Anti-tumor active compounds and pharmaceutical compositions based on cpf-st3 and uses

Abstract

The application relates to the technical field of medicines, in particular to an anti-tumor active compound based on CPF-ST3, a pharmaceutical composition and application. The active compound specifically refers to a cyclic peptide active molecule with a structure shown in formula (I); the cyclic peptide active molecule with the structure of formula (I) and a pharmaceutically acceptable salt or ester thereof: GX1X2GPX3X4KIX5X6KVGSNX7X8 (I). The cyclic peptide anti-tumor active compound can enhance cell permeability, improve enzyme stability and anti-tumor activity.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to antitumor active compounds and pharmaceutical compositions based on CPF-ST3 and their applications. Background Technology

[0002] Liver cancer is one of the most common malignant digestive tract tumors in my country, accounting for as much as 40% of all cases worldwide. The incidence rate is higher in men (13.79%) than in women (6.26%), and higher in rural areas (12.20%) than in urban areas (9.11%). Due to its insidious onset and high malignancy, 70%-80% of patients are diagnosed at an advanced stage, often with underlying conditions such as hepatitis and cirrhosis. Liver cancer has become the second leading cause of cancer death after lung cancer. Each treatment option for liver cancer has its advantages and disadvantages: partial hepatectomy is considered a potential radical treatment with a 5-year survival rate exceeding 70%, but this method is currently only suitable for early-stage liver cancer patients, and some patients still have the possibility of recurrence after surgery; local ablation therapy has achieved the same effect as surgery in treating small liver cancers ≤3 cm in diameter, but the treatment effect is not ideal in single liver cancers >5 cm in diameter; arterial chemoembolization is the best choice for treating liver cancer when surgery is not feasible, but repeated procedures may cause damage to the arterial intima and liver, interfere with normal treatment, and even cause lung metastasis; most chemotherapy drugs have problems with poor specificity and sensitivity in the treatment of liver cancer, and are rarely used clinically; the molecularly targeted drug sorafenib has significant significance in improving the survival of patients with advanced liver cancer, but its high cost limits its widespread clinical application. Although the above-mentioned treatment methods for liver cancer are constantly being improved and perfected, the 5-year survival rate of liver cancer patients remains between 30% and 40%, and the overall efficacy of liver cancer treatment has not yet significantly improved. Therefore, it is imperative to find efficient and inexpensive treatment technologies and drugs for liver cancer.

[0003] In recent years, host-defense peptides (HDPs) from amphibian skin secretions have become a new hot topic in anti-tumor research due to their unique biological activities. These peptides exhibit broad-spectrum antibacterial activity and selective cytotoxicity against various tumor cells, while showing low cytotoxicity against normal cells. CPF-ST3 (also known as XT-7) is a host-defense peptide isolated from the skin secretions of the Xenopus laevis (Silurana tropicalis). It has an amphiphilic α-helical structure and exhibits moderate cytotoxicity against the HepG2 liver cancer cell line (LC50 = 75 µM). Through systematic structure-activity relationship studies, the research team rationally designed this peptide and found that moderately increasing its cationicity can significantly enhance its anti-tumor activity. Among them, the [P5K, S15K, N16K] CPF-ST3 mutant showed 15-fold increased activity compared to the natural peptide (LC50 = 5 µM), while maintaining low hemolytic activity against normal erythrocytes, indicating a good therapeutic window. CPF-ST3 peptides primarily kill tumor cells rapidly through membrane action, circumventing multidrug resistance. Furthermore, their highly modifiable structure allows them to be combined with targeting peptides or nanocarriers to construct targeted-membrane bifunctional peptide systems, providing new strategies and drug candidates for precision treatment of liver cancer. However, linear peptides also suffer from conformational instability, poor membrane permeability, and weak resistance to hydrolytic enzymes, issues that urgently need to be addressed. Therefore, cyclic peptide antitumor active compounds, their preparation methods, and applications are proposed to solve these problems. Summary of the Invention

[0004] The purpose of this invention is to provide antitumor active compounds and pharmaceutical compositions based on CPF-ST3 and their applications, in order to solve the problems mentioned in the background art.

