WO2019008154A1 - 3-(azolylmethoxy)biphenyl derivatives as inhibitors of the pd-1/pd-l1 protein/protein interaction - Google Patents

Abstract

The present invention provides novel compounds of formula (I) that are useful as inhibitors of the PD-1/PD-L1 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-(AZOLYLMETHOXY)BI PHENYL 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-l/PD-Ll 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-l/PD-Ll 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) :

(I)

wherein

X is an oxygen atom or a group of formula NR⁵; 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 -0-CH₂-CH₂-0- , -0-CH2- CH₂-N(Me)-, -N(Me) -CH2-CH2-O-, -0-CF₂-0- or -0-CH₂-0-;

R³ is a methyl group, CN or a halogen atom; and

R⁴ is a group of formula NR⁶R⁷ or OR⁸; R⁵ is a hydrogen atom or a methyl group; the groups R⁶, R⁷ and R⁸ are independently from each other a hydrogen atom 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; or R⁶ and R⁷ together with the nitrogen atom to which they are bound are part of an optionally substituted heterocycloalkyl group containing 3 , 4 , 5 , 6 or 7 ring atoms ; and

R⁹ 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 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, X is an oxygen atom.

Further preferably, R¹ and R² together are a group of formula -O-CH2-CH2-O-, -O-CF2- O- or -O-CH2-O-.

Moreover preferably, R¹ and R² are both hydrogen atoms. Further preferably, R¹ and R² are both chlorine atoms. Moreover preferably, R³ is a methyl group.

Further preferably, R⁴ is a group of formula NR⁶R⁷.

Moreover preferably, R⁶ is a hydrogen atom or a methyl group (especially a hydrogen atom) and R⁷ is a hydrogen atom or a Ci- Cg 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 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, 0 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 C±-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, R⁶ is a hydrogen atom or a methyl group (especially a hydrogen atom) and R⁷ is a Ci-s alkyl group (which may optionally be substituted by an OH group) , a phenyl group or a -CH2 -phenyl group.

Further preferably, R^s and R⁷ 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 optionally be substituted (preferably by a C1-C4 alkyl group or a group of formula -C(=0)-CH3 or -CH2OH) .

Moreover preferably, R⁵ is a methyl group.

Further preferably, R⁸ is a hydrogen atom.

Moreover preferably, R⁹ is a methyl group.

Further preferably, R⁹ is a phenyl group.

Especially preferred are compounds of formula (II) :

(ID wherein R¹, R², R⁶ and R⁷ are as defined above.

Further especially preferred are compounds of formula (III) :

(III) wherein R¹, R², R⁶ and R⁷ are as defined above.

Most preferred compounds of formula (I) 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 Cl) .

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 O, S and/or N (especially 0 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 O, 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: R^a-0-Y^a-, R^a-S-Y^a-, R^a- S0-Y^a-, R^a-S0₂-Y^a-, R^a-N (R^b) -Y^a- , R^a-CO-Y^a-, R -0-CO-Y^a-, R^a- C0-0-Y^a-, R^a- CO-N (R^b) -Y^a- , R^a-N (R^b) -CO-Y^a- , Ra-o- CO-N (R^b) -Y^a- , R^a-N (R^b) -CO-0-Y^a- , R^a-N (R^b) -CO-N (R^c ) -Y^a- ,

_Ra_₀-CO-0-Y^a-, R^a-N (R ) -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-Y³- , R^a-0- CS -N (R^b) -Y^a- , R^a-N (R^b) -CS-0-Y^a- , R^a-N (R^b) -CS-N ( R^c) -Y^a- , R^a-0-CS-0-Y^a- ,

R^a-S-CO-Y^a-, R^a-CO-S-Y^a-, R^a- S - CO-N (R^b) -Y^a- , R^a-N (R^b) -CO-S-Y³-, R^a-S-CO-0-Y^a-, R^a-0-CO-S-Y^a-, R^a-S-CO-S-Y^a- , R^a-S-CS-Y^a-, R^a-CS-S-Y^a-, R^a- S - CS -N (R^b) -Y^a-, R^a-N (R^b) -CS-S-Y^a- , R^a-S-CS-0-Y^a- , R^a-0-CS-S-Y^a- , wherein R^a being a hydrogen atom, a C1-C6 alkyl, a C2-C6 alkenyl or a C2-C6 alkynyl group; R^b being a hydrogen atom, a C1-C6 alkyl, a C2-C6 alkenyl or a C2-C6 alkynyl group; R^c being a hydrogen atom, a Ci-Ce alkyl, a C2-C6 alkenyl or a C₂-C6 alkynyl group; R^d being a hydrogen atom, a C1-C6 alkyl, a C2-C6 alkenyl or a C2-C6 alkynyl group and Y^a being a direct bond, a C1-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, NH₂, =NH, N₃ or O2 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 [ , 5] decanyl, norbornyl, cyclohexyl, cyclopentenyl, cyclohexadienyl, decalinyl, bicyclo [4.3.0] nonyl, tetraline, cyclopentylcyclohexyl, fluorocyclohexyl or cyclohex-2-enyl group .

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, N¾, =NH, 3 or NO2 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 O, 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, Ν¾, 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, oxadiazolyl, thiadiazolyl, 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, IH-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 grou .

