CN113117100B - Fluorescent molecular probe for targeting PSMA and preparation method and application thereof - Google Patents
Fluorescent molecular probe for targeting PSMA and preparation method and application thereof Download PDFInfo
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- CN113117100B CN113117100B CN202011561337.1A CN202011561337A CN113117100B CN 113117100 B CN113117100 B CN 113117100B CN 202011561337 A CN202011561337 A CN 202011561337A CN 113117100 B CN113117100 B CN 113117100B
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
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- A61K49/0063—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres
- A61K49/0069—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres the agent being in a particular physical galenical form
- A61K49/0089—Particulate, powder, adsorbate, bead, sphere
- A61K49/0091—Microparticle, microcapsule, microbubble, microsphere, microbead, i.e. having a size or diameter higher or equal to 1 micrometer
- A61K49/0093—Nanoparticle, nanocapsule, nanobubble, nanosphere, nanobead, i.e. having a size or diameter smaller than 1 micrometer, e.g. polymeric nanoparticle
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0013—Luminescence
- A61K49/0017—Fluorescence in vivo
- A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
- A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
Abstract
The invention relates to the technical field of disease detection, in particular to a fluorescent molecular probe targeting PSMA, a preparation method and application thereof. In the fluorescent molecular probe, a target head is a PSMA small molecule inhibitor or an oligopeptide substrate; the load is a fluorescent group; with or without albumin binding groups. The fluorescent molecular probe enhances the binding capacity of the probe and PSMA, and increases the internalization mediated by PSMA; the half-life period is long, the tumor enrichment time is long, and the accumulation of the targeting probe in the prostate tumor tissue is increased; the tumor fluorescence signal is remarkable and stable for a long time; the fluorescent molecular probe provided by the invention has excellent TBR in-vivo and in-vitro imaging.
Description
Technical Field
The invention belongs to the technical field of fluorescent molecular probes, and particularly relates to a PSMA-targeted fluorescent molecular probe, and a preparation method and application thereof.
Background
Prostate cancer (PCa) is the most common malignancy in men, the third leading cause of cancer-related death in men worldwide. It is counted that about 1-2% of men die from prostate cancer. This disease, which threatens global male health, will become increasingly serious as humans enter the aging society. Although early diagnosis and treatment techniques for prostate cancer have progressed rapidly in recent years, there are still many patients who have developed serum prostate-specific antigen (prostate specific antigen, PSA) elevation or imaging progression again after primary treatment. Methods of treatment for prostate cancer include surgery, radiation therapy, and endocrine therapy. Patients diagnosed with early stage focal prostate cancer are generally curable, but patients diagnosed with or progressing to castration resistant prostate cancer (mCRPC) have no clinical choice for cure. More than 90% of prostate tumors found in the primary screening are clinically localized and these patients are suitable for radical prostatectomy. The major challenge of radical prostatectomy is that the degree of tumor infiltration can only be determined by post-operative pathological assessment of the resected tissue, which is difficult for the surgeon to discover and assess tumor cell invasion. Thus, about 20% of prostatectomy does not achieve complete resection (post-operative pathology confirms a positive margin), resulting in disease recurrence in these patients exceeding 60%. There is a serious unmet need in the field of prostate cancer diagnosis and treatment.
Existing technologyDiagnostic techniques such as 18 F-fluorodeoxyglucose, 11 C-choline, 18 The nonspecific PET/CT molecular probes such as F-choline and the like have limited detection sensitivity and specificity on PCa primary foci and metastasis, and have a plurality of influencing factors such as hormone dependence, tumor size, grading, position, serum prostate specific antigen level and the like, which are not beneficial to early detection of tumors. Therefore, the traditional diagnosis and treatment scheme cannot meet clinical requirements. In recent years, prostate Specific Membrane Antigen (PSMA) has received widespread attention. PSMA is highly expressed in the epithelial cell membrane of prostate cancer, and its expression level is positively correlated with the number and invasiveness of tumor cells. In order to improve accurate diagnosis and treatment of PCa, particularly metastatic castration-resistant prostate cancer (mCRPC), molecular probe development results with PSMA specificity have attracted clinical widespread attention in recent years.
