CN116999570A - Tumor-targeted polypeptide drug conjugate and application thereof - Google Patents

Tumor-targeted polypeptide drug conjugate and application thereof Download PDF

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Publication number
CN116999570A
CN116999570A CN202310986826.9A CN202310986826A CN116999570A CN 116999570 A CN116999570 A CN 116999570A CN 202310986826 A CN202310986826 A CN 202310986826A CN 116999570 A CN116999570 A CN 116999570A
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tumor
mmol
pdc
drug conjugate
polypeptide
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王锐
张海龙
王聪
胡宽
王坤
易娟
杨瀚舸
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Lanzhou University
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Lanzhou University
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    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
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    • A61K31/337—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having four-membered rings, e.g. taxol
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    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/65—Peptidic linkers, binders or spacers, e.g. peptidic enzyme-labile linkers
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    • A61K49/0017—Fluorescence in vivo
    • A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
    • A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
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    • A61K49/0017—Fluorescence in vivo
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    • A61K49/0056—Peptides, proteins, polyamino acids
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    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
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    • A61K51/04—Organic compounds
    • A61K51/041—Heterocyclic compounds
    • A61K51/044—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine, rifamycins
    • A61K51/0459—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine, rifamycins having six-membered rings with two nitrogen atoms as the only ring hetero atoms, e.g. piperazine
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    • A61K51/04—Organic compounds
    • A61K51/0474—Organic compounds complexes or complex-forming compounds, i.e. wherein a radioactive metal (e.g. 111In3+) is complexed or chelated by, e.g. a N2S2, N3S, NS3, N4 chelating group
    • A61K51/0482—Organic compounds complexes or complex-forming compounds, i.e. wherein a radioactive metal (e.g. 111In3+) is complexed or chelated by, e.g. a N2S2, N3S, NS3, N4 chelating group chelates from cyclic ligands, e.g. DOTA
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    • A61K51/04—Organic compounds
    • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
    • A61K51/088—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins conjugates with carriers being peptides, polyamino acids or proteins
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Abstract

The invention belongs to the field of development of antitumor drugs, and particularly relates to design synthesis and application of a tumor-targeted polypeptide drug conjugate. The tumor targeting polypeptide drug conjugate structure comprises: tumor targeting peptides (targeting plectin-1), targeting/metabolic regulatory functional groups, linkers and cytotoxic anti-tumor drug/radionuclide chelating groups; according to the polypeptide coupling drug provided by the invention, the cytotoxic anti-tumor drug/radionuclide chelating group is connected with the polypeptide, and the functional regulating group is inserted, so that the tumor targeting capability of the polypeptide drug conjugate is improved, the in-vivo metabolism of the drug is prolonged, the high concentration enrichment and long-time retention of tumor tissue parts can be realized, the targeting of the chemotherapeutic drug/radionuclide is improved, the high-dose chemotherapeutic drug/radionuclide can accurately kill/diagnose tumor cells, and the better and safer pancreatic cancer treatment function is realized.

Description

Tumor-targeted polypeptide drug conjugate and application thereof
Technical Field
The invention belongs to the field of development of antitumor drugs, and particularly relates to a tumor-targeted polypeptide drug conjugate and application thereof.
Background
In recent years, cancer has evolved into one of the most leading causes of disease death in the global population, and among many malignant tumors, pancreatic cancer (Pancreatic Cancer, PC) has the highest mortality-to-morbidity ratio, and thus pancreatic cancer is also called "cancer king". Pancreatic cancer is one of the most malignant tumors, is highly invasive and metastatic, and lacks typical early symptoms, resulting in pancreatic cancer patients already in advanced stages or metastasized when diagnosed. In the united states, the 5-year survival rate of pancreatic cancer is about 10%, mainly because of the fact that of diagnosed patients, less than 20% of patients are judged to be resectable early in pancreatic cancer, whereas about 80% -85% of pancreatic cancer patients have advanced to the advanced stage, and surgical resection does not significantly improve prognosis of patients, they still need to receive long-term systemic chemotherapy to control, improve the condition. Therefore, more diagnostic methods for early detection of pancreatic cancer and improving discrimination between benign and malignant tumors need to be actively developed to address the clinical needs of pancreatic cancer patients.
The clinic treatment method for pancreatic cancer mainly comprises surgical excision, radiotherapy, chemotherapy or the combination therapy of the above methods, and the chemotherapy means are most widely applied. Although various therapeutic methods are being tried and developed, the current clinical needs of pancreatic cancer patients are far from being met, and therefore more and more effective pancreatic cancer diagnosis and treatment methods need to be explored and developed to solve the dilemma.
Plectin-1 is a high molecular weight protein (about 500 kDa) comprising a central rod domain flanked by a globular N-terminal end domain and a C-terminal tail domain, the head domain comprising an actin binding domain and a plakin domain, the tail consisting of a plakin repeat domain having 6 repeats, a linker subdomain and a glycine-serine-arginine domain. Plectrin-1 is widely present in various cancer types including pancreatic cancer, breast cancer, lung cancer, prostate cancer, etc., and is currently being used in many studies of pancreatic cancer. Plectin-1 acts as a cell-linking agent, linking and stabilizing cytoskeletal proteins (microtubules, microfilaments and intermediate filaments), which play an important role in the normal physiological function of cells. It has specific abnormal localization on the surface of pancreatic cancer cells, and in normal pancreatic tissue or normal fibroblasts, it is localized in the nucleus and cytoplasm. The pleectin-1 targeting polypeptide sequence KTLLPTP.
Dirk Bausc et al designed and synthesized a targeting molecule t-PTP (KTLLPTP) based on pleectin-1, and the polypeptide sequence can well target and combine pleectin-1 at the molecular level. There have also been some researchers who use this sequence of polypeptides in an attempt to perform fluorescence imaging diagnosis of pleectin-1 based pancreatic cancer. However, the targeting and metabolic stability of the sequence polypeptide in animals limit further intensive research and application.
Disclosure of Invention
In order to solve the problems in the prior art, the invention adopts the strategy of polypeptide drug coupling, and prepares a series of polypeptide drug conjugates based on pleectin-1 by a chemical synthesis method, wherein the structural general formula (I) is P-F-C-D,
p is plectin-1 targeting polypeptide for specifically recognizing and binding to a corresponding target;
f is a functional regulating group, which is used for improving the targeting of the whole conjugate molecule, prolonging the in vivo circulation time of the conjugate molecule, and simultaneously regulating the water solubility, the fat solubility and the like of the whole conjugate molecule so as to improve the PK value;
c is a linker linking the targeting polypeptide-functional regulatory moiety and the payload moiety;
d is a payload group comprising a chemical antitumor drug, a radionuclide chelating group or a fluorescent group, and plays a role in tumor killing or imaging.
According to the tumor targeted polypeptide drug conjugate, the P unit in the general formula (I) is a polypeptide sequence or a polypeptide derivative with the amino acid sequence of KTLLPTP (SEQ ID NO. 1) or 1-3 amino acids replaced, deleted and inserted on the basis.
According to the tumor targeted polypeptide drug conjugate, F units in the general formula (I), wherein the functional regulatory group is selected from folic acid, RGD targeting peptide, albumin targeting ligand and pleectin-1 targeting peptide (monomer and dimer), and the structure is as follows:
A tumor targeted polypeptide drug conjugate according to an embodiment of the invention, the functional regulatory group is selected from the structures shown below:
according to the tumor targeted polypeptide drug conjugate, the linker in the general formula (I) is a connecting cleavable linker or a non-cleavable linker; preferably, the linker takes a chain shape which is formed by covalent bond connection between carbon atoms and is not ring-formed as a carbon frame;
the tumor targeted polypeptide drug conjugate according to the specific embodiment of the invention has a linker selected from one of the following structures:
in the tumor targeted polypeptide drug conjugate in the general formula (I), the cytotoxic anti-tumor drug comprises paclitaxel and/or MMAE.
