2019 Volume 67 Issue 10 Pages 1116-1122
In recent studies, combinations of histone deacetylases (HDACs) inhibitor with kinase inhibitor showed additive and synergistic effects. BRafV600E as an attractive target in many diseases treatments has been studied extensively. Herein, we present a novel design approach though incorporating the pharmacophores of BRafV600E inhibitor and HDACs inhibitor in one molecule. Several synthesized compounds exhibited distinct BRafV600E and HDAC1 inhibitory activities. The representative dual Raf/HDAC inhibitor, 7a, showed better antiproliferative activities against A549 and SK-Mel-2 in cellular assay than SAHA and sorafenib, with IC50 values of 9.11 µM and 5.40 µM, respectively. This work may lay the foundation for the further development of dual Raf/HDAC inhibitors as potential anticancer agents.
During the last few decades, developing mechanism-based targeted anticancer drugs has made great achievement. However, unsustainable clinical effectiveness and acquired drug resistance always limits the use of these agents.1) Since cancer is a disease involving complex signaling networks, blocking a single biological target may not completely shut off the core hallmark capability, allowing residual cancer cells to go on working. To address these problems, one particularly promising approach is incorporating the elements that simultaneously tackle multiple cancer-fighting targets into one molecule to obtain new chemical entity. Compared to single-target treatment, this kind of therapeutic regimens have superior efficacy and fewer side effects.2)
Protein kinases are important participants in the processes of governing cellular proliferation, differentiation and evasion from apoptosis. BRaf, as one member of the Raf family, plays a crucial role in the Ras/Raf/Mek/Erk (mitogen-activated protein kinase, MAPK) signaling pathway.3,4) The Mutation of BRaf, especially BRafV600E, is the most common in human cancers. Up to data, a lot of BRafV600E inhibitors have been developed such as sorafenib,5) dabrafenib, and vemurafenib (Fig. 1). They effectively block the MAPK signaling pathway and inhibit proliferation of tumor cells expressing BRafV600E. Histone deacetylases (HDACs) are important targets for tumor therapy.6–9) The HDACs family containing 18 isoforms is categorized into Class I (HDAC1, 2, 3, and 8), Class IIa (HDAC4, 5, 7, and 9), Class IIb (HDAC6 and 10), Class III (sirtuin1–7), and Class IV (HDAC11). Class I, II, and IV HDACs are all zinc-dependent deacetylases, and Class III HDACs are mechanistically distinct from other HDACs, which require nicotine adenine dinucleotide as a cofactor. The pharmacophore of HDACs inhibitors were generally composed of three parts: cap, linker, and zinc binding group (ZBG). Hydroxamic acid is the most frequently used ZBG group, three of which (i.e., vorinostat,10) belinostat,11) panobinostat12) have gained U.S. Food and Drug Administration approvals for clinical treatment.
A lot of literatures have described the synergistic and additive effects by the joint use of HDACs inhibitors and various antitumor agents.13–15) Recently, Emmons et al. revealed that HDACs inhibitor could enhance the durability of BRaf inhibitor therapy.16) Hence, the development of BRafV600E and HDACs dual inhibitors maybe a valuable strategy to circumvent resistance. Our group had previously reported a 2-(1H-imidazol-2-yl) pyridine derivative CLW27 possessing comparable antiproliferative activities to sorafenib in vitro or in vivo, and it showed inhibitory effect on BRaf and KDR-VEGFR2 kinases.17) In that compound, imidazole motief in the hinge region played a beneficial role for antitumor potency. For another, considering the genotoxicity of hydroxamic acid group and generated chromosomal aberrations in many cases,18,19) an alternative to hydroxamate as ZBG was tried in our design of novel Raf/HDAC dual inhibitors. Vasudevan et al. reported the use of acyl imidazole as ZBG in HDAC inhibitors, but it did not show potent inhibitory activity.20) However, the HDAC activity is not be determined only by the ZBG group, the spatial structures of cap and linker parts also have influence on activity. In this study, we incorporated acyl imidazole motif as ZBG and retained key urea group in sorafenib, then synthesized a series of novel derivatives (Fig. 2).
