WO2025136134A1 - Dimethylbiphenyl covalent pd-l1/pd-1 interaction inhibitor compound, pharmaceutical composition containing said inhibitor and the use thereof - Google Patents

Abstract

In the first aspect, the invention relates to a compound of general formula (I) and/or its pharmaceutically acceptable salts. The invention also relates to a pharmaceutical composition comprising a compound of general formula (I) and/or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier. The invention also relates to the use of said compound as a covalent inhibitor of PD-L1/PD-1 interaction its use for the preparation of a drug for the prevention and/or treatment of PD-1/PD-L1 pathway-dependent disease.

Description

DIMETHYLBIPHENYL COVALENT PD-L1/PD-1 INTERACTION INHIBITOR COMPOUND, PHARMACEUTICAL COMPOSITION CONTAINING SAID INHIBITOR AND THE USE THEREOF

The invention relates to novel 2,2'-dimethyl[l,l'-biphenyl]-3,3'-diamine derivatives constituting small-molecule covalent inhibitors targeting PD-1/PD-L1 interaction for possible use in cancer immunotherapy.

The PD-L1/PD-1 interaction is one of the so-called immune checkpoints. Immune checkpoints ensure strict regulation of the immune response by delivering co-stimulatory or inhibitory signals to T lymphocytes. As the result, the balance of the immune system is maintained and allows the elimination of pathogens while preventing the immune system from attacking its own cells. On the other hand, activation of immune checkpoints is used by cancer cells to evade immune surveillance.

PD-L1/PD-1 is the central checkpoint of the immune system. The programmed death receptor 1 (PD-1, CD279) is expressed on activated T cells, and interacts with two programmed death ligands: PD-L1 (CD274, B7-H1) and PD-L2 (CD273, B7-DC). Increased expression of PD-L1 and PD-L2 ligands is observed in many types of cancer cells. Binding between PD-1 and its ligands attenuates the body's natural immune response leading to T-cell exhaustion. In clinical settings, the blocking of the PD-L1/PD-1 interaction as well as other immune checkpoints was confirmed to result in immune activation and elimination of cancer cells.

Over the past decade, therapies involving antibodies that block the immune checkpoints showed impressive outcomes in the treatment of selected types of cancer, thereby revolutionizing the treatment of cancer patients. Nevertheless, antibody-based therapies carry the risk of immune-related adverse events. Moreover, in some patients, immunotherapy does not show a therapeutic effect. . Other obvious disadvantages of antibodies include their requirement to be administered by injection and high cost of production.

Pharmaceuticals based on small-molecule inhibitors could contribute to the overcoming of the above-mentioned disadvantages of antibody-based immunotherapy. The advantages of small- molecule inhibitors include lower manufacturing costs, the possibility of oral instead of intravenous administration, better and more predictable pharmacokinetics, as well as the potential lack of adverse effects from the patient's immune system. Over the past decade, a number of small-molecule inhibitors have been developed for the PD-L1/PD-1 interaction. To date, six small-molecule compounds are in various phases of clinical trials.

In the context of small-molecule compounds, in recent years there has been an increasing interest in drugs capable of interacting with their molecular targets in a covalent manner. This is evidenced by the increasing number of drugs registered by the FDA for the treatment of various conditions, mainly cancer. This is due to the additional advantages of covalent drugs, including prolonged duration of action and increased drug activity. Most covalent drugs are designed to target the cysteine residue in the vicinity of the binding molecule. However, some proteins being potential molecular targets for disease treatment, including PD-L1, lack solvent- exposed cysteine residues on their surface. In recent years, the advances in medicinal chemistry facilitated the discovery of electrophilic moieties capable of forming covalent bonds with the side chains of other amino acids, including lysine, tyrosine, histidine and methionine.

US patent application US2021040118 describes immunomodulators and pharmaceutical compositions containing 2,2'-dimethyl[l,l'-biphenyl]-3,3'-diamine derivatives for the treatment, prevention or alleviation of diseases or disorders such as cancer or infections. Likewise, another U.S. patent application, US2021032270, describes biphenyl structure derivatives that can be used in the preparation of drugs for the prevention and/or treatment of cancer or tumors, immune-related diseases and disorders, infectious diseases, or metabolic diseases mediated by the PD-1/PD-L1 signaling pathway.

European patent application EP4001274 discloses biphenyl derivatives containing a cyclopropyl substituent capable of blocking PD-1/PD-L1 interaction. They can be widely used in the preparation of drugs for the prevention and/or treatment of cancer or tumors, immune- related diseases and disorders, infectious diseases, or metabolic diseases mediated by the PD- 1/PD-L1 signaling pathway. Another European patent description, EP4086253, discloses asymmetrically structured biphenyl derivatives that may find application in the preparation of drugs for the treatment of cancer, infectious diseases and autoimmune diseases.

In the context of the above, it is rationale to develop irreversible PD-L1 inhibitors capable of inhibiting the ligand's interaction with the PD-1 receptor in a permanent manner. To date, no small-molecule compounds capable of inhibiting PD-L1/PD-1 interaction by covalently binding the PD-L1 have been described in the prior art.

In the first aspect, the invention relates to a compound of general formula (I) and/or its pharmaceutically acceptable salts

wherein:

- L₁ is -(CH₂)- or -C(O)-;

- A is an aryl or a heterocycle containing at least one nitrogen atom;

- R₁ is hydrogen or alkyl;

- R₂ is hydrogen or alkyl, wherein both R₁ and R₂ can be hydrogens;

- R₃ is selected from the group comprising -SO₂F, -OSO₂F,

wherein:

- L₂ is -(CH₂)-, -C(O)- or -S(O₂)-;

- X₁ is a hydrogen or a halogen;

- X₂ is CH₂ or -CHC(O)OCH₃;

- X₃ is a halogen or -CH₂SO₂F; wherein R₃ and R₄ can be the same or different.

In a preferred embodiment, the compound is defined by formula (II)

wherein:

- L is -C(O)-;

- R₁and R₂ are hydrogens;

- R₃ and R4 are selected from the group comprising

where

- L₂ is -(CH₂)-, -C(O)- or -S(O₂)-;

- X₁ is a hydrogen or a halogen;

- X₂ is CH₂ or -CHC(O)OCH₃;

- X₃ is a halogen or -CH₂SO₂F;

In another preferred embodiment, the compound is defined by formula (III)

wherein:

- R₃ and R4 are selected from the group comprising

In yet another preferred embodiment, the compound is defined by formula (IV)

- L₁ is -(CH₂)- or -C(O)-;

- A is an aryl or a heterocycle containing at least one nitrogen atom;

- R₁ is hydrogen or alkyl;

- R₂ is hydrogen or alkyl, wherein both R₁ and R₂ can be hydrogens;

- R₃ is selected from the group comprising -SO₂F, -OSO₂F.

Preferably, A is selected from group comprising

In another preferred embodiment of the invention, the compound as defined by formula (I) is selected from the group comprising the derivatives as listed below.

In yet another preferred embodiment of the invention, the compound as defined by formula (I) is selected from the group comprising the derivatives as listed below.

In the second aspect, the invention relates to the use of the compound of the general formula (I) as a PD-L1/PD-1 interaction inhibitor. The invention also relates to a pharmaceutical composition comprising a compound of general formula (I) and/or a pharmaceutically acceptable salt thereof according to the first aspect of the invention, and a pharmaceutically acceptable carrier.

In another aspect, the invention relates to a compound of general formula (I) and/or a pharmaceutically acceptable salt thereof according to the first aspect of the invention for use in the preparation of a drug for the prevention and/or treatment of a disease dependent on the PD-1/PD-L1 pathway.

In view of state of the art described above, the invention provides a number of PD-L1/PD-1 interaction inhibitors containing specific functional groups capable of forming a covalent bond with specific amino acids within the PD-L1 protein structure, in particular lysine 124 (Lysl24), tyrosine 56 (Tyr56) or tyrosine 123 (Tyrl23), which can be described by formulae (II) and (IV), respectively:

Selected PD-L1/PD-1 interaction inhibitors targeting the Lysl24 or Tyrl23 residue according to the invention cause permanent and irreversible dimerization of the PD-L1 protein, preventing its interaction with PD-1. Selected PD-L1/PD-1 interaction inhibitors targeting tyrosine 56 bind covalently to PD-L1 to trigger its dimerization. Due to the covalent nature of the interaction, inhibitors enhance and prolong the duration of the therapeutic effect. This allows for a reduction in the therapeutic dose used, which in turn creates a basis for developing combinations with other immunotherapeutics and extending therapeutic use in patients who do not respond to treatment with monotherapy. Compounds of the invention demonstrate the ability to inhibit PD-L1/PD-1 interaction by covalently binding to PD-L1, both in isolated protein and cell-system assays. The presented results provide a basis for their use as immune activators in cancer immunotherapy. For a better understanding of the invention, this description shall now be followed by some example embodiments presented in more detail.

The invention is illustrated in the following figures, where:

- Figs. 1A-1C present the HTRF signal vs. concentration relationship curves for inhibitor compounds

11 (1A) and 17 (IB) as well as the reference compound - compound A (1C); compounds 11 and 17 show a characteristic shift in IC₅₀ value dependent on incubation time - typical of covalent compounds; for compound A, the IC₅₀ remains constant regardless of incubation time;

- Fig. 2 illustrates the formation of covalently bound PD-L1 dimers as induced by compound 17;

- Fig. 3 confirms the covalent binding to PD-L1 (MW=14.8 kDa) for compound 42; the left panel shows the MS spectrum of PD-L1 whereas the right panel shows the MS spectrum of PD-L1 treated with compound 42; covalent adduct marked by arrow;

- Fig. 4 confirms the covalent binding to PD-L1 (MW=25.4 kDa) for compound 48; the left panel shows the MS spectrum of PD-L1 whereas the right panel shows the MS spectrum of PD-L1 treated with compound 43; covalent adduct marked by arrow;

- Fig. 5 confirms the covalent binding to PD-L1 (MW=25.4 kDa) for compound 17; the left panel shows the MS spectrum of PD-L1 whereas the right panel shows the MS spectrum of PD-L1 treated with compound 17; covalent adduct marked by arrow;

- Fig. 6 confirms the covalent binding to PD-L1 (MW=25.4 kDa) for compound 17 - a time- dependent experiment; the covalent adduct corresponding to PD-L1 dimer marked by an arrow;

- Fig. 7 confirms a non-obvious feature of the solution in the form of covalent dimerization of PD-L1 being induced within the cellular system using compound 17 with no dimerization taking place when using compound A (cmpd A), being a representative of known compounds with a very similar structure (A); compound 17 binds the surface of PD-L1 at the binding site of therapeutic antibodies as evidenced by the presence of therapeutic antibodies (A - atezolizumab, D - durvalumab) clearly impairing the ability of compound 17 to covalently dimerize PD-L1 (B);

- Fig. 8 confirms a non-obvious feature of the solution in the form of covalent dimerization of PD-L1 being induced using compound 17 within three cell lines to represent (i) dimerization of overproduced PD-L1 protein (CHO/TCRAct cells), (ii) dimerization of endogenous human PD-L1 (RKO/TCRAct cells) and (iii) dimerization of endogenous human PD-L1 produced in response to interferon (IFN) gamma (U-2 OS cells).

