CN116162127A - Granzyme B targeted inhibitor, probe and application - Google Patents

Abstract

The invention belongs to the field of nuclear medicine, and relates to a granzyme B targeted inhibitor, a probe and application. The granzyme B targeted inhibitor has a structure shown in a formula I. The molecular probes contain structural units derived from the granzyme B targeted inhibitor. The inhibitor and the probe have good granzyme B inhibition effect, the probes marked by nuclides are concentrated in tumor areas, have obvious advantages in the aspect of tumor contrast, and are a very potential granzyme B targeting molecular probe.

Description

Granzyme B targeted inhibitor, probe and application

Technical Field

The invention belongs to the field of nuclear medicine, and particularly relates to a granzyme B targeting inhibitor, a molecular probe formed by the inhibitor, a nuclear medicine probe and application thereof.

Background

GRANZYME (GRANZYME) is a serine protease, including A, B, H, K, M, which is believed to be a key mediator of cell death mediated by Cytotoxic T Lymphocytes (CTLs) and Natural Killer (NK) granule exocytosis. Granzyme B mediated apoptosis generally works with perforin, both released from the lysed granules of CTL and NK cells. Under the action of perforin, the target cell membrane is destroyed and pores with an inner diameter of 16-22 nm are formed, so that granzyme enters the target cell, and once internalized, the granzyme initiates apoptosis through Caspase-dependent and Caspase-independent pathways. Granzyme B, one of the most prominent effector molecules of granzyme, is currently studied mainly on granule-induced apoptosis pathways, emphasizing the response to tumor and virus-infected cells, and is one of the important markers of cell killing.

In recent years granzyme B has gained widespread attention as a biomarker and therapeutic target for various chronic inflammatory and damaging conditions (e.g. atherosclerosis, aortic aneurysm, heat (burn), aging, pathological angiogenesis, dermatitis, asthma and sepsis) as well as tumor models. Among them, granzyme B is thought to play a pathological role in aortic aneurysm progression, being widely distributed in thoracic and abdominal aortic aneurysm tissues of patients compared to normal human aorta, continuous infusion of Ang II for 28 days promoted formation of aortic aneurysms on the kidneys in preclinical mouse models of abdominal aortic aneurysms, and diffuse granzyme B staining was shown in adventitia and thrombus. Diffuse granzyme B staining was associated with increased mortality and rupture of aneurysms in mice. Therefore, early and timely prediction of the correlation of granzyme B with various immune diseases such as aneurysms, organ transplants and the like, and timely establishment of other treatment strategies are of great importance to patients.

The presently recommended imaging examinations include multiparameter nuclear magnetic imaging (multiparametric magnetic resonance imaging, mpMRI), computerized tomography (computed tomography, CT). However, the traditional imaging method only provides relevant information of disease morphology, and prognosis cannot be predicted.

The expression quantity of granzyme B is closely related to immune related diseases, so PET imaging of granzyme B can reflect the damage of immune response to tissue repair and tumor cells, and has important value in predicting disease prognosis. If it is possible to develop targeted granzyme B nuclide imaging/therapeutic agents with good target affinity and in vivo metabolic capacity, in particular ⁶⁸ Ga、 ¹⁸ F、 ^99m Tc and ¹⁷⁷ the Lu and other nuclide labeled reagent provides more efficient tools for immune related disease monitoring and has wide application prospect.

Disclosure of Invention

The object of the present invention is to provide a novel granzyme B targeted inhibitor and probes further formed therefrom.

Specifically, the first aspect of the present invention provides a granzyme B targeted inhibitor, which has a structure shown in formula I:

formula I.

In a second aspect, the present invention provides the use of a granzyme B targeted inhibitor as described above in the preparation of a nuclear medicine probe.

In a third aspect the present invention provides a molecular probe comprising a structural unit derived from a granzyme B targeted inhibitor as described above.

Specifically, the molecular probe contains one or two structural units derived from the compound of formula I, as well as a linking unit and a nuclide chelating unit.

When the molecular probe contains a structural unit derived from the compound of the formula I, the connecting unit is of a linear structure, and the two ends of the connecting unit are respectively connected with the structural unit derived from the compound of the formula I and the nuclide chelating unit.

