Double-target conjugate synthesized based on PARP1 inhibitor and Rigosertib as well as preparation method and application of double-target conjugate

Abstract

The invention belongs to the field of medical chemistry, and particularly discloses a double-target conjugate synthesized on the basis of a PARP1 inhibitor and Rigosertib and a preparation method and application of the double-target conjugate synthesized on the basis of the PARP1 inhibitor and the Rigosertib, the structural general formula of the double-target conjugate synthesized on the basis of the PARP1 inhibitor and the Rigosertib is as shown in the formula I: in the # imgabs0 # formula (I), Linker is any one of an alkyl chain and a PEG chain; n is equal to 1-6. Compared with the prior art, the dual-target conjugate synthesized based on the PARP1 inhibitor and Rigosertib is designed and synthesized through a drug coupling strategy, the application of PARPis in the field of treatment of BRCA wild type TNBC is enhanced, and the dual-target conjugate has potential anti-proliferative activity on BRCA mutant or wild type TNBC and has the advantages that the inhibitory activity on PARP-1 is high, RAS-RAF signal transduction is inhibited, DNA damage is induced, and the anti-proliferative activity on BRCA mutant or wild type TNBC is improved. And a mitochondrial dependent apoptosis pathway is activated, and a good effect is achieved in a test of resisting the BRCA wild type triple negative breast cancer.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical chemistry, and particularly relates to a dual-target conjugate synthesized based on a PARP1 inhibitor and Rigosertib, and a preparation method and application thereof. Background Art

[0002] Breast cancer (BC) is one of the most malignant tumors worldwide. To date, there are numerous treatment strategies for BC, including surgery, chemotherapy, radiotherapy, and endocrine therapy. However, the cure rates of both early and advanced BC remain unsatisfactory. In addition, triple-negative cancer (TNBC), characterized by negative expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 protein (HER-2), is one of the most aggressive subtypes of BC. Therefore, its complex pathological characteristics determine that TNBC is insensitive to the above-mentioned therapies, resulting in low survival rates and high morbidity in TNBC patients. Currently, the treatment of TNBC is limited to traditional chemotherapy, but this approach cannot eliminate the disease or avoid the systemic toxicity of chemotherapeutic drugs. Therefore, the development of new and potent targeted drugs to treat TNBC is urgent.

[0003] BRCA1 and BRCA2 proteins play important roles in homologous recombination (HR) repair and double-strand break (DSBs) repair. However, BRCA1 / 2 deletion or mutation in TNBC inactivates HR and makes HR dependent on poly (ADP-ribose) polymerase 1 (PARP1) to complete DNA repair. Therefore, TNBC patients with BRCA mutations or HR repair defects can benefit from PARP1 inhibitors. To date, the reported PARP inhibitors (PARPis), such as Olaparib (Ola), Niraparib and Fluzoparib, have shown significant therapeutic effects on TNBC patients with BRCA mutations. However, about 80% of TNBC patients do not have BRCA mutations, which cannot benefit from the synthetic lethal strategy, limiting the further application of PARPis. Drug combination strategies can enhance the anti-cancer efficacy of PARPis by using two or more drugs, but this strategy is often limited by drug dosage uncertainty, pharmacokinetics complexity or unpredictable drug interactions. Another strategy is to conjugate the pharmacophore of PARPis with other targeted drugs to construct multi-target drugs. Recently, a large number of multi-target molecules have been reported, such as dual PARP and CDK6 inhibitors, dual PARP and EZH2 inhibitors, dual PARP and PI3K inhibitors, etc. These multi-target molecules are more potent than PARPis in inhibiting BRCA wild-type TNBC in vitro and in vivo. Obviously, the development of multi-target molecules based on PARPis provides a method for treating BRCA wild-type TNBC.

[0004] The RAS / RAF / MEK / ERK mitogen-activated signaling cascade pathway plays a crucial role in regulating cell proliferation, differentiation, and apoptosis, and is highly expressed in many aggressive cancers, including TNBC. Therefore, disrupting the signal transduction between RAS proteins and downstream effectors may help develop new anti-TNBC drugs. A RAS analog (Rigosertib) has been reported, which can effectively block the downstream signaling pathways regulated by RAS and exert anti-cancer effects. In addition, these studies have shown that Rigosertib exhibits strong anti-proliferative efficacy in BRCA wild-type cancer cell lines (such as MDA-MB-231, MCF-7, and SKOV-3). Therefore, it is attractive to construct multi-target molecules based on Rigosertib and provide ideas for the design of some anti-TNBC drugs.

[0005] Based on this, the inventors combined the pharmacophores of Ola and Rigosertib in one molecule to design and synthesize an anti-TNBC candidate. Subsequently, relevant biological tests were performed to verify whether the obtained compound exerts anti-cancer effects through a multifunctional mechanism.

[0006] Summary of the invention

[0007] In view of the shortcomings of the prior art, the purpose of the present invention is to provide a dual-target conjugate synthesized based on a PARP1 inhibitor and Rigosertib, and a preparation method and application thereof, which can enhance the application of PARPis in the treatment of BRCA wild-type TNBC, has potential anti-proliferative activity against BRCA mutant or wild-type TNBC, and also has multiple anti-cancer effects such as high inhibitory activity against PARP-1, inhibition of RAS-RAF signal transduction, induction of DNA damage, and activation of mitochondria-dependent apoptosis pathways. Good results have been achieved in the test of anti-BRCA wild-type triple-negative breast cancer, and can effectively solve the problems in the background technology.

[0008] The present invention is achieved through the following technical solutions: A dual-target conjugate synthesized based on a PARP1 inhibitor and Rigosertib, the general structural formula of which is shown in Formula I:

[0009] Wherein, Linker is any one of an alkyl chain and a PEG chain; n = 1-6.

