CN112457297B

CN112457297B - PARP protein degradation agent and preparation method and application thereof

Info

Publication number: CN112457297B
Application number: CN202011328048.7A
Authority: CN
Inventors: 刘洋; 林珊珊; 吴丽贤; 黄秀旺; 许建华; 刘全裕; 林友文
Original assignee: Fujian Medical University
Current assignee: Fujian Medical University
Priority date: 2020-11-24
Filing date: 2020-11-24
Publication date: 2023-04-28
Anticipated expiration: 2040-11-24
Also published as: CN112457297A

Abstract

A PARP protein degradation agent shown as X-Y-Z or X-Z, and its preparation method and application are provided: X-Y-Z (I-1) or X-Z (I-2), X represents the ligand of PARP protein, Z represents the ligand of E3 ligase, Y represents the chain connecting X and Z, the compound can target and degrade PARP protein, and can be used for treating or preventing tumor.

Description

PARP protein degradation agent and preparation method and application thereof

Technical Field

The invention relates to the field of antitumor drug preparation, in particular to a compound for targeted degradation of PARP protein, a preparation method and application thereof.

Background

Poly (ADP-ribose) polymerase, PARP, is a ribozyme that is closely related to DNA repair, particularly DNA single strand damage BER repair, and includes 18 subtypes, with the highest levels of PARP-1 in eukaryotic cells. When tumor cell DNA is damaged by chemotherapy or ionizing radiation, PARP-1 is activated quickly, and NAD+ is used as a substrate to transfer poly (ADP) to specific protein, PARP-1 catalyzes nuclear saccharification (PAR) of poly (ADP) of specific protein and starts DNA repair process. Studies have shown that PARP-1 is involved in DNA repair pathways such as BER, HR, and NHEJ.

The PARP inhibitor can inhibit PAR function of PAPR to prevent damaged DNA from being repaired effectively, or the PARP inhibitor is combined with an NAD+ binding pocket of PARP1 (or PARP 2) to cause conformational isomerism, so that PARP is captured at the damaged DNA and cannot be repaired subsequently, and both PARP and PARP can kill tumor cells. Although PARP inhibitors have achieved remarkable clinical results, conventional PARP small molecule inhibitors do not affect PARP protein levels, which quickly recover activity upon withdrawal; drug resistance is easy to occur. And the PARP protein degradation agent based on the PROTAC technology can effectively and permanently inhibit the growth of tumors because the tumor cells need a certain time to recover the content of the PARP protein in the nucleus even after stopping the drug.

Several PARP1/2 degrading agents have been reported, including compound 1zhao, q.; lan, t.; su, s.; rao, y. Instruction of apoptosis in MDA-MB-231breast cancer cells by a PARP1-targeting PROTAC small molecular. Chem. Comm.2019,55,369-372. Compound 2 (icaparib-AP 5), 3 (icaparib-AP 6), and 4 (icaparib AP 6) Wang, s.; han, l.; han, j.; li, P; ding, q; zhang, q. -j; liu, z. -p; chen, c.; yu, y.unctupling of PARP1 trapping and inhibition using selective PARP degradation, nature, chem. Biol.2019,15,1223-1231. Compound 5, cao, c. -g; yang, j.; chen, y. -w.; discovery of SK-575as a Highly Potent and Efficacious Proteolysis Targeting Chimera (PROTAC) Degrader of PARP1 for treatment treatments cancer J.Med.chem 2020.DOI:10.1021/acs. Jmed chem.0c00821.

