阅读说明：本技术 一种双酶体系探针及其应用 (Double-enzyme system probe and application thereof ) 是由 杨兴 刘昭飞 徐红闯 王琰璞 张宁 范岩 杨志 高献书 于 2020-12-21 设计创作，主要内容包括：本发明属于核医学领域,涉及一种双酶体系探针及其应用。该探针具有式I所示结构,其中,QD为荧光基团或药物基团；L为linker,具有式II所示结构；T为PSMA特异性靶向基团；Z为H、核素螯合基团、放射性核素或螯合有放射性核素的核素螯合基团。本发明基于前列腺特异性膜抗原PSMA靶向和Caspase-3识别策略来递送药物分子的双酶体系核医学探针,可以靶向前列腺癌进行放疗增敏治疗,有助于解决去势抗性前列腺癌患者放疗抵抗和靶向核素治疗抗性等难题,并有望实现前列腺癌术中导航。为肿瘤治疗的检测和治疗提供更多高效的工具,具有广阔的应用前景。(The invention belongs to the field of nuclear medicine, and relates to a double-enzyme system probe and application thereof. The probe has a structure shown in a formula I, wherein QD is a fluorophore or a drug group; l is linker and has a structure shown in a formula II; t is a PSMA specific targeting group; z is H, a nuclide chelating group, a radionuclide or a nuclide chelating group chelated with a radionuclide. The invention relates to a double-enzyme system nuclear medicine probe for delivering medicine molecules based on the PSMA (prostate specific membrane antigen) targeting and Caspase-3 recognition strategies, which can be used forThe targeted prostate cancer is subjected to radiotherapy sensitization treatment, so that the problems of radiotherapy resistance and targeted nuclide treatment resistance of castration-resistant prostate cancer patients are solved, and the navigation in the prostate cancer operation is expected to be realized. Provides more efficient tools for the detection and treatment of tumor treatment, and has wide application prospect.)

1. A probe of a double enzyme system is characterized in that the probe has a structure shown in a formula I,

wherein the content of the first and second substances,

QD is a fluorophore or drug group;

l is linker and has a structure shown in formula II:

R₁is C₅-C₈Cycloalkylene or arylene of (a); r₂Is substituted or unsubstituted aryl; n is an integer of 1 to 6;

t is a PSMA specific targeting group;

z is H, a nuclide chelating group, a radionuclide or a nuclide chelating group chelated with a radionuclide.

2. The two-enzyme system probe according to claim 1, wherein in the structure represented by formula II, R is₁Is phenylene or cyclohexylene; a is 1, 2,3 or 4; r₂Is substituted or unsubstituted phenyl or naphthyl, the substituted group is halogen, hydroxyl, amino, carboxyl or C₁-C₄An alkyl group; the substitution is preferably monosubstituted.

3. The two-enzyme system probe according to claim 2, wherein R₂Is para-substituted phenyl or naphthyl, preferably para-halogen-substituted phenyl or naphthyl; the halogen is preferably chlorine, bromine, iodine.

4. The two-enzyme system probe according to claim 1, wherein the PSMA-specific targeting group is represented by formula III or formula IV:

5. the two-enzyme system probe according to claim 1, wherein the fluorescent group is derived from a fluorescent compound selected from the group consisting of 7- (ethylamino) -4, 6-dimethylcoumarin, 7-amino-4-trifluoromethylcoumarin, 7-amino-4-methylcoumarin, coumarin 307, 7-aminocoumarin, 3-aminocoumarin, 6-aminocoumarin, 7-amino-4-chloromethylcoumarin, 3-amino-7-hydroxycoumarin, 7-amino-4-carboxymethylcoumarin, 7-amino-4-methyl-3-coumarin acetic acid, 7-amino-4-methoxymethylcoumarin, 7-amino-4-chloro-3-methoxyisocoumarin, and, 4-amino-benzopyran-2-one, rhodamine 123, dihydrorhodamine 123, 6-aminotetramethylrhodamine, 5-aminotetramethylrhodamine, (E) -2- (2- (6-amino-2, 3-dihydro-1H-xanthine-4-yl) vinyl) -1,3, 3-trimethyl-3H-indol-1-ium, (E) -2- (2- (6-amino-6, 3-dihydro-1H-thioxanthen-4-yl) vinyl) -1,1, 3-trimethyl-1H-benzo [ E ] indol-3-ium, (E) -2- (2- (4-aminostyryl) -4H-chromium-4-methylenemethylene Yl) malononitrile, indocyanine green-amino, neoindocyanine green-amino, aminomethylene blue, 5-aminolevulinic acid, 5-FITC cadaverine;

the drug moiety is derived from a drug selected from the group consisting of venenum Bufonis, paclitaxel, vinorelbine, docetaxel, hydroxycamptothecin, vinblastine, vincristine, gemcitabine, cytarabine, epirubicin, pirarubicin, idarubicin, mitomycin, mitoxantrone, dacarbazine, 6-mercaptopurine, hydroxyurea, melphalan, daunorubicin, amsacrine, mitoxantrone, etoposide, teniposide, cephalotaxine, licarparide, nilapali, and taraxazolpalit.

6. The two-enzyme system probe according to claim 5, wherein QD is R¹、R²、R³Or R⁴The group shown:

7. the two-enzyme system probe according to claim 1, wherein the nuclide-chelating group is a group formed by a bifunctional chelating agent selected from DOTA, NOTA, NODA, nodaa, DOTP, TETA, ATSM, PTSM, EDTA, EC, HBEDCC, DTPA, SBAD, BAPEN, Df, DFO, TACN, NO2A/NOTAM, CB-DO2A, Cyclen, NOTA-AA, DO3A, DO3AP, HYNIC, MAS3, MAG3, or isonitrile.

8. The two-enzyme system Probe according to claim 1, wherein the two-enzyme system Probe is Probe B, Probe C, Probe D, Probe E, Probe G, or a nuclide Probe formed by labeling a radionuclide with any of them:

9. the dual enzyme system probe according to claim 1 or 8, wherein the radionuclide is a diagnostic radionuclide or a therapeutic radionuclide; the diagnostic radionuclide is preferably⁶⁸Ga、⁶⁴Cu、¹⁸F、⁸⁶Y、⁹⁰Y、⁸⁹Zr、¹¹¹In、^99mTc、¹¹C、¹²³I、¹²⁵I and¹²⁴at least one of I; the therapeutic radionuclide is preferably¹⁷⁷Lu、¹²⁵I、¹³¹I、²¹¹At、¹¹¹In、¹⁵³Sm、¹⁸⁶Re、¹⁸⁸Re、⁶⁷Cu、²¹²Pb、²²⁵Ac、²¹³Bi、²¹²Bi and²¹²at least one of Pb.

10. Use of the two-enzyme system probe according to any one of claims 1 to 9 for the preparation of an imaging agent or a therapeutic agent.

Technical Field

The invention belongs to the field of nuclear medicine, and particularly relates to a double-enzyme system probe and application thereof.

