Compound as well as preparation method and application thereof

Abstract

The invention provides a camphinol modified peptide compound, a preparation method, a pharmaceutical composition and application thereof. The compound is prepared from camphinol and polypeptide through chemical coupling. The compound can dissolve thrombus and has the application of cerebral ischemia protection.

Description

Technical Field

[0001] The present disclosure belongs to the field of biomedicine and relates to a variety of camphorol-modified peptide compounds, preparation methods thereof, and their applications in pharmaceuticals and disease treatment. Background Art

[0002] In recent years, the incidence of thrombotic diseases, especially ischemic stroke, has been increasing. The recurrence of stroke patients will lead to the aggravation of existing neurological dysfunctions, disability, and a significant increase in mortality, seriously threatening human health. Therefore, the drug treatment of thrombotic diseases is the focus and hotspot of ischemic stroke treatment.

[0003] Ischemic stroke, also known as stroke, triggers multiple mechanisms in its development, including the activation of glutamate receptors, assisting glutamate release and calcium ion influx, thereby activating nitric oxide, cysteine protease, and protease. This causes inflammation, the generation of free radicals, and protein damage leading to neuronal cell apoptosis (Rogalewski et al., 2006). Several other key features of stroke include blood-brain barrier (BBB) damage, oxidative stress, cytokine-mediated toxicity, excitotoxicity, and neuronal function loss (Kuriakose and Xiao, 2020). The simultaneous activation of multiple pathways makes the treatment of ischemic stroke and neurological function damage challenging. Therefore, cerebral ischemia protection is of great value for the treatment of thrombotic diseases, and there is an urgent need for a compound suitable for thrombotic diseases to provide cerebral ischemia protection. Summary of the Invention

[0004] The present disclosure provides a camphorol-modified peptide compound, which has an application in cerebral ischemia protection and can relieve or reduce neurological symptoms and brain tissue damage caused by cerebral ischemia.

[0005] In a first aspect of the present disclosure, there is provided a compound or its prodrug, tautomer, optical isomer, geometric isomer, solvate, or a pharmaceutically acceptable salt thereof, which comprises a camphorol moiety and a polypeptide moiety coupled to the camphorol, and the polypeptide moiety comprises Pro-Ala-Lys (PAK), Ala-Lys-Pro (AKP), or Lys-Ala-Pro (KAP).

[0006] In certain embodiments, the compound has a structure as shown in Formula I:

[0007]

[0008] Wherein, L represents a linker, and P 1 represents the polypeptide moiety.

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

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

[0011] In some embodiments, the linker L of the compound forms an ester bond with the hydroxyl group of borneol.

[0012] In some embodiments, the borneol of the compound is (+)-borneol.

[0013] In some embodiments, the compound has the structure shown in Formula II:

[0014]

[0015] 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 cyclic nitrogen atom at the N-terminus of the polypeptide moiety;

[0016] P represents the polypeptide residues of the polypeptide moiety other than the -N(H)- 0,1 other than the -N(H)- at the N-terminus.

[0017] In some embodiments, the polypeptide moiety comprises one, two or three repeating sequence peptides having at least one of Pro-Ala-Lys, Ala-Lys-Pro and Lys-Ala-Pro as a structural unit.

[0018] In some embodiments, the polypeptide moiety of the compound is a tripeptide, tetrapeptide, pentapeptide, hexapeptide, heptapeptide, octapeptide, nonapeptide, decapeptide or undecapeptide.

[0019] In some embodiments, 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.

[0020] In some embodiments, the polypeptide moiety further comprises a Lys residue covalently linked to the linker, optionally, the C-terminus of the Lys residue is linked to an Arg-Gly-Asp-Ser or Ile-Glu-Ser-Asp-Val fragment.

[0021] In some embodiments, the peptide fragment comprising Lys-Ala-Pro is linked to the Lys residue.

[0022] In certain embodiments, one or more sites of the C-terminus, N-terminus, and intermediate residues of the polypeptide moiety are modified with polyethylene glycol (PEG). Optionally, the PEG is selected from one or more of PEG2 to PEG24.

[0023] In certain embodiments, the N-terminal residue of the polypeptide moiety includes a PEG modification selected from PEG2, PEG4, PEG8, PEG12, or PEG24. Optionally, the PEG modification is linked to a linker.

[0024] In certain 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.

[0025] The second aspect of the present disclosure provides a method for preparing the aforementioned compound, comprising: preparing an intermediate comprising an ester bond from borneol and a coupling agent under reaction conditions, sequentially bonding the intermediate with corresponding amino acids, and purifying to obtain the compound.

[0026] The third aspect of the present disclosure provides a pharmaceutical composition comprising the aforementioned compound or a compound prepared by the aforementioned method, and one or more pharmaceutically acceptable excipients.

[0027] The fourth aspect of the present disclosure provides the use of the aforementioned compound, a compound prepared by the aforementioned method, or the aforementioned pharmaceutical composition in the preparation of a drug for dissolving thrombus, scavenging free radicals, or anti-inflammatory.

[0028] In certain embodiments, the use includes the use of the compound or pharmaceutical composition in the preparation of a drug for cerebral ischemia protection.

[0029] In certain embodiments, the compound and the pharmaceutical composition are useful in the preparation of a drug for treating thrombotic diseases.

[0030] In some embodiments, the thrombotic diseases include ischemic stroke, myocardial infarction, stroke, venous embolism, pulmonary embolism, peripheral arterial obstructive disease, venous catheter occlusion, arteriovenous fistula and shunt occlusion, and carotid artery stenosis.

[0031] The fifth aspect of the present disclosure provides a method for treating a subject suffering from a thrombotic disease, comprising administering to the subject an effective amount of the foregoing compound, a compound prepared by the foregoing method, or the foregoing pharmaceutical composition.

[0032] The sixth aspect of the present disclosure provides a method for improving cerebral ischemia, cerebral thrombosis, free radical status, or inflammatory status in a subject, comprising administering to a subject in need thereof an effective amount of the foregoing compound, a compound prepared by the foregoing method, or the foregoing pharmaceutical composition. Description of the Drawings

[0033] Figure 1 Showing the mass spectrum of borneol-linker-Pro-Ala-Lys in Example 2;

[0034] Figure 2 Showing the mass spectrum of borneol-linker-Lys-Ala-Pro in Example 3;

[0035] Figure 3 Showing the mass spectrum of borneol-linker-β-Ala-His-Lys-Ala-Pro in Example 4;

[0036] Figure 4 Showing the mass spectrum of borneol-linker-β-Ala-His-Pro-Ala-Lys in Example 5;

[0037] Figure 5 Showing the mass spectrum of borneol-linker-Gly-Arg-Pro-Ala-Lys in Example 6;

[0038] Figure 6 Showing the mass spectrum of borneol-linker-PEG4-Pro-Ala-Lys in Example 7;

[0039] Figure 7 Showing the mass spectrum of borneol-linker-Lys(Lys-Ala-Pro)-Arg-Gly-Asp-Ser in Example 8;

[0040] Figure 8 Showing the mass spectrum of borneol-linker-Lys(Lys-Ala-Pro-His-β-Ala)-Arg-Gly-Asp-Ser in Example 9;

[0041] Figure 9Shows the mass spectrometry of borneol-linker-Lys(Lys-Ala-Pro-Arg-Gly)-Arg-Gly-Asp-Ser in Example 10;

[0042] Figure 10 Shows the mass spectrometry of borneol-linker-Lys(Lys-Ala-Pro)-Ile-Glu-Ser-Asp-Val in Example 11;

[0043] Figure 11 Shows the mass spectrometry of borneol-linker-Lys(Lys-Ala-Pro-His-Ala-β)-Ile-Glu-Ser-Asp-Val in Example 12;

[0044] Figure 12 Shows the mass spectrometry of borneol-linker-Lys(Lys-Ala-Pro-Arg-Gly)-Ile-Glu-Ser-Asp-Val in Example 13;

[0045] Figure 13 Shows the effect of the test drug on the neurological symptom score of MCAO rats in Example 14. Detailed implementation mode

[0046] The following specific examples illustrate the implementation modes of the present application. Those familiar with this technology can easily understand other advantages and effects of the present application from the content disclosed in this specification.

[0047] Unless otherwise specified, all numbers representing contents, concentrations, ratios, masses, volumes, times, temperatures, thicknesses, technical effects, etc. used in this specification and claims should be understood to be modified by the term "about" or "substantially" in any case. Therefore, unless there is a contrary indication, the numerical parameters listed in the following specification and appended claims are approximate values. For those skilled in the art, they can vary according to the desired properties and effects sought through this disclosure, and each numerical parameter should be interpreted according to the number of significant digits and the conventional rounding method or the manner understood by those skilled in the art.

[0048] Although the numerical ranges and parameters of the broad scope of the present disclosure are approximate values, the numerical values set forth in the specific examples are provided as precisely as possible. However, any numerical value will inherently contain certain errors, which are necessarily caused by the standard deviations found in their corresponding test measurements. Each numerical range given in this specification will include each narrower numerical range falling within that broader numerical range, as if these narrower numerical ranges were explicitly written herein.

[0049] In this application, the term "comprising" generally means including, encompassing, containing, or incorporating. In some cases, it also means "consisting of" or "composed of".

[0050] 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 meanings of similar expressions can be deduced accordingly.

[0051] Unless otherwise specified, all numbers representing contents, concentrations, ratios, masses, volumes, times, temperatures, thicknesses, technical effects, etc. used in this specification and claims shall in all cases be understood to be modified by the term "about" or "substantially". Therefore, unless otherwise indicated, the numerical parameters listed in the following specification and appended claims are approximate values. For those skilled in the art, they can vary according to the desired properties and effects sought through this disclosure and should be interpreted according to the number of significant digits and conventional rounding methods or the manner understood by those skilled in the art for each numerical parameter.

[0052] Although the numerical ranges and parameters that set forth the broad scope of this disclosure are approximate values, the numerical values set forth in the specific examples are provided as precisely as possible. However, any numerical value will inherently contain certain errors that are necessarily caused by the standard deviations found in their corresponding test measurements. Each numerical range given in this specification will include every narrower numerical range that falls within that broader numerical range, as if these narrower numerical ranges were all expressly written herein.

[0053] [Compound]

[0054] The treatment of thrombotic diseases, especially those occurring in the brain, is relatively complex. Due to the existence of the blood-brain barrier, many thrombolytic drugs cannot penetrate the blood-brain barrier and enter the brain. In addition, a large number of free radicals generated during the cerebral ischemia-reperfusion process can also damage brain tissue and cause impaired brain function. Therefore, compounds that can penetrate the blood-brain barrier and have a brain-protective effect are very important for the treatment of brain thrombotic diseases.

[0055] Generally, the "blood-brain barrier" refers to the barrier between the cerebral capillary wall and glial cells that forms between the plasma and brain cells and the barrier between the plasma and cerebrospinal fluid formed by the choroid plexus, and these barriers can prevent certain substances from entering the brain tissue from the blood.

[0056] The present application provides a bornane-modified peptide compound or a prodrug, tautomer, optical isomer, geometric isomer, solvate or pharmaceutically acceptable salt thereof that can be used for thrombotic diseases. The compound includes a bornane moiety and a polypeptide moiety covalently coupled (chemically covalently bonded) to the bornane.

[0057] As used herein, the term "polypeptide" refers to a compound formed by dehydration condensation of three or more amino acids linked together by peptide bonds. Polypeptides can be prepared by chemical synthesis or by proteolytic hydrolysis. The types of amino acids in polypeptides can include, but are not limited to, natural or unnatural amino acids such as L-amino acids, D-amino acids, α-amino acids or β-amino acids.

