COMPOUND TARGETING PROSTATE SPECIFIC MEMBRANE ANTIGEN, AND PREPARATION METHOD AND APPLICATION THEREOF

Abstract

The present disclosure provides a compound targeting prostate specific membrane antigen (PSMA), wherein the compound has the following structure shown in Formula (I); R.sub.1 is a compound structure targeting prostate specific membrane antigen; L.sub.1 is (X).sub.n(CH.sub.2).sub.m(Y).sub.q, X and Y are independently selected from lysine, glutamic acid or a derivative structure containing lysine and glutamic acid, n is an integer from 0 to 12, m is an integer from 0 to 60, q is an integer from 0 to 12, and each CH.sub.2 may be individually substituted with O, NH(CO), or (CO)NH; L.sub.2 is (CH.sub.2).sub.p, p is an integer from 0 to 30, and each CH.sub.2 may be individually substituted with O, NH(CO), or (CO)NH; and R.sub.2 is a nuclide chelating group. The present disclosure also provides a radiolabeled complex based on the structure of the compound. The compound and the radiolabeled complex have appropriate blood circulation time, high uptake in tumors and long retention time, and are suitable for nuclide therapy and imaging of tumors with high expression of PSMA.

##STR00001##

Claims

1-13. (canceled)

14. A compound targeting prostate specific membrane antigen or a pharmaceutically available salt thereof, wherein the molecular structure of the compound targeting prostate specific membrane antigen is shown in Formula (II-1): ##STR00114##

15. A method for preparing a compound targeting prostate specific membrane antigen, comprising the following steps: introducing a Boc protective group to one end of 4,4-diamino-3,3-dimethyl biphenyl, followed by a reaction with 4,6-diamino-5-hydroxy-1,3-naphthalenedisulfonic acid to prepare a truncated Evans Blue derivative; removing the Boc protective group, followed by an amide condensation reaction with N?-Fmoc-N?-Boc-L-lysine; next, removing the Boc protective group under the action of TFA, followed by an amide condensation reaction with COOH-PEG.sub.2-COOH and a reaction with PSMA-617 under the presence of EDC and NETS; then removing an Fmoc protective group using piperazine; and finally, carrying out a reaction with DOTA-NHS to obtain a compound having the following structure shown in Formula (II-1): ##STR00115##

16. A radiolabeled compound targeting prostate specific membrane antigen, wherein the compound is a complex obtained by using the compound shown in Formula (I) according to claim 14 as a ligand and labeling the ligand with a radionuclide.

17. The radiolabeled compound according to claim 16, wherein the radionuclide is any one of .sup.177Lu, .sup.90Y, .sup.18F, .sup.64Cu, .sup.68Ga, .sup.62Cu, .sup.67Cu, .sup.86Y, .sup.89Zr, .sup.99mTc, .sup.89Sr, .sup.153Sm, .sup.149Tb, .sup.161Tb, .sup.186Re, .sup.188Re, .sup.212Pb, .sup.213Bi, .sup.223Ra, .sup.225Ac, .sup.226Th, .sup.227Th, .sup.131I, .sup.211At, or .sup.111In.

18. The radiolabeled compound according to claim 16, wherein the radionuclide is .sup.68Ga, .sup.177Lu, or .sup.90Y.

19. A method for preparing a radiolabeled compound targeting prostate specific membrane antigen, wherein the method comprises the following steps: dissolving the compound shown in Formula (II-1) according to claim 14 in a buffer solution or deionized water; and adding a radionuclide solution to a resulting solution for a reaction under closed conditions for 5-40 min to produce a radionuclide labeled complex; or the method comprises the following steps: dissolving an appropriate amount of the compound shown in Formula (II-1) according to claim 14 in a buffer solution or deionized water; treating the obtained solution to aseptic filtration, followed by loading into a container, freeze-drying and sealing with a stopper to obtain a freeze-dried medicine box; and then adding an appropriate amount of an acetic acid solution or a buffer solution to the freeze-dried medicine box for dissolution, and adding a corresponding radionuclide solution for a reaction under closed conditions for 5-40 min to produce a radionuclide labeled complex.

