Compound and preparation method, composition, kit and application thereof

Abstract

The present invention discloses a compound and a preparation method, a composition, a kit and an application thereof, wherein the compound structural formula is as follows: wherein the cation M is selected from an alkali metal cation, R is a C2-C5 alkyl group, and n is an integer in the range of 2-5. The compound and its application products have good recognition ability for PSMA receptors and good targeting. In practical applications, they can be widely used in multiple scenarios such as flow cytometry analysis and in vivo imaging, and have the advantages of fast signal response speed, high resolution, good safety, excellent metabolic capacity, etc.

Description

Technical Field

[0001] The present invention relates to biological detection technology, in particular to a compound and its preparation method, composition, kit and application. Background Art

[0002] Prostate cancer (PCa) is one of the most common tumors that threaten men's health. In recent years, the incidence of prostate cancer in my country has increased significantly, and it has become the fastest growing type of malignant tumor. Due to the lack of sensitive imaging agents for the diagnosis and efficacy monitoring of prostate cancer, choosing an appropriate treatment plan is still a major challenge facing the clinic.

[0003] Accurate detection of early prostate cancer is crucial to the treatment effect and survival of patients. Prostate cancer is often difficult to detect early due to its relatively slow progression. Prostate-specific antigen (PSA) combined with magnetic resonance imaging and puncture biopsy can effectively diagnose PCa, but there is currently a lack of highly sensitive and specific functional imaging equipment that can define its biological behavior. In the past decade, PET / CT has made great progress. PET / CT integrates PET's nuclear medicine molecular imaging with high sensitivity and specificity and CT's fine anatomical images, and has exerted great potential in many aspects such as preoperative diagnosis and staging of primary prostate cancer and treatment decision-making. In terms of treatment, radical prostatectomy is one of the most effective methods for treating localized and locally advanced prostate cancer. The surgeon mainly relies on preoperative imaging examinations, intraoperative naked eye findings and exploration, and the surgeon's own experience to determine the scope of the resection margin. If the scope of surgical resection is too large, normal tissue will be damaged and normal functions such as urinary control will be affected; while if the scope of resection is too small, positive resection margins will occur, and patients are prone to recurrence. Therefore, how to preserve normal tissue and function as much as possible during surgery while completely removing the area invaded by prostate cancer is a problem that clinicians often need to face and solve.

[0004] Targeted fluorescence intraoperative navigation technology is a good way to solve this problem. In the field of prostate cancer, there is a highly specific molecular marker for prostate cancer - prostate-specific membrane antigen (PSMA). PSMA is overexpressed in 90% of prostate cancers, while only a small amount is expressed in normal tissues such as lacrimal glands, salivary glands, and renal proximal tubules. The expression level of PSMA in prostate cancer is highly correlated with the invasion and malignancy of the tumor. Prostate-specific membrane antigen (PSMA) is a multifunctional type II transmembrane protein present in the prostate cell membrane, consisting of 750 amino acid residues. PSMA has high prostate tissue specificity, and its sensitivity and specificity in distinguishing prostate cancer from other types of malignant tumors are 65.9% and 94.5% respectively. Therefore, PSMA is still a very sensitive and highly specific antigen on prostate cancer cells. PSMA is superior to PSA in distinguishing benign and malignant prostate tissues, and the later the clinical stage, the stronger its expression, which is positively correlated with the malignancy of the tumor. Therefore, PSMA is a more specific prostate cancer marker than PSA and can be used as an important indicator for the prognosis evaluation of prostate cancer. Therefore, PSMA has become an ideal biomarker for precise positioning and imaging of prostate cancer lesions and intraoperative navigation. Near-infrared fluorescence surgical navigation technology targeting PSMA mainly uses the advantages of near-infrared dyes such as longer wavelength, stronger tissue penetration, and less scattering. It can illuminate the lesions during surgery, allowing the surgeon to clearly understand the scope of the lesions, thereby performing a more complete and effective resection. Therefore, near-infrared fluorescence surgical navigation targeting PSMA will be a very promising surgical method for treating prostate cancer. Furthermore, optical-nuclear medicine dual-modality molecular imaging probes can combine the advantages of two imaging modalities, with high sensitivity and high accuracy, to identify and locate diseased tissues through nuclear medicine modalities before surgery, and to accurately fluorescently mark the location of lesions during surgery to guide surgical resection.

[0005] The radionuclide diagnostic and surgical navigation drugs targeting PSMA currently used in clinical trials are all based on glutamic acid-urea groups as the basic skeleton. These probes have good targeting and affinity for PSMA, but there are problems of severe radionuclide retention in the bladder and high radionuclide background in the kidneys. The retention of a large amount of radionuclide in the bladder seriously affects the accuracy of early diagnosis of prostate cancer, and the high radionuclide background in both kidneys has certain toxic side effects on the kidneys. Therefore, it is urgent to develop PSMA molecular imaging probes with high specificity and low renal excretion by changing the drug structure, which is of great significance for the precise positioning and grading of prostate cancer lesions. However, the shortcomings of the existing probes in terms of targeting and metabolic safety limit the prospects for application and promotion.

[0006] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention and should not be regarded as an acknowledgment or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art. Summary of the invention

[0007] The object of the present invention is to provide a compound and a preparation method, a composition, a kit and an application thereof. The compound and its application products have good recognition ability for PSMA receptors and good targeting. In practical applications, they can be widely used in multiple scenarios such as flow cytometry analysis and in vivo imaging, and have advantages such as fast signal response speed and high resolution.

[0008] To achieve the above object, the embodiments of the present invention provide a compound, the structural formula of which is as follows:

[0009] Wherein the cation M is selected from an alkali metal cation, R is a C2-C5 alkyl group, and n is an integer in the range of 2-5.

[0010] In one or more embodiments of the present invention, the cation M is selected from Na + , K + , Li + .