[0058] In some embodiments, the compound has the structure shown in Formula I:

[0059]

[0060] Wherein, L represents a linker, and P 1 represents the polypeptide moiety.

[0061] Existing studies have shown that inflammation is a potential therapeutic target for ischemic stroke, and it can interact with factors such as excitotoxicity and oxidative stress to form a vicious cycle pathway, leading to aggravated injury, and further resulting in enlarged infarcts, damaged nerve cells, brain edema, etc.

[0062] Bornane (or borneol) is a small molecule bicyclic monoterpene compound with good lipophilicity, which can easily cross the blood-brain barrier and enter the brain to exert its anti-inflammatory and brain-protective effects. The bornane moiety in the compound shown in Formula I can improve the lipophilicity of the compound, facilitate the compound to cross the blood-brain barrier, and exert anti-inflammatory and brain-protective effects.

[0063] In some embodiments, the bornane is (-)-borneol. In some embodiments, the bornane is (+)-borneol, which can inhibit the expression of inflammatory cytokines such as TNF-α (Tumor Necrosis Factor-α) and IL-1β (Interleukin-1β) and pro-inflammatory proteins such as COX-2 (Cyclooxygenase-2) and iNOS (inducible nitric oxide synthase) during cerebral ischemia-reperfusion, and activate the γ-aminobutyric acid type A receptor (GABAa) receptor, blocking the above vicious cycle pathway, thereby reducing apoptosis and necrosis of cells, protecting the blood-brain barrier, reducing brain edema, and thus alleviating ischemia-reperfusion injury.

[0064] The linker is used to connect the bornane alcohol moiety and the polypeptide moiety. In some embodiments, the bornane alcohol is connected to the polypeptide moiety via a cleavable or non-cleavable linker L. In some embodiments, the bornane alcohol is connected to the polypeptide moiety via a cleavable linker L. Under appropriate cleavage conditions, the compound shown in Formula I is cleaved to release free bornane alcohol and / or polypeptide, thereby exerting a therapeutic effect.

[0065] In some embodiments, the bornane alcohol is bonded to the linker L via an ester bond. In some embodiments, the linker L forms an ester bond connection with the hydroxyl group of the bornane alcohol. In some embodiments, the linker L is bonded to the N-terminus of the polypeptide moiety via an amide bond.

[0066] In some embodiments, the linker L is -C(=O)-.

[0067] In some embodiments, the compound has the structural formula shown in Formula II:

[0068]

[0069] 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 of the polypeptide moiety other than the N-terminal -N(H) 0,1 - other than.

[0070] 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" means that atoms or groups in a certain original substance (or compound) are treated by at least one of substitution, removal, condensation, and addition reactions to form a more complex or simpler product with a chemical bond change compared to the original substance.

[0071] 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 and free radical scavenging activities, and synergistically acts with bornane alcohol in vivo to simultaneously achieve the effects of dissolving thrombus, anti-inflammation, and eliminating free radicals.

[0072] In some embodiments, the polypeptide moiety includes a polypeptide fragment that is Pro-Ala-Lys, Ala-Lys-Pro, or Lys-Ala-Pro from the N-terminus to the C-terminus.

[0073] As used herein, a "fragment" of a polypeptide includes a complete and separable polypeptide segment, and also includes a polypeptide residue segment that is covalently bonded and inseparable from other molecules, such as P in this application 1, those skilled in the art can easily understand and distinguish the specific meanings represented by the fragments under different conditions.

[0074] In this article, standard three-letter or single-letter coding rules are used to represent amino acids. 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).

[0075] In the borneol-modified peptide compound provided by the present disclosure, the polypeptide part may include 1, 2, 3, 4, 5, 6 or more repeating sequence peptides with at least one of Pro-Ala-Lys, Ala-Lys-Pro, and Lys-Ala-Pro as the structural unit. The structural units may be distributed continuously or at intervals.

[0076] In some embodiments, the polypeptide part contains 1, 2, or 3 repeating sequence peptides with Pro-Ala-Lys, Ala-Lys-Pro, or Lys-Ala-Pro as the structural unit.

[0077] In some embodiments, the polypeptide part contains 1 repeating sequence peptide with Pro-Ala-Lys, Ala-Lys-Pro, or Lys-Ala-Pro as the structural unit.

[0078] In this article, the polypeptide part may be a tripeptide, tetrapeptide, pentapeptide, hexapeptide, heptapeptide, octapeptide, nonapeptide, decapeptide, undecapeptide or a peptide with a greater number. In some embodiments, the polypeptide part is a tripeptide, tetrapeptide, pentapeptide, hexapeptide, heptapeptide, octapeptide, nonapeptide, decapeptide, or undecapeptide.

[0079] In this article, a "tripeptide" refers to a peptide fragment formed by connecting three natural or unnatural amino acids through peptide bonds, which can be a linear peptide fragment or a peptide fragment containing a branched chain. It can be understood that dipeptides, tetrapeptides, pentapeptides, hexapeptides, heptapeptides, octapeptides, nonapeptides, decapeptides, and undecapeptides have similar understandings.

[0080] In some embodiments, in the polypeptide fragment, β-Ala-His, His-β-Ala or Arg-Gly is linked to the N-terminus or C-terminus of the Pro-Ala-Lys, Ala-Lys-Pro or Lys-Ala-Pro fragment.

[0081] In some embodiments, 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.

[0082] In some embodiments, the polypeptide moiety may further comprise a polypeptide fragment that is Arg-Gly-Asp-Ser or Ile-Glu-Ser-Asp-Val from the N-terminus to the C-terminus.

[0083] In some embodiments, the polypeptide moiety further comprises a Lys residue covalently linked to a linker. Optionally, the C-terminus of the Lys residue is linked to an Arg-Gly-Asp-Ser or Ile-Glu-Ser-Asp-Val fragment. In some embodiments, the polypeptide moiety comprises a polypeptide fragment that is Lys-Arg-Gly-Asp-Ser or Lys-Ile-Glu-Ser-Asp-Val from the N-terminus to the C-terminus.

[0084] In some embodiments, in the polypeptide moiety, a peptide fragment comprising Lys-Ala-Pro is linked to the Lys residue. In this case, the peptide fragment may exist in the form of a branched peptide fragment. For example, the polypeptide moiety comprises a polypeptide fragment of Lys(Lys-Ala-Pro)-, wherein the peptide fragment Lys-Ala-Pro within "()" represents a branched peptide chain and is covalently linked to the Lys immediately adjacent to the left of "()".

[0085] 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 can be understood that other polypeptide moieties with branched peptide chains in the present disclosure have similar understandings and meanings.

[0086] In the compounds provided by the present disclosure, the amino acids (or amino acid residues) of the polypeptide moiety can be natural or chemically modified naturally or artificially, including but not limited to phosphorylation, acetylation, methylation, glycosylation, ubiquitination, succinylation, crotonylation, 2-hydroxyisobutyrylation, lactylation, polyethylene glycol (PEG)ylation. 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, include polyethylene glycol (PEG), glycosylation, and phosphorylation modifications.

[0087] In some embodiments, one or more sites of the C-terminus, N-terminus, and intermediate residues of the polypeptide moiety have polyethylene glycol (PEG) modifications. In some embodiments, the N-terminal residue of the polypeptide moiety includes a polyethylene glycol (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, and PEG24. In some embodiments, the N-terminal residue of the polypeptide moiety includes a polyethylene glycol (PEG4) modification. The PEG modification can extend the half-life of the compounds or polypeptide moieties provided in this application in vivo, reduce or eliminate immunogenicity, and reduce toxic and side effects.

[0088] In some embodiments, the PEG modification is covalently linked to the 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.

[0089] In some embodiments, the polypeptide moiety includes or has one or more of the following groups (1) to (12):

[0090] (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。

[0091] The polypeptide portions of the above groups (1) to (12) have the main chain and side chain structures shown in the following table:

[0092]

[0093]

[0094] The borneol-modified peptide compound provided by the present disclosure has suitable physical properties, can penetrate the blood-brain barrier well, and can self-degrade in the brain to release borneol and the polypeptide with thrombolytic activity, playing the roles of thrombolysis, anti-inflammation and free radical scavenging. The two work synergistically to reduce cell necrosis and tissue damage, and play a role in protecting against cerebral ischemia.

[0095] [Preparation method]

[0096] The present disclosure provides a preparation method of the aforementioned compound, including: preparing an intermediate including an ester bond from borneol and a coupling agent under reaction conditions, and sequentially bonding the intermediate with corresponding amino acids, and purifying to obtain the compound.

[0097] As used herein, the term "coupling agent" refers to a class of substances having at least two reactive groups, and the reactive groups can participate in chemical reactions and form chemical bonds, thereby combining two substances.

[0098] In some embodiments, the coupling agent can be phenyl chloroformate 4-nitrophenyl ester.

[0099] In some embodiments, solid-phase peptide synthesis techniques can be used to sequentially bond intermediates with corresponding amino acids. Solid-phase peptide synthesis techniques are well-known and can be practiced proficiently by those skilled in the art. In this method, the synthesis of the peptides in the compounds of the present disclosure can be carried out by sequentially binding the desired amino acid residues one by one to the growing peptide chain according to the general principles of the solid-phase method. 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, Volume 2, Gross, E. and Meienhofer, J. editors, Academic Press 1-284 (1980).

[0100] In the chemical synthesis of peptides, the reactive side-chain groups of each amino acid residue are protected with suitable protecting groups to prevent chemical reactions at this site before the protecting groups are removed. For example, when reacting at the carboxyl group of an amino acid or fragment, the α-amino group is protected with a protecting group (e.g., 9-fluorenylmethoxycarbonyl (Fmoc), tert-butoxycarbonyl (Boc)), and then the α-amino protecting group is selectively removed, thereby allowing subsequent reactions at this site.

[0101] Orthogonal protecting groups can be used as appropriate to prepare polypeptides containing side chains, for example, Fmoc-Ser(tBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Arg(pbf)-OH.

[0102] Any suitable reagent can be used to cleave the compound from the solid phase, for example, a combination of trifluoroacetic acid (TFA), triisopropylsilane (TIS), and water. The final product is precipitated by adding cold ether and collected by filtration.

[0103] Purification can be carried out using any one of the known methods in the prior art, such as reverse-high performance liquid chromatography.

[0104] Although the synthesis is mainly described with reference to solid-phase peptide synthesis methods, it should be understood that other chemical and synthetic methods can be employed to prepare the compounds of the present invention.

[0105] [Pharmaceutical Composition]

[0106] The present disclosure provides a pharmaceutical composition comprising the aforementioned compound or a compound prepared by the aforementioned method, further comprising a pharmaceutically acceptable excipient.

[0107] The pharmaceutical compositions of the present disclosure can be formulated in any manner known in the art, including but not limited to solid, semi-solid or liquid system dosage forms such as tablets, capsules, caplets, suspensions, powders, lyophilized formulations, suppositories, eye drops, skin patches, orally soluble formulations, sprays, aerosols, etc.

[0108] The pharmaceutical compositions can be immediate release and / or modified release formulations, including delayed release, sustained release, pulsatile release, controlled release, targeted release and programmed release formulations.

