Compounds as well as preparation method therefor and use thereof

Abstract

Provided are borneol-modified peptide compounds, a preparation method therefor, a pharmaceutical composition thereof and the use thereof. The compounds are prepared by means of chemical coupling of borneol and polypeptides. The compounds can dissolve thrombi and thus can be used for cerebral ischemia protection.

Description

Compounds, methods of making and uses thereof TECHNICAL FIELD

[0001] The present disclosure belongs to the field of biological medicine, and relates to a plurality of menthol-modified peptide compounds, methods of preparation and uses thereof in pharmaceuticals and disease treatment. BACKGROUND

[0002] In recent years, the incidence of thrombotic diseases, especially ischemic stroke, has been increasing. Recurrence of stroke in patients can lead to aggravation of existing neurological dysfunction, disability, and significantly increased mortality, which seriously threatens human health. Therefore, drug treatment of thrombotic diseases is the focus and hotspot of ischemic stroke treatment.

[0003] Ischemic stroke, also known as stroke, is triggered by a variety of mechanisms, including activation of glutamate receptors, glutamate release and calcium influx, thereby activating nitric oxide, cysteine proteases and proteases. This causes inflammation, production of free radicals and protein damage leading to apoptosis of neuronal cells (Rogalewski et al., 2006). Other key features of stroke include blood-brain barrier (BBB) damage, oxidative stress, cytokine-mediated toxicity, excitotoxicity and loss of neuronal function (Kuriakose and Xiao, 2020). Simultaneous activation of multiple pathways makes the treatment of ischemic stroke and neurological damage challenging. Therefore, cerebral ischemia protection is of great value for the treatment of thrombotic diseases, and an appropriate compound for thrombotic diseases is urgently needed to provide cerebral ischemia protection.

[0004] SUMMARY

[0005] The present disclosure provides a menthol-modified peptide compound, which has a cerebral ischemia protection application and can relieve or alleviate neurological symptoms and brain tissue damage caused by cerebral ischemia.

[0006] A first aspect of the present disclosure provides a compound or a prodrug, tautomer, optical isomer, geometric isomer, solvate thereof, or a pharmaceutically acceptable salt thereof, comprising a menthol moiety and a polypeptide moiety coupled to the menthol, the polypeptide moiety comprising Pro-Ala-Lys (PAK), Ala-Lys-Pro (AKP) or Lys-Ala-Pro (KAP).

[0007] In certain embodiments, the compound has a structure as shown in Formula I:

[0008] wherein L represents a linker, P 1 represents a polypeptide moiety.

[0009] In certain embodiments, the polypeptide portion of the compound is derived from a polypeptide having thrombolytic activity and / or free radical scavenging activity.

[0010] In certain embodiments, the linker L of the compound is covalently linked to the N-terminus of the polypeptide portion P. 1

[0011] In certain embodiments, the linker L of the compound is ester-bonded to the hydroxyl group of the camphor.

[0012] In certain embodiments, the camphor of the compound is dextrocamphor.

[0013] In certain embodiments, the compound has a structure as shown in Formula II:

[0014] wherein 0 and 1 represent the number of H; when the number of H is 1, -NH- represents the imino group at the N-terminus of the polypeptide portion; when the number of H is 0, N represents the ring nitrogen atom at the N-terminus of the polypeptide portion;

[0015] P represents the polypeptide residues of the polypeptide portion other than the N-terminus -N(H) 0,1 - of the polypeptide portion.

[0016] In certain embodiments, the polypeptide portion comprises one, two or three repeat sequence peptides of at least one of Pro-Ala-Lys, Ala-Lys-Pro and Lys-Ala-Pro.

[0017] In certain embodiments, the polypeptide portion of the compound is a tripeptide, a tetrapeptide, a pentapeptide, a hexapeptide, a heptapeptide, an octapeptide, a nonapeptide, a decapeptide or an undecapeptide.

[0018] In certain embodiments, the polypeptide portion comprises a dipeptide fragment β-Ala-His, Arg-Gly at the N-terminus of the Pro-Ala-Lys, Ala-Lys-Pro or Lys-Ala-Pro fragment, or His-β-Ala, Arg-Gly at the C-terminus.

[0019] In certain embodiments, the polypeptide portion further comprises a Lys residue covalently linked to the linker, and optionally, the C-terminus of the Lys residue is linked to a Arg-Gly-Asp-Ser or Ile-Glu-Ser-Asp-Val fragment.

[0020] In certain embodiments, the peptide fragment comprising Lys-Ala-Pro is linked to the Lys residue.

[0021] ​In some embodiments, the polypeptide moiety has one or more PEG modifications at one or more sites of the C-terminus, N-terminus, or internal residues of the polypeptide moiety, optionally, the PEG is selected from one or more of PEG2 to PEG24.

[0022] In some embodiments, the polypeptide moiety has a PEG modification at the N-terminus residue of the polypeptide moiety, the PEG modification is selected from PEG2, PEG4, PEG8, PEG12, or PEG24, optionally, the PEG modification is linked to a linker.

[0023] In some embodiments, the polypeptide of the compound comprises one or more of the following polypeptide fragments: Pro-Ala-Lys, Lys-Ala-Pro, β-Ala-His-Lys-Ala-Pro, β-Ala-His-Pro-Ala-Lys, Gly-Arg-Pro-Ala-Lys, PEG4-Pro-Ala-Lys, Lys(Lys-Ala-Pro)-Arg-Gly-Asp-Ser, Lys(Lys-Ala-Pro-His-β-Ala)-Arg-Gly-Asp-Ser, Lys(Lys-Ala-Pro-Arg-Gly)-Arg-Gly-Asp-Ser, Lys(Lys-Ala-Pro)-Ile-Glu-Ser-Asp-Val, Lys(Lys-Ala-Pro-His-Ala-β)-Ile-Glu-Ser-Asp-Val, or Lys(Lys-Ala-Pro-Arg-Gly)-Ile-Glu-Ser-Asp-Val.

[0024] The second aspect of the present disclosure provides a method for preparing the aforementioned compound, comprising: preparing an intermediate comprising an ester bond from camphor and a coupling agent under reaction conditions, sequentially bonding the intermediate to corresponding amino acids, purifying to obtain the compound.

[0025] The third aspect of the present disclosure provides a pharmaceutical composition, comprising the aforementioned compound or the compound prepared by the aforementioned method, and one or more pharmaceutically acceptable excipients.

[0026] The fourth aspect of the present disclosure provides use of the aforementioned compound, the compound prepared by the aforementioned method, or the aforementioned pharmaceutical composition in the preparation of a thrombolytic, free radical scavenging, or anti-inflammatory drug.

[0027] In some embodiments, the use comprises use of the compound or the pharmaceutical composition in the preparation of a cerebral ischemia protective drug.

[0028] In some embodiments, the compound or the pharmaceutical composition has use in the preparation of a drug for treating thrombotic diseases.

[0029] In some embodiments, the thrombotic disease comprises ischemic stroke, myocardial infarction, stroke, venous embolism, pulmonary embolism, peripheral arterial occlusive disease, venous catheter occlusion, arteriovenous fistula and shunt occlusion, and carotid stenosis.

[0030] The fifth aspect of the present disclosure provides a method for treating a subject having a thrombotic disease, comprising administering to the subject an effective amount of the aforementioned compound, the compound prepared by the aforementioned method, or the aforementioned pharmaceutical composition.

[0031] The sixth aspect of the present disclosure provides a method for improving cerebral ischemia, cerebral thrombosis, free radical state, or inflammatory state in a subject, comprising administering to the subject in need thereof an effective amount of the aforementioned compound, the compound prepared by the aforementioned method, or the aforementioned pharmaceutical composition. BRIEF DESCRIPTION OF DRAWINGS

[0032] Figure 1 shows the mass spectrum of camphor-linker-Pro-Ala-Lys in Example 2;

[0033] Figure 2 shows the mass spectrum of camphor-linker-Lys-Ala-Pro in Example 3;

[0034] Figure 3 shows the mass spectrum of camphor-linker-β-Ala-His-Lys-Ala-Pro in Example 4;

[0035] Figure 4 shows the mass spectrum of camphor-linker-β-Ala-His-Pro-Ala-Lys in Example 5;

[0036] Figure 5 shows the mass spectrum of camphor-linker-Gly-Arg-Pro-Ala-Lys in Example 6;

[0037] Figure 6 shows the mass spectrum of camphor-linker-PEG4-Pro-Ala-Lys in Example 7;

[0038] Figure 7 shows the mass spectrum of camphor-linker-Lys(Lys-Ala-Pro)-Arg-Gly-Asp-Ser in Example 8;

[0039] Figure 8 shows the mass spectrum of camphor-linker-Lys(Lys-Ala-Pro-His-β-Ala)-Arg-Gly-Asp-Ser in Example 9;

[0040] Figure 9 shows the mass spectrum of camphor-linker-Lys(Lys-Ala-Pro-Arg-Gly)-Arg-Gly-Asp-Ser in Example 10;

[0041] Figure 10 shows the mass spectrum of camphor-linker-Lys(Lys-Ala-Pro)-Ile-Glu-Ser-Asp-Val in Example 11;

[0042] Figure 11 shows the mass spectrum of camphor-linker-Lys(Lys-Ala-Pro-His-Ala-β)-Ile-Glu-Ser-Asp-Val in Example 12;

[0043] Figure 12 shows the mass spectrum of camphor-linker-Lys(Lys-Ala-Pro-Arg-Gly)-Ile-Glu-Ser-Asp-Val in Example 13;

[0044] Figure 13 shows the effect of the test drug on the neurological symptom score of MCAO rats in Example 14. DETAILED DESCRIPTION

[0045] The embodiments of the present application are illustrated by the following specific examples, and other advantages and effects of the present application can be easily understood by those skilled in the art from the disclosure.

[0046] Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as molecular weight, percent, ratios, amounts, time, temperatures, thicknesses, and so forth, used in the specification and claims are to be understood as being modified in all instances by the term "about." Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations. At the very least, each numerical parameter should at least be construed in light of the number of significant digits and by applying ordinary rounding techniques, or by varying the numerical parameter a little arithmetic from the stated value — this according to the application at hand and the normal of the art to which the application pertains.

[0047] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the disclosure are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

[0048] In this application, the term "comprise" generally means include, encompass, contain or consist of, in some cases, it also means "consist essentially of".

[0049] As used herein, the expression "A and / or B" includes three cases: (1) A; (2) B; and (3) A and B. The expression "A, B and / or C" includes seven cases: (1) A; (2) B; (3) C; (4) A and B; (5) A and C; (6) B and C; and (7) A, B and C. The meaning of similar expressions can be extended in this way.

[0050] Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as molecular weight, percent, ratios, amounts, volumes, times, temperatures, thicknesses, and so forth, used in the specification and claims are to be understood as being modified in all instances by the term "about." Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations. Any numerical value, however, can inherently contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements and / or the experimentation by a person of ordinary skill in the art, according to the state of the art, and can, accordingly, be contemplated as modifying the numerical parameters set forth in the following specification and attached claims.

[0051] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the disclosure are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, can inherently contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements and / or the experimentation by a person of ordinary skill in the art, according to the state of the art. Every numerical range given throughout this specification will include every narrower numerical range falling within such broader numerical range, as if such narrower numerical ranges were all expressly written herein.

[0052] [Compound]

[0053] The treatment of thrombotic diseases, especially those occurring in the brain, is complicated by the presence of the blood-brain barrier, which prevents many thrombolytic drugs from crossing the blood-brain barrier and entering the brain. In addition, a large number of free radicals generated during cerebral ischemia-reperfusion can also cause damage to brain tissue, resulting in impaired brain function. Therefore, compounds that can cross the blood-brain barrier and have a brain protective effect are very important for the treatment of thrombotic diseases in the brain.

[0054] Generally, the "blood-brain barrier" refers to the barrier between plasma and brain cells formed by the walls of brain capillaries and glial cells and the barrier between plasma and cerebrospinal fluid formed by the choroid plexus, which prevents certain substances from entering the brain tissue from the blood.

[0055] The present application provides a camphor-modified peptide compound or a prodrug, tautomer, optical isomer, geometric isomer, solvate or pharmaceutically acceptable salt thereof, which can be used for the treatment of thrombotic diseases, the compound comprising a camphor moiety and a polypeptide moiety covalently coupled (chemically covalently linked) to the camphor.

[0056] As used herein, the term "polypeptide" refers to a compound formed by the dehydration condensation of three or more amino acids linked together by peptide bonds. The polypeptide can be prepared by chemical synthesis or by protein hydrolysis. The types of amino acids in the polypeptide can include, but are not limited to, L-amino acids, D-amino acids, alpha-type amino acids or beta-type amino acids, and the like natural or unnatural amino acids.

[0057] In some embodiments, the compound has a structure as shown in Formula I:

[0058] wherein L represents a linker, P 1 represents a polypeptide moiety.

[0059] Previous studies have shown that inflammation is a potential therapeutic target for ischemic stroke, which can promote a vicious cycle pathway with excitotoxicity, oxidative stress and other factors, leading to increased damage, and further leading to infarct expansion, nerve cell damage, brain edema, and the like.

[0060] Camphor (or borneol) is a small molecule bicyclic monopeptide compound with good fat solubility, which can easily penetrate the blood-brain barrier into the brain and play its anti-inflammatory and brain protective role. The camphor moiety in the compound shown in Formula I can improve the fat solubility of the compound, facilitate the penetration of the compound through the blood-brain barrier, and play an anti-inflammatory and brain protective role.

