CN111574592B - Cyclic peptide compounds with antagonistic PD-1/PD-L1 interaction and application thereof - Google Patents
Cyclic peptide compounds with antagonistic PD-1/PD-L1 interaction and application thereof Download PDFInfo
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Abstract
The invention discloses a cyclopeptide compound and application thereof, wherein the cyclopeptide compound and pharmaceutically acceptable salts, esters or solvent compounds thereof can be used for preparing PD-1/PD-L1 pathway inhibitors, and have good application prospects in preparation of medicaments for treating tumors. Compared with monoclonal antibody research and development, the peptide inhibitor in the field of PD-1/PD-L1 inhibitors is slow in progress, so that research and development of inhibitors for inhibiting PD-1/PD-L1 interaction have important clinical significance.
Description
Technical Field
The invention relates to a compound and application thereof, in particular to a cyclic peptide compound and pharmaceutically acceptable salt, ester or solvent compound thereof, and application of the cyclic peptide compound as a PD-1/PD-L1 inhibitor in preparation of a medicine for treating malignant tumor.
Background
PD-1 is also named CD279, is a type I transmembrane protein with a relative molecular weight of 55000-60000, belongs to immunoglobulin superfamily members, and mainly comprises an extracellular immunoglobulin variable region (IgV) like structure domain, a hydrophobic transmembrane region and an intracellular region. The intracellular domain includes C-terminal and N-terminal amino acid residues, contains 2 independent phosphorylation sites, which are an immunoreceptor tyrosine based inhibition motif (ITIM) and an immunoreceptor tyrosine based switch motif (ITSM), respectively. PD-1 is mainly expressed on activated CD4+ T cells, CD8+ T cells, B cells, NK cells, monocytes, dendritic cells and other immune cells, and promotes the maturation of T cells. The ligands of PD-1 are members of the B7 family, including PD-L1 (also known as B7-H1, CD 274) and PD-L2 (also known as B7-DC), both of which are highly expressed in placental tissue; low expression in spleen, lymph node, thymus; no expression in brain tissue. Wherein PD-L1 is I type transmembrane protein and is mainly expressed in antigen presenting cells, B cells, T cells, epithelial cells, muscle cells, endothelial cells and the like. PD-1 and PD-L1 jointly form a PD-1/PD-L1 signal channel, inhibit the generation of growth factors and cell proliferation, and play an important role in the activation of T cells and the regulation of immune response. Following activation of the PD-1/PD-L1 pathway, the immune system is suppressed in cancer, pregnancy, tissue transplantation and autoimmune diseases. At present, the development of PD-1/PD-L1 inhibitors mainly focuses on the field of monoclonal antibodies, and monoclonal antibodies such as Nivolumab, lamborlizumab, atezolizumab, durvalumab, avelumab and the like are sold on the market at home and abroad, so that the inhibitor can be used for treating diseases with poor effect in conventional treatment methods such as non-small cell lung cancer, melanoma and the like, and has obvious treatment effect. Compared with monoclonal antibody research and development, the peptide inhibitor in the field is slow to progress. Therefore, the research and development of the inhibitor for inhibiting the PD-1/PD-L1 interaction have important clinical significance.
Disclosure of Invention
The purpose of the invention is as follows: the invention aims to provide a cyclic peptide compound and application thereof, wherein the compound is a PD-1/PD-L1 inhibitor and influences the generation and cell proliferation of growth factors. The invention also aims to indicate that the compound has good application prospect in preparing medicaments for treating breast cancer and melanoma.
The technical scheme is as follows: the cyclic peptide compound and the pharmaceutically acceptable salt, ester or solvent compound thereof have the structure shown as the general formula (I):
R 1 selected from one of the following structures or stereoisomers or racemates thereof:
-Me[Ala]、-CH 2 CH(CH 3 ) 2 [Leu]、Me(Et)CH-[Ile]、-CH 2 NMe 2 [4-aza-leu]、CH 3 CH 2 CH 2 CH 2 -[Nle]-CH 2 CH 2 CH(CH 3 ) 2 [HOLeu]、
[Cpa]；
R 2 selected from one of the following structures or stereoisomers or racemates thereof:
-Me[Ala]、-CH 2 CH(CH 3 ) 2 [Leu]、Me(Et)CH-[Ile]、-CH 2 NMe 2 [4-aza-leu]、CH 3 CH 2 CH 2 CH 2 -[Nle]-CH 2 CH 2 CH(CH 3 ) 2 [HOLeu]、
[Cpa]、Ph(4-Ph)-CH 2 -[4-Ph-Phe]、PhCH 2 -[Phe]、4-OH-Phe[Tyr]；
R 3 selected from the following structures or one of its stereoisomers or racemates:
-CH 2 NH 2 [Dap]、-CH 2 CH 2 NH 2 [Dab]、-CH 2 CH 2 CH 2 NH 2 [Orn]、-CH 2 CH 2 CH 2 CH 2 NH 2 [Lys]、
[His]；
R 4 、R 5 selected from the following structures or one of its stereoisomers or racemates:
R 6 selected from one of the following structures or stereoisomers or racemates thereof:
-CH 2 CONH 2 [Asn]、-CH 2 CH 2 CONH 2 [Gln]、CH 2 CH 2 CH 2 NHC(N)NH 2 [Arg]、CH 2 CH- 2 CH 2 NHCONH 2 [Cit]、CH 2 NHCONH 2 [Alb]；
R 7 ，R 8 selected from one of the following structures or stereoisomers or racemates thereof:
R 9 one selected from the following structures:
-H[Phe]，-NH 2 [4-NH 2 -Phe]，-NHMe[4-NHMe-Phe]，-NMe 2 [4-NMe 2- Phe]，-OH[Tyr]，-OMe[OMe-Tyr]，-Ph[4-Ph-Phe]；
l is selected from one of the following structures:
the wavy line indicates the connection manner.
The cyclic peptide compound and a pharmaceutically acceptable salt, ester or solvent compound thereof are selected from the following compounds:
pharmaceutical compositions comprising said cyclic peptide compounds and pharmaceutically acceptable salts, esters or solvent compounds thereof.
The pharmaceutical composition, the cyclic peptide compound and the pharmaceutically acceptable salt, ester or solvent compound thereof are added with one or more pharmaceutically acceptable auxiliary materials to prepare a preparation.
The pharmaceutical composition comprises the pharmaceutical auxiliary materials including diluents, excipients, fillers, binders, wetting agents, absorption enhancers, surfactants, lubricants or stabilizers.
The preparation of the pharmaceutical composition is tablets, capsules, granules, powder, syrup, oral liquid or injection.
The cyclopeptide compound and the pharmaceutically acceptable salt, ester or solvent compound thereof are applied to the preparation of PD-1/PD-L1 pathway inhibitors.
The application of the cyclopeptide compound and the pharmaceutically acceptable salt, ester or solvent compound thereof in preparing the T cell immune activity enhancer.
The cyclopeptide compound and pharmaceutically acceptable salts, esters or solvent compounds thereof or the application of the pharmaceutical composition in preparing medicines for preventing or treating breast cancer and melanoma.
The medicine for preventing or treating breast cancer and melanoma is breast cancer and melanoma immunotherapy medicine or breast cancer and melanoma chemotherapy medicine or breast cancer and melanoma targeted therapy medicine.
Has the advantages that: the cyclopeptide compound provided by the invention can be used for preparing a PD-1/PD-L1 inhibitor, influences the generation and cell proliferation of growth factor inhibition, plays an important role in activation of T cells and regulation and control of immune response, and has a good application prospect in preparation of medicines for treating breast cancer and melanoma.
