PD-1 and PD-l1 inhibitors and process for preparation thereof

Abstract

The present invention provides the family of heteroarylated biaryl compounds useful as PD-1 and PD-L1 inhibitors and process for the preparation of these biaryl moiety attached isoxazole, pyrazole and furan derivatives.

Description

[0001] PT / 2026 / 10546

[0002] PD-1 AND PD-L1 INHIBITORS AND PROCESS FOR PREPARATION THEREOF FIELD OF THE INVENTION

[0003] The present invention relates to substituted Biaryl derivatives as PD-1 and PD-L1 inhibitors. More particularly, it provides the series of new aromatic 5-membered heterocyclic substituted biaryl derivatives or pharmaceutically acceptable salts or solvates thereof, and method of preparation thereof. Further it also provides the PD-1 and PD-L1 inhibition studies.

[0004] BACKGROUND OF THE INVENTION

[0005] Biaryl moieties are important structural skeletons among a series of various pharmaceutically effective substances and natural products. These compounds synthetic strategies and exhibition of wide range of biological have been reported (W02005019211A; W00073292A; WO2019149183A1; US20050096359A; US4912276; US005922898A; US008895741B2; US008236799B2; CN101429170; RU2736511C1). A certain category of biaryl group are reported to exhibit PD-1 / PD-L1 inhibition properties (WO2019149183A1); however, the small biaryl compound showing selective PD1 / PD-L1 inhibition are very rarely noticed. Therefore, the design and construction of biaryl group that are capable of selective PD1 / PDL1 inhibition properties is quite challenging and essential assignment. Even though, many types of synthesis of biaryl groups are reported using a variety of strategies towards the synthesis of biaryl architecture, (US4912276; W02005019211A; Chem. Soc. Rev., 2016, 45, 6766; Top. Curr. Chem. 2020, 378, 23; Chem. Soc. Rev., 2004, 33, 274) there are certain factions of heteroaryl substituted biphenyl which are of interest and have not been evaluated for biological properties and also trivial focused on synthesis. Heteroaryl substitution based biaryl compounds of this invention are examples of this kind which are of rare occurrence and the strategies for the synthesis of this complexes and studies on biological activities is the most required task. In this regard, this invention aims towards the synthesis and systematic screening of the structurally diverse heterocyclic core substituted on biaryl skeleton. In this context, a large number of new biaryl derivatives have been synthesized and evaluated for PD-1 andPD-Ll inhibition activity.

[0006] OBJECTIVE OF THE INVENTION

[0007] The present invention is the applicability of the hetero group substitution on biaryl ring (majorly at ortho and meta position) towards the inhibition of PD-1 and PD-L1 cell lines. The main objective of the invention is to provide novel heteroaryl (viz. isoxazole, pyrazole andPT / 2026 / 10546

[0008] furan) substituted biaryl derivatives as PD-1 / PD-L1 inhibitors.

[0009] Another objective of this claimed invention is to provide process and synthetic routes for the preparation of a family of biaryl heterocyclic compounds, s

[0010] Further, another objective of current invention is to provide the PD-1 & PD-L1 inhibition studies of synthesized novel aromatic 5-membered heterocyclic substituted Biaryl derivatives.

[0011] SUMMARY OF THE INVENTION

[0012] The present invention provides an immunotherapeutic compound of Formula I, pharmaceutically acceptable salts or solvates thereof,

[0013]

[0014] Formula I

[0015] wherein

[0016] A is O or N-Rl, wherein R1 is H or Cl to C3 alkyl;

[0017] B is N or C-R2, wherein R2 is Cl to C3 alkyl;

[0018] X and Y are independently

[0019] when X

[0020]

[0021] R3 is selected from the group consisting of halo, alkoxy, trifluoroalkyl, CN and C1-C5 alkyl, Y is H;

[0022] when Y is

[0023]

[0024] wherein R3 is selected from the group consisting of halo and alkoxy, X is o II

[0025] wherein R4 is selected from the group consisting of hydroxy, alkoxy, and C1-C5 alkyl; and

[0026] Z is selected from the group consisting of C1-C5 alkyl, Bn and

[0027]

[0028] , wherein R5 is CN,

[0029] tetrazole,

[0030]

[0031] In a preferred embodiment the present invention provides a compound of formula la a pharmaceutically acceptable salt or solvate thereof,P 172026 / 10546

[0032]

[0033] Formula la

[0034] wherein A is O or N-Rl, wherein R1 is H or Cl to C3 alkyl;

[0035] B is N or C-R2, wherein R2 is Cl to C3 alkyl;

[0036] R3 is selected from the group consisting of halo, alkoxy, trifluoroalkyl, CN and C1-C5 alkyl; and

[0037] Z is selected from the group consisting of C1-C5 alkyl, Bn, and

[0038]

[0039] , wherein Rs is CN,

[0040] tetrazole,

[0041]

[0042] In a preferred embodiment the present invention provides a compound of Formula lb, a pharmaceutically acceptable salt or solvate thereof,

[0043]

[0044] Formula lb

[0045] wherein A is O; B is N or C-R2, wherein R2 is Cl to C3 alkyl;

[0046] R4 is selected from the group consisting of hydroxy, alkoxy, and C1-C5 alkyl;

[0047] R3 is selected from the group consisting of halo and alkoxy, and

[0048] Z is selected from the group consisting of C1-C5 alkyl, Bn, and

[0049]

[0050] , wherein Rs is CN,

[0051] tetrazole,

[0052]

[0053] In a preferred embodiment the present invention provides a compound selected from the group consisting of:

[0054] 4-(3-([l,l'-Biphenyl]-3-yl)isoxazol-5-yl)benzonitrile (1);

[0055] 4-(3-(4'-methyl-[l,r-biphenyl]-3-yl)isoxazol-5-yl)benzonitrile (2);

[0056] 4-(3-(4'-(Trifluoromethoxy)-[l,r-biphenyl]-3-yl)isoxazol-5-yl)benzonitrile (3);

[0057] 3'-(5-(4-Cyanophenyl)isoxazol-3-yl)-3-fluoro-[l,r-biphenyl]-4-carbonitrile (4);

[0058] (4-(3-([l,l'-Biphenyl]-3-yl)isoxazol-5-yl)phenyl)methanol (5);

[0059] 4-(4-(3-([l,l'-Biphenyl]-3-yl)isoxazol-5-yl)benzyl)morpholine (6);

[0060] 5-(4-(2JH-Tetrazol-5-yl)phenyl)-3-([l,l'-biphenyl]-3-yl)isoxazole (7);PT / 2026 / 10546

[0061] 4-(3-([l,l '-Biphenyl]-3-yl)-l / / -pyrazol-5-yl)benzonitrile (8);

[0062] 4-(3-([ 1 , 1 '-Biphenyl]-3-yl)-l -methyl- 17 / -pyrazol-5-yl)benzonitrile (9);

[0063] 3-([l,l'-Biphenyl]-2-yl)-5-propylisoxazole (10);

[0064] 3-(4'-Methoxy-[l,l'-biphenyl]-2-yl)-5-phenylisoxazole (11);

[0065] 3-(4'-Bromo-[l,l'-biphenyl]-2-yl)-5-phenylisoxazole (12);

[0066] 3-(4'-Methoxy-[l, l'-biphenyl]-2-yl)-5-phenyl-l / / -pyrazole (13);

[0067] 4-(4'-ethyl-[l,l'-biphenyl]-2-yl)-2-phenyl-lH-pyrrole (14);

[0068] 3-(4'-Bromo-[l, l'-biphenyl]-2-yl)-5-phenyl-l / / -pyrazole (15);

[0069] 3 -([ 1 , 1 '-Biphenyl] -2-yl)-5-propyl- 1 / Z-pyrazole (16);

[0070] Ethyl 5-benzyl-4-(4'-methoxy-[l,r-biphenyl]-2-yl)-2-methylfuran-3-carboxylate (17);

[0071] Ethyl 5-benzyl-4-(4'-ethyl-[l,r-biphenyl]-2-yl)-2-methylfuran-3-carboxylate (18);

[0072] Ethyl 5-benzyl-4-(4'-bromo-[l,r-biphenyl]-2-yl)-2-methylfuran-3-carboxylate (19);

[0073] 5-Benzyl-4-(4'-methoxy-[l,l'-biphenyl]-2-yl)-2-methylfuran-3-carboxylic acid (20); and l-(5-benzyl-4-(4'-methoxy-[l,l'-biphenyl]-2-yl)-2-methylfuran-3-yl)ethan-l-one (21).

[0074] Methyl (4-(3-([l,l'-biphenyl]-3-yl)isoxazol-5-yl)benzoyl)serinate(35)

[0075] 4-(3-([l,l'-Biphenyl]-3-yl)isoxazol-5-yl)benzoic acid (36)

[0076] 4-(3-([l,r-Biphenyl]-3-yl)-l,2,4-oxadiazol-5-yl)benzonitrile (37)

[0077] 5-(4-(l / 7-Tetrazol-5-yl)phenyl)-3-([l,r-biphenyl]-3-yl)-l,2,4-oxadiazole (38)

[0078] Methyl 1 -(4-(3 -([ 1 , r-biphenyl]-3-yl)isoxazol-5-yl)benzoyl)piperidine-2-carboxylate (39) Methyl l-(4-(3-([l,l'-biphenyl]-3-yl)isoxazol-5-yl)benzoyl)-4-hydroxypyrrolidine-2-carboxylate (40)

[0079] 4-(3-([l,l'-Biphenyl]-3-yl)isoxazol-5-yl)benzamide (41)

[0080] 4-(3-([l,l'-Biphenyl]-3-yl)isoxazol-5-yl)-N-hydroxybenzamide (42)

[0081] 3-([l,l'-Biphenyl]-3-yl)-5-(4-(l-methyl-lH-tetrazol-5-yl)phenyl)isoxazole (43)

[0082] The present invention provides a process for the preparing isoxazole substituted biaryl compound of formula la

[0083]

[0084] >

[0085] Formula la

[0086] wherein A is O, B is N, R3 is selected from the group consisting of halo, alkoxy, trifluoroalkyl, CN and C1-C5 alkyl; andPT / 2026 / 10546

[0087] Z is selected from the group consisting of C1-C5 alkyl, Bn, and

[0088]

[0089] , wherein R5 is CN,

[0090] tetrazole, -CH2OH and

[0091]

[0092] , comprising the steps of:

[0093] i. reacting benzaldehyde compound 22 with boronic acid compound 23, wherein R3 is selected from the group consisting of halo, alkoxy, trifluoroalkyl, CN and C1-C5 alkyl, under Suzuki coupling reaction in presence of palladium catalyst and base in a solvent at 80 °C for 3-5 hours to obtain the compound 24, wherein R3 is as defined above;

[0094]

[0095] ii. treating the compound 24 with Hydroxylamine hydrochloride in presence of NazCOs in ethanol and water solvent mixture at 25 - 40 °C for 2-5 hours to produce the compound 25, wherein R3 is as defined above;

[0096]

[0097] iii. reacting compound 25 with alkyne 33, wherein Z is selected from the group

[0098] consisting of C1-C5 alkyl, Bn, and

[0099]

[0100] , wherein R6 is CN, tetrazole, -CH2OH; in the presence of base and chlorinating reagent in polar solvent at 25 - 40 °C for 12- 15 hours to produce isoxazole substituted biaryl compound of formula la (compound 26).

[0101]

[0102] iv. alternatively, the isoxazole substituted biaryl derivatives 26 obtained by reacting the compound 27 with tosyl hydroxylamine in presence of acid in polar solvent followed by N-tosyl deprotection with TBAF.

[0103] In a preferred embodiment the base in step (i) is selected from Na2COs, NaHCCh, CsF, Et3N, K2CO3, TBAF and KOtBu and the solvent in step (i) selected from EtOH, H2O, DCM, EtOAc, THF, CH3CN.

[0104] In a preferred embodiment the chlorinating reagent used in step (iii) is NCS and acid in stepPT / 2026 / 10546

[0105] (iv) is selected from BF3.ET2O and / ?TSA.

