CN114369084A - Truncated Evans blue modified fibroblast activation protein inhibitor and preparation method and application thereof - Google Patents

Truncated Evans blue modified fibroblast activation protein inhibitor and preparation method and application thereof Download PDF

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CN114369084A
CN114369084A CN202110182478.0A CN202110182478A CN114369084A CN 114369084 A CN114369084 A CN 114369084A CN 202110182478 A CN202110182478 A CN 202110182478A CN 114369084 A CN114369084 A CN 114369084A
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Abstract

The invention provides a truncated Evans blue modified fibroblast activation protein inhibitor compound, which is composed of a connecting group L1、L2、L3、L4And X is formed by connecting the truncated Evans blue, the fibroblast activation protein inhibitor and the nuclide chelating group togetherThe structure is shown as formula (I); r1Is a fibroblast activation protein inhibitor; l is1Is lysine, glutamic acid or derivative structure thereof; l is2Is- (CH)2)n-, where n is an integer from 0 to 30, where each CH2May be individually replaced with-O-, -NH (CO) -or- (CO) -NH-; l is3Is- (CH)2)m-, where m is an integer of 0 to 30, where each CH2Can be replaced by-O-, or- (CO) -alone; l is4Is- (CH)2)p-, where p is an integer from 0 to 30, where each CH2May be individually replaced with-O-, -NH (CO) -or- (CO) -NH-; x is selected from N, C, O, S or
Figure DDA0002942507470000011
Figure DDA0002942507470000012
R2Is a nuclide chelating group. The invention also provides a radioactive marker based on the structure of the compound, and the compound and the radioactive marker have the advantages of obviously prolonged blood circulation half-life period, enhanced tumor uptake enrichment and retention time and are suitable for nuclide treatment and imaging of FAP protein high-expression tumors.

Description

Truncated Evans blue modified fibroblast activation protein inhibitor and preparation method and application thereof
Technical Field
The invention relates to the field of nuclear medicine and molecular imaging, in particular to a truncated Evans blue modified fibroblast activation protein inhibitor, a preparation marker and application thereof.
Background
Fibroblast Activation Protein (FAP) is a membrane serine peptidase, which is expressed on the surface of Fibroblast activated by tumor stroma and plays an important role in the process of generating and developing tumors. Previous studies show that FAP is not expressed in normal human tissues generally, but is selectively expressed on the surface of stromal fibroblasts of more than 90% of epithelial malignant tumors, including breast cancer, ovarian cancer, lung cancer, colorectal cancer, gastric cancer, pancreatic cancer and the like. In view of its wide expression and important role in tumors, FAP has become an important target for tumor imaging and therapy.
At present, radionuclide-labeled inhibitors of Fibroblast Activation Protein (FAPI) represented by quinolinic acid derivatives have made important progress in the field of precise imaging of tumors. For example, more than 30 different types of tumor-specific imaging have been achieved with PET/CT imaging agents such as FAPI-02 and FAPI-04. Compared with FDG imaging, FAPI imaging has lower background in brain, liver and oropharynx mucosa and higher detection rate for tumor focus. FAPI, as currently reported, is rapidly cleared in the blood circulation and rapidly eluted at the tumor site. This metabolic property is beneficial for imaging because it provides a cleaner background. But is disadvantageous for treatment because rapid metabolism and elution result in a lower effective dose at the tumor site, too short a retention time, the need to use higher doses or more frequent administration to meet the therapeutic needs, and increased likelihood of adverse reactions.
In the case of FAPI-02, which is completely cleared from the blood circulation within one hour, the retained dose at the tumor site decreases by about 75% after 24 h. Although recent research efforts have optimized the non-pharmacophore moiety in the FAPI structure, improvements in tumor uptake dose and retention time for FAPI are limited and do not meet the needs of therapeutic use. It is known to those skilled in the art that if the small molecule drug circulates in the blood vessel too short or is rapidly cleared by the body, insufficient binding of the drug to the target will result. Therefore, in preparing the FAPI probe, if the circulation half-life of the probe can be properly prolonged, the uptake dose and the retention time of the probe at a target site can be possibly improved.
Therefore, new strategies are needed to extend the circulating half-life of FAPI probes with appropriate metabolic kinetics, higher tumor uptake doses and longer tumor retention times, meeting the nuclide therapy and imaging needs.
Disclosure of Invention
Based on the background, the primary object of the present invention is to develop a short-cut evans blue (tEB) and Fibroblast Activation Protein Inhibitor (FAPI) conjugate, which is characterized in that the short-cut evans blue is effectively combined with serum albumin to realize that albumin is used as a FAPI delivery vector, so as to prolong the half-life period of the short-cut evans blue in peripheral blood and improve the uptake, enrichment and retention time of the short-cut evans blue in tumors. The tEB-FAPI connector developed by the invention can overcome the defects of over-rapid metabolism of small molecular FAPI and over-short retention time of target organs, improves the therapeutic and imaging effects of targeted FAP protein nuclide, and has the potential of clinical popularization and application.
It is another object of the present invention to provide a class of radiolabeled truncated evans blue-modified fibroblast activation protein inhibitors (tEB-FAPI) having a long circulating half-life;
the invention also aims to provide a preparation method of the radiolabeled tEB-FAPI complex;
the invention further aims to provide application of the complex in targeted FAP protein tumor nuclide imaging and treatment.
The technical scheme for realizing the above primary object of the invention has the following aspects of ligand synthesis and radioactive labeling.
