Introduction

Cancer is one of the major health concerns in the world that represents the leading cause of death worldwide as evidenced by 8.2 million cancer-related deaths in 2012 (WHO, 2015). Because of high cancer morbidity and mortality, new approaches in cancer therapy are needed that require development of novel, more effective antitumor agents (Chong and Jänne, 2013; Holohan et al., 2013). Modification of known, clinically approved drugs, is one of the possible strategies in drug discovery. In the present study, we have directed our attention to sorafenib, the first multikinase inhibitor approved in 2005 for the therapy of advanced renal cell carcinoma and hepatocellular carcinoma. Sorafenib supresses tumor growth through the inhibition of C-RAF and B-RAF serine/threonine kinase activities and tumor angiogenesis by abrogating vascular endothelial growth factor receptor (VEGFR) and platelet-derived growth factor receptors signaling (Wilhelm et al., 2006, 2008). Because of its multi-antitumoral mechanisms, a broad spectrum of anticancer activity, good results in combination trials, poor physicochemical properties, inactivity against mutated BRAF, toxicity and drug resistance (Schutz et al., 2011; Villaneuva and Llovet, 2012), many research efforts have been focused on the optimization of sorafenib molecule (Sun et al., 2010; Yao et al., 2012; Babić et al., 2012; Zhan et al., 2012; Lu et al., 2014; Qin et al., 2014; Jiao et al., 2015). As a result, a closely related analog of sorafenib, regorafenib, was registered in 2012, and is currently approved for the treatment of metastatic colorectal cancer and advanced gastrointestinal stromal tumors (Crona et al., 2013).

The widespread use of pyrazine heterocycle as an important pharmacophore in medicinal chemistry establishes this moiety as a member of a privileged structures class. As a part of ongoing studies focused on the development of new heterocycle anticancer agents, novel sorafenib analogs bearing pyrazine scaffold were prepared. Herein, we report their synthesis, chemical characterization, in vitro screening, and the effects on the activity of known sorafenib target, namely, c-Raf kinase.

Results and discussion

Chemistry

Novel compounds 6a–i described in this paper are derived from the cytostatic drug sorafenib. Crucial parts of the structure (diarylurea, ether, heterocycle, amide) are preserved, but a different heterocycle (pyrazine instead of pyridine) and various substituents in urea and/or amide regions were introduced. Comparison of the title sorafenib analogs and the parent molecule is given in Fig. 1.

Fig. 1
figure 1

Comparison of sorafenib analogs 6a–i and the parent molecule. The regions exposed to changes are cycled

Full size image

A pyridine heterocycle was replaced by its isoster pyrazine in the hope that the additional nitrogen atom would increase the binding interactions between the drug and c-Raf kinase. The central bis-aryl urea and the amide functionalities were left intact, since it was shown that they played key roles in the interaction with Raf (Wan et al., 2004), but various substituents in these two regions were introduced.

Majority of the pyrazine sorafenib analogs are fully in agreement with the Lipinski’s and Gelovani’s rules for prospective small molecular drugs (MW ≤ 500, log P ≤ 5, number of H-bond donors ≤ 5, number of H-bond acceptors ≤ 10, molecular polar surface area < 140 Å2, molar refractivity within the range of 40 and 130 cm3/mol, the number of atoms 20–70) and some analogs showed minimal aberrations of the rules (Luzina and Popov, 2012). The parameters are calculated with Chemicalize.org program and presented in Table 1.

Table 1 Properties of pyrazine sorafenib analogs 6a–i: the Lipinski’s and Gelovani’s parameters
Full size table

Two alternative strategies for the preparation of the title compounds 6a–i were applied (Scheme 1). The first strategy involved ether formation between 4-hydroxyphenyl urea 3a–c and 5-chloro-pyrazine-2-carboxamides 4a–f. The reactions were performed in dimethylformamide (DMF) in the presence of K2CO3. Ureas 3 were prepared from N-(4-hydroxyphenyl)-1H-benzo[d][1,2,3]triazole-1-carboxamide (2) and the corresponding amine [cyclopentylamine, 4-bromoaniline or 4-chloro-3-(trifluoromethyl)aniline], while compound 2 was synthesized from BtcCl (1) and 4-aminophenol according to our slightly modified procedure (Butula et al., 1978). On the other hand, amides 4 were obtained by the reaction of the corresponding amine [methylamine, ethanolamine, cyclopentylamine, cyclohexylamine, O-benzylhydroxylamine, or 4-chloro-3-(trifluoromethyl)aniline] with 5-chloropyrazine-2-carboxylic chloride, prepared from 5-hydroxy-pyrazine-2-carboxylic acid and thionyl chloride. The amidation step was performed at room temperature in the presence of triethylamine (TEA) as HCl acceptor.

Scheme 1
scheme 1

Synthetic pathways of intermediates 2–5 and target compounds 6a–i

Full size image

In the second strategy, leading to sorafenib pyrazine analogs 6, ether functionality was introduced in the molecule before the urea moiety. Amides 4 were first converted to 5-(4-aminophenoxy)-N-alkylpyrazine-2-carboxamides 5. Ether bond formation was performed by the reaction of precursors 4 with p-aminophenol in the presence of K2CO3 and potassium t-butoxyde. Under the basic conditions employed, the alkoxyde derived from 4-aminophenol was a stronger nucleophile than the amino group; therefore the main product was the ether, rather than the secondary amine. The last step in the preparation of products 6 involved urea formation between the amino group of compounds 5 and 4-chloro-3-(fluoromethyl)phenyl isocyanate. Urea bond formation was performed at room temperature, using a slight excess of isocyanate.

Preparation of compounds 3b and 3c has been previously published (Winum et al., 2012; Yang et al., 2013), but their analytical and spectral data were not given. Compound 4a is commercially available and all other compounds are new compounds. They are isolated and fully characterized by the usual spectroscopic methods [infrared (IR), 1H-, 13C nuclear magnetic resonance (NMR), and MS] and elemental analyses. Spectral data are consistent with the proposed structures and are given in short in the Experimental section and in detail in Supporting information. As could be expected, the signals of carbon atoms bearing three fluoro atoms and aromatic carbon atoms adjacent to CF3 group split in 13C NMR spectra and appeared as quartets.

Biological evaluation

Cytostatic activity

Eight human tumor cell lines derived from seven human cancer types: acute lymphoblastic leukemia (CEM, Molt4/C8), cervical carcinoma (HeLa), lung adenocarcinoma (A549), hepatocellular carcinoma (HepG2), metastatic colorectal adenocarcinoma (SW620), breast carcinoma (MCF-7), murine cancer cell line (L1210), and normal human diploid fibroblasts (WI-38) derived from embryonic lung tissue were used in antiproliferative evaluation assay. The results of in vitro screening of sorafenib analogs 6a–i against selected cell lines are presented in Table 2. The strongest cytostatic activity was exerted by 5-/4-[3-(4-chloro-3-trifluoromethyl-phenyl)-ureido]-phenoxy/-pyrazine-2-carboxylic acid (4-chloro-3-trifluoromethylphenyl)-amide (6g), which showed comparable or even more potent activity than sorafenib (IC50 0.9–7.5 μM). 6e, 6f, and 6i were also very potent against all tested cell lines, including WI-38, like sorafenib itself. The common structural feature for these compounds is 4-chloro-3-trifluoromethylphenyl moiety in urea part. The most active compound 6g have 4-chloro-3-trifluoromethylphenyl residue on both wings of the molecule, e.g., both in urea and amide region. This compound was also the most liphophilic, suggesting that lipohilicity is important for anticancer activity.

