CN115873014A - Controllable proteolysis targeting chimera and application thereof - Google Patents

Controllable proteolysis targeting chimera and application thereof Download PDF

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CN115873014A
CN115873014A CN202211579088.8A CN202211579088A CN115873014A CN 115873014 A CN115873014 A CN 115873014A CN 202211579088 A CN202211579088 A CN 202211579088A CN 115873014 A CN115873014 A CN 115873014A
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pamam
tco
degradation
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tcb
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尤启冬
姜正羽
范静
金雨辉
王茹嫣
毕欣宇
朱志伟
王轩宇
徐晓莉
郭小可
王磊
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China Pharmaceutical University
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Abstract

The invention discloses a controllable proteolysis targeting chimera with a general formula I and application thereof, and belongs to the technical field of biochemistry. The controllable proteolysis targeting chimera is a compound with tetrazine group as mother nucleus, and the compound can realize the termination regulation of targeted protein degradation through bio-orthogonal reaction, thereby providing a feasible scheme for terminating the degradation process of PROTACs. The invention also discloses a connection-adsorption system for instantly terminating the degradation of the target protein, which is a systemThe system consists of a controlled proteolytic targeting chimera and PAMAM-G5-TCO, which can terminate the degradation of BET family proteins in living cells.
Figure DDA0003987367330000011

Description

Controlled proteolysis targeting chimera and application thereof
Technical Field
The invention belongs to the technical field of biochemistry, and particularly relates to a controllable proteolysis targeting chimera and application thereof.
Background
In recent years, a targeted induced protein degradation technology (PROTACs) has been vigorously developed as a novel drug development strategy by its unique mode of action and is receiving attention from the global academia and pharmaceutical industry. The PROTACs technology is mainly characterized in that a bifunctional conjugate is utilized to regulate an in-vivo protein ubiquitination degradation system and induce the degradation of in-vivo specific proteins in a targeted manner, so that the content of the proteins in cells is directly reduced. The technology fully utilizes the in vivo ubiquitin-proteasome system to realize the degradation of the specific induction target protein, solves the selectivity problem of the induced protein degradation, and is expected to solve the problem that a plurality of traditional small molecules are difficult to target the target which is difficult to form a drug.
The original aim of the development of PROTACs technology is to degrade the traditional meaning of 'non-druggable' target, and the target comprises most of framework proteins and partial transcription factors. The PROTACs belong to event-driven molecules, and the catalytic degradation characteristic of the technology also brings excessive degradation risk to functional targets in the degradation process of target proteins. At present, excessive degradation of target protein caused by the irreducible degradation process is one of the important problems that the PROTACs technology can not solve in the application process.
In recent years, there has been considerable research effort devoted to the development of regulatable PROTACs by the introduction of stimulus responsive elements. A number of intracellular and extracellular stimulatory signals have been applied to the construction of activatable PROTACs, including: (1) an external optical signal; (2) changes in the tumor microenvironment: hypoxia, redox factors (such as GSH) and specific enzymes (such as cathepsin b, hydrolase, dehydrogenase, etc.) overexpressed in tumors, which can effectively alleviate potential extratissue side effects of PROTAC drugs.
To date, studies on controllable PROTACs have focused on stimulus-responsive elements, mainly on activated PROTACs, while studies on termination processes have been blank.
Disclosure of Invention
The purpose of the invention is as follows: the invention aims to provide a controllable proteolysis targeting chimera based on a bioorthogonal principle and application thereof aiming at the defects of the prior art. The controllable proteolysis targeting chimeras (hereinafter referred to as Tz-PROTACs) are compounds taking tetrazine groups as mother nuclei, can realize the termination regulation and control of targeted protein degradation through bio-orthogonal reaction, and further provide a feasible scheme for terminating the degradation process of PROTACs.
The present invention develops an effective immediate termination strategy for event-driven protein degradation processes. The ideal termination strategy requires a high response rate and sensitivity to rapidly and completely eliminate the intracellular sub-stoichiometric amount of PROTACs molecules. To achieve this objective, the present invention designs a "link-adsorption" system based on PAMAM dendrimers and the inverse electron Diels-Alder reaction (IEDDA): wherein, the PAMAM dendrimer forms a perfect bracket, and the surface of the PAMAM dendrimer can be modified by different functional groups. The modified PAMAM dendrimer can absorb the PROTACs through bio-orthogonal reaction, theoretically, the high reaction rate of IEDDA and a large number of groups on the surface of the PAMAM molecule can rapidly and thoroughly eliminate the intracellular free PROTACs, thereby completely stopping the event-driven protein degradation in living cells.
The technical scheme is as follows: the purpose of the invention is realized by the following technical scheme:
the invention provides a controllable proteolysis targeting chimera shown as a general formula I,
wherein,
Figure BDA0003987367310000021
general formula IR 1 Is selected from
Figure BDA0003987367310000022
R 2 Is selected from
Figure BDA0003987367310000023
Figure BDA0003987367310000024
n＝1～3。
The controlled proteolytic targeting chimeras of the invention of general formula I are preferably the following compounds:
Figure BDA0003987367310000025
/>
Figure BDA0003987367310000031
wherein n =1 to 3.
The controllable proteolytic targeting chimeras of the invention are further preferably compounds with n =1.
The invention also provides application of the controllable proteolysis targeting chimera in termination of targeted protein degradation.
The controllable proteolysis targeting chimera is used for preparing a PROTAC or a small molecule inhibitor with molecular biological activity and capability of terminating targeted protein degradation.
The invention also provides a connection-adsorption system for instantly terminating the degradation of the target protein, which consists of the controllable proteolysis target chimera and the PAMAM-G5-TCO, wherein the PAMAM-G5-TCO is trans-cyclooctene modified polyamide PAMAM dendrimer, and the PAMAM-PROTACs are formed through inverse electron Diels-Alder reaction to completely absorb the controllable proteolysis target chimera.
The invention designs a connection-adsorption system based on the bioorthogonal reaction of tetrazine and trans-cyclooctene. The tetrazine group is a connecting fragment with small volume, and can be easily embedded into PROTACs molecules to construct heterobifunctional molecules. Meanwhile, the fifth generation PAMAM dendrimer is a commercial, highly branched, monodisperse spherical nanomaterial. The material has a perfect spherical skeleton, and a large number of functional segments can be modified on the surface of a huge molecule. Theoretically, PAMAM-G5-TCO can form PAMAM-PROTACs through IEDDA reaction, thereby completely absorbing Tz-PROTACs. Due to the rapid reactivity of IEDDA and the numerous groups on the surface of PAMAM-G5-TCO macromolecules, intracellular free PROTACs can be rapidly and completely eliminated, thereby stopping event-driven degradation in living cells.
The PAMAM-G5-TCO is synthesized according to the following method:
mixing PAMAM-G5-NH 2 Dissolving in an organic solvent, adding a TCO-NHS-Ester solution, stirring at room temperature, dialyzing the reaction solution with pure water after the reaction is finished, collecting the dialyzate, and freeze-drying to obtain the PAMAM-G5-TCO product.
In particular, the organic solvent is selected from methanol.
A specific preferred embodiment of the present invention is:
weighing 50mg of PAMAM-G5-NH 2 Dissolving in 3mL of methanol, adding 1mL of TCO-NHS-Ester (40 eq.) methanol solution, stirring and reacting for 0.5 hour at room temperature, transferring the reaction solution into a dialysis bag (with molecular weight cutoff of 2 kDa) after the reaction is finished, dialyzing for 24 hours by pure water, collecting the dialysate, and freeze-drying to obtain the PAMAM-G5-TCO product.
The invention also provides trans-cyclooctene modified polyamide PAMAM dendrimer PAMAM-G5-TCO, which has the following structural formula:
Figure BDA0003987367310000041
has the beneficial effects that:
(1) The invention provides a controllable proteolysis targeting chimera based on a bioorthogonal principle, which can controllably regulate the level of a specific target protein in a cell. Compared with the reported proteolysis targeting chimera, the compound can immediately terminate the degradation process on the basis of high-efficiency degradation of the target protein, and can realize free regulation of the level of the target protein in the cell.
(2) The present invention provides a link-adsorption system for instantly terminating degradation of a target protein. The invention selects the BET family protein as a target protein to verify the concept of a 'connection-adsorption' strategy, and experimental results show that the 'connection-adsorption' system can stop the degradation of the BET family protein in living cells.
(3) The invention provides a trans-cyclooctene modified polyamide PAMAM dendrimer PAMAM-G5-TCO, which is a dendrimer with bio-orthogonal activity. The molecule is a spherical structure and has certain rigidity and stable tertiary structure. Meanwhile, the molecule has low toxicity, high water solubility, good membrane permeability and good biocompatibility. Can be used to terminate event-driven processes in vivo.
Drawings
FIG. 1 is a TEM image of PAMAM-G5-TCO dendrimer;
FIG. 2 is a Zeta potential diagram of PAMAM-G5-TCO;
FIG. 3 is a table showing the immunoblotting method for screening advantageous compounds of TCB and TCP series;
FIG. 4 is an immunoblot assay of TCB-2 for BET family protein concentration-dependent degradation;
FIG. 5 is an immunoblot assay of TCP-1 for concentration-dependent degradation of PARP protein;
FIG. 6 is a immunoblot of TCB-2 for time dependent degradation of BET family proteins;
FIG. 7 is a diagram of immunoblot detection of TCP-1 for time-dependent degradation of PARP protein;
FIG. 8 is an immunoblot assay of TCB-2 induced BET family protein degradation mechanism;
FIG. 9 shows the effect of flow cytometry on the apoptosis of MV-4-11 cell line by TCB-2 at different concentrations;
FIG. 10 shows the anti-cell proliferative activity of TCB-2 and (+) -JQ-1 on MV-4-11 cell line;
FIG. 11 is a HPLC quantitative analysis of click reaction between PAMAM-G5-TCO and TCB-2;
FIG. 12 is a graph of the effect of different equivalents of PAMAM-G5-TCO on TCB-2 target inhibitory activity;
FIG. 13 is a FP assay of (+) -JQ-1, TCB-2 and TCB-2+ PAMAM-G5-TCO;
FIG. 14 is a blot of the immunodetection of BET family proteins in MV-4-11 cells treated with TCB-2+ PAMAM-G5-TCO at various ratios for 24 hours;
FIG. 15 shows TCB-2 and TCB-2: immunoblot assay of PAMAM-G5-TCO (1;
FIG. 16 is TCB-2 and TCB-2: immunoblot assay of PAMAM-G5-TCO (1;
FIG. 17 shows TCB-2 and TCB-2: immunoblot assay of PAMAM-G5-TCO (1;
FIG. 18 is TCB-2 and TCB-2: immunoblot assay of PAMAM-G5-TCO (1;
FIG. 19 is an immunoblot assay of PAMAM-G5-TCO to stop degradation of PARP protein in SW620 cells.
FIG. 20 shows flow cytometry to detect the effect of (+) -JQ-1, TCB-2 and TCB-2+ PAMAM-G5-TCO on apoptosis of MV-4-11 cell line;
FIG. 21 shows the anti-cell proliferative activity of (+) -JQ-1, TCB-2 and TCB-2+ PAMAM-G5-TCO on MV-4-11 cell line.
Detailed Description
The technical solution of the present invention is described in detail by the following specific examples, but the scope of the present invention is not limited to the examples.
All chemicals purchased from commercial suppliers were used as received, unless otherwise indicated. The chemical reagents are all commercially available chemical pure or analytical pure. All solvents were reagent grade, purified and dried by standard methods if necessary. All reactions were monitored by Thin Layer Chromatography (TLC) on a visual silica gel plate (GF-254).
