TRUNCATED EVANS BLUE MODIFIED FIBROBLAST ACTIVATION PROTEIN INHIBITOR, PREPARATION METHOD AND APPLICATION THEREOF

Abstract

The present disclosure provides a truncated Evans Blue modified fibroblast activation protein inhibitor compound. The compound is formed by connecting truncated Evans Blue, a fibroblast activation protein inhibitor and a nuclide chelating group by means of connecting groups L.sub.1, L.sub.2, L.sub.3, L.sub.4 and X. The compound has the following structure shown in Formula (I), where R.sub.1 is a fibroblast activation protein inhibitor; L.sub.1 is lysine, glutamic acid, or a derivative structure thereof; L.sub.2 is —(CH.sub.2).sub.n—, n is an integer from 0 to 30, and each —CH.sub.2— may be individually substituted or unsubstituted with —O—, —NH—, —(CO)—, —NH(CO)—, or —(CO)—NH—; L.sub.3 is —(CH.sub.2).sub.m—, m is an integer from 0 to 30, and each —CH.sub.2— may be individually substituted or unsubstituted with —O— or —(CO)—; L.sub.4 is —(CH.sub.2).sub.p—, p is an integer from 0 to 30, and each —CH.sub.2— may be individually substituted or unsubstituted with —O—, —NH—, —(CO)—, —NH(CO)—, or —(CO)—NH—; X is selected from N, C, O, S, or

##STR00001##

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 the characteristics of significantly prolonging the half-life in blood circulation, improving the uptake and enrichment in tumors and prolonging the retention time, and are suitable for nuclide therapy and imaging of tumors with high expression of FAP.

##STR00002##

Claims

1-20. (canceled)

21. A truncated Evans Blue modified fibroblast activation protein inhibitor compound or a pharmaceutically available salt thereof, wherein the molecular structure of the compound has the following structures shown in Formula (II-1): ##STR00167##

22. A truncated Evans Blue modified fibroblast activation protein inhibitor compound or a pharmaceutically available salt thereof, wherein the molecular structure of the compound has any one of the following structures shown in Formula (II-2) to Formula (II-8): ##STR00168## ##STR00169##

23. A method for preparing a truncated Evans Blue modified fibroblast activation protein inhibitor, comprising the following steps: (1) reacting 6-hydroxy-4-quinolinecarboxylic acid with tert-butyl glycinate by amide condensation, followed by reactions with 1-bromo-3-chloropropane and tert-butyl 1-piperazinecarboxylate in sequence; then, removing Boc and tert-butyl protective groups under the action of TFA, and introducing a Boc protective group to amino, followed by an amide condensation reaction with (S)-pyrrolidene-2-carbonitrile hydrochloride; then, removing the Boc protective group using p-toluenesulfonic acid, followed by a condensation reaction with 5,8,11,14-tetraoxa-2-azaheptadecanedioic acid-1-tert-butyl ester; and removing the Boc protective group again under the action of p-toluenesulfonic acid to obtain an intermediate compound A; (2) introducing a Boc protective group to one end of 4,4′-diamino-3,3′-dimethyl biphenyl, followed by a reaction with monosodium 1-amino-8-naphthol-2,4-disulfonate to prepare a truncated Evans Blue derivative; removing the Boc protective group, followed by an amide condensation reaction with N-tert-butyloxycarbonyl-L-glutamic acid-1-tert-butyl ester; then, removing Boc and tert-butyl protective groups under the action of TFA; and then carrying out a reaction with di-tert-butyl dicarbonate, and introducing a Boc protective group to amino to obtain an intermediate compound B; and (3) reacting the intermediate compound A obtained in step (1) with the intermediate compound B obtained in step (2) by amide condensation; then removing the Boc protective group using p-toluenesulfonic acid; and finally, carrying out a reaction with DOTA-NHS to obtain a truncated Evans Blue modified fibroblast activation protein inhibitor compound having the following structure shown in Formula (II-1) ##STR00170##

24. A radiolabeled complex of truncated Evans Blue modified Fibroblast activation protein inhibitor, having the following structure shown in Formula (IV): ##STR00171## wherein L.sub.1 is a glutamic acid structure; L.sub.2 is —(CH.sub.2).sub.0—, —NH—CH.sub.2—(CO)—, —NH—CH.sub.2—(CH.sub.2OCH.sub.2).sub.2—CH.sub.2—(CO)—, —NH—CH.sub.2—(CH.sub.2OCH.sub.2).sub.3—CH.sub.2(CO)—; L.sub.3 is —(CH.sub.2).sub.3—; X is ##STR00172## R.sub.3 and R.sub.4 are both H or both F; and M is a radionuclide selected from any one of .sup.68Ga, .sup.177Lu, or .sup.90Y.

