Polypeptide-coupled camptothecin derivative and Anti-tumour use thereof

Abstract

Provided are a polypeptide-coupled drug and an anti-tumour use thereof. The polypeptide-coupled drug is a compound of formula (I) or a pharmaceutically acceptable salt thereof. In formula (I), P is a cyclic peptide containing 4-9 amino acids, L is a linking group, and D is selected from topoisomerase I inhibitors; the polypeptide-coupled drug has better tumour growth inhibitory activity. P-L-D (I)

Description

A polypeptide-coupled camptothecin derivative and its anti-tumor application

[0001] This application claims priority to Chinese patent application No. 2023116479049, filed on December 1, 2023, and cites the full text of the aforementioned Chinese patent application. Technical Field

[0002] The present invention relates to the field of medical technology, and in particular to a polypeptide-coupled drug and its anti-tumor application. Background Art

[0003] In recent years, the development of antibody-drug conjugates (ADCs) has been in full swing. Many domestic and foreign pharmaceutical companies have entered the ADC market, and the number of ADC drugs approved for marketing worldwide has reached 14. Many multinational pharmaceutical companies such as Roche, Gilead, AbbVie, Daiichi Sankyo, and Pfizer have begun to develop ADC pipelines. Currently, the mature anti-tumor targets targeted by ADC drugs on the market include HER2, TROP2, Nectin4, EGFR, CD22, CD20, etc. Although ADC treatment continues to make progress, some problems still exist: (1) As cytotoxic drugs, ADCs may produce severe toxicity, hindering further treatment; (2) The large molecular weight of ADCs affects their ability to penetrate solid tumors, thereby limiting their efficacy; (3) The complex structure of ADCs leads to unstable toxin loading, making drug dosage difficult to control; (4) The production cost of ADCs is high and highly dependent on antibody pharmaceutical companies.

[0004] At present, the competition for peptide-drug conjugates (PDCs) on the market is not as fierce as that for antibody-drug conjugates (ADCs). Compared with ADCs, PDCs have the advantages of small molecular weight, low immunogenicity, high metabolic safety, strong tumor penetration, controllable toxin load, and low production cost. Novartis, Bicycle Therapeutics, Tongyi Pharmaceutical, and many other well-known domestic and foreign pharmaceutical companies have inherited and optimized the technical experience accumulated over many years of exploration and accumulation of antibody-drug conjugates, and have begun to lay out PDC R&D pipelines. Currently, two PDC drugs are on the market in the United States, namely Lutathera, which was approved in 2018, and Pluvicto, which was approved in 2022. Lutathera is an octreotide-conjugated 177Lu targeting somatostatin receptors. It is used to treat adult somatostatin receptor-positive gastroenteropancreatic neuroendocrine tumors. This is also the first peptide-conjugated drug approved by the FDA for targeted tumor treatment. Pluvicto is a peptide derivative ligand conjugated to 177Lu targeting prostate-specific membrane antigen (PSMA). It is used to treat patients with PSMA-positive metastatic castration-resistant prostate cancer who have undergone chemotherapy and androgen receptor signaling pathway inhibitors. In addition, a large number of PDC drugs are in clinical trials, targeting a variety of tumor types with clear targets and established ligands.

[0005] Currently, the two peptide-drug conjugates currently on the market are both radioactive conjugates, while small molecule chemotherapy drugs, which are widely used in antibody drugs, have not yet been successfully applied in the field of peptide-drug conjugate research and development. Therefore, it is necessary to develop a targeted anti-tumor drug that uses a targeted peptide instead of an antibody.

[0006] Summary of the Invention

[0007] The present application provides a polypeptide-coupled drug for inhibiting tumor growth.

[0008] Based on preliminary research, we selected a peptide-containing cleavable linker that has good serum stability and is only cut intracellularly, a topoisomerase I inhibitor as the toxin payload, and a cyclic peptide containing multiple amino acids as a tumor-targeting ligand. We designed and synthesized multiple peptide-coupled molecules, and the HPLC purity of all of them was above 98%.

[0009] In one aspect of the present invention, there is provided a compound of formula (I) or a pharmaceutically acceptable salt thereof:

[0010] PLD(I)

[0011] Wherein, P is a cyclic peptide containing 4-9 amino acids;

[0012] L is a linking group;

[0013] D is selected from topoisomerase I inhibitors.

[0014] In one embodiment, D is selected from camptothecin and its derivatives;

[0015] Preferably, D is

[0016] In one embodiment, the amino acids constituting the cyclic peptide are selected from one or more of cysteine, phenylalanine, tryptophan, lysine, threonine, arginine, and aspartic acid;

[0017] Preferably, P is a cyclic peptide comprising 4, 5, 6, 7 or 8 amino acids.

[0018] In one embodiment, P is a polypeptide targeting one or more of somatostatin receptor (SSTR) and integrin β (ITB).

[0019] In one embodiment, P is a sequence polypeptide consisting of one or more amino acids selected from phenylalanine, cysteine, tryptophan and threonine.

[0020] In one embodiment, P is a polypeptide consisting of one or more amino acids selected from arginine, glycine and aspartic acid.

[0021] In one embodiment, P is an Arg-Gly-Asp containing polypeptide.

[0022] In one embodiment, P is a polypeptide selected from a polypeptide targeting SSTR, and further can be a polypeptide targeting SSTR2 or SSTR5, for example, octreotide Its derivatives and their free radicals.

[0023] The octreotide derivative may be an alkyl derivative in which the amino group on the lysine in the octreotide structure is substituted by an alkyl group.

[0024] In one embodiment, P is selected from a polypeptide targeting ITB, and may further be a polypeptide targeting ITB3, for example, an RGD cyclic peptide.

[0025] In one embodiment, P is selected from the compounds represented by the following formula (IIa) or formula (IIb):

[0026] Among them, R 2 、R 3 、R 4 、R 5 、R 6 、R 7 、R 8Each independently selected from hydrogen, -(CH2)3-N=C(NH2)2, -CH2-C(=O)OH, -CH2-phenyl, -CH2-phenyl-OH, -CH(CH3)-OH or

[0027] A 1 、A 2 Each independently selected from amino acid residues that do not contain a hydroxyl group at the carboxyl terminus;

[0028] R 9 Selected from hydrogen or C1-C6 alkyl, further selected from hydrogen, methyl, ethyl or propyl.