[0109] As used herein, "pharmaceutically acceptable excipients" refers to components other than the active ingredient in the pharmaceutical composition that are non-toxic to the subject. Pharmaceutically acceptable excipients include but are not limited to excipients (such as diluents, carriers, etc.) and additives (such as stabilizers, preservatives, solubilizers, buffers, etc.). Excipients can include polyvinylpyrrolidone, gelatin, hydroxypropyl cellulose (HPC), gum arabic, polyethylene glycol, mannitol, sodium chloride and sodium citrate. For injectable formulations or other liquid dosage forms for administration, water containing at least one or more buffering components is preferred, and stabilizers, preservatives and solubilizers can also be used. For solid dosage forms for administration, any of a variety of thickeners, fillers, extenders and carrier additives can be used, such as starches, sugars, cellulose derivatives, fatty acids, etc. For topical dosage forms for administration, any of a variety of creams, ointments, gels, lotions, etc. can be used. For most pharmaceutical formulations, the inactive ingredients can constitute a major portion of the formulation by weight or volume. For pharmaceutical formulations, the use of any of a variety of metered release, sustained release or controlled release formulations and additives is also contemplated, such that the dosage can be formulated to deliver the compounds of the present disclosure over a period of time.

[0110] The compounds of the present disclosure can be administered by mucosal administration, intrabuccal administration, oral administration, transdermal administration, inhalation administration, intranasal administration, urethral administration, vaginal administration, and intravenous, subcutaneous, intramuscular, intraperitoneal injection, etc. The excipients in the pharmaceutical composition are adapted to the route of administration.

[0111] In some embodiments, the compounds of the present disclosure can be delivered orally, such as in tablets or capsules. The compounds can be packaged in enteric protectants, preferably such that the compounds are not released until the tablets or capsules reach the stomach and optionally further reach a portion of the small intestine.

[0112] In some embodiments, the compounds of the present disclosure can be administered by injection. Pharmaceutical forms suitable for injection 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 must be fluid to the extent that it can be administered by syringe. The form must be stable under the conditions of preparation 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, polyols (e.g., glycerol, propylene glycol, or liquid polyethylene glycol), suitable mixtures thereof, and vegetable oils.

[0113] Therapeutic administration can also be achieved through sustained-release injection formulations, such as formulations that permit subcutaneous injection, including: nanospheres / microspheres, liposomes, emulsions, gels, insoluble salts, or suspensions.

[0114] 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 common excipients or preservatives. It can also be in the form of a dry formulation or powder.

[0115] [Use]

[0116] The present disclosure provides the use of the aforementioned compounds, compounds prepared by the aforementioned methods, or the aforementioned pharmaceutical compositions in the preparation of drugs for dissolving thrombi, scavenging free radicals, or having anti-inflammatory effects.

[0117] The borneol moiety and the thrombolytic polypeptide moiety in the compound contribute to the compound's ability to cross the blood-brain barrier and exert the anti-inflammatory effect of borneol and the thrombolytic and free radical scavenging effects of the polypeptide.

[0118] Furthermore, the present disclosure provides the use of the aforementioned compounds, compounds prepared by the aforementioned methods, or the aforementioned pharmaceutical compositions in the preparation of drugs for protecting against cerebral ischemia.

[0119] Thrombotic diseases can cause cerebral ischemic injury and lead to cerebral ischemia-reperfusion injury. After the compound crosses the blood-brain barrier and enters the brain, it spontaneously cleaves to release borneol and thrombolytic peptides. Borneol inhibits the expression of inflammatory cytokines and inflammatory proteins, activates the GABAa receptor, reduces apoptosis and necrosis of cells, and alleviates cerebral edema and ischemia-reperfusion injury; while the polypeptide can dissolve the thrombus in the brain and scavenge the free radicals generated during cerebral ischemia-reperfusion, alleviating the cerebral ischemia condition and ischemia-reperfusion injury, which together play a protective role on the ischemic site of the brain.

[0120] Furthermore, the present disclosure provides the use of the aforementioned compounds, compounds prepared by the aforementioned methods, or the aforementioned pharmaceutical compositions in the preparation of drugs for treating thrombotic diseases.

[0121] As used herein, the term "thrombotic disease" refers to a disease caused by the formation of abnormal blood clots within the blood vessels of the formed elements in the circulating blood of humans and animals during their survival.

[0122] Among them, the thrombotic diseases include but are not limited to: ischemic stroke, myocardial infarction, stroke, venous embolism, pulmonary embolism, peripheral arterial obstructive disease, venous catheter occlusion, arteriovenous fistula and shunt occlusion, and carotid artery stenosis.

[0123] In another aspect, the present application provides a method for treating a subject suffering from a thrombotic disease, the method comprising: administering to the subject an effective amount of the foregoing compound, a compound prepared by the foregoing method, or the foregoing pharmaceutical composition.

[0124] In another aspect, the present application provides a method for improving cerebral ischemia, cerebral thrombosis, free radical status or inflammatory status of a subject, the method comprising: administering to the subject an effective amount of the foregoing compound, a compound prepared by the foregoing method, or the foregoing pharmaceutical composition.

[0125] As used herein, an "effective amount" is an amount sufficient to elicit the desired therapeutic effect administered by any of the above methods or any other methods known in the art.

[0126] Generally, the actual amount of the compound of the present disclosure administered to a patient can vary within a wide range, depending on the mode of administration, the patient's condition (including weight, sex, health status, and diet), the formulation used, and the desired response.

[0127] The compounds of the present disclosure have good biological activity. For example, depending on the specific compound selected, the desired therapeutic response, the route of administration, the formulation, and other factors known to those skilled in the art, the compound can be administered at about 0.1, 0.5, 1, 5, 50, 100, 500, 1000, or 5000 μg / kg body weight (as a single dose or in divided daily doses).

[0128] The various embodiments and preferred options 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 such combination are regarded as part of the disclosure of the present application.

[0129] The technical solutions of the present disclosure will be more clearly and definitely described below by way of examples. It should be understood that these examples are only for illustrative purposes and are in no way intended to limit the protection scope of the present disclosure.

[0130] Examples

[0131] Hereinafter, embodiments of the present application will be described. The embodiments described below are exemplary and are only used to explain the present application, and should not be construed as a limitation to the present application. For those without specific technical or conditions noted in the embodiments, the techniques or conditions described in the literature in the art or according to the product specifications are followed. For reagents or instruments without the manufacturer noted, they are all conventional products that can be obtained through commercial purchase.

[0132] The Chinese explanations of the abbreviations or English full names used in the present application are shown in Table 1 below:

[0133] Table 1 Comparison of Chinese Explanations of Abbreviations or English Full Names Used

[0134]

[0135]

[0136] The resin raw material used in Examples 2 to 12 is 2-chlorotrimethylbenzene resin (CTC Resin), and the substitution constant is 0.5 mmol / g.

[0137] Example 1: Synthesis of Camphenol-Linker

[0138]

[0139] Weigh 9.65 g (48 mmol) of phenyl chloroformate-4-nitro and 6.16 g (40 mmol) of camphenol into a 250 ml eggplant flask, add dichloromethane for dissolution, and dropwise add 5.5 ml (40 mmol) of triethylamine under ice bath conditions. After naturally rising to room temperature (25 ± 5 °C), react for 3 h. After the reaction is complete, concentrate the reaction solution under reduced pressure to dryness, add petroleum ether for dissolution, mix with silica gel, and purify by column chromatography to obtain 9.31 g (yield 73%) of the camphenol-linker target product, [M+H] + : 319.08. The 1H-NMR analysis is shown in Table 2.

[0140] Table 2: 1 1H-NMR Analysis of Camphenol-Linker Target Product

[0141] δ, ppm (DMSO) Type of H Assignment 0.872-0.892 <![CDATA[C H 3]]> <![CDATA[C on borneol H 3]]> 1.224-2.405 <![CDATA[C H 2 and C H > <![CDATA[C on borneol H 2]]> 4.840-4.878 <![CDATA[C H > <![CDATA[The C on borneol that is connected to the hydroxyl group H > 7.563-7.603、8.295-8.335 <![CDATA[C H > <![CDATA[C on the benzene ring H >

[0142] Example 2: Synthesis of Camphenol-Linker-PAK (PDC-1)

[0143]

[0144] (1) Synthesis of Fmoc-Lys(Boc)-CTC Resin

[0145] Weigh 1.0 g of 2-CTC Resin into a reactor, add 10 ml of DMF for swelling for 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, wash the resin alternately with DMF and DCM, 3 times each, then add 20% methanol dichloromethane solution and 0.25 ml of DIEA, and shake the reaction for 1 h.

[0146] (2) Synthesis of Fmoc-Ala-Lys(Boc)-CTC Resin

[0147] 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, wash the resin alternately with DMF and DCM, 3 times each; weigh 0.93 g (3 mmol) of Fmoc-Ala-OH and 1.14 g (3 mmol) of HATU, dissolve them in DMF and add them 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, then remove the reaction solution, and add DMF and DCM to wash the resin alternately, 3 times each.

[0148] (3) Synthesis of Fmoc-Pro-Ala-Lys(Boc)-CTC Resin

[0149] 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-Pro-OH according to the synthesis method mentioned in step (2). After the reaction, add DMF and DCM to wash the resin alternately, 3 times each.

[0150] (4) Synthesis of Camphenol-Linker-Pro-Ala-Lys(Boc)-CTC Resin

[0151] 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 camphenol-linker, dissolve it in DCM, and then add 0.5 ml (4 mmol) of DIEA to start the reaction. After the reaction is complete (determine the end point by ninhydrin detection), remove the reaction solution, and wash the resin successively with DMF 3 times, methanol 2 times, DCM 2 times, and methanol 2 times, and then dry it under vacuum to obtain the peptide resin.

[0152] (5) Synthesis of Camphenol-Linker-Pro-Ala-Lys

[0153] The dried peptide resin was added to a reactor, and cleavage solution (TFA:TIS:H2O = 95:2.5:2.5), 10 mL per gram of peptide resin, was added. The mixture was stirred at room temperature for 2 h. After the reaction, the mixture was filtered, and the filtrate was collected. The resin was washed twice with a small amount of TFA. Centrifuge tubes were prepared, ice-cold methyl tert-butyl ether was added, and then the collected filtrate was poured into the methyl tert-butyl ether. The mixture was stirred until the polypeptide precipitated, centrifuged, and the supernatant was decanted. The white solid at the bottom of the centrifuge tube was washed twice with methyl tert-butyl ether and then placed in a vacuum dryer to dry for more than 12 h to obtain the crude product.

[0154] (6) Purification of borneol-linker-Pro-Ala-Lys

[0155] The obtained crude product was dissolved in acetonitrile and water, and the solution was loaded onto a reverse-high performance liquid chromatography column for gradient elution. The position of the product was confirmed by detecting mass spectrometry (MS). The correct product fractions were collected and lyophilized 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 。

[0156] Example 3: Synthesis of borneol-linker-KAP (PDC-2)

[0157]

[0158] (1) Synthesis of Fmoc-Pro-CTC Resin

[0159] 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-Pro-OH was added, 0.25 mL (2 mmol) of DIEA was added, and the mixture was shaken for reaction for 1 h. The solvent was removed, and the resin was washed alternately with DMF and DCM, 3 times each. Then, 20% methanol-dichloromethane solution and 0.25 mL of DIEA were added, and the mixture was shaken for reaction for 1 h.

[0160] (2) Synthesis of Fmoc-Ala-Pro-CTC Resin

[0161] Remove the Fmoc group at the N-terminus of the resin with 25% 4-methylpiperidine / DMF (by volume) and react for 30 min. Evacuate the solvent, and wash the resin alternately with DMF and DCM, 3 times each; Weigh 0.93 g (3 mmol) of Fmoc-Ala-OH and 1.14 g (3 mmol) of HATU, dissolve them in an appropriate amount of 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, evacuate the reaction solution, and wash the resin alternately with DMF and DCM, 3 times each.

