PROTAC compound for targeted degradation of SPT5-Pol II as well as preparation method and application of PROTAC compound

Abstract

The invention discloses a PROTAC compound for targeted degradation of SPT5-Pol II as well as a preparation method and application of the PROTAC compound, the PROTAC compound comprises a target protein ligand, a connector and an E3 ubiquitin ligase ligand which are connected in sequence, and the target protein ligand has a structure as shown in a formula 1; the type of the linker is PEG (Polyethylene Glycol), and the linker comprises an active end group which is selected from one of amino, carboxyl and sulfydryl; the E3 ubiquitin ligase ligand is selected from one of a CRBN ligand, a VHL ligand and an MDM2 ligand. The PROTAC compound provided by the invention has an inhibition effect on inflammatory factor release mediated by inflammasome, and can be applied to treatment and prevention of immune diseases; the compound has an obvious inhibition effect on the tumor forming ability of cancer cells, has dose dependence, and can be applied to treatment and prevention of tumor diseases.

Description

Technical Field

[0001] This application belongs to the field of biomedical technology, and particularly relates to a PROTAC compound for targeted degradation of SPT5-Pol II, its preparation method and application. Background Art

[0002] Transcription mediated by RNA polymerase II (Pol II) is a highly regulated dynamic process. The precise regulation of transcription depends on a set of core factors or complexes that control each step of transcription and coordinate the transition from one step to another. Among these factors, the DRB sensitivity-inducing factor (DSIF) is an evolutionarily conserved and ubiquitously expressed heterodimeric transcription elongation factor composed of two subunits, SPT4 and SPT5, which plays a key role in almost all transcription steps. Studies have shown that SPT5 plays a special role in the transcription of inflammatory and stress-induced genes. Therefore, the regulation of SPT5 activity is of great significance for immune diseases. In addition, SPT5 can support the continuous cell proliferation of cancer cells by activating hTERT related to telomere maintenance. The human homolog of SPT5, SUPT5H, also interacts with the Paf complex involved in tumor development. Therefore, the therapy targeting the degradation of SPT5 can be applied in the anti-tumor field.

[0003] However, the existing SPT5 activity regulation strategies still cannot meet the needs of clinical applications. Research has confirmed that inhibiting SPT5 with shRNA can reduce the proliferation and metastasis ability of colon cancer cells. However, the poor cell membrane permeability of oligonucleotides limits their delivery in biological systems, and they have poor drug-likeness and are difficult to be applied in clinical treatment. On the other hand, there is little research work on small molecule inhibitors of SPT5. So far, only two literatures have reported small molecule inhibitors of SPT5. Among them, the lowest IC50 value of the existing small molecule inhibitors of SPT5 is 5 μM, and no nanomolar small molecule inhibitors of SPT5 have been found. The high dosing concentrations required for these small molecule inhibitors of SPT5 increase the risk of off-target toxicity. Therefore, more efficient, safe and drug-like SPT5 activity regulation strategies are needed. Summary of the Invention

[0004] The purpose of this application is to provide a PROTAC compound for targeted degradation of SPT5-Pol II, its preparation method and application, so as to solve the technical problems existing in the prior art that the SPT5 activity regulation strategy has poor drug-likeness, is difficult to be applied in clinical treatment, has a high dosing concentration, and increases the risk of off-target toxicity.

[0005] To achieve the above object, a first aspect of the present application provides a PROTAC compound for targeted degradation of SPT5-Pol II, comprising a target protein ligand, a linker, and an E3 ubiquitin ligase ligand connected in sequence, wherein the target protein ligand has the structure shown in Formula 1; the type of the linker is PEG-based, and the linker comprises an active end group selected from one of amino group, carboxyl group, and mercapto group; the E3 ubiquitin ligase ligand is selected from one of a CRBN ligand, a VHL ligand, and an MDM2 ligand;

[0006]

Formula 1

[0007] In Formula 1 represents the position connected to the linker.

[0008] In one or more embodiments, the active end group is an amino group, and the linker has the structure shown in Formula 2;

[0009]

Formula 2

[0010] In Formula 2, n is 3 or 4.

