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WO2019008152A1 - 3-cyanothiophene derivatives as inhibitors of the pd-1/pd-l1 protein/protein interaction - Google Patents

3-cyanothiophene derivatives as inhibitors of the pd-1/pd-l1 protein/protein interaction Download PDF

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WO2019008152A1
WO2019008152A1 PCT/EP2018/068411 EP2018068411W WO2019008152A1 WO 2019008152 A1 WO2019008152 A1 WO 2019008152A1 EP 2018068411 W EP2018068411 W EP 2018068411W WO 2019008152 A1 WO2019008152 A1 WO 2019008152A1
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Alexander Dömling
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Rijksuniversiteit Groningen
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D333/00—Heterocyclic compounds containing five-membered rings having one sulfur atom as the only ring hetero atom
    • C07D333/02—Heterocyclic compounds containing five-membered rings having one sulfur atom as the only ring hetero atom not condensed with other rings
    • C07D333/04—Heterocyclic compounds containing five-membered rings having one sulfur atom as the only ring hetero atom not condensed with other rings not substituted on the ring sulphur atom
    • C07D333/26—Heterocyclic compounds containing five-membered rings having one sulfur atom as the only ring hetero atom not condensed with other rings not substituted on the ring sulphur atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D333/38—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
    • 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
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • 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
    • C07D409/12—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing two hetero rings linked by a chain containing hetero atoms as chain links
    • 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
    • C07D413/04—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 directly linked by a ring-member-to-ring-member bond
    • 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
    • C07D413/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing three or more hetero 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
    • C07D417/12—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 chain containing hetero atoms as chain links
    • 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

  • the present invention provides novel compounds that are useful as inhibitors of the PD-1/PD-L1 protein/protein interaction.
  • the PD-l/PD-Ll axis is hijacked by viruses, bacteria and uncontrolled fast growing cells to suppress the immune surveillance.
  • the malignant cells express PD-L1 which bind to the PD1 receptor expressed on immune T-cells. Binding of PD-1 to PD-1L determines a downregulation of T-cell effector functions in cancer patients inhibiting the antitumor immune response and leading to T-cell exhaustion.
  • a similar mechanism is used by viruses and bacteria to undermine the effective immune recognitions and answer.
  • the present invention provides compounds of formula (I) :
  • R 1 is a hydrogen atom or a halogen atom
  • R 2 is a hydrogen atom or a halogen atom
  • R 1 and R 2 together are a group of formula -O-CH2-CH2-O- , -O-CH2- CHa-N(Me)-, -N(Me) -CH2-CH2-O-, -O-CF2-O- or -O-CH2-O-;
  • R 4 is a hydrogen atom or an alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, alkylcycloalkyl, hetero- alkylcycloalkyl, aryl, heteroaryl, aralkyl or heteroaralkyl group; all of which groups may optionally be substituted; and
  • R 5 is a hydrogen atom or an alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, alkylcycloalkyl, hetero- alkylcycloalkyl, aryl, heteroaryl, aralkyl or heteroaralkyl group; all of which groups may optionally be substituted; or
  • R 4 and R 5 together with the nitrogen atom to which they are bound are part of an optionally substituted heterocycloalkyl group containing 5 , 6 or 7 ring atoms ; or a pharmaceutically acceptable salt, ester, solvate or hydrate or a pharmaceutically acceptable formulation thereof.
  • R 1 and R 2 together are a group of formula -O- CH2 - CH2-0- or -O- CH2-O-.
  • R 1 and R 2 are both hydrogen atoms.
  • R 1 and R 2 are both chlorine atoms.
  • X is a bond, an oxygen atom or a group of formula -(CH 2 ) n - wherein n is 1, 2 or 3 or a group of formula - ( CH 2 ) 3 -0- .
  • X is a bond.
  • Ar is a phenyl group or a heteroaryl group containing one ring and 5 or 6 ring atoms selected from C, O, N and S, wherein Ar may be substituted by 1, 2, 3, 4 or 5 groups R 3 and/or wherein Ar may be substituted by a group of formula -CH 2 -N (R 4 ) -R 5 .
  • Ar is substituted by one or two groups R 3 .
  • the groups R 3 are independently halogen, NO2 , N 3 , OH, SH, NH 2 or a Ci - C 6 alkyl group; a Ci - Ce heteroalkyl group; a cycloalkyl group containing one ring having from 3 to 7 ring atoms; a heterocycloalkyl group containing one ring having from 3 to 7 ring atoms comprising 1 or 2 heteroatoms selected from S, O and N; an alkylcycloalkyl group containing a C 1 -C6 alkyl group and a cycloalkyl group containing one ring having from 3 to 7 ring atoms; a heteroalkylcycloalkyl group containing a Ci - C 6 alkyl group or a C1-C6 heteroalkyl group and a cycloalkyl group containing one ring having from 3 to 7 ring atoms or a heterocycloalkyl group containing one ring having from 3 to 7 ring atoms
  • Ar is substituted by a group of formula - CH 2 -N ( R 4 ) - R 5 .
  • Ar is substituted by a group of formula - CH2 -N ( R 4 ) - R 5 and further by one group R 3 , which is preferably selected from F, CI , -OMe and - CHO .
  • R 4 is a hydogen atom and R 5 is a hydrogen atom or a C1-C6 alkyl group; a C1-C6 heteroalkyl group; a cycloalkyl group containing one ring having from 3 to 7 ring atoms; a heterocycloalkyl group containing one ring having from 3 to 7 ring atoms comprising 1 or 2 heteroatoms selected from S, O and N; an alkylcycloalkyl group containing a Ci - Ce alkyl group and a cycloalkyl group containing one ring having from 3 to 7 ring atoms; a heteroalkylcycloalkyl group containing a C1 - C6 alkyl group or a C1 - C6 heteroalkyl group and a cycloalkyl group containing one ring having from 3 to 7 ring atoms or a heterocycloalkyl group containing one ring having from 3 to 7 ring atoms
  • alkyl refers to a saturated, straight-chain or branched hydrocarbon group that contains from 1 to 20 carbon atoms, preferably from 1 to 12 carbon atoms, especially from 1 to 6 (e.g. 1, 2, 3 or 4) carbon atoms, for example a methyl, ethyl, propyl, iso-propyl, n-butyl, iso-butyl, sec-butyl, tert-butyl, n-pentyl, iso-pentyl, n-hexyl, 2 , 2-dimethylbutyl or n-octyl group.
  • alkyl refers to groups in which one or more hydrogen atoms have been replaced by a halogen atom (preferably F or CI) such as, for example, a 2 , 2 , 2-trichloroethyl or a trifluoromethyl group.
  • a halogen atom preferably F or CI
  • alkenyl and alkynyl refer to at least partially unsaturated, straight-chain or branched hydrocarbon groups that contain from 2 to 20 carbon atoms, preferably from 2 to 12 carbon atoms, especially from 2 to 6 (e.g. 2, 3 or 4) carbon atoms, for example an ethenyl (vinyl) , propenyl (allyl) , iso-propenyl, butenyl, ethinyl, propinyl, butinyl, acetylenyl, propargyl, isoprenyl or hex-2-enyl group.
  • alkenyl groups have one or two (especially preferably one) double bond(s)
  • alkynyl groups have one or two (especially preferably one) triple bond(s)
  • alkenyl and alkynyl refer to groups in which one or more hydrogen atoms have been replaced by a halogen atom (preferably F or CI) .
  • heteroalkyl refers to an alkyl, alkenyl or alkynyl group in which one or more (preferably 1, 2 or 3) carbon atoms have been replaced by an oxygen, nitrogen, phosphorus, boron, selenium, silicon or sulfur atom (preferably by an oxygen, sulfur or nitrogen atom) or a group of formula SO or SO2.
  • the expression heteroalkyl furthermore refers to a carboxylic acid or to a group derived from a carboxylic acid, such as, for example, acyl, acylalkyl, alkoxycarbonyl , acyloxy, acyloxyalkyl , carboxyalkylamide or alkoxycarbonyloxy.
  • a heteroalkyl group contains from 1 to 12 carbon atoms and from 1 to 4 hetero atoms selected from oxygen, nitrogen and sulphur (especially oxygen and nitrogen) .
  • a heteroalkyl group contains from 1 to 6 (e.g. 1, 2, 3 or 4) carbon atoms and 1, 2 or 3 (especially 1 or 2) hetero atoms selected from oxygen, nitrogen and sulphur (especially oxygen and nitrogen) .
  • the term C1-C6 heteroalkyl refers to a heteroalkyl group containing from 1 to 6 carbon atoms and 1, 2 or 3 heteroatoms selected from 0, S and/or N (especially O and/or N) .
  • C1-C4 heteroalkyl refers to a heteroalkyl group containing from 1 to 4 carbon atoms and 1, 2 or 3 heteroatoms selected from 0, S and/or N (especially 0 and/or N) .
  • heteroalkyl refers to groups in which one or more hydrogen atoms have been replaced by a halogen atom (preferably F or Cl) .
  • heteroalkyl groups are groups of formulae: R a _ 0 _ Y a_ ; Ra _ s _ Y a _ i R a -N (R b ) -Y a - , R a -C0-Y a - , R a -0-C0-Y a - , R a -CO- 0-Y a -, R a -CO-N(R b ) -Y a -, R a -N (R b ) -CO-Y a - , R a -0-CO-N (R b ) -Y a - , R a -N(R b ) -CO- 0-Y a - , R a -N (R b ) -CO- 0-Y a - , R a -N (R b ) -CO- 0-Y a - , R a
  • R a -N (R b ) -C ( NR d ) -N (R c ) -Y a - , R a -CS-Y a - ( R a - 0-CS-Y a -, R a -CS-0-Y a -, R a - CS-N(R b ) -Y a - , R a -N(R b ) - CS-Y a -, R a - 0- CS-N (R b ) -Y a - ,
  • heteroalkyl groups are methoxy, trifluoromethoxy, ethoxy, n-propyloxy, isopropyloxy, butoxy, tert-butyloxy, methoxymethyl , ethoxymethyl , -CH2CH2OH, -CH2OH, methoxyethyl , 1-methoxyethyl , 1-ethoxyethyl, 2 -methoxyethyl or 2-ethoxyethyl, methylamino, ethylamino, propylamino, isopropylamino, dimethylamino, diethylamino, isopropyl- ethylamino, methylamino methyl, ethylamino methyl, diiso- propylamino ethyl, methylthio, ethylthio, isopropylthio, enol ether, dimethylamino methyl, dimethylamino
  • cycloalkyl refers to a saturated or partially unsaturated (for example, a cycloalkenyl group) cyclic group that contains one or more rings (preferably 1 or 2) , and contains from 3 to 14 ring carbon atoms, preferably from 3 to 10 (especially 3, 4, 5, 6 or 7) ring carbon atoms.
  • cycloalkyl groups are a cyclopropyl , cyclobutyl , cyclopentyl , spiro [4,5] decanyl , norbornyl, cyclohexyl, cyclopentenyl, cyclohexadienyl, decalinyl, bicyclo [4.3.0] nonyl, tetraline, cyclopentylcyclohexyl, fluorocyclohexyl or cyclohex-2-enyl grou .
  • heterocycloalkyl refers to a cycloalkyl group as defined above in which one or more (preferably 1, 2, 3 or 4) ring carbon atoms have been replaced by an oxygen, nitrogen, silicon, selenium, phosphorus or sulfur atom (preferably by an oxygen, sulfur or nitrogen atom) .
  • a heterocycloalkyl group has preferably 1 or 2 ring(s) containing from 3 to 10 (especially 3, 4, 5, 6 or 7) ring atoms (preferably secected from C, O, N and S) .
  • Examples are a piperidyl, prolinyl, imidazolidinyl, piperazinyl, morpholinyl, urotropinyl, pyrrolidinyl, tetrahydrothiophenyl , tetrahydropyranyl , tetrahydrofuryl or 2-pyrazolinyl group and also lactames, lactones, cyclic imides and cyclic anhydrides.
  • alkylcycloalkyl refers to groups that contain both cycloalkyl and also alkyl, alkenyl or alkynyl groups in accordance with the above definitions, for example alkylcycloalkyl, cycloalkylalkyl, alkylcycloalkenyl , alkenylcycloalkyl and alkynylcycloalkyl groups.
  • An alkylcycloalkyl group preferably contains a cycloalkyl group that contains one or two rings having from 3 to 10 (especially 3, 4, 5, 6 or 7) ring carbon atoms, and one or two alkyl, alkenyl or alkynyl groups (especially alkyl groups) having 1 or 2 to 6 carbon atoms .
  • heteroalkylcycloalkyl refers to alkylcycloalkyl groups as defined above in which one or more (preferably 1, 2, 3, 4 or 5) carbon atoms have been replaced by an oxygen, nitrogen, silicon, selenium, phosphorus or sulfur atom (preferably by an oxygen, sulfur or nitrogen atom) .
  • a heteroalkylcycloalkyl group preferably contains 1 or 2 rings having from 3 to 10 (especially 3, 4, 5, 6 or 7) ring atoms, and one or two alkyl, alkenyl, alkynyl or heteroalkyl groups (especially alkyl or heteroalkyl groups) having from 1 or 2 to 6 carbon atoms (the heteroalkyl groups having preferably 1, 2 or 3 heteroatoms selected from 0, S and N) .
  • alkylheterocycloalkyl alkylheterocycloalkenyl, alkenylheterocycloalkyl, alkynylheterocycloalkyl, heteroalkylcycloalkyl, heteroalkylheterocycloalkyl and hetero- alkylheterocycloalkenyl, the cyclic groups being saturated or mono-, di- or tri-unsaturated.
  • aryl refers to an aromatic group that contains one or more rings containing from 5 or 6 to 14 ring carbon atoms, preferably from 5 or 6 to 10 (especially 6) ring carbon atoms.
  • aryl refers furthermore to groups in which one or more hydrogen atoms have been replaced by fluorine, chlorine, bromine or iodine atoms or by OH, SH, NH 2 , N 3 or NO2 groups .
  • aryl refers furthermore to groups in which one or more hydrogen atoms have been replaced by fluorine, chlorine, bromine or iodine atoms or by OH, SH, NH 2 , N 3 or NO2 groups .
  • phenyl naphthyl , biphenyl , 2-fluorophenyl , anilinyl, 3 -nitrophenyl or 4-hydroxyphenyl group .
  • heteroaryl refers to an aromatic group that contains one or more rings containing from 5 to 14 ring atoms, preferably from 5 to 10 (especially 5 or 6 or 9 or 10) ring atoms, and contains one or more (preferably 1, 2, 3, 4 or 5) oxygen, nitrogen, phosphorus or sulfur ring atoms (preferably O, S or N) .
  • the expression heteroaryl refers furthermore to groups in which one or more hydrogen atoms have been replaced by fluorine, chlorine, bromine or iodine atoms or by OH, SH, N3, NH2 or NO2 groups. Examples are pyridyl (e.g. 4-pyridyl) , imidazolyl (e.g.
  • 2-imidazolyl) phenylpyrrolyl (e.g. 3- phenylpyrrolyl) , thiazolyl, isothiazolyl, 1, 2 , 3-triazolyl, 1, 2 , 4-triazolyl, oxadxazolyl, thiadxazolyl, indolyl, indazolyl, tetrazolyl, pyrazinyl, pyrimidinyl, pyridazinyl, oxazolyl, isoxazolyl, triazolyl, tetrazolyl, isoxazolyl, indazolyl, indolyl, benzimidazolyl, benzoxazolyl , benzisoxazolyl, benzthiazolyl, pyridazinyl, quinolinyl, isoquinolinyl, pyrrolyl , purinyl , carbazolyl , acridinyl,
  • aralkyl refers to groups containing both aryl and also alkyl, alkenyl, alkynyl and/or cycloalkyl groups in accordance with the above definitions, such as, for example, arylalkyl, arylalkenyl, arylalkynyl, arylcycloalkyl, aryl- cycloalkenyl, alkylarylcycloalkyl and alkylarylcycloalkenyl groups.
  • aralkyls are toluene, xylene, mesitylene, styrene, benzyl chloride, o-fluorotoluene, lH-indene, tetraline, dihydronaphthalene , indanone, phenylcyclopentyl , cumene, cyclohexylphenyl, fluorene and indane .
  • An aralkyl group preferably contains one or two aromatic ring systems (1 or 2 rings) containing from 6 to 10 carbon atoms and one or two alkyl, alkenyl and/or alkynyl groups containing from 1 or 2 to 6 carbon atoms and/or one or two cycloalkyl groups containing 5 or 6 ring carbon atoms.
  • heteroaralkyl refers to an aralkyl group as defined above in which one or more (preferably 1, 2, 3 or 4) carbon atoms have been replaced by an oxygen, nitrogen, silicon, selenium, phosphorus, boron or sulfur atom (preferably oxygen, sulfur or nitrogen) , that is to say to groups containing both aryl or heteroaryl, respectively, and also alkyl, alkenyl, alkynyl and/or heteroalkyl and/or cycloalkyl and/or heterocycloalkyl groups in accordance with the above definitions.
  • a heteroaralkyl group preferably contains one or two aromatic ring systems (1 or 2 rings) containing from 5 or 6 to 10 ring carbon atoms and one or two alkyl, alkenyl and/or alkynyl groups containing 1 or 2 to 6 carbon atoms and/or one or two cycloalkyl groups containing 5 or 6 ring carbon atoms, wherein 1, 2, 3, 4, 5 or 6 of these carbon atoms have been replaced by oxygen, sulfur or nitrogen atoms .
  • Examples are arylheteroalkyl, arylheterocycloalkyl, aryl- heterocycloalkenyl, arylalkylheterocycloalkyl, arylalkenyl- heterocycloalkyl, arylalkynylheterocycloalkyl , arylalkyl- heterocycloalkenyl, heteroarylalkyl , heteroarylalkenyl , heteroarylalkynyl, heteroarylheteroalkyl , heteroaryl- cycloalkyl, heteroarylcycloalkenyl, heteroarylhetero- cycloalkyl, heteroarylheterocycloalkenyl , heteroarylalkyl- cycloalkyl, heteroarylalkylheterocycloalkenyl , heteroarylalkyl- cycloalkyl, heteroarylalkylheterocycloalkenyl, heteroarylalkyl-
  • halogen or halogen atom refers to F , CI , Br or I .
  • This expression refers furthermore to groups that may be substituted by one, two, three or more preferably unsubstituted Ci - Cio alkyl, C2 - C10 alkenyl, C2 - C10 alkynyl, C1 - C10 heteroalkyl, C3-C18 cycloalkyl, C 2 - Ci 7 heterocycloalkyl, C4- C20 alkylcycloalkyl , C2-C 1 9 heteroalkylcycloalkyl, C6-C18 aryl, C 1 -C 1 7 heteroaryl, C7- C20 aralkyl or C2-C19 heteroaralkyl groups.
  • This expression refers furthermore especially to groups that may be substituted by one, two, three or more preferably unsubstituted C1-C6 alkyl, C 2 - C 6 alkenyl, C 2 - C 6 alkynyl, Ci - C 6 heteroalkyl, C 3 - C10 cycloalkyl, C2-C9 heterocycloalkyl, C7- C12 alkylcycloalkyl, C 2 - Cn heteroalkylcycloalkyl, C6- C10 aryl, C1-C9 heteroaryl, C7- C 1 2 aralkyl or C 2 - Cu heteroaralkyl groups.
  • a substituent contains a ring
  • this ring may be bonded to the respective substituted group via a single or double bond (especially a single bond) or, if the substituted group also contains a ring, the ring of the substituent may also be annulated to the ring of the substituted group.
