Fulvestrant polylysine conjugate

Abstract

Disclosed in the present invention is a fulvestrant polymer conjugate. Specifically, provided in the present invention are a compound represented by formula (I), a preparation method therefor, a pharmaceutical composition containing the compound, and use thereof in the preparation of a drug for treating related diseases such as breast cancer.

Description

fulvestrant polylysine conjugate

[0001] This application claims priority to Chinese patent application 2025102783733, filed on March 10, 2025. The entire contents of the aforementioned Chinese patent application are incorporated herein by reference. Technical Field

[0002] This invention belongs to the field of pharmaceutical technology and relates to a polylysine conjugate containing fulvestrant, its preparation method, and its use in treating cancers such as breast cancer. Background Technology

[0003] Breast cancer is one of the most common malignant tumors among women worldwide, and its incidence is rising year by year. It is also a leading cause of cancer death in women, seriously threatening women's health. Treatment methods for breast cancer include surgery, chemotherapy, radiotherapy, endocrine therapy, targeted therapy, and immunotherapy. Because hormone receptor-positive breast cancer is the most common type, accounting for approximately 60-70% of all breast cancers, and endocrine therapy has fewer adverse reactions, it plays a crucial role in the treatment of hormone receptor (HR)-positive breast cancer.

[0004] Fulvestrant is an endocrine therapy drug, a selective estrogen receptor degrader (SERD). It works by binding to and degrading estrogen receptors (ER), blocking estrogen signaling pathways and thus inhibiting the proliferation of breast cancer cells to achieve therapeutic effects. Fulvestrant injection was launched in the United States in 2002 and has been widely used to treat postmenopausal estrogen receptor-positive (ER+) advanced breast cancer with significant efficacy.

[0005] Although fulvestrant has demonstrated good efficacy in the treatment of breast cancer, it, or its existing formulations, still suffer from the following major drawbacks: High-viscosity injections with castor oil as the main excipient can lead to numerous adverse reactions, including injection site reactions and joint pain. In particular, the intense injection pain caused by high-viscosity, large-volume, and prolonged intramuscular injections severely impacts patients' quality of life and medication adherence. High-viscosity formulations also limit the increase in clinical dosage, thus affecting treatment efficacy. Furthermore, fulvestrant's poor pharmacokinetic properties, low bioavailability, and low tumor tissue delivery efficiency limit its clinical application as a monotherapy.

[0006] To overcome the aforementioned drawbacks, the development of fulvestrant drug conjugates has become a promising drug delivery strategy. Summary of the Invention

[0007] The object of this invention is to provide a drug-polymer conjugate as shown in formula (I):

[0008] Where R is

[0009] n represents the degree of polymerization of polylysine, selected from 25-35;

[0010] m represents the degree of polymerization of polyethylene glycol, selected from 17-25.

[0011] In some embodiments, the drug-polymer conjugate of formula (I) of the present invention is a drug-polymer conjugate of formula (II):

[0012] In some embodiments, the drug-polymer conjugate of formula (I) of the present invention is a drug-polymer conjugate of formula (III):

[0013] In some embodiments, the drug-polymer conjugate of formula (I) of the present invention is a drug-polymer conjugate of formula (IV):

[0014] In some embodiments, n in the drug-polymer conjugate is 25-35, for example 25, 29, 30, 31 or 35.

[0015] In some embodiments, n is 29-31 in the drug-polymer conjugate.

[0016] In some embodiments, m in the drug-polymer conjugate is 17-25, for example 17, 20, 21, 22 or 25.

[0017] In some embodiments, m is 20-22 in the drug-polymer conjugate. In some embodiments, n is 25-35 and m is 17-25 in the drug-polymer conjugate.

[0018] In some embodiments, the drug-polymer conjugate satisfies any of the following conditions: (1) n is 29, m is 21; (2) n is 30, m is 21; (3) n is 31, m is 21; (4) n is 29, m is 20; (5) n is 29, m is 22; (6) n is 25, m is 21; (7) n is 35, m is 21; (8) n is 29, m is 17; (9) n is 29, m is 25; (10) n is 17, m is 25; (11) n is 25, m is 35; (12) n is 17, m is 35; (13) n is 25, m is 35; (14) n is 21, m is 29.

[0019] In some embodiments, a drug-polymer conjugate as shown in formula (III) is used, where n is 29-31 and m is 20-22.

[0020] In some implementations, a drug-polymer conjugate as shown in formula (III) is used, where n is 29 and m is 21.

[0021] In some embodiments, the average nanoparticle size of the drug-polymer conjugate of formula (I) of the present invention ranges from 6 to 30 nanometers.

[0022] In some embodiments, the average nanoparticle size of the drug-polymer conjugate of formula (I) of the present invention ranges from 10 to 20 nanometers.

[0023] In some embodiments, the average nanoparticle size of the drug-polymer conjugate is in the range of 14-16 nanometers.

[0024] In some embodiments, the average nanoparticle size of the drug-polymer conjugate is 15.19 nm, 14.89 nm, or 15.56 nm.

[0025] In some embodiments, the drug-polymer conjugate satisfies any of the following conditions: (1) the average nanoparticle size is 15.19 nm, where n is 29 and m is 21; (2) the average nanoparticle size is 14.89 nm, where n is 30 and m is 21; (3) the average nanoparticle size is 15.56 nm, where n is 31 and m is 21.

[0026] Those skilled in the art should understand that, due to potential process fluctuations during preparation and inherent measurement errors in nanoparticle size detection methods, the average nanoparticle size of the drug-polymer conjugates described in this invention is not strictly limited to the absolute values ​​listed herein. Based on the disclosure of this invention, any particle size deviating from the specific values ​​described herein within ±10% (e.g., due to measurement errors or fluctuations in conventional processes) should be considered to achieve substantially the same function and effect and fall within the protection scope of this invention.

[0027] In some embodiments, in the drug-polymer conjugate, PEG(-(OCH2CH2)) m The molecular weight of OCH3 is 700-1200, for example 750-1150.

[0028] The present invention also provides a method for preparing a drug-polymer conjugate, the method comprising the following route:

[0029] Intermediate A or its stereoisomer and intermediate B are obtained by reacting a condensation reagent and a base in a solvent at a certain temperature and then undergoing some purification methods.

[0030] The present invention also provides a method for preparing a drug-polymer conjugate as described in any one of the present invention, comprising the following steps: in a solvent, in the presence of a condensing agent and a base, intermediate A or its stereoisomer and intermediate B undergo a condensation reaction to obtain the drug-polymer conjugate shown in formula (I);

[0031] In some specific embodiments, the condensing agent in the preparation method includes, but is not limited to, one or more of the following: dicyclohexylcarbodiimide (DCC), N-hydroxybenzotriazole (HOBt), benzotriazole-N,N,N′,N′-tetramethylurea hexafluorophosphate (HBTU), 2-(7-azobenzotriazole)-N,N,N′,N′-tetramethylurea hexafluorophosphate (HATU), N-hydroxy-7-azobenzotriazole (HOAt), and Carter's condensing agent (BOP). reagent), benzotriazine-1-yl-oxytripyrrolidinephosphide hexafluorophosphate (PyBOP), N,N′-diisopropylcarbodiimide (DIC), N,N,N′,N′-tetramethyl-O-(N-succinimide)urea hexafluorophosphate (TSTU), 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine chloride (DMTMM), O-(7-azabenzotriazine-1-yl)-N,N,N′,N′-tetramethylureatetrafluoroboric acid Ester (TATU), (1-cyano-2-ethoxy-2-oxoethyleneaminooxy)dimethylaminomorpholine carbium hexafluorophosphate (COMU), hexafluorophosphate-O-(6-chlorobenzotriazol-1-yl)-N,N,N′,N′-tetramethylurea (HOTU), propyl phosphoric anhydride (T3P), 2-chloro-4,6-dimethoxy-1,3,5-triazine-2-yl-trimethylammonium chloride (TCFH) or PyAOP (7-azabenzotriazol-1-oxo)tripyrrolephos hexafluorophosphate;

[0032] The base includes, but is not limited to, one or more of the following: triethylamine, pyridine, 2,6-dimethylpyridine, N,N-diisopropylethylamine, dimethylaminopyridine, N-methylmorpholine, or N-methylimidazole;

[0033] The solvents include, but are not limited to, one or more of the following: water, methanol, dichloromethane, chloroform, ethanol, N,N-dimethylformamide, dimethyl sulfoxide, N,N-dimethylacetamide, acetonitrile, tetrahydrofuran, methyltetrahydrofuran, acetone, isopropanol, 1,4-dioxane, ethyl acetate or butyl acetate.

[0034] The reaction temperature is -20℃ to 100℃, preferably -5℃ to 50℃;

[0035] The purification methods include, but are not limited to, one or more of the following: recrystallization, pulping, precipitation, membrane separation (including nanofiltration, microfiltration, and ultrafiltration), dialysis, size exclusion chromatography (gel filtration chromatography), or ion exchange chromatography.

[0036] In some embodiments, the condensing agent in the preparation method is N-hydroxybenzotriazole (HOBt) and benzotriazole-1-yl-oxytripyrrolidinephosphine hexafluorophosphate (PyBOP). The base is N-methylmorpholine. The solvent is DMF. The reaction temperature is 20°C.

[0037] The present invention also provides a pharmaceutical composition comprising the compound described in any of the above embodiments and one or more pharmaceutically acceptable carriers, diluents or excipients.

[0038] The present invention also provides a pharmaceutical composition comprising the drug-polymer conjugate described in any one of the present invention and one or more pharmaceutically acceptable carriers, diluents or excipients.

[0039] In some embodiments, the pharmaceutical composition is configured for intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, or local administration.

[0040] In some embodiments, the pharmaceutical composition is configured for intravenous injection, subcutaneous injection, or intramuscular injection.

[0041] In some embodiments, the pharmaceutical composition is in the form of a solution. The solvent for the solution is, for example, physiological saline. The drug concentration of the solution is, for example, 40–60 mg / mL, preferably 50 mg / mL.

[0042] The present invention also provides a pharmaceutical composition comprising one or more additional therapeutic agents.

[0043] The present invention also provides a pharmaceutical combination comprising any of the pharmaceutical-polymer conjugates or pharmaceutical compositions of the present invention, and one or more other therapeutic agents.

[0044] In some embodiments, the other therapeutic agents include: chemotherapeutic agents, CDK inhibitors, PI3K inhibitors, mTOR inhibitors, AKT inhibitors, MEK inhibitors, HDAC inhibitors, HER2 inhibitors, EGFR inhibitors, VEGFR inhibitors, SERD agents, PARP inhibitors, WEE1 inhibitors, ATR inhibitors, PD-1 / PD-L1 inhibitors, KAT6 inhibitors, aromatase inhibitors, KRAS inhibitors, PROTAC agents, monoclonal antibodies, and antibody-drug conjugates (ADCs); wherein chemotherapeutic agents include topoisomerase inhibitors, microtubule inhibitors, alkylating agents, antimetabolites, and anthracycline antibiotics.

[0045] In some implementations, the other therapeutic agent is an anticancer agent.

[0046] In some specific embodiments, the drug-polymer conjugates provided herein may be used alone or in combination with one or more other anticancer agents selected from the following: palbociclib, ribociclib, abemaciclib, trilaciclib, alpelisib, everolimus, letrozole, anastrozole, and capivasert. The following drugs are listed: ib), olaparib, niraparib, rucaparib, talazoparib, ceralasertib (AZD-6738), berzosertib (VE-822), elacestrant, ARV-471, CTx-648 (PF-9363), irinotecan, paclitaxel, docetaxel, cabazitaxel, gemcitabine, doxorubicin, belintecan (BL0020), sacituzumab govitecan, trastuzumab (DS-8201), and trastuzumab emtansine (T-DM1).

[0047] In some embodiments, the anticancer agent is selected from palbociclib and abemaciclib.

[0048] In this invention, those skilled in the art will understand that the components in the pharmaceutical combination can be formulated separately into pharmaceutical compositions, or some or all of them can be formulated together into a pharmaceutical composition for administration; the pharmaceutical compositions can each independently contain a pharmaceutically acceptable carrier, diluent, or excipient. The components in the pharmaceutical combination can be administered individually, or some or all of them can be administered together. The components in the pharmaceutical combination can be administered independently by a variety of suitable routes, including but not limited to oral or parenteral administration (e.g., intravenous injection, subcutaneous injection, or intramuscular injection). The components in the combination of this invention can each be independently a suitable dosage form, including but not limited to injections (intravenous, subcutaneous, or intramuscular) or solutions.