25. A method for preparing a radiolabeled complex of truncated Evans Blue modified Fibroblast activation protein inhibitor, comprising the following steps: dissolving the compound shown in Formula (II-1) according to claim 21 in a buffer solution or deionized water; and adding a radionuclide solution to the obtained solution for a reaction under closed conditions for 5-40 min to produce a radionuclide labeled complex. or, comprising the following steps: dissolving the compound shown in Formula (II-1) according to claim 21 in a buffer solution or deionized water; treating the obtained solution by 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.

26. Application of the compound according to claim 21 or a pharmacologically acceptable salt thereof in preparation of medicines in nuclide therapy or imaging of tumors with high expression of FAP.

27. Application of the complex according to claim 24 in nuclide therapy or imaging of tumors with high expression of FAP.

28. The application according to claim 26, wherein the compound or the complex is formulated as an injection and then intravenously injected into patients with tumors with high expression of FAP; and the tumors with high expression of FAP comprise, but are not limited to, breast cancer, ovarian cancer, lung cancer, colorectal cancer, gastric cancer or pancreatic cancer.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0062] FIG. 1 is a diagram showing mass spectrum of compound 2 in Example 1 of the present disclosure.

[0063] FIG. 2 shows nuclear magnetic hydrogen spectrum of compound 2 in Example 1 of the present disclosure.

[0064] FIG. 3 shows nuclear magnetic carbon spectrum of compound 2 in Example 1 of the present disclosure.

[0065] FIG. 4 is a diagram showing mass spectrum of compound 3 in Example 1 of the present disclosure.

[0066] FIG. 5 shows nuclear magnetic hydrogen spectrum of compound 3 in Example 1 of the present disclosure.

[0067] FIG. 6 is a diagram showing mass spectrum of compound 4 in Example 1 of the present disclosure.

[0068] FIG. 7 shows nuclear magnetic hydrogen spectrum of compound 4 in Example 1 of the present disclosure.

[0069] FIG. 8 shows nuclear magnetic carbon spectrum of compound 4 in Example 1 of the present disclosure.

[0070] FIG. 9 is a diagram showing mass spectrum of compound 7 in Example 1 of the present disclosure.

[0071] FIG. 10 shows nuclear magnetic hydrogen spectrum of compound 7 in Example 1 of the present disclosure.

[0072] FIG. 11 shows nuclear magnetic carbon spectrum of compound 7 in Example 1 of the present disclosure.

[0073] FIG. 12 is a diagram showing mass spectrum of compound 10 in Example 1 of the present disclosure.

[0074] FIG. 13 is a diagram showing mass spectrum of compound 20 in Example 1 of the present disclosure.

[0075] FIG. 14 is a diagram showing mass spectrum of compound in Example 10 of the present disclosure.

[0076] FIG. 15 is a diagram showing the mass spectrum of a compound in Example 11 of the present disclosure.

[0077] FIG. 16 is an HPLC chromatogram of compound 10 in Example 1 of the present disclosure.

[0078] FIG. 17 is an HPLC chromatogram of compound 17 in Example 1 of the present disclosure.

[0079] FIG. 18 is an HPLC chromatogram of a reaction system of compound 17 and compound 10 in Example 1 of the present disclosure.

[0080] FIG. 19 is an HPLC chromatogram of compound 19 in Example 1 of the present disclosure.

[0081] FIG. 20 is an HPLC chromatogram of a reaction system of compound 19 and DOTA-NHS in Example 1 of the present disclosure.

[0082] FIG. 21A and FIG. 21B show MicroPET imaging of a .sup.68Ga labeled tEB-FAPI complex of the present disclosure and .sup.68Ga labeled FAPI-02 in normal mice.

[0083] FIG. 22 shows SPECT imaging of .sup.177Lu-tEB-FAPI prepared in Example 40 of the present disclosure in normal mice at different time points.

[0084] FIG. 23 shows SPECT imaging of .sup.177Lu-tEB-FAPI prepared in Example 40 of the present disclosure in xenograft model mice with human pancreatic cancer at different time points.

