INHIBITORS OF THE PD-1/PD-L1 PROTEIN/PROTEIN INTERACTION

Abstract

The present invention provides novel compounds of formula (I) that are useful as inhibitors of the PD-1/PD-L1 protein/protein interaction.

##STR00001##

Claims

1. A compound of formula (I): ##STR00073## wherein R.sup.1 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; R.sup.10 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 z is a bond or a C.sub.1-C.sub.6 alkylene, a C.sub.2-C.sub.6 alkenylene, a C.sub.2-C.sub.6 alkynylene or a heteroalkylene group containing from 2 to 8 atoms selected from C, N, O and S; or R.sup.1 and R.sup.10 together are part of a heterocycloalkyl or heteroaryl group, both of which groups may optionally be substituted; or R.sup.10 and z together are part of a heterocycloalkyl or heteroaryl group, both of which groups may optionally be substituted; or R.sup.10 is bound to Ar.sup.1 to form a heterocycloalkyl or heteroaryl group which is annulated to Ar.sup.1, both of which groups may optionally be substituted; Ar.sup.1 is an aryl, heteroaryl, aralkyl or heteroaralkyl group, all of which may optionally be substituted; Ar.sup.2 is a phenylene group or a heteroarylene group having 5 or 6 ring atoms and 1, 2, 3 or 4 heteroatoms selected from O, S and N, which group Ar.sup.2 is unsubstituted or substituted by one group R.sup.2 which is preferably bound to a carbon or a nitrogen atom of Ar.sup.2 which carbon or nitrogen atom is adjacent to an atom at which group Ar.sup.3 is bound to Ar.sup.2 and to an atom at which group Y is bound to Ar.sup.2; Ar.sup.3 is a phenylene group or a heteroarylene group having 5 or 6 ring atoms and 1, 2, 3 or 4 heteroatoms selected from O, S and N which group Ar.sup.3 is unsubstituted or substituted by one, two or three group(s) R.sup.3; X is O, S, CHOMe, NOMe, CFH, CF.sub.2, NH or CH.sub.2; Y is O, S, CHOMe, NOMe, CFH, CF.sub.2, NH or CH.sub.2; R.sup.2 is methyl, CN or halogen; the group(s) R.sup.3 is/are independently halogen, CN, hydroxy or a C.sub.1-C.sub.6 alkyl, a C.sub.2-C.sub.6 alkenyl, a C.sub.2-C.sub.6 alkynyl or a heteroalkyl group containing from 2 to 8 atoms selected from C, N, O and S or a C.sub.3-C.sub.7 cycloalkyl group or a heterocycloalkyl group containing 3 to 7 ring atoms selected from O, S, N and C; or two groups R.sup.3 together are part of a C.sub.3-C.sub.7 cycloalkyl group, a heterocycloalkyl group containing 3 to 7 ring atoms selected from O, S, N and C, a phenyl group or a heteroaryl group having 5 or 6 ring atoms and 1, 2, 3 or 4 heteroatoms selected from O, S and N; or a pharmaceutically acceptable salt, ester, solvate or hydrate or a pharmaceutically acceptable formulation thereof.

2. A compound according to claim 1, wherein R.sup.10 is a hydrogen atom.

3. A compound according to claim 1, wherein X is CH.sub.2 and Y is O or X is O and Y is CH.sub.2.

4. A compound according to claim 1, wherein z is a bond, CH.sub.2, CH(CH.sub.3), CH.sub.2NH or CO; especially wherein z is CH.sub.2.

5. A compound according to claim 1, wherein R.sup.1 is a hydrogen atom, a C.sub.1-6 alkyl group or a heteroalkyl group having from 1 to 6 carbon atoms and from 1 to 5 heteroatoms selected from O, S and N (especially O and N).

6. A compound according to claim 1, wherein R.sup.1 is hydrogen, methyl or a group of formula CH.sub.2CH.sub.2OH, CH.sub.2CH.sub.2NH.sub.2 or CH.sub.2CH.sub.2NHCOCH.sub.3.

7. A compound according to claim 1, wherein Ar.sup.1 is selected from the following groups: ##STR00074## wherein (z) denotes the bond to group z; (X) denotes the bond to group X; E is selected from O, S and NR.sup.6; and R.sup.4, R.sup.5 and R.sup.6 are independently selected from a hydrogen atom, a halogen atom, NO.sub.2, N.sub.3, OH, SH, NH.sub.2, SO.sub.3H or an alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, alkylcycloalkyl, heteroalkylcycloalkyl, aryl, heteroaryl, aralkyl or heteroaralkyl group, all of which groups may optionally be substituted.

8. A compound according to claim 1, wherein, Ar.sup.2 is selected from the following groups: ##STR00075## wherein (Y) denotes the bond to group Y; (Ar.sup.3) denotes the bond to group Ar.sup.3; A is selected from O, S and NH and R.sup.2 is as defined above.

9. A compound according to claim 1, wherein Ar.sup.3 is selected from the following groups: ##STR00076## wherein (Ar.sup.2) denotes the bond to group Ar.sup.2; D is selected from O, S and NR.sup.9; G is selected from O, S and NR.sup.7; R.sup.7, R.sup.8 and R.sup.9 are independently hydrogen, halogen, CN, hydroxy or a C.sub.1-C.sub.6 alkyl, a C.sub.2-C.sub.6 alkenyl, a C.sub.2-C.sub.6 alkynyl or a heteroalkyl group containing from 2 to 8 atoms selected from C, N, O and S or a C.sub.3-C.sub.7 cycloalkyl group or a heterocycloalkyl group containing 3 to 7 ring atoms selected from O, S, N and C; or R.sup.7 and R.sup.8 together are part of a C.sub.3-C.sub.7 cycloalkyl group, a heterocycloalkyl group containing 3 to 7 ring atoms selected from O, S, N and C, a phenyl group or a heteroaryl group having 5 or 6 ring atoms and 1, 2, 3 or 4 heteroatoms selected from O, S and N.

10. A compound according to claim 1, wherein Ar.sup.3 is unsubstituted or wherein Ar.sup.3 is substituted by a halogen atom.

11. A compound according to claim 1, wherein Ar.sup.3 is substituted by two groups that together are part of a C.sub.5-C.sub.6 cycloalkyl group or a heterocycloalkyl group containing 5 or 6 ring atoms selected from O, S, N and C.

12. A compound according to claim 1, wherein Ar.sup.3 is substituted by two groups which together form a group of formula OCH.sub.2CH.sub.2O, OCH.sub.2O or OCF.sub.2O.

