WO2020248908A1 - Bifunctional immunomodulator, and pharmaceutically acceptable salt and pharmaceutical composition thereof - Google Patents

Abstract

Disclosed are a bifunctional immunomodulator, and a pharmaceutically acceptable salt and pharmaceutical composition thereof. The modulator has a structure represented by formula I. The bifunctional immunomodulator comprises a benzyl phenyl ether molecular skeleton that blocks PD-1/PD-L1 interaction, and a 4-(4-pyrazole) quinoline molecular skeleton that blocks a TGF-β signaling pathway. The two molecular skeletons are connected via molecular chain fragments. The bifunctional immunomodulator can simultaneously block PD-1/PD-L1 interaction and a TGF-β signaling pathway.

Description

Bifunctional immunomodulator and its pharmaceutically acceptable salt and pharmaceutical composition Technical field

This application belongs to the technical field of medicine, and relates to a bifunctional immunomodulator and a pharmaceutically acceptable salt and pharmaceutical composition thereof.

Background technique

Immunotherapy refers to a treatment method that achieves the purpose of treatment by inducing, enhancing or suppressing immune response. In recent years, significant progress has been made in the treatment of various diseases, especially cancer, and has become the most advanced drug therapy in addition to chemotherapy and targeted therapy. Important treatment options. In the process of cancer occurrence and development, cancer cells have evolved various protective mechanisms to avoid the recognition and killing of the body's immune system, that is, cancer immune escape. The more in-depth study of cancer immune escape signaling pathway is PD-1/PD-L1 (Programmed death 1/Programmed death-ligand 1). At present, many drugs have entered clinical applications. For example, in 2014, the FDA approved the listing of PD-1 The monoclonal antibodies Pembrolizumab (Keytruda, Merck) and Nivolumab (Opdivo, Bristol-Myers Squibb) have become blockbuster drugs for the treatment of various cancers. The PD-1/PD-L1 interaction is a negative signaling pathway that regulates the immune response. Cancer cells or stromal cells in the cancer microenvironment inhibit T cells through high expression of PD-L1 protein and PD-1 protein on the immune T cell membrane The function and promote its apoptosis. PD-1/PD-L1 modulators, such as the monoclonal antibody Pembrolizumab, disrupt the interaction between PD-L1 on cancer cells and T cell PD-1, block the negative regulatory function of the pathway, and reactivate T cells to cancer The immune response of the cells restores the vitality of T cells to achieve the purpose of killing cancer cells to treat cancer. Therefore, the PD-1/PD-L1 interaction has been widely studied and has played an important role in the clinical treatment of cancer in recent years.

The TGF-β (transforming growth factor-β) signaling pathway is widely involved in a variety of cellular processes, including cell growth, differentiation, migration and adhesion, apoptosis, etc., in the embryonic development and tissue and organ formation, tissue repair, immune supervision and Play an important role in adult homeostasis. The TGF-β signaling pathway is very complex, and the TGF-β/SMAD pathway has been extensively studied. The TGF-β/SMAD pathway consists of three subtypes (β1, β2, β3) of free ligands that bind to cell surface type I and type II TGF-β transmembrane serine/threonine kinase receptors to form heterologous complexes Activating receptor-specific SMAD protein (R-SAMD), R-SAMD combines with common SMAD (co-SMAD) to enter the nucleus and interact with other cytokines to mediate gene transcription. Abnormal TGF-β signaling pathway can lead to many diseases, such as cancer occurrence and metastasis, tissue fibrosis, cartilage dysplasia, abnormal inflammatory response, and pulmonary hypertension. In particular, in the advanced stages of various cancers, tumor cells and intratumoral stromal cells generally promote the immune escape and metastasis of cancer through high expression of TGF-β, leading to cancer deterioration and increasing the difficulty of cancer treatment. Studies have shown that inhibiting the TGF-β signaling pathway can significantly enhance the immune response of cancer and reduce tumor metastasis; the combination of TGF-β signaling pathway inhibitors and PD-1/PD-L1 pathway monoclonal antibodies has also improved in the laboratory The survival time of experimental animals.

Tumor immunotherapy has made a lot of progress in all aspects, but various drugs still have many shortcomings. The most prominent is that the effective response rate of patients is very low. For example, for various solid tumors, PD-1/PD-L1 drugs are only effective for 10-20% of patients. Therefore, it is urgent to improve the existing PD-1/PD-L1 drugs to increase the remission rate and survival rate of patients. As mentioned earlier, the TGF-β signaling pathway is generally highly expressed in the tumor microenvironment at the same time. Therefore, while inhibiting the PD-1/PD-L1 pathway, blocking the tumor microenvironment TGF-β pathway can theoretically promote PD-1 /PD-L1 drug effect. Based on this design concept, German Merck has developed a new type of tumor immune drug M7824. M7824 is a bifunctional fusion protein, which is formed by the fusion of an IgG1 monoclonal antibody targeting PD-L1 protein and the extramembrane domain of TGF-β receptor type II. Therefore, the protein can inhibit both the PD-1/PD-L1 interaction and the TGF-β signaling pathway, which can significantly promote the killing of tumor cells by immune cells. Furthermore, one drug targeting two pathways with different functions at the same time may overcome the resistance of tumor cells to single pathway therapies, and greatly improve drug benefits and reduce the side effects of combined drugs. Existing experimental data and clinical results have confirmed that M7824 is effective in a variety of solid tumors. For example, in the American Society of Clinical Oncology (ASCO) in 2018, the results of a phase I clinical study of M7824 in the second-line treatment of non-small cell lung cancer showed a very positive effect: when the dose was 500 mg, PD-L1 was positive (≥1% ) The overall response rate (ORR) of patients was 22.6%, and the ORR of patients with PD-L1 high expression (≥80%) was 33.3%; when the dose was 1200 mg, the overall response rate of PD-L1 positive patients reached 40.7%, and In patients with high PD-L1 expression, the ORR was as high as 71.4%.

Currently, bifunctional fusion proteins targeting PD-1/PD-L1 and TGF-β signaling pathways have been disclosed in some patents, such as WO2006074451A2, WO2009152610A1, WO2011109789A2, WO2013164694A1, WO2014164427A1, WO2015077540A2, WO2015118175A2, WO2018205985A1, etc., but PD-1 There are no reports on the bifunctional small molecule immunomodulators of PD-L1 and TGF-β signaling pathway. Compared with protein drugs, small molecules have many unique advantages: relatively stable, can be taken orally, and convenient to use; product quality is easy to control, and batch stability is good; the production process, transportation and storage are relatively easy, and the cost is low; and it is less likely to produce immune crossover reaction. Therefore, the study of bifunctional small molecule immunomodulators is a better choice for immunotherapy and will achieve better therapeutic effects. In view of the fact that there are no dual-functional small molecules that target PD-1/PD-L1 and TGF-β signaling pathways, this situation needs to be resolved urgently.

Summary of the invention

In the first aspect, this application provides a dual-function immunomodulator having a structure as shown in formula I:

R _{1 is} selected from

In this application,

Indicates the position where the group is attached.

R ₂ and R ₃ are each independently selected from hydrogen, halogen, cyano (-CN), substituted or unsubstituted C1-C3 alkyl, substituted or unsubstituted C1-C3 alkoxy, trifluoromethyl or ethylene base.

R _{4 is} selected from hydrogen, substituted or unsubstituted C1-C6 linear or branched alkyl, substituted or unsubstituted C3-C7 cyclic hydrocarbon group, substituted or unsubstituted C1-C4 alkoxy, substituted or unsubstituted Substituted C1-C4 alkyl acyl, substituted or unsubstituted C1-C4 alkylsulfonyl, substituted or unsubstituted C1-C4 alkyl acyl methylene, substituted or unsubstituted benzyl, substituted or unsubstituted Aroyl acyl methylene, substituted or unsubstituted 5-7 membered ring heteroarylmethylene or substituted or unsubstituted 5-7 membered ring heteroaroyl methylene.

R ₅ and R ₆ are each independently selected from hydrogen, halogen, hydroxyl, cyano, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 alkoxy, trifluoromethyl, substituted or unsubstituted Substituted C1-C4 alkyl acyl, substituted or unsubstituted C1-C4 alkylsulfonyl, substituted or unsubstituted C1-C4 alkyl acyl methylene, substituted or unsubstituted benzyl, substituted or unsubstituted Aroyl acylmethylene, substituted or unsubstituted 5-7 membered ring heteroarylmethylene or substituted or unsubstituted 5-7 membered ring heteroaryl acylmethylene, or R ₅ and R _{6 are} connected with Pyrazole forms a 5-7 membered ring. In this application, the

It means that R ₅ and R ₆ can be connected to each other to form a ring structure.

R _{7 is} selected from hydrogen, halogen, cyano, C1-C6 alkyl, C1-C6 alkoxy, aryl or 5-7 membered heteroaryl.

A is selected from a C1-C16 alkyl group, a C1-C16 polyethylene glycol alkoxy group, a C1-C16 sulfur atom or nitrogen atom-containing alkyl group, or a C1-C16 amino acid-containing alkyl group; wherein, A It is connected to the benzylidene group through a CN bond, and is connected to the benzene ring of the quinoline ring through an oxygen atom, and the connection position is the 7 or 8 position of the quinoline ring.

The bifunctional immunomodulator provided in this application includes a benzyl phenyl ether molecular skeleton that simultaneously blocks the interaction of PD-1/PD-L1 and a 4-(4-pyrazole) quinoline molecular skeleton that blocks the TGF-β signaling pathway , The two are connected by molecular chain fragments. Therefore, the dual-function immunomodulator provided in this application can simultaneously block the PD-1/PD-L1 interaction and the TGF-β signaling pathway.

In the present application, the alkyl group refers to a linear or branched saturated or unsaturated alkyl group, for example, C1-C6 alkyl includes C1-C6 linear alkyl, C3-C6 branched alkyl , C2-C6 linear alkene groups, C3-C6 branched alkene groups, and alkynyl groups.

In this application, the alkoxy group is -OR-, where R is an alkyl group.

In this application, the trifluoromethyl group is -CF ₃ .

In this application, the alkyl acyl group refers to -C(O)R, where R is an alkyl group.

In this application, the alkylsulfonyl group refers to -S(O) ₂ R, wherein R represents an alkyl group.

In this application, the alkylacylmethylene refers to -CH ₂ C(O)R, where R represents an alkyl group.

In this application, the arylacylmethylene refers to -CH ₂ C(O)Ar, where Ar represents an aryl group.

In this application, the heteroarylmethylene group refers to -CH ₂ A ₁ , where A ₁ represents a heteroaryl group.

In this application, the heteroaryl acyl methylene group refers to -CH ₂ C(O)A ₁ , where A ₁ represents a heteroaryl group.

In this application, the polyethylene glycol alkoxy group refers to -(CH ₂ -CH ₂ -O) _n -, where n≥1.

In this application, the alkyl group containing sulfur atom or nitrogen atom refers to containing S or N in the main chain of the alkyl group.

In this application, the amino acid-containing alkoxy group refers to amino acids such as glycine, proline, alanine, etc.

