CN114573563A - Bifunctional molecular compound for inducing PD-L1 protein degradation and preparation and application thereof - Google Patents
Bifunctional molecular compound for inducing PD-L1 protein degradation and preparation and application thereof Download PDFInfo
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- CN114573563A CN114573563A CN202210300097.2A CN202210300097A CN114573563A CN 114573563 A CN114573563 A CN 114573563A CN 202210300097 A CN202210300097 A CN 202210300097A CN 114573563 A CN114573563 A CN 114573563A
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- 150000001875 compounds Chemical class 0.000 title claims abstract description 110
- 108010074708 B7-H1 Antigen Proteins 0.000 title claims abstract description 80
- 102000008096 B7-H1 Antigen Human genes 0.000 title claims abstract description 80
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- 230000001939 inductive effect Effects 0.000 title claims abstract description 21
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- 230000015572 biosynthetic process Effects 0.000 claims description 67
- 238000003786 synthesis reaction Methods 0.000 claims description 67
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 63
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 claims description 60
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 claims description 46
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-Diisopropylethylamine (DIPEA) Chemical compound CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 claims description 36
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Images
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- C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
- C07D401/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing three or more hetero rings
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Abstract
The invention discloses a bifunctional molecular compound for inducing PD-L1 protein degradation and a preparation method and application thereof, belonging to the technical field of chemical biology. The compound takes PD-L1 as a target protein binding ligand, can degrade PD-L1 protein in a targeted manner, can be used as a PD-L1 protein targeted degradation agent, and can provide a very potential treatment scheme for PD-L1 mediated infection, tumor, autoimmune disease and/or other diseases through research. The structural formula (I) and B and L in each structural formula are described in the specification.
Description
Technical Field
The invention belongs to the technical field of chemical biology, and particularly relates to a bifunctional molecular compound for inducing the degradation of PD-L1 protein, and preparation and application thereof. The compounds can degrade PD-L1 in a375 and B16F 10 cell lines and can increase the tumor killing effect of T cells.
Background
Programmed cell death protein 1 ligand 1(PD-L1, B7-H1, CD274) is a member of the B7 family of cell surface ligands, which regulate T cell activation and immune responses. PD-L1 is structurally similar to members of the B7 family, in that it contains extracellular IgV and IgC domains and a short cell plasma region. PD-L1 ligand binds to PD-L1 transmembrane receptor and inhibits T cell activation. Studies have shown that PD-L1 is expressed in tissues such as antigen presenting cells, activated T cells, placenta, heart and lung. In addition, PD-L1 is expressed in cells of many tumor types, melanoma, ovarian cancer, colon cancer, lung cancer, breast cancer, and renal cell carcinoma. Expression of PD-L1 in cancer cells is associated with tumor infiltrating lymphocytes and can mediate PD-L1 expression by releasing interferon gamma. Other studies have shown that PD-L1 expression is associated with cancers associated with viral infections.
The concept of protein targeting chimera gradually enters the field of drug development, and target protein is degraded by a ubiquitination mechanism to achieve the inhibition of the target protein. Traditional small molecules, antibodies and the like inhibit the function of target proteins to play a role in treating diseases through an 'occupation driving' action mode, and the action mode requires that an inhibitor or a monoclonal antibody has higher concentration to be capable of occupying active sites of targets and blocking the transduction of downstream signal paths. And PROTAC plays a role through an event-driven mechanism, does not influence the function of protein, and mediates the degradation of target protein.
At present, the medicines for tumor immunotherapy are mainly monoclonal antibody medicines, which is mainly because the curative effect of the PD-L1 small molecule inhibitor is not ideal. The effect of a small molecule inhibitor on poor effect of PD-L1 can be well solved by using the PROTAC technology, so that the design of the PD-L1 related small molecule degradation agent can obviously improve the clinical immunotherapy effect.
Disclosure of Invention
The invention aims to complement the prior art and provides a bifunctional molecular compound for inducing the degradation of PD-L1 protein and preparation and application thereof, in particular to a non-natural PD-L1 degradation agent synthesized based on biphenyl compounds and application thereof in tumor immunotherapy. The direct killing effect of T cells on tumor cells is enhanced by degrading the intracellular PD-L1. Researches show that the PROTAC molecule taking biphenyl compounds BMS-37 as PD-L1 ligands has certain degradation effect on PD-L1 no matter CRBN or VHL is selected as an E3 ligase ligand. The compound synthesized in the study can degrade PD-L1 by combining two E3 ligands, and the phenomenon can be reversed by a proteasome inhibitor MG132, which indicates that the compound plays a role through a ubiquitin proteasome pathway. In addition, research results show that the compounds can obviously increase the killing effect of T cells. Therefore, designing the PD-L1 protein degradation agent based on the biphenyl compound provides a new treatment strategy for tumor immunotherapy.
The specific scheme of the invention is as follows:
the invention provides five bifunctional molecular compounds which are shown as a formula I, a formula II, a formula III, a formula IV and a formula V and can induce the degradation of PD-L1 protein, or pharmaceutically acceptable salts, hydrates or prodrugs thereof;
wherein B is ubiquitin ligase E3 ligand, more preferably one of CRBN, VHL, MDM2, cIAP, UBR7, RNF114, CBLB and KEAP 1.
Wherein the CRBN is preferably one of the following structural formulas:
wherein:
w is selected from CH2、C=O、SO2One of NH and N-alkyl; the alkyl group is preferably a C1-C4 alkyl group;
x is selected from one or two of O, S;
z is selected from one of hydrogen, C1-C4 alkyl, C3-C6 cycloalkyl and halogen;
G. each G' independently represents H, C1-C4 alkyl, -OH, C1-C4 alkyl substituted 5-10 membered heterocyclyl containing 1-3 heteroatoms of N, O or S;
R1is selected from H, D, halogen, nitryl, amino, cyano, hydroxyl, C1-C4 alkyl, halogenated C1-C4 alkyl and deuterated C1-C4 alkyl.
The structural formula of VHL is as follows:
wherein:
R2is selected from CH3And H.
The MDM2 has a structural formula as follows:
wherein:
R3is a piperazinyl, piperidinyl, heterocyclic group or one of the linking groups of the following structures:
in the above linking group, n is an integer of 0 to 3.
Wherein:
the heterocyclic group is one of piperazinonyl, pyrrolyl, pyrazolyl, furyl, thienyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, pyridyl, pyrimidinyl, pyrazinyl or pyridazinyl.
The cIAP has the structure as follows:
R4h or Boc.
The L is preferably any one of the structures shown below:
wherein: m is an integer selected from 1 to 10.
The invention preferably relates to a bifunctional molecular compound which induces the degradation of PD-L protein and is shown in the following formulas VI and VII:
among the bifunctional molecular compounds for inducing the degradation of PD-L1 protein represented by formula VI and formula VII above, any one of the following structures is more preferable:
m is selected from integers between 1 and 10.
The invention is more preferably a bifunctional molecular compound which induces the degradation of PD-L1 protein and is shown in the following formula VIII:
in the bifunctional molecular compound shown in formula VIII above, which induces the degradation of PD-L1 protein, L preferably has the following structure:
m is an integer selected from 1 to 5.
Preferred bifunctional molecular compounds of the present invention that induce the degradation of PD-L1 protein, represented by the following structural formula, include, but are not limited to:
according to the present invention, pharmaceutically acceptable salts of bifunctional molecular compounds inducing the degradation of PD-L1 protein include addition salts with the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, benzenesulfonic acid, naphthalenedisulfonic acid, acetic acid, propionic acid, lactic acid, trifluoroacetic acid, maleic acid, citric acid, fumaric acid, oxalic acid, tartaric acid, benzoic acid, and the like. Hydrochloric acid, hydrobromic acid, sulfuric acid, citric acid, tartaric acid, phosphoric acid, lactic acid, pyruvic acid, acetic acid, trifluoroacetic acid, maleic acid, benzenesulfonic acid, succinic acid, and similar known acceptable acid salts.
In addition, the present invention also includes prodrugs of the derivatives of the present invention. They may themselves have a weak or even no activity, but are converted to the corresponding biologically active form under physiological conditions (e.g., by metabolism, solvolysis, or otherwise) after administration.
