Compound for targeted degradation of TSPO protein as well as preparation method and application of compound

Abstract

The invention belongs to the technical field of biological medicine, and discloses application of a reagent for detecting TSPO expression in preparation of a product for diagnosing and treating cardiac insufficiency caused by sepsis. A mouse test finds that the TSPO molecule is used as a marker in sepsis cardiac dysfunction and is highly expressed in cardiac insufficiency caused by sepsis; meanwhile, septicopyemia is simulated by injecting LPS into the small abdominal cavity, then the heart function and the heart damage condition of the mouse are observed, and TSPO knockout can improve the survival rate of the lipopolysaccharide-mediated septicopyemia cardiac insufficiency mouse and improve the heart function. The invention further discloses a PROTAC degradation agent compound of the targeted TSPO. The PROTAC degradation agent compound can selectively utilize a ubiquitin-proteasome system to induce targeted degradation of the TSPO protein. Meanwhile, LPS is injected into the abdominal cavity of a mouse to simulate a septicopyemia model, and it is found that the TSPO-PROTAC is used for treating the SICD to improve the septicopyemic multi-organ injury.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a compound for targeted degradation of TSPO protein, and a preparation method and application thereof. Background Art

[0002] Sepsis-induced cardiac dysfunction (SICD) is one of the common complications of sepsis and septic shock. Cardiac dysfunction plays a vital role in the clinical outcomes of severe sepsis and septic shock. More than 40% of sepsis patients experience cardiac dysfunction, which is associated with high mortality and poor prognosis. How to effectively control SICD to reduce mortality remains a major challenge in clinical practice. At present, the treatment of sepsis-induced cardiac dysfunction mainly involves the use of broad-spectrum antibiotics to fight infection, maintain blood pressure, correct hypoxia, etc., but there is a lack of targeted and effective drugs. Therefore, there is an urgent need to develop drugs that can specifically treat sepsis-induced cardiac dysfunction.

[0003] The pathophysiological mechanism of SICD is composed of multiple factors. Among them, myocardial inhibitory factors, mitochondrial dysfunction, myocardial cell apoptosis, and calcium ion homeostasis imbalance all play a role. LPS (bacterial lipopolysaccharide) promotes the innate immune system to be inert, produces excessive inflammatory mediators (TNF-a, IL-1β and IL-6), directly inhibits myocardial function, and leads to abnormal mitochondrial structure and function, increases mitochondrial reactive oxygen species (ROS) levels, and triggers heart failure. Mitochondrial outer membrane protein (TSPO) is a component of a macromolecular protein complex that regulates cell survival and death pathways. It exists in most organs in the body (heart, kidney, liver, brain) and other tissues. It is expressed in large quantities during inflammation and tissue damage. TSPO has been used as a diagnostic marker for inflammation in clinical practice. The specific ligand 11C-(R)-pk11195 is used for PET / CT myocardial and vascular imaging, which can image and quantitatively analyze arterial wall inflammation in patients with myocarditis and large vessel inflammation. Specific knockout of cardiac TSPO can prevent heart failure caused by pressure overload by regulating mitochondrial calcium uptake and restoring mitochondrial function. 10 TSPO ligands can improve ischemia-reperfusion injury by limiting the opening of the mitochondrial permeability transition pore (mPTP) and inhibiting oxidative stress after ischemia-reperfusion. TSPO can also reduce the incidence of atrial fibrillation by acting on the cell membrane anion channel (IMAC) and permeability transition pore (PTP). However, the role of TSPO in sepsis-induced cardiac dysfunction is still unclear. Summary of the invention

[0004] The present invention aims to solve one of the technical problems in the related art at least to a certain extent. To this end, the main purpose of the present invention is to provide a compound for targeted degradation of TSOP protein and a preparation method and application thereof.

[0005] The objective of the present invention is achieved through the following technical solutions:

[0006] The inventors found in the study that the TSPO content in the heart tissue of the model of heart failure caused by sepsis was significantly increased, and TSPO knockout can improve the survival rate and improve the cardiac function of mice with septic heart failure mediated by lipopolysaccharide; and then found that antagonizing or degrading TSPO may be a strategy for treating SICD. However, there are currently no clear TSPO agonists and antagonists, only ligands that act on TSPO. The present invention will use the method of proteolysis targeting chimeras (PROTAC) to construct a drug that can directly degrade TSPO, which is of great significance.