According to the tumor targeted polypeptide drug conjugate of the specific embodiment of the invention, in D in the general formula (I), the fluorescent group is selected from the group consisting of:
according to the tumor targeted polypeptide drug conjugate of the specific embodiment of the invention, the radionuclide chelating group in the general formula (I) is selected from any one of the following structures:
the tumor targeted polypeptide drug conjugate according to the specific embodiment of the invention has the structural general formula (I) of P-F-C-D, wherein,
P is pleectin-1 targeting polypeptide, and the amino acid sequence of the targeting polypeptide is KTLLPTP;
f is a functional regulatory group selected from folic acid, RGD targeting ligand, albumin ligand or plectin-1;
c is a linker selected from one of the following structures;
d is a payload group selected from a chemical antitumor drug or radionuclide chelating group, preferably MMAT, DOTA- 68 Ga。
A tumor-targeted polypeptide drug conjugate according to an embodiment of the invention is any one or more of the following structures:
in the above structure, the linker moiety C may be substituted with a corresponding substituent selected from the group consisting of non-hydrogen substituents consisting of: c3-10 cycloalkyl, C5-12 aryl, C5-12 heteroaryl, hydroxy, halogen and C1-4 alkoxycarbonyl, C1-12 alkyl substituted by at least one non-hydrogen substituent, C3-10 cycloalkyl, C1-12 alkoxy, C5-12 aryl, C5-12 heteroaryl, hydroxy and halogen, and said aryl and heteroaryl groups are unsubstituted or substituted by at least one non-hydrogen substituent selected from the group consisting of C1-4 alkyl, C1-4 alkoxy, hydroxy and halogen.
As used herein, a "substituted" group refers to a group in which at least one hydrogen atom is replaced with at least one non-hydrogen atom group, provided that the group must meet valence requirements and that the substitution results in a chemically stable compound. In this specification, unless specifically described as "unsubstituted", it is understood that all substituents may be substituted or unsubstituted.
"alkyl" refers to straight and branched chain saturated hydrocarbon groups, typically having a specified number of carbon atoms (e.g., 1 to 12 carbon atoms). Examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, n-hexyl, and n-heptyl. Alkyl groups may be attached to the parent group or substrate through any ring atom unless such attachment would disrupt the valence requirements. Likewise, an alkyl or alkenyl group may contain at least one non-hydrogen substituent unless such substitution would disrupt the valence requirements.
"cycloalkyl" refers to saturated monocyclic and polycyclic hydrocarbon rings, typically having a specific number of carbon atoms including the ring (e.g., C3-10 cycloalkyl refers to cycloalkyl having 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms as ring members). Cycloalkyl groups may be attached to the parent or substrate through any ring atom unless such attachment would disrupt the valence requirements. Likewise, cycloalkyl groups may contain at least one non-hydrogen substituent unless such substitution would disrupt the valence requirements.
"aryl" refers to monovalent and divalent aromatic radicals, including 5-membered and 6-membered monocyclic aromatic radicals, respectively, and "heteroaryl" refers to monovalent and divalent aromatic radicals, including 5-membered and 6-membered monocyclic aromatic radicals containing 1 to 4 heteroatoms independently selected from nitrogen, oxygen and sulfur, respectively. Examples of monocyclic aryl and heteroaryl groups include, but are not limited to, phenyl, pyridyl, furyl, pyrrolyl, thienyl, thiazolyl, isothiazolyl, imidazolyl, triazolyl, tetrazolyl, pyrazolyl, oxazolyl, isoxazolyl, pyrazinyl, pyridazinyl, pyrimidinyl, and the like. Aryl and heteroaryl also include bicyclic groups, tricyclic groups, and the like, including fused 5-and 6-membered rings as described above. Examples of polycyclic aryl and heteroaryl groups include, but are not limited to, isoquinolinyl, naphthyl, biphenyl, anthracenyl, pyrenyl, carbazolyl, benzoxazolyl, benzodioxazolyl, benzimidazolyl, benzothienyl, quinolinyl, indolyl, benzofuranyl, purinyl, indolizinyl, and the like. Aryl and heteroaryl groups may be attached to the parent group or matrix through any ring atom unless such attachment would disrupt the valence requirements. Likewise, aryl and heteroaryl groups can contain at least one non-hydrogen substituent unless such substitution would disrupt the valence requirements. The non-hydrogen substituents of the aryl and heteroaryl groups may also be substituted with additional non-hydrogen substituents.
"carbonyl" refers to a C (O) R'. As used herein "(O)" refers to oxygen attached to an atom such as carbon or sulfur through a double bond.
Herein, "R" refers to a non-hydrogen substituent such as lower alkyl, lower alkoxy, and the like. Examples of carbonyl groups include, but are not limited to, 2-methoxyoxoethyl, 3-methoxyoxopropyl, and the like. Carbonyl groups may be attached to the parent group or matrix through any ring atom unless such attachment would disrupt the valence requirements. Likewise, a carbonyl group can contain at least one non-hydrogen substituent unless such substitution would disrupt the valence requirements.
"alkoxy" refers to alkyl-O-. Here, the alkyl group is the same as defined above. Examples of alkoxy groups include, but are not limited to, methoxy, ethoxy, and the like. Alkoxy groups may be attached to the parent group or matrix through any ring atom unless such attachment would disrupt the valence requirements. Likewise, an alkoxy group may contain at least one non-hydrogen substituent unless such substitution would disrupt the valence requirements.
"hydroxy" refers to-OH, "halogen" refers to fluorine, chlorine, bromine and iodine, and "oxo" refers to =o.
The preparation method of the tumor targeting polypeptide drug conjugate comprises the following steps:
Step 1, synthesizing the Boc-protected plectain-1 tumor targeting polypeptide by adopting an Fmoc solid-phase synthesis strategy.
Step 2, connecting the pleectin-1 tumor targeting polypeptide in the step 1 with a functional regulating group through solid phase or liquid phase condensation to obtain a P-F fragment.
And 3, connecting the P-F fragment obtained in the step 2 with a chemical toxic drug with a linker through click or condensation reaction, or connecting the P-F fragment with a radionuclide chelating group through condensation reaction, and purifying the P-F fragment by HPLC to obtain the P-F-C-D polypeptide drug conjugate.
The polypeptide conjugate with excellent anti-tumor effect is obtained through in vitro and in vivo anti-tumor activity tests, so the invention provides application of the polypeptide drug conjugate (comprising chelate and radionuclide) based on clathrin plectasin-1 in preparation of anti-tumor diagnosis and treatment drugs. Wherein the tumor preferentially selects pancreatic cancer and other tumors with abnormal expression of plectrin-1.
In another aspect of the present invention there is provided a medicament comprising as an active ingredient a tumor targeting polypeptide drug conjugate as described above or a pharmaceutically acceptable salt thereof.
The pharmaceutical compositions of the present invention comprise at least one pharmaceutically acceptable carrier in addition to the active ingredient. As used herein, "pharmaceutically acceptable carrier" refers to known pharmaceutically acceptable excipients that are useful in formulating a pharmaceutically active compound for administration to a subject and are substantially non-toxic and non-irritating under the conditions of use. The exact amount of excipient is determined by standard pharmaceutical practice, the solubility, chemical identity and the chosen route of administration of the active compound.
The pharmaceutical compositions of the present invention may be formulated in an appropriate form for the desired method of administration using suitable and physiologically acceptable adjuvants (e.g., excipients, disintegrants, sweeteners, binders, coating agents, bulking agents, lubricants, brightness agents, flavoring agents, and the like).
The preparation of the pharmaceutical composition taking the tumor targeting polypeptide drug conjugate (P-F-C-D) as an active ingredient comprises various forms such as freeze-dried powder injection, tablet, liposome, nano preparation and the like.
The invention has the beneficial effects that:
the tumor targeting (targeting pleectin-1) polypeptide drug conjugate provided by the invention connects a cytotoxic drug (MMAE, taxol) or a radionuclide with pancreatic cancer targeting polypeptides, so that the high-concentration enrichment and killing of the cytotoxic drug on tumor tissue parts can be realized, the PET imaging diagnosis of tumors can be realized, and the diagnosis and treatment integrated drug development of pancreatic cancer can be realized.
Drawings
In order to more clearly illustrate the embodiments of the invention or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, it being obvious that the drawings in the following description are only some embodiments of the invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a chart of the product KTLLPTPG (without BOC) HRMS analysis.
FIG. 2 is a chart of the HRMS analysis of the product KTLLPTPG-EDA (Free BOC).
FIG. 3 is a chart of PDC-1 HRMS analysis.
FIG. 4 is a chart of PDC-2 HRMS analysis.
FIG. 5 is a chart of PDC-3 HRMS analysis.
FIG. 6 is a chart of PDC-4 HRMS analysis.
FIG. 7 is a PDC-5 HRMS analysis chart
FIG. 8 is a PDC-6 HRMS analysis chart
FIG. 9 is a chart of PDC-7 HRMS analysis.
FIG. 10 is a chart of PDC-8 HRMS analysis.
FIG. 11 is a chart of PDC-9 HRMS analysis.
FIG. 12 is a chart of PDC-10 HRMS analysis.
FIG. 13 is a chart of PDC-11 HRMS analysis.
FIG. 14 is a chart of PDC-12 HRMS analysis.
FIG. 15 is a chart of PDC-15 HRMS analysis.
FIG. 16 shows that the level of labeling of the polypeptide-drug conjugate (fluorophore) for PANC-1 in both competitive and non-competitive situations, is significantly reduced compared to the non-competitive state in the competition of the targeting polypeptide.