[0011] In one or more embodiments of the present invention, the method for preparing the compound comprises the following steps:

[0012] Prepare intermediate A and intermediate B, intermediate A is R is a C2-C5 alkyl group, n is an integer in the range of 2-5, and the intermediate B is The cation Q is selected from Na + , K + , Li + ; Intermediate A and intermediate B are stirred and reacted, and then separated to obtain the target compound.

[0013] In one or more embodiments of the present invention, intermediate A is prepared as follows:

[0014] A1, prepare swollen CTC resin, react with Fmoc-Tyr(tBu)-OH / DIEA, and then deprotect to obtain intermediate 1;

[0015] B1, intermediate 1 reacts with Fmoc-NH-PEG2-CH2CH2COOH / HOBt after activation and deprotection to obtain intermediate 2;

[0016] C1, intermediate 2 and compound 1 / HBTU / HOBt react after activation, shrink, cleave and deprotect to obtain intermediate A, wherein compound 1 is

[0017] In one or more embodiments of the present invention, intermediate B is prepared as follows:

[0018] A2, phosphorus trichloride reacts with DMF and cyclohexanone, the resulting mixture is mixed with ice and stirred for reaction, the precipitate is separated, and the precipitate is washed, purified, and dried to obtain compound 2;

[0019] After hydrazinobenzenesulfonic acid, sodium acetate and 3-methyl-2-butanone are stirred in acetic acid under protective gas atmosphere for reaction, ether is added for precipitation, and the mixture is washed and dried to obtain compound 3; compound 3 is reacted with 1,4-butanesulfone under o-dichlorobenzene and protective gas conditions, and then washed, separated and dried to obtain compound 4;

[0020] B2. After the compound 2, compound 4 and sodium acetate are heated in methanol, acetic anhydride is added for reaction, and the intermediate B is obtained by separation and purification at room temperature.

[0021] In one or more embodiments of the present invention, the stirring reaction condition is 65-75° C. for 0.5-1.5 h. Preferably, the stirring reaction condition is 70° C. for 1 h.

[0022] In one or more embodiments of the present invention, the composition includes the aforementioned compound and may also include other auxiliary components such as water or an appropriate amount of sodium chloride. The auxiliary components may also include a carrier and the like.

[0023] In one or more embodiments of the present invention, the kit includes the aforementioned compound or the aforementioned composition.

[0024] In one or more embodiments of the present invention, the aforementioned compound, the aforementioned composition, or the aforementioned kit is used in a fluorescent detection material. The fluorescent detection material includes but is not limited to reagents and the like.

[0025] In one or more embodiments of the present invention, the aforementioned compound, the aforementioned composition, or the aforementioned kit is used in a PSMA fluorescence detection reagent.

[0026] In one or more embodiments of the present invention, the aforementioned compound or the aforementioned composition is used in a flow cytometry detection reagent.

[0027] Compared with the prior art, the compounds and preparation methods, compositions, kits and applications according to the embodiments of the present invention have good recognition ability for PSMA receptors and good targeting. In practical applications, they can be widely used in multiple scenarios such as flow cytometry analysis and in vivo imaging, and have advantages such as fast signal response speed and high resolution. In addition, they show high resolution in flow cytometry experiments and have good metabolic safety and metabolic efficiency, which is conducive to the wide application of products in PSMA detection technology. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 is a flow cytometry detection result according to an embodiment of the present invention;

[0029] Figure 2 is a fluorescence metabolism map according to one embodiment of the present invention;

[0030] Figure 3 is a comparison diagram of a product according to an embodiment of the present invention and an existing product OTL78;

[0031] Figure 4 The fluorescence metabolism comparison diagram of the product according to one embodiment of the present invention and the existing product OTL78: (a) is a 1h metabolism comparison diagram; (b) is a 2h metabolism comparison diagram; (c) is a 4h metabolism comparison diagram; (d) is an 8h metabolism comparison diagram; (e) is a 24h metabolism comparison diagram; (f) is a 72h metabolism comparison diagram; the circled part in each diagram is the tumor location;

[0032] Figure 5 This is a 2-hour in vitro tissue comparison chart of a product according to one embodiment of the present invention and an existing product OTL78. DETAILED DESCRIPTION

[0033] The specific implementation modes of the present invention are described in detail below in conjunction with the accompanying drawings, but it should be understood that the protection scope of the present invention is not limited by the specific implementation modes.

[0034] Unless explicitly stated otherwise, throughout the specification and claims, the term “comprise” or variations such as “include” or “comprising”, etc., will be understood to include the stated elements or components but not to exclude other elements or components.

[0035] The prior art OTL78 refers to the compound in Figure 25 of the prior art “201780067507.0”.

[0036] Including but not limited to the following embodiments: Refers to the carrier resin itself that does not participate in the reaction. eq refers to molar multiple. "V" represents the volume / mass ratio, that is, taking the table as an example, 7V means that when the amount of CTC resin is 1g, the amount of the corresponding reagent is 7mL. Mice refer to BALB / c nude mice, 6-8 weeks old, weighing 18-20 grams; rats refer to SD rats: 8 weeks old, about 250g.