[0162] (3) Synthesis of Fmoc-Lys(Boc)-Ala-Pro-CTC Resin

[0163] Add 25% 4-methylpiperidine / DMF (by volume) 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, wash the resin alternately with DMF and DCM, 3 times each.

[0164] (4) Synthesis of Camphenol-Linker-Lys(Boc)-Ala-Pro-CTC Resin

[0165] Add 25% 4-methylpiperidine / DMF (by volume) to the resin to remove the Fmoc group at the N-terminus of the resin, then add 0.64 g (2 mmol) of camphenol-linker, dissolve it in DCM, and then add 0.5 ml (4 mmol) of DIEA to start the reaction. After the reaction is complete (determine the end point by ninhydrin detection), evacuate the reaction solution. Wash the resin successively with DMF 3 times, methanol 2 times, DCM 2 times, and methanol 2 times, and then dry it under vacuum to obtain the peptide resin.

[0166] (5) Synthesis of Camphenol-Linker-Lys-Ala-Pro

[0167] Add the dried peptide resin to the reactor, add the cleavage solution (TFA:TIS:H2O = 95:2.5:2.5), 10 ml per gram of peptide resin, stir and react at room temperature for 2 h. Filter the reaction mixture, collect the filtrate, and wash the resin with a small amount of TFA 2 times; Prepare a centrifuge tube, add ice-cold methyl tert-butyl ether, then pour the collected filtrate into the methyl tert-butyl ether, stir until the polypeptide precipitates, centrifuge, pour off the supernatant, wash the white substance at the bottom of the centrifuge tube with methyl tert-butyl ether 2 more times, and then place it in a vacuum dryer to dry for more than 12 hours to obtain the crude product.

[0168] (6) Purification of Camphenol-Linker-Lys-Ala-Pro

[0169] The obtained crude product was dissolved in acetonitrile and water, and the solution was loaded onto a reverse-high performance liquid chromatography column for gradient elution. The position of the product was confirmed by detecting MS; the correct product fraction was collected and freeze-dried to obtain the final product. Purity: 99.9% (HPLC, 220 nm, C18, linear gradient); [M+H] + : 495.3, see Figure 2 .

[0170] Example 4: Synthesis of borneol-linker-β-AHKAP (PDC-3)

[0171]

[0172] (1) Synthesis of Fmoc-Pro-CTC Resin

[0173] Weigh 1.0 g of 2-CTC Resin into a reactor, add 10 ml of DMF for swelling 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. The solvent was removed by suction, and the resin was washed alternately with DMF and DCM, 3 times each. Then, 20% methanol dichloromethane solution and 0.25 ml of DIEA were added, and the reaction was shaken for 1 h.

[0174] (2) Synthesis of Fmoc-Ala-Pro-CTC Resin

[0175] Add 25% 4-methylpiperidine / DMF (volume ratio) to remove the Fmoc group at the N-terminus of the resin, and react for 30 min. The solvent was removed by suction, and the resin was washed alternately with DMF and DCM, 3 times each; weigh 0.93 g (3 mmol) of Fmoc-Ala-OH and 1.14 g (3 mmol) of HATU, dissolve them in DMF and add them to the resin, then add 0.75 ml (6 mmol) of DIEA to start the reaction. The reaction end point was judged by ninhydrin detection. If the detection result showed that the reaction was complete, the reaction solution was removed by suction, and the resin was washed alternately with DMF and DCM, 3 times each.

[0176] (3) Synthesis of Fmoc-β-Ala-His(trt)-Lys(Boc)-Ala-Pro-CTC Resin

[0177] 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, Fmoc-His(trt)-OH, and Fmoc-β-Ala-OH in the same method as in step (2) in sequence. After the reaction, the resin was washed alternately with DMF and DCM, 3 times each.

[0178] (4) Synthesis of Camphenol - linker - β - Ala - His(trt) - Lys(Boc) - Ala - Pro - CTC Resin

[0179] 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 camphenol - linker, dissolve it in DCM, and then add 0.5 ml (4 mmol) of DIEA to start the reaction. After the reaction is complete (determined by ninhydrin detection endpoint), drain the reaction solution. Wash the resin successively with DMF three times, methanol twice, DCM twice, and methanol twice, and then dry it under vacuum to obtain the peptide resin.

[0180] (5) Synthesis of Camphenol - linker - β - Ala - His - Lys - Ala - Pro

[0181] Add the dried peptide resin to the reactor, add the cleavage solution (TFA:TIS:H2O = 95:2.5:2.5), 10 ml per gram of peptide resin, stir and react at room temperature for 2 h. Filter the reaction mixture, collect the filtrate, and wash the resin with a small amount of TFA twice. Prepare a centrifuge tube, add ice - cold methyl tert - butyl ether, and then pour the collected filtrate into the methyl tert - butyl ether, stir until the polypeptide precipitates, centrifuge, pour off the supernatant. Wash the white solid at the bottom of the centrifuge tube twice with methyl tert - butyl ether, and then place it in a vacuum dryer to dry for more than 12 hours to obtain the crude product.

[0182] (6) Purification of Camphenol - linker - β - Ala - His - Lys - Ala - Pro

[0183] Dissolve the obtained crude product in acetonitrile and water, load the solution onto a reverse - high performance liquid chromatography column for gradient elution, and confirm the position of the product by detecting MS; collect the correct product fractions and lyophilize them to obtain the final product. Purity: 99.1% (HPLC, 220 nm, C18, linear gradient); [M + H] + : 704.4, [M + 2H] 2+ : 352.3, see Figure 3 .

[0184] Example 5: Synthesis of Camphenol - linker - β - AHPAK (PDC - 4)

[0185]

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

[0187] Weigh 1.0 g of 2-CTC Resin into a reactor, add 10 ml of DMF for swelling for 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, wash the resin alternately with DMF and DCM, 3 times each, then add 20% methanol dichloromethane solution and 0.25 ml of DIEA, and shake the reaction 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, wash the resin alternately with DMF and DCM, 3 times each; weigh 0.93 g (3 mmol) of Fmoc-Ala-OH and 1.14 g (3 mmol) of HATU, dissolve them in DMF and add them 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, then remove the reaction solution, and add DMF and DCM to wash the resin alternately, 3 times each.

[0190] (3) Synthesis of Fmoc-β-Ala-His(trt)-Pro-Ala-Lys(Boc)-CTC Resin

[0191] Add 25% 4-methylpiperidine / DMF (volume ratio) to the resin to remove the Fmoc group at the N-terminus of the resin, and then according to the synthesis method in step (2), successively connect Fmoc-Pro-OH, Fmoc-His(trt)-OH, and Fmoc-β-Ala-OH. After the reaction is completed, wash the resin alternately with DMF and DCM, 3 times each.

[0192] (4) Synthesis of borneol-linker-β-Ala-His(trt)-Pro-Ala-Lys(Boc)-CTC Resin

[0193] 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 borneol-linker, dissolve it in DCM, 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 wash the resin successively with DMF 3 times, methanol 2 times, DCM 2 times, and methanol 2 times, and then vacuum dry to obtain the peptide resin.

[0194] (5) Synthesis of Camphenol - Linker - β - Ala - His - Pro - Ala - Lys

[0195] Add the dried peptide resin into the reactor, add the cleavage solution (TFA:TIS:H2O = 95:2.5:2.5), 10 mL per gram of peptide resin, stir and react at room temperature for 2 h. Filter the reaction mixture, collect the filtrate, and wash the resin with a small amount of TFA twice; Prepare a centrifuge tube, add ice-cold methyl tert-butyl ether, then pour the collected filtrate into the methyl tert-butyl ether, stir until the polypeptide precipitates, centrifuge, pour off the supernatant. After washing the white substance at the bottom of the centrifuge tube with methyl tert-butyl ether twice, place it in a vacuum dryer and dry for more than 12 hours to obtain the crude product.

[0196] (6) Purification of Camphenol - Linker - β - Ala - His - Pro - Ala - Lys

[0197] Dissolve the obtained crude product in acetonitrile and water, load the solution onto a reverse - high performance liquid chromatography column for gradient elution, confirm the position of the product by detecting MS; Collect the correct product components and freeze-dry to obtain the final product. Purity: 99.8% (HPLC, 220 nm, C18, linear gradient); [M + H] + : 703.4, [M + 2H] 2+ : 352.4, see Figure 4 。

[0198] Example 6: Synthesis of Camphenol - Linker - GRPAK (PDC - 5)

[0199]

[0200] (1) Synthesis of Fmoc - Lys(Boc) - CTC Resin

[0201] Weigh 1.0 g of 2 - CTC Resin into the reactor, add 10 mL of DMF to swell for 1 h, add 0.47 g (1 mmol) of Fmoc - Lys(Boc) - OH, add 0.25 mL (2 mmol) of DIEA, and shake and react for 1 h. Evacuate the solvent, wash the resin alternately with DMF and DCM, 3 times each, then add 20% methanol dichloromethane solution and 0.25 mL of DIEA, and shake and react for 1 h.

[0202] (2) Synthesis of Fmoc - Ala - Lys(Boc) - CTC Resin

[0203] Remove the Fmoc group at the N-terminus of the resin with 25% 4-methylpiperidine / DMF (volume ratio), and react for 30 min. Evaporate the solvent, and wash the resin alternately with DMF and DCM, 3 times each; Weigh 0.93 g (3 mmol) of Fmoc-Ala-OH and 1.14 g (3 mmol) of HATU, dissolve them 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, evaporate the reaction solution, and wash the resin alternately with DMF and DCM, 3 times each.

[0204] (3) Synthesis of Fmoc-Gly-Arg(pbf)-Pro-Ala-Lys(Boc) CTC Resin

[0205] 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-Pro-OH, Fmoc-Arg(pbf)-OH, and Fmoc-Gly-OH in sequence according to the synthesis method in step (2). After the reaction, wash the resin alternately with DMF and DCM, 3 times each.

[0206] (4) Synthesis of borneol-linker-Gly-Arg(pbf)-Pro-Ala-Lys(Boc) CTC Resin

[0207] 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 borneol-linker, dissolve it in DCM, and then add 0.5 ml (4 mmol) of DIEA to start the reaction. After the reaction is complete (ninhydrin detection end point), evaporate the reaction solution, and wash the resin with DMF 3 times, methanol 2 times, DCM 2 times, and methanol 2 times in sequence, and then dry it under vacuum to obtain the peptide resin.

[0208] (5) Synthesis of borneol-linker-Gly-Arg-Pro-Ala-Lys

[0209] Add the dried peptide resin to the reactor, add the cleavage solution (TFA:TIS:H2O = 95:2.5:2.5), 10 ml per gram of peptide resin, stir and react at room temperature for 2 h. Filter the reaction mixture, collect the filtrate, and wash the resin with a small amount of TFA 2 times; Prepare a centrifuge tube, add ice-cold methyl tert-butyl ether, and then pour the collected filtrate into the methyl tert-butyl ether, stir until the polypeptide precipitates, centrifuge, pour off the supernatant, wash the white solid at the bottom of the centrifuge tube with methyl tert-butyl ether 2 more times, and then place it in a vacuum dryer to dry for more than 12 h to obtain the crude product.