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, N%, =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-C17 heterocycloalkyl, C4- C20 alkylcycloalkyl, C2-C₁9 heteroalkylcycloalkyl, C6-C18 aryl, C1-C17 heteroaryl, C₇ - C2o 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 - Ce heteroalkyl , C3 - C10 cycloalkyl, C2-C9 heterocycloalkyl, C7- C12 alkylcycloalkyl, C2 - C11 heteroalkylcycloalkyl, C6- C10 aryl, C1-C9 heteroaryl, C₇ - Ci2 aralkyl or C2 - C11 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, NO2 , C1-4 alkyl (e.g. methyl, ethyl, t-butyl) , NMe_2/ NHMe, CONH₂, CH₂NMe₂, HS0₂Me, C(CH₃)₂CN, COMe, OMe, SMe, COOMe, COOEt, CH₂C00H , OCH₂COOH , COOH, SOMe, S0₂Me, cyclopropyl, S0₂NH₂, S0₂NHMe, SO2CH₂CH₂OH , SFs, S0₂NMe₂, CHO, OCF₃, SO2CF3, COMe, CH2OH, CN or CF₃.

Especially preferred substituents are F, CI, Br, OH, N¾, Me, Ethyl, NMe₂, 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) or (III) 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) or (III) 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) or (III) 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) or (III) 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) or (III) . Compounds of formula (I) , (II) or (III) 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) or (III) . 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) or (III) 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) or (III) 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) or (III) , 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) or (III) 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) or (III) 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) or (III) , 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) or (III).

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) or (III), their solvates, salts or formulations are also comprised in the scope of the present invention. In general, compounds of formula (I) , (II) or (III) 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) or (III) , 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) or (III) , 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) or (III) 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-Hodgkin¹ 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

Synthesis of building block (1)

Table 1

General Procedure

3-Bromo-2-methylphenol (16.0 mraol) was dissolved in monoglyme (84 mL) and the appropriate arylboronic acid (19.3 mmol) was added followed by demi-water (29 mL) , sodium carbonate (40.1 mmol) and finally tetrakis triphenylphosphine palladium (1.6 mmol) . The reaction mixture was heated at reflux overnight and allowed to cool to room temperature . Saturated aqueous ammonium chloride solution (200 mL) was added and the aqueous phase extracted with DCM (2 x 100 mL) . The organic layer was washed with water (2 x 100 mL) and brine (100 mL) , dried with sodium sulfate, filtered and concentrated to get a brown oil which was purified by ISCO (EtOAc/heptanes gradient) to give building blocks 1.

S nthesis of Building Block (3)

General rocedure for compounds (2)

The appropriate building block 1 (3.10 mmol) was dissolved in acetonitrile (31 mL) , to which were added successively anhydrous potassium carbonate (7.74 mmol) and ethyl chloroacetate (4.65 mmol). The suspension was heated to an external temperature of 92 °C under reflux and nitrogen atmosphere overnight. The yellow suspension was allowed to cool to room temperature and the solvent removed in vacuo. The solids were dissolved in water and ethyl acetate and the layers separated. The aqueous layer (40 mL) was extracted with further ethyl acetate (2 x 20 mL) and the combined organic layers washed with brine (2 x 15 mL) , dried with sodium sulfate, filtered and concentrated to afford 1.05 g (quant.) of a yellow oil which was used without further purification.

General procedure for compounds

The appropriate ethyl ester 2 (1.27 mmol) was dissolved in THF (10 mL) , demi water (10 mL) was then added followed by lithium hydroxide monohydrate (2.55 mmol). The reaction mixture became clear after several minutes and was stirred at room temperature overnight. The reaction mixture was acidified to pH = 2 with 3M aqueous HC1 solution, which resulted in an emulsion. The mixture was extracted with ethyl acetate (1 x 15 mL and 2 x 10 mL) the combined organic layers were washed with brine (1 x 10 mL) , dried with sodium sulfate, filtered and concentrated in vacuo to afford compounds 3 as a shiny white solid.

Table 2

S nthesis of compounds (5)

General rocedure for compounds

The appropriate building block 3 (1.17 mmol) and phenylglyoxal monohydrate (1.28 mmol) were suspended in DCM (1.5 mL) . The appropriate isocyanide (1.28 mmol) was added and the reaction mixture stirred at room temperature overnight. The reaction mixture was filtered over sodium sulfate and concentrated. The resulting sticky oil was used for the next step without further purification. neral procedure for compounds (5)

The appropriate Passerini product 4 (0.7 mmol) was dissolved in acetic acid (4.8 mL) , ammonium acetate (0.734 mmol) was added and the reaction mixture was refluxed overnight under N2 atmosphere. The reaction mixture was then cooled to room temperature and poured into ice with DCM. The mixture was neutralized with aqueous sodium hydroxide (5N) . The layers were separated and the organic layer extracted with DCM (2x) . The organic layer was washed with brine (2x) , dried with sodium sulfate, filtered and concentrated carefully. The crude material was purified by ISCO (EtOAc/heptane gradient) to afford compound 5.