13. A compound according to claim 1, wherein R.sup.2 is methyl or CN.

14. A compound of formula (II) according to claim 1: ##STR00077## wherein R.sup.1 is hydrogen, methyl or a group of formula CH.sub.2CH.sub.2OH, CH.sub.2CH.sub.2NH.sub.2 or CH.sub.2CH.sub.2NHCOCH.sub.3; X is CH.sub.2 and Y is O or X is O and Y is CH.sub.2; z is CH.sub.2; Ar.sup.1 is selected from the following groups: ##STR00078## wherein (z) denotes the bond to group z; (X) denotes the bond to group X; E is selected from O, S and NR.sup.6; and R.sup.4, R.sup.5 and R.sup.6 are independently selected from a hydrogen atom, a halogen atom, NO.sub.2, N.sub.3, OH, SH, NH.sub.2, SO.sub.3H or an alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, alkylcycloalkyl, heteroalkylcycloalkyl, aryl, heteroaryl, aralkyl or heteroaralkyl group, all of which groups may optionally be substituted; Ar.sup.2 is selected from the following groups: ##STR00079## wherein (Y) denotes the bond to group Y; (Ar.sup.3) denotes the bond to group Ar.sup.3; R.sup.2 is methyl, CN or halogen; and A is selected from O, S and NH; Ar.sup.3 is selected from the following groups: ##STR00080## wherein (Ar.sup.2) denotes the bond to group Ar.sup.2; D is selected from O, S and NR.sup.9; G is selected from O, S and NR.sup.7; R.sup.7, R.sup.8 and R.sup.9 are independently hydrogen or halogen, or R.sup.7 and R.sup.8 together form a group of formula OCH.sub.2CH.sub.2O, OCH.sub.2O, or OCF.sub.2O; or a pharmaceutically acceptable salt, ester, solvate or hydrate or a pharmaceutically acceptable formulation thereof.

15. A compound of formula (I) according to claim 1: ##STR00081## wherein R.sup.1 is a hydrogen atom, a C.sub.1-6 alkyl group or a heteroalkyl group having from 1 to 6 carbon atoms and from 1 to 5 heteroatoms selected from O, S and N and R.sup.10 is a hydrogen atom; or R.sup.1 and R.sup.10 together are part of a heterocycloalkyl group having 5 or 6 ring atoms which are selected from C, O, N and S, and which group may optionally be substituted; X is CH.sub.2 and Y is O or X is O and Y is CH.sub.2; z is a bond, CH.sub.2, CH(CH.sub.3), CH.sub.2NH or CO Ar.sup.1 is selected from the following groups: ##STR00082## wherein (z) denotes the bond to group z; (X) denotes the bond to group X; E is selected from O, S and NR.sup.6; and R.sup.4, R.sup.5 and R.sup.6 are independently selected from a hydrogen atom, a halogen atom, NO.sub.2, N.sub.3, OH, SH, NH.sub.2, SO.sub.3H or an alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, alkylcycloalkyl, heteroalkylcycloalkyl, aryl, heteroaryl, aralkyl or heteroaralkyl group, all of which groups may optionally be substituted; Ar.sup.2 is selected from the following groups: ##STR00083## wherein (Y) denotes the bond to group Y; (Ar.sup.3) denotes the bond to group Ar.sup.3; A is selected from O, S and NH; Ar.sup.3 is selected from the following groups: ##STR00084## wherein (Ar.sup.2) denotes the bond to group Ar.sup.2; D is selected from O, S and NR.sup.9; G is selected from O, S and NR.sup.7; R.sup.7, R.sup.8 and R.sup.9 are independently hydrogen, halogen, CN, hydroxy or a C.sub.1-C.sub.6 alkyl, a C.sub.2-C.sub.6 alkenyl, a C.sub.2-C.sub.6 alkynyl or a heteroalkyl group containing from 2 to 8 atoms selected from C, N, O and S or a C.sub.3-C.sub.7 cycloalkyl group or a heterocycloalkyl group containing 3 to 7 ring atoms selected from O, S, N and C; or R.sup.7 and R.sup.8 together are part of a C.sub.3-C.sub.7 cycloalkyl group, a heterocycloalkyl group containing 3 to 7 ring atoms selected from O, S, N and C, a phenyl group or a heteroaryl group having 5 or 6 ring atoms and 1, 2, 3 or 4 heteroatoms selected from O, S and N; and R.sup.2 is methyl, CN or halogen; or a pharmaceutically acceptable salt, ester, solvate or hydrate or a pharmaceutically acceptable formulation thereof.

16. A compound of formula (I) according to claim 1: ##STR00085## wherein R.sup.1 is a hydrogen atom, a C.sub.1-6 alkyl group or a heteroalkyl group having from 1 to 6 carbon atoms and from 1 to 5 heteroatoms selected from O, S and N and R.sup.10 is a hydrogen atom; or R.sup.1 and R.sup.10 together are part of a heterocycloalkyl group having 5 or 6 ring atoms which are selected from C, O, N and S, and which group may optionally be substituted; X is CH.sub.2 and Y is O or X is O and Y is CH.sub.2; z is a bond, CH.sub.2, CH(CH.sub.3), CH.sub.2NH or CO Ar.sup.1 is selected from the following groups: ##STR00086## wherein (z) denotes the bond to group z; (X) denotes the bond to group X; E is selected from O, S and NR.sup.6; and R.sup.4, R.sup.5 and R.sup.6 are independently selected from a hydrogen atom, a halogen atom, NO.sub.2, N.sub.3, OH, SH, NH.sub.2, SO.sub.3H or an alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, alkylcycloalkyl, heteroalkylcycloalkyl, aryl, heteroaryl, aralkyl or heteroaralkyl group, all of which groups may optionally be substituted; Ar.sup.2 is selected from the following groups: ##STR00087## wherein (Y) denotes the bond to group Y; (Ar.sup.3) denotes the bond to group Ar.sup.3; A is selected from O, S and NH; Ar.sup.3 is selected from the following groups: ##STR00088## wherein (Ar.sup.2) denotes the bond to group Ar.sup.2; D is selected from O, S and NR.sup.9; G is selected from O, S and NR.sup.7; R.sup.7, R.sup.8 and R.sup.9 are independently hydrogen, halogen, CN, hydroxy or a C.sub.1-C.sub.6 alkyl, a C.sub.2-C.sub.6 alkenyl, a C.sub.2-C.sub.6 alkynyl or a heteroalkyl group containing from 2 to 8 atoms selected from C, N, O and S or a C.sub.3-C.sub.7 cycloalkyl group or a heterocycloalkyl group containing 3 to 7 ring atoms selected from O, S, N and C; or R.sup.7 and R.sup.8 together are part of a C.sub.3-C.sub.7 cycloalkyl group, a heterocycloalkyl group containing 3 to 7 ring atoms selected from O, S, N and C, a phenyl group or a heteroaryl group having 5 or 6 ring atoms and 1, 2, 3 or 4 heteroatoms selected from O, S and N; and R.sup.2 is methyl, CN or halogen; or a pharmaceutically acceptable salt, ester, solvate or hydrate or a pharmaceutically acceptable formulation thereof.

