing)
p is 1
Figure imgf000039_0001
o is 1 and R = C
p is 1. EXAMPLES
Synthesis of compounds
SYNTHESIS OF INTERMEDIATES
Figure imgf000039_0002
Methyl 2-(thiophen-3-yl)acetate (Intermediate A)
Acteyl chloride (7.50 ml, 106 mmol) was added dropwise to a solution of 3-thiopheneacetic acid (5.00 g, 35.3 mmol) in dry MeOH (150 ml) at 0 °C. The mixture was left to slowly reach room temperature and stirred overnight. Solvents were evaporated and residue filtered through a short plug of silica gel (DCM) to give a quantitative yield of the title compound an oil. 1H MR (400 MHz, CDC13):□ 3.64 (s, 2H), 3.70 (s, 3H), 7.03-7.05 (m, 1H), 7.14 7.15 (m, 1H), 7.27-7.29 (m, 1H).
Figure imgf000039_0003
Methyl 2-(2-bromothiophen-3-yl)acetate (Intermediate B)
BS (6.28 g, 35.3 mmol) was added portion-wise to a solution of intermediate A (5.51 g, 35.3 mmol) in CHC13 (15 ml) and acetic acid (15 ml) at 0 °C. The mixture was left to slowly reach ambient temperature and stirred for 3 d. Et20 (100 ml) was added and mixture was filtered and concentrated. The residue was purified by flash chromatography using 2-10% EtOAc in iso-hexane as eluent. Yield: 5.74 g (69%); colourless oil. 1H MR (400 MHz, CDC13): δ 3.64 (s, 2H), 3.72 (s, 3H), 6.93 (d, J 6 Hz, 1H), 7.24 (d, J 6 Hz, 1H).
Figure imgf000040_0001
Methyl 2-(2-{5-[3-(2-methoxy-2-oxoethynthiophen-2-yllthiophen-2-yl}thiophen-3-ynacetate (Intermediate C)
Argon was bubbled through a mixture of intermediate B (16.3 g, 69.1 mmol), 2,5- thiophenediboronic acid (5.93 g, 34.5 mmol) and K2CO3 (28.6 g, 207 mmol) in toluene (100 ml) and MeOH (100 ml). After 10 min PEPPSI-iPr™ (475 mg, 0.70 mmol) was added and the mixture heated at 50 °C for 1.5 h. The mixture was filtered through a short plug of silica gel and concentrated. The residue was purified by flash chromatography using iso- hexane/EtOAc 9: 1 and 4: 1 as eluents. Yield: 9.07 g (67%); slightly yellow oil. 1H MR (400 MHz, CDCI3): δ 3.74 (s, 6H), 3.80 (s, 4H), 7.07 (d, J 5.3 Hz, 2H), 7.16 (s, 2H), 7.26 (d, J 5.3 Hz, 2H).
Figure imgf000040_0002
Methyl 2-(5-bromo-2-{5-[5-bromo-3-(2-methoxy-2-oxoethyl)thiophen-2-yl]thiophen-2- yl}thiophen-3-yl)acetate (Intermediate D)
BS (2.96 g, 16.6 mmol) was added portion-wise to a solution of intermediate C (3.11 g, 7.92 mmol) in acetic acid (50 ml). The mixture was stirred at rt for 2 h. Solvent was evaporated and residue dissolved in DCM (200 ml) and water was added. The organic layer was washed with sat. NaHCC"3 and organic layer concentrated. The residue was purified by flash chromatography using 20% EtOAc in iso-hexane as eluent and then purified again using pet.ether/ Et20 2: 1 and 1 : 1 as eluents. Yield: 3.65 g (83%); yellow solid. 1H MR (400 MHz, CDCI3): δ 3.71 (s, 4H), 3.74 (s, 6H), 7.03 (s, 2H), 7.09 (s, 2H).
Figure imgf000040_0003
Methyl 2-(2-(5-[5-bromo-3-(2-methoxy-2-oxoethvnthiophen-2-yllthiophen-2-yl|thiophen-3- yPacetate (Intermediate E) BS (1.88 g, 10.5 mmol) was added portion-wise to intermediate C (4.37 g, 11.4 mmol) in CHCI3 (30 ml) and AcOH (30 ml). The mixture was stirred at rt for 1 h. DCM and water was added. The organic phase was washed with water and sat. NaHC03. The organic layer was evaporated and residue purified by flash chromatography using 100% DCM and once more purification using DCM/CHCI3 (2: 1) as eluent. Yield: 2.02 g (38%); pale yellow oil. 1H NMR (400 MHz, CDC13): δ 3.74 (s, 2H), 3.75 (s, 3H), 3.76 (s, 3H), 3.80 (s, 2H), 7.05 (s, 1H), 7.08 (d, J 5.2 Hz, 1H), 7.13 (d, J4 Hz, 1H), 7.16 (d, J 4 Hz, 1H), 7.27 (d, J 5.2 Hz, 1H).
Figure imgf000041_0001
2-(2-Bromothiophen-3-yl)ethan-l-ol (Intermediate F)
NBS (9.53 g, 53.6 mmol) was added portionwise to a solution of 3-thiopheneethanol 6.87 g, 53.6 mmol) in AcOH (30 ml). The temperature was raised to 40 °C and mixture was stirred for 1 h. DCM (400 ml) and water was added. The organic layer was washed with water and sat. NaHC03. The organic layer evaporated and residue purified by flash chromatography using 25-33% EtOAc in iso-hexane. Yield: 5.39 g (49%); colourless oil. 1H NMR (400 MHz, CDC13): δ 2.89 (t, J 6.4 Hz, 2H), 3.85 (t, J 6.4 Hz, 2H), 6.89 (d, J 5.6 Hz, 1H), 7.26 (d, J 5.6 Hz, 1H).
Figure imgf000041_0002
2-(2-(5-[3-(2-Hvdroxyethvnthiophen-2-yllthiophen-2-yl|thiophen-3-vnethan-l-ol
(Intermediate G)
Nitrogen was bubbled through a mixture of intermediate F (1.148 g, 5.54 mmol), 2,5- thiophenediboronic acid (476 mg, 2.77 mmol) and K2C03 (1.92 g, 13.9 mmol) in toluene (8 ml) and MeOH (8 ml). PEPPSI-iPr™ (19 mg, 0.0277 mmol) was added and mixture heated at 50 °C for 45 min under nitrogen. Chloroform was added and mixture filtered. The residue after evaporation was dissolved in MeOH. Silica was added and solvent evaporated. The dry silica was applied on a flash column and product eluted using iso-hexane/EtOAc 1 : 1 and 1 :2. Yield: 542 mg (58%); yellow oil. HPLC: RT = 2.49 min, 95% (254 nm, 10-90% MeCN in 3 min, XBridge). 1H NMR (400 MHz, CDC13): 6 3.10 (t, J 6.8 Hz, 4H), 3.92 (t, J 6.8 Hz, 4H), 7.02 (d, J 5.2 Hz, 2H), 7.13 (S, 2H), 7.27 (d, J 5.2 Hz, 2H).
Figure imgf000042_0001
2-(5-Bromo-2-{5-[5-bromo-3-(2-hydroxyethyl)thiophen-2-yl]thiophen-2-yl}thiophen-3- yl)ethan-l-ol (Intermediate H)
BS (324 mg, 1.82 mmol) was added portion-wise to a solution of intermediate G (307 mg, 0.911 mmol) in CHC13 (8 ml) and AcOH (8 ml). The mixture was stirred at rt overnight. DCM (100 ml) and water was added. The organic layer was washed with 1 M NaOH until the aqueous layer was alkaline. The organic layer was separated and residue purified by flash chromatography using EtOAc/iso-hexane 3 :2 as eluent. Yield: 254 mg (56%); yellow solid. 1H NMR (400 MHz, CDC13) δ 3.02 (t, J 6.8 Hz, 4H), 3.89 (d, J 6.8 Hz, 4H), 7.00 (s, 2H), 7.07 (s, 2H).
Figure imgf000042_0002
2-(Thiophen-3-yl)ethyl methanesulfonate (Intermediate I)
Methyl sulfonyl chloride (2.22 ml, 28.75 mmol) was added dropwise to a solution of 3- thiophene-ethanol (2.46 g, 19.2 mmol) and Et3N (5.3 ml) in DCM (50 ml) as 0 °C. The mixture was stirred for 15 min before DCM and water was added. Organic phase was separated and residue purified by flash chromatography using 20-25% EtOAc in iso-hexane as eluents. Yield: 3.93 g (99%); colourless oil. HPLC: RT = 2.20 min, 96% (254 nm, 10-90% MeCN in 0.1% TFA, 3 min ACE). 1H NMR (400 MHz, CDC13): δ 2.90 (s, 3H), 3.13 (t, J6.8 Hz, 2H), 4.44 (t, J6.8 Hz, 2H), 7.00-7.02 (m, 1H), 7.11-7.12 (m, 1H), 7.32-7.36 m, 1H).
Figure imgf000042_0003
2-(2-Bromothiophen-3-yl)ethyl methanesulfonate (Intermediate J) BS (3.38 g, 19.0 mmol) was added portion-wise to a solution of intermediate I (3.92 g, 19.0 mmol) in AcOH (50 ml). The mixture was stirred at rt for 1 h. Solvent was evaporated and residue dissolved in DCM. The solution was washed with sat. NaHC03 and evaporated. The residue was purified by flash chromatograpy using iso-hexane:EtOAC 3 : 1 as eluent. Yield: 4.51 g (83%); colourless oil. HPLC: RT = 2.41 min, 97% (10-90% MeCN in 10 mM buffer, XBridge). 1H MR (400 MHz, CDC13): δ 2.93 (s, 3H), 3.07 (t, J 6.8 Hz, 2H), 4.39 (t, J 6.8 Hz, 2H), 6.90 (d, J4.4 Hz, 1H), 7.28 (d, J4.4 Hz, 1H).
Figure imgf000043_0001
fert-Butyl N- [2-(2-bromothiophen-3 -yPethyl] -N- [(tert-butoxy)carbonyl] carb amate
(Intermediate K)
Cs2C03 (7.70 g, 23.7 mmol) was added to a solution of di-t-butyl iminodicarboxylate (5.14 g, 23.7 mmol) in DMSO (100 ml) at rt and the mixture was stirred for 30 min before
intermediate J (4.50 g, 15.8 mmol) in DMSO (5 ml) was added. The mixture was stirred at rt overnight. Et20 and water was added. Organic phase was washed with water and then evaporated. The residue was purified by flash chromatography using 5-10% EtOAc in iso- hexane, Yield: 4.44 g (69%); colourless oil. HPLC: RT = 2.08 min, 100% (60-90% MeCN in 10 mM buffer, 3 min, XBridge). 1H NMR (400 MHz, CDC13): 6 1.51 (s, 18H), 2.88-2.92 (m, 2H), 3.78-3.81 (m, 2H), 6.83 (d, J 5.6 Hz, 1H), 7.21 (d, J 5.6 Hz, 1H).
Figure imgf000043_0002
tert-Butyl N-[2-(2-(5-[3-(2-(bis[(tert-butoxy)carbonyllaminolethvnthiophen-2-yllthiophen- 2-yl}thiophen-3-yl)ethyl]-N-[(tert-butoxy)carbonyl]carbamate (Intermediate L).
Nitrogen was bubbled through a mixture of intermediate K (2.10 g, 5.17 mmol), 2,5- thiophenediboronic acid (444 mg, 2.58 mmol) and K2C03 (1.43 g, 10.4 mmol) in toluene (40 ml) and MeOH (20 ml). PEPPSI-iPr™ (88 mg, 0.129 mmol) was added and the mixture stirred at 60 °C for 45 min. Toluene and water were added. Aqueous layer was extracted with toluene and combined organic layers concentrated. The crude material was purified by flash chromatography using 0-5% EtOAc in toluene. Yield: 1.65 g (87%); yellow oil which solidifies. 1H MR (400 MHz, CDC13): δ 1.47 (s, 36H), 3.06-3.10 (m,4H), 3.85-3.88 (m, 4H), 6.98 (d, J 5.2 Hz, 2H), 7.14 (s, 2H), 7.22 (d, J 5.2 Hz, 2H).
Figure imgf000044_0001
tert-Butyl N-[2-(2-{5-[3-(2-{bis[(tert-butoxy)carbonyl]amino}ethyl)-5-bromothiophen-2- yl]thiophen-2-yl}-5-bromothiophen-3-yl)ethyl]-N-[(tert-butoxy)carbonyl]carbamate
(Intermediate M)
BS (310 mg, 1.74 mmol) was added portionwise to a solution of intermediate L (642 mg, 0.873 mmol) in CHC13 (8 ml) and AcOH (8 ml). The mixture was stirred at rt for 2.5 h.
CHC13 and water was added. Organic layer was washed with 1 M NaOH until aqueous layer was alkaline and then concentrated. The crude material was purified by flash chromatography using 3% EtOAc in toluene. Yield: 582 mg (75%); yellow oil which solidifies. 1H NMR (400 MHz, CDC13): δ 1.47 (s, 36H), 3.00 (t, J6.8 Hz, 4H), 3.83 (t, J 6.8 Hz, 4H), 6.94 (s, 2H), 7.09 (s, 2H).
Figure imgf000044_0002
Methyl 5 -(5 - ( 5 - [5 -bromo-3 -(2-methoxy-2-oxoethvnthiophen-2-yllthiophen-2-yl I -4-(2- methoxy-2-oxoethyl)thiophen-2-yl)thiophene-2-carboxylate (Intermediate N)
Acetyl chloride (0.5 ml) was added to a solution of 5-carboxy-2-thiopheneboronic acid (554 mg. 3.22 mmol) in methanol (10 ml) and the mixture was stirred at ambient temperature overnight. More acetyl chloride (0.5 ml) was added dropwise and the mixture refluxed for 3 h. Solvents were evaporated. Yield: 538 mg (90%); white solid. 1H NMR (400 MHz. DMSO- d6): δ 3.81 (s, 3H), 7.67 (d, 3.6 Hz, 1H), 7.80 (d, J3.6 Hz, 1H), 8.55 (s, 1H). HPLC: RT = 3.09 min, 98% (254 nm, 0520A3).
Argon was bubbled through a mixture of intermediate E (897 mg, 1.902 mmol), 5- (methoxycarbonyl)thiophen-2-ylboronic acid (424 mg, 2.28 mmol), potassium carbonate (788 mg, 5.71 mmol) in toluene (10 ml) and MeOH (10 ml). PEPPSI-iPr™ (24 mg, 0.035 mmol) was added and the mixture was heated at 60 °C under argon for 45 min. Water and toluene was added. The aqueous layer was extracted with toluene. Silica gel was added to the combined organic layers and solvents evaporated. The material absorbed on silica gel was applied on a flash column which was eluted with 5-10% EtOAc in toluene. Yield: 840 mg (83%); orange solid. 1H NMR (400 MHz, DMSO-i¾): δ 3.65 (s, 3H), 3.66 (s, 3H), 3.84 (bs, 5H), 3.88 (s, 2H), 7.10 (d, J 5.2 Hz, IH), 7.25 (d, J4.0 Hz, IH), 7.31 (d, J4.0 Hz, IH), 7.43 (d, J4.0 Hz, IH), 7.50 (s, IH), 7.57 (d, J 5.2 Hz, IH), 7.77 (d, J4.0 Hz, IH). HPLC: RT = 1.90 min, 99% (254 mn, 6090X3).
BS (278 mg, 1.56 mmol) was added portion-wise to a solution of the material from above (832 mg, 1.56 mmol) in CHC13 (5 ml) and acetic acid (5 ml). The mixture was stirred at rt for 3 h. Toluene and silica gel was added to the mixture and solvents evaporated. The material absorbed on silica was applied on a flash column and eluted with 7.5% EtOAc in toluene. Yield: 917 mg (96%); orange solid. 1H NMR (400 MHz, OMSO-d6): δ 3.65-3.66 (m, 6H), 3.82-3.87 (m, 7H), 7.24 (s, IH), 7.26 (d, J4.0 Hz, IH), 7.31 (d, J 4.0 Hz, IH), 7.44 (d, J4.0 Hz, IH), 7.50 (s, IH), 7.77 (d, J 4.0 Hz, IH).
Figure imgf000045_0001
Methyl 2- [5 -(5 -bromothiophen-2-yl)-2- ( 5 - [5 -(5 -bromothiophen-2-y0-3 -(2-methoxy-2- oxoethyl)thiophen-2-yl]thiophen-2-yl}thiophen-3-yl]acetate (Intermediate O)
Argon was bubbled through a mixture of intermediate D (2.52 g, 4.58 mmol), 2- thiophenboronic acid (1.64 g, 11.4 mmol) and K2CO3 (3.16 g, 22.0 mmol) in toluene (50 ml) and MeOH (50 ml). PEPPSI-iPr™ (77 mg, 0.114 mmol) was added and the mixture heated at 60 °C for 45 under argon. Toluene and water was added. The organic layer was concentrated and residue purified by flash chromatography using 1.5-2% EtOAc in toluene as eluent. Yield: 1.86 g (73%); orange solid.
The material from above (570 mg, 1.02 mmol) was dissolved in CHCI3 (20 ml) and AcOH (20 ml). NBS (364 mg, 2.05 mmol) was added portion-wise and stirring continued for 3 d at rt. DCM (50 ml) and water was added. Organic phase washed with water and sat. Na2C03. Organic layer concentrated. Yield: 707 mg (97%). 1H NMR (400 MHz, CDC13): δ 3.75 (s,
6H), 3.77 (s, 4H), 6.93 (d, J 3.8 Hz, 2H), 6.98 (d, J3.8 Hz, 2H), 7.06 (s, 2H), 7.18 (s, 2H).
Figure imgf000046_0001
2-[5-(5-bromothiophen-2-yl)-2-{5-[5-(5-bromothiophen-2-yl)-3-(carboxymethyl)thiophen-2- yl]thiophen-2-yl}thiophen-3-yl] acetic acid (Intermediate P)
Intermediate O (707 mg, 0.989 mmol) was dissolved in dioxane (5 ml) and 2 M NaOH (5 ml) was added. The mixture was heated at 80 °C for 1 h and dioxane (10 ml) and 2 M NaOH (10 ml) was added. The mixture was stirred for 1 h at 80 °C. Water was added and the mixture acidified using 6 M HCl. The precipitated material was isolated by centrifugation, washed with water and dried at high vacuum overnight. Yield: 679 mg (100%); orange solid. 1H
NMR (400 MHz, DMSO-i¾): δ 3.75 (s, 4H), 7.21 (d, J 3.8 Hz, 2H), 7.25 (d, J 3.8 Hz, 2H), 7.29 s, 2H), 7.31 (s, 2H),
Figure imgf000046_0002
2-(5-Bromo-2-{5-[5-bromo-3-(carboxymethyl)thiophen-2-yl]thiophen-2-yl}thiophen-3- yPacetic acid (Intermediate Q)
Intermediate D (1.10 g, 2.00 mmol) was dissolved in dioxane (5 ml) and 2 M NaOH (5 ml) was added. The mixture was heated at 80 °C for lh. 6 M HCl was added and the aqueous mixture extracted with EtOAc. Combined organic layers were dried (MgS04) and evaporated.
Yield: 982 mg (94%); pale yellow solid. 1H NMR (400 MHz, DMSO-i¾): δ 3.77 (s, 4H), 7.30 s, 2H), 7.31 (s, 2H).
Figure imgf000046_0003
Methyl 2-(2-(4-[3-(2-methoxy-2-oxoethvnthiophen-2-yllphenyl|thiophen-3-vnacetate (Intermediate R)
Argon was bubbled through a mixture of 1,4-benzenediboronic acid (0.45 g, 2.72 mmol), intermediate B (1.34 g, 5.71 mmol) and K2C03 (1.18 g, 8.57 mmol) in toluene/MeOH (1 : 1, 16 ml). After 15 min PEPPSI-iPr™ (57 mg, 0.08 mmol) was added and the mixture heated at 60 °C for 60 min. The reaction mixture was cooled to RT, diluted with Et20 (35 ml), washed with water (40 ml), dried with MgS04 (s), filtered and evaporated to dryness to obtain the crude product. The crude product was dissolved in hot acetone (15 ml) followed by drop wise addition of iso-hexane (17 ml) and stirred at 4 °C for 20 hrs. The obtained solid was filtered, washed with iso-hexane (6 ml), washed with Et20 (2 x 7 ml) and dried under vacuum. Yield: 0.48 g (46%); white solid. 1H MR (400 MHz, CDC13): δ 3.70 (s, 4H), 3.73 (s, 6H), 7.10 (d, J 5.3 Hz, 2H), 7.30 (d, J 5.3 Hz, 2H), 7.53 (s, 4H).
Figure imgf000047_0001
Methyl 2-(5-bromo-2-{4-[5-bromo-3-(2-methoxy-2-oxoethyl)thiophen-2-yl]phenyl}thiophen- 3-yl)acetate (Intermediate S)
Intermediate R (0.48 g, 1.24 mmol) was dissolved in CHC13 (4.5 ml), acetic acid (4.5 ml) was added and the solution was cooled to 0 °C. NBS (0.46 g, 2.54 mmol) was added and the mixture was left to slowly reach ambient temperature. After 16 hrs, the reaction mixture was diluted with CH2C12 (20 ml), washed with water (40 ml), dried with MgS04 (s), filtered and evaporated to dryness to obtain the crude product. The crude product was dissolved in hot EtOAc (15 ml) and stirred at 4 °C for 18 hrs. The obtained solid was filtered, washed with iso- hexane (6 ml), washed with Et20 (6 ml) and dried under vaccum. Yield: 0.54 g (80%); off- white solid. 1H MR (400 MHz, CDC13): δ 3.62 (s, 4H), 3.74 (s, 6H), 7.07 (s, 2H), 7.47 (s, 4H).
Figure imgf000047_0002
Methyl 2-(2-{3-[3-(2-methoxy-2-oxoethynthiophen-2-yllphenyl}thiophen-3-ynacetate
(Intermediate T)
Argon was bubbled through a mixture of 1,3-benzenediboronic acid (0.38 g, 2.27 mmol), intermediate B (1.17 g, 4.99 mmol) and K2C03 (1.00 g, 7.26 mmol) in toluene/MeOH (1 : 1, 12 ml). After 15 min PEPPSI-iPr™ (63 mg, 0.09 mmol) was added and the mixture heated at 60 °C for 60 min. The reaction mixture was cooled to RT, diluted with CH2C12 (40 ml), washed with water (40 ml), dried with MgS04 (s), filtered and evaporated to dryness to obtain the crude product. The residue was purified by flash chromatography using toluene/EtOAc 100: 1→50: 1→40: 1) as eluent. Yield: 0.77 g (87%); yellowish oil. 1H MR (400 MHz, CDCI3): δ 3.70 (s, 10H), 7.09 (d, J 5.3 Hz, 2H), 7.29 (d, J 5.3 Hz, 2H), 7.46 - 7.48 (m, 3H), 7.57 (m, 1H).
Figure imgf000048_0001
Methyl 2-(5-bromo-2-{3-[5-bromo-3-(2-methoxy-2-oxoethyl)thiophen-2-yl]phenyl}thiophen- 3-yl)acetate (Intermediate U)
Intermediate T (0.81 g, 2.10 mmol) was dissolved in CHCI3 (8 ml), acetic acid (8 ml) was added and the solution was cooled to 0 °C. BS (0.77 g, 4.30 mmol) was added and the mixture was left to slowly reach ambient temperature. After 18 hrs, the reaction mixture was diluted with CH2C12 (30 ml), washed with water (40 ml), dried with MgS04 (s), filtered and evaporated to dryness to obtain the crude product. The residue was purified by flash chromatography using toluene/EtOAc 100: 1→40: 1) as eluent. Yield: 0.94 g (87%); off-white solid. 1H MR (400 MHz, CDC13): δ 3.61 (s, 4H), 3.72 (s, 6H), 7.06 (s, 2H), 7.41 - 7.47 (m, 4H).
Figure imgf000048_0002
Methyl 2-[5-(5-cvanothiophen-2-vn-2-(5-[5-(5-cvanothiophen-2-vn-3-(2-methoxy-2- oxoethyl)thiophen-2-yl]thiophen-2-yl}thiophen-3-yl]acetate (Intermediate V)
Argon was bubbled through a mixture of intermediate D (161 mg, 0.293 mmol), 5- cyanothiophene-2-boronic acid (112 mg, 0.731 mmol) and K2C03 (122 mg, 0.879 mmol) in toluenen (2 ml) and MeOH (2 ml). PEPPSI-iPr™ (8 mg, 0.0118 mmol) was added and the mixture heated at 70 °C for 10 min in a microwave reactor. Silica was added to the mixture and solvents evaporated. The dry silica was applied on a flash column which was eluted with 5-10% EtOAc in toluene. Yield: 115 mg (64%); bright orange solid. 1H MR (400 MHz,
CDC13): δ 3.77 (s, 6H), 3.80 (s, 4H), 7.14 (d, J4.0 Hz, 2H), 7.23 (s, 2H), 7.25 (s, 2H), 7.54 (d, J 4.0 Hz, 2H).
Figure imgf000049_0001
Methyl 2-(2-{4-[5-bromo-3-(2-methoxy-2-oxoethyl)thiophen-2-yl]phenyl}thiophen-3- yPacetate (Intermediate X)
Intermediate R (1.13 g, 2.92 mmol) was dissolved in CHCI3 (5 ml), acetic acid (5 ml) was added and the solution was cooled to 0 °C. BS (0.53 g, 2.92 mmol) was added and the mixture was left to slowly reach ambient temperature. After 14 hrs, the reaction mixture was diluted with CH2C12 (20 ml), washed with water/brine (30: 1 - 60 ml), dried with MgS04 (s), filtered and evaporated to dryness to obtain the crude product. The residue was purified by flash chromatography using toluene/EtOAc 100: 1→40: 1) as eluent. Yield: 0.54 g (40%); off- white solid.1 H MR (400 MHz, CDC13): δ 3.63 (s, 2H), 3.69 (s, 2H), 3.73 (s, 3H), 3.74 (s, 3H), 7.06 (s, 1H), 7.09 (d, J 5.3 Hz, 1H), 7.30 (d, J 5.3 Hz, 1H), 7.45-7.48 (m, 2H), 7.51-7.54 (m, 2H).
Figure imgf000049_0002
Methyl 2-[2-(4-methanesulfonylphenyl)thiophen-3-yl]acetate (Intermediate Y)
Intermediate B (2.30 g, 9.78 mmol), 4-methylsulfonylphenylboronic acid (1.96 g, 9.78 mmol), K2CC"3 (4.15 g, 30.0 mmol), tetrakis (triphenylphosphine)palladium(O) (150 mg, 0.130 mmol), MeCN (30 ml) and water (10 ml) were heated at 70 °C for 1 hour. The organic phase was removed in vacuo and the crude material was dissolved in DCM. The mixture was purified by flash chromatography using 30% and 50% EtOAc in iso-hexane as eluent. Yield: 2.25 g (74%); white solid. 1H MR (400 MHz, CDC13): 5 3.11 (s, 3H), 3.67 (s, 2H), 3.74 (s, 3H), 7.13 (d, J 5.0 Hz, 1H), 7.39 (d, J 5.0 Hz, 1H), 7.67 - 7.72 (m, 2H), 7.98 - 8.04 (m, 2H).
Figure imgf000049_0003
Methyl 2-[5-bromo-2-(4-methanesulfonylphenyl)thiophen-3-yl]acetate (Intermediate Z)
BS (209 mg, 1.18 mmol) was added in small portions to a stirred solution of Intermediate Y (365 mg, 1.18 mmol) in a mixture of MeOH (10 ml) and chloroform (5 ml) at 0 °C. AcOH (0.250 ml) was added and the reaction was stirred at 0 °C for 1 hour then at r.t. for 3 hours. More NBS (35 mg, 0.20 mmol) was added at 0 °C and the reaction was stirred at r.t. over night.
A new reaction was also started. NBS (342 mg, 1.92 mmol) was added in small portions to a stirred solution of Intermediate Y (519 mg, 1.67 mmol) in a mixture of MeOH (15 ml) and chloroform (7 ml) at 0 °C. AcOH (0.50 ml) was added and the reaction was stirred at r.t. over night.
The two reaction mixtures were combined and the solvents were removed in vacuo. The crude material was dissolved in toluene and the mixture was purified by flash chromatography using 50% EtOAc in iso-hexane as eluent. Yield: 1.03 g (93%); white solid. 1H NMR (400 MHz, CDC13): δ 3.10 (s, 3H), 3.60 (s, 2H), 3.75 (s, 3H), 7.10 (s, 1H), 7.62 - 7.66 (m, 2H), 7.98 - 8.03 (m, 2H)
Figure imgf000050_0001
Methyl 2-[2-(5-chlorothiophen-2-yl)thiophen-3-yl]acetate (Intermediate A2)
Argon was bubbled through a mixture of intermediate B (475 mg, 2.02 mmol), 5-chloro-2- thiopheneboronic acid (395 mg, 2.42 mmol) and K2CO3 (838 mg, 6.06 mmol) in toluene (5 ml) and methanol (5 ml). PEPPSI-iPr™ (27 mg, 0,0396 mmol) was added and the mixture heated at 60 °C for 30 min under argon. CHCI3 was added, mixture filtered and concentrated The residue was purified by flash chromatography using 4% EtOAc in iso-hexane as eluent. Yield: 448 mg (81%); colourless oil. 1H NMR (400 MHz, CDC13): δ 3.72 (s, 5H), 6.89 (d, J 3.8 Hz, 1H), 6.94 (d, J4.0 Hz, 1H), 7.03 (d, J 5.3 Hz, 1H), 7.25 (d, J 5.3 Hz, 1H).
Figure imgf000050_0002
Methyl 3 -(2- { 5-[3 -(3 -methoxy-3 -oxopropyl)thiophen-2-yl]thiophen-2-yl }thiophen-3 - yPpropanoate (Intermediate B2)
10% Pd/C (12.5 g) was added into a solution of trans-3-(3-thienyl)acrylic acid (25.0 g, 16.2 mmol) in methanol (300 ml) and acetic acid (150 ml). The flask was evacuated and flushed with H2. The reaction was stirred for 12 hrs at 50°C then filtered through Celite. The filtrate was poured into ice water and then extracted with ethyl acetate. The organic extracts were washed with water, sat. NaHC03, brine, dried with anhydrous Na2S04, filtered, concentrated in vacuo. Yield: 25.0 g (99%); pale yellow oil.
The material from above (15.6 g, 10 mmol) was dissolved in methanol (200 ml) at 0°C, then thionyl chloride (11.8 g, 10 mmol) was added dropwise to maintain the temperature below 5°C. The reaction was warmed to RT and stirred for 3 hrs. The reaction mixture was poured into ice water and extracted with ethyl acetate. The organic extracts were washed with water, sat. NaHC03, brine, dried with anhydrous Na2S04, filtered, concentrated in vacuo. Yield: 17.0 g (100%); oil.
NBS (37.5 g, 21 mmol) was added in small portions into a solution of the material from above (40.0 g, 24 mmol) in chloroform (300 ml) and acetic acid (300 ml) below 5°C and then the reaction was stirred at RT. The completion of the reaction was indicated by the disappearance of starting material by HPLC. The reaction mixture was concentrated and ice-water was added. The mixture was extracted with dichloromethane and the organic extracts were washed with water, sat. NaHC03, brine, dried with anhydrous Na2S04, filtered and concentrated in vacuo . Yield: 52.0 g (87%); pale brown oil.
2,5-Bis(tributylstannyl)thiophene (20.0 g, 30 mmol), the bromide from above (12.5 g, 50 mmol) and toluene (200 ml) were charged into a flask, Pd(PPh3)4 was added and the reaction was stirred for 12 hrs at RT. The reaction mixture was poured in to ice water and extracted with ethyl acetate, washed with water, sat. NaHC03, brine, dried with anhydrous Na2S04, filtered, concentrated in vacuo. The residue was purified by flash chromatography (Petroleum ether: Ethyl acetate = 10 : 1). Yield: 2.1 g; yellow oil. 1H MR (300 MHz, CDC13): δ 7.21 (d, J 5.9 Hz, 2H), 7.09 (s, 2H), 6.95 (d, J 5.9 Hz, 2H), 3.69 (s, 6H), 3.14 (t, J 7.8 Hz, 4H), 2.66 (t, J 7.8 Hz, 4H). MS (ESI, positive): 438.1 [M+NH4]+.
Figure imgf000052_0001
Methyl 3-(5-bromo-2-{5-[5-bromo-3-(3-methoxy-3-oxopropyl)thiophen-2-yl]thiophen-2- yl}thiophen-3-yl)propanoate (Intermediate C2)
BS (3.4 g, 19 mmol) was added in small portions into a solution of intermediate B2 (4.2 g, 10
mmol) in chloroform (50 ml) and acetic acid (50 ml) below 5°C and then the reaction was stirred at RT. The completion of the reaction was indicated by the disappearance of starting material by HPLC. The reaction mixture was concentrated and ice water was added. The mixture was extracted with dichloromethane. The organic extracts were washed with water, sat. NaHC03, brine, dried with anhydrous Na2S04, filtered, concentrated in vacuo. The residue was purified by flash chromatography (Petroleum ether: Ethyl acetate = 7 : 1). Yield: 5.3 g (92%); yellow solid. 1H MR (CDC13, 300 MHz) δ: 7.03 (s, 2H), 6.92 (s, 2H), 3.69 (s, 6H), 3.05 (t, J 7.8 Hz, 4H), 2.61 (t, J 7.8 Hz, 4H). MS (ESI, positive): 595.9 [M+ H4]+.
Figure imgf000052_0002
Methyl 2-{2-[3-(2-methoxy-2-oxoethyl)thiophen-2-yl]thiophen-3-yl}acetate (Intermediate 1)2)
Argon was flushed through a mixture of intermediate B (1.83 g, 7.78 mmol),
bispinacolatodiboron (0.989 g, 3.89 mmol) and KF (2.27 g, 39.0 mmol) in toluene (10 ml) and MeOH (10 ml). l, -Bis(diphenylphosphino)ferrocene-palladium(II)dichloride (171 mg, 0.234 mmol) was added and the mixture was heated in a sealed tube at 80 °C for 30 min. Toluene and water were added. The organic phase was concentrated and residue purified by flash chromatography using 15% EtOAc in iso-hexane as eluent. Yield: 591 mg (49%); colourless oil. 1H MR (400 MHz, CDC13): δ 3.54 (s, 4H), 3.66 (s, 6H), 7.07 (d, J 5.3 Hz, 2H), 7.36 (d, J 5.3 Hz, 2H).