The invention also provides a preparation method of the bifunctional molecular compound for inducing the degradation of the PD-L1 protein, which comprises the following steps:
the synthetic route of the PD-L1 protein degradation agent shown in the formula I is as follows:
step 1-1: synthesis of Compound 3
4-hydroxy-2, 6-dimethoxybenzaldehyde (910mg, 5mmol), triphenylphosphine (1.44g, 5.5mmol) and (2-methyl- [1,1' -biphenyl) were weighed out]-3-yl) methanol (990mg, 5mmol) was added to anhydrous THF, a solution of diisopropyl azodicarboxylate (1.11g, 5.5mmol) in anhydrous THF was added dropwise to the above reaction system under ice bath conditions, the reaction was continued overnight at room temperature, and the reaction mixture was poured into H2O, then the mixture was extracted with DCM, the organic phase was washed with brine, dried over anhydrous sodium sulfate and concentrated in vacuo to give a residue which was purified by silica gel chromatography eluting with an ethyl acetate/petroleum ether gradient to give compound 3;
step 1-2: synthesis of Compound 6
Dissolving 4-pentanoic acid (200mg, 2mmol) with DCM, then adding N-Boc-ethylenediamine (320mg, 2mmol), EDCI (400mg, 2.1mmol), HOBt (270mg, 2mmol) and DIPEA (0.4mL) to the round bottom flask, stirring the mixture at room temperature overnight, diluting the reaction system with DCM, washing the organic phase twice with saturated sodium bicarbonate solution, drying over anhydrous sodium sulfate, concentrating the mixture under reduced pressure, purifying the product by silica gel chromatography, then deprotecting with trifluoroacetic acid to give compound 6;
step 1-3: synthesis of Compound 7
Adding a compound 3(3.4mmol, 1.23g) and a compound 6(5.1mmol, 715mg) into a round-bottom flask in sequence, reacting for 2 hours at room temperature, adding sodium triacetoxyborohydride (4.1mmol, 870mg) and a drop of acetic acid, stirring at room temperature overnight, diluting the reaction system with dichloromethane, washing the organic phase twice with saturated sodium bicarbonate solution, drying with anhydrous sodium sulfate, and purifying by silica gel chromatography to obtain a compound 7;
step 1-4: synthesis of Compounds of formula I
Mixing compound 7(0.035mmol, 17mg), B-linker, CuSO4(0.044mmol, 7mg) and VcNa (0.105mmol, 21mg) are sequentially added into a mixed solution (1:1) of THF and water, stirred at room temperature for 2 hours, concentrated to remove tetrahydrofuran, added with water and dichloromethane for 3 times, an organic phase is dried by anhydrous sodium sulfate and concentrated to obtain a crude product, and the crude product is purified by silica gel column chromatography to obtain a compound shown in the formula I;
(II) the synthesis routes of the PD-L1 protein degradation agents shown in the formula II and the formula III are as follows:
step 2-1: synthesizing a compound 9;
5-chloro-2, 4-dihydroxybenzaldehyde (863mg, 5mmol), triphenylphosphine (1.44g, 5.5mmol) and (2-methyl- [1,1' -biphenyl) were weighed]-3-yl) methanol (990mg, 5mmol) was added to anhydrous THF, a solution of diisopropyl azodicarboxylate (1.11g, 5.5mmol) in anhydrous THF was added dropwise to the above reaction system under ice bath conditions, the reaction was continued overnight at room temperature, and the reaction mixture was poured into H2O, the mixture was then extracted with DCM, the organic phase was washed with brine, dried over anhydrous sodium sulfate,concentration in vacuo afforded a residue which was purified by silica gel chromatography eluting with an ethyl acetate/petroleum ether gradient to afford compound 3;
step 2-2: synthesis of Compound 11
Dissolving a compound 9(0.21mmol, 74mg) with DMF, sequentially adding 3- (bromomethyl) benzonitrile (0.25mmol, 49mg), reacting at 80 ℃ for 30min, adding water into the reaction system, extracting with ethyl acetate, washing an organic phase with saturated brine for 3 times, drying with anhydrous sodium sulfate, evaporating under reduced pressure to obtain a crude product, purifying the crude product with tert-butyl methyl ether, and filtering to obtain a compound 11;
step 2-3: synthesis of Compound 13
Compound 11(0.13mmol, 63mg), 2-amino-2-methylpropanoic acid (0.54mmol, 56mg), AcOH (2d) and NaBH3CN (0.67mmol, 42mg) in 5mL DMF was left to react at 80 ℃ for 1h, ethyl acetate and water were added to the reaction mixture, the organic extract was concentrated and the crude product was chromatographed on silica gel eluting with methanol and dichloromethane to give compound 13;
step 2-4: synthesis of Compound 13y
Dissolving compound 13(0.02mmol, 13mg) in DMF, taking propargylamine (0.03mmol, 2 μ L), HATU (0.03mmol, 11mg) and DIPEA (0.04mmol, 6 μ L) to react for 2 hours at room temperature, and performing silica gel chromatography on a product, namely a petroleum ether ethyl acetate system to obtain compound 13 y;
step 2-5: synthesis of Compounds of formula II
Mixing compound 13y (0.035mmol, 17mg), B-linker, CuSO4(0.044mmol, 7mg) and VcNa (0.105mmol, 21mg) are sequentially added into a mixed solution (1:1) of THF and water, stirred at room temperature for 2 hours, concentrated to remove tetrahydrofuran, added with water and dichloromethane for 3 times, an organic phase is dried by anhydrous sodium sulfate and concentrated to obtain a crude product, and the crude product is purified by silica gel column chromatography to obtain a compound shown in a formula II;
step 2-6: synthesis of Compounds of formula III
Compound 13(0.04mmol, 22mg), EDCI (0.08mmol, 15mg), HOBt (0.08mmol, 11mg), DIPEA (0.16mmol, 28 μ L) and B-linker were dissolved in DCM and stirred at room temperature for 2h, the reaction mixture was extracted with water and DCM, the organic layer was washed with water 3 times, dried over anhydrous sodium sulfate, concentrated by distillation under reduced pressure to give a residue which was purified by silica gel chromatography to give formula III;
(III) the synthetic route of the PD-L1 protein degradation agent shown in the formula IV is as follows:
step 3-1: synthesis of Compound 16
To a solution of 2-methylbiphenyl-3-methanol (1.45g, 7.31mmol) in dichloromethane at room temperature was added portionwise dess-martin reagent (3.26g, 7.68mmol), the resulting mixture was stirred at room temperature for 30min, then quenched with sodium bicarbonate solution and sodium sulfate solution, the mixture was extracted with dichloromethane, the combined extracts were dried over anhydrous sodium sulfate and concentrated, and the residue was purified by column chromatography to give compound 16;
step 3-2: synthesis of Compound 18y
Methyl 3-amino-4-hydroxybenzoate (49mg, 0.29mmol), compound 16(69mg, 0.35mmol) and zinc trifluoromethanesulfonate (10mg, 0.03mmol) were dissolved in ethanol (1.5mL) and refluxed overnight, after which the reaction mixture was cooled to room temperature and then concentrated, the residue was dissolved in dichloromethane, then dichlorodicyanoquinone (100mg, 0.6mmol) was added, the mixture was stirred at room temperature for 0.5 hour, then diluted with ethyl acetate and washed with sodium bicarbonate solution, sodium sulfate solution, water and brine, the organic layer was dried over anhydrous sodium sulfate and concentrated, and the residue was purified by column chromatography to give compound 18 y;
step 3-3: synthesis of Compound 18
Treating the compound 18y under the conditions of LiOH, methanol/water, and then extracting and concentrating by ethyl acetate to obtain a compound 18;
step 3-4: synthesis of compound 19, compound 13y, synthesis of compound 19;
step 3-5: synthesis of compounds of formula IV;
mixing compound 19(0.035mmol, 13mg), B-linker, CuSO4(0.044mmol, 7mg) and VcNa (0.105mmol, 21mg) are sequentially added into a mixed solution (1:1) of THF and water, stirred at room temperature for 2 hours, concentrated to remove tetrahydrofuran, added with water and dichloromethane for 3 times, an organic phase is dried by anhydrous sodium sulfate and concentrated to obtain a crude product, and the crude product is purified by silica gel column chromatography to obtain a compound shown in a formula II;
(IV) the synthetic route of the PD-L1 protein degradation agent shown in the formula V is as follows:
step 4-1: synthesis of Compound 22
6-Chloropyridazin-3-amine (5mmol, 648mg) was dissolved in 10mL dioxane, and NaHCO was added sequentially3(7.5mmol, 630mg) and ethyl 3-bromo-2-oxopropanoate (5.5mmol, 1.07g), reacting the mixture at 100 ℃ for 3h, filtering the reaction system, diluting the filtrate with water, extracting with ethyl acetate, drying over anhydrous sodium sulfate, concentrating to obtain a crude black colloid, and purifying the product by silica gel column chromatography;
step 4-2: synthesis of Compound 24
Compound 22(10.1mmol, 2.5g), K2CO3(30.5mmol, 4.2g), (R) -pyrrolidin-3-ylcarbamic acid tert-butyl ester (10.1mmol, 1.9g) and 40mL DMF are reacted at 110 ℃ for 12 hours, diluted with water, extracted 3 times with ethyl acetate, the organic phase is washed 3 times with saturated brine, dried over anhydrous sodium sulfate, concentrated to give a crude product, which is purified by column chromatography to give compound 24;
step 4-3: synthesis of compound 25, synthesis of compound 25 and compound 18;
step 4-4: synthesis of Compound 27
Boc-D-histidine (2mmol, 511mg) and K were added to 20mL DMF2CO3(6mmol, 829mg) followed by the dropwise addition of benzyl bromide (2.2mmol, 261 μ L), reaction at room temperature for 4h and purification of the residue by flash chromatography on silica gel to give compound 27;
and 4-5: synthesizing a compound 28, namely removing a protecting group Boc of the compound 27 by trifluoroacetic acid to obtain a compound 28;
and 4-6: synthesis of Compound 29
Dissolving 2-methyl- [1,1' -biphenyl ] -3-methanol (1g, 5mmol) in dichloromethane, adding triethylamine (5.5mmol, 0.7mL) and a dichloromethane solution of triphosgene (600mg, 2mmol) at-20 ℃, then turning to 0 ℃ for reaction for 1.5 hours, adding 30mL of n-hexane into the reaction system, stirring for 10 minutes, filtering, washing a filter cake for 3 times by using a mixed solution of 10mL of dichloromethane and n-hexane (1:2), concentrating the filtrate to obtain a compound 29, and directly putting the compound into the next reaction without further purification due to instability of a product;
and 4-7: synthesis of Compound 30
Compound 24(1.86mmol, 650mg) was dissolved in a mixed solution of THF and water, NaHCO was added3(5.6mmol, 470mg), compound 29 in THF (6.5mL) was added dropwise at 0 ℃, transferred to room temperature and stirred for 2 hours more, quenched with water and extracted 3 times with ethyl acetate, the organic phase was dried over anhydrous sodium sulfate, filtered and concentrated, and the residue was purified by silica gel column chromatography to give compound 30;
and 4-8: synthesis of compound 31;
compound 28(0.04mmol, 15mg), compound 30(0.04mmol, 19mg), EDCI (0.08mmol, 15mg), HOBt (0.08mmol, 11mg), DIPEA (0.16mmol, 28 μ L) were dissolved in DCM and stirred at room temperature for 2h, the reaction mixture was extracted with water and DCM, the organic layer was washed with water 3 times, dried over anhydrous sodium sulfate, concentrated by distillation under reduced pressure to give a residue which was purified by chromatography on silica gel to give compound 31;
and 4-9: synthesis of compound 31 y;
compound 31(0.04mmol, 32mg), propynylamine (0.08mmol, 4mg), EDCI (0.08mmol, 15mg), HOBt (0.08mmol, 11mg), DIPEA (0.16mmol, 28 μ L) were dissolved in DCM and stirred at room temperature for 2h, the reaction mixture was extracted with water and DCM, the organic layer was washed with water 3 times, dried over anhydrous sodium sulfate, concentrated by distillation under reduced pressure to give a residue, which was purified by chromatography on silica gel to give compound 31;
step 4-10: synthesis of compounds of formula V.