[0049] The present invention also provides the use of any of the above-described drug-polymer conjugates or the above-described pharmaceutical compositions in the preparation of a medicament for treating patients with estrogen receptor-mediated conditions.

[0050] The present invention also provides the use of any of the drug-polymer conjugates, drug compositions, or drug combinations described in any of the present invention in the preparation of medicaments for treating conditions mediated by estrogen receptors.

[0051] The present invention also provides a method for treating estrogen receptor-mediated conditions in a subject in need, comprising: administering to the subject an effective amount of any of the drug-polymer conjugates of the present invention or any of the drug compositions of the present invention or any of the drug combinations of the present invention.

[0052] The present invention also provides a drug-polymer conjugate or a drug composition or combination thereof according to any one of the present invention for treating conditions mediated by estrogen receptors.

[0053] In any aspect or implementation described herein, the disease or condition is cancer.

[0054] In any aspect or implementation described herein, the condition referred to is breast cancer.

[0055] In any aspect or implementation described herein, the breast cancer is MCF-7.

[0056] In some embodiments, the drug-polymer conjugate is administered via, for example, parenteral administration (e.g., subcutaneous, intravenous, or intramuscular injection). Other therapeutic agents are administered via, for example, oral administration.

[0057] In some embodiments, the drug-polymer conjugate is administered, for example, once a week or once every two weeks. Other therapeutic agents are administered, for example, once daily.

[0058] In some embodiments, the drug-polymer conjugate is administered for, for example, at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 6 weeks, or for several months or longer as needed for maintenance therapy. The other therapeutic agents are administered for at least 3 weeks, or for several months or longer as needed for maintenance therapy.

[0059] In some embodiments, the drug-polymer conjugate is administered to mammals that have or are suspected of having an estrogen receptor-mediated condition. The mammals are, for example, humans or non-human mammals. The non-human mammals are, for example, mice (e.g., rats) or dogs (e.g., Beagle dogs).

[0060] In some embodiments, the other therapeutic agents are administered to mammals that have or are suspected of having an estrogen receptor-mediated condition. The mammals are, for example, humans or non-human mammals. The non-human mammals are, for example, mice (e.g., rats) or dogs (e.g., Beagle dogs).

[0061] In some embodiments, when the drug-polymer conjugate is administered to mice, the dosage of the drug-polymer conjugate is from 20 mg / kg body weight per dose to 120 mg / kg body weight per dose, for example from 25 mg / kg body weight per dose to 100 mg / kg body weight per dose, for example 25 mg / kg body weight per dose, 50 mg / kg body weight per dose, or 100 mg / kg body weight per dose. The dosage of the other therapeutic agent is from 20 mg / kg body weight per dose to 40 mg / kg body weight per dose, for example 25 mg / kg body weight per dose or 30 mg / kg body weight per dose.

[0062] In some embodiments, when the drug-polymer conjugate is administered to dogs, the dosage of the drug-polymer conjugate is from 10 mg / kg body weight per dose to 100 mg / kg body weight per dose, for example from 15 mg / kg body weight per dose to 90 mg / kg body weight per dose, for example 15 mg / kg body weight per dose, 45 mg / kg body weight per dose or 90 mg / kg body weight per dose.

[0063] Those skilled in the art will understand that the above-described routes of administration, dosages, frequencies, cycles, and target populations can be combined to form various specific treatment regimens. All regimens combined in the foregoing manner that effectively prevent and / or treat immune-mediated inflammatory diseases are included within the scope of this invention.

[0064] In some embodiments, the drug-polymer conjugate has one or more of the following effects:

[0065] (1) Its water solubility is superior to existing fulvestrant injections (e.g., fulvestrant injections made from castor oil). Product Name: Fushide;

[0066] (2) Drug-polymer conjugates with better water solubility than other PEG polymerization degrees (e.g., drug-polymer conjugates that differ only in PEG polymerization degree, such as m being 11 or 44);

[0067] (3) Its tumor-suppressing effect (e.g., in breast cancer) is superior to existing fulvestrant injections (e.g., fulvestrant injections formulated with castor oil, for example). Product Name: Fushide;

[0068] (4) The antitumor effect (e.g., breast cancer) is superior to drug-polymer conjugates with other PEG polymerization degrees (e.g., drug-polymer conjugates that differ only in PEG polymerization degree, such as m being 11 or 44);

[0069] (5) It has better tumor tissue targeting than existing fulvestrant injections (e.g., fulvestrant injections made from castor oil). Product Name: Fushide;

[0070] (6) The tumor tissue targeting is superior to drug-polymer conjugates with other PEG polymerization degrees (e.g., drug-polymer conjugates that differ only in PEG polymerization degree, such as m being 11 or 44);

[0071] (7) When used in combination with other therapeutic agents (e.g., abecitabine or palbociclib), its antitumor effect (e.g., in breast cancer) is superior to existing fulvestrant injections (e.g., fulvestrant injections in castor oil formulations, for example...). Product Name: Fushide;

[0072] (8) It has low toxicity and high safety.

[0073] Unless otherwise stated, the terms used herein have the following meanings.

[0074] The term "degree of polymerization" refers to the number of repeating units in a polymer. In this invention, the degree of polymerization n refers to the number of repeating units in lysine, and the degree of polymerization m refers to the number of repeating units in ethylene glycol. Unless otherwise specified, both refer to the number-average degree of polymerization, which is the average number of repeating units contained in the polymer macromolecular chain. When the ε-polylysine backbone is synthesized through solid-phase synthesis, the number of repeating units n is a single value. When ε-polylysine is synthesized through bio-fermentation or other chemical polymerization methods, the number of repeating units n has a certain distribution, and its value is expressed as a number average. When the PEG backbone is synthesized stepwise through different glycol raw materials, the number of repeating units m is a single value. However, when synthesized through chemical polymerization, the number of repeating units m has a certain distribution, and its value is expressed as a number average.

[0075] "Pharmaceutical composition" means a mixture containing one or more of the compounds described herein or their physiologically pharmaceutically acceptable salts or prodrugs, along with other chemical components, such as physiologically pharmaceutically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration to a living organism, thereby promoting the absorption of the active ingredient and the exertion of its biological activity.

[0076] Unless otherwise stated, any abbreviations for protecting groups, amino acids and other compounds used in this invention shall be the commonly used and recognized abbreviations, or refer to the IUPAC-IUBC Commission on Biochemical Nomenclature (see Biochem. 1972, 11, 942-944).

[0077] The positive and progressive effects of the present invention are as follows: the fulvestrant drug conjugate of the present invention has one or more advantages such as reducing the viscosity of the formulation, improving pharmacokinetic properties, enhancing tumor tissue targeting, controlling release and improving drug stability, and reducing the pain of administration.

[0078] Our previous work (patent WO2023078464A1) developed a polylysine drug conjugate for precise drug delivery. Building on this technology, we discovered that optimizing the hydrophilic-hydrophobic balance of the delivery system and the drug itself is crucial for fulvestrant delivery. Through extensive experimental research, we optimized the delivery system and developed a novel fulvestrant conjugate by reducing the molecular weight of the hydrophilic polyethylene glycol from ~2K to ~1K. This change resulted in the following unexpected advantages for the entire drug delivery system: 1. Significantly increased drug loading, and unexpectedly, it was found that this compound could achieve the same drug concentration... 1. The formulation exhibits better solubility, allowing for the further development of high-concentration formulations suitable for intramuscular or subcutaneous injection, thus improving patient compliance; 2. Compared to the control compound of PEG2K, the compound of this patent has superior tissue distribution parameters, significantly improving tumor tissue targeting, and unexpectedly, the release rate of the compound in tumor tissue was nearly doubled; 3. Due to the high targeting of the compound of this invention to tumor tissue and drug release specificity on PK, its efficacy in animal models is significantly superior to conjugates with other PEG chain lengths, and the compound of this invention also exhibits significant synergistic effects when used in combination with targeted drugs such as abexilide. It is hoped that the implementation of this patent will provide breast cancer patients with a more efficient, safer, and more compliant treatment option, promoting further development in the field of breast cancer treatment and bringing new research directions to related therapeutic areas. Attached Figure Description

[0079] Figure 1. Pharmacodynamics of compounds 1-5 and control compounds 16 and 17 in a human breast cancer MCF-7 tumor model.

[0080] Figure 2. Plasma drug concentration-time curve of compound 1 in a human breast cancer MCF-7 tumor model.

[0081] Figure 3. Tissue drug concentration-time curve of compound 1 in human breast cancer MCF-7 tumor model.

[0082] Figure 4. Drug concentration-time curve of compound 1 in tumor tissue of human breast cancer MCF-7 tumor model.

[0083] Figure 5. Combined pharmacodynamics of compound 1 and abexicillin in a human breast cancer MCF-7 tumor model.

[0084] Figure 6. Combined pharmacodynamics of compound 1 and abexicillin in a human breast cancer MCF-7 tumor model.

[0085] Figure 7. Combined pharmacodynamics of compound 1 and abexicillin in a human breast cancer MCF-7 tumor model.

[0086] Figure 8. Particle size diagram of the representative compound of the present invention.

[0087] Figure 9. Pharmacodynamics of compound 1 in a human breast cancer MCF-7 tumor model by different administration routes.

[0088] Figure 10. Pharmacodynamics of compound 1 and palbociclib in a human breast cancer MCF-7 tumor model. Detailed Implementation

[0089] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0090] The abbreviations used in this invention are shown in the table below:

[0091] Since the invention has been described according to its specific embodiments, certain modifications and equivalent variations will be apparent to those skilled in the art and are included within the scope of the invention.

[0092] High-performance liquid chromatography (HPLC) analysis was performed using an Agilent 1260 HPLC system.

[0093] The structures of all compounds in this invention can be determined by nuclear magnetic resonance (NMR) or mass spectrometry (MS). NMR shifts (δ) are expressed in 10⁻¹⁰ increments. -6 Units recorded in ppm. NMR measurements were performed using a Bruker Avance-400 spectrometer. The deuterated solvents used in the tests included deuterated chloroform (CDCl3), deuterated methanol (MeOD), deuterated dimethyl sulfoxide (DMSO-D6), and heavy water (D2O), with tetramethylsilane (TMS) as the internal standard.

[0094] Low-resolution mass spectrometry (MS) was performed using an Agilent 6120 quadruple LCMS mass spectrometer.

[0095] Unless otherwise specified in the examples, all reactions can be carried out under argon or nitrogen atmosphere; argon or nitrogen atmosphere refers to the reaction flask being connected to an approximately 1L volume argon or nitrogen balloon; hydrogen atmosphere refers to the reaction flask being connected to an approximately 1L volume hydrogen balloon; the reaction temperature is room temperature, between 20°C and 30°C; the reaction progress in the examples is monitored using thin-layer chromatography (TLC), LCMS, HPLC, or... 1 1H NMR; The developing solvent used in thin-layer chromatography (TLC) is usually a dichloromethane / methanol or petroleum ether / ethyl acetate system, which can be adjusted by adding a small amount of triethylamine, acetic acid or water.

[0096] Alkaline hydrolysis method for drug content: Weigh an appropriate compound and dissolve it in methanol, add 4N NaOH solution, heat to 50-60℃ and shake or stir for 5-6 hours, restore to room temperature, add acetic acid to neutralize, make up to volume with methanol, and perform content test (HPLC) using fulvestrant as standard.

[0097] The degree of polymerization of the polymer of this invention is calculated by nuclear magnetic resonance integration. The NMR integral of the terminal group (such as benzyl, benzoyl, methyl, Boc or acetyl, etc.) is A, the number of hydrogen atoms of the functional group is p, the NMR integral of the α-hydrogen of the amino acid is B, and the degree of polymerization is n = (B*p / A).