[0029] In one embodiment, R 2 、R 3 、R 4 Each independently selected from hydrogen, -CH2-phenyl-OH, -CH(CH3)-OH or

[0030] In one embodiment, R 5 、R 6 、R 7 、R 8 Each is independently selected from -(CH2)3-N=C(NH2)2, -CH2-C(=O)OH or -CH2-phenyl.

[0031] In one embodiment, A 1 、A 2 Each independently selected from

[0032] In one embodiment, P is selected from the compound represented by the following formula (IIa):

[0033] Among them, R 2 、R 3 、R 4 Each independently selected from hydrogen, -CH2-phenyl-OH, -CH(CH3)-OH or

[0034] A 1 、A 2 Each is independently selected from amino acid residues that do not contain a hydroxyl group at the carboxyl terminus.

[0035] Furthermore, A 1 、A 2 Each independently selected from

[0036] R 9 Selected from hydrogen or C1-C6 alkyl, further selected from hydrogen, methyl, ethyl or propyl.

[0037] In one embodiment, P is selected from the compound represented by the following formula (IIb):

[0038] Among them, R 5 、R 6 、R 7 、R 8 Each is independently selected from -CH2-CH2-N=C(NH2)2, -CH2-C(=O)OH or -CH2-phenyl.

[0039] R 9 Selected from hydrogen or C1-C6 alkyl, further selected from hydrogen, methyl, ethyl, propyl or cyclopropyl.

[0040] In one embodiment, P is selected from

[0041] Preferably, P is selected from

[0042] In one embodiment, L is selected from The position shown indicates connection with P; The position shown indicates connection with phase D;

[0043] in,

[0044] m1, m2, and m3 are each independently selected from an integer of 0 to 6, such as 0, 1, 2, 3, or 4;

[0045] L P Selected from peptide residues comprising 2-7 amino acids, wherein the amino acids in the peptide residues are optionally substituted by one or more substituents selected from C1-C6 alkyl; preferably a peptide residue comprising 2, 3, 4, 5 or 6 amino acids, further preferably a peptide residue consisting of 2, 3, 4 or 5 amino acids.

[0046] Z is selected from -L Z -L j -, L Z Selected from a linker that can connect to the amino group of the peptide, L j Selected from -(CH2) n1 -、-NH-(CH2) n1 -or-(CH2) n2 -C(O)-NH-(CH2) n3 -(OCH2CH2) n4 -;

[0047] n1 is an integer selected from 0-8; for example, 0, 1, 2, 3, 4, 5, 6 or 7.

[0048] n2, n3, and n4 are each independently selected from an integer of 0 to 4, such as 0, 1, 2, or 3;

[0049] X is selected from -O- or -(CH2)-;

[0050] Preferably, Lz is selected from -C(O)- or

[0051] Preferably, L j Selected from -(CH2)2-, -(CH2)4-, -(CH2)5-, -(CH2)6-, -(CH2)2-C(O)-NH-(CH2)2-(OCH2CH2)2-, -(CH2)2-C(O)-NH-(CH2)2-(OCH2CH2)4- or -NH-(CH2)2-.

[0052] Preferably, L P Selected from q is selected from 2, 3, 4, 5 or 6;

[0053] q R in the fragment 1 are independently the same or different, R 1 is selected from hydrogen, isopropyl, isobutyl, benzyl, -(CH2)3-NH-C(O)NH2, -(CH2)2-C(O)OH or -(CH2)4-NH2.

[0054] In one embodiment, L P for

[0055] In one embodiment, Z is selected from -C(O)-(CH2) n1 -, n1 is selected from 0, 1, 2, 3, 4, 5, 6 or 7.

[0056] In one embodiment, L is selected from:

[0057] The position shown indicates connection with P; The position shown indicates connection with phase D;

[0058] wherein n1 is selected from an integer of 1-8; for example, 0, 1, 2, 3, 4, 5, 6 or 7.

[0059] m2 and m3 are each independently selected from an integer of 0-6, such as 0, 1, 2, 3 or 4;

[0060] q is selected from 2, 3, 4, 5 or 6;

[0061] q R in the fragment1 are independently the same or different, R 1 is selected from hydrogen, isopropyl or benzyl.

[0062] In one embodiment, L is selected from:

[0063] In one embodiment, -LD is selected from:

[0064] In one embodiment, the compound of formula (I) is selected from the compound represented by the following formula (IA):

[0065] Wherein, P is defined as above.

[0066] The present invention provides the following compounds or pharmaceutically acceptable salts thereof:

[0067] The present invention provides the following intermediate compounds:

[0068] In one embodiment, the compound of formula (I) is obtained by coupling the intermediate compound with a cyclic peptide.

[0069] In another aspect of the present invention, a method for preparing the above-mentioned compound is provided, which comprises reacting and linking a compound comprising the cyclic peptide structure with a compound comprising the aforementioned topoisomerase I inhibitor-linker structure by a method commonly used in the art.

[0070] In another aspect of the present invention, there is provided a method for preparing the compound of formula (IA), comprising the following steps:

[0071] Compound 6 is reacted with compound PH to obtain a compound of formula (IA); wherein P is as defined above;

[0072] Wherein, m is selected from an integer of 0-7, and can further be 3 or 5.

[0073] Furthermore, the compound 6 is prepared by a method comprising the following steps:

[0074] Furthermore, the compound 5 is prepared by a method comprising the following steps:

[0075] In one embodiment, the compound 03 is prepared by a method comprising the following steps:

[0076] Furthermore, the compound 4 is prepared by a method comprising the following steps:

[0077] Furthermore, the compound 3 is prepared by a method comprising the following steps:

[0078] Furthermore, the compound 2 is prepared by a method comprising the following steps:

[0079] In the above preparation method, the raw materials and abbreviations used are consistent with those of Examples 1-3 of the present application.