In a first aspect, the present invention provides a truncated form of evans blue (tEB) -modified Fibroblast Activation Protein Inhibitor (FAPI), wherein the structure of said compound is represented by the following formula (I), and is denoted as "tEB-FAPI";
Figure BDA0002942507450000021
wherein:
L1is lysine, glutamic acid or derivative compounds containing lysine or glutamic acid structures;
L2is- (CH)2)n-, where n is an integer of 0 to 30 (preferably an integer of 0 to 12), where each CH2Can be replaced by-O-, -NH (CO) -or- (CO) -NH-alone, provided that there are no two adjacent CH' s2The group is replaced;
L3is- (CH)2)m-, where m is an integer of 0 to 30 (preferably an integer of 1 to 6), where each CH2Can be replaced by-O-, or- (CO) -alone, provided that there are no two adjacent CH' s2The group is replaced;
L4is- (CH)2)p-, where p is an integer of 0 to 30 (preferably an integer of 0 to 12), where each CH2Can be replaced by-O-, -NH (CO) -or- (CO) -NH-alone, provided that there are no two adjacent CH' s2The group is replaced;
x is selected from N, C, O, S or the following structure
Figure BDA0002942507450000022
R1Is a fibroblast activation protein inhibitor or a polypeptide ligand thereof, selected from
Figure BDA0002942507450000031
R2Is a nuclide chelating group selected from
Figure BDA0002942507450000032
Or a pharmaceutically acceptable salt thereof.
In the scheme of the invention, when R in the formula (I)1Is composed of
Figure BDA0002942507450000033
When said L is1Preferably a glutamic acid structure, X is preferably
Figure BDA0002942507450000034
L3Is preferably- (CH)2)3-，L4Is preferably- (CH)2)0-，R3Preferably DOTA, i.e., a preferred compound of the invention, tEB-FAPI, has the structure shown in formula II below:
Figure BDA0002942507450000035
wherein R is3And R4Both are H or both are F atoms, L2Is- (CH)2)0、-NH-CH2-(CO)-、-NH-CH2-(OCH2CH2)-O-CH2(CO) -or-NH-CH2-(OCH2CH2)3-O-CH2(CO)-。
In a further preferred embodiment of the present invention, the structure of the compound, tEB-FAPI, is any one of the following formulas (II-1) to (II-8):
Figure BDA0002942507450000041
Figure BDA0002942507450000051
or
Figure BDA0002942507450000052
In the scheme of the invention, when R in the formula (I)1Is composed of
Figure BDA0002942507450000053
When said L is1Preferably a glutamic acid structure, X is preferably S, R2Preferably DOTA, i.e., a preferred compound of the invention, tEB-FAPI, has the structure shown in formula (III) below:
Figure BDA0002942507450000061
L2is- (CH)2)0、-NH-CH2-(CO)-、-NH-CH2-(OCH2CH2)-O-CH2(CO) -or-NH-CH2-(OCH2CH2)3-O-CH2(CO)-。
In a further preferred embodiment of the present invention, the structure of the compound, tEB-FAPI, is any one of the following formulas (III-1) to (III-4):
Figure BDA0002942507450000062
Figure BDA0002942507450000071
on the basis, the invention further provides a method for preparing the compound tEB-FAPI shown as the formula (II-1), which comprises the following steps:
carrying out amide condensation reaction on 7-hydroxy-4-quinoline carboxylic acid and glycine tert-butyl ester; then sequentially reacting with 1-bromo-3-chloropropane and 1-tert-butyloxycarbonyl piperazine; then removing Boc and tert-butyl protecting groups under the action of TFA; introducing Boc protection on amino; then carrying out amide condensation reaction with (S) -pyrrolidine-2-carbonitrile hydrochloride; removing Boc protection by utilizing p-toluenesulfonic acid; then carrying out condensation reaction with 5,8,11, 14-tetraoxa-2-aza heptadeca diacid-1-tert-butyl ester; removing Boc protection under the action of p-toluenesulfonic acid again to obtain an intermediate compound A;
secondly, introducing single side of 4,4 '-diamino-3, 3' -dimethyl biphenyl into Boc for protection, and then reacting with 4, 6-diamino-5-hydroxy-1, 3-naphthalenedisulfonic acid to prepare a truncated Evans blue derivative; removing Boc protection, and then carrying out amide condensation reaction with N-tert-butyloxycarbonyl-L-glutamic acid-1-tert-butyl ester; then removing Boc and tert-butyl protecting groups under the action of TFA; then reacting with di-tert-butyl dicarbonate, and introducing Boc protection on amino to obtain an intermediate compound B;
thirdly, the intermediate compound A and the intermediate compound B are subjected to amide condensation reaction; then, removing Boc protection by utilizing p-toluenesulfonic acid; finally reacting with DOTA-NHS to obtain the compound shown in the formula (III).
The invention discloses a preferable method for preparing a compound tEB-FAPI shown as a formula (II-1), which comprises the following steps:
dissolving 7-hydroxy-4-quinolinecarboxylic acid (compound 1) and tert-butyl glycinate in N, N-dimethylformamide, and adding HATU to obtain compound 2; dissolving the compound 2 in N, N-dimethylformamide, adding 1-bromo-3-chloropropane and potassium carbonate, and heating the reaction system to 60 ℃ for a certain time to obtain a compound 3; dissolving the compound 3 in N, N-dimethylformamide, adding 1-tert-butyloxycarbonyl piperazine and potassium iodide, and reacting to obtain a compound 4; dissolving the compound 4 in a trifluoroacetic acid solution to remove a protecting group, thereby obtaining a compound 5; dissolving the compound 5 in N, N-dimethylformamide, and adding di-tert-butyl dicarbonate and an acid-binding agent to obtain a compound 6; carrying out condensation reaction on the compound 6 and (S) -pyrrolidine-2-carbonitrile hydrochloride under the action of HATU and DIPEA to obtain a compound 7; removing protection of the compound 7 under the action of p-toluenesulfonic acid to obtain a compound 8; carrying out condensation reaction on the compound 8 and 5,8,11, 14-tetraoxa-2-aza-heptadecadioic acid-1-tert-butyl ester under the action of HATU and DIPEA to obtain a compound 9; removing protection from the compound 9 under the action of p-toluenesulfonic acid to obtain a compound 10 (namely the intermediate compound A);
reacting 4,4 '-diamino-3, 3' -dimethylbiphenyl (compound 11) with di-tert-butyl dicarbonate to obtain a compound 12; reacting the compound 12 with 4, 6-diamino-5-hydroxy-1, 3-naphthalene disulfonic acid and sodium nitrite to prepare a truncated Evans blue derivative (compound 13); removing Boc protection from the compound 13 to obtain a compound 14; carrying out condensation reaction on the compound 14 and N-tert-butyloxycarbonyl-L-glutamic acid-1-tert-butyl ester under the action of HATU and DIPEA to obtain a compound 15; dissolving the compound 15 in a trifluoroacetic acid solution to remove a protecting group, thereby obtaining a compound 16; dissolving a compound 16 in N, N-dimethylformamide, and adding di-tert-butyl dicarbonate and an acid-binding agent to obtain a compound 17 (namely the intermediate compound B);
carrying out condensation reaction on the compound 17 and the compound 10 under the action of HATU and DIPEA to obtain a compound 18; removing protection of the compound 18 under the action of p-toluenesulfonic acid to obtain a compound 19; reacting the compound 19 with DOTA-NHS to obtain a final compound 20 shown as a formula (II-1);
the synthetic route of the specific steps is as follows:
Figure BDA0002942507450000091
the other tieb-FAPI compounds of the scheme of the present invention may be prepared in a manner similar to that of compound 20, and may be prepared substantially by reference to the synthetic route for compound 20, based on existing conventional means.