[0061] In some embodiments, the camphor is levorotatory camphor. In some embodiments, the camphor is dextrorotatory camphor, which can inhibit the expression of inflammatory cytokines such as TNF-α (Tumor Necrosis Factor-α), IL-1β (Interleukin-1β) and proinflammatory proteins such as COX-2 (Cyclooxygenase-2) and iNOS (inducible nitric oxide sythase) during cerebral ischemia-reperfusion, and activate GABAa receptor, block the above vicious cycle pathway, and thus reduce cell apoptosis and cell necrosis, protect the blood-brain barrier, reduce brain edema, and thus reduce ischemia-reperfusion injury.

[0062] The linker is used to connect the camphor moiety and the polypeptide moiety. In some embodiments, the camphor is connected to the polypeptide moiety through a cleavable or non-cleavable linker L. In some embodiments, the camphor is connected to the polypeptide moiety through a cleavable linker L, and under suitable cleavage conditions, the compound shown in Formula I is cleaved to release free camphor and / or polypeptide, thereby exerting a therapeutic effect.

[0063] In some embodiments, the camphor is bonded to the linker L through an ester bond. In some embodiments, the linker L forms an ester bond connection with the hydroxyl group of the camphor. In some embodiments, the linker L is bonded to the N-terminus of the polypeptide moiety through an amide bond.

[0064] In some embodiments, the linker L is -C(=0)-.

[0065] In some embodiments, the compound has a structural formula as shown in Formula II:

[0066] wherein 0 and 1 represent the number of H; when the number of H is 1, -NH- represents the imino group at the N-terminus of the polypeptide moiety; when the number of H is 0, N represents the ring nitrogen atom at the N-terminus of the polypeptide moiety; P represents the polypeptide residues other than the N-terminal -N(H) 0,1 .

[0067] In the compounds provided by the present disclosure, the polypeptide moiety is derived from a polypeptide having thrombolytic activity and / or free radical scavenging activity. The term "derived" refers to the processing of atoms or groups in a certain original substance (or compound) through at least one of the reactions including substitution, removal, condensation and addition, to form a more complex or simpler product with a change in chemical bond compared to the original substance.

[0068] In some embodiments, the polypeptide of the polypeptide moiety has thrombolytic activity. In some embodiments, the polypeptide of the polypeptide moiety has free radical scavenging activity. In some embodiments, the polypeptide of the polypeptide moiety has both thrombolytic activity and free radical scavenging activity, and cooperates with camphor in vivo, thereby achieving the effects of thrombolysis, anti-inflammation and free radical elimination.

[0069] In some embodiments, the polypeptide moiety comprises a polypeptide fragment of Pro-Ala-Lys, Ala-Lys-Pro or Lys-Ala-Pro from the N-terminus to the C-terminus.

[0070] Herein, the "fragment" of a polypeptide includes an intact, separable polypeptide segment, and also includes a polypeptide residue segment covalently bonded to other molecules, which is not separable, such as P 1 , which can be easily understood and distinguished by those skilled in the art in terms of specific meanings of fragments under different conditions.

[0071] As used herein, the standard three-letter or one-letter code rules for amino acids are used, specifically, alanine-Ala (A), cysteine-Cys (C), aspartic acid-Asp (D), glutamic acid-Glu (E), phenylalanine-Phe (F), glycine-Gly (G), histidine-His (H), isoleucine-Ile (I), lysine-Lys (K), leucine-Leu (L), methionine-Met (M), asparagine-Asn (N), proline-Pro (P), glutamine-Gln (Q), arginine-Arg (R), serine-Ser (S), threonine-Thr (T), valine-Val (V), tryptophan-Trp (W), tyrosine-Tyr (Y).

[0072] In the camphor-modified peptide compounds provided by the present disclosure, the polypeptide moiety can comprise one, two, three, four, five, six or more repeat sequence peptides having at least one of Pro-Ala-Lys, Ala-Lys-Pro and Lys-Ala-Pro as a structural unit. The structural units can be distributed continuously or intermittently.

[0073] In some embodiments, the polypeptide moiety comprises one, two or three repeat sequence peptides having Pro-Ala-Lys, Ala-Lys-Pro or Lys-Ala-Pro as a structural unit.

[0074] In some embodiments, the polypeptide moiety comprises one repeat sequence peptide having Pro-Ala-Lys, Ala-Lys-Pro or Lys-Ala-Pro as a structural unit.

[0075] In the present disclosure, the polypeptide moiety can be a tripeptide, a tetrapeptide, a pentapeptide, a hexapeptide, a heptapeptide, an octapeptide, a nonapeptide, a decapeptide or an undecapeptide, or a peptide having more than eleven amino acids. In some embodiments, the polypeptide moiety is a tripeptide, a tetrapeptide, a pentapeptide, a hexapeptide, a heptapeptide, an octapeptide, a nonapeptide, a decapeptide or an undecapeptide.

[0076] In the present disclosure, a "tripeptide" refers to a peptide fragment formed by linking three natural or unnatural amino acids via peptide bonds, which can be a straight-chain peptide fragment or a peptide fragment containing a branch. It can be understood that a dipeptide, a tetrapeptide, a pentapeptide, a hexapeptide, a heptapeptide, an octapeptide, a nonapeptide, a decapeptide and an undecapeptide have similar understanding.

[0077] In some embodiments, the N-terminal or C-terminal of the Pro-Ala-Lys, Ala-Lys-Pro or Lys-Ala-Pro fragment in the polypeptide fragment is linked to β-Ala-His, His-β-Ala or Arg-Gly.

[0078] In some embodiments, the polypeptide moiety comprises a dipeptide fragment of β-Ala-His, Arg-Gly, at the N-terminus of a Pro-Ala-Lys, Ala-Lys-Pro, or Lys-Ala-Pro fragment, or His-β-Ala, Arg-Gly, at the C-terminus.

[0079] In some embodiments, the polypeptide moiety can further comprise a polypeptide fragment of Arg-Gly-Asp-Ser or Ile-Glu-Ser-Asp-Val, from N-terminus to C-terminus.

[0080] In some embodiments, the polypeptide moiety further comprises a Lys residue covalently linked to the linker, and optionally, the C-terminus of the Lys residue is linked to a Arg-Gly-Asp-Ser or Ile-Glu-Ser-Asp-Val fragment. In some embodiments, the polypeptide moiety comprises a polypeptide fragment of Lys-Arg-Gly-Asp-Ser or Lys-Ile-Glu-Ser-Asp-Val, from N-terminus to C-terminus.

[0081] In some embodiments, the polypeptide moiety comprises a peptide fragment of Lys-Ala-Pro linked to the Lys residue, in which case the peptide fragment can exist as a branched peptide fragment. For example, the polypeptide moiety comprises a polypeptide fragment of Lys(Lys-Ala-Pro)-, in which the peptide fragment Lys-Ala-Pro within the parentheses represents a branched peptide chain and is covalently linked to the Lys immediately to the left of the parentheses.

[0082] In some embodiments, the polypeptide moiety comprises a polypeptide fragment of Lys(Lys-Ala-Pro-His-β-Ala)- or Lys(Lys-Ala-Pro-Arg-Gly)-. It is understood that other polypeptide moieties in the disclosure having branched peptide chains have similar understanding and meaning.

[0083] In the compounds provided by the disclosure, the amino acids (or amino acid residues) of the polypeptide moiety can be natural or chemically modified, either naturally or artificially, including but not limited to, phosphorylation, acetylation, methylation, glycosylation, ubiquitination, succinylation, crotonylation, 2-hydroxyisobutyrylation, lactic acidation, polyethylene glycol (PEG)ation. In some embodiments, one or more sites of the amino acids (or amino acid residues) of the polypeptide moiety, including the C-terminus, N-terminus, and intermediate residues of the polypeptide moiety, comprise polyethylene glycol PEG, glycosylation, phosphorylation modification.

[0084] In some embodiments, the polypeptide moiety has one or more PEG modifications at one or more sites of the C-terminal end, N-terminal end, and / or internal residues. In some embodiments, the N-terminal residue of the polypeptide moiety comprises a PEG modification. Optionally, the PEG is selected from any one or more of PEG2 to PEG24, including but not limited to PEG2, PEG4, PEG8, PEG12, PEG24. In some embodiments, the N-terminal residue of the polypeptide moiety comprises a PEG4 modification. The PEG modification can extend the half-life of the compound or polypeptide moiety provided herein in a living organism, reduce or eliminate immunogenicity, and reduce toxic side effects.

[0085] In some embodiments, the PEG modification is covalently linked to a linker. In some embodiments, one end of the PEG contains a carboxyl group and / or an amino group. In some embodiments, the PEG used in the PEG modification is NH2-PEG4-COOH, wherein the amino group forms an amide bond with the linker and the carboxyl group forms an amide bond with the N-terminal amino group of the amino acid.

[0086] In some embodiments, the polypeptide moiety comprises or has one or more of the following (1) to (12) groups:

[0087] (1) Pro-Ala-Lys; (2) Lys-Ala-Pro; (3) β-Ala-His-Lys-Ala-Pro; (4) β-Ala-His-Pro-Ala-Lys; (5) Gly-Arg-Pro-Ala-Lys; (6) PEG4-Pro-Ala-Lys; (7) Lys(Lys-Ala-Pro)-Arg-Gly-Asp-Ser; (8) Lys(Lys-Ala-Pro-His-β-Ala)-Arg-Gly-Asp-Ser; (9) Lys(Lys-Ala-Pro-Arg-Gly)-Arg-Gly-Asp-Ser; (10) Lys(Lys-Ala-Pro)-Ile-Glu-Ser-Asp-Val; (11) Lys(Lys-Ala-Pro-His-Ala-β)-Ile-Glu-Ser-Asp-Val; (12) Lys(Lys-Ala-Pro-Arg-Gly)-Ile-Glu-Ser-Asp-Val.

[0088] The polypeptide moieties of the above (1) to (12) groups have the backbone and side chain structures shown in the following table:

[0089] The camphor-modified peptide compound provided by the present disclosure has suitable physical properties, can well penetrate the blood-brain barrier, and can self-degrade in the brain to release camphor and a polypeptide having thrombolytic activity, thereby exerting thrombolytic, anti-inflammatory and free radical scavenging effects, and reducing cell necrosis and tissue damage through synergistic effects, thereby exerting a cerebral ischemia protective effect.

[0090] [Preparation method]

[0091] The present disclosure provides a preparation method of the aforementioned compound, comprising: preparing an intermediate comprising an ester bond by reacting camphor with a coupling agent under reaction conditions, sequentially bonding the intermediate to corresponding amino acids, and purifying to obtain the compound.

[0092] In this context, the term "coupling agent" refers to a class of substances having at least two reactive groups that can participate in chemical reactions and form chemical bonds, thereby combining two substances.

[0093] In some embodiments, the coupling agent can be 4-nitrophenyl chloroformate.

[0094] In some embodiments, the intermediate can be sequentially bonded to the corresponding amino acids using solid-phase peptide synthesis technology. Solid-phase peptide synthesis technology is well known to those skilled in the art and can be practiced skillfully. In this method, the synthesis of the peptide in the compound of the present disclosure can be carried out by sequentially combining the desired amino acid residues into a growing peptide chain one at a time according to the general principles of solid-phase methods. These methods are disclosed in a number of references, including Merrifield, R. B., Solid phase synthesis (Nobel lecture). Angew Chem 24: 799-810 (1985); and Barany et al., The Peptides, Analysis, Synthesis and Biology, Vol. 2, Gross, E. and Meienhofer, J. Eds., Academic Press 1-284 (1980).

[0095] In the chemical synthesis of peptides, the reactive side chain groups of each amino acid residue are protected with appropriate protecting groups to prevent chemical reactions at that site before the protecting group is removed. For example, a protecting group is used to protect the alpha amino group (e.g., 9-fluorenylmethyloxycarbonyl (Fmoc), tert-butyloxycarbonyl (Boc)) when reacting at the carboxyl group of the amino acid or fragment, followed by selective removal of the alpha amino protecting group, thereby allowing the site to undergo subsequent reactions.

[0096] Orthogonal protecting groups can be used as appropriate to make polypeptides containing side chains, e.g., Fmoc-Ser(tBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Arg(pbf)-OH.

[0097] The compounds can be cleaved from the solid phase using any appropriate reagent, e.g., a combination of trifluoroacetic acid (TFA), triisopropylsilane (TIS), water. The final product is precipitated by the addition of cold ether and collected by filtration.

[0098] Purification can be performed using any of the methods known in the art, e.g., reverse-phase high performance liquid chromatography.

[0099] While the synthesis is described primarily with reference to solid phase peptide synthesis methods, it is understood that other chemical and synthetic methods can be employed to make the compounds of the present application.

[0100] [Pharmaceutical compositions]

[0101] The present disclosure provides a pharmaceutical composition comprising the aforementioned compound or a compound prepared by the aforementioned method, and further comprises a pharmaceutically acceptable excipient.

[0102] The pharmaceutical composition of the present disclosure can be formulated by any means known in the art, including but not limited to dosage forms formulated as tablets, capsules, caplets, suspensions, powders, lyophilized preparations, suppositories, eye drops, skin patches, orally dissolvable preparations, sprays, aerosols, etc. solid, semi-solid or liquid systems.

[0103] The pharmaceutical composition can be an immediate release and / or modified release formulation, including delayed release, sustained release, pulsed release, controlled release, targeted release, and programmed release formulations.

