Fluorescent probe for targeting prostatic cancer PSMA as well as preparation method and application of fluorescent probe

Abstract

The invention discloses a fluorescent probe for targeting prostatic cancer PSMA as well as a preparation method and application of the fluorescent probe. On the basis of a PSMA ligand Glu-Urea-Lys structure, a novel indocyanine green (IR820) derivative is adopted to develop a PSMA fluorescent targeting probe PSMA-F, and structures such as 6-aminocaproic acid and tyrosine iodide are added into a connecting arm, so that the distance between a target molecule and a fluorophore is prolonged, steric hindrance is reduced, the solubility and rigidity of the probe are adjusted, and the affinity of the probe and a PSMA target spot is improved. Evaluation on the detection efficiency, in-vivo metabolism and the like of the PSMA-F probe proves that the PSMA-F probe has remarkable tumor targeting property and has the potential of clinical transformation research.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to a fluorescent probe targeting prostate cancer PSMA and a preparation method and application thereof. BACKGROUND

[0002] The treatment method of prostate cancer (PCa) depends on the disease stage at the initial diagnosis. For early localized prostate cancer patients who need surgical resection, radical prostatectomy (RP) is still the main treatment method. However, due to the infiltrative growth of the tumor during surgical treatment, irregular tumor margins can make it difficult to completely remove the cancerous tissue without damaging normal tissue. Although complete removal of the prostate can ensure complete removal of the primary lesion, it can cause damage to functional tissues such as surrounding nerves, thereby causing side effects including urinary incontinence and erectile dysfunction. Clinicians need to face the challenge of balancing complete removal and preservation of nerve function and the challenge of surgical margin judgment.

[0003] Prostatic specific membrane antigen (PSMA) is highly expressed in prostate cancer cells and positively correlated with Gleason score, making PSMA an important biomarker for prostate cancer and a target with great application potential for diagnosis and treatment. In actual use, the small molecules targeting PSMA that are widely studied mainly include glutamic acid urea-based types, including EuK types (glutamic acid-urea-lysine) and EuE types (glutamic acid-urea-glutamic acid). In view of the actual problems encountered during surgery, the development of near-infrared (NIR) fluorescent molecules targeting PSMA for fluorescence navigation in prostate cancer surgery has great application potential.

[0004] In recent years, intraoperative imaging techniques that assist in positioning and correctly identifying tumor boundaries have been continuously tried in basic research and clinical practice. Among them, ICG has been used for imaging of blood vessels and lymphatic drainage due to its high body clearance rate and good biological safety, and has been widely used for intraoperative navigation and postoperative evaluation. However, as a non-specific fluorescent probe, ICG does not have targeting specificity. Passive uptake of such a probe can result in a high imaging background, and the detection results are often unsatisfactory. Therefore, it is of great significance to develop a fluorescent probe with better targeting specificity for intraoperative navigation of RP. SUMMARY

[0005] To make up for the deficiencies of the prior art, the application provides a fluorescent probe targeting prostate cancer PSMA and a preparation method and application thereof.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: The first aspect of this invention provides a fluorescent probe targeting PSMA in prostate cancer, the fluorescent probe having a structure as shown in formula (I):

[0007] Equation (I) Wherein, F is a fluorescent group.

[0008] In some implementations, the fluorescent probe is a type of molecule with characteristic fluorescence that can sensitively change its own fluorescence properties (such as excitation and emission wavelengths, intensity, lifetime, polarization, etc.) according to changes in the properties of its environment (such as polarity, refractive index, viscosity, etc.), thereby enabling the detection and labeling of different substances or biological processes. A fluorescent probe typically consists of three important components: a recognition-binding group (R): a group with a reactive site with the analyte, responsible for targeting; a signal reporter group (F): also known as a fluorophore or chromophore, responsible for converting chemical signals into optical signals; and a linking group (S): generally an alkyl chain, which connects the various groups and maintains structural stability. Fluorescent probes are affected by the surrounding environment; after chemical or physical interaction with a substance, their fluorescence signal changes accordingly, thereby revealing the characteristics of the surrounding environment or specific information present in the environment. By utilizing the contrast between specific molecular probe signal changes and the intrinsic properties of in vivo tissues, fluorescence imaging can demonstrate high spatial resolution in vivo.

[0009] In some embodiments, the fluorescent group refers to a compound structure capable of absorbing photons of a certain energy (wavelength) and emitting fluorescence. Fluorescent groups are typically aromatic groups or planar heterocyclic molecules with π bonds. Based on emission wavelength, fluorescent groups can be classified into blue fluorescent groups, green fluorescent groups, orange-red fluorescent groups, near-infrared fluorescent groups, and far-infrared fluorescent groups. Near-infrared fluorescent groups can be further divided into near-infrared region I (NIR-I, 700-900 nm) and near-infrared region II (NIR-II, 1000-1700 nm). Near-infrared region I fluorescent groups include indocyanine green (ICG), cyanine dyes (Cy series), BODIPY dyes, and rhodamine dyes; near-infrared region II fluorescent groups include the IR-1080 series and BF-MTPA / BF-TPA dyes.

[0010] Furthermore, the fluorescent group is a near-infrared fluorescent group.

[0011] Furthermore, the fluorescent group is a near-infrared first-region fluorescent group.

