A polypeptide dimer, pharmaceutical composition and use thereof

Abstract

The present application relates to the technical field of medicine, and provides a polypeptide dimer, a pharmaceutical composition and application. The polypeptide dimer provided by the present application can inhibit the binding of IL-23 and its receptor IL-23R, has good inhibitory activity, and has a wide application prospect in the preparation of drugs for preventing and / or treating IL-23 and / or IL-23R mediated diseases.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and more particularly to a polypeptide dimer, a pharmaceutical composition, and its application. Background Technology

[0002] Interleukin-23 (IL-23) is an important pro-inflammatory cytokine belonging to the interleukin-12 family. It is produced by specific cells in the immune system (such as macrophages and dendritic cells), and its main role is to promote inflammatory responses in the immune response. The structure of IL-23 consists of two subunits, IL-12p40 and IL-23p19. IL-23 binds to its receptor IL-23R, thereby initiating cell signaling and promoting the proliferation and differentiation of Th17 cells, a process crucial for the development of autoimmune diseases.

[0003] IL-23 is a key cytokine linking innate and adaptive immune responses, crucial for driving early local immune responses. IL-23 was initially shown to induce IFN-γ production, which is essential in Th1 responses and cell-mediated immunity against intracellular pathogens. Furthermore, IL-23 plays a leading role in activating NK cells, enhancing T cell proliferation, and regulating antibody production. Extensive evidence suggests that increased IL-23 is closely associated with various autoimmune and chronic inflammatory diseases, such as inflammatory bowel disease (IBD), psoriasis, rheumatoid arthritis (RA), and multiple sclerosis (MS). In these diseases, aberrant expression or dysfunction of IL-23 promotes inflammatory responses and disease progression. For example, in psoriasis, elevated IL-23 expression levels in patient skin lesions exacerbate skin inflammation by activating Th17 cells to release inflammatory factors. Simultaneously, IL-23 has been well-established as a maturation factor for Th17 cells, which are also key participants in autoimmunity. Therefore, IL-23 is also an important component of autoimmune disease signaling pathways. As an upstream target of IL-17, IL-23 has become an important target for the development of autoimmune drugs. In summary, the IL-23 pathway can induce a cascade of cytokines, activate immune cells, and induce an immune response.

[0004] With the rapid development of the treatment of autoimmune diseases, IL-23, as a key target, has seen breakthroughs in antibody drug development. Current antibody drugs include ustekinumab, briakinumab, tildrakizumab, guselkumab, MEDI2070, and BI-655066. Tiltaximab, guselkumab, MEDI2070, and BI-655066 target the p19 subunit of IL-23, while ustekinumab and briakinumab target the p40 subunit. Although these antibody drugs can inhibit the binding of IL-23 and IL-23R to some extent, their inhibitory activity is low, and the therapeutic effect remains poor. Summary of the Invention

[0005] In view of this, the present invention provides a polypeptide dimer, a pharmaceutical composition, and its application. The polypeptide dimer provided by the present invention inhibits the binding of IL-23 to its receptor IL-23R, exhibiting high inhibitory activity.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] A polypeptide dimer and its stereoisomers, solvates, or pharmaceutically acceptable salts, said polypeptide dimer having the structure shown in Formula I:

[0008]

[0009] In Formula I, R1 is selected from N6-acetyl-L-lysine (Ac-Lys), L-cyclohexylalanine (Cha), or (S)-2-amino-4,4-dimethylvaleric acid (tBu-Ala) residues after removing the amino and carboxyl groups.

[0010] R2 is selected from the residues of 4-aminotetrahydro-2H-pyran-4-carboxylic acid (Thp), α-methyl-phenylalanine (Amf), or 2-amino-2-methylpropionic acid (Aib) after removing the amino and carboxyl groups;

[0011] R3 is selected from residues other than amino and carboxyl groups of glutamic acid (Glu), (S)-2-amino-3-cyclopropylpropionic acid (Cpa), (S)-2-amino-3-morpholinopropionic acid (Oxa), (S)-2-amino-3-piperidinylpropionic acid (3-Ppa), (S)-2-amino-3-(4-methoxyphenyl)propionic acid (Phe(4-OMe)) or (S)-2-amino-3-(4-(trifluoromethyl)phenyl)propionic acid (Phe(4-CF3)).