[0037] Example 1

[0038] Synthesis of intermediate A:

[0039] A solid phase synthesis method was used: CTC resin was used as a fixed carrier raw material (1 g), which was first swollen in DMF for 1 h, and 3.0 equivalents (equivalent in this embodiment refers to the mass multiple relative to CTC resin, the same below) of Fmoc-Tyr(tBu)-OH were dissolved in 7V DMF relative to the resin, and then 6.0 equivalents (equivalent here refers to the mass multiple relative to CTC resin, the same below) of DIEA were added to hang the resin, react for 2 h, and washed with DMF 5 times, 6 eq (eq in this embodiment is the molar multiple relative to Fmoc-Tyr(tBu)-OH, the same below) of methanol and 10 eq (eq in this embodiment is the molar multiple relative to Fmoc-Tyr(tBu)-OH, the same below) of DIEA were added for end-capping, and washed with DMF 4 to 5 times; 20% piperidine / DMF solution was then added to remove the N-terminal Fmoc protecting group to make the N-terminal a free amino group, and washed with DMF 5 to 6 times to obtain intermediate 1;

[0040] 3.0 equivalents (equivalent in this embodiment refers to the mass multiple relative to CTC resin, the same below) of Fmoc-NH-PEG2-CH2CH2COOH were dissolved in 7V DMF relative to the resin, 3.0 equivalents of HOBt were added, 3.8 equivalents of DIC were activated for 5 min, and then added to the resin, and the reaction was carried out under nitrogen protection for 1 h, and washed with DMF 4 to 5 times; then, the N-terminal Fmoc protecting group was removed again with 20% piperidine / DMF solution to make the N-terminal a free amino group, and washed with DMF 5 times to obtain intermediate 2;

[0041] The intermediate 1 (200 mg) obtained by the solid phase synthesis was placed in a three-necked flask, and 3.0 equivalents (equivalent in this example refers to the mass multiple relative to CTC resin, the same below) of

[0042] C221211A-int2 (3-BOC-DUPA) was dissolved, 3.0 equivalents of HBTU, 1.5 equivalents of HOBt, and 4.5 equivalents of DIEA were added for activation for 5 min, and then added to the resin, reacted for 1 h under nitrogen protection, and rinsed with DMF 4 to 5 times;

[0043] Methanol was added for concentration and then dried.

[0044] The target polypeptide was cleaved from the resin using a cleavage solution (trifluoroacetic acid: H2O: triisopropylsilane = 90:5:5, v / v) and the side chain protecting groups were removed (reacted at 20-30°C for 3 hours). The mixture was filtered and the filtrate was added to methyl tert-butyl ether to precipitate. The crude product of C221211A-19 was obtained by centrifugation and prepared for separation. After freeze-drying, C221211A-19 (70 mg), i.e., intermediate A, was obtained.

[0045] Purity 62.18%, LCMS: [M+H] + =643.2

[0046]

[0047]

[0048] Synthesis of intermediate B (dye) Synthesis of compound 2: (E)-2-chloro-3-(hydroxymethylene)cyclohex-1-enecarbaldehyde (I).

[0049]

[0050] At 0°C, phosphorus oxychloride (35.0 mL, 58.8 g, 0.38 mol) was added dropwise to a dry flask containing anhydrous DMF (50 mL). Stir for 30 min, add cyclohexanone (10.5 mL, 9.98 g, 0.10 mol), and heat to 50°C for 6 hours. Subsequently, the red mixture was poured into 150 g of ice and stirred for 5 hours. The yellow precipitate was collected, washed with water, and purified with CH2Cl2. The product was filtered, collected, and dried in vacuo. Compound 2 (13.5 g, 78.4 mmol, yield 77%) was obtained. LCMSm / z=173 (m+H) + .

[0051]

[0052] Synthetic compound 3: Chemical name: 2,3,3-trimethyl-3H-indole-5-sulfonic acid (II)

[0053] Add p-hydrazinobenzenesulfonic acid (10.00 g, 53.2 mmol), sodium acetate (8.72 g, 106.4 mmol) and 3-methyl 2-butanone (16.80 mL, 13.50 g, 156.80 mmol), glacial acetic acid (60 mL) in sequence. Stir for 5 hours at 130 ° C under argon protection. Cool to room temperature, add ether to precipitate the product. Filter and dry in vacuo to obtain compound 3 (11.2 g, 46.8 mmol, 88%). LCMS: m / z = 240, (m+H) + .

[0054] Synthesis of compound 4: 5-sulfo-1-(4-sulfobutyl)-2,3,3-trimethyl-3H-indolium betaine (III)

[0055] Compound 3 (5.00 g, 20.92 mmol) and 1,4-butane sultone (4.91 mL, 6.54 g, 48.11 mmol) were added to 1,2-dichlorobenzene (125 mL) and stirred at 110 ° C for 12 hours under argon protection. After cooling to room temperature, deionized water was added to the reaction mixture to dissolve the solid matter. The aqueous phase was washed with CH2Cl2 and the organic layer was discarded. Freeze-dried to obtain compound 4 (5.21 g, 13.89 mmol, 66% yield). LCMS: m / z=375.9, (m+H) +

[0056] Synthetic intermediate B (dye).

[0057]

[0058] Compound 4 (3.00 g, 8.00 mmol), compound 2 (688 mg, 4.00 mmol,) and sodium acetate (656 mg, 8.00 mol) were heated to 70°C in methanol (80 mL). Acetic anhydride (80 mL) was added and stirred for 8 hours. After cooling to room temperature, ether was added to the solution to precipitate. Filter and collect the solid precipitate, dissolve the solid precipitate in deionized water, and precipitate with 2-propanol. Filter and collect the solid, and repeat the process twice. After vacuum drying, (dye) intermediate B (1.18 g, 1.33 mmol, 33%) was obtained. LC-MS m / z: 886.8 (M) + .

[0059] 1 H NMR (400MHz, D2O): δ = 8.35 (d, J = 14.0Hz, 2H), 7.88 (s, 2H), 7.79 (d, J = 8.4Hz, 2H), 7.34 (d, J = 8.4Hz, 2H), 6.25(d,J=14.0Hz,2H),4.17(s,4H),3.07-2.86(m,4H),2.59(s,4H),2.03-1.76(m,10H),1.72(s,12H).