1 H-NMR、 13 The C-NMR spectra were determined by a Bruker model AV 300 (300 MHz) nuclear magnetic resonance apparatus with TMS as internal standard and CDCl3, CD3OD or DMSO-d6 as solvents, and the chemical shifts (δ) reported were parts per million (ppm) of Tetramethylsilane (TMS). Mass spectra were measured by an Advion Expression LCMS type mass spectrometer (ESI-MS) and a Water Q-Tof type mass spectrometer (HRMS).
Purity was determined by Shimadzu Reservoir Tray type HPLC. The chromatographic column is an Agilent C18 (4.6X 250mm,5 μm) reverse phase column; the mobile phase was chromatographed methanol with water =8 and a flow rate of 1.0mL/min. The eluent is petroleum ether (boiling range 60-90 ℃) and ethyl acetate, dichloromethane and methanol.
Examples 1-9 below are for the synthesis and structural confirmation of the PROTAC molecule.
Figure BDA0003987367310000061
Example 1: synthesis of 2- ((S) -4- (4-chlorophenyl) -2,3, 9-trimethyl-6H-thieno [3,2-f ] [1,2,4] triazolo [4,3-a ] [1,4] diazepin-6-yl) -N- ((6- (4- (2- (2- (2, 6-dioxopiperidin-3-yl) -1, 3-dioxoisoindol-4-yl) amino) ethyl) amino) -2-oxyethyl) phenyl) -1,2,4, 5-tetrazin-3-yl-methyl) aminocarboxamide (TCB-1, compound 11 a)
1. 2- (2, 6-dioxopiperidin-3-yl) -5-fluoroisoindoline-1, 3-dione (Compound 2)
Compound 1 (1.0 g,6 mmol), 3-aminopiperidine-2, 6-dione hydrochloride (1.0 g,6 mmol) and sodium acetate (0.6 g, 7.2mmol) were dissolved in glacial acetic acid (15 mL). After 10 hours of reaction at 60 ℃, the reaction was poured into saturated brine (500 mL), the pH was adjusted to neutral with sodium bicarbonate, extracted with ethyl acetate (100 mL × 3), and the organic phase was purified by column chromatography (DCM) to give compound 2 as a gray solid (1.04g, 39.2%).
1 HNMR(300MHz,DMSO-d 6 )δ11.18(s,1H),7.97(ddd,J＝8.4,7.3,4.6Hz,1H),7.84–7.71(m,2H),5.18(dd,J＝12.9,5.4Hz,1H),2.91(ddd,J＝17.3,14.0,5.3Hz,1H),2.67–2.57(m,1H),2.55(d,J＝4.4Hz,1H),2.07(ddt,J＝13.2,5.6,2.7Hz,1H)。
2. Tert-butyl (2- ((2- (2, 6-dioxopiperidin-3-yl) -1, 3-dioxoisoindolin-4-yl) amino) ethyl) carbamate (Compound 3)
Compound 2 (500mg, 1.81mmol) and tert-butyl (2-aminoethyl) carbamate (289mg, 1.81mmol) were dissolved in DMF (7 mL), and potassium carbonate (750mg, 5.4 mmol) was added. After 10 hours of reaction at 90 ℃, the reaction was poured into saturated brine (500 mL), extracted with ethyl acetate (100 mL × 3), and the organic phase was purified by column chromatography (DCM: meOH =200 1) to give compound 3 as a green solid (399.04mg, 53.05%).
1 H NMR(300MHz,DMSO-d 6 )δ11.12(s,1H),7.60(t,J＝7.8Hz,1H),7.16(d,J＝8.6Hz,1H),7.08–6.97(m,2H),6.74(t,J＝6.1Hz,1H),5.07(dd,J＝12.8,5.4Hz,1H),3.38(d,J＝6.0Hz,2H),3.13(q,J＝6.1Hz,2H),2.91(ddd,J＝18.6,14.1,5.2Hz,1H),2.61(d,J＝12.4Hz,1H),2.57(d,J＝3.7Hz,1H),2.03(d,J＝12.1Hz,1H),1.38(s,9H)。
3. 2- (4- (6- (((tert-butoxycarbonyl) amino) methyl) -1,2,4, 5-tetrazin-3-yl) phenyl) acetic acid (Compound 5)
Tert-butyl (cyanomethyl) carbamate (14.0 g, 89.74mmol) and compound 4 (1.9g, 11.8mmol) were dissolved in DMSO (4.5 mL). 3-mercaptopropionic acid (1.25g, 11.8mmol) and hydrazine hydrate (9.4g, 188.82mmol) were added dropwise under ice bath, and stirred for 12 hours under nitrogen protection. The reaction solution was dropped into a solution of sodium nitrite (18.9g, 177.02mmol), adjusted in pH =4 with 1M dilute hydrochloric acid, extracted with ethyl acetate (100 mL × 3), then washed with a sodium bicarbonate solution, adjusted in pH =3 with 1M dilute hydrochloric acid, and extracted with ethyl acetate (100 mL × 3). The organic phase was purified by column chromatography (DCM: meOH = 200) to afford compound 5 as a pink solid (1.23g, 30.16%).
1 H NMR(300MHz,DMSO-d 6 )δ12.51(s,1H),8.46(d,J＝8.0Hz,2H),7.72(t,J＝6.0Hz,1H),7.59(d,J＝8.1Hz,2H),4.78(d,J＝5.9Hz,2H),3.77(s,2H),1.42(s,9H)。
4. (6- (4- (2- ((2- (2, 6-dioxopiperidin-3-yl) -1, 3-dioxoisoindolin-4-yl) amino) ethyl) amino) -2-oxoethyl) phenyl) -1,2,4, 5-tetrazin-3-yl) methyl) carbamic acid tert-butyl ester (Compound 6)
Compound 3 (3, 81mg, 0.19mmol) was dissolved in DCM (2 mL) and trifluoroacetic acid (1 mL) was added. After stirring at room temperature for 8 hours, the reaction solution was distilled under reduced pressure to give a green solid. The green solid and Compound 5 (5.67mg, 0.19mmol) were dissolved in DMF (5 mL) and a solution of DIEA (49mg, 0.38mmol) and pyBop (128mg, 0.24mmol) in DMF (5 mL) was added dropwise over an ice bath. After stirring at room temperature for 12 hours, the reaction mixture was poured into saturated brine (100 mL) and extracted with ethyl acetate (30 mL. Times.3). The organic phase was purified by column chromatography (DCM: meOH = 50) to give compound 6 as a pink solid (76.72mg, 62.29%).
1 H NMR(300MHz,DMSO-d 6 )δ11.12(s,1H),8.42(d,J＝7.9Hz,3H),7.73(t,J＝5.9Hz,1H),7.61–7.52(m,3H),7.18(d,J＝8.6Hz,1H),7.03(d,J＝7.0Hz,1H),6.78(t,J＝6.1Hz,1H),5.08(dd,J＝12.8,5.3Hz,1H),4.78(d,J＝5.9Hz,2H),3.58(s,2H),3.46–3.41(m,2H),3.31(d,J＝6.0Hz,2H),3.00–2.83(m,1H),2.65(s,1H),2.58(s,1H),2.13–1.99(m,1H),1.42(s,9H)。
5. (S) -2- (2- (4- (4-chlorophenyl) -2,3, 9-trimethyl-6H-thieno [3,2-f ] [1,2,4] triazolo [4,3-a ] [1,4] diazepin-6-yl) acetamido) acetic acid (Compound 10 b)
Compound 7 (200mg, 0.44mmol) was dissolved in DCM (2 mL) and trifluoroacetic acid (1 mL) was added. After stirring at room temperature for 8 hours, the reaction mixture was distilled under reduced pressure to give a colorless oily liquid (Compound No. 8). The colorless oily liquid (compound 8), DIEA (227.04mg, 1.76mmol) and glycine methyl ester hydrochloride (55mg, 0.44mmol) were dissolved in DMF (5 mL). A solution of pyBop (457.6 mg, 0.88mmol) in DMF (5 mL) was added dropwise in ice bath and reacted at room temperature for 12 hours. The reaction mixture was poured into saturated brine (100 mL) and extracted with ethyl acetate (30 mL. Times.3). The organic phase was purified by column chromatography (DCM: meOH = 50) to afford compound 9 as a colorless oily liquid. Compound 9 (0.17 mmol) was dissolved in methanol and LiOH (31mg, 0.76mmol) solution was added. After 6 hours of reaction at room temperature, the pH was adjusted to 2 to 4 with dilute hydrochloric acid, and the mixture was extracted with ethyl acetate (30 mL. Times.3). The organic phase was purified by column chromatography (DCM: meOH = 50).
1 H NMR(300MHz,Methanol-d 4 )δ7.54–7.47(m,2H),7.40(d,J＝8.3Hz,2H),4.64(dd,J＝9.4,4.6Hz,1H),4.00(q,J＝17.9Hz,2H),3.16(td,J＝6.7,3.5Hz,2H),2.70(s,3H),2.45(s,3H),1.70(s,3H)。
6. (S) -3- (2- (4- (4-chlorophenyl) -2,3, 9-trimethyl-6H-thieno [3,2-f ] [1,2,4] triazolo [4,3-a ] [1,4] diazepin-6-yl) acetamido) propanoic acid (Compound 10 c)
Compound 7 (200mg, 0.44mmol) was dissolved in DCM (2 mL) and trifluoroacetic acid (1 mL) was added. After stirring at room temperature for 8 hours, the reaction mixture was distilled under reduced pressure to give a colorless oily liquid (Compound No. 8). The colorless oily liquid (Compound 8), DIEA (227.04mg, 1.76mmol) and methyl 3-aminopropionate hydrochloride (61mg, 0.44mmol) were dissolved in DMF (5 mL). A solution of pyBop (457.6 mg, 0.88mmol) in DMF (5 mL) was added dropwise in ice bath and reacted at room temperature for 12 hours. The reaction mixture was poured into saturated brine (100 mL) and extracted with ethyl acetate (30 mL. Times.3). The organic phase was purified by column chromatography (DCM: meOH = 50) to afford compound 9 as a colorless oily liquid. Compound 9 (0.17 mmol) was dissolved in methanol and LiOH (31mg, 0.76mmol) solution was added. After 6 hours of reaction at room temperature, the pH was adjusted to 2 to 4 with dilute hydrochloric acid, and the mixture was extracted with ethyl acetate (30 mL. Times.3). The organic phase was purified by column chromatography (DCM: meOH = 50) to give compound 10c as a white solid (68mg, 32.95%).
1 H NMR(300MHz,Chloroform-d)δ7.82(t,J＝6.1Hz,1H),7.41(d,J＝8.3Hz,2H),7.33(d,J＝8.4Hz,2H),4.77–4.72(m,1H),3.72(ddd,J＝30.3,14.1,7.5Hz,2H),3.43(ddd,J＝30.7,14.4,6.3Hz,2H),2.69(s,3H),2.55(t,J＝5.7Hz,2H),2.42(s,3H)。
7. (S) -4- (2- (4- (4-chlorophenyl) -2,3, 9-trimethyl-6H-thieno [3,2-f ] [1,2,4] triazolo [4,3-a ] [1,4] diazepin-6-yl) acetamido) butanoic acid (Compound 10 d)
Compound 7 (200mg, 0.44mmol) was dissolved in DCM (2 mL) and trifluoroacetic acid (1 mL) was added. After stirring at room temperature for 8 hours, the reaction mixture was distilled under reduced pressure to give a colorless oily liquid (Compound No. 8). The colorless oily liquid (Compound 8), DIEA (227.04mg, 1.76mmol) and methyl 4-aminobutyrate hydrochloride (67mg, 0.44mmol) were dissolved in DMF (5 mL). A solution of pyBop (457.6 mg, 0.88mmol) in DMF (5 mL) was added dropwise in ice bath and reacted at room temperature for 12 hours. The reaction mixture was poured into saturated brine (100 mL) and extracted with ethyl acetate (30 mL. Times.3). The organic phase was purified by column chromatography (DCM: meOH = 50) to afford compound 9 as a colorless oily liquid. Compound 9 (0.17 mmol) was dissolved in methanol and LiOH (31mg, 0.76mmol) solution was added. After 6 hours reaction at room temperature, the pH was adjusted to 2 to 4 with dilute hydrochloric acid, and the mixture was extracted with ethyl acetate (30 mL. Times.3). The organic phase was purified by column chromatography (DCM: meOH = 50) to give compound 10d as a white solid (66mg, 30.68%).