- Fig. 9 confirms a non-obvious feature of the solution in the form of compound 17-induced covalent dimerization of human PD-L1 (left panel) and the absence of this dimerization in murine PD-L1 (right panel). At the same time, dimerization of human and murine PD-L1 is not observed when using compound A (labeled A), being a representative of known compounds with a very similar structure.

- Fig. 10 confirms the blocking of the surface of human PD-L1 protein by compound 17 as manifested by a decrease in the fluorescence intensity of MC38/hPD-Ll cells labeled with a PD-Ll-binding antibody in the presence of compound 17 at concentrations of 5 μM (short- dashed line) and 20 μM (long-dashed line) as compared to control cells (continuous line). A similar decrease in fluorescence intensity is observed in the presence of the control PD-L1- blocking antibody, durvalumab (dotted line). Staining of cells with the isotype control antibody is shown with a double dotted line (first graph from the top).

Example 1. 2,2'-Dimethyl[1,1'-bisphenyl]-3,3'-diamine (3)

A two-necked round-bottom flask was charged with 3-bromo-2-methylaniline (3.00 g, 16.12 mmol, 1.0 eq.), 2-methyl-3-(4,4,5,5-tetramethyl-l,3-dioxolan-2-yl)aniline (4.53 g, 19.45 mmol, 1.2 eq.), K₂CO₃ (6.71 g, 48.64 mmol, 3.0 eq.) and dioxane/water mixture (60 mL, 2:1 v/v). The resulting solution was degassed by flushing with argon while in an ultrasonic bath for 30 minutes. The flask was placed in an oil bath heated to 95°C, and Pd(dppf)CI(2)-DCM (0.66 g, 0.81 mmol, 0.05 eq.) was added. The flask was heated at 95°C for 3 h in an inert gas atmosphere. After disappearance of substrate 1 (TLC analysis), the reaction mixture was cooled to room temperature. The contents of the flask were transferred to a separatory funnel and extracted three times with ethyl acetate (50 mL each). The organic phases were combined, dried over anhydrous NazSCU, and concentrated with silica gel under reduced pressure on a rotary evaporator. The crude product was purified by column chromatography (SiCh, hexane/ethyl acetate, 2:1) to obtain 2.74 g (80%) of product 3 as a bright orange solid.

¹H NMR (400 MHz, DMSO) δ 6.84 (t, J = 7.6 Hz, 2H), 6.55 (d, J = 7.9 Hz, 2H), 6.21 (d, J = 7.3 Hz, 2H), 4.77 (s, 4H), 1.68 (s, 6H).

¹³C NMR (101 MHz, DMSO) δ 146.95, 143.06, 125.98, 119.48, 117.91, 113.22, 25.49.

Example 2. Dimethyl 6,6'-(((2,2-dimethyl-Iljl'-biphenyn-3,3- diyl)bis(azanediyl))bis(carbonyl))dinicotinate (6)

In the first step, 5-(methoxycarbonyl)picolinic acid was placed in a round-bottom flask followed by the addition of SOCb (53.8 g, 32.8 mL, 452.19 mmol, 32 eq). The flask was equipped with a reflux condenser and the reaction mixture was heated to 90°C. The reaction was continued for 4 h. After this time, the reaction mixture was cooled to room temperature and residual SOCb was evaporated on a rotary evaporator under reduced pressure. The crude product was dissolved in anhydrous THF (10 mL). A separate flask was charged with compound 3 (3.00g, 14.13 mmol, 1 eq.) followed by the addition of anhydrous THF (40 mL) and triethylamine (TEA, 22.8 g, 30.8 mL, 226.10 mmol, 16 eq.). The flask was equipped with a dropping funnel and placed in an ice bath. The dropping funnel was charged with crude solution of acid chloride 5 for subsequent slow, dropwise addition to the reaction mixture. The reaction was continued for 16 h. After completion of the reaction, the mixture was poured into cooled ethyl acetate. The product was filtered on a vacuum funnel and washed with cooled ethyl acetate (100 mL) and diethyl ether (100 mL). The compound 6 (3.51 g), obtained as a light yellow precipitate with 46% yield, was used in the next reaction without purification.

¹H NMR (400 MHz, CDCI3) δ 10.14 (s, 2H), 9.21 (dd, J = 2.0, 0.7 Hz, 2H), 8.51 (dd, J = 8.2, 2.0 Hz, 2H), 8.40 (dd, J = 8.2, 0.7 Hz, 2H), 8.28 (dd, J = 8.1, 0.8 Hz, 2H), 7.33 (t, J = 7.9 Hz, 2H), 7.00 (dd, J = 7.6, 1.0 Hz, 2H), 3.99 (s, 6H), 2.13 (s, 6H).

¹³C NMR (101 MHz, CDCI3) δ 165.12, 161.14, 153.12, 149.51, 142.31, 139.03, 135.82, 128.39, 126.73, 126.44, 126.42, 122.16, 120.85, 52.83, 14.47.

Example 3. N,N'-(2,2'-Dimethyl-[1,1'-biphenyl]-3,3'-diyl)bis(5- (hydroxymethyl)picolinamide) (7)

Compound 6 (3.5 g, 6.5 mmol, 1 eq.) was placed in a round-bottom flask and suspended in 75 mL of THF/MeOH mixture (2:1, v/v). The flask was placed in an ice bath. LiBH4 (1.42 g, 65 mmol, 10 eq.) suspended in THF (5 mL) was added to the reaction mixture. The reaction was continued for 16 h. After the reaction was completed, saturated NH₄CI solution (100 mL) was added and the resulting mixture was extracted with ethyl acetate (3 x 50 mL). The organic phases were combined, dried over anhydrous Na₂SO₄, and concentrated with silica gel under reduced pressure. The product was purified by column chromatography (SiCh, chloroform/methanol, 20:1) to obtain 1.54 g (50%) of product 7 as a white solid.

¹H NMR (400 MHz, DMSO) δ 10.31 (s, 2H), 8.63 (d, J = 1.2 Hz, 2H), 8.12 (d, J = 8.0 Hz, 2H), 7.96

(dd, J = 8.0, 2.0 Hz, 2H), 7.85 (d, J = 8.0 Hz, 2H), 7.29 (t, J = 7.8 Hz, 2H), 6.96 (d, J = 7.5 Hz, 2H),

5.49 (s, 2H), 4.63 (s, 4H), 1.99 (s, 6H). ¹³C NMR (101 MHz, DMSO) δ 162.65, 148.86, 147.39, 142.35, 141.93, 136.77, 136.49, 129.19,

126.61, 126.33, 123.10, 122.40, 60.89, 15.05.

Example 4. N,N'-(2,2'-Dimethyl-[1,1'-biphenyl]-3,3'-diyl)bis(5-(chloromethyl)picolinamide)

(8)

7 8

Compound 7 (1.54 g, 3.19 mmol, 1 eq.) was placed in a round-bottom flask and suspended in 30 mL DCM. The flask was placed in an ice bath. 10 drops of DMF were added to the reaction mixture. Next, SOCI₂ (2.28 g, 1.4 mL, 19.15 mmol, 6 eq.) was added dropwise. The reaction was continued for 16 h. After the reaction was completed, DCM was evaporated. The residual unreacted thionyl chloride was removed by concentrating the mixture on a rotary evaporator in the presence of toluene to obtain 1.69 g (89%) of crude product 8 (in the form of hydrochloride) as a light yellow solid to be used in subsequent steps without further purification.

¹H NMR (600 MHz, DMSO) δ 10.37 (s, 1H), 8.81 (d, J = 1.7 Hz, 1H), 8.20 (d, J = 8.1 Hz, 1H), 8.15 (dd, J = 8.1, 2.1 Hz, 1H), 7.84 (d, J = 7.6 Hz, 1H), 7.33 (t, J = 7.8 Hz, 1H), 7.01 (d, J = 7.5 Hz, 1H), 4.95 (s, 2H), 2.02 (s, 3H).

¹³C NMR (151 MHz, DMSO) δ 162.27, 149.95, 149.17, 142.29, 138.94, 137.41, 136.62, 129.53, 126.75, 126.27, 123.40, 122.73, 43.04, 15.01.

Example 4. N,N'-(2,2'-Dimethyl-[1,1'-biphenyl]-3,3'-diyl)bis(5-(azidomethyl)picolinamide)

(9)

A round-bottom flask was charged with compound 8 (1.68, 2.84, 1 eq.) and DMF (30 mL).

Sodium azide (1.11 g, 17.02 mmol, 6 eq.) was added. The reaction mixture was heated at 100°C for 2 h. After completion of the reaction, the mixture was poured into ice/water mixture. The resulting precipitate was filtered under vacuum through a glass fritted funnel, washed with diethyl ether (50 mL) and dried to obtain 1.34 g (78%) of product 9 as a light yellow solid for use in the subsequent step without further purification.

1H NMR (600 MHz, CDCI₃) δ 10.10 (s, 2H), 8.59 (d, J = 1.6 Hz, 2H), 8.35 (d, J = 8.0 Hz, 2H), 8.28 (d, J = 8.0 Hz, 2H), 7.89 (dd, J = 8.0, 2.0 Hz, 2H), 7.33 (t, J = 7.9 Hz, 2H), 7.00 (d, J = 7.4 Hz, 2H), 4.51 (s, 4H), 2.13 (s, 6H).

