WO2025189832A1 - Anti-tumor compound and preparation method therefor and use thereof - Google Patents

Abstract

Provided is an anti-tumor compound. The anti-tumor compound is a polypeptide whose N-terminus links to a protective group and C-terminus links to a molecular fragment having cytotoxic activity, and the polypeptide has an amino acid sequence of RGDAAN or RGDKTAN. In the anti-tumor compound, a polypeptide is successfully coupled with a molecule having cytotoxicity. The anti-tumor compound can not only realize tumor targeting, but also release a drug only in a tumor environment having legumain after entering a cell, and has good selectivity and good anti-tumor activity.

Description

An antitumor compound and its preparation method and application Technical Field

The present invention belongs to the field of medicine, and specifically relates to an anti-tumor coupling compound in which a polypeptide is covalently bonded to a small molecule drug, a preparation method thereof, and an application thereof in preparing tumor therapeutic drugs.

Background Art

Cancer is currently one of the biggest diseases affecting human health worldwide, posing a serious threat to human life and health. Melanoma is a highly malignant tumor originating from melanocytes and mostly occurs in the skin. Conventional chemotherapy remains the main method for treating cancer in clinical practice. High doses of cytotoxic drugs are usually necessary to kill tumor cells, but traditional chemotherapy drugs have difficulty distinguishing between cancer cells and normal cells, thereby aggravating systemic toxicity in areas such as the heart, hair follicles, skin, bone marrow, digestive tract, and reproductive system. Therefore, improving the targeting of drug tumor delivery is one of the important factors to improve the therapeutic effect of cytotoxic drugs and reduce the toxicity of chemotherapy drugs.

In recent years, peptide–drug conjugates (PDCs) have garnered widespread attention as targeted anti-tumor drugs. Compared to normal cells, tumor cells often overexpress certain receptors. The PDC development strategy involves designing a short peptide that specifically binds to the receptor and then combining it with a toxic payload to enhance the targeted delivery of the drug to the tumor. These short targeting peptides are typically composed of dozens of amino acids and can be chemically synthesized, making them easy to produce.

PDCs consist of peptides specifically conjugated to drugs via linkers. Peptides offer numerous advantages as tumor-targeting vectors. The peptide moiety preferentially targets tumor cells and limits off-target cytotoxicity by releasing the cytotoxic payload at or within the tumor site rather than in healthy tissues.

Currently, several PDCs have been approved by the FDA. The earliest approved PDC was ^111In -DTPA-Octreotide ( ^Octreoscan® ), a diagnostic drug for locating cancerous tumors via intravenous injection. It was launched in the United States in 1994. Octreotide is a tumor-targeting peptide that targets the somatostatin receptor (SSTR), with ^111In as the payload chelated to DTPA (Diethylene triamino pentaacetic acid). Although high-dose ^Octreoscan® can relieve symptoms in patients with metastatic neuroendocrine tumors, tumor size reduction is unsatisfactory, resulting in its approval only for diagnostic imaging of SSTR-positive tumors. The most successful radioactive PDC therapy to date is ^177Lu -DOTATATE (Lutathera ^® ), developed by Novartis subsidiary Advanced Accelerator Applications SA. This is the first peptide receptor radionuclide therapy (PRRT) drug, using somatostatin as a homing peptide. It is indicated for the treatment of SST receptor-positive gastroenteropancreatic neuroendocrine tumors by intramuscular injection and received FDA approval on January 26, 2018. Melflufen (Pepaxto ^® ), the lead drug in Oncopeptides' PDC platform, is an aminopeptidase-targeting PDC that rapidly delivers the alkylating agent melphalan as a payload into tumor cells for the treatment of multiple myeloma. It received FDA approval on February 27, 2021.

The RGD sequence is a short peptide composed of arginine, glycine, and aspartic acid that specifically binds to the extracellular domain of integrin receptors on the cell surface. The specific expression of these receptors in various human tissues, and the close correlation between their expression profiles and various pathophysiological conditions, make them suitable targetable drug candidates for the diagnosis and treatment of a wide range of diseases. RGD-based integrin ligands have been widely used in biomedical research. Because certain integrin receptors, such as the avβ3 receptor, are overexpressed on tumor cells and tumor vascular endothelial cells, existing technologies utilize RGD sequences to specifically deliver therapeutic or diagnostic agents to cancer cells. However, the integrin receptors bound by RGD also exist and exert physiological functions in normal tissues, blood, and other body fluids. Therefore, the safety of RGD-based targeted drug delivery significantly affects the development of related anti-tumor products. The first integrin-targeted drug, abciximab, failed to meet study endpoints due to potential direct toxicity induced by a prothrombotic mechanism. Efalizumab was approved in 2003 but was withdrawn in 2009 due to adverse reactions associated with progressive multifocal leukoencephalopathy. In 2013, Cilengitide failed in a clinical trial for the treatment of glioblastoma. To date, there are no approved drugs targeting av-integrin.

