2013 Volume 61 Issue 7 Pages 747-756
Design and synthesis of a new series of quinolinylaminoisoquinoline derivatives as conformationally restricted bioisosteres of Sorafenib are described. Their in vitro antiproliferative activity against A375P melanoma cell line was tested. Compounds 1b, 1d, 1g, and 1j showed the highest potency against A375P cell line with IC50 values in sub-micromolar scale. In addition, compound 1d exerted high selectivity towards RAF1 serine/threonine kinase with 96.47% inhibition at 10 µM, and IC50 of 0.96 µM. This compound can possess antiproliferative activity against melanoma cells through inhibition of RAF1 kinase.
The RAS/RAF/MEK/extraccellular signal-regulated kinase (ERK) (ERK pathway) is a well-known cell signaling network essential for cell survival, growth, and proliferation.1) The RAS proteins are membrane-bound small G-protein, whereas RAF, MEK, and ERK are cytosolic protein kinases which compose a sequential signaling cascade. Among these four kinases, RAF kinases have been the most studied drug targets since mutations of the RAF protein were found in approximately 7% of human cancers2,3) with particularly high frequency in melanoma (50–70%), ovarian cancer (35%), thyroid cancer (30%), and colorectal cancer (10%). Due to high incidence of RAF overexpression in melanoma, it has been a potential drug target for treatment of melanoma.
Melanoma is a malignant tumor of melanocytes. It is considered as the most aggressive form of skin cancer. The major risk factors for melanoma development include exposure to solar UV irradiation, fair skin, dysplastic nevi syndrome, and a family history of melanoma. Melanomas can metastasize either by the lymphatic or by the hematogenous route.4) Early stage melanoma (stage I/II) can be cured surgically with more than 95% success rate. But melanoma metastasizing to major organs (stage IV) is virtually incurable.5) Patients with advanced melanoma have a median survival time of less than one year, and the estimated 5-year survival rate is less than 15%.6,7) With the incidence of melanoma rapidly rising in the United States and other developed countries, there has been an urgent need to develop more effective drugs.
Sorafenib (Nexavar®, Fig. 1) targets ERK pathway. It inhibits basal phosphorylation of ERK (pERK) in numerous cancer cell lines in vitro, including melanoma cell lines, independent of their K-RAS and B-RAF mutational status.8) Numerous compounds with structural similarity to Sorafenib have been recently reported as potential antiproliferative agents against melanoma.9–23) In addition, amino-heterocycles as urea bioisosteres have been reported as RAF kinase inhibitors. The 2-aminoquinazoline and 2-aminoquinoline derivatives illustrated in Fig. 1 are bioisosteres of Sorafenib with RAF kinase inhibitory activity.24) In the present investigation, the target compounds were designed as conformationally restricted bioisosteres of Sorafenib. The pyridine-2-carboxamide moiety of Sorafenib was replaced with 8′-aminoquinoline nucleus in our target compounds. The oxygen linker of Sorafenib was isosterically replaced with NH. And the phenylurea scaffold of Sorafenib was replaced with substituted 1′-aminoisoquinoline moiety (Fig. 1). The antiproliferative activity of the target compounds was tested over A375P human melanoma cell line. And one potential compound was examined for inhibitory effect over a panel of 38 kinases. The procedures and results are reported in details.
The target compounds 1a–o and 2a–m were synthesized by the pathways illustrated in Charts 1 and 2. Nitration of 1-chloroisoquinoline (3) using a mixture of nitric acid and sulfuric acid yielded 1-chloro-5-nitroisoquinoline (4).25) Nucleophilic substitution of the chloro group of compound 4 with different (hetero)aromatic amines was carried out by heating in the presence of 2,2′-bis(diphenylphosphino)-1,1′-binaphthyl (BINAP), palladium acetate, and anhydrous potassium carbonate to produce compounds 5a–o. Reduction of the nitro group of 5a–o to the corresponding amino compounds 6a–o was done using Pd/C in hydrogen atmosphere. Nitration of 4-chloroquinoline (9) was carried out in the same way as preparation of compound 4 using a mixture of nitric and sulfuric acids to produce 4-chloro-8-nitroquinoline (7).9,26) Reaction between the amino compounds 6a–o and the chloro compound 7 afforded the quinolinylaminoisoquinoline compounds 8a–o. Finally, the nitro group of 8a–o was reduced with Pd/C in hydrogen atmosphere to give the final compounds 1a–o (Chart 1).
Reagents and reaction conditions: (i) HNO3, H2SO4, rt, 3 h; (ii) BINAP, Pd(OAc)2, K2CO3, dioxane, 100°C, 40 h; (iii) Pd/C, H2, MeOH, rt, 2 h; (iv) BINAP, Pd(OAc)2, K2CO3, dioxane, 100°C, 19 h; (v) HNO3, H2SO4, rt, 4 h.
For synthesis of the diarylamide target compounds 2a–m, the pathway drawn in Chart 2 was utilized. Substitution of chloro group of compound 4 with ethanolic ammonia yielded 1-amino-5-nitroisoquinoline (10). Condensation of the amino compound 10 with the appropriate aromatic carboxylic acids in the presence of O-benzotriazole-N,N,N′,N′-tetramethyl-uronium-hexafluoro-phosphate (HBTU) and diisopropylethylamine (Hünig’s base) produced the diarylamides 11a–m possessing nitro group. Reduction of nitro group of 11a–m to the corresponding amino analogues 12a–m was achieved using Pd/C in hydrogen atmosphere. Similar to preparation of compounds 8a–o, the amino compounds 12a–m were heated with 4-chloro-8-nitroquinoline (7) in the presence of BINAP, palladium acetate, and anhydrous potassium carbonate to afford compounds 13a–m. The nitro group of 13a–m was reduced to amino with Pd/C in hydrogen atmosphere to produce the final compounds 2a–m.
Reagents and reaction conditions: (i) NH3 (7 N solution in EtOH), 80°C, 40 h; (ii) HBTU, DIPEA, THF, 80°C, 30 h; (iii) Pd/C, H2, MeOH, rt, 2 h; (iv) BINAP, Pd(OAc)2, K2CO3, dioxane, 100°C, 40 h.
The antiproliferative activity of the target compounds was tested against A375P human melanoma cell line. Their ability to inhibit the growth of A375P cell line is summarized in Tables 1 and 2. Sorafenib was utilized as a reference standard due to structural similarity with the target compounds, and it has shown high potency against different melanoma cell lines.27)
The results showed that compounds 1c, d with amine linker at position 1 of the isoquinoline nucleus were more potent than the corresponding amide analogues 2d, e. This may be rationalized to appropriate fitting of the amine derivatives at the receptor site. The amine derivatives are exact bioisosteres of Sorafenib, but amide analogues possess additional carbonyl group. Among all the target compounds, compounds 1b, 1d, 1g, and 1j with amine linker were the most potent with IC50 values in sub-micromolar scale. Compounds 2c, 2e, and 2k with amide linker, and compound 1c with amine linker showed also superior potency to Sorafenib but with IC50 values in micromolar scale.
Upon investigating the influence of the terminal aryl ring on potency, it was found that heteroaromatics were unfavorable for activity of compounds 1k–o and 2l. For instance, compound 1c with 3′-(trifluoromethyl)phenyl terminal moiety showed higher potency than the corresponding pyrimidine isostere 1m. The increased bulkiness on that terminal aryl ring as in compounds 1h, i, 2h–j, and 2m was unfavorable for activity. Compound 1b with 4′-(trifluoromethyl)phenyl terminal ring demonstrated higher potency than 1e possessing 2′,4′-bis(trifluoromethyl)phenyl terminal moiety. Opposite finding was encountered in case of compounds 2d and 2g. In addition, compound 2g with 3′,5′-bis(trifluoromethyl)phenyl terminal ring was more potent than its positional isomer 2f containing 2′,4′-bis(trifluoromethyl)phenyl terminal moiety.
In case of compounds with NH linker, compounds 1b–d and 1g with electron-withdrawing groups on the terminal aryl ring were more potent than compound 1a with 3′,4′-dimethylphenyl terminal moiety. This can be attributed to steric and/or electronic differences between the substituents on terminal aryl ring, and their influence(s) on affinity and potency. And in case of diarylamide derivatives, dimethoxyphenyl (compounds 2a, b) and benzo[b]dioxine (compound 2i) terminal moieties were unfavorable for activity.
