CN115154419B - Nanometer micelle precursor, nanometer micelle, preparation method and application thereof - Google Patents

Nanometer micelle precursor, nanometer micelle, preparation method and application thereof Download PDF

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CN115154419B
CN115154419B CN202110360627.8A CN202110360627A CN115154419B CN 115154419 B CN115154419 B CN 115154419B CN 202110360627 A CN202110360627 A CN 202110360627A CN 115154419 B CN115154419 B CN 115154419B
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nano
micelle
protein degradation
precursor
group
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CN115154419A (en
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于海军
裴明亮
祝奇文
高晶
李俊豪
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Shanghai Institute of Materia Medica of CAS
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    • 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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    • 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
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    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
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Abstract

The application belongs to the field of medicines, and in particular relates to a nano micelle precursor, a nano micelle, a preparation method and application thereof. The nano micelle is formed by self-assembly of nano micelle precursors. The nano micelle precursor comprises a protein degradation agent, a connecting group and a polymer framework; one end of the polymer skeleton is connected with the protein degradation agent through a connecting group in a covalent bond way, the other end of the polymer skeleton is connected with a ligand or a terminal group, and the ligand is selected from ligand groups targeting tumor cells or tumor matrixes. The nano micelle can selectively target tumor cells, improve the uptake capacity of the tumor cells on the protein degradation agent, improve the tissue distribution condition and the pharmacokinetic property of the protein degradation agent molecules in organisms while maintaining the protein degradation activity, widen the treatment window and improve the tumor inhibition effect.

Description

Nanometer micelle precursor, nanometer micelle, preparation method and application thereof
Technical Field
The application belongs to the field of medicines, and in particular relates to a nano micelle precursor and a nano micelle, and a preparation method and application thereof.
Background
The protein hydrolysis targeting chimera (PROTAC) technology is taken as a new generation protein degradation technology, the target protein can be subjected to ubiquitination marking through simultaneous combination of molecules, the target protein and E3 ubiquitin ligase, and the target protein is degraded by an intracellular ubiquitin-proteasome system, so that the target protein can be cleared conveniently, and the protein is a hot spot in the field of research and development of the current medicines. Compared with the traditional small molecule inhibitors, protein degradation agent molecules such as PROTAC have catalytic activity, the toxicity is relatively low, the realization of degradation is not independent of the affinity between the molecules and the target protein, and good selectivity can be realized at the cellular level, so that the occurrence of drug resistance of the small molecule inhibitors in organisms, especially the drug resistance in tumor treatment, can be improved. However, such protein degradation agent molecules generally have larger molecular weight, relatively poor film permeability and relatively low bioavailability, and toxic and side effects caused by off-target effects of the protein degradation agent molecules prevent the development of protein degradation technology in clinical application.
In view of this, the present application has been made.
Disclosure of Invention
The primary object of the present application is to propose a nano-micelle precursor.
The second application aims to provide a preparation method of the nano micelle precursor.
A third object of the present application is to propose a nanomicelle.
The fourth application aims at providing a preparation method of the nano micelle.
A fifth object of the present application is to propose the use of the nanomicelle.
In order to achieve the aim of the application, the technical scheme adopted is as follows:
the first aspect of the application provides a nano micelle precursor comprising a protein degradation agent, a connecting group and a polymer skeleton; the polymer backbone is selected from polyethylene glycol or polyethylene glycol derivatives and comprises at least one of the structural units as described by formula i1, formula i2 and formula i 3:
wherein m is selected from integers from 10 to 145, preferably from 100 to 140, more preferably 113; q is selected from integers of 0 to 100, preferably 20 to 50, more preferably 38; p is an integer from 0 to 100, preferably an integer from 20 to 50, more preferably 38; and m, q, p are not zero at the same time or are not 0 at the same time;
one end of the polymer skeleton is connected with the protein degradation agent through a connecting group in a covalent bond; the other end of the polymer skeleton is connected with a ligand or a terminal group; the ligand is selected from ligands targeting tumor cells or tumor stroma; the terminal group is preferably methoxy.
The second aspect of the present application provides a method for preparing a nano micelle precursor, which at least comprises the following steps:
reacting the precursor of the linking group with a protein degradation agent molecule to form covalent connection between the precursor and the protein degradation agent molecule and obtain a protein degradation agent molecule with a derivative functional group;
covalently connecting the functional group-derivatized protein degradation agent molecules with a polymer skeleton with end groups or an uncapped polymer skeleton to obtain a polymer-protein degradation agent conjugate with end groups or an uncapped polymer-protein degradation agent conjugate;
the non-end-capped polymer-protein degradation agent conjugate and the ligand are subjected to biological orthogonal reaction, and the polymer-protein degradation agent conjugate with the terminal group is the nano micelle precursor.
The third aspect of the application provides a nano micelle, which is formed by self-assembling the nano micelle precursor.
The fourth aspect of the present application provides a preparation method of the nano micelle, at least comprising the following steps: and (3) dissolving the nano micelle precursor in an organic solvent to obtain a solution, dispersing the solution in water, and carrying out ultrafiltration or dialysis to obtain the nano micelle.
In a fifth aspect, the present application proposes the use of the above nanomicelle for the preparation of a medicament for the treatment of a malignancy, preferably selected from breast cancer, cervical cancer, liver cancer, gastric cancer, pancreatic cancer, ovarian cancer, colon cancer or prostate cancer.
The technical scheme of the application has at least the following technical effects:
the nano micelle obtained by self-assembling the nano micelle precursor can selectively target tumor cells, improve the uptake capacity of the tumor cells on protein degradation agent molecules, improve the tissue distribution condition and the pharmacokinetic properties of the protein degradation agent in organisms, widen the treatment window, improve the tumor inhibition effect and lay a foundation for the clinical application of the protein degradation agent.