[0012] Furthermore, the fluorescent group has a structure as shown in formula (II):

[0013] Formula (II) Further, the fluorescent probe has a structure as shown in Formula (III):

[0014] Formula (III) The second aspect of the present application provides a preparation method of a fluorescent probe targeting prostate cancer PSMA, the method comprising: taking compound 1 as a substrate, adding a fluorescent group after reaction, to obtain a fluorescent probe shown in Formula (III), wherein the compound 1 is a product obtained after reaction of EUK with Ahx-OH, Lys(Dde)-OH, Phe-OH, 3-Iodo-Tyr(OtBu)-OH, Gly-OH and Asp(OtBu)-OH.

[0015] Further, the method comprises: taking compound 2 as a substrate, adding Asp(OtBu)-OH and a fluorescent group after reaction, to obtain a fluorescent probe shown in Formula (III), wherein the compound 2 is a product obtained after reaction of EUK with Ahx-OH, Lys(Dde)-OH, Phe-OH and 3-Iodo-Tyr(OtBu)-OH.

[0016] Further, the method comprises: taking compound 3 as a substrate, adding Gly-OH, Asp(OtBu)-OH and a fluorescent group after reaction, to obtain a fluorescent probe shown in Formula (III), wherein the compound 3 is a product obtained after reaction of EUK with Ahx-OH, Lys(Dde)-OH and Phe-OH.

[0017] Further, the method comprises: taking compound 4 as a substrate, adding 3-Iodo-Tyr(OtBu)-OH, Gly-OH, Asp(OtBu)-OH and a fluorescent group after reaction, to obtain a fluorescent probe shown in Formula (III), wherein the compound 4 is a product obtained after reaction of EUK with Ahx-OH and Lys(Dde)-OH.

[0018] Further, the method comprises: taking compound 5 as a substrate, adding Phe-OH, Iodo-Tyr(OtBu)-OH, Gly-OH, Asp(OtBu)-OH and a fluorescent group after reaction, to obtain a fluorescent probe shown in Formula (III), wherein the compound 5 is a product obtained after reaction of EUK with Ahx-OH.

[0019] Further, the method comprises: taking compound 6 as a substrate, adding Lys(Dde)-OH, Phe-OH, 3-Iodo-Tyr(OtBu)-OH, Gly-OH, Asp(OtBu)-OH and a fluorescent group to react, to obtain the fluorescent probe shown in formula (III), wherein the compound 6 is a product obtained by reacting EUK with Ahx-OH.

[0020] Further, the method comprises: taking resin Resin-EUK as a substrate, sequentially synthesizing a peptide resin by using standard Fmoc chemistry on Fmoc-Ahx-OH, Fmoc-Lys(Dde)-OH, Fmoc-Phe-OH, Fmoc-3-Iodo-Tyr(OtBu)-OH, Fmoc-Gly-OH, Fmoc-Asp(OtBu)-OH; removing the Fmoc protecting group of the peptide resin, adding a fluorescent group to react to synthesize a fluorescent peptide resin; and treating the fluorescent peptide resin with a cleavage mixture to obtain the fluorescent probe shown in formula (III).

[0021] In some embodiments, the resin is mainly polystyrene resins and modified bodies thereof, and common types include Wang Resin, Rink Amide Resin, Mermaid Resin, PAL Resin, CTC Resin, etc. These resins are usually connected with a solid carrier through a connecting arm to provide a cleavable polypeptide connection point, facilitating subsequent deprotection and condensation reactions, and finally cleaving to obtain the target polypeptide chain under acidic conditions.

[0022] In some embodiments, the standard Fmoc chemistry is a method for synthesizing a polypeptide by protecting the α-amino group of an amino acid with a 9-fluorenylmethyloxycarbonyl (Fmoc) group on a solid carrier. The core of the method is to utilize the basic removal characteristics of the Fmoc group in combination with stepwise condensation reactions to realize the extension of the peptide chain. The Fmoc group is a protecting group that is stable to acid but unstable to base, and can be conveniently removed under alkaline conditions (such as piperidine / DMF solution) to expose the free amino group, providing an active site for the next coupling reaction. In solid-phase synthesis, the C-terminal amino acid of the peptide chain is first fixed on an insoluble resin, and then the steps of deprotection, washing, activation, coupling, etc. are performed in cycles to gradually extend the peptide chain to the target length.

[0023] In some embodiments, the steps of the standard Fmoc chemistry include selecting an appropriate resin (such as CTC resin, Wang resin, etc.), covalently attaching the carboxyl group of the first amino acid to the resin. The activation process usually involves the reaction of the resin with an activating agent (such as dithioaminimidazole) to introduce a reactive group. The resin is treated with a basic solution (such as a 20% piperidine / DMF solution) to remove the Fmoc group, exposing the free amino group. The resin is washed with a suitable solvent (such as DMF, DCM, methanol, etc.) to remove unreacted materials and byproducts. The next Fmoc-protected amino acid is activated by an activating agent (such as DCC / HOBt, HBTU / DIPEA, etc.) to activate its carboxyl end, and then undergoes a condensation reaction with the free amino group on the resin to form a peptide bond. The temperature and time of this step are strictly controlled to ensure the efficiency and selectivity of the activation. The above steps are repeated, and the deprotection, washing, activation, coupling, etc. are repeated according to the sequence of the target peptide chain, until the peptide chain is extended to the desired length. The resin is treated with a strong acid (such as TFA) or a specific cleavage solution to simultaneously remove the side chain protecting groups and cleave the polypeptide from the resin, obtaining a free polypeptide. The polypeptide is purified by liquid chromatography, and the target peak liquid is collected for freeze-drying treatment to obtain a fluffy powder-like solid polypeptide. The polypeptide is identified by mass spectrometry, liquid chromatography, etc. to confirm its purity and molecular weight.

