Cleaved Evans Blue modified fibroblast activation protein inhibitor and its preparation method and application

Abstract

The present invention provides a truncated Evans Blue-modified fibroblast activation protein inhibitor compound, which is constructed by linking groups L1, L2, L3, L4, and X linking together a truncated Evans Blue, a fibroblast activation protein inhibitor, and a nuclide chelating group, and has the structure shown in formula (I), wherein R1 is a fibroblast activation protein inhibitor, L1 is a lysine, glutamic acid, or derivative thereof, and L2 is -(CH2) n -, n is an integer of 0 to 30, and each -CH2- may be independently substituted or unsubstituted with -O-, -NH-, -(CO)-, -NH(CO)-, or -(CO)-NH-; L3 is -(CH2)m-, and m is an integer of 0 to 30, and each -CH2- may be independently substituted or unsubstituted with -O- or -(CO)-; L4 is -(CH2)m-, and p -, p is an integer from 0 to 30, each -CH2- may be independently substituted or unsubstituted with -O-, -NH-, -(CO)-, -NH(CO)-, or -(CO)-NH-, and X is N, C, O, S, or JPEG2023545213000078.jpg61170 formula (I)

Description

[Technical field]

[0001] The present invention relates to the field of nuclear medicine and molecular imaging, in particular to a truncated Evans Blue-modified fibroblast activation protein inhibitor, its preparation and labeling, and its applications. [Background technology]

[0002] Fibroblast activation protein (FAP) is a membrane serine peptidase expressed on the surface of activated fibroblasts in tumor stroma and plays a key role in tumor initiation and progression. Previous studies have shown that FAP is not usually expressed in normal human tissues, but is selectively and highly expressed on the surface of stromal fibroblasts in more than 90% of epithelial malignancies, including breast, ovarian, lung, colorectal, gastric, and pancreatic cancers. Given its widespread expression and important role in tumors, FAP has become an important target in tumor imaging and therapy.

[0003] At present, radionuclide-labeled fibroblast activation protein inhibitors (FAPIs), represented by quinolinic acid derivatives, have made important progress in the field of precise tumor imaging. For example, PET / CT contrast agents such as FAPI-02 and FAPI-04 have realized specific imaging of more than 30 types of tumors. Compared with FDG imaging, FAPI imaging has a lower background in the brain, liver, and oropharyngeal mucosa and a higher detection rate of tumor lesions. The currently reported FAPIs are rapidly removed from the blood circulation and simultaneously rapidly eluted at the tumor site. This metabolic characteristic is advantageous for imaging because it provides a cleaner background. However, it is highly unfavorable for treatment because the rapid metabolism and elution result in a lower effective dose and shorter retention time at the tumor site, which requires higher doses or more frequent administration to meet the needs of treatment, and increases the possibility of side effects.

[0004] Take FAPI-02 as an example, it is completely removed from the blood circulation within 1 hour, and the retention dose at the tumor site is reduced by about 75% after 24 hours. In recent studies, the non-pharmacophore part of the FAPI structure has been optimized, but the improvement of the tumor uptake and retention time of FAPI is very limited and cannot meet the needs of therapeutic applications. Those skilled in the art know that if the time for a small molecule drug to circulate in the blood vessels is too short or it is rapidly removed by the body, the binding of the drug to the target will be insufficient. Therefore, when preparing a FAPI probe, if the blood circulation half-life of the probe can be appropriately extended, it will be possible to increase the uptake and retention time of the probe at the target site.

[0005] Therefore, to meet the needs of radionuclide therapy and imaging, new strategies are needed to extend the blood circulation half-life of FAPI probes so that they have suitable metabolic kinetics, higher tumor uptake, and longer tumor retention time. Summary of the Invention

[0006] Based on the above background, the main objective of the present invention is to develop a kind of conjugate of cleaved Evans Blue (tEB) and fibroblast activation protein inhibitor (FAPI), the feature of which is to realize the use of albumin as a FAPI delivery carrier by effectively combining cleaved Evans Blue with serum albumin, thereby extending the half-life in peripheral blood, and increasing the uptake, accumulation and retention time in tumors. The tEB-FAPI conjugate developed by the present invention overcomes the shortcomings of the fast metabolism and short retention time of small molecule FAPI in target organs, and can improve the radionuclide therapy and imaging effect targeting FAP protein, and has the potential for clinical application.

[0007] Another object of the present invention is to provide a radiolabeled truncated Evans Blue-modified fibroblast activation protein inhibitor (tEB-FAPI) with a long blood circulation half-life.

[0008] Another object of the present invention is to provide a method for preparing the above radiolabeled tEB-FAPI conjugate.

[0009] Yet another object of the present invention is to provide an application of said conjugate in tumor nuclide imaging and therapy targeting FAP protein.

[0010] The technical solutions for achieving the main objective of the present invention as mentioned above include the following two aspects: ligand synthesis and radiolabeling.

[0011] In a first aspect, the present invention provides a truncated Evans Blue (tEB) modified fibroblast activation protein inhibitor (FAPI), the structure of which is shown in formula (I) below, designated as "tEB-FAPI": [ka] Formula (I) wherein L1 is a lysine or glutamic acid structure, or a derivative compound structure containing a lysine or glutamic acid structure; L2 is -(CH2) n -, n is an integer from 0 to 30, and each CH2 may be independently substituted or unsubstituted with -O-, -NH-, -(CO)-, -NH(CO)-, or -(CO)-NH-, with the substitution condition that two adjacent CH2 groups are unsubstituted; L3 is -(CH2) m -, m is an integer from 0 to 30, and each CH2 may be independently substituted or unsubstituted with -O- or -(CO)-, with the substitution condition that two adjacent CH2 groups are unsubstituted; L4 is -(CH2) p -, p is an integer from 0 to 30, and each CH2 may be independently substituted or unsubstituted with -O-, -NH-, -(CO)-, -NH(CO)-, or -(CO)-NH-, with the substitution condition that two adjacent CH2 groups are unsubstituted; X is selected from N, C, O, S or any of the following structures: [ka] R1 is the following structure of fibroblast activation protein inhibitor: [ka] R2 is a nuclide chelating group selected from any of the following structures: [ka] R3 and R4 are the same or different and are each independently selected from H or F.

