Peptide-conjugated t2 toxin and Anti-tumor application thereof

Abstract

Provided are a peptide-conjugated drug and an anti-tumor application thereof. The peptide-conjugated drug is the compound as shown in formula (I) or a pharmaceutically acceptable salt thereof. In formula (I): P is a peptide including 2-9 amino acids; L is a linking group; and D is selected from a T2 toxin, or a derivative, prodrug or metabolite thereof. The peptide-conjugated drug has better tumor growth inhibitory activity. P-L-D (I)

Description

A peptide-coupled T2 toxin and its anti-tumor application

[0001] This application claims priority to Chinese patent application No. 2023116453405, 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 peptide-coupled drug for inhibiting tumor growth.

[0008] Based on preliminary research, we selected a peptide-cleavable linker with good serum stability that is only cut intracellularly, selected T2 toxin as the toxin payload, and selected a peptide that targets tumor cells 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 peptide containing 2-9 amino acids;

[0012] L is a linking group;

[0013] D is selected from T2 toxin, a derivative, a prodrug or a metabolite thereof.

[0014] In one embodiment, the T2 toxin, its derivative, prodrug or metabolite is selected from the group consisting of:

[0015] In one embodiment, D is selected from Among them, T2 is selected from

[0016] In one embodiment, D is

[0017] In one embodiment, the amino acids constituting P are selected from one or more of cysteine, phenylalanine, tryptophan, lysine, threonine, arginine, aspartic acid, and glutamic acid.

[0018] In one embodiment, P is selected from a cyclic peptide comprising 4, 5, 6, 7, 8 or 9 amino acids.

[0019] In one embodiment, P is a peptide selected from one or more of prostate-specific membrane antigen (PSMA), somatostatin receptor (SSTR) and integrin β (ITB).

[0020] In one embodiment, P is a polypeptide selected from a PSMA-targeting polypeptide, for example, a polypeptide comprising Glu-Urea-Lys or peptides of its free radicals.

[0021] In one embodiment, P is a sequence polypeptide consisting of one or more amino acids selected from phenylalanine, cysteine, tryptophan and threonine.

[0022] In one embodiment, P is a polypeptide consisting of one or more amino acids selected from arginine, glycine and aspartic acid.

[0023] In one embodiment, P is an Arg-Gly-Asp containing polypeptide.

[0024] 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.

[0025] 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.

[0026] 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.

[0027] In one embodiment, P is selected from the compounds represented by the following formula (IIa) or formula (IIb):

[0028] 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

[0029] A 1 、A 2 Each independently selected from amino acid residues that do not contain a hydroxyl group at the carboxyl terminus;

[0030] R 9 Selected from hydrogen or C1-C6 alkyl.

[0031] In one embodiment, R 9 is selected from hydrogen, methyl, ethyl or propyl.

[0032] In one embodiment, R 2 、R 3 、R 4 Each independently selected from -CH2-phenyl-OH, -CH(CH3)-OH or

[0033] In one embodiment, R 5 、R 6 、R 7 、R 8 Each is independently selected from hydrogen, -(CH2)3-N=C(NH2)2, -CH2-C(=O)OH or -CH2-phenyl.

[0034] In one embodiment, A 1 、A 2 Each independently selected from

[0035] In one embodiment, P is selected from

[0036] In one embodiment, P is selected from

[0037] In one embodiment, P is selected from a peptide comprising 2 or 3 amino acids and / or a urea group.

[0038] In one embodiment, P is

[0039] In one embodiment, L is selected from The position shown indicates connection with P; The position shown indicates connection with phase D;

[0040] in,

[0041] m1, m2, and m3 are each independently selected from an integer of 0 to 6, such as 1, 2, 3, or 4;

[0042] 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.

[0043] 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 -、-(CH2) n2 -C(O)-NH-(CH2) n3 -(OCH2CH2) n4 -;

[0044] n1 is an integer selected from 0-8; for example 0, 1, 2, 3, 4, 5, 6 or 7;

[0045] X is selected from -O- or -(CH2)-.

[0046] n2, n3, n4 are each independently selected from an integer of 0-4; for example, 0, 1, 2 or 3.

[0047] In one embodiment, Lz is selected from -C(O)- or

[0048] In one embodiment, L jSelected 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-.

[0049] In one embodiment, L P Selected from q is selected from 2, 3, 4, 5 or 6;

[0050] 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.