[0060] Synthesis of DGPR1008:

[0061]

[0062] C221211A-19 (68.6 mg, 0.1068 mmol), C221211A-int1 (dye)

[0063] (104.2mg, 0.1068mmol) was placed in a 25ml single-mouth bottle, and Na2CO3 (67.8mg, 0.6403mmol) was dissolved in purified water (40V relative to the resin), added to the reaction bottle, stirred at 70℃ for 1h, and sampled for HPLC detection, RT=6.930; purity: 40.52%. The reaction solution was prepared and purified, and the mobile phase was 0.5% ammonium bicarbonate in methanol / water solution, and the product 69.2mg was lyophilized.

[0064] 1 H NMR(400MHz,D2O)δ7.92(s,2H),7.79(s,4H),7.43-7.22(m,4H),7.19-7.02(m,2H),6.29-6.09(m,1H),4.52-4.33(m,1H),4.23-3.97(m,6H ),3.64-3.14(m,13H),3.04–2.56(m,9H),2.48–2.33(m,4H),2.33-2. 23(m,2H),2.19-1.99(m,2H),1.99-1.70(m,12H),1.43-1.30(m,12H).

[0065] Product testing:

[0066] 1. In vitro flow cytometry to detect receptor binding:

[0067] Experimental Principle

[0068] Prostate-specific membrane antigen (PSMA) is overexpressed on prostate cancer cells. Both DUPA-FITC and DGPR1008 can target PSMA receptors in prostate cancer cells. The difference is that DUPA-FITC is connected to a visible light dye (Ex=495nm, Em=520nm), while DGPR1008 is connected to a near-infrared dye (Ex=776nm, Em=793nm). Since most of the laser types equipped in flow cytometers are within the visible light range, it is impossible to directly measure the binding force of DGPR1008 to the receptor. Therefore, DGPR1008 and DUPA-FITC are used to compete for binding to PSMA to indirectly react to the binding force between the test compound and the receptor.

[0069] Experimental Materials

[0070] DGPR1008, DUPA-FITC, 22RV1 (PSMA+), PC3 (PSMA-), 1640 culture medium, FBS, double antibody, trypsin, centrifuge, 4°C constant temperature incubator, flow cytometer.

[0071] Experimental methods:

[0072] Drug preparation: 100 nmol / L DUPA-FITC was prepared in culture medium, and the test compound was diluted to 0.00098 μM, 0.002 μM, 0.008 μM, 0.01 μM, 0.03 μM, 0.05 μM, 0.09 μM, 0.1 μM, and 0.125 μM with culture medium containing 100 nmol / L DUPA-FITC, for a total of nine concentrations, with three replicate wells for each concentration.

[0073] Cell culture: cells were inoculated in T75 culture flasks. After 48 hours, cells in logarithmic growth phase were obtained and digested with trypsin for 1-2 minutes. Fresh culture medium was added to terminate digestion. After counting the cells, the cells were transferred into 96-well plates (1*10 5 Cells / well), 27 wells in total. Centrifuge (800rpm, 5min) to discard the culture medium, rinse the cells twice with PBS and collect the cell precipitate by centrifugation. Add the prepared 9 concentrations of culture medium to the wells with cells, 3 replicates for each concentration. Incubate at 4℃ in the dark for 30min, discard the culture medium in the wells, add 300μL fresh culture medium (2 times) and saline (1 time) to each well to wash the cells, then resuspend the cells with 300μL saline for flow cytometry detection.

[0074] Experiments show that: Figure 1 As shown, the negative cell line PC3 has no binding ability to the PSMA receptor, while the positive cell line 22RV1 has a certain binding ability to the PSMA receptor, and the target compound shows good affinity. 2. In vivo live imaging efficacy verification experiment

[0075] Experimental Principle

[0076] Prostate-specific membrane antigen (PSMA) is overexpressed on prostate cancer cells. The test compound is a PSMA-targeting molecule with a near-infrared I dye attached to the molecule. The test compound can bind to PSMA and "light up" cancer cells under the stimulation of near-infrared light sources. This experiment uses in vivo live imaging to detect the tissue distribution of the test compound in mice.

[0077] Experimental Materials

[0078] DGPR1008, 22RV1 (PSMA+), PC3 (PSMA-), 1640 culture medium, FBS, double antibody, trypsin, Matrigel (BD), vernier caliper, centrifuge, IVIS live imager.

[0079] Experimental methods

[0080] Cell line: 22RV1 (PSMA+)

[0081] Place Matrigel on ice to melt in advance, and pre-cool the culture medium on ice. After centrifugation of the cells, resuspend the cells in pre-cooled culture medium and add HC Matrigel gel in an equal volume to the culture medium. Place the centrifuge tube containing the cells on ice and transfer it to the animal room. Blow the cells thoroughly before inoculation. Disinfect the right shoulder of the mouse with an alcohol cotton ball and administer a single intravenous dose with an inoculation volume of 200 μL / mouse. All operations are performed on ice. The tumor volume reaches 300-400mm 3 The drug was administered in groups, and 9 mice with good tumor growth and regular shape were selected for inclusion. The 9 mice were randomly divided into 3 groups: G125nmol / mouse group, G250nmol / mouse group, and G3100nmol / mouse group, with 3 mice in each group. The scanning time was 1, 2, 4, 8, 24, and 48h.

[0082] Experiments show that: Figure 2 As shown, at the three different doses of high, medium and low, the target compound can be enriched in the prostate cancer tumor site within a certain period of time. The DGPR1008 compound showed good targeting.

[0083] 3. Comparison with existing product OTL78

[0084] ① Results and data of in vivo imaging of drug efficacy (mice were used as experimental subjects in this part)

[0085] like Figure 4 As shown, in vivo imaging data comparison of OTL78 and DGPR1008:

[0086]

[0087]

[0088] Combining images and data, we can see that:

[0089] 1) At every time point, the fluorescence value of DGPR1008 in the tumor site was greater than that of OTL78;

[0090] 2) The elimination metabolism of DGPR1008 in mice is slower than that of OTL78;

[0091] 3) The compound of this example showed good TBR (tumor to background ratio) within 2-4 hours.

