2021 Volume 69 Issue 7 Pages 620-629
Poly(ADP-ribose)polymerase (PARP) is a significant therapeutic target for the treatment of numerous human diseases. Olaparib has been approved as a PARP inhibitor. In this paper, a series of new compounds were designed and synthesized with Olaparib as the lead compound. In order to evaluate the inhibitory activities against PARP1 of the synthesized compounds, in vitro PARP1 inhibition assay and intracellular PARylation assay were conducted. The results showed that the inhibitory activities of the derivatives were related to the type of substituent and the length of alkyl chain connecting the aromatic ring. 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT)-based assay also proved that these compounds demonstrating strong inhibition to PARP1 also have high anti-proliferative activities against BRCA2-deficient cell line (Capan-1). Analysis of the entire results suggest that compound 23 with desirable inhibitory efficiency may hold promise for further in vivo exploration of PARP inhibition.
Poly(ADP-ribose)polymerase (PARP) inhibitors recently revealed a prospective activity for the treatment of breast and ovarian cancer related to mutations in the BRCA1/2.1,2) PARP enzyme family has been regarded as a vital role in a variety of biological processes, including maintaining chromosomal structural integrity and genome stability, participating in DNA replication and transcription, regulating apoptosis and immune response.3–6) PARP1 and its close relative PARP2 are members of PARP family, which are responsible for the repair of DNA single-strand breaks (SSBs).7–9) PARP1 protein, a 113 kDa nuclear protein, utilizes C-terminal domain to recognize damaged DNA and binds to broken DNA, thereby facilitating C-terminal domain to decompose oxidized form of nicotinamide adenine dinucleotide (NAD+) into nicotinamide and ADP ribose which further lead to the production of PAR polymers.10–14) Poly-ADP ribosylated PARP1 promotes the recruitment of DNA repair proteins to the site of SSBs and enables damaged DNA to be repaired by base excision repair.15,16) PARP2 owns similar function as PARP1, but accounting for 5–10% of total PARP activity.17,18)
The initial antitumor mechanism of PARP inhibitors was to promote tumor cell apoptosis by impeding DNA repair.19,20) However, subsequent studies have found a synthetic mortality rate (SL) between PARP inhibition and BRCA1/2 mutations, providing a new strategy for the treatment of BRCA mutated tumor patients.20–22)
Several PARP inhibitors have entered the clinic, most of which imitate the structure of nicotinamide (PARP1 endogenous substrate) and exert inhibitory activity through binding to the donor site of the catalytic domain of PARP1 protein.23,24) With the continuous optimization of the structure of PARP inhibitors, there are currently more than 30 skeletons of PARP inhibitors.25) In 2014, Olaparib (AZD-2281, Fig. 1) as the first PARP inhibitor, was approved by the Food and Drug Administration (FDA) for treating breast and ovarian cancer.26) To date, four small-molecule PARP inhibitors including Olaparib,27) Rucaparib (AG-014699),28) Niraparib (MK-4827),29) and Talazoparib (BMN 673) have been commercially available30) (Fig. 1). Apart from these, many other small molecular PARP inhibitors are at various stages of clinical trials against different cancers. Over the past several years, PARP inhibitors that possess synergistic or additive antitumor effects in rational combinations with anticancer drugs have also attracted significant scientific attention.
Olaparib is a selective PARP1 and PARP2 inhibitor with IC50 values of 5 and 1 nM, respectively. The eutectic structures of Olaparib and PARP1 protein reported from PDB database were used to analyze the structure–activity relationship (PDB code: 5DS3). The center of the structure of PARP1 is a groove-shaped binding pocket, and the 1 (2H) phthalazinone structure in the Olaparib molecule is inserted into the interior of the PARP1 protein. His862, Gly863, Ser904 and Tyr907, crucial amino acid residues in the binding pocket, constitute the most important catalytic substrate binding center in the binding pocket which can specifically recognize the phthalazinone structure. Among them, Gly863 and Ser904 can form three hydrogen bonds with the oxygen atom on the carbonyl group and the free hydrogen on the amide. The 4-fluorobenzyl structure in the molecule enhances the binding strength of Olaparib and PARP1, while tail of the molecular, piperazine and cyclopropyl structures, have a lower binding strength to PARP1 protein, thus indicating that 1 (2H) phthalazinone and 4-fluorobenzyl parts are the main pharmacophore, while the piperazine and cyclopropyl structures are non-essential pharmacophores.
Consequently, on the basis of the knowledge of the binding mode of Olaparib and PARP1, we chose Olaparib as a lead compound for the development of the novel PARP inhibitors. We retained the main pharmacophore of Olaparib and tried to introduce an aromatic ring at the tail part (the red dotted frame in Fig. 2) to provide additional interactions between the molecule and the amino acid residues. Meanwhile, the introduced aromatic group and the amide bond are connected through alkyl groups of different chain lengths, which is to explore the best alkyl chain length. To further analyze the structure–activity relationship (SAR) on the tail part, the electronic effect of the aryl ring was investigated by introducing phenyl rings or heterocycles with various electronics (Fig. 2). Based on this design concept, a series of new derivatives were synthesized. The study aimed to explore whether the introduction of new groups can make new compounds occupy the PARP1 binding pocket more stably, and further enhance the binding strength of molecules and PARP1 protein, thereby improving the inhibitory activity and drug efficacy.
(Color figure can be accessed in the online version.)