[0080] In another aspect of the present invention, a pharmaceutical composition is provided, comprising the compound and a pharmaceutically acceptable carrier.

[0081] In another aspect of the present invention, there is provided a use of the compound or a pharmaceutically acceptable salt thereof or the pharmaceutical composition in the preparation of a drug for treating cancer.

[0082] In one embodiment, provided is the use of the compound or a pharmaceutically acceptable salt thereof or the pharmaceutical composition in the preparation of a medicament for treating liver cancer, prostate cancer, melanoma, ovarian cancer and / or histiosarcoma.

[0083] In another aspect of the present invention, there is provided a use of the compound or a pharmaceutically acceptable salt thereof or the pharmaceutical composition in the preparation of a medicament for treating diseases mediated by one or more of ITB and SSTR.

[0084] In another aspect of the present invention, a method for treating a disease is provided, comprising the step of administering the compound or a pharmaceutically acceptable salt thereof or the pharmaceutical composition to a patient in need thereof, wherein the disease is selected from cancers that highly express SSTR and / or ITB.

[0085] In one embodiment, the compound or its pharmaceutically acceptable salt or the pharmaceutical composition is administered in a therapeutically effective amount.

[0086] In one embodiment, the ITB is ITB3.

[0087] In one embodiment, the SSTR is SSTR2 or SSTR5.

[0088] In one embodiment, the disease is selected from liver cancer, prostate cancer, melanoma, ovarian cancer and / or histiosarcoma. BRIEF DESCRIPTION OF THE DRAWINGS

[0089] FIG1 shows the HPLC spectrum of DXD-PDC1 in Example 1 (purity>99%).

[0090] FIG2 shows the MS spectrum of DXD-PDC1 in Example 1.

[0091] FIG3 shows the binding force between DXD-PDC1 and the target protein in Test Example 1, wherein 3A shows the binding force between c(RGDfK) and the target protein, and 3B shows the binding force between DXD-PDC1 and the target protein.

[0092] Figure 4 shows the proliferation inhibition of DXD-PDC on tumor cell lines with high and no target expression in Test Example 2, where 4A and 4B are flow cytometry test results, and 4C is the calculated IC 50 value.

[0093] FIG5 shows the serum stability of DXD-PDC in Test Example 3. ...

[0094] FIG6 shows the in vivo efficacy evaluation results of DXD-PDC in Test Example 5, wherein 6A is the tumor inhibition result detected by IVIS in vivo imaging (*** indicates P < 0.0001), and 6B is the weight change of mice. DETAILED DESCRIPTION

[0095] I. Definition

[0096] Unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the relevant terms and laboratory procedures used herein are those widely used in the relevant fields and routine procedures. To facilitate a better understanding of the present invention, definitions and explanations of relevant terms are provided below.

[0097] As used herein and unless otherwise indicated, the term "about" or "approximately" means within plus or minus 10% of a given value or range. Where an integer is required, the term means within plus or minus 10% of a given value or range, rounded up or down to the nearest integer.

[0098] In the description herein, references to “some embodiments,” “some implementation schemes,” or “some implementation plans” describe a subset of all possible embodiments, but it will be understood that “some embodiments” may be the same subset or different subsets of all possible embodiments and may be combined with each other without conflict.

[0099] As used herein and unless otherwise specified, the terms "comprises," "includes," "has," "contains," and their grammatical equivalents should generally be understood as open-ended and non-limiting, e.g., not excluding other unlisted elements or steps.

[0100] As used herein, the term "linker" refers to a bifunctional moiety that connects the topoisomerase I inhibitor to the cyclic peptide in the compound of formula (I). It is a polypeptide that has good serum stability and is only cleaved inside cells.

[0101] As used herein, the term "cyclic peptide" refers to a sequence of 4-9 amino acids in which the two terminal amino acids are linked together by a covalent bond, which can be a peptide bond, a disulfide bond, or a synthetic non-peptide bond (e.g., a thioether bond, a phosphodiester bond, a disilyl bond, or a urethane bond). The term "amino acid" refers to an L-amino acid, a D-amino acid, or an amino acid analog, which can optionally be optically pure (i.e., a single enantiomer, and therefore chiral) or a mixture of enantiomers. Preferred amino acids of the present invention are optically pure.

[0102] Suitable peptides for use in the present invention include: RGD cyclic peptide, octreotide and analogs thereof.

[0103] As used herein, the term "topoisomerase I inhibitor" includes, but is not limited to, topotecan, gimatecan, irinotecan, camptothecin, and analogs or derivatives thereof.

[0104] Refers to the chemical bond connection.

[0105] As used herein, the term "alkyl" refers to a straight-chain or branched saturated aliphatic hydrocarbon group. 1- "C6 alkyl" refers to a straight or branched chain alkyl group having 1 to 6 carbon atoms. Preferably, it is C 1-4 Alkyl. More preferably C 1-3 Alkyl. Specific examples include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, and various branched chain isomers thereof.

[0106] Benzyl:

[0107] The term "treating" includes inhibiting, alleviating, preventing or eliminating one or more symptoms or side effects associated with the disease, condition or disorder being treated.

[0108] The term "inhibit" is used relative to a control. One skilled in the art will readily determine the appropriate control for each experiment. For example, a reduced response in a subject or cell treated with a compound is compared to a response in a subject or cell not treated with the compound.

[0109] The term "pharmaceutical composition" means a composition comprising a compound of the present disclosure or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable ingredient selected from the following depending on the mode of administration and the nature of the dosage form, including but not limited to: carriers, diluents, adjuvants, excipients, preservatives, fillers, disintegrants, wetting agents, emulsifiers, suspending agents, sweeteners, flavoring agents, fragrances, antibacterial agents, antifungal agents, lubricants, dispersants, temperature-sensitive materials, temperature regulators, adhesives, stabilizers, suspending agents, etc.