Currently, molecular probes targeting PSMA mainly include three major classes, nuclide probes, fluorescent probes, and multi-modal probes. Nuclide probes are main strategies for developing and treating prostate cancer clinically by using molecules with high affinity to PSMA for nuclide labeling for diagnosis, staging, re-staging and treatment of prostate cancer. Urea-based PSMA inhibitors play an important role in the development of nuclide probes and PET imaging. The only FDA-approved PSMA-targeting prostate cancer imaging agent is prosatascint, manufactured by 111 In-labeled murine antibody 7E11 (Prostate 1991; 18:229-41). Second generation antibody J591 binding to the extracellular domain of PSMA has been described 111 In、 89 Zr、 90 Y and 177 lu radiolabels have shown excellent binding properties and tumor background ratios in clinical trials with metastatic and castration resistant prostate. The main disadvantage of antibodies is that the recognition of the target and the background clearance rate are both slow in the appropriate time, reducing their utility as intra-operative image navigation. In recent years, a variety of PSMA-targeted SPECT and PET small molecule imaging agents have been developed, with varieties such as ureido-based DCFBC, MIP-1095, MIP-1072, PSMA-617, and the like having entered the clinical research stage.
Because of the radioactivity of the nuclide probe, the nuclide probe is only applied to preoperative imaging and treatment of the prostate cancer clinically at present and is not used for intraoperative real-time imaging. The extent of tumor infiltration beyond the surgical margin of a prostate cancer procedure can only be determined post-operatively, and thus there is a need for an imaging modality that can improve visualization of tissue during the procedure to help define the tumor margin. Fluorescence Guided Surgery (FGS) is a technique that uses fluorescence to highlight cancer cells and guide surgeons in real-time tumor resection, meeting this need. To achieve this, there is a need to develop excellent PSMA-targeted fluorescent probes. These probes should be able to accumulate selectively in prostate tumors and have an improved Tumor Background Ratio (TBR). PSMA-targeted fluorescent probes are a new development direction in the field, but development progress slowly, and no product has entered the clinical research stage yet. Moreover, the related reports at present are relatively few, and if only patent CN 111362971A discloses a PSMA-targeted bisbenzothiadiazole compound through searching, the structure of the bisbenzothiadiazole compound is shown as the following formula (I) or formula (II):
the benzothiadiazole derivative provided by the patent has high affinity to PSMA protein, can be used for preparing fluorescent molecular probes (fluorescent probes for near infrared two-region optical imaging of prostate cancer, photoacoustic imaging probes and photodynamic therapy probes) of the targeted PSMA protein so as to realize early diagnosis of the prostate cancer, and can also be used for navigation or cleaning in a fluorescent operation in prostate cancer excision. However, currently, there are many limitations to the development of fluorescent molecular probes, such as:
1) The small molecular fluorescent probe has short half-life, quick metabolism and short residence time in tumor, and ideal TBR is difficult to realize;
2) The fluorescent probe based on the antibody has long half-life and slow clearance, the high molecular weight of the antibody leads to poor permeability of tumor tissues, and the ideal TBR can be achieved only by delaying imaging (delaying for 2-4 days) after injection, so that nursing cost and difficulty are increased. In addition, monoclonal antibodies are potentially immunogenic and are prone to safety concerns;
3) The method can not well meet the requirement of tumor specific imaging in operation, and has a larger improvement space in sensitivity and specificity.
Therefore, it is important to develop a new PSMA-targeted fluorescent probe with excellent optical, pharmacokinetic and biological characteristics.
Disclosure of Invention
Accordingly, it is an object of the present invention to provide a fluorescent molecular probe targeting PSMA. The fluorescent molecular probe increases the accumulation of the targeting probe in tumor tissues; enhancing the ability of the probe to bind to PSMA, increasing internalization mediated by PSMA; the signal to noise ratio is enhanced.
Another object of the present invention is to provide a method for preparing the PSMA-targeted fluorescent molecular probe.
The invention also aims to provide the application of the PSMA-targeted fluorescent molecular probe in preparing prostate cancer diagnosis and treatment medicines.
