CN113117099B - PSMA (patterned beam-induced fluorescence) targeted fluorescent molecular probe as well as preparation method and application thereof - Google Patents
PSMA (patterned beam-induced fluorescence) targeted fluorescent molecular probe as well as preparation method and application thereof Download PDFInfo
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- 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
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- 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
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Abstract
The invention relates to the technical field of disease detection, in particular to a PSMA (patterned beam splitter) -targeted fluorescent molecular probe and a preparation method and application thereof. In the fluorescent molecular probe, a target head is a PSMA small molecular 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 internalization mediated by PSMA; the half-life period is long, the tumor enrichment time is long, and the accumulation of the targeting probe in prostate tumor tissues is increased; the tumor fluorescence signal is significant and stable for a long time; the fluorescent molecular probe disclosed by the invention shows 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 (patterned fluorescent dye array) -targeted fluorescent molecular probe as well as 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. Statistically, about 1-2% of men currently die from prostate cancer. As humans enter an aging society, this disease threatening the health of men worldwide will become increasingly serious. Although early diagnosis and treatment techniques for prostate cancer have been developed rapidly in recent years, there are still many patients who experience a rise in serum Prostate Specific Antigen (PSA) or imaging progress after the initial treatment. Methods of treatment of prostate cancer include surgery, radiation therapy and endocrine therapy. Patients diagnosed with early stage focal prostate cancer are usually curable, but patients diagnosed with or progressing to castration-resistant prostate cancer (mCRPC) have no cure option clinically. Over 90% of prostate tumors found in primary screening are clinically localized and these patients are eligible for radical prostatectomy. The major challenge in radical prostatectomy is that the degree of tumor infiltration can only be determined by pathological assessment of the resected tissue after surgery, where it is difficult for the surgeon to find and assess the invasion of tumor cells. Thus, complete resection of the prostatectomy (post-operative pathology confirmed positive margins) is not achieved in about 20% of these patients, resulting in disease recurrence in excess of 60%. There is a serious unmet need in the field of prostate cancer diagnosis and treatment.
The existing diagnostic techniques such as18F-fluorodeoxyglucose,11C-choline,18F-choline and other non-specific PET/CT molecular probes have limited detection sensitivity and specificity on PCa primary foci and metastasis foci to a certain extent, and are more in influence factors such as hormone dependence, tumor size, classification, position, serum prostate specific antigen level and the like, so that the F-choline and other non-specific PET/CT molecular probes are not beneficial to early detection of tumors. Therefore, the traditional diagnosis and treatment scheme can not meet the clinical requirement. In recent years, the prostate-specific membrane antigen (PSMA) has received much attention. PSMA is highly expressed in prostate cancer epithelial cell membrane, and the expression level of PSMA is positively correlated with the number and invasiveness of tumor cells. For improving PCa especiallyAccurate diagnosis and treatment of metastatic castration-resistant prostate cancer (mCRPC), a molecular probe development result with PSMA specificity has attracted extensive clinical attention in recent years.
At present, molecular probes targeting PSMA mainly comprise three major types, namely a nucleon probe, a fluorescent probe and a multi-modal probe. The nuclide probe carries out nuclide labeling on a molecule with high affinity with PSMA for diagnosis, staging, re-staging and treatment of the prostate cancer, and is a main strategy for clinical prostate cancer imaging and treatment. Urea-based PSMA inhibitors play an important role in the development of nuclear probes and PET imaging. The only PSMA-targeting prostate cancer imaging agent approved by the FDA is ProstaScint, manufactured by111In-labeled murine antibody 7E11 was obtained (Prostate 1991; 18: 229-41). A second generation antibody J591 binding to the extracellular domain of PSMA has been shown111In、89Zr、90Y and177lu radiolabeling has 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 background clearance speed are slow in a proper time, reducing their utility as intra-operative image navigation. In recent years, a plurality of PSMA-targeted SPECT and PET small molecule imaging agents are developed, wherein urea-based DCFBC, MIP-1095, MIP-1072, PSMA-617 and other varieties enter the clinical research stage.
