Active compound for treating prostatic cancer and preparation method thereof

Abstract

The invention belongs to the field of biological medicine, and provides an active compound for treating prostatic cancer and a preparation method thereof. A-B-C-D-P five-module linear connection design is adopted, wherein the A module is a prostate specific membrane antigen targeting binding unit, the B module is a prostate specific antigen enzyme digestion recognition sequence HSSKLQ, the C module is a cathepsin B-responsive Val-Cit-p-aminobenzyl self-destruction unit, the D module is a reduction-responsive disulfide bond connecting arm, and the P is an anti-tumor pharmacodynamic fragment. A coupling reaction is accurately controlled through a continuous flow microchannel technology, controllable self-assembly of the 20-60-nanometer nano aggregate is achieved, the average hydration diameter dispersion coefficient is not larger than 0.20, and the nano aggregate stably exists in plasma and selectively releases drugs in a tumor microenvironment. Multiple contradictions among prodrug system process manufacturability, intracellular response release and stable dispersion of the preparation are solved, and wide clinical application value is achieved.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to a prostate cancer therapeutic active compound and its preparation method. Background Technology

[0002] Prostate cancer is one of the most common malignant tumors in men, with its incidence increasing significantly with age, posing a serious threat to patients' quality of life and survival. Prostate-specific membrane antigen (PSA) is highly expressed on the surface of prostate cancer cells, making it an ideal tumor-targeting biomarker. Precision treatment strategies targeting this marker have significant clinical implications. Targeted drug delivery systems need to achieve highly selective enrichment in tumor tissues, reduce systemic toxicity, and ensure drug stability in the bloodstream and efficient release within the tumor microenvironment. The prostate cancer tumor microenvironment has unique biological characteristics, including high expression of PSA and cathepsin B, and a high concentration of reduced glutathione within cells. These characteristics provide a biological basis for designing multiple-response release mechanisms. The particle size distribution, surface charge, and colloidal stability of nanomedicine carriers directly affect their in vivo pharmacokinetic behavior and tumor penetration ability. Nanosystems with particle sizes controlled in the 20-60 nm range and low dispersion coefficients can achieve effective penetration into tumor blood vessels and deep tissue distribution. Developing prostate cancer therapeutics that combine targeted recognition, responsive release, and formulation stability has significant scientific value and clinical application prospects for improving treatment efficacy, patient prognosis, and promoting the development of precision medicine.

[0003] Currently, targeted prodrug systems for prostate cancer face multiple technical challenges in clinical translation. For example, Chinese patent CN119735685B discloses an antibody targeting prostate-specific membrane antigens, its antigen-binding fragment, preparation method, and applications, but it suffers from drawbacks such as large antibody molecular weight, complex preparation process, and poor batch stability. Existing small-molecule prodrug systems, although with small molecular weights, often employ a single-response mechanism, leading to high systemic toxicity due to non-specific release in the bloodstream. Multi-response prodrug design can theoretically improve targeting selectivity, but there are mutual constraints among the chemical stability, enzymatic cleavage efficiency, and release kinetics of each response unit. In terms of process preparation, traditional batch reactions involving multi-step coupling reactions suffer from problems such as imprecise control of reagent molar ratios, difficulty in suppressing side reactions, and unstable product purity, making industrial scale-up difficult. Hydrophobic antitumor drugs have extremely low solubility in aqueous solutions. Although prodrug modification can improve water solubility, high drug loading can easily lead to hydrophobic aggregation, forming non-uniform particles with wide particle size distribution and high polydispersity index. The formation of nanoaggregates involves complex intermolecular interactions. Achieving self-assembly with controllable particle size and uniform dispersion while maintaining chemical stability requires systematic optimization at the levels of molecular design, solvent system, and ionic strength. Current technologies lack a holistic solution that unifies targeted recognition, multiple responses, process manufacturability, and formulation stability. Summary of the Invention

[0004] The purpose of this invention is to provide a therapeutically active compound for prostate cancer and its preparation method, resolving the coupling contradictions faced by a three-trigger prostate-specific membrane antigen-targeted prodrug system in its large-scale and clinical translation: First, the solvation and interfacial tension conflict between the continuous flow manufacturability of high solids content and low viscosity at the process end and the precise self-assembly stability of 20-60 nm low polydispersity coefficient in the aqueous phase; Second, the contradiction between the electronic effects and steric hindrance regulation between the structural and fluid stability requirements of microchannel reactions to resist shear and adsorption and the release of highly sensitive disulfide bonds within cells; Third, the colloidal thermodynamics and optical scattering conflict between the hydrophobic aggregation tendency brought about by high loading of hydrophobic pharmacodynamic fragments and the requirements of transparent, filterable, small-particle-size stable dispersions at the formulation end.

[0005] This invention achieves tumor targeting and hierarchical activation through a quadruple synergistic release mechanism involving prostate-specific membrane antigen recognition, prostate-specific antigen enzymatic cleavage, cathepsin B self-destruction, and glutathione reduction. The negative charge of module A and the hydrophobicity of the P fragment create an amphiphilic balance driving nano-assembly, while the flexible hexapeptide of module B provides steric hindrance stability, inhibiting excessive aggregation. Continuous flow microchannels precisely control the coupling reaction, reducing byproducts to one-tenth of traditional methods. An acetonitrile-ethyl acetate mixed solvent balances reactivity and solubility, enabling high-purity, narrow-distribution, and reproducible preparation. This quadruple synergistic response and two-phase interface regulation overcome the technical bottleneck of single-method approaches simultaneously achieving targeting, responsiveness, and stability.

[0006] To achieve the above objectives, the present invention provides the following technical solution: A method for preparing an active compound for the treatment of prostate cancer, wherein the active compound is composed of five modules ABCDP linked in a linear sequence, wherein A is a prostate-specific membrane antigen binding unit, B is the amino acid sequence HSSKLQ, C is an L-valine-L-citrulline-p-aminobenzyl self-destructing unit, D is a linker arm containing a disulfide bond, and P is a pharmacodynamic fragment having a hydroxyl or primary amine linker site; the method includes the following steps: S1, Prepare module A; S2, synthesize module B; S3, Prepare module C; S4, introduce a D module containing disulfide bonds and connect it to the C module; S5, connecting the P and the D module by a carbonate bond or a urethane bond; S6, assemble in the following order: first, connect A and B through an amide bond to form AB; then connect AB and C through an amide bond to form ABC; then connect ABC and module D to form ABCD; finally, connect ABCD and P through a carbonate bond or a carbamate bond to form ABCDP; wherein the molar ratio of substrate to coupling agent in each condensation or coupling step is 1:1.0-1.2, and the single-step reaction time is 0.5–4.0 h.

[0007] Further, the A module is obtained by the following steps: L-glutamic acid and N,N'-carbonyldiimidazole are activated in N,N-dimethylformamide for 0.5–3.0 h; then L-lysine or L-ornithine is added and the reaction is continued for 1.0–4.0 h to obtain L-glutamic acid-urea-L-lysine or L-glutamic acid-urea-L-ornithine, with an unreacted amine residue of no more than 0.20 wt%.

[0008] Furthermore, in module B, L-histidine, L-serine, L-serine, L-lysine, L-leucine, and L-glutamine are sequentially condensed on a polystyrene-based solid-phase carrier resin in the order of HSSKLQ. The loading amount of the solid-phase carrier resin is 0.8–1.2 mmol / g, and the condensation time for each step is 0.5–3.0 h.

[0009] Furthermore, the C module is obtained by the following steps: reacting L-valine with L-citrulline for 2.0–4.0 h to form Val-Cit; activating 4-aminobenzyl alcohol with p-nitrophenyl chloroformate for 0.5–3.0 h and linking it with Val-Cit to form Val-Cit-p-aminobenzyl self-destruct unit, with the unreacted hydroxyl or primary amine residue not exceeding 0.30 wt%.

[0010] Furthermore, the D module is a connecting arm containing a disulfide bond, which is introduced by reacting N-hydroxysuccinimide-3-pyridine dithiopropionate with the amine or carboxyl derivative site on the C module for 0.5–2.0 h, so that the C is linked to the D and forms —S—S— in the resulting connecting arm.

[0011] Furthermore, P is 7-ethyl-10-hydroxycamptothecin or monomethylauratestatin E, and is connected to the D module by a carbonate bond or a carbamate bond.

[0012] As another concept of this invention, the present invention employs a quadruple response mechanism and a synergistic design with nano-self-assembly, primarily to enhance the tumor-targeting enrichment capacity, microenvironment-responsive release efficiency, and blood circulation stability of therapeutic compounds for prostate cancer. In module A, the L-glutamic acid-urea-L-lysine structure carries a negative charge at physiological pH, enhancing water solubility. The urea bond recognizes prostate-specific membrane antigens at two points. Compared to ornithine derivatives, the lysine derivative exhibits a 1.5-2.3 times higher binding affinity and a dissociation constant of 8-15 nanomolars, resulting in a tumor tissue-targeting enrichment rate of 35-52%. In module B, the all-L configuration HSSKLQ hexapeptide exhibits significantly higher enzymatic cleavage efficiency against prostate-specific antigens than the mixed configuration containing D-amino acids, with a 2.1-3.6 times higher ratio of catalytic efficiency constant to Michaelis constant. Its 12-28 hour enzymatic cleavage half-life in human plasma ensures stable blood circulation. Module C, Val-Cit-p-aminobenzyl, is a cathepsin B-specific substrate. After enzymatic cleavage, p-aminobenzyl undergoes a 1,6-elimination reaction, spontaneously releasing the active drug fragment with a half-life of less than 30 minutes and a drug release completeness of not less than 92%. Module D, the pyridine disulfide linker, cleaves at intracellular glutathione concentrations of 2-10 mM, with a half-life of 0.5-3.0 hours. In plasma, at glutathione concentrations less than 2 μM, the retention rate is not less than 85% within 24 hours, demonstrating significant intracellular and extracellular selectivity. Module A, with its negatively charged carboxyl group, forms an amphiphilic molecule with the hydrophobic drug fragment P, which self-assembles in water into 20-60 nm aggregates with a dispersion coefficient not greater than 0.20. Module B, a flexible hexapeptide, provides steric stabilization, with a zeta potential of -15 to -25 mV. Water solubility is increased to over 5.0 mg / mL, more than 500 times higher than free camptothecin. In a simulated plasma environment, the integrity retention rate is not less than 82% within 24 hours, with a particle size change of less than 10%.

[0013] This invention also discloses an active compound for the treatment of prostate cancer, prepared using the method described above. The active compound consists of five modules (ABCDP) linked in a linear sequence, where A is L-glutamic acid-urea-L-lysine or L-glutamic acid-urea-L-ornithine, B is the amino acid sequence HSSKLQ, C is L-valine-L-citrulline-p-aminobenzyl self-destructing unit, D is a disulfide-containing linker connected to C, and P is a pharmacodynamic fragment with a hydroxyl or primary amine linker site connected to D via a carbonate or carbamate bond. It is stable in an aqueous neutral environment; releases the pharmacodynamic fragment under the action of prostate-specific antigen or cathepsin B; breaks the disulfide bond in a reducing environment; and self-assembles in water to form aggregates with an average hydrated diameter of 20–60 nm and a dispersion coefficient not greater than 0.20.

[0014] Furthermore, P is 7-ethyl-10-hydroxycamptothecin or monomethylauratestatin E.

[0015] Furthermore, the average hydration diameter of the aggregate is 20–40 nm and the dispersion coefficient is not greater than 0.15.

[0016] Furthermore, in the preparation method, the carbonate or carbamate step employs continuous flow reaction technology with a residence time of 1.0–5.0 min; the continuous flow reactor is a microchannel reactor with a channel equivalent diameter of 0.3–1.5 mm and a flow rate of 0.5–3.0 mL·min. -1 Back pressure was controlled at 0.5–2.0 MPa; reaction temperature was controlled at 15–35 °C. Continuous flow technology can significantly improve reaction selectivity, reduce by-product content to below 0.05 wt%, and achieve batch-to-batch reproducibility coefficient of variation of less than 3%.

[0017] Furthermore, in the preparation method, the organic solvent used in the reaction is a mixture of acetonitrile and ethyl acetate in a volume ratio of 1:1–4:1; the water content in the solvent is less than 200 ppm, determined by the Karl Fischer method; the amount of the solvent system used in the coupling reaction, i.e., step S6, is 8–15 times the mass of the substrate. The acetonitrile-ethyl acetate mixed solvent system can improve the solubility of the intermediate, shorten the reaction time to 0.5–2.0 h, and increase the reaction rate by 40–65% compared to the pure acetonitrile system.

[0018] Furthermore, in the preparation method described above, the content of the main peak after purification is not less than 98.0 wt%, determined by high performance liquid chromatography (HPLC). The chromatographic conditions are: C18 column with specifications of 250 mm × 4.6 mm and a particle size of 5 μm; mobile phase: gradient elution of acetonitrile-0.1% trifluoroacetic acid aqueous solution; detection wavelength: 254 nm. The total residual solvent content is not greater than 0.10 wt%, of which N,N-dimethylformamide does not exceed 880 ppm, acetonitrile does not exceed 410 ppm, and ethyl acetate does not exceed 5000 ppm, determined by headspace gas chromatography (HGC). The water content is not greater than 0.50 wt%, determined by Karl Fischer method. The residual carbamate is less than 50 ppm, and the residual N-hydroxysuccinimide is less than 30 ppm.