Example 5. N,N'-(2,2'-Dimethyl-[1,1'-biphenyl]-3,3'-diyl)bis(5-(aminomethyl)picolinamide) (10)

A round-bottom flask was charged with compound 9 (1.34, 2.52, 1 eq.) and a mixture of THF/H2O (36 mL, 5:1 v/v). The reaction mixture was cooled in an ice bath and triphenylphosphine (2.64 g, 10.06 mmol, 4 eq.) was added. The reaction was continued overnight at room temperature. After completion of the reaction, the solvent was evaporated under reduced pressure on a rotary evaporator. DCM (100 mL) and H2O (100 mL) were added to the residue in the flask. The mixture was transferred to a separatory funnel and extracted with DCM (3 x 100 mL). The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated with silica gel under reduced pressure on a rotary evaporator. The product was purified by column chromatography using DCM/MeOH (20:1 v/v) + 0.25 M NH3 as the eluent to obtain product 10 as light brown solid in 63% yield.

¹H NMR (600 MHz, DMSO) δ 10.34 (s, 2H), 8.68 (d, J = 1.4 Hz, 2H), 8.13 (d, J = 8.0 Hz, 2H), 8.02 (dd, J = 8.0, 2.1 Hz, 2H), 7.91 (d, J = 7.6 Hz, 2H), 7.33 (t, J = 7.8 Hz, 2H), 7.00 (dd, J = 7.5, 0.9 Hz, 2H), 3.86 (s, 4H), 3.33 (s, 4H), 2.03 (s, 6H).

¹³C NMR (101 MHz, DMSO ) δ 162.69, 148.38, 148.24, 143.56, 142.35, 137.12, 136.79, 129.03, 126.54, 126.34, 122.96, 122.31, 43.42, 15.01. General procedure (1) for the preparation of compounds of general formula III (11-14)

A round-bottom flask was charged with compound 10 (0.25-0.31 mmol, 1 eq.) followed by the addition of anhydrous DCM (10 mL) and triethylamine (1.25-1.56 mmol, 5 eq.). The reaction mixture was cooled in an ice bath. Then, an acylating agent was added (0.55-0.69 mmol, 2.2 eq.). The reaction was continued overnight at room temperature. Afterthe reaction was completed, the mixture was diluted with DCM and H2O was added (50 mL). The mixture was extracted three times with DCM. The organic layers were combined, dried over anhydrous sodium sulfate and concentrated with silica gel under reduced pressure on a rotary evaporator. The crude product was purified by column chromatography using a DCM/MeOH eluent system.

Example 6. N,N'-(2,2'-Dimethyl-[1,1'-biphenyl]-3,3'-diyl)bis(5

(vinylsulfonylamidomethyl)picolinamide) (11)

Following the general procedure described in Example 7, starting from compound 10 (0.12 g, 0.25 mmol), triethylamine (0.126 g, 0.174 mL, 1.25 mmol) and 2-chloroethylsulfonyl chloride (0.09 g, 0.55 mmol, 0.06 mL) in DCM (10 mL), product 11 (0.119 g) was obtained as a white precipitate in72% yield.

¹H NMR (600 MHz, DMSO) δ 10.32 (s, 2H), 8.65 (s, 2H), 8.15 (d, J = 8.1 Hz, 2H), 8.07-7.96 (m, 4H), 7.84 (d, J = 7.9 Hz, 2H), 7.31 (t, J = 7.8 Hz, 2H), 6.98 (d, J = 7.0 Hz, 2H), 6.74 (dd, J = 16.5, 10.0 Hz, 2H), 6.06 (d, J = 16.5 Hz, 2H), 5.98 (d, J = 10.0 Hz, 2H), 4.22 (d, 4H), 2.00 (s, 6H). ¹³C NMR (151 MHz, DMSO) δ 162.45, 149.18, 148.34, 142.30, 137.93, 137.75, 137.33, 136.68,

129.27, 126.63, 126.43, 126.27, 123.17, 122.42, 43.67, 14.98.

Example 7. N,N'- (2,2'-Dimethyl-[1,1^,-biphenyl]-3,3^,-diyl)bis(5-

(hydroxymethyl)picolinamide)) (12)

Following the general procedure described in Example 7, and starting from compound 10 (0.13 g, 0.27 mmol), triethylamine (0.137 g, 0.190 mL, 1.35 mmol) and acroyl chloride (0.053 g, 0.59 mmol, 0.048 mL) in DCM (10 mL), product 12 (0.086 g) was obtained as white solid in 54% yield.

¹H NMR (600 MHz, DMSO) δ 10.38 (s, 2H), 8.83 (t, J = 6.0 Hz, 2H), 8.70 (s, 2H), 8.21 (d, J = 8.0 Hz, 2H), 7.96 (dd, J = 36.1, 12 Hz, 4H), 7.38 (t, J = 7.8 Hz, 2H), 7.05 (d, J = 7.4 Hz, 2H), 6.34 (m, 2H), 6.20 (d, J = 18 Hz, 2H), 5.71 (dd, J = 10.2, 1.8 Hz, 2H), 4.55 (d, J = 5.8 Hz, 4H), 2.07 (s, 6H).

¹³C NMR (151 MHz, DMSO) δ 165.38, 162.46, 148.92, 148.23, 142.29, 138.97, 137.42, 136.68, 131.80, 129.14, 126.58, 126.33, 123.04, 122.50, 14.95.

Example 8. N,N'-(2,2'-Dimethyl-[1,1'-biphenyl]-3,3-diy)bis(5-((2- chloroacetamido)methyl)picolinamide) (13)

Following the general procedure described in Example 7, starting from compound 10 (0.15 g, 0.31 mmol), triethylamine (0.16 g, 0.22 mL, 1.56 mmol) and chloroacetyl chloride (0.08 g, 0.55 mmol, 0.069 mL) in DCM (10 mL), product 13 (0.056 g) was obtained as yellow solid in28% yield. ¹H NMR (400 MHz, DMSO) δ 10.29 (s, 2H), 8.87 (s, 2H), 8.60 (s, 2H), 8.11 (d, J = 7.7 Hz, 2H), 7.87 (dd, J = 25.5, 7.5 Hz, 4H), 7.28 (t, J = 7.2 Hz, 2H), 6.95 (d, J = 7.0 Hz, 2H), 4.42 (d, J = 4.6 Hz, 4H), 4.13 (s, 4H), 1.98 (s, 6H).

¹³C NMR (101 MHz, DMSO) δ 166.96, 162.50, 149.01, 148.21, 142.34, 138.62, 137.43, 136.73, 129.22, 126.65, 126.34, 123.12, 122.51, 43.11, 15.02.

Example 9. N,N'-(2,2'-Dimethyl-[1,1'-biphenyl]-3,3-diy)bis(5-((2- bromoacetamido)methyl)picolinamide) (14)

Following the general procedure described in Example 7, and starting from compound 10 (0.13 g, 0.27 mmol), triethylamine (0.14 g, 0.19 mL, 1.35 mmol) and bromoacetyl bromide (0.12 g, 0.6 mmol, 0.052 mL) in DCM (10 mL), product 14 (0.080 g) was obtained as white solid in 40% yield.

¹H NMR (400 MHz, DMSO) δ 10.29 (s, 2H), 8.92 (s, 2H), 8.59 (s, 2H), 8.11 (s, 2H), 7.86 (d, J = 30.8 Hz, 4H), 7.28 (s, 2H), 6.96 (s, 2H), 4.41 (s, 4H), 3.91 (s, 2H), 1.97 (s, 6H).

¹³C NMR (101 MHz, DMSO) δ 168.07, 167.03, 162.52, 149.03, 148.14, 142.34, 138.61, 137.37, 136.73, 129.33, 126.68, 126.33, 123.22, 122.53, 29.83, 15.04.

General procedure (2) for the preparation of compounds of general formula III (15-16)

A round-bottom flask was charged with corresponding carboxylic acid derivative (0.25-0.31 mmol, 3 eq.) followed by the addition of anhydrous DMF (10 mL) and diisopropylethylamine (6 eq.). Thus obtained reaction mixture was treated by the addition of HATU (1.25-1.56 mmol, 3 eq.). The contents of the flask were stirred vigorously for 30 min. After this time, the mixture was placed in an ice bath and, after cooling, compound 10 (0.55-0.69 mmol, 1 eq.) was added to the reaction. The reaction was continued overnight at room temperature. After completion of the reaction, the mixture was poured into cooled brine (50 mL). The mixture was extracted three times with DCM (3 x 50 mL). The organic layers were combined, dried over anhydrous sodium sulfate, and concentrated with silica gel under reduced pressure on a rotary evaporator. The crude product was purified by column chromatography using a DCM/MeOH or chloroform/MeOH eluent system.

Example 10. N,N'-(2,2'-Dimethyl-[1,1'-biphenyl]-3,3-diy)bis(5-((2- fluoroacrylamido))methyl)picolinamide) (15)

Following the general procedure 2, 2-fluoroacrylic acid (0.17 g, 1.87 mmol, 0.119 mL) was dissolved in DMF (10 mL) followed by the addition of diisopropylethylamine (0.484 g, 3.75 mmol, 0.652 mL) and HATU (0.71 g, 1.87 mmol). After 30 min., compound 10 (0.3 g, 0.62 mmol, 1 eq.) was added to afford product 15 (0.193 g) as a white solid in 53% yield. The product was purified by column chromatography using a 50:1 (v/v) DCM/methanol mixture as the eluent.

¹H NMR (400 MHz, DMSO) δ 10.29 (s, 2H), 9.20 (t, J = 5.9 Hz, 2H), 8.61 (d, J = 1.4 Hz, 2H), 8.11 (d, J = 8.0 Hz, 2H), 7.91 (dd, J = 8.1, 1.9 Hz, 2H), 7.83 (d, J = 7.9 Hz, 2H), 7.28 (t, J = 7.8 Hz, 2H), 6.95 (d, J = 7.2 Hz, 2H), 5.54 (dd, J = 48.0, 3.5 Hz, 2H), 5.27 (dd, J = 15.7, 3.5 Hz, 2H), 4.46 (d, J = 6.0 Hz, 4H), 1.98 (s, 6H).

¹³C NMR (101 MHz, DMSO) δ 162.51, 159.93, 159.61, 158.04, 155.37, 149.05, 148.33, 142.34, 138.46, 137.55, 136.73, 129.27, 126.67, 126.33, 123.17, 122.58, 99.68, 99.54, 15.02.

Example 11. N,N'-(2,2'-Dimethyl-[1,1'-biphenyl]-3,3-diy)bis(5-((E-)-3- methoxyacrylamido)methyl)picolinamide) (16)

Following the general procedure 2, monomethyl fumarate (0,19 g, 1.44 mmol) was dissolved in DMF (10 mL) followed by the addition of diisopropylethylamine (0.371 g, 2.87 mmol, 0.5 mL) and HATU (0.55 g, 1.44 mmol). After 30 min., compound 10 (0.23 g, 0.48 mmol, 1 eq.) was added to afford product 16 (0.273 g) as a white solid in 81% yield. The product was purified by column chromatography using a 30:1 (v/v) chloroform/methanol mixture as the eluent.