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|---|---|---|---|---|---|
| Comp. No. | R | A375P (IC50, µM) | Comp. No. | R | A375P (IC50, µM) |
| 1a | ;%0A%09%09%09newWindow.document.open();%0A%09%09%09newWindow.document.write('<img src=%22./Graphics/FT1a.png%22>');%0A%09%09%09newWindow.document.close();%0A%09%09) | >10 | 1i | ;%0A%09%09%09newWindow.document.open();%0A%09%09%09newWindow.document.write('<img src=%22./Graphics/FT1i.png%22>');%0A%09%09%09newWindow.document.close();%0A%09%09) | >10 |
| 1b | ;%0A%09%09%09newWindow.document.open();%0A%09%09%09newWindow.document.write('<img src=%22./Graphics/FT1b.png%22>');%0A%09%09%09newWindow.document.close();%0A%09%09) | 0.94 | 1j | ;%0A%09%09%09newWindow.document.open();%0A%09%09%09newWindow.document.write('<img src=%22./Graphics/FT1j.png%22>');%0A%09%09%09newWindow.document.close();%0A%09%09) | 0.30 |
| 1c | ;%0A%09%09%09newWindow.document.open();%0A%09%09%09newWindow.document.write('<img src=%22./Graphics/FT1c.png%22>');%0A%09%09%09newWindow.document.close();%0A%09%09) | 1.0 | 1k | ;%0A%09%09%09newWindow.document.open();%0A%09%09%09newWindow.document.write('<img src=%22./Graphics/FT1k.png%22>');%0A%09%09%09newWindow.document.close();%0A%09%09) | >10 |
| 1d | ;%0A%09%09%09newWindow.document.open();%0A%09%09%09newWindow.document.write('<img src=%22./Graphics/FT1d.png%22>');%0A%09%09%09newWindow.document.close();%0A%09%09) | 0.99 | 1l | ;%0A%09%09%09newWindow.document.open();%0A%09%09%09newWindow.document.write('<img src=%22./Graphics/FT1l.png%22>');%0A%09%09%09newWindow.document.close();%0A%09%09) | >10 |
| 1e | ;%0A%09%09%09newWindow.document.open();%0A%09%09%09newWindow.document.write('<img src=%22./Graphics/FT1e.png%22>');%0A%09%09%09newWindow.document.close();%0A%09%09) | >10 | 1m | ;%0A%09%09%09newWindow.document.open();%0A%09%09%09newWindow.document.write('<img src=%22./Graphics/FT1m.png%22>');%0A%09%09%09newWindow.document.close();%0A%09%09) | >10 |
| 1f | ;%0A%09%09%09newWindow.document.open();%0A%09%09%09newWindow.document.write('<img src=%22./Graphics/FT1f.png%22>');%0A%09%09%09newWindow.document.close();%0A%09%09) | >10 | 1n | ;%0A%09%09%09newWindow.document.open();%0A%09%09%09newWindow.document.write('<img src=%22./Graphics/FT1n.png%22>');%0A%09%09%09newWindow.document.close();%0A%09%09) | >10 |
| 1g | ;%0A%09%09%09newWindow.document.open();%0A%09%09%09newWindow.document.write('<img src=%22./Graphics/FT1g.png%22>');%0A%09%09%09newWindow.document.close();%0A%09%09) | 0.98 | 1o | ;%0A%09%09%09newWindow.document.open();%0A%09%09%09newWindow.document.write('<img src=%22./Graphics/FT1o.png%22>');%0A%09%09%09newWindow.document.close();%0A%09%09) | >10 |
| 1h | ;%0A%09%09%09newWindow.document.open();%0A%09%09%09newWindow.document.write('<img src=%22./Graphics/FT1h.png%22>');%0A%09%09%09newWindow.document.close();%0A%09%09) | >10 | Sorafenib | 3.4 | |
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|---|---|---|---|---|---|
| Comp. No. | R3 | A375P (IC50, µM) | Comp. No. | R3 | A375P (IC50, µM) |
| 2a | ;%0A%09%09%09newWindow.document.open();%0A%09%09%09newWindow.document.write('<img src=%22./Graphics/FT2a.png%22>');%0A%09%09%09newWindow.document.close();%0A%09%09) | >10 | 2h | ;%0A%09%09%09newWindow.document.open();%0A%09%09%09newWindow.document.write('<img src=%22./Graphics/FT2h.png%22>');%0A%09%09%09newWindow.document.close();%0A%09%09) | >10 |
| 2b | ;%0A%09%09%09newWindow.document.open();%0A%09%09%09newWindow.document.write('<img src=%22./Graphics/FT2b.png%22>');%0A%09%09%09newWindow.document.close();%0A%09%09) | 7.0 | 2i | ;%0A%09%09%09newWindow.document.open();%0A%09%09%09newWindow.document.write('<img src=%22./Graphics/FT2i.png%22>');%0A%09%09%09newWindow.document.close();%0A%09%09) | >10 |
| 2c | ;%0A%09%09%09newWindow.document.open();%0A%09%09%09newWindow.document.write('<img src=%22./Graphics/FT2c.png%22>');%0A%09%09%09newWindow.document.close();%0A%09%09) | 2.7 | 2j | ;%0A%09%09%09newWindow.document.open();%0A%09%09%09newWindow.document.write('<img src=%22./Graphics/FT2j.png%22>');%0A%09%09%09newWindow.document.close();%0A%09%09) | >10 |
| 2d | ;%0A%09%09%09newWindow.document.open();%0A%09%09%09newWindow.document.write('<img src=%22./Graphics/FT2d.png%22>');%0A%09%09%09newWindow.document.close();%0A%09%09) | >10 | 2k | ;%0A%09%09%09newWindow.document.open();%0A%09%09%09newWindow.document.write('<img src=%22./Graphics/FT2k.png%22>');%0A%09%09%09newWindow.document.close();%0A%09%09) | 2.6 |
| 2e | ;%0A%09%09%09newWindow.document.open();%0A%09%09%09newWindow.document.write('<img src=%22./Graphics/FT2e.png%22>');%0A%09%09%09newWindow.document.close();%0A%09%09) | 1.4 | 2l | ;%0A%09%09%09newWindow.document.open();%0A%09%09%09newWindow.document.write('<img src=%22./Graphics/FT2l.png%22>');%0A%09%09%09newWindow.document.close();%0A%09%09) | >10 |
| 2f | ;%0A%09%09%09newWindow.document.open();%0A%09%09%09newWindow.document.write('<img src=%22./Graphics/FT2f.png%22>');%0A%09%09%09newWindow.document.close();%0A%09%09) | >10 | 2m | ;%0A%09%09%09newWindow.document.open();%0A%09%09%09newWindow.document.write('<img src=%22./Graphics/FT2m.png%22>');%0A%09%09%09newWindow.document.close();%0A%09%09) | 7.0 |
| 2g | ;%0A%09%09%09newWindow.document.open();%0A%09%09%09newWindow.document.write('<img src=%22./Graphics/FT2g.png%22>');%0A%09%09%09newWindow.document.close();%0A%09%09) | 2.8 | Sorafenib | 3.4 | |
In order to study the mechanism of action at molecular level and the kinase inhibitory profile of this series of compounds, compound 1d was selected as a representative example due to its high potency against A375P human melanoma cell line, and due to high structural similarity to the lead compound, Sorafenib, especially at the terminal moiety. Compound 1d was tested at a single dose concentration of 10 µM over a panel of 38 oncogenic kinases. As illustrated in Fig. 2, it exerted strong inhibitory effect, 96.47%, with high selectivity against RAF1 kinase. So it can be concluded that this compound may inhibit melanoma cell proliferation due to RAF1 kinase inhibition.
% Activity in each enzyme is the mean of two different readings; test compound was used in a single dose concentration of 10 µM; 100% activity refers to enzyme activity in negative control (DMSO); % inhibition was calculated by subtracting % activity from 100.
Compound 1d was further tested over RAF1 and B-RAF (V600E) kinases in order to determine its IC50, where a 10-doses IC50 mode with threefold serial dilutions starting at 2 µM concentration was applied. Table 3 illustrates the IC50 values of compound 1d over RAF1 and V600E B-RAF kinases. It was 9.8 times more potent against RAF1 kinase than B-RAF (V600E). So compound 1d could inhibit melanoma cell growth through RAF1 kinase inhibition rather than B-RAF (V600E) kinase inhibition.
| Kinase | IC50 (µM) |
|---|---|
| B-RAF (V600E) | 9.4 |
| RAF1 | 0.96 |
The selectivity of compound 1d towards RAF1 kinase may be attributed to restricted conformation in both aminoquinoline and aminoisoquinoline nuclei. The difference in geometry between the binding pocket of RAF1 (C-RAF) serine/threonine kinase and other kinases may enable the selectivity of compound 1d for RAF1.
A new series of quinolinylaminoisoquinoline derivatives was designed as conformationally restricted bioisosteres of Sorafenib. They were synthesized as a continuation of our ongoing anticancer development research project. The target compounds were evaluated for antiproliferative activity against A375P human melanoma cell line. A structure–activity relationship (SAR) study has been made to correlate between the compounds structures and antiproliferative activities. Compounds 1b, 1d, 1g, and 1j possessing amine linker showed the highest potency against A375P cell line with IC50 values in sub-micromolar scale. Their terminal aryl rings were 4′-(trifluoromethyl)phenyl, 4′-chloro-3′-(trifluoromethyl)phenyl, 2′-morpholino-5′-(trifluoromethyl)phenyl, and benzo[d][1,3]dioxole moieties, respectively. So these terminal aryl moieties together with amine linker are the most optimum for antiproliferative activity of this series of compounds. In addition, compounds 1c, 2c, 2e, 2g, and 2k exerted higher potency than Sorafenib against A375P, but with IC50 values in micromolar scale.
In order to examine the possible mechanism of antiproliferative activity of this series of compounds at molecular level, compound 1d was tested for inhibitory activity against 38 oncogenic kinase panel. It demonstrated high selectivity towards RAF1 serine/threonine kinase over the other 37 tested kinases. Compound 1d inhibited the activity of RAF1 kinase by 96.47% at 10 µM, and its IC50 value was 0.96 µM. So this compound can inhibit melanoma cell proliferation through inhibition of RAF1 kinase which is overexpressed in most of melanoma cases. This compound can be utilized for future development of selective RAF1 inhibitors for treatment of melanoma.
1H- and 13C-NMR spectra were recorded on a Bruker Avance 400 spectrometer (400 MHz for 1H and 100 MHz for 13C) using tetramethylsilane as an internal standard. LC-mass spectra were determined on a Waters Quattro Micro System. Column chromatography was carried out using silica gel (230–400 mesh). Solvents and liquid reagents were transferred using hypodermic syringes. Purity % of all the target compounds were determined by LC-MS and found to be > 95%. All solvents and reagents were commercially available and used without further purification.