[0019] Furthermore, in the active compound, module A is L-glutamic acid-urea-L-lysine, with the structural formula Glu-CO-NH-CO-NH-Lys; the ε-amino group of the L-lysine side chain is connected to the N-terminus of module B via an amide bond; the α-carboxyl group of L-glutamic acid remains in a free state and carries a negative charge at physiological pH, enhancing water solubility. Compared with L-ornithine derivatives, L-lysine derivatives have a 1.5–2.3-fold increased affinity for prostate-specific membrane antigens, with a dissociation constant Kd of 8–15 nM, as determined by surface plasmon resonance technology. The tumor tissue targeting enrichment rate is increased to 35–52%, quantified by fluorescence imaging.

[0020] Furthermore, in the active compound, module B is a fully L-configured HSSKLQ hexapeptide, with all six amino acid residues in the L-configuration; the HSSKLQ is linked to module A by an amide bond, with the linking site being the N-terminus of HSSKLQ and the ε-amino group of the L-lysine or L-ornithine side chain in module A; in the HSSKLQ sequence, L-histidine (H) is located at the N-terminus, and L-glutamine (Q) is located at the C-terminus. The cleavage efficiency (Kcat / Km) of the fully L-configured hexapeptide against prostate-specific antigen is 3.2–5.8 × 10⁻⁶. 5 M -1 ·s -1 It exhibits 2.1–3.6 times higher cleavage efficiency than mixed-configuration hexapeptides containing D-amino acids; its enzymatic half-life in human plasma is 12–28 h, demonstrating good biological stability.

[0021] Furthermore, in the active compound, module C comprises a Val-Cit dipeptide and a p-aminobenzyl self-destruct group. The Val-Cit dipeptide is linked to the C-terminus of module B via an amide bond. The amino group of the p-aminobenzyl group forms an amide bond with the carboxyl group of Val. The benzyl alcohol hydroxyl group of the p-aminobenzyl group is linked to module D via a carbamate bond. Val-Cit-p-aminobenzyl is a substrate of cathepsin B, with the cleavage site at the C-terminus of Cit, and a Kcat / Km value of 1.5–3.2 × 10⁻⁶. 4 M -1 ·s -1 After enzymatic digestion, the aminobenzyl group undergoes a 1,6-elimination reaction, spontaneously releasing the pharmacodynamic fragment P with a half-life of less than 30 min. The assay conditions are pH 7.4 and temperature 37 ℃. The completeness of drug release is not less than 92%, as determined by high performance liquid chromatography.

[0022] Furthermore, in the active compound, module D is derived from a pyridine disulfide-derived linker arm, specifically 3-pyridine disulfide propionyl. Module D is linked to the benzyl alcohol hydroxyl group of the p-aminobenzyl group in module C, with the following connection method: the carboxyl group of module D is first converted to the active ester, namely N-hydroxysuccinimide ester, and then forms a carbamate bond or carbonate bond with the p-aminobenzyl hydroxyl group. The -S-S-disulfide bond breaks in a reducing environment within cells, where the glutathione concentration is 2–10 mM and the disulfide bond breakage half-life is 0.5–3.0 h. It remains stable when the glutathione concentration in plasma is less than 2 μM, and the disulfide bond retention rate is not less than 85% within 24 h. After the disulfide bond breaks, a thiol-containing fragment is released, further triggering the p-aminobenzyl self-destruction mechanism.

[0023] Furthermore, the active compound is stable in an aqueous neutral environment with a pH of 6.8–7.6 and a temperature of 25 °C. After 7 days of storage, the main peak content remains at no less than 95%, and no obvious degradation products are detected by high-performance liquid chromatography. When incubated in a simulated plasma environment for 24 h with a pH of 7.4, containing 4.5% bovine serum albumin, and a temperature of 37 °C, the compound retains at least 82% integrity. The aggregate particle size change is less than 10%, the zeta potential is −15 to −25 mV, and the polydispersity index is less than 0.18. Its water solubility in phosphate buffer is at least 5.0 mg / mL. -1 The buffer solution has a pH of 7.4 and exhibits no precipitation or turbidity; its water solubility is less than 0.01 mg / mL compared to the free drug 7-ethyl-10-hydroxycamptothecin. -1 Its water solubility is increased by more than 500 times.

[0024] Furthermore, the logarithmic partition coefficient of the active compound at pH 7.4 is 1.5–3.5, determined by the shake-flask method using a two-phase system of n-octanol-phosphate buffer at 25 °C. Controlling the logarithmic partition coefficient within this range balances cell membrane permeability and plasma stability. When the logarithmic partition coefficient is less than 1.5, cellular uptake efficiency decreases by more than 40%; when the logarithmic partition coefficient is greater than 3.5, the non-specific binding rate to plasma proteins exceeds 75%, resulting in decreased bioavailability. Preferably, the logarithmic partition coefficient is 2.0–3.0, at which point the tumor cell uptake rate is highest, with a half-maximal inhibitory concentration (IC50) of 0.5–3.0 nM. This was tested on PC-3, DU145, and LNCaP prostate cancer cell lines, showing an IC50 of 8–25 nM compared to free 7-ethyl-10-hydroxycamptothecin, and a 5–20-fold increase in cytotoxicity.

[0025] The present invention also discloses the use of an active compound in the preparation of a medicament for treating prostate cancer that is positive for prostate-specific membrane antigen, wherein the dosage of the medicament is 0.1–10 mg / kg and the route of administration is intravenous injection or intraperitoneal injection.

[0026] In this invention, the respective focuses and synergistic effects of module A (L-glutamic acid-urea-L-lysine) and module B (HSSKLQ hexapeptide) in the prostate cancer targeted prodrug system are manifested at multiple levels. Module A is mainly responsible for tumor cell surface recognition and initial enrichment. The glutamic acid urea bond structure provides bidentate chelate recognition of prostate-specific membrane antigens, with a dissociation constant reaching the nanomolar level. The positive charge of the lysine side chain electrostatic attraction with the negative charge of the tumor cell membrane promotes cellular uptake, while the negative charge of the glutamic acid carboxyl group enhances water solubility and drives the assembly of both components into nanoaggregates. Module B is mainly responsible for tumor microenvironment response release and circulation stability regulation. The HSSKLQ sequence is a highly specific substrate for prostate-specific antigens, resisting protease degradation in plasma and being rapidly cleaved by prostate-specific antigens in prostate cancer tissue. The flexible hexapeptide segment provides steric hindrance to inhibit excessive aggregation of nanoaggregates. The two-module synergistic mechanism enhances targeting performance as follows: Module A recognizes and aggregates the drug on the surface of tumor cells, while Module B's enzymatic cleavage triggers the first-order response, allowing the drug to enter the cells, increasing the target enrichment rate to 35-52%, which is 1.8-2.5 times higher than that of a single targeting ligand. Regarding stability, the negatively charged carboxyl group of Module A and the flexible peptide of Module B jointly stabilize the nanoaggregates. A zeta potential of -15 to -25 mV provides electrostatic repulsion, and the steric hindrance of the hexapeptide prevents particle fusion. The aggregates retain at least 82% integrity in plasma for 24 hours, with a particle size change of less than 10%. In terms of release kinetics, endocytosis following recognition by Module A allows the prodrug to enter the lysosome. Enzymatic cleavage by Module B, initiated by prostate-specific antigen, initiates a cascade release. Subsequently, Module C performs secondary cleavage by cathepsin B, and Module D performs reduction cleavage by cytoplasmic glutathione, forming a triple synergistic effect of enzymatic cleavage-self-destruction-reduction. The complete drug release is at least 92%, which is 3.2-4.8 times higher than that of a single-response mechanism.

[0027] (3) Beneficial technical effects Achieving synergistic effects of tumor-specific targeted enrichment and multiple-response release: Through a cascade design of a quadruple response mechanism—module A (prostate-specific membrane antigen recognition), module B (prostate-specific antigen enzymatic cleavage), module C (cathepsin B self-destruction), and module D (glutathione reduction)—the drug is first enriched on the surface of prostate cancer cells. Subsequently, it is sequentially activated by enzymatic cleavage in the tumor microenvironment, undergoes secondary enzymatic cleavage in lysosomes, and is reduced and broken down in the cytoplasm, forming a progressively amplified response release. The drug enrichment rate in tumor tissue reaches 35-52%, and the intracellular drug release completeness is not less than 92%. Compared with single-target or single-response prodrugs, tumor selectivity is increased by 3-5 times, and systemic toxicity is reduced by 60-75%, achieving synergistic effects of targeting and responsiveness.

[0028] Overcoming technical bottlenecks in manufacturability and product quality stability: Continuous flow microchannel reaction technology is employed to precisely control the molar ratio of substrate to coupling reagent at 1:1.0-1.2, with a residence time of 1.0-5.0 minutes, significantly improving reaction selectivity, reducing byproduct content to below 0.05%, and achieving a batch-to-batch reproducibility coefficient of variation of less than 3%. The acetonitrile-ethyl acetate mixed solvent system balances reactivity and intermediate solubility, shortening reaction time by 40-65%. The microchannel anti-adsorption design with an equivalent diameter of 0.3-1.5 mm and back pressure control of 0.5-2.0 MPa ensure fluid stability. After purification, the main peak content is not less than 98.0%, and residual solvent, unreacted reagents, and catalyst residues are strictly controlled below pharmacopoeia limits. This achieves high-purity, high-yield, and high-reproducibility large-scale preparation, laying a technological foundation for clinical translation.

[0029] To resolve the contradiction between the poor water solubility of hydrophobic drugs and the stability of nanoaggregates: A module, using the amphiphilic balance design of the negatively charged carboxyl group of glutamate and the P-fragment hydrophobic drug, drives the self-assembly of the aqueous phase to form uniform nanoaggregates with an average hydrated diameter of 20-60 nm and a dispersion coefficient of no more than 0.20. Module B, the flexible HSSKLQ hexapeptide, provides steric hindrance to inhibit excessive aggregation, while a Zeta potential of -15 to -25 mV provides electrostatic repulsion stability. The active compound achieves a water solubility of over 5.0 mg / mL in phosphate buffer, compared to less than 0.01 mg / mL for free 7-ethyl-10-hydroxycamptothecin, representing a more than 500-fold improvement in water solubility. After 24 hours of incubation in a simulated plasma environment, the compound retains at least 82% integrity, with aggregate particle size change of less than 10%, thus solving the problem of solubility and formulation stability of potent hydrophobic drugs.

[0030] Balancing blood circulation stability with rapid intracellular release: After 7 days of storage in an aqueous neutral environment (pH 6.8-7.6, 25°C), the main peak content remains no less than 95%. In plasma, when the glutathione concentration is less than 2 μmol, the disulfide bond retention rate is no less than 85% within 24 hours. Module B has an enzymatic cleavage half-life of 12-28 hours in human plasma, ensuring the drug's chemical and biological stability in blood circulation. Upon entering tumor cells, it rapidly responds to prostate-specific antigen, cathepsin B, and 2-10 mmol glutathione, shortening the disulfide bond cleavage half-life to 0.5-3.0 hours and the p-aminobenzyl self-destruction half-life to less than 30 minutes, achieving a kinetic balance between circulatory stability and rapid intracellular release, thus improving the therapeutic index.

[0031] Enhanced antitumor cytotoxicity and expanded therapeutic window: Through the cascade amplification of a quadruple response mechanism and the tumor penetration enhancement effect of nanocarriers, the half-maximal inhibitory concentration (IC50) of the active compound against PC-3, DU145, and LNCaP prostate cancer cells was reduced to 0.5-3.0 nanomolars, compared to 8-25 nanomolars for free 7-ethyl-10-hydroxycamptothecin, resulting in a 5-20 fold increase in cytotoxicity. The logarithmic partition coefficient was controlled within the range of 1.5-3.5 to balance cell membrane permeability and plasma stability, with the highest tumor cell uptake rate observed at an optimal range of 2.0-3.0. Effective treatment was achieved at a dosage of 0.1-10 mg / kg, with significantly reduced systemic toxicity, expanded therapeutic window, and improved drug safety, providing a new strategy for precision treatment of prostate cancer. Attached Figure Description

[0032] Figure 1 This invention relates to the effect of the molar ratio of substrate to coupling reagent on the content of main peak and by-product.

[0033] Figure 2 This invention relates to the effect of continuous flow residence time on batch-to-batch coefficient of variation and by-product content.

[0034] Figure 3 This invention relates to the effect of reaction temperature on the content of the main peak and the reaction time.

[0035] Figure 4 The transmission mode Fourier transform infrared spectrum of the ABCDP active compound of Example 1 of the present invention is shown.

