AU2020104403A4 - Compound and application thereof in synthesis of pdl1 antagonist drug molecules - Google Patents

Abstract

The disclosure provides a compound. The compound of the disclosure, as an intermediate, can be used for synthesizing a PDL1 antagonist. A compound prepared by using the compound of the disclosure as the intermediate has relatively high inhibition activity on PD-i/PD-Li signals in a cell level.

Description

AUSTRALIA

Patents Act 1990

COMPLETE SPECIFICATION INNOVATION PATENT COMPOUND AND APPLICATION THEREOF IN

SYNTHESIS OF PDL1 ANTAGONIST DRUG

MOLECULES

The following statement is a full description of this invention, including the best

method of performing it

known to me:

COMPOUND AND APPLICATION THEREOF IN SYNTHESIS OF PDL1 ANTAGONIST DRUG MOLECULES

TECHNICAL FIELD

[0001] The disclosure relates to a PD-Li antagonist compound and a method for

treating/preventing immune-associated diseases using the PD-Li antagonist

compound.

BACKGROUND

[0002] Due to its excellent curative effect and innovation, tumor immunotherapy

was named the most important scientific breakthrough annually by Science in 2013.

Tumor immunotherapy is expected to become an innovation in the field of tumor

treatment following surgery, chemotherapy, radiotherapy and targeted therapy. Tumor

immunotherapy applies an immunology principle and method to improve the

immunogenicity of tumor cells and sensitivity on effector cell killing, and stimulate

and enhance body antitumor immunity response, and applies immune cells and

effector molecules to be injected into a host and cooperates a body immune system to

kill tumors and inhibit growth of tumors. Tumor immunotherapy has received much

attentions recently, and is a focus for tumor treatment. In recent years, tumor

immunotherapy gains increasing good news and has currently exhibited strong

antitumor activity in treatment of some tumor types such as melanoma and non-small

cell lung cancer. Furthermore, drugs for tumor immunotherapy have been approved

by Food and Drug Administration (FDA) to be clinically applied.

[0003] PD-i (programmed death 1) is a member of CD28 superfamily.

Immunomodulation targeting PD-i is of great significance in the aspects of

anti-tumors, anti-infection, anti-autoimmune diseases, organ transplantation and

survival and the like. Its ligand PD-Li can also be used as a target spot, and a

corresponding antibody can also plays the same role. PD-Li (programmed death-Ligand 1) is a type 1 transmembrane protein having a size of 40 kDa. Under normal circumstances, the immune system generates reaction on foreign antigens gathered in lymph glands or spleen to promote the proliferation of antigen-specific T cells. The binding of PD-i and PD-L can conduct inhibitory signals and reduce the proliferation of T cells.

[0004] One way for tumor cells to escape T cell destruction is to produce PD-Li on

its surface. After PD-i on the surfaces of immune cells T cells recognizes PD-Li, it

can conduct inhibitory signals, and T cells cannot detect tumor cells and give out

attack signals to tumor cells. PD-i is a new immunotherapy by relieving tumor cells

to escape the immune system. The mechanism of PD-i immunotherapy is to design a

specific protein antibody against PD-i or PD-Li, prevent the recognition process of

PD-i and PD-Li and partially restore the function of T cells, so that T cells can kill

tumor cells.

[0005] PD-i is expressed in activated T cells, B cells and myeloid cells. It has two

ligands, namely PD-Li and PD-L2. PD-Li/L2 is expressed in antigen presenting cells,

and PD-Li is also expressed in multiple tissues. The binding of PD-i and PD-Li

mediates a co-inhibitory signal for T cell activation, regulates activation and

proliferation of T cells, and plays a negative regulatory role similar to CTLA-4.

Liping Chen, a Chinese scientist, first discovered high expression of PD-Li in tumor

tissues and a function of regulating the function of tumor infiltrating CD8 T cells.

Therefore, immunomodulation targeting PD-i/PD-Li is greatly significant in

anti-tumors.