Because the nuclide probe has radioactivity, the nuclide probe is only clinically applied to preoperative imaging and treatment of prostate cancer at present and is not applied to intraoperative real-time imaging. The extent of tumor infiltration beyond the surgical margin in prostate cancer surgery can only be determined post-operatively, and there is a need for an imaging modality that can improve visualization of tissue during surgery to help define the tumor margin. Fluorescence Guided Surgery (FGS) is a technique that uses fluorescence to highlight cancer cells and guide surgeons in resecting tumors in real-time, and can meet this need. To realize this technology, an excellent PSMA-targeted fluorescent probe needs to be developed. These probes should be able to selectively accumulate in prostate tumors and have an improved Tumor Background Ratio (TBR). The PSMA targeted fluorescent probe is a new development direction in the field, but the development is slow, and no product enters the clinical research stage. Moreover, relatively few reports are reported, and only patent CN 111362971a was found to disclose a bisbenzothiadiazole compound targeting PSMA after search, and the structure thereof is shown as follows:
the benzothiadiazole derivative provided by the patent has high affinity to PSMA protein, can be used for preparing a fluorescent molecular probe (a fluorescent probe for prostate cancer near-infrared two-region optical imaging, a photoacoustic imaging probe and a photodynamic therapy probe) for targeting PSMA protein so as to realize early diagnosis of prostate cancer, and can also be used for navigation or cleaning in fluorescence in prostatectomy. However, the fluorescent molecular probes under investigation have many limitations, such as:
1) the half-life period of the small-molecule fluorescent probe is short, the metabolism is fast, the retention time in the tumor is short, and the ideal TBR is difficult to realize;
2) the fluorescent probe based on the antibody has long half-life and slow clearance, the large molecular weight of the antibody causes poor permeability of tumor tissues, and the imaging needs to be delayed (2-4 days delayed) after injection to reach the ideal TBR, thereby increasing the nursing cost and difficulty. In addition, the monoclonal antibody has potential immunogenicity, and is easy to generate safety problems;
3) can not well meet the requirements of tumor specific imaging in the operation, and has larger promotion space in the aspects of sensitivity and specificity.
Therefore, the development of a novel PSMA (PSMA-targeted fluorescent probe) with excellent optical, pharmacokinetic and biological characteristics is of great significance.
Disclosure of Invention
In view of the above, it is an object of the present invention to provide a PSMA-targeted fluorescent molecular probe. The fluorescent molecular probe increases the accumulation of the targeted probe in tumor tissues; the binding capacity of the probe and the PSMA is enhanced, and the internalization mediated by the PSMA is increased; the signal-to-noise ratio is enhanced.
The invention also aims to provide a preparation method of the PSMA-targeted fluorescent molecular probe.
The invention also aims to provide application of the PSMA-targeted fluorescent molecular probe in preparation of a prostate cancer diagnosis and treatment medicine.
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 1:
the target head is a PSMA small molecule inhibitor or oligopeptide substrate;
the load is a fluorophore;
the joint 1 is-CH2-、-CH2CH2O-、-C(＝O)-、-C(＝O)O-、-C(＝O)NH2-、-S-S-、-O-、-S-、-NH-、-SC-、-HC＝CH-、-HC≡CH-、
Or a repeating unit thereof;
wherein R is-H or-CH3(ii) a -SC-is the residue of 20 natural and partially unnatural amino acids, selected from the following structures:
for a targeted fluorescent probe, sufficient binding time of the probe molecule to the target is required to achieve a sufficient TBR (tumor to background ratio, T/B). Studies have shown that the binding time is largely dependent on the dissociation rate of the probe-target complexRate, i.e. koff. The effectiveness of the probe depends on the dissociation process, probe-target binding rate constant konLimited by concentration and diffusion rate and thus difficult to control. And k isoffIt 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 to the target in solution and binds to the receptor with the free energy of interaction ag ═ Δ H-T Δ S, where Δ G is the free energy binding force, which is the sum of the enthalpy (Δ H) and entropy (-T Δ S) contributions. Only two states, bound and unbound, exist in a monovalent system. In a multivalent system, the scaffold itself and the attachment of multiple ligands to the scaffold, the entropy penalty required for binding of multivalent probes to the target can be reduced by judicious design of the linkers.