Compounds 7a–h were synthesized as follows. Nucleophilic reaction of ethyl 4-hydroxybenzoate (1) with 1-fluoro-4-nitrobenzene yielded intermediate 2 using potassium carbonate with acetonitrile as solvent. Compound 4 was obtained by mannich reaction with imidazole (3), formaldehyde and dimethylamine hydrochloride as materials. Nucleophilic addition of 2 and 4 in tetrahydrofuran yielded key intermediate 5 with n-BuLi as the base. Subsequently, reduction of 5 with iron powder as catalyst in ethanol afforded 6. Reaction of 6 with various arylamines in the presence of 1,1′-carbonyldiimidazole at room temperature gave the target compounds 7a–h (Chart 1).
Reagents and conditions: (a) 1-fluoro-4-nitrobenzene, K2CO3, CH3CN, Reflux, 4 h; (b) dimethylamine hydrochloride, CH2O, H2O, HCl, r.t., 48 h; (c) n-BuLi, THF, −78°C, 4 h; (d) iron powder, NH4Cl, H2O, C2H5OH, reflux, 4 h; (e) CDI, CH2Cl2, r.t. 12 h, then R-NH2, overnight.
The antitumor effect of the inhibition of the class I HDAC isoforms, especially HDAC1, was well confirmed.21–23) Therefore, all prepared derivatives were tested for their inhibitory ability against HDAC1, using SAHA as the reference compound. As shown in Table 1, IC50 values illustrated that compound 7c bearing a terminal 4-methoxylphenyl, exhibited the best inhibitory activity against HDAC1 (IC50 = 635 nM). However, it was much weaker than that of SAHA. While changing methoxyl from para-position to meta-position, compound 7f also showed a comparative activity (IC50 = 679 nM). Additionally, compounds with groups such as –Me (7b, 7g), –Cl or –CF3 (7a, 7d, and 7e) on phenyl inhibited HDAC1 with IC50 values in submicromolar or micromolar range. The activities against BRafV600E of these derivatives were also evaluated. 7a, which reserved the same 4-chloro-3-trifluoromethyl substituent as sorafenib, had the strongest activity (IC50 = 86 nM), although it was slightly less potent than sorafenib (IC50 = 38 nM). Compound 7d and 7e with electron-withdrawing substitutions also showed moderate inhibitory activities. However, electron-donating substitutions such as -Me or -OMe, were detrimental to BRafV600E inhibition, exemplified by 7b, 7c, 7f, and 7g, with IC50 values ranging from 0.840 to 4.727 µM. When the phenyl was replaced with pyridyl, obvious declines on both enzymatic activities were observed.
 |
Based on the enzymatic results, representative compounds 7a and 7c with better HDAC1 or BRafV600E activity were further evaluated against Raf and HDAC isoforms. As shown in Table 2, 7a also exhibited potent ARaf, BRafWT, and CRaf inhibitions. However, 7a and 7c showed no activities against HDAC6 and HDAC8.
| Cpd. | ARaf | BRafV600E | BRafWT | CRaf | HDAC1 | HDAC6 | HDAC8 |
|---|---|---|---|---|---|---|---|
| 7a | 0.045 | 0.086 | 0.134 | 0.102 | 1.710 | NAb) | NA |
| 7c | 1.103 | 1.853 | 0.226 | 2.374 | 0.635 | NA | NA |
| Sorafenib | 0.010 | 0.038 | 0.024 | 0.041 | / | / | / |
| SAHA | / | / | / | / | 0.037 | 0.026 | 3.21 |
a) We ran experiments in duplicate, S.D. <15%. b) NA: no activity was observed at 50 µM concentration.
The most potent compound 7a was further tested against another six kinases which were frequently used in our lab to evaluate its selectivity and to discovery potential other molecular targets. The initial screening of 7a was conducted at 10 µM concentration for the inhibitory rates. As summarized in Table 3, compound 7a showed weak inhibitory effects against KDR-VEGFR2 and EGFR with IC50 values of 19.210 µM and 35.470 µM, respectively, and had no obvious inhibition against CDK4/6, FLT3, and BCR/ABL kinases.