[0006] Multiple therapeutic monoclonal antibodies (mAbs) targeting PD-i/PD-Li

interaction have been approved by FDA. In addition to the development of relevant

monoclonal antibodies, searching small molecular compounds that are convenient for

oral administration of patients to perform targeted inhibition of immune checkpoints

is also a frontier field of tumor immunotherapy. Small molecule compounds can pass

through cytomembranes to act on intracellular target spots, and therefore have a

widely application scope. Next, the chemically modified small molecules often have good bioavailability and compliance, so as to effectively avoid the decomposition and inactivation of enzymes in a digestive tract. Finally, in multiple levels such as production processes, dosage form design and administration modes, researches on small molecules are also quite mature.

[0007] The use way of the most monoclonal antibodies (mAbs) is intravenous administration at high doses. Small molecule drugs are more suitable for oral administration, which can reduce severe immune related adverse events. Compared with monoclonal antibodies, small molecule drug inhibitors have many other advantages, such as more economical and stable manufacturing costs, and better organ and tumor permeability. Considering the many advantages of small molecule pharmacokinetics, it will show dose flexibility in single therapy or other combination solutions. The small molecule compound of the disclosure can provide an attractive treatment choice for patients and doctors.

SUMMARY

[0008] The disclosure provides an intermediate 12 for synthesizing a PDL1 antagonist compound, having the following structure:

0 Br C1

-'t' o 0

12 N CN

[0009] In addition, the disclosure also provides a method for synthesizing the intermediate 12, comprising the following steps:

[0010] The synthesis route of the intermediate 12 is as follows: 0

NC Cl CI Br CO I. 0 OH __ _ _ _N I TsOH, MeOH K2C03, KI, DMF 12 N

CN

[0011] Specifically, the disclosure provides the preparation process of the intermediate 12, comprising the following steps: adding an intermediate 11 (1.3 g, 3.68 mmol) into methanol (10 mL), adding trimethyl orthoformate (1.96 g, 18.42 mmol) and p-toluenesulfonic acid (31.73 mg, 0.184 mmol), stirring the reaction mixture for overnight at 50°C until reaction solution is concentrated, dissolving residues into

N,N-dimethyl formamide (10 mL), adding potassium carbonate (1.53 g, 11.05 mmol),

potassium iodide (61.17 mg, 0.368 mmol) and 4-choromethyl-2-cyanopridine (1.3 g,

3.68 mmol), stirring the reaction mixture for 6 h at 70°C, cooling the reaction solution

to room temperature, adding 4 mol/L hydrochloric acid (5 mL), continuously stirring

for 20 min, filtering, washing a filter cake three times with 10 mL of water, washing

twice with 10 mL of ethyl acetate, and drying to obtain the intermediate 12.

[0012] In the technical solution of the disclosure, synthesis of the intermediate Il

comprises the following steps: HO OH 0 Br 0 r CI HA Br KI I Br H2 N ~ NaNO 2 , HCI NBS, BPO Br K- -0 O H Br OH OH

|1

[0013] Specifically, synthesis of the intermediate Il comprises the following steps:

[0014] Step 1: adding 2-bromo-3-methylaniline (31.5 g, 169.32 mmol) into

concentrated hydrochloric acid (100 mL), stirring for 20 min at room temperature,

adding 250 g of broken ice into a system, slowly adding sodium nitrite (26.25 g,

380.43 mmol) under ice bath, controlling the temperature of reaction solution to be

less than 5C, keeping stirring for 1 h under ice bath after addition is completed,

subsequently slowly adding potassium iodide (112.89 g, 679.99 mmol) under ice bath,

continuously stirring the reaction solution for 2 h, slowly heating to room temperature,

then stirring for 1 h at 60°C, cooling the reaction solution to room temperature,

quenching with 200 mL of sodium bisulfite, extracting with petroleum ether (3 x 200

mL), combining organic phases, washing once with 200 mL of saturated sodium

bicarbonate, then washing once with 150 mL of saturated salt solution, drying the

organic phase with anhydrous sodium sulfate, filtering, concentrating, separating

residues with a rapid column loading machine to obtain 2-bromo-3-methyl iodobenzene;

[0015] Step 2: dissolving 2-bromo-3-methyl iodobenzene (33.0 g, 111.14 mmol) into carbon tetrachloride (400 mL), adding N-bromosuccinimide (39.6 g, 222.27 mmol) and benzoyl peroxide (5.0 g, 20.66 mmol), refluxing the reaction mixture at a nitrogen atmosphere for overnight, then filtering, concentrating filtrate, separating a crude product with a rapid column loading machine to obtain 2-bromo-3-bromomethyliodobenzene (28.0 g, 74.50 mmol); and