When the molecular probe contains two structural units derived from the compound of formula I, the linking unit is a structural unit having three arms, each of which may be a peptide chain, the three arms linking two structural units derived from the compound of formula I and a nuclide chelating unit, respectively.

The nuclide chelating unit is a group formed by a bifunctional chelating agent selected from DOTA, NOTA, NODA, NODAGA, DOTP, TETA, ATSM, PTSM, EDTA, EC, HBEDCC, DTPA, SBAD, BAPEN, df, DFO, TACN, NO A/NOTAM, CB-DO2A, cyclen, NOTA-AA, DO3A, DO3AP, HYNIC, MAS3, MAG3 or isonitrile.

According to a preferred embodiment of the present invention, the molecular probe has a structure represented by formula II or formula III:

formula II->

Formula III.

In a fourth aspect, the present invention provides a nuclear medicine probe which is a radionuclide labelled molecular probe as described above. The radionuclide is chelated on the nuclide chelating unit.

The radionuclide may be a diagnostic radionuclide or a therapeutic radionuclide; wherein the diagnostic radionuclide is ⁶⁸ Ga、 ⁶⁴ Cu、 ¹⁸ F、 ⁸⁶ Y、 ⁹⁰ Y、 ⁸⁹ Zr、 ¹¹¹ In、 ^99m Tc、 ¹¹ C、 ¹²³ I、 ¹²⁵ I and ¹²⁴ at least one of I; the therapeutic radionuclide is ¹⁷⁷ Lu、 ¹²⁵ I、 ¹³¹ I、 ²¹¹ At、 ¹¹¹ In、 ¹⁵³ Sm、 ¹⁸⁶ Re、 ¹⁸⁸ Re、 ⁶⁷ Cu、 ²¹² Pb、 ²²⁵ Ac、 ²¹³ Bi、 ²¹² Bi and Bi ²¹² At least one of Pb.

In a fifth aspect, the present invention provides the use of a nuclear medicine probe as described above for the preparation of an imaging diagnostic or therapeutic agent targeting granzyme B.

The inhibitor and the probe have good granzyme B inhibition effect, the probes marked by nuclides are concentrated in tumor areas, have obvious advantages in the aspect of tumor contrast, and are a very potential granzyme B targeting molecular probe.

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

Drawings

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

FIG. 1 shows the overall route for the preparation of G1-10.

Fig. 2-1 to 2-10 are mass spectrograms of G1-10.

FIG. 3 shows the overall route for the preparation of G11.

Fig. 4 is a mass spectrum of G11.

FIG. 5 shows the overall route for the preparation of G12.

Fig. 6 is a mass spectrum of G12.

FIG. 7 is a diagram of ¹⁸ PET map of F-labeled G11 tumor-bearing mice.

FIG. 8 is a diagram of ¹⁸ Flabeled G11 was injected for 30min (left) and 60min (right) tumor bearing mouse biodistribution.

Detailed Description

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

Synthesis of GZMB targeted inhibitors

Preparation of inhibitors G1, G2, G3, G4, G5, G6, G7, G8, G9, G10 and G11:

g1-10 structural formula

The specific structures of G1-10 are shown above, (A) shows the general structure, and (B) shows the respective A part structures of different inhibitors.

The synthetic route is shown in FIG. 1. Reaction conditions: (a) DMF solution of 20% piperidine, DMF solution of Fmoc- (2S, 5S) -5-amino-1,2,4,5,6,7-hexahydroazepino [3,2,1-Hi ] endo-4-one-2-carboxilic acid, HBTU, HOBt and DIPEA; (b) DMF solution of 20% piperidine, DMF solution of Fmoc-L-isoleucine, HBTU, HOBt and DIPEA; (c) 20% piperidine in DMF, fmoc-A-OH, HBTU, HOBt and EIPEA in DMF; (d) 20% piperidine in DMF, mono-tert-butyl succinate, HBTU, HOBt and EIPEA in DMF; (e) trifluoroacetic acid, water and triisopropylsilane. The amino acid coupling was performed according to standard Fmoc solid phase synthesis.