[0051] In one embodiment, L P for (Val-Cit dipeptide) or (Glu-Val-Cit tripeptide).

[0052] In one embodiment, Z is selected from -C(O)-(CH2) n1 -, n1 is selected from 0, 1, 2, 3, 4, 5, 6 or 7.

[0053] In one embodiment, L is selected from

[0054] In another aspect of the present invention, the following compound or a pharmaceutically acceptable salt thereof is provided:

[0055] In another aspect of the present invention, the following intermediate compounds are provided:

[0056] 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 peptide structure with a compound comprising the aforementioned T2 toxin, its derivative, prodrug or metabolite-linker structure by a method commonly used in the art.

[0057] Specifically, it includes making the selected Peptides and React Connect.

[0058] In another aspect of the present invention, a pharmaceutical composition is provided, which comprises the compound and a pharmaceutically acceptable carrier.

[0059] 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.

[0060] 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.

[0061] 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 PSMA, ITB, and SSTR.

[0062] 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 PSMA, ITB, and / or SSTR.

[0063] In one embodiment, the compound or its pharmaceutically acceptable salt or the pharmaceutical composition is administered in a therapeutically effective amount.

[0064] In one embodiment, the ITB is ITB3.

[0065] In one embodiment, the SSTR is SSTR2 or SSTR5.

[0066] In one embodiment, the disease is selected from liver cancer, prostate cancer, melanoma, ovarian cancer and / or histiosarcoma. BRIEF DESCRIPTION OF THE DRAWINGS

[0067] FIG1 shows the MS spectrum of T2-PDC1.

[0068] Figure 2 shows the T2-PDC2 1 H NMR spectrum.

[0069] FIG3 shows the MS spectrum of T2-PDC2.

[0070] FIG4 shows the MS spectrum of T2-PDC3.

[0071] FIG5 shows the MS spectrum of T2-PDC2G.

[0072] FIG6 shows the binding force between T2-PDC and the target protein in Test Example 1, wherein 6A shows the binding force between c(RGDfK) and the target protein, and 6B shows the binding force between T2-PDC2 and the target protein.

[0073] Figure 7 shows the proliferation inhibition of T2-PDC on tumor cell lines with high and no target expression in Test Example 2, where 7A and 7B are flow cytometry test results, and 7C is the calculated IC 50 value.

[0074] FIG8 shows the in vitro serum stability of T2-PDC in Test Example 3, wherein FIG8A shows the in vitro serum stability of T2-PDC2, and FIG8B shows the in vitro serum stability of T2-PDC2G.

[0075] FIG9 shows the in vivo efficacy evaluation results of T2-PDC in Test Example 5, wherein 9A is the tumor inhibition result detected by IVIS in vivo imaging (*** indicates P < 0.0001), and 9B is the weight change of mice. DETAILED DESCRIPTION

[0076] I. Definition

[0077] 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.

[0078] 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.

[0079] 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.

[0080] 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.

[0081] As used herein, the term "linker" refers to a bifunctional moiety (P) that connects T2 toxin, its derivatives, prodrugs or metabolites to the peptide in the compound of formula (I). It is a polypeptide with good serum stability and is only cleaved inside cells.

[0082] 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.

[0083] Suitable peptides for use in the present invention include: RGD cyclic peptide, octreotide, its analogs, and peptidomimetics containing Glu-Urea-Lys.

[0084] Refers to the chemical bond connection.

[0085] 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.

[0086] Benzyl:

[0087] 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.

[0088] 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.

[0089] 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.

[0090] The term "effective amount" or "therapeutically effective amount" refers to a sufficient amount of a non-toxic drug or pharmaceutical agent that achieves the desired effect. In embodiments of the present invention, when treating a patient 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., weight) of the subject or host to be treated, but, depending on specific surrounding circumstances, including, for example, the specific drug, route of administration, the condition to be treated, and the subject or host to be treated, the dosage can be conventionally determined by methods known in the art. Typically, for adult treatment, the dosage is typically in the range of 0.02-5000 mg / day, such as about 1-1500 mg / day. The desired dose 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.

[0091] 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 antibody acts as a carrier to transport the small molecule drug into the target cells.