[0016] Step 3: dissolving 2-bromo-3-bromomethyliodobenzene (14.0 g, 37.25 mmol) and 5-chloro-2,4-dihydroxybenzaldehyde (6.43 g, 37.25 mmol) into acetonitrile (150 mL), adding sodium bicarbonate (10.0 g, 119.05 mmol), stirring the reaction mixture and refluxing for overnight, cooling the reaction solution to room temperature, adding 150 mL of water under the stirring, continuously stirring for 20 min, filtering, washing a filter cake three times with 20 mL of water, and drying to obtain the intermediate Il.

[0017] Further, the disclosure also provides a method for preparing a PDL1 antagonist drug molecule 1 utilizing the intermediate compound 12, the PDL1 antagonist drug molecule 1 having the following structural formula: OH

HN

~' Br O

N C O N N HO CN

[0018] wherein, the method comprises the following steps:

0 0

Br Pd(dppf)C, K 2C0 3 N Br N~ 0 0 0 0

12 CN HO Ia CN

OH

HN O CI OH 1. L-serine, HOAc Br CO 2. NaBH(OAc) 3 N O H N

HO6 CN

[0019] Step 1:dissolving the intermediate 12 (200 mg, 0.34 mmol) and an

intermediate 16 (190.86 mg, 0.51 mmol) into 1,4-dioxane (5 mL), adding potassium

carbonate (94.73 mg, 0.68 mmol) and [1,1'-bis(diphenylphosphino) ferrocene]

palladium dichloride (25.08 mg, 0.034 mmol), stirring the reaction mixture for

overnight at 95C at a nitrogen atmosphere, cooling the reaction solution to room

temperature, adding 30 mL of ethyl acetate, filtering with diatomite, washing the

filtrate three times with 10 mL of water, washing once with 10 mL of saturated salt

solution, drying with anhydrous sodium sulfate, filtering, concentrating, and

separating residues with a rapid column loading machine to obtain compound la (80

mg, 0.11 mmol) which has a yield of 33.3% and is a light yellow solid. MS (ESI): mlz

700.3 (M+H) +.

[0020] Step 2: dissolving the compound la (80 mg, 0.11 mmol) into a mixture of

N,N-dimethylformamide (3 mL) and acetic acid (0.2 mL), adding L-serine (23.99 mg,

0.23 mmol), stirring the reaction mixture for overnight at 45C, then adding sodium

triacetoxyborohyride (90.67 mg, 0.43 mmol), continuing to stir for 3 h, cooling the

reaction solution to room temperature, dropping water for quenching, filtering, and

purifying the filtrate with a reverse preparative chromatography to obtain compound

1.

[0021] Wherein, the intermediate 16 has the following structure:

X0 / N 16 HO

[0022] In the method of the disclosure, the synthesis method of the intermediate 16 comprises the following steps:

[0023] the synthesis route of the intermediate 16 is as follows:

Br'- -Cl- HO ' NH

HN BrK2CO3, MeCN C__N Br K2CO3,23N Br

H6

B-B dr 0 N

KOAc, Pd(dppf)C1 2 16 N

HO

[0024] Specifically, the synthesis method of the intermediate 16 comprises the following steps:

[0025] Step 1: dissolving 1-bromo-3-chloropropane (5.99 g, 38.06 mmol) and 4-bromo-1H-indazole (5.0 g, 25.38 mmol) into acetonitrile (50 mL), adding potassium carbonate (7.01 g, 50.75 mmol), stirring the reaction mixture for overnight at 60°C, cooling the reaction solution to room temperature, filtering, concentrating, and separating the residues with a rapid column loading machine to obtain 4-bromo-1-(4-chloropropyl)-1H-indazole;

[0026] Step 2: dissolving 4-bromo-1-(4-chloropropyl)-1H-indazole (1.5 g, 5.48 mmol) and (R)-3-hydroxypyrrolidine (955.42 mg, 10.97 mmol) into acetonitrile (20 mL), adding potassium carbonate (3.03 g, 21.93 mmol) and potassium iodide (0.25 g, 1.48 mmol), stirring the reaction mixture for overnight at 60°C, cooling the reaction solution to room temperature, filtering, concentrating the filtrate, then adding 50 mL of ethyl acetate, washing three times with 20 mL of water, then washing once with saturated salt solution, drying with anhydrous sodium sulfate, filtering, and concentrating to obtain (R)-1-(4-(4-bromo-1H-indazole-1-yl) propyl) pyrrolidine-3-ol; and

