Synthesis and antiproliferative activities against Hep-G2 of salicylanide derivatives: potent inhibitors of the epidermal growth factor receptor (EGFR) tyrosine kinase

Abstract

A series of salicylanilide derivatives (compounds 1–32) were synthesised by reacting substituted salicylic acids and anilines. The chemical structures of these compounds were determined by ¹H-NMR, electrospray ionisation mass spectrometry (ESI-MS) and elemental analysis. The compounds were assayed for their antiproliferative activities against the Hep-G2 cell line by the 3-(4,5-dimethylthylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. Among the compounds tested, 22 and 28 showed the most favouable antiproliferative activities with 50% inhibitory concentration (IC₅₀) values of 1.7 and 1.3 μM, respectively, which were comparable to the positive control of 5-fluorouracil (IC₅₀ = 1.8 μM). A solid-phase ELISA assay was also performed to evaluate the ability of compounds 1–32 to inhibit the autophosphorylation of the epidermal growth factor receptor tyrosine kinase (EGFR TK). Docking simulations of 22 and 28 were carried out to illustrate the binding mode of the molecule into the EGFR active site, and the result suggested that both compounds 22 and 28 could bind the EGFR kinase well.

Keywords::

• Salicylanilide derivatives
• antiproliferative activities
• EGFR TK
• structure-activity relationship
• docking simulations

Introduction

Liver cancer is one of the most threatening diseases in the world today. A study made by the International Agency for Research on Cancer (IARC) indicated that liver cancer was the third common cancer and caused more than 620,000 deaths per year all over the world. There are several drugs for the treatment of liver cancer such as doxorubicin, fluorouracil, cisplatin, and α-interferon, but all of them have serious side effects and therefore limit their clinical applications. Due to the shortage of effective drugs, the discovery of new compounds with potent anti-hepatoma activities is a very important task. Here, we synthesised a series of salicylanilide derivatives (compounds 1–32). Salicylanilides are usually synthesised from substituted salicylic acids and anilines. Salicylanilide derivatives isolated from natural plants have been reported to possess antiproliferative activities against the Hep-G2 cell line, a type of liver cancer cell often selected for research to test compounds that may possess potent activity for liver cancer treatment [1–3]. Pei-Wen Hsieh et al. reported the isolation of 4-methoxydianthramide B, a salicylanilide derivative, from the traditional Chinese medicinal plant Dianthus superbus. The compound 4-methoxydianthramide B showed antiproliferative activities against the Hep-G2 cancer cell line with a 50% inhibitory concentration (IC₅₀) value of 4.08 μg/mL [4]. This investigation led to the idea that salicylanilide derivatives could possess potential antiproliferative activities against the Hep-G2 cell line, and by structure activity relationship (SAR), the results may be useful to gain more understanding about the antiproliferative activities of salicylanilide derivatives. In this paper, we report the synthesis of some salicylanilide compounds and their antiproliferative activities against the Hep-G2 cell line.

Docking simulations of compounds 22 and 28 were carried out to give structural insights into the binding mode with epidermal growth factor receptor tyrosine kinase (EGFR TK) [5–8], to illustrate the antiproliferative activities against the Hep-G2 cancer cell line. Molecules designed to block EGFR TK, a class of potent, selective, ATP-competitive inhibitors of EGFR TK, induced signaling on their own and further degrade to a DNA damaging species, should induce significant cell-killing in tumours [9,10]. The first molecular probe designed to verify the combi-targeting postulates, was shown to strongly block the EGFR TK activity on its own in a short exposure enzyme assay [11,12]. The receptor protein tyrosine kinases played a key role in signal transduction pathways that regulate cell division and differentiation. The interaction of growth factors with these receptors was a necessary event in the normal regulation of cell growth. However, under certain conditions, as a result of overexpression, mutation, or coexpression of the ligand and the receptor, these receptors could become hyperactivated and induce uncontrolled cell proliferation [13]. Among the growth factor receptor kinases, EGFR kinase (also known as erb-B1 or HER-1) was important in cancer deregulation of growth-factor signaling due to that hyperactivation of EGFR was seen in several cancer types [14,15]. Activation of EGFR might be a result of overexpression, mutations resulting in constitutive activation, or autocrine expression of the ligand. EGFR overexpression was often seen in various cancers [16–18]. Compounds that inhibit the kinase activity of EGFR after binding of its cognate ligand were of potential interest as new therapeutic antitumor agents [19,20].