Structure, where R represents the side chain of an amino acid, and n≥1.

In this application, in R ₂ -R ₆ , substitution means that one or more hydrogens on the carbon are substituted, but does not include the original heteroatoms. For example, substituted or unsubstituted alkylsulfonyl refers to Except for the sulfonyl group, other hydrogen atoms on the carbon atoms of the alkyl group are substituted.

In this application, the C1-C3 can be C1, C2, or C3.

In this application, the C1-C6 can be C1, C2, C3, C4, C5, or C6.

In this application, the C3-C7 can be C3, C4, C5, C6, or C7.

In this application, the C1-C4 can be C1, C2, C3, or C4.

In this application, the 5-7 membered ring may be a 5-membered ring, a 6-membered ring or a 7-membered ring.

In this application, the C1-C16 may be C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, or C16, etc.

The 7 or 8 positions of the quinoline ring are as follows:

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the technical field of this application. The terms used in the specification of this application are only for the purpose of explanation and description of specific embodiments, and are not intended to limit the application.

In one embodiment, the modulator has a structure as shown in Formula II:

Wherein, M is selected from a C1-C16 alkyl group, a C1-C16 polyethylene glycol alkoxy group, a C1-C16 sulfur atom or nitrogen atom-containing alkyl group, or a C1-C16 amino acid-containing alkyl group; oxygen The connection position of the atom and the quinoline derivative is the 7 or 8 position of the quinoline ring.

R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , R ₆ and R ₇ have the same ranges as described above.

Preferably, the R ₂ is methyl.

Preferably, when referring to substituted groups, the substituents are selected from halogen (-X), hydroxyl (-OH), amino (-NH ₂ ), nitro (-NO ₂ ), mercapto (-SH), cyano Group (-CN), C1-C3 alkylsulfonyl (-R-SO ₃ H), C1-C3 alkoxy (-OR) or substituted heteroaromatic methoxy group.

Preferably, the R _{3 is} selected from methyl, trifluoromethyl, fluorine, chlorine, bromine, cyano, methanesulfonyl or trifluoromethanesulfonyl. The methylsulfonyl group is -S(O) ₂ -CH ₃ . The trifluoromethanesulfonyl group is -S(O) ₂ -CF ₃ .

Preferably, R _{4 is} selected from methyl, methoxy, benzyl, benzyloxy, ortho or meta cyanobenzyloxy, pyridinemethyleneoxy, or ortho or meta cyanopyridinemethyleneoxy base. The structural formula of the ortho-cyanobenzyloxy group is

The meta-cyanobenzyloxy group is

The ortho-cyanopyridinemethyleneoxy group is

Meta-cyanopyridinemethyleneoxy is

Indicates the position where the group is attached.

Preferably, R ₅ and R _{6 are} connected and form a 5-6 membered ring with pyrazole.

As a preferred technical solution of this application, the bifunctional regulator is selected from

Any one or a combination of at least two of them.

In the second aspect, this application provides a preparation method of the bifunctional immunomodulator according to the first aspect, the preparation method includes the following steps:

Performing a reductive condensation reaction between compound A having a structure of formula III and compound B having a structure of formula IV to obtain the bifunctional immunomodulator;

Among them, the structural formula of compound A is as follows:

The structural formula of the compound B is as follows:

Among them, R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , R ₆ , R ₇ and M have the same ranges as described above.

Preferably, the molar ratio of compound A and compound B is 1:1;

Preferably, the reagent for the condensation reduction reaction is sodium cyanoborohydride;

Preferably, the added amount of sodium cyanoborohydride is more than 2 times the molar added amount of compound B;

Preferably, the reaction temperature of the condensation reduction reaction is 10-30°C, and the reaction time is 12-48h;

Preferably, the preparation method of the compound A is as follows:

Substituting compound C and compound D to obtain compound A, the reaction equation is as follows:

Preferably, the molar ratio of the compound C to the compound D is (1.1-1.3):1, such as 1.15:1, 1.2:1, 1.25:1, and the like.

Preferably, the reagent for the substitution reaction is any one of sodium hydrogen, sodium hydroxide, potassium hydroxide or sodium carbonate.

Preferably, the reaction temperature of the substitution reaction is 25°C or more, such as 30°C, 40°C, 45°C, 50°C, 55°C, etc., and the reaction time is 2h or more, such as 2.5h, 3h, 4h, 5h, etc.

Preferably, the reagent is first added to the compound D solution and mixed, and then compound C is added to the compound D solution for substitution reaction.

Preferably, the adding temperature of the reagent is below 0°C, such as -1°C, -2°C, -5°C, -10°C and the like.

Preferably, the preparation method of the compound C is as follows:

Preferably, the preparation method of the compound D is as follows:

Preferably, step (a) is the reduction reaction of the carboxyl group, namely: -COOH→-CH ₂ OH, and the reduction is carried out using borane.

Preferably, step (b) is the reaction of -Br with R ₁ boron reagent.

Preferably, step (c) is the bromination reaction of the hydroxyl group, that is, -OH→-Br.

Preferably, step (d) is an R ₃ derivatization reaction such as chlorination.

Preferably, the preparation method of the compound B includes deprotecting the amine group protected by the tert-butoxycarbonyl group, and the reagent used for the deprotection of the amine group is an acidic reagent.

which is:

Among them, the

It has the following two synthesis processes:

Both step (e) and step (f) are condensation reactions.

Preferably, the reaction of step (e) is carried out in the presence of an organic base or an inorganic base. The organic base is preferably triethylamine, and the inorganic base is preferably sodium hydroxide, cesium carbonate or sodium bicarbonate.

Preferably, the reaction in step (e) is carried out in the presence of a condensing agent.

Preferably, the reaction of step (f) is carried out in the presence of an inorganic base, and the inorganic base is preferably sodium hydrogen, sodium hydroxide, cesium carbonate or sodium bicarbonate.

In the third aspect, the application provides an enantiomer, diastereomer or pharmaceutically acceptable salt of the bifunctional immunomodulator according to the first aspect.

Enantiomers in this application refer to chiral molecules that are optically mirror images of each other and cannot overlap; diastereomers refer to all stereoisomers that are not enantiomers, that is, those that do not have a mirror image relationship. Optical isomers.

Preferably, the pharmaceutically acceptable salt is an acid addition salt or a base addition salt.

When the compound of the present application has a free base form, reacting the free base form of the compound with a pharmaceutically acceptable inorganic or organic acid can prepare a pharmaceutically acceptable acid addition salt of the bifunctional immunomodulator of the present application.

Preferably, the acid addition salt includes hydrochloride, hydrobromide, hydroiodide, phosphate, sulfate, nitrate, ethanesulfonate, tosylate, benzenesulfonate, acetic acid Salt, maleate, tartrate, succinate, citrate, benzoate, ascorbate, salicylate, malonate, adipate, caproate, arginine, Any one or a combination of at least two of fumarate, nicotinate, phthalate, or oxalate.

When the benzyl phenyl ether derivatives of the present application are in the form of free acid, the free acid form is reacted with a pharmaceutically acceptable inorganic or organic base to prepare the bifunctional immunomodulator of the present application in a pharmaceutically acceptable base addition. A salt.

Preferably, the base addition salt includes lithium salt, sodium salt, potassium salt, barium salt, calcium salt, magnesium salt, aluminum salt, iron salt, ferrous salt, copper salt, zinc salt, or the bifunctional immune The modifier is a salt composed of morpholine, diethylamine, triethylamine, isopropylamine, trimethylamine, lysine or histidine.

In the fourth aspect, this application provides a pharmaceutical composition, the pharmaceutical composition comprising the bifunctional immunomodulator described in the first aspect or the enantiomers of the bifunctional immunomodulator described in the third aspect, Diastereomers or pharmaceutically acceptable salts and pharmaceutically acceptable carriers.

In this application, the pharmaceutically acceptable carrier can be in liquid, semi-liquid or solid form, formulated in a manner suitable for the administration route used, and can be used for in vivo treatment and has biocompatibility. The pharmaceutical composition can be administered according to the following modes of administration: oral, parenteral, intraperitoneal, intravenous, transdermal, sublingual, intramuscular, rectal, oral, intranasal, liposome, etc. Kind of form.

The oral pharmaceutical composition can be solid, gel or liquid. Examples of solid preparations include, but are not limited to, tablets, capsules, granules, and bulk powders. The preparation may optionally contain binders, diluents, disintegrants, lubricants, glidants, sweeteners, and flavoring agents. Examples of binders include, but are not limited to, microcrystalline cellulose, glucose solution, gum arabic, gelatin solution, sucrose and starch paste; examples of lubricants include, but are not limited to, talc, starch, magnesium stearate, calcium stearate , Stearic acid; examples of diluents include but are not limited to lactose, sucrose, starch, mannitol, dicalcium phosphate; examples of glidants include but are not limited to silicon dioxide; examples of disintegrants include but are not limited to Sodium carboxymethyl cellulose, sodium starch glycolate, alginic acid, corn starch, potato starch, methyl cellulose, agar, and carboxymethyl cellulose.

The pharmaceutical composition of the present application is administered parenterally, generally by injection, including subcutaneous, intramuscular or intravenous injection. The injection can be made into any conventional form, such as a liquid solution or suspension, a solid form suitable for dissolving or suspending in a liquid before injection, or an emulsion. Examples of pharmaceutically acceptable carriers that can be used in the injections of this application include, but are not limited to, aqueous carriers, non-aqueous carriers, antimicrobial agents, isotonic agents, buffers, antioxidants, suspending and dispersing agents, emulsifiers, chelating agents And other pharmaceutically acceptable substances. Examples of aqueous carriers include Sodium Chloride Injection, Ringer's Injection, Isotonic Dextrose Injection, Sterile Water Injection, Dextrose and Lactated Ringer's Injection; Examples of non-aqueous carriers include fixed oils of plant origin, Cottonseed oil, corn oil, sesame oil, and peanut oil; examples of antimicrobial agents include m-cresol, benzyl alcohol, chlorobutanol, benzalkonium chloride, etc.; examples of isotonic agents include sodium chloride and glucose; buffers include phosphate And citrate.

The composition of the present application can also be prepared into a sterile freeze-dried powder injection by dissolving the compound in a sodium phosphate buffer solution, which contains glucose or other suitable excipients, and then dissolving the solution under standard conditions known to those skilled in the art. Sterile filtration followed by freeze drying to obtain the desired formulation.

In the fifth aspect, this application provides the bifunctional immunomodulator according to the first aspect or the enantiomers, diastereomers or pharmaceutically acceptable The accepted salt is used in the preparation of dual inhibitors of PD-1/PD-L1 and TGF-β.

The dual-function immunomodulator provided in this application can inhibit the PD-1/PD-L1 pathway while blocking the TGF-β signaling pathway.

In the sixth aspect, this application provides the bifunctional immunomodulator according to the first aspect or the enantiomers, diastereomers or pharmaceutically acceptable The application of the accepted salt in the preparation of drugs for diseases related to PD-1/PD-L1 and/or TGF-β signaling pathway.