[0012] In a preferred embodiment of the present invention, L2 in formula (I) is -(CH2) n -, n is an integer of 0 to 16, more preferably an integer of 0 to 12, and even more preferably 0, 3, or 10, wherein each -CH2- may be independently substituted or unsubstituted with -O-, -NH-, or -(CO)-, and the substitution condition is that two adjacent -CH2- groups are unsubstituted.

[0013] In a preferred embodiment of the present invention, L3 in formula (I) is -(CH2) m and m is an integer of 0 to 20, more preferably an integer of 1 to 6, and even more preferably 2 or 3, in which each -CH2- may be independently substituted or unsubstituted with -O-, and the substitution condition is that two adjacent -CH2- groups are unsubstituted.

[0014] In a preferred embodiment of the present invention, L4 in formula (I) is -(CH2) p and p is an integer of 0 to 20, more preferably an integer of 0 to 12, further preferably 3, 4, 9 or 12, and most preferably 3, wherein each -CH2- may be independently substituted or unsubstituted with -O-, -NH-, -(CO)-, -NH(CO)- or -(CO)-NH-, and the substitution condition is that two adjacent -CH2- groups are unsubstituted.

[0015] JPEG0007651785000005.jpg23170 [ka] Formula (II) wherein R3 and R4 are both H or F, L1 is a glutamic acid or lysine structure, and L2 is -(CH2)0-, -NH-CH2-(CO)-, -NH-CH2-(CHOCH2)2-CH2-(CO)-, -NH-CH2-(CHOCH2)4-CH2(CO)-, -(CO)-CH2-(CO)-, -(CO)-(CH2)2-(CO)-, -(CO)-CH2-(CHOCH2)2-CH2(CO)-, or -(CO)-CH2-(CHOCH2)4-CH2(CO)-.

[0016] JPEG0007651785000007.jpg15170-NH-CH2-(CO)-, -NH-CH2-(CH2OCH2)2-CH2-(CO)- or -NH-CH2-(CH2OCH2)4-CH2(CO)-, L3 is -(CH2)3-, L4 is -(CH2)0-, R2 is DOTA, and R3 and R4 are both H or F.

[0017] JPEG0007651785000008.jpg28170

[0018] In a further preferred embodiment of the present invention, the structure of the above compound tEB-FAPI is any one of the following formulas (II-1) to (II-16). [ka] Formula (II-1) JPEG0007651785000010.jpg55170 formula (II-2) JPEG0007651785000011.jpg55170 formula (II-3) JPEG0007651785000012.jpg57170 formula (II-4) JPEG0007651785000013.jpg50170 formula (II-5) JPEG0007651785000014.jpg54170 formula (II-6) JPEG0007651785000015.jpg61170 formula (II-7) JPEG0007651785000016.jpg62170 formula (II-8) JPEG0007651785000017.jpg62170 formula (II-9) JPEG0007651785000018.jpg58170 formula (II-10) JPEG0007651785000019.jpg54170 formula (II-11) JPEG0007651785000020.jpg52170 formula (II-12) JPEG0007651785000021.jpg55170 formula (II-13) JPEG0007651785000022.jpg56170 formula (II-14) JPEG0007651785000023.jpg55170 formula (II-15) or JPEG0007651785000024.jpg52170 formula (II-16)

[0019] Based on the above, the present invention further provides a method for preparing a compound tEB-FAPI represented by formula (II-1), the method comprising: (1) amide condensation reaction of 6-hydroxy-4-quinoline carboxylic acid and glycine tert-butyl ester, followed by sequential reaction with 1-bromo-3-chloropropane and 1-tert-butoxycarbonylpiperazine, followed by removal of Boc and tert-butyl protecting groups under the action of TFA, introduction of Boc protection to amino group, followed by amide condensation reaction with (S)-pyrrolidine-2-carbonitrile hydrochloride, removal of Boc protection using p-toluenesulfonic acid, followed by condensation reaction with 5,8,11,14-tetraoxa-2-azaheptadecanedioic acid-1-tert-butyl ester, removal of Boc protection using p-toluenesulfonic acid again to obtain intermediate compound A; (2) introducing Boc protection at one end of 4,4'-diamino-3,3'-dimethylbiphenyl, reacting with 1-amino-8-naphthol-2,4-disulfonic acid monosodium salt to obtain truncated Evans Blue derivative, then removing Boc protection, carrying out amide condensation reaction with N-tert-butoxycarbonyl-L-glutamic acid-1-tert-butyl ester, then removing Boc and tert-butyl protecting groups under the action of TFA, and further reacting with di-tert-butyl dicarbonate to introduce Boc protection to the amino group to obtain intermediate compound B; (3) subjecting intermediate compound A and intermediate compound B to an amide condensation reaction, then removing the Boc protection using p-toluenesulfonic acid, and finally reacting with DOTA-NHS to obtain the compound represented by formula (II-1).

[0020] A preferred method for preparing the compound tEB-FAPI represented by formula (II-1) of the present invention specifically includes the following steps:

[0021] 6-Hydroxy-4-quinoline carboxylic acid (compound 1) and glycine tert-butyl ester are dissolved in N,N-dimethylformamide, and HATU is added to obtain compound 2. Compound 2 is dissolved in N,N-dimethylformamide, 1-bromo-3-chloropropane and potassium carbonate are added, and the reaction system is heated to 60°C for a certain period of time to obtain compound 3. Compound 3 is dissolved in N,N-dimethylformamide, and 1-tert-butoxycarbonylpiperazine and potassium iodide are added to react with compound 4. Compound 4 is dissolved in trifluoroacetic acid solution to remove the protecting group to obtain compound 5. Compound 5 is dissolved in N,N-dimethylformamide, and di-tert-butyl dicarbonate and an acid binder are added to obtain compound 6. Compound 6 is condensed with (S)-pyrrolidine-2-carbonitrile hydrochloride under the action of HATU and DIPEA to obtain compound 7. Compound 7 is deprotected under the action of p-toluenesulfonic acid to obtain compound 8. Compound 8 is condensed with 5,8,11,14-tetraoxa-2-azaheptadecanedioic acid-1-tert-butyl ester under the action of HATU and DIPEA to give compound 9. Compound 9 is deprotected under the action of p-toluenesulfonic acid to give compound 10 (i.e., intermediate compound A).