  • Ci- 4 alkyl e.g. methyl, ethyl, t-butyl
  • substituents are F, CI, Br, OH, NH 2 , Me, Ethyl, Me 2 , CONH 2 , OMe, CN or CF 3 .
  • all alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, alkylcycloalkyl, heteroalkylcycloalkyl, aralkyl and heteroaralkyl groups described herein may optionally be substituted.
  • aryl, heteroaryl, cycloalkyl, alkylcycloalkyl, heteroalkylcycloalkyl, heterocycloalkyl, aralkyl or heteroaralkyl group contains more than one ring, these rings may be bonded to each other via a single or double bond or these rings may be annulated.
  • the present invention further provides pharmaceutical compositions comprising one or more compounds of formula (I) , (II) , (III) or (IV) as defined herein or a pharmaceutically acceptable ester, prodrug, hydrate, solvate or salt thereof, optionally in combination with a pharmaceutically acceptable carrier.
  • the compounds of the present invention may be used for the treatment and/or prevention of cancer, viral diseases and infectious diseases .
  • the compounds of the present invention may be used for the treatment and/or prevention of neurodegenerative diseases such as: Schizophrenia, Alzheimer, Multiples Sclerosis, Parkinson, Corea Huntington, Spinocerebellar ataxia type 1 (SCA1) , Amyotrophic lateral sclerosis, Batten disease.
  • neurodegenerative diseases such as: Schizophrenia, Alzheimer, Multiples Sclerosis, Parkinson, Corea Huntington, Spinocerebellar ataxia type 1 (SCA1) , Amyotrophic lateral sclerosis, Batten disease.
  • a therapeutically effective amount of a compound in accordance with this invention means an amount of compound that is effective to prevent, alleviate or ameliorate symptoms of disease or prolong the survival of the subject being treated. Determination of a therapeutically effective amount is within the skill in the art.
  • the therapeutically effective amount or dosage of a compound according to this invention can vary within wide limits and may be determined in a manner known in the art. Such dosage may be adjusted to the individual requirements in each particular case including the specific compound being administered, the route of administration, the condition being treated, as well as the patient being treated.
  • Examples of pharmacologically acceptable salts of sufficiently basic compounds of formula (I) , (II) , (III) or (IV) are salts of physiologically acceptable mineral acids like hydrochloric, hydrobromic, sulfuric and phosphoric acid; or salts of organic acids like methanesulfonic, p-toluenesulfonic, lactic, acetic, trifluoroacetic, citric, succinic, fumaric, maleic and salicylic acid.
  • a sufficiently acidic compound of formula (I) , (II) , (III) or (IV) may form alkali or earth alkali metal salts, for example sodium, potassium, lithium, calcium or magnesium salts; ammonium salts; or organic base salts, for example methylamine, dimethylamine, trimethylamine, triethylamine, ethylenediamine , ethanolamine, choline hydroxide, meglumin, piperidine, morpholine, tris- (2- hydroxyethyl) amine, lysine or arginine salts; all of which are also further examples of salts of formula (I) , (II) , (III) or (IV) .
  • alkali or earth alkali metal salts for example sodium, potassium, lithium, calcium or magnesium salts; ammonium salts; or organic base salts, for example methylamine, dimethylamine, trimethylamine, triethylamine, ethylenediamine , ethanolamine, choline hydroxide
  • Compounds of formula (I) , (II) , (III) or (IV) may be solvated, especially hydrated.
  • the hydratization/hydration may occur during the process of production or as a consequence of the hygroscopic nature of the initially water free compounds of formula (I) , (II) , (III) or (IV) .
  • the solvates and/or hydrates may e.g. be present in solid or liquid form.
  • (I) , (II) , (III) or (IV) may contain asymmetric C-atoms, they may be present either as achiral compounds, mixtures of diastereomers, mixtures of enantiomers or as optically pure compounds.
  • the present invention comprises both all pure enantiomers and all pure diastereomers, and also the mixtures thereof in any mixing ratio.
  • compositions according to the present invention comprise at least one compound of formula (I) , (II) ,
  • the present invention also relates to pro-drugs which are composed of a compound of formula (I) , (II) , (III) or (IV) and at least one pharmacologically acceptable protective group which will be cleaved off under physiological conditions, such as an alkoxy- , arylalkyloxy- , acyl-, acyloxymethyl group (e.g. pivaloyloxymethyl) , an 2-alkyl-, 2-aryl- or 2-arylalkyl- oxycarbonyl-2-alkylidene ethyl group or an acyloxy group as defined herein, e.g.
  • the present invention also relates to a prodrug, a biohydrolyzable ester, a biohydrolyzable amide, a polymorph, tautomer, stereoisomer, metabolite, N-oxide, biohydrolyzable carbamate, biohydrolyzable ether, physiologically functional derivative, atropisomer, or in vivo-hydrolysable precursor, diastereomer or mixture of diastereomers, chemically protected form, affinity reagent, complex, chelate and a stereoisomer of the compounds of formula (I) , (II) , (III) or (IV) .
  • ester especially refers to esters which hydrolyze in vivo and include those that break down readily in the human body to leave the parent compound or a salt thereof.
  • Suitable ester groups include, for example, those derived from pharmaceutically acceptable aliphatic carboxylic acids, particularly alkanoic, alkenoic, cycloalkanoic and alkanedioic acids, in which each alkyl or alkenyl moiety advantageously has not more than 6 carbon atoms .
  • esters include, but are not limited to, formates, acetates, propionates, butyrates, acrylates and ethylsuccinates .
  • therapeutically useful agents that contain compounds of formula (I), (II), (III) or (IV), their solvates, salts or formulations are also comprised in the scope of the present invention.
  • compounds of formula (I) , (II) , (III) or (IV) will be administered by using the known and acceptable modes known in the art, either alone or in combination with any other therapeutic agent.
  • oral administration such therapeutically useful agents can be administered by one of the following routes: oral, e.g.
  • TDS transdermal delivery system
  • the therapeutically useful product may be mixed with pharmaceutically inert, inorganic or organic excipients as are e.g. lactose, sucrose, glucose, gelatine, malt, silica gel, starch or derivatives thereof, talc, stearinic acid or their salts, dried skim milk, and the like.
  • pharmaceutically inert, inorganic or organic excipients as are e.g. lactose, sucrose, glucose, gelatine, malt, silica gel, starch or derivatives thereof, talc, stearinic acid or their salts, dried skim milk, and the like.
  • excipients e.g. vegetable, petroleum, animal or synthetic oils, wax, fat, polyols.
  • liquid solutions, emulsions or suspensions or syrups one may use as excipients e.g.
  • excipients as are e.g. vegetable, petroleum, animal or synthetic oils, wax, fat and polyols .
  • compressed gases suitable for this purpose, as are e.g.
  • the pharmaceutically useful agents may also contain additives for conservation, stabilization, e.g. UV stabilizers, emulsifiers, sweetener, aromatizers, salts to change the osmotic pressure, buffers, coating additives and antioxidants .
  • additives for conservation stabilization, e.g. UV stabilizers, emulsifiers, sweetener, aromatizers, salts to change the osmotic pressure, buffers, coating additives and antioxidants .
  • the daily dosage can be administered as a single dose or in divided doses, or for parenteral administration, it may be given as continuous infusion or subcutaneous injection.
  • the present invention moreover provides a method of inhibiting growth, proliferation, or metastasis of cancer cells in a subject in need thereof, said method comprising administering to the subject a therapeutically effective amount of a compound of formula (I), (II), (III) or (IV), or a pharmaceutically acceptable salt .
  • the cancer is selected from melanoma, renal cell carcinoma, squamous non- small cell lung cancer (NSCLC) , non-squamous NSCLC, colorectal cancer, castration-resistant prostate cancer, ovarian cancer, gastric cancer, hepatocellular carcinoma, pancreatic carcinoma, squamous cell carcinoma of the head and neck, carcinomas of the esophagus, gastrointestinal tract and breast, cancer of the genital organs, penis and vagina, and a hematological malignancy.
  • NSCLC non- small cell lung cancer
  • NSCLC non-squamous NSCLC
  • colorectal cancer castration-resistant prostate cancer
  • ovarian cancer gastric cancer, hepatocellular carcinoma, pancreatic carcinoma, squamous cell carcinoma of the head and neck, carcinomas of the esophagus, gastrointestinal tract and breast, cancer of the genital organs, penis and vagina, and a hematological malignancy.
  • the present invention provides a method of treating an infectious disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a compound of formula (I) , (II) , (III) or (IV), or a pharmaceutically acceptable salt thereof.
  • the infectious disease is caused by a virus.
  • the virus is selected from HIV, Hepatitis A, Hepatitis B, Hepatitis C, Hepatitis D, herpes viruses, papillomaviruses and influenza.
  • a method for treating cancer comprising administering to a patient in need thereof, a therapeutically effective amount of a compound of formula (I) , (II), (III) or (IV) or a salt thereof.
  • cancers include those whose growth may be inhibited using compounds of the disclosure include cancers typically responsive to immunotherapy.
  • Non-limiting examples of preferred cancers for treatment include melanoma (e.g., metastatic malignant melanoma) , renal cancer (e.g. clear cell carcinoma) , prostate cancer (e.g. hormone refractory prostate adenocarcinoma), breast cancer, colon cancer and lung cancer (e.g. non-small cell lung cancer) .
  • the disclosure includes refractory or recurrent malignancies whose growth may be inhibited using the compounds of the present invention.
  • cancers examples include bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin's Disease, non-Hodgkin' s lymphoma, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, chronic or acute leukemias including acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia
  • pathogenic viruses causing infections treatable by methods of the present invention include HIV, hepatitis (A, B, C, or D) , herpes viruses (e.g., VZV, HSV-1 , HAV-6, HHv-7, HHV-8, HSV-2, CMV, and Epstein Barr virus), adenovirus, influenza virus, fiaviviruses, echovirus, rhinovirus, coxsackie virus, cornovirus, respiratory syncytial viras, mumps viras, rotaviras, measles viras, rabella viras, parvovirus, vaccinia virus, HTLV viras, dengue viras, papillomavirus, molluscum viras, poliovirus, rabies viras, JC viras and arboviral encephalitis viras.
  • herpes viruses e.g., VZV, HSV-1 , HAV-6
  • pathogenic bacteria causing infections treatable by methods of the present invention include chlamydia, rickettsial bacteria, mycobacteria, staphylococci, streptococci, pneumonococci, meningococci and conococci, lebsiella, proteus, serratia, pseudomonas, legionella, diphtheria, salmonella, bacilli, cholera, tetanus, botulism, anthrax, plague, leptospirosis, and Lymes disease bacteria.
  • pathogenic fungi causing infections treatable by methods of the present invention include Candida (albicans, krasei, glabrata, tropicalis, etc.), Cryptococcus neoformans, Aspergillus (fumigatus, niger, etc.), Genus Mucorales (mucor, absidia, rhizophus) , Sporothrix schenkii, Blastomyces dermatitidis , Paracoccidioides brasiliensis, Coccidioides immitis and Histoplasma capsulatum.
  • Candida albicans, krasei, glabrata, tropicalis, etc.
  • Cryptococcus neoformans Aspergillus (fumigatus, niger, etc.)
  • Genus Mucorales micor, absidia, rhizophus
  • Sporothrix schenkii Blastomyces dermatitidis
  • Paracoccidioides brasiliensis Coccidioides immit
  • pathogenic parasites causing infections treatable by methods of the present invention include Entamoeba histolytica, Balantidium coli, Naegleriafowleri, Acanthamoeba sp., Giardia lambia, Cryptosporidium sp., Pneumocystis carinii, Plasmodium vivax, Babesia microti, Trypanosoma bracei, Trypanosoma crazi, Leishmania donovani, Toxoplasma gondi, and Nippostrongylus brasiliensis.
  • Example 5a ethyl 2-amino-4- (4-methyl-3 , -dihydro-2H- benzo [b] [1,4] oxazin-7-yl) thiophene-3-carboxylate
  • Example 5b ethyl 2-amino-4- (4-methyl-3 , 4-dihydro-2H- benzo [b] [1,4] oxazin-6-yl) thiophene-3 -carboxylate
  • Example 20 4-phenyl-2- ( (3- (trifluoromethyl) benzyl) oxy) - thiophene-3 -carbonitril
  • Example 35 4-phenyl-2- ( (3- (trifluoromethoxy) benzyl) oxy) - thiophene-3 -carbonitri
  • Example 50 4 - (2 , 3-dihydrobenzo [b] [1 , 4] dioxin-6-yl) -2- iodobenzyl) oxy) thiophene-3-carbonitrile
  • Example 51 4- (2 , 3-dihydrobenzo [b] [1, 4] dioxin-6-yl) -2- (4- nitrophenethoxy) thiophene-3-carbonitrile
  • Example 52 4- (2 , 3-dihydrobenzo [b] [1, 4] dioxin-6-yl) -2- ( (4- nitrobenzyl) oxy) thiophene-3-carbonitrile
  • Example 54 4- (2 , 3-dihydrobenzo [b] [1, 4] dioxin-6-yl) -2- (naphthalen-2-ylmethoxy) thiophene-3-carbonitrile
  • Example 55 4- (2, 3 -dihydrobenzo [b] [1, 4] dioxin-6-yl) -2- ( (3- fluorobenzyl) oxy) thiophene-3-carbonitrile
  • Example 56 4 - (2 , 3 -dihydrobenzo [b] [1 , 4] dioxin-6-yl) -2- (3- phenoxypropoxy) thiophene-3 -carbonitrile
  • Example 58 4 - (2 , 3 -dihydrobenzo [b] [1 , 4] dioxin-6-yl) -2 - ( (4 - (hydroxymethyl) benzyl) oxy) thiophene-3 -carbonitrxle
  • Example 60 4- (2, 3-dihydrobenzo [b] [1, 4] dioxin-6-yl) -2- (2-oxo- 2 -pheny1ethoxy) thiophene-3 -carbonitri1e
  • Example 62 4- (2 , 3 -dihydrobenzo [b] [1, 4] dioxin-6-yl) -2- ( (4- formylbenzyl) oxy) thiophene-3-carbonitrile
  • Example 64 2 - ( (5-chlorothiophen-2-yl) methoxy) - - (2 , 3- dihydrobenzo [b] [1, 4] dioxin-6-yl) thiophene -3 -carbonitrile
  • Example 65 4- (2 , 3 -dihydrobenzo [b] [1, 4] dioxin-6-yl) -2- ( thiophen- 3 -ylmethoxy) thiophene- 3 -carbonitrile
  • Example 66 4- (2 , 3-dihydrobenzo [b] [1, 4] dioxin-6-yl) -2- ( yridin-4 -ylmethoxy) thiophene-3-carbonitrile
  • Example 70 4- (2 , 3 -dihydrobenzo [b] [1, 4] dioxin-6-yl) -2- ( (perfluorophenyl) methoxy) thiophene-3-carbonitrile
  • Example 73 4- (2, 3-dihydrobenzo [b] [1, 4] dioxin-6-yl) -2- ( (4- (methylsulfonyl) benzyl) oxy) thiophene-3 -carbonitrile
  • Example 74 4- (2 , 3-dihydrobenzo [b] [1, 4] dioxin-6-yl) -2- (quinolin-8-ylmethoxy) thiophene-3 -carbonitrile
  • Example 75 4- (2 , 3-dihydrobenzo [b] [1, 4] dioxin-6-yl) -2- ( (2- fluoropyridin-4-yl) methoxy) thiophene-3 -carbonitrile
  • Example 76 4- (2 , 3-dihydrobenzo [b] [1 , 4] dioxin-6-yl) -2 -( (3- formylbenzyl) oxy) thiophene-3-carbonitrile
  • Example 77 4- (2 , 3-dihydrobenzo [b] [1, 4] dioxin-6-yl) -2- ( (6- methoxypyridin-2-yl)methoxy) thiophene-3-carbonitrile
  • Example 82 2- ( (3-chlorobenzyl) oxy) -4- (3 , 4-dichlorophenyl) - thiophene-3 -carbonitrile
  • Example 84 4- (3 , 4-dichlorophenyl) -2- (4 -nitrophenethoxy) - thiophene- 3 -carbonitrile
  • Example 85 4- (3 , 4-dichlorophenyl) -2- ( (4-nitrobenzyl) oxy) - thiophene-3 -carbonitrxle
  • Example 86 4- (3 , -dichlorophenyl) -2- ( (3- (trifluoromethyl) - benzyl) oxy) thiophene-3-carbonitrile
  • Example 87 4- (3 , 4 -dichlorophenyl) -2- (naphthalen-2-ylmethoxy) - thiophene- 3 -carbonitrile
  • Example 88 4- (3 , 4 -dichlorophenyl) -2- ( (3-fluorobenzyl) oxy) - thiophene- 3 -carbonitrile
  • Example 90 2- ( (3 , 4 -dichlorobenzyl) oxy) -4- (3 , 4-dichloro- phenyl) thiophene-3 -carbonitrile
  • Example 91 4- (3 , -dichlorophenyl) -2- ( (4- (hydroxymethyl) benzyl) oxy) thiophene- 3 -carbonitrile
  • Example 93 4- (3 , 4-dichlorophenyl) -2- (2-oxo-2-phenylethoxy) - thiophene-3 -carbonitrile
  • Example 100 4- (3 , 4-dichlorophenyl) -2- (pyridin-3 -ylmethoxy) - thiophene-3 -carbonitrile
  • Example 101 4- (3 , 4-dichlorophenyl) -2- ( (3- (trifluoromethoxy) - benzyl) oxy) thiophene-3 -carbonitrile
  • Example 102 2- ( ( - (1 , 2 , 3-thiadiazol-4-yl) benzyl) oxy) -4 - (3 , 4 - dichlorophenyl) thiophene-3 -carbonitrile
  • Example 103 4- (3 , 4 -dichlorophenyl) -2- ( (perfluorophenyl) methoxy) thiophene-3 -carbonitrile
  • Example 104 2- ( (4- (tert-butyl) benzyl) oxy) -4- (3 , 4-dichloro- phenyl) thiophene-3-carbonitrile
  • Example 105 4- (3 , 4 -dichlorophenyl) -2- ( (4- (trifluoromethoxy) - benzyl) oxy) thiophene-3-carbonitrile
  • Example 106 4- (3 , 4 -dichlorophenyl) -2- ( (4- (methylsulfonyl) - benzyl) oxy) thiophene-3-carbonitrile
  • Example 107 4- (3 , 4-dichlorophenyl) -2- (quinolin-8-ylmethoxy) - thiophene-3 -carbonitrile
  • Example 108 4- (3 , 4-dichlorophenyl) -2- ( (2 -fluoropyridin-4 - yl) methoxy) thiophene-3 -carbonitrile
  • Example 109 4- ( , 4 -dichlorophenyl) -2- ( (3-formylbenzyl) oxy) - thiophene-3 -carbonitrile
  • Example 110 4- (3 , 4 -dichlorophenyl) -2- ( (6-methoxypyridin-2- yl) -ethoxy) thiophene-3-carbonitrile
  • Example 111 Example 4: 4- (benzo [d] [1, 3] dioxol-5-yl) -2- ( (4- bromobenzyl) oxy) thiophene- 3 -carbonitrile
  • Example 112 tert-butyl 4- ( ( (4- (benzo [d] [1, 3] dioxol-5-yl) -3- cyanothiophen-2-yl) oxy) methyl) benzylcarbamate
  • Example 113 4- (benzo [d] [1, 3] dioxol-5-yl) -2- (benzyloxy) - thiophene-3 -carbonitrile
  • Example 114 4- (benzo [d] [1, 3] dioxol-5-yl) -2- ( (2-bromobenzyl) - oxy) thiophene-3 -carbonitrile
  • Example 115 4- (benzo [d] [1, 3] dioxol-5-yl) -2- ( (3 -chlorobenzyl) - oxy) thiophene-3-carbonitrile
  • Example 116 4- (benzo [d] [1, 3] dioxol-5-yl) -2- ( (4 -iodobenzyl) - oxy) thiophene-3-carbonitrile
  • Example 118 4- (benzo [d] [1, 3] dioxol-5-yl) -2- ( (4-nitrobenzyl) - oxy) thiophene-3 -carbonitrile
  • Example 119 4- (benzo [d] [1, 3] dioxol-5-yl) -2- ( (3- (trifluoro- methyl) benzyl) oxy) thiophene-3 -carbonitrile
  • Example 120 4- (benzo [d] [1, 3] dioxol-5-yl) -2- (naphthalen-2 -yl- methoxy) thiophene-3 -carbonxtrile
  • Example 121 4- (benzo [d] [1, 3] dioxol-5-yl) -2- ( (3-fluorobenzyl) - oxy) thiophene-3 -carbonitrile
  • Example 122 4- (benzo [d] [1, 3] dioxol-5-yl) -2- (3-phenoxy- propoxy) thiophene-3 -carbonitri1e
  • Example 123 4- (benzo [d] [1, 3] dioxol-5-yl) -2- ( (3 , 4-dichloro- benzyl) oxy) thiophene-3-carbonitrile
  • Example 124 4 - (benzo [d] [1 , 3] dioxol-5-yl) -2 - ( (4 - (hydroxy- methyl) benzyl) oxy) thiophene-3-carbonitrile
  • Example 125 4- (benzo [d] [1,3] dioxol-5-yl) -2- ( (2,4-dichloro- benzyl) oxy) thiophene-3 -carbonitrile
  • Example 126 4- (benzo [d] [1, 3] dioxol-5-yl) -2- (2-oxo-2-phenyl- ethoxy) thiophene-3 -carbonitrile
  • Example 127 4- (benzo [d] [1, 3] dioxol-5-yl) -2- ( (4 -cyanobenzy1) - oxy) thiophene-3-carbonitrile
  • Example 128 4- (benzo [d] [1, 3] dioxol-5-yl) -2- ( (4-formylbenzyl) - oxy) thiophene-3-carbonitrile
  • Example 129 4- (benzo [d] [1, 3] dioxol-5-yl) -2- (thiophen-2-yl- methoxy) thiophene-3 -carbonitrile
  • Example 130 4- (benzo [d] [1, 3] dioxol-5-yl) -2- ( (5-chloro- t iophen-2 -yl) methoxy) thiophene-3-carbonitrile
  • Example 131 4- (benzo [d] [1, 3] dioxol-5-yl) -2- (thiophen-3-yl- methoxy) thiophene-3-carbonitrile
  • Example 134 4- (benzo [d] [1, 3] dioxol-5-yl) -2- ( (3- (trifl oro- methoxy) benzyl) oxy) thiophene-3 -carbonitrile
  • Example 135 2- ( (4- (1, 2 , 3 -thiadiazol-4 -yl) benzyl) oxy) -4- (benzo [d] [1,3] dioxol-5-yl) thiophene-3-carbonitrile
  • Example 136 4- (benzo [d] [1 , 3] dioxol-5-yl) -2- ( (perfluoro- phenyl) methoxy) thiophene-3 -carbonitrile
  • Example 137 4- ⁇ benzo[d] [1, 3] dioxol-5-yl) -2- ( (4- (tert-butyl) - benzyl) oxy) thiophene-3-carbonitrile
  • Example 138 4- (benzo [d] [1, 3] dioxol-5-yl) -2- ( (4- (trifluoro- methoxy) benzyl) oxy) thiophene-3-carbonitrile
  • Example 139 4-(benzo[d] [1, 3] dioxol-5-yl) -2- ( (4- (methylsulfonyl) benzyl) oxy) thiophene-3-carbonitrile
  • Example 140 4- (benzo [d] [1, 3] dioxol-5-yl) -2- (guinolin-8-yl- methoxy) thiophene-3-carbonitrile
  • Example 141 4- (benzo [d] [1, 3] dioxol-5-yl) -2- ( (2-fluoropyridin- 4 -yl) methoxy) thiophene-3-carbonitrile
  • Example 142 4- (benzo [d] [1, 3] dioxol-5-yl) -2- ( (3-formylbenzyl) - oxy) thiophene-3 -carbonitrile
  • Example 143 4- (benzo [d] [1, 3] dioxol-5-yl) -2- ( (6-methoxy- pyridin-2 -yl) methoxy) thiophene-3 -carbonitrile
  • O-alkylation specific scaffolds where selected for further functionalization (e.g. examples 13, 46, 79 and 112) towards compounds D.