Preferably, the diseases related to the PD-1/PD-L1 and/or TGF-β signaling pathway include cancer, infectious diseases or autoimmune diseases.

Preferably, the diseases related to the PD-1/PD-L1 and/or TGF-β signaling pathway include multiple myeloma, melanoma, glioma, glioblastoma, leukemia, sarcoma, smooth muscle Tumor, mesothelioma, breast cancer, cervical cancer, lung cancer, stomach cancer, rectal cancer, colon cancer, pancreatic cancer, brain cancer, skin cancer, oral cancer, prostate cancer, bone cancer, kidney cancer, ovarian cancer, bladder cancer, liver cancer , Bacterial infection, viral infection, chronic lymphocytic thyroiditis, hyperthyroidism, insulin-dependent diabetes mellitus, myasthenia gravis, ulcerative colitis, pernicious anemia with chronic atrophic gastritis, pulmonary hemorrhage nephritis syndrome, primary bile Liver cirrhosis, multiple cerebrospinal sclerosis, rheumatoid arthritis, dermatomyositis, polymyositis, osteoporosis, ulcers, vascular damage, glomerulonephritis, diabetic nephropathy, lupus nephritis, hypertension- Induced nephropathy, renal interstitial fibrosis, renal fibrosis, graft nephropathy, liver fibrosis, liver dysfunction, alcohol-induced hepatitis, cystic fibrosis, interstitial lung disease, acute lung injury, adult respiratory distress Syndrome, idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease, lung disease caused by infectious or toxic factors, heart fibrosis after infarction, congestive heart failure, dilated cardiomyopathy, myocarditis, intimal thickening, vascular stenosis , Pulmonary hypertension, coronary artery restenosis, peripheral artery restenosis, carotid artery restenosis, stent-induced restenosis, atherosclerosis, ocular scar formation, corneal scar formation, proliferative vitreoretinopathy, glaucoma, intraocular pressure High, excessive or hypertrophic dermal scars or keloid formation, peritoneal and subcutaneous adhesions, scleroderma, fibrosclerosis, progressive systemic sclerosis, decreased nervous system function, Peyronie's disease, Dupuytren's Contractures, Alzheimer's disease, Raynaud's syndrome, or radiation-induced fibrotic disease.

Because the bifunctional immunomodulator provided in this application can inhibit the PD-1/PD-L1 pathway while blocking the TGF-β signaling pathway, the bifunctional immunomodulator provided in this application can be used to prepare application drugs. The main role of drugs is to treat or prevent diseases related to PD-1/PD-L1 signaling pathway or/and TGF-β signaling pathway.

Compared with the prior art, this application has the following beneficial effects:

(1) The bifunctional immunomodulator provided in this application includes a benzyl phenyl ether molecular skeleton that simultaneously blocks the interaction of PD-1/PD-L1 and 4-(4-pyrazole)quine that blocks the TGF-β signaling pathway The morpholino molecular skeleton, the two are connected by molecular chain fragments. Therefore, the dual-function immunomodulator provided in this application can simultaneously block the PD-1/PD-L1 interaction and the TGF-β signaling pathway.

(2) The pharmaceutically acceptable salt of the bifunctional immunomodulator provided in this application contains one or more bifunctional immunomodulators. Therefore, it has a good application effect in preparing medicines for treating and/or preventing diseases related to PD-1/PD-L1 and/or TGF-β signaling pathway.

Detailed ways

The technical solution of the present application will be further explained through specific implementations below. It should be understood by those skilled in the art that the described embodiments are only to help understand the application and should not be regarded as specific limitations to the application.

Unless otherwise specified, the starting materials and reaction reagents used in the following examples are all commercially available products. The reagents and solvents used in the experiment are handled according to the specific conditions of the reaction.

The reagent abbreviations used in this application are commonly used expressions in the field and have the following meanings:

Petroleum ether (PE), dichloromethane (DCM), ethyl acetate (EA), tetrahydrofuran (THF), methanol (MeOH), N,N-dimethylformamide (DMF), ethylene glycol dimethyl ether ( DME), N,N-dimethylacetamide (DMA), N,N-diisopropylethylamine (DIEA), triethylamine (TEA), dicyclohexylcarbodiimide (DCC), 4- N,N-dimethylaminopyridine (DMAP), 1-hydroxybenzotriazole (HOBt), 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC·HCl ), di-tert-butyl dicarbonate (Boc anhydride).

In the following examples, the analysis data of the samples are measured by the following instruments: NMR is measured by Bruker AMX-400 and Bruker AMX-500 nuclear magnetic resonance instruments, TMS (tetramethylsilane) is the internal standard, and the chemical shift unit is ppm, the coupling constant unit is Hz; the mass spectrum is measured by an Agilent1200/MSD mass spectrometer.

200-300 mesh silica gel for column chromatography (produced by Qingdao Ocean Chemical Plant); TLC silica gel plate is HSGF-254 thin-layer chromatography prefabricated plate produced by Yantai Chemical Plant; petroleum ether boiling range 60-90℃; UV lamp is used , Iodine cylinder and phosphomolybdic acid, etc.

Example 1

A bifunctional immunomodulator, its chemical structure is as follows:

The preparation method is as follows:

Synthesis of compound 1-1:

Dissolve 2-methyl 3-bromobenzoic acid (64.5 g, 0.3 mol) in 450 mL of dry ether, protect with argon, and add 1 M BH3-THF complex (450 mL, 0.45 mol) dropwise at 0°C. After the completion, it was gradually raised to room temperature and stirred overnight. After TLC detected that there was no raw material, most of the solvent was evaporated under reduced pressure, and 500 mL of saturated sodium bicarbonate aqueous solution was added. A large amount of white solid precipitated out. Stir thoroughly, filter, wash with water, drain, and then use petroleum ether. After washing, a dry white solid was obtained as compound 1-1 (56.4 g, yield 93.5%).

¹ H NMR (400MHz, CDCl ₃ ): δ = 7.51 (d, J = 8.0 Hz, 1H), 7.31 (d, J = 7.6 Hz, 1H), 7.05 (t, J = 7.6 Hz, 1H), 4.71 ( s, 2H), 2.42 (s, 3H), 1.75 (s, 1H).

Synthesis of compound 1-2:

Compound 1-1 (20.1g, 0.10mol) was dissolved in 150mL of dichloromethane, phenylboronic acid (14.6g, 0.12mol), potassium carbonate (22.1g, 0.16mol) and palladium chloride (5.8g, 0.005mol), replace the air in the reaction system with argon protection, gradually increase to 90℃ and stir overnight. After TLC detects that there is no raw material, add it to 200mL water after cooling down, then extract with 250mL×3 ethyl acetate, and combine the organic layers , Washed with water and saturated brine successively, dried with anhydrous sodium sulfate, filtered with suction, concentrated the filtrate and purified by column chromatography to obtain compound 1-2 (16.8 g, yield 84.7%) as a white solid.

¹ H NMR (400MHz, CDCl ₃ ): δ=7.44-7.39 (m, 3H), 7.37-7.33 (m, 1H), 7.31-7.28 (m, 2H), 7.26-7.25 (m, 1H), 7.22 7.20 (m, 1H), 4.78 (s, 2H), 2.25 (s, 3H), 1.65 (brs, 1H).

MS (MM-ES+APCI) m/z: 495.5 [2M+Na] ⁺ .

Synthesis of compound 1-3:

Compound 1-2 (15.8g, 0.08mol) was dissolved in 150mL of dry methanol, protected by argon, and phosphorus tribromide (15.8g, 0.08mol) was added dropwise at 0°C. After the addition, it was raised to room temperature and stirred overnight. After TLC detects that there is no raw material, the solvent and excess phosphorus tribromide are evaporated under reduced pressure, and the remaining oil mixture is directly purified by flash chromatography to obtain compound 1-3 (18.9g, yield 90.5%) ).

¹ H NMR (400MHz, CDCl ₃ ): δ=7.45-7.40 (m, 2H), 7.36-7.35 (m, 1H), 7.34-7.33 (m, 1H), 7.31-7.29 (m, 2H), 7.25 7.17(m, 2H), 4.60(s, 2H), 2.31(s, 3H).

¹³ C NMR (500MHz, CDCl ₃ ): δ=143.4, 141.8, 136.3, 134.9, 130.7, 129.3, 128.1, 127.0, 125.8, 33.1, 16.1.

Synthesis of compound 1-4:

The compound 4-hydroxy-2-methoxybenzaldehyde (5.1g, 0.033mol) was dissolved in 250mL of dry tetrahydrofuran solvent, protected by argon, and sulfone chloride (5.4g, 0.040mol) was added dropwise at 0℃. Then it was raised to room temperature and stirred for 30 minutes, and most of the solvent was evaporated under reduced pressure. The remaining mixture was poured into stirring ice water. A large amount of off-white solid was precipitated. The filter cake was washed once with petroleum ether and filtered to obtain a white solid. It is compound 1-4 (5.4 g, yield 87.7%).

¹ H NMR (400MHz, CDCl ₃ ): δ = 10.25 (s, 1H), 7.83 (s, 1H), 6.64 (s, 1H), 6.10 (s, 1H), 3.91 (s, 3H).

¹³ C NMR (500 MHz, CDCl ₃ ): δ=187.2, 162.5, 157.5, 129.0, 119.6, 112.9, 99.6, 56.0.

MS(MM-ES+APCI) m/z: 185.4[MH] ^- .

Synthesis of compound 1-5:

Dissolve compound 1-4 (5.4g, 0.029mol) in 150mL of dry ether, add 60% sodium hydrogen (1.8g, 0.045mol) at 0°C, stir for about 30min until no gas is generated, protect it with argon, Add compound 1-3 (9.0g, 0.035mol) in 100mL of dry tetrahydrofuran solution dropwise, raise to 55°C and stir overnight after the dropwise addition is complete. After TLC detects that there is no raw material, filter to remove the insoluble matter, and evaporate the low boiling point solvent under reduced pressure. The remaining mixture was poured into stirring ice water, and a large amount of white solid was precipitated. After suction filtration, the filter cake was washed once with petroleum ether and suction filtered to obtain the white solid compound 1-5 (7.0 g, yield 65.8%).

¹ H NMR (400MHz, CDCl ₃ ): δ = 10.27 (s, 1H), 7.87 (s, 1H), 7.48 (d, J = 6.4 Hz, 1H), 7.42 (d, J = 7.2 Hz, 2H), 7.37 (d, J = 7.2Hz, 2H), 7.32-7.28 (m, 5H), 6.61 (s, 1H), 3.94 (s, 3H); 2.85 (s, 3H).

¹³ C NMR (500MHz, CDCl ₃ ): δ = 187.2, 162.2, 160.0, 143.2, 141.6, 134.3, 133.8, 130.5, 130.0, 129.4, 128.1, 127.8, 127.0, 125.7, 119.0, 116.1, 97.3, 70.4, 56.1, 16.3.