Figure imgf000053_0001
Methyl 2-{5-bromo-2-[5-bromo-3-(2-methoxy-2-oxoethyl)thiophen-2-yl]thiophen-3- yl} acetate (Intermediate E2)
BS (170 mg, 0.960 mmol) was added portion-wise to a solution of intermediate D2 (149 mg, 0.480 mmol) in AcOH (5 ml) and CHC13 (5 ml) at ambient temperature. The mixture was stirred overnight, solvents evaporated and residue purified by flash chromatography using 10% EtOAc in iso-hexanes as eluent. Yield: 161 mg (72%); colourless oil.1H NMR (400 MHz, CDCI3): δ 3.49 (s, 4H), 3.68 (s, 6H), 7.04 (s, 2H).
Figure imgf000053_0002
Methyl 2- { 5 -bromo-2- [3 -(2-methoxy-2-oxoethyl)thiophen-2-yl]thiophen-3 -yl } acetate (Intermediate F2)
Br2 (0.022 ml, 0.425 mmol) in DCM (1 ml) was added to a stirred solution of intermediate D2 (132 mg, 0.425 mmol) in DCM (3 ml) at 0°C. The reaction was stirred at this temperature for 15 min. DCM (-10 ml) was added and the mixture was extracted with saturated Na2S203 and diluted IQCO3. The organic phase was dried over MgS04 and removed in vacuo. The crude material was purified by flash chromatography (3% iso-propanol in toluene and withl5% EtOAc in isohexane) Yield: 66 mg (40%); colourless oil. 1H NMR (400 MHz, CDC13): δ 3.50 (s, 2H), 3.57 (s, 2H), 3.69 (s, 6H), 7.07-7.10 (m, 2H), 7.40 (d, 1H).
Me02C C02Me MeO,C CO.Me Methyl 2-(5-iodo-2-(5-[5-iodo-3-(2-methoxy-2- oxoethyl)thiophen-2-yl]thiophen-2-yl}thiophen-3-yl)acetate (Intermediate G2) and methyl 2- (5-iodo-2-{5-[3-(2-methoxy-2-oxoethyl)thiophen-2-yl]thiophen-2-yl}thiophen-3-yl)acetate (Intermediate H2).
NIS (2.31 g, 10.3 mmol) was added portion-wise to a solution of intermediate C (4.03 g, 10.3 mmol) in AcOH (15 ml) and CHCI3 (15 ml) at 0 °C. The mixture was allowed to slowly reach ambient temperature overnight. DCM and sat. Na2C03 was added. Organic phase was evaporated and residue purified by flash chromatography using 2% EtOAc in toluene as eluent. First eluted methyl 2-(5-iodo-2-{5-[5-iodo-3-(2-methoxy-2-oxoethyl)thiophen-2- yl]thiophen-2-yl}thiophen-3-yl)acetate (G2). Yield: 1.010 g (15%); yellow oil. 1H MR (400 MHz, CDCI3): δ 3.75 (s, 4H), 3.76 (s, 6H), 7.11 (s, 2H), 7.23 (s, 2H).
Second eluted methyl 2-(5-iodo-2-{5-[3-(2-methoxy-2-oxoethyl)thiophen-2-yl]thiophen-2- yl}thiophen-3-yl)acetate (H2). Yield: 2.368 g (44%); yellow oil. 1H NMR (400 MHz, CDC13): δ 3.75 (s, 3H), 3.76 (s, 3H), 3.76 (s, 2H), 3.80 (s, 2H), 7.08 (d, J 5.3 Hz, 1H), 7.13 (d, J4.0 Hz, 1H), 7.16 (d, J4.0 Hz, 1H), 7.23 (s, 1H), 7.29 (d, J 5.3 Hz, 1H)
SYNTHESIS OF ACTIVE COMPOUNDS (LCOs)
Figure imgf000054_0001
Example 1. 2-(2-(5-[3-(Carboxymethvn-5-(5-[4-(carboxymethvn-5-(5-[3- (carboxymethyl)thiophen-2-yl]thiophen-2-yl}thiophen-2-yl]thiophen-2-yl}thiophen-2- yllthiophen-2-yl|thiophen-3-yl)acetic acid (9708 003)
Nitrogen was bubbled through a mixture of intermediate E (2.00 g, 4.24 mmol) and 2,5- thiophenediboronic acid (346 mg, 2.02 mmol) and K2CO3 (1.67 g, 12.1 mmol) in toluene (50 ml) and MeOH (50 ml). After 10 min PEPPSI-iPr™ (13.7 mg, 0.02 mmol) was added and the mixture heated at 50 °C for 1 h. Water and toluene were added. The aqueous layer was extracted with toluene and DCM. Organic layer was evaporated and residue purified by flash chromatography using 0-5% MeOH in DCM as eluent and a second time using 10-20%) EtOAc in toluene as eluent. Yield: 1.22 g (70%>); orange solid. The ester from above (989 mg, 1.14 mmol) was dissolved in dioxane (60 ml) and 2 M NaOH (60 ml) was added. The mixture was stirred at rt for 1 h and at 60 °C for 2 h. The mixture was cooled and cone. HCl was added dropwise (pH 2). The red precipitate was collected by centrifugation and washed several times with water. The material was dried at high vacuum overnight. Yield: 870 mg (94%); dark orange metallic crystals. 1H (400 MHz, DMSO-<¾): δ 3.74 (s, 4H), 3.77 (s, 4H), 7.11 (d, J 5.4 Hz, 2H), 7.27 (d, J4.4 Hz, 2H), 7.31 (d, J4.4 Hz, 2H), 7.33 (s, 2H), 7.35 (s, 2H), 7.55 (d, J 5.4 Hz, 2H). LC-MS: m/z = 807 (M-l). HPLC: RT = 2.48 min, 95% (254 nm, 10-40% MeCN in 10 mM buffer, 3 min, XBridge) and RT = 2.50 min, 95% (400 nm, 10-40% MeCN in 10 mM buffer, 3 min, XBridge).
Figure imgf000055_0001
Example 2. 2-(2-{5-[3-(Carboxymethyl)thiophen-2-yl]thiophen-2-yl}thiophen-3-yl)acetic acid (9708_004)
Intermediate C (71 mg, 0.181 mmol) was dissolved in dioxane (2 ml) and 1 M NaOH (0.40 ml) and the mixture was stirred at rt overnight. The mixture was acidified by 1 M HCl and aqueous layer extracted with EtOAc. The organic layer was dried (MgS04) and filtered.
Yield: 57 mg (87%); yellow soid. 1H NMR (400 HMz, DMSO-i¾): δ 3.73 (s, 4H), 7.09 (d, J 5.2 Hz, 2H), 7.23 (s, 2H), 7.53 (d, J 5.2 Hz, 2H), 12.52 (s, 2H).
Figure imgf000055_0002
Example 3. 5-[4-(Carboxymethyl)-5-{5-[3-(carboxymethyl)thiophen-2-yl]thiophen-2- yl}thiophen-2-yl]thiophene-2-carboxylic acid (9708_005)
Nitrogen was bubbled through a mixture of intermediate E (172 mg, 0.37 mmol), 5- carboxythiophene-2-boronic acid (94 mg, 0.73 mmol) and K2CO3 (202 mg, 1.46 mmol) in toluene (2.5 ml) and MeOH (2.5 ml). PEPPSI-iPr™ (4.96 mg, 0.0073 mmol) was added and the mixture heated at 50 °C for 1 h. Toluene and 1 M HCl was added. The organic layer was separated and evaporated. The crude material was purified by flash chromatography using 2% AcOH in DCM as eluent. Yield: 156 mg. 1H NMR (500 MHz, CDC13): δ 3.65 (s, 3H), 3.68 (s, 3H), 3.70 (s, 2H), 3.71 (s, 2H), 6.97 (d, J 5.5 Hz, IH), 7.05 (d, J4.0 Hz, IH), 7.09 (d, J4.0 Hz, IH), 7.15 (s, IH), 7.17 (d, J 5.5 Hz, IH), 7.67 (s, IH).
The material from above (156 mg, 0.301 mmol) was dissolved in dioxane (10 ml) and 1 M NaOH (10 ml) was added. The mixture was stirred at rt for 2 h. Solid material precipitated upon addition of 1 M HC1 which was separated by centrifugation, washed with water and dried at high vacuum to give a quantitative yield of the title compound. 1H NMR (400 MHz, DMSO-i¾): δ 3.73 (s, 2H), 3.75 (s, 2H), 7.09 (d, IH), 7.25 (d, IH), 7.27-7.29 (m, 2H), 7.35 (d, IH), 7.54 (d, IH), 7.56 (d, IH). LC-MS: 489 (M-l).
Figure imgf000056_0001
Example 4. 2-(2-(5-[3-(Carboxymethvnthiophen-2-yllthiophen-2-yl|-5-(thiophen-2- vnthiophen-3-yl)acetic acid (9708 006)
Nitrogen was bubbled through a mixture of intermediate E (58 mg, 0.12 mmol), thiophene-2- boronic acid (32 mg, 0.25 mmol) and K2CO3 (68 mg, 0.49 mmol) in toluene (2 ml) and MeOH (2 ml). PEPPSI-iPr™ (1.7 mg, 0.0025 mmol) was added and the mixture heated at 50 °C for 1 h under N2. Solvents were evaporated and residue purified by flash chromatography using DCM. Yield: 58.4 mg.
The material from above (148 mg) was dissolved in dioxane (15 ml) and 1 M NaOH (15 ml) and the mixture was stirred at rt for 2h. 1 M HC1 (25 ml) was added and the precipitated material was separated by centrifugation, washed with water and dried under high vacuum. Yield: 134 mg; yellow solid. 1 H NMR (400 MHz, DMSO-i¾): δ 3.77 (s, 2H), 3.78 (s, 2H), 7.10 (d, IH), 7.27 (d, IH), 7.35 (d, IH), 7.40 (d, IH), 7.46 (s, IH), 7.54 (d, IH). LC-MS: m/z = 445 (M-l).
Figure imgf000056_0002
Example 5. 5-(5-{5-[5-(5-Carboxythiophen-2-yl)-3-(2-hydroxyethyl)thiophen-2-yl]thiophen- 2-yl|-4-(2-hvdroxyethvnthiophen-2-vnthiophene-2-carboxylic acid (9708_008)
Nitrogen was bubbled through a mixture of intermediate H (250 mg, 0.506 mmol), 5- carboxythiopheneboronic acid (217 mg, 1.26 mmol) and K2CO3 (349 mg, 2.52 mmol) in toluene (10 ml) and MeOH (10 ml). PEPPSI-iPr1M (17 mg, 0.025 mmol) was added and the mixture stirred at 60 °C for lh. MeOH (70 ml) was added. The suspension was acidified with 1 M HCl and a red solid precipitated, which was isolated by centrifugation. The solid material was washed with MeOH and water several times. Yield: 277 mg (93%); red solid. LC-MS: m/z = 587 (M-l). HPLC: RT = 1.47 min, 99% (254 nm, 10-90% MeCN in 10 mM buffer, 3 min, XBridge) and RT = 2.13 min, 97% (400 nm, 10-40% MeCN in 10 mM buffer, 3 min, XB rid e).
Figure imgf000057_0001
Example 6. 5-[3-(Carboxymethyl)-5-{5-[4-(carboxymethyl)-5-(5-carboxythiophen-2- v0thiophen-2-yllthiophen-2-yl |thiophen-2-yllthiophene-2-carboxylic acid (9708 009)
A mixture of intermediate B (705 mg, 3.00 mmol), 2-carboxythiophene-5-boronic acid (619 mg, 3.60 mmol) and potassium carbonate (1.24 g, 9.00 mmol) in toluene (10 ml) and methanol (10 ml) was degassed by bubbling nitrogen through the mixture. PEPPSI-iPr™ (102 mg, 0.150 mmol) was added and the mixture heated at 60 °C for 45 min under nitrogen. Water and EtOAc were added. The aqueous layer was acidified using 2 M HCl (pH 2). The organic layer was separated and concentrated. The residue was purified by flash chromatography using 5% MeOH + 0.1% AcOH in DCM as eluent. Yield: 715 mg (85%); beige solid. HPLC: RT = 2.08 min, 92%, 254 nm (1040X3). 1H NMR (400 MHz, CDC13): δ 3.75 (s, 3H), 3.83 (s, 2H), 7.10 (d, J 5.2 Hz, 1H), 7.24 (d, J 4.0 Hz, 1H), 7.35 (d, J 5.2 Hz, 1H), 7.86 (d, J 3.6 Hz, 1H).
Methyl iodide (232 μΐ, 3.74 mmol) was added to a mixture of the carboxylic acid from above (705 mg, 2.50 mmol) and sodium carbonate (529 mg, 4.99 mmol) in DMF (10 ml). The mixture was stirred at ambient temperature overnight. Water and diethyl ether were added. The aqueous layer was extracted with diethyl ether, The combined organic layers were concentrated and residue purified by flash chromatography using 5-20% EtOAc in iso- hexane. Yield: 364 mg (49%); white solid. HPLC: RT = 2.73 min, 99%, 254 nm (1090X3) 1H NMR (400 MHz, CDC13): δ 3.74 (s, 3H), 3.81 (s, 2H), 3.92 (s, 3H), 7.09 (d, J 5.6 Hz, 1H), 7.19 (d, J 4.0 Hz, 1H), 7.32 (d, J 5.2 Hz, 1H), 7.77 (d, J4.0 Hz, 1H).
NBS (216 mg, 1.21 mmol) was added portionwise to a solution of the ester from above (360 mg, 1.21 mmol) in CHC13 (5 ml) and AcOH (5 ml). The mixture was stirred at ambient overnight. DCM and water was added. The organic layer was washed with sat. NaHC03 and concentrated. The residue was purified by flash chromatography using 15-20% EtOAc in iso- hexane as eluent. Yield: 349 mg (77%); white solid. HPLC: RT = 2.21 min, 98%, (254 nm, 4090A3) and RT = 3.04 min, 99%, 254 nm (1090A3). 1H NMR (400 MHz, CDC13): δ 3.74 (s, 2H), 3.76 (s, 3H), 3.92 (s, 3H), 7.07 (s, 1H), 7.14 (d, J 3.6 Hz, 1H), 7.76 (d, J 4.0 Hz, 1H). Argon was bubbled through a mixture of the bromide from above (342 mg, 0.911 mmol), 2,5- thiophenediboronic acid (78 mg, 0.455 mmol) and K2C03 (314 mg, 2.27 mmol) in toluene (8 ml) and MeOH (8 ml). PEPPSI-iPr™ (15 mg, 0.0228 mmol) was added and the mixture heated at 60 °C for 45 min under argon. Orange material precipitated during the reaction. Water and CHC13 were added. The organic layer was separated and silica was added to the mixture and the solvents were evaporated. The dry silica was applied on a flash column and eluted with 0-3% MeOH in DCM. Yield: 230 mg (75%); orange solid.
The ester from above (180 mg, 0.268 mmol) was suspended in dioxane (5 ml) and 1 M NaOH (10 ml). The mixture was stirred at rt overnight. 1 M NaOH (5 ml) was added and the mixture heated at 80 °C for 4 h. The solution was transferred to Falcon tubes, and 1 M HC1 (20 ml) was added. The precipitate was isolated by centrifugation, and washed three times with water. The solid was dried under high vacuum overnight. Yield: 143 mg (87%); red solid. 1H NMR (400 MHz. DMSO-i¾): δ 3.79 (s, 4H), 7.31 (d, J 3.8 Hz, 2H), 7.38 (s, 2H), 7.39 (s, 2H), 7.74 (d, J3.8 Hz, 2H). HPLC: RT = 2.36 min, 91% (254 nm, 0520X3). LC-MS: m/z = 615 (M-l).
Figure imgf000058_0001
Example 7. 5-(5-{5-[5-(5-Carboxythiophen-2-yl)-3-(2-acetamidoethyl)thiophen-2- yl]thiophen-2-yl}-4-(2-acetamidoethyl)thiophen-2-yl)thiophene-2-carboxylic acid (9708 010) Intermediate M (165 mg, 0.185 mmol) was dissolved in DCM (2 ml) and TFA (2 ml) was added. The mixture was stirred at rt for 3 h. Solvents were evaporated. The crude mixture was suspended in DCM (10 ml). Et3N (201 μΐ, 1.45 mmol) and acetyl chloride (52 μΐ, 0.726 mmol) was added. The mixture was stirred at rt for 2 h, solvent evaporated and residuel purified by flash chromatography using 0-5% MeOH in DCM as eluent. Yield: 73 mg (68%); yellow solid. 1H NMR (400 MHz, CDC13): δ 1.96 (s, 6H), 2.98 (m, 4H), 3.47 (m, 4H), 6.68 (br s, 2H), 6.95 (s, 2H), 7.02 (s, 2H). The dibromide (73 mg, 0.126 mmol) from above was dissolved in toluene (2 ml) and MeOH (2 ml). 5-Carboxythiophene-2-bornic acid (54.4 mg, 0.317 mmol) and K2C03 (110 mg, 0.792 mmol) were added. The suspension was degassed by bubbling argon through the mixture. PEPPSI-iPr™ (ca. 2 mg) was added and the mixture heated at 60 °C for 1 h. MeOH (20 ml) and 1 M HC1 (4 ml) was added. Solid material was isolated by centrifugation, washed with water and dried. Yield: 78 mg (92%); orange solid. HPLC: RT = 2.07 min, 96% (254 nm, 10- 40% MeCN in 10 mM buffer, 3 min, XBridge) and RT = 2.09 min, 97% (400 nm, 10-40% MeCN in 10 mM buffer, 3 min, XBridge)
Figure imgf000059_0001
Example 8. 2-(2-(5-[3-(2-Aminoethvnthiophen-2-yllthiophen-2-yl|thiophen-3-yl)ethan-l- amine (9708 011)
Intermediate L (208 mg, 0.283 mmol) was dissolved DCM (2 ml) and TFA (2 ml) was added.
The mixture was stirred at rt for lh. Solvents evaporated and residue dissolved in water. The solution was added 1 M NaOH (2 ml) and the aqueous layer extracted with CHC13. The combined organic layers were dried (MgS04) and 1.2 M HC1 in EtOH (2 ml) was added.
Solvents were evaporated to give the HCl-salt of the title compound. Yield: 105 mg (99%); yellow-orange solid. HPLC: RT = 2.68 min, 95% (254 nm, 10-40% MeCN in 0.1% TFA, 3 min, ACE) and RT = 2.68 min, 94% (350 nm, 10-40% MeCN in 0.1% TFA, 3 min, ACE). 1H NMR (400 MHz, OMSO-d6): δ 3.06 (br s, 12H), 7.14 (d, J 5.2 Hz, 2H), 7.27 (s, 2H), 7.58 (d,
J 5.2 Hz, 2H). LC-MS: m/z = 335 (M+l).
Figure imgf000059_0002
Example 9. 4-[4-(Carboxymethyl)-5-{5-[3-(carboxymethyl)-5-(4-carboxyphenyl)thiophen-2- yllthiophen-2-yl|thiophen-2-yllbenzoic acid (9708 012)
Argon was bubbled through a mixture of intermediate D (100 mg, 0.188 mmol), 4- methoxycarbonylphenylboronic acid (82 mg, 0.454 mmol) and K2C03 (100 mg, 0.728 mmol) in toluene (2 ml) and MeOH (2 ml). PEPPSI-iPr™ (ca 2 mg) was added and the tube sealed and heated in a microwave reactor at 100 °C for 15 min. CHC13 was added and the mixture filtered and concentrated. The residue was purified by flash chromatography using 0-1% MeOH in DCM. Yield: 96.1 mg (80%); orange solid. 1H NMR (400 MHz, OMSO-d6): δ 3.69 (s, 6H), 3.88 (s, 6H), 3.91 (s, 4H), 7.36 (s, 2H), 7.69 (s, 2H), 7.82 (d, J 8.4 Hz, 4H), 8.01 (d, J 8.4 Hz, 4H). HPLC: RT = 2.77 min, 92% (350 nm, 60-90% MeCN in 10 mM buffer, 3 min, XB ridge).
The ester from above (94 mg, 0.142 mmol) was dissolved in dioxane (2 ml) and 2 M NaOH (2 ml) was added. The mixture was heated at 140 °C for 10 min in a microwave reactor. Aqueous phase was acidified using 6 M HCl, precipitate isolated by centrifugation and washed twice with water. The material was dried under high vacuum for 2d. Yield: 44 mg (51%); yellow-orange solid. 1H NMR (DMSO-i¾): δ 3.81 (s, 4H), 7.37 (s, 2H), 7.67 (s, 2H), 7.80 (d, J 8 Hz, 4H), 8.00 (d, J 8 Hz, 4H). HPLC: HPLC: RT = 1.88 min, 93% (254 nm, 5- 20% MeCN in 10 mM buffer, 3 min, XBridge) and RT = 1.93 min, 94% (400 nm, 5-20% MeCN in 10 mM buffer, 3 min, XBridge). LC-MS: m/z = 603 (M-l)
Figure imgf000060_0001
Example 10. 3-[4-(Carboxymethyl)-5-{5-[3-(carboxymethyl)-5-(3-carboxyphenyl)thiophen- 2-yllthiophen-2-yl|thiophen-2-yllbenzoic acid (9708 013)
Argon was bubbled through a mixture of intermediate D (168 mg, 0.305 mmol), 3- carboxybenzeneboronic acid (127 mg, 0.763 mmol) and K2CO3 in toluene (2.5 ml) and
MeOH (2.5 ml). PEPPSI-iPr™ (ca 2 mg) was added to the degassed mixture and the mixture heated at 100 °C for 10 min in a microwave reactor. Solvents were evaporated and the crude material dissolved in dioxane (2.5 ml) and 2 M NaOH (2.5 ml). The mixture was stirred at rt overnight. Organic layer was removed and aqueous layer acidified by addition of 6 M HCl. Solid material was isolated by centrifugation and dissolved in 1 M Na2C03 and purified by preparative HPLC (Xterra 19 x 50 mm, 5-20% MeCN in 50 mM NH4HCO3 buffer). Pure fractions were collected and amount of solvents reduced. The wanted product was precipitated by addition of 1 M HCl, isolated by centrifugation and washed with water. The material was dried at high vacuum for 3 d. Yield: 69 mg (38%, two steps); yellow solid. 1H NMR (400 MHz, MeOD-i¾): δ 3.84 (s, 4H), 7.31 (s, 2H), 7.47 (s, 2H), 7.53 (d, J 7.8 Hz, 2H)m 7.89 (d, J 8.3 Hz, 2H), 7.94-7.98 (m, 2H), 8.26-8.29 (m, 2H). LC-MS: m/z =622 (M + H4 +).
Figure imgf000061_0001
Example 11. 2-[4-(Carboxymethyl)-5-{5-[3-(carboxymethyl)-5-(2-carboxyphenyl)thiophen- 2-yllthiophen-2-yl|thiophen-2-yllbenzoic acid (9708 014)
Argon was bubbled through a mixture of intermediate D (152 mg, 0.276 mmol), 2- methoxycarboxyphenylboronic acid (138 mg, 0.690 mmol) and K2CO3 (190 mg, 1.38 mmol) in toluene (4 ml) and MeOH (4 ml). PEPPSI-iPr™ (15 mg) was added and the mixture was heated in a microwave reactor at 70 °C for 15 min. Solvent evaporated and residue purified by flash chromatography using 10-15% EtOAc in toluene as eluent. Yield: 124 mg (68%);
orange-yellow solid. 1H MR (400 MHz, CDC13): δ 3.75-3.81 (m, 16H), 6.99 (s, 2H)), 7.19 (s, 2H), 7.40-7.43 (m, 2H), 7.50-7.52 (m, 4H), 7.73 (d, J 7.3 Hz, 2H). The ester (124 mg, 0.187 mmol) was dissolved in dioxane (5 ml) and 2 M NaOH (5 ml) was added. The mixture was stirred at rt for 3 h and at 80 °C for 1 h. EtOAc was added and organic layer removed.
The aqueous layer was acidified using 6 M HCl. Solid material was isolated by centrifugation, washed with 0.1 M HCl (three times) and dried at high vacuum for 2 d. Yield: 105 mg (93%); yellow solid. 1H MR (400 MHz, methanol-^): δ 3.79 (s, 4H), 7.10 (s, 2H), 7.27 (s, 2H), 7.44-7.47 (m, 2H), 7.50-7.59 (m, 4H), 7.72 (d , J 7.5 Hz, 2H). HPLC: Rt = 2.67 min, 97% at 254 nm (5-20% MeCN in buffer, XBridge) and Rt = 2.59 min. 97% at 400 nm (5-20% MeCN in buffer, XBridge). LC-MS: m/z = 622 (M + H4 +)
Figure imgf000061_0002
Example 12. 5-[4-(Carboxymethyl)-5-{5-[3-(carboxymethyl)-5-(thiophen-2-yl)thiophen-2- yllthiophen-2-yl |thiophen-2-yllthiophene-2-carboxylic acid (9708 015)
Argon was bubbled through a mixture of intermediate N (162 mg, 0.26 mmol), 2- thiopheneboronic acid (51 mg, 0.397 mmol) and K2CO3 (91 mg, 0.660 mmol) in toluene (4 ml) and MeOH (4 ml). PEPPSI-iPr™ (5 mg) was added and the mixture stirred at 60 +C for 45 min. Silica was added and solvents evaporated. The dry silica was applied on a flash column and product eluted using 10% EtOAc in toluene. Yield: 154 mg (95%); orange solid. The material from above was dissolved in dioxane (5 ml) and 2 M NaOH (5 ml) was added. The mixture was heated at 100 °C for 45 min. EtOAc was added and organic layer removed. The aqueous layer was acidified by addition of 6 M HCl and precipitated material isolated by centrifugation. The material was dissolved in 1 M Na2C03 and purified by preparative hplc (10-40% MeCN in 50 mM buffer, big XBridge). Pure fractions were combined and amount of solvents was reduced. The product was precipitated by addition of 6 M HCl and isolated by centrifugation. The material was washed with water and dried at high vacuum for 2 d. Yield: 72 mg (50%); orange solid. 1H NMR (400 MHz, OMSO-d6): δ 3.76 (s, 2H), 3.78 (s, 2H), 7.12 (dd, J 5.1 Hz, 3.6 Hz), 7.29 (s, 1H), 7.32 (d, J 3.8 Hz, 1H), 7.34 (d, J 3.8 Hz, 1H), 7.36 (dd, J 3.6 Hz, 1.1 Hz), 7.40 (d, J3.8 Hz, 1H), 7.47 (s, 1H), 7.57 (dd, J 5.1 Hz, 1.1 Hz, 1H), 7.68 (d, J 4.0 Hz, 1H), 12.43-12.99 (m, 3H). HPLC: Rt = 2.18 min, 98% at 254 nm (10-40% MeCN in buffer, XBridge). Rt = 2.16 min. 98% at 400 nm (10-40% MeCN in buffer, XBrid e). LC-MS: m/z = 590 (M + NH4 +)
Figure imgf000062_0001
Example 13. 5-[4-(Carboxymethyl)-5-{5-[3-(carboxymethyl)-5-(pyridin-3-yl)thiophen-2- yl]thiophen-2-yl }thiophen-2-yl]thiophene-2-carboxylic acid (9708 016)
Intermediate N (700 mg, 1.14 mmol) was dissolved in dioxane (5 ml) and 2 M NaOH (5 ml) was added. The mixture was heated at 100 °C for 30 min. EtOAc was added and organic layer removed. Aqueous phase was acidified using 6 M HCl and precipitated material isolated by centrifugation and washed with water, the dried at high vacuum for 2 d.
The crude material from above (170 mg, 0.298 mmol) was dissolved in MeOH (6 ml) and toluene (4 ml). Pyridine-3-boronic acid (74 mg, 0.597 mmol) and K2C03 (206 mg, 1.49 mmol) were added. The mixture was degassed by bubbling argon through the mixture.
PEPPSI-iPr™ (22 mg, 0.0323 mmol) was added, and the mixture heated in a microwave reactor at 100 °C for 30 min. Solvents were evaporated and residue dissolved in 1 M Na2C03 and filtered. The product was isolated by preparative hplc (10-35% MeCN in 50 mM buffer, big XBridge). Pure fractions were combined and amount of solvents reduced and solid material precipitated by addition of 6 M HCl. Solid material was isolated by centrifugation, washed with water and dried under high vacuum for 2 d. Yield: 65 mg (36%); red shiny solid. 1H MR (400 MHz, OMSO-d6): δ 3.80 (s, 2H), 3.81 (s, 2H), 7.37 (s, 2H), 7.42 (d, J3.8 Hz, 1H), 7.49 (s, 1H), 7.62 (dd, J 7.8, 5.0 Hz, 1H), 7.69 (d, J 3.8 Hz, 1H), 7.71 (s, 1H), 8.24 (d, J 8.0 Hz, 1H), 8.60 (dd, J4.9 Hz, 1.4 Hz, 1H), 9.00 (J2.3 Hz, 1H). HPLC: Rt = 1.67 min, 98% at 254 nm (10-40% MeCN in buffer, XBridge) Rt = 1.68 min. 97% at 400 nm (10-40% MeCN in buffer, XBridge). LC-MS: m/z = 568 (M + 1).
Figure imgf000063_0001
Example 14. 5-[4-(Carboxymethyl)-5-{5-[3-(carboxymethyl)-5-(3-carboxyphenyl)thiophen- 2-yllthiophen-2-yl |thiophen-2-yllthiophene-2-carboxylic acid (9708 017)
Intermediate N (700 mg, 1.14 mmol) was dissolved in dioxane (5 ml) and 2 M NaOH (5 ml) was added. The mixture was heated at 100 °C for 30 min. EtOAc was added and organic layer removed. Aqueous phase was acidified using 6 M HCl and precipitated material isolated by centrifugation and washed with water, then dried at high vacuum for 2 d.
The crude material from above (166 mg, 0.291 mmol) was dissolved in MeOH (6 ml) and toluene (4 ml). 3-Carboxybenzeneboronic acid (97 mg, 0.583 mmol) and K2CO3(201 mg, 1.46 mmol) was added and the mixture was degassed by bubbling argon through the mixture. PEPPSI-iPr™ (15 mg, 0.022 mmol) was added and mixture heated at 100 °C for 30 min in a microwave reactor. Solvents were evaporated and residue dissolved in 1 M Na2C03, filtered and purified by preparative hplc (5-25% MeCN in 50 mM buffer, XBridge). Pure fractions were combined and amount of solvents reduced. The wanted product precipitated by addition of 6 M HCl, separated by centrifugation, washed with water and dried at high vacuum for 2 d. Yield: 54.6 mg (31%); deep red solid with metallic shine. 1H NMR (400 MHz, OMSO-d6): δ 3.80 (s, 2H), 3.80 (s, 2H), 7.36 (s, 2H), 7.41 (d, J3.8 Hz, 1H), 7.48 (s, 1H), 7.59 (t, J 7.8 z, 1H), 7.63 (s, 1H), 7.69 (s, J3.8 Hz, 1H), 7.86-7.99 (m, 2H), 8.14-8.18 (m, 1H). HPLC: Rt = 2.36 min, 98% at 254 nm (5-20% MeCN in buffer, XBridge) and Rt = 2.36 min. 98% at 400 nm (5-20% MeCN in buffer, XBridge). LC-MS: m/z = 628 (M + NH4
Figure imgf000064_0001
Example 15. 2-(2-{5-[3-(CarboxymethylV5-(pyridin-3-ynthiophen-2-yllthiophen-2-yl}-5- (pyridin-3-yl)thiophen-3-yl)acetic acid (9708 018)
Argon was bubbled through a mixture of intermediate D (156 mg, 0.283 mmol), pyridine-3- boronic acid (87 mg, 0.708 mmol) and potassium carbonate (196 mg, 1.417 mmol) in toluene (2.5 ml) and MeOH (2.5 ml). PEPPSI-iPr™ (10 mg, 0.283 mmol) was added and the mixture heated at 100 °C for 10 min in a microwave reactor. TLC indicated not full conversion. Silica was added and solvents evaporated. The material was purified twice by flash chromatography using 5% and 3% MeOH in DCM as eluents. Yield: 39.3 mg (25%); yellow solid. 1H MR (400 MHz, CDC13): δ 3.77 (s, 6H), 3.84 (s, 4H), 7.24 (s, 2H), 7.33-7.34 (m, 2H), 7.34 (s, 2H), 7.87 (dt, 78.0 Hz, 1.9 Hz, 2H), 8.54 (dd, 74.8, 1.2 Hz, 1H), 8.88 (d, 7 1.8 Hz, 1H).
The material from above (39 mg, 0.071 mmol) was dissolved in dioxane (3 ml) and 2 M NaOH (3 ml) was added. The mixture was stirred at rt for 4 h and heated at 60 °C for lh. EtOAc was added and organic layer removed. The aqueous layer was adjusted to pH 3 by addition of 1 M HCl. The solid material was isolated by centrifugation, washed with water and dried at high vacuum for 5 d. Yield: 38 mg; orange/yellow solid. HPLC: Rt = 2.38 min, 95% at 254 nm (10-40% MeCN in buffer, XBridge) and Rt = 2.35 min. 95% at 400 nm (10- 40% MeCN in buffer, XBridge).MS: m/z = 519 (M + 1).
Figure imgf000064_0002
Example 16. 2-(2-{5-[3-(Carboxymethyl)-5-(4-methanesulfonylphenyl)thiophen-2- yl]thiophen-2-yl}-5-(4-methanesulfonylphenyl)thiophen-3-yl)acetic acid (9708 019)
Argon was bubbled through a mixture of intermediate D (165 mg, 0.300 mmol), 4- methylsulfonylphenylboronic acid (150 mg, 0.750 mmol) and K2CO3 (207 mg, 1.50 mmol) in toluene (5 ml) and MeOH (5 ml). PEPPSI-iPr™ (10 mg, 0.0147 mmol) was added and the mixture heated at 80 °C for 30 min in a microwave reactor. Silica gel was added and solvents evaporated. The dry silica was applied on a flash column and product eluted using
toluene/EtOAc 2: 1 and 1 : 1. Yield: 191 mg (91%); orange solid.