The invention aims to design a novel fluorescent molecular probe, wherein the improvement of TBR (tumor background ratio, T/B) is to improve TBR, increase T or reduce B, the effective method for increasing T is to prolong the action time of the probe and a target, and the effective method for prolonging the action time of the probe and the target is to reduce koff. The joint in the monovalent probe enhances the affinity between the targeted fluorescent molecular probe and the target, and the multivalent probe can further enhance the affinity. (polyvalent vs. monovalent, k)offThis is greatly reduced, as seen in Multivalency: Concepts, 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:
or a repeating unit thereof;
preferably, the load is:
Further preferably, the load is:
as a preferred embodiment, the structure of the fluorescent molecular probe is shown as formula I:
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 the fluorescent molecular probe.
The invention also provides a fluorescent molecular probe with a structure 2 as shown in the specification:
the target head is a PSMA small molecule inhibitor or oligopeptide substrate;
the load is a fluorophore;
the albumin binding group is one of octadecanedioic acid and 4- (p-tolyl) butyric acid;
the joint 1 is defined in the structure 1;
the joint 2 is-CH2-、-CH2CH2O-、-C(＝O)-、-C(＝O)O-、-C(＝O)NH2-、-S-S-、-O-、-S-、-NH-、-SC-、-HC＝CH-、-HC≡CH-、
Or a repeating unit thereof;
wherein, the linker 1 and the linker 2 may be the same or different.
Preferably, the joint 2 is:
or a repeating unit thereof;
as a preferred embodiment, the structure of the fluorescent molecular probe is shown in formula II:
as a preferred embodiment, the structure of the fluorescent molecular probe is 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 the load through the joint 1 to obtain a target head-joint 1-load compound; the albumin binding group is attached to the target head-linker 1-position of linker 1 of the load complex via linker 2, resulting in a fluorescent molecular probe with the following structure.
The invention also provides application of the fluorescent molecular probe in preparation of a prostate cancer diagnosis and treatment medicine.
The invention also provides a composition, which comprises the fluorescent molecular probe and a pharmaceutically acceptable carrier. The carrier is conventionally selected by those skilled in the art as needed, 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 medicinal preparation which comprises the PSMA-targeted fluorescent molecular probe.
Compared with the prior art, the invention has the following beneficial effects:
1) the fluorescent molecular probe has an albumin binding group, can be combined with albumin to form a compound, has a remarkably increased volume, is easy to form an Enhanced Permeability and Retentivity (EPR) effect, and enables the probe to be deposited in tumor tissues more, so that the binding capacity of the probe and PSMA is enhanced, and internalization mediated by PSMA is increased;
2) the fluorescent molecular probe has long half-life period and long tumor enrichment time, and increases the accumulation of the targeted probe in prostate tumor tissues; the tumor fluorescence signal is significant and stable for a long time;
3) the fluorescent molecular probe of the invention shows excellent TBR in vivo and in vitro imaging.
Drawings
FIG. 1 is a schematic representation of a monovalent PSMA targeting probe bound to 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;
figure 5 compound 9MS spectrum;
figure 6 compound 10HPLC profile;
figure 7 compound 10MS spectrum;
FIG. 8 Compound 11MS profile;
figure 9 compound 12MS spectrum;
figure 10 in vivo imaging of compound 6LnCaP subcutaneous tumor model;
figure 11 compound 6 ex vivo tissue and tumor fluorescence imaging;
FIG. 12 Compound 12-HSA 22Rv1 subcutaneous tumor model in vivo imaging;
FIG. 13 in vivo imaging of the Compound 11-HSA 22Rv1 subcutaneous tumor model;
FIG. 14 fluorescence imaging of compound 12-HSA and compound 11-HSA in vitro tissue and tumors.
Detailed Description
The invention discloses a PSMA-targeted fluorescent molecular probe, and a preparation method and application thereof, and can be realized by appropriately improving process parameters by referring to the content. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.
The invention provides a fluorescent molecular probe, which has the following structure:
the "target" carries the "load" (fluorophore) to the "target".
Wherein the target comprises a PSMA small molecule inhibitor or an oligopeptide substrate.