Reduction of amide in compound

N- ( (2- ( (3- (benzotd] [1, 3] dioxol-5-yl) -2 -methylphenoxy) methyl) - 4 -phenyloxazol- 5-yl) methyl) butan- 1-amine (6.3)

2- ( (3- (Benzo [d] [1,3] dioxol-5-yl) -2 -methylphenoxy) methyl) -N- butyl-4-phenyloxazole-5-carboxamide (0.053 mmol) was dissolved in dry DCM (0.7 mL) in a heatgun dried flask under N2 atmosphere. 2 -Fluoropyridine (0.058 mmol) was added and the reaction mixture cooled to -78 °C external for 20 minutes. Triflic anhydride (0.55 mmol) was added dropwise and the reaction mixture stirred for 10 minutes. The reaction mixture was then warmed to 0 °C external and triethylsilane (0.058 mmol) was added. After stirring at 0 °C for 10 minutes, cooling was removed. Methanol (0.3 mL) was added and sodium borohydride (0.159 mmol) was added, after 10 minutes further sodium borohydride was added (0.159 mmol) and after 10 minutes, the reaction mixture was concentrated. The residue was partitioned between DCM and water and layers were mixed and separated. The aqueous layer was extracted with DCM and combined organics were dried with sodium sulfate, filtered and concentrated. The crude material was first purified by ISCO (EtOAc/heptanes gradient) and then by RP-ISCO to give the product. MS (ESI) m/z: calc . for C29H30N2O4, [M+H]⁺: 471.23 found: All.20.

Synthesis of compounds (8)

General rocedure for compounds

Phenylglyoxal monohydrate (0.87 mmol) was dissolved in MeOH (1.75 mL) and the appropriate amine (0.87 mmol) was added. The reaction mixture was stirred at room temperature for 30 minutes. Compound 3 (0.87 mmol) and the appropriate isocyanide were then added and the reaction mixture heated to 60 °C external under reflux for 38 hours under N2 atmosphere. The reaction mixture was concentrated and the residue purified partially by ISCO (EtOAc/heptane gradient) to give the product which was used for the next step without further purification. for compounds (8)

The appropriate Ugi product 7 (0.35 mmol) was dissolved in acetic acid (0.7 mL) and ammonium acetate was added (5.25 mmol) . The reaction mixture was heated at 120 °C external for one hour and then cooled. The reaction mixture was diluted with DCM and ice and carefully neutralized with aqueous sodium bicarbonate solution. The aqueous layer was extracted with DCM (3 x) and the combined organics were dried with sodium sulfate, filtered, concentrated and the residue purified by ISCO (EtOAc/heptane gradient) to afford product 8.

Synthesis of building block (9)

Method 1

9

General procedure for the synthesis of building block (9)

The appropriate oxazole alcohol (7.63 mmol) and building block 1 (7.63 mmol) were dissolved in dry THF (89 mL) , tri-n- butylphosphine (11.44 mmol) was added and the reaction mixture cooled to 0 °C in an ice/water bath under nitrogen atmosphere. ADDP (11.44 mmol) was added in portions. After a precipitate formed, the cooling bath was removed and the suspension stirred for overnight, reaction progress was monitored with HPLC-MS. The reaction mixture was filtered over a P4 glass filter and the filtrate concentrated. The resulting material was suspended in DCM (50 mL) and filtered over a P4 glass filter. The filtrate was concentrated and purified by silica gel flash chromatography (coated on 7 g hydromatrix with DCM, gradient EtOAc in heptanes) to afford compound 9 as a white solid.

Method 2

The appropriate amide 10 (1.75 mmol) was suspended in EtOAc (4 mL) , silver triflate (5.70 mmol) was added followed by 2- bromoacetophenone (5.70 mmol). The reaction mixture was heated at 70 °C for 1 h, then allowed to cool to room temperature. EtOAc (3 mL) and brine (4 mL) were added and the mixture was stirred at room temperature overnight . Reaction mixture was filtered over Celite on a P4 glass filter. The organic layer was washed with water (10 mL) and very carefully with saturated aqueous NaHCCh solution (10 mL) , further water plus NaCl (10 mL) and 1 N aqueous HC1 solution (10 mL) followed by brine (10 mL) . The organic layer was dried with sodium sulfate, filtered and concentrated. The residue was purified by silica gel flash chromatography (EtOAc/heptanes 0->20% gradient) to afford compound 9 as a pale yellow solid.

S nthesis of building block 15

1-methyl-4 -phenyl-1H-imidazole (12) Sodium hydride (60% dispersion in mineral oil, 10.83 mmol) was suspended in dry n-pentane under N2 and the solids were allowed to settle. The standing solution was removed via pipette. The solids were suspended in dry DMF (4 mL) and cooled with an ice/water bath. A solution of 4-phenylimidazole (9.85 mmol) in DMF (6 mL) was added dropwise to the stirred suspension over the course of 40 minutes. Addition turned the reaction mixture yellow. After stirring for 30 minutes at room temperature, all cloudiness had disappeared. Methyl iodide (10.34 mmol) was added dropwise but quickly, which turned the mixture cloudy again. The reaction mixture was stirred for 3 hours at room temperature. Water (10 mL) was added, and the clear solution was extracted with EtOAc (3 x 20 mL) , the combined organic layers washed with brine (6 x 20 mL) . The organic layers were dried with sodium sulfate, filtered and concentrated in vacuo to give an orange crystalline solid, a mixture of l-methyl-4- phenyl-lH-imidazole and 1 -methyl-5-phenyl-1H- imidazole

(88:11), which was used without further purification. MS (ESI) m/z: calc. for CxoHi₀N₂ , [M+H]⁺: 159,09; found: 159.10.