Drawings
FIG. 1 is a graph showing a dynamic light scattering particle size distribution of CPEG-PLGA-ssV-JQ1 nano-micelle prepared in preparation example 6;
FIG. 2 is a graph showing the cumulative release profile of JQ1-p-VHL in vitro simulated normal and tumor cell physiological microenvironments of CPEG-PLGA-ssV-JQ1 nanomicelles prepared in preparation example 6, wherein squares represent 0.5% Tween 80+10. Mu.M GSH (normal cell physiological microenvironment) and dots represent 0.5% Tween 80+10mM GSH (tumor cell physiological microenvironment);
FIG. 3 shows uptake of CPEG-PLGA-ssV-JQ1 fluorescent nanomicelle and PEG-PLGA-ssV-JQ1 fluorescent nanomicelle prepared in preparation example 7 in MDA-MB-231 breast cancer cells. Wherein, the bar graph of the black background represents PEG-PLGA-ssV-JQ1 fluorescent nano-micelle, and the bar graph of the white background represents CPEG-PLGA-ssV-JQ1 fluorescent nano-micelle;
FIG. 4 shows the results of experiments for inhibiting the proliferation potency of CPEG-PLGA-ssV-JQ1 nanomicelle (right panel) prepared in preparation example 1, JQ1-p-VHL (left panel) and preparation example 6 in MDA-MB-231 breast cancer cells;
FIG. 5 shows the degradation of BRD4 protein in MDA-MB-231 breast cancer cells by the CPEG-PLGA-ssV-JQ1 nanomicelle (right panel) prepared in preparation example 1 (left panel) and the JQ1-p-VHL molecule prepared in preparation example 6.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the embodiments of the present application more apparent, the technical solutions in the embodiments of the present application will be clearly and completely described below, and it is apparent that the described embodiments are some embodiments of the present application, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the application without making any inventive effort, are intended to be within the scope of the application. In addition, numerous specific details are set forth in the following description in order to provide a better illustration of the application. It will be understood by those skilled in the art that the present application may be practiced without some of these specific details. In some embodiments, materials, elements, methods, means, etc. well known to those skilled in the art are not described in detail in order to highlight the gist of the present application.
Throughout the specification and claims, unless explicitly stated otherwise, the term "comprise" or variations thereof such as "comprises" or "comprising", etc. will be understood to include the stated element or component without excluding other elements or components.
Aiming at improving the application of the protein degradation technology in tumor treatment, the embodiment of the application provides a nano micelle of a protein degradation agent and a precursor thereof, so as to realize targeted delivery of medicines. By forming the drug-loaded nano-particles, the endocytosis of tumor cells to nano-micelles can improve the drug uptake capacity of cells, and the influence of the membrane permeability of drug molecules on the cell uptake is reduced; improving the pharmacokinetics property of the protein degradation agent and finally improving the tumor treatment effect. Meanwhile, under the protection of the nano particles, the half life of the drug can be effectively prolonged, and the tissue distribution and the pharmacokinetic property of the drug can be improved. The nano micelle of the embodiment of the application is formed by self-assembling a nano micelle precursor. The nano micelle precursor of the embodiment of the application is prepared by coupling a protein degradation agent group, a connecting group and a polymer framework in a covalent connection mode through a connecting group, so that the polymer framework is used for regulating the ratio of a lipophilic segment to a hydrophilic segment of a conjugate to promote the formation of nano prodrug. One end of the polymer skeleton is connected with the protein degradation agent through a connecting group in a covalent bond, and can trigger response in a specific physiological microenvironment (comprising pH value difference and glutathione level (GSH)) to release the protein degradation agent; the other end of the polymer skeleton is connected with a ligand or a terminal group; the ligand group may be selected from ligands targeting tumor cells or tumor stroma; the terminal group is an inert group which is not reactive. Compared with the micelle obtained by adopting the conventional encapsulation technology, the protein degradation agent is connected to the polymer skeleton through the covalent bond, thereby being beneficial to realizing the targeted delivery and the slow release of the protein degradation agent and reducing the influence of concentration on the drug action effect.
In certain embodiments, the structural formula of the nano-micelle precursor of the present embodiments is shown in formula I and formula II:
wherein, ligands represent ligands, F-PEG represents polymer skeletons for functionalization, linker represents a linking group, PROTAC represents a protein degradation agent group, R represents a terminal group, and all the groups are connected through covalent bonds.
Specifically, the ligand is at least one selected from fusion proteins with tumor cell or tumor matrix targeting, nanobodies, targeting peptide molecules and small molecule targeting ligands.
Preferably, the terminal group is methoxy.
In certain embodiments, the fusion protein is selected from at least one of an antibody fusion protein (e.g., an Fc fusion protein), a diphtheria toxin fusion protein (e.g., DT389-hIL13-13E 13K), an immunoglobulin fusion protein (e.g., an Lg fusion protein);
preferably, the nanobody is selected from the group consisting of, but not limited to: at least one of an anti-CD 44 nanobody, an anti-Carbonic Anhydrase IX (CAIX) nanobody, and an anti-Epidermal Growth Factor Receptor (EGFR) nanobody;
wherein, the targeting peptide molecule is at least one peptide with the amino acid sequence shown in SEQ ID NO. 1-5, and the amino acid sequence is shown in Table 1:
TABLE 1
In certain embodiments, the small molecule targeting ligand and derivatives thereof are selected from at least one of the following structural formulas:
in certain embodiments, the ligand comprises a linking group A for coupling to the polymer backbone,
alternatively, the linking group a is used to attach to a derivatized functional group on the polymer backbone, selected from the group formed after the reaction of a sulfhydryl or alkynyl functional group. For example, the thiol-forming linker A is-S-, the alkynyl-functional-forming linker A is
The free sulfhydryl or disulfide bond in the ligand such as fusion protein, nano antibody and targeting peptide molecule is reduced to generate sulfhydryl, and alkynyl functional group at the tail end of the small molecule ligand can react with the derivatization functional group on the polymer skeleton.