[0104] As used herein, "pharmaceutically acceptable excipient" means an ingredient other than the active ingredient of a pharmaceutical composition which is nontoxic to the subject. A pharmaceutically acceptable excipient includes, but is not limited to, excipients (e.g., diluents, carriers, and the like) and additives (e.g., stabilizers, preservatives, solubilizers, buffers, and the like). Excipients can include polyvinylpyrrolidone, gelatin, hydroxypropyl cellulose (HPC), acacia, polyethylene glycol, mannitol, sodium chloride, and sodium citrate. For injectable or other liquid administration formulations, it is preferred that water containing at least one or more buffering components be used, and stabilizers, preservatives, and solubilizers can also be employed. For solid administration formulations, any of a variety of thickening agents, fillers, extenders, and carrier additives can be employed, such as starch, sugar, cellulose derivatives, fatty acids, and the like. For topical administration formulations, any of a variety of creams, ointments, gels, lotions, and the like can be employed. For most pharmaceutical formulations, the non-active ingredients can comprise a large portion of the formulation by weight or volume. For pharmaceutical formulations, any of a variety of metered release, sustained release, or continuous release formulations and additives can also be employed, such that the dosage can be formulated to deliver the compounds of the disclosure over a period of time.

[0105] The compounds of the disclosure can be administered by a variety of routes including, but not limited to, mucosally, buccally, orally, transdermally, by inhalation, intranasally, urethrally, vaginally, and intravenously, subcutaneously, intramuscularly, intraperitoneally, and the like. The excipients in the pharmaceutical composition are compatible with the route of administration.

[0106] In some embodiments, the compounds of the disclosure can be delivered orally, for example, as tablets or capsules. The compounds can be packaged in an enteric coating, preferably such that the tablet or capsule does not release the compound until it reaches the stomach, and optionally further until it reaches the small intestine.

[0107] In some embodiments, the compounds of the disclosure can be delivered by injection, and the pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and fluid to the extent that it can be easily administered in the form of an injectable solution or suspension. The form must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol or liquid polyethylene glycol), suitable mixtures thereof, and vegetable oils.

[0108] Therapeutic administration can also be by injection of sustained release formulations, for example, formulations that allow for subcutaneous injection, including: nanospheres / microspheres, liposomes, emulsions, gels, insoluble salts or suspensions.

[0109] In some embodiments, the compounds of the present disclosure can be administered intranasally. The pharmaceutical composition can be in the form of an aqueous solution, such as a solution including saline, citrate or other commonly used excipients or preservatives. It can also be in the form of a dry preparation or powder.

[0110] [Uses]

[0111] The present disclosure provides use of the aforementioned compound, the compound prepared by the aforementioned method or the aforementioned pharmaceutical composition in the preparation of a thrombolytic, free radical scavenging or anti-inflammatory drug.

[0112] The camphor moiety and the thrombolytic polypeptide moiety in the compound help the compound to break through the blood-brain barrier, exert the anti-inflammatory effect of camphor and the thrombolytic and free radical scavenging effect of the polypeptide.

[0113] Further, the present disclosure provides use of the aforementioned compound, the compound prepared by the aforementioned method or the aforementioned pharmaceutical composition in the preparation of a cerebral ischemia protective drug.

[0114] Thrombotic diseases can cause cerebral ischemic injury, leading to cerebral ischemia-reperfusion injury. After the compound penetrates into the brain through the blood-brain barrier, it self-cleaves to release camphor and thrombolytic peptides. Camphor inhibits the expression of inflammatory cytokines and inflammatory proteins, activates GABAa receptors, reduces cell apoptosis and cell necrosis, and reduces brain edema and ischemia-reperfusion injury. The polypeptide can dissolve the thrombus in the brain and scavenge the free radicals generated during the cerebral ischemia-reperfusion process, thereby reducing the cerebral ischemia and ischemia-reperfusion injury, which collectively protects the ischemic site in the brain.

[0115] Further, the present disclosure provides use of the aforementioned compound, the compound prepared by the aforementioned method or the aforementioned pharmaceutical composition in the preparation of a drug for treating thrombotic diseases.

[0116] Herein, the term "thrombotic disease" refers to a disease caused by the formation of abnormal blood clots in the formed blood vessels in the circulating blood of humans and animals during their lifetime.

[0117] The thrombotic diseases include, but are not limited to, ischemic stroke, myocardial infarction, stroke, venous embolism, pulmonary embolism, peripheral arterial occlusive disease, venous catheter obstruction, arteriovenous fistula and shunt obstruction, and carotid artery stenosis.

[0118] In another aspect, the present disclosure provides a method for treating a subject suffering from a thrombotic disease, the method comprising: administering to the subject an effective amount of the aforementioned compound, the compound prepared by the aforementioned method or the aforementioned pharmaceutical composition.

[0119] In another aspect, the present application provides a method for improving cerebral ischemia, cerebral thrombosis, free radical state or inflammatory state in a subject, the method comprising: administering to the subject an effective amount of the aforementioned compound, the compound prepared by the aforementioned method or the aforementioned pharmaceutical composition.

[0120] As used herein, an "effective amount" is an amount sufficient to elicit the desired therapeutic effect by any of the above-mentioned modes of administration or any other mode known in the art.

[0121] In general, the actual amount of a compound of the present disclosure administered to a patient will vary depending on the mode of administration, the condition of the patient (including weight, sex, health, and diet), the formulation used, and the response desired.

[0122] The compounds of the present disclosure have good biological activity. For example, depending on the specific compound chosen, the desired therapeutic response, the route of administration, the formulation, and other factors known to those of skill in the art, the compound can be administered (in a single dose or in divided doses) at about 0.1, 0.5, 1, 5, 50, 100, 500, 1000, or 5000 μg / kg body weight.

[0123] The various embodiments and preferences disclosed above can be combined with each other (as long as they are not inherently contradictory to each other), and the various embodiments formed by the combination are all considered to be part of the disclosure of the present application.

[0124] The technical solutions of the present disclosure will be described more clearly and explicitly below by way of examples. It should be understood that these examples are for illustrative purposes only and are by no means intended to limit the protection scope of the present disclosure.

[0125] Examples

[0126] The following describes the examples of the present application. The examples described below are exemplary and are for the purpose of explanation only and are not to be construed as limiting the present application. If a specific technique or condition is not specified in the examples, the technique or condition described in the literature in the art or according to the product manual is used. If the manufacturer of the reagent or instrument is not specified, it is a conventional product that can be obtained commercially.

[0127] The Chinese explanations of the abbreviations or English full names used in the present application are shown in the following Table 1:

[0128] Table 1: Chinese explanations of the abbreviations or English full names used

[0129] The resin raw material used in Examples 2 to 12 was 2-chlorotrimesyl resin (CTC Resin) with a substitution constant of 0.5 mmol / g.

[0130] Example 1: Synthesis of Camphor-linker

[0131] Pheoc-Lys(Boc)-CTC Resin was synthesized as follows: 1.0 g of 2-CTC Resin was weighed into a reactor and swelled in 10 ml of DMF for 1 h. 0.47 g (1 mmol) of Fmoc-Lys(Boc)-OH was added, followed by 0.25 ml (2 mmol) of DIEA, and the reaction was shaken for 1 h. The solvent was removed, and the resin was washed with DMF and DCM, each for 3 times. Then 20% methanol in dichloromethane and 0.25 ml of DIEA were added, and the reaction was shaken for 1 h. + : 319.08. The 1H-NMR analysis is shown in Table 2.

[0132] Table 2: 1H-NMR analysis of Camphor-linker target 1 1H-NMR analysis

[0133] Example 2: Synthesis of Camphor-linker-PAK (PDC-1)

[0134] (1) Synthesis of Fmoc-Lys(Boc)-CTC Resin

[0135] Fmoc-Lys(Boc)-CTC Resin was synthesized as follows: 1.0 g of 2-CTC Resin was weighed into a reactor and swelled in 10 ml of DMF for 1 h. 0.47 g (1 mmol) of Fmoc-Lys(Boc)-OH was added, followed by 0.25 ml (2 mmol) of DIEA, and the reaction was shaken for 1 h. The solvent was removed, and the resin was washed with DMF and DCM, each for 3 times. Then 20% methanol in dichloromethane and 0.25 ml of DIEA were added, and the reaction was shaken for 1 h.

[0136] (2) Synthesis of Fmoc-Ala-Lys(Boc)-CTC Resin

[0137] Fmoc-Ala-Lys(Boc)-CTC Resin was synthesized as follows: 1.0 g of Fmoc-Lys(Boc)-CTC Resin was weighed into a reactor and swelled in 10 ml of DMF for 1 h. 25% 4-methylpiperidine / DMF (volume ratio) was added to remove the N-terminal Fmoc group, and the reaction was allowed to proceed for 30 min. The solvent was removed, and the resin was washed with DMF and DCM, each for 3 times. 0.93 g (3 mmol) of Fmoc-Ala-OH and 1.14 g (3 mmol) of HATU were dissolved in DMF and added to the resin, followed by the addition of 0.75 ml (6 mmol) of DIEA. The reaction was allowed to proceed, and the reaction endpoint was determined by ninhydrin detection. If the detection result showed that the reaction was complete, the reaction was stopped, and the resin was washed with DMF and DCM, each for 3 times.

[0138] (3) Synthesis of Fmoc-Pro-Ala-Lys(Boc)-CTC Resin

[0139] The Fmoc group at the N-terminus of the resin was removed by adding 25% 4-methylpiperidine / DMF (volume ratio) to the resin, and then Fmoc-Pro-OH was coupled according to the synthetic method mentioned in step (2). After the reaction was completed, the resin was washed with DMF and DCM alternately, each for 3 times.

[0140] (4) Synthesis of Camphor-linker-Pro-Ala-Lys(Boc)-CTC Resin

[0141] The Fmoc group at the N-terminus of the resin was removed by adding 25% 4-methylpiperidine / DMF (volume ratio) to the resin, and then 0.64 g (2 mmol) of camphor-linker was added, dissolved in DCM, and 0.5 ml (4 mmol) of DIEA was added to start the reaction. After the reaction was completed (the end point was detected by ninhydrin), the reaction solution was removed, and the resin was washed with DMF for 3 times, methanol for 2 times, DCM for 2 times, and methanol for 2 times, and then vacuum dried to obtain the peptide resin.

[0142] (5) Synthesis of Camphor-linker-Pro-Ala-Lys

[0143] The dried peptide resin was added to a reactor, and a cleavage solution (TFA:TIS:H2O = 95:2.5:2.5) was added at 10 ml per gram of peptide resin, and the reaction was stirred at room temperature for 2 h. After the reaction, the mixture was filtered, and the filtrate was collected. The resin was washed with a small amount of TFA for 2 times. A centrifuge tube was prepared, ice methyl tert-butyl ether was added, and then the collected filtrate was poured into the methyl tert-butyl ether, stirred until the polypeptide was precipitated, centrifuged, and the supernatant was poured away. The white object at the bottom of the centrifuge tube was washed with methyl tert-butyl ether for 2 times, and then placed in a vacuum dryer for drying for more than 12 hours to obtain a crude product.

[0144] (6) Purification of Camphor-linker-Pro-Ala-Lys

[0145] The obtained crude product was dissolved with acetonitrile and water, and the solution was loaded into a reverse-high performance liquid chromatography column for gradient elution. The position of the product was confirmed by mass spectrometry (MS). The correct product component was collected and freeze-dried to obtain the final product. Purity: 95.1% (HPLC, 220 nm, C18, linear gradient); [M+H] + : 495.3, [2M+H] + : 990.6, see Figure 1.

[0146] Example 3: Synthesis of Camphor-linker-KAP (PDC-2)

[0147] (1) Synthesis of Fmoc-Pro-CTC Resin

[0148] Weigh 1.0 g of 2-CTC Resin in a reactor, add 10 ml of DMF to swell for 1 h, add 0.34 g (1 mmol) of Fmoc-Pro-OH, add 0.25 ml (2 mmol) of DIEA, and shake the reaction for 1 h. Remove the solvent and wash the resin with DMF and DCM alternately, each for 3 times, then add 20% methanol in dichloromethane and 0.25 ml of DIEA, and shake the reaction for 1 h.

[0149] (2) Synthesis of Fmoc-Ala-Pro-CTC Resin

[0150] Add 25% 4-methylpiperidine / DMF (volume ratio) to remove the Fmoc group at the N-terminus of the resin, and react for 30 min. Remove the solvent and wash the resin with DMF and DCM alternately, each for 3 times; weigh 0.93 g (3 mmol) of Fmoc-Ala-OH and 1.14 g (3 mmol) of HATU, dissolve in an appropriate amount of DMF, and then add to the resin, followed by the addition of 0.75 ml (6 mmol) of DIEA to start the reaction. Determine the end point of the reaction by ninhydrin detection, and if the detection result shows that the reaction is complete, remove the reaction solution, and wash the resin with DMF and DCM alternately, each for 3 times.

[0151] (3) Synthesis of Fmoc-Lys(Boc)-Ala-Pro-CTC Resin

[0152] Add 25% 4-methylpiperidine / DMF (volume ratio) to the resin to remove the Fmoc group at the N-terminus of the resin, and then connect Fmoc-Lys(Boc)-OH according to the synthesis method in step (2). After the reaction is complete, wash the resin with DMF and DCM alternately, each for 3 times.

[0153] (4) Synthesis of Camphor-linker-Lys(Boc)-Ala-Pro-CTC Resin

[0154] Add 25% 4-methylpiperidine / DMF (volume ratio) to the resin to remove the Fmoc group at the N-terminus of the resin, then add 0.64 g (2 mmol) of camphor-linker, dissolve in DCM, and then add 0.5 ml (4 mmol) of DIEA to start the reaction. After the reaction is complete (end point of ninhydrin detection), remove the reaction solution, and wash the resin with DMF for 3 times, methanol for 2 times, DCM for 2 times, and methanol for 2 times, and then vacuum dry to obtain the peptide resin.