Compound 1, based on the niraparib derivative and the nutlin-3 derivative, specifically induces PARP1 cleavage and apoptosis in MDA-MB-231 cells. Compounds 2 and 3, prepared by using all polyethylene glycol (PEG) linkers of varying lengths, were reported to be potent and effective PARP1 degrading agents with compounds having maximum degradation concentrations (DC 50) of 36nM and 82nM, respectively, for rat neonatal cardiomyocytes. However, these compounds do not completely degrade intracellular PARP1 proteins, thereby limiting their therapeutic efficacy.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention provides a PARP protein degradation agent, a preparation method and application thereof, the compound adopts a double-target molecular structure, and target degradation of target protein is achieved by utilizing a PROTAC (protein-targeting chimeras) technology, namely a ubiquitination degradation system which is responsible for clearing defective proteins in cells. The structure of one end of the molecule is targeted to bind E3 ligase MDM2, the structure of the other end is targeted to bind PARP protein, the structures of the two ends are connected through a chain (linker) to form a complete compound molecule, and the compound ubiquitinates target protein through E3 ligase and guides the target protein to enter a proteasome degradation system to specifically degrade the target protein.

The compounds provided herein may have a proliferation inhibiting effect in breast cancer cells such as MDA-MB-231 at a lower concentration than the PARP inhibitor nilaparib. Therefore, the compound has potential application value in the field of anti-tumor.

Specifically, a PARP protein degradation agent is shown as a formula I-1 or I-2:

X-Y-Z (I-1) or X-Z (I-2)

Wherein X represents a ligand of PARP protein, Z represents a ligand of E3 ligase, Y represents a chain connecting X and Z;

x is a compound shown in a formula II-1, Z is a compound shown in a formula II-2,

the Y is a compound shown in a formula III-1, the Y is a compound shown in the formula III-1,

each n is independently an integer of 1 to 10

In detail, the PARP protein degradation agent is:

formula 1-1, structure of X-Z type

1-2, n=1

1-3, n=2

1-4, n=3

1-5, n=4

1-6, n=5

The preparation process of the compound comprises the following steps:

drawings

FIG. 1 is a schematic diagram showing the PARP protein degradation of PARP protein degradation agent of example 4 of the present invention on different tumor cell lines, and it can be seen that the compounds NN3 of formulas 1-4 significantly degrade MDA-MB-231 and MCF-7PARP proteins.

Detailed Description

Embodiments of the present invention will be described in further detail below, which are exemplary, intended to be illustrative of the present invention, and not to be construed as limiting the present invention.

Example 1 preparation of Compounds of formula 1-1

1-1

A mixture of Nutlin acid intermediate (123.31 mg,0.25 mmol), HATU (70 mg,0.184 mmol) and DIEA (90. Mu.L) was stirred in a round bottom flask for 0.5 h at room temperature. Nilapatinib (79 mg,0.25m was then addedmol) of 0.5mL anhydrous DMF solution was slow. The reaction mixture was stirred at room temperature for 12 hours. The reaction was quenched, the water and mixture were washed once with saturated aqueous NaCl solution and ethyl acetate was extracted with diethyl ether. The organic layer was treated with anhydrous Na ₂ The SO4 was dried and evaporated in vacuo to give the crude product which was further purified by chromatography on a silica gel column (DCM: meoh=40:1) to give the product.

¹ H NMR(400MHz,Chloroform-d)δ9.18–8.96(m,1H),8.54(d,J＝5.4Hz,1H),8.32(dd,J＝6.7,3.6Hz,1H),7.99–7.75(m,3H),7.61(dd,J＝8.4,3.4Hz,1H),7.41(t,J＝7.6Hz,2H),7.30(d,J＝4.3Hz,1H),7.27(d,J＝7.4Hz,1H),7.15–7.01(m,4H),7.00–6.81(m,4H),6.71–6.44(m,2H),6.20(dd,J＝10.8,3.3Hz,1H),5.67–5.18(m,2H),4.65(t,J＝13.0Hz,2H),4.25(dd,J＝37.8,15.8Hz,1H),4.11(s,1H),3.96(d,J＝15.9Hz,1H),3.92–3.68(m,6H),3.47(s,2H),3.27–3.06(m,3H),3.05–2.72(m,2H),2.65(t,J＝12.1Hz,1H),2.14(d,J＝10.6Hz,1H),1.83–1.57(m,2H),1.46–1.33(m,6H).[M-H]939.80。