In certain embodiments, the polymeric backbone used to modulate the ratio of lipophilic to hydrophilic segments of the conjugate to promote the formation of the nanomicelles is selected from polyethylene glycol or polyethylene glycol derivatives, including at least one of the structural units shown as formula i1, formula i2, and formula i 3:
wherein m is selected from integers from 10 to 145, preferably from 100 to 140, more preferably 13;
q is selected from integers of 0 to 100, preferably 20 to 50, more preferably 38;
p is an integer from 0 to 100, preferably an integer from 20 to 50, more preferably 38;
and m, q, p are not zero at the same time or are not 0 at all.
In certain embodiments the polymer backbone may have structural units as shown in formula ii1 or formula ii2,
wherein m is the number of ethylene glycol units in the polymer, and is preferably an integer of 10 to 145, more preferably an integer of 100 to 140, and still more preferably 113;
q is the number of units of lactic acid in the polymer, preferably an integer of 0 to 100, more preferably an integer of 20 to 50, still more preferably 38;
p is the unit number of glycolic acid in the polymer, and is an integer of 0 to 100, preferably an integer of 20 to 50, more preferably 38;
and m, q, p are not zero at the same time or are not zero at all.
R a Represents the residue on the polymer after the derivatizing functional group has reacted with the ligand to form a covalent bond, R b Represents a terminal group.
In certain embodiments, R a Selected from: -NH-,R b Selected from methoxy;
wherein R is a The functional groups on the ligand are in one-to-one correspondence, and specifically:
when the polymer derivatizing functionality is NH 2 When it is a carboxyl function on the ligand, which forms a covalent bond with it, R on the polymer a forming-NH-CO-with the linking group A on the ligand, thereby connecting the polymer and the ligand through covalent bonds;
when the polymer derivatizing functional group isIn the case of covalent bonds, the functional groups are mercapto groups on the ligand, or mercapto groups produced by intramolecular reduction of disulfide bonds, R on the polymer a With a linking group A on the ligandThereby covalently linking the polymer to the ligand;
when the polymer derivatizing functional group is-N 3 Covalent bond with it is a ligand-derivatized alkynyl group, R on the polymer a With a linking group A on the ligandThereby covalently linking the polymer to the ligand.
In certain embodiments, when p is 0 and q is not 0, the polymer backbone is polyethylene glycol-polylactic acid;
when q is 0 and p is not 0, the polymer skeleton is polyethylene glycol-polyglycolic acid;
when q and p are not 0, the polymer skeleton is polyethylene glycol-polylactic acid-polyglycolic acid.
Preferably m is preferably 113, q is preferably 38, and p is preferably 0 or 38.
In certain embodiments, the precursor of the polymer backbone has a structure as shown in formula iii1 or formula iii 2:
therein, m, q, p, R b The meaning of the expression is the same as that of the formulas ii1 and ii 2; r is R a-p Represents a derivatising functional group for covalent binding to a ligand, wherein R a-p Selected from: NH (NH) 2 -、-N 3 。
In certain embodiments, the linking group is selected from the group shown by formulas iv1, iv2, and iv 3;
wherein M is 1 、M 2 、R 3 Independently selected from alkylene groups having 1 to 4 carbon atoms, preferably R 1 、R 2 Independently selected from carbonyl, -O-C (O) -, -NH-C (O) -;
preferably, R 3 is-C (CH) 3 ) 2 -，
Further preferably, M 1 And M is as follows 2 The same applies.
In certain embodiments, the group of formula iv2 may be obtained by reacting a dithiodiacid as a starting material, such as 4, 4-dithiodibutyric acid. The group of formula iv1 can be obtained by reacting a thiodiacid as starting material, for example thiodiacetic acid. The group of formula iv3 may be prepared starting from thioketal diacid, for example 2,2' - [ propane-2, 2-diylbis (thio) ] diacetic acid.
In certain embodiments, the structural formula of the linking group may be selected from the group shown in the following chemical formulas, without being limited thereto:
in certain embodiments, the precursor of the linking group is selected from the group consisting of compounds represented by formulas iv1-p, iv2-p, and iv 3-p;
wherein M is 1 、M 2 、R 3 The meaning of the expression is the same as that of iv1 to iv 3;
L 1 、L 2 each independently selected from-COOH, -NCO, -NH 2 、–OH；
Further, the compound shown by iv1-p is preferably thiodiacetic acid, the compound shown by iv2-p is preferably 4, 4-dithiodibutyric acid, and the compound shown by iv3-p is preferably 2,2' - [ propane-2, 2-diylbis (thio) ] diacetic acid.
In certain embodiments, the protein degrading agent is selected from protein degrading agent molecules that bind to E3 ubiquitin ligases containing the Von Hippel-Lindau receptor and are covalently linked to the linking group through the proline hydroxy site present in the molecule.
In certain embodiments, the protein degrading agent is selected from the following molecules, without limitation:
the embodiment of the application also relates to a preparation method of the nano micelle precursor, which comprises the following two modes:
a mode one,
S1, reacting a protein degradation agent molecule with a precursor of a connecting group to form covalent connection between the protein degradation agent molecule and the precursor of the connecting group, so as to obtain a protein degradation agent molecule derivatized by a functional group;
s2, carrying out covalent connection on the protein degradation agent molecules obtained by derivatization of the functional groups and an unblocked polymer skeleton to obtain an unblocked polymer-protein degradation agent conjugate;
s3, enabling the unblocked polymer-protein degradation agent conjugate to react with a ligand in a bio-orthogonal manner to form covalent connection, and obtaining the ligand modified polymer-protein degradation agent conjugate.