Concretely, G1-10 is prepared: resin 1 (0.25 mmol) was taken in a mass in a 10mL solid phase synthesis tube, swollen with 2 mL Dichloromethane (DCM) and repeated three times for 5 minutes each, followed by the use ofN,NDimethylformamide (DMF) three washes of 5 min each. The amino protecting group Fmoc was deprotected using 20% piperidine in DMF (v/v) and the procedure was 2 mL 20% piperidine in DMF for 2 min, 10 min followed by 3-5 washes with 2 mL DMF for 2 min each. Fmoc- (2S, 5S) -5-amino-1,2,4,5,6,7-hexahydroazepino [3,2,1-Hi ] in 3-fold chemical amount relative to resin (0.02 mmol)]The endole-4-one-2-carboxilic acid is added into a synthesis tube after being activated by HBTU with the chemical quantity of 3.6 times in the presence of DIPEA with the chemical quantity of 7.2 times, and is reacted for 1 hour under electromagnetic stirring; followed by 2 mLN,NDimethylformamide (DMF) 6 washes for 1 min each. The amino protecting group Fmoc was deprotected using 20% piperidine in DMF (v/v) and the procedure was 2 mL 20% piperidine in DMF for 2 min, 10 min followed by 3-5 washes with 2 mL DMF for 2 min each. Fmoc-L-isoleucine of 3 times the chemical amount was activated with HBTU of 3.6 times the chemical amount in the presence of DIPEA of 7.2 times the chemical amount relative to the resin (0.02 mmol) and then added to the synthesis tube for reaction under electromagnetic stirring for 1 hour; followed by 2 mLN, NDimethylformamide (DMF) 6 washes for 1 min each. Deamination of protecting group Fm using 20% piperidine in DMF (v/v)oc, specific procedure was 2 mL of 20% piperidine in DMF for 2 min, 10 min followed by 3-5 washes with 2 mL DMF for 2 min each. Fmoc-A-OH of 3 times the chemical amount was activated with HBTU of 3.6 times the chemical amount in the presence of DIPEA of 7.2 times the chemical amount relative to the resin (0.02 mmol) and then added to the synthesis tube for reaction for 1 hour under electromagnetic stirring; followed by 2 mLN,NDimethylformamide (DMF) 6 washes for 1 min each. The amino protecting group Fmoc was deprotected using 20% piperidine in DMF (v/v) and the procedure was 2 mL 20% piperidine in DMF for 2 min, 10 min followed by 3-5 washes with 2 mL DMF for 2 min each. Relative to the resin (0.02 mmol), 3 times the chemical amount of mono-tert-butyl succinate was activated with 3.6 times the chemical amount of HBTU in the presence of 7.2 times the chemical amount of DIPEA and then added to the synthesis tube for reaction for 1 hour under electromagnetic stirring. Dissociation of the ligand from the resin and removal of the tert-butyl ester was accomplished using 5mL trifluoroacetic acid/triisopropylsilane/water (95:2.5:2.5, v/v/v) with stirring for 2 hours, and the resin was washed with 2 mL trifluoroacetic acid, all filtrates were collected, after removal of the trifluoroacetic acid under reduced pressure, the crude product was reverse prepared by HPLC and lyophilized to afford inhibitor G1-10. The structure was identified by mass spectrometry. The mass spectra of G1-10 are shown in FIGS. 2-1 through 2-10.

Synthesis of DOTA-GZMB targeting ligand

Preparation of ligand G11:

g11 structural formula

The specific structure of G11 is as described above. The synthesis procedure is shown in FIG. 3. Reaction conditions: (a) DMF solution of 20% piperidine, DMF solution of Fmoc- (2S, 5S) -5-amino-1,2,4,5,6,7-hexahydroazepino [3,2,1-Hi ] endo-4-one-2-carboxilic acid, HBTU, HOBt and DIPEA; (b) DMF solution of 20% piperidine, DMF solution of Fmoc-L-isoleucine, HBTU, HOBt and DIPEA; (c) DMF solution of 20% piperidine, fmoc-L-aspartic acid-1-tert-butyl ester, HBTU, HOBt and DIPEA; (d) DMF solution of 20% piperidine, fmoc-L-aspartic acid-1-tert-butyl ester, HBTU, HOBt and EIPEA; (e) trifluoroacetic acid, water and triisopropylsilane; (f) a DMF solution of bis-NHS-Fmoc-L-glutamic acid and DIPEA; (g) 20% piperidine in DMF; (h) DMF solution of NHS-NOTA and DIPEA. The amino acid coupling was performed according to standard Fmoc solid phase synthesis.