| Kinase | Inhibition rate% | IC50 (µM) |
|---|---|---|
| KDR-VEGFR2 | 41.75% | 19.210 |
| CDK4/cyclin D1 | 0.08% | / |
| CDK6/cyclin D1 | −2.05% | / |
| FLT3 | 9.20% | / |
| BCR-ABL | −0.13% | / |
| EGFR | 38.60% | 35.470 |
We chose compounds 7a, 7b, 7c, and reference compounds sorafenib and SAHA to evaluate antiproliferative activities against tumor cell lines K562 (leukemia), HCT116 (colon cancer) and A549 (non-small cell lung cancer). As shown in Table 4, all three compounds demonstrated obvious antiproliferative activities against all tumor cells, especially 7a, although it had the weakest HDAC1 inhibition. Compared to sorafenib, 7a showed overall more potent antiproliferative activity. 7a also exhibited superior activity in solid tumor cell line A549 (IC50 = 9.11 µM) to that of SAHA (IC50 = 18.13 µM). Besides, 7a was further tested against another two tumor cells SK-Mel-2 (malignant melanoma) and MV4-11 (leukemia) which had extraordinary expression of BRafV600E or BRafWT. 7a displayed submicromolar inhibiton toward MV4-11 with IC50 value of 0.38 µM. Especially, 7a had more potent activity against SK-Mel-2 than those of SAHA and sorafenib.
| Cpd. | K562 | HCT116 | A549 | SK-Mel-2 | MV4-11 |
|---|---|---|---|---|---|
| 7a | 9.13 | 10.87 | 9.11 | 5.40 | 0.38 |
| 7b | 12.09 | 14.24 | 16.25 | / | / |
| 7c | 10.10 | 11.26 | 13.37 | / | / |
| SAHA | 0.65 | 6.21 | 18.13 | 8.20 | 0.11 |
| Sorafenib | 11.20 | 12.25 | 10.63 | 9.30 | 0.42 |
a) IC50 values are averages of three independent experiments, S.D.<15%.
To further understand the interaction between the inhibitors and two proteins, we docked 7c in the active site of HDAC1 (PDB code: 5ICN) and 7a in BRafV600E (PDB code: 1UWJ) respectively. As shown in Fig. 3A, 7c exhibited excellent shape complementarity with the binding pocket of HDAC1. The acyl imidazole moiety could form bidentate coordination with zinc ion at the bottom of the pocket, with O-Zn2+ distance of 2.510 Å for C=O and N-Zn2+ distance of 2.731 Å for imidazole –NH–, respectively. Additionally, the O atom of carbonyl and the N atom of imidazole generated H-bond interactions with His178 and Cys151. Moreover, the phenyl linkage connected to acyl imidazole formed stacking π–π interactions with the residues of Phe150 and Phe205. The urea group and two adjacent phenyls occupied the surface groove and came into close contact with the residues (Gln26, Gly27, and His28) at the rim region. Another H-bond was formed between NH of urea and Glu98 to enhance their binding (Fig. 3B). Comparing the binding modes of sorafenib (Fig. 3C) and 7a (Fig. 3D) in BRafV600E, 7a reserved three key H-bond interactions between the urea group and the residues of Glu500 and Asp593, while the H-bond in the hinge was missed. This might be responsible for reduced potency against BRafV600E of 7a.
The zinc ion appears as a large gray sphere. Metal coordination between inhibitor and the catalytic zinc ion were shown in solid black lines. The H-bonding interactions with residues were labeled in dash line; (B) Docked position obtained for 7c in HDAC1. The HDAC1 protein was shown as a surface representation in gray. (C) The binding mode of sorafenib (wathet) in BRafV600E; (D) The binding mode of 7a in BRafV600E. For clarity, all hydrogen atoms were hidden.
In summary, we designed a series of novel Raf/HDAC dual inhibitors bearing acyl imidazole as ZBG. Eight target compounds were synthesized and tested against BRafV600E and HDAC1, and most compounds exhibited obvious inhibitory activities. Among these, compounds 7a and 7c were the most potent compounds against BRafV600E and HDAC1, respectively. Then 7a and 7c were submitted to evaluate the isoforms selectivities. The results showed these derivatives to be pan-Raf and selective HDAC1 inhibitors. Further in vitro antiproliferative assay showed that 7a, 7b, and 7c had inhibitory effects against several tumor cell lines including K562, HCT116, and A549. Moreover, 7a exhibited more potent antiproliferative activities against A549 and SK-Mel-2 cell lines than sorafenib and SAHA. Subsequently, molecular docking was performed to explain the structure–activity relationship. The demonstration of dual Raf/HDAC inhibitors in this paper provided useful tool compounds for further studies of multiple pathway inhibition achieved with a single molecule.