In another aspect, the invention further provides a radiolabelled tEB-FAPI complex, which is obtained by labelling a radionuclide with a compound of formula (I) as described in the invention as a ligandAnd (3) a complex. The radioactive labeling complex can be used as a novel tumor radioactive diagnosis and treatment probe, namely a radionuclide diagnostic probe or a radionuclide therapeutic probe. The nuclide can be selected177Lu、90Y、18F、64Cu、68Ga、62Cu、67Cu、86Y、89Zr、99mTc、89Sr，153Sm、149Tb、161Tb、186Re、188Re、212Pb、213Bi、223Ra、225Ac、226Th、227Th、131I、211At or111Any one of In; preference is given to68Ga、177Lu or90Y。
The structure of the complex which is preferred in the invention is shown as the following formula (IV):
Figure BDA0002942507450000101
wherein L is2Is- (CH)2)n-, where n is an integer of 0 to 30 (preferably an integer of 0 to 12), where each CH2Can be replaced by-O-, -NH (CO) -or- (CO) -NH-alone, provided that there are no two adjacent CH' s2The group is replaced; l is3Is- (CH)2)m-, where m is an integer of 0 to 30 (preferably an integer of 1 to 6), where each CH2Can be replaced by-O-, or- (CO) -alone, provided that there are no two adjacent CH' s2The group is replaced; x is selected from N, C, O, S or the following structure:
Figure BDA0002942507450000102
R1a fibroblast activation protein inhibitor structure selected from any one of:
Figure BDA0002942507450000103
m is a radionuclide selected from68Ga、177Lu or90Y。
The radioactive labeling complex can be prepared by a compound containing radioactive nuclide and the compound of the formula (I) according to the prior various labeling methods; the preferred labeling method of the present invention is the following wet or freeze-drying method:
a wet marking scheme comprising: dissolving a proper amount of the compound shown as the formula (I) in a buffer solution or deionized water; adding a radionuclide solution into the obtained solution, and carrying out closed reaction for 5-40min to generate a radionuclide-labeled complex;
alternatively, a lyophilization labeling protocol comprising: dissolving a proper amount of the compound shown as the formula (I) in a buffer solution or deionized water; the obtained solution is aseptically filtered, subpackaged in containers, freeze-dried, plugged and sealed to obtain a freeze-dried medicine box; adding a proper amount of acetic acid solution or buffer solution into the freeze-dried medicine box for dissolving, then adding corresponding radionuclide solution, and carrying out closed reaction for 5-40min to generate the radionuclide-labeled complex. Wherein, the container for split charging is preferably a freezing storage tube or a tube-type antibiotic bottle. Excipients, such as mannitol, ascorbic acid and the like, can be added into the medicine box according to the forming condition of the freeze-dried powder of the medicine box, and the forming of the medicine box is optimized by adjusting the dosage of the compound shown as the formula (I) and the excipients.
The products obtained by the wet labeling scheme and the freeze-drying labeling scheme can be further prepared into injection by conventional treatment (such as chromatographic separation and purification, solvent removal by rotary evaporation, residue dissolution with PBS or water or physiological saline, sterile filtration and the like).
In a preferred embodiment of the present invention, the compound 20 represented by the formula (II-1) is used as a ligand, and the preferred method for preparing the radiolabeled compound 20 is a wet labeling method comprising the steps of: dissolving compound 20 in a buffer solution or deionized water; adding fresh radioactive solution, sealing at 37-90 deg.C for 5-40min, and cooling; adding water to diluteSeparating and purifying the reaction solution by a Sep-Pak C18 chromatographic column, washing the chromatographic column with buffer solution or water to remove unreacted radioactive ions, leaching with hydrochloric acid ethanol solution or ethanol solution, diluting with normal saline or PBS, and performing sterile filtration to obtain the injection of the radioactive labeled complex with the structure as shown in the formula (IV-1); wherein the radionuclide M is68Ga、177Lu or90Y, and the like.
Figure BDA0002942507450000111
Another preferred method of preparing the radiolabeled compound 20 of the invention is by lyophilization labeling, which comprises: dissolving the compound 20 and other necessary reagents in a buffer solution, performing sterile filtration on the obtained solution, subpackaging the sterile filtered solution in a freezing storage tube, and performing freeze drying and sealing to obtain a freeze-dried medicine box; adding appropriate amount of buffer solution into the lyophilized kit, dissolving, adding fresh radioactive solution, sealing, reacting at 37-120 deg.C for 5-40min, and cooling; diluting the reaction solution with water, separating and purifying by a Sep-Pak C18 chromatographic column, washing the chromatographic column with buffer solution or water to remove unreacted radioactive ions, leaching with hydrochloric acid ethanol solution or ethanol solution, diluting with normal saline or PBS, and performing sterile filtration to obtain the injection of the radioactive labeled complex with the structure shown as the formula (VI-1); wherein the radionuclide M is68Ga、177Lu or90Y, and the like.
Other chemicals used in the above synthesis steps are commercially available.
The buffer solution is a substance for stabilizing the pH value of the reaction solution, and can be acetate, lactate, tartrate, malate, maleate, succinate, ascorbate, carbonate, phosphate, a mixture thereof and the like.