[0012] In some specific embodiments, the polypeptide dimer and its stereoisomers, solvates or pharmaceutically acceptable salts are preferably any one of the polypeptide dimer structures shown in Table 1;

[0013] Table 1. Structure of polypeptide dimers

[0014]

[0015]

[0016]

[0017] The present invention also provides a pharmaceutical composition comprising an active ingredient and pharmaceutically acceptable excipients; said active ingredient being one or more of the polypeptide dimers and their stereoisomers, solvates, and pharmaceutically acceptable salts described in the above-described scheme.

[0018] Preferably, the pharmaceutically acceptable excipient is a carrier and / or a propellant.

[0019] The present invention also provides the use of one or more of the polypeptide dimers and stereoisomers described in the above-described schemes, solvates, pharmaceutically acceptable salts, and pharmaceutical compositions described in the above-described schemes in the preparation of inhibitors that inhibit the binding of IL-23 and IL-23R.

[0020] The present invention also provides the use of one or more of the polypeptide dimers and their stereoisomers, solvates, pharmaceutically acceptable salts, and pharmaceutical compositions described above in the preparation of medicaments for treating and / or preventing IL-23 and / or IL-23R-mediated diseases.

[0021] Preferably, the IL-23 and / or IL-23R-mediated diseases are diseases associated with overexpression of IL-23.

[0022] Preferably, the diseases that overexpress IL-23 include one or more of inflammatory bowel disease, Crohn's disease, and psoriasis.

[0023] This invention provides a polypeptide dimer and its stereoisomers, solvates, or pharmaceutically acceptable salts, the structure of which is shown in Formula I. The polypeptide dimer provided by this invention can inhibit the binding of IL-23 to its receptor IL-23R, exhibiting good inhibitory activity, and has broad application prospects in the preparation of drugs for the prevention and / or treatment of IL-23 and / or IL-23R-mediated diseases. Attached Figure Description

[0024] Figure 1 This is a schematic diagram of the synthesis process for preparing polypeptide dimers (compound 1) in Example 1 of the present invention. Detailed Implementation

[0025] Unless otherwise specified, the terminology used in this invention has the following meanings:

[0026] In this invention, the term "peptide" broadly refers to a sequence of two or more amino acids linked together by peptide bonds. It should be understood that this term does not imply a polymer of amino acids of a specific length, nor is it intended to imply or distinguish whether a polypeptide is produced using recombinant technology, chemical synthesis, or enzymatic synthesis, or whether it is naturally occurring.

[0027] The term "dimer" as used in this invention broadly refers to a peptide comprising two or more monomeric subunits. The dimers of this invention include homodimers and heterodimers. The monomeric subunits of the dimer may be linked at their C-terminus or N-terminus, or they may be linked via internal amino acid residues. The monomeric subunits of the dimer may be linked through the same site, or each may be linked through different sites (e.g., C-terminus, N-terminus, or internal sites).

[0028] The term "derivative" or "analyte" as used in this invention refers to a product derived from a compound by replacing a hydrogen atom or group of atoms with other atoms or groups of atoms. It should be understood that amino acid analogs of peptide compounds as defined in this invention are within the scope of this invention. Examples of such suitable modified amino acid derivatives include one or more modifications selected from: N-terminal and / or C-terminal modifications; replacement of one or more amino acid residues with one or more non-natural amino acid residues (e.g., replacement of one or more polar amino acid residues with one or more isosteric or isoelectronic amino acids; replacement of one or more non-polar amino acid residues with other non-natural isosteric or isoelectronic amino acids); replacement of one or more amino acid residues with alanine; replacement of one or more L-amino acid residues with one or more D-amino acid residues; N-alkylation of one or more amide bonds in a bicyclic peptide ligand; peptide backbone length modification.

[0029] "Pharmaceutically acceptable salt" means that the compound of the present invention retains the bioavailability and properties of a free acid or a free base, wherein the free acid is obtained by reacting with a non-toxic inorganic or organic base, and the free base is obtained by reacting with a non-toxic inorganic or organic acid.

[0030] "Pharmaceutical composition" means one or more of the compounds described in this invention or their stereoisomers, solvates, pharmaceutically acceptable salts or eutectics.

[0031] Unless otherwise specified, all amino acids are used in the L-configuration.

[0032] The names, abbreviations, and corresponding structural formulas of the non-natural amino acids involved in this invention are shown in Table 2:

[0033] Table 2. Names, abbreviations, and corresponding structural formulas of non-natural amino acids.