[0106] The present invention provides a process for the preparing pyrazole substituted biaryl compound of formula I

[0107]

[0108] Formula I

[0109] wherein A is N-Rl, wherein R1 is H or Cl to C3 alkyl; B is N; R3 is selected from the group consisting of halo, alkoxy, trifluoroalkyl, CN and C1-C5 alkyl; and Z is selected from the

[0110] group consisting of C1-C5 alkyl, Bn, and,

[0111]

[0112] Rs is CN, tetrazole, -CH2OH and

[0113]

[0114] , comprising the steps of:

[0115] i. reacting compound 24, wherein R3 is selected from the group consisting of halo, alkoxy, trifluoroalkyl, CN and C1-C5 alkyl with alkyne 33, wherein Z is selected from the group consisting of C1-C5 alkyl, Bn, and, wherein R6 is CN, tetrazole, -CH2OH in presence of base in THF at -78°C for 2 hours to produce the compound 27, wherein R3 and Z are as defined above;

[0116]

[0117] ii. oxidation of the secondary alcohol of compound 27 using oxidizing agent in DCM at 0 - 25 °C for 2 hours to obtain the compound 28, wherein R3 and Z are as defined above;

[0118]

[0119] iii. treating the compound 28 with tosyl hydrazine in Methanol at 25 - 40 °C for 3 hours to produce the compound 29 wherein R3 and Z are as defined above;PT / 2026 / 10546

[0120]

[0121] iv. subjecting the compound 29 for base mediated cyclisation reaction in the presence of Triethylsilane, KF and catalyst in DMF at 25 - 40 °C for 12-15 hours to obtain the compound 30, wherein R3 and Z are as defined above;

[0122]

[0123] v. deprotection of tosyl group of compound 30 in DMSO at 25 - 40 °C for 12 hours to produce the pyrazole substituted biaryl compound of formula I (compound 31); and

[0124]

[0125] vi. alternatively reacting the compound 27 with tosyl Hydrazine in presence of acid in acetonitrile followed by addition KOtBu to produce the pyrazole substituted biaryl compound of formula la (compound 31).

[0126] The present invention provides a process for the preparation of furan substituted biaryl derivatives of formula I as claimed in claim 1 comprising reacting compound 27 and 1,3- diketoester 34 in presence of acid

[0127]

[0128] in acetonitrile stirred for 2h and then addition of K2CO3 to the reaction mixture and then further stirred at 80°C for lOh to produce the furan substituted biaryl derivatives 32.

[0129] The present invention provides a pharmaceutical composition comprising at least one compound as claimed in claim 1, or a pharmaceutically acceptable salt, or prodrug thereof, inPT / 2026 / 10546

[0130] combination with a pharmaceutically acceptable carrier or excipient.

[0131] BRIEF DESCRIPTIONS OF THE DRAWINGS

[0132] Fig 1: TSA / DSF utilized to identify the PD-L1 binders initially. It has been identified that compounds 1 (Fig IB) and 7 (Fig 1C) as initial binders to PD-L1 in opposite mechanisms (One that binds and gives stability to protein folding and other that binds and disrupts the protein folding). Other set of series showed no binding to PD-L1 (Fig D and E).

[0133] Fig 2. Cell-based PD-1 / PD-L1 interaction mediated luciferase bioassay employed to identify the small molecule that inhibit the PD-1 / PD-L1 interaction.

[0134] Fig 3. Dose-dependent PD-1 / PD-L1 blockade bioassay was performed to determine the ECso.

[0135] Figure 4. ITC (Isothermal Titration Calorimetry) assay was further performed to confirm the identified inhibitors orthogonally. BMS compounds BMS-l / PDI-l(Fig.4A), BMS 202(Fig.4B) with KD values of 1.68pM and 1.21pM and compounds 1 (Fig.4C), 7 (Fig.4D), with KD values of 1.37pM, 2.08pM, respectively showed significant inhibition.

[0136] Figure 5: In vitro-cytokine induction assay was performed to assess immune activation ability of the identified inhibitors.

[0137] Figure 6. In vivo Syngeneic mice experiments were performed to observe the potency of inhibitors to reduce tumor mass upon treatment.

[0138] DETAILED DESCRIPTIONS OF THE INVENTION

[0139] Heteroaryl derivates are diversely substituted architecture and are prevalence in diverse molecules across the chemical sciences, in natural products, pharmaceuticals, agrochemicals and ligands. These functionalities are major structural motifs of new medicines from different pharmacological groups. This resulted in design and synthesis of a large number of heteroaryl substituted biaryls derivatives as illustrated in Figure 1. These new class of hetero-arylated biaryls are useful as selective PD-1 and PD-L1 inhibitors.

[0140] The present invention also providing a process for the preparation of a family of biaryl family substituted with heterocycle as described in the general formula I, la & lb The biaryl structure possessing diversely substituted architecture is fundamental to diverse molecules across the chemical sciences, being prevalent in natural products, pharmaceuticals,PT / 2026 / 10546

[0141] agrochemicals and ligands. These functionalities are major structural motifs of new medicines from different pharmacological groups. To enhance the drug discovery process, the attachment of heterocyclic scaffolds onto the biaryl skeleton is very important. In this connection, a family of biaryl motifs were developed as depicted in the above general formula I, la and lb.

[0142] Scheme 1:

[0143]

[0144] Scheme II:

[0145]

[0146] Scheme IIIPT / 2026 / 10546

[0147]

[0148] The process for the preparation of isoxazole substituted biaryl derivatives wherein the said process comprising the steps of:

[0149] a) Suzuki reaction / coupling using boronic acid derivative 23, palladium catalyst, base in solvent mixture at 80 °C for 3-5 hours;

[0150] b) Installation of oxime group using amine salts and a base at 24 in polar solvent mixture at 25 - 40 °C for 2-5 hours;

[0151] c) Cyclization of oxime 25 with substituted alkyne in the presence of base and chlorinating reagent in polar solvent at 25 - 40 °C for 12-15 hours;

[0152] d) In the case of pyrazole functionality:

[0153] i. Installation of alkyne functionality in the presence of base and substituted acetylene in polar solvent at -78 °C for 2 hours;

[0154] ii. Oxidation of the secondary alcohol 27 using oxidizing agent in polar solvent at 0 - 25 °C for 2 hours;

[0155] iii. Installation of hydrazide functionality to 28 using hydrazine hydrate in polar solvent at 25 - 40 °C for 3 hours;

[0156] iv. Intramolecular-cyclization of hydrazide with alkyne 29, through a base mediated reaction in the presence of silylating reagent and catalyst in polar non protonated solvent at 25 - 40 °C for 12-15 hours;

[0157] v. Deprotection on nitrogen of 30 in polar non protonated solvent at 25 - 40 °C forPT / 2026 / 10546

[0158] 12 hours to produce the compounds 31

[0159] In yet another embodiment of the present invention, polar solvents are selected from EtOH, H2O, DCM, EtOAc, polar non protonated solvents taken from DMF and DMSO and nonpolar solvents are selected from Toluene.

[0160] In yet another embodiment of the present invention, bases are selected from NazCOs, NaHCOs, CsF and Et3N.

[0161] In yet another embodiment of the present invention, amine salts are selected from hydroxylamine hydrochloride, palladium catalysts is selected as Pd(PPh3)4 and copper triflate as the catalyst

[0162] In yet another embodiment of the present invention, boronic acid derivatives are selected from aryl boronic acids with substitutions R1is hydrogen, o-tri fl uorom ethoxy and cyano.

[0163] In yet another embodiment of the present invention, alkyne is selected from substituted phenyl acetylene with substitutions R is cyano, primary alcohol and heteroaryl.

[0164] In still another embodiment of the present invention, the heteroaryl substituted at the meta position of biaryl derivatives prepared are tested for efficiency towards PD-1 and PD-L1 inhibition properties.

[0165] The process for the preparation of ortho- heteroaryl substituted biaryl derivatives wherein the said process comprising the steps of:

[0166] a) Suzuki reaction / coupling using boronic acid derivative 23, palladium catalyst, base in solvent mixture at 80 °C for 3-5 hours;

[0167] b) Installation of alkyne functionality on 24 in the presence of base and acetylene 33 in polar solvent at -78 °C for 2 hours;

[0168] c) Annulation strategy of alkynyl secondary alcohol 27 with 1,3 -diketoester 34, hydrazine salts and hydroxylamine catalyzed by Lewis acids in the presence of base in polar solvents at 0 - 80 °C for 2-12 hours;

[0169] In yet another embodiment of the present invention, polar solvents are selected from EtOH, H2O, THF, CH3CN and DCM.PT / 2026 / 10546

[0170] In yet another embodiment of the present invention, bases are selected from Na2COs, K2CO3, TBAF, / / -BuLi and KOThi.

[0171] In yet another embodiment of the present invention, lewis acids are selected from BF3.ET2O and / ?TSA.

[0172] In yet another embodiment of the present invention, boronic acid derivatives are selected from aryl boronic acids with substitutions R2is hydrogen, methoxy, ethyl and bromo.

[0173] In yet another embodiment of the present invention, alkyne is selected from substituted acetylene with substitutions, R3is aromatic and aliphatic.

[0174] In still another embodiment of the present invention, the heteroaryl substituted at the ortho position of biaryl derivatives prepared are tested for efficiency towards PD-1 and PD-L1 inhibition properties.

[0175] EXAMPLES

[0176] The present invention will be more specifically explained by following examples. However, the scope of the present invention is not limited to the scope of the examples stated below.

[0177] A. For synthesis of isoxazole substituted biaryls:

[0178] Step-1: Synthesis of [l,l'-biphenyl]-3-carbaldehyde:

[0179] To a stirred solution of 3 -bromo-benzaldehyde (1 equiv.) in mixture of solvents (Toluene: H2O: EtOH::2:2:l) was added phenyl boronic acid (1.3 equiv.), tetrakis (triphenylphospine)palladium (5 mol%), Na2CC>3 (3 equiv.) and stirred for 3 h at an oil bath temperature of 80 °C. After completion of reaction, the mixture was filtered over a pad of celite and the filtrate was extracted with EtOAc and H2O. Then organic extract was dried over anhydrous sodium sulfate, concentrated and purified with silica gel column chromatography to compound 4'-methoxy-[l,l'-biphenyl]-2-carbaldehyde

[0180] Step-2: Synthesis of [l,l'-biphenyl]-3-carbaldehyde oxime:

[0181] To a stirred solution of [l,l'-biphenyl]-3-carbaldehyde (1 equiv.) in a mixture of solvents (EtOH:H2O::5:l) was added hydroxylamine hydrochloride (1.2 equiv.), Na2CC>3 (1.2 equiv.) and stirred for 2 h. After completion of reaction, the mixture was extracted with EtOAc and H2O, organic extract was dried over anhydrous sodium sulfate, concentrated and purified with silica gel column chromatography, to afford [l,l'-biphenyl]-3-carbaldehyde oxime.PT / 2026 / 10546

[0182] Step-3: Synthesis of 4-(3-([l,l'-biphenyl]-3-yl)isoxazol-5-yl)benzonitrile:

[0183] To a stirred solution of [l,l'-biphenyl]-3-carbaldehyde oxime (1 equiv.) in DCM was added A-chlorosuccinamide (NCS) (1.2 equiv.), and left it to stir for 2 h. After that triethylamine (1.5 equiv.), and 4-ethynylbenzonitrile (0.6 equiv.) was added and stirred for 12 h. After completion of reaction, the mixture was extracted with DCM and H2O, and organic extract was dried over anhydrous sodium sulfate, concentrated and purified with silica gel column chromatography, to afford 4-(3-([l,l'-biphenyl]-3-yl)isoxazol-5-yl)benzonitrile.

[0184] B. For synthesis of pyrazole substituted biaryls:

[0185] Step-1: Synthesis of [l,l'-biphenyl]-3-carbaldehyde:

[0186] To a stirred solution of 3 -bromo-benzaldehyde (1 equiv.) in mixture of solvents (Toluene:H2O:EtOH::2:2:l) was added phenyl boronic acid (1.3 equiv.), tetrakis(triphenylphospine)palladium (5 mol%), Na2COs (3 equiv.) and stirred for 3 h at an oil bath temperature of 80 °C. After completion of reaction, the mixture was filtered over a pad of celite and the filtrate was extracted with EtOAc and H2O. Then organic extract was dried over anhydrous sodium sulfate, concentrated and purified with silica gel column chromatography to compound 4'-methoxy-[l,l'-biphenyl]-2-carbaldehyde.