In still another aspect, the invention also provides application of the tEB-FAPI compound shown in the formula (I) or pharmaceutically acceptable salt thereof in preparing a nuclide treatment or imaging drug of FAP protein high-expression tumor.
The invention also provides application of the radiolabeled tEB-FAPI complex shown in the formula (IV) in nuclide treatment and imaging of FAP protein high-expression tumors.
In the preferable application of the invention, the complex is prepared into an injection and is administrated by intravenous injection, and the injection is used for patients with tumors with high FAP protein expression.
In the application, the FAP protein high-expression tumor comprises but is not limited to breast cancer, ovarian cancer, lung cancer, colorectal cancer, gastric cancer or pancreatic cancer.
The invention provides a truncated Evans blue modified fibroblast activation protein inhibitor tEB-FAPI and a radionuclide labeled complex thereof, and provides a preparation method and a labeling method of the compound. Biological test results show that the compound has the advantages of obviously prolonged blood circulation half-life, enhanced tumor uptake enrichment and retention time. The novel performance is not possessed by other FAPI imaging agents at present, and the FAPI imaging agent is suitable for nuclide treatment and imaging of FAP protein high-expression tumors.
Drawings
FIG. 1 is a mass spectrum of Compound 2 in example 1 of the present invention.
FIG. 2 shows a nuclear magnetic hydrogen spectrum of Compound 2 in example 1 of the present invention.
FIG. 3 is a nuclear magnetic carbon spectrum of Compound 2 in example 1 of the present invention.
FIG. 4 is a mass spectrum of Compound 3 in example 1 of the present invention.
FIG. 5 shows a nuclear magnetic hydrogen spectrum of Compound 3 in example 1 of the present invention.
FIG. 6 is a mass spectrum of Compound 4 in example 1 of the present invention.
FIG. 7 shows a nuclear magnetic hydrogen spectrum of Compound 4 in example 1 of the present invention.
FIG. 8 is a nuclear magnetic carbon spectrum of Compound 4 in example 1 of the present invention.
FIG. 9 is a mass spectrum of Compound 7 in example 1 of the present invention.
FIG. 10 shows a nuclear magnetic hydrogen spectrum of Compound 7 in example 1 of the present invention.
FIG. 11 is a nuclear magnetic carbon spectrum of Compound 7 in example 1 of the present invention.
FIG. 12 is a mass spectrum of Compound 10 in example 1 of the present invention.
FIG. 13 is a mass spectrum of Compound 20 in example 1 of the present invention.
FIG. 14 is an HPLC chromatogram of Compound 10 in example 1 of the present invention.
FIG. 15 is an HPLC chromatogram of Compound 17 in example 1 of the present invention.
FIG. 16 is an HPLC chromatogram of a reaction system of Compound 17 and Compound 10 in example 1 of the present invention.
FIG. 17 is an HPLC chromatogram of Compound 19 in example 1 of the present invention.
FIG. 18 is an HPLC chromatogram of the reaction system of Compound 19 with DOTA-NHS of example 1 of the present invention.
FIGS. 19A and 19B embody the present invention68Ga-labelled tEB-FAPI complexes and68MicroPET imaging of Ga-labeled FAPI-02 in normal mice.
FIG. 20 shows three embodiments of the present invention177And (3) taking a tumor uptake value of a human pancreatic cancer xenograft model mouse 48 hours after the Lu-labeled tEB-FAPI complex and FAPI-02 are injected.
Detailed Description
The technical solution of the present invention will be further illustrated and described below with reference to the accompanying drawings by means of specific embodiments.
Example 1: preparation of tEB-FAPI linker (Compound 20)
Synthesis of Compound 2:
a100 mL flask was charged with compound 1 (7-hydroxy-4-quinolinecarboxylic acid, 1.89g, 10.0mmol), tert-butyl glycinate (1.89g, 10.0mmol), HATU (3.8g, 10.0mmol) and N, N-diisopropylethylamine (2.6g, 20.0mmol) in that order to 30mL of N, N-dimethylformamide. The reaction mixture was stirred overnight and the solvent was distilled off under reduced pressure to give the crude product. Purification on a silica gel column (dichloromethane/methanol ═ 30:1) gave compound 2 as a white solid in 87% yield, fig. 1 is a mass spectrum of compound 2, fig. 2 shows a nuclear magnetic hydrogen spectrum of compound 2, and fig. 3 shows a nuclear magnetic carbon spectrum of compound 2.
Synthesis of Compound 3:
in a 100mL flask, compound 2(1.51g, 5.0mmol), 1-bromo-3-chloropropane (1.55g, 10.0mmol), and potassium carbonate (1.38g, 10.0mmol) were charged in this order into 50mL of N, N-dimethylformamide. Heating the system to 60 ℃, keeping the system at 60 ℃, stirring overnight, and removing the solvent by reduced pressure distillation to obtain a crude product. Purification on a silica gel column (dichloromethane/methanol ═ 50:1) gave compound 3 as a white solid in 63% yield, fig. 4 is a mass spectrum of compound 3, and fig. 5 shows a nuclear magnetic hydrogen spectrum of compound 3.
Synthesis of Compound 4:
in a 100mL flask, compound 3(0.76g, 2.0mmol), 1-tert-butoxycarbonylpiperazine (0.55g, 3.0mmol), and potassium iodide (0.49g, 3.0mmol) were charged in this order into 30mL of acetonitrile. And (3) heating the system to 60 ℃, keeping the system at 60 ℃, stirring overnight, and distilling under reduced pressure to remove the solvent to obtain a crude product. Purification on a silica gel column (dichloromethane/methanol ═ 30:1) afforded compound 4 as a white solid in 58% yield. MS (ESI) m/z calcelledfor [ C ]28H40N4O6]:528.29；found:529.10[M+H]+Fig. 6 is a mass spectrum of compound 4, fig. 7 shows a nuclear magnetic hydrogen spectrum of compound 4, and fig. 8 shows a nuclear magnetic carbon spectrum of compound 4.
Synthesis of Compound 5:
compound 4(0.52g, 1.0mmol) was dissolved in 10mL of a mixed solution of dichloromethane and trifluoroacetic acid (volume ratio 9:1) under ice-bath conditions, the system was warmed to room temperature and reacted for 2 hours, after the reaction was completed, the solvent was distilled off under reduced pressure and dissolved in 10mL of N-dimethylformamide for further use.