1-Chloro-5-nitroisoquinoline (4)To a solution of 1-chloroisoquinoline (3, 1.0 g, 6.11 mmol) in sulfuric acid (5.0 mL, 84.4 mmol) was added nitric acid (0.9 mL, 21.4 mmol) dropwise at 0°C. The mixture was stirred at room temperature for 3 h. Upon completion, the reaction mixture was cooled to 0°C and neutralized with 10% NaHCO3 aqueous solution. The resulting precipitate was collected by filtration and dried to give the title compound (1.26 g, 99%) as a white solid. 1H-NMR (DMSO-d6) δ: 8.74–8.69 (m, 2H), 8.53 (d, J=6.1 Hz, 1H), 8.29 (d, J=6.1 Hz, 1H), 8.00 (t, J=8.1 Hz, 1H); 13C-NMR (DMSO-d6) δ: 152.6, 145.3, 144.8, 133.3, 130.3, 128.9, 127.5, 127.0, 115.8.
General Procedure for the Preparation of 5-Nitro-N-substituted Isoquinolin-1-amines (5a–o)To a mixture of 4-chloro-5-nitroisoquinoline (3, 1.0 g, 4.8 mmol), BINAP (62 mg, 0.10 mmol), palladium(II) acetate (22 mg, 0.10 mmol), and anhydrous potassium carbonate (0.263 g, 1.9 mmol) in dioxane, the appropriate amine (0.48 mmol) was added. The mixture was refluxed for 40 h. Upon completion, the reaction mixture was filtered through celite. The filtrate was evaporated under reduced pressure, and the resulting residue was purified by column chromatography to afford the corresponding compounds 5a–o.
N-(3,4-Dimethylphenyl)-5-nitroisoquinolin-1-amine (5a): Brown solid, yield 50%; 1H-NMR (DMSO-d6) δ: 9.38 (s, 1H), 8.92 (d, J=8.5 Hz, 1H), 8.49 (dd, J=0.7, 0.8 Hz, 1H), 8.16 (d, J=6.2 Hz, 1H), 7.77 (t, J=8.1 Hz, 1H), 7.55–7.51 (m, 2H), 7.49 (d, J=6.1 Hz, 1H), 7.11 (d, J=8.8 Hz, 1H), 2.22 (d, J=11.0 Hz, 6H).
5-Nitro-N-(4-(trifluoromethyl)phenyl)isoquinolin-1-amine (5b): Orange solid, yield 49%; 1H-NMR (DMSO-d6) δ: 9.86 (s, 1H), 8.96 (d, J=8.5 Hz, 1H), 8.55 (d, J=7.8 Hz, 2H), 8.28 (d, J=6.1 Hz, 1H), 8.08 (d, J=8.5 Hz, 2H), 7.85 (t, J=8.1 Hz, 1H), 7.72–7.67 (m, 2H).
5-Nitro-N-(3-(trifluoromethyl)phenyl)isoquinolin-1-amine (5c): Brown solid, yield 44%; 1H-NMR (DMSO-d6) δ: 9.80 (s, 1H), 8.96 (d, J=8.3 Hz, 1H), 8.54 (d, J=7.8 Hz, 1H), 8.29–8.25 (m, 2H), 8.19 (d, J=8.3 Hz, 1H), 7.85 (t, J=8.1 Hz, 1H), 7.63 (d, J=6.2 Hz, 1H), 7.59 (t, J=8.0 Hz, 1H), 7.37 (d, J=7.7 Hz, 1H).
N-(4-Chloro-3-(trifluoromethyl)phenyl)-5-nitroisoquinolin-1-amine (5d): Brown solid, yield 60%; 1H-NMR (DMSO-d6) δ: 9.89 (s, 1H), 8.94 (d, J=8.3 Hz, 1H), 8.55 (d, J=7.7 Hz, 1H), 8.42 (s, 1H), 8.27 (d, J=6.0 Hz, 2H), 7.86 (t, J=8.2 Hz, 1H), 7.70–7.67 (m, 2H).
N-(2,4-Bis(trifluoromethyl)phenyl)-5-nitroisoquinolin-1-amine (5e): Brown solid, yield 40%; 1H-NMR (DMSO-d6) δ: 9.32–9.27 (m, 2H), 9.13 (d, J=7.9 Hz, 1H), 8.59 (d, J=7.9 Hz, 1H), 8.14 (s, 1H), 8.02–7.96 (m, 3H), 7.85 (d, J=7.8 Hz, 1H).
N-(4-Morpholinophenyl)-5-nitroisoquinolin-1-amine (5f): Brown solid, yield 54%; 1H-NMR (DMSO-d6) δ: 9.38 (s, 1H), 8.90 (d, J=8.4 Hz, 1H), 8.49 (t, J=7.9 Hz, 2H), 8.11 (d, J=6.1 Hz, 1H), 8.01 (d, J=6.2 Hz, 1H), 7.75 (t, J=8.2 Hz, 1H), 7.44 (d, J=6.2 Hz, 1H), 6.96 (d, J=9.0 Hz, 2H), 3.75 (t, J=4.6 Hz, 4H), 3.08 (t, J=4.6 Hz, 4H).
N-(2-Morpholino-5-(trifluoromethyl)phenyl)-5-nitroisoquinolin-1-amine (5g): Orange solid, yield 50%; 1H-NMR (DMSO-d6) δ: 9.29 (s, 1H), 8.75 (d, J=8.4 Hz, 1H), 8.55 (d, J=7.5 Hz, 1H), 8.47 (d, J=1.8 Hz, 1H), 8.24 (d, J=6.3 Hz, 1H), 7.89 (t, J=8.1 Hz, 1H), 7.59 (d, J=6.1 Hz, 1H), 7.46 (dd, J=1.7, 1.8 Hz, 1H), 7.40 (d, J=8.4 Hz, 1H), 3.67 (t, J=4.3 Hz, 4H), 2.94 (t, J=4.5 Hz, 4H).
N1-(5-Nitroisoquinolin-1-yl)-N4-phenylbenzene-1,4-diamine (5h): Brown solid, yield 43%; 1H-NMR (DMSO-d6) δ: 9.42 (s, 1H), 8.91 (d, J=8.4 Hz, 1H), 8.49 (t, J=9.0 Hz, 2H), 8.35–8.31 (m, 2H), 8.13 (d, J=6.1 Hz, 1H), 8.07 (s, 1H), 8.01 (d, J=6.1 Hz, 1H), 7.76 (t, J=8.3 Hz, 1H), 7.69–7.64 (m, 2H), 7.21 (t, J=7.5 Hz, 3H), 6.90 (d, J=8.4 Hz, 1H).
5-Nitro-N-(3-phenoxyphenyl)isoquinolin-1-amine (5i): Orange solid, yield 28%; 1H-NMR (DMSO-d6) δ: 9.58 (s, 1H), 8.91 (d, J=8.4 Hz, 1H), 8.51 (d, J=7.8 Hz, 1H), 8.19 (dd, J=1.4, 1.5 Hz, 1H), 7.79 (t, J=8.3 Hz, 1H), 7.67 (d, J=8.1 Hz, 1H), 7.63–7.60 (m, 1H), 7.56 (d, J=6.2 Hz, 1H), 7.38–7.32 (m, 3H), 7.15 (t, J=7.2 Hz, 1H), 7.10–7.06 (m, 2H), 6.69 (d, J=8.0 Hz, 1H).
N-(Benzo[d][1,3]dioxol-5-yl)-5-nitroisoquinolin-1-amine (5j): Brown solid, yield 22%; 1H-NMR (DMSO-d6) δ: 9.43 (s, 1H), 8.89 (d, J=8.4 Hz, 1H), 8.49 (d, J=7.7 Hz, 1H), 8.15 (d, J=6.2 Hz, 1H), 7.77 (t, J=8.2 Hz, 1H), 7.49 (d, J=6.2 Hz, 1H), 7.45 (d, J=2.0 Hz, 1H), 7.16 (dd, J=2.1, 2.2 Hz, 1H), 6.91 (d, J=8.4 Hz, 1H), 6.01 (s, 2H).
N-(3-Chloro-5-(trifluoromethyl)pyridin-2-yl)-5-nitroisoquinolin-1-amine (5k): Brown solid, yield 35%; 1H-NMR (DMSO-d6) δ: 9.09 (s, 1H), 8.76–8.71 (m, 2H), 8.59–8.52 (m, 3H), 8.02 (t, J=8.1 Hz, 1H), 7.80 (t, J=8.0 Hz, 1H).
N-(4,6-Dimethylpyrimidin-2-yl)-5-nitroisoquinolin-1-amine (5l): Brown solid, yield 48%; 1H-NMR (DMSO-d6) δ: 10.15 (s, 1H), 9.57–8.53 (m, 2H), 8.45 (s, 1H), 7.99 (s, 1H), 7.74 (t, J=8.1 Hz, 1H), 6.75 (s, 1H), 2.25 (s, 6H).
5-Nitro-N-(4-(trifluoromethyl)pyrimidin-2-yl)isoquinolin-1-amine (5m): Brown solid, yield 33%; 1H-NMR (DMSO-d6) δ: 11.08 (s, 1H), 8.44 (d, J=4.9 Hz, 1H), 8.65–8.61 (m, 2H), 8.45 (br s, 1H), 8.12 (br s, 1H), 7.78–7.75 (m, 1H), 7.40 (d, J=4.9 Hz, 1H).