The "linker" may be a part of a chain, cyclic, saturated, unsaturated, aromatic ring or aromatic heterocycle (containing 1-4N, O or S atoms), composed of 0-500 carbon, nitrogen, oxygen, sulfur or hydrogen atoms, and the units constituting the "linker" may be: -CH2-、-CH2CH2O-、-C(＝O)-、-C(＝O)O-、-C(＝O)NH2-units such as-S, -O, -S, -NH, -C (SC) -C (O) -, -HC-CH-or-HC ≡ CH-SC- (Side Chain) are residues of 20 natural amino acids and some unnatural amino acids, and may be:
or a repeating unit thereof;
wherein R may be-H or-CH3. These units may or may not be repeating in the "linker".
"linker 1" and "linker 2" may be the same or different.
"Supported" is a fluorophore (near infrared fluorophore and near infrared two-region NIR-II fluorophore):
Wherein, the preferred structure of load, joint and target head respectively is as follows:
the load is as follows:
the joint is as follows:
one kind of (1).
The target head is as follows:
an "Albumin binding group" can be introduced by, but is not limited to, reaction of Octadecanedioic Acid, 4- (p-tolyl) butyric Acid, with the free amino group on "linker 1" (1.Alessandro Zorzi, Simon J. Middendorp, Jonas Wilbs, Kayce Deyle & Christian Heinis "Acylated peptide complexes with high affinity and application as tag nucleic acids long-acting peptides" NATURE COMME COMMUNICATIONS |8:16092| DOI:10.1038/ncomms 16092; 2.Cassandra E. Callmann et al, anticancer Activity of 1, 18-Octageonic Acid-bound complex with Human Serum Albumin, 10.1021/JAC. 048. Sobj. 048. the stability of the molecule is enhanced).
The preferred molecular probe structure (compound 6) is:
more preferably, the structure of the fluorescent molecular probe is shown as formula I:
the preferred molecular probe structure containing linker 2 and albumin binding group (compound 12) is:
the preferred molecular probe structure containing linker 2 and albumin binding group (compound 11) is:
the invention designs a synthesized targeted fluorescent molecular probe for but not limited to: (1) solid tumor identification, border identification and lymph node visualization of prostate cancer; (2) solid tumor identification, 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 cancer.
The PSMA-targeted fluorescent molecular probe provided by the invention, and the preparation method and the reagent used in the application thereof can be purchased from the market.
The invention is further illustrated by the following examples:
EXAMPLE 1 Synthesis of fluorescent molecular Probe (Compound 6)
Resin activation: compound 1(Fmoc-Lys (Dde) -Wang resin, available from Nanjing peptide Biotechnology Ltd., R61124, 0.3-0.8mmol/g, 100-200mesh, 1g) was first freed from the N-terminal Fmoc protecting group by removing the N-terminal amino group with piperidine/DMF mixed solvent I (volume ratio 25%: 75%).
And (3) amino acid inoculation: n, N-disuccinimidyl carbonate (3.2mmol), DIPEA (10mmol) and H-Glu (OtBu) -OtBu (3.2mmol) were dissolved in 20ml of DMF and added to the above resin (Fmoc-deprotected Compound 1) for 12 hours to react for 24 hours to give Compound 2.
compound 3: after removing the side chain Dde protecting group of Lys in the resin (i.e., compound 2) with 2% hydrazine hydrate/DMF solution (hydrazine hydrate: DMF: 2%: 98%), 3 times equivalent of Fmoc-2-Nal-OH/HOBt/DIC (i.e., the amount of Fmoc-2-Nal-OH, HOBt and DIC is 3 times of the molar amount of the resin) was added and reacted at room temperature for 24 hours, and a grafting reaction was performed to introduce a 2-Nal amino acid residue.
Compound 4: the Fmoc protecting group on the resin (i.e., compound 3) was removed with 25% piperidine/DMF (piperidine/DMF volume ratio 25%: 75%) to make the N-terminus of 2-Nal a free amino group, and 3 equivalents of N-Boc-trans-4-aminomethylcyclohexanecarboxylic acid/HOBt/DIC (i.e., N-Boc-trans-4-aminomethylcyclohexanecarboxylic acid: HOBt: DIC used in an amount of 3 times the molar amount of the resin) were added and reacted at room temperature for 24 hours to perform a grafting reaction to introduce trans-4-aminomethylcyclohexanecarboxylic acid.