Specifically, G11 preparation: resin 1 (0.25 mmol) was taken in a mass in a 10mL solid phase synthesis tube, swollen with 2 mL Dichloromethane (DCM) and repeated three times for 5 minutes each, followed by the use ofN,NDimethylformamide (DMF) three washes of 5 min each. The amino protecting group Fmoc was deprotected using 20% piperidine in DMF (v/v) and the procedure was 2 mL 20% piperidine in DMF for 2 min, 10 min followed by 3-5 washes with 2 mL DMF for 2 min each. Fmoc amino acid of 3 times the chemical amount was activated with HBTU of 3.6 times the chemical amount in the presence of DIPEA of 7.2 times the chemical amount relative to the resin (0.02 mmol) and then added to the synthesis tube for reaction for 1 hour with electromagnetic stirring. Dissociation of the ligand from the resin and removal of the tert-butyl ester was accomplished using 5mL trifluoroacetic acid/triisopropylsilane/water (95:2.5:2.5, v/v/v) with stirring for 2 hours, and the resin was washed with 2 mL trifluoroacetic acid, all filtrate was collected, after removal of trifluoroacetic acid under reduced pressure, the crude product was reverse prepared by HPLC and lyophilized to give intermediate compound 2, compound 2 was reacted with 0.5-fold chemical amount of bis NHS-Fmoc-L-glutamic acid and 2-fold chemical amount of DIPEA to give intermediate compound 3, compound 3 was reacted with 2 mL of 20% piperidine in DMF for 10 minutes to give intermediate compound 4, and compound 4 was reacted with 1-fold chemical amount of bis NHS-NOTA, 2-fold chemical amount of DIPEA to give the target ligand G11. The ligand structure was identified by mass spectrometry as shown in figure 4.

Preparation of ligand G12:

g12 structural formula

The specific structure of G12 is as described above. The synthesis procedure is shown in FIG. 5. Reaction conditions: (a) DMF solution of 20% piperidine, DMF solution of Fmoc- (2S, 5S) -5-amino-1,2,4,5,6,7-hexahydroazepino [3,2,1-Hi ] endo-4-one-2-carboxilic acid, HBTU, HOBt and DIPEA; (b) DMF solution of 20% piperidine, DMF solution of Fmoc-L-isoleucine, HBTU, HOBt and DIPEA; (c) DMF solution of 20% piperidine, fmoc-L-aspartic acid-1-tert-butyl ester, HBTU, HOBt and DIPEA; (d) trifluoroacetic acid, water and triisopropylsilane; (e) a DMF solution of bis-NHS-Fmoc-L-glutamic acid and DIPEA; (f) 20% piperidine in DMF; (g) DMF solution of NHS-NOTA and DIPEA. The amino acid coupling was performed according to standard Fmoc solid phase synthesis.