[0036] Figure 5 To illustrate the surface plasmon resonance (SPR) sensing image and 1:1 Langmuir kinetic global fitting curve of Embodiment 1 of the present invention, the binding and dissociation processes and residual analysis at a concentration of 1.0 nM are presented. Detailed Implementation

[0037] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Example

[0038] A method for preparing an active compound for the treatment of prostate cancer, wherein the active compound of this embodiment is composed of five modules ABCDP linked in a linear sequence, wherein A is a prostate-specific membrane antigen binding unit, B is the amino acid sequence HSSKLQ, C is an L-valine-L-citrulline-p-aminobenzyl self-destructing unit, D is a linker arm containing a disulfide bond, and P is a pharmacodynamic fragment having a hydroxyl or primary amine linker site, comprising the following steps: S1, Preparation of Module A: L-glutamic acid and N,N'-carbodiimidazole were activated in N,N-dimethylformamide for 1.5 h; then L-lysine was added and the reaction continued for 2.5 h to obtain L-glutamic acid-urea-L-lysine, with an unreacted amine residue of 0.12 wt%. Module A in this embodiment is L-glutamic acid-urea-L-lysine, with the structural formula Glu-CO-NH-CO-NH-Lys; the ε-amino group of the L-lysine side chain is connected to the N-terminus of Module B via an amide bond; the α-carboxyl group of L-glutamic acid remains in a free state and carries a negative charge at physiological pH, enhancing water solubility. Compared with L-ornithine derivatives, L-lysine derivatives have a 1.9-fold increased affinity for prostate-specific membrane antigens, with a dissociation constant Kd of 11 nM. This data was determined by surface plasmon resonance technology. The tumor tissue targeting enrichment rate was 43%, quantified by fluorescence imaging.

[0039] S2, Synthesis of Module B: L-histidine, L-serine, L-serine, L-lysine, L-leucine, and L-glutamine were sequentially condensed on a polystyrene-based solid-phase support resin in the order of HSSKLQ. In this embodiment, the solid-phase support resin loading was 1.0 mmol / g, and the condensation time for each step was 1.5 h. Module B in this embodiment is a fully L-configured HSSKLQ hexapeptide, with all six amino acid residues in the L-configuration. The HSSKLQ in this embodiment is linked to Module A via an amide bond, with the linking site being the N-terminus of HSSKLQ and the ε-amino group of the L-lysine side chain in Module A. In the HSSKLQ sequence of this embodiment, L-histidine (H) is located at the N-terminus, and L-glutamine (Q) is located at the C-terminus. The cleavage efficiency (Kcat / Km) of the fully L-configured hexapeptide against prostate-specific antigen was 4.5 × 10⁻⁶. 5 M -1 ·s -1 It exhibits a 2.8-fold increase in efficiency compared to mixed-configuration hexapeptides containing D-amino acids; its enzymatic cleavage half-life in human plasma is 20 h, demonstrating good biological stability.

[0040] S3, Preparation of Module C: L-valine and L-citrulline were reacted for 3.0 h to form Val-Cit; 4-aminobenzyl alcohol was activated with p-nitrophenyl chloroformate for 1.5 h and linked with Val-Cit of this embodiment to form Val-Cit-p-aminobenzyl self-destructing unit, with an unreacted hydroxyl or primary amine residue of 0.18 wt%. Module C of this embodiment contains a Val-Cit dipeptide and a p-aminobenzyl self-destructing group. The Val-Cit dipeptide is linked to the C-terminus of Module B of this embodiment via an amide bond. The amino group of p-aminobenzyl forms an amide bond with the carboxyl group of Val. The benzyl alcohol hydroxyl group of p-aminobenzyl is linked to Module D of this embodiment via a carbamate bond. Val-Cit-p-aminobenzyl is a substrate of cathepsin B, with the cleavage site at the C-terminus of Cit, and a Kcat / Km value of 2.3 × 10⁻⁶. 4 M -1 ·s -1 After enzymatic digestion, the aminobenzyl group undergoes a 1,6-elimination reaction, spontaneously releasing the pharmacodynamic fragment P with a half-life of 22 min. The assay conditions are pH 7.4 and temperature 37 ℃. The drug release completeness is 95%, as determined by high performance liquid chromatography.

[0041] S4, introduce a D module containing disulfide bonds to connect it to the C module of this embodiment: The D module of this embodiment is a connecting arm containing disulfide bonds, which is introduced by reacting N-hydroxysuccinimide-3-pyridine dithiopropionate with the amine or carboxyl derivative site on the C module of this embodiment for 1.2 h, so that the C of this embodiment is connected to the D of this embodiment and a —S—S— is formed in the resulting connecting arm. In this embodiment, module D is derived from a pyridine disulfide-derived linker arm, specifically 3-pyridine disulfide propionyl. Module D in this embodiment is connected to the benzyl alcohol hydroxyl group of the p-aminobenzyl group in module C of this embodiment. The connection method is as follows: the carboxyl group of module D is first converted into an active ester, namely N-hydroxysuccinimide ester, and then forms a carbamate bond with the p-aminobenzyl hydroxyl group. In this embodiment, the -S-S- disulfide bond breaks in a reducing environment within the cell, where the glutathione concentration is 6 mM and the disulfide bond breakage half-life is 1.8 h. It remains stable when the glutathione concentration in plasma is less than 2 μM, and the disulfide bond retention rate is 88% within 24 h. After the disulfide bond breaks, a thiol-containing fragment is released, further triggering the p-aminobenzyl self-destruction mechanism.

[0042] S5, so that P in this embodiment is connected to module D in this embodiment by a carbamate bond: P in this embodiment is 7-ethyl-10-hydroxycamptothecin, and is connected to module D in this embodiment by a carbamate bond.

[0043] S6, assemble in the following order: first, connect A and B of this embodiment through amide bonds to form AB; then connect AB and C of this embodiment through amide bonds to form ABC; then connect ABC and D of this embodiment to form ABCD; finally, connect ABCD of this embodiment with P of this embodiment through urethane bonds to form ABCDP; wherein the molar ratio of substrate to coupling agent in each condensation or coupling step is 1:1.1, and the single-step reaction time is 2.0 h.

[0044] In the preparation method of this embodiment, the carbamate esterification step adopts continuous flow reaction technology with a residence time of 3.0 min; the continuous flow reactor is a microchannel reactor with a channel equivalent diameter of 0.9 mm and a flow rate of 1.5 mL·min. -1 The back pressure was controlled at 1.2 MPa, and the reaction temperature was controlled at 25 °C. Continuous flow technology significantly improved reaction selectivity, reduced by-product content to 0.03 wt%, and achieved a batch-to-batch reproducibility coefficient of variation of 2.1%.

[0045] In the preparation method of this embodiment, the organic solvent used in the reaction is a mixture of acetonitrile and ethyl acetate in a volume ratio of 2:1; the water content in the solvent is 120 ppm, determined by the Karl Fischer method; the amount of solvent system used in this embodiment in the coupling reaction, i.e., step S6, is 11 times the amount of substrate. The acetonitrile-ethyl acetate mixed solvent system can improve the solubility of the intermediate, shorten the reaction time to 1.5 h, and increase the reaction rate by 52% compared with the pure acetonitrile system.

[0046] In the preparation method of this embodiment, the content of the main peak after purification was 98.5 wt%, which was determined by high performance liquid chromatography. The chromatographic conditions were: C18 column with specifications of 250 mm × 4.6 mm and particle size of 5 μm; mobile phase: gradient elution of acetonitrile-0.1% trifluoroacetic acid aqueous solution; detection wavelength: 254 nm; the total residual solvent was 0.06 wt%, of which N,N-dimethylformamide was 520 ppm, acetonitrile was 280 ppm, and ethyl acetate was 2800 ppm, which was determined by headspace gas chromatography; the water content was 0.32 wt%, which was determined by Karl Fischer method; the residual carbamate was 28 ppm, and the residual N-hydroxysuccinimide was 18 ppm.

[0047] The active compound in this embodiment is composed of five modules ABCDP linked in a linear sequence, where A is L-glutamic acid-urea-L-lysine, B is the amino acid sequence HSSKLQ, C is L-valine-L-citrulline-p-aminobenzyl self-destructing unit, D is a disulfide-bonded linker connected to C in this embodiment, and P is 7-ethyl-10-hydroxycamptothecin connected to D in this embodiment via a carbamate bond. It is stable in an aqueous neutral environment (pH 7.2, temperature 25 °C), maintaining a main peak content of 96% after 7 days of storage. High-performance liquid chromatography (HPLC) analysis showed no significant degradation products. In a simulated plasma environment (pH 7.4, containing 4.5% bovine serum albumin, temperature 37 °C), the compound retained 85% of its integrity after 24 h of incubation. It releases the pharmacodynamic fragment under the action of prostate-specific antigen or cathepsin B. Disulfide bonds break in a reducing environment. It self-assembles in water to form aggregates; the average hydrated diameter of the aggregates in this embodiment is 30 nm, and the dispersion coefficient is 0.12. The aggregate size variation was 6%, the zeta potential was −20 mV, and the polydispersity index was 0.12; the water solubility in phosphate buffer was 6.5 mg·mL⁻¹. -1 The buffer solution has a pH of 7.4 and exhibits no precipitation or turbidity; its water solubility is less than 0.01 mg / mL compared to the free drug 7-ethyl-10-hydroxycamptothecin. -1 Its water solubility is increased by 650 times.

[0048] The logarithmic partition coefficient of the active compound in this embodiment was 2.5 at pH 7.4, determined by the shake-flask method using a two-phase system of n-octanol-phosphate buffer at 25 °C. Controlling the logarithmic partition coefficient within this range balances cell membrane permeability and plasma stability. When the logarithmic partition coefficient is less than 1.5, cellular uptake efficiency decreases by more than 40%; when the logarithmic partition coefficient is greater than 3.5, the non-specific binding rate to plasma proteins exceeds 75%, resulting in decreased bioavailability. A logarithmic partition coefficient of 2.0-3.0 is preferred, as this range indicates the highest uptake rate by tumor cells, with a half-maximal inhibitory concentration (IC50) of 1.5 nM. The test subjects were PC-3, DU145, and LNCaP prostate cancer cell lines. Compared to free 7-ethyl-10-hydroxycamptothecin, its IC50 concentration was 8-25 nM, demonstrating a 12-fold increase in cytotoxicity.

[0049] The use of the active compound in the preparation of a medicament for treating prostate cancer that is positive for prostate-specific membrane antigen (PSM) in this embodiment. The dosage of the medicament in this embodiment is 3.0 mg / kg, and the route of administration is intravenous injection.

[0050] Example 1 Features: This example employs moderately conservative parameter configurations to ensure the stability and reproducibility of the preparation process. L-lysine was selected as the A module, exhibiting higher PSMA binding affinity compared to L-ornithine. 7-Ethyl-10-hydroxycamptothecin was used as the pharmacodynamic fragment, linked via a carbamate bond, achieving efficient drug release while ensuring stability. Continuous flow reaction parameters were balanced, with a residence time of 3.0 min and a reaction temperature of 25°C, ensuring batch-to-batch consistency. The resulting aggregates had a particle size of 30 nm and a dispersion factor of 0.12, demonstrating excellent homogeneity. This example is suitable for large-scale production scenarios requiring stable processes and high reproducibility, and is particularly suitable as a standard formulation for clinical trials. The logarithmic partition coefficient of 2.5 is within the optimal range, and the cytotoxicity is 12-fold higher than that of the free drug. It is suitable for metastatic castration-resistant prostate cancer patients with high expression of prostate-specific membrane antigens. The recommended dosage is 3.0 mg / kg intravenously, administered every 3 weeks. Example

[0051] A method for preparing an active compound for the treatment of prostate cancer, wherein the active compound of this embodiment is composed of five modules ABCDP linked in a linear sequence, wherein A is a prostate-specific membrane antigen binding unit, B is the amino acid sequence HSSKLQ, C is an L-valine-L-citrulline-p-aminobenzyl self-destructing unit, D is a linker arm containing a disulfide bond, and P is a pharmacodynamic fragment having a hydroxyl or primary amine linker site, comprising the following steps: S1, Preparation of Module A: L-glutamic acid and N,N'-carbodiimidazole were activated in N,N-dimethylformamide for 1.0 h; then L-lysine was added and the reaction continued for 1.5 h to obtain L-glutamic acid-urea-L-lysine, with an unreacted amine residue of 0.08 wt%. Module A in this embodiment is L-glutamic acid-urea-L-lysine, with the structural formula Glu-CO-NH-CO-NH-Lys; the ε-amino group of the L-lysine side chain is connected to the N-terminus of Module B via an amide bond; the α-carboxyl group of L-glutamic acid remains in a free state, carrying a negative charge at physiological pH, enhancing water solubility. Compared with L-ornithine derivatives, L-lysine derivatives have a 2.1-fold increased affinity for prostate-specific membrane antigens, with a dissociation constant Kd of 9 nM. This data was determined by surface plasmon resonance technology. The tumor tissue targeting enrichment rate was 48%, quantified by fluorescence imaging.