[0092] The amino acid or polypeptide structural formula described in this application is as follows:

[0093] Cysteine ​​(Cys): Phenylalanine (Phe): Tryptophan (Trp): Lysine (Lys): Threonine (Thr): Arginine (Arg): Aspartic acid (Asp): Glutamate (Glu): It is understood that individual amino acids are linked via peptide bonds formed by condensation of the amino and carboxyl groups.

[0094] c(RGDfK):

[0095] II. Examples

[0096] 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.

[0097] 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.

[0098] 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.

[0099] Verification of the binding ability of PDC molecules to target proteins (SPR method)

[0100] 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.

[0101] In vitro efficacy evaluation of PDC

[0102] 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.

[0103] Evaluation of the stability of PDC small animal serum

[0104] 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.

[0105] PDC small animal acute toxicity assessment

[0106] 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.

[0107] Example 1: Synthesis of T2-PDC-1

[0108] Step 1: Synthesis of T2-BB-7

[0109] Step 1: Synthesis of T2-BB-1

[0110] 6-(tert-Butoxy)-6-oxohexanoic acid (336 mg, 1.68 mmol, 1.1 eq) was dissolved in dry DMF (10 ml), and HATU (714 mg, 1.86 mmol, 1.2 eq) and DIPEA (606 mg, 4.68 mmol, 3.0 eq) were added. The mixture was stirred at room temperature for 10 min. (S)-2-[(S)-2-amino-3-methylbutanamido]-N-[4-(hydroxymethyl)phenyl]-5-ureapentanamide (600 mg, 1.56 mmol, 1.0 eq) was added to the reaction mixture, and the mixture was stirred at room temperature for 2 h. LC-MS confirmed the reaction was complete. The residue was concentrated under reduced pressure and dissolved in DCM, washed three times with saturated NaCl, dried over anhydrous NaSO₄, and the organic phase was concentrated under reduced pressure to yield T2-BB-1 (750 mg, 85%) as an off-white solid. 1 H NMR (400MHz, DMSO-d6) δ9.89 (s, 1H), 8.07 (d, J = 7.7Hz, 1H), 7.83 (d, J = 8.6Hz, 1H), 7.58-7.51 (m, 2H), 7.23(d,J=8.4Hz,2H),5.97(t,J=5.8Hz,1H),5.41(s,2H),5.09(s,1H),4.43(s,2H),4.20(dd,J=8.6, 6.8Hz,1H),3.12-2.87(m,2H),2.71(d,J=16.7Hz,1H),2.25-2.08(m,3H),1.98(h,J=6.7Hz,1H),1.47 (d,J=6.9Hz,1H),1.39(s,9H),1.24(d,J=6.5Hz,3H),0.85(dd,J=12.0,6.8Hz,6H).LCMS: 564.33[M+H] + .

[0111] Step 2: Synthesis of T2-BB-2

[0112] T2-BB-1 (750 mg, 1.33 mmol, 1.0 eq) was dissolved in DMF (10 ml), and DIPEA (207 mg, 1.59 mmol, 1.2 eq) was added. The mixture was stirred at room temperature for 10 min. Di(p-nitrobenzene) carbonate (1213 mg, 3.99 mmol, 3.0 eq) was added to the reaction mixture, and the mixture was stirred at room temperature for 2 h. LC-MS confirmed the reaction was complete, and the mixture was concentrated under reduced pressure. The residue was washed with dry EtOAc to obtain T2-BB-2 (756 mg, 78%) as a white solid. LC-MS: 729.34 [M+H] + .