[0110] The term "effective amount" or "therapeutically effective amount" refers to a non-toxic but sufficient amount of a drug or medicament that can achieve the desired effect. In embodiments of the present invention, when a patient is treated according to the present invention, the amount of a given drug depends on many factors, such as a specific dosage regimen, the type of disease or condition and its severity, the uniqueness (e.g., body weight) of the patient or host in need of treatment, but, according to specific surrounding circumstances, including, for example, the specific drug, route of administration, the condition to be treated, and the patient or host to be treated, the dosage can be conventionally determined by methods known in the art. Typically, with respect to the dosage used for adult treatment, the dosage is typically in the range of 0.02-5000 mg / day, for example, about 1-1500 mg / day. The desired dosage can be conveniently expressed as a single dose, or simultaneously administered (or in a short period of time) or in divided doses at appropriate intervals, such as two, three, four, or more divided doses per day. It will be appreciated by those skilled in the art that, although the above-mentioned dosage range has been given, the specific effective amount can be appropriately adjusted according to the patient's condition and in conjunction with the physician's diagnosis.

[0111] The term "peptide-drug conjugate (PDC)" refers to the connection of a biologically active small molecule drug (toxin payload) to a peptide through a chemical link. The peptide acts as a carrier to transport the small molecule drug into the target cells.

[0112] The amino acid or polypeptide structural formula described in this application is as follows:

[0113] Cysteine ​​(Cys): Phenylalanine (Phe): Tryptophan (Trp): Lysine (Lys): Threonine (Thr): Arginine (Arg): Aspartic acid (Asp): It will be appreciated that the individual amino acids are linked via peptide bonds.

[0114] c(RGDfK):

[0115] II. Examples

[0116] In order to make the purpose, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below. The described embodiments should not be regarded as limiting the present invention. All other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0117] Before further explaining the embodiments of the present invention in detail, the nouns and terms involved in the embodiments of the present invention are explained. The nouns and terms involved in the embodiments of the present invention are subject to the following interpretations.

[0118] The raw materials and equipment used in the specific embodiments of the present disclosure are all known products and are obtained by purchasing commercially available products.

[0119] Verification of the binding ability of PDC molecules to target proteins (SPR method)

[0120] Surface Plasmon Resonance (SPR) is used to measure the binding affinity between peptides and target proteins. Surface Plasmon Resonance (SPR) is an optical phenomenon that can be used to track interactions between biomolecules in their native state in real time. This method is biomolecule-safe and does not require any labeling.

[0121] In vitro efficacy evaluation of PDC

[0122] The toxicity of these peptide-drug conjugates was tested in multiple tumor cell lines, and the expression of the target protein in these tumor cells was simultaneously verified by flow cytometry. This experiment will verify whether the synthesized PDC potential molecules have specific killing toxicity against tumor cell lines with high expression of the target protein.

[0123] Evaluation of the stability of PDC small animal serum

[0124] Drug stability is a crucial parameter during drug administration, directly determining the duration of a drug's effective concentration in the body. We evaluated the stability of potential PDC molecules in mouse serum and simultaneously developed a preliminary method for PDC mass spectrometry detection.

[0125] PDC small animal acute toxicity assessment

[0126] In order to confirm the dosage for animal experiments, small animal acute toxicity tests were performed to evaluate the maximum tolerated dose of the drug in small animals.

[0127] Example 1: Synthesis of DXD-PDC1

[0128] Step 1: Synthesis of intermediate DXD-BB-1

[0129] At room temperature, DXD-BB-0 (1.52 g, 3.96 mmol, 1.05 eq.) and DXD-BB-01 (2 g, 3.77 mmol, 1.00 eq.) were dissolved in DMF (15 mL) and stirred for 5 min to obtain a mixed solution. HATU (2.14 g, 5.63 mmol, 1.50 eq.) was then added to the mixture and stirred evenly. Finally, DIPEA (2.2 mL, 11.3 mmol, 3.00 eq.) was added and stirred for 0.5 hours, and the reaction progress was monitored by LCMS. After the reaction was completed, the reaction mixture was slowly added dropwise to 20 mL of ice-pure water to precipitate the solid and stirred continuously. The solid was then filtered out by standing chromatography. After washing the impurities with a small amount of ethanol, the solid was filtered and air-dried to obtain DXD-BB-1 (3.2 g, quant.). LC-MS m / z: 802 (M+H) + .

[0130] Step 2: Synthesis of intermediate DXD-BB-2

[0131] DXD-BB-1 (3.2 g, 4.00 mmol, 1.00 eq.) was dissolved in THF (30.0 mL) and stirred for 5 min. Diethylamine (25 mL, 240.00 mmol, 60.00 eq.) was then added and stirred for 0.5 h. The reaction progress was monitored by LCMS. After the reaction was complete, a small amount of EA was added and stirred for 10 min. The solid was filtered and air-dried to obtain DXD-BB-2 (1.92 g, quant.). LC-MS m / z: 580 (M+H) + .

[0132] Step 3: Synthesis of intermediate DXD-BB-3

[0133] At room temperature, DXD-BB-2 (1.92 g, 3.32 mmol, 1.00 eq.) and DXD-BB-02 (1.58 g, 3.15 mmol, 0.95 eq.) were dissolved in DMF (20.0 mL) and stirred for 5 min to obtain a mixture. HATU (1.39 g, 3.66 mmol, 1.10 eq.) was then added to the mixture and stirred evenly. Finally, DIPEA (1.94 mL, 9.93 mmol, 3.00 eq.) was added and stirred for 0.5 h. The reaction progress was monitored by LCMS. After completion of the reaction, the reaction mixture was slowly added dropwise to 15 mL of ice-pure water to precipitate a solid. The mixture was stirred continuously and allowed to stand for chromatography, followed by filtration of the solid and air-drying to obtain DXD-BB-3 (2.0 g). LC-MS m / z: 1063 (M+H) + .