Detailed Description
1. General procedure for solid phase Synthesis
Solid phase amino acid synthesis was performed in a solid phase reaction synthesis tube using rink amide-AM resin (Merrifield polymer supported (2, 4-dimethoxyphenyl) (4-alkoxyphenyl) methane, where the 4-alkoxy group is the position and type of bond attached to the resin, with a loading of 0.7 mmol/g). After the reagents used in the reaction were dissolved in DMF and DCM, they were added to the reaction tube along the tube wall and shaken for the desired time after passing nitrogen through the tube. Thereafter, the reaction solution was evacuated from the lower side of the reaction tube by a vacuum pump. The solvents and reagents used in the reaction were: DMF = N, N-dimethylformamide; DCM = dichloromethane; HATU =1- [ bis (dimethylamino) methylene]-3-oxo-1H-1, 2, 3-triazolo [4,5-b]Pyridine hexafluorophosphate; DIPEA = diisopropylethylamine. The swelling process of the resin is described below in "resin swelling step". The condensation step is described below as the "condensation step". The amino acid derivatives and terminal carboxylic acids used are shown below (side chain protecting groups placed in parentheses): fmoc-L-Gly-OH, fmoc-L-Cys (Trt) -OH, fmoc-L-Leu-OH, fmoc-L- [ N-Me]Nle-OH，Fmoc-L-Trp(CH 2 COOtBu)-OH，Fmoc-L-Trp(Boc)-OH，Fmoc-L-Dab(Boc)-OH，Fmoc-L-[O-tBu]Hyp-OH，Fmoc-L-Dap(Boc)-OH，Fmoc-L-Pro-OH，Fmoc-L-Asn(Trt)-OH，Fmoc-L-[N-Me]Ala-OH，Fmoc-L-[O-tBu]Tyr-OH，ClCH 2 COOH。
In some embodiments, the following unnatural amino acids or derivatives thereof are used:
2. resin swelling step
Rink Amide-AM resin (286mg, 0.2mmol) was added to a 10mL solid phase synthesis reaction tube, 8mL DCM was added, and the mixture was allowed to stand for half an hour. After that, the DCM was evacuated by a vacuum pump to complete the swelling of the resin.
3. Step of condensation
6mL of 20% piperidine/DMF solution was added to the reaction tube and shaken for 30 minutes. The reaction solution was drained, and the resin was washed 3 times with anhydrous DMF (10 mL), anhydrous methanol (10 mL) and anhydrous DCM (10 mL), after which the sample was colored with chloranil, and the blue color of the resin indicated the end of the removal of the protecting group.
After deprotection was complete, the desired amino acid (0.6 mmol), HATU (228mg, 0.6 mmol), DMF (6 mL) and DIPEA (210. Mu.L, 1.2 mmol) were added sequentially to a dry round bottom flask and clarified by ultrasonic-assisted dissolution. Adding the mixed solution into a solid-phase synthesis reaction tube, reacting for 3 hours at 25 ℃, sampling, and developing color through chloranil, wherein the resin is colorless and transparent and can indicate that the condensation reaction is finished. The resin was washed 3 times with dry DMF (10 mL), dry methanol (10 mL) and dry DCM (10 mL) respectively for the next condensation.
4. Peptide liberation procedure
After completion of the desired linear peptide synthesis, the resin was washed with anhydrous DMF 1 time and then pumped to dryness. To the dried resin was added a cleavage solution (trifluoroacetic acid: thioanisole: 1, 2-ethanedithiol: anisole =90, volume ratio. The mixture was shaken at 10 ℃ for 3 hours. After the reaction is finished, the cutting fluid is filtered and concentrated to 1/5 of the original volume, and the concentrated solution is dropwise added into 10 times of anhydrous ether at the temperature of-20 ℃. And (3) carrying out suction filtration to form a precipitate to obtain the crude peptide, and directly carrying out the next reaction without treatment.
5. Step of cyclization of linear peptide
About 100mg of the crude peptide obtained in step 4 was taken and dissolved in 50mL of acetonitrile, and 50mL of 0.1M aqueous ammonium carbonate was slowly added to the solution. The reaction solution was stirred at room temperature for 24 hours. After the reaction is monitored by RP-HPLC, 0.6mL of glacial acetic acid is added into the reaction solution, the reaction solution is concentrated until acetonitrile is completely evaporated, and a water layer is freeze-dried to obtain a crude cyclopeptide product.
RP-HPLC separation procedure
Dissolving the obtained crude peptide in a certain amount of purified water, adjusting the pH value to 7 by using triethylamine or 2M HCl, adding acetonitrile to clarify, and freeze-drying to obtain a crude solid. An amount of acetonitrile was added to dissolve it completely, and the mixture was filtered through a 0.33. Mu.M filter. Separation was performed using an agent eclipse XDB-C18 column, mobile phase A:0.1% of TFA/H 2 O; mobile phase B:0.1% TFA/MeCN. The chromatographic condition is 10% B-100% B for 20min.
Example 1
the synthetic route is shown as follows:
a25 mL reaction tube was purged with nitrogen. Fmoc- (4-Br) -Phe-OH (1, 233mg, 0.5mmol), phB (OH) were added in succession 2 (122mg，1mmol)，Pd(OAc) 2 (5.1mg, 4.5 mol%), dtBuPF (11.9mg, 5 mol%) and K 3 PO 4 (265mg, 1.25mmol). After the addition was complete, the reaction was purged with nitrogen to remove other gases and then heated to 50 ℃. After 16 hours, 20% acetic acid aqueous solution was added to adjust the pH to 4-5, the organic layer was separated, and the aqueous layer was extracted 3 times with ethyl acetate. The organic layers were combined and concentrated to dryness, and the crude product was separated by silica gel column chromatography (petroleum ether: ethyl acetate = 10). The nuclear magnetic resonance hydrogen spectrum data of the compound is completely consistent with the literature report (J.org.chem.2016, 81, 9499-9506) and can be considered as the target product。
Example 2
The synthetic route is as follows:
compound 3 (250mg, 1.3mmol) was dissolved in 13mL of acetone, and 13mL of 10% aqueous sodium carbonate solution was slowly added. The reaction mixture was transferred to 0 ℃ and Fmoc-OSu (526 mg, 1.56mmol) was added. After stirring at 0 ℃ for 5 minutes, the reaction was allowed to warm to room temperature and stirred for 24 hours. After the reaction is finished, 2M hydrochloric acid solution is added to adjust the pH value to 2-3, ethyl acetate is added to extract for 3 times, water washing is carried out for 2 times, and saturated salt water washing is carried out for 1 time. The organic layer was dried over anhydrous sodium sulfate and concentrated to dryness. The crude product was separated by silica gel column chromatography (dichloromethane: methanol =150 = 1) to obtain 368mg of the target product in 75% yield. 1 H NMR(500MHz,Chloroform-d)δ7.81(dd,J＝7.8,1.1Hz,2H),7.70(dd,J＝7.7,1.5Hz,2H),7.65(td,J＝7.6,1.5Hz,2H),7.56(td,J＝7.6,1.2Hz,2H),4.46(d,J＝5.4Hz,2H),4.35–4.29(m,1H),4.21(d,J＝6.5Hz,1H),3.71(dd,J＝10.3,4.2Hz,1H),3.42(dd,J＝10.3,3.3Hz,1H),2.40–2.28(m,2H),1.77(m,1H),1.72–1.46(m,4H),1.39(m,1H). 13 C NMR(125MHz,Chloroform-d)δ172.84,156.53,144.92,144.83,139.80,127.04,126.92,125.02,124.92,122.20,122.09,69.76,67.37,54.22,47.92,47.21,46.36,34.04,33.48,25.64.HRESIMS:378.1633[M+H] + ,calcd 378.1626.