Wherein the unblocked polymer backbone is a polymer backbone having derivatized functional groups.
In certain embodiments, the ligand is modified prior to reaction with the derivatized functionality on the polymer backbone, preferably the modification comprises reduction of disulfide bonds to produce sulfhydryl groups.
A second mode,
S1, reacting a protein degradation agent molecule with a precursor of a connecting group to form covalent connection between the protein degradation agent molecule and the precursor of the connecting group, so as to obtain a protein degradation agent molecule derivatized by a functional group;
s2, carrying out covalent connection on the protein degradation agent molecules with the obtained functional groups and the polymer with the terminal groups.
The embodiment of the application also provides a nano micelle which is formed by self-assembling the nano micelle precursor.
In certain embodiments, the average hydrated particle size of the nanomicelles is 10 to 150nm, more preferably 50 to 60nm.
In some embodiments, the application of the nano micelle polymer dispersion index is 0.01-0.3.
In certain embodiments, the nanomicelles have fluorescent probes supported therein for labeling.
The embodiment of the application also provides a preparation method of the nano micelle, which comprises the following steps:
dissolving the nano micelle in an organic solvent to obtain a solution, dispersing the polymer-protein degradation agent conjugate solution in water under the ultrasonic condition, and obtaining the nano micelle through ultrafiltration or dialysis.
According to the application, the ultrasound conditions are conventional preparation conditions of nano-micelles in the art.
In certain embodiments, the water is deionized water and the organic solvent is selected from at least one of haloalkane organic solvents, alcohol organic solvents, ether organic solvents, ketone organic solvents, nitrile organic solvents, and N, N-dimethylformamide and dimethylsulfoxide; the halogenated alkane organic solvent is selected from dichloromethane and chloroform, the alcohol organic solvent is selected from methanol and ethanol, the nitrile organic solvent is selected from acetonitrile, ether organic solvent diethyl ether and tetrahydrofuran, and the ketone organic solvent is selected from acetone. The organic solvent is preferably dimethyl sulfoxide.
The cut-off molecular weight of the dialysis bag in the dialysis process can be 800, 1000, 3500 and 14000, and one of the cut-off molecular weight and the cut-off molecular weight is reasonably selected. The solvent used for dialysis can be at least one selected from deionized water, methanol, tetrahydrofuran, and N, N-dimethylformamide.
The ultrafiltration process can be carried out by adopting an ultrafiltration tube centrifugal ultrafiltration or an ultrafiltration machine ultrafiltration mode, the molecular weight cut-off of an ultrafiltration tube or an ultrafiltration membrane bag used in the process can be 1500D, 3kD, 5kD and 10kD, and one of the ultrafiltration tube, the ultrafiltration membrane bag and the ultrafiltration membrane bag is reasonably selected.
In another aspect, the application also provides the use of the nanomicelle in a medicament for treating malignant tumors, wherein the malignant tumors comprise, but are not limited to, breast cancer, cervical cancer, liver cancer, gastric cancer, pancreatic cancer, ovarian cancer, colon cancer or prostate cancer.
Detailed Description
The reagents and apparatus used in the following examples are as follows:
the CRGDK small peptides used in the examples were purchased from calico biotechnology limited; mal-PEG 5k -PLGA 5k -OH (PEG, mn=5000 Da; plga, mn=5000 Da, lactic acid: glycolic acid=50:50), mPEG 5k -PLGA 5k -OH (mPEG, mn=5000 Da; plga, mn=5000 Da, lactic acid: glycolic acid=50:50) purchased from jinan dai biotechnology limited; mPEG (polyethylene glycol) 5k -NH 2 Available from Sibao Biotech Co.Ltd, (S) - (+) -2- (4- (4-chlorophenyl) -2,3, 9-trimethyl-6H-thieno [3, 2-F)][1,2,4]Triazolo [4,3-A ]][1,4]Diazepin-6-yl) acetic acid ((+) -JQ 1) was purchased from sigma aldrich (Shanghai) trade company, inc, (2S, 4 r) -1- [ (2S) -2-amino-3, 3-dimethylbutyryl]-4-hydroxy-N- [ (1S) -1- [4- (4-methyl-1, 3-thiazol-5-yl) phenyl]Ethyl group]Pyrrolidine-2-carboxamide hydrochloride was purchased from Jiangsu ai Kang Shengwu pharmaceutical development Co., ltd, tert-butyl 2- (2- (2- (2-aminoethoxy) ethoxy) acetate was purchased from Aimad biotechnology Co., st.O., 4-dithiodibutyric acid, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDCI), 4-Dimethylaminopyridine (DMAP), diisopropylethylamine (DIEA), dichloromethane (DCM), tetrahydrofuran (THF), N, N-Dimethylformamide (DMF), dimethyl sulfoxide, 2- (7-azabenzotriazole) -N, N, N ', N' -tetramethylurea Hexafluorophosphate (HATU) was purchased from Shanghai lare technologies Co., ltd; the remaining reagents and solvents were purchased from the national drug group (Shanghai) chemical reagent Co., ltd unless otherwise specified.
MDA-MB-231 breast cancer cells were purchased from Shanghai cell Bank of the Chinese sciences, and RPIM1640 medium for cell culture and fetal bovine serum were purchased from Gibco corporation.