[0027] Step 3: dissolving (R)-1-(4-(4-bromo-1H-indazole-1-yl)propyl)

pyrrolidine-3-ol (1.78 g, 5.49 mmol) and pinacol bis (borate) (2.09 g, 8.24 mmol) into

1,4-dioxane (20 mL), adding potassium acetate (1.62 g, 16.47 mmol) and 1,1'-bis

(diphenylphosphino) ferrocene] palladium dichloride (401.72 mg, 0.549 mmol),

stirring the reaction mixture for overnight at 90°C at a nitrogen atmosphere, cooling

the reaction solution to room temperature, adding 100 mL of ethyl acetate, filtering

with diatomite, washing the filtrate three times with 50 mL of water, washing once

with 50 mL of saturated salt solution, drying with anhydrous sodium sulfate, filtering,

concentrating, and separating the residues with a rapid column machine to obtain the

intermediate 16.

DETAILED DESCRIPTION

[0028] Intermediate Il: 0 Br CI

O OH

|1

[0029] The synthesis route of the intermediate I1 was as follows: HO OH0 Br KI Br BrBr C H 2N NaNO 2 . HCI NBS, BPO " - Br Br

[0030] Step 1: 2-bromo-3-methylaniline (31.5 g, 169.32 mmol) was added into

concentrated hydrochloric acid (100 mL) and stirred for 20 min at room temperature,

250 g of broken ice was added into a system, sodium nitrite (26.25 g, 380.43 mmol)

was slowly added under ice bath, the temperature of reaction solution was controlled

to be less than 5C, stirring was kept for 1 h under ice bath after addition was

completed, subsequently, potassium iodide (112.89 g, 679.99 mmol) was slowly

added, the reaction solution was continuously stirred for 2 h and slowly heated to room temperature, the stirring was performed for 1 h at 60°C, the reaction solution was cooled to room temperature, quenched with 200 mL of sodium bisulfite and extracted with petroleum ether (3 x 200 mL), organic phases were combined, washed once with 200 mL of saturated sodium bicarbonate, then washed once with 150 mL of saturated salt solution, the organic phase was dried with anhydrous sodium sulfate, filtered and concentrated, and residues were separated with a rapid column loading machine to obtain 2-bromo-3-methyl iodobenzene (36.0 g, 121.23 mmol) which was light yellow liquid and had a yield of 71.6%.

[0031] Step 2: 2-bromo-3-methyl iodobenzene (33.0 g, 111.14 mmol) was dissolved

into carbon tetrachloride (400 mL), N-bromosuccinimide (39.6 g, 222.27 mmol) and

benzoyl peroxide (5.0 g, 20.66 mmol) were added, refluxing was carried out on a

reaction mixture at a nitrogen atmosphere for overnight, then the reaction mixture was

filtered, the filtrate was concentrated, and a crude product was separated with a rapid

column loading machine to obtain 2-bromo-3-bromomethyliodobenzene (28.0 g,

74.50 mmol) which was light yellow liquid and had a yield of 67.0%; and

[0032] Step 3: 2-bromo-3-bromomethyliodobenzene (14.0 g, 37.25 mmol) and

5-chloro-2,4-dihydroxybenzaldehyde (6.43 g, 37.25 mmol) were dissolved into

acetonitrile (150 mL), sodium bicarbonate (10.0 g, 119.05 mmol) was added, the

reaction mixture was stirred and refluxed for overnight, the reaction solution was

cooled to room temperature, 150 mL of water was added under the stirring,

continuously stirred for 20 min and filtered, and a filter cake was washed three times

with 20 mL of water and dried to obtain the intermediate 11 (17.0 g, 36.36 mmol)

which was a white solid and had a yield of 97.6%. MS (ESI): m/z 464.9 (M-H)-.