[0210] (6) Purification of Bornol-Linker-Gly-Arg-Pro-Ala-Lys

[0211] Dissolve the obtained crude product in acetonitrile and water, load the solution onto a reverse-high performance liquid chromatography column for gradient elution, and confirm the position of the product by detecting MS; collect the correct product fractions and lyophilize to obtain the final product. Purity: 98.5% (HPLC, 220 nm, C18, linear gradient); [M+H] + : 709.4, [M+2H] 2+ : 354.8, see Figure 5 。

[0212] Example 7: Synthesis of Bornol-Linker-PEG4-PAK (PDC-6)

[0213]

[0214] (1) Synthesis of Fmoc-Lys(Boc)-CTC Resin

[0215] Weigh 1.0 g of 2-CTC Resin into a reactor, add 10 ml of DMF for swelling for 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. Drain the solvent, wash the resin alternately with DMF and DCM, 3 times each, then add 20% methanol dichloromethane solution and 0.25 ml of DIEA, and shake the reaction for 1 h.

[0216] (2) Synthesis of Fmoc-Ala-Lys(Boc)-CTC Resin

[0217] Add 25% 4-methylpiperidine / DMF (volume ratio) to remove the Fmoc group at the N-terminus of the resin, and react for 30 min. Drain the solvent, wash the resin alternately with DMF and DCM, 3 times each; weigh 0.93 g (3 mmol) of Fmoc-Ala-OH and 1.14 g (3 mmol) of HATU, dissolve them in DMF and add them 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, drain the reaction solution, and wash the resin alternately with DMF and DCM, 3 times each.

[0218] (3) Synthesis of Fmoc-PEG4-Pro-Ala-Lys(Boc)-CTC Resin

[0219] Add 25% 4-methylpiperidine / DMF (by volume) to the resin to remove the Fmoc group at the N-terminus of the resin. Then, according to the synthesis method in step (2), successively connect Fmoc-Pro-OH and Fmoc-NH-PEG4-COOH. After the reaction is completed, wash the resin alternately with DMF and DCM, 3 times for each.

[0220] (4) Synthesis of borneol-linker-PEG4-Pro-Ala-Lys(Boc)-CTC Resin

[0221] Add 25% 4-methylpiperidine / DMF (by volume) to the resin to remove the Fmoc group at the N-terminus of the resin. Then add 0.64 g (2 mmol) of borneol-linker, dissolve it in an appropriate amount of DCM, and then add 0.5 ml (4 mmol) of DIEA to start the reaction. After the reaction is complete (determined by ninhydrin detection), draw off the reaction solution. Wash the resin successively with DMF 3 times, methanol 2 times, DCM 2 times, and methanol 2 times, and then dry it under vacuum to obtain the peptide resin.

[0222] (5) Synthesis of borneol-linker-PEG4-Pro-Ala-Lys

[0223] Add the dried peptide resin to the reactor, add the cleavage solution (TFA:TIS:H2O = 95:2.5:2.5), 10 ml per gram of peptide resin, stir and react at room temperature for 2 h. Filter the reaction mixture, collect the filtrate, and wash the resin with a small amount of TFA 2 times; prepare a centrifuge tube, add ice-cold methyl tert-butyl ether, then pour the collected filtrate into the methyl tert-butyl ether, stir until the polypeptide precipitates, centrifuge, pour off the supernatant. Wash the white substance at the bottom of the centrifuge tube with methyl tert-butyl ether two more times, and then place it in a vacuum dryer to dry for more than 12 hours to obtain the crude product.

[0224] (6) Purification of borneol-linker-PEG4-Pro-Ala-Lys

[0225] Dissolve the obtained crude product in acetonitrile and water, load the solution onto a reverse-high performance liquid chromatography column for gradient elution, and confirm the position of the product by detecting MS; collect the correct product fractions and freeze-dry them to obtain the final product. Purity: 98.6% (HPLC, 220 nm, C18, linear gradient); [M+H] + : 742.4, see Figure 6 。

[0226] Example 8: Synthesis of borneol-linker-K(KAP)RGDS(PDC-7)

[0227]

[0228] (1) Synthesis of Fmoc-Ser(tBu)-CTC Resin

[0229] Weigh 1.0 g of 2-CTC Resin into a reactor, add 10 ml of DMF to swell 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. Pump out the solvent, wash the resin alternately with DMF and DCM, 3 times each, then add 20% methanol dichloromethane solution and 0.25 ml of DIEA, and shake the reaction for 1 h.

[0230] (2) Synthesis of Fmoc-Asp(OtBu)-Ser(tBu)-CTC Resin

[0231] Add 25% 4-methylpiperidine / DMF (volume ratio) to remove the Fmoc group at the N-terminus of the resin, and react for 30 min. Pump out the solvent, wash the resin alternately with DMF and DCM, 3 times each; weigh 1.23 g (3 mmol) of Fmoc-Asp(OtBu)-OH and 1.14 g (3 mmol) of HATU, dissolve them 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, pump out the reaction solution, and add DMF and DCM to wash the resin alternately, 3 times each.

[0232] (3) Synthesis of Fmoc-Arg(pbf)-Gly-Asp(OtBu)-Ser(tBu)-CTC Resin

[0233] 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-Gly-OH and Fmoc-Arg(pbf)-OH in sequence according to the synthesis method mentioned in step (2). After the reaction, add DMF and DCM to wash the resin alternately, 3 times each.

[0234] (4) Synthesis of Alloc-Lys(Fmoc)-Arg(pbf)-Gly-Asp(OtBu)-Ser(tBu)-CTC Resin

[0235] Remove the Fmoc group at the N-terminus of the resin with 25% 4-methylpiperidine / DMF (by volume) and react for 30 min. Evacuate the solvent and wash the resin alternately with DMF and DCM, 3 times each; Weigh 1.27 g (3 mmol) of Alloc-Lys(Fmoc)-OH and 1.14 g (3 mmol) of HATU, dissolve them 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, evacuate the reaction solution and wash the resin alternately with DMF and DCM, 3 times each.

[0236] (5) Synthesis of Camphenol-Linker-Lys[Lys(Boc)-Ala-Pro]-Arg(pbf)-Gly-Asp(OtBu)-Ser(tBu)-CTC Resin

[0237] Add 25% 4-methylpiperidine / DMF (by volume) to the resin to remove the Fmoc group at the N-terminus of the resin, and then successively couple Fmoc-Lys(Boc)-OH, Fmoc-Ala-OH, and Fmoc-Pro-OH according to the method in step (2). After the reaction is completed, wash the resin alternately with DMF and DCM, 3 times each.

[0238] (6) Synthesis of Camphenol-Linker-Lys[Lys(Boc)-Ala-Pro]-Arg(pbf)-Gly-Asp(OtBu)-Ser(tBu)-CTC Resin

[0239] Add a dichloromethane solution of 20% tetrakis(triphenylphosphine)palladium and 10 mmol of phenylsilane to the resin to remove the Alloc group at the N-terminus of the resin, then add 0.64 g (2 mmol) of camphenol-linker, dissolve it in DCM, and then add 0.5 ml (4 mmol) of DIEA to start the reaction. After the reaction is complete (determine the end point by ninhydrin detection), evacuate the reaction solution, wash the resin successively with DMF 3 times, methanol 2 times, DCM 2 times, and methanol 2 times, and then dry it under vacuum to obtain the peptide resin.

[0240] (7) Synthesis of Camphenol-Linker-Lys(Lys-Ala-Pro)-Arg-Gly-Asp-Ser

[0241] Add the drained peptide resin to the reactor, add the cleavage solution (TFA:TIS:H2O = 95:2.5:2.5), 10 ml per gram of peptide resin, stir and react at room temperature for 2 h. Filter the reaction mixture, collect the filtrate, and wash the resin with a small amount of TFA twice. Prepare a centrifuge tube, add ice-cold methyl tert-butyl ether, then pour the collected filtrate into the methyl tert-butyl ether, stir until the polypeptide precipitates, centrifuge, pour off the supernatant. After washing the white substance at the bottom of the centrifuge tube twice with methyl tert-butyl ether, place it in a vacuum dryer and dry for more than 12 hours to obtain the crude product.

[0242] (8) Purification of borneol-linker-Lys(Lys-Ala-Pro)-Arg-Gly-Asp-Ser

[0243] Dissolve the obtained crude product in acetonitrile and water, load the solution onto a reverse-high performance liquid chromatography column for gradient elution, and confirm the position of the product by detecting MS; collect the correct product fractions and lyophilize to obtain the 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 Figure 7 。

[0244] Example 9: Synthesis of borneol-linker-K(KAPHA-β)RGDS(PDC-8)

[0245]

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

[0247] Weigh 1.0 g of 2-CTC Resin into the reactor, add 10 ml of DMF to swell for 1 h, add 0.38 g (1 mmol) of Fmoc-Ser(tBu)-OH, add 0.25 ml (2 mmol) of DIEA, and shake and react for 1 h. Evacuate the solvent, wash the resin alternately with DMF and DCM, 3 times each, then add 20% methanol dichloromethane solution and 0.25 ml of DIEA, and shake and react 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 with 25% 4-methylpiperidine / DMF (volume ratio), and react for 30 min. Evaporate the solvent, and wash the resin alternately with DMF and DCM, 3 times each; Weigh 1.23 g (3 mmol) of Fmoc-Asp(OtBu)-OH and 1.14 g (3 mmol) of HATU, dissolve them 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, evaporate the reaction solution, and wash the resin alternately with DMF and DCM, 3 times each.

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

[0251] 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-Gly-OH and Fmoc-Arg(pbf)-OH in sequence according to the synthesis method in step (2). After the reaction, wash the resin alternately with DMF and DCM, 3 times each.

[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 the resin to remove the Fmoc group at the N-terminus of the resin, and react for 30 min. Evaporate the solvent, and wash the resin alternately with DMF and DCM, 3 times each; Weigh 1.27 g (3 mmol) of Alloc-Lys(Fmoc)-OH and 1.14 g (3 mmol) of HATU, dissolve them 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, evaporate the reaction solution, and wash the resin alternately with DMF and DCM, 3 times each.

[0254] (5) Synthesis of Alloc-Lys[Lys(Boc)-Ala-Pro-His(trt)-β-Ala]-Arg(pbf)-Gly-Asp(OtBu)-Ser(tBu)-CTC Resin

[0255] Add 25% of 4-methylpiperidine / DMF (volume ratio) to the resin to remove the Fmoc group at the N-terminus of the resin. Then, according to the synthesis method used in step (2), successively connect Fmoc-Lys(Boc)-OH, Fmoc-Ala-OH, Fmoc-Pro-OH, Fmoc-His(trt)-OH, and Fmoc-β-Ala-OH. After the reaction, add DMF and DCM to the resin alternately to wash the resin, and wash each three times.

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

[0257] Add a dichloromethane solution of 20% tetrakis(triphenylphosphine)palladium and 10 mmol of phenylsilane to the resin to remove the Alloc group at the N-terminus of the resin. Then add 0.64 g (2 mmol) of borneol-linker, dissolve it in DCM, and then add 0.5 ml (4 mmol) of DIEA to start the reaction. After the reaction is complete (detect the end point with ninhydrin), draw off the reaction solution. Wash the resin successively with DMF three times, methanol twice, DCM twice, and methanol twice, and then dry it under vacuum to obtain the peptide resin.

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

[0259] Add the dried peptide resin to the reactor, add the cleavage solution (TFA:TIS:H2O = 95:2.5:2.5), 10 ml per gram of peptide resin, stir and react at room temperature for 2 h. Filter the reaction mixture, collect the filtrate, and wash the resin with a small amount of TFA twice; prepare a centrifuge tube, add ice-cold methyl tert-butyl ether, and then pour the collected filtrate into the methyl tert-butyl ether, stir until the polypeptide precipitates, centrifuge, pour off the supernatant. Wash the white substance at the bottom of the centrifuge tube twice with methyl tert-butyl ether, and then place it in a vacuum dryer to dry for more than 12 hours to obtain the crude product.