The material from above (188 mg, 0.268 mmol) was dissolved in dioxane (3 ml) and 2 M NaOH (3 ml) was added. The mixture was heated at 50 °C for 2 h before 6 M HC1 was added (0.5 ml). The precipitate was washed several times with water and MeOH and dried at high vacuum for 3 d. Yield: 100 mg (55%); orange solid. 1H NMR (400 MHz, CDC13): δ 3.26 (s, 6H), 3.82 (s, 4H), 7.24 (s, 2H), 7.40 (s, 2H), 7.74 (s, 2H), 7.91-8.00 (m, 8H). HPLC: Rt = 2.75 min, 91% at 254 nm (10-40% MeCN in buffer, XBridge) and Rt = 2.74 min. 92% at 400 nm (10-40% MeCN in buffer, XBridge). LC-MS: m/z = 690 (M + NH4 +)
Figure imgf000065_0001
Example 17. 2-(2-(5-r3-(Carboxymethvn-5-r4-(carboxymethvn-5-(5-r3- (carboxymethyl)thiophen-2-yl]thiophen-2-yl}thiophen-2-yl]thiophen-2-yl]thiophen-2- yl}tmophen-3-yl)acetic acid (9708_020)
Kosher's reagent (35 mg, 0.089 mmol) was added to a solution of intermediate C (350 mg, 0.892 mmol) and N-iodosuccinimide (180 mg, 0.803 mmol) in MeOH (20 ml). The mixture was stirred at rt for 1.5 h before DCM and water was added. The organic phase was washed with 5% Na2S203 and concentrated. The residue was purified by flash chromatography using 1-2% EtOAc in toluene and a second time using DCM/CHC13 2: 1 as eluent. Yield: 138 mg (30%); colourless oil. 1H NMR (400 MHz, CDC13): δ 3.73 (s, 3H), 3.73 (s, 3H), 3.74 (s, 2H), 3.78 (s, 2H), 7.06 (d, J 5 Hz, 1H), 7.10 (d, J4 Hz, 1H), 7.13 (d, J4 Hz, 1H), 7.21 (s, 1H), 7.26 (d, J 5 Hz, 1H).
The iodide from above (137 mg, 0.264 mmol) in toluene (5 ml) was added palladium acetate (3 mg, 0.0133 mmol), tetrabutylammonium bromide (43 mg, 0.132 mmol) and
diisopropylethylamine (46 μΐ, 0.264 mmol). The mixture was heated at 105 °C for 4 h. The material was applied on a flash column wich was eluted with 5-15% EtOAc in toluene. Yield: 48.6 mg (47%); orange oil. 1H NMR (400 MHz, CDC13): δ 3.74 (s, 6H), 3.75 (s, 6H), 3.78 (s, 4H), 3.80 (s, 4H), 7.06 (d, J 5 Hz, 2H), 7.13 (s, 2H), 7.16-7.19 (m 4H), 7.25-7.28 (m, 2H). The ester from above (48 mg, 0.061 mmol) was dissolved in dioxane (2 ml) and 2 M NaOH (2 ml) was added. The mixture was heated at 80 °C for lh. Water and 6 M HC1 was added. The precipitated was isolated by centrifugation, washed with water and dried at high vacuum for 2 d. Yield: 35.8 mg (81%); orange solid. 1H NMR (400 MHz, OMSO-d6): δ 3.74 (s, 4H), 3.77 (s, 4H), 7.10 (d, J 5.3 Hz, 2H), 7.27 (d, J4.0 Hz, 2H), 7.32 (d, J4.0 Hz, 2H), 7.33 (s, 2H), 7.54 (d, J 5.0 Hz, 2H). HPLC: Rt = 2.01 min, 85% at 254 nm (10-40% MeCN in buffer, XBridge) and Rt = 2.01 min. 93% at 400 nm (10-40% MeCN in buffer, XBridge). LC-MS: m/z = 744 (M + NH4 +)
Figure imgf000066_0001
Example 18. 5-{5-[4-(Carboxymethyl)-5-{5-[3-(carboxymethyl)-5-[5-(5-carboxythiophen-2- yl)thiophen-2-yl]thiophen-2-yl]thiophen-2-yl}thiophen-2-yl]thiophen-2-yl}thiophene-2- carboxylic acid (9708_021)
Argon was bubbled through a mixture of intermediate P (102 mg, 0.149 mmol), 2- carboxythiophene-5-boronic acid (64 mg, 0.371 mmol) and K2CO3 (103 mg, 0.75 mmol) in MeOH (8 ml) and toluene (6 ml). PEPPSI-iPr™ (5 mg, 0.0073 mg) was added and mixture heated at 100 °C for 30 min in microwave reactor. Solvents were evaporated, residue dissolved in 1 M Na2C03 and purified by preparative hplc (10-40% MeCN in 50 mM buffer, Xterra). Pure fractions were combined. Some solvents evaporated and 6 M HC1 added. The solid material was separated by centrifugation, washed with water and dried at high vacuum for 2 d. Yield: 25.2 mg (22%); red solid. 1H NMR (400 MHz, OMSO-d6): δ 3.78 (s, 4H), 7.33 (s, 2H), 7.38-7.43 (m, 6H), 7.49 (d, J 3.8 Hz, 2H), 7.67 (d, J3.8 Hz, 2H). HPLC: Rt = 2.06 min, 99% at 254 nm (10-40% MeCN in buffer, XBridge) and Rt = 2.04 min. 98% at 400 nm (10-40% MeCN in buffer, XBridge). LC-MS: m/z = 798 (M + NH4 +)
Figure imgf000066_0002
Example 19. 4-(5-r4-(Carboxymethvn-5-(5-r3-(carboxymethvn-5-r5-(4- carboxyphenyl)thiophen-2-yl]thiophen-2-yl]thiophen-2-yl}thiophen-2-yl]thiophen-2- yllbenzoic acid (9708_022)
Argon was bubbled through a mixture of intermediate P (125 mg, 0.168 mmol), 4- carboxyphenylboronic acid (69 mg, 0.419 mmol) and K2CO3 (196 mg, 1.18 mmol) in MeOH (8 ml) and toluene (6 ml), PEPPSI-iPr™ (5 mg, 0.0073 mg) was added and mixture heated at 100 °C for 30 min in microwave reactor. Solvents were evaporated, residue dissolved in 1 M Na2C03 and purified by preparative hplc (10-40% MeCN in 50 mM buffer, XBridge). Pure fractions were combined and some solvents evaporated. Solid material precipitated by addition of 6 M HC1. The material was isolated by centrifugation, washed with water and dried at high vacuum for 3 d. Yield: 59.2 mg (46%); red solid. 1H NMR (400 MHz, DMSO- d6): δ 3.79 (s, 4H), 7.34 (s, 2H), 7.39 (s, 2H), 7.45 (d, J 3.8 Hz, 2H), 7.70 (d, J 4.0 Hz, 2H), 7.82 (d, J 8.5 Hz, 4H), 7.98 (d, J 8.5 Hz, 4H. HPLC: Rt = 2.08 min, 96% at 254 nm (10-40% MeCN in buffer, XBridge) and Rt = 2.07 min. 98% at 400 nm (10-40% MeCN in buffer, XBridge). LC-MS: m/z = 786 (M + NH4 +).
Figure imgf000067_0001
Example 20. 3-(5-r4-(Carboxymethvn-5-(5-r3-(carboxymethvn-5-r5-(3- carboxyphenyl)thiophen-2-yl]thiophen-2-yl]thiophen-2-yl}thiophen-2-yl]thiophen-2- yllbenzoic acid (9708_023)
Argon was bubbled through a mixture of intermediate P (128 mg, 0.186 mmol),3- carboxybenzeneboronic acid (77 mg, 0.466 mmol), K2C03 (180 mg, 1.30 mmol) in MeOH (8 ml) and toluene (6 ml). PEPPSI-iPr™ (5 mg, 0.0073 mg) was added and mixture heated at 100 °C for 30 min in a microwave reactor. Solvents were evaporated, residue dissolved in 1 M Na2C03 and purified by preparative hplc (10-40% MeCN in 50 mM buffer, XBridge). Yield: 62 mg (43%); red solid. 1H NMR (400 MHz, DMSO-i¾): δ 3.79 (s, 4H), 7.33 (s, 2H), 7.38 (s, 2H), 7.42 (d, J 3.8 Hz, 2H), 7.58 (t, J7.9 Hz, 2H), 7.64 (d, J4.0 Hz, 2H), 7.85-7.91 (m, 2H), 7.94 - 8.00 (m 2H), 8.17 (t, J 1.6 Hz, 2H). HPLC: Rt = 2.35 min, 99% at 254 nm (10-40% MeCN in buffer, XBridge) and Rt = 2.36 min. 97% at 400 nm (10-40% MeCN in buffer, XBridge). LC-MS: m/z = 786 (M + NH4 +)
Figure imgf000068_0001
Example 21. 2-(2-(5-[3-(Carboxymethvn-5-phenylthiophen-2-yllthiophen-2-yl|-5- phenylthiophen-3-yl)acetic acid (9708 024)
Argon was bubbled through a mixture of intermediate Q (123 mg, 0.236 mmol),
benzeneboronic acid (72 mg, 0.589 mmol) and K2C03 (195 mg, 1.41 mmol) in MeOH (2 ml) and toluene (2 ml). PEPPSI-iPr™ (15 mg, 0.022 mmol) was added and the mixture was heated at 90 °C for 30 min in a microwave reactor. Solvents were evaporated and residue dissolved in 0.5 M Na2C03 / MeOH and purified by preparative hplc (20-50% MeCN in 50 mM buffer, XBridge). Pure fractions were combined and some solvents were evaporated. 6 M HCl was added and precipitated material collected by centrifugation, washed with water and dried at high vacuum Yield: 22.7 mg (19%); yellow solid. 1H MR (400 MHz, DMSO-i¾): δ 3.79 (s, 4H), 7.33 (s, 2H), 7.33-7.38 (m, 2H), 7.41-7.48 (m, 4H), 7.51 (s, 2H), 7.65-7.71 (m, 4H). HPLC: Rt = 2.64 min, 99% at 254 nm (20-50% MeCN in buffer, XBridge) and Rt = 2.64 min. 99% at 400 nm (20-50% MeCN in buffer, XBridge). LC-MS: m/z = 534 (M + NH4 +).
Figure imgf000068_0002
Example 22. 2-(2-{5-[3-(Carboxymethyl)-5-(3-methanesulfonylphenyl)thiophen-2- yl]thiophen-2-yl}-5-(3-methanesulfonylphenyl)thiophen-3-yl)acetic acid (9708 025)
Argon was bubbled through a mixture of intermediate Q (123 mg, 0.236 mmol), 3- (methylsulfonyl)phenylboronic acid (118 mg, 0.589 mmol) and K2C03 (196 mg, 1.41 mmol) in MeOH (2 ml) and toluene (2 ml). PEPPSI™ (15 mg, 0.0221 mmol) was added and the mixture heated at 90 °C for 30 min in a microwave reactor. Solvents were evaporated, residue dissolved in 0.5 M Na2C03/water/MeOH and purified by preparative hplc (15-45% MeCN in 50 mM buffer). Pure fractions were combined and solid material precipitated using 6 M HCl. The precipitate was isolated by centrifugation, washed with water and dried at high vacuum for 2 d. Yield: 68.5 mg (43%); red solid. 1H NMR (400 MHz, DMSO-i¾): δ 3.33 (s, 6H), 3.82 (s, 4H), 7.39 (s, 2H), 7.68-7.77 (m, 4H), 7.88 (d, J= 8.3 Hz, 2H), 7.99-8.04 (m, 2H), 8.16 (t, J = 1.76 H, 2H). HPLC: Rt = 2.83 min, 96% at 254 nm (10-40% MeCN in buffer, XBridge) and Rt = 2.83 min. 95% at 400 nm (10-40% MeCN in buffer, XBridge). LC-MS: m/z = 690 (M +
NH4 +)
Figure imgf000069_0001
Example 23. 2-(2-(5-[3-(Carboxymethvn-5-[4-(methylsulfamovnphenyllthiophen-2- yl]thiophen-2-yl}-5-[4-(methylsulfamoyl)phenyl]thiophen-3-yl)acetic acid (9708 026)
Argon was bubbled through a mixture of intermediate Q (123 mg, 0.236 mmol), methyl 4- boronobenzene sulfonamide (127 mg, 0.589 mmol) and K2CO3 (195 mg, 1.41 mmol) in MeOH (2 ml) and toluene (2 ml). PEPPSI-iPr™(15 mg, 0.0221 mmol) was added and the mixture heated at 90 °C for 30 min in a microwave reactor. Solvents were evaporated, residue dissolved in 0.2 M Na2C03 and purified by preparative hplc (15-45% MeCN in 50 mM buffer, XBridge). Pure fractions were combined, some solvents evaporated and 6 M HC1 added. Precipitate was isolated by centrifugation, washed with water and dried at high vacuum for 2 d. Yield: 45.3 mg (27%); yellow solid. HPLC: Rt = 2.83 min, 97% at 254 nm (10-40% MeCN in buffer, XBridge) and Rt = 2.83 min. 98% at 400 nm (10-40% MeCN in buffer, XBridge). LC-MS: m/z = 720 (M + NH4 +).
Figure imgf000069_0002
Example 24. 2-(2-{5-[3-(Carboxymethyl)-5-(3-methanesulfinylphenyl)thiophen-2- yl]thiophen-2-yl}-5-(3-methanesulfinylphenyl)thiophen-3-yl)acetic acid (9708 027)
Argon was bubbled through a mixture of intermediate Q (123 mg, 0.236 mmol), 3- methylsulfinylphenylboronic acid (109 mg, 0.589 mmol) and K2C03 (195 mg, 1.41 mmol) in MeOH (2 ml) and toluene (2 ml) PEPPSI-iPr™ (15 mg, 0.0221 mmol) was added and the mixture heated at 90 °C for 30 min in a microwave reactor. Solvents were evaporated, residue dissolved in 0.5 M Na2C03/water and purified by preparative hplc (10-40% MeCN in 50 mM buffer, XBridge). Yield: 48.1 mg (32%); yellow solid. 1H NMR (400 MHz,DMSO-i¾): δ 2.82 (s, 6H), 3.80 (s, 4H), 7.37 (s, 2H), 7.62-7.65 (m, 6H), 7.81 (m, 2H), 7.95 (d, J 1.0 Hz, 2H). HPLC: Rt = 2.44 min, 99% at 254 nm (10-40% MeCN in buffer, XBridge) and Rt 99% at 400 nm (10-40% MeCN in buffer, XBridge). LC-MS: m/z = 641 (M + 1).
Figure imgf000070_0001
Example 25. 5-[4-(Carboxymethyl)-5-{4-[3-(carboxymethyl)-5-(5-carboxythiophen-2- yl)thiophen-2-yl]phenyl }thiophen-2-yl]thiophene-2-carboxylic acid (9708 028)
Argon was bubbled through a mixture of intermediate S (202 mg, 0.37 mmol), 2- carboxythiophene-5-boronic acid (160 mg, 0.93 mmol) and K2CO3 (184 mg, 1.33 mmol) in toluene/MeOH (1 : 1, 5 ml). After 15 min, PEPPSI-iPr™ (10 mg, 0.014 mmol) was added and the mixture heated to 70 °C. After 30 min, the reaction mixture was cooled to RT and evaporated to dryness. The residue was purified by flash chromatography using CHCl3/MeOH (containing 1% Et3N) 9: 1→6: 1) as eluent. Yield: 90 mg (38%); green solid. 1H NMR (400 MHz, DMSO-i¾, Et3N-salt): δ 3.68 (s, 4H), 3.75 (s, 6H), 7.12 (d, J4 Hz, 2H), 7.19 (s, 2H), 7.49 (d, J4Hz, 2H), 7.55 (s, 4H).
The ester from above (84 mg, 0.13 mmol) was dissolved in dioxane (4 ml) and 2 M NaOH (4 ml) was added. After 18 hrs at RT, the reaction mixture was diluted with water (15 ml) and acidified using 1 M HC1 (approx. 12 ml). The formed precipitate was isolated by
centrifugation and washed three times with water. The material was dried under high vacuum for 2d. Yield: 47 mg (59%); yellow-green solid. 1H NMR (DMSO-i¾): δ 3.69 (s, 4H), 7.40 (d, J 4 Hz, 2H), 7.49 (s, 2H), 7.63 (s, 4H), 7.70 (d, J 4 Hz, 2H). HPLC: RT = 2.11 min, 98% (254 nm, 5-20% MeCN in 10 mM buffer, 3 min, XBridge) and RT = 2.08 min, 98% (400 nm, 5- 20% MeCN in 10 mM buffer, 3 min, XBridge). LC-MS: m/z = 609 (M-l).
Figure imgf000070_0002
Example 26. 2-(2-{4-[3-(Carboxymethyl)-5-(4-methanesulfonylphenyl)thiophen-2- yl]phenyl}-5-(4-methanesulfonylphenyl)thiophen-3-yl)acetic acid (9708 029)
Argon was bubbled through a mixture of intermediate S (172 mg, 0.32 mmol), 4- methylsulfonylphenylboronic acid (170 mg, 0.85 mmol) and K2C03 (162 mg, 1.17 mmol) in toluene/MeOH/DMF (2: 1 : 1, 6 ml). After 15 min, PEPPSI-iPr™ (9 mg, 0.013 mmol) was added and the mixture heated to 65 °C. After 45 min, the reaction mixture was cooled to RT and evaporated to dryness, the residue was dissolved in CH2CI2 (40 ml), washed with water (40 ml) and evaporated to dryness. The residue was purified by flash chromatography using CHCl3/MeOH 9: 1→6: 1) as eluent. Yield: 99 mg (45%); green solid. 1 H MR (400 MHz, DMSO-i¾): δ 3.26 (s, 6H), 3.66 (s, 6H), 3.83 (s, 4H), 7.65 (s, 4H), 7.73 (s, 2H), 7.94 (d, J 12 Hz, 4H), 7.98 (d, J 12 Hz, 4H).
The ester from above (87 mg, 0.13 mmol) was dissolved in dioxane (4 ml) and 2 M NaOH (4 ml) was added. After 18 hrs at RT, the reaction mixture was diluted with water (15 ml) and acidified using 1 M HC1 (approx. 12 ml). The formed precipitate was isolated by
centrifugation and washed three times with water. The material was dried under high vacuum for 2d. Yield: 52 mg (62%); off-white solid. 1H NMR (DMSO-i¾): 3.26 (s, 6H), 3.73 (s, 4H), 7.68 (s, 4H), 7.74 (s, 2H), 7.95 (d, J 8 Hz, 4H), 7.98 (d, J 8 Hz, 4H). HPLC: RT = 2.81 min, 97% (254 nm, 10-40% MeCN in 10 mM buffer, 3 min, XBridge) and RT = 2.81 min, 100% (400 nm, 10-40% MeCN in 10 mM buffer, 3 min, XBridge). LC-MS: m/z = 665 (M-l).
Figure imgf000071_0001
Example 27. 2-(2-{5-[3-(Carboxymethyl)-5-(2-methanesulfonylphenyl)thiophen-2- yl]thiophen-2-yl}-5-(2-methanesulfonylphenyl)thiophen-3-yl)acetic acid (9708 030)
Argon was bubbled through a mixture of intermediate D (1 15 mg, 0.209 mmol), 2-
(methylsulfonylphenyl)boronic acid (105 mg, 0.523 mmol) and K2C03 (1 15 mg, 0.836 mmol) in MeOH (4 ml) and toluene (4 ml). PEPPSI-iPr™(10 mg, 0.0147 mmol) was added and the mixture heated at 60 °C for 3 h under argon. Solvents were evaporated and residue purified by flash chromatography using 10-20%) EtOAc as eluent. Yield: 108 mg (74%>); slightly yellow oil. 1H NMR (400 MHz, CDC13): 2.89 (s, 6H), 3.76 (s, 6H), 3.85 (s, 4H), 7.22 (s, 2H), 7.47 (s, 2H), 7.54-7.67 (m, 6H), 8.23 - 8.31 (m, 2H).
The material from above (108 mg, 0.154 mmol) was dissolved in dioxane (3 ml) and 2 M NaOH (3 ml) was added. The mixture was heated at 80 °C for 3h before 6 M HC1 was added and yellow precipitate isolated by centrifugation, washed and dried at high vacuum for 3 d. Yield: 100.3 mg (97%); pale yellow solid. 1H NMR (400 MHz,DMSO-i¾): δ 3.05 (s, 6H), 3.81(s, 4H), 7.36 (s, 2H), 7.37 (s, 2H), 7.66 (dd, J7.6 Hz, 1.1 Hz, 2H), 7.69-7.76 (m, 2H), 7.79 (dd, J7.5 Hz, 1.5 Hz, 2H), 8.14 (dd, J 7.9 Hz, 1.4 Hz, 2H). HPLC: Rt = 2.91 min, 97% at 254 nm (10-40% MeCN in buffer, XBridge) and Rt = 2.90 min. 95% at 400 nm (10-40% MeCN in buffer, XBridge). LC-MS: m/z = 690 (M + H4 +).
Figure imgf000072_0001
Example 28. 2-r5-(l-benzothiophen-2-vn-2-(5-r5-(l-benzothiophen-2-vn-3- (carboxymethyl)thiophen-2-yl]thiophen-2-yl}thiophen-3-yl]acetic acid (9708 031)
Argon was bubbled though a mixture of intermediate D (115 mg, 0.209 mmol),
benzo(b)thiophene-2-boronicacid (93 mg, 0.522 mmol) and K2CO3 (115 mg, 0.832 mmol) in toluene (4 ml) and MeOH (4 ml). PEPPSI-iPr™ (10 mg, 0.0147 mmol) was added and the mixture heated at 60 °C for 20 min. Solvents were evaporated and residue purified by flash chromatography using 2% EtOAc in toluene as eluent. Yield: 105 mg (76%); yellow solid. 1H MR (400 MHz, CDC13): δ 3.78 (s, 6H), 3.81 (s, 4H), 7.23 (s, 2H), 7.25 (s, 2H), 7.31-7.35 (m, 4H), 7.41 (s, 2H), 7.33-7.80 (m, 4H).
The ester from above (105 mg, 0.160 mmol) was dissolved in dioxane (3 ml) and 2 M NaOH (3 ml) was added. The mixture was heated at 80 °C for 3 h. 6 M HC1 (ca 2 ml) was added and the precipitate isolated by centrifugation, washed several times with water and dried at high vacuum. Yield: 81 mg (81%). 1H NMR (400 MHz, OMSO-d6): δ 3.75 (s, 4H), 7.37-7.41 (m, 6H), 7.42 (s, 2H), 7.69 (s, 2H), 7.80-7.85 (m, 2H), 7.96-7.98 (m, 2H).
Figure imgf000072_0002
Example 29. 5-[4-(Carboxymethyl)-5-{3-[3-(carboxymethyl)-5-(5-carboxythiophen-2- yl)thiophen-2-yl]phenyl}thiophen-2-yl]thiophene-2-carboxylic acid (9708 032)
Argon was bubbled through a mixture of intermediate U (162 mg, 0.30 mmol), 2- carboxythiophene-5-boronic acid (133 mg, 0.77 mmol) and K2CO3 (144 mg, 1.04 mmol) in toluene/MeOH (1 : 1, 4 ml). After 15 min, PEPPSI-iPr™ (10 mg, 0.015 mmol) was added and the mixture heated to 45 °C. After 2.5 hrs, the reaction mixture was cooled to RT and evaporated to dryness. The residue was purified by flash chromatography using CHCl3/MeOH (containing 0.5% Et3N) 9: 1→5: 1) as eluent. Yield: 138 mg (72%); yellow solid. 1H MR (400 MHz, MeOH-i¾, Et3N-salt): δ 3.73 (s, 6H), 3.75 (s, 4H), 7.24 (d, J 4 Hz, 2H), 7.31 (s, 2H), 7.54 - 7.57 (m, 5H), 7.63 (m, 1H).
The ester from above (98 mg, 0.15 mmol) was dissolved in dioxane (2.5 ml) and 2 M NaOH (2.5 ml) was added. After 18 hrs at RT, the reaction mixture was diluted with water (15 ml) and acidified using 1 M HC1 (approx. 13 ml). The formed precipitate was isolated by centrifugation and washed three times with water. The material was dried under high vacuum for 2d. Yield: 68 mg (72%); yellow solid. 1H MR (DMSO-i¾): δ 3.67 (s, 4H), 7.39 (d, J4 Hz, 2H), 7.48 (s, 2H), 7.54 - 7.57 (m, 2H), 7.61 - 7.65 (m, 2H), 7.69 (d, J4 Hz, 2H), 12.96 (bs, 4H). HPLC: RT = 2.26 min, 100% (254 nm, 5-20% MeCN in 10 mM buffer, 3 min, XB ridge) and RT = 2.27 min, 100% (400 nm, 5-20% MeCN in 10 mM buffer, 3 min,
XBridge). LC-MS: m/z = 628 (M + NH4 +).
Figure imgf000073_0001
Example 30. 2-(2-{3-[3-(Carboxymethyl)-5-(4-methanesulfonylphenyl)thiophen-2- yl]phenyl}-5-(4-methanesulfonylphenyl)thiophen-3-yl)acetic acid (9708 033)
Argon was bubbled through a mixture of intermediate U (185 mg, 0.34 mmol), 4- methylsulfonylphenylboronic acid (176 mg, 0.88 mmol) and K2C03 (162 mg, 1.17 mmol) in toluene/MeOH (1 : 1, 4 ml). After 15 min, PEPPSI-iPr™ (10 mg, 0.014 mmol) was added and the mixture heated to 50 °C. After 1.5 hrs, the reaction mixture was cooled to RT, diluted with CH2C12 (20 ml), washed with water (40 ml) and evaporated to dryness. The residue was purified by flash chromatography using toluene/EtOAc 3 : 1→2: 1→1 : 1) as eluent. Yield: 202 mg (86%); off-white solid. 1H NMR (400 MHz, CDC13): δ 3.09 (s, 6H), 3.75 (s, 10H), 7.47 (s, 2H), 7.55 - 7.56 (m, 3H), 7.67 (m, 1H), 7.79 (d, J 8 Hz, 4H), 7.96 (d, J 8 Hz, 4H).
The ester from above (200 mg, 0.29 mmol) was dissolved in dioxane (2.5 ml) and 2 M NaOH (2.5 ml) was added. After 18 hrs at RT, the reaction mixture was diluted with water (15 ml) and acidified using 1 M HC1 (approx. 13 ml). The formed precipitate was isolated by centrifugation and washed three times with water. The material was dried under high vacuum for 2d. Yield: 133 mg (69%); off-white solid. 1H NMR (DMSO-i¾): δ 3.26 (s, 6H), 3.71 (s, 4H), 7.58 - 7.69 (m, 4H), 7.74 (s, 2H), 7.95 (d, J 8 Hz, 4H), 7.98 (d, J 8 Hz, 4H), 12.63 (bs, 2H). HPLC: RT = 2.66 min, 99% (254 nm, 10-40% MeCN in 10 mM buffer, 3 min, XBridge) and RT = 2.65 min, 70% (400 nm, 10-40% MeCN in 10 mM buffer, 3 min, XBridge). LC-MS: m/z = 684 (M + NH4 +).
Figure imgf000074_0001
Example 31. 5-(4-[2-(Acetyloxy)ethyll-5-(5-(3-[2-(acetyloxy)ethyll-5-(5-carboxythiophen-
2- yl)thiophen-2-yl}thiophen-2-yl)thiophen-2-yl}thiophene-2-carboxylic acid (9708 034)
Nitrogen was bubbled through a mixture of intermediate H (250 mg, 0.506 mmol), 5- carboxythiopheneboronic acid (217 mg, 1.26 mmol) and K2CO3 (349 mg, 2.52 mmol) in toluene (10 ml) and MeOH (10 ml). PEPPSI-iPr™ (17 mg, 0.025 mmol) was added and the mixture stirred at 60 °C for lh. MeOH (70 ml) was added. The suspension was acidified 1 M HCl and a red solid precipitated, which was isolated by centrifugation. The solid material was washed with MeOH and water several times. Yield: 277 mg (93%); red solid. LC-MS: m/z = 587 (M-l). HPLC: RT = 1.47 min, 99% (254 nm, 10-90% MeCN in 10 mM buffer, 3 min, XBridge) and RT = 2.13 min, 97% (400 nm, 10-40% MeCN in 10 mM buffer, 3 min,
XB ridge).
The diol above (60 mg, 0.10 mmol) was dissolved in pyridine (2 ml), cooled to 0 °C followed by addition of Ac20 (2 ml). After 16 hrs, the reaction mixture was evaporated to dryness, dissolved in H20/NaHC03 (sat.) (9: 1, 10 ml) and acidified using 1 M HCl (approx. 15 ml). The formed precipitate was isolated by centrifugation and washed three times with water. Yield: 39 mg (57%); dark red solid. 1H NMR (DMSO-i¾): δ 1.98 (s, 6H), 3.09 (t, J 8 Hz, 4H), 4.32 (t, J 8 Hz, 4H), 7.36 (s, 2H), 7.38 (d, J4 Hz, 2H), 7.51 (s, 2H), 7.66 (d, J4 Hz, 2H). HPLC: RT = 2.87 min, 100% (254 nm, 10-40% MeCN in 10 mM buffer, 3 min, XBridge) and RT = 2.89 min, 100% (400 nm, 10-40% MeCN in 10 mM buffer, 3 min, XBridge). LC-MS: m/z = 690 (M + NH4 +).
Figure imgf000075_0001
Example 32. 2-(2-{5-[3-(2-hydroxyethyl)-5-(4-methanesulfonylphenyl)thiophen-2- yl]thiophen-2-yl}-5-(4-methanesulfonylphenyl)thiophen-3-yl)ethan-l-ol (9708 035)
Argon was bubbled through a mixture of intermediate H (120 mg, 0.24 mmol), 4- methylsulfonylphenylboronic acid (126 mg, 0.63 mmol) and K2CO3 (116 mg, 0.84 mmol) in toluene/MeOH (3 :2, 5 ml). After 15 min, PEPPSI-iPr™ (7 mg, 0.009 mmol) was added and the mixture heated to 55 °C. After 45 min, the reaction mixture was cooled to RT, diluted with CHCI3 (50 ml), washed with water/brine (1 : 1, 60 ml) and evaporated to dryness. The crude product was dissolved in dioxane/H20 (2: 1, 30 ml) under reflux followed by dropwise addition of iPrOH (30 ml) and stirred at 4 °C for 4 days. The obtained solid was filtered, washed with cold MeOH (6 ml), EtOAc (6 ml), iso-hexane (2 x 6 ml) and dried under vaccum. Yield: 108 mg (69%); orange solid. 1H MR (400 MHz, DMSO-i¾): δ 2.96 (t, J 8 Hz, 4H), 3.25 (s, 6H), 3.75 (dt, J 8 Hz, 12 Hz, 4H), 4.93 (t, J 8 Hz, 2H), 7.40 (s, 2H), 7.75 (s, 2H), 7.93 (d, J 8 Hz, 4H), 7.96 (d, J 8 Hz, 4H). HPLC: RT = 2.64 min, 100% (254 nm, 30- 60% MeCN in 10 mM buffer, 3 min, XBridge) and RT = 2.64 min, 100% (400 nm, 30-60% MeCN in 10 mM buffer, 3 min, XBridge). LC-MS: m/z = 662 (M + H4 +).
Figure imgf000075_0002
Example 33. 2-(2-(5-r3-(Carboxymethvn-5-(4-r4-(carboxymethvn-5-(5-r3- (carboxymethyl)thiophen-2-yl]thiophen-2-yl}thiophen-2-yl]phenyl}thiophen-2-yl]thiophen-2- yllthiophen-3-vnacetic acid (9708 036)
Argon was bubbled through a mixture of 1,4-benzenediboronic acid (24 mg, 0.15 mmol), intermediate E (150 mg, 0.32 mmol) and K2C03 (64 mg, 0.46 mmol) in toluene/MeOH (1 : 1, 5 ml). After 15 min PEPPSI-iPr™ (4 mg, 0.006 mmol) was added and the mixture heated to 60 °C for 60 min. The reaction mixture was cooled to RT, diluted with CH2C12 (25 ml) and washed with water (30 ml). The aqueous layer was washed with CH2C12 (2 x 15 ml), the combined organic layers were evaporated to dryness to obtain the crude product. The residue was purified by flash chromatography using toluene/EtOAc 25: 1→20: 1→15: 1) as eluent. Yield: 34 mg (28%); Orange solid. 1H MR (400 MHz, CDC13): δ 3.74 (s,6H), 3.76 (s, 6H), 3.81 (s, 4H), 3.82 (s, 4H), 7.07 (d, J4 Hz, 2H), 7.17 (d, J4 Hz, 2H), 7.20 (d, J4 Hz, 2H), 7.27 (d, J4 Hz, 2H), 7.31 (s, 2H), 7.60 (s, 4H).
The ester from above (43 mg, 0.04 mmol) was dissolved in dioxane (2.5 ml) and 2 M NaOH (2.5 ml) was added. After 18 hrs at RT, the reaction mixture was diluted with water (15 ml) and acidified using 1 M HC1 (approx. 13 ml). The formed precipitate was isolated by centrifugation and washed three times with water. The material was dried under high vacuum for 2d. Yield: 27 mg (86%); dark red solid. 1H NMR (DMSO-i¾): 3.74 (s,4H), 3.78 (s, 4H), 7.10 (d, J4 Hz, 2H), 7.27 (d, J4 Hz, 2H), 7. 32 (d, J4 Hz, 2H), 7.55 (d, J4 Hz, 2H), 7.57 (s, 2H), 7.73 (s, 4H), 12.60 (bs, 4H). HPLC: RT = 2.43 min, 96% (254 nm, 10-40% MeCN in 10 mM buffer, 3 min, XBridge) and RT = 2.44 min, 96% (400 nm, 10-40% MeCN in 10 mM buffer, 3 min, XBridge). LC-MS: m/z = 820 (M + NH4 +).
