Temporin-1CEa-based antitumor active compounds, pharmaceutical compositions and applications

Abstract

This invention relates to the field of pharmaceutical technology, specifically to an antitumor active compound based on Temporin-1CEa, a pharmaceutical composition thereof, and its application. The compound specifically refers to a cyclic peptide active molecule having the structure shown in formula (I), specifically a cyclic peptide active molecule having the structure of formula (I) and its pharmaceutically acceptable salt or ester: FX1X2LKX3X4AX5X6X7NSIFX8X9 (I). The antitumor active compound based on Temporin-1CEa obtained in this invention, and pharmaceutical compositions containing it, can be used to prepare antitumor drugs, aiming to enhance their cell permeability, improve enzyme stability, and enhance antitumor activity.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to an antitumor active compound and pharmaceutical composition based on Temporin-1CEa, and its application. Background Technology

[0002] Breast cancer is the most common malignant tumor among women worldwide. Triple-negative breast cancer (TNBC) accounts for approximately 15-20% of all breast cancer subtypes. Due to the lack of expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2), it is insensitive to both endocrine therapy and targeted therapy, resulting in an extremely poor prognosis with a 5-year survival rate of less than 15%. Traditional chemotherapy drugs such as paclitaxel have some efficacy, but they are prone to multidrug resistance and have significant toxic side effects. Therefore, the development of novel, highly effective, and low-toxicity anti-tumor drugs is an urgent priority.

[0003] In recent years, host-defense peptides (HDPs) in the skin secretions of amphibians have attracted widespread attention due to their unique membrane-targeting mechanism and selective antitumor activity. Unlike conventional chemotherapeutic drugs that act through intracellular targets, these cationic amphiphilic peptides mainly kill tumor cells rapidly through membrane disruption, circumventing multidrug resistance mechanisms and exhibiting natural selectivity for rapidly proliferating cancer cells. Temporin-1CEa is a cationic antimicrobial peptide isolated and purified from the skin secretions of the Chinese forest frog (Ranachensinensis). It has a molecular weight of 1751.1 Da, consists of 16 amino acid residues (sequence: FVDLKKIANIINSIFGK-NH2), and possesses a typical amphiphilic α-helix structure. This peptide exhibited significant dose-dependent cytotoxicity against two human breast cancer cell lines—the estrogen receptor-positive MCF-7 cells and the estrogen receptor-negative, more invasive MDA-MB-231 cells—with IC50 values ​​of 31.91 µM (MCF-7) and 57.94 µM (MDA-MB-231) after 1 hour of treatment, respectively, demonstrating a biased killing effect on hormone receptor-positive breast cancer.

[0004] Chinese invention patent application number 202210132674.1 discloses an optimized method for Temporin-1CEa based on a full hydrocarbon stapling strategy. However, its synthesis uses the expensive non-natural amino acid Fmoc-S5-OH as a raw material and employs a transition metal catalyst for cyclization, resulting in the introduction of heavy metals and making large-scale production impossible.

[0005] Therefore, it is necessary to provide a novel antitumor active compound to address the shortcomings of existing technologies. Summary of the Invention

[0006] The purpose of this invention is to provide an antitumor active compound and pharmaceutical composition based on Temporin-1CEa, and its application, in order to solve the problems mentioned in the background art.

[0007] To achieve the above objectives, the present invention provides the following technical solution: Antitumor active compounds based on Temporin-1CEa 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: FX1X2LKX3X4AX5X6X7NSIFX8X9(I) In this fragment, X1 represents valine or cysteine; X2 represents aspartic acid or cysteine; X3 represents lysine or cysteine; X4 represents isoleucine or cysteine; X5 represents asparagine or cysteine; X6 represents isoleucine or cysteine; X7 represents isoleucine or cysteine; X8 represents glycine or cysteine; and X9 represents lysine or cysteine. The paired cysteine ​​residues in the fragment are cyclized via a halogenated thiol click chemistry reaction.

[0008] 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".

[0009] 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.

[0010] 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. Unless otherwise specified, amino acids in this article generally refer to L-type amino acids.

[0011] Table 1: Amino Acid Abbreviations alanine Ala A Leucine Leu L Arginine Arg R Lysine Lys K Asparagine Asn N Methionine Met M Aspartic acid Asp D Phenylalanine Phe F Cysteine Cys C proline Pro P glutamine Gln Q Serine Ser S glutamic acid Glu E threonine Thr T glycine Gly G Tryptophan Trp W Histidine His H Tyrosine Tyr Y Isoleucine Ile I Valine Val V "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.

[0012] 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.

[0013] 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.

[0014] 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.

[0015] The composition may contain one or more of the cyclic peptide active molecules of the present invention, preferably only one.

[0016] 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.

[0017] 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.

[0018] 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.