FIG. 17 shows that the tumor inhibitory activity of the polypeptide drug conjugate on cell-derived pancreatic cancer tumor-bearing mouse model, and that the difference in average tumor volume of 3 PDC-administered groups after the end of administration compared to the blank control group was statistically significant.
FIG. 18 shows the effect of the compounds prepared according to the invention on the body weight of mice, with a marked decrease in body weight in the PDC-3-dosed group compared to the other 2-dosed groups and the placebo group.
FIG. 19 shows a radionuclide 68 Ga-labeled PDC-12PET/CT imaging of mouse models of pancreatic cancer with tumors by PDC-14 and PDC-16 shows that compared with the mouse models 68 Ga-PDC-12 68 Ga-PDC-14， 68 Ga-PDC-16 tail vein injection for 0.5h has best uptake and imaging at tumor site.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be described in detail below. It will be apparent that the described embodiments are only some, but not all, embodiments of the invention. All other embodiments, based on the examples herein, which are within the scope of the invention as defined by the claims, will be within the scope of the invention as defined by the claims.
In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be described in detail below. It will be apparent that the described embodiments are only some, but not all, embodiments of the invention. All other embodiments, based on the examples herein, which are within the scope of the invention as defined by the claims, will be within the scope of the invention as defined by the claims.
In certain embodiments of the invention, cells that may be used include:
human pancreatic cancer cells: PANC-1;
human normal pancreatic duct cells; hTERT-HPNE;
all of the above cells are commercially available, for example, from the cell bank of the national academy of sciences.
EXAMPLE 1 chemical Synthesis
(1) Synthesis of KTLLPTPG
The synthesis method comprises the following steps:
the peptide washing method comprises the following steps: DMF 4X 10mL X2 min.
Fmoc protecting group removal method: 20% piperidine +80% DMF 2X 10mL X5 min.
Indene detection: add the prepared ninhydrin, phenol, pyridine in an amount = 1 drop: 1 drop: 2 drops, heating at 100deg.C for 30-90s, if there is exposed amino system, it is blue or light brown; if no exposed amino group exists, the system is colorless.
The first amino acid access method: 3eq amino acid +6eq DIEA,DMF 10mL.
Other amino acid access methods: 3eq amino acid+3 eq HBTU (different condensing agents may be selected according to different conditions) +4eq HOBT+6eq DIEA,DMF 10mL, and shaking for 1-4h at room temperature.
Peptide cutting: 1% TFA/DCM, and shaking for 3h.
Extraction of peptide and HPLC preparation: the aqueous phase was collected by extraction with 10mL of glacial diethyl ether and 2X 10mL of water. HPLC preparation: 0.1% TFA acetonitrile/0.1% TFA water, 0-80 min acetonitrile 20% -100%, flow rate: 8ml/min.
First (103 mg,0.1 mmol) of the dichloro resin was swollen in 10mL of DCM for 30min, washed with DCM for 3X 10mL X2 min, washed with DMF for 3X 10mL X2 min, then Fmoc-Gly-OH was added, and the peptide was washed in DCM: meOH: diea=8.5 mL:1mL: blocking 0.5mL for 30min, washing peptide, removing Fmoc, washing peptide, indening, then sequentially accessing PTPLLTK, cutting peptide, extracting peptide and preparing by HPLC according to the sequence of (accessing amino acid, indening, removing Fmoc, washing peptide, indening, accessing next amino acid: 0.1% TFA acetonitrile/0.1% TFA water, 0-80 min acetonitrile 20% -100%, flow rate: 8ml/min, rt=45 min, dried under reduced pressure to give the product KTLLPTPG (With BOC) (41 mg,0.04mmol, yield: 40%). The former was added 20% TFA/DCM, stirred at room temperature for 2h, HPLC preparation: 0.1% TFA acetonitrile/0.1% TFA water, 0-80 min acetonitrile 20% -100%, flow rate: 8ml/min, rt=20 min, dried under reduced pressure to give product KTLLPTPG (Free BOC) (37.7 mg,0.037mmol, yield: 92%) HRMS (ESI): C 38 H 67 N 9 O 11 [M+H] 2+ calcd:413.7553,found:413.7553.Purity:99.03％.
The product KTLLPTPG (no BOC) HRMS analysis spectrum is shown in fig. 1:
(2) Synthesis of KTLLPTPG-EDA
The synthesis method comprises the following steps:
(25 mg,0.0242 mmol) KTLLPTPG (With BOC), (3.3 mg,0.029 mmol) N-hydroxysuccinimide was dissolved in 1mL DCM, followed by the addition of (6 mg,0.029 mmol) DCC and (0.3 mg,0.00242 mmol) DMAP, (4.68 mg,0.0363 mmol) DIEA, stirred at room temperature for 1h, followed by the addition of (3 mg,0.0484 mmol) ethylenediamine, stirred at room temperature for 1h, concentrated under reduced pressure, dissolved in acetonitrile, water, HPLC preparation: 0.1% TFA acetonitrile/0.1% TFA water, 0-80 min acetonitrile 20% -100%, flow rate: 8ml/min, rt=30 min, dried under reduced pressure to give the product KTLLPTPG-EDA (With BOC) (16 mg,0.015mmol, 62% yield). The former was added 20% TFA/DCM, stirred at room temperature for 2h, HPLC preparation: 0.1% TFA acetonitrile/0.1% TFA water, 0-80 min acetonitrile 20% -100%, flow rate: 8ml/min, rt=20 min, dried under reduced pressure to give the product KTLLPTPG-EDA (Free BOC) (14.9 mg,0.014mmol, 93% yield) HRMS (ESI): C 40 H 73 N 11 O 10 [M+H] 2+ calcd:434.7844,found:434.7845.Purity:96.10％。
The product KTLLPTPG-EDA (Free BOC) HRMS analysis is shown in FIG. 2.
(3) Synthesis of PDC-1
The synthesis method comprises the following steps:
(50 mg,0.06 mmol) of Compound 1, (12 mg,0.12 mmol) of succinic anhydride, (7 mg,0.06 mmol) of DMAP was added to the reaction flask, dried in vacuo for 2h, followed by 2mL of DCM and 1mL of dried pyridine, and stirred at room temperature for 3h. 15mL of DCM was added, the organic phases were washed with 1N HCl (3X 15 mL), the combined organic phases were washed with saturated aqueous NaCl (3X 10 mL), and concentrated under reduced pressure to give SA-PTX (Compound 2) which was directly taken into the next step.
(9.6 mg,0.01 mmol) SA-PTX (Compound 2), (11 mg,0.01 mmol) KTLLPTPG-EDA (With BOC), (3 mg,0.012 mmol) BOP-Cl was added to the reaction flask followed by 1.5mL DCM and (2 mg,0.015 mmol) DIEA was added and stirred at room temperature for 2h. Concentrating under reduced pressure, adding 20% TFA/DCM, stirring at room temperature for 2h,concentrating under reduced pressure, adding acetonitrile and water for dissolving, and preparing by HPLC: 0.1% TFA acetonitrile/0.1% TFA water, 0-80 min acetonitrile 20% -100%, flow rate: 8ml/min, rt=30 min, dried under reduced pressure to give the product PDC-1 (5.4 mg,0.003mmol, 30% yield), HRMS (ESI): C 91 H 126 N 12 O 26 [M+H] 2+ calcd:902.4526,found:902.4527.Purity:99.05％。
The PDC-1HRMS analysis spectrum is shown in FIG. 3.
(4) Synthesis of PDC-2
The synthesis method comprises the following steps:
(720 mg,1.95 mmol) of Compound 3, (488 mg,3.9 mmol) of amino-polyethylene glycol-propionic acid tert-butyl ester was dissolved in 15mL of DMF, followed by addition of (1.12 g,5.85 mmol) of EDCI, (79mg, 5.85 mmol) of HOBT and (75 mg,5.85 mmol) of DIEA at 0℃and stirring at room temperature for 4h. Concentrating under reduced pressure, extracting (30 mL of water, 3X 50mL of ethyl acetate), washing with 2X 70mL of water, 2X 70mL of saturated aqueous NaCl solution, and anhydrous Na 2 SO 4 Drying, concentrating under reduced pressure, purifying by column chromatography, and petroleum ether: ethyl acetate = 1: 4-1: 10. compound 4 (704 mg,0.99mmol, 50.8% yield) is obtained. Compound 4 (20 mg,0.028 mmol) was dissolved in 2mL of 20% TFA/DCM and stirred at room temperature for 3h, DCM 3X 3mL and taken directly into the next reaction.