As summarized in Table 1, fifteen target compounds (11–27) were synthesized. The synthetic routes are illustrated in Chart 1. Starting from 2-Carboxybenzaldehyde 5, treatment with dimethyl hydrogen phosphite in methanol (CH3OH) at room temperature (r.t.) in the presence of sodium methoxide (CH3ONa) to give 3-oxo-1,3-dihydroisobenzofuran-1-ylphosphonic acid 6 as previously reported. At room temperature, compound 6 reacted with 2-fluoro-5-formylbenzonitrile in tetrahydrofuran for 12 h to obtain intermediate 8, which was then hydrolyzed and cyclized in the presence of hydrazine hydrate and sodium hydroxide solution to obtain 9 with satisfactory yield. Under the protection of nitrogen, compound 9 was treated with oxalochloride to obtain intermediate 10, which was condensed with straight-chain amine containing a benzene ring and then treated with saturated sodium bicarbonate solution to produce the target compounds (11–27). The structures of all derivatives were characterized by 1H- and 13C-NMR spectra and mass spectra (see Supplementary Materials).
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a) Values are averages of three independent experiments, standard deviation (S.D.) < 10%. b) Values are averages of three independent experiments, S.D. < 10%.
(a) (CH3O)2POH, MeOH, MeONa, methanesulfonic acid (MSA), r.t., 7 h, 99%; (b) Tetrahydrofuran (THF), Et3N, r.t., 12 h, 97%; (c) NaOH, N2H4·H2O, 8 h, 65%; (d) Dichloromethane (DCM), (COCl)2, N,N-dimethylformamide (DMF), 0 °C, 2 h, 98%; (e) CH3CN, K2CO3, 4-dimethylaminopyridine (DMAP), r.t., 12 h, 47–75%.
To explore the potency of the newly synthesized compounds, 11–27 were evaluated for enzymatic inhibition against PARP1 enzyme and intracellular inhibition against PARP1. In addition, Olaparib was also assayed as a comparison. The results from the PARP1 enzyme inhibition assay and intracellular PARylation assay are summarized in Table 1.
Obviously, the majority of the compounds 11–27 exhibited potent PARP1 inhibition activities. Among them, compound 23 expressed better inhibitory activities than Olaparib in the PARP1 inhibition assay and intracellular PARylation assay (IC50 = 3.24 nM, EC50 = 0.47 nM). Compounds 13 and 14 showed similar inhibitory activities to Olaparib both in two activity evaluation experiments. Compound 14 showed a relatively superior inhibitory effect in the in vitro PARP1 enzyme inhibition experiment (IC50 = 4.74 nM), but it did not perform well in the intracellular PARylation experiment. By comparing the IC50 and EC50 values of compounds 11, 12, 22, 27 with phenyl as tail aromatic ring but connected to alkyl chain of different lengths (n = 0, 1, 2, 3), it can be found that when the range of n is between 0–2, the PARP1 inhibitory abilities of compounds increased with the growth of the alkyl chain. As the value of n increased to 3, the inhibitory ability of the compound showed a slight decrease. To draw a conclusion, ethyl as linker chain (n = 2) may be the optimal choice. It can be seen that the compounds containing halogen-substituted benzene rings generally owed stronger inhibitory activities against PARP1 than other compounds in both of the two bioactivity tests by analyzing the activity evaluation results of compounds. Among compounds 11–27, compounds containing fluorine atoms substituted benzene ring, such as compounds 13–15, 23, showed better inhibitory activities compared to other compounds with the same alkyl chain length, indicating that the presence of fluorine atoms substituted benzene ring in the compound is beneficial to the inhibition against PARP1. While, compounds 20 and 21 displayed weak inhibitory potency in the PARP1 enzyme inhibition assays and intracellular PARylation assays (IC50 > 50 nM, EC50 > 50 nM), which may be attributed to the stronger electron-withdrawing substituents on the benzene ring. Meanwhile, we tried to investigate whether replacing the benzene ring with an aromatic heterocycle can further enhance its inhibitory activities. However, compound 26 with a thiophene ring instead of a benzene ring manifested decreased inhibitory activities (IC50 = 18.52 nM, EC50 = 31.21 nM respectively), it is, therefore, reasonable to conclude that the use of thiophene ring instead of benzene ring maybe not a kind of advisable method. Simultaneously, it is worth pointing out that the compound 18 displayed inferior inhibitory activities against PARP1, which can be attributed to steric hindrance. 18 has a larger substituent on the benzene ring, which may make the compound not easy to enter the binding pocket.
Studies have shown that some PARP inhibitors exhibited single agent activity against tumors that lack a BRCA1/2 dependent DNA double-stranded repair mechanism.31,32) Therefore, compounds displayed strong PARP1 inhibitory activities namely 13, 14, 15, 22, 23 were investigated their anti-proliferative activities employing the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT)-based assay against BRCA2-deficient cell line: human pancreatic cancer cell (Capan-1). Their anti-proliferative activities are indicated by IC50 values that were calculated by non-linear regression analysis of the concentration–response curves obtained for each compound. The results from the MTT-based assay are listed in Table 2. We found that most of test compounds showed moderate anti-proliferative activities against Capan-1 cell line with IC50 value ranging from 7.5–19 μmoL/L. Two compounds 14 and 23 exhibited enhanced antitumor activities compared to the standard Olaparib (IC50 = 10.412 µM). Compound 22 displayed moderate activity and compounds 13 and 15 showed weak activities. The most active compound was 23 with IC50 = 7.532 µM, which corresponded to the results of PARP1 enzyme inhibition assay and intracellular PARylation assay. Taken the biological data together, the conclusion drawn is that some target compounds can be identified as potent PARP enzyme inhibitors.
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a) Values are averages of three independent experiments, S.D. < 10%.