[0024] Further, the EUK is glutamic acid-urea-lysine.

[0025] Further, the fluorescent group is shown as formula (II).

[0026] Further, the amount of Fmoc-Ahx-OH, Fmoc-Lys(Dde)-OH, Fmoc-Phe-OH, Fmoc-3-Iodo-Tyr(OtBu)-OH, Fmoc-Gly-OH, Fmoc-Asp(OtBu)-OH and the fluorescent group is 4 / 3 molar equivalents based on the amount of the substrate resin Resin-EUK.

[0027] Further, the cleavage mixture includes 90% trifluoroacetic acid, 5% triisopropylsilane, and 5% H2O by weight percentage.

[0028] The third aspect of the present application provides a detection composition comprising the fluorescent probe of the first aspect of the present application.

[0029] Further, the detection composition further includes other products that can be used to detect PSMA.

[0030] Further, the other products that can be used to detect PSMA include PSMA-targeting imaging agents or anti-PSMA antibodies.

[0031] In some embodiments, the PSMA-targeting imaging agents include, but are not limited to, Posluma (flotufolastat F 18), fluciclatide injection (XTR020), TLX007-CDx, PSMA11, PSMA617. The other products that can be used to detect PSMA are not limited to the specific products listed above, and any product that can be used to detect PSMA will fall within the scope of the present application.

[0032] The fourth aspect of the present application provides a reagent for detecting the level of PSMA in a sample, which comprises the fluorescent probe of the first aspect of the present application or the detection composition of the third aspect of the present application and a solvent.

[0033] In some embodiments, the solvent can be any one or more of water, physiological saline, a buffer or ethanol. The buffer refers to a mixed solution composed of a weak acid and its salt, a weak base and its salt, which can offset or reduce the influence of an added strong acid or strong base on the pH of the solution to some extent, thereby maintaining the pH of the solution relatively stable. Common buffer systems include: a weak acid and its salt (such as HAc— NaAc); a weak base and its salt (NH3·H2O— NH4Cl); an aqueous solution composed of an acid salt of a polybasic weak acid and its corresponding secondary salt (such as NaH2PO4— Na2HPO4). Common buffers include phosphate buffered saline (PBS), Tris buffer, HEPES buffer, citric acid buffer, acetic acid buffer, etc.

[0034] The fifth aspect of the present application provides a kit for detecting the level of PSMA in a sample, which comprises the fluorescent probe of the first aspect of the present application, the detection composition of the third aspect of the present application or the reagent of the fourth aspect of the present application.

[0035] In some embodiments, the kit further comprises instructions or a label, a positive control, a negative control, a buffer, an adjuvant or a solvent, and further comprises one or more containers for containing the compositions contained in the kit. The compositions can be in liquid form or can be lyophilized. Suitable containers for the compositions include, for example, bottles, vials, syringes, and test tubes. Containers can be formed from a variety of materials, including glass or plastic. The instructions or label detail how to use the kit for detecting the sample and the kit for detecting the level of PSMA.

[0036] In some embodiments, the components of the kit may be packaged in an aqueous medium or in lyophilized form. The container for the kit typically includes at least one vial, test tube, flask, bottle, syringe, or other container in which the components can be placed, and preferably appropriately aliquoted. In cases where the kit contains more than one component, the kit typically also includes a second, third, or other additional container in which the additional components can be placed individually. However, various combinations of components can be contained in the vial.

[0037] The sixth aspect of the present invention provides any of the following applications: 1) The use of the fluorescent probe described in the first aspect of the present invention in the preparation of reagents or kits for detecting PSMA levels in samples.

[0038] 2) The use of the fluorescent probe described in the first aspect of the present invention in the preparation of a product for fluorescent navigation in prostate cancer surgery.

[0039] 3) The use of the reagents described in the fourth aspect of the present invention or the kits described in the fifth aspect of the present invention in the preparation of products for diagnosing prostate cancer.

[0040] In some embodiments, the term "sample" refers to a sample obtained or derived from a biological source of interest as described herein (e.g., tissue or organism or cell culture). In some embodiments, the biological sample is or comprises biological tissue or fluid. In some embodiments, the biological sample may be or comprise bone marrow; blood; blood cells; ascites; tissue or fine-needle biopsy samples; cell-containing body fluids; free-floating nucleic acids; sputum; saliva; urine; cerebrospinal fluid, peritoneal fluid; pleural fluid; lymph; gynecological fluids; skin swabs; vaginal swabs; oral swabs; nasal swabs; rinsing or lavage fluids, such as catheter lavage or bronchoalveolar lavage fluid; aspirates; scrapings; bone marrow samples; tissue biopsy samples; surgical samples; feces, other body fluids, secretions and / or excretions; and / or cells derived from them, etc. In some embodiments, the biological sample is or comprises cells obtained from an individual. In some embodiments, the obtained cells are or comprise cells from the individual from whom the sample was obtained. In some embodiments, the sample is a "primary sample" obtained directly from the source of interest by any appropriate means. For example, in some implementations, primary biological samples are obtained by methods selected from the group consisting of: biopsy (e.g., fine-needle aspiration or tissue biopsy), surgery, collection of bodily fluids (e.g., blood, lymph, feces, cerebrospinal fluid, etc.).