In order to achieve the above object, the present invention provides the following technical solutions:
a PSMA-targeted fluorescent molecular probe having the following structure:
target head-joint 1-load
The target head is a PSMA small molecule inhibitor or an oligopeptide substrate;
the load is a fluorescent group;
the linker 1 is-CH 2 -、-CH 2 CH 2 O-、-C(＝O)-、-C(＝O)O-、-C(＝O)NH 2 -、-S-S-、 -O-、-S-、-NH-、-SC-、-HC＝CH-、-HC≡CH-、
One of (a) or a repeating unit thereof;
wherein R is-H or-CH 3 The method comprises the steps of carrying out a first treatment on the surface of the SC-20 natural AmmoniaResidues of a base acid and a partially unnatural amino acid selected from the following structures:
for targeted fluorescent probes, sufficient TBR (tumor background ratio, T/B) is achieved, requiring sufficient binding time of the probe molecule to the target. Studies have shown that the binding time is largely dependent on the dissociation rate of the probe-target complex, i.e. k off . The effectiveness of the probe depends on the dissociation process, the probe-target binding rate constant k on Limited by concentration and diffusion rate, and thus difficult to control. And k is off It is entirely dependent on the specific interaction between the probe and its target binding. Monovalent ligand-receptor interactions are primarily enthalpy-driven processes in which the ligand diffuses in solution to the target and binds to the receptor with the free energy of interaction Δg=Δh-tΔs, where Δg is the free energy binding force, which is the sum of the enthalpy (Δh) and entropy (-tΔs) contributions. There are only two states of bound and unbound in the monovalent system. In multivalent systems, the scaffold itself and the multiple ligands attached to the scaffold, the entropy penalty required for multivalent probe binding to the target can be reduced by rational design of the linker.
The invention aims at designing a novel fluorescent molecular probe, and the effective method for increasing TBR (tumor background ratio, T/B) to increase TBR, increase T or decrease B is to prolong the action time of the probe and a target point and the effective method for prolonging the action time of the probe and the target point is to decrease k off . The linker in the monovalent probe of the invention enhances the affinity between the targeted fluorescent molecular probe and the target, while the multivalent probe may further enhance the affinity. (multivalent vs. monovalent, k) off Greatly reduced, see multitvalency: peptides, research and Applications, edited by Jurriaan Huskens, leonard J.Prins, rainer Haag, bart Jan Ravoo, pp 209).
Preferably, the target head is
Preferably, the joint 1 is:
one of (a) or a repeating unit thereof;
preferably, the load is:
one of them.
Further preferably, the load is:
as a preferred embodiment, the fluorescent molecular probe has a structure shown in formula I:
the invention also provides a preparation method of the fluorescent molecular probe, which comprises the following steps:
the target head is connected with a load through a connector 1, and the fluorescent molecular probe is obtained.
The invention also provides a fluorescent molecular probe as shown in fig. 15:
the target head is a PSMA small molecule inhibitor or an oligopeptide substrate;
the load is a fluorescent group;
the albumin binding group is one of octadecanedioic acid and 4- (p-tolyl) butyric acid;
the joint 1 is defined in the structure 1;
linker 2 is-CH 2 -、-CH 2 CH 2 O-、-C(＝O)-、-C(＝O)O-、-C(＝O)NH 2 -、-S-S-、-O-、-S-、-NH-、-SC-、-HC＝CH-、-HC≡CH-、
One of (a) or a repeating unit thereof;
wherein the joint 1 and the joint 2 may be the same or different.
Preferably, the joint 2 is:
one of (a) or a repeating unit thereof;
as a preferred embodiment, the fluorescent molecular probe has a structure shown in formula II:
as a preferred embodiment, the fluorescent molecular probe has a structure shown in formula III:
the invention also provides a preparation method of the fluorescent molecular probe, which comprises the following steps: connecting the target head with a load through a joint 1 to obtain a target head-joint 1-load compound; the albumin binding group was attached to the linker 1 position of the target-linker 1-supported complex via linker 2, yielding a fluorescent molecular probe with the following structure.
The invention also provides application of the fluorescent molecular probe in preparation of prostate cancer diagnosis and treatment medicines.