Figure imgf000076_0001
Example 34. 2-(2-(5-r3-(Carboxymethvn-5-(3-r4-(carboxymethvn-5-(5-r3-
(carboxymethyl)thiophen-2-yl]thiophen-2-yl}thiophen-2-yl]phenyl}thiophen-2-yl]thiophen-2- yllthiophen-3-vnacetic acid (9708 037)
Argon was bubbled through a mixture of 1,3-benzenediboronic acid (26 mg, 0.15 mmol), intermediate E (157 mg, 0.33 mmol) and K2C03 (67 mg, 0.48 mmol) in toluene/MeOH (1 : 1, 5 ml). After 15 min PEPPSI-iPr™ (5 mg, 0.008 mmol) was added and the mixture heated to 55 °C for 2 hrs. The reaction mixture was cooled to RT, diluted with CH2C12 (30 ml) and washed with water (30 ml). The aqueous layer was washed with CH2C12 (2 x 15 ml), the combined organic layers was evaporated to dryness to obtain the crude product. The residue was purified by flash chromatography using toluene/EtOAc 25 : 1→20: 1→15 : 1) as eluent. Yield: 95 mg (73%); dark green solid. 1H NMR (400 MHz, CDC13): δ 3.74 (s,6H), 3.77 (s, 6H), 3.81 (s, 4H), 3.83 (s, 4H), 7.06 (d, J 4 Hz, 2H), 7.18 (d, J 4 Hz, 2H), 7.22 (d, J 4 Hz, 2H), 7.27 (d, J 4 Hz, 2H), 7.33 (s, 2H), 7.40 (m, 1H), 7.51 - 7.53 (m, 2H), 7.79 (m, 1H). The ester from above (95 mg, 0.1 1 mmol) was dissolved in dioxane (2.5 ml) and 2 M NaOH (2.5 ml) was added. After 18 hrs at RT, the reaction mixture was diluted with water (20 ml) and acidified using 1 M HC1 (approx. 13 ml). The formed precipitate was isolated by centrifugation and washed three times with water. The material was dried under high vacuum for 2d. Yield: 43 mg (49%); green-yellow solid. 1H NMR (OMSO-d6): 3.75 (s, 4H), 3.79 (s, 4H), 7.10 (d, J4 Hz, 2H), 7.28 (d, J4 Hz, 2H), 7. 33 (d, J4 Hz, 2H), 7.45 - 7.62 (m, 5H), 7.67 (s, 2H), 7.92 (m, 1H), 12.59 (bs, 4H). HPLC: RT = 2.60 min, 96% (254 nm, 10-40% MeCN in 10 mM buffer, 3 min, XBridge) and RT = 2.60 min, 97% (400 nm, 10-40% MeCN in 10 mM buffer, 3 min, XBridge). LC-MS: m/z = 820 (M + NH4 +).
Figure imgf000077_0001
Example 35. 2-(2-{5-[3-(Carboxymetfayl)-5-[5-(lH-1.2 .4 e razol-5-yl)tMophen-2- yllthiophen-2-yllthiophen-2-yl}-5-[5-(lH-1.2.3.4-tetrazol-5-ynthiophen-2-yllthiophen-3- vDacetic acid (9708 038)
Sodium azide (37 mg, 0.563 mmol) and Et3N-HCl (71 mg, 0.563 mmol) was added to a solution of intermediate V (114 mg, 0.188 mmol) in DMF (5 ml). The mixture was heated at 50 °C for 30 min and at 100 °C overnight. Solvent was evaporated and residue dissolved in dioxane (3 ml). 2 M NaOH (3 ml) was added and the mixture heated at 80 °C for 1 h. 6 M HCl was added and solid material isolated by centrifugation. The product was purified by preparative hplc (10-30% MeCN in 50 mM buffer, XBridge). Pure fractions were combined and some solvents evaporated. 1 M HCl was added, precipitate collected by centrifugation, washed with water and dried at high vacuum. Yield: 88 mg (71%); deep orange solid, metallic shine. 1H NMR (400 MHz, OMSO-d6): δ 3.80 (s, 4H), 7.36 (s, 2H), 7.48 (s, 4H), 7.52 (d, J 3.8 Hz, 2H), 7.75 (d, J4.0 Hz, 2H). HPLC: Rt = 1.78 min, 98% (400 nm, 10-30% MeCN in 10 mM buffer, 3 min, XBridge) and Rt = 1.52 min, 97% (254 nm, 10-40% MeCN in 10 mM buffer, 3 min, XBridge). LC-MS: m/z = 665 (M+l).
Figure imgf000077_0002
Example 36. 2-(5-{5-[4-(Carboxymethyl)-5-(4-methanesulfonylphenyl)thiophen-2- yl]thiophen-2-yl}-2-(4-methanesulfonylphenyl)thiophen-3-yl)acetic acid (9708 039)
Argon was bubbled through a solution of intermediate Z (206 mg, 0.53 mmol) and 2,5- thiophenediylbisboronic acid (45 mg, 0.26 mmol) in MeOH (3 ml) and toluene (3 ml) for 5 min. PEPPSI-iPr™ (10 mg, 0.014 mmol) and K2C03 (220, 1.06 mmol) were added and the mixture heated to 60 °C for 2 hours. The mixture was then concentrated under vacuum and the crude material was dissolved in DCM with some MeOH. The solution was purified by flash chromatography using 40% and 100% EtOAc in iso-hexane as eluent. Yield: 80 mg (42%); yellow solid. Ή MR (400 MHz, CDC13): δ 3.12 (s, 6H), 3.67 (s, 4H), 3.78 (s, 6H), 7.15 (s, 2H), 7.22 (s, 2H), 7.69 - 7.76 (m, 4H), 8.00 - 8.06 (m, 4H).
The ester from above (70 mg, 0.10 mmol) was added dioxane (3 ml) and 2 M NaOH (3 ml) and the mixture was heated at 80 °C for 90 min. The solution was cooled to r.t. and added 6 M HC1 (1.5 ml). The formed precipitate was isolated by centrifugation and washed three times with water. The material was added some MeOH and the solvent was removed in vacuo. The yellow solid was then dried under high vacuum for 2 days. Yield: 55 mg (82%); yellow solid. Ή NMR (400 MHz, DMSO-de): δ 3.28 (s, 6H), 3.68 (s, 4H), 7.39 (s, 2H), 7.40 (s, 2H), 7.75 - 7.80 (m, 4H), 8.01 - 8.05 (m, 4H). R, = 2.83 min, 98% (254 nm, 10-40% MeCN in 10 mM buffer, XBridge) Rt = 2.83 min, 98% (400 nm, 10-40% MeCN in 10 mM buffer, XBridge). LC-MS: m/z = 690 (M + NH4 +).
Figure imgf000078_0001
Example 37. 2-(5-{4-[4-(Carboxymethyl)-5-(4-methanesulfonylphenyl)thiophen-2- yl]phenyl}-2-(4-methanesulfonylphenyl)thiophen-3-yl)acetic acid (9708 040)
Argon was bubbled through a solution of intermediate Z (205 mg, 0.53 mmol) and 2,5-T thiophenediylbisboronic acid (44 mg, 0.26 mmol) in MeOH (3 ml) and toluene (3 ml) for 5 min. PEPPSI-iPr™ (10 mg, 0.014 mmol) and K2C03 (220, 1.6 mmol) were added and the mixture heated to 60 °C for 2 hours. The mixture was then concentrated under vacuum and the crude material was dissolved in DCM with some MeOH. The solution was purified by flash chromatography using 40% and 100% EtOAc in iso-hexane as eluent. Yield: 104 mg (54%)); pale yellow solid.
The ester from above (100 mg, 0.15 mmol) was added dioxane (4 ml) and 2 M NaOH (4 ml) and the mixture was heated at 80 °C for 90 min. The solution was cooled to r.t. and added 6 M HC1 (2 ml). The formed precipitate was isolated by centrifugation and washed three times with water. The material was added some MeOH and the solvent was removed in vacuo. The yellow solid was then dried under high vacuum for 2 days. Yield: 59 mg (61%>); yellow solid. Ή NMR (400 MHz, DMSO-de): δ 3.28 (s, 6H), 3.70 (s, 4H), 7.62 (s, 2H), 7.77 (s, 4H), 7.78 - 7.81 (m, 4H), 8.02 - 8.06 (m, 4H). R, = 2.76 min, 95% (254 nm, 10-40% MeCN in 10 mM buffer, XBridge) Rt = 2.76 min, 95% (400 nm, 10-40% MeCN in 10 mM buffer, XBridge). LC-MS: m/z = 684 (M + NH4 +).
Figure imgf000079_0001
Example 38. 2-(2-(4-r3-(Carboxymethvn-5-(4-r4-(carboxymethvn-5-(4-r3- (carboxymethyl)thiophen-2-yl]phenyl }thiophen-2-yl]phenyl }thiophen-2-yl]phenyl }thiophen- 3-yl)acetic acid (9708_041)
Argon was bubbled through a mixture of 1,4-benzenediboronic acid (24 mg, 0.15 mmol), intermediate X (151 mg, 0.32 mmol) and K2C03 (65 mg, 0.47 mmol) in toluene/MeOH (1 : 1, 4 ml). After 15 min PEPPSI-iPr™ (5 mg, 0.007 mmol) was added and the mixture heated to 55 °C for 60 min. The reaction mixture was cooled to RT, diluted with CH2C12 (30 ml) and washed with water (30 ml). The aqueous layer was washed with CH2C12 (2 x 15 ml), the combined organic layers was evaporated to dryness to obtain the crude product. The residue was purified by flash chromatography using toluene/EtOAc (20: 1→14: 1→9: 1) as eluent. Yield: 64 mg (51%); Off-white solid. 1H NMR (400 MHz, CDC13): δ 3.73 (s, 4H), 3.74 (s, 4H), 3.76 (s, 6H), 3.78 (s, 6H), 7.12 (d, J 5.0 Hz, 2H), 7.33 (d, J 5.0 Hz, 2H), 7.37 (s, 2H), 7.55 - 7.61 (m, 8H), 7.65 (s, 4H).
The ester from above (64 mg, 0.08 mmol) was dissolved in dioxane (2 ml) and 2 M NaOH (2 ml) was added. After 2 hrs at 80 °C followed by 18 hrs at RT, the reaction mixture was diluted with water (20 ml) and acidified using 2 M HC1 (approx. 5 ml). The formed precipitate was isolated by centrifugation and washed three times with water. The material was dried under high vacuum for 2d. Yield: 50 mg (79%); Yellow solid. 1H NMR (DMSO- d6): 3.65 (s, 4H), 3.69 (s, 4H), 7.12 (d, J 5.3 Hz, 2H), 7.55 - 7.64 (m, 12H), 7.74 (s, 4H). HPLC: RT = 2.55 min, 92% (254 nm, 10-40% MeCN in 10 mM buffer, 3 min, XBridge) and RT = 2.54 min, 94% (400 nm, 10-40% MeCN in 10 mM buffer, 3 min, XBridge). LC-MS: m/z = 808 (M + NH4 +).
Figure imgf000080_0001
Example 39. 2-(5-{3-[4-(Carboxymethyiy5-{4-[3-(carboxym
yl]phenyl}thiophen-2-yl]phenyl}-2-{4-[3-(carboxymethyl)thiophen-2-yl]phenyl}thioph vDacetic acid (9708_042)
Argon was bubbled through a mixture of 1,3-benzenediboronic acid (25 mg, 0.15 mmol), intermediate X (154 mg, 0.33 mmol) and K2C03 (75 mg, 0.54 mmol) in toluene/MeOH (1 : 1, 4 ml). After 15 min PEPPSI-iPr™ (5 mg, 0.008 mmol) was added and the mixture heated to 55 °C for 90 min. The reaction mixture was cooled to RT, diluted with CH2C12 (30 ml) and washed with water/brine (3 :2 - 50 ml). The organic layer was evaporated to dryness to obtain the crude product. The residue was purified by flash chromatography using toluene/EtOAc 14: 1 as eluent. Yield: 68 mg (52%); Yellowish solid. 1 H NMR (400 MHz, CDC13): δ 3.73 (s, 4H), 3.75 (s, 10H), 3.79 (s, 6H), 7.12 (d, J 5.3 Hz, 2H), 7.32 (d, J 5.3 Hz, 2H), 7.39 (s, 2H), 7.42 (m, 1H), 7.55 - 7.62 (m, 10H), 7.86 (m, 1H).
The ester from above (68 mg, 0.08 mmol) was dissolved in dioxane (2 ml) and 2 M NaOH (2 ml) was added. After 18 hrs at RT, the reaction mixture was diluted with water (20 ml) and acidified using 2 M HC1 (approx. 5 ml). The formed precipitate was isolated by centrifugation and washed three times with water. The material was dried under high vacuum for 2d. Yield: 57 mg (90%); Yellow solid. 1H NMR (DMSO-<¾): 3.65 (s, 4H), 3.71 (s, 4H), 7.13 (d, J4 Hz, 2H), 7.50 - 7.67 (m, 15H), 7.95 (m, 1H). HPLC: RT = 2.68 min, 95% (254 nm, 10-40% MeCN in 10 mM buffer, 3 min, XBridge) and RT = 2.67 min, 50% (400 nm, 10-40% MeCN in 10 mM buffer, 3 min, XBridge). LC-MS: m/z = 808 (M + NH4 +).
Figure imgf000080_0002
Example 40. 2-(5-{5-[4-(Carboxymethyl)-5-{4-[3-(carboxymethyl)thiophen-2- yl]phenyl}thiophen-2-yl]thiophen-2-yl}-2-{4-[3-(carboxymethyl)thiophen-2- vllphenyl|thiophen-3-yl)acetic acid (9708 043)
Argon was bubbled through a mixture of 2,5-thiophenediboronic acid (25 mg, 0.15 mmol), intermediate X (151 mg, 0.32 mmol) and K2C03 (65 mg, 0.47 mmol) in toluene/MeOH (1 : 1, 6 ml). After 15 min PEPPSI-iPr™ (5 mg, 0.008 mmol) was added and the mixture heated to 50 °C for 90 min. The reaction mixture was cooled to RT, diluted with CH2C12 (30 ml) and washed with water/brine (10: 1 - 55 ml). The organic layer was evaporated to dryness to obtain the crude product. The residue was purified by flash chromatography using toluene/EtOAc (25: l→20: l→18: l→12: l) as eluent. Yield: 46 mg (37%); Green oil. 1H NMR (400 MHz,
CDC13): δ 3.71 (s, 4H), 3.73 (s, 4H), 3.75 (s, 6H), 3.78 (s, 6H), 7.12 (d, J 5.0 Hz, 2H), 7.12 (s, 2H), 7.20 (s, 2H), 7.32 (d, J 5.0 Hz, 2H), 7.56 (s, 8H).
The ester from above (42 mg, 0.05 mmol) was dissolved in dioxane (2 ml) and 2 M NaOH (2 ml) was added. After 18 hrs at RT, the reaction mixture was diluted with water (20 ml) and acidified using 2 M HC1 (approx. 5 ml). The formed precipitate was isolated by centrifugation and washed three times with water. The material was dried under high vacuum for 2d. Yield: 23 mg (59%); Green-yellow solid. 1H MR (DMSO-i¾): 3.65 (s, 4H), 3.68 (s, 4H), 7.12 (d, J 4 Hz, 2H), 7.35 (s, 2H), 7.36 (s, 2H), 7.57 (d, J4 Hz, 2H), 7.57-7.63 (m, 8H). HPLC: RT = 2.61 min, 91% (254 nm, 10-40% MeCN in 10 mM buffer, 3 min, XBridge) and RT = 2.61 min, 93% (400 nm, 10-40% MeCN in 10 mM buffer, 3 min, XBridge). LC-MS: m/z = 797 (M + H+).
Figure imgf000081_0001
Example 41. 2-(2-{5-[3-(2-Aminoethyl)-5-(4-methanesulfonylphenyl)thiophen-2- yl]thiophen-2-yl}-5-(4-methanesulfonylphenyl)thiophen-3-yl)ethan-l -amine (9708 044)
Argon was flushed through a stirred solution of intermediate M (190 mg, 0.213 mmol) and 4- methylsulfonylphenylboronic acid (110 mg, 0.550 mmol) in a mixture of toluene (3 ml) and MeOH (3 ml). K2C03 (250 mg, 1.81 mmol) and PEPPSI-iPr™ (7.8 mg, 0.011 mmol) were added and the reaction heated in a sealed flask at 60°C for 1 hour. The solvents were removed in vacuo and the crude material was added water (-10 ml) and extracted with DCM (~5 ml). The organic phase was purified by flash chromatography (30% EtOAc in toluene, 200 ml silica). Yield: 185 mg, (83%) as a orange solid. 1H MR (400 MHz, OMSO-d6): δ 1.36 (s, 36H), 3.06 (t, J 6.90 Hz, 4H), 3.26 (s, 6H), 3.85 (t, J 6.90 Hz, 4H), 7.33 (s, 2H), 7.61 (s, 2H), 7.88 - 7.93 (m, 4H), 7.95 - 8.00 (m, 4H)
TFA (1 ml) was added to a stirred solution of the material from above (185 mg, 0.177 mmol) in DCM (3 ml). The reaction was stirred at r.t. for 2 hours and the solvents were removed in vacuo. 1 M NaOH (15 ml) was added and the mixture was extracted with DCM (250 ml). The organic phase was dried over MgS04 and removed in vacuo. The material was dissolved in AcOH (~5 ml) and cone HC1 (-0.2 ml) was added. The solvents were removed in vacuo and the compound was placed under high vacuum over the weekend. Yield: 65 mg (51%) as a red solid, HCl-salt of title compound. 1H MR (400 MHz, DMSO-d6): δ 3.08 - 3.25 (m, 8H), 3.27 (s, 6H), 7.44 (s, 2H), 7.83 (s, 2H), 7.92 - 8.03 (m, 8H), . HPLC: Rt = 1.98 min, 100% (254 nm, 10-90% MeCN in 0.1% TFA) and Rt = 2.65 min, 100% (254 nm, 05-60% MeCN in 0.1% TFA). LC-MS: m/z = 643 (M + 1).
Figure imgf000082_0001
Example 42. 2-(2-(5-r3-(2-Aminoethvn-5-(5-r4-(2-aminoethvn-5-(5-r3-(2- aminoethyl)thiophen-2-yl]thiophen-2-yl}thiophen-2-yl]thiophen-2-yl}thiophen-2- yllthiophen-2-yl lthiophen-3 -yPethan- 1 -amine (9708 045)
BS (39 mg, 0.22 mmol) was added to a stirred solution of intermediate L (160 mg, 0.22 mmol) in a mixture of CHC13 (2 ml) and AcOH (2 ml) at r.t. The reaction was stirred for 1 hour and poured on a stirred mixture of 5 M NaOH (15 ml) and ice. DCM (5 ml) was added and the organic phase was dried over MgS04 and removed in vacuo. The crude material was dissolved in MeCN (2 ml) and purified by prep-HPLC (90-100% MeCN, in 50 mM
NH3/NH4HC03 buffer). The pure fractions were combined and concentrated to dryness.
Yield: 81 mg (46%) as a yellow gum. 1H NMR (400 MHz, CDC13): δ 1.46 (s, 18H), 1.46 (s, 18H), 2.97 - 3.09 (m, 4H), 3.79 - 3.88 (m, 4H), 6.93 (s, 1H), 6.97 (d, J 5.3 Hz, 1H), 7.07 - 7.10 (m, 1H), 7.1 1 - 7.13 (m, 1H), 7.22 (d, J 5.3 Hz, 1H)
Argon was flushed through a solution of the material from above (150 mg, 0.18 mmol) and 2,5-thiopenediboronic acid (15 mg, 0.092 mmol) in a mixture of toluene (2 ml) and MeOH (2 ml). K2CO3 (150 mg, 1,1 mmol) and PEPPSI-iPr1M (10 mg, 0.015 mmol) were added and the reaction heated at 55°C for 90 min. The solvents were removed in vacuo and the crude material was added water (-10 ml) and extracted with DCM (~5 ml). The organic phase was dried over MgS04 and added TFA (2 ml). The reaction was stirred at r.t. for 2 hours and concentrated. The crude material was dissolved in MeOH (8 ml), filtered and purified by prep-HPLC (30-70% MeCN in 0.1% TFA aq). The pure fractions were combined and freeze dried. Not pure fractions containing product were combined and concentrated to dryness. The red solid was dissolved in MeOH (2 ml) and purified on prep-HPLC (30-70% MeCN in 0.1% TFA aq). The pure fractions were combined and freeze dried. The 2 batches were combined. 1H NMR (400 MHz, DMSO-i¾): δ 3.00 - 3.22 (m, 16H), 7.17 (d, J 5.3 Hz, 2H), 7.30 (d J4.0 Hz, 2H), 7.34 (d, J4.0 Hz, 2H), 7.38 (s, 2H), 7.42 (s, 2H), 7.64 (d, J 5.3 Hz, 2H), 7.84 - 8.02 (m, 12H). HPLC: Rt = 1.95 min, 100% (254 nm, 10-90% MeCN in 0.1% TFA) R, = 1.25 min, 100% (305 nm, 10-90% MeCN in 0.1% TFA) LC-MS: m/z = 749 (M + 1).
Figure imgf000083_0001
Example 43. 2-(5-r4-(Carboxymethvn-5-(5-r3-(carboxymethvn-5-r5-(2- carboxyphenyl)thiophen-2-yl]thiophen-2-yl]thiophen-2-yl}thiophen-2-yl]thiophen-2- yllbenzoic acid (9708 046)
Argon was bubbled through a mixture of intermediate O (174 mg, 0.244 mmol), 2- methoxycarbonylphenylboronic acid (131 mg, 0.731 mmol) and K2CO3 (168 mg, 1.22 mmol) in toluene (10 ml) and methanol (10 ml). PEPPSI-iPr™ (3 mg, 0.005 mmol) was added and the mixture was heated at 70 °C for 30 min. Solvents were evaporated and residue dissolved in dioxane (5 ml) and 2 M NaOH (5 ml) was added and the mixture heated at 80 °C for 1 h. The mixture was acidified by addition of 2 M HCl and solid material was isolated by centrifugation, dissolved in 1 M Na2C03 and purified by prep. hplc. Pure fractions were combined and solid material precipitated by addition of 1 M HCl. Solid material was isolated by centrifugation and washed three times with water. Freeze-dried under high vacuum for 3 d. Yield: 54.4 mg (29%); orange solid. Rt = 2.17 min, 96% at 254 nm (10-40% MeCN in buffer, XB ridge) and Rt = 2.18 min. 98% at 400 nm (10-40% MeCN in buffer, XBridge). MS: m/z = 767 (M - 1) (neg. ionization)
Figure imgf000084_0001
Example 44. 2-(2-{5-[3-(Carboxymethyl)-5-[3-(hydroxymethyl)phenyl]thiophen-2- yl]thiophen-2-yl}-5-[3-(hydroxymethyl)phenyl]thiophen-3-yl)acetic acid (9708 047)
Argon was bubbled through a mixture of intermediate D (150 mg, 0.273 mmol), 3-
(hydroxymethyl)phenylboronic acid (104 mg, 0.681 mmol) and K2CO3 (150 mg, 1.09 mmol) in toluene (2 ml) and methanol (2 ml). PEPPSI-iPr™ (4 mg, 0.005 mmol) was added and the mixture heated in a microwave reactor at 80 °C for 15 min. Solvents were evaporated and residue added dioxane (2 ml) and 2 M NaOH (2 ml) and the mixture was heated at 80 °C for 1 h. 2 M HCl (ca 5 ml) was added and the precipitated material isolated by centrifugation and dissolved in 1 M Na2C03/water , and purified by prep, hplc using 10-40% MeCN in 50 mM buffer (XBridge). Yield: 7.6 mg (5%); yellow solid. Rt = 2.73 min, 99% at 254 nm (10-40% MeCN in buffer, XBridge) and Rt = 2.73 min. 98% at 400 nm (10-40% MeCN in buffer, XBridge). MS: m/z = 575 (M - 1) (neg. ionization)
Figure imgf000084_0002
Example 45. 2-(2-{5-[3-(Carboxymethyl)-5-[4-(hydroxymethyl)phenyl]thiophen-2- yl]thiophen-2-yl}-5-[4-(hydroxymethyl)phenyl]thiophen-3-yl)acetic acid (9708 048)
Argon was bubbled through a mixture of intermediate D (162 mg, 0.294 mmol), 4- (hydroxymethyl)phenylboronic acid (111 mg, 0.736 mmol) and K2C03 (162 mg, 1.78 mmol) in toluene (2 ml) and methanol (2 ml). PEPPSI-iPr™ (4 mg, 0.005 mmol) was added and the mixture heated in a microwave reactor at 80 °C for 15 min. Solvents were evaporated and the residue added dioxane (2 ml) and 2 M NaOH (2 ml). The mixture was heated at 80 °C for 1 h. 2 M HCl (ca 5 ml) was added and the precipitated material isolated by centrifugation and purified by prep, hplc using 10-40%) MeCN in 50 mM buffer. Pure fractions were collected and some solvents were evaporated. 1 M HCl was added and solid precipitate extracted with EtOAc, dried with MgS04 filtered and concentrated. Yield: 61.1 mg (36%>); yellow solid. Rt = 2.46 min, 99% at 254 nm (10-40% MeCN in buffer, XBridge) and Rt = 2.47 min. 98% at 400 nm (10-40% MeCN in buffer, XBridge). 1H NMR (400 MHz, CDC13): δ 3.77 (s, 4H), 4.52 (s, 4H), 7.31 (s, 2H), 7.38 (d, J 8 Hz, 4H), 7.47 (s, 2H), 7.62 (d, J8 Hz, 4H). MS: m/z = 575 (M - 1) (neg. ionization)
Figure imgf000085_0001
Example 46. 2-(2-{5-[3-(Carboxymethyn-5-(lH-indol-5-ynthiophen-2-yllthiophen-2-yl}-5- (lH-indol-5-yl)thiophen-3-yl)acetic acid (9708 049)
Argon was bubbled through a mixture of intermediate D (193 mg, 0.351 mmol), lH-Indole-5- boronic acid (141 mg, 0.0.877 mmol) and K2C03 (150 mg, 1.09 mmol) in toluene (2 ml) and methanol (2 ml). PEPPSI-iPr™ (5 mg, 0.007 mmol) was added and the mixture heated in a microwave reactor at 90 °C for 10 min. Solvents were evaporated, the residue added dioxane (2 ml) and 2 M NaOH (2 ml), and the mixture heated at 80 °C for 1 h. 2 M HC1 (ca 5 ml) was added and the precipitated material isolated by centrifugation and purified by prep, hplc using 20-45%) MeCN in 50 mM buffer as eluent. Pure fractions were collected and some solvents were evaporated. 1 M HC1 was added and mixture was extracted with EtOAc. Combined organic layers were dried (MgS04) and concentrated. Yield: 50.6 mg (24%); yellow-brown solid. Rt = 2.27 min, 90% at 254 nm (20-50% MeCN in buffer, XBridge) and Rt = 2.27 min. 90% at 400 nm (20-50% MeCN in buffer, XBridge). MS: m/z = 593 (M - 1) (neg. ionization).
Figure imgf000085_0002
Example 47. 2-(2-{5-[3-(Carboxymethyl)-5-[2-(carboxymethyl)phenyl]thiophen-2- yl]thiophen-2-yl}-5-[2-(carboxymethyl)phenyl]thiophen-3-yl)acetic acid (9708 050)
Argon was flushed through a stirred solution of intermediate D (80.2 mg, 0.146 mmol) and 2- (methoxycarbonylmethyl)phenylboronic acid pinacol ester (115 mg, 0.416 mmol) in a mixture of toluene (3 ml) and MeOH (3 ml). K2C03 (489 mg, 3.54 mmol) and PEPPSI-iPr™ (15.2 mg, 0.0210 mmol) were added and the reaction heated at 65°C for 1 hour. The solvents were removed in vacuo and the crude material was added water (1 ml) and 2 M NaOH (1 ml). The reaction was stirred at 80°C for 1 hour. The mixture was cooled to r.t. and filtered. Some (-0.5 ml) water was added and the mixture was purified by prep-HPLC (5-40% MeCN, in 50 mM H3/NH4HCO3 buffer). The combined pure fractions were concentrated to dryness. The compound was dissolved in water (20 ml) and some 1 M HC1 was added. The solid was collected and washed several times with water and dried. Yield: 55 mg (60%) as a yellow solid. 1H MR (400 MHz, OMSO-d6): δ 3.7-3.8 (m, 8H), 7.07-7.13 (m, 2H), 7.22-7.47 (m, 9H), 7.51-7.56 (m, 1H), 12.5 (s, 4H). HPLC Rt = 1.78 min, 100% (254 nm, 10-40% MeCN in 10 mM buffer, XBridge) and ¾ = 1.13 min, 100% (254 nm, 10-90% MeCN in 10 mM buffer, XBridge). LC-MS: m/z = 650 (M + 18).
Figure imgf000086_0001
Example 48. 2-{5-[4-(Carboxymethyl)-5-(4-methanesulfonylphenyl)thiophen-2-yl]-2-(4- methanesulfonylphenyl)thiophen-3-yl} acetic acid (9708 051)
NIS (638 mg, 2.84 mmol) was added in portions to a stirred solution of intermediate Y (800 mg, 2.58 mmol) in a mixture of AcOH (5 ml) and CHCI3 (5 ml) at 0 °C. The reaction was stirred at this temperature for 5 min and then at r.t. for 3 days. The mixture was concentrated, added some DCM (~5 ml), washed with diluted NaOH and purified by flash chromatography (50% EtOAc in toluene). Yield: 1.06 g (94%). HPLC Rt = 2.71 min, 92% (254 nm, 10-90% MeCN in 10 mM buffer, XBridge).
Argon was bubbled through a mixture of material from above (109 mg, 0.250 mmol), K2CO3 (104 mg, 0,750 mmol) and DMSO (2 ml) for 5 min. Bis(pinacolato)diboron (254 mg, 0.125 mmol) and l, l'-bis(diphenylphosphino)ferrocene-palladium(II)di chloride (9.1 mg, 0,013 mmol) were added and the reaction was stirred at 80°C in a sealed tube overnight. The reaction was added water (0.60 ml) and 5 M NaOH (0.60 ml) and stirred at 80°C for 1 hour. The mixture was cooled and filtered. Some (-0.5 ml) water was added and the mixture was purified by prep-HPLC (5-45% MeCN, in 50 mM NH3/NH4HCO3 buffer). The combined pure fractions were concentrated to dryness. The compound was dissolved in water (20 ml) and some 1 M HC1 was added. The solid was collected and washed several times with water and dried. Yield: 47 mg (32%) as a pale yellow solid. 1H NMR (400 MHz, DMSO-^): δ 3.28 (s, 6H), 3.70 (s, 4H), 7.41 (s, 2H), 7.76 - 7.81 (m, 2H), 8.00 - 8.05 (m, 2H), 12.62 (s br, 2H). HPLC: Rt = 2.14 min, 90% (254 nm, 10-40% MeCN in 10 mM buffer, XBridge) and Rt = 1.47 min, 90% (254 nm, 10-90% MeCN in 10 mM buffer, XBridge). LC-MS: m/z = 608 (M + 18 .
Figure imgf000087_0001
Example 49. 2-{2-[3-(Carboxymethyl)-l-benzothiophen-2-yl]-l-benzothiophen-3-yl}acetic acid (9708_052)
Acetyl chloride (0.882 ml, 12.4 mmol) was added to a stirred solution of benzothiophene-3 - acetic (950 mg, 4.94 mmol) in MeOH (10 ml) at 0°C. The reaction was then stirred at r.t. over night and the solvent was removed in vacuo. The crude material was dissolved in EtOAc (-50 ml) and washed with 0.5 M NaOH. The organic phase was dried over MgS04 and removed in vacuo. Yield: 0.865 g (85%). HPLC Rt = 2.62 min, 100% (254 nm, 10-90% MeCN in 10 mM buffer, XBridge).
Br2 (0.121 ml, 2.36 mmol) in DCM (1 ml) was slowly added to a stirred solution of the material from above (0.486 g, 2.36 mmol) in DCM (7 ml) at 0°C. The reaction was stirred at this temperature for 1 hour. The mixture was added sat. Na2S203 (1 ml) and washed with (-20 ml) water. The organic phase was purified on a plug of silica using 15% EtOAc in toluene as eluent. Yield: 622 mg (93%) as a colourless oil. 1H NMR (400 MHz, CDC13): δ 3.71 (s, 3H), 3.90 (s, 2H), 7.32 - 7.41 (m, 2H), 7.66 - 7.70 (m, 1H), 7.72 - 7.76 (m, 1H)
Argon was bubbled through a stirred mixture of the material from above (113 mg, 0.396 mmol), K2C03 (164 mg, 1.19 mmol) and DMSO (1 ml) for 5 min. Bis(pinacolato)diboron (50.3 mg, 0.198 mmol) and l, -bis(diphenylphosphino)ferrocene-palladium(II)dichloride (14.5 mg, 0.0200 mmol) were added and the reaction was stirred at 80°C in a sealed tube overnight. The reaction was added water (1,5 ml) and 5 M NaOH (0,5 ml) and heated at 80°C for 30 min. The mixture was cooled and filtered. Water (-0.5 ml) was added and the mixture was purified by prep-HPLC (5-45% MeCN, in 50 mM NH3/NH4HC03 buffer). The combined pure fractions were concentrated to dryness. Water and some HCl were added and the mixture was extracted with EtOAc. The organic phase was dried over MgS04 and removed in vacuo. Yield: 37 mg, (24%) as a white solid. 1H NMR (400 MHz, DMSO-i¾): δ 3.80 (s, 4H), 7.42- 7.51 (m, 4H), 7.78-7.84 (m, 2H), 8.01-8.07 (m, 2H), 12.51 (s, 2H), HPLC: Rt = 1.86 min, 100% (254 nm, 10-40% MeCN in 10 mM buffer, XBridge) and Rt = 1.36 min, 100% (254 nm, 10-90% MeCN in 10 mM buffer, XBridge). LC-MS: m/z = 400 (M + 18).