[0098] Example 1: Synthesis of Compound 1

[0099] Synthesis of Compound 1-1

[0100] At room temperature (20°C), 200 g (200 mmol, 1.0 eq) of polyethylene glycol monomethyl ether (degree of polymerization m = 21) 1-SM2 was added to a 3 L jacketed flask, and 2 L of water was added and stirred to dissolve. The temperature was then lowered to approximately 5°C. Sodium carbonate (16.96 g, 160 mmol, 0.8 eq), potassium bromide (16.66 g, 140 mmol, 0.7 eq), and TEMPO (1.56 g, 10 mmol, 0.05 eq) were added sequentially, and the mixture was stirred to dissolve for 10 minutes. A sodium hypochlorite solution (≥7.5%, 600 mL, 3.0 eq) was slowly added dropwise to the reaction flask, and the reaction was continued for 2 hours while maintaining an internal temperature of 5°C. The mixture was then slowly raised to room temperature and stirred for 16 hours. The reaction solution was then poured into a sodium bisulfite solution (200 g dissolved in 2.0 L of water) and stirred for 0.5 hours. Extracted with dichloromethane (repeated 3 times, 1 L each time), the organic phase was washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated to give compound 1-1, 195 g, yield 96%, purity 99% as determined by HPLC-CAD.

[0101] Synthesis of Compounds 1-2

[0102] Compound 1-1 (190 g, 187.37 mmol, 1.0 eq) was dissolved in dichloromethane (1.4 L) under nitrogen protection at room temperature, followed by the addition of NHS (32 g, 281.05 mmol, 1.5 eq), and then EDCI (54 g, 281.05 mmol, 1.5 eq) in portions. The reaction mixture was stirred at room temperature for 16 hours. 1 N hydrochloric acid solution (2 L) was added to the reaction mixture, and the mixture was extracted and separated. The organic phase was washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated to give 196 g of waxy solid compound 1-2, in 94% yield.

[0103] Synthesis of compounds 1-3

[0104] ε-poly-L-lysine hydrochloride (degree of polymerization n = 29) (24 g, 145.5 mmol, 1.0 eq, stoichiometry by polymerization unit) was suspended in 500 mL DMSO. Triethylamine (45 g, 436.5 mmol, 3.0 eq) and BOC-LYS(Z)-ONP (109 g, 218.2 mmol) were added. The mixture was stirred at 30 °C for 12 hours under nitrogen protection. 4 L of acetonitrile was added to the reaction mixture, and the mixture was filtered. The filter cake was washed successively with acetonitrile, water, and acetonitrile, and then dried under vacuum to give compounds 1-3 as white solids, 61 g, yield 85%.

[0105] 1 H NMR(400MHz,DMSO-d6)7.74(m,62H),7.27(m,179H),6.90(m,28H),5.16–4.80(s,60H) ,4.31–4.01(br,30H),4.00–3.68(br,30H),3.12–2.82(m,120H),1.91–0.58(m,642H).

[0106] Synthesis of compounds 1-4

[0107] Compounds 1-3 (60 g, 3.97 mmol, 1.0 eq) were added to acetic acid (630 mL) at room temperature, and the mixture was heated to 45 °C and stirred until dissolved. Then, methanol (630 mL) and palladium on carbon (10.2 g, 10 wt%, 60% water content) were added. The mixture was purged with hydrogen three times, and the reaction mixture was stirred at 35 °C for 48 hours. The mixture was filtered through diatomaceous earth, concentrated, and slurried with MTBE to give 55 g of a white solid product, with a yield of 93%.

[0108] 1H NMR (400MHz, D2O) δ4.20–3.98(m,30H),3.88(t,J=7.0Hz,31H),3.04(s,61H),2.87(t,J=7.6Hz,59H),1.84(s,122H),1.78–1.10(m,594H).

[0109] Synthesis of compounds 1-5

[0110] At room temperature, a solution of compound 1-4 (56 g, 134.29 mmol, 1.0 eq) in methanol (1.3 L) and DIPEA (78 g, 604.30 mmol, 4.5 eq) were added to a solution of compound 1-2 (194 g, 174.58 mmol, 1.3 eq) in acetonitrile (680 mL) and methanol (600 mL). The mixture was stirred at room temperature for 16 hours. The reaction solution was concentrated, and the residue was dissolved in methanol (400 mL). The solution was then added dropwise to MTBE and stirred for 1 hour. The supernatant was discarded, and the residue was concentrated under reduced pressure. It was dissolved in methanol:water at a ratio of 1:4 and purified by ultrafiltration (MW 10 kDa, methanol and water system). The concentrated product yielded 153 g of compound 1-5 as an oil, with a yield of 84% and a GPC purity of 99.2%.

[0111] Synthesis of compounds 1-6

[0112] Under nitrogen protection, TFA (500 mL) was added in portions to a DCM (1.5 L) solution of compounds 1-5 (152 g, 112.38 mmol, 1.0 eq). After the addition was complete, the mixture was stirred at room temperature for 16 hours. The reaction solution was concentrated under reduced pressure, dissolved in dichloromethane (1 L), and concentrated under reduced pressure again. This process was repeated three times to obtain 450 g of crude product. MTBE (1.2 L) was added and the mixture was sonicated for 20 minutes. The supernatant was discarded, and this process was repeated three times. The residue was dried by an oil pump to obtain 148 g of oil, with a yield of 96%.

[0113] Synthesis of compounds 1-7

[0114] At room temperature, ethyl acetate (1.5 L) and DIPEA (83 g, 641.1 mmol, 6.0 eq) were added to compounds 1-6 (146 g, 106.85 mmol, 1.0 eq), and the mixture was stirred to dissolve. Succinic anhydride (21 g, 213.7 mmol, 2.0 eq) was then added, and the reaction mixture was stirred at room temperature for 16 hours. The reaction solution was concentrated, dissolved in ethanol:water = 1:9 (total volume 4 L), purified by ultrafiltration (MW 10 kDa, methanol and water system), and concentrated to give compounds 1-7 as a waxy solid, 137 g, yield 87%.

[0115] Synthesis of Compound 1

[0116] At room temperature, compounds 1-7 (10 g, 7.39 mmol, 1.0 eq) were added to DMF (20 mL), followed by a DMF (50 mL) solution of 1-SM3 (synthesized according to compound 40-2 in Example 77 of patent WO2024222960A1) (9.0 g, 8.05 mmol, 1.1 eq). Then, HOBt (1.89 g, 14.04 mmol, 1.9 eq), PyBOP (7.31 g, 14.04 mmol, 1.9 eq), and N-methylmorpholine (3.19 g, 31.55 mmol, 4.27 eq) were added sequentially. The reaction mixture was stirred at 20 °C for 3 hours. The reaction solution was added dropwise to MTBE (500 mL), precipitating out a solid. The mixture was stirred for 0.5 hours, and the solid was collected by filtration as the crude product. The crude product was dissolved in methanol:water = 1:1 (500 mL), filtered, purified by ultrafiltration (MW 30 kDa, methanol and water system), concentrated, and lyophilized to obtain compound 1 as a white solid, 14.7 g, yield 86%, HPLC purity 98.6%, and alkaline hydrolysis yielded fulvestrant with a drug content of 24.0%.

[0117] 1 H NMR (400MHz, DMSO) δ9.96 (s, 31H), 8.40–8.03 (m, 56H), 8.03–7.77 (m, 38H), 7.77–7.39 (m, 95H), 7. 39–7.08(m,98H),6.88–6.59(m,58H),6.02(s,31H),5.46(s,57H),5.13–4.75(m,53H),4.50(s,30 H),4.46–4.30(m,31H),4.29–3.99(m,86H),3.85(s,60H),3.66–3.40(m,2630H),3.23(s,95H),3. 14–2.76(m,568H),2.46–2.16(m,201H),2.07–1.01(m,1370H),0.96–0.73(m,201H),0.66(s,89H).

[0118] Example 2: Synthesis of Compound 2

[0119] Synthesis of Compound 2-1

[0120] ε-poly-L-lysine hydrochloride (degree of polymerization n = 30) 2-SM1 (2.4 g, 14.6 mmol, 1.0 eq, stoichiometry of polymerization units) was suspended in 50 mL of DMSO. Triethylamine (4.5 g, 43.7 mmol, 3.0 eq) and BOC-LYS(Z)-ONP (10.9 g, 21.8 mmol) were added. The mixture was stirred at 30°C for 12 hours under nitrogen protection. 400 mL of acetonitrile was added to the reaction mixture, and the mixture was filtered. The filter cake was washed successively with acetonitrile, water, and acetonitrile, and then dried under vacuum to give compound 2-1 as a white solid, 6.0 g, yield 84%.

[0121] 1 H NMR(400MHz,DMSO-d6)7.74(m,64H),7.27(m,185H),6.90(m,29H),5.16–4.80(s,62H) ,4.31–4.01(br,31H),4.00–3.68(br,31H),3.12–2.82(m,124H),1.91–0.58(m,659H).

[0122] Synthesis of compound 2-2

[0123] Compound 2-1 (6.0 g, 0.4 mmol, 1.0 eq) was added to acetic acid (60 mL) at room temperature, and the mixture was heated to 45 °C and stirred until dissolved. Then, methanol (60 mL) and palladium on carbon (1.0 g, 10 wt%, 60% water content) were added. The mixture was purged with hydrogen three times, and the reaction mixture was stirred at 35 °C for 48 hours. The mixture was filtered through diatomaceous earth, concentrated, and slurried with MTBE to give 5.6 g of a white solid product, 95% yield.

[0124] 1 H NMR (400MHz, D2O) δ4.20–3.98(m,31H),3.88(t,J=7.0Hz,33H),3.04(s,64H),2.87(t,J=7.6Hz,64H),1.84(s,133H),1.78–1.10(m,650H).

[0125] Synthesis of Compounds 2-3

[0126] At room temperature, a solution of compound 2-2 (5.7 g, 13.4 mmol, 1.3 eq) in methanol (150 mL) and DIPEA (7.8 g, 60.4 mmol, 4.5 eq) were added to a solution of compound 1-2 (19.4 g, 17.5 mmol, 1.3 eq) in acetonitrile (70 mL) and methanol (60 mL). The mixture was stirred at room temperature for 16 hours. The reaction solution was concentrated, and the residue was dissolved in methanol (40 mL). This solution was then added dropwise to MTBE and stirred for 1 hour. The supernatant was discarded, and the residue was concentrated under reduced pressure. It was dissolved in methanol:water at a ratio of 1:4 and purified by ultrafiltration (MW 10 kDa, methanol and water system). The concentrated product yielded 15 g of compound 2-3 as an oily product, with a yield of 85% and a GPC purity of 99.5%.

[0127] Synthesis of compounds 2-4

[0128] Under nitrogen protection, TFA (50 mL) was added in portions to a DCM (150 mL) solution of compounds 2-3 (15 g, 11.2 mmol, 1.0 eq). After the addition was complete, the mixture was stirred at room temperature for 16 hours. The reaction solution was concentrated under reduced pressure, dissolved in dichloromethane (100 mL), and concentrated under reduced pressure again, repeated three times. The crude product was added to MTBE (150 mL) and sonicated for 20 minutes. The supernatant was discarded, and this process was repeated three times. The residue was dried by an oil pump to give 14.5 g of an oily substance, with a yield of 95%.

[0129] Synthesis of compounds 2-5

[0130] At room temperature, ethyl acetate (200 mL) and DIPEA (8.5 g, 64.2 mmol, 6.0 eq) were added to compounds 2-4 (14.5 g, 10.7 mmol, 1.0 eq), and the mixture was stirred to dissolve. Succinic anhydride (2.2 g, 21.4 mmol, 2.0 eq) was then added, and the reaction mixture was stirred at room temperature for 16 hours. The reaction solution was concentrated, dissolved in ethanol:water at a ratio of 1:9 (v / v), purified by ultrafiltration (MW 10 kDa, methanol and water system), and concentrated to give compounds 2-5 as a waxy solid, 14.5 g, yield 88%.

[0131] Synthesis of Compound 2

[0132] At room temperature, compound 2-5 (10 g, 7.3 mmol, 1.0 eq) was added to DMF (20 mL), followed by a DMF (50 mL) solution of 1-SM3 (9.0 g, 8.05 mmol, 1.1 eq). Then, HOBt (1.89 g, 14.04 mmol, 1.9 eq), PyBOP (7.31 g, 14.04 mmol, 1.9 eq), and N-methylmorpholine (3.19 g, 31.55 mmol, 4.27 eq) were added sequentially. The reaction mixture was stirred at 20 °C for 3 hours. The reaction solution was added dropwise to MTBE (500 mL), precipitating out a solid. The mixture was stirred for 0.5 hours, and the solid was collected by filtration as the crude product. The crude product was dissolved in methanol:water = 1:1 (500 mL), filtered, purified by ultrafiltration (MW 30 kDa, methanol and water system), concentrated, and lyophilized to obtain compound 2 as a white solid, 13.9 g, yield 84%, HPLC purity 99.6%, and alkaline hydrolysis yielded fulvestrant with a drug content of 23.9%.