[0010] Further, selected from the following compounds: 13a: 13b: 13c: 14a: 14b: 14c:

[0011] A further embodiment of the present invention is: A method for preparing a dual-target conjugate synthesized based on a PARP1 inhibitor and Rigosertib, comprising the following steps: (1) subjecting compound 10 to a nucleophilic substitution reaction or an acylation reaction to obtain compounds 11a-11c or 12a-12c; (2) subjecting compounds 11a-11c or 12a-12c to a condensation reaction to obtain target compounds 13a-13c or 14a-14c; (3) Dissolving Compound 13a-14c in an acid or base solution and drying to obtain a pharmaceutically acceptable salt of Compound 13a-14c.

[0012] The synthetic route is as follows:

[0013] Furthermore, in step (1), MeOH is used as solvent and NaOH is used as catalyst, compound 10 reacts with an acid to undergo a nucleophilic substitution reaction to obtain compounds 11a-11c, wherein MeOH represents methanol; and the acid is one of bromoacetic acid, bromobutyric acid or bromohexanoic acid.

[0014] Furthermore, in step (1), DMF is used as solvent, Et 3 N is a catalyst, and compound 10 undergoes acylation reaction with anhydride to obtain compounds 12a~12c, DMF represents N,N-dimethylformamide, Et 3 N represents triethylamine; the acid anhydride is one of succinic anhydride, glutaric anhydride or adipic anhydride.

[0015] Furthermore, in step (2), DMF is used as solvent, Et 3 N is a catalyst, and compounds 11a~11c or 12a~12c react with 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethylurea tetrafluoroborate to obtain target compounds 13a~13c or 14a~14c, DMF represents N,N-dimethylformamide, Et 3 N represents triethylamine.

[0016] Furthermore, in step (3), the acid is one of hydrochloric acid, phosphoric acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, citric acid, tartaric acid or lactic acid; and the base is one of an inorganic base containing an alkaline metal cation, an alkaline earth metal cation or an ammonium cation salt.

[0017] A further embodiment of the present invention is: A dual-target conjugate synthesized based on a PARP1 inhibitor and Rigosertib is used in the preparation of a drug for treating breast cancer.

[0018] Furthermore, the breast cancer is BRCA wild-type triple-negative breast cancer. Beneficial Effects

[0019] Compared with the prior art, the present invention has the following obvious advantages: 1. The present invention designs and synthesizes a dual-target conjugate based on PARP1 inhibitor and Rigosertib through a drug conjugation strategy, which enhances the application of PARPis in the treatment of BRCA wild-type TNBC and has potential anti-proliferative activity against BRCA mutant or wild-type TNBC.

[0020] 2. The conjugate obtained by the present invention has multiple anti-cancer effects such as high inhibitory activity on PARP-1, inhibition of RAS-RAF signal transduction, induction of DNA damage, and activation of mitochondria-dependent apoptosis pathway.

[0021] 3. The conjugate obtained by the present invention has achieved good results in the test of anti-BRCA wild-type triple-negative breast cancer. Studies have shown that this type of compound has the use of treating BRCA wild-type TNBC. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a graph showing the results of the PARP1 activity test of the compound in Example 5 of the present invention.

[0023] Figure 2 is a cell apoptosis diagram in Example 6 of the present invention. MDA-MB-231 cells were treated with control (PBS, A), 3 (5 μM, B), 11b (5 μM, C), mixture (3+11b, 5+5 μM, D) or 13b (5+5 μM, E) for 24 h and collected for flow cytometry analysis. (F) A graph of the proportion of apoptotic cells, and is shown as the mean ± SD of three independent experiments. Compared with the 13b treatment group, **p<0.01, *p<0.05.

[0024] Figure 3 This is a figure of DNA comet assay in Example 7 of the present invention. MDA-MB-231 cells were treated with control (PBS, A), 3 (5 μM, B), 11b (5 μM, C), mixture (3+11b, 5+5 μM, D) or 13b (5+5 μM, E) for 24 h and collected for DNA comet assay. (F) The relative length of the comet tail is shown as the mean ± standard deviation of three independent experiments. Compared with the 13b treatment group, **p<0.01, *p<0.05.

[0025] Figure 4 : This is a graph of the expression of related proteins in the RAS-RAF pathway in Example 8 of the present invention. MDA-MB-231 cells were treated with control (PBS), 3 (5 μM), 11b (5 μM), mixture (3+11b, 5+5 μM) or 13b (5+5 μM) for 24 h and collected for western blot experiments. (F) The relative values ​​of protein expression are the mean ± standard deviation of three independent experiments. Compared with the 13b treatment group, **p<0.01, *p<0.05.

[0026] Figure 5 This is a graph of the expression of related proteins in the mitochondrial apoptosis pathway in Example 9 of the present invention. MDA-MB-231 cells were treated with control (PBS), 3 (5 μM), 11b (5 μM), mixture (3+11b, 5+5 μM) or 13b (5+5 μM) for 24 h and collected for western blot experiments. (F) The relative values ​​of protein expression are the mean ± standard deviation of three independent experiments. Compared with the 13b treatment group, **p<0.01, *p<0.05.

[0027] Figure 6 This is an in vivo anti-tumor effect diagram in Example 10 of the present invention. 15 MDA-MB-231 xenografted female BALB / c nude mice were randomly divided into five groups and received intraperitoneal injection of saline, Ola (20 mg / kg), 11b (23 mg / kg), mixture (Ola+11b, 20+23 mg / kg) or 13b (38 mg / kg) every 2 days for a total of 28 days. (A) Tumor picture, (B) Tumor volume change diagram, (C) Tumor weight diagram, (D) Nude mouse weight change diagram, compared with the 13b treatment group, **p<0.01, *p<0.05.