Compound 5: cleavage reagent (trifluoroacetic acid: H) was used2Triisopropylsilane-90: 5:5, v/v) the desired product was cleaved from the resin and the side chain protecting groups were removed (cleavage at 30 ℃ for 3 hours). Adding the filtrate into a large amount of cold anhydrous ether to precipitate polypeptide, centrifuging, washing with ether for several times, and drying to obtain crude product.
The crude product was purified by reverse phase HPLC (95% purity, 65% yield). Type of chromatographic column: AgelaC18(10 μm,
50X 250 mm). Chromatographic operating conditions are as follows: the mobile phase A was (aqueous solution containing 0.05% trifluoroacetic acid, 2% acetonitrile) and the mobile phase B was 90% acetonitrile/water (acetonitrile/water volume ratio 90%: 10%), flow rate 25 ml/min and ultraviolet detection wavelength 220 nm. The solvent was lyophilized to give a fluffy pure polypeptide product whose chemical structure was characterized by MALDI-TOF mass spectrometry and purity was given by analytical high performance liquid chromatography (Agela C18-10X 250mm, flow rate: 1 ml/min). The HPLC and MS spectra of compound 5 are shown in FIGS. 2 and 3.
Compound 6: to 50. mu.L of an aqueous solution of Compound 5 (concentration: 0.01 mg/. mu.L), 0.01M Na was added2HPO4(150. mu.L), IRDye800CW NHS (0.5mg) was dissolved in 10. mu.L of DMSO, added to an aqueous solution of Compound 5, stirred at room temperature in the absence of light for 2 hours, and purified by high performance liquid chromatography (Agilent 588915. mu.m, HC-C18(2)4.6X150mm, 5. mu.m, 0-5 min: 5% acetonitrile, 5-45 min: 5-95% acetonitrile, flow rate: 1 ml/min) (purity 90%, yield 85%).
EXAMPLE 2 Synthesis of fluorescent molecular probes (Compounds 11 and 12)
Compound 7: the Fmoc protecting group in compound 3 was removed with 25% piperidine/DMF (volume ratio, as defined above) to make the N-terminus of 2-Nal free amino, 3 equivalents of N-Fmoc-trans-4-aminomethylcyclohexanecarboxylic acid/HOBt/DIC (i.e., N-Fmoc-trans-4-aminomethylcyclohexanecarboxylic acid, HOBt, DIC were used in amounts 3 times the molar amount of the resin) were added and reacted at room temperature for 24 hours to effect grafting reaction and to introduce trans-4-aminomethylcyclohexanecarboxylic acid.
Compound 8: the Fmoc protecting group of compound 7 was removed with 25% piperidine/DMF (vol/vol), and 3 equivalents 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 in 3 molar amounts) were added and reacted at room temperature for 24 hours to effect grafting and to introduce lysine residues.
Compound 9: first, after removing the side chain Dde protecting group of Lys in Compound 8 with a 2% hydrazine hydrate/DMF (hydrazine hydrate: DMF: 2%: 98%), 10-fold equivalent of 1, 18-octanedioic Acid/HOBt/DIC (i.e., 1, 18-octanedioic Acid, HOBt, DIC are used in amounts of 10-fold molar amount of the resin) was added and reacted at room temperature for 24 hours to perform a grafting reaction and introduce a 2-Nal amino Acid residue.
Next, the Fmoc protecting group at the end of Compound 9 was removed using 25% piperidine/DMF (by volume).