[0034]

[0035]

[0036] This invention provides a polypeptide dimer and its stereoisomers, solvates, or pharmaceutically acceptable salts, wherein the polypeptide dimer has the structure shown in Formula I:

[0037]

[0038] In some specific embodiments of the present invention, R1 in Formula I is selected from residues of Ac-Lys, Cha, or tBu-Ala after removing amino and carboxyl groups; R2 is selected from residues of Thp, Amf, or Aib after removing amino and carboxyl groups; and R3 is selected from residues of Glu, Cpa, Oxa, Ppa, Phe(4-OMe), or Phe(4-CF3) after removing amino and carboxyl groups.

[0039] In this invention, the structure of the polypeptide dimer is any one of the structures shown in Table 1 above.

[0040] In this invention, the preparation method of the polypeptide dimer adopts the standard Fmoc chemical method.

[0041] In this invention, the method for preparing the polypeptide dimer preferably includes the following steps:

[0042] Based on the monomeric amino acid sequence of the polypeptide dimer described in the above technical solution, a peptide sequence is prepared on a resin to obtain a peptide resin;

[0043] The peptide resin was subjected to solid-phase cyclization and then cleaved to obtain the polypeptide dimer.

[0044] This invention prepares peptide sequences on resin based on the monomeric amino acid sequences of the polypeptide dimers described in the above technical solution, thereby obtaining peptide resin. In this invention, the resin can be MBHAResin, which can be subjected to swelling and deprotection of Fmoc protecting groups sequentially before use; the swelling reagent can be dichloromethane; the deprotection reagent is preferably a solution of piperidine in N,N-dimethylformamide, wherein the volume ratio of piperidine to N,N-dimethylformamide is preferably 1:4; the deprotection preferably includes a first-stage reaction and a second-stage reaction sequentially; the temperature of the first-stage reaction is preferably room temperature, and the time is preferably 5 min; the temperature of the second-stage reaction is preferably room temperature, and the time is preferably 10 min.

[0045] This invention does not specifically limit the method for preparing peptide sequences on resin; any method well-known to those skilled in the art can be used. In specific embodiments of this invention, Fmoc-protected amino acids are sequentially coupled to the resin according to the monomeric amino acid sequence of the polypeptide dimer, followed by removal of the Fmoc protecting groups, and finally end-capping to obtain the peptide resin. In specific embodiments of this invention, Fmoc-protected amino acids, 2-(1H-benzotriazo-L-1-yl)-1,1,3,3-tetramethylurea tetrafluoroborate (TBTU), N,N-diisopropylethylamine (DIEA), and N,N-dimethylformamide (DMF) are mixed and activated; then the resulting activated solution is mixed with the resin after removing the Fmoc protecting groups for a coupling reaction, and the above operation is repeated to sequentially couple other Fmoc-protected amino acids.

[0046] In this embodiment of the invention, after the coupling of Fmoc-protected amino acids is completed, deprotection is performed. The present invention does not have special requirements for the operation method of deprotection, and any method known in the art can be used.

[0047] After obtaining the peptide resin, the present invention performs solid-phase cyclization and then cleaves the peptide resin to obtain the polypeptide dimer. The solid-phase cyclization is preferably carried out in the presence of DMF, HATU (2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate), HOBT (1-hydroxybenzotriazole), and DIEA, with the molar ratio of HATU, HOBT, and DIEA preferably being 1:2:2; the solid-phase cyclization is preferably carried out at room temperature, and the reaction time is preferably 2 hours.

[0048] After obtaining the peptide resin, the present invention preferably uses a lysis buffer to lyse the solid-phase cyclized peptide resin. In the present invention, the lysis buffer preferably comprises trifluoroacetic acid (TFA), phenylthiomethane (PhsMe), 1,8-octanedithiol, phenol (PhOH), and water, wherein the volume ratio of trifluoroacetic acid, phenylthiomethane, 1,8-octanedithiol, phenol, and water is preferably 82.5:5:2.5:5:5; the lysis is preferably carried out in the presence of tris(2-carbonylethyl)phosphohydrochloride (TCEP), and the concentration of TCEP in the system during lysis can be 1 mg / mL; the lysis is preferably carried out at room temperature, and the lysis time is preferably 2 h.