20. A pharmaceutical composition, comprising a compound targeting prostate specific membrane antigen or a pharmaceutically available salt thereof, wherein the molecular structure of the compound targeting prostate specific membrane antigen is shown in Formula (II-1): ##STR00116## or a radiolabeled compound targeting prostate specific membrane antigen according to claim 16 and a pharmaceutically acceptable carrier.

21. The composition according to claim 20, wherein the pharmaceutically acceptable carrier is selected from an adhesive, a buffer, a colorant, a diluent, a disintegrator, an emulsifier, a flavoring agent, a flow aid, a lubricant, a preservative, a stabilizer, a surfactant, a tableting agent, a wetting agent, or a combination thereof.

22-23. (canceled)

Description

BRIEF DESCRIPTION OF DRAWINGS

[0050] FIG. 1 shows uptake results in tumors 24 h after different medicines are injected into subcutaneous transplanted tumor model mice with prostate cancer.

[0051] FIG. 2 shows comparison of uptake in the blood of normal mice at different time points after compound in Example 31, .sup.177Lu-PSMA and .sup.177Lu-EB-PSMA 617 are injected.

[0052] FIG. 3 shows distribution results in tissues 24 h after compound in Example 31 is injected into subcutaneous transplanted tumor model mice with prostate cancer.

[0053] FIG. 4 shows distribution results in tissues 24 h after .sup.177Lu-EB-PSMA 617 is injected into subcutaneous transplanted tumor model mice with prostate cancer.

[0054] FIG. 5 is a diagram showing SPECT-CT imaging of normal mice at different time points after the compound in Example 31 is injected.

[0055] FIG. 6 is a diagram showing SPECT-CT imaging of normal mice at different time points after .sup.177Lu labeled compound (II-2) in an example is injected.

[0056] FIG. 7 shows uptake results in the blood of normal mice at different time points after .sup.177Lu labeled compound (II-2) is injected.

[0057] FIG. 8 is a diagram showing the mass spectrum of compound 10 prepared in Example 1.

[0058] FIG. 9 is a diagram showing the mass spectrum of compound (II-3) prepared in Example 3.

[0059] FIG. 10 is an HPLC chromatogram of compound 5 prepared in Example 1.

[0060] FIG. 11 is an HPLC chromatogram of compound 6 prepared in Example 1.

[0061] FIG. 12 is an HPLC chromatogram of compound 7 prepared in Example 1.

[0062] FIG. 13 is an HPLC chromatogram of compound 8 prepared in Example 1.

[0063] FIG. 14 is an HPLC chromatogram of compound 9 prepared in Example 1.

[0064] FIG. 15 is an HPLC chromatogram of compound 10 prepared in Example 1.

DETAILED DESCRIPTION OF EMBODIMENTS

[0065] Technical solutions of the present disclosure are further explained and described below in conjunction with specific embodiments and attached drawings.

EXAMPLE 1: PREPARATION OF COMPOUND 10 SHOWN IN FORMULA (II-1)

Synthesis of Compound 2:

[0066] 4,4-diamino-3,3-dimethyl biphenyl (compound 11) (2.12 g, 10.0 mmol), di-tert-butyl dicarbonate (2.2 g, 10.0 mmol), N,N-diisopropylethylamine (1.3 g, 10.0 mmol) and 20 mL of dichloromethane were separately put into a 100 mL flask, and stirred overnight at room temperature. After monitoring by HPLC that a reaction was completed (r.t. was 10.13 min), reduced pressure distillation was conducted to remove the solvent to obtain a crude product. Then purification was conducted with a silica gel column (a ratio of petroleum ether to ethyl acetate was 5:1) to obtain white solid compound 2 with a yield of 59%.