All of the starting materials were obtained commercially and were used without further purification. All of the reported yields were for isolated products and were not optimized. Melting points were determined in open capillaries on a WRS-1A digital melting point apparatus (Shenguang). 1H-NMR spectra was recorded in dimethyl sulfoxide (DMSO)-d6 on a Bruker DRX-500 (500 MHz) using TMS as internal standard. The chemical shifts were reported in ppm (δ) and coupling constants (J) values were given in Hertz (Hz). Mass spectra were obtained from Agilent 1100 LC/MSD (Agilent, U.S.A.) or Q-tof micro MS (Micromass, U.S.A.). The IR spectra were performed on a FTIR-8400S (Shimadzu, Japan) in KBr pellets; the frequencies are expressed in cm−1. The purities of all tested compounds were established by HPLC to be >95.0%. HPLC analysis was performed at room temperature using an Agilent Eclipse XDB-C18 (250 × 4.6 mm) and 20% MeOH/H2O as a mobile phase and plotted at 254 nm.
Ethyl 4-(4-Nitrophenoxy)benzoate (2)To a solution of ethyl 4-hydroxybenzoate (16.6 g, 0.1 mol), potassium carbonate (55.2 g, 0.4 mol) in anhydrous acetonitrile (300 mL) under nitrogen atmosphere was added fluoro-4-nitrobenzene (15.5 g, 0.11 mol). The mixture was refluxed for 4 h, then filtered. The mixture was concentrated in vacuo, then washed with water. The mother liquid was extracted with ethyl acetate (30 mL × 2). The combined organic extracts were dried over anhydrous MgSO4 and concentrated under reduced pressure. The product was obtained as a white solid by chromatography on a silica gel column (24.3 g, 84.4%); mp: 110–112°C. 1H-NMR (500 MHz, DMSO-d6) δ: 8.57 (dd, J = 7.0, 2.5 Hz, 2H), 8.31 (dd, J = 7.0, 2.3 Hz, 2H), 7.25–7.89 (m, 4H), 4.23 (q, J = 7.2 Hz, 2H), 1.25 (t, J = 7.2 Hz, 3H);
1-(1H-Imidazol-1-yl)-N,N-dimethylmethanamine (4)Imidazole (20.4 g, 0.3 mol) and dimethylamine hydrochloride (26.0 g, 0.3 mol) were added in 50 mL water at 0°C, then conc. hydrochloric acid was added to adjust pH to 4. Then resulting mixture was stirred for 10 min, and formalin (37%, 27 g, 0.33 mol) was added. The resulting mixture was stirred for additional 48 h at room temperature, then 20% KOH solution was added to adjust pH to 10. The reaction mixture was extracted with chloroform (20 mL × 3). The combined organic extracts were dried over anhydrous Na2SO4 and concentrated under reduced pressure. The product was obtained as a liquid (28.4 g, 75.6%). 1H-NMR (500 MHz, DMSO-d6) δ: 8.03 (s, 1H), 7.53 (d, J = 1.9 Hz, 1H), 7.14 (d, J = 1.8 Hz, 1H), 4.82 (s, 2H), 2.51 (s, 6H).
(1H-Imidazol-2-yl)(4-(4-nitrophenoxy)phenyl)methanone (5)Under nitrogen atmosphere, to a solution of 4 (1.25 g, 10 mmol) in THF (40 mL) at −78°C was added n-BuLi 2.5 M solution in n-hexane (4.2 mL), then the resulting mixture was stirred for 1 h at −78°C. A solution of 2 (3.01 g, 10.5 mmol) in THF (10 mL) was added. The mixture was stirred for 4 h at −78°C and monitored end by TLC. The mixture was acidified with 2 N HCl (80 mL) to adjust pH to 6–7 and extracted with ethyl acetate (40 mL × 3). The combined organic extracts were dried over anhydrous Na2SO4 and concentrated under reduced pressure. The product was obtained as a white solid by chromatography on a silica gel column (2.20 g, 71.2%); mp: 177–179°C; 1H-NMR (500 MHz, DMSO-d6) δ: 13.46 (br, 1H), 8.65 (dd, J = 7.0, 2.5 Hz, 2H), 8.31 (dd, J = 7.0, 2.3 Hz, 2H), 7.53 (d, J = 1.9 Hz, 1H), 7.34–7.29 (m, 5H).