[0133] 1 H NMR(400MHz,DMSO)δ9.96(s,32H),8.40–8.03(m,58H),8.03–7.77(m,39H),7.77–7.39(m,98H),7. 39–7.08(m,101H),6.88–6.59(m,60H),6.02(s,32H),5.46(s,59H),5.13–4.75(m,55H),4.50(s,31 H),4.46–4.30(m,32H),4.29–3.99(m,89H),3.85(s,62H),3.66–3.40(m,2705H),3.23(s,98H),3. 14–2.76(m,585H),2.46–2.16(m,207H),2.07–1.01(m,1410H),0.96–0.73(m,207H),0.66(s,92H).

[0134] Example 3: Synthesis of Compound 3

[0135] Synthesis of compound 3-1

[0136] ε-poly-L-lysine hydrochloride (degree of polymerization n = 31) 3-SM1 (2.5 g, 14.5 mmol, 1.0 eq, stoichiometry of polymerization units) was suspended in 50 mL of DMSO. Triethylamine (4.5 g, 43.7 mmol, 3.0 eq) and BOC-LYS(Z)-ONP (10.9 g, 21.8 mmol) were added. The mixture was stirred at 30 °C for 12 hours under nitrogen protection. 500 mL of acetonitrile was added to the reaction mixture, and the mixture was filtered. The filter cake was washed successively with acetonitrile, water, and acetonitrile, and then dried under vacuum to give compound 3-1 as a white solid, 5.6 g, yield 82%.

[0137] 1 H NMR(400MHz,DMSO-d6)7.74(m,66H),7.27(m,190H),6.90(m,30H),5.16–4.80(s,64H) ,4.31–4.01(br,32H),4.00–3.68(br,32H),3.12–2.82(m,127H),1.91–0.58(m,680H).

[0138] Synthesis of compound 3-2

[0139] Compound 3-1 (6.0 g, 0.4 mmol, 1.0 eq) was added to acetic acid (80 mL) at room temperature, and the mixture was heated to 45 °C and stirred until dissolved. Then, methanol (80 mL) and palladium on carbon (1.0 g, 10 wt%, 60% water content) were added. The mixture was purged with hydrogen three times, and the reaction mixture was stirred at 35 °C for 48 hours. The mixture was filtered through diatomaceous earth, concentrated, and slurried with MTBE to give 5.7 g of a white solid product, 96% yield.

[0140] 1 H NMR (400MHz, D2O) δ4.20–3.98(m,32H),3.88(t,J=7.0Hz,34H),3.04(s,66H),2.87(t,J=7.6Hz,66H),1.84(s,137H),1.78–1.10(m,668H).

[0141] Synthesis of compound 3-3

[0142] At room temperature, a solution of compound 3-2 (5.8 g, 13.4 mmol, 1.0 eq) in methanol (150 mL) and DIPEA (7.8 g, 60.4 mmol, 4.5 eq) were added to a solution of compound 1-2 (19.4 g, 17.5 mmol, 1.3 eq) in acetonitrile (70 mL) and methanol (60 mL). The mixture was stirred at room temperature for 16 hours. The reaction solution was concentrated, and the residue was dissolved in methanol (40 mL). This solution was then added dropwise to MTBE and stirred for 1 hour. The supernatant was discarded, and the residue was concentrated under reduced pressure. It was dissolved in methanol:water at a ratio of 1:4 and purified by ultrafiltration (MW 10 kDa, methanol and water system). The concentrated product yielded 16.2 g of compound 3-3 as an oily product, with a yield of 86% and a GPC purity of 99.1%.

[0143] Synthesis of compounds 3-4

[0144] Under nitrogen protection, TFA (50 mL) was added in portions to a DCM (150 mL) solution of compound 3-3 (16 g, 11.2 mmol, 1.0 eq). After the addition was complete, the mixture was stirred at room temperature for 16 hours. The reaction solution was concentrated under reduced pressure, dissolved in dichloromethane (100 mL), and concentrated under reduced pressure again, repeated three times. The crude product was added to MTBE (200 mL) and sonicated for 20 minutes. The supernatant was discarded, and this process was repeated three times. The residue was dried by an oil pump to give 15.0 g of an oily substance, with a yield of 95%.

[0145] Synthesis of compounds 3-5

[0146] At room temperature, ethyl acetate (200 mL) and DIPEA (8.5 g, 64.2 mmol, 6.0 eq) were added to compounds 3-4 (14.8 g, 10.7 mmol, 1.0 eq), and the mixture was stirred to dissolve. Succinic anhydride (2.2 g, 21.4 mmol, 2.0 eq) was then added, and the reaction mixture was stirred at room temperature for 16 hours. The reaction solution was concentrated, dissolved in ethanol:water at a ratio of 1:9 (v / v), purified by ultrafiltration (MW 10 kDa, methanol and water system), and concentrated to give compounds 3-5 as a waxy solid, 15.1 g, yield 92%.

[0147] Synthesis of Compound 3

[0148] At room temperature, compounds 3-5 (10.3 g, 7.3 mmol, 1.0 eq) were added to DMF (20 mL), followed by a DMF (50 mL) solution of 1-SM3 (9.0 g, 8.05 mmol, 1.1 eq). Then, HOBt (1.89 g, 14.04 mmol, 1.9 eq), PyBOP (7.31 g, 14.04 mmol, 1.9 eq), and N-methylmorpholine (3.19 g, 31.55 mmol, 4.27 eq) were added sequentially. The reaction mixture was stirred at 20 °C for 3 hours. The reaction solution was added dropwise to MTBE (500 mL), precipitating out a solid. The mixture was stirred for 0.5 hours, and the solid was collected by filtration as the crude product. The crude product was dissolved in methanol:water = 1:1 (500 mL), filtered, purified by ultrafiltration (MW 30 kDa, methanol and water system), concentrated, and lyophilized to obtain compound 3 as a white solid, 13.9 g, yield 84%, HPLC purity 99.6%, and alkaline hydrolysis yielded fulvestrant with a drug content of 23.5%.

[0149] 1 H NMR (400MHz, DMSO) δ9.96 (s, 33H), 8.40–8.03 (m, 59H), 8.03–7.77 (m, 40H), 7.77–7.39 (m, 100H), 7. 39–7.08(m,103H),6.88–6.59(m,61H),6.02(s,32H),5.46(s,60H),5.13–4.75(m,56H),4.50(s,32 H),4.46–4.30(m,33H),4.29–3.99(m,91H),3.85(s,64H),3.66–3.40(m,2790H),3.23(s,100H),3. 14–2.76(m,602H),2.46–2.16(m,213H),2.07–1.01(m,1452H),0.96–0.73(m,215H),0.66(s,95H).

[0150] Example 4: Synthesis of Compound 4

[0151] Synthesis of compound 4-1

[0152] At room temperature and in an ice-water bath, sodium carbonate (1.7 g, 16.0 mmol, 0.8 eq), potassium bromide (1.7 g, 14.0 mmol, 0.7 eq), and TEMPO (0.16 g, 1.0 mmol, 0.05 eq) were added sequentially to 200 mL of an aqueous solution of polyethylene glycol monomethyl ether (degree of polymerization m = 20) 4-SM (19.2 g, 20 mmol, 1.0 eq), and stirred for 10 minutes to dissolve. Sodium hypochlorite solution (≥7.5%, 60 mL, 3.0 eq) was then slowly added dropwise to the mixture into the reaction flask, and the reaction was continued with stirring at 5°C for 2 hours. The mixture was then allowed to warm naturally to room temperature and stirred for 12 hours. The reaction solution was then poured into a sodium bisulfite solution (20.0 g dissolved in 200 mL of water) and stirred for 0.5 hours. Extracted with dichloromethane (repeated 3 times, 100 mL each time), washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated to give compound 4-1, 19.0 g, yield 97%, purity 98.5% as determined by HPLC-CAD.

[0153] Synthesis of compound 4-2

[0154] Compound 4-1 (19.0 g, 19.5 mmol, 1.0 eq) was dissolved in dichloromethane (150 mL) under nitrogen protection at room temperature. NHS (3.2 g, 28.1 mmol, 1.5 eq) was added, followed by the fractional addition of EDCI (5.4 g, 28.1 mmol, 1.5 eq). The reaction mixture was stirred at room temperature for 12 hours. 200 mL of 1 N hydrochloric acid solution was added to the reaction mixture, and the mixture was extracted and separated. The organic phase was washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated to give 19.7 g of a waxy solid, compound 4-2, in 95% yield.

[0155] Synthesis of compound 4-3

[0156] At room temperature, a solution of compound 1-4 (5.6 g, 13.4 mmol, 1.0 eq) in methanol (150 mL) and DIPEA (7.8 g, 60.4 mmol, 4.5 eq) were added to a solution of compound 4-2 (18.6 g, 17.5 mmol, 1.3 eq) in acetonitrile (60 mL) and methanol (60 mL). The mixture was stirred at room temperature for 12 hours. The reaction solution was concentrated, and the residue was dissolved in methanol (50 mL). The solution was then added dropwise to MTBE and stirred for 1 hour. The supernatant was discarded, and the residue was concentrated under reduced pressure. The residue was dissolved in methanol:water at a ratio of 1:4 and purified by ultrafiltration (MW 10 kDa, methanol and water system). The concentrated product yielded 15.0 g of compound 4-3 as an oily product, with a yield of 83% and a GPC purity of 98.8%.

[0157] Synthesis of compound 4-4

[0158] Under nitrogen protection, TFA (50 mL) was added in portions to a DCM (150 mL) solution of compound 4-3 (14.5 g, 1.0 eq). After the addition was complete, the mixture was stirred at room temperature for 16 hours. The reaction solution was concentrated under reduced pressure, dissolved in dichloromethane (150 mL), and concentrated under reduced pressure again, repeated three times. The crude product was added to MTBE (150 mL) and sonicated for 20 minutes. The supernatant was discarded, and this process was repeated three times. The residue was dried by an oil pump to obtain 14.5 g of an oily substance, with a yield of 95%.

[0159] Synthesis of compounds 4-5

[0160] At room temperature, ethyl acetate (150 mL) and DIPEA (8.3 g, 64.1 mmol, 6.0 eq) were added to compound 4-4 (14.5 g, 10.5 mmol, 1.0 eq), and the mixture was stirred to dissolve. Succinic anhydride (2.1 g, 21.4 mmol, 2.0 eq) was then added, and the reaction mixture was stirred at room temperature for 12 hours. The reaction solution was concentrated, dissolved in ethanol:water = 1:9 (total volume 500 mL), purified by ultrafiltration (MW 10 kDa, methanol and water system), and concentrated to give compound 4-5 as a waxy solid, 14.0 g, yield 93%.

[0161] Synthesis of Compound 4

[0162] At room temperature, compounds 4-5 (10.1 g, 7.3 mmol, 1.0 eq) were added to DMF (20 mL), followed by a DMF (50 mL) solution of 1-SM3 (9.0 g, 8.1 mmol, 1.1 eq). Then, HOBt (1.9 g, 14.0 mmol, 1.9 eq), PyBOP (7.3 g, 14.0 mmol, 1.9 eq), and N-methylmorpholine (3.2 g, 31.6 mmol, 4.3 eq) were added sequentially. The reaction mixture was stirred at 20 °C for 4 hours. The reaction solution was added dropwise to MTBE (500 mL), precipitating out a solid. The mixture was stirred for 0.5 hours, and the solid was collected by filtration as the crude product. The crude product was dissolved in methanol:water = 1:1 (500 mL), filtered, purified by ultrafiltration (MW 30 kDa, methanol and water system), concentrated, and lyophilized to obtain compound 4 as a white solid, 12.8 g, yield 81%, HPLC purity 99.7%, and alkaline hydrolysis yielded fulvestrant with a drug content of 24.1%.

[0163] 1H NMR (400MHz, DMSO) δ9.96 (s, 31H), 8.40–8.03 (m, 56H), 8.03–7.77 (m, 38H), 7.77–7.39 (m, 95H), 7. 39–7.08(m,98H),6.88–6.59(m,58H),6.02(s,31H),5.46(s,57H),5.13–4.75(m,53H),4.50(s,30 H),4.46–4.30(m,31H),4.29–3.99(m,86H),3.85(s,60H),3.66–3.40(m,2505H),3.23(s,95H),3. 14–2.76(m,570H),2.46–2.16(m,202H),2.07–1.01(m,1368H),0.96–0.73(m,202H),0.66(s,90H).

