(S)-3-(methylamino) pyrrolidine cyclic peptide compound and application thereof

Abstract

The invention discloses a (S)-3-(methylamino) pyrrolidine cyclic peptide compound or a salt thereof and application of the (S)-3-(methylamino) pyrrolidine cyclic peptide compound or the salt thereof. The invention also discloses a pharmaceutical composition containing the (S)-3-(methylamino) pyrrolidine cyclic peptide compound. The invention further discloses a preparation method of the (S)-3-(methylamino) pyrrolidine cyclic peptide compound. Molecular and cellular level anti-tumor activity experiments show that the (S)-3-(methylamino) pyrrolidine cyclic peptide compound disclosed by the invention can play a role in resisting colon cancer metastasis by targeted inhibition of protein-protein interaction of APC / Asef protein in colon cancer cells. The (S)-3-(methylamino) pyrrolidine cyclic peptide compound disclosed by the invention is high in target specificity, strong in membrane permeability, stable and not easy to inactivate.

Description

Technical Field

[0001] The invention belongs to the technical field of medicinal chemistry and relates to a (S)-3-(methylamino)pyrrolidine cyclic peptide compound and application thereof. Background Art

[0002] Colon cancer originates from the pathological transformation of normal colon epithelial cells into adenomatous polyps, which then transform into invasive tumors. Early studies have found that mutations in the APC gene are not only closely related to familial adenomatous polyposis (FAP), but also play an important role in sporadic colon cancer. In more than 85% of congenital colon cancer patients and 80% of acquired colon cancer patients, the APC gene undergoes frameshift mutations or deletion mutations, expressing truncated APC proteins. The ARM domain at the N-terminus of the truncated APC protein is completely exposed and no longer performs normal physiological functions, but can effectively bind to the guanylate exchange factor Asef. Under normal physiological conditions, Asef is in an autoinhibitory state. After the truncated APC binds, the conformation of the Asef protein changes, the autoinhibition is released, and the activity of the guanine nucleotide exchange factor is activated. After APC activates Asef, Asef can bind to Cdc42, thereby forming an APC / Asef / Cdc42 ternary complex, activating the Rho family GTPase-Cdc42 to convert GTP into GDP, and upregulating MMP9 (matrixmetalloproteinase 9) through the JNK pathway, thereby causing abnormal cell flattening, cell membrane wrinkling, pseudopodia, reduced cell-to-cell adhesion, promoted cell migration and angiogenesis, and formed polyps, leading to the proliferation and invasion of colon cancer cells. Therefore, the truncated APC / Asef protein-protein interaction under pathological conditions (hereinafter referred to as APC / Asef interaction) plays an important role in the occurrence and development of colon cancer, and blocking the APC / Asef interaction is a new target in the treatment of colon cancer.

[0003] Based on this, people try to find specific active peptides that can block the APC / Asef interaction and use these peptides as cancer treatment drugs to solve the current situation of drug resistance and lack of effective treatment drugs. However, the problems of poor membrane permeability and stability of peptide drugs need to be solved urgently. Summary of the invention

[0004] In order to solve the above technical problems, the present invention provides a (S)-3-(methylamino)pyrrolidine cyclic peptide compound or a pharmaceutically acceptable salt thereof represented by the following formula (1), wherein two Cys form a disulfide bond:

[0005]

[0006] Where R1 is -COR A1 、-SO2R A1 or-CO-R A1 R A2 , and R A1 and R A2 R2 is isopropyl, cyclopropyl, 2-methylpropyl, 3-methylbutyl, cyclopentyl, cyclohexyl, cyclopentylmethyl or cyclohexylmethyl.

[0007] In detail, the (S)-3-(methylamino)pyrrolidine cyclic peptide compound of the present invention is derived from an APC / Asef protein interaction polypeptide inhibitor, and the (S)-3-(methylamino)pyrrolidine cyclic peptide compound of the present invention is obtained by cyclization.

[0008] In one embodiment, R1 is 4-methoxybenzoyl or 4-phenoxybenzoyl.

[0009] In one embodiment, R2 is 2-methylpropyl, 3-methylbutyl, cyclopentylmethyl or cyclohexylmethyl.