  • the gene encoding human PD-Ll (amino acids 18-134) was cloned into the pET-21b, the gene encoding human PD-L2 (20-220) was cloned into pET28a and that of human PD-1 (33-150, Cys93 exchanged to serine) into pET-24d. Proteins were expressed in the E. coli BL21 (DE3) . Cells were cultured in LB at 37°C. The protein production was induced with 1 mM IPTG at OD600 of 1.0 and the cells were cultured for additional 5h. For hPD-1, after induction the temperature was lowered to 30°C.
  • Proteins were expressed as inclusion bodies which were collected by centrifugation, washed twice with 50 mM Tris-HCl pH 8.0 containing 200 mM NaCl, 0.5% Triton X-100, 10 mM EDTA and 10 mM 2 -mercaptoethanol and once more with the same buffer with no detergent. The inclusion bodies were stirred overnight in 50 mM Tris pH 8.0 containing 6M GuHCl, 200 mM NaCl and 10 mM 2-mercaptoethanol . Solubilized fraction was clarified by high speed centrifugation.
  • hPD-Ll and hPD-L2 were refolded by drop- wise dilution into 0.1 M Tris pH 8.0 containing 1 M L-Arg hydrochloride, 0.25 mM oxidized glutathione and 0.25 mM reduced glutathione for hPD-Ll and 0.1 M Tris pH 8.5 containing 1 M NDSB201, 0.2 M NaCl, 5 mM cysteamine and 0.5 mM cystamine for hPD-L2.
  • hPD-1 was refolded in similar manner in 0.1 M Tris pH 8.0 containing 0.4 M L-Arg hydrochloride, 2 mM EDTA, 5 mM cystamine and 0.5 mM cysteamine.
  • the proteins were dialyzed 3 times against 10 mM Tris pH 8.0 containing 20 mM NaCl, and purified by size exclusion chromatography on Superdex 75 (GE Healthcare) in 10 mM Tris pH 8.0 containing 20 mM NaCl. The purity and protein folding were evaluated by SDS-PAGE and NMR, respectively. Analytical size-exclusion chromatography
  • the oligomeric state of tested proteins was analyzed by size exclusion chromatography.
  • Superdex 75 10/30 HR (GE Healthcare) was equilibrated with PBS pH 7.4 and calibrated using globular proteins of known molecular weight .
  • Approximate molecular weight of apo-hPD-Ll and hPD-Ll-small molecule complex (3:1 compound: protein molar ratio) were estimated using the calibration curve.
  • DFS Differential scanning fluorimetry
  • DSF analysis was performed according to Niesen and colleagues (24). In brief hPD-Ll and hPD-L2 (both 12.5 ⁇ ) were incubated alone, with compound BMS-202 or compound 8 (both at 37.5 ⁇ ) in the presence of SYPRO Orange Dye (Life Technologies, final concentration 20x) . Constant temperature gradient of 0.2°C/min was applied and changes in fluorescence were monitored using real time thermocycler (BioRad) . Melting temperature (Tm) was estimated from first derivative of fluorescence intensity as a function of temperature .
  • Uniform 15N labeling was obtained by expressing the protein in the M9 minimal medium containing 15NH4C1 as the sole nitrogen source. Unlabeled proteins were prepared as for crystallization.
  • the buffer was exchanged by gel filtration to PBS pH 7.4 (hPD-Ll) or 25 mM sodium phosphate containing 100 mM NaCl pH 6.4 (hPD-1) . 10% (v/v) of D 2 O was added to the samples to provide lock signal. All spectra were recorded at 300K using a Bruker Avance 600 MHz spectrometer.
  • Binding of the compounds was analyzed by titrating the 15N- labeled PD-Ll (0.3 mM) and recording the 1H-15N HMQC spectra prior and after addition of the compound (Supplementary Figs. SI, S2 and S3) .
  • MST Microscale Thermophoresis
  • Monolith NT.115 NanoTemper technologies, Miinchen
  • MST Microscale Thermophoresis
  • Monolith NT.115 NanoTemper technologies, Miinchen
  • the PD-1 construct is a 13.2 kDa protein. While PD-L1 is 14.5 kDa containing a His6-tag. Labeling of PD-L1 can be performed in several ways, for example, by attaching a fluorescent label to free amine groups, cysteine's or by binding to the His6-tag. PD-L1 was labeled using the Monolith NTtm His-tag labeling kit RED-tris-NTA (Nano temper technologies, Munchen) .
  • the RED-tris-NTA dye (100 nm) was mixing with PD-L1 (200 nM) in a 1:1 ratio and incubated for 30 min at room temperature followed by centrifugation at 13k rpm for 15 min. Binding of the dye to the PD-L1 His6-tag was confirmed by measuring the KD ( ⁇ 8 nm) .
  • Compound stocks were prepared at 100 mM in 100% DMSO and were subsequently diluted in PBS-t buffer to a concentration of 2- 0.25 mM, depending on their solubility, while keeping the DMSO concentration at 2%.
  • the PD-1/PD-L1 complex is prepared by mixing PD-Ll and PD-1 in a 1:10 ratio the complex was labeled a described above for PD- Ll.
  • the dissociation of the PD-1/ PD-Ll complex is measured by adding the labeled complex to a final PD-Ll concentration of 50 nm to a 2 -fold dilution series of the compound and measuring using the Monolith NT.115.
  • MST Rapid screening of PD-Ll binding without a full KD determination is also possible using MST. This requires preparation of two samples, one at low compound concentration and one at high compound concentration. If the MST measurements show a significant change in fluorescence signal between the two samples after the temperature jump it is a good indication if the compound is binding or not . Subsequently, a full KD determination as described above can be performed on the hits .

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Abstract

The present invention provides novel compounds of formula (I) that are useful as inhibitors of the PD-l/PD-Ll protein/protein interaction. The compounds may be used in the treatment of cancer, infectious diseases and neurodegenerative diseases such as schizophrenia, Alzheimer, multiple sclerosis or Parkinson.

Description

3-CYANOTHIOPHENE DERIVATIVES AS INHIBITORS OF THE PD-1/PD-L1 PROTEIN/PROTEIN INTERACTION
The present invention provides novel compounds that are useful as inhibitors of the PD-1/PD-L1 protein/protein interaction.
The PD-l/PD-Ll axis is hijacked by viruses, bacteria and uncontrolled fast growing cells to suppress the immune surveillance. In cancer for example, the malignant cells express PD-L1 which bind to the PD1 receptor expressed on immune T-cells. Binding of PD-1 to PD-1L determines a downregulation of T-cell effector functions in cancer patients inhibiting the antitumor immune response and leading to T-cell exhaustion. In viral and infectious diseases a similar mechanism is used by viruses and bacteria to undermine the effective immune recognitions and answer.
Current medication directed towards the PD-l/PD-Ll axis includes monoclonal antibodies . These have shown impressive clinical results in the treatment of several types of tumours. Therapeutic antibodies however exhibit several disadvantages such as limited tissue and tumour penetration, very long half life time, lacking oral bioavailability, immunogenicity, and difficult and expensive production. The current PD-l/PD-Ll axis directed monoclonal antibodies lead to a tumour response only in a fraction of cases and tumour types. Recently small molecules have been described to bind to PD-L1 in WO 2015/160641 and WO 2015/034820. The compounds described therein, however, display a high lipophilicity (cLogP) .
A high cLogP is often associated with extensive metabolism, poor water solubility, fast excretion and toxicity and reduced target selectivity. Therefore PD-1/PD-L1 axis targeted drugs are needed which overcome the above disadvantages and which further lead to a high tumour response, are fast and efficient to produce and can penetrate tumour tissue and have favourable half-life times to be able to adequately react on drug induced immunological adverse side effects. These objects are solved by the compounds of the present invention.
The present invention provides compounds of formula (I) :
Figure imgf000003_0001
(I)
wherein
R1 is a hydrogen atom or a halogen atom; and R2 is a hydrogen atom or a halogen atom; or
R1 and R2 together are a group of formula -O-CH2-CH2-O- , -O-CH2- CHa-N(Me)-, -N(Me) -CH2-CH2-O-, -O-CF2-O- or -O-CH2-O-;
X is a bond, an oxygen atom or a group of formula ~C(=0)-, or -(CH2)n- wherein n is 1, 2 or 3 or a group of formula -(CH2)m-0- wherein m is 2, 3 or 4 ; and Ar is a phenyl group, a naphthyl group or a heteroaryl group containing 1 or 2 rings and from 5 to 10 ring atoms selected from C, 0, N and S, wherein Ar may be substituted by 1, 2, 3, 4 or 5 groups R3 and/or wherein Ar may be substituted by a group of formula -CH2-N (R4) -R5 ; the groups R3 are independently from each other halogen, NO2, N3, OH, SH, NH2 or an alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl , alkylcycloalkyl , heteroalkyl- cycloalkyl, aryl, heteroaryl, aralkyl or heteroaralkyl group; all of which groups may optionally be substituted;
R4 is a hydrogen atom or an alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, alkylcycloalkyl, hetero- alkylcycloalkyl, aryl, heteroaryl, aralkyl or heteroaralkyl group; all of which groups may optionally be substituted; and
R5 is a hydrogen atom or an alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, alkylcycloalkyl, hetero- alkylcycloalkyl, aryl, heteroaryl, aralkyl or heteroaralkyl group; all of which groups may optionally be substituted; or
R4 and R5 together with the nitrogen atom to which they are bound are part of an optionally substituted heterocycloalkyl group containing 5 , 6 or 7 ring atoms ; or a pharmaceutically acceptable salt, ester, solvate or hydrate or a pharmaceutically acceptable formulation thereof.
In the following, preferred embodiments of the present invention are disclosed. It is preferred that the preferred embodiments may be combined in any manner: Preferably, R1 and R2 together are a group of formula -O- CH2 - CH2-0- or -O- CH2-O-.
Further preferably, R1 and R2 are both hydrogen atoms.
Moreover preferably, R1 and R2 are both chlorine atoms.
Further preferably, X is a bond, an oxygen atom or a group of formula -(CH2)n- wherein n is 1, 2 or 3 or a group of formula - ( CH2)3-0- .
Especially preferably, X is a bond.
Moreover preferably, Ar is a phenyl group or a heteroaryl group containing one ring and 5 or 6 ring atoms selected from C, O, N and S, wherein Ar may be substituted by 1, 2, 3, 4 or 5 groups R3 and/or wherein Ar may be substituted by a group of formula -CH2-N (R4) -R5.
Further preferably, Ar is substituted by one or two groups R3.
Moreover preferably, the groups R3 are independently halogen, NO2 , N3, OH, SH, NH2 or a Ci - C6 alkyl group; a Ci - Ce heteroalkyl group; a cycloalkyl group containing one ring having from 3 to 7 ring atoms; a heterocycloalkyl group containing one ring having from 3 to 7 ring atoms comprising 1 or 2 heteroatoms selected from S, O and N; an alkylcycloalkyl group containing a C1-C6 alkyl group and a cycloalkyl group containing one ring having from 3 to 7 ring atoms; a heteroalkylcycloalkyl group containing a Ci - C6 alkyl group or a C1-C6 heteroalkyl group and a cycloalkyl group containing one ring having from 3 to 7 ring atoms or a heterocycloalkyl group containing one ring having from 3 to 7 ring atoms comprising 1 or 2 heteroatoms selected from S, O and N with the proviso that a heteroalkylcycloalkyl group contains at least one heteroatom; a phenyl group; a heteroaryl group containing 5 or 6 ring atoms comprising 1, 2, 3 or 4 heteroatoms selected from 0, S and N; an aralkyl group containing a Ci - Ce alkyl group and a phenyl group; or a heteroaralkyl group containing a Ci - Ce alkyl group or a Ci - Ce heteroalkyl group and a phenyl group or a heteroaryl group containing 5 or 6 ring atoms comprising 1, 2, 3 or 4 heteroatoms selected from O, S and N with the proviso that a heteroaralkyl group contains at least one heteroatom; all of which groups may optionally be substituted.
Especially preferably, Ar is substituted by a group of formula - CH2 -N ( R4 ) - R5 .
Further preferably, Ar is substituted by a group of formula - CH2 -N ( R4 ) - R5 and further by one group R3 , which is preferably selected from F, CI , -OMe and - CHO .
Moreover preferably, R4 is a hydogen atom and R5 is a hydrogen atom or a C1-C6 alkyl group; a C1-C6 heteroalkyl group; a cycloalkyl group containing one ring having from 3 to 7 ring atoms; a heterocycloalkyl group containing one ring having from 3 to 7 ring atoms comprising 1 or 2 heteroatoms selected from S, O and N; an alkylcycloalkyl group containing a Ci - Ce alkyl group and a cycloalkyl group containing one ring having from 3 to 7 ring atoms; a heteroalkylcycloalkyl group containing a C1 - C6 alkyl group or a C1 - C6 heteroalkyl group and a cycloalkyl group containing one ring having from 3 to 7 ring atoms or a heterocycloalkyl group containing one ring having from 3 to 7 ring atoms comprising 1 or 2 heteroatoms selected from S, O and N with the proviso that a heteroalkylcycloalkyl group contains at least one heteroatom; a phenyl group; a heteroaryl group containing 5 or 6 ring atoms comprising 1, 2, 3 or 4 heteroatoms selected from O, S and N; an aralkyl group containing a C1-C6 alkyl group and a phenyl group; or a heteroaralkyl group containing a C1-C6 alkyl group or a Ci-Cs heteroalkyl group and a phenyl group or a heteroaryl group containing 5 or 6 ring atoms comprising 1, 2, 3 or 4 heteroatoms selected from O, S and N with the proviso that a heteroaralkyl group contains at least one heteroatom; all of which groups may optionally be substituted.
Further preferably, R4 and R5 together with the nitrogen atom to which they are bound are part of a heterocycloalkyl group containing 5, 6 or 7 ring atoms and optionally one further heteroatom selected from O and N, which heterocycloalkyl group may be substituted (preferably by a C1-C4 alkyl group or a group of formula -C(=0)-CH3 or -CH2OH) .
Especially preferred are compounds of formula (II) :
Figure imgf000007_0001
(ID wherein R1, R2, R4 and R5 are as defined above. Further especially preferred are compounds of formula (III) :
Figure imgf000008_0001
(III] wherein R1, R2, R4 and R5 are as defined above and Rs is H or OMe.
Moreover especially preferred are compounds of formula (IV) :
Figure imgf000008_0002
(IV) wherein R1, R2, R4 and Rs are as defined above.
According to a preferred embodiment, the following compounds are excluded from the scope of the present application:
Figure imgf000009_0001
The most preferred compounds of formula (I) of the present invention are the compounds disclosed in the examples .