17. A compound of formula (I) according to claim 1: ##STR00089## wherein R.sup.1 is a hydrogen atom, a C.sub.1-6 alkyl group or a heteroalkyl group having from 1 to 6 carbon atoms and from 1 to 5 heteroatoms selected from O, S and N and R.sup.10 is a hydrogen atom; or R.sup.1 and R.sup.10 together are part of a heterocycloalkyl group having 5 or 6 ring atoms which are selected from C, O, N and S, and which group may optionally be substituted; X is CH.sub.2 and Y is O or X is O and Y is CH.sub.2; z is a bond, CH.sub.2, CH(CH.sub.3), CH.sub.2NH or CO Ar.sup.1 is selected from the following groups: ##STR00090## wherein (z) denotes the bond to group z; (X) denotes the bond to group X; E is selected from O, S and NR.sup.6; and R.sup.4, R.sup.5 and R.sup.6 are independently selected from a hydrogen atom, a halogen atom, NO.sub.2, N.sub.3, OH, SH, NH.sub.2, SO.sub.3H or an alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, alkylcycloalkyl, heteroalkylcycloalkyl, aryl, heteroaryl, aralkyl or heteroaralkyl group, all of which groups may optionally be substituted; Ar.sup.2 is selected from the following groups: ##STR00091## wherein (Y) denotes the bond to group Y; (Ar.sup.3) denotes the bond to group Ar.sup.3; A is selected from O, S and NH; Ar.sup.3 is selected from the following groups: ##STR00092## wherein (Ar.sup.2) denotes the bond to group Ar.sup.2; D is selected from O, S and NR.sup.9; G is selected from O, S and NR.sup.7; R.sup.7, R.sup.8 and R.sup.9 are independently hydrogen, halogen, CN, hydroxy or a C.sub.1-C.sub.6 alkyl, a C.sub.2-C.sub.6 alkenyl, a C.sub.2-C.sub.6 alkynyl or a heteroalkyl group containing from 2 to 8 atoms selected from C, N, O and S or a C.sub.3-C.sub.7 cycloalkyl group or a heterocycloalkyl group containing 3 to 7 ring atoms selected from O, S, N and C; or R.sup.7 and R.sup.8 together are part of a C.sub.3-C.sub.7 cycloalkyl group, a heterocycloalkyl group containing 3 to 7 ring atoms selected from O, S, N and C, a phenyl group or a heteroaryl group having 5 or 6 ring atoms and 1, 2, 3 or 4 heteroatoms selected from O, S and N; and R.sup.2 is methyl, CN or halogen; or a pharmaceutically acceptable salt, ester, solvate or hydrate or a pharmaceutically acceptable formulation thereof.

18. A pharmaceutical composition comprising a compound according to claim 1 or a pharmaceutically acceptable ester, prodrug, hydrate, solvate or salt thereof, optionally in combination with a pharmaceutically acceptable carrier.

19. Use of a compound or a pharmaceutical composition according to claim 1 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.

20. A compound or a pharmaceutical composition according to claim 1 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.

21. A method of treating a subject suffering from or susceptible to cancer, a viral disease, and infectious disease and/or a neurodegenerative disease, the method comprising: administering to the subject an effective amount of a compound of claim 1.

22. The method of claim 21 wherein the subject is suffering from cancer, a viral disease, and infectious disease and/or a neurodegenerative disease.

23. The method of claim 21 wherein the subject is suffering from Schizophrenia, Alzheimer, Multiples Sclerosis, Parkinson, Corea Huntington, Spinocerebellar ataxia type 1 (SCA1), Amyotrophic lateral sclerosis or Batten disease.

Description

EXAMPLES

Example 1: 1-(benzyloxy)-3-bromo-2-methylbenzene

[0080] ##STR00011##

[0081] Sodium benzyloxide was prepared by adding benzyl alcohol (20 mmol) to a suspension of NaH (20 mmol) in N-methylpyrrolidone. The freshly prepared solution (10.8 mmol) was added to 1-bromo-3-fluoro-2-methylbenzene (5.41 mmol) in N-methylpyrrolidone. The reaction was heated at 100 C. and was monitored by TLC until complete consumption of the starting material. Water and ethyl acetate were added, the aqueous layer was separated and the organic layer was washed with water, dried over MgSO4, filtered and concentrated. The residue was chromatographed with hexane/EtOAc mixture. Yield: 92%. 1H NMR (500 MHz, CDCl3) 7.49-7.39 (m, 4H), 7.37 (d, J=7.0 Hz, 1H), 7.20 (d, J=8.0 Hz, 1H), 7.02 (t, J=8.1 Hz, 1H), 6.86 (d, J=8.2 Hz, 1H), 5.09 (s, 2H), 2.41 (d, J=3.3 Hz, 3H). 13C NMR (126 MHz, CDCl3) 157.5, 137.0, 128.6, 128.0, 127.3, 127.3, 127.2, 126.0, 125.0, 110.7, 70.5, 16.0. MS (EI) m/z 277(M+).

[0082] Ref: J. R. Rodriguez, J. Agejas, A. Bueno, Tet. Lett. 2006, 47, 5661-5663

Example 2: 3-(benzyloxy)-2-methyl-1,1-biphenyl

[0083] ##STR00012##

[0084] A solution of 1-(benzyloxy)-3-bromo-2-methylbenzene (3.60 mmol), phenylboronic acid (4.32 mmol), sodium carbonate (9 mmol) and tetrakis(triphenylphosphine) palladium (0.36 mmol) in DME (19.5 ml) and water (6.5 ml) were stirred at reflux overnight. The reaction mixture was cooled to room temperature, poured into 100 mL of 1 N NH4Cl, and extracted with ethyl acetate (2100 mL). The combined organic layers were washed with water, washed with brine, dried over sodium sulfate, concentrated, and purified on silica to yield 65% as a colorless oil. 1H NMR (500 MHz, DMSO-d6) 7.49 (d, J=7.6 Hz, 2H), 7.46-7.28 (m, 8H), 7.21 (t, J=7.9 Hz, 1H), 7.05 (d, J=8.2 Hz, 1H), 6.82 (d, J=7.6 Hz, 1H), 5.16 (s, 2H), 2.10 (s, 3H). 13C NMR (126 MHz, DMSO-d6) 157.0, 143.1, 141.7, 137.9, 129.5, 129.2, 128.9, 128.6, 128.2, 127.9, 127.4, 126.7, 123.9, 122.5, 111.2, 69.9. MS (EI) m/z 275(M+).