[0010] Preferably, the structure of the (S)-3-(methylamino)pyrrolidine cyclic peptide compound of the present invention is as shown in the following formula (II), formula (III), formula (IV), formula (V), formula (VI) or formula (VII):

[0011]

[0012] Any of the (S)-3-(methylamino)pyrrolidine cyclic peptide compounds described above can significantly inhibit the APC / Asef interaction, thereby inhibiting the migration and invasion of colon cancer cells, and playing an anti-colon cancer effect.

[0013] In one embodiment, the present invention provides a use of any one of the aforementioned (S)-3-(methylamino)pyrrolidine cyclic peptide compounds in the preparation of an inhibitor of APC-Asef protein interaction.

[0014] In one embodiment, the present invention provides a use of any one of the aforementioned (S)-3-(methylamino)pyrrolidine cyclic peptide compounds in the preparation of a drug for treating and preventing colon cancer.

[0015] In one embodiment, the present invention provides a pharmaceutical composition for treating and preventing colon cancer, comprising: the (S)-3-(methylamino)pyrrolidine cyclic peptide compound or its salt as described above, or a mixture containing the (S)-3-(methylamino)pyrrolidine cyclic peptide compound or its salt as described above; and a pharmaceutically acceptable carrier.

[0016] The pharmaceutical composition of the present invention can be prepared into various conventional dosage forms known in the art, including but not limited to: tablets suitable for oral administration (including various coated tablets, sustained-release or controlled-release tablets), lozenges, capsules (including soft capsules and hard capsules), granules, dispersible powders, aqueous or oily suspensions, emulsions, elixirs or syrups, etc.; creams, ointments, gels, aqueous or oily solutions or suspensions, etc. suitable for topical use; powders or liquid aerosols suitable for inhalation, sterile aqueous or oily intravenous, subcutaneous or intramuscular injections, suppositories, etc. suitable for parenteral administration.

[0017] Those skilled in the art know that in the pharmaceutical composition of the present invention, the appropriate amount of the anti-tumor cyclic peptide as an active ingredient and the pharmaceutically acceptable carrier can be determined according to conventional methods in the art. Those skilled in the art also know how to prepare a pharmaceutical composition containing the anti-tumor cyclic peptide of the present invention.

[0018] The (S)-3-(methylamino)pyrrolidine cyclic peptide compound of the present invention can be synthesized by a solid phase synthesis method known to those skilled in the art, and purified by a high performance liquid chromatography method known to those skilled in the art.

[0019] The advantages of the present invention are as follows: the present invention provides a (S)-3-(methylamino)pyrrolidine cyclic peptide compound, which inhibits the APC / Asef interaction in colon cancer cells to play an anti-colon cancer metastasis role. The (S)-3-(methylamino)pyrrolidine cyclic peptide compound of the present invention is derived from an APC / Asef protein interaction polypeptide inhibitor, and the inhibitor is obtained by cyclization to obtain the (S)-3-(methylamino)pyrrolidine cyclic peptide compound of the present invention, thereby improving the membrane penetration efficiency and in vitro plasma stability. The cyclic peptide stereo configuration of the (S)-3-(methylamino)pyrrolidine cyclic peptide compound of the present invention can fit with the APC protein pocket, so it can strongly block the APC / Asef interaction and inhibit the migration of colon cancer cells, thereby playing a therapeutic effect on colon cancer. The (S)-3-(methylamino)pyrrolidine cyclic peptide compound of the present invention has high target specificity, strong membrane permeability, and is stable and not easy to inactivate. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 In vitro binding affinity experiments of (S)-3-(methylamino)pyrrolidine cyclic peptide compounds II-VII;

[0021] Figure 2 The results of immunoblotting assay for the inhibitory effects of (S)-3-(methylamino)pyrrolidine cyclic peptide compounds III, IV, V, VI and VII on APC / Asef interaction are shown; and

[0022] Figure 3 The results of the Transwell assay for the inhibitory effects of (S)-3-(methylamino)pyrrolidine cyclic peptide compounds III, IV, V, VI and VII on cell migration and invasion are shown. Specific implementation plan

[0023] The present invention will be further described below in conjunction with specific examples. The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial sources. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0024] In one embodiment, the present invention provides a (S)-3-(methylamino)pyrrolidine cyclic peptide compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof:

[0025]