Example 3
Preparation of S3
According to the aforementioned "resin swelling step", after swelling the resin, according to the "condensation step", the amino acids selected were Fmoc-L-Gly-OH, fmoc-L-Cys (Trt) -OH, fmoc-L-Leu-OH, boc-L-Trp-OH, fmoc-L-Trp (Boc) -OH, fmoc-L-Dab (Boc) -OH, fmoc-L- [ O-tBu [ ], respectively]Hyp-OH，Fmoc-L-Dap(Boc)-OH，Fmoc-L-Pro-OH，Fmoc-L-Asn(Trt)-OH，Fmoc-L-[N-Me]Ala-OH，Fmoc-L-Ala-OH，Fmoc-L-[O-tBu]Tyr-OH. After completion of the condensation, a crude linear peptide precursor can be obtained according to the aforementioned "peptide liberation step", and a crude cyclic peptide can be obtained according to the aforementioned "linear peptide cyclization step". The mixture was dissolved in acetonitrile: water =1 (volume ratio), and the cyclic peptide compound can be purified according to the aforementioned "RP-HPLC separation procedure". t is t R ＝14.892min。HRESIMS:[M+H] + ＝1844.9095。
Example 4
Preparation of S4
According to the aforementioned "resin swelling step", after swelling the resin, according to the "condensation step", the amino acids selected are Fmoc-L-Gly-OH, fmoc-L-Cys (Trt) -OH, fmoc-L-Leu-OH, boc-L-Trp-OH, fmoc-L-Trp (Boc) -OH, fmoc-L-Dab (Boc) -OH, fmoc-L- [ O-tBu ], respectively]Hyp-OH，Fmoc-L-Dap(Boc)-OH，Fmoc-L-Pro-OH，Fmoc-L-Asn(Trt)-OH，Fmoc-L-[N-Me]Ala-OH，Fmoc-L-Val-OH，Fmoc-L-[O-tBu]Tyr-OH. After completion of the condensation, a crude linear peptide precursor can be obtained according to the aforementioned "peptide liberation step", and a crude cyclic peptide can be obtained according to the aforementioned "linear peptide cyclization step". The mixture was dissolved in acetonitrile: water =1 (volume ratio), and the cyclic peptide compound can be purified according to the aforementioned "RP-HPLC separation procedure". t is t R ＝14.635min。
HRESIMS:[M+H] + ＝1879.9401。
Example 5
Preparation of S5
According to the aforementioned "resin swelling step", after swelling the resin, according to the "condensation step", the amino acids selected were Fmoc-L-Gly-OH, fmoc-L-Cys (Trt) -OH, fmoc-L-Leu-OH, boc-L-Trp-OH, fmoc-L-Trp (Boc) -OH, fmoc-L-Dab (Boc) -OH, fmoc-L- [ O-tBu [ ], respectively]Hyp-OH，Fmoc-L-Dap(Boc)-OH，Fmoc-L-Pro-OH，Fmoc-L-Asn(Trt)-OH，Fmoc-L-[N-Me]Ala-OH，Fmoc-L-Val-OH，Fmoc-L-[O-tBu]Tyr-OH. After completion of the condensation, a crude linear peptide precursor can be obtained according to the aforementioned "peptide liberation step", and a crude cyclic peptide can be obtained according to the aforementioned "linear peptide cyclization step". The mixture was dissolved in acetonitrile: water =1 (volume ratio), and the cyclic peptide compound can be obtained in pure form according to the aforementioned "RP-HPLC separation procedure". t is t R ＝14.812min。HRESIMS:[M+H] + ＝1886.9555。
Example 6
Preparation of S6
According to the aforementioned "resin swelling step", after swelling the resin, according to the "condensation step", the amino acids selected are Fmoc-L-Gly-OH, fmoc-L-Cys (Trt) -OH, fmoc-L-Leu-OH, boc-L-Trp-OH, fmoc-L-Trp (Boc) -OH, fmoc-L-Dab (Boc) -OH, fmoc-L- [ O-tBu ], respectively]Hyp-OH，Fmoc-L-Dap(Boc)-OH，Fmoc-L-Pro-OH，Fmoc-L-Asn(Trt)-OH，Fmoc-L-[N-Me]Ala-OH，Fmoc-L-Val-OH，Fmoc-L-[O-tBu]Tyr-OH. After completion of the condensation, a crude linear peptide precursor can be obtained according to the aforementioned "peptide liberation step", and a crude cyclic peptide can be obtained according to the aforementioned "linear peptide cyclization step". The mixture was dissolved in acetonitrile: water =1 (volume ratio), and the cyclic peptide compound can be purified according to the aforementioned "RP-HPLC separation procedure". t is t R ＝14.812min。HRESIMS:[M+H] + ＝1886.9557。
Example 7
Preparation of S7
According to the aforementioned "resin swelling step", after swelling the resin, according to the "condensation step", the amino acids selected are Fmoc-L-Gly-OH, fmoc-L-Cys (Trt) -OH, fmoc-L-Leu-OH, boc-L-Trp-OH, fmoc-L-Trp (Boc) -OH, fmoc-L-Dab (Boc) -OH, fmoc-L- [ O-tBu ], respectively]Hyp-OH，Fmoc-L-Dap(Boc)-OH，Fmoc-L-Pro-OH，Fmoc-L-Asn(Trt)-OH，Fmoc-L-[N-Me]Ala-OH，Fmoc-L-Val-OH，Fmoc-L-[O-tBu]Tyr-OH. After completion of the condensation, a crude linear peptide precursor can be obtained according to the aforementioned "peptide liberation step", and a crude cyclic peptide can be obtained according to the aforementioned "linear peptide cyclization step". The mixture was dissolved in acetonitrile: water =1 (volume ratio), and the cyclic peptide compound can be purified according to the aforementioned "RP-HPLC separation procedure". t is t R ＝15.022min。HRESIMS:[M+H] + ＝1996.9712。
Example 8
Preparation of S8
According to the aforementioned "resin swelling step", after swelling the resin, according to the "condensation step", the amino acids selected were Fmoc-L-Gly-OH, fmoc-L-Cys (Trt) -OH, fmoc-L-Leu-OH, boc-L-Trp-OH, fmoc-L-Trp (Boc) -OH, fmoc-L-Dab (Boc) -OH, fmoc-L- [ O-tBu [ ], respectively]Hyp-OH，Fmoc-L-Dap(Boc)-OH，Fmoc-L-Pro-OH，Fmoc-L-Asn(Trt)-OH，Fmoc-L-[N-Me]Ala-OH，Fmoc-L-Leu-OH，Fmoc-L-[O-tBu]Tyr-OH. After completion of the condensation, a crude linear peptide precursor can be obtained according to the aforementioned "peptide liberation step", and a crude cyclic peptide can be obtained according to the aforementioned "linear peptide cyclization step". The mixture was dissolved in acetonitrile: water =1 (volume ratio), and the cyclic peptide compound can be purified according to the aforementioned "RP-HPLC separation procedure". t is t R ＝15.022min。HRESIMS:[M+H] + ＝1996.9712。
Example 9
Preparation of S9
According to the aforementioned "resin swelling step", after swelling the resin, according to the "condensation step", the amino acids selected were Fmoc-L-Gly-OH, fmoc-L-Cys (Trt) -OH, fmoc-L-Leu-OH, boc-L-Trp-OH, fmoc-L-Trp (Boc) -OH, fmoc-L-Dab (Boc) -OH, fmoc-L- [ O-tBu [ ], respectively]Hyp-OH，Fmoc-L-Dap(Boc)-OH，Fmoc-L-Pro-OH，Fmoc-L-Asn(Trt)-OH，Fmoc-L-[N-Me]Ala-OH，Fmoc-Oic-OH，Fmoc-L-[O-tBu]Tyr-OH. After completion of the condensation, a crude linear peptide precursor can be obtained according to the aforementioned "peptide liberation step", and a crude cyclic peptide can be obtained according to the aforementioned "linear peptide cyclization step". The mixture was dissolved in acetonitrile: water =1 (volume ratio), and the cyclic peptide compound can be purified according to the aforementioned "RP-HPLC separation procedure". t is t R ＝14.922min。HRESIMS:[M+H] + ＝1941.0002。
Example 10
Preparation of S10