[0113] Step 3: Synthesis of T2-BB-5

[0114] T2-toxin (130 mg, 0.28 mmol, 1.0 eq) was dissolved in DMF (5 ml), and DMAP (34 mg, 0.28 mmol, 1.00 eq) and DIPEA (73 mg, 0.56 mmol, 2.0 eq) were added. The mixture was stirred at room temperature for 10 min. Di(p-nitrobenzene) carbonate (170 mg, 0.56 mmol, 2.0 eq) was added to the reaction mixture, and the mixture was stirred at room temperature for 1 h. LC-MS confirmed the completion of the reaction, yielding intermediate T2-BB-3. Dimethylethylenediamine (25 mg, 0.28 mmol, 1.0 eq) and DIPEA (73 mg, 0.56 mmol, 2.0 eq) were added to the reaction mixture, and the mixture was stirred at room temperature for 1 h. The reaction was completed by LC-MS detection, and the mixture was concentrated under reduced pressure. The residue was dissolved in DCM, washed three times with 1 M hydrochloric acid and saturated NaCl, and dried over anhydrous NaSO4. The organic phase was concentrated under reduced pressure to obtain T2-BB-5 (150 mg, 92%). 1 H NMR (400MHz, DMSO-d6) δ5.80(d,J=3.3Hz,1H),5.72(d,J=5.6Hz,1H),5.24(d,J=5.4Hz,1H),4.97-4.88 (m,1H),4.15(s,10H),3.96(dd,J=12.7,4.6Hz,1H),3.69(dd,J=9.6,4.8Hz,1H),3.09(d,J=3.9Hz,1H) ,2.94(s,1H),2.90(d,J=3.9Hz,1H),2.87(s,2H),2.47(s,3H),2.28-2.17(m,1H),2.10(d,J=4.9Hz,1H ),2.05(s,3H),1.98(s,3H),1.67(s,3H),0.90(dd,J=6.6,3.3Hz,6H),0.66(s,3H).LC-MS: 581.30[M+H] +.

[0115] Step 4: Synthesis of T2-BB-6

[0116] T2-BB-2 (400 mg, 0.55 mmol, 1.0 eq) was dissolved in DMF (5 ml), and HOBt (19 mg, 0.28 mmol, 0.3 eq) and pyridine (1.1 ml) were added. The mixture was stirred at room temperature for 10 min. T2-BB-5 (300 mg, 0.52 mmol, 0.9 eq) was added to the reaction mixture, and the mixture was stirred at room temperature overnight. LC-MS confirmed the reaction was complete, and the mixture was concentrated under reduced pressure. The residue was dissolved in EtOAc, washed three times with saturated NaHCO₃, dried over anhydrous NaSO₄, and the organic phase was concentrated under reduced pressure. The residue was washed with ether to give T2-BB-6 (500 mg, 77%). LC-MS: 1171.61 [M+H] + .

[0117] Step 5: Synthesis of T2-BB-7

[0118] T2-BB-6 (500 mg, 0.43 mmol, 1.0 eq) was dissolved in formic acid (2 ml) and stirred at room temperature for 3 h. The reaction was completed by LC-MS and concentrated under reduced pressure to give T2-BB-7 (396 mg, 82%). LC-MS: 1115.55 [M+H] + .

[0119] Step 2: Synthesis of T2-BB-7a

[0120] Compared with T2-BB-7, T2-BB-7a introduces an acidic amino acid glutamic acid at the N-terminus of valine, which can increase the stability of the linker in mouse plasma without affecting the intracellular cathepsin-mediated cleavage reactivity.

[0121] Step 3: Synthesis of T2-PDC-1

[0122] T2-BB-7 (50 mg, 0.04 mmol, 1.0 eq) was dissolved in DMF (2 ml), and HATU (15 mg, 0.04 mmol, 1.0 eq), DIPEA (21 mg, 0.16 mmol, 5.0 eq) and PSMA ligand (29 mg, 0.06 mmol, 1.5 eq) were added and stirred at room temperature for 1 h. The reaction was detected by LC-MS and concentrated under reduced pressure to obtain the intermediate T-2-BB-8. T2-BB-8 was dissolved in formic acid (2 ml) and stirred at room temperature for 1 hour. The reaction was detected by LC-MS and concentrated under reduced pressure. The residue was purified by reverse phase preparative chromatography to obtain T2-PDC-1 (16 mg, 25%), LC-MS: 709.67 (M+2H). 2+ (Figure 1).