Mixing compound 31y (0.035mmol, 25.8mg), B-linker, CuSO4(0.044mmol, 7mg) and VcNa (0.105mmol, 21mg) are sequentially added into a mixed solution (1:1) of THF and water, stirred at room temperature for 2 hours, concentrated to remove tetrahydrofuran, added with water and extracted with dichloromethane for 3 times, an organic phase is dried by anhydrous sodium sulfate and concentrated to obtain a crude product, and the crude product is purified by silica gel column chromatography to obtain a compound shown in a formula V;
the synthetic route of ubiquitin ligase E3 ligand and L (linker) is as follows:
(1) when the ubiquitin ligase E3 ligand is CRBN, Th (thalidomide derivative) is preferred, and the synthesis method comprises the following steps:
dissolving a compound Th (thalidomide derivative) in DMF, adding DIPEA and 1.0mol of linker into a reaction system, reacting for 2 hours at 90 ℃, adding water, extracting with ethyl acetate, collecting an organic layer, drying with anhydrous sodium sulfate, concentrating under reduced pressure, and purifying by silica gel column chromatography to obtain a compound Th-L;
(2) when the ubiquitin ligase E3 ligand is MDM2, the synthesis method comprises the following steps:
dissolving 1.0mol of MDM2 in DCM, adding 1.1mol of EDCI, 1.1mol of HOBt and 1.5mol of DIPEA under ice bath, adding 1.0mol of linker, reacting for 5h, adding a proper amount of DCM for dilution and extraction, collecting an organic layer, drying with anhydrous sodium sulfate, concentrating under reduced pressure, and purifying by silica gel column chromatography to obtain a compound MDM 2-L;
(3) when the ubiquitin ligase E3 ligand is VHL, the synthetic route is the same as that of MDM2-L, and the difference is that MDM2 is replaced by VHL, and VHL-L is prepared correspondingly;
(4) when the ubiquitin ligase E3 ligand is cIAP, the synthetic route is the same as MDM2-L, and the difference is that VHL is replaced by cIAP, and the cIAP-L is correspondingly prepared.
A pharmaceutical composition comprising as an active ingredient a therapeutically effective amount of a bifunctional molecular compound which induces the degradation of PD-L1 protein.
A pharmaceutical composition further comprising a pharmaceutically acceptable carrier, diluent, adjuvant, vehicle, or combination thereof.
Wherein the dosage form of the pharmaceutical composition is any one of injection, tablet and capsule.
The invention also provides application of the bifunctional molecular compound for inducing the PD-L1 protein degradation and pharmaceutically acceptable salts thereof or the pharmaceutical composition in preparing medicines for treating and/or preventing infection, cancer or autoimmune diseases.
The infection is one or more of skin infection, gastrointestinal tract infection, urogenital system infection, systemic infection, or virus infection caused by one or more of influenza, hepatitis C virus, human papilloma virus, cytomegalovirus, Epstein Barr virus, poliovirus, hydrozymia ribbon-shaped cross-sectional virus, coxsackie virus and human immunodeficiency virus;
the cancer is one or more of bone cancer, lung cancer, stomach cancer, colon cancer, membrane adenocarcinoma, breast cancer, prostate cancer, lung cancer, brain cancer, ovarian cancer, cancer of the shoulder, cervical cancer, cancer of the innocent pill, kidney cancer, head and neck cancer, lymphoma, leukemia and skin cancer.
The autoimmune disease is one or more of rheumatoid arthritis, systemic lupus erythematosus, mixed connective tissue disease, systemic scleroderma, dermatomyositis, nodular vasculitis, nephropathy, endocrine related diseases, liver disease, psoriasis and autoimmune reaction caused by infection.
The invention has the beneficial effects that:
the invention takes PROTAC technology as support and PD-L1 inhibitor as raw material to synthesize bifunctional molecular compounds which induce PD-L1 protein degradation and have different Linker lengths.
The present invention effectively targets and degrades PD-L1; similar to catalytic reaction, the medicament has low effective dose; only provides binding activity, is event-driven, is different from the traditional occupation drive, and does not need to directly inhibit the functional activity of the target protein; the drug does not require long-term and high-strength binding to the target protein. The bifunctional molecular compound for inducing the PD-L1 protein degradation provides a new treatment mode for treating PD-L1-mediated tumors and/or other diseases.
Drawings
FIG. 1 shows the degradation of PD-L1 by BMS-37-C1-C5, BMS37-3C-V2, BMS37-5C-V2 and BMS 37-7C-V2;
FIG. 2 is a time-dependent degradation of PD-L1 and MG132 reversal experiments with BMS-37-C1-C5 and BMS37-3C-V2, BMS37-5C-V2, BMS37-7C-V2 of the present invention;
FIG. 3 shows that BMS-37-C1, BMS-37-C3 and BMS37-5C-V2 of the present invention detect changes in PD-L1 expression using flow cytometry;
FIG. 4 is a graph showing the effect of BMS-37-C1 and BMS-37-C3 of the present invention on T cell killing of tumor cells;
FIG. 5 is a graph of the affinity binding curves of BMS-37-C1, BMS-37-C3 and BMS37-5C-V2 to PD-L1 measured by MST in capillaries treated with standards according to an example of the invention.
Detailed Description
The invention is further illustrated by the examples. It should be understood, however, that the scope of the present invention is not limited to the following examples.
Example 1 Synthesis of intermediate Compound Th-L2 (m-2)
b is CRBN, the synthetic route is as follows:
58mg of compound Th (a derivative of thalidomide, commercially available) is put in a solanaceous bottle, 3mL of DMF is added, 50mg of azido-2 PEG-amine and 47 muL of DIPEA are sequentially added under stirring, the mixture reacts for 3 to 4 hours at 90 ℃, 30mL of water and 30mL of ethyl acetate are added for extraction, an organic layer is dried by anhydrous sodium sulfate and concentrated to obtain a crude product, the crude product is purified by silica gel column chromatography, and petroleum ether-ethyl acetate is eluted in a gradient from 1:2 to 1:4 to obtain 33.2mg of yellow oily matter with the yield of 41%.
Th-L2,1H NMR(400MHz,CDCl3)δ9.06(br s,1H),7.49(t,J=7.8Hz,1H),7.09(dd,J=
7.2,2.0Hz,1H),6.93(d,J=8.4Hz,1H),6.50(t,J=5.6Hz,1H),4.96–4.92(m,1H),3.74(t,J=5.2Hz,2H),3.70–3.67(m,6H),3.48(q,J=5.6Hz,2H),3.38(t,J=4.4Hz,2H),2.80–2.73(m,3H),2.13–2.10(m,1H);13C NMR(100MHz,CDCl3)δ171.7,169.4,168.8,167.7,146.9,136.1,132.6,116.9,111.6,110.3,70.75,70.72,70.1,69.6,50.7,48.9,42.4,31.5,22.8。
Example 2 synthesis of intermediate compound M-L1(M ═ 1)
In this embodiment, B is MDM 2;
adding 320mg of compound M into a solanaceous bottle, adding 3mL of DCM, sequentially adding 65mg of azido-1 PEG-amine, 132 mu L of DIPEA, 74mg of EDCI and 92mg of HOBt under ice bath, reacting for 3-4 hours, adding 30mL of water and 30mL of DCM for extraction, drying an organic layer by anhydrous sodium sulfate, concentrating to obtain a crude product, and purifying by silica gel column chromatography to obtain 237.5mg of a white solid, wherein the yield is as follows: and (3.2).