The invention also provides a composition comprising the fluorescent molecular probe and a pharmaceutically acceptable carrier. The carrier may be selected as desired by one skilled in the art, and may be selected from, for example, sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium chloride, citric acid, sodium citrate, polysorbate 20, polysorbate 80, water for injection, and the like.
The invention also provides a pharmaceutical formulation comprising the PSMA-targeted fluorescent molecular probe.
Compared with the prior art, the invention has the following beneficial effects:
1) The fluorescent molecular probe has albumin binding groups, can be combined with albumin to form a compound, has obviously increased volume and is easy to form an EPR effect (enhancedpermeability and retention effect), so that the probe is more deposited in tumor tissues, the binding capacity of the probe and PSMA is enhanced, and the internalization mediated by the PSMA is increased;
2) The fluorescent molecular probe has long half-life and long tumor enrichment time, and increases the accumulation of the targeting probe in prostate tumor tissues; the tumor fluorescence signal is remarkable and stable for a long time;
3) The fluorescent molecular probe provided by the invention has excellent TBR in-vivo and in-vitro imaging.
Drawings
FIG. 1 is a schematic diagram of a monovalent PSMA targeting probe combined with albumin to form a multivalent probe;
figure 2 HPLC profile of compound 5;
FIG. 3 Compound 5MS profile;
FIG. 4 HPLC profile of Compound 9;
FIG. 5 Compound 9MS profile;
figure 6 compound 10HPLC profile;
FIG. 7 Compound 10MS profile;
FIG. 8 Compound 11MS profile;
FIG. 9 Compound 12MS profile;
FIG. 10 in vivo imaging of compound 6LnCaP subcutaneous tumor model;
FIG. 11 fluorescence imaging of compound 6 in vitro tissues and tumors;
FIG. 12 in vivo imaging of compound 12-HSA 22Rv1 subcutaneous tumor model;
FIG. 13 in vivo imaging of compound 11-HSA 22Rv1 subcutaneous tumor model;
FIG. 14 fluorescence imaging of compound 12-HSA and compound 11-HSA in vitro tissues and tumors;
FIG. 15 is a schematic structural diagram of one fluorescent probe molecule of the present invention.
Detailed Description
The invention discloses a PSMA-targeted fluorescent molecular probe, a preparation method and application thereof, and a person skilled in the art can properly improve the technological parameters by referring to the content of the present disclosure. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included in the present invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that variations and modifications can be made in the methods and applications described herein, and in the practice and application of the techniques of this invention, without departing from the spirit or scope of the invention.
The invention provides a fluorescent molecular probe, which has the following structure:
target-adapter 1-load or structure as shown in FIG. 15
The "target" brings the "load" (fluorophore) to the "target".
Wherein the target head comprises a PSMA small molecule inhibitor or an oligopeptide substrate.
The "linker" may be part of a chain, ring, saturated, unsaturated, aromatic ring or aromatic heterocyclic ring (containing 1-4N, O or S atoms), consisting of 0-500 carbon, nitrogen, oxygen, sulfur or hydrogen atoms, and the "linker" unit may be: -CH 2 -、 -CH 2 CH 2 O-、-C(＝O)-、-C(＝O)O-、-C(＝O)NH 2 -S-, -O-, -S-, -NH-C (SC) -C (=o) -, -hc=ch-, or-hc≡ch-, and the like, -SC- (Side Chain) is a residue of 20 natural amino acids and a part of unnatural amino acids, and may also be:
one of (a) or a repeating unit thereof;
wherein R may be-H or-CH 3 . These units may or may not be repeating in the "linker".
The "linker 1" and the "linker 2" may be the same or different.
"Supported" is a fluorophore (near infrared and near infrared two-region NIR-II fluorophores):
one of them. Wherein, the preferable structure of load, joint and target head respectively is following:
the load is:
the joint is as follows:
one of them.