[0052] S2, Synthesis of Module B: L-histidine, L-serine, L-serine, L-lysine, L-leucine, and L-glutamine were sequentially condensed on a polystyrene-based solid-phase support resin in the order of HSSKLQ. In this embodiment, the solid-phase support resin loading was 0.9 mmol / g, and the condensation time for each step was 1.0 h. Module B in this embodiment is a fully L-configured HSSKLQ hexapeptide, with all six amino acid residues in the L-configuration. The HSSKLQ in this embodiment is linked to Module A via an amide bond, with the linking site being the N-terminus of HSSKLQ and the ε-amino group of the L-lysine side chain in Module A. In the HSSKLQ sequence of this embodiment, L-histidine (H) is located at the N-terminus, and L-glutamine (Q) is located at the C-terminus. The cleavage efficiency (Kcat / Km) of the fully L-configured hexapeptide against prostate-specific antigen was 5.2 × 10⁻⁶. 5 M -1 ·s -1 It exhibits a 3.2-fold increase in efficiency compared to mixed-configuration hexapeptides containing D-amino acids; its enzymatic cleavage half-life in human plasma is 16 h, demonstrating good biological stability.

[0053] S3, Preparation of Module C: L-valine and L-citrulline were reacted for 2.5 h to form Val-Cit; 4-aminobenzyl alcohol was activated with p-nitrophenyl chloroformate for 1.0 h and linked with Val-Cit of this embodiment to form Val-Cit-p-aminobenzyl self-destructing unit, with an unreacted hydroxyl or primary amine residue of 0.10 wt%. Module C of this embodiment contains a Val-Cit dipeptide and a p-aminobenzyl self-destructing group. The Val-Cit dipeptide is linked to the C-terminus of Module B of this embodiment via an amide bond. The amino group of p-aminobenzyl forms an amide bond with the carboxyl group of Val. The benzyl alcohol hydroxyl group of p-aminobenzyl is linked to Module D of this embodiment via a carbamate bond. Val-Cit-p-aminobenzyl is a substrate of cathepsin B, with the cleavage site at the C-terminus of Cit, and a Kcat / Km value of 2.8 × 10⁻⁶. 4 M -1 ·s -1 After enzymatic digestion, the aminobenzyl group undergoes a 1,6-elimination reaction, spontaneously releasing the pharmacodynamic fragment P with a half-life of 18 min. The assay conditions are pH 7.4 and temperature 37 ℃. The drug release completeness is 96%, as determined by high performance liquid chromatography.

[0054] S4, introduce a D module containing disulfide bonds to connect it to the C module of this embodiment: The D module of this embodiment is a connecting arm containing disulfide bonds, which is introduced by reacting N-hydroxysuccinimide-3-pyridine dithiopropionate with the amine or carboxyl derivative site on the C module of this embodiment for 0.8 h, so that the C of this embodiment is connected to the D of this embodiment and a —S—S— is formed in the resulting connecting arm. In this embodiment, module D is derived from a pyridine disulfide-derived linker arm, specifically 3-pyridine disulfide propionyl. Module D in this embodiment is connected to the benzyl alcohol hydroxyl group of the p-aminobenzyl group in module C of this embodiment. The connection method is as follows: the carboxyl group of module D is first converted into an active ester, namely N-hydroxysuccinimide ester, and then forms a carbamate bond with the p-aminobenzyl hydroxyl group. In this embodiment, the -S-S-disulfide bond breaks in a reducing environment within the cell, where the glutathione concentration is 8 mM and the disulfide bond breakage half-life is 1.2 h. It remains stable when the glutathione concentration in plasma is less than 2 μM, and the disulfide bond retention rate is 90% within 24 h. After the disulfide bond breaks, a thiol-containing fragment is released, further triggering the p-aminobenzyl self-destruction mechanism.

[0055] S5, connect P and D module of this embodiment with a carbamate bond: P in this embodiment is monomethylaurestatin E, and is connected to D module of this embodiment with a carbamate bond.

[0056] S6, assemble in the following order: first, connect A and B of this embodiment through amide bonds to form AB; then connect AB and C of this embodiment through amide bonds to form ABC; then connect ABC and D of this embodiment to form ABCD; finally, connect ABCD of this embodiment with P of this embodiment through urethane bonds to form ABCDP; wherein the molar ratio of substrate to coupling agent in each condensation or coupling step is 1:1.2, and the single-step reaction time is 1.0 h.

[0057] In the preparation method of this embodiment, the carbamate esterification step adopts continuous flow reaction technology with a residence time of 2.0 min; the continuous flow reactor is a microchannel reactor with a channel equivalent diameter of 0.6 mm and a flow rate of 2.5 mL·min. -1 The back pressure was controlled at 1.5 MPa, and the reaction temperature was controlled at 30 °C. Continuous flow technology significantly improved reaction selectivity, reduced by-product content to 0.02 wt%, and achieved a batch-to-batch reproducibility coefficient of variation of 1.8%.

[0058] In the preparation method of this embodiment, the organic solvent used in the reaction is a mixture of acetonitrile and ethyl acetate in a volume ratio of 3:1; the water content in the solvent is 95 ppm, determined by the Karl Fischer method; the amount of solvent system used in this embodiment in the coupling reaction, i.e., step S6, is 10 times the amount of substrate. The acetonitrile-ethyl acetate mixed solvent system can improve the solubility of the intermediate, shorten the reaction time to 0.8 h, and increase the reaction rate by 58% compared with the pure acetonitrile system.

[0059] In the preparation method of this embodiment, the main peak content after purification was 98.8 wt%, which was determined by high performance liquid chromatography (HPLC). The chromatographic conditions were: C18 column with specifications of 250 mm × 4.6 mm and particle size of 5 μm; mobile phase: gradient elution of acetonitrile-0.1% trifluoroacetic acid aqueous solution; detection wavelength: 254 nm. The total residual solvent content was 0.04 wt%, of which N,N-dimethylformamide was 380 ppm, acetonitrile was 180 ppm, and ethyl acetate was 1800 ppm, which was determined by headspace gas chromatography (HGC). The water content was 0.25 wt%, which was determined by Karl Fischer method. The residual carbamate was 22 ppm, and the residual N-hydroxysuccinimide was 15 ppm.

[0060] The active compound in this embodiment is composed of five modules ABCDP linked in a linear sequence, where A is L-glutamic acid-urea-L-lysine, B is the amino acid sequence HSSKLQ, C is L-valine-L-citrulline-p-aminobenzyl self-destructing unit, D is a disulfide-bonded linker connected to C in this embodiment, and P is monomethylaurestatin E connected to D in this embodiment via a carbamate bond. It is stable in an aqueous neutral environment (pH 7.2, temperature 25 °C), maintaining a main peak content of 97% after 7 days of storage. High-performance liquid chromatography (HPLC) analysis showed no significant degradation products. In a simulated plasma environment (pH 7.4, containing 4.5% bovine serum albumin, temperature 37 °C), the compound retained 87% of its integrity after 24 hours of incubation. It releases the pharmacodynamic fragment under the action of prostate-specific antigen or cathepsin B. Disulfide bonds break in a reducing environment. It self-assembles into aggregates in water; the average hydrated diameter of the aggregates in this embodiment is 25 nm and the dispersion coefficient is 0.10. The aggregate size variation was 5%, the zeta potential was -18 mV, and the polydispersity index was 0.10; the water solubility in phosphate buffer was 7.2 mg·mL⁻¹. -1 The buffer solution has a pH of 7.4 and exhibits no precipitation or turbidity; its water solubility is less than 0.01 mg / mL compared to the free drug, monomethylaurestatin E. -1 Its water solubility is increased by 720 times.

[0061] The logarithmic partition coefficient of the active compound in this embodiment was 2.8 at pH 7.4, determined by the shake-flask method using an octanol-phosphate buffer two-phase system at 25 °C. Controlling the logarithmic partition coefficient within this range balances cell membrane permeability and plasma stability. When the logarithmic partition coefficient is less than 1.5, cellular uptake efficiency decreases by more than 40%; when the logarithmic partition coefficient is greater than 3.5, the non-specific binding rate to plasma proteins exceeds 75%, resulting in decreased bioavailability. A logarithmic partition coefficient of 2.0-3.0 is preferred, as this range indicates the highest uptake rate by tumor cells, with a half-maximal inhibitory concentration (IC50) of 0.8 nM. The test subjects were PC-3, DU145, and LNCaP prostate cancer cell lines. Compared to free monomethylaurestatin E, its IC50 concentration was 5-18 nM, demonstrating a 15-fold increase in cytotoxicity.

[0062] The use of the active compound in the preparation of a medicament for treating prostate cancer with prostate-specific membrane antigen positivity, wherein the dosage of the medicament in this embodiment is 1.5 mg / kg, and the route of administration is intravenous injection.

[0063] Example 2 Features: This example employs a rapid and efficient preparation strategy, significantly shortening the preparation cycle through optimized reaction conditions. The reaction time is reduced to 1.0 h, the molar ratio of coupling reagents is increased to 1:1.2, the continuous flow residence time is only 2.0 min, and the channel diameter of 0.6 mm enhances mass transfer efficiency. Monomethylaurestatin E is selected as the pharmacodynamic fragment, exhibiting stronger tubulin inhibitory activity, 15-fold improved cytotoxicity compared to the free drug, and a low half-maximal inhibitory concentration (IC50) of 0.8 nM. PSMA binding affinity is increased by 2.1 times, and tumor-targeting enrichment rate reaches 48%. The main peak content reaches 98.8 wt%, with a batch-to-batch coefficient of variation of only 1.8%, demonstrating high-quality control through rapid process. The aggregate particle size is 25 nm, and the dispersion factor is 0.10, exhibiting excellent uniformity. This example is suitable for scenarios requiring rapid preparation and high-throughput production, particularly suitable for rapid iteration and small-batch customized production in preclinical studies. Recommended for prostate cancer patients with high PSMA expression and sensitivity to microtubule inhibitors, the dosage is 1.5 mg / kg intravenously, administered once every 2 weeks, which can achieve more frequent dosing cycles to maintain tumor suppression. Example

[0064] A method for preparing an active compound for the treatment of prostate cancer, wherein the active compound of this embodiment is composed of five modules ABCDP linked in a linear sequence, wherein A is a prostate-specific membrane antigen binding unit, B is the amino acid sequence HSSKLQ, C is an L-valine-L-citrulline-p-aminobenzyl self-destructing unit, D is a linker arm containing a disulfide bond, and P is a pharmacodynamic fragment having a hydroxyl or primary amine linker site, comprising the following steps: S1, Preparation of Module A: L-glutamic acid and N,N'-carbodiimidazole were activated in N,N-dimethylformamide for 2.5 h; then L-ornithine was added and the reaction continued for 3.5 h to obtain L-glutamic acid-urea-L-ornithine, with an unreacted amine residue of 0.16 wt%. Module A in this embodiment is L-glutamic acid-urea-L-ornithine, with the structural formula Glu-CO-NH-CO-NH-Orn; the δ-amino group of the L-ornithine side chain is connected to the N-terminus of Module B via an amide bond; the α-carboxyl group of L-glutamic acid remains in a free state, carrying a negative charge at physiological pH, enhancing water solubility; the binding affinity of the L-ornithine derivative to prostate-specific membrane antigen is increased by 1.6 times, and the dissociation constant Kd is 14 nM, which was determined by surface plasmon resonance technology. The tumor tissue targeting enrichment rate was 37%, quantified by fluorescence imaging.

[0065] S2, Synthesis of Module B: L-histidine, L-serine, L-serine, L-lysine, L-leucine, and L-glutamine were sequentially condensed on a polystyrene-based solid-phase support resin in the order of HSSKLQ. In this embodiment, the solid-phase support resin loading was 1.1 mmol / g, and the condensation time for each step was 2.5 h. Module B in this embodiment is a fully L-configured HSSKLQ hexapeptide, with all six amino acid residues in the L-configuration. The HSSKLQ in this embodiment is linked to Module A via an amide bond, with the linking site being the N-terminus of HSSKLQ and the δ-amino group of the L-ornithine side chain in Module A. In the HSSKLQ sequence of this embodiment, L-histidine (H) is located at the N-terminus, and L-glutamine (Q) is located at the C-terminus. The cleavage efficiency (Kcat / Km) of the fully L-configured hexapeptide against prostate-specific antigen was 3.8 × 10⁻⁶. 5 M -1 ·s -1 It exhibits a 2.4-fold increase in efficiency compared to mixed-configuration hexapeptides containing D-amino acids; its enzymatic cleavage half-life in human plasma is 24 h, demonstrating good biological stability.

[0066] S3, Preparation of Module C: L-valine and L-citrulline were reacted for 3.5 h to form Val-Cit; 4-aminobenzyl alcohol was activated with p-nitrophenyl chloroformate for 2.5 h and linked with Val-Cit of this embodiment to form Val-Cit-p-aminobenzyl self-destructing unit, with an unreacted hydroxyl or primary amine residue of 0.25 wt%. Module C of this embodiment contains a Val-Cit dipeptide and a p-aminobenzyl self-destructing group. The Val-Cit dipeptide is linked to the C-terminus of Module B of this embodiment via an amide bond. The amino group of p-aminobenzyl forms an amide bond with the carboxyl group of Val. The benzyl alcohol hydroxyl group of p-aminobenzyl is linked to Module D of this embodiment via a carbonate bond. Val-Cit-p-aminobenzyl is a substrate of cathepsin B, with the cleavage site at the C-terminus of Cit, and a Kcat / Km value of 1.8 × 10⁻⁶.4 M -1 ·s -1 After enzymatic digestion, the aminobenzyl group undergoes a 1,6-elimination reaction, spontaneously releasing the pharmacodynamic fragment P with a half-life of 26 min. The assay conditions are pH 7.4 and temperature 37 ℃. The drug release completeness is 93%, as determined by high performance liquid chromatography.

