Synthesis of Novel (S)-3-(1-Aminoethyl)-8-pyrimidinyl-2-phenylisoquinolin-1(2H)-ones by Suzuki–Miyaura Coupling and Their Cell Toxicity Activities

A series of (S)-3-(1-aminoethyl)-8-pyrimidinyl-2-phenylisoquinoline-1(2H)-ones 3a–3k was synthesized in 40–98% yield through Suzuki–Miyaura coupling using Pd(PPh3)2Cl2, Sphos, and K2CO3 in THF/H2O mixed solvent. All newly synthesized compounds were evaluated for cell viability (IC50) against MDA-MB-231, HeLa, and HepG2 cells. The antitumor activities of 3a–3k were improved when various pyrimidine motifs were introduced at position C-8 of the isoquinolinone ring.


Synthesis of Compounds
First, we compared the SMC reaction efficiencies of (S)-3-(1-aminoethyl)-8-chloro-2phenylisoquinolin-1(2H)-one (1) prepared in a known method [15] with (2-methoxypyrimidin-5-yl)boronic acid (2a) on various Pd catalysts in dioxane/H 2 O for 12 h at 80 • C with reference to a previously reported mixed solvent system [16,17]. To screen Pd catalysts in SMC reactions, the synthesis efficiencies of 3a for various Pd catalyst systems were compared under the above reaction conditions, including 5 mol% of Pd complex and 10 mol% of ligand (Table 1, Entries 1-5). When Pd(PPh 3 ) 2 Cl 2 and PPh 3 (Entry 5) were used, 3a was obtained in a low yield of 39%. Lower yields were also observed when using other common catalysts, such as Pd(PPh 3 ) 4 , Pd(OAc) 2 /PPh 3 , or PdCl 2 /PPh 3 .   SMC reactions with Pd(PPh3)2Cl2 (2.5 mol%), selected as a Pd catalyst, and various phosphine ligands (5 mol%) were studied under conditions in which the amount of catalyst used was reduced by half of that given above. First, the reaction efficiency was investigated by increasing the bulkiness of the ligand using mono-phosphine ligands (PPh3 < P(O-tol)3 < P(Cy)3) (Entries 6-8) and di-phosphine ligands (dppf < Xantphos) (Entries 9,11). It was observed that mono-phosphine ligands or di-phosphine ligands [19] showed low reactivity (Entries 1-9,11). However, the yield increased in the electron-rich phosphine ligands, such as Aphos (Entry 10). Unlike the mono-aryl type Aphos, the yield as well as the reaction rate increased dramatically when using Buchwald ligands, such as Sphos, Ruphos, and Davephos, which are biaryl types (Entries 13-15). We attributed this to the structural characteristics of the electron-rich and bulky phosphine ligands substituted with cyclohexyl or biaryl groups. This is consistent with reports that electron-rich and bulky phosphine Buchwald ligands accelerate reductive elimination and oxidative addition in SMC reactions, resulting in increased reactivity [20][21][22].
Subsequently, base screening with various solvents was performed in SMC (Table 2). SMC reactions with Pd(PPh 3 ) 2 Cl 2 (2.5 mol%), selected as a Pd catalyst, and various phosphine ligands (5 mol%) were studied under conditions in which the amount of catalyst used was reduced by half of that given above. First, the reaction efficiency was investigated by increasing the bulkiness of the ligand using mono-phosphine ligands (PPh 3 < P(Otol) 3 < P(Cy) 3 ) (Entries 6-8) and di-phosphine ligands (dppf < Xantphos) (Entries 9,11). It was observed that mono-phosphine ligands or di-phosphine ligands [18] showed low reactivity (Entries 1-9,11). However, the yield increased in the electron-rich phosphine ligands, such as Aphos (Entry 10). Unlike the mono-aryl type Aphos, the yield as well as the reaction rate increased dramatically when using Buchwald ligands, such as Sphos, Ruphos, and Davephos, which are biaryl types (Entries 13-15). We attributed this to the structural characteristics of the electron-rich and bulky phosphine ligands substituted with cyclohexyl or biaryl groups. This is consistent with reports that electron-rich and bulky phosphine Buchwald ligands accelerate reductive elimination and oxidative addition in SMC reactions, resulting in increased reactivity [19][20][21].
Subsequently, base screening with various solvents was performed in SMC ( Table 2). The SMC reaction occurred in the highest yield in the THF/H 2 O mixed solvent system. In addition, phase separation occurred in the THF/H 2 O solvent system after the reaction was completed, but phase separation was not observed in the aqueous solution system mixed with other solvents. Therefore, in this solvent system, the separated organic layer was extracted with an acidic aqueous solution, neutralized, and then crystallized to easily obtain a high-purity product without a chromatographic separation, which is very advantageous for a large-scale synthesis process. To further develop this process, a design of experiments (DoE; using Design Expert 12) was performed and optimized. The optimal conditions established through the DoE were Pd(PPh 3 ) 2 Cl 2 (0.5 mol%), Sphos (1.5 mol%), and K 2 CO 3 (3.0 eq) in THF and H 2 O mixed solvent. It was found that the amount of solvent THF/H 2 O used to facilitate phase separation between the organic layer and the aqueous layer after the reaction was more than 10 mL per gram of substrate 1.  The SMC reaction occurred in the highest yield in the THF/H2O mixed solvent system. In addition, phase separation occurred in the THF/H2O solvent system after the reaction was completed, but phase separation was not observed in the aqueous solution system mixed with other solvents. Therefore, in this solvent system, the separated organic layer was extracted with an acidic aqueous solution, neutralized, and then crystallized to easily obtain a high-purity product without a chromatographic separation, which is very advantageous for a large-scale synthesis process. To further develop this process, a design of experiments (DoE) was performed and optimized [23]. The optimal conditions established through the DoE were Pd(PPh3)2Cl2 (0.5 mol%), Sphos (1.5 mol%), and K2CO3 (3.0 eq) in THF and H2O mixed solvent. It was found that the amount of solvent THF/H2O used to facilitate phase separation between the organic layer and the aqueous layer after the reaction was more than 10 mL per gram of substrate 1.
Novel (S)-3-(1-aminoethyl)-8-pyrimidinyl-2-phenylisoquinolin-1(2H)-one derivatives (3) were synthesized by using SMC reactions under these optimized conditions ( Table 3). In the case of unstable boronic acid, pinacol boronate (in case of 3g and 3k) was used as a reactant. Electron-rich pyrimidinyl boronic acid substituted with methoxy, ethoxy, dimethylamino, and piperidinyl groups at the para position of the pyrimidine ring gave the product in good yield (3a, 3b, 3i and 3j). In the case of other boronic acids with low reactivity, the reaction could be completed in good yield by increasing the amount of catalyst and ligand (3c, 3e-3g). Boronic acids with steric hindrance, such as 2,4-dimethoxypyrimidinyl boronic acid, still showed low yield even when the amount of catalyst and ligand was increased (3d). In addition, electron-deficient boronic acid (e.g., 2-cyanopyrimidinyl boronic acid) showed the lowest reactivity (3k). Novel (S)-3-(1-aminoethyl)-8-pyrimidinyl-2-phenylisoquinolin-1(2H)-one derivatives (3) were synthesized by using SMC reactions under these optimized conditions ( Table 3). The structure of 3a-3k were confirmed by 1 H-NMR, 13 C-NMR spectrosco-py (Supplementary Materials Figures S1-S22). In the case of unstable boronic acid, pinacol boronate (in case of 3g and 3k) was used as a reactant. Electron-rich pyrimidinyl boronic acid substituted with methoxy, ethoxy, dimethylamino, and piperidinyl groups at the para position of the pyrimidine ring gave the product in good yield (3a, 3b, 3i and 3j). In the case of other boronic acids with low reactivity, the reaction could be completed in good yield by increasing the amount of catalyst and ligand (3c, 3e-3g). Boronic acids with steric hindrance, such as 2,4-dimethoxypyrimidinyl boronic acid, still showed low yield even when the amount of catalyst and ligand was increased (3d). In addition, electron-deficient boronic acid (e.g., 2-cyanopyrimidinyl boronic acid) showed the lowest reactivity (3k).