(4-(4-Aminophenoxy)phenyl)(1H-imidazol-2-yl)methanone (6)To a solution of 5 (2.00 g, 7 mmol), ammonium chloride (0.37 g, 7 mmol) in water (20 mL) and ethanol (1 mL) was added iron powder (3.70 g, 70 mmol) in batches. The mixture was refluxed for 4 h, then filtered. The mother liquid was extracted with ethyl acetate (20 mL × 3). The combined organic extracts were dried over anhydrous MgSO4 and concentrated under reduced pressure. The product was obtained as a white solid by chromatography on a silica gel column (1.66 g, 85.0%); mp: 192–194°C; 1H-NMR (500 MHz, DMSO-d6) δ: 13.36 (br, 1H), 8.53 (dd, J = 8.8, 2.7 Hz, 2H), 7.48 (s, 1H), 7.27 (s, 1H), 6.97 (d, J = 8.9 Hz, 2H), 6.85 (d, J = 8.7 Hz, 2H), 6.63 (d, J = 8.7 Hz, 2H), 5.08 (s, 2H).
General Procedure for the Preparation of Target Compounds 7a–hTo a solution of 6 (3 mmol) in anhydrous dichloromethane (5 mL) was added CDI (0.58 g, 3.6 mmol) at room temperature. After the mixture was stirred for 12 h, different arylamine (3.6 mmol) was added. The mixture was stirred for overnight. After completion of the reaction as monitored by TLC, the reaction mixture was filtered. The filter cake was washed with CH2Cl2 (2 mL × 2) and dried in vacuo. The crude product was recrystallizated in mixed solvent of ethyl acetate and tetrahydrofuran (v/v = 1 : 1) to give the purified target compound.
1-(4-(4-(1H-Imidazole-2-carbonyl)phenoxy)phenyl)-3-(4-chloro-3-(trifluoromethyl)phenyl)urea (7a)White solid; yield 73.6%; mp: 235–237°C; 1H-NMR (500 MHz, DMSO-d6) δ: 13.37 (s, 1H), 9.16 (s, 1H), 8.91 (s, 1H), 8.58 (d, J = 9.0 Hz, 2H), 8.11 (d, J = 2.4 Hz, 1H), 7.66 (dd, J = 8.8, 2.4 Hz, 1H), 7.61 (d, J = 8.8 Hz, 1H), 7.55 (d, J = 8.9 Hz, 2H), 7.49 (d, J = 2.4 Hz, 1H), 7.28 (s, 1H), 7.12 (d, J = 8.9 Hz, 2H), 7.06 (d, J = 9.0 Hz, 2H); ESI-MS m/z: 501.1 ([M + H]+), 523.1 ([M + Na]+); IR (KBr, cm−1): 3344, 1674, 163, 1597, 1541, 1489, 1323, 906, 775, 611.
1-(4-(4-(1H-Imidazole-2-carbonyl)phenoxy)phenyl)-3-(p-tolyl)urea (7b)White solid; yield 75.7%; mp: 246–247°C; 1H-NMR (500 MHz, DMSO-d6) δ: 13.39 (s, 1H), 8.70 (s, 1H), 8.58 (d, J = 8.9 Hz, 2H), 8.55 (s, 1H), 7.53 (d, J = 8.9 Hz, 2H), 7.50 (d, J = 2.4 Hz, 1H), 7.35 (d, J = 8.3 Hz, 2H), 7.28 (s, 1H), 7.12-7.04 (m, 6H), 2.25 (s, 3H); ESI-MS m/z: 413.3 ([M + H]+); IR (KBr, cm−1): 3304, 1635, 1603, 1553, 1501, 1302, 1217, 823, 744, 665.
1-(4-(4-(1H-Imidazole-2-carbonyl)phenoxy)phenyl)-3-(4-methoxyphenyl)urea (7c)White solid; yield 76.1%; mp: 239–240°C; 1H-NMR (500 MHz, DMSO-d6) δ: 13.39 (s, 1H), 8.67 (s, 1H), 8.57 (d, J = 8.9 Hz, 2H), 8.47 (s, 1H), 7.54 (d, J = 9.0 Hz, 2H), 7.50 (s, 1H), 7.36 (d, J = 8.3 Hz, 2H), 7.28 (s, 1H), 7.10 (s, 2H), 7.05 (s, 2H), 6.88 (d, J = 8.9 Hz, 2H), 3.71 (s, 3H); ESI-MS m/z: 429.3 ([M + H]+); IR (KBr, cm−1): 3348, 1634, 1591, 1501, 1475, 1389, 1225, 903, 773, 662.