1-methyl -4 -phenyl- 1H-imidazole-2 -carbaldehyde ( 13 )

A mixture of 1-methyl-4 -phenyl-1H- imidazole and 1-methyl-5- phenyl-lH-imidazole (88:11) (200 mg, 1.26 mmol, 1.0 equiv) was dissolved in dry THF (5.8 mL) and cooled to -47 °C with a dry ice/acetonitrile bath under N₂ atmosphere. n-BuLi (2.5 M solution in hexanes, 4.5 mmol) was added dropwise, keeping internal temperature below -40 °C. The reaction mixture was cooled to -78 °C with a dry ice/acetone bath and dry DMF (0.13 mL, 1.64 mmol, 1.3 equiv) was added dropwise keeping internal temperature below -74 °C. This changed the reaction mixture from orange to yellow. Let the reaction mixture cool to -75 °C and then warm to room temperature slowly over the course of 75 minutes by lowering the cooling bath stepwise, this gave a solid in suspension. The reaction mixture was then stirred for 2 hours and EtOAc was added (10 mL) . The organic phase was washed with water (2 x 15 mL) and brine (10 mL) , then dried with sodium sulfate, filtered and concentrated to give 200 mg of a yellow solid. The solid was recrystallized from 2- propanol, which gave 52.3 mg of the desired 1-methyl-4 -phenyl- 1H- imidazole-2 -carbaldehyde . The mother liquor was concentrated and the two isomers separated by ISCO (gradient to 50% EtOAc/heptanes) . Giving the desired 1-methyl- -phenyl- 1H-imidazole-2 -carbaldehyde as a white solid and l-methyl-5- phenyl-lH- imidazole-2 -carbaldehyde as a colorless oil that solidified overnight. MS (ESI) m/z: calc. for CnHioN₂0, [M+H] ⁺ : 187,09; found: 187.4.

(1-methyl-4 -phenyl- 1H-imidazol-2 -yl) methanol (14)

l-Methyl-4-phenyl-lH-imidazole-2-carbaldehyde (0.505 mmol) was dissolved in MeOH (5 mL) and sodium borohydride (1.01 mmol) was added. The reaction mixture was stirred at room temperature for 1 hour and then concentrated by rotary evaporation. The solids were taken up in EtOAc and water, layers were separated and the aqueous layer was extracted with EtOAc (3 x 10 mL) . The combined organic layers were washed with brine (10 mL) , dried with sodium sulfate, filtered and concentrated to give the product as a shiny white solid. ¾ NMR (300 MHz , CDC1₃) : δ 3.61 (s, 3H) , 4.12 (br s, 1H) , 4.78 (s, 2H) 7.05 (s, 1H) , 7.2-7.3 (t, 2H, overlaps with GHCI3) , 7.32-7.42 t, 2H) , 7.70 (d, 2H) ppm.

2- ({3- (benzo [d] [1, 3] dioxol-5-yl) -2-methylphenoxy) methyl) -1-methyl-4- phenyl-1H-imidazole (15) Was prepared according to the method for preparing building block 9. MS (ESI) m/z: calc. for C25H22N2O3, [M+H] + : 399,17; found: 399.2.

Mannich

15

General procedure

The appropriate oxazole (or imidazole) (0.075 mmol) and paraformaldehyde (1.13 mmol) were stirred in acetic acid (1 mL) . The amine (0.150 mmol) was added dropwise and the mixture stirred at 120 °C (external) overnight. The acetic acid was evaporated in vacuo, the residue neutralized with saturated aqueous sodium carbonate solution and the aqueous phase extracted with DCM (3 x) . The combined organic layers were dried with sodium sulfate, filtered and concentrated in vacuo. The resulting orange/brown oil was purified with silica gel flash chromatography (EtOAc/heptane or MeOH/DCM gradient) to give a colorless or pale yellow solid.

1- ( (2- ( {3- (benzo [d] [1,3] dioxol-5-yl) -2 -methylphenoxy) methyl) - 1-methyl-4 -phenyl- 1H-imidazol - 5-yl) methyl) - -methylpiperazine (16)

Synthesized according to the general procedure, MS (ESI) m/z: calc. for C31H34N4O3, [M+H]⁺: 511.27; found: 511.40.

2- ( (3- (benzo [d] [1,3] dioxol-5-yl) -2 -methylphenoxy) methyl) -5- ( (4-methylpiperazin-l-yl)methyl) -4-phenyloxazole (17)

Synthesized according to the general procedure, MS (ESI) calc. for C30H3₁N3O₄, [M+H]⁺: 498.24; found: 498.20.

4- ( (2- ((3- (benzo [d] [1, 3] dioxol-5-yl) -2 -methylphenoxy) methyl) - 4 -phenyloxazol- 5-yl) methyl) morpholine (18)

Synthesized according to the general procedure, MS (ESI) m/z: calc. for C29H28N2O5, [M+H]⁺: 485.21; found: 485.20.

2- ( (3- (benzo [d] [1,3] dioxol-5-yl) -2 -methylphenoxy) methyl) -4- phenyl-5- (piperidin-l-ylmethyl) oxazole (19)

Synthesized according to the general procedure, MS (ESI) ra/z: calc. for C30H30N2O4, [M+H]⁺: 483.23; found: 483.35.