According to the aforementioned "resin swelling step", after swelling the resin, according to the "condensation step", the amino acids selected were Fmoc-L-Gly-OH, fmoc-L-Cys (Trt) -OH, fmoc-L-Leu-OH, boc-L-Trp-OH, fmoc-L-Trp (Boc) -OH, fmoc-L-Dab (Boc) -OH, fmoc-L- [ O-tBu [ ], respectively]Hyp-OH，Fmoc-L-Dap(Boc)-OH，Fmoc-L-Pro-OH，Fmoc-L-Asn(Trt)-OH，Fmoc-L-[N-Me]Ala-OH，Fmoc-L-[O-tBu]Tyr-OH and Compound 4 of example 2. After completion of the condensation, a crude linear peptide precursor can be obtained according to the aforementioned "peptide liberation step", and a crude cyclic peptide can be obtained according to the aforementioned "linear peptide cyclization step". The mixture was dissolved in acetonitrile: water =1 (volume ratio), and the cyclic peptide compound can be purified according to the aforementioned "RP-HPLC separation procedure". t is t R ＝14.910min。HRESIMS:[M+H] + ＝1926.9855。
Example 11
Preparation of S11
According to the aforementioned "resin swelling step", after swelling the resin, according to the "condensation step", the amino acids selected were Fmoc-L-Gly-OH, fmoc-L-Cys (Trt) -OH, fmoc-L-Leu-OH, boc-L-Trp-OH, fmoc-L-Trp (Boc) -OH, fmoc-L-Dab (Boc) -OH, fmoc-L- [ O-tBu [ ], respectively]Hyp-OH，Fmoc-L-Dap(Boc)-OH，Fmoc-L-Pro-OH，Fmoc-L-Asn(Trt)-OH，Fmoc-L-[N-Me]Ala-OH，Fmoc-L-[O-tBu]Tyr-OH and Compound 4 of example 2. After completion of the condensation, a crude linear peptide precursor can be obtained according to the aforementioned "peptide liberation step", and a crude cyclic peptide can be obtained according to the aforementioned "linear peptide cyclization step". The mixture was dissolved in acetonitrile: water =1 (volume ratio), and the cyclic peptide compound can be purified according to the aforementioned "RP-HPLC separation procedure". t is t R ＝15.202min。HRESIMS:[M+H] + ＝1904.2346。
Example 12
Preparation of S12
According to the aforementioned "resin swelling step", after swelling the resin, according to the "condensation step", the amino acids selected are Fmoc-L-Gly-OH, fmoc-L-Cys (Trt) -OH, fmoc-L-Leu-OH, boc-L-Trp-OH, fmoc-L-Trp (Boc) -OH, fmoc-L-Dab (Boc) -OH, fmoc-L- [ O-tBu ], respectively]Hyp-OH，Fmoc-L-Dap(Boc)-OH，Fmoc-L-Pro-OH，Fmoc-L-Asn(Trt)-OH，Fmoc-L-[N-Me]Ala-OH，Fmoc-L-[O-tBu]Tyr-OH and S-Fmoc-piperidine-2-carboxylic acid. After completion of the condensation, a crude linear peptide precursor can be obtained according to the aforementioned "peptide liberation step", and a crude cyclic peptide can be obtained according to the aforementioned "linear peptide cyclization step". The mixture was dissolved in acetonitrile: water =1 (volume ratio), and the cyclic peptide compound can be purified according to the aforementioned "RP-HPLC separation procedure". t is t R ＝14.961min。HRESIMS:[M+H] + ＝1900.9766。
Example 13
Preparation of S13
After swelling the resin according to the "resin swelling step" described above, amino acids were selected according to the "condensation step" as Fmoc-L-Gly-OH, fmoc-L-Cys (Trt) -OH, fmoc-L-Leu-OH, boc-L-Trp-OH, fmoc-L-Trp (Boc) -OH, fmoc-L-Dab (Boc)-OH，Fmoc-L-[O-tBu]Hyp-OH，Fmoc-L-Dap(Boc)-OH，Fmoc-L-Pro-OH，Fmoc-L-Asn(Trt)-OH，Fmoc-L-[N-Me]Ala-OH，Fmoc-L-[O-tBu]Tyr-OH. After completion of the condensation, a crude linear peptide precursor can be obtained according to the aforementioned "peptide liberation step", and a crude cyclic peptide can be obtained according to the aforementioned "linear peptide cyclization step". The mixture was dissolved in acetonitrile: water =1 (volume ratio), and the cyclic peptide compound can be obtained in pure form according to the aforementioned "RP-HPLC separation procedure". t is t R ＝14.929min。HRESIMS:[M+H] + ＝1898.9523。
Example 14
Preparation of S14
According to the aforementioned "resin swelling step", after swelling the resin, according to the "condensation step", the amino acids selected are Fmoc-L-Gly-OH, fmoc-L-Cys (Trt) -OH, fmoc-L-Leu-OH, boc-L-Trp-OH, fmoc-L-Trp (Boc) -OH, fmoc-L-Dab (Boc) -OH, fmoc-L- [ O-tBu ], respectively]Hyp-OH，Fmoc-L-Dap(Boc)-OH，Fmoc-L-Pro-OH，Fmoc-L-Asn(Trt)-OH，Fmoc-L-[N-Me]Ala-OH，Fmoc-L-[O-tBu]Tyr-OH. After completion of the condensation, a crude linear peptide precursor can be obtained according to the aforementioned "peptide liberation step", and a crude cyclic peptide can be obtained according to the aforementioned "linear peptide cyclization step". The mixture was dissolved in acetonitrile: water =1 (volume ratio), and the cyclic peptide compound can be purified according to the aforementioned "RP-HPLC separation procedure". t is t R ＝15.000min。HRESIMS:[M+H] + ＝1953.0001。
Example 15
Preparation of S15
According to the aforementioned "resin swelling step", after swelling the resin, according to the "condensation step", the amino acids selected were Fmoc-L-Gly-OH, fmoc-L-Cys (Trt) -OH, fmoc-L-Leu-OH, boc-L-Trp-OH, fmoc-L-Trp (Boc) -OH, fmoc-L-Dab (Boc) -OH, fmoc-L- [ O-tBu [ ], respectively]Hyp-OH，Fmoc-L-Dap(Boc)-OH，Fmoc-L-Pro-OH，Fmoc-L-Asn(Trt)-OH，Fmoc-L-[N-Me]Ala-OH，Fmoc-L-[O-tBu]Tyr-OH. After completion of the condensation, a crude linear peptide precursor can be obtained according to the aforementioned "peptide liberation step", and a crude cyclic peptide can be obtained according to the aforementioned "linear peptide cyclization step". The mixture was dissolved in acetonitrile: water =1 (volume ratio), and the cyclic peptide compound can be obtained in pure form according to the aforementioned "RP-HPLC separation procedure". t is t R ＝14.992min。HRESIMS:[M+H] + ＝1953.0001。
Example 16
Preparation of S16
According to the aforementioned "resin swelling step", after swelling the resin, according to the "condensation step", the amino acids selected are Fmoc-L-Gly-OH, fmoc-L-Cys (Trt) -OH, fmoc-L-Leu-OH, boc-L-Trp-OH, fmoc-L-Trp (Boc) -OH, fmoc-L-Dab (Boc) -OH, fmoc-L- [ O-tBu ], respectively]Hyp-OH，Fmoc-L-Dap(Boc)-OH，Fmoc-L-Pro-OH，Fmoc-L-Asn(Trt)-OH，Fmoc-L-[N-Me]Ala-OH，Fmoc-L-[O-tBu]Tyr-OH. After completion of the condensation, a crude linear peptide precursor can be obtained according to the aforementioned "peptide liberation step", and a crude cyclic peptide can be obtained according to the aforementioned "linear peptide cyclization step". The mixture was dissolved in acetonitrile: water =1 (volume ratio), and the cyclic peptide compound can be purified according to the aforementioned "RP-HPLC separation procedure". t is t R ＝15.101min。HRESIMS:[M+H] + ＝1914.9496。
Example 17
Preparation of S17