[0134] Step 4: Synthesis of intermediate DXD-BB-4

[0135] DXD-BB-3 (2.0 g, 1.88 mmol, 1.00 eq.) was dissolved in THF (20.0 mL) and stirred for 5 min at room temperature. Diethylamine (12 mL, 113.00 mmol, 60.00 eq.) was then added and stirred for 0.5 h. The reaction progress was monitored by LCMS. After completion of the reaction, a small amount of EA was added and stirred for 20 min. The solid was filtered and air-dried to obtain DXD-BB-4 (2.3 g). 1 H NMR(400MHz,DMSO-d6)δ8.68(t,J=4.0Hz,1H),8.63-8.40(m,3H),8.39-8.32(m ,2H),7.74-7.63(m,2H),7.26-7.21(m,6H),5.63-5.50(m,2H),5.43-5.38(m,3H ),5.16-4.97(m,3H),4.65(d,J=20.0Hz,2H),4.54-4.47(m,2H),3.20-2.98(m,5 H),2.82-2.70(m,2H),2.32(s,4H),1.89-1.81(m,3H),0.88-0.86(m,5H).LC-MS m / z:841(M+H) + .

[0136] Step 5: Synthesis of Intermediate 1

[0137] SM1 (5.0 g, 28.74 mmol, 1.00 eq.) was dissolved in THF (50.0 mL) and stirred for 10 min to obtain a mixed solution. NHS (3.3 g, 5.74 mmol, 1.00 eq.) was added under an ice-water bath at 0°C. DCC (6.2 g, 30 mmol, 1 eq.) and DMAP (350 mg, 2.87 mmol, 0.01 eq.) were then added to the mixture. The ice bath was removed and the mixture was stirred for 4 hours. The reaction progress was monitored by LCMS. After completion of the reaction, the solvent was distilled off under reduced pressure, and the resulting residue was purified by silica gel column chromatography [PE:EA = 0:1 (v / v)]. The crude product was then slurried with deionized water to obtain crude product 1 (2.0 g). 1 H NMR (400MHz, DMSO-d6) δ2.81(s,4H),2.65(t,J=4.0Hz,2H),2.17(t,J=4.0Hz,2H),1.66-1.56(m,2H),1.53-1.44(m,2H),1.37-1.26(m,4H).LC-MS m / z:270(MH) + .

[0138] Step 6: Synthesis of intermediate DXD-BB-5

[0139] DXD-BB-4 (2.0 g, 2.38 mmol, 1.00 eq.) and 1 (1.61 g, 5.95 mmol, 2.50 eq.) were dissolved in DMF (20.0 mL) and stirred for 10 min at room temperature. DIPEA (1.4 mL, 1.38 mmol, 3.00 eq.) was then added and stirred for 0.5 h. The reaction progress was monitored by LCMS. After completion of the reaction, the solvent was evaporated under reduced pressure, and the resulting residue was purified by silica gel column chromatography [DCM:MeOH = 10:1 (v / v)]. The crude product was obtained by distillation under reduced pressure, yielding DXD-BB-5 (930 mg). LC-MS m / z: 997 (M+H).

[0140] Step 7: Synthesis of intermediate DXD-BB-6

[0141] At room temperature, DXD-BB-5 (910.0 mg, 0.914 mmol, 1.00 eq.) was dissolved in DMF (10.0 mL) and stirred for 5 min before adding NHS (210 mg, 1.83 mmol, 2 eq.). HATU (382 mg, 1.01 mmol, 1.10 eq.) dissolved in DMF (10 mL) was added and stirred. Finally, DIPEA (0.53 mL, 2.74 mmol, 3.00 eq.) was added and stirred for 0.5 h. The reaction progress was monitored by LCMS. After completion of the reaction, EA and water were added to the reaction system, and the EA phase was extracted, dried over anhydrous magnesium sulfate, and the solvent was removed by distillation under reduced pressure to obtain DXD-BB-6 (1.0 g). LC-MS m / z: 1094 (M+H) + .

[0142] Step 8: DXD-PDC1 synthesis

[0143] DXD-BB-6 (1.0 g, 0.91 mmol, 1.00 eq.) was dissolved in DMF (10.0 mL) at room temperature and stirred for 5 minutes. RDG (552 mg, 0.91 mmol, 1.00 eq.) dissolved in DMF (10.0 mL) was added to the mixture, followed by DIPEA (5.3 mL, 27.43 mmol, 30.00 eq.) and stirred for 0.5 hours. LCMS monitored the reaction progress. After completion of the reaction, the target compound was isolated using high pressure preparative separation, with a purity of >99.0% as determined by HPLC (Figure 1). 1H NMR(400MHz, DMSO-d6)δ8.62(t,J=8.0Hz,1H),8.50(d,J=12.0Hz,1H),8.43-8.15(m,5H),8.14-7.86(m,4H),7.84-7.73(m,3H),7.45-6.97 (m,12H),6.75-6.42(m,1H),5.63-5.54(m,1H),5.46-5.34(m,2H),5.25-5.12(m,2H),4.70-4.54(m,3H),4.48-4.42(m,2H),4.31-4.21(m, 2H),4.02(s,2H),3.80-3.55(m,6H),3.35-3.14(m,15H),2.82-2.72( m,1H),2.69-2.58(m,2H),2.36(s,3H),2.24-2.14(m,3H),2.09(t,J= 8.0Hz,2H),2.01(t,J=8.0Hz,2H),1.91-1.79(m,2H),1.78-1.51(m,2H),1.50-1.29(m,7H),1.28-1.15(m,7H),0.86(t,J=8.0Hz,3H).LC-MS m / z: 792(M+2H) 2+ (Figure 2).

[0144] Example 2: Synthesis of DXD-PDC2

[0145] At room temperature, DXD-BB-6 (100 mg, 0.1 mmol, 1.00 eq.) was dissolved in DMF (1.0 mL). Tyr3-Octreotate (100 mg, 0.1 mmol, 1.00 eq.) dissolved in 1 mL DMF was added to the mixture. DIPEA (0.4 mL, 3.00 mmol, 30.00 eq.) was then added and stirred for 1 hour. The reaction was monitored by LCMS. After completion of the reaction, the reaction system was directly purified to obtain the target molecule, with a purity of >98.5% as determined by HPLC. LC-MS m / z: 1014 (M+2H). 2+ .