The hydrodynamic particle size of the protein degradation agent nano-micelle is measured by a Markov laser particle sizer (ZEN 3690, malven, USA). Electronic balances (quinmix 224-1,Sartorius Germany); rotary evaporator (B-40, buchi, switzerland); constant temperature magnetic stirrer (DF-101S, shanghai pre-treatment instruments Co., ltd.); nuclear magnetic resonance spectroscopy (mercuryplus 400, varian, usa); separation and purification was performed by Waters preparative liquid chromatograph (Waters e2695 chromatography pump, xbridge C18 μm 19×250mm column, waters 2998 uv detector). Flow assay data were measured by BD FACS CALIBUR flow cytometer. Unless otherwise indicated, the equipment and testing methods used are those conventional in the art.
In the present application, unless otherwise specified, the equipment and test methods used are those conventional in the art.
Preparation example 1: synthesis of protein degradation agent molecule (JQ 1-p-VHL)
JQ1 (1.37 g) was dissolved in 12mL 50% trifluoroacetic acid/dichloromethane and reacted at room temperature for 4h. After the completion of the reaction, the solvent was distilled off under reduced pressure, and the resulting pale yellow solid powder was separated by column chromatography to obtain Compound 1 (1.15 g, 95.83%). 1 H NMR(400MHz,CDCl 3 )δ10.19(s,1H),7.45(d,J＝8.4Hz,2H),7.35(d,J＝8.6Hz,2H),4.64(t,J＝7.0Hz,1H),3.73(dd,J＝17.0,7.0Hz,1H),3.61(dd,J＝17.0,6.9Hz,1H),2.73(s,3H),2.43(s,3H),1.71(s,3H).
Compound 1 (596 mg,1.48 mmol) was dissolved in 5.0mL of Dichloromethane (DCM), and 2- (7-azabenzotriazol) -N, N, N ', N' -tetramethyluronium hexafluorophosphate (HATU, 678mg,1.78 mmol), N, N-diisopropylethylamine (DIEA, 479mg,3.72 mmol) and compound 2 (411 mg,1.56 mmol) were added thereto and reacted at room temperature for 24h. After the reaction, the reaction mixture was diluted with methylene chloride, washed with water and saturated ammonium chloride solution, dried over anhydrous sodium sulfate, and separated by column to give compound 3 (756.5 mg,79.11％)。 1 H NMR(500MHz,CDCl 3 )δ7.42(d,J＝8.5Hz,2H),7.33(d,J＝8.8Hz,2H),7.08(s,1H),4.66(t,J＝7.0Hz,1H),4.04(s,2H),3.77–3.71(m,4H),3.71–3.65(m,4H),3.65–3.58(m,2H),3.57–3.48(m,3H),3.39(dd,J＝14.6,7.2Hz,1H),2.67(s,3H),2.41(s,3H),1.68(s,3H),1.47(s,9H).
660mg of Compound 3 (1.02 mmol) was weighed out and dissolved in 10mL of 50% trifluoroacetic acid/dichloromethane and reacted at room temperature for 4h. After the completion of the reaction, the solvent was removed by spin separation to obtain Compound 4 (532)
mg,88.27％)。 1 H NMR(500MHz,CDCl 3 )δ7.77(t,J＝4.9Hz,1H),7.43(d,J＝
8.5Hz,2H),7.35(d,J＝8.8Hz,2H),4.75(t,J＝7.1Hz,1H),4.26–4.14(m,2H),3.86–3.82(m,1H),3.78(dd,J＝8.6,3.7Hz,2H),3.76–3.75(m,2H),3.74–3.71(m,1H),3.70–3.68(m,2H),3.65(dd,J＝6.8,3.8Hz,1H),3.62(dd,J＝7.8,4.5Hz,2H),3.60–3.55(m,1H),3.52–3.47(m,2H),2.70(s,3H),2.47–2.38(m,3H),1.76–1.65(m,3H).
Compound 4 (1.0 g,1.7 mmol) and compound 5 (8238 mg,1.86 mmol) were dissolved in 15mL of methylene chloride, and 2- (7-azabenzotriazol) -N, N, N ', N' -tetramethylurea hexafluorophosphate (965 mg,2.54 mmol), N, N-diisopropylethylamine (1.47 mL,8.47 mmol) was added thereto and reacted at room temperature for 12 hours. After the reaction, the reaction mixture was diluted with methylene chloride, washed with water and saturated ammonium chloride solution, and dried over anhydrous sodium sulfate, followed by column separation to obtain JQ1-p-VHL (1.12 g, 64.85%). 1 H NMR(500MHz,CDCl 3 )δ8.67(s,1H),7.87(s,1H),7.55(d,J＝7.8Hz,1H),7.41(d,J＝8.4Hz,2H),7.36(dd,J＝8.8,2.5Hz,4H),7.32(dd,J＝9.0,4.3Hz,3H),5.12–5.05(m,1H),4.82(t,J＝7.7Hz,1H),4.73(d,J＝9.3Hz,1H),4.68(dd,J＝8.6,5.5Hz,1H),4.46(s,1H),4.31(d,J＝15.8Hz,1H),4.13(d,J＝15.9Hz,1H),4.08(d,J＝11.1Hz,1H),3.75–3.60(m,12H),3.59–3.47(m,2H),3.42–3.33(m,2H),2.64(s,3H),2.52(s,3H),2.46–2.41(m,1H),2.40(s,3H),2.10(t,J＝10.1Hz,1H),1.67(s,3H),1.47(d,J＝6.9Hz,3H),1.07(s,9H).