[0092] ②2h in vitro tissue distribution results (rats were used as experimental subjects in this part):

[0093] Fluorescence ratio of tumor and normal prostate in situ:

[0094]

[0095] Fluorescence Units:

[0096] photon / second

[0097] in conclusion:

[0098] 1) From the above table and Figure 5 It can be seen that when the compound of this example is used as a recognition aid, there is an obvious signal intensity ratio in tumor / normal prostate tissue, which can fully reflect the ability of the drug to distinguish normal prostate from diseased prostate;

[0099] 2) Compared with OTL78, DGPR1008 of this embodiment exhibits stronger differentiation ability (about 5 times) and higher TBR. The higher TBR can make the tumor boundary line clearer during surgery and obtain more accurate resection margins.

[0100] 3) From the pharmacokinetic parameters of 2 mg / kg plasma in rats, it can be seen that the average clearance of OTL78 is 3.50 mL / min / kg, and the average clearance of DGPR1008 in this example is 5.23 mL / min / kg, indicating that DGPR1008 is metabolized faster in rats and the safety factor will be increased accordingly.

[0101] 4) In the acute toxicity experiment on rats, no toxic reactions were observed in the three dose groups of high, medium and low doses, which proved that the DGPR1008 compound of this example has good safety.

[0102] In summary, the compound of this example has a high signal-to-noise ratio in fluorescent display applications, which means it is more sensitive during surgery; and it is cleared quickly, which means it is cleared more quickly after surgery, and has a greater safety advantage.

[0103] Example 2

[0104] The only difference from Example 1 is: The other corresponding reagents can be adaptively adjusted by those skilled in the art.

[0105] The target compound prepared in this example is It is detected by the test method shown in Example 1: the negative cell line PC3 has no binding ability to the PSMA receptor, the positive cell 22RV1 has a certain binding ability to the PSMA receptor, and the target compound shows good affinity. The target compound can be enriched in the prostate cancer tumor site within a certain period of time. The target compound shows good targeting. In the experiment with mice as experimental subjects, the fluorescence value of the compound of this embodiment at the tumor site is greater than OTL78; the elimination metabolism in the mouse body is slower than OTL78; and a good TBR (tumor background ratio) is shown within 2-4h. In the experiment with rats as experimental subjects, a good TBR (tumor background ratio) is shown within 2-4h. It shows a stronger ability to distinguish (about 5 times), a higher TBR, and a higher TBR can make the tumor boundary line clearer during surgery and obtain a more accurate resection margin; it is metabolized faster in rats, and the safety factor will be correspondingly increased; in the acute toxicity experiment of rats, no toxic reactions were observed in the three dose groups of high, medium and low. These all indicate that the compound of this example has a high signal-to-noise ratio in fluorescent display applications, which means it is more sensitive during surgery; and a fast clearance rate, which means it is cleared more quickly after surgery, and has a greater safety advantage.

[0106] Example 3

[0107] The only difference from Example 1 is: The other corresponding reagents can be adaptively adjusted by those skilled in the art.

[0108] The target compound prepared in this example is:

[0109]

[0110] It is detected by the test method shown in Example 1: the negative cell line PC3 has no binding ability to the PSMA receptor, the positive cell 22RV1 has a certain binding ability to the PSMA receptor, and the target compound shows good affinity. The target compound can be enriched in the prostate cancer tumor site within a certain period of time. The target compound shows good targeting. In the experiment with mice as experimental subjects, the fluorescence value of the compound of this embodiment at the tumor site is greater than OTL78; the elimination metabolism in the mouse body is slower than OTL78; and a good TBR (tumor background ratio) is shown within 2-4h. In the experiment with rats as experimental subjects, a good TBR (tumor background ratio) is shown within 2-4h. It shows a stronger ability to distinguish (about 5 times), a higher TBR, and a higher TBR can make the tumor boundary line clearer during surgery and obtain a more accurate resection margin; it is metabolized faster in rats, and the safety factor will be correspondingly increased; in the acute toxicity experiment of rats, no toxic reactions were observed in the three dose groups of high, medium and low. These all indicate that the compound of this example has a high signal-to-noise ratio in fluorescent display applications, which means it is more sensitive during surgery; and a fast clearance rate, which means it is cleared more quickly after surgery, and has a greater safety advantage.

[0111] The intermediate 1 used in the following examples, including but not limited to, can be realized by using the technical means listed in Example 1, including but not limited to.

[0112] Example 4

[0113] The target compound prepared in this example is:

[0114]

[0115] The preparation process is as follows:

[0116]

[0117] The intermediate 1 (200 mg) obtained by the solid phase synthesis was placed in a three-necked flask, and 3.0 equivalents (the equivalent in this example refers to the mass multiple of CTC resin, the same below) of

[0118] Fmoc-NH-PEG3-CH2CH2COOH was dissolved, 3.0 equivalents of HOBt and 3.8 equivalents of DIC were added for activation for 6 min, nitrogen protection was used for reaction for 1 h, and DMF was used for washing 4 times; then, the N-terminal Fmoc protecting group was removed with 20% piperidine / DMF solution to make the N-terminal a free amino group, and DMF was used for washing 5 times to obtain intermediate 22;

[0119] 3.0 equivalents of 3-BOC-DUPA were dissolved in 7V DMF relative to the resin, 3.0 equivalents of HBTU, 1.5 equivalents of HOBt, and 4.5 equivalents of DIEA were added for activation for 5 min, and then added to the intermediate 22, reacted for 1 h under nitrogen protection, and rinsed with DMF 5 times;

[0120] Methanol was added for concentration and then dried.