Table 2 Inhibitory effects of pyrazine sorafenib analogs on proliferation of cancer cell lines and fibroblast
Full size table

Two compounds, namely, 5-/4-[3-(4-bromophenyl)-ureido]-phenyloxy/-pyrazine-2-carboxylic acid cyclopentylamide (6c) and 5-/4-[3-(4-chloro-3-trifluoromethyl-phenyl)-ureido]-phenoxy/-pyrazine-2-carboxylic acid cyclopentylamide (6h), exerted cytostatic activities (IC50 0.6–0.9 μM) that significantly surpassed the effects observed with sorafenib in three cancer cell lines (HepG2, HeLa, A549). Similar to sorafenib, compound 6h proved to be cytotoxic to normal human fibroblast cells, whereas compound 6c did not diminish proliferative capacity of these cells (IC50 > 100 μM). Compounds 6a and 6b showed also high cytostatic activity against the same cancer cell lines (6a: HeLa and HepG2; 6b: HeLa and A549). All these compounds have either cyclopentyl residue in amide part or bromophenyl group in urea part or both these structural features (compound 6c).

All compounds exerted cytostatic activity against HeLa cell line. However, most of the compounds were active toward WI-38 as well and showed comparable activity to that observed for sorafenib. Compounds 6b–d were exceptions: 6b and 6d showed low toxicity toward fibroblast WI-38 (IC50 58.1 and 21.9 μM, respectively) and 6c no toxicity. It is interesting to note that compound 6d, the most similar compound to sorafenib, with the same urea and amide part, but different heterocycle, showed much higher selectivity than sorafenib itself: it was very active against three cancer cell lines (HepG2, HeLa, and A549 again) and practically inactive against other five. It also showed lower toxicity to fibroblast WI-38 than sorafenib. The observed selectivity could be fully attributed to pyrazine moiety.

In conclusion, all the tested compounds showed strong antiproliferative activity in the low micromolar range or high selectivity against the selected cancer cell lines. Compound 6c may be considered as the most promising pyrazine sorafenib analog, due to more potent anticancer activity against three cancer cell lines, reduced toxicity in comparison with sorafenib, and full agreement with the Lipinski’s and Gelovani’s rules for prospective small molecular drugs. It could be used as a scaffold for development of new, more effective, and safer drugs for treating hepatocellular, lung, and cervical carcinoma.

c-Raf kinase inhibition

In vitro antiproliferative screening identified two compounds, 6h and 6c, with potent cytostatic activity that surpassed the effects observed with sorafenib in three cancer cell lines (HepG2, HeLa, A549). To investigate if compounds 6c and 6h share the common cytostatic mechanism with sorafenib, we analyzed the effects of these compounds on the activity of c-Raf kinase, known sorafenib target, using Western blot method with specific phospho-c-Raf (Ser259) antibody.

The method detects endogenous levels of c-Raf only when phosphorylated at Ser259, indicating its activation. Western blot analysis clearly showed marked downregulation of phospho-c-Raf in A549 and SW620 cell lines treated with 6h at its IC50 value for 48 h, which suggests that 6h acts as c-Raf inhibitor. On contrary, compound 6c did not elicit significant change in the expression level of phospho-c-Raf in HepG2 cells, which suggests that other mechanism than c-Raf inhibition may account for its observed cytostatic activity in hepatocellular carcinoma cells. This striking difference between compounds 6h and 6c in the mechanism underlying their antiproliferative effects as well as opposing cytotoxic effects in normal human fibroblasts could be attributed to their structural differences. Both compounds possess the same cyclopentyl substituent in amide region and differ only in the urea part of the molecules: 6h has trifluoromethyl and chloro-substituted benzene ring as sorafenib, while 6c is a bromophenyl derivative. The results suggest that the urea part of the molecule is essential for the interaction between 6h and c-Raf (Fig. 2).

Fig. 2
figure 2

Representative Western blots for expression of phospho-c-Raf (Ser259), 75kDa in cancer cell lines treated with compounds 6h and 6c after 24h. Downregulation of phospho-c-Raf is visible in A549 and SW 620 cells treated with 6h

Full size image

Experimental

Chemistry

Melting points were determined on a Stuart Melting Point Apparatus SMP3 and were uncorrected. IR spectra were recorded on a Fourier transform-infrared spectroscopy Perkin Elmer Paragon 500 spectrometer (Perkin-Elmer, UK). 1H and 13C NMR spectra were recorded on a Bruker AV-600 spectrometer (Bruker, USA) operating at 300 and 600 MHz for 1H and 75 and 150 MHz for 13C nuclei. Samples were measured in dimethyl sulfoxide (DMSO)-d 6 solutions at 20 °C in 5-mm NMR tubes. Chemical shifts (δ) in ppm were referred to tetramethylsilane. Mass spectra were taken on a high-performance liquid chromatography–mass spectrometry (HPLC, Agilent Technologies 1200 Series; MS, Agilent Technologies 6410 Triple Quad). Elemental analyses were performed on a CHNS LECO analyzer (LECO Corporation, USA). Analyses indicated by the symbols of the elements were within ± 0.4 % of the theoretical values. All compounds are routinely checked by thin-layer chromatography (TLC) with Merck silica gel 60F-254 glass plates using the following solvent systems: cyclohexane/ethyl acetate/methanol 3:1:0.5, cyclohexane/ethyl acetate 3:1, 1:1, and 1:2, ethyl acetate/methanol 2:0.1, dichloromethane/methanol/cyclohexane/ethyl acetate 9:1:5:5, dichloromethane/methanol 9.5:0.5, ethyl acetate/petrolether/methanol 2:2:0.1 and 3:1:0.1, petrolether/ethyl acetate 1:1, and ethyl acetate. Spots were visualized by short-wave UV light and iodine vapour. Column chromatography was performed on silica gel (0.063−0.200 mm), with the same eluents used in TLC. 1H-benzo[d][1,2,3]triazole (BtH), 5-hydroxy-pyrazine-2-carboxylic acid, methylamine hydrochloride, cyclopentylamine, cyclohexylamine, ethanolamine, 4-bromoaniline, 4-chloro-3-(fluoromethyl)aniline, O-benzylhydroxylamine hydrochloride, and potassium t-butoxide (KO t Bu) were purchased from Aldrich (Germany), 4-aminophenol and 4-chloro-3-(fluoromethyl)phenyl isocyanate from Acros Organics (Belgium), triphosgene from Alpha Aesar (USA), and TEA from Sigma Chemical Co. (USA). 1-benzotriazole carboxylic acid chloride (BtcCl, 1) was prepared from BtH and triphosgene following our published procedure (Butula et al., 1977; Butula and Jadrijević-Mladar Takač, 2000).

N-(4-hydroxyphenyl)-1H-benzo[d][1,2,3]triazole-1-carboxamide (2)

A solution of 1.086 g (6.0 mmol) BtcCl (1) in 25 ml of dry dioxane was added dropwise to a solution of equimolar amount of p-aminophenol (0.655 g) and TEA (0.606 g) in 30 ml of dry dioxane. Reaction mixture was stirred 30 min at room temperature. TEA × HCl was filtered off and the mother liquor was evaporated under reduced pressure. After purification by column chromatography (mobile phase dichloromethane/methanol 9.5:0.5) and crystallization from toluene and acetone/water, 0.915 g (60 %) of 2 was obtained; mp. 240 °C (decomp.); IR (KBr): ν max 3456, 3252, 1726, 1544, 1616, 1544, 1618, 1488, 1446, 1370, 1236, 1212, 1060, 926, 825, 146, 583 cm−1; 1H NMR (DMSO, 300 MHz): δ = 10.89 (s, 1H, 1), 9.41 (s, 1H, 8), 8.24 (d, J = 9.12, 2H, arom.), 7.76–7.73 (m, 1H, arom.), 7.60–7.57 (m, 3H, arom.), 6.83–6.81 (m, 2H, arom.); 13C NMR (DMSO, 75 MHz): δ = 154.61 (9), 146.99 (2), 145.50 (15), 131.47 (10), 129.94 (13), 128.40 (5), 125.62 (12), 123.19 (4, 6), 119.18 (14), 115.18 (3, 7), 113.69 (11); ESIMS m/z (pos): 277.1, C13H10N4NaO2 (calcd. 277.2).