According to the aforementioned "resin swelling step", after swelling the resin, according to the "condensation step", the amino acids selected were Fmoc-L-Gly-OH, fmoc-L-Cys (Trt) -OH, fmoc-L-Leu-OH, boc-L-Trp-OH, fmoc-L-Trp (Boc) -OH, fmoc-L-Dab (Boc) -OH, fmoc-L- [ O-tBu [ ], respectively]Hyp-OH，Fmoc-L-Dap(Boc)-OH，Fmoc-L-Pro-OH，Fmoc-L-Asn(Trt)-OH，Fmoc-L-[N-Me]Ala-OH，Fmoc-L-[O-tBu]Tyr-OH. After completion of the condensation, a crude linear peptide precursor can be obtained according to the aforementioned "peptide liberation step", and a crude cyclic peptide can be obtained according to the aforementioned "linear peptide cyclization step". The mixture was dissolved in acetonitrile: water =1 (volume ratio), and the cyclic peptide compound can be purified according to the aforementioned "RP-HPLC separation procedure". t is t R ＝15.101min。HRESIMS:[M+H] + ＝1914.9487。
Example 18
Preparation of S18
According to the aforementioned "resin swelling step", after swelling the resin, according to the "condensation step", the amino acids selected are Fmoc-L-Gly-OH, fmoc-L-Cys (Trt) -OH, fmoc-L-Leu-OH, boc-L-Trp-OH, fmoc-L-Trp (Boc) -OH, fmoc-L-Dab (Boc) -OH, fmoc-L- [ O-tBu ], respectively]Hyp-OH，Fmoc-L-Dap(Boc)-OH，Fmoc-L-Pro-OH，Fmoc-L-Asn(Trt)-OH，Fmoc-L-[N-Me]Ala-OH，Fmoc-L-[O-tBu]Tyr-OH. After completion of the condensation, a crude linear peptide precursor can be obtained according to the aforementioned "peptide liberation step", and a crude cyclic peptide can be obtained according to the aforementioned "linear peptide cyclization step". The mixture was dissolved in acetonitrile: water =1 (volume ratio), and the cyclic peptide compound can be purified according to the aforementioned "RP-HPLC separation procedure". t is t R ＝14.892min。HRESIMS:[M+H] + ＝1912.9755。
Example 19
Preparation of S19
According to the aforementioned "resin swelling step", after swelling the resin, according to the "condensation step", the amino acids selected were Fmoc-L-Gly-OH, fmoc-L-Cys (Trt) -OH, fmoc-L-Leu-OH, boc-L-Trp-OH, fmoc-L-Trp (Boc) -OH, fmoc-L-Dab (Boc) -OH, fmoc-L- [ O-tBu [ ], respectively]Hyp-OH，Fmoc-L-Dap(Boc)-OH，Fmoc-L-Pro-OH，Fmoc-L-Asn(Trt)-OH，Fmoc-L-[N-Me]Ala-OH，Fmoc-L-[O-tBu]Tyr-OH. After completion of the condensation, a crude linear peptide precursor can be obtained according to the aforementioned "peptide liberation step", and a crude cyclic peptide can be obtained according to the aforementioned "linear peptide cyclization step". The mixture was dissolved in acetonitrile: water =1 (volume ratio), and the cyclic peptide compound can be purified according to the aforementioned "RP-HPLC separation procedure". t is t R ＝14.999min。HRESIMS:[M+H] + ＝1812.9623。
Example 20
Preparation of S20
According to the aforementioned "resin swelling step", after swelling the resin, according to the "condensation step", the amino acids selected were Fmoc-L-Gly-OH, fmoc-L-Cys (Trt) -OH, fmoc-L-Leu-OH, boc-L-Trp-OH, fmoc-L-Trp (Boc) -OH, fmoc-L-Dab (Boc) -OH, fmoc-L- [ O-tBu [ ], respectively]Hyp-OH，Fmoc-L-Dap(Boc)-OH，Fmoc-L-Pro-OH，Fmoc-L-Asn(Trt)-OH，Fmoc-L-[N-Me]Ala-OH，Fmoc-L-[O-tBu]Tyr-OH. After completion of the condensation, a crude linear peptide precursor can be obtained according to the aforementioned "peptide liberation step", and a crude cyclic peptide can be obtained according to the aforementioned "linear peptide cyclization step". The mixture was dissolved in acetonitrile: water =1 (volume ratio), and the cyclic peptide compound can be purified according to the aforementioned "RP-HPLC separation procedure". t is t R ＝15.102min。HRESIMS:[M+H] + ＝1897.9724。
Example 21
Preparation of S21
According to the aforementioned "resin swelling step", after swelling the resin, according to the "condensation step", the amino acids selected were Fmoc-L-Gly-OH, fmoc-L-Cys (Trt) -OH, fmoc-L-Leu-OH, boc-L-Trp-OH, fmoc-L-Trp (Boc) -OH, fmoc-L-Dab (Boc) -OH, fmoc-L- [ O-tBu [ ], respectively]Hyp-OH，Fmoc-L-Dap(Boc)-OH，Fmoc-L-Pro-OH，Fmoc-L-Asn(Trt)-OH，Fmoc-L-[N-Me]Ala-OH，Fmoc-L-[O-tBu]Tyr-OH. After completion of the condensation, a crude linear peptide precursor can be obtained according to the aforementioned "peptide liberation step", and a crude cyclic peptide can be obtained according to the aforementioned "linear peptide cyclization step". The mixture was dissolved in acetonitrile: water =1 (volume ratio), and the cyclic peptide compound can be purified according to the aforementioned "RP-HPLC separation procedure". t is t R ＝14.986min。HRESIMS:[M+H] + ＝1911.9880。
Example 22
Preparation of S22
According to the aforementioned "resin swelling step", after swelling the resin, according to the "condensation step", the amino acids selected were Fmoc-L-Gly-OH, fmoc-L-Cys (Trt) -OH, fmoc-L-Leu-OH, boc-L-Trp-OH, fmoc-L-Trp (Boc) -OH, fmoc-L-Dab (Boc) -OH, fmoc-L- [ O-tBu [ ], respectively]Hyp-OH，Fmoc-L-Dap(Boc)-OH，Fmoc-L-Pro-OH，Fmoc-L-Asn(Trt)-OH，Fmoc-L-[N-Me]Ala-OH，Fmoc-L-[O-tBu]Tyr-OH. After completion of the condensation, a crude linear peptide precursor can be obtained according to the aforementioned "peptide liberation step", and a crude cyclic peptide can be obtained according to the aforementioned "linear peptide cyclization step". The mixture was dissolved in acetonitrile: water =1 (volume ratio), and the cyclic peptide compound can be obtained in pure form according to the aforementioned "RP-HPLC separation procedure". t is t R ＝15.001min。HRESIMS:[M+H] + ＝1898.9466。
Example 23
Preparation of S23
According to the aforementioned "resin swelling step", after swelling the resin, according to the "condensation step", the amino acids selected are Fmoc-L-Gly-OH, fmoc-L-Cys (Trt) -OH, fmoc-L-Leu-OH, boc-L-Trp-OH, fmoc-L-Trp (Boc) -OH, fmoc-L-Dab (Boc) -OH, fmoc-L- [ O-tBu ], respectively]Hyp-OH，Fmoc-L-Dap(Boc)-OH，Fmoc-L-Pro-OH，Fmoc-L-Asn(Trt)-OH，Fmoc-L-[N-Me]Ala-OH，Fmoc-L-[O-tBu]Tyr-OH. After completion of the condensation, a crude linear peptide precursor can be obtained according to the aforementioned "peptide liberation step", and a crude cyclic peptide can be obtained according to the aforementioned "linear peptide cyclization step". The mixture was dissolved in acetonitrile: water =1 (volume ratio), and the cyclic peptide compound can be obtained in pure form according to the aforementioned "RP-HPLC separation procedure". t is t R ＝14.886min。HRESIMS:[M+H] + ＝1912.9733。
Example 24
Preparation of S24