[0155] (5) Synthesis of Camphor-linker-Lys-Ala-Pro

[0156] The drained peptide resin was added to a reactor, and cleavage solution (TFA:TIS:H2O = 95:2.5:2.5) was added at 10 ml per gram of peptide resin. The reaction was stirred at room temperature for 2 h. The reaction mixture was filtered, and the filtrate was collected. The resin was washed with a small amount of TFA twice. A centrifuge tube was prepared, and ice methyl tert-butyl ether was added. The collected filtrate was then poured into the methyl tert-butyl ether, and the polypeptide was allowed to precipitate by stirring. The supernatant was decanted, and the white material at the bottom of the centrifuge tube was washed with methyl tert-butyl ether twice. The material was then dried in a vacuum desiccator for more than 12 h to obtain the crude product.

[0157] (6) Purification of Camphor-linker-Lys-Ala-Pro

[0158] The obtained crude product was dissolved in acetonitrile and water, and the solution was loaded onto a reverse-phase high-performance liquid chromatography column for gradient elution. The position of the product was confirmed by MS detection. The correct product component was collected and lyophilized to obtain the final product. Purity: 99.9% (HPLC, 220 nm, C18, linear gradient); [M+H] + : 495.3, as shown in FIG. 2.

[0159] Example 4: Synthesis of Camphor-linker-β-AHKAP (PDC-3)

[0160] (1) Synthesis of Fmoc-Pro-CTC Resin

[0161] A 1.0 g sample of 2-CTC Resin was weighed into a reactor, and 10 ml of DMF was added for swelling for 1 h. Then, 0.34 g (1 mmol) of Fmoc-Pro-OH and 0.25 ml (2 mmol) of DIEA were added, and the reaction was shaken for 1 h. The solvent was removed, and the resin was washed with DMF and DCM alternately, each for 3 times. Then, 20% methanol in dichloromethane and 0.25 ml of DIEA were added, and the reaction was shaken for 1 h.

[0162] (2) Synthesis of Fmoc-Ala-Pro-CTC Resin

[0163] The Fmoc group at the N-terminus of the resin was removed by adding 25% 4-methylpiperidine / DMF (by volume) and reacting for 30 min. The solvent was removed, and the resin was washed with DMF and DCM alternately, each for 3 times. Then, 0.93 g (3 mmol) of Fmoc-Ala-OH and 1.14 g (3 mmol) of HATU were dissolved in DMF and added to the resin, followed by the addition of 0.75 ml (6 mmol) of DIEA. The reaction was started, and the reaction endpoint was determined by ninhydrin detection. If the detection result showed that the reaction was complete, the reaction solution was removed, and the resin was washed with DMF and DCM alternately, each for 3 times.

[0164] (3) Synthesis of Fmoc-β-Ala-His(trt)-Lys(Boc)-Ala-Pro-CTC Resin

[0165] The Fmoc group at the N-terminus of the resin was removed by adding 25% 4-methylpiperidine in DMF (v / v) to the resin, and then Fmoc-Lys(Boc)-OH, Fmoc-His(trt)-OH, Fmoc-β-Ala-OH were sequentially coupled to the resin according to the same method as in step (2). After the reaction was completed, the resin was washed with DMF and DCM alternately, each for 3 times.

[0166] (4) Synthesis of Camphor-linker-β-Ala-His(trt)-Lys(Boc)-Ala-Pro-CTC Resin

[0167] The Fmoc group at the N-terminus of the resin was removed by adding 25% 4-methylpiperidine in DMF (v / v) to the resin, and then 0.64 g (2 mmol) of camphor-linker was added, dissolved in DCM, and 0.5 ml (4 mmol) of DIEA was added to start the reaction. After the reaction was completed (detection of end point by ninhydrin), the reaction solution was removed, and the resin was washed with DMF for 3 times, methanol for 2 times, DCM for 2 times, and methanol for 2 times, and then vacuum dried to obtain the peptide resin.

[0168] (5) Synthesis of Camphor-linker-β-Ala-His-Lys-Ala-Pro

[0169] The dried peptide resin was added to a reactor, and a cleavage solution (TFA:TIS:H2O = 95:2.5:2.5) was added at 10 ml per gram of peptide resin, and the reaction was stirred at room temperature for 2 h. After the reaction, the mixture was filtered, and the filtrate was collected. The resin was washed with a small amount of TFA for 2 times. A centrifuge tube was prepared, ice methyl tert-butyl ether was added, and then the collected filtrate was poured into the methyl tert-butyl ether, and the polypeptide was precipitated by stirring, centrifuged, and the supernatant was poured away. The white substance at the bottom of the centrifuge tube was washed with methyl tert-butyl ether for 2 times, and then placed in a vacuum dryer for drying for more than 12 hours to obtain a crude product.

[0170] (6) Purification of Camphor-linker-β-Ala-His-Lys-Ala-Pro

[0171] The obtained crude product was dissolved with acetonitrile and water, and the solution was loaded into a reverse-high performance liquid chromatography column for gradient elution. The position of the product was confirmed by MS detection. The correct product component was collected and freeze-dried to obtain the final product. Purity: 99.1% (HPLC, 220 nm, C18, linear gradient); [M+H] + : 704.4, [M+2H] 2+:352.3, see Fig. 3.

[0172] Example 5: Synthesis of Camphor-linker-β-AHPAK (PDC-4)

[0173] (1) Synthesis of Fmoc-Lys(Boc)-CTC Resin

[0174] Weigh 1.0 g of 2-CTC Resin in a reactor, add 10 ml of DMF for swelling 1 h, add 0.47 g (1 mmol) of Fmoc-Lys(Boc)-OH, add 0.25 ml (2 mmol) of DIEA, and shake the reaction for 1 h. Remove the solvent and wash the resin with DMF and DCM alternately, each for 3 times, then add 20% methanol in dichloromethane and 0.25 ml of DIEA, and shake the reaction for 1 h.

[0175] (2) Synthesis of Fmoc-Ala-Lys(Boc)-CTC Resin

[0176] Remove the Fmoc group at the N-terminus of the resin with 25% 4-methylpiperidine / DMF (volume ratio) for 30 min. Remove the solvent and wash the resin with DMF and DCM alternately, each for 3 times; weigh 0.93 g (3 mmol) of Fmoc-Ala-OH and 1.14 g (3 mmol) of HATU, dissolve them in DMF, and then add them to the resin, followed by the addition of 0.75 ml (6 mmol) of DIEA to start the reaction. Determine the reaction endpoint by ninhydrin detection. If the detection result shows that the reaction is complete, remove the reaction solution, and wash the resin with DMF and DCM alternately, each for 3 times.

[0177] (3) Synthesis of Fmoc-β-Ala-His(trt)-Pro-Ala-Lys(Boc)-CTC Resin

[0178] Remove the Fmoc group at the N-terminus of the resin with 25% 4-methylpiperidine / DMF (volume ratio), and then sequentially connect Fmoc-Pro-OH, Fmoc-His(trt)-OH, and Fmoc-β-Ala-OH according to the synthesis method in step (2). After the reaction is complete, wash the resin with DMF and DCM alternately, each for 3 times.

[0179] (4) Synthesis of Camphor-linker-β-Ala-His(trt)-Pro-Ala-Lys(Boc)-CTC Resin

[0180] The Fmoc group at the N-terminus of the resin was removed by adding 25% 4-methylpiperidine / DMF (volume ratio) to the resin, followed by adding 0.64 g (2 mmol) of camphor-linker, adding DCM to dissolve, and then adding 0.5 ml (4 mmol) of DIEA to start the reaction. After the reaction was completed (the end point was detected by ninhydrin), the reaction solution was removed, and the resin was washed with DMF three times, methanol two times, DCM two times, and methanol two times, and then was dried by vacuum suction to obtain the peptide resin.

[0181] (5) Synthesis of Camphor-linker-β-Ala-His-Pro-Ala-Lys

[0182] The dried peptide resin was added to a reactor, and a cleavage solution (TFA:TIS:H2O = 95:2.5:2.5) was added at 10 ml per gram of the peptide resin. The reaction was stirred at room temperature for 2 h, and the mixture after the reaction was filtered. The filtrate was collected, and the resin was washed with a small amount of TFA twice. A centrifuge tube was prepared, ice methyl tert-butyl ether was added, and then the collected filtrate was poured into the methyl tert-butyl ether. The polypeptide was allowed to precipitate by stirring, and was centrifuged. The supernatant was discarded, and the white substance at the bottom of the centrifuge tube was washed with methyl tert-butyl ether twice. After that, the substance was dried in a vacuum desiccator for more than 12 h to obtain a crude product.

[0183] (6) Purification of Camphor-linker-β-Ala-His-Pro-Ala-Lys

[0184] The obtained crude product was dissolved with acetonitrile and water, and the solution was loaded into a reverse-phase high-performance liquid chromatography column for gradient elution. The position of the product was confirmed by MS detection. The correct product component was collected and freeze-dried to obtain the final product. Purity: 99.8% (HPLC, 220 nm, C18, linear gradient); [M+H] + : 703.4, [M+2H] 2+ : 352.4, as shown in FIG. 4.

[0185] Example 6: Synthesis of Camphor-linker-GRPAK (PDC-5)

[0186] (1) Synthesis of Fmoc-Lys(Boc)-CTC Resin

[0187] A 1.0 g of 2-CTC Resin was weighed into a reactor, and 10 ml of DMF was added to swell for 1 h. Then, 0.47 g (1 mmol) of Fmoc-Lys(Boc)-OH was added, 0.25 ml (2 mmol) of DIEA was added, and the reaction was shaken for 1 h. The solvent was removed, and the resin was washed with DMF and DCM alternately, each for three times. Then, 20% methanol in dichloromethane and 0.25 ml of DIEA were added, and the reaction was shaken for 1 h.

[0188] (2) Synthesis of Fmoc-Ala-Lys(Boc)-CTC Resin

[0189] Add 25% 4-methylpiperidine / DMF (volume ratio) to remove the Fmoc group at the N-terminus of the resin, and react for 30 min. Remove the solvent and wash the resin with DMF and DCM alternately, 3 times each. Weigh 0.93 g (3 mmol) of Fmoc-Ala-OH and 1.14 g (3 mmol) of HATU, dissolve in DMF, and add to the resin, then add 0.75 ml (6 mmol) of DIEA to start the reaction. Determine the end point of the reaction by ninhydrin detection. If the detection result shows that the reaction is complete, remove the reaction solution, and wash the resin with DMF and DCM alternately, 3 times each.

[0190] (3) Synthesis of Fmoc-Gly-Arg(pbf)-Pro-Ala-Lys(Boc) CTC Resin

[0191] Add 25% 4-methylpiperidine / DMF (volume ratio) to remove the Fmoc group at the N-terminus of the resin, then follow the synthesis method in step (2) to sequentially connect Fmoc-Pro-OH, Fmoc-Arg(pbf)-OH, and Fmoc-Gly-OH. After the reaction is complete, wash the resin with DMF and DCM alternately, 3 times each.

[0192] (4) Synthesis of Camphor-linker-Gly-Arg(pbf)-Pro-Ala-Lys(Boc) CTC Resin

[0193] Add 25% 4-methylpiperidine / DMF (volume ratio) to remove the Fmoc group at the N-terminus of the resin, then add 0.64 g (2 mmol) of camphor-linker, dissolve in DCM, and add 0.5 ml (4 mmol) of DIEA to start the reaction. After the reaction is complete (end point of ninhydrin detection), remove the reaction solution, and wash the resin with DMF 3 times, methanol 2 times, DCM 2 times, and methanol 2 times, then dry under vacuum to obtain the peptide resin.

[0194] (5) Synthesis of Camphor-linker-Gly-Arg-Pro-Ala-Lys

[0195] The drained peptide resin was added to a reactor, and cleavage solution (TFA:TIS:H2O = 95:2.5:2.5) was added, 10 ml per gram of peptide resin, and the reaction was stirred at room temperature for 2 h. The reaction mixture was filtered, and the filtrate was collected. The resin was washed with a small amount of TFA twice. A centrifuge tube was prepared, ice methyl tert-butyl ether was added, and then the collected filtrate was poured into the methyl tert-butyl ether. The polypeptide was precipitated by stirring, and was centrifuged. The supernatant was discarded, and the white substance at the bottom of the centrifuge tube was washed with methyl tert-butyl ether twice. After that, the white substance was dried in a vacuum desiccator for more than 12 h to obtain a crude product.

[0196] (6) Purification of Camphor-linker-Gly-Arg-Pro-Ala-Lys

[0197] The obtained crude product was dissolved with acetonitrile and water, and the solution was loaded into a reverse-high performance liquid chromatography column for gradient elution. The position of the product was confirmed by MS detection. The correct product component was collected and freeze-dried to obtain a final product. Purity: 98.5% (HPLC, 220 nm, C18, linear gradient); [M+H] + : 709.4, [M+2H] 2+ : 354.8, as shown in FIG. 5.