[0049] This invention also provides a pharmaceutical composition comprising an active ingredient and pharmaceutically acceptable excipients; the active ingredient is one or more of the polypeptide dimer and its stereoisomers, solvates, and pharmaceutically acceptable salts described in the above-described scheme. In this invention, the pharmaceutically acceptable excipients may be carriers and / or excipients; this invention does not have specific requirements regarding the specific types of carriers and / or excipients, and those well known to those skilled in the art can be used.

[0050] The present invention also provides the use of one or more of the polypeptide dimers and stereoisomers described in the above-described schemes, solvates, pharmaceutically acceptable salts, and pharmaceutical compositions described in the above-described schemes in the preparation of inhibitors that inhibit the binding of IL-23 and IL-23R.

[0051] The present invention also provides the use of one or more of the polypeptide dimers and their stereoisomers, solvates, pharmaceutically acceptable salts, and pharmaceutical compositions described above in the preparation of medicaments for the prevention and / or treatment of IL-23 and / or IL-23R-mediated diseases; wherein the IL-23 and / or IL-23R-mediated diseases are preferably diseases associated with overexpression of IL-23, specifically including one or more of inflammatory bowel disease, Crohn's disease, and psoriasis.

[0052] The present invention also provides a method for treating a disease in mammals or humans, the method comprising treatment by administering a therapeutically effective amount of the polypeptide dimer, its stereoisomer, dimer, solvate, or pharmaceutically acceptable salt or the pharmaceutical composition described above; wherein the disease is an IL-23 and / or IL-23R mediated disease, specifically a disease associated with overexpression of IL-23, preferably one or more of inflammatory bowel disease, Crohn's disease, and psoriasis.

[0053] The technical solutions of this invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0054] Example 1

[0055] The polypeptide dimer (compound 1) was synthesized using standard Fmoc chemical methods, and the synthesis process is as follows: Figure 1 As shown, the specific steps are as follows:

[0056] 1. Swelling: Add MBHAResin (0.54 g, 0.28 mmol / g, 0.15 mmol) to a peptide synthesis tube and swell with dichloromethane (DCM) (15 mL) for 15 minutes at room temperature. Drain the solvent. Dry the tube and wash three times with DMF (15 mL) to obtain Fmoc peptide resin.

[0057] 2. Deprotection of Fmoc protecting groups: Add 15 mL of piperidine DMF solution (V) to the Fmoc peptide resin obtained above. 哌啶 V DMF=1:4), react at room temperature for 5 minutes, dry under vacuum, and add piperidine DMF solution (15 mL, V) again. 哌啶 V DMF The reaction mixture (1:4) was reacted at room temperature for 10 minutes. After the reaction was complete, the reaction solution was drained, and the resin was washed 6 times with 15 mL of DMF, and the solvent was drained. A small amount of resin was heated at 110 °C for 3 minutes with a 5% ninhydrin ethanol solution. The resin turned deep blue, indicating that Fmoc had been removed and it could be used directly for the next reaction.

[0058] 3. Coupling: Fmoc-Sar-OH (70 mg, 0.225 mmol, 1.5 eq) and 2-(1H-benzotriazo-L-1-yl)-1,1,3,3-tetramethylurea tetrafluoroborate (TBTU) (72 mg, 0.225 mmol, 1.5 eq) were dissolved in DMF (10 mL). DIEA (0.078 mL, 0.45 mmol, 3 eq) was added to the reaction solution under ice bath conditions, and the mixture was stirred and activated for 5 minutes. The activated solution was added to the Fmoc-free resin, and the reaction was carried out at room temperature for 1 hour. A small amount of resin was then added to a 5% ninhydrin ethanol solution and heated at 110 °C for 3 minutes; the resin turned colorless. The reaction solution was dried under vacuum and washed three times with DMF (10 mL). The resulting resin was used directly in the next reaction step.

[0059] 4. Couplement cycle: Repeat the above operation to couple the following amino acids one by one:

[0060] Fmoc-L-3-Pya-OH (0.087g, 0.225mmol, 1.5eq);

[0061] Fmoc-Asn(Trt)-OH (0.180g, 0.3mmol, 2.0eq);

[0062] Fmoc-Glu(OtBu)-OH (0.128g, 0.3mmol, 2.0eq);

[0063] Fmoc-Thp-Gly-OH (0.082g, 0.225mmol, 1.5eq);

[0064] Fmoc-2-Nal-OH (0.098g, 0.225mmol, 1.5eq);

[0065] Fmoc-Phe(4-AE)(Boc)-OH (0.13g, 0.225mmol, 1.5eq);