In order to further investigate the interactions between these compounds and PARP1, we performed docking simulations to model the possible binding modes using Autodock vina. The three dimensional (3D) structure of PARP1 (5DS3) used was downloaded from the Protein Data Bank. As observed in Fig. 3, the oxygen atom and free hydrogen atom of the phthalazinone skeleton could form hydrogen bonds with Gly863 and Ser904 residues, which is similar to Olaparib. In addition to these hydrogen bonding interactions, the aromatic rings in compound 23 can also form π–π interactions with Tyr907, His862, and Tyr896, respectively. Simultaneously, Ile895 forms a hydrogen bond with the oxygen of amide bond connecting the tail structure, and the fluorine atom on the benzene ring at the tail forms a hydrogen bond with Arg878 residue and a halogen bond with Gly871 residue. On the other hand, compounds 13 and 14, which are slightly less active than 23, bind to the protein in a similar way to 23, but the fluorine attached to the benzene ring at the tail cannot form hydrogen bonds with the amino acid residues. Therefore, it can be deduced that the hydrogen bond and halogen bond formed by the fluorine atom of the tail benzene ring of compound 23 and the amino acid residues effectively enhanced the binding affinity to PARP1.
(C) Model of 13 bound to PARP1. (D) Model of 14 bound to PARP1. Hydrogen bonds are displayed as green dashed lines. Hydrophobic interactions are displayed as red dashed lines. Halogen bonds are displayed as blue dashed lines. (Color figure can be accessed in the online version.)
In summary, on the basis of retaining the main pharmacophore of Olaparib (phthalazinone part), a series of new derivatives were designed and synthesized. These newly synthesized compounds were explored for their activities through in vitro PARP1 inhibition assay, intracellular PARylation assay and anti-proliferative test. The majority of compounds 11–27 have a certain ability to inhibit PARP1 enzyme, which indicates that the structural modification of the tail of the Olaparib molecule can still maintain the ability to inhibit PARP1 activity. At the same time, the inhibitory activities of the target derivatives may be related to the electron withdrawing ability and steric hindrance of the substituents. Excessive electron-withdrawing ability of the substituted group on the benzene ring and large steric hindrance are not conducive to the inhibition against PARP1. Introducing the aromatic ring structure of fluorine-containing groups of different chain lengths at the tail of the lead compound Olaparib can make the derivatives expressed increased inhibitory potency by stably occupying the PARP binding pocket. Especially when the number of the atom of fluorine is one, it owed the most excellent inhibitory activities against PARP1. The ethyl alkyl chain is regarded as a suitable linker segment since it owed optimal inhibitory potency by comparing compounds with alky chain (n from 0 to 3) linked with benzene ring. MTT test also proved compounds 13, 14, 15, 22, 23 have evident anti-proliferative activities against BRCA2-deficient cell line: human pancreatic cancer cell (Capan-1). Among them, anti-proliferative activity of compound 23 was better than Olaparib, consistent with the results of PARP1 inhibition assay and intracellular PARylation assay. In conclusion, our data demonstrate that compound 23 maybe a better source for the future development of new PARP1 inhibitors.
All reagents were purchased from commercial sources and used without further purification. Column chromatography: silica gel (200–300 mesh; Qingdao Makall Group Co., Ltd., Qingdao, China) was used for purifying the crude product. 1H-NMR spectra were recorded in dimethyl sulfoxide (DMSO)-d6 on a Bruker-ACF 300/500 spectrometer. 13C-NMR spectra were recorded in DMSO-d6 at 125 or 75 MHz with tetramethylsilane (TMS) as the internal standard. Chemical shifts are quoted in ppm downfield from TMS; coupling constants (J) are quoted in hertz (Hz). Mass spectra (MS) were obtained from Agilent technologies 6520 Accurate-Mass Q-TOF LC/MS instruments. Melting points were measured on an RY-1 hot-stage microscope, and the thermometer was uncorrected. All compounds were routinely checked by TLC with silica gel GF-254 glass plates and viewed under UV light at 254 nm.
Synthesis of the Compound 6sodium methoxide (2.28 g, 0.042 mol) was dissolved in methanol (40 mL) into a 150 mL round-bottom flask. To this solution, dimethyl hydrogen phosphite (4.8 mL, 0.049 mol) and 2-carboxybenzaldehyde (compound 5, 5.00 g, 0.033 mol) were added by dripping at 0 °C for 30 min. Then the mixture was stirred at room temperature for 8 h. After the reaction was completed, methanesulfonic acid (3.1 mL, 0.047 mol) was added dropwise and stirred for 30 min. The solvent was removed under reduced pressure, following by adding 40 mol water. The aqueous phase was extracted with dichloromethane for three times, the combined organic phase was washed with water until neutral and dried with anhydrous sodium sulfate. Dichloromethane was evaporated under reduced pressure to afford product 6 (4.93 g, 99%) as white solid. 1H-NMR (500 MHz, DMSO-d6) δ: 3.62 (3H, d, J = 8 Hz), 3.86 (3H, d, J = 12 Hz), 6.36 (1H, d, J = 8 Hz), 7.74–7.70 (2H, m), 7.91–7.88 (1H, m), 7.98–7.96 (1H, m).
Synthesis of the Compound 8Compound 6 (5.00 g, 0.021 mol) was dissolved in tetrahydrofuran (30 mL) into a 150 mL round-bottom flask. To this solution, triethylamine (5.6 mL, 0.042 mol) was added. 2-Fluoro-5-formyl benzonitrile (compound 7, 3.08 g, 0.021 mol) was dissolved in tetrahydrofuran into another round-bottom flask, following by added with triethylamine (2.9 mL, 0.021 mol) and stirring evenly. Then the reaction fluids in the two flasks were mixed and reacted at room temperature for 12 h. The product was concentrated under reduced pressure and 50 mL water was added. The suspension was stirred for 30 min until the solids were not precipitated. Then, filtration was performed, filter cake was collected and dried to obtain compound 8 (5.41 g, 97%). 1H-NMR (500 MHz, DMSO-d6) δ: 8.23–8.10 (2H, m), 8.07 (1H, d, J = 7.8 Hz), 7.98 (1H, d, J = 7.7 Hz), 7.92 (1H, t, J = 7.5 Hz), 7.72 (1H, t, J = 7.5 Hz), 7.64 (1H, t, J = 9.0 Hz), 6.96 (1H, s).