[0198] Example 7: Synthesis of Camphor-linker-PEG4-PAK (PDC-6)

[0199] (1) Synthesis of Fmoc-Lys(Boc)-CTC Resin

[0200] 1.0 g of 2-CTC Resin was weighed into a reactor, and 10 ml of DMF was added for swelling for 1 h. 0.47 g (1 mmol) of Fmoc-Lys(Boc)-OH was added, and 0.25 ml (2 mmol) of DIEA was added. The reaction was shaken for 1 h. The solvent was drained, and the resin was washed with DMF and DCM alternately, 3 times for each. Then, 20% methanol dichloromethane solution and 0.25 ml of DIEA were added, and the reaction was shaken for 1 h.

[0201] (2) Synthesis of Fmoc-Ala-Lys(Boc)-CTC Resin

[0202] Add 25% 4-methylpiperidine / DMF (volume ratio) to remove the Fmoc group at the N-terminus of the resin, and react for 30 min. Remove the solvent, and wash the resin with DMF and DCM alternately, each for 3 times; weigh 0.93 g (3 mmol) of Fmoc-Ala-OH and 1.14 g (3 mmol) of HATU, dissolve in DMF, and then add to the resin, and then add 0.75 ml (6 mmol) of DIEA to start the reaction, and determine the reaction end point by ninhydrin detection; if the detection result shows that the reaction is complete, remove the reaction solution, and wash the resin with DMF and DCM alternately, each for 3 times.

[0203] (3) Synthesis of Fmoc-PEG4-Pro-Ala-Lys(Boc)-CTC Resin

[0204] Add 25% 4-methylpiperidine / DMF (volume ratio) to remove the Fmoc group at the N-terminus of the resin, and then follow the synthesis method in step (2) to sequentially connect Fmoc-Pro-OH and Fmoc-NH-PEG4-COOH; after the reaction is completed, wash the resin with DMF and DCM alternately, each for 3 times.

[0205] (4) Synthesis of Camphor-linker-PEG4-Pro-Ala-Lys(Boc)-CTC Resin

[0206] Add 25% 4-methylpiperidine / DMF (volume ratio) to remove the Fmoc group at the N-terminus of the resin, and then add 0.64 g (2 mmol) of camphor-linker, add an appropriate amount of DCM to dissolve, and then add 0.5 ml (4 mmol) of DIEA to start the reaction; after the reaction is complete (ninhydrin detection end point), remove the reaction solution, and then wash the resin with DMF for 3 times, methanol for 2 times, DCM for 2 times, and methanol for 2 times, and then vacuum dry to obtain the peptide resin.

[0207] (5) Synthesis of Camphor-linker-PEG4-Pro-Ala-Lys

[0208] Add the vacuum-dried peptide resin to a reactor, add a cleavage solution (TFA:TIS:H2O = 95:2.5:2.5) of 10 ml per gram of peptide resin, and stir to react at room temperature for 2 h; filter the reaction mixture after the reaction, collect the filtrate, and wash the resin with a small amount of TFA for 2 times; prepare a centrifuge tube, add ice methyl tert-butyl ether, and then pour the collected filtrate into the methyl tert-butyl ether, stir until the polypeptide is precipitated, centrifuge, and pour away the supernatant; wash the white substance at the bottom of the centrifuge tube with methyl tert-butyl ether for 2 times, and then place it in a vacuum dryer to dry for 12 hours or more to obtain the crude product.

[0209] (6) Purification of Camphor-linker-PEG4-Pro-Ala-Lys

[0210] The resulting crude product was dissolved in acetonitrile and water, and the solution was loaded onto a reverse-phase high performance liquid chromatography column for gradient elution. The product was identified by MS detection. The correct product fraction was collected and lyophilized to obtain the final product. Purity: 98.6% (HPLC, 220 nm, C18, linear gradient); [M+H] + : 742.4, see Figure 6.

[0211] Example 8: Synthesis of Camphor-linker-K (KAP) RGDS (PDC-7)

[0212] (1) Synthesis of Fmoc-Ser(tBu)-CTC Resin

[0213] Weigh 1.0 g of 2-CTC Resin into a reactor, add 10 ml of DMF for swelling for 1 h, add 0.38 g (1 mmol) of Fmoc-Ser(tBu)-OH, add 0.25 ml (2 mmol) of DIEA, and shake the reaction for 1 h. Remove the solvent and wash the resin with DMF and DCM alternately, each for 3 times, then add 20% methanol in dichloromethane and 0.25 ml of DIEA, and shake the reaction for 1 h.

[0214] (2) Synthesis of Fmoc-Asp(OtBu)-Ser(tBu)-CTC Resin

[0215] Add 25% 4-methylpiperidine / DMF (volume ratio) to remove the Fmoc group at the N-terminus of the resin, and react for 30 min. Remove the solvent and wash the resin with DMF and DCM alternately, each for 3 times; weigh 1.23 g (3 mmol) of Fmoc-Asp(OtBu)-OH and 1.14 g (3 mmol) of HATU into DMF, then add them to the resin, and then add 0.75 ml (6 mmol) of DIEA to start the reaction. Determine the reaction endpoint by ninhydrin detection. If the detection result shows that the reaction is complete, remove the reaction solution, and wash the resin with DMF and DCM alternately, each for 3 times.

[0216] (3) Synthesis of Fmoc-Arg(pbf)-Gly-Asp(OtBu)-Ser(tBu)-CTC Resin

[0217] Add 25% 4-methylpiperidine / DMF (volume ratio) to the resin to remove the Fmoc group at the N-terminus of the resin, and then sequentially connect Fmoc-Gly-OH and Fmoc-Arg(pbf)-OH according to the synthesis method mentioned in step (2). After the reaction is completed, wash the resin with DMF and DCM alternately, each for 3 times.

[0218] (4) Synthesis of Alloc-Lys(Fmoc)-Arg(pbf)-Gly-Asp(OtBu)-Ser(tBu)-CTC Resin

[0219] Add 25% 4-methylpiperidine / DMF (volume ratio) to remove the Fmoc group at the N-terminus of the resin, and react for 30 min. Remove the solvent and wash the resin with DMF and DCM alternately, 3 times each. Weigh 1.27 g (3 mmol) of Alloc-Lys(Fmoc)-OH and 1.14 g (3 mmol) of HATU, dissolve in DMF, and add to the resin, then add 0.75 ml (6 mmol) of DIEA to start the reaction. Determine the end point of the reaction by ninhydrin detection. If the detection result shows that the reaction is complete, remove the reaction solution, and wash the resin with DMF and DCM alternately, 3 times each.

[0220] (5) Synthesis of Alloc-Lys[Lys(Boc)-Ala-Pro]-Arg(pbf)-Gly-Asp(OtBu)-Ser(tBu)-CTC Resin

[0221] Add 25% 4-methylpiperidine / DMF (volume ratio) to remove the Fmoc group at the N-terminus of the resin, then follow the method of step (2) to sequentially connect Fmoc-Lys(Boc)-OH, Fmoc-Ala-OH, and Fmoc-Pro-OH. After the reaction is complete, wash the resin with DMF and DCM alternately, 3 times each.

[0222] (6) Synthesis of Camphor-linker-Lys[Lys(Boc)-Ala-Pro]-Arg(pbf)-Gly-Asp(OtBu)-Ser(tBu)-CTC Resin

[0223] Add 20% tetrakis(triphenylphosphine)palladium in dichloromethane and 10 mmol of phenylsilane to remove the Alloc group at the N-terminus of the resin, then add 0.64 g (2 mmol) of camphor-linker, dissolve in DCM, and add 0.5 ml (4 mmol) of DIEA to start the reaction. After the reaction is complete (end point of ninhydrin detection), remove the reaction solution, and wash the resin with DMF 3 times, methanol 2 times, DCM 2 times, and methanol 2 times, then dry under vacuum to obtain the peptide resin.

[0224] (7) Synthesis of Camphor-linker-Lys(Lys-Ala-Pro)-Arg-Gly-Asp-Ser

[0225] The drained peptide resin was added to a reactor, and cleavage solution (TFA:TIS:H2O=95:2.5:2.5) was added, 10 ml per gram of peptide resin, and the reaction was stirred at room temperature for 2 h. The reaction mixture was filtered, and the filtrate was collected. The resin was washed with a small amount of TFA twice. A centrifuge tube was prepared, ice methyl tert-butyl ether was added, and then the collected filtrate was poured into the methyl tert-butyl ether. The polypeptide was precipitated by stirring, and centrifugation was performed. The supernatant was discarded, and the white substance at the bottom of the centrifuge tube was washed with methyl tert-butyl ether twice. Then, the white substance was placed in a vacuum dryer and dried for more than 12 h to obtain a crude product.

[0226] (8) Purification of Camphor-linker-Lys(Lys-Ala-Pro)-Arg-Gly-Asp-Ser

[0227] The obtained crude product was dissolved with acetonitrile and water, and the solution was loaded into a reverse-high performance liquid chromatography column for gradient elution. The position of the product was confirmed by MS detection. The correct product component was collected and freeze-dried to obtain a final product. Purity: 95.66% (HPLC, 220 nm, C18, linear gradient); [M+H] + : 1039.5, [M+2H] 2+ : 519.8, [M+3H] 3+ : 347.0, see FIG. 7.

[0228] Example 9: Synthesis of Camphor-linker-K(KAPHA-β)RGDS (PDC-8)

[0229] (1) Synthesis of Fmoc-Ser(tBu)-CTC Resin

[0230] 1.0 g of 2-CTC Resin was weighed into a reactor, 10 ml of DMF was added for swelling for 1 h, 0.38 g (1 mmol) of Fmoc-Ser(tBu)-OH was added, 0.25 ml (2 mmol) of DIEA was added, and the reaction was shaken for 1 h. The solvent was removed, and the resin was washed with DMF and DCM alternately, each for 3 times. Then, 20% methanol dichloromethane solution and 0.25 ml of DIEA were added, and the reaction was shaken for 1 h.

[0231] (2) Synthesis of Fmoc-Asp(OtBu)-Ser(tBu)-CTC Resin

[0232] Add 25% 4-methylpiperidine / DMF (v / v) to remove the Fmoc group at the N-terminus of the resin for 30 min. Remove the solvent and wash the resin with DMF and DCM alternately for 3 times each; weigh 1.23 g (3 mmol) of Fmoc-Asp(OtBu)-OH and 1.14 g (3 mmol) of HATU into DMF and then add them to the resin, followed by the addition of 0.75 ml (6 mmol) of DIEA to start the reaction. Determine the end point of the reaction by the ninhydrin test. If the test result shows that the reaction is complete, remove the reaction solution and wash the resin with DMF and DCM alternately for 3 times each.

[0233] (3) Synthesis of Fmoc-Arg(pbf)-Gly-Asp(OtBu)-Ser(tBu)-CTC Resin

[0234] Add 25% 4-methylpiperidine / DMF (v / v) to remove the Fmoc group at the N-terminus of the resin, and then sequentially connect Fmoc-Gly-OH and Fmoc-Arg(pbf)-OH according to the synthesis method in step (2). After the reaction is completed, wash the resin with DMF and DCM alternately for 3 times each.

[0235] (4) Synthesis of Alloc-Lys(Fmoc)-Arg(pbf)-Gly-Asp(OtBu)-Ser(tBu)-CTC Resin

[0236] Add 25% 4-methylpiperidine / DMF (v / v) to remove the Fmoc group at the N-terminus of the resin for 30 min. Remove the solvent and wash the resin with DMF and DCM alternately for 3 times each; weigh 1.27 g (3 mmol) of Alloc-Lys(Fmoc)-OH and 1.14 g (3 mmol) of HATU into DMF and then add them to the resin, followed by the addition of 0.75 ml (6 mmol) of DIEA to start the reaction. Determine the end point of the reaction by the ninhydrin test. If the test result shows that the reaction is complete, remove the reaction solution and wash the resin with DMF and DCM alternately for 3 times each.

[0237] (5) Synthesis of Alloc-Lys[Lys(Boc)-Ala-Pro-His(trt)-β-Ala]-Arg(pbf)-Gly-Asp(OtBu)-Ser(tBu)-CTC Resin

[0238] The resin was washed with 25% 4-methylpiperidine / DMF (v / v) to remove the N-terminal Fmoc group, then Fmoc-Lys(Boc)-OH, Fmoc-Ala-OH, Fmoc-Pro-OH, Fmoc-His(trt)-OH, Fmoc-β-Ala-OH were sequentially coupled according to the synthetic procedure used in step (2). After the reaction was completed, the resin was washed with DMF and DCM alternately, each for 3 times.

[0239] (6) Synthesis of Camphor-linker-Lys[Lys(Boc)-Ala-Pro-His(trt)-β-Ala]-Arg(pbf)-Gly-Asp(OtBu)-Ser(tBu)-CTC Resin

[0240] The resin was washed with 20% palladium tetrakis(triphenylphosphine) in dichloromethane, 10 mmol phenylsilane to remove the N-terminal Alloc group, then 0.64 g (2 mmol) of camphor-linker was added, dissolved in DCM, and 0.5 ml (4 mmol) of DIEA was added to start the reaction. After the reaction was completed (indanetrione detection end point), the reaction solution was removed, and the resin was washed with DMF for 3 times, methanol for 2 times, DCM for 2 times, and methanol for 2 times, and then vacuum dried to obtain the peptide resin.

[0241] (7) Synthesis of Camphor-linker-Lys(Lys-Ala-Pro-His-β-Ala)-Arg-Gly-Asp-Ser

[0242] The dried peptide resin was added to a reactor, and a cleavage solution (TFA:TIS:H2O = 95:2.5:2.5) was added at 10 ml per gram of peptide resin. The reaction was stirred at room temperature for 2 h, and the mixture after the reaction was filtered, and the filtrate was collected. The resin was washed with a small amount of TFA for 2 times. A centrifuge tube was prepared, ice methyl tert-butyl ether was added, and then the collected filtrate was poured into methyl tert-butyl ether. The polypeptide was stirred until it precipitated, centrifuged, and the supernatant was poured away. The white object at the bottom of the centrifuge tube was washed with methyl tert-butyl ether for 2 times, and then placed in a vacuum dryer for drying for more than 12 hours to obtain a crude product.