General procedure for preparation of 4-hydroxyphenyl ureas 3a–c

Method A: A suspension of 0.280 g (1.1 mmol) benzotriazole-1-carboxylic acid (4-hydroxyphenyl)-amide (2) and corresponding amine (2.2 mmol) in 20 ml of ethanol was heated 1 h at 50 °C. The reaction mixture was evaporated under reduced pressure. Method B: A suspension of 0.280 g (1.1 mmol) benzotriazole-1-carboxylic acid (4-hydroxyphenyl)-amide (2) and corresponding amine (2.2 mmol) in 25 ml of DMF was heated 3 h at 115 °C. The reaction mixture was evaporated under reduced pressure. Products 3a–c were purified by column chromatography or by crystallization.

1-cyclopentyl-3-(4-hydroxyphenyl)urea (3a)

This compound was prepared by the reaction of 0.280 g (1.1 mmol) amide 2 and 0.113 g (2.2 mmol) cyclopentylamine. After purification by column chromatography (mobile phase petrolether/etyl acetate 1:1), 0.208 g (86 %) of a white solid was obtained; mp. 175–177 °C; IR (KBr): ν max 3243, 2960, 2870, 1608, 1574, 1517, 1442, 1309, 1259, 1220, 1163, 835, 805, 727, 667, 523 cm−1; 1H NMR (DMSO, 300 MHz): δ = 8.88 (s, 1H, 1), 7.86 (s, 1H, 8), 7.13–7.11 (m, 2H, arom.), 6.63–6.60 (m, 2H, arom.), 5.94 (d, J = 7.20, 1H, 10), 3.93–3.88 (m, 1H, 11), 1.84–1.78 (m, 2H, 12), 1.64–1.59 (m, 2H, 15), 1.55–1.50 (m, 2H, 13), 1.36–1.30 (m, 2H, 12); 13C NMR (DMSO, 75 MHz): δ = 155.11 (9), 151.79 (2), 132.07 (5), 119.60 (4, 6), 115.02 (3, 7), 50.82 (11), 32.86 (12, 15), 23.10 (13, 14); ESIMS m/z (pos): 221.1, C12H17N2O2 (calcd. 221.3); Anal. Calcd for C12H16N2O2: C, 65.43; H, 7.32; N, 12.72. Found: C, 65.16, H, 6.96; N, 12.35.

1-(4-bromophenyl)-3-(4-hydroxyphenyl)urea (3b)

This compound was prepared by the reaction of 0.280 g (1.1 mmol) amide 2 and 0.378 g (2.2 mmol) 4-bromoaniline. After crystallization from dichloromethane, 0.301 g (89 %) of a white solid was obtained; mp. 244–245 °C (decomp.); IR (KBr): ν max 3302, 1637, 1590, 1565, 1509, 1488, 1464, 1393, 1302, 1224, 1102, 1071, 1010, 853, 824, 793, 649 cm–1; 1H NMR (DMSO, 300 MHz): δ = 9.05 (s, 1H, 1), 8.65 (s, 1H, 8), 8.33 (s, 1H, 10), 7.41 (s, 4H, 12, 13, 15, 16), 7.23–7.18 (m, 2H, 3, 7), 6.71–6.66 (m, 2H, 4, 6); 13C NMR (DMSO, 75 MHz): δ = 152.68 (2), 152.61 (9), 139.42 (11), 131.39 (12, 16), 130.82 (5), 120.58 (4, 6), 119.89 (13, 15), 115.16 (3, 7), 112.77 (14); ESIMS m/z (pos): 309.1, C13H12 81BrN2O2 (calcd. 309.1); 307.2, C13H12 79BrN2O2 (calcd. 307.1); Anal. Calcd for C13H11BrN2O2: C, 50.85; H, 3.61; N, 9.12. Found: C, 50.65, H, 3.99; N, 9.02.

1-(4-chloro-3-(trifluoromethyl)phenyl)-3-(4-hydroxyphenyl)urea (3c)

This compound was prepared by the reaction of 0.280 g (1.1 mmol) amide 2 and 0.430 g (2.2 mmol) 4-chloro-3-(trifluoromethyl)aniline. After purification by column chromatography (mobile phase dichloromethane/methanol 9.5:0.5), 0.109 g (30 %) of a white solid was obtained; mp. 210–215 °C; IR (KBr): ν max 3305, 3112, 2930, 1673, 1625, 1591, 1560, 1482, 1436, 1328, 1268, 1185, 1147, 1126, 832, 684 cm–1; 1H NMR (DMSO, 300 MHz): δ = 9.09 (s, 1H, 1), 9.00 (s, 1H, 8), 8.46 (s, 1H, 10), 8.09 (d, J = 2.13, 1H, 16), 7.64–7.56 (m, 2H, 12, 13), 7.24–7.19 (m, 2H, 3, 7), 6.72–6.67 (m, 2H, 4, 6); 13C NMR (DMSO, 75 MHz): δ = 152.95 (1), 152.60 (9), 139.66 (11), 131.88 (13), 130.45 (5), 126.93–126.32 (q, J = 30.57, 15), 125.53–120.10 (q, J = 272.90, 17), 122.77 (12), 121.84 (14), 120.99 (4, 6), 116.57–116.45 (q, J = 5.72, 16), 115.17 (3, 7); ESIMS m/z (pos): 330.7, C14H10ClF3N2O2 (calcd. 330.7); Anal. Calcd for C14H10ClF3N2O2: C, 50.85; H, 3.05; N, 8.47. Found: C, 50.49, H, 3.22; N, 8.88.

General procedure for preparation of 5-chloro-pyrazine-2-carboxamides 4a–f

A suspension of 0.308 g (2.2 mmol) 5-hydroxy-2-pyrazine carboxylic acid, 1.666 g (14.0 mmol) thionyl chloride, and 2 drops of DMF in 30 ml of dry toluene was heated at 100 °C 70 min. The reaction mixture was cooled, filtrated, and evaporated under reduced pressure. The red oil residue was dissolved in 10 ml of dry dichloromethane and added dropwise to a cold solution (0 °C) of corresponding amine (1.7 mmol) and 0.404 g (4.0 mmol) TEA in 20 ml of dry dichloromethane. The reaction mixture was stirred 30 min and evaporated under reduced pressure. Products 4a–f were purified by column chromatography or by crystallization.

5-chloro-N-methylpyrazine-2-carboxamide (4a)

This compound was prepared by the reaction of 0.308 g (2.2 mmol) 5-hydroxy-2-pyrazine carboxylic acid, 1.666 g (14.0 mmol) thionyl chloride, 0.144 g (1.7 mmol) methylamine hydrochloride, and 0.606 g (6.0 mmol) TEA. After purification by column chromatography (mobile phase ethyl acetate/petrolether/methanol 3:1:0.1), 0.198 g (68 %) of a white solid was obtained; mp. 153–156 °C; IR (KBr): ν max 3369, 3078, 2944, 1660, 1573, 1536, 1517, 1456, 1412, 1278, 1181, 1126, 1032, 1003, 932, 852, 664, 632, 522, 491 cm–1; 1H NMR (DMSO, 300 MHz): δ = 8.99 (d, J = 1.32, 1H, 5), 8.89–8.86 (m, 2H, 3, 1′), 2.83 (d, 3H, J = 4.83, 2′); 13C NMR (DMSO, 75 MHz): δ = 162.40 (1), 150.54 (4), 143.59 (2), 143.17 (5), 143.00 (3), 25.98 (2′); ESIMS m/z (pos): 172.1, C6H7ClN3O (calcd. 171.6); Anal. Calcd for C6H6ClN3O: C, 42.00; H, 3.52; N, 24.49. Found: C, 42.33, H, 3.00; N, 24.11.