5-Nitro-N-(pyrazin-2-yl)isoquinolin-1-amine (5n): Brown solid, yield 45%; 1H-NMR (DMSO-d6) δ: 9.89 (s, 1H), 9.28 (br s, 1H), 9.0 (d, J=8.38 Hz, 1H), 8.6 (d, J=7.72 Hz, 1H), 8.41–8.38 (m, 1H), 8.26 (d, J=2.6 Hz, 2H), 7.80 (t, J=8.1 Hz, 1H), 7.66 (br s, 1H).
4-Methyl-N-(5-nitroisoquinolin-1-yl)thiazol-2-amine (5o): Orange solid, yield 45%; 1H-NMR (DMSO-d6) δ: 12.29 (s, 1H), 9.10 (d, J=8.2 Hz, 1H), 8.52 (d, J=7.8 Hz, 1H), 8.32 (d, J=5.4 Hz, 1H), 7.77 (t, J=8.0 Hz, 1H), 7.62 (s, 1H), 6.65 (s, 1H), 2.30 (s, 3H).
General Procedure for the Preparation of N1-Substituted isoquinolin-1,5-diamines (6a–o)A mixture of the appropriate nitroisoquinolinylamine 5a–o (0.27 mmol) and 5% Pd/C (40 mg) in MeOH (5 mL) was stirred in hydrogen atmosphere at room temperature for 2 h. Upon completion, the reaction mixture was filtered through celite. The filtrate was evaporated under reduced pressure, and the resulting residue was purified by column chromatography to afford the corresponding compounds 6a–o in 14–91% yield.
4-Chloro-8-nitroquinoline (7)To a solution of 4-chloroisoquinoline (9, 5.34 g, 32.7 mmol) in sulfuric acid (24.0 mL, 84.4 mmol) was added nitric acid (4.8 mL, 114.6 mmol) dropwise at 0°C. The mixture was stirred at room temperature for 4 h. Upon completion, the reaction mixture was cooled to 0°C and neutralized with 10% NaHCO3 aqueous solution. The resulting precipitate was collected by filtration and dried to give 7 (4.21 g, 62%) as a white solid. 1H-NMR (DMSO-d6) δ: 8.97 (d, J=4.7 Hz, 1H), 8.48 (d, J=8.5 Hz, 1H), 8.40 (d, J=7.5 Hz, 1H), 8.00 (d, J=4.7 Hz, 1H), 7.93 (t, J=8.3 Hz, 1H); 13C-NMR (CDCl3) δ: 152.0, 149.0, 143.2, 140.8, 128.2, 127.4, 126.3, 124.3, 122.9.
General Procedure for the Preparation of N5-(8-Nitroquinolin-4-yl)-N1-substituted Isoquinolin-1,5-diamines (8a–o)According to the procedure described for 5a–o, compounds 8a–o were obtained from the corresponding 6a–o. 8a: 46%; 8b: 86%; 8c: 48%; 8d: 59%; 8e: 44%; 8f: 41%; 8g: 60%; 8h: 44%; 8i: 35%; 8j: 24%; 8k: 21%; 8l: 14%; 8m: 26%; 8n: 16%; 8o: 8%.
General Procedure for the Preparation of Target Compounds (1a–o)A mixture of the appropriate nitroquinolinylisoquinolinylamine 8a–o (0.20 mmol) and 5% Pd/C (30 mg) in MeOH (5 mL) was stirred in hydrogen atmosphere at room temperature for 2 h. Upon completion, the reaction mixture was filtered through celite. The filtrate was evaporated under reduced pressure, and the resulting residue was purified by column chromatography to afford the corresponding compounds 1a–o.
N1-(3,4-Dimethylphenyl)-N5-(8-nitroquinolin-4-yl)isoquinoline-1,5-diamine (1a): Pale yellow solid, yield 83%; MS m/z: 406 [M+1]+; 1H-NMR (DMSO-d6) δ: 9.12 (s, 1H), 8.88 (s, 1H), 8.50 (t, J=4.7 Hz, 1H), 8.17 (d, J=5.1 Hz, 1H), 7.89 (d, J=5.9 Hz, 1H), 7.69–7.64 (m, 2H), 7.62–7.56 (m, 3H), 7.24 (t, J=7.9 Hz, 1H), 7.08 (d, J=8.7 Hz, 1H), 6.97 (d, J=5.9 Hz, 1H), 6.85 (d, J=7.4 Hz, 1H), 6.05 (d, J=5.1 Hz, 1H), 5.80 (s, 2H), 2.22 (d, J=13.2 Hz, 6H); 13C-NMR (DMSO-d6) δ: 153.4, 149.7, 147.1, 145.1, 141.0, 138.5, 137.9, 135.8, 135.7, 134.0, 129.8, 129.2, 127.9, 126.3, 125.6, 122.3, 121.3, 119.6, 119.3, 118.6, 109.1, 108.0, 107.4, 101.8, 19.6, 18.7.
N4-(1-(4-(Trifluoromethyl)phenylamino)isoquinolin-5-yl)quinoline-4,8-diamine (1b): Pale green solid, yield 59%; MS m/z: 446 [M+1]+; 1H-NMR (DMSO-d6) δ: 9.63 (s, 1H), 8.91 (s, 1H), 8.52 (d, J=5.3 Hz, 1H), 8.18 (d, J=4.9 Hz, 1H), 8.14 (d, J=8.2 Hz, 2H), 8.02 (d, J=5.6 Hz, 1H), 7.23 (s, 2H), 7.68 (d, J=8.2 Hz, 2H), 7.61 (d, J=8.2 Hz, 1H), 7.26 (t, J=7.4 Hz, 1H), 7.16 (d, J=5.5 Hz, 1H), 6.86 (d, J=7.3 Hz, 1H), 6.08 (d, J=4.8 Hz, 1H), 5.81 (s, 2H); 13C-NMR (DMSO-d6) δ: 152.5, 149.7, 147.1, 145.0, 144.8, 140.5, 137.9, 136.0, 134.1, 128.1, 126.8, 126.0, 125.7, 125.6, 123.3, 121.5, 121.3, 120.0, 119.6, 119.3, 109.3, 109.2, 108.0, 101.9.
N4-(1-(3-(Trifluoromethyl)phenylamino)isoquinolin-5-yl)quinoline-4,8-diamine (1c): Pale green solid, yield 86%; MS m/z: 446 [M+1]+; 1H-NMR (DMSO-d6) δ: 9.58 (s, 1H), 8.91 (s, 1H), 8.53 (d, J=6.9 Hz, 1H). 8.37 (s, 1H), 8.25 (d, J=8.0 Hz, 1H), 8.18 (d, J=4.8 Hz, 1H), 8.00 (d, J=5.7 Hz, 1H), 7.75–7.70 (m, 2H), 7.61 (d, J=8.2 Hz, 1H), 7.56 (t, J=7.6 Hz, 1H), 7.32 (d, J=7.4 Hz, 1H), 7.25 (t, J=7.8 Hz, 1H), 7.13 (d, J=5.7 Hz, 1H), 6.86 (d, J=7.3 Hz, 1H), 6.08 (d, J=4.5 Hz, 1H), 5.81 (s, 2H); 13C-NMR (DMSO-d6) δ: 153.2, 150.1, 147.5, 145.8, 142.5, 141.0, 138.6, 136.6, 134.6, 129.9, 128.4, 127.1, 126.1, 124.0, 123.6, 121.8, 120.3, 119.9, 118.1, 116.5, 110.3, 109.5, 109.3, 108.4, 102.5.
N4-(1-(4-Chloro-3-(trifluoromethyl)phenylamino)isoquinolin-5-yl)quinoline-4,8-diamine (1d): Pale green solid, yield 64%; MS m/z: 480 [M+1]+; 1H-NMR (DMSO-d6) δ: 9.78 (s, 1H), 8.93 (s, 1H), 8.52 (s, 2H), 8.34 (d, J=6.5 Hz, 1H), 8.18 (s, 1H), 8.00 (s, 1H), 7.73 (s, 2H), 7.66 (d, J=9.0 Hz, 1H), 7.61 (d, J=7.4 Hz, 1H), 7.25 (s, 1H), 7.16 (s, 1H), 6.86 (d, J=6.1 Hz, 1H), 6.07 (s, 1H), 5.81 (s. 2H); 13C-NMR (DMSO-d6) δ: 158.5, 152.4, 145.5, 144.0, 140.7, 135.4, 134.0, 131.5, 129.8, 128.4, 126.7, 126.4, 126.1, 124.4, 121.9, 121.6, 119.8, 119.1, 118.6, 110.5, 109.1, 108.7, 108.2, 107.0, 101.5.
N4-(1-(2,4-Bis(trifluoromethyl)phenylamino)isoquinolin-5-yl)quinoline-4,8-diamine (1e): Brown solid, yield 49%; MS m/z: 514 [M+1]+; 1H-NMR (DMSO-d6) δ: 9.57 (s, 1H), 9.20 (s, 1H), 9.08–9.06 (m, 2H), 8.72 (d, J=7.6 Hz, 1H), 8.22 (d, J=5.1 Hz, 1H), 8.11 (s, 1H), 7.90 (t, J=7.6 Hz, 2H), 7.84 (d, J=7.6 Hz, 1H), 7.63 (d, J=8.3 Hz, 1H), 7.35 (d, J=7.6 Hz, 1H), 7.29 (t, J=7.9 Hz, 1H), 6.88 (d, J=7.5 Hz, 1H), 6.22 (d, J=5.0 Hz, 1H), 5.84 (s, 2H).