[0243] (8) Purification of Camphor-linker-Lys(Lys-Ala-Pro-His-β-Ala)-Arg-Gly-Asp-Ser

[0244] The obtained crude product was dissolved with acetonitrile and water, and the solution was loaded into a reverse-high performance liquid chromatography column for gradient elution. The position of the product was confirmed by MS detection. The correct product component was collected and freeze-dried to obtain the final product. Purity: 99.4% (HPLC, 220 nm, C18, linear gradient); [M+H] +: 1246.6, [M+2H] 2+ : 623.9, [M+3H] 3+ : 416.4, see Figure 8.

[0245] Example 10: Synthesis of Camphor-Linker-K (KAPRG) RGDS (PDC-9)

[0246] (1) Synthesis of Fmoc-Ser(tBu)-CTC Resin

[0247] Weigh 1.0 g of 2-CTC Resin in the reactor, add 10 ml of DMF for swelling for 1 h, add 0.38 g (1 mmol) of Fmoc-Ser(tBu)-OH, add 0.25 ml (2 mmol) of DIEA, and shake the reaction for 1 h. Remove the solvent and wash the resin with DMF and DCM alternately, each for 3 times, then add 20% methanol in dichloromethane and 0.25 ml of DIEA, and shake the reaction for 1 h.

[0248] (2) Synthesis of Fmoc-Asp(OtBu)-Ser(tBu)-CTC Resin

[0249] Remove the Fmoc group at the N-terminus of the resin by adding 25% 4-methylpiperidine / DMF (volume ratio) for 30 min. Remove the solvent and wash the resin with DMF and DCM alternately, each for 3 times; weigh 1.23 g (3 mmol) of Fmoc-Asp(OtBu)-OH and 1.14 g (3 mmol) of HATU, add DMF to dissolve, then add to the resin, and then add 0.75 ml (6 mmol) of DIEA to start the reaction. Determine the end point of the reaction by ninhydrin detection. If the detection result shows that the reaction is complete, remove the reaction solution, and wash the resin with DMF and DCM alternately, each for 3 times.

[0250] (3) Synthesis of Fmoc-Arg(pbf)-Gly-Asp(OtBu)-Ser(tBu)-CTC Resin

[0251] Remove the Fmoc group at the N-terminus of the resin by adding 25% 4-methylpiperidine / DMF (volume ratio) to the resin, then sequentially connect Fmoc-Gly-OH and Fmoc-Arg(pbf)-OH according to the synthesis method used in step (2), and after the reaction is completed, wash the resin with DMF and DCM alternately, each for 3 times.

[0252] (4) Synthesis of Alloc-Lys(Fmoc)-Arg(pbf)-Gly-Asp(OtBu)-Ser(tBu)-CTC Resin

[0253] Add 25% 4-methylpiperidine / DMF (volume ratio) to remove the Fmoc group at the N-terminus of the resin, and react for 30 min. Remove the solvent, and wash the resin with DMF and DCM alternately, 3 times each. Weigh 1.27 g (3 mmol) of Alloc-Lys(Fmoc)-OH and 1.14 g (3 mmol) of HATU, dissolve in DMF, and add to the resin, then add 0.75 ml (6 mmol) of DIEA to start the reaction. Determine the end point of the reaction by ninhydrin detection, and if the detection result shows that the reaction is complete, remove the reaction solution, and wash the resin with DMF and DCM alternately, 3 times each.

[0254] (5) Synthesis of camphor-linker-Lys[Lys(Boc)-Ala-Pro-Arg(pbf)-Gly-Boc]-Arg(pbf)-Gly-Asp(OtBu)-Ser(tBu)-CTC Resin

[0255] Add 25% 4-methylpiperidine / DMF (volume ratio) to remove the Fmoc group at the N-terminus of the resin, then sequentially connect Fmoc-Lys(Boc)-OH, Fmoc-Ala-OH, Fmoc-Pro-OH, Fmoc-Arg(pbf)-OH, and Fmoc-Gly-OH using the synthesis method used in step (2), and after the reaction is complete, wash the resin with DMF and DCM alternately, 3 times each.

[0256] (6) Synthesis of camphor-linker-Lys[Lys(Boc)-Ala-Pro-Arg(pbf)-Gly-Boc]-Arg(pbf)-Gly-Asp(OtBu)-Ser(tBu)-CTC Resin

[0257] Add 20% tetrakis(triphenylphosphine)palladium in dichloromethane and 10 mmol of phenylsilane to remove the Alloc group at the N-terminus of the resin, then add 0.64 g (2 mmol) of camphor-linker, dissolve in DCM, and add 0.5 ml (4 mmol) of DIEA to start the reaction. After the reaction is complete (end point of ninhydrin detection), remove the reaction solution, and sequentially wash the resin with DMF 3 times, methanol 2 times, DCM 2 times, and methanol 2 times, then vacuum dry to obtain the peptide resin.

[0258] (7) Synthesis of camphor-linker-Lys(Lys-Ala-Pro-Arg-Gly)-Arg-Gly-Asp-Ser

[0259] The drained peptide resin was added to a reactor, and cleavage solution (TFA:TIS:H2O = 95:2.5:2.5) was added, 10 ml per gram of peptide resin, and the reaction was stirred at room temperature for 2 h. The reaction mixture was filtered, and the filtrate was collected. The resin was washed with a small amount of TFA twice. A centrifuge tube was prepared, ice methyl tert-butyl ether was added, and then the collected filtrate was poured into the methyl tert-butyl ether. The polypeptide was precipitated by stirring, and was centrifuged. The supernatant was discarded, and the white substance at the bottom of the centrifuge tube was washed with methyl tert-butyl ether twice. Then, the white substance was placed in a vacuum desiccator and dried for more than 12 h to obtain a crude product.

[0260] (8) Purification of Camphor-linker-Lys(Lys-Ala-Pro-Arg-Gly)-Arg-Gly-Asp-Ser

[0261] The obtained crude product was dissolved with acetonitrile and water, and the solution was loaded into a reverse-high performance liquid chromatography column for gradient elution. The position of the product was confirmed by MS detection. The correct product component was collected and freeze-dried to obtain a final product. Purity: 99.2% (HPLC, 220 nm, C18, linear gradient); [M+H] + : 1252.1, [M+2H] 2+ : 626.5, [M+3H] 3+ : 418.1, see Figure 9.

[0262] Example 11: Synthesis of Camphor-linker-K(KAP)IESDV(PDC-10)

[0263] (1) Synthesis of Fmoc-Val-CTC Resin

[0264] 1.0 g of 2-CTC Resin was weighed into a reactor, 10 ml of DMF was added for swelling for 1 h, 0.34 g (1 mmol) of Fmoc-Ser(tBu)-OH was added, 0.25 ml (2 mmol) of DIEA was added, and the reaction was shaken for 1 h. The solvent was removed, and the resin was washed with DMF and DCM alternately, each for 3 times. Then, 20% methanol dichloromethane solution and 0.25 ml of DIEA were added, and the reaction was shaken for 1 h.

[0265] (2) Synthesis of Fmoc-Asp(OtBu)-Val-CTC Resin

[0266] Add 25% 4-methylpiperidine / DMF (v / v) to remove the Fmoc group at the N-terminus of the resin for 30 min. Remove the solvent and wash the resin with DMF and DCM alternately for 3 times each; weigh 1.23 g (3 mmol) of Fmoc-Asp(OtBu)-OH and 1.14 g (3 mmol) of HATU into DMF and then add them to the resin, followed by the addition of 0.75 ml (6 mmol) of DIEA to start the reaction. Determine the end point of the reaction by ninhydrin test. If the test result shows that the reaction is complete, remove the reaction solution and wash the resin with DMF and DCM alternately for 3 times each.

[0267] (3) Synthesis of Fmoc-Ile-Glu(OtBu)-Ser(tBu)-Asp(OtBu)-Val-CTC Resin

[0268] Add 25% 4-methylpiperidine / DMF (v / v) to remove the Fmoc group at the N-terminus of the resin, and then sequentially connect Fmoc-Ser(tBu)-OH, Fmoc-Glu(OtBu)-OH and Fmoc-Ile-OH according to the synthetic method used in step (2). After the reaction is completed, wash the resin with DMF and DCM alternately for 3 times each.

[0269] (4) Alloc-Lys(Fmoc)-Ile-Glu(OtBu)-Ser(tBu)-Asp(OtBu)-Val-CTC Resin

[0270] Add 25% 4-methylpiperidine / DMF (v / v) to remove the Fmoc group at the N-terminus of the resin for 30 min. Remove the solvent and wash the resin with DMF and DCM alternately for 3 times each; weigh 1.27 g (3 mmol) of Alloc-Lys(Fmoc)-OH and 1.14 g (3 mmol) of HATU into DMF and then add them to the resin, followed by the addition of 0.75 ml (6 mmol) of DIEA to start the reaction. Determine the end point of the reaction by ninhydrin test. If the test result shows that the reaction is complete, remove the reaction solution and wash the resin with DMF and DCM alternately for 3 times each.

[0271] (5) Synthesis of Alloc-Lys[Lys(Boc)-Ala-Pro-Boc]-Ile-Glu(OtBu)-Ser(tBu)-Asp(OtBu)-Val-CTC Resin

[0272] The resin was washed with 25% 4-methylpiperidine / DMF (v / v) to remove the N-terminal Fmoc group of the resin, then Fmoc-Lys(Boc)-OH, Fmoc-Ala-OH, Fmoc-Pro-OH were sequentially coupled according to the synthetic procedure used in step (2). After the reaction was completed, the resin was washed with DMF and DCM alternately, 3 times each.

[0273] (6) Synthesis of Camphor-linker-Lys[Lys(Boc)-Ala-Pro-Boc]-Ile-Glu(OtBu)- Ser(tBu)-Asp(OtBu)-Val-CTC Resin

[0274] The resin was washed with 20% palladium tetrakis(triphenylphosphine) in dichloromethane, 10 mmol of phenylsilane to remove the N-terminal Alloc group of the resin, then 0.64 g (2 mmol) of camphor-linker was added, dissolved in DCM, and 0.5 ml (4 mmol) of DIEA was added to start the reaction. After the reaction was completed (ninhydrin test end point), the reaction solution was removed, and the resin was washed with DMF 3 times, methanol 2 times, DCM 2 times, and methanol 2 times, and then vacuum dried to obtain the peptide resin.

[0275] (7) Synthesis of Camphor-linker-Lys(Lys-Ala-Pro)-Ile-Glu-Ser-Asp-Val

[0276] The dried peptide resin was added to a reactor, and cleavage solution (TFA:TIS:H2O = 95:2.5:2.5) was added at 10 ml per gram of peptide resin, and the reaction was stirred at room temperature for 2 h. The reaction mixture was filtered after the reaction, and the filtrate was collected. The resin was washed with a small amount of TFA twice. A centrifuge tube was prepared, ice methyl tert-butyl ether was added, and then the collected filtrate was poured into methyl tert-butyl ether. The polypeptide was precipitated by stirring, centrifuged, and the supernatant was poured off. The white object at the bottom of the centrifuge tube was washed with methyl tert-butyl ether twice, and then placed in a vacuum dryer for drying for more than 12 hours to obtain a crude product.

[0277] (8) Purification of Camphor-linker-Lys(Lys-Ala-Pro)-Ile-Glu-Ser-Asp-Val

[0278] The obtained crude product was dissolved with acetonitrile and water, and the solution was loaded into a reverse-high performance liquid chromatography column for gradient elution. The position of the product was confirmed by MS detection. The correct product component was collected and freeze-dried to obtain the final product. Purity: 99.6% (HPLC, 220 nm, C18, linear gradient); [M+H] + : 1167.6, [M+2H] 2+ : 584.0, see Figure 10.

[0279] Example 12: Synthesis of Camphor-linker-K (KAPHA-beta) IESDV (PDC-11)

[0280] (1) Synthesis of Fmoc-Val-CTC Resin

[0281] Weigh 1.0 g of 2-CTC Resin in a reactor, add 10 ml of DMF for swelling for 1 h, add 0.34 g (1 mmol) of Fmoc-Ser(tBu)-OH, add 0.25 ml (2 mmol) of DIEA, and shake the reaction for 1 h. Remove the solvent and wash the resin with DMF and DCM alternately, each for 3 times, then add 20% methanol dichloromethane solution and 0.25 ml of DIEA, and shake the reaction for 1 h.

[0282] (2) Synthesis of Fmoc-Asp(OtBu)-Val-CTC Resin

[0283] Remove the Fmoc group at the N-terminus of the resin with 25% 4-methylpiperidine / DMF (volume ratio) for 30 min. Remove the solvent and wash the resin with DMF and DCM alternately, each for 3 times; weigh 1.23 g (3 mmol) of Fmoc-Asp(OtBu)-OH and 1.14 g (3 mmol) of HATU dissolved in DMF and then added to the resin, followed by the addition of 0.75 ml (6 mmol) of DIEA to start the reaction, and the reaction endpoint was determined by ninhydrin detection. If the detection result shows that the reaction is complete, remove the reaction solution, and wash the resin with DMF and DCM alternately, each for 3 times.