Example 2 preparation of Compounds of formulas 1-2

1-2

A mixture of Nutlin acid intermediate (123.31 mg,0.25 mmol), HATU (70 mg,0.184 mmol) and DIEA (90. Mu.L) was stirred in a round bottom flask for 0.5 h at room temperature. Then, NH is added ₂ -PEG ₁ A solution of COOtBu (33.26 mg,0.25 mmol) in 0.5mL anhydrous DMF was slow. The reaction mixture was stirred at room temperature for 12 hours. The reaction was quenched, the water and mixture were washed once with saturated aqueous NaCl solution and ethyl acetate was extracted with diethyl ether. The organic layer was treated with anhydrous Na ₂ The SO4 was dried and evaporated in vacuo to give the crude product which was further purified by chromatography on a silica gel column (DCM: meoh=40:1) to give the product.

To a solution of Nutlin derivative ester (140 mg, 0.020mmol) in DCM (1 mL) was added TFA (2 mL) at 0deg.C. After stirring at room temperature for 17 hours, the resulting mixture was diluted with toluene (10 mL) and evaporated. The residue was purified by flash column chromatography (initially 3:99 methanol-dichloromethane, fractionated to 1:9 methanol-dichloromethane) to give 123mg of Nutlin derivative acid intermediate.

A mixture of Nutlin derivative acid intermediate (123.31 mg,0.25 mmol), HATU (70 mg,0.184 mmol) and DIEA (90. Mu.L) was stirred in a round bottom flask for 0.5 h at room temperature. Then, a solution of Nilapatinib (79 mg,0.25 mmol) in 0.5mL anhydrous DMF was added slowly. The reaction mixture was stirred at room temperature for 12 hours. The reaction was quenched, the water and mixture were washed once with saturated aqueous NaCl solution and ethyl acetate was extracted with diethyl ether. The organic layer was dried over anhydrous Na 2so 4 and evaporated in vacuo to give the crude product which was further purified by chromatography on a silica gel column (DCM: meoh=40:1) to give the product.

¹ H NMR(400MHz,Chloroform-d)δ9.05(s,1H),8.54(d,J＝3.6Hz,1H),8.32(d,J＝7.0Hz,1H),8.03–7.74(m,3H),7.59(t,J＝8.6Hz,1H),7.41(dd,J＝8.3,6.0Hz,3H),7.26(s,1H),7.16–6.79(m,8H),6.68–6.35(m,2H),6.20(s,1H),5.69–5.41(m,2H),5.31(s,1H),4.83–4.48(m,2H),4.01–3.70(m,9H),3.62–3.40(m,7H),3.13(s,3H),2.84–2.61(m,3H),2.14(d,J＝12.3Hz,1H),1.86–1.54(m,3H),1.44–1.29(m,6H).[M+H]1056.50。

Example 3 the compounds of formulas 1-3 were prepared according to the preparation method described above.

1-3

¹ H NMR(400MHz,Chloroform-d)δ9.07(s,1H),8.56(s,1H),8.33(d,J＝7.1Hz,1H),8.04(s,1H),7.88(dq,J＝25.5,9.0,8.1Hz,3H),7.61(d,J＝8.5Hz,1H),7.41(dd,J＝19.7,8.8Hz,2H),7.15–6.78(m,8H),6.56(d,J＝8.0Hz,1H),6.49(s,1H),6.07(s,1H),5.59(d,J＝9.5Hz,1H),5.49(d,J＝9.4Hz,1H),4.85–4.67(m,1H),4.66–4.53(m,1H),4.13(d,J＝7.5Hz,1H),3.99(q,J＝14.5,12.8Hz,2H),3.86(d,J＝8.0Hz,6H),3.74(d,J＝14.1Hz,1H),3.64(dd,J＝12.2,5.1Hz,4H),3.58–3.38(m,5H),3.30(s,1H),3.13(d,J＝10.6Hz,3H),2.98(s,1H),2.91(s,1H),2.67(dd,J＝21.5,9.3Hz,4H),2.25–1.99(m,2H),1.37(d,J＝16.6Hz,6H).[M+H]1100。

Example 4 Compounds of formulas 1-4 were prepared according to the preparation methods described above.