[0146] Example 3: Synthesis of DXD-PDC3

[0147] Step 1: Synthesis of intermediate DXD-BB-7

[0148] DXD-BB-4 (845 mg, 1 mmol, 1.00 eq.) and SM3 (152 mg, 0.6255 mmol, 0.6255 eq.) were dissolved in 3 mL of DMF at room temperature. DIPEA (521 μL, 3 mmol, 3.00 eq.) was added and stirred for 1 hour. Completion was monitored by LCMS. The reaction solution was added dropwise to EA and centrifuged to obtain the supernatant. The solid was dissolved in 20 mL of acetonitrile and water (1:1 volume ratio) and centrifuged. The insoluble material was discarded and the combined supernatants were rotary evaporated to yield 860 mg of crude DXD-BB-7. 1 H NMR(400MHz, DMSO-d6)δ8.72-8.59(m,1H),8.48(d,J=8.0Hz,1H),8.35-8.25(m,1H),8.19-8.00(m,3H), 7.71-7.57(m,1H),7.25-7.17(m,5H),6.74-6.31(m,1H),5.61-5.49(m,1H),5.46-5.29(m,2H),5.21-4. 91(m,2H),4.64(d,J=8.0Hz,2H),4.51-4.40(m,1H),3.22-2.96(m,4H),2.77(dd,J=12.0,4.0Hz,1H),2. 43-1.96(m,10H),1.91-1.78(m,2H),1.47-1.45(m,5H),1.25-1.20(m,7H),0.85(t,J=8.0Hz,3H).LC-MS m / z:969(M+H) + .

[0149] Step 2: Synthesis of intermediate DXD-BB-8

[0150] At room temperature, the crude DXD-BB-7 product (210 mg, 0.2169 mmol, 1.00 eq.) was dissolved in 400 μL of DMF and stirred until uniform. HATU (82 mg, 0.2169 mmol, 1.00 eq.) and HOSU (24.9 mg, 0.2169 mmol, 1.00 eq.) were then added. After half an hour of reaction, the reaction was monitored by LC-MS. Ethyl acetate was added, and the organic phase was washed three times with saturated brine. The combined organic phases were dried over anhydrous sodium sulfate and decompressed to obtain 200 mg of a solid. LC-MS m / z: 1066 (M+H) + .

[0151] Step 3: DXD-PDC3 synthesis

[0152] At room temperature, DXD-BB-8 (100 mg, 0.10 mmol, 1.00 eq.) was dissolved in 1 mL of DMF. A 1.0 mL solution of RDG cyclic peptide (60 mg, 0.10 mmol, 1.00 eq.) in DMF was added. DIPEA (0.4 mL, 3.0 mmol, 30.00 eq.) was then added and stirred for 1 hour. The reaction progress was monitored by LCMS. After completion of the reaction, a sample was separated by preparative chromatography to obtain the target molecule, which had a purity >98.5% as determined by HPLC. LC-MS m / z: 789 (M+2H). 2+ .

[0153] DXD-PDC4-6 was prepared by referring to the preparation method of Examples 1-3 above:

[0154] Test Case

[0155] Test Example 1: Target protein binding ability of targeting peptides (SPR method)

[0156] Surface Plasmon Resonance (SPR) is used to detect the binding force between peptides and target proteins. Surface Plasmon Resonance (SPR) is an optical phenomenon that can be used to track the interactions between biomolecules in their natural state in real time. First, a biomolecule (target molecule, specifically ITB3) is bonded to the surface of the biosensor, and then a solution containing another biomolecule that can interact with the target molecule (analyte, specifically c(RGDfK) or DXD-PDC1) is injected and flows through the surface of the biosensor. The binding between biomolecules causes an increase in the mass of the biosensor surface, resulting in an increase in the refractive index in the same proportion, and the change in the reaction between biomolecules is observed. This reaction is measured in response units (RU): 1RU = 1pg protein / mm 2 =1x 10 -6 RIU (Refractive Index Unit).

[0157] During injection, the analyte flows through the interaction surface by convection and diffusion, forming a complex with the target molecule, resulting in a change in analyte concentration. The analyte-target complex forms on the biosensor surface, leading to an enhanced reaction. After the analyte is injected, the analyte-target complex dissociates, resulting in a weakened reaction. By fitting this reaction curve with a binding interaction model, the kinetic constants can be determined. The test results are shown in Table 1 below.

[0158] Table 1

[0159] Results: The binding affinity between cRDGfK and the target protein was 1.57uM, while that between DXD-PDC1 and the target protein was 0.75uM. After coupling, DXD-PDC1 had a stronger binding affinity with ITB3 protein than cRDGfK (Figure 3).

[0160] Test Example 2: PDC in vitro efficacy evaluation

[0161] The cell lines selected were involved in the in vitro pharmacological evaluation of the PDC molecules prepared in the examples (Table 2) in melanoma, renal cancer, colorectal cancer, liver cancer, and prostate cancer cells (Table 3). The target information of the PDC molecules involved is shown in Table 2 below.

[0162] Table 2: PDC molecule number and related information

[0163] Table 3: Cell lines and culture conditions used in this experiment

[0164] Experimental design:

[0165] One day in advance, expand the cells to be tested to the desired number and plate them into a 96-well plate at a density of 5,000-10,000 cells / well, depending on the cell growth rate. For example, for a PDC drug to be tested, three groups are required: the PDC molecule, the targeting peptide molecule used by the PDC, and the toxin molecule used by the PDC. Each group has eight 10-fold serial dilutions and at least three replicate wells for each condition, plus three solvent control wells and three empty medium control wells, for a total of 78 wells.

[0166] On the day of the experiment, drug-containing culture medium was prepared according to the designed concentration gradient for each group. The cells were then treated with the drugs and cultured for 72 hours at 37°C and 5% CO2. The cell plates were observed and photographed, and cell viability in each well was assessed using the MTT assay kit (II). The half-maximal inhibitory dose (50%) of PDC and the corresponding toxins for each cell line was evaluated. This project used the Shanghai Sangon MTT Cell Proliferation Assay Kit (E606334). The experimental results are shown in Table 4 below.