Figure imgf000088_0001
Example 50. 2-{5-[4-(Carboxymethyl)-5-[5-(4-methanesulfonylphenyl)thiophen-2- yl]thiophen-2-yl]-2-[5-(4-methanesulfonylphenyl)thiophen-2-yl]thiophen-3-yl}acetic acid (9708_053)
Argon was bubbled through a mixture of intermediate A2 (360 mg, 1.320 mmol), 4- methylsulfonylphenylboronic acid (328 mg, 1.64 mmol) and K2CO3 (454 mg, 3.29 mmol) in toluene (4 ml) and MeOH (4 ml). PEPPSI-iPr™(17 mg, 0.025 mmol) was added and the mixture heated at 70 °C for 30 min in a sealed tube. Water and toluene were added. The aqueous layer was extracted with toluene. Combined organic layers were concentrated and residue purified by flash chromatography using 10% EtOAc in toluene as eluent. Yield: 507 mg (98%); yellow oil which solidifies when stored in the fridge. 1H NMR (400 MHz, CDC13): δ 3.10 (s, 3H), 3.76 (s, 3H), 3.84 (s, 2H), 7.10 (d, J 5.2 Hz, 1H), 7.23 (d, J 3.6 Hz, 1H), 7.31 (d, J 5.2 Hz, 1H), 7.44 (d, J 3.6 Hz, 1H), 7.79 (d, J 8.0 Hz, 2H), 7.97 (d, J 8.0 Hz, 2H).
The material from above (570 mg, 1.29 mmol) was dissolved in CHCI3 (10 ml) and AcOH (10 ml). NBS (253 mg, 1.42 mmol) was added portion-wise at 0 °C. The mixture was stirred at ambient temperature for 3 h. Solvents were evaporated and residue purified by flash chromatography using 10-15%) EtOAc in toluene as eluent. Yield: 509 mg (83%>); yellow solid. 1H NMR (400 MHz, CDC13): δ 3.10 (s, 3H), 3.77 (s, 5H), 7.08 (s, 1H), 7.19 (d, J4 Hz, 1H), 7.43 (d, J 4.0 Hz, 1H), 7.78 (d, J 8 Hz, 2H), 7.97 (d, J 8 Hz, 2H).
The material from above (209 mg, 0.443 mmol) and bispinacolatodiboron (56 mg, 0.222 mmol) was dissolved in DMSO (5 ml). Potassium carbonate (245 mg, 1.77 mmol) was added and the mixture was degassed by bubbling argon through the mixture. 1, 1 '- bis(diphenylphosphino)ferrocene dichloropalladium (II) (16 mg, 0.022 mmol) was added and the mixture heated at 80 °C for 22 h in a sealed tube. 2 M NaOH (3 ml) was added and heating continued for 2 h. The mixture was filtered and purified by prep, hplc using 15-45% MeCN in 50 mM buffer as eluent. Pure fractions were combined, some solvent evaporated and 1 M HCl added. Precipitated material was isolated by centrifugation and washed with water three times. The material was freeze-dried for 2 days. Yield: 42.5 mg (25%); orange solid. Rt = 3.09 min, 99% at 254 nm (10-40% MeCN in buffer, XBridge) and Rt = 3.10 min. 99% at 400 nm (10-40% MeCN in buffer, XBridge) 1H NMR (400 MHz, OMSO-d6): δ 3.24 (s, 6H, 7.35 (s, 2H), 7.37 (d, J4 Hz, 2H), 7.79 (d,J4 Hz, 2H), 7.93-7.98 (m, 8H). MS: m/z = 753 (M - 1) (neg. ionization).
Figure imgf000089_0001
Example 51. 3-(2-{5-[3-(2-Carboxyethyl)-5-(4-methanesulfonylphenyl)thiophen-2- yl]thiophen-2-yl}-5-(4-methanesulfonylphenyl)thiophen-3-yl)propanoic acid (9708 054) The intermediate C2 (201.9 mg, 0.35 mmol) and 4-(methylsulfonyl)phenylboronic acid (177.6 mg, 0.89 mmol) were dissolved in methanol (5 ml) and toluene (5 ml). Potassium carbonate (242 mg, 2 mmol) was added and argon (g) was bubbled through the reaction mixture before the PEPPSI-iPr™ (12.2 mg, 0.018 mmol) was added. The reaction mixture was heated at 60 °C for 30 minutes. Silica gel was added and the mixture was concentrated under reduced pressure. The silica-adsorbed compound was purified using column
chromatography, eluent: toluene-ethyl acetate (2: 1).
The formed diester was hydrolyzed in 2 M NaOH (3,5 ml) and 1,4-dioxane (3 ml) at 60 °C for 1,5 h. Ethyl acetate (2 ml) was added and the phases were separated. The desired compound precipitated upon addition of 6 M HCl. The obtained crystals were collected by centrifugation and washed with water two times. Further purification using preparative HPLC was necessary. Yield: 26.5 mg, (4 %); orange solid. 1H NMR (400 MHz, CDC13): δ 2.70 (t, J 7.2 Hz, 4H), 3.04 (t, J 7.2 Hz, 4H), 3.25 (s, 6H), 7.39 (s, 2H), 7.57 (s, 2H), 7.95 (q, J 8.4 Hz, 8H).
Figure imgf000090_0001
Example 52. 2-(2-{5-[3-(Carboxymethyl)-5-[3-(carboxymethyl)phenyl]thiophen-2- yl]thiophen-2-yl}-5-[3-(carboxymethyl)phenyl]thiophen-3-yl)acetic acid (9708 055)
Argon was flushed through a solution of intermediate D (91.7 mg, 0.167 mmol) and 3-(2- methoxy-2-oxoethyl)phenylboronic acid, pinacol ester (115 mg, 0.416 mmol) in a stirred mixture of toluene (3 ml) and MeOH (3 ml). K2C03 (345 mg, 2.50 mmol) and PEPPSI-iPr™ (15.2 mg, 0.0210 mmol) were added and the reaction heated at 60°C for 2 hours. The solvents were removed in vacuo and the crude material was added dioxane (2 ml), water (2 ml) and 5 M NaOH (1 ml). The reaction was stirred at 80°C for 1 hour, then cooled and concentrated to dryness. Some water and 1 M H4HCO3 were added and the mixture was purified by prep- HPLC (5-45% MeCN, in 50 mM H3/NH4HC03 buffer). The combined pure fractions were concentrated to dryness. The compound was dissolved in water (20 ml) and some 1 M HC1 was added. The solid was collected and washed several times with water and dried. Yield: 31 mg (29%) as a yellow solid. 1H NMR (400 MHz, DMSO-i¾): δ 3.65 (s, 4H), 3.79 (s, 4H), 7.24 (d, 2H), 7.33 (s, 2H), 7.39 (t, 2H), 7.50 (s, 2H), 7.55 (d, 2H), 7.58 (s, 2H). HPLC: R, = 1.56 min, 100% (254 nm, 10-40% MeCN in 10 mM buffer, XBridge) and ¾ = 1.13 min, 100% (214 nm, 10-90% MeCN in 10 mM buffer, XBridge). LC-MS: m/z = 650 (M + 18).
Figure imgf000090_0002
Example 53. 5-[4-(2-Carboxyethvn-5-(5-[3-(2-carboxyethvn-5-(5-carboxythiophen-2- yl)thiophen-2-yl]thiophen-2-yl}thiophen-2-yl]thiophene-2-carboxylic acid (9708 056)
The intermediate C2 (199 mg, 0.34 mmol) and (5-methoxycarbonyl-2-thiophenyl) boronic acid (166 mg, 0.89 mmol) were dissolved in methanol (5 ml) and toluene (5 ml). Potassium carbonate (241 mg, 1.74 mmol) was added and argon (g) was bubbled through the reaction mixture before the PEPPSI-iPr™ (12 mg, 0.017 mmol) was added. The reaction mixture was heated for 30 minutes at 60 °C and temperature was then elevated to 80 °C for another 30 minutes. Solution went from bright yellow to brown. The solvents were removed under reduced pressure.
The diester was hydrolyzed using 1,4-dioxane (7 ml) and 2 M NaOH (7 ml) and the mixture was left to stir over night. The temperature was raised to 80 °C and stirred for 30 minutes and the solution was dark and transparent. H20 and EtOAc were added to the reaction and the phases were separated and the acid was precipitated using 6 M HC1. The crystals were collected by centrifugation and washed twice with water and purified by preparative HPLC. The desired compound was obtained as a red solid in 23 % yield (51 mg). 1H NMR (400
MHz, DMSO-i¾): δ 2.67 (t, J7.6 Hz, 4H), 3.00 (t, J7.6 Hz, 4H), 7.36-7.38 (m, 4H), 7.48 (br s, 2H), 7.67 (d, J3.6 Hz, 2H)
Figure imgf000091_0001
Example 54. 4-{5-[3-(Carboxymethyl)-5-{5-[4-(carboxymethyl)-5-[5-(4- carboxyphenyl)thiophen-2-yl]thiophen-2-yl]thiophen-2-yl}thiophen-2-yl]thiophen-2- yllbenzoic acid (9708_057)
Argon was bubbled through a mixture of intermediate A2 (331 mg, 1.21 mmol), 4- methoxycarboxyphenylboronic acid (327 mg, 1.82 mmol) and K2CO3 (503 mg, 3.64 mmol) in toluene (4 ml) and MeOH (4 ml). PEPPSI-iPr™ (16 mg, 0.024 mmol) was added and the mixture heated at 60 °C for lh under argon. EtOAc and water were added. Organic layer was separated and evaporated. The residue was purified by flash chromatography using 0-1%
EtOAc in toluene as eleuent, Yield: 424 mg (93%); yellow oil which solidifies. 1H NMR (400 MHz, CDCI3): δ 3.75 (s, 3H), 3.81 (s, 2H), 3.93 (s, 3H), 7.07 (d, J 5.2 Hz, IH), 7.18 (d, J 3.6 Hz, IH), 7.27 (d, J 5.6 Hz, IH), 7.39 (d, J4.0 Hz, IH), 7.65 (d, J 8.4 Hz, 2H), 8.04 (d, J 8.4 Hz, 2H).
NBS (260 mg, 1.46 mmol) was added portion-wise to a solution of the material from above (546 mg, 1.46 mmol) in chloroform (5 ml) and AcOH (5 ml) at 0 °C and the mixture was stirred at 4 °C overnight. Solvents were evaporated and residue purified by flash
chromatography using 2% EtOAc in toluene as eluent. Yield: 512 mg (78%); pale yellow solid. 1H NMR (400 MHz, CDC13): δ 3.74 (s, 5H), 3.93 (s, 3H), 7.04 (s, IH), 7.14 (d, J 3.6 Hz, IH), 7.37 (d, J 3.6 Hz, IH), 7.65 (d, J 8.4 Hz, 2H), 8.05 (d, J 8.4 Hz, 2H). Argon was bubbled through a mixture of the material from above (204 mg, 0.452 mmol), 2,5- thiophenediboronic acid (38 mg, 0.226 mol) and K2CO3 (78 mg, 0.565 mmol) in toluene (8 ml) and MeOH (8 ml). PEPPSI-iPr™ (6 mg, 0.009 mmol) was added and mixture heated at 60 °C under argon for 45 min. Solvents were evaporated and residue added dioxane (5 ml) and 2 M NaOH (5 ml). The mixture was heated and 80 °C for 1 h. Material was precipitated by addition of 2 M HCl and purified by prep, hplc using 10-30% MeCN in 50 mM buffer as eluent. Pure fractions were combined and some solvents evaporated. Solid material was precipitated by addition of 2 M HCl, isolated by centrifugation, washed three times with water and dried at high vacuum for 3 d. Yield: 24.7 mg (14%); deep red solid. Rt = 2.07 min, 95% at 254 nm (10-40% MeCN in buffer, XBridge) and Rt = 2.09 min. 95% at 400 nm (10-40% MeCN in buffer, XBridge). 1H NMR (400 MHz, OMSO-d6): 6 3.81 (s, 4H), 7.34 (d, J4.0 Hz, 2H), 7.37 (s, 2H), 7.74 (d, 73.8 Hz, 2H), 7.82 (d, 78.5 Hz, 4H), 7.99 (d, 78.5 Hz, 4H). MS: m/z = 767 (M-l) (neg. ionization).
Figure imgf000092_0001
Example 55. 4-{5-[3-(Carboxymethyl)-5-[4-(carboxymethyl)-5-[5-(4- carboxyphenyl)thiophen-2-yl]thiophen-2-yl]thiophen-2-yl]thiophen-2-yl}benzoic acid (9708_058)
Argon was bubbled through a mixture of intermediate A2 (331 mg, 1.21 mmol), 4- methoxycarboxyphenylboronic acid (327 mg, 1.82 mmol) and K2CO3 (503 mg, 3.64 mmol) in toluene (4 ml) and MeOH (4 ml). PEPPSI-iPr™ (16 mg, 0.024 mmol) was added and the mixture heated at 60 °C for lh under argon. EtOAc and water was added. Organic layer was separated and evaporated. The residue was purified by flash chromatography using 0-1 % EtOAc in toluene as eleuent. Yield: 424 mg (93%); yellow oil which solidifies. 1H NMR (400 MHz, CDCI3): 6 3.75 (s, 3H), 3.81 (s, 2H), 3.93 (s, 3H), 7.07 (d, 75.2 Hz, IH), 7.18 (d, 73.6 Hz, IH), 7.27 (d, 75.6 Hz, IH), 7.39 (d, 74.0 Hz, IH), 7.65 (d, 78.4 Hz, 2H), 8.04 (d, 78.4 Hz, 2H).
NBS (260 mg, 1.46 mmol) was added portion-wise to a solution of the material from above (546 mg, 1.46 mmol) in chloroform (5 ml) and AcOH (5 ml) at 0 °C and the mixture was stirred at 4 °C overnight. Solvent was evaporated and residue purified by flash chromatography using 2% EtOAc in toluene as eluent. Yield: 512 mg (78%); pale yellow solid. 1H MR (400 MHz, CDC13): δ 3.74 (s, 5H), 3.93 (s, 3H), 7.04 (s, 1H), 7.14 (d, J 3.6 Hz, 1H), 7.37 (d, J 3.6 Hz, 1H), 7.65 (d, J 8.4 Hz, 2H), 8.05 (d, J 8.4 Hz, 2H).
Argon was bubbled through a mixture of the material from above (174 mg, 0.386 mmol), bispinacolatodiboron (49 mg, 0.193 mg) and K2C03 (266 mg, 1.93 mmol) in DMSO (2 ml). l, l '-Bis(diphenylphosphino)ferrocene dichloropalladium (II) (49 mg, 0.193 mmol) was added and the mixture was heated at 80 °C in a sealed tube overnight. 2 M NaOH (2 ml) was added and heating was continued at 80 °C for 1 h. Solid material was precipitated by addition of 2 M HC1 and isolated by centrifugation. The solid was purified by prep, hplc using 5-25% MeCN in buffer as eluent. Pure fractions were combined and some solvents evaporated. Solid material precipitated upon addition of 2 M HC1 and this was isolated by centrifugation, washed three times with water and dried at high vacuum for 3 d. Yield: 45.5 mg (34%); red solid. Rt = 2.96 min, 96% at 400 nm (5-20% MeCN in buffer, XBridge) and Rt = 1.74 min. 97% at 400 nm (10-30% MeCN in buffer, XBridge). 1H NMR (400 MHz, OMSO-d6): δ 3.82 (s, 4H), 7.35 (s, 2H), 7.37 (s, 2H), 7.75 (d, J4.0 Hz, 2H), 7.82 (d, J 8.5 Hz, 4H), 8.00 (d, J 8.5 Hz, 4H). MS: m/z = 685 (M-l) (neg. ionization).
Figure imgf000093_0001
Example 56. 2-(2-{5-[3-(Carboxymethyl)-5-{5-[2-(carboxymethyl)phenyl]thiophen-2- yl}thiophen-2-yl]thiophen-2-yl}-5-{5-[2-(carboxymethyl)phenyl]thiophen-2-yl}thiophen-3- vDacetic acid (9708_059)
Argon was bubbled through the mixture of intermediate P (144 mg, 0.210 mmol), 2- (methoxycarbonylmethyl)phenylboronic acid pinacol ester (144 mg, 0.524 mmol) in dioxane (3 ml) and 1 M K2C03 (3 ml). PEPPSI-iPr™ (5 mg, 0.007 mmol) was added and the mixture heated at 100 °C for 2.5 h. 2 M NaOH (3 ml) was added and the mixture was heated at 80 °C for 1 h. Mixture was added water and 2 M HC1 (ca 10 ml) the precipitate was filtered and washed with water. Solid material was purified by prep, hplc using 10-40% MeCN in buffer, Pure fractions were combined and some solvents were evaporated. 2 M HC1 was added and the precipitate isolated by centrifugation and washed three times with water. The material was dried under high vacuum for 2 d. Yield: 35.7 mg (22%); red solid. Rt = 2.43 min, 95% at 254 nm (10-40% MeCN in buffer, XBridge) and Rt = 2.43 min, 95% at 400 nm (10-40% MeCN in buffer, XBridge). 1H NMR (400 MHz, OMSO-d6): δ 3.75 (s, 4H), 3.78 (s, 4H), 7.12 (d, J 3.8 Hz, 2H), 7.31 (s, 2H), 7.33 (s, 2H), 7.34-7.47 (m, 10H). MS: m/z = 795 (M-l) (neg.
ionization).
Figure imgf000094_0001
Example 57. 2-(2-{5-[3-(Carboxymethyl)-5-{5-[3-(carboxymethyl)phenyl]thiophen-2- yl}thiophen-2-yl]thiophen-2-yl}-5-{5-[3-(carboxym
vDacetic acid (9708 060)
Argon was bubbled through a mixture of intermediate O (118 mg, 0.165 mmol), 3-(2- methoxy-2-oxoethyl)phenylboronic acid, pinacol ester (114 mg, 0.413 mmol) and K2C03 (114 mg, 0.826 mmol) in toluene (8 ml) and MeOH (8 ml). PEPPSI-iPr™ (5 mg, 0.0074 mmol) was added and the mixture heated at 70 °C in a sealed tube for 45 min. Solvents were evaporated and residue added dioxane (5 ml) and 2 M NaOH (5 ml). The mixture was heated at 80 °C for 1 h.2 M HCl was added and precipitated material isolated by centrifugation, and purified by prep. hplc. Pure fractions were combined and some solvents evaporated. 2 M HCl was added and precipitate isolated by centrifugation and washed with water (three times). The material was dried at high vacuum for 2 d. Yield: 37.4 mg (26%); dark red almost black solid. Rt = 2.40 min, 97% at 400 nm (10-40% MeCN in buffer, XBridge) and Rt = 2.40 min. 96% at 254 nm (10-40% MeCN in buffer, XBridge). 1H NMR (400 MHz, OMSO-d6): δ 3.65 (s, 4H),
3.79 (s, 4H), 7.24 (d, J 7.5 Hz, 2H), 7.33 (s, 2H),7.35 (s, 2H), 7.37-7.41 (m, 4H), 7.52 (d, J 4.0 Hz, 2H), 7.58-7.60 (m, 4H). MS: m/z = 795 (M-l) (neg. ionization).
Figure imgf000094_0002
Example 58. 2-(2-(5-r3-(Carboxymethvn-5-(5-r4-(carboxymethvn-5-(5-r3- (carboxymethyl)-5-chlorothiophen-2-yl]thiophen-2-yl}thiophen-2-yl]thiophen-2-yl}thiophen- 2-yllthiophen-2-yl|-5-chlorothiophen-3-yl)acetic acid (9708 061)
NCS (73 mg, 0.547 mmol) was added to a solution of intermediate E (258 mg, 0.547 mmol) in AcOH (5 ml). The mixture was stirred at rt for 3 days. Solvent was evaporated and residue purified by flash chromatography using 2% EtOAc in toluene. Yield: 205.8 mg (74%); yellow oil.1H MR (400 MHz, CDC13): δ 3.69 (s, 2H), 3.71 (s, 2H), 3.74 (s, 6H), 6.90 (s, 1H), 7.03 (s, 1H), 7.09 (s, 2H).
Argon was bubbled through a mixture of the material from above (205 mg, 0.407 mmol), 2,5- thiophenediboronic acid and K2C03 (224 mg, 1.63 mmol) in toluene (4 ml) and MeOH (4 ml). PEPPSI-iPr™ was added and the mixture heated at 60 °C for 30 min under argon.
Solvents were evaporated and dioxane (5 ml) and 2 M NaOH (5 ml) was added. The mixture was heated at 80 °C for 2 h. Solid material was precipitated by addition of 2 M HC1 and separated by centrifugation. The solid material was purified by prep. hplc. Pure fractions were combined, some solvents evaporated and material precipitated by addition of 2 M HC1. The solid was isolated by centrifugation, washed three times with water and dired at high vacuum for 2 d. Yield: 19.0 mg (10%); black solid. Rt = 2.98 min, 96% at 254 nm (10-40% MeCN in buffer, XBridge) and Rt = 2.98 min. 96% at 400 nm (10-40% MeCN in buffer, XBridge). 1H NMR (400 MHz, OMSO-d6): δ 3.71 (s, 4H), 3.76 (s, 4H), 7.15 (s, 2H), 7.27 (d, J 4 Hz, 2H), 7.31 (d, J4 Hz, 2H), 7.35 (s, 4H). MS: m/z = 877 (M-l) (neg. ionization).
Figure imgf000095_0001
Example 59. 2-(2-{5-[2-(Carboxymethyl)-4-chlorophenyllthiophen-2-yl|-5- chlorophenyPacetic acid (9708 062)
Acetyl chloride (1.43 ml, 20.0 mmol) was added to a stirred solution of 2-bromo-5- chlorophenyl acetic acid (2.00 g, 8.00 mmol) in MeOH (50 ml) at 0°C. The reaction was then stirred at r.t. overnight and the solvent was removed in vacuo. The crude material was dissolved in Et02 (-100 ml) and washed with diluted Na2C03. The organic phase was dried over MgS04 and removed in vacuo. Yield: 1.93 g (91%). LC-MS: m/z = 263, 265 (M + 17, 19)
The material from above (866 mg, 3.29 mmol) in dry 1,4 dioxane (15 ml) was added to bis(pinacolato)diboron (918 mg, 3.62 mmol) and AcOK (1.19 g, 12.2 mmol). The stirred mixture was flushed with argon and l,l'-bis(diphenylphosphino)ferrocene- palladium(II)dichloride (120 mg, 0.164 mmol) was added. The reaction was heated in a sealed tube at 100°C for 3 hours. Cooled and diluted with Et20. The organic phase was washed with water and brine, dried over MgS04 and removed in vacuo. The crude material was dissolved in toluene and purified by flash chromatography (5% EtOAc in toluene, 200 ml silica). Yield: 511 mg (50%) as a colourless oil. LC-MS: m/z = 311 (M + 1).
Argon was flushed through a stirred solution of the material from above (140 mg, 0.451 mmol) and 2,5-dibromothiophene (32.7 mg, 0.135 mmol) in a mixture of toluene (3 ml) and MeOH (3 ml). K2C03 (374 mg, 2.71 mmol) and PEPPSI-iPr™ (16.5 mg, 0.0230 mmol) were added and the reaction heated at 60°C for 3 hours. The solvents were removed in vacuo. The crude material was dissolved in a mixture of water, MeOH and some 1 M H4HC03 and purified by prep-HPLC (5-40% MeCN, in 50 mM H3/NH4HC03 buffer). The combined pure fractions were concentrated to dryness. The compound was dissolved in water and some 1 M HC1 was added. The mixture was extracted with EtOAc. The organic phase was dried over MgS04 and concentrated in vacuo. Yield: 15 mg (8%) as a white solid. 1H NMR (400 MHz, DMSO-i¾): δ 3.77 (s, 4H), 7.15 (s, 2H), 7.42 (d, J 2.0 Hz, 0.4H), 7.44 (d, J 2.0 Hz, 1.6H), 7.45 (s, 1.6H), 7.47 (s, 0.4 H), 7.51 (d, J 2.3 Hz, 2 H). HPLC: Rt = 2.59 min, 95% (254 nm, 10-40% MeCN in 10 mM buffer, XBridge) and Rt = 1.65 min, 97% (214 nm, 10-90% MeCN in 10 mM buffer, XBridge). LC-MS: m/z = 438 (M + 18).
Figure imgf000096_0001
Example 60. 2-(5-{5-[5-(2-Carboxy-4-methylphenyl)-3-(carboxymethyl)thiophen-2- yl]thiophen-2-yl}-4-(carboxymethyl)thiophen-2-yl)-5-methylbenzoic acid (9708 063)
Acetyl chloride (1.66 ml, 23.3 mmol) was added to a stirred solution of 2-bromo-5- methylbenzoic acid (2.00 g, 9.30 mmol) in MeOH (50 ml) at 0°C. The reaction was then stirred at r.t. over night. More acetyl chloride (1 ml, 14.0 mmol) was added and the reaction was stirred for 24 hours. The solvent was removed in vacuo and the crude material was dissolved in Et02 (-100 ml) and washed with 0.5 M NaOH. The organic phase was dried over MgS04 and removed in vacuo. Yield: 1.97 g (92%). 1H NMR (400 MHz, DMSO-i¾): δ 2.33 (s, 3H), 3.94 (s, 3H), 7.13 (d, 1H), 7.52 (d, 1H), 7.60 (s, 1H)
The ester from above (800 mg, 3.49 mmol) in dry 1,4 dioxane (15 ml) was added to bis(pinacolato)diboron (976 mg, 3.84 mmol) and AcOK (1.27 g, 12.9 mmol). The stirred mixture was flushed with argon and 1, l'-bis(diphenylphosphino)ferrocene- palladium(II)dichloride (25.6 mg, 0.035 mmol) was added. The reaction was heated in a sealed flask at 100°C for 3 hours then cooled and diluted with Et20. The organic phase was washed with water and brine, dried over MgS04 and removed in vacuo. The crude material was diluted with iso-hexane and purified by flash chromatography (20% EtOAc in iso- hexane, 200 ml silica). Yield: 697 mg (72%) as a colourless oil. 1H NMR (400 MHz, DMSO-- d6): δ 1.42 (s, 12H), 2.38 (s, 3H), 3.91 (s, 3H), 7.31-7.35 (m, 1H), 7.38-7.43 (m, 1H), 7.76 (s, 1H).
Argon was flushed through a stirred solution of intermediate D (120 mg, 0.217 mmol) and the pinacolester from above (150 mg, 0.543 mmol) in a mixture of toluene (3 ml) and MeOH (3 ml). K2C03 (150 mg, 0.543 mmol) and PEPPSI-iPr™ (19.8 mg, 0.027 mmol) were added and the reaction heated at 60°C for 2 hours in a sealed flask. The solvents were removed in vacuo and the crude material was added dioxane (2 ml), water (2 ml) and 5 M NaOH (1 ml). The reaction was stirred at 80° C for 30 min. The mixture was cooled and concentrated. Some water and 1 M H4HCO3 (until pH~10) were added and the mixture was purified by prep- HPLC (5-40% MeCN, in 50 mM H3/NH4HCO3 buffer). The combined pure fractions were concentrated to dryness. The compound was dissolved in water (20 ml) and some 2 M HC1 was added. The solid was collected and washed several times with water and dried. Yield: 25 mg (18%); yellow solid. 1H NMR (400 MHz, DMSO-i ): δ 2.37 (s, 6 H), 3.74 (s, 4 H), 7.10 (s, 2 H), 7.29 (s, 2 H), 7.36 - 7.44 (m, 4 H), 7.47 - 7.49 (m, 2 H). HPLC: Rt =1.82 min, 100% (254 nm, 10-40% MeCN in 10 mM buffer, XBridge) and Rt =1.35 min, 97% (400 nm, 10- 90% MeCN in 10 mM buffer, XBridge). LC-MS: m/z = 650 (M + 18).
Figure imgf000097_0001
Example 61. 2-[4-(2-Carboxyethyl)-5-{5-[3-(2-carboxyethyl)-5-(2-carboxyphenyl)thiophen- 2-yllthiophen-2-yl|thiophen-2-yllbenzoic acid (9708 064)
Intermediate B2 (198.5 mg, 0.34 mmol) and (2-methoxycarbonylbenzene)boronic acid (149.9 mg, 0.83 mmol) were dissolved in methanol (5 ml) and toluene (5 ml). Potassium carbonate (239.4 mg, 1.73 mmol) was added and argon was bubbled through the solution before PEPPSI-iPr™ was added. The reaction was stirred at 60 °C for 2 h. The solvents were removed under reduced pressure. Column chromatography using toluene/ethyl acetate (3 : 1→ 2: 1) as eluent was performed. 132 mg of the desired material was afforded (not completely pure) and 45 mg of the starting material was retrieved.
The coupled product was dissolved in 1,4-dioxane (3 ml) and NaOH 2 M (3 ml) and heated at 60 °C for 30 minutes. The acid was precipitated by adding HC1 6 M (0.5 ml) and solid material purified by preparative HPLC (Xbridge 5%-50% MeCN) was necessary. The compound was precipitated using 6 M HC1 and collected 38.2 mg (18 %) pure compound. 1H NMR (400 MHz, OMSO-d6): δ 2.62 (t, J7.8 Hz, 4H), 3.00 (t, J 7.6 Hz, 4H), 7.13 (s, 2H), 7.29 (s, 2H), 7.42-7.49 (m, 2H), 7.50-7.59 (m, 4H), 7.64 (d, J7.5 Hz, 2H).
Figure imgf000098_0001
Example 62. 3-(2-(5-[3-(2-Carboxyethvn-5-(5-[4-(2-carboxyethvn-5-(5-[3-(2- carboxyethyl)thiophen-2-yl]thiophen-2-yl}thiophen-2-yl]thiophen-2-yl}thiophen-2- yl]thiophen-2-yl}thiophen-3-yl)propanoic acid (9708 065).
The intermediate B2 (373.6 mg, 0.89 mmol) was dissolved chloroform (4 ml) and acetic acid (4 ml). The solution was cooled to 0 °C and N-iodosuccinimide (199.8 mg, 0.89 mmol) was added. The reaction was slowly warmed to room temperature over night. The reaction mixture was poured onto Na2S203 and extracted with DCM (x2). The organic phases were combined and washed with water and brine and dried with MgS04. Concentration under reduced pressure gave a 519 mg red residue. The residue was purified by column chromatography, 2% ethyl acetate in toluene: 191.5 mg (38%) of a yellow oil. 1H NMR (400 MHz, CDC13): δ 2.64 (dt, J 1 1.5, 7.8 Hz, 4H), 3.10 (dt, J15.9, 7.8 Hz, 4H), 3.69 (d, J 1.5 Hz, 6H), 6.96 (d, J 5.3 Hz, 1H), 7.02-7.06 (m, 1H), 7.07-7.12 (m, 2H), 7.22 (d, J 5.3 Hz, 1H).
The monoido-product (138.9 mg, 0.25 mmol) from above, 2,5-thiophenediboronic acid (21.6 mg, 0.13 mmol) and K2C03 (52.4 mg, 0.38 mmol) was suspended in toluene (3 ml) and methanol (3 ml). Argon was bubbled through the mixture before PEPPSI-iPr™ (4.9 mg, 0.007 mmol) was added. The reaction was stirred at 70 °C for 45 minutes; temperature was lowered to 55 °C and stirred for another hour. More K2C03 (51.5 mg, 0.37 mmol) was added and reaction continued for 45 minutes. The solvents were removed under reduced pressure. The red solid was dissolved in 1,4-dioxane (3.5 ml) and 2 M NaOH (3 ml) and heated at 60 °C for 50 minutes. Precipitated with 6 M HCl and collected the solid by centrifugation.
Purification: prep HPLC (5-40% MeCN in H4COO3)→ 80% pure; Prep HPLC (15-45% MeCN in H4COO3)→ 99% pure. The pure fractions were combined and the product was precipitated using 6 M HCl and washed twice with H20. Compound dried on freeze-drier for
3 d. The dark red compound was afforded in 21 mg (19 %) yield.
1H MR (400 MHz, OMSO-d6): δ 2.55-2.69 (m, 8H), 2.99 (t, J 7.6 Hz, 8H), 7.09 (d, J 5.3 Hz, 2H), 7.26 (d, J3.8 Hz, 2H), 7.30 (d, J 3.8 Hz, 2H), 7.34 (d, J 7.8 Hz, 4H), 7.52 (d, J 5.0 Hz, 2H).
Figure imgf000099_0001
Example 63. 2-(5-r5-(5-(5-r5-(2-Carboxy-4-methylphenvnthiophen-2-yl1-3- (carboxymethyl)thiophen-2-yl}thiophen-2-yl)-4-(carboxymethyl)thiophen-2-yl]thiophen-2- yl|-5-methylbenzoic acid (9708 066)
Acetyl chloride (1.66 ml, 23.3 mmol) was added to a stirred solution of 2-bromo-5- methylbenzoic acid (2.00 g, 9.30 mmol) in MeOH (50 ml) at 0°C. The reaction was then stirred at r.t. over night. More acetyl chloride (1 ml, 14.0 mmol) was added and the reaction was stirred for 24 hours. The solvent was removed in vacuo and the crude material was dissolved in Et02 (-100 ml) and washed with 0.5 M NaOH. The organic phase was dried over MgSC-4 and removed in vacuo. Yield: 1.97 g (92%). 1H NMR (400 MHz, DMSO-i¾): δ 2.33 (s, 3H), 3.94 (s, 3H), 7.13 (d, 1H), 7.52 (d, 1H), 7.60 (s, 1H).
The ester from above (800 mg, 3.49 mmol) in dry 1,4 dioxane (15 ml) was added to bis(pinacolato)diboron (976 mg, 3.84 mmol) and AcOK (1.27 g, 12.9 mmol). The stirred mixture was flushed with argon and l,l'-bis(diphenylphosphino)ferrocene- palladium(II)dichloride (25.6 mg, 0.035 mmol) was added. The reaction was heated in a sealed flask at 100°C for 3 hours then cooled and diluted with Et20. The organic phase was washed with water and brine, dried over MgS04 and concentrated in vacuo. The crude material was diluted with iso-hexane and purified by flash chromatography (20% EtOAc in iso-hexane, 200 ml silica). Yield: 697 mg (72%) as a colourless oil. 1H NMR (400 MHz, DMSO-i¾): δ 1.42 (s, 12H), 2.38 (s, 3H), 3.91 (s, 3H), 7.31-7.35 (m, 1H), 7.38-7.43 (m, 1H), 7.76 (s, 1H).