Chemistry

A series of salicylanilides were synthesised by reacting four kinds of substituted salicylic acids and several kinds of substituted anilines (Scheme 1). All the compounds gave satisfactory chemical analysis. The chemical structures of these compounds were determined by ¹H-NMR, electrospray ionisation mass spectrometry (ESI-MS) spectra and elemental analyses.

Scheme 1.  Synthesis of salicylanilide derivatives 1–32. (a) EDC, CH₂Cl₂, reflux, 8 h, 70–86% yield.

Results and discussion

Biological activity

The in vitro antiproliferative activities of the synthesised salicylanilide derivatives 1–32 were studied using the human liver cancer cell line Hep-G2 by applying the 3-(4,5-dimethylthylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay. The compounds were tested over a range of concentrations from 0.5 to 300 μM, and the calculated IC50 values i.e. the concentration (μM) of compounds which were able to cause 50% of cell death with respect to the control culture, are reported in Table 2.

Compounds 1–32 could be classed into 4 series: a) salicylic acid series; b) 4-methyl salicylic acid series; c) 5-iodo salicylic acid series; d) 3,5-dibromo salicylic acid series (Table 1). Among these salicylanilide derivatives tested, compounds 22 and 28 were found to show the most potent activities with IC50 values of 1.8 and 1.3 μM, respectively. Compounds 21, 24, 25, 26, 27, 30, 31 and 32 showed the best antiproliferative activities with IC50 values ranging from 5-50 μM. The remaining compounds showed weak antiproliferative activities with IC50 values ranging from 50–300 μM (compounds 2–7, 9–12, 14, 15, 18, 19 and 29), or not active with IC50 values over 300 μM (compounds 1, 8, 13, 16, 17, 20, 23).

Table 1.  Physical Properties of salicylanilide derivatives 1–32.

Compound	R1	R2	R3	R4	R5	Formula	Mp °C	Yield, %
1	H	H	H	H	H	C13H11NO2	127–129	74
2	H	H	H	F	H	C13H10FNO2	120–122	78
3	H	H	H	Cl	H	C13H10ClNO2	161–162	77
4	H	H	H	Br	H	C13H10BrNO2	155–157	85
5	H	H	H	H	F	C13H10FNO2	157–158	79
6	H	H	H	H	Cl	C13H10ClNO2	157–159	83
7	H	H	H	H	Br	C13H10BrNO2	159–161	71
8	H	H	H	F	F	C13H9F2NO2	183–185	75
9	H	H	H	Cl	Cl	C13H9Cl2NO2	191–193	70
10	H	H	H	H	CH3	C14H13NO2	145–147	81
11	H	H	H	H	OCH3	C14H13NO3	156–157	86
12	H	H	H	H	CH(CH3)2	C16H17NO2	90–92	72
13	H	CH3	H	H	H	C14H13NO2	193–195	77
14	H	CH3	H	Br	H	C14H12BrNO2	165–167	83
15	H	CH3	H	H	F	C14H12FNO2	135–137	81
16	H	CH3	H	H	Cl	C14H12ClNO2	158–160	80
17	H	CH3	H	F	F	C14H11F2NO2	196–198	71
18	H	CH3	H	Cl	Cl	C14H11Cl2NO2	171–173	76
19	H	CH3	H	H	CH3	C15H15NO2	154–156	73
20	H	CH3	H	H	CH(CH3)2	C17H19NO2	173–175	82
21	H	H	I	Cl	H	C13H9ClINO2	194–195	85
22	H	H	I	Br	H	C13H9BrINO2	181–182	70
23	H	H	I	H	F	C13H9FINO2	230–232	81
24	H	H	I	H	Cl	C13H9ClINO2	238–240	77
25	H	H	I	H	Br	C13H9BrINO2	236–237	74
26	H	H	I	Cl	Cl	C13H8Cl2INO2	209–211	86
27	H	H	I	H	CH3	C14H12INO2	241–243	79
28	H	H	I	H	OCH3	C14H12INO3	232–234	75
29	Br	H	Br	H	F	C13H8Br2FNO2