[0005] To achieve the above objectives, the present invention provides the following technical solution: Antitumor active compounds based on CPF-ST3 specifically refer to cyclic peptide active molecules having the structure shown in formula (I); Cyclic peptide active molecules having the structure of formula (I) and their pharmaceutically acceptable salts or esters: GX1X2GPX3X4KIX5X6KVGSNX7X8(I) Where X1 represents leucine or (2R)-2-amino-2-methyl-6-heptenoic acid; X2 represents leucine or (2R)-2-amino-2-methyl-6-heptenoic acid; X3 represents leucine or (2R)-2-amino-2-methyl-6-heptenoic acid; X4 represents leucine or (2R)-2-amino-2-methyl-6-heptenoic acid; and X5 represents alanine or (2R)-2-amino-2-methyl-6-heptenoic acid or (2R)-2-amino-2-methyl-9-decenoic acid. X6 represents alanine or (2R)-2-amino-2-methyl-6-heptenoic acid or (2R)-2-amino-2-methyl-9-decenoic acid; X7 represents leucine or (2R)-2-amino-2-methyl-6-heptenoic acid; X8 represents leucine or (2R)-2-amino-2-methyl-6-heptenoic acid; the paired (2R)-2-amino-2-methyl-6-heptenoic acid or (2R)-2-amino-2-methyl-9-decenoic acid in the fragment undergoes cyclization via olefin metathesis.

[0006] A pharmaceutical composition comprising a cyclic peptide active molecule having the structure of formula (I) above, wherein the pharmaceutical composition comprises any one of the cyclic peptide active molecules having the structure of formula (I) above.

[0007] In this document, "cyclic peptide active molecules of the present invention" refers to polypeptides having the structure shown in formula (I) in the present invention. In this document, such polypeptides may be referred to as "cyclic peptide active molecules", "peptide fragments" or "polypeptides of the present invention".

[0008] The N-terminal amino group and C-terminal carboxyl group of the polypeptide of formula (I) and the amino acid side chain group may not be modified, or they may be modified without substantially affecting the activity of the polypeptide of the present invention, such as forming a "pharmaceuticalally acceptable ester". The modification of the N-terminal amino group includes, but is not limited to, de-amino, N-lower alkyl, N-dilower alkyl and N-acyl modification. The modification of the C-terminal carboxyl group includes, but is not limited to, amide, lower alkyl amide, dialkyl amide and lower alkyl ester modification. The N-terminal amino group of the polypeptide of the present invention is acetylated, i.e., -Ac, and the C-terminal carboxyl group is amidated, i.e. -NH2.

[0009] The methods used in this article to represent polypeptides, amino acids, and chemical groups are all recognized in the relevant fields. The abbreviations for amino acids can be found in Table 1. Specific amino acid structures can be found in Table 2. Unless otherwise specified, amino acids in this article generally refer to L-type amino acids.

[0010] Table 1: Amino Acid Abbreviations

[0011] Table 2: List of Special Amino Acid Abbreviations

[0012] "Pharmaceutical acceptable salts" refer to salts formed by small molecule acidic or basic compounds and peptides. These salts generally increase the solubility of peptides, and the formed salts do not significantly alter the activity of the peptides.

[0013] For example, acids that can typically form salts with the polypeptides of the present invention include hydrochloric acid, phosphoric acid, sulfuric acid, acetic acid, succinic acid, maleic acid, and citric acid; bases that can form salts with the polypeptides of the present invention include hydroxides of alkali metals or alkaline earth metals, ammonium, and carbonates.

[0014] The antitumor effect of the polypeptides of the present invention can be verified by conventional experimental methods in the field, such as cell experiments. In the specific embodiments of the present invention, cell experiments such as the CCK-8 method are preferred. Through this experiment, it was found that the cyclic peptides of formula (I) involved in the present invention all have in vitro antitumor effects.