[0026] Where R1 is -COR A1 、-SO2R A1 or-CO-R A1 R A2 , and R A1 and R A2 R is independently hydrogen, an optionally substituted hydrocarbon group, an optionally substituted heterocyclic group or an optionally substituted phenyl group; R is isopropyl, cyclopropyl, 2-methylpropyl, 3-methylbutyl, cyclopentyl, cyclohexyl, cyclopentylmethyl or cyclohexylmethyl. In one embodiment, the optionally substituted hydrocarbon group includes alkyl, alkenyl, alkynyl and aryl and any combination thereof. In another embodiment, the optionally substituted hydrocarbon group is C1-C 18 In yet another embodiment, the optionally substituted hydrocarbyl group is C1-C 10 Hydrocarbon.

[0027] In this article, the term "optionally" means that the event or situation described later may or may not occur, and the description includes the case where the event or situation occurs, as well as the case where it does not occur. For example, the phrase "optionally substituted hydrocarbyl" means that the hydrocarbyl group may or may not be substituted, and the description includes unsubstituted hydrocarbyl and hydrocarbyl with substituents. For another example, the term "optionally substituted phenyl" refers to unsubstituted phenyl or to phenyl with 1, 2, 3, 4 or 5, especially 1, 2 or 3 substituents that are inert under the reaction conditions.

[0028] Substituents will not or will have little adverse effect on reaction rate or stereoselectivity.Optional substituents include halogen, cyano, nitro, amino, imino, sulfhydryl, acyl, alkyl, heterocyclic, alkoxy, hydroxyl, alkylthio, ester, carboxyl, amide and sulfonyl.

[0029] As used herein, the term "hydrocarbyl" refers to a group formed by removing one or more hydrogen atoms from a hydrocarbon carbon atom (when multiple hydrogen atoms are removed they need not be removed from the same carbon atom).

[0030] As used herein, the term "heterocyclic group" refers to a ring in which a carbon atom in the carbocyclic ring is replaced by at least one heteroatom independently selected from a N atom, an oxygen atom or a sulfur atom.

[0031] In one embodiment, R1 is 4-methoxybenzoyl or 4-phenoxybenzoyl.

[0032] In one embodiment, R2 is 2-methylpropyl, 3-methylbutyl, cyclopentylmethyl or cyclohexylmethyl.

[0033] In one embodiment, the (S)-3-(methylamino)pyrrolidine cyclic peptide compound of the present invention is represented by the following formula II, formula III, formula IV, formula V, formula VI or formula VII:

[0034]

[0035] In one embodiment, the present invention also provides a pharmaceutical composition for treating and preventing colon cancer, the pharmaceutical composition comprising: any one of the (S)-3-(methylamino)pyrrolidine cyclic peptide compounds or their salts as described above, or a mixture containing any one of the (S)-3-(methylamino)pyrrolidine cyclic peptide compounds or their salts as described above; and a pharmaceutically acceptable carrier.

[0036] The pharmaceutical composition of the present invention can be prepared into various conventional dosage forms known in the art, including but not limited to, tablets suitable for oral administration (including various coated tablets, sustained-release or controlled-release tablets), lozenges, capsules (including soft capsules and hard capsules), granules, dispersible powders, aqueous or oily suspensions, emulsions, elixirs or syrups, etc.; creams, ointments, gels, aqueous or oily solutions or suspensions, etc. suitable for topical use; powders or liquid aerosols suitable for inhalation, sterile aqueous or oily intravenous, subcutaneous or intramuscular injections, suppositories, etc. suitable for parenteral administration.

[0037] As used herein, the term "cancer" or "tumor" refers to an abnormal tissue mass, wherein the growth of the mass exceeds the growth of normal tissue, and is not coordinated with the growth of normal tissue. According to the following characteristics, cancer or tumor can be defined as benign or malignant: degree of cell differentiation, including morphology and function, growth rate, local invasion and metastasis. Benign tumors can be well differentiated, grow slower than malignant tumors, and are still confined to the primary site. In addition, in some cases, benign tumors do not have the ability to infiltrate, invade or transfer to distant sites. Malignant tumors can be poorly differentiated (anaplasia), with characteristic rapid growth, accompanied by the progressive infiltration, invasion and destruction of surrounding tissues. In addition, malignant tumors can have the ability to transfer to distant sites.