1-(4-(4-(1H-Imidazole-2-carbonyl)phenoxy)phenyl)-3-(4-chlorophenyl)urea (7d)White solid; yield 70.1%; mp: 268–270°C; 1H-NMR (500 MHz, DMSO-d6) δ: 13.40 (s, 1H), 8.82 (s, 1H), 8.80 (s, 1H), 8.56 (d, J = 9.0 Hz, 2H), 7.54 (d, J = 8.9 Hz, 2H), 7.51–7.47 (m, 3H), 7.33 (d, J = 8.9 Hz, 2H), 7.28 (s, 1H), 7.10 (d, J = 8.9 Hz, 2H), 7.06 (d, J = 8.9 Hz, 2H); ESI-MS m/z: 433.2 ([M + H]+); IR (KBr, cm−1): 3360, 1686, 1595, 1497, 1389, 1229, 905, 839, 768, 598.
1-(4-(4-(1H-Imidazole-2-carbonyl)phenoxy)phenyl)-3-(3,4-dichlorophenyl)urea (7e)White solid; yield: 78.1%; mp: 263–265°C; 1H-NMR (500 MHz, DMSO-d6) δ: 13.38 (s, 1H), 9.00 (s, 1H), 8.88 (s, 1H), 8.57 (d, J = 9.0 Hz, 2H), 7.88 (d, J = 2.4 Hz, 1H), 7.53 (d, J = 9.0 Hz, 2H), 7.51 (d, J = 9.0 Hz, 1H), 7.49 (d, J = 2.4 Hz, 1H), 7.35 (dd, J = 8.9, 2.5 Hz, 1H), 7.28 (s, 1H), 7.12 (d, J = 8.9 Hz, 2H), 7.06 (d, J = 9.0 Hz, 2H); ESI-MS m/z: 467.2 ([M + H]+); IR (KBr, cm−1): 3348, 1634, 1593, 1543, 1386, 1225, 903, 847, 771, 662.
1-(4-(4-(1H-Imidazole-2-carbonyl)phenoxy)phenyl)-3-(3-methoxyphenyl)urea (7f)White solid; yield: 70.7%; mp: 219–221°C; 1H-NMR (500 MHz, DMSO-d6) δ: 13.36 (s, 1H), 8.72 (s, 1H), 8.66 (s, 1H), 8.58 (d, J = 9.0 Hz, 2H), 7.55 (d, J = 9.0 Hz, 2H), 7.52 (s, 1H), 7.28 (s, 1H), 7.20–7.19 (m, 2H), 7.12 (d, J = 8.9 Hz, 2H), 7.07 (d, J = 9.0 Hz, 2H), 6.94 (d, J = 8.0 Hz, 1H), 6.55 (d, J = 7.5 Hz, 1H), 3.29 (s, 3H); ESI-MS m/z: 429.3 ([M + H]+); IR (KBr, cm−1): 3265, 1695, 1572, 1499, 1420, 1393, 1232, 901, 771, 656.
1-(4-(4-(1H-Imidazole-2-carbonyl)phenoxy)phenyl)-3-(m-tolyl)urea (7g)White solid; yield: 70.2%; mp: 233–235°C; 1H-NMR (500 MHz, DMSO-d6) δ: 13.38 (s, 1H), 8.72 (s, 1H), 8.58 (s, 1H), 8.56 (d, J = 9.0 Hz, 2H), 7.53 (d, J = 9.0 Hz, 2H), 7.49 (d, J = 2.4 Hz, 1H), 7.30 (s, 1H), 7.28 (s, 1H), 7.23 (d, J = 9.0 Hz, 1H), 7.16 (s, J = 8.0 Hz, 1H), 7.12 (d, J = 8.9 Hz, 2H), 7.05 (d, J = 9.0 Hz, 2H), 6.79 (d, J = 7.5 Hz, 1H), 2.28 (s, 3H); ESI-MS m/z: 413.3 ([M + H]+); IR (KBr, cm−1): 3300, 1639, 1568, 1398, 1240, 906, 847, 775, 658, 517.