[0011] In one or more embodiments, the E3 ubiquitin ligase ligand is pomalidomide and has the structure shown in Formula 3:

[0012]

Formula 3

[0013] In one or more embodiments, the PROTAC compound has the structure shown in Formula 4:

Formula 4

[0014] In one or more embodiments, the PROTAC compound is selected from the following structures:

[0015]

[0016] To achieve the above object, a second aspect of the present application provides a preparation method of a PROTAC compound for targeted degradation of SPT5-Pol II, comprising:

[0017] Using 2,5-dimethoxyacetophenone and p-acetamidobenzaldehyde as raw materials to react to obtain a first intermediate: (2E)-3-(4-aminophenyl)-1-(2,5-dimethoxyphenyl)prop-2-en-1-one;

[0018] The first intermediate is subjected to a demethylation reaction to obtain a second intermediate: (2E)-3-(4-aminophenyl)-1-(2,5-dihydroxyphenyl)prop-2-en-1-one;

[0019] The pomalidomide-PEG-acid having the structure of Formula 5 and the second intermediate are subjected to a condensation reaction using a HATU / DIEA / DMF system. After the reaction is completed, extraction and purification are carried out in sequence to obtain the PROTAC compound having the structure of Formula 4;

[0020] [Formula 4]

[0021] [Formula 5]

[0022] In the above Formulas 4 and 5, n is 3 or 4.

[0023] In one or more embodiments, the step of reacting 2,5-dimethoxyacetophenone and p-acetamidobenzaldehyde as raw materials is specifically as follows:

[0024] 2,5-Dimethoxyacetophenone and p-acetamidobenzaldehyde are dispersed in absolute ethanol to obtain a suspension;

[0025] An aqueous NaOH solution is added dropwise to the suspension, and the mixture is stirred for reaction. After the reaction is completed, dilution, extraction, separation, washing, drying, filtration, evaporation and purification are carried out in sequence to obtain the first intermediate.

[0026] In one or more embodiments, the step of subjecting the first intermediate to a demethylation reaction to obtain a second intermediate is specifically as follows:

[0027] The first intermediate is dissolved in dichloromethane, and boron tribromide is added dropwise under ice bath conditions, and the mixture is stirred for reaction. After the reaction is completed, dilution, extraction, separation, washing, drying, filtration, evaporation and purification are carried out in sequence to obtain the second intermediate.

[0028] In one or more embodiments, the molar ratio of 2,5-dimethoxyacetophenone to p-acetamido benzene is 1:1.1 to 1.3; and / or,

[0029] The molar ratio of the pomalidomide-PEG-acid to the second intermediate is 1:1.4 to 1.6.

[0030] To achieve the above object, a third aspect of the present application provides an application of the PROTAC compound according to any one of the above embodiments or the PROTAC compound prepared by the preparation method according to any one of the above embodiments in the preparation of a drug for preventing or treating diseases related to SPT5-Pol II.

[0031] In one or more embodiments, the disease includes immune diseases and tumor diseases;

[0032] To achieve the above object, a fourth aspect of the present application provides a pharmaceutical composition, comprising the PROTAC compound described in any of the above embodiments or the PROTAC compound prepared by the preparation method described in any of the above embodiments, and a pharmaceutically acceptable carrier.

[0033] Different from the prior art, the beneficial effects of the present application are as follows:

[0034] The PROTAC compound of the present application can target and bind to SPT5 through the target protein ligand, and targetedly degrade SPT5 through the recruited E3 ligase, so as to achieve the purpose of reducing the activity level of SPT5 in the biological system. It has the advantages of high efficiency, strong selectivity, low toxicity, etc., and is more suitable for being developed into a clinical drug, solving the technical problems of the existing SPT5 inhibitors, such as high required dosing concentration, poor selectivity for SPT5, and poor drug-likeness;

[0035] The preparation method of the PROTAC compound of the present application has mild reaction conditions, a simple and efficient synthesis route, and strong popularity;

[0036] The PROTAC compound of the present application has an inhibitory effect on the release of inflammatory factors mediated by inflammasomes and can be applied to the treatment and prevention of immune diseases, such as autoimmune diseases like rheumatoid disease;