[0066] Fmoc-Glu(OAll)-OH (0.12g, 0.3mmol, 2.0eq);

[0067] Fmoc-Lys(Ac)-OH (0.09g, 0.225mmol, 1.5eq);

[0068] Fmoc-7-Me-Trp-OH (0.10g, 0.225mmol, 1.5eq);

[0069] Fmoc-Thr(tBu)-OH (0.12g, 0.3mmol, 2.0eq);

[0070] Fmoc-Asn(Trt)-OH (0.18g, 0.3mmol, 2.0eq);

[0071] Fmoc-trans-4-Amc-OH (0.11g, 0.3mmol, 2.0eq);

[0072] After all amino acid coupling is completed, deprotection is performed.

[0073] 5. Deprotection: Add DCM (15 mL) to the synthesis tube, add benzyl silane (0.222 mL, 1.8 mmol, 12 eq) and triphenylphosphine (0.008 g, 0.03 mmol, 0.2 eq) to the mixture, and finally add Pd(PPh3)4 catalyst (0.017 g, 0.015 mmol, 0.1 eq). After bubbling under nitrogen for 2 hours, perform a small-scale pyrolysis test (the pyrolysis solution consists of TFA, PhSMe, 1,8-octanedithiol, PhOH and H2O, with a volume ratio of 82.5:5:2.5:5:5). Monitor the pyrolysis by LCMS until complete removal is achieved. Finally, perform deprotection of Fmoc as described above.

[0074] 6. Solid-phase cyclization: DMF (15 mL), HATU (0.114 g, 0.3 mmol, 2.0 eq), and HOBT (0.081 g, 0.6 mmol, 4.0 eq) were added, followed by DIEA (0.105 mL, 0.6 mmol, 4.0 eq). The mixture was bubbled under nitrogen for 2 hours, then washed three times alternately with 10 mL each of DCM and EtOH. The mixture was then dried under vacuum and transferred to a vacuum oven at 35 °C for 2 hours to obtain 0.89 g of intermediate resin. A small-scale cyclization test was conducted (the cyclization solution consisted of TFA, PhSMe, 1,8-octanedithiol, PhOH, and H2O, with a volume ratio of 82.5:5:2.5:5:5) to monitor complete cyclization. The cyclization was then directly used in the next reaction.

[0075] 7. Cleavage: Add lysis buffer (6.5 mL, 10 mL / g resin, composed of TFA, PhSMe, 1,8-octanedithiol, PhOH, and H2O in a volume ratio of 82.5:5:2.5:5:5) to 0.65 g of cyclized resin. Then add TCEP (7 mg, 1 mg / mL) and react for 2 hours. Filter and wash the resin with lysis buffer (1 mL). Combine the filtrates and add methyl tert-butyl ether (MTBE) (50 mL). Allow to settle in an ice bath for 1 hour, then centrifuge. Wash three times with MTBE (45 mL) to obtain a white solid. Transfer the solid to a vacuum drying oven and dry under vacuum at 35 °C for 2 hours to obtain 170 mg of crude peptide. The crude peptide (170 mg) was dissolved in 22% ACN and then purified. The purified solution was obtained by pre-HPLC, concentrated, and lyophilized to obtain the target polypeptide dimer, designated as compound 1, with a yield of 45.68 mg (26.87%) and HPLC purity of 98.03%. MS (ESI) m / z: [M+3H]. + / 3 = 1231.0.

[0076] Using a similar synthetic method, polypeptide dimer compounds 2 to 11 were synthesized. The specific structures of the compounds are shown in Table 1, and the purity and mass spectrometry test results are shown in Table 3.

[0077] Table 3. Purity and mass spectrometry results of compounds 2 to 11

[0078] Compound numbering molecular weight Purity (HPLC) Actual LCMS test Compound 2 3655.8 90.25％ (M+3H+) / 3=1220.0, (M+4H+) / 4=915.3 Compound 3 3603.77 95.24％ (M+3H+) / 3=1202.3, (M+3H+) / 4=902.1 Compound 4 3572.14 98.89％ (M+3H+) / 3=1191.4, (M+4H+) / 4=893.7 Compound 5 3608.08 96.12％ (M+3H+) / 3=1203.0, (M+4H+) / 4=902.7 Compound 6 3656.22 92.66％ (M+3H+) / 3=1219.5, (M+3H+) / 4=914.8 Compound 7 3757.82 94.22％ (M+3H+) / 3=1253.2, (M+3H+) / 4=940.5 Compound 8 3785.85 91.61％ (M+3H+) / 3=1262.95, (M+3H+) / 4=974.2 Compound 9 3861.80 96.83％ (M+3H+) / 3=1288.0, (M+3H+) / 4=966.4 Compound 10 3739.91 93.59％ (M+3H+) / 3=1247.2, (M+3H+) / 4=936.1 Compound 11 3743.87 97.24％ (M+3H+) / 3=1249.3, (M+3H+) / 4=936.8