[0028] Figure 7 It is a schematic diagram of in vivo anti-tumor activity in Example 10 of the present invention. DETAILED DESCRIPTION

[0029] Example 1

[0030] The synthesis method of compound 3 is as follows:

[0031] Compounds 4-10 were synthesized according to the method reported in the literature (J. Med. Chem. 2020; 63(1): 186–204.) The synthetic route is as follows:

[0032] General synthesis method of compounds 11a~11c: To a solution of compound 10 (2.0 g, 5.10 mmol) in MeOH (40 mL) were added bromoacetic acid / bromobutyric acid / bromohexanoic acid (6.6 mmol) and NaOH (10%, 16.5 mmol) at room temperature. The solution was then stirred at 55 °C for 4 h. The reaction process was monitored by TLC until completion. Subsequently, the solvent was evaporated under reduced pressure, the pH was adjusted to 6.0, and the residue was collected. The crude product was purified by silica gel chromatography to obtain 11a~11c.

[0033] ( E )-(2-methoxy-5-((2,4,6-trimethoxyphenylvinyl)sulfonyl)methyl)phenyl)glycine (11a) as pale yellow solid (845.1 mg, 1.87 mmol), yield 36.7%. 1 H NMR (500 MHz, DMSO- d 6 ) δ 12.26 (s,1H), 7.58 (d, J = 15.7 Hz, 1H), 7.14 (d, J = 15.7 Hz, 1H), 6.79 (d, J = 8.6Hz, 1H), 6.58 (d, J = 6.6 Hz, 2H), 6.31 (s, 2H), 4.90 (s, 1H), 4.32 (s, 2H), 3.87 – 3.86 (m, 9H), 3.78 (s, 3H), 3.22 (s, 2H). ( E )-4-((2-methoxy-5-((2,4,6-trimethoxyphenylvinyl)sulfonyl)methyl)phenyl)amino)butanoic acid (11b) as pale yellow solid (1.03 g, 2.16 mmol), yield 42.3%. 1 H NMR (500 MHz, DMSO- d 6 ) δ12.08 (s, 1H), 7.53 (d, J = 15.7 Hz, 1H), 7.10 (d, J = 15.7 Hz, 1H), 6.78 –6.74 (m, 1H), 6.52 – 6.49 (m, 2H), 6.28 (s, 2H), 4.87 (s, 1H), 4.26 (s, 2H), 3.84 – 3.83 (m, 9H), 3.75 (s, 3H), 2.95 (t,J = 6.3 Hz, 2H), 2.21 (t, J = 7.3Hz, 2H), 1.75 – 1.69 (m, 2H). ( E )-6-((2-methoxy-5-((2,4,6-trimethoxyphenylvinyl)sulfonyl)methyl)phenyl)amino)hexanoic acid (11c) as a pale yellow solid (1.24 g, 2.61 mmol), yield 51.1%. 1 H NMR (500 MHz, DMSO- d 6 ) δ11.99 (s, 1H), 7.52 (d, J = 15.7 Hz, 1H), 7.10 (d, J = 15.7 Hz, 1H), 6.74 (d, J = 8.2 Hz, 1H), 6.51 – 6.50 (m, 1H), 6.46 (d, J = 1.9 Hz, 1H), 6.28 (s, 2H), 4.72 (s, 1H), 4.26 (s, 2H), 3.83 (s, 9H), 3.75 (s, 3H), 2.90 (t, J = 6.8 Hz,2H), 2.16 (t, J = 7.4 Hz, 2H), 1.49 – 1.41 (m, 4H), 1.25 – 1.20 (m, 2H). Example 2 General method for the synthesis of compounds 12a~12c: To a solution of compound 10 (2.0 g, 5.10 mmol) in DMF (40 mL) were added succinic anhydride / glutaric anhydride / adipic anhydride (12.75 mmol) and Et 3 N (1.28 g, 12.7 mmol). The solution was then stirred at 65 °C for 4 h. The reaction process was monitored by TLC until completion. Subsequently, the solvent was removed and the residue was collected. The crude product was purified by silica gel chromatography to give 12a~12c.

[0034] ( E )-4-((2-methoxy-5-((2,4,6-trimethoxyphenylvinyl)sulfonyl)methyl)phenyl)amino)-4-oxobutanoic acid (12a) was obtained as a white solid (1.25 g, 2.53 mmol). Yield: 49.6%. 1H NMR (500 MHz, DMSO- d 6 ) δ12.08 (s, 1H), 9.15 (s, 1H), 8.02 (s, 1H), 7.58 (d, J = 15.6 Hz, 1H), 7.10(d, J = 15.6 Hz, 1H), 7.03 – 6.99 (m, 2H), 6.28 (s, 2H), 4.33 (s, 2H), 3.84 –3.81 (m, 12H), 2.62 (t, J = 6.6 Hz, 2H), 2.48 – 2.46 (m, 2H). ( E )-5-((2-methoxy-5-((2,4,6-trimethoxyphenylvinyl)sulfonyl)methyl)phenyl)amino)-5-oxopentanoic acid (12b) as a white solid (1.38 g, 2.72 mmol), yield 53.4%. 1 H NMR (500 MHz, DMSO- d 6 ) δ9.01 (s, 1H), 7.97 (s, 1H), 7.58 (d, J = 15.7 Hz, 1H), 7.10 (d, J = 15.5 Hz,1H), 7.08 – 7.00 (m, 2H), 6.29 (s, 2H), 4.33 (s, 2H), 3.85 – 3.82 (m, 12H),2.40 (t, J = 6.3 Hz, 2H), 2.26 (t, J = 7.0 Hz, 2H), 1.81 – 1.74 (m, 2H). ( E )-6-((2-methoxy-5-((2,4,6-trimethoxyphenylvinyl)sulfonyl)methyl)phenyl)amino)-6-oxohexanoic acid (12c) was obtained as a white solid (1.08 g, 2.07 mmol). Yield: 40.7%. 1 H NMR (500 MHz, DMSO- d 6 ) δ12.01 (s, 1H), 9.05 (s, 1H), 7.97 (s, 1H), 7.58 (d, J= 15.6 Hz, 1H), 7.11(d, J = 15.7 Hz, 1H), 7.04 – 6.99 (m, 2H), 6.28 (s, 2H), 4.33 (s, 2H), 3.84 –3.83 (m, 9H), 3.81 (s, 3H), 2.36 (t, J = 6.7 Hz, 2H), 2.22 (t, J = 7.0 Hz, 2H), 1.55 – 1.51 (m, 4H). Example 3 General method for the synthesis of compounds 13a-14c: To a solution of compounds 11a-12c (0.45 mmol) in DMF (20 mL) were added 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetrafluoroborate tetramethylurea (216.7 mg, 0.68 mmol) and Et 3 N (68.7 mg, 0.68 mmol). Then, the solution was stirred at 45 °C for 4 h. The reaction process was monitored by TLC until completion. The solvent was then removed under reduced pressure, and the residue was dissolved in 200 mL DCM, washed with brine and concentrated under reduced pressure. The crude product was further purified by silica gel chromatography to give 13a~14c.