DETAILED DESCRIPTION OF EMBODIMENTS

[0085] Technical solutions of the present disclosure are further explained and described below in conjunction with specific embodiments and attached drawings.

Example 1: Preparation of a tEB-FAPI Conjugates Connector (Compound 20)

Synthesis of Compound 2

[0086] Compound 1 (6-hydroxy-4-quinolinecarboxylic acid, 1.89 g, 10.0 mmol), tert-butyl glycinate (1.89 g, 10.0 mmol), HATU (3.8 g, 10.0 mmol) and N,N-diisopropylethylamine (2.6 g, 20.0 mmol) were sequentially put into 30 mL of N,N-dimethylformamide in a 100 mL flask. A reaction mixture was stirred overnight, and 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 dichloromethane to methanol was 30:1) to obtain a white solid compound 2 with a yield of 87%. FIG. 1 is a diagram showing the mass spectrum of compound 2. FIG. 2 shows nuclear magnetic hydrogen spectrum of the compound 2. FIG. 3 shows the nuclear magnetic carbon spectrum of compound 2.

Synthesis of Compound 3

[0087] Compound 2 (1.51 g, 5.0 mmol), 1-bromo-3-chloropropane (1.55 g, 10.0 mmol) and potassium carbonate (1.38 g, 10.0 mmol) were sequentially put into 50 mL of N,N-dimethylformamide in a 100 mL flask. The system was heated to 60° C. and stirred overnight at 60° C., and 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 dichloromethane to methanol was 50:1) to obtain a white solid compound 3 with a yield of 63%. FIG. 4 is a diagram showing the mass spectrum of compound 3. FIG. 5 shows nuclear magnetic hydrogen spectrum of compound 3.

Synthesis of Compound 4

[0088] Compound 3 (0.76 g, 2.0 mmol), tert-butyl 1-piperazinecarboxylate (0.55 g, 3.0 mmol) and potassium iodide (0.49 g, 3.0 mmol) were sequentially put into 30 mL of acetonitrile in a 100 mL flask. The system was heated to 60° C. and stirred overnight at 60° C., and 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 dichloromethane to methanol was 30:1) to obtain a white solid compound 4 with a yield of 58%. MS(ESI).sub.m/z calculated for [C.sub.28H.sub.40N.sub.4O.sub.6]: 528.29; found: 529.10 [M+H].sup.+. FIG. 6 is a diagram showing the mass spectrum of compound 4. FIG. 7 shows nuclear magnetic hydrogen spectrum of compound 4. FIG. 8 shows the nuclear magnetic carbon spectrum of compound 4.

Synthesis of Compound 5

[0089] Compound 4 (0.52 g, 1.0 mmol) was dissolved in 10 mL of a mixed solution of dichloromethane and trifluoroacetic acid (at a volume ratio of 9:1) in an 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. Then the resulting product was dissolved in 10 mL of N,N-dimethylformamide for later use.

Synthesis of Compound 6

[0090] Di-tert-butyl dicarbonate (0.22 g, 1.0 mmol) and N,N-diisopropylethylamine (0.39 g, 3.0 mmol) were separately added to an N,N-dimethylformamide solution of the compound 5. The system was stirred overnight at room temperature, and 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 dichloromethane to methanol was 10:1) to obtain a white solid compound 6 with a yield of 72%.

Synthesis of Compound 7

[0091] Compound 6 (0.47 g, 1.0 mmol), (S)-pyrrolidene-2-carbonitrile hydrochloride (0.13 g, 10.0 mmol), HATU (0.38 g, 1.0 mmol) and N,N-diisopropylethylamine (0.26 g, 2.0 mmol) were sequentially put into 10 mL of N,N-dimethylformamide in a 100 mL flask. A reaction mixture was stirred at room temperature until a reaction was completed, and 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 dichloromethane to methanol was 50:1) to obtain a white solid compound 7 with a yield of 85%. FIG. 9 is a diagram showing mass spectrum of compound 7. FIG. 10 shows nuclear magnetic hydrogen spectrum of compound 7. FIG. 11 shows the nuclear magnetic carbon spectrum of compound 7.

Synthesis of Compound 8

[0092] Compound 7 (0.55 g, 1.0 mmol) and p-toluenesulfonic acid monohydrate (0.27 g, 1.5 mmol) were sequentially put into 10 mL of acetonitrile in a 100 mL flask. The reaction system was heated to 60° C. and stirred until a reaction was completed, and reduced pressure distillation was conducted to remove the solvent to obtain a crude product.