N4-(1-(4-Morpholinophenylamino)isoquinolin-5-yl)quinoline-4,8-diamine (1f): Pale yellow solid, yield 25%; MS m/z: 463 [M+1]+; 1H-NMR (DMSO-d6) δ: 9.09 (s, 1H), 8.85 (s, 1H), 8.48–8.46 (m, 1H), 8.17 (d, J=4.3 Hz, 1H), 7.85 (d, J=5.5 Hz, 1H), 7.72–7.64 (m, 4H), 7.61 (d, J=8.7 Hz, 1H), 7.24 (t, J=7.4 Hz, 1H), 6.94 (t, J=7.9 Hz, 3H), 6.85 (d, J=7.1 Hz, 1H), 6.05 (d, J=4.1 Hz, 1H), 5.80 (s, 2H), 3.75 (t, J=4.3 Hz, 4H), 3.07 (t, J=4.5 Hz, 4H); 13C-NMR (DMSO-d6) δ: 153.6, 149.6, 147.0, 146.6, 145.3, 141.1, 138.1, 135.8, 134.1, 133.3, 127.7, 126.0, 125.5, 122.4, 121.4, 119.5, 119.3, 115.4, 108.7, 107.8, 107.0, 101.8, 66.2, 49.2.
N4-(1-(2-Morpholino-5-(trifluoromethyl)phenylamino)isoquinolin-5-yl)quinoline-4,8-diamine (1g): Pale yellow solid, yield 53%; MS m/z: 531 [M+1]+; 1H-NMR (DMSO-d6) δ: 9.24 (s, 1H), 8.98 (s, 1H), 8.93 (s, 1H), 8.28–8.23 (m, 2H), 8.09 (d, J=5.9 Hz, 1H), 7.88 (t, J=7.7 Hz, 1H), 7.79 (d, J=7.4 Hz, 1H), 7.67 (d, J=8.3 Hz, 1H), 7.52 (d, J=8.2 Hz, 1H), 7.45 (d, J=8.2 Hz, 1H), 7.31 (t, J=7.9 Hz, 1H), 7.19 (d, J=5.9 Hz, 1H), 6.92 (d, J=7.5 Hz, 1H), 6.17 (d, J=5.1 Hz, 1H), 5.87 (s, 2H), 3.88 (t, J=4.0 Hz, 4H), 3.04 (t, J=4.3 Hz, 4H); 13C-NMR (DMSO-d6) δ: 152.0, 149.4, 147.0, 145.7, 145.3, 140.7, 138.1, 136.6, 135.3, 133.8, 127.7, 127.4, 126.3, 125.6, 124.9, 124.4, 122.7, 121.1, 119.7, 119.5, 119.0, 116.5, 108.8, 107.9, 102.2, 66.7, 51.4.
N4-(1-(4-(Phenylamino)phenylamino)isoquinolin-5-yl)quinoline-4,8-diamine (1h): Brown solid, yield 64%; MS m/z: 469 [M+1]+; 1H-NMR (DMSO-d6) δ: 9.16 (s, 1H), 8.87 (s, 1H), 8.49 (t, J=4.7 Hz, 1H), 8.17 (d, J=5.1 Hz, 1H), 8.03 (s, 1H), 7.86 (d, J=6.0 Hz, 1H), 7.71 (d, J=8.8 Hz, 2H), 7.66 (d, J=4.4 Hz, 2H), 7.61 (d, J=8.3 Hz, 1H), 7.21 (m, 3H), 7.09 (d, J=8.8 Hz, 2H), 7.02 (d, J=7.8 Hz, 2H), 6.94 (d, J=6.0 Hz, 1H), 6.85 (d, J=6.9 Hz, 1H), 6.75 (t, J=7.2 Hz, 1H), 6.06 (d, J=5.2 Hz, 1H), 5.80 (s, 2H); 13C-NMR (DMSO-d6) δ: 153.5, 149.6, 147.0, 144.5, 141.1, 138.1, 137.8, 135.8, 134.1, 129.1, 127.8, 126.1, 125.5, 122.5, 122.1, 121.4, 119.5, 119.3, 118.6, 118.3, 118.1, 115.4, 108.8, 107.8, 107.1, 101.9.
N4-(1-(3-Phenoxyphenylamino)isoquinolin-5-yl)quinoline-4,8-diamine (1i): Pale brown solid, yield 68%; MS m/z: 470 [M+1]+; 1H-NMR (DMSO-d6) δ: 9.35 (s, 1H), 8.89 (s, 1H), 8.49 (s, 1H), 8.17 (d, J=4.2 Hz, 1H), 7.92 (d, J=5.3 Hz, 1H), 7.74–7.67 (m, 4H), 7.61 (d, J=8.0 Hz, 1H), 7.42–7.37 (m, 2H), 7.32 (t, J=7.9 Hz, 1H), 7.25 (t, J=7.4 Hz, 1H), 7.13 (t, J=6.7 Hz, 1H), 7.10–7.05 (m, 3H), 6.85 (d, J=6.8 Hz, 1H), 6.64 (d, J=7.3 Hz, 1H), 6.06 (d, J=4.1 Hz, 1H), 5.80 (s, 2H); 13C-NMR (DMSO-d6) δ: 157.3, 157.1, 152.3, 150.3, 147.5, 145.8, 144.2, 143.3, 141.1, 139.4, 136.5, 134.6, 130.5, 130.0, 128.4, 126.9, 126.0, 123.7, 120.3, 120.0, 119.0, 115.8, 112.2, 110.9, 109.2, 108.9, 108.4, 102.4.
N4-(1-(Benzo[d][1,3]dioxol-5-ylamino)isoquinolin-5-yl)quinoline-4,8-diamine (1j): Pale green solid, yield 94%; MS m/z: 422 [M+1]+; 1H-NMR (DMSO-d6) δ: 9.20 (s, 1H), 8.89 (s, 1H), 8.48 (d, J=4.1 Hz, 1H), 8.16 (d, J=4.9 Hz, 1H), 7.87 (d, J=5.9 Hz, 1H), 7.64–7.58 (m, 4H), 7.24 (t, J=7.8 Hz, 2H), 6.97 (d, J=5.9 Hz, 1H), 6.89–6.85 (m, 2H), 6.04 (d, J=5.1 Hz, 1H), 6.00 (s, 2H), 5.80 (s, 2H); 13C-NMR (DMSO-d6) δ: 153.4, 149.6, 147.0, 146.8, 145.3, 142.2, 140.9, 138.1, 135.9, 135.4, 134.0, 127.8, 126.2, 125.5, 121.3, 119.5, 119.4, 113.8, 108.8, 107.9, 107.7, 107.5, 103.5, 101.9, 100.7.
N4-(1-(3-Chloro-5-(trifluoromethyl)pyridin-2-ylamino)isoquinolin-5-yl)quinoline-4,8-diamine (1k): Pale green solid, yield 47%; MS m/z: 481 [M+1]+; 1H-NMR (DMSO-d6) δ: 8.90 (s, 1H), 8.75 (s, 1H), 8.55 (s, 1H), 8.26–8.22 (m, 2H), 7.73–7.64 (m, 5H), 7.26 (s, 1H), 6.87 (m, 2H), 6.11 (s, 1H), 5.82 (s, 2H).
N4-(1-(4,6-Dimethylpyrimidin-2-ylamino)isoquinolin-5-yl)quinoline-4,8-diamine (1l): Pale green solid, yield 27%; MS m/z: 408 [M+1]+; 1H-NMR (DMSO-d6) δ: 9.89 (s, 1H), 8.94 (s, 1H), 8.20–8.16 (m, 2H), 8.07 (d, J=8.2 Hz, 1H), 7.70 (d, J=7.5 Hz, 1H), 7.65–7.61 (m, 2H), 7.42 (d, J=5.8 Hz, 1H), 7.26 (t, J=7.7 Hz, 1H), 6.86 (d, J=7.0 Hz, 1H), 6.70 (s, 1H), 6.08 (d, J=5.1 Hz, 1H), 5.81 (s, 2H), 2.25 (s, 6H).
N4-(1-(4-(Trifluoromethyl)pyrimidin-2-ylamino)isoquinolin-5-yl)quinoline-4,8-diamine (1m): Pale yellow solid, yield 80%; MS m/z: 448 [M+1]+; 1H-NMR (DMSO-d6) δ: 10.89 (s, 1H), 8.96 (s, 1H), 8.76 (d, J=4.8 Hz, 1H), 8.20 (d, J=5.1 Hz, 2H), 8.16 (br s, 1H), 7.74 (d, J=6.8 Hz, 1H), 7.68 (t, J=8.1 Hz, 1H), 7.62 (d, J=8.3 Hz, 1H), 7.45 (br s, 1H), 7.31 (d, J=4.8 Hz, 1H), 7.26 (t, J=7.9 Hz, 1H), 6.86 (d, J=7.5 Hz, 1H), 6.11 (d, J=5.1 Hz, 1H), 5.82 (s, 2H).
N4-(1-(Pyrazin-2-ylamino)isoquinolin-5-yl)quinoline-4,8-diamine (1n): Orange solid, yield 74%; MS m/z: 380 [M+1]+; 1H-NMR (DMSO-d6) δ: 10.15 (s, 1H), 9.47 (s, 1H), 8.93 (s, 1H), 8.56 (d, J=4.6 Hz, 1H), 8.38 (s, 1H), 8.23 (s, 1H), 8.18 (d, J=5.0 Hz, 1H), 8.07 (d, J=5.6 Hz, 1H), 7.74–7.71 (m, 2H), 7.61 (d, J=8.8 Hz, 1H), 7.27 (d, J=5.7 Hz, 2H), 6.86 (d, J=7.4 Hz, 1H), 6.09 (s, 1H), 5.82 (s, 2H).