[0019] The names, structural formulas, and mass spectrometry data of some of the preferred compounds synthesized in this invention are shown in Table 2: Table 2: Names, structural formulas, and mass spectrometry data of preferred cyclic peptide active molecules temporin-1CEa-Sp1 like Figure 1 As shown <![CDATA[[M+2H] 2+ =987.6422[M+3H] 3+ =658.7686]]> temporin-1CEa-Sp2 like Figure 2 As shown <![CDATA[[M+2H] 2+ =987.1703[M+3H] 3+ =658.3493]]> temporin-1CEa-Sp3 like Figure 3 As shown <![CDATA[[M+2H] 2+ =943.6416[M+3H] 3+ =629.1701]]> temporin-1CEa-Sp4 like Figure 4 As shown <![CDATA[[M+2H] 2+ =951.1938[M+3H] 3+ =634.4649]]> temporin-1CEa-Sp5 like Figure 5 As shown <![CDATA[[M+2H] 2+ =978.1175[M+3H] 3+ =652.7858]]> temporin-1CEa-Sp6 like Figure 6 As shown <![CDATA[[M+2H] 2+ =943.6625[M+3H] 3+ =652.7774]]> Compared with the prior art, the beneficial effects of the present invention are: 1. This invention relates to a novel cyclic peptide antitumor active compound based on a stapling cyclization modification strategy, its preparation method, and its applications. Existing research has confirmed that chemically modifying the α-helix conformation of peptides is an effective strategy to enhance their membrane permeability, enzyme stability, and drug-likeness; among these strategies, stapling cyclization is the most widely reported. This strategy typically utilizes olefin metathesis reactions between the side chains of specific non-natural amino acids to achieve cyclization, which can significantly enhance the structural rigidity of the peptide, stabilize its α-helix conformation, and thus improve its tolerance to enzymatic degradation and cell permeability.

[0020] 2. In the synthesis of this invention, inexpensive and readily available 1,2-bis(bromomethyl)benzene and 4,4'-bis(bromomethyl)-1,1'-biphenyl are used to replace the expensive Fmoc-S5-OH. Cyclone ligation is achieved through a highly efficient alkylation reaction. Using inexpensive raw materials to replace expensive non-natural amino acids significantly reduces synthesis costs, providing economic feasibility for large-scale production. The invention completely eliminates transition metal catalysts, removing the risk of heavy metal introduction at the source, resulting in higher product purity and better compliance with pharmaceutical safety production standards. While optimizing cost and safety, this cyclization strategy also effectively stabilizes the α-helix conformation of the peptide, thereby maintaining or even enhancing its enzyme stability, cell permeability, and antitumor activity. This invention, while retaining the inherent advantages of stapling cyclic peptides, successfully solves the two key bottlenecks of cost and safety through innovation in raw materials and processes, making this type of cyclic peptide drug more promising for clinical translation. Attached Figure Description

[0021] Figure 1 This is a schematic diagram of the temporin-1CEa-Sp1 structure in Table 2 of this invention; Figure 2 This is a schematic diagram of the temporin-1CEa-Sp2 structure in Table 2 of this invention; Figure 3 This is a schematic diagram of the temporin-1CEa-Sp3 structure in Table 2 of this invention; Figure 4 This is a schematic diagram of the temporin-1CEa-Sp4 structure in Table 2 of this invention; Figure 5 This is a schematic diagram of the temporin-1CEa-Sp5 structure in Table 2 of this invention; Figure 6 This is a schematic diagram of the temporin-1CEa-Sp6 structure in Table 2 of this invention. Detailed Implementation

[0022] 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.

[0023] Please see Figures 1 to 6 The present invention provides: Example 1: Preparation method of cyclic peptide antitumor active compound, solid-phase synthesis of temporin-1CEa-Sp1, the specific steps are as follows: The α-amino group of the amino acid is protected with a 9-fluorenylmethoxycarbonyl (Fmoc) group, and the amino acids are 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). Cysteine ​​is used to replace the amino acids at positions 2 and 6. 6-chlorobenzotriazole-1,1,3,3-tetramethylurea hexafluorophosphate (HCTU) and N,N-diisopropylethylamine (DIPEA) are used as activating agents to sequentially protect the above-mentioned amino acids. Coupled with 20% piperidine / DMF for 10 minutes each time, the peptide was cleaved from the resin by reacting with TFA / EDT / TIPs / Water (95:2:2:1, v / v / v / v) at room temperature for 2 hours after coupling, simultaneously removing side-chain protecting groups. The crude peptide was then precipitated with anhydrous diethyl ether and purified by reversed-phase HPLC within 30 minutes, followed by lyophilization to obtain a white lyophilized powder with a purity ≥97.0%. The purified peptide was dissolved in saturated NaHCO3, and 1.2 eq of 1,2-bis(bromomethyl)benzene was dissolved in acetonitrile. The mixtures were then in equal volumes and reacted at room temperature for 1 hour. The supernatant was then purified by preparative HPLC and lyophilized to obtain a white lyophilized powder with a purity ≥97.0%.