M-L1,1H NMR(600MHz,DMSO-d6)δ7.96(t,J=5.7Hz,1H),7.56–7.51(m,1H),7.17–
7.14(m,2H),7.12(d,J=8.3Hz,2H),7.07–7.02(m,2H),6.98(d,J=8.0Hz,2H),6.62(d,J=7.3Hz,2H),5.66(d,J=9.7Hz,1H),5.58(d,J=9.7Hz,1H),4.72(hept,J=6.0Hz,1H),3.83(s,4H),3.76–3.67(m,2H),3.65–3.55(m,3H),3.43(t,J=5.9Hz,2H),3.38(t,J=4.9Hz,3H),3.21(q,J=5.8Hz,3H),3.00(h,J=7.3Hz,2H),1.27(d,J=6.0Hz,3H),1.22(d,J=6.0Hz,3H).HRMS(ESI+):m/z calculated for C36H40Cl2N8O6[M+H]+,751.2448;found,751.2443。
EXAMPLE 3 Synthesis of intermediate Compound V-2L
In this example, B is VHL;
215mg of compound V is put in an eggplant-shaped bottle, 3mL of DCM is added, 57.5mg of 3-azidopropionic acid, 132 μ L of DIPEA, 74mg of EDCI and 92mg of HOBt are sequentially added under ice bath, reaction is carried out for 3-4 hours, 30mL of water and 30mL of DCM are added for extraction, an organic layer is dried by anhydrous sodium sulfate and concentrated to obtain a crude product, and the crude product is purified by silica gel column chromatography to obtain 279mg of a white solid, wherein the yield is as follows: 53 percent.
V-2L,1H NMR(600MHz,DMSO-d6)δ8.98(s,1H),8.57(t,J=6.0Hz,1H),8.12(d,J=9.4
Hz,1H),7.47–7.36(m,4H),5.13(d,J=3.6Hz,1H),4.58(d,J=9.4Hz,1H),4.47–4.40(m,2H),4.39–4.33(m,1H),4.21(dd,J=15.8,5.5Hz,1H),3.69(dd,J=10.5,4.2Hz,1H),3.62(dt,J=10.8,1.8Hz,1H),3.52(ddd,J=12.2,7.9,5.6Hz,1H),3.47(dt,J=12.2,6.1Hz,1H),2.63–2.53(m,1H),2.46–2.45(m,1H),2.45(s,3H),2.07–1.99(m,1H),1.91(ddd,J=12.9,8.5,4.6Hz,1H),0.95(s,9H).HRMS(ESI+):m/z calculated for C25H33N7O4S[M+H]+,528.2315;found,528.2320。
EXAMPLE 4 Synthesis of Compound 3
The specific operation process is shown in step 1-1, light yellow solid with the yield of 58%.1H NMR(400MHz,CDCl3)δ10.37(s,1H),7.42–7.27(m,8H),6.20(s,2H),5.15(s,2H),3.88(s,6H),2.27(s,3H).
EXAMPLE 5 Synthesis of Compound 6
See step 1-2 for a yellow oil.1H NMR(400MHz,CDCl3)δ6.29(s,1H),4.92(s,1H),3.40–3.36(m,2H),3.31–3.28(m,2H),2.55–2.51(m,2H),2.42–2.39(m,2H),2.01(t,J=2.4Hz,1H),1.44(s,9H).
EXAMPLE 6 Synthesis of Compound 7
The specific operation process is shown in steps 1-3, and the yield is as follows: 48 percent.1H NMR(400MHz,CDCl3)δ7.48–7.27(m,8H),6.24(s,2H),5.08(s,2H),4.61(s,2H),3.96(s,2H),3.84(s,6H),3.47–3.44(m,2H),2.82(t,J=4.8Hz,2H),2.51–2.49(m,2H),2.47–2.43(m,2H),2.27(s,3H),1.94(t,J=2.4Hz,1H).
EXAMPLE 7 Synthesis of Compound 11
See step 2-2 for a detailed procedure, white solid product 70mg, yield: 71 percent.1H NMR(600MHz,CDCl3)δ10.32(s,1H),7.91(s,1H),7.72(td,J=1.8,0.8Hz,1H),7.68(ddt,J=7.8,6.2,1.3Hz,2H),7.54(t,J=7.8Hz,1H),7.46–7.40(m,3H),7.40–7.34(m,1H),7.32–7.27(m,4H),6.63(s,1H),5.20(d,J=9.6Hz,4H),2.27(s,3H).
EXAMPLE 8 Synthesis of Compound 13
See step 2-3 for a specific procedure, 28mg of white solid, yield 38%.1H NMR(600MHz,DMSO-d6)δ8.03(s,1H),7.89(d,J=7.9Hz,1H),7.83(d,J=7.8Hz,1H),7.62(t,J=7.8Hz,1H),7.56(s,1H),7.50–7.44(m,3H),7.42–7.36(m,1H),7.33–7.26(m,3H),7.21(dd,J=7.6,1.4Hz,1H),7.12(s,1H),5.29(d,J=3.5Hz,4H),3.87(s,2H),2.23(s,3H),1.90(s,1H),1.27(s,6H).
EXAMPLE 9 Synthesis of Compound 13y
See steps 2-4 for a white solid, 8mg, 70%.1H NMR(600MHz,DMSO-d6)δ8.13(s,1H),7.96(s,1H),7.84–7.79(m,2H),7.62(t,J=7.8Hz,1H),7.49–7.42(m,4H),7.40–7.37(m,1H),7.33–7.30(m,2H),7.27(t,J=7.6Hz,1H),7.21(dd,J=7.7,1.5Hz,1H),7.06(s,1H),5.31(s,2H),5.24(s,2H),3.83(dd,J=6.0,2.6Hz,2H),3.49(s,2H),3.02(t,J=2.5Hz,1H),2.23(s,3H),1.99(dq,J=15.9,6.8,6.2Hz,1H),1.25–1.22(m,3H),1.20(s,3H).
EXAMPLE 10 Synthesis of Compound 16
See step 3-1 for a detailed procedure, 430mg of white solid, yield: 86 percent.1H NMR(400MHz,CDCl3)δ10.39(s,1H),7.84(dd,J=7.6,1.6Hz,1H),7.48-7.38(m,5H),7.29-7.27(m,2H),2.55(s,3H).
EXAMPLE 11 Synthesis of Compound 22
The specific operation process is shown in step 4-1, and the yield is as follows: 52 percent.1H NMR(600MHz,DMSO-d6)δ8.89(s,1H),8.30(d,J=9.6Hz,1H),7.50(d,J=9.6Hz,1H),4.35(q,J=7.1Hz,3H),1.33(t,J=7.1Hz,4H).
EXAMPLE 12 Synthesis of Compound 24
The specific operation process is shown in step 4-2, wherein the yield of the black colloid is 1.9g and 44.8%.1H NMR(600MHz,DMSO)δ8.35(d,J=0.7Hz,1H),7.83(d,J=10.0Hz,1H),7.25(d,J=6.7Hz,1H),6.98(d,J=9.9Hz,1H),4.27(q,J=7.1Hz,2H),4.13(d,J=6.8Hz,1H),3.65(dd,J=10.8,6.3Hz,1H),3.57(dt,J=10.2,7.2Hz,1H),3.47(ddd,J=10.3,7.9,5.7Hz,1H),3.28(dd,J=10.8,4.6Hz,1H),2.14(dq,J=13.6,6.9Hz,1H),1.91(dq,J=12.7,6.2Hz,1H),1.39(s,10H),1.30(t,J=7.1Hz,3H).
EXAMPLE 13 Synthesis of Compound 27
The specific operation process is shown in step 4-4, and the yield is as follows: 49 percent.1H NMR(600MHz,DMSO-d6)δ7.33(t,J=7.7Hz,1H),7.28(dd,J=8.3,6.4Hz,5H),7.24–7.16(m,3H),6.82(s,3H),5.11(d,J=1.9Hz,2H),5.03(q,J=12.5Hz,2H),4.31–4.24(m,1H),2.84–2.80(m,2H),1.34(s,9H).