[0187] Step-2: Synthesis of 4-(3-([l,l'-biphenyl]-3-yl)-3-hydroxyprop-l-yn-l-yl)benzonitrile: To a stirred solution of 4-ethynylbenzonitrile (1.2 equiv.), in THF at -78° c was added n-BuLi (2.5 M, 1.5 equiv.), and stirred for 30 min then slowly added [l,l'-biphenyl]-3-carbaldehyde (1 equiv.), and stirred for 2 h. After completion of reaction, the mixture was quenched with saturated solution of aq. NH4CI and extracted with EtOAc (3 x 20 mL) and H2O. Then organic extract dried over anhydrous sodium sulfate, concentrated and purified with silica gel column chromatography which gives compound 4-(3-([ 1 , 1 '-biphenyl] -3 -yl)-3 -hydroxyprop- 1-yn- 1 -yl)benzonitrile.

[0188] Step-3: Synthesis of 4-(3-([l,l'-biphenyl]-3-yl)-3-oxoprop-l-yn-l-yl)benzonitrile:

[0189] To a stirred solution of 4-(3-([l,l'-biphenyl]-3-yl)-3-hydroxyprop-l-yn-l-yl)benzonitrile (1 equiv.), in DCM was added DMP (1.5 equiv.), and stirred for 2 h. After completion of reaction, the mixture was quenched with NaHCOs and extracted with DCM (3 x 20 mL) and H2O. Then organic extract was dried over anhydrous sodium sulfate, concentrated and purified with silica gel column chromatography to afford 4-(3-([l, l'-biphenyl]-3-yl)-3-oxoprop- 1 -yn- 1 -yl)benzonitrile.PT / 2026 / 10546

[0190] Step-4: Synthesis of (Z)-7V-(l-([l,r-biphenyl]-3-yl)-3-(4-cyanophenyl)prop-2-yn-l-ylidene)-4-methylbenzenesulfonohydrazide:

[0191] To a stirred solution of 4-(3-([l,l'-biphenyl]-3-yl)-3-oxoprop-l-yn-l-yl)benzonitrile (1 equiv.), in methanol was added TsNHNFL (1. 5 equiv.), and stirred for 3 h. After completion of reaction the reaction mixture was concentrated and extracted with EtOAc and H2O. Then organic extract was dried over anhydrous sodium sulfate, concentrated and purified with silica gel column chromatography which gives (Z)-A-(l-([l,l'-biphenyl]-3-yl)-3-(4-cyanophenyl)prop-2-yn-l-ylidene)-4-methylbenzenesulfonohydrazide.

[0192] Step-5: Synthesis of 4-(3-([l,l'-biphenyl]-3-yl)-l-(p-tolyl)-lH-pyrazol-5-yl)benzonitrile:

[0193] To a stirred solution of (Z)-A-(l-([l,l'-biphenyl]-3-yl)-3-(4-cyanophenyl)prop-2-yn-l-ylidene)-4-methylbenzenesulfonohydrazide (1 equiv.), in DMF was added triethyl silane (1.2 equiv.), copper triflate (10 mol%), potassium fluoride (0.6 equiv.), and was allowed to stir for 15 h. After completion of reaction (monitored by TLC), the crude product was extracted with EtOAc (3 x20 mL) and H2O. Then organic extract dried over anhydrous sodium sulfate, concentrated and purified with silica gel column chromatography which affords 4-(3-([l,l'-biphenyl]-3-yl)-l -( / 2-tolyl)-l / / -pyrazol-5-yl)benzonitrile.

[0194] Step-6: Synthesis of 4-(3-([l,l'-biphenyl]-3-yl)-lH-pyrazol-5-yl)benzonitrile:

[0195] 4-(3-([l,l'-biphenyl]-3-yl)-l-( / ?-tolyl)-lH-pyrazol-5-yl)benzonitrile (1 equiv.), was dissolved in 5 mL of DMSO and was allowed to stir for 12 h. After completion of reaction, water (5 mL) was added to the reaction and the crude product was extracted with EtOAc and H2O. Then the organic extract was dried over anhydrous sodium sulfate, concentrated and purified with silica gel column chromatography to afford 4-(3-([l,l'-biphenyl]-3-yl)-lJ / -pyrazol-5-yl)benzonitrile.

[0196] C. For synthesis of 2-furan substituted biaryls:

[0197] Step-1: Synthesis of 4'-methoxy-[l,l'-biphenyl]-2-carbaldehyde:

[0198] To a stirred solution of 2-bromo-benzaldehyde (1 equiv.) in mixture of solvents (Toluene:H2O:EtOH::2:2:l) was added phenyl boronic acid (1.3 equiv.), tetrakis(triphenylphospine)palladium (5 mol%), Na2CO3 (3 equiv.) and stirred for 3 h at an oil bath temperature of 80 °C. After completion of reaction, the mixture was filtered over a pad of celite and the filtrate was extracted with EtOAc and H2O. Then organic extract was dried over anhydrous sodium sulfate, concentrated and purified with silica gel column chromatography to compound 4'-methoxy-[l,l'-biphenyl]-2-carbaldehyde (xx%)PT / 2026 / 10546

[0199] Step-2: Synthesis of l-(4'-methoxy-[l,l'-biphenyl]-2-yl)-3-phenylprop-2-yn-l-ol:

[0200] To a stirred solution of phenyl acetylene (1.2 equiv.), in THF at -78° c was added n-BuLi (2.5 M, 1.3 equiv.), and stirred for 30 min and then was slowly added 4'-methoxy-[l,l'-biphenyl]-2-carbaldehyde (1 equiv.), and stirred for 2 h. After completion of reaction, the reaction mixture was quenched with saturated solution of NH4CI and extracted with EtOAc and H2O. Then organic extract was dried over anhydrous sodium sulfate, concentrated and purified with silica gel column chromatography which gives compound l-(4'-methoxy-[l,l'-biphenyl] -2-yl)-3 -phenylprop-2-yn- 1 -ol .

[0201] Step-3: Synthesis of Ethyl 5-benzyl-4-(4'-methoxy-[l,l'-biphenyl]-2-yl)-2-methylfuran-3-carboxylate:

[0202] l-(4'-methoxy-[l,l'-biphenyl]-2-yl)-3-phenylprop-2-yn-l-ol (1 equiv.) and ethyl acetoacetate (1.2 equiv.) were dissolved in 2 mL of acetonitrile followed by addition of BF3 Et2O (5 mol%) and the reaction mixture was stirred at room temperature, until the starting material disappeared, as monitored by TLC. Then K2CO3 (0.93 mmol, 3 equiv.) was then added and allowed to stir at an oil bath temperature of 80 °C until complete consumption of starting material (as indicated by TLC). Upon completion, 10 mL of water was added, and the reaction mixture was extracted thrice with 10 mL of ethyl acetate. The ethyl acetate was evaporated under reduced pressure, and the crude product was purified by column chromatography on silica gel, yielding 82% of the product, ethyl 5 -benzyl -4-(4'-methoxy-[1,1 '-biphenyl]-2-yl)-2-methylfuran-3 -carboxylate.

[0203] D. For synthesis of 2-pyrazole substituted biaryls:

[0204] After following Step 1 and Step 2, 1 -(4'-methoxy-[ 1,1 '-biphenyl] -2-yl)-3-phenylprop-2-yn-l-ol (1 equiv.) and tosyl hydrazine (1 equiv.) were dissolved in 2 mL of acetonitrile and to it, BF3 Et2O (5 mol%) was added, and the reaction mixture was stirred at room temperature, until the starting material consumption. Then, KO’Bu (3 equiv.) was then added at room temperature and was allowed to stir at an oil bath temperature of 80 °C until the starting material was completely consumed (as indicated by TLC). Upon completion, 10 mL of water was added, and the mixture was extracted three times with 10 mL of ethyl acetate. The ethyl acetate was evaporated under reduced pressure, and the crude product was purified by column chromatography on silica gel, yielding 3-(4'-methoxy-[l,l'-biphenyl]-2-yl)-5-phenyl-IH-pyrazole in 70% yield.

[0205] E. For synthesis of 2-isoxazole substituted biaryls:PT / 2026 / 10546

[0206] After following Step 1 and Step 2, 1 -(4'-methoxy-[ 1,1 '-biphenyl] -2-yl)-3-phenylprop-2-yn-l-ol la (1 equiv.) and tosyl hydroxylamine (1 equiv.) were dissolved in 5 mL of dichloromethane at room temperature and / / ra- toluene sulfonic acid monohydrate ( TSA.H2O) (5 mol%) was added, and allowed to stir until the starting material is fully consumed (monitored by TLC). After the reaction was complete, 10 mL of water was added and extracted with DCM (1 x 10 mL). Organic layer was collected and dried over sodium sulfate. The organic layer collected was cooled to 0 °C and 1.0 M TBAF in THF solution (0.96 mmol, 3 equiv.) was added to it under nitrogen atmosphere. The reaction mixture was then allowed to stir at room temperature until the starting material was completely consumed, as indicated by TLC. After the reaction was complete, 10 mL of saturated ammonium chloride was added, and the mixture was extracted three times with 10 mL of dichloromethane. Dichloromethane was evaporated under reduced pressure, and the crude product was purified by column chromatography on silica gel, yielding 80% of product 3-(4'-methoxy-[l,l'-biphenyl]-2-yl)-5-phenylisoxazole.

[0207] F. For synthesis of isoxazole substituted functional groups:

[0208] Step-1: Synthesis of (E)-3-nitrobenzaldehyde oxime:

[0209] To a stirred solution of 3-nitro benzaldehyde (1 equiv.) in a mixture of solvents (EtOH:H2O::5:l) was added hydroxylamine hydrochloride (1.2 equiv.), Na2COs (1.2 equiv.) and stirred for 2 h. After completion of reaction, the mixture was extracted with EtOAc and H2O, organic extract was dried over anhydrous sodium sulfate, concentrated and purified with silica gel column chromatography, to afford (E)-3 -nitrobenzaldehyde oxime.

[0210] Step-2: Synthesis of4-(3-(3-nitrophenyl)isoxazol-5-yl)benzonitrile:

[0211] To a stirred solution of (E)-3 -nitrobenzaldehyde oxime (1 equiv.) in DCM was added N-chlorosuccinamide (NCS) (1.2 equiv.), and left it to stir for 2 h. After that triethylamine (1.5 equiv.), and 4-ethynylbenzonitrile (0.6 equiv.) was added and stirred for 12 h. After completion of reaction, the mixture was extracted with DCM and H2O, and organic extract was dried over anhydrous sodium sulfate, concentrated and purified with silica gel column chromatography, to afford 4-(3-(3-nitrophenyl)isoxazol-5-yl)benzonitrile.

[0212] G. Synthesis of 4-(3-([l,l'-biphenyl]-3-yl)isoxazol-5-yl)benzoic acid:

[0213] To a stirred solution of 4-(3-([l,l'-biphenyl]-3-yl)isoxazol-5-yl)benzonitrile (1 equiv.) in a mixture of solvents (EtOELELO::!:!) was add KOH (4 equiv.) and stirred for 12 h. After completion of reaction, the mixture was acidified with 2N HC1 (PH=1) then extracted withPT / 2026 / 10546

[0214] EtOAc and H2O, organic extract was dried over anhydrous sodium sulfate, concentrated and purified with silica gel column chromatography, to afford 4-(3-([l,l'-biphenyl]-3-yl)isoxazol-5-yl)benzoic acid.

[0215] H. Synthesis of methyl (4-(3-([l,l'-biphenyl]-3-yl)isoxazol-5-yl)benzoyl)serinate:

[0216] To a stirred solution of 4-(3-([l,l'-biphenyl]-3-yl)isoxazol-5-yl)benzoic acid (1 equiv.) in DMF was added EDC.HC1 (1.5 equiv.), HOBT (1.5 equiv.), DIPEA (3 equiv.) and left it to stirred for 1 h. After that serine was added. The mixture was stirred for 12 h. After completion of reaction, the mixture was extracted with EtoAc and H2O, and organic extract was dried over anhydrous sodium sulfate, concentrated and purified with silica gel column chromatography, to afford methyl (4-(3-([l,l'-biphenyl]-3-yl)isoxazol-5-yl)benzoyl)serinate.