[0024] Example 2: Preparation method of cyclic peptide antitumor active compound, solid-phase synthesis of temporin-1CEa-Sp6, the specific steps are as follows: The α-amino group of the amino acid is protected with a 9-fluorenylmethoxycarbonyl (Fmoc) group, and the amino acids are 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). Cysteine ​​is used to replace the amino acids at positions 3 and 10. 6-chlorobenzotriazole-1,1,3,3-tetramethylurea hexafluorophosphate (HCTU) and N,N-diisopropylethylamine (DIPEA) are used as activating agents to sequentially protect the above-mentioned amino acids. Coupled with 20% piperidine / DMF for 10 minutes each time, the peptide was cleaved from the resin by reacting with TFA / EDT / TIPs / Water (95:2:2:1, v / v / v / v) at room temperature for 2 hours after coupling, simultaneously removing side chain protecting groups. The crude peptide was then precipitated with anhydrous diethyl ether and purified by reversed-phase HPLC within 30 minutes, followed by lyophilization to obtain a white lyophilized powder with a purity ≥97.0%. The purified peptide was dissolved in saturated NaHCO3, and 1.2 eq of 4,4'-bis(bromomethyl)-1,1'-biphenyl was dissolved in acetonitrile. The mixtures were then in equal volumes and reacted at room temperature for 1 hour. The supernatant was then purified by preparative HPLC and lyophilized to obtain a white lyophilized powder with a purity ≥97.0%.

[0025] Experimental example: 1) Cell biology experiments CCK-8 in vitro tumor suppression assay: Breast cancer MCF-7 cells were cultured in high-glucose D-MEM containing fetal bovine serum (10%), penicillin (100 KU·L⁻¹), and streptomycin (100 mg·L⁻¹) in a 37°C, 5% CO₂ incubator using standard methods. C42B cells in logarithmic growth phase were then cultured 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 peptide was 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. 50 ).

[0026] 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 temporin-1CEa. The results are shown in Table 3.

[0027] 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.

[0028] Experimental results: The results of the chymotrypsin stability test showed that temporin-1CEa-Sp3 and temporin-1CEa-Sp6 exhibited stronger antichymotrypsin ability than the negative control temporin-1CEa. The results are shown in Table 3.

[0029] Table 3: Degradation half-life of cyclic peptide bioactive molecules chymotrypsin (t) 1 / 2 ) and the half-maximal inhibitory concentration (IC50) of anti-breast cancer cells MCF-7. 50 ) temporin-1CEa 1.2 31.91 temporin-1CEa-Sp1 6.3 23.26 temporin-1CEa-Sp2 8.1 28.26 temporin-1CEa-Sp3 >12 7.37 temporin-1CEa-Sp4 3.7 21.38 temporin-1CEa-Sp5 4.3 32.55 temporin-1CEa-Sp6 >12 16.21 This invention relates to antitumor active compounds and pharmaceutical compositions based on Temporin-1CEa and their applications. Research reports indicate that enhancing the α-helix configuration of peptides through chemical means, thereby increasing their membrane permeability and enzyme stability, 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 via an olefin metathesis reaction of the pentenyl side chain of a specific amino acid can effectively improve the structural rigidity of the peptide and consolidate its α-helix configuration, thereby enhancing enzyme tolerance and cell permeability. Therefore, we designed and synthesized a series of novel temporin-1CEa cyclic peptide active molecules using a 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. This addresses the problems of conformational instability, poor membrane permeability, and weak resistance to hydrolytic enzymes in existing anticancer drugs.

[0030] 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. Anti-tumour active compounds based on Temporin-1CEa, 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: FX1X2LKX3X4AX5X6X7NSIFX8X9(I) In this fragment, X1 represents valine or cysteine; X2 represents aspartic acid or cysteine; X3 represents lysine or cysteine; X4 represents isoleucine or cysteine; X5 represents asparagine or cysteine; X6 represents isoleucine or cysteine; X7 represents isoleucine or cysteine; X8 represents glycine or cysteine; and X9 represents lysine or cysteine. The paired cysteine ​​residues in the fragment are cyclized via a halogenated thiol click chemistry reaction.

2. A pharmaceutical composition comprising the cyclic peptide active molecule having the structure of formula (I) as defined above, characterized in that, The composition comprises the polypeptide according to any one of claims 1.

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

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

5. Application of antitumor active compounds based on Temporin-1CEa: Drug compositions of cyclic peptide active molecules having the structure of formula (I) are used to prepare antitumor drugs.

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