Argon was flushed through a solution of intermediate O (155 mg, 0.217 mmol) and the boronic ester from above (150 mg, 0.543 mmol) in a mixture of toluene (5 ml) and MeOH (5 ml). K2C03 (450 mg, 3.26 mmol) and PEPPSI-iPr™ (19.8 mg, 0.027 mmol) were added and the reaction heated at 60°C for 3 hours. The solvents were removed in vacuo and the crude material was added dioxane (1 ml) and 5 M NaOH (1 ml). The reaction was stirred at 80°C for 30 min. The mixture was cooled and water and 1 M H4HCO3 were added. The mixture was purified by prep-HPLC (20-50% MeCN, in 50 mM H3/NH4HCO3 buffer). The combined pure fractions were concentrated to dryness. The compound was dissolved in water (20 ml) and some 2 M HC1 was added. The solid was collected and washed several times with water and dried. Yield: 25 mg (18%) as a orange solid. 1H NMR (400 MHz, DMSO-i¾): δ 2.38 (s, 6H), 3.78 (s, 4H), 7.08 (d, J 3.8 Hz, 2H), 7.31 (s, 2H), 7.32 (s, 2H), 7.34 (d, J 3.5 Hz, 2H), 7.36 - 7.41 (m, 2H), 7.43 - 7.47 (m, 2H), 7.47 - 7.50 (m, 2H). HPLC: Rt = 2.42 min, 97% (254 nm, 10-40% MeCN in 10 mM buffer, XBridge) and Rt = 1.55 min, 100% (400 nm, 10- 90% MeCN in 10 mM buffer, XBridge). LC-MS: m/z = 797 (M + 1).
Figure imgf000100_0001
Example 64. (2E)-3-(5-(5-[5-(5-(5-[(lE)-2-Carboxyeth-l-en-l-yllthiophen-2-yll-3- carboxymethyl)thiophen-2-yl)thiophen-2-yl]-4-(carboxymethyl)thiophen-2- yl}thiophen-2-yl)prop-2-enoic acid (9708 067)
Argon was bubbled though a mixture of intermediate D (480 mg, 0.873 mmol), 5- formylthiophene-2-boronic acid (409 mg, 2.62 mmol) and K2CO3 (724 mg, 5.24 mmol) in MeOH (10 ml) and toluene (10 ml). PEPPSI-iPr™ (30 mg, 0.0437 mmol) was added and the mixture heated under argon at 80 °C for 30 min. Chloroform was added and mixture filtered. Silica was added to the filtrate and solvents evaporated. The silica was applied on a flash column which was eluted with 1% MeOH in DCM. Yield: 430 mg (80%). 1 H NMR (400 MHz, CDCI3): δ 3.77 (s, 6H), 3.81 (s, 4H), 7.24 (s, 2H), 7.27 (d, J4.0 Hz, 2H), 7.32 (s, 2H), 7.68 (d, J4.0 Hz, 2H), 9.88 (s, 2H). The material from above (103 mg, 0.168 mmol), methyl triphenylphosphoranylidene)acetate (115 mg, 0.345 mmol) and toluene (15 ml) were stirred at 90°C for 3 hours. More methyl (triphenylphosphoranylidene)acetate (130 mg, 0.389 mmol) was added and the reaction stirred for 3 hours. The solvent was removed in vacuo and the crude material was dissolved in dioxane (2 ml) and water (2 ml). 5 M NaOH (2 ml) was added the reaction was stirred at 70 °C for 30 min. The solvents were removed in vacuo and the crude material was dissolved in water (~8 ml). Solid H4HCO3 was added. The mixture was stirred, filtered and purified by prep-HPLC (10-40% MeCN, in 50 mM H3/NH4HCO3 buffer). The combined pure fractions were concentrated to dryness. The compound was dissolved in water (20 ml) and some 2 M HC1 was added. The solid was collected and washed several times with water and dried. Yield: 85 mg, (76%) as a orange solid. 1H NMR (400 MHz, DMSO-i¾): δ 3.78 (s, 4H), 6.17 (d, J 15.8 Hz, 2H), 7.34 (s, 2H), 7.38 - 7.42 (m, 4H), 7.51 (d, J 3.8 Hz, 2H), 7.73 (d, J 15.6 Hz, 2H). HPLC: Rt = 1.50 min, 95% (254 nm, 10-40% MeCN in 10 mM buffer, XBridge) and Rt = 1.49 min, 95% (400 nm, 10-40% MeCN in 10 mM buffer, XBridge). LC-MS: m/z = 669 (M + 1).
Figure imgf000101_0001
Example 65. 3-{5-[3-(Carboxymethyl)-5-{5-[4-(carboxymethyl)-5-[5-(3- carboxyphenyl)thiophen-2-yl]thiophen-2-yl]thiophen-2-yl}thiophen-2-yl]thiophen-2- yllbenzoic acid (9708 068)
Argon was bubbled through a mixture of intermediate A2 (607 mg, 2.23 mmol) and 3- methoxycarboxyphenylboronic acid (480 mg, 2.67 mmol) and K2CO3 (738 mg, 5.34 mmol) in toluene (8 ml) and MeOH (8 ml). PEPPSI-iPr™ (30 mg, 0.045 mmol) was added and mixture was heated in a sealed tube for 45 min. Chloroform was added and mixture filtered and concentrated. The residue was purified by flash chromatography using 2% EtOAc in toluene as eluent. Yield: 749 mg (90%); pale yellow oil. 1H NMR (400 MHz, CDC13) δ 3.74 (s, 3H), 3.82 (s, 2H), 3.96 (s, 3H), 7.06 (d, J 5.2 Hz, IH), 7.17 (d, J 4.0 Hz, IH), 7.26 (d, J 5.2 Hz, IH), 7.35 (d, J4.0 Hz, IH), 7.46 (t, J 8 Hz, IH), 7.78 (d, J 8 Hz, IH), 7.95 (d, J 8 Hz, IH), 8.27 (s, IH).
NBS (358 mg, 2.01 mmol) was added portion-wise to the material from above (749 mg, 2.01 mmol) in CHCI3 (8 ml) and AcOH (8 ml) and the mixture was stirred at ambient temperature overnight. Solvents were evaporated and residue purified by flash chromatography using 2% EtOAc in toluene.Yield: 702 mg, pure on tic, pale yellow solid. 1H NMR (400 MHz, CDC13): δ 3.74 (s, 5H), 3.96 (s, 3H), 7.04 (s, 1H), 7.12 (d, J4 Hz, 1H), 7.34 (d, J4 Hz, 1H), 7.47 (t, J 8 Hz, 1H), 7.77 (d, J 8 Hz, 1H), 7.96 (d, J 8 Hz, 1H), 8.23 (s, 1H).
Argon was bubbled through a mixture of material from above (204 mg, 0.452 mmol), 2,5- thiophenediboronic acid (35 mg, 0.204 mmol) and K2C03 (140 mg, 1.02 mmol) in toluene (8 ml) and MeOH (8 ml). PEPPSI-iPr™ was added and mixture heated at 60 °C under argon for 2h. Solvents were evaporated and residue added dioxane (5 m,l) and 2 M NaOH (5 ml) the mixture was heated at 80 °C for 1 h. Material was purified by prep. hplc. Pure fractions were combined and some solvents evaporated. 2 M HC1 was added and solid material isolated by centrifugation, washed with water and freeze-dried for 2 d. Yield: 36.8 mg (24%); deep red solid. Rt = 2.32 min, 91% at 400 nm (10-40% MeCN in buffer, XBridge) and Rt = 2.32 min. 91% at 254 nm (10-40% MeCN in buffer, XBridge). 1H NMR (400 MHz, OMSO-d6): 6 3.81 (s, 4H), 7.33 /d, J3.8 Hz, 2H), 7.36 (s, 4H), 7.59 (t, J 7.7 Hz, 2H), 7.69 (d, J 3.8 Hz, 2H), 7.91 (d, J7.8 Hz, 2H), 7.97 (d, J 7.8 Hz, 2H), 8.17 (s, 2H). MS: m/z = 767 (M-l) (neg. ionization).
Figure imgf000102_0001
Example 66. 3-{5-[3-(Carboxymethyl)-5-[4-(carboxymethyl)-5-[5-(3- carboxyphenyl)thiophen-2-yl]thiophen-2-yl]thiophen-2-yl]thiophen-2-yl}benzoic acid (9708_069)
Argon was bubbled through a mixture of intermediate A2 (607 mg, 2.23 mmol) and 3- methoxycarboxyphenylboronic acid (480 mg, 2.67 mmol) and K2C03 (738 mg, 5.34 mmol) in toluene (8 ml) and MeOH (8 ml). PEPPSI-iPr™ (30 mg, 0.045 mmol) was added and mixture was heated in a sealed tube for 45 min. Chloroform was added and mixture filtered and concentrated. The residue was purified by flash chromatography using 2% EtOAc in toluene as eluent. Yield: 749 mg (90%); pale yellow oil. 1H NMR (400 MHz, CDC13): δ 3.74 (s, 3H), 3.82 (s, 2H), 3.96 (s, 3H), 7.06 (d, J 5.2 Hz, 1H), 7.17 (d, J4.0 Hz, 1H), 7.26 (d, J 5.2 Hz, 1H), 7.35 (d, J4.0 Hz, 1H), 7.46 (t, J 8 Hz, 1H), 7.78 (d, J 8 Hz, 1H), 7.95 (d, J 8 Hz, 1H), 8.27 (s, 1H). BS (358 mg, 2.01 mmol) was added portion-wise to the material from above (749 mg, 2.01 mmol) in CDC13 (8 ml) and AcOH (8 ml) and the mixture was stirred at ambient temperature overnight. Solvents were evaporated and residue purified by flash chromatography using 2% EtOAc in toluene.Yield: 702 mg, pure on tic, pale yellow solid. 1H NMR (400 MHz, CDC13): δ 3.74 (s, 5H), 3.96 (s, 3H), 7.04 (s, 1H), 7.12 (d, J4 Hz, 1H), 7.34 (d, J4 Hz, 1H), 7.47 (t, J 8 Hz, 1H), 7.77 (d, J 8 Hz, 1H), 7.96 (d, J 8 Hz, 1H), 8.23 (s, 1H).
Argon was flushed though the mixture of the material from above (174 mg, 0.386 mmol), bispinacolatodiboron (49 mg, 0.193 mmol) and K2C03 in DMSO (3 ml). 1, 1 '- bis(diphenylphosphino)ferrocene dichloropalladium (II) (14 mg, 0.019 mmol) was added and the mixture heated at 80 °C overnight in a sealed tube. 2 M NaOH (2 ml) was added and the mixture was stirred at 80 °C for 1 h. 2 M HCl and water was added. Solid material was isolated by centrifugation and purified by prep, hplc using 10-30% MeCN in buffer.
Pure fractions were collected and some solvents evaporated. 2 M HCl was added and solid material was isolated by centrifugation. The solid material was washed with water (three times) and freeze-dried for 3 d. Yield: 15.9 mg (6%); deep red solid. Rt = 1.78 min, 95% at 254 nm (10-40% MeCN in buffer, XBridge) and Rt = 2.14 min. 95% at 400 nm (10-30% MeCN in buffer, XBridge). 1H NMR (400 MHz, OMSO-d6): 6 3.81 (s, 4H), 7.33 (d, J4 Hz, 2H), 7.34 (s, 2H), 7.59 (t, J 8 Hz, 2H), 7.69 (d, J 3.2 Hz, 2H), 7.90 (d, J 8 Hz, 2H), 7.97 (d, J 8 Hz, 2H), 8.17 (s, 2H). MS: m/z = 685 (M-l) (neg. ionization)
Figure imgf000103_0001
Example 67. 2-(2-{5-[3-(Carboxymethyl)-5-(6-chloropyridin-3-yl)thiophen-2-yl]thiophen-2- yl}-5-(6-chloropyridin-3-yl)thiophen-3-yl)acetic acid (9708 070)
Argon was bubbled through a mixture of intermediate G2 (134 mg, 0.208 mmol), 2- chloropyridine-5-boronic acid (72 mg, 0.458 mmol) and K2C03 (144 mg, 1.04 mmol) in toluene (3 ml) and MeOH (3 ml). PEPPSI-iPr™ (1.4 mg, 0.002 mmol) was added and the mixture heated in a sealed tube at 80 °C for 45 min. Chloroform was added and mixture filtered and evaporated. The residue was purified by flash chromatography using 10-20% EtOAc in toluene. Yield: 91.2 mg (71%); bright yellow solid. 1H NMR (400 MHz, CDC13): δ 3.77 (s, 6H), 3.83 (s, 4H), 7.23 (s, 2H, 7.32 (s, 2H), 7.36 (d, J 8.4 Hz, 2H), 7.82 (dd, J 8 Hz, 2 Hz, 2H), 8.63 (d, J2 Hz, 2H)..
The material from above (91.2 mg, 0.148 mmol) was dissolved in dioxane (3 ml) and 2 M NaOH (3 ml) was added. The mixture was heated at 80 °C for 1 h. EtOAc was added and aqueous layer separated. The organic layer was filtered, colourless solution, yellow solid on filter which was suspended in 2 M HCl and solid material washed with water (three times). The material was dried at high vacuum for 3 d. Yield: 72.6 mg (83%); yellow solid. Rt = 2.18 min, 98% at 254 nm (20-50% MeCN in buffer, XBridge) and Rt = 2.20 min. 99% at 400 nm (20-50% MeCN in buffer, XBridge). 1H NMR (400 MHz, OMSO-d6): δ 3.72 (s, 4H), 7.36 (s, 2H), 7.57 (d, J 8 Hz, 2H), 7.65 (s, 2H), 8.11 (d, J 9 Hz, 2H), 8.72 (s, 2H). MS: m/z = 589 (M+l)
Figure imgf000104_0001
Example 68. 2-(2-{5-[3-(Carboxymethyl)-5-(4-hydroxy-3-methoxyphenyl)thiophen-2- yl]thiophen-2-yl}-5-(4-hydroxy-3-methoxyphenyl)thiophen-3-yl)acetic acid (9708 071) Argon was flushed through a solution of intermediate D (130 mg, 0.236 mmol) and 4- hydroxy-3-methoxyphenylboronic acid (120 mg, 0.714 mmol) in a mixture of toluene (3 ml) and MeOH (3 ml). PEPPSI-iPr™ (26.1 mg, 0.036 mmol) and KF (125 mg, 2.14 mmol) were added and the reaction heated at 55 °C for 120 min. The solvents were removed in vacuo and the crude was added water (3 ml), 5 M NaOH (1 ml) and dioxane (2 ml). The reaction was heated to 70°C for 30 min then cooled, filtered and washed with water. 1 M NH4HCO3 was added and the product was collected as a solid by filtration. Water and 2 M HCl were added. The solid was once more collected by filtration and washed with water. Yield: 110 mg (77%) as a off white solid. 1H NMR (400 MHz, DMSO-i¾): δ 3.74 (s, 4H), 3.85 (s, 6H), 6.82 (d, J 8.3 Hz, 2H), 7.05 (dd, J8.28, 2.01 Hz, 2H), 7.17 (d, J2.0 Hz, 2H), 7.26 (s, 2H), 7.34 (s, 2H), 9.32 (s, 2H), 12.54 (br. s., 2H). HPLC: Rt = 2.61 min, 99% (254 nm, 10-40% MeCN in 10 mM buffer, XBridge) and Rt = 2.60 min, 98% (400 nm, 10-40% MeCN in 10 mM buffer, XBridge). LC-MS: m/z = 626 (M + 18).
Figure imgf000105_0001
Example 69. 5-(5-{5-[5-(5-Carboxythiophen-2-yl)-3-(2-methoxy-2-oxoethyl)thiophen-2- yl]thiophen-2-yl}-4-(2-methoxy-2-oxoethyl)thiophen-2-yl)thiophene-2-carboxylic acid (9708_072)
Argon was bubbled through a mixture of intermediate D (208 mg, 0.378 mmol) and 5- carboxythiophene-2-boronic acid (162 mg, 0.945 mmol) and KF (109 mg, 1.89 mmol) in toluene (5 ml) and MeOH (5 ml). PEPPSI-iPr™ (5.2 mg, 0.008 mmol) and the mixture heated at 60 °C for 1 h. Solvents were removed and residue dissolved in 1 M
NHtHCCVwater/dioxane and purified by prep, hplc using 15-45% MeCN in 50 mM buffer as eluent. Pure fractions were combined and some solvent evaporated. 1 M HCl was added and the precipitate isolated by centrifugation and washed twice with water. The material was freeze-dried for 3 d. Yield: 51.3 mg (21%); dark red solid. Rt = 2.70 min, 98% at 400 nm (10- 40% MeCN in buffer, XBridge) and Rt = 2.70 min. 98% at 254 nm (10-40% MeCN in buffer, XBridge). 1H NMR (400 MHz, DMSO-i¾): δ 3.68 (s, 6H), 3.90 (s, 4H), 7.34 (s, 2H), 7.41 (d, J4 Hz, 2H), 7.48 (s, 2H), 7.68 (d, J4 Hz, 2H). MS: m/z = 662 (M+NH4).
Figure imgf000105_0002
Example 70. 2-(2-{5-[3-(Carboxymethyl)-5-[5-(methoxycarbonyl)thiophen-2-yl]thiophen-2- yl]thiophen-2-yl}-5-[5-(methoxycarbonyl)thiophen-2-yl]thiophen-3-yl)acetic acid (9708 073) Argon was bubbled through a mixture of intermediate Q (90 mg, 0.172 mmol), 5- methoxycarbonyl)thiophen-2-ylboronic acid (80 mg, 0.431 mmol) and KF (50 mg, 0.862 mmol) in toluene (5 ml) and MeOH (10 ml). PEPPSI-iPr™ was added and the mixture heated at 60 °C for 30 min. Solvents were evaporated and the residue purified by prep. hplc. Pure fractions were combined, some solvents evaporated and 1 M HCl added. The precipitate was filtered and washed several times with water. The material was dried at high vacuum for 1 d. Yield: 6.2 mg (6%); red solid. Rt = 2.57 min, 99% at 400 nm (20-50% MeCN in buffer, XBridge) and Rt = 2.57 min. 98% at 254 nm (20-50% MeCN in buffer, XBridge). 1H NMR (400 MHz, DMSO-i¾j: δ 3.78 (s, 4H), 3.84 (s, 6H), 7.36 (s, 2H), 7.44 (d, J4 Hz, 2H), 7.51 (s, 2H), 7.77 (d, J4 Hz, 2H). MS: m/z = 643 (M-l).
Figure imgf000106_0001
Example 71. 4- { 2-[5 -(4-Methanesulfonylphenyl)-2- { 5 - [5 -(4-methanesulfonylphenyl)-3 - [2- (morpholin-4-yl)ethyl]thiophen-2-yl]m^
(9708_074)
Argon was bubbled through a stirred mixture of intermediate J (660 mg, 2.31 mmol), 2,5- thiopenediboronic acid (240 mg, 1.40 mmol), KF (530 mg, 9.12 mmol), MeOH (7 ml) and toluene (7 ml) for 10 min. PEPPSI-iPr™ (40 mg, 0.055 mmol) was added and the reaction was heated at 55°C in a sealed flask for 90 min. The solvents were removed in vacuo and DCM (~7 ml) was added. The mixture was washed with water and the organic phase was purified by flash chromatography (12.5% EtOAc in toluene, 100 ml silica). Yield: 390 mg (68%) as a yellow solid. 1H NMR (400 MHz, CDC13): δ 2.92 (s, 6H), 3.27 (t, J 6.8 Hz, 4H), 4.45 (t, J6.9 Hz, 4H), 7.02 (d, J 5.3 Hz, 2H), 7.12 (s, 2H), 7.28 (d, J 5.3 Hz, 2H).
Br2 (0.082 ml, 1.58 mmol) in DCM (1 ml) was added to a stirred solution of the material from above (390 mg, 0.792 mmol) in DCM (10 ml) at 0°C. The reaction was stirred at this temperature for 15 min. DCM (-10 ml) was added and the mixture was washed with saturated Na2S203 and diluted NaHC03. The organic phase was dried over MgS04 and removed in vacuo. Yield: 486 mg LC-MS: m/z = 668 (M + 18).
Argon was bubbled through a stirred mixture of the material from above (258 mg, 0.396 mmol), 4-methylsulfonylphenylboronic acid (240, 1.20 mmol), KF (209 mg, 3.60 mmol) in MeOH (10 ml) and toluene (10 ml) for 10 min. PEPPSI-iPr™ (26.3 mg, 0.036 mmol) was added and the reaction was heated at 55°C in a sealed flask for 3 hours. The mixture was added MeCN (-40 ml) and heated until most material had dissolved and then filtered. The solvents were removed in vacuo and the solid was dissolved in boiling MeCN (-15-20 ml). MeOH (-10 ml) were added and the mixture left at r.t. over the weekend. The product was collected, washed with some MeOH and dried. Yield: 137 mg (43%) as a red solid. 1H NMR (400 MHz, DMSO- ): δ 3.18 (s, 4H), 3.22 - 3.28 (m, 4H), 3.26 (s, 6H), 4.58 (t, J6.8 Hz, 4H), 7.42 (s, 2H), 7.85 (s, 2H), 7.92 - 8.00 (m, 8H). The material from above (100 mg, 0.125 mmol) and morpholine (0.406 ml, 4.71 mmol) were stirred neat in a small tube at 55°C for 1 hour and at 70°C for 1 hour in a sealed tube. The mixture was cooled and water (-20 ml) was added. A solid was collected by filtration, washed with water and dried at reduced pressure. The compound was added water (-20 ml) and 6 M HC1 (0.5 ml). The mixture was heated to 70°C, added MeOH (-10 ml) and filtered. The solution was left at r.t. for 5 days and the product was collected as a red solid. The solid was dried in the rotavapor and under high vacuum for 2 days. Yield: 71 mg (66%). 1H MR (400 MHz, OMSO-de): δ 3.27 (s, 6H), 3.30-3.42 (m, 8H), 3.56 (s, 8H), 3.84 (t, J 11.8 Hz, 4H), 4.02 (d, J 11.0 Hz, 4H), 7.49 (s, 2H), 7.81 (s, 2H), 7.91 - 7.96(m, 4H), 7.98 - 8.03 (m, 4H), 11.37 (br. s., 2H). HPLC: Rt = 2.82 min, 99% (254 nm, 10-90% MeCN in 10 mM buffer,
Ace) and Rt = 2.82 min, 98% (400 nm, 10-90% MeCN in 10 mM buffer, Ace). LC-MS: m/z = 783 (M + 1).
Figure imgf000107_0001
Example 72. 2-(5-{5-[5-(Carboxymethyl)thiophen-2-yl]thiophen-2-yl}thiophen-2-yl)acetic acid (9708_075)
Acetyl chloride (2.43 ml, 34.1 mmol) was added to a solution of the 2-thiopheneacetic acid (1.94 g, 13.7 mmol) in MeOH (100 ml) at 0°C. The reaction was then stirred at r.t. over night and the solvent was removed in vacuo. The crude material was dissolved in Et20 (-50 ml) and washed with diluted K2CO3. The organic phase was dried over MgS04 and removed in vacuo. Yield: 1.81g (85%). LC-MS: m/z = 174 (M + 1).
Br2 (0.333 ml, 6.47 mmol) in DCM (3 ml) was added to a solution of the ester from above (1.01 g, 6.47 mmol) in DCM (20 ml) at 0°C. The reaction was stirred at this temp for 30 min. DCM (-10 ml) was added and the mixture was washed with saturated Na2S203 and diluted Na2C03. The organic phase was dried over MgS04 and removed in vacuo. The crude product was dissolved in DCM and run though a plug of silica with 5% EtOAc in iso-hexane as eluent. Yield: 1.21 g (80%) as a pale yellow oil. 1H NMR (400 MHz, CDC13): δ 3.74 (s, 3H), 3.78 (s, 2H), 6.70 (d, J 3.8 Hz, 1H), 6.91 (d, J3.8 Hz, 1H).
Argon was bubbled through a stirred mixture of bromide from above (274 mg, 1.16 mmol), 2,5-thiopenediboronic acid (80 mg, 0.466 mmol), KF (162 mg, 2.79 mmol), MeOH (3 ml) and toluene (3 ml) for 10 min. PEPPSI-iPr™(13.6 mg, 0.019 mmol) was added and the reaction was heated at 55°C for 3 hours. The mixture was concentrated and added water and DCM (~4 ml). The DCM mixture was purified by flash chromatography (2.5% EtOAc in toluene, 12g Redisep silica). The solid was dissolved in dioxane (2 ml) and water (1 ml) and 1 M NaOH (0,600 ml) were added. The reaction was heated at 75°C for 30 min. The solvents were removed in vacuo and the crude was dissolved in water with some H4HCO3. The mixture was filtered and purified by prep-HPLC (5-40% MeCN, in 50 mM H3/NH4HCO3 buffer). The combined pure fractions were concentrated to dryness. Water and HC1 were added and the mixture extracted with EtOAc. The organic phase was dried over MgS04 and removed in vacuo. Yield: 30 mg (52%) as a pale yellow solid. 1H NMR (400 MHz, DMSO- d6): δ 3.84 (s, 4H), 6.91 (d, J3.5 Hz, 2H), 7.16 (d, J3.5 Hz, 2H), 7.20 (s, 2H), 12.63 (br. s., 2H). HPLC: Rt = 1.48 min, 100% (254 nm, 10-40% MeCN in 10 mM buffer, XBridge) and Rt = 1.45 min, 100% (400 nm, 10-40% MeCN in 10 mM buffer, XBridge). LC-MS: m/z = 365 (M + 1).
Figure imgf000108_0001
Example 73. 4-(5-{5-[5-(4-Carboxyphenyl)-3-(2-hydroxyethyl)thiophen-2-yl]thiophen-2-yl}- 4-(2-hvdroxyethv0thiophen-2-v0benzoic acid (9708 076)
Argon was bubbled through a mixture of intermediate H (150 mg, 0.303 mmol), 4- carboxyphenylboronic acid (126 mg, 0.759 mmol) and KF (88 mg, 1.52 mmol) in toluene (5 ml), MeOH (5 ml) and dioxane (1 ml). PEPPSI-iPr™ (4.1 mg, 0.006 mmol) was added and the mixture heated in a sealed tube at 80 °C for 30 min. Solvents were evaporated and residue purified by prep, hplc, Pure fractions were combined. Some solvents were evaporated and 2 M HC1 was added. Precipitate was isolated by centrifugation, washed with water (twice) and freeze-dried for 3 d. Yield: 9.6 mg (5%); yellow solid. Rt = 2.10 min, 98% at 254 nm (10-
40% MeCN in buffer, XBridge) and Rt = 2.07 min. 98% at 400 nm (10-40% MeCN in buffer, XBridge). 1H NMR (400 MHz, OMSO-d6): δ 2.96 (t, J7 Hz, 4H), 3.73-3.76 (m, 4H), 4.89 (t, J 5 Hz, 2H), 7.38 (s, 2H), 7.67 (s, 2H), 7.79 (d, J 8 Hz, 4H), 7.97 (d, J 8 Hz, 4H). MS: m/z = 575 (M-l).
Figure imgf000109_0001
Example 74. 3-(5-{5-[5-(3-Carboxyphenyl)-3-(2-hydroxyethyl)thiophen-2-yl]thiophen-2-yl}- 4-(2-hvdroxyethyl)thiophen-2-v0benzoic acid (9708_077)
Argon was bubbled through a mixture of intermediate H (150 mg, 0.303 mmol), 3- carboxyphenylboronic acid (126 mg, 0.759 mmol) and KF (88 mg, 1.52 mmol) in toluene (5 ml), MeOH (5 ml) and dioxane (1 ml). PEPPSI-iPr™ (4.1 mg, 0.006 mmol) was added and the mixture heated in a sealed tube at 80 °C for 90 min. Solvents were evaporated and residue purified by prep. hplc. Pure fractions were combined. Some solvents were evaporated and 2 M HC1 was added. Precipitate was isolated by centrifugation, washed with water (twice) and freeze-dried for 3 d. Yield: 37.6 mg (22%); yellow solid. Rt = 2.44 min, 98% at 254 nm (10-
40% MeCN in buffer, XBridge) and Rt = 2.44 min. 99% at 400 nm (10-40% MeCN in buffer, XBridge). 1H MR (400 MHz, OMSO-d6): δ 2.96 (t, J7 Hz, 4H), 3.72-3.80 (m, 4H), 7.37 (s, 2H), 7.58 (t, J 8 Hz, 2H), 7.63 (s, 2H), 7.87-7.91 (m, 2H), 7.91-7.93 (m, 2H), 8.17 (t, J2 Hz, 2H). MS: m/z = 575 (M-l).
Figure imgf000109_0002
Example 75. 5-[4-(Carboxymethyl)-5-{5-[3-(carboxymethyl)-5-(4- methanesulfonylphenyl)thiophen-2-yl]thiophen-2-yl}thiophen-2-yl]thiophene-2-carboxylic acid (9708_078)
Argon was bubbled through a mixture of intermediate N (104 mg,0.170 mmol), 4- methylsolfonylboronic acid (51 mg, 0.255 mmol) and KF (30 mg, 0.510 mmol) in toluene (2 ml) and MeOH (2 ml). PEPPSI-iPr™ (2.3 mg, 0.003 mmol) was added and the mixture heated in a sealed tube at 80 °C for 30 min. Solvents were evaporated and residue dissolved in dioxane (4 ml), 2 M NaOH (2 ml) was added and the mixture was heated at 80 °C for 2 h. Solvents were evaporated 6 M HC1 (0.2 ml) and water (3 ml) was added the mixture was filtered and solid purified by prep, hplc Pure fractions were combined, some solvent removed and 1 M HC1 added. The precipitate was isolated by centrifugation, washed with water three times and freeze dried for 2 d. Yield: 39.7 mg (36%); deep red powder. Rt = 1.76 min, 99% at 254 nm (10-40% MeCN in buffer, XBridge) and Rt = 1.77 min. 99% at 400 nm (10-40% MeCN in buffer, XBridge). 1H NMR (400 MHz, OMSO-d6): δ 3.28 (s, 3H), 3.80 (br s, 4H), 7.32- 7.52 (m, 4H), 7.64-7.96 (m, 6H). MS: LC-MS: m/z = 643 (M-l).
Figure imgf000110_0001
Example 76. 2-{2-[3-(Carboxymethyl)-5-(4-methanesulfonylphenyl)thiophen-2-yl]-5-(4- methanesulfonylphenyl)thiophen-3-yl} acetic acid (9708 079)
Argon was bubbled through a mixture of intermediate E2 (80 mg, 0171 mmol), 4- methylsulfonylphenylboronic acid (85 mg, 0.427 mmol) and KF (49 mg, 0.854 mmol) in toluene (2 ml) and MeOH (2 ml). PEPPSI-iPr™ (2.3 mg, 0.003 mmol) was added and the mixture heated at 60 °C for 30 min under argon. Solvents were evaporated and dioxane (2 ml) and 2 M NaOH (2 ml) was added. The mixture was heated at 80 °C for 1 h. EtOAc and 6 M HCl (0.5 ml) was added. The aqueous phase was diluted with some 1 M H4HCO3 filtered and purified by prep. hplc. Pure fractions were combined, some solvents were removed, the compound was precipitated by addition of 1 M HCl, isolated by centrifugation, washed three times with water and freeze-dried for 2 d. Yield: 45.3 mg (45%); bright yellow powder. Rt = 1.98 min, 99% at 254 nm (10-40% MeCN in buffer, XBridge) and Rt = 1.98 min. 99% at 400 nm (10-40% MeCN in buffer, XBridge). 1H NMR (400 MHz, OMSO-d6): δ 3.25 (s, 6H), 3.57 (s, 4H), 7.75 (s, 2H), 7.91-8.00 (m, 8H). MS: m/z = 608 (M + NH4 +).
Figure imgf000110_0002
Example 77. 2-(2-{5-[3-(Carboxymethyl)-5-[l-methyl-3-(trifluoromethyl)-lH-pyrazol-5- yl]thiophen-2-yl]thiophen-2-yl}-5-[l-methyl-3-(trifluoromethyl)-lH-pyrazol-5-yl]thiophen-3- vDacetic acid (9708_080)
Argon was bubbled through a mixture of intermediate D (55 mg, 0.100 mmol), l-methyl-3- trifluoromethylpyrazole-5-boronic acid (48.4 mg, 0.250 mmol) and KF (29 mg, 0.500 mmol) in toluene (1 ml) and MeOH (1 ml). PEPPSI-iPr™ (2 mg, 0.003 mmol) was added and the mixture heated at 80 °C for 30 min in a sealed tube. Solvents were evaporated and dioxane (1 ml) and 2 M NaOH (1 ml) was added. The mixture was heated at 80 °C for 2 h. The product was isolated by prep, hplc using 25-55% MeCN in 50 mM buffer as eluent. Pure fractions were combined, product precipitated by addition of 6 M HC1 and isolated by centrifugation. The precipitate was washed several times with water and freeze dried for 2 d. Yield: 27.0 mg (41%); bright yellow solid. Rt = 2.72 min, 99% at 254 nm, (20-50% MeCN in buffer, XBridge) and Rt = 2.72 min, 98% at 400 nm (20-50% MeCN in buffer, XBridge). 1H NMR (400 MHz, DMSO-i¾): δ 3.82 (s, 4H), 4.07 (s, 6H), 7.09 (d, J0.5 Hz, 2H), 7.38 (s, 2H), 7.54 (s, 2H). LC-MS: m/z = 661 (M + 1)
Figure imgf000111_0001
Example 78. 2-(2-(5-r3-(Carboxymethyl)-5-r3-fluoro-4-(pyrrolidine-l- carbonyl)phenyl]thiophen-2-yl]thiophen-2-yl}-5-[3-fluoro-4-(pyrrolidine-l- carbonyl)phenyl]thiophen-3-yl)acetic acid (9708 081)
Argon was bubbled through a mixture of intermediate D (55 mg, 0.100 mmol), 3-fluoro-4- (pyrrolidine-l-carbonyl)phenylboronic acid (59 mg, 0.250 mmol) and KF (29 mg, 0.500 mmol) in toluene (1 ml) and MeOH (1 ml). PEPPSI-iPr™ (2 mg, 0.003 mmol) was added and the mixture heated at 80 °C for 30 min in a sealed tube. Solvents were evaporated and dioxane (1.5 ml) and 2 M NaOH (1.5 ml) was added. The mixture was heated at 80 °C for 1 h. The product was isolated by prep, hplc using 20-50% MeCN in 50 mM buffer as eluent. Pure fractions were combined, product precipitated by addition of 6 M HC1 and isolated by centrifugation. The precipitate was washed several times with water and freeze dried for 2 d. Yield: 24.7 mg (33%); bright yellow solid. Rt = 2.16 min, 99% at 254 nm, (20-50% MeCN in buffer, XBridge) and Rt = 2.16 min, 98% at 400 nm (20-50% MeCN in buffer, XBridge). 1H NMR (400 MHz, OMSO-d6): δ 1.80-1.93 (m, 8H), 3.25-3.50 (m, 8H), 3.79 (s, 4H), 7.36 (s, 2H), 7.45-7.51 (m, 2H), 7.53-7.58 (m, 2H), 7.74 (dd, J= 10.9 Hz, J= 1.6 Hz, 2H), 7.66 (s, 2H).