The expression alkyl refers to a saturated, straight-chain or branched hydrocarbon group that contains from 1 to 20 carbon atoms, preferably from 1 to 12 carbon atoms, especially from 1 to 6 (e.g. 1, 2, 3 or 4) carbon atoms, for example a methyl, ethyl, propyl, iso-propyl, n-butyl, iso-butyl, sec-butyl, tert-butyl, n-pentyl, iso-pentyl, n-hexyl, 2 , 2-dimethylbutyl or n-octyl group. Furthermore, the term alkyl refers to groups in which one or more hydrogen atoms have been replaced by a halogen atom (preferably F or CI) such as, for example, a 2 , 2 , 2-trichloroethyl or a trifluoromethyl group.
The expressions alkenyl and alkynyl refer to at least partially unsaturated, straight-chain or branched hydrocarbon groups that contain from 2 to 20 carbon atoms, preferably from 2 to 12 carbon atoms, especially from 2 to 6 (e.g. 2, 3 or 4) carbon atoms, for example an ethenyl (vinyl) , propenyl (allyl) , iso-propenyl, butenyl, ethinyl, propinyl, butinyl, acetylenyl, propargyl, isoprenyl or hex-2-enyl group. Preferably, alkenyl groups have one or two (especially preferably one) double bond(s) , and alkynyl groups have one or two (especially preferably one) triple bond(s) . Furthermore, the terms alkenyl and alkynyl refer to groups in which one or more hydrogen atoms have been replaced by a halogen atom (preferably F or CI) .
The expression heteroalkyl refers to an alkyl, alkenyl or alkynyl group in which one or more (preferably 1, 2 or 3) carbon atoms have been replaced by an oxygen, nitrogen, phosphorus, boron, selenium, silicon or sulfur atom (preferably by an oxygen, sulfur or nitrogen atom) or a group of formula SO or SO2. The expression heteroalkyl furthermore refers to a carboxylic acid or to a group derived from a carboxylic acid, such as, for example, acyl, acylalkyl, alkoxycarbonyl , acyloxy, acyloxyalkyl , carboxyalkylamide or alkoxycarbonyloxy.
Preferably, a heteroalkyl group contains from 1 to 12 carbon atoms and from 1 to 4 hetero atoms selected from oxygen, nitrogen and sulphur (especially oxygen and nitrogen) . Especially preferably, a heteroalkyl group contains from 1 to 6 (e.g. 1, 2, 3 or 4) carbon atoms and 1, 2 or 3 (especially 1 or 2) hetero atoms selected from oxygen, nitrogen and sulphur (especially oxygen and nitrogen) . The term C1-C6 heteroalkyl refers to a heteroalkyl group containing from 1 to 6 carbon atoms and 1, 2 or 3 heteroatoms selected from 0, S and/or N (especially O and/or N) . The term C1-C4 heteroalkyl refers to a heteroalkyl group containing from 1 to 4 carbon atoms and 1, 2 or 3 heteroatoms selected from 0, S and/or N (especially 0 and/or N) . Furthermore, the term heteroalkyl refers to groups in which one or more hydrogen atoms have been replaced by a halogen atom (preferably F or Cl) .
Examples of heteroalkyl groups are groups of formulae: Ra _ 0 _ Ya_ ; Ra_ s _Ya _ i Ra-N (Rb) -Ya- , Ra-C0-Ya- , Ra-0-C0-Ya- , Ra-CO- 0-Ya-, Ra-CO-N(Rb) -Ya-, Ra-N (Rb) -CO-Ya- , Ra-0-CO-N (Rb) -Ya- , Ra-N(Rb) -CO- 0-Ya- , Ra-N (Rb) -CO-N (Rc) -Ya- , Ra - 0-CO- 0-Y - ,
Ra-N (Rb) -C ( =NRd) -N (Rc) -Ya- , Ra-CS-Ya-( Ra - 0-CS-Ya-, Ra-CS-0-Ya-, Ra- CS-N(Rb) -Ya- , Ra-N(Rb) - CS-Ya-, Ra - 0- CS-N (Rb) -Ya- ,
Ra-N (Rb) -CS-0-Ya- , Ra-N (Rb) - CS -N (Rc) -Ya- , Ra -0 - CS - 0-Ya- ,
Ra-S-CO-Ya-, Ra-CO-S-Ya-, Ra-S-CO-N(Rb) -Ya-, Ra-N (Rb) -CO-S-Ya- , Ra-S-CO-0-Ya- , Ra-0-CO-S-Ya- , Ra-S-CO-S-Ya- , Ra-S-CS-Ya-, Ra-CS-S-Ya-, Ra-S-CS-N(Rb) -Ya-, Ra-N (Rb) - CS - S -Ya- , Ra-S-CS-0-Ya- , R - 0-CS-S-Ya- , wherein Ra being a hydrogen atom, a Ci-C~6 alkyl, a C2-C6 alkenyl or a C2-C6 alkynyl group; Rb being a hydrogen atom, a Ci-C6 alkyl, a C2-C6 alkenyl or a C2- C6 alkynyl group; Rc being a hydrogen atom, a C1-C6 alkyl, a C -C6 alkenyl or a C2-C6 alkynyl group; Rd being a hydrogen atom, a Ci-C& alkyl, a C2-C6 alkenyl or a C2-C6 alkynyl group and Ya being a direct bond, a Ci-C6 alkylene, a C2-C6 alkenylene or a C2-C6 alkynylene group, wherein each heteroalkyl group contains at least one carbon atom and one or more hydrogen atoms may be replaced by fluorine or chlorine atoms.
Specific examples of heteroalkyl groups are methoxy, trifluoromethoxy, ethoxy, n-propyloxy, isopropyloxy, butoxy, tert-butyloxy, methoxymethyl , ethoxymethyl , -CH2CH2OH, -CH2OH, methoxyethyl , 1-methoxyethyl , 1-ethoxyethyl, 2 -methoxyethyl or 2-ethoxyethyl, methylamino, ethylamino, propylamino, isopropylamino, dimethylamino, diethylamino, isopropyl- ethylamino, methylamino methyl, ethylamino methyl, diiso- propylamino ethyl, methylthio, ethylthio, isopropylthio, enol ether, dimethylamino methyl, dimethylamino ethyl, acetyl, propionyl, butyryloxy, acetyloxy, methoxycarbonyl , ethoxy- carbonyl, propionyloxy, acetylamino or propionylamino, carboxymethyl, carboxyethyl or carboxypropyl , N-ethyl-N- methylcarbamoyl or N-methylcarbamoyl . Further examples of heteroalkyl groups are nitrile, isonitrile, cyanate, thio- cyanate, isocyanate, isothiocyanate and alkylnitrile groups.
The expression cycloalkyl refers to a saturated or partially unsaturated (for example, a cycloalkenyl group) cyclic group that contains one or more rings (preferably 1 or 2) , and contains from 3 to 14 ring carbon atoms, preferably from 3 to 10 (especially 3, 4, 5, 6 or 7) ring carbon atoms. The expression cycloalkyl refers furthermore to groups in which one or more hydrogen atoms have been replaced by fluorine, chlorine, bromine or iodine atoms or by OH, =0, SH, =S, N¾, =NH, N3 or NO2 groups, thus, for example, cyclic ketones such as, for example, cyclohexanone, 2-cyclohexenone or cyclopenta- none. Further specific examples of cycloalkyl groups are a cyclopropyl , cyclobutyl , cyclopentyl , spiro [4,5] decanyl , norbornyl, cyclohexyl, cyclopentenyl, cyclohexadienyl, decalinyl, bicyclo [4.3.0] nonyl, tetraline, cyclopentylcyclohexyl, fluorocyclohexyl or cyclohex-2-enyl grou .
The expression heterocycloalkyl refers to a cycloalkyl group as defined above in which one or more (preferably 1, 2, 3 or 4) ring carbon atoms have been replaced by an oxygen, nitrogen, silicon, selenium, phosphorus or sulfur atom (preferably by an oxygen, sulfur or nitrogen atom) . A heterocycloalkyl group has preferably 1 or 2 ring(s) containing from 3 to 10 (especially 3, 4, 5, 6 or 7) ring atoms (preferably secected from C, O, N and S) . The expression heterocycloalkyl refers furthermore to groups that may be substituted by one or more fluorine, chlorine, bromine or iodine atoms or by one or more OH, =0, SH, =S, NH2, =NH, N3 or N02 groups. Examples are a piperidyl, prolinyl, imidazolidinyl, piperazinyl, morpholinyl, urotropinyl, pyrrolidinyl, tetrahydrothiophenyl , tetrahydropyranyl , tetrahydrofuryl or 2-pyrazolinyl group and also lactames, lactones, cyclic imides and cyclic anhydrides.
The expression alkylcycloalkyl refers to groups that contain both cycloalkyl and also alkyl, alkenyl or alkynyl groups in accordance with the above definitions, for example alkylcycloalkyl, cycloalkylalkyl, alkylcycloalkenyl , alkenylcycloalkyl and alkynylcycloalkyl groups. An alkylcycloalkyl group preferably contains a cycloalkyl group that contains one or two rings having from 3 to 10 (especially 3, 4, 5, 6 or 7) ring carbon atoms, and one or two alkyl, alkenyl or alkynyl groups (especially alkyl groups) having 1 or 2 to 6 carbon atoms .
The expression heteroalkylcycloalkyl refers to alkylcycloalkyl groups as defined above in which one or more (preferably 1, 2, 3, 4 or 5) carbon atoms have been replaced by an oxygen, nitrogen, silicon, selenium, phosphorus or sulfur atom (preferably by an oxygen, sulfur or nitrogen atom) . A heteroalkylcycloalkyl group preferably contains 1 or 2 rings having from 3 to 10 (especially 3, 4, 5, 6 or 7) ring atoms, and one or two alkyl, alkenyl, alkynyl or heteroalkyl groups (especially alkyl or heteroalkyl groups) having from 1 or 2 to 6 carbon atoms (the heteroalkyl groups having preferably 1, 2 or 3 heteroatoms selected from 0, S and N) . Examples of such groups are alkylheterocycloalkyl, alkylheterocycloalkenyl, alkenylheterocycloalkyl, alkynylheterocycloalkyl, heteroalkylcycloalkyl, heteroalkylheterocycloalkyl and hetero- alkylheterocycloalkenyl, the cyclic groups being saturated or mono-, di- or tri-unsaturated. The expression aryl refers to an aromatic group that contains one or more rings containing from 5 or 6 to 14 ring carbon atoms, preferably from 5 or 6 to 10 (especially 6) ring carbon atoms. The expression aryl refers furthermore to groups in which one or more hydrogen atoms have been replaced by fluorine, chlorine, bromine or iodine atoms or by OH, SH, NH2, N3 or NO2 groups . Examples are the phenyl , naphthyl , biphenyl , 2-fluorophenyl , anilinyl, 3 -nitrophenyl or 4-hydroxyphenyl group .
The expression heteroaryl refers to an aromatic group that contains one or more rings containing from 5 to 14 ring atoms, preferably from 5 to 10 (especially 5 or 6 or 9 or 10) ring atoms, and contains one or more (preferably 1, 2, 3, 4 or 5) oxygen, nitrogen, phosphorus or sulfur ring atoms (preferably O, S or N) . The expression heteroaryl refers furthermore to groups in which one or more hydrogen atoms have been replaced by fluorine, chlorine, bromine or iodine atoms or by OH, SH, N3, NH2 or NO2 groups. Examples are pyridyl (e.g. 4-pyridyl) , imidazolyl (e.g. 2-imidazolyl) , phenylpyrrolyl (e.g. 3- phenylpyrrolyl) , thiazolyl, isothiazolyl, 1, 2 , 3-triazolyl, 1, 2 , 4-triazolyl, oxadxazolyl, thiadxazolyl, indolyl, indazolyl, tetrazolyl, pyrazinyl, pyrimidinyl, pyridazinyl, oxazolyl, isoxazolyl, triazolyl, tetrazolyl, isoxazolyl, indazolyl, indolyl, benzimidazolyl, benzoxazolyl , benzisoxazolyl, benzthiazolyl, pyridazinyl, quinolinyl, isoquinolinyl, pyrrolyl , purinyl , carbazolyl , acridinyl , pyrimidyl , 2,3'- bifuryl, pyrazolyl (e.g. 3-pyrazolyl) and isoquinolinyl groups .
The expression aralkyl refers to groups containing both aryl and also alkyl, alkenyl, alkynyl and/or cycloalkyl groups in accordance with the above definitions, such as, for example, arylalkyl, arylalkenyl, arylalkynyl, arylcycloalkyl, aryl- cycloalkenyl, alkylarylcycloalkyl and alkylarylcycloalkenyl groups. Specific examples of aralkyls are toluene, xylene, mesitylene, styrene, benzyl chloride, o-fluorotoluene, lH-indene, tetraline, dihydronaphthalene , indanone, phenylcyclopentyl , cumene, cyclohexylphenyl, fluorene and indane . An aralkyl group preferably contains one or two aromatic ring systems (1 or 2 rings) containing from 6 to 10 carbon atoms and one or two alkyl, alkenyl and/or alkynyl groups containing from 1 or 2 to 6 carbon atoms and/or one or two cycloalkyl groups containing 5 or 6 ring carbon atoms.
The expression heteroaralkyl refers to an aralkyl group as defined above in which one or more (preferably 1, 2, 3 or 4) carbon atoms have been replaced by an oxygen, nitrogen, silicon, selenium, phosphorus, boron or sulfur atom (preferably oxygen, sulfur or nitrogen) , that is to say to groups containing both aryl or heteroaryl, respectively, and also alkyl, alkenyl, alkynyl and/or heteroalkyl and/or cycloalkyl and/or heterocycloalkyl groups in accordance with the above definitions. A heteroaralkyl group preferably contains one or two aromatic ring systems (1 or 2 rings) containing from 5 or 6 to 10 ring carbon atoms and one or two alkyl, alkenyl and/or alkynyl groups containing 1 or 2 to 6 carbon atoms and/or one or two cycloalkyl groups containing 5 or 6 ring carbon atoms, wherein 1, 2, 3, 4, 5 or 6 of these carbon atoms have been replaced by oxygen, sulfur or nitrogen atoms .
Examples are arylheteroalkyl, arylheterocycloalkyl, aryl- heterocycloalkenyl, arylalkylheterocycloalkyl, arylalkenyl- heterocycloalkyl, arylalkynylheterocycloalkyl , arylalkyl- heterocycloalkenyl, heteroarylalkyl , heteroarylalkenyl , heteroarylalkynyl, heteroarylheteroalkyl , heteroaryl- cycloalkyl, heteroarylcycloalkenyl, heteroarylhetero- cycloalkyl, heteroarylheterocycloalkenyl , heteroarylalkyl- cycloalkyl, heteroarylalkylheterocycloalkenyl, heteroaryl- heteroalkylcycloalkyl, heteroarylheteroalkylcycloalkenyl and heteroarylheteroalkylheterocycloalkyl groups, the cyclic groups being saturated or mono-, di- or tri-unsaturated. Specific examples are a tetrahydroisoquinolinyl, benzoyl, 2- or 3-ethylindolyl, 4 -methylpyridino, 2-, 3- or
4-methoxyphenyl, 4-ethoxyphenyl, 2-, 3- or 4-carboxy- phenylalkyl group.
The term halogen or halogen atom refers to F , CI , Br or I .
The expression "optionally substituted" especially refers to groups in which one, two, three or more hydrogen atoms may have been replaced by fluorine, chlorine, bromine or iodine atoms or by OH, =0, SH, =S, H2, =NH, N3 or N02 groups. This expression refers furthermore to groups that may be substituted by one, two, three or more preferably unsubstituted Ci - Cio alkyl, C2 - C10 alkenyl, C2 - C10 alkynyl, C1 - C10 heteroalkyl, C3-C18 cycloalkyl, C2 - Ci7 heterocycloalkyl, C4- C20 alkylcycloalkyl , C2-C19 heteroalkylcycloalkyl, C6-C18 aryl, C1-C17 heteroaryl, C7- C20 aralkyl or C2-C19 heteroaralkyl groups. This expression refers furthermore especially to groups that may be substituted by one, two, three or more preferably unsubstituted C1-C6 alkyl, C2 - C6 alkenyl, C2 - C6 alkynyl, Ci - C6 heteroalkyl, C3 - C10 cycloalkyl, C2-C9 heterocycloalkyl, C7- C12 alkylcycloalkyl, C2 - Cn heteroalkylcycloalkyl, C6- C10 aryl, C1-C9 heteroaryl, C7- C12 aralkyl or C2 - Cu heteroaralkyl groups. If a substituent contains a ring, this ring may be bonded to the respective substituted group via a single or double bond (especially a single bond) or, if the substituted group also contains a ring, the ring of the substituent may also be annulated to the ring of the substituted group.
Preferred substituents are F, CI, Br, I, OH, =0, NH2, N02, Ci-4 alkyl (e.g. methyl, ethyl, t-butyl ) , NMe2, NHMe, CONH2, CH2NMe2, NHS02Me, C(CH3)2CN, COMe, OMe, SMe, COOMe, COOEt, CH2COOH, OCH2COOH, COOH, SOMe, S02Me, cyclopropyl, S02NH2, S02NHMe, S02CH2CH2OH, SF5, S02NMe2, CHO, OCF3, S02CF3, COMe, CH2OH, CN or CF3.
Especially preferred substituents are F, CI, Br, OH, NH2, Me, Ethyl, Me2, CONH2, OMe, CN or CF3.
According to a preferred embodiment, all alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, alkylcycloalkyl, heteroalkylcycloalkyl, aralkyl and heteroaralkyl groups described herein may optionally be substituted.
When an aryl, heteroaryl, cycloalkyl, alkylcycloalkyl, heteroalkylcycloalkyl, heterocycloalkyl, aralkyl or heteroaralkyl group contains more than one ring, these rings may be bonded to each other via a single or double bond or these rings may be annulated.
The present invention further provides pharmaceutical compositions comprising one or more compounds of formula (I) , (II) , (III) or (IV) as defined herein or a pharmaceutically acceptable ester, prodrug, hydrate, solvate or salt thereof, optionally in combination with a pharmaceutically acceptable carrier.
It is a further object of the present invention to provide a compound of formula (I) , (II) , (III) or (IV) as defined herein or a pharmaceutical composition as defined herein for the preparation of a medicament for the treatment of one or more diseases mentioned herein.
Preferably the compounds of the present invention may be used for the treatment and/or prevention of cancer, viral diseases and infectious diseases .
Further preferably, the compounds of the present invention may be used for the treatment and/or prevention of neurodegenerative diseases such as: Schizophrenia, Alzheimer, Multiples Sclerosis, Parkinson, Corea Huntington, Spinocerebellar ataxia type 1 (SCA1) , Amyotrophic lateral sclerosis, Batten disease.
A therapeutically effective amount of a compound in accordance with this invention means an amount of compound that is effective to prevent, alleviate or ameliorate symptoms of disease or prolong the survival of the subject being treated. Determination of a therapeutically effective amount is within the skill in the art.
The therapeutically effective amount or dosage of a compound according to this invention can vary within wide limits and may be determined in a manner known in the art. Such dosage may be adjusted to the individual requirements in each particular case including the specific compound being administered, the route of administration, the condition being treated, as well as the patient being treated.