1 H NMR(300MHz,Chloroform-d)δ7.41(d,J＝8.2Hz,2H),7.32(d,J＝2.7Hz,2H),7.27(d,J＝1.5Hz,1H),4.86–4.62(m,1H),3.86–3.58(m,2H),3.54–3.15(m,4H),2.69(s,3H),2.41(d,J＝4.9Hz,3H),1.88(td,J＝13.9,13.4,6.9Hz,2H)。
8. Synthesis of TCB-1 (Compound 11 a)
Tert-butyl (S) -2- (4- (4-chlorophenyl) -2,3, 9-trimethyl-6H-thieno [3,2-f ] [1,2,4] triazolo [4,3-a ] [1,4] diazepin-6-yl) acetate (56mg, 0.12mmol,1 eq) was dissolved in DCM (2 mL) and trifluoroacetic acid (1 mL) was added. After stirring at room temperature for 8 hours, the reaction solution was distilled under reduced pressure to obtain a colorless oily liquid. Compound 6 (80mg, 0.12mmol) was dissolved in DCM (2 mL) and trifluoroacetic acid (1 mL) was added. After stirring at room temperature for 8 hours, the reaction solution was distilled under reduced pressure to obtain an orange solid. The colorless oily liquid and the orange solid were dissolved in DMF (5 mL) and a solution of DIEA (32mg, 0.24mmol), pyBop (129mg, 0.24mmol) in DMF (5 mL) was added dropwise over an ice bath. After stirring at room temperature for 12 hours, the reaction solution was poured into saturated brine (100 mL) and extracted with ethyl acetate (30 mL. Times.3). The organic phase was purified by column chromatography (DCM: meOH = 500) to give compound 11a as an orange solid (24mg, 20.70%).
1 HNMR(300MHz,DMSO-d6)δ11.10(s,1H),9.20(t,J＝5.6Hz,1H),8.40(dd,J＝11.5,6.8Hz,3H),7.54(d,J＝8.0Hz,3H),7.46(s,4H),7.17(d,J＝8.7Hz,1H),7.01(d,J＝7.0Hz,1H),6.76(t,J＝6.1Hz,1H),5.11–4.98(m,2H),4.92(dd,J＝16.4,5.4Hz,1H),4.51(dd,J＝8.9,5.2Hz,1H),3.57(s,2H),3.44(t,J＝4.7Hz,2H),3.26(s,2H),2.97–2.82(m,1H),2.62–2.60(m,1H),2.59(s,3H),2.56(d,J＝3.0Hz,1H),2.39(s,3H),2.02(dd,J＝11.6,6.0Hz,1H),1.59(s,3H)。
HR-MS(ESI,C 46 H 41 ClN 14 O 7 S):Calcd.for[M+H] + 926.26998; found:926.27065. Example 2:2- ((S) -4- (4-chlorophenyl) -2,3, 9-trimethyl-6H-thieno [3, 2-f)][1,2,4]Triazolo [4,3-a][1,4]Synthesis of diaza-6-yl) -N- (2- (((6- (4- (2- (2- (2, 6-dioxopiperidin-3-yl) -1, 3-dioxoisoindol-4-yl) amino) ethyl) amino) -2-oxoethyl) phenyl) -1,2,4, 5-tetrazin-3-ylmethyl) amino) -2-oxoethyl) acetamide (TCB-2, compound 11 b)
Compound 6 (80mg, 0.12mmol) was dissolved in DCM (2 mL) and trifluoroacetic acid (1 mL) was added. After stirring at room temperature for 8 hours, the reaction solution was distilled under reduced pressure to obtain an orange solid. Compound 10b (55mg, 0.12mmol) and the orange solid were dissolved in DMF (5 mL) and a solution of DIEA (32mg, 0.24mmol), pyBop (129mg, 0.24mmol) in DMF (5 mL) was added dropwise over an ice bath. After stirring at room temperature for 12 hours, the reaction solution was poured into saturated brine (100 mL) and extracted with ethyl acetate (30 mL. Times.3). The organic phase was purified by column chromatography (DCM: meOH = 500) to afford compound 11b as an orange solid (19mg, 15.75%).
1 HNMR(300MHz,DMSO-d6)δ11.12(s,1H),8.77(dt,J＝12.3,5.9Hz,2H),8.40(d,J＝8.3Hz,3H),7.55(t,J＝8.2Hz,3H),7.44(s,4H),7.17(d,J＝8.6Hz,1H),7.01(d,J＝7.0Hz,1H),6.77(t,J＝5.9Hz,1H),5.07(dd,J＝12.9,5.3Hz,1H),4.95(d,J＝5.5Hz,2H),4.52(t,J＝7.3Hz,1H),4.08–3.71(m,2H),3.57(s,2H),3.44–3.41(m,2H),3.30(s,2H),2.90(ddd,J＝18.0,13.6,5.3Hz,1H),2.75(d,J＝15.4Hz,1H),2.64(d,J＝15.3Hz,2H),2.50(s,3H),2.39(s,3H),2.03(d,J＝12.0Hz,1H),1.60(s,3H)。
13 CNMR(75MHz,DMSO-d6)δ174.63,172.75,171.19,170.00,169.66,168.61,167.18,166.80,163.79,163.10,155.08,149.80,146.19,141.22,136.59,136.11,135.09,132.08,132.04,130.57,130.14,130.10,129.80,129.66,129.46,128.30,127.38,117.02,110.47,109.12,72.22,64.82,53.75,48.41,42.70,42.12,41.97,41.82,41.19,38.16,37.54,30.88,22.07,13.93,12.55,11.15。
HR-MS(ESI,C 47 H 43 ClN 14 O 7 S):Calcd.for[M+Na] + :1005.27283；Found:1005.27406。
Example 3:3- (2- ((S) -4- (4-chlorophenyl) -2,3, 9-trimethyl-6H-thieno [3,2-f ] [1,2,4] triazolo [4,3-a ] [1,4] diazepin-6-yl) acetamide) -N- ((6- (4- (2- ((2- (2, 6-dioxopiperidin-3-yl) -1, 3-dioxoisoindolin-4-yl) amino) ethyl) amino) -2-oxoethyl) phenyl) -1,2,4, 5-tetrazin-3-yl) methyl) propanamide (Compound 11c, TCB-3)
Compound 6 (80mg, 0.12mmol) was dissolved in DCM (2 mL) and trifluoroacetic acid (1 mL) was added. After stirring at room temperature for 8 hours, the reaction solution was distilled under reduced pressure to obtain an orange solid. Compound 10c (61mg, 0.12mmol) and the orange solid were dissolved in DMF (5 mL) and a solution of DIEA (32mg, 0.24mmol), pyBop (129mg, 0.24mmol) in DMF (5 mL) was added dropwise over an ice bath. After stirring at room temperature for 12 hours, the reaction solution was poured into saturated brine (100 mL) and extracted with ethyl acetate (30 mL. Times.3). The organic phase was purified by column chromatography (DCM: meOH = 500) to afford compound 11c as an orange solid (22mg, 17.99%).
1 H NMR(300MHz,DMSO-d 6 )δ11.08(s,1H),8.88(t,J＝5.6Hz,1H),8.47–8.32(m,3H),8.23(t,J＝5.7Hz,1H),7.53(dd,J＝15.1,7.9Hz,3H),7.48–7.37(m,4H),7.15(d,J＝8.6Hz,1H),7.00(d,J＝7.0Hz,1H),6.73(d,J＝6.3Hz,1H),5.04(dd,J＝12.8,5.4Hz,1H),4.89(d,J＝5.5Hz,2H),4.49(t,J＝7.0Hz,1H),3.55(s,2H),3.39(s,2H),3.20(s,1H),2.97–2.79(m,1H),2.61–2.55(m,5H；-CH 3 ,-CH 2 ),2.44(d,J＝7.2Hz,2H),2.39(s,3H),2.01(d,J＝13.3Hz,1H),1.60(s,3H),1.22(s,2H)。
HR-MS(ESI,C 48 H 45 ClN 14 O 7 S):Calcd.for[M+Na] + :1019.28839；Found is 1019.28971. Example 4:4- (2- ((S) -4- (4-chlorophenyl) -2,3, 9-trimethyl-6H-thieno [3, 2-f)][1,2,4]Triazolo [4,3-a][1,4]Diaza-6-yl) acetamide-N-N- ((6- (4- (2- ((2- (2, 6-dioxopiperidin-3-yl) -1, 3-dioxoisoindolin-4-yl) amino) ethyl) amino) -2-oxoethyl) phenyl) -1,2,4, 5-tetrazin-3-yl) methyl) butyramide (Compound 11d, TCB-4)
Compound 6 (80mg, 0.12mmol) was dissolved in DCM (2 mL) and trifluoroacetic acid (1 mL) was added. After stirring at room temperature for 8 hours, the reaction solution was distilled under reduced pressure to obtain an orange solid. Compound 10d (67mg, 0.12mmol) and the orange solid were dissolved in DMF (5 mL) and a solution of DIEA (32mg, 0.24mmol), pyBop (129mg, 0.24mmol) in DMF (5 mL) was added dropwise over an ice bath. After stirring at room temperature for 12 hours, the reaction solution was poured into saturated brine (100 mL) and extracted with ethyl acetate (30 mL. Times.3). The organic phase was purified by column chromatography (DCM: meOH = 500) to afford compound 11d as an orange solid (14mg, 11.29%).
1 H NMR(300MHz,DMSO-d 6 )δ11.14(s,1H),8.83(t,J＝5.7Hz,1H),8.42(dd,J＝10.6,7.0Hz,3H),8.29(d,J＝6.0Hz,1H),7.62–7.49(m,3H),7.44(q,J＝8.6Hz,4H),7.19(d,J＝8.7Hz,1H),7.03(d,J＝7.0Hz,1H),6.79(t,J＝6.0Hz,1H),5.08(dd,J＝12.8,5.4Hz,1H),4.91(d,J＝5.5Hz,2H),4.52(dd,J＝8.1,6.1Hz,1H),4.02(dt,J＝18.6,6.6Hz,1H),3.58(s,2H),3.33–3.27(m,2H),3.26–3.19(m,2H),3.19–3.13(m,1H),3.02–2.83(m,1H),2.61(s,3H),2.42(s,3H),2.30(t,J＝7.6Hz,2H),2.11–1.96(m,1H),1.74(t,J＝7.6Hz,2H),1.63(s,3H),1.38–1.21(m,2H)。
HR-MS(ESI,C 49 H 47 ClN 14 O 7 S):Calcd.for[M+Na] + 1033.30536; found:1033.30406. Example 5:2- ((S) -4- (4-chlorophenyl) -2,3, 9-trimethyl-6H-thieno [3, 2-f)][1,2,4]Triazolo [4,3-a ]][1,4]Synthesis of diaza-6-yl) -N- (2- (((6- (4- (2- (2- (1-methyl-2, 6-dioxopiperidin-3-yl) -1, 3-dioxoisoindol-4-yl) amino) ethyl) amino) -2-oxyethyl) phenyl) -1,2,4, 5-tetrazin-3-ylmethyl) amino) -2-oxyethyl) acetamide (TCB-Neg, compound 15)
Figure BDA0003987367310000101
1. 4-fluoro-2- (1-methyl-2, 6-dioxopiperidin-3-yl) isoindole-1, 3-dione (Compound 12)
2- (2, 6-dioxopiperidin-3-yl) -4-fluoroisoindoline-1, 3-dione (630mg, 2.32mmol) and cesium carbonate (1.13g, 3.49mmol) were dissolved in DMF (5 mL). Methyl iodide (396mg, 2.79mmol) was added dropwise under ice-cooling. After stirring in an ice bath for 8 hours, the reaction mixture was poured into saturated brine (100 mL) and extracted with ethyl acetate (30 mL. Times.3). The organic phase was distilled under reduced pressure to give crude product, which was recrystallized from ethanol to give compound 12 (282mg, 42.70%).