General
All commercially available materials from Sigma-Aldrich (Burlington, MA, USA), Daejung (Siheung, Korea), TCI (Tokyo, Japan), Chemieliva (Chongqing, China) and solvents were used without further purification. All small-scale screening reactions (≤10 mL of solvent) were performed in 50 mL round bottom flasks on a Radleys Carousel 6 Plus Reaction Station under an air atmosphere. HPLC was performed on a Hitachi LaChrom Elite HPLC system. 1 H NMR (400 MHz) and 13 C NMR (100 MHz) were measured on a Bruker Avance 400 spectrometer system. 1 H NMR spectra chemical shifts were expressed in parts per million (ppm) downfield from tetramethylsilane, and coupling constants were reported in Hertz (Hz). Splitting patterns are indicated as follows: s, singlet; d, doublet; t, triplet; and q, quartet; m, multiplet. 13 C NMR spectra were reported in ppm, referenced to deuterochloroform (77.16 ppm). Melting points were determined by DSC (Mettler Toledo). High resolution mass spectra (HRMS, JEOL MStation JMS-700) were obtained using an electron impact (EI) ionization technique (magnetic sector-electric sector double focusing mass analyzer) from the KBSI (Korea Basic Science Institute Daegu Center).

Conclusions
In this study, we developed an effective method of introducing various pyrimidine groups into (S)-3-(1-aminoethyl)-8-chloro-2-phenylisoquinolin-1(2H)-one (1) via SMC to provide new pyrimidine-substituted isoquinoline derivatives 3. To evaluate the activity of the compounds 3, their cell viability (IC 50 ) was measured in cancer cell lines of MDA-MB-231, HeLa, and HepG2. From the antitumor activity of compounds 3, it was found that antitumor activity was increased when various pyrimidine rings were introduced instead of Cl at position 8 of the isoquinoline derivative 1. This is further evidence that the pyrimidine functional group is a very good pharmacophore. In the future, we hope to identify more novel compounds with enhanced pharmacological activity using this synthetic method.