Synthesis of compound 1-6:

Dissolve tetraethylene glycol (7.8g, 0.040mol) in 250mL of dry ethyl ether, add 60% sodium hydrogen (1.8g, 0.045mol) at 0°C, stir for about 30min until no gas is generated, then argon protection, drip Add tert-butyl bromoacetate (7.8g, 0.040mol), raise to room temperature and stir overnight after dripping, the reaction solution was diluted with 500mL of dichloromethane, washed twice with 100mL of saturated brine, and the organic layer was washed with anhydrous sulfuric acid After drying with sodium, filtering with suction, the filtrate was concentrated and purified by column chromatography to obtain compound 1-6 (8.0 g, yield 65.3%) as a colorless oil.

¹ H NMR (400MHz, CDCl ₃ ): δ = 3.95 (s, 2H), 3.65-3.64 (m, 6H), 3.63-3.60 (m, 8H), 3.55-3.52 (m, 2H), 1.41 (s, 9H).

MS (MM-ES+APCI) m/z: 331.4 [M+Na] ⁺ .

Synthesis of compounds 1-7:

Compound 1-6 (6.2g, 0.020mol) was dissolved in 100mL of tetrahydrofuran, and an equal volume of trifluoroacetic acid was added at 0°C. The mixture was stirred overnight at room temperature. After TLC detected no raw materials, the solvent and excess reagents were evaporated under reduced pressure to obtain The brown oil is crude compound 1-7 (4.2 g, yield 83.2%).

MS(MM-ES+APCI) m/z: 251.2[MH] ^- .

Synthesis of compounds 1-8:

The crude compound 1-7 (4.2g, 0.017mol) was dissolved in 150mL of dry dichloromethane, and 1-hydroxybenzotriazole (2.3g, 0.017mol), N-tert-butoxycarbonylethylenediamine ( 2.7g, 0.017mol), triethylamine (3.4g, 0.034mol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (3.0g, 0.017mol), room temperature After stirring overnight, the reaction solution was added with 150 mL saturated aqueous ammonium chloride solution, and then extracted with 200×3 mL ethyl acetate. The organic layers were combined, washed with water and saturated brine successively, dried over anhydrous sodium sulfate, filtered with suction, and the filtrate was concentrated and then column chromatography After purification, the colorless oily substance was compound 1-8 (3.4 g, yield 50.5%).

¹ H NMR (400MHz, CDCl ₃ ): δ = 7.53 (s, 1H), 5.26 (s, 1H), 3.99 (s, 2H), 3.71-3.70 (m, 2H), 3.67-3.66 (m, 12H) , 3.61-3.58 (m, 2H), 3.41-3.37 (m, 2H), 3.29-3.26 (m, 2H), 2.22 (s, 1H), 1.42 (s, 9H).

¹³ C NMR (500MHz, CDCl ₃ ): δ=170.7, 156.2, 79.3, 72.9, 72.6, 70.8, 70.5, 70.4, 70.2, 70.0, 61.6, 40.7, 39.1,28.4.

MS(MM-ES+APCI) m/z: 417.5[M+Na] ⁺ .

Synthesis of compounds 1-9:

The crude compound 1-8 (3.0g, 7.6mmol) was dissolved in 50mL of dry tetrahydrofuran, triethylamine (1.2g, 11.4mmol) was added at room temperature, protected by argon, and methanesulfonyl chloride (1.1g, 9.5 mmol), kept at 0°C and stirred for 10 min, then warmed to room temperature and stirred overnight. The reaction solution was added with 50 mL of saturated aqueous ammonium chloride solution, and then extracted with 100×3 mL of ethyl acetate. The organic layers were combined, washed successively with water and saturated brine, dried with anhydrous sodium sulfate, suction filtered, and the filtrate was concentrated and purified by column chromatography to obtain The colorless oil is compound 1-9 (1.8 g, yield 50.1%).

¹ H NMR (400MHz, CDCl ₃ ): δ = 4.37-4.35 (m, 2H), 3.99 (s, 1H), 3.77-3.75 (m, 2H), 3.71-3.59 (m, 14H), 3.41-3.78 ( m, 2H), 3.27-3.26 (m, 2H), 3.06 (s, 3H), 1.42 (s, 9H).

MS (MM-ES+APCI) m/z: 495.3 [M+Na] ⁺ .

Synthesis of compounds 1-10:

The compound 1-9 (0.61g, 1.3mmol), 4-(2-(pyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl ) Quinoline-7-ol (0.33g, 1.0mmol) and cesium carbonate (0.72g, 2.2mmol) were dissolved in 10mL of dry DMF, protected by argon, and stirred at 85°C overnight. After cooling, the reaction solution was added to 50 mL of water, and then extracted with 100×3 mL of ethyl acetate. The organic layers were combined, washed with water and saturated brine successively, dried with anhydrous sodium sulfate, filtered with suction, and the filtrate was concentrated and purified by column chromatography. The obtained yellow solid was compound 1-10 (0.45 g, yield 49.1%).

¹ H NMR (400MHz, CDCl ₃ ): δ = 8.71 (d, J = 4.4 Hz, 1H), 8.36 (d, J = 4.4 Hz, 1H), 8.58 (d, J = 8.8 Hz, 1H), 7.17 ( d,J=8.0Hz,1H), 7.10(d,J=4.4Hz,1H),7.01-6.97(m,3H),5.30(s,1H), 4.28(t,J=7.2Hz,2H), 4.22(t,J=4.0Hz,2H),3.92(s,1H), 3.86(t,J=8.0Hz,2H), 3.68(t,J=2.4Hz,2H),3.66-3.61(m,6H ), 3.59-3.53 (m, 4H), 3.35-3.19 (m, 2H), 2.80-2.77 (m, 2H), 2.66-2.59 (m, 2H), 1.36 (s, 9H).

¹³ C NMR (500MHz, CDCl ₃ ): δ=170.7, 159.6, 159.5, 156.2, 153.2, 152.2, 150.1, 149.5, 146.7, 141.1, 136.0, 127.3, 122.5, 122.1, 121.9, 119.5, 110.2, 1085.2, 79.1, 77.4, 77.3, 77.1, 76.9, 72.6, 70.9, 70.8, 70.5, 70.3, 70.1, 69.5, 67.5, 48.2, 40.6, 39.1,28.4, 25.88, 23.21.

MS(MM-ES+APCI) m/z: 705.8[M+H+], 727.6[M+Na] ⁺ .

Synthesis of compounds 1-11:

Dissolve compound 1-10 (0.35g, 0.5mmol) in 10mL of dichloromethane, add an equal volume of trifluoroacetic acid at 0°C, and stir overnight at room temperature. After TLC detects that there is no raw material, the solvent and excess reagents are evaporated under reduced pressure After adding methanol to dissolve, add sodium bicarbonate (0.13g, 1.5mmol) and stir for 30 minutes. After filtering to remove the solid, the filtrate is concentrated and purified by column chromatography to obtain the crude compound 1-11 as a yellow solid. The molecular weight is identified by mass spectrometry. Go directly to the next step.

MS(MM-ES+APCI) m/z: 605.4[M+H] ⁺ .

Synthesis of compound 1:

The crude compound 1-11 and compound 1-5 (0.20 g, 0.55 mmol) were dissolved in 10 mL of DMF, sodium cyanoborohydride (0.14 g, 2.2 mmol) was added, and the mixture was stirred at room temperature overnight. After TLC detects that there is no raw material, the reaction solution is added to 50mL of water, and then extracted with 100×3mL of ethyl acetate. The organic layers are combined, washed with water and saturated brine successively, dried with anhydrous sodium sulfate, filtered with suction, and the filtrate is concentrated and the column layer After analysis and purification, the yellow solid was obtained as compound 1 (0.45 g, yield 49.1%).

¹ H NMR (400MHz, CDCl ₃ ): δ = 8.71 (d, J = 4.4 Hz, 1H), 8.38 (d, J = 4.4 Hz, 1H), 7.88 (t, J = 4.0 Hz, 1H), 7.62 ( d,J=9.2Hz,1H),7.47(d,J=2.4Hz,1H),7.44(d,J=2.0Hz,1H),7.39(d,J=7.6Hz,2H),7.32(d, J = 10.4 Hz, 1H), 7.29 (d, J = 1.2 Hz, 1H), 7.27 (d, J = 0.8 Hz, 1H), 7.25-7.23 (m, 2H), 7.16-7.15 (d, J = 8.4 Hz,1H),7.04-7.02(m,2H),6.57(s,1H),5.13(s,2H),4.33(t,J=7.2Hz,2H), 4.26(t,J=8.4Hz,2H ),3.95(s,2H),3.93(s,2H),3.89(t,J=4.4Hz,2H),3.81(t,J=2.4Hz,2H),3.73-3.71(m,2H),3.66 -3.65(m,2H),3.63-3.61(m,8H),3.56(d,J=5.2Hz,1H), 2.99(t,J=5.2Hz,1H), 2.81(t,J=7.2Hz, 1H), 2.69-2.62 (m, 2H), 2.25 (s, 3H).

¹³ C NMR (500MHz, CDCl ₃ ): δ1771.3, 159.8, 157.4, 155.6, 153.2, 152.1, 149.6, 149.5, 149.4, 146.9, 143.0, 141.8, 136.2, 134.5, 134.2, 132.2, 130.2, 129.4, 128.1, 127.7, 127.3,126.9,125.6,122.6,122.2,122.0,120.5,119.8,115.0,114.6,110.2,107.7,70.9,70.8,70.5,69.5,67.6,55.8,48.3,46.8,46.2,36.8,29.7,25.9,23.12, 22.8, 16.2.

MS (MM-ES+APCI) m/z: 955.8 [M+H] ⁺ .

Example 2

A bifunctional immunomodulator, its chemical structure is as follows:

The preparation method is as follows:

Synthesis of compound 2-1:

Dissolve D-serine (5.25g, 0.050mol) in a mixed solvent of 60mL water and 60mL ethanol, add sodium carbonate (2.20g, 0.055mol) at 0℃, and add di-tert-butyl dicarbonate (12.00g, 0.055mol) dropwise ), keep at 0°C and stir for 1h, then warm to room temperature and stir overnight. The tert-butanol was evaporated under reduced pressure, the aqueous layer was extracted once with 50 mL of n-pentane, the aqueous layer was slowly added with cold 10% sodium bisulfate aqueous solution, adjusted to pH 1-2, and then extracted with 250×3 mL of ethyl acetate. The organic layers were combined, washed with water and saturated brine in turn, dried over anhydrous sodium sulfate, filtered with suction, and the filtrate was concentrated and crystallized with n-hexane to obtain compound 2-1 (9.35 g, yield 91.1%) as a white solid.

¹ H NMR (400MHz, CDCl ₃ ): δ = 5.23 (s, 1H), 4.35 (s, 1H), 4.08 (d, J = 8.4 Hz, 1H), 3.87 (dd, J = 11.6, 4.0 Hz, 2H ), 1.47(s, 9H).

MS(MM-ES+APCI) m/z: 204.0[MH] ^- .