[0164] Example 5: Synthesis of Compound 5

[0165] Synthesis of Compound 5-1

[0166] At room temperature and in an ice-water bath, sodium carbonate (1.7 g, 16.0 mmol, 0.8 eq), potassium bromide (1.7 g, 14.0 mmol, 0.7 eq), and TEMPO (0.16 g, 1.0 mmol, 0.05 eq) were added sequentially to 200 mL of an aqueous solution of polyethylene glycol monomethyl ether (degree of polymerization m = 22) 5-SM (20.9 g, 20 mmol, 1.0 eq), and stirred for 10 minutes to dissolve. Sodium hypochlorite solution (≥7.5%, 60 mL, 3.0 eq) was then slowly added dropwise to the mixture into the reaction flask, and the reaction was continued with stirring at 5°C for 2 hours. The mixture was then allowed to warm naturally to room temperature and stirred for 14 hours. The reaction solution was then poured into a sodium bisulfite solution (20.0 g dissolved in 200 mL of water) and stirred for 0.5 hours. Extracted with dichloromethane (repeated 3 times, 100 mL each time), washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated to give compound 5-1, 19.5 g, yield 92%, purity 97.8% as determined by HPLC-CAD.

[0167] Synthesis of Compound 5-2

[0168] Compound 5-1 (20.5 g, 19.5 mmol, 1.0 eq) was dissolved in dichloromethane (150 mL) under nitrogen protection at room temperature, followed by the addition of NHS (3.2 g, 28.1 mmol, 1.5 eq), and then EDCI (5.4 g, 28.1 mmol, 1.5 eq) in portions. The reaction mixture was stirred at room temperature for 12 hours. 200 mL of 1 N hydrochloric acid solution was added to the reaction mixture, and the mixture was extracted and separated. The organic phase was washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated to give 20.2 g of a waxy solid compound 5-2 (90% yield).

[0169] Synthesis of compound 5-3

[0170] At room temperature, a solution of compound 1-4 (5.6 g, 13.4 mmol, 1.0 eq) in methanol (150 mL) and DIPEA (7.8 g, 60.4 mmol, 4.5 eq) were added to a solution of compound 5-2 (20.2 g, 17.5 mmol, 1.3 eq) in acetonitrile (60 mL) and methanol (60 mL). The mixture was stirred at room temperature for 14 hours. The reaction solution was concentrated, and the residue was dissolved in methanol (50 mL). The solution was then added dropwise to MTBE and stirred for 1 hour. The supernatant was discarded, and the residue was concentrated under reduced pressure. It was dissolved in methanol:water at a ratio of 1:4 and purified by ultrafiltration (MW 10 kDa, methanol and water system). The concentrated product yielded 16.0 g of compound 5-3 as an oil, with a yield of 84% and a GPC purity of 99.2%.

[0171] Synthesis of Compound 5-4

[0172] Under nitrogen protection, TFA (50 mL) was added in portions to a DCM (150 mL) solution of compound 5-3 (15.0 g, 1.0 eq). After the addition was complete, the mixture was stirred at room temperature for 16 hours. The reaction solution was concentrated under reduced pressure, dissolved in dichloromethane (150 mL), and concentrated under reduced pressure again, repeated three times. The crude product was added to MTBE (150 mL) and sonicated for 20 minutes. The supernatant was discarded, and this process was repeated three times. The residue was dried by an oil pump to obtain 14.8 g of an oily substance, with a yield of 93%.

[0173] Synthesis of compound 5-5

[0174] At room temperature, ethyl acetate (150 mL) and DIPEA (8.3 g, 64.1 mmol, 6.0 eq) were added to compound 5-4 (14.8 g, 10.5 mmol, 1.0 eq), and the mixture was stirred to dissolve. Succinic anhydride (2.1 g, 21.4 mmol, 2.0 eq) was then added, and the reaction mixture was stirred at room temperature for 12 hours. The reaction solution was concentrated, dissolved in ethanol:water = 1:9 (total volume 500 mL), purified by ultrafiltration (MW 10 kDa, methanol and water system), and concentrated to give compound 5-5 as a waxy solid, 14.3 g, yield 92%.

[0175] Synthesis of Compound 5

[0176] At room temperature, compound 5-5 (10.5 g, 7.3 mmol, 1.0 eq) was added to DMF (20 mL), followed by a DMF (50 mL) solution of 1-SM3 (9.0 g, 8.1 mmol, 1.1 eq). Then, HOBt (1.9 g, 14.0 mmol, 1.9 eq), PyBOP (7.3 g, 14.0 mmol, 1.9 eq), and N-methylmorpholine (3.2 g, 31.6 mmol, 4.3 eq) were added sequentially. The reaction mixture was stirred at 20 °C for 4 hours. The reaction solution was added dropwise to MTBE (500 mL), precipitating out a solid. The mixture was stirred for 0.5 hours, and the solid was collected by filtration as the crude product. The crude product was dissolved in methanol:water = 1:1 (500 mL), filtered, purified by ultrafiltration (MW 30 kDa, methanol and water system), concentrated, and lyophilized to obtain compound 5 as a white solid, 13.4 g, yield 83%, HPLC purity 99.2%, and alkaline hydrolysis yielded fulvestrant with a drug content of 24.2%.

[0177] 1 H NMR (400MHz, DMSO) δ9.96 (s, 31H), 8.40–8.03 (m, 56H), 8.03–7.77 (m, 38H), 7.77–7.39 (m, 95H), 7. 39–7.08(m,98H),6.88–6.59(m,58H),6.02(s,31H),5.46(s,57H),5.13–4.75(m,53H),4.50(s,30 H),4.46–4.30(m,31H),4.29–3.99(m,86H),3.85(s,60H),3.66–3.40(m,2760H),3.23(s,95H),3. 14–2.76(m,568H),2.46–2.16(m,201H),2.07–1.01(m,1370H),0.96–0.73(m,201H),0.66(s,89H).

[0178] Examples 6-13: Synthesis of Compounds 6-13

[0179] Following the synthesis described in Examples 1-5, synthesis was carried out using different raw materials:

[0180] Example 14: Synthesis of Compound 14

[0181] Synthesis of Compound 14-1

[0182] ε-poly-L-lysine hydrochloride (degree of polymerization n = 29) 1-SM2 (2.4 g, 14.5 mmol, 1.0 eq, stoichiometry of polymerization units) was suspended in 50 mL of DMSO. Triethylamine (4.5 g, 43.7 mmol, 3.0 eq) and ZD-Lys(Boc)-ONP (10.9 g, 21.8 mmol) were added. The reaction mixture was stirred at 30°C for 12 hours under nitrogen protection. 500 mL of acetonitrile was added to the reaction mixture, and the mixture was filtered. The filter cake was washed successively with acetonitrile, water, and acetonitrile, and then dried under vacuum to give compound 14-1 as a white solid, 5.8 g, yield 83%.

[0183] 1 H NMR(400MHz,DMSO-d6)7.74(m,62H),7.27(m,178H),6.90(m,28H),5.18–4.75(s,60H) ,4.29–4.05(br,30H),4.00–3.65(br,30H),3.12–2.82(m,118H),1.91–0.58(m,638H).

[0184] Synthesis of Compound 14-2

[0185] Compound 14-1 (5.9 g, 0.4 mmol, 1.0 eq) was added to acetic acid (60 mL) at room temperature, and the mixture was heated to 45 °C and stirred until dissolved. Then, methanol (60 mL) and palladium on carbon (1.0 g, 10 wt%, 60% water content) were added. The mixture was purged with hydrogen three times, and the reaction mixture was stirred at 35 °C for 45 hours. The mixture was filtered through diatomaceous earth, concentrated, and slurried with MTBE to give 5.5 g of a white solid product, 94% yield.

[0186] Synthesis of Compound 14-3

[0187] At room temperature, a solution of compound 14-2 (0.6 g, 13.4 mmol, 1.0 eq) in methanol (20 mL) and DIPEA (0.8 g, 6.0 mmol, 4.5 eq) were added to a solution of compound 1-2 (2.0 g, 1.8 mmol, 1.3 eq) in acetonitrile (20 mL) and methanol (15 mL). The mixture was stirred at room temperature for 16 hours. The reaction solution was concentrated, and the residue was dissolved in methanol (10 mL). The solution was then added dropwise to MTBE and stirred for 1 hour. The supernatant was discarded, and the residue was concentrated under reduced pressure. It was dissolved in methanol:water at a ratio of 1:4 and purified by ultrafiltration (MW 10 kDa, methanol and water system). The concentrated product yielded 1.6 g of compound 2-3 as an oily product, with a yield of 90% and a GPC purity of 99.0%.

[0188] Synthesis of compound 14-4

[0189] Under nitrogen protection, TFA (5 mL) was added in portions to a DCM (15 mL) solution of compound 14-3 (1.5 g, 1.1 mmol, 1.0 eq). After the addition was complete, the mixture was stirred at room temperature for 12 hours. The reaction solution was concentrated under reduced pressure, dissolved in dichloromethane (10 mL), and concentrated under reduced pressure again, repeated three times. MTBE (20 mL) was added to the crude product and sonicated for 10 minutes. The supernatant was discarded, and this process was repeated three times. The residue was dried by an oil pump to obtain 1.5 g of an oily substance, with a yield of 96%.

[0190] Synthesis of Compound 14-5

[0191] At room temperature, ethyl acetate (20 mL) and DIPEA (0.9 g, 6.4 mmol, 6.0 eq) were added to compound 14-4 (1.5 g, 1.1 mmol, 1.0 eq), and the mixture was stirred to dissolve. Succinic anhydride (0.2 g, 2.1 mmol, 2.0 eq) was then added, and the reaction mixture was stirred at room temperature for 12 hours. The reaction solution was concentrated, dissolved in ethanol:water at a ratio of 1:9 (v / v), purified by ultrafiltration (MW 10 kDa, methanol and water system), and concentrated to give compound 14-5 as a waxy solid, 1.4 g, yield 85%.

[0192] Synthesis of Compound 14

[0193] At room temperature, compound 14-5 (1.0 g, 0.7 mmol, 1.0 eq) was added to DMF (5 mL), followed by a DMF (5 mL) solution of 1-SM3 (0.9 g, 0.8 mmol, 1.1 eq). Then, HOBt (0.19 g, 1.4 mmol, 1.9 eq), PyBOP (0.7 g, 1.4 mmol, 1.9 eq), and N-methylmorpholine (0.3 g, 3.0 mmol, 4.0 eq) were added sequentially. The reaction mixture was stirred at 20 °C for 3 hours. The reaction solution was added dropwise to MTBE (50 mL), precipitating out a solid. The mixture was stirred for 0.5 hours, and the solid was collected by filtration as the crude product. The crude product was dissolved in methanol:water = 1:1 (50 mL), filtered, purified by ultrafiltration (MW 30 kDa, methanol and water system), concentrated, and lyophilized to obtain compound 14 as a white solid, 1.2 g, yield 81%, HPLC purity 98.8%, and alkaline hydrolysis yielded fulvestrant with a drug content of 23.8%.

[0194] Example 15: Synthesis of Compound 15

[0195] Synthesis of Compound 15-1

[0196] ε-polylysine hydrochloride (degree of polymerization n = 29) 2-SM1 (2.4 g, 14.5 mmol, 1.0 eq, stoichiometry of polymerization units) was suspended in 50 mL of DMSO. Triethylamine (4.5 g, 43.7 mmol, 3.0 eq) and BOC-LYS(Z)-ONP (10.9 g, 21.8 mmol) were added. The mixture was stirred at 30°C for 12 hours under nitrogen protection. 400 mL of acetonitrile was added to the reaction mixture, and the mixture was filtered. The filter cake was washed successively with acetonitrile, water, and acetonitrile, and then dried under vacuum to give compound 15-1 as a white solid, 6.1 g, yield 84%.

[0197] 1 H NMR(400MHz,DMSO-d6)7.74(m,63H),7.27(m,185H),6.90(m,29H),5.16–4.80(s,62H) ,4.32–4.01(br,31H),4.00–3.68(br,31H),3.12–2.82(m,122H),1.91–0.58(m,658H).