N4-(1-(4-Methylthiazol-2-ylamino)isoquinolin-5-yl)quinoline-4,8-diamine (1o): Pale green solid, yield 38%; MS m/z: 399 [M+1]+; 1H-NMR (DMSO-d6) δ: 11.76 (s, 1H), 8.91 (s, 1H), 8.70 (s, 1H), 8.17 (s, 1H), 8.09 (br s, 1H), 7.71 (s, 2H), 7.60 (d, J=7.5 Hz, 1H), 7.25 (t, J=6.8 Hz, 1H), 7.16 (br s, 1H), 6.86 (d, J=6.5 Hz, 1H), 6.66 (s, 1H), 6.06 (s, 1H), 5.81 (s, 2H), 2.32 (s, 3H); 13C-NMR (DMSO-d6) δ: 158.7, 151.4, 150.0, 147.5, 145.8, 141.7, 140.9, 138.6, 136.5, 134.3, 128.6, 127.5, 126.1, 121.9, 119.9, 109.3, 108.3, 106.9, 102.5, 17.3.
5-Nitroisoquinolin-1-amine (10)1-Chloro-5-nitroisoquinoline (4) (500 mg, 2.40 mmol) was added to ammonia (7 N solution in EtOH) (10 mL), and the mixture was refluxed for 40 h. After completion of the reaction, the solvent was removed under reduced pressure. The residue was purified by column chromatography (silica gel, ethyl acetate–hexane 2 : 1 v/v) to give 10 (350 mg, 77%) as an orange solid. 1H-NMR (CDCl3) δ: 8.41 (d, J=7.8 Hz, 1H), 8.16–8.13 (m, 2H), 7.76 (d, J=6.3 Hz, 1H), 7.59 (t, J=7.7 Hz, 1H), 5.32 (s, 2H); 13C-NMR (100 MHz, DMSO-d6) δ: 167.4, 157.8, 146.1, 144.4, 129.3, 127.6, 118.0, 104.1, 102.2.
General Procedure for the Preparation of N-(5-Nitroisoquinolin-1-yl)-substituted 2-Carboxamides (11a–m)A mixture of compound 10 (21 mg, 0.11 mmol), HBTU (102 mg, 0.27 mmol), and N,N-diisopropylethylamine (71 mg, 0.55 mmol) in dry THF (5 mL) was stirred at 0°C for 30 min. The appropriate acid (0.22 mmol) was then added. After stirring under reflux for 30 h, the reaction mixture was diluted with saturated aqueous NaHCO3, and extracted with ethyl acetate. The organic layer was washed with brine, dried over anhydrous MgSO4, and concentrated in vacuo. The residue was purified by column chromatography to afford the corresponding compounds 11a–m.
3,4-Dimethoxy-N-(5-nitroisoquinolin-1-yl)benzamide (11a): Yellow solid, yield 29%; 1H-NMR (DMSO-d6) δ: 11.09 (s, 1H), 8.63 (s, 1H), 8.33–8.30 (m, 1H), 7.81–7.74 (m, 4H), 7.44 (s, 1H), 7.12 (s, 1H), 3.86 (s, 6H).
3,5-Dimethoxy-N-(5-nitroisoquinolin-1-yl)benzamide (11b): Yellow solid, yield 41%; 1H-NMR (400 MHz, DMSO-d6) δ: 10.92 (s, 1H), 8.64 (d, J=7.7 Hz, 1H), 7.93–7.88 (m, 2H), 7.46–7.41 (m, 2H), 7.31 (s, 1H), 6.77 (t, J=4.5 Hz, 1H), 6.41 (d, J=2.2 Hz, 1H), 3.76 (s, 6H).
4-Methyl-N-(5-nitroisoquinolin-1-yl)benzamide (11c): Yellow solid, yield 47%; 1H-NMR (DMSO-d6) δ: 11.14 (s, 1H), 8.64 (d, J=6.2 Hz, 2H), 8.42 (d, J=8.3 Hz, 1H), 8.24 (d, J=6.0 Hz, 1H), 8.00 (d, J=7.8 Hz, 2H), 7.84 (t, J=8.3 Hz, 1H), 7.38 (d, J=7.7 Hz, 2H), 2.42 (s, 3H).
N-(5-Nitroisoquinolin-1-yl)-3-(trifluoromethyl)benzamide (11d): Yellow solid, yield 31%; 1H-NMR (DMSO-d6) δ: 8.66 (dd, J=0.9, 1.0 Hz, 2H), 8.49 (s, 3H), 8.02 (d, J=7.5 Hz, 2H), 7.89 (d, J=8.0 Hz, 1H), 8.85 (d, J=5.3 Hz, 1H), 7.81 (d, J=7.7 Hz, 1H).
4-Chloro-N-(5-nitroisoquinolin-1-yl)-3-(trifluoromethyl)benzamide (11e): Yellow solid, yield 65%; 1H-NMR (CDCl3) δ: 15.3 (br s, 1H), 9.38 (s, 1H), 8.76 (s, 1H), 8.58 (d, J=6.9 Hz, 1H), 8.50 (d, J=8.1 Hz, 1H), 7.91 (d, J=15.0 Hz, 1H), 7.82 (t, J=8.1 Hz, 1H), 7.63 (d, J=8.1 Hz, 2H).
N-(5-Nitroisoquinolin-1-yl)-2,4-bis(trifluoromethyl)benzamide (11f): Pale yellow solid, yield 35%; 1H-NMR (DMSO-d6) δ: 11.72 (s, 1H), 8.83–8.81 (m, 1H), 8.66 (d, J=7.7 Hz, 1H), 8.60 (br s, 1H), 8.22 (s, 2H), 8.14 (s, 2H), 7.91 (t, J=8.1 Hz, 1H).
N-(5-Nitroisoquinolin-1-yl)-3,5-bis(trifluoromethyl)benzamide (11g): Yellow solid, yield 69%; 1H-NMR (CDCl3) δ: 15.63 (s, 1H), 9.40 (d, J=8.1 Hz, 1H), 8.86 (s, 2H), 8.61 (dd, J=1.3, 1.4 Hz, 1H), 8.04 (s, 1H), 7.93 (d, J=6.9 Hz, 1H), 7.86–7.83 (m, 1H), 7.67–7.65 (m, 1H).
3-Morpholino-N-(5-nitroisoquinolin-1-yl)-5-(trifluoromethyl)benzamide (11h): Yellow solid, yield 45%; 1H-NMR (DMSO-d6) δ: 11.41 (s, 1H), 8.66 (s, 2H), 8.46 (s, 1H), 8.28 (s, 1H), 7.87–7.82 (m, 3H), 7.46 (s, 1H), 3.79 (t, J=4.4 Hz, 4H), 3.32 (t, J=4.8 Hz, 4H).
N-(5-Nitroisoquinolin-1-yl)-2,3-dihydrobenzo[b][1,4]dioxine-6-carboxamide (11i): Yellow solid, yield 32%; 1H-NMR (DMSO-d6) δ: 11.05 (s, 1H), 8.63 (d, J=7.2 Hz, 1H), 8.17 (d, J=8.4 Hz, 1H), 7.89 (d, J=8.3 Hz, 1H), 7.80 (dd, J=2.1, 2.2 Hz, 1H), 7.74 (d, J=2.1 Hz, 1H), 7.66 (d, J=7.5 Hz, 1H), 7.54–7.51 (m, 1H), 7.18 (d, J=8.5 Hz, 1H), 4.44–4.36 (m, 4H).
4,7-Dimethoxy-N-(5-nitroisoquinolin-1-yl)-1-naphthamide (11j): Yellow solid, yield 23%; 1H-NMR (DMSO-d6) δ: 11.41 (s, 1H), 8.65 (d, J=7.6 Hz, 1H), 8.56 (br s, 1H), 8.18 (d, J=9.2 Hz, 3H), 8.06 (br s, 1H), 7.90–7.85 (m, 2H), 7.22 (dd, J=2.5, 2.6 Hz, 1H), 6.98 (d, J=8.2 Hz, 1H), 4.05 (s, 3H), 3.80 (s, 3H).
2,5-Dimethyl-N-(5-nitroisoquinolin-1-yl)furan-3-carboxamide (11k): Pale yellow solid, yield 17%; 1H-NMR (DMSO-d6) δ: 10.65 (s, 1H), 8.65–8.61 (m, 2H), 8.38 (d, J=8.4 Hz, 1H), 8.23 (d, J=6.0 Hz, 1H), 7.85 (t, J=8.1 Hz, 1H), 6.76 (s, 1H), 2.49 (s, 3H), 2.30 (s, 3H).
N-(5-Nitroisoquinolin-1-yl)thiazole-4-carboxamide (11l): Yellow solid, yield 31%; 1H-NMR (DMSO-d6) δ: 11.11 (s, 1H), 9.33 (s, 1H), 8.66 (d, J=7.5 Hz, 2H), 8.61 (s, 1H), 8.47 (d, J=8.3 Hz, 1H), 8.27 (d, J=6.0 Hz, 1H), 7.86 (t, J=8.0 Hz, 1H).