The target head is as follows:
the "albumin binding group" may be introduced by, but is not limited to, reaction of octadecanedioic acid, 4- (p-tolyl) butanoic acid with a free amino group on "linker 1" (1.Alessandro Zorzi,Simon J.Middendorp,Jonas Wilbs,Kaycie Deyle&Christian Heinis"Acylated heptapeptide binds albumin with high affinity and application as tag furnishes long-acting peptides" NATURE COMMUNICATIONS |8:16092|DOI: 10.1038/ncomms16092;2.Cassandra E.Callmann et al, antitumor Activity of 1,18-Octadecanedioic Acid-Paclitaxel Complexed with Human Serum Albumin, 10.1021/jacs.9b04272 J.Am.chem.Soc.), enhancing the cycling stability of the molecule.
Preferred molecular probe structures (compound 6) are:
more preferably, the fluorescent molecular probe has a structure as shown in formula I:
preferred molecular probe structures containing linker 2 and albumin binding group (compound 12) are:
preferred molecular probe structures containing linker 2 and albumin binding group (compound 11) are:
the invention designs a synthesized targeted fluorescent molecular probe for use in, but not limited to: (1) Solid tumor identification, boundary identification and lymph node visualization of prostate cancer; (2) Identification of solid tumors, boundary identification and lymph node visualization of bladder cancer; (3) identification of renal tumor entities and boundaries; (4) identification of adrenal tumors; (5) sentinel lymph node visualization of penile carcinoma.
The PSMA-targeted fluorescent molecular probe provided by the invention, the preparation method thereof and the reagent used in the application can be purchased from the market.
The invention is further illustrated by the following examples:
example 1 Synthesis method of fluorescent molecular Probe (Compound 6)
Compound 2:
resin activation: compound 1 (Fmoc-Lys (Dde) -Wang resin, available from Nanjing peptide Biotechnology Co., ltd., R61124,0.3-0.8mmol/g,100-200mesh,1 g) was first freed of the N-terminal Fmoc protecting group with a hexahydropyridine/DMF mixed solvent I (25% by volume: 75%) to make the N-terminal free amino.
Access amino acid: n, N disuccinimidyl carbonate (3.2 mmol) and DIPEA (10 mmol) and H-Glu (OtBu) -OtBu (3.2 mmol) were dissolved in 20ml DMF and added to the above 12H resin (Fmoc deprotected compound 1) and reacted for 24H to give compound 2.
Compounds 3, 4, 5 were grafted using a similar grafting procedure as above:
compound 3: after removal of the side chain Dde protecting group of Lys on the resin (i.e. Compound 2) with a 2% hydrazine hydrate/DMF solution (hydrazine hydrate: DMF=2%: 98%), 3 times the equivalent of Fmoc-2-Nal-OH/HOBt/DIC (i.e. Fmoc-2-Nal-OH, HOBt and DIC were used in an amount 3 times the molar amount of the resin) was added and reacted for 24 hours at normal temperature, followed by grafting reaction to introduce 2-Nal amino acid residues.
Compound 4: fmoc protecting groups on the resin (namely, the compound 3) are removed by 25% of hexahydropyridine/DMF (the volume ratio of the hexahydropyridine/DMF is 25% to 75%) so that the N end of 2-Nal is free amino, 3 times of N-Boc-trans-4-aminomethylcyclohexane carboxylic acid/HOBt/DIC (namely, the N-Boc-trans-4-aminomethylcyclohexane carboxylic acid: HOBt: DIC is 3 times of the molar amount of the resin) are added for reaction for 24 hours at normal temperature, and grafting reaction is carried out to introduce the trans-4-aminomethylcyclohexane carboxylic acid.
Compound 5: using cleavage reagent (trifluoroacetic acid: H) 2 Triisopropylsilane=90:5:5, v/v) cleaves the target product from the resin and removes the side chain protecting group (cleavage at 30 ℃ C. For 3 hours). Adding the filtrate into a large amount of cold anhydrous diethyl ether to precipitate polypeptide, centrifuging, washing with diethyl ether for several times, and drying to obtain crude product.