Preparation example 2: synthesis of JQ1-p-VHL-ssCOOH and Mal-PEG-PLGA-ssJQ1-p-VHL
4, 4-Dithiobutanedioic acid (140 mg,0.59 mmol), N, N-dimethylaminopyridine (24 mg, 0.197mmol), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (113 mg,0.59 mmol) and diisopropylethylamine (205. Mu.L, 1.18 mmol) were dissolved in 2.0mL of anhydrous dichloromethane and reacted at room temperature for 1.5 hours, and then JQ1-p-VHL (200 mg, 0.197mmol) prepared in preparation example 1 was added thereto and reacted at room temperature for 48 hours. After the reaction, washing with water and 1M hydrochloric acid aqueous solution in turn, combining organic phases, anhydrous Na 2 SO 4 Drying and column chromatography gave the compound JQ1-p-VHL-ssCOOH (115 mg, 47.32%). 1 H NMR(500MHz,CDCl 3 )δ8.69(s,1H),7.69(d,J＝7.6Hz,1H),7.43(d,J＝8.3Hz,2H),7.35(dd,J＝12.0,7.9Hz,7H),5.39(s,1H),5.14–5.06(m,1H),4.82(t,J＝7.7Hz,1H),4.70(t,J＝6.9Hz,1H),4.63(d,J＝9.1Hz,1H),4.16(dd,J＝15.8,6.9Hz,3H),3.87(dd,J＝11.5,4.2Hz,1H),3.79–3.68(m,8H),3.64(s,2H),3.55(ddd,J＝13.8,12.1,5.8Hz,3H),3.45(dd,J＝14.8,7.3Hz,1H),2.75(dt,J＝10.5,7.1Hz,4H),2.67(s,3H),2.65–2.58(m,1H),2.53(s,3H),2.52–2.45(m,3H),2.43(s,3H),2.30–2.24(m,1H),2.02(dd,J＝18.9,11.8Hz,5H),1.70(s,3H),1.46(d,J＝4.9Hz,4H),1.30(d,J＝14.6Hz,3H),1.09(s,9H),0.91–0.86(m,4H).
Compound JQ1-p-VHL-ssCOOH (24.5 mg,0.02 mmol), N, N-dimethylaminopyridine (2.5 mg,0.02 mmol), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (3.8 mg,0.02 mmol) was dissolved in 5.0mL of anhydrous dichloromethane, diisopropylethylamine (3.3. Mu.L) was added, and the reaction was carried out at room temperature for 1.5h. Mal-PEG-PLGA-OH (67 mg) was then added and the reaction continued for 48h. After the reaction, removing impurities by a dialysis method, and freeze-drying to obtain 45mg of a polymer Mal-PEG-PLGA-ssJQ1-p-VHL (abbreviated as PEG-PLGA-ssV-JQ 1).
Preparation example 3: synthesis of mPEG-PLGA-ssJQ1-p-VHL
The compound JQ1-p-VHL-ssCOOH (24 mg,0.02 mmol) produced in production example 2, N, N-dimethylaminopyridine (2.5 mg,0.02 mmol), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (7.7 mg,0.04 mmol) was dissolved in anhydrous dichloromethane, and diisopropylethylamine (10. Mu.L) was added and reacted at room temperature for 1.5 hours. Next, a solution of mPEG-PLGA-OH (67 mg) was added dropwise thereto, and the reaction was continued for 48 hours. After the reaction, removing impurities by a dialysis method, and freeze-drying to obtain 50mg of polymer mPEG-PLGA-ssJQ1-p-VHL (abbreviated as mPEG-PLGA-ssV-JQ 1).
Preparation example 4: synthesis of mPEG-ssJQ1-p-VHL
The compound JQ1-p-VHL-ssCOOH (24 mg,0.02 mmol) produced in production example 2, N, N-dimethylaminopyridine (2.5 mg,0.02 mmol), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (3.8 mg,0.04 mmol) was dissolved in anhydrous dichloromethane, and diisopropylethylamine (10. Mu.L) was added and reacted at room temperature for 1.5 hours. Subsequent addition of mPEG 5k -NH 2 (50 mg) solution, the reaction was continued for 48h. After the reaction, removing impurities by a dialysis method, and freeze-drying to obtain 35mg of a polymer mPEG-ssJQ1-p-VHL (abbreviated as mPEG-ssV-JQ 1).
Preparation example 5: preparation of CRGDK-modified PEG-PLGA-protein degradation agent
CRGDK (12.6 mg,0.03 mmol) was dissolved in 5.0mL of N, N-Dimethylformamide (DMF) with Mal-PEG-PLGA-ssJQ1-p-VHL (100 mg) prepared in preparation example 2 and reacted under nitrogen atmosphere at 40℃for 8 hours. Removing impurities by dialysis, and freeze-drying to obtain compound CRGDK-PEG-PLGA-ssJQ1-p-VHL (CPEG-PLGA-ssV-JQ 1).
Preparation example 6: preparation of nanomicelle
CPEG-PLGA-ssV-JQ1 (20 mg) prepared in preparation example 5 was dissolved in 1mL of dimethylsulfoxide solvent, and then 50. Mu.L of the above solution (20 mg/mL) was slowly dropped into 1mL of deionized water in an ultrasonic (power 70W,2 min) instrument. Subsequently, the obtained solution was dialyzed in deionized water (dialysis bag cut-off molecular weight 3500), and CPEG-PLGA-ssV-JQ1 nano-micelle was obtained. And measuring the hydrodynamic radius and the particle size distribution of the nano micelle by using dynamic light scattering.
As shown in FIG. 1, the average hydration particle size of the CPEG-PLGA-ssV-JQ1 nano-micelle constructed is about 55.5nm, and the PDI is 0.096.