[0038] For any formulation used in the methods of the present invention, the therapeutically effective amount or dosage can be initially estimated from in vitro and cell culture assays. For example, a dosage can be formulated in an animal model to achieve a desired concentration or titer. This information can be used to more accurately determine a useful dosage for humans.

[0039] The dosage may vary depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage may be chosen by the individual physician according to the patient's condition.

[0040] Dosage and interval can be adjusted individually to provide levels of the active ingredient sufficient to induce or inhibit a biological effect (minimal effective concentration, MEC). The MEC varies for each preparation and can be estimated based on in vitro data. Dosages required to achieve the MEC depend on individual characteristics and route of administration. Detection assays can be used to determine plasma concentrations.

[0041] Depending on the severity and responsiveness of the condition to be treated, administration may be single or multiple administrations, with the course of treatment lasting from several days to several weeks, or until a cure is effected or a diminution of the disease state is achieved.

[0042] Reference is now made to the following examples, which together with the above descriptions illustrate some embodiments of the invention in a non limiting fashion.

[0043] Example 1: Solid phase synthesis, cleavage, cyclization, purification and identification of the (S)-3-(methylamino)pyrrolidine cyclic peptide compound (II) of the present invention:

[0044] 1.1 Solid phase synthesis of the (S)-3-(methylamino)pyrrolidine cyclic peptide compound (II) of the present invention:

[0045] The (S)-3-(methylamino)pyrrolidine cyclic peptide compound of the present invention is synthesized by solid phase synthesis using a peptide synthesizer. With DMF as solvent, the concentration of various amino acid solutions with α-amino groups protected by Fmoc is 0.25 mol / L, the concentration of HBTU solution and HOBt solution is 0.33 mol / L, the concentration of piperidine solution is 200 ml / L, and the concentration of DIEA solution is 174.2 ml / L.

[0046] 1.1.1 Activated resin:

[0047] Take 1g of Fmoc (fluorenylbenzyloxycarbonyl) protected butyramide resin and put it into the reactor. Add an appropriate amount of dichloromethane to swell the resin. Remove the excess dichloromethane in the reactor and put the dichloromethane swelled resin back into the reactor. Add 6.00mL of N,N-dimethylformamide solution containing 20% ​​piperidine and shake for 5 minutes. Remove the solvent. Add 6.00mL of N,N-dimethylformamide solution containing 20% ​​piperidine and shake for 15 minutes. Remove the solvent. Wash with methanol and dichloromethane in sequence. Remove the solvent again. Take 15 resins and test with ninhydrin. If the test result shows blue, stop the activation step. If it does not change color, repeat the above steps.

[0048] 1.1.2 Polypeptide condensation:

[0049] Weigh 2.1 g of (S)-3-(methylamino)pyrrolidine non-natural amino acid raw material 2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-((S)-3-(((R)-4-(allyloxy)-3-(((allyl oxy)carbonyl)amino)-4-oxobutyl)(methyl)amino)pyrrolidin-1-yl)butanoic acid and 0.14 g of 1-hydroxybenzotriazole (HOBT) into the above reaction kettle. Add 6.00 mL of N,N-dimethylformamide solution (containing 0.33 mL of 2,4,6-trimethylpyridine and 0.16 mL of N,N-diisopropylcarbodiimide) and shake for reaction. After the reaction is completed, wash and filter the reaction solution, wash with N, N-dimethylformamide, methanol and dichloromethane in turn, and take 15 resins for detection with ninhydrin. If it is blue, repeat the above process until the test result is colorless. Then add 6.00mL of N, N-dimethylformamide solution containing 20% ​​piperidine and shake for 5 minutes, then shake for another 15 minutes. After filtration, wash the resin with N, N-dimethylformamide, methanol and dichloromethane for 3 times respectively. After filtering and removing the filtrate, take 15 resins for detection with ninhydrin. If it is blue, end the above steps. If it is colorless, repeat the above Fmoc removal step. According to the above method, condense from C-terminus to N-terminus one by one. After the synthesis is completed, cap with p-methoxybenzoyl. Then wash the resin with N, N-dimethylformamide and dichloromethane respectively, and place it in a vacuum drying oven at 50°C for 48 hours for use.