[0007] Specifically:

[0008] A compound that targets the degradation of TSPO protein. Based on the design principle of PROTAC, we designed TSPO-PROTAC from the following points:

[0009] 1. Selection of Linker and target protein ligand: Linker uses a widely used and commonly used polyethylene glycol chain. There are many ligands for TSPO, including isoquinolinecarboxamide, among which 1-(2-chlorophenyl)-n-methyl-n-(1-methylpropyl)-1-isoquinolinecarboxamide (PK11195) is widely regarded as a typical TSPO ligand, used as a reference compound in biological experiments, and radiolabeled for imaging studies; In view of the wide clinical application of PK11195, which fully demonstrates its clinical safety, we selected the TSPO ligand PK11195 as the binding part of the target protein;

[0010] 2. Connection between Linker and target protein ligand: Regarding the choice of connection position, since the binding of PK11195 to TSPO involves the aromatic heterocycle of PK11195, it cannot be connected from the site with the aromatic ring; some studies have modified the methyl end of PK11195 and coupled it with a fluorescent group to synthesize a contrast agent for diagnosing cancer; therefore, we referred to the above method and connected the modified PK11195 (compound 4) to the Linker at the methyl end.

[0011] 3. Selection of E3 ligases and their ligands: The main E3 ligases are CRBN, VHL, cIAP and MDM2; the E3 ligase with better effect and highest frequency of use is mainly CRBN; Regarding the ligand of E3 ligase CRBN: There are currently three main ligands: thalidomide, lenalidomide and pomalidomide (Kd values ​​are 249.2nM, 177.8nM, 156.6nM, respectively). Among the three ligands, pomalidomide has the strongest affinity and has an amino group, which makes it easier to connect to the other end of the polyethylene glycol chain. Therefore, we selected pomalidomide (compound 6) as the ligand of E3 ligase CRBN.

[0012] 4. Linker to E3 ligase ligand: The amino part of pomalidomide is conventionally selected to connect with compound 5. The commonly used Click chemistry method with milder conditions and higher efficiency is used to connect the molecules at both ends to obtain TSPO-PROTAC.

[0013] Application of reagents for detecting TSPO expression in the preparation of products for diagnosing and treating cardiac insufficiency caused by sepsis.

[0014] In certain embodiments, the TSOP is upregulated in patients with sepsis-induced cardiac insufficiency.

[0015] In certain embodiments, the product comprises a TSOP detection reagent.

[0016] A pharmaceutical composition for diagnosing and treating cardiac insufficiency caused by sepsis, the pharmaceutical composition comprising the reagent for inhibiting TSOP expression in the aforementioned application.

[0017] In certain embodiments, the agent comprises a small molecule PROTAC that targets and degrades TSOP.

[0018] In certain specific embodiments, the preparation method of the small molecule PROTAC targeting the degradation of TSOP is prepared as follows:

[0019]

[0020] The specific steps include:

[0021] 1) Synthesis of compound 3

[0022] Compound 2 (1.0 g, 3.06 mmol) was weighed into a 50 mL round-bottom flask, and DCM (30 mL) was added and stirred to dissolve. Subsequently, DCC (820.5 mg, 3.98 mmol) and compound 1 (359.8 mg, 3.67 mmol) were added, and the reaction was carried out at room temperature overnight. After the reaction was completed, DCM was removed under vacuum, and the residue was subjected to silica gel column chromatography (eluent, DCM:MeOH=125:1-60:1), and the product was collected and concentrated under reduced pressure to obtain product 3.