[0121] The target polypeptide was cleaved from the resin using a cleavage solution (trifluoroacetic acid: H2O: triisopropylsilane = 90:5:5, v / v), and the side chain protecting groups were removed (reacted at 25°C for 3 hours). The filtrate was filtered and added to methyl tert-butyl ether, a precipitate was precipitated, and centrifuged to obtain a crude product of peptide-linked DUPA, which was prepared and separated, and freeze-dried to obtain intermediate 23.

[0122] Condensation of intermediate 23 and dye: 50 mg of intermediate 23 and dye were placed in a 25 ml single-necked bottle at a molar ratio of 1:1, 6 equivalents of Na2CO3 were dissolved in purified water (40V relative to the resin), added to the reaction bottle, stirred at 70°C for 1 h, and the reaction solution was purified to obtain 23 mg of the product.

[0123] LCMS: [M-7Na+4H] 3+ =511.2, molecular weight of target compound sodium salt: 1690.33

[0124] 1 H NMR (400MHz, D2O): δ7.92(s,2H),7.79(s,4H),7.43-7.22(m,4H),7.19-7.02(m,2H),6.29-6.09(m,1H),4.52-4.33(m,1H),4.23-3.97(m,6H) ),3.64-3.14(m,17H),3.04–2.56(m,9H),2.48–2.33(m,4H),2.33-2. 23(m,2H),2.19-1.99(m,2H),1.99-1.70(m,12H),1.43-1.30(m,12H).

[0125] It is detected by the test method shown in Example 1: the negative cell line PC3 has no binding ability to the PSMA receptor, the positive cell 22RV1 has a certain binding ability to the PSMA receptor, and the target compound shows good affinity. The target compound can be enriched in the prostate cancer tumor site within a certain period of time. The target compound shows good targeting. In the experiment with mice as experimental subjects, the fluorescence value of the compound of this embodiment at the tumor site is greater than OTL78; the elimination metabolism in the mouse body is slower than OTL78; and a good TBR (tumor background ratio) is shown within 2-4h. In the experiment with rats as experimental subjects, a good TBR (tumor background ratio) is shown within 2-4h. It shows a stronger ability to distinguish (about 5 times), a higher TBR, and a higher TBR can make the tumor boundary line clearer during surgery and obtain a more accurate resection margin; it is metabolized faster in rats, and the safety factor will be correspondingly increased; in the acute toxicity experiment of rats, no toxic reactions were observed in the three dose groups of high, medium and low. These all indicate that the compound of this example has a high signal-to-noise ratio in fluorescent display applications, which means it is more sensitive during surgery; and a fast clearance rate, which means it is cleared more quickly after surgery, and has a greater safety advantage.

[0126] Example 5

[0127] The target compound prepared in this example is:

[0128]

[0129] The preparation process is as follows:

[0130]

[0131] The intermediate 1 obtained by the solid phase synthesis was placed in a three-necked flask, and 3.0 equivalents (the equivalent in this example refers to the mass multiple of CTC resin, the same below) of DMF were added to the resin at 7V.

[0132] Fmoc-NH-(CH2CH2CH2O)2-CH2CH2COOH was dissolved, 3.0 equivalents of HOBt and 3.8 equivalents of DIC were added for activation for 6 min, and the reaction was carried out under nitrogen protection for 1 h, and then rinsed with DMF 4 times; then the N-terminal Fmoc protecting group was removed with 20% piperidine / DMF solution to make the N-terminal a free amino group, and then rinsed with DMF 5 times to obtain intermediate 32;

[0133] 3.0 equivalents of 3-BOC-DUPA were dissolved in 7V DMF relative to the resin, 3.0 equivalents of HBTU, 1.5 equivalents of HOBt, and 4.5 equivalents of DIEA were added for activation for 5 min, and then added to the intermediate 32, reacted for 1 h under nitrogen protection, and rinsed with DMF 5 times;

[0134] Methanol was added for concentration and then dried.

[0135] The target polypeptide was cleaved from the resin using a cleavage solution (trifluoroacetic acid: H2O: triisopropylsilane = 90:5:5, v / v), and the side chain protecting groups were removed (reacted at 30°C for 3 hours). The filtrate was filtered and added to methyl tert-butyl ether, a precipitate was precipitated, and centrifuged to obtain a crude product of peptide-linked DUPA, which was prepared and isolated, and freeze-dried to obtain intermediate 33.

[0136] Condensation of intermediate 33 and dye: 50 mg of intermediate 33 and dye were placed in a 25 ml single-mouth bottle at a molar ratio of 1:1, 6 equivalents of Na2CO3 were dissolved in purified water (40V relative to the resin), added to the reaction bottle, stirred at 70°C for 1 h, and the reaction solution was purified to obtain 35 mg.

[0137] LCMS: [M-7Na+4H] 3+ =505.6, molecular weight of target compound sodium salt: 1674.34

[0138] 1 H NMR (400MHz, D2O): δ7.92(s,2H),7.79(s,4H),7.43-7.22(m,4H),7.19-7.02(m,2H),6.29-6.09(m,1H),4.52-4.33(m,1H),4.23-3.97(m,6H) ),3.64-3.14(m,17H),3.04–2.56(m,9H),2.48–2.33(m,4H),2.33-2. 23(m,2H),2.19-1.99(m,2H),1.99-1.70(m,16H),1.43-1.30(m,12H).

