Phosphorylated peptidomimetic compound and medical application thereof

Abstract

The invention discloses a phosphorylated peptidomimetic compound and a medical application thereof. The invention provides a phosphorylated peptidomimetic compound with a novel structure. Activity research shows that the phosphorylated peptidomimetic compound has excellent Cbl-b inhibitory activity and is an effective Cbl-b inhibitor. Technicians in the field know that Cbl-b overexpression can inhibit T cell immune response, and inhibition of Cbl-b can activate killing of an immune system on tumor cells. Therefore, the phosphorylated peptidomimetic compound or pharmaceutically acceptable salts and solvates thereof provided by the invention can be developed and prepared into drugs for treating diseases (such as non-small cell lung cancer, breast cancer, prostate cancer, head and neck squamous cell carcinoma, liver cancer, pancreatic cancer, colorectal cancer, ovarian cancer and other tumors) treated or relieved by inhibiting Cbl-b (such as non-small cell lung cancer, breast cancer, prostate cancer, head and neck squamous cell carcinoma, liver cancer, pancreatic cancer, colorectal cancer, ovarian cancer and the like) the medicine prospect is promising.

Description

Technical Field

[0001] This invention belongs to the field of medicinal chemistry and relates to the synthesis and application of new compounds, specifically to a phosphorylated peptide compound and its pharmaceutical uses. Background Technology

[0002] The Casitas B-lineage lymphoma proto-oncogene b (Cbl-b) is expressed in various immune cells and is a key regulatory molecule in the immune system. Structurally, it consists of four domains: a tyrosine kinase-binding domain, an LHR domain, a proline-rich domain, and a ubiquitin-associated domain. The N-terminal tyrosine kinase-binding domain and the LHR domain are associated with the E3 ligase function of Cbl-b. The tyrosine kinase-binding domain comprises three parts: a four-helix bundle domain, a calcium-binding EF domain, and an SH2 domain. The tyrosine kinase-binding domain recognizes specific Cbl-b substrates and signals them by binding to the zeta chain of proteins containing specific phosphorylated tyrosine motifs, such as spleen tyrosine kinase (Syk) and the associated protein kinase 70. The LHR domain mediates the transfer of ubiquitin proteins between E2 and target protein substrates; this domain plays a crucial role in maintaining E3 activity.

[0003] Cbl-b, as a key negative regulator of the T cell receptor (TCR) signaling pathway, regulates the threshold of T cell activation and modulates peripheral T cell tolerance through multiple mechanisms. Cbl-b overexpression suppresses T cell immune responses; conversely, the absence of Cbl-b in T cells, even in the absence of CD28 co-stimulation, significantly enhances IL-2 production. Studies have shown that knocking out the Cbl-b gene or knocking in the inactive Cbl-b (C373A) mutant in mice leads to T cell activation and enhanced autoimmune responses. Therefore, Cbl-b is involved in the development and progression of immune-mediated diseases, and blocking Cbl-b function could serve as a novel immunotherapy strategy.

[0004] Several types of preclinical and clinical Cbl-b modulators have been reported, making it possible to develop novel immune checkpoint inhibitors targeting Cbl-b. However, to date, no small molecule inhibitors targeting Cbl-b have been approved for clinical treatment. Therefore, developing small molecule Cbl-b inhibitors is a promising but challenging goal in cancer immunotherapy.

[0005] The phosphorylated peptide-like Cbl-b small molecule inhibitor provided by this invention has a novel backbone structure, can effectively inhibit Cbl-b, activate the immune system, and can be used for the treatment of various cancers. Based on this, this invention is proposed. Summary of the Invention

[0006] The first objective of this invention is to provide a phosphorylated peptide compound, and the second objective is to provide the pharmaceutical use of the phosphorylated peptide compound.

[0007] The above-mentioned objective of this invention is achieved through the following technical solution:

[0008] A phosphorylated peptide compound or a pharmaceutically acceptable salt or solvate thereof, wherein the general structural formula of the phosphorylated peptide compound is shown below:

[0009]

[0010] in:

[0011] R1 is selected from C1-C3 alkyl and —OCH2R4, wherein: R4 is a substituted or unsubstituted aryl or fluorenyl group;

[0012] R2 and R3 are independently selected from hydrogen, substituted or unsubstituted 5-7 membered N-containing or N-free carbon rings and substituted or unsubstituted aryl or benzyl groups, or R2 and R3 together with the N attached to them form substituted or unsubstituted 5-7 membered N-containing carbon rings or azabicyclic rings.

[0013] Preferably, the structural formula of the phosphorylated peptide-like compound is shown in Figures 1-12:

[0014]

[0015]

[0016] The above-mentioned phosphorylated peptide compounds or their pharmaceutically acceptable salts or solvates are used in the preparation of Cbl-b inhibitor drugs.

[0017] The above-mentioned phosphorylated peptide compounds or their pharmaceutically acceptable salts or solvates are used in the preparation of medicaments for treating or alleviating diseases by inhibiting Cbl-b.

[0018] Preferably, the disease that is treated or alleviated by inhibiting Cbl-b is cancer.

[0019] More preferably, the cancer is selected from non-small cell lung cancer, breast cancer, prostate cancer, head and neck squamous cell carcinoma, liver cancer, pancreatic cancer, colorectal cancer, and ovarian cancer.