Specifically, the preparation of G12: resin 1 (0.25 mmol) was taken in a mass in a 10mL solid phase synthesis tube, swollen with 2 mL Dichloromethane (DCM) and repeated three times for 5 minutes each, followed by the use ofN,NDimethylformamide (DMF) three washes of 5 min each. The amino protecting group Fmoc was deprotected using 20% piperidine in DMF (v/v) and the procedure was 2 mL 20% piperidine in DMF for 2 min, 10 min followed by 3-5 washes with 2 mL DMF for 2 min each. Fmoc amino acid of 3 times the chemical amount was activated with HBTU of 3.6 times the chemical amount in the presence of DIPEA of 7.2 times the chemical amount relative to the resin (0.02 mmol) and then added to the synthesis tube for reaction for 1 hour with electromagnetic stirring. Dissociation of the ligand from the resin and removal of the tert-butyl ester was accomplished using 5mL trifluoroacetic acid/triisopropylsilane/water (95:2.5:2.5, v/v/v) with stirring for 2 hours, and the resin was washed with 2 mL trifluoroacetic acid, all filtrates were collected, after removal of trifluoroacetic acid under reduced pressure, the crude product was reverse prepared by HPLC and lyophilized to give intermediate compound 2, compound 2 was reacted with 0.5-fold chemical amount of bis NHS-Fmoc-L-glutamic acid and 2-fold chemical amount of DIPEA to give intermediate compound 3, compound 3 was reacted with 2 mL of 20% piperidine in DMF for 10 minutes to give intermediate compound 4, and compound 4 was reacted with 1-fold chemical amount of bis NHS-NOTA, 2-fold chemical amount of DIPEA to give the target ligand G12. The ligand structure was identified by mass spectrometry as shown in figure 6.

Inhibition constant measurement

The test method is as follows:

1. fully and uniformly mixing the murine cathepsin C (10 mg/mL) and the human granzyme B (100 mg/mL) by using an activating buffer solution (50 mM MES,50 mM NaCl,pH 5.5), and incubating at 37 ℃ for 4 hours to activate the activity of the human granzyme B;

2. diluting the human granzyme B to 0.25 mg/mL with test buffer (50 mM Tris,pH 7.5), and diluting the substrate Ac-IEPD-AMC to 100mM;

3. 10mL of human granzyme B,5mL of substrate Ac-IEPD-AMC and 5mL of inhibitors with different concentrations are respectively added into a 384-hole PCR plate, and after being uniformly mixed, the mixture is placed in a dark place at 37 ℃ for incubation for 1 hour;

4. fluorescence intensity of each well was measured with a microplate reader at Ex/em=380/460 nm, and the data was fitted with Prism software to calculate Ki values.

Inhibition constant of the inhibitors of Table 1

As can be seen from Table 1, each of G9, G11 and G12 of the present invention has a very good granzyme B inhibitory effect.

Marking and quality control

Marking:

¹⁸ f: 1.0mg of G11 was precisely weighed into a sample bottle, 100. Mu.L of DMSO (dimethyl sulfoxide) was added to dissolve it, and then pure water was added to dilute the ligand concentration to 10. Mu.g/. Mu.L. Taking 5 mu L of ligand solution in a bottle, and taking 20 mu L of KHP of 0.5mol/L and 7 mu L of AlCl of 20mmol/L ₃ Solution and 100 [ mu ] L ¹⁸ F-sodium fluoride is added into a bottle, the bottle is placed for 5 min at room temperature after shaking, 100 mu L of ethanol is added into a reaction bottle, and the mixture is reacted for 10 min at 110 ℃ after mixing uniformly. After the reaction, 10mL injection water is added for dilution, and the activated Sep-pak VAC C C-18 column is used for eluting, and then 5.0 mL pure water is used for eluting impurities and discarding the impurities. The product was collected in a bottle with 0.5 mL of 80% ethanol solution, and 5.0. 5.0 mL physiological saline was added to the system for use. Quality control was analyzed by HPLC.

And (3) quality control:

¹⁸ the radiochemical purity of the F-G11 complex was determined using HPLC (high Performance liquid chromatography) and the mobile phase was an aqueous solution containing 20% acetonitrile (containing 0.1% TFA) with a radiochemical purity of greater than 95%.

Imaging of the labeled product

Taking a new preparation of 0.1 mL ¹⁸ F-G11 complex (5.6 MBq-7.4 MBq) is injected into a mouse with female MC38 tumor (tumor diameter is about 1 cm) through tail vein, 1 h is anesthetized by isoflurane, PET/CT (SUPER-NOVA, ping-Sheng technology, china) imaging is carried out on a small animal, and delineating SUV with standard uptake value is carried out on a region of interest.