Preparation example 7: preparation of fluorescent probe ICG marked nano micelle
CPEG-PLGA-ssV-JQ1 prepared in preparation example 5 or PEG-PLGA-ssV-JQ1 (20 mg) prepared in preparation example 2 was mixed with ICG (0.1 mg) and dissolved in 1mL of dimethyl sulfoxide solvent, and then 50. Mu.L of the above solution was slowly dropped into 1mL of deionized water solution under ultrasonic conditions (power 70W,2 min). Then, the obtained solution is dialyzed in deionized water (dialysis bag cut-off molecular weight is 3500), and the obtained CPEG-PLGA-ssV-JQ1/PEG-PLGA-ssV-JQ1 nano micelle marked by the fluorescent probe ICG is used for detecting the cell uptake condition of the nano micelle.
Test example 1: release of JQ1-p-VHL in CPEG-PLGA-ssV-JQ1 nanomicelle
CPEG-PLGA-ssV-JQ1 nano-micelles prepared in preparation example 6 were dispersed in a dialysis bag (14 kD), and 2 different buffer solutions were prepared to simulate the normal cell physiological environment (0.5% Tween 80+10. Mu.M GSH, control group) and the tumor cell physiological microenvironment (0.5% Tween 80+10mM GSH), respectively. The buffer was collected at 0, 0.5, 1, 2, 4, 6, 8, 12, 24h with shaking at 37℃and the cumulative release of JQ1-p-VHL was calculated by high performance analytical liquid chromatography.
As shown in FIG. 2, CPEG-PLGA-ssV-JQ1 nano-micelle can realize efficient sustained release of protein degradation agent molecules in the presence of 10mM GSH, which indicates that the micelle can effectively release JQ1-p-VHL molecules under the tumor microenvironment condition, and the accumulated drug release amount can reach 80% at 36 h.
Test example 2: uptake capacity measurement of tumor cells on CPEG-PLGA-ssV-JQ1 nano-micelle and PEG-PLGA-ssV-JQ1 nano-micelle
MDA-MB-231 cells were cultured according to 5X 10 4 Is seeded in 24-well plates and after 24h, is used when the cells are in the logarithmic phase of growth. Fluorescent nanomicelle of CPEG-PLGA-ssV-JQ1 (product of preparation example 5) containing ligand CRGDK prepared in preparation example 7 and PEG-PLGA-ssV containing no ligand CRGDKJQ1 (product of preparation example 2) fluorescent nanomicelle was incubated with cells for 2h, 4h, 8h, 12h and 24h, respectively, after incubation was completed, the cells were washed 3 times with PBS and their fluorescence intensity was then detected using a flow cytometer.
As shown in the test result in FIG. 3, compared with PEG-PLGA-ssV-JQ1 nano-micelle without ligand CRGDK, the uptake of the CPEG-PLGA-ssV-JQ1 nano-micelle by MDA-MB-231 breast cancer cells is obviously increased along with the time extension, which shows that the targeting capability of the nano-micelle to tumors can be effectively improved by the modification of ligand CRGDK, and the targeting of tumor cells of protein degradation agent molecules can be improved.
Test example 3: determination of anti-tumor cell proliferation ability of CPEG-PLGA-ssV-JQ1 nano-micelle
MDA-MB-231 cells were cultured according to 2X 10 3 After 24 hours, CPEG-PLGA-ssV-JQ1 nanomicelle and molecular JQ1-p-VHL prepared in preparation example 6 were incubated with cells at a concentration (0.0001. Mu.M, 0.001. Mu.M, 0.01. Mu.M, 0.1. Mu.M, 0.5. Mu.M and 1.0. Mu.M) for 72 hours, respectively, the medium was removed, washed 3 times with PBS, a medium containing 10% CCK8 was added, and after incubation for a suitable period, absorbance was measured at 450nm with an enzyme-labeled instrument, and cytotoxicity of the nanomicelle was calculated.
As shown in the test result in FIG. 4, CPEG-PLGA-ssV-JQ1 nano micelle shows cytotoxicity to MDA-MB-231 breast cancer cells, which is stronger than JQ1-p-VHL small molecules, and shows that the effective release of JQ1-p-VHL molecules in tumor cells is realized by improving the uptake capacity of the tumor cells to JQ1-p-VHL, thereby being beneficial to the growth and proliferation inhibition of protein degradation agent molecules JQ1-p-VHL to the tumor cells.
Test example 4: determination of BRD4 protein degradation ability of CPEG-PLGA-ssV-JQ1 nano-micelle
MDA-MB-231 cells were cultured according to 5X 10 5 After 24 hours of culture in 6-well plates, the nanomicelle CPEG-PLGA-ssV-JQ1 and the molecule JQ1-p-VHL prepared in preparation example 6 were incubated with cells at a certain concentration for a certain period of time (4 hours and 16 hours: 0.1. Mu.M, 0.5. Mu.M, 1. Mu.M; 24 hours: 0.1. Mu.M, 0.25. Mu.M, 0.5. Mu.M, 1. Mu.M and 5.0. Mu.M), respectively), the medium was removed, and the corresponding cells were collected after washing with PBS. Cell channelAnd (3) obtaining a corresponding protein sample after the lysate is treated, and analyzing the change of the expression quantity of the related protein by a gel electrophoresis mode.
The test results are shown in fig. 5, the nanomicelle (right) has BRD4 protein degradation ability as the protein degradation agent molecule (left), and the protein degradation also has time and concentration dependence.
While the application has been described in terms of the preferred embodiment, it is not intended to limit the scope of the claims, and any person skilled in the art can make many variations and modifications without departing from the spirit of the application, so that the scope of the application shall be defined by the claims.