N-(2-hydroxyethyl)-5-chloropyrazine-2-carboxamide (4b)

This compound was prepared by the reaction of 0.308 g (2.2 mmol) 5-hydroxy-2-pyrazine carboxylic acid, 1.666 g (14.0 mmol) thionyl chloride, and 0.267 g (4.3 mmol) ethanolamine. After purification by column chromatography (mobile phase ethyl acetate/petrolether/methanol 3:1:0.1), 0.218 g (59 %) of a white solid was obtained; mp. 94–96 °C; IR (KBr): ν max 3356, 3079, 2942, 2879, 1665, 1573, 1529, 1459, 1439, 1365, 1302, 1278, 1210, 1182, 1127, 1055, 1031, 872, 794, 742, 692, 519 cm–1; 1H NMR (DMSO, 300 MHz): δ = 9.00 (d, J = 1.32, 1H, 5), 8.87 (d, J = 1.32, 1H, 3), 8.76 (t, J = 4.63, 1H, 1′), 4.77 (t, J = 5.51, 1H, 4′), 3.54 (q, J = 5.90, 2H, 3′), 3.39 (q, J = 5.96, 2H, 2′); 13C NMR (DMSO, 75 MHz): δ = 161.97 (1), 150.64 (4), 143.46 (2), 143.27 (5), 143.00 (3), 59.41 (3′), 41.67 (2′); ESIMS m/z (pos): 224.2, C7H9ClN3NaO2 (calcd. 224.6); 202.2, C7H9ClN3O2 (calcd. 201.6); Anal. Calcd for C7H8ClN3O2: C, 41.70; H, 4.00; N, 20.84. Found: C, 41.99, H, 3.68; N, 20.61.

N-cyclopentyl-5-chloropyrazine-2-carboxamide (4c)

This compound was prepared by the reaction of 0.308 g (2.2 mmol) 5-hydroxy-2-pyrazine carboxylic acid, 1.666 g (14.0 mmol) thionyl chloride, 0.145 g (1.7 mmol) cyclopentylamine, and 0.404 g (4.0 mmol) TEA. After purification by column chromatography (mobile phase dichloromethane/methanol 9.5:0.5), 0.384 g (89 %) of a white solid was obtained; mp. 107–108 °C; IR (KBr): ν max 3373, 3079, 2951, 2870, 1660, 1571, 1528, 1466, 1295, 1176, 1161, 1125, 1027, 927, 632, 526, 492 cm–1; 1H NMR (DMSO, 300 MHz): δ = 8.97 (d, J = 1.32, 1H, 5), 8.84 (d, J = 1.52, 1H, 3), 8.79 (d, J = 7.56, 1H, 1′), 4.29–4.23 (m, 1H, 2′), 1.91–1.86 (m, 2H, 3′), 1.72–1.67 (m, 2H, 6′), 1.63–1.51 (m, 4H, 4′, 5′); 13C NMR (DMSO, 75 MHz): δ = 161.56 (1), 150.45 (4), 143.80 (2), 143.37 (5), 142.84 (3), 50.68 (2′), 31.85 (3′, 6′), 23.52 (4′, 5′); ESIMS m/z (pos): 248.1, C10H12ClN3NaO (calcd. 248.7); 226.1, C10H13ClN3O (calcd. 226.7); Anal. Calcd for C10H12ClN3O: C, 53.22; H, 5.36; N, 18.62. Found: C, 53.67, H, 5.02; N, 18.33.

N-cyclohexyl-5-chloropyrazine-2-carboxamide (4d)

This compound was prepared by the reaction of 0.308 g (2.2 mmol) 5-hydroxy-2-pyrazine carboxylic acid, 1.666 g (14.0 mmol) thionyl chloride, 0.169 g (1.7 mmol) cyclohexylamine, and 0.404 g (4.0 mmol) TEA. After two crystallization from ether, 0.277 g (68 %) of a white solid was obtained; mp. 153–154 °C; IR (KBr): ν max 3377, 3081, 2923, 2851, 1657, 1570, 1530, 1465, 1454, 1310, 1169, 1124, 1079, 1026, 925, 895, 861, 633, 524, 439 cm–1; 1H NMR (DMSO, 300 MHz): δ = 8.99 (d, J = 1.35, 1H, 5), 8.84 (d, J = 1.32, 1H, 3), 8.60 (d, J = 8.37, 1H, 1′), 3.85–3.74 (m, 1H, 2′), 1.81–1.69, 1.63–1.58, 1.48–1.24, 1.20–1.06 (m, 10H, 3′–7′); 13C NMR (DMSO, 75 MHz): δ = 160.96 (1), 150.49 (4), 143.77 (2), 143.38 (5), 142.84 (3), 48.19 (2′), 31.93 (3′, 7′), 25.01 (5′), 24.76 (4′, 6′); ESIMS m/z (pos): 262.1, C11H14ClN3NaO (calcd. 262.7); 240.1, C11H15ClN3O (calcd. 240.7); Anal. Calcd for C11H14ClN3O: C, 55.12; H, 5.89; N, 17.53. Found: C, 54.96, H, 5.56; N, 17.77.

5-chloro-N-(4-chloro-3-(trifluoromethyl)phenyl)pyrazine-2-carboxamide (4e)

This compound was prepared by the reaction of 0.308 g (2.2 mmol) 5-hydroxy-2-pyrazine carboxylic acid, 1.666 g (14.0 mmol) thionyl chloride, 0.332 g (1.7 mmol) 4-chloro-3-trifluormethylaniline, and 0.444 g (4.4 mmol) TEA. After crystallization with ether and recrystallization from acetone/water, 0.371 g (65 %) of a white solid was obtained; mp. 149–151 °C; IR (KBr): ν max 3376, 3360, 3122, 3083, 1693, 1590, 1530, 1423, 1332, 1262, 1251, 1180, 1136, 1116, 1023, 830, 663 cm–1; 1H NMR (DMSO, 300 MHz): δ = 11.22 (s, 1H, 1′), 9.13 (d, J = 1.20, 1H, 5), 8.96 (d, J = 1.26, 1H, 3), 8.52 (d, J = 2.37, 1H, 3′), 8.23 (dd, J = 2.31, J = 6.51, 1H, 7′), 7.74 (d, J = 8.79, 1H, 6′); 13C NMR (DMSO, 75 MHz): δ = 161.53 (1), 151.24 (4), 144.19 (5), 143.22 (2), 142.99 (3), 137.66 (2′), 132.01 (7′), 126.97–126.36 (q, J = 30.94, 4′), 125.43 (6′), 125.09 (5′), 125.40–119.97 (q, J = 273.67, 8′), 119.56–119.45 (q, J = 5.58, 3′); ESIMS m/z (pos): 336.2, C12H6Cl2F3N3O (calcd. 336.1); Anal. Calcd for C12H6Cl2F3N3O: C, 42.88; H, 1.80; N, 12.50. Found: C, 42.65, H, 2.06; N, 12.09.