As shown in fig. 7 and table 2, in each mouse ¹⁸ The F-G11 complex can be obviously concentrated in the tumor area, the SUVmax of the tumor is 0.33+/-0.02, the SUVmax of the muscle is 0.11+/-0.02, and the ratio of the SUVmax of the tumor to the SUVmax of the muscle is 3.03+/-0.40. It can be seen that the light source is, ¹⁸ F-G11 has a significant advantage in terms of tumor aspect ratio.

Table 2 Complex ¹⁸ SUVmax value and ratio of F-G11 in tumor and muscle (mean+ -SD, n=4)

Biodistribution experiments

For a pair of ¹⁸ The biodistribution data of F-G11 in tumor-bearing mice were analyzed and the results are shown in FIG. 8, from which it can be seen that after 30min of injection, ¹⁸ F-G11 is concentrated in tumor area, and has certain uptake in liver, kidney and other metabolic organs, ¹⁸ the uptake of F-G11 in tumors was 0.64.+ -. 0.15, in liver and kidney was 2.45.+ -. 0.35 and 8.45.+ -. 0.65, respectively, whereas the ratios of tumor to blood, tumor to muscle were 1.13.+ -. 0.36, 2.61.+ -. 0.47, respectively. These results also indicate that the results show that, ¹⁸ F-G11 has obvious advantages in terms of tumor meat ratio and is very potential ¹⁸ F labeling GZMB targeting molecular probes.

The foregoing description of embodiments of the invention has been presented for purposes of illustration and description, and is not intended to be exhaustive or limited to the embodiments disclosed. Many modifications and variations will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the various embodiments described.

Claims (10)

1. A granzyme B targeted inhibitor, wherein the granzyme B targeted inhibitor has a structure according to formula I:

formula I.

2. Use of a granzyme B targeted inhibitor of claim 1 in the preparation of a nuclear medicine probe.

3. A molecular probe comprising a structural unit derived from the granzyme B targeted inhibitor of claim 1.

4. A molecular probe according to claim 3, wherein the molecular probe comprises one or two structural units derived from a compound of formula I, as well as a linking unit and a nuclide chelating unit.

5. The molecular probe according to claim 4, wherein the molecular probe comprises two structural units derived from the compound of formula I, and the linking unit is a peptide chain having three arms, and the two structural units derived from the compound of formula I and the nuclide chelating unit are linked, respectively.

6. The molecular probe of claim 5, wherein the nuclide chelating unit is a group formed by a bifunctional chelating agent selected from DOTA, NOTA, NODA, NODAGA, DOTP, TETA, ATSM, PTSM, EDTA, EC, HBEDCC, DTPA, SBAD, BAPEN, df, DFO, TACN, NO a/NOTAM, CB-DO2A, cyclen, NOTA-AA, DO3A, DO3AP, HYNIC, MAS3, MAG3 or isonitrile.

7. The molecular probe according to claim 6, wherein the molecular probe has a structure represented by formula II or formula III:

formula II->

Formula III.

8. A nuclear medicine probe, characterized in that it is a radionuclide labelled molecular probe according to any of claims 3 to 7.

9. The nuclear medicine probe according to claim 8, wherein the radionuclide is a diagnostic radionuclide or a therapeutic radionuclide; the diagnostic radionuclide is ⁶⁸ Ga、 ⁶⁴ Cu、 ¹⁸ F、 ⁸⁶ Y、 ⁹⁰ Y、 ⁸⁹ Zr、 ¹¹¹ In、 ^99m Tc、 ¹¹ C、 ¹²³ I、 ¹²⁵ I and ¹²⁴ at least one of I; the therapeutic radionuclide is ¹⁷⁷ Lu、 ¹²⁵ I、 ¹³¹ I、 ²¹¹ At、 ¹¹¹ In、 ¹⁵³ Sm、 ¹⁸⁶ Re、 ¹⁸⁸ Re、 ⁶⁷ Cu、 ²¹² Pb、 ²²⁵ Ac、 ²¹³ Bi、 ²¹² Bi and Bi ²¹² At least one of Pb.

10. Use of a nuclear medicine probe according to claim 8 or 9 for the preparation of an imaging diagnostic or therapeutic agent targeting granzyme B.

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