[0033] Intermediate 12: 0 Br C1

12 CN

[0034] The synthesis route of the intermediate 12 was as follows:

0BNC B C Br ci CBr

TsOH, MeOH K2C0 3 , KI, DMF

II 12 CN

[0035] The intermediate 11 (1.3 g, 3.68 mmol) was added into methanol (10 mL), trimethyl orthoformate (1.96 g, 18.42 mmol) and p-toluenesulfonic acid (31.73 mg, 0.184 mmol) were added, the above reaction mixture was stirred for overnight at 50°C until reaction solution was concentrated, residues were dissolved into N,N-dimethyl formamide (10 mL), potassium carbonate (1.53 g, 11.05 mmol), potassium iodide (61.17 mg, 0.368 mmol) and 4-choromethyl-2-cyanopridine (1.3 g, 3.68 mmol) were added, the above reaction mixture was stirred for 6 h at 70°C, the reaction solution was cooled to room temperature, 4 mol/L hydrochloric acid (5 mL) was added, the above solution was continuously stirred for 20 min and filtered, and a filter cake was washed three times with 10 mL of water, washed twice with 10 mL of ethyl acetate and dried to obtain the intermediate 12 (0.97 g, 2.07 mmol) which was a light yellow solid and had a yield of 56.1%. MS (ESI): m/z 583.3 (M+H)+.

[0036] Intermediate 16:

6 HO

[0037] The synthesis route of the intermediate 16 was as follows:

I Br "' C I HO, KINH HN Br K2 C0, MeCN N Br K2 C0 3 , KI N Br

HO B-BN

KOAc, Pd(dppf)C12 6

HO

[0038] Step 1: 1-bromo-3-chloropropane (5.99 g, 38.06 mmol) and

4-bromo-1H-indazole (5.0 g, 25.38 mmol) were dissolved into acetonitrile (50 mL),

potassium carbonate (7.01 g, 50.75 mmol) was added, the reaction mixture was stirred

for overnight at 60°C, the reaction solution was cooled to room temperature, filtered

and concentrated, and the residues were separated with a rapid column loading

machine to obtain 4-bromo-1-(4-chloropropyl)-1H-indazole (3.0 g, 10.97 mmol),

which was light yellow liquid and had a yield of 43.2%. MS (ESI): mz 273.4

(M+H)+.

[0039] Step 2: 4-bromo-1-(4-chloropropyl)-1H-indazole (1.5 g, 5.48 mmol) and

(R)-3-hydroxypyrrolidine (955.42 mg, 10.97 mmol) were dissolved into acetonitrile

(20 mL), potassium carbonate (3.03 g, 21.93 mmol) and potassium iodide (0.25 g,

1.48 mmol) were added, the reaction mixture was stirred for overnight at 60°C, the

reaction solution was cooled to room temperature and filtered, the filtrate was

concentrated, then 50 mL of ethyl acetate was added, and the above solution was

washed three times with 20 mL of water, then washed once with saturated salt

solution, dried with anhydrous sodium sulfate, filtered and concentrated to obtain

(R)-1-(4-(4-bromo-1H-indazole-1-yl) propyl) pyrrolidine-3-ol (1.78 g, 5.49 mmol)

which was light yellow liquid and had a yield of 100%. MS (ESI): mz 324.4

(M+H)+.

[0040] Step 3: (R)-1-(4-(4-bromo-1H-indazole-1-yl) propyl) pyrrolidine-3-ol (1.78 g,

5.49 mmol) and pinacol bis (borate) (2.09 g, 8.24 mmol) were dissolved into

1,4-dioxane (20 mL), potassium acetate (1.62 g, 16.47 mmol) and 1,1'-bis

(diphenylphosphino) ferrocene] palladium dichloride (401.72 mg, 0.549 mmol) were

added, the reaction mixture was stirred for overnight at 90°C at a nitrogen atmosphere,

the reaction solution was cooled to room temperature, 100 mL of ethyl acetate was

added, the above solution was filtered with diatomite, the filtrate was washed three

times with 50 mL of water, washed once with 50 mL of saturated salt solution, dried

with anhydrous sodium sulfate, filtered and concentrated, and the residues were

separated with a rapid column machine to obtain the intermediate 16 (1.38 g, 3.74

mmol) which was light yellow liquid and had a yield of 68.1%. MS (ESI): m/z 372.4

(M+H)+.