[0023] 2) Synthesis of compound 5

[0024] Compound 4 (349.0 mg, 1.23 mmol) was weighed into a 100 mL round-bottom flask, and DCM (45 mL) was added and stirred to dissolve. Subsequently, EDCI (306.7 mg, 1.60 mmol) and HOBT (232.6 mg, 1.72 mmol) were added. After stirring at room temperature for 10 min, compound 3 (500.0 mg, 1.23 mmol) was added and reacted at 55 °C overnight. After the reaction was completed, DCM was removed under vacuum, and the residue was purified by silica gel column chromatography to obtain compound 5;

[0025] 3) Synthesis of Compound 7

[0026] Weigh 2.73g of compound 6 into a 50mL round-bottom flask, add an appropriate amount of THF, stir and suspend. Use a pipette to measure 2.50mL of bromoacetyl chloride and add it, reflux and react overnight; after the reaction is completed, cool the reaction system to room temperature, remove THF under vacuum, add ether, and precipitate a large amount of off-white solids. Collect the solids, wash with ether, and vacuum dry to obtain the target product compound 7;

[0027] 4) Synthesis of Compound 8

[0028] Compound 7 (1.5 g, 2.55 mmol) was weighed into a 100 mL round-bottom flask, and DMF (50 mL) was added to stir and dissolve, followed by the addition of NaN3 (489.8 mg, 7.65 mmol) and the reaction was carried out at 55°C overnight. After the reaction was completed, DMF was removed under vacuum, and the residue was subjected to silica gel column chromatography (eluent, PE:EA:DCM=(10:1:5-8:1:5), the product was collected, and the product was concentrated under reduced pressure to obtain product 8;

[0029] 5) Synthesis of Compound 9

[0030] Compound 8 (280.0 mg, 0.78 mmol) and compound 5 (524.2 mg, 0.78 mmol) were weighed into a 100 mL round-bottom flask, and DMF (15 mL) and methanol (10 mL) were added to stir and dissolve, and then copper acetate (10 mg, 0.05 mmol) and ascorbic acid (17.6 mg, 0.10 mmol) were added, and the mixture was reacted at 35° C. for 2 h. After the reaction was completed, DMF was removed under vacuum, and the residue was subjected to silica gel column chromatography (eluent, DCM:MeOH=80:1-30:1), and the product was collected and concentrated under reduced pressure to obtain product 9.

[0031] Application of TSOP in the preparation of a pharmaceutical composition for diagnosing and treating cardiac insufficiency caused by sepsis.

[0032] Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, such carriers include (but are not limited to): diluents, buffers, suspensions, emulsions, granules, encapsulation agents, excipients, fillers, adhesives, sprays, transdermal absorbents, wetting agents, disintegrants, absorption enhancers, surfactants, colorants, flavoring agents or adsorption carriers.

[0033] The pharmaceutical composition of the present invention can be prepared into various dosage forms as required, including but not limited to tablets, solutions, granules, patches, ointments, capsules, aerosols or suppositories.

[0034] The administration route of the pharmaceutical composition of the present invention is not limited as long as it can exert the desired therapeutic or preventive effect, including but not limited to oral administration, intravenous injection, intramuscular injection, subcutaneous injection, sublingual dissolution, rectal irrigation, nasal spray, oral spray, topical skin administration or systemic transdermal administration.

[0035] The pharmaceutical composition of the present invention can also be used in combination with other drugs for treating SICD. The combined use of multiple drugs can greatly increase the success rate of treatment.

[0036] Compared with the prior art, the present invention has at least the following advantages:

[0037] 1) The present invention found through mouse experiments that TSPO molecules are used as markers in septic cardiac dysfunction and are highly expressed in cardiac dysfunction caused by sepsis;

[0038] 2) The present invention simulates sepsis by intraperitoneal injection of LPS, and then observes the cardiac function and heart damage of mice. TSPO knockout can increase the survival rate of mice with lipopolysaccharide-mediated sepsis and heart failure and improve cardiac function.

[0039] 3) Based on the PROTAC technology principle, the present invention synthesized and developed a new PROTAC degrader compound targeting TSPO; it can selectively use the ubiquitin-proteasome system to induce the targeted degradation of TSPO protein. At the same time, the mouse intraperitoneal injection of LPS was used to simulate the sepsis model, and it was found that TSPO-PROTAC treatment of SICD improved septic multi-organ damage. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] In order to more clearly illustrate the specific embodiments of the present invention, the drawings required for use in the specific embodiments or the description of the prior art will be briefly introduced below.