According to the aforementioned "resin swelling step", after swelling the resin, according to the "condensation step", the amino acids selected were Fmoc-L-Gly-OH, fmoc-L-Cys (Trt) -OH, fmoc-L-Leu-OH, boc-L-Trp-OH, fmoc-L-Trp (Boc) -OH, fmoc-L-Dab (Boc) -OH, fmoc-L- [ O-tBu [ ], respectively]Hyp-OH，Fmoc-L-Dap(Boc)-OH，Fmoc-L-Pro-OH，Fmoc-L-Asn(Trt)-OH，Fmoc-L-[N-Me]Ala-OH，Fmoc-L-[O-tBu]Tyr-OH. After completion of the condensation, a crude linear peptide precursor can be obtained according to the aforementioned "peptide liberation step", and a crude cyclic peptide can be obtained according to the aforementioned "linear peptide cyclization step". The mixture was dissolved in acetonitrile: water =1 (volume ratio), and the cyclic peptide compound can be obtained in pure form according to the aforementioned "RP-HPLC separation procedure". t is t R ＝15.102min。HRESIMS:[M+H] + ＝1953.0002。
Example 25
Preparation of S25
According to the aforementioned "resin swelling step", after swelling the resin, according to the "condensation step", the amino acids selected were Fmoc-L-Gly-OH, fmoc-L-Cys (Trt) -OH, fmoc-L-Leu-OH, boc-L-Trp-OH, fmoc-L-Trp (Boc) -OH, fmoc-L-Dab (Boc) -OH, fmoc-L- [ O-tBu [ ], respectively]Hyp-OH，Fmoc-L-Dap(Boc)-OH，Fmoc-L-Pro-OH，Fmoc-L-Asn(Trt)-OH，Fmoc-L-[N-Me]Ala-OH，Fmoc-L-[O-tBu]Tyr-OH. After completion of the condensation, a crude linear peptide precursor can be obtained according to the aforementioned "peptide liberation step", and a crude cyclic peptide can be obtained according to the aforementioned "linear peptide cyclization step". The mixture was dissolved in acetonitrile: water =1 (volume ratio), and the cyclic peptide compound can be obtained in pure form according to the aforementioned "RP-HPLC separation procedure". t is t R ＝15.220min。HRESIMS:[M+H] + ＝1958.9986。
Example 26
Preparation of S26
According to the aforementioned "resin swelling step", after swelling the resin, according to the "condensation step", the amino acids selected are Fmoc-L-Gly-OH, fmoc-L-Cys (Trt) -OH, fmoc-L-Leu-OH, boc-L-Trp-OH, fmoc-L-Trp (Boc) -OH, fmoc-L-Dab (Boc) -OH, fmoc-L- [ O-tBu ], respectively]Hyp-OH，Fmoc-L-Dap(Boc)-OH，Fmoc-L-Pro-OH，Fmoc-L-Asn(Trt)-OH，Fmoc-L-[3-F]-Pro-OH，Fmoc-L-[O-tBu]Tyr-OH. After completion of the condensation, a crude linear peptide precursor can be obtained according to the aforementioned "peptide liberation step", and a crude cyclic peptide can be obtained according to the aforementioned "linear peptide cyclization step". The mixture was dissolved in acetonitrile: water =1 (volume ratio), and the cyclic peptide compound can be purified according to the aforementioned "RP-HPLC separation procedure". t is t R ＝14.993min。HRESIMS:[M+H] + ＝1915.9503。
Example 27
Preparation of S27
According to the aforementioned "resin swelling step", after swelling the resin, according to the "condensation step", the amino acids selected were Fmoc-L-Gly-OH, fmoc-L-Cys (Trt) -OH, fmoc-L-Leu-OH, boc-L-Trp-OH, fmoc-L-Trp (Boc) -OH, fmoc-L-Dab (Boc) -OH, fmoc-L- [ O-tBu [ ], respectively]Hyp-OH，Fmoc-L-Dap(Boc)-OH，Fmoc-L-Pro-OH，Fmoc-L-Asn(Trt)-OH，Fmoc-L-[3-F]-Pro-OH，Fmoc-L-[O-tBu]Tyr-OH. After completion of the condensation, a crude linear peptide precursor can be obtained according to the aforementioned "peptide liberation step", and a crude cyclic peptide can be obtained according to the aforementioned "linear peptide cyclization step". The mixture was dissolved in acetonitrile: water =1 (volume ratio), and the cyclic peptide compound can be purified according to the aforementioned "RP-HPLC separation procedure". t is t R ＝15.230min。HRESIMS:[M+H] + ＝1915.9798。
Example 28
Preparation of S28
According to the aforementioned "resin swelling step", after swelling the resin, according to the "condensation step", the amino acids selected were Fmoc-L-Gly-OH, fmoc-L-Cys (Trt) -OH, fmoc-L-Leu-OH, boc-L-Trp-OH, fmoc-L-Trp (Boc) -OH, fmoc-L-Dab (Boc) -OH, fmoc-L- [ O-tBu [ ], respectively]Hyp-OH，Fmoc-L-Dap(Boc)-OH，Fmoc-L-Pro-OH，Fmoc-L-Asn(Trt)-OH，Fmoc-L-[3-F]-Pro-OH，Fmoc-L-[O-tBu]Tyr-OH. Condensation ofAfter completion, a crude linear peptide precursor can be obtained according to the aforementioned "peptide liberation step", and a crude cyclic peptide can be obtained according to the aforementioned "linear peptide cyclization step". The mixture was dissolved in acetonitrile: water =1 (volume ratio), and the cyclic peptide compound can be purified according to the aforementioned "RP-HPLC separation procedure". t is t R ＝15.230min。HRESIMS:[M+H] + ＝1930.9497。
Example 29
Preparation of S29
According to the aforementioned "resin swelling step", after swelling the resin, according to the "condensation step", the amino acids selected were Fmoc-L-Gly-OH, fmoc-L-Cys (Trt) -OH, fmoc-L-Leu-OH, boc-L-Trp-OH, fmoc-L-Trp (Boc) -OH, fmoc-L-Dab (Boc) -OH, fmoc-L- [ O-tBu [ ], respectively]Hyp-OH，Fmoc-L-Dap(Boc)-OH，Fmoc-L-Pro-OH，Fmoc-L-Asn(Trt)-OH，Fmoc-L-[3-F]-Pro-OH，Fmoc-L-[O-tBu]Tyr-OH. After completion of the condensation, a crude linear peptide precursor can be obtained according to the aforementioned "peptide liberation step", and a crude cyclic peptide can be obtained according to the aforementioned "linear peptide cyclization step". The mixture was dissolved in acetonitrile: water =1 (volume ratio), and the cyclic peptide compound can be obtained in pure form according to the aforementioned "RP-HPLC separation procedure". t is t R ＝15.298min。HRESIMS:[M+H] + ＝1966.0170。
Example 30
The synthetic route for Fmoc-4-aza-Leu is shown below:
compound 5 (1.1g, 5 mmol) was dissolved in 70mL of methanol, and 2.3mL of a 37% aqueous solution of formaldehyde was added to replace the gas in the reaction flask with nitrogen. Then 0.22g of 10% Pd/C was added and the reaction flask was purged with hydrogen balloon. The reaction solution was stirred at room temperature for 24 hours. After the reaction was monitored by TLC (DCM: meOH =5, ninhydrin color) the reaction solution was filtered through celite, the filtrate was concentrated to near dryness, and the remaining solvent was removed by lyophilization to give crude compound 6, which was used in the next reaction without purification.
The crude compound 6 (about 5 mmol) was dissolved in 22mL of DCM, 5mL of triethylsilane was added, 22mL of trifluoroacetic acid was slowly added, and the reaction solution was reacted at room temperature for 1 hour. After the reaction is finished, concentrating the reaction solution to be dry, adding anhydrous ether, pouring supernatant liquid to obtain crude products of the oily compounds 7, and directly carrying out the next reaction without purification.