[0041] Advantages and beneficial effects of the present invention: The present application is based on the PSMA ligand Glu-Urea-Lys structure, and a PSMA fluorescent targeting probe PSMA-F is developed by using a new indocyanine green (IR820) derivative, and 6-aminohexanoic acid, iodinated tyrosine and other structures are added in the connecting arm, so as to prolong the distance between the target molecule and the fluorescent group, reduce the steric hindrance, adjust the solubility and rigidity of the probe, and increase the affinity of the probe to the PSMA target point. Through the evaluation of the detection efficiency and in-vivo metabolism of the PSMA-F probe, it is proved that the PSMA-F probe has significant tumor targeting property and has the potential for clinical transformation research. BRIEF DESCRIPTION OF DRAWINGS

[0042] Figure 1 A flow chart for preparing part of raw materials of the PSMA-F probe, wherein A is a preparation flow chart of Fmoc-3-Iodo-Tyr(tBu)-OH, B is a preparation flow chart of Resin-EuK, and C is a preparation flow chart of a fluorescent group.

[0043] Figure 2 A flow chart for preparing the PSMA-F probe.

[0044] Figure 3 A graph of optical properties of the PSMA-F, wherein A is an absorption spectrum, B is an emission spectrum, C is a graph of linear relationship between the concentration of the PSMA-F and the absorption of light, the slope is a molar absorption coefficient, and D is a light stability graph.

[0045] Figure 4 A graph of in-vivo biodistribution results of the PSMA-F, wherein A is a fluorescence image of 24 h tumor and other tissue (or organ) biodistribution (1. tumor; 4. liver; 7. kidney), B is an in-vivo distribution graph of the PSMA-F at each time point, and C is a graph of tumor muscle fluorescence intensity ratio and tumor liver fluorescence intensity ratio.

[0046] Figure 5 A graph of in-vivo biodistribution of the fluorescent group.

[0047] Figure 6 A fluorescence imaging graph of 22Rv1 tumor-bearing mice after injection of the PSMA-F for 24 h. DETAILED DESCRIPTION

[0048] In order to make the purpose, technical scheme and advantages of the present application more clear, the technical scheme in the present application will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the protection scope of the present application.

[0049] Unless otherwise indicated, technical or conditions in the examples were carried out according to the techniques or conditions described in the literature or according to the product manual. Unless otherwise indicated, the reagents or instruments used were conventional products that can be purchased through regular channels.

[0050] Example 1 Preparation method of fluorescent probe PSMA-F 1. Experimental materials: All chemicals were purchased from commercial suppliers and used without further purification. 2-CTC Resin (BD302542, Bide Pharmatech Co., Ltd.), N6-Cbz-L-lysine benzyl ester hydrochloride (BD21948, Bide Pharmatech Co., Ltd.), Glu(OtBu)2 (BD731434, Bide Pharmatech Co., Ltd.), triethylamine (BD155212, Bide Pharmatech Co., Ltd.), DCM (BD91203, Bide Pharmatech Co., Ltd.), HOBt (BD9926, Bide Pharmatech Co., Ltd.), HBTU (BD23222, Bide Pharmatech Co., Ltd.), DMF (BD105599, Bide Pharmatech Co., Ltd.), Fmoc-Tyr(tbu)-OH (BD8611, Bide Pharmatech Co., Ltd.), Fmoc-Ahx-OH (BD22987, Bide Pharmatech Co., Ltd.), Fmoc-Lys(Dde)-OH (BD32025, Bide Pharmatech Co., Ltd.), Fmoc-Phe-OH (BD8605, Bide Pharmatech Co., Ltd.), Fmoc-Asp(OtBu)-OH (BD8581, Bide Pharmatech Co., Ltd.), Fmoc-Gly-OH (BD8594, Bide Pharmatech Co., Ltd.), DIPEA (N741860, Shanghai McLean Biotechnology Co., Ltd.), new indocyanine green (N815030, Shanghai McLean Biotechnology Co., Ltd.).

[0051] The preparation process is shown in Figure 1 , Figure 2 .

[0052] 2. Preparation of Fmoc-3-Iodo-Tyr(OtBu)-OH: (a) A mixture of iodine (0.129 g, 0.50 mmol) and silver sulfate (0.158 g, 0.50 mmol) was dissolved in methanol (8 mL) and stirred at 50 °C for 10 min. Fmoc-Tyr(tbu)-OH (1, 0.193 g, 0.42 mmol) was then added and stirred at 50 °C for an additional 2 h. The reaction mixture was filtered and concentrated at low pressure. The resulting cream solid was dissolved in ethyl acetate (10 mL) and washed with sodium thiosulfate (0.1 M, 3 x 10 mL), then the combined organic layers were dried over magnesium sulfate, filtered, and evaporated at low pressure to yield a white solid. Purification via column chromatography (Si02; 100% hexanes to 50 / 50 hexanes / EtOAc) yielded compound 2 as a colorless crystalline solid and compound 3 as a white crystalline solid.