[0041] Figure 1 This is a diagram showing the cardiac function test of the mouse SICD model constructed in Example 1 of the present invention;

[0042] Figure 2 The expression changes of TSPO after LPS intervention in Example 1 of the present invention;

[0043] Figure 3 This is a schematic diagram of constructing TSPO gene knockout (OK) mice in Example 2 of the present invention;

[0044] Figure 4 This is a diagram showing the effect of TSPO gene knockout on cardiac function and survival rate in mice in Example 2 of the present invention;

[0045] Figure 5 This is the nuclear magnetic resonance hydrogen spectrum test spectrum of compound 7 in Example 3 of the present invention;

[0046] Figure 6 This is the high-resolution mass spectrometry identification test spectrum of compound 9 (TSOP-PROTAC) in Example 3 of the present invention;

[0047] Figure 7 This is the nuclear magnetic resonance hydrogen spectrum test spectrum of compound 9 (TSOP-PROTAC) in Example 3 of the present invention;

[0048] Figure 8 This is the effect of TSPO-PROTAC on TSPO degradation in Example 4 of the present invention;

[0049] Fig. 9 This is the effect of TSPO-PROTAC in Example 5 of the present invention on the survival rate and cardiac function of mice. DETAILED DESCRIPTION

[0050] In order to make the purpose, technical solution and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments, but it should not be understood as limiting the scope of protection of the present invention. Any equivalent changes in form made based on the concept of the present invention should be regarded as the scope of the present invention.

[0051] The present invention provides a general and / or specific description of the materials and test methods used in the experiment. The test methods or test methods involved are all conventional methods unless otherwise specified; the reagents or instruments used are all conventional products available on the market, prepared or used in a conventional manner, unless the manufacturer is specified.

[0052] Example 1: TSOP is highly expressed in animals with sepsis-induced cardiac dysfunction

[0053] This example found and verified that TSOP is highly expressed in cardiac insufficiency caused by sepsis, specifically:

[0054] 1) Establishing SICD model

[0055] The animals used in the experiment were provided by the Experimental Animal Center of Daping Hospital of Army Medical University. C57BL / 6 mice weighed 22-25 g and were raised in the SPF Animal Experimental Center of Army Special Medical Center. The indoor temperature was 23-25°C and the relative humidity was 40%-60%. The mice had free access to water and food.

[0056] SICD model was successfully established in C57BL / 6 mice by intraperitoneal injection of LPS (25 mg / kg, intraperitoneal injection), and the cardiac function decline was determined by gene expression detection and ELISA kit detection of myocardial injury markers (including left ventricular ejection fraction (LVEF), left ventricular short axis shortening index (LVFS), left ventricular end-diastolic diameter (LVEDD) and left ventricular end-systolic diameter (LVESD)), as shown in the following figure. Figure 1 As shown in the figure, it can be seen that the levels of cardiac TNF-α, IL-1β and IL-6 mRNA in mice after LPS intervention (abbreviated as LPS) were increased, and the levels of serum cTn-T and CK-MB were increased, indicating that the SICD model was successfully established by intraperitoneal injection of LPS (25 mg / kg).

[0057] 2) Detection method

[0058] Gene expression detection: Total RNA was extracted using Trizol reagent (Invitrigen); two micrograms of RNA was used to synthesize cDNA using a commercial kit (TAKARA, Kusatsu, Japan); quantitative PCR was performed using SYBR Green dye (TAKARA), and mRNA expression levels were measured on a CFX-Connect real-time system (Bio-Rad, San Diego, CA).

[0059] ELISA kits (Nanjing Jiancheng Bioengineering Institute) and (Shanghai Shenggong Biotechnology Co., Ltd.) were used to detect the concentrations of creatine kinase isoenzyme MB (CK-MB) and troponin T (cTnT) in plasma.

[0060] Western blot: Protein samples were processed by 4% to 20% SDS-PAGE and transferred to PVDF membrane. After blocking, primary antibodies: TSPO (1:1000, Abcam, ab109497) and GAPDH (1:800, CUSABIO) were used; placed at 4°C for 12 hours. Secondary antibodies (Li-Cor Biosciences); incubated at 25°C for 1 hour; then all membranes were washed and detected using the Odyssey infrared imaging system.