[0139] It is detected by the test method shown in Example 1: the negative cell line PC3 has no binding ability to the PSMA receptor, the positive cell 22RV1 has a certain binding ability to the PSMA receptor, and the target compound shows good affinity. The target compound can be enriched in the prostate cancer tumor site within a certain period of time. The target compound shows good targeting. In the experiment with mice as experimental subjects, the fluorescence value of the compound of this embodiment at the tumor site is greater than OTL78; the elimination metabolism in the mouse body is slower than OTL78; and a good TBR (tumor background ratio) is shown within 2-4h. In the experiment with rats as experimental subjects, a good TBR (tumor background ratio) is shown within 2-4h. It shows a stronger ability to distinguish (about 5 times), a higher TBR, and a higher TBR can make the tumor boundary line clearer during surgery and obtain a more accurate resection margin; it is metabolized faster in rats, and the safety factor will be correspondingly increased; in the acute toxicity experiment of rats, no toxic reactions were observed in the three dose groups of high, medium and low. These all indicate that the compound of this example has a high signal-to-noise ratio in fluorescent display applications, which means it is more sensitive during surgery; and a fast clearance rate, which means it is cleared more quickly after surgery, and has a greater safety advantage.

[0140] Example 6

[0141] The target compound prepared in this example is:

[0142]

[0143] The preparation process is as follows:

[0144]

[0145] The intermediate 1 (240 mg) obtained by the solid phase synthesis was placed in a three-necked flask, and 3.0 equivalents (the equivalent in this example refers to the mass multiple of CTC resin, the same below) were added with 12 mL DMF.

[0146] Fmoc-NH-PEG3-(CH2CH2CH2O)2-CH2CH2COOH was dissolved, 3.0 equivalents of HOBt and 3.8 equivalents of DIC were added for activation for 6 min, nitrogen protection was used for reaction for 1 h, and DMF was used for washing 4 times; then, the N-terminal Fmoc protecting group was removed with 20% piperidine / DMF solution to make the N-terminal a free amino group, and DMF was used for washing 5 times to obtain intermediate 42;

[0147] 3.0 equivalents of 3-BOC-DUPA were dissolved in 7V DMF relative to the resin, 3.0 equivalents of HBTU, 1.5 equivalents of HOBt, and 4.5 equivalents of DIEA were added for activation for 5 min, and then added to the intermediate 42, reacted for 1 h under nitrogen protection, and rinsed with DMF 5 times;

[0148] Methanol was added for concentration and then dried.

[0149] The target polypeptide was cleaved from the resin using a cleavage solution (trifluoroacetic acid: H2O: triisopropylsilane = 90:5:5, v / v), and the side chain protecting groups were removed (reacted at 20°C for 4 hours). The filtrate was filtered and added to methyl tert-butyl ether, a precipitate was precipitated, and the crude product of peptide-linked DUPA was obtained by centrifugation. The intermediate 43 was obtained after preparative separation and freeze-drying.

[0150] Condensation of intermediate 43 and dye: 50 mg of intermediate 43 and dye were placed in a 25 ml single-necked bottle at a molar ratio of 1:1, 6 equivalents of Na2CO3 were dissolved in purified water (40V relative to the resin), added to the reaction bottle, and stirred at 70°C for 1 h. The reaction solution was purified to obtain 27 mg of product.

[0151] LCMS: [M-7Na+4H] 3+ =549.6, molecular weight of target compound sodium salt: 1806.42

[0152] 1 H NMR (400MHz, D2O): δ7.92(s,2H),7.79(s,4H),7.43-7.22(m,4H),7.19-7.02(m,2H),6.29-6.09(m,1H),4.52-4.33(m,1H),4.23-3.97(m,6H) ),3.64-3.14(m,29H),3.04–2.56(m,9H),2.48–2.33(m,4H),2.33-2. 23(m,2H),2.19-1.99(m,2H),1.99-1.70(m,16H),1.43-1.30(m,12H).

[0153] It is detected by the test method shown in Example 1: the negative cell line PC3 has no binding ability to the PSMA receptor, the positive cell 22RV1 has a certain binding ability to the PSMA receptor, and the target compound shows good affinity. The target compound can be enriched in the prostate cancer tumor site within a certain period of time. The target compound shows good targeting. In the experiment with mice as experimental subjects, the fluorescence value of the compound of this embodiment at the tumor site is greater than OTL78; the elimination metabolism in the mouse body is slower than OTL78; and a good TBR (tumor background ratio) is shown within 2-4h. In the experiment with rats as experimental subjects, a good TBR (tumor background ratio) is shown within 2-4h. It shows a stronger ability to distinguish (about 5 times), a higher TBR, and a higher TBR can make the tumor boundary line clearer during surgery and obtain a more accurate resection margin; it is metabolized faster in rats, and the safety factor will be correspondingly increased; in the acute toxicity experiment of rats, no toxic reactions were observed in the three dose groups of high, medium and low. These all indicate that the compound of this example has a high signal-to-noise ratio in fluorescent display applications, which means it is more sensitive during surgery; and a fast clearance rate, which means it is cleared more quickly after surgery, and has a greater safety advantage.

[0154] Example 7

[0155] The target compound prepared in this example is:

[0156]

[0157] The preparation process is as follows:

[0158]

[0159] The intermediate 1 (200 mg) obtained by the solid phase synthesis was placed in a three-necked flask, and 3.0 equivalents (the equivalent in this embodiment refers to the mass multiple of the CTC resin, the same below) of Fmoc-NH-PEG2-(CH2)5O-(CH2)3O-CH2CH2COOH were dissolved in 7V DMF relative to the resin, and 3.0 equivalents of HOBt and 3.8 equivalents of DIC were added for activation for 6 min, and the reaction was carried out under nitrogen protection for 1 h, and washed with DMF 4 times; then, the N-terminal Fmoc protecting group was removed with 20% piperidine / DMF solution to make the N-terminal a free amino group, and washed with DMF 5 times to obtain intermediate 52;

[0160] 3.0 equivalents of 3-BOC-DUPA were dissolved in 7V DMF relative to the resin, 3.0 equivalents of HBTU, 1.5 equivalents of HOBt, and 4.5 equivalents of DIEA were added for activation for 5 min, and then added to the intermediate 52, reacted for 1 h under nitrogen protection, and rinsed with DMF 5 times;

[0161] Methanol was added for concentration and then dried.