[0079] Example 2: Binding activity assay of polypeptide dimer compounds against the IL-23R target

[0080] In this field, in vitro affinity is an important evaluation indicator for assessing the effect of a molecule on a target in the early stages of drug development. This embodiment uses surface plasmon resonance (SPR) technology to test the affinity between the polypeptide dimer described in this invention and the target protein IL-23R. The specific steps are as follows:

[0081] To analyze the affinity activity of the peptide dimer, this experiment used an SPR instrument (GE Healthcare, model: Biacore 8K+) and a CM5 chip (GE Healthcare). Using standard amine coupling chemistry, recombinant human IL-23R protein (AcroBiosystems Inc., catalog number ILR-H82F3) was coupled to the chip at 25°C with HBS-EP+ (10 mM HEPES, 0.15 M NaCl, pH 7.4) as the run buffer. In short, equal volumes of EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; 0.4 M aqueous solution) and NHS (N-hydroxysuccinimide; 0.1 M aqueous solution) were mixed and injected at a flow rate of 10 μL / min for 20 min to activate the Fc1 and Fc2 channels. The IL23R protein was diluted to a final concentration of 5 μg / mL with 10 mM sodium acetate at pH 5.5. The Fc2 channel was injected at a flow rate of 10 μL / min for 10 min, with an expected protein conjugation amount of approximately 5000 RU. The Fc1 channel served as a control. After conjugation, both channels were blocked with ethanolamine solution at a flow rate of 10 μL / min for 10 min. Then, a running buffer containing 0.5% DMSO (HBS-EP+) was used, and a series of diluted peptides were prepared in this buffer, with a final DMSO concentration of 0.5%. Different concentrations of DMSO solutions were also prepared as calibration curves. The SPR program was run at 25°C at a flow rate of 30 μL / min, with a contact time of 180 s and a dissociation time of 1800 s. All data were processed and kinetic fitted using standard procedures to obtain the affinity constant KD between the polypeptide dimer and the target protein IL23R. The same method was used to test the affinity constant KD between PN235 and the target protein IL23R. The results are shown in Table 4.

[0082] Table 4 Binding activity of peptide dimers against IL-23R target

[0083] Compound Name <![CDATA[IC 50 (pM)]]> Compound 1 0.059 Compound 2 12.35 Compound 3 6.23 Compound 4 3.84 Compound 5 5.88 Compound 6 0.37 Compound 7 1.26 Compound 8 0.09 Compound 9 0.51 Compound 10 2.55 Compound 11 1.61 PN235 (Reference Standard) 4.43

[0084] As can be seen from Table 4, the polypeptide dimer compounds of the present invention can inhibit the binding of IL-23 to its receptor IL-23R, and the inhibitory activity of most compounds is better than that of the control PN235.

[0085] Example 3: Competitive inhibition ELISA assay of polypeptide dimer compounds against the IL-23R target.

[0086] The competitive ELISA technique was used to test the competition effect between the compounds in the examples and the control PN235 and the target protein IL-23R. The specific steps are as follows:

[0087] Striptigin-plus (recombinant) was diluted to 5 μg / mL with coating buffer (pH = 9.6), and 50 μL / well was added to a 384-well plate and incubated overnight at 4°C. The next day, the plate was dried by shaking off the excess liquid, and then washed three times with 1×TBST. 100 μL of TBST solution containing 2% BSA was added to each well, and the plate was incubated at room temperature for 60 min. Afterward, the plate was dried by shaking off the excess liquid, and then washed three times with 1×TBST.

[0088] Prepare IL-23R at a concentration of 1.2 μg / mL using 1×TBST (containing 0.5% BSA), add 50 μL / well to a 384-well plate, and incubate at room temperature for 1 h. Then wash 3 times with 1×TBST.