[0061] 3) Results and Discussion

[0062] The experimental results are as follows Figure 2 As shown, by immunofluorescence staining, red is a myocardial marker, green is TSPO, and blue is DAPI, and it can be seen that TSPO expression is increased; by western blot detection, it was found that the expression of TSPO protein in SICD mice was higher than that in the control group mice; by qPCR detection of TSPO mRNA expression, it was found that the expression of TSPO mRNA in SICD mice was higher than that in the control group mice (referred to as Vehicle).

[0063] Example 2: Effect of TSOP on improving the survival rate and cardiac function of mice

[0064] This example further verifies through TSOP knockout experiments that TSOP knockout can effectively improve the survival rate and cardiac function of mice, specifically:

[0065] 1) Materials and methods

[0066] To demonstrate the role of TSPO in SICD, we constructed TSPO knockout (TSPO- / -) mice confirmed by PCR. The schematic diagram of constructing TSPO knockout (OK) mice is shown in Figure 3As shown: The gray box represents the non-coding region of the TSPO gene, and the blue box represents the coding region of the TSPO gene exon. The yellow box represents the gene deletion region. The primer combination and PCR product length used for genotyping are as follows: F1: forward primer 1, R1: reverse primer 1; F2: forward primer 2, R2: reverse primer 2.

[0067]

[0068] Model establishment, survival rate, and cardiac function testing: The SICD model was established by intraperitoneal injection of lipopolysaccharide (LPS) 25 mg / kg (dissolved in sterile saline) as the LPS group; the WT group was injected with an equal amount of saline; at the same time, the TSPO- / - group (specifically, TSPO- / - mice were intraperitoneally injected with saline) and the TSPO- / -+LPS group (specifically, TSPO- / - mice were intraperitoneally injected with lipopolysaccharide (LPS) 25 mg / kg) were introduced. 12 hours after injection, echocardiography was used to evaluate cardiac function. The mice were then killed, and blood and organ samples were collected for further experiments. The survival rate of the remaining mice was observed 72 hours after LPS injection.

[0069] 2) Detection method

[0070] Echocardiography: Cardiac function was measured using the Vevo3100 imaging system (Fujifilm VisualSonics, Inc., Toronto, Canada). Mice were anesthetized (2% isoflurane) and fixed on an examination table. Two-dimensional and M-mode imaging was performed in the parasternal long and short axes, respectively. Cardiac function indices, including left ventricular ejection fraction (LVEF), left ventricular short axis shortening index (LVFS), left ventricular end-diastolic dimension (LVEDD), and left ventricular end-systolic dimension (LVESD), were calculated. Images were analyzed blindly using VisualSonics software.

[0071] 3) Results and Discussion

[0072] From the experimental results Figure 4 As shown, from Figure 4 It can be seen that the survival rate of the WT group and TSPO- / - group did not change, but decreased significantly in the LPS group. Systemic knockout of TSPO (TSPO- / -) improved the survival rate of mice treated with LPS (25 mg / kg), accompanied by improved cardiac function compared with WT mice.

[0073] Example 3: Synthesis of TSOP-PROTAC

[0074] This embodiment provides a method for synthesizing TSOP-PROTAC, which specifically comprises the following steps:

[0075] 1) Synthesis of compound 3

[0076] Compound 2 (1.0 g, 3.06 mmol) was weighed into a 50 mL round-bottom flask, DCM (30 mL) was added and stirred to dissolve, and then DCC (820.5 mg, 3.98 mmol) and compound 1 (359.8 mg, 3.67 mmol) were added, and the reaction was allowed to proceed overnight at room temperature; after the reaction was completed, DCM was removed by vacuum decompression, and the residue was subjected to silica gel column chromatography (eluent, DCM:MeOH=125:1-60:1), the product was collected, and the product was concentrated under reduced pressure to obtain product 3 (665.3 mg), with a yield of 53.5%;