HATU (32 mg,0.084 mmol) was added to the above product, 3mL of DMF was dissolved, DIEA (11 mg,0.084 mmol) was added, stirring was carried out at 0deg.C for 20min, followed by KTLLPTPG-EDA (With BOC) (60 mg,0.056 mmol) and stirring was carried out at room temperature for 3h. Concentrated under reduced pressure, 2mL of 5% DBU/DCM was added, stirred at room temperature for 2h, DCM 3X 3mL to give Dimer-KTLLPTPG (compound 5) which was directly taken into the next reaction.
(2 mg,0.00209 mmol) of SA-PTX (Compound 2), (1 mg,0.00209 mmol) of HATU was dissolved in 1mL of DMF, and (1 mg,0.0077 mmol) of DIEA was added, followed by stirring at 0℃for 20min, then in (5.2 mg,0.00209 mmol) of Dimer-KTLLPTPG (Compound 5), stirring at room temperature for 2h, concentrating under reduced pressure, adding 2mL of 20% TFA/DCM, stirring at room temperature for 3h, DCM 3X 3mL. Adding BNitrile, water dissolution, HPLC preparation: 0.1% TFA acetonitrile/0.1% TFA water, 0-80 min acetonitrile 20% -100%, flow rate: 8ml/min, rt=23 min, dried under reduced pressure to give the product PDC-2 (1 mg,0.00033mmol, 15.78% yield). HRMS (ESI) C 146 H 222 N 26 O 42 [M+H] 2+ calcd:1506.8120,found:1506.8099.Purity:>99.00％.
The PDC-2HRMS analysis spectrum is shown in FIG. 4.
(5) Synthesis of PDC-3
The synthesis method comprises the following steps:
(1 g,2.635 mmol) of Compound 7, (325 mg,2.899 mmol) of Compound 6 and (1.099 g,2.899 mmol) of HBTU, (399mg, 2.899 mmol) of HOBT were dissolved in 20mL of DMF, followed by the addition of (264 mg,2.899 mmol) of DIEA, stirred at room temperature for 6h and concentrated under reduced pressure. Acetonitrile/water dissolution, HPLC preparation: 0.1% TFA acetonitrile/0.1% TFA water, 0-90 min acetonitrile 10% -100%, flow rate: 8ml/min, rt=25 min, drying under reduced pressure gives compound 8 (1.16 g,2.4505mmol, 93% yield).
Compound 8 (500 mg,1.05 mmol), compound 9 (384 mg,3.15 mmol) was dissolved in 10mL DMF, followed by addition of DIEA (30 mg,0.231 mmol) and stirring at room temperature for 5h. Concentrated under reduced pressure, 40mL of ethyl acetate was added, filtered, and washed with 3X 30mL of ethyl acetate to give compound 10 (637 mg,0.9975mmol, yield 95%).
Compound 10 (300 mg,0.4697 mmol), compound 11 (321 mg,0.4473 mmol) and HOBT (25 mg,0.1878 mmol) were dissolved in 6mL DMF, followed by addition of pyridine (883 mg,11.18 mmol) and stirring overnight. Concentrating under reduced pressure, dissolving in acetonitrile/water, and preparing by HPLC: 0.1% TFA acetonitrile/0.1% TFA water, 0-80 min acetonitrile 20% -100%, flow rate: 8ml/min, rt=40 min, drying under reduced pressure gives compound 12 (283 mg,0.2325mmol, 52% yield).
The (10 mg,0.0082 mmol) of Compound 12, (7.3 mg,0.0082 mmol) KTLLPTPG-N 3 (Compound 13), (1 mg,0.0041 mmol) CuSO 4 ·5H 2 O and (3.3 mg,0.0164 mmol) sodium ascorbateAdded to the reaction flask, followed by DMF: h 2 O=0.75 mL:0.25mL, stirred overnight at room temperature, concentrated under reduced pressure, HPLC preparation: 0.1% TFA acetonitrile/0.1% TFA water, 0-80 min acetonitrile 20% -100%, flow rate: 8ml/min, rt=27 min, dried under reduced pressure to give the product PDC-3 (5.7 mg,0.0027mmol, yield: 32.9%). HRMS (ESI) C 104 H 171 N 23 O 23 [M+H] 2+ calcd:1056.1532,found:1056.1533.Purity:96.50％.
The PDC-3HRMS analysis spectrum is shown in FIG. 5.
(6) Synthesis of PDC-4
The synthesis method comprises the following steps:
compound 14 (100 mg,0.217 mmol), compound 15 (75 mg,0.2387 mmol), HBTU (50 mg,0.2604 mmol) and HOBT (35 mg,0.2604 mmol) were dissolved in 6mL DMF, DIEA (42 mg,0.3255 mmol) was added, stirred at room temperature for 6h, extracted (15 mL, ethyl acetate 3X 20 mL), the organic phase was collected, washed once with 50mL of water, washed once with 50mL of saturated aqueous NaCl solution, anhydrous Na 2 SO 4 Drying), concentrating under reduced pressure, and purifying by column chromatography: dichloromethane: methanol=30: 1-10: 1 to give the product (134 mg,0.1844mmol, yield 84.97%), 60mg,0.0825 mmol) was taken, 20% TFA/DCM 2mL was added, stirred at room temperature for 3h, DCM. Times.3 to give compound 16, which was directly taken for the next reaction.
To (47 mg,0.0825 mmol) of Compound 16 was added (102 mg,0.09075 mmol) of KTLLPTPG-NHS (Compound 17), 3mL of DMF was dissolved, followed by the addition of (22 mg,0.165 mmol) of DIEA and stirring at room temperature for 4h. Concentrating under reduced pressure, dissolving in acetonitrile/water, and preparing by HPLC: 0.1% TFA acetonitrile/0.1% TFA water, 0-70 min acetonitrile 30% -100%, flow rate: 8ml/min, rt=40 min, drying under reduced pressure gave compound 18 (56 mg,0.0354mmol, 43% yield).
Take (25 mg,0.0158 mmol) compound 18, add 5% DBU/DCM 1mL, stir at room temperature for 1h, DCM X3, add (20 mg,0.01738 mmol) KTLLPTPG-NHS (compound 17), DMF 1.5mL, (4 mg,0.0316 mmol) DIEA, stir at room temperature for 4h, concentrate under reduced pressure, and directly put into the next reaction. To the former was added (2 mg,0.01422 mmol) of azidoethylamine, (4 mg,0.02133 mmol) of HBTU and (3 mg,0.02133 mmol) of HOBT, 1mL of DMF, (4 mg,0.02844 mmol) of DIEA, stirred overnight at room temperature, concentrated under reduced pressure, and prepared by HPLC with acetonitrile/water: 0.1% TFA acetonitrile/0.1% TFA water, 0-70 min acetonitrile 30% -100%, flow rate: 8ml/min, rt=40 min, drying under reduced pressure gave compound 19 (8.5 mg,0.0035mmol, 24.6% yield).
(8.5 mg,0.0035 mmol) of the compound 19 was added to 2mL of 30% TFA/DCM and stirred at room temperature for 3h, DCM. Times.3, and the mixture was directly introduced into the next reaction. Add (4.5 mg,0.0035 mmol) HA-Val-Cit-PABC-MMAE (0.5 mg,0.00175 mmol) CuSO 4 ·5H 2 O and (1.5 mg, 0.0070 mmol) sodium ascorbate, H 2 O: dmf=0.25 mL:0.75mL. Stirring overnight at room temperature, concentrating under reduced pressure, HPLC preparation: 0.1% TFA acetonitrile/0.1% TFA water, 0-90 min acetonitrile 10% -100%, flow rate: 8ml/min, rt=34.5 min, freeze-dried to give the product (6.8 mg,0.0021mmol, 60% yield). HRMS (ESI) C 157 H 265 N 35 O 38 [M+H] 2+ calcd:1625.4993,found:1625.5014.Purity:>99.00％.
The PDC-4HRMS analysis spectrum is shown in FIG. 6.
(7) Synthesis of PDC-5
The synthesis method comprises the following steps:
(20 mg,0.238 mmol) of compound 20, (87 mg,0.2856 mmol) of compound 9 was dissolved in 2mL of DMF, and (37 mg,0.2856 mmol) of DIEA was added thereto and stirred at room temperature for 5 hours. Concentrating under reduced pressure, purifying by column chromatography, and petroleum ether: ethyl acetate = 20: 1-10: 1 to give the product (50 mg,0.2015mmol, 84.7% yield). The product (20 mg,0.0803 mmol) was taken, MMAE (52 mg,0.073 mmol), HOBT (5 mg,0.0365 mmol) was added, 1mL DMF was taken, pyridine (144 mg, 1.8235 mmol) was added, stirred overnight at room temperature, concentrated under reduced pressure, purified by column chromatography, dichloromethane: methanol=25: 1-5: 1 to give compound 21 (42 mg,0.0507mmol, 69.5% yield).