[0284] (3) Synthesis of Fmoc-Ile-Glu(OtBu)-Ser(tBu)-Asp(OtBu)-Val-CTC Resin

[0285] Remove the Fmoc group at the N-terminus of the resin with 25% 4-methylpiperidine / DMF (volume ratio), and then sequentially connect Fmoc-Ser(tBu)-OH, Fmoc-Glu(OtBu)-OH, and Fmoc-Ile-OH according to the synthesis method in step (2). After the reaction is completed, wash the resin with DMF and DCM alternately, each for 3 times.

[0286] (4) Synthesis of Alloc-Lys(Fmoc)-Ile-Glu(OtBu)-Ser(tBu)-Asp(OtBu)-Val-CTC Resin

[0287] Remove the Fmoc group from the N-terminus of the resin with 25% 4-methylpiperidine / DMF (v / v) for 30 min. Wash the resin with DMF and DCM alternately for 3 times each. Weigh 1.27 g (3 mmol) of Alloc-Lys(Fmoc)-OH and 1.14 g (3 mmol) of HATU into DMF and add to the resin. Then add 0.75 ml (6 mmol) of DIEA to start the reaction. The reaction is monitored by ninhydrin test. If the test shows that the reaction is complete, remove the reaction solution and wash the resin with DMF and DCM alternately for 3 times each.

[0288] (5) Synthesis of camphor-linker-Lys[Lys(Boc)-Ala-Pro-His(trt)-Ala-β-Boc]-Ile-Glu(OtBu)-Ser(tBu)-Asp(OtBu)-Val-CTC Resin

[0289] Remove the Fmoc group from the N-terminus of the resin with 25% 4-methylpiperidine / DMF (v / v) for 30 min. Then follow the procedure of step (2) to sequentially attach Fmoc-Lys(Boc)-OH, Fmoc-Ala-OH, Fmoc-Pro-OH, Fmoc-His(trt)-OH, and Boc-β-Ala-OH. After the reaction is completed, wash the resin with DMF and DCM alternately for 3 times each.

[0290] (6) Synthesis of camphor-linker-Lys[Lys(Boc)-Ala-Pro-His(trt)-Ala-β-Boc]-Ile-Glu(OtBu)-Ser(tBu)-Asp(OtBu)-Val-CTC Resin

[0291] Remove the Alloc group from the N-terminus of the resin with 20% tetrakis(triphenylphosphine)palladium in dichloromethane and 10 mmol of phenylsilane. Then add 0.64 g (2 mmol) of camphor-linker, dissolve in DCM, and add 0.5 ml (4 mmol) of DIEA to start the reaction. After the reaction is completed (ninhydrin test), remove the reaction solution and wash the resin with DMF for 3 times, methanol for 2 times, DCM for 2 times, and methanol for 2 times. Then dry the resin under vacuum to obtain the peptide resin.

[0292] (7) Synthesis of camphor-linker-Lys(Lys-Ala-Pro-His-Ala-β)-Ile-Glu-Ser-Asp-Val

[0293] The drained peptide resin was added to a reactor, and cleavage solution (TFA:TIS:H2O = 95:2.5:2.5) was added at 10 ml per gram of peptide resin. The reaction was stirred at room temperature for 2 h. The reaction mixture was filtered, and the filtrate was collected. The resin was washed with a small amount of TFA twice. A centrifuge tube was prepared, and ice methyl tert-butyl ether was added. The collected filtrate was then poured into the methyl tert-butyl ether, and the polypeptide was allowed to precipitate by stirring. The mixture was centrifuged, and the supernatant was discarded. The white material at the bottom of the centrifuge tube was washed with methyl tert-butyl ether twice and then dried in a vacuum desiccator for more than 12 h to obtain the crude product.

[0294] (8) Purification of Camphor-linker-Lys(Lys-Ala-Pro-His-Ala-β)-Ile-Glu-Ser-Asp-Val

[0295] The obtained crude product was dissolved in acetonitrile and water, and the solution was loaded into a reverse-phase high-performance liquid chromatography column for gradient elution. The position of the product was confirmed by MS detection. The correct product component was collected and lyophilized to obtain the final product. Purity: 98.9% (HPLC, 220 nm, C18, linear gradient); [M+H] + : 1380.7, [M+2H] 2+ : 690.5, [M+3H] 3+ : 460.8, see Figure 11.

[0296] Example 13: Synthesis of Camphor-linker-K(KAPRG)IESDV(PDC-12)

[0297] (1) Synthesis of Fmoc-Val-CTC Resin

[0298] A 1.0 g sample of 2-CTC Resin was weighed into a reactor, and 10 ml of DMF was added for swelling for 1 h. Then, 0.34 g (1 mmol) of Fmoc-Ser(tBu)-OH was added, followed by 0.25 ml (2 mmol) of DIEA. The reaction was shaken for 1 h. The solvent was drained, and the resin was washed with DMF and DCM alternately, 3 times for each. Then, 20% methanol in dichloromethane and 0.25 ml of DIEA were added, and the reaction was shaken for 1 h.

[0299] (2) Synthesis of Fmoc-Asp(OtBu)-Val-CTC Resin

[0300] Add 25% 4-methylpiperidine / DMF (v / v) to remove the Fmoc group at the N-terminus of the resin for 30 min. Remove the solvent and wash the resin with DMF and DCM alternately for 3 times; weigh 1.23 g (3 mmol) of Fmoc-Asp(OtBu)-OH and 1.14 g (3 mmol) of HATU into DMF and then add them to the resin, followed by adding 0.75 ml (6 mmol) of DIEA to start the reaction. Determine the end point of the reaction by ninhydrin test. If the test result shows that the reaction is complete, remove the reaction solution and wash the resin with DMF and DCM alternately for 3 times.

[0301] (3) Synthesis of Fmoc-Ile-Glu(OtBu)-Ser(tBu)-Asp(OtBu)-Val-CTC Resin

[0302] Add 25% 4-methylpiperidine / DMF (v / v) to remove the Fmoc group at the N-terminus of the resin, and then sequentially connect Fmoc-Ser(tBu)-OH, Fmoc-Glu(OtBu)-OH and Fmoc-Ile-OH according to the synthesis method in step (2). After the reaction is completed, wash the resin with DMF and DCM alternately for 3 times.

[0303] (4) Synthesis of Alloc-Lys(Fmoc)-Ile-Glu(OtBu)-Ser(tBu)-Asp(OtBu)-Val-CTC Resin

[0304] Add 25% 4-methylpiperidine / DMF (v / v) to remove the Fmoc group at the N-terminus of the resin for 30 min. Remove the solvent and wash the resin with DMF and DCM alternately for 3 times; weigh 1.27 g (3 mmol) of Alloc-Lys(Fmoc)-OH and 1.14 g (3 mmol) of HATU into DMF and then add them to the resin, followed by adding 0.75 ml (6 mmol) of DIEA to start the reaction. Determine the end point of the reaction by ninhydrin test. If the test result shows that the reaction is complete, remove the reaction solution and wash the resin with DMF and DCM alternately for 3 times.

[0305] (5) Synthesis of Alloc-Lys[Lys(Boc)-Ala-Pro-Arg(pbf)-Gly-Boc]-Ile-Glu(OtBu)-Ser(tBu)-Asp(OtBu)-Val-CTC Resin

[0306] The resin was washed with 25% 4-methylpiperidine / DMF (volume ratio) to remove the Fmoc group at the N-terminus of the resin, and then Fmoc-Lys(Boc)-OH, Fmoc-Ala-OH, Fmoc-Pro-OH, Fmoc-Arg(pbf)-OH, Fmoc-Gly-OH were sequentially coupled according to the synthesis method in step (2). After the reaction was completed, the resin was washed with DMF and DCM alternately, each for 3 times.

[0307] (6) Synthesis of camphor-linker-Lys[Lys(Boc)-Ala-Pro-Arg(pbf)-Gly-Boc]-Ile-Glu(OtBu)-Ser(tBu)-Asp(OtBu)-Val-CTC Resin

[0308] The resin was washed with 20% tetrakis(triphenylphosphine)palladium in dichloromethane and 10 mmol phenylsilane to remove the Alloc group at the N-terminus of the resin, and then 0.64 g (2 mmol) of camphor-linker was added, dissolved in DCM, and 0.5 ml (4 mmol) of DIEA was added to start the reaction. After the reaction was completed (indanetrione detection end point), the reaction solution was removed, and the resin was washed with DMF for 3 times, methanol for 2 times, DCM for 2 times, and methanol for 2 times, and then vacuum dried to obtain the peptide resin.

[0309] (7) Synthesis of camphor-linker-Lys(Lys-Ala-Pro-Arg-Gly)-Ile-Glu-Ser-Asp-Val

[0310] The dried peptide resin was added to a reactor, and a cleavage solution (TFA:TIS:H2O=95:2.5:2.5) was added at 10 ml per gram of peptide resin, and the reaction was stirred at room temperature for 2 h. After the reaction, the mixture was filtered, and the filtrate was collected. The resin was washed with a small amount of TFA for 2 times. A centrifuge tube was prepared, ice methyl tert-butyl ether was added, and then the collected filtrate was poured into the methyl tert-butyl ether, stirred until the polypeptide was precipitated, centrifuged, and the supernatant was poured away. The white object at the bottom of the centrifuge tube was washed with methyl tert-butyl ether for 2 times, and then placed in a vacuum dryer for drying for more than 12 hours to obtain a crude product.

[0311] (8) Purification of camphor-linker-Lys(Lys-Ala-Pro-Arg-Gly)-Ile-Glu-Ser-Asp-Val

[0312] The obtained crude product was dissolved with acetonitrile and water, and the solution was loaded into a reverse-high performance liquid chromatography column for gradient elution. The position of the product was confirmed by MS (mass spectrum) detection. The correct product component was collected and freeze-dried to obtain the final product. Purity: 99.5% (HPLC, 220 nm, C18, linear gradient); [M+H]+ 1375.7, [M+2H] 2+ 688.0, [M+3H] 3+ 459.0, see Figure 12.

[0313] Example 14: Detection of the therapeutic effect of the chamaejasmine polypeptide conjugate on rats with stroke

[0314] 14.1. Drug and solvent

[0315] 14.1.1 Test drug:

[0316] Positive control drug: Edaravone and dextromethorphan (4:1) combination

[0317] Test product: PDC-1, PDC-2, PDC-3, PDC-4, PDC-5, PDC-6 prepared in Examples 2-7 respectively

[0318] Solvent: 20% propylene glycol aqueous solution

[0319] 14.1.2 Preparation method:

[0320] Positive control drug: Edaravone 40 mg and dextromethorphan 10 mg were added to 3.0 mL of propylene glycol, and after complete dissolution, water for injection was slowly added to 15.0 mL to obtain the administration solution of the positive control drug. Among them, the final concentration of edaravone was 2.67 mg / mL, and the final concentration of dextromethorphan was 0.67 mg / mL.

[0321] Test product: Similar to the preparation method of the positive control drug, PDC-1, PDC-2, PDC-3, PDC-4, PDC-5, PDC-6 were dissolved in propylene glycol respectively, and water for injection was slowly added to dilute the propylene glycol by 4 times the volume to obtain the administration solution of the test product. Among them, the final concentration of PDC-1 was 0.82 mg / mL, the final concentration of PDC-2 was 1.17 mg / mL, the final concentration of PDC-3 was 0.82 mg / mL, the final concentration of PDC-4 was 1.17 mg / mL, the final concentration of PDC-5 was 1.24 mg / mL, and the final concentration of PDC-6 was 1.18 mg / mL.

[0322] 14.2 Experimental materials and reagents

[0323] 2,3,5 chlorinated triphenyl tetrazolium (TTC): purchased from Sigma-Aldrich (Sigma-Aldrich (Shanghai) Trading Co., Ltd.), prepared into a 2% TTC solution with normal saline, and stored in the dark. Isoflurane inhalation anesthesia, MCAO plug line: purchased from Beijing Xilong Technology Co., Ltd.; 0.36 mm series, A2636-A4 suitable for 250-280 g rats, plug line length about 45 mm, head end diameter 0.36±0.02 mm.

[0324] 14.3 Experimental animals

[0325] Adult SD rats, SPF level (specific pathogen-free animals, not carrying major potential infections or conditionally pathogenic and pathogenic to scientific experiments of large pathogenic animals), male, body weight: the body weight at the time of the experiment was maintained at 260-280 g; source: Beijing Vito Lihua Experimental Animal Technology Co., Ltd. Environmental adaptation period: at least 2 days, the experimenter observed the cage once a day during the adaptation period.

[0326] 14.4 Rearing conditions

[0327] No more than 5 per cage. The environmental parameters of the animal room during the feeding period were recorded. During the experiment, no other species of animals were fed in the same room. Temperature: 18-24℃; relative humidity: 40%-70%; ventilation: not less than 8 times per hour, using an IVC independent air supply system (independent ventilation cage system); light: automatic light, 12 hours light and dark alternation, lights off at 8:00 am, lights on at 8:00 am the next day.