[0050] 1.2 Cutting of redundant groups and resin:

[0051] The dried resin was cut at the joint with a scraper and placed in an eggplant-shaped bottle. 15 mL of resin cutting solution (containing 92.5% trifluoroacetic acid, .5% water, 2.5% p-cresol and 2.5% 1,2-ethanedithiol) was slowly dripped in an ice-water bath. The mixture was shaken at room temperature for 60 min. After the reaction was completed, the resin was filtered off and the collected filtrate was placed in an eggplant-shaped bottle. The mixture was precipitated with ice ether and centrifuged at 4°C and 7000×g for 15 min. The supernatant was discarded and the operation was repeated 3 times. Finally, 0.2 g of crude peptide was obtained by drying.

[0052] 1.3 Peptide purification:

[0053] The peptide was purified by reversed-phase high-performance liquid chromatography. The stationary phase was a C18 semi-preparative column, and the mobile phase consisted of an aqueous phase and an organic phase. The aqueous phase was deionized water (containing 0.1% trifluoroacetic acid by mass); the organic phase was an 80% acetonitrile aqueous solution (containing 0.1% trifluoroacetic acid by mass); the flow rate was 6.00 mL / min; the elution gradient was an aqueous phase (55% to 10%, 1% / min), an organic phase (45% to 90%, 1% / min). The eluate of the highest peak in the high-performance liquid chromatogram was collected, the acetonitrile in the eluate was removed, and the remaining aqueous solution was quickly frozen and the water was removed to obtain 35 mg of a white solid with a purity of 97.2%, ESI: 950.09 [MH] - .

[0054] Example 2: The (S)-3-(methylamino)pyrrolidine cyclic peptide compound of formula (III) of the present invention was prepared in the same manner as in Example 1. The solid purity was 95%, ESI: 976.13 [MH] - .

[0055] Example 3: (S)-3-(methylamino)pyrrolidine cyclic peptide compound (IV) was prepared in the same manner as in Example 1. The solid purity was greater than 95%, ESI: 964.12 [MH] - .

[0056] Example 4: (S)-3-(methylamino)pyrrolidine cyclic peptide compound (V) was prepared in the same manner as in Example 1. The solid purity was greater than 95%, ESI: 1012.16 [MH] - .

[0057] Example 5: (S)-3-(methylamino)pyrrolidine cyclic peptide compound (VI) was prepared in the same manner as in Example 1. The solid purity was greater than 95%, ESI: 1038.20 [MH] - .

[0058] Example 6: (S)-3-(methylamino)pyrrolidine cyclic peptide compound (VII) was prepared in the same manner as in Example 1. The solid purity was greater than 95%, ESI: 1026.19 [MH] - .

[0059] Embodiment 7:

[0060] The (S)-3-(methylamino)pyrrolidine cyclic peptide compounds II-VII of the present invention inhibit APC / Asef interaction at the molecular level.

[0061] An in vitro screening system for APC / Asef interaction inhibitors was established using fluorescence polarization (FP) assay to evaluate the ability of (S)-3-(methylamino)pyrrolidine cyclic peptide compounds to inhibit APC / Asef interaction at the molecular level.

[0062] (1) Expression and purification of APC (303-739) and Asef (170-271). Prokaryotic expression vectors pET28a-APC and pET28a-Asef were constructed, and APC and Asef were expressed in large quantities in the strain BL-21. Then, chromatographic methods such as affinity ion exchange and gel filtration were used to obtain purified proteins APC and Asef expressed by the recombinant plasmids;

[0063] (2) Preparation of mother solution: Weigh 1 mg of each of the above (S)-3-(methylamino)pyrrolidine cyclic peptide compounds and dissolve them in DMSO to an initial concentration of 100 mM. Prepare a fluorescent peptide mother solution with a concentration of 100 μM.

[0064] (3) Reaction conditions: APC (303-739) and fluorescent peptide (Tracer) were diluted to their respective concentrations in reaction buffer (50 mM Hepes 7.5, 300 mM NaCl, 1 mM EDTA, 1 mM DTT). The reaction was performed at room temperature in a 96-well plate. Three replicate wells were prepared for each experimental group.