[0015] In addition, another technical problem to be solved by the present invention is to provide a pharmaceutical composition containing a polypeptide fragment of formula (I) that can be used for anti-tumor therapy.

[0016] The composition may contain one or more of the cyclic peptide active molecules of the present invention, preferably only one.

[0017] The composition may contain one or more pharmaceutically acceptable diluents, excipients or carriers, preferably in unit dose form, such as tablets, films, pills, capsules (including sustained-release or delayed-release forms), powders, granules, syrups or emulsions, sterilized solutions for injection, suspensions or lyophilized powder injections, aerosols or liquid sprays, automated dropper injection devices or suppositories.

[0018] The above-mentioned active pharmaceutical components can be combined with a non-toxic, pharmaceutically acceptable inert carrier, such as ethanol, glycerol, water, or a combination thereof. The cyclic peptide active molecules of formula (I) of the present invention are preferably prepared using sterilized aqueous solutions for injection.

[0019] The pharmaceutical compositions of the present invention can be administered via methods of administration well known to those skilled in the art, such as oral, rectal, sublingual, pulmonary, transdermal, iontophoresis, vaginal, and intranasal administration. The pharmaceutical compositions of the present invention are preferably administered parenterally, such as subcutaneously, intramuscularly, or intravenously.

[0020] The names, structural formulas, and mass spectrometry data of some of the preferred compounds synthesized in this invention are shown in Table 3: Table 3: List of preferred cyclic peptide active molecules, their structural formulas, and mass spectrometry data.

[0021] Compared with the prior art, the beneficial effects of the present invention are: In this invention, enhancing the α-helix configuration of peptides through chemical means to increase their membrane permeability and enzyme stability is an effective strategy to address the poor drug-likeness of linear peptides. Among these strategies, the stapling cyclization modification strategy has been reported most frequently. Cyclization is achieved through an olefin metathesis reaction of the pentenyl side chain of a specific amino acid, which can effectively improve the structural rigidity of the peptide and consolidate the α-helix configuration, thereby improving enzyme tolerance and cell permeability. Therefore, we designed and synthesized a series of novel CPF-ST3 cyclic peptide active molecules using the stapling cyclization modification strategy, aiming to enhance their cell permeability, improve enzyme stability and antitumor activity, making them more suitable for safer use in cancer patients. Attached Figure Description

[0022] Figure 1 This is a schematic diagram of the CPF-ST3-Sp1 structure in Table 3 of the present invention; Figure 2 This is a schematic diagram of the CPF-ST3-Sp2 structure in Table 3 of the present invention; Figure 3 This is a schematic diagram of the CPF-ST3-Sp3 structure in Table 3 of the present invention; Figure 4 This is a schematic diagram of the CPF-ST3-Sp4 structure in Table 3 of the present invention; Figure 5 This is a schematic diagram of the CPF-ST3-Sp5 structure in Table 3 of the present invention; Figure 6 This is a schematic diagram of the CPF-ST3-Sp6 structure in Table 3 of the present invention. Detailed Implementation

[0023] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0024] Please see Figures 1 to 6 The present invention provides: Example 1: Preparation method of cyclic peptide antitumor active compound, solid-phase synthesis of CPF-ST3-Sp1, the specific steps are as follows: The α-amino group of amino acids is protected with a 9-fluorenylmethoxycarbonyl (Fmoc) group, and the amino acids are also protected by side chains: the side chain protecting group of Ser is tert-butyl (tBu), and the side chain protecting group of Lys is tert-butyloxycarbonyl (Boc). The amino acids at positions 2 and 6 are replaced with Fmoc-S5-OH. 6-Chlorobenzotriazole-1,1,3,3-tetramethylurea hexafluorophosphate (HCTU) and N,N-diisopropylethylamine (DIPEA) are used as activating agents to sequentially couple the above-mentioned protected amino acids. Each coupling takes 40 minutes and is performed with 20% piperidine / DMF was used as the de-Fmoc reagent, with each reaction lasting 10 minutes. After peptide ligation was complete, phenylmethylene bis(triphenylhexylphosphine) ruthenium dichloride (first-generation Grubbs catalyst) was used as the cyclization reagent, and the reaction was carried out overnight. Then, TFA / EDT / TIPs / Water (95:2:2:1, v / v / v / v) was used to react at room temperature for 2 hours to cleave the peptide from the resin and remove the side chain protecting groups. The crude peptide was then precipitated with anhydrous diethyl ether and purified by reversed-phase HPLC within 30 minutes. The crude peptide was then lyophilized to obtain a white lyophilized powder with a purity ≥97.0%.