[0085] Ref: H. A. Harris et al. J. Med. Chem., 2005, 48, 3953-3979

Example 3: 2-methyl-[1,1-biphenyl]-3-ol

[0086] ##STR00013##

[0087] 3-(benzyloxy)-2-methyl-1,1-biphenyl was hydrogenated under atmospheric hydrogen with 10% Pd/C on carbon (containing 50% water, 50 mg) as catalyst in MeOH (5 ml) and THF (2 ml) at room temperature for 72 hours. Filtration of precipitated, wash with ethyl acetate, then concentration gave the colorless oil 299 mg (95%). 1H NMR (500 MHz, CDCl3) 7.40 (m, 2H), 7.36-7.27 (m, 3H), 7.10 (td, J=7.8, 2.0 Hz, 1H), 6.89-6.82 (m, 1H), 6.79 (m, 1H). 13C NMR (126 MHz, CDCl3) 154.1, 143.8, 141.7, 129.3, 128.1, 126.9, 126.3, 122.5, 113.9, 113.9, 13.10. MS (EI) m/z 185 (M+), 207 [M+Na]+.

[0088] Ref: S. Mikami et al. J. Med. Chem., 2012, 55, 3756-3776

Example 4: 2-oxo-4-phenyl-1,2-dihydropyridine-3-carbonitrile

[0089] ##STR00014##

[0090] Addition of 4 mol equiv. of t-BuOK, in one portion, to a 0.5 M DMSO solution of cinnamaldehyde (1 equiv.) and 2-cyanoacetamide (1.1. equiv.), at room temperature, and under an oxygen atmosphere (O2 balloon), induced an exothermic reaction. After stirring for 30 min without external cooling, the reaction mixture was diluted with 4 volumes of water followed by 5 volumes of 4N aqueous HCI, added slowly and with good stirring. The precipitate was filtered to give the desired pyridine, which was further washed with water, and dried in air, giving a crude product of 65% yield. Pure product was recrystallized from MeOH. 1H NMR (500 MHz, DMSO-d6) 12.62 (br, 1H), 7.82 (d, J=6.7 Hz, 1H), 7.63 (m, 2H), 7.56 (m, 3H), 6.44 (d, J=6.7 Hz, 1H). 13C NMR (126 MHz, CDCl3) 160.8, 160.6, 140.4, 135.9, 130.5, 128.9, 128.0, 116.3, 106.7, 100.9. MS (EI) m/z 197 (M+).

[0091] Ref: R. Jain, F. Roschangar, M. A. Ciufolini, Tet. Lett. 1995, 36, 330-3310

Example 5: Ethyl 2-((3-cyano-4-phenylpyridin-2-yl)oxy)acetate

[0092] ##STR00015##

[0093] Ethyl 2-chloroacetate (1 mmol) was added dropwise to a well-stirred mixture of powdered K2CO3 (1.5 mmol) and 2-oxo-4-phenyl-1,2-dihydropyridine-3-carbonitrile (1 mmol) in acetone (25 mL). The reaction mixture was heated under reflux for 10-12 h. After completion of the reaction water and ethyl acetate were added, the aqueous layer was separated and the organic layer was washed with brine, dried over MgSO4, filtered and concentrated. The residue was chromatographed with petroleum ether/ethyl acetate, to give a brown solid in 75% yield. 1H NMR (500 MHz, CDCl3) 7.62 (m, 2H), 7.57-7.45 (m, 4H), 6.40 (d, J=7.1 Hz, 1H), 4.72 (s, 2H), 4.26 (q, J=7.1 Hz, 2H), 1.30 (t, J=7.2 Hz, 3H). 13C NMR (126 MHz, CDCl3) 167.0, 160.5, 160.3, 141.6, 135.3, 130.90, 129.0, 128.1, 115.4, 107.5, 102.6, 62.4, 50.6, 14.1. MS (EI) m/z 283 (M+), 305 [M+Na]+.

[0094] Ref: a) N. S. El-Gohary, M I. Shaaban Arch. Pharm., 2015, 348, 666-680 b) H. C. Shah, V. H. Shah, N. D. Desai, Synth. Comm. 2009, 39, 3126-3140

Example 6: 2-chloro-3-methoxy-1,1-biphenyl

[0095] ##STR00016##

[0096] A solution of 1-bromo-3-methoxybenzene (5.34 mmol), (2-chlorophenyl)boronic acid (6.4 mmol), sodium carbonate (13.35 mmol) and tetrakis (triphenylphosphine)palladium (0.53 mmol) in DME (19.5 ml) and water (6.5 ml) were stirred under reflux overnight. The reaction mixture was cooled to room temperature, poured into 100 mL of 1 N NH4Cl, and extracted with ethyl acetate (2100 mL). The combined organic layers were washed with water, washed with brine, dried over sodium sulfate, concentrated, and purified on silica to yield 81% of a colorless oil. 1H NMR (500 MHz, CDCl3) 7.52 (d, J=7.5 Hz, 1H), 7.39 (m, 2H), 7.33 (m, 2H), 7.11-7.03 (m, 2H), 6.99 (d, J=8.2 Hz, 1H), 3.88 (s, 3H). 13C NMR (126 MHz, CDCl3) 159.3, 140.8, 140.4, 132.5, 131.3, 130.0, 129.1, 128.6, 126.8, 121.9, 115.2, 113.3, 55.32. MS (EI) m/z 219 (M+).

[0097] Ref: H. A. Harris et al. J. Med. Chem., 2005, 48, 3953-3979

Example 7: 2-amino-4-phenylthiophene-3-carbonitrile

[0098] ##STR00017##

[0099] First Step: Malononitrile (32 mmol) and the acetophenone (28 mmol) were dissolved in 20 mL of toluene containing ammonium acetate (500 mg, 6.5 mmol) and glacial acetic acid (2 mL) in a 50 mL flask. By refluxing vigorously, the water formed in the reaction was removed by a Dean and Stark trap placed under the reflux condenser. Evaporation of the toluene left a residue that was recrystallized from alcohol or distilled under vacuum to give pure product. 2-(1-Phenylethylidene) malononitrile Yield: 61%. 1H NMR (CDCl3, 300 MHz), (ppm) 7.56-7.50 (m, 5H, ArH), 2.63 (s, 3H); 13C NMR (CDCl3, 75 MHz), (ppm) 175.4, 135.7, 132.1, 128.9, 127.2, 112.7, 112.6, 84.5, 24.1.

[0100] Second step: A 20 mL vial is charged with 2-(1-Phenylethylidene) malononitrile (5 mmol), sulfur (5 mmol), and triethylamine (5 mmol) in ethanol (5 mL, 1.0 M solution). The reaction is heated 60 C. for 12 h. Then, the reaction was cooled down to room temperature. Evaporation of ethanol left a residue that was purified by column chromatography Yield: 78%. 1H NMR (500 MHz, CDCl3) 7.60-7.50 (m, 2H), 7.39 (m, 2H), 7.36-7.29 (m, 1H), 6.29 (s, 1H), 5.06 (br, 2H). 13C NMR (126 MHz, CDCl3) 164.0, 139.8, 134.1, 128.7, 128.1, 127.1, 116.1, 105.8, 87.9. MS (EI) m/z 201 (M+).