[0162] The target polypeptide was cleaved from the resin using a cleavage solution (trifluoroacetic acid: H2O: triisopropylsilane = 90:5:5, v / v), and the side chain protecting groups were removed (reacted at 30°C for 3 hours). The filtrate was filtered and added to methyl tert-butyl ether, a precipitate was precipitated, and centrifuged to obtain a crude product of peptide-linked DUPA, which was prepared and separated, and freeze-dried to obtain intermediate 53.

[0163] Condensation of intermediate 53 and dye: 50 mg of intermediate 53 and dye were placed in a 25 ml single-necked bottle at a molar ratio of 1:1, 7 equivalents of Na2CO3 were dissolved in purified water (40V relative to the resin), added to the reaction bottle, and stirred at 70°C for 1 h. The reaction solution was purified to obtain 35 mg of product.

[0164] LCMS: [M-7Na+4H] 3+ =544.3, molecular weight of target compound sodium salt: 1790.42

[0165] 1 H NMR (400MHz, D2O): δ7.92(s,2H),7.79(s,4H),7.43-7.22(m,4H),7.19-7. 02(m,2H),6.29-6.09(m,1H),4.52-4.33(m,1H),4.23-3.97(m,6H),3.64- 3.14(m,25H),3.04–2.56(m,9H),2.48–2.33(m,4H),2.33-2.23(m,2H),2. 19-1.99(m,2H),1.99-1.70(m,14H),1.50-1.56(m,6H)1.43-1.30(m,12H).

[0166] It is detected by the test method shown in Example 1: the negative cell line PC3 has no binding ability to the PSMA receptor, the positive cell 22RV1 has a certain binding ability to the PSMA receptor, and the target compound shows good affinity. The target compound can be enriched in the prostate cancer tumor site within a certain period of time. The target compound shows good targeting. In the experiment with mice as experimental subjects, the fluorescence value of the compound of this embodiment at the tumor site is greater than OTL78; the elimination metabolism in the mouse body is slower than OTL78; and a good TBR (tumor background ratio) is shown within 2-4h. In the experiment with rats as experimental subjects, a good TBR (tumor background ratio) is shown within 2-4h. It shows a stronger ability to distinguish (about 5 times), a higher TBR, and a higher TBR can make the tumor boundary line clearer during surgery and obtain a more accurate resection margin; it is metabolized faster in rats, and the safety factor will be correspondingly increased; in the acute toxicity experiment of rats, no toxic reactions were observed in the three dose groups of high, medium and low. These all indicate that the compound of this example has a high signal-to-noise ratio in fluorescent display applications, which means it is more sensitive during surgery; and a fast clearance rate, which means it is cleared more quickly after surgery, and has a greater safety advantage.

[0167] The foregoing description of specific exemplary embodiments of the present invention is for the purpose of illustration and demonstration. These descriptions are not intended to limit the present invention to the precise form disclosed, and it is clear that many changes and variations can be made based on the above teachings. The purpose of selecting and describing the exemplary embodiments is to explain the specific principles of the present invention and its practical application, so that those skilled in the art can realize and utilize various different exemplary embodiments of the present invention and various different selections and changes. The scope of the present invention is intended to be limited by the claims and their equivalents.

Claims

1. A compound, the simplified structural formula of which is as follows: , wherein the cation M is selected from an alkali metal cation, R is ethylene, and n is an integer in the range of 2-5.

2. The compound according to claim 1, characterized in that The cation M is selected from Na + , K + , Li + .

3. A method for preparing the compound according to claim 1 or 2, comprising the steps of: Prepare intermediate A and intermediate B, intermediate A is , R is ethylene, n is an integer in the range of 2-5, and the intermediate B is , the cation Q is selected from Na + , K + , Li + ; Intermediate A and intermediate B are stirred and reacted, and then separated to obtain the target compound.

4. The method for preparing the compound according to claim 3, characterized in that: The preparation of the intermediate A is as follows: A1, prepare swollen CTC resin, react with Fmoc-Tyr(tBu)-OH / DIEA, and then deprotect to obtain intermediate 1; B1, intermediate 1 reacts with Fmoc-NH-PEG2-CH2CH2COOH / HOBt after activation and deprotection to obtain intermediate 2; C1, intermediate 2 and compound 1 / HBTU / HOBt react after activation, shrink, cleave and deprotect to obtain intermediate A, wherein compound 1 is .

5. The method for preparing the compound according to claim 3, characterized in that: The preparation of the intermediate B is: A2, phosphorus trichloride reacts with DMF and cyclohexanone, the obtained mixture is mixed with ice and stirred for reaction, the precipitate is separated, and the precipitate is washed, purified and dried to obtain compound 2; After hydrazinobenzenesulfonic acid, sodium acetate and 3-methyl-2-butanone are stirred in acetic acid under protective gas atmosphere for reaction, ether is added for precipitation, and the mixture is washed and dried to obtain compound 3; compound 3 is reacted with 1,4-butanesulfone under o-dichlorobenzene and protective gas conditions, and then washed, separated and dried to obtain compound 4; B2. After heating the compound 2, compound 4 and sodium acetate in methanol, acetic anhydride is added to react, and the intermediate B is obtained by separation and purification at room temperature.

6. The method for preparing the compound according to claim 3, characterized in that: The stirring reaction condition is 65-75° C. for 0.5-1.5 h.

7. A composition comprising the compound according to claim 1 or 2.

8. A kit comprising the compound according to claim 1 or 2 or the composition according to claim 7.

9. Use of the compound according to claim 1 or 2 or the composition according to claim 7 in a flow cytometry detection reagent.

10. Use of the compound according to claim 1 or 2, the composition according to claim 7, or the kit according to claim 8 in a fluorescent detection material.

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