2- ( (3- (2, 3-dihydrobenzo [b] [1,4] dioxin-6-yl) -2- methylphenoxy) methyl) -5- ( (4 -methylpiperazin-l-yl) methyl) -4 phenyloxazole (20)

Synthesized according to the general procedure, MS (ESI) calc. for C31H33N3O4, [M+H]⁺: 512.26; found: 512.25.

4- ( (2- ( (3- (2,3-dihydrobenzo[b] [1, 4] dioxin-6-yl) -2- methylphenoxy) methyl) -4 -phenyloxazol-5-yl) methyl) morpholxne (21)

Synthesized according to the general procedure, MS (ESI) m/z: calc. for C30H30N2O5, [M+H]⁺: 499.22; found: 499.20.

2- ( (3- (2, 3-dihydrobenzo [b] [1,4] dioxin-6-yl) -2- methylphenoxy) methyl) -4 -phenyl- 5- (piperidin-l-ylmethyl) oxazole (22)

Synthesized according to the general procedure, MS (ESI) m/z: ca2c. for C31H32N2O4, [M+H]⁺: 497.24; found: 497.20.

2- ( (3- (2, 3-dihydrobenzo [b] [1,4] dioxin-6-yl) -2- methylphenoxy) methyl) -4 -phenyl- 5- (pyrrolidin-1- ylmethyl) oxazole (23)

Synthesized according to the general procedure

calc. for C₃oH₃oN₂0₄, [M+H]⁺: 483.23; found: 483.2

Synthesis of alcohol

Compound 9 (0.26 mmol) and paraformaldehyde (3.74 mmol) were suspended in glacial acetic acid (3.4 mL) , N-ethylbenzylamine (0.52 mmol) was added and the mixture refluxed overnight at 120 °C external temperature. The reaction mixture was allowed to cool down to room temperature and the acetic acid was removed in vacuo. The resulting residue was quenched with saturated aqueous sodium bicarbonate solution (15 mL) and extracted with DCM (3x) . The combined organic extracts were washed with brine (lx) , dried with sodium sulfate, filtered and concentrated. The acetate ester was purified by silica gel flash chromatography (EtOAc/Heptane gradient 0->40%) . The residue was dissolved in THF/demi-water (1:1) 10 mL and lithium hydroxide monohydrate (0.45 mmol) was added. The reaction mixture was stirred overnight at room temperature. EtOAc (10 mL) , demi-water (5 mL) and NaCl were added and the layers were mixed and separated. The aqueous phase was extracted with EtOAc (2 x) , the combined organic extracts were washed with brine (1 x) dried with sodium sulfate, filtered and concentrated. The residue was purified by silica gel flash chromatography (EtOAc/Heptane gradient 0->40%) to give the product alcohol 24 as a sticky white solid.

(2- ( (3- (benzo [d] [1,3] dioxol-5-yl) -2 -methylphenoxy) methyl) -4- phenyloxazol-5 -yl) methanol (24)

MS (ESI) m/z: calc. for C₂5H₂iN0₅, [M+H]⁺: 416.15; found: 416.20.

Synthesis of building block (25)

9 25

Compound 9 (2.0 mmol) was dissolved in dry THF (9.4 mL) and cooled to -42 °C internal temperature using a dry ice/MeCN bath under a nitrogen atmosphere. 2.5 M N-Butyl lithium solution in hexane (2.39 mmol) was added dropwise over 25 minutes, keeping internal temperature below -40 °C. The reaction mixture was allowed to warm to -29 °C over the course of 1 hour and then cooled to -76 °C using a dry ice/acetone bath. Dry DMF (10.13 mmol) was added dropwise, which increased internal temperature to -73 °C. Temperature was maintained for 0.5 h, then the reaction mixture was allowed to warm to room temperature over the course of 1 h and stirred for 3 h. The reaction mixture was diluted with EtOAc (10 mL) and brine (20 mL) . The layers were mixed and separated and the organic layer washed with brine (2 x 20 mL) , dried with sodium sulfate, filtered and concentrated. The residue was purified by silica gel column chromatography (EtOAc/heptane gradient) to afford compound 25 as a sticky yellow solid.

Synthesis of building block (32)

32 31

Ethyl 4 -methyloxazole- 5 -carboxylate (26)

Ethyl 2-chloro-3-oxobutanoate (Ethyl 2-chloroacetoacetate) (0.243 mol) was dissolved in formic acid (240 mL) and ammonium formate (1.26 mol) was added portionwise. The reaction mixture was stirred at 115 °C external temperature under reflux and N2 atmosphere for 17 hours. The reaction mixture was concentrated until no more formic acid could be removed (60 °C @ 50 mbar) . The resulting suspension was diluted with water (300 mL) and was neutralized with sodium carbonate (1.63 mol) or 30% aqueous NaOH in batches while cooling in ice/water. At pH 8, the two layers were separated and the aqueous layer was extracted with EtOAc (2 x 150 mL) . The combined organic layers were dried with sodium sulfate, filtered and concentrated to give a brown oil. The product was purified by manual silicagel chromatography (250 g, eluting with 40% EtOAc/heptanes) to give the product as an orange oil. ¾ NMR (300 MHz, CDCI3) : δ 1.42 (t, 3H) , 2.53 (s, 3H) , 4.42 (q, 2H) , 7.89 (s, 1H) ppm.