[0020] A pharmaceutical preparation comprising, as an active ingredient, the aforementioned phosphorylated peptide compound or its pharmaceutically acceptable salt or solvate, and formulated into a pharmaceutically acceptable dosage form using pharmaceutically acceptable excipients.

[0021] Preferably, the excipient is a solid, liquid, or semi-solid.

[0022] Preferably, the dosage form is a tablet, capsule, or injection.

[0023] Beneficial effects:

[0024] This invention provides a novel phosphorylated peptide-like compound; activity studies have shown that this phosphorylated peptide-like compound exhibits excellent Cbl-b inhibitory activity, making it an effective Cbl-b inhibitor. Those skilled in the art know that Cbl-b overexpression suppresses T-cell immune responses, and that inhibiting Cbl-b can activate the immune system to kill tumor cells. Therefore, the phosphorylated peptide-like compound provided by this invention, or its pharmaceutically acceptable salts or solvates, has the potential to be developed into medicaments for treating or alleviating diseases (such as non-small cell lung cancer, breast cancer, prostate cancer, head and neck squamous cell carcinoma, liver cancer, pancreatic cancer, colorectal cancer, and ovarian cancer) that are treated or alleviated by inhibiting Cbl-b. Detailed Implementation

[0025] The following describes the substantive content of the present invention in detail with reference to embodiments, but this does not limit the scope of protection of the present invention.

[0026] Example 1: Synthesis and structural confirmation of compounds

[0027] I. General Experimental Rules:

[0028] All chemical reagents used in this invention are commercially available chemically pure or analytically pure and are used directly without further purification. 1 H-NMR, 13 C-NMR, 31 P-NMR spectra were determined using a Bruker AV300 (300MHz) NMR spectrometer (TMS as internal standard), mass spectrometry was performed using a Water Q-Tof mass spectrometer (HRMS), purity was determined using a Shimadzu LC-20AT, and the chromatographic column was an Agilent C18 reversed-phase column. Column chromatography separation of the compounds was performed using 200-300 mesh silica gel (Qingdao Ocean Chemical Plant Branch), with ethyl acetate, petroleum ether, dichloromethane, and methanol as eluents. Solvent concentration under reduced pressure was performed using a rotary evaporator (Shanghai Yarong RE52CS-1) at temperatures below 50℃. The reaction was monitored using 25×75mm HSGF254 silica gel plates (Yantai Jiangyou), with ethyl acetate, petroleum ether, dichloromethane, and methanol as developing solvents. Fluorescence was observed using a darkroom-type ultraviolet analyzer (Nanjing Keer Instrument Equipment Co., Ltd.). Room temperature refers to ambient temperature conditions.

[0029] II. Preparation of intermediates and final products

[0030] Preparation of (S)-2-((benzyloxycarbonyl)amino)-3-(4-((bis(tert-butoxyphosphoryl)oxy)phenyl)propionic acid

[0031]

[0032] Benzyloxycarbonyl-L-tyrosine (5.0 g, 15.8 mmol), tert-butyldimethylchlorosilane (2.38 g, 15.8 mmol), and anhydrous tetrahydrofuran (100 mL) were added to a 250 mL round-bottom flask under nitrogen protection. N-methylmorpholine (1.78 mL, 15.8 mmol) was slowly added dropwise, and the reaction was carried out at room temperature for 1 hour. N,N-diisopropylphosphonamide di-tert-butyl ester (8.5 mL, 29.2 mmol) and tetrazolium (2.78 g, 39.6 mmol) were added, and the mixture was stirred at room temperature for 2 hours. 70% tert-butyl hydroperoxide (6.59 mL, 48.6 mmol) was added dropwise under ice bath conditions, and the reaction was carried out at room temperature for 6 hours. The reaction system was cooled to 0 °C, and saturated sodium thiosulfate aqueous solution (50 mL) was added. The aqueous layer was extracted with ethyl acetate (3 × 80 mL), and the ethyl acetate extract was dried over anhydrous magnesium sulfate. The solvent was removed under reduced pressure to obtain a crude product, which was recrystallized from ethyl acetate / petroleum ether (1:5) to give a white solid (5.16 g, yield 64%). 1 H NMR (300MHz, DMSO-d6) δ7.38–7.31(m,5H),7.24(d,J=8.1Hz,1H),7.12–7.03(m,4H),5.00(d,J=5.9Hz ,2H),4.13–4.07(m,1H),3.17–3.03(m,1H),2.90–2.83(m,1H),1.46(s,18H).ESI-MSm / z:530.3[M+Na] + .