[0035] ( E )-4-(4-fluoro-3-(4-(2-methoxy-5-((2,4,6-trimethoxyphenylvinyl)sulfonyl)methyl)phenyl)glyceryl)piperazine-1-carbonyl)benzyl)phthalazin-1(2H)-one (13a) as a white solid (207.0 mg, 0.26 mmol), yield 57.5%. 1 H NMR (500 MHz, CDCl 3 ) δ 11.12 – 11.08 (d, 1H), 8.46 – 8.44 (m, 1H), 7.79 – 7.72 (m, 4H), 7.34 (d, J= 5.4 Hz, 2H), 7.05 – 6.96 (m, 2H), 6.72 –6.61 (m, 2H), 6.52 (s, 1H), 6.06 – 6.04 (m, 2H), 5.29 (s, 1H), 4.28 (s, 2H),4.17 – 4.12 (m, 2H), 3.83 – 3.82 (m, 9H), 3.78 (s, 3H), 3.76 (s, 4H), 3.61 –3.57 (m, 2H), 3.44 (s, 1H), 3.36 (s, 1H), 3.30 (s, 2H). 13 C NMR (125 MHz, DMSO- d 6 ) δ 168.06, 164.52 (d, J = 10.0 Hz), 164.08, 161.40 (2C), 159.89,157.84, 155.90, 146.90, 145.32, 137.46 (d, J = 6.2 Hz), 135.34, 134.00,133.51, 133.43, 132.32, 132.06, 129.68, 129.42, 128.45, 126.57, 125.87,124.08, 121.87 (d, J = 18.8 Hz), 119.54, 113.20 (d, J = 21.3 Hz), 109.82,103.03 (2C), 91.39, 61.09, 56.51 (2C), 56.08, 55.94, 55.40, 44.77, 44.29,42.07, 41.60, 36.92. ( E )-4-(4-fluoro-3-(4-(4-(2-methoxy-5-((2,4,6-trimethoxyphenylvinyl)sulfonyl)methyl)phenyl)amino)butanoyl)piperazine-1-carbonyl)benzyl)phthalazin-1(2H)-one (13b) as a white solid (241.4 mg, 0.29 mmol), yield 64.8%. 1 H NMR (500 MHz, CDCl 3 ) δ 10.95 – 10.91 (m, 1H), 8.45 – 8.44(m, 1H), 7.82 (t, J= 15.4 Hz, 1H), 7.75 – 7.69 (m, 3H), 7.33 – 7.32 (m, 2H),7.08 – 7.02 (m, 2H), 6.71 – 6.61 (m, 3H), 6.07 (s, 2H), 5.29 (s, 1H), 4.26(d, J = 11.8 Hz, 2H), 4.14 (d, J = 12.7 Hz, 2H), 3.83 (s, 3H), 3.82 – 3.80(m, 9H), 3.77 – 3.76 (m, 1H), 3.69 – 3.66 (m, 1H), 3.56 (s, 1H), 3.51 (s,1H), 3.37 (s, 1H), 3.26 (s, 2H), 3.18 – 3.17 (m, 2H), 2.44 (t, J = 7.0 Hz,1H), 2.39 (t, J = 6.7 Hz, 1H), 1.98 – 1.94 (m, 2H), 1.86 – 1.79 (m, 1H). 13 CNMR (125 MHz, CDCl 3 ) δ 171.28, 165.22, 165.11, 163.73 (d, J = 7.5 Hz), 161.64(2C), 160.52, 157.89, 156.00, 147.14, 145.45, 135.26, 134.36 (d, J = 13.7Hz), 133.68, 131.61, 129.54, 129.31 (d, J = 12.5 Hz), 128.28, 127.13 (d, J =7.5 Hz), 125.11, 123.94, 123.65, 123.03, 121.23, 120.91, 116.38 (d, J = 12.5Hz), 109.24, 103.85, 90.34 (2C), 62.08, 55.77 (2C), 55.50 (2C), 53.46, 47.19,46.80, 45.07, 43.03, 42.13, 41.77, 37.72. ( E)-4-(4-fluoro-3-(4-(6-(2-methoxy-5-((2,4,6-trimethoxyphenylvinyl)sulfonyl)methyl)phenyl)amino)hexanoyl)piperazine-1-carbonyl)benzyl)phthalazin-1(2H)-one (13c) as a white solid (294.3 mg, 0.34 mmol), yield 76.4%. 1 H NMR (500 MHz, CDCl 3 ) δ 11.00 – 10.89 (m, 1H), 8.46 – 8.45(m, 1H), 7.85 – 7.73 (m, 2H), 7.71 – 7.64 (m, 2H), 7.33 – 7.29 (m, 2H), 7.08– 6.98 (m, 1H), 6.70 – 6.69 (m, 1H), 6.63 – 6.61 (m, 2H), 6.06 (s, 1H), 6.04(s, 1H), 5.29 (s, 1H), 4.57 (d, J = 7.8 Hz, 1H), 4.28 (s, 2H), 3.83 – 3.82(m, 4H), 3.81 – 3.73 (m, 12H), 3.56 – 3.54 (m, 2H), 3.42 (s, 1H), 3.30 – 3.27(m, 1H), 3.09 – 3.06 (m, 2H), 2.37 – 2.30 (m, 2H), 1.80 – 1.76 (m, 1H), 1.66 – 1.59 (m, 4H), 1.43 – 1.39 (m, 2H), 1.28 – 1.24 (s, 1H). 13 C NMR (126 MHz, CDCl 3 ) δ 171.71, 165.25, 165.18, 164.34 (d, J = 6.2 Hz), 161.46, 160.61,160.03 (2C), 158.01, 156.05, 147.06, 145.56, 139.22, 135.04, 134.43, 133.73,131.78, 131.70 (d, J = 6.2 Hz), 129.55, 128.99, 128.33, 127.17 (d, J = 5.0Hz), 125.09, 123.08, 121.19 (d, J= 13.7 Hz), 116.35 (d, J = 12.5 Hz),108.95, 103.93, 90.21 (2C), 61.30, 55.76, 55.56 (2C), 55.42, 53.50, 52.19,46.92, 45.16, 43.43, 42.24, 37.73, 33.05, 29.73, 26.98. ( E )-4-(4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)piperazin-1-yl)-N-(2-methoxy-5-((2,4,6-trimethoxyphenylvinyl)sulfonyl)methyl)phenyl)-4-oxobutanamide (14a) was obtained as a white solid (271.7 mg, 0.32 mmol). Yield: 71.7%. 1 H NMR (500 MHz, CDCl 3 ) δ 11.16 – 11.11(m, 1H), 8.44 (d, J = 6.7 Hz, 1H), 8.35 (s, 1H), 8.09 (d, J = 13.2 Hz, 1H),7.84 – 7.80 (m, 1H), 7.75 – 7.70 (m, 3H), 7.34 – 7.29 (m, 2H), 7.11 – 7.00(m, 3H), 6.83 (d, J = 8.4 Hz, 1H), 6.05 (s, 2H), 5.28 (s, 1H), 4.27 (s, 2H), 4.18 (s, 2H), 3.85 – 3.84 (m, 2H), 3.81 – 3.79 (m, 12H), 3.59 (s, 2H), 3.47(s, 1H), 3.34 – 3.27 (m, 1H), 2.74 – 2.67 (m, 4H), 2.08 – 1.92 (m, 1H). 13 CNMR (125 MHz, DMSO- d 6 ) δ 171.43, 171.02, 164.90, 164.74, 164.32 (d, J= 6.3Hz), 161.38, 160.10, 159.79, 157.79, 155.81, 150.27, 145.45, 138.52, 135.42,133.98, 133.36, 132.25 (d, J = 8.7 Hz), 132.06, 129.65, 128.37, 127.61,127.47, 126.61, 125.94, 124.11 (d, J = 16.2 Hz), 120.74, 116.42 (d, J = 20.0Hz), 111.20, 103.05, 91.40, 91.24, 60.29, 56.53, 56.33, 56.17, 56.13, 52.58,47.21, 46.76, 41.87, 41.58, 36.92, 32.23. ( E )-5-(4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)piperazin-1-yl)-N-(2-methoxy-5-((2,4,6-trimethoxyphenylvinyl)sulfonyl)methyl)phenyl)-5-oxopentamide (14b) as white solid (200.0 mg, 0.23 mmol), yield 51.9%. 1 H NMR (500 MHz, DMSO- d 6 ) δ 12.60 (s,1H), 9.01 (d, J = 28.8 Hz, 1H), 8.26 (d, J = 7.8 Hz, 1H), 7.99 – 7.95 (m,2H), 7.89 (t, J = 7.5 Hz, 1H), 7.83 (t, J = 7.5 Hz, 1H), 7.44 – 7.39 (m, 1H), 7.36 (s, 1H), 7.23 (t, J= 9.0 Hz, 1H), 7.10 – 6.92 (m, 2H), 6.29 (s, 1H), 6.24 (s, 1H), 4.62 (s, 1H), 4.33 (s, 2H), 3.85 – 3.83 (m, 7H), 3.76 (s, 3H),3.75 (s, 1H), 3.69 (s, 2H), 3.63 – 3.57 (m, 2H), 3.52 (s, 2H), 3.41 – 3.37(m, 2H), 3.17 – 3.14 (m, 2H), 2.36 – 2.30 (m, 4H), 1.65 – 1.46 (m, 4H). 13 CNMR (125 MHz, CDCl 3 ) δ 170.46, 170.12, 165.25 (d, J = 18.7 Hz), 163.72,161.50 (2C), 160.66, 158.00, 156.03, 148.13, 145.56, 135.35, 134.46, 133.72,131.64, 129.54, 129.32 (d, J = 12.5 Hz), 128.29, 127.73, 127.13, 126.31,125.10, 123.65 (d, J =17.5 Hz), 122.90 (d, J = 9.0 Hz), 122.48, 120.95,116.22 (d, J = 22.5 Hz), 110.07, 103.91, 90.31 (2C), 61.54, 55.86, 55.76(2C), 55.49, 53.51, 46.77, 45.56, 45.01, 41.96, 37.69, 32.53, 28.34. ( E )-6-(4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)piperazin-1-yl)-N-(2-methoxy-5-((2,4,6-trimethoxyphenylvinyl)sulfonyl)methyl)phenyl)-6-oxohexanamide (14c) as a white solid (267.4 mg, 0.31 mmol), yield 68.3%. 1H NMR (500 MHz, DMSO) δ 12.60 (s,1H), 9.02 (d, J = 28.8 Hz, 1H), 8.26 (d, J = 7.8 Hz, 1H), 7.99 – 7.95 (m,2H), 7.89 (t, J = 7.6 Hz, 1H), 7.83 (t, J = 7.5 Hz, 1H), 7.47 –7.44 (m, 1H),7.39 – 7.36 (s, 1H), 7.33 (s, 1H), 7.23 (t, J = 9.0 Hz, 1H), 7.13 –7.00 (m,2H), 6.29 (s, 1H), 6.24 (s, 1H), 4.62 (s, 1H), 4.33 (s, 2H), 3.85 – 3.76 (m,12H), 3.69 (s, 2H), 3.63 (s, 1H), 3.60 – 3.57 (m, 1H), 3.52 (s, 2H), 3.41 –3.37 (m, 2H), 3.17 – 3.16 (m, 2H), 2.36 – 2.30 (m, 4H), 1.57 – 1.52 (m, 4H). 13 C NMR (125 MHz, DMSO- d 6 ) δ 171.64, 171.21, 165.01, 164.79, 164.50 (d, J =10.0 Hz), 164.07, 161.58, 160.15, 159.88, 155.81, 150.02, 145.34, 138.42,135.30, 133.86, 133.45, 132.25 (d, J = 6.3 Hz), 132.00, 129.56, 128.37,127.52, 126.56, 125.88, 125.17, 124.05, 120.82, 116.43 (d, J= 10.0 Hz),111.22, 102.99, 91.41, 91.25, 60.19, 56.53, 56.34, 56.18, 56.09, 52.58,45.45,45.03, 42.08, 41.71, 36.91, 32.53, 25.43, 24.80. Example 4 In vitro cytotoxicity assay: Experimental method: All test cells were seeded into 96-well plates and treated with different compounds for 72 hours. Before the experiment, the test compounds were dissolved in DMSO and diluted to the required concentration with PBS. After incubation, 20 μL of MTT (5 mg / mL) was added to each well and incubated for another 4 hours. The formed formazan was further dissolved in 100 μL of DMSO, and the absorbance at 570 nm was recorded by a microplate reader. IC 50 The values ​​were calculated using GraphPad Prism software.