[0217] I. Synthesis of 4-(3-([l,l'-biphenyl]-3-yl)-l,2,4-oxadiazol-5-yl)benzonitrile:

[0218] Step-1: Synthesis of [l,l'-biphenyl]-3-carbonitrile:

[0219] To a stirred solution of 3-bromo benzonitrile (1 equiv.) in mixture of solvents (Toluene:H2O:EtOH::2:2:l) was added phenyl boronic acid (1.3 equiv.), tetrakis(triphenylphospine)palladium (5 mol%), Na2CC>3 (3 equiv.) and stirred for 3 h at an oil bath temperature of 80 °C. After completion of reaction, the mixture was filtered over a pad of celite and the filtrate was extracted with EtOAc and H2O. Then organic extract was dried over anhydrous sodium sulfate, concentrated and purified with silica gel column chromatography to compound [l,l'-biphenyl]-3-carbonitrile.

[0220] Step-2: Synthesis of (Z)-N'-hydroxy-[l,l'-biphenyl]-3-carboximidamide:

[0221] To a stirred solution of [l,l'-biphenyl]-3-carbonitrile (1 equiv.) in a Methanol was added hydroxylamine hydrochloride (1.2 equiv.), Et3N (2 equiv.) at an oil bath temperature 60 °C and stirred for 2 h. After completion of reaction, the mixture was concentrated and extracted with EtOAc and H2O, organic extract was dried over anhydrous sodium sulfate, concentrated and purified with silica gel column chromatography, to afford (Z)-N'-hydroxy-[l,l'-biphenyl]-3-carboximidamide.

[0222] Step-3 Synthesis of 4-(3-([l,l'-biphenyl]-3-yl)-l,2,4-oxadiazol-5-yl)benzonitrile:

[0223] To a stirred solution of (Z)-N'-hydroxy-[l,l'-biphenyl]-3-carboximidamide (1 equiv.) in DMSO was added 4-cyanobenzoic acid (1.1 equiv.), CDI (1.2 equiv.) and stirred for 12 h. After that was added NaOH (1.2 equiv.) and stirred for 2 h then the reaction mixture was acidified with 2N HC1 (PH=1) then extracted with EtOAc and H2O, organic extract was driedPT / 2026 / 10546

[0224] over anhydrous sodium sulfate, concentrated and purified with silica gel column chromatography, to afford 4-(3-([l,r-biphenyl]-3-yl)-l,2,4-oxadiazol-5-yl)benzonitrile.

[0225] J. Synthesis of 3-([l,l'-biphenyl]-3-yl)-5-(4-(l-methyl-lH-tetrazol-5-yl)phenyl)isoxazole:

[0226] To a stirred solution of 5-(4-(lH-tetrazol-5-yl)phenyl)-3-([l,l'-biphenyl]-3-yl)isoxazole (1 equiv.) in DMF was added NaH (1.2 equiv.), Mel (1.5 equiv.) and stirred for 2 h. After completion of reaction mixture was extracted with with EtOAc and H2O, organic extract was dried over anhydrous sodium sulfate, concentrated and purified with silica gel column chromatography,toafford3-([l,l'-biphenyl]-3-yl)-5-(4-(l-methyl-lH-tetrazol-5-yl)phenyl)isoxazole.

[0227] K. Synthesis of 4-(3-([l,l'-biphenyl]-3-yl)isoxazol-5-yl)benzamide:

[0228] To a stirred solution of 4-(3-([l,l'-biphenyl]-3-yl)isoxazol-5-yl)benzoic acid (1 equiv. ) in DMF was added NH4CI (3 equiv.), HATU (1.5 equiv.), DIPEA (3 equiv.). and stirred for 2 h. After completion of reaction mixture was extracted with with EtOAc and H2O, organic extract was dried over anhydrous sodium sulfate, concentrated and purified with silica gel column chromatography, to afford 4-(3-([l,l'-biphenyl]-3-yl)isoxazol-5-yl)benzamide.

[0229] L. Synthesis of 4-(3-([l,l'-biphenyl]-3-yl)isoxazol-5-yl)-N-hydroxybenzamide:

[0230] To a stirred solution of methyl 4-(3-([l,l'-biphenyl]-3-yl)isoxazol-5-yl)benzoate (1 equiv.) in 1,4-dioxane was added NaOH (4.8 equiv.), hydroxylamine hydrochloride (1.2 equiv.) and stirred for 3 h .then the reaction mixture was acidified with 2N HC1 (PH=1) then extracted with EtOAc and H2O, organic extract was dried over anhydrous sodium sulfate, concentrated and purified with silica gel column chromatography, to afford 4-(3-([l,l'-biphenyl]-3-y 1 )i sox azol - 5 -yl)-N -hydroxyb enzami de .

[0231] Example 1: 4-(3-([l,l'-Biphenyl]-3-yl)isoxazol-5-yl)benzonitrile (1):XH NMR (500 MHz, CDCh) 38.09 (s, 1H), 7.97 (d, J= 8.2 Hz, 2H), 7.84 (d, J= 7.6 Hz, 1H), 7.80 (d, J= 8.2 Hz, 1H), 7.72 (d, J= 7.6 Hz, 1H), 7.66 (d, J= 7.4 Hz, 2H), 7.58 (d, J= 7.7 Hz, 1H), 7.48 (t, J = 7.5 Hz, 2H), 7.41 (d, J= 13 Hz, 1H). Mass (M+H)+: 322

[0232] Example 3: 4-(3-(4'-(Trifluoromethoxy)-[l,l'-biphenyl]-3-yl)isoxazol-5-yl)benzonitrile (3): 'H NMR (400 MHz, DMSO) 3 8.15 (s, 1H), 8.07 (s, 2H), 7.91 (d, J= 7.7 Hz, 2H), 7.78 (d, J = 7.8 Hz, 2H), 7.69 (d, J = 7.2 Hz, 1H), 7.60 (d, J = 7.9 Hz, 1H), 7.51 (d, J = 7.1 Hz, 3H), 7.42 (t, J= 7.2 Hz, 1H); Mass (M+H)+: 306PT / 2026 / 10546

[0233] Example 4: 3'-(5-(4-Cyanophenyl)isoxazol-3-yl)-3-fluoro-[l,l'-biphenyl]-4-carbonitrile (4): 'H NMR (500 MHz, CDCh) 3 8.09 (s, 1H), 7.97 (d, J= 8.2 Hz, 2H), 7.82 (dd, J= 19.6, 7.9 Hz, 3H), 7.75 - 7.54 (m, 4H), 7.48 (t, J = 7.5 Hz, 2H), 7.41 (d, J = 7.3 Hz, 1H), 7.02 (s, 1H); Mass (M+H)+: 365

[0234] Example 5: (4-(3-([l,l'-Biphenyl]-3-yl)isoxazol-5-yl)phenyl)methanol (5): 1H NMR (400 MHz, DMSO) 3 8.19 (t, J = 1.5 Hz, 1H), 7.98 - 7.86 (m, 3H), 7.86 - 7.71 (m, 4H), 7.65 (t, J = 7.7 Hz, 1H), 7.58 - 7.50 (m, 4H), 7.44 (d, J = 7.3 Hz, 1H), 5.37 (s, 1H), 4.59 (s, 2H); Mass (M+H)+: 327

[0235] Example 6: 4-(4-(3-([l,l'-Biphenyl]-3-yl)isoxazol-5-yl)benzyl)morpholine (6): 'l l NMR (400 MHz, DMSO) 3 8.19 (s, 1H), 7.94 (d, J= 7.8 Hz, 1H), 7.90 (d, J= 8.2 Hz, 1H), 7.85 -7.81 (m, 1H), 7.80 - 7.77 (m, 1H), 7.75 (s, 1H), 7.65 (t, J = 7.7 Hz, 1H), 7.52 (d, J= 7.0 Hz, 1H), 7.44 (d, J= 7.4 Hz, 1H), 3.63 - 3.57 (m, 1H), 3.55 (s, 1H), 2.51 - 2.49 (m, J= 3.5, 1.8 Hz, 1H); Mass (M+H)+: 396

[0236] Example 7: 5-(4-(2H-Tetrazol-5-yl)phenyl)-3-([l,l'-biphenyl]-3-yl)isoxazole (7): II MIK (400 MHz, DMSO) 3 8.26 (d, J = 8.5 Hz, 2H), 8.21 (s, 1H), 8.16 (d, J = 8.5 Hz, 2H), 7.98 -7.91 (m, 2H), 7.87 - 7.82 (m, 1H), 7.81 - 7.76 (m, 2H), 7.67 (t, J= 7.7 Hz, 1H), 7.54 (dd, J= 10.4, 4.8 Hz, 2H), 7.47 - 7.41 (m, 1H); Mass (M+H)+: 365

[0237] Example 8: 4-(3-([l,l'-Biphenyl]-3-yl)-lH-pyrazol-5-yl)benzonitrile (8): 'l l NMR (400 MHz, DMSO) 3 13.71 (s, 1H), 8.15 (s, 1H), 8.07 (s, 1H), 8.01 (d, J= 10.3 Hz, 1H), 7.91 (d, J = 7.7 Hz, 2H), 7.78 (d, J= 7.8 Hz, 3H), 7.69 (d, J= 7.2 Hz, 1H), 7.60 (d, J= 7.9 Hz, 1H), 7.56 - 7.47 (m, 3H), 7.42 (t, J= 7.2 Hz, 1H) ; Mass (M+H)+: 321

[0238] Example 9: 4-(3-([l,l'-Biphenyl]-3-yl)-l-methyl-lH-pyrazol-5-yl)benzonitrile (9): 'l l NMR (400 MHz, DMSO) 3 8.04 (d, J= 8.5 Hz, 2H), 7.88 (dd, J= 7.5, 5.1 Hz, 3H), 7.80 -7.73 (m, 3H), 7.62 (dd, J= 5.3, 4.3 Hz, 2H), 7.51 (t, J= 7.5 Hz, 2H), 7.42 (d, J= 7.3 Hz, 1H), 7.19 (s, 1H); Mass (M+H)+: 335

[0239] Example 10: 3-([l,l'-Biphenyl]-2-yl)-5-propylisoxazole (10): 'l l NMR (400 MHz, CDCh) 3 7.83 - 7.69 (m, 1H), 7.52 - 7.40 (m, 3H), 7.32 (dd, J= 4.1, 2.4 Hz, 3H), 7.26 (dd, J= 4.5, 2.6 Hz, 2H), 5.22 (s, 1H),2.57 (t, J= 7.4 Hz, 2H), 1.58 (dd, J= 10.3, 4.3 Hz, 2H), 0.85 (t, J= 7.4 Hz, 3H).; Mass (M+H)+: 264.1383.P 172026 / 10546

[0240] Example 11: 3-(4'-Methoxy-[l,l'-biphenyl]-2-yl)-5-phenylisoxazole (11): 'l l NMR (400 MHz, CDCh) 37.80 (dd, J= 7.8, 1.5 Hz, 1H), 7.64 - 7.60 (m, 2H), 7.49 (dd, J= 7.0, 1.9 Hz, 1H), 7.46 - 7.38 (m, 5H), 7.25 - 7.21 (m, 2H), 6.90 - 6.85 (m, 2H), 5.85 (s, 1H), 3.81 (s, 3H); Mass (M+H)+: 328.1329.

[0241] Example 12: 3-(4'-Bromo-[l,l'-biphenyl]-2-yl)-5-phenylisoxazole (12): 'l l NMR (400 MHz, CDCh) 37.79 (dd, J= 7.5, 1.3 Hz, 1H), 7.64 (dd, J= 7.5, 2.1 Hz, 2H), 7.55 - 7.40 (m, 8H), 7.19 (d, J= 8.4 Hz, 2H), 5.90 (s, 1H); Mass (M+H)+: 376.0325.

[0242] Example 13: 3-(4'-Methoxy-[l,l'-biphenyl]-2-yl)-5-phenyl-lH-pyrazole (13): 'l l NMR (400 MHz, CDCh) 37.72 (d, J= 7.2 Hz, 2H), 7.66 (d, J = 2.8 Hz, 1H), 7.40 (d, J= 6.6 Hz, 5H), 7.31 (d, J= 7.3 Hz, 1H),7.22 (d, J= 8.7 Hz, 2H), 6.90 (d, J = 8.7 Hz, 2H), 6.54 (s, 1H), 3.82 (s, 3H).; Mass (M+H)+: 327.1490.