[0033] Preparation of benzyl((S)-1-(((2S,3S)-1-amino-3-hydroxy-1-oxobutane-2-yl)amino)-3-(4-((di-tert-butoxyphosphoryl)oxy)phenyl)-1-oxopropane-2-yl)carbamate

[0034]

[0035] Anhydrous tetrahydrofuran (60 mL) was added to a 100 mL reaction flask and placed in an ice bath. Then, (S)-2-((benzyloxycarbonyl)amino)-3-(4-((bis(tert-butoxyphosphoryl)oxy)phenyl)propionic acid (3.0 g, 5.91 mmol), threonamide hydrochloride (1.08 g, 7.09 mmol), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (3.02 g, 8.28 mmol) and sodium bicarbonate (1.99 g, 23.6 mmol) were added. The mixture was reacted at room temperature for 1 hour, then poured into ice water (100 mL), filtered, and the collected solid was washed and dried to give a white solid product (1.85 g, yield 61%). 1 HNMR(300MHz,DMSO-d6)δ7.75(s,1H),7.44(s,1H),7.40–7.34(m,6H),7.33–7.27(m,3H),7.18(s,1H),7.03(s,1H),5.08–4. 97(m,3H),4.65(s,1H),4.20(s,1H),3.93(s,1H),3.02(s,2H),1.40(s,18H),1.12(d,J=5.7Hz,3H).ESI-MSm / z:630.2[M+Na] + .

[0036] Preparation of 4-((S)-2-amino-3-(((2S,3S)-1-amino-3-hydroxy-1-oxobut-2-yl)amino)-3-oxopropyl)phenyl di-tert-butyl phosphate

[0037]

[0038] In a 100 mL reaction flask, benzyl((S)-1-(((2S,3S)-1-amino-3-hydroxy-1-oxobutane-2-yl)amino)-3-(4-((di-tert-butoxyphosphoryl)oxy)phenyl)-1-oxopropane-2-yl)carbamate (0.8 g, 1.32 mmol) was dissolved in 30 mL of anhydrous ethanol, and 10% palladium on carbon (200 mg) was added. The reaction was carried out at room temperature under hydrogen atmosphere at 1 atm for 6 hours. The mixture was filtered through diatomaceous earth, and the filtrate was concentrated to give a crude product, a light green solid (0.57 g, yield 82%), which was used directly in the next reaction.

[0039] Preparation of 4-((S)-2-((S)-2-acetamide-5-guanidinebutyramide)-3-(((2S,3S)-1-amino-3-hydroxy-1-oxobutane-2-yl)amino)-3-oxopropyl)phenylphosphine dihydrogen phosphate (compound 1)

[0040]

[0041] Anhydrous dichloromethane (20 mL) was added to a 100 mL reaction flask. Under ice bath conditions, N-acetyl-N'-((2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-yl)sulfonyl)-L-arginine (50 mg, 0.106 mmol), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (50.6 mg, 0.138 mmol), sodium bicarbonate (27.2 mg, 0.32 mmol), and 4-((S)-2-amino-3-(((2S,3S)-1-amino-3-hydroxy-1-oxobutyl-2-yl)amino)-3-oxopropyl)phenyl di-tert-butyl phosphate (65.6 mg, 0.138 mmol) were added. The mixture was stirred at room temperature for 2 hours. The reaction mixture was then poured into 100 mL of water. The aqueous layer was extracted with dichloromethane (3 × 50 mL), the dichloromethane extract was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to approximately 5 mL. Trifluoroacetic acid (3 mL) and triethylsilane (0.15 mL) were added to the concentrate, and the mixture was reacted at room temperature for 2 hours, followed by removal of the solvent under reduced pressure. The product was purified by preparative thin-layer chromatography to give a white solid (14 mg, 21% yield). 1 H NMR(300MHz,DMSO-d6)δ8.62(d,J=8.8Hz,1H),8.15(t,J=8.2Hz,1H),8.02(d,J=8.2Hz,1H), 7.73(d,J=8.5Hz,1H),7.31(s,2H),7.24–7.13(m,4H),7.12–6.94(m,4H),5.00(s,1H),4.54 (m,1H),4.21–4.00(m,3H),3.18–2.89(m,3H),2.81(m,1H),2.67(m,1H),1.84(d,J=7.2Hz,3 H),1.43(q,J=7.5Hz,1H),1.34–1.21(m,2H),1.04(d,J=3.3Hz,4H).ESI-MSm / z:560.2[M+H] + .HPLC(70:30acetonitrile / water with 1‰TFA):t R = 9.8 min, 99.7%.

[0042] Preparation of benzyl((S)-1-(((S)-1-(((2S,3S)-1-amino-3-hydroxy-1-oxobutane-2-yl)amino)-1-oxo-3-(4-(phosphooxy)phenyl)prop-2-yl)amino)-5-guanidinyl-1-oxo-2-yl)amino (compound 2)

[0043]

[0044] The synthesis method is basically the same as that of compound 1, except that N-acetyl-N'-((2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-yl)sulfonyl)-L-arginine is replaced with N-((benzyloxy)carbonyl)-N'-((2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-yl)sulfonyl)-L-arginine. White solid, yield 18%. 1 H NMR(300MHz, Methanol-d4)δ7.39(d,J=5.5Hz,4H),7.35(s,1H),7.22(d,J=8.4Hz,2H), 7.15(d,J=8.4Hz,2H),5.21–5.06(m,2H),4.77–4.72(m,1H),4.34(d,J=3.8Hz,1H),4.2 4(d,J=6.4Hz,1H),4.00(t,J=6.7Hz,1H),3.25(d,J=8.7Hz,1H),3.08(q,J=6.4Hz,2H), 3.03–2.92(m,1H),1.54(t,J=8.0Hz,2H),1.39(d,J=8.1Hz,2H),1.21(d,J=6.4Hz,3H). 31 P NMR(243MHz,DMSO-d6)δ-4.56.HRMS(ESI):calcd forC 27 H 38 N7O 10 P[M+H] + 652.2496,found652.2490.HPLC(70:30acetonitrile / water with1‰TFA):t R = 5.61 min, 98.2%.