Examples of pharmacologically acceptable salts of sufficiently basic compounds of formula (I) , (II) , (III) or (IV) are salts of physiologically acceptable mineral acids like hydrochloric, hydrobromic, sulfuric and phosphoric acid; or salts of organic acids like methanesulfonic, p-toluenesulfonic, lactic, acetic, trifluoroacetic, citric, succinic, fumaric, maleic and salicylic acid. Further, a sufficiently acidic compound of formula (I) , (II) , (III) or (IV) may form alkali or earth alkali metal salts, for example sodium, potassium, lithium, calcium or magnesium salts; ammonium salts; or organic base salts, for example methylamine, dimethylamine, trimethylamine, triethylamine, ethylenediamine , ethanolamine, choline hydroxide, meglumin, piperidine, morpholine, tris- (2- hydroxyethyl) amine, lysine or arginine salts; all of which are also further examples of salts of formula (I) , (II) , (III) or (IV) . Compounds of formula (I) , (II) , (III) or (IV) may be solvated, especially hydrated. The hydratization/hydration may occur during the process of production or as a consequence of the hygroscopic nature of the initially water free compounds of formula (I) , (II) , (III) or (IV) . The solvates and/or hydrates may e.g. be present in solid or liquid form.
It should be appreciated that certain compounds of formula (I) , (II) , (III) or (IV) may have tautomeric forms from which only one might be specifically mentioned or depicted in the following description, different geometrical isomers (which are usually denoted as cis/trans isomers or more generally as (E) and (Z) isomers) or different optical isomers as a result of one or more chiral carbon atoms (which are usually nomenclatured under the Cahn-Ingold-Prelog or R/S system) . All these tautomeric forms, geometrical or optical isomers (as well as racemates and diastereomers) and polymorphous forms are included in the invention. Since the compounds of formula
(I) , (II) , (III) or (IV) may contain asymmetric C-atoms, they may be present either as achiral compounds, mixtures of diastereomers, mixtures of enantiomers or as optically pure compounds. The present invention comprises both all pure enantiomers and all pure diastereomers, and also the mixtures thereof in any mixing ratio.
The therapeutic use of compounds according to formula (I) ,
(II) , (III) or (IV) , their pharmacologically acceptable salts, solvates and hydrates, respectively, as well as formulations and pharmaceutical compositions also lie within the scope of the present invention.
The pharmaceutical compositions according to the present invention comprise at least one compound of formula (I) , (II) ,
(III) or (IV) as an active ingredient and, optionally, carrier substances and/or adjuvants.
The present invention also relates to pro-drugs which are composed of a compound of formula (I) , (II) , (III) or (IV) and at least one pharmacologically acceptable protective group which will be cleaved off under physiological conditions, such as an alkoxy- , arylalkyloxy- , acyl-, acyloxymethyl group (e.g. pivaloyloxymethyl) , an 2-alkyl-, 2-aryl- or 2-arylalkyl- oxycarbonyl-2-alkylidene ethyl group or an acyloxy group as defined herein, e.g. ethoxy, benzyloxy, acetyl or acetyloxy or, especially for a compound of formula (I) , (II) , (III) or (IV), carrying a hydroxy group (-OH): a sulfate, a phosphate (-OPO3 or -OCH2OPO3) or an ester of an amino acid.
Preferably, the present invention also relates to a prodrug, a biohydrolyzable ester, a biohydrolyzable amide, a polymorph, tautomer, stereoisomer, metabolite, N-oxide, biohydrolyzable carbamate, biohydrolyzable ether, physiologically functional derivative, atropisomer, or in vivo-hydrolysable precursor, diastereomer or mixture of diastereomers, chemically protected form, affinity reagent, complex, chelate and a stereoisomer of the compounds of formula (I) , (II) , (III) or (IV) .
As used herein, the term pharmaceutically acceptable ester especially refers to esters which hydrolyze in vivo and include those that break down readily in the human body to leave the parent compound or a salt thereof. Suitable ester groups include, for example, those derived from pharmaceutically acceptable aliphatic carboxylic acids, particularly alkanoic, alkenoic, cycloalkanoic and alkanedioic acids, in which each alkyl or alkenyl moiety advantageously has not more than 6 carbon atoms . Examples of particular esters include, but are not limited to, formates, acetates, propionates, butyrates, acrylates and ethylsuccinates .
As mentioned above, therapeutically useful agents that contain compounds of formula (I), (II), (III) or (IV), their solvates, salts or formulations are also comprised in the scope of the present invention. In general, compounds of formula (I) , (II) , (III) or (IV) will be administered by using the known and acceptable modes known in the art, either alone or in combination with any other therapeutic agent. For oral administration such therapeutically useful agents can be administered by one of the following routes: oral, e.g. as tablets, dragees, coated tablets, pills, semisolids, soft or hard capsules, for example soft and hard gelatine capsules, aqueous or oily solutions, emulsions, suspensions or syrups, parenteral including intravenous, intramuscular and subcutaneous injection, e.g. as an injectable solution or suspension, rectal as suppositories, by inhalation or insufflation, e.g. as a powder formulation, as microcrystals or as a spray (e.g. liquid aerosol), transdermal, for example via an transdermal delivery system (TDS) such as a plaster containing the active ingredient or intranasal . For the production of such tablets, pills, semisolids, coated tablets, dragees and hard, e.g. gelatine, capsules the therapeutically useful product may be mixed with pharmaceutically inert, inorganic or organic excipients as are e.g. lactose, sucrose, glucose, gelatine, malt, silica gel, starch or derivatives thereof, talc, stearinic acid or their salts, dried skim milk, and the like. For the production of soft capsules one may use excipients as are e.g. vegetable, petroleum, animal or synthetic oils, wax, fat, polyols. For the production of liquid solutions, emulsions or suspensions or syrups one may use as excipients e.g. water, alcohols, aqueous saline, aqueous dextrose, polyols, glycerin, lipids, phospholipids, cyclodextrins , vegetable, petroleum, animal or synthetic oils. Especially preferred are lipids and more preferred are phospholipids (preferred of natural origin; especially preferred with a particle size between 300 to 350 nm) preferred in phosphate buffered saline (pH = 7 to 8 , preferred 7.4). For suppositories one may use excipients as are e.g. vegetable, petroleum, animal or synthetic oils, wax, fat and polyols . For aerosol formulations one may use compressed gases suitable for this purpose, as are e.g. oxygen, nitrogen and carbon dioxide. The pharmaceutically useful agents may also contain additives for conservation, stabilization, e.g. UV stabilizers, emulsifiers, sweetener, aromatizers, salts to change the osmotic pressure, buffers, coating additives and antioxidants .
In general, in the case of oral or parenteral administration to adult humans weighing approximately 80 kg, a daily dosage of about 10 mg to about 10,000 mg, preferably from about 20 mg to about 1,000 mg, should be appropriate, although the upper limit may be exceeded when indicated. The daily dosage can be administered as a single dose or in divided doses, or for parenteral administration, it may be given as continuous infusion or subcutaneous injection.
The present invention moreover provides a method of inhibiting growth, proliferation, or metastasis of cancer cells in a subject in need thereof, said method comprising administering to the subject a therapeutically effective amount of a compound of formula (I), (II), (III) or (IV), or a pharmaceutically acceptable salt . In one embodiment the cancer is selected from melanoma, renal cell carcinoma, squamous non- small cell lung cancer (NSCLC) , non-squamous NSCLC, colorectal cancer, castration-resistant prostate cancer, ovarian cancer, gastric cancer, hepatocellular carcinoma, pancreatic carcinoma, squamous cell carcinoma of the head and neck, carcinomas of the esophagus, gastrointestinal tract and breast, cancer of the genital organs, penis and vagina, and a hematological malignancy. Further the present invention provides a method of treating an infectious disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a compound of formula (I) , (II) , (III) or (IV), or a pharmaceutically acceptable salt thereof. In one embodiment the infectious disease is caused by a virus. In a further embodiment the virus is selected from HIV, Hepatitis A, Hepatitis B, Hepatitis C, Hepatitis D, herpes viruses, papillomaviruses and influenza.
In one embodiment, a method is provided for treating cancer comprising administering to a patient in need thereof, a therapeutically effective amount of a compound of formula (I) , (II), (III) or (IV) or a salt thereof. Examples of cancers include those whose growth may be inhibited using compounds of the disclosure include cancers typically responsive to immunotherapy. Non-limiting examples of preferred cancers for treatment include melanoma (e.g., metastatic malignant melanoma) , renal cancer (e.g. clear cell carcinoma) , prostate cancer (e.g. hormone refractory prostate adenocarcinoma), breast cancer, colon cancer and lung cancer (e.g. non-small cell lung cancer) . Additionally, the disclosure includes refractory or recurrent malignancies whose growth may be inhibited using the compounds of the present invention.
Examples of other cancers that may be treated using the methods of the present invention include bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin's Disease, non-Hodgkin' s lymphoma, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, chronic or acute leukemias including acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, solid tumors of childhood, lymphocytic lymphoma, cancer of the bladder, cancer of the kidney or urethra, carcinoma of the renal pelvis, neoplasm of the central nervous system (CNS) , primary CNS lymphoma, tumor angiogenesis, spinal axis tumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid cancer, squamous cell cancer, T-cell lymphoma, environmentally induced cancers including those induced by asbestos, and combinations of said cancers. The present invention is also useful for treatment of metastatic cancers, especially metastatic cancers that express PD-L1.
Some examples of pathogenic viruses causing infections treatable by methods of the present invention include HIV, hepatitis (A, B, C, or D) , herpes viruses (e.g., VZV, HSV-1 , HAV-6, HHv-7, HHV-8, HSV-2, CMV, and Epstein Barr virus), adenovirus, influenza virus, fiaviviruses, echovirus, rhinovirus, coxsackie virus, cornovirus, respiratory syncytial viras, mumps viras, rotaviras, measles viras, rabella viras, parvovirus, vaccinia virus, HTLV viras, dengue viras, papillomavirus, molluscum viras, poliovirus, rabies viras, JC viras and arboviral encephalitis viras. Some examples of pathogenic bacteria causing infections treatable by methods of the present invention include chlamydia, rickettsial bacteria, mycobacteria, staphylococci, streptococci, pneumonococci, meningococci and conococci, lebsiella, proteus, serratia, pseudomonas, legionella, diphtheria, salmonella, bacilli, cholera, tetanus, botulism, anthrax, plague, leptospirosis, and Lymes disease bacteria.
Some examples of pathogenic fungi causing infections treatable by methods of the present invention include Candida (albicans, krasei, glabrata, tropicalis, etc.), Cryptococcus neoformans, Aspergillus (fumigatus, niger, etc.), Genus Mucorales (mucor, absidia, rhizophus) , Sporothrix schenkii, Blastomyces dermatitidis , Paracoccidioides brasiliensis, Coccidioides immitis and Histoplasma capsulatum.
Some examples of pathogenic parasites causing infections treatable by methods of the present invention include Entamoeba histolytica, Balantidium coli, Naegleriafowleri, Acanthamoeba sp., Giardia lambia, Cryptosporidium sp., Pneumocystis carinii, Plasmodium vivax, Babesia microti, Trypanosoma bracei, Trypanosoma crazi, Leishmania donovani, Toxoplasma gondi, and Nippostrongylus brasiliensis.
Examples
I. Intermediates A and B:
(In the following Scheme, R represents R1 and R2)
Figure imgf000027_0001
General procedure for the preparation of substituted 2-amino- 4-phenylthiophene-3-carbonitrile (Building block A) :
To a stirred solution of dry dichloromethane (DCM) (200 mL) , the corresponding acetophenone (50.2 mmol) , and ethyl cyanoacetate (6.42 ml, 60.3 mmol) were added. The reaction mixture was cooled to 0°C and neat TiCl4 (11.02 ml, 100.5 mmol) was added dropwise. After completion of the addition at 0°C, the reaction mixture was stirred for 0.5 h. Then dry pyridine (3.4 ml) was added dropwise and stirred at room temperature (rt) for 1 h. Additional dry pyridine (10 ml) was added dropwise and the mixture was allowed to stir overnight. The mixture was poured into 3 M HCl (200 ml) and the organic layer was separated. The aqueous layer was further extracted with DCM (2 x 50 mL) , and the combined organic phases were washed with water and brine, dried with MgSC>4, filtered and concentrated yielding a viscous amber oil. The oil is dissolved in THF (40 mL) and elemental sulfur (2.0 g, 62.38 mmol) was added followed by Et2NH (10 mL) dropwise, and the solution was stirred at room temperature for 2 h. The mixture was diluted with ether (200 mL) and washed with water (2 x 100 mL) and finally with brine. The organic layer was separated, dried (MgS04) , filtered, and evaporated in vacuo.
The following specific intermediates A have been prepared according to the above procedure: Example 1: ethyl 2-amino-4 -phenylthiophene-3 -carboxylate
Figure imgf000028_0001
¾ NMR (500 MHz , CDC13): δ 4.91 (br s, 2H) , 6.35 (s, 1H) , 7.36- 7.37 (m, 1H) , 7.40-7.43 (m, 2H) , 7.57-7.59 (m, 2H) ppm. MS (ESI) m/z: calc. for Ci3Hi3N02S, [M+H]+: 248.31; found: 248.32.
Example 2: ethyl 2-amino-4- (2, 3-dihydrobenzo [b] [1, 4] dioxin-6- yl) thiophene-3 -carboxylate
Figure imgf000028_0002
¾ NMR (500 MHz, CDC13) : δ 1.04 (t, J" = 10 Hz, 3H) , 4.09 (q, J
= 10 Hz, 2H) , 6.02 (s, 1H) , 6.06 br s, 2H) , 6.77-6.83 (m, 3H) ppm. MS (ESI) m/z: calc. for Ci5Hi5N04S, [M+H]+: 248.31; found: 248.37.
Example 3: ethyl 2-amino-4- (3 , 4-dichlorophenyl) thiophene-3- carboxylate
Figure imgf000028_0003
¾ NMR {500 MHz , CDCI3) : δ 1.02 (t, J" = 10 Hz, 3H) , 4.08 (q, J = 10 Hz, 2H) , 6.07 (s, 1H) , 6.15 (br s, 2H) , 7.14 (dd, J" = 5 , 0.5 Hz, 1H) , 7.38 (d, J = 10 Hz, 1H) , 7.40 (d, J = 5 Hz, 1H) ppm. MS (ESI) m/z: calc. for C13HiiCl2N02S , [M+H]+: 317.20; found: 317.28.
Example 4: ethyl 2-amino-4- (benzo [d] [1, 3] dioxol-5- yl) thiophene-3 -carboxylate
Figure imgf000029_0001
XH NMR (500 MHz, CDCI3) : δ 1.04 (t, J = 10 Hz, 3H) , 4.08 (q, J" = 10 Hz, 2H) , 5.96 (s, 2H) , 6.02 (s, 1H) , 6.08 (br s, 2H) , 6.77 (s, 2H) , 6.79 (s, 1H) ppm. MS (ESI) m/z: calc. for C14H13NO4S, [M+H]+: 292.32; found: 292.39.
Example 5a: ethyl 2-amino-4- (4-methyl-3 , -dihydro-2H- benzo [b] [1,4] oxazin-7-yl) thiophene-3-carboxylate
Figure imgf000029_0002
MS (ESI) m/z: calc. for CieHisNaOsS, [M+H]+: 319.10; found:
319.48.
Example 5b: ethyl 2-amino-4- (4-methyl-3 , 4-dihydro-2H- benzo [b] [1,4] oxazin-6-yl) thiophene-3 -carboxylate
Figure imgf000030_0001
MS (ESI) m/z: calc. for CieHisNaOsS , [M+H] + : 319.10; found: 319.21.
General procedure for the preparation of substituted 2- hydroxy-4 -phenylthiophene-3 -carbonitrile (Building block B) : To a boiling mixture of the corresponding building block A (40 mmol) in absolute ethanol (40 ml), a solution of Na (1.8 g) in EtOH (40 ml) was added dropwise. Then, the reaction mixture was refluxed for 4 h. Afterwards, 50 to 90 ml of the solvent were evaporated, the mixture was cooled, water (150 ml) was added and stirred for 1 h at rt. The residue was filtered off and the extract was acidified with HC1 (37%) . The solid was filtered and dried, providing the desired building blocks B.
The following specific intermediates B have been prepared according to the above procedure:
Example 6: 2-hydroxy-4-phenylthiophene-3-carbonitrile
Figure imgf000030_0002
¾ MR (500 MHz, CDCl3) : (equilibrium with the keto form) δ 4.59 (br s, 2H) , 7.56-7.59 (m, 2H) , 7.62-7.63 (m, 1H) , 7.89- 7.91 (m, 2H) ppm. MS (ESI) m/z: calc. for C11H7NOS, [M+H]+: 202.24; found: 202.35. Example 7: 4- (2 , 3-dihydrobenzo [b] [1, 4] dioxin-6-yl) -2- hydroxythiophene-3 -carbonitrile
Figure imgf000031_0001
1H NMR (500 MHz , CDC13) : (equilibrium with the keto form) δ 4.32 (t, J = 0.5 Hz, 2H) , 4.36 (t, J = 0.5 Hz, 2H) , 4.51 (s, 2H) , 7.01 (d, J = 10 Hz, 1H) , 7.47 (s, 1H) , 7.55 (d, J = 10 Hz, 1H) ppm. MS (ESI) m/z: calc. for C13H9NO3S , [M+H]+: 260.28; found: 260.32.
- (3 , -dichlorophenyl) -2 -hydroxythiophene-
Figure imgf000031_0002
XH NMR (500 MHz, CDCI3 ) : (equilibrium with the keto form) δ 4.54 (s, 2H) , 7.66 (d, J = 10 Hz, 1H) , 7.80 (dd, J = 10, 0.5 Hz, 1H) , 7.92 (d, J = 0.5 Hz, 1H) ppm. MS (ESI) m/z: calc. for C11H5CI2NOS , [M+H]+: 271.13; found: 271.19.
Example 9: 4- (benzo [d] [1, 3] dioxol-5-yl) -2 -hydroxythiophene-3- carbonitrile
Figure imgf000032_0001
Example 10: 2-hydroxy-4- (4 -methyl-3 , 4 -dihydro-2H- benzo [b] [1,4] oxazin-7-yl) thiophene-3-carbonitrile
Figure imgf000032_0002
MS (ESI) m/z: calc. for Ci4Hi2N202S, [M+H]+: 273.06; found:
273.12.
Example 11: 2-hydroxy-4- (4-methyl-3 , 4-dihydro-2H- benzo [b] [1, 4] oxazin-6-yl) thio hene-3-carbonitrile
Figure imgf000032_0003
MS (ESI) m/z: calc . for Q H^OaS , [M+H]+: 273.06; found:
273.12.
II. General procedure for the O-alkylation of building block C: (In the following scheme, R represents R1 and R2 and X represents one or more groups R3 or -C¾-N (R4) -R5)
Figure imgf000033_0001
B Z= Br, CI
In a microwave vial (4 ml) , the corresponding building block B (0.5 mmol) was mixed with K2CO3 (2.0 mmol) and the corresponding benzyl bromide (1.1 mmol) in MeCN (1 ml). The reaction mixture was irradiated at 130 °C for 20 min and purified by column chromatography.
The following compounds have been prepared according to the above procedure :
Example 12: 2- ( (4-bromobenzyl) oxy) -4 -phenylthiophene-3 - carbonitrile
Figure imgf000033_0002
¾ NMR (500 MHz , CDC13) : δ 5.29 (s, 2H) , 6.59 (s, 1H) , 7.34- 7.43 (m, 5H) , 7.44-7.58 (m, 4H) ppm. MS (ESI) m/z: calc. for Ci8Hi2BrNOS, CM+HJ+: 371.26; found: 371.32.