Cleavage reagent (trifluoroacetic acid: H) was used2Triisopropylsilane-90: 5:5, v/v) the desired product was cleaved from the resin and the side chain protecting groups were removed (cleavage at 30 ℃ for 3 hours). Adding the filtrate into a large amount of cold anhydrous ether to precipitate out 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 (95% purity, 54% yield). The type of the chromatographic column: AgelaC18(10 μm,
50X 250 mm). The chromatographic operation conditions are as follows: mobile phase a was (aqueous solution containing 0.05% trifluoroacetic acid, 2% acetonitrile) and mobile phase B was 90% acetonitrile/water (acetonitrile: water 90%: 10%), flow rate 25 ml/min, uv detection wavelength 220 nm. Freeze drying the solvent to obtain fluffy polypeptide pure product, wherein the chemical structure of the fluffy polypeptide pure product is characterized by MALDI-TOF mass spectrum, and the purity of the fluffy polypeptide pure product isIt is given by an analytical high performance liquid chromatograph (Agela C18-10X 250mm, flow rate: 1 ml per minute). The HPLC and MS spectra of compound 9 are shown in FIGS. 4 and 5.
Compound 10: first, after removing the side chain Dde protecting group of Lys in Compound 8 with a 2% hydrazine hydrate/DMF (hydrazine hydrate: DMF volume ratio: 2%: 98%), 10 equivalents of 4- (p-Tolyl) butyl ic acid/HOBt/DIC (i.e., the amounts of 4- (p-Tolyl) butyl ic acid, HOBt, DIC are 10 times of the molar amount of the resin) were added and reacted at room temperature for 24 hours to carry out a grafting reaction, and 2-Nal amino acid residue was introduced.
Next, the Fmoc protecting group at the end was removed with 25% piperidine/DMF (25% by volume: 75%).
Cleavage reagent (trifluoroacetic acid: H) was used2Triisopropylsilane-90: 5:5, v/v) the desired product was cleaved from the resin and the side chain protecting groups were removed (cleavage at 30 ℃ for 3 hours). Adding the filtrate into a large amount of cold anhydrous 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 HPLC (95% purity, 47% yield). The type of the chromatographic column: AgelaC18(10 μm,
50X 250 mm). The chromatographic operation conditions are as follows: mobile phase a (0.05% trifluoroacetic acid in 2% acetonitrile in water) and mobile phase B90% acetonitrile/water (acetonitrile: water volume ratio 90%: 10%), flow rate 25 ml/min, uv detection wavelength 220 nm. The solvent was lyophilized to give a fluffy pure polypeptide, whose chemical structure was characterized by MALDI-TOF mass spectrometry and purity was given by analytical high performance liquid chromatography (Agela C18-10X 250mm, flow rate: 1 ml/min). The HPLC and MS spectra of compound 10 are shown in fig. 6 and 7.
Compound 11: to 50. mu.L of an aqueous solution of Compound 9 (concentration: 0.01 mg/. mu.L) was added 0.01M Na2HPO4(150. mu.L, pH 8.4). IRDye800CW NHS (1eq) 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, purified by high performance liquid chromatography (Agilent 588915-902, HC-C18(2)4.6X150mm, 5. mu.m, 0-5 min: 5% acetonitrile, 5-45 min: 5-95% acetonitrile, flow rate: 1 ml per minute) (product purity 95%, yield 85%) and chemical structure characterized by LC-MS from Waters, where the MS (partial) spectrum is shown in FIG. 8.
Compound 12: to 50. mu.L of an aqueous solution (concentration: 0.01 mg/. mu.L) of Compound 10 was added 0.01M Na2HPO4(150. mu.L, pH 8.4). IRDye800CW NHS (1eq) 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)4.6X150mm, 5. mu.m, 0-5 min: 5% acetonitrile, 5-45 min: 5-95% acetonitrile, flow rate: 1 ml per minute) (product purity 95%, yield 90%). The chemical structure is characterized by LC-MS of Waters, and the map is shown in FIG. 9.
Compound 11-HSA and compound 12-HSA:
1. compound 11 was dissolved in deionized water. HSA (Sigma A1887) was dissolved in deionized water at a concentration of 0.03M. To a 5mL centrifuge tube, 20 μ L (0.06M) of 11 in water was added followed by a rapid addition of 200 μ L of HSA solution to yield a 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 A1887) was dissolved in deionized water at a concentration of 0.03M. To a 5mL centrifuge tube, 20 μ L (0.06M) of 12 in water was added followed by a rapid addition of 200 μ L of HSA solution to yield a 5: 1 molar ratio of 12-HSA solution. Lyophilizing to obtain white powder
Test examples
1. Establishing a tumor model
All animal experimental procedures were performed with approval from the experimental animal center at suzhou university and the animal protection and use committee at suzhou university. In order to establish a subcutaneous tumor model of the Hold LNCaP/22Rv1, healthy male Balb/C nude mice (18-22 g) were used in the experiment, and 50. mu.L of LNCaP/22Rv1 cell suspension (1X 10) was injected into the right axilla of the mice7Individual cells). The tumor volume reaches about 70-200mm after one month3At that time, a live 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 0.24. mu. mol/mL solution, and a portion of the solution was diluted with physiological saline to prepare 0.12. mu. mol/mL and 0.012. mu. mol/mL solutions.