Synthesis of Compound 3:

[0067] The compound 2 (0.31 g, 1.0 mmol) and 4 mL of acetonitrile were separately put into 50 mL flask in ice bath, 1.5 mL of 2 M hydrochloric acid was added dropwise to the reaction flask for a reaction for 15 min, and sodium nitrite (0.068 g, 1.0 mmol) was added to 2 mL of water for dissolution and then added dropwise to the reaction flask for a reaction for half an hour to obtain solution A for later use. 4,6-diamino-5-hydroxy-1,3-naphthalenedisulfonic acid (0.33 g, 1.0 mmol), sodium carbonate (0.105 g, 1.0 mmol) and 5 mL of water were added to another 50 mL reaction flask in ice bath to obtain solution B, and the solution A was slowly added dropwise to the solution B and stirred for a reaction for 2 h in the ice bath. Then purification was conducted with a reversed phase column, followed by freeze-drying to obtain pure compound 3 with yield of 47%.

Synthesis of a Compound 4:

[0068] The compound 3 (0.52 g, 1.0 mmol) was dissolved in trifluoroacetic acid in ice bath. The system was heated to room temperature for a reaction for 2 h, and after the reaction was completed, reduced pressure distillation was conducted to remove the solvent to obtain a crude product. Then purification was conducted on the crude product with a reversed phase column, followed by freeze-drying to obtain pure compound 4 with yield of 73%.

Synthesis of a Compound 5:

[0069] The compound 4 (0.54 g, 1.0 mmol), N?-Fmoc-N?-Boc-L-lysine (0.46 g, 1.0 mmol), HATU (0.38 g, 1.0 mmol), N,N-diisopropylethylamine (0.26 g, 2.0 mmol) and 10 mL of N,N-dimethylformamide were separately put into 100 mL flask. A reaction mixture was stirred until a reaction was completed, and reduced pressure distillation was conducted to remove the solvent to obtain a crude product. Then purification was conducted on the crude product with a reversed phase column, followed by freeze-drying to obtain pure compound 5 with yield of 57%.

Synthesis of a Compound 6:

[0070] Tert-butyl and Boc protective groups were removed from compound 5 using a mixture of thioanisole, 1,2-ethanedithiol, anisole and TFA (at ratio of 5:3:2:90) at room temperature to obtain compound 6. After a reaction was completed, the TFA was removed by an argon flow, and the resulting product was dissolved in 10 mL of N,N-dimethylformamide for later use.

Synthesis of Compound 7:

[0071] COOH-PEG.sub.2-COOH (0.23 g, 1.10 mmol), HATU (0.38 g, 1.0 mmol) and N,N-diisopropylethylamine (0.39 g, 3.0 mmol) were separately added to N,N-dimethylformamide solution of the compound 6. The system was stirred overnight at room temperature, and a reaction was completed according to monitoring by HPLC (r.t. was 10.84 min). Reduced pressure distillation was conducted to remove the solvent to obtain a crude product. Then purification was conducted on the crude product with a reversed phase column, followed by freeze-drying to obtain pure compound 7 with yield of 50% in two steps.

Synthesis of a Compound 8:

[0072] The compound 7 (0.21 g, 0.2 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (0.04 g, 0.2 mmol), NHS (0.02 g, 0.2 mmol) and 10 mL of N,N-dimethylformamide were separately put into 50 mL flask. After a reaction was carried out for 4 h, N,N-diisopropylethylamine (0.06 g, 0.5 mmol) and PSMA-617 (0.13 g, 0.2 mmol) were added. A reaction mixture was stirred for a reaction, and the reaction was completed according to monitoring by HPLC (r.t. was 12.16 min). Reduced pressure distillation was conducted to remove the solvent to obtain a crude product. Then purification was conducted on the crude product with a reversed phase column, followed by freeze-drying to obtain pure compound 8 with yield of 59%.

Synthesis of a Compound 9:

[0073] The compound 8 (0.16 g, 0.1 mmol) and piperidine (0.08 g, 10.0 mmol) were separately put into 5 mL of DMF in 25 mL flask. The process of removing protective groups was monitored by HPLC until a reaction was completed (r.t. was 10.47 min). Reduced pressure distillation was conducted to remove the solvent to obtain a crude product. Then purification was conducted on the crude product with a reversed phase column, followed by freeze-drying to obtain pure compound 9 with yield of 63%.