PDCs provide targeted delivery of small molecules to diseased tissues, increasing local drug concentrations and mitigating toxic effects caused by systemic exposure and accumulation in non-lesioned tissues. However, the drug and peptide that comprise the conjugate must synergize through different pathways to achieve optimal therapeutic efficacy. Tissue-specific peptide delivery is influenced by multiple factors. Extracellular peptide receptors must internalize sufficient small drug molecules in a ligand-dependent manner and further release them from the PDC for the drug to exert its pharmacological effect. Furthermore, the toxicity of PDCs in normal tissues, which can lead to adverse drug reactions, must be considered. Therefore, designing a structure that balances efficacy and selectivity is crucial. (moleclues, 2019, 24: 1855)

Summary of the Invention

In response to the above-mentioned problems of the prior art, the present invention provides an anti-tumor compound, a preparation method and application thereof. The anti-tumor compound successfully couples a polypeptide with a cytotoxic molecule, which not only achieves tumor targeting, but also, after entering the cell, can release the drug only in the tumor environment with legume protein, has good selectivity, and has good anti-tumor activity.

To achieve the above object, the present invention adopts the following technical solutions:

An antitumor compound is a polypeptide with a protective group connected to the N-terminus and a molecular fragment with cytotoxic activity connected to the C-terminus, and the amino acid sequence of the polypeptide is RGDAAN or RGDKTAN.

Preferably, the protecting group is acetyl (Ac) or 9-fluorenylmethoxycarbonyl (Fmoc).

Preferably, the molecular fragment having cytotoxic activity is derived from daunorubicin or doxorubicin.

Preferably, the antitumor compound is Ac-RGDKTAN-daunorubicin or a pharmaceutically acceptable salt thereof, or Fmoc-RGDAAN-doxorubicin or a pharmaceutically acceptable salt thereof.

The structural formula of the Ac-RGDKTAN-daunorubicin is:

.

The structural formula of the Fmoc-RGDAAN-adriamycin is:

.

The present invention also provides a method for preparing the above-mentioned antitumor compound, comprising the following steps:

(1) preparing the polypeptide with a protective group connected to the N-terminus by solid phase synthesis;

(2) The polypeptide with a protective group connected to the N-terminus obtained in step (1) and a molecule with cytotoxic activity are added to a solvent, and benzotriazole tetramethyl tetrafluoroboric acid (TBTU) and N,N-diisopropylethylamine (DIEA) are added to react to obtain the product.

Preferably, the molar ratio of the polypeptide with the N-terminal protective group to the molecule having cytotoxic activity is 1:1.

Preferably, the reaction time in step (2) is 2 h.

The present invention also provides a pharmaceutical composition containing the anti-tumor compound.

The present invention also provides the use of the anti-tumor compound in the preparation of tumor therapeutic drugs.

The tumor is colorectal cancer, breast cancer, liver cancer, pancreatic cancer, bladder cancer, prostate cancer, gastric cancer, lung cancer, melanoma, osteosarcoma or hematological cancer.

The beneficial effects of the present invention are:

The anti-tumor compound prepared by the present invention successfully couples the polypeptide with a cytotoxic molecule, which not only achieves tumor targeting, but also releases free drugs only in the tumor environment with leguminous proteins after entering the cell, and has excellent tumor cell selectivity.

The antitumor compound prepared by the invention has significant therapeutic effect in antitumor treatment.

The antitumor compound prepared by the present invention releases free drugs only in the tumor environment, so the toxicity to tissues is significantly reduced.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a HPLC analysis graph of Fmoc-RGDAAN-doxorubicin prepared in Example 2 at 214 nm and 480 nm, respectively.

FIG2 is a flow cytometric analysis of different concentrations of Fmoc-RGDAAN-doxorubicin in 293T cells and B16-F10 cells in Example 4.