[0067] S4, introduce a D module containing disulfide bonds to connect it to the C module of this embodiment: The D module of this embodiment is a connecting arm containing disulfide bonds, which is introduced by reacting N-hydroxysuccinimide-3-pyridine dithiopropionate with the amine or carboxyl derivative site on the C module of this embodiment for 1.5 h, so that the C of this embodiment is connected to the D of this embodiment and a —S—S— is formed in the resulting connecting arm. In this embodiment, module D is derived from a pyridine disulfide-derived linker arm, specifically 3-pyridine disulfide propionyl. Module D in this embodiment is connected to the benzyl alcohol hydroxyl group of the p-aminobenzyl group in module C of this embodiment. The connection method is as follows: the carboxyl group of module D is first converted into an active ester, namely N-hydroxysuccinimide ester, and then forms a carbonate bond with the p-aminobenzyl hydroxyl group. In this embodiment, the -S-S- disulfide bond breaks in a reducing environment within the cell, where the glutathione concentration is 5 mM and the disulfide bond breakage half-life is 2.2 h. It remains stable when the glutathione concentration in plasma is less than 2 μM, and the disulfide bond retention rate is 92% within 24 h. After the disulfide bond breaks, a thiol-containing fragment is released, further triggering the p-aminobenzyl self-destruction mechanism.

[0068] S5, connecting P and D modules of this embodiment by carbonate bonds: P in this embodiment is 7-ethyl-10-hydroxycamptothecin, and is connected to D modules of this embodiment by carbonate bonds.

[0069] S6, assemble in the following order: first, connect A and B of this embodiment through amide bonds to form AB; then connect AB and C of this embodiment through amide bonds to form ABC; then connect ABC and D of this embodiment to form ABCD; finally, connect ABCD of this embodiment with P of this embodiment through carbonate bonds to form ABCDP; wherein the molar ratio of substrate to coupling agent in each condensation or coupling step is 1:1.05, and the single-step reaction time is 3.5 h.

[0070] In the preparation method of this embodiment, the carbonate step adopts continuous flow reaction technology with a residence time of 4.0 min; the continuous flow reactor is a microchannel reactor with a channel equivalent diameter of 1.2 mm and a flow rate of 1.0 mL·min. -1The back pressure was controlled at 0.8 MPa, and the reaction temperature was controlled at 20 °C. Continuous flow technology significantly improved reaction selectivity, reduced by-product content to 0.04 wt%, and achieved a batch-to-batch reproducibility coefficient of variation of 2.5%.

[0071] In the preparation method of this embodiment, the organic solvent used in the reaction is a mixture of acetonitrile and ethyl acetate in a volume ratio of 1.5:1; the water content in the solvent is 160 ppm, determined by the Karl Fischer method; the amount of solvent system used in this embodiment in the coupling reaction, i.e., step S6, is 13 times the amount of substrate. The acetonitrile-ethyl acetate mixed solvent system can improve the solubility of the intermediate, shorten the reaction time to 1.8 h, and increase the reaction rate by 48% compared with the pure acetonitrile system.

[0072] In the preparation method of this embodiment, the content of the main peak after purification was 98.3 wt%, which was determined by high performance liquid chromatography. The chromatographic conditions were: C18 column with specifications of 250 mm × 4.6 mm and particle size of 5 μm; mobile phase: gradient elution of acetonitrile-0.1% trifluoroacetic acid aqueous solution; detection wavelength: 254 nm; the total residual solvent was 0.08 wt%, of which N,N-dimethylformamide was 620 ppm, acetonitrile was 350 ppm, and ethyl acetate was 3500 ppm, which was determined by headspace gas chromatography; the water content was 0.42 wt%, which was determined by Karl Fischer method; the residual carbamate was 35 ppm, and the residual N-hydroxysuccinimide was 22 ppm.

[0073] The active compound in this embodiment is composed of five modules ABCDP linked in a linear sequence, where A is L-glutamic acid-urea-L-ornithine, B is the amino acid sequence HSSKLQ, C is L-valine-L-citrulline-p-aminobenzyl self-destructing unit, D is a disulfide-bonded linker connected to C in this embodiment, and P is 7-ethyl-10-hydroxycamptothecin connected to D in this embodiment by a carbonate bond. It is stable in an aqueous neutral environment (pH 7.2, temperature 25 °C), maintaining a main peak content of 97% after 7 days of storage. High-performance liquid chromatography (HPLC) analysis showed no significant degradation products. In a simulated plasma environment (pH 7.4, containing 4.5% bovine serum albumin, temperature 37 °C), the compound retained 83% of its integrity after 24 hours of incubation. It releases the pharmacodynamic fragment under the action of prostate-specific antigen or cathepsin B. Disulfide bonds break in a reducing environment. It self-assembles into aggregates in water; the average hydrated diameter of the aggregates in this embodiment is 38 nm and the dispersion coefficient is 0.16. The aggregate size variation was 8%, the Zeta potential was −22 mV, and the polydispersity index was 0.16; the water solubility in phosphate buffer was 5.8 mg·mL⁻¹. -1The buffer solution has a pH of 7.4 and exhibits no precipitation or turbidity; its water solubility is less than 0.01 mg / mL compared to the free drug 7-ethyl-10-hydroxycamptothecin. -1 Its water solubility is increased by 580 times.

[0074] The logarithmic partition coefficient of the active compound in this embodiment was 2.2 at pH 7.4, determined by the shake-flask method using a two-phase system of n-octanol-phosphate buffer at 25 °C. Controlling the logarithmic partition coefficient within this range balances cell membrane permeability and plasma stability. When the logarithmic partition coefficient is less than 1.5, cellular uptake efficiency decreases by more than 40%; when the logarithmic partition coefficient is greater than 3.5, the non-specific binding rate to plasma proteins exceeds 75%, resulting in decreased bioavailability. A logarithmic partition coefficient of 2.0-3.0 is preferred, as this range yields the highest uptake rate in tumor cells, with a half-maximal inhibitory concentration (IC50) of 2.0 nM. The test subjects were PC-3, DU145, and LNCaP prostate cancer cell lines. Compared to free 7-ethyl-10-hydroxycamptothecin, its IC50 concentration was 8-25 nM, demonstrating an 8-fold increase in cytotoxicity.

[0075] The use of the active compound in the preparation of a medicament for treating prostate cancer that is positive for prostate-specific membrane antigen (PSM) in this embodiment. The dosage of the medicament in this embodiment is 5.0 mg / kg, and the route of administration is intraperitoneal injection.

[0076] Example 3 Features: This example emphasizes the stability and long-lasting performance of the preparation process. Sufficient reaction and high stability are ensured by extending the reaction time and selecting a lower reaction temperature. L-ornithine is used as module A, with the pharmacodynamic fragment 7-ethyl-10-hydroxycamptothecin linked by a carbonate bond. The disulfide bond cleavage half-life is extended to 2.2 h, and the disulfide bond retention rate in plasma reaches 92% within 24 h, demonstrating excellent plasma stability. The longer reaction time (3.5 h in step S6) and lower reaction temperature (20°C) ensure high purity and stability of the product. The aggregate particle size is 38 nm, and the dispersion coefficient is 0.16, suitable for slow release and long-circulation applications. This example is particularly suitable for clinical applications requiring long-lasting release and is recommended for patients who need to reduce the frequency of dosing. Intraperitoneal injection at a dose of 5.0 mg / kg can achieve a longer in vivo circulation time and sustained tumor suppression effect. Carbonate bonds have better hydrolytic stability than urethane bonds, making them suitable for therapeutic scenarios with high requirements for metabolic stability. Example

[0077] A method for preparing an active compound for the treatment of prostate cancer, wherein the active compound of this embodiment is composed of five modules ABCDP linked in a linear sequence, wherein A is a prostate-specific membrane antigen binding unit, B is the amino acid sequence HSSKLQ, C is an L-valine-L-citrulline-p-aminobenzyl self-destructing unit, D is a linker arm containing a disulfide bond, and P is a pharmacodynamic fragment having a hydroxyl or primary amine linker site, comprising the following steps: S1, Preparation of Module A: L-glutamic acid and N,N'-carbodiimidazole were activated in N,N-dimethylformamide for 0.6 h; then L-ornithine was added and the reaction continued for 3.8 h to obtain L-glutamic acid-urea-L-ornithine, with an unreacted amine residue of 0.18 wt%. Module A in this embodiment is L-glutamic acid-urea-L-ornithine, with the structural formula Glu-CO-NH-CO-NH-Orn; the δ-amino group of the L-ornithine side chain is connected to the N-terminus of Module B via an amide bond; the α-carboxyl group of L-glutamic acid remains in a free state, carrying a negative charge at physiological pH, enhancing water solubility; the binding affinity of the L-ornithine derivative to prostate-specific membrane antigen is increased by 1.7 times, and the dissociation constant Kd is 13 nM. This data was determined by surface plasmon resonance technology, and the tumor tissue targeting enrichment rate was 39%, quantified by fluorescence imaging.

[0078] S2, Synthesis of Module B: L-histidine, L-serine, L-serine, L-lysine, L-leucine, and L-glutamine were sequentially condensed on a polystyrene-based solid-phase support resin in the order of HSSKLQ. In this embodiment, the solid-phase support resin loading was 1.15 mmol / g, and the condensation time for each step was 2.8 h. Module B in this embodiment is a fully L-configured HSSKLQ hexapeptide, with all six amino acid residues in the L-configuration. The HSSKLQ in this embodiment is linked to Module A via an amide bond, with the linking site being the N-terminus of HSSKLQ and the δ-amino group of the L-ornithine side chain in Module A. In the HSSKLQ sequence of this embodiment, L-histidine (H) is located at the N-terminus, and L-glutamine (Q) is located at the C-terminus. The cleavage efficiency (Kcat / Km) of the fully L-configured hexapeptide against prostate-specific antigen was 4.0 × 10⁻⁶. 5 M -1 ·s -1 It exhibits a 2.6-fold increase in efficiency compared to mixed-configuration hexapeptides containing D-amino acids; its enzymatic cleavage half-life in human plasma is 26 h, demonstrating good biological stability.

[0079] S3, Preparation of Module C: L-valine and L-citrulline were reacted for 2.2 h to form Val-Cit; 4-aminobenzyl alcohol was activated with p-nitrophenyl chloroformate for 0.6 h and linked with Val-Cit of this embodiment to form Val-Cit-p-aminobenzyl self-destructing unit, with an unreacted hydroxyl or primary amine residue of 0.28 wt%. Module C of this embodiment contains a Val-Cit dipeptide and a p-aminobenzyl self-destructing group. The Val-Cit dipeptide is linked to the C-terminus of Module B of this embodiment via an amide bond. The amino group of p-aminobenzyl forms an amide bond with the carboxyl group of Val. The benzyl alcohol hydroxyl group of p-aminobenzyl is linked to Module D of this embodiment via a carbonate bond. Val-Cit-p-aminobenzyl is a substrate of cathepsin B, with the cleavage site at the C-terminus of Cit, and a Kcat / Km value of 1.6 × 10⁻⁶. 4 M -1 ·s -1 After enzymatic digestion, the aminobenzyl group undergoes a 1,6-elimination reaction, spontaneously releasing the pharmacodynamic fragment P with a half-life of 28 min. The assay conditions are pH 7.4 and temperature 37 ℃. The drug release completeness is 92%, as determined by high performance liquid chromatography.

[0080] S4, introduce a D module containing disulfide bonds to connect it to the C module of this embodiment: The D module of this embodiment is a connecting arm containing disulfide bonds, which is introduced by reacting N-hydroxysuccinimide-3-pyridine dithiopropionate with the amine or carboxyl derivative site on the C module of this embodiment for 1.9 h, so that the C of this embodiment is connected to the D of this embodiment and a —S—S— is formed in the resulting connecting arm. In this embodiment, module D is derived from a pyridine disulfide-derived linker arm, specifically 3-pyridine disulfide propionyl. Module D in this embodiment is connected to the benzyl alcohol hydroxyl group of the p-aminobenzyl group in module C of this embodiment. The connection method is as follows: the carboxyl group of module D is first converted into an active ester, namely N-hydroxysuccinimide ester, and then forms a carbonate bond with the p-aminobenzyl hydroxyl group. In this embodiment, the -S-S- disulfide bond breaks in a reducing environment within the cell, where the glutathione concentration is 9 mM and the disulfide bond breakage half-life is 0.7 h. It remains stable when the glutathione concentration in plasma is less than 2 μM, and the disulfide bond retention rate is 86% within 24 h. After the disulfide bond breaks, a thiol-containing fragment is released, further triggering the p-aminobenzyl self-destruction mechanism.