(4 -methyloxazol - 5 -yl ) methanol (27)

Ethyl 4 -methyloxazole-5-carboxylate (24.6 mmol) was dissolved in MeOH (30 mL) and sodium borohydride (73.4 mmol) was added portionwise. Addition gave gas evolution and warmed up the reaction mixture, no cooling or nitrogen was used. After stirring at room temperature for 1 hour, gas evolution had ceased and a second portion of sodium borohydride was added (13.2 mmol} and the reaction mixture was stirred for an additional 30 minutes. The solvent was then carefully removed by rotary evaporation. The residue was dissolved in water and DCM (20 mL each) and the layers separated. The aqueous phase was neutralized with 3 M aqueous HC1 and extracted with DCM (3 x 20 mL) . The combined organic layers were dried with sodium sulfate, filtered and concentrated. The product was purified by ISCO (EtOAc/heptanes gradient) to give the product as a colorless oil. ¾ NMR (300 MHz , CDC1₃) : δ 1.77 (t, 1H) , 2.21 (s, 3H) , 4.64 (s, 2H) , 7.77 (s, 1H) ppm.

5- ( ( (tert-butyldimethylsilyl) oxy) methyl) -4 -methyloxazole (28)

(4 -methyloxazol- 5-yl) methanol (4.42 mmol) was dissolved in DCM (40 mL) , imidazole (13 mmol) was added followed by TBDMSC1 (6.63 mmol in 5 mL DCM). Addition of TBDMSC1 almost immediately gave a precipitate. The reaction mixture was stirred at room temperature for 1 hour. Water (25 mL) was then added and the layers separated. The aqueous layer was extracted with DCM (2 x 15 mL) and the combined organic layers were washed with water (30 mL) and brine (30 mL) , dried with sodium sulfate, filtered and concentrated. The crude material was filtered over silica (10 g) with 50 mL of EtOAc to give 1.23 g of the product which was used in the next step without further purification. MS (ESI) m/z: calc . for CiiH₂iN0₂Si, [M+H]+: 228.14; found: 228.4.

(5- ( ( (tert-butyldimethylsilyl) oxy) methyl) -4-methyloxazol-2- yl) methanol (29)

Dry THF (10 mL) was cooled to -46 °C internal temperature under N2 with a MeCN/C02 cooling bath. n-BuLi (2.5 M in hexanes, 6.38 mmol) was added dropwise, allowing internal temperature to rise to -30 °C and drop to -46 °C again. 5- ( ( (tert-butyldimethylsilyl) oxy) methyl) -4-methyloxazole (4.42 mmol) was dissolved in dry THF (2-3 mL) and added dropwise to the n-BuLi solution, keeping internal T below -40 °C. The reaction mixture was stirred for 35 minutes at -43 °C and then dry DMF (27.0 mmol) was added dropwise. The reaction mixture was then allowed to warm up to room temperature overnight and quenched with water (20 mL) . The layers were separated and the aqueous phase was extracted with EtOAc (3 x 10 mL) , the combined organic layers were washed with brine, dried with sodium sulfate, filtered and concentrated to give an orange oil containing product aldehyde and starting material. The crude material was dissolved in MeOH (10 mL) and NaBH4 (5.4 mmol) was added and the reaction mixture was stirred for 1 hour. The solvent was removed by rotary evaporation and the solids were taken up in DCM/water (20 mL) . The layers were separated, the aqueous layer extracted with DCM (2 x 15 mL) and combined organics dried thoroughly until clear with Na2S0₄, filtered and concentrated to give an opaque oil. ISCO chromatography (0->100% EtOAc in heptane) gave the product alcohol as a crystalline solid. ¾ NMR (300 MHz, CDC1₃) : δ 0.11 (s, 6H) , 0.92 (s, 9H) , 2.17 (s, 3H) , 2.60 (t, 1H) 4.46 (s, 2H) , 4.71 (d, 2H) ppm.

2- ( (3- (benzo [d] [1,3] dioxol-5-yl) -2 -methylphenoxy) methyl) -5- ( { (tert-butyldimethylsilyl) oxy) methyl) -4-methyloxazole (30)

(5- ( ( (tert-butyldimethylsilyl) oxy) methyl) -4-methyloxazol-2- yl) methanol (0.73 mmol) and Building Block D (0.73 mmol) were dissolved in dry THF (10 mL) and tri-n-butylphosphine (1.09 mmol) was added. The reaction mixture cooled in ice/water under N2 atmosphere. 1, 1 ' - (azodicarbonyl) dipiperidine (1.09 mmol) was added portionwise and the deep orange solution was stirred for five minutes. A brown suspension then formed, the ice/water bath was removed and stirring speed was increased. After stirring at room temperature for 85 minutes, the reaction was finished (monitored by HPLC-MS) . The reaction mixture was then filtered over glass P4 and the filtrate concentrated. The residue was then taken up in DCM and filtered over glass P4. This second filtrate was purified poorly by ISCO (0->30% EtOAc in heptanes) and then by RP-ISCO, to give the product as a colorless oil, 220 mg, 65%. MS (ESI) m/z: calc. for C26H33NO5S1 , [M+H]⁺: 468.22; found: 468.2.