Background

Prostate cancer ranks second as a malignancy of male lethality, and dominates the incidence of cancer in western developed countries. With the continuous development of economic society of China, the aging problem of population is prominent, the process of urbanization and industrialization is accelerated, unhealthy life style and chronic infection factors are accumulated gradually, and the morbidity and the mortality of the prostatic cancer are increased every year. The focus of the prostate cancer can be treated by adopting radiotherapy and operation, and endocrine treatment of the prostate cancer at the middle and late stages can also obtain very good curative effect, but Castration Resistant Prostate Cancer (CRPC) is not sensitive to endocrine treatment and radiotherapy, and the prognosis is very poor; nuclide-targeted therapy has shown up to 80% effective control in targeted therapy against late castration-resistant prostate cancer, but slow treatment cycles can lead to tumor cell mutation and thus resistance. Therefore, how to solve the problem of radiotherapy resistance of castration resistant prostate cancer patients, improve the radiobiological effect and increase the killing capacity to tumor cells is the key for solving the clinical requirements of the patients.

Prostate Specific Membrane Antigen (PSMA) is a II transmembrane glycoprotein that transports its bound inhibitor from the cell membrane into the cell, while PSMA is only highly expressed in the neovasculature of prostate cancer as well as many cancers, with only a small amount expressed in normal tissues. This makes PSMA an ideal biomarker for nuclear imaging and targeted therapy of prostate cancer, and in recent years, small molecule agents based on PSMA targeting have made great progress in the field of prostate cancer diagnosis and treatment.

Caspase-3 is cysteine protease, exists in cytoplasm, the expression quantity of the cysteine protease is closely related to apoptosis, radiotherapy-mediated apoptosis can cause the expression quantity of Caspase-3 to be increased, and the current targeted radiation-induced tumor apoptosis drug delivery strategy based on the Caspase-3 achieves good results. Therefore, the double-enzyme system nuclear medicine probe based on the prostate specific membrane antigen PSMA targeting and Caspase-3 recognition functional molecule delivery (medicine and fluorescent dye) can achieve the purpose of radiotherapy and targeted nuclide treatment synergy, is helpful for solving the problems of radiotherapy resistance and targeted nuclide treatment resistance of castration-resistant prostate cancer patients and the like, and is expected to realize navigation in prostate cancer operation.

For example, the development of a prostate specific membrane antigen PSMA-targeted and Caspase-3 recognized nuclear imaging/therapeutic agent with good target affinity and in vivo metabolic capacity, in particular⁶⁸Ga、¹⁸F、^99mTc and¹⁷⁷lu and other nuclide labeled reagents provide more efficient tools for detection and treatment of tumor treatment, and have wide application prospects.

Disclosure of Invention

The invention aims to provide a two-enzyme system probe for targeting prostate specific membrane antigen PSMA and delivering Caspase-3 recognition functional molecules (drugs or optical dyes).

Specifically, the invention provides a double-enzyme system probe, which has a structure shown in a formula I,

wherein the content of the first and second substances,

QD is a fluorophore or drug group;

l is linker and has a structure shown in formula II:

R₁is C₅-C₈Cycloalkylene or arylene of (a); r₂Is substituted or unsubstituted aryl; n is an integer of 1 to 6;

t is a PSMA specific targeting group;

z is H, a nuclide chelating group, a radionuclide or a nuclide chelating group chelated with a radionuclide.

According to the invention, preferably, in the structure of formula II, R₁Is phenylene or cyclohexylene; a is 1, 2,3 or 4; r₂Is substituted or unsubstituted phenyl or naphthyl, the substituted group is halogen, hydroxyl, amino, carboxyl or C₁-C₄An alkyl group; the substitution is preferably monosubstituted.

Further preferably, R₂Is para-substituted phenyl or naphthyl, preferably para-halogen-substituted phenyl or naphthyl; the halogen is preferably chlorine, bromine, iodine.

According to the present invention, the PSMA-specific targeting group may be any group known in the art to specifically target PSMA, preferably the PSMA-specific targeting group is represented by formula III or formula IV:

in the present invention, DEVD is a known Caspase-3 recognition site, and the QD group does not affect the recognition of DEVD by Caspase-3, so that QD can be any conventional fluorophore or drug group, especially a small molecule drug group, from the viewpoint of the delivery by recognition of Caspase-3.

In the present invention, the meaning of "fluorescent group" or "drug group" is well known to those skilled in the art and refers to a group derived from a fluorescent compound or drug, usually obtained by removing a portion of the original structure by reaction with the fluorescent compound or drug.

Specifically, the fluorescent compound is selected from 7- (ethylamino) -4, 6-dimethylcoumarin, 7-amino-4-trifluoromethylcoumarin, 7-amino-4-methylcoumarin, coumarin 307, 7-aminocoumarin, 3-aminocoumarin, 6-aminocoumarin, 7-amino-4-chloromethylcoumarin, 3-amino-7-hydroxycoumarin, 7-amino-4-carboxymethylcoumarin, 7-amino-4-methyl-3-coumarin acetic acid, 7-amino-4-methoxymethylcoumarin, 7-amino-4-chloro-3-methoxyisocoumarin, 4-amino-benzopyran-2-one, and a pharmaceutically acceptable salt thereof, Rhodamine 123, dihydrorhodamine 123, 6-aminotetramethylrhodamine, 5-aminotetramethylrhodamine, (E) -2- (2- (6-amino-2, 3-dihydro-1H-xanthine-4-yl) vinyl) -1,3, 3-trimethyl-3H-indol-1-ium, (E) -2- (2- (6-amino-6, 3-dihydro-1H-thioxanthen-4-yl) vinyl) -1,1, 3-trimethyl-1H-benzo [ E ] indol-3-ium, (E) -2- (2- (4-aminostyryl) -4H-chromium-4-methylene) propanedinitrile, Indocyanine green-amino, neoindocyanine green-amino, aminomethylene blue, 5-aminolevulinic acid, 5-FITC cadaverine toxin. The drug moiety is derived from a drug selected from the group consisting of venenum Bufonis, paclitaxel, vinorelbine, docetaxel, hydroxycamptothecin, vinblastine, vincristine, gemcitabine, cytarabine, epirubicin, pirarubicin, idarubicin, mitomycin, mitoxantrone, dacarbazine, 6-mercaptopurine, hydroxyurea, melphalan, daunorubicin, amsacrine, mitoxantrone, etoposide, teniposide, cephalotaxine, licarparide, nilapali, and taraxazolpalit.

In one embodiment, QD is R¹、R²、R³Or R⁴The group shown:

in the present invention, the position of the Z substituent may or may not be linked to a nuclide. The linked nuclide may be a diagnostic nuclide or a therapeutic nuclide. When the double-enzyme system probe is connected with a nuclide for treatment, the double-enzyme system probe is an endogenous probe, and when the double-enzyme system probe is not connected with the nuclide or is connected with a nuclide for diagnosis, the double-enzyme system probe is an exogenous probe and can be matched with radiotherapy for use.

When a nuclide is attached, it may be directly attached, or it may be attached through a nuclide chelating group, said nuclide chelating group being a group formed by a bifunctional chelating agent selected from DOTA, NOTA, NODA, NODAGA, DOTP, TETA, ATSM, PTSM, EDTA, EC, HBEDCC, DTPA, SBAD, BAPEN, Df, DFO, TACN, NO2A/NOTAM, CB-DO2A, Cyclen, NOTA-AA, DO3A, DO3AP, HYNIC, MAS3, MAG3, or isonitrile.