[0037] The PROTAC compound of the present application has an obvious inhibitory effect on the tumorigenic ability of cancer cells and has a dose-dependence, and can be applied to the treatment and prevention of tumor diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] To more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only some embodiments recorded in the present application. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0039] Figure 1 It is the electrophoresis experimental result diagram of each group in Effect Example 1 of the present application;

[0040] Figure 2 It is the data diagram of the expression levels of IL-1β and Caspase-1 in each group in Effect Example 1 of the present application;

[0041] Figure 3 It is the microscopic picture of the cell colonies cultured in each group in Effect Example 2 of the present application;

[0042] Figure 4 It is a data graph comparing the number and area of cell colonies cultured in each group in Example 2 of the effects of this application. Detailed implementation manners

[0043] To enable those skilled in the art to better understand the technical solutions in this application, the following will clearly and completely describe the technical solutions in the embodiments of this application with reference to the accompanying drawings in the embodiments of this application. Obviously, the described embodiments are only a part of the embodiments of this application, rather than all the embodiments. Based on the embodiments in this application, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of this application.

[0044] To solve the problem that the current SPT5 activity regulation strategy still cannot meet the needs of clinical applications, the applicant has developed a PROTAC compound that can target and degrade SPT5-Pol II. Compared with traditional small molecule inhibitors, this PROTAC compound has high efficiency and persistence, and requires a low dosing concentration, thus avoiding problems such as poor selectivity, high toxicity, and easy drug resistance caused by high dosing concentrations of small molecule inhibitors, and is more suitable for being developed into a clinical drug.

[0045] Specifically, the PROTAC compound of this application includes a target protein ligand, a linker, and an E3 ubiquitin ligase ligand connected in sequence.

[0046] Among them, the target protein ligand has the structure shown in Formula 1:

[0047]

Formula 1

[0048] In Formula 1 represents the position connected to the linker.

[0049] The type of the linker can be PEG-based and includes an active end group, and the active end group is selected from one of amino group, carboxyl group, and mercapto group.

[0050] In one embodiment, the active end group of the linker can be an amino group, and the linker can have the structure shown in Formula 2:

[0051]

Formula 2

[0052] In Formula 2, n is 3 or 4.

[0053] In one embodiment, the E3 ubiquitin ligase ligand can be selected from one of a CRBN ligand, a VHL ligand, and an MDM2 ligand.

[0054] In one embodiment, the E3 ubiquitin ligase ligand can be pomalidomide and has the structure shown in Formula 3:

[0055]

Formula 3

Formula 4

[0056] Specifically, the PROTAC compounds of the present application may be selected from the following structures:

[0057]

[0058] The PROTAC compounds of the present application include a target protein ligand that can target and bind to SPT5, and an E3 ligase ligand that can recruit the E3 ligase family. The two are connected by a linker. Through the target protein ligand, the PROTAC compound can target and bind to SPT5, and through the recruited E3 ligase, the targeted degradation of SPT5 can be carried out, so as to achieve the purpose of reducing the activity level of SPT5 in the biological system. It has the advantages of high efficiency, strong selectivity, low toxicity, etc., and solves the technical problems existing in the existing SPT5 inhibitors, such as high required dosing concentration, poor selectivity for SPT5, and poor drug-likeness.

[0059] The present application also provides a preparation method of the above-mentioned PROTAC compound, and the preparation method includes:

[0060] S100: Using 2,5-dimethoxyacetophenone and p-acetamidobenzaldehyde as raw materials to react to obtain a first intermediate: (2E)-3-(4-aminophenyl)-1-(2,5-dimethoxyphenyl)prop-2-en-1-one.

[0061] In one embodiment, the above reaction process may specifically be:

[0062] Disperse 2,5-dimethoxyacetophenone and p-acetamidobenzaldehyde in absolute ethanol to obtain a suspension;

[0063] Dropwise add an aqueous NaOH solution to the suspension, stir and react. After the reaction is completed, dilute, extract, separate, wash, dry, filter, evaporate and purify in sequence to obtain the first intermediate.

[0064] In one embodiment, the concentration of NaOH in the aqueous NaOH solution may be 40%.

[0065] In one embodiment, the reaction time of the above stirring reaction may be 8 - 12 h, and the reaction temperature may be room temperature.

[0066] In one embodiment, the molar ratio of 2,5-dimethoxyacetophenone to p-acetamido benzene may be 1:1.1 - 1.3.