1-(4-Methoxyphenyl)-N-(5-nitroisoquinolin-1-yl)-5-(trifluoromethyl)-1H-pyrazole-4-carboxamide (11m): Yellow solid, yield 50%; 1H-NMR (DMSO-d6) δ: 11.42 (s, 1H), 8.64 (d, J=7.6 Hz, 1H), 8.54–8.47 (m, 3H), 8.46 (s, 1H), 7.88 (t, J=8.0 Hz, 1H), 7.48 (d, J=8.7 Hz, 2H), 7.12 (d, J=8.8 Hz, 2H).
General Procedure for the Preparation of N-(5-Aminoisoquinolin-1-yl)-substituted 2-Carboxamides (12a–m)According to the procedure described for 6a–o, compounds 12a–m were obtained from the corresponding 11a–m in 38–85% yield.
General Procedure for the Preparation of N-(5-((8-Nitroquinolin-4-yl)amino)isoquinolin-1-yl)-substituted 2-Carboxamides (13a–m)According to the procedure described for 5a–o, compounds 13a–m were obtained from the corresponding 12a–m. 13a: 27%; 13b: 25%; 13c: 22%; 13d: 25%; 13e: 23%; 13f: 28%; 13g: 19%; 13h: 22%; 13i: 25%; 13j: 31%; 13k: 26%; 13l: 20%; 13m: 17%.
General Procedure for the Preparation of Target Compounds (2a–m)The target compounds 2a–m were prepared from the corresponding 13a–m as described for 1a–o.
N-(5-(8-Aminoquinolin-4-ylamino)isoquinolin-1-yl)-3,4-dimethoxybenzamide (2a): Brown solid, yield 16%; MS m/z: 466 [M+1]+; 1H-NMR (DMSO-d6) δ: 10.91 (s, 1H), 8.99 (s, 1H), 8.20 (d, J=5.1 Hz, 1H), 7.79–7.71 (m, 7H), 7.62 (d, J=8.4 Hz, 1H), 7.27 (t, J=7.8 Hz, 1H), 7.12 (d, J=8.5 Hz, 1H), 6.87 (d, J=6.9 Hz, 1H), 6.12 (d, J=5.1 Hz, 1H), 5.82 (s, 2H), 3.86 (s, 6H); 13C-NMR (DMSO-d6) δ: 167.0, 162.7, 155.8, 150.3, 146.1, 140.8, 134.2, 130.8, 130.1, 129.6, 129.0, 128.5, 127.3, 127.1, 126.9, 126.6, 125.2, 125.0, 124.6, 123.7, 123.3, 122.9, 121.0, 115.8, 109.8, 56.5.
N-(5-(8-Aminoquinolin-4-ylamino)isoquinolin-1-yl)-3,5-dimethoxybenzamide (2b): Brown solid, yield 9%; MS m/z: 466 [M+1]+; 1H-NMR (DMSO-d6) δ: 10.97 (s, 1H), 8.96 (s, 1H), 8.20 (d, J=5.1 Hz, 2H), 7.75–7.71 (m, 3H), 7.62 (d, J=8.0 Hz, 1H), 7.35 (s, 1H), 7.26 (t, J=7.7 Hz, 1H), 7.08–7.06 (m, 1H), 6.87 (d, J=7.6 Hz, 1H), 6.73 (s, 1H), 6.11 (d, J=5.1 Hz, 1H), 5.82 (s, 2H); 13C-NMR (DMSO-d6) δ: 166.9, 161.6, 160.8, 157.7, 152.6, 150.0, 148.0, 146.0, 144.7, 140.6, 140.0, 138.3, 135.4, 132.6, 128.1, 126.8, 125.0, 124.4, 122.5, 118.5, 117.2, 108.8, 105.7, 56.9.
N-(5-(8-Aminoquinolin-4-ylamino)isoquinolin-1-yl)-4-methylbenzamide (2c): Brown solid, yield 27%; MS m/z: 420 [M+1]+; 1H-NMR (DMSO-d6) δ: 10.94 (s, 1H), 8.99 (s, 1H), 8.34 (s, 1H), 8.21 (d, J=4.8 Hz, 1H), 8.04–8.01 (m, 1H), 7.93 (d, J=8.5 Hz, 1H), 7.73–7.66 (m, 5H), 7.40–7.36 (m, 2H), 7.27 (t, J=6.8 Hz, 1H), 6.87 (d, J=7.4 Hz, 1H), 6.13 (s, 1H), 5.82 (s, 2H), 2.42 (s, 3H); 13C-NMR (DMSO-d6) δ: 166.9, 160.5, 152.6, 150.0, 149.0, 146.6, 144.7, 142.0, 139.0, 138.0, 134.0, 130.6, 129.8, 127.7, 126.2, 125.0, 124.6, 123.7, 122.5, 118.2, 117.6, 112.6, 107.7, 22.7.
N-(5-(8-Aminoquinolin-4-ylamino)isoquinolin-1-yl)-3-(trifluoromethyl)-benzamide (2d): Green solid, yield 57%; MS m/z: 474 [M+1]+; 1H-NMR (DMSO-d6) δ: 8.95 (s, 1H), 8.57–8.52 (m, 2H), 8.48 (br s, 1H), 8.19 (t, J=8.4 Hz, 2H), 8.05 (d, J=5.2 Hz, 1H), 7.92–7.88 (m, 2H), 7.75–7.70 (m, 2H), 7.63 (d, J=7.3 Hz, 1H), 7.26 (t, J=7.8 Hz, 2H), 6.86 (d, J=7.3 Hz, 1H), 6.08 (d, J=4.9 Hz, 1H), 5.81 (s, 2H); 13C-NMR (DMSO-d6) δ: 166.3, 149.6, 147.0, 145.3, 138.1, 135.7, 135.1, 134.2, 132.4, 131.5, 129.6, 129.3, 128.9, 128.8, 127.8, 127.0, 126.1, 125.6, 125.3, 124.8, 124.6, 124.1, 119.4, 108.8, 107.9, 101.9.
N-(5-(8-Aminoquinolin-4-ylamino)isoquinolin-1-yl)-4-chloro-3-(trifluoromethyl)benzamide (2e): Brown solid, yield 91%; MS m/z: 508 [M+1]+; 1H-NMR (CDCl3) δ: 15.61 (s, 1H), 8.98 (d, J=7.8 Hz, 1H), 8.81 (s, 1H), 8.55 (d, J=8.9 Hz, 1H), 8.38 (d, J=5.2 Hz, 1H), 7.83 (d, J=7.5 Hz, 1H), 7.78–7.75 (m, 2H), 7.63 (d, J=8.0 Hz, 1H), 7.41–7.36 (m, 2H), 7.17 (d, J=7.6 Hz, 1H), 6.98 (d, J=7.4 Hz, 1H), 6.65 (s, 1H), 6.32 (s, 1H), 5.06 (s, 2H); 13C-NMR (DMSO-d6) δ: 166.8, 165.1, 150.2, 149.3, 146.3, 142.6, 140.8, 139.8, 137.9, 135.9, 133.0, 130.8, 128.5, 127.6, 125.3, 122.9, 122.0, 119.7, 116.6, 112.8, 110.8, 108.2, 106.8, 104.2, 103.9, 102.2.
N-(5-(8-Aminoquinolin-4-ylamino)isoquinolin-1-yl)-2,4-bis(trifluoromethyl)benzamide (2f): Yellow solid, yield 79%; MS m/z: 542 [M+1]+; 1H-NMR (DMSO-d6) δ: 12.06 (s, 1H), 9.18 (s, 1H), 8.29 (d, J=6.7 Hz, 1H), 8.23–8.16 (m, 3H), 8.11 (d, J=4.8 Hz, 2H), 7.79–7.76 (m, 2H), 7.61 (d, J=8.2 Hz, 1H), 7.46 (s, 1H), 7.26 (t, J=7.6 Hz, 1H), 6.86 (d, J=7.3 Hz, 1H), 6.10 (d, J=4.9 Hz, 1H), 5.82 (s, 2H); 13C-NMR (DMSO-d6) δ: 167.7, 149.5, 147.0, 145.3, 138.1, 136.2, 134.2, 130.3, 130.1, 129.7, 129.5, 128.5, 127.6, 127.2, 126.9, 126.8, 125.6, 125.0, 125.0, 123.5, 123.4, 123.3, 121.3, 119.5, 108.8, 107.9, 102.1.
N-(5-(8-Aminoquinolin-4-ylamino)isoquinolin-1-yl)-3,5-bis(trifluoromethyl)benzamide (2g): Pale brown solid, yield 92%; MS m/z: 542 [M+1]+; 1H-NMR (CDCl3) δ: 14.31 (s, 1H), 8.97 (d, J=7.9 Hz, 1H), 8.89 (s, 2H), 8.35 (d, J=5.0 Hz, 1H), 8.00 (s, 1H), 7.83 (d, J=7.1 Hz, 1H), 7.78 (d, J=8.0 Hz, 1H), 7.46 (d, J=7.1 Hz, 1H), 7.35–7.31 (m, 2H), 7.20 (d, J=6.9 Hz, 1H), 6.96 (d, J=6.2 Hz, 1H), 6.81 (s, 1H), 6.30 (d, J=4.9 Hz, 1H), 5.06 (s, 2H); 13C-NMR (DMSO-d6) δ: 167.3, 165.8, 152.3, 152.0, 148.0, 145.7, 144.0, 140.5, 137.9, 135.0, 132.8, 130.9, 127.7, 125.9, 125.5, 123.6, 122.9, 120.6, 119.9, 115.7, 110.0, 107.8, 106.8, 102.9.