1-4 (NN 3)

¹ H NMR(400MHz,Chloroform-d)δ9.07(s,1H),8.55(s,1H),8.37–8.21(m,1H),7.96–7.74(m,3H),7.58(d,J＝8.4Hz,1H),7.41(d,J＝7.7Hz,3H),7.26(d,J＝7.6Hz,2H),7.08(d,J＝7.5Hz,2H),7.03(d,J＝7.5Hz,2H),6.93(d,J＝7.8Hz,2H),6.88(d,J＝7.7Hz,2H),6.54(d,J＝8.4Hz,1H),6.48(s,1H),6.31(d,J＝18.1Hz,1H),5.57(d,J＝9.6Hz,1H),5.48(d,J＝9.6Hz,1H),4.78–4.52(m,2H),4.04–3.89(m,2H),3.85(s,5H),3.81(d,J＝4.4Hz,2H),3.73–3.55(m,10H),3.52(s,3H),3.41(s,2H),3.27(s,2H),2.98–2.86(m,1H),2.80(d,J＝12.1Hz,1H),2.72–2.58(m,3H),1.85–1.56(m,4H),1.35(dd,J＝13.8,4.4Hz,6H).[M+Na]1166.60。

Example 5 Compounds of formulas 1-5 were prepared according to the preparation methods described above.

1-5

¹ H NMR(400MHz,Chloroform-d)δ9.11(s,2H),8.55(d,J＝4.6Hz,2H),8.33(dd,J＝8.3,2.1Hz,2H),8.20–8.00(m,2H),7.99–7.75(m,6H),7.67–7.49(m,1H),7.49–7.33(m,5H),7.18–6.99(m,1H),6.90(d,J＝8.4Hz,1H),6.63–6.41(m,1H),6.21(d,J＝7.9Hz,3H),5.84–5.07(m,2H),4.91–4.43(m,3H),4.26–

3.55(m,6H),3.48–3.05(m,5H),3.05–2.44(m,6H),2.42–2.04(m,8H),1.30(d,J＝18.5Hz,9H),1.03–0.68(m,6H).[M+Na]1212.40。

Example 6 Compounds of formulas 1-6 were prepared according to the preparation methods described above.

1-6

¹ H NMR(400MHz,Chloroform-d)δ9.07(d,J＝2.9Hz,1H),8.61(s,1H),8.31(d,J＝6.4Hz,1H),8.17(s,1H),8.05(d,J＝8.7Hz,3H),7.98(d,J＝8.7Hz,2H),7.93(d,J＝8.3Hz,1H),7.60(d,J＝8.5Hz,1H),7.30(s,1H),7.27(s,1H),7.09(d,J＝8.4Hz,4H),7.03(d,J＝8.5Hz,3H),6.94(d,J＝8.4Hz,2H),6.89(d,J＝8.3Hz,2H),6.64(s,2H),6.48(d,J＝2.2Hz,1H),6.31(d,J＝2.9Hz,1H),5.58(d,J＝9.7Hz,1H),5.49(d,J＝9.7Hz,1H),4.62(dt,J＝9.9,5.5Hz,3H),4.00–3.92(m,4H),3.85(s,5H),3.77(d,J＝15.6Hz,1H),3.71–3.64(m,6H),3.63(s,5H),3.60–3.54(m,5H),3.54–3.44(m,5H),3.39(q,J＝5.1Hz,3H),3.28(dt,J＝13.3,5.9Hz,2H),3.12(p,J＝7.1,6.2Hz,2H),1.40–1.34(m,6H).[M+H]1234.55。

Example 4 the compounds of the present invention have a high PARP degrading activity and are tested using small molecule compounds of formulas 1-1 to 1-6 as examples.

1. The main solution preparation:

configuration of test compounds and positive control drugs nilaparib and Nutlin: weighing a proper amount of test compound powder, dissolving in DMSO to obtain a final concentration of 10mmol/L,10 μl/tube, storing at-20deg.C in dark place, thawing in the future, and diluting at the required concentration.