[0022] 4,4'-diamino-3,3'-dimethylbiphenyl (compound 11) is reacted with di-tert-butyl dicarbonate to obtain compound 12. Compound 12 is reacted with 1-amino-8-naphthol-2,4-disulfonic acid monosodium salt and sodium nitrite to obtain truncated Evans Blue derivative (compound 13). Boc protection of compound 13 is removed to obtain compound 14. Compound 14 is condensed with N-tert-butoxycarbonyl-L-glutamic acid-1-tert-butyl ester under the action of HATU and DIPEA to obtain compound 15. Compound 15 is dissolved in trifluoroacetic acid solution to remove the protecting group to obtain compound 16. Compound 16 is dissolved in N,N-dimethylformamide, and di-tert-butyl dicarbonate and an acid binding agent are added to obtain compound 17 (i.e., intermediate compound B).

[0023] Compound 17 and compound 10 are condensed under the action of HATU and DIPEA to give compound 18. Compound 18 is deprotected under the action of p-toluenesulfonic acid to give compound 19. Compound 19 is reacted with DOTA-NHS to give the final compound 20 represented by formula (II-1).

[0024] The specific synthetic routes for the above steps are as follows: [ka]

[0025] The preparation methods of other tEB-FAPI compounds in the present invention are similar to the preparation method of compound 20, and can basically be prepared based on existing conventional means with reference to the synthetic route of compound 20.

[0026] In another aspect, the present invention further provides a radiolabeled tEB-FAPI conjugate, which is obtained by labeling the compound of formula (I) according to the present invention with a radionuclide as a ligand. The radiolabeled conjugate can be used as a new type of tumor radiodiagnosis and therapy probe, i.e., as a radionuclide diagnostic probe or a radionuclide therapy probe. The radionuclide is preferably: 177 Lu, 90 Y, 18 F, 64 Cu, 68 Ga, 62 Cu, 67 Cu, 86 Y, 89 Zr, 99m Tc, 89 Sr., 153 Sm, 149 Tb, 161 Tb, 186 Re, 188 Re, 212 Pb, 213 Bi, 223 Ra, 225 Ac, 226 Th, 227 Th, 131 I, 211 At or111 In, preferably 68 Ga, 177 Lu or 90 It's Y.

[0027] The structure of a preferred conjugate of the present invention is as shown in formula (IV): [ka] Formula (IV) wherein L1 is a lysine or glutamic acid structure, or a derivative compound structure containing a lysine or glutamic acid structure; L2 is -(CH2) n -, n is an integer from 0 to 30, and each CH2 may be independently substituted or unsubstituted with -O-, -NH-, -(CO)-, -NH(CO)-, or -(CO)-NH-, with the substitution condition that two adjacent CH2 groups are unsubstituted; L3 is -(CH2) m -, m is an integer from 0 to 30, and each CH2 may be independently substituted or unsubstituted with -O- or -(CO)-, with the substitution condition that two adjacent CH2 groups are unsubstituted; X is selected from N, C, O, S or the following structures: [ka] R3 and R4 are the same or different and are each independently selected from H or F; M is 68 Ga, 177 Lu or 90 Y is a radionuclide selected from any one of

[0028] In a preferred embodiment of the conjugate of the present invention, L2 in formula (IV) is -(CH2) n-, n is an integer of 0 to 16, more preferably an integer of 0 to 12, and even more preferably 0, 3, or 10, where each -CH2- may be independently substituted or unsubstituted with -O-, -NH-, or -(CO)-, and the substitution condition is that two adjacent -CH2- groups are unsubstituted. More preferred L2 is -(CH2)0, -NH-CH2-(CO)-, -NH-CH2-(CHOCH2)2-CH2-(CO)-, -NH-CH2-(CHOCH2)4-CH2(CO)-, -(CO)-CH2-(CO)-, -(CO)-(CH2)2-(CO)-, -(CO)-CH2-(CHOCH2)2-CH2(CO)-, or -(CO)-CH2-(CHOCH2)4-CH2(CO)-.

[0029] In a preferred embodiment of the complex of the present invention, L3 in formula (IV) is -(CH2) m and m is an integer of 0 to 20, more preferably an integer of 1 to 6, and even more preferably 2 or 3, where each -CH2- may be independently substituted or unsubstituted with -O-, and the substitution condition is that two adjacent -CH2- groups are unsubstituted. More preferably, L3 is -(CH2)3-.

[0030] The radiolabeled complex of the present invention can be prepared from a compound containing a radionuclide and a compound of formula (I) according to the present invention according to various existing labeling methods, but the preferred labeling method of the present invention is the following wet method or freeze-drying method.

[0031] The wet labeling method includes the steps of dissolving an appropriate amount of the compound of formula (I) according to the present invention in a buffer solution or deionized water, adding a radionuclide solution to the obtained solution, and reacting the solution in a sealed state for 5 to 40 minutes to produce a radionuclide-labeled complex.

[0032] Alternatively, the freeze-drying labeling method includes the steps of dissolving an appropriate amount of the compound of formula (I) described in the present invention in a buffer solution or deionized water, sterile filtering the resulting solution, dispensing it into a container, freeze-drying it, and sealing it to obtain a freeze-dried kit, and adding an appropriate amount of acetic acid solution or buffer solution to the freeze-dried kit to dissolve it, adding a corresponding radioactive nuclide solution, and reacting it in a sealed state for 5 to 40 minutes to generate a radioactive nuclide-labeled complex. Here, the above-mentioned dispensing container is preferably a cryopreservation tube or an antibiotic tube vial. In addition, the kit can be optimized in its molding state by adding an excipient such as mannitol or ascorbic acid to the kit according to the molding state of the freeze-dried powder in the kit and adjusting the dosage of the compound of formula (I) described in the present invention and the excipient.

[0033] The products obtained by the above wet labeling method and freeze-dried labeling method can be further prepared into injection solutions by conventional processing (e.g., separation and purification by chromatography, rotary evaporation to remove the solvent, dissolving the residue in PBS or water or saline, sterile filtration, etc.).