Synthesis of the Compound 9Compound 8 (4.00 g, 0.015 mol) was added into a 250 mL round-bottom flask, following by added with water (22 mL) and sodium hydroxide solution (6.0 mL, 0.078 mol). The mixture was heated to 90 °C for 1 h. The reaction was cooled to 70 °C and hydrazine hydrate (10.2 mL, 0.214 mol) was added, stirring for 8 h. Then the mixture was cooled to room temperature, appropriate amount of 2 mol/L hydrochloric acid was added to adjust pH until no solid was precipitated. The resulting precipitate was vacuum filtered and washed with water. Then, the solid was dried in vacuum to afford the compound 9 (3.21 g, 65%). 1H-NMR (500 MHz, DMSO-d6) δ: 13.13 (1H, s), 12.60 (1H, s), 8.31–8.27 (1H, m), 8.00 (1H, d, J = 8.0 Hz), 7.91 (1H, t, J = 7.4 Hz), 7.85 (2H, t, J = 7.5 Hz), 7.62–7.57 (1H, m), 7.28–7.21 (1H, m), 4.38 (2H, s). MS: [M + H]+ m/z: 299.0826 (Calcd for C16H11FN2O3: 299.0826).
Synthesis of the Compound 10Compound 9 (2.00 g, 0.0067 mol) was dissolved in anhydrous dichloromethane (40 mL). The mixture was cooled to 0 °C under nitrogen atmosphere. To the solution, the oxalyl chloride (2.4 mL, 0.025 mol) was added dropwise slowly, following by adding two drops of DMF. Then the reaction was transferred to room temperature for 2 h. Solvent was removed under reduced pressure to afford the pale yellow solid (2.12 g) which was directly put into the next reaction.
Synthesis of the Compounds 11–25Aniline (0.5 g, 0.005 mol) and potassium carbonate (2.5 g, 0.018 mol) was dissolved in anhydrous acetonitrile (20 mL) into a 150 mL round-bottom flask. Then the mixture was cooled to 0 °C, following by adding the solution of compound 10 (1.6 g, 0.005 mol) in anhydrous acetonitrile (20 mL). The reaction was transferred to room temperature and catalyst 4-dimethylaminopyridine (DMAP) (1.00 g, 0.008 mol) was added. The mixture stirred at room temperature for 12 h until TLC indicated that the reactants reacted completely. Solvent was removed under reduced pressure to afford the crudes, which water (40 mL) was added. The pH of the aqueous phase was adjusted to weakly basic with the solution of sodium bicarbonate, and extracted with ethyl acetate. Combine the organic layers, dry over sodium sulfate (Na2SO4), filter, and concentrate. The crude residue was purified by column chromatography (SiO2, DCM : methanol = 120 : 1) to afford the white product 11 (1.21 g, 67%). Preparation of 12–25 were followed the procedure for 11 described above.
2-Fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)-N-phenylbenzamide (11)White solid, mp: 249–253 °C, yield: 58%. 1H-NMR (300 MHz, DMSO-d6) δ: 12.59 (1H, s), 10.36 (1H, s), 8.29 (1H, dd, J = 7.7, 1.5 Hz), 8.02 (1H, d, J = 7.8 Hz), 7.85 (2H, dtd, J = 21.2, 7.4, 1.4 Hz), 7.78–7.61 (3H, m), 7.51 (1H, ddd, J = 7.8, 5.0, 2.4 Hz), 7.41–7.21 (3H, m), 7.10 (1H, t, J = 7.4 Hz), 4.37 (2H, s). 13C-NMR (75 MHz, DMSO-d6) δ: 162.98, 159.75, 159.65, 156.37, 145.22, 139.15, 134.86, 133.84, 132.97, 131.87, 130.27, 129.45, 129.07, 128.31, 126.43, 125.84, 125.33, 124.21, 120.16, 116.70, 36.81. MS: [M + H]+ m/z: 374.1297 (Calcd for C22H16FN3O2: 374.1299).
N-Benzyl-2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzamide (12)White solid, mp: 185–186 °C, yield: 52.3%. 1H-NMR (500 MHz, DMSO-d6) δ: 12.62 (1H, d, J = 5.5 Hz), 8.86 (1H, t, J = 6.3 Hz), 8.27 (1H, d, J = 7.7 Hz), 7.98 (1H, d, J = 8.0 Hz), 7.89 (1H, q, J = 6.8, 6.3 Hz), 7.83 (1H, t, J = 7.4 Hz), 7.62 (1H, dd, J = 6.7, 2.3 Hz), 7.47 (1H, q, J = 5.1, 2.6 Hz), 7.36–7.29 (4H, m), 7.24 (2H, q, J = 9.5, 8.5 Hz), 4.46 (2H, d, J = 6.0 Hz), 4.34 (2H, d, J = 5.7 Hz). 13C-NMR (125 MHz, DMSO-d6) δ: 164.12, 161.53, 159.88, 159.21, 157.57, 145.43, 139.69, 134.87, 134.00, 132.98, 132.04, 130.57, 129.54, 128.77, 128.35, 127.58, 127.25, 126.54, 126.00, 124.27, 116.75, 43.07, 36.91. ESI-MS: [M + H]+ m/z: 388.1471 (Calcd for C23H18FN3O2: 388.1483).