[0067] In one embodiment, dilution can be carried out using deionized water, extraction can be carried out using an organic solvent such as ethyl acetate, washing can be carried out using saturated brine, drying can be carried out using anhydrous sodium sulfate, evaporation can specifically be rotary evaporation, and purification can be carried out using silica gel column chromatography.

[0068] The reaction formula of the above reaction is specifically as follows:

[0069]

[0070] S200. Perform a demethylation reaction on the first intermediate to obtain a second intermediate: (2E)-3-(4-aminophenyl)-1-(2,5-dihydroxyphenyl)prop-2-en-1-one.

[0071] Furthermore, the first intermediate prepared in S100 can be subjected to a demethylation reaction to further obtain a second intermediate.

[0072] In one embodiment, the demethylation reaction can specifically be:

[0073] Dissolve the first intermediate in dichloromethane, add boron tribromide dropwise under ice bath conditions, stir and react. After the reaction is completed, carry out dilution, extraction, separation, washing, drying, filtration, evaporation and purification in sequence to obtain the second intermediate.

[0074] In one embodiment, the molar ratio of the first intermediate to boron tribromide can be 1:1.4 - 1.6.

[0075] In one embodiment, the reaction time of the above stirring reaction can be 8 - 14 h, and the reaction temperature can be room temperature.

[0076] In one embodiment, dilution can be carried out using deionized water, extraction can be carried out using dichloromethane, washing can be carried out using saturated brine, drying can be carried out using anhydrous sodium sulfate, evaporation can specifically be rotary evaporation, and purification can be carried out using silica gel column chromatography.

[0077] The reaction formula of the above reaction is specifically as follows:

[0078]

[0079] S300. Carry out a condensation reaction on pomalidomide-PEG-acid with the structure of formula 5 and the second intermediate in the HATU / DIEA / DMF system. After the reaction is completed, carry out extraction and purification in sequence to obtain the PROTAC compound with the structure of formula 4.

[0080]

Formula 4

[0081]

Formula 5

[0082] In the above formulas 4 and 5, n is 3 or 4.

[0083] Based on the second intermediate obtained in S200, the second intermediate and pomalidomide-PEG-acid can be subjected to a condensation reaction to obtain the final PROTAC compound.

[0084] Among them, the condensation reaction uses a classical HATU / DIEA / DMF system, that is, using O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) as a condensing agent, using N,N-dimethylformamide (DMF) as a reaction solvent, and using N,N-diisopropylethylamine (DIEA) as an organic base to condense the second intermediate and pomalidomide-PEG-acid.

[0085] In one embodiment, the molar ratio of pomalidomide-PEG-acid to the second intermediate can be 1:1.4 - 1.6.

[0086] In one embodiment, purification can be carried out by silica gel column chromatography, and the mobile phase can be dichloromethane and methanol with a volume ratio of 10:1.

[0087] The reaction formula of the above reaction is specifically as follows:

[0088]

[0089] Based on the above preparation method, a PROTAC compound capable of targeting the degradation of SPT5-Pol II was prepared. The reaction conditions are mild, the synthesis route is simple and efficient, and the popularity is strong.

[0090] This application also provides a pharmaceutical composition, which includes the PROTAC compound of any one of the above embodiments and a pharmaceutically acceptable carrier.

[0091] The effects of the technical solution of this application will be further elaborated in detail below with specific examples.

[0092] Example 1:

[0093] A PROTAC compound SPT5-Protac 1 targeting the degradation of SPT5-Pol II has the following structural formula:

[0094]

[0095] The preparation method of SPT5-Protac 1 includes:

[0096] Step 1: Preparation of (2E)-3-(4-aminophenyl)-1-(2,5-dimethoxyphenyl)prop-2-en-1-one

[0097] 2 g of 2,5-dimethoxyacetophenone (11.10 mmol, 1.0 eq) and 2.17 g of p-acetamidobenzaldehyde (13.32 mmol, 1.2 eq) were dispersed in 100 mL of anhydrous ethanol to obtain a white suspension. Under the condition of 0 °C, 40% aqueous NaOH solution was added dropwise to the above white suspension, and the mixture was stirred at room temperature overnight.