[0034] In a preferred specific embodiment of the present invention, when compound 20 represented by formula (II-1) is used as a ligand, the preferred method for preparing radiolabeled compound 20 is a wet labeling method, which includes the steps of dissolving compound 20 in a buffer solution or deionized water, adding fresh radioactive solution, reacting at 37-90°C for 5-40 minutes in a sealed state, cooling, diluting the reaction solution with water, separating and purifying it on a Sep-Pak C18 chromatography column, washing the chromatography column with buffer solution or water to remove unreacted radioactive ions, rinsing with a hydrochloric acid ethanol solution or ethanol solution, diluting with saline or PBS, and sterile filtering to obtain an injection solution of a radiolabeled complex having the structure described in formula (IV-1), wherein the radionuclide M is 68 Ga, 177 Lu or 90 Y, etc. [ka] Formula (IV-1)

[0035] Another preferred method for preparing the radiolabeled compound 20 of the present invention is a freeze-drying labeling method, which includes the steps of dissolving compound 20 and other necessary reagents in a buffer solution, sterile filtering the resulting solution, dispensing it into cryopreservation tubes, freeze-drying and sealing to obtain a freeze-dried kit; adding an appropriate amount of buffer solution to the freeze-dried kit to dissolve it, then adding a freshly prepared radioactive solution, reacting it at 37-120°C for 5-40 minutes in a sealed state, and cooling it; adding water to dilute the reaction solution, separating and purifying it on a Sep-Pak C18 chromatography column, washing the chromatography column with buffer solution or water to remove unreacted radioactive ions, rinsing with a hydrochloric acid ethanol solution or an ethanol solution, diluting with physiological saline or PBS, and sterile filtering to obtain an injection solution of a radiolabeled complex having the structure described in formula (IV-1), wherein the radionuclide M is 68 Ga, 177 Lu or 90 Y, etc.

[0036] Other chemicals used in the above synthetic steps are commercially available.

[0037] The buffer is a substance for stabilizing the pH value of the reaction solution, and may be acetate, lactate, tartrate, malate, maleate, succinate, ascorbate, carbonate, phosphate, and mixtures thereof.

[0038] In another aspect, the present invention also provides the use of the tEB-FAPI compound of formula (I) or a pharma- ceutically acceptable salt thereof in radionuclide therapy or in the preparation of an imaging agent for tumors with high expression of FAP protein.

[0039] The present invention also provides the application of the radiolabeled tEB-FAPI conjugate represented by formula (IV) in radionuclide therapy or imaging for tumors with high expression of FAP protein.

[0040] In a preferred application of the present invention, the above complex is prepared as an injection and administered intravenously to a patient having a tumor with high expression of FAP protein.

[0041] In a preferred application of the present invention, tumors with high expression of FAP proteins include, but are not limited to, breast, ovarian, lung, colorectal, gastric or pancreatic cancer.

[0042] The present invention provides a truncated Evans Blue modified fibroblast activation protein inhibitor tEB-FAPI and its radionuclide-labeled complex, and also provides a method for preparing and labeling this type of compound. Biological testing shows that the inhibitor has the properties of significantly extending blood circulation half-life, and increasing tumor uptake, accumulation and retention time. This novelty is not found in other FAPI contrast agents at present, making it suitable for radionuclide therapy and imaging of tumors with high expression of FAP protein. [Brief description of the drawings]

[0043] [Figure 1] 1 is a mass spectrum of compound 2 in Example 1 of the present invention. [Diagram 2] 1 is a hydrogen nuclear magnetic resonance spectrum of compound 2 in Example 1 of the present invention. [Diagram 3] 1 is a carbon nuclear magnetic resonance spectrum of compound 2 in Example 1 of the present invention. [Figure 4] 1 is a mass spectrum of compound 3 in Example 1 of the present invention. [Diagram 5] 1 is a hydrogen nuclear magnetic resonance spectrum of compound 3 in Example 1 of the present invention. [Figure 6] 1 is a mass spectrum of compound 4 in Example 1 of the present invention. [Figure 7] 1 is a hydrogen nuclear magnetic resonance spectrum of compound 4 in Example 1 of the present invention. [Figure 8] 1 is a carbon nuclear magnetic resonance spectrum of compound 4 in Example 1 of the present invention. [Figure 9]1 is a mass spectrum of compound 7 in Example 1 of the present invention. [Figure 10] 1 is a hydrogen nuclear magnetic resonance spectrum of compound 7 in Example 1 of the present invention. [Figure 11] 1 is a carbon nuclear magnetic resonance spectrum of compound 7 in Example 1 of the present invention. [Figure 12] 1 is a mass spectrum of compound 10 in Example 1 of the present invention. [Figure 13] 1 is a mass spectrum of compound 20 in Example 1 of the present invention. [Figure 14] 1 is a mass spectrum of the compound in Example 10 of the present invention. [Figure 15] 1 is a mass spectrum of the compound in Example 11 of the present invention. [Figure 16] 1 is an HPLC chromatogram of Compound 10 in Example 1 of the present invention. [Figure 17] 1 is an HPLC chromatogram of compound 17 in Example 1 of the present invention. [Figure 18] 1 is an HPLC chromatogram of the reaction system of Compound 17 and Compound 10 in Example 1 of the present invention. [Figure 19] 1 is an HPLC chromatogram of compound 19 in Example 1 of the present invention. [Figure 20] 1 is an HPLC chromatogram of the reaction system of Compound 19 and DOTA-NHS in Example 1 of the present invention. [Figure 21A-21B] FIG. 1 shows MicroPET imaging of the 68Ga-labeled tEB-FAPI complex of the present invention and 68Ga-labeled FAPI-02 in a normal mouse. [Figure 22] FIG. 13 shows SPECT imaging of 177Lu-tEB-FAPI prepared in Example 40 of the present invention in normal mice at different time points. [Diagram 23] FIG. 13 shows SPECT imaging of 177Lu-tEB-FAPI prepared in Example 40 of the present invention at different time points in a human pancreatic cancer xenograft model mouse. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0044] The technical solutions of the present invention will be further illustrated and explained below through specific embodiments in conjunction with the accompanying drawings. Example 1: Preparation of tEB-FAPI linker (compound 20)