(2- ( (3- (benzo [d] [1,3] dioxol-5-yl) -2 -methylphenoxy) methyl) -4- methyloxazol-5 -yl) methanol (31)

2- ( (3- (benzo [d] [1,3] dioxol-5-yl) -2 -methylphenoxy) methyl) -5- ( ( (tert-butyldimethylsilyl) oxy) methyl) -4-methyloxazole (0.47 mmol) was dissolved in dry THF (7 mL) and TBAF (1 M solution in THF, 0.94 mmol) was added dropwise. This very slightly gave a yellow color to the reaction mixture, which deepened over time. After 3 hours of stirring, full conversion of starting material was achieved and the reaction mixture was concentrated by rotary evaporation and directly purified by ISCO (1 -> 100% EtOAc in heptanes) to give the product alcohol as a colorless oil. ¾ NMR (300 MHz , CDC13): δ 1.70 (t, 1H) , 2.17 (s, 3H) , 2.23 (s, 3H) , 4.67 (br d, 2H) , 5.13 (s, 2H) , 6.01 (s, 2H) , 6.82-6.71 (m, 2H) , 6.94-6.82 (m, 2H) , 6.99 (d, 1H) , 7.18 (t, 1H) ppm.

2- ( (3- (benzo [d] [1,3] dioxol-5-yl) -2 -methylphenoxy) methyl) -4- methyloxazole- 5-carbaldehyde (32)

(2- ( (3- (benzo [d] [1,3] dioxol-5-yl) -2-methylphenoxy) methyl) -4- methyloxazol-5 -yl) methanol (0.35 mmol) was dissolved in DCM (5 mL) and cooled in an ice/water bath under N2 atmosphere. Dess- Martin periodinane (15wt% solution in DCM, 0.424 mmol) was added dropwise over 3 minutes. After stirring at 0 °C for 22 minutes, the cooling bath was removed and the reaction mixture stirred for 1 hour at room temperature. Further Dess-Martin periodinane (15wt% solution in DCM, 0.177 mmol) was then added and the reaction mixture stirred for an additional 90 minutes. The reaction was then quenched with saturated aqueous sodium bicarbonate and diluted with EtOAc until the aqueous layer was below the organic layer. The organic layer was washed with water (10 mL) and brine (10 mL) , dried with sodium sulfate, filtered and concentrated. The crude material was purified by ISCO (0->50% EtOAc in heptanes) to give the product as a partially opaque sticky oil (73 mg, 59%) . ^-H-NMR (300 MHz, CDCI3) : δ 2.19 (s, 3H) , 2.55 (s, 3H) , 5.23 (s, 2H) , 6.02 (s, 2H) , 6.83-6.69 (m, 2H) , 6.99-6.83 (m, 3H) , 7.17 (t, 1H) , 9.89

(s, 1H) ppm.

Reductive aminations using building blocks (25) and (32)

32

General Procedure

The appropriate aldehyde (building block 25 or 32) (0.073 mmol) was dissolved in DCM (0.5 mL) and diluted with MeOH (1 mL) . One drop of glacial acetic acid was added and the amine (0.109 mmol) was added dropwise to the stirred solution. The reaction mixture was stirred at room temperature for 1-3 hours until HPLC-MS showed full conversion of the aldehyde. Sodium borohydride (0.219 mmol) was then added and the reaction mixture stirred for 1-3 hours until HPLC-MS showed full conversion of the imine. The reaction mixture was concentrated in vacuo and partitioned between DCM and water. The layers were separated and the aqueous phase extracted with DCM (2 x) . The combined organic layers were dried with sodium sulfate, filtered and concentrated in vacuo. The residue was purified by silica gel flash chromatography (EtOAc/heptane or MeOH/DCM gradient) to give a colorless or pale yellow solid.

N- ( (2- ( (3- (benzo [d] [1,3] dioxol-5-yl) -2 -methylphenoxy) methyl) - 4 -phenyloxazol-5 -yl) methyl) propan- 1-amine (33)

Synthesized according to the general procedure, ^XH NMR (300 MHz, CDC1₃) : δ 0.88-0.94 (t, 3H) , 1.46-1.58 (m, 5H, overlaps with H₂0) , 2.19 (s, 3H) , 2.63 (t, 2H) , 4.06 (s, 2H) , 5.22 (s, 2H) , 6.01 (s, 2H) , 6.75-6.80 (m, 2H) , 6.86-6.92 (m, 2H) , 7.02- 7.05 (d, 1H) , 7.19 (t, 1H) , 7.33-7.39 (m, 1H) , 7.43-7.48 (m, 2H) , 7.70-7.32 (m, 2H) ppm. MS (ESI) m/z: calc. for C28H28N2O4, [M+H]⁺: 457.21; found: 457.25.

2- ( ( (2- ( (3- (benzo [d] [1, 3] dioxol-5-yl) -2 -methylphenoxy) methyl) - 4 -phenyloxazol-5 -yl) methyl) (methyl) amino) ethan-l-ol (34)

Synthesized according to the general procedure, MS (ESI) m/z: calc. for C28H28 2O5, [M+H]⁺: 473.21; found: 473.30.

2- ( ( (2- ( (3- (benzo [d] [1,3] dioxol-5-yl) -2 -methylphenoxy) methyl) - 4 -phenyloxazol-5 -yl) methyl) amino) ethan-l-ol (35) Synthesized according to the general procedure

calc. for C₂7H26 ₂0₅, [M+H]⁺: 459.19 ; found: 459

N- ( (2- ( (3- (benzo [d] [1,3] dioxol-5-yl) -2 -methylphenoxy) methyl) - 4-phenyloxazol-5-yl)ntethyl) aniline (36)

Synthesized according to the general procedure, MS (ESI) m/ calc. for C H₂6N20₄, [M+H]⁺: 491.20; found: 491.20.