[0243] Example 14: 3-(4'-Ethyl-[l,l'-biphenyl]-2-yl)-5-phenyl-lH-pyrazole (14): 'l l NMR (400 MHz, CDCh) 3 7.76 - 7.65 (m, 3H), 7.40 (ddd, J= 15.0, 7.2, 3.6 Hz, 5H), 7.31 (d, J= 7.3 Hz, 1H), 7.22 (s, 4H), 6.56 (d, J = 6.6 Hz, 1H), 2.69 (q, J = 7.6 Hz, 2H), 1.28 (t, J= 7.6 Hz, 3H); Mass (M+H)+: 325.1699.

[0244] Example 15: 3-(4'-Bromo-[l,l'-biphenyl]-2-yl)-5-phenyl-lH-pyrazole (15):XH NMR (400 MHz, CDCh) 37.66 (dd, J= 8.1, 3.2 Hz, 3H), 7.50 - 7.42 (m, 4H), 7.36 (ddd, J= 16.7, 14.2, 7.4 Hz, 4H), 7.15 (d, J= 8.2 Hz, 2H), 6.41 (s, 1H).; Mass (M+H)+: 375.0486.

[0245] Example 16: 3-([ l,l'-Biphenyl]-2-yl)-5-propyl-lH-pyrazole (16): 'l l NMR (400 MHz, CDC13) 3 7.70 - 7.59 (m, 1H), 7.35 (dddd, J= 9.9, 8.8, 5.1, 3.1 Hz, 6H), 7.25 (dd, J= 5.2, 2.5 Hz, 2H), 5.73 (s, 1H), 2.50 (t, J= 7.5 Hz, 2H), 1.56 (dd, J= 14.9, 7.4 Hz, 2H), 0.88 (t, J= 7.4 Hz, 3 H) ; Mass (M+H)+: 263.1540.

[0246] Example 17: Ethyl 5-benzyl-4-(4'-methoxy-[l,l'-biphenyl]-2-yl)-2-methylfuran-3-carboxylate (17): 'H NMR (500 MHz, CDCh): 37.31 - 7.27 (m, 2H), 7.24 - 7.21 (m, 1H), 7.17 (d, J = 7.4 Hz, 1H), 7.13 - 7.05 (m, 3H), 7.03 - 6.99 (m, 2H), 6.80 (d, J = 6.9 Hz, 2H),6.70 -6.66 (m, 2H), 3.95 (dd, J = 26.0, 7.1 Hz, 2H), 3.72 (s, 3H), 3.52 - 3.40 (m, 2H), 2.41 (s, 3H), 0.92 (t, J = 7.1 Hz, 3H).; Mass (M+H)+: 427.1895

[0247] Example 18: Ethyl 5-benzyl-4-(4'-ethyl-[l,l'-biphenyl]-2-yl)-2-methylfuran-3-carboxylate (18): 'H NMR (500 MHz, CDCh) 3 7.38 (dt, J= 4.1, 2.7 Hz, 2H), 7.32 - 7.29PT / 2026 / 10546

[0248] (m, 1H), 7.25 - 7.24 (m, 1H), 7.15 (ddd, J= 6.2, 5.1, 2.1 Hz, 3H), 7.10 - 7.08 (m, 2H), 7.05 (d, J = 8.3 Hz, 2H),6.84 (dd, J = 7.8, 1.6 Hz, 2H), 4.02 (dt, J = 10.8, 3.6 Hz, 2H), 3.51 (dd, J = 48.9, 16.0 Hz, 2H), 2.63 (d, J = 7.6 Hz, 2H), 2.47 (s, 3H), 1.23 (d, J = 7.6 Hz, 3H), 0.98 (t, J= 7.1 Hz, 3H).; Mass (M+H)+: 425.2103.

[0249] Example 19: Ethyl 5-benzyl-4-(4'-bromo-[l,l'-biphenyl]-2-yl)-2-methylfuran-3-carboxylate (19): 'H NMR (300 MHz, CDCh) 37.26 (dd, J= 8.9, 4.6 Hz, 6H), 7.10 (s, 3H), 6.94 (d, J= 8.5 Hz, 2H), 6.77 (dd, J= 7.6, 1.8 Hz, 2H), 4.02 - 3.90 (m, 2H), 3.46 (d, J= 12.9 Hz, 2H), 2.42 (s, 3H), 0.94 (t, J= 7.1 Hz, 3H).; Mass (M+H)+: 475.0895.

[0250] Example 20: 5-Benzyl-4-(4'-methoxy-[l,l'-biphenyl]-2-yl)-2-methylfuran-3-carboxylic acid (20): 'H NMR (500 MHz, CDCh) 37.35 (s, 2H), 7.26 (d, J = 2.4 Hz, 2H), 7.17 (d, J = 7.4 Hz, 3H), 7.07 (d, J= 8.5 Hz, 2H), 6.86 (d, J= 6.7 Hz, 2H), 6.73 (d, J= 8.6 Hz, 2H), 3.77 (s, 3H), 3.55 (d, J= 16.0 Hz, 1H), 3.47 (s, 1H), 2.46 (s, 3H);Mass (M+H)+: 399.1587

[0251] Example 21: l-(5-Benzyl-4-(4'-methoxy-[l,l'-biphenyl]-2-yl)-2-methylfuran-3-yl)ethan-1-one (21): 'H NMR (300 MHz, CDCh) 37.45 - 7.41 (m, 2H), 7.36 (s, 1H), 7.30 - 7.28 (m, 1H), 7.24 - 7.16 (m, 3H), 7.08 - 7.03 (m, 2H), 6.97 - 6.92 (m, 2H),6.78 - 6.73 (m, 2H), 3.79 (s, 3H), 3.59 (s, 2H), 2.46 (s, 3H), 1.90 (s, 3H).; Mass (M+H)+: 397.1793.

[0252] Example 24: Methyl (4-(3-([l,l'-biphenyl]-3-yl)isoxazol-5-yl)benzoyl)serinate (35) : 'l l NMR (300 MHz, DMSO) 3 8.83 (d, J= 7.4 Hz, 1H), 8.24 (s, 1H), 8.12 (q, J= 8.6 Hz, 4H), 8.05 - 7.94 (m, 1H), 7.86 (dd, J= 17.6, 7.6 Hz, 2H), 7.72 (d, J= 7.7 Hz, 1H), 7.58 (t, J= 7.4 Hz, 2H), 7.49 (d, J = 13 Hz, 1H), 5.16 (t, J= 6.1 Hz, 1H), 4.62 (dd, J= 12.6, 5.4 Hz, 1H), 3.87 (t, J= 5.7 Hz, 2H), 3.71 (s, 3H). Mass (M+H)+: 442.1529.

[0253] Example 25: 4-(3-([l,l'-biphenyl]-3-yl)isoxazol-5-yl)benzoic acid (36): 'l l NMR (300 MHz, DMSO) 3 8.20 (s, 1H), 8.14 (d, J= 5.3 Hz, 1H), 8.05 (q, J = 8.6 Hz, 2H), 7.95 (d, J = 8.0 Hz, 1H), 7.89 (s, 1H), 7.85 (d, J= 8.3 Hz, 1H), 7.82 - 7.77 (m, 2H), 7.67 (t, J = 7.7 Hz, 1H), 7.54 (dd, J= 10.1, 4.7 Hz, 2H), 7.44 (dd, J= 8.4, 6.2 Hz, 1H). Mass (M+H)+: 341.1052.

[0254] Example 26: 4-(3-([l,l'-biphenyl]-3-yl)-l,2,4-oxadiazol-5-yl)benzonitrile (37): 'l l NMR (500 MHz, CDCh) 38.37 (d, J= 8.4 Hz, 2H), 8.31 (s, 1H), 8.13 (d, J = 8.4 Hz, 3H), 8.09 (d, J = 7.7 Hz, 1H), 7.92 (d, J = 7.9 Hz, 1H), 7.77 - 7.68 (m, 3H), 7.53 (t, J= 7.6 Hz, 2H), 7.44 (t, J= 13 Hz, 1H). Mass (M+H)+: 323.1059.PT / 2026 / 10546

[0255] Example 27: 5-(4-(lH-tetrazol-5-yl)phenyl)-3-([l,l'-biphenyl]-3-yl)-l,2,4-oxadiazole (38) : 'H NMR (400 MHz, DMSO) 3 8.26 (d, J= 8.5 Hz, 1H), 8.21 (t, J= 1.6 Hz, 1H), 8.16 (d, J = 8.5 Hz, 2H), 8.00 - 7.90 (m, 2H), 7.85 (d, J= 8.2 Hz, 1H), 7.82 - 7.76 (m, 2H), 7.67 (t, J = 7.7 Hz, 1H), 7.54 (t, J= 7.6 Hz, 2H), 7.44 (t, J= 7.3 Hz, 1H). Mass (M+H)+: 366.1229.

[0256] Example 28: Methyl l-(4-(3-([l,l'-biphenyl]-3-yl)isoxazol-5-yl)benzoyl)piperidine-2-carboxylate (39) : 'H NMR (400 MHz, DMSO) 3 8.23 (s, 1H), 8.01 (dd, J= 30.8, 7.1 Hz, 3H), 7.93 - 7.78 (m, 4H), 7.68 (t, J= 7.7 Hz, 1H), 7.65 - 7.53 (m, 4H), 7.50 - 7.43 (m, 1H), 5.33 (s, 1H), 4.49 (s, 1H), 3.75 (d, J= 19.4 Hz, 3H), 3.56 (d, J= 12.4 Hz, 1H), 3.19 (t, J = 11.8 Hz, 1H), 2.29 - 2.03 (m, 1H), 1.74 (d, J= 10.1 Hz, 2H), 1.53 (dd, J = 40.1, 10.4 Hz, 2H), 1.31 (dd, J = 30.2, 17.6 Hz, 1H). Mass (M+H)+: 466.1893.

[0257] Example29 : Methyl l-(4-(3-( [1 , 1 '-biphenyl]-3-yl)isoxazol-5-yl)benzoyl)-4-hydroxy pyrrolidine- 2-carboxylate (40) : 'H NMR (400 MHz, DMSO) 3 8.25 (s, 1H), 8.07 (d, J = 8.2 Hz, 2H), 8.00 (d, J= 7.4 Hz, 1H), 7.95 (s, 1H), 7.89 (d, J= 7.6 Hz, 1H), 7.82 (dd, J = 11.4, 8.0 Hz, 4H), 7.70 (t, J = 7.7 Hz, 1H), 7.57 (t, J = 7.5 Hz, 2H), 7.49 (d, J = 7.3 Hz, 1H), 5.22 (d, J = 2.9 Hz, 1H), 4.65 (t, J= 8.5 Hz, 1H), 4.36 (s, 1H), 3.85 (dd, J = 10.8, 3.4 Hz, 1H), 3.73 (s, 3H), 2.34 - 2.21 (m, 1H), 2.14 - 2.00 (m, 1H), 1.27 (s, 1H). Mass (M+H)+: 468.1685

[0258] Example 30: 4-(3-([l,l'-biphenyl]-3-yl)isoxazol-5-yl)benzamide (41):LHNMR (400 MHz, DMSO) 3 8.23 (s, 1H), 8.17 (s, 1H), 8.11 (d, J = 8.5 Hz, 2H), 8.05 (d, J = 8.5 Hz, 2H), 7.98 (d, J= 7.8 Hz, 1H), 7.92 (s, 1H), 7.87 (d, J= 8.2 Hz, 1H), 7.84 - 7.80 (m, 2H), 7.69 (t, J = 7.7 Hz, 1H), 7.57 (dd, J = 10.2, 4.8 Hz, 3H), 7.47 (t, J = 7.3 Hz, 1H). Mass (M+H)+: 340.1212.

[0259] Example 31: 4-(3-([l,l'-biphenyl]-3-yl)isoxazol-5-yl)-7V-hydroxybenzamide (42): 'l l NMR (500 MHz, DMSO) 3 8.20 (s, 1H), 8.13 (d, J = 8.3 Hz, 2H), 8.06 (d, J = 8.3 Hz, 2H), 7.95 (d, J= 7.7 Hz, 1H), 7.92 (s, 1H), 7.84 (d, J= 7.7 Hz, 1H), 7.78 (d, J= 7.5 Hz, 2H), 7.66 (t, J = 7.7 Hz, 1H), 7.53 (t, J = 7.6 Hz, 2H), 7.44 (t, J = 7.3 Hz, 1H). Mass (M+H)+: 356.1161.