[0045] Synthesis of compound 2: Compound 1 (6-hydroxy-4-quinoline carboxylic acid, 1.89 g, 10.0 mmol), glycine tert-butyl ester (1.89 g, 10.0 mmol), HATU (3.8 g, 10.0 mmol) and N,N-diisopropylethylamine (2.6 g, 20.0 mmol) were added in sequence to 30 mL of N,N-dimethylformamide in a 100 mL flask. The reaction mixture was stirred overnight and the solvent was removed by distillation under reduced pressure to obtain the crude product. The product was purified on a silica gel column (dichloromethane / methanol=30:1) to obtain a white solid compound 2 in 87% yield. Figure 1 shows the mass spectrum of compound 2, Figure 2 shows the hydrogen nuclear magnetic resonance spectrum of compound 2, and Figure 3 shows the carbon nuclear magnetic resonance spectrum of compound 2.

[0046] Synthesis of compound 3: 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 added in order to 50 mL of N,N-dimethylformamide in a 100 mL flask. The temperature of the system was raised to 60°C and stirred overnight while maintaining it at 60°C. The solvent was removed by distillation under reduced pressure to obtain a crude product. The crude product was purified by a silica gel column (dichloromethane / methanol = 50:1) to obtain a white solid compound 3 in a yield of 63%. Figure 4 shows the mass spectrum of compound 3, and Figure 5 shows the hydrogen nuclear magnetic resonance spectrum of compound 3.

[0047] Synthesis of compound 4: Compound 3 (0.76 g, 2.0 mmol), 1-tert-butoxycarbonylpiperazine (0.55 g, 3.0 mmol), and potassium iodide (0.49 g, 3.0 mmol) were added in sequence to 30 mL of acetonitrile in a 100 mL flask. The temperature of the system was raised to 60°C and stirred overnight at 60°C. The solvent was removed by distillation under reduced pressure to obtain the crude product. The crude product was purified by a silica gel column (dichloromethane / methanol = 30:1) to obtain a white solid compound 4 in 58% yield. MS(ESI)m / z calculated for [C 28 H 40 N4O6]:528.29;found:529.10 [M+H] + FIG. 6 is a mass spectrum of compound 4, FIG. 7 is a hydrogen nuclear magnetic resonance spectrum of compound 4, and FIG. 8 is a carbon nuclear magnetic resonance spectrum of compound 4.

[0048] Synthesis of compound 5: Compound 4 (0.52 g, 1.0 mmol) was dissolved in 10 mL of a mixed solution of dichloromethane and trifluoroacetic acid (volume ratio 9:1) under ice bath conditions, the temperature of the system was raised to room temperature, and the reaction was carried out for 2 hours. After the reaction was completed, the solvent was removed by distillation under reduced pressure, and the compound was dissolved in 10 mL of N,N-dimethylformamide in preparation for the next reaction.

[0049] Synthesis of compound 6: To the N,N-dimethylformamide of compound 5, di-tert-butyl dicarbonate (0.22 g, 1.0 mmol) and N,N-diisopropylethylamine (0.39 g, 3.0 mmol) were added, stirred at room temperature overnight, and the solvent was removed by distillation under reduced pressure to obtain the crude product. The crude product was purified by silica gel column (dichloromethane / methanol = 10:1) to obtain white solid compound 6 in 72% yield.

[0050] Synthesis of compound 7: Compound 6 (0.47 g, 1.0 mmol), (S)-pyrrolidine-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 added in sequence to 10 mL of N,N-dimethylformamide in a 100 mL flask. The reaction mixture was stirred at room temperature until the reaction was completed, and the solvent was removed by distillation under reduced pressure to obtain a crude product. The product was purified by a silica gel column (dichloromethane / methanol=50:1) to obtain a white solid compound 7 in 85% yield. Figure 9 shows the mass spectrum of compound 7, Figure 10 shows the hydrogen nuclear magnetic resonance spectrum of compound 7, and Figure 11 shows the carbon nuclear magnetic resonance spectrum of compound 7.

[0051] Synthesis of compound 8: Compound 7 (0.55 g, 1.0 mmol) and p-toluenesulfonic acid monohydrate (0.27 g, 1.5 mmol) were added in turn to 10 mL of acetonitrile in a 100 mL flask. The temperature of the reaction system was raised to 60°C and stirred until the reaction was completed, and the solvent was removed by distillation under reduced pressure to obtain a crude product.

[0052] Synthesis of compound 9: 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 added to the reaction flask of compound 8, respectively. The reaction mixture was stirred overnight and the solvent was removed by distillation under reduced pressure to obtain the crude product. It was purified by silica gel column (dichloromethane / methanol=50:1) to obtain white solid compound 9 in 64% yield.

[0053] Synthesis of compound 10: Compound 9 (0.61 g, 1.0 mmol) and p-toluenesulfonic acid monohydrate (0.27 g, 1.5 mmol) were added in turn to 10 mL of acetonitrile in a 100 mL flask. The temperature of the reaction system was raised to 60°C and stirred until the reaction was completed. The solvent was removed by distillation under reduced pressure to obtain a crude product. The product was purified by a silica gel column (dichloromethane / methanol = 10:1) to obtain a white solid compound 10 in 59% yield. MS(ESI) m / z calculated for [C 35 H 51 N7O8]:697.38;found:698.43 [M+H] + Figure 12 shows the mass spectrum of compound 10.

[0054] The synthetic route for the above steps is as follows: [ka]

[0055] Synthesis of compound 12: 4,4'-Diamino-3,3'-dimethylbiphenyl (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 added to a 100 mL flask, and the mixture was stirred at room temperature overnight. The completion of the reaction was monitored by HPLC (rt was 10.13 min), and the solvent was removed by distillation under reduced pressure to obtain the crude product, which was purified by silica gel column (petroleum ether / ethyl acetate=5:1) to obtain white solid compound 12 in 59% yield.