Example 13: tert-butyl 4- ( ( (3-cyano-4-phenylthiophen-2- yl) oxy) methyl) benzylcarbamate
Figure imgf000034_0001
MS (ESI) m/z: calc. for C24H24N2O3S , [M+H]+: 421.15; found: 421.17
Example 14: 2- (benzyloxy) -4 -phenylthiophene-3 -carbonitrile
Figure imgf000034_0002
MS (ESI) m/z: calc. for Ci8Hi3NOS, [M+H]+: 292.07; found: 292.08
Example 15: 2- ( (2-bromobenzyl) oxy) -4 -phenylthiophene-3 - carbonitrile
Figure imgf000034_0003
MS (ESI) m/z: calc. for Ci8Hi2BrNOS, [M+H]+: 369.98; found: 369.99
Example 16: 2- ( (3-chlorobenzyl) oxy) -4-phenylthiophene-3- carbonitrile
Figure imgf000035_0001
MS (ESI) m/z: calc. for Ci8Hi2ClNOS, [M+H]+: 326.03; found: 327.10
Example 17: 2- ( (4-iodobenzyl) oxy) -4 -phenylthiophene-3 - carbonitrile
Figure imgf000035_0002
MS (ESI) m/z: calc. for Ci8Hi2INOS, [M+H]+: 417.97; found: 417.99
Example 18: 2- (4 -nitrophenethoxy) -4 -phenylthiophene-3 - carbonitrile
Figure imgf000035_0003
MS (ESI) m/z: calc. for C19H14N2O3S , [M+H]+: 351.07; found: 351.17
Example 19: 2- ( (4-nitrobenzyl) oxy) -4-phenylthiophene-3 - carbonitrile
Figure imgf000036_0001
MS (ESI) m/z: calc. for Ci8Hi2N203S, [M+H]+: 337.06; found 337.08
Example 20: 4-phenyl-2- ( (3- (trifluoromethyl) benzyl) oxy) - thiophene-3 -carbonitril
Figure imgf000036_0002
MS (ESI) m/z: calc. for C19H12F3NOS , [M+H]+: 360.06; found: 360.12
Example 21: 2- (naphthalen-2-ylmethoxy) - -phenylthiophene-3- carbonitrile
Figure imgf000036_0003
MS (ESI) m/z: calc. for C22H15NOS, [M+H]+: 342.09; found: 342.13
Example 22: 2- ( (3 -fluorobenzyl) oxy) -4 -phenylthiophene-3- carbonitrile
Figure imgf000037_0001
MS (ESI) m/z: calc. for Ci8Hi2FNOS, [M+H] + : 310.06; found: 310.16
Example 23: 2- (3-phenoxypropoxy) -4 -phenylthiophene-3- carbonitrile
Figure imgf000037_0002
MS (ESI) m/z: calc. for C20H17NO2S, [M+H]+: 336.10; found: 336.20
Example 24: 2- ( (3 , 4-dichlorobenzyl) oxy) -4-phenylthiophene-3- carbonitrile
Figure imgf000037_0003
(ESI) m/z: calc. for Ci8HnCl2NOS, [M+H]+: 359.99; found .05 Example 25: 2- ( (4- (hydroxymethy1) benzyl) oxy) -4-phenyl- thiophene- 3 -carbonxtrile
Figure imgf000038_0001
MS (ESI) m/z: calc. for Ci9Hi5N02S, [M+H]+: 322.08; found: 322.12
Example 26: 2- ( (2 , 4-dichlorobenzyl) oxy) -4 -phenylthiophene-3 - carbonitrile
Figure imgf000038_0002
MS (ESI) m/z: ca2c. for Ci8HiiCl2NOS , [M+H]+: 359.99; found: 360.04
Example 27: 2- (2-oxo-2-phenylethoxy) -4 -phenylthiophene-3 - carbonitrile
Figure imgf000038_0003
MS (ESI) m/z: calc. for C19HX3NO2S, [M+H]+: 320.07; found: 320.27 Example 28: 2- ( (4-cyanobenzyl) oxy) -4 -phenylthiophene-3- carbonitrile
Figure imgf000039_0001
MS (ESI) m/z: calc. for Ci9Hi2N2OS, [M+H]+: 317.07; found: 317.09
Example 29: 2- ( (4-formylbenzyl) oxy) -4-phenylthiophene-3- carbonitrile
Figure imgf000039_0002
MS (ESI) m/z: calc. for Ci9Hi3N02S, [M+H]+: 320.07; found: 320.12
Example 30: -phenyl-2 - (thiophen-2 -ylmethoxy) thiophene-3- carbonitrile
Figure imgf000039_0003
MS ( ESI ) m/z: calc. for CiSHiiNOS2 i [M+H] + : 298.03; found: 298.08 Example 31: 2- ( (5-chlorothiophen-2-yl)methoxy) -4-phenyl- thiophene-3 -carbonitril
Figure imgf000040_0001
MS (ESI) m/z: calc. for Ci6Hi0ClNOS2 , [M+H]+: 331.99; found: 332.02
Example 32: 4 -phenyl-2 - (thiophen-3 -ylmethoxy) thiophene- 3- carbonitrile
Figure imgf000040_0002
MS (ESI) m/z: calc. for Ci6HuNOS2 , [M+H]+: 298.03; found: 298.09
Example 33: 4 -phenyl-2 - (pyridin-4-ylmethoxy) thiophene-3- carbonitrile
Figure imgf000040_0003
MS (ESI) m/z: calc. for C17H12N2OS, [M+H]+: 293.07; found: 293.14 Example 34: 4-phenyl-2- (pyridin-3 -ylmethoxy) thiophene-3- carbonitrile
Figure imgf000041_0001
MS (ESI) m/z: calc. for C17H12N2OS , [M+H]+: 293.07; found 293.12
Example 35: 4-phenyl-2- ( (3- (trifluoromethoxy) benzyl) oxy) - thiophene-3 -carbonitri
Figure imgf000041_0002
MS (ESI) m/z: calc. for C19H12F3NO2S , [M+H]+: 376.05
376.09
Example 36: 2- ( (4 - (1, 2 , 3 -thiadiazol-4-yl) benzyl) oxy) -4
phenylthiophene-3-carbonitrile
Figure imgf000041_0003
MS (ESI) m/z: calc. for C20H13N3OS2 , [M+H]+: 376.05; found: 376.12 Example 37: 2- ( (perfluorophenyl) methoxy) -4 -phenylthiophene-3 - carbonitrile
Figure imgf000042_0001
MS (ESI) m/z: calc. for CisHsFsNOS , [M+H] + : 382.02; found: 382.08
Example 38: 2- ( (4- (tert-butyl) benzyl) oxy) -4 -phenylthiophene-3 - carbonitrile
Figure imgf000042_0002
MS (ESI) m/z: calc. for C22H2iNOS , [M+H]+: 348.13; found: 348.16
Example 39 : 4-phenyl-2- ( (4- (trifluoromethoxy) benzyl) oxy) - thiophene-3 -carbonitr
Figure imgf000042_0003
F3
MS (ESI) m/z: calc. for C19H12F3NO2S , [M+H]+: 376.05; found: 376.07 Example 40: 2 -( (4 - (methylsulfonyl) enzyl) oxy) -4 -phenyl- thiophene- 3 -carbonitri
Figure imgf000043_0001
MS (ESI) m/z: calc. for CigHisNOaSa , [M+H]+: 370.05; found: 370.12
Example 41: 4-phenyl-2- (guinolin-8-ylmethoxy) thiophene-3- carbonitrile
Figure imgf000043_0002
MS (ESI) m/z: calc. for C2iHi4N2OS( [M+H]+: 343.08; found: 343.09
Example 42: 2- ( (2-fluoropyridin-4-yl) methoxy) -4-phenyl- thiophene-3 -carbonitri
Figure imgf000043_0003
MS (ESI) m/z: calc. for C18Hi3FN202S , [M+H]+: 341.07; found: 341.12
Example 43 : 2- ( (3-formylbenzyl) oxy) -4 -phenylthiophene-3 - carbonitrile
Figure imgf000044_0001
MS (ESI) m/z: calc. for Ci9Hi3N02S, [M+H]+: 320.07; found: 320.11
Example 44: 2- ( (6-methoxypyridin-2-yl)methoxy) -4-phenyl- thiophene-3 -carbonitril
Figure imgf000044_0002
MS (ESI) m/z: calc. for Ci8H14N202S, [M+H]+: 323.08; found: 323.09
Example 45: 2- ( (4-bromobenzyl) oxy) -4- (2 , 3 -dihydrobenzo- [b] [1, 4] dioxin-6-yl) thiophene-3 -carbonitrile
Figure imgf000045_0001
¾ NMR (500 MHz , CDC13) : δ 4.26 (s, 4H) , 5.20 (s, 2H) , 6.47 (s, 1H) , 6.90 (d, J" = 10 Hz, 1H) , 7.05-7.08 (m, 2H) , 7.33 (d, J = 5 Hz, 2H) , 7.54 (d, J = 5 Hz, 2H) ppm. MS (ESI) m/z: calc. for C2oHi4BrN03S, [M+H]+: 429.30; found: 429.36
Example 46: tert-butyl 4 - ( ( (3 -cyano-4 - (2 , 3 -dihydrobenzo-
[b] [1,4] dioxin-6-yl) thiophen-2-yl) oxy) methyl) benzylcarbamate
Figure imgf000045_0002
HBoc
MS (ESI) m/z: calc. for C26H26N2O5S , [M+HJ+: 479.16; found: 479.17
Example 47: 2- (benzyloxy) -4- (2 , 3-dihydrobenzo [b] [1, 4] dioxin-6- yl) thiophene-3-carbonitrile
Figure imgf000045_0003
MS (ESI) m/z: calc. for C2oHi5N03S, [M+H]+: 350.08; 350.12
Example 48: 2- ( (2-bromobenzyl) oxy) -4- (2, 3 -dihydrobenzo- [b] [1, 4] dioxiri-6-yl) thiophene-3 -carbonitrile
Figure imgf000046_0001
MS (ESI) m/z: calc. for C2oHiBrN03S , [M+H]+: 427.99; found: 428.01
Example 49: 2 - ( (3 -chlorobenzyl) oxy) -4 - (2 , 3 -dihydrobenzo- [b] [1, 4] dioxin-6-yl) thiophene-3 -carbonitrile
Figure imgf000046_0002
MS (ESI) m/z: calc. for C20H14CINO3S , [M+H]+: 384.04
384.12
Example 50: 4 - (2 , 3-dihydrobenzo [b] [1 , 4] dioxin-6-yl) -2- iodobenzyl) oxy) thiophene-3-carbonitrile
Figure imgf000047_0001
MS (ESI) m/z: calc. for C20H14 INO3S , [M+H]+: 475.97; found: 475.99
Example 51: 4- (2 , 3-dihydrobenzo [b] [1, 4] dioxin-6-yl) -2- (4- nitrophenethoxy) thiophene-3-carbonitrile
Figure imgf000047_0002
MS (ESI) m/z: calc. for C2iHi6N20sS, [M+H]+: 409.08; found: 409.11
Example 52: 4- (2 , 3-dihydrobenzo [b] [1, 4] dioxin-6-yl) -2- ( (4- nitrobenzyl) oxy) thiophene-3-carbonitrile
Figure imgf000047_0003
MS (ESI) m/z: calc. for C20Hi4N2O5S , [M+H]+: 395.06; found: 395.13 Example 53: 4- (2 , 3 -dihydrobenzo [b] [1, 4] dioxin-6-yl) -2- ( (3- (trifluoromethyl) benz l) oxy) thiophene-3-carbonitrile
Figure imgf000048_0001
MS (ESI) m/z: calc. for C21H14F3NO3S , [M+H]+: 418.06
418.16
Example 54: 4- (2 , 3-dihydrobenzo [b] [1, 4] dioxin-6-yl) -2- (naphthalen-2-ylmethoxy) thiophene-3-carbonitrile
Figure imgf000048_0002
MS (ESI) m/z: calc. for C24Hi7N03S, [M+H]+: 400.09; found: 400.11
Example 55: 4- (2, 3 -dihydrobenzo [b] [1, 4] dioxin-6-yl) -2- ( (3- fluorobenzyl) oxy) thiophene-3-carbonitrile
Figure imgf000049_0001
MS (ESI) m/z: calc. for C20H14FNO3S , [M+H]+: 368.07; found: 368.13
Example 56: 4 - (2 , 3 -dihydrobenzo [b] [1 , 4] dioxin-6-yl) -2- (3- phenoxypropoxy) thiophene-3 -carbonitrile
Figure imgf000049_0002
MS (ESI) m/z: calc. for C22H19NO4S , [M+H]+: 394.10; found: 394.17
Example 57: 2- ( (3 , 4-dichlorobenzyl) oxy) -4- (2 , 3 -dihydrobenzo- [b] [1,4] dioxin-6-yl) thiophene-3 -carbonitrile
Figure imgf000049_0003
MS (ESI) m/z: calc. for C2oHi3Cl23 ro3S, [M+H]+: 418.00; found: 418.02
Example 58: 4 - (2 , 3 -dihydrobenzo [b] [1 , 4] dioxin-6-yl) -2 - ( (4 - (hydroxymethyl) benzyl) oxy) thiophene-3 -carbonitrxle
Figure imgf000050_0001
MS (ESI) m/z: calc. for C21H17NO4S, [M+H]+: 380.09; found: 380.11
Example 59: 2- ( (2 , 4 -dichlorobenzyl) oxy) -4- (2 , 3 -dihydrobenzo- [b] [1,4] dioxin-6-yl) thiophene-3 -carbonitrxle
Figure imgf000050_0002
MS (ESI) m/z: calc. for CaoHiaClaNOaS , [M+H]+: 418.00; found: 418.02
Example 60: 4- (2, 3-dihydrobenzo [b] [1, 4] dioxin-6-yl) -2- (2-oxo- 2 -pheny1ethoxy) thiophene-3 -carbonitri1e
Figure imgf000051_0001
MS (ESI) m/z: calc. for C2iHi5N04S, [M+H]+: 378.07;
378.11
Example 61: 2- ( (4-cyanobenzyl) oxy) -4- (2, 3-dihydrobenzo- [b] [1,4] dioxin-6-yl) thiophene-3-carbonitrile
Figure imgf000051_0002
MS (ESI) m/z: calc. for C21H14N2O3S , [M+H]+: 375.07; found: 375.08
Example 62: 4- (2 , 3 -dihydrobenzo [b] [1, 4] dioxin-6-yl) -2- ( (4- formylbenzyl) oxy) thiophene-3-carbonitrile
Figure imgf000051_0003
MS (ESI) m/z: calc. for C2iHi5N04S, [M+H]+: 378.07; found: 378.10 Example 63: 4- (2 , 3 -dihydrobenzo [b] [1, 4] dioxin-6-yl) -2- (thiophen- 2 -ylmethoxy) thiophene- 3 -carbonitrile
Figure imgf000052_0001
MS (ESI) m/z: calc. for CieHisNC^, [M+H]+: 356.03;
356.12
Example 64: 2 - ( (5-chlorothiophen-2-yl) methoxy) - - (2 , 3- dihydrobenzo [b] [1, 4] dioxin-6-yl) thiophene -3 -carbonitrile
Figure imgf000052_0002
MS (ESI) m/z: calc. for C18H12CINO3S2 , [M+H]+: 389.99; found: 390.05
Example 65: 4- (2 , 3 -dihydrobenzo [b] [1, 4] dioxin-6-yl) -2- ( thiophen- 3 -ylmethoxy) thiophene- 3 -carbonitrile
Figure imgf000053_0001
MS (ESI) m/z: calc. for C18H13 O3S2 , [M+H]+: 356.03; found: 356.08
Example 66: 4- (2 , 3-dihydrobenzo [b] [1, 4] dioxin-6-yl) -2- ( yridin-4 -ylmethoxy) thiophene-3-carbonitrile
Figure imgf000053_0002
MS (ESI) m/z: calc. for C19H14N2O3S , [M+H]+: 351.07; found 351.11
Example 67: 4- (2, 3-dihydrobenzo [b] [1, 4] dioxin- 6 -yl) -2- (pyridin-3 -ylmethoxy) thiophene-3 -carbonitrile
Figure imgf000053_0003
MS (ESI) m/z: calc. for C19H14N2O3S , [M+H]+: 351.07; found: 351.10 Example 68: 4- (2 , 3 -dihydrobenzo [b] [1, ] dioxin-6-yl) -2- ( (3- (trifluoromethoxy) benzyl) oxy) thiophene-3-carbonitrile
Figure imgf000054_0001
MS (ESI) m/z: calc. for C21H14F3NO4S , [M+H]+: 434.06; found: 434.12
Example 69: 2 - ( (4 - (1, 2 , 3-thiadiazol-4-yl) benzyl) oxy) -4 - (2 , 3 - dihydrobenzo [b] [1,4] dioxin-6-yl) thiophene-3-carbonitrile
Figure imgf000054_0002
MS (ESI) m/z: calc. for C22Hi5N303S2 , [M+H]+: 434.06; found: 434.08
Example 70: 4- (2 , 3 -dihydrobenzo [b] [1, 4] dioxin-6-yl) -2- ( (perfluorophenyl) methoxy) thiophene-3-carbonitrile
Figure imgf000055_0001
MS (ESI) m/z: calc. for C2oHioF5N03S , [M+H]+: 440.03; found: 440.03
Example 71: 2- ( (4- (tert-butyl) benzyl) oxy) -4- (2 , 3-dihydrobenzo- [b] [1,4] dioxin-6-yl) thiophene-3 -carbonitrile
Figure imgf000055_0002
MS (ESI) m/z: calc. for C24H23N03S, [M+H]+: 406.14;
406.13
Example 72: 4 - (2 , 3 -dihydrobenzo [b] [1 , 4] dioxin-6-yl) -2- (
(trifluoromethoxy) benzyl) oxy) thiophene-3 -carbonitrile
Figure imgf000055_0003
MS (ESI), m/z: calc. for C21H14F3NO4S , [M+H]+: 434.06; found: 434.10
Example 73: 4- (2, 3-dihydrobenzo [b] [1, 4] dioxin-6-yl) -2- ( (4- (methylsulfonyl) benzyl) oxy) thiophene-3 -carbonitrile
Figure imgf000056_0001
MS (ESI) m/z: calc. for C2iHi7NOsS2, [M+H]+: 428.05; found: 428.11
Example 74: 4- (2 , 3-dihydrobenzo [b] [1, 4] dioxin-6-yl) -2- (quinolin-8-ylmethoxy) thiophene-3 -carbonitrile
Figure imgf000056_0002
MS (ESI) m/z: calc. for C23Hi6N203S, [M+H]+: 401.09; found: 401.11
Example 75: 4- (2 , 3-dihydrobenzo [b] [1, 4] dioxin-6-yl) -2- ( (2- fluoropyridin-4-yl) methoxy) thiophene-3 -carbonitrile
Figure imgf000057_0001
MS (ESI) m/z: calc. for C20H15FN2O4S , [M+H]+: 399.07; found: 400.02
Example 76: 4- (2 , 3-dihydrobenzo [b] [1 , 4] dioxin-6-yl) -2 -( (3- formylbenzyl) oxy) thiophene-3-carbonitrile
Figure imgf000057_0002
MS (ESI) m/z: calc. for C2iHisN04S, [M+H]+: 378.07; found: 378.09
Example 77: 4- (2 , 3-dihydrobenzo [b] [1, 4] dioxin-6-yl) -2- ( (6- methoxypyridin-2-yl)methoxy) thiophene-3-carbonitrile
Figure imgf000057_0003
MS (ESI) m/z: calc. for C20H16 2O4S , [M+H]+: 381.08; found: 381.14
Example 78: 2- ( (4-bromobenzyl) oxy) -4- (3 , 4-dichlorophenyl) - thiophene- 3 -carbonxtrile
Figure imgf000058_0001
¾ NMR (500 MHz, CDCI3 ) : δ 5.24 (s, 1H) , 6.62 (s, 1H) , 7.35 (d, J = 10 Hz, 2H) , 7.45 (dd, J = 5 , 0.5 Hz, 1H) , 7.50 (dd, J = 5, 0.5 Hz, 1H) , 7.55 (d, J = 10 Hz, 2H) , 7.44 (d, J = 5 Hz, 1H) ppm. MS (ESI) m/z: calc. for Ci8Hi0BrCl2NOS , [M+H]+: 440.15; found: 440.35
Example 79: tert-butyl 4- (( (3-cyano-4- (3 , 4-dichlorophenyl) - thiophen-2-yl) oxy) methyl) benzylcarbamate
Figure imgf000058_0002
HBoc