RPMI1640 medium: each bag of RPMI1640 powder is dissolved in about 800ml double distilled water, 1.5g nahco3,2.5g glucose and 0.11g sodium pyruvate are added, and the mixture is fully dissolved and is supplemented to 1000ml by double distilled water. Filtering and sterilizing with 0.22 μm microporous membrane, packaging in equal amount, and storing at 4deg.C for use. 10% fetal bovine serum, 1 Xdiabody, was added prior to use.

DMEM broth: each bag of DMEM powder is dissolved in about 800ml of double distilled water, 1.5g of NaHCO3,2.5g of glucose and 0.11g of sodium pyruvate are added, and the mixture is fully dissolved and supplemented to 1000ml by the double distilled water. Filtering and sterilizing with 0.22 μm microporous membrane, packaging in equal amount, and storing at 4deg.C for use. 10% fetal bovine serum, 1 Xdiabody, was added prior to use.

PBS buffer (pH 7.4):8.0g of NaCl, 0.2g of KCl and Na are weighed ₂ HPO4·12H ₂ O 1.44g，KH ₂ PO4·12H ₂ O0.24 g, dissolved in 900ml double distilled water, HCl adjusted pH to 7.4, double distilled water was used to fix volume to 1000ml, autoclaved and stored at room temperature.

2. Immunoblotting analysis to detect protein expression level of cells

(1) Electrophoresis: the denatured protein samples were equally loaded into 5% concentrated gel and 10% separation gel (ddH 2O,30% polyacrylamide, 1.5M Tris-HCl (pH 8.8) or 1.0M Tris-HCl (pH 6.8), 10% SDS,10% ammonium persulfate, TEMED) as the main components, the loading amount of each pore protein was about 40. Mu.g, 100V constant pressure electrophoresis, and the electrophoresis was ended when bromophenol blue was fast run to the bottom of the gel.

(2) Transferring: after electrophoresis, the glass plate is disassembled to take out gel, the concentrated gel is cut off, then the separation gel is properly sheared according to the molecular weight of the target protein and then soaked in the pre-cooling film transferring liquid so as not to dry, and meanwhile, the filter paper and the sponge are soaked in the pre-cooling film transferring liquid. Cutting a PVDF film with the size similar to that of the gel, soaking in methanol for 2min to activate the film, and preparing a sandwich according to the following arrangement sequence: the negative electrode is sequentially stacked with 1 sponge pad, 2 pieces of filter paper, gel, PVDF film, 2 pieces of filter paper and one sponge pad. Note that: the PVDF film must drive off the bubbles after it is attached to the gel. Then, the electrophoresis tank is arranged in the correct positive and negative electrode direction for wet rotation, ice cubes are put on the periphery for cooling, and the voltage is regulated to be constant current of 200V (1.5 h).

(3) Closing: after the transfer, the PVDF membrane is cleaned for 5min in TBST, and then put into 5% sealing liquid to be sealed for 0.5-1h on a decolorizing shaker.

(4) Antibody incubation: primary antibodies were diluted with blocking solution or primary antibody dilution at 1:500-1:1000 and covered on PVDF membranes and incubated at room temperature for 2h or overnight at 4 ℃, followed by three washes with TBST for 5min each. After dilution of the secondary antibody at 1:5000, incubation with PVDF membrane on a shaker for 1h at room temperature was carried out, followed by three washes with TBST for 5min each.

(5) Chemiluminescence, developing and fixing: mixing the reagent A and the reagent B in equal volume, dripping the mixture onto an ImageStation 4000MM imager, then covering the PVDF film on the ECL reagent with the front face downwards, and exposing according to the operation instruction of the imager to obtain a protein band result.

3. Results: the PARP protein degradation agent has the following degradation activity on PARP, namely, in MDA-MB-231 and MCF-7 cell lines, after the compound NN3 obtained in the example 4 is treated for 48 hours, the degradation effect of the compound NN3 on PARP protein under different doses can be obviously observed through Western blotting result analysis. The degradation of PARP protein by compound NN3 in the MDA-MB-231 cell line at 12.5uM,25uM is shown.