Synthesis of a Compound 9

[0093] 5,8,11,14-tetraoxa-2-azaheptadecanedioic acid-1-tert-butyl ester (0.19 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 the reaction flask of compound 8. A reaction mixture was stirred overnight, and reduced pressure distillation was conducted to remove the solvent to obtain a crude product. Then purification was conducted with a silica gel column (ratio of dichloromethane to methanol was 50:1) to obtain a white solid compound 9 with a yield of 64%.

Synthesis of Compound 10

[0094] Compound 9 (0.61 g, 1.0 mmol) and p-toluenesulfonic acid monohydrate (0.27 g, 1.5 mmol) were sequentially put into 10 mL of acetonitrile in a 100 mL flask. The reaction system was heated to 60° C. and 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 with a silica gel column (ratio of dichloromethane to methanol was 10:1) to obtain a white solid compound 10 with a yield of 59%. MS(ESI) m/z calculated for [C.sub.35H.sub.51N.sub.7O.sub.8]: 697.38; found: 698.43 [M+H].sup.+. FIG. 12 is a diagram showing the mass spectrum of the compound 10.

[0095] A synthesis route in the above steps is as follows:

##STR00021##

Synthesis of Compound 12

[0096] 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 (ratio of petroleum ether to ethyl acetate was 5:1) to obtain a white solid compound 12 with a yield of 59%.

Synthesis of Compound 13

[0097] Compound 12 (0.31 g, 1.0 mmol) and 4 mL of acetonitrile were separately put into a 50 mL flask in an 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 reaction for half an hour to obtain a solution A for later use. Monosodium 1-amino-8-naphthol-2,4-disulfonate (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 an ice bath to obtain a solution B, and the solution A was slowly added dropwise to the solution B and stirred for 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 13 with a yield of 47%.

Synthesis of Compound 14

[0098] Compound 13 (0.52 g, 1.0 mmol) was dissolved in trifluoroacetic acid in an 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 14 with a yield of 73%.

Synthesis of Compound 15

[0099] Compound 14 (0.54 g, 1.0 mmol), N-tert-butyloxycarbonyl-L-glutamic acid-1-tert-butyl ester (0.30 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 a 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 15 with a yield of 52%.

Synthesis of Compound 16

[0100] Tert-butyl and Boc protective groups were removed using a mixture of thioanisole, 1,2-ethanedithiol, anisole and TFA (at a ratio of 5:3:2:90) at room temperature. 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 17

[0101] Di-tert-butyl dicarbonate (0.22 g, 1.0 mmol) and N,N-diisopropylethylamine (0.39 g, 3.0 mmol) were separately added to an N,N-dimethylformamide solution of the compound 16. 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 17 with a yield of 43% in two steps.

Synthesis of Compound 18

[0102] Compound 17 (0.77 g, 1.0 mmol), compound 10 (0.51 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 a 50 mL flask. 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 18 with a yield of 55%.

Synthesis of Compound 19

[0103] Compound 15 (0.13 g, 0.1 mmol) and p-toluenesulfonic acid monohydrate (0.05 g, 0.3 mmol) were sequentially put into 5 mL of acetonitrile in a 25 mL flask. The reaction system was heated to 60° C. and stirred for reaction, and the process of removing protective groups was monitored by HPLC until the 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 19 with a yield of 61%.

Synthesis of Compound 20

[0104] Compound 19 (0.12 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 a 25 mL flask. The reaction system was stirred for 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 20 with a yield of 53%. MS(ESI).sub.m/z calculated for [C.sub.80H.sub.104N.sub.16O.sub.24S.sub.2]: 1736.69; found: 1737.743 [M+H].sup.+. FIG. 13 is a diagram showing mass spectrum of compound 20.

[0105] A synthesis route in the above steps is as follows:

##STR00022## ##STR00023## ##STR00024##

Examples 2-Examples 16

[0106] Compounds in Examples 2-Examples 16 have structures shown in Formula (II-2) to Formula (II-16) respectively, and preparation methods of the compounds can refer to the preparation method in Example 1. The glutamic acid structure reacting with the compound 14 was substituted with a lysine structure, or the 5,8,11,14-tetraoxa-2-azaheptadecanedioic acid-1-tert-butyl ester reacting with the compound 8 was substituted with 5,8,11-trioxa-2-azatridecanediic acid-1-tert-butyl ester, tert-butyl 9-amino-4,7-dioxazononate, tert-butyl glycinate or other suitable compounds, or the (S)-pyrrolidene-2-carbonitrile hydrochloride reacting with the compound 6 was substituted with 3,3-difluoropyrrolidene hydrochloride, or the above compounds were substituted at the same time to obtain corresponding structures as follows:

##STR00025## ##STR00026## ##STR00027## ##STR00028##

[0107] The mass spectrum of compound (11-10) in Example 10 is shown in FIG. 14. The mass spectrum of compound (II-11) in Example 11 is shown in FIG. 15.