[0056] Synthesis of compound 13: Compound 12 (0.31 g, 1.0 mmol) and 4 mL of acetonitrile were placed in a 50 mL flask, and 1.5 mL of 2 M hydrochloric acid was added dropwise to the reaction flask in an ice bath, and the mixture was allowed to react for 15 minutes. Sodium nitrite (0.068 g, 1.0 mmol) was added and dissolved in 2 mL of water, and the mixture was added dropwise to the reaction flask again and allowed to react for 30 minutes to obtain liquid A. Another 50 mL reaction flask was prepared, and 1-amino-8-naphthol-2,4-disulfonic acid monosodium salt (0.33 g, 1.0 mmol), sodium carbonate (0.105 g, 1.0 mmol) and 5 mL of water were added, and liquid A was slowly added dropwise to liquid B in an ice bath, and the mixture was allowed to react for 2 hours while stirring in an ice bath. The mixture was purified using a reverse phase column and freeze-dried to obtain pure compound 13 in 47% yield.

[0057] Synthesis of compound 14: Compound 13 (0.52 g, 1.0 mmol) was dissolved in trifluoroacetic acid under ice bath conditions, and the temperature of the system was raised to room temperature and reacted for 2 hours. After the reaction was completed, the solvent was removed by vacuum distillation to obtain a crude product. The crude product was purified by reverse phase column and lyophilized to obtain pure compound 14 in 73% yield.

[0058] Synthesis of compound 15: Compound 14 (0.54 g, 1.0 mmol), N-tert-butoxycarbonyl-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 added to a 100 mL flask. The reaction mixture was stirred until the reaction was complete, and the solvent was removed by distillation under reduced pressure to obtain the crude product. The crude product was purified by reverse phase column and lyophilized to obtain pure compound 15 in 52% yield.

[0059] Synthesis of compound 16: The tert-butyl ester and Boc protection were removed at room temperature using thioanisole:1,2-ethanedithiol:anisole:TFA (5:3:2:90). After the reaction was completed, the TFA was removed by a stream of argon and then dissolved in 10 mL of N,N-dimethylformamide for the next reaction.

[0060] Synthesis of compound 17: To the N,N-dimethylformamide of compound 16, di-tert-butyl dicarbonate (0.22 g, 1.0 mmol) and N,N-diisopropylethylamine (0.39 g, 3.0 mmol) were added, respectively, and stirred at room temperature overnight. The completion of the reaction was monitored by HPLC (rt was 10.84 min). The solvent was removed by vacuum distillation to obtain the crude product. The crude product was purified by reverse phase column and lyophilized to obtain pure compound 17 in 43% two-step yield.

[0061] Synthesis of compound 18: 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 added to a 50 mL flask. The reaction mixture was stirred and reacted, and the completion of the reaction was monitored by HPLC (rt was 12.16 min). The solvent was removed by vacuum distillation to obtain the crude product. The crude product was purified by reverse phase column and lyophilized to obtain pure compound 18 in 55% yield.

[0062] Synthesis of compound 19: Compound 15 (0.13 g, 0.1 mmol) and p-toluenesulfonic acid monohydrate (0.05 g, 0.3 mmol) were added in turn to 5 mL of acetonitrile in a 25 mL flask. The temperature of the reaction system was raised to 60°C, and the reaction was stirred and the deprotection process was monitored by HPLC until the reaction was completed (rt was 10.47 min), and the solvent was removed by vacuum distillation to obtain the crude product. The crude product was purified by reverse phase column and lyophilized to obtain pure compound 19 in 61% yield.

[0063] Synthesis of compound 20: 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 added in sequence to 5 mL of N,N-dimethylformamide in a 25 mL flask. The reaction was stirred at room temperature and monitored by HPLC until completion (rt was 11.35 min), and the solvent was removed by vacuum distillation to obtain the crude product. The crude product was purified by reverse phase column and lyophilized to obtain pure compound 20 in 53% yield. MS(ESI)m / z calculated for [C 80 H 104 N 16 O 24 S2]:1736.69;found:1737.743 [M+H] + Figure 13 shows the mass spectrum of compound 20.

[0064] The synthetic route for the above steps is as follows: [ka] Examples 2 to 16

[0065] The structures of the compounds of Examples 2 to 16 are as shown in Formula (II-2) to Formula (II-16), respectively, and the preparation methods thereof can all be referred to in Example 1. The glutamic acid structure reacted with Compound 14 was replaced with a lysine structure, or the 5,8,11,14-tetraoxa-2-azaheptadecanedioic acid-1-tert-butyl ester reacted with Compound 8 was replaced with 5,8,11-trioxa-2-azatridecanedioic acid-1-tert-butyl ester, 9-amino-4,7-dioxanoic acid tert-butyl ester, glycine tert-butyl ester or other suitable compounds, or the (S)-pyrrolidine-2-carbonitrile hydrochloride reacted with Compound 6 was replaced with 3,3-difluoropyrrolidine hydrochloride, or replaced simultaneously, to obtain the corresponding structures below. [ka] Formula (II-2) JPEG0007651785000032.jpg51170 formula (II-3) JPEG0007651785000033.jpg55170 formula (II-4) JPEG0007651785000034.jpg48170 formula (II-5) JPEG0007651785000035.jpg49170 formula (II-6) JPEG0007651785000036.jpg53170 formula (II-7) JPEG0007651785000037.jpg53170 formula (II-8) JPEG0007651785000038.jpg51170 formula (II-9) JPEG0007651785000039.jpg47170 formula (II-10) JPEG0007651785000040.jpg54170 formula (II-11) JPEG0007651785000041.jpg45170 formula (II-12) JPEG0007651785000042.jpg55170 formula (II-13) JPEG0007651785000043.jpg53170 formula (II-14) JPEG0007651785000044.jpg48170 formula (II-15) or JPEG0007651785000045.jpg49170 formula (II-16)

[0066] Here, the mass spectrum of the compound (II-10) of Example 10 is shown in FIG. 14, and the mass spectrum of the compound (II-11) of Example 11 is shown in FIG. Examples 17 to 38:

[0067] With reference to the preparation methods of Examples 1 to 16, a tEB-FAPI compound represented by the following formula (I) was prepared. [ka] Formula (I) [Table 1] JPEG0007651785000048.jpg210170 Example 39: Preparation of radioactive Ga-68 labeled tEB-FAPI complex

[0068] Wet method: In a centrifuge tube containing 0.5 mL of the acetic acid-acetate solution (1.0 g / L) of compound 20 prepared in Example 1, about 18.5 to 1850 megabecquerels (MBq) of 68 GaCl3 hydrochloric acid solution (eluted from a germanium gallium generator) was added and reacted at 37°C for 20 minutes. The C18 separation cartridge was taken and rinsed slowly with 10 mL of absolute ethanol first, then with 10 mL of water. The labeling solution was diluted with 10 mL of water, and then loaded onto the separation column, and the unlabeled eluate was first rinsed with 10 mL of water. 68 Ga ions were removed, and then the plate was rinsed with 0.3 mL of 10 mM HCl in ethanol. 68 The eluate was diluted with saline and sterile filtered to obtain Ga-labeled tEB-FAPI complex. 68 Injection of the Ga-labeled tEB-FAPI complex was obtained.