1 H NMR(300MHz,DMSO-d 6 )δ7.96(td,J＝7.9,4.5Hz,1H),7.82–7.71(m,2H),5.23(dd,J＝13.0,5.3Hz,1H),3.00–2.85(m,1H),2.78(ddd,J＝18.1,5.1,2.8Hz,1H),2.54–2.49(m,1H),2.09(dtd,J＝13.3,5.5,2.6Hz,1H)。
2. (2- ((2- (1-methyl-2, 6-dioxopiperidin-3-yl) -1, 3-dioxoisoindolin-4-yl) amino) ethyl) carbamic acid tert-butyl ester (Compound 13)
Tert-butyl (2-aminoethyl) carbamate (825.6 mg, 5.16mmol) and compound 12 (500mg, 1.72mmol) were dissolved in 1, 4-dioxane (10 mL). After 8 hours of reaction at 100 ℃, the reaction mixture was poured into saturated brine (100 mL) and extracted with ethyl acetate (30 mL. Times.3). The organic phase was purified by column chromatography (DCM: meOH = 500) to afford compound 13 as a green solid (340mg, 45.10%).
1 H NMR(300MHz,DMSO-d 6 )δ7.60(dd,J＝8.6,7.1Hz,1H),7.17(d,J＝8.6Hz,1H),7.07(dd,J＝9.1,6.2Hz,2H),6.76(t,J＝6.2Hz,1H),5.15(dd,J＝13.0,5.3Hz,1H),3.38(d,J＝4.4Hz,2H),3.13(q,J＝6.0Hz,2H),3.00–2.90(m,1H),2.77(dt,J＝16.7,3.7Hz,1H),2.58(dd,J＝13.0,4.4Hz,1H),2.12–1.97(m,1H),1.38(s,9H)。
3. (6- (4- (2- ((2- ((2- (1-methyl-2, 6-dioxopiperidin-3-yl) -1, 3-dioxoisoindolin-4-yl) amino) ethyl) amino) -2-oxoethyl) phenyl) -1,2,4, 5-tetrazin-3-yl) methyl) carbamic acid tert-butyl ester (Compound 14)
Compound 13 (266mg, 0.62mmol) was dissolved in DCM (2 mL) and trifluoroacetic acid (1 mL) was added. After stirring at room temperature for 8 hours, the reaction solution was distilled under reduced pressure to give a green solid. The green solid and 2- (4- (6- (((tert-butoxycarbonyl) amino) methyl) -1,2,4, 5-tetrazin-3-yl) phenyl) acetic acid (5, 213mg, 0.62mmol) were dissolved in DMF (5 mL). DIEA (160mg, 1.24mmol) and pyBop (645mg, 1.24mmol) in DMF (5 mL) were added dropwise in an ice bath. After stirring at room temperature for 12 hours, the reaction mixture was poured into saturated brine (100 mL) and extracted with ethyl acetate (30 mL. Times.3). The organic phase was purified by column chromatography (DCM: meOH = 50) to afford compound 14 as a pink solid (198mg, 48.60%).
1 H NMR(300MHz,DMSO-d 6 )δ8.40(dd,J＝6.4,3.0Hz,3H),7.71(t,J＝6.3Hz,1H),7.61–7.49(m,3H),7.17(d,J＝8.6Hz,1H),7.02(d,J＝7.0Hz,1H),6.76(t,J＝6.1Hz,1H),5.13(dd,J＝12.9,5.3Hz,1H),4.76(d,J＝5.9Hz,2H),3.56(s,2H),3.41(t,J＝6.2Hz,2H),3.30(d,J＝5.8Hz,2H),3.02(s,3H),2.99–2.87(m,1H),2.83–2.69(m,1H),2.64–2.52(m,1H),2.19–1.99(m,1H),1.40(s,9H)。
4. Synthesis of TCB-Neg (Compound 15)
Compound 14 (40mg, 0.064mmol) was dissolved in DCM (2 mL) and trifluoroacetic acid (1 mL) was added. After stirring at room temperature for 8 hours, the reaction solution was distilled under reduced pressure to give an orange solid. The orange solid and compound 10b (30mg, 0.064 mmol) were dissolved in DMF (5 mL). A solution of DIEA (17mg, 0.13mmol) and pyBop (68mg, 0.13mmol) in DMF (5 mL) was added dropwise over an ice bath. After stirring at room temperature for 12 hours, the reaction mixture was poured into saturated brine (100 mL) and extracted with ethyl acetate (30 mL. Times.3). The organic phase was purified by column chromatography (DCM: meOH = 500) to afford compound 15 as an orange solid (11mg, 17.50%).
1 H NMR(300MHz,DMSO-d 6 )δ8.76(dd,J＝13.0,6.6Hz,2H),8.40(d,J＝7.7Hz,3H),7.54(dd,J＝7.9,3.2Hz,3H),7.44(s,4H),7.17(d,J＝8.6Hz,1H),7.02(d,J＝7.0Hz,1H),6.76(t,J＝6.1Hz,1H),5.13(dd,J＝12.9,5.3Hz,1H),4.95(d,J＝5.5Hz,2H),4.53(t,J＝7.3Hz,1H),4.00–3.76(m,2H),3.57(s,2H),3.42(d,J＝6.1Hz,2H),3.30(d,J＝7.8Hz,2H),2.99–2.89(m,1H),2.82–2.71(m,1H),2.62–2.54(m,1H),2.39(s,3H),2.04(d,J＝13.1Hz,1H),1.60(s,3H)。
HR-MS(ESI,C 48 H 45 ClN 14 O 7 S):Calcd.for[M+Na] + :1019.28971；Found:1019.28760。
Example 6: synthesis of 2- ((2- (2, 6-dioxopiperidin-3-yl) -1, 3-dioxoisoindolin-4-yl) amino) -N- ((6- (4- (2- (4- (2- (2-fluoro-5- ((4-oxo-3, 4-dihydrophthalazin-1-yl) methyl) benzoyl) piperazin-1-yl) -2-oxoethyl) phenyl) -1,2,4, 5-tetrazin-3-yl) methyl) acetamide (TCP-1, compound 20)
Figure BDA0003987367310000121
1. 4- (2-fluoro-5- ((4-oxo-3, 4-dihydrophthalazin-1-yl) methyl) benzoyl) piperazine-1-carboxylic acid tert-butyl ester (Compound 17)
2-fluoro-5- (4-oxo-3, 4-dihydrophthalazin-1-yl) benzoic acid (1.19g, 4mmol) and tert-butylpiperazine-1-carboxylate (890mg, 4mmol) were dissolved in 5mL of DMF, and DIEA (1032mg, 8mmol) was added. A solution of pyBop (2.7mg, 5.2mmol) in DMF (5 mL) was added dropwise over an ice bath. After 8 hours of reaction at room temperature, the reaction solution was poured into saturated brine (100 mL), extracted with ethyl acetate (30 mL × 3), and the organic phase was purified by column chromatography (DCM: meOH = 50).
1 H NMR(300MHz,DMSO-d 6 )δ12.61(s,1H),8.26(dd,J＝7.5,1.7Hz,1H),7.96(d,J＝7.2Hz,1H),7.89(td,J＝7.5,1.7Hz,1H),7.83(td,J＝7.4,1.5Hz,1H),7.44(ddd,J＝8.3,5.1,2.2Hz,1H),7.35(dd,J＝6.5,2.3Hz,1H),7.24(t,J＝9.0Hz,1H),4.33(s,2H),3.59(s,2H),3.38(t,J＝3.9Hz,2H),3.22(d,J＝6.1Hz,2H),3.14(s,2H),1.40(s,9H)。
2. ((6- (4- (2- (4- (2-fluoro-5- ((4-oxo-3, 4-dihydrophthalazin-1-yl) methyl) benzoyl) piperazin-1-yl) -2-oxoethyl) phenyl) -1,2,4, 5-tetrazin-3-yl) methyl) carbamic acid tert-butyl ester (Compound 18)
Compound 17 (100mg, 0.21mmol) was dissolved in DCM (2 mL), and trifluoroacetic acid (1 mL) was added. After stirring at room temperature for 8 hours, the reaction mixture was distilled under reduced pressure to give a colorless oily liquid. The colorless oily liquid and Compound 5 (74mg, 0.21mmol) were dissolved in 5mL of DMF, DIEA (227.04mg, 0.64mmol) was added, and a solution of pyBop (145.06mg, 0.27mmol) in DMF (5 mL) was added dropwise over ice, followed by reaction at room temperature for 12 hours. The reaction was poured into saturated brine (100 mL), extracted 3 times with ethyl acetate (30 mL × 3), and the organic phase was purified by column chromatography (DCM: meOH = 50) to give compound 18 as a pink oil (60mg, 41.37%).
1 H NMR(300MHz,DMSO-d 6 )δ12.62(s,1H),8.43(d,J＝6.0Hz,2H),8.30–8.21(m,1H),7.97(d,J＝7.7Hz,1H),7.84(q,J＝8.1,7.1Hz,2H),7.74(t,J＝5.9Hz,1H),7.53(t,J＝7.1Hz,2H),7.43(d,J＝7.0Hz,1H),7.39(d,J＝6.9Hz,1H),7.24(t,J＝9.0Hz,1H),4.76(d,J＝5.9Hz,2H),4.33(s,2H),3.90(d,J＝19.8Hz,2H),3.62(s,4H),3.46(d,J＝14.2Hz,2H),3.18(s,2H),1.40(s,9H)。
3. (2- (2, 6-dioxopiperidin-3-yl) -1, 3-dioxoisoindolin-4-yl) glycine tert-butyl ester (Compound 19)
To a DMSO solution (7 mL) of 2- (2, 6-dioxopiperidin-3-yl) -4-fluoroisoindole-1, 3-dione (2, 500mg, 1.81mmol) was added tert-butyl (2-aminoethyl) carbamate (474mg, 3.62mmol) and DIEA (750mg, 5.43mmol). After 10 hours reaction at 90 ℃, the reaction mixture was poured into 500mL of saturated brine, extracted 3 times with ethyl acetate (100 mL × 3), and the organic phase was purified by column chromatography (DCM: meOH =200 1) to give compound 19 as a green solid (387mg, 51.39%).
1 H NMR(300MHz,DMSO-d 6 )δ11.13(s,1H),7.59(dd,J＝8.5,7.1Hz,1H),7.08(d,J＝7.1Hz,1H),6.98(d,J＝8.5Hz,1H),6.86(t,J＝5.9Hz,1H),5.08(dd,J＝12.8,5.3Hz,1H),4.10(d,J＝6.0Hz,2H),2.97–2.83(m,1H),2.62(s,1H),2.56(s,1H),2.11–1.99(m,1H),1.43(s,9H)。
4. Synthesis of TCP-1 (Compound 20)
Compound 18 (50mg, 0.072mmol) was dissolved in DCM (2 mL) and trifluoroacetic acid (1 mL) was added. The mixture was stirred at room temperature for 8 hours, and the reaction mixture was distilled under reduced pressure to obtain a pink oily liquid. Compound 19 (27.86mg, 0.072mmol) was dissolved in DCM (2 mL) and trifluoroacetic acid (1 mL) was added. The mixture was stirred at room temperature for 8 hours, and the reaction mixture was distilled under reduced pressure to obtain a green oily liquid. The pink and green liquids and DIEA (27.86mg, 0.216mmol) were dissolved in DMF (5 mL) and a solution of pyBop (48.67mg, 0.094mmol) in DMF (5 mL) was added dropwise over ice. After 12 hours of reaction at room temperature, the reaction mixture was poured into saturated brine (100 mL) and extracted with ethyl acetate (30 mL. Times.3). The organic phase was purified by column chromatography (DCM: meOH = 50) to afford compound 20 as an orange solid (28mg, 41.89%).