[0053] (b) Compound 2 (0.102 g, 0.17 mmol) was dissolved in THF (1.5 mL). Lithium hydroxide dissolved in ice water (2 mL) (0.014 g, 0.33 mmol) was added to the THF solution and stirred at 0 °C for 30 min. One equivalent of LiOH in water was added and the solution was stirred for an additional 30 min. The reaction was quenched with ice-cold HC1 (0.1 M, 20 mL) and extracted in ethyl acetate (3 x 15 mL). The organic layers were combined, dried over magnesium sulfate, filtered, and concentrated under reduced pressure to yield 4 as a white crystalline solid.

[0054] 3. Preparation of Resin-EUK N6-Cbz-L-lysine benzyl ester hydrochloride (5, 1 g, 2.46 mmol) and triethylamine (997 mg, 9.85 mmol) were dissolved in 50 mL DCM.

[0055] (a) Under ice-salt bath conditions, add triphosgene (243 mg, 0.82 mmol), and continue to react at room temperature for 3 hours after dropwise addition is complete.

[0056] (b) Add Glu(OtBu)2 (638 mg, 2.46 mmol) and triethylamine (747 mg, 7.38 mmol) to the reaction solution, and purify the reaction via column chromatography to obtain compound 6.

[0057] (c) Compound 6 was deprotected with benzyl ester and Cbz in methanol under catalysis of 100 mg 10% Pd / C and hydrogen. The crude product was purified by silica gel column to obtain compound 7.

[0058] (d) Compound ⑦ (280 mg, 0.65 mmol) was dissolved in 20 mL of dioxane / water (2.5 / 1, v / v), sodium bicarbonate (NaHCO3, 164 mg, 1.95 mmol) and Fmoc-Cl (201 mg, 0.78 mmol) were added, and stirred at room temperature for 15 minutes. After the reaction was completed, 200 mL of ethyl acetate (EA) was added to the reaction solution, washed with water twice, the organic phase was collected, dried over anhydrous sodium sulfate (Na2SO4), and the solvent was removed under reduced pressure. The crude product was purified by silica gel column to obtain compound ⑧.

[0059] (e) 1 g of 2-CTC Resin was taken in a 50 mL solid phase synthesis tube, swelled with dichloromethane for 1 hour, and after the solvent was removed by suction, a DCM / DMF solution (1:1, v / v) of compound ⑧ was added. After reacting at room temperature for 3 hours, it was capped four times with DCM / MeOH / DIPEA (10:10:1, v / v / v) for 10 minutes each time, washed with methanol, and dried to constant weight to obtain compound ⑨ (Resin-EuK).

[0060] 4. Preparation of a fluorescent group: Synthesis structure: 4-[2-[(E)-2-[(E)-2-(4-carboxyphenoxy)-3-[(E)-2-(1,1-dimethyl-3-(4-sulfonatobutyl)-1,3-dihydro-2H-benzo[e]indol-2-ylidene)ethylidene]cyclohex-1-en-1-yl]vinyl]-1,1-dimethyl-1H-benzo[e]indol-3-ium-3-yl]butane-1-sulfonate.

[0061] 4-(2-((E)-2-((E)-2-(4-carboxyphenoxy)-3-((E)-2-(1,1-dimethyl-3-(4-sulfonatobutyl)-1,3-dihydro-2H-benzo[e]indol-2-ylidene)ethylidene)cyclohex-1-en-1-yl)vinyl)-1,1-dimethyl-1H-benzo[e]indol-3-ium-3-yl)butane-1-sulfonate. Preparation method: (a) Take new indocyanine green (ICG, 200 mg, 0.24 mmol) and tert-butyl p-hydroxybenzoate (45 mg, 0.26 mmol) in a 50 ml round bottom flask, add 20 ml acetonitrile (ACN) to dissolve, add potassium carbonate (K2CO3, 50 mg, 0.36 mmol), heat to 50 °C with electromagnetic stirring, stop the reaction after 20 hours of reaction. After reverse phase high performance liquid chromatography (HPLC), green solid (134 mg) is obtained.

[0062] (b) Take the green solid obtained in the previous step in a round bottom flask, add 4 ml trifluoroacetic acid (TFA) and 4 ml dichloromethane (DCM), react for 3 hours with electromagnetic stirring. After the reaction is complete, remove the solvent to obtain green solid (120 mg), which is the fluorescent group.

[0063] 5. Preparation of PSMA-F: Synthesis structure: 4-[2-[(E)-2-[(E)-2-(4-[[[(3S,7S,21S,29S)-21-[(R)-2-[(R)-2-(2- aminoacetamido)-3-(4-hydroxy-3-iodophenyl)propanamido]-3- phenylpropanamido]-1,3,7,29-tetracarboxy-5,13,20,27-tetraoxo-4,6,12,19,26- pentaazanonacosa-29-yl]formamido]phenoxy]-3-[(E)-2-(1,1-dimethyl-3-(4- sulfobutyl)-1,3-dihydro-2H-benzo[e]indol-2-ylidene)ethylidene]cyclohex-1-en-1- yl]vinyl]-1,1-dimethyl-1H-benzo[e]indol-3-ium-3-yl]butane-1-sulfonate.