[0045] Preparation of (9H-fluorene-9-yl)methyl((S)-1-((S)-1-(((2S,3S)-1-amino-3-hydroxy-1-oxobutane-2-yl)amino)-1-oxo-3-(4-(phosphooxy)phenyl)propane-2-yl)amino)-5-guanidinyl-1-oxopentan-2-yl)carbamate (compound 3)

[0046]

[0047] The synthesis method is basically the same as that of compound 1, except that N-acetyl-N'-((2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-yl)sulfonyl)-L-arginine is replaced with N-(((9H-fluorene-9-yl)methoxy)carbonyl)-N'-((2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-yl)sulfonyl)-L-arginine. White solid, yield 19%. 1 HNMR(300MHz,Methanol-d4)δ7.79(d,J=7.5Hz,2H),7.70–7.63(m,2H),7.38(t,J=7.4Hz,2H),7 .31(t,J=7.4Hz,2H),7.13(s,4H),4.69(s,1H),4.49(t,J=8.6Hz,1H),4.40(t,J=8.5Hz,1H),4.3 0(d,J=3.6Hz,1H),4.21(d,J=6.5Hz,2H),3.89(t,J=6.5Hz,1H),3.23(d,J=9.2Hz,1H),3.08–2. 97(m,2H),2.95–2.87(m,1H),1.45(s,2H),1.32(s,2H),1.16(d,J=6.4Hz,3H).HRMS(ESI):calcd for C 34 H 42 N7O 10 P[M+H] + 740.2809,found 740.2805.HPLC(70:30acetonitrile / water with 1‰TFA):t R = 5.02 min, 98.4%.

[0048] Preparation of (S)-2-((benzyloxycarbonyl)amino)-3-(4-((di-tert-butoxyphosphoryl)oxy)phenyl)propionyl-L-allethreonine methyl ester

[0049]

[0050] (S)-2-((benzyloxycarbonyl)amino)-3-(4-((bis(tert-butoxyphosphoryl)oxy)phenyl)propionic acid (3.0 g, 5.91 mmol), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (2.81 g, 7.68 mmol), sodium bicarbonate (1.99 g, 23.64 mmol), and methylthreonine hydrochloride (0.99 g, 5.91 mmol) were added to anhydrous tetrahydrofuran (30 mL) under ice bath conditions. The mixture was stirred at room temperature for 1 hour, and the reaction mixture was poured into ice water and filtered. The collected solid was washed and dried to give a white solid (2.92 g, yield 79%). 1 HNMR(300MHz,DMSO-d6)δ8.06(s,1H),7.61(d,J=8.8Hz,1H),7.39–7.30(m, 5H),7.28–7.23(m,2H),7.08(d,J=7.7Hz,2H),5.01(s,1H),4.96(s,2H),4.4 5(s,1H),4.35(d,J=5.5Hz,1H),4.23–4.15(m,1H),3.65(s,3H),3.04(s,1H) ,2.75(s,1H),1.45(s,18H),1.12(d,J=6.7Hz,3H).ESI-MSm / z:645.5[M+Na] + .

[0051] Preparation of (S)-2-amino-3-(4-((di-tert-butoxyphosphoryl)oxy)phenyl)propionyl-L-allothreonine methyl ester

[0052]

[0053] (S)-2-((benzyloxycarbonyl)amino)-3-(4-((di-tert-butoxyphosphoryl)oxy)phenyl)propionyl-L-allothreonine methyl ester (1.0 g, 1.61 mmol) was dissolved in 30 mL of anhydrous ethanol. 10% palladium on carbon (300 mg) was added, and the reaction was carried out at room temperature under hydrogen atmosphere at 1 atm for 6 hours. The mixture was filtered, and the filtrate was concentrated to give a light green solid (0.68 g, 87% yield), which was used directly in the next reaction.

[0054] Preparation of methyl(S)-2-((S)-2-(((benzyloxy)carbonyl)amino)-5-(3-(((2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-yl)sulfonyl)guanidinyl)pentanoylamino)-3-(4-((di-tert-butoxy)phosphoryloxy)phenyl)propionate-L-allothreonine ester

[0055]

[0056] Cbz-Arg(Pbf)-OH (0.6 g, 0.91 mmol), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (0.43 g, 1.18 mmol), NaHCO3 (0.22 g, 2.37 mmol), and (S)-2-amino-3-(4-((di-tert-butoxyphosphoryl)oxy)phenyl)propionyl-L-altothreonine methyl ester (0.44 g, 0.91 mmol) were added to anhydrous tetrahydrofuran (40 mL) under ice bath conditions. The mixture was stirred at room temperature for 3 hours, and then the reaction mixture was poured into ice water (150 mL). The aqueous layer was extracted with ethyl acetate (3 × 60 mL), and the ethyl acetate extracts were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to give the crude product, a white solid (0.67 g, 71% yield).