[0077] 2) Synthesis of compound 5

[0078] Compound 4 (349.0 mg, 1.23 mmol) was weighed into a 100 mL round-bottom flask, DCM (45 mL) was added and stirred to dissolve, then EDCI (306.7 mg, 1.60 mmol) and HOBT (232.6 mg, 1.72 mmol) were added, and after stirring at room temperature for 10 min, compound 3 (500.0 mg, 1.23 mmol) was added, and the mixture was reacted at 55 °C overnight. After the reaction was completed, DCM was removed under vacuum, and the residue was purified by silica gel column chromatography (eluent, DCM:MeOH=80:1-30:1) to obtain compound 5;

[0079] 3) Synthesis of Compound 7

[0080] Weigh 2.73g of compound 6 into a 50mL round-bottom flask, add an appropriate amount of THF, stir and suspend. Use a pipette to measure 2.50mL of bromoacetyl chloride and add it, reflux and react overnight; after the reaction is completed, cool the reaction system to room temperature, remove THF under vacuum, add ether, and a large amount of off-white solid precipitates. The solid is collected, washed with ether, and dried under vacuum to obtain 3.8g of the target product compound 7, with a yield of 97.2%.

[0081] The prepared compound 7 was subjected to H NMR spectrum test, and the results were as follows: Figure 5 As shown:

[0082] 1 H-NMR (ppm, 600MHz, DMSO-d6): δ11.11 (s, 1H), δ10.27 (s, 1H), δ8.47 (d, J=6.0Hz, 1H), δ7.88 (t, J=12.0Hz, 1H), δ7.69 (d, J=6.0Hz, 1H), δ5.18 (q, J=12.0Hz, 1H), δ4.34 (s, 2H), δ2.90 (m, 1H), δ2.55 (m, 2H), δ2.08 (m, 1H); indicating that compound 7 was successfully prepared;

[0083] 4) Synthesis of Compound 8

[0084] Compound 7 (1.5 g, 2.55 mmol) was weighed into a 100 mL round-bottom flask, and DMF (50 mL) was added to stir and dissolve, followed by the addition of NaN3 (489.8 mg, 7.65 mmol) and the reaction was carried out at 55°C overnight. After the reaction was completed, DMF was removed under vacuum, and the residue was subjected to silica gel column chromatography (eluent, PE:EA:DCM=(10:1:5-8:1:5) to collect the product, and concentrated under reduced pressure to obtain product 8 (800.0 mg), with a yield of 59.2%.

[0085] 5) Synthesis of Compound 9

[0086] Compound 8 (280.0 mg, 0.78 mmol) and compound 5 (524.2 mg, 0.78 mmol) were weighed into a 100 mL round-bottom flask, and DMF (15 mL) and methanol (10 mL) were added to stir and dissolve, and then copper acetate (10 mg, 0.05 mmol) and ascorbic acid (17.6 mg, 0.10 mmol) were added, and the mixture was reacted at 35° C. for 2 h. After the reaction was completed, DMF was removed under vacuum, and the residue was subjected to silica gel column chromatography (eluent, DCM:MeOH=80:1-30:1), and the product was collected and concentrated under reduced pressure to obtain product 9 (282.8 mg), with a yield of 35.3%.

[0087] The compound 9 (TSOP-PROTAC) prepared in this example was subjected to nuclear magnetic resonance hydrogen spectrum and high-resolution mass spectrometry identification tests, and the results were as follows Figure 6 and Figure 7 As shown:

[0088] 1H NMR (600MHz, CDCl3) δ9.36 (s, 1H), 9.08 (s, 1H), 8.71 (d, J = 8.5Hz, 1H), 8.63 (s, 1H), 8.03 (d, J = 8.2Hz, 1H), 7.78–7. 75(m,1H),7.68(t,J=7.9Hz,2H),7.64–7.62(m,2H),7.55(d,J=7.3Hz,1H),7.50–7.46(m,2H),7.45–7.39(m,2H),5. 37–5.34(m,1H),5.27–5.24(m,1H),4.90–4.87(m,1H),4.62–4.56(m,2H),4.28–4.22(m,2H),3.89–3.86(m,2H),3. 71–3.69(m,2H),3.61–3.58(m,18H),3.13–3.05(m,2H),2.87–2.80(m,2H),2.75–2.68(m,2H),2.14–2.04(m,4H).MS calcd for[C 50 H 54 ClN7O15]1028.3found 1028.4; indicating that compound 9 (TSOP-PROTAC) was successfully prepared.