[0089] The compound was serially diluted to appropriate concentrations using 1×TBST (containing 0.5% BSA) solution, and the ligand protein human IL23A / 12B-His protein was diluted to 2.5 μg / mL. The compound and ligand protein were mixed at a 1:1 volume ratio, and 50 μL / well was added to a 384-well plate and incubated at room temperature for 60 min. Each well was washed three times with 100 μL of 1×TBST. Anti-his-HRP antibody was added to 50 μL / well of the 384-well plate, and after incubation at room temperature for 60 min, the plate was washed three times.

[0090] Under light-protected conditions, add 50 μL TMB to each well. After 20-30 minutes, read the OD650 value using a microplate reader (add stop solution when the OD650 reading at the "0 point" reaches between 0.85 and 1.0). Add 25 μL of stop solution to each well, then read the OD450 value using the microplate reader. After the experiment, analyze the results using GraphPad Prism 8.0.2 software to obtain the IC50 values ​​of the compound and the target protein. 50 The results are shown in Table 5.

[0091] Table 5 Competitive inhibition of IL-23R target by polypeptide dimer compounds

[0092] Compound Name <![CDATA[IC 50 (nM)]]> Compound 1 3.38 Compound 2 52.12 Compound 3 26.55 Compound 4 13.75 Compound 5 10.56 Compound 6 2.81 Compound 7 6.66 Compound 8 3.47 Compound 9 8.90 Compound 10 6.35 Compound 11 11.27 PN235 (Reference Standard) 7.063

[0093] As can be seen from Table 5, the polypeptide dimer compounds provided by the present invention have good inhibitory activity against the IL-23R target, and the activities of compounds 1, 6, 7, 8 and 10 are all better than those of the control PN235.

[0094] Example 4: IL-23R reporter gene assay

[0095] This embodiment utilizes an IL-23 reporter gene cell line expressing luciferase, a downstream of IL-23R, to evaluate the inhibitory effect of IL-23-mediated cell signaling. The specific experimental steps are as follows:

[0096] 24 hours before the experiment, cells were removed from the culture flask, digested, centrifuged to collect the cell pellet, resuspended in an appropriate amount of complete culture medium, cell viability was assessed and counted, and the cell concentration was adjusted to 2 × 10⁻⁶ cells / mL with complete culture medium. 5 Add 100 μL of cells / mL to the 11 central wells using a multi-channel pipette. Add 100 μL of PBS to the surrounding wells. Cover the plate and incubate overnight in an incubator before use.

[0097] The compound was serially diluted using Assaybuffer (DMEM + 1% FBS + 1% P / S) to a final DMSO concentration of 0.5%. After incubation overnight, the plate was removed, the culture medium was discarded, and 100 μL of the prepared compound serial dilution was added to each well. The plate was capped and incubated at 37°C in a 5% CO2 incubator for 1 h. Then, the agonist hIL-23 (Acro, ILB-H52W5) diluted with Assaybuffer was added to a final agonist concentration of 30 ng / mL, and the plate was incubated at 37°C for 16 h.

[0098] After removing the cell culture plate from the incubator and bringing it to room temperature, add 100 μL of luciferase assay reagent (Giman, GM040505C) to each well (analyte:cell culture medium = 1:1). Mix by pipetting 5 times, vortex horizontally for 10-30 seconds, and incubate at room temperature for 10 minutes. Then, transfer 100 μL of cell lysis buffer per well to a 96-well white microplate using a multipipe. Detect at 570 nm. After the experiment, analyze the results using GraphPad Prism 8.0.2 software to obtain the IC50 values ​​of the compounds. 50 The results are shown in Table 6.

[0099] Table 6. Inhibitory activity of polypeptide dimer compounds against IL-23R

[0100] Compound Name <![CDATA[IC 50 (nM)]]> Compound 1 25.88 Compound 2 285.90 Compound 3 80.65 Compound 4 47.03 Compound 5 36.56 Compound 6 12.04 Compound 7 15.87 Compound 8 22.61 Compound 9 18.71 Compound 10 10.53 Compound 11 14.31 PN235 (Reference Standard) 40.98

[0101] As shown in Table 6, the dipeptide compounds provided by this invention can effectively block IL-23-induced cell signaling, with compounds 1, 5-11 exhibiting the highest half-maximum inhibitory concentrations (IC50). 50 It is superior to the control molecule PN-235.