Synthesis of Compound 6:
di-tert-butyl dicarbonate (0.22g, 1.0mmol) and N, N-diisopropylethylamine (0.39g, 3.0mmol) were added to N, N-dimethylformamide of Compound 5, respectively, and stirred at room temperature overnight, and the solvent was distilled off under reduced pressure to give a crude product. Purification on a silica gel column (dichloromethane/methanol ═ 10:1) afforded compound 6 as a white solid in 72% yield.
Synthesis of compound 7:
a100 mL flask was charged with compound 6(0.47g, 1.0mmol), (S) -pyrrolidine-2-carbonitrile hydrochloride (0.13g, 10.0mmol), HATU (0.38g,1.0mmol) and N, N-diisopropylethylamine (0.26g, 2.0mmol) in that order to 10mL of N, N-dimethylformamide. The reaction mixture was stirred at room temperature until the reaction was completed, and the solvent was removed by distillation under the reduced pressure to obtain a crude product. Purification on a silica gel column (dichloromethane/methanol ═ 50:1) afforded compound 7 as a white solid in 85% yield. Fig. 9 is a mass spectrum of compound 7, fig. 10 shows a nuclear magnetic hydrogen spectrum of compound 7, and fig. 11 shows a nuclear magnetic carbon spectrum of compound 7.
Synthesis of compound 8:
in a 100mL flask, compound 7(0.55g, 1.0mmol) and p-toluenesulfonic acid monohydrate (0.27g, 1.5mmol) were put in 10mL of acetonitrile in this order. Heating the reaction system to 60 ℃, stirring until the reaction is finished, and removing the solvent by reduced pressure distillation to obtain a crude product.
Synthesis of compound 9:
5,8,11, 14-Tetraoxa-2-azaheptadecanedioic acid-1-tert-butyl ester (0.19g, 1.0mmol), HATU (0.38g,1.0mmol), N-diisopropylethylamine (0.26g, 2.0mmol) and 10mL of N, N-dimethylformamide were charged into the reaction flask for Compound 8. The reaction mixture was stirred overnight and the solvent was distilled off under reduced pressure to give the crude product. Purification on a silica gel column (dichloromethane/methanol 50:1) gave compound 9 as a white solid in 64% yield.
Synthesis of compound 10:
in a 100mL flask, compound 9(0.61g, 1.0mmol) and p-toluenesulfonic acid monohydrate (0.27g, 1.5mmol) were put in 10mL of acetonitrile in this order. Heating the reaction system to 60 ℃, stirring until the reaction is finished, and removing the solvent by reduced pressure distillation to obtain a crude product. Purification on a silica gel column (dichloromethane/methanol ═ 10:1) afforded compound 10 as a white solid in 59% yield. MS (ESI) m/z calcelledfor [ C ]35H51N7O8]:697.38；found:698.43[M+H]+Fig. 12 is a mass spectrum of compound 10.
The synthetic route of the steps is as follows:
Figure BDA0002942507450000151
synthesis of compound 12:
in a 100mL flask, 4 '-diamino-3, 3' -dimethylbiphenyl (compound 11) (2.12g, 10.0mmol), di-tert-butyl dicarbonate (2.2g, 10.0mmol), N-diisopropylethylamine (1.3g, 10.0mmol) and 20mL of dichloromethane were charged, respectively, stirred at room temperature overnight, the completion of the reaction was monitored by HPLC (r.t. 10.13 minutes), and the solvent was distilled off under reduced pressure to give a crude product, which was purified by a silica gel column (petroleum ether/ethyl acetate ═ 5:1) to give compound 12 as a white solid in 59% yield.
Synthesis of compound 13:
compound 12(0.31g, 1.0mmol) and 4mL of acetonitrile were put into a 50mL flask, and 1.5mL of 2M hydrochloric acid was added dropwise into the flask in an ice bath to react for 15min, and sodium nitrite (0.068g, 1.0mmol) was added and dissolved in 2mL of water, and the mixture was added dropwise into the flask again to react for half an hour to prepare solution A for future use. A50 mL reaction flask was prepared, and 4, 6-diamino-5-hydroxy-1, 3-naphthalenedisulfonic acid (0.33g, 1.0mmol), sodium carbonate (0.105g, 1.0mmol) and 5mL of water were added thereto, followed by dropwise addition of solution A to solution B in an ice bath, followed by stirring in an ice bath for 2 hours. Reverse phase column chromatography, freeze drying gave pure compound 13 in 47% yield.
Synthesis of compound 14:
under ice bath conditions, compound 13(0.52g, 1.0mmol) was dissolved in trifluoroacetic acid, the system was warmed to room temperature for 2h, and after the reaction was completed, the solvent was distilled off under reduced pressure to obtain a crude product. The crude product was reverse phase pillared and lyophilized to give pure compound 14 in 73% yield.
Synthesis of compound 15:
in a 100mL flask, compound 14(0.54g, 1.0mmol), N-t-butoxycarbonyl-L-glutamic acid-1-t-butyl ester (0.30g, 1.0mmol), HATU (0.38g,1.0mmol), N-diisopropylethylamine (0.26g, 2.0mmol), and 10mL of N, N-dimethylformamide were placed, respectively. The reaction mixture was stirred until the reaction was completed, and the solvent was removed by distillation under the reduced pressure to obtain a crude product. The crude product was reverse phase pillared and lyophilized to give pure compound 15 in 52% yield.
Synthesis of compound 16:
the use of thioanisole: deprotection of tert-butyl ester and Boc protection of 1, 2-ethanedithiol, anisole, TFA (5:3:2:90) was performed at room temperature. After the reaction was completed, TFA was removed by a stream of argon gas, followed by dissolution in 10mL of N, N-dimethylformamide, and the resulting solution was used.
Synthesis of compound 17:
di-tert-butyl dicarbonate (0.22g, 1.0mmol) and N, N-diisopropylethylamine (0.39g, 3.0mmol) were added to N, N-dimethylformamide of Compound 16, respectively, stirred at room temperature overnight, and the reaction was monitored by HPLC for completion (r.t. 10.84 min). The solvent was distilled off under reduced pressure to obtain a crude product. The crude product was reverse phase pillared and lyophilized to give pure compound 17 in 43% yield over two steps.