N-(benzyloxy)-5-chloropyrazine-2-carboxamide (4f)

This compound was prepared by the reaction of 0.308 g (2.2 mmol) 5-hydroxy-2-pyrazine carboxylic acid, 1.666 g (14.0 mmol) thionyl chloride, 0.271 g (1.7 mmol) O-benzylhydroxylamine hydrochloride, and 0.444 g (4.4 mmol) TEA. After crystallization with ether and recrystallization from acetone/water, 0.319 g (67 %) of a white solid was obtained; mp. 124–125 °C; IR (KBr): ν max 3246, 3089, 3062, 3032, 2963, 1678, 1491, 1452, 1276, 1121, 1016, 906, 751, 699, 505 cm–1; 1H NMR (DMSO, 300 MHz): δ = 12.33 (s, 1H, 1′), 8.98 (d, J = 0.99, 1H, 5), 8.85 (d, J = 1.23, 1H, 3), 7.49–7.37 (m, 5H, 4′–8′), 4.96 (s, 2H, 2′); 13C NMR (DMSO, 75 MHz): δ = 160.05 (1), 151.42 (4), 143.83 (5), 143.72 (3), 143.54 (2), 136.04 (3′), 129.30 (5′, 7′), 128.83 (4′), 128.78 (6′, 8′), 77.18 (2′); ESIMS m/z (pos): 286.2, C12H10ClN3NaO2 (calcd. 286.7); 264.2, C12H11ClN3O2 (calcd. 264.7); Anal. Calcd for C12H10ClN3O2: C, 54.66; H, 3.82; N, 15.94. Found: C, 54.29, H, 3.80; N, 16.03.

General procedure for preparation of 5-(4-aminophenoxy)-N-alkylpyrazine-2-carboxamides 5a, b

A suspension of 0.112 g (1 mmol) KO t Bu and 0.11 g (1 mmol) p-aminophenol in 10 ml of dry DMF was stirred at room temperature for 30 min. Amide 4 (1 mmol) and 0.07 g (0.5 mmol), K2CO3 was added and the reaction mixture was stirred at 80 °C for 2 h. DMF was evaporated under reduced pressure. Products 5 were purified by column chromatography and crystallization from ether.

5-(4-aminophenoxy)-N-cyclopentylpyrazine-2-carboxamide (5a)

This compound was prepared by the reaction of 0.226 g (1 mmol) amide 4c, 0.112 g (1 mmol) KO t Bu, and 0.11 g (1 mmol) p-aminophenol. After purification by column chromatography (mobile phase dichloromethane/methanol 95:5) and crystallization from ether, 0.176 g (59 %) of a white solid was obtained; mp. 142–144 °C; IR (KBr): ν max 3458, 3380, 3079, 3020, 2938, 2864, 1652, 1619, 1587, 1506, 1470, 1352, 1326, 1298, 1268, 1196, 1025, 836, 594 cm–1; 1H NMR (DMSO, 300 MHz): δ = 8.67 (d, J = 1.29, 1H, 3), 8.46 (d, J = 7.95, 1H, 1′), 8.40 (d, J = 1.29, 1H, 5), 6.91–6.86 (m, 2H, 7, 11), 6.63–6.58 (m, 2H, 8, 10), 5.09 (s, 2H, 12), 4.31–4.19 (m, 1H, 2′), 1.93–1.81 (m, 2H, 3′), 1.75–1.65 (m, 2H, 6′), 1.63–1.51 (m, 4H, 4′, 5′); 13C NMR (DMSO, 75 MHz): δ = 162.11 (4), 161.72 (1), 146.54 (6), 142.44 (9), 140.93 (5), 139.22 (2), 133.13 (3), 121.69 (7, 11), 114.41 (8, 10), 50.48 (2′), 32.02 (3′, 6′), 23.53 (4′, 5′); ESIMS m/z (pos): 299.3, C16H19N4O2 (calcd. 299.3); Anal. Calcd for C16H18N4O2: C, 64.411; H, 6.08; N, 18.78. Found: C, 64.86, H, 5.66; N, 18.53.

5-(4-aminophenoxy)-N-cyclohexylpyrazine-2-carboxamide (5b)

This compound was prepared by the reaction of 0.240 g (1 mmol) amide 4d, 0.112 g (1 mmol) KO t Bu, and 0.11 g (1 mmol) p-aminophenol. After purification by column chromatography (mobile phase cyclohexane/ethyl acetate 1:2) and crystallization from ether, 0.140 g (45 %) of a white solid was obtained; mp. 167–170 °C; IR (KBr): ν max 3404, 3336, 3018, 2929, 2855, 1661, 1609, 1579, 1522, 1504, 1459, 1344, 1326, 1262, 1181, 1022, 890, 834, 593, 509 cm–1; 1H NMR (DMSO, 300 MHz): δ = 8.67 (d, J = 1.29, 1H, 3), 8.39 (d, J = 1.32, 1H, 5), 8.32 (d, J = 8.52, 1H, 1′), 6.91–6.86 (m, 2H, 7, 11), 6.63–6.58 (m, 2H, 8, 10), 5.06 (s, 2H, 12), 3.84–3.72 (m, 1H, 2′), 1.80–1.70, 1.62–1.56, 1.47–1.25, 1.20–1.11 (m, 10H, 3′–7′); 13C NMR (DMSO, 75 MHz): δ = 161.70 (4), 161.47 (1), 146.48 (6), 142.45 (9), 140.92 (5), 139.17 (2), 133.09 (3), 121.60 (7, 11), 114.38 (8, 10), 47.85 (2′), 32.10 (3′, 7′), 25.04 (5′), 24.76 (4′, 6′); ESIMS m/z (pos): 313.3, C17H21N4O2 (calcd. 313.4); Anal. Calcd for C17H20N4O2: C, 65.37; H, 6.45; N, 17.94. Found: C, 65.29, H, 6.56; N, 17.61.

General procedure for preparation of pyrazine sorafenib analogs 6a–i

Method A: A suspension of 5-chloro-pyrazine-2-carboxamide 4 (0.4 mmol), urea 3 (0.4 mmol), and 0.111 g (0.8 mmol) potassium carbonate in 4 ml of dry DMF was heated at 100 °C for 3–4.5 h. The reaction mixture was cooled to room temperature, diluted with 30 ml ethyl acetate, and extracted three times with brine. Organic layer was dried over anhydrous sodium sulfate, filtered, and evaporated under reduced pressure. The crude products were purified by column chromatography on silica gel using petrolether/ethyl acetate/methanol (3:1:0.5, v/v) (6b), petrolether/ethyl acetate/methanol (2:2:01, v/v) (6d and 6f), dichloromethane/methanol (9.5:0.5, v/v) (6g) or by crystallization from ether (6a, 6h–i) or methanol (6e). Method B: A suspension of 5-(4-aminophenoxy)-N-alkylpyrazine-2-carboxamide 5 (0.2 mmol) and 0.066 g (0.3 mmol) 4-chloro-3-(trifluoromethyl)phenylisocyanate in 3 ml of dry dichloromethane was stirred at room temperature for 2 h. The precipitated products 6h or 6i were filtered off and crystallized from ether.

5-[4-(3-cyclopentyl-ureido)-phenyloxy]-pyrazine-2-carbocylic acid cyclopentylamide (6a)

This compound was prepared by the reaction of 0.090 g (0.4 mmol) amide 4c and 0.088 g (0.4 mmol) urea 3a (Method A). After crystallization from ether, 0.067 g (41 %) of a white solid was obtained; mp. 235–237 °C; IR (KBr): ν max 3341, 3070, 3052, 2952, 2867, 1660, 1606, 1571, 1505, 1461, 1408, 1349, 1316, 1284, 1242, 1194, 1023, 897, 835, 626, 527, 502 cm–1; 1H NMR (DMSO, 300 MHz): δ = 8.67 (d, J = 1.29, 1H, 3), 8.48 (d, J = 1.32, 1H, 5), 8.43 (d, J = 2.88, 1H, 1′), 8.32 (s, 1H, 12), 7.46–7.41 (m, 2H, 8, 10), 7.12–7.07 (m, 2H, 7, 11), 6.15 (d, J = 7.23, 1H, 14), 4.31–4.19 (m, 1H, 2′), 4.00–3.89 (m, 1H, 15), 1.93–1.79, 1.72–1.50, 1.42–1.32 (3m, 16H, 3′–6′, 16–19); 13C NMR (DMSO, 75 MHz): δ = 162.01 (4), 161.21 (1), 154.78 (13), 146.18 (6), 140.82 (5), 139.57 (2), 138.06 (9), 133.87 (3), 121.45 (8, 10), 118.70 (7, 11), 50.86 (15), 50.48 (2′), 32.78 (3′, 6′), 31.99 (16, 19), 23.49 (4′, 5′), 23.62 (17, 18); ESIMS m/z (pos): 432.2, C22H27N5NaO3 (calcd. 432.5), 410.2, C22H28N5O3 (calcd. 410.5); Anal. Calcd for C22H27N5O3: C, 64.53; H, 6.65; N, 17.10. Found: C, 64.72, H, 6.60; N, 16.82.