1 H NMR(300MHz,DMSO-d 6 )δ12.61(s,1H),11.11(s,1H),9.06(t,J＝5.7Hz,1H),8.43(dd,J＝8.3,3.2Hz,2H),8.30–8.23(m,1H),7.96(d,J＝7.8Hz,1H),7.89(s,1H),7.86–7.80(m,1H),7.61(t,J＝7.8Hz,1H),7.53(t,J＝7.1Hz,2H),7.49–7.41(m,1H),7.38(s,1H),7.24(t,J＝9.0Hz,1H),7.09(d,J＝7.1Hz,1H),7.02(t,J＝5.8Hz,1H),6.94(d,J＝8.5Hz,1H),5.07(dd,J＝12.7,5.3Hz,1H),4.95(d,J＝5.5Hz,2H),4.33(s,2H),4.11(d,J＝5.7Hz,2H),3.90(d,J＝19.8Hz,2H),3.62(s,4H),3.46(d,J＝13.1Hz,2H),3.38(d,J＝7.0Hz,2H),3.19(s,2H),2.98–2.79(m,1H),2.61(s,1H),2.55(s,1H),2.02(d,J＝12.0Hz,1H)。
13 C NMR(75MHz,DMSO)δ172.71,169.94,169.46,168.56,168.50,167.20,166.70,163.98,163.89,159.28,145.72,144.75,140.89,136.05,134.73,133.39,131.89,131.73,131.62,131.47,130.34,129.70,128.97,128.85,127.78,127.46,125.97,125.35,117.55,115.98,115.68,111.00,109.85,48.44,46.41,46.11,45.32,45.12,41.93,41.36,41.06,36.32,30.87,22.04。
HR-MS(ESI,C 46 H 39 FN 12 O 8 ):Calcd.for[M+Na] + 929.28901; found 929.28947. Example 7:2- ((2- (2, 6-dioxopiperidin-3-yl) -1, 3-dioxoisoindolin-4-yl) amino) -N- ((6- (4- (2- ((2- (4- (2-fluoro-5- ((4-oxo-3, 4-dihydrophthalazin-1-yl) methyl) benzoyl) piperazin-1-yl) -2-oxoethyl) amino) -2-oxoethyl) phenyl) -1,2,4, 5-tetrazin-3-yl) methyl) acetamide (TCP-2, compound 23 a)
Figure BDA0003987367310000131
1. (tert-butyl 2- (4- (2-fluoro-5- ((4-oxo-3, 4-dihydrophthalazin-1-yl) methyl) benzoyl) piperazin-1-yl) -2-oxoethyl) carbamate (Compound 21 a)
Compound 17 (200mg, 0.42mmol) was dissolved in DCM (2 mL) and trifluoroacetic acid (1 mL) was added. After stirring at room temperature for 8 hours, the reaction mixture was distilled under reduced pressure to obtain a colorless oily liquid. A colorless oily liquid and N-Boc-glycine (74mg, 0.42mmol) and DIEA (162.54mg, 1.26mmol in DMF (5 mL) were added dropwise under ice bath a solution of pyBop (283.92mg, 0.55mmol) in DMF (5 mL) after stirring at room temperature for 12 hours, the reaction was poured into saturated brine (100 mL) and extracted with ethyl acetate (30 mL × 3) the organic phase was purified by column chromatography (DCM: meOH = 50) to give compound 21a as a colorless oily liquid (146mg, 66.67%).
1 H NMR(300MHz,DMSO-d 6 )δ12.61(s,1H),8.30–8.23(m,1H),7.97(d,J＝7.8Hz,1H),7.92(d,J＝7.5Hz,1H),7.87–7.79(m,1H),7.49–7.42(m,1H),7.38(s,1H),7.24(t,J＝9.0Hz,1H),6.80(s,1H),4.34(s,2H),3.80(dd,J＝17.3,5.8Hz,2H),3.62(s,2H),3.50(s,2H),3.18(s,2H),1.38(s,9H)。
2. (3- (4- (2-fluoro-5- ((4-oxo-3, 4-dihydrophthalazin-1-yl) methyl) benzoyl) piperazin-1-yl) -3-oxopropyl) carbamic acid tert-butyl ester (Compound 21 b)
Compound 17 (200mg, 0.42mmol) was dissolved in DCM (2 mL) and trifluoroacetic acid (1 mL) was added. After stirring at room temperature for 8 hours, the reaction mixture was distilled under reduced pressure to obtain a colorless oily liquid. The colorless oily liquid and N-Boc- β -alanine (79mg, 0.42mmol) and DIEA (162.54mg, 1.26mmol in DMF (5 mL) were dissolved in ice bath and a solution of pyBop (283.92mg, 0.55mmol) in DMF (5 mL) was added dropwise under ice bath after stirring at room temperature for 12 hours, the reaction was poured into saturated brine (100 mL) and extracted with ethyl acetate (30 mL × 3) the organic phase was purified by column chromatography (DCM: meOH = 50) to give compound 21b as a colorless oily liquid (128mg, 57.14%).
1 H NMR(300MHz,DMSO-d 6 )δ12.60(s,1H),8.29–8.23(m,1H),7.97(d,J＝7.9Hz,1H),7.90(t,J＝7.4Hz,1H),7.83(td,J＝7.4,1.5Hz,1H),7.43(d,J＝6.9Hz,1H),7.36(s,1H),7.24(t,J＝9.0Hz,1H),6.72(s,1H),4.33(s,2H),3.61(d,J＝19.0Hz,2H),3.50(s,2H),3.14(s,4H),3.01(h,J＝3.8Hz,4H),1.38(s,9H)。
3. (4- (4- (2-fluoro-5- ((4-oxo-3, 4-dihydrophthalazin-1-yl) methyl) benzoyl) piperazin-1-yl) -4-oxobutyl) carbamic acid tert-butyl ester (Compound 21 c)
Compound 17 (200mg, 0.42mmol) was dissolved in DCM (2 mL) and trifluoroacetic acid (1 mL) was added. After stirring at room temperature for 8 hours, the reaction mixture was distilled under reduced pressure to obtain a colorless oily liquid. The colorless oily liquid and N-Boc- γ -aminobutyric acid (85mg, 0.42mmol) and DIEA (162.54mg, 1.26mmol) were dissolved in DMF (5 mL), a solution of pyBop (283.92mg, 0.55mmol) in DMF (5 mL) was added dropwise under ice bath, after stirring at room temperature for 12 hours, the reaction was poured into saturated brine (100 mL), extracted with ethyl acetate (30 mL × 3), and the organic phase was purified by column chromatography (DCM: meOH = 50) to give compound 21c as a colorless oily liquid (132mg, 57.03%).
1 H NMR(300MHz,DMSO-d 6 )δ12.61(s,1H),8.28(d,J＝7.6Hz,1H),7.98(d,J＝7.9Hz,1H),7.91(t,J＝7.8Hz,1H),7.85(t,J＝7.7Hz,1H),7.45(d,J＝6.8Hz,1H),7.37(s,1H),7.25(t,J＝9.0Hz,1H),6.82(s,1H),4.35(s,2H),3.62(d,J＝19.3Hz,2H),3.52(s,2H),3.18(s,2H),3.03(td,J＝6.6,3.7Hz,2H),2.94(s,2H),2.38–2.22(m,2H),1.61(s,2H),1.38(s,9H)。
4. ((6- (4- (2- ((2- (4- (2-fluoro-5- ((4-oxo-3, 4-dihydrophthalazin-1-yl) methyl) benzoyl) piperazin-1-yl) -2-oxoethyl) amino) -2-oxoethyl) phenyl) -1,2,4, 5-tetrazin-3-yl) methyl) carbamic acid tert-butyl ester (22 a)
Compound 21a (100mg, 0.19mmol) was dissolved in DCM (2 mL), and trifluoroacetic acid (1 mL) was added. After stirring at room temperature for 8 hours, the reaction mixture was distilled under reduced pressure to obtain a colorless oily liquid. The colorless oily liquid and Compound 5 (65.97mg, 0.19mmol) were dissolved in DMF (5 mL), DIEA (73.99mg, 0.57mmol) was added, and a solution of pyBop (128.44mg, 0.25mmol) in DMF (5 mL) was added dropwise over an ice bath. After stirring at room temperature for 12 hours, the reaction mixture was poured into saturated brine (100 mL) and extracted with ethyl acetate (30 mL. Times.3). The organic phase was purified by column chromatography (DCM: meOH = 125) to give compound 22a as a pink solid (90mg, 63.15%).
1 H NMR(300MHz,DMSO-d 6 )δ12.62(s,1H),8.42(d,J＝7.3Hz,2H),8.35(d,J＝5.4Hz,1H),8.26(dd,J＝7.7,1.6Hz,1H),7.97(d,J＝7.9Hz,1H),7.93–7.87(m,1H),7.83(td,J＝7.4,1.4Hz,1H),7.73(t,J＝6.0Hz,1H),7.59(d,J＝7.9Hz,2H),7.44(s,1H),7.38(d,J＝6.1Hz,1H),7.24(t,J＝8.9Hz,1H),4.76(d,J＝5.9Hz,2H),4.33(s,2H),4.02(dd,J＝17.8,5.1Hz,2H),3.68(s,2H),3.64(s,2H),3.54(s,2H),3.40(s,2H),3.18(s,2H),1.40(s,9H)。
5. ((6- (4- (2- ((3- (4- (2-fluoro-5- ((4-oxo-3, 4-dihydrophthalazin-1-yl) methyl) benzoyl) piperazin-1-yl) -3-oxopropyl) amino) -2-oxoethyl) phenyl) -1,2,4, 5-tetrazin-3-yl) methyl) carbamic acid tert-butyl ester (22 b)
Compound 21b (102mg, 0.19mmol) was dissolved in DCM (2 mL) and trifluoroacetic acid (1 mL) was added. After stirring at room temperature for 8 hours, the reaction mixture was distilled under reduced pressure to obtain a colorless oily liquid. The colorless oily liquid and Compound 5 (65.97mg, 0.19mmol) were dissolved in DMF (5 mL), DIEA (73.99mg, 0.57mmol) was added, and a solution of pyBop (128.44mg, 0.25mmol) in DMF (5 mL) was added dropwise over an ice bath. After stirring at room temperature for 12 hours, the reaction mixture was poured into saturated brine (100 mL) and extracted with ethyl acetate (30 mL. Times.3). The organic phase was purified by column chromatography (DCM: meOH = 125) to give compound 22b as a pink solid (77mg, 53.04%).