2-Fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)-N-(3-phenylpropyl)benzamide (13)White solid, mp: 194–198 °C, yield: 67%. 1H-NMR (300 MHz, DMSO-d6) δ: 12.59 (1H, s), 8.83 (1H, s), 8.26 (1H, d, J = 7.5 Hz), 7.97 (1H, d, J = 7.6 Hz), 7.92–7.79 (2H, m), 7.60 (1H, dd, J = 6.9, 2.4 Hz), 7.46 (1H, ddd, J = 7.7, 4.8, 2.4 Hz), 7.40–7.30 (2H, m), 7.28–7.08 (3H, m), 4.42 (2H, d, J = 5.9 Hz), 4.33 (2H, s). 13C-NMR (75 MHz, DMSO-d6) δ: 163.95, 163.14, 159.94, 159.73,156.66, 145.23, 135.75, 134.73, 133.84, 132.86, 131.87, 130.46, 129.54, 128.27, 126.41, 125.83, 124.18, 116.68, 116.37, 115.47, 115.19, 42.33, 36.80. MS: [M + H]+ m/z: 406.1358 (Calcd for C23H17F2N3O2: 406.1361).
N-(3,4-Difluorobenzyl)-2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzamide (14)White solid, mp: 191–199 °C, yield: 63%. 1H-NMR (500 MHz, DMSO-d6) δ: 12.57 (1H, s), 8.85 (1H, s), 8.27 (1H, d, J = 7.8 Hz), 7.98 (1H, d, J = 8.0 Hz), 7.91–7.87 (1H, m), 7.83 (1H, t, J = 7.5 Hz), 7.61 (1H, dd, J = 6.8, 1.9 Hz), 7.47 (1H, ddd, J = 7.2, 4.5, 2.0 Hz), 7.41–7.32 (2H, m), 7.23 (1H, dd, J = 10.2, 8.7 Hz), 7.16 (1H, s), 4.43 (2H, d, J = 5.9 Hz), 4.34 (2H, s). 13C-NMR (125 MHz, DMSO-d6) δ: 164.19, 159.43, 157.46, 149.78, 145.37, 137.58, 134.95, 133.98, 133.20, 132.02, 130.60, 129.57, 128.39, 126.55, 125.97, 124.35, 124.08, 117.82, 116.78, 116.60, 116.53, 42.22, 36.91. MS: [M + H]+ m/z: 424.1270 (Calcd for C23H16F3N3O2: 424.1267).
2-Fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)-N-(2,3,4-trifluorobenzyl)benzamide (15)White solid, mp: 201–211 °C, yield: 58%. 1H-NMR (300 MHz, DMSO-d6) δ: 12.57 (1H, s), 8.88 (1H, td, J = 6.1, 2.4 Hz), 8.26 (1H, dd, J = 7.7, 1.6 Hz), 7.98 (1H, dd, J = 7.6, 1.5 Hz), 7.85 (2H, dtd, J = 18.7, 7.3, 1.5 Hz), 7.63 (1H, dd, J = 6.9, 2.4 Hz), 7.48 (1H, ddd, J = 8.4, 4.9, 2.4 Hz), 7.23 (3H, ddd, J = 9.6, 7.8, 2.8 Hz), 4.43 (2H, d, J = 6.0 Hz), 4.34 (2H, s). 13C-NMR (125 MHz, DMSO-d6) δ: 164.16, 159.99, 159.73, 156.71, 145.23, 137.26, 134.87, 134.83, 133.83, 133.22, 133.10, 131.88, 130.51, 129.43, 128.25, 126.41, 125.82, 123.78, 123.58, 116.73, 116.43, 112.00, 111.72, 42.04, 36.78. MS: [M + H]+ m/z: 442.1173 (Calcd for C23H15F4N3O2: 442.1173).
N-(4-Chlorobenzyl)-2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzamide (16)White solid, mp: 218–223 °C, yield: 68%. 1H-NMR (300 MHz, DMSO-d6) δ: 12.58 (1H, s), 8.84 (1H, t, J = 4.7 Hz), 8.30–8.24 (1H, m), 7.97 (1H, d, J = 7.4 Hz), 7.91–7.80 (2H, m), 7.61 (1H, dd, J = 6.9, 2.2 Hz), 7.47 (1H, ddd, J = 7.5, 4.8, 2.3 Hz), 7.36 (4H, q, J = 8.6 Hz), 7.22 (1H, dd, J = 10.3, 8.6 Hz), 4.43 (2H, d, J = 6.0 Hz), 4.34 (2H, s). 13C-NMR (75 MHz, DMSO-d6) δ: 163.99, 159.96, 159.72, 156.67, 145.22, 138.66, 134.79, 133.84, 133.02, 131.87, 131.72, 130.48, 129.40, 128.58, 128.29, 126.42, 125.83, 124.11, 123.91, 116.69, 116.39, 42.40, 36.80. MS: [M + H]+ m/z: 422.1065 (Calcd for C23H17ClFN3O2: 422.1066).
N-(3,4-Dichlorobenzyl)-2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzamide (17)White solid, mp: 189–195 °C, yield: 63.8%. 1H-NMR (500 MHz, DMSO-d6) δ: 12.62 (1H, d, J = 31.5 Hz), 8.88 (1H, s), 8.33–8.20 (1H, m), 7.96 (1H, d, J = 7.6 Hz),7.84 (2H, m, J = 18.9, 7.2, 1.3 Hz), 7.68–7.52 (3H, m), 7.52–7.43 (1H, m), 7.27–7.25 (2H, m), 4.45 (2H, s), 4.43 (2H, s). 13C-NMR (75 MHz, DMSO-d6) δ: 164.10, 159.74, 156.71, 145.23, 140.89, 134.85, 133.85, 133.16, 131.89, 131.25, 130.84, 130.48, 129.70, 129.55, 128.29, 127.92, 126.43, 125.83, 123.91, 116.74, 116.44, 42.04, 36.79. MS: [M + H]+ m/z: 456.0672 (Calcd for C23H16Cl2FN3O2: 456.0676).