Examples 17-Examples 38

[0108] With reference to the preparation methods in Examples 1-Examples 16, a tEB-FAPI compound shown in the following Formula (I) was prepared.

TABLE-US-00001 Formula (I) [00029] Ex- am- ple X L.sub.1 17 [00030] [00031] 18 [00032] [00033] 19 [00034] [00035] 20 [00036] [00037] 21 [00038] [00039] 22 [00040] [00041] 23 [00042] [00043] 24 [00044] [00045] 25 [00046] [00047] 26 [00048] [00049] 27 [00050] [00051] 28 [00052] [00053] 29 N [00054] 30 S [00055] 31 O [00056] 32 C [00057] 33 [00058] [00059] 34 [00060] [00061] 35 [00062] [00063] 36 [00064] [00065] 37 [00066] [00067] 38 [00068] [00069] Ex- am- ple L.sub.2 L.sub.3 18 [00070] [00071] 19 [00072] [00073] 20 [00074] [00075] 21 [00076] [00077] 22 [00078] [00079] 23 [00080] [00081] 24 [00082] [00083] ` 25 [00084] [00085] 26 [00086] [00087] 27 [00088] [00089] 28 [00090] [00091] 29 — — 30 [00092] [00093] 31 [00094] [00095] 32 [00096] [00097] 33 — [00098] 34 [00099] [00100] 35 [00101] [00102] 36 — [00103] 37 [00104] [00105] 38 [00106] [00107] Ex- am- ple L.sub.4 R.sub.1 17 [00108] [00109] 18 [00110] [00111] 19 [00112] [00113] 20 [00114] [00115] 21 [00116] [00117] 22 [00118] [00119] 23 [00120] [00121] 24 [00122] [00123] 25 [00124] [00125] 26 [00126] [00127] 27 [00128] [00129] 28 [00130] [00131] 29 — [00132] 30 [00133] [00134] 31 [00135] [00136] 32 [00137] [00138] 33 — [00139] 34 — [00140] 35 — [00141] 36 — [00142] 37 — [00143] 38 — [00144] Ex- am- ple R.sub.2 17 [00145] 18 [00146] 19 [00147] 20 [00148] 21 [00149] 22 [00150] 23 [00151] 24 [00152] 25 [00153] 26 [00154] 27 [00155] 28 [00156] 29 [00157] 30 [00158] 31 [00159] 32 [00160] 33 [00161] 34 [00162] 35 [00163] 36 [00164] 37 [00165] 38 [00166]

Example 39: Preparation of a Radioactive .SUP.68.Ga Labeled tEB-FAPI Complex

[0109] Wet method: A hydrochloric acid solution of about 18.5-1,850 MBq of .sup.68GaCl.sub.3 (rinsed from a germanium-gallium generator) was added to an acetic acid-acetate solution (1.0 g/L) containing 0.5 mL of compound 20 prepared in Example 1 in a centrifuge tube, and the reaction was carried out at 37° 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. A resulting labeled solution was diluted with 10 mL of water, and then sampled to the separation column. Unlabeled .sup.68Ga 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.68Ga labeled tEB-FAPI complex. The rinsed solution was diluted with normal saline, followed by aseptic filtration to obtain an injection of the .sup.68Ga labeled tEB-FAPI complex.

[0110] Freeze-drying method: A hydrochloric acid solution of about 18.5-1,850 MBq of .sup.68GaCl.sub.3 (rinsed with a germanium-gallium generator) was added to a freeze-dried medicine box containing the compound 20, and uniformly mixed for a reaction at 37° 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. A resulting labeled solution was diluted with 10 mL of water, and then sampled to the separation column. Unlabeled .sup.68Ga 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 a rinsed solution of a complex. The rinsed solution was diluted with normal saline, followed by aseptic filtration to obtain an injection of the .sup.68Ga labeled tEB-FAPI complex.