[0036] Experimental results: The in vitro antitumor activity of target compounds 13a to 14c was evaluated by MTT assay using BRCA1 mutant breast cancer cell lines (HCC1937, MDA-MB-436) and BRCA wild-type TNBC cell lines (MDA-MB-231, MDA-MB-468). The human normal breast cell line (MCF-10A) was selected to study its potential cytotoxicity to normal tissue cells. After 72 h of incubation, the cytotoxicity of the tested compounds is listed in Table 1. Olaparib showed strong antiproliferative activity against BRCA-deficient cells, with an IC of 1.54 in HCC1937. 50 The value is 5.37 μM, and the IC 50 was 4.25 μM; however, it showed moderate inhibitory activity in BRCA wild-type cells. The IC 50 The value is 25.46 μM, and the IC of MDA-MB-468 50 The value was 14.76 μM. The effects of compounds 13a~14c on these TNBC cells (IC 50 values ​​ranged from 0.75 to 4.85 μM) were greater than Ola (IC 50 The values ​​ranged from 4.25 to 25.46 μM), indicating that 13a~13c showed strong antiproliferative activity against both BRCA wild-type and BRCA1-deficient breast cancer cells. In particular, 13b showed significant cell activity in MDA-MB-231, MDA-MB-468, HCC1937, and MDA-MB-436, with IC 50The values ​​were 0.75, 1.24, 1.7 and 0.97 μM, respectively, and were more effective than Ola. In addition, the physical mixture of 3 and 11b also showed enhanced cellular activity against the tested cancer cells compared with Ola. In addition, the potential toxicity results against the human normal breast cell line MCF-10A showed that all tested compounds had good safety at the cellular level.