After compound 7 (about 5 mmol) was dissolved in 70mL of water and sodium carbonate (1.6 g, 15mmol) was added in portions, the reaction was stirred until no gas was produced, the reaction was transferred to an ice bath, fmoc-OSu (1.9 g,5.5 mmol) was dissolved in 35mL of dioxane and added dropwise to the reaction. The reaction was stirred at 0 ℃ for 1 hour and then at room temperature for 16 hours. After TLC monitoring the reaction was complete (DCM: meOH =5, ninhydrin color, uv color) ether was added. The organic layer was discarded, and the aqueous layer was adjusted to pH 3 with 2M HCl and extracted 3 times with ethyl acetate. The organic layers were combined, washed 2 times with water and 1 time with saturated sodium chloride. The organic layer was concentrated to dryness and separated by silica gel column chromatography (DCM: meOH = 8. 1 H NMR(300MHz,Methanol-d 4 )δ7.80(d,J＝7.4Hz,2H),7.67(t,J＝6.5Hz,2H),7.49–7.36(m,2H),7.31(m,2H),4.61–4.28(m,2H),4.22(t,J＝6.7Hz,1H),3.37(s,2H),3.61–3.15(m,1H),2.92(s,6H). 13 C NMR(125MHz,Methanol-d 4 )δ172.6,158.7,144.8,139.8,127.0,126.9,124.9,122.1,66.8,61.2,50.8,47.6,46.2。MS(ESI)＝354.2(M+H) + 。
Example 31
Preparation of S31
According to the aforementioned "resin swelling step", after swelling the resin, according to the "condensation step", the amino acids selected are Fmoc-L-Gly-OH, fmoc-L-Cys (Trt) -OH, fmoc-L-Leu-OH, boc-L-Trp-OH, fmoc-L-Trp (Boc) -OH, fmoc-L-Dab (Boc) -OH, fmoc-L- [ O-tBu ], respectively]Hyp-OH，Fmoc-L-Dap(Boc)-OH，Fmoc-L-HoPro-OH，Fmoc-L-Asn(Trt)-OH，Fmoc-L-[3-F]-Pro-OH，Fmoc-L-[O-tBu]Tyr-OH. After completion of the condensation, a crude linear peptide precursor can be obtained according to the aforementioned "peptide liberation step", and a cyclic condensation of the linear peptide according to the aforementioned "linear peptideThe step can obtain a crude cyclic peptide product. The mixture was dissolved in acetonitrile: water =1 (volume ratio), and the cyclic peptide compound can be purified according to the aforementioned "RP-HPLC separation procedure". t is t R ＝14.864min。HRESIMS:[M+H] + ＝1927.9645。
Example 32
Preparation of S32
According to the aforementioned "resin swelling step", after swelling the resin, according to the "condensation step", the amino acids selected were Fmoc-L-Gly-OH, fmoc-L-Cys (Trt) -OH, fmoc-L-Leu-OH, fmoc-L-4-aza-Leu-OH, boc-L-Trp-OH, fmoc-L-Trp (Boc) -OH, fmoc-L-Dab (Boc) -OH, fmoc-L- [ O-tBu]Hyp-OH，Fmoc-L-Dap(Boc)-OH，Fmoc-L-HoPro-OH，Fmoc-L-Asn(Trt)-OH，Fmoc-L-[3-F]-Pro-OH，Fmoc-L-[O-tBu]Tyr-OH. After completion of the condensation, a crude linear peptide precursor can be obtained according to the aforementioned "peptide liberation step", and a crude cyclic peptide can be obtained according to the aforementioned "linear peptide cyclization step". The mixture was dissolved in acetonitrile: water =1 (volume ratio), and the cyclic peptide compound can be obtained in pure form according to the aforementioned "RP-HPLC separation procedure". t is t R ＝13.992min。HRESIMS:[M+H] + ＝1928.9611。
Example 33
Preparation of S33
According to the aforementioned "resin swelling step", after swelling the resin, according to the "condensation step", the amino acids selected are Fmoc-L-Gly-OH, fmoc-L-Cys (Trt) -OH, fmoc-L-Leu-OH, fmoc-L-4-aza-Leu-OH, boc-L-Trp-OH, fmoc-L-Trp (Boc) -OH, fmoc-L-Dab (Boc) -OH, fmoc-L- [ O-tBu]Hyp-OH，Fmoc-L-Dap(Boc)-OH，Fmoc-L-HoPro-OH，Fmoc-L-Asn(Trt)-OH，Fmoc-L-[3-F]-Pro-OH，Fmoc-L-[O-tBu]Tyr-OH. After completion of the condensation, a crude linear peptide precursor can be obtained according to the aforementioned "peptide liberation step", and a crude cyclic peptide can be obtained according to the aforementioned "linear peptide cyclization step". The mixture was dissolved in acetonitrile: water =1 (volume ratio), and the cyclic peptide compound can be purified according to the aforementioned "RP-HPLC separation procedure". t is t R ＝13.992min。HRESIMS:[M+H] + ＝1928.9610。
Example 34
Preparation of S34
According to the aforementioned "resin swelling stepStep "after swelling the resin, according to the" condensation step ", the amino acids selected were Fmoc-L-Gly-OH, fmoc-L-Cys (Trt) -OH, fmoc-L-Leu-OH, fmoc-L-4-aza-Leu-OH, boc-L-Trp-OH, fmoc-L-Trp (Boc) -OH, fmoc-L-Dab (Boc) -OH, fmoc-L- [ O-tBu]Hyp-OH，Fmoc-L-Dap(Boc)-OH，Fmoc-L-HoPro-OH，Fmoc-L-Asn(Trt)-OH，Fmoc-L-[3-F]-Pro-OH，Fmoc-L-[O-tBu]Tyr-OH. After completion of the condensation, a crude linear peptide precursor can be obtained according to the aforementioned "peptide liberation step", and a crude cyclic peptide can be obtained according to the aforementioned "linear peptide cyclization step". The mixture was dissolved in acetonitrile: water =1 (volume ratio), and the cyclic peptide compound can be purified according to the aforementioned "RP-HPLC separation procedure". t is t R ＝13.501min。HRESIMS:[M+H] + ＝1929.9598。
Example 35
Evaluation of homogeneous time fluorometric assay example inhibition of the PD-1/PD-L1 interaction:
the interaction of PD-1 and PD-L1 can be determined using recombinant proteins of the ectodomain portions of the two proteins. The PD-1 and PD-L1 protein extracellular domains are expressed as fusion proteins with a detection tag, for PD-1 the Fc portion of an immunoglobulin (PD-1-Ig), for PD-L1 it is a 6 histidine motif (PD-L1-His). Human PD-1 (25-167), C-terminal human Fc domain with immunoglobulin G (Ig) epitope tag [ hPD-1 (25-167) -3S-Ig ], and human PD-L1 (18-239) with C-terminal His epitope tag [ hPD-L1 (18-239) -TVMV-His1] were expressed in HEK293T cells and purified sequentially by protein a affinity chromatography and size exclusion chromatography.
The interaction studies were all performed in HTRF assay buffer consisting of an additional addition of 0.1% (containing) bovine serum albumin and 0.05% (v/v) Tween-20 in dPBS. For the hPD-L1-His binding assay, the inhibitor was preincubated with PD-L1-His (final concentration 10 nM) in 4ul assay buffer for 15 min before the addition of PD-1-Ig (final concentration 20 nM). In 1uL assay buffer and further incubation for 15 min. HTRF detection was achieved using europium calcium phosphate-labeled anti-Ig (final concentration 1 nM) and isonicotina phthalocyanine (APC) -labeled anti-His (final 20 nM). Antibodies were diluted in HTRF detection buffer and 5ul taken. The reaction mixture was allowed to equilibrate for 30 minutes and the resulting signal was obtained using an EnVision fluorometer (665 nm/620 nm).
The results of the determination of the inhibition of the PD-1/PD-L1 interaction by the respective cyclic peptide compounds are shown in Table 1.
A：IC 50 <100nM；B：100nM<IC 50 <10μM；C：10μM<IC 50 <100μM
Table 1: results of homogeneous time fluorescence assay for inhibition of PD-1/PD-L1 interaction in examples
As shown in the above table, the cyclic peptide compounds in the examples all have a certain ability to inhibit the PD-1/PD-L1 interaction.