[0057] Preparation of ((S)-2-((S)-2-((benzyloxy)carbonyl)amino)-5-(3-((2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-yl)sulfonyl)guanidinyl)pentamido)-3-(4-((di-tert-butoxyphosphoryl)oxy)phenyl)propionyl)-L-allethreonine

[0058]

[0059] Methyl(S)-2-((S)-2-(((benzyloxy)carbonyl)amino)-5-(3-(((2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-yl)sulfonyl)guanidinyl)pentanoyl)-3-(4-((di-tert-butoxy)phosphoryloxy)phenyl)propionate-L-allothreonine ester (0.6 g, 0.58 mmol) was dissolved in 5 mL of methanol, followed by 5 mL of an aqueous solution of lithium hydroxide (0.27 g, 1.16 mmol), and then 5 mL of tetrahydrofuran. The mixture was stirred at room temperature for 2 hours. The reaction mixture was poured into ice water (50 mL), extracted with ethyl acetate (3 × 30 mL), and the ethyl acetate extracts were combined, washed with saturated brine, and dried over anhydrous sodium sulfate. The solvent was removed under reduced pressure to give the crude product, a white solid (0.50 g, 86% yield).

[0060] Preparation of benzyl((S)-5-guanidinyl-1-(((S)-1-(((2S,3S)-3-hydroxy-1-oxo-1-(((S)-piperidin-3-yl)amino)butan-2-yl)amino)-1-oxo-3-(4-(phosphooxy)phenyl)propan-2-yl)amino)-1-oxopentane-2-yl)carbamate (compound 4)

[0061]

[0062] Under ice bath conditions, ((S)-2-((S)-2-(((benzyloxycarbonyl)amino)-5-(3-((2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-yl)sulfonyl)guanidinyl)pentanamide)-3-(4-((di-tert-butoxyphosphoryl)oxy)phenyl)propionyl)-L-threonine (100 mg, 0.098 mmol), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (46.6 mg, 0.12 mmol), and sodium bicarbonate (24.7 mg, 0.29 mmol) were added. (1 mmol) and (S)-3-aminopiperidine (14.7 mg, 0.14 mmol) were added to anhydrous tetrahydrofuran (10 mL), and the mixture was reacted at room temperature for 3 hours. The reaction mixture was then poured into ice water and extracted with dichloromethane (3 × 30 mL). The dichloromethane extracts were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to approximately 5 mL. Trifluoroacetic acid (3 mL) and triethylsilane (0.15 mL) were then added, and the mixture was reacted at room temperature for 2 hours. The solvent was then removed under reduced pressure. The product was purified by preparative thin-layer chromatography to give a white solid (6 mg, yield 24.4%). 1 H NMR (300MHz, DMSO-d6) δ7.98(d,J=8.4Hz,1H),7.90–7.84(m,1H),7.72(d,J=7.4Hz,1H),7.54(t,J=7.3Hz,1H) ,7.40–7.37(m,5H),7.19(s,2H),7.12(s,3H),7.01(d,J=8.1Hz,3H),5.12–4.96(m,4H),4.93(s,1H),4.68(s,1 H),4.10–4.06(m,1H),3.97(s,1H),3.88(q,J=10.4,9.5Hz,3H),3.18(s,1H),3.07–2.87(m,4H),2.82–2.70(m, 2H),2.54(s,1H),1.80(s,2H),1.62(s,1H),1.41(s,3H),1.23(s,2H),1.02(d,J=5.9Hz,3H).HRMS(ESI):calcd for C 32 H 47 N8O 10 P[M+H] + 735.3231,found 735.3227.HPLC(70:30acetonitrile / water with 1‰TFA):t R =4.78 min, 98.0%.

[0063] Preparation of benzyl(S)-3-((5S,8S,11S)-5-(3-guanidinopropyl)-11-((S)-1-hydroxyethyl)-3,6,9-trioxo-1-phenyl-8-(4-(phosphooxy)benzyl)-2-oxa-4,7,10-triazadodecane-12-amido)piperidine-1-carboxylate (compound 5)

[0064]

[0065] The synthesis method is basically the same as that of compound 4, except that (S)-3-aminopiperidine is replaced with (S)-1-benzyloxycarbonyl-3-aminopiperidine. White solid, yield 23%. 1 H NMR (300MHz, DMSO-d6) δ8.28(s,1H),8.04–7.83(m,2H),7.70(d,J=7.4Hz,1H),7.51(d,J=7.7Hz,1H),7.37(d,J=4.0Hz,4H),7 .34–7.30(m,6H),7.16(d,J=8.4Hz,4H),7.01(d,J=8.1Hz,2H),5.04(d,J=7.7Hz,4H),4.89(s,1H),4.66(t,J=9.5Hz,1H),4.15 (q,J=7.4Hz,1H),3.96(s,1H),3.92–3.79(m,2H),3.77(s,1H),3.63(s,2H),3.04(s,1H),3.00(s,1H),2.98–2.84(m,3H),2.8 1–2.68(m,2H),2.54(s,1H),1.77–1.69(m,2H),1.49–1.30(m,4H),1.23–1.19(m,2H),1.03(d,J=4.2Hz,3H).HRMS(ESI):calcd for C 40 H 53 N8O 12 P[M+H] + 869.3599,found 869.3589.HPLC(70:30acetonitrile / water with 1‰TFA):t R =8.71 min, 95.0%.