Synthesis of compound 2-2:

Compound 2-1 (4.10g, 0.020mol) was dissolved in 100mL DMF, imidazole (4.10g, 0.060mol) and TBDPSCl (7.15g, 0.026mol) were added at 0°C, and then heated to room temperature and stirred overnight. After TLC detected no raw materials, the reaction solution was added to 150mL saturated ammonium chloride aqueous solution, and then extracted with 250×3mL ethyl acetate. The organic layers were combined, washed with water and saturated brine successively, dried with anhydrous sodium sulfate, filtered with suction, and the filtrate Concentrated and crystallized with n-hexane/ether system to obtain compound 2-2 (5.60 g, yield 63.1%) as a white solid.

¹ H NMR (400MHz, CDCl ₃ ): δ = 8.34 (brs, 1H), 7.64 (d, J = 5.6 Hz, 4H), 7.43-7.36 (m, 6H), 5.43 (d, J = 4.0 Hz, 1H ), 4.46 (d, J = 8.0 Hz, 1H), 4.13 (d, J = 11.6 Hz, 1H), 3.93 (d, J = 10.4 Hz, 1H), 1.48 (s, 9H), 1.75 (s, 9H) ).

¹³ C NMR (500MHz, CDCl ₃ ): δ=175.4, 155.5, 135.6, 135.5, 129.9, 127.8, 80.2, 61.4, 55.4, 28.3, 26.7, 19.3.

MS(MM-ES+APCI) m/z: 442.5[MH] ^- .

Synthesis of compound 2-3:

Dissolve ethylene glycol (62.07g, 1.00mol) and triethylamine (50.50g, 0.50mol) in 50mL of dichloromethane, add methanesulfonyl chloride (57.27g, 0.50mol) dropwise at 0°C, then warm to room temperature and stir overnight. The reaction solution was added to 150 mL saturated aqueous ammonium chloride solution, and then extracted with 300×3 mL ethyl acetate. The organic layers were combined, washed with water and saturated brine successively, dried over anhydrous sodium sulfate, filtered with suction, and the filtrate was concentrated and then column chromatography. The colorless oily substance is compound 2-3 (10.43 g, yield 14.9%).

¹ H NMR (400MHz, CDCl ₃ ): δ = 4.34 (t, J = 4.4 Hz, 2H), 3.89 (t, J = 4.0 Hz, 2H), 3.07 (s, 3H), 2.41 (s, 1H).

Synthesis of compound 2-4:

Compound 2-3 (0.67g, 4.8mmol), 4-(2-(pyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl ) Quinoline-7-ol (0.66g, 2.0mmol) and sodium carbonate (1.43g, 4.4mmol) were dissolved in 20mL of dry DMSO, protected by argon, and stirred at 85°C overnight. After cooling, the reaction solution was added to 50 mL of water, and then extracted with 100×3 mL of dichloromethane. The organic layers were combined, washed with water and saturated brine successively, dried over anhydrous sodium sulfate, filtered with suction, and the filtrate was concentrated and purified by column chromatography. The yellow solid is compound 2-4 (0.37 g, yield 49.7%).

¹ H NMR (500MHz, CDCl ₃ ): δ = 8.74 (dd, J = 5.0, 1.5 Hz, 1H), 8.37 (d, J = 5.0 Hz, 1H), 7.60 (d, J = 9.5 Hz, 1H), 7.43(s,1H),7.39(t,J=5.5Hz,1H),7.23(d,J=9.0Hz,1H),7.14-7.13(m,1H),7.02-6.97(m,2H),4.31 (t,J=9.0Hz,2H), 4.20(t,J=7.0Hz,2H), 3.99(t,J=3.5Hz,2H), 2.79(t,J=6.5Hz,2H),2.66-2.63 (m,2H).

MS (MM-ES+APCI) m/z: 373.5 [M+H] ⁺ , 746.0 [2M+H] ⁺ .

Synthesis of compound 2-5:

Compound 2-2 (0.22g, 0.50mmol), compound 2-4 (0.17g, 0.46mmol) and 4-dimethylaminopyridine (0.05g, 0.4mmol) were dissolved in 20mL of dry tetrahydrofuran, protected by argon, Dicyclohexylcarbodiimide (0.20 g, 1.0 mmol) in 10 mL of dry dichloromethane solution was added dropwise at 0°C, after the addition was completed, stirred at room temperature overnight. The insoluble matter in the reaction solution was removed by filtration, and the filtrate was concentrated and purified by column chromatography to obtain compound 2-5 (0.28 g, yield 73.1%) as a colorless oil.

¹ H NMR (400MHz, CDCl ₃ ): δ = 8.78 (d, J = 4.4 Hz, 1H), 8.40 (d, J = 4.4 Hz, 1H), 7.62 (d, J = 9.2 Hz, 1H), 7.60- 7.57(m,4H),7.42-7.39(m,3H),7.37-7.30(m,5H),7.23(d,J=8.0Hz,1H),7.17(d,J=8.4Hz,1H),7.02 (td,J=5.4,0.8Hz,1H), 6.96(dd,J=9.2,2.4Hz,1H), 4.93(d,J=7.2Hz,1H), 4.64-4.57(m,1H), 4.48- 4.44(m,2H), 4.34(t,J=7.2Hz,2H), 4.29-4.26(m,2H), 4.10(d,J=7.2Hz,1H), 3.90(d,J=7.2Hz,1H ), 2.82 (t, J = 7.2Hz, 2H), 2.67 (m, 2H), 1.44 (s, 9H), 1.01 (s, 9H).

¹³ C NMR (500MHz, CDCl ₃ ): δ=170.7, 159.2, 155.3, 153.3, 152.2, 150.2, 149.6, 146.7, 141.1, 136.0, 135.5, 132.9, 129.9, 127.8, 127.4, 122.7, 122.1, 121.9, 120.7, 119.4, 110.2, 108.4, 79.9, 65.47, 64.6, 63.5, 55.6, 48.3, 28.3, 26.7, 25.9, 23.1, 19.3.

MS(MM-ES+APCI) m/z: 798.5[M+H] ⁺ , 820.7[M+Na] ⁺ .

Synthesis of compound 2-6:

Dissolve compound 2-5 (0.28g, 0.35mmol) in 10mL of DMF, add an equal volume of trifluoroacetic acid at 0°C, and stir at room temperature for 5h. After TLC detects that there is no raw material, the solvent and excess reagents are evaporated under reduced pressure. After the methanol was dissolved, sodium bicarbonate (0.13 g, 1.5 mmol) was added and stirred for 30 min. After the solid was removed by filtration, the filtrate was concentrated to obtain the crude compound 2-6 as a yellow oil. After the molecular weight was identified by mass spectrometry, proceed directly to the next step.

MS: 698.8 [M+H] ⁺ , 720.5 [M+Na] ⁺ .

Synthesis of compounds 2-7:

The crude compound 2-6 and compound 1-5 (0.13 g, 0.35 mmol) were dissolved in 10 mL of tetrahydrofuran, sodium cyanoborohydride (0.14 g, 2.2 mmol) was added, and the mixture was stirred at room temperature overnight. After TLC detects that there is no raw material, the reaction solution is added to 50 mL of water, and a white solid is separated out, filtered with suction, and the filter cake is sucked dry and purified by column chromatography to obtain compound 2-7 (0.15 g, yield 40.9%) as a yellow solid.

¹ H NMR (400MHz, CDCl ₃ ): δ = 8.78 (d, J = 4.0 Hz, 1H), 8.40 (d, J = 3.6 Hz, 1H), 7.62-7.61 (m, 5H), 7.47 (d, J =3.6Hz,1H),7.47(d,J=3.6Hz,1H),7.43-7.38(m,5H),7.34-7.28(m,8H),7.23(s,2H),7.18(d,J= 2.8Hz, 1H), 7.01-6.97 (m, 2H), 6.55 (s, 1H), 5.14 (s, 2H), 4.47 (d, J = 13.2Hz, 2H), 4.33 (t, J = 6.8 Hz, 2H), 4.29 (s, 2H), 3.90 (dd, J = 10.8, 4.4 Hz, 2H), 3.74 (s, 3H), 3.72 (dd, J = 20.4, 9.6 Hz, 2H), 2.81 (d, J ＝6.4Hz,2H),2.68-2.65(m,2H),2.27(s,3H),1.01(s,3H).

¹³ C NMR (500MHz, CDCl ₃ ): δ = 173.2, 159.3, 157.0, 154.0, 153.3, 152.2, 150.1, 149.6, 146.8, 142.9, 141.9, 141.3, 136.0, 135.6, 134.9, 134.1, 133.1, 130.7, 130.1, 129.7, 129.4, 127.8, 127.4, 126.8, 125.5, 122.7, 122.1, 122.0, 121.9, 120.7, 119.5, 114.4, 110.2, 108.2, 98.9, 70.6, 65.9, 65.2, 62.8, 62.4, 55.7, 48.3, 46.3, 26.9, 23.1, 19.2, 16.2.

MS (MM-ES+APCI) m/z: 1049.2 [M+H] ⁺ .

Synthesis compound 2:

Compound 2-7 (0.12g, 0.11mmol) was dissolved in 10mL of dichloromethane, protected by argon, and 1M tetrabutylammonium fluoride in tetrahydrofuran solution (0.16mL) was added dropwise at 0°C. After the addition, keep Stir at 0°C for 10 min, and then at room temperature for 1 h. After TLC detected no raw material, the reaction solution was concentrated and purified by column chromatography to obtain compound 2 (0.05 g, yield 56.1%) as a white solid.

¹ H NMR (400MHz, CDCl ₃ ): δ8.77 (d, J = 4.4Hz, 1H), 8.41 (d, J = 4.8Hz, 1H), 7.65 (d, J = 9.2Hz, 1H), 7.48- 7.45(m,2H),7.43-7.40(m,3H),7.36-7.35(d,J=6.8Hz,2H),7.31-7.28(m,2H),7.24(d,J=1.6Hz,3H) ,7.22(s,1H),7.18-7.17(d,J=4.8Hz,1H),7.06-7.01(m,2H),6.58(s,1H),5.15(s,2H),4.52-4.48(m ,2H),4.36-4.33(m,4H),3.82-3.79(m,4H),3.74(s,3H),3.68-3.66(m,2H),3.47(t,J=7.2Hz,2H), 2.70-2.66 (m, 2H), 2.68-2.65 (m, 2H), 2.27 (s, 3H).

¹³ C NMR (500MHz, CDCl ₃ ): δ=172.9, 159.2, 157.1, 154.3, 152.2, 150.1, 149.9, 149.6, 146.8, 142.9, 141.9, 141.5, 136.1, 134.8, 134.2, 130.9, 130.2, 129.4, 128.1, 127.8, 127.6, 126.9, 125.6, 122.8, 122.2, 121.9, 121.2, 120.7, 119.4, 114.3, 110.1, 108.2, 70.6, 65.9, 63.3, 62.3, 61.8, 55.8, 48.3, 46.6, 25.9, 23.2.

MS (MM-ES+APCI) m/z: 810.8 [M+H] ⁺ .