[0069] Freeze-drying method: A freeze-drying kit containing compound 20 was prepared containing approximately 18.5 to 1850 megabecquerels (MBq). 68 GaCl3 hydrochloric acid solution (eluted from a germanium gallium generator) was added, mixed uniformly, and reacted at 37 °C for 20 min. The C18 separation cartridge was taken and rinsed slowly with 10 mL of absolute ethanol first, then with 10 mL of water. The labeling solution was diluted with 10 mL of water, and then loaded onto the separation column, and the unlabeled solution was first washed with 10 mL of water. 68 The Ga ions were removed, and then the complex eluate was obtained by rinsing with 0.3 mL of 10 mM HCl ethanol solution. The eluate was diluted with saline and sterile filtered to obtain the complex eluate. 68 Injection of the Ga-labeled tEB-FAPI complex was obtained.

[0070] Example 40: Preparation of Lu-177 labeled tEB-FAPI complex

[0071] Wet method: Approximately 18.5 to 1850 MBq 177 The LuCl3 sodium acetate solution was added to three centrifuge tubes containing 0.5 mL of acetic acid-acetate solutions (1.0 g / L) of compound 20 of Example 1, compound 2 (compound of formula II-2), and compound 3 (compound of formula II-3), respectively, and reacted at 90°C for 20 minutes. The C18 separation cartridge was taken and first rinsed slowly with 10 mL of absolute ethanol, then rinsed with 10 mL of water. The labeling solution was diluted with 10 mL of water, and then loaded onto the separation column, and unlabeled was first rinsed with 10 mL of water. 177 Lu ions were removed, and then the three were rinsed with 0.3 mL of 10 mM HCl in ethanol. 177 The Lu-labeled tEB-FAPI complex was obtained. The eluate was diluted with saline and sterile filtered to give three fractions. 177 An injection of Lu-labeled tEB-FAPI complex was obtained.

[0072] Freeze-drying method: Approximately 18.5 to 1850 MBq 177 The LuCl3 sodium acetate solution was added to three freeze-dried kits containing compound 20 of Example 1, compound 2 of Example 2 (compound of formula II-2) and compound 3 of Example 3 (compound of formula II-3), mixed uniformly and reacted at 90°C for 20 minutes. The C18 separation cartridge was taken out and rinsed slowly with 10 mL of absolute ethanol first, then with 10 mL of water. The labeling solution was diluted with 10 mL of water, and then loaded onto the separation column, and unlabeled was first washed with 10 mL of water. 177 Lu ions were removed, and then the three were rinsed with 0.3 mL of 10 mM HCl in ethanol. 177 The eluate was diluted with saline and sterile filtered to obtain three fractions. 177 An injection of Lu-labeled tEB-FAPI complex was obtained.

[0073] Example: Analysis and application effects

[0074] 1. HPLC analysis and identification

[0075] HPLC system: SHIMADZULC-20A, analytical C18 chromatography column (YMC, 3 μm, 4.6 × 150 mm). Detection wavelength 254 nm, flow rate 1 mL / min, elution gradient: 0-3 min: 10% acetonitrile and 90% water (50 mM ammonium acetate) maintained. 3-16 min: increased to 90% acetonitrile and 10% water (50 mM ammonium acetate). 16-18 min: 90% acetonitrile and 10% water (50 mM ammonium acetate) maintained. 18-20 min: reduced to 10% acetonitrile and 90% water (50 mM ammonium acetate). 20-22 min: 10% acetonitrile and 90% water (50 mM ammonium acetate) maintained.

[0076] Compound 10, compound 17, the reaction system of compound 10 and compound 17, compound 19, and the reaction system of compound 19 and DOTA-NHS in Example 1 were identified and analyzed according to the above system, and the results obtained are shown in Figures 16 to 20.

[0077] Hereinafter, the two radioactively labeled probes prepared in Example 39 and Example 40 are used as experimental agents, and their performance measurements are described as follows.

[0078] 2. In normal mice 68 MicroPET imaging of Ga-labeled tEB-FAPI complex

[0079] Purity greater than 95% according to the method of Example 39 68 Ga-tEB-FAPI was prepared and administered to normal FVB mice at 3.7 MBq. 68 Ga-tEB-FAPI or 68 Ga-FAPI-02 (as a control) was injected via the tail vein, and then MicroPET imaging was performed under isoflurane anesthesia at 0 to 120 minutes after administration. The results are shown in Figures 21A and 21B. The results are consistent with those of the complex of Example 39. 68 Whereas Ga-tEB-FAPI showed high uptake in the cardiac blood pool in mice (Figure 21A). 68The results showed that Ga-FAPI-02 was almost completely eliminated within the test time (FIG. 21B), indicating that the introduction of cleaved Evans Blue can significantly extend the blood circulation half-life.

[0080] 3. Human pancreatic cancer xenograft model in mice 177 Tumor uptake experiment of Lu-labeled tEB-FAPI complex

[0081] Purity greater than 95% according to the method of Example 40 177 Prepare Lu-tEB-FAPI, 1.3 MBq 177 Lu-tEB-FAPI was injected into normal mice and human pancreatic cancer xenograft model mice via the tail vein. SPECT imaging was performed at different time points after injection, and the results are shown in Figures 22 and 23. 177 We demonstrated that Lu-tEB-FAPI had good pharmacokinetics in normal mice and could be continuously taken up into tumor tissues and maintained for more than 48 h in human pancreatic cancer xenograft model mice, indicating that tEB-FAPI has significantly increased tumor uptake and retention time and can be used as a tumor therapeutic and imaging agent.