¹H NMR (600 MHz, DMSO-d6) δ 10.32 (s, 2H), 9.18 (t, J = 5.8 Hz, 2H), 8.67 (s, 2H), 8.16 (d, J = 8.0 Hz, 2H), 7.96 (dd, J = 8.0, 1.6 Hz, 2H), 7.89 (d, J = 8.0 Hz, 2H), 7.33 (t, J = 7.8 Hz, 2H), 7.08 (d, J = 15.5 Hz, 2H), 7.00 (d, J = 7.5 Hz, 2H), 6.66 (d, J = 15.5 Hz, 2H), 4.55 (d, J = 5.8 Hz, 4H), 3.74 (s, 6H), 2.02 (s, 6H).

¹³C NMR (151 MHz, DMSO-d6) δ 165.93, 163.64, 162.42, 149.05, 148.26, 142.30, 138.35, 137.61, 137.48, 136.68, 129.12, 129.04, 126.58, 126.27, 123.03, 122.52, 52.51, 40.56, 14.93.

Example 12. 1-((6-((3'-(5-((4-(Fluorosulfonyl)-lH-pyrazol-1-yl)methyl)picolinamido)-2,2'- dimethyl-[1,1'-biphenyl]-3-yl)carbamoyl)pyridin-3-yl)methyl)-1H-pyrazole-4-sulfonyl fluoride (17)

8 17

A round-bottom flask was charged with compound 8 (0.15 g, 0.29 mmol, 1 eq.) and K₂CO₃ (0.16 g, 1.16 mmol, 4 eq.), and DMF (4 mL) was added. The reaction mixture was cooled in an ice bath and 4-(l/7-pyrazole)sulfonyl fluoride (0.17 g, 1.16 mmol, 4 eq.) was added. The reaction was continued for 16 h. After this time, the reaction mixture was poured into ice/water mixture. DCM (50 mL) was added and the mixture was transferred to a separatory funnel. The mixture was extracted three times with DCM (3 x 50 mL). The organic layers were combined and washed with cold brine (2 x 50 mL). The organic layer was dried over sodium sulfate, filtered and concentrated with silica gel under reduced pressure on a rotary evaporator. The product was purified by column chromatography using DCM/MeOH (40:1 v/v) as the eluent, to obtain 0.208 g (96%) of product 17 as light brown solid.

¹H NMR (400 MHz, DMSO-d6) δ 10.31 (s, 2H), 9.12 (s, 2H), 8.71 (d, J = 1.9 Hz, 2H), 8.59 (s, 2H), 8.15 (d, J = 8.1 Hz, 2H), 7.98 (dd, J = 8.1, 2.1 Hz, 2H), 7.79 (d, J = 7.9 Hz, 2H), 7.28 (t, J = 7.8 Hz, 2H), 6.96 (d, J = 7.4 Hz, 2H), 5.62 (s, 4H), 1.97 (s, 6H).

¹³C NMR (101 MHz, DMSO-d6) δ 162.32, 150.12, 148.93, 142.33, 141.05, 138.57, 136.73, 135.02, 129.55, 126.80, 126.33, 123.42, 122.84, 113.15, 112.85, 112.75, 112.46, 53.43, 15.04.

Example 13. Methyl 6-((3-bromo-2-methylphenyl)carbamoyl)nicotinate (18)

5-(Methoxycarbonyl)picolinic acid 4 (3.98 g, 22.0 mmol, 1.2 eq.) was dissolved in 15 mL (207.0 mmol) of thionyl chloride and the mixture was heated to 85°C in an oil bath. The reaction was continued for 5 hours under a reflux condenser protected on the top with a calcium chloride drying tube. After the reaction was completed, the mixture was concentrated under reduced pressure on a rotary evaporator and dried under vacuum. The resulting acid chloride in the form of yellow solid was dissolved in 25 mL of anhydrous tetrahydrofuran and placed in a dropping funnel. At the same time, 3-bromo-2-methylaniline (2.26 mL, 18.3 mmol, 1.0 eq.) was dissolved in 10 mL of anhydrous tetra hydrofuran along with triethylamine (36.7 mmol, 5.11 mL, 2.0 eq.), and the resulting solution was placed in a round-bottom flask with a magnetic stirring bar. The reaction flask was purged with argon and cooled in an ice bath. An acid chloride solution was slowly added dropwise into the flask. The reaction was stirred for 12 h while being allowed to heat to ambient temperature and quenched by diluting with dichloromethane for subsequent extraction with brine (2 x 50 mL) and saturated sodium bicarbonate solution (1 x 50 mL). The organic phases were combined, dried over magnesium sulfate, filtered and concentrated under reduced pressure on a rotary evaporator. The crude product was purified by column chromatography on silica gel, using hexane/ethyl acetate 30:1-1:1 (v/v) as the eluent to obtain product 18 as a white solid in 92% yield.

¹H NMR (600 MHz, DMSO-d6) δ 10.67 (s, 1H), 9.19 (d, J = 1.4 Hz, 1H), 8.54 (dd, J = 8.1, 2.1 Hz, 1H), 8.30-8.23 (m, 1H), 7.58 (d, J = 7.9 Hz, 1H), 7.53 (d, J = 8.0 Hz, 1H), 7.20 (t, J = 8.0 Hz, 1H), 3.94 (s, 3H), 2.32 (s, 3H).

¹³C NMR (151 MHz, DMSO-d6) δ 164.60, 161.83, 152.71, 148.97, 138.95, 137.23, 132.45, 129.86, 128.06, 127.45, 125.11, 124.62, 122.56, 52.77, 18.10

Example 14. N -(3-Bromo-2-methylphenyl)-5-(hydroxymethyl)picolinamide (19)

Compound 18 (5.89 g, 16.86 mmol, 1.0 eq.) was dissolved in 60 mL of a mixture of anhydrous tetrahydrofuran and anhydrous methanol (2:1, v/v, 40:20 mL). The solution was cooled in an ice bath and purged with argon. Lithium borohydride (1.25 g, 57.32 mmol, 3.4 eq.) was dissolved in 5 mL of anhydrous tetrahydrofuran, placed in a dropping funnel, and slowly added dropwise into the cooled reaction flask. The reaction was stirred for 12 h and then quenched by dilution with ethyl acetate. The mixture was washed with saturated ammonium chloride solution. The organic layer was dried over magnesium sulfate, filtered and concentrated under reduced pressure on a rotary evaporator. The crude product was purified by column chromatography on silica gel, using the chloroform/methanol 70:1-20:1 (v/v) eluent system, to obtain product 19 as a white solid in 89% yield.

¹H NMR (600 MHz, DMSO-d6) δ 10.48 (s, 1H), 8.68 (dd, J = 2.0, 0.6 Hz, 1H), 8.13 (d, J = 7.9 Hz, 1H), 8.00-7.98 (m, 1H), 7.67 (d, J = 7.8 Hz, 1H), 7.50 (dd, J = 8.0, 0.9 Hz, 1H), 7.19 (t, J = 8.0 Hz, 1H), 5.53 (t, J = 5.7 Hz, 1H), 4.67 (d, J = 5.6 Hz, 2H), 2.35 (s, 3H).

¹³C NMR (151 MHz, DMSO-d6) δ 164.60, 161.83, 152.71, 148.97, 138.95, 137.23, 132.45, 129.86, 128.06, 127.45, 125.11, 124.62, 122.56, 52.77, 18.10. Example 15. N,N'- (3'-Amino-2,2'-dimethyl-[l,r-biphenyl]-3-yl)-5-

(hydroxymethyl)picolinamide) (20)

A three-neck round-bottom flask was charged with compound 19 (5.48 g, 17.1 mmol, 1.0 eq.), potassium carbonate (3.54 g, 25.6 mmol, 1.5 eq.), PdfdppfhCl-DCM (1.115 g, 1.36 mmol, 0.08 eq.) and 100 mL of dioxane/water mixture (1:1, v/v, 50:50 mL). The flask was flushed three times with argon. The mixture was placed in an oil bath at 90°C and stirred for 15 minutes. Next, compound 2 (5.53 g, 23.7 mmol, 1.4 eq.) dissolved in 50 mL of 1,4-dioxane, was placed in a dropping funnel and slowly added dropwise into the reaction flask. The reaction was stirred for another 40 h and quenched by the addition of ethyl acetate. The mixture was washed with brine (1 x 100 mL). The organic layer was separated, dried over magnesium sulfate, filtered and concentrated under reduced pressure on a rotary evaporator. The product was purified by column chromatography on silica gel using a 70:1-40:1 (v/v) dichloromethane/methanol mixture as the eluent, to obtain product 20 as a white solid in 84% yield.

¹H NMR (600 MHz, DMSO-d6) δ 10.33 (s, 1H), 8.69 (d, J = 1.2 Hz, 1H), 8.19 (d, J = 8.0 Hz, 1H), 8.03 (dd, J = 8.0, 1.9 Hz, 1H), 7.88 (d, J = 7.8 Hz, 1H), 7.30 (t, J = 7.8 Hz, 1H), 6.98 (d, J = 7.7 Hz, 1H), 6.96 (d, J = 7.0 Hz, 1H), 6.69 (d, J = 7.4 Hz, 1H), 6.35 (d, J = 6.9 Hz, 1H), 5.56 (t, J = 5.7 Hz, 1H), 4.95 (s, 2H), 4.70 (d, J = 5.6 Hz, 2H), 2.04 (s, 3H), 1.79 (s, 3H).

¹³C NMR (151 MHz, DMSO-d6) δ 162.51, 148.84, 147.32, 147.17, 143.54, 141.81, 136.43, 128.89, 126.48, 126.22, 125.97, 122.36, 122.30, 119.40, 117.74, 113.62, 60.84, 14.87, 14.38.

Example 16. N -(3'-Amino-2,2'-dimethyl-[l,l'-biphenyl]-3-yl)-5-(((tert- butyldimethylsilyl))(oxy)methyl)picolinamide (21)

A round-bottom flask was charged with compound 20 (2.06 g, 5.93 mmol, 1.0 eq.), which was then dissolved in 35 mL of anhydrous acetonitrile and 10 mL of anhydrous dimethylformamide in an argon atmosphere. The solution was cooled to 0°C in an ice bath, and imidazole (0.807 g, 11.86 mmol, 2.0 eq.) was added. A solution of tert-butyl dimethylsilyl chloride (1.21 g, 8.0 mmol, 1.35 eq., TBDMSCI) dissolved in 10 mL of anhydrous acetonitrile was added dropwise into the flask. The reaction was continued for 14 hours and quenched by the addition of cold water. The resulting precipitate was filtered-off, washed with cold water and dried under vacuum to afford product 21 as a white solid in 89% yield.