[0328] 14.5 Dose design

[0329] Table 3 Dose design table

[0330] Dose = concentration x dose amount

[0331] 14.6 Experimental method

[0332] The MCAO (middle cerebral artery occlusion) reperfusion model was made according to the Longa method. The rats were fasted for 12 hours before the operation and allowed to drink freely. Anesthesia was induced with isoflurane inhalation. A midline incision was made in the neck to expose the common carotid artery (CCA), external carotid artery (ECA) and the pterygopalatine artery. A thread was inserted through the right ECA incision in all rats (a special thread was purchased from Beijing Xilong, A2636-A4 was suitable for rats weighing 250-280 g, and the thread was marked at a length of 20 mm). The pterygopalatine artery was temporarily clamped to prevent misinsertion. The length of the thread was about 18-20 mm from the CCA bifurcation, which was determined according to the weight of the animal. The right middle cerebral artery was occluded, then the skin was sutured, and the tail end of the thread was fixed to the skin. After 2 hours of ischemia, the thread was carefully removed to form reperfusion. The sham operation group only did not insert the thread, and the rest of the steps were the same as the operation group. The body temperature was maintained at (37±0.5)℃ during ischemia and 2 hours after reperfusion. The success of the model was indicated by the appearance of left limb paralysis, unsteady standing, and turning to one side when lifting the tail after the rats woke up from anesthesia. After the rats woke up, the Zea-Longa 5-point scoring standard was used: 0 points represented no neurological deficit symptoms; 1 point represented an inability to fully extend the contralateral forelimb; 2 points represented turning to the paretic side when walking; 3 points represented falling to the paretic side when walking; 4 points represented an inability to walk, and consciousness disorder; 5 points represented death. A cumulative score of 1 point or more was considered a successful model; any score of 5 points, subarachnoid hemorrhage, or no neurological deficit symptoms (0 points) were considered a failed model.

[0333] The rats were randomly divided into groups according to the score, and the number of effective animals in each group was not less than 8; 1 hour after ischemia, 20% propylene glycol solution, positive control drug (edaravone 8 mg / kg + dextromethorphan 2 mg / kg), and 5 μmol / kg of the test product were injected into the tail vein of each group of rats. The animals were scored for signs of neurological deficit according to the Zea-Longa five-point standard 24 hours after administration to evaluate the efficacy. The brain infarction volume was determined by 2,3,5-chlorinated triphenyl tetrazolium (TTC) staining. The animals were sacrificed by decapitation after excessive inhalation of carbon dioxide, and the rat brain was removed quickly and placed in ice water for 10 minutes. The brain was cut into 2 mm thick slices in the coronal plane, and then quickly placed in a 2% TTC solution (37℃) for 30 minutes, and then fixed with 4% polyformaldehyde. After 24 hours, a digital camera was used to take pictures, which were input into a computer, and the infarction area was calculated using image processing software (ADOBE, PHOTOSHOP). The pink area was normal brain tissue, and the white area was infarction area. To reduce the influence of cerebral ischemia on the results, the infarction volume was calculated by subtracting the volume of the ipsilateral normal tissue from the volume of the contralateral normal tissue. The results were expressed as a percentage of the infarction volume. The cerebral infarction rate = (contralateral normal tissue volume - ipsilateral normal tissue volume) / contralateral normal tissue volume x 100%. All statistical processes were performed using GraphPad Prism software.

[0334] 14.7 Experimental results

[0335] 14.7.1. Effect of the test drug on the behavior of rats with acute cerebral ischemia

[0336] After cerebral ischemia, the anesthetized rats showed varying degrees of focal neurological dysfunction after waking up, manifested as weakness of the left lower limb, turning to the left side when walking straight, severe tilting to the left side, and even being unable to walk, and even consciousness disorder. When the tail was raised, the left forelimb was flexed and adducted, the hindlimb was straightened and rotated to the right side. The model group animals showed obvious neurological injury symptoms after cerebral ischemia, and the neurological function score was significantly increased (P < 0.05). Table 4 shows the number of animals alive after the end of the experiment and the score of the behavior score. It can be seen that compared with the solvent group, the behavior score of the test product group was significantly reduced, indicating that the test product can significantly improve the function of the behavior of rats with acute cerebral ischemia. The behavior scores of compounds PDC-1, PDC-3 and the positive control group are basically the same, indicating that PDC-1, PDC-3 can significantly improve the function of the behavior of rats with acute cerebral ischemia.

[0337] 14.7.2. Effect of the test drug on the volume of cerebral infarction in rats with acute cerebral ischemia

[0338] After staining, normal brain tissue was rose pink throughout, while infarcted tissue was white and had a clear boundary. Except for the sham operation group, the brains of rats in other groups showed obvious infarction. Table 4 shows the cerebral infarction rate of the experimental animals, and the cerebral infarction rate of the test product group was significantly reduced compared with the solvent group, indicating that the test product can significantly reduce the volume of cerebral infarction in rats. The cerebral infarction rate of compounds PDC-1 and PDC-4 was lower than that of the positive control group, indicating that PDC-1 and PDC-4 significantly improved the infarction of brain tissue and were better than the positive control drug.

[0339] Table 4

[0340] The values represent the mean ± SD. One-way ANOVA, Dunnett's multiple comparisons test analysis method was used, *P < 0.05 compared with the sham operation group; #P < 0.05 compared with the solvent group.

[0341] In summary, compound PDC-1 can effectively prevent rats with cerebral ischemia from developing motor disorders and cerebral tissue infarction.

[0342] Example 15: Detection of the continuous administration treatment effect of the chamaejasmine polypeptide conjugate on rats with stroke

[0343] The method of making the MCAO reperfusion rat model, the model scoring standard and Example 14 are basically the same, the difference is that the ischemia time is 1 h, and the drug is administered through the tail vein. After 2 h of ischemia, the plug line is carefully pulled out, which forms reperfusion.

[0344] According to the model score, the animals are randomly grouped, and the animal grouping and administration method are shown in Table 5:

[0345] Table 5

[0346] The first administration after ischemia is 1 h, and 20% propylene glycol aqueous solution, positive control drug (edaravone 8 mg / kg + dextromethorphan 2 mg / kg) and 5 μmol / kg of the test product are respectively injected into the tail vein of each group of rats, and then continuously administered for 5 days. At 1 hour after modeling (d0, initial score), the animals are scored according to the Zea-Longa five-point method standard, and at the same time points of 1 day after modeling (d1, 24 hours after reperfusion), 2 days after modeling (d2), 3 days after modeling (d3), 4 days after modeling (d4), 5 days after modeling (d5), and 6 days after modeling (d6), the animals are scored respectively, and the Zea-Longa score reduction rate at the end of the experiment is calculated to investigate the efficacy.

[0347] Figure 13 shows the effect of the test drugs on the neurological behavior of acute cerebral ischemia rats, wherein the initial score of PDC-1, PDC-2, PDC-7 and PDC-10 relative to the vehicle group, and the reduction at the end point are 0.58, 0.64, 0.89 and 0.92 respectively, showing a trend of improving the neurological behavior of rats.

[0348] In addition, the same method as in Example 14 is used to stain the rat brain tissue and calculate the cerebral infarction rate to investigate the effect of the test drugs on the cerebral infarction volume of acute cerebral ischemia rats, and the results are shown in Table 6. It can be found that after modeling, the rats have obvious infarction foci, and the cerebral infarction rate of the vehicle group is significantly higher than that of the sham operation group. After 5 days of continuous administration, the positive control drug, PDC-1 and PDC-2 can effectively alleviate the cerebral infarction volume, and there is a statistical difference (P<0.05).

[0349] Table 6

[0350] The numerical value represents the mean ± SD. One-way ANOVA, Dunnett's multiple comparisons test is used for analysis, and *P<0.05 compared with the vehicle group.

[0351] The results show that the compound provided by the disclosure can obviously improve and increase the function of the neurological symptom behavior of an acute cerebral ischemia rat, can significantly reduce the volume of cerebral infarction, shows a good cerebral ischemia protection effect, shows excellent curative effect for treating stroke, and has a good clinical application prospect.

Claims

1. A compound or a prodrug, tautomer, optical isomer, geometric isomer, solvate thereof, or a pharmaceutically acceptable salt thereof, characterized in that, comprising a camphor moiety and a polypeptide moiety coupled to the camphor, the polypeptide moiety comprising a Pro-Ala-Lys, Ala-Lys-Pro or Lys-Ala-Pro fragment.

2. The compound of claim 1, wherein The compounds have the structure shown in Formula I: wherein L represents a linker, P 1 represents a polypeptide moiety.

3. The compound of claim 1 or 2, wherein The polypeptide moiety is derived from a polypeptide having thrombolytic activity and / or free radical scavenging activity.

4. The compound of any one of claims 1 to 3, wherein, (1) the linker L is covalently attached to the N-terminus of the polypeptide moiety P 1 ; (2) the linker L is connected to the hydroxyl group of camphor by an ester bond; and / or, (3) the camphor is dextrocamphor.

5. The compound of any one of claims 1 to 4, wherein The compounds have a structural formula shown in Formula II: wherein 0 and 1 represent the number of H; when the number of H is 1, -NH- represents an imino group at the N-terminus of the polypeptide moiety; when the number of H is 0, N represents a ring nitrogen atom at the N-terminus of the polypeptide moiety; P represents a polypeptide residue other than N-terminal -N(H) 0,1 - except N-terminal -N(H) 6. The compound of any one of claims 1 to 5, wherein The polypeptide moiety comprises one, two or three repeating sequence peptides with at least one of Pro-Ala-Lys, Ala-Lys-Pro and Lys-Ala-Pro as a structural unit.

7. The compound of any one of claims 1 to 6, wherein The polypeptide moiety is a tripeptide, a tetrapeptide, a pentapeptide, a hexapeptide, a heptapeptide, an octapeptide, a nonapeptide, a decapeptide or an undecapeptide.

8. The compound of any one of claims 1 to 7, wherein The polypeptide moiety comprises a dipeptide fragment β-Ala-His, Arg-Gly at the N-terminus of the Pro-Ala-Lys, Ala-Lys-Pro or Lys-Ala-Pro fragment, or His-β-Ala, Arg-Gly at the C-terminus.

9. The compound of any one of claims 1 to 8, wherein The polypeptide moiety further comprises a Lys residue covalently linked to the linker, and optionally, the C-terminus of the Lys residue is linked to an Arg-Gly-Asp-Ser or Ile-Glu-Ser-Asp-Val fragment.

10. The compound of claim 9, wherein The peptide fragment comprising Lys-Ala-Pro is linked to the Lys residue.

11. The compound of any one of claims 1 to 10, wherein One or more sites of the C-terminus, the N-terminus, the middle residues of the polypeptide moiety are modified with polyethylene glycol (PEG), and optionally, the PEG is selected from one or more of PEG2 to PEG24.

12. The compound of any one of claims 1 to 11, wherein, The N-terminal residue of the polypeptide moiety comprises a PEG modification selected from PEG2, PEG4, PEG8, PEG12 or PEG24, and optionally, the PEG modification is covalently linked to the linker.

13. The compound of any one of claims 1 to 12, wherein The polypeptide moiety comprises one or more of: Pro-Ala-Lys, Lys-Ala-Pro, beta-Ala-His-Lys-Ala-Pro, beta-Ala-His-Pro-Ala-Lys, Gly-Arg-Pro-Ala-Lys, PEG4-Pro-Ala-Lys, Lys(Lys-Ala-Pro)-Arg-Gly-Asp-Ser, Lys(Lys-Ala-Pro-His-beta-Ala)-Arg-Gly-Asp-Ser, Lys(Lys-Ala-Pro-Arg-Gly)-Arg-Gly-Asp-Ser, Lys(Lys-Ala-Pro)-Ile-Glu-Ser-Asp-Val, Lys(Lys-Ala-Pro-His-beta-Ala)-Ile-Glu-Ser-Asp-Val, Lys(Lys-Ala-Pro-Arg-Gly)-Ile-Glu-Ser-Asp-Val.

14. Process for the preparation of a compound according to any one of claims 1 to 13, characterized in that, Comprising: The intermediate comprising an ester bond is prepared from camphor and a coupling agent under reaction conditions, the intermediate is sequentially bonded with corresponding amino acids, purified to obtain the compound.

15. A pharmaceutical composition, characterized by, The compound of any one of claims 1 to 13 or the compound prepared by the method of claim 14, and a pharmaceutically acceptable excipient.

16. Use of the compound of any one of claims 1 to 13, the compound prepared by the method of claim 14 or the pharmaceutical composition of claim 15 in the preparation of a drug for dissolving thrombus, scavenging free radicals or anti-inflammatory.

17. Use according to claim 16, characterized in that, The use comprises use of the compound or the pharmaceutical composition in the preparation of a drug for protecting against cerebral ischemia.

18. Use according to claim 16 or 17, characterized in that, The compound or the pharmaceutical composition has use in the preparation of a drug for treating thrombotic diseases.

19. The use according to claim 18, characterized in that, The thrombotic diseases comprise ischemic stroke, myocardial infarction, stroke, venous embolism, pulmonary embolism, peripheral arterial occlusive disease, venous catheter occlusion, arteriovenous fistula and shunt occlusion, and carotid stenosis.

20. A method of treating a subject having a thrombotic disorder, characterized in that, The use comprises administering to the subject an effective amount of the compound of any one of claims 1 to 13, the compound prepared by the method of claim 14 or the pharmaceutical composition of claim 15.

21. The method of claim 20, wherein, The thrombotic diseases comprise ischemic stroke, myocardial infarction, stroke, venous embolism, pulmonary embolism, peripheral arterial occlusive disease, venous catheter occlusion, arteriovenous fistula and shunt occlusion, and carotid stenosis.

22. A method of improving cerebral ischemia, cerebral thrombosis, free radical status, or inflammatory status in a subject, comprising administering to the subject a therapeutically effective amount of a compound of any one of claims 1-21. The use comprises administering to the subject in need thereof an effective amount of the compound of any one of claims 1 to 13, the compound prepared by the method of claim 14 or the pharmaceutical composition of claim 15.

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