[0101] Ref: a) K. Wang, K. Nguyen, Y. Huang, A. Dmling, J. Comb. Chem., 2009, 11, 920-927 b) K. Wang, D. Kim A. Dmling, J. Comb. Chem., 2010, 12, 111-118

Example 8: 2-((3-cyano-4-phenylthiophen-2-yl)amino)acetic acid

[0102] ##STR00018##

[0103] A solution of 2-amino-4-phenylthiophene-3-carbonitrile (10 mmol), glyoxylic acid hydrate (15 mmol) and sodium methoxide (15 mmol) in 100 ml. Of absolute methanol was stirred at 65 C. for 30 min. After cooling to room temperature, the reaction was further cooled in an ice bath and the imine reduced with portionwise additions of sodium borohydride (15 mmol). Upon complete addition, the ice bath was removed and the mixture stirred for an additional 20 minutes. Methanol was removed in vacuo. The residue was dissolved in a saturated solution of sodium bicarbonate, filtered, and acidified with hydrochloric acid. This method resulted in a yield of Yield: 60%. 1H NMR (500 MHz, CDCl3) 7.53 (m, 2H), 7.37-7.28 (m, 2H), 7.29-7.20 (m, 1H), 6.65 (s, 1H), 4.11 (d, J=6.2 Hz, 2H). 13C NMR (125 MHz, CDCl3) 171.5, 162.0, 133.5, 132.3, 129.0, 128.8, 128.3, 115.2, 105.1, 91.3, 51.9. MS (EI) m/z 257 (M).

[0104] Ref: R. A. Crochet, J. T. Boatright, C. D. Blanton, J. Het. Chem., 1974, 11, 143-150

Example 9: 2-amino-5-methyl-4-phenylthiophene-3-carbonitrile

[0105] ##STR00019##

[0106] First Step: Malononitrile (32 mmol) and the propiophenone (28 mmol) were dissolved in 20 mL of toluene containing ammonium acetate (500 mg, 6.5 mmol) and glacial acetic acid (2 mL) in a 50 mL flask. By refluxing vigorously, the water formed in the reaction was removed by a Dean and Stark trap placed under the reflux condenser. Evaporation of the toluene left a residue that was recrystallized from alcohol or distilled under vacuum to give pure product. 2-(1-Phenylpropylidene)malononitrile Yield: 65%; solid 1H NMR (CDCl3, 300 MHz), (ppm) 7.53-7.40 (m, 5H, ArH), 2.98 (q, J=7.5 Hz, 2H, CH2), 1.11 (t, J=7.5 Hz, 3H, CH3); 13C NMR (CDCl3, 75 MHz), (ppm) 181.5, 134.5, 131.9, 129.1, 127.4, 112.6, 112.3, 31.0, 12.7.

[0107] Second step: A 20 mL vial is charged with 2-(1-Phenyl-propylidene)malononitrile (5 mmol), sulfur (5 mmol), and triethylamine (5 mmol) in ethanol (5 mL, 1.0 M solution). The reaction is heated 60 C. for 12 h. Then, the reaction was cooled down to room temperature. Evaporation of ethanol left a residue that was purified by column chromatography Yield: 81%. 1H NMR (500 MHz, CDCl3) 7.46-7.40 (m, 2H), 7.38-7.33 (m, 3H), 4.70 (br, 2H), 2.24 (s, 3H). 13C NMR (126 MHz, CDCl3) 159.9, 135.1, 133.7, 129.1, 128.5, 127.8, 119.2, 115.9, 89.8, 13.4. MS (EI) m/z 215 (M+).

[0108] Ref: a) K. Wang, K. Nguyen, Y. Huang, A. Dmling, J. Comb. Chem., 2009, 11, 920-927 b) K. Wang, D. Kim A. Dmling, J. Comb. Chem., 2010, 12, 111-118

Example 10: 2-amino-5-(chloromethyl)-4-phenylthiophene-3-carbonitrile

[0109] ##STR00020##

[0110] 2-amino-5-methyl-4-phenylthiophene-3-carbonitrile (30 mmol) was dissolved in CCl4 (150 mL) and the solution was heated to reflux. Benzoyl peroxide (60 mg, 0.25 mmol) was added to the refluxing mixture. After 5 min another batch of benzoyl peroxide (60 mg, 0.25 mmol) and N-bromosuccinimide (5.34 g, 30 mmol) were added. The solution was refluxed for 1 h. After cooling to rt, the reaction mixture was diluted by hexanes. The precipitate was removed via filtration. 2-amino-5-(chloromethyl)-4-phenylthiophene-3-carbonitrile was obtained upon solvent removal.

[0111] Ref: A. H. Younes, L. Zhang, R. J. Clark, M. W. Davidson, L. Zhu, Org. Biomol. Chem., 2010, 8, 5431-5441

Example 11: Methyl 2-((2-methyl-[1,1-biphenyl]-3-yl)oxy)acetate

[0112] ##STR00021##

Example 12: 2-((2-methyl-[1,1-biphenyl]-3-yl)oxy)acetic acid

[0113] ##STR00022##

Example 13: 2-((3-cyano-4-phenylpyridin-2-yl)oxy)acetic acid

[0114] ##STR00023##

Example 14: 4-fluoro-3-(2-oxoethoxy)benzonitrile

[0115] ##STR00024##

[0116] LiCl (3.36 g, 79.6 mmol) was added to a solution of 4-fluoro-3-methoxybenzonitrile (3.0 g, 19.9 mmol) in DMF (25 mL). The reaction mixture was refluxed for 16 h. After cooling to rt, the reaction mixture was poored into water, acidified with 6 N HCl and extracted with with EtOAc (350 mL). The organic layer was sequentially wash with brine (275 mL), dried over anhydrous Na2SO4, and concentrated in vacuo. The residue was purified by column chromatography (SiO2, hexane/EtOAc 60:40) to give 4-fluoro-3-hydroxybenzonitrile (2.6 g, 96%).

[0117] Ref: W. L. Jorgensen, et al J. Med. Chem., 2011, 54, 8582-8591

[0118] Synthesis of reference compounds from US 2015 0291549:

Example 15: Preparation of (3-bromo-2-methylphenyl)-methanol (2)

[0119] ##STR00025##

[0120] Compound 1 (5.0 g, 23.2 mmol) was dissolved in anhydrous THF (25 mL) under argon and the reaction vessel was cooled to 0 C. in an ice bath. To this cooled solution BH3-THF complex (1M in THF, 35 mL) was added dropwise over a 3 h period. The reaction mixture was warmed to room temperature and stirred for an additional 12 h. The mixture was then poured into 1M hydrochloric acid (126 mL) and then extracted with Et2O (3mL). The organic extracts were combined, dried over anhydrous MgSO4, filtered, and concentrated to afford the intermediate (4.6 g; 99%) as a colorless solid. The crude product was subjected to the subsequent reaction without further purification.