[0066] Preparation of benzyl((S)-1-(((S)-1-(((2S,3S)-1-(cyclohexylamino)-3-hydroxy-1-oxobut-2-yl)amino)-1-oxo-3-(4-(phosphoryloxy)phenyl)prop-2-yl)amino)-5-guanidinyl-1-oxopent-2-yl)carbamate (compound 6)

[0067]

[0068] The synthesis method is basically the same as that of compound 4, except that (S)-3-aminopiperidine is replaced with cyclohexylamine. White solid, yield 22%. 1 HNMR (300MHz, DMSO-d6) δ7.91(d,J=8.6Hz,1H),7.83(s,1H),7.51(d,J=7.7Hz,2H),7.37(s,5H),7.20(s,2 H),7.14(d,J=8.2Hz,3H),7.00(d,J=8.0Hz,2H),5.16–4.97(m,2H),4.86(s,1H),4.66(s,1H),4.13(s,1H) ,3.95(s,1H),3.84(d,J=7.2Hz,1H),3.52(s,2H),3.04–3.00(m,2H),2.91(s,2H),2.74–2.70(m,1H),2.56 (s,1H),1.68(s,4H),1.51(s,1H),1.35(s,2H),1.27–1.13(m,7H),1.03(d,J=6.2Hz,3H).HRMS(ESI):calcd forC 33 H 48 N7O 10 P[M+H] + 734.3279,found 734.3275.HPLC(70:30acetonitrile / water with 1‰TFA):t R = 5.76 min, 95.0%.

[0069] Preparation of benzyl((S)-5-guanidinyl-1-(((S)-1-(((2S,3S)-3-hydroxy-1-oxo-1-(phenylamino)but-2-yl)amino)-1-oxo-3-(4-(oxophosphono)phenyl)prop-2-yl)amino)-1-oxopent-2-yl)carbamate (compound 7)

[0070]

[0071] The synthesis method is basically the same as that of compound 4, except that (S)-3-aminopiperidine is replaced with aniline. White solid, yield 22%. 1H NMR (300MHz, DMSO-d6) δ9.80 (s, 1H), 8.07 (d, J = 8.1Hz, 1H), 7.82 (d, J = 8.9Hz, 1H), 7.62 (d, J = 8.0Hz, 2H),7.54(d,J=7.0Hz,1H),7.37(d,J=3.7Hz,4H),7.30(t,J=7.9Hz,4H),7.15(d,J=8.7Hz,4H),7.08– 6.98(m,3H),5.11–5.00(m,3H),4.73(s,1H),4.39–4.30(m,1H),4.11(s,1H),3.83(s,1H),3.47(s,2H ),3.09–3.05(m,2H),2.90(s,2H),2.71(s,1H),1.33(s,2H),1.11(d,J=6.1Hz,5H).HRMS(ESI):calcd for C 33 H 42 N7O 10 P[M+H] + 728.2809,found 728.2801.HPLC(70:30acetonitrile / water with 1‰TFA):t R = 6.22 min, 96.5%.

[0072] Preparation of benzoyl((4S,7S,10S)-15-amino-4-((S)-1-hydroxyethyl)-15-imino-3,6,9-trioxo-1-phenyl-7-(4-(phosphoryloxy)benzyl)-2,5,8,14-tetraazapentadecan-10-yl)carbamate (compound 8)

[0073]

[0074] The synthesis method is basically the same as that of compound 4, except that (S)-3-aminopiperidine is replaced with benzylamine. White solid, yield 17%. 1HNMR (300MHz, DMSO-d6) δ8.22(s,1H),7.99(d,J=8.5Hz,1H),7.91(d,J=8.9Hz,1H),7.51(d,J=7.8Hz,1H),7.38(s ,4H),7.30(d,J=2.6Hz,5H),7.23(s,2H),7.16(d,J=8.4Hz,3H),7.02(d,J=8.1Hz,2H),5.14–4.92(m,3H),4.71(s ,1H),4.34(d,J=5.9Hz,2H),4.27–4.18(m,1H),4.08(s,1H),3.87(d,J=7.6Hz,1H),3.59(s,2H),3.08–3.03(m,1H ),2.94(s,2H),2.79–2.70(m,1H),2.56(s,1H),1.38(s,2H),1.19(s,2H),1.06(d,J=6.3Hz,3H).HRMS(ESI):calcd for C 34 H 44 N7O 10 P[M+H] + 742.2966,found 742.2960.HPLC(70:30acetonitrile / water with 1‰TFA):t R = 6.37 min, 96.5%.

[0075] Preparation of benzyl (S)-5-guanidinyl-1-((S)-1-((2S,3S)-3-hydroxy-1-oxo-1-(piperidin-1-yl)butan-2-ylamino)-1-oxo-3-(4-(phosphoryloxy)phenyl)propan-2-ylamino)-1-oxopentane-2-yl)carbamate (compound 9)

[0076]

[0077] The synthesis method is basically the same as that of compound 4, except that (S)-3-aminopiperidine is replaced with piperidine. White solid, yield 16%. 1H NMR (300MHz, DMSO-d6) δ8.14(d,J=8.4Hz,1H),7.89(s,2H),7.49(d,J=7.9Hz,1H),7.37 (d,J=3.7Hz,5H),7.15(d,J=8.4Hz,3H),7.00(d,J=8.1Hz,3H),5.12–4.97(m,2H),4.79 (s,1H),4.66(d,J=6.6Hz,2H),3.90(s,4H),3.31(s,4H),2.98(s,3H),2.77–2.69(m,1H ),1.66–1.53(m,2H),1.45(s,6H),1.27(s,2H),1.02(d,J=6.1Hz,3H).HRMS(ESI):calcd for C 32 H 46 N7O 10 P[M+H] + 720.3122,found 720.3119.HPLC(70:30acetonitrile / water with 1‰TFA):t R = 6.32 min, 98.3%.