[0089] Example 4: Effect of TSOP-PROTAC on TSOP

[0090] This example provides the experimental methods and results of the effects of TSOP-PROTAC on TSOP, specifically:

[0091] 1) Materials and methods

[0092] The effects of TSPO-PROTAC on TSPO expression were studied in vitro and in vivo using H9c2 cardiomyocytes and C57BL / 6 mice. Effect of TSPO-PROTAC on TSPO degradation in H9c2 cardiomyocytes: H9c2 cardiomyocytes were treated with TSPO-PROTAC (100, 200, 500, 1000 nM) for 12 or 24 h; TSPO expression was detected by Western blot.

[0093] Effect of TSPO-protac on TSPO expression in mouse heart: C57BL / 6 mice were given intraperitoneal injection of TSPO-PROTAC (5, 10, 20 mg / kg;) for 12 hours; Western blot was used to detect TSPO expression.

[0094] 2) Detection method

[0095] Cell culture: H9c2 cardiomyocytes (CL-0089; Procell, Wuhan) and HEK-293 cells (CL-0001; Procell) were cultured with H9c2 cardiomyocyte-specific medium (CM-0089; Procell) and HEK-293 cell-specific medium (CM-0001; Procell) at 37°C in a 5% CO2 and 95% CO2 air incubator, respectively. Cells were passaged regularly until about 80-90% confluence and treated with 10 μg / mL LPS or TSPO-PROTAC (100-1000 nM, dissolved in dimethyl sulfoxide) for 12-24 h. Untreated cells served as the control group (Vehicle).

[0096] Western blot: Protein samples were processed by 4% to 20% SDS-PAGE and transferred to PVDF membrane. After blocking, primary antibodies: TSPO (1:1000, Abcam, ab109497) and GAPDH (1:800, CUSABIO); placed at 4°C for 12 hours. Secondary antibodies (Li-Cor Biosciences); incubated at 25°C for 1 hour. All membranes were then washed and detected using the Odyssey infrared imaging system.

[0097] 3) Results and Discussion

[0098] The results are as follows Figure 8 As shown in the figure, it can be seen that TSPO-PROTAC (100-1000nM) treatment effectively reduced TSPO expression in H9c2 cardiomyocytes in a concentration-dependent manner at 12 or 24 hours; TSPO-PROTAC also reduced TSPO expression in the heart of SICD mice.

[0099] Example 5: Effect of TSOP-PROTAC on the survival rate and cardiac function of C57BL / 6 mice

[0100] This example provides the experimental method and results of the effect of TSOP-PROTAC on the survival rate and cardiac function of C57BL / 6 mice, specifically:

[0101] Model establishment and survival rate: SICD model was established by intraperitoneal injection of lipopolysaccharide (LPS) 25 mg / kg (dissolved in sterile saline), which was the LPS group. The vehicle group was injected with an equal amount of saline. At the same time, the TSPO-PROTAC group (specifically, C57BL / 6 mice were intraperitoneally injected with TSPO-PROTAC 20 mg / kg) and the TSPO- / -+LPS group (specifically, TSPO- / - mice were intraperitoneally injected with lipopolysaccharide (LPS) 25 mg / kg and TSPO-PROTAC 20 mg / kg) were introduced. At 12 hours after injection, cardiac function was evaluated by echocardiography, and the survival rate of the remaining mice was observed 72 hours after LPS injection.

[0102] 2) Detection method

[0103] Echocardiography: Cardiac function was measured using the Vevo3100 imaging system (Fujifilm VisualSonics, Inc., Toronto, Canada). Mice were anesthetized (2% isoflurane) and fixed on an examination table. Two-dimensional and M-mode imaging was performed in the parasternal long and short axes, respectively. Cardiac function indices, including left ventricular ejection fraction (LVEF), left ventricular short axis shortening index (LVFS), left ventricular end-diastolic dimension (LVEDD), and left ventricular end-systolic dimension (LVESD), were calculated. Images were analyzed blindly using VisualSonics software.