Example 16: Preparation of (2-Methyl-3-biphenylyl)methanol (4)

[0121] ##STR00026##

[0122] A mixture of compound 2 (4.6 g, 22.8 mmol), phenylboronic acid 3 (5.65 g, 46.3 mmol) and [1,1-bis(diphenylphosphino)-ferrocene]dichloropalladium(II)dichloromethane complex (0.188 g, 0.103 mmol) in toluene (34.5 mL) and ethanol (11.3 mL) was placed under argon. To this solution sodium bicarbonate, 2M (34.5 mL, 69.0 mmol) was added and the mixture was heated at 80 C. for 30 min. Ethyl acetate (44 mL) and (11 mL) water were added to the reaction mixture. The organic extract was concentrated by rotatory evaporation. The crude product was chromatographed on silica gel eluting with 0-40% ethyl acetate in hexane to afford 4.58 g of an off-white solid. mp: 58.0-59.5 C.; 1H NMR (600 MHz, CDCl3) [ppm]: 7.43-7.40 (m, 3H), 7.35 (m, 1H), 7.31-7.29 (m, 2H), 1H), 7.26 (t, J=7.6 Hz, 1H), 7.20 (dd, J1=7.6 Hz, J2=1.3 Hz, 1H), 4.78 (s, 2H), 2.25 (s, 3H); 13C NMR (151 MHz, DMSO-d6) [ppm]: 143.0, 142.2, 140.0, 133.8, 129.7, 129.5, 128.2, 127.0, 126.9, 125.7, 64.2, 16.0; IR (ATR cm-1): 3365, 3054, 1601, 1469, 1047, 757.

Example 17: Preparation of 2-chloro-6-methoxy-3-pyridine-carboxaldehyde (6)

[0123] ##STR00027##

[0124] Compound 5 was added (4.14 mL, 34.8 mmol) over 5 min to the solution of tert-butyllithium (1.7M in pentane, 22.5 mL, 38.3 mmol) in 69 mL of THF at 78 C. The reaction mixture was stirred at 78 C. for 1 h, then dimethylformamide (3.5 mL, 45.0 mmol) was added and the mixture was stirred at 78 C. for 1.5 h. After the addition of glacial acetic acid (4.0 mL, 69.0 mmol), the reaction mixture was allowed to warm to room temperature over a 30-min period and diethyl ether (200 mL) was added. The organic phase was washed with saturated aqueous sodium bicarbonate (50 mL) and brine (50 mL), and was dried over Na2SO4. Concentration under reduced pressure afforded the product which was recrystallized from hexane to yield 4.6 g (76%) of compound 6 as light yellow solid. mp: 67.0-68.0 C.; 1H NMR (300 MHz, CDCl3) [ppm]: 10.29 (d, J=0.8 Hz, 1H), 8.05 (d, J=7.9 Hz, 1H), 7.02 (dd, J1=7.9 Hz, J2=0.8 Hz, 1H), 4.08 (s, 3H); 13C NMR (75 MHz, CDCl3) [ppm]: 188.0, 164.2, 154.6, 140.0, 117.6, 117.5, 54.8; IR (ATR cm-1): 3103, 2869, 1683, 1567, 1468, 1378, 1273, 1005.

Example 18: Preparation of 2-methoxy-6-[ (2-methyl-3-phenyl-phenyl) methoxy]pyridine-3-carbaldehyde (7)

[0125] ##STR00028##

[0126] Palladium (II) acetate (0.08 g, 0.36 mmol), caesium carbonate (2.23 g, 6.83 mmol), 2-di-tert-butylphosphino-2,4,6-triisopropylbiphenyl (t-Butyl XPhos) (0.290 g, 0.68 mmol), compound 6 (0.59 g, 3.41 mmol), and 4 (0.88 g, 4.44 mmol) and toluene (30 mL) were combined and purged by a stream of argon for 3 minutes. The reaction was sealed and heated at 80 C. for 4 h. The mixture was filtered through a pad of Celite. The filtrate was concentrated under reduced pressure. The product was purified by flash chromatography on silica gel using 0-60% ethyl acetate in hexane. The product was recrystallized from diethyl ether (488 mg, 35%). mp: 132.0-133.0 C.; 1H NMR (600 MHz, CDCl3) [ppm]: 10.22 (d, J=0.8 Hz, 1H), 8.06 (d, J=8.4 Hz, 1H), 7.44-7.41 (m, 3H), 7.37-7.34 (m, 1H), 7.32-7.30 (m, 2H), 7.28 (d, J=7.5 Hz, 1H), 7.26 (m, 1H), 6.45 (dd, J1=8.4 Hz, J2=0.8 Hz, 1H), 5.52 (s, 2H), 4.08 (3H), 2.28 (s, 3H); 13C NMR (151 MHz, DMSO-d6) [ppm]: 187.8, 166.6, 165.2, 143.2, 142.0, 140.5, 135.0, 134.8, 130.5, 129.5, 128.7, 128.3, 127.1, 125.7, 112.5, 103.9, 67.8, 54.1, 16.5; IR (ATR cm-1): 3063, 2961, 2870, 1671, 1591, 1460, 1330, 1277; HRMS (ESI-TOF) Calcd for C21H19NO3Na [M+Na]+: 356.1263; found [M+Na]+: 356.1256.

Example 19: Preparation of N-{2-[ ({2-methoxy-6-[ (2-methyl-3-phenylphenyl) methoxy]pyridin-3-yl}methyl)amino]ethyl}acetamide hydrochloride BMS-202 (9)

[0127] ##STR00029##

[0128] Combined sodium cyanoborohydride (200 mg, 3.18 mmol), N-(2-aminoethyl)acetamide 8 (250 mg, 2.45 mmol), and compound 9 (200 mg, 0.60 mmol) in DMF (20 mL) and acetic acid (5 drops) were stirred at room temperature for 16 h. The mixture was concentrated under reduced pressure. The product was purified by flash chromatography on silica gel (0-60% methanol in ethyl acetate) as yellow oil. The product 9 was converted into the corresponding hydrochloride salt and recrystallized from acetone (76 mg, yield: 27%). mp: 139.5-140.5 C.; 1H NMR (600 MHz, DMSO-d6) [ppm]: 8.97 (br. s., 2H), 8.20 (t, J=5.6 Hz, 1H), 7.82 (d, J=8.0 Hz, 1H), 7.47-7.44 (m, 3H), 7.39-7.36 (m, 1H), 7.30 (m, 2H), 7.21 (t, J=7.6, 1H), 7.19 (dd, J1=7.7 Hz, J2=1.2 Hz, 1H), 6.53 (d, J=8.0 Hz, 1H), 5.45 (s, 2H), 4.04 (s, 2H), 3.95 (s, 3H), 3.36 (q, J=6.3 Hz, 2H), 2.95 (t, J=6.3 Hz, 2H), 2.22 (s, 3H), 1.83 (s, 3H); 13C NMR (151 MHz, DMSO-d6) [ppm]: 170.2, 162.5, 160.5, 144.0, 142.2, 141.4, 135.6, 133.9, 129.7, 129.2, 128.3, 128.3, 127.0, 125.5, 105.3, 101.5, 66.5, 53.7, 46.1, 44.1, 35.2, 22.6, 15.9; IR (ATR cm-1) 3253, 3062, 2934, 2702, 1651, 1605, 1587, 1463, 1309, 1003; HRMS (ESI-TOF) Calcd for C25H29N3O3 [M+H]+: 420.2287; found [M+H]+: 420.2299.