[0081] S5, connect P and D modules of this embodiment with carbonate bonds: P in this embodiment is monomethylaurestatin E, and is connected to D module of this embodiment with carbonate bonds.

[0082] S6, assemble in the following order: first, connect A and B of this embodiment through amide bonds to form AB; then connect AB and C of this embodiment through amide bonds to form ABC; then connect ABC and D of this embodiment to form ABCD; finally, connect ABCD of this embodiment with P of this embodiment through carbonate bonds to form ABCDP; wherein the molar ratio of substrate to coupling agent in each condensation or coupling step is 1:1.0, and the single-step reaction time is 3.8 h.

[0083] In the preparation method of this embodiment, the carbonate step adopts continuous flow reaction technology with a residence time of 1.2 min; the continuous flow reactor is a microchannel reactor with a channel equivalent diameter of 1.4 mm and a flow rate of 0.6 mL·min. -1 The back pressure was controlled at 1.9 MPa, and the reaction temperature was controlled at 33 °C. Continuous flow technology significantly improved reaction selectivity, reduced by-product content to 0.04 wt%, and achieved a batch-to-batch reproducibility coefficient of variation of 2.7%.

[0084] In the preparation method of this embodiment, the organic solvent used in the reaction is a mixture of acetonitrile and ethyl acetate in a volume ratio of 3.8:1; the water content in the solvent is 185 ppm, determined by the Karl Fischer method; the amount of solvent system used in this embodiment in the coupling reaction, i.e., step S6, is 14 times the amount of substrate. The acetonitrile-ethyl acetate mixed solvent system can improve the solubility of the intermediate, shorten the reaction time to 1.9 h, and increase the reaction rate by 62% compared with the pure acetonitrile system.

[0085] In the preparation method of this embodiment, the content of the main peak after purification was 98.2 wt%, which was determined by high performance liquid chromatography. The chromatographic conditions were: C18 column with specifications of 250 mm × 4.6 mm and particle size of 5 μm; mobile phase: gradient elution of acetonitrile-0.1% trifluoroacetic acid aqueous solution; detection wavelength: 254 nm; the total residual solvent was 0.09 wt%, of which N,N-dimethylformamide was 750 ppm, acetonitrile was 390 ppm, and ethyl acetate was 4200 ppm, which was determined by headspace gas chromatography; the water content was 0.48 wt%, which was determined by Karl Fischer method; the residual carbamate was 42 ppm, and the residual N-hydroxysuccinimide was 28 ppm.

[0086] The active compound in this embodiment is composed of five modules ABCDP linked in a linear sequence, where A is L-glutamic acid-urea-L-ornithine, B is the amino acid sequence HSSKLQ, C is L-valine-L-citrulline-p-aminobenzyl self-destructing unit, D is a disulfide-bonded linker connected to C in this embodiment, and P is monomethylaurestatin E connected to D in this embodiment via a carbonate bond. It is stable in an aqueous neutral environment (pH 7.2, temperature 25 °C), maintaining a main peak content of 95% after 7 days of storage. High-performance liquid chromatography (HPLC) analysis showed no significant degradation products. In a simulated plasma environment (pH 7.4, containing 4.5% bovine serum albumin, temperature 37 °C), the compound retained 82% of its integrity after 24 h of incubation. It releases the pharmacodynamic fragment under the action of prostate-specific antigen or cathepsin B. Disulfide bonds break in a reducing environment. It self-assembles in water to form aggregates; the average hydrated diameter of the aggregates in this embodiment is 54 nm and the dispersion coefficient is 0.19. The aggregate size variation was 9%, the zeta potential was −24 mV, and the polydispersity index was 0.17; the water solubility in phosphate buffer was 5.2 mg·mL⁻¹. -1 The buffer solution has a pH of 7.4 and exhibits no precipitation or turbidity; its water solubility is less than 0.01 mg / mL compared to the free drug, monomethylaurestatin E. -1 Its water solubility is increased by 520 times.

[0087] The active compound in this embodiment has a logarithmic partition coefficient of 3.2 at pH 7.4, determined by the shake-flask method using an octanol-phosphate buffer two-phase system at 25 °C. Maintaining the logarithmic partition coefficient within this range balances cell membrane permeability and plasma stability. When the logarithmic partition coefficient is less than 1.5, cellular uptake efficiency decreases by more than 40%; when the logarithmic partition coefficient is greater than 3.5, non-specific binding to plasma proteins exceeds 75%, resulting in decreased bioavailability. A logarithmic partition coefficient of 2.0-3.0 is preferred, as this range indicates the highest tumor cell uptake rate and a half-maximal inhibitory concentration (IC50) of 1.2 nM. The test subjects were PC-3, DU145, and LNCaP prostate cancer cell lines. Compared to free monomethylaurestatin E, its IC50 is 5-18 nM, demonstrating a 10-fold increase in cytotoxicity.

[0088] The use of the active compound in the preparation of a medicament for treating prostate cancer that is positive for prostate-specific membrane antigen (PSM) in this embodiment. The dosage of the medicament in this embodiment is 9.0 mg / kg, and the route of administration is intraperitoneal injection.

[0089] Example 4 Features: This example employs a diversified parameter range configuration strategy, selecting key parameter combinations covering the entire scope of the claims. A substrate to coupling reagent molar ratio of 1:1.0 demonstrates sufficient reactivity with a stoichiometric ratio of 1:1; a CDI activation time of 0.6 h verifies the feasibility of rapid activation; a Val-Cit reaction time of 2.2 h demonstrates the effectiveness of a shorter reaction time; a D module introduction time of 1.9 h verifies complete reactivity with a longer reaction time; a solid resin loading of 1.15 mmol / g verifies the feasibility of a high loading; a back pressure of 1.9 MPa verifies stable operation under high pressure; and a solvent volume ratio of 3.8:1 demonstrates the applicability of a high acetonitrile ratio. The aggregate particle size of 54 nm is within the optimized range, and the dispersion factor of 0.19 meets the requirements. The combination of L-ornithine and monomethylaurestatin E, linked by a carbonate bond, with a dosage of 9.0 mg / kg, verifies the safety and efficacy of the high dose. This embodiment is particularly suitable for patients with advanced prostate cancer requiring high-dose treatment. The intraperitoneal injection route achieves a balance between high local concentration and systemic distribution, making it suitable for clinical scenarios involving peritoneal metastasis or requiring high drug exposure. This embodiment fully demonstrates the complete feasibility of the parameter range described in the claims, providing solid technical support for the scope of patent protection.

[0090] Comparative Example 1: Basically the same as Example 1, except that module A uses L-glutamic acid-urea-L-ornithine, while the amounts of other components and preparation conditions remain unchanged.

[0091] Comparative Example 2: Basically the same as Example 1, except that module B uses a mixed configuration hexapeptide containing D-amino acids, in which the second L-serine is replaced with D-serine, and the amounts of other components and preparation conditions remain unchanged.

[0092] Comparative Example 3: It is basically the same as Example 1, except that the P module uses camptothecin (without 7-ethyl and 10-hydroxyl modification), and the amounts of other components and preparation conditions remain unchanged.

[0093] Comparative Example 4: It is basically the same as Example 1, except that the activation time of L-glutamic acid by N,N'-carbonyldiimidazole in step S1 is 0.3 h, while the amount of other components and preparation conditions remain unchanged.

[0094] Comparative Example 5: It is basically the same as Example 1, except that the reaction time of L-lysine and activated L-glutamic acid in step S1 is 0.8 h, while the amount of other components and preparation conditions remain unchanged.

[0095] Comparative Example 6: It is basically the same as Example 1, except that the loading of polystyrene-based solid carrier resin in step S2 is 0.6 mmol / g, while the amount of other components and preparation conditions remain unchanged.

[0096] Comparative Example 7: It is basically the same as Example 1, except that the solid phase condensation time in step S2 is 0.3 h, while the amount of other components and preparation conditions remain unchanged.

[0097] Comparative Example 8: It is basically the same as Example 1, except that the reaction time of L-valine and L-citrulline in step S3 is 1.5 h, while the amount of other components and preparation conditions remain unchanged.

[0098] Comparative Example 9: It is basically the same as Example 1, except that the reaction time for introducing N-hydroxysuccinimide-3-pyridinedithiopropionate into module D in step S4 is 0.3 h, while the amount of other components and preparation conditions remain unchanged.

[0099] Comparative Example 10: Basically the same as Example 1, except that the molar ratio of substrate to coupling reagent in step S6 is 1:0.85, while the amounts of other components and preparation conditions remain unchanged.

[0100] Comparative Example 11: Basically the same as Example 1, except that the residence time in the continuous flow reactor is 6.5 min, while the amounts of other components and preparation conditions remain unchanged.

[0101] Comparative Example 12: Basically the same as Example 1, except that the continuous flow reaction temperature is 10 °C, while the amounts of other components and preparation conditions remain unchanged.

[0102] Comparative Example 13: Basically the same as Example 1, except that the volume ratio of organic solvent acetonitrile to ethyl acetate is 0.6:1, and the amounts of other components and preparation conditions remain unchanged.

[0103] Comparative Example 14: It is basically the same as Example 1, except that in steps S5-S6, a batch reaction is used instead of a continuous flow reaction technology. Carbonation or carbamate is completed and assembled in a conventional reactor at 25 °C for 4.5 h with stirring. The amount of other components and preparation conditions remain unchanged.

[0104] Performance testing: Experiment 1: Determination of Dynamic Light Scattering Particle Size and Dispersion Coefficient Test Object: Aggregates formed by the self-assembly of the active compound of this invention in aqueous solution. Test Objective: To evaluate the average hydrated diameter and particle size distribution uniformity of the aggregates, and to verify the technical characteristics of an average hydrated diameter of 20-60 nm and a polydispersity index ≤0.20 in claim 7. Test Principle: Dynamic light scattering technology measures the intensity fluctuations of scattered light caused by the Brownian motion of particles in solution, and calculates the particle hydrated diameter and polydispersity index using the Stokes-Einstein equation. Experimental Method: The active compound was dissolved in phosphate buffer (pH 7.4) at a concentration of 1.0 mg / mL, filtered through a 0.22 μm filter, and transferred to a cuvette. Measurements were taken using a Malvern Zetasizer Nano ZS dynamic light scattering instrument at a detection angle of 173° and a temperature of 25 ℃. Each sample was measured in triplicate. Standard Basis: Refer to ISO 22412 or similar standard "Particle size analysis - Dynamic light scattering". Key parameters: laser wavelength 633 nm, automatic attenuation factor adjustment, equilibration time 120 s, number of measurements ≥ 12 per sample. Data processing: The Z-mean particle size and polydispersity index were obtained by fitting the correlation function using the cumulative method. The results are expressed as mean ± standard deviation (n=3). A polydispersity index < 0.2 indicates a narrow and uniform particle size distribution.

[0105] Experiment 2: Surface Plasmon Resonance (SPR) Measurement of PSMA Binding Affinity Test Subject: The binding of module A of the active compound of this invention to prostate-specific membrane antigen (PSMA). Test Objective: To quantitatively determine the binding affinity (dissociation constant Kd) of the active compound to PSMA, verifying the targeting function of the PSMA binding unit in the core technical features of the claims. Test Principle: SPR technology is used to detect the refractive index change caused by the binding of the ligand to the immobilized receptor, monitoring the binding and dissociation processes in real time. The binding rate constant (kon), dissociation rate constant (koff), and dissociation constant Kd = koff / kon are calculated through kinetic fitting. Experimental Methods: Recombinant human PSMA protein (purity >95%) was immobilized on the surface of a CM5 sensor chip (immobilization density approximately 8000 RU) using a Biacore T200 surface plasmon resonance spectrometer via amine coupling. Active compounds were flowed through the chip surface at serial concentrations (1.25-40 nM, 2-fold dilution) in HBS-EP+ buffer at pH 7.4 (flow rate 30 μL / min). The binding phase lasted 360 s, followed by a dissociation phase of 600 s. Each concentration was repeated twice. The chip surface was regenerated for 30 s using 10 mM glycine-HCl (pH 2.0). Standards: Refer to "Drug Evaluation Research" or other commonly used methods in the biopharmaceutical field. Key Parameters: Temperature 25 ℃, data acquisition frequency 10 Hz, reference cell excluding non-specific binding signals. Data processing: The Biacore T200 evaluation software was used, and the sensorgram curve was fitted using a 1:1 Langmuir binding model. Kon, koff, and Kd were calculated. The results are expressed as mean ± standard deviation (n=2). Kd < 15 nM indicates high affinity binding.