Synthesis of a Compound 10:

[0074] The compound 9 (0.13 g, 0.1 mmol), DOTA-NHS (0.05 g, 0.1 mmol) and N,N-diisopropylethylamine (0.04 g, 0.3 mmol) were sequentially put into 5 mL of N,N-dimethylformamide in 25 mL flask. The reaction system was stirred for a reaction at room temperature, and the process of removing protective groups was monitored by HPLC until the reaction was completed (r.t. was 11.35 min). Reduced pressure distillation was conducted to remove the solvent to obtain a crude product. Then purification was conducted on the crude product with a reversed phase column, followed by freeze-drying to obtain pure compound 10 with yield of 61%. Characterization of the structure of the resulting compound is shown in FIG. 8.

[0075] A synthesis route in the above steps is as follows:

##STR00018## ##STR00019##

EXAMPLES 2-EXAMPLES 6

[0076] Compounds in Examples 2-Examples 6 have structures shown in Formula (II-2) to Formula (II-6) respectively, and preparation methods of the compounds can refer to the preparation method in Example 1. For example, the COOH-PEG.sub.2-COOH reacting with the compound 6 in Example 1 was substituted with COOH-PEG.sub.4-COOH, malonic acid or other suitable compounds for preparation of the compounds shown in Formula (II-2) and Formula (II-3). The N?-Fmoc-N?-Boc-L-lysine reacting with the compound 4 in Example 1 was substituted with Boc-glycine for preparation of the compounds shown in Formula (II-4) to Formula (II-6), or the PSMA-617 reacting with the compound 7 in Example 1 was substituted with PSMA-617-(Fmoc)Lys- to obtain corresponding structures as follows:

##STR00020## ##STR00021##

[0077] Characterization of the structure of the compound (II-3) is shown in FIG. 9.

EXAMPLES 7-EXAMPLES 30

[0078] With reference to the preparation methods in Examples 1-Examples 6, compound shown in the following Formula (I) was prepared.

##STR00022##

TABLE-US-00001 Ex- ample number R1 R2 L1 L2 7 [00023] [00024] [00025] [00026] 8 [00027] [00028] [00029] [00030] 9 [00031] [00032] [00033] [00034] 10 [00035] [00036] [00037] [00038] 11 [00039] [00040] [00041] [00042] 12 [00043] [00044] [00045] [00046] 13 [00047] [00048] [00049] 14 [00050] [00051] [00052] [00053] 15 [00054] [00055] [00056] [00057] 16 [00058] [00059] [00060] [00061] 17 [00062] [00063] [00064] [00065] 18 [00066] [00067] [00068] 19 [00069] [00070] [00071] [00072] 20 [00073] [00074] [00075] [00076] 21 [00077] [00078] [00079] [00080] 22 [00081] [00082] [00083] [00084] 23 [00085] [00086] [00087] [00088] 24 [00089] [00090] [00091] 25 [00092] [00093] [00094] [00095] 26 [00096] [00097] [00098] [00099] 27 [00100] [00101] [00102] 28 [00103] [00104] [00105] [00106] 29 [00107] [00108] [00109] 30 [00110] [00111] [00112] [00113]

EXAMPLE 31: PREPARATION OF .SUP.177.Lu LABELED COMPLEX

[0079] Wet method: Sodium acetate solution of about 18.5-1,850 MBq of .sup.177LuCl.sub.3 was added to acetic acid-acetate solution (1.0 g/L) containing 0.5 mL of the compound 10 in Example 1 in a centrifuge tube, and a reaction was carried out at 90? C. for 20 min. A small C18 separation column was slowly rinsed with 10 mL of anhydrous ethanol first, and then rinsed with 10 mL of water. Resulting labeled solution was diluted with 10 mL of water, and then sampled to the separation column. Unlabeled .sup.177Lu ions were removed with 10 mL of water, and rinsing was conducted with 0.3 mL of a 10 mM ethanol solution of HCl to obtain .sup.177Lu labeled complex. The rinsed solution was diluted with normal saline, followed by aseptic filtration to obtain injection of the .sup.177Lu labeled complex.