[0198] Synthesis of Compound 15-2

[0199] Compound 2-1 (6.0 g, 0.4 mmol, 1.0 eq) was added to acetic acid (60 mL) at room temperature, and the mixture was heated to 45 °C and stirred until dissolved. Then, methanol (60 mL) and palladium on carbon (1.0 g, 10 wt%, 60% water content) were added. The mixture was purged with hydrogen three times, and the reaction mixture was stirred at 35 °C for 48 hours. The mixture was filtered through diatomaceous earth, concentrated, and slurried with MTBE to give 5.8 g of a white solid product, 96% yield.

[0200] Synthesis of Compound 15-3

[0201] At room temperature, a solution of compound 15-2 (5.7 g, 13.4 mmol, 1.0 eq) in methanol (150 mL) and DIPEA (7.8 g, 60.4 mmol, 4.5 eq) were added to a solution of compound 1-2 (19.4 g, 17.5 mmol, 1.3 eq) in acetonitrile (60 mL) and methanol (60 mL). The mixture was stirred at room temperature for 16 hours. The reaction solution was concentrated, and the residue was dissolved in methanol (50 mL). The solution was then added dropwise to MTBE and stirred for 1 hour. The supernatant was discarded, and the residue was concentrated under reduced pressure. The residue was dissolved in methanol:water at a ratio of 1:4 and purified by ultrafiltration (MW 10 kDa, methanol and water system). The concentrated product yielded 15-3 as an oily product, 15.1 g, with a yield of 85% and a GPC purity of 99.2%.

[0202] Synthesis of Compound 15-4

[0203] Under nitrogen protection, TFA (50 mL) was added in portions to a DCM (150 mL) solution of compound 15-3 (15.0 g, 11.2 mmol, 1.0 eq). After the addition was complete, the mixture was stirred at room temperature for 16 hours. The reaction solution was concentrated under reduced pressure, dissolved in dichloromethane (100 mL), and concentrated under reduced pressure again, repeated three times. The crude product was added to MTBE (150 mL) and sonicated for 20 minutes. The supernatant was discarded, and this process was repeated three times. The residue was dried by an oil pump to give 14.0 g of an oily substance, with a yield of 94%.

[0204] Synthesis of Compound 15-5

[0205] At room temperature, ethyl acetate (200 mL) and DIPEA (8.2 g, 64.0 mmol, 6.0 eq) were added to compound 15-4 (14.0 g, 10.6 mmol, 1.0 eq), and the mixture was stirred to dissolve. Succinic anhydride (2.1 g, 21.0 mmol, 2.0 eq) was then added, and the reaction mixture was stirred at room temperature for 16 hours. The reaction solution was concentrated, dissolved in ethanol:water at a ratio of 1:9 (v / v), purified by ultrafiltration (MW 10 kDa, methanol and water system), and concentrated to give compound 15-5 as a waxy solid, 13.7 g, yield 86%.

[0206] Synthesis of Compound 15

[0207] At room temperature, compound 15-5 (10.0 g, 7.3 mmol, 1.0 eq) was added to DMF (20 mL), followed by a DMF (50 mL) solution of 1-SM3 (9.0 g, 8.05 mmol, 1.1 eq). Then, HOBt (1.89 g, 14.04 mmol, 1.9 eq), PyBOP (7.31 g, 14.04 mmol, 1.9 eq), and N-methylmorpholine (3.19 g, 31.55 mmol, 4.27 eq) were added sequentially. The reaction mixture was stirred at 20 °C for 5 hours. The reaction solution was added dropwise to MTBE (500 mL), precipitating out a solid. The mixture was stirred for 1 hour, and the solid was collected by filtration as the crude product. The crude product was dissolved in methanol:water = 1:1 (500 mL), filtered, purified by ultrafiltration (MW 30 kDa, methanol and water system), concentrated, and lyophilized to obtain compound 15 as a white solid, 12.8 g, yield 80%, HPLC purity 98.6%, and alkaline hydrolysis yielded fulvestrant with a drug content of 24.2%.

[0208] 1 H NMR(400MHz,DMSO)δ9.95(s,32H),8.40–8.03(m,57H),8.03–7.77(m,39H),7.78–7.39(m,96H),7. 40–7.08(m,100H),6.88–6.60(m,60H),6.02(s,32H),5.46(s,59H),5.13–4.75(m,55H),4.50(s,31 H),4.46–4.30(m,32H),4.29–3.99(m,89H),3.85(s,62H),3.64–3.42(m,2698H),3.23(s,98H),3. 15–2.75(m,590H),2.47–2.15(m,206H),2.08–1.02(m,1398H),0.98–0.73(m,208H),0.67(s,90H).

[0209] Synthesis of Compound 16

[0210] Using raw materials PEG2K-NHS and 1-SM2, compound 16 was synthesized according to compound 40 in Example 77 of patent WO2024222960A1, with a purity of 99.1%. Alkaline hydrolysis yielded fulvestrant with a content of 16.1%.

[0211] Synthesis of Compound 17

[0212] Using the raw materials PEG550-NHS and 1-SM2, compound 17 was obtained by following the synthesis method of compound 16. The purity was 98.2%, and the fulvestrant content was 27.5% after alkaline hydrolysis.

[0213] Evaluation Example 1: Formulation Development Study of Compounds 1-5 and Comparative Compounds 16 and 17

[0214] Experimental protocol: Prepare 5 mL of the drug formulation, using physiological saline as an excipient. The concentration of fulvestrant injection is set to 50 mg / mL. Calculate and weigh the corresponding compounds 1-5 and control compounds 16 and 17 (weights shown in Table 1) according to different drug contents. Add 4.5 mL of physiological saline. Heat the mixture to 30-40℃ and shake or stir for 2 hours. Then, make up to the target volume of 5 mL. Visually check whether it is completely dissolved. Take samples of the dissolved compounds, filter them, and perform HPLC analysis to determine the content, in order to examine the feasibility of the physiological saline formulation.

[0215] Test results: The formulation studies of the compounds of this invention are shown in Table 1.

[0216] Table 1. Results of research on saline formulations of the compounds of this invention.

[0217] The solubility study results in Table 1 show that the representative compound of this invention has a higher drug loading (>23%), significantly greater than that of the comparative compound 16 (16.1%). When preparing injection solutions with the same drug concentration (50 mg / mL), all the representative compounds of this invention were completely dissolved. Surprisingly, the comparative compound 16 (PEG degree of polymerization m = 44, PEG molecular weight 2K), with a longer PEG chain, was not completely dissolved, as was compound 17 (PEG degree of polymerization m = 11, PEG molecular weight 550).

[0218] The experimental results show that the compound of this invention has the following significant advantages in formulation development: 1. In clinical practice, fulvestrant injection is administered via intramuscular injection, requiring a dose of up to 500 mg (dissolved in 10 ml of mixed organic solvent) to achieve a certain clinical effect. Due to the limited injection volume of high-viscosity formulations, the dosage cannot be increased. Therefore, a high drug loading can maximize clinical efficacy. The drug loading of the compound of this invention is significantly greater than that of other compounds, allowing for the preparation of high-concentration formulations for various injection methods, such as intravenous, intramuscular, and subcutaneous injections; 2. The compound of this invention has good water solubility, lower viscosity than fulvestrant injection prepared from castor oil, lower toxicity, fewer side effects, and better patient compliance.

[0219] Evaluation Example 2: Pharmacodynamic study of compounds 1-5 and comparative compounds 16 and 17 in a nude mouse model of human breast cancer cells MCF-7.

[0220] Laboratory animals and inoculation methods:

[0221] BALB / c nude mice, female, 4-5 weeks old, weighing 20-25 grams, were housed in the experimental environment for one week after arrival. Estrogen-releasing microspheres were subcutaneously injected into the midline of the back of each mouse. Two days later, an MCF-7 tumor was transplanted in situ into the right second nipple fat pad. The tumors were allowed to grow to an average volume of 160 mm². 3 At approximately 10:00 AM, mice were randomly divided into groups of 6 mice each, based on tumor size and mouse weight.

[0222] Sample preparation:

[0223] Prepare a saline injection solution of compound 1 with a drug concentration of 50 mg / mL. The control fulvestrant injection solution is... (Product name: Fushide).

[0224] Dosage of test drug:

[0225] The dosage and administration regimen are shown in Table 2. The subcutaneous tumor volume of nude mice was measured 2-3 times per week (the formula for calculating tumor volume is: V = 0.5a × b). 2 (where a and b represent the long and short diameters of the tumor, respectively). Weigh the rat and record the data.

[0226] Table 2 Dosage Regimen

[0227] Evaluation indicators: The relative tumor proliferation rate (T / C) (%) or tumor growth inhibition rate (TGI) (%) were used for evaluation, where T represents the experimental group and C represents the control group.

[0228] The formula for calculating the relative tumor proliferation rate T / C (%) is as follows: T / C% = T RTV / C RTV ×100% (T) RTV : Mean RTV of the treatment group; C RTV (Negative control group RTV mean). The relative tumor volume (RTV) was calculated based on tumor measurements using the formula: RTV = V0 / V0. t / V0, where V0 is the tumor volume measured at the time of group administration (i.e., d0), V t T represents the tumor volume at a given measurement. RTV With C RTV Take data from the same day.

[0229] Tumor inhibition rate (TGI) (%) = (1 - T / C) × 100%.

[0230] The evaluation criteria were as follows: T / C (%) > 40 was considered invalid; T / C (%) ≤ 40, and after statistical processing, P < 0.05 was considered valid.

[0231] Results of the pharmacodynamic experiment:

[0232] Figure 1 shows the inhibitory effects of compounds 1-5 and control compounds 16 and 17 on a nude mouse model of human breast cancer cells MCF-7.

[0233] The experimental results are shown in Figure 1. Subcutaneous injection was administered once weekly for a total of two weeks. After 28 days of observation, the tumor inhibition rates of compounds 1-5 at a dose of 50 mg / kg were 88.65%, 89.90%, 91.51%, 93.15%, and 94.04%, respectively, which were significantly different from the blank control group and significantly superior to the fulvestrant injection (Fusend) group (dose of 100 mg / kg, tumor inhibition rate of 41.76%). Unexpectedly, they were also superior to control compound 16 (tumor inhibition rate of 70.66%, PEG2K) and control compound 17 (tumor inhibition rate of 60.07%, PEG550). The results demonstrate that the compounds of this invention exhibit extremely strong tumor-inhibiting activity in this model. Compound 1 of this invention, containing PEG1K, unexpectedly showed superior efficacy compared to control compounds 16 (containing PEG2K) and 17 (containing PEG550).

[0234] Evaluation Example 3: Tissue distribution of compound 1 in NOD / SCID mice after orthotopic xenografting of human breast cancer cell line MCF-7.

[0235] Laboratory animals and inoculation methods:

[0236] NOD / SCID mice, female, 5-6 weeks old, with an average weight >16 grams, underwent subcutaneous implantation of estrogen-releasing microspheres in their backs. The following day, tumors with a diameter of 2-3 mm were inoculated subcutaneously into the right breast pad of the NOD / SCID mice. Tumors were allowed to grow to an average volume of 440 mm². 3 At approximately 10:00 AM, mice were randomly divided into groups of 24 each, based on tumor size and mouse weight.

[0237] Sample preparation:

[0238] Prepare a saline injection solution of compound 1 with a drug concentration of 50 mg / mL. The control fulvestrant injection solution is... (Product name: Fushide).

[0239] Dosage of test drug:

[0240] The dosage and administration regimen are shown in Table 3.

[0241] Table 3. Dosage regimens for Compound 1 and fulvestrant injection. Note: The injection site is the back of the mouse.

[0242] Experimental methods:

[0243] Blood and tumor samples were collected at 3h, 8h, 24h, 48h, 72h, 168h, 240h, and 336h after drug administration. Three mice were euthanized at each time point. Tumors were flash-frozen and blood samples were placed in anticoagulant tubes and immediately placed on ice. The tubes were centrifuged at 4°C for 10 minutes, and plasma was collected. All plasma samples were stored in a freezer at approximately -80°C until LCMS / MS analysis.

[0244] Experimental results:

[0245] The tissue distribution of compound 1 and fulvestrant injection in a human breast cancer cell MCF-7 nude mouse tumor model is shown in Figures 2-4.