[0041] Synthesis of compound 1

OH 0 HN

Nr CO OH

N K HO CN

0 0 Br CI Br CI C Pd(dppf)C, K2C03 N NN C

12 CN Ho 1a CN

OH

HN O CI OH 1. L-serine, HOAc Br C 2. NaBH(OAc) 3

N N

Ho CN

[0042] Step 1: the intermediate 12 (200 mg, 0.34 mmol) and the intermediate 16

(190.86 mg, 0.51 mmol) were added into 1,4-dioxane (5 mL), potassium carbonate

(94.73 mg, 0.68 mmol) and [1,1'-bis(diphenylphosphino) ferrocene] palladium

dichloride (25.08 mg, 0.034 mmol) were added, the reaction mixture was stirred for

overnight at 95°C at a nitrogen atmosphere, the reaction solution was cooled to room

temperature, 30 mL of ethyl acetate was added, the above solution was filtered with

diatomite, the filtrate was washed three times with 10 mL of water, washed once with

10 mL of saturated salt solution, dried with anhydrous sodium sulfate, filtered and

concentrated, and residues were separated by using a rapid column loading machine to

obtain compound la (80 mg, 0.11 mmol) which had a yield of 33.3% and was a light

yellow solid. MS (ESI): m/z 700.3 (M+H) +.

[0043] Step 2: the compound la (80 mg, 0.11 mmol) was dissolved into a mixture of

N,N-dimethylformamide (3 mL) and acetic acid (0.2 mL), L-serine (23.99 mg, 0.23

mmol) was added, the reaction mixture was stirred for overnight at 45C, then sodium

triacetoxyborohydride (90.67 mg, 0.43 mmol) was added and continued to stir for 3 h,

the reaction solution was cooled to room temperature, water was dropwise added to

the reaction solution, the above reaction solution was quenched and filtered, and the

filtrate was purified with a reverse preparative chromatography to obtain compound 1

(4.6 mg, 5.82 umol) which had a yield of 5.1% and was a white solid. MS (ESI): mlz

789.3 (M+H)+. 1H NMR (500 MHz, d6-DMSO) 6 8.75 (d, J= 5.0 Hz, 1H), 8.17 (s,

1H), 7.89 (d, J = 5.0 Hz, 1H), 7.74 (d, J= 8.5 Hz, 1H), 7.70 (d, J= 10.0 Hz, 2H), 7.56 (q, J= 7.0, 5.5 Hz, 2H), 7.53-7.43 (m, 2H), 7.09 (d, J = 7.0 Hz, 1H), 7.04 (s, 1H), 5.39

(d, J= 4.5 Hz, 2H), 5.33 (s, 2H), 4.83 (s, 1H), 4.49 (s, 3H), 4.21 (s, 1H), 4.03 (s, 2H),

3.77-3.60 (m, 2H), 3.21 (s, 1H), 2.72 (d, J = 7.5 Hz, 3H), 2.36 (s, 1H), 2.10-1.91 (m,

5H), 1.59 (s, 1H).

[0044] Bioactivity detection of PD-i/PD-Li signal inhibition in cell level

[0045] This detection method is used for estimation of bioactivity of the compound

1 of the disclosure in a cell level

[0046] Experimental principle

[0047] In this method, a luciferase reporter gene method was used to detect the

biological activity of a compound in PD-i/PD-Li signal inhibition in a cell level.

PD-1/NFAT-Reporter-Jurkat cells stably expressed human PD-i and expressed the

luciferase reporter gene regulated by an NFAT element; TCR activator/PD-LI-CHO

cells stably expressed human PD-Li and TCR activating elements. When two cell

strains were co-cultured, the binding of PD-I/PD-Li inhibited a TCR signal pathway,

thus inhibiting the expression of luciferase reporter genes controlled by downstream

NFAT. When the PD-i/PD-Li antibody or inhibitor was added, this inhibitory effect

was reversed and luciferase was expressed, thereby detecting the influence of the

PD-i/PD-Li inhibitor on luciferase activity.