Solid powders of compounds 11 and 12 were weighed, dissolved in physiological saline to prepare a 0.06. mu. mol/mL solution, and a portion of the solution was diluted with physiological saline to prepare a 0.03mol/mL solution.
3. Administration and imaging
Nude mice of the 6LNCaP subcutaneous tumor model were randomly divided into 3 groups of 2 mice each. The prepared 3 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, 200. mu.L each, by tail vein administration. The 12 nude mice of the 22Rv1 subcutaneous tumor model were randomly divided into 4 groups of 3 mice each. Two prepared solutions of 11 and 12 each at different concentrations (0.03. mu. mol/mL, 0.06. mu. mol/mL) were injected into mice, 200. mu.L each, by tail vein administration. Anesthetized with isoflurane, scanned for images and analyzed using a small animal near-infrared imaging system (IVIS luminea II) (ex.740nm and em.820 nm). During the imaging procedure, mice were anesthetized with 3% isoflurane all the way through the nose cone system. After 48 hours, the mice were sacrificed by cervical dislocation, and the tumors, heart, liver, spleen, lung, kidney, stomach, muscle and tumors were collected, washed and dried. Fluorescence pictures were acquired by the IVIS luminea II imaging system.
Images of the mice are shown in FIGS. 10-14.
The results show that compound 6 was significantly enriched at the tumor site starting 24 hours after injection and that the tumor contrast to background was more intense 48 hours later. After 4 hours of injection, the compound 12-HSA is obviously enriched in the tumor part, and the compound 11-HSA is also obviously enriched after 4 hours of injection at low concentration of 0.3 mu mol/kg, which shows that the above 3 preferable compounds have targeting property on PSMA positive tumors and can be used for tumor near-infrared optical imaging, wherein the compound 12-HSA and the compound 11-HSA can obviously shorten the tumor imaging time.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (5)
1. The PSMA-targeted fluorescent molecular probe is characterized in that the fluorescent molecular probe forms a complex with albumin, and the structure of the fluorescent molecular probe is shown as the following formula I and formula II:
2. the method for preparing a fluorescent molecular probe according to claim 1, comprising the steps of: through the joint 1
Will the target head
And a load
Connecting to obtain a target head-joint 1-load compound; through the joint 2
The albumin binding group octadecanedioic acid or 4- (p-tolyl) butyric acid was attached to the tarmac-linker 1-loading complex.
3. The use of the fluorescent molecular probe according to claim 1 for the preparation of a diagnostic reagent for diseases comprising: the disease diagnosis includes: solid tumor identification, border identification and lymph node visualization of prostate cancer; solid tumor identification, boundary identification and lymph node visualization of bladder cancer; identification of renal tumor entities and boundaries; recognition of adrenal tumors and visualization of sentinel lymph nodes of penile cancer.
4. The fluorescent molecular probe of claim 1 is applied to preparation of a prostate cancer diagnosis and treatment reagent.
5. A composition comprising the fluorescent molecular probe of claim 1 and a pharmaceutically acceptable carrier.
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| CN113979912B (en) * | 2021-11-16 | 2023-04-07 | 中国医学科学院肿瘤医院 | Two prostate specific membrane antigen targeted fluorescent probes and preparation method and application thereof |
| CN115925586A (en) * | 2022-11-01 | 2023-04-07 | 青岛蓝谷多肽生物医药科技有限公司 | Preparation method of parent of targeting PSMA and derivative thereof |
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| CN113117100B (en) | 2024-03-01 |
| CN113117099A (en) | 2021-07-16 |
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