Synthesis of compound 18:
a50 mL flask was charged with compound 17(0.77g, 1.0mmol), compound 10(0.51g, 1.0mmol), HATU (0.38g,1.0mmol), N-diisopropylethylamine (0.26g, 2.0mmol), and 10mL of N, N-dimethylformamide, respectively. The reaction mixture was stirred and the reaction was monitored by HPLC for completion (r.t. 12.16 min). The solvent was distilled off under reduced pressure to obtain a crude product. The crude product was reverse phase pillared and lyophilized to give pure compound 18 in 55% yield.
Synthesis of compound 19:
in a 25mL flask, compound 15(0.13g, 0.1mmol) and p-toluenesulfonic acid monohydrate (0.05g, 0.3mmol) were put in 5mL of acetonitrile in this order. The reaction system is heated to 60 ℃ and stirred for reaction, the deprotection process is monitored by HPLC until the reaction is finished (r.t. is 10.47 minutes), and the solvent is removed by reduced pressure distillation to obtain a crude product. The crude product was reverse phase pillared and lyophilized to give pure compound 19 in 61% yield.
Synthesis of compound 20:
a25 mL flask was charged with compound 19(0.12g, 0.1mmol), DOTA-NHS (0.05g, 0.1mmol) and N, N-diisopropylethylamine (0.04g, 0.3mmol) in that order, and then charged with 5mL of N, N-dimethylformamide. The reaction system was stirred at room temperature, monitored by HPLC until the reaction was complete (r.t. 11.35 min), and the solvent was distilled off under reduced pressure to give a crude product.The crude product was reverse phase pillared and lyophilized to give pure compound 20 in 53% yield. MS (ESI) m/z calcelledfor [ C ]80H104N16O24S2]:1736.69；found:1737.743[M+H]+Figure 13 is a mass spectrum of compound 20.
The synthetic route of the steps is as follows:
Figure BDA0002942507450000171
examples 2 to 8
The structures of the compounds of examples 2 to 8 are shown by the formulae (II-2) to (II-8), and their preparation methods are all as in example 1, wherein 5,8,11, 14-tetraoxa-2-azaheptadecanedioic acid-1-tert-butyl ester reacted with compound 8 is replaced by 5,8, 11-trioxa-2-azatridecanedioic acid-1-tert-butyl ester, 9-amino-4, 7-dioxanonanoic acid tert-butyl ester, glycine tert-butyl ester or other suitable compounds, or (S) -pyrrolidine-2-carbonitrile hydrochloride reacted with compound 6 is replaced by 3, 3-difluoropyrrolidine hydrochloride, or both, to give the corresponding structures as follows:
Figure BDA0002942507450000181
Figure BDA0002942507450000191
examples 9 to 13:
the compounds of examples 9-13 have the structures shown in formulas (III-1) to (III-4), wherein
Figure BDA0002942507450000192
Partially according to the method for preparing the compound 3BP-4089 disclosed in WO2021005131A1, and the remaining preparation steps are referred to examples 1 and 2 to 8.
Figure BDA0002942507450000193
Figure BDA0002942507450000201
Examples 14 to 35:
with reference to the preparation methods of examples 1 to 13, a tEB-FAPI compound expressed by the following formula (I) was prepared:
Figure BDA0002942507450000211
Figure BDA0002942507450000212
Figure BDA0002942507450000221
Figure BDA0002942507450000231
example 36 preparation of radioactive Ga-68-labelled tEB-FAPI complex:
and (2) wet method: about 18.5 to 1850 million Beech (MBq)68GaCl3The hydrochloric acid solution (eluted from the germanium gallium generator) was added to a centrifuge tube containing 0.5mL of an acetate-acetate solution (1.0g/L) of Compound 20 prepared in example 1, and the mixture was allowed to react at 37 ℃ for 20 min. A C18 separation cartridge was loaded and rinsed slowly with 10mL of absolute ethanol and then 10mL of water. The labeling solution was diluted with 10mL of water and applied to a column, and the unlabeled fraction was removed with 10mL of water68Ga ions, then 0.3mL of 10mM HCl in ethanol is used for leaching to obtain68Ga-labelled tEB-FAPI complexes. Diluting the eluate with normal saline, and sterile filtering to obtain the final product68Ga-labeled tEB-FAPI complex injection.
The freeze-drying method comprises the following steps: about 18.5 to 1850 million Beech (MBq)68GaCl3Hydrochloric acid solution (eluted from germanium gallium generator) was added to the lyophilized kit containing compound 20, mixed well and reacted at 37 ℃ for 20 min. A C18 separation cartridge was loaded and rinsed slowly with 10mL of absolute ethanol and then 10mL of water. The labeling solution was diluted with 10mL of water and applied to a column, and the unlabeled fraction was removed with 10mL of water68Ga ions are eluted by 0.3mL of 10mM HCl in ethanol to obtain complex eluent. Diluting the eluate with normal saline, and sterile filtering to obtain the final product68Ga-labeled tEB-FAPI complex injection.
Example 37 preparation of a Lu-177-labeled tEB-FAPI Complex:
and (2) wet method: about 18.5 to 1850MBq177LuCl3The sodium acetate solution was added to three centrifuge tubes containing 0.5mL of an acetate-acetate solution (1.0g/L) of the compound of example 1, compound 20, example 2 (compound of formula II-2) and example 3 (compound of formula II-3), respectively, and the mixture was allowed to react at 90 ℃ for 20 min. A C18 separation cartridge was loaded and rinsed slowly with 10mL of absolute ethanol and then 10mL of water. The labeling solution was diluted with 10mL of water and applied to a column, and the unlabeled fraction was removed with 10mL of water177Lu ion, then 0.3mL of 10mM HCl in ethanol to obtain three177Lu-labeled tEB-FAPI complexes. Diluting the eluate with normal saline, and sterile filtering to obtain three solutions177Lu-labeled tEB-FAPI complex injection.