Example 3

A bifunctional immunomodulator, its chemical structure is as follows:

The preparation method is as follows:

Synthesis of compound 3-1:

Compound 1-1 (20.1g, 0.10mol) was dissolved in 150 mL of a mixture solvent of 1,3-dioxane and water with a volume ratio of 2:1, and benzo-1,4-dioxane was added at room temperature. Hexacyclic-6-boronic acid (21.6g, 0.12mol), potassium carbonate (22.1g, 0.16mol) and tetrakistriphenylphosphine palladium (5.8g, 0.005mol), replace the air in the reaction system with argon protection, gradually Raise to 90℃ and stir overnight. After TLC detects that there is no raw material, add to 200mL water after cooling down, then extract with 250mL×3 ethyl acetate. Combine the organic layers, wash with water and saturated brine in turn, dry with anhydrous sodium sulfate, and pump After filtration, the filtrate was concentrated and purified by column chromatography to obtain compound 3-1 (24.2 g, yield 94.4%) as a colorless oil.

¹ H NMR (400MHz, CDCl ₃ ): δ = 7.37 (dd, J = 7.2, 0.8 Hz, 1H), 7.25-7.18 (m, 2H), 6.93 (d, J = 8.4 Hz, 1H), 6.84 (d ,J=2.0Hz,1H),6.93(d,J=8.4Hz,1H),6.77(dd,J=8.0,2.0Hz,1H),4.72(s,2H),4.28(s,4H),2.83 (s, 1H), 2.26 (s, 3H).

MS (MM-ES+APCI) m/z: 279.5 [M+Na] ⁺ , 535.6 [2M+Na] ⁺ .

Synthesis of compound 3-2:

Compound 3-1 (12.8g, 0.05mol) was dissolved in 250mL of dry tetrahydrofuran, protected by argon, and phosphorus tribromide (15.8g, 0.10mol) was added dropwise at 0°C. After the addition, it was raised to room temperature and stirred overnight. After TLC detects that there is no raw material, the solvent and excess phosphorus tribromide are evaporated under reduced pressure, and the remaining oil mixture is directly purified by flash chromatography to obtain compound 1-3 (15.4g, yield 96.5%) ).

¹ H NMR (400MHz, CDCl ₃ ): δ = 7.31 (m, 1H), 7.20-7.18 (m, 2H), 6.90 (d, J = 8.0 Hz, 1H), 6.81 (d, J = 2.0 Hz, 1H ), 6.77(dd,J=8.0,2.0Hz,1H), 4.72(s,3H), 4.31(s,4H), 2.32(s,3H).

¹³ C NMR (500MHz, CDCl ₃ ): δ=143.1, 142.8, 142.7, 136.3, 135.1, 135.0, 130.8, 129.1, 125.8, 122.5, 118.2, 116.9.

Synthesis of compound 3-3:

Dissolve the compound 2,4-dihydroxybenzaldehyde (5.52g, 0.04mol) in 150mL of dry ether, protect it with argon, add sulfone chloride (6.74g, 0.05mol) dropwise at 0℃, and warm to room temperature after the addition is complete After stirring for 30 minutes, most of the solvent was evaporated under reduced pressure, the remaining mixture was poured into stirring ice water, a large amount of milky white solid precipitated out, after suction filtration, the filter cake was washed again with petroleum ether and suction filtered to obtain the milky white solid as compound 3. -3 (4.58g, yield 66.4%).

¹ H NMR (400MHz, CDCl ₃ ): δ = 11.25 (s, 1H), 9.69 (s, 1H), 7.52 (s, 1H), 6.61 (s, 1H), 6.26 (s, 1H).

MS(MM-ES+APCI) m/z: 171.0[MH] ^- .

Synthesis of compound 3-4:

Compound 3-3 (4.28g, 24.8mol) and sodium bicarbonate (6.25g, 74.4mmol) were dissolved in 250mL of dry acetonitrile, stirred at room temperature for about 30min until no gas was generated, protected by argon, and compound 3- was added dropwise. 2 (3.96g, 12.4mmol) of 50mL dry DMF solution, after the dropwise addition is complete, raise to 60℃ and stir for 3 hours. After TLC detects that there is no raw material, the low boiling point solvent is evaporated under reduced pressure, and the remaining mixture is poured into the stirred A large amount of white solid precipitated in ice water. After suction filtration, the filter cake was crystallized with ethyl acetate, and the filtrate was purified by column chromatography to obtain a white solid as compound 3-4 (2.4 g, yield 47.1%).

¹ H NMR (400MHz, CDCl ₃ ): δ = 11.43 (s, 1H), 9.70 (s, 1H), 7.55 (s, 1H), 7.44 (dd, J = 4.8, 2.8 Hz, 2H), 7.25 (d ,J=2.8Hz,2H), 6.91(d,J=4.8Hz,1H), 6.83(d,J=2.8Hz,1H), 6.78(dd,J=8.4,2.8Hz,1H), 6.63(s ,1H), 5.20(s, 2H), 4.31(s, 4H), 2.27(s, 3H).

MS (MM-ES+APCI) m/z: 409.8 [M+H] ⁺ .

Synthesis of compound 3-5:

Compound 3-4 (1.23g, 3.0mol) and cesium carbonate (1.47g, 4.5mmol) were dissolved in 50mL of dry DMF, stirred at room temperature for about 10min until no gas was generated, protected by argon, and added dropwise m-cyanobenzyl A solution of bromine (0.88g, 4.5mmol) in 10mL of dry DMF. After the addition is complete, keep it at room temperature and stir overnight. After TLC detects that there is no raw material, the reaction solution is poured into stirring ice water. A large amount of white solid precipitates out. After suction filtration, filter The cake was washed once with petroleum ether and drained to obtain a white solid compound 3-5 (2.40 g, yield 91.4%).

¹ H NMR (400MHz, CDCl ₃ ): δ10.31 (s, 1H), 7.91 (s, 1H), 7.72 (s, 1H), 7.67 (d, J = 3.6 Hz, 2H), 7.56-7.52 (m ,1H),7.38(s,1H),7.25(s,2H),6.91(d,J=8.0Hz,2H),6.81(s,1H),6.77(d,J=8.0,1H),6.60( s, 1H), 5.20 (s, 2H), 5.18 (s, 2H), 4.31 (s, 4H), 2.28 (s, 3H).

MS(MM-ES+APCI) m/z: 524.2[MH] ^- .

Synthesis of compound 3-6:

The crude compound 2-6 (0.26 g, 0.37 mmol) and compound 3-5 (0.20 g, 0.37 mmol) were dissolved in 10 mL of tetrahydrofuran, sodium cyanoborohydride (0.09 g, 1.84 mmol) was added, and the mixture was stirred at room temperature overnight. After TLC detects that there is no raw material, the reaction solution is added to 50 mL of water, and a white solid is precipitated. The filter cake is drained and purified by column chromatography. The white solid is obtained as compound 3-6 (0.10g, yield 22.3) %).

¹ H NMR (400MHz, CDCl ₃ ): δ = 8.78 (d, J = 4.4 Hz, 1H), 8.40 (d, J = 4.0 Hz, 1H), 7.69 (s, 1H), 7.61-7.55 (m, 6H) ),7.42(s,1H),7.40-7.35(m,6H),7.33-7.29(m,5H),7.24-7.20(m,3H),7.17(d,J=4.4Hz,1H),7.05- 7.02(m,1H), 6.94(dd,J=9.2,2.4Hz,1H), 6.90(d,J=8.0Hz,1H), 6.80(s,1H), 6.76(d,J=8.4,1H) ,6.53(s,1H),5.08(s,2H),5.01(s,2H),4.49-4.44(s,4H),4.36-4.32(m,3H),4.30(s,4H),4.26(s ,2H),3.92-3.87(m,3H),3.66(d,J=13.6Hz,1H), 3.47(s,1H), 2.81(t,J=6.8Hz,2H), 2.69-2.66(m, 2H), 2.26(s, 3H), 0.97(s, 9H).

MS (MM-ES+APCI) m/z: 1209.1 [M+H] ⁺ .

Synthesis of compound 3:

Compound 3-6 (0.10g, 0.08mmol) was dissolved in 10mL of DMF, protected by argon, and 1M tetrabutylammonium fluoride solution in tetrahydrofuran (0.16mL) was added dropwise at 0°C. After the addition, keep at 0°C. Stir for 10 minutes and then at room temperature for 1 hour. After TLC detects that there is no raw material, the reaction solution is concentrated and purified by column chromatography to obtain compound 3 (0.04 g, yield 51.6%) as a white solid.

¹ H NMR (400MHz, CDCl ₃ ): δ = 8.77 (d, J = 4.4 Hz, 1H), 8.41 (d, J = 4.0 Hz, 1H), 7.77 (s, 1H), 7.65-7.60 (m, 3H) ),7.48(d,J=3.6Hz,1H),7.43(s,2H),7.41-7.38(m,2H),7.28(s,1H),7.23-7.21(m,3H),7.17(d, J=4.0Hz,1H),7.05-6.99(m,2H), 6.90(d,J=8.0Hz,1H), 6.80(s,1H), 6.76(d,J=8.0,1H), 6.56(s ,1H),5.09(s,2H),5.06(s,2H),4.54-4.46(s,4H),4.36-4.33(m,3H),4.30(s,4H),3.80-3.73(m,3H) ), 3.67-3.65 (m, 1H), 3.48-3.47 (m, 1H), 2.83-2.81 (m, 2H), 2.70-2.67 (m, 2H), 2.26 (s, 3H).

¹³ C NMR (500MHz, CDCl ₃ ): δ172.9, 159.1, 155.6, 154.2, 153.3, 152.2, 150.3, 150.1, 149.6, 146.8, 143.1, 142.7, 142.4, 141.3, 138.0, 136.1, 135.1, 134.6, 134.1, 131.8, 131.3, 130.7, 130.2, 129.5, 127.5, 127.4, 125.6, 122.7, 122.5, 122.2, 121.9, 121.8, 120.8, 119.4, 118.5, 118.2, 116.9, 115.4, 112.9, 110.1, 108.3, 100.3, 70.6, 69.4, 65.9, 64.4, 63.37, 62.4, 61.9, 48.3, 46.7, 31.6, 25.9, 23.2, 16.2.

MS (MM-ES+APCI) m/z: 970.2 [M+H] ⁺ .

Example 4

A bifunctional immunomodulator, its chemical structure is as follows:

The preparation method is as follows:

Synthesis of compound 4-1:

Dissolve N-(tert-butoxycarbonyl)ethanolamine (4.03g, 0.025mol) and triethylamine (3.30g, 0.032mol) in 50mL of dichloromethane, add methanesulfonyl chloride (3.10g, 0.027mol) dropwise at 0°C , Then warm to room temperature and stir overnight. The reaction solution was added to 150 mL saturated aqueous ammonium chloride solution, and then extracted with 200×3 mL ethyl acetate. The organic layers were combined, washed with water and saturated brine successively, dried with anhydrous sodium sulfate, filtered with suction, and the filtrate was concentrated and then column chromatography. The colorless oil was obtained as compound 4-1 (2.35 g, yield 39.3%).