Example 36
Examples evaluation of the inhibition of the PD-1/PD-L1 interaction at the cellular level:
recombinant murine PD-L1 was used in this experiment as the source of PD-L1 in the cell experiments. Mouse splenocytes were obtained by triturating mouse spleen in a 40um cell strainer and further treating with 1mL ACK lysis buffer for 5 minutes at room temperature. After washing with 9mL lrpmi complete medium, cells were resuspended in 3mL of 1xPBS in a 15mL tube. Carefully add 3mL to the bottom of the tube without disturbing the overlaid splenocyte suspension. After centrifugation at 800 Xg for 20 minutes at room temperature, an opaque layer of splenocytes was collected. The resulting splenocytes were washed twice more with cold PBS solution and the total cell number was counted using trypan blue staining for later cell-level testing. Spleen cells were cultured in RPMI complete medium (RPMI +10% fetal bovine serum +1mM sodium pyruvate +10,000u/mL penicillin and 10,000ug/mL streptomycin) and maintained at 37 ℃ at 5% CO 2 CO of (2) 2 An incubator.
CFSE is a dye that passively diffuses into cells and binds to intracellular proteins. 1x10 6 cells/mL of splenocytes was treated with 5uM CFSE at 37 deg.C in pre-warmed 1 XPBS/0.1% BSA solutionThe treatment is carried out for 10 minutes. Excess CFSE quenched 5 volumes of 0 ℃ media into the cells and incubated on ice for 5 minutes. CFSE-labeled splenocytes were further washed three times with 0 ℃ complete RPMI medium. Marking CFSE 1 × 10 5 Splenocytes were added to wells containing either MDA-MB231 cells (1X 10 cells cultured in high glucose DMEM medium) or recombinant human PD-L1 (100 ng/mL) and test compound. Stimulation of splenocytes with anti-mouse CD3 and anti-mouse CD28 antibodies (1 ug/mL each), and incubation at 37 deg.C, 5% CO 2 The culture was further continued for 72 hours. Cells were harvested and washed three times with ice cold FACS buffer and analyzed for percent proliferation by flow cytometry with 488nm excitation and 521nm emission filters.
The percentage of splenocyte proliferation was analyzed using FACS program and the percentage of compound reversion to splenocyte proliferation was calculated after subtracting background proliferation value (%) and normalizing stimulated splenocyte proliferation (%, positive control) to 100%.
Stimulated splenocytes: splenocytes + anti-CD3/CD28
Stimulating background proliferation: splenocytes + anti-CD3/CD28+ PD-L1
Proliferation of the compound: splenocyte + anti-CD3/CD28+ PD-L1+ compound
Compound effects were tested by adding the required concentrations. The expression of anti-CD3/CD28 stimulated splenocytes by compounds in the presence of ligand (PD-L1).
The results of the assay for each cyclic peptide compound at a concentration of 100nM for inhibition of the PD-1/PD-L1 interaction are shown in Table 2.
A：>90％；B：70％<IC 50 <90％；C：50％<IC 50 <70％；D：<50％
Table 2: EXAMPLES results of cell-level inhibition of PD-1/PD-L1 interactions
| Compound (I) | IC 50 | Compound (I) | IC 50 | Compound (I) | IC 50 |
| S3 | A | S14 | A | S25 | A |
| S4 | A | S15 | A | S26 | A |
| S5 | A | S16 | A | S27 | A |
| S6 | A | S17 | A | S28 | A |
| S7 | A | S18 | A | S29 | A |
| S8 | A | S19 | A | S31 | A |
| S9 | A | S20 | A | S32 | A |
| S10 | A | S21 | A | S33 | A |
| S11 | A | S22 | A | S34 | A |
| S12 | A | S23 | A | ||
| S13 | A | S24 | A |
As shown in the above table, the cyclic peptide compounds in the examples all have a certain ability to inhibit the PD-1/PD-L1 interaction.
Example 37
Example experiments to restore IFN-. Gamma.secretion from human Peripheral Blood Mononuclear Cells (PBMC)
Human Peripheral Blood Mononuclear Cells (PBMC) can secrete cytokines such as IFN-gamma, IL-2 and TNF-alpha after being activated, and hPD-L1 (human PD-L1) protein is combined with PD-1 protein on human PBMC to inhibit the activation of the PBMC and reduce the secretion of the cytokines. In this experiment, the human breast cancer cell line MDA-MB-231 or the human melanoma line B16F10 was used as a human PD-L1 protein donor. The aim of this experiment was to test the ability of compounds to block hPD-L1 protein inhibition of PBMC activation. The specific operation is as follows: human PBMC were seeded in 96-well plates at 1X10 per well 5 And (4) cells. The experiment was divided into six groups, one group was blank control (PBMC alone), one group was activation group (PBMC + anti-CD3/anti-CD28 antibody), and two groups were inhibition group, one was PBMC + anti-CD3/anti-CD28 antibody + additional inoculation MDA-MB-231, and co-incubation, and the other was PBMC + anti-CD3/anti-CD28 antibody + additional inoculation B16F10, and co-incubation. The last two groups were the cyclic peptide compounds of the examples added at 1uM and 10nM, respectively, based on the inhibitory group. After 48 hours, the content of IFN-gamma in the cell supernatant was measured by using IFN-gamma ELISA kit from Biolegend. The results of the experiments show that PBMC in the activation group are subjected toafter the anti-CD3/anti-CD28 antibody acts, IFN-gamma secretion is obviously increased, which indicates that PBMC is in an activated state; the hPD-L1 protein in the inhibition group inhibits the PBMC from secreting IFN-gamma, which indicates that the activation state of the PBMC is inhibited; the compounds of examples 3-29 and 31-35 have a significant effect of restoring IFN- γ secretion from PBMC cells at 1uM and 10nM concentrations. The result shows that the compound has the effect of inhibiting PD-1/PD-L1 interaction in the presence of human breast cancer cells and human melanoma so as to enhance the immune activity of T cells.
Claims (9)
1. A cyclic peptide compound and pharmaceutically acceptable salts thereof, wherein the cyclic peptide compound is selected from the group consisting of:
2. a pharmaceutical composition comprising a cyclic peptide compound of claim 1 and pharmaceutically acceptable salts thereof.
3. The pharmaceutical composition of claim 2, wherein the cyclic peptide compound and the pharmaceutically acceptable salt thereof are formulated with one or more pharmaceutically acceptable excipients.
4. The pharmaceutical composition of claim 3, wherein the pharmaceutical excipient comprises a diluent, an excipient, a filler, a binder, a wetting agent, an absorption enhancer, a surfactant, a lubricant, or a stabilizer.
5. The pharmaceutical composition of claim 3, wherein the formulation is a tablet, capsule, granule, powder, syrup, oral liquid, or injection.
6. The cyclic peptide compounds and pharmaceutically acceptable salts thereof as claimed in claim 1, for use in the preparation of PD-1/PD-L1 pathway inhibitors.
7. Use of cyclic peptides and pharmaceutically acceptable salts thereof as claimed in claim 1 for the preparation of T cell immune activity enhancing agents.
8. Use of cyclic peptide compounds and pharmaceutically acceptable salts thereof according to claim 1 or pharmaceutical compositions according to claim 2 for the preparation of medicaments for the prevention or treatment of breast cancer and melanoma.
9. The use of claim 8, wherein the drug for preventing or treating breast cancer or melanoma is a breast cancer or melanoma immunotherapy drug or a breast cancer or melanoma chemotherapy drug or a breast cancer or melanoma targeted therapy drug.
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| CN106999536A (en) * | 2014-09-11 | 2017-08-01 | 百时美施贵宝公司 | The macrocyclic hcv inhibitors of PD 1/PD L1 and CD80 (B7 1)/PD L1 protein/protein interaction |
| CN108409830A (en) * | 2018-02-05 | 2018-08-17 | 郑州大学 | A kind of people PD-1 albumen extracellular fragments are affine cyclic peptide C8 and its application |
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