Example 40: Preparation of a .SUP.177.Lu Labeled tEB-FAPI Complex

[0111] Wet method: A sodium acetate solution of about 18.5-1,850 MBq of .sup.177LuCl.sub.3 was separately added to an acetic acid-acetate solution (1.0 g/L) containing 0.5 mL of compound 20 in Example 1, the compound (Formula (II-2)) in Example 2 and the compound (Formula (II-3)) in Example 3 in three centrifuge tubes, and 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 three .sup.177Lu labeled tEB-FAPI complexes. The rinsed solutions were diluted with normal saline, followed by aseptic filtration to obtain injections of the three .sup.177Lu labeled tEB-FAPI complexes.

[0112] Freeze-drying method: A sodium acetate solution of about 18.5-1,850 MBq of .sup.177LuCl.sub.3 was separately added to three freeze-dried medicine boxes containing compound 20 in Example 1, the compound (Formula (II-2)) in Example 2 and the compound (Formula (II-3)) in Example 3, and uniformly mixed for reactions at 90° C. for 20 min. A small C18 separation column was taken, slowly rinsed with 10 mL of anhydrous ethanol first, and then rinsed with 10 mL of water. Resulting labeled solutions were 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 rinsed solutions of three .sup.177Lu labeled tEB-FAPI complexes. The rinsed solutions were diluted with normal saline, followed by aseptic filtration to obtain injections of the three .sup.177Lu labeled tEB-FAPI complexes.

Experimental Example: Analysis and Application Effect

[0113] 1. HPLC Analysis and Identification

[0114] An HPLC system was as follows: SHIMADZULC-20A; and a C18 chromatographic column (YMC, 3 μm, 4.6*150 mm) was used for analysis. Detection was conducted at a wavelength of 254 nm and a 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.

Compound 10, compound 17, a reaction system of compound 10 and compound 17, compound 19 and a reaction system of compound 19 and DOTA-NHS in Example 1 were identified and analyzed according to the above system. Results obtained are shown in FIG. 16 to FIG. 20.
The two radiolabeled probes prepared in Example 39 and Example 40 were used as experimental agents below, and determination of properties of the probes is described as follows.

[0115] 2. MicroPET Imaging of a .sup.68Ga Labeled tEB-FAPI Complex in Normal Mice

[0116] .sup.68Ga-tEB-FAPI with a purity of greater than 95% was prepared by the method in Example 39. 3.7 MBq of the .sup.68Ga-tEB-FAPI or .sup.68Ga-FAPI-02 (as a control) was intravenously injected into tails of normal FVB mice anesthetized with isoflurane. Then MicroPET imaging was conducted after administration for 0-120 min. Results are shown in FIG. 21A and FIG. 21B. The results show that the .sup.68Ga-tEB-FAPI complex in Example 39 has higher uptake in the cardiac blood pool of the mice (FIG. 21A), while the .sup.68Ga-FAPI-02 is almost completely cleared in the test period (FIG. 21B), indicating that the half-life in blood circulation can be obviously prolonged by introducing truncated Evans Blue.

[0117] 3. Uptake Experiment of a .sup.177Lu Labeled tEB-FAPI Complex in Tumors in Xenograft Model Mice with Human Pancreatic Cancer

[0118] .sup.177Lu-tEB-FAPI with a purity of greater than 95% was prepared by the method in Example 40. 1.3 MBq of the .sup.177Lu-tEB-FAPI was intravenously injected into tails of normal mice and xenograft model mice with human pancreatic cancer separately. SPECT imaging was conducted at different time points after injection. Results are shown in FIG. 22 and FIG. 23. The results show that the .sup.177Lu-tEB-FAPI has good pharmacokinetics in the normal mice, and can be continuously taken up by tumor tissues in the xenograft model mice with human pancreatic cancer and maintained for more than 48 h, indicating that the tEB-FAPI has the characteristics of significantly improving the uptake in tumors and prolonging the retention time, and can be used as a therapeutic agent and an imaging agent for tumors.

[0119] In summary, the truncated Evans Blue modified fibroblast activation protein inhibitor provided by the present disclosure can significantly prolong the half-life in blood circulation, improve the uptake and accumulation in tumors and prolong the tumor retention. Such novel properties are not available to other FAPI imaging agents. According to further preclinical animal level studies and clinical studies, it is proven that the inhibitor is expected to be used in radionuclide therapy and imaging of tumors with high expression of FAP.

[0120] 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.