[0123] Example 2: Synthesis of T2-PDC-2

[0124] T2-BB-7 (20 mg, 0.02 mmol, 1.0 eq) was dissolved in DMF (2 ml), and HATU (7 mg, 0.02 mmol, 1.0 eq), DIPEA (13 mg, 0.10 mmol, 5.0 eq) and HOSu (4 mg, 0.03 mmol, 1.5 eq) were added and stirred at room temperature for 1 h. The reaction was detected to be complete by LCMS, and DIPEA (13 mg, 0.10 mmol, 5.0 eq) and cRGD (18 mg, 0.02 mmol, 1.5 eq) were added to the mixture and stirred at room temperature overnight. The reaction was detected to be complete by LCMS, and T2-PDC-2 (11.3 mg, 37%) was obtained by purification using reverse phase preparative chromatography. 1 The H NMR spectrum is shown in Figure 2 , and the MS spectrum is shown in Figure 3 . 11H NMR (400 MHz, DMSO-d6) δ 10.01 (s, 1H), 8.39 (d, J = 9.0 Hz, 1H), 8.29 - 8.17 (m, 3H), 8.13 (d, J = 7.9 Hz, 2H), 7.82 (d, J = 6.4 Hz, 1H), 7.75 (d, J = 8.9 Hz, 1H), 7.58 (d, J = 8.2 Hz, 2H), 7.30 - 7.11 (m, 12H), 6.01 (t, J = 5.9 Hz, 1H), 5.80 (d, J = 3.4 Hz, 1H), 5.70 (s, 1H), 5.42 (s, 2H), 5.23 (d, J = 5.5 Hz, 1H), 5.09 - 4.98 (m, 3H), 4.67 - 4.56 (m, 1H), 4.48 (q, J = 7.4 Hz, 1H), 4.38 (q, J = 7.8 Hz, 1H), 4.32 - 4.10 (m, 6H), 3.96 (d, J = 12.4 Hz, 1H), 3.66 (d, J = 4.9 Hz, 1H), 3.12 - 3.04 (m, 2H), 2.98 (d, J = 6.3 Hz, 1H), 2.88 (s, 2H), 2.84 (s, 4H), 2.77 - 2.68 (m, 1H), 2.70 - 2.64 (m, 1H), 2.67 - 2.59 (m, 1H), 2.24 (dd, J = 15.2, 5.6 Hz, 1H), 2.20 - 2.08 (m, 2H), 2.08 - 1.98 (m, 3H), 1.97 (d, J = 5.6 Hz, 3H), 1.93 - 1.82 (m, 1H), 1.65 (s, 3H), 1.46 (s, 3H), 1.44 - 1.31 (m, 2H), 1.26 - 1.11 (m, 1H), 0.92 - 0.86 (m, 6H), 0.86 (s, 3H), 0.85 (s, 3H), 0.84 (s, 3H), 0.82 (s, 3H), 0.65 (s, 3H). LC-MS: 851.83 (M + 2H) 2+ .

[0125] Example 3: Synthesis of T2-PDC-3

[0126] T2-BB-7 (80 mg, 0.07 mmol, 1.0 eq) was dissolved in DMF (2 ml), and HATU (27 mg, 0.07 mmol, 1.0 eq), DIPEA (45 mg, 0.35 mmol, 5.0 eq) and HOSu (12 mg, 0.11 mmol, 1.5 eq) were added and stirred at room temperature for 1 h. The reaction was detected by LCMS. DIPEA (45 mg, 0.35 mmol, 5 eq) and Ty3-Octreotate (115 mg, 0.11 mmol, 1.5 eq) were added to the mixture and stirred at room temperature overnight. The reaction was detected by LCMS and purified by reverse phase preparative chromatography to give T2-PDC-3 (20 mg, 13%). LC-MS: 1073.93 (M+2H) 2+ (Figure 4).

[0127] Example 4: Synthesis of T2-PDC-2G

[0128] Step 1: Evcit-Fmoc synthesis

[0129] Fmoc-O-tert-butyl-L-glutamic acid (617 mg, 1.45 mmol, 1.1 eq) was dissolved in dry DMF (10 ml), and HATU (602 mg, 1.58 mmol, 1.2 eq) and DIPEA (512 mg, 3.96 mmol, 3.0 eq) were added. The mixture was stirred at room temperature for 10 min. (S)-2-[(S)-2-amino-3-methylbutanamido]-N-[4-(hydroxymethyl)phenyl]-5-ureapentanamide (500 mg, 1.32 mmol, 1.0 eq) was added to the reaction mixture, and the mixture was stirred at room temperature for 2 h. LC-MS confirmed the reaction was complete, and DCM was added to precipitate the product. The residue was filtered and washed with DCM to obtain Evcit-Fmoc as a reddish-brown solid (850 mg, 82%). LCMS: 787.39 [M+H] + .