[0098] After the reaction system was diluted with water, it was extracted with ethyl acetate, separated, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and rotary evaporated to obtain a crude product. The crude product was separated and purified by silica gel column chromatography to obtain 0.9 g of a pure target product in the form of a yellow solid, namely (2E)-3-(4-aminophenyl)-1-(2,5-dimethoxyphenyl)prop-2-en-1-one, with a yield of 28.62%.

[0099] The product was analyzed by 1H NMR, and the following data were obtained, which were consistent with the characteristics of the first intermediate:

[0100] 1 H NMR (500 MHz, DMSO-d 6 ) δ 7.40–7.30 (m, 3H), 7.11–7.00 (m, 3H), 6.95 (d, J = 3.0 Hz, 1H), 6.60–6.53 (m, 2H), 5.86 (s, 2H), 3.78 (s, 3H), 3.73 (s, 3H).

[0101] Step 2: Preparation of (2E)-3-(4-aminophenyl)-1-(2,5-dihydroxyphenyl)prop-2-en-1-one

[0102] 0.9 g of the first intermediate prepared in Step 1 (3.18 mmol, 1.0 eq) was dissolved in 10 mL of dichloromethane, and 25.4 mL of boron tribromide (25.41 mmol, 8.0 eq) was added dropwise under ice bath conditions, and the mixture was stirred at room temperature overnight;

[0103] After the reaction system was diluted with water, it was extracted with dichloromethane, separated, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and rotary evaporated to obtain a crude product. The crude product was separated and purified by silica gel column chromatography to obtain 0.36 g of a pure target product in the form of a red-brown solid, namely (2E)-3-(4-aminophenyl)-1-(2,5-dihydroxyphenyl)prop-2-en-1-one, with a yield of 44.40%.

[0104] The product was analyzed by 1H NMR, and the following data were obtained, which were consistent with the characteristics of the second intermediate:

[0105] 1 H NMR (400 MHz, DMSO-d 6)δ9.22(s,1H),7.72(d,J=15.1Hz,1H),7.62–7.47(m,4H),7.00(dd,J=8.8,2.9Hz,1H),6.80(d,J=8.8Hz,1H),6.61(d,J=8.5Hz,2H),6.04(s,2H).

[0106] Step 3:

[0107] 150 mg of pomalidomide - triethylene glycol - acid (314.16 μmol, 1.0 eq) was condensed with 120.29 mg of (2E)-3-(4-aminophenyl)-1-(2,5-dihydroxyphenyl)prop-2-en-1-one (471.23 μmol, 1.5 eq) obtained from Step 2 using a classical HATU / DIEA / DMF system;

[0108] After the reaction was complete, the crude product was obtained by extraction. The crude product was separated and purified by silica gel column chromatography (mobile phase: dichloromethane:methanol = 10:1, V / V) to obtain 30 mg of the pure target product as an orange solid, N-{4-[(1E)-3-(2,5-dihydroxyphenyl)-3-oxopropylidene]phenyl}-3-[(11-{[2-(2,6-dioxohexahydropyridin-3-yl)-1,3-dioxo-2,3-dihydro-1H-isoindol-4-yl]amino}-3,6,9-trioxaundecan-1-yl)oxy]propanamide, namely SPT5-Protac1, with a yield of 13.36%.

[0109] 1H NMR analysis of the product gave the following data, which is consistent with the characteristics of SPT5-Protac 1.

[0110] 1 H NMR(500MHz,DMSO-d 6)δ13.00(s,1H),11.10(s,1H),7.96(d,J=2.8Hz,1H),7.78(d,J=15.1Hz,1H),7.65–7.54(m,4H),7.27(dd,J=8.9,2.8Hz,1H),7.12(d,J=8.7Hz,1H),7.03(d,J=7.0Hz,1H),6.98(d,J=8.9Hz,1H),6.61(dd,J=7.0,5.0Hz,3H),6.10(s,2H),5.05(dd,J=12.8,5.4Hz,1H),3.76(t,J=6.3Hz,2H),3.61(t,J=5.4Hz,2H),3.45(q,J=5.6Hz,2H),2.91–2.81(m,3H),2.65–2.52(m,3H).

