CN112457297A

CN112457297A - PARP protein degradation agent and preparation method and application thereof

Info

Publication number: CN112457297A
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: 2021-03-09
Anticipated expiration: 2040-11-24
Also published as: CN112457297B

Abstract

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

Description

PARP protein degradation agent and preparation method and application thereof

Technical Field

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

Background

Poly (ADP-ribose) polymerase, PARP, is a ribozyme that is closely related to DNA repair, particularly to BER repair of single strand damage of DNA, and includes 18 subtypes, of which PARP-1 is the highest in eukaryotic cells. When tumor cell DNA is damaged by chemotherapeutics or ionizing radiation and the like, PARP-1 is quickly activated, NAD + is used as a substrate, poly (adenosine diphosphate) ribosyl (ADP) is transferred to a specific protein, and the PARP-1 catalyzes the nuclear saccharification (PAR) of the poly (adenosine diphosphate) of the specific protein to start a 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 ensure that damaged DNA cannot be timely and effectively repaired, or the PARP inhibitor is combined with NAD + combination pocket of PARP1 (or PARP2) to cause conformational isomerism, so that PARP is captured at damaged DNA and cannot be subsequently repaired, and both of the PARP inhibitor and the PARP inhibitor can kill tumor cells. Although the PARP inhibitor has achieved surprising results in clinic, the content of PARP protein cannot be affected by the conventional PARP small molecule inhibitor, and the PARP protein can rapidly recover the activity after drug withdrawal; the phenomenon of drug resistance is easy to appear. Even after the medicine is stopped, the tumor cells need a certain time to recover the content of the nuclear PARP protein, so that the PARP protein degrading agent based on the PROTAC technology can effectively and durably inhibit the growth of the tumor.

Currently, several PARP1/2 degradants have been reported, including compound 1Zhao, q.; lan, t.; su, s.; rao, Y.Industtion of apoptosis in MDA-MB-231Breast cancer cells by a PARP1-targeting PROTAC small molecule. chem.Comm.2019,55,369-372 Compound 2(iRucaparib-AP5), 3(iRucaparib-AP6), and 4(iVeliparib AP6) Wang, S.; han, L.; han, J.; li, P.; ding, q.; zhang, q. -j.; liu, z. -p.; chen, c.; yu, Y.Ungroupling of PARP1 mapping and inhibition using selective PARP1 degradation. Nat.Chem.biol.2019,15, 1223-; yang, j.; chen, y. -w.; discovery of SK-575as a high hly post and efficiency proteins Targeting kernel (PROTAC) digrader of PARP1 for Targeting cancer.J.Med.chem 2020.DOI: 10.1021/acs.jmedchem.0c00821.

Compound 1, based on the niraparib derivative and the nutlin-3 derivative, specifically induced lysis and apoptosis of PARP1 in MDA-MB-231 cells. Compounds 2 and 3, prepared by using all polyethylene glycol (PEG) linkers of different lengths, were reported to be potent and effective PARP1 degradants with maximum compound degradation concentrations (DC50) of 36nM and 82nM, respectively, on rat neonatal cardiomyocytes. However, these compounds do not completely degrade the intracellular PARP1 protein, 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 and a preparation method and application thereof, the compound adopts a double-target-point molecular structure, and target protein is degraded in a targeted way by using a PROTAC (protein-targeting chimeras) technology, namely, an ubiquitination degradation system which is responsible for removing defective proteins in cells. One end of the molecule is targeted and combined with E3 ligase MDM2, the other end of the molecule is targeted and combined with PARP protein, the structures at the two ends are connected through a chain (linker) to form a complete compound molecule, and the compound is used for ubiquitinating a target protein through E3 ligase and guiding the target protein to enter a proteasome degradation system to specifically degrade the target protein.

Compared with a PARP inhibitor, the nilapanib provided by the invention can play a role in proliferation inhibition in breast cancer cells such as MDA-MB-231 at a lower concentration. Therefore, the compound has potential application value in the anti-tumor field.

In particular, a PARP protein degradation agent represented by formula I-1 or I-2:

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

Wherein X represents a ligand for PARP protein, Z represents a ligand for E3 ligase, and Y represents a chain linking X and Z;

the X is a formula II-1, the Z is a compound shown as a formula II-2,

the Y is a compound shown as a 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 X-Z type

Formula 1-2, n ═ 1

Formula 1-3, n ═ 2

Formula 1-4, n ═ 3

Formula 1-5, n ═ 4

Formula 1-6, n ═ 5

The preparation process of the compound comprises the following steps:

drawings

FIG. 1 is a schematic diagram of the degradation of PARP protein of different tumor cell lines by the PARP protein degrading agent shown in example 4 of the present invention, and it can be seen that the compound NN3 shown in formulas 1-4 significantly degrades MDA-MB-231 and MCF-7PARP proteins.

Detailed Description

The following examples of the present invention are described in further detail, and are intended to be illustrative, but not limiting, of the present invention.