[0130] Step 2: Evcit-Fmoc-1 synthesis

[0131] Evcit-Fmoc (850 mg, 1.08 mmol, 1.0 eq) was dissolved in DMF (10 ml), and DIPEA (176 mg, 1.29 mmol, 1.2 eq) was added. The mixture was stirred at room temperature for 10 min. Di(p-nitrobenzene) carbonate (986 mg, 3.24 mmol, 3.0 eq) was added to the reaction mixture, and the mixture was stirred at room temperature for 2 h. LC-MS confirmed the reaction was complete. The product was precipitated by adding water, filtered, and the residue was washed with acetonitrile to obtain Evcit-Fmoc-1 (748 mg, 73%) as a white solid. LC-MS: 952.40 [M+H] + .

[0132] Step 3: Evcit-Fmoc-3 synthesis

[0133] Evcit-Fmoc-1 (748 mg, 0.79 mmol, 1.0 eq) was dissolved in DMF (5 ml), and HOBt (32 mg, 0.24 mmol, 0.3 eq) and pyridine (1.1 ml) were added. The mixture was stirred at room temperature for 10 min. T2-BB-5 (412 mg, 0.72 mmol, 0.9 eq) was added to the reaction mixture, and the mixture was stirred at room temperature overnight. LC-MS confirmed the completion of the reaction. The product was precipitated by adding water, filtered, and washed with ether to obtain Evcit-Fmoc-2 (901 mg, 82%). Evcit-Fmoc-2 (901 mg, 0.64 mmol, 1.0 eq) was dissolved in THF (2 ml), and diethylamine (2 ml) was added. The mixture was stirred at room temperature for 1 h. The reaction was completed by LC-MS, and the product was concentrated under reduced pressure and purified by reverse phase chromatography to obtain Evcit-Fmoc-3 (569 mg, 76%). LC-MS: 1171.60 [M+H] + .

[0134] Step 4: Evcit-Fmoc-6 synthesis

[0135] Evcit-Fmoc-3 (569 mg, 0.48 mmol, 1.0 eq) was dissolved in dry DMF (5 ml), and adipic acid (77 mg, 0.52 mmol, 1.1 eq), HATU (219 mg, 0.58 mmol, 1.2 eq), and DIPEA (186 mg, 1.44 mmol, 3.0 eq) were added. The mixture was stirred at room temperature for 2 h. LC-MS confirmed the reaction was complete to yield Evcit-Fmoc-4. HOSu (83 mg, 0.72 mmol, 1.5 eq) was added to the reaction mixture, and the mixture was stirred at room temperature for 1 h. LC-MS confirmed the reaction was complete to yield Evcit-Fmoc-5. cRGD (434 mg, 0.72 mmol, 1.5 eq) was added to the reaction solution and stirred at room temperature for 2 h. The reaction was completed by LC-MS. The product was purified by reverse phase chromatography to obtain Evcit-Fmoc-6 (379 mg, 42%). LC-MS: 943.95 [M+2H] 2+ .

[0136] Step 5: T2-PDC2-G synthesis

[0137] Evcit-Fmoc-6 (50 mg, 0.02 mmol, 1.0 eq) was dissolved in formic acid (2 ml) and stirred at room temperature for 3 h. The reaction was detected by LCMS and purified by reverse phase preparative chromatography to give T2-PDC2-G (14 mg, 39%). LC-MS: 915.89 [M+2H] 2+ (Figure 5).

[0138] Test Case

[0139] Test Example 1: Target protein binding ability of targeting peptides (SPR method)

[0140] 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 T2-PDC2) 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).

[0141] 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.

[0142] Table 1

[0143] Results: The binding affinity of c(RGDfK) molecule to the target protein was 1.57uM, while that of T2-PDC2 to the target protein was 0.46uM. The binding affinity of T2-PDC2 to the target protein was better than that of c(RGDfK) ( Figure 6 ).

[0144] Test Example 2: PDC in vitro efficacy evaluation

[0145] The in vitro pharmacology of the PDC molecules prepared in the examples (Table 2) was evaluated on melanoma, renal cancer, colorectal cancer, liver cancer, prostate cancer and other cells (Table 3). The target information of the PDC molecules involved is shown in Table 1 below.

[0146] Table 2: PDC molecule number and related information

[0147] Table 3: Cell lines and culture conditions used in this experiment

[0148] Experimental design:

[0149] 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.

[0150] 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, 5% CO2. The plates were then photographed and the 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 and Figure 7 below.