1 H NMR(300MHz,DMSO-d 6 )δ12.61(s,1H),8.49–8.38(m,2H),8.32–8.17(m,2H),7.98(d,J＝8.1Hz,1H),7.94–7.81(m,2H),7.76–7.69(m,1H),7.60–7.52(m,2H),7.45(s,1H),7.40–7.32(m,1H),7.24(t,J＝9.0Hz,1H),4.77(d,J＝5.5Hz,2H),4.35(s,2H),3.62(s,2H),3.57(s,4H),3.50(s,2H),3.40(s,2H),3.31(s,2H),3.17(s,2H),1.41(s,9H)。
6. ((6- (4- (2- ((4- (4- (2-fluoro-5- ((4-oxo-3, 4-dihydrophthalazin-1-yl) methyl) benzoyl) piperazin-1-yl) -4-oxobutyl) amino) -2-oxoethyl) phenyl) -1,2,4, 5-tetrazin-3-yl) methyl) carbamic acid tert-butyl ester (22 c)
Compound 21c (105mg, 0.19mmol) was dissolved in DCM (2 mL) and trifluoroacetic acid (1 mL) was added. After stirring at room temperature for 8 hours, the reaction mixture was distilled under reduced pressure to obtain a colorless oily liquid. The colorless oily liquid and Compound 5 (65.97mg, 0.19mmol) were dissolved in DMF (5 mL), DIEA (73.99mg, 0.57mmol) was added, and a solution of pyBop (128.44mg, 0.25mmol) in DMF (5 mL) was added dropwise over an ice bath. After stirring at room temperature for 12 hours, the reaction mixture was poured into saturated brine (100 mL) and extracted with ethyl acetate (30 mL. Times.3). The organic phase was purified by column chromatography (DCM: meOH = 125) to give compound 22c as a pink solid (82mg, 55.47%).
1 H NMR(300MHz,DMSO-d 6 )δ12.59(s,1H),8.41(dd,J＝14.0,7.9Hz,2H),8.30–8.21(m,1H),8.17(s,1H),7.96(d,J＝8.0Hz,1H),7.90(d,J＝6.7Hz,1H),7.86–7.79(m,1H),7.70(s,1H),7.55(t,J＝7.7Hz,2H),7.43(s,1H),7.35(d,J＝6.5Hz,1H),7.23(t,J＝9.0Hz,1H),4.76(d,J＝5.9Hz,2H),4.33(s,2H),3.63–3.56(m,2H),3.55(s,2H),3.49(d,J＝13.5Hz,2H),3.41–3.34(m,2H),3.18–3.04(m,4H),2.41–2.22(m,2H),1.65(t,J＝7.2Hz,2H),1.40(s,9H)。
7. Synthesis of TCP-2 (Compound 23 a)
Compound 22a (54mg, 0.072mmol) was dissolved in DCM (2 mL) and trifluoroacetic acid (1 mL) was added. After stirring at room temperature for 8 hours, the reaction solution was distilled under reduced pressure to obtain a pink oily liquid. Compound 19 (27.86mg, 0.072mmol) was dissolved in DCM (2 mL) and trifluoroacetic acid (1 mL) was added. The mixture was stirred at room temperature for 8 hours, and the reaction mixture was distilled under reduced pressure to obtain a green oily liquid. The pink and green liquids and DIEA (27.86mg, 0.216mmol) were dissolved in DMF (5 mL) and a solution of pyBop (48.67mg, 0.094mmol) in DMF (5 mL) was added dropwise over ice. After 12 hours of reaction at room temperature, the reaction mixture was poured into saturated brine (100 mL) and extracted with ethyl acetate (30 mL. Times.3). The organic phase was purified by column chromatography (DCM: meOH = 50) to afford compound 23a as an orange solid (37mg, 53.36%).
1 H NMR(300MHz,DMSO-d 6 )δ12.63(s,1H),11.14(s,1H),9.08(t,J＝5.8Hz,1H),8.43(d,J＝7.9Hz,2H),8.37(s,1H),8.30–8.24(m,1H),7.98(d,J＝7.8Hz,1H),7.91(t,J＝7.4Hz,1H),7.88–7.81(m,1H),7.76–7.64(m,1H),7.64–7.57(m,3H),7.44(d,J＝7.3Hz,1H),7.39(d,J＝6.0Hz,1H),7.25(t,J＝9.0Hz,1H),7.10(d,J＝7.1Hz,1H),7.02(d,J＝6.1Hz,1H),6.95(d,J＝8.6Hz,1H),5.14–5.04(m,1H),4.96(d,J＝5.5Hz,2H),4.34(s,2H),4.12(d,J＝5.4Hz,2H),4.03(dd,J＝18.1,5.2Hz,2H),3.69(s,2H),3.64(s,2H),3.53(s,2H),3.36(s,2H),3.20(s,2H),2.98–2.83(m,1H),2.62(d,J＝3.4Hz,1H),2.16(d,J＝16.6Hz,1H),2.04(dd,J＝11.8,6.2Hz,1H)。
HR-MS(ESI,C 48 H 42 FN 13 O 9 ):Calcd.for[M+Na] + :986.31047；Found:986.30863。
Example 8:2- ((2- (2, 6-dioxopiperidin-3-yl) -1, 3-dioxoisoindolin-4-yl) amino) -N- ((6- (4- (2- ((3- (4- (2-fluoro-5- ((4-oxo-3, 4-dihydrophthalazin-1-yl) methyl) benzoyl) piperazin-1-yl) -3-oxo-propyl) amino) -2-oxoethyl) phenyl) -1,2,4, 5-tetrazin-3-yl) methyl) acetamide (Compound 23b, TCP-3)
Compound 22b (55mg, 0.072mmol) was dissolved in DCM (2 mL) and trifluoroacetic acid (1 mL) was added. The mixture was stirred at room temperature for 8 hours, and the reaction mixture was distilled under reduced pressure to obtain a pink oily liquid. Compound 19 (27.86mg, 0.072mmol) was dissolved in DCM (2 mL) and trifluoroacetic acid (1 mL) was added. The mixture was stirred at room temperature for 8 hours, and the reaction mixture was distilled under reduced pressure to obtain a green oily liquid. The pink and green liquids and DIEA (27.86mg, 0.216mmol) were dissolved in DMF (5 mL) and a solution of pyBop (48.67mg, 0.094 mmol) in DMF (5 mL) was added dropwise over ice. After 12 hours of reaction at room temperature, the reaction mixture was poured into saturated brine (100 mL) and extracted with ethyl acetate (30 mL. Times.3). The organic phase was purified by column chromatography (DCM: meOH = 50) to afford compound 23b as an orange solid (24mg, 33.34%).
1 H NMR(300MHz,DMSO-d 6 )δ12.62(s,1H),11.13(s,1H),9.08(s,1H),8.42(d,J＝4.8Hz,3H),8.27(d,J＝8.4Hz,3H),7.97(d,J＝7.8Hz,1H),7.89(dd,J＝16.4,7.4Hz,2H),7.63(d,J＝7.6Hz,1H),7.61–7.52(m,3H),7.45(s,1H),7.37(d,J＝6.6Hz,1H),7.24(t,J＝9.0Hz,1H),7.10(d,J＝7.1Hz,1H),7.01(d,J＝6.0Hz,1H),6.95(d,J＝8.5Hz,1H),5.09(dd,J＝12.9,5.2Hz,1H),4.96(d,J＝5.6Hz,2H),4.34(s,2H),4.12(d,J＝5.7Hz,3H),3.57(s,6H),2.88(d,J＝13.1Hz,1H),2.62(s,1H),2.03(s,1H)。
HR-MS(ESI,C 49 H 44 FN 13 O 9 ):Calcd.for[M+Na] + :1000.32612；Found:1000.32351。
Example 9:2- ((2- (2, 6-dioxopiperidin-3-yl) -1, 3-dioxoisoindolin-4-yl) amino) -N- ((6- (4- (2- ((4- (4- (2-fluoro-5- ((4-oxo-3, 4-dihydrophthalazin-1-yl) methyl) benzoyl) piperazin-1-yl) -4-oxobutyl) amino) -2-oxoethyl) phenyl) -1,2,4, 5-tetrazin-3-yl) methyl) acetamide (Compound 23c, TCP-4)
Compound 22b (55mg, 0.072mmol) was dissolved in DCM (2 mL) and trifluoroacetic acid (1 mL) was added. The mixture was stirred at room temperature for 8 hours, and the reaction mixture was distilled under reduced pressure to obtain a pink oily liquid. Compound 19 (27.86mg, 0.072mmol) was dissolved in DCM (2 mL) and trifluoroacetic acid (1 mL) was added. The mixture was stirred at room temperature for 8 hours, and the reaction mixture was distilled under reduced pressure to obtain a green oily liquid. The pink and green liquids and DIEA (27.86mg, 0.216mmol) were dissolved in DMF (5 mL) and a solution of pyBop (48.67mg, 0.094mmol) in DMF (5 mL) was added dropwise over ice. After 12 hours of reaction at room temperature, the reaction mixture was poured into saturated brine (100 mL) and extracted with ethyl acetate (30 mL. Times.3). The organic phase was purified by column chromatography (DCM: meOH = 50) to afford compound 23c as an orange solid (32mg, 43.83%).
1 H NMR(300MHz,DMSO-d 6 )δ12.65(s,1H),11.16(s,1H),9.10(s,1H),8.47(dd,J＝13.4,8.1Hz,2H),8.31(d,J＝7.6Hz,1H),8.24(s,1H),8.02(d,J＝8.0Hz,1H),7.96(d,J＝7.0Hz,1H),7.90(d,J＝9.1Hz,1H),7.67(d,J＝7.8Hz,1H),7.62(d,J＝7.9Hz,2H),7.49(s,1H),7.40(t,J＝8.5Hz,1H),7.28(t,J＝8.9Hz,1H),7.14(d,J＝7.1Hz,1H),7.06(s,1H),6.99(d,J＝8.5Hz,1H),5.13(dd,J＝12.8,5.4Hz,1H),5.01(d,J＝5.3Hz,2H),4.39(s,2H),4.16(d,J＝5.1Hz,2H),3.66(s,2H),3.61(s,4H),3.51(s,2H),3.49(s,1H),3.19(s,2H),2.92(d,J＝12.0Hz,1H),2.67(s,1H),2.61(s,1H),2.40(s,1H),2.07(d,J＝12.5Hz,1H)。
HR-MS(ESI,C 49 H 44 FN 13 O 9 ):Calcd.for[M+Na] + :1014.34177；Found:1014.34048。
Example 10 Synthesis and structural confirmation of PAMAM-G5-TCO
Figure BDA0003987367310000171
Weighing 50mg of PAMAM-G5-NH 2 (CAS number: 163442-68-0) is dissolved in 3mL of methanol, 1mL of TCO-NHS-Ester (CAS number: 1191901-33-3) (40 eq.) methanol solution is added, the mixture is stirred and reacted for 0.5 hour at room temperature, the reaction solution is transferred into a dialysis bag (molecular weight cut-off of 2 kDa) after the reaction is finished, pure water dialysis is carried out for 24 hours, and the dialyzate is collected and freeze-dried to obtain the PAMAM-G5-TCO product.
A novel dendrimer of PAMAM-G5-TCO, whose surface amine groups are modified with functionalized TCO groups (1. The structure of the compound is shown in the specification 1 HNMR confirmed: the skeleton of PAMAM shows 5 broad peaks, the characteristic peak of amido bond of modified TCO is 6.92ppm, the characteristic peak of double bond is 5.39-5.61 ppm corresponding to-NHCO-connecting TCO and PAMAM bracket, the characteristic peak of aliphatic hydrogen on TCO ring is 1.39-4.22 ppm corresponding to-CH = CH-of trans-cyclooctene. Of PAMAM-G5-TCO 1 HNMR spectra confirmed that trans-cyclooctene was successfully modified on the surface of PAMAM-G5-NH 2.
Transmission Electron Microscope (TEM) images show that the nanoparticles are very dense and the shape of the conjugate is mostly spherical (see fig. 1). The particle size observed by TEM is about 20-40 nm.
The Zeta potential of PAMAM-G5-TCO was determined by Dynamic Light Scattering (DLS) and Phase Analysis Light Scattering (PALS) using a nanoZsizer (Malvern Zetasizer Nano ZS 90), respectively (see FIG. 2). The results showed that the zeta potential was positive and was about 36.7mV. The result shows that after the TCO group is modified, the size of the dendritic macromolecule is increased, but the dendritic macromolecule still has positive charge; this indicates that the PAMAM has lost some of the positive amino end groups but remains stable in solution.