The crude product was purified by reverse phase high performance liquid chromatography (purity 95%, yield 65%). Chromatographic column model: agela C18 (10 μm,50X 250 mm). Chromatographic operating conditions: mobile phase a (aqueous solution containing 0.05% trifluoroacetic acid, 2% acetonitrile) and mobile phase B (90% acetonitrile/water (90%: 10% acetonitrile/water by volume) were prepared, the flow rate was 25 ml/min, and the uv detection wavelength was 220nm. The solvent was lyophilized to give a pure polypeptide in a fluffy state, the chemical structure of which was characterized by MALDI-TOF mass spectrometry, and the purity of which was given by analytical high performance liquid chromatography (Agela C18-10X 250mm, flow rate: 1 ml per minute). Compound 5HPLC and MS profiles are shown in figures 2 and 3.
Compound 6: to 50. Mu.L of an aqueous solution (concentration: 0.01 mg/. Mu.L) of Compound 5 was added 0.01M Na 2 HPO 4 IRDye800CW NHS (0.5 mg) was dissolved in 10. Mu.L DMSO and added to an aqueous solution of Compound 5, and the mixture was stirred at room temperature under light-shielding conditions for 2 hours, followed by purification by high performance liquid chromatography (Agilent 588915-902, HC-C18 (2) 4.6X105 mm,5 μm,0-5 min: 5% acetonitrile, 5-45 min: 5-95% acetonitrile, flow rate: 1 ml per minute) (purity 90%, yield 85%).
Example 2 Synthesis of fluorescent molecular probes (Compounds 11 and 12)
Compound 7: fmoc protecting groups in the compound 3 are removed by 25% of hexahydropyridine/DMF (volume ratio, the meaning is the same as above) so that the N end of 2-Nal becomes free amino, 3 times of equivalent N-Fmoc-trans-4-aminomethylcyclohexane carboxylic acid/HOBt/DIC (namely, the dosage of N-Fmoc-trans-4-aminomethylcyclohexane carboxylic acid, HOBt and DIC is 3 times of the molar amount of resin) are added for reaction for 24 hours at normal temperature, and grafting reaction is carried out to introduce trans-4-aminomethylcyclohexane carboxylic acid.
Compound 8: fmoc protecting group on compound 7 was removed with 25% piperidine/DMF (volume ratio), 3-fold equivalent of N-Fmoc-N '- [1- (4, 4-dimethyl-2, 6-dioxocyclohexylidene) ethyl ] -lysine/HOBt/DIC (i.e., N-Fmoc-N' - [1- (4, 4-dimethyl-2, 6-dioxocyclohexylidene) ethyl ] -lysine/HOBt/DIC was used in an amount 3-fold the molar amount of resin) was added and the grafting reaction was carried out for 24 hours at normal temperature to introduce lysine residues.
Compound 9: first, after removing the side chain Dde protecting group of Lys on the compound 8 with a 2% hydrazine hydrate/DMF (hydrazine hydrate: DMF=2%: 98%) solution, 10-fold equivalent of 1,18-Octadecanedioic Acid/HOBt/DIC (namely, 1,18-Octadecanedioic Acid, HOBt, DIC are all 10-fold the molar amount of the resin) was added for reaction at normal temperature for 24 hours, and grafting reaction was carried out to introduce 2-Nal amino acid residues.
Next, the Fmoc protecting group at the end of compound 9 was removed with 25% piperidine/DMF (volume ratio).
Using cleavage reagent (trifluoroacetic acid: H) 2 Triisopropylsilane=90:5:5, v/v) cleaves the target product from the resin and removes the side chain protecting group (cleavage at 30 ℃ C. For 3 hours). Adding the filtrate into a large amount of cold anhydrous diethyl ether to precipitate polypeptide, and centrifuging. Washing with diethyl ether for several times, and drying to obtain crude product.
The crude product was purified by reverse phase high performance liquid chromatography (purity 95%, yield 54%). Chromatographic column model: agela C18 (10 μm,50X 250 mm). Chromatographic operating conditions: mobile phase a was (0.05% trifluoroacetic acid in 2% acetonitrile in water) and mobile phase B was 90% acetonitrile/water (acetonitrile: water=90%: 10%), flow rate was 25 ml per minute, and uv detection wavelength was 220nm. The solvent was lyophilized to give a pure polypeptide in a fluffy state, the chemical structure of which was characterized by MALDI-TOF mass spectrometry, and the purity of which was given by analytical high performance liquid chromatography (Agela C18-10X 250mm, flow rate: 1 ml per minute).Compound 9HPLC and MS profiles are shown in figures 4 and 5.