1-(4-(4-(1H-Imidazole-2-carbonyl)phenoxy)phenyl)-3-(pyridin-2-yl)urea (7h)White solid; yield: 73.9%; mp: 230–232°C; 1H-NMR (500 MHz, DMSO-d6) δ: 13.40 (s, 1H), 10.61 (s, 1H), 9.47 (s, 1H), 8.58 (d, J = 8.8 Hz, 2H), 8.29 (d, J = 8.0 Hz, 1H), 7.76 (d, J = 8.5 Hz, 1H), 7.62 (d, J = 8.8 Hz, 2H), 7.49 (m, 2H), 7.28 (s, 1H), 7.14 (s, 2H), 7.07 (d, J = 8.9 Hz, 2H), 7.02 (t, J = 6.9 Hz, 1H); ESI-MS m/z: 400.3 ([M + H]+); IR (KBr, cm−1): 3275, 1639, 1603, 1558, 1402, 1234, 1159, 908, 849, 773.
In Vitro HDAC Enzyme AssayThe HDAC activity was determined using the HDAC fluorimetric activity assay kit (Biomol, Plymouth Meeting, PA, U.S.A.). Briefly, recombinant proteins of HDAC1, 6 and 8 was incubated with test compounds, and HDAC reaction was initiated by addition of Fluor-de-Lys substrate. Samples were incubated for 10 min at room temperature, followed by adding developer to stop the reaction. Fluorescence was measured by a fluorimetric reader with excitation at 360 nm and emission at 460 nm. The HDAC activity was expressed as arbitrary fluorescence units (AFU). The HDAC activity was calculated as a percentage of activity compared with the control group. The IC50 values for the test compounds were calculated using SigmaPlot software.
In Vitro Kinase Enzyme AssayActivity of full length BRafV600E was determined using Hot-SpotSM kinase assay. 5 nM of human GST-tagged BRafV600E protein (AA416–766) (Invitrogen, Cat# PV3894) was mixed with 20 µM of the substrate His 6-Tagged Full-length Human MEK1 (K97R) in reaction buffer (20 mM Hepes pH 7.5, 10 mM MgCl2, 1 mM ethylene glycol bis(2-aminoethyl ether)-N,N,N′,N′-tetra acetic acid (EGTA), 0.02% Brij35, 0.02 mg/mL BSA (albumin from bovine serum), 0.1 mM Na3VO4, 2 mM DTT, 1% DMSO at room temperature, the compounds dissolved in 100% DMSO at indicated doses (starting at 30 µM with 3-fold dilution) was delivered into the kinase reaction mixture by Acoustic technology (Echo550; nanoliter range), incubate for 20 min at room temperature. After 10 µM 33P-γ-ATP (specific activity 10 µCi/µL) (Perkin Elmer, NEG302H001 MC) was added to initiate the reaction, the reactions were carried out at 25°C for 2 h. The kinase activities were detected by filter-binding method. IC50 values and curve fits were obtained by Prism (GraphPad Software).
The Pan-Raf and other kinases inhibitory assay were in a manner same as BRafV600E.
Cell Culture and Cytotoxicity/Proliferation AssayThe antiproliferative activitives of compounds 7a, 7b, and 7c were evaluated against different tumor cell lines by the standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay in vitro, with SAHA and sorafenib as the positive control. The cancer cell lines were cultured in RPMI 1640 medium with 10% fetal bovine serum (FBS). Approximate 2.5 × 103 cells, suspended in RPMI 1640 medium, were plated into each well of a 96-well plate and incubated in 5% CO2 at 37°C for 24 h. The tested compounds at the indicated final concentrations were added to the culture medium and incubated for 48 h. Fresh MTT was added to each well at the terminal concentration of 0.5 mg/mL, and incubated with cells at 37°C for 4 h. After the supernatant was discarded, 150 µL DMSO was added to each well, and the absorbance values were determined by a microplate reader (Bio-Rad, Hercules, CA, U.S.A.) at 490 nm.
Computational MethodsAll computational work was performed in Discovery Studio 3.0 software (BIOVIA, 5005 Wateridge Vista Drive, San Diego, CA92121 U.S.A.). Docking was conducted using Cdocker module based on the cocrystal of BRafV600E (PDB: 1UWJ) and HDAC1 (PDB: 5ICN). Water molecules outside the binding pockets were excluded. The energy minimization for compounds was performed by Powell’s method for 1000 iterations using tripos force field and with Gasteiger–Hückel charge. The other docking parameters were kept as default.
This work was supported by Chinese Universities Scientific Fund (Grant No. 2452017174), the Scientific Startup Foundation for Doctors of Northwest A&F University (Grant No. 2452016146) and National Natural Science Foundation of China (Grant No. 31800031).
The authors declare no conflict of interest.
The online version of this article contains supplementary materials.