FIG3 is a graph showing changes in body weight of animals during the experimental period in Example 5.

FIG4 is a graph showing the tumor volume of the animals in Example 5 at day 11.

FIG5 is a graph showing the tumor weights of the animals in Example 5 at day 11.

FIG6 shows the tumor morphology of the animals in Example 5 at day 11.

FIG7 is a diagram of tumor, liver, and heart tissue sections obtained in Example 5 (scale bar: 50 mm).

DETAILED DESCRIPTION Example 1

Preparation of Ac-RGDKTAN-daunorubicin:

(1) Soak Wang resin in DCM solution to swell it, and then rinse the resin with DMF solution. Weigh Fmoc-Asn(Trt)-OH equivalent to 6 times the resin loading, and add appropriate amounts of DIEA and DCM solution to the reaction tube. Then add DMF solution (make DCM:DMF=1:1) and react for 2 h. Wash the resin, remove the Fmoc group, and detect with ninhydrin. Weigh the corresponding Fmoc-protected amino acid equivalent to 3 times the resin loading, and add appropriate amounts of HOBt, TBTU, and DIEA to the reaction tube. Add DMF solution to react. Repeat the washing, Fmoc group removal, and detection operations.

Repeat the above steps according to the peptide sequence RGDKTAN to complete the peptide chain coupling. Finally, the N-terminus is protected with acetic anhydride to obtain Ac-RGDKTAN-Wang resin, which is then washed and vacuum dried.

The resin was added to the cutting liquid trifluoroacetic acid TFA: triisopropylsilane: water = 95:2.5:2.5 (v/v/v) for reaction. The final white flocculent solid obtained was the polypeptide Ac-RGDKTAN-OH, which was stored at -20°C, purified by high performance liquid chromatography, and characterized by mass spectrometry.

(2) The synthesized peptide and daunorubicin hydrochloride were added to DMF at a molar ratio of 1:1. TBTU and DIEA were added and the reaction was carried out for 2 h. The resulting crude Ac-RGDKTAN-daunorubicin was purified by HPLC and lyophilized to obtain Ac-RGDKTAN-daunorubicin. The product was stored at -20°C and characterized by HPLC and mass spectrometry. ESI-MS was used for structural characterization.

The ESI-MS experimental conditions were as follows: end plate offset of −500 V, capillary voltage of 4000 V, nebulizer pressure of 1.5 bar, drying gas flow rate of 6.0 L/min, drying gas temperature of 180°C, and primary scan mass range of m/z 400–2000, yielding [M+2H] ²⁺ of 656.92 and [M+H] ⁺ of 1311.57.

Example 2

Preparation of Fmoc-RGDAAN-doxorubicin:

(1) The crude peptide Fmoc-RGDAAN-OH was prepared using 2-Cl resin as a solid phase support, Fmoc-amino acid as a raw material, DIC/4-dimethylaminopyridine and HOBt/DIC as condensing agents, 20% piperidine as a de-Fmoc reagent, ninhydrin as a reaction indicator, and a mixed solution of trifluoroacetic acid (TFA): triisopropylsilane: water = 95:2.5:2.5 (v/v/v) as a cleavage agent. The crude peptide sample was purified and analyzed for purity by RP-HPLC, and its structure was characterized by ESI-MS.

The experimental conditions of RP-HPLC were as follows: Phenomenex PEPTIDE (C18, 250 mm × 4.6 mm, 5 mm) column, column temperature of 25 °C, detection wavelength of 214 nm, mobile phase A was water containing 1‰ trifluoroacetic acid, mobile phase B was acetonitrile containing 1‰ trifluoroacetic acid, flow rate was 1 ml/min, gradient elution was B: 20%-70% (0-20 min), and injection volume was 20 mL.

The ESI-MS experimental conditions were as follows: end plate offset of -500 V, capillary voltage of 4000 V, nebulizer pressure of 1.5 bar, drying gas flow rate of 6.0 L/min, drying gas temperature of 180°C, primary scan range of m/z 400-2000, and the obtained [M+H] was 826.09.

(2) The synthesized polypeptide Fmoc-RGDAAN-OH and doxorubicin hydrochloride were added to DMF at a molar ratio of 1:1, and TBTU and DIEA were added. The reaction was carried out for 2 h. The obtained crude Fmoc-RGDAAN-doxorubicin was purified by high performance liquid chromatography, lyophilized to obtain Fmoc-RGDAAN-doxorubicin, stored at -20°C, and characterized by high performance liquid chromatography and mass spectrometry.