The (10 mg,0.012 mmol) of Compound 21 and (11 mg,0.012 mmol) of KTLLPTPG-N were combined 3 (Compound 13), (1.5 mg, 0.006mmol) CuSO 4 ·H 2 O and (5 mg,0.024 mmol) sodium ascorbate were added to a reaction flask, and DMF was added: h2o=0.75 mL:0.25mL, stirred overnight at room temperature, concentrated under reduced pressure, HPLC preparation: 0.1% TFA acetonitrile/0.1% TFA water, 0-80 min acetonitrile 20% -100%, flow rate: 8ml/min, rt=25 min, dried under reduced pressure to give the product PDC-5 (6.5 mg,0.0037mmol, 30.8% yield). HRMS (ESI) C 85 H 144 N 18 O 19 [M+H] 2+ calcd:861.5500,found:861.5510.Purity:98.96％.
The PDC-5HRMS analysis spectrum is shown in FIG. 7:
(8) Synthesis of PDC-6
The synthesis method comprises the following steps:
the azidoamine hydrochloride (200 mg,1.64 mmol), the compound 22 (92mg, 1.968 mmol) and HBTU (806 mg,5.985 mmol) were dissolved in 15mL DMF followed by the addition of DIEA (211 mg,1.64 mmol), stirring at room temperature for 4h, adding water 20mL, ethyl acetate 3X 40mL extraction, saturated NaCl 100mL wash, anhydrous NaSO 4 Drying, column chromatography purification, dichloromethane: methanol=40: 1 to give the product (748 mg, 1.390 mmol, 85% yield). The product was taken (100 mg,0.1863 mmol), 20% TFA/DCM 3mL was added, stirred at room temperature for 2h, DCM. Times.3, boc-NH-PEG3-COOH (59 mg,0.2019 mmol), HBTU (141 mg,0.3726 mmol), 6mL DMF (48 mg,0.3726 mmol) DIEA, stirred at room temperature for 2h, concentrated under reduced pressure, purified by column chromatography, dichloromethane: methanol=20: 1 to give compound 23.
Take (74 mg,0.1 mmol) compound 23,5% DBU/DCM 2mL, stir at room temperature for 1h, DCM. Times.3, add (65 mg,0.11 mmol) compound 24, (76 mg,0.2 mmol) HBTU, (27 mg,0.2 mmol) HOBT, DMF 3mL, (26 mg,0.2 mmol) DIEA, stir at room temperature for 2h, concentrate under reduced pressure, purify by column chromatography, dichloromethane: methanol=20: 1 to give compound 25.
Take (30 mg,0.0275 mmol) compound 25, 20% TFA/DCM 2mL, stir at room temperature for 2h, DCM. Times.3, add (34 mg,0.03025 mmol) KTLLPTPG-NHS (compound 17), DMF 2mL, (11 mg,0.0825 mmol) DIEA, stir at room temperature for 2h, concentrate under reduced pressure, HPLC preparation: 0.1% TFA acetonitrile/0.1% TFA water, 0-80 min acetonitrile 20% -100%, flow rate: 8ml/min, rt=40 min. Compound 26 (24 mg,0.0126mmol, 45.8% yield) is obtained. The (11 mg,0.00576 mmol) of Compound 26 was taken, 20% TFA/DCM was added, stirred at room temperature for 2h, DCM. Times.3 was directly put into the next reaction, and (7 mg,0.00576 mmol) of HA-Val-Cit-PABC-MMAE (0.75 mg, 0.003mmol) of CuSO was added 4 ·5H 2 O, (2.5 mg, 0.011115 mmol) sodium ascorbate, DMF: h 2 O=0.75 mL:0.25mL, stirred overnight at room temperature, concentrated under reduced pressure, HPLC preparation: 0.1% TFA acetonitrile/0.1% TFA water, 0-80 min acetonitrile 20% -100%, flow rate: 8ml/min, rt=35 min, and freeze-drying to give the product PDC-6 (6 mg,0.00207mmol, 36% yield). HRMS (ESI) C 140 H 227 IN 28 O 33 [M+H] 2+ calcd:1478.8048,found:1478.8069.Purity:95.34％.
The PDC-6HRMS analysis spectrum is shown in FIG. 8.
(9) Synthesis of PDC-7
The synthesis method comprises the following steps:
(87 mg,0.4 mmol) of Compound 27, (49 mg,0.4 mmol) of azidoethylamine hydrochloride, (303 mg,0.7994 mmol) of HBTU, (108 mg,0.8 mmol) of HOBT was added to a reaction flask followed by 15mL of DMF, and (103 mg,0.8 mmol) of DIEA was added, stirred at room temperature for 4h, concentrated under reduced pressure, purified by column chromatography, petroleum ether: ethyl acetate = 1:2, directly feeding the obtained product into the next step. The product was taken (100 mg,0.1863 mmol), 20% TFA/DCM 3mL was added, stirred at room temperature for 2h, DCM. Times.3, boc-NH-PEG3-COOH (66 mg,0.2049 mmol), HBTU (141 mg,0.3726 mmol), HOBT (50 mg,0.3726 mmol), 6mL DMF was added for dissolution, followed by (48 mg,0.3726 mmol) DIEA, stirred at room temperature for 2h, concentrated under reduced pressure, purified by column chromatography, dichloromethane: methanol=20: 1, compound 28 (120 mg,0.1621mmol, 87% yield) was obtained.
Compound 28 (120 mg,0.1621 mmol) was added to 20% TFA/DCM 2mL and stirred at room temperature for 2h, DCM. Times.3, and the reaction was directly put into the next reaction. To the former was added (273 mg,0.2432 mmol) KTLLPTPG-NHS, 3mL dissolved in DMF, followed by (104 mg,0.8105 mmol) DIEA, stirred at room temperature for 4h, concentrated under reduced pressure, 5% DBU/DCM 3mL added, stirred at room temperature for 1h, DCM X3. Acetonitrile/water dissolution, HPLC preparation: 0.1% TFA acetonitrile/0.1% TFA water, 0-70 min acetonitrile 30% -100%, flow rate: 8mL/min, rt=40 min, dried under reduced pressure to give compound 29 (152 mg,0.107mmol, 66% yield).
(15 mg,0.0108 mmol) of compound 29, (6.5 mg,0.0119 mmol) FA-NHS (compound 30) was added to 1mL of DMF, and (2.8 mg,0.0216 mmol) of DIEA was added and stirred at room temperature for 2h. Concentrating under reduced pressure, and preparing by HPLC: 0.1% TFA acetonitrile/0.1% TFA water, 0-80 min acetonitrile 20% -100%, flow rate: 8mL/min, rt=33 min, and drying under reduced pressure to obtain the compound 31. Take (5.6 mg,0.00303 mmol) Compound 31 and add 20% TFA/DCM, stir at RT for 2h, DCM X3, add directly (3.7 mg,0.00303 mmol) HA-Val-Cit-PABC-MMAE, (0.5 mg,0.0015 mmol) CuSO 4 ·5H 2 O and (1.2 mg,0.00606 mmol) sodium ascorbate, DMF: h 2 O=0.75 mL:0.25mL, stirred overnight at room temperature, concentrated under reduced pressure, HPLC preparation: 0.1% TFA acetonitrile/0.1% TFA water, 0-90 min acetonitrile 10% -100%, flow rate: 8mL/min, rt=33 min, dried under reduced pressure to give the product PDC-7 (2 mg,0.0007mmol, 23% yield). HRMS (ESI) C 138 H 217 N 33 O 33 [M+H] 2+ calcd:1433.3247,found:1433.3231.Purity:95.34％.
The PDC-7HRMS analysis is shown in FIG. 9.