[0260] Example 32: 3-([ l,l'-biphenyl ]-3-yl)-5-(4-(l -methyl- lZ / -tetrazol-5-yl)phenyl)isoxazole (43): 'H NMR (400 MHz, DMSO) 3 8.20 (d, J = 8.6 Hz, 2H), 8.14 (s, 1H), 8.06 (d, J = 8.6 Hz, 2H), 7.89 (d, J= 7.8 Hz, 1H), 7.84 (s, 1H), 7.81 - 7.75 (m, 1H), 7.74 - 7.70 (m, 2H), 7.60 (t, J= 7.7 Hz, 1H), 7.47 (t, J= 7.6 Hz, 2H), 7.37 (t, J = 7.3 Hz, 1H), 4.40 (s, 3H). MassPT / 2026 / 10546

[0261] (M+H)+: 379.1433.

[0262] Biological activity:

[0263] Cloning, Expression, Refolding and purification:

[0264] The full-length coding sequences of human PD-1 and PD-L1 were retrieved from the NCBI website (https: / / www.ncbi.nlm.nih.gov / gene), and primers for the full-length and active domains were designed using Primer3web (http: / / primer3.ut.ee). cDNA from WPMY cells and PD-l-misFIT-4x (Addgene) was used for amplifying the PD-L1 and PD-1 genes. The PCR products and the destination vector (pET28a) were double-digested with Ncol and Xhol restriction enzymes. The PCR products were then ligated into the pET28a vector using T4 DNA ligase and transformed into the DH5a strain. Positive clones were confirmed by sequence-specific PCR, restriction digestion, and DNA sequencing. The pET28a-PD-l and pET28a-PD-Ll plasmids, which contained the full-length and their IgV extracellular domain inserts, were transformed into BL21(DE3) expression strains. Protein expression was induced with 1 mM IPTG for 6 hours at 30°C. The target proteins, which formed inclusion bodies (insoluble fraction), were harvested using a lysis buffer at pH 8 (50 mM Tris, 200 mM NaCl, 2 mM EDTA, 10 mM P-mercaptoethanol, and 6 M urea) and purified using Ni-NTA Sepharose beads. The denatured proteins were refolded by drop-wise dilution of the denatured protein into a refolding buffer (50 mM HEPES, 200 mM NaCl, 1 mM reduced glutathione, 600 mM L-arginine, and 1 mM cysteamine) and concentrated using 10 kDa centrifugal filtration units.

[0265] Thermal Shift Assay / Differential Scanning Fluorometry:

[0266] Small molecules dissolved in DMSO at a final concentration of 10 mM were diluted to 2 mM and dispensed into bioassay plates containing PD-L1 / PD-1 proteins, along with appropriate controls, at a final concentration of 50 pM. As positive controls, reported small-molecule inhibitors such as BMS-1 and BMS-202 were used. For the sample control, protein alone was incubated with DMSO at a maximum dose of 2%. Sypro Orange was diluted 1:50 from a lOOx stock solution and added to the protein-molecule mixtures, which were then incubated at room temperature for 5 minutes. The assay sample volume was adjusted to 40 pL for triplicate measurements in the temperature range of 25°C to 95°C using a real-time thermal cycler (CFX Opus 96, Bio-Rad). Data were normalized and plotted using GraphPad Prism 9.5.1, and the melting points (Tm) were calculated using the CFX Opus 96, Bio-Rad analysis software.PT / 2026 / 10546

[0267] PD-1 / PD-L1 mediated Luciferase activation assay:

[0268] To identify inhibitors of the PD-1 / PD-L1 interaction, two engineered cell lines were used: PD-L1 aAPC / CHO-Kl cells, which act as antigen-presenting cells, and PD-1 Effector cells, which act as T-cells. Prior to assay setup, small molecules were diluted in assay buffer (Gibco RPMI media + 1% FBS). PD-L1 aAPC / CHO-Kl cells (20,000 cells / well) were seeded into a 96-well plate and cultured in a 5% CO2 incubator for 24 hours. PD-1 Effector cells (40,000 cells / well) were then added to the plate and treated with the compounds, along with appropriate controls. Following treatment, the co-culture plates were incubated for 6 hours in the 5% CO2 incubator. After the co-culture period, the plates were allowed to equilibrate at room temperature for 10 minutes before adding Bio-Gio™ Reagent. Luminescence was measured using a plate reader (Infinite M200 Pro, TECAN), and EC50 values were calculated using GraphPad Prism 9.5.1.

[0269] Isothermal Citation Calorimetry (ITC):

[0270] Isothermal Titration Calorimetry (ITC) was used as an orthogonal assay to determine the binding of small molecules to target proteins. In this assay, BMS-1 and BMS-202 compounds purchased from Abeam were used as positive controls. The ITC assay was performed with recombinant PD-L1 using the MicroCai PEAQ-ITC. A 20 pM solution of recombinant PD-L1 protein (300 pL) was injected into the sample cell and 80 pL solution of 200 pM ligand was loaded into the syringe for performing the binding assay. The ligand was titrated against the target protein sample in 2 pL increments every 150 seconds, and the heat released was measured. The reaction data were analyzed using MicroCai PEAQ-ITC Analysis Software vl.41 for determination of kD values for estimating the binding of small molecules to target proteins.

[0271] PD-1 / PD-L1 inhibitor screening ELISA assay:

[0272] Small molecule mediated inhibitory effect of CRR SS-03 was determined by employing competitive ELISA assay kit (PD-1 [Biotinylated]: PD-L1 Inhibitor Screening ELISA Kit, Acrobiosystems). Firstly, 96-well plate was coated with human PD-L1 and incubated at 4°C for overnight. To the same wells, biotinylated PD-1 was added and incubated at room temperature for 20 min. Further treated with CRR-SS-03 in concentration dependent manner and incubated at 37°C for 1 hr, and washed thrice with wash buffer. Later plates were incubated by adding streptavidin bound HRP and washed again. Then TMB was added and TECAN infinite M200PRO used to measure the absorbance at 450nm.PT / 2026 / 10546

[0273] Immune activation-cytokine induction assay:

[0274] To validate the immune activation and T-cell rescue capability of the identified small molecule inhibitors against the PD-1 / PD-L1 interaction, human normal PBMCs were activated using CD3 and CD28 antibodies. Independently, MDA-MB-231 cells (10,000 cells / well) were then seeded in a 96-well plate and incubated in a 5% CO2 incubator for 24 hours. Then, activated PBMCs were added to the MDA-MB-231 cells in the plates, and the mixture was treated with small molecules and appropriate controls. The co-culture was incubated for 48-72 hours in the 5% CO2 incubator. After incubation, media were collected to measure cytokine levels. Cytokines including IL- la, IL-ip, IL-2, and perforin were quantified using Krishgen ELISA kits according to the manufacturer’s instructions, and the results were plotted using GraphPad Prism 9.5.1.

[0275] T cell cytotoxicity assay:

[0276] Small molecule mediated T cell anticancer activity was determined by Flow cytometry analysis and CCk-8 cell viability assay. Briefly, MDAMB-231 (IxlO5) cells were labelled using CFSE (carboxyfluorescein diacetate succinimidyl ester) and seeded into 6 well plate and incubated overnight and on the next day co-cultured with CD-3 / CD-28 activated T cells (5xl05), and treated with BMS-1 (4pM) and CRR-SS-03 (4pM). After 24 hours, cells were harvested and stained with Propidium Iodide (PI), and then analysed by flow cytometry. Similarly, co-cultured cells along with treated small molecules were incubated for 72 hours and cell viability assay was performed using Cell Counting Kit-8 (CCK-8) Medchemexpress, USA).

[0277] Syngeneic mice experiments:

[0278] The mouse triple-negative breast cancer (TNBC) cell line 4T1 (50,000 cells per mouse) was orthotopically injected with 100 pL of DMEM media. Mice were then divided into six groups when the tumor volume reached approximately 100 mm3. Following grouping, small molecules BMS-1 (4 mg / kg), CRR-SS-02 / RJ-6 (4 mg / kg and 8 mg / kg), and CRR-SS-03 (4 mg / kg and 8 mg / kg) were administered intraperitoneally (i.p.) and orally daily for 14 days. Anti PD-L1 (200pg / animal) antibody was administered twice a week through interveinal (IV) injections. Tumor volume was measured every 3 days using the formula LxW*Hx0.52. After 14 days, when the tumor volume in the vehicle control group reached approximately 1000 mm3, blood was collected and the mice were sacrificed. Tumor and spleen weights werePT / 2026 / 10546

[0279] measured and collected for further experiments. Tumor volume and weight data were plotted using GraphPad Prism 9.5.1.

[0280] Tumor single cell dissociation and Flow cytometry:

[0281] TILs population was assessed by analysing the expression levels of CD3, CD4 and CD8. Tumor samples were collected at the time of necropsy and chopped into small pieces by placing it on 60mm dishes. Then the tissue samples were digested using 250pl of trypsin and 20 pl of DNase-I (2000U) at 37°C for 10 min, and further chopped into small pieces. This step was repeated for 3 times. After thorough digestion, cells were strained using 100pm cell strainer. Strained cells were counted and IxlO5cells stained using the antibodies of antimouse CD3 conjugated with APC, PE-conjugated anti-mouse CD4 and anti-mouse CD8 conjugated with PerCP / Cyanine5.5. Antibody-stained cells were incubated on ice for 45min and washed with ice cold PBS by centrifugation at 3000rpm for 5 min. Supernatant was discarded and pellet was dissolved in lOOpl PBS and analysed by Flow cytometry.

[0282] Histopathology and Fluorescent Immunohistochemistry (F-IHC):

[0283] To determine the TILs infiltration and cessation of activated Tregs in the tumor microenvironment (TME), murine CD8a, FoxP3 and PD-L1 expression was analysed by employing histopathology and fluorescent immunohistochemistry (F-IHC). Tissue sections were prepared from tumor samples and deparaffinized by sequential washing in xylene, rehydrated with descending alcohol concentrations (100%, 90%, 70% and 50%,) to distilled water, and the antigen was unmasked with IX citrate buffer by heating in a microwave oven for 20min. Endogenous peroxidase and non-specific binding was blocked by 3% H2O2 and 10% Normal Horse Serum (NHS). Afterwards, murine CD8a, FoxP3 and PD-L1 primary antibodies were added onto the tissue slides and incubated for overnight at 4°C. Followed by, fluorophore-conjugated secondary antibodies were added and mounted with DAPI. Then images were captured and analysed by confocal microscopy.

[0284] Pharmacokinetic analysis:

[0285] Compounds 1 and 7 were evaluated for pharmacokinetic profiling following oral and intravenous administration. Male Sprague Dawley rats with 12 weeks old, were used in the study. After randomization, compounds 1 and 7 were administered orally 50 mg / kg and intravenously 10 mg / kg, and blood samples were collected at 0.0 h, 5 min, 30 min, 1.0 h, 2.0 h, 4.0 h, 8.0 h, 12.0 h, and 24.0 h intervals and processed for plasma isolation and subsequent bioanalytical analysis.PT / 2026 / 10546

[0286] MTD (Maximum Tolerated Dose) for compound 7:

[0287] To evaluate the drug toxicity and determine the oral MTD, female Balb / C with 8 weeks old were administered compound 7 orally. All animals allocated into different groups and observed for 14 days, followed by sacrificed on the day 15. Compound 7 was administered via the oral route as a dose level one is 175 mg / kg (n=3), dose level two is 550 mg / kg (n=3) and dose level three is 1750 mg / kg (n=3). Prior to dosing, animals were fasted and observed for clinical signs of toxicity during the first 0 to 4 hours and the subsequent days daily for 14 days. Body weight was measured on the day of 0, 7 and 14. On the day of 15, all animals were subjected necropsy and blood was collected for hematology analysis.

[0288] Results:

[0289] A family of heterocyles substituted biaryl compounds, comprising of both a biaryl moiety and one heterocyclic moiety (isoxazole, pyrazole and furan) linked at the meta (series-I) or ortho (Series-II) position of the biaryls were synthesized.