5-/4-[3-(4-bomophenyl)-ureido]-phenoxy/-pyrazine-2-carboxylic acid methylamide (6b)

This compound was prepared by the reaction of 0.069 g (0.4 mmol) amide 4a and 0.123 g (0.4 mmol) urea 3b (Method A). After purification by column chromatography (mobile phase petrolether/ethyl acetate/methanol 3:1:0.5) and crystallization from ether, 0.058 g (33 %) of a white solid was obtained; IR (KBr): ν max 3311, 1662, 1646, 1591, 1551, 1505, 1457, 1348, 1309, 1271, 1236, 1194, 1008, 831 cm–1; 1H NMR (DMSO, 300 MHz): δ = 8.85 (s, 1H, 14), 8.80 (s, 1H, 12), 8.73–8.69 (m, 2H, 3, 1′), 8.53 (d, J = 1.25, 1H, 5), 7.55–7.49 (m, 2H, 8, 10), 7.45 (s, 4H, 16, 17, 19, 20), 7.21–7.16 (m, 2H, 7, 11), 2.82 (d, J = 4.87, 3H, 2′); 13C NMR (DMSO, 75 MHz): δ = 162.95 (4), 161.21 (1), 152.45 (13), 146.98 (6), 140.66 (5), 139.58 (15), 139.11 (2), 137.01 (9), 133.63 (3), 131.50 (17, 19), 121.76 (16, 20), 120.13 (8, 10), 119.68 (7, 11), 113.21 (18), 25.91 (2′); ESIMS m/z (neg): 442.1, C19H15 81BrN5O3 (calcd. 442.3), 440.0, C19H15 79BrN5O3 (calcd. 440.3); Anal. Calcd for C19H16BrN5O3: C, 51.60; H, 3.65; N, 15.84. Found: C, 51.99, H, 3.36; N, 15.72.

5-/4-[3-(4-bromophenyl)-ureido]-phenyloxy/-pyrazine-2-carboxylic acid cyclopentylamide (6c)

This compound was prepared by the reaction of 0.090 g (0.4 mmol) amide 4c and 0.123 g (0.4 mmol) urea 3b (Method A). After crystallization from ether/petrolether and recrystallization from acetone/water, 0.107 g (54 %) of a white solid was obtained; mp. 238–241 °C (decomp.); IR (KBr): ν max 3306, 2951, 2870, 1660, 1640, 1591, 1533, 1505, 1469, 1348, 1302, 1277, 1240, 1198, 1071, 1024, 1011, 903, 831, 657 cm–1; 1H NMR (DMSO, 300 MHz): δ = 8.84 (s, 1H, 14), 8.79 (s, 1H, 12), 8.69 (d, J = 1.21, 1H, 3), 8.51 (d, J = 1.17, 1H, 5), 8.47 (d, J = 7.93, 1H, 1′), 7.55–7.51 (m, 2H, 8, 10), 7.45 (s, 4H, 16, 17, 19, 20), 7.19–7.17 (m, 2H, 7, 11), 4.28–4.22 (m, 1H, 2′), 1.91–1.87 (m, 2H, 3′), 1.73–1.67 (m, 2H, 6′), 1.61–1.51 (m, 4H, 4′, 5′); 13C NMR (DMSO, 75 MHz): δ = 162.01 (4), 161.14 (1), 152.43 (13), 147.02 (6), 140.83 (5), 139.66 (15), 139.10 (2), 136.97 (9), 133.47 (3), 131.47 (17, 19), 121.67 (16, 20), 120.13 (8, 10), 119.67 (7, 11), 113.20 (18), 50.49 (2′), 32.00 (3′, 6′), 23.51 (4′, 5′); ESIMS m/z (neg): 496.1, C23H21 81BrN5O3 (calcd. 496.4), 494.1, C23H21 79BrN5O3 (calcd. 494.4); Anal. Calcd for C23H22BrN5O3: C, 55.65; H, 4.47; N, 14.11. Found: C, 55.29, H, 4.05; N, 14.55.

5-/4-[3-chloro-3-trifluoromethyl-phenyl)-ureido]-phenoxy/-pyrazine 2-carboxylic acid methylamide (6d)

This compound was prepared by the reaction of 0.068 g (0.4 mmol) amide 4a and 0.132 g (0.4 mmol) urea 3c (Method A). After purification by column chromatography (mobile phase petrolether/ethyl acetate/methanol 2:2:0.1) and crystallization from ether, 0.093 g (50 %) of a white solid was obtained; mp. 233–239 °C; IR (KBr): ν max 3415, 3289, 3134, 3103, 1710, 1662, 1608, 1586, 1550, 1510, 1487, 1460, 1402, 1301, 1176, 1125, 1029, 837, 664, 509 cm–1; 1H NMR (DMSO, 600 MHz): δ = 9.18 (s, 1H, 14), 8.91 (s, 1H, 12), 8.69 (s, 2H, 3, 1′), 8.53 (s, 1H, 5), 8.12 (s, 1H, 20), 7.68–7.60 (m, 2H, 16, 17), 7.54 (d, J = 8.73, 2H, 8, 10), 7.19 (d, J = 8.70, 2H, 7, 11), 2.82 (d, J = 4.56, 3H, 2′); 13C NMR (DMSO, 150 MHz): δ = 162.94 (4), 161.16 (1), 152.47 (13), 147.25 (6), 140.64 (5), 139.61 (15), 139.35 (2), 136.68 (9), 133.63 (3), 131.96 (17), 126.99–126.38 (q, 19, J = 30.48), 125.51–120.28 (q, 21, J = 273.27), 123.10 (16), 122.27 (18), 121.75 (8, 10), 120.04 (7, 11), 116.82–116.71 (q, 20, J = 5.44), 25.89 (2′); ESIMS m/z (pos): 466.1, C20H16ClF3N5O3 (calcd. 466.1); Anal. Calcd for C20H15ClF3N5O3: C, 51.57; H, 3.25; N, 15.03. Found: C, 51.22, H, 3.11; N, 15.44.

5-/4-[3-(4-chloro-3-trifluoromethyl-phenyl)-ureido]-phenoxy/-pyrazine-2-carboxylic acid (2-hydroxyethyl)amide (6e)