Fluoro-N-(4-isopropylbenzyl)-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzamide (18)White solid, mp: 207–213 °C, yield: 47%. 1H-NMR (300 MHz, DMSO-d6) δ: 12.59 (1H, s), 8.81–8.72 (1H, m), 8.27 (1H, dd, J = 7.7, 1.6 Hz), 7.97 (1H, d, J = 8.1 Hz), 7.85 (2H, dtd, J = 18.7, 7.2, 1.5 Hz), 7.60 (1H, dd, J = 6.9, 2.3 Hz), 7.46 (1H, ddd, J = 8.1, 4.7, 2.3 Hz), 7.21 (5H, q, J = 7.2, 6.2 Hz), 4.40 (2H, d, J = 5.9 Hz), 4.33 (2H, s), 2.84 (1H, h, J = 6.8 Hz), 1.19 (3H, s), 1.17 (3H, s). 13C-NMR (75 MHz, DMSO-d6) δ: 163.91, 159.74, 147.30, 145.27, 136.92, 134.72, 134.68, 133.86, 132.87, 132.76, 131.88, 130.42, 129.44, 128.25, 127.56, 126.52, 125.85, 124.34, 124.14, 116.67, 116.37, 42.75, 36.81, 33.45, 24.26. MS: [M + Na]+ m/z: 452.1744 (Calcd for C26H24FN3O2: 452.1744).
2-Fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)-N-(4-(trifluoromethyl)benzyl)benzamide (19)White solid, mp: 178–183 °C, yield: 68.1%. 1H-NMR (300 MHz, DMSO-d6) δ: 12.57 (1H, s), 8.92 (1H, s), 8.43–8.12 (1H, m), 7.97 (1H, d, J = 7.7 Hz), 7.84 (2H, m, J = 19.6, 7.3, 1.2 Hz), 7.71 (2H, d, J = 8.1 Hz), 7.62 (1H, dd, J = 6.9, 2.2 Hz), 7.56–7.45 (3H, m), 7.42 (1H, dd, J = 10.4, 8.6 Hz), 4.53 (2H, d, J = 5.9 Hz), 4.34 (2H, s). 13C-NMR (75 MHz, DMSO-d6) δ: 164.11, 159.75, 156.73, 145.24, 144.52, 134.82, 133.85, 133.14, 133.02, 131.88, 130.51, 129.46, 128.28, 128.18, 127.69, 126.43, 125.83, 125.56, 123.97, 123.77, 116.73, 116.42, 42.72, 36.79. MS: [M + H]+ m/z: 456.1325 (Calcd for C24H17F4N3O2: 456.1330).
2-N-(4-Cyanobenzyl)-2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzamide (20)White solid, mp: 182–186 °C, yield: 53.7%. 1H-NMR (300 MHz, DMSO-d6) δ: 12.61 (1H, d, J = 10.0 Hz), 8.93 (1H, dd, J = 5.7, 3.8 Hz), 8.27 (1H, dd, J = 7.7, 1.1 Hz), 7.97 (1H, d, J = 7.6 Hz), 7.92–7.85 (1H, m), 7.85–7.76 (3H, m), 7.62 (1H, dd, J = 6.9, 2.2 Hz), 7.48 (3H, dd, J = 10.4, 5.3 Hz), 7.24 (1H, dd, J = 10.4, 8.6 Hz), 4.52 (2H, d, J = 5.9 Hz), 4.32 (2H, d, J = 14.5 Hz). 13C-NMR (75 MHz, DMSO-d6) δ: 164.16, 159.77, 156.72, 145.51, 145.26, 134.83, 133.87, 133.19, 133.08, 132.62, 131.90, 130.50, 129.45, 128.32, 126.43, 125.83, 123.86, 123.66, 119.21, 116.74, 116.44, 109.97, 42.85, 36.79. MS: [M + H]+ m/z: 413.1406 (Calcd for C24H17FN4O2: 413.1408).
2-Fluoro-N-(4-nitrobenzyl)-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzamide (21)White solid, mp: 173–179 °C, yield: 60%. 1H-NMR (300 MHz, DMSO-d6) δ: 12.57 (1H, s), 8.83 (1H, t, J = 4.7 Hz), 8.30-8.24 (1H, m), 7.97 (1H, d, J = 7.4 Hz), 7.91–7.80 (2H, m), 7.61 (1H, dd, J = 6.9, 2.2 Hz), 7.47 (1H, m, J = 7.5, 4.8, 2.3 Hz), 7.36 (4H, q, J = 8.6 Hz), 7.22 (1H, d, J = 10.3, 8.6 Hz), 4.43 (2H, d, J = 6.0 Hz), 4.34 (2H, s). 13C-NMR (75 MHz, DMSO-d6) δ: 163.99, 159.96, 159.72, 156.67, 145.22, 138.66, 134.79, 133.84, 133.02, 131.87, 131.72, 130.48, 129.40, 128.58, 128.29, 126.42, 125.83, 124.11, 123.91, 116.69, 116.39, 42.40, 36.80. MS: [M + H]+ m/z: 432.1406 (Calcd for C23H17FN4O4: 432.1408).
2-Fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)-N-phenethylbenzamide (22)White solid, mp: 157–158 °C, yield: 64.5%. 1H-NMR (500 MHz, DMSO) δ: 12.64 (1H, s), 8.35 (1H, d, J = 37.1 Hz), 8.28 (1H, dd, J = 7.8, 0.8 Hz), 7.97 (1H, d, J = 8.0 Hz), 7.92–7.87 (1H, m), 7.86–7.81 (1H, m), 7.52 (1H, dd, J = 6.7, 2.1 Hz), 7.50–7.45 (1H, m), 7.28 (2H, t, J = 7.4 Hz), 7.25–7.13 (4H, m), 4.33 (2H, s), 3.46 (2H, dd, J = 13.5, 6.9 Hz), 2.81 (2H, t, J = 7.4 Hz). 13C-NMR (125 MHz, DMSO-d6) δ: 163.92, 159.88, 159.13, 157.49, 145.38, 139.79, 134.80, 133.99, 132.89, 132.03, 130.40, 129.53, 129.14, 128.76, 128.38, 126.55, 126.00, 124.54, 124.44, 116.70, 116.55, 41.28, 36.98, 35.39. ESI-MS: [M + H]+ m/z: 402.1637 (Calcd for C24H20FN3O2: 402.1640).