[0078] Preparation of benzyl (S)-5-guanidinyl-1-((S)-1-((2S,3S)-3-hydroxy-1-oxo-1-(pyrrolidone-1-yl)but-2-ylamino)-1-oxo-3-(4-(phosphoryloxy)phenyl)prop-2-ylamino)-1-oxopent-2-yl)carbamate (compound 10)

[0079]

[0080] The synthesis method is basically the same as that of compound 4, except that (S)-3-aminopiperidine is replaced with tetrahydropyrrole. White solid, yield 19%. 1HNMR (300MHz, DMSO-d6) δ8.18(d,J=8.0Hz,1H),7.90(d,J=8.2Hz,1H),7.79(s,1H),7.52(d,J=7.9Hz,1H),7. 45–7.28(m,6H),7.15(d,J=8.3Hz,3H),7.02(d,J=8.2Hz,2H),5.14–4.99(m,2H),4.68–4.61(m,1H),4.49–4. 41(m,1H),3.96–3.88(m,2H),3.63–3.48(m,2H),3.40–3.23(m,2H),3.06–2.97(m,3H),2.82–2.74(m,1H),2. 56(s,1H),1.90–1.77(m,4H),1.60–1.40(m,2H),1.39–1.26(m,2H),1.07(d,J=6.2Hz,3H).HRMS(ESI):calcd for C 31 H 44 N7O 10 P[M+H] + 706.2966,found 706.2954.HPLC(70:30acetonitrile / water with1‰TFA):t R =6.25min, 98.7%.

[0081] Preparation of benzyl((S)-1-(((S)-1-(((2S,3S)-1-(azacyclohexyl)-3-hydroxy-1-oxobut-2-yl)amino)-1-oxo-3-(4-(phosphoryloxy)phenyl)prop-2-yl)amino)-5-guanidinyl-1-oxopent-2-yl)carbamate (compound 11)

[0082]

[0083] The synthesis method is basically the same as that of compound 4, except that (S)-3-aminopiperidine is replaced with cycloheximine. White solid, yield 22%. 1HNMR (300MHz, DMSO-d6) δ8.21(d,J=8.7Hz,1H),7.82(d,J=8.1Hz,2H),7.51(d,J=7.8Hz,1H),7.37(d,J =3.7Hz,5H),7.12(d,J=8.2Hz,3H),6.99(d,J=8.2Hz,3H),5.09–5.00(m,2H),4.80(s,1H),4.63(t,J=7 .2Hz,2H),3.94–3.85(m,2H),3.57(s,2H),3.48(s,3H),3.40(s,1H),3.01–2.98(m,3H),2.79–2.71(m, 1H),2.54(s,1H),1.72–1.59(m,4H),1.48(s,6H),1.31(s,2H),1.05(d,J=6.2Hz,3H).HRMS(ESI):calcd forC 33 H 48 N7O 10 P[M+H] + 734.3279,found 734.3277.HPLC(70:30acetonitrile / water with 1‰TFA):t R = 6.04 min, 98.9%.

[0084] Preparation of benzyl((2S)-1-(((2S)-1-(((2S,3S)-1-(2-azabicyclo[2.2.1]hept-2-yl)-3-hydroxy-1-oxobut-2-yl)amino)-1-oxo-3-(4-(phosphoryloxy)phenyl)prop-2-yl)amino)-5-guanidinyl-1-oxopent-2-yl)carbamate (compound 12)

[0085]

[0086] The synthesis method is basically the same as that of compound 4, except that (S)-3-aminopiperidine is replaced with (1S,4S)-2-azabicyclo[2.2.1]heptane. White solid, yield 26%. 1HNMR (300MHz, DMSO-d6) δ8.21(d,J=7.9Hz,2H),7.78(t,J=10.6Hz,1H),7.49(t,J=7.5Hz,1H),7.40–7.30( m,5H),7.17–7.08(m,4H),6.99(d,J=8.1Hz,2H),5.14–4.98(m,2H),4.66(s,1H),4.59(s,1H),4.39(d,J=7. 2Hz,1H),4.20(t,J=7.0Hz,1H),3.95(s,1H),3.86(s,2H),3.38(d,J=8.6Hz,2H),3.21(s,1H),2.96(s,3H) ,2.74–2.67(m,1H),2.54(s,1H),1.67–1.51(m,4H),1.44–1.28(m,5H),1.21(s,2H),1.04(d,J=6.1Hz,3H). 13 C NMR (151MHz, DMSO) δ170.86,170.60,167.55,166.94,156.61,155.87,151.17,136.69,131.66,129.97,128.28,127.77,1 19.20,66.81,65.61,57.30,55.23,54.52,53.12,52.43,40.09,37.72,36.77,35.58,30.53,28.94,26.90,24.77,19.30. 31 P NMR(243MHz,DMSO-d6)δ-4.84.HRMS(ESI):calcd for C 33 H 46 N7O 10 P[M+H] + 732.3122,found 732.3118.HPLC(70:30acetonitrile / water with 1‰TFA):t R =7.54 min, 98.7%.