Compound 10: firstly, removing a side chain Dde protecting group of Lys on a compound 8 by using a 2% hydrazine hydrate/DMF (hydrazine hydrate: DMF volume ratio is 2%: 98%) solution, adding 10 times equivalent of 4- (p-Tolyl) butyl acid/HOBt/DIC (namely, the dosage of 4- (p-Tolyl) butyl acid, HOBt and DIC is 10 times of the molar weight of resin), reacting for 24 hours at normal temperature, and carrying out grafting reaction to introduce 2-Nal amino acid residues.
Next, the terminal Fmoc protecting group was removed with 25% piperidine/DMF (25% by volume: 75%).
Using cleavage reagent (trifluoroacetic acid: H) 2 Triisopropylsilane=90:5:5, v/v) cleaves the target product from the resin and removes the side chain protecting group (cleavage at 30 ℃ C. For 3 hours). Adding the filtrate into a large amount of cold anhydrous diethyl ether to precipitate polypeptide, and centrifuging. Washing with diethyl ether for several times, and drying to obtain crude product.
The crude product was purified by reverse phase high performance liquid chromatography (purity 95%, yield 47%). Chromatographic column model: agela C18 (10 μm,50X 250 mm). Chromatographic operating conditions: mobile phase a was (0.05% trifluoroacetic acid in 2% acetonitrile in water) and mobile phase B was 90% acetonitrile/water (acetonitrile: water volume 90%: 10%) at a flow rate of 25 ml per minute and an ultraviolet detection wavelength of 220nm. The solvent was lyophilized to give a pure polypeptide in a fluffy state, the chemical structure of which was characterized by MALDI-TOF mass spectrometry, and the purity of which was given by analytical high performance liquid chromatography (Agela C18-10X 250mm, flow rate: 1 ml per minute). Compound 10HPLC and MS profiles are shown in figures 6 and 7.
Compound 11: to an aqueous solution of compound 9 (concentrationDegree: 0.01 mg/. Mu.L) 50. Mu.L of 0.01M Na 2 HPO 4 (150. Mu.L, pH 8.4). IRDye800CW NHS (1 eq) was dissolved in 10. Mu.L DMSO, added to an aqueous solution of Compound 9, stirred at room temperature in the dark for 2 hours and purified by high performance liquid chromatography (Agilent 588415-902, HC-C18 (2) 4.6X105 mm,5 μm,0-5 min: 5% acetonitrile, 5-45 min: 5-95% acetonitrile, flow rate: 1 ml per minute) with a chemical structure characterized by LC-MS of Waters, wherein the pattern (part) is shown in FIG. 8.
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Compound 12: to 50. Mu.L of an aqueous solution (concentration: 0.01 mg/. Mu.L) of Compound 10 was added 0.01M Na 2 HPO 4 (150. Mu.L, pH 8.4). IRDye800CW NHS (1 eq) was dissolved in 10. Mu.L DMSO, added to an aqueous solution of compound 10, stirred at room temperature in the dark for 2 hours and purified by high performance liquid chromatography (Agilent 588915-902, HC-C18 (2) 4.6X150 mm,5 μm,0-5 min: 5% acetonitrile, 5-45 min: 5-95% acetonitrile, flow rate: 1 ml per minute) with a product purity of 95% and a yield of 90%). The chemical structure was characterized by LC-MS of Waters, and the pattern is shown in fig. 9.
Compound 11-HSA and compound 12-HSA:
1. compound 11 was dissolved in deionized water. HSA (Sigma a 1887) at a concentration of 0.03M was dissolved in deionized water. To a 5mL centrifuge tube was added 20 μl (0.06M) of 11 in water, followed by rapid addition of 200 μl of HSA solution to yield 5:1 molar ratio of 11-HSA solution. After lyophilization, a white powder was obtained.
2. Compound 12 was dissolved in deionized water. HSA (Sigma a 1887) at a concentration of 0.03M was dissolved in deionized water. To a 5mL centrifuge tube was added 20 μl (0.06M) of 12 in water, followed by rapid addition of 200 μl of HSA solution to yield 5:1 molar ratio of 12-HSA solution. After lyophilization, a white powder was obtained.