This compound was prepared by the reaction of 0.087 g (0.4 mmol) amide 4b and 0.132 g (0.4 mmol) urea 3c (Method A). After purification by column chromatography (mobile phase ethyl acetate/methanol 2:0.1) and crystallization from methanol, 0.070 g (35 %) of a white solid was obtained; mp. 177–180 °C; IR (KBr): ν max 3437, 3352, 3140, 2929, 1712, 1647, 1608, 1561, 1509, 1486, 1462, 1420, 1354, 1329, 1304, 1279, 1258, 1225, 1192, 1143, 1076, 1032, 1019, 915, 894, 845, 688, 664, 641, 513 cm–1; 1H NMR (DMSO, 600 MHz): δ = 9.26 (s, 1H, 14), 9.00 (s, 1H, 12), 8.70 (d, J = 1.30, 1H, 3), 8.56 (t, J = 5.85, 1H, 1′), 8.54 (d, J = 1.31, 1H, 5), 8.12 (d, J = 2.32, 1H, 20), 7.68–7.59 (m, 2H, 16, 17), 7.56–7.51 (m, 2H, 8, 10), 7.22–7.17 (m, 2H, 7, 11), 4.77 (t, J = 5.47, 1H, 4′), 3.56–3.50 (q, J = 5.85, 2H, 3′), 3.41–3.35 (q, J = 5.74, 2H, 2′); 13C NMR (DMSO, 150 MHz): δ = 162.39 (4), 161.17 (1), 152.42 (13), 147.24 (6), 140.73 (5), 139.40 (15), 139.30 (2), 136.63 (9), 133.58 (3), 131.91 (17), 126.55 (t, 19, J = 30.33), 125.46–120.03 (q, 21, J = 272.95), 123.01 (16), 122.23 (18), 121.63 (8, 10), 120.01 (7, 11), 116.80–116.68 (q, 20, J = 5.68), 59.55 (3′), 41.48 (2′); ESIMS m/z (pos): 518.2, C21H17ClF3N5NaO4 (calcd. 518.8), 496.8, C21H18ClF3N5O4 (calcd. 496.8); Anal. Calcd for C21H17ClF3N5O4: C, 50.87; H, 3.46; N, 14.12. Found: C, 50.50, H, 3.41; N, 13.78.

5-/4-[3-(4-chloro-3-trifluoromethylphenyl)-ureido]-phenoxy/-pyrazine-2-carboxylic acid benzyloxy-amide (6f)

This compound was prepared by the reaction of 0.112 g (0.4 mmol) amide 4f and 0.132 g (0.4 mmol) urea 3c (Method A). After purification by column chromatography (mobile phase petrolether/ethyl acetate/methanol 2:2:0.1) and crystallization from ether/petrolether, 0.040 g (18 %) of a white solid was obtained; mp. 208–212 °C; IR (KBr): ν max 3351, 3073, 1678, 1595, 1547, 1506, 1486, 1460, 1420, 1349, 1330, 1280, 1230, 1194, 1137, 1033, 1017, 912, 839, 753, 701, 664, 636, 532, 516 cm–1; 1H NMR (DMSO, 600 MHz): δ = 12.10 (s, 1H, 1′), 9.20 (s, 1H, 14), 8.94 (s, 1H, 12), 8.68 (d, J = 1.23, 1H, 3), 8.52 (d, J = 1.29, 1H, 5), 8.12 (d, J = 2.28, 1H, 20), 7.68–7.60 (m, 2H, 16, 17), 7.57–7.51 (m, 2H, 8, 10), 7.49–7.45, 7.42–7.35 (2m, 5H, 4′–8′), 7.22–7.17 (m, 2H, 7, 11), 4.94 (s, 2H, 2′); 13C NMR (DMSO, 150 MHz): δ = 161.35 (4), 160.17 (1), 152.48 (13), 147.15 (6), 140.93 (5), 139.35 (15), 138.83 (2), 136.74 (9), 135.73 (3′), 133.92 (3), 131.99 (17), 128.78 (4′, 8′), 128.28 (5′–7′), 128.24–117.38 (q, 18, J = 272.87), 127.30–126.09 (q, 19, J = 30.54), 123.07 (16), 121.78 (8, 10), 120.05 (7, 11), 116.88–116.66 (q, 20, J = 5.53), 77.11 (2′); ESIMS m/z (pos): 558.2, C26H20ClF3N5O4 (calcd. 558.1); Anal. Calcd for C26H19ClF3N5O4: C, 55.97; H, 3.43; N, 12.55. Found: C, 56.14, H, 3.40; N, 12.17.

5-/4-[3-(4-chloro-3-trifluoromethyl-phenyl)-ureido]-phenoxy/-pyrazine-2-carboxylic acid (4-chloro-3-trifluoromethylphenyl)-amide (6g)

This compound was prepared by the reaction of 0.134 g (0.4 mmol) amide 4e and 0.132 g (0.4 mmol) urea 3c (Method A). After purification by column chromatography (mobile phase dichloromethane/methanol 9.5:0.5) and crystallization from ether/petrolether, 0.068 g (27 %) of a white solid was obtained; 141−144 °C; IR (KBr): ν max 3353, 3123, 1689, 1593, 1532, 1506, 1482, 1463, 1421, 1353, 1321, 1274, 1261, 1230, 1185, 1138, 1113, 1034, 1020, 835, 665 cm–1; 1H NMR (DMSO, 600 MHz): δ = 11.09 (s, 1H, 1′), 9.20 (s, 1H, 14) 8.95 (s, 1H, 12), 8.86 (d, J = 1.26, 1H, 3), 8.65 (d, J = 2.46, 1H, 5), 8.24 (dd, J = 2.40, J = 6.42, 1H, 3′), 8.12 (d, J = 2.22, 1H, 7′), 7.73 (d, J = 8.89, 1H, 6′), 7.68−7.60 (m, 2H, 16, 17), 7.58−7.53 (m, 2H, 8, 10), 7.26−7.21 (m, 2H, 7, 11); 13C NMR (DMSO, 150 MHz): δ = 161.97 (4) 161.60 (1) 152.47 (13), 147.10 (6) 142.02 (5), 139.34 (15), 138.91 (2), 137.97 (2′), 136.83 (9), 133.76 (3), 131.98 (17), 131.96 (6′), 128.23−117.37 (q, 21, J = 273.33), 128.18−117.32 (q, J = 273.33, 8′), 127.30−126.08 (q, J = 30.59, 19), 127.24−126.02 (q, J = 30.77, 4′), 125.26 (16) 124.71 (d, J = 1.66, 5′) 123.06 (7′), 122.29 (d, J = 1.65, 18), 121.79 (8, 10), 120.04 (7, 11) 119.44−119.21 (q, J = 5.71, 20), 116.88−116.65 (q, 3′, J = 5.71); ESIMS m/z (pos): 630.2, C26H15Cl2F6N5O3 (calcd. 630.3); Anal. Calcd for C26H15Cl2F6N5O3: C, 49.54; H, 2.40; N, 11.11. Found: C, 49.75, H, 2.76; N, 10.83.

5-/4-[3-(4-chloro-3-trifluoromethyl-phenyl)-ureido]-phenoxy/-pyrazine-2-carboxylic acid cyclopentylamide (6h)

This compound was prepared by the reaction of 0.090 g (0.4 mmol) amide 4c and 0.132 g (0.4 mmol) urea 3c (Method A). After crystallization from ether, 0.094 g (45 %) of a white solid was obtained. Product 6h was also prepared by the reaction of 0.060 g (0.2 mmol) compound 5a and 0.066 g (0.3 mmol) 4-chloro-3-(trifluoromethyl)phenylisocyanate (Method B). After crystallization from ether 0.051 g (49 %) of pure product was obtained; mp. 231–233 °C; IR (KBr): ν max 3382, 3294, 3115, 3081, 2963, 2875, 1711, 1658, 1604, 1549, 1509, 1485, 1460, 1327, 1195, 1133, 1023, 840, 661, 513 cm–1; 1H NMR (DMSO, 600 MHz): δ = 9.20 (s, 1H, 14), 8.95 (s, 1H, 12), 8.69 (d, J = 1.20, 1H, 3), 8.53 (d, J = 1.26, 1H, 5), 8.49 (d, J = 7.98, 1H, 1′), 8.12 (d, J = 2.46, 1H, 20), 7.67–7.65 (m, 1H, 16), 7.63–7.61 (m, 1H, 17), 7.55–7.53 (m, 2H, 8, 10), 7.21–7.18 (m, 2H, 7, 11), 4.27–4.22 (m, 1H, 2′), 1.91–1.85 (m, 2H, 3′), 1.72–1.68 (m, 2H, 6′), 1.62–1.53 (m, 4H, 4′, 5′); 13C NMR (DMSO, 150 MHz): δ = 162.02 (4), 161.14 (1), 152.48 (13), 147.28 (6), 140.84 (5), 139.70 (15), 139.35 (2), 136.67 (9), 133.51 (3), 131.97 (17), 126.99-126.39 (q, J = 30.50, 19), 125.52–120.09 (q, J = 273.44, 21), 123.05 (16), 122.28 (18), 121.71 (8, 10), 120.04 (7, 11), 116.81–116.70 (q, J = 4.39, 20), 50.50 (2′), 32.01 (3′, 6′), 23.52 (4′, 5′); ESIMS m/z (pos): 520.1, C24H22ClF3N5O3 (calcd. 520.1); Anal. Calcd for C24H21ClF3N5O3: C, 55.44; H, 4.07; N, 13.47. Found: C, 55.10, H, 3.77; N, 13.10.