[0048] Experimental materials and devices

[0049] PD-1/NFAT-Reporter-Jurkat cells (Art 60535) and TCR

activator/PD-LI-CHO cells (Art 60536) were purchased from BPS Bioscience

company; the PD-Li antibody (Atezolizumab, Art A2004) was purchased from

Selleck company; and luciferase detection reagent (ONE-GloTM Luciferase Assay

System (Art E6120) was purchased from Promega company, and a multifunctional

microplate detector (model Spectramax i3x) was purchased from Molecular Devices

company.

[0050] The main experimental process

[0051] PD-1/NFAT-Reporter-Jurkat cells and TCR activator/PD-Li-CHO cells were

cultured according to the routine cell culture procedure.

[0052] TCR activator/PD-Li-CHO cells were collected and inoculated into a

96-well plate with 35000 cells/well for incubation overnight at 37°C, and the medium

volume was 100 L. On the next day, the medium was discarded and the compounds

were added to be incubated for 30 minutes. At the same time, solvent control (DMSO,

final concentration was 0.1%) and PD-L antibody (Atezolizumab, final concentration

was about 10 nM) positive control were set. PD-/NFAT-reporter-Jurkat cells were

added. After continuous culture for 6 h, the activity of luciferase was detected

according to the instructions of the luciferase detection reagent.

[0053] The PD-Li antibody was used as the positive control to calculate the

following value of the tested compound: PD-i/PD-Li binding inhibition rate(%)=

(average chemiluminescence value of compound treatment pore/average

chemiluminescence value of solvent control pore-1)/(average chemiluminescence

value of PD-Li antibody pore/average chemiluminescence value of solvent control

pore-1) x 100%.

[0054] According to the above detection method, the biological activity estimation

was carried out on the compound I of the disclosure in the cell level. Test results are

as follows.

Compound Absolute EC50 (UM) number

1 0.34

Claims (10)

The claims for defining the invention are as follows:

1. An intermediate 12 for synthesizing a PDL1 antagonist compound, having the following structure:

0 Br C1

B 0 0

12 N CN

2. A method for preparing the intermediate 12 according to claim 1, comprising the following steps:

0 0 NC C1 C1 C1 CI Br Br / N I I -~ 0 O 0K 1 O TsOH,MeOH K2 C0 3 , KI, DMF

11 12 CN

3. The method according to claim 2, comprising the following steps: adding an intermediate Il into methanol, adding trimethyl orthoformate and p-toluenesulfonic acid, stirring the reaction mixture at 50°C for overnight until reaction solution is concentrated, dissolving residues into N,N-dimethylformamide, adding potassium carbonate, potassium iodide and 4-choromethyl-2-cyanopridine, stirring the above reaction mixture for 6 h at 70°C, cooling the reaction solution to room temperature, adding 4 mol/L hydrochloric acid, continuously stirring, filtering, washing a filter cake three times with water, washing twice with ethyl acetate, and drying to obtain an intermediate 12.

4. The method according to claim 3, wherein synthesis of the intermediate Il comprises the following steps:

HO OH 0 Br Br BrC i H Br KI H2N NaNO2 ,HCI NBS, BPO I Br HO HO C O

II

5. The method according to claim 4, wherein the preparation process of the intermediate

Il comprises the following steps:

Step 1: adding 2-bromo-3-methylaniline into concentrated hydrochloric acid, stirring for 20 min at room temperature, adding 250 g of broken ice into a system, slowly adding sodium nitrite under ice bath, controlling the temperature of reaction solution to be less than 5°C, keeping stirring for 1 h under ice bath after addition is completed, subsequently slowly adding potassium iodide under ice bath, continuously stirring the reaction solution for 2 h, slowly heating to room temperature, then stirring for 1 h at 60°C, cooling the reaction solution to room temperature, quenching with 200 mL of sodium bisulfite, extracting with petroleum ether (3 x 200 mL), combining organic phases, washing once with 200 mL of saturated sodium bicarbonate, then washing once with 150 mL of saturated salt solution, drying the organic phase with anhydrous sodium sulfate, filtering, concentrating, separating residues with a rapid column loading machine to obtain 2-bromo-3-methyl iodobenzene;

Step 2: dissolving 2-bromo-3-methyl iodobenzene into carbon tetrachloride, adding N-bromosuccinimide and benzoyl peroxide, refluxing a reaction mixture at a nitrogen atmosphere for overnight, then filtering, concentrating filtrate, separating a crude product with a rapid column loading machine to obtain 2-bromo-3-bromomethyliodobenzene; and

Step 3: dissolving 2-bromo-3-bromomethyliodobenzene and -chloro-2,4-dihydroxybenzaldehyde into acetonitrile, adding sodium bicarbonate, stirring the reaction mixture and refluxing for overnight, cooling the reaction solution to room temperature, adding 150 mL of water under the stirring, continuously stirring for 20 min, filtering, washing a filter cake three times with 20 mL of water, and drying to obtain the intermediate Il.