The structures of the above bifunctional chelating agents are well known to those skilled in the art, for example, the DOTA and NOTA structures are shown below, respectively:

according to a preferred embodiment of the present invention, the two-enzyme system Probe is Probe B, Probe C, Probe D, Probe E, Probe G, or a nuclide Probe formed by labeling a radionuclide with any of these probes:

according to the invention, the radionuclide may be a diagnostic radionuclide or a therapeutic radionuclide; the diagnostic radionuclide is preferably⁶⁸Ga、⁶⁴Cu、¹⁸F、⁸⁶Y、⁹⁰Y、⁸⁹Zr、¹¹¹In、^99mTc、¹¹C、¹²³I、¹²⁵I and¹²⁴at least one of I; the therapeutic radionuclide is preferably¹⁷⁷Lu、¹²⁵I、¹³¹I、²¹¹At、¹¹¹In、¹⁵³Sm、¹⁸⁶Re、¹⁸⁸Re、⁶⁷Cu、²¹²Pb、²²⁵Ac、²¹³Bi、²¹²Bi and²¹²at least one of Pb. One skilled in the art can select the appropriate species according to particular needs.

The probe with the double enzyme system can be used for preparing an imaging reagent or a therapeutic agent.

The double-enzyme nuclear medicine probe for delivering the medicine molecules based on the prostate specific membrane antigen PSMA targeting and Caspase-3 recognition strategies can target prostate cancer to carry out radiotherapy sensitization treatment, is beneficial to solving the problems of radiotherapy resistance and targeted nuclide treatment resistance of castration-resistant prostate cancer patients and the like, and is expected to realize navigation in prostate cancer operation. Provides more efficient tools for the detection and treatment of tumor treatment, and has wide application prospect.

Interpretation of terms

Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the presently described subject matter belongs.

As used herein, the term "substituted", whether preceded by the term "optionally", and substituents, refers to the ability to change one functional group on a molecule to another, provided that the valency of all atoms is maintained. When more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituents may be the same or different at each position. The substituents may also be further substituted. As used herein, C₁-C₄Alkyl includes, but is not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutylAnd (4) a base. The term "alkylene" by itself or as part of another substituent refers to a straight or branched chain divalent aliphatic hydrocarbon group derived from an alkyl group; "arylene" by itself or as part of another substituent refers to a straight or branched chain divalent aromatic hydrocarbon radical derived from an aromatic radical. Unless otherwise stated, the term "aryl" means an aromatic substituent which may be a single ring or a fused polycyclic ring.

Additional features and advantages of the invention will be set forth in the detailed description which follows.

Drawings

The above and other objects, features and advantages of the present invention will become more apparent by describing in more detail exemplary embodiments thereof with reference to the attached drawings.

FIG. 1 shows the design concept of the two-enzyme system probe of the present invention, and the groups in the boxes are only examples.

FIG. 2 shows the structure of Probe A-D.

FIG. 3 shows a general scheme for the preparation of probes A-D.

FIGS. 4 to 8 are mass spectrograms of Compound 2, Probe A, Probe B, Probe C and Probe D, respectively.

FIGS. 9A and 9B show the results of co-incubation of Probe A with caspase-3 protein. A photograph under fluorescent illumination after 100. mu.M of Probe A incubated with 0U,0.25U,0.5U,1U,2U and 4U of human Caspase-3 is shown in FIG. 9A; FIG. 9B is a graph showing the change in fluorescence intensity of 100. mu.M Probe A incubated with caspase-3 protein.

FIG. 10 is a drawing showing⁶⁸Tumor-bearing mouse PET images of Ga-labeled Probe A-D.

FIG. 11 shows the structural formulae of Probe E and Probe F.

FIG. 12 shows the general routes for preparation of Probe E and Probe F.

Fig. 13 is a mass spectrum of compound 7.

FIGS. 14, 15 and 16 are each for Compound 8¹H NMR chart,¹C NMR chart and mass spectrum chart.

FIGS. 17-18 show the mass spectra of Probe E and Probe F, respectively.

FIGS. 19A and 19B show the results of protein incubation of Caspase-3 with Probe E and Probe F. FIG. 19A shows photographs of 100. mu.M Probe E (DEVD) and 100. mu.M Probe F (D-DEVD) incubated with 0U,0.25U,0.5U,1U,2U and 4U of human Caspase-3 under fluorescent irradiation; FIG. 19B is a graph showing the change in fluorescence intensity of 100. mu.M Probe E and 100. mu.M Probe F protein incubation.

FIG. 20 shows the results of Probe E incubation with 22RV1(PSMA high expression) cells and PC-3(PSMA low expression) cells, respectively. Panel a, among others, corresponds to 22RV1 cells: nuclear staining (control group); 100 μ M Probe E was incubated with 22RV1 cells and the nuclei were stained (Probe E group); after 48h of 8GyX irradiation, the cells were incubated with 100. mu.M Probe E for 12h and stained for nuclei (Probe E + RT panel). Panel B corresponds to PC-3 cells: nuclear staining (control group); 100 μ M Probe E was incubated with PC-3 cells and the nuclei were stained (Probe E group); after 48h of 8GyX irradiation, the cells were incubated with 100. mu.M Probe E for 12h and stained for nuclei (Probe E + RT panel).

FIG. 21 shows⁶⁸Tumor-bearing mouse PET image of Ga-labeled Probe E.

FIG. 22 shows fluorescence images of tumor-bearing mice of Probe E in vivo. Wherein, the graph A shows a tumor-bearing mouse of a xenograft tumor model of 22RV1 cell line, 200nmol of Probe E is injected into tail vein, and the tumor-bearing mouse is subjected to fluorescence imaging (Before RT) in 1h, 4h, 12h, 24h and 48h respectively; radiotherapy of 10Gy is carried out on the same batch of mice, 200nmol of Probe E is injected After 48 hours, and fluorescence imaging (After RT) is carried out on the mice respectively for 1 hour, 4 hours, 12 hours, 24 hours and 48 hours; panel B shows the change of the fluorescence intensity of the tumor tissue before and after radiotherapy for 1h, 4h, 12h, 24h and 48 h.

FIG. 23 shows the SPECT/CT visualization of the 22RV1 tumor-bearing mouse in vivo of Probe E. A: tail vein injection of 500uCi¹⁷⁷Lu-labeled Probe E, SPECT imaging at 1h, 4h, 8h, 12h and 24h after injection; b: tail vein co-injection of 500uCi¹⁷⁷Lu-labeled Probe E and nuclide-unlabeled Probe E200 nmol; c: tumor pairs with different injection modes and different time points¹⁷⁷Uptake of Lu Probe E; d: liver pairs with two injection modes and different time points¹⁷⁷Uptake of Lu Probe E.