Test examples
1. Establishing a tumor model
All animal experiment procedures were performed with the university of su laboratory animal center and the university of su animal protection and use committee approval. To establish LNCaP/22Rv 1-loaded subcutaneous tumor model, experiments were performed using healthy male Balb/C nude mice (18-22 g), with 50. Mu.L of LNCaP/22Rv1 cell suspension (1X 10) 7 Individual cells). Tumor volume reaches about 70-200mm after one month 3 At this time, a fluorescence imaging experiment and a biodistribution experiment were started.
2. Preparing injection
The solid powder of Compound 6 was weighed, dissolved in physiological saline to prepare a solution of 0.24. Mu. Mol/mL, and a part of the solution was diluted with physiological saline to prepare a solution of 0.12. Mu. Mol/mL and 0.012. Mu. Mol/mL.
The solid powders of the compounds 11 and 12 were weighed, dissolved in physiological saline, and prepared as a solution of 0.06. Mu. Mol/mL, and a part of the solution was diluted with physiological saline to prepare a solution of 0.03 mol/mL.
3. Administration and imaging
The 6 nude mice of the LNCaP subcutaneous tumor model were randomly divided into 3 groups of 2. The 3 prepared groups of compound 6 solutions of different concentrations (0.012. Mu. Mol/kg, 0.12. Mu. Mol/kg, 0.24. Mu. Mol/kg) were injected into mice by tail vein administration, each 200. Mu.L. The 12 nude mice of the 22Rv1 subcutaneous tumor model were randomly divided into 4 groups of 3. Two different concentrations (0.03. Mu. Mol/mL, 0.06. Mu. Mol/mL) of each of the formulated 11 and 12 solutions were injected into mice via tail vein administration, 200. Mu.L each. Anesthesia with isoflurane was performed, photographed and analyzed using a small animal near infrared imaging system (IVIS luminea II) scan (ex.740 nm and em.820 nm). During imaging, mice were anesthetized by the nose cone system with 3% isoflurane all the way through. After 48 hours, the mice were sacrificed by cervical dislocation, and tumors and heart, liver, spleen, lung, kidney, stomach, muscle and tumors were collected, washed and dried. Fluorescence pictures were acquired by an IVIS luminea II imaging system.
The imaging of mice is shown in FIGS. 8-12.
The results show that compound 6 starts to be significantly enriched at the tumor site 24 hours after injection and that the tumor is more strongly compared to the background 48 hours later. The compound 12-HSA starts to be obviously enriched at the tumor part after 4 hours of injection, and the compound 11-HSA also has obvious enrichment after 4 hours of injection at a low concentration of 0.3 mu mol/kg, which shows that the above 3 preferred compounds all have targeting effect on PSMA positive tumors and can be used for near infrared optical imaging of tumors, wherein the compound 12-HSA and the compound 11-HSA can obviously shorten the tumor imaging time.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (5)
1. The fluorescent molecular probe for targeting PSMA is characterized in that the fluorescent molecular probe forms a complex with albumin, the albumin is HSA, and the structure of the fluorescent molecular probe is shown in the following formula I and formula II:
2. the fluorescent molecular probe according to claim 1, wherein the preparation method of the fluorescent molecular probe comprises the following steps:
through the joint 1Target head->And a load
Ligation gives a target-linker 1-cargo, which is then combined with albumin to form a complex.
3. The use of a fluorescent molecular probe according to claim 1 for the preparation of a disease diagnostic reagent, wherein the disease diagnosis comprises: solid tumor identification, boundary identification and lymph node visualization of prostate cancer; identification of solid tumors, boundary identification and lymph node visualization of bladder cancer; identifying kidney tumor entity and boundary; identification of adrenal tumors and sentinel lymph node visualization of penile carcinoma.
4. The use of the fluorescent molecular probe according to claim 1 for preparing a diagnosis and treatment reagent for prostate cancer.
5. A composition comprising the fluorescent molecular probe of claim 1 and a pharmaceutically acceptable carrier.
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