¹ H NMR (400MHz, CDCl ₃ ): δ = 5.13 (s, 1H), 4.20 (t, J = 5.2 Hz, 2H), 3.38 (d, J = 4.0 Hz, 1H), 2.97 (s, 3H), 1.37(s, 9H).

Synthesis of compound 4-2:

The compound 4-1 (0.72g, 3.0mmol), 4-(2-(pyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl ) Quinoline-7-ol (0.33g, 1.0mmol) and cesium carbonate (0.72g, 2.2mmol) were dissolved in 20mL of dry tetrahydrofuran, protected by argon, and stirred at 85°C overnight. After cooling, the reaction solution was added to 50 mL of water, and then extracted with 100×3 mL of dichloromethane. The organic layers were combined, washed with 10% sodium carbonate aqueous solution, water and saturated brine in turn, dried with anhydrous sodium sulfate, filtered with suction, and the filtrate After concentration, it was purified by column chromatography to obtain compound 4-2 (0.25 g, yield 53.0%) as a pale yellow solid.

¹ H NMR (400MHz, CDCl ₃ ): δ = 8.81 (d, J = 4.4 Hz, 1H), 8.43 (d, J = 4.4 Hz, 1H), 7.64 (d, J = 9.2 Hz, 1H), 7.42 ( td,J=8.0,2.0Hz,2H), 7.22(d,J=8.0Hz,1H), 7.17(d,J=4.4Hz,1H), 7.05(m,1H), 7.00(dd,J=9.2 ,2.4Hz,1H), 4.99(s,1H), 4.35(t,J=7.2Hz,2H), 4.17(t,J=5.2Hz,2H), 3.60(d,J=4.8Hz,2H), 2.84 (d, J = 7.2 Hz, 1H), 2.73-2.66 (m, 2H), 1.45 (s, 9H).

Synthesis of compound 4-3:

Dissolve compound 4-2 (0.25g, 0.53mmol) in 10mL of tetrahydrofuran, add an equal volume of saturated methanolic hydrogen chloride solution at 0°C, and stir overnight at room temperature. After TLC detects that there is no raw material, the solvent and excess reagents are evaporated under reduced pressure After adding methanol to dissolve, add sodium bicarbonate (0.13g, 1.5mmol) and stir for 30 minutes. After filtering to remove the solid, the filtrate is concentrated to obtain the crude product of compound 2-6 as a yellow oil. The molecular weight is identified by mass spectrometry and the next step is directly carried out.

MS (MM-ES+APCI) m/z: 372.5 [M+H] ⁺ .

Synthesis of compound 4-4:

Dissolve compound 4-3 (0.15g, 0.40mmol) and compound 2-1 (0.12g, 0.60mmol) in 10mL ether, add 1-hydroxybenzotriazole (0.09g, 0.60mmol), triethyl Amine (0.15g, 1.5mmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (0.12g, 0.60mol) were stirred at room temperature overnight. The reaction solution was added to 150 mL of saturated aqueous ammonium chloride solution, and then extracted with 100×3 mL of ethyl acetate. The organic layers were combined, washed with sodium bicarbonate aqueous solution, water and saturated brine successively, dried over anhydrous sodium sulfate, filtered with suction, and the filtrate was concentrated After purification by column chromatography, compound 4-4 was obtained as a colorless oil (0.18 g, yield 80.5%).

¹ H NMR (400MHz, CDCl ₃ ): δ = 8.77 (d, J = 4.4 Hz, 1H), 8.31 (s, 1H), 7.69 (d, J = 9.2 Hz, 1H), 7.54-7.52 (m, 1H) ),7.51(s,1H),7.35-7.28(m,2H),7.07-7.05(m,2H),5.70(d,J=7.2Hz,1H), 4.36(t,J=7.2Hz,2H) ,4.29(t,J=5.6Hz,2H),4.23(s,1H),4.09-4.05(m,1H),3.77-3.76(m,2H),3.73-3.70(m,2H),2.87-2.83 (m, 2H), 2.75-2.69 (m, 2H), 1.40 (s, 9H).

MS (MM-ES+APCI) m/z: 559.3 [M+H] ⁺ .

Synthesis of compound 4-5:

Dissolve compound 4-4 (0.18g, 0.53mmol) in 10mL of DMF, add an equal volume of saturated methanolic hydrogen chloride solution at 0°C, stir at room temperature overnight, after TLC detects no raw materials, evaporate the solvent and excess reagents under reduced pressure After adding ethyl acetate to dissolve, add sodium bicarbonate (0.13g, 1.5mmol) and stir for 30min. After filtering to remove the solid, the filtrate is concentrated to obtain the crude compound 4-5 as a yellow oil. After the molecular weight is identified by mass spectrometry, proceed directly to the next step .

MS: 459.4 [M+H] ⁺ .

Synthesis of compound 4:

The crude compound 4-5 and compound 3-5 (0.18 g, 0.35 mmol) were dissolved in 5 mL of dichloromethane, sodium cyanoborohydride (0.14 g, 2.2 mmol) was added, and the mixture was stirred at room temperature overnight. After TLC detects that there is no raw material, the reaction solution is added to 50 mL of water, and a white solid is separated out, filtered with suction, and the filter cake is sucked dry and purified by column chromatography to obtain a white solid as compound 4 (0.08 g, yield 23.6%).

¹ H NMR (400MHz, CDCl ₃ ): δ = 8.76 (d, J = 4.4 Hz, 1H), 8.39 (d, J = 3.2 Hz, 1H), 7.72 (s, 1H), 7.69 (s, 1H), 7.65(s,1H),7.62(d,J=11.6Hz,1H),7.59-7.57(m,2H),7.46-7.44(m,1H),7.41(s,2H),7.36-7.35(m, 1H), 7.24 (s, 1H), 7.22-7.21 (m, 2H), 7.16-7.15 (m, 1H), 7.02-7.01 (m, 1H), 7.05-6.99 (m, 2H), 6.90 (d, J = 8.4Hz, 1H), 6.80 (s, 1H), 6.76 (d, J = 8.4, 1H), 6.53 (s, 1H), 5.06 (s, 2H), 5.05-5.01 (m, 2H), 4.34 -4.33 (m, 2H), 4.30 (s, 4H), 4.18 (s, 2H), 3.76-3.75 (m, 2H), 3.74 (s, 2H), 3.72-3.68 (m, 2H), 3.27 (s ,1H), 2.83-2.79 (m, 2H), 2.68-2.64 (m, 2H), 2.25 (s, 3H), 2.04 (s, 1H).

¹³ C NMR (500MHz, CDCl ₃ ): δ=172.7, 159.3, 155.5, 153.3, 152.2, 150.3, 150.1, 149.6, 146.8, 143.1, 142.7, 142.4, 141.3, 137.9, 136.1, 135.1, 134.5, 134.1, 131.9, 131.3, 131.1, 130.6, 130.3, 129.6, 127.5, 127.4, 125.6, 122.7, 122.5, 122.1, 121.9, 121.7, 120.8, 119.2, 118.5, 118.2, 116.9, 115.5, 112.9, 110.1, 108.5, 100.3, 70.6, 69.4, 66.9, 64.4, 63.0, 62.9, 48.3, 47.0, 38.5, 29.7, 25.9, 23.2, 16.2.

MS(MM-ES+APCI) m/z: 969.1[M+H] ⁺ .

Example 5

A bifunctional immunomodulator, its chemical structure is as follows:

Compound 5 can be synthesized via a similar route to compound 1.

Example 6

A bifunctional immunomodulator, its chemical structure is as follows:

Compound 6 can be synthesized via a similar route to compound 1.

Example 7

A bifunctional immunomodulator, its chemical structure is as follows:

Compound 7 can be synthesized via a similar route to compound 3.

Example 8

A bifunctional immunomodulator, its chemical structure is as follows:

Compound 8 can be synthesized via a similar route to compound 4.

Example 9

A bifunctional immunomodulator, its chemical structure is as follows:

Compound 9 can be synthesized via a similar route to compound 4.

Example 10

A bifunctional immunomodulator, its chemical structure is as follows:

Using unnatural D-serine as the starting material, compound 10, the enantiomer of compound 4, can be synthesized by a similar route.

Comparative example 1

Compound 2-4 provided in Example 1.

Comparative example 2

Refer to the literature (J. Med. Chem. 2017, 60, 5857-5867) to prepare synthetic PD-L1 inhibitor 86 as Comparative Example 2.

Comparative example 3

Refer to the literature (J. Med. Chem. 2017, 60, 5857-5867) to prepare and synthesize PD-L1 inhibitor 87 as Comparative Example 3.

Biological evaluation test:

Test Example 1: Test of the inhibitory effect of the compounds of the present application and comparative examples on PD-L1 protein at the cellular level

Experimental principle: Use a cell line with high expression of PD-L1 protein, add DMSO or compound to incubate for a certain period of time, then add PE-Labeled Human PD-1 protein, and then use flow cytometry to detect whether the cells are labeled with PD-1 protein. In order to determine whether the tested compound is consistent with the interaction of PD-L1 and PD-1 protein. The specific test procedure is as follows: first add the prepared compound (20 mM DMSO solution) to PBS, and dilute it to 1 mM. After counting the CR1-PDL1 cells with high expression of PDL1, adjust the cells to 5x10 ⁵ /100μl, and then spread the cells (100μl) into a 96-well plate. Add 1μl of 1mM compound to each well (diluted 100 times, final compound concentration 10μM), mix well with a gun and stand for 15min at room temperature in the dark. Then add 0.25μl PD1-PE (PE-Labeled Human PD-1, Fc tag, His tag) to each well, mix well, avoid light, and let stand at 4°C for 1 hour. Finally, add 1ml PBS, centrifuge at 1600rpm, 5min to wash the cells, add PBS solution to the washed cells and collect data by flow cytometry.

The inhibitory activities of compounds 1-4 and comparative examples on PD-L1 protein measured according to the above method are shown in Table 1.

Table 1. Inhibitory activity of compounds (10μM) on PD-L1 protein

Compound number	Inhibition activity value (%)	Compound number	Inhibition activity value (%)
Compound 1	78.6	DMSO	0
Compound 2	55.7	Comparative example 1	5.7
Compound 3	10.3	Comparative example 2	85.7
Compound 4	67.6	Comparative example 3	80.2

It can be seen from Table 1 that the compound of the present application has a significant inhibitory effect on PD-L1 protein, and compound 1 has the better activity.

Test Example 2: Test of the inhibitory effect of the compounds of the present application and comparative examples on the TGF-β signal pathway at the cellular level

Experimental principle: The TGF-β signaling pathway is directly related to the phagocytic ability of macrophages. When the signaling pathway is inhibited, the phagocytic ability of macrophages is significantly enhanced. Therefore, the phagocytic ability of macrophages can be improved by detecting the compound treatment. The above test compound inhibits the activity of the TGF-β signaling pathway. The specific test procedure is: spread macrophages at 50,000 cells/well in a 24-well plate, add compound and anti-CD47 antibody after 24 hours, and add TGF-β protein after 2 hours. After the above system was incubated in an incubator for 48 hours, L1210 tumor cells labeled with CFSE fluorescently labeled were added to the macrophage wells at 200,000 cells/well. After incubating for 2 hours at 37°C, they were photographed under a fluorescence microscope to calculate the phagocytosis rate ( The number of phagocytic tumor cells in each field/total number of macrophages in each field×100%).