Figure imgf000112_0001
Example 79. 2-(2-{5-[3-(Carboxymethyl)-5-[5-methyl-6-(morpholin-4-yl)pyridin-3- yl]thiophen-2-yl]thiophen-2-yl}-5-[5-methyl-6-(m
vDacetic acid (9708_082)
Argon was bubbled through a mixture of intermediate D (55 mg, 0.100 mmol), 5-methyl-6- morpholin-4-yl)pyridine-3-boronic acid (64 mg, 0.250 mmol) and KF (29 mg, 0.500 mmol) in toluene (1 ml) and MeOH (1 ml). PEPPSI-iPr™ (2 mg, 0.003 mmol) was added and the mixture heated at 80 °C for 30 min in a sealed tube. Solvents were evaporated and dioxane (1.5 ml) and 2 M NaOH (1.5 ml) was added. The mixture was heated at 80 °C for 1 h. The material was dissolved in HCl/water and filtered. 2 M NaOH was carefully added and precipitate filtered, washed with water and dried at high vacuum for 3 d. Yield: 41.9 mg (58%); yellow solid. Rt = 2.27 min, 90% at 254 nm (20-50% MeCN in buffer, XBridge) and Rt = 2.27 min. 93% at 400 nm (20-50% MeCN in buffer, XBridge). 1H NMR (400 MHz, DMSO-i¾): δ 2.31 (s, 6H), 3.05-3.18 (m, 8H), 3.66-3.82 (m, 12H), 7.30 (s, 2H), 7.46 (s, 2H), 7.77-7.89 (m, 2H), 8.41 (d, J2.3 Hz, 2H). MS: m/z = 717 (M+l)
Figure imgf000112_0002
Example 80. 2-(2-{5-[3-(Carboxymethyl)-5-[3-(cyclopropylcarbamoyl)phenyl]thiophen-2- yl]thiophen-2-yl}-5-[3-(cyclopropylcarbamoyl)phenyl]thiophen-3-yl)acetic acid (9708 083) Argon was bubbled through a mixture of intermediate D (55 mg, 0.100 mmol), 3-
(cyclopropylaminocarbonyl)benzeneboronic acid (51 mg, 0.250 mmol) and KF (29 mg, 0.500 mmol) in toluene (1 ml) and MeOH (1 ml). PEPPSI-iPr™ (2 mg, 0.003 mmol) was added and the mixture heated at 80 °C for 30 min in a sealed tube. Solvents were evaporated and dioxane (1.5 ml) and 2 M NaOH (1.5 ml) were added. The mixture was heated at 80 °C for 1 h.
Mixture was acidified and mixture extracted with EtOAc. The organic phase was evaporated and residue purified by prep. hplc. Pure fractions were combined and material precipitated by addition of 6 M HC1. The solid material was isolated by centrifugation and washed three times with water. The material was freeze-dried for 2 d. Yield: 7.9 mg (12%); yellow solid. Rt = 1.82 min, 97% at 254 nm (20-50% MeCN in buffer, XBridge) and Rt = 2.27 min. 97% at 400 nm (20-50% MeCN in buffer, XBridge). 1H NMR (400 MHz, OMSO-d6): δ 0.57-0.78 (m, 8H), 2.87-2.88 (m, 2H), 3.80 (s, 4H), 7.36 (s, 2H), 7.52 (t, J 7.8 Hz, 2H), 7.59 (s, 2H), 7.74-7.85 (m, 4H), 8.07 (t, J 1.5 Hz, 2H), 8.60 (d, J4.0 Hz, 2H).MS: m/z = 681 (M-l).
Figure imgf000113_0001
Example 81. 2-(2-r3-(Carboxymethvnthiophen-2-yl1-5-(4-hvdroxy-3- methoxyphenyl)thiophen-3-yl} acetic acid (9708 084)
Argon was bubbled through a mixture of 4-hydroxy-3-methoxyphenylboronic acid (0.186 g, 1.11 mmol), KF (0.172 g, 2.95 mmol) and intermediate F2 in MeOH/toluene (1 : 1, 8 ml).
After 15 min PEPPSI-iPr™ (15 mg, 0.022 mmol) was added and the mixture heated to 55 °C. After 60 min, the reaction mixture was evaporated to dryness, dissolved in CH2CI2 (30 ml) and washed with water (30 ml). The organic layer was evaporated to dryness to obtain the crude product. The residue was purified by flash chromatography using toluene/EtOAc (9: 1→7: 1) as eluent. Yield: 256 mg (80%); 1H NMR (400 MHz, CDC13): δ 3.55 (s, 2H), 3.62 (s, 2H), 3.69 (s, 3H), 3.69 (s, 3H), 3.96 (s, 3H), 6.93 (d, J 8.0 Hz, 1H), 7.06 (d, J 4 Hz, 1H), 7.10 (d, J 4.0 Hz, 1H), 7.12 (dd, J4.0, 8.0 Hz, 1H), 7.17 (s, 1H), 7.39 (d, J 4.0 Hz, 1H).
The ester from above (54 mg, 0.13 mmol) was dissolved in dioxane (2 ml) and 2 M NaOH (2 ml) was added. After stirring 45 min at 70 °C, the reaction was cooled to RT, diluted with water (6 ml) and acidified using 2 M HC1 (approx. 3 ml). The formed precipitate was isolated by centrifugation and washed two times with water. The material was dried under high vacuum for 48 hrs. Yield: 26 mg (52%); White solid. 1H NMR (DMSO-i¾): δ 3.42 (s, 2H), 3.49 (s, 2H), 3.84 (s, 3H), 6.80 (d, J 8.2 Hz, 1H), 7.01 (dd, J 8.2, 2.0 Hz, 1H), 7.10 (d, J 5.3 Hz, 1H), 7.15 (d, J2.0 Hz, 1H), 7.32 (s, 1H), 7.62 (d, J 5.2 Hz, 1H), 9.28 (s, 1H), 12.39 (bs, 2H). HPLC: RT = 1.20 min, 99% (254 nm, 10-40% MeCN in 10 mM buffer, 3 min, XBridge). LC-MS: m/z = 422 (M + NH4 +).
Figure imgf000114_0001
Example 82. 5-[4-(Carboxymethyl)-5-{5-[3-(carboxymethyl)-5-(4-hydroxy-3- methoxyphenyl)thiophen-2-yl]thiophen-2-yl}thiophen-2-yl]thiophene-2-carboxylic acid (9708_085)
Argon was bubbled through a mixture of intermediate N (60 mg, 0.10 mmol), 4-hydroxy-3- methoxyphenylboronic acid (25 mg, 0.15 mmol) and KF (19 mg, 0.32 mmol) in
MeOH/toluene (1 : 1, 4 ml). After 10 min, PEPPSI-iPr™ (2 mg, 0.003 mmol) was added and the mixture stirred at 80 °C in a sealed tube. After 10 min, the reaction was cooled to RT, diluted with CH2CI2 (30 ml), washed with water (30 ml) and the aqueous layer was subsequently extracted with DCM (15 ml x 2). The organic layers were combined and evaporated to dryness to obtain the crude product. The residue was purified by flash chromatography using toluene/EtOAc (6: 1) as eluent. Yield: 55 mg (85%)
The ester from above (55 mg, 0.08 mmol) was dissolved in dioxane (2 ml) and 2 M NaOH (2 ml) was added. After stirring 45 min at 70 °C, the reaction was cooled to RT, diluted with water (11 ml) and acidified using 2 M HC1 (approx. 3 ml). The formed precipitate was isolated by centrifugation and washed two times with water. The material was dried under high vacuum for 48 hrs. Yield: 44 mg (86%); Red solid; 1H MR (DMSO-i¾): δ 3.74 (s, 2H), 3.78 (s, 2H), 3.85 (s, 3H), 6.82 (d, J 8.0 Hz, 1H), 7.06 (dd, J 8.0, 2.1 Hz, 1H), 7.17 (d, J2.1 Hz, 1H), 7.27 (d, J4.0 Hz, 1H), 7.33 (d, J4.0 Hz, 1H), 7.35 (s, 1H), 7.40 (d, J4.0 Hz, 1H), 7.47 (s, 1H), 7.68 (d, J4.0 Hz, 1H), 9.33 (s, 1H), 12.74 (bs, 3H). HPLC: RT = 1.76 min, 97% (254 nm, 10-40% MeCN in 10 mM buffer, 3 min, XBridge) and RT = 1.77 min, 97% (400 nm, 10-40% MeCN in 10 mM buffer, 3 min, XBridge). LC-MS: m/z = 630 (M + NH4 +).
Figure imgf000114_0002
Example 83. 2-(2-(5-[3-(Carboxymethvn-5-(3-cvanophenvnthiophen-2-yllthiophen-2-yl|-5- (3-cyanophenyl)thiophen-3-yl)acetic acid (9708 086)
Argon was bubbled through a mixture of intermediate Q (57 mg, 0.109 mmol) and 3- cyanophenylboronic acid (40.1 mg, 0.273 mmol) in dioxane (1.5 ml) and 1 M Κ^03(1.5 ml). PEPPSI-iPr (3.7 mg, 0.005 mmol) was added and the mixture heated in a sealed tube at 100 °C for 30 min. Mixture was filtered and purified by prep, hplc using 25-50% MeCN in buffer. Pure fractions were combined and some solvents evaporated. 6 M HCl (ca 2 ml) was added and the aqueous layer extracted with EtOAc. The organic layer was washed with water (twice) and solvent concentrated. The material was dried under high vacuum for 6 d. Yield:
22.1 mg (36%.); light yellow solid. Rt = 2.37 min, 100% at 254 nm (20-50% MeCN in buffer, XBridge) and Rt = 2.37 min. 100% at 400 nm (20-50% MeCN in buffer, XBridge) 1H NMR (400 MHz, DMSO-i¾):□ 3.85 (s, 4H), 7.43 (s, 2H), 7.69- 7.73 (m, 2H), 7.76 (s, 2H), 7.85 (d, J 7.5 Hz, 2H), 8.05 (d, J 8.0 Hz, 2H), 8.25 (s, 2H). MS: m/z = 565 (M-l).
Figure imgf000115_0001
Example 84. 2-(2-{5-[3-(Carboxymethyl)-5-{5-[3-(carboxymethyl)thiophen-2-yl]thiophen-2- yl}thiophen-2-yl]thiophen-2-yl}-5-{5-[3-(carboxymethyl)thiophen-2-yl]thiophen-2- yl}tmophen-3-yl)acetic acid (9708 087)
Intermediate B (500 mg, 2.13 mmol) in dry 1,4-dioxane (4 ml) was added to
bis(pinacolato)diboron (594 mg, 2.34 mmol) and dry AcOK (772 mg, 7.87 mmol). The mixture was flushed with argon and l,l'-bis(diphenylphosphino)ferrocene- palladium(II)dichloride (31.1 mg, 0.043 mmol) was added. The reaction was heated in a sealed tube at 95°C for 3 hours. After cooling the mixture was filtered trough celite and new AcOK (600 mg, 6.11 mmol) was added. Argon was flushed trough the mixture for 10 min and l, -bis(diphenylphosphino)ferrocene-palladium(II)dichloride (-50 mg, 0.068 mmol) was added. The reaction was heated in a sealed tube at 95°C for 1 hour then cooled and diluted with Et20 (-150 ml). The organic phase was washed with water and brine, dried over MgS04 and removed in vacuo. The crude was dissolved in toluene (-3 ml) and purified by flash chromatography (10% diisopropyl ether in iso-hexane, 24 gram silica redisep colon). Yield:
55 mg (9%). 1H NMR (400 MHz, CDC13) δ 1.33 (s, 12H), 3.70 (s, 3H), 4.00 (s, 2H), 7.12 (d, J 4.77 Hz, 1H), 7.55 (d, J 4.52 Hz, 1H).
Argon was flushed through a solution of the boronic ester from above (52.1 mg, 0.185 mmol), intermediate O (60 mg, 0.084 mmol) and KF (36.6 mg, 0.0630 mmol) in a mixture of toluene (5 ml) and MeOH (5 ml) for 10 min. PEPPSI-iPr™ (3.0 mg, 0.0042 mmol) was added and the reaction heated at 60°C for 3 hours. The solvents were removed in vacuo. The crude material was added dioxane (1 ml), 5 M NaOH (1 ml) and water (2 ml) and the reaction was heated to 65°C for 30 min. The solution was filtered and purified by prep-HPLC (5-45% MeCN, in 50 mM NH3/NH4HCO3 buffer). The pure fractions were combined and concentrated to dryness. The solid was dissolved in water and 2 M HC1 (6 ml) was added. A red solid was collected, washed several times with water and dried in the rotavapor and at high vacuum. Yield: 33 mg (49%) as a red solid. 1H NMR (400 MHz, DMSO-^6) δ 3.75 (s, 4H), 3.78 (s, 4H), 7.10 (d, J 5.0 Hz, 2H), 7.20 (d, J3.7 Hz, 2H), 7.33 (s, 2H), 7.33 (s, 2H), 7.39 (d, J.3.7 Hz, 2H), 7.54 (d, J 5.3 Hz, 2H). HPLC: Rt = 2.45 min, 99% (254 nm, 10-40% MeCN in 10 mM buffer, XBridge) and Rt = 2.44 min, 98% (400 nm, 10-40% MeCN in 10 mM buffer, XBridge). LC- MS: m/z = 826 (M + 18).
Figure imgf000116_0001
Example 85. 2-(2-{5-[3-(Carboxymethyl)-5-(5-cyanothiophen-2-yl)thiophen-2-yl]thiophen- 2-yl|-5-(5-cvanothiophen-2-yl)thiophen-3-yl)acetic acid (9708 088)
Argon was bubbled through a mixture of intermediate Q (55 mg, 0.105 mmol), 5- cyanothiophene-2-boronic acid (64 mg, 0.421 mmol) and KF (37 mg, 0.032 mmol) in toluene (2 ml) and MeOH (2 ml). The mixture was heated at 60 °C for 2 h. Solvents were evaporated and residue purified by prep, hplc using 20-50% MeCN in 50 mM buffer as eluent. Pure fractions were combined and 2 M HC1 was added. The aqueous phase was extracted with EtOAc and dried under high vacuum for 3 d. Yield: 22.5 mg (37%); orange solid. Rt = 2.36 min, 99% at 254 nm (20-50% MeCN in buffer, XBridge) and Rt = 2.38 min. 100% at 400 nm (20-50% MeCN in buffer, XBridge). 1H NMR (400 MHz, DMSO-i¾): δ 3.79 (s, 4H), 7.38 (s, 2H), 7.52 (d, J 3.8 Hz, 7.55 (s, 2H), 7.97 (d, J4.0 Hz, 2H). MS: m/z = 577 (M - 1).
Figure imgf000116_0002
Example 86. 2-(2-{5-[3-(Carboxymethyn-5-ethynylthiophen-2-yllthiophen-2-yl}-5- ethynylthiophen-3-yOacetic acid (9708 089) Argon was bubbled through a stirred mixture of the intermediate G2 (50 mg, 0.078 mmol), Cul (3.0 mg, 0.016 mmol) water (0.056 ml, 3.1 mmol) and toluene (10 ml) for 10 min.
PdCl2(PPh3)2 (3.3 mg, 0.0047 mmol) and trimethylsilylacetylene (0.0215 ml, 0.155 mmol) were added and the mixture stirred in a sealed tube at r.t. over night. The solvent was removed in vacuo and the residue purified by flash chromatography (1% EtOAc in toluene, 24 g silica). The solid was dissolved in dioxane (2 ml) and 5 M NaOH (0,5 ml) and water (1 ml) were added. The mixture was stirred at r.t. overnight, filtered and purified by prep-HPLC (10-50% MeCN, in 50 mM H3/NH4HC03 buffer). The combined pure fractions were concentrated, added water and HC1 and extracted with EtOAc. The organic phase was dried over MgS04 and removed in vacuo. Yield: 21 mg (85%), as a yellow solid. 1H NMR (400 MHz, DMSO- d6): δ 3.73 (s, 4H), 4.71 (s, 2H), 7.31 (s, 2H), 7.34 (s, 2H), 12.58 (br. s., 2H). HPLC: Rt = 2.54 min, 95% (254 nm, 10-40% MeCN in 10 mM buffer, XBridge) and Rt = 2.53 min, 95% (400 nm, 10-40% MeCN in 10 mM buffer, XBridge). LC-MS: m/z = 430 (M + 18).
Figure imgf000117_0001
Example 87. 2-(2-{5-[3-(Carboxymethyl)-5-[3-(trifluoromethoxy)phenyl]thiophen-2- yl]thiophen-2-yl}-5-[3-(trifluoromethoxy)phenyl]thiophen-3-yl)acetic acid (9708 090)
Argon was flushed through a solution of intermediate D (60.0 mg, 0.109 mmol), 3- (trifluoromethoxy)phenylboronic acid (50.0 mg, 0.248 mmol) and KF (47.5 mg, 0.818 mmol) in a mixture of toluene (2 ml) and MeOH (2 ml) for 10 min. PEPPSI-iPr™ (4.0 mg, 0.0055 mmol) was added and the reaction heated at 60°C for 3 hours. The solvents were removed in vacuo and the crude material was purified by flash chromatography (1% EtOAc in toluene, 24 g silica). The product was dissolved in dioxane (2 ml) and 5 M NaOH (0.5 ml) and water (1 ml) were added. The mixture was stirred at r.t. over night. The mixture was concentrated to dryness added water (~2 ml) and MeCN (~2 ml). The water phase was removed and new water was added (~2 ml). The homogen solution was filtered and purified by prep-HPLC (10- 60% MeCN, in 50 mM NH3/NH4HC03 buffer). The combined pure fractions were
concentrated and added water and 2 M HC1. The solid was collected washed several times with water and dried. Yield: 24 mg (37%) as a yellow solid. 1H NMR (400 MHz, DMSO-d6): δ 3.73 (s, 4H), 4.71 (s, 2H), 7.31 (s, 2H), 7.34 (s, 2H), 12.58 (br. s., 2H). HPLC: Rt = 2.45 min, 99% (254 nm, 10-90% MeCN in 10 mM buffer, XBridge) and Rt = 2.45 min, 99% (400 nm, 10-90% MeCN in 10 mM buffer, XBridge). LC-MS: m/z = 702 (M + 18).
Figure imgf000118_0001
Example 88. 2-{2-[3-(Carboxymethyl)-5-(4-hydroxy-3-methoxyphenyl)thiophen-2-yl]-5-(4- hydroxy-3-methoxyphenyl)thiophen-3-yl} acetic acid (9708 091)
Argon was bubbled through a mixture of 4-hydroxy-3-methoxyphenylboronic acid (0.171 g, 1.02 mmol), KF (0.158 g, 3.00 mmol) and Intermediate E2 (0, 159 g, 0,34 mmol) in
MeOH/toluene (1 : 1, 5 ml). After 15 min PEPPSI-iPr™ (12 mg, 0.017 mmol) was added and the mixture heated to 55 °C. After 90 min, the reaction mixture was evaporated to dryness. The residue was purified by flash chromatography using toluene/EtOAc
(6: 1→3 : 1→1 :3→1 :6) as eluent. Yield: 101 mg (54%); 1H NMR (400 MHz, CDC13): δ 3.61 (s, 4H), 3.71 (s, 6H), 3.96 (s, 6H), 5.70 (bs, 2H), 6.94 (d, J 8.4 Hz, 2H), 7.07 (d, J2.0 Hz, 2H), 7.13 (dd, J 8.4, 2.0 Hz, 2H), 7.18 (s, 2H).
The ester from above (101 mg, 0.18 mmol) was dissolved in dioxane (3 ml), followed by addition of 2 M NaOH (3 ml) and water (1 ml). After stirring 45 min at 70 °C, the reaction was cooled to RT, diluted with water (8 ml) and acidified using 1 M HC1 (approx. 6 ml). The formed precipitate was isolated by centrifugation and washed two times with water. The material was dried under high vacuum for 48 hrs. Yield: 82 mg (86%); brown solid. 1H NMR (DMSO-i¾): δ 3.48 (s, 4H), 3.84 (s, 6H), 6.82 (d, J 8.3 Hz, 2H), 7.01 (dd, J 8.3 , 2.0 Hz, 2H), 7.16 (d, J2.0 Hz, 2H), 7.33 (s, 2H), 9.29 (s, 2H), 12.42 (bs, 2H). HPLC: RT = 1.91 min. 96% at 254 nm (10-40% MeCN in buffer, 3 min, XBridge) and RT = 1.91 min, 100% at 400 nm (10-40% MeCN in buffer, 3 min, XBridge). ). LC-MS: m/z = 544 (M + ).
Figure imgf000118_0002
Example 89. 5-(5-{5-[5-(5-Carboxy-4-methylthiophen-2-yl)-3-(carboxymethyl)thiophen-2- yl]thiophen-2-yl}-4-(carboxymethyl)thiophen-2-yl)-3-methylthiophene-2-carboxyli acid (9708_092)
Argon was bubbled through a mixture of intermediate D (61 mg, 0.11 mmol), 2-carboxy-4- metylthiophene-5-boronic acid (60 mg, 0.30 mmol) and KF (42 mg, 0.72 mmol) in
MeOH/toluene (1 : 1, 4 ml). After 10 min, PEPPSI-iPr™ (4 mg, 0.006 mmol) was added and the mixture stirred at 80 °C in a sealed tube. After 10 min, the reaction was cooled to RT, diluted with CH2CI2 (30 ml), washed with water/brine (1 : 1, 30 ml) and the aqueous layer was subsequently washed with CH2CI2 (15 ml x 2). The organic layers were combined and evaporated to dryness to obtain the crude product. The residue was purified by flash chromatography using toluene/EtOAc (100: 1→19: 1→9: 1) as eluent. Yield: 60 mg (77%); 1H NMR (400 MHz, CDC13): δ 2.55 (s, 6H), 3.77 (s, 6H), 3.79 (s, 4H), 3.88 (s, 6H), 7.01 (s, 2H), 7.21 (s, 2H), 7.22 (s, 2H).
The ester from above (60 mg, 0.09 mmol) was dissolved in dioxane (2 ml), followed by addition of 2 M NaOH (2 ml) and water (1 ml). After stirring 35 min at 70 °C, the reaction was cooled to RT, diluted with water (10 ml) and acidified using 1 M HCl (approx. 4 ml). The formed precipitate was isolated by centrifugation and washed two times with water. The material was dried under high vacuum for 48 hrs. The obtained solid was dissolved in 1 M Na2C03 and purified by preparative hplc (5-40% MeCN in 50 mM buffer, XBridge). Pure fractions were combined and some solvents were evaporated. Solid material precipitated by addition of 6 M HCl, centrifuged, washed with two times with water. The material was dried under high vacuum for 4 d. Yield: 33 mg (57%); Dark red solid. 1H NMR (400 MHz, DMSO- d6): δ 2.46 (s, 6H), 3.77 (s, 4H), 7.29 (s, 2H), 7.33 (s, 2H), 7.42 (s, 2H), 12.90 (bs, 4H).
HPLC: RT = 1.41 min. 98% at 254 nm (10-40% MeCN in buffer, 3 min, XBridge) and RT = 1.41 min, 99% at 400 nm (10-40% MeCN in buffer, 3 min, XBridge). LC-MS: m/z = 662 (M + NH4 +).
Figure imgf000119_0001
Example 90. 2-[5-(3-Carbamoylphenyl)-2-{5-[5-(3-carbamoylphenyl)-3- (carboxymethyl)thiophen-2-yl]thiophen-2-yl}thiophen-3-yl]acetic acid (9708 093) Argon was flushed through a solution of intermediate D (60.0 mg, 0.109 mmol), 3- aminocarbonylphenylboronic acid (41.0 mg, 0.249 mmol) and KF (47.5 mg, 0.818 mmol) in a mixture of toluene (2 ml) and MeOH (2 ml) for 10 min. PEPPSI-iPr™ (4.0 mg, 0.0054 mmol) was added and the reaction heated at 60°C for 3 hours. The solvents were removed in vacuo and the crude was dissolved in dioxane (2 ml) and 5 M NaOH (0,5 ml) and water (1 ml) were added. The mixture was stirred at r.t. overnight, concentrated to dryness and dissolved in 0.1 M NaOH solution. 2 M HC1 was added and the solid was collected by centrifugation. -1/3 of the solid was dissolved in a mixture of water (~7 ml), MeCN (~1 ml) and a small amount of cone NH3. The mixture were filtered and purified by prep-HPLC (5-50% MeCN, in 50 mM NH3/NH4HCO3 buffer). The combined pure fractions were concentrated and added water and HC1. The solid was washed several times with water and collected by centrifugation. The solid was dried on the rotavapor and under high vacuum. Yield: 7 mg (11%) as a yellow solid. 1H NMR (400 MHz, DMSO- ,) δ 3.80 (s, 4H), 7.35 (s, 2H), 7.49 (s, 2H), 7.53 (t, J7.8 Hz, 2H), 7.60 (s, 2H), 7.78 - 7.86 (m, 4H), 8.14 - 8.18 (m, 4H). HPLC: Rt = 1,52 min, 100% (254 nm, 10-90% MeCN in 10 mM buffer, XBridge) and Rt = 1.52 min, 100% (400 nm, 10-90% MeCN in 10 mM buffer, XBridge). LC-MS: m/z = 620 (M + 18).
Figure imgf000120_0001
Example 91. 2-[5-(4-Carbamoylphenyl)-2-{5-[5-(4-carbamoylphenyl)-3- (carboxymethyl)thiophen-2-yl]thiophen-2-yl}thiophen-3-yl]acetic acid (9708 094)
Argon was flushed through a solution of intermediate D (60.0 mg, 0.109 mmol), 4- Aminocarbonylphenylboronic acid (41.0 mg, 0.249 mmol) and KF (47.5 mg, 0.818 mmol) in a mixture of toluene (2 ml) and MeOH (2 ml) for 10 min. PEPPSI-iPr™ (4.0 mg, 0.0054 mmol) was added and the reaction heated in a sealed tube at 60°C for 3 hours. The solvents were removed in vacuo and the crude was dissolved in dioxane (2 ml) and 5 M NaOH (0.5 ml) and water (1 ml) were added. The mixture was stirred at r.t. over night, concentrated to dryness and dissolved in 0.1 M NaOH solution. 2 M HC1 was added and the solid was collected by centrifugation. -1/3 of the solid was dissolved in a mixture of water (~7 ml) MeCN (~1 ml) and some cone NH3. The mixture were filtered and purified by prep-HPLC (5- 45% MeCN, in 50 mM NH3/NH4HCO3 buffer). The combined pure fractions were concentrated and added water and HCl. The solid was washed several times with water and collected by centrifugation. The solid was dried on the rotavapor and under high vacuum.
Yield: 11 mg (16%) as a yellow solid. 1H NMR (400 MHz, DMSO-^6) δ 3.80 (s, 4H), 7.35 (s, 2H), 7.40 (br. s., 2H), 7.63 (s, 2H), 7.71 - 7.78 (m, 4H), 7.76 (s, 3H), 7.91 - 7.97 (m, 4H), 8.03 (br. s., 1H). HPLC: Rt = 1,39 min, 100% (254 nm, 10-90% MeCN in 10 mM buffer, XBridge) and Rt = 1.38 min, 100% (400 nm, 10-90% MeCN in 10 mM buffer, XBridge). LC-MS: m/z = 603 (M + 1).
Figure imgf000121_0001
Example 92 and 93. 2-(2-(5-r3-(Carboxymethvn-5-(thiophene-2-carbonyl)thiophen-2- yllthiophen-2-yl|thiophen-3-yl)acetic acid (9708 095) and 2-(2-(5-[3-(carboxymethyl)-5- (thiophene-2-carbonyl)thiophen-2-yl]thiophen-2-yl}-5-(thiophene-2-carbonyl)thiophen-3- vDacetic acid (9708 096)
Intermediate C (146 mg, 0.372 mmol) was dissolved in dry CH2CI2 (3 ml), 2- thiophenecarbonyl chloride (92 μΐ^, 0.856 mmol) and AICI3 (114 mg, 0.856 mmol) were added and the mixture was sealed under Ar-atmosphere. After 26 hrs, the reaction was diluted with CH2CI2 (10 ml), washed with water (20 ml) and the aqueous layer was subsequently washed with CH2CI2 (10 ml x 2). The organic layers were combined and evaporated to dryness to obtain the crude product. The residue was purified by flash chromatography using toluene/EtOAc (19: 1→9: 1→6: 1) as eluent to first obtain the monoacylated product methyl 2- (2-{5-[ 3-(2-methoxy-2-oxoethyl)-5-( thiophene-2-carbonyl)thiophen-2-yl Jthiophen-2- yl}thiophen-3-yl)acetate Yield: 63 mg (34%). 1H NMR (400 MHz, CDC13): δ 3.75 (s, 3H), 3.77 (s, 3H), 3.81 (s, 2H), 3.87 (s, 2H), 7.08 (d, J4.0Hz, 1H), 7.20 - 7.23 (m, 2H), 7.29 - 7.31 (m, 2H), 7.72 (dd, J2.0, 4.0 Hz, 1H), 7.87 (s, 1H), 7.94 (dd, J2.0, 4.0 Hz, 1H). Second eluted compound; the diacylated product methyl 2-(2-{5-[3-(2-methoxy-2-oxoethyl)-5-(thiophene-2- carbonyl)thiophen-2-yl]thiophen-2-yl}-5-(thiophene-2-carbonyl)thio^ Yield: 90 mg (39%) 1H NMR (400 MHz, CDC13): δ 3.79 (s, 6H), 3.87 (s, 4H), 7.23 (dd, J 4.0, 4.0 Hz, 2H), 7.36 (s, 2H), 7.73 (dd, J2.0, 4.0 Hz, 2H), 7.88 (s, 2H), 7.94 (dd, J2.0, 4.0, 2H). Methyl 2-(2-{5-[3-(2-methoxy-2-oxoethyl)-5-(thiophene-2-carbonyl)thiophen-2-yl]thiophen- 2-yl}thiophen-3-yl)acetate (63 mg, 0.125 mmol) was dissolved in dioxane (2 ml), followed by addition of 2 M NaOH (2 ml) and water (1 ml). After stirring 45 min at 70 °C, the reaction was cooled to RT, diluted with water (10 ml) and acidified using 1 M HC1 (approx. 4 ml). The reaction mixture was evaporated to dryness, dissolved in EtOAc (15 ml), washed with water (15 ml). The organic layer was dried with MgS04 (s), filtered and evaporated to dryness to obtain the product. Yield: 58 mg (98%); orange solid. 1H NMR (400 MHz, OMSO-d6): δ 3.75 (s, 2H), 3.88 (s, 2H), 7.12 (d, J 5.3 Hz, IH), 7.31 (d, J 3.8 Hz, IH), 7.35 (dd, J4.9, 3.8 Hz, IH), 7.47 (d, J 3.8 Hz, IH), 7.57 (d, J 5.3 Hz, IH), 8.06 - 8.11 (m, 2H), 8.13 (dd, J4.9, 1.1 Hz), 12.63 (bs, 2H). HPLC: RT = 2.35 min. 97% at 254 nm (10-40% MeCN in buffer, 3 min, XBridge) and RT = 2.33 min, 99% at 400 nm (10-40% MeCN in buffer, 3 min, XBridge). LC- MS: m/z = 475 (M + H).
Methyl 2-(2-{5-[3-(2-methoxy-2-oxoethyl)-5-(thiophene-2-carbonyl)thiophen-2-yl]thiophen- 2-yl}-5-(thiophene-2-carbonyl)thiophen-3-yl)acetate. (90 mg, 0.146 mmol) was dissolved in dioxane (3 ml), followed by addition of 2 M NaOH (3 ml) and water (1 ml). After stirring 45 min at 70 °C, the reaction was cooled to RT, diluted with water (8 ml) and acidified using 1 M HC1 (approx. 6 ml). The formed precipitate was isolated by centrifugation and washed two times with water. The material was dried under high vacuum for 48 hrs. Yield: 71 mg (83%); orange solid. 1H NMR (400 MHz, OMSO-d6): δ 3.91 (s, 4H), 7.35 (dd, J4.9, 3.9 Hz, 2H), 7.54 (s, 2H), 8.08 - 8.11 (m, 4H), 8.14 (dd, J 4.9, 1.1 Hz, 2H), 1.72 (bs, 2H). HPLC: RT = 2.96 min. 97% at 254 nm (10-40% MeCN in buffer, 3 min, XBridge) and RT = 2.97 min, 96% at 400 nm (10-40% MeCN in buffer, 3 min, XBridge). LC-MS: m/z = 585 (M + H).