[0082] In summary, the truncated Evans Blue modified fibroblast activation protein inhibitor provided by the present invention can significantly extend its blood circulation half-life, and increase its uptake, accumulation and retention time in tumors, which is a novelty not found in other FAP contrast agents at present. Further preclinical animal and clinical studies have confirmed that this inhibitor is expected to be applied to radionuclide therapy and imaging of tumors with high expression of FAP protein.

[0083] Although the present invention has been described in detail above through general description, specific embodiments and tests, it is obvious to those skilled in the art that some modifications or improvements can be made based on the present invention. Therefore, all such modifications or improvements made within the scope of the present invention without departing from the spirit of the present invention belong to the protection scope of the present invention.

Claims

1. A truncated Evans Blue modified fibroblast activation protein inhibitor compound or a pharma- ceutically acceptable salt thereof, the molecular structure of which is formed by a linking group linking together a truncated Evans Blue, a fibroblast activation protein inhibitor and a nuclide chelating group, the structure of which is as shown in formula (II) below: Formula (II) Here, R ３ and R ４ are both H or F, and L １ is a glutamic acid or lysine structure, L １ is a glutamic acid structure, L ２ Ha-(CH ２ ) ０ --NH-CH ２ -(CO)-, -NH-CH ２ - (CH ２ OCH ２ ) ２ -CH ２ -(CO)-, -NH-CH ２ - (CH ２ OCH ２ ) ４ -CH ２ (CO)-, When L 1 is a lysine structure, L 2 is —(CO)—CH ２ -(CO)-, -(CO)-(CH ２ ) ２ -(CO)-, -(CO)-CH ２ - (CH ２ OCH ２ ) ２ -CH ２ (CO)- or -(CO)-CH ２ - (CH ２ OCH ２ ) ４ -CH ２ A compound characterized in that it is (CO)-.

2. The compound according to claim 1, characterized in that the structure of the compound is any one of the following formulas (II-1) to (II-16). Formula (II-1) Formula (II-2) Formula (II-3) Formula (II-4) Formula (II-5) Formula (II-6) Formula (II-7) Formula (II-8) Formula (II-9) Formula (II-10) Formula (II-11) Formula (II-12) Formula (II-13) Formula (II-14) Formula (II-15) or Formula (II-16)

3. A method for preparing a truncated Evans Blue-modified fibroblast activation protein inhibitor, comprising: (1) amide condensation reaction of 6-hydroxy-4-quinoline carboxylic acid and glycine tert-butyl ester, followed by sequential reaction with 1-bromo-3-chloropropane and 1-tert-butoxycarbonylpiperazine, followed by removal of Boc and tert-butyl protecting groups under the action of TFA, introduction of Boc protection to amino group, followed by amide condensation reaction with (S)-pyrrolidine-2-carbonitrile hydrochloride, removal of Boc protection using p-toluenesulfonic acid, further condensation reaction with 5,8,11,14-tetraoxa-2-azaheptadecanedioic acid-1-tert-butyl ester, and removal of Boc protection using p-toluenesulfonic acid again to obtain intermediate compound A; (2) introducing Boc protection to one end of 4,4'-diamino-3,3'-dimethylbiphenyl, reacting with 1-amino-8-naphthol-2,4-disulfonic acid monosodium salt to obtain a truncated Evans Blue derivative, then removing Boc protection, carrying out amide condensation reaction with N-tert-butoxycarbonyl-L-glutamic acid-1-tert-butyl ester, then removing Boc and tert-butyl protecting groups under the action of TFA, and further reacting with di-tert-butyl dicarbonate to introduce Boc protection to the amino group to obtain intermediate compound B; (3) subjecting the intermediate compound A obtained in (1) and the intermediate compound B obtained in (2) to an amide condensation reaction, then removing the Boc protection using p-toluenesulfonic acid, and finally reacting with DOTA-NHS to obtain a truncated Evans Blue-modified fibroblast activation protein inhibitor compound having the structure shown in the following formula (II-1). Formula (II-1)

4. A radiolabeled truncated Evans Blue modified fibroblast activation protein inhibitor conjugate, which is obtained by labeling the compound of formula (II) according to claim 1 as a ligand with a radionuclide, said radionuclide being １７７ Lu, ９０ Y. １８ F. ６４ Cu, ６８ G.A. ６２ Cu, ６７ Cu, ８６ Y. ８９ Zr, ９９ｍ Tc, ８９ Sr, １５３ Sm, １４９ Tb, １６１ Tb, １８６ Re, １８８ Re, ２１２ Pb, ２１３ Bi, ２２３ Ra, ２２５ A c, ２２６ Th, ２２７ Th, １３１ I, ２１１ At or １１１ In, and the radionuclide is ６８ G.A. １７７ Lu or ９０ Y, a complex.

5. A method for preparing a radiolabeled truncated Evans Blue modified fibroblast activation protein inhibitor conjugate, comprising the steps of dissolving the compound of formula (II) according to claim 1 in a buffer solution or deionized water, and adding a radionuclide solution to the resulting solution and reacting for 5-40 minutes under sealed conditions to produce a radionuclide labeled conjugate.

6. A method for preparing a radiolabeled truncated Evans Blue-modified fibroblast activation protein inhibitor complex, comprising the steps of: dissolving the compound of formula (II) according to claim 1 in a buffer solution or deionized water; sterile filtering the resulting solution, dispensing it into containers, freeze-drying and sealing it to obtain a freeze-dried kit; and adding an appropriate amount of acetic acid solution or buffer solution to the freeze-dried kit to dissolve it, adding a corresponding radionuclide solution, and reacting it in a sealed state for 5 to 40 minutes to produce a radionuclide-labeled complex.

7. The compound according to any one of claims 1 to 2, or a pharma- ceutically acceptable salt thereof, in the preparation of a radionuclide therapy or imaging agent for tumors with high expression of the FAP protein.

8. The conjugate according to claim 4 in radionuclide therapy or imaging for tumors with high expression of FAP protein.

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