[0111] Example 2:

[0112] A PROTAC compound SPT5-Protac 2 that targets the degradation of SPT5-Pol II, with the structural formula as follows:

[0113]

[0114] The preparation method of SPT5-Protac 2 includes:

[0115] Step 1: The second intermediate, namely (2E)-3-(4-aminophenyl)-1-(2,5-dihydroxyphenyl)prop-2-en-1-one, was prepared by the same method as in Example 1;

[0116] Step 2:

[0117] 250 mg of pomalidomide-tetraethylene glycol-acid (481.18 μmol, 1.0 eq) was subjected to a condensation reaction with 184.25 mg of (2E)-3-(4-aminophenyl)-1-(2,5-dihydroxyphenyl)prop-2-en-1-one (721.78 μmol, 1.5 eq) obtained in Step 1 using the classical HATU / DIEA / DMF system;

[0118] After the reaction was completed, the crude product was obtained by extraction. The crude product was separated and purified by silica gel column chromatography (mobile phase: dichloromethane: methanol = 10:1, V / V) to obtain 65 mg of the pure target product in the form of an orange solid, N-{4-[(1E)-3-(2,5-dihydroxyphenyl)-3-oxopropyl-1-enyl]phenyl}-3-[(8-{[2-(2,6-dioxohexahydropyridin-3-yl)-1,3-dioxo-2,3-dihydro-1H-isoindol-4-yl]amino}-3,6-dioxooct-1-yl)oxy]propanamide, namely SPT5-Protac 2, with a yield of 17.80%.

[0119] The 1H NMR analysis of the product was performed, and the following data were obtained, which were consistent with the characteristics of SPT5-Protac 2.

[0120] 1 H NMR(500MHz,DMSO-d 6 )δ13.01(s,1H),11.09(s,1H),7.97(d,J=2.8Hz,1H),7.65–7.54(m,4H),7.27(dd,J=8.9,2.8Hz,1H),7.13(d,J=8.6Hz,1H),7.01(dd,J=25.8,8.0Hz,2H),6.65–6.56(m,3H),6.10(s,2H),5.05(dd,J=12.8,5.4Hz,1H),3.76(t,J=6.2Hz,2H),3.61(t,J=5.5Hz,2H),3.57–3.51(m,12H),3.46(q,J=5.5Hz,2H),2.93–2.81(m,3H),2.66–2.52(m,3H).

[0121] Effect Example 1: Study on the inhibitory effect on the release of inflammatory factors

[0122] SPT5-Protac1 and SPT5-Protac2 prepared in Examples 1 and 2 were respectively selected as samples to study their inhibitory effects on the release of inflammatory factors in murine macrophages J7774a.1. The specific experimental procedures were as follows:

[0123] (1) J774a.1 cells were seeded in 12-well plates overnight. Subsequently, the medium was changed to opti-MEM, and samples with concentrations of 0.25 μM, 2.5 μM, and 25 μM were added to each well plate. Then, the cells were stimulated with lipopolysaccharide LPS (500 ng / mL) for 3 hours, and then stimulated with the inflammasome activator ATP (5 mM) for 1 h. The supernatant was collected, and a control group ATP without adding the sample and a blank group sham without adding the sample and not being primed with LPS were set up;

[0124] (2) Collect the supernatant into a 2 mL EP tube, centrifuge at 4 °C and 3000 r / min for 5 minutes to remove residual cells. Use chemical purification method (a mixture of anhydrous methanol and chloroform in proportion) to separate the proteins in the supernatant, centrifuge at 4 °C and 13000 r / min for 10 minutes, discard the supernatant, and collect the precipitate;

[0125] (3) Dry the protein precipitate, add 1X Loading butter (containing 1 mM PMSF), mix well, and boil on a 99 °C metal bath for 5 min to completely dissolve the precipitate;

[0126] (4) Use Western blot method to detect the expression levels of IL-1β and Caspase-1 proteins in the precipitate, and obtain Figure 1 and Figure 2 , Figure 1 which are the electrophoresis experimental result graphs of each group in Effect Example 1 of this application, Figure 2 and

[0127] As Figure 1 and Figure 2 shown, compared with the control group, SPT5-Protac1 at a concentration of 0.25 μM showed an obvious inhibition of the expression level of IL-1β (P17), and the inhibition rate gradually increased with the increase of the concentration;