[0037]

[0038] IC 50 Expressed as mean ± SD of three independent experiments; b 3+11b, physical mixture of 3 and 11b (mol / mol, 1:1); c Ola, Olaparib. Example 5 Test of compound activity on PARP1: Experimental Methods: The effect of the test compounds on PARP1 activity was determined by histone-based ELISA assay using the PARP1 Colorimetric Assay Kit (BPS Bioscience, catalog number 80580). The histone mixture was added to a 96-well plate and allowed to adhere overnight, followed by the addition of blocking buffer (200 μL) and washing with PBST. The test compound (25 μL) and PARP enzyme in PARP buffer (20 μL, 1.0 ng / uL) were then added sequentially. After incubation for 60 min at room temperature, the wells were washed twice with PBS, and 20 μL of streptavidin-HRP blocking buffer was added for another 30 min at room temperature. Finally, the wells were washed twice with PBST, and the colorimetric HRP substrate (100 μL / well) was added. After incubation for 20 min, 100 μL of sulfuric acid (2 M) was added to each well. The absorbance at OD450 nm was recorded by a microplate reader, and the IC was calculated by GrapPad Prism software. 50 value.

[0039] Experimental results: With Ola and 3 as controls, the effect of 13b on PARP1 was studied by ELISA. Figure 1 As shown, 13b showed good efficacy against PARP1, with an IC 50 The value was 112.2 nM, which was comparable to Ola (IC50, 39.5 nM) and 3 (IC 50 , 57.2 nM), indicating that 13b inherited the inhibitory activity of Ola on PARP1.