[0064] 4-(2-((E)-2-((E)-2-(4-(((3S,7S,21S,29S)-21-((R)-2-((R)-2-(2-aminoacetamido)-3-(4-hydroxy-3-iodophenyl)propanamido)-3-phenylpropanamido)-1,3,7,29-tetracarboxy-5,13,20,27-tetraoxo-4,6,12,19,26-pentaazanonacosan-29-yl)carbamoyl)phenoxy)-3-((E)-2-(1,1-dimethyl-3-(4-sulfobutyl)-1,3-dihydro-2H-benzo[e]indol-2-ylidene)ethylidene)cyclohex-1-en-1-yl)vinyl)-1,1-dimethyl-1H-benzo[e]indol-3-ium-3-yl)butane-1-sulfonate.

[0065] Preparation method: solid phase synthesis method. Take the resin Resin-EUK (9, 100 mg, 0.15 mmol) in the solid phase synthesis tube, dichloromethane (DCM) swelling washing three times (2 ml each time, 5 min each time), N, N-dimethylformamide (DMF) washing three times (2 ml each time, 5 min each time). Then according to the repeated method of solid phase peptide synthesis.

[0066] (a) Take the above resin, use DMF containing 20% piperidine to remove the protecting group Fmoc of amino group (repeat twice, 2 ml each time, 10 min each time), DMF washing three to five times (2 ml each time, 2 min each time). In DMF (3 ml) as the reaction solvent, add Fmoc-Ahx-OH (70.68 mg, 0.20 mmol), pre-activated by O-benzotriazole-N, N, N', N'-tetramethyluronium hexafluorophosphate (HBTU, 91.02 mg, 0.24 mmol), 1-hydroxybenzotriazole (HOBt, 32.43 mg, 0.24 mmol) and N, N-diisopropyl ethylamine (DIPEA, 100 μl, 0.6 mmol) for 15 min, electromagnetic stirring reaction at room temperature for 1 hour. The resin is washed with DMF three to five times (2 ml each time, 2 min each time).

[0067] (b) The resin from step (a) was stirred in 20% piperidine in DMF to remove Fmoc (twice for 2 ml each for 10 min), washed with DMF three to five times (2 ml each for 2 min). Fmoc-Lys(Dde)-OH (106.53 mg, 0.20 mmol) was added in DMF (3 ml) as reaction solvent, pre-activated by HBTU (91.02 mg, 0.24 mmol), HOBt (32.43 mg, 0.24 mmol) and DIPEA (100 μΐ, 0.6 mmol) for 15 min, and the reaction was stirred magnetically at room temperature for 1 h. The resin was washed with DMF three to five times (2 ml each for 2 min).

[0068] (c) The resin from step (b) was stirred in 20% piperidine in DMF to remove Fmoc (twice for 2 ml each for 10 min), washed with DMF three to five times (2 ml each for 2 min). Fmoc-Phe-OH (77.49 mg, 0.20 mmol) was added in DMF (3 ml) as reaction solvent, pre-activated by HBTU (91.02 mg, 0.24 mmol), HOBt (32.43 mg, 0.24 mmol) and DIPEA (100 μΐ, 0.6 mmol) for 15 min, and the reaction was stirred magnetically at room temperature for 1 h. The resin was washed with DMF three to five times (2 ml each for 2 min).

[0069] (d) The resin from step (c) was stirred in 20% piperidine in DMF to remove Fmoc (twice for 2 ml each for 10 min), washed with DMF three to five times (2 ml each for 2 min). Compound 4 (117.09 mg, 0.20 mmol) was added in DMF (3 ml) as reaction solvent, pre-activated by HBTU (91.02 mg, 0.24 mmol), HOBt (32.43 mg, 0.24 mmol) and DIPEA (100 μΐ, 0.6 mmol) for 15 min, and the reaction was stirred magnetically at room temperature for 1 h. The resin was washed with DMF three to five times (2 ml each for 2 min).

[0070] (e) The resin from step (d) was stirred in a solution of 20% piperidine in DMF to remove Fmoc (twice for 2 ml each for 10 min) and washed with DMF three to five times (2 ml each for 2 min). Fmoc-Gly-OH (59.46 mg, 0.20 mmol) was added in DMF (3 ml) as reaction solvent, pre-activated by HBTU (91.02 mg, 0.24 mmol), HOBt (32.43 mg, 0.24 mmol) and DIPEA (100 μΐ, 0.6 mmol) for 15 min, and the reaction was magnetically stirred at room temperature for 1 h. The resin was washed with DMF three to five times (2 ml each for 2 min).

[0071] (f) The resin from step (e) was stirred in a solution of 2% hydrazine hydrate in DMF to remove Dde group (twice for 2 ml each for 10 min) and washed with DMF three to five times (2 ml each for 2 min). Fmoc-Asp(OtBu)-OH (82.29 mg, 0.20 mmol) was added in DMF (3 ml) as reaction solvent, pre-activated by HBTU (91.02 mg, 0.24 mmol), HOBt (32.43 mg, 0.24 mmol) and DIPEA (100 μΐ, 0.6 mmol) for 15 min, and the reaction was magnetically stirred at room temperature for 1 h. The resin was washed with DMF three to five times (2 ml each for 2 min).

[0072] (g) The resin from step (f) was stirred in a solution of 20% piperidine in DMF to remove Fmoc (twice for 2 ml each for 10 min) and washed with DMF three to five times (2 ml each for 2 min). (h) The resin from step (g) was stirred in a solution of 20% piperidine in DMF to remove Fmoc (twice for 2 ml each for 10 min) and washed with DMF three to five times (2 ml each for 2 min).