FIG. 24 shows¹⁷⁷Lu Probe E22 RV1 tumor-bearing miceIn vivo fluorescence imaging. A: tail vein injection of 500uCi¹⁷⁷Lu-labeled Probe E24 h fluorescence live body imaging; b: tail vein injection of 500uCi¹⁷⁷After Lu labeled Probe E24 h, injecting 200nmol Probe E24 h for in vivo fluorescence imaging; c: tail vein co-injection of 500uCi¹⁷⁷Lu labeled Probe E and nuclide unlabeled Probe E200nmol 24h fluorescence live body imaging; d: tail vein co-injection of 500uCi¹⁷⁷Lu labeled Probe E and non-nuclide labeled Probe E were visualized in 200nmol 48h fluorescent living bodies.

FIG. 25 shows the structural formulae of Probe E and Probe F.

FIG. 26 shows a general route for preparation of Probe E and Probe F.

Fig. 27 and 28 are mass spectra of compound 13 and compound 14, respectively.

FIGS. 29, 30 and 31 are each for Compound 16¹H NMR chart,¹C NMR chart and mass spectrum chart.

FIG. 32 is a mass spectrum of compound Probe G.

Fig. 33 shows 22RV1 cell viability. A: 22RV1 cell survival rate for Probe G (Venenu bufonis-linker-PSMA), Compound 14(Venenu bufonis-linker) and Bufonis Venenum (Venenu bufonis); b: x-ray irradiation to 22RV1 cell viability at 10Gy, Probe G (Venenu bufonis-linker-PSMA), Compound 14(Venenu bufonis-linker) and Bufonis Venenum (Venenu bufonis).

FIG. 34 shows the results of in vivo tumor suppression experiments in Probe G22 RV1 tumor-bearing mice. A: a tumor growth curve; b: mouse body weight change curve. 200nmol Probe G (Drugs group) was administered by tail vein injection; radiotherapy dose 10Gy (RT group); radiotherapy 10Gy, 200nmol Probe G injected in the tail vein after 48h (RT + Drugs group).

Detailed Description

Preferred embodiments of the present invention will be described in more detail below. While the following describes preferred embodiments of the present invention, it should be understood that the present invention may be embodied in various forms and should not be limited by the embodiments set forth herein.

Preparation of probes Probe A, Probe B, Probe C and Probe D

The specific structure of Probe A-D is shown in FIG. 2. The synthesis procedure is shown in FIG. 3. The coupling of the amino acids was performed according to standard Fmoc solid phase synthesis.

FIG. 3 shows a general scheme for the preparation of probes A-D. Reaction conditions are as follows: (a) DCM solution of Fmoc-L-aspartic acid 4-tert-butyl ester, HATU and DIPEA; (b) TFA in DCM; (c) 2-Chlorotriphenylchloride resin and DIPEA in DMF/DCM; (d) 20% piperidine in DMF, Fmoc-L-valine, HBTU, HOBt and DIPEA in DMF; (e) 20% piperidine in DMF, N-Fmoc-L-glutamic acid-5-tert-butyl ester, HBTU, HOBt and DIPEA in DMF; (f) 20% piperidine in DMF, Fmoc-L-aspartic acid 4-tert-butyl ester, HBTU, HOBt and EIPEA in DMF; (g) 20% piperidine in DMF, Fmoc-L-phenylalanine, HBTU, HOBt and EIPEA in DMF; (h) 20% piperidine in DMF, N-Fmoc-N' - [1- (4, 4-dimethyl-2, 6-dioxocyclohexylidene) ethyl ] -D-lysine, HBTU, HOBt and EIPEA in DMF; (i) 20% piperidine in DMF, 1,4,7, 10-tetraazacyclododecane-1, 4,7, 10-tetraacetic acid tri-tert-butyl ester, HBTU, HOBt and EIPEA in DMF; (j) 2% hydrazine hydrate in DMF, Fmoc-a-OH, HBTU, HOBt and EIPEA in DMF; (k) 20% piperidine in DMF, Fmoc-b-OH, HBTU, HOBt and EIPEA in DMF; (l) 20% piperidine in DMF, and (S) -5- (tert-butoxy) -4- (3- ((S) -1,5-di-tert-butoxy-1, 5-diopentotan-2-yl) ureido) -5-oxopentanoic acid, HBTU, HOBt and EIPEA in DMF; (h) trifluoroacetic acid, water and triisopropylsilane. Wherein, the steps (j) and (k) are selectively present according to different Probe A-D structures.

Synthesis of Compound 2: in a 50mL round-bottomed flask, 7-amino-4-methylcoumarin (1, 200.00mg, 1.14mmol), HATU (560.88mg, 1.37mmol) and Fmoc-L-aspartic acid 4-tert-butyl ester (560.88mg, 1.37mmol) were taken, dissolved by addition of 10mL DCM, followed by addition of DIPEA (394.12mg,2.28mmol), stirring magnetically at room temperature, and reaction was stopped after 4 hours. The dichloromethane was removed under reduced pressure, the crude product was taken for the next reaction without further treatment, 10mL DCM was added and dissolved, 5mL trifluoroacetic acid was added and stirred at room temperature for 1h, dichloromethane was removed under reduced pressure, and the product was purified by silica gel column to give white solid 2.

Synthesis of resin 3: 2-Chlorotriphenyl chloride resin (1.00g) was placed in a solid phase synthesis tube and swollen with 2mL of Dichloromethane (DCM) and repeated three times for 5 minutes each, followed by three washes with N, N-Dimethylformamide (DMF) for 5 minutes each. Dissolving compound 2(256.26mg, 0.50mmol) in a mixed solvent of DCM and DMF, adding DIPEA (130mg, 1.00mmol), adding the above mixture into a solid phase synthesis tube, electromagnetically stirring at room temperature, and stopping after reacting for 2 hours; 2mL of Dichloromethane (DCM) wash was repeated three times for 5 minutes each; the resin was blocked with 7mL of mixed solvent (DCM: MeOH: DIPEA ═ 10mL:1mL) three times for 5 minutes each; washing with 2mL of Dichloromethane (DCM) was repeated three times for 5 minutes each and the solvent was removed under reduced pressure to give yellow resin 3.

Preparation of Probe A-D: a mass of resin 3(0.05mmol) was taken in a 10mL solid phase synthesis tube, swollen with 2mL Dichloromethane (DCM) and repeated three times for 5 minutes each, followed by three washes with N, N-Dimethylformamide (DMF) for 5 minutes each. The amino protecting group Fmoc was removed using 20% piperidine in DMF (v/v) in 2mL 20% piperidine in DMF followed by 2 min 2-5 washes with 2mL DMF for 2 min each, 10 min. 3 times the chemical amount of Fmoc amino acid to resin (0.02mmol) was activated with 3.6 times the chemical amount of HBTU in the presence of 7.2 times the chemical amount of DIPEA, and then added to a synthesis tube, followed by reaction for 1 hour with electromagnetic stirring. The removal of the protecting group Dde was repeated twice using a 2% hydrazine in DMF (v/v) solution for 3 minutes each, followed by 3-5 washes with DMF for 2 minutes each. The cleavage of the ligand from the resin and the removal of the tert-butyl ester was done with 5mL trifluoroacetic acid/triisopropylsilane/water (95:2.5:2.5, v/v/v) with stirring for 2h and the resin was washed with 2mL trifluoroacetic acid, all filtrates were collected, after removal of the trifluoroacetic acid under reduced pressure, the crude product was prepared back by HPLC and lyophilized to give the target ligand Probe A-D. Ligand structure was identified by high resolution mass spectrometry.