5-/4-[3-(4-chloro-3-trifluoromethyl-phenyl)-ureido]-phenoxy/-pyrazine-2-carboxylic acid cyclohexylamide (6i)

This compound was prepared by the reaction of 0.096 g (0.4 mmol) amide 4d and 0.132 g (0.4 mmol) urea 3c (Method A). After several crystallizations from ether, 0.135 g (63 %) of a white solid was obtained. Product 6i was also prepared by the reaction of 0.062 g (0.2 mmol) compound 5b and 0.066 g (0.3 mmol) 4-chloro-3-(trifluoromethyl)phenylisocyanate (Method B). After crystallization from ether, 0.065 g (61 %) of pure product was obtained; mp. 217–220 °C; IR (KBr): ν max 3394, 3322, 3122, 3082, 2928, 2857, 1711, 1660, 1595, 1513, 1481, 1421, 1310, 1227, 1143, 1033, 844, 576, 515 cm–1; 1H NMR (DMSO, 600 MHz): δ = 9.19 (s, 1H, 14), 8.93 (s, 1H, 12), 8.69 (d, J = 1.20, 1H, 3), 8.52 (d, J = 1.20, 1H, 5), 8.40 (d, J = 8.52, 1H, 1′), 8.12 (d, J = 2.13, 1H, 20), 7.68–7.60 (m, 1H, 16, 17), 7.56–7.51 (m, 2H, 8, 10), 7.22–7.17 (m, 2H, 7, 11), 3.84–3.73 (m, 1H, 2′), 1.80–1.70, 1.62–1.58, 1.48–1.25, 1.19–1.07 (4m, 10H, 3′–7′); 13C NMR (DMSO, 150 MHz): δ = 161.43 (4), 161.17 (1), 152.48 (13), 147.30 (6) 140.90 (5) 139.67 (15) 139.36 (2), 136.68 (9) 133.55 (3), 131.99 (17) 128.25–117.41 (q, J = 273.26, 21) 127.31–126.09 (q, J = 30.29, 19), 123.54 (16), 122.27 (18), 122.18 (8, 10), 120.53 (7, 11) 116.88–116.65 (q, J = 5.82, 20), 47.93 (2′), 32.13 (3′, 7′), 25.08 (5′), 24.82 (4′, 6′); ESIMS m/z (pos): 534.4, C25H24ClF3N5O3 (calcd. 534.9); Anal. Calcd for C25H23ClF3N5O3: C, 56.24; H, 4.34; N, 13.12. Found: C, 56.65, H, 4.01; N, 12.74.

Biological evaluation

Antiproliferative evaluation assay

The cell lines (CEM, HeLa, A549, HepG2, SW620, MCF-7, Molt4/C8, L1210,WI-38) were cultured as monolayers and maintained in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10 % fetal bovine serum (FBS), 2 mM L-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin in a humidified atmosphere with 5 % CO2 at 37 °C. The cell lines were inoculated onto a series of standard 96-well microtiter plates on day 0 at seeding density of 3000–6000 cells per well depending upon their specific doubling times. Freshly prepared dilutions of test compounds in culture medium in the concentration range from 10−8 to 10−4 M were added to the microtiter plates, and the cells were grown for further 3–4 days. Working dilutions of the compounds were freshly prepared on the day of testing. The solvent (DMSO) was also tested for its potential inhibitory activity by adjusting its concentration to the values used for the preparation of the working concentrations (DMSO concentration never exceeded 0.1 %). After 3–4 days of incubation, the cell growth rate was evaluated first light microscopically, then by performing the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. Experimentally determined absorbance values were transformed into the cell percentage of growth (PG) using the formulas proposed by NIH and described previously (Gazivoda et al., 2008). This method directly relies on control cells behaving normally at the day of assay commencement because it compares the growth of treated cells with that of untreated cells in control wells on the same plate. The results are therefore a percentile difference from the calculated expected value. The IC50 values for each compound are calculated from dose-response curves using linear regression analysis by fitting the mean test concentrations that give PG values above and below the reference value. However, if all of the tested concentrations produce PGs exceeding the respective reference level of effect (e.g., PG value of 50) for a given cell line, then the highest tested concentration is assigned as the default value (in the screening data report that default value is preceded by a “>” sign). Each test point was performed in quadruplicate in three individual experiments. The results were statistically analyzed (Analysis of variance, Tukey post hoc test at p < 0.05).

Determination of c-Raf activity using Western blotting

In order to study the effects of selected compounds on c-Raf activity, known protein target of multikinase inhibitor sorafenib, cells were cultured in 6-well plates at seeding density of 2 × 105 cells/well and subjected to treatment for indicated time points. Cells were lysed in RIPA buffer containing 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM Na2EDTA, 1 mM ethyleneglycol tetraacetic acid (EGTA), 1 % NP-40, and 1 % sodium deoxycholate supplemented with protease inhibitor cocktail (Roche). Total proteins (50 μg) were resolved on 12 % Tris-glycine polyacrylamide gels and transferred to polyvinylidene fluoride (PVDF) membranes. Subsequently, membranes were blocked for 1 h at room temperature with 4 % bovine serum albumin (BSA) in TBST [50 mmol/L Tris base, 150 mM NaCl, 0.1 % Tween 20 (pH 7.5)] and probed overnight at 4 °C with primary antibody against Phospho-c-Raf (Ser259) (Cell Signaling Technology; dilution 1:500). Membranes were washed with TBST and incubated with an anti-rabbit (DakoCytomation) horseradish peroxidase-conjugated secondary antibody at room temperature for 1 h. Individual proteins were visualized by the BM Chemiluminescence Western Blotting Substrate (POD) (Roche, Switzerland).

Conclusions

Nine novel pyrazine sorafenib analogs 6a–i differing in amide and/or urea regions were prepared and evaluated as potential cytostatic agents. The strongest cytostatic activity to all tested cell lines was observed for compound 6g, followed by 6e, 6f, and 6i, all having 4-chloro-3-trifluoromethylphenyl moiety in urea part. Their antiproliferative activity, as well as cytotoxicity to normal human fibroblasts cells, was comparable to sorafenib. Compounds 6c and 6h exerted cytostatic activities that surpassed the effects observed with sorafenib in three cancer cell lines (HepG2, HeLa, A549). Similar to sorafenib, compound 6h proved to be cytotoxic to normal human fibroblast cells, whereas compound 6c did not diminish proliferative capacity of these cells. Antiproliferative activity of 6h could be, at least partially, attributed to its inhibitory effect on c-Raf activation. On contrary, 6c did not inhibit the activity of c-Raf, which implies that other cell signaling pathways govern its antiproliferative effects. In conclusion, due to its reduced adverse effects on the growth of normal cells and more potent anticancer activity in particular cancer cell lines in comparison with sorafenib, compound 6c, e.g., 5-/4-[3-(4-bromophenyl)-ureido]-phenyloxy/-pyrazine-2-carboxylic acid cyclopentylamide, may be considered a potential scaffold for development of new, more effective, and safer drugs for treating hepatocellular, lung, and cervical carcinoma.