EXAMPLE 1 preparation of Compound represented by formula 1-1

Formula 1-1

Mixture of Nutlin acid intermediate (123.31mg,0.25mmol), HATU (70mg,0.184mmol) and DIEA (90. mu.L) dried DMF (3.78mL) was stirred at room temperature in a round bottom flask for 0.5 h. Then, a solution of nilapanib (79mg,0.25mmol) in 0.5mL anhydrous DMF was added slowly. The reaction mixture was stirred at room temperature for 12 hours. The water and mixture were washed once with saturated aqueous NaCl solution by quenching the reaction and the ethyl acetate was extracted with diethyl ether. The organic layer was washed with anhydrous Na₂SO4 was dried and evaporated in vacuo to give the crude product, which was further purified by chromatography on silica gel (DCM: MeOH ═ 40: 1) to give the product.

¹H 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 the Compound represented by the formula 1-2

Formula 1-2

Mixture of Nutlin acid intermediate (123.31mg,0.25mmol), HATU (70mg,0.184mmol) and DIEA (90. mu.L) dried DMF (3.78mL) was stirred at room temperature in a round bottom flask for 0.5 h. Then, NH is added₂-PEG₁A solution of-COOtBu (33.26mg,0.25mmol) in 0.5mL anhydrous DMF was slow. The reaction mixture was stirred at room temperature for 12 hours. The water and mixture were washed once with saturated aqueous NaCl solution by quenching the reaction and the ethyl acetate was extracted with diethyl ether. The organic layer was washed with anhydrous Na₂SO4 was dried and evaporated in vacuo to give the crude product, which was further purified by chromatography on silica gel (DCM: MeOH ═ 40: 1) to give the product.

To a solution of the Nutlin derivative ester (140mg, 0.020mmol) in DCM (1mL) was added TFA (2mL) at 0 ℃. After stirring at room temperature for 17 hours, the resulting mixture was diluted with toluene (10mL) 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.

Mixture of Nutlin derivative acid intermediate (123.31mg,0.25mmol), HATU (70mg,0.184mmol) and DIEA (90. mu.L) dried DMF (3.78mL) was stirred at room temperature in a round bottom flask for 0.5 h. Then, a solution of nilapanib (79mg,0.25mmol) in 0.5mL anhydrous DMF was added slowly. The reaction mixture was stirred at room temperature for 12 hours. The water and mixture were washed once with saturated aqueous NaCl solution by quenching the reaction and the 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 silica gel (DCM: MeOH ═ 40: 1) to give the product.

¹H 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 represented by formulas 1 to 3 were prepared according to the above preparation method.

Formulas 1 to 3

¹H 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 represented by formulas 1 to 4 were prepared according to the above preparation method.

Formulas 1-4(NN3)

¹H 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 represented by formulae 1 to 5 were prepared according to the above preparation method.

Formulas 1 to 5

¹H 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 represented by formulae 1 to 6 were prepared according to the above preparation method.

Formulas 1 to 6

¹H 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 strong PARP degrading activity, and the following tests were conducted by taking the small molecule compounds represented by formulas 1-1 to 1-6 as examples.

1. Preparing a main solution:

configuration of test compounds and positive control drugs nilapani and Nutlin: an appropriate amount of test compound powder was weighed, dissolved in DMSO to give a final concentration of 10mmol/L, stored at-20 ℃ in a dark place, thawed immediately, and diluted to the desired concentration.

RPMI1640 culture solution: each bag of RPMI1640 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 solution is fully dissolved and supplemented to 1000ml by the double distilled water. Filtering with 0.22 μm microporous membrane for sterilization, packaging, and storing at 4 deg.C. Before use, 10% fetal bovine serum, 1 × double antibody was added.

DMEM culture solution: each bag of DMEM powder is dissolved in about 800ml of double distilled water, and 1.5g of NaHCO3, 2.5g of glucose and 0.11g of sodium pyruvate are added and fully dissolved, and the solution is supplemented to 1000ml with double distilled water. Filtering with 0.22 μm microporous membrane for sterilization, packaging, and storing at 4 deg.C. Before use, 10% fetal bovine serum, 1 × double antibody was added.

PBS buffer (pH 7.4): weighing 8.0g of NaCl, 0.2g of KCl and Na₂HPO4·12H₂O 1.44g，KH₂PO4·12H₂Dissolving O0.24 g in 900ml double distilled water, adjusting pH to 7.4 with HCl, diluting to 1000ml with double distilled water, autoclaving, and storing at room temperature.

2. Immunoblot analysis for detecting protein expression level of cells

(1) Electrophoresis: the denatured protein samples were loaded in equal amounts onto 5% concentrated gel and 10% separation gel (mainly composed of ddH2O, 30% polyacrylamide, 1.5M Tris-HCl (pH8.8) or 1.0M Tris-HCl (pH6.8), 10% SDS, 10% ammonium persulfate, TEMED), with a loading of about 40. mu.g per well of protein, run at 100V at constant pressure, and stop the electrophoresis when bromophenol blue runs to the bottom of the gel.