MS (ESI) m/z: calc. for C24H22CI2N2O3S , [M+H]+: 489.07; found: 489.12 Example 80: 2- (benzyloxy) -4- (3 , 4-dichlorophenyl) thiophene-3- carbonitrile
Figure imgf000059_0001
MS (ESI) m/z: calc. for Ci8HuCl2NOS , [M+H]+: 359.99; found: 360.04
Example 81: 2- ( (2 -bromobenzyl) oxy) -4- (3 , 4-dichlorophenyl) - thiophene-3 -carbonitrile
Figure imgf000059_0002
MS (ESI) m/z: calc. for Ci8HioBrCl2NOS , [M+H]+: 437.90; found: 437.99
Example 82: 2- ( (3-chlorobenzyl) oxy) -4- (3 , 4-dichlorophenyl) - thiophene-3 -carbonitrile
Figure imgf000059_0003
MS (ESI) m/z: calc. for CisHioClsNOS, [M+H]+: 393.95; found: 393.99 Example 83: 4- (3 , 4-dichlorophenyl) -2- ( (4-iodobenzyl) oxy) - thiophene-3 -carbonitrile
Figure imgf000060_0001
MS (ESI) m/z: calc. for Ci8Hi0Cl2INOS , [M+H]+: 485.89; found: 485.99
Example 84 : 4- (3 , 4-dichlorophenyl) -2- (4 -nitrophenethoxy) - thiophene- 3 -carbonitrile
Figure imgf000060_0002
MS (ESI) m/z: calc. for C19H12CI2NO3S , [M+H]+: 418.99; found: 419.06
Example 85: 4- (3 , 4-dichlorophenyl) -2- ( (4-nitrobenzyl) oxy) - thiophene-3 -carbonitrxle
Figure imgf000060_0003
MS (ESI) m/z: calc. for Ci8HioCl2N203S , [M+H]+: 404.98; found: 405.02
Example 86: 4- (3 , -dichlorophenyl) -2- ( (3- (trifluoromethyl) - benzyl) oxy) thiophene-3-carbonitrile
Figure imgf000061_0001
MS (ESI) m/z: calc. for C19H10CI2F3NOS , [M+H]+: 427.98; found: 428.03
Example 87 : 4- (3 , 4 -dichlorophenyl) -2- (naphthalen-2-ylmethoxy) - thiophene- 3 -carbonitrile
Figure imgf000061_0002
MS (ESI) m/z: calc. for C22H13CI2NOS , [M+HJ+: 410.01; found: 410.12
Example 88: 4- (3 , 4 -dichlorophenyl) -2- ( (3-fluorobenzyl) oxy) - thiophene- 3 -carbonitrile
Figure imgf000062_0001
MS (ESI) m/z: calc. for Ci8Hi0Cl2FNOS , [M+H]+: 377.98; found: 378.06
Example 89: 4 - (3 , 4-dichlorophenyl) -2- (3 -phenoxypropoxy) - thiophene-3 -carbonxtrile
Figure imgf000062_0002
MS (ESI) m/z: calc. for C20H15CI2NO2S , [M+H]+: 404.02;
404.09
Example 90: 2- ( (3 , 4 -dichlorobenzyl) oxy) -4- (3 , 4-dichloro- phenyl) thiophene-3 -carbonitrile
Figure imgf000062_0003
MS (ESI) m/z: calc. for Ci8H9Cl4NOS, [M+H]+: 427.92; 427.99
Example 91: 4- (3 , -dichlorophenyl) -2- ( (4- (hydroxymethyl) benzyl) oxy) thiophene- 3 -carbonitrile
Figure imgf000063_0001
MS (ESI) m/z: calc. for C19H13CI2NO2S , [M+H]+: 389.00;
389.11
Example 92: 2- ( (2, 4-dichlorobenzyl) oxy) -4- (3, 4-dichlorophenyl) thiophene-3 -carbonitrile
Figure imgf000063_0002
MS (ESI) m/z: calc. for Ci8H9Cl4NOS, [M+H]+: 427.92; found: 427.99
Example 93: 4- (3 , 4-dichlorophenyl) -2- (2-oxo-2-phenylethoxy) - thiophene-3 -carbonitrile
Figure imgf000064_0001
MS (ESI) m/z: calc. for C19H11CI2NO2S , [M+H]+: 387.99; found: 388.05
Example 9 : 2- ( (4-cyanobenzyl) oxy) -4- (3 , 4-dichlorophenyl) - thiophene-3 -carbonitrile
Figure imgf000064_0002
MS (ESI) m/z: calc. for C19H10CI2N2OS , [M+H]+: 384.99; found: 385.03
Example 95: 4- (3 , 4-dichlorophenyl) -2- ( (4-formylbenzyl) oxy) - thiophene-3 -carbonitrile
Figure imgf000064_0003
(ESI) m/z: calc. for C19H11CI2NO2S , [M+H]+: 387.99; found .03 Example 96: 4- (3 , 4 -dichlorophenyl) -2- (thiophen-2-ylmethoxy) - thiophene-3 -carbonitrile
Figure imgf000065_0001
MS (ESI) m/z: calc. for Ci6H9Cl2NOS2 , [M+H]+: 365.95; found: 365.99
Example 97: 2- ( (5-chlorothiophen-2-yl) methoxy) -4- (3 , 4- dichlorophenyl) thiophene-3 -carbonitrile
Figure imgf000065_0002
MS (ESI) m/z: calc. for Ci6H8Cl3 OS2 , [M+H]+: 399.91; found: 399.99
Example 98: - (3 , 4 -dichlorophenyl) -2- (thiophen-3 -ylmethoxy) - thiophene-3 -carbonitrile
Figure imgf000065_0003
MS (ESI) m/z: calc. for CX6H9CI2NOS2 , [M+H]+: 365.95; found: 365.99
Example 99: 4- (3 , -dichlorophenyl) -2- (pyridin-4 -ylmethoxy) - thiophene-3 -carbonitrile
Figure imgf000066_0001
MS (ESI) m/z: calc. for Ci7Hi0Cl2N2OS , [M+H]+: 359.99; found: 360.02
Example 100: 4- (3 , 4-dichlorophenyl) -2- (pyridin-3 -ylmethoxy) - thiophene-3 -carbonitrile
Figure imgf000066_0002
MS (ESI) m/z: ca2c. for Ci7Hi0Cl2N2OS, [M+H]+: 360.99; found: 361.02
Example 101: 4- (3 , 4-dichlorophenyl) -2- ( (3- (trifluoromethoxy) - benzyl) oxy) thiophene-3 -carbonitrile
Figure imgf000067_0001
MS (ESI) m/z: calc. for Ci9HioCl2F3N02S , [M+H]+: 443.98; found: 444.09
Example 102: 2- ( ( - (1 , 2 , 3-thiadiazol-4-yl) benzyl) oxy) -4 - (3 , 4 - dichlorophenyl) thiophene-3 -carbonitrile
Figure imgf000067_0002
MS (ESI) m/z: calc. for C2oHiiCl2N3OS2 , [M+H]+: 443.97;
443.99
Example 103: 4- (3 , 4 -dichlorophenyl) -2- ( (perfluorophenyl) methoxy) thiophene-3 -carbonitrile
Figure imgf000067_0003
MS (ESI) m/z: calc. for Ci8H6Cl2F5NOS , [M+H]+: 449.95; found: 450.08
Example 104: 2- ( (4- (tert-butyl) benzyl) oxy) -4- (3 , 4-dichloro- phenyl) thiophene-3-carbonitrile
Figure imgf000068_0001
MS (ESI) m/z: calc. for C22H19CI2NOS , [M+H]+: 415.06; found: 415.12
Example 105: 4- (3 , 4 -dichlorophenyl) -2- ( (4- (trifluoromethoxy) - benzyl) oxy) thiophene-3-carbonitrile
Figure imgf000068_0002
F3
MS (ESI) m/z: calc. for Ci9Hi0Cl2F3NO2S , [M+H]+: 443.98; found: 444.05
Example 106: 4- (3 , 4 -dichlorophenyl) -2- ( (4- (methylsulfonyl) - benzyl) oxy) thiophene-3-carbonitrile
Figure imgf000069_0001
MS (ESI) m/z: calc. for C19H13CI2NO3S2 , [M+H]+: 437.97; found: 438.05
Example 107: 4- (3 , 4-dichlorophenyl) -2- (quinolin-8-ylmethoxy) - thiophene-3 -carbonitrile
Figure imgf000069_0002
MS (ESI) m/z: calc. for C2iHi2Cl2N2OS, [M+H]+: 411.00; found: 411.05
Example 108: 4- (3 , 4-dichlorophenyl) -2- ( (2 -fluoropyridin-4 - yl) methoxy) thiophene-3 -carbonitrile
Figure imgf000069_0003
MS (ESI) m/z: calc. for C17H13CI2NOS , [M+H]+: 371.26; found: 371.32
Example 109: 4- ( , 4 -dichlorophenyl) -2- ( (3-formylbenzyl) oxy) - thiophene-3 -carbonitrile
Figure imgf000070_0001
MS (ESI) m/z: calc. for C19H11CI2NO2S , [M+H]+: 387.99; found: 388.05
Example 110: 4- (3 , 4 -dichlorophenyl) -2- ( (6-methoxypyridin-2- yl) -ethoxy) thiophene-3-carbonitrile
Figure imgf000070_0002
MS (ESI) m/z: calc. for Ci8Hi2Cl2N202S , [M+H]+: 391.00; found: 391.05
Example 111: Example 4: 4- (benzo [d] [1, 3] dioxol-5-yl) -2- ( (4- bromobenzyl) oxy) thiophene- 3 -carbonitrile
Figure imgf000071_0001
Example 112: tert-butyl 4- ( ( (4- (benzo [d] [1, 3] dioxol-5-yl) -3- cyanothiophen-2-yl) oxy) methyl) benzylcarbamate
Figure imgf000071_0002
MS (ESI) m/z: calc. for Ci8Hi2BrNOS, [M+H] + : 371.26; found: 371.32
Example 113: 4- (benzo [d] [1, 3] dioxol-5-yl) -2- (benzyloxy) - thiophene-3 -carbonitrile
Figure imgf000071_0003
(ESI) m/z: calc. for Ci8Hi2BrNOS, [M+H]+: 371.26; found
Example 114: 4- (benzo [d] [1, 3] dioxol-5-yl) -2- ( (2-bromobenzyl) - oxy) thiophene-3 -carbonitrile
Figure imgf000072_0001
MS (ESI) m/z: calc. for Ci8Hi2BrNOS, [M+H]+: 371.26; found: 371.32
Example 115: 4- (benzo [d] [1, 3] dioxol-5-yl) -2- ( (3 -chlorobenzyl) - oxy) thiophene-3-carbonitrile
Figure imgf000072_0002
MS (ESI) m/z: calc. for Ci8Hi2BrNOS, [M+H]+: 371.26; found: 371.32
Example 116: 4- (benzo [d] [1, 3] dioxol-5-yl) -2- ( (4 -iodobenzyl) - oxy) thiophene-3-carbonitrile
Figure imgf000072_0003
MS (ESI) m/z: calc. for Ci8Hi2BrNOS, [M+H]+: 371.26; found: 371.32
Example 117: 4- (benzo [d] [1, 3] dioxol-5-yl) -2- (4-nitro- phenethoxy) thiophene-3 -carbonitrile
Figure imgf000073_0001
MS (ESI) m/z: calc. for C18Hi2BrNOS, [M+H]+: 371.26; found: 371.32
Example 118: 4- (benzo [d] [1, 3] dioxol-5-yl) -2- ( (4-nitrobenzyl) - oxy) thiophene-3 -carbonitrile
Figure imgf000073_0002
MS (ESI) m/z: calc. for Ci8Hi2BrNOS, [M+H]+: 371.26; found: 371.32
Example 119: 4- (benzo [d] [1, 3] dioxol-5-yl) -2- ( (3- (trifluoro- methyl) benzyl) oxy) thiophene-3 -carbonitrile
Figure imgf000074_0001
MS (ESI) m/z: calc. for Ci8Hi2BrNOS, [M+H]+: 371.26; found: 371.32
Example 120: 4- (benzo [d] [1, 3] dioxol-5-yl) -2- (naphthalen-2 -yl- methoxy) thiophene-3 -carbonxtrile
Figure imgf000074_0002
MS (ESI) m/z: calc. for Ci8Hi2BrNOS, [M+H]+: 371.26; found: 371.32
Example 121: 4- (benzo [d] [1, 3] dioxol-5-yl) -2- ( (3-fluorobenzyl) - oxy) thiophene-3 -carbonitrile
Figure imgf000074_0003
MS (ESI) m/z: calc. for Ci8Hi2BrNOS, [M+H]+: 371.26 371.32
Example 122: 4- (benzo [d] [1, 3] dioxol-5-yl) -2- (3-phenoxy- propoxy) thiophene-3 -carbonitri1e
Figure imgf000075_0001
MS (ESI) m/z: calc. for CisHizBrNOS , [M+H]+: 371.26; found: 371.32
Example 123: 4- (benzo [d] [1, 3] dioxol-5-yl) -2- ( (3 , 4-dichloro- benzyl) oxy) thiophene-3-carbonitrile
Figure imgf000075_0002
MS (ESI) m/z: calc. for Ci8Hi2BrNOS, [M+H]+: 371.26; found: 371.32
Example 124: 4 - (benzo [d] [1 , 3] dioxol-5-yl) -2 - ( (4 - (hydroxy- methyl) benzyl) oxy) thiophene-3-carbonitrile
Figure imgf000076_0001
MS (ESI) ra/z: calc. for Ci8Hi2BrNOS, [M+H]+: 371.26; found: 371.32
Example 125: 4- (benzo [d] [1,3] dioxol-5-yl) -2- ( (2,4-dichloro- benzyl) oxy) thiophene-3 -carbonitrile
Figure imgf000076_0002
MS (ESI) m/z: calc. for
Figure imgf000076_0003
[M+H]+: 371.26; found: 371.32
Example 126: 4- (benzo [d] [1, 3] dioxol-5-yl) -2- (2-oxo-2-phenyl- ethoxy) thiophene-3 -carbonitrile
Figure imgf000076_0004
MS (ESI) m/z: calc. for Ci8H12BrNOS, [M+H]+: 371.26; found: 371.32
Example 127: 4- (benzo [d] [1, 3] dioxol-5-yl) -2- ( (4 -cyanobenzy1) - oxy) thiophene-3-carbonitrile
Figure imgf000077_0001
MS (ESI) m/z: calc. for Ci8Hi2BrNOS, [M+H]+: 371.26; found: 371.32
Example 128: 4- (benzo [d] [1, 3] dioxol-5-yl) -2- ( (4-formylbenzyl) - oxy) thiophene-3-carbonitrile
Figure imgf000077_0002
MS (ESI) m/z: calc. for Ci8Hi2BrNOS, [M+H]+: 371.26; found: 371.32
Example 129: 4- (benzo [d] [1, 3] dioxol-5-yl) -2- (thiophen-2-yl- methoxy) thiophene-3 -carbonitrile
Figure imgf000078_0001
MS (ESI) m/z: calc. for Ci8Hi2BrNOS, [M+H]+: 371.26; found: 371.32
Example 130: 4- (benzo [d] [1, 3] dioxol-5-yl) -2- ( (5-chloro- t iophen-2 -yl) methoxy) thiophene-3-carbonitrile
Figure imgf000078_0002
MS (ESI) m/z: calc. for Ci8Hi2BrNOS, [M+H]+: 371.26; found: 371.32
Example 131: 4- (benzo [d] [1, 3] dioxol-5-yl) -2- (thiophen-3-yl- methoxy) thiophene-3-carbonitrile
Figure imgf000078_0003
MS (ESI) m/z: calc. for CisHiaBrNOS, [M+H]+: 371.26; found: 371.32 Example 132: 4- (benzo [d] [1, 3] dioxol-5-yl) -2- (pyridin-4 -yl- methoxy) thiophene-3 -carbonitrile
Figure imgf000079_0001
MS (ESI) m/z: calc . for Ci8Hi2BrNOS, [M+H]+: 371.2C
371.32
Example 133: 4- (benzo [d] [1, 3] dioxol-5-yl) -2- (pyridin-3
methoxy) thiophene-3 -carbonitrile
Figure imgf000079_0002
MS (ESI) m/z: calc. for Ci8Hi2BrNOS, [M+H]+: 371.26; found: 371.32
Example 134: 4- (benzo [d] [1, 3] dioxol-5-yl) -2- ( (3- (trifl oro- methoxy) benzyl) oxy) thiophene-3 -carbonitrile
Figure imgf000080_0001
MS (ESI) m/z: calc. for Ci8Hi2BrNOS, [M+H]+: 371.26; found: 371.32
Example 135: 2- ( (4- (1, 2 , 3 -thiadiazol-4 -yl) benzyl) oxy) -4- (benzo [d] [1,3] dioxol-5-yl) thiophene-3-carbonitrile
Figure imgf000080_0002
MS (ESI) m/z: calc. for Ci8Hi2BrNOS, [M+H]+: 371.26;
371.32
Example 136: 4- (benzo [d] [1 , 3] dioxol-5-yl) -2- ( (perfluoro- phenyl) methoxy) thiophene-3 -carbonitrile
Figure imgf000080_0003
MS (ESI) ra/z: calc. for Ci8Hi2BrNOS, [M+H]+: 371.26; found: 371.32
Example 137: 4-{benzo[d] [1, 3] dioxol-5-yl) -2- ( (4- (tert-butyl) - benzyl) oxy) thiophene-3-carbonitrile
Figure imgf000081_0001
MS (ESI) m/z: calc. for Ci8Hi2BrNOS, [M+H]+: 371.26; found: 371.32
Example 138: 4- (benzo [d] [1, 3] dioxol-5-yl) -2- ( (4- (trifluoro- methoxy) benzyl) oxy) thiophene-3-carbonitrile
Figure imgf000081_0002
F3
MS (ESI) m/z: calc. for Ci8Hi2BrNOS, [M+H]+: 371.26; found: 371.32
Example 139: 4-(benzo[d] [1, 3] dioxol-5-yl) -2- ( (4- (methylsulfonyl) benzyl) oxy) thiophene-3-carbonitrile
Figure imgf000082_0001
MS (ESI) m/z: calc. for CisHiaBrNOS, [M+H]+: 371.26; found: 371.32
Example 140: 4- (benzo [d] [1, 3] dioxol-5-yl) -2- (guinolin-8-yl- methoxy) thiophene-3-carbonitrile
Figure imgf000082_0002
MS (ESI) m/z: calc. for Ci8Hi2BrNOS, [M+H]+: 371.26; found: 371.32
Example 141: 4- (benzo [d] [1, 3] dioxol-5-yl) -2- ( (2-fluoropyridin- 4 -yl) methoxy) thiophene-3-carbonitrile
Figure imgf000082_0003
MS (ESI) m/z: calc. for Ci8Hi2BrNOS, [M+H]+: 371.26; found: 371.32
Example 142: 4- (benzo [d] [1, 3] dioxol-5-yl) -2- ( (3-formylbenzyl) - oxy) thiophene-3 -carbonitrile
Figure imgf000083_0001
MS (ESI) m/z: calc. for Ci8Hi2BrNOS, [M+H]+: 371.26; found: 371.32
Example 143: 4- (benzo [d] [1, 3] dioxol-5-yl) -2- ( (6-methoxy- pyridin-2 -yl) methoxy) thiophene-3 -carbonitrile
Figure imgf000083_0002
MS (ESI) m/z: calc. for Ci8Hi2BrNOS, [M+H]+: 371.26; found: 371.32
Example 144 : 2- ( (5-formylthiophen-3-yl) methoxy) -4- pheny1thiophene-3-carbonitri1e
Figure imgf000084_0001
MS (ESI) m/z: calc. for Ci7HiiN02S2, [ +H] Λ
326.10
Example 145: 2- ( (5-formyl-2-methoxybenzyl) oxy)
phenylthiophene-3-carbonitrile
(ESI) m/z: calc
Figure imgf000084_0002
350.08; found:
350.09
III. General procedures for the reductive amination (In the following schemes, X represents R1 and R2 and R represents R5)
From the compounds produced from the general procedure of the
O-alkylation specific scaffolds where selected for further functionalization (e.g. examples 13, 46, 79 and 112) towards compounds D.