[0290] Initially, small molecules were screened using Differential Scanning Fluorometry (DSF), also known as the Thermal Shift Assay (TSA), to identify compounds that bind to recombinant human PD-L1 (amino acids 18-133). In this target-based assay, the melting temperature (Tm) of the target protein changes upon ligand binding. Specifically, the Tm can increase if the ligand stabilizes the protein, or decrease if the ligand disrupts the protein's native structure. As part of the primary screening, all the compounds prepared were tested for the thermal shift assay at an initial concentration of 10 pM. From the results, it was identified that compound 7 and 27 belong to the series-I interact with the target protein PD-L1 in vitro (see Figure 1A-1B). In the presence of 1% DMSO, recombinant human PD-L1 exhibited a Tm of 44.5°C. Among the tested compounds,, compound 7 was found to destabilize the native folding of PD-L1, as indicated by a decrease in Tm to 29°C compared to the DMSO control. Dosedependent TSA / DSF assay for the identified compounds 7 and 27 against PD-L1 showed concentration-dependent unfolding of PD-L1, indicating the robust binding of compounds 7 and 27 toPD-Ll (Fig. 1C, ID).

[0291] A cell-based TCR-NFAT-dependent luciferase activation assay was employed to identify small molecules that inhibit the PD-1 / PD-L1 interaction. This assay utilized two engineered cell lines: PD-L1 aAPC / CHO-Kl and PD-1 Effector (Jurkat) cells. The PD-L1 aAPC / CHO-K1 cells serve as antigen-presenting cells, while the PD-1 Effector (Jurkat) cells function asPT / 2026 / 10546

[0292] T-cells. In this assay system, engagement of CD3 / CD28 with the T-cell receptor (TCR) triggers a signaling cascade that activates the luciferase gene. The interaction between PD-1 and PD-L1 disrupts this CD3 / CD28 / TCR-mediated signaling cascade, thereby inhibiting luciferase activation. Consequently, inhibition of PD-1 / PD-L1 interaction leads to enhanced luciferase expression, which can be quantitatively measured using a luminescence plate reader. First, the assay was standardized using appropriate positive controls and all the selected compounds were tested at a single concentration 8p (Figure IE). For the compounds showing activity against PD-1 and PD1-1 interaction comparable to positive control BMS-1, determined EC50 was have by assaying dose dependent effect of the test compounds (Fig. 1 F, 1G). The half-maximal effective concentration (EC50) was calculated using the "Log (agonist) vs. normalized response-variable slope" analysis in GraphPad Prism 9.5.1, and the results are presented in Table 1. From the results obtained, it is clear compound 1, 6, 7, 5, 16 and 10 showed good inhibition on PDL-1 interaction with PD-1.

[0293] <>

[0294]

[0295] To confirm the identified binders through an orthogonal approach, Isothermal Titration Calorimetry (ITC) assay was employed. ITC is advantageous because it measures the binding affinity of interacting partners in their native states, eliminating the need for fluorescent tags or any form of immobilization. This assay allows us to assess various thermodynamic parameters, including entropy (AS), enthalpy (AH), reaction stoichiometry (n), and binding constants (KD) for the interaction. ITC was used to evaluate the binding affinities of the compounds identified from Thermal Shift Assays (TSA) / Differential Scanning Fluorimetry (DSF) and cell-based assays. Initially, the assay was optimized using positive controls, BMS- 1 which had a binding constant (KD) of 1.68 pM (Fig. 4A), and BMS-202, which had a KD of 1.21 pM (Fig. 4B). From the results obtained it is clear that compounds 1 and 7 bound to target protein PDL-1 which exhibited KD values of 1.37pM and 2.08pM, respectively (Fig.

[0296] 2C, 2D). The other compounds demonstrated moderate to weak inhibition, as indicated by their binding affinities.PT / 2026 / 10546

[0297] Similarly, compounds from series-II have been screened and found that no compounds showed binding to PD-L1 in the TSA / DSF assay (Fig. 3A). PD-1 / PD-L1 blockade bioassay showed that compounds 10, 15, and 16 exhibited increased luciferase activity with significant IC-50 values as shown from the series I (Fig. 3B, 3C). However, these same compounds showed moderate to low binding affinity in the orthogonal ITC assay (Fig. 3D).

[0298] To showcase the small-molecule-mediated inhibitory effect of compound 7 on PD-1 / PD-L1 interaction, we have employed a competitive ELISA assay where soluble PD-L1 can bind with solid-phase PD-1; hence, we can determine at which IC 50 concentration a small molecule can effectively block this interaction. Here we observe that positive control BMS-1 and compound 7 were able to inhibit the PD-1 / PD-L1 interaction at similar IC-50 values i.e., 1.07 pM and 1.03 respectively (Fig. 2E).

[0299] Further, to assess the small molecule induced immune activation by blocking the PD-l / PD-L1 mediated immune suppression, the induction cytokines by the compounds were determined. In this assay, PBMC were activated with anti-CD3 and anti-CD28 antibodies, cocultured with MDAMB-231 cells and treated with small molecules and clinical antibody nivolumab and, incubated for 48 hrs. Then the supernatant was collected to measure pro-inflammatory cytokines such as IL-la, IL-ip, IL-2 (Fig A, B, C, D) and the cytolytic protein perforin (Fig 5E) using ELISA. From the results, it is clearly shown that cytokine levels were increased markedly in cells treated with test compounds 1 and 7 compared to respective vehicle control and as efficiently as nivolumab and BMS-1.

[0300] T cell cytotoxicity assay was performed to demonstrate the phenotypic effect of small molecule mediated T cell anticancer activity ex vivo. CD-3 / CD-28 T cells were cocultured with MDAMB-231 and treated with small molecules and incubated for 24 hours to perform flow cytometry analysis, and the same was incubated for 3 days to perform CCK-8 cell viability assay. Flow cytometry analysis revealed that CRR-SS-02 and CRR-SS-03 treatment exhibited increased cell death i.e, 32.94% and 34.83% compared to the control (CD-3 / CD-28 T cells alone) with 24.43%, which is comparable to the effect of possible control, BMS-1 with 28.62% cell death (Fig. 5A). CCK-8 cell viability assay also revealed a similar effect when treated with small molecules compared to the controls (Fig. 5B).PT / 2026 / 10546

[0301] Taken together, it was observed that compound 1 and compound 7 effectively blocks PD-1 / PD-L1 interaction mediated suppression of immune activation. Further, to determine efficacy of these small molecules on tumor growth in-vivo, syngeneic BCa model was employed.

[0302] The 4T1 triple-negative breast cancer (TNBC) cell line, known for its high PD-L1 expression, was used to induce tumors in Balb / C mice. The mice were injected orthotopically with 4T1 cells and allowed to grow until the tumors reached a volume of 100 mm3. Subsequently, the mice were grouped and treated daily for 14 days with either a vehicle control, BMS-1 (4 mg / kg), compound 1 (4 mg / kg and 8 mg / kg), or compound 7 (4 mg / kg and 8 mg / kg). At the end of the treatment period, the mice were sacrificed, and tumors were excised. During the treatment period, the tumor volume was measured and recorded on every 3rdday. On termination of the experiment, significant tumor reduction was observed in the small molecule-treated groups compared to the vehicle control. From the results, it is clearly shown that both the compounds 1 and 7 notably reduced tumor volume and weight (Figure 6 A, 6B, and 6C). As positive control, BMS-1 also demonstrated a strong response against 4T1 tumors. However, compound 7 exhibited more attenuation of tumor growth. Post-sacrifice analysis of tissue samples, including the liver and spleen, as well as body weight measurements, revealed no significant toxicity induced by the small molecules.

[0303] To investigate the small molecule-mediated immune mechanism of action in vivo, we measured serum cytokine levels. We have observed that both Compound 1 and Compound 7 were able to increase the pro-inflammatory serum cytokine levels, notably INF-y, IL-ip, TNF-a and IL-la compared to vehicle control (Fig. 7A -7D) . BMS-l / PDI-1 was used as positive control.

[0304] However, it is essential to identify the T-cell subtypes recruited in the tumors to induce cell death upon inhibition of the interaction between PD-1 and PDL-1. To determine the T-cell subtypes in the tumor microenvironment, we have performed FACS using the dissociated tumor samples. FACS analysis of intratumoral tissue shows increased percentage of CD3+, CD4+, and CD8+ T cells in the treated animals compared to vehicle control. Dot plots shows the representative FACS images of the CD3+, CD4+ and CD8+ T cells derived from dissociated tumor samples (Fig. 8A- 8C).PT / 2026 / 10546

[0305] Ultimately, it is important to determine the recruitment of T cells in native tumor tissues harvested from the in-vivo efficacy study. We have performed H&E staining of all the tumor tissue sections and found that the control group exhibited dense proliferation of neoplastic cells with minimal necrotic regions observed within the tumor stroma whereas the BMS-1 (4 mg / kg) treated group displayed multifocal necrosis of moderate severity, accompanied by a moderate infiltration of lymphocytes into the tumor stroma. Compound 1 (4&8 mg / kg) treated group showed multifocal necrotic foci, with a mild to moderate degree of necrosis and a moderate degree of lymphocytic infiltration into the tumor stroma. Compound 7 (4 mg / kg) treated group revealed diffused necrosis of moderate to marked severity, with a marked degree of lymphocytic infiltration diffusely present within the tumor stroma. However, the Compound 7 (8mg / kg) treated group revealed large, diffuse necrotic foci with minimal residual proliferating tumor cell population. Extensive lymphocytic infiltration was prominent in the necrotic areas within the stroma (Fig. 9A).

[0306] In the tumor microenvironment (TME), the ratio of CD8+ T cells, FoxP3 -positive (Tregs) cells, and PD-L1 expression is a strong predictor of response to immunotherapy. Hence, we performed Fluorescent immunohistochemical analysis (F-IHC) to determine the CD8, FoxP3, and PD-L1 expression in the Compound 1 (4 mg / kg, 8 mg / kg) and Compound 7 (4 mg / kg, 8 mg / kg) treated 4T1 tumors. As anticipated, small molecule treatment increased the CD8+ T cells and decreased the FoxP3 (treg) and PD-L1 positive cells, suggesting these small molecules function through the mechanism by recruiting cytotoxic T cells and suppressing the activation of inhibitory Treg cells in the TME (Fig. 9B).

[0307] The pharmacokinetics and absolute bioavailability of Compound 1 and Compound 7 was performed in the male Sprague Dawley Rats though oral (50mg / kg) and IV (5mg / kg) route. Here, we have observed that Compound 1 showed poor absorption of drug when administered through oral route of administration. While intravenous administration provides rapid systemic exposure, it is accompanied by faster clearance and a shorter half-life. The absence of detectable oral absorption underscores a potential limitation in oral bioavailability. However, when Compound 7 was administered orally at a dose of 50 mg / kg, it exhibited rapid gastrointestinal absorption achieving a mean peak plasma concentration (Cmax) of 16112.41 ± 1395.73 ng / mL within 2.53 ± 0.39 hours (Tmax). The area under the concentration-time curve (AUCO-t) of 138324.05 ± 6226.64 hr*ng / mL and (AUC0- last) of 138773.55 ± 6054.44 hr*ng / mL indicated substantial systemic exposure. With a highPT / 2026 / 10546

[0308] bioavailability of 40.16%, Compound 7 presents as a promising candidate for oral administration. The terminal half-life of 2.53 ± 0.39 hours suggests that Compound 7 could support less frequent dosing regimens enhancing its therapeutic potential. Intravenous administration of Compound 7 at a dose of 10 mg / kg resulted in Cmax of 17351.79 ± 2474.28 ng / mL. The AUCO-t was found to be 68887.18 ± 5465.89 hr*ng / mL and (AUC0-last) of 69025.13 ± 5484.75 hr*ng / mL further demonstrating the dose-dependent pharmacokinetics of the compound. Compound 7 showed a elimination rate with a half-life of 2.7 ± 0.11 hours. The clearance rate was measured at 2.43 ± 0.19 mL / min / kg and the volume of distribution at steady state (Vss) was 0.57 ± 0.06 L / kg indicating moderate distribution primarily within the circulatory system with a mean residence time (MRT) of 3.92 ± 0.24 hours (Table 2). Overall, the pharmacokinetic characteristics of Compound 7 indicate significant therapeutic potential, especially for oral delivery due to its high bioavailability and prolonged systemic presence. Intravenous administration provides more rapid systemic exposure but is accompanied by moderate clearance and a shorter half-life (Fig. 10A, Table 2).