1- (2 - { (3 - (benzo [d] [1,3] dioxol -5-yl) -2 -methylphenoxy) methyl ) -4 phenyloxazol-5-yl) -N-benzylmethanamine (37)

Synthesized according to the general procedure, MS (ESI) m/ calc. for C32H28N2O4, [M+H]⁺: 505.21; found: 505.30.

N- ( (2- ( (3- (benzo [d] [1,3] dioxol-5-yl) -2 -methylphenoxy) methyl) - 4 -phenyloxazol-5-yl) methyl) ropan-1-amine (38)

Synthesized according to the general procedure, MS (ESI) m/: calc. for C₂8H2₈N₂04, [M+H]⁺: 457.21; found: 457.25.

N- ( (2- ( (3- (benzo [d] [1,3] dioxol-5-yl) -2 -methylphenoxy) methyl) - 4-phenyloxazol-5-yl) methyl) ethanamine (39)

Synthesized according to the general procedure, MS (ESI) m/z: calc. for C27H26 2O4, [M+H]⁺: 443.20; found: 443.20.

N- ( (2- ( (3- (benzo [d] [1, 3] dioxol-5-yl) -2 -methylphenoxy) methyl) - 4-methyloxazol-5-yl)methyl) aniline (40)

Synthesized according to the general procedure, MS (ESI) m/z: calc. for C26H24 2O4, [M+H]⁺: 429,18; found: 429.20.

1- (2- ( (3- (benzo [d] [1,3] dioxol-5-yl) -2 -methylphenoxy) methyl) -4- methyloxazol-5-yl) -N-benzylmethanaraine (41)

Synthesized according to the general procedure, MS (ESI) m/z: calc. for C27H26 2O4, [M+H]⁺: 443.20; found: 443.20.

Screening:

Expression and purification of recombinant PD-L1, PD-L2 and PD-1 The gene encoding human PD-L1 (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-L1 (0.3 mM) and recording the -^Ν 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 (ICso) 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, Munchen) 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 PD1/PD-L1 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-Ll and PD-1 proteins were used for all experiments. The PD-1 construct is a 13.2 kDa protein. While PD-Ll is 14.5 kDa containing a His6-tag. Labeling of PD-Ll 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-Ll was labeled using the Monolith NTtm His-tag labeling kit RED-tris-NTA (Nano temper technologies, Miinchen) . The RED-tris-NTA dye (100 nm) was mixing with PD-Ll (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-Ll 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-Ll vs compounds:

For determining affinity of the test compounds to PD-Ll a 2- fold dilution series was made using the compound stocks and dilution with PBS-t buffer with 2% DMSO. The labeled PD-Ll was added to a final concentration of 50 nm and the samples were loaded into Monolith NT115 standard treated capillaries (NanoTemper technologies, Miinchen) and measured using the Monolith NT.115. The obtained binding curves were analyzed using the MO. Affinity analysis software (NanoTemper technologies, Miinchen) .

Dissociation of the PD-1/ PD-Ll complex:

The PD-l/PD-Ll 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-L1 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. A compound of formula (I) :

:i)

wherein

X is an oxygen atom or a group of formula NR⁵;

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) - , -N(Me) -CH₂-CH₂-0-, -0-CF₂-0- or -O-CH2-O-;

R³ is a methyl group, CN or a halogen atom; and

R⁴ is a group of formula NR⁶R⁷ or OR⁸;

R⁵ is a hydrogen atom or a methyl group; the groups R⁶, R⁷ and R⁸ are independently from each other a hydrogen atom 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; or R⁶ and R⁷ together with the nitrogen atom to which they are bound are part of an optionally substituted heterocycloalkyl group containing 3, 4, 5, 6 or 7 ring atoms; and

R⁹ is a hydrogen atom or an alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, alkylcyclo- alkyl, heteroalkylcycloalkyl, aryl, heteroaryl, aralkyl or heteroaralkyl group; all of which groups may optionally be substituted; 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- , -O- CF2-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 an oxygen atom.

4. A compound according to any one of the preceding claims , wherein R³ is a methyl group.

5. A compound according to any one of the preceding claims, wherein, R⁴ is a group of formula NR⁶R⁷.

6. A compound according to any one of the preceding claims, wherein R⁶ is a hydrogen atom or a methyl group and R⁷ 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, 0 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 C1-C6 alkyl group and a phenyl group; or a heteroaralkyl group containing a C1-C6 alkyl group or a C1-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 claim 6, wherein R⁶ is a hydrogen atom.

A compound according to any one of the preceding claims 1 to 5, wherein R⁶ and R⁷ 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 0 and N, which heterocycloalkyl group may optionally be substituted.

9. A compound according to any one of the preceding claims, wherein R⁹ is a methyl group or a phenyl group.

10. A compound according to any one of claims 1, 2 and 6 to 8 of formula (II) :

(ID . compound according to any one of claims 1, 2 and 6 f formula (III) :

(III) .

12. 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.

13. 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.

1 . A compound or a pharmaceutical composition according to anyone of claims 1 to 12 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.