(10) Synthesis of PDC-8
The synthesis method comprises the following steps:
the compound (100 mg,0.229 mmol) 32, (283 mg,0.2519 mmol) KTLLPTPG-NHS (compound 17) was dissolved in 5mL DMF followed by (59 mg,0.458 mmol) DIEA, stirred at room temperature for 2h, concentrated under reduced pressure to give compound 33, 5% DBU/DCM 3mL, stirred at room temperature for 1h, DCM X3, HPLC preparation: 0.1% TFA acetonitrile/0.1% TFA water, 0-70 min acetonitrile 30% -100%, flow rate: 8mL/min, rt=40 min, and the product is obtained by drying under reduced pressure and is put into the next reaction. The product, (2.8 mg,0.0245 mmol) glutaric anhydride was taken and dissolved in 2mL DCM, TEA (5 mg,0.049 mmol) was added and stirred at room temperature for 1h to give compound 34, which was directly put into the next reaction, DCC (5.5 mg,0.02646 mmol) was added to compound 34, (3 mg,0.02646 mmol) NHS (1.5 mg, 0.01102mmol) DMAP was added, stirred at room temperature for 6h, concentrated under reduced pressure, compound 35 (11 mg,0.01804 mmol) was directly added and stirred at room temperature for 4h, concentrated under reduced pressure, HPLC preparation: 0.1% TFA acetonitrile/0.1% TFA water, 0-80 min acetonitrile 20% -100%, flow rate: 8mL/min, rt=30 min, dried under reduced pressure to give the product (10 mg,0.0052mmol, 28.8% yield). To the product (10 mg,0.0052 mmol) was added 20% TFA/DCM 2mL, stirred at room temperature for 2h, DCM X3, HA-Val-Cit-PABC-MMAE (6.3 mg,0.0052 mmol) and CuSO (0.65 mg,0.0026 mmol) 4 ·5H 2 O and (2 mg,0.0104 mmol) sodium ascorbate, DMF: h 2 O=0.75 mL:0.25mL, and stirred overnight at room temperature. Concentrating under reduced pressure, and preparing by HPLC: 0.1% TFA acetonitrile/0.1% TFA water, 0-80 min acetonitrile 20% -100%, flow rate: 8mL/min, rt=16 min, dried under reduced pressure to give the product PDC-8 (5 mg,0.0017mmol, 32.7% yield). HRMS (ESI) C 142 H 228 N 34 O 33 [M+H] 3+ calcd:980.2475,found:980.2478.Purity:96.13％.
The PDC-8HRMS analysis spectrum is shown in FIG. 10.
(11) Synthesis of PDC-9
The synthesis method comprises the following steps:
the preparation was performed by dissolving (10 mg,0.0093 mmol) KTLLPTPG-EDA (With BOC) and (3.6 mg,0.0093 mmol) FITC (36) in 1mL DMF followed by (1.2 mg,0.0093 mmol) DIEA, stirring at room temperature for 2h, concentrating under reduced pressure, adding 20% TFA/DCM 1mL, stirring at room temperature for 2h, DCM X3, HPLC: 0.1% TFA acetonitrile/0.1% TFA water, 0-90 min acetonitrile 10% -100%, flow rate: 8mL/min, rt=28 min, dried under reduced pressure to give the product (6 mg,0.0048mmol, 51.6% yield). HRMS (ESI) C 61 H 84 N 12 O 15 S[M+H] 2+ calcd:629.3023,found:629.3027.Purity:99.12％.
The PDC-9HRMS analysis is shown in FIG. 11.
(12) Synthesis of PDC-10
The synthesis method comprises the following steps:
the peptide washing method comprises the following steps: DMF 4X 10mL X2 min.
Fmoc protecting group removal method: 3% DBU+3% piperidine+94% DMF 1X 5min, 1X 15min.
The Dde protecting group removal method comprises the following steps: 2% hydrazine hydrate/DMF 3X 3min.
Indene detection: add the prepared ninhydrin, phenol, pyridine in an amount = 1 drop: 1 drop: 2 drops, heating at 100deg.C for 30-90s, if there is exposed amino system, it is blue or light brown; if no exposed amino group exists, the system is colorless.
The following amino acid method is accessed: 3eq amino acid+3 eq HBTU (different condensing agents may be selected according to different conditions) +4eq HOBT+6eq DIEA,DMF 10mL, and shaking for 1-4h at room temperature.
Method of accessing FA-NHS (compound 30): 3eq of FA-NHS (Compound 30) +3eq HBTU+4eq HOBT+6eq DIEA,DMF 10mL, shaking at room temperature for 1-4h.
FITC (compound 36) access method: 1eq FITC (Compound 36) +3eq DIEA,DMF 10mL, and shaking at room temperature for 1-4h.
Peptide cutting: 95% TFA/H 2 O, oscillate for 3h.
Extraction of peptide and HPLC preparation: the aqueous phase was collected by extraction with 10mL of glacial diethyl ether and 2X 10mL of water. HPLC preparation: 0.1% TFA acetonitrile/0.1% TFA water, 0-80 min acetonitrile 20% -100%, flow rate: 8ml/min.
Swelling (217 mg,0.1 mmol) of the amino resin in 10mL of DCM for 30min, DCM for 2X 10mL X2 min, DMF for 3X 10mL X2 min, indene (the system should be colorless), fmoc-Lys (Dde) -OH, peptide-washing, indene, fmoc-washing, peptide-washing, indene-washing, then PTPLLTK, peptide-washing, dde-washing, peptide-washing, indene-washing, fmoc-Lys (Dde) -OH, peptide-washing, indene-washing, fmoc-washing, FA-NHS (compound 30), peptide-washing, indene-washing, dde-washing, FITC (compound 36), peptide-washing, indene-washing, and the following the sequence of (amino acid-washing, indene-washing, the following). Cleavage of peptides, extraction of peptides, HPLC preparation: 0.1% TFA acetonitrile/0.1% TFA water, 0-80 min acetonitrile 20% -100%, flow rate: 8ml/min, rt=26 min, dried under reduced pressure to give the product PDC-10 (55 mg,0.03mmol, yield: 30%). HRMS (ESI) C 38 H 67 N 9 O 11 [M+H] 2+ calcd:918.9316C 88 H 117 N 21 O 21 S,found:918.9301.Purity:96.11％.
The PDC-10HRMS analysis spectrum of the product is shown in FIG. 12.
(13) Synthesis of PDC-11
The synthesis method comprises the following steps:
the peptide washing method comprises the following steps: DMF 4X 10mL X2 min.
Fmoc protecting group removal method: 3% DBU+3% piperidine+94% DMF 1X 5min, 1X 15min.
The Dde protecting group removal method comprises the following steps: 2% hydrazine hydrate/DMF 3X 3min.
Indene detection: add the prepared ninhydrin, phenol, pyridine in an amount = 1 drop: 1 drop: 2 drops, heating at 100deg.C for 30-90s, if there is exposed amino system, it is blue or light brown; if no exposed amino group exists, the system is colorless.
The following amino acid method is accessed: 3eq amino acid+3 eq HBTU (different condensing agents may be selected according to different conditions) +4eq HOBT+6eq DIEA,DMF 10mL, and shaking for 1-4h at room temperature.
Access compound 24 method: 3eq (Compound 24) +3eq HBTU+4eq HOBT+6eq DIEA,DMF 10mL, and shaking at room temperature for 1-4h.
FITC (compound 36) access method: 1eq FITC (Compound 36) +3eq DIEA,DMF 10mL, and shaking at room temperature for 1-4h.
Peptide cutting: 95% TFA/H 2 O, oscillate for 3h.
Extraction of peptide and HPLC preparation: the aqueous phase was collected by extraction with 10mL of glacial diethyl ether and 2X 10mL of water. HPLC preparation: 0.1% TFA acetonitrile/0.1% TFA water, 0-80 min acetonitrile 20% -100%, flow rate: 8ml/min.
The amino resin (217 mg,0.1 mmol) was first swollen in 10mL DCM for 30min, DCM for 2X 10mL X2 min, DMF for 3X 10mL X2 min, indene (the system should be colorless), fmoc-removed, DMF for 4X 10mL X2 min, indene (the system should be blue), fmoc-Lys (Dde) -OH, peptide-removed, indene, fmoc-removed, peptide-removed, indene-removed, then PTPLLTK, peptide-removed, dde, peptide-removed, indene-removed, fmoc-Lys (Dde) -OH, peptide-removed, indene-removed, fmoc-removed, compound 24, peptide-removed, indene-removed, dde-removed, ACP-removed, indene-removed, fmoc-removed, FITC (compound 36), peptide-removed, indene-removed, and the next amino acid sequence was sequentially introduced. Cleavage of peptides, extraction of peptides, HPLC preparation: 0.1% TFA acetonitrile/0.1% TFA water, 0-80 min acetonitrile 20% -100%, flow rate: 8ml/min, rt=28.5 min, dried under reduced pressure to give the product PDC-11 (73 mg,0.036mmol, yield: 40%). HRMS (ESI) C 38 H 67 N 9 O 11 [M+H] 2+ calcd:1020.9557C 96 H 138 IN 17 O 22 S,found:1020.9554.Purity:98.69％.
The PDC-11HRMS analysis spectrum of the product is shown in FIG. 13.