[0309] Table 2. Pharmacokinetic and bioavailability analysis of compound 7 in male Sprague Dawley rats.

[0310]

[0311] In our previous in vivo efficacy study, we compared the effect of Compound 1 and Compound 7 to the known PD-L1 inhibitor BMS-1, where we observed that our molecules reduced the 4T1 tumor mass in Balb / C mice. To further compare the efficacy of our moleculePT / 2026 / 10546

[0312] with the anti-PD-Ll antibody, and the oral efficacy as indicated by the pharmokinetic analysis, we have performed similar efficacy study using the 4T1 tumor induced Balb / C mice. In this efficacy study, we have performed dose-dependent treatment of Compound 7, i.e, 4 mg / kg, 8 mg / kg, and 16 mg / kg to compare with the anti-PD-Ll antibody (200 pg / kg) and BMS-1 (4 mg / kg). Here, we have demonstrated that oral dosing of Compound 7 treatment has reduced the tumor mass significantly, which is as good as BMS-1 and the effect was comparable to anti-PD-Ll antibody. And no change in body weight was observed in the compound 7 treated group compound to the vehicle control or anti-PD-Ll -treated groups (Fig 11A-11C). As anticipated, no changes in body weight (Fig. 11D) or hematology was observed in treatment groups compared to control group (Table 3) .

[0313] Table 3. Hematological analysis of compound 7 in oral efficacy study

[0314]

[0315] Primary criterias to evaluate MTD for compound 7 were dose-limiting toxicities (DLT) and mortality. No DLTs were observed at the 175 mg / kg dose group, and all animals remain alive and clinically healthy. In the 1750 mg / kg dosed group, critical dose-limiting toxicities were observed, and mortality was found on the 2ndday of administration, indicating a potential drug toxicity. However, based on these findings, 550 mg / kg was identified as the maximumPT / 2026 / 10546

[0316] tolerated dose for oral administration. No abnormalities were revealed in the gross pathology examination. No hematological changes were seen in any of the dosed animals (Table 4). Biochemical analysis also revealed no significant changes in the compound 7-treated groups compared to the control group animals (Table 5).

[0317] Table 4. Hematological analysis in the MTD of compound 7 in female Balb / C mice

[0318]

[0319] Table 5. Biochemical analysis in the MTD of compound 7 in female Balb / C mice

[0320]

[0321] PT / 2026 / 10546

[0322]

Claims

1. PT / 2026 / 10546WE CLAIM:

1. An immunotherapeutic compound of Formula I, pharmaceutically acceptable salts or solvates thereof,Formula IwhereinA is O or N-Rl, wherein R1 is H or Cl to C3 alkyl;B is N or C-R2, wherein R2 is Cl to C3 alkyl;X and Y are independentlywhen X iswherein R3 is selected from the group consisting of halo, alkoxy, trifluoroalkyl, CN and C1-C5 alkyl, Y is H;whenwherein R3 is selected from the group consisting of halo and alkoxy, X O11is wherein R4 is selected from the group consisting of hydroxy, alkoxy, and Cl- C5 alkyl; andZ is selected from the group consisting of C1-C5 alkyl, Bn and, wherein R5 isCN, tetrazole,2. The compound as claimed in claim lof formula la a pharmaceutically acceptable salt or solvate thereof,Formula lawherein A is O or N-Rl, wherein R1 is H or Cl to C3 alkyl;B is N or C-R2, wherein R2 is Cl to C3 alkyl;R3 is selected from the group consisting of halo, alkoxy, trifluoroalkyl, CN and C1-C5 alkyl; andPT / 2026 / 10546Z is selected from the group consisting of C1-C5 alkyl, Bn, and, wherein R5 isCN, tetrazole,3. The compound as claimed in claim 1 of Formula lb, a pharmaceutically acceptable salt or solvate thereof,Formula lbwherein A is O; B is N or C-R2, wherein R2 is Cl to C3 alkyl;R4 is selected from the group consisting of hydroxy, alkoxy, and C1-C5 alkyl;R3 is selected from the group consisting of halo and alkoxy, andZ is selected from the group consisting of C1-C5 alkyl, Bn,wherein R5 isCN, tetrazole,4. The compound as claimed in claims 1, is selected from the group consisting of:4-(3-([l,l'-Biphenyl]-3-yl)isoxazol-5-yl)benzonitrile (1);4-(3-(4'-methyl-[l,r-biphenyl]-3-yl)isoxazol-5-yl)benzonitrile (2);4-(3-(4'-(Trifluoromethoxy)-[l,r-biphenyl]-3-yl)isoxazol-5-yl)benzonitrile (3);3'-(5-(4-Cyanophenyl)isoxazol-3-yl)-3-fluoro-[l,r-biphenyl]-4-carbonitrile (4);(4-(3-([l,l'-Biphenyl]-3-yl)isoxazol-5-yl)phenyl)methanol (5);4-(4-(3-([l,l'-Biphenyl]-3-yl)isoxazol-5-yl)benzyl)morpholine (6);5-(4-(2J / -Tetrazol-5-yl)phenyl)-3-([l,r-biphenyl]-3-yl)isoxazole (7);4-(3-([l,r-Biphenyl]-3-yl)-17 / -pyrazol-5-yl)benzonitrile (8);4-(3-([ 1 , 1 '-Biphenyl]-3-yl)-l -methyl- 17 / -pyrazol-5-yl)benzonitrile (9);3-([l,l'-Biphenyl]-2-yl)-5-propylisoxazole (10);3-(4'-Methoxy-[l,l'-biphenyl]-2-yl)-5-phenylisoxazole (11);3-(4'-Bromo-[l,l'-biphenyl]-2-yl)-5-phenylisoxazole (12);3-(4'-Methoxy-[l,r-biphenyl]-2-yl)-5-phenyl-17 / -pyrazole (13);4-(4'-ethyl-[l,l'-biphenyl]-2-yl)-2 -phenyl- IH-pyrrole (14);3-(4'-Bromo-[l,r-biphenyl]-2-yl)-5-phenyl-17 / -pyrazole (15);P 172026 / 105463 -([ 1 , 1 ’-Biphenyl] -2-yl)-5-propyl- 1 / Z-pyrazole (16);Ethyl 5-benzyl-4-(4'-methoxy-[l,l'-biphenyl]-2-yl)-2-methylfuran-3-carboxylate (17); Ethyl 5-benzyl-4-(4'-ethyl-[l, l'-biphenyl]-2-yl)-2-methylfuran-3-carboxylate (18);Ethyl 5-benzyl-4-(4'-bromo-[l,l'-biphenyl]-2-yl)-2-methylfuran-3-carboxylate (19);5-Benzyl-4-(4'-methoxy-[l,l'-biphenyl]-2-yl)-2-methylfuran-3-carboxylic acid (20);l-(5-benzyl-4-(4'-methoxy-[l,l'-biphenyl]-2-yl)-2-methylfuran-3-yl)ethan-l-one (21) Methyl (4-(3-([l,l'-biphenyl]-3-yl)isoxazol-5-yl)benzoyl)serinate(35)4-(3-([l,l'-Biphenyl]-3-yl)isoxazol-5-yl)benzoic acid (36)4-(3-([l,l'-Biphenyl]-3-yl)-l,2,4-oxadiazol-5-yl)benzonitrile (37)5-(4-(lJH-Tetrazol-5-yl)phenyl)-3-([l,l'-biphenyl]-3-yl)-l,2,4-oxadiazole (38) Methyl l-(4-(3-([l,l'-biphenyl]-3-yl)isoxazol-5-yl)benzoyl)piperidine-2-carboxylate (39) Methyl l-(4-(3-([l,l'-biphenyl]-3-yl)isoxazol-5-yl)benzoyl)-4-hydroxypyrrolidine-2- carboxylate (40)4-(3-([l,l'-Biphenyl]-3-yl)isoxazol-5-yl)benzamide (41)4-(3-([l,l'-Biphenyl]-3-yl)isoxazol-5-yl)-N-hydroxybenzamide (42) and3-([l,l'-Biphenyl]-3-yl)-5-(4-(l-methyl-lH-tetrazol-5-yl)phenyl)isoxazole (43).

5. A process for the preparing isoxazole substituted biaryl compound of formula laFormula lawherein A is O, B is N, R3 is selected from the group consisting of halo, alkoxy, trifluoroalkyl, CN and C1-C5 alkyl; and|j 2LR5Z is selected from the group consisting of C1-C5 alkyl, Bn, and5, wherein Rs isCN, tetrazole, -CH2OH and, comprising the steps of:i. reacting benzaldehyde compound 22 with boronic acid compound 23, wherein R3 is selected from the group consisting of halo, alkoxy, trifluoroalkyl, CN and C1-C5 alkyl, under Suzuki coupling reaction in presence of palladium catalyst and base in a solvent at 80 °C for 3-5 hours to obtain the compound 24, wherein R3 is as defined above;PT / 2026 / 10546ii. treating the compound 24 with Hydroxylamine hydrochloride in presence of ISfeCCh in ethanol and water solvent mixture at 25 - 40 °C for 2-5 hours to produce the compound 25, wherein R3 is as defined above;iii. reacting compound 25 with alkyne 33, wherein Z is selected from the groupconsisting of C1-C5 alkyl, Bn, and, wherein R6 is CN, tetrazole, -CH2OH; in the presence of base and chlorinating reagent in polar solvent at 25 - 40 °C for 12-15 hours to produce isoxazole substituted biaryl compound of formula la (compound 26); andiv. alternatively, the isoxazole substituted biaryl derivatives 26 obtained by reacting the compound 27 with tosyl hydroxylamine in presence of acid in polar solvent followed by N-Tosyl deprotection with TBAF.

6. The process as claimed in claim 5 wherein the base in step (i) is selected from ISfeCCh, NaHCCh, CsF, Et3N, K2CO3, TBAF and KOtBu and the solvent in step (i) selected from EtOH, H2O, DCM, EtOAc, THF, CH3CN.

7. The process as claimed in claim 5 wherein the chlorinating reagent used in step (iii) is NCS and acid in step (iv) is selected from BF3.ET2O and / ?TSA.

8. A process for the preparing pyrazole substituted biaryl compound of formula IFormula IPT / 2026 / 10546wherein A is N-Rl, wherein R1 is H or Cl to C3 alkyl; B is N; R3 is selected from the group consisting of halo, alkoxy, trifluoroalkyl, CN and C1-C5 alkyl; and Z is selectedfrom the group consisting of C1-C5 alkyl, Bn, and,R5 is CN,tetrazole,i. reacting compound 24, wherein R3 is selected from the group consisting of halo, alkoxy, trifluoroalkyl, CN and C1-C5 alkyl with alkyne 33, wherein Z is selected from the group consisting of C1-C5 alkyl, Bn, and, wherein R6 is CN, tetrazole, -CH2OH in presence of base in THF at -78°C for 2 hours to produce the compound 27, wherein R3 and Z are as defined above;ii. oxidation of the secondary alcohol of compound 27 using oxidizing agent in DCM at 0 - 25 °C for 2 hours to obtain the compound 28, wherein R3 and Z are as defined above;iii. treating the compound 28 with tosyl hydrazine in Methanol at 25 - 40 °C for 3 hours to produce the compound 29 wherein R3 and Z are as defined above;iv. subjecting the compound 29 for base mediated cyclisation reaction in the presence of Triethylsilane, KF and catalyst in DMF at 25 - 40 °C for 12-15 hours to obtain the compound 30, wherein R3 and Z are as defined above;P 172026 / 10546v. deprotection of tosyl group of compound 30 in DMSO at 25 - 40 °C for 12 hours to produce the pyrazole substituted biaryl compound of formula I (compound 31); andvi. alternatively reacting the compound 27 with tosyl Hydrazine in presence of acid in acetonitrile followed by addition KOtBu to produce the pyrazole substituted biaryl compound of formula la (compound 31).

9. A process for the preparation of furan substituted biaryl derivatives of formulareacting compound 27 and 1,3 -diketoester 34 in presence of acidin acetonitrile stirred for 2h and then addition of K2CO3 to the reaction mixture and then further stirred at 80°C for lOh to produce the furan substituted biaryl derivatives 32.

10. A pharmaceutical composition comprising at least one compound as claimed in claim 1, or a pharmaceutically acceptable salt, or prodrug thereof, in combination with a pharmaceutically acceptable carrier or excipient.

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