Figure imgf000122_0001
Example 94 and 95. 2-(2-{5-[5-Benzoyl-3-(carboxymethyl)thiophen-2-yllthiophen-2- yllthiophen-3-vnacetic acid (9708_097) and 2-(5-benzoyl-2-(5-[5-benzoyl-3- (carboxymethyl)thiophen-2-yl]thiophen-2-yl}thiophen-3-yl)acetic acid (9708 098)
Intermediate C (144 mg, 0.367 mmol) was dissolved in dry CH2CI2 (3 ml), benzoyl chloride (98 \L, 0.844 mmol) and AICI3 (113 mg, 0.844 mmol) were added and the mixture was sealed under Ar-atmosphere. After 19 hrs, the reaction was diluted with CH2CI2 (10 ml), washed with water (20 ml) and the aqueous layer was subsequently washed with CH2CI2 (10 ml x 2). The organic layers were combined and evaporated to dryness to obtain the crude product. The residue was purified by flash chromatography using toluene/EtOAc (19: 1→14: 1→9: 1) as eluent to first obtain the monoacylated product; methyl 2-(2-{5-[5- benzoyl-3-(2-methoxy-2-oxoethyl)thiophen-2-yl]thiophen-2-yl}thiophen- Yield: 81 mg (45%), followed by the diacylated product methyl 2-(5-benzoyl-2-{5-[5-benzoyl-3-(2- methoxy-2-oxoethyl)thiophen-2-ylJthiophen-2-yl}thiophen-3-yl)acetate and 47 mg (21%) Methyl 2-(2-{5-[5-benzoyl-3-(2-methoxy-2-oxoethyl)thiophen-2-yl]thiophen-2-yl}thiophen- 3-yl)acetate (81 mg, 0.163 mmol) was dissolved in dioxane (3 ml), followed by addition of 2 M NaOH (3 ml) and water (1 ml). After stirring 45 min at 70 °C, the reaction was cooled to RT, diluted with water (10 ml) and acidified using 1 M HC1 (approx. 4 ml). The reaction mixture was evaporated to dryness, dissolved in EtOAc (15 ml), washed with water (15 ml). The organic layer was dried with MgS04 (s), filtered and evaporated to dryness to obtain the product. Yield: 51 mg (63%); orange solid. 1H MR (400 MHz, OMSO-d6): δ 3.75 (s, 2H), 3.86 (s, 2H), 7.12 (d, J 5.3 Hz, IH), 7.32 (d, J3.9 Hz, IH), 7.47 (d, J3.9 Hz, IH), 7.57 (d, J 5.3 Hz, IH), 7.61 (m, 2H), 7.70 (m, IH), 7.74 (s, IH), 7.82 - 7.88 (m, 2H), 12.62 (bs, 2H). HPLC: RT = 2.50 min. 98% at 254 nm (10-40% MeCN in buffer, 3 min, XBridge) and RT = 2.49 min, 96% at 400 nm (10-40% MeCN in buffer, 3 min, XBridge). LC-MS: m/z = 469 (M + H).
Methyl 2-(5-benzoyl-2-{5-[5-benzoyl-3-(2-methoxy-2-oxoethyl)thiophen-2-yl]thiophen-2- yl}thiophen-3-yl)acetate (47 mg, 0.078 mmol) was dissolved in dioxane (2 ml), followed by addition of 2 M NaOH (2 ml) and water (1 ml). After stirring 45 min at 70 °C, the reaction was cooled to RT, diluted with water (10 ml) and acidified using 1 M HC1 (approx. 4 ml). The reaction mixture was evaporated to dryness, dissolved in EtOAc (15 ml), washed with water (15 ml). The organic layer was dried with MgS04 (s), filtered and evaporated to dryness to obtain the product. Yield: 47 mg (94%); yellow oil. 1H NMR (400 MHz, OMSO-d6): δ 3.88 (s, 4H), 7.55 (s, 2H), 7.57 - 7.64 (m, 4H), 7.71 (m, 2H), 7.76 (s, 2H), 7.84 - 7.88 (m, 4H), 12.72 (bs, 2H). HPLC: RT = 3.10 min. 95% at 254 nm (10-40% MeCN in buffer, 3 min, XBridge) and RT = 3.09 min, 96% at 400 nm (10-40% MeCN in buffer, 3 min, XBridge). LC- MS: m/z = 573 (M + H).
Figure imgf000124_0001
Example 96. 5-[5-(5-(5-[5-(5-Carboxythiophen-2-vO-3-(2-hvdroxye^
yl]thiophen-2-yl}thiophen-2-yl)-4-(2-hydroxyethyl)thiophen-2-yl]thiophene-2-carb acid (9708 099)
Argon was bubbled through a mixture of intermediate F (633 mg, 3.06 mmol), 2,2'- bithiophene-5-5'-diboronic acid pinacol ester (575 mg, 1.38 mmol) and KF (532 mg, 9.17 mmol) in toluene (8 ml) and MeOH (8 ml). PEPPSI-iPr™ (41 mg, 0.061 mmol) was added and the mixture heated at 55 °C for 45 min. Silica was added to the mixture and solvents evaporated. The dry silica was applied on a column which was eluted with 30-50% ethyl acetate in toluene. Yield: 324 mg (56%); yellow solid. 1H NMR (400 MHz, CDC13): δ 3.09 (t, J 6.7 Hz, 4H), 3.92 (t, 6.5 Hz, 4H), 7.01 (d, J 5.3 Hz, 2H), 7.08 (d, J3.8 Hz, 2H), 7.13 (d, 3.8 Hz, 2H), 7.24 (d, J 5.3 Hz, 2H).
NBS (275 mg, 1.54 mmol) was added portion-wise to the solution of the material from above (324 mg, 0.774 mmol) in CHC13 (10 ml) and AcOH (10 ml). The mixture was stirred at ambient temperature overnight. 2 M NaOH and EtOAc were added. Organic phase was separated, washed with water and evaporated. Yield: 421 mg (94%).
Argon was bubbled through the solution of the crude material from above (50 mg, 0.0867 mmol) and 5-carboxythiophene-2-boronic acid (48 mg, 0.481 mmol) in dioxane (1 ml) and 1 M K2C03 (1 ml). PEPPSI-iPr™ (2 mg, 0.003 mmol) was added and the mixture heated at 80 °C for 20 min. Mixture was filtered and product isolated by prep. hplc. Pure fractions were combined and some solvents were evaporated. Solid material was precipitated with 2 M HCl, collected by centrifugation and washed with water three times. Yield: 23.2 mg (40%); red solid. Rt = 2.75 min, 100% at 254 nm (10-40% MeCN in buffer, XBridge) and Rt = 2.74 min, 100% at 400 nm (10-40% MeCN in buffer, XBridge). 1H NMR (400 MHz, OMSO-d6): δ 2.93
(t, J6.8 Hz, 4H), 3.69.3.77 (m, 4H), 4.87 (t, J 5.0 Hz, 2H), 7.33 (d, J3.8 Hz, 2H), 7.38 (d, J 4.0 Hz, 2H), 7.43 (d, J4.0 Hz, 2H), 7.67 (d, J 3.8 Hz, 2H). LC-MS: m/z = 669 (M-l).
Further compounds The following compounds may further be synthesized.
Figure imgf000125_0001
Figure imgf000126_0001
Figure imgf000127_0001
Figure imgf000128_0001
Figure imgf000129_0001
Figure imgf000129_0002
Figure imgf000130_0001
Figure imgf000131_0001
Figure imgf000131_0002
Figure imgf000131_0003
Figure imgf000131_0004
Figure imgf000132_0001
LABELLING OF COMPOUNDS MRI:
Examples of compounds for MRI
A deuteron analogue of Example 21 can easily be prepared using commercially available phenyl-d5-boronic acid according to the scheme below.
Figure imgf000132_0002
Other commercially available deuterio boronic acids may be used analagously.
19F containing compounds already prepared:
Example 77 Example 78
Example 87
Other examples easily prepared:
Figure imgf000133_0001
2. NaOH , dioxane/H20
Suggested synthesis of fluonnated analogue of Example 1 :
Figure imgf000133_0002
Selectfluor
Bu4NF
Figure imgf000133_0003
[Selectfluor = l-Chloromethyl-4-fluoro-l,4-diazoniabicyclo[2.2.2]octane bis(tetrafluoroborat] using the nitro group to induce π-stacking in terthiophenes (Sears, Wendy A. et al. Canadian Journal of Chemistry, 88(4), 309-317; 2010) or
Figure imgf000134_0001
PET:
18F may be introduced using methods described in Chun, Joong-Hyun; Lu, Shuiyu; Pike, Victor W. Rapid and Efficient Radiosyntheses of meta- Substituted [18F]Fluoroarenes from [18F]Fluoride Ion and Diaryliodonium Tosylates within a Microreactor. European Journal of Organic Chemistry (201 1 ), 2011(23), 4439-4447, S4439/1-S4439/67.
Figure imgf000134_0002
Figure imgf000134_0003
Nucleophilic [18F]fluorination and subsequent decarbonylation of methoxy-substituted nitro- and halogen-benzenes activated by one or two formyl groups. Shen, Bin et al. Journal of Labelled Compounds and Radiopharmaceuticals, 53(3), 113-119; 2010.
Microwave-accelerated fluorodenitrations and nitrodehalogenations: expeditious routes to labeled PET ligands and fluoropharmaceuticals. La Beaume, Paul, et al. Tetrahedron Letters, 51(14), 1906-1909; 2010.
1.
Figure imgf000135_0001
toluene/ eOH 80 °C
2. NaOH, dioxane/H20
2- 18Fluorothiophene :
Fluorine-labeling of thiophene and N-methylpyrrole. Crestoni, Maria Elisa. Journal of Labelled Compounds and Radiopharmaceuticals, 28(9), 1109-12; 1990.
Figure imgf000135_0002
3 - 18Fluorothiophene :
Fluorine-labeling of thiophene and N-methylpyrrole. Crestoni, Maria Elisa. Journal of Labelled Compounds and Radiopharmaceuticals, 28(9), 1 109-12; 1990.
I labeled compound:
Figure imgf000136_0001
1. Bis(neopentyl glycolato)diboron
AcOK, Pd(dppf)CI2, DMSO, 80 °C
2. NaOH, p-MeC6H4S02NCI- «Na+, Na123l, H20, THF, 5 min, rt
Figure imgf000136_0002
Synthesis of radioiodinated aryl iodides via boronate precursors. Kabalka, George W. et al. Nuclear Medicine and Biology, 29(8), 841-843; 2002. IMAGING EXPERIMENTS
IN VITRO IMAGING OF AGGREGATED PROTEINS: STAINING General information
• Staining of brain sections from an APPSL x TMHT double-transgenic mouse that features several hallmarks of familial Alzheimer's disease (AD) that contain pathological material such as AB deposits.
• The human APP with London (717) and Swedish (670/671) mutations, hAPP751 SweLon, is expressed in high levels, resulting in an age-dependent increase of B-amyloidl-40 and B-amyloidl-42, the pathologically relevant forms of amyloid protein. The mice develop plaques consisting of amyloid depositions in early age, starting at 3 - 6 months in the frontal cortex.
• Staining of brain sections brain sections from R6/2 mice that feature several hallmarks of Huntington's Disease (HD) that contain pathological material such as Htt deposits.
• Staining of brain sections brain sections from a SOD1-G93A transgenic mouse that
features several hallmarks of familial inherited Amyotrophic Lateral Sclerosis (ALS) that contain pathological material such as SOD deposits
• Staining of brain sections brain sections from TDP43 +/+ transgenic mouse that features several hallmarks of familial inherited Amyotrophic Lateral Sclerosis (ALS). The staining capacity of compounds prepared in Examples above on slices of Tg mouse models in different clinical indications (Alzheimer's Disease (AD), Amyotrophic Lateral Sclerosis (ALS) and Huntington's Disease (HD)) was investigated. This study provides important information on in vitro target engagement that indicates therapeutic possibilities to use the compounds of the present invention for staining and treatment of pathological features in various neurodegenerative disorders.
Material
Material: Alzheimer's Disease (AD) - APPSL x TMHT
Brain slices from an APPSL x TMHT mouse at 6 months of age were obtained. They are crossbreds of APPSL mice (Rockenstein et al. 2001) and TMHT mice. Mice were bred and maintained in a certified animal facility until sacrification. At sacrifice, mice were transcardially perfused with 0.9% NaCl to wash out the majority of erythrocytes. After extraction, brains were post-fixed for one hour in 4% Paraformaldehyde in PBS at room temperature and then transferred to 15% sucrose over night for cryoprotection. Thereafter brains were shock-frozen in dry ice cooled liquid Isopentane. Mounted in OCT medium, brains were cut systematically on a Leica CM3050 S cryotome with 10 μπι slice thickness.
Material: Alzheimer's Disease (AD) - hAPP751 SweLon
Brain slices from hAPP751 SweLon mouse at 9.5 - 12 months of age were obtained. Mice were bred and maintained in a certified animal facility until sacrification. At sacrifice, mice were transcardially perfused with 0.9% NaCl to wash out the majority of erythrocytes. After extraction, brains were post-fixed for one hour in 4% Paraformaldehyde in PBS at room temperature and then transferred to 15% sucrose over night for cryoprotection. Thereafter brains were shock-frozen in dry ice cooled liquid Isopentane. Mounted in OCT medium, brains were cut systematically on a Leica CM3050 S cryotome with 10 μπι slice thickness.
Material: Huntington's Disease (HD)
Brain slices from a R6/2 mouse at 4.5 months of age were obtained. Mice were bred and maintained in the AAALAC certified animal facility of JSW Life Sciences until sacrification. At sacrifice, mice were transcardially perfused with 0.9% NaCl to wash out the majority of erythrocytes. After extraction, brains were post-fixed for 24 hours in 4% Paraformaldehyde in PBS at 4°C and then paraffin embedded. Brains were cut systematically on a Leica SM2000 R slide microtome with 5 μπι slice thickness.
Material: Amyotrophic Lateral Sclerosis (ALS) - SODl
Brain slices from SOD1-G93A Tg mice, further referred to as SODl mice, at 17 weeks of age were obtained. SODl transgenic mice were first introduced by Gurney et al. in 1994. Animals express high levels of mutated human SODl that carries a G93→A substitution. The SOD1- G93 A mice were purchased from the Jackson Laboratory
(http://jaxmice.jax.org/strain/002726.html) and maintained in the AAALAC certified animal facility of JSW Life Sciences until sacrifice. At sacrifice, mice were transcardially perfused with 0.9% NaCl to wash out the majority of erythrocytes. After extraction, brains were post- fixed for one hour in 4% Paraformaldehyde in PBS at room temperature and then transferred to 15%) sucrose over night for cryoprotection. Thereafter brains were shock-frozen in dry ice cooled liquid Isopentane. Mounted in OCT medium, brains were cut systematically on a Leica CM3050 S cryotome with 10 μπι slice thickness. Material: Amyotrophic Lateral Sclerosis (ALS) - TDP43
Brain sections from TDP43 +/+ transgenic mouse that features several hallmarks of familial inherited Amyotrophic Lateral Sclerosis (ALS). Overexpressses hTAR DNA binding protein- 43 hThy-1 promotor (constitutive neuronal expression). Ubiqutinated, phosphorylated TDP43 inclusions in nucleus and cytoplasm.
Histochemical Staining Protocol
1) Pre-treatment:
la) Cryosections: Dry cryosections 60 min at room-temperature
lb) Paraffin sections: Deparaffinize and rehydrate as following: 10 min with Tissue Clear, 05 min with Tissue Clear/100% Ethanol (1 : 1), 02 min with 100% Ethanol, 02 min with 96% Ethanol, 02 min with 70% Ethanol, 02 min with 50% Ethanol and finally 02 min with PBS.
2) Post-fixation in 4% Paraformaldehyde: Fixate air-dried cryosections for 10 min with 4% paraformaldehyde at room temperature
3) Washing: 2 times for 5 min with PBS at room temperature
4) Fixation: 10 min in ice-cold 100% Ethanol
5) Fixation: 5 min in ice-cold 70% Ethanol
6) Washing 1 min in ddH20 7) Washing: 10 min in 100 mM Sodium-Carbonate Puffer (pH=10).
8) Compound incubation: Incubate for 30 min at room temperature light-protected in a damp chamber.
9) Washing: 2 times for 5 min with 100 mM Sodium-Carbonate Puffer (pH=10) (light- protected). 10) Washing: 5 min in PBS (light-protected).
11) Labeling the cell nuclei: Incubate for 15 min with DAPI [(25mg/ml in ddH20) and methanol] at room temperature (light-protected).
Imaging
A fully automated Zeiss Axio. Imager Zl fluorescent microscope equipped with LED illumination (Colibri) and AxioCam MRm B&W camera served to record the images.
Images of cortical plaques were systematically recorded.
Results: Alzheimer's Disease (AD) - APPSL x TMHT
The table summarizes the results of compound staining in brain slices of APPSL mice.
Figure imgf000140_0001
Results: Alzheimer's Disease (AD) - hAPPSweLon
The table summarizes the results of compound staining in brain slices of hAPPSweLon.
Compound Plaque cores Mature fibers Immature fibers p-FTAA ++ +
Ex. 1 +++ ++ +
Ex. 21 ++ + Results: Huntington's Disease (HD)
The table summarizes the results of compound staining in brain slices of HD/htt related pathology in R6/2 mice.
Figure imgf000141_0001
Results: Amyotrophic Lateral Sclerosis (ALS) - SOD1
The table summarizes the results of compound staining in brain slices of ALS/SOD related pathology in SOD1-G93A Tg mice.
Compound Neuronal Fibers Astrocytes Amyloid SOD1 somata aggregate p-FTAA + + + + +
Ex. 1 +++ +++ +++ +++ ++
Ex. 13 + + + + +
Ex. 15 + + +
Ex. 19 +++ +++ +++ +++ ++
Ex. 35 + ++ ++ + +
Ex. 47 +
Ex. 52 ++ ++ ++ ++ +
Ex. 56 +++ +++ +++ +++ ++
Ex. 62 +++ +++ +++ +++ +++ Results: Amyotrophic Lateral Sclerosis (ALS) - TDP43
The compounds of the present invention stain both human TARDBP (nuclear) and phospho TDP-43 (extra-nuclear aggregates). The table below summarizes the obtained results in various parts of the brain.
Figure imgf000142_0001
IN VIVO IMAGING OF AGGREGATED PROTEINS
General information
Brain localization of compounds prepared in Examples 1 and 21 above was investigated using in vivo two photon microscopic imaging. Prior to in vivo imaging, an in vitro test was performed by Neurotar to characterize fluorescent properties of Example 1 compounds under two-photon excitation conditions. Vehicle: Saline (155 mM NaCl.
Test Item (T.I): Compounds of Example 1 and Example 2, respectively, diluted in vehicle from stock solution in deionized water (10 mg/ml).
Material Material: Alzheimer's Disease (AD) - hAPP751 SweLon
hAPP751 SweLon mouse at 10 months of age were obtained. Mice were bred and maintained in a certified animal facility. The human APP with London (717) and Swedish (670/671) mutations, hAPP751 SweLon, is expressed in high levels, resulting in an age-dependent increase of B-amyloidl-40 and B-amyloidl-42, the pathologically relevant forms of amyloid protein. The mice develop plaques consisting of amyloid depositions in early age, starting at 3 - 6 months in the frontal cortex. Treatment
Compounds were injected into blood circulation of the TG mice (APP751 SL, female, 10 months old). Acquisition of Z-stacks was carried out at 30-40 minute intervals for the whole duration of the imaging session. The animal was kept anaesthetized and its vital functions closely monitored throughout the imaging session. 150 μΐ^ of solutions of compounds prepared in Example 1 or Example 2 were injected intravenously in the mouse tail vein. First image stack was acquired within 10 minutes after the intravenous injection.
Experimental settings and data analysis
Image acquisition setup
Illumination: 800 nm, femtosecond laser Mai-Tai Broad Band DeepSee.
Emission filters
Cube B:
Channel 1 - band pass 526-557 nm for Example 1 (predominantly intravascular fluorescence) Channel 2 - band pass 580-638 nm for Example 1 (predominantly fluorescence in plaques) Cube A:
Channel 1 - band pass 515-560 nm for Example 1 (predominantly intravascular fluorescence) Channel 2 - band pass 590-650 nm for Example 1 (predominantly fluorescence in plaques) Stacks of images were collected with the vertical step of 1 μπι (total depth of stack 400 μπι) at the temporal interval of 30-35 minutes. Each imaging field covered 500 X 500 μπι2 of space in xy coordinates.
Data Analysis
After acquisition, the data were analyzed using Fiji/ImageJ software. For analyzing the localization of Example 1 and Example 21 at least three ROIs were selected in each of the studied locations: neuropil (parenchyma), blood vessels and Abeta plaques (or other suspicious intracortical fluorescent objects). The data were transferred to Origin 7.5 software for statistical analysis and plotting of graphs. Data points from vascular, parenchymal and plaque ROIs were normalized to the pre-injection or first imaging timepoint values.
Fluorescence intensity values from ROIs were pooled and presented as mean ± SEM. Experiment 1
During in vivo Experiment 1, 10 mg/kg of Example 1 solution were injected i.v. in mouse and followed by during 4 hours of imaging session. As shown in Figure 1, Example 1 compound labels plaque efficiently and the fluorescence of these plaques increased with accelerated rate until the end of the imaging session. Total number of individual plaques in volume of imaged cortical tissue was approximately 12-15. Plaques varied in shape and size: their diameter ranged from 10 to 20 μπι. Synchronously with accumulation in parenchymal Abeta plaques was also noticed accumulation of Example 1 compound in perivascular zones (presumably plaques) around many large and medium vessels.
Main conclusions from Experiment 1 :
1) Example 1 compound starts to accumulate inside intracortical and perivascular plaques.
2) Total number of intracortical plaques is around 15 in the imaged volume.
Experiment 2
Compound of Example 1 for its ability to cross BBB and stain Abeta plaques. For this purpose 10 mg/kg were injected i.v. followed by 15 mg/kg of compound of Example 21 and monitored. As shown in Figure 2, the compound of Example 21 labels plaque and the fluorescence of these plaques increases until the end of the imaging session.
Similarly to Experiment 1 with the Example 1 compound, observed both bright
autofluorescent spots (presumably lipofuscin) as well as a large amount of large (up to 50 um in diameter) and relatively dim intraparenchymal objects (plaques, Figure 2) were observed. Main conclusions from Experiment 2:
1) The mouse used in Experiment 2 contained a high number of large objects (plaques) and small bright objects (presumably lipofuscin), which are not related to staining produced by the
Example 21 compound.
2) Example 21 compound stains intracortical and perivascular Abeta plaques.
Confirmation of in vivo results: in vitro staining
A follow up study was performed to correlatively trace amyloid labeling of in vivo
administered and imaged Example 1 and Example 21 compounds by histological methods ex vivo in the brain of APPSL transgenic mice. Furthermore, the general distribution of Example 1/Example 21 staining in the brain was investigated by multiple immunofluorescent co- labeling with 6E10 and LOC antibodies.
After brains were imaged in vivo in Experiments 1 and 2, and after a certain time mice were sacrificed. The brains were removed, frozen in liquid nitrogen and histological analysis was done.
Plaque load was determined with 6E10 antibody (Covance®, #SIG-39320) directed against the human amyloid peptide (amino acids 1-16) and an anti-amyloid fibrils LOC antibody (Millipore®, #AB2287) against amyloid fibrils in a double incubation.
Both the compound of Example 1 and the compound of Example 21 did show in vitro fluorescence on aggregated Abeta in the brain with different degrees of intensities leading to a broad spectrum of red and green spectrum color mix. With the narrow band filter sets used in this study red and green color channels of the Example 1 compound rather overlap leading to a seemingly uniform color mix at lower magnification. However, at higher magnification color gradations on plaques are visible. The compound of Example 21 showed different spectral mixes for parenchymal plaques and CAA.

Claims

1. A compound of formula (I)
(C)n-B-(A)m-B-(C> 'n (I) wherein m is 0 or 1, and n is independently 0, 1, 2 or 3,
A, each B and each C are independently selected from phenylene and five- and six-membered heteroaromatic rings, and for a terminal ring B or C also from bicyclic heteroaromatic fused rings having seven to ten ring members, wherein the bond between at least two of the rings A to C may be replaced by a carbonyl group ( -CO- ), wherein at least two of the rings A to C are substituted with one or two groups R, and wherein each ring A to C further optionally is substituted with one or two groups R , wherein each group R is independently selected from hydroxy, hydroxyalkyl, hydroxyalkoxy, hydroxyalkoxyalkyl, hydroxypolyoxyalkylene, alkoxy, alkoxyalkyl, polyoxyalkylene, carboxy, carboxyalkyl, carboxyalkoxy, carboxyalkoxyalkyl, carboxypolyoxyalkylene, alkoxycarbonyl, alkoxycarbonylalkyl, alkoxycarbonylalkoxy, alkoxycarbonylalkoxyalkyl, alkoxycarbonylpolyoxyalkylene, amino, alkylamino, dialkylamino, aminoalkyl,
alkylaminoalkyl, dialkylaminoalkyl, aminoalkoxy, alkylaminoalkoxy, dialkylaminoalkoxy, aminopolyoxyalkylene, alkylaminopolyoxyalkylene, dialkylaminopolyoxyalkylene, aminoalkoxyalkyl, alkylaminoalkoxyalkyl, dialkylaminoalkoxyalkyl, (amino)(carboxy)alkyl, (alkylamino)(carboxy)alkyl, (dialkylamino)(carboxy)alkyl, (amino)(carboxy)alkoxy,
(alkylamino)(carboxy)alkoxy, (dialkylamino)(carboxy)alkoxy, (amino)(carboxy)alkoxyalkyl, (alkylamino)(carboxy)alkoxyalkyl, (dialkylamino)(carboxy)alkoxyalkyl,
(amino)(carboxy)polyoxyalkylene, (alkylamino)(carboxy)polyoxyalkylene, (dialkylamino)(carboxy)polyoxyalkylene, (alkoxycarbonyl)(amino)alkyl,
(alkoxycarbonyl)(alkylamino)alkyl, (alkoxycarbonyl)(dialkylamino)alkyl,
(alkoxycarbonyl)(amino)alkoxy, (alkoxycarbonyl)(alkylamino)alkoxy,
(alkoxycarbonyl)(dialkylamino)alkoxy, (alkoxycarbonyl)(amino)alkoxyalkyl,
(alkoxycarbonyl)(alkylamino)alkoxyalkyl, (alkoxycarbonyl)(dialkylamino)alkoxyalkyl, (alkoxycarbonyl)(amino)polyoxyalkylene, (alkoxycarbonyl)(alkylamino)polyoxyalkylene, (alkoxycarbonyl)(dialkylamino)polyoxyalkylene, acylamino, acylaminoalkyl,
acylaminoalkoxy, acylaminoalkoxyalkyl, acylaminopolyoxyalkylene, acylalkylamino, acylalkylaminoalkyl, acylalkylaminoalkoxy, acylalkylaminoalkoxyalkyl,
acylalkylaminopolyoxyalkylene, hydrazinocarbonyl, hydrazinocarbonylalkyl,
hydrazinocarbonylalkoxy, hydrazinocarbonylalkoxyalkyl,
hydrazinocarbonylpolyoxyalkylene, nitro, nitroalkyl, nitroalkoxy, nitroalkoxyalkyl, nitropolyoxyalkylene, cyano, cyanoalkyl, cyanoalkoxy, cyanoalkoxyalkyl,
cyanopolyoxyalkylene, sulfo, sulfoalkyl, sulfoalkoxy, sulfoalkoxyalkyl,
sulfopolyoxyalkylene, alkylsulphonyl, alkyl sulphinyl, alkylaminosulphonyl, acyloxyalkyl, carboxyalkenyl, pyrrolidincarbonyl, morpholinoalkyl, cycloalkylcarbamoyl, trifluoroxy, carbamoyl, hydroxyalkylaminoalkyl, carboxyalkylaminoalkyl, aminoalkylaminoalkyl, piperidinealkyl, piperazinealkyl, or any two groups R attached to the same thiophene ring taken together are alkylenedioxy, optionally substituted with sulfoalkyl, sulfoalkoxy, sulfoalkoxyalkyl or sulfopolyoxyalkylene; and wherein each group R1 is independently selected from halogen, alkyl, alkenyl, alkynyl, trifluoro, with the provisos that:
(i) when m=0, then one n is other than 0 or both B represent a bicyclic heteroaromatic fused ring, and, preferably,
(ii) when m=l and each n=l, A, B and C are thienylene, and C is unsubstituted or mono- substituted with carboxy or iodo, then a substituent R on a ring B in ortho-position to A is other than carboxymethyl, methoxycarbonylmethyl and aminoethyl; wherein any H2 group may optionally be protected as a tert-butyl carbamate, benzyl carbamate or 9-fluorenylmethyl carbamate or substituted with a biotinyl moiety; wherein any alkyl or alkylene moiety is Ci-6-alkyl or Ci-6-alkylene, and any alkenyl and alkynyl moiety is C2-6-alkenyl and C2-6-alkynyl, respectively; or a pharmaceutically acceptable salt thereof, wherein the compound is labelled with a label detectable with an in vivo imaging method.
2. The compound according to claim 1, wherein rings A, B and C are selected from phenylene, pyridinylene, pyrazinylene, pyridazinylene, pyrimidinylene, thienylene, thiazolylene, thiadiazolylene, oxazolylene, furanylene, triazolylene, pyrazolylene, imidazolylene and pyrrolylene.
3. The compound according to claim 1 or 2, wherein the bicyclic heteroaromatic fused rings for B and C are selected from benzothienylene, benzodiazolylene and indolylene.
4. The compound according to claim 1, 2 or 3, wherein said group R for A is a
2 3 4 2 3 4
group R , for B a group R , and for C a group R , wherein each R , R and R are
independently selected from carboxy, carboxyalkyl, aminoalkyl, acylaminoalkyl,
acyloxyalkyl, hydroxyalkyl, alkylsulphonyl, alkylsulphinyl, alkylaminosulphonyl, alkoxycarbonylalkyl, morpholinoalkyl, carboxyalkenyl, hydroxy, alkoxycarbonyl, pyrrolidinecarbonyl, morpholino, cycloalkylcarbamoyl, cyano, trifluoroxy, carbamoyl, hydroxyalkoxyalkyl, carbamoylalkyl, hydroxyalkylaminoalkyl, cyanoalkyl, alkylaminoalkyl, aminoalkylaminoalkyl, carboxyalkylaminoalkyl, piperidinealkyl, and piperazinealkyl.
5. The compound according to claim 4, wherein each R2 and R3 are independently selected from carboxy, carboxyalkyl, alkoxycarbonylalkyl, morpholinoalkyl, aminoalkyl, alkylcarbonylaminoalkyl, hydroxyalkoxyalkyl, carbamoylalkyl, hydroxyalkylaminoalkyl, cyanoalkyl, aminoalkylaminoalkyl, carboxyalkylaminoalkyl, piperidyl and piperazinyl, and each R4 is independently selected from carboxy, alkylsulphonyl, alkylaminosulphonyl, hydroxyalkyl, carboxyalkenyl, hydroxy, alkoxycarbonyl, pyrrolidinoyl, morpholino, cycloalkylcarbamoyl, cyano, trifluoroxy, and carbamoyl.
6. The compound according to any one of claims 1 to 5, wherein A is phenylene or thienylene.
7. The compound according to any one of claims 1 to 6, wherein each B is thienylene or each B is phenylene.
8. The compound according to any one of claims 1 to 6, wherein each ring C independently is unsubstituted, monosubstituted or di substituted.
9. The compound according to any one of claims 1 to 8, wherein ring A is unsubstituted.
10. The compound according to any one of claims 1 to 9, wherein each group R3 is independently selected from carboxy-Ci-4-alkyl, hydroxy-Ci-4-alkyl, amino-Ci-4-alkyl,
Ci-6-acylamino-Ci-4-alkyl, Ci-4-alkoxycarbonylamino-Ci-4-alkyl, and morpholino-Ci-4-alkyl.
11. The compound according to any one of claims 1 to 10, wherein each R4 is independently selected from carboxy, carboxy-Ci-4-alkyl, Ci-4-alkylsulphonyl, carboxy-Ci-4- alkylene, hydroxy, Ci-4-alkyl, Ci-4-alkoxycarbonyl, pyrrolidinecarbonyl, morpholino, C3-6-cycloalkylcarbamoyl, cyano, trifluoroxy, carbamoyl and amino-Ci-4-alkyl.
12. A compound according to any one of claims 1 to 11, wherein said label is detectable with a method selected from the group consisting of PET, SPECT, NMR, MRS, MRI, NIR, MPM and CAT.
13. A compound according to any one of claims lto 12 wherein the label comprises a detectable isotope selected from 18F, 19F, 123I, nC 2H, nC, 13C, 14C, 18C, 13N, 15N, 150 170, 180, 18F, 35S, 36C1, 75Br, 76Br, 77Br, 82Br, 120I, 123I, 124I, 125I, 131I, 67Ga, 81mKr, 82Rb, mIn, 133Xe, 201T1 90Y and 99mTc.
14. A compound according to claim 12, wherein the compound comprises a detectable micro- or nanoparticle, such as a gold particle, a magnetic, supramagnetic or ferromagnetic particle, a lanthanide particle (e.g. Gd, Eu or Nd) optionally doped with a metal, or a nanocrystal.
15. A composition comprising a compound according to any of claims 1 to 14, and optionally a pharmaceutically acceptable buffer, diluent, excipient and/or carrier.
16. A method for imaging of misfolded or aggregated forms of proteins in a sample or subject, comprising the steps of:
- bringing the sample in contact with, or administering to the subject, a compound of formula I as defined in any one of claims 1 to 14, optionally labelled with a label detectable with an in vivo imaging method;
- allowing said compound to bind to said misfolded or aggregated forms of proteins; and
- detecting the presence of said compound;
- optionally localizing said compound in said sample or subject;
- optionally quantifying the amount of said compound in said sample or subject.
17. A method for diagnosis of a disease involving misfolded or aggregated forms of proteins in a mammalian subject, comprising the steps of
- bringing a sample from said subject in contact with, or administering to the subject, a compound of formula I as defined in any one of claims 1 to 14, optionally labeled with a label detectable with an in vivo imaging method;
- allowing said compound to bind to said misfolded or aggregated forms of proteins in said sample or subject;
- allowing unbound amounts of said compound to be cleared from the subject, or
washing unbound amounts of said compound from said sample; and
- detecting the presence of said compound in said subject or sample;
- optionally localizing said compound in said subject or sample;
- optionally quantifying the amount of said compound in said subject or sample;
wherein the presence of said compound in said subject or sample is indicative of a disease involving misfolded or aggregated forms of protein.
18. A method according to claim 16 or 17, wherein the detection step is performed with a method selected from the group consisting of PET, SPECT, NMR, MRS, MRI, CAT, NIR and MPM.
19. Use of a compound of formula I as defined in any one of claims 1 to 14 in a method according to any of claims 16 to 18.
20. Use of a compound of formula I as defined in any one of claims 1 to 14, labeled or unlabeled, in imaging.
21. A compound of formula I in any one of claims 1 to 14, labelled or unlabeled, for use in imaging.
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