Example 20: Preparation of 3-bromo-4-[(2-methyl-3-phenyl-phenyl)methoxy]benzaldehyde (11)

[0129] ##STR00030##

[0130] To the ice-cooled solution of 3-bromo-4-hydroxybenzaldehyde 10 (0.71 g, 3.51 mmol), triphenylphosphine (1.02 g, 3.89 mmol) and compound 4 (0.70 g, 0.52 mmol) in dry THF (21 mL), diisopropyl azodicarboxylate (DIAD) (0.735 mL, 3.89 mmol) in THF (21 mL) was added dropwise. The resulting yellow solution was allowed to warm to room temperature and was stirred for additional 20 h. The mixture was concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel using 0-60% ethyl acetate in hexane. Recrystallization from MeOH yielded colourless solid (0.47 g, yield: 35%). mp: 108.0-110.0 C.; 1H NMR (600 MHz, DMSO-d6) [ppm]: 9.87 (s, 1H), 8.14 (d, J=2.0 Hz, 1H), 7.97 (dd, J1=6.5 Hz, J2=2.0 Hz, 1H), 7.54 (m, 2H), 7.46 (m, 2H), 7.39 (m, 1H), 7.33-7.30 (m, 3H), 7.22 (dd, J1=6.5 Hz, J2=1 Hz, 1H), 5.39 (s, 2H), 2.23 (s, 3H); 13C NMR (151 MHz, DMSO-d6) [ppm]: 190.6, 159.2, 142.2, 141.2, 134.6, 134.0, 133.9, 131.2, 130.8, 129.8, 129.2, 128.3, 127.5, 127.0, 125.6, 114.1, 111.9, 69.8, 15.9; IR (ATR cm-1) 3063, 2852, 1689, 1594, 1278, 1254, 1189, 1048; HRMS (ESI-TOF) Calcd C21H17BrO2 [M+Na]+: 403.0310; found [M+Na]+: 403.0302.

Example 21: Preparation of 1-({3-bromo-4-[(2-methyl-3-phenyl-phenyl)methoxy]phenyl}methyl)piperidine-2-carboxylic acid BMS-8 (13)

[0131] ##STR00031##

[0132] A solution of 3-bromo-4-[(2-methyl-3-phenylphenyl)methoxy]-benzaldehyde 11 (150 mg, 0.39 mmol), piperidine-2-carboxylic acid (148 mg, 1.17 mmol), sodium cyanoborohydride (74 mg, 1.17 mmol) and acetic acid (2 drops) in DMF (4 mL) was stirred at 80 C. for 3 h. The mixture was concentrated under reduced pressure. The residue was purified by silica gel flash chromatography (0-60% methanol in ethyl acetate). The product was recrystallized from ethyl acetate (50 mg, yield: 26%). mp: 119.5-121.0 C.; 1H NMR (300 MHz, DMSO-d6) [ppm]: 17.45 (br.s, 1H), 7.73 (d, J=2.0 Hz, 1H), 7.55 (dd, J1=7.5 Hz, J2=1.2 Hz, 1H), 7.49-7.35 (m, 8H), 7.22 (dd, J1=7.5 Hz, J2=1.2 Hz, 1H), 5.29 (s, 2H), 4.62 (s, 2H), 3.78 (m, 1H), 3.21 (m, 2H), 2.23 (s, 3H), 2.03-1.93 (m, 2H), 1.86-1.56 (m, 3H), 1.50-1.34 (m, 1H); 13C NMR (75 MHz, DMSO-d6) [ppm]: 170.2, 155.7, 137.1, 134.9, 133.8, 133.7, 129.7, 129.2, 128.3, 127.6, 127.0, 125.6, 122.0, 113.7, 110.8, 69.9, 69.4, 68.6, 59.9, 25.0, 20.1, 19.6, 15.8; IR (ATR cm-1) 3329, 2946, 2520, 1728, 1605, 1500, 1452, 1290, 1265, 1056; HRMS (ESI-TOF) Calcd C25H29N3O3 [M+Na]+: 516.1150; found [M+Na]+: 516.1137.

Example 21: Preparation of 2,6-dimethoxy-4-[ (2-methyl-3-phenylphenyl) methoxy]benzaldehyde (15)

[0133] ##STR00032##

[0134] To the ice-cooled solution of 2,6-dimethoxy-4-[(2-methyl-3-phenylphenyl)methoxy]benzaldehyde 14 (0.92 g, 5.04 mmol), triphenylphosphine (1.45 g, 5.55 mmol) and compound 4 (1.0 g, 5.04 mmol) in dry THF (21 mL), diisopropyl azodicarboxylate (DIAD) (1.08 mL, 5.55 mmol) in THF (21 mL) was added dropwise. The resulting yellow solution was allowed to warm to room temperature and stirred for additional 20 h. The mixture was concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel using 0-60% ethyl acetate in hexane as colourless solid (1.02 g, yield: 56%). mp: 161.5-162.0 C.; 1H NMR (600 MHz, DMSO-d6) [ppm]: 10.37 (s, 1H), 7.44-7.40 (m, 3H), 7.37 (m, 1H), 7.32-7.28 (m, 4H), 6.20 (s, 2H), 5.16 (s, 2H), 3.89 (s, 6H), 2.27 (s, 3H); 13C NMR (151 MHz, DMSO-d6) [ppm]: 187.9, 165.6, 164.3, 143.4, 141.9, 134.7, 134.3, 130.8, 129.5, 128.5, 128.3, 127.1, 125.9, 109.1, 91.1, 69.7, 56.2, 16.4; IR (ATR cm-1) 3013, 2936, 1668, 1607, 1607, 1582, 1465, 1166; HRMS (ESI-TOF) Calcd C23H22O4 [M+Na]+: 385.1416; found [M+Na]+: 385.1420.