Figure imgf000085_0001
Compound C (1.0 mmol) was dissolved in a solution of HC1 in butanol 5 N. After stirring at rt for 2 h, the solid that was obtained was filtered off and washed with ether.
The corresponding hydrochloric salt (0.5 mmol) was transferred into a round bottom flask and dissolved into MeOH (5 mL) . Addition of Et3 (1.1 eq) was followed and the reaction mixture was stirred at rt for 30 min. Afterwards, the corresponding carbonyl compound (1.1 eq) was added and after 1 h stirring at rt, NaBH4 (1.1 eq) was added and the reaction mixture was stirred at rt overnight. The reaction was quenched with water, extractions followed with DCM and finally purification by column chromatography.
From the compounds produced from the general procedure of the O-alkylation specific scaffolds where selected for further functionalization (e.g. examples 29, 43, 62, 76, 95, 109, 128 and 142) towards compounds E.
Figure imgf000085_0002
To a stirred solution of compound C (0.5 mmol) in MeOH (5 mL) , the corresponding carbonyl compound (1.1 eq) was added and after 1 h stirring at rt, NaCNBH3 (1.1 eq) was added and the reaction mixture was stirred at rt overnight. The reaction was quenched with water, extractions followed with DCM and finally purification by column chromatography.
Figure imgf000086_0001
To a stirred solution of compound F (e.g. example 144) (0.5 mmol) in MeOH (5 mL) , the corresponding carbonyl compound (1.1 eq) was added and after 1 h stirring at rt, NaCNBH3 (1.1 eq) was added and the reaction mixture was stirred at rt overnight. The reaction was quenched with water, extractions followed with DCM and finally purification by column chromatography .
Figure imgf000086_0002
To a stirred solution of compound H (e.g. example 145) (0.5 mmol) in MeOH (5 mL) , the corresponding carbonyl compound (1.1 eq) was added and after 1 h stirring at rt, NaCNB¾ (1.1 eq) was added and the reaction mixture was stirred at rt overnight. The reaction was quenched with water, extractions followed with DCM and finally purification by column chromatography.
Further examples which have been prepared according to the general procedures described above:
Figure imgf000087_0001
Figure imgf000088_0001
Figure imgf000089_0001
Figure imgf000090_0001
Figure imgf000091_0001
Figure imgf000092_0001
Figure imgf000093_0001
Figure imgf000094_0001
Figure imgf000095_0001
Figure imgf000096_0001
Figure imgf000097_0001
Figure imgf000098_0001
Figure imgf000099_0001
Figure imgf000100_0001
Figure imgf000101_0001
Figure imgf000102_0001
Figure imgf000103_0001
Figure imgf000104_0001
Figure imgf000105_0001
Figure imgf000106_0001
Figure imgf000107_0001
Figure imgf000108_0001
Figure imgf000109_0001
Figure imgf000110_0001
Figure imgf000111_0001
Figure imgf000112_0001
Figure imgf000113_0001
Figure imgf000114_0001
Figure imgf000115_0001
Screening: Expression and purification of recombinant PD-Ll, PD-L2 and PD-1
The gene encoding human PD-Ll (amino acids 18-134) was cloned into the pET-21b, the gene encoding human PD-L2 (20-220) was cloned into pET28a and that of human PD-1 (33-150, Cys93 exchanged to serine) into pET-24d. Proteins were expressed in the E. coli BL21 (DE3) . Cells were cultured in LB at 37°C. The protein production was induced with 1 mM IPTG at OD600 of 1.0 and the cells were cultured for additional 5h. For hPD-1, after induction the temperature was lowered to 30°C. Proteins were expressed as inclusion bodies which were collected by centrifugation, washed twice with 50 mM Tris-HCl pH 8.0 containing 200 mM NaCl, 0.5% Triton X-100, 10 mM EDTA and 10 mM 2 -mercaptoethanol and once more with the same buffer with no detergent. The inclusion bodies were stirred overnight in 50 mM Tris pH 8.0 containing 6M GuHCl, 200 mM NaCl and 10 mM 2-mercaptoethanol . Solubilized fraction was clarified by high speed centrifugation. hPD-Ll and hPD-L2 were refolded by drop- wise dilution into 0.1 M Tris pH 8.0 containing 1 M L-Arg hydrochloride, 0.25 mM oxidized glutathione and 0.25 mM reduced glutathione for hPD-Ll and 0.1 M Tris pH 8.5 containing 1 M NDSB201, 0.2 M NaCl, 5 mM cysteamine and 0.5 mM cystamine for hPD-L2. hPD-1 was refolded in similar manner in 0.1 M Tris pH 8.0 containing 0.4 M L-Arg hydrochloride, 2 mM EDTA, 5 mM cystamine and 0.5 mM cysteamine. After refolding, the proteins were dialyzed 3 times against 10 mM Tris pH 8.0 containing 20 mM NaCl, and purified by size exclusion chromatography on Superdex 75 (GE Healthcare) in 10 mM Tris pH 8.0 containing 20 mM NaCl. The purity and protein folding were evaluated by SDS-PAGE and NMR, respectively. Analytical size-exclusion chromatography
The oligomeric state of tested proteins was analyzed by size exclusion chromatography. Superdex 75 10/30 HR (GE Healthcare) was equilibrated with PBS pH 7.4 and calibrated using globular proteins of known molecular weight . Approximate molecular weight of apo-hPD-Ll and hPD-Ll-small molecule complex (3:1 compound: protein molar ratio) were estimated using the calibration curve.
Differential scanning fluorimetry (DSF)
DSF analysis was performed according to Niesen and colleagues (24). In brief hPD-Ll and hPD-L2 (both 12.5 μΜ) were incubated alone, with compound BMS-202 or compound 8 (both at 37.5 μΜ) in the presence of SYPRO Orange Dye (Life Technologies, final concentration 20x) . Constant temperature gradient of 0.2°C/min was applied and changes in fluorescence were monitored using real time thermocycler (BioRad) . Melting temperature (Tm) was estimated from first derivative of fluorescence intensity as a function of temperature .
NMR methods
Uniform 15N labeling was obtained by expressing the protein in the M9 minimal medium containing 15NH4C1 as the sole nitrogen source. Unlabeled proteins were prepared as for crystallization. For NMR measurements the buffer was exchanged by gel filtration to PBS pH 7.4 (hPD-Ll) or 25 mM sodium phosphate containing 100 mM NaCl pH 6.4 (hPD-1) . 10% (v/v) of D2O was added to the samples to provide lock signal. All spectra were recorded at 300K using a Bruker Avance 600 MHz spectrometer. Binding of the compounds was analyzed by titrating the 15N- labeled PD-Ll (0.3 mM) and recording the 1H-15N HMQC spectra prior and after addition of the compound (Supplementary Figs. SI, S2 and S3) .
The ability of tested compounds to dissociate hPD-Ll / hPD-1 was evaluated using AIDA (27). 15N-labeled hPD-1 (0.2 mM) was slightly overtitrated with unlabeled hPD-Ll. Compound was aliquoted into the resulting mixture. During the experiment the 1H-15N signals were monitored by HMQC experiment.
Changes in the oligomeric state of hPD-Ll in the presence of tested compounds were monitored by titration of unlabeled hPD- Ll (0.3 mM) while recording 1H spectra prior and after addition of the compound. The approximate molecular weights of protein populations present in the sample were determined by analyzing the linewidth (relaxation time) of well separated NMR signals.
All compounds showed activity (IC50) in the range of from 0.001 to 1000 μΜ.
Screening for PD-Ll binders using Microscale Thermophoresis
MST (Microscale Thermophoresis) using the Monolith NT.115 (NanoTemper technologies, Miinchen) is a technique that allows measuring of the interaction between biomolecules and the designed inhibitors. It can be used in several ways in the hit discovery and lead identification process. It can be used to determine if the designed compounds show binding to PD-Ll and determine their KD for PD-Ll. In addition, it allows measuring of the dissociation of the PDl/PD-Ll complex upon addition of the compounds and it permits rapid screening of compounds using two point measurements.
Protein preparation, labeling and compound preparation:
Refolded PD-L1 and PD-1 proteins were used for all experiments. The PD-1 construct is a 13.2 kDa protein. While PD-L1 is 14.5 kDa containing a His6-tag. Labeling of PD-L1 can be performed in several ways, for example, by attaching a fluorescent label to free amine groups, cysteine's or by binding to the His6-tag. PD-L1 was labeled using the Monolith NTtm His-tag labeling kit RED-tris-NTA (Nano temper technologies, Munchen) . The RED-tris-NTA dye (100 nm) was mixing with PD-L1 (200 nM) in a 1:1 ratio and incubated for 30 min at room temperature followed by centrifugation at 13k rpm for 15 min. Binding of the dye to the PD-L1 His6-tag was confirmed by measuring the KD (~8 nm) .
Compound stocks were prepared at 100 mM in 100% DMSO and were subsequently diluted in PBS-t buffer to a concentration of 2- 0.25 mM, depending on their solubility, while keeping the DMSO concentration at 2%.
PD-L1 vs compounds:
For determining affinity of the test compounds to PD-L1 a 2- fold dilution series was made using the compound stocks and dilution with PBS-t buffer with 2% DMSO. The labeled PD-L1 was added to a final concentration of 50 nm and the samples were loaded into Monolith NT115 standard treated capillaries (NanoTemper technologies, Munchen) and measured using the Monolith NT.115. The obtained binding curves were analyzed using the MO. Affinity analysis software (NanoTemper technologies, Munchen) . Dissociation of the PD-1/ PD-Ll complex:
The PD-1/PD-L1 complex is prepared by mixing PD-Ll and PD-1 in a 1:10 ratio the complex was labeled a described above for PD- Ll. The dissociation of the PD-1/ PD-Ll complex is measured by adding the labeled complex to a final PD-Ll concentration of 50 nm to a 2 -fold dilution series of the compound and measuring using the Monolith NT.115.
Screening of compounds using two point measurements:
Rapid screening of PD-Ll binding without a full KD determination is also possible using MST. This requires preparation of two samples, one at low compound concentration and one at high compound concentration. If the MST measurements show a significant change in fluorescence signal between the two samples after the temperature jump it is a good indication if the compound is binding or not . Subsequently, a full KD determination as described above can be performed on the hits .

Claims

Claims
1. compound of formula
Figure imgf000121_0001
(I)
wherein
R1 is a hydrogen atom or a halogen atom; and
R2 is a hydrogen atom or a halogen atom; or
R1 and R2 together are a group of formula -O-CH2-CH2-O- , - 0-CH2-CH2-N(Me) -, - (Me) -CH2-CH2-0- , -O-CF2-O- or -O-CH2-O-;
X is a bond, an oxygen atom or a group of formula -C(=0)-, or -(CH2)n- wherein n is 1, 2 or 3 or a group of formula -(CH2)m-0- wherein m is 2, 3 or 4; and
Ar is a phenyl group, a naphthyl group or a heteroaryl group containing 1 or 2 rings and from 5 to 10 ring atoms selected from C, O, N and S, wherein Ar may be substituted by 1, 2, 3, 4 or 5 groups R3 and/or wherein Ar may be substituted by a group of formula -CH2-N(R4) -R5; the groups R3 are independently from each other halogen, N02, N3, OH, SH, NH2 or an alkyl, alkenyl, alkynyl, hetero- alkyl, cycloalkyl, heterocycloalkyl, alkylcycloalkyl , heteroalkylcycloalkyl, aryl, heteroaryl, aralkyl or heteroaralkyl group; all of which groups may optionally be substituted;
R4 is a hydrogen atom or an alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, alkylcycloalkyl, heteroalkylcycloalkyl, aryl, heteroaryl, aralkyl or heteroaralkyl group; all of which groups may optionally be substituted; and
R5 is a hydrogen atom or an alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, alkylcycloalkyl, heteroalkylcycloalkyl, aryl, heteroaryl, aralkyl or heteroaralkyl group; all of which groups may optionally be substituted; or
R4 and R5 together with the nitrogen atom to which they are bound are part of an optionally substituted heterocycloalkyl group containing 5, 6 or 7 ring atoms; or a pharmaceutically acceptable salt, ester, solvate or hydrate or a pharmaceutically acceptable formulation thereof .
2. A compound according to claim 1, wherein R1 and R2 together are a group of formula -O-CH2-CH2-O- or -O-CH2-O-; or wherein R1 and R2 are both hydrogen atoms; or wherein R1 and R2 are both chlorine atoms.
3. A compound according to claim 1 or 2, wherein X is a bond.
4. A compound according to any one of the preceeding claims, wherein Ar is a phenyl group or a heteroaryl group containing one ring and 5 or 6 ring atoms selected from C, O, N and S, wherein Ar may be substituted by 1, 2, 3, 4 or 5 groups R3 and/or wherein Ar may be substituted by a group of formula -CH2-N (R4) -R5.
5. A compound according to any one of the preceeding claims, wherein, Ar is substituted by one or two groups R3.
6. A compound according to any one of the preceeding claims, wherein the groups R3 are independently halogen, NO2, N3, OH, SH, NH2 or a Ci-C6 alkyl group; a Ci-C6 heteroalkyl group; a cycloalkyl group containing one ring having from 3 to 7 ring atoms; a heterocycloalkyl group containing one ring having from 3 to 7 ring atoms comprising 1 or 2 heteroatoms selected from S, O and N; an alkylcycloalkyl group containing a C1-C6 alkyl group and a cycloalkyl group containing one ring having from 3 to 7 ring atoms; a heteroalkylcycloalkyl group containing a C1-C6 alkyl group or a C1-C6 heteroalkyl group and a cycloalkyl group
containing one ring having from 3 to 7 ring atoms or a heterocycloalkyl group containing one ring having from 3 to 7 ring atoms comprising 1 or 2 heteroatoms selected from S, O and N with the proviso that a
heteroalkylcycloalkyl group contains at least one
heteroatom; a phenyl group; a heteroaryl group containing
5 or 6 ring atoms comprising 1, 2, 3 or 4 heteroatoms selected from O, S and N; an aralkyl group containing a Ci-Cs alkyl group and a phenyl group; or a heteroaralkyl group containing a Ci-C6 alkyl group or a Ci-C6 heteroalkyl group and a phenyl group or a heteroaryl group containing 5 or 6 ring atoms comprising 1, 2, 3 or 4 heteroatoms selected from O, S and N with the proviso that a
heteroaralkyl group contains at least one heteroatom; all of which groups may optionally be substituted.
A compound according to any one of the preceeding claims, wherein Ar is substituted by a group of formula -C¾- N (R4) -R5.
A compound according to any one of the preceeding claims, wherein R4 is a hydogen atom and R5 is a hydrogen atom or a C1-C6 alkyl group; a Ci-Cs heteroalkyl group; a
cycloalkyl group containing one ring having from 3 to 7 ring atoms; a heterocycloalkyl group containing one ring having from 3 to 7 ring atoms comprising 1 or 2
heteroatoms selected from S, O and N; an alkylcycloalkyl group containing a Ci-Ce alkyl group and a cycloalkyl group containing one ring having from 3 to 7 ring atoms; a heteroalkylcycloalkyl group containing a C1-C6 alkyl group or a Ci-Ce heteroalkyl group and a cycloalkyl group containing one ring having from 3 to 7 ring atoms or a heterocycloalkyl group containing one ring having from 3 to 7 ring atoms comprising 1 or 2 heteroatoms selected from S, O and N with the proviso that a
heteroalkylcycloalkyl group contains at least one
heteroatom; a phenyl group; a heteroaryl group containing 5 or 6 ring atoms comprising 1, 2, 3 or 4 heteroatoms selected from O, S and N; an aralkyl group containing a Ci-C6 alkyl group and a phenyl group; or a heteroaralkyl group containing a C1-C6 alkyl group or a Ci-C3 heteroalkyl group and a phenyl group or a heteroaryl group containing 5 or 6 ring atoms comprising 1, 2, 3 or 4 heteroatoms selected from 0, S and N with the proviso that a heteroaralkyl group contains at least one heteroatom; all of which groups may optionally be substituted.
9. A compound according to any one of the preceeding claims, wherein R4 and R5 together with the nitrogen atom to which they are bound are part of a heterocycloalkyl group containing 5, 6 or 7 ring atoms and optionally one further heteroatom selected from O and N, which heterocycloalkyl group may be substituted.
10. A compound according to any one of claims 1, 2, 8 and 9 of formula (II) :
Figure imgf000125_0001
(ID wherein R1, R2, R4 and R5 are as defined above.
A compound according to any one of claims 1, 2, 8 and formula (III) :
Figure imgf000126_0001
(III) wherein R1, R2, R4 and R5 are as defined above and R6 is H or OMe .
12. A compound according to any one of claims 1, 2, 8 and 9 of formula (IV) :
Figure imgf000126_0002
(IV) wherein R1, R2, R4 and R5 are as defined above.
A pharmaceutical composition comprising a compound according to anyone of the preceding claims or a pharmaceutically acceptable ester, prodrug, hydrate, solvate or salt thereof, optionally in combination with a pharmaceutically acceptable carrier.
14. Use of a compound or a pharmaceutical composition according to anyone of the preceding claims for the preparation of a medicament for the treatment of cancer, viral diseases and infectious diseases and neurodegenerative diseases such as: Schizophrenia, Alzheimer, Multiples Sclerosis, Parkinson, Corea Huntington, Spinocerebellar ataxia type 1 (SCA1) , Amyotrophic lateral sclerosis, Batten disease.
15. A compound or a pharmaceutical composition according to anyone of claims 1 to 13 for use in the treatment of cancer, viral diseases and infectious diseases and neurodegenerative diseases such as Schizophrenia, Alzheimer, Multiples Sclerosis, Parkinson, Corea Huntington, Spinocerebellar ataxia type 1 (SCA1) , Amyotrophic lateral sclerosis, Batten disease.
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