[0246] Table 4. Tissue distribution AUC parameters of compound 1

[0247] The tissue distribution parameters of Compound 1 injection and the reference formulation fulvestrant injection (Fusect) are shown in Table 4, and the drug concentration curves are shown in Figures 2-4. The tissue distribution results of a single subcutaneous injection of the same dose (100 mg / kg) in mice showed:

[0248] 1. Compound 1 is highly stable in plasma, with a fulvestrant release rate of only 0.08% (Figure 2);

[0249] 2. Compound 1 has the property of being highly enriched in tumor tissue, with the total fulvestrant in the tumor accounting for as much as 42% of the total in the plasma (Figure 3).

[0250] 3. Compound 1 exhibits highly specific release, with free fulvestrant accounting for up to 38.7% of the release in the tumor microenvironment (Figure 4);

[0251] 4. Compared to the reference fulvestrant injection (Fusted), compound 1 released a fulvestrant drug concentration (AUC) in the tumor that was 140 times higher (Figure 4).

[0252] Meanwhile, the tissue distribution of compound 16 in tumor-bearing mice was also studied under the same conditions, and the comparison with compound 1 is shown in Table 5 below.

[0253] Table 5. Comparison of tissue distribution of compound 1 and control compound 16 in tumor-bearing mice.

[0254] As can be seen from the comparison table 5, compound 1 with PEG1K has better tumor tissue targeting than the control compound with PEG2K, and the drug release rate in tumor tissue is also higher. This shows that compound 1 has better pharmacokinetic parameters, thereby improving efficacy.

[0255] In summary, compound 1 exhibits high stability in blood circulation and high targeting and specificity to tumor tissues, demonstrating a significant advantage in tissue distribution compared to fulvestrant injection (Fustrasin) and other comparative compounds.

[0256] Evaluation Example 4: Pharmacodynamic study of the combination of compound 1 and abemaciclib in a nude mouse model of human breast cancer cells MCF-7.

[0257] Laboratory animals and inoculation methods:

[0258] BALB / c nude mice, female, 4-5 weeks old, weighing 20-25 grams, were housed in the experimental environment for one week after arrival. Estrogen-releasing microspheres were subcutaneously injected into the midline of the back of each mouse. Two days later, an MCF-7 tumor was transplanted in situ into the right second nipple fat pad. The tumors were allowed to grow to an average volume of 440 mm². 3 At approximately 10:00 AM, mice were randomly divided into groups of 9 mice each, based on tumor size and mouse weight.

[0259] Sample preparation:

[0260] Prepare a saline injection solution of compound 1 with a drug concentration of 50 mg / mL. The control fulvestrant injection solution is... (Product name: Fushide).

[0261] Abemaciclib solvent preparation: Weigh 500 mg of lactic acid and dissolve it in 80 mL of sterile distilled water. After vortexing, adjust the pH to 4.0 ± 0.1 with 2N NaOH solution, and finally bring the volume to 100 mL to obtain a 50 mmol / L sodium lactate solution. Weigh 283.5 mg of abemaciclib powder, add 94.5 mL of 50 mmol / L sodium lactate solution, stir to dilute, vortex, and sonicate to dissolve.

[0262] Dosage of test drug:

[0263] The dosage and administration regimen are shown in Table 6. The subcutaneous tumor volume of nude mice was measured 2-3 times per week (the formula for calculating tumor volume is: V = 0.5a × b). 2 (where a and b represent the long and short diameters of the tumor, respectively). Weigh the rat and record the data.

[0264] Table 6 Combined Dosing Regimens

[0265] Experimental evaluation indicators: Same as those for Example 2.

[0266] Results of the pharmacodynamic experiment:

[0267] Figure 5 shows the combined pharmacodynamic effects of high-dose compound 1, fulvestrant injection, and abemaciclib in a human breast cancer cell MCF-7 mouse tumor model.

[0268] The experimental results are shown in Figure 5. In the single-drug group, compound 1 and fulvestrant injection were administered subcutaneously once every two weeks for a total of two administrations. In the abexicillin group, oral administration was administered daily for 21 days. At day 28, the tumor inhibition rate of compound 1 (dose 100 mg / kg) was 90.44%, which was significantly different from the blank control group and significantly superior to the fulvestrant injection (Fushide) group (dose 100 mg / kg, tumor inhibition rate 41.98%) and the abexicillin group (dose 30 mg / kg, tumor inhibition rate 16.97%). The combination therapy groups also showed a superior synergistic effect. The tumor inhibition rate of compound 1 combined with abexicillin was as high as 93.07%, while the tumor inhibition rate of fulvestrant (Fushide) combined with abexicillin was only 68.61%.

[0269] Evaluation Example 5: Pharmacodynamic study of the combination of compound 1 and abemaciclib in a large tumor model of human breast cancer cells MCF-7 in nude mice.

[0270] Laboratory animals and inoculation methods:

[0271] BALB / c nude mice, female, 4-5 weeks old, weighing 20-25 grams, were housed in the experimental environment for one week after arrival. Estrogen-releasing microspheres were subcutaneously injected into the midline of the back of each mouse. Two days later, an MCF-7 tumor was transplanted in situ into the right second nipple fat pad. The tumors were allowed to grow to an average volume of 850 mm². 3 At approximately 10:00 AM, mice were randomly divided into groups of 6 mice each, based on tumor size and mouse weight.

[0272] Sample preparation: Same as in Evaluation Example 4

[0273] Dosage of test drug:

[0274] The dosage and administration regimen are shown in Table 7. The subcutaneous tumor volume of nude mice should be measured 2-3 times per week (the formula for calculating tumor volume is: V = 0.5a × b). 2 (where a and b represent the long and short diameters of the tumor, respectively). Weigh the rat and record the data.

[0275] Table 7 Combined Dosing Regimens

[0276] Experimental evaluation indicators: Same as those for Example 2.

[0277] Results of the pharmacodynamic experiment:

[0278] The combined pharmacodynamic effects of medium-dose compound 1, fulvestrant injection (Fusted), and abemaciclib in a large human breast cancer cell MCF-7 mouse model are shown in Figure 6.

[0279] The experimental results are shown in Figure 6. In the single-drug group, compound 1 and fulvestrant injection were administered subcutaneously once every two weeks for a total of two administrations. In the abexicillin group, oral administration was given daily for 21 days. At day 22, compound 1 (50 mg / kg) showed a tumor inhibition rate of 70.72% in the large tumor model, which was significantly different from the blank control group and significantly superior to the fulvestrant injection group (100 mg / kg, tumor inhibition rate 56.23%) and the abexicillin group (30 mg / kg, tumor inhibition rate 7.54%). The tumor inhibition rate of compound 1 (50 mg / kg) combined with abexicillin increased to 85.93%, significantly better than the efficacy of the fulvestrant combined with abexicillin group (tumor inhibition rate 64.21%).

[0280] Evaluation Example 6: Combined pharmacodynamic study of compound 1 and abemaciclib in a female NOD / SCID mouse model of orthotopic xenograft of human breast cancer cell line MCF-7.

[0281] Laboratory animals and inoculation methods:

[0282] NOD / SCID mice, female, 5-6 weeks old, with an average weight >16 grams, had estrogen-releasing microspheres implanted subcutaneously in their backs. The next day, tumors with a diameter of 2-3 mm were inoculated subcutaneously into the right breast pad of the NOD / SCID mice. The tumors were allowed to grow to an average volume of 150 mm². 3 At approximately 10:00 AM, mice were randomly divided into groups of 9 mice each, based on tumor size and mouse weight.

[0283] Sample preparation: Same as in evaluation example 4.

[0284] Dosage of test drug:

[0285] The dosage and administration regimen are shown in Table 8. The subcutaneous tumor volume of nude mice should be measured 2-3 times per week (the formula for calculating tumor volume is: V = 0.5a × b). 2 (where a and b represent the long and short diameters of the tumor, respectively). Weigh the rat and record the data.

[0286] Table 8 Combined Dosing Regimens

[0287] Experimental evaluation indicators: Same as those for Example 2.

[0288] Results of the pharmacodynamic experiment:

[0289] Figure 7 shows the combined pharmacodynamic effects of low-dose compound 1, fulvestrant injection, and abemaciclib in a human breast cancer cell MCF-7 mouse tumor model.

[0290] The experimental results are shown in Figure 7. In the low-dose monotherapy group, compound 1 and fulvestrant injection were administered subcutaneously once every two weeks for a total of two administrations. In the abexicillin group, oral administration was given daily for 21 days. At day 27, the tumor inhibition rate of compound 1 (25 mg / kg) was 57.12%, significantly superior to the fulvestrant injection group (100 mg / kg, tumor inhibition rate 17.63%) and the abexicillin group (30 mg / kg, tumor inhibition rate 47.22%). The tumor inhibition rate of the low-dose compound 1 (25 mg / kg) combined with abexicillin group was 78.32%, still higher than the high-dose fulvestrant injection combined with abexicillin group (tumor inhibition rate only 67.47%).

[0291] Evaluation Example 7: Nanoparticle Size Study of the Compounds of the Invention

[0292] Sample preparation: Weigh an appropriate amount of sample, add water for injection to dissolve, filter through a 0.22μm aqueous phase filter membrane, and bring the volume up to a certain level. The final compound concentration is 5-10 mg / mL.

[0293] Sample detection: The above sample solution was slowly transferred to a cuvette using a dropper for sample detection. The detection instrument was a Zeta sizer Pro Blue nanoparticle size potentiometer.

[0294] Test results: The nanoparticle size data of the compounds of this invention are shown in Figure 8 and Table 9.

[0295] Table 9 Nanoparticle size data for some compounds of this invention

[0296] The results in Table 8 show that the average particle size of the compounds of the present invention is around 15 nm, and they are all unimodal (see Figure 8), with a narrow particle size dispersion coefficient and no aggregates were observed.

[0297] Evaluation Example 8: Toxicological Study of Compound 1

[0298] Experimental animals: Beagle dogs, half female and half male, 6-8 months old, 6.3-9.1 kg, randomly grouped according to sex and weight.

[0299] Sample preparation:

[0300] The saline solution for compound 1 was prepared with a fulvestrant content of 53.1%. The solvent control group was 0.9% saline.

[0301] Dosage of test drug:

[0302] The dosage and administration regimen are shown in Table 10. Low, medium, and high doses of the test sample were set at 15, 45, and 90 mg / kg, respectively. A solvent control group was also included, receiving 0.9% sodium chloride injection. Administration was via intramuscular injection in the left hind limb (no more than 3.0 mL per injection site), administered on days 1, 15, and 29. The administration period was four weeks, and the observation period was four weeks. Animal mortality and near-death experiences were observed, along with general observation, irritation at the administration site, body weight, food intake, and relevant clinical indicators.

[0303] Table 10 Dosing Regimen

[0304] Toxicological test results:

[0305] Beagle dogs were administered Compound 1 via intramuscular injection at doses of 15, 45, and 90 mg / kg, once every two weeks for a total of three administrations. After a four-week recovery period, the animals tolerated the medication well. No unplanned moribund or death was observed during the study. The highest non-severely toxic dose (HNSTD) was 90 mg / kg (90 mg / kg as fulvestrant, 373 mg / kg as Compound 1). The no-observed-adverse-effect level (NOAEL), excluding fulvestrant-related reproductive system changes and administration site irritation, was 90 mg / kg (90 mg / kg as fulvestrant, 373 mg / kg as Compound 1). All animals survived to the planned necropsy during the study. Based on the above toxicological data, Compound 1 demonstrates a favorable safety profile and is expected to exhibit low toxicity and high safety in further clinical studies.

[0306] Evaluation Example 9: Pharmacodynamic study of compound 1 by different administration routes in a nude mouse model of human breast cancer cells MCF-7.

[0307] Laboratory animals and inoculation methods:

[0308] BALB / c nude mice, female, 4-5 weeks old, weighing 20-25 grams, were housed in the experimental environment for one week after arrival. Estrogen-releasing microspheres were subcutaneously injected into the midline of the back of each mouse. Two days later, an MCF-7 tumor was transplanted in situ into the right second nipple fat pad. The tumor was allowed to grow to an average volume of 441 mm². 3At approximately 10:00 AM, mice were randomly divided into groups of 6 mice each, based on tumor size and mouse weight.

[0309] Sample preparation:

[0310] Prepare a saline injection solution of compound 1 with a drug concentration of 50 mg / mL. The control fulvestrant injection solution is... (Product name: Fushide).

[0311] Dosage of test drug:

[0312] The dosage and administration regimen are shown in Table 11. The subcutaneous tumor volume of nude mice should be measured 2-3 times per week (the formula for calculating tumor volume is: V = 0.5a × b). 2 (where a and b represent the long and short diameters of the tumor, respectively). Weigh the rat and record the data.