[0073] (h) The resin from step (g) was stirred in a solution of 20% piperidine in DMF to remove Fmoc (twice for 2 ml each for 10 min) and washed with DMF three to five times (2 ml each for 2 min).

[0074] (i) The final product was cleaved from the resin obtained in (h) using a solution of 5 ml trifluoroacetic acid (TFA) / triisopropylsilane (TIPS) / H2O (v / v / v: 90% / 5% / 5%) for 4 hours. After HPLC purification, PSMA-F was obtained as a green solid. i.e. PSMA-F.

[0075] Example 2 Determination of the optical properties of PSMA-F I. Experimental methods The UV absorption spectrum of PSMA-F was detected using a fluorescence spectrophotometer (Shimadzu RF-6000, Shimadzu, Japan). The fluorescence spectrum and fluorescence quantum yield were collected using a fluorescence spectrometer (FLS 980, Edinburgh, UK).

[0076] The UV absorption spectrum of PSMA-F was detected using a fluorescence spectrophotometer (Shimadzu RF-6000, Shimadzu, Japan). The fluorescence spectrum and fluorescence quantum yield were collected using a fluorescence spectrometer (FLS 980, Edinburgh, UK). The UV absorption spectrum of PSMA-F was recorded in the range of 550-950 nm at different concentrations of 10 µM, 8 µM, 6 µM, 5 µM, 4 µM, respectively. The emission spectrum of PSMA-F was recorded in the range of 750-900 nm at a concentration of 10 µM. The absolute quantum yield (Φ, %) of PSMA-F (5 µM) was recorded on a Hamamatsu Quantaurus-QY C11347-11 absolute quantum yield spectrometer (Hamamatsu, Japan). The PBS dilutions (3 ml) of the above five concentrations were used to measure the molar absorption coefficient (ε, L / mol / cm). The light stability was determined by continuous irradiation at 810 nm for 60 min.

[0077] II. Experimental results The main peak of the absorption spectrum of PSMA-F was at 808 nm, and there was a peak at 736 nm, which was lower than the peak at 808 nm. Figure 3 A). The emission peak was at 822 nm Figure 3 B). The molar absorption coefficient was 63491 L / mol / cm Figure 3 C). The fluorescence quantum yield was 0.75%, and the fluorescence stability was good Figure 3 D).

[0078] Example 3 Study on the effect of PSMA-F in targeting PSMA in vivo I. Experimental methods 1. Experimental animals were purchased from Charles River Laboratories (Beijing, China). The experimental study selected 3-4 weeks old, body weight about 16-18 g BALB / c male nude mice. The mice were fed in accordance with the requirements of SPF level animal feeding. The animals were given a 7-day adaptation period before the experiment. The 22Rv1 cell suspension (about 5 x 10 6 was injected subcutaneously into the right shoulder of the nude mice (100 μl), forming a 22Rv1 tumor xenograft. The tumor length and short diameter were measured by vernier caliper and the tumor volume was estimated according to the formula (V = 1 / 2 x length x short diameter 2 ). When the tumor volume reached 200-300 mm 3 , the imaging experiment was performed.

[0079] 2. Live fluorescence images of tumor-bearing nude mice were collected. According to the optical properties, PSMA-F fluorescence data was collected. The same parameters (exposure height, exposure intensity, exposure time, etc.) were used during collection. The collected images were uniformly processed, and the fluorescence intensity was analyzed using the average fluorescence emission efficiency [(ph / s / cm 2 / sr) / (μW / cm 2 )]. 22Rv1 tumor-bearing nude mice (n = 5) were injected with PSMA-F (40 μg / kg, 150 μl) via the tail vein, and live fluorescence imaging was performed at 24 h and 48 h. Another 22Rv1 tumor-bearing nude mice were divided into 4 groups (n = 3), and the experiment was terminated 1 h, 2 h, 24 h, and 48 h after PSMA-F injection via the tail vein. The tumor and main tissues and organs were dissected, and in vitro imaging was performed using a fluorescence imaging system. The ROI region was outlined, and the average fluorescence intensity value was measured.

[0080] II. Experimental results The results showed that 24 h after injection of PSMA-F, the 22Rv1 tumor showed specific targeting, except for a certain fluorescence signal in the kidney, other normal tissues did not show fluorescence signal. Figure 5

[0081] In addition, ex vivo fluorescence imaging of tissues and organs was performed to analyze the in vivo distribution. The in vitro distribution results and data of 22Rv1 tumor-bearing nude mice are shown in Figure 4 A. 1 h after injection of PSMA-F, 22Rv1 tumor uptake was high, and other normal tissues and organs also had varying degrees of uptake, and as time went on, PSMA-F was gradually cleared, while the tumor continued to maintain a high uptake. Figure 4 B). The tumor muscle fluorescence intensity ratio and tumor liver fluorescence intensity ratio both increased with time, representing the increasing contrast between tumor and background. Figure 4

[0082] ​​It was found that the tumor uptake and retention of the simple fluorescent group was significantly lower than that of PSMA-F when the fluorescent group without the PSMA ligand was injected into the mice. Figure 6 ].