The inhibitory activities of compounds 1-4 and comparative examples on the TGF-β signal pathway measured according to the above method are shown in Table 2.

Table 2. Inhibitory activity of compounds (2uM) on PD-L1 protein

Compound number	Phagocytosis rate (%)	Compound number	Phagocytosis rate (%)
Compound 1	7.45	DMSO	4.88
Compound 2	13.13	Comparative example 1	26.84
Compound 3	7.31	Comparative example 2	6.52
Compound 4	11.21	Comparative example 3	6.59

It can be seen from Table 2 that the compound of the present application has a significant inhibitory effect on the TGF-β signaling pathway, and compound 2 has the better activity.

The applicant declares that this application uses the above-mentioned examples to illustrate the bifunctional immunomodulator of this application and its pharmaceutically acceptable salts and pharmaceutical compositions, but this application is not limited to the above detailed methods, which does not imply this The application must rely on the above detailed methods to be implemented. Those skilled in the art should understand that any improvement to this application, the equivalent replacement of each raw material of the product of this application, the addition of auxiliary components, the selection of specific methods, etc., fall within the scope of protection and disclosure of this application.

Claims (15)

1. A bifunctional immunomodulator, which has a structure as shown in formula II:

   

   Where R _{1 is} selected from

   

   R ₂ and R ₃ are each independently selected from hydrogen, halogen, cyano, substituted or unsubstituted C1-C3 alkyl, substituted or unsubstituted C1-C3 alkoxy, trifluoromethyl or vinyl;

   R _{4 is} selected from hydrogen, substituted or unsubstituted C1-C6 linear or branched alkyl, substituted or unsubstituted C3-C7 cyclic hydrocarbon group, substituted or unsubstituted C1-C4 alkoxy, substituted or unsubstituted Substituted C1-C4 alkyl acyl, substituted or unsubstituted C1-C4 alkylsulfonyl, substituted or unsubstituted C1-C4 alkyl acyl methylene, substituted or unsubstituted benzyl, substituted or unsubstituted Aroyl acyl methylene, substituted or unsubstituted 5-7 membered ring heteroarylmethylene or substituted or unsubstituted 5-7 membered ring heteroaroyl methylene;

   R ₅ and R ₆ are each independently selected from hydrogen, halogen, hydroxyl, cyano, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 alkoxy, trifluoromethyl, substituted or unsubstituted Substituted C1-C4 alkyl acyl, substituted or unsubstituted C1-C4 alkylsulfonyl, substituted or unsubstituted C1-C4 alkyl acyl methylene, substituted or unsubstituted benzyl, substituted or unsubstituted Aroyl acyl methylene, substituted or unsubstituted 5-7 membered ring heteroarylmethylene or substituted or unsubstituted 5-7 membered ring heteroaryl acylmethylene, or R ₅ and R _{6 are} connected and Form 5-7 membered ring with pyrazole;

   R _{7 is} selected from hydrogen, halogen, cyano, C1-C6 alkyl, C1-C6 alkoxy, aryl or 5-7 membered heteroaryl; and M is selected from C1-C16 alkyl, C1- C16 polyethylene glycol alkoxy group, C1-C16 sulfur atom or nitrogen atom-containing alkyl group, or C1-C16 amino acid-containing alkyl group; and M is connected to the 7 or 8 position of the quinoline ring through an oxygen atom Bit connection.
2. The bifunctional immunomodulator according to claim 1, wherein the R ₂ is a methyl group.
3. The bifunctional immunomodulator according to claim 1, wherein when referring to substituted groups, the substituents are selected from halogen, hydroxyl, amino, nitro, mercapto, cyano, C1-C3 alkyl, C1 -C3 alkoxy or substituted heteroaromatic ring methyleneoxy.
4. The bifunctional immunomodulator according to claim 1, wherein the R _{3 is} selected from methyl, trifluoromethyl, fluorine, chlorine, bromine, cyano, methanesulfonyl or trifluoromethanesulfonyl.
5. The bifunctional immunomodulator according to claim 1, wherein R _{4 is} selected from methyl, methoxy, benzyl, benzyloxy, ortho- or meta-cyanobenzyloxy, pyridinemethyleneoxy, Or ortho or meta cyanopyridinemethyleneoxy.
6. The bifunctional immunomodulator according to claim 1, wherein R ₅ and R _{6 are} connected to form a 5-6 membered ring with pyrazole.
7. The bifunctional immunomodulator according to any one of claims 1 to 6, wherein the bifunctional immunomodulator is selected from

   

   

   Any one or a combination of at least two of them.
8. The preparation method of the bifunctional immunomodulator according to any one of claims 1 to 7, which comprises the following steps:

   Performing a condensation reduction reaction between compound A having a structure of formula III and compound B having a structure of formula IV to obtain the bifunctional immunomodulator;

   Among them, the structural formula of compound A is as follows:

   

   The structural formula of the compound B is as follows:

   

   Wherein, R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , R ₆ , R ₇ and M have the same definitions as in claim 1.
9. The preparation method according to claim 8, wherein the molar ratio of the compound A and the compound B is 1:1;

   Preferably, the reagent for the condensation reduction reaction is sodium cyanoborohydride;

   Preferably, the added amount of sodium cyanoborohydride is more than 2 times the molar added amount of compound B;

   Preferably, the reaction temperature of the condensation reduction reaction is 10-30°C, and the reaction time is 12-48h.
10. The preparation method according to claim 8, wherein the preparation method of the compound A is as follows:

    Substituting compound C and compound D to obtain compound A, the reaction equation is as follows:

    

    Preferably, the molar ratio of compound C and compound D is (1.1-1.3):1;

    Preferably, the reagent for the substitution reaction is any one of sodium hydrogen, sodium hydroxide, potassium hydroxide or sodium carbonate;

    Preferably, the reaction temperature of the substitution reaction is 25° C. or more, and the reaction time is 2 h or more;

    Preferably, the reagent is first added to the compound D solution and mixed, and then compound C is added to the compound D solution for substitution reaction;

    Preferably, the adding temperature of the reagent is below 0°C;

    Preferably, the preparation method of the compound B is as follows:

    

    Preferably, in the preparation method of compound B, the reagent used for amine group deprotection is an acidic reagent;

    Preferably, the acidic reagent includes inorganic acid or organic acid;

    Preferably, the

    

    The preparation method is as follows:

    

    Preferably, the reaction of step (e) is carried out in the presence of an organic base or an inorganic base;

    Preferably, the reaction in step (e) is carried out in the presence of a condensing agent;

    Preferably, the reaction of step (f) is carried out in the presence of an inorganic base.
11. The enantiomer, diastereomer or pharmaceutically acceptable salt of the bifunctional immunomodulator according to any one of claims 1 to 7.
12. The enantiomer, diastereomer or pharmaceutically acceptable salt of the bifunctional immunomodulator according to claim 11, wherein the pharmaceutically acceptable salt is an acid addition salt Or alkali addition salt;

    Preferably, the acid addition salt includes hydrochloride, hydrobromide, hydroiodide, phosphate, sulfate, nitrate, ethanesulfonate, tosylate, benzenesulfonate, acetic acid Salt, maleate, tartrate, succinate, citrate, benzoate, ascorbate, salicylate, malonate, adipate, caproate, arginine, Any one or a combination of at least two of fumarate, nicotinate, phthalate or oxalate;

    Preferably, the base addition salt includes lithium salt, sodium salt, potassium salt, barium salt, calcium salt, magnesium salt, aluminum salt, iron salt, ferrous salt, copper salt, zinc salt, or the bifunctional immune The modifier is a salt composed of morpholine, diethylamine, triethylamine, isopropylamine, trimethylamine, lysine or histidine.
13. A pharmaceutical composition comprising the bifunctional immunomodulator according to any one of claims 1 to 7 or the enantiomers, diastereomers of the bifunctional immunomodulator according to claim 11 or 12 Isomers or pharmaceutically acceptable salts and pharmaceutically acceptable carriers.
14. The bifunctional immunomodulator according to any one of claims 1 to 7 or the enantiomers, diastereomers or pharmacologically of the bifunctional immunomodulator according to claim 11 or 12 Application of acceptable salts in the preparation of dual inhibitors of PD-1/PD-L1 and TGF-β.
15. The bifunctional immunomodulator according to any one of claims 1 to 7 or the enantiomers, diastereomers or pharmacologically of the bifunctional immunomodulator according to claim 11 or 12 Use of acceptable salts in the preparation of drugs for diseases related to PD-1/PD-L1 and/or TGF-β signaling pathway;

    Preferably, the disease related to PD-1/PD-L1 and/or TGF-β signaling pathway includes cancer, infectious disease or autoimmune disease;

    Preferably, the diseases related to the PD-1/PD-L1 and/or TGF-β signaling pathway include multiple myeloma, melanoma, glioma, glioblastoma, leukemia, sarcoma, smooth muscle Tumor, mesothelioma, breast cancer, cervical cancer, lung cancer, stomach cancer, rectal cancer, colon cancer, pancreatic cancer, brain cancer, skin cancer, oral cancer, prostate cancer, bone cancer, kidney cancer, ovarian cancer, bladder cancer, liver cancer , Bacterial infection, viral infection, chronic lymphocytic thyroiditis, hyperthyroidism, insulin-dependent diabetes mellitus, myasthenia gravis, ulcerative colitis, pernicious anemia with chronic atrophic gastritis, pulmonary hemorrhage nephritis syndrome, primary bile Liver cirrhosis, multiple cerebrospinal sclerosis, rheumatoid arthritis, dermatomyositis, polymyositis, osteoporosis, ulcers, vascular damage, glomerulonephritis, diabetic nephropathy, lupus nephritis, hypertension- Induced nephropathy, renal interstitial fibrosis, renal fibrosis, graft nephropathy, liver fibrosis, liver dysfunction, alcohol-induced hepatitis, cystic fibrosis, interstitial lung disease, acute lung injury, adult respiratory distress Syndrome, idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease, lung disease caused by infectious or toxic factors, heart fibrosis after infarction, congestive heart failure, dilated cardiomyopathy, myocarditis, intimal thickening, vascular stenosis , Pulmonary hypertension, coronary artery restenosis, peripheral artery restenosis, carotid artery restenosis, stent-induced restenosis, atherosclerosis, ocular scar formation, corneal scar formation, proliferative vitreoretinopathy, glaucoma, intraocular pressure High, excessive or hypertrophic dermal scars or keloid formation, peritoneal and subcutaneous adhesions, scleroderma, fibrosclerosis, progressive systemic sclerosis, decreased nervous system function, Peyronie's disease, Dupuytren's Contractures, Alzheimer's disease, Raynaud's syndrome, or radiation-induced fibrotic disease.