FIGS. 4 to 8 are mass spectrograms of Compound 2, Probe A, Probe B, Probe C and Probe D, respectively.

Caspase-3 protein incubation

In order to verify the reasonability of the molecular design of the compound, Probe A and activated human recombinant Caspase-3 protein are incubated, and whether DEVD in the molecular structure can be identified and cut off or not is verified, so that the release of a fluorescent group is promoted, and the fluorescent signal is enhanced.

Weighing a certain mass of Probe A, dissolving the Probe A in 50mM HEPES buffer solution containing 50mM NaCl, 10mM EDTA, 5% glycerol and 10mM DTT, adding human Caspase-3 recombinant proteins with different enzyme activities of 0U,0.25U,0.5U,1U,2U and 4U, and incubating for 8 hours at 37 ℃. In FIGS. 9A and 9B, it can be very intuitively found that the fluorescence signal is significantly increased with the increase of the protease activity, confirming that Caspase-3 can recognize DEVD structural fragment in the molecular structure and cleave it to promote the release of the fluorophore. Therefore, the reasonability of molecular design is verified, and the human Caspase-3 protein can recognize and cut specific groups in the molecular structure of the probe.

Marking and quality control

Marking:

⁶⁸ga: a certain mass of ligand was precisely weighed into the sample, dissolved by adding 20. mu.L of DMSO (dimethyl sulfoxide), and then diluted to 1 nmol/. mu.L by adding pure water. Pipetting 30. mu.L of ligand solution and 65. mu.L of NaOAc solution (1mol/L) into a vial, and adding 1mL of freshly eluted ligand solution⁶⁸Ga³⁺An ionic solution (hydrochloric acid solution with a solvent of 0.05mol/L and a radioactivity of 10-17mCi) was shaken up, sealed and reacted at 85 ℃ for 10 minutes. The reaction solution was cooled to room temperature and analyzed by HPLC for quality control.

Quality control:

⁶⁸the radiochemical purity of the Ga complexes was determined by HPLC (high performance liquid chromatography) with a mobile phase of 20% acetonitrile in water (with 0.1% TFA), all complexes having a radiochemical purity of more than 90% and being studied without purification.

⁶⁸Imaging of Ga-labelled products

Taking 0.1mL of the newly prepared⁶⁸Ga-labeled complex (5.6-7.4 MBq) is injected into Balb/c nude mice with male 22RV1 tumor (the tumor diameter is about 1 cm) by tail vein, 1.After 0h, the patient is anesthetized by isoflurane, small animal PET (SUPER-NOVA, Pingsheng science and technology, China) imaging is carried out, and the SUV (standard uptake value) is sketched for the region of interest.

As shown in figure 10 and in table 1,⁶⁸ga-labeled Probe A is concentrated in a tumor region, but the uptake in the tumor is only 0.14 +/-0.03, the effect is not ideal, the uptake of Probe B, Probe C and Probe D introduced with fat-soluble groups in the tumor tissue is obviously improved, wherein the uptake of Probe B and Probe C in the tumor tissue is respectively 0.43 +/-0.06 and 0.40 +/-0.09, the uptake of Probe D in the tumor region is obviously concentrated, the tumor uptake is optimal, the uptake in the tumor is 0.85 +/-0.35, and is basically equivalent to the uptake of PSMA-11, namely 0.89 +/-0.01; the muscle and kidney intake is 0.11 + -0.03 and 9.52 + -2.42, respectively, which is slightly better than that of PSMA-11, muscle and kidney intake of 0.15 + -0.07 and 10.18 + -3.46. It can be seen that Probe D is a very potent candidate⁶⁸The Ga labels PSMA targeting molecular probe.

Table 1 SUVmax values and their ratios (mean ± SD, n ═ 4) of the complexes in tumor, muscle, liver and kidney

The rationality of the molecular design of the two-enzyme system is verified from two aspects of protein incubation and living body PET imaging in the above experiment.

Preparation of probes Probe E and Probe F

The specific structures of Probe E and Probe F are shown in FIG. 11. The synthesis procedure is shown in FIG. 12. The coupling of the amino acids was performed according to standard Fmoc solid phase synthesis.

FIG. 12 shows the general routes for preparation of Probe E and Probe F. Reaction conditions are as follows: (a) acetonitrile solution of m-nitrophenol and potassium carbonate; (b) SnCl₂HCl in methanol; (c) DCM solution of Fmoc-L-aspartic acid 4-tert-butyl ester, HATU and DIPEA; (d) TFA in DCM; (e) 2-Chlorotriphenylchloride resin and DIPEA in DMF/DCM; (f) 20% piperidine in DMF, Fmoc-valine, HBTU, HOBt and DIPEA in DMF; (g)20 of the formula% piperidine in DMF, N-Fmoc-glutamic acid-5-tert-butyl ester, HBTU, HOBt and DIPEA in DMF; (h) 20% piperidine in DMF, Fmoc-aspartic acid 4-tert-butyl ester, HBTU, HOBt and EIPEA in DMF; (i) 20% piperidine in DMF, Fmoc-L-phenylalanine, HBTU, HOBt and EIPEA in DMF; (j) 20% piperidine in DMF, N-Fmoc-N' - [1- (4, 4-dimethyl-2, 6-dioxocyclohexylidene) ethyl]-DMF solutions of D-lysine, HBTU, HOBt and EIPEA; (k) 20% piperidine in DMF, 1,4,7, 10-tetraazacyclododecane-1, 4,7, 10-tetraacetic acid tri-tert-butyl ester, HBTU, HOBt and EIPEA in DMF; (l) 2% hydrazine hydrate in DMF, Fmoc-a-OH, HBTU, HOBt and EIPEA in DMF; (m) 20% piperidine in DMF, Fmoc-b-OH, HBTU, HOBt and EIPEA in DMF; (n) 20% piperidine in DMF, and (S) -5- (tert-butoxy) -4- (3- ((S) -1,5-di-tert-butoxy-1, 5-diopentotan-2-yl) ureido) -5-oxopentanoic acid, HBTU, HOBt and EIPEA in DMF; (o) trifluoroacetic acid, water and triisopropylsilane.

Synthesis of compound 7: IR-775 chloride (6, 200.00mg, 0.38mmol), m-nitrophenol (133.88mg, 0.96mmol) and potassium carbonate (132.81mg, 0.96mmol) were taken in a 50mL round-bottomed flask, dissolved by adding 10mL acetonitrile, stirred magnetically at room temperature, and the reaction was stopped after 4 hours. Removing acetonitrile under reduced pressure, dissolving with dichloromethane, washing with saturated saline solution, distilling under reduced pressure with organic phase, and directly carrying out the next reaction without treatment on the crude product; anhydrous SnCl₂(1.44g,7.60mmol) and concentrated hydrochloric acid (4mL) were added to a 50mL round bottom flask, the crude product was dissolved using 20mL methanol, added to the round bottom flask, refluxed at 80 ℃ for 12h, cooled to room temperature, the reaction diluted with 50mL DCM, filtered with suction and extracted with dichloromethane. The product was purified by silica gel column to give green solid 7.