[0025] Example 2: Preparation method of cyclic peptide antitumor active compound, solid-phase synthesis of CPF-ST3-Sp6, the specific steps are as follows: The α-amino group of amino acids was protected with a 9-fluorenylmethoxycarbonyl (Fmoc) group, and the amino acids were also protected by side chains: the side chain protecting group of Ser was tert-butyl (tBu), and the side chain protecting group of Lys was tert-butyloxycarbonyl (Boc). The third amino acid was replaced by Fmoc-R8-OH, and the tenth amino acid was replaced by Fmoc-S5-OH. 6-Chlorobenzotriazole-1,1,3,3-tetramethylurea hexafluorophosphate (HCTU) and N,N-diisopropylethylamine (DIPEA) were used as activating agents to sequentially couple the above-mentioned protected amino acids, with each coupling lasting 40 minutes. 20% piperidine / DMF was used as the de-Fmoc reagent, with each reaction lasting 10 minutes. After peptide ligation was complete, phenylmethylene bis(triphenylhexylphosphine) ruthenium dichloride (first-generation Grubbs catalyst) was used as the cyclization reagent, and the reaction was carried out overnight. The peptide was then cleaved from the resin using TFA / EDT / TIPs / Water (95:2:2:1, v / v / v / v) at room temperature for 2 hours, while removing the side chain protecting groups. The crude peptide was then precipitated with anhydrous diethyl ether and purified by reversed-phase HPLC within 30 minutes. The crude peptide was then lyophilized to obtain a white lyophilized powder with a purity ≥97.0%.

[0026] Experimental example: 1) Cell biology experiments CCK-8 in vitro tumor suppression experiment: HepG2 liver cancer cells were treated with fetal bovine serum (10%) and penicillin (100 KU·L⁻¹). -1C42B cells were cultured in high-glucose D-MEM containing streptomycin (100 mg·L⁻¹) at 37°C in a 5% CO₂ incubator using conventional methods. Cells in the logarithmic growth phase were then cultured and passaged at 2 × 10⁻⁶ cells / cells. 4 mL -1 Cells were seeded at a density of 100 μL per well in 96-well plates, with three replicates per group. The peptides were administered at concentrations of 0.39, 0.78, 1.56, 3.125, 6.25, 12.5, 25, and 50 μM. After 96 h of cell culture, 100 μL of complete culture medium containing 10% CCK-8 reagent was added to each well. The cells were incubated at 37°C in a 5% CO2 incubator for 2 h in the dark. The absorbance (OD) of each well was measured at 450 nm using a microplate reader (BioTek, Vermont, USA). Cell viability (VR) was calculated based on the OD values: VR = (OD value of drug-treated group - OD value of blank group) / (OD value of control group - OD value of blank group). The average VR of the three parallel wells was calculated. Based on the drug VR, the half-maximal inhibitory concentration (IC50) was determined by linear regression of the logarithm of the drug concentration with VR.

[0027] Experimental results: The in vitro tumor inhibition experiment of CCK-8 showed that all peptide fragments exhibited good in vitro tumor cell inhibition effects, which were higher than those of the negative control CPF-ST3. The results are shown in Table 4.