Example 11 screening for dominant Tz-PROTAC Using immunoblotting
The invention adopts an immunoblotting experimental method to carry out activity screening on the Tz-PROTAC molecules (TCB-1-TCB-4 and TCP-1-TCP-4) given in the embodiments 1-9. TCB-1 to TCB-4 at formulated concentrations of 0.5nM, 5nM and 50nM adding MV-4-11 cells (purchased from the cell Bank of the Committee for type culture Collection of the Chinese academy of sciences) at 37 ℃ 5% 2 In an incubator of 24 hoursAnd detecting the concentration of the target protein by using an immunoblotting method. Or TCP-1 to TCP-4 were added at concentrations of 0.5nM, 5nM and 50nM to SW620 cells (purchased from cell bank of the tissue culture Collection of Chinese academy of sciences) and incubated in an incubator at 37 ℃ and 100% air for 24 hours, and then the concentration of the target protein was determined by immunoblotting.
The immunoblotting experiment was performed as follows:
first, mix and wash cells with 1 × PBS (Gibco, 10010023), aspirate, add weak RIPA lysate (100 μ L) (Biyun day, P0013) to lyse cells for 30 minutes, centrifuge at 13000rpm for 15 minutes at 4 deg.C, take 80 μ L of supernatant and add 20 μ L of loading buffer (Biyun day, P0015) and heat at 100 deg.C for 8 minutes, cool on ice, load 10 μ L onto 10% SDS-PAGE gel, run at constant pressure of 60V on Bio-Rad protein electrophoresis apparatus until the marker is separated, and run 120V to the bottom edge. And after electrophoresis, taking down the glue, shearing a PVDF film with a proper size according to the size, and sequentially arranging filter paper, the PVDF film, the glue, the filter paper and a black gauze from the white board to the blackboard. Protein was transferred to PVDF membrane (Perkin Elmer, northwalk, CT, USA) using a wet transfer membrane for 2 h. After completion, the PVDF membrane was immersed in ponceau staining solution, stained for 2 to 5min, washed with distilled water, the target band was cut off, and ponceau was washed away with 1 × tbst (cloudy day, ST 671). Blocking with blocking buffer (1 × TBST containing 5% w/v skimmed milk powder) for 1h at 37 ℃. After blocking was complete, three washes of 15ml 1 × tbst were performed, 5 minutes each. The primary antibodies used were BRD2 (Abcam, ab 139690), BRD3 (Abcam, ab 50818), BRD4 (Abcam, ab 128874), PARP (CST, # 9532S), β -actin (Proteintech, 66009-1-Ig), placed at the appropriate dilution as recommended in the product specification in 10mL primary Anti-dilution buffer (1 tbst containing 5% skimmed milk powder, to prepare 20mL, add 1.0g skimmed milk powder to 20ml 1 tbst, then mix well), incubated overnight at 4 ℃ and gently shaken from time to time, washed three times with 1 tbst for 10 minutes each after completion, then diluted Anti-rabbitigg, cell Signaling Antibody HRP (Cell Signaling #7074, in the proportion of 1 st) and Anti-HRP, mote, igG, cell Signaling Antibody HRP (Cell Signaling # 68810), incubated with the membrane for three times at room temperature, and incubated with the protein, after incubation, the protein was incubated with the incubation for 1000 hours, the protein was incubated with the # 1 st, incubated for 10 minutes, and incubated with the protein was washed with the membrane # 1 lrgene # 1, incubated for three times, after incubation, the incubation with the incubation, the protein was incubated for 10 hours, and the protein was kept at room temperature, incubated with the # 1 st, and the protein was incubated for 10 hours, and the following incubation with the incubation time, incubated with the sequence # 1, incubated for 10 hours, the following incubation of the membrane, the incubation of the membrane was carried out, the incubation of the membrane, the membrane was carried out, the following 1. The immunoblotting results are shown in FIG. 3, in which TCB-2 and TCP-1 are the two series of advantageous compounds, respectively.
EXAMPLE 12 establishment of "connection-adsorption" System
The connection-adsorption system consists of controlled proteolysis target chimeras Tz-PROTACs and PAMAM-G5-TCO, wherein the PAMAM-G5-TCO is trans-cyclooctene modified Polyamide (PAMAM) dendrimer, and the PAMAM-PROTACs are formed through inverse electron Diels-Alder reaction to completely absorb the controlled proteolysis target chimeras.
According to the invention, (+) -JQ-1 is combined with CRBN ligand through tetrazine group modified linkers with different lengths, so that a series of Tz-PROTACs (TCB series and TCP series) targeting BET family proteins are constructed. 1,2,4, 5-tetrazine is a small linker fragment that can be easily incorporated into the linker of PROTACs.
The fifth generation PAMAM dendrimer is a commercial, highly branched, monodisperse, spherical nanomaterial. The material has a perfect spherical skeleton, and a large number of functional segments can be modified on the surface of a huge molecule. The present invention prepared a novel dendrimer named PAMAM-G5-TCO (see example 10).
Theoretically, PAMAM-G5-TCO can form PAMAM-PROTACs through IEDDA reaction, thereby completely absorbing Tz-PROTACs. Due to the rapid reactivity of IEDDA and the numerous groups on the surface of the PAMAM-G5-TCO macromolecule, intracellular free PROTACs can be rapidly and completely eliminated, thereby stopping the event-driven protein degradation in living cells.
Example 13 evaluation of Tz-PROTACs degradation Activity
The invention utilizes the immunoblotting experiment to evaluate the degradation effect of TCB-2 and TCP-1 on the target protein.
TCB-2 was prepared as a 10. Mu.M solution in DMSO, diluted 3-fold at 12 concentrations and added to MV-4-11 cells. At the temperature of 37 ℃ and under the temperature of the water,5％CO 2 after 24 hours of incubation in the incubator of (1), the BET proteins (BRD 2, BRD3 and BRD 4) in MV-4-11 cells were detected by immunoblotting, the immunoblotting experiment referring to example 11. The experimental results show that TCB-2 can induce the degradation of the BET protein in MV-4-11 in a concentration-dependent manner. (see fig. 4).
A10. Mu.M solution of TCP-1 was prepared in DMSO, diluted 3-fold at 12 concentrations and added to SW-620 cells. After 24 hours of incubation, the SW620 cells were detected for PARP protein using immunoblotting, an immunoblotting experiment referring to example 11. The results of the experiments show that TCP-1 is able to induce the degradation of PARP protein in SW620 cells in a concentration-dependent manner. (see FIG. 5)
5nM TCB-2 solution in DMSO was added to MV-4-11 cells. After incubation for 0, 2,4, 8, 12 and 24 hours in sequence, BET proteins (BRD 2, BRD3 and BRD 4) were detected in MV-4-11 cells using immunoblotting, an immunoblotting experiment being referred to in example 11. The experimental results show that TCB-2 can induce the degradation of the BET protein in MV-4-11 in a time-dependent manner. (see FIG. 6).
A10 nM solution of TCP-1 was prepared in DMSO and added to SW620 cells. After incubation for 0, 2,4, 8, 12 and 24 hours in sequence, the SW620 cells were detected for PARP protein using immunoblotting, which is an experiment referred to in example 11. Experimental results show that TCP-1 can induce the degradation of the BET protein in MV-4-11 in a time-dependent manner.
(see FIG. 7).
Example 14 investigation of degradation mechanism of TCB-2
The invention utilizes an immunoblotting experiment to verify the degradation mechanism of TCB-2 induced BET family protein.
(+) -JQ-1 (1. Mu.M) (MCE: HY-13030) and MLN4924 (1. Mu.M) (MCE: HY-70062) solutions in DMSO solutions were added to MV-4-11 cells at 37 ℃ with 5% CO 2 After 4h incubation in the incubator of (1), the negative compounds TCB-Neg (50nM, TCB-2 negative control) and TCB-2 (5 nM) were added, and samples were collected after 16h, and the BET family proteins (BRD 2, BRD3 and BRD 4) were detected in MV-4-11 cells by immunoblotting, the procedure of which was described in reference example 11. The results of the experiments showed that NAE inhibitor (MLN-4924) completely blocked the degradation of BRD2 and BRD3 by TCB-2, indicating that the degradation process is egg-dependentThe proteasome. At the same time, the addition of the BET inhibitors (+) -JQ-1 and TCB-Neg effectively blocked the degradation of BRD2 and BRD3 by TCB-2. This series of results indicates that TCB-2 induces the degradation of BET family proteins by inducing the formation of ternary complexes and using the UPS system (see FIG. 8).
Example 15TCB-2 Induction of MV-4-11 apoptosis by degradation of BET proteins
Preparing TCB-2 (5 nM), TCB-2 (50 nM) and (+) -JQ-1 (50 nM) solutions in DMSO solutions, adding MV-4-11 cells at 37 ℃,5% CO 2 After the cells are incubated in the incubator for 24 hours, the cells are analyzed by a BD FACS Calibur flow cytometer, and the experimental result shows that concentration-dependent apoptosis occurs after the MV-4-11 cells are acted by TCB-2 for 24 hours. No significant apoptosis was observed in the BET inhibitor group at the same concentration as in the TCB-2 group (see FIG. 9).
Example 16TCB-2 inhibition of MV-4-11 cell proliferation by degradation of BET proteins
Cell viability assays were used to evaluate the level of inhibition of AML cell lines by inhibitors and degradants targeting BET family proteins. MV-4-11 cells were cultured at 5X 10 3 Cell/well density was plated in 96-well plates for culture. TCB-2 (2. Mu.M), (+) -JQ-1 (2. Mu.M) solutions were prepared in DMSO solutions, diluted three-fold in 9 concentration gradients and MV-4-11 cells were added. Cells and different concentrations of compounds at 37 ℃ 5% CO 2 Was incubated in the incubator of (1) for 48 hours. Then, 100. Mu.L of CellTiter-Lumi Plus (Beyotime C0068M) detection reagent was added to each well, and shaken for 5min. Fluorescence values (RLU) were measured using a spectrophotometer (multifunctional microplate reader). The fluorescence value of the blank (medium, gibco 11875093) in the test is recorded as RLU blank The fluorescence value of the negative control (medium + cells) is recorded as RLU control The fluorescence of the sample is recorded as RLU treated . The inhibition rate of the compound at each concentration is calculated as follows:
Figure BDA0003987367310000191
IC was calculated using GraphPad Prim 8.0 software 50 The value is obtained.
Cell viability assay showed, TCInhibition of MV-4-11 cells by B-2 (IC) 50 <5 nM) is much greater than (+) -JQ1 (IC) 50 =211 nM), which indicates that TCB-2 inhibits cell viability by degrading BET (see fig. 10). As noted above, TCB-2 is a typical event-driven degradation agent.
Example 17 qualitative/quantitative analysis of Click reaction between TCB-2 and PAMAM-G5-TCO
The invention carries out qualitative and quantitative analysis aiming at the reaction between PAMAM-G5-TCO and Tz-PROTACs, and the concentration of the PAMAM-G5-TCO mentioned in the invention refers to the apparent concentration of trans-cyclooctene modified on the surface of the PAMAM framework.
TCB-2 (DCM solution) and PAMAM-G5-TCO (PBS solution) were mixed in equal volumes at 500. Mu.M concentration, stirred at 37 ℃ for 5 minutes and observed for experimental phenomenon. The qualitative analysis result shows that: TCB-2 dissolved in organic phase (DCM) was able to be absorbed by PAMAM-G5-TCO dissolved in aqueous Phase (PBS) changing from colorless to yellow and the organic phase (DCM) becoming pale in color within 5min by IEDDA reaction at 37 ℃.