(14) Synthesis of PDC-12
The synthesis method comprises the following steps:
the preparation was performed by dissolving (5 mg,0.0125 mmol) NOTA-NHS (Compound 37), (13 mg,0.0119 mmol) KTLLPTPG-EDA (With BOC) in 0.5mL DMF followed by (3 mg,0.0238 mmol) DIEA, stirring at room temperature for 2h, concentrating under reduced pressure, adding 20% TFA/DCM, stirring at room temperature for 2h, DCM X3, HPLC: 0.1% TFA acetonitrile/0.1% TFA water, 0-80 min acetonitrile 20% -100%, flow rate: 8ml/min, rt=20 min, (4 mg,0.00347mmol, 29.16% yield). HRMS (ESI) C 52 H 92 N 14 O 15 [M+H] 2+ calcd:577.3506,found:577.3508.Purity:87.20％.
The PDC-12HRMS analysis spectrum of the product is shown in FIG. 14.
(15) Synthesis of PDC-13
The synthesis method comprises the following steps:
(5 mg,0.01 mmol) of Compound 38, (13.5 mg,0.0121 mmol) KTLLPTPG-NHS (Compound 17) was dissolved in 0.5mL DMF followed by (2.8 mg,0.022 mmol) of DIEA, stirred at room temperature for 2h, concentrated under reduced pressure, 20% TFA/DCM was added, stirred at room temperature for 2h, DCM. Times.3, HPLC preparation: 0.1% TFA acetonitrile/0.1% TFA water, 0-80 min acetonitrile 20% -100%, flow rate: 8ml/min, rt=25 min, (4.5 mg,0.0036mmol, yield 32.5%).
(16) Synthesis of PDC-14
The synthesis method comprises the following steps:
(5 mg,0.01 mmol) of Compound 39, (12.5 mg,0.01 mmol) KTLLPTPG-NHS (Compound 17) was dissolved in 0.5mL DMF followed by (2.6 mg,0.02 mmol) DIEA, stirred at room temperature for 2h, concentrated under reduced pressure, 20% TFA/DCM was added, stirred at room temperature for 2h, DCM. Times.3, HPLC preparation: 0.1% TFA acetonitrile/0.1% TFA water, 0-80 min acetonitrile 20% -100%, flow rate: 8ml/min, rt=22.5 min, (4 mg,0.0031mmol, 31% yield).
(17) Synthesis of PDC-15
The synthesis method comprises the following steps:
(720 mg,1.95 mmol) of Compound 3, (488 mg,3.9 mmol) of amino-polyethylene glycol-propionic acid tert-butyl ester was dissolved in 15mL of DMF, followed by addition of (1.12 g,5.85 mmol) of EDCI, (79mg, 5.85 mmol) of HOBT and (75 mg,5.85 mmol) of DIEA at 0℃and stirring at room temperature for 4h. Concentrating under reduced pressure, extracting (30 mL of water, 3X 50mL of ethyl acetate), washing with 2X 70mL of water, 2X 70mL of saturated aqueous NaCl solution, and anhydrous Na 2 SO 4 Drying, concentrating under reduced pressure, purifying by column chromatography, and petroleum ether: ethyl acetate = 1: 4-1: 10. the product was obtained (704 mg,0.99mmol, 50.8% yield). The product (20 mg,0.028 mmol) was dissolved in 2mL of 20% TFA/DCM and stirred at room temperature for 3h, DCM 3X 3mL and taken directly into the next reaction.
HATU (32 mg,0.084 mmol) was added to the above product, 3mL of DMF was dissolved, DIEA (11 mg,0.084 mmol) was added, stirring was carried out at 0deg.C for 20min, followed by KTLLPTPG-EDA (With BOC) (60 mg,0.056 mmol) and stirring was carried out at room temperature for 3h. Concentrated under reduced pressure, 2mL of 5% DBU/DCM was added, stirred at room temperature for 2h, DCM 3X 3mL to give Dimer-KTLLPTPG (compound 5) which was directly taken into the next reaction.
(5 mg,0.012 mmol) of NOTA-NHS (Compound 37), (27 mg, 0.0111 mmol) of Dimer-KTLLPTPG (Compound 5) was dissolved in 1mL of DMF, DIEA (3 mg,0.024 mmol) was added, stirred at room temperature for 2h, concentrated under reduced pressure, 2mL of 20% TFA/DCM was added, stirred at room temperature for 3h, DCM 3X 3mL. Acetonitrile and water are added for dissolution, and HPLC preparation: 0.1% TFA acetonitrile/0.1% TFA water, 0-90 min acetonitrile 10% -100%, flow rate: 8ml/min, rt=25 min, dried under reduced pressure to give the product PDC-15 (5.6 mg,0.0024mmol, 22% yield). HRMS (ESI) C 107 H 188 N 28 O 31 [M+H] 2+ calcd:1181.7070,found:1181.7072.Purity:>99.00％.
The PDC-15HRMS analysis spectrum is shown in FIG. 15.
(18) Synthesis of PDC-16
The synthesis method comprises the following steps:
the peptide washing method comprises the following steps: DMF 4X 10mL X2 min.
Fmoc protecting group removal method: 3% DBU+3% piperidine+94% DMF 1X 5min, 1X 15min.
The Dde protecting group removal method comprises the following steps: 2% hydrazine hydrate/DMF 3X 3min.
Indene detection: add the prepared ninhydrin, phenol, pyridine in an amount = 1 drop: 1 drop: 2 drops, heating at 100deg.C for 30-90s, if there is exposed amino system, it is blue or light brown; if no exposed amino group exists, the system is colorless.
The following amino acid method is accessed: 3eq amino acid+3 eq HBTU (different condensing agents may be selected according to different conditions) +4eq HOBT+6eq DIEA,DMF 10mL, and shaking for 1-4h at room temperature.
Access DOTA (compound 40) method: 3eqDOTA (Compound 30) +3eq HBTU+4eq HOBT+6eq DIEA,DMF 10mL, and shaking at room temperature for 1-4h.
Method of accessing FA-NHS (compound 30): 3eq of FA-NHS (Compound 30) +3eq HBTU+4eq HOBT+6eq DIEA,DMF 10mL, shaking at room temperature for 1-4h.
FITC (compound 36) access method: 1eq FITC (Compound 36) +3eq DIEA,DMF 10mL, and shaking at room temperature for 1-4h.
Peptide cutting: 95% TFA/H 2 O, oscillate for 3h.
Extraction of peptide and HPLC preparation: the aqueous phase was collected by extraction with 10mL of glacial diethyl ether and 2X 10mL of water. HPLC preparation: 0.1% TFA acetonitrile/0.1% TFA water, 0-80 min acetonitrile 20% -100%, flow rate: 8mL/min.
Swelling (217 mg,0.1 mmol) amino resin in 10mL DCM for 30min, DCM washing 2X 10mL 2min, DMF washing 3X 10mL 2min, indene detection (the system should be colorless), fmoc-removing, DMF washing 4X 10mL 2min, indene detection (the system should be blue), fmoc-Lys (Dde) -OH, peptide washing, indene detection, fmoc removing, peptide washing, indene detection, then sequentially accessing GPTPLLTK according to the sequence of (accessing amino acid- & gt indene detection- & gt Fmoc- & gt peptide washing- & gt indene detection- & gt accessing the next amino acid,De-Dde, wash peptide, indene assay, fmoc-Lys (Dde) -OH, wash peptide, indene assay, fmoc-removal, DOTA (Compound 40), wash peptide, indene assay, de-Dde, FA-NHS (Compound 30), wash peptide, indene assay. Cleavage of peptides, extraction of peptides, HPLC preparation: 0.1% TFA acetonitrile/0.1% TFA water, 0-95 min acetonitrile 5% -100%, flow rate: 8mL/min, rt=22.5 min, dried under reduced pressure to give the product PDC-16 (115 mg,0.061mmol, yield: 61%). LCMS (ESI) C 85 H 135 N 25 O 24 [M+H] 2+ found:946.5Rt＝2.77min.Purity:97.81％.
Example 2 in vitro experiments: testing the Activity of each polypeptide drug conjugate against tumor cells and imaging
1) Activity experiments
PANC-1 (human pancreatic cancer cells), hTERT-HPNE (human normal pancreatic ductal cells) cells were isolated at a ratio of 3.0X10 3 Inoculating each cell/100 mu L/hole into a 96-well plate, placing the 96-well plate into an incubator, adding medicines after overnight, setting different medicine concentrations, adding the stock solution into a complete culture medium, diluting the stock solution 100 times, acting on the cells, placing the stock solution into the incubator for 48 hours, adding 10 mu L of CCK-8 solution into each hole, setting a blank control group (only containing the CCK-8 solution and the complete culture medium), incubating the stock solution in the incubator for 1h,Flex Station III Services type enzyme-linked immunosorbent assay (ELISA) to measure absorbance at 450nm, and repeating the same experiment for at least 3 times.
The inhibition of the tested cancer cells by the compounds of the present invention was calculated.
Inhibitory effect and IC of each compound on cancer cells and normal cells at different concentrations 50 The values (half inhibition concentration) are shown in table 1.