Example 5 half inhibition of tumor cell lines of different origins

Different tumor cell lines: human breast cancer cell lines were selected experimentally: MCF-7, HCC1937, MDA-MB-231, SKBR3, MX-1 normal breast cells MCF-10A in order to examine half-inhibitory activity of compound NN3 against tumor cell lines of different origins. The corresponding cells were cultured in RPMI1640 medium containing 10% fetal bovine serum, penicillin 100IU/ml and streptomycin 100. Mu.g/ml. Cells were cultured in a saturated humidity incubator at 37℃with 5% CO2, and cells in the logarithmic growth phase were taken for the experiment. Cells in the logarithmic growth phase were inoculated at 4X 104 cells/ml into 96-well plates at 180. Mu.l per well.

The initial concentration of 200uM prepared in examples 1-6 was added to each of the experimental groups and 20. Mu.l of the half-diluted compound was used without the addition of the negative control, the positive control, nilapatinib, nutlin-3a (from Shanghai Boban), and 3 parallel wells were placed in each group and incubated at 37℃for 48h. After adding 20. Mu.l/well of 5mg/ml MTT solution and further culturing for 4 hours, the supernatant was removed, 150. Mu.l of DMSO was added, and the mixture was shaken by a micro-shaker for 10 minutes, and absorbance (OD value) was measured at a wavelength of 570nm by an enzyme-labeled instrument. Cell growth inhibition rate was calculated from absorbance [ cell growth inhibition rate= (control OD-experimental OD)/control od×100% ], IC50 value was calculated by Logit method, experiment was repeated 3 times, and average value was taken.

Example 4 of the present invention shows different biological activities for different breast cancer cell lines. Compared with a positive control medicine nilaparib, nutlin-3a, the compound NN3 shows stronger anti-tumor effect, and has no cytotoxicity to normal breast cells MCF-10A.

TABLE 1 biological Activity detection at cellular level of PARP degrading agent (maximum inhibition or IC50 (uM) under the action of 200uM of 48h Compound; NS: not tested)

Compounds of formula (I)

Naming the name

Molecular weight

MDA-MB-231

MX-1

MCF-7

SKBR3

HCC1937

MCF-10A

1-1

NN0

1011

5.59％

34.97％

10.19％

18.63％

Has no inhibiting effect

NS

1-2

NN1

1055

Has no inhibiting effect

11.39％

Has no inhibiting effect

NS

1-3

NN2

1099.41

99.03uM

16.71％

42.401uM

40.14％

40.06％

NS

1-4

NN3

1143

22.15uM

24.6％

20.88uM

45.33％

47.02％

38.21％

1-5

NN4

1187

161.52uM

80.26uM

54.32uM

199.11uM

29.63％

NS

1-6

NN5

1231

159uM

59.50uM

117.93uM

266.47uM

243.45uM

NS

Nilapatinib

320

70.40uM

49.91uM

38.69uM

70.23uM

97.15uM

87.16uM

Nutlin

581.49

64.04uM

50.45uM

42.66uM

75.61uM

66.44uM

63.82uM

Claims

1. A PARP protein degradation agent, characterized by the formula i-1 or i-2:

X-Y-Z (I-1) or X-Z (I-2)

X is a structure shown in a formula II-1 and represents a ligand of PARP protein;

y is a structure represented by formula III-1, and represents a chain connecting X and Z;

z is a structure shown in a formula II-2 and represents a ligand of E3 ligase;

Ⅱ-1

Ⅱ-2

Ⅲ-1

n is each independently an integer between 1 and 10.

2. The PARP protein degradation agent according to claim 1, wherein the compound is:

formula 1-1, structure of X-Z type

1-2, n=1

1-3, n=2

1-4, n=3

1-5, n=4

Formulas 1-6, n=5.

3. The process for preparing PARP protein degradation agent according to claim 1, wherein the reaction formula is as follows:

。

4. use of the PARP protein degradation agent according to claim 1 for the preparation of a medicament for the treatment or prevention of tumors.

5. The use according to claim 4, wherein the tumor is breast cancer.