200 μ M of TCB-2 was incubated with 0, 0.5, 1, 1.5, 2 and 3 equivalents of PAMAM-G5-TCO at 37 ℃ for 5 minutes and the residual amount of free TCB-2 was evaluated by High Performance Liquid Chromatography (HPLC). The results show that more than 2 times equivalent of PAMAM-G5-TCO can completely absorb and completely eliminate free TCB-2 within 5min at 37 ℃ (see FIG. 11). The above results demonstrate that PAMAM-G5-TCO is able to rapidly and completely absorb free Tz-PROTACs by IEDDA reaction.
Example 18 inhibition of target binding Activity of TCB-2 by the "ligation-adsorption" System
The inhibitory activity of TCB-2 on BD1 in BRD4 was evaluated by fluorescence polarization. Compound stock (10 mM DMSO) was buffered (150mM NaCl,50mM Na) 3 PO 4 4mM chaps,0.3mM DTT, pH 7.4) were diluted in 12-concentration gradient, and the 5-FAM- (+) -JQ-1 probe and BRD4-BD1 proteins (5-FAM- (+) -JQ-1 probe, BRD4-BD1 proteins were prepared as described in the references: doi.org/10.1016/j.ejmech.2022.114423) was diluted in buffer to the indicated concentration. Equal volumes (20. Mu.L) of the compound, BD1 from BRD4 (final concentration 60 nM) and 5-FAM- (+) -JQ-1 solution (final concentration 3.7 nM) were added sequentially to a black 384 well plate (Corning 3575) in a final volume of 60. Mu.L. Will be provided withThe plates were covered and shaken in the dark at 4 ℃ for 4 hours, and then the polarization values were measured using a SpectraMax multimode microplate monitoring system (Molecular Devices) with an excitation wavelength of 485nm and an emission wavelength of 535 nm. FP values for the blank control (5-FAM- (+) -JQ-1 only) in the test were recorded as mP min The FP value of the negative control (5-FAM- (+) -JQ-1 and BRD4-BD1 protein) was recorded as mP max FP values for the test wells (Compound, 5-FAM- (+) -JQ-1 and BRD4-BD1 protein) were recorded as mP test . The formula for calculating the inhibition rate of the compound is as follows:
Figure BDA0003987367310000201
IC was calculated using GraphPad Prim 8.0 software 50 The value is obtained.
The fluorescence polarization experiment (FP) results show: TCB-2 has strong inhibitory activity to BRD4 protein (the inhibition rate is over 80% at 300 nM). Compared with the free state, after the incubation of TCB-2 and PAMAM-G5-TCO with different equivalent weight for 5 minutes at 37 ℃, the inhibitory activity on BRD4 is obviously reduced, and more than 2 times of the equivalent weight of the PAMAM-G5-TCO can completely absorb the free TCB-2 (see figure 12). Concentration gradient assay showed that the inhibitory activity of TCB-2 on BRD4 (1188 nM) after PAMAM-G5-TCO treatment was reduced by about 15-fold compared to free TCB-2 (76 nM) (see FIG. 13).
Example 19 quantitative relationship study of intracellular PAMAM-G5-TCO and Tz-PROTAC
To MV-4-11 cells were added TCB-2 (5 nM) and different equivalents of PAMAM-G5-TCO (TCB-2. Levels of the BET protein in MV-4-11 cells were determined by immunoblotting. Immunoblot assay methods referring to example 11, after collection of cells, cells were lysed in lysis buffer (2 mM HEPES,150mM NaCl,2mM EDTA,1% Triton X-100, pH 7.4) under ice for 30 minutes, and phosphatase inhibitor (Bilun day, 1045) and protease inhibitor (Bilun day, 1005) without EDTA were added. At 4 ℃ 12000g for 10 min, the supernatant was aspirated and the protein content was tested by BCA. The primary antibodies used in the test were BRD2 (Abcam, ab 139690), BRD3 (Abcam, ab 50818), BRD4 (Abcam, ab 128874), PARP (CST, # 9532S), beta-actin (Proteintetech, 66009-1-Ig). Protein content was detected and analyzed using Tanon5500 Multi-magic system.
The analysis result shows that the addition of equimolar amount of PAMAM-G5-TCO can effectively inhibit the degradation of TCB-2, and more than 2 times of equivalent amount of PAMAM-G5-TCO can completely stop the degradation of TCB-2 (see FIG. 14).
Example 20 "ligation-adsorption" System termination of intracellular TCB-2 induced protein degradation
TCB-2 (100 nM) and TCB-2 (100 nM) were prepared in DMSO solutions with a PAMAM-G5-TCO of 1:3, 3 times diluted to 7 concentrations, added to MV-4-11 cells. At 37 ℃,5% CO 2 After 24 hours of incubation in the incubator of (1), the BET proteins (BRD 2, BRD3 and BRD 4) in MV-4-11 cells were detected by immunoblotting, the immunoblotting experiment referring to example 11. The experimental results showed that there was no significant degradation of the BET family proteins after TCB-2 was absorbed by PAMAM-G5-TCO, whereas the degradation of the BET family proteins of the TCB-2 group was concentration dependent (see FIG. 15).
TCB-2 (5 nM) and TCB-2 (5 nM) were prepared in DMSO solutions with a PAMAM-G5-TCO of 1:3, to MV-4-11 cells. Successively, 5% CO at 37 ℃ 2 After incubation in the incubator of (1) for 0, 2,4, 8, 12 and 24 hours, the BET family proteins (BRD 2, BRD3 and BRD 4) were detected in MV-4-11 cells by immunoblotting, an immunoblotting experiment being referred to in example 11. The experimental results showed that there was no significant degradation of the BET family proteins after TCB-2 was absorbed by PAMAM-G5-TCO, whereas the degradation of the BET family proteins of the TCB-2 group was time dependent (see FIG. 16).
Example 21 "ligation-adsorption" System to terminate TCP-1-induced protein degradation in cells
TCP-1 (150 nM) and TCP-1 (150 nM) were made up in DMSO solutions with a PAMAM-G5-TCO of 1:3, 3-fold diluted to 7 concentrations, added to SW620 cells. After incubation at 37 ℃ for 24 hours in an incubator with 100% air, the PARP protein in SW620 cells was detected by immunoblotting, which is an experiment according to reference example 11. The experimental results showed that there was no significant degradation of PARP protein after TCP-1 was absorbed by PAMAM-G5-TCO, whereas the degradation of PARP protein of TCP-1 group was concentration dependent (see FIG. 17).
TCP-1 (10 nM) and TCP-1 (10 nM) were prepared in DMSO solutions with a PAMAM-G5-TCO of 1:3, to SW620 cells. PARP protein was detected in SW620 cells by immunoblotting after incubation for 0, 2,4, 8, 12 and 24 hours at 37 ℃ in a 100% air incubator in sequence, reference example 11 of the immunoblotting experiment. The experimental results showed that after TCP-1 was absorbed by PAMAM-G5-TCO, there was no significant degradation of PARP protein, whereas the degradation of PARP protein of TCP-1 group was time dependent (see FIG. 18).
A solution of TCP-1 (10 nM) was prepared in DMSO, added to SW620 cells, and cell samples were collected after incubation sequentially at 37 ℃ in an incubator with 100% air for 0, 2,4, 8, 12 hours, and SW620 cells incubated for 12 hours with TCP-1 (10 nM) were incubated with TCP-1: 3, and then sequentially incubating in an incubator at 37 ℃ and 100% air for 0, 2,4, 6, 8, 12, 24, 36, and 48 hours, collecting cell samples, and detecting the level of PARP protein by immunoblotting, which is the experiment of reference example 11. The results of the experiments show that the "link-adsorption" system can stop the degradation of PARP protein in living cells (see figure 19).
Example 22 uptake of TCB-2 by the "Link-adsorb" System to terminate apoptosis
TCB-2 (5 nM), (+) -JQ-1 (5 nM) and TCB-2 (5 nM) were prepared in DMSO solutions: the result of the flow cytometry analysis of the PAMAM-G5-TCO (1.
Example 23 termination of the proliferation-inhibiting Effect of TCB-2 on cells by the "ligation-adsorption" System
MV-4-11 cells were cultured at 5X 10 3 Cell/well density was plated in 96-well plates for culture. TCB-2 (2. Mu.M), (+) -JQ-1 (2. Mu.M), PAMAM-G5-TCO (2. Mu.M) and TCB-2 (5 nM) were prepared in DMSO solutions: PAMAM-G5-TCO (1. The experimental procedure was the same as in example 16.
Cell viability assay showed that TCB-2 incubated with PAMAM-G5-TCO had significantly less inhibition of MV-4-11 cell viability than (+) -JQ1 and free TCB-2 (see FIG. 21). This indicates that: the "ligation-adsorption" system successfully terminated the proliferation-inhibiting effect of TCB-2 on the cells.
The "link-adsorb" system of the invention can rapidly eliminate intracellular molecules of PROTACs and terminate event-driven degradation. This strategy has the following advantages: first, the system is able to rapidly and completely terminate event-driven degradation, which benefits from the fast reactivity of the IEDDA reaction and the numerous groups on the surface of the PAMAM-G5-TCO macromolecule. Secondly, the tetrazine is a functional fragment with small volume, has good biocompatibility, can be easily applied to different molecular designs, and has good universality; thirdly, the system is simple to operate, the target protein degradation process can be started/closed only by adding Tz-ProTACs or PAMAM-G5-TCO, and no additional operation is needed. In conclusion, the present invention provides an unprecedented tool for studying protein degradation, making it possible to flexibly adjust the level of target protein in living cells as desired.
As noted above, while the present invention has been shown and described with reference to certain preferred embodiments, it is not to be construed as limited thereto. Various changes in form and detail may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.

Claims (9)

1. A controllable proteolysis targeting chimera shown as a general formula I,
Figure FDA0003987367300000011
wherein,
R 1 is selected from
Figure FDA0003987367300000012
R 2 Is selected from
Figure FDA0003987367300000013
Figure FDA0003987367300000014
n＝1～3。
2. The controlled proteolytic targeting chimera according to claim 1, characterized by being selected from the group consisting of:
Figure FDA0003987367300000015
/>
Figure FDA0003987367300000021
wherein n =1 to 3.
3. The controlled proteolytic targeting chimera according to claim 1, characterized in that: n =1.
4. Use of a controlled proteolysis targeting chimera according to any one of claims 1-3 to terminate targeted protein degradation.
5. The use of claim 4, wherein the controlled proteolytic targeting chimera is used to produce molecular biologically active, terminable targeted proteolytic degradation ProTAC or small molecule inhibitors.
6. A link-adsorption system for instantly terminating target protein degradation, which is characterized by consisting of a controlled proteolysis targeting chimera and PAMAM-G5-TCO, wherein the PAMAM-G5-TCO is trans-cyclooctene modified polyamide PAMAM dendrimer which forms PAMAM-PROTACs through inverse electron Diels-Alder reaction to completely absorb the controlled proteolysis targeting chimera.
7. The ligation-adsorption system for instantly terminating the degradation of a target protein according to claim 6, wherein the PAMAM-G5-TCO is synthesized by the following method:
mixing PAMAM-G5-NH 2 Dissolving in an organic solvent, adding a TCO-NHS-Ester solution, stirring at room temperature, dialyzing the reaction solution with pure water after the reaction is finished, collecting the dialyzate, and freeze-drying to obtain the PAMAM-G5-TCO product.
8. The ligation-adsorption system for instantly terminating the degradation of a target protein as claimed in claim 7, wherein the organic solvent in the synthesis of PAMAM-G5-TCO is selected from methanol.
9. A trans-cyclooctene modified polyamide PAMAM dendrimer PAMAM-G5-TCO is characterized in that,
has the following structural formula:
Figure FDA0003987367300000031
/>
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