Synthesis of compound 8: compound 7(437.20mg, 1.14mmol), HATU (560.88mg, 1.37mmol) and Fmoc-L-aspartic acid 4-tert-butyl ester (560.88mg, 1.37mmol) were taken in a 50mL round-bottomed flask, dissolved by addition of 10mL DCM, DIPEA (394.12mg,2.28mmol) was added, stirred magnetically at room temperature, and the reaction was stopped after 4 hours. The dichloromethane was removed under reduced pressure, the crude product was taken on to the next reaction without further treatment, 10mL of DCM was added to dissolve it, 5mL of trifluoroacetic acid was added, stirring was carried out at room temperature for 1h, dichloromethane was removed under reduced pressure, and the product was purified by silica gel column to give blue solid 8.

Synthesis of resin 9: 2-Chlorotriphenyl chloride resin (1.00g) was placed in a solid phase synthesis tube and swollen with 2mL of Dichloromethane (DCM) and repeated three times for 5 minutes each, followed by three washes with N, N-Dimethylformamide (DMF) for 5 minutes each. Dissolving compound 8(360.42mg, 0.50mmol) in a mixed solvent of DCM and DMF, adding DIPEA (130mg, 1.00mmol), adding the above mixture into a solid phase synthesis tube, electromagnetically stirring at room temperature, and stopping after reacting for 2 hours; 2mL of Dichloromethane (DCM) wash was repeated three times for 5 minutes each; the resin was blocked with 7mL of mixed solvent (DCM: MeOH: DIPEA ═ 10mL:1mL) three times for 5 minutes each; washing with 2mL of Dichloromethane (DCM) was repeated three times for 5 minutes each and the solvent was removed under reduced pressure to give yellow resin 9.

Synthesis of Probe E-F: a mass of resin 9(0.05mmol) was taken in a 10mL solid phase synthesis tube, swollen with 2mL Dichloromethane (DCM) and repeated three times for 5 minutes each, followed by three washes with N, N-Dimethylformamide (DMF) for 5 minutes each. The amino protecting group Fmoc was removed using 20% piperidine in DMF (v/v) in 2mL 20% piperidine in DMF followed by 2 min 2-5 washes with 2mL DMF for 2 min each, 10 min. 3 times the chemical amount of Fmoc amino acid to resin (0.02mmol) was activated with 3.6 times the chemical amount of HBTU in the presence of 7.2 times the chemical amount of DIPEA, and then added to a synthesis tube, followed by reaction for 1 hour with electromagnetic stirring. The removal of the protecting group Dde was repeated twice using a 2% hydrazine in DMF (v/v) solution for 3 minutes each, followed by 3-5 washes with DMF for 2 minutes each. The dissociation of the ligand from the resin and the removal of the tert-butyl ester was done with 5mL trifluoroacetic acid/triisopropylsilane/water (95:2.5:2.5, v/v/v) stirred for 2 hours and the resin was washed with 2mL trifluoroacetic acid, all filtrates were collected, after removal of the trifluoroacetic acid under reduced pressure, the crude product was prepared back by HPLC and lyophilized to give the target ligands Probe E, F. Ligand structure was identified by high resolution mass spectrometry.

Fig. 13 is a mass spectrum of compound 7. FIGS. 14, 15 and 16 are each for Compound 8¹H NMR chart,¹C NMR diagram and mass spectrum. FIGS. 17-18 show the mass spectra of Probe E and Probe F, respectively.

Caspase-3 protein incubation

In order to verify the specificity of the series of compounds to Caspase-3, Probe E (DEVD) is designed, and amino acids in a DEVD sequence of the Probe E are all in an L configuration and can be normally recognized and cut by Caspase-3; and Probe F (D-DEVD), wherein only one aspartic acid in the DEVD sequence is in an L configuration, and the other three amino acids in the DEVD sequence are in a D configuration and cannot be normally recognized by Caspase-3.

Weighing certain mass of Probe E and Probe F, dissolving in 50mM HEPES buffer solution containing 50mM NaCl, 10mM EDTA, 5% glycerol and 10mM DTT, adding human Caspase-3 recombinant proteins with different enzyme activities of 0U,0.25U,0.5U,1U,2U and 4U, and incubating at 37 ℃ for 8 h. As shown in FIGS. 19A and 19B, it was found that in the Probe E (DEVD) test group, a significant increase in fluorescence signal occurred with an increase in protease activity; in the Probe F (D-DEVD) control group, the fluorescence intensity did not change as the protease activity increased. The enhancement of the fluorescence signal in the Probe E experimental group is proved to be caused by the cleavage of DEVD structural fragments in the molecular structure by Caspase-3, and the specific recognition and cleavage of the series of compounds by Caspase-3 are further proved.

Cell radiotherapy

Carrying out X-ray radiation irradiation on 22RV1 cells with high expression of prostate specific membrane antigen PSMA and PC-3 cells with low expression, wherein the dose is 8Gy, continuously culturing the cells for 48h, adding 100 mu M Probe E, incubating for 12h, and detecting under a laser confocal microscope. As shown in FIG. 20, in the 710nm fluorescence signal channel of 22RV1 cells, there was a significant increase in the fluorescence signal in the radiotherapy experimental group (Probe E + RT group in A of FIG. 20) compared to the Control group (Control group in A of FIG. 20 and Probe E group in A of FIG. 20). It was confirmed that Probe E was transported to cytoplasm via PSMA and DEVD structure in the molecule was cleaved by Caspase-3 up-regulated by X-ray induction, releasing fluorophore and increasing fluorescence signal. In addition, a small amount of fluorescent signal was found in 22RV1 cells incubated with only Probe E and not irradiated with X-rays, due to the self-background signal of Probe E transported by PSMA but not cleaved by Caspase-3. In PC-3 cells, due to low expression of PSMA on the cell surface, the Probe could not be transported into the cytoplasm, and thus no fluorescence signal and no up-regulation of fluorescence signal could be detected in the cytoplasm, which is reflected in that no fluorescence signal was observed in the PC-3 cells Probe E incubation radiotherapy experimental group (Probe E + RT group in B of FIG. 20) and Probe E incubation group (Probe E group in B of FIG. 20). Further proves that the compound Probe E can be transported by the prostate specific membrane antigen PSMA in living cells, and is specifically identified and cut by the apoptosis-related protease Caspase-3 with the expression quantity up-regulated by X-ray mediation, thereby achieving the purpose of fluorescent imaging in the living cells. Thereby realizing the delivery of specific functional molecules under a dual-enzyme system in living cells.

⁶⁸Imaging of Ga-labeled Probe E products

As shown in figure 21 and in table 2,⁶⁸the Ga complex Probe E can be obviously concentrated in a tumor area, and the intake in the tumor is 0.56 +/-0.10; the uptake in muscle and kidney is 0.28 +/-0.05 and 4.25 +/-1.06 respectively, and Probe E is slightly weaker than Probe D in the uptake of tumor after 1h of tail intravenous injection, but can still contain partial compound in blood pool and still can increase the continuous uptake of tumor; it is also noted that the kidney intake of Probe E is 4.25 + -1.06 better than the kidney intake of Probe D of 9.52 + -2.42. Thus, Probe E is expected to develop into a substance with low renal metabolism⁶⁸The Ga labels PSMA targeting molecular probe. In addition, the fluorescent group in Probe E is near infrared dye of methine cyanine analogue at 710nm, which is more favorable for fluorescent imaging and surgical navigation in living body than the fluorescent fragment AMC (emission wavelength of 450nm) in Probe D.