The freeze-drying method comprises the following steps: about 18.5 to 1850MBq177LuCl3The sodium acetate solution was added to three lyophilized cartridges containing compound 20 of example 1, compound of example 2 (formula II-2) and compound of example 3 (formula II-3), respectively, mixed well and reacted at 90 ℃ for 20 min. A C18 separation cartridge was loaded and rinsed slowly with 10mL of absolute ethanol and then 10mL of water. The labeling solution was diluted with 10mL of water and applied to a column, and the unlabeled fraction was removed with 10mL of water177Lu ion, then 0.3mL of 10mM HCl in ethanol to obtain three77Lu-labeled tEB-FAPI complex leacheate. Diluting the eluate with normal saline, and sterile filtering to obtain three solutions177Lu-labeled tEB-FAPI pairingThe compound injection.
Experimental examples, analysis and application effects
1. HPLC analysis identification
The HPLC system is as follows: Shimadzulc-20A; a C18 column (YMC, 3 μm, 4.6X 150mm) was used for the analysis. Detection wavelength 254nm, flow rate 1mL/min, elution gradient: 0-3 minutes: 10 % acetonitrile 0 and 90% water (50mM ammonium acetate) were kept unchanged; 3-16 minutes: increase to 90% acetonitrile and 10% water (50mM ammonium acetate); 16-18 min: maintaining 90% acetonitrile and 10% water (50mM ammonium acetate); 18-20 min: reduced to 10% acetonitrile and 90% water (50mM ammonium acetate); 20-22 min: 10% acetonitrile and 90% water (50mM ammonium acetate) were maintained.
The results of the identification analysis of compound 10, compound 17, the reaction system of compound 10 with compound 17, compound 19, and the reaction system of compound 19 with DOTA-NHS in example 1 according to the above system are shown in fig. 14 to fig. 18.
The following two radiolabeled probes prepared in example 36 and example 37 were used as experimental agents, and their performance measurements are described below:
2、68MicroPET imaging of Ga-labeled tEB-FAPI complex in normal mice
Prepared by the method of example 36 to a purity of greater than 95%68Ga-tEB-FAPI 3.7MBq by tail vein injection in normal FVB mice68Ga-tEB-FAPI or68Ga-FAPI-02 (as a control) was followed by MicroPET imaging under isoflurane anesthesia 0-120 min post-dose, respectively, and the results are shown in FIG. 19A and FIG. 19B. The results show that the complex of example 3668Ga-tEB-FAPI had higher uptake in mouse cardiovasculary (FIG. 19A), while68Ga-FAPI-02 was almost completely eliminated during the test time (fig. 19B), indicating that the introduction of truncated evans blue can significantly extend the circulation half-life.
3、177Lu-labeled tEB-FAPI complex is used for tumor uptake experiments in mice of human pancreatic cancer xenograft models.
Three of the compounds of example 37 were prepared at a purity of greater than 95%177Lu-tEB-FAPI for human pancreatic cancer xenograftingIn model mice, three of 1.3MBq of the preparation of example 37 were injected via the tail vein177Lu-tEB-FAPI, and injecting a control group with 1.3MBq177Lu-FAPI-02. After 48h of injection, the eyeballs of the tumor-bearing nude mice were removed, blood was taken and then killed by cervical dislocation, tumor tissue samples were obtained by dissection, weighed, and counted for radioactivity using a gamma counter. The measurement data were background subtracted, corrected for decay time, and then averaged. Data are expressed as the percentage of the amount ingested per gram of tissue to the injected dose (% ID/g) and the results are shown in figure 20. The results show that 48 hours after injection, three of the preparations of example 37177The Lu-tEB-FAPI group (corresponding to A, B and C in the figure) had tumor uptake>5% ID/g, control group177Tumor uptake by Lu-FAPI-02 (D in the corresponding panel)<0.1% ID/g, indicating that tEB-FAPI has significantly enhanced tumor uptake and retention time and can be used as a tumor imaging agent.
In conclusion, the modified fibroblast activation protein inhibitor of the truncated Evans blue provided by the invention can remarkably prolong the circulation half-life period of the inhibitor, and can enhance the uptake enrichment and retention time of tumors, and the novel performance is not possessed by other FAPI imaging agents at present. The animal level before clinical use and clinical research prove that the protein is expected to become nuclide treatment and imaging of FAP protein high-expression tumor.
Although the invention has been described in detail hereinabove by way of general description, specific embodiments and experiments, it will be apparent to those skilled in the art that many modifications and improvements can be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Claims (13)

1. A truncated Evans blue modified fibroblast activation protein inhibitor compound or a pharmaceutically acceptable salt thereof, comprising: the molecular structure is formed by a connecting group L1、L2、L3、L4And X is formed by connecting truncated Evans blue, a fibroblast activation protein inhibitor and a nuclide chelating group togetherAs shown in the following formula (I)
Figure FDA0002942507440000011
Wherein:
L1is lysine, glutamic acid or derivative compounds containing lysine or glutamic acid structures;
L2is- (CH)2)n-, where n is an integer of 0 to 30 (preferably an integer of 0 to 12), where each CH2Can be replaced by-O-, -NH (CO) -or- (CO) -NH-alone, provided that there are no two adjacent CH' s2The group is replaced;
L3is- (CH)2)m-, where m is an integer of 0 to 30 (preferably an integer of 1 to 6), where each CH2Can be replaced by-O-, or- (CO) -alone, provided that there are no two adjacent CH' s2The group is replaced;
L4is- (CH)2)p-, where p is an integer of 0 to 30 (preferably an integer of 0 to 12), where each CH2Can be replaced by-O-, -NH (CO) -or- (CO) -NH-alone, provided that there are no two adjacent CH' s2The group is replaced;
x is selected from N, C, O, S or the following structure:
Figure FDA0002942507440000012
R1a fibroblast activation protein inhibitor structure selected from any one of:
Figure FDA0002942507440000013
R2is a nuclide chelating group selected from any one of the following structures:
Figure FDA0002942507440000021
2. the compound of claim 1, wherein: r in the formula (I)1Is composed of
Figure FDA0002942507440000022