[0102] Example 5: Assay on the inhibitory activity of polypeptide dimer compounds against pSTAT3 in PBMC cells

[0103] 400g of frozen human peripheral blood mononuclear cells (PBMCs) were resuscitated in a 37°C water bath, centrifuged for 10 min to collect cells, resuspended in RPMI 1640 complete medium, and human IL-1β was added to a final concentration of 100 ng / mL. These cells were then seeded in culture flasks coated with 5 μg / mL CD3 antibody and induced at 37°C in a 5% CO2 incubator for 5 days. After induction, the cells were collected, resuspended in serum-free medium, and seeded at 1×10⁶ cells / well. 5 Cells were starved at 37°C for 4 hours in 96-well plates. Then, diluted compounds were added, and the cells were incubated at 37°C in a 5% CO2 incubator for 1 hour. Diluted hIL-23 (final concentration 20 ng / mL) was then added, and the cells were incubated at 37°C for 30 min. The cell plates were centrifuged at 1000 rpm for 1 min, and cell lysis and pSTAT3 detection were performed according to the pSTAT3 ELISA Kit (CST, 7300CA) instructions. The results are shown in Table 7.

[0104] Table 7. Inhibition of pSTAT3 in PBMC cells by polypeptide dimer compounds.

[0105] Compound Name <![CDATA[IC 50 (pM)]]> Compound 1 1.83 Compound 2 62.34 Compound 3 67.51 Compound 4 5.68 Compound 5 11.65 Compound 6 1.02 Compound 7 3.33 Compound 8 6.52 Compound 9 13.74 Compound 10 10.90 Compound 11 8.66 Reference standard PN235 15.01

[0106] As shown in Table 7, the polypeptide dimer compounds provided by this invention can effectively inhibit IL-23-induced proximal signaling. In human peripheral blood mononuclear cells (PBMCs), the compounds inhibited IL-23-induced STAT3 phosphorylation in a concentration-dependent manner, with compounds 1, 4, 5-11 showing the highest half-maximal inhibitory concentrations (IC50). 50 It is superior to the control molecule PN-235.

[0107] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A polypeptide dimer and its stereoisomers, solvates, or pharmaceutically acceptable salts, characterized in that, The polypeptide dimer has the structure shown in Formula I: In Formula I, R1 is selected from N6-acetyl-L-lysine, L-cyclohexylalanine, or (S)-2-amino-4,4-dimethylvaleric acid residues after removing the amino and carboxyl groups. R2 is selected from the residues of 4-aminotetrahydro-2H-pyran-4-carboxylic acid, α-methyl-phenylalanine, or 2-amino-2-methylpropionic acid after removing the amino and carboxyl groups. R3 is selected from glutamic acid, (S)-2-amino-3-cyclopropylpropionic acid, (S)-2-amino-3-morpholinopropionic acid, (S)-2-amino-3-piperidinylpropionic acid, (S)-2-amino-3-(4-methoxyphenyl)propionic acid, or (S)-2-amino-3-(4-(trifluoromethyl)phenyl)propionic acid residues after removing the amino and carboxyl groups.

2. The polypeptide dimer and its stereoisomers, solvates, or pharmaceutically acceptable salts according to claim 1, characterized in that, The polypeptide dimer has any one of the following structures:

3. A pharmaceutical composition, characterized in that, It includes an active ingredient and pharmaceutically acceptable excipients; the active ingredient is one or more of the polypeptide dimer and its stereoisomers, solvates and pharmaceutically acceptable salts as described in claim 1 or 2.

4. The pharmaceutical composition according to claim 3, characterized in that, The pharmaceutically acceptable excipients are carriers and / or excipients.

5. The use of one or more of the polypeptide dimer and its stereoisomers, solvates, pharmaceutically acceptable salts, and pharmaceutical compositions according to any one of claims 1 or 2 in the preparation of inhibitors that inhibit the binding of IL-23 and IL-23R.

6. Use of one or more of the polypeptide dimer and its stereoisomers, solvates, pharmaceutically acceptable salts, and pharmaceutical compositions according to any one of claims 1 or 2 in the preparation of medicaments for the treatment and / or prevention of IL-23 and / or IL-23R-mediated diseases.

7. The application according to claim 6, characterized in that, The diseases mediated by IL-23 and / or IL-23R are diseases associated with overexpression of IL-23.

8. The application according to claim 7, characterized in that, The diseases that overexpress IL-23 include one or more of inflammatory bowel disease, Crohn's disease, and psoriasis.

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