[0167] Table 4: Inhibitory effect of DXD-PDC1 on tumor cells

[0168] Results: DXD-PDC1 showed cytotoxicity against A375 and HepG2 tumor cell lines with high expression of ITB3 target protein. 50 The concentration can reach the micromolar level (less than 1 uM), which is about 10 times higher than that of the cell line with low target expression, indicating that the PDC compound designed and synthesized in this application has the expected tumor cell targeting effect (Figure 4).

[0169] Test Example 3: Serum Stability Assessment

[0170] ① Based on the results of in vitro cell experiments, a 10-fold effective dose of the drug to be tested is prepared, with an estimated vaccination dose of 7.5 mg / kg body weight;

[0171] ② The two test drugs were divided into two experimental groups, with 5 replicates in each group. A total of 10 SD rats weighing 200-250 g were purchased;

[0172] ③ After the rats arrive, they are divided into groups and caged in the observation room. After continuous observation for 3 days to confirm that there are no abnormalities, they are transferred to the rat room to start the experiment;

[0173] ④ Take the corresponding concentration of drugs according to the group and rat weight, and inject the corresponding dose of the drug to be tested by tail vein injection;

[0174] ⑤ Blood samples were collected from the orbital venous plexus at 0h, 0.5h, 1h, 2h, 4h, 8h, and 72h after inoculation, with each collection amount of approximately 50ul.

[0175] ⑥Separate the serum from the rat blood sample, precipitate to remove protein and freeze-dry to concentrate the sample. 5500LC / MS / MS liquid chromatography-mass spectrometry is used to quantify the drug to be tested;

[0176] ⑦Draw the concentration according to the drug concentration in serum at different time points

[0177] curve and calculate the half-life of the drug in rat blood.

[0178] Results: The test results are shown in Figure 5. The half-life of Dxd-PDC1 in mouse serum was measured to be approximately 2 days.

[0179] Test Example 4: Acute Toxicity Assessment

[0180] ① Based on the results of in vitro cell experiments, 10, 25, and 50 times the effective dose of the drug to be tested were prepared, and the expected vaccination doses were 7.5, 15, and 37.5 mg / kg body weight, respectively;

[0181] ② After dissolution, the two test drugs were divided into three dose groups: high, medium, and low. Together with the normal saline control group, there were a total of 7 experimental groups. Each group had 5 replicates of animals. A total of 35 Balb / c mice aged 5-8 weeks were purchased.

[0182] ③ After the mice arrive, they are divided into groups and caged in the observation room. After continuous observation for 3 days to confirm that there are no abnormalities, they are transferred to the mouse room to start the experiment;

[0183] ④ Take the corresponding concentration of drugs according to the group and mouse weight, and inject the corresponding dose of the drug to be tested by tail vein injection;

[0184] ⑤ Observe the mice for two weeks after inoculation and record in detail any abnormal reactions such as death, lethargy, and stress levels;

[0185] ⑥ Two weeks later, the mice were euthanized and the liver and kidney toxicity pathologies were observed by autopsy;

[0186] ⑦ Evaluate the maximum tolerated dose of mice based on the abnormal reaction records and organ lesions of mice.

[0187] Results: The maximum dose of Dxd-PDC1 reached 75 mg / kg body weight (equivalent to the IC 50 At the highest dose tested, mice exhibited symptoms of intense stress and excitement, but recovered completely after three days and showed normal weight gain. No mice died. This suggests that the maximum tolerated dose of Dxd-PDC1 is above 75 mg / kg.

[0188] Test Example 5: In vivo efficacy evaluation

[0189] Construction of orthotopic liver tumor-bearing mouse model:

[0190] ① Cell culture: HepG2-LG cells (human liver cancer cells) were revived and cultured in culture medium at 37°C and 5% CO2, and the cell state was adjusted to the optimal state;

[0191] ② Tumor subcutaneous inoculation: Five 3-4 week old Balb / c nude mice were inoculated subcutaneously (cell count 1*10^7 / mouse, inoculation volume 200ul) into the subcutaneous area on the back.

[0192] ③ Measure the long diameter (a) and short diameter (b) of the tumor with a vernier caliper twice a week and record them. Calculate the tumor volume (v) = ab 2 / 2, when HepG2-LG subcutaneous tumors grew to 300mm 3 Orthotopic transplantation was performed when left and right;

[0193] ④ Orthotopic liver transplantation: The established subcutaneous tumor was dissected and divided into small tumor tissue fragments in culture medium. The tumor tissue fragments were surgically transplanted uniformly into the liver of new Balb / c nude mice. A total of 36 3-4 week old Balb / c nude mice were transplanted in situ.

[0194] ⑤ Perform IVIS imaging of the tumor twice a week. When the animals show obvious fluorescent signals, the tumor-bearing mice are divided into groups according to the tumor fluorescence signal value and body weight to begin evaluating the anti-tumor effect of the test drug.

[0195] Evaluation of the anti-tumor efficacy of the test drug in an orthotopic liver tumor-bearing mouse model:

[0196] ① Based on the results of in vitro cell experiments, 10 and 25 times the effective dose of the drug to be tested were prepared, with an estimated vaccination dose of 7.5 mg / kg and 15 mg / kg body weight;

[0197] ② The drug to be tested was divided into two dose groups (10 mpk and 20 mpk), plus saline (Vehicle) and a control group (doxorubicin, 4 mpk), for a total of 4 experimental groups. Each group had 5 replicates, and a total of 30 nude mice were required to successfully establish the Balb / c liver in situ model.

[0198] ③ Take the corresponding concentration of drug according to the group and mouse weight, and inject the corresponding dose of the test drug by tail vein injection. The day of injection is day 0, and it is expected to be administered once every 3 days for 4 weeks (it can be adjusted appropriately according to the drug stability and the maximum tolerated dose of mice);

[0199] ④ Clinical status observation: After the start of the experiment, the clinical status of the animals should be observed daily, including mental state, activity level, diet, drinking water, etc.