[0128] Compared with the control group, SPT5-Protac1 at a concentration of 2.5 μM could significantly reduce the expression level of Caspase-1 (P20), and lower concentrations of SPT5-Protac1 also showed a certain inhibitory effect, and the overall inhibition rate increased with the increase of the concentration of SPT5-Protac1;

[0129] Compared with the control group, SPT5-Protac2 at a concentration of 2.5 μM could significantly reduce the expression level of IL-1β (P17), and lower concentrations of SPT5-Protac2 also showed a certain inhibitory effect, and the overall inhibition rate increased with the increase of the concentration of SPT5-Protac2;

[0130] Compared with the control group, SPT5-Protac2 at a concentration of 2.5 μM could significantly reduce the expression level of Caspase-1 (P20), and lower concentrations of SPT5-Protac2 also showed a certain inhibitory effect, and the overall inhibition rate increased with the increase of the concentration of SPT5-Protac2.

[0131] As can be seen from the above data, both SPT5-Protac1 and SPT5-Protac2 can dose-dependently reduce the expression of IL-1β (p17) and Caspase-1 (p20) in the cell supernatant, indicating that both of them have inhibitory effects on the release of inflammatory factors mediated by inflammasomes and can be applied to the treatment and prevention of immune diseases, such as autoimmune diseases like rheumatoid arthritis.

[0132] Effect Example 2: Study on the inhibitory effect on the tumorigenic ability of cancer cells

[0133] Select SPT5-Protac1 with the best inhibitory effect in Effect Example 1 for further experiments to study its inhibitory effect on the tumorigenic ability of cancer cells. The specific experimental process is as follows:

[0134] (1) Preparation of cell suspension: Take pancreatic cancer KPC cells in the logarithmic growth phase, digest them with 0.25% trypsin and then terminate the digestion. Centrifuge at 1000 rpm for 5 min to collect the cells;

[0135] (2) Cell seeding: Seed the cells evenly on a 6-well plate at a density of 2000 / well, and add SPT5-Protac1 with concentrations of 0.025 μM, 0.25 μM, 2.5 μM, and 25 μM for culture. At the same time, set up a control group control without adding SPT5-Protac1;

[0136] (3) Cell culture: Place the culture plate in an incubator at 37 °C and 5% CO 2 and incubate statically for 10 days. During this period, change the fresh medium every 2 days and supplement the corresponding concentration of SPT5-Protac1;

[0137] (4) Cell fixation and staining: After visible clones are formed (i.e., the diameter > 0.5 mm) to the naked eye, discard the medium and gently wash twice with pre-cooled PBS. Add 4% paraformaldehyde to fix for 15 min (or use methanol to fix for 10 min), discard the fixative, and wash three times with PBS; Add 0.5% crystal violet staining solution to cover the cell layer and stain at room temperature for 30 min. Discard the staining solution and gently rinse with deionized water until the background is colorless.

[0138] (5) Colony counting and analysis: Invert the culture plate and observe it under a microscope (4× objective lens) to obtain Figure 3 After that, scan the culture plate and use ImageJ software for analysis to obtain Figure 4 Figure 3 is the microscopic picture of the cell colonies cultured in each group in Effect Example 2 of this application, Figure 4 is the data graph of the comparison of the number and area of the cell colonies cultured in each group in Effect Example 2 of this application.

[0139] AsFigure 3 and Figure 4 As shown, 0.025 μM of SPT5-Protac1 exhibited an obvious inhibitory effect on the aggregation number of KPC cells, and the inhibitory effect increased with the increase of SPT5-Protac1, indicating that SPT5-Protac1 has an obvious inhibitory effect on the tumorigenic ability of pancreatic cancer cells and has a dose-dependence, and can be applied to the treatment and prevention of tumor diseases.

[0140] For those skilled in the art, it is obvious that the present application is not limited to the details of the above exemplary embodiments, and can be implemented in other specific forms without departing from the spirit or basic characteristics of the present application. Therefore, from any point of view, the embodiments should be regarded as exemplary and non-limiting. The scope of the present application is defined by the appended claims rather than the above description. Therefore, all changes falling within the meaning and scope of the equivalent elements of the claims are intended to be included in the present application. Any reference signs in the claims should not be construed as limiting the claimed rights.