[0151] Table 4: Inhibitory effect of T2-PDC1 on tumor cells

[0152] Results: T2-PDC2 showed cytotoxicity against A375 and HepG2, two tumor cell lines with high expression of ITB3. 50 The concentration of the PDC compound can be 5-6 micromolar, which is about 7-8 times higher than that of the target low-expressing cell line, indicating that the PDC compound designed and synthesized in this application has the expected tumor cell targeting effect.

[0153] However, in in vitro toxicity tests, T2-PDC2 had no targeting effect on A498 cells that lowly expressed ITB3 protein.

[0154] Test Example 3: Serum Stability Assessment

[0155] 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;

[0156] 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;

[0157] 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;

[0158] 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;

[0159] ⑤ Blood samples were collected from the orbital venous plexus at 0h, 0.5h, 1h, 2h, 4h, and 8h after inoculation, with each collection amount of approximately 50ul.

[0160] ⑥Separate the serum from the mouse 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;

[0161] ⑦ Draw a concentration curve based on the drug concentration in serum at different time points and calculate the half-life of the drug to be tested in rat blood.

[0162] Results: The test results are shown in Figure 8. The half-life of T2-PDC2 in mouse serum was measured to be approximately 1 hour. The half-life of T2-PDC2G obtained by introducing glutamic acid into the peptide linker Val-Cit was significantly extended to more than 8 hours.

[0163] Test Example 4: Acute Toxicity Assessment

[0164] 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;

[0165] 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.

[0166] 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;

[0167] 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;

[0168] ⑤ Observe the mice for two weeks after inoculation and record in detail any abnormal reactions such as death, lethargy, and stress levels;

[0169] ⑥ Two weeks later, the mice were euthanized and the liver and kidney toxicity pathologies were observed by autopsy;

[0170] ⑦ Evaluate the maximum tolerated dose of mice based on the abnormal reaction records and organ lesions of mice.

[0171] Results: The maximum dose of T2-PDC2 reached 37.5 mg / kg body weight (equivalent to 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 T2-PDC2 is above 37.5 mg / kg.

[0172] Test Example 5: In vivo efficacy evaluation

[0173] Construction of orthotopic liver tumor-bearing mouse model:

[0174] 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;

[0175] 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.

[0176] 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 300mm3 Orthotopic transplantation was performed when left and right;

[0177] 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.

[0178] 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.

[0179] Evaluation of the anti-tumor efficacy of the test drug in an orthotopic liver tumor-bearing mouse model:

[0180] 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;

[0181] ② The drug to be tested was divided into two dose groups (10 mpk and 20 mpk), plus a saline negative (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.

[0182] 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);

[0183] 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.

[0184] ⑤ Body weight data collection (twice a week): Weigh the animals twice a week and record the weight changes, and draw the weight change curve (Figure 9B);

[0185] 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 9A);

[0186] ⑦The trial endpoint is the completion of 4 weeks of dosing;

[0187] ⑧Sample collection and analysis at the test endpoint:

[0188] a) After euthanasia of the animals, serum was collected and stored at -80°C;

[0189] b) Liver tumors were measured and photographed, then snap-frozen in liquid nitrogen and stored at -80°C;

[0190] c) Collect heart, liver, spleen, lung, and kidney tissues and freeze them in liquid nitrogen and store at -80°C;

[0191] 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.

[0192] As can be seen from the results in FIG9 , both the T2-PDC-2 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.

[0193] 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 variations and modifications 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 realize and utilize a variety of exemplary embodiments of the invention and various options and modifications. 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 peptide containing 2-9 amino acids; L is a linking group; D is selected from T2 toxin, its derivatives, prodrugs or metabolites.

2. The compound according to claim 1, wherein D is selected from T2 is selected from Preferably, D is 3. The compound according to claim 1 or 2, wherein The amino acids constituting P are selected from one or more of cysteine, phenylalanine, tryptophan, lysine, threonine, arginine, aspartic acid, and glutamic acid.

4. The compound according to claim 1 or 2, wherein P is selected from a cyclic peptide comprising 4-9 amino acids; Preferably, 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 -CH2-phenyl-OH, -CH(CH3)-OH or R 5 , R 6 , R 7 , R 8 Each is independently selected from hydrogen, -(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 claim 1 or 2, wherein P is selected from a peptide comprising 2 or 3 amino acids and / or a urea group; Preferably, P is 6. The compound according to any one of claims 1 to 5, 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 Selected from a peptide residue comprising 2-7 amino acids; 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 -、-(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.

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 PSMA, ITB and / or SSTR.

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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