¹H NMR (600 MHz, DMSO) δ 10.18 (s, 1H), 8.55 (s, 1H), 8.05 (d, J = 8.0 Hz, 1H), 7.89-7.83 (m, J = 8.0, 1.7 Hz, 1H), 7.73 (d, J = 8.0 Hz, 1H), 7.15 (t, J = 7.8 Hz, 1H), 6.82 (q, J = 14.6, 7.1 Hz, 2H), 6.54 (d, J = 7.9 Hz, 1H), 6.20 (d, J = 7.0 Hz, 1H), 4.80 (s, 2H), 4.76 (s, 2H), 1.88 (s, 3H), 1.64 (s, 3H), 0.80 (s, 9H), -0.00 (s, 6H).

¹³C NMR (151 MHz, DMSO) δ 162.41, 149.08, 147.18, 146.88, 143.54, 141.79, 140.59, 136.42, 136.05, 128.88, 126.49, 126.22, 125.96, 122.38, 119.39, 117.72, 113.62, 62.38, 26.25, 18.44, 14.87, 14.38, -4.86.

General procedure (3) for the preparation of compounds of general formula V

Depending on the substrate, derivatives of arylfluorosulfonyl acids of general formula V were obtained as follows:

1) starting from an amine derivative, in two steps involving the Sandmeyer reaction with thionyl chloride and the halide exchange reaction with potassium hydrogen fluoride (Examples 17-19); or

2) starting from chlorosulfonyl acid in the direct reaction with potassium hydrogen fluoride (Example 20); or

3) starting from a phenolic derivative in reaction with l,l'-sulfonylbis(l/7-imidazole) and silver fluoride (Example 21)

In the first step, an aqueous solution of thionyl chloride was prepared by 3 mL (1.83 mmol) of thionyl chloride being slowly added dropwise into 12 mL of water cooled in an ice bath without stirring. The solution was left to stand for 12 h. After this time, the solution was cooled again in an ice bath and 70 mg (0.7 mmol, 0.11 eq.) of copper(l) chloride was added. The solution was slowly stirred for 20 minutes. At the same time, the corresponding acid (6.2 mmol, 1.0 eq.) was dissolved in 2 mL of glacial acetic acid and 5 mL of 36% HCI solution. The mixture was cooled to -5°C. A solution of 450 mg (6.52 mmol, 1.05 eq.) of sodium nitrite in 6 mL of water cooled to 0°C was slowly added in a dropwise fashion and the reaction mixture was stirred for 0.5 hours. The resulting diazonium salt solution was added dropwise using a cooled syringe into a previously prepared and cooled thionyl chloride solution. The reaction was continued for 3 hours, with the temperature of the mixture being maintained at 0-5°C. The precipitated solid was filtered on a Buchner funnel, washed with cold water and dried under vacuum. Potassium hydrogen fluoride (3.0 eq.) was suspended in 2 mL of acetonitrile and 1.5 mL of water. The product was then dissolved in 1 mL of glacial acetic acid and 2 mL of acetonitrile and added to the reaction mixture. The reaction was stirred for 16 h at ambient temperature. The solution was combined with ethyl acetate and washed with 2M HCI solution. The organic layer was separated and dried over magnesium sulfate, filtered and concentrated under reduced pressure on a rotary evaporator and dried under vacuum.

Example 17. 4-(Fluorosulfonyl)benzoic acid (23)

Following the general procedure 3, starting from p-aminobenzoic acid 22, the product 23 was obtained as a white solid in 43% yield.

¹H NMR (600 MHz, DMSO) δ 8.27 (s, 4H).

¹³C NMR (151 MHz, DMSO) δ 166.04, 138.25, 131.41, 129.29.

Example 18. 5-(Fluorosulfonyl)pyridine-2-carboxylic acid (26)

Following the general procedure 3, starting from 2-(5-amino)pyridinic acid 24, the product 25 was obtained as a white solid in 48% yield.

¹H NMR (600 MHz, DMSO) δ 9.40 (s, 1H), 8.79 (t, J=23.1 Hz, 1H), 8.45-8.28 (m, 1H). Example 19. 4-(Fluorosulfonyl)-2-methylbenzoic acid (27)

Following the general procedure 3, starting from 4-amino-2-benzoic acid 26, the product 27 was obtained as a white solid in 48% yield.

¹H NMR (400 MHz, DMSO) δ 7.98 (s, 1H), 7.94-7.86 (m, 2H), 2.52 (s, 3H).

¹³C NMR (101 MHz, DMSO) δ 172.57 , 168.83 , 142.63 , 140.17 , 132.53 (d, J = 23.2 Hz), 130.81 (d, J = 17.2 Hz), 126.16 , 20.77.

Example 20. 3-(Fluorosulfonyl)benzoic acid (29)

(3-Chlorosulfonyl)benzoic acid (7g, 31.73 mmol, 1 eq.) was dissolved in 35 mL of 1,4-dioxane. Next, 35 mL of 2M aqueous KHF2 solution (5.7 g, 73 mmol, 2.3 eq.) was added. The reaction was continued overnight at room temperature. After the reaction was completed, the mixture was diluted with ethyl acetate and extracted three times. The organic layers were combined and dried over anhydrous sodium sulfate. The solvent was evaporated to give (3- fluorosulfonyl)benzoic acid 29 as a light brown solid in 90% yield (5.81 g).

¹H NMR (400 MHz, DMSO) δ 8.45-8.39 (m, J = 5.2 Hz), 8.38-8.33 (m), 7.90 (t, J = 7.8 Hz).

¹³C NMR (101 MHz, DMSO) δ 165.69, 137.41, 133.38, 132.78, 132.52, 131.87, 129.10.

¹⁹F NMR (376 MHz, DMSO) δ 66.48. Example 21. 4-(Fluorosulfonyloxy)benzoic acid (32)

30 31 32 p-Hydroxybenzoic acid 30 (1.90 mmol, 1.0 eq.) and l,l'-sulfonylbis(l/7-imidazole) (31) (753 mg, 3.80 mmol, 2.0 eq.) were dissolved in 10 mL of THF. The solution was stirred vigorously and cesium carbonate (309 mg, 0.95 mmol, 0.5 eq.) was introduced. The reaction was continued for 24 h. After completion, the reaction mixture was diluted with ethyl acetate and washed with IM HCI solution. The organic layer was dried with sodium sulfate, filtered and concentrated on a rotary evaporator. The resulting white solid was dried under vacuum (yield 97%). The resulting solid was then dissolved in 10 mL of a mixture of acetonitrile and water (5:1, v/v) and silver fluoride (723 mg, 5.7 mmol, 3.0 eq.) was added. The reaction was stirred for 6 h at 60°C. After the reaction was completed, the mixture was diluted with ethyl acetate and washed with IM HCI solution. The precipitated silver chloride was removed by filtration. The organic layer was dried over sodium sulfate, filtered and concentrated under reduced pressure on a rotary evaporator. The product was purified by column chromatography on silica gel eluting with a 150:1 (v/v) dichloromethane/methanol mixture. The resulting white solid was dried under vacuum to afford the product in 72% yield.

¹H NMR (400 MHz, DMSO) δ 7.98 (s, 1H), 7.94-7.86 (m, 2H), 2.52 (s, 3H).

¹³C NMR (101 MHz, DMSO) δ 172.57 , 168.83, 142.63, 140.17, 132.53 (d, J = 23.2 Hz), 130.81, 126.16, 20.77.

General procedure (4) for the preparation of intermediate compounds of general formula

VI

In the first step, the corresponding compound V (1.1 mmol, 1.7 eq.) was dissolved in 10 mL of anhydrous dichloromethane and thionyl chloride (1.64 mL, 22.6 mmol) was slowly added dropwise. The solution was stirred for 16 h, after which the solvents were evaporated under reduced pressure. The resulting yellow substance (acid chloride) was dissolved under argon in a round-bottom flask in 2 mL of anhydrous THF. The flask was cooled in an ice bath. Anhydrous pyridine (1.95 mmol, 157 pL, 3.0 eq.) was then added and the solution was stirred for 20 minutes. A solution of compound 21 (300 mg, 0.65 mmol, 1.0 eq.) and anhydrous TEA (182 pL, 1.3 mmol, 2.0 eq.) in anhydrous THF was slowly added dropwise (1.5 h) into the cooled acid chloride solution. The reaction was continued for 7 hours and stopped by dilution with ethyl acetate. The organic layer was washed with brine and water. After separation from the aqueous layer, the organic layer was dried over magnesium sulfate and concentrated on a rotary evaporator under reduced pressure. The resulting solid was dissolved in a small volume of chloroform and purified by column chromatography on silica gel. The product was eluted with a 120:1 (v/v) chloroform/acetonitrile mixture to obtain a white solid product. Example 22. 4-((3'-(5-((((tert-Butyldimethylsilyl)oxy)methyl)picolinamido)-2,2^,-dimethyl-

[l,l'-biphenyl]-3-yl)carbamoyl)benzenesulfonyl fluoride (33)

Following the general procedure 4, starting from 4-(fluorosulfonyl)benzoic acid 23, the product 33 was obtained as a white solid in 35% yield.

¹H NMR (600 MHz, CDCI₃) δ 10.00 (s, 1H), 8.44 (d, J = 1.2 Hz, 1H), 8.14-8.11 (m, 2H), 8.01 (s, 4H), 7.75 (s, 2H), 7.74-7.70 (m, 1H), 7.23-7.15 (m, 2H), 6.96-6.94 (m, 1H), 6.82 (d, J = 6.9 Hz, 1H), 4.71 (s, 2H), 1.98 (s, 3H), 1.91 (s, 3H), 0.83-0.81 (m, 12H), O.Ol-(-O.Ol) (m, 6H).

Example 23. 6-((3'-(5-(((tert-Butyldimethylsilyl)oxy)methyl)picolinamido)-2,2'-dimethyl-

[l,l'-biphenyl]-3-yl)carbamoyl)pyridine-3-sulfonyl fluoride (34)

Following the general procedure 4, starting from 5-(fluorosulfonyl)pyridine-2-carboxylic acid 25, the product 34 was obtained as a white solid in 30% yield.