Example 22: Preparation of N-{2-[({2,6-dimethoxy-4-[(2-methyl-3-phenylphenyl)methoxy]phenyl}methyl)amino]ethyl}acetamide BMS-37 (16)

[0135] ##STR00033##

[0136] A solution of 2,6-dimethoxy-4-[(2-methyl-3-phenylphenyl)-methoxy]benzaldehyde 15 (90 mg, 0.25 mmol), N-(2-aminoethyl)-acetamide 8 (104 mg, 1.02 mmol), sodium cyanoborohydride (83.5 mg, 1.3 mmol) and acetic acid (2 drops) in DMF (5 mL) was stirred at 80 C. for 3 h. The mixture was concentrated under reduced pressure. The residue was purified by silica gel flash chromatography (0-60% methanol in CHCl3) as colourless solid. (41 mg, yield: 36%). mp: 112.0-113.5 C.; 1H NMR (600 MHz, DMSO-d6) [ppm]: 7.75 (t, J=5.4 Hz, 1H), 7.47 (dd, J1=7.6 Hz, J2=1.05 Hz, 1H), 7.45 (m, 2H), 7.38 (m, 1H), 7.32 (m, 2H), 7.29 (t, J=7.6 Hz, 1H), 7.21 (dd, J1=7.6 Hz, J2=1.20 Hz, 1H), 6.36 (s, 2H), 5.15 (s, 2H), 3.76 (s, 6H), 3.59 (s, 2H), 3.08 (q, J=6.3 Hz, 2H), 2.43 (t, J=6.4 Hz, 2H), 2.22 (s, 3H), 1.77 (s, 3H); 13C NMR (151 MHz, DMSO-d6) [ppm]: 169.0, 159.2, 158.9, 142.2, 141.4, 135.6, 134.0, 129.7, 129.2, 128.2, 127.0, 125.5, 108.7, 91.5, 68.6, 55.7, 47.9, 40.5, 40.1, 38.8, 22.6, 15.9; IR (ATR cm-1) 3317, 2934, 2836, 1647, 16155, 1597, 1498, 1200, 1147, 1033; HRMS (ESI-TOF) Calcd C27H32N2O4 [M+H]+: 449.2440; found [M+H]+: 449.2440.

Example 23: Preparation of (2R)-2-[({2,6-dimethoxy-4-[(2-methyl-3-phenylphenyl)methoxy]-phenyl}methyl)amino]-3-methyl-butan-1-ol BMS-242 (17)

[0137] ##STR00034##

[0138] A solution of 2,6-dimethoxy-4-[(2-methyl-3-phenylphenyl)-methoxy]-benzaldehyde 15 (181 mg, 0.5 mmol), L-valinol 8 (210 mg, 2.03 mmol), sodium cyanoborohydride (167 mg, 2.6 mmol) and acetic acid (2 drops) in DMF (4 mL) was stirred at 80 C. for 3 h and in room temperature overnight. The mixture was concentrated under reduced pressure. The residue was purified by silica gel flash chromatography (0-60% methanol in ethyl acetate). The product 17 was converted into the corresponding hydrochloride salt and recrystallized from isopropanol and diisopropyl ether (55 mg, yield: 48%). mp: 74.0-76.0 C.; 1H NMR (600 MHz, DMSO-d6) [ppm]: 8.08 (d br., J=54.5 Hz, 2H), 7.5 (dd, J1=6.6 Hz, J2=1.0 Hz, 1H), 7.46 (m, 2H), 7.38 (m, 1H), 7.31 (m, 3H), 7.21 (dd, J1=6.6 Hz, J2=1.20 Hz, 1H), 6.46 (s, 2H), 5.34 (t, J=4.9 Hz, 1H), 5.21 (s, 2H), 4.12 (t, J=5.6 Hz, 2H), 3.83 (s, 6H), 3.70 (m, 1H), 2.80 (m, 1H), 2.22 (s, 3H), 2.08 (m, 1H), 1.23 (s, 1H), 0.96 (d, J=6.9 Hz, 3H), 0.92 (d, J=6.9 Hz, 3H); (Peaks observed at 1.04 and 3.61 ppm corresponds to residual amount of diisopropyl ether) 13C NMR (151 MHz, DMSO-d6) [ppm]: 161.9, 159.9, 142.7, 141.8, 135.8, 134.6, 130.3, 129.6, 128.8, 128.7, 127.5, 126.0, 100.2, 92.0, 69.3, 67.8, 64.5, 57.7, 56.5, 38.7, 26.8, 23.3, 19.6, 18.1, 16.4; IR (ATR cm-1) 3289 (br), 2965, 1611, 1595, 1463, 1150; HRMS (ESI-TOF) Calcd C28H35NO4 [M+H]+: 450.2644; found [M+H]+: 450.2640.

[0139] Further Examples:

TABLE-US-00001 ex- am- ple- structure and smiles MH.sup.+ 23 [00035] 321.1 24 [00036] 411.2 25 [00037] 379.2 26 [00038] 325.1 27 [00039] 395.2 28 [00040] 390.2 29 [00041] 391.1 30 [00042] 395.5 31 [00043] 337.1 32 [00044] 422.2 33 [00045] 564.2 34 [00046] 480.2 35 [00047] 546.2 36 [00048] 556.2 37 [00049] 381.1 38 [00050] 367.1 39 [00051] 337.1 40 [00052] 337.1 41 [00053] 338.1 42 [00054] 466.2 43 [00055] 468.1 44 [00056] 451.1 44 [00057] 483.1 46 [00058] 499.1 47 [00059] 527.1 48 [00060] 542.2 49 [00061] 520.2 50 [00062] 503.1 51 [00063] 510.2 52 [00064] 496.2 53 [00065] 514.2 54 [00066] 514.2 55 [00067] 514.2 56 [00068] 420.2 57 [00069] 468.2 58 [00070] 599.3 59 [00071] 479.2 60 [00072] 572.3

Screening:

Expression and Purification of Recombinant PD-L1, PD-L2 and PD-1

[0140] 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 5 h. 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-L1 and hPD-L2 were refolded by dropwise 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-L1 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

[0141] 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-L1 and hPD-L1-small molecule complex (3:1 compound:protein molar ratio) were estimated using the calibration curve.

Differential Scanning Fluorimetry (DSF)

[0142] DSF analysis was performed according to Niesen and colleagues (24). In brief hPD-L1 and hPD-L2 (both 12.5 M) were incubated alone, with compound BMS-202 or compound 8 (both at 37.5 M) in the presence of SYPRO Orange Dye (Life Technologies, final concentration 20). 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

[0143] Uniform 15N labeling was obtained by expressing the protein in the M9 minimal medium containing 15NH4Cl 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-L1) 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.

[0144] Binding of the compounds was analyzed by titrating the 15N-labeled PD-L1 (0.3 mM) and recording the 1H-15N HMQC spectra prior and after addition of the compound (Supplementary FIGS. S1, S2 and S3).

[0145] The ability of tested compounds to dissociate hPD-L1/hPD-1 was evaluated using AIDA (27). 15N-labeled hPD-1 (0.2 mM) was slightly overtitrated with unlabeled hPD-L1. Compound was aliquoted into the resulting mixture. During the experiment the 1H-15N signals were monitored by HMQC experiment.

[0146] Changes in the oligomeric state of hPD-L1 in the presence of tested compounds were monitored by titration of unlabeled hPD-L1 (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.

[0147] All compounds showed activity (IC.sub.50) in the range of from 0.001 to 1000 M.