N-(5-(8-Aminoquinolin-4-ylamino)isoquinolin-1-yl)-3-morpholino-5-(trifluoromethyl)benzamide (2h): Brown solid, yield 21%; MS m/z: 559 [M+1]+; 1H-NMR (DMSO-d6) δ: 8.95 (s, 1H), 8.23 (d, J=5.2 Hz, 1H), 8.12 (br s, 1H), 8.02 (s, 1H), 7.91 (s, 1H), 7.71 (m, 4H), 7.62 (d, J=8.2 Hz, 1H), 7.42–7.37 (m, 2H), 7.26 (t, J=7.8 Hz, 1H), 6.86 (d, J=7.4 Hz, 1H), 6.09 (d, J=5.0 Hz, 1H), 5.82 (s, 2H), 3.80 (t, J=4.2 Hz, 4H), 3.32 (t, J=3.8 Hz, 4H); 13C-NMR (DMSO-d6) δ: 165.9, 164.8, 150.8, 149.5, 146.9, 145.8, 140.3, 138.7, 136.2, 134.3, 130.6, 130.2, 129.6, 129.2, 128.2, 128.1, 127.3, 126.6, 126.1, 124.4, 124.1, 123.3, 120.0, 118.4, 112.8, 107.8, 66.8. 54.5.
N-(5-(8-Aminoquinolin-4-ylamino)isoquinolin-1-yl)-2,3-dihydrobenzo[b][1,4]dioxine-6-carboxamide (2i): Pale brown solid, yield 42%; MS m/z: 464 [M+1]+; 1H-NMR (DMSO-d6) δ: 10.71 (s, 1H), 8.97 (s, 1H), 8.19 (d, J=4.8 Hz, 2H), 8.17–8.15 (m, 1H), 7.77–7.70 (m, 5H), 7.62 (d, J=8.4 Hz, 1H), 7.26 (t, J=8.0 Hz, 1H), 6.99 (d, J=8.2 Hz, 1H), 6.86 (d, J=7.3 Hz, 1H), 6.10 (d, J=4.9 Hz, 1H), 5.82 (s, 2H), 4.36–4.29 (m, 4H); 13C-NMR (DMSO-d6) δ: 166.5, 164.7, 149.6, 147.1, 146.8, 145.3, 143.0, 142.9, 138.1, 136.5, 135.6, 134.2, 127.5, 127.4, 127.0, 125.6, 124.3, 122.5, 119.4, 117.3, 116.7, 114.8, 110.0, 109.0, 108.8, 107.9, 102.0.
N-(5-(8-Aminoquinolin-4-ylamino)isoquinolin-1-yl)-4,7-dimethoxy-1-naphthamide (2j): Pale brown solid, yield 31%; MS m/z: 516 [M+1]+; 1H-NMR (DMSO-d6) δ: 11.01 (s, 1H), 9.01 (s, 1H), 8.30 (d, J=7.2 Hz, 1H), 8.23–8.16 (m, 3H), 8.08–8.01 (m, 3H), 7.81–7.75 (m, 2H), 7.64 (d, J=8.3 Hz, 1H), 7.27 (t, J=7.7 Hz, 1H), 7.22 (dd, J=2.2, 2.4 Hz, 1H), 6.98 (d, J=8.1 Hz, 1H), 6.87 (d, J=7.6 Hz, 1H), 6.13 (d, J=5.1 Hz, 1H), 5.83 (s, 2H), 4.05 (s, 3H), 3.83 (s, 3H); 13C-NMR (DMSO-d6) δ: 169.8, 165.7, 160.8, 158.7, 152.6, 150.0, 145.1, 140.9, 138.0, 135.7, 134.4, 132.4, 131.5, 129.7, 129.1, 128.9, 128.8, 127.2, 127.0, 126.6, 125.4, 125.3, 124.0, 123.6, 122.6, 120.4, 115.6, 110.3, 105.9, 56.8, 55.5.
N-(5-(8-Aminoquinolin-4-ylamino)isoquinolin-1-yl)-2,5-dimethylfuran-3-carboxamide (2k): Brown solid, yield 32%; MS m/z: 424 [M+1]+; 1H-NMR (DMSO-d6) δ: 10.43 (s, 1H), 8.96 (s, 1H), 8.19 (d, J=5.0 Hz, 2H), 7.74–7.67 (m, 3H), 7.61 (d, J=8.3 Hz, 1H), 7.34 (br s, 1H), 7.26 (t, J=8.0 Hz, 1H), 6.86 (d, J=7.4 Hz, 1H), 6.68 (br s, 1H), 6.09 (d, J=5.0 Hz, 1H), 5.82 (s, 2H), 2.52 (s, 3H), 2.29 (s, 3H); 13C-NMR (DMSO-d6) δ: 165.8, 161.8, 159.0, 155.6, 153.9, 150.87, 148.1, 146.8, 142.3, 137.8, 135.0, 131.3, 128.7, 127.9, 125.7, 123.2, 120.5, 118.6, 117.7, 113.3, 110.0, 108.3, 107.9, 13.2, 11.6.
N-(5-(8-Aminoquinolin-4-ylamino)isoquinolin-1-yl)thiazole-4-carboxamide (2l): Pale yellow solid, yield 17%; MS m/z: 413 [M+1]+; 1H-NMR (DMSO-d6) δ: 10.89 (s, 1H), 9.33 (s, 1H), 8.99 (s, 1H), 8.59 (s, 1H), 8.34 (s, 1H), 8.21 (d, J=5.1 Hz, 1H), 7.97 (d, J=7.3 Hz, 1H), 7.77–7.72 (m, 2H), 7.69–7.66 (m, 1H), 7.62 (d, J=8.9 Hz, 1H), 7.27 (t, J=7.8 Hz, 1H), 6.87 (d, J=7.6 Hz, 1H), 6.13 (d, J=5.2 Hz, 1H), 5.82 (s, 2H); 13C-NMR (DMSO-d6) δ: 162.6, 155.4, 149.0, 146.5, 140.5, 135.2, 130.8, 129.9, 129.4, 128.9, 127.6, 127.1, 126.1, 126.0, 125.2, 124.9, 124.0, 123.8, 122.4, 120.6, 114.7, 108.7.
N-(5-(8-Aminoquinolin-4-ylamino)isoquinolin-1-yl)-1-(4-methoxyphenyl)-5-(trifluoromethyl)-1H-pyrazole-4-carboxamide (2m): Pale brown solid, yield 18%; MS m/z: 570 [M+1]+; 1H-NMR (DMSO-d6) δ: 12.72 (s, 1H), 8.98 (s, 1H), 8.43 (s, 1H), 8.32 (s, 1H), 8.20 (d, J=5.1 Hz, 1H), 8.07–8.04 (m, 1H), 7.78 (d, J=7.9 Hz, 2H), 7.61 (d, J=8.2 Hz, 1H), 7.49 (d, J=8.9 Hz, 2H), 7.27 (t, J=7.8 Hz, 1H), 7.13 (d, J=8.9 Hz, 2H), 7.10–7.07 (m, 1H), 6.87 (d, J=7.5 Hz, 1H), 6.12 (d, J=5.1 Hz, 1H), 5.82 (s, 2H), 3.86 (s, 3H); 13C-NMR (DMSO-d6) δ: 167.3, 160.5, 155.1, 152.6, 150.0, 149.0, 146.1, 142.5, 138.4, 134.6, 130.3, 129.9, 129.5, 128.0, 127.7, 127.3, 126.9, 126.1, 125.2, 125.1, 124.8, 123.7, 123.7, 122.0, 120.6, 118.0, 115.6, 108.4, 56.2.
Evaluation of the Antiproliferative ActivityA375P cells were purchased from American Type Culture Collection (ATCC, Rockville, MD, U.S.A.) and maintained in Dulbecco’s modified Eagle’s medium (DMEM, Welgene, Daegu, Korea) supplemented with 10% foetal bovine serum (FBS, Welgene, Daegu, Korea) and 1% penicillin/streptomycin (Welgene, Daegu, Korea) in a humidified atmosphere with 5% CO2 at 37°C. A375P cells were taken from culture substrate with 0.05% trypsin–0.02% ethylenediaminetetraacetic acid (EDTA) and plated at a density of 5×103 cells/well in 96 well plates and then incubated at 37°C for 24 h in a humidified atmosphere with 5% CO2 prior to treatment with various concentrations (3-fold serial dilution, 12 points) of test compounds. The cells were incubated for 48 h after treatment with the test compounds. The A357P cell viability was assessed by the conventional 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay. MTT assays were carried out with CellTiter 96® (Promega) according to the manufacturer’s instructions. The absorbance at 590 nm was recorded using EnVision 2103 (Perkin Elmer, Boston, MA, U.S.A.). The IC50 was calculated using GraphPad Prism 4.0 software.
Kinase ScreeningReaction Biology Corp. Kinase HotSpotSM service28) was used for screening of compound 1d, and IC50 Profiler Express for IC50 measurement. Assay protocol: In a final reaction volume of 25 µL, kinase (5–10 mU) is incubated with 25 mM Tris pH 7.5, 0.02 mM ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA), 0.66 mg/mL myelin basic protein, 10 mM magnesium acetate and [γ33P-ATP] (specific activity approx. 500 cpm/pmol, concentration as required). The reaction is initiated by the addition of the Mg-ATP mix. After incubation for 40 min at room temperature, the reaction is stopped by the addition of 5 µL of a 3% phosphoric acid solution. Ten microliter of the reaction is then spotted onto a P30 filtermat and washed three times for 5 min in 75 mM phosphoric acid and once in methanol prior to drying and scintillation counting.
We are grateful to the Korea Institute of Science and Technology (KIST) and Korea Research Council of Fundamental Science and Technology (KRCF) for financial support.