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

[0261] Dissolve the obtained crude product in acetonitrile and water, load the solution onto a reverse-high performance liquid chromatography column for gradient elution, and confirm the position of the product by detecting MS; collect the correct product components and freeze-dry them 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 .

[0262] Example 10: Synthesis of Bornol-Connector-K(KAPRG)RGDS(PDC-9)

[0263]

[0264] (1) Synthesis of Fmoc-Ser(tBu)-CTC Resin

[0265] Weigh 1.0 g of 2-CTC Resin into a reactor, add 10 ml of DMF to swell it 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, wash the resin alternately with DMF and DCM, 3 times each, then add 20% methanol-dichloromethane solution and 0.25 ml of DIEA, and shake the reaction for 1 h.

[0266] (2) Synthesis of Fmoc-Asp(OtBu)-Ser(tBu)-CTC Resin

[0267] 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, wash the resin alternately with DMF and DCM, 3 times each; weigh 1.23 g (3 mmol) of Fmoc-Asp(OtBu)-OH and 1.14 g (3 mmol) of HATU, dissolve them in DMF and add to the resin, then add 0.75 ml (6 mmol) of DIEA to start the reaction. Judge the end point of the reaction by ninhydrin detection. If the detection result shows that the reaction is complete, then remove the reaction solution, and add DMF and DCM to wash the resin alternately, 3 times each.

[0268] (3) Synthesis of Fmoc-Arg(pbf)-Gly-Asp(OtBu)-Ser(tBu)-CTC Resin

[0269] 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-Gly-OH and Fmoc-Arg(pbf)-OH in sequence according to the synthesis method used in step (2). After the reaction, add DMF and DCM to wash the resin alternately, 3 times each.

[0270] (4) Synthesis of Alloc-Lys(Fmoc)-Arg(pbf)-Gly-Asp(OtBu)-Ser(tBu)-CTC Resin

[0271] Add 25% 4-methylpiperidine / DMF (by volume) to remove the Fmoc group at the N-terminus of the resin, and react for 30 min. Pump out the solvent, and wash the resin alternately with DMF and DCM, 3 times each; Weigh 1.27 g (3 mmol) of Alloc-Lys(Fmoc)-OH and 1.14 g (3 mmol) of HATU, dissolve them 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, pump out the reaction solution, and wash the resin alternately with DMF and DCM, 3 times each.

[0272] (5) Synthesis of Alloc-Lys[Lys(Boc)-Ala-Pro-Arg(pbf)-Gly-Boc]-Arg(pbf)-Gly-Asp(OtBu)-Ser(tBu)-CTC Resin

[0273] Add 25% 4-methylpiperidine / DMF (by volume) to the resin to remove the Fmoc group at the N-terminus of the resin, and then connect Fmoc-Lys(Boc)-OH, Fmoc-Ala-OH, Fmoc-Pro-OH, Fmoc-Arg(pbf)-OH, and Fmoc-Gly-OH in sequence according to the synthesis method used in step (2). After the reaction is completed, wash the resin alternately with DMF and DCM, 3 times each.

[0274] (6) Synthesis of Camphenol-Linker-Lys[Lys(Boc)-Ala-Pro-Arg(pbf)-Gly-Boc]-Arg(pbf)-Gly-Asp(OtBu)-Ser(tBu)-CTC Resin

[0275] Add a 20% solution of tetrakis(triphenylphosphine)palladium in dichloromethane and 10 mmol of phenylsilane to the resin to remove the Alloc group at the N-terminus of the resin, then add 0.64 g (2 mmol) of camphenol-linker, dissolve it in DCM, and then add 0.5 ml (4 mmol) of DIEA to start the reaction. After the reaction is complete (determine the end point by ninhydrin detection), pump out the reaction solution, wash the resin with DMF 3 times, methanol 2 times, DCM 2 times, and methanol 2 times in sequence, and then dry it under vacuum to obtain the peptide resin.

[0276] (7) Synthesis of Camphenol-Linker-Lys(Lys-Ala-Pro-Arg-Gly)-Arg-Gly-Asp-Ser

[0277] The dried peptide resin was added to a reactor, and a cleavage solution (TFA:TIS:H2O = 95:2.5:2.5) was added at a rate of 10 mL per gram of peptide resin. The mixture was stirred at room temperature for 2 h, and the reaction mixture was filtered. The filtrate was collected, and the resin was washed twice with a small amount of TFA. Centrifuge tubes were prepared and ice-cold methyl tert-butyl ether was added. Then the collected filtrate was poured into the methyl tert-butyl ether and stirred until the polypeptide precipitated. After centrifugation, the supernatant was decanted. The white solid at the bottom of the centrifuge tube was washed twice with methyl tert-butyl ether and then placed in a vacuum dryer to dry for more than 12 h to obtain the crude product.

[0278] (8) Purification of borneol-linker-Lys(Lys-Ala-Pro-Arg-Gly)-Arg-Gly-Asp-Ser

[0279] The obtained crude product was dissolved in acetonitrile and water, and the solution was loaded onto a reverse-high performance liquid chromatography column for gradient elution. The position of the product was confirmed by detecting MS. The correct product fractions were collected and lyophilized to obtain the 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 。

[0280] Example 11: Synthesis of borneol-linker-K(KAP)IESDV(PDC-10)

[0281]

[0282] (1) Synthesis of Fmoc-Val-CTC Resin

[0283] Weighed 1.0 g of 2-CTC Resin into a reactor, added 10 mL of DMF for swelling for 1 h, added 0.34 g (1 mmol) of Fmoc-Ser(tBu)-OH, added 0.25 mL (2 mmol) of DIEA, and oscillated for reaction for 1 h. The solvent was removed by suction, and the resin was washed alternately with DMF and DCM, 3 times each. Then, 20% methanol dichloromethane solution and 0.25 mL of DIEA were added, and the mixture was oscillated for reaction for 1 h.

[0284] (2) Synthesis of Fmoc-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 react for 30 min. Evacuate the solvent, and wash the resin alternately with DMF and DCM, 3 times each; Weigh 1.23 g (3 mmol) of Fmoc-Asp(OtBu)-OH and 1.14 g (3 mmol) of HATU, dissolve them 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, evacuate the reaction solution, and wash the resin alternately with DMF and DCM, 3 times each.

[0286] (3) Synthesis of Fmoc-Ile-Glu(OtBu)-Ser(tBu)-Asp(OtBu)-Val-CTC Resin

[0287] 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-Ser(tBu)-OH, Fmoc-Glu(OtBu)-OH, and Fmoc-Ile-OH in sequence according to the synthesis method used in step (2). After the reaction, wash the resin alternately with DMF and DCM, 3 times each.

[0288] (4) Alloc-Lys(Fmoc)-Ile-Glu(OtBu)-Ser(tBu)-Asp(OtBu)-Val-CTC Resin

[0289] Add 25% 4-methylpiperidine / DMF (volume ratio) to the resin to remove the Fmoc group at the N-terminus of the resin, and react for 30 min. Evacuate the solvent, and wash the resin alternately with DMF and DCM, 3 times each; Weigh 1.27 g (3 mmol) of Alloc-Lys(Fmoc)-OH and 1.14 g (3 mmol) of HATU, dissolve them 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, evacuate the reaction solution, and wash the resin alternately with DMF and DCM, 3 times each.

[0290] (5) Synthesis of Alloc-Lys[Lys(Boc)-Ala-Pro-Boc]-Ile-Glu(OtBu)-Ser(tBu)-Asp(OtBu)-Val-CTC Resin

[0291] Add 25% 4-methylpiperidine / DMF (volume ratio) to the resin to remove the Fmoc group at the N-terminus of the resin. Then, according to the synthesis method used in step (2), sequentially connect Fmoc-Lys(Boc)-OH, Fmoc-Ala-OH, and Fmoc-Pro-OH. After the reaction is completed, wash the resin alternately with DMF and DCM, 3 times for each.

[0292] (6) Synthesis of borneol-linker-Lys[Lys(Boc)-Ala-Pro-Boc]-Ile-Glu(OtBu)-Ser(tBu)-Asp(OtBu)-Val-CTC Resin

[0293] Add a dichloromethane solution of 20% tetrakis(triphenylphosphine)palladium and 10 mmol of phenylsilane to the resin to remove the Alloc group at the N-terminus of the resin. Then add 0.64 g (2 mmol) of borneol-linker, dissolve it in DCM, and then add 0.5 ml (4 mmol) of DIEA to start the reaction. After the reaction is complete (detect the end point with ninhydrin), suck out the reaction solution. Wash the resin successively with DMF 3 times, methanol 2 times, DCM 2 times, and methanol 2 times, and then dry it under vacuum to obtain the peptide resin.

[0294] (7) Synthesis of borneol-linker-Lys(Lys-Ala-Pro)-Ile-Glu-Ser-Asp-Val

[0295] Put the dried peptide resin into a reactor, add the cleavage solution (TFA:TIS:H2O = 95:2.5:2.5), 10 ml per gram of peptide resin, stir and react at room temperature for 2 h. Filter the reaction mixture, collect the filtrate, and wash the resin with a small amount of TFA 2 times; Prepare a centrifuge tube, add ice-cold methyl tert-butyl ether, and then pour the collected filtrate into the methyl tert-butyl ether, stir until the polypeptide precipitates, centrifuge, pour off the supernatant. Wash the white substance at the bottom of the centrifuge tube with methyl tert-butyl ether two more times, and then dry it in a vacuum dryer for more than 12 hours to obtain the crude product.

[0296] (8) Purification of borneol-linker-Lys(Lys-Ala-Pro)-Ile-Glu-Ser-Asp-Val

[0297] Dissolve the obtained crude product in acetonitrile and water, load the solution onto a reverse-high performance liquid chromatography column for gradient elution, and confirm the position of the product by detecting MS; Collect the correct product fractions and freeze-dry them 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 .

[0298] Example 12: Synthesis of Camphenol - Linker - K (KAPHA - β) IESDV (PDC - 11)

[0299]

[0300] (1) Synthesis of Fmoc - Val - CTC Resin

[0301] Weigh 1.0 g of 2 - CTC Resin into 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. Drain the solvent, wash the resin alternately with DMF and DCM, 3 times each, then add 20% methanol - dichloromethane solution and 0.25 ml of DIEA, and shake the reaction for 1 h.

[0302] (2) Synthesis of Fmoc - Asp(OtBu) - Val - CTC Resin

[0303] Add 25% 4 - methylpiperidine / DMF (volume ratio) to remove the Fmoc group at the N - terminus of the resin, and react for 30 min. Drain the solvent, wash the resin alternately with DMF and DCM, 3 times each; weigh 1.23 g (3 mmol) of Fmoc - Asp(OtBu) - OH and 1.14 g (3 mmol) of HATU, dissolve them 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, drain the reaction solution, and wash the resin alternately with DMF and DCM, 3 times each.

[0304] (3) Synthesis of Fmoc - Ile - Glu(OtBu) - Ser(tBu) - Asp(OtBu) - Val - CTC Resin

[0305] 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 - Ser(tBu) - OH, Fmoc - Glu(OtBu) - OH, and Fmoc - Ile - OH in sequence according to the synthesis method in step (2). After the reaction, wash the resin alternately with DMF and DCM, 3 times each.