[0104] 3) Results and Discussion

[0105] The results are as follows Fig. 9 As shown in the figure, compared with the normal group, intraperitoneal injection of TSPO-PROTAC (20 mg / kg) in mice had no significant effect on cardiac function and survival rate, while the cardiac function and survival rate in the LPS group decreased significantly. TSPO-PROTAC significantly reversed the decrease in survival rate and cardiac function (LVEF, LVFS, LEVDD and LVESD) induced by LPS.

[0106] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein by equivalents. These modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention, and they should all be included in the scope of the claims and specification of the present invention.

Claims

1. Application of reagents for detecting TSPO expression in the preparation of products for diagnosing and treating cardiac insufficiency caused by sepsis.

2. The use according to claim 1, characterized in that: The TSOP is upregulated in patients with sepsis-induced cardiac dysfunction.

3. The use according to claim 1, characterized in that: The products include TSOP detection reagents.

4. A pharmaceutical composition for diagnosing and treating cardiac insufficiency caused by sepsis, characterized in that: The pharmaceutical composition comprises the agent for inhibiting TSOP expression for use in any one of claims 1 to 3.

5. The pharmaceutical composition according to claim 4, characterized in that The reagents include small molecule PROTACs that target degradation of TSOP.

6. The method for preparing a small molecule PROTAC targeting the degradation of TSOP according to claim 5, characterized in that: The steps include: 1) Synthesis of compound 3 1.0 g, 3.06 mmol of compound 2 was weighed into a 50 mL round-bottom flask, 30 mL of DCM was added and stirred to dissolve, then 820.5 mg, 3.98 mmol of DCC and 359.8 mg, 3.67 mmol of compound 1 were added, and the reaction was carried out at room temperature overnight; after the reaction was completed, DCM was removed by vacuum decompression, the residue was subjected to silica gel column chromatography, the product was collected, and the product was concentrated under reduced pressure to obtain product 3; 2) Synthesis of Compound 5 349.0 mg, 1.23 mmol of compound 4 was weighed into a 100 mL round-bottom flask, 45 mL of DCM was added and stirred to dissolve, then 306.7 mg, 1.60 mmol of EDCI and 232.6 mg, 1.72 mmol of HOBT were added, and after stirring at room temperature for 10 min, compound 3 (500.0 mg, 1.23 mmol) was added, and the mixture was reacted at 55 °C overnight. After the reaction was completed, DCM was removed under vacuum, and the residue was purified by silica gel column chromatography to obtain compound 5; 3) Synthesis of Compound 7 Weigh 2.73g of compound 6 into a 50mL round-bottom flask, add an appropriate amount of THF, stir and suspend. Use a pipette to measure 2.50mL of bromoacetyl chloride and add it, reflux and react overnight; after the reaction is completed, cool the reaction system to room temperature, remove THF under vacuum, add ether, and precipitate a large amount of off-white solids. Collect the solids, wash with ether, and vacuum dry to obtain the target product compound 7; 4) Synthesis of Compound 8 1.5 g, 2.55 mmol of compound 7 was weighed into a 100 mL round-bottom flask, 50 mL of DMF was added and stirred to dissolve, then 489.8 mg, 7.65 mmol of NaN3 was added and reacted at 55°C overnight; after the reaction was completed, DMF was removed under vacuum, the residue was subjected to silica gel column chromatography, the product was collected, and the product was concentrated under reduced pressure to obtain product 8; 5) Synthesis of Compound 9 Weigh 280.0 mg, 0.78 mmol of compound 8 and 524.2 mg, 0.78 mmol of compound 5 into a 100 mL round-bottom flask, add 15 mL of DMF and 10 mL of methanol and stir to dissolve, then add 10 mg, 0.05 mmol of copper acetate and 17.6 mg, 0.10 mmol of ascorbic acid, and react at 35 °C for 2 h; after the reaction is completed, DMF is removed under vacuum, the residue is chromatographed on a silica gel column, the product is collected, and concentrated under reduced pressure to obtain product 9.

7. Application of TSOP in the preparation of a pharmaceutical composition for diagnosing and treating cardiac insufficiency caused by sepsis.

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