[0106] Experiment 3: Determination of disulfide bond breaking kinetics Test Subject: The cleavage behavior of disulfide bonds in the active compound D module of this invention under a reducing environment. Test Objective: To evaluate the cleavage kinetics of disulfide bonds in a simulated intracellular reducing environment and to verify the effectiveness of the disulfide bond cleavage release mechanism. Test Principle: Under the action of reducing agents such as glutathione, disulfide bonds undergo a thiol-disulfide bond exchange reaction, cleaving into free thiols. The retention time of the active compound and the amount of cleavage products are monitored by HPLC to calculate the cleavage half-life. Experimental Methods: The active compound (final concentration 0.2 mM) was dissolved in phosphate buffer (pH 7.4, with 4% DMSO for solubilization), and glutathione (final concentration 5 mM or 10 mM to simulate the intracellular reducing environment) was added. The mixture was incubated at 37 °C, and samples were taken at 0, 0.5, 1, 2, 4, 6, 8, 12, and 24 h. Immediately after incubation, an equal volume of acetonitrile containing 0.1% TFA was added to terminate the reaction and precipitate the protein. After centrifugation, the supernatant was collected for HPLC analysis. Chromatographic conditions were the same as in Experiment 6, with a detection wavelength of 254 nm. The peak area of ​​the intact compound was recorded over time. Standards: Referencing commonly used methods in the field of bioconjugate drug development. Key Parameters: Temperature 37 °C ± 0.5 °C, pH 7.4 ± 0.1, and glutathione concentrations of 5 mM or 10 mM representing the intracellular and normal extracellular environments, respectively. Data processing: Plot the percentage of peak area of ​​intact compounds against time, fit the data using the first-order kinetic equation C=C0e^(-kt), calculate the fracture rate constant k and half-life t1 / 2=ln2 / k, and express the results as mean ± standard deviation (n=3).

[0107] Experiment 4: Cytotoxicity test (MTT assay) Test subject: The in vitro antiproliferative activity of the active compound of this invention against prostate cancer cell lines. Test objective: To evaluate the cytotoxicity of the active compound against PSMA-positive prostate cancer cells and determine the half-maximal inhibitory concentration (IC50). 50This study aimed to verify the tumor-killing activity of the drug fragments after release. The MTT assay utilizes the reduction of yellow MTT to purple formazan crystals by mitochondrial succinate dehydrogenase in living cells. The number of surviving cells is quantified by measuring the absorbance at 570 nm; a decrease in absorbance reflects cell proliferation inhibition or death. The experimental method involved seeding logarithmically growing prostate cancer cells (PC-3, DU145, and LNCaP, all PSMA-positive) into 96-well plates (5 × 10³ cells / well). After 24 h of culture, a series of concentrations of the active compound (0.01-100 nM, 10-fold dilution, 3 replicates per concentration) were added, and the cells were cultured for another 72 h. Then, MTT solution (5 mg / mL, 20 μL / well) was added and incubated for 4 h. The culture medium was removed, and 150 μL / well of DMSO was added to dissolve the formazan crystals. The mixture was shaken for 10 min, and the absorbance at 570 nm was measured using a microplate reader. The standard was based on the General Chapters of the Chinese Pharmacopoeia or similar evaluation methods for antitumor drugs. Key parameter: Cell density 5×10 3 / well, drug treatment time 72 h, MTT incubation time 4 h, negative control (no drug) and solvent control (with equal volume of DMSO) were set up simultaneously. Data processing: cell viability = (drug A - blank A) / (control A - blank A) × 100%, and the IC50 was calculated by nonlinearly fitting the log(concentration) versus viability curve using GraphPad Prism software. 50 Values ​​were calculated, and results were expressed as mean ± standard deviation (n=3). The IC50 of the free pharmacodynamic fragments (7-ethyl-10-hydroxycamptothecin or monomethylauratestatin E) was also determined. 50 As a positive control.

[0108] Experiment 5: Determination of water solubility and partition coefficient Test Subjects: Water solubility and lipid-water partition coefficient of the active compound of this invention. Test Objective: To evaluate the solubility and lipophilicity of the active compound in the aqueous phase, and to verify that the water solubility is significantly improved after encapsulation of the hydrophobic pharmacodynamic fragment, and that the partition coefficient is controlled within the range of 1.5-3.5 to balance membrane permeability and plasma stability. Test Principle: Water solubility is determined by the saturated solubility method, and the n-octanol-water partition coefficient (logP) is determined by the shake-flask method. LogP reflects the compound's partition tendency between the lipid and aqueous phases. Experimental methods: Water solubility determination - Excess active compound powder was gradually added to phosphate buffer (pH 7.4, 10 mL), shaken at 25 ℃ for 24 h until dissolution equilibrium was reached. After centrifugation (12000 rpm, 10 min), the supernatant was collected, diluted appropriately, and the compound concentration was determined by HPLC under the same chromatographic conditions as in Experiment 6. Partition coefficient determination - The active compound was dissolved in octanol-saturated phosphate buffer (pH 7.4) (initial concentration approximately 0.5 mg / mL), and an equal volume of phosphate buffer-saturated octanol was added. The mixture was shaken at 25 ℃ for 24 h until partition equilibrium was reached. After standing and separation, the aqueous phase and octanol phase were collected separately, diluted, and the compound concentration in both phases was determined by HPLC. The calculation was logP = log(Coctanol / Cwater). Standards: Water solubility was referenced from General Chapter 0902 of the Chinese Pharmacopoeia, Part IV, "Determination of Solubility". Partition coefficient was referenced from OECD Test No. 107, "Partition Coefficient (n-octanol / water): Shake Flask Method". Key parameters: temperature 25 ℃±0.5 ℃, shaking time 24 h to ensure equilibrium, pH 7.4±0.1. Data processing: water solubility is expressed as mg / mL, and the results are mean ± standard deviation (n=3); logP is expressed as mean ± standard deviation (n=3), and the water solubility improvement factor is calculated by comparing it with the water solubility of the free pharmacodynamic fragment and logP.

[0109] Experiment 6: Determination of Purity and Residual Solvents by High Performance Liquid Chromatography (HPLC) Test Subjects: The chemical purity and residual solvent content of the active compounds of this invention. Test Objective: To determine the content of the main peak (verified to be ≥98.0 wt%) and the content of residual solvents (N,N-dimethylformamide, acetonitrile, ethyl acetate) in the purified product to ensure compliance with quality standards. Test Principle: HPLC reversed-phase chromatography is used to separate the main peak and impurity peaks; the main peak content is calculated using the area normalization method; headspace gas chromatography is used to determine the residual volatile organic solvents. Experimental methods: HPLC – C18 column (250 mm × 4.6 mm, 5 μm), mobile phase A: 0.1% trifluoroacetic acid aqueous solution, mobile phase B: acetonitrile, gradient elution (0-5 min, 20% B; 5-25 min, 20%-80% B; 25-30 min, 80% B; 30-35 min, 80%-20% B), flow rate: 1.0 mL / min, column temperature: 30 ℃, detection wavelength: 254 nm, injection volume: 10 μL (sample concentration 1.0 mg / mL in mobile phase), chromatogram recorded and peak areas integrated; Headspace gas chromatography – approximately 100 mg of sample was accurately weighed into a 20 mL headspace vial, N,N-dimethylformamide internal standard solution was added, equilibrated at 80 ℃ for 30 min, and then 1 mL was injected. Gas chromatographic conditions: DB-624 capillary column (30 m × 0.32 mm, 1.8 μm). μm), programmed temperature ramp, flame ionization detector (FID), external standard method for quantification. Standards: HPLC method referenced Chinese Pharmacopoeia, Part IV, General Chapter 0512 "High Performance Liquid Chromatography"; residual solvent referenced Chinese Pharmacopoeia, Part IV, General Chapter 0861 "Residual Solvent Determination Method" Method II (Headspace Gas Chromatography) and ICH Q3C guidelines. Key parameters: HPLC detection wavelength 254 nm (maximum absorption of the compound), headspace equilibration temperature 80 ℃, equilibration time 30 min. Data processing: Main peak content = main peak area / (sum of all peak areas) × 100%, result is mean ± standard deviation (n=2); residual solvent concentration was calculated based on the standard curve, unit is ppm, result is mean (n=2), to determine whether DMF ≤ 880 ppm, acetonitrile ≤ 410 ppm, and ethyl acetate ≤ 5000 ppm meet the requirements.

[0110] Figure 1To investigate the effect of the substrate-to-coupling reagent molar ratio on the content of the main peak and byproducts, the experiment fixed the following parameters: CDI activation time for module A was 1.5 h, Lys reaction time was 2.5 h; solid resin loading for module B was 1.0 mmol / g, condensation time for each step was 1.5 h; Val-Cit reaction time for module C was 3.0 h, 4-aminobenzyl alcohol activation time was 1.5 h; and reaction time for module D was 1.2 h. 7-Ethyl-10-hydroxycamptothecin was used as module P, connected via carbamate bonds. The continuous flow residence time was 3.0 min, channel diameter was 0.9 mm, flow rate was 1.5 mL / min, back pressure was 1.2 MPa, reaction temperature was 25 °C, and the solvent was acetonitrile / ethyl acetate (2:1, water content 120%). The concentration was ppm (11 times the substrate mass), and the single-step reaction time was 2.0 hours. The effect of varying the molar ratio of substrate to coupling reagent in step S6 (1:0.85 to 1:1.35) on the main peak content and by-product content was investigated. Experimental results showed that when the molar ratio was 1:1.15, the main peak content reached a peak value of 98.6 ± 0.2 wt%, and the by-product content decreased to a minimum of 0.03 ± 0.01 wt%. This optimal point is approximately 20% away from the lower limit of the claim (1:1.0) and approximately 25% away from the upper limit (1:1.2), both meeting the requirement of being ≥10% from the boundary. However, when the molar ratio was below the lower limit (1:1.0), the main peak content decreased to below 98.2 wt%, and the by-product content increased to above 0.05 wt% (e.g., at 1:0.85, the main peak was only 96.5 wt%, and the by-product content reached 0.15 wt%). The percentage of unreacted residues (wt%) indicates that insufficient coupling reagent leads to incomplete reaction and accumulation of unreacted residues. When the molar ratio is higher than the upper limit of 1:1.2, the main peak content drops below 98.5 wt% and the byproduct increases to above 0.04 wt% (e.g., at 1:1.35, the main peak drops to 97.9 wt% and the byproduct reaches 0.06 wt%), indicating that excessive coupling reagent triggers an increase in side reactions. Therefore, the molar ratio range of 1:1.0-1.2 defined in the claims can ensure that the main peak content is ≥98.2 wt% and the byproduct is ≤0.05 wt%. Optimizing this parameter plays a key role in achieving the preparation of high-purity products.

[0111] Figure 2 To illustrate the effect of continuous flow residence time on batch-to-batch coefficient of variation and byproduct content, this experiment was conducted... Figure 1With fixed parameters, an optimal substrate / coupling reagent molar ratio of 1:1.1 was used, maintaining a continuous flow channel diameter of 0.9 mm, a back pressure of 1.2 MPa, and a reaction temperature of 25 °C. The flow rate was adjusted to match different residence times (0.6–6.0 min) to investigate its effect on batch-to-batch reproducibility coefficient of variation and byproduct content. Experimental results showed that when the residence time was 2.0–4.0 min, the batch-to-batch coefficient of variation decreased to a minimum of 2.0–2.2 ± 0.2–0.3%, and the byproduct content remained stable at 0.03 ± 0.01 wt%. The optimal value was 3.0 min (coefficient of variation 2.1 ± 0.2%), which was 50% away from both the lower limit (1.0 min) and the upper limit (5.0 min) of the claim, located in the central region of the parameter space. However, when the residence time was shorter than the lower limit (1.0 min), the coefficient of variation increased sharply to over 2.8%, and the byproduct content increased to 0.04 wt%. A residence time exceeding wt% (e.g., a coefficient of variation of 4.2 ± 0.5% and byproducts of 0.08 wt% at 0.6 minutes) indicates insufficient mixing time leading to uneven reaction and increased batch-to-batch variation. When the residence time exceeds the upper limit of 5.0 minutes, the coefficient of variation rises to above 2.4% and byproducts increase slightly (e.g., a coefficient of variation of 2.8 ± 0.4% and byproducts of 0.04 wt% at 6.0 minutes), possibly due to slight side reactions caused by excessively long residence time. Therefore, the residence time range of 1.0-5.0 minutes specified in the claims can ensure that the coefficient of variation between batches is ≤2.8% and byproducts are ≤0.04 wt%. This parameter optimization plays a key role in achieving process stability and product batch consistency. Compared to the coefficient of variation of 3-5% in traditional batch reactions, continuous flow technology can reduce batch-to-batch variation by 30-50% under optimized residence time conditions.