[0040] Example 6 Cell apoptosis test: Experimental method: MDA-MB-231 cells (3.0×10 5Cells were seeded into six-well plates (5 μM cells / well) and allowed to adhere overnight. Cells were then treated with PBS, 11b (5 μM), 3 (5 μM), a mixture (3+13b, 5+5 μM, or 13b (5 μM)) for 24 h. Thereafter, cells were harvested, stained with Annexin V-FITC and PI, and analyzed by flow cytometry. Apoptosis data were analyzed by FlowJo, and representative images of three independent experiments are shown.

[0041] Experimental results: To further determine the effect of compound 13b on cell death, MDA-MB-231 cells were stained with Annexin-V FITC and PI and analyzed by flow cytometry. Figure 2 A, After incubation with 11b (5 μM), 3 (5 μM), mixed groups (3+13b, 5+5 μM, or 13b (5 μM) for 24 h, 5 μM 3, 11b, mixed groups, or 13b induced apoptosis in MDA-MB-231 cells, with apoptosis values ​​of 21.14%, 31.1%, 43.94%, and 48.26%, respectively. It is worth noting that 13b induced the highest apoptosis rate, which was about 2.3 or 1.6 times higher than 3 and 11b, but only about 1.1 times higher than the mixture ( Figure 2 B). In conclusion, 13b has a great potency in inducing apoptosis in MDA-MB-231 cells compared with monotherapy or combination therapy, which may be partly attributed to its strong anticancer ability.

[0042] Example 7 DNA Comet Assay: Experimental method: MDA-MB-231 cells (3.0×10 5 Cells were seeded into six-well plates (5 μM cells / well) and allowed to adhere overnight. Cells were then treated with PBS, 11b (5 μM), 3 (5 μM), a mixture (3+11b, 5+5 μM, or 13b (5 μM)) for 24 h. Thereafter, cells were collected and processed according to the instructions of the Comet Assay Kit (Beyotime, C2041S), and DNA images were recorded by laser confocal microscopy. The experimental results are representative images of three independent experiments.

[0043] Experimental results: Figure 2 A, After 24 h of treatment with 3 (5 μM), cells were collected for DNA electrophoresis analysis, and cells treated with 11b (5 μM), mixed groups (3+11b, 5+5 μM, or 13b (5 μM) were used as controls. Figure 2As shown in B, cells treated with 3 showed a modest increase in comet-like tails compared to untreated cells, indicating that single treatment with 3 did not significantly induce severe DNA damage in MDA-MB-231 cells. However, treatment with 11b resulted in a 1.7-fold increase in comet-like tails compared to 3, indicating that 11b was more potent than 3 in inducing DNA damage ( Figure 3 ). In general, the length of the comet tail is positively correlated with DNA damage. In addition, the results of the comet assay showed that the comet-like tails of the mixed group and 13b were 2.4 and 6.2 times higher than those of 3, respectively. The study showed that both the mixed group and 13b induced more severe DNA damage than 3 or 11b, which was beneficial to their anticancer activity.

[0044] Example 8 Detection of related protein expression in RAS-RAF pathway: Experimental methods: MDA-MB-231 cells (3.0×105 cells / well) were seeded into six-well plates and allowed to adhere overnight. The cells were then treated with PBS, 11b (5 μM), 3 (5 μM), a mixture (3+11b, 5+5 μM, or 13b (5 μM) for 24 h. Thereafter, the cells were collected and lysed with cell lysis buffer. The concentration of total protein was determined by BCA protein assay kit (P0010S), and equal amounts of total protein were separated by SDS-PAGE gel. Afterwards, the tested proteins were transferred to nitrocellulose membranes, blocked with 5% skim milk powder, and labeled with the designated primary antibodies and horseradish peroxidase-labeled secondary antibodies.

[0045] Experimental results: Blocking the signaling cascade between RAF and RAF can effectively interfere with the expression of its downstream effectors, including the phosphorylation of MEK and ERK. Considering the importance of the MAPK pathway in regulating the biological activity of cancer cells, we examined the effect of 13b on the expression levels of key proteins involved in the MAPK pathway. Figure 4 The results showed that 11b had no significant effect on the expression level of RAS in MDA-MB-231 cells, but it could effectively reduce the expression level of CRAF compared with the untreated group, which may benefit from Rigosertib. In addition, 3 had no activity on the expression levels of RAS and CRAF, indicating that 3 is not an inhibitor or activator of these proteins. The mixed group (3+11b) or 13b significantly inhibited the expression of CRAF protein, but had little effect on RAS protein. Figure 3As shown in A and B, 11b treatment for 24 h significantly reduced the phosphorylation of MEK and ERK 1 / 2 (p-MEK, p-ERK1 / 2) compared with untreated cells, but had no significant effect on the expression of total MEK or ERK 1 / 2. As mentioned above, 3 had no significant activity on p-MEK and p-ERK1 / 2, but its activity was enhanced when it was combined with 11b or combined with 11b into one entity. Taken together, these findings suggest that 13b can interfere with signal transduction in the MAPK pathway, thereby providing powerful anticancer activity.

[0046] Example 9 Detection of related protein expression in mitochondrial apoptosis pathway: Experimental method: Same as Example 8 Experimental results: Mitochondria play a vital role in maintaining cell homeostasis. In order to study the effect of 13b on the mitochondrial apoptosis signaling pathway, Western blot analysis of apoptosis-related proteins was performed. MDA-MB-231 cells were incubated with 13b (5 μM) for 24 h and collected for Western blot analysis. 3 (5 μM), 11b (5 μM) and mixed groups (3+11b, 5+5 μM") were used as controls ( Figure 5 A and B). In general, the Bcl-2 family (such as Bax and Bcl-2) is important for high mitochondrial membrane potential, however, 24 h treatment with 3, 11b, mixed group (3+11b), or 13b significantly altered the expression of Bax and Bcl-2 in MDA-MB-231 cells compared with the control group, showing upregulation of Bax and downregulation of Bcl-2. In addition, compared with the untreated group or the 3-treated group, 11b, mixed group (3+11b), and 13b effectively promoted the expression level of cleaved PARP in MDA-MB-231 cells, indicating that 11b, mixed group, and 13b were more potent than 3 in inducing DNA damage. In addition, the changes in Bax, Bcl-2, and cleaved PARP promoted the release of cytoplasmic Cyt c and initiated the caspase cascade pathway, which was conducive to cell apoptosis. In summary, these results indicate that 13b activates the mitochondria-mediated apoptosis pathway and induces apoptosis in MDA-MB-231 cells.