X is
Figure FDA0002942507440000023
L1Is a glutamic acid structure, L3Is- (CH)2)3-，L4Is- (CH)2)0-，R3Is composed of
Figure FDA0002942507440000024
Namely, the structure of the compound is shown as the following formula (II):
Figure FDA0002942507440000025
wherein R is3And R4Both are H or both are F atoms, L2Is- (CH)2)0、-NH-CH2-(CO)-、-NH-CH2-(OCH2CH2)-O-CH2(CO) -or-NH-CH2-(OCH2CH2)3-O-CH2(CO)-。
3. The compound of claim 2, wherein: the structure of the compound is shown as the following formula (II-1):
Figure FDA0002942507440000026
4. the compound of claim 2, wherein: the structure of the compound is any one of the following formulas (II-2) to (II-8):
Figure FDA0002942507440000031
Figure FDA0002942507440000041
or
Figure FDA0002942507440000042
5. The compound of claim 1, wherein: r in the formula (I)1Is composed of
Figure FDA0002942507440000043
L1Is a glutamic acid structure, X is S, R2Is composed of
Figure FDA0002942507440000044
Namely, the structure of the compound is shown as the following formula (III):
Figure FDA0002942507440000045
wherein L is2Is- (CH)2)0、-NH-CH2-(CO)-、-NH-CH2-(OCH2CH2)-O-CH2(CO) -or-NH-CH2-(OCH2CH2)3-O-CH2(CO)-。
6. The compound of claim 5, wherein: the structure of the compound is any one of the following formulas (III-1) to (III-4):
Figure FDA0002942507440000051
or
Figure FDA0002942507440000061
7. The method for preparing the truncated Evans blue modified fibroblast activation protein inhibitor is characterized by comprising the following steps of: the method comprises the following steps:
carrying out amide condensation reaction on 7-hydroxy-4-quinoline carboxylic acid and glycine tert-butyl ester; then sequentially reacting with 1-bromo-3-chloropropane and 1-tert-butyloxycarbonyl piperazine; then removing Boc and tert-butyl protecting groups under the action of TFA; introducing Boc protection on amino; then carrying out amide condensation reaction with (S) -pyrrolidine-2-carbonitrile hydrochloride; removing Boc protection by utilizing p-toluenesulfonic acid; then carrying out condensation reaction with 5,8,11, 14-tetraoxa-2-aza heptadeca diacid-1-tert-butyl ester; removing Boc protection under the action of p-toluenesulfonic acid again to obtain an intermediate compound A;
secondly, introducing single side of 4,4 '-diamino-3, 3' -dimethyl biphenyl into Boc for protection, and then reacting with 4, 6-diamino-5-hydroxy-1, 3-naphthalenedisulfonic acid to prepare a truncated Evans blue derivative; removing Boc protection, and then carrying out amide condensation reaction with N-tert-butyloxycarbonyl-L-glutamic acid-1-tert-butyl ester; then removing Boc and tert-butyl protecting groups under the action of TFA; then reacting with di-tert-butyl dicarbonate, and introducing Boc protection on amino to obtain an intermediate compound B;
thirdly, carrying out amide condensation reaction on the intermediate compound A obtained in the step one and the intermediate compound B obtained in the step two; then, removing Boc protection by utilizing p-toluenesulfonic acid; finally reacting with DOTA-NHS to obtain the truncated Evans blue modified fibroblast activation protein inhibitor compound with the structure shown as the following formula (II-1)
Figure FDA0002942507440000062
8. A radiolabeled truncated evans blue-modified fibroblast activation protein inhibitor complex, which is a complex obtained by labeling a radionuclide with a compound of formula (I) according to claim 1 as a ligand; the radionuclide is preferably177Lu、90Y、18F、64Cu、68Ga、62Cu、67Cu、86Y、89Zr、99mTc、89Sr，153Sm、149Tb、161Tb、186Re、188Re、212Pb、213Bi、223Ra、225Ac、226Th、227Th、131I、211At or111Any one of In; further preferred radionuclides are68Ga、177Lu or90Y。
9. A radiolabeled truncated Evans blue modified fibroblast activation protein inhibitor complex having the structure shown in formula (IV):
Figure FDA0002942507440000071
wherein the content of the first and second substances,
L2is- (CH)2)n-, where n is an integer of 0 to 30 (preferably an integer of 0 to 12), where each CH2Can be replaced by-O-, -NH (CO) -or- (CO) -NH-alone, provided that there are no two adjacent CH' s2The group is replaced;
L3is- (CH)2)m-, where m is an integer of 0 to 30 (preferably an integer of 1 to 6), where each CH2Can be replaced by-O-, or- (CO) -alone, provided that there are no two adjacent CH' s2The group is replaced;
x is selected from N, C, O, S or the following structure:
Figure FDA0002942507440000072
R1a fibroblast activation protein inhibitor structure selected from any one of:
Figure FDA0002942507440000073
m is a radionuclide selected from68Ga、177Lu or90Any one of Y.
10. A method of preparing a radiolabeled truncated evans blue-modified fibroblast activation protein inhibitor complex, comprising the steps of: dissolving a compound of formula (I) according to claim 1 in a buffer solution or deionized water; adding a radionuclide solution into the obtained solution, and carrying out closed reaction for 5-40min to generate a radionuclide-labeled complex;
or comprises the following steps: dissolving a compound of formula (I) according to claim 1 in a buffer solution or deionized water; the obtained solution is aseptically filtered, subpackaged in containers, freeze-dried, plugged and sealed to obtain a freeze-dried medicine box; adding a proper amount of acetic acid solution or buffer solution into the freeze-dried medicine box for dissolving, then adding corresponding radionuclide solution, and carrying out closed reaction for 5-40min to generate the radionuclide-labeled complex.
11. Use of a compound of any one of claims 1 to 6 or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the nuclide treatment or imaging of tumors highly expressing FAP protein.
12. Use of the complex of any one of claims 8 or 9 for nuclide therapy or imaging of tumors with high expression of FAP protein.
13. The use of any one of claims 11 or 12, wherein: the compound or the complex is prepared into an injection and is administered by intravenous injection, and the injection is used for patients with tumors with high FAP protein expression; the FAP protein high-expression tumor comprises but is not limited to breast cancer, ovarian cancer, lung cancer, colorectal cancer, gastric cancer or pancreatic cancer.
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