6. Application of the intermediate 12 according to claim 1 in preparation of a PDL1 antagonist drug molecule 1, wherein the PDL1 antagonist drug molecule 1 has the following structure formula:

OH

C1 HN~0 CI OH Br

N 0 0 N N

HO CN

7. The application according to claim 6, comprising the following steps:

0 0

Br C1 P , Br C1 I ~~ Pd(dPPf)C1 2 , K 2C0 3 N

N N

N 01- N

12 CN HO 1a CN

OH

HN O C1 OH 1. L-serine, HOAc - Br 2. NaBH(OAc) 3 0 N N0 N

H CN

8. The application according to claim 7, comprising the following steps:

Step 1:dissolving the intermediate 12 and an intermediate 16 into 1,4-dioxane, adding

potassium carbonate and [1,1'-bis (diphenylphosphino) ferrocene] palladium dichloride, stirring

the reaction mixture for overnight at 95C at a nitrogen atmosphere, cooling the reaction solution

to room temperature, adding 30 mL of ethyl acetate, filtering with diatomite, washing filtrate three

times with 10 mL of water, washing once with 10 mL of saturated salt solution, drying with

anhydrous sodium sulfate, filtering, concentrating, and separating residues with a rapid column

loading machine to obtain compound la;

wherein, the intermediate 16 has the following structure:

N N- O

. 16 HO

Step 2: dissolving the compound la into a mixture of N,N-dimethylformamide and acetic

acid, adding L-serine, stirring the reaction mixture for overnight at 45°C, then adding sodium

triacetoxyborohyride, continuing to stir for 3 h, cooling the reaction solution to room temperature, dropping water for quenching, filtering, and purifying the filtrate with a reverse preparative chromatography to obtain compound 1.

9. The application according to claim 8, wherein the synthesis method of the intermediate 16 comprises the following steps:

Br '- C HO NH Br NBB HN K2C0 3 , MeCN CI N Br K2C03, KI N

Ho

B-N

KOAc, Pd(dppf)C1 2 0

Ho5

10. The application according to claim 9, wherein the synthesis method of the intermediate 16 comprises the following steps:

Step 1: dissolving 1-bromo-3-chloropropane and 4-bromo-1H-indazole into acetonitrile, adding potassium carbonate, stirring the reaction mixture for overnight at 60°C, cooling the reaction solution to room temperature, filtering, concentrating, and separating the residues with a rapid column loading machine to obtain 4-bromo--(4-chloropropyl)-1H-indazole;

Step 2: dissolving 4-bromo-1-(4-chloropropyl)-1H-indazole and (R)-3-hydroxypyrrolidine into acetonitrile, adding potassium carbonate and potassium iodide, stirring the reaction mixture for overnight at 60°C, cooling the reaction solution to room temperature, filtering, concentrating the filtrate, then adding 50 mL of ethyl acetate, washing three times with 20 mL of water, then washing once with saturated salt solution, drying with anhydrous sodium sulfate, filtering, and concentrating to obtain (R)-1-(4-(4-bromo-1H-indazole-1-yl) propyl) pyrrolidine-3-ol; and

Step 3: dissolving (R)-1-(4-(4-bromo-1H-indazole-1-yl) propyl) pyrrolidine-3-ol and pinacol

bis (borate) into 1,4-dioxane, adding potassium acetate and 1,1'-bis (diphenylphosphino)

ferrocene] palladium dichloride, stirring the reaction mixture for overnight at 90°C at a nitrogen

atmosphere, cooling the reaction solution to room temperature, adding 100 mL of ethyl acetate,

filtering with diatomite, washing the filtrate three times with 50 mL of water, washing once with mL of saturated salt solution, drying with anhydrous sodium sulfate, filtering, concentrating, and separating the residues with a rapid column machine to obtain the intermediate 16.