[0087] Example 2: Activity test of the compound (competitive inhibition experiment of fluorescence polarization)

[0088] The protein used in the fluorescence polarization experiment was the wild-type protein of the N-terminal TKB domain (36-344) of Cbl-b, and a polypeptide linked to the FITC group was used as the fluorescent probe (FITC-acp-RpYTPEP-NH2). The instrument used in the experiment was a SpectraMax Multi-Mode Microplate Reader (Molecular Devices), and detection was performed using a black Corning 3676384-well plate.

[0089] The experimental system for each well plate was 60 μL, containing 20 μL of the corresponding gradient concentration of the analyte, 20 μL of a final concentration of 100 nM protein, and 20 μL of a 5 nM fluorescent probe, all prepared using phosphate-buffered saline (1×PBS). The reaction mixture was added to the wells before detection and incubated with shaking at room temperature for 30 min. For detection, the fluorescence intensity in the horizontal and vertical directions was measured using an excitation wavelength of 485 nm and an emission wavelength of 535 nm, and the milli-biased values ​​for the corresponding wells were calculated. The inhibition rate of the analyte at a given concentration was calculated using the formula: inhibition = 1 - (P0)0 obs -P min ) / (P max -P min )Calculations show that P max P represents the millivariance value of a well containing only protein and probe. min P represents the millibias value containing only the probe hole. obs This represents the millivariance value of the pores containing different concentrations of the analyte. IC50 of the inhibitor. 50 The Ki values ​​were calculated using a sigmoid regression fitting curve of concentration-inhibition rate, with GraphPadPrism 5.0 as the software. Table 1 shows the Ki values ​​for each compound.

[0090] Table 1 IC50 of each compound 50 value

[0091]

[0092]

[0093]

[0094]

[0095] In summary of the above embodiments:

[0096] This invention provides a novel phosphorylated peptide-like compound; activity studies have shown that this phosphorylated peptide-like compound exhibits excellent Cbl-b inhibitory activity, making it an effective Cbl-b inhibitor. Those skilled in the art know that Cbl-b overexpression suppresses T-cell immune responses, and that inhibiting Cbl-b can activate the immune system to kill tumor cells. Therefore, the phosphorylated peptide-like compound provided by this invention, or its pharmaceutically acceptable salts or solvates, has the potential to be developed into medicaments for treating or alleviating diseases (such as non-small cell lung cancer, breast cancer, prostate cancer, head and neck squamous cell carcinoma, liver cancer, pancreatic cancer, colorectal cancer, and ovarian cancer) that are treated or alleviated by inhibiting Cbl-b.

[0097] The purpose of the above embodiments is to specifically illustrate the substantive content of the present invention, but those skilled in the art should know that the scope of protection of the present invention should not be limited to the specific embodiments.

Claims

1. A phosphonated peptidomimetic compound or a pharmaceutically acceptable salt, solvate thereof, characterized in that, The structural general formula of the phosphorylated peptoid compound is as follows: Among them: R1 is selected from C1-C3 alkyl and —OCH2R4, wherein: R4 is substituted or unsubstituted aryl or fluorenyl; R2, R3 are independently selected from hydrogen, substituted or unsubstituted 5-7 membered N-containing or non-N-containing carbocyclic ring and substituted or unsubstituted aryl or benzyl, or R2, R3 together with the N to which they are connected form a substituted or unsubstituted 5-7 membered N-containing carbocyclic ring, azabicyclo ring.

2. The phosphorylated peptoid or pharmaceutically acceptable salt, solvate thereof according to claim 1, wherein, The structural formula of the phosphorylated peptoid compound is as shown in 1-12:

3. Use of the phosphorylated peptoid compound or a pharmaceutically acceptable salt, solvate thereof according to claim 1 or 2 for the preparation of a Cbl-b inhibitor drug.

4. Use of the phosphorylated peptoid compound or a pharmaceutically acceptable salt, solvate thereof according to claim 1 or 2 for the preparation of a drug for treating or alleviating a disease by inhibiting Cbl-b.

5. Use according to claim 4, characterized in that: The disease treated or alleviated by inhibiting Cbl-b is cancer.

6. Use according to claim 5, characterized in that: The cancer is selected from non-small cell lung cancer, breast cancer, prostate cancer, head and neck squamous cell carcinoma, liver cancer, pancreatic cancer, colorectal cancer and ovarian cancer.

7. A pharmaceutical preparation characterized in that: The phosphorylated peptoid compound or a pharmaceutically acceptable salt, solvate thereof according to claim 1 or 2 is used as an active ingredient, and a pharmaceutically acceptable excipient is used to prepare a pharmaceutically acceptable dosage form.

8. The pharmaceutical preparation according to claim 7, characterized in that: The excipient is solid, liquid or semi-solid.

9. The pharmaceutical preparation according to claim 7, characterized in that: The dosage form is tablet, capsule or injection.

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