¹H NMR (600 MHz, CDCI₃) δ 10.05 (s, 1H), 9.86 (s, 1H), 9.10 (d, J = 2.1 Hz, 1H), 8.48 (d, J = 8.3 Hz, 1H), 8.45 (d, J = 1.3 Hz, 1H), 8.40 (dd, J = 8.3, 2.2 Hz, 1H), 8.17 (d, J = 8.0 Hz, 1H), 8.12 (td, J = 11.3, 4.0 Hz, 2H), 7.75 (dd, 1H), 7.20 (dt, 2H), 6.92 (dd, J = 7.5, 0.9 Hz, 1H), 6.85 (dd, J = 7.5, 0.9 Hz, 1H), 4.72 (s, 2H), 1.99 (s, 6H), 0.83-0.81 (m, 12H), 0.02-(-0.01) (m, 6H).

¹³C NMR (151 MHz, CDCI3) δ 161.94, 159.53, 155.48, 148.72, 147.63, 145.88, 142.60, 141.89, 140.27, 138.27, 136.12, 135.60, 135.21 , 132.59-132.19, 126.97, 126.82, 126.37, 126.04, 123.11, 122.36, 120.98, 77.24, 77.03, 76.82, 62.54, 25.88, 18.37, 14.42 , -5.30.

Example 24. 4-((3'-(5-(((tert-Butyldimethylsilyl)oxy)methyl)picolinamido)-2,2^,-dimethyl- [l,l'-biphenyl]-3-yl)carbamoyl)-3-methylbenzenesulfonyl fluoride (35)

Following the general procedure 4, starting from 4-(fluorosulfonyl)-2-methylbenzoic acid 27, the product 35 was obtained as a white solid in 69% yield.

¹H NMR (400 MHz, CDCI3) δ 10.12 (s, 1H), 8.57 (d, J = 1.0 Hz, 1H), 8.25 (dd, J = 7.8, 4.3 Hz, 2H), 7.92 (t, J = 9.8 Hz, 3H), 7.84 (dd, J = 8.0, 1.8 Hz, 1H), 7.75 (d, J = 7.8 Hz, 1H), 7.48 (s, 1H), 7.32 (dt, J = 16.0, 7.8 Hz, 2H), 7.08 (d, J = 7.4 Hz, 1H), 6.95 (d, J = 7.4 Hz, 1H), 4.84 (s, 2H), 2.64 (s, 3H), 2.11 (s, 3H), 2.02 (s, 3H), 0.95 (s, 12H), 0.13 (s, 6H).

¹³C NMR (101 MHz, ) δ 162.27, 148.91, 146.28, 143.39-142.80, 141.88, 141.69, 140.23,

138.97, 138.83, 136.23, 135.35, 135.10, 130.87, 127.89, 126.46, 126.30, 126.23, 126.03,125.95, 123.03,122.57, 122.14, 121.02, 77.43, 77.11, 76.80, 62.64, 25.96, 19.95, 18.45,

14.83, 14.55, 1.12, -5.22.

Example 25. 3-((3'-(5-(((tert-Butyldimethylsilyl)oxy)methyl)picolinamido)-2,2'-dimethyl-

[l,l'-biphenyl]-3-yl)carbamoyl)benzenesulfonyl fluoride (36)

Following the general procedure 4, starting from 3-(fluorosulfonyl)-benzoic acid 29, the product 36 was obtained as a white solid in 65% yield.

¹H NMR (400 MHz, DMSO) δ 10.40 (s), 10.30 (s), 8.69-8.62 (m), 8.50 (d, J = 7.8 Hz), 8.33 (d, J = 8.0 Hz), 8.13 (d, J = 8.0 Hz), 7.97-7.90 (m), 7.83 (d, J = 8.0 Hz), 7.37 (d, J = 7.7 Hz), 7.29 (t, J = 7.9 Hz), 7.03 (d, J = 7.4 Hz), 6.96 (d, J = 7.5 Hz), 4.84 (s), 1.98 (s), 1.91 (s), 0.88 (s), 0.07 (s).

¹³C NMR (101 MHz, DMSO) δ 163.55, 162.59, 149.10, 146.96, 142.58, 142.29, 140.69, 136.78, 136.75, 136.60, 136.14, 136.12, 132.61, 131.66, 131.52, 129.22, 127.98, 127.84, 126.56, 126.37, 126.29, 123.24, 122.50, 62.43, 26.31, 18.50, 15.59, 15.01, -4.80.

Example 26. 4-((3'-(5-((((tert-Butyl-dimetholsilyl)oxy)methyl)picolinamido)-2,2'-dimethyl- [l,l'-biphenyl]-3-yl)carbamoyl)phenyl fluorosulfate (37)

37

Following the general procedure 4, starting from 4-(fluorosulfonyloxy)benzoic acid 32, the product 37 was obtained as a white solid in 34% yield.

Example 28. 4-((3^,-(5-(((tert-Butyl-dimetholsilyl)oxy)methyl)picolinamido)-2,2' dimethyl- [l,l'-biphenyl]-3-yl)carbamoyl)-2-methylbenzenesulfonyl fluoride (39)

Following the general procedure 4, starting from commercially available 4-(fluorosulfonyl)-3- methylbenzoic acid 38, the product 39 was obtained as a white solid in 80% yield.

¹H NMR (400 MHz, CDCI₃) δ 10.12 (s, 1H), 8.56 (d, J = 0.7 Hz, 1H), 8.28-8.21 (m, 2H), 8.14 (d, = 8.2 Hz, 1H), 7.98-7.79 (m, 4H), 7.36-7.27 (m, 2H), 7.10-7.04 (m, 1H), 6.95 (t, J = 6.9 Hz, 1H), 4.84 (s, 2H), 2.75 (s, 2H), 2.10 (s, 3H), 2.03 (s, 3H), 0.95 (s, 9H), 0.13 (s, 6H).

Example 29. 4-(((3'-(5-((((tert-Butyl-dimetholsilyl)oxy)methyl)picolinamido)-2,2^,-dimethyl-

[l,l'-biphenyl]-3-yl)amino)methyl)benzenesulfonyl fluoride (41)

Compound 21 (0.15 g, 0.32 mmol, 1 eq.) was dissolved in anhydrous DMF (2 mL) in a 5 mL vial. Potassium carbonate (0.033 g, 0.49 mmol, 1.5 eq.) was added. The reaction mixture was cooled in an ice bath. Then. 4-(bromomethyl)benzenesulfonyl fluoride 40 (0.123 g, 0.49 mmol, 1.5 eq.) was injected into the mixture. The reaction was continued for 16 h. After completion, the reaction was poured into ice/water mixture and extracted twice with ethyl acetate. The organic layers were washed with cold brine, dried over magnesium sulfate, concentrated, and applied onto the chromatography column. The crude product was purified by column chromatography on silica gel. The product was eluted with a 120:1 (v/v) chloroform/MeOH mixture to obtain product 41 (0.081 g) as a white solid in 39% yield.

¹H NMR (600 MHz, CDCI₃) δ 9.99 (s, 1H), 8.44 (d, J = 1.3 Hz, 1H), 8.18-8.12 (m, 2H), 7.86 (d, J = 8.4 Hz, 2H), 7.74-7.70 (m, 1H), 7.54 (d, J = 8.2 Hz, 2H), 7.16 (t, J = 7.8 Hz, 1H), 6.95 (t, J = 7.8 Hz, 1H), 6.85 (dd, J = 7.5, 1.0 Hz, 1H), 6.48 (d, J = 7.2 Hz, 1H), 6.32 (d, J = 8.0 Hz, 1H), 4.71 (s, 2H), 4.46 (s, 2H), 1.99 (s, 3H), 1.80 (s, 3H), 0.82 (s, 7H), 0.00 (s, 5H).

¹³C NMR (151 MHz, CDCI3) δ 162.15, 149.02, 148.78, 146.22, 145.18, 142.79, 142.19, 140.04, 135.98, 135.37, 135.19, 128.99, 128.83, 128.31, 128.12, 126.58, 126.35, 126.17, 126.03, 122.15, 122.05, 120.60, 120.49, 120.33, 119.88, 109.18, 108.93, 62.75, 62.44, 47.96, 25.89, 18.38, 14.49, 14.40, 13.97, 13.77, -5.22, -5.36.

General procedure (5) for the preparation of final compounds of general formula IV

IV

Final compounds of general formula IV were obtained using one of the two variants of general procedure 5:

Variant 1: The appropriate intermediate compound VI (0.50 mmol, 1.0 eq.) was dissolved in 4 mL of anhydrous tetra hydrofuran and 70 pL of glacial acetic acid in an argon atmosphere. The reaction flask was cooled to -5°C and IM TBAF (1.1 mmol, 2.2 eq.) was slowly added dropwise. The solution was slowly stirred for 6 h while maintaining the temperature of about 0°C. The reaction was quenched by diluting the mixture with dichloromethane. The organic layer was washed twice with saturated ammonium chloride solution. The organic layer was separated, dried over magnesium sulfate, filtered and concentrated on a rotary evaporator. The resulting crude product was dissolved in a small volume of chloroform and purified by column chromatography on silica gel. The products were obtained as white solids in yields of 70 to 75%.

Variant 2: The appropriate intermediate compound VI (0.09-4.0 mmol, 1.0 eq.) was dissolved in anhydrous acetonitrile. The reaction mixture was cooled down to 0 °C. A solution of Olah reagent (HF-pyridine 70%, 35 M) was added dropwise into the mixture. The reaction was continued overnight. After completion, the reaction mixture was diluted with cold water and extracted three times with dichloromethane. The organic layers were combined and washed with 0.5 M HCI solution. The organic layer was dried over anhydrous sodium sulfate. After evaporation of the solvent, the crude product was purified by column chromatography. Example 30. 4-((3'-(5-(Hydroxymethyl)picolinamido)-2,2'-dimethyl-[l,l'-biphenyl]-3- yl)carbamoyl)benzenesulfonyl fluoride (42)

Following variant 1 of the general procedure 5 and starting from compound 33, product 42 was obtained as a white solid in 71% yield.

¹H NMR (600 MHz, DMSO) δ 10.40 (s, 1H), 10.35 (s, 1H), 8.67 (d, J = 1.2 Hz, 1H), 8.33 (s, 4H), 8.16 (d, J = 8.0 Hz, 1H), 8.00 (dd, J = 8.0, 1.9 Hz, 1H), 7.88 (d, J = 7.9 Hz, 1H), 7.43 (d, J = 7.8 Hz, 1H), 7.34 (td, J = 7.7, 2.7 Hz, 2H), 7.08 (d, J = 7.3 Hz, 1H), 7.00 (d, J = 7.4 Hz, 1H), 5.53 (t, J = 5.7 Hz, 1H), 4.67 (d, J = 5.6 Hz, 2H), 2.03 (s, 3H), 1.97 (s, 3H).