[0112] Figure 3 To illustrate the effect of reaction temperature on the main peak content and reaction time in this invention, this experiment was conducted in... Figure 1 and Figure 2With fixed parameters, an optimal substrate / coupling reagent molar ratio of 1:1.1 and an optimal continuous flow residence time of 3.0 min were used (channel diameter 0.9 mm, flow rate 1.5 mL / min, back pressure 1.2 MPa). The effects of varying reaction temperatures (10-42℃) on the main peak content and the reaction time required to reach 98% main peak content were investigated. Experimental results showed that when the reaction temperature was 25-30℃, the main peak content reached a peak value of 98.5 ± 0.3 wt%, and the reaction time was moderate (1.8-2.0 ± 0.2 hours). 25℃ was the optimal temperature used in Example 1 (main peak 98.5 wt%, reaction time 2.0 hours). This optimal range was 50-75% away from the lower limit of the claim (15℃) and 25-50% away from the upper limit (35℃), both meeting the requirement of ≥10% from the boundary. However, when the temperature was below the lower limit (15℃), the reaction rate decreased significantly, resulting in an excessively long required time (e.g., 4.5 ± 0.5 hours at 10℃, and the main peak content was only 97.8%). The percentage of the main peak content (wt%) indicates that insufficient activation energy at low temperatures affects conversion efficiency. When the temperature exceeds the upper limit of 35℃, the main peak content drops below 98.3 wt%, and although the reaction time is shortened, the product quality deteriorates (e.g., at 42℃, the reaction time is only 1.2 hours, but the main peak drops to 97.5±0.5wt%). This indicates that high temperature triggers side reactions (such as ester bond hydrolysis and carbamate decomposition), leading to the loss of the main product. Therefore, the temperature range of 15-35℃ defined in the claims can ensure that the main peak content is ≥98.2 wt% and the reaction time is ≤3.2 hours. This parameter optimization plays a key role in balancing the reaction rate and product purity. Within the optimal range of 25-30℃, a synergistic optimization of high purity (98.5wt%) and high efficiency (≤2.0 hours) can be achieved.

[0113] Figure 4 The Fourier transform infrared (FTIR) spectrum of the ABCDP active compound of Example 1 of this invention is shown. The ABCDP active compound of Example 1 exhibits absorption of key functional groups in FTIR consistent with the structural design, demonstrating the rationality of the scheme and the integrity of the structure. N–H / O–H stretching of hydrogen bonding occurs in the high wavenumber region, reflecting the presence of urea groups and peptide residues; 1700 cm⁻¹ -1 The simultaneous discrete absorptions of lactone C=O, carbamate C=O, and urea C=O nearby confirm the camptothecin core, the carbamate bond between P and D, and the urea structure of module A; 1650 cm⁻¹ -1 With 1540 cm -1 Typical amide I and amide II bands appear at the respective locations, supporting the formation of the peptide bond and Val-Cit linkage in module B; 1400 cm -1 Symmetrical stretching of the carboxylate occurred nearby, consistent with the ionization state of Glu-urea-Lys and the peptide under neutral conditions; 1260–1170 cm⁻¹ -1The C–N / C–O vibration of the carbamate in the region corresponds to 1100–1000 cm⁻¹ -1 C–O absorptions jointly indicate that DP is linked via a carbamate pathway; the aromatic ring characteristics of the self-destructive aminobenzyl group are observed at 1600, 1510, and 860 cm⁻¹. -1 Clearly visible in the region; C–S and S–S absorptions appear in the low wavenumber range, supporting the introduction of disulfide bond linkers. The same peak series is reproduced consistently in the ATR and transmission modes, with only the relative intensity and low wavenumber enhancement varying with the ATR penetration depth, further confirming the correctness of the coupling sequence and functional group configuration and the stability of the system under neutral conditions.

[0114] Figure 5 The SPR results of Example 1 of this invention show a clear concentration-dependent binding and reversible dissociation with the target. The sensing curve rises rapidly during the injection period and decays exponentially during the elution period. The 1:1 Langmuir model global fitting can accurately cover the measured response with low residuals at a concentration of 1.25 nM. The residual distribution shows no systematic deviation or trend with time / concentration, suggesting the absence of significant multivalent effects, mass transfer limitations, or nonspecific adsorption. The binding rate constant and dissociation rate constant obtained from the fitting provide nanomolar apparent affinity, and the parameters are stable across repeated cycles and different concentrations, proving that the interaction is specific, reversible, and kinetically consistent. These results are consistent with the unit-point recognition and receptor targeting mechanism of the formulation design, indicating that the construction strategy is reasonable at the molecular recognition level and can achieve reliable binding control at physiologically relevant concentrations, providing a solid basis for subsequent in vivo and in vitro functional verification.

[0115] The performance of the examples and comparative examples is summarized in Tables 1 and 2. In Comparative Example 1, L-ornithine was used instead of L-lysine as the A-module targeting ligand. Due to its shorter side chain (4 carbons vs. 5 carbons), the spatial matching with the PSMA receptor active pocket was reduced, resulting in a deterioration of the binding dissociation constant Kd from 11 nM in Example 1 to 16 nM. The tumor enrichment rate also decreased accordingly from 43% to 32%, ultimately leading to a decrease in the half-maximal inhibitory concentration (IC50) of the three prostate cancer cell lines. 50 All values ​​increased to the range of 2.4-2.7 nM, demonstrating that the precise design of the targeting module has a crucial impact on the overall therapeutic effect. Comparative Example 2 introduced a D-amino acid configuration into the hexapeptide sequence of module B. Although it maintained PSMA targeting performance comparable to Example 1 (Kd 11 nM, enrichment rate 42%), the substrate cleavage efficiency Kcat / Km containing D-amino acids decreased from 4.5 × 10⁻⁶ in Example 1 due to the strict stereoselectivity of prostate-specific antigen (PSA) to its substrates. 5 M -1 ·s -1 It plummeted to 1.6 × 10 5 M -1 ·s-1 The plasma half-life was also shortened from 20 hours to 8 hours. The loss of enzyme selectivity prevented the drug from being effectively released at the tumor site, ultimately reducing the IC50 value. 50 The degradation to 4.2-4.8 nM indicates that the full L configuration is indispensable for maintaining the cleavage triggering function; Comparative Example 3 replaced the payload of the P module from 7-ethyl-10-hydroxycamptothecin with unmodified camptothecin, although its PSMA targeting, cleavage release, and self-assembly performance were close to those of Example 1 (Kd 11 nM, Kcat / Km 4.5 × 10⁻⁶). 5 M -1 ·s -1 (Particle size 30 nm), but due to the loss of the 10-position hydroxyl modification, the binding affinity with DNA topoisomerase I is reduced, resulting in significantly weakened cytotoxicity and IC50. 50 The degradation from 1.4-1.7 nM in Example 1 to 6.2-7.0 nM demonstrates that chemical modification of the drug molecule plays a decisive role in the final antitumor activity. Comparative Examples 4-10 deviated from the optimal process parameters of Example 1 in steps S2, S3, S4, S6, S7, S8, and S9 (e.g., shortened reaction time, reduced solvent volume, insufficient continuous flow residence time, etc.). Although these deviations had a relatively small impact on the core functions of target recognition and enzymatic release (Kd remained at 11 nM, and Kcat / Km remained at 4.2-4.5 × 10⁻⁶), they were not significant. 5 M -1 ·s -1 However, this resulted in a decrease in the main peak content from 98.5% in Example 1 to the range of 96.5-97.8%, and an increase in the byproduct content from 0.03% to 0.05-0.15%. The purity degradation was most pronounced in Comparative Example 4 (S2 step reaction time 1.0 h vs. optimal 1.5 h) and Comparative Example 10 (S9 step reaction time 1.0 h vs. optimal 2.0 h) (96.8% and 96.5%), indicating that insufficient reaction time leads to the accumulation of unreacted residues and increased side reactions. Comparative Example 11 used the exact same formulation and process parameters as Example 1 but did not utilize continuous flow technology for optimization. Although its targeting, enzymatic cleavage, and antitumor activities were comparable to Example 1 (Kd 11 nM, IC50), it still showed significant improvement. 50The concentration of the main peak decreased slightly to 98.1% due to uneven mixing and local concentration fluctuations in the batch reaction (1.4-1.7 nM), while the byproduct concentration increased to 0.04%. Comparative Examples 12-14 represent the non-optimal conditions of steps S10 (purification), S11 (lyophilization solvent), and S12 (batch reaction vs. continuous flow), respectively. Their performance was between that of Comparative Examples 4-10 and Comparative Example 11, with a main peak concentration of 97.5-98.0%. This indicates that downstream process optimization is equally important for product purity. Overall, the examples achieved high affinity binding of PSMA (Kd < 15 nM) and efficient PSA cleavage release (Kcat / Km > 3.5 × 10⁻⁶) through the synergistic optimization of L-lysine-targeting ligand, all-L-configuration hexapeptide, hydroxylated camptothecin, and continuous flow precision process. 5 M -1 ·s -1 Nanoscale uniform self-assembly (particle size 20-60 nm, PDI < 0.20) and potent antitumor activity (IC50). 50 The comprehensive performance advantage of <2.0 nM) and the fact that the replacement of any single module or deviation of process parameters will lead to the deterioration of at least one key performance indicator fully demonstrates the indispensability of each element of the technical solution of this invention and the necessity of overall optimization.

[0116] Table 1. Target recognition, enzyme release, and self-assembly performance data.

[0117] Table 2 Physicochemical properties, antitumor activity, and purity data

[0118] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that any equivalent structural transformations made under the concept of the present invention and using the contents of the specification and drawings of the present invention should be covered within the scope of protection of the claims of the present invention.

Claims

1. A process for the preparation of a prostate cancer therapeutically active compound, said active compound being linked in linear order from A-B-C-D-P, wherein A is a prostate specific membrane antigen binding unit, B is the amino acid sequence HSSKLQ, C is a L-valine-L-citrulline-p-aminobenzyl self-immolative unit, D is a disulfide bond containing linker arm, and P is a pharmacophore fragment having a hydroxyl or primary amine attachment site; characterized in that, comprising the steps of: S1, preparing an A module; S2, synthesizing a B module; S3, preparing a C module; S4, introducing a D module containing a disulfide bond to be connected to the C module; S5, connecting the P to the D module with a carbonate or carbamate bond; S6, assembling in the following order: first connecting the A to the B with an amide bond to form A-B, then connecting the A-B to the C with an amide bond to form A-B-C, then connecting the A-B-C to the D module to form A-B-C-D, and finally connecting the A-B-C-D to the P with a carbonate or carbamate bond to form A-B-C-D-P; wherein the molar ratio of the substrate to the coupling reagent in each condensation or coupling step is 1:1.0-1.2, and the reaction time for each step is 0.5-4.0 h.

2. The production method according to claim 1, characterized by, The A module is obtained by activating L-glutamic acid with N,N'-carbonyldiimidazole in N,N-dimethylformamide for 0.5-3.0 h, then adding L-lysine or L-ornithine and continuing the reaction for 1.0-4.0 h to obtain L-glutamic acid-urea-L-lysine or L-glutamic acid-urea-L-ornithine, with an unreacted amine residual amount of no more than 0.20 wt%.

3. The production method according to claim 1, characterized by, The B module is obtained by sequentially condensing L-histidine, L-serine, L-serine, L-lysine, L-leucine, and L-glutamine in the order of H-S-S-K-L-Q on a polystyrene-based solid-phase carrier resin with a loading capacity of 0.8-1.2 mmol / g, and each condensation time being 0.5-3.0 h.

4. The production method according to claim 1, characterized by, The C module is obtained by reacting L-valine with L-citrulline for 2.0-4.0 h to form Val-Cit, activating 4-aminobenzyl alcohol with p-nitrophenyl chloroformate for 0.5-3.0 h, and connecting it to the Val-Cit to form a Val-Cit-p-aminobenzyl self-destruction unit, with an unreacted hydroxyl or primary amine residual amount of no more than 0.30 wt%.

5. The production method according to claim 1, characterized by, The D module is a disulfide-containing linking arm, which is introduced by reacting N-hydroxysuccinimide-3-pyridine disulfide propionate with the amine or carboxyl derivative site on the C module for 0.5-2.0 h to connect the C to the D and form —S—S— in the resulting linking arm.

6. The method of claim 1, wherein, The P is 7-ethyl-10-hydroxy camptothecin or monomethyl auristatin E, and is connected to the D module with a carbonate or carbamate bond.

7. A prostate cancer therapeutically active compound prepared by the method of any one of claims 1 to 6; said active compound is composed of five modules A-B-C-D-P connected in linear order, wherein A is L-glutamic acid-urea-L-lysine or L-glutamic acid-urea-L-ornithine, B is the amino acid sequence HSSKLQ, C is L-valine-L-citrulline-p-aminobenzyl self-immolative unit, D is a disulfide bond containing linker connected to said C, and P is a pharmacophore fragment having a hydroxyl or primary amine attachment site connected to said D via a carbonate or carbamate linkage; stable in aqueous neutral environment; releases the pharmacophore fragment upon action of prostate specific antigen or cathepsin B; disulfide bond is cleaved in reducing environment; self-assembles in water to form aggregates having an average hydrated diameter of 20-60 nm and a polydispersity index of no more than 0.

20.

8. Active compounds according to claim 7, characterized in that Said P is 7-ethyl-10-hydroxy camptothecin or monomethyl auristatin E.

9. Active compound according to claim 7, characterized in that Said aggregates have an average hydrated diameter of 20-40 nm and a polydispersity index of no more than 0.

15.

10. Use of an active compound according to any one of claims 7 to 9 for the manufacture of a medicament for the treatment of prostate cancer positive for prostate-specific membrane antigen, characterized in that, Said drug is administered at a dose of 0.1-10 mg / kg by intravenous or intraperitoneal injection.

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