Table 1 IC of Compounds for PANC-1, hTERT-HPNE 50 Value of
IC 50 /nM PDC-1 PDC-2 PDC-3 PDC-4 PDC-5 PDC-6 PDC-7 PDC-8 PTX MMAE
PANC-1 ＞50000 4667±577.4 171.3±14.01 3087±580.1 2583±591.8 170±19.92 999.3±1.155 168.7±34.21 10.67±1.155 3.333±1.528
HPNE / / 274.3±44.28 1514±538.5 1365±188.4 401.5±82.31 5433±416.3 1523±336.3 / 10.67±3.512
As shown in table 1: PTX, MMAE, lack selectivity for normal and cancer cells as cytotoxic drugs. In contrast, the PDC molecules of the invention, PDC-1 and PDC-2 taking PTX as cytotoxic drugs, have significantly reduced antiproliferative activity on PANC-1 cells compared with PTX alone: two-component compound IC 50 Value: PDC-1: > 50. Mu.M; PDC-2: 4667.+ -. 577.4nM. This indicates that after the targeting polypeptide has been conjugated to the cytotoxic drug, the way the entire conjugate molecule enters the cell has been altered: the free diffusion of cytotoxic drugs into cells is transformed into a recognition of the entry into cells by receptor-ligand. Meanwhile, due to the addition of the cleavable linker, the release degree of the cytotoxic drug in the cell can be influenced by the corresponding hydrolase which plays a role in cleavage. Thus, at the same molar concentration administered, the targeted polypeptide-drug conjugate will have a reduced activity compared to the chemical drug alone.
The inhibitory activity of PDC-3-PDC-8 with MMAE as cytotoxic drug and Val-Cit-PABC as linker on PANC-1 cells is basically maintained at nanomolar concentration, and each compound IC 50 Value: PDC-3: 171.3+ -14.01 nM; PDC-4: 3087+ -580.1 nM; PDC-6: 170+ -19.92 nM; PDC-7:999.3 + -1.155 nM; PDC-8: 168.7.+ -. 34.21nM, which indicates that MMAE is better released in the cell. The PDC-5 linker is replaced by fatty chain alkynol, so that the sensitivity to cathepsin B (specifically recognizing and cutting Val-Cit-PABC) is reduced, the release efficiency of MMAE in cells is reduced, and the in-vitro anti-tumor activity of PDC-5 is obviously lower than that of PDC-3 on the basis of taking a polypeptide monomer as a targeting part. However, these compounds lack selectivity between PANC-1 and hTERT-HPNE.
PDC-6, PDC-7 and PDC-8 with dual-specific ligands show better selectivity between PANC-1 and hTERT-HPNE cells, and target polypeptide and albumin ligand, target polypeptide and folic acid, target polypeptide and integrin ligand are respectively used as dual-specific ligands, and are respectively opposite to IC of PANC-1 50 The values are respectively: PDC-6: 170+ -19.92 nM; PDC-7:999.3 + -1.155 nM; PDC-8: 168.7+ -34.21 nM; IC for hTERT-HPNE 50 The values are respectively: PDC-6:401.5 + -82.31 nM; PDC-7: 5433+ -416.3 nM; PDC-8: 1523+ -336.3 nM.
2) Fluorescence imaging
According to 2X 10 5 Inoculating cells to special dish for laser confocal, adding 1.5mL complete culture medium, standing overnight in incubator, adding pleectin-1 targeting peptide in competition group at 5 μm concentration for 2 hr, removing culture medium, adding PDC-9 simultaneously with dosing group, adding PDC-9 at 1 μm concentration at 37deg.C and 5% CO 2 Incubation for 4h at room temperature followed by PBS wash (3X 1mL X2 min), paraformaldehyde fixation for 10min, PBS wash (2X 2 min), DAPI action for 5min at room temperature, PBS wash (3X 2 min) and complete medium addition. Laser confocal imaging was then performed with Zeiss LSM800, german as shown in fig. 16.
Example 3 testing of polypeptide-drug conjugates for anti-tumor Activity in vivo
An immunodeficient mouse was used as a subject for cancer cell transplantation, and the mice were initially vaccinated with PANC-1 cells at 7-8 weeks of age. The specific operation flow is as follows:
preparing cells, adding PBS to matrigel=2:1, mixing, and placing in ice at a ratio of 1.0X10 7 The cells are inoculated under the armpit skin of the mice at a concentration of 150 mu L/mouse, and the average tumor volume reaches 70-90 mm on the 20 th day 3 The administration was started on the day in 5 groups, 3 administration groups (PDC-3, PDC-6, PDC-7), 1 positive control group (PTX), 1 blank control group. Intraperitoneal injection, dose: administration group (PDC-3, PDC-6, PDC-7): 0.3mg/kg. PTX positive control group: 8mg/kg. Blank control group: 200. Mu.L of physiological saline/patient.
Tumor volumes and mouse weights were recorded 1 time every 2 days, tumor volume calculation method: v (mm) 3 )＝(L×W 2 2), L: a long diameter; w: and (3) a short diameter. When the average tumor volume of the blank control group reaches 1200mm 3 About, mice were sacrificed, tumor was taken and weighed.
The tumor inhibitory effect of each dominant compound and the effect on the body weight of mice are shown in fig. 17 and 18.
Example 4 PET/CT imaging of cell-derived pancreatic cancer tumor-bearing mouse model by polypeptide-drug conjugate
According to volume ratio V (0.25M sodium acetate solution) ∶V (0.05M hydrochloric acid solution) Mixing the two solutions in a ratio of 1:4, and eluting the germanium gallium generator with 8mL of the buffer solution to obtain 68 Ga hydrochloric acid-sodium acetate buffer. 3 portions of 2mL of the buffer solution were taken in parallel, 20. Mu.g of PDC-12, 14 and 16, respectively, were added and the reaction mixture was heated to 100℃for 10min. The post-reaction solution was then passed through a C18 column to remove free ions, and the C18 column was then rinsed with 70% ethanol solution. Obtaining marked 68 Ga-PDC-12、 68 Ga-PDC-14 68 Ga-PDC-16. Will be marked 68 Ga-PDC-12、 68 Ga-PDC-14 68 Ga-PDC-16 is diluted by normal saline, mice are divided into 3 groups, each mouse in the group is injected with 100 muCi, PET/CT scanning imaging is carried out at 0.5h, reconstruction is carried out by PET/CT image processing software, and the image is shown in figure 19. Compared with 68 Ga-PDC-12 68 Ga-PDC-14， 68 Ga-PDC-16 has good uptake and imaging at tumor sites.
The foregoing is merely illustrative of the present invention, and the present invention is not limited thereto, and any person skilled in the art will readily recognize that variations or substitutions are within the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.

Claims (9)

1. The tumor-targeted polypeptide drug conjugate is characterized by having a structure shown in a formula (I): P-F-C-D, wherein,
p is tumor targeting polypeptide to specifically identify tumor specific target plectain-1;
f is a functional regulatory group;
c is a linker;
d is a payload group comprising a cytotoxic anti-tumor drug, a radionuclide chelating group, or a fluorescent group.
2. The tumor-targeted polypeptide drug conjugate of claim 1, wherein the amino acid sequence of the tumor-targeted polypeptide is shown in SEQ ID No.1, or
A polypeptide sequence or polypeptide derivative with 1 to 3 amino acids replaced, deleted and inserted on SEQ ID NO. 1.
3. The tumor targeted polypeptide drug conjugate of claim 1, wherein the functional regulatory group is selected from one or more of folic acid, RGD cyclic peptide, 4-iodophenyl end group, evans blue, plectin-1 targeting peptide.
4. The tumor targeted polypeptide drug conjugate of claim 1, wherein the linker is a linked cleavable linker or a non-cleavable linker; preferably, the linker takes a chain shape which is formed by covalent bond connection between carbon atoms and is not ring-formed as a carbon frame; preferably, the linker is selected from one of the following structures:
5. the tumor targeted polypeptide drug conjugate of claim 1, wherein the cytotoxic anti-tumor drug comprises paclitaxel and/or MMAE.
6. The tumor targeted polypeptide drug conjugate of claim 1, wherein the fluorophore is selected from the group consisting of:
7. the tumor targeted polypeptide drug conjugate of claim 1, wherein the radionuclide chelating group is selected from one of the following structures:
8. the polypeptide drug conjugate of claim 1 wherein the polypeptide drug conjugate is any one or more of the following structures:
9. use of a tumor-targeted polypeptide drug conjugate according to any one of claims 1-8 in the preparation of an anticancer drug.
CN202310986826.9A 2023-08-07 2023-08-07 Tumor-targeted polypeptide drug conjugate and application thereof Pending CN116999570A (en)

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