[0313] Table 11 Dosing Regimen

[0314] Experimental evaluation indicators: Same as those for Example 2.

[0315] Results of the pharmacodynamic experiment:

[0316] Figure 9 shows the inhibitory effects of different administration routes of compound 1 and fulvestrant injection on human breast cancer cell MCF-7 nude mouse tumor models.

[0317] The experimental results are shown in Figure 9. Compound 1 was administered subcutaneously or intravenously once a week for a total of three weeks. After 21 days of observation, the tumor inhibition rates of compound 1 administered subcutaneously and intravenously at a dose of 100 mg / kg were essentially the same, at 89.93% and 88.89%, respectively. These rates were significantly different from the blank control group and significantly superior to the fulvestrant injection group (dose of 100 mg / kg, tumor inhibition rate of 6.39%). The results demonstrate that the two administration methods (intravenous and subcutaneous injection) of the compound of this invention exhibit consistent efficacy, providing greater flexibility and choice for clinical application.

[0318] Evaluation Example 10: Pharmacodynamic Study of the Combination of Compound 1 and Palbociclib in a Human Breast Cancer Cell MCF-7 Nude Mouse Tumor Model

[0319] BALB / c nude mice, female, 4-5 weeks old, weighing 20-25 grams, were housed in the experimental environment for one week after arrival. Estrogen-releasing microspheres were subcutaneously injected into the midline of the back of each mouse. Two days later, an MCF-7 tumor was transplanted in situ into the right second nipple fat pad. The tumors were allowed to grow to an average volume of 200 mm². 3At approximately 10:00 AM, mice were randomly divided into groups of 6 mice each, based on tumor size and mouse weight.

[0320] Sample preparation: Same as in Evaluation Example 4

[0321] Dosage of test drug:

[0322] The dosage and administration regimen are shown in Table 12. The subcutaneous tumor volume of nude mice was measured 2-3 times per week (the formula for calculating tumor volume is: V = 0.5a × b). 2 (where a and b represent the long and short diameters of the tumor, respectively). Weigh the rat and record the data.

[0323] Table 12 Combined Dosing Regimens

[0324] Experimental evaluation indicators: Same as those for Example 2.

[0325] Results of the pharmacodynamic experiment:

[0326] Figure 10 shows the combined pharmacodynamic effects of low-dose compound 1 and palbociclib in a human breast cancer cell MCF-7 mouse tumor model.

[0327] The experimental results are shown in Figure 10. Compound 1 was administered subcutaneously every two weeks for a total of two administrations in the monotherapy group, while palbociclib was administered orally daily for 21 days. By day 28, the tumor inhibition rate of the compound 1 (25 mg / kg) combined with palbociclib increased from 72.07% in the monotherapy group to 85.21%, significantly superior to the palbociclib monotherapy group (25 mg / kg, tumor inhibition rate of only 8.5%). This demonstrates that the compounds of this invention, when combined with CDK4 / 6 inhibitors, are significantly effective in the MCF-7 mouse model.

Claims

1. A compound as shown in formula (I), in, R is: n represents the degree of polymerization of polylysine, selected from 25-35; m represents the degree of polymerization of polyethylene glycol, selected from 17-25.

2. The compound according to claim 1, wherein the compound is a compound of formula (II), a compound of formula (III), or a compound of formula (IV).

3. The compound according to claim 1 or 2, wherein the compound satisfies one or more of the following conditions: (1) n is 25-35, for example 25, 29, 30, 31 or 35; n is preferably 29-31, for example 29; (2) m is 17-25, for example 17, 20, 21, 22 or 25; m is preferably 20-22, for example 21; (3) The average nanoparticle size of the compound is in the range of 6-30 nanometers, preferably 10-20 nanometers, more preferably 14-16 nanometers, for example 15.19 nanometers, 14.89 nanometers or 15.56 nanometers; Preferably, the compound satisfies any of the following conditions: (1) n is 29, m is 21; (2) n is 30, m is 21; (3) n is 31, m is 21; (4) n is 29, m is 20; (5) n is 29, m is 22; (6) n is 25, m is 21; (7) n is 35, m is 21; (8) n is 29, m is 17; (9) n is 29, m is 25; (10) n is 17, m is 25; (11) n is 25, m is 35; (12) n is 17, m is 35; (13) n is 25, m is 35; (14) n is 21, m is 29; More preferably, the compound satisfies any of the following conditions: (1) the average nanoparticle size is 15.19 nm, n is 29, and m is 21; (2) the average nanoparticle size is 14.89 nm, n is 30, and m is 21; (3) the average nanoparticle size is 15.56 nm, n is 31, and m is 21.

4. The method for preparing the compound according to any one of claims 1-3, It includes the following steps: In a solvent, in the presence of a condensing agent and a base, intermediate A or its stereoisomer and intermediate B undergo a condensation reaction to obtain the drug-polymer conjugate shown in formula (I); Wherein, m and n are defined as described in any one of claims 1-3.

5. The preparation method according to claim 4, wherein it satisfies one or more of the following conditions: (1) The condensing agent is selected from one or more of the following: dicyclohexylcarbodiimide, N-hydroxybenzotriazole, benzotriazole-N,N,N′,N′-tetramethylurea hexafluorophosphate, 2-(7-azobenzotriazole)-N,N,N′,N′-tetramethylurea hexafluorophosphate, N-hydroxy-7-azobenzotriazole, Carter condensing agent, benzotriazole-1-yl-oxytripyrrolidinephosphide hexafluorophosphate, N,N′-diisopropylcarbodiimide, N,N,N′,N′-tetramethyl-O-(N-succinimide)urea hexafluorophosphate, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine Chloride, O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethylurea tetrafluoroborate, (1-cyano-2-ethoxy-2-oxoethyleneaminooxy)dimethylaminomorpholine carbium hexafluorophosphate, hexafluorophosphate-O-(6-chlorobenzotriazol-1-yl)-N,N,N′,N′-tetramethylurea, propylphosphoric anhydride, 2-chloro-4,6-dimethoxy-1,3,5-triazin-2-yl-trimethylammonium chloride or PyAOP (7-azabenzotriazol-1-oxo)tripyrrolephos hexafluorophosphate), preferably N-hydroxybenzotriazole and benzotriazol-1-yl-oxytripyrrolephosphosiphosphate; (2) The base is selected from one or more of the following: triethylamine, pyridine, 2,6-dimethylpyridine, N,N-diisopropylethylamine, dimethylaminopyridine, N-methylmorpholine and N-methylimidazolium, preferably N-methylmorpholine; (3) The solvent is selected from one or more of the following: water, methanol, dichloromethane, chloroform, ethanol, N,N-dimethylformamide, dimethyl sulfoxide, N,N-dimethylacetamide, acetonitrile, tetrahydrofuran, methyltetrahydrofuran, acetone, isopropanol, 1,4-dioxane, ethyl acetate or butyl acetate, preferably DMF; (4) The reaction temperature is -20℃ to 100℃, preferably -5℃ to 50℃, for example 20℃.

6. A pharmaceutical composition comprising a compound as described in any one of claims 1-3 and one or more pharmaceutically acceptable carriers, diluents or excipients.

7. The pharmaceutical composition according to claim 6, wherein it satisfies one or more of the following conditions: (1) The pharmaceutical composition is configured for intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection or local administration, such as intravenous injection, subcutaneous injection or intramuscular injection; (2) The dosage form of the pharmaceutical composition is a solution; the solvent of the solution is, for example, physiological saline; the drug concentration of the solution is, for example, 40-60 mg / mL, preferably 50 mg / mL.

8. A pharmaceutical combination comprising a compound as claimed in any one of claims 1-3 or a pharmaceutical composition as claimed in any one of claims 6-7, and one or more other therapeutic agents.

9. The pharmaceutical combination of claim 8, wherein the other therapeutic agent comprises: Chemotherapy drugs, CDK inhibitors, PI3K inhibitors, mTOR inhibitors, AKT inhibitors, MEK inhibitors, HDAC inhibitors, HER2 inhibitors, EGFR inhibitors, VEGFR inhibitors, SERD drugs, PARP inhibitors, WEE1 inhibitors, ATR inhibitors, PD-1 / PD-L1 inhibitors, KAT6 inhibitors, aromatase inhibitors, KRAS inhibitors, PROTAC drugs, monoclonal antibodies, and antibody-drug conjugates (ADCs); among which, chemotherapy drugs include topoisomerase inhibitors, microtubule inhibitors, alkylating agents, antimetabolites, and anthracycline antibiotics; or The other therapeutic agents are anticancer agents; preferably, the other anticancer agents are selected from the following drugs: palbociclib, ribociclib, abeciclib, trobracilib, apeliximab, everolimus, letrozole, anastrozole, capapasetinib, olaparib, niraparib, rucaparib, taraparib, AZD-6738, VE-822, ellastrant, ARV-471, CTx-648, irinotecan, paclitaxel, docetaxel, cabazitaxel, gemcitabine, doxorubicin, berintec, goxatuzumab, detrastuzumab, and trastuzumab emtansine, for example selected from palbociclib and abeciclib.

10. Use of the compound according to any one of claims 1-3, or the pharmaceutical composition according to any one of claims 6-7, or the pharmaceutical combination according to any one of claims 8-9, in the preparation of a medicament for treating conditions mediated by estrogen receptors.

11. The use according to claim 10, wherein it satisfies one or more of the following conditions: (1) The disease is cancer, preferably breast cancer, such as MCF-7; (2) The compound is administered via parenteral administration, such as subcutaneous injection, intravenous injection or intramuscular injection. (3) The compound is administered once a week or once every two weeks; (4) The administration period of the compound is at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks or at least 6 weeks; (5) The compound is administered to mammals that have or are suspected of having a disease mediated by estrogen receptors; the mammals are, for example, humans or non-human mammals; the non-human mammals are, for example, mice (e.g., rats) or dogs (e.g., Beagle dogs); when the compound is administered to mice, the dosage of the compound is, for example, from 20 mg / kg body weight / time to 120 mg / kg body weight / time, preferably from 25 mg / kg body weight / time to 100 mg / kg body weight / time, such as 25 mg / kg body weight / time, 50 mg / kg body weight / time or 100 mg / kg body weight / time; when the compound is administered to dogs, the dosage of the compound is, for example, from 10 mg / kg body weight / time to 100 mg / kg body weight / time, preferably from 15 mg / kg body weight / time to 90 mg / kg body weight / time, such as 15 mg / kg body weight / time, 45 mg / kg body weight / time or 90 mg / kg body weight / time; (6) The other therapeutic agents are administered orally; (7) The other therapeutic agents shall be administered once daily; (8) The administration period for the other therapeutic agents is at least 3 weeks; (9) The other therapeutic agents are administered to mammals that have or are suspected of having a disease mediated by estrogen receptors; the mammals are, for example, humans or non-human mammals; the non-human mammals are, for example, mice (e.g., rats) or dogs (e.g., Beagle dogs); when the drug-polymer conjugate is administered to mice, the dosage of the other therapeutic agents is, for example, from 20 mg / kg body weight per dose to 40 mg / kg body weight per dose, preferably 25 mg / kg body weight per dose or 30 mg / kg body weight per dose; (10) The compound has one or more of the following effects: a. It has better water solubility than existing fulvestrant injections (e.g., fulvestrant injections made from castor oil); b. Drug-polymer conjugates with superior water solubility compared to other PEG degrees of polymerization (e.g., drug-polymer conjugates that differ only in PEG degree of polymerization); c. Its tumor-suppressing effect (e.g., in breast cancer) is superior to existing fulvestrant injections (e.g., fulvestrant injections in castor oil formulations); d. The antitumor effect (e.g., in breast cancer) is superior to drug-polymer conjugates with other PEG polymerization degrees (e.g., drug-polymer conjugates that differ only in PEG polymerization degree); e. It has better tumor tissue targeting than existing fulvestrant injections (e.g., fulvestrant injections made from castor oil); f. Superior tumor tissue targeting compared to drug-polymer conjugates with other PEG polymerization degrees (e.g., drug-polymer conjugates differing only in PEG polymerization degree); g. When used in combination with other therapeutic agents (such as abecitabine or palbociclib), the tumor-suppressing effect (e.g., in breast cancer) is superior to existing fulvestrant injections (e.g., fulvestrant injections in castor oil formulations). h. has low toxicity and high safety.

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