[0083] The above examples are only used to illustrate the technical solutions of the present application, but not to limit it; although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that the technical solutions recorded in the foregoing examples can be modified, or some technical features can be replaced by equivalents; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. A fluorescent probe targeting PSMA in prostate cancer, characterized in that, The fluorescent probe has the structure shown in formula (I): Equation (I) Wherein, F is a fluorescent group.

2. The fluorescent probe according to claim 1, characterized in that, The fluorescent group is a near-infrared fluorescent group; Preferably, the fluorescent group is a near-infrared I region fluorescent group; Preferably, the fluorescent group has a structure as shown in formula (II): Formula (II).

3. The fluorescent probe according to claim 1 or 2, characterized in that, The fluorescent probe has the structure shown in Formula (III): Formula (III).

4. A method for preparing a fluorescent probe targeting PSMA in prostate cancer, characterized in that, The method includes: using compound 1 as a substrate, adding a fluorescent group and reacting to obtain a fluorescent probe as shown in formula (Ⅲ), wherein compound 1 is the product obtained by reacting EUK with Ahx-OH, Lys(Dde)-OH, Phe-OH, 3-Iodo-Tyr(OtBu)-OH, Gly-OH, and Asp(OtBu)-OH; Preferably, the method includes: using compound 2 as a substrate, adding Asp(OtBu)-OH and a fluorescent group to react, and then obtaining the fluorescent probe shown in formula (Ⅲ), wherein compound 2 is the product obtained by reacting EUK with Ahx-OH, Lys(Dde)-OH, Phe-OH, 3-Iodo-Tyr(OtBu)-OH and Gly-OH; Preferably, the method includes: using compound 3 as a substrate, adding Gly-OH, Asp(OtBu)-OH and a fluorescent group to react and obtain the fluorescent probe shown in formula (Ⅲ), wherein compound 3 is the product obtained by reacting EUK with Ahx-OH, Lys(Dde)-OH, Phe-OH and 3-Iodo-Tyr(OtBu)-OH; Preferably, the method includes: using compound 4 as a substrate, adding 3-Iodo-Tyr(OtBu)-OH, Gly-OH, Asp(OtBu)-OH and a fluorescent group to react and obtain the fluorescent probe shown in formula (III), wherein compound 4 is the product obtained by reacting EUK with Ahx-OH, Lys(Dde)-OH and Phe-OH; Preferably, the method includes: using compound 5 as a substrate, adding Phe-OH, Iodo-Tyr(OtBu)-OH, Gly-OH, Asp(OtBu)-OH and a fluorescent group to react and obtain the fluorescent probe shown in formula (Ⅲ), wherein compound 5 is the product obtained by reacting EUK with Ahx-OH and Lys(Dde)-OH; Preferably, the method includes: using compound 6 as a substrate, adding Lys(Dde)-OH, Phe-OH, 3-Iodo-Tyr(OtBu)-OH, Gly-OH, Asp(OtBu)-OH and a fluorescent group to react and obtain the fluorescent probe shown in formula (Ⅲ), wherein compound 6 is the product obtained by reacting EUK with Ahx-OH; Preferably, the method includes: using Resin-EUK resin as a substrate, sequentially synthesizing peptide resins from Fmoc-Ahx-OH, Fmoc-Lys(Dde)-OH, Fmoc-Phe-OH, Fmoc-3-Iodo-Tyr(OtBu)-OH, Fmoc-Gly-OH, and Fmoc-Asp(OtBu)-OH using the standard Fmoc chemical method; removing the Fmoc protecting group from the peptide resins; adding fluorescent groups to synthesize fluorescent peptide resins; and treating the fluorescent peptide resins with a lysis mixture to obtain the fluorescent probe shown in formula (III).

5. The preparation method according to claim 4, characterized in that, The EUK is glutamic acid-urea-lysine; Preferably, the fluorescent group is as shown in formula (II); Preferably, based on the amount of resin Resin-EUK, the amounts of Fmoc-Ahx-OH, Fmoc-Lys(Dde)-OH, Fmoc-Phe-OH, Fmoc-3-Iodo-Tyr(OtBu)-OH, Fmoc-Gly-OH, Fmoc-Asp(OtBu)-OH and the fluorescent group are 4 / 3 molar equivalents; Preferably, the pyrolysis mixture comprises, by weight percentage, 90% trifluoroacetic acid, 5% triisopropylsilane, and 5% H2O.

6. A detection composition, characterized in that, The detection composition comprises the fluorescent probe as described in any one of claims 1-3.

7. The detection composition according to claim 6, characterized in that, The detection composition also includes other products that can be used to detect PSMA; Preferably, the other products that can be used to detect PSMA include PSMA-targeting imaging agents or anti-PSMA antibodies.

8. A reagent for detecting PSMA levels in a sample, characterized in that, The reagent comprises the fluorescent probe as described in any one of claims 1-3 or the detection composition as described in claim 6, and a solvent.

9. A kit for detecting PSMA levels in a sample, characterized in that, The kit comprises the fluorescent probe of any one of claims 1-3, the detection composition of claim 6, or the reagent of claim 7.

10. Any of the following applications: 1) The use of the fluorescent probe according to any one of claims 1-3 in the preparation of reagents or kits for detecting PSMA levels in samples; 2) The use of the fluorescent probe according to any one of claims 1-3 in the preparation of a product for fluorescent navigation in prostate cancer surgery; 3) Use of the reagent of claim 7 or the kit of claim 8 in the preparation of products for diagnosing prostate cancer.

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