Example 31. 6-((3'-(5-(Hydroxymethyl)picolinamido)-2,2'-dimethyl-[l,l'-biphenyl]-3- yl)carbamoyl)pyridine-3-sulfonyl fluoride (43)

Following variant 1 of the general procedure 5 and starting from compound 34, product 43 was obtained as a white solid in 70% yield. ¹H NMR (600 MHz, DMSO) δ 10.46 (s, 1H), 10.46 (s, 1H), 10.23 (s, 1H), 10.23 (s, 1H), 9.30 (d, J = 2.1 Hz, 1H), 8.72 (dd, J = 8.4, 2.3 Hz, 1H), 8.55 (d, J = 1.3 Hz, 1H), 8.35 (d, J = 8.4 Hz, 1H), 8.04 (d, J = 8.0 Hz, 1H), 7.88 (dd, J = 8.0, 2.0 Hz, 1H), 7.76 (d, J = 7.9 Hz, 1H), 7.59 (d, J = 7.9 Hz, 1H), 7.22 (dt, J = 10.9, 7.8 Hz, 2H), 6.94 (d, J = 7.5 Hz, 1H), 6.88 (d, J = 7.5 Hz, 1H), 5.40 (t, J = 5.6 Hz, 1H), 4.54 (d, J = 5.3 Hz, 2H), 1.91 (s, 3H), 1.89 (s, 3H).

Example 32. 4-((3'-(5-(Hydroxymethyl)picolinamido)-2,2^,-dimethyl-[l,l'-biphenyl]-3- yl)carbamoyl)-3-methylbenzenesulfonyl fluoride (44)

Following variant 1 of the general procedure 5 and starting from compound 35, product 44 was obtained as a white solid in 75% yield.

¹H NMR (400 MHz, DMSO) δ 10.32 (s, 1H), 10.14 (s, 1H), 8.63 (d, J = 1.1 Hz, 1H), 8.12 (d, J = 7.6

Hz, 2H), 8.06 (d, J = 8.1 Hz, 1H), 7.96 (dd, J = 8.0, 1.8 Hz, 1H), 7.88-7.81 (m, 2H), 7.49 (d, J = 7.8

Hz, 1H), 7.29 (t, J = 7.9 Hz, 2H), 7.02 (d, J = 7.3 Hz, 1H), 6.95 (d, J = 7.3 Hz, 1H), 5.49 (t, J = 5.7

Hz, 1H), 4.63 (d, J = 5.6 Hz, 2H), 2.54 (s, 3H), 1.97 (d, J = 6.9 Hz, 6H).

¹³C NMR (101 MHz, ) δ 166.69, 162.68, 148.87, 147.39, 145.11, 142.60, 142.26, 141.94, 138.63, 136.80, 136.49, 136.28, 132.66,32.34, 131.68, 130.38, 129.44, 129.20, 127.77, 126.55, 126.31, 125.90,

Example 33. 3-((3'-(5-(Hydroxymethyl)picolinamido)-2,2'-dimethyl-[l,l'-biphenyl]-3- yl)carbamoyl)benzenesulfonyl fluoride (45)

Following variant 2 of the general procedure 5 and starting from compound 36 (3,94 mmol, 2,55 g), product 45 was obtained as a white solid in 96% yield.

¹H NMR (400 MHz, DMSO) δ 10.40 (s), 10.31 (s), 8.69-8.60 (m), 8.50 (d, J = 7.8 Hz), 8.34 (d, J = 8.0 Hz), 8.11 (d, J = 8.0 Hz), 7.98-7.90 (m), 7.83 (d, J = 7.9 Hz), 7.37 (d, J = 7.6 Hz), 7.29 (t, J = 7.8 Hz), 7.05-7.02 (m), 6.98-6.93 (m), 5.48 (t, J = 5.7 Hz), 4.62 (d, J = 5.6 Hz), 1.99 (s), 1.92(s).

¹³C NMR (101 MHz, DMSO) δ 163.55, 162.67, 148.86, 147.38, 142.59, 142.28, 141.93, 136.81, 136.74, 136.60, 136.48, 136.16, 132.62, 132.35, 131.68, 131.53, 129.19, 127.99, 127.85, 126.53, 126.37, 126.30, 123.23, 122.41, 79.71, 60.88, 15.60, 15.02.

Example 34. 4-((3'-(5-(Hydroxymethyl)picolinamido)-2,2^,-dimethyl-[l,l'-biphenyl]-3- yl)carbamoyl)phenyl fluorosulfate (46)

Following variant 1 of the general procedure 5 and starting from compound 37, product 46 was obtained as a white solid in 73% yield. ¹H NMR (600 MHz, DMF) δ 10.40 (s, 1H), 10.27 (s, 1H), 8.72 (d, J = 1.3 Hz, 1H), 8.35 (d, J = 8.8 Hz, 2H), 8.24 (d, J = 8.0 Hz, 1H), 8.17-8.13 (m, 1H), 8.10 (dd, J = 8.0, 2.1 Hz, 1H), 8.03 (s, 1H), 7.85 (d, J = 8.8 Hz, 2H), 7.56 (d, J = 7.8 Hz, 1H), 7.38 (dt, J = 10.2, 7.8 Hz, 2H), 7.12 (dd, J = 7.5, 0.9 Hz, 1H), 7.04 (dd, J = 7.5, 0.9 Hz, 1H), 5.71 (t, J = 5.7 Hz, 1H), 4.81 (d, J = 5.6 Hz, 2H), 2.15 (s, 3H), 2.08 (s, 3H).

Example 35. 4-((3'-(5-(Hydroxymethyl)picolinamido)-2,2^,-dimethyl-[l,l'-biphenyl]-3- yl)carbamoyl)-2-methylbenzenesulfonyl fluoride (47)

Following variant 1 of the general procedure 5 and starting from compound 39, product 47 was obtained as a white solid in 70% yield.

¹H NMR (400 MHz, DMSO) δ 10.30 (d, J = 6.7 Hz, 2H), 8.62 (s, 1H), 8.24-8.01 (m, 4H), 7.95 (d, J = 7.6 Hz, 1H), 7.83 (d, J = 8.0 Hz, 1H), 7.37 (d, J = 7.7 Hz, 1H), 7.29 (t, J = 7.8 Hz, 2H), 7.03 (d, J = 7.3 Hz, 1H), 6.95 (d, J = 7.4 Hz, 1H), 5.48 (t, J = 5.6 Hz, 1H), 4.62 (d, J = 5.6 Hz, 2H), 2.68 (s, 3H), 1.95 (d, J = 26.5 Hz, 6H).

Example 36. 4-(((3'-(5-(Hydroxymethyl)picolinamido)-2,2^,-dimethyl-[l,l'-biphenyl]-3- yl)amino)methyl)benzenesulfonyl fluoride (48)

48

Following variant 2 of the general procedure 5 and starting from compound 41, product 48 was obtained as a white solid in 73% yield.

¹H NMR (400 MHz, CDCI₃) δ 10.11 (s, 1H), 8.59 (s, 1H), 8.25 (dd, J = 12.4, 8.1 Hz), 7.99 (d, J = 8.2 Hz, 2H), 7.90 (d, J = 8.0 Hz, 2H), 7.67 (d, J = 8.1 Hz, 1H), 7.29 (t, J = 7.8 Hz, 2H), 7.07 (t, J = 7.8 Hz, 1H), 6.98 (d, J = 7.5 Hz, 1H), 6.60 (d, J = 7.5 Hz, 1H), 6.44 (d, J = 8.0 Hz, 1H), 4.83 (s, 2H), 4.59 (s, 2H), 2.12 (s, 3H), 1.93 (s, 3H).

Example 37. Determination of in vitro activity of compounds - inhibition of PD-L1/PD-1 interaction

The inhibitory activity of PD-1/PD-L1 compounds was determined using a Cisbio reagent kit (W64PD1PEG) and the homogeneous time-resolved fluorescence (HTRF) method.

Experiments were conducted in 96-well white plates in total volumes of 20 pL. Compounds were tested at two (5 and 50 nM) or five concentrations to determine IC₅₀ values. The tested compounds were mixed with PD-L1 (5 nM) and PD-l-Fc (50 nM) proteins. In the next step, prepared mixtures were treated with Anti-6HIS-Eu cryptate Gold antibody solution (HTRF donor) and Anti Human lgG-XL665 antibody solution (HTRF acceptor). The reaction was continued for 1 h at room temperature. Measurements were carried out using a microplate reader (TECAN). The excitation wavelength was 340 nm, and the emission wavelengths were 620 nm and 665 nm. The results were normalized for positive-control signals (PD-L1/PD-1 complex without inhibitor) and negative-control signals (no hPD-1 protein). The results of three independent measurements were averaged to afford single values. The results are presented as the undissociated

PD-1/PD-L1 complex percentage contents (Table 1).

Table 1. Activity of compounds - inhibition of PD-L1/PD-1 interaction

The I C50 values were also determined for selected compounds. The measurements were taken at lh, 4h, 8h, and 24h to confirm the covalent nature of bonding (a decrease in IC₅₀ overtime). The results were plotted as HTRF signal vs. compound concentration relationships and compared to the reference PD-L1/PD-1 interaction inhibitor, compound A - cmpd A (Park et al. 2021, doi.org/10.1038/s41467-021-21410-l) (Fig. 1A-1C). Both compound 11 (Fig. 1A) and compound 17 (Fig. IB) of this invention demonstrated incubation time-dependent changes in IC₅₀ values (shifts toward lower values) typical of covalent inhibitors. No such effect was demonstrated for compound A (Fig. 1C), which does not interact with PD-L1 in a covalent manner. Compounds of the invention may constitute one of the active ingredients, or the only one active ingredient of a pharmaceutical composition, wherein derivatives and/or pharmaceutically acceptable salts, preferably hydrochloride, are also contemplated. Pharmaceutical agent according to the invention may be in a form suitable for oral, parenteral, intranasal, or sublingual administration. Specifically, the agent may be in the form of a tablet, pill, capsule, powder, orgranules. Suitable pharmaceutical forms of the agent according to the invention are prepared by methods known in the art of pharmacy.