[0065] (4) Gradient dilution: Add 8 μL of (S)-3-(methylamino)pyrrolidine cyclic peptide compound to each well of row A, and add 4 μL of DMSO to each well of rows B to H. Take 4 μL of (S)-3-(methylamino)pyrrolidine cyclic peptide compound from row A and add it to row B for dilution, then take out 4 μL and add it to row C, and so on until row G. After dilution of row G, take out 4 μL of (S)-3-(methylamino)pyrrolidine cyclic peptide compound and discard it.

[0066] (5) Add 91 μL of APC protein dilution and incubate at room temperature for 1.5 hours (h). Add 5 μL of fluorescent peptide dilution and continue incubating at room temperature for 1.5 h.

[0067] (6) Signal value detection: The fluorescence polarization value was detected using a multifunctional microplate reader (Synergy H4 Hybrid Reader). The excitation light was 485 nm, the emission light was 525 nm, the G factor was 1, and the sensitivity was set to 65.

[0068] (7) Data processing: (1) Calculate the average fluorescence value of the sample, including "protein + fluorescent peptide" as the positive control and "Tracer" as the negative control; (2) Then calculate according to the following formula: Inhibition (%) = 100*(positive control - sample) / (positive control - negative control). The log value of the final concentration of the (S)-3-(methylamino)pyrrolidine cyclic peptide compound (inhibitor) is used as the horizontal axis, and the inhibition rate (%) is used as the vertical axis. Graphpad Prism 7 software is used to fit the inhibition curve and calculate the IC 50 The results are shown in Table 1. The corresponding affinity curves are drawn. Figure 1 shown.

[0069] Table 1: IC of (S)-3-(methylamino)pyrrolidine cyclic peptide compounds II-VII for inhibiting APC / Asef interaction 50 (Unit: μM)

[0070]

[0071] It can be seen from Table 1 that among the (S)-3-(methylamino)pyrrolidine cyclic peptide compounds II-VII, the IC values ​​of (S)-3-(methylamino)pyrrolidine cyclic peptide compounds IV, V, VI and VII are 50 The inhibitory effects of (S)-3-(methylamino)pyrrolidine cyclic peptide compounds IV, V, VI and VII on APC / Asef interaction were better.

[0072] Embodiment 8:

[0073] The (S)-3-(methylamino)pyrrolidine cyclic peptide compound of the present invention is used for detecting the activity of inhibiting APC / Asef interaction at the cell level.

[0074] Flag-APC (303-876) and HA-Asef (170-632) plasmids were constructed and transiently transfected into 293T cells. (Group 1) was transfected with HA-Asef+vector; (Group 2) was transfected with HA-Asef and Flag-APC; (Group 3) was transfected with HA-Asef and Flag-APC. 48 hours after transfection, different concentrations of (S)-3-(methylamino)pyrrolidine cyclic peptide compounds were added and incubated for 6 hours. After cell lysis, Anti-Flag M2 Affinity Gel was added and incubated overnight. Western blot was then performed using the corresponding antibodies to detect changes in the amount of Asef pulled down by APC. Figure 2As shown, (S)-3-(methylamino)pyrrolidine cyclic peptide compounds IV, V, VI and VII can enter the cell membrane at 25 μM and significantly inhibit the intracellular APC / Asef interaction.

[0075] Embodiment 9:

[0076] The (S)-3-(methylamino)pyrrolidine cyclic peptide compound of the present invention inhibits the migration and invasion of colon cancer cells at the cell level.