[0200] ⑤ Body weight data collection (twice a week): The animals were weighed and recorded twice a week, and the weight change curve was plotted as Figure 6B;

[0201] ⑥ Tumor IVIS imaging (twice a week): In orthotopic tumor animal models, tumor fluorescence signals were detected by IVIS in vivo imaging twice a week, and the tumor fluorescence curve was recorded and plotted (Figure 6A);

[0202] ⑦The trial endpoint is the completion of 4 weeks of dosing;

[0203] ⑧Sample collection and analysis at the test endpoint:

[0204] a) After euthanasia of the animals, serum was collected and stored at -80°C;

[0205] b) Liver tumors were measured and photographed, then snap-frozen in liquid nitrogen and stored at -80°C;

[0206] c) Collect heart, liver, spleen, lung, and kidney tissues and freeze them in liquid nitrogen and store at -80°C;

[0207] d) Mouse serum, tumor tissue homogenate, and heart, liver, spleen, lung, and kidney tissue homogenate were precipitated to remove protein and lyophilized to concentrate the samples. The 5500LC / MS / MS liquid spectrometer was used for quantitative analysis of the drug to be tested.

[0208] As can be seen from the results in FIG6 , both the DXD-PDC-1 10 mpk and 20 mpk dose groups had a significant inhibitory effect on tumor growth in tumor-bearing mice (significantly different from the Vehicle group, P < 0.0001), and there was no significant change in the weight of the mice.

[0209] The foregoing descriptions of specific exemplary embodiments of the present invention are for purposes of illustration and description. These descriptions are not intended to limit the invention to the precise forms disclosed, and it is apparent that many modifications and variations are possible in light of the foregoing teachings. The exemplary embodiments have been selected and described for the purpose of explaining the specific principles of the invention and their practical application, thereby enabling those skilled in the art to make and utilize a variety of exemplary embodiments of the invention and various options and variations. The scope of the invention is intended to be defined by the claims and their equivalents.

Claims

1. A compound of formula (I) or a pharmaceutically acceptable salt thereof: PLD (I) Wherein, P is a cyclic peptide containing 4-9 amino acids; L is a linking group; D is selected from topoisomerase I inhibitors.

2. The compound according to claim 1, wherein D is selected from camptothecin and its derivatives; Preferably, D is 3. The compound according to claim 1 or 2, wherein The amino acids constituting the cyclic peptide are selected from one or more of cysteine, phenylalanine, tryptophan, lysine, threonine, arginine and aspartic acid; Preferably, P is a cyclic peptide comprising 4, 5, 6, 7 or 8 amino acids.

4. The compound according to claim 1 or 2, wherein P is selected from the compounds represented by the following formula (IIa) or formula (IIb): Among them, R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 Each independently selected from hydrogen, -(CH2)3-N=C(NH2)2, -CH2-C(=O)OH, -CH2-phenyl, -CH2-phenyl-OH, -CH(CH3)-OH or A 1 , A 2 Each is independently selected from amino acid residues that do not contain a hydroxyl group at the carboxyl terminus; R 9 Selected from hydrogen or C1-C6 alkyl; Preferably, R 2 , R 3 , R 4 Each independently selected from hydrogen, -CH2-phenyl-OH, -CH(CH3)-OH or R 5 , R 6 , R 7 , R 8 Each independently selected from -(CH2)3-N=C(NH2)2, -CH2-C(=O)OH or -CH2-phenyl; Preferably, A 1 , A 2 Each independently selected from Preferably, P is selected from 5. The compound according to any one of claims 1 to 4, wherein L is selected from The position shown indicates connection with P; The position shown indicates connection with phase D; in, m1, m2, and m3 are each independently selected from an integer of 0 to 6; L P is selected from a peptide residue comprising 2 to 7 amino acids, wherein the amino acids in the peptide residue are optionally substituted by one or more substituents selected from C1-C6 alkyl; Z is selected from -L Z -L j -, L Z is selected from a linker that can connect to the amino group of the peptide, L j Selected from -(CH2) n1 -、-NH-(CH2) n1 -or-(CH2) n2 -C(O)-NH-(CH2) n3 -(OCH2CH2) n4 -; n1 is an integer selected from 0-8; n2, n3, n4 are each independently selected from an integer selected from 0-4; X is selected from -O- or -(CH2)-; Preferably, Lz is selected from -C(O)- or Preferably, L P Selected from q is selected from 2, 3, 4, 5 or 6; q R in the fragment 1 are independently the same or different, R 1 Selected from hydrogen, isopropyl, isobutyl, benzyl, -(CH2)3-NH-C(O)NH2, -(CH2)2-C(O)OH or -(CH2)4-NH2.

6. The compound according to any one of claims 1 to 4, wherein L is selected from: The position shown indicates connection with P; The position shown indicates connection with phase D; Wherein, n1 is selected from an integer of 1-8; m2 and m3 are each independently selected from an integer of 0-6; q is selected from 2, 3, 4, 5 or 6; q R in the fragment 1 are independently the same or different, R 1 is selected from hydrogen, isopropyl or benzyl.

7. The compound according to any one of claims 1 to 6, wherein L is selected from 8. The following compound or a pharmaceutically acceptable salt thereof:

9. A pharmaceutical composition comprising the compound according to any one of claims 1 to 8 and a pharmaceutically acceptable carrier.

10. Use of the compound according to any one of claims 1 to 8 or a pharmaceutically acceptable salt thereof or the pharmaceutical composition according to claim 9 in the preparation of a medicament for treating cancer.

11. A method for treating a disease, comprising the step of administering the compound or a pharmaceutically acceptable salt thereof or the pharmaceutical composition to a patient in need thereof, wherein the disease is selected from cancers that highly express SSTR and / or ITB.

12. The use according to claim 10 or the method according to claim 11, wherein: The cancer is selected from liver cancer, prostate cancer, melanoma, ovarian cancer and / or histiosarcoma.

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