Solid forms, such as tablets, pills, powders, granules or capsules, are prepared by mixing the active ingredient with a pharmaceutical carrier. The carrier can be selected from corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate, or rubbers (natural and synthetic). When mixed with a pharmaceutically acceptable solvent, such as water, a homogeneous mixture of the compound or its pharmaceutically acceptable salt is formed for subsequent tableting, sugar coating or encapsulation.

Example 38. Confirmation of in vitro covalent dimerization of PD-L1 by means of polyacrylamide gel electrophoresis (SDS-PAGE)

The selected inhibitors of the invention were designed to be able to form covalently bound dimers of the PD-L1 by reacting with the side chain groups of lysine 124 (Lysl24) or tyrosine 123 (Tyr 123) (Table 2). To verify this hypothesis, SDS-PAGE analysis was performed on protein samples following the treatment with selected inhibitors. Compounds showing the postulated mechanism of action were expected to form PD-L1 dimers, which should result in the appearance of an additional stripe having a molecular weight equivalent to twice that of the PD-L1 monomer. The intensity of the dimer band should increase with the incubation time.

Method Description:

A solution of PD-L1 (15 μM, 200 pL) in 10 mM Tris pH 8.0 buffer, 20 mM NaCI, was treated with compound 17. The final concentration of the compound was 75 μM. The mixture was sampled at time intervals of 0, 1, 2, 4, 6, 8, 16 and 24 h. Samples were resuspended in loading buffer (10 pL) and heated to 95 °C immediately after collection. The samples, along with the molecular weight standard, were introduced into the wells of the polyacrylamide gel. Samples were separated in a 10% gel. Electrophoresis was carried out in a BioRad apparatus at a constant voltage of 100 V. After completion, the gels were stained with a staining solution to visualize the bands. The described activity of compound 17 is demonstrated in Fig. 2.

Example 39. Confirmation of covalent binding of inhibitors to PD-L1 - determination protein mass by intact mass spectrometry

Mass spectrometry (MS) is a technique for determining the exact mass of a evaluated protein. To confirm the covalent binding of the inhibitor to the PD-L1, samples of the protein treated with the compounds of the invention were subjected to intact MS analysis.

Method Description:

Aliquots of the PD-L1 solution (50 μM, 200 pL) in 10 mM Tris buffer pH 8.0, 20 mM NaCI were treated with the tested compounds (17, 42, and 48). The final concentration of compounds was 250 μM. The mixture was sampled at the beginning (0 h) and at the end (24 h) of the experiment. Samples were analyzed using a microTOF-Q II mass spectrometer (Bruker Daltonics, Germany) equipped with an electrospray ionization source. The instrument was calibrated prior to measurements with ESI-L Low Concentration Tuning Mix (Agilent Technologies). Samples were desalted on Amicon Ultra-0.5 3K centrifugal filters (Millipore) using 0.05% formic acid (FA) as a washing solution. Desalted samples were mixed with 50% MeCN in 0.05% FA in a 1:1 ratio and directly injected into the mass spectrometer using a syringe pump at a flow rate of 3 pL/min. The mass spectrometer was operated in positive mode, with electron spray ionization voltage of 4500 V and dry gas temperature of 180 °C. MS scans were taken in the mass range of m/z 50-3000. MS spectra were processed using the Maximum Entropy Deconvolution algorithm as implemented in the Data Analysis 4.1 software (Bruker Daltonics, Germany). Selected results are presented in Figs. 3-6

Example 40. Confirmation of biological activity of compounds within a cell system

Compounds showing the ability to covalently dimerize PD-L1 have been tested for this activity in a cell system. The results are illustrated using compound 17 as an example.

Method Description: U2OS G8 cells were seeded onto 12-well plates at 120,000 cells/well. After 24h, the medium was replaced and IFN-y was added at the concentration of 20 mg/mL to all wells except the controls. Compound 17 and compound A (cmpd A) were then added to the appropriate wells at a concentration of 5 μM. The control wells were filled with the carrier in an amount corresponding to the volume in which the added compounds were suspended. After 24h, protein lysates were harvested and subjected to SDS-PAGE separation and transfer onto a membrane. Lysates were incubated with antibodies as shown in Table 2 below.

Table 2:

In addition, an experiment as described below was performed to demonstrate that the inhibitors covalently dimerize PD-L1 at the binding sites of anti-PD-Ll antibodies. The results are illustrated using compound 17 as an example.

Method Description: CHO cells were seeded onto 12-well plates at 100,000 cells/well. After 24 hours, antibodies (Atezolizumab or Durvalumab) at a concentration of 5 pg/mLwere added to the appropriate wells and incubated for 30 min. Compound 17 was then added to the appropriate wells at a concentration of 5 μM. The control wells were filled with the carrier in an amount corresponding to the volume in which the added compounds were suspended. After another 24 h, protein lysates were harvested and subjected to SDS-PAGE separation and transfer onto a

The lysates were incubated with antibodies according to the method above. The results of the experiments are illustrated in Fig. 7

The reproducibility of the observed non-obvious effect of covalent dimerization of PD-L1 was verified by similar tests being performed in additional cell lines expressing either human PD- L1 protein (CHO/TCRAct, RKO/TCRAct, U-2 OS and Mc38 hPD-Ll lines) or murine PD-L1 protein (Mc38 WT line). The results are illustrated using compound 17 as an example.

Method Description: CHO/TCRAct, RKO/TCRAct, U-2 OS, Mc38 hPD-Ll or Mc38 WT cells were seeded onto 12-well plates. After 24 h, the medium was replaced and (for the U-2 OS line) new medium containing human IFN-y at the concentration of 20 mg/mL was added to all wells except the controls for 48 h, or (for the Mc38 WT line) new medium containing murine IFN-y at the concentration of 20 mg/mL was added to all wells except the controls for 48 h. The remaining lines were not subjected to interferon treatment. Next, compound 17 at concentrations of either 1, 5 or 10 μM and compound A (cmpd A) at a concentration of 5 μM were then added to the corresponding wells. The control wells were filled with the carrier in an amount corresponding to the volume in which the added compounds were suspended. After another 24 h, protein lysates were harvested and subjected to SDS-PAGE separation and transfer onto a membrane. The membranes were incubated with antibodies according to the method above. The results of the experiments are illustrated in Fig. 8 and Fig. 9

To test the ability of the presented compounds to block the surface of PD-L1 protein, a flow cytometry analysis was carried out using compound 17 as an example inhibitor. In the experiment, the compound was first covalently bound to the PD-L1 protein on the surface of Mc38 hPD-Ll cells; the binding was followed by an attempt to bind a fluorescently labeled antibody to the same protein. Lower cell fluorescence values were observed in the presence of compound 17, comparable to those observed for the control therapeutic antibody (durvalumab). The results of the experiment are illustrated in Fig. 10

Method Description: Mc38 hPD-Ll cells were seeded onto 12-well plates. After 24 hours, the medium was replaced and either compound 17 at a concentration of 5 μM or durvalumab at a concentration of 5 pg/mL was added to the appropriate wells for 24 h or 30 minutes, respectively. The control wells were filled with the carrier in an amount corresponding to the volume in which the added compounds were suspended. Cells were harvested and stained using either an anti-PD-Ll antibody (MIH1 clone) stained with APC fluorescent dye or isotype control. A flow cytometry assay was performed. The results of the experiments are illustrated in Fig. 10

LIST OF ABBREVIATIONS:

EtOAc, ethyl acetate;

AcOH, acetic acid;

CHCh, chloroform;

DMSO, dimethylsulfoxide; DCM, dichloromethane;

DMF, dimethylformamide;

HTRF, homogeneous time-resolved fluorescence

DIPEA, diisopropylethylamine;

MW, molecular weight;

MeOH, methanol;

MeCN, acetonitrile;

MgSOa, magnesium sulfate;

MS, mass spectrometry;

NMR, nuclear magnetic resonance;

PD-1, programmed death receptor 1;

PD-L1, programmed death receptor ligand;

SDS-PAGE, polyacrylamide gel electrophoresis under denaturing conditions;

SOCh, thionyl chloride;

THF, tetrahydrofuran;

TLC, thin-layer chromatography.

Claims

Claims

1. A compound of general formula (I) and/or its pharmaceutically acceptable salts

wherein:

- L₁ is -(CH₂)- or -C(O)-;

- A is an aryl or a heterocycle containing at least one nitrogen atom,

- R₁ is hydrogen or alkyl; R₂ is hydrogen or alkyl, wherein both R₁ and R₂ can be hydrogens at the same time;

- R₃ is selected from the group comprising -SO₂F, -OSO₂F,

- Rds selected from the group comprising -(CH₂)OH,

wherein:

- L₂ is -(CH₂)-, -C(O)- or -S(O₂)-;

- X₁ is a hydrogen or a halogen;

- X₂ is CH₂ or -CHC(O)OCH₃;

- X₃ is a halogen or -CH₂SO₂F; wherein R₃ and Rzican be the same or different.

2. A compound according to claim 1 as defined by formula (II)

wherein:

- L is -C(O)-;

- R₁and Rz are hydrogens;

- Rsand R4 are selected from the group comprising

where

- X₁ is a hydrogen or a halogen;

- X₂ is a hydrogen or -CHC(O)OCH₃;

- X₃ is a halogen or -CH₂SO₂F;

3. A compound according to claim 1 or 2 as defined by the formula (III)

wherein:

- A is an aryl;

- R₁and R2 are hydrogens;

- Rsand R4 are selected from the group comprising

4. A compound according to claim 1. of formula (IV)

- L₁ is -(CH₂)- or -C(O)-;

- A is an aryl or a heterocycle containing at least one nitrogen atom;

- R₁ is hydrogen or alkyl;

- R₂ is hydrogen or alkyl, wherein both R₁ and R₂ can be hydrogens;

- R₃ is selected from the group comprising -SO₂F, -OSO₂F;

5. A compound according to claim 4, wherein A is selected from group comprising

6. A compound according to claim 1, 2, or 3 selected from a group comprising

7. A compound according to claim 1 or 4 selected from a group comprising

8. A compound of general formula (I) for use as a PD-L1/PD-1 interaction inhibitor.

9. A pharmaceutical composition comprising a compound of general formula (I) and/or a pharmaceutically acceptable salt thereof according to claims 1 to 7, and a pharmaceutically acceptable carrier.

10. A compound of general formula (I) and/or a pharmaceutically acceptable salt thereof according to claims 1 to 7 for use in the preparation of a drug for the prevention and/or treatment of a disease dependent on the PD-1/PD-L1 pathway.