The RP-HPLC experimental conditions were the same as above, with detection wavelengths of 214 nm and 480 nm. The spectrum is shown in Figure 1.

The ESI-MS experimental conditions were as follows: end plate offset of −500 V, capillary voltage of 4000 V, nebulizer pressure of 1.5 bar, drying gas flow rate of 6.0 L/min, drying gas temperature of 180°C, primary scan and automatic secondary full scan mass scanning range of m/z 400–2000, and [M+2H] ²⁺ was 675.27 and [M+H] ⁺ was 1349.53.

The ESI-MS/MS experimental conditions were as follows: spray voltage (+) was 1500.00 V, capillary temperature was 320.00 °C, maximum spray current was 50.00 V, RF level was 40.00, ion source was NSI, and the scan range was m/z 300-1800. The fragment mass numbers of Fmoc-RGDAAN-doxorubicin were 551.2249, 622.2617, 693.2983, and 807.3405.

Preparation of Boc-RGDPTN-epirubicin:

(1) The polypeptide Boc-RGDPTN-OH was prepared, cleaved and purified using the method of step (1) of Example 1.

(2) The synthesized peptide and epirubicin were added to DMF at a molar ratio of 1:1. TBTU and DIEA were added and the reaction was continued for 2 h to obtain Boc-RGDPTN-epirubicin. The mass spectrometry results were [M+2H] ²⁺ 642.81, [M+H] ⁺ 1284.52.

Example 3

(1) In vitro antitumor activity of Fmoc-RGDAAN-doxorubicin:

Cytotoxicity was assessed using the MTT assay. Trypsin-digested 293T and B16-F10 cells were plated in 96-well plates at a density of 4 × ¹⁰ cells per well (200 mL). After cell attachment, the medium was removed and various concentrations of drugs diluted in fresh medium were added. Sterile water was added to the negative control group, while the experimental groups received different concentrations of Fmoc-RGDAAN-doxorubicin prepared in Example 2 at concentrations of 33 nM, 100 nM, 333 nM, 1000 nM, 3333 nM, and 10,000 nM. A positive control group received doxorubicin at concentrations comparable to those in the experimental group. After 72 hours of incubation, 100 mL of each group was removed from the cells and 10 mL of 5 mg/mL MTT solution was added. The cells were then incubated in an incubator for 4 hours. The cells were then removed and 100 mL of formazan solution was added to each well, followed by a further 4 hours of incubation until the formazan was completely dissolved under a standard optical microscope. The OD values of the 96-well plate at 570 nm were measured using a multifunctional microplate reader. Each group was repeated three times. The _IC50 of Fmoc-RGDAAN-doxorubicin against B16-F10 cells at 72 hours was 3650 nM, while the _IC50 of the positive control was 69.92 nM. The 293T cell survival rates in the doxorubicin group and the Fmoc-RGDAAN-doxorubicin group were 48% and 101%, respectively, at 48 h.

(2) In vitro antitumor activity of Boc-RGDPTN-epirubicin:

Cytotoxicity was measured using the MTT assay. Trypsin-digested B16-F10 cells were added to 96-well plates at a density of 200 mL per well, for a total of 4 × ¹⁰ cells. After cell attachment, the culture medium was removed and various concentrations of the drug diluted in fresh culture medium were added. Sterile water was added to the negative control group. The experimental groups were treated with different concentrations of Boc-RGDPTN-epirubicin prepared in Comparative Example 1, at concentrations of 33 nM, 100 nM, 333 nM, 1000 nM, 3333 nM, and 10,000 nM. The positive control group was treated with epirubicin at a concentration similar to that of the experimental group. After 72 hours of incubation, 100 mL was removed from all groups and 10 mL of 5 mg/mL MTT solution was added. The cells were then incubated in an incubator for 4 hours. The cells were then removed and 100 mL of formazan solution was added to each well, followed by a further 4 hours of incubation until the formazan was completely dissolved under a conventional optical microscope. The OD value of the 96-well plate at 570 nm was measured using a multifunctional microplate reader. Each group was repeated three times. Within the tested concentration range, no IC ₅₀ was obtained for Boc-RGDPTN-epirubicin against B16-F10 cells at 72 h. The IC ₅₀ of the positive control group was 90.67 nM.