(2) Film transfer: after electrophoresis is finished, the glass plate is disassembled, the gel is taken out, the concentrated gel is cut off, the separation gel is properly sheared according to the molecular weight of the target protein and then is soaked in the precooled membrane transferring liquid so as to avoid drying, and meanwhile, the filter paper and the sponge are also soaked in the precooled membrane transferring liquid. Cutting a PVDF membrane with the size similar to that of the gel, soaking in methanol for 2min to activate the membrane, and then making a sandwich according to the following arrangement sequence: and sequentially stacking 1 spongy cushion, 2 pieces of filter paper, gel, a PVDF (polyvinylidene fluoride) membrane, 2 pieces of filter paper and one spongy cushion on the negative electrode. Note that: the PVDF membrane must be allowed to drive off the bubbles after application to the gel. And then, installing an electrophoresis tank in the correct positive and negative electrode directions for wet rotation, putting ice blocks around the electrophoresis tank for cooling, and adjusting the voltage to be constant current 200V (1.5 h).

(3) And (3) sealing: and after the membrane transfer is finished, the PVDF membrane is washed for 5min in TBST, and then is put into 5 percent of confining liquid to be confined on a decoloring shaking table for 0.5-1 h.

(4) Antibody incubation: diluting the primary antibody blocking solution or the primary antibody dilution solution according to a ratio of 1:500-1:1000, covering the diluted primary antibody blocking solution or the diluted primary antibody dilution solution on a PVDF membrane, incubating for 2h or overnight at 4 ℃ at room temperature, and then washing for 5min with TBST for three times. After diluting the secondary antibody according to the ratio of 1:5000, incubating the secondary antibody with a PVDF membrane for 1h at room temperature on a shaking table, and then washing the secondary antibody with TBST for three times, 5min each time.

(5) Chemiluminescence, development, fixation: and mixing the reagent A and the reagent B in equal volume, dripping the mixture on an ImageStation 4000MM imager, covering the ECL reagent with the PVDF membrane with the front surface facing downwards, and exposing according to the operation instruction of the imager to obtain a protein band result.

3. As a result: the PARP protein degradation activity of the PARP protein degradation agent of the invention is as follows, after the cell lines MDA-MB-231 and MCF-7 are treated for 48 hours by the compound NN3 obtained in example 4, the result of Western blot analysis can obviously observe the degradation effect of the compound NN3 on the PARP protein under different dosages. The degradation of PARP protein by compound NN3 at 12.5uM, 25uM in the MDA-MB-231 cell line is shown.

Example 5 half-maximal inhibition of tumor cell lines of different origins

Different tumor cell lines: human breast cancer cell lines were selected: MCF-7, HCC1937, MDA-MB-231, SKBR3, MX-1 normal mammary cells MCF-10A, so as to examine the half inhibition rate activity of the compound NN3 on tumor cell lines of different sources. The corresponding cells were cultured in RPMI1640 medium containing 10% fetal bovine serum, 100IU/ml penicillin and 100. mu.g/ml streptomycin. The cells were cultured in an incubator at 37 ℃ and saturated humidity with 5% CO2, and cells in the logarithmic growth phase were used for the experiment. Cells in logarithmic growth phase were seeded at 4X 104 cells/ml in 96-well plates at 180. mu.l per well.

The experimental groups were each added 20. mu.l of the compound prepared in examples 1-6 at an initial concentration of 200uM and diluted in half, the negative control group was not dosed, and the positive control agents Nilaparib and Nutlin-3a (purchased from Shanghai Bopont) were each provided with 3 parallel wells and incubated at 37 ℃ for 48 h. 20. mu.l/well of 5mg/ml MTT solution was added, and after further culturing for 4 hours, the supernatant was removed, 150. mu.l DMSO was added, shaking was carried out on a micro-shaker for 10 minutes, and the absorbance (OD value) was measured at a wavelength of 570nm with a microplate reader. The cell growth inhibition rate [ cell growth inhibition rate (control OD-experimental OD)/control OD × 100% ]wascalculated from the absorbance, and IC50 values were calculated by the Logit method, and the experiment was repeated 3 times to obtain an average value.

Example 4 of the present invention shows different biological activities for different breast cancer cell lines. Compared with the positive control drug Nilaparib, Nutlin-3a, the compound NN3 shows stronger antitumor effect, but has no cytotoxicity to normal mammary cells MCF-10A.

TABLE 1 PARP degrader cellular level bioactivity assay (48h maximum inhibition of Compound 200uM or IC50 (uM); NS: not tested)

Compound (I)

Name of

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

Nilaparib

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 degrading agent represented by 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, and Y represents a chain connecting X and Z.

2. The PARP protein degrading agent according to claim 1, wherein X is represented by formula II-1, Z is represented by formula II-2,

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

n is independently an integer of 1 to 10.

3. The PARP protein degrading agent of claim 2, wherein said compound is:

。

4. the process for preparing a PARP protein degrading agent according to claim 1, wherein the reaction formula is as follows:

。

5. use of the PARP protein degrading agent of claim 1 for the preparation of a medicament for the treatment or prevention of tumors.

6. The use according to claim 5, wherein the neoplasm is breast cancer.