[0080] Freeze-drying method: Sodium acetate solution of about 18.5-1,850 MBq of .sup.177LuCl.sub.3 was added to a freeze-dried medicine box containing the compound 10 in Example 1, and uniformly mixed for a reaction at 90? C. for 20 min. A small C18 separation column was slowly rinsed with 10 mL of anhydrous ethanol first, and then rinsed with 10 mL of water. Resulting labeled solution was diluted with 10 mL of water, and then sampled to the separation column. Unlabeled .sup.177Lu ions were removed with 10 mL of water, and rinsing was conducted with 0.3 mL of 10 mM ethanol solution of HCl to obtain rinsed solution of .sup.177Lu labeled complex. The rinsed solution was diluted with normal saline, followed by aseptic filtration to obtain injection of the .sup.177Lu labeled complex.

EXPERIMENTAL EXAMPLE: ANALYSIS AND APPLICATION EFFECT

1. HPLC Analysis and Identification

[0081] An HPLC system was as follows: SHIMADZULC-20A; and a C18 chromatographic column (YMC, 3 ?m, 4.6*150 mm) was used for analysis. Detection was conducted at wavelength of 254 nm and flow rate of 1 mL/min according to the following rinsing gradient: at 0-3 min, 10% of acetonitrile and 90% of water (50 mM ammonium acetate) were remained unchanged; at 3-16 min, the system was increased to include 90% of acetonitrile and 10% of water (50 mM ammonium acetate); at 16-18 min, 90% of acetonitrile and 10% of water (50 mM ammonium acetate) were remained; at 18-20 min, the system was reduced to include 10% of acetonitrile and 90% of water (50 mM ammonium acetate); and at 20-22 min, 10% of acetonitrile and 90% of water (50 mM ammonium acetate) were retained.

[0082] Compound 5, compound 6, compound 7, compound 8, compound 9 and compound 10 in Example 1 were identified and analyzed according to the above system. Identification and analysis results are shown in FIG. 10, FIG. 11, FIG. 12, FIG. 13, FIG. 14 and FIG. 15, respectively.

[0083] The radiolabeled probe prepared in Example 31 is used as an experimental agent below, and experiments for determining properties of the probe are described as follows.

2. Uptake Experiment of .sup.177Lu Labeled Complex in Subcutaneous Transplanted Tumor Model Mice with Prostate Cancer

[0084] The compound in Example 31 or another prior radioactive probe targeting PSMA was injected into subcutaneous transplanted tumor model mice with prostate cancer, and uptake results in tumors and distribution results in tissues were compared. A specific plan is as follows.

[0085] The subcutaneous transplanted tumor model mice with prostate cancer (22RV1) were randomly divided into 3 groups including an experimental group, a control group A and a control group B with 3 mice in each group.

[0086] .sup.177Lu complex with purity of greater than 95% was prepared according to the method in Example 31. The complex is obtained by labeling compound 10 in Example 1 with .sup.177Lu, which was used as medicine B for the experimental group in the experiment.

[0087] .sup.177Lu-PSMA 617 with purity of greater than 95% was prepared according to a prior method, which was used as medicine A for the control group A in the experiment.

[0088] .sup.177Lu-EB-PSMA 617 with purity of greater than 95% was prepared according to the method in Example 8 in WO2019/165200, which was used as medicine C for the control group B in the experiment.

[0089] 5 MBq of medicine B, the medicine A and the medicine C were intravenously injected into tails of the mice in the experimental group, the control group A and the control group B, respectively. After the injection was completed for 24 h, the mice in each group were sacrificed and dissected to obtain tumor tissues, blood or other tissues. The obtained tissues were weighed, and measured by a y counter to obtain the radioactive counting of samples in the experimental group, the control group A and the control group B. Measured data were subtracted from the background, the decay time was corrected, and then average values were obtained. The data was expressed as the percentage of injected dose per gram tissue (% ID/g) of the dose uptake in tissues per gram in the injected dose. Results are shown in FIG. 1, FIG. 3 and FIG. 4.