[0306] (4) Synthesis of Alloc - Lys(Fmoc) - Ile - Glu(OtBu) - Ser(tBu) - Asp(OtBu) - Val - CTC Resin

[0307] Remove the Fmoc group at the N-terminus of the resin with 25% 4-methylpiperidine / DMF (volume ratio) and react for 30 min. Evacuate the solvent and wash the resin alternately with DMF and DCM, 3 times each; Weigh 1.27 g (3 mmol) of Alloc-Lys(Fmoc)-OH and 1.14 g (3 mmol) of HATU, dissolve them 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, evacuate the reaction solution and wash the resin alternately with DMF and DCM, 3 times each.

[0308] (5) Synthesis of Camphenol-Linker-Lys[Lys(Boc)-Ala-Pro-His(trt)-Ala-β-Boc]-Ile-Glu(OtBu)-Ser(tBu)-Asp(OtBu)-Val-CTC Resin

[0309] 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 couple Fmoc-Lys(Boc)-OH, Fmoc-Ala-OH, Fmoc-Pro-OH, Fmoc-His(trt)-OH, Boc-β-Ala-OH according to the method of step (2). After the reaction is completed, wash the resin alternately with DMF and DCM, 3 times each.

[0310] (6) Synthesis of Camphenol-Linker-Lys[Lys(Boc)-Ala-Pro-His(trt)-Ala-β-Boc]-Ile-Glu(OtBu)-Ser(tBu)-Asp(OtBu)-Val-CTC Resin

[0311] Add a dichloromethane solution of 20% tetrakis(triphenylphosphine)palladium and 10 mmol of phenylsilane to the resin to remove the Alloc group at the N-terminus of the resin, then add 0.64 g (2 mmol) of camphenol-linker, dissolve it in DCM, and then add 0.5 ml (4 mmol) of DIEA to start the reaction. After the reaction is complete (ninhydrin detection end point), evacuate the reaction solution, wash the resin with DMF 3 times, methanol 2 times, DCM 2 times, methanol 2 times in sequence, and then dry it under vacuum to obtain the peptide resin.

[0312] (7) Synthesis of Camphenol-Linker-Lys(Lys-Ala-Pro-His-Ala-β)-Ile-Glu-Ser-Asp-Val

[0313] The dried peptide resin was added to a reactor, and cleavage solution (TFA:TIS:H2O = 95:2.5:2.5), 10 mL per gram of peptide resin, was added. The mixture was stirred at room temperature for 2 h, and the reaction mixture was filtered. The filtrate was collected, and the resin was washed twice with a small amount of TFA. Centrifuge tubes were prepared, ice-cold methyl tert-butyl ether was added, and then the collected filtrate was poured into the methyl tert-butyl ether. The mixture was stirred until the polypeptide precipitated, centrifuged, and the supernatant was decanted. The white solid at the bottom of the centrifuge tube was washed twice with methyl tert-butyl ether and then placed in a vacuum dryer for drying for more than 12 h to obtain the crude product.

[0314] (8) Purification of borneol-linker-Lys(Lys-Ala-Pro-His-Ala-β)-Ile-Glu-Ser-Asp-Val

[0315] The obtained crude product was dissolved in acetonitrile and water, and the solution was loaded onto a reverse-high performance liquid chromatography column for gradient elution. The position of the product was confirmed by detecting MS. The correct product fractions were 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 .

[0316] Example 13: Synthesis of borneol-linker-K(KAPRG)IESDV(PDC-12)

[0317]

[0318] (1) Synthesis of Fmoc-Val-CTC Resin

[0319] 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 mixture was shaken for reaction for 1 h. The solvent was removed, and the resin was washed alternately with DMF and DCM, 3 times each, and then 20% methanol dichloromethane solution and 0.25 mL of DIEA were added, and the mixture was shaken for reaction for 1 h.

[0320] (2) Synthesis of Fmoc-Asp(OtBu)-Val-CTC Resin

[0321] Remove the Fmoc group at the N-terminus of the resin with 25% 4-methylpiperidine / DMF (volume ratio), and react for 30 min. Evaporate the solvent, and wash the resin alternately with DMF and DCM, 3 times each; Weigh 1.23 g (3 mmol) of Fmoc-Asp(OtBu)-OH and 1.14 g (3 mmol) of HATU, dissolve them in DMF and add them 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, evaporate the reaction solution, and wash the resin alternately with DMF and DCM, 3 times each.

[0322] (3) Synthesis of Alloc-Lys(Fmoc)-Ile-Glu(OtBu)-Ser(tBu)-Asp(OtBu)-Val-CTC Resin

[0323] 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-Ser(tBu)-OH, Fmoc-Glu(OtBu)-OH, and Fmoc-Ile-OH in sequence according to the synthesis method in step (2). After the reaction is completed, wash the resin alternately with DMF and DCM, 3 times each.

[0324] (4) Synthesis of Alloc-Lys(Fmoc)-Ile-Glu(OtBu)-Ser(tBu)-Asp(OtBu)-Val-CTC Resin

[0325] Add 25% 4-methylpiperidine / DMF (volume ratio) to the resin to remove the Fmoc group at the N-terminus of the resin, and react for 30 min. Evaporate the solvent, and wash the resin alternately with DMF and DCM, 3 times each; Weigh 1.27 g (3 mmol) of Alloc-Lys(Fmoc)-OH and 1.14 g (3 mmol) of HATU, dissolve them in DMF and add them 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, evaporate the reaction solution, and wash the resin alternately with DMF and DCM, 3 times each.

[0326] (5) Synthesis of Alloc-Lys[Lys(Boc)-Ala-Pro-Arg(pbf)-Gly-Boc]-Ile-Glu(OtBu)-Ser(tBu)-Asp(OtBu)-Val-CTC Resin

[0327] Add 25% of 4-methylpiperidine / DMF (volume ratio) to the resin to remove the Fmoc group at the N-terminus of the resin. Then, according to the synthesis method in step (2), sequentially connect Fmoc-Lys(Boc)-OH, Fmoc-Ala-OH, Fmoc-Pro-OH, Fmoc-Arg(pbf)-OH, and Fmoc-Gly-OH. After the reaction, add DMF and DCM to the resin and wash the resin alternately, 3 times for each.

[0328] (6) Synthesis of borneol-linker-Lys[Lys(Boc)-Ala-Pro-Arg(pbf)-Gly-Boc]-Ile-Glu(OtBu)-Ser(tBu)-Asp(OtBu)-Val-CTC Resin

[0329] Add a dichloromethane solution of 20% tetrakis(triphenylphosphine)palladium and 10 mmol of phenylsilane to the resin to remove the Alloc group at the N-terminus of the resin. Then add 0.64 g (2 mmol) of borneol-linker, dissolve it in DCM, and then add 0.5 ml (4 mmol) of DIEA to start the reaction. After the reaction is complete (detect the end point with ninhydrin), suck out the reaction solution. Wash the resin with DMF 3 times, methanol 2 times, DCM 2 times, and methanol 2 times in sequence, and then dry it under vacuum to obtain the peptide resin.

[0330] (7) Synthesis of borneol-linker-Lys(Lys-Ala-Pro-Arg-Gly)-Ile-Glu-Ser-Asp-Val

[0331] Add the dried peptide resin to the reactor, add the cleavage solution (TFA:TIS:H2O = 95:2.5:2.5), 10 ml per gram of peptide resin, stir and react at room temperature for 2 h. Filter the reaction mixture, collect the filtrate, and wash the resin with a small amount of TFA 2 times; prepare a centrifuge tube, add ice-cold methyl tert-butyl ether, and then pour the collected filtrate into the methyl tert-butyl ether, stir until the polypeptide precipitates, centrifuge, pour off the supernatant. Wash the white substance at the bottom of the centrifuge tube with methyl tert-butyl ether twice, and then place it in a vacuum dryer to dry for more than 12 hours to obtain the crude product.

[0332] (8) Purification of borneol-linker-Lys(Lys-Ala-Pro-Arg-Gly)-Ile-Glu-Ser-Asp-Val

[0333] Dissolve the obtained crude product in acetonitrile and water, load the solution onto a reverse-high performance liquid chromatography column for gradient elution, and confirm the position of the product by detecting MS (mass spectrometry); collect the correct product components and freeze-dry them 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 .

[0334] Example 14: Detection of the Therapeutic Effect of Camphene Polypeptide Conjugates on Stroke in Rats

[0335] 14.1 Drugs and Solvents

[0336] 14.1.1 Tested Drugs:

[0337] Positive Control Drug: Edaravone and (+)-borneol (4:1) combination

[0338] Test Articles: PDC-1, PDC-2, PDC-3, PDC-4, PDC-5, PDC-6 prepared in Examples 2-7 respectively

[0339] Solvent: 20% propylene glycol aqueous solution

[0340] 14.1.2 Preparation Method:

[0341] Positive Control Drug: Add 40 mg of edaravone and 10 mg of (+)-borneol to 3.0 mL of propylene glycol. After complete dissolution, slowly add water for injection to 15.0 mL to obtain the administration solution of the positive control drug. Among them, the final concentration of edaravone is 2.67 mg / mL, and the final concentration of (+)-borneol is 0.67 mg / mL.

[0342] Test Articles: Similar to the preparation method of the positive control drug, dissolve PDC-1, PDC-2, PDC-3, PDC-4, PDC-5, PDC-6 in propylene glycol respectively, and slowly add water for injection with a volume 4 times that of propylene glycol for dilution to obtain the administration solution of the test articles. Among them, the final concentration of PDC-1 is 0.82 mg / mL, the final concentration of PDC-2 is 1.17 mg / mL, the final concentration of PDC-3 is 0.82 mg / mL, the final concentration of PDC-4 is 1.17 mg / mL, the final concentration of PDC-5 is 1.24 mg / mL, and the final concentration of PDC-6 is 1.18 mg / mL.

[0343] 14.2 Experimental Consumables and Reagents

[0344] 2,3,5-Triphenyltetrazolium Chloride (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 suture: Purchased from Beijing Xinong Technology Co., Ltd.; 0.36 mm series, A2636 - A4 is applicable to rats weighing 250 - 280 g, suture length is about 45 mm, and the head diameter is 0.36 ± 0.02 mm.

[0345] 14.3 Experimental animals

[0346] Adult male SD rats, SPF grade (specific pathogen-free animals, not carrying major potential infectious or opportunistic pathogens and pathogens that interfere greatly with scientific experiments), body weight: maintained at 260 - 280 g during the experiment; source: Beijing Vital River Laboratory Animal Technology Co., Ltd. Environmental adaptation period: at least 2 days, and the experimental personnel conduct cage-side observations once a day during the adaptation period.

[0347] 14.4 Breeding conditions

[0348] No more than 5 animals per cage. Record the environmental parameters of the animal house during the breeding period. During the experiment, no other types of animals are bred in the same room. Temperature: 18 - 24 °C; relative humidity: 40% - 70%; ventilation: the number of air changes per hour is not less than 8 times, using an IVC independent air supply system (independent ventilation cage system); lighting: automatic lighting, with 12-hour light and dark alternation, turning off the lights at 8:00 am and turning on the lights at 8:00 am the next day.