The comparison shows that legume protein has different enzymatic hydrolysis efficiencies for Fmoc-RGDAAN-doxorubicin and Boc-RGDPTN-epirubicin, and Fmoc-RGDAAN-doxorubicin has better cytotoxicity against B16-F10 cells.

Example 4

In vitro cellular uptake of Fmoc-RGDAAN-doxorubicin:

The fluorescence of doxorubicin in cells was quantitatively analyzed by flow cytometry to investigate the cell uptake capacity of Fmoc-RGDAAN-doxorubicin.

Figure 2 shows the uptake capacity of 293T cells and B16-F10 cells for different concentrations of Fmoc-RGDAAN-doxorubicin over 24 hours. For B16-F10 cells, the relative fluorescence intensity increased with increasing doxorubicin concentration in Fmoc-RGDAAN-doxorubicin, indicating that both cell types increased their uptake of Fmoc-RGDAAN-doxorubicin in a concentration-dependent manner. At high concentrations, the uptake capacity of B16-F10 cells for Fmoc-RGDAAN-doxorubicin was 8.5 times that of 293T cells.

Example 5

In vivo antitumor activity of Fmoc-RGDAAN-doxorubicin:

Mice bearing tumors 80-120 ^mm³ in size and well-proportioned morphology were randomly divided into five groups: a negative control group, a positive control group (doxorubicin 5 mg/kg), and groups receiving high-dose Fmoc-RGDAAN-doxorubicin (equivalent to 8 mg/kg), medium-dose Fmoc-RGDAAN-doxorubicin (equivalent to 5 mg/kg), and low-dose Fmoc-RGDAAN-doxorubicin (equivalent to 2.5 mg/kg), with four mice per group. The day of the first tail vein injection was designated Day 0 of the efficacy study, with subsequent administration on Days 2, 4, 6, and 8. Tumor volumes were calculated using the formula V = L × ^H² /2 by measuring the long and short diameters (L) and short diameters (H) of the tumors every other day using a vernier caliper. Body weights of the mice were monitored and recorded daily, and tumor tissue was removed and weighed on the final day. Figures 3 and 4 show the time course of tumor changes in mice treated with the drug. Hearts, livers, and tumors were harvested, fixed with 4% paraformaldehyde, dehydrated, embedded in paraffin, and sectioned for hematoxylin and eosin staining to observe histopathological changes.

The data were compared among the groups using one-way ANOVA. **** indicates statistical significance at p < 0.0001.

Claims (10)

An anti-tumor compound, characterized in that the anti-tumor compound is a polypeptide with a protective group connected to the N-terminus and a molecular fragment with cytotoxic activity connected to the C-terminus, and the amino acid sequence of the polypeptide is RGDAAN or RGDKTAN. The anti-tumor compound according to claim 1 is characterized in that the protecting group is acetyl or 9-fluorenylmethoxycarbonyl. The anti-tumor compound according to claim 1 is characterized in that the molecular fragment with cytotoxic activity is derived from daunorubicin or doxorubicin. The anti-tumor compound according to any one of claims 1-3 is characterized in that the anti-tumor compound is Ac-RGDKTAN-daunorubicin or a pharmaceutically acceptable salt thereof, or Fmoc-RGDAAN-doxorubicin or a pharmaceutically acceptable salt thereof. The method for preparing the anti-tumor compound according to any one of claims 1 to 4 is characterized in that it comprises the following steps: (1) preparing the polypeptide with a protective group connected to the N-terminus by solid phase synthesis; (2) The polypeptide with a protective group connected to the N-terminus obtained in step (1) and a molecule with cytotoxic activity are added to a solvent, and benzotriazole tetramethyl tetrafluoroboric acid and N,N-diisopropylethylamine are added for reaction to obtain the product. The preparation method according to claim 5 is characterized in that the molar ratio of the polypeptide with the N-terminal connected protective group to the molecule with cytotoxic activity is 1:1. The preparation method according to claim 5 is characterized in that the reaction time in step (2) is 2 hours. A pharmaceutical composition containing the anti-tumor compound described in any one of claims 1-4. The use of the anti-tumor compound described in any one of claims 1-4 in the preparation of tumor treatment drugs. The use according to claim 9 is characterized in that the tumor is colorectal cancer, breast cancer, liver cancer, pancreatic cancer, bladder cancer, prostate cancer, gastric cancer, lung cancer, melanoma, osteosarcoma or hematologic malignancy.