[0077] SW480 cells were cultured in 10 cm culture dishes. The culture medium was replaced with serum-free DMEM medium before the experiment, and serum starvation was performed for 8 h. 100 μL of serum-free DMEM medium was added to the upper chamber of the Transwell chamber of the 24-well plate and placed in an incubator for pre-equilibration for 1 h. SW480 cells were digested with 0.05% trypsin / EDTA solution, and then the digestion was terminated with trypsin inhibitor DTI. The cells were then washed twice with serum-free DMEM medium, and finally the cells were resuspended in serum-free DMEM medium for cell counting. Cell suspensions containing different (S)-3-(methylamino)pyrrolidine cyclic peptide compounds were prepared using serum-free DMEM medium at a concentration of 5×10 4 Cell / well density Add 200 μL of cell suspension to the upper chamber of the pre-equilibrated Transwell chamber. Then, add 600 μL of complete medium (DMEM medium containing 10% FBS) containing the same (S)-3-(methylamino)pyrrolidine cyclic peptide compound to the lower chamber, let it stand for 10 minutes, and then place it in the incubator for 48 hours. Discard the culture medium in the upper and lower chambers, wash twice with ice PBS, then gently wipe with a cotton swab to remove the cells in the upper chamber, and then wash once with PBS. Add 600 μL of methanol to the lower chamber and let it stand for 10 minutes to fix and permeabilize the cells that migrated to the lower chamber. Discard the methanol, wait for the transwell membrane to dry, add 600 μL of 0.2% crystal violet solution to the lower chamber, and place it at 4°C overnight. Then, discard the crystal violet solution, rinse repeatedly with a large amount of distilled water, wipe the cells in the upper chamber further with a cotton swab, and place the chamber in an oven to dry. Finally, the chamber was placed on a glass slide and photographed with an inverted microscope. The entire field of view was photographed at 4 times, and five different fields of view (up, down, left, right, and center) were photographed at 10 times. The cells were counted at 10 times using ImageJ software, and the mean and standard deviation were calculated. One-way ANOVA was used for statistical analysis. Figure 3 As shown, (S)-3-(methylamino)pyrrolidine cyclic peptide compounds IV, V, VI and VII can significantly inhibit the migration of colon cancer cells SW480 at 25 μM, and pyrrolidine cyclic peptide compound VI is the most active in inhibiting cell migration.

[0078] In summary, the (S)-3-(methylamino)pyrrolidine cyclic peptide compounds of the present invention play an anti-colon cancer metastasis role by inhibiting the APC / Asef interaction in colon cancer cells. The cyclic peptide stereo configuration of the (S)-3-(methylamino)pyrrolidine cyclic peptide compounds of the present invention can fit with the APC protein pocket to strongly block the APC / Asef interaction and inhibit the migration of colon cancer cells, thereby achieving a therapeutic effect on colon cancer. The (S)-3-(methylamino)pyrrolidine cyclic peptide compounds of the present invention have high target specificity, strong membrane permeability, and are stable and not easily inactivated.

[0079] The above is only a preferred embodiment of the present invention. It should be pointed out that ordinary technicians in this technical field can make several improvements and modifications without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A (S)-3-(methylamino)pyrrolidine cyclic peptide compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof: Where R 1 For-COR A1 、-SO 2 R A1 or-CO-R A1 R A2 , and R A1 and R A2 are each independently hydrogen, an optionally substituted hydrocarbon group, an optionally substituted heterocyclic group or an optionally substituted phenyl group; R 2 is isopropyl, cyclopropyl, 2-methylpropyl, 3-methylbutyl, cyclopentyl, cyclohexyl, cyclopentylmethyl or cyclohexylmethyl.

2. The (S)-3-(methylamino)pyrrolidine cyclic peptide compound according to claim 1, Features: R 1 It is 4-methoxybenzoyl or 4-phenoxybenzoyl.

3. The (S)-3-(methylamino)pyrrolidine cyclic peptide compound according to claim 1, Features: R 2 It is 2-methylpropyl, 3-methylbutyl, cyclopentylmethyl or cyclohexylmethyl.

4. Use of the (S)-3-(methylamino)pyrrolidine cyclic peptide compound according to any one of claims 1 to 3 in the preparation of an inhibitor of APC-Asef protein interaction.

5. Use of the (S)-3-(methylamino)pyrrolidine cyclic peptide compound according to any one of claims 1 to 3 in the preparation of a medicament for treating and preventing colon cancer.

6. A pharmaceutical composition for treating and preventing colon cancer, It is characterized in that The pharmaceutical composition comprises: a (S)-3-(methylamino)pyrrolidine cyclic peptide compound or a salt thereof as described in any one of claims 1 to 3, or a mixture containing a (S)-3-(methylamino)pyrrolidine cyclic peptide compound or a salt thereof as described in any one of claims 1 to 3; and a pharmaceutically acceptable carrier.

7. The pharmaceutical composition according to claim 6, Features: The pharmaceutical composition is in the form of tablets, capsules, granules, patches, subcutaneous implants, injections, controlled-release preparations or sustained-release preparations.

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