[0349] 14.5 Dose design

[0350] Table 3 Dose design table

[0351]

[0352] Dose = Concentration × Dosage

[0353] 14.6 Experimental methods

[0354] The MCAO (middle cerebral artery occlusion) reperfusion model was made with reference to the Longa method. Food was withheld 12 hours before surgery, and free access to water was allowed. The rats were anesthetized by inhaling isoflurane. A midline cervical incision was made to expose the common carotid artery (CCA), external carotid artery (ECA), and pterygopalatine artery. A suture thread (specially purchased suture thread, Beijing Xinong, A2636 - A4 suitable for rats weighing 250 - 280 g, and marked at 20 mm) was inserted through the incision of the right ECA in all rats. The pterygopalatine artery was briefly clamped to prevent misinsertion. The length of the suture thread was about 18 - 20 mm from the bifurcation of the CCA, depending on the body weight of the animal. The right middle cerebral artery was embolized, and then the skin was sutured. The end of the suture thread was fixed to the skin. After 2 hours of ischemia, the suture thread was carefully withdrawn to form reperfusion. In the sham operation group, the suture thread was not inserted, and the other steps were the same as those in the operation group. The body temperature was maintained at (37 ± 0.5) °C during ischemia and 2 hours after reperfusion. The criteria for a successful model were that the rats showed left limb paralysis, unstable standing, and circling to one side when the tail was lifted after the anesthesia was lifted. After the rats woke up, they were scored according to the Zea - Longa 5 - point scoring standard: 0 points represented no neurological deficit symptoms; 1 point represented incomplete extension of the contralateral forelimb; 2 points represented circling towards the hemiplegic side when walking; 3 points represented falling towards the hemiplegic side when walking; 4 points represented inability to walk spontaneously and consciousness disorder; 5 points represented death. A cumulative score of 1 point and above was considered a successful model; any score of 5 points, subarachnoid hemorrhage found during dissection, or no neurological deficit symptoms (0 points) were considered failed models.

[0355] The animals were randomly grouped according to the scores, and the number of valid animals in each group was not less than 8. At 1 hour after ischemia, 20% propylene glycol aqueous solution, positive control drug (edaravone 8 mg / kg + dextrancamphor sulfonic acid 2 mg / kg), and the test article at 5 μmol / kg were injected into the rats in each group via the tail vein. At 24 hours after administration, the neurological deficit signs of the animals were scored according to the Zea - Longa five - point method to evaluate the drug efficacy. 2,3,5 - triphenyltetrazolium chloride (TTC) staining was used to determine the cerebral infarction volume. The animals were decapitated after excessive inhalation of carbon dioxide, and the rat brains were quickly removed and placed in ice - saline for 10 minutes. Coronal sections with a thickness of 2 mm were evenly cut in a brain trough and quickly placed in a 2% TTC solution (37 °C) for staining for 30 minutes, and then fixed with 4% paraformic acid. After 24 hours, digital photos were taken with a digital camera, input into a computer, and the infarct area was calculated using image - processing software (ADOBE, PHOTOSHOP). The pink area was normal brain tissue, and the white area was the infarct area. To reduce the influence of cerebral ischemia - hemisphere edema on the results, the infarct volume was calculated by subtracting the volume of the normal tissue on the ipsilateral side from the volume of the normal tissue on the contralateral side of the injury, and the results were expressed as a percentage of the infarct volume. The cerebral infarction rate = (volume of normal tissue on the contralateral side - volume of normal tissue on the ipsilateral side) / volume of normal tissue on the contralateral side × 100%. All statistical processes were performed using GraphPad Prism software.

[0356] 14.7 Experimental Results

[0357] 14.7.1 Effects of the Test Drugs on the Neurological Symptom Behavior of Rats with Acute Cerebral Ischemia

[0358] After cerebral ischemia, the anesthetized rats showed varying degrees of focal neurological dysfunction after waking up, manifested as weakness in the left lower limb, circling to the left when walking straight, falling to the left or even unable to walk severely, and even developing consciousness disorders. When the tail was lifted, it was manifested as flexion and adduction of the left forelimb, and straightening and rotation to the right of the hind limb. After cerebral ischemia, obvious neurological injury symptoms appeared in the animals of the model group, and the neurological function score increased significantly (P<0.05). Table 4 shows the number of surviving animals and the scores of the behavioral evaluation after the experiment. It can be seen that compared with the solvent group, the behavioral scores of the test article groups were significantly reduced, indicating that the test article could significantly improve the neurological symptom behavior function of rats with acute cerebral ischemia. The behavioral scores of compounds PDC-1 and PDC-3 were basically equivalent to those of the positive control group, indicating that PDC-1 and PDC-3 could significantly improve the neurological symptom behavior function of rats with acute cerebral ischemia.

[0359] 14.7.2 Effects of the Test Drugs on the Infarct Volume of Rats with Acute Cerebral Ischemia

[0360] After staining, the normal brain tissue showed a whole rose red color, while the infarcted tissue showed a white color and the boundary was distinct. Except for the sham operation group, obvious infarct lesions appeared in the brains of rats in other groups. Table 4 shows the cerebral infarction rates of the experimental animals. Compared with the solvent group, the cerebral infarction rates of the test article groups were significantly reduced, indicating that the test article could significantly reduce the infarct volume of rats. The cerebral infarction rates of compounds PDC-1 and PDC-4 were lower than those of the positive control group, indicating that PDC-1 and PDC-4 significantly improved cerebral tissue infarction and the effect was better than the positive control drug.

[0361] Table 4

[0362]

[0363] Values are expressed as mean ± SD. One-way ANOVA and Dunnett's multiple comparisons test were used for analysis. Compared with the sham operation group, *P<0.05; compared with the solvent group, #P<0.05.

[0364] In summary, compound PDC-1 can effectively prevent the occurrence of motor disorders and cerebral tissue infarction in rats with cerebral ischemia.

[0365] Example 15: Detection of the Continuous Administration Therapeutic Effect of Camphenol Polypeptide Conjugate on Stroke Rats

[0366] The method for making the MCAO reperfusion rat model, the model scoring criteria are basically the same as those in Example 14, with the differences being: the ischemia time is 1 h, and the drug is administered via the caudal vein of the rat. After ischemia reaches 2 h, the suture is carefully withdrawn to form reperfusion.

[0367] The animals were randomly grouped according to the model scores, and the animal grouping and drug administration methods are shown in Table 5 below:

[0368] Table 5

[0369]

[0370]

[0371] The first drug administration was performed 1 h after ischemia. A 20% propylene glycol aqueous solution, a positive control drug (edaravone 8 mg / kg + dextrancamphor 2 mg / kg), and the test article at 5 μmol / kg were respectively injected into the caudal veins of the rats in each group, and then the drug was continuously administered for 5 days. The neurological deficit signs of the animals were scored according to the Zea-Longa five-point method standard 1 h after modeling (d0, starting score), and the scores were also taken at the same time points on the 1st day (d1, i.e., 24 h after reperfusion), the 2nd day (d2), the 3rd day (d3), the 4th day (d4), the 5th day (d5), and the 6th day (d6) after modeling. The decrease rate of the Zea-Longa score at the end point of the experiment was calculated to investigate the drug efficacy.

[0372] Figure 13 The effects of the test drugs on the neurological symptom behavior of rats with acute cerebral ischemia are shown. Among them, the decreases in the PDC-1, PDC-2, PDC-7, and PDC-10 groups at the end point compared with the starting score of the solvent group were 0.58, 0.64, 0.89, and 0.92 respectively, showing a trend of improving the neurological symptom behavior of rats.

[0373] In addition, the rat brain tissues were stained and the cerebral infarction rate was calculated using the same method as in Example 14 to investigate the effects of the test drugs on the cerebral infarction volume of rats with acute cerebral ischemia. The results are shown in Table 6. It can be found that obvious infarction foci appeared in the brains of the rats after modeling, and the cerebral infarction rate of the rats in the solvent group was significantly higher than that in the sham operation group. After 5 days of continuous drug administration treatment, the positive control drug, PDC-1, and PDC-2 could effectively relieve the cerebral infarction volume, and there were statistical differences (P < 0.05).

[0374] Table 6

[0375]

[0376] The numerical values represent mean ± SD. Analyzed by One-way ANOVA and Dunnett's multiple comparisons test, *P < 0.05 compared with the vehicle group.

[0377] The results indicate that the compounds provided by the present disclosure can significantly improve and enhance the neurological symptom behavior function of rats with acute cerebral ischemia, can significantly reduce the volume of cerebral infarction, show good cerebral ischemia protection, show excellent curative effects for the treatment of stroke, and have good clinical application prospects.

Claims

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

2. The compound according to claim 1, characterized in that, The compound has the structure shown in Formula I: Among them, L represents the linker, and P 1 represents the polypeptide part.

3. The compound according to claim 1 or 2, characterized in that, The polypeptide moiety is derived from a polypeptide having thrombolytic activity and / or free radical scavenging activity.

4. The compound according to any one of claims 1 to 3, characterized in that (1) The linker L and the polypeptide moiety P 1 are covalently linked to the N-terminus; (2) The linker L forms an ester bond connection with the hydroxyl group of borneol; and / or, (3) The borneol is (+)-borneol.

5. The compound according to any one of claims 1 to 4, characterized in that, The compound has the structural formula shown in Formula II: 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 cyclic nitrogen atom at the N-terminus of the polypeptide moiety; P represents the polypeptide residues of the polypeptide moiety other than the N-terminal -N(H) 0,1 - other than;.

6. The compound according to any one of claims 1 to 5, characterized in that, The polypeptide moiety comprises 1, 2 or 3 repeat sequence peptides with at least one of Pro-Ala-Lys, Ala-Lys-Pro and Lys-Ala-Pro as the structural unit.

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

8. The compound according to any one of claims 1 to 7, characterized in that, 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 according to any one of claims 1 to 8, characterized in that, The polypeptide moiety further comprises a Lys residue covalently linked to the linker, 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 according to claim 9, wherein The peptide fragment comprising Lys-Ala-Pro is linked to the Lys residue.

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

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

13. The compound according to any one of claims 1 to 12, characterized in that, The polypeptide moiety includes one or more of the following: 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-β-Ala)-Ile-Glu-Ser-Asp-Val, Lys(Lys-Ala-Pro-Arg-Gly)-Ile-Glu-Ser-Asp-Val.

14. A process for preparing the compound according to any one of claims 1 to 13, characterized in that, Comprising: Borneol and a coupling agent are used to prepare an intermediate containing an ester bond under reaction conditions, and the intermediate is sequentially bonded to the corresponding amino acids and purified to obtain the compound.

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

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

17. The application according to claim 16, wherein The application includes the use of the compound and the pharmaceutical composition in the preparation of a drug for cerebral ischemia protection.

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

19. The application according to claim 18, characterized in that The thrombotic diseases include ischemic stroke, myocardial infarction, stroke, venous embolism, pulmonary embolism, peripheral arterial obstructive disease, venous catheter obstruction, arteriovenous fistula and shunt tube obstruction, and carotid artery stenosis.

20. A method for treating a subject suffering from a thrombotic disease, characterized in that, Comprising administering to the subject an effective amount of the compound according to any one of claims 1 to 13, the compound prepared by the method according to claim 14, or the pharmaceutical composition according to claim 15.

21. The method according to claim 20, characterized in that, The thrombotic diseases include ischemic stroke, myocardial infarction, stroke, venous embolism, pulmonary embolism, peripheral arterial obstructive disease, venous catheter obstruction, arteriovenous fistula and shunt tube obstruction, and carotid artery stenosis.

22. A method for improving cerebral ischemia, cerebral thrombosis, free radical status or inflammatory status in a subject, characterized in that, Comprising administering to a subject in need thereof an effective amount of the compound according to any one of claims 1 to 13, the compound prepared by the method according to claim 14, or the pharmaceutical composition according to claim 15.

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