EXAMPLE 14 Synthesis of Compound 30
See steps 4-7 for a detailed procedure, 512mg yellow solid, yield: and 55 percent.1H NMR(600MHz,DMSO-d6)δ8.35(s,1H),7.84(dd,J=10.0,0.7Hz,1H),7.73(d,J=6.6Hz,1H),7.44(t,J=7.5Hz,2H),7.40–7.33(m,2H),7.28–7.26(m,2H),7.24(d,J=7.6Hz,1H),7.16(d,J=7.6Hz,1H),6.98(d,J=9.9Hz,1H),5.12(s,2H),4.28(q,J=7.1Hz,2H),4.23(q,J=6.0Hz,1H),3.68(dd,J=10.9,6.2Hz,1H),3.58(dt,J=10.3,7.3Hz,1H),3.50(ddd,J=10.1,7.7,5.2Hz,1H),3.37(dd,J=11.1,4.4Hz,1H),2.22–2.17(m,1H),2.16(s,3H),2.00–1.91(m,1H),1.30(t,J=7.1Hz,3H).
Example 15 synthesis of compound BMS37-Cn (n ═ 1-5, exemplified by n ═ 1)
Mixing compound 7(0.035mmol, 17mg), Th-L1(0.035mmol, 13.5mg), CuSO4(0.044mmol, 7mg) and VcNa (0.105mmol, 21mg) were successively added to a mixed solution of THF and water (1:1), and stirred at room temperature for 2 hours. Concentrating to remove tetrahydrofuran, adding water, extracting with dichloromethane for 3 times, drying the organic phase with anhydrous sodium sulfate, concentrating to obtain crude product, and purifying by silica gel column chromatography to obtain 13mg solid with yield: 42.5 percent.1H NMR(400MHz,CDCl3)δ7.72(s,1H),7.62(s,1H),7.52–7.48(m,1H),7.43–7.41(m,3H),7.38–7.28(m,4H),7.12(d,J=7.2Hz,1H),6.88(d,J=8.4Hz,1H),6.47(t,J=5.2Hz,1H),6.24(s,2H),5.10(s,2H),4.97–4.42(m,1H),4.54–4.51(m,2H),4.12(s,2H),3.88–3.83(m,2H),3.82(s,6H),3.67(t,J=4.8Hz,2H),3.50–3.48(m,2H),3.43(q,J=5.2Hz,2H),3.03(td,J=6.8,2.8Hz,3H),2.92(t,J=4.8Hz,2H),2.86–2.73(m,4H),2.55(t,J=7.6Hz,2H),2.27(s,3H),2.09–2.07(m,1H).HRMS m/z calcd for C47H53N8O9 873.3936,found873.3930[M+H+].
Example 16 Synthesis of NP19-nP-Th (n-2-3, for example n-2)
The concrete operation and ratio are referred to in example 15.
NP19-2P-Th, yellow solid, yield 48%.1H NMR(600MHz,DMSO-d6)δ11.09(s,1H),8.25(s,1H),7.93(s,1H),7.83(s,1H),7.80–7.75(m,2H),7.59(t,J=7.8Hz,1H),7.54(dd,J=8.6,7.1Hz,1H),7.46(q,J=7.9Hz,3H),7.40–7.34(m,2H),7.32–7.29(m,2H),7.26(t,J=7.6Hz,1H),7.20(dd,J=7.7,1.5Hz,1H),7.07–6.99(m,3H),6.55(t,J=5.9Hz,1H),5.26(s,2H),5.21(s,2H),5.07(dd,J=12.7,5.6Hz,1H),4.48(t,J=5.2Hz,2H),4.30(d,J=5.7Hz,2H),3.80(t,J=5.2Hz,2H),3.56(t,J=5.5Hz,2H),3.48(s,2H),3.41–3.38(m,2H),3.17(dd,J=5.3,1.1Hz,1H),2.88(ddd,J=16.8,13.6,5.4Hz,1H),2.62–2.52(m,2H),2.22(s,3H),2.01(tdd,J=12.6,8.0,5.1Hz,2H),1.46(s,1H),1.32–1.25(m,2H),1.23(s,3H),1.21(s,3H).HRMS m/z calcd for C55H57ClN9O9 1022.3962,found 1022.4005[M+H+].
Example 17 Synthesis of MP-PC-nC compound (n ═ 4,6, exemplified by n ═ 4)
Compound 13(0.04mmol, 22mg), EDCI (0.08mmol, 15mg), HOBt (0.08mmol, 11mg), DIPEA (0.16mmol, 28. mu.L) and Th-4L (0.04mmol, 13.76mg) were dissolved in DCM and stirred at room temperature for 2 h. The mixture was diluted with water and extracted with DCM. The combined organic layers were washed with water, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give a residue. Purify by silica gel chromatography to obtain 15mg of yellow solid with a yield of 43%.1H NMR(600MHz,CDCl3)δ8.17(s,1H),7.68(d,J=1.7Hz,1H),7.62(ddt,J=10.8,7.8,1.4Hz,2H),7.50(t,J=7.7Hz,2H),7.46–7.40(m,4H),7.38–7.35(m,1H),7.31(d,J=1.5Hz,1H),7.29(d,J=0.9Hz,2H),7.26(d,J=1.4Hz,1H),7.25(s,1H),7.04(d,J=7.1Hz,1H),6.85(d,J=8.5Hz,1H),6.59(s,1H),6.19(s,1H),5.09(s,4H),4.89(dd,J=12.5,5.3Hz,1H),3.61(s,2H),3.25(q,J=6.6Hz,2H),3.19(q,J=6.7Hz,2H),2.85(ddd,J=16.9,3.6,2.4Hz,1H),2.79–2.66(m,2H),2.25(s,3H),2.12–2.07(m,1H),1.63–1.60(m,2H),1.55(dt,J=10.5,4.4Hz,2H),1.35(s,6H).HRMS m/z calcd for C50H50ClN6O7 881.3424,found 881.3432[M+H+].
EXAMPLE 18 Synthesis of NP19-2C-V2 Compound
The concrete operation and proportion are referred to in example 18.
NP19-2C-V2 as a yellow solid in 42% yield.1H NMR(600MHz,DMSO-d6)δ8.98(s,1H),8.38(d,J=7.8Hz,1H),7.98–7.91(m,2H),7.89(s,1H),7.81(dd,J=7.8,1.7Hz,2H),7.62(d,J=7.7Hz,1H),7.49(s,1H),7.48–7.45(m,2H),7.44(d,J=5.4Hz,1H),7.42(s,1H),7.40–7.35(m,3H),7.32–7.29(m,2H),7.27(s,1H),7.21(d,J=1.4Hz,1H),7.06(s,1H),5.35–5.32(m,1H),5.31(d,J=4.9Hz,2H),5.24(s,2H),4.91(s,1H),4.53(d,J=9.3Hz,1H),4.42(t,J=8.0Hz,1H),4.27(p,J=3.3Hz,1H),3.60(d,J=3.4Hz,2H),3.54–3.49(m,2H),3.27(p,J=6.5Hz,1H),3.21(p,J=6.5Hz,2H),2.45(s,3H),2.35–2.25(m,1H),2.23(s,3H),2.05–1.95(m,4H),1.23(s,6H),1.18(d,J=4.5Hz,3H),0.90(s,9H).HRMS m/z calcd for C59H66ClN7O7S 1052.4506,found 1052.4512[M+H+]
EXAMPLE 19 Synthesis of NB series Compounds (exemplified by NB-C1)
The concrete operation and ratio are referred to in example 15.
NB-C1, yellow solid, yield 85%.1H NMR(400MHz,CDCl3)δ9.28(s,1H),8.24(d,J=1.2Hz,1H),8.10(dd,J=6.8,2.4Hz,1H),7.93(s,1H),7.90(dd,J=8.8,1.6Hz,1H),7.58(d,J=8.4Hz,1H),7.47-7.33(m,9H),7.10(d,J=6.8Hz,1H),6.85(d,J=8.4Hz,1H),6.47(t,J=5.2Hz,1H),5.08(dd,J=12.0,5.2Hz,1H),4.84-4.72(m,2H),4.62-4.58(m,2H),3.90-3.88(m,2H),3.70(t,J=5.2Hz,2H),3.45-3.42(m,2H),2.88-2.76(m,3H),2.62(s,3H),2.18-2.14(m,1H).HRMS m/z calcd for C41H36N8O7Na 775.2599,found 775.2613[M+Na+].
Example 20 synthesis of Pd-nC-Th compound (n ═ 2,4, 6, exemplified by n ═ 2)
The concrete operation and proportion are referred to in example 18.
Pd-2C-Th, yellow solid, yield 53%.1H NMR(600MHz,DMSO-d6)δ11.09(s,1H),8.24–8.18(m,2H),8.15(s,1H),7.84(d,J=9.9Hz,1H),7.72(d,J=6.6Hz,2H),7.58–7.53(m,1H),7.44(t,J=7.5Hz,2H),7.40–7.33(m,2H),7.29–7.26(m,2H),7.25–7.21(m,2H),7.21–7.18(m,2H),7.16(dd,J=8.8,2.8Hz,2H),7.15–7.11(m,2H),7.01(dd,J=7.0,1.3Hz,1H),6.97(d,J=9.9Hz,1H),6.91(s,1H),6.75(d,J=4.9Hz,1H),5.11(d,J=15.4Hz,4H),5.04(ddd,J=12.8,5.5,1.8Hz,1H),4.67–4.62(m,1H),4.23(q,J=5.7Hz,1H),3.70(dd,J=10.8,6.1Hz,1H),3.59(q,J=7.8,5.4Hz,1H),3.53–3.49(m,1H),3.37(dd,J=10.8,4.1Hz,2H),3.27(t,J=6.5Hz,2H),3.25–3.15(m,2H),2.96(s,2H),2.92–2.83(m,1H),2.60–2.54(m,1H),2.20(dt,J=12.6,6.9Hz,1H),2.16(s,3H),2.04–1.92(m,4H).HRMS m/z calcd for C54H53N12O8 997.4104,found 997.4083[M+H+].