[0028] 2) Enzyme stability experiment Chymotrypsin stability experiment: 1-2 mg of peptide was weighed and dissolved in a certain amount of DMSO to prepare a 1 nM stock solution. A certain amount of chymotrypsin was weighed and dissolved in 50 mM phosphate buffer solution containing 2 mM calcium chloride (pH=7.4) until the concentration of chymotrypsin was 0.5 ng / μl. 1950 μl of phosphate buffer solution containing chymotrypsin and 50 μl of peptide stock solution were added to 2 ml centrifuge tubes to carry out enzymatic degradation reaction. 50 μl of reaction solution at 0, 1, 2, 4, 8 and 12 hours was added to 50 μl of hydrochloric acid (1 M) to quench the activity of chymotrypsin. The residual amount of peptide at different time points was analyzed by HPLC.

[0029] Experimental results: The results of the chymotrypsin stability test showed that CPF-ST3-Sp3 and CPF-ST3-Sp6 exhibited stronger antichymotrypsin ability than the negative control CPF-ST3, as shown in Table 4.

[0030] Table 4: Degradation half-life of cyclic peptide bioactive molecules chymotrypsin (t) 1 / 2 ) and the half-maximal inhibitory concentration (IC50) of the anti-hepatocellular carcinoma cell line HepG2.50 )

[0031] The antitumor active compounds in this invention enhance the α-helix configuration of peptides through chemical means, thereby increasing their membrane permeability and enzyme stability. This is an effective strategy to address the poor drug-likeness of linear peptides. Among these strategies, stapling cyclization modification is the most frequently reported. Cyclization is achieved through an olefin metathesis reaction of the pentenyl side chain of a specific amino acid, which can effectively improve the structural rigidity of the peptide and consolidate the α-helix configuration, thereby improving enzyme tolerance and cell permeability. Therefore, we designed and synthesized a series of novel CPF-ST3 cyclic peptide active molecules using stapling cyclization modification strategy, aiming to enhance their cell permeability, improve enzyme stability and antitumor activity, making them more suitable for safe use in cancer patients. This solves the problems of conformational instability, poor membrane permeability and weak resistance to hydrolytic enzymes in existing anticancer drugs.

[0032] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A compound with antitumor activity based on CPF-ST3, characterized in that, Specifically, it refers to cyclic peptide active molecules with the structure shown in formula (I); Cyclic peptide active molecules having the structure of formula (I) and their pharmaceutically acceptable salts or esters: GX1X2GPX3X4KIX5X6KVGSNX7X8(I) Where X1 represents leucine or (2R)-2-amino-2-methyl-6-heptenoic acid; X2 represents leucine or (2R)-2-amino-2-methyl-6-heptenoic acid; X3 represents leucine or (2R)-2-amino-2-methyl-6-heptenoic acid; X4 represents leucine or (2R)-2-amino-2-methyl-6-heptenoic acid; and X5 represents alanine or (2R)-2-amino-2-methyl-6-heptenoic acid or (2R)-2-amino-2-methyl-9-decenoic acid. X6 represents alanine or (2R)-2-amino-2-methyl-6-heptenoic acid or (2R)-2-amino-2-methyl-9-decenoic acid; X7 represents leucine or (2R)-2-amino-2-methyl-6-heptenoic acid; X8 represents leucine or (2R)-2-amino-2-methyl-6-heptenoic acid; the paired (2R)-2-amino-2-methyl-6-heptenoic acid or (2R)-2-amino-2-methyl-9-decenoic acid in the fragment undergoes cyclization via olefin metathesis.

2. A pharmaceutical composition containing a cyclic peptide active molecule having the structure of formula (I) described above, characterized in that, The pharmaceutical composition contains the polypeptide as described in any one of claims 1.

3. The pharmaceutical composition of cyclic peptide active molecules according to claim 2, characterized in that, It also contains pharmaceutically acceptable diluents, excipients, or carriers.

4. The pharmaceutical composition of the cyclic peptide active molecule according to claim 3, characterized in that, The carrier is one or more of ethanol, glycerol, or water.

5. The application of antitumor active compounds based on CPF-ST3, characterized in that, Pharmaceutical compositions using cyclic peptide active molecules having the structure of formula (I) can be used to prepare antitumor drugs.

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