TABLE 2 SUVmax values and ratios of Probe E complexes in tumors, muscles, liver and kidney

Radiotherapy-mediated in vivo imaging

In order to further verify the property of Probe E in vivo, whether in vivo imaging can be realized or not, and prostate cancer operation navigation is carried out. Tumor-bearing mice of 22RV1 were injected with 200nmol of Probe E by tail vein injection and subjected to fluorescent imaging. First, near-infrared fluorescence imaging was performed on mice Before radiotherapy at different time points 1h, 4h, 12h, 24h and 48h after probe injection, and no increase in fluorescence signal was detected at the tumor site (beforee RT in a-B of fig. 22). After 10GyX radiation is subsequently carried out on the tumor part of the same batch of tumor-bearing mice, 200nmol of Probe E is injected in a tail vein injection mode After 48 hours, and fluorescence imaging is carried out on the tumor part at different time points of 1h, 4h, 12h, 24h and 48h After injection, so that obvious fluorescence signals can be found at the tumor part and are obviously enhanced compared with the fluorescence signals before radiotherapy (After RT in A-B of figure 22). The Probe E is proved to be capable of being transported by the prostate specific membrane antigen PSMA in a tumor-bearing mouse body and being specifically identified and cut by the apoptosis-related protease Caspase-3 with the expression quantity up-regulated by X-ray mediation, thereby achieving the purpose of fluorescence imaging in a living body and realizing the delivery of specific functional molecules under a double-enzyme system.

The experiments prove that the expression quantity of Caspase-3 in the tumor cells is positively correlated with the fluorescence signal intensity, and the method has higher time and space resolution. Lays a foundation for intuitively understanding the activity of Caspase-3 in the tumor cell apoptosis pathway after radiotherapy.

¹⁷⁷Visualization of Lu-labeled products

In order to further verify the property of Probe E as a nuclear medicine therapeutic agent targeting PSMA in vivo, the SPECT imaging of Probe E is carried out on a tumor-bearing mouse of 22RV1 by a tail vein injection mode. As shown in figure 23 of the drawings,¹⁷⁷the Lu Probe E was significantly concentrated in the tumor region 1h after injection, and there was no decrease in signal intensity at the time points 4h, 8h, 12h and 24h (A in FIG. 23), further confirming that Probe E can remain in the animal for a longer period of time and can be used as a nuclear medicine targeting PSMAThe use of a chemotherapeutic probe.

In B of FIG. 23, the¹⁷⁷The co-injection of Lu-labeled Probe E and non-nuclide-labeled Probe E to compensate for the insufficient chemical dosage of Probe E has been found¹⁷⁷There was significant concentration in the tumor area 1h after Lu Probe E injection, and there was no significant decrease in signal intensity at the 4h, 8h, 12h and 24h time points. Tumor pairs under two injection modes simultaneously¹⁷⁷There was no significant difference in the uptake of Lu Probe E; the tumor and the liver do not show the improvement of the dosage of the Probe E¹⁷⁷Inhibition of Lu Probe E intake (C of fig. 23 and D of fig. 23). Further illustrates that in vivo imaging of Probe E can be performed by co-injection, which helps to increase patient compliance.

Nuclide-targeted mediated in vivo imaging

For further confirmation¹⁷⁷Whether the Lu Probe E can up-regulate the expression quantity of the tumor cell caspase-3 or not so as to realize the delivery of functional molecules. Go on to¹⁷⁷Lu-labeled Probe E was subjected to in vivo fluorescence imaging in tumor-bearing mice. Will be provided with¹⁷⁷Lu Probe E was injected into tumor-bearing mice, and¹⁷⁷lu Probe E was co-injected with 200nmol Probe E into tumor-bearing mice and subjected to fluorography after 24h (FIGS. 24A and B), and it can be seen that due to the chemical dose of Probe E, it was injected separately¹⁷⁷Lu Probe E was in tumor tissue and did not have a fluorescent signal (A in FIG. 24); whereas in the coinjected group there was a significant increase in fluorescence signal (B in fig. 24). Then only to proceed¹⁷⁷Lu Probe E injection group, and then injected with 200nmol of Probe E. After 24h, in vivo fluorescence imaging was performed on both groups of mice, and it was found that there was a significant increase in the fluorescence signal in both tumor tissues (C and D in FIG. 24). Description of the invention¹⁷⁷The Lu marked Probe E as a nuclear medicine treatment reagent can induce and up-regulate the expression quantity of Caspase-3 of tumor cells, further realize the delivery of functional molecules, realize the fluorescence imaging of living bodies, detect the expression quantity of Caspase-3 in the apoptosis process of the tumor cells and realize the operation navigation of prostate cancer. In summary,¹⁷⁷lu-labeled Probe E can be used asIs used as a nuclear medicine treatment probe targeting PSMA.

Preparation of probes Probe G and Probe H

The specific structure of Probe G-H is shown in FIG. 25. The synthesis procedure is shown in FIG. 26. The coupling of the amino acids was performed according to standard Fmoc solid phase synthesis.

FIG. 26 shows a general route for preparation of Probe G-H. Reaction conditions are as follows: (a) DCM solution of p-nitrophenyl chloroformate and triethylamine; (b) a solution of propylene diamine in DCM; (c) DCM solutions of fluorenylmethyloxycarbonyl-aspartic acid- β benzyl ester, HATU and DIPEA; (d) h₂Pd/C, methanol solution (e) 2-chlorotriphenylchloride resin and DIPEA in DMF/DCM; (f) 20% piperidine in DMF, Fmoc-valine, HBTU, HOBt and DIPEA in DMF; (g) 20% piperidine in DMF, N-Fmoc-glutamic acid-5-tert-butyl ester, HBTU, HOBt and DIPEA in DMF; (h) 20% piperidine in DMF, Fmoc-aspartic acid 4-tert-butyl ester, HBTU, HOBt and EIPEA in DMF; (i) 20% piperidine in DMF, Fmoc-L-phenylalanine, HBTU, HOBt and EIPEA in DMF; (j) 20% piperidine in DMF, N-Fmoc-N' - [1- (4, 4-dimethyl-2, 6-dioxocyclohexylidene) ethyl]-DMF solutions of D-lysine, HBTU, HOBt and EIPEA; (k) 20% piperidine in DMF, 1,4,7, 10-tetraazacyclododecane-1, 4,7, 10-tetraacetic acid tri-tert-butyl ester, HBTU, HOBt and EIPEA in DMF; (l) 2% hydrazine hydrate in DMF, Fmoc-a-OH, HBTU, HOBt and EIPEA in DMF; (m) 20% piperidine in DMF, Fmoc-b-OH, HBTU, HOBt and EIPEA in DMF; (n) 20% piperidine in DMF, and (S) -5- (tert-butoxy) -4- (3- ((S) -1,5-di-tert-butoxy-1, 5-diopentotan-2-yl) ureido) -5-oxopentanoic acid, HBTU, HOBt and EIPEA in DMF; (o) trifluoroacetic acid, water and triisopropylsilane; (p) Compound 13, a DMSO solution of triethylamine.