Example 21 Western Blot to detect changes in related proteins
Change in expression of PD-L1 protein
(1) Cells that grew logarithmically and reached 90% confluence were collected by digestion, adjusted for cell concentration by adding fresh medium, and then counted using a cell counting plate. Uniformly inoculating the cells into a large dish, culturing the cells in an incubator to adhere to the wall and start to be in a logarithmic division phase, replacing a culture medium containing the medicament, and then putting the cells into the incubator to continue culturing for 48 hours. After the culture is finished, the cells are digested by pancreatin and collected, and marked. The pancreatin was washed twice with PBS to remove excess EDTA, 5min each time and the cells were collected in 1.5mL EP tubes. After the washing is finished, adding a proper amount of RIPA lysate according to the number of cells, adding a phosphatase inhibitor, a holoenzyme inhibitor and PMSF in advance into the lysate. PMSF is a serine protease irreversible inhibitor, and can inhibit serine proteases (trypsin and chymotrypsin), and also inhibit cysteine protease and acetylcholinesterase, thereby preventing degradation of proteins. After addition of lysis buffer, cells were mixed well using vortex shaker and protein was lysed on ice to prevent protein degradation, for 20 min. After the lysis was completed, centrifugation was performed using a 4 ℃ centrifuge at 11500rpm for 10 min. After the centrifugation is finished, sucking the supernatant, wherein the supernatant is protein;
(2) protein content detection was performed by BCA method in this experiment. Under alkaline conditions, the protein converts Cu into2+Reduction to Cu+,Cu+Forming a purple blue complex with the BCA reagent, measuring the absorption value of the complex at 562nm, and comparing the absorption value with a standard curve to calculate the concentration of the protein to be detected;
(3) adding 6 XLoading buffer with corresponding volume according to the volume of the protein sample with the end of quantification. Before use, DTT is added into the 6 Xloading buffer, and the DTT can open the disulfide bond on cys to make it become a thread-bound polypeptide, thus facilitating the recognition of the antibody and the binding of the antibody to a specific site. Mixing the sample with vortex oscillator, and boiling in 100 deg.C water bath for 5-10min to completely denature protein. If the amino acid composition of one protein contains more hydrophobic amino acids (tryptophan W, alanine A, valine V, leucine L, isoleucine I, proline P, phenylalanine F and the like), and the protein has strong hydrophobicity, the protein can be easily formed into aggregates after being boiled, and for the protein rich in the hydrophobic amino acids, the protein can be incubated for 1h at 37 ℃, or the protein rich in the hydrophobic amino acids can be boiled in a water bath kettle at 60 ℃ for 30min instead of 100 ℃ for 5-10 min;
(4) preparing SDS-PAGE gel, and loading the prepared sample;
(5) the PVDF membrane was placed in anhydrous methanol for 30s to activate the PVDF membrane. The bubble is then introduced into the wet-converting fluid. And sequentially placing the sponge, the filter paper and the PVDF film into the white surface of the electric transfer nip, and then sequentially placing the glue, the filter paper and the sponge to manufacture the sandwich structure. Air bubbles are avoided between the glue and the membrane. Insert the sandwich structure in the electricity commentaries on classics groove to put into the ice box that ice-water mixes with the electricity commentaries on classics device, avoid producing too much heat at the commentaries on classics membrane in-process and lead to changeing the membrane failure. Protein was transferred using 300mA for 2 h;
(6) after the albumen rendition is ended, open sandwich structure, use the spring red to dye the membrane and look over the rendition effect and use the pure water to wash the membrane clean. The membrane was cut according to protein molecular weight, then the PVDF membrane was air dried with filter paper, and secondary activation was performed again with methanol. After activation, cleaning methanol on the surface with pure water, then putting the cleaned methanol into 5% skimmed milk, and sealing the skimmed milk for 2 hours by using a shaking table;
(7) the surface of the closed membrane is washed clean with TBST solution. The desired antibody was formulated using a 5% BSA liquid to make a primary anti-dilution according to the dilution ratio specified in the antibody specification. Then putting the PVDF membrane in the TBST on a wet box for primary antibody incubation, and putting the wet box in a refrigerator at 4 ℃ for overnight incubation of the primary antibody;
(8) the wet box was removed and the membranes were washed three times with TBST on a shaker for 5min each. Different secondary antibodies were selected according to the primary antibody instructions. The washed membrane was placed on a wet box and secondary antibody liquid was added and blocked at room temperature for two hours;
(9) the target strip was exposed using a gel imager.
As shown in FIGS. 1 and 2, the results of the experiments showed that the compounds BMS-37-C3 and BMS-37-C1 degraded most efficiently in the A375 cell line. Both compounds began to inhibit the expression level of PD-L1 protein at 0.3. mu.M. C2 also has degradability but inferior ability to C3. C4 and C5 had no effect in the a375 cell line. Similarly, BMS-37-C3 and C1 in the B16F 10 cell line still had stronger inhibition effect on PD-L1 than other compounds. BMS-37-VHL series compounds also have inhibitory effects on the expression of PD-L1. After further MG132 was applied to the cells, the inhibition of PD-L1 by BMS-37-C1 and BMS-37-C3 was reversed, which further indicates that these compounds degrade proteins via the ubiquitination pathway to decrease the expression of PD-L1.
Example 22 flow cytometry detection of alterations in related proteins
(1) Cells that grew logarithmically and reached 90% confluence were collected by digestion, adjusted for cell concentration by adding fresh medium, and then counted using a cell counting plate. The cells are evenly inoculated in a six-hole plate, the cells adhere to the wall in an incubator and begin to be in the logarithmic division phase, and the cells are placed in the incubator for continuous culture for 48h after a culture medium containing the medicine is replaced.
(2) After the end of the culture, the cells were digested with pancreatin and collected in 1.5mL sample tubes, labeled. Centrifuging the sample at 4 ℃ for 5min, discarding the supernatant, adding PBS for rinsing to remove the redundant EDTA in pancreatin and serum contained in the culture medium, dividing the control group into two groups, one group is a negative control and the other group is a positive control, centrifuging at 4 ℃ for 400g again for 5min, and discarding the supernatant.
(3) PBS solution containing 5% BSA was prepared, and the antibody and PBS were mixed at a volume ratio of 1:60 (murine source) to 1:40 (human source) under dark conditions, and finally IgG and PD-L1 antibody were added to the sample tube in a volume of 30. mu.L, respectively.
(4) Incubating at 4 ℃ in the dark for 40min, again incubating at 4 ℃ at 400g, centrifuging for 5min, discarding the supernatant, adding 200 mu L of PBS solution into the sample tube, and detecting on a machine.
As shown in FIG. 3, the experimental results show that BMS-37-C1 and BMS-37-VHL both reduced the expression of cell surface PD-L1 between 0.1 and 1 μ M in B16-F10 and A375 cells, but the HOCK effect occurred at 3-10 μ M; BMS-37-C3 can reduce the content of PD-L1 on the cell surface in B16-F10 and A375 cells in a concentration-dependent manner, and the effect is better.
EXAMPLE 23 Effect of Compounds on T cell killing of tumor cells
(1) Cells that grew logarithmically and reached 90% confluence were collected by digestion, adjusted for cell concentration by addition of fresh medium, and counted using a cell counting plate. Uniformly inoculating the cells into a ninety-six-well plate, adding the T cells after the cells adhere to the wall, and finally continuously co-culturing the tumor cells and the T cells for 48h according to the proportion of 1:0, 1:1, 1:2, 1:3, 1:4 and 1: 6.
(2) After the co-culture is finished, taking out ninety-six-hole plates, slowly sucking out the supernatant, adding 100 mu L PBS into each hole to rinse two sides, adding 4% paraformaldehyde to fix at room temperature for 20min, rinsing two sides with PBS again, dyeing for 15-20 min at room temperature with DAPI staining solution, rinsing with PBS, and detecting on a machine to determine the final ratio of co-culture.
(3) Cells that grew logarithmically and reached 90% confluence were collected by digestion, adjusted for cell concentration by adding fresh medium, and then counted using a cell counting plate. Uniformly inoculating the cells into a ninety-six-well plate, simultaneously adding a corresponding culture medium containing the medicine, placing the culture medium into an incubator, after the cells adhere to the wall, replacing the culture medium containing the medicine and the T cells in the proportion, and then placing the culture medium into the incubator to continue culturing for 48 hours.
(4) And taking out the co-culture plate, slowly collecting supernatant, adding 100 mu L PBS into each hole to rinse two sides, adding 4% paraformaldehyde to fix at room temperature for 20min, rinsing two sides with PBS again, dyeing with DAPI staining solution at room temperature for 15-20 min, rinsing with PBS, and detecting in an on-machine manner.