[0047] Example 10 In vivo anti-tumor effect detection: Experimental method: MDA-MB-231 cells (0.1 mL, 5×10 6 When the tumor volume reached approximately 200 mm 3At the time of the experiment, mice were randomly divided into five groups and intraperitoneally injected with saline, Ola (20 mg / kg), 11b (23 mg / kg), a mixture (Ola+11b, 20+23 mg / kg), or 13b (38 mg / kg) every two days for 28 days. Body weight and tumor volume were measured every two days. After the last dose was completed, the tumor solids were collected and weighed. The tumor volume was obtained by the following formula: V (mm 3 )=L×W 2 / 2, L is the length and W is the width.

[0048] Results: We evaluated the in vivo antitumor efficacy of 13b in MDA-MAB-231 xenografts. 3 Afterwards, 15 BALB / c nude mice were randomly divided into five groups and intraperitoneally injected with saline, Ola (20 mg / kg), 11b (23 mg / kg), mixed groups (Ola+11b, 20+23 mg / kg), or 13b (38 mg / kg). The drugs were administered every two days for 28 days. During the test period, tumor volume and body weight were measured every two days. Figure 6 As shown in A-D, the tumor volume of the control group grew rapidly during treatment, reaching approximately 920 mm3. However, at a dose of 20 mg / kg, Ola showed moderate inhibitory efficacy on the growth of MDA-MB-231 cells, with a tumor growth inhibition rate (TGI) of 38.5%. In addition, at the same dose, the anticancer activity of 11b (TGI, 51.8%) was higher than that of Ola. 13b (38 mg / kg) further enhanced the efficacy (TGI, 61.3%), even exceeding the combination group (Ola+11b, 20+23 mg / kg; TGI, 56.7%), which indirectly explains why 13b achieved greater anticancer activity than Ola or 11b monotherapy. In addition, no significant effect on body weight was found between the test group and the control group, indicating that these tested compounds were well tolerated and biocompatible. In summary, these results indicate that 13b shows great potential as a promising candidate for the treatment of MDA-MB-231.

[0049] The above embodiments are only for illustrating the technical concept and features of the present invention, and their purpose is to enable people familiar with the technology to understand the content of the present invention and implement it accordingly, and they cannot be used to limit the protection scope of the present invention. Any equivalent transformation or modification made according to the spirit of the present invention should be included in the protection scope of the present invention.

Claims

1. A dual-target conjugate synthesized based on PARP1 inhibitor and Rigosertib, characterized in that: The general structural formula is shown in Formula I: Wherein, Linker is any one of an alkyl chain and a PEG chain; n = 1-6.

2. A dual-target conjugate synthesized based on a PARP1 inhibitor and Rigosertib according to claim 1, characterized in that: Selected from the following compounds: 13a： 13b： 13c： 14a： 14b： 14c： 。 3. The method for preparing a dual-target conjugate synthesized based on a PARP1 inhibitor and Rigosertib according to claim 2, characterized in that: The following steps are involved: (1) subjecting compound 10 to a nucleophilic substitution reaction or an acylation reaction to obtain compounds 11a-11c or 12a-12c; (2) subjecting compounds 11a-11c or 12a-12c to a condensation reaction to obtain target compounds 13a-13c or 14a-14c; (3) Dissolve compounds 13a-14c in an acid or base solution and dry to obtain pharmaceutically acceptable salts of compounds 13a-14c. The synthetic route is as follows: 。 4. The method for preparing a dual-target conjugate synthesized based on a PARP1 inhibitor and Rigosertib according to claim 3, characterized in that: In step (1), MeOH is used as solvent and NaOH is used as catalyst, compound 10 reacts with an acid to undergo a nucleophilic substitution reaction to obtain compounds 11a-11c; the acid is one of bromoacetic acid, bromobutyric acid or bromohexanoic acid.

5. The method for preparing a dual-target conjugate synthesized based on a PARP1 inhibitor and Rigosertib according to claim 3, characterized in that: In step (1), using DMF as solvent and Et3N as catalyst, compound 10 undergoes acylation reaction with an acid anhydride to obtain compounds 12a-12c; the acid anhydride is one of succinic anhydride, glutaric anhydride or adipic anhydride.

6. The method for preparing a dual-target conjugate synthesized based on a PARP1 inhibitor and Rigosertib according to claim 3, characterized in that: In step (2), using DMF as solvent and Et3N as catalyst, compounds 11a~11c or 12a~12c react with 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethylurea tetrafluoroborate to obtain target compounds 13a~13c or 14a~14c.

7. The method for preparing a dual-target conjugate synthesized based on a PARP1 inhibitor and Rigosertib according to claim 3, characterized in that: In step (3), the acid is one of hydrochloric acid, phosphoric acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, citric acid, tartaric acid or lactic acid; and the base is one of an inorganic base containing an alkaline metal cation, an alkaline earth metal cation or an ammonium cation salt.

8. Use of a dual-target conjugate synthesized based on a PARP1 inhibitor and Rigosertib as claimed in claim 1 in the preparation of a drug for treating breast cancer.

9. The use according to claim 8, characterized in that: The breast cancer is BRCA wild-type triple-negative breast cancer.

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