Synthesis of compound 13: bufonis venenum (12, 146.88mg, 0.38mmol), p-nitrophenyl chloroformate (193.50mg, 0.96mmol) and triethylamine (97.14mg, 0.96mmol) were taken in a 50mL round-bottomed flask, dissolved by addition of 10mL DCM, stirred magnetically at room temperature, and the reaction was stopped after 4 hours. DCM was removed under reduced pressure. The product was purified by silica gel column to give white solid 13.

Synthesis of compound 14: compound 13(209.62mg, 0.38mmol) and propylenediamine (56.33mg, 0.96mmol) were placed in a 50mL round-bottomed flask, and 10mL of DCM was added and dissolved, followed by magnetic stirring at room temperature, and the reaction was stopped after 4 hours. DCM was removed under reduced pressure. The product was purified on a silica gel column to give a white solid 14.

Synthesis of compound 16: compound 15(198.63mg, 1.14mmol), HATU (560.88mg, 1.37mmol) and fluorenylmethoxycarbonyl-aspartic acid-. beta.benzyl ester (610.29mg, 1.37mmol) were taken in a 50mL round-bottomed flask, 10mL of DCM was added and dissolved, DIPEA (394.12mg,2.28mmol) was added, and the reaction was stopped after 4 hours. Removing dichloromethane under reduced pressure, directly carrying out the next reaction on the crude product without treatment, adding 10mL of anhydrous methanol to dissolve the crude product, adding 10% by mass of Pd/C, introducing hydrogen, stirring at room temperature for 12h, removing dichloromethane under reduced pressure, and purifying the product by a silica gel column to obtain a white solid 16.

Synthesis of resin 17: 2-Chlorotriphenyl chloride resin (1.00g) was placed in a solid phase synthesis tube and swollen with 2mL of Dichloromethane (DCM) and repeated three times for 5 minutes each, followed by three washes with N, N-Dimethylformamide (DMF) for 5 minutes each. Dissolving compound 16(255.78mg, 0.50mmol) in a mixed solvent of DCM and DMF, adding DIPEA (130mg, 1.00mmol), adding the above mixture into a solid phase synthesis tube, electromagnetically stirring at room temperature, and stopping after 2 hours of reaction; 2mL of Dichloromethane (DCM) wash was repeated three times for 5 minutes each; the resin was blocked with 7mL of mixed solvent (DCM: MeOH: DIPEA ═ 10mL:1mL) three times for 5 minutes each; washing with 2mL of Dichloromethane (DCM) was repeated three times for 5 minutes each and the solvent was removed under reduced pressure to give yellow resin 17.

Synthesis of Probe G-H: a mass of resin 17(0.05mmol) was taken in a 10mL solid phase synthesis tube, swollen with 2mL Dichloromethane (DCM) and repeated three times for 5 minutes each, followed by three washes with N, N-Dimethylformamide (DMF) for 5 minutes each. The amino protecting group Fmoc was removed using 20% piperidine in DMF (v/v) in 2mL 20% piperidine in DMF followed by 2 min 2-5 washes with 2mL DMF for 2 min each, 10 min. 3 times the chemical amount of Fmoc amino acid to resin (0.02mmol) was activated with 3.6 times the chemical amount of HBTU in the presence of 7.2 times the chemical amount of DIPEA, and then added to a synthesis tube, followed by reaction for 1 hour with electromagnetic stirring. The removal of the protecting group Dde was repeated twice using a 2% hydrazine in DMF (v/v) solution for 3 minutes each, followed by 3-5 washes with DMF for 2 minutes each. The cleavage of the ligand from the resin and the removal of the tert-butyl ester was done with 5mL trifluoroacetic acid/triisopropylsilane/water (95:2.5:2.5, v/v/v) stirred for 2h and the resin was washed with 2mL trifluoroacetic acid, all filtrates were collected, after removal of the trifluoroacetic acid under reduced pressure, the crude product was prepared back by HPLC and lyophilized to give compound 19. Compound 19(40.00mg, 0.02mmol) and compound 13(10.67mg, 0.03mmol) were dissolved in a 50mL round-bottomed flask by the addition of 3mL DMSO, DIPEA (5.2mg,0.04mmol) was added, and the reaction was stopped after 12 hours by magnetic stirring at room temperature. And carrying out reverse preparation on the crude product by HPLC, and freeze-drying to obtain target ligands Probe G and H. Ligand structure was identified by mass spectrometry.

Fig. 27 and 28 are mass spectra of compound 13 and compound 14, respectively. FIGS. 29, 30 and 31 are each for Compound 16¹H NMR chart,¹C NMR chart and mass spectrum chart. FIG. 32 is a mass spectrum of compound Probe G.

Cell survival rate

The survival assay for 22RV1 cells was first determined for Probe G, compound 14 and venenum bufonis, and the results are shown in fig. 33. In the Control group (Control, a in fig. 33), the toxicity to 22RV1 was not significant and the safety was better as the concentration of Probe G drug was increased from 0nM to 80 nM; the compound 14 and the toad venom have obvious reduction of cell survival rate along with the increase of concentration, which indicates that the toad venom and the derivatives have higher killing effect on prostate cancer cells. In the RT group (fig. 33B), compound 14 and bufonis were similar in trend to the control group, but the toxicity of the Probe G drug was gradually increased toward 22RV1 with increasing concentration. Further illustrates that compound 14 can be used as a cytotoxic drug to kill tumor cells. Probe G can be used as a nuclear medicine Probe for killing tumor cells by targeting prostate specific membrane antigen PSMA, Caspase-3 mediated compound 14.

Radiotherapy sensitization treatment experiment of Probe G

The radiotherapy sensitization experiment research is carried out on Probe G, namely the drug effect research is carried out on a xenograft tumor model of the 22RV1 cell line. Firstly, radiotherapy is carried out on a radiotherapy group (RT group) and a radiotherapy medicine group (RT + Drugs group) on the 0 th day, and the dose is 10 Gy; on days 2 and 5, 200nmol of Probe G was injected by tail vein injection into the drug group (Drugs group) and the radiotherapy drug group (RT + Drugs group). Tumor size and mouse body weight were then monitored for different groups of tumor-bearing mice. As shown in fig. 34, in the experimental group of radiotherapy Drugs (RT + Drugs group), it was found that the tumor volume was significantly smaller than that of the control drug group and the radiotherapy group, and the tumor-suppressing effect was significant (a of fig. 34). Meanwhile, the weight of the mice in the radiotherapy drug experimental group is better than that of the mice in the simple radiotherapy group (B in figure 34). Experimental results show that the Probe G serving as a bifunctional reagent for radiotherapy sensitization of the prostate cancer and sensitization of targeted nuclide therapy has an obvious curative effect and small side effect.

Having described embodiments of the present invention, the foregoing description is intended to be exemplary, not exhaustive, and not limited to the embodiments disclosed. Many modifications and variations will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the described embodiments.