2-Fluoro-N-(4-fluorophenethyl)-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzamide (23)White solid, mp: 195–205 °C, yield: 75%. 1H-NMR (300 MHz, DMSO-d6) δ: 12.60 (1H, s), 8.27 (2H, d, J = 7.4 Hz), 7.99–7.80 (3H, m), 7.48 (2H, ddt, J = 15.5, 7.3, 3.3 Hz), 7.29–7.17 (3H, m), 7.09 (2H, t, J = 8.8 Hz), 4.32 (2H, s), 3.44 (2H, q, J = 6.8 Hz), 2.80 (2H, t, J = 7.2 Hz). 13C-NMR (75 MHz, DMSO-d6) δ: 163.94, 162.29, 160.37, 159.88, 159.32 157.35, 145.37, 135.97, 134.83, 134.80, 133.97, 132.90, 132.02, 130.88, 130.42, 128.41, 126.56, 125.97, 124.39, 116.71, 115.48, 41.25, 36.98, 34.50. MS: [M + H]+ m/z: 420.1516 (Calcd for C24H19F2N3O2: 420.1518).
N-(4-Chlorophenethyl)-2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzamide (24)White solid, mp: 193–201 °C, yield: 53%. 1H-NMR (500 MHz, DMSO-d6) δ: 12.63 (1H, s), 8.35–8.30 (2H, m), 8.00 (1H, d, J = 8.0 Hz), 7.93 (1H, t, J = 7.2 Hz), 7.87 (1H, d, J = 7.5 Hz), 7.55 (1H, d, J = 6.6 Hz), 7.50 (1H, d, J = 3.2 Hz), 7.36 (2H, d, J = 8.3 Hz), 7.29 (2H, d, J = 8.3 Hz), 7.25–7.21 (1H, m), 4.36 (2H, s), 3.51–3.47 (2H, m), 2.84 (2H, t, J = 7.1 Hz). 13C-NMR (125 MHz, DMSO-d6) δ: 163.96, 159.87, 159.31, 157.34, 145.37, 138.88, 134.82, 133.98, 132.91, 132.84, 132.02, 131.25, 131.05, 130.42, 129.56, 128.65, 128.41, 126.56, 125.97, 116.71, 116.53, 41.01, 36.97, 34.64. MS: [M + H]+ m/z: 436.1220 (Calcd for C24H19ClFN3O2: 436.1222).
N-(3,4-Dimethylphenethyl)-2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzamide (25)White solid, mp: 206–214 °C, yield: 51%. 1H-NMR (300 MHz, DMSO-d6) δ: 12.59 (1H, s), 8.27 (2H, dd, J = 7.5, 1.7 Hz), 7.96 (1H, dd, J = 7.7, 1.5 Hz), 7.86 (2H, dtd, J = 17.8, 7.2, 1.4 Hz), 7.52 (1H, dd, J = 6.9, 2.3 Hz), 7.46 (1H, ddd, J = 7.6, 4.9, 2.4 Hz), 7.20 (1H, dd, J = 10.4, 8.4 Hz), 7.04–6.97 (2H, m), 6.92 (1H, dd, J = 7.6, 1.9 Hz), 4.32 (2H, s), 3.45–3.37 (2H, m), 2.72 (2H, t, J = 7.4 Hz), 2.16 (6H, d, J = 2.3 Hz). 13C-NMR (75 MHz, DMSO-d6) δ: 163.75, 159.74, 156.58, 145.24, 136.91, 136.22, 134.68, 134.64, 134.04, 133.85, 132.77, 132.67, 131.89, 130.19, 129.72, 129.43, 128.26, 126.42, 126.30, 125.84, 124.47, 116.64, 116.34, 41.34, 36.86, 34.87, 19.67, 19.26. MS: [M + Na]+ m/z: 452.1744 (Calcd for C26H24FN3O2: 452.1744).
2-Fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)-N-(2-(thiophen-2-yl)ethyl)benzamide (26)White solid, mp: 169–176 °C, yield: 47%. 1H-NMR (500 MHz, DMSO-d6) δ: 12.62 (1H, s), 8.39 (1H, s), 8.29 (1H, d, J = 7.7 Hz), 7.97 (1H, d, J = 8.0 Hz), 7.90 (1H, t, J = 7.6 Hz), 7.84 (1H, d, J = 7.2 Hz), 7.56–7.54 (1H, m), 7.50 (1H, td, J = 4.8, 2.3 Hz), 7.35–7.32 (1H, m), 7.25–7.21 (1H, m), 6.97–6.95 (1H, m), 6.91 (1H, d, J = 2.9 Hz), 4.35 (2H, s), 3.50–3.46 (2H, m), 3.04 (2H, t, J = 7.1 Hz).13C-NMR (125 MHz, DMSO-d6) δ: 164.02, 159.89, 159.36, 157.39, 145.36, 141.83, 134.86, 133.99, 132.98, 132.02, 130.37, 129.56, 128.41, 127.38, 126.56, 125.99, 125.69, 124.50, 116.74, 41.41, 37.00, 29.52. MS: [M + H]+ m/z: 408.1176 (Calcd for C22H18FN3O2S: 408.1176).