Sequence listing
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Claims (25)

1. A nano-micelle precursor, characterized in that the nano-micelle precursor comprises a protein degradation agent, a connecting group and a polymer backbone; wherein,
the polymer skeleton is selected from polyethylene glycol or polyethylene glycol derivatives, and comprises at least one of structural units shown in formula i1, formula i2 and formula i 3:
wherein m is an integer from 10 to 145;
q is an integer from 0 to 100;
p is an integer of 0 to 100; and m, q, p are not zero at the same time;
one end of the polymer skeleton is connected with the protein degradation agent through the connecting group in a covalent bond;
the other end of the polymer skeleton is connected with a ligand or a terminal group;
the ligand is selected from ligands targeting tumor cells or tumor matrixes and is selected from targeting peptide molecules of at least one of peptides with amino acid sequences shown as SEQ ID NO. 1-5;
the terminal group is methoxy;
the linking group is selected from the group represented by formula iv1, formula iv2 and formula iv 3;
wherein M is 1 、M 2 、R 3 Each independently selected from alkylene groups having 1 to 4 carbon atoms, R 1 、R 2 Each independently selected from carbonyl, -O-C (O) -, -NH-C (O) -;
the protein degrading agent is selected from protein degrading agent molecules capable of being combined with E3 ubiquitin ligase of Von Hippel-Lindau receptor, and is connected with the connecting group through a covalent bond through the hydroxyl of proline in the protein degrading agent molecules.
2. The nano-micelle precursor according to claim 1, wherein,
m is selected from integers of 100 to 140; q is an integer from 20 to 50; p is an integer of 20 to 50.
3. The nano-micelle precursor according to claim 1, wherein,
m is 113; q is 38; p is 38.
4. The nanomicelle precursor according to claim 1 wherein the ligand comprises a linking group a for coupling to the polymer backbone, the linking group a being for linking to a derivatized functional group residue on the polymer backbone selected from thiol, azide or alkyne functional groups formed upon reaction.
5. The nano-micelle precursor according to claim 1, wherein the polymer backbone has a structural unit represented by formula ii1 or formula ii2,
wherein m, q, p are not zero at the same time or are not 0, R a Represents a residue of a derivatising functional group, R b Represents an end group;
the derivative functional group is used for forming a covalent bond with the ligand, and the derivative functional group residue is a residue after the derivative functional group forms a covalent bond with the ligand.
6. The nano-micelle precursor according to claim 5, wherein,
R a selected from: -NH-,R b Selected from methoxy groups.
7. The nano-micelle precursor according to claim 5, wherein,
m is 113, q is 38, p is 0 or 38.
8. The nanomicelle precursor of claim 1 wherein R 3 is-C (CH) 3 ) 2 -。
9. The nanomicelle precursor of claim 1 wherein M 1 And M is as follows 2 The same applies.
10. The nanomicelle precursor according to claim 1 wherein the linking group is selected from the group represented by the following formula:
11. the nanomicelle precursor of claim 1 wherein R 1 、R 2 Before the reaction, independently selected from-COOH, -NCO, -NH 2 or-OH.
12. The nano-micelle precursor according to claim 1, wherein;
the protein degrading agent is selected from the following molecules:
13. the method of preparing a nano-micelle precursor according to any one of claims 1 to 12, comprising at least the steps of:
reacting the precursor of the linking group with a protein degradation agent molecule to form covalent connection between the precursor and the protein degradation agent molecule and obtain a protein degradation agent molecule with a derivatized functional group;
covalently connecting the protein degradation agent molecules derivatized by the functional groups with a polymer skeleton with terminal groups or an uncapped polymer skeleton to obtain a polymer-protein degradation agent conjugate with terminal groups or an uncapped polymer-protein degradation agent conjugate;
the non-end capped polymer-protein degradation agent conjugate and the ligand are subjected to biological orthogonal reaction or condensation reaction to obtain a product, and the polymer-protein degradation agent conjugate with the terminal group is the nano micelle precursor.
14. The method of claim 13, wherein the unblocked polymer backbone is a polymer backbone having derivatized functional groups.
15. The method of claim 14, wherein the ligand is modified and reacted with a derivatized functional group on the polymer backbone.
16. The method of claim 15, wherein the modification comprises reducing disulfide bonds to produce sulfhydryl groups.
17. A nano-micelle, characterized in that it is self-assembled from the nano-micelle precursor according to any one of claims 1 to 12.
18. The nanomicelle according to claim 17, wherein the average hydrated particle size of the nanomicelle is 10 to 150nm.
19. The nanomicelle according to claim 17, wherein the average hydrated particle size of the nanomicelle is 50 to 60nm.
20. The nanomicelle according to claim 17, wherein the nanomicelle has a dispersibility index of 0.01 to 0.30.
21. The nanomicelle of claim 17 wherein the nanomicelle is further loaded with a fluorescent probe.
22. The method for preparing nano-micelle according to claim 17, comprising at least the steps of:
and dispersing the solution obtained by dissolving the nano micelle precursor in an organic solvent in water, and obtaining the nano micelle through ultrafiltration or dialysis.
23. The method of claim 22, wherein the solution is dispersed in water under ultrasonic conditions.
24. The method according to claim 22, wherein a fluorescent probe is added to the organic solvent.
25. Use of the nanomicelle of claim 17 in the manufacture of a medicament for the treatment of a malignancy selected from breast cancer, cervical cancer, liver cancer, gastric cancer, pancreatic cancer, ovarian cancer, colon cancer or prostate cancer.
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CN109952299A (en) * 2016-09-14 2019-06-28 邓迪大学 It is used to prepare the fluoro hydroxyproline derivative of protein degradation targeting chimera
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CN111646977A (en) * 2020-03-12 2020-09-11 杭州医学院 Compound for target ubiquitination degradation of TGF-beta 1 and preparation method and application thereof

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CN107698657A (en) * 2017-09-26 2018-02-16 中国药科大学 Bifunctional molecule and its preparation and application based on VHL parts and the induction BET degradeds of BET inhibitor
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