[0141] In addition, it should be understood that although this specification is described according to embodiments, not every embodiment only contains an independent technical solution. This narrative way of the specification is only for clarity. Those skilled in the art should regard the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A PROTAC compound targeting degradation of SPT5-Pol II, characterized in that It comprises a target protein ligand, a linker and an E3 ubiquitin ligase ligand which are connected in sequence, wherein the target protein ligand has a structure as shown in Formula 1; the linker is of the PEG type, and the linker comprises an active terminal group, wherein the active terminal group is selected from one of an amino group, a carboxyl group and a thiol group; and the E3 ubiquitin ligase ligand is selected from one of a CRBN ligand, a VHL ligand and an MDM2 ligand; [Formula 1] In Formula 1, "-------" indicates the position where the linker is connected.

2. The PROTAC compound according to claim 1, characterized in that The active terminal group is an amino group, and the linker has a structure shown in Formula 2; and / or, The E3 ubiquitin ligase ligand is pomalidomide, which has a structure shown in Formula 3: [Formula 2] [Formula 3] In Formula 2, n is 3 or 4, and in Formula 3, "-------" indicates the position of connection with the linker.

3. The PROTAC compound according to claim 1 or 2, characterized in that It has the structure shown in Formula 4: [Formula 4] In formula 4, n is 3 or 4.

4. The PROTAC compound according to claim 3, characterized in that Select from the following structures:

5. A method for preparing a PROTAC compound that targets and degrades SPT5-Pol II, characterized in that: include: Using 2,5-dimethoxyacetophenone and 4-acetylaminobenzaldehyde as raw materials for reaction, a first intermediate product is obtained: (2E)-3-(4-aminophenyl)-1-(2,5-dimethoxyphenyl)prop-2-en-1-one; The first intermediate product is subjected to a demethylation reaction to obtain a second intermediate product: (2E)-3-(4-aminophenyl)-1-(2,5-dihydroxyphenyl)prop-2-en-1-one; The pomalidomide-PEG-acid of the structure of Formula 5 and the second intermediate product are subjected to a condensation reaction using a HATU / DIEA / DMF system, and after the reaction is completed, extraction and purification are performed in sequence to obtain the PROTAC compound of the structure of Formula 4; [Formula 4] [Formula 5] In the above formula 4 and formula 5, n is 3 or 4.

6. The preparation method according to claim 5, characterized in that: The specific steps of reacting 2,5-dimethoxyacetophenone and 4-acetaminobenzaldehyde as raw materials are as follows: Dispersing 2,5-dimethoxyacetophenone and p-acetaminobenzaldehyde in anhydrous ethanol to obtain a suspension; A NaOH aqueous solution is added dropwise to the suspension, and the mixture is stirred for reaction. After the reaction is completed, the mixture is diluted, extracted, separated, washed, dried, filtered, evaporated and purified in sequence to obtain the first intermediate product.

7. The preparation method according to claim 5, characterized in that: The step of subjecting the first intermediate product to a demethylation reaction to obtain a second intermediate product is specifically: The first intermediate product is dissolved in dichloromethane, boron tribromide is added dropwise in an ice bath, and the reaction is stirred. After the reaction is completed, dilution, extraction, separation, washing, drying, filtration, evaporation and purification are carried out in sequence to obtain the second intermediate product.

8. The preparation method according to claim 5, characterized in that: The molar ratio of 2,5-dimethoxyacetophenone to 4-acetaminobenzene is 1:1.1-1.3; and / or, The molar ratio of the pomalidomide-PEG-acid to the second intermediate product is 1:1.4-1.

6.

9. Use of a PROTAC compound according to any one of claims 1 to 4 or a PROTAC compound prepared by the preparation method according to any one of claims 5 to 8 in the preparation of a medicament for preventing or treating a disease associated with SPT5-Pol II.

10. The use according to claim 9, characterized in that: The diseases include immune diseases and tumor diseases.

11. A pharmaceutical composition, characterized in that It comprises a PROTAC compound according to any one of claims 1 to 4 or a PROTAC compound prepared by the preparation method according to any one of claims 5 to 9, and a pharmaceutically acceptable carrier.

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