2-Fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)-N-(3-phenylpropyl)benzamide (27)White solid, mp: 186–192 °C, yield: 63%. 1H-NMR (300 MHz, DMSO-d6) δ: 12.59 (1H, s), 8.45–8.14 (2H, m), 7.99 (1H, s), 7.82 (2H, s), 7.54 (1H, dd, J = 6.8, 2.1 Hz), 7.45 (1H, ddd, J = 7.4, 4.7, 2.2 Hz), 7.21 (6H, ddd, J = 18.3, 13.9, 7.4 Hz), 4.33 (2H, s), 3.24 (2H, q, J = 6.7 Hz), 2.61 (2H, t, J = 7.7 Hz), 1.79 (2H, p, J = 7.2 Hz). 13C-NMR (75 MHz, DMSO-d6) δ: 163.90, 159.82, 159.72, 156.54, 145.23, 142.03, 134.69, 133.82, 132.61, 132.50, 131.86, 130.32, 129.45, 128.62, 128.29, 126.42, 126.06, 125.84, 124.79,124.58, 116.59, 116.28, 36.83, 32.87, 31.04. MS: [M + H]+ m/z: 416.1770 (Calcd for C25H22FN3O2: 416.1768).
In Vitro PARP1 Inhibition AssayThe PARP1 enzymatic assay was carried out using a highly sensitive fluorescence assay (HT-F Homogeneous Inhibition Assay; Trevigen, Cat # 4690-096-K) according to the manufacturer’s instructions. The various test compounds were dissolved in DMSO and then diluted to the desired concentration. First of all, each well was added with 50 µL of PARP1 buffer and incubated at 23 °C for 20 min. After incubation, the liquid in the wells was discarded to ensure thorough drying. Then 10 µL of the inhibitor solution, 15 µL of diluted PARP1 enzyme, and 25 µL of PARP cocktail was added into the wells successively. Enzyme solution and inhibitor solution were not required to add in the case of the blank reading. Olaparib was used to positive control. The wells were incubated at room temperature for 1 h, following by draining the liquid from the well as mentioned before. Then the wells were washed with 0.1% Triton x-100/phosphate buffered saline (PBS) solution (2 × 100 µL) and PBS (2 × 100 µL). Avidin reagent Strep-horseradish peroxidase (HRP) was added to each well and incubated at room temperature for 1 h for the detection of ribosylation. After discarding the liquid, the wells were washed with 0.1% Weix-100/PBS (200 µL) and PBS (200 µL). Subsequently, 50 µL of TACS-Sapphire colorimetric substrate was added to each well and allowed to be incubated in the dark at room temperature for 15 min. Finally, 0.2 N hydrochloric acid (50 µL) stop solution was added in order to stop the reaction and the absorbance was measured at 450 nm. IC50 value of each compound was calculated according to the above results. All experiments were independently performed at least three times.
Intracellular PARylation AssayThe aim of intracellular PARylation assay is to assess the ability of test compounds to inhibit poly(ADP)-ribose (PAR) polymerization. Hela cells in 100 µL culture medium (containing 0.1 mg/mL Penicillin-streptomycin, 10% fetal calf serum (FCS) in Dulbecco’s modified Eagle’s medium (DMEM) and 2 mM L-glutamine) were plated in 96-well microtiter plates, and allowed to adhere at 37 °C with 5% CO2 for 4 h. The test compounds were diluted to 0.2–1000 nM with 5% DMSO/H2O, which were used to treat the Hela cells. After the target compounds were added, the cells were incubated at 37 °C with 5% CO2 for another 18 h. Then the culture medium was discarded, 100 µL serum-free medium (containing 500 µM H2O2 solution) was added. The solution in wells was poured out, and prechilled methanol (100 µL) was used to treat cells at − 20 °C for 20 min. The methanol was discarded before the plate was washed ten times with PBS buffer. Each well was added with 80 µL detection buffer including 5000 µL PBS buffer, 60 µL second antibody antimouse Alexa Fluor 488 antibody (1 : 1000), 70 µL bovine serum albumin (BSA) and 3 µL primary antibody PAR monoclonal antibody (mAb) (1 : 2000), and allowed to adhere for 4 h at room temperature in dark. Nuclei were stained with propidium iodide. The final results were expressed by T/C%, where T was the total fluorescence intensity of PARP-positive nuclei and C was the total number of nuclei labeled with propidium iodide. EC50 values were calculated using GraphPad Prism5.
MTT AssayThe selected compounds were evaluated against BRCA2- deficient cell line: human pancreatic cancer cell (Capan-1) employing the MTT-based assay. First of all, cells in Iscove’s modified dulbecco medium (IMDM) medium with 10% fetal bovine serum were plated in 96-well microtiter plates (5.0 × 103 cells/well), and allowed to adhere at 37 °C with 5% CO2 for 24 h. After the target compounds were added, the cells were incubated at 37 °C with 5% CO2 for another 48 h. The next step is to remove the cell growth medium. Subsequently, 5 mg/mL MTT was added to each well, and incubation was carried out for 4 h at 37 °C. The absorbance of each well was measured with a plate reader at 490 nm later on. The final results were expressed by T/C%, where T was the optical density (OD) value of the injected cells and C was the OD value of the control cells. IC50 was calculated using GraphPad Prism 5.0.
Molecular DockingAll of the molecular docking studies were performed using AutoDock vina in combination with Discovery Studio software. The co-crystal structure of Olaparib with PARP-1 (PDB ID code: 5DS3) was used as the docking model. Firstly, the three-dimensional structures of compound 11–27 were built using ChemDraw12.0, followed by MM2 energy minimization. Then the protein target was prepared for the molecular docking simulation by removing the water molecules and bound ligands and adding hydrogen atoms. The site sphere was defined based on the binding site of Olaparib in its crystal pose in 5DS3 (center_x = − 2.5932, center_y = 39.9170, center_z = 14.6137). All of the other parameters used in the docking process were the default values of the system.
This work was supported by Jiangsu Natural Science Foundation (No. BK20161438).
The authors declare no conflict of interest.
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