[0090] From FIG. 1, it can be seen that the uptake in tumors after the .sup.177Lu complex (B) in Example 31 of the present disclosure is injected for 24 his 23.46?0.63% ID/g, which is much higher than that after the .sup.177Lu-PSMA 617 (A) is injected in the control group A (7.60?1.22% ID/g) and lower than that after the .sup.177Lu-EB-PSMA 617 (C) is injected in the control group B (48.97?7.77% ID/g).

[0091] FIG. 3 and FIG. 4 show the distribution of major tissues 24 h after the .sup.177Lu complex (B) in Example 31 of the present disclosure is injected in the experimental group and the .sup.177Lu-EB-PSMA 617 (C) is injected in the control group B, respectively. It can be observed that the uptake of the .sup.177Lu complex in Example 31 of the present disclosure (FIG. 3) in the kidneys 24 h after the injection is much lower than that in the .sup.177Lu-EB-PSMA-617 group (FIG. 4).

3. Experiment of .SUP.177.Lu Labeled Complex in Normal Mice

[0092] The normal mice were randomly divided into experimental group I, experimental group II, control group A and control group B with 3 mice in each group.

[0093] .sup.177Lu complex with purity of greater than 95% was prepared according to the method in Example 31. The complex is obtained by labeling compound 10 in Example 1 with .sup.177Lu, which was used as medicine B for the experimental group I in the experiment.

[0094] With reference to the method in Example 31, .sup.177Lu labeled compound (II-2) was prepared by substituting compound 10 with the compound (II-2) in Example 2, which was used as medicine D for the experimental group II in the experiment.

[0095] .sup.177Lu-PSMA 617 with purity of greater than 95% was prepared according to a prior method, which was used as medicine A for the control group A in the experiment.

[0096] .sup.177Lu-EB-PSMA 617 with purity of greater than 95% was prepared according to the method in Example 8 in WO2019/165200, which was used as medicine C for the control group B in the experiment.

[0097] 5 MBq of medicine B, medicine D, medicine A and medicine C were intravenously injected into tails of the mice in the first experimental group, the second experimental group, the control group A and the control group B, respectively. The uptake in the blood was measured 1 h, 4 h and 24 h after the injection was completed. Results are shown in FIG. 2 and FIG. 7. SPECT-CT imaging was conducted 1 h, 4 h, 24 h, and 48 h after the injection was completed. Results are shown in FIG. 5 and FIG. 6.

[0098] From FIG. 2, the uptake of the .sup.177Lu complex (B) in Example 31 of the present disclosure in the blood is higher than that in the .sup.177Lu-PSMA-617 (A) group but much lower than that in the .sup.177Lu-EB-PSMA-617 (C) group at all tested time points (1 h, 4 h and 24 h). From the comparison of FIG. 7 and FIG. 2, the uptake of the .sup.177Lu labeled compound (II-2) in the blood is much lower than that in the .sup.177Lu-EB-PSMA-617 (C) group at all tested time points (1 h, 4 h and 24 h).

[0099] FIG. 5 and FIG. 6 are diagrams showing SPECT-CT imaging of normal mice after the .sup.177Lu complex in Example 31 and the .sup.177Lu labeled compound (II-2) are injected, respectively.

[0100] In summary, compared with existing PSMA-targeting probes, the compound targeting prostate specific membrane antigen provided by the present disclosure has high uptake in tumors, and more importantly has appropriate blood circulation time, so that when the radionuclide labeled compound targeting prostate specific membrane antigen of the present disclosure is used in therapy of prostate cancer, not only can the therapeutic needs of uptake in the blood and uptake in tumors be met, but also hematotoxicity and myelosuppression risks are greatly reduced. The compound has a higher value in clinical application and popularization and is expected to be applied to nuclide therapy and imaging of prostate cancer.

[0101] Although the present disclosure has been described in detail by general descriptions, specific embodiments and tests above, it is obvious to persons skilled in the field that some modifications or improvements can be made on the basis of the present disclosure. Therefore, all the modifications or improvements made without departing from the spirit of the present disclosure shall fall within the protection scope of the present disclosure.