As shown in fig. 4, wherein fig. 4A is a microscopic observation of stimulated T cells after primary culture; b is the discussion of the co-culture ratio of T cells and tumor cells; c is the cytotoxicity effect on A375 cells after BMS-37, BMS-37-C1, BMS-37-C3 and T cells are co-cultured; d is a statistical chart of the T cell killing activity of BMS-37, BMS-37-C1, BMS-37-C3 and the acitizumab; the experimental results show that: when the co-culture effective target ratio of the tumor cells and the T cells is explored and the tumor cells and the T cells are co-cultured in a ratio of 1:3, the T cells can kill about 50% of the tumor cells, so that the fixed use effective target ratio in subsequent experiments is 1: 3. When corresponding drugs are added and the drugs are co-cultured with T cells, PD-L1 single PROTAC BMS-37-C1 and BMS-37-C3 can promote anti-tumor immunity within 0.3-1 mu M, and have concentration dependence, and the effect is stronger than that of raw drug BMS-37 and PD-L1 monoclonal antibody Atezolizumab.
EXAMPLE 24 microcalorimetric surge experiment
With Monolith NTTMProtein Labeling Kit RED (Cat # L001) to PD-L1 Protein (50 n)M) and the samples were diluted in 20mM Tris-HCl (pH 7.5) and 0.5 (v/v)% Tween-20. BMS-37-C1/BMS-37-C3/BMS-37-5C-V2 powder was dissolved in 5% DMSO at a concentration of 200. mu.M, diluted in duplicate. After 10 minutes of incubation at room temperature, the samples were loaded into Monolith TM standard treated capillaries and after 30 minutes of incubation on a Monolith NT.115 instrument (NanoTemper Technologies, Munich, Germany) thermophoresis was measured at 25 ℃. The laser power was set to 40% using a 30 second on time. The power of the LED is set to 100%. The dissociation constant Kd values were fitted by using NTAnalysis software (NanoTemper Technologies, minchen, Germany).
As shown in FIG. 5, the results of the experiment revealed that the equilibrium dissociation constants Kd of BMS-37-C1/BMS-37-C3/BMS-37-5C-V2 were 28.70. + -. 4.94. mu.M, 1.61. + -. 0.37. mu.M and 9.10. + -. 2.11. mu.M, respectively, indicating that the synthesized PD-L1 PROTACs could be well combined with PD-L1.
In general, the invention detects the degradation level of PD-L1 protein through in vitro binding force, intracellular Western Blot and flow cytometry, finally finds out a strong and effective PD-L1 degradation agent BMS-37-C3, and the compound has the effect on immunotherapy, can enhance the killing capacity of T cells and provides a new treatment mode for tumor immunotherapy.
Claims (10)
1. A bifunctional molecular compound inducing the degradation of PD-L1 protein, as shown in the following formula I, formula II, formula III, formula IV and formula V, or a pharmaceutically acceptable salt, hydrate or prodrug thereof;
wherein, B is ubiquitin ligase E3 ligand;
the L is any one of the following structures:
wherein: m is an integer selected from 1 to 10.
2. A bifunctional molecular compound inducing degradation of PD-L1 protein according to claim 1, or a pharmaceutically acceptable salt, hydrate or prodrug thereof, wherein B is one of CRBN, VHL, MDM2, cIAP, UBR7, RNF114, CBLB, KEAP 1.
3. The bifunctional molecular compound for inducing degradation of PD-L1 protein according to claim 2, or a pharmaceutically acceptable salt, hydrate or prodrug thereof, wherein the CRBN is one of the following structural formulas:
wherein:
w is selected from CH2、C=O、SO2One of NH and N-alkyl; the alkyl is C1-C4 alkyl;
x is selected from one or two of O, S;
z is selected from one of hydrogen, C1-C4 alkyl, C3-C6 cycloalkyl and halogen;
G. g' independently represents H, C1-C4 alkyl, -OH, C1-C4 alkyl substituted 5-10 member heterocyclic group, the heterocyclic group contains 1-3 hetero atoms of N, O or S;
R1one selected from H, D, halogen, nitro, amino, cyano, hydroxyl, C1-C4 alkyl, halogenated C1-C4 alkyl, deuterated C1-C4 alkyl;
the structural formula of VHL is as follows:
wherein: r2Is selected from CH3And H;
the MDM2 has a structural formula as follows:
wherein:
R3is a piperazinyl, piperidinyl, heterocyclic group or one of the linking groups of the following structures:
in the above-mentioned linking group, n is an integer of 0 to 3;
wherein: the heterocyclic group is one of piperazinone, pyrrolyl, pyrazolyl, furyl, thienyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, pyridyl, pyrimidinyl, pyrazinyl or pyridazinyl;
the cIAP has the structure as follows:
R4h or Boc.
6. The bifunctional molecular compound inducing degradation of PD-L1 protein according to claim 1, or a pharmaceutically acceptable salt, hydrate or prodrug thereof, which is specifically a compound of formula VIII:
in the bifunctional molecular compound shown in formula VIII for inducing the degradation of PD-L1 protein, L has the following structure:
m is an integer selected from 1 to 5.
8. the method for preparing the bifunctional molecular compound for inducing the degradation of PD-L1 protein according to claim 1, which comprises the following steps:
the synthetic route of the PD-L1 protein degradation agent shown in the formula I is as follows:
step 1-1: synthesis of Compound 3
Weighing 4-hydroxy-2, 6-dimethoxybenzaldehyde, triphenylphosphine and (2-methyl- [1,1' -biphenyl)]-3-yl) methanol was added to anhydrous THF, a solution of diisopropyl azodicarboxylate in anhydrous THF was added dropwise to the above reaction system under ice bath conditions, the reaction was continued overnight at room temperature, and the reaction mixture was poured into H2O, then the mixture is extracted, the organic phase is washed, dried and concentrated in vacuo to give a residue which is purified by chromatography on silica gel eluting with ethyl acetate/petroleum ether gradient to give compound 3;
step 1-2: synthesis of Compound 6
Dissolving 4-pentanoic acid with DCM, then adding N-Boc-ethylenediamine, EDCI, HOBt and DIPEA to a round bottom flask, stirring the mixture at room temperature overnight, diluting the reaction system with DCM, washing the organic phase with saturated sodium bicarbonate solution, drying over anhydrous sodium sulfate, concentrating the mixture under reduced pressure, purifying the product by silica gel chromatography, then deprotecting with trifluoroacetic acid to give compound 6;
step 1-3: synthesis of Compound 7
Adding the compound 3 and the compound 6 into a round-bottom flask in sequence, reacting at room temperature, adding sodium triacetoxyborohydride and a drop of acetic acid, stirring at room temperature overnight, diluting the reaction system with dichloromethane, washing an organic phase with a saturated sodium bicarbonate solution, drying with anhydrous sodium sulfate, and purifying by silica gel chromatography to obtain a compound 7;
step 1-4: synthesis of Compounds of formula I
Mixing the compound 7, B-linker and CuSO4Adding VcNa and the mixture into a mixed solution of THF and water in sequence, stirring at room temperature, concentrating to remove tetrahydrofuran, adding water, extracting with dichloromethane, drying an organic phase with anhydrous sodium sulfate, concentrating to obtain a crude product, and purifying by silica gel column chromatography to obtain a compound shown in the formula I;
(II) the synthetic route of the PD-L1 protein degradation agent shown in the formula II and the formula III is as follows:
step 2-1: synthesizing a compound 9;
weighing 5-chloro-2, 4-dihydroxybenzaldehyde, triphenylphosphine and (2-methyl- [1,1' -biphenyl)]-3-yl) methanol was added to anhydrous THF, a solution of diisopropyl azodicarboxylate in anhydrous THF was added dropwise to the above reaction system under ice bath conditions, the reaction was continued overnight at room temperature, and the reaction mixture was poured into H2O, then the mixture was extracted with DCM, the organic phase was washed with brine, dried over anhydrous sodium sulfate and concentrated in vacuo to give a residue which was purified by silica gel chromatography eluting with an ethyl acetate/petroleum ether gradient to give compound 3;
step 2-2: synthesis of Compound 11
Dissolving a compound 9 with DMF, sequentially adding 3- (bromomethyl) benzonitrile, reacting at 80 ℃, adding water into a reaction system, extracting with ethyl acetate, washing an organic phase with saturated saline, drying with anhydrous sodium sulfate, evaporating under reduced pressure to obtain a crude product, purifying the crude product with tert-butyl methyl ether, and filtering to obtain a compound 11;
step 2-3: synthesis of Compound 13
Reacting compound 11, 2-amino-2-methylpropionic acid, AcOH (2d) and NaBH3CN in DMF is placed at 80 ℃ for reaction, ethyl acetate and water are added into the reaction mixture, the organic extract is concentrated, and the crude product is chromatographed on silica gel and eluted by methanol and dichloromethane solution to obtain compound 13;
step 2-4: synthesis of Compound 13y
Dissolving the compound 13 in DMF, taking propargylamine, HATU and DIPEA to react at room temperature, and performing silica gel chromatography on a petroleum ether ethyl acetate system to obtain a compound 13 y;
step 2-5: synthesis of Compounds of formula II
Mixing the compound 13y, B-linker and CuSO4Adding VcNa and the mixture into a mixed solution of THF and water in sequence, stirring at room temperature, concentrating to remove tetrahydrofuran, adding water, extracting with dichloromethane, drying an organic phase with anhydrous sodium sulfate, concentrating to obtain a crude product, and purifying by silica gel column chromatography to obtain a compound shown in a formula II;
step 2-6: synthesis of Compounds of formula III
Dissolving compound 13, EDCI, HOBt, DIPEA and B-linker in DCM, stirring at room temperature, extracting the reaction mixture with water and DCM, washing the organic layer with water, drying over anhydrous sodium sulfate, distilling under reduced pressure and concentrating to obtain residue, and purifying by silica gel chromatography to obtain formula III;
(III) the synthetic route of the PD-L1 protein degradation agent shown in the formula IV is as follows:
step 3-1: synthesis of Compound 16
Adding dess-martin reagent to a dichloromethane solution of 2-methylbiphenyl-3-methanol at room temperature in portions, stirring the resulting mixture at room temperature, then quenching with sodium bicarbonate solution and sodium sulfate solution, extracting the mixture with dichloromethane, drying the combined extracts with anhydrous sodium sulfate and concentrating, purifying the residue by column chromatography to give compound 16;
step 3-2: synthesis of Compound 18y
Dissolving methyl 3-amino-4-hydroxybenzoate, compound 16 and zinc trifluoromethanesulfonate in ethanol under reflux overnight, then cooling the reaction mixture to room temperature, then concentrating, dissolving the residue in dichloromethane, then adding dichlorodicyanobenzoquinone, stirring the mixture at room temperature, diluting with ethyl acetate and washing with sodium bicarbonate solution, sodium sulfate solution, water and brine, drying the organic layer over anhydrous sodium sulfate and concentrating, and purifying the residue by column chromatography to give compound 18 y;
step 3-3: synthesis of Compound 18
Treating the compound 18y under the conditions of LiOH, methanol/water, and then extracting and concentrating by ethyl acetate to obtain a compound 18;
step 3-4: synthesis of compound 19, compound 13y, synthesis of compound 19;
step 3-5: synthesis of compounds of formula IV;
mixing the compound 19, B-linker and CuSO4Adding VcNa and the mixture into a mixed solution of THF and water in sequence, stirring at room temperature, concentrating to remove tetrahydrofuran, adding water, extracting with dichloromethane, drying an organic phase with anhydrous sodium sulfate, concentrating to obtain a crude product, and purifying by silica gel column chromatography to obtain a compound shown in a formula II;
(IV) the synthetic route of the PD-L1 protein degradation agent shown in the formula V is as follows:
step 4-1: synthesis of Compound 22
6-Chloropyridazin-3-amine was dissolved in dioxane, then NaHCO was added in order3Reacting the mixture with ethyl 3-bromo-2-oxopropionate at 100 deg.C for 3h, filtering the reaction system, diluting the filtrate with water, extracting with ethyl acetate, drying with anhydrous sodium sulfate, concentrating to obtain black colloid crude product, and purifying the product by silica gel column chromatography;
step 4-2: synthesis of Compound 24
Reacting the compound 22, K2CO3Reacting a mixture of tert-butyl (R) -pyrrolidin-3-ylcarbamate and DMF at 110 ℃, diluting with water, extracting with ethyl acetate, washing the organic phase with saturated brine, drying over anhydrous sodium sulfate, concentrating to obtain a crude product, and purifying by column chromatography to obtain a compound 24;
step 4-3: synthesis of compound 25, synthesis of compound 25 and compound 18;
step 4-4: synthesis of Compound 27
Adding Boc-D-histidine and K to DMF2CO3Then, benzyl bromide is added dropwise to react at room temperature, and the residue is purified by silica gel flash chromatography to obtain a compound 27;
and 4-5: synthesizing a compound 28, namely removing a protecting group Boc of the compound 27 by trifluoroacetic acid to obtain a compound 28;
and 4-6: synthesis of Compound 29
Dissolving 2-methyl- [1,1' -biphenyl ] -3-methanol in dichloromethane, adding dichloromethane solution of triethylamine and triphosgene, dropwise adding at-20 ℃, then turning to 0 ℃ for reaction, adding n-hexane into a reaction system, stirring, filtering, washing a filter cake by using a mixed solution of dichloromethane and n-hexane, concentrating the filtrate to obtain a compound 29, and directly putting the compound in the next step for reaction without further purification due to instability of a product;
and 4-7: synthesis of Compound 30
Compound 24 is dissolved in a mixed solution of THF and water, NaHCO is added3The THF solution of compound 29 is added dropwise at 0 ℃ and transferred to room temperature with further stirring, and water is usedQuenching reaction, extracting with ethyl acetate, drying organic phase with anhydrous sodium sulfate, filtering, concentrating, and purifying residue with silica gel column chromatography to obtain compound 30;
and 4-8: synthesizing a compound 31;
dissolving compound 28, compound 30, EDCI, HOBt and DIPEA in DCM, stirring at room temperature, extracting the reaction mixture with water and DCM, washing the organic layer with water, drying over anhydrous sodium sulfate, distilling under reduced pressure and concentrating to obtain residue, and purifying by silica gel chromatography to obtain compound 31;
and 4-9: synthesis of compound 31 y;
dissolving compound 31, propynylamine, EDCI, HOBt and DIPEA in DCM, stirring at room temperature, extracting the reaction mixture with water and DCM, washing the organic layer with water, drying over anhydrous sodium sulfate, distilling under reduced pressure and concentrating to obtain residue, and purifying by silica gel chromatography to obtain compound 31;
step 4-10: synthesis of Compounds of formula V
Mixing the compound 31y, B-linker and CuSO4Adding VcNa and the mixture into a mixed solution of THF and water in sequence, stirring at room temperature, concentrating to remove tetrahydrofuran, adding water, extracting with dichloromethane, drying an organic phase with anhydrous sodium sulfate, concentrating to obtain a crude product, and purifying by silica gel column chromatography to obtain a compound shown in the formula V;
the synthetic route of ubiquitin ligase E3 ligand and L is as follows:
(1) when the ubiquitin ligase E3 ligand is CRBN, the specific is Th, and the synthesis method comprises the following steps:
dissolving a compound Th in DMF, adding DIPEA and a linker into a reaction system, reacting at 90 ℃, adding water, extracting with ethyl acetate, collecting an organic layer, drying with anhydrous sodium sulfate, concentrating under reduced pressure, and purifying by silica gel column chromatography to obtain a compound Th-L;
(2) when the ubiquitin ligase E3 ligand is MDM2, the synthesis method comprises the following steps:
dissolving MDM2 in DCM, adding EDCI, HOBt and DIPEA in ice bath, adding linker, reacting, adding DCM for dilution and extraction, collecting organic layer, drying with anhydrous sodium sulfate, concentrating under reduced pressure, and purifying by silica gel column chromatography to obtain MDM 2-L;
(3) when the ubiquitin ligase E3 ligand is VHL, the synthetic route is the same as that of MDM2-L, and the difference is that MDM2 is replaced by VHL, and VHL-L is correspondingly prepared;
(4) when the ubiquitin ligase E3 ligand is cIAP, the synthetic route is the same as MDM2-L, and the difference is that VHL is replaced by cIAP, and the cIAP-L is correspondingly prepared.
9. A pharmaceutical composition comprising a therapeutically effective amount of the bifunctional molecular compound inducing degradation of PD-L1 protein according to any one of claims 1 to 7 as an active ingredient, further comprising a pharmaceutically acceptable carrier, diluent, adjuvant, vehicle or combination thereof.
10. The use of the bifunctional molecular compound inducing PD-L1 protein degradation, or a pharmaceutically acceptable salt, hydrate or prodrug thereof according to any one of claims 1-7, or the pharmaceutical composition according to claim 9, for the preparation of a medicament for the treatment and/or prevention of infection, cancer or autoimmune disease, wherein the infection is one or more of a skin infection, a gastrointestinal infection, a genitourinary infection, a systemic infection, or a viral infection caused by one or more of influenza, hepatitis c virus, human papilloma virus, cytomegalovirus, epstein-barr virus, poliovirus, aquo-zonate virus, coxsackie virus and human immunodeficiency virus;
the cancer is one or more of bone cancer, lung cancer, stomach cancer, colon cancer, membrane adenocarcinoma, breast cancer, prostate cancer, lung cancer, brain cancer, ovarian cancer, cancer of the arm, cervical cancer, cancer of the innocent pill, kidney cancer, head and neck cancer, lymph cancer, leukemia and skin cancer;
the autoimmune disease is one or more of rheumatoid arthritis, systemic lupus erythematosus, mixed connective tissue disease, systemic scleroderma, dermatomyositis, nodular vasculitis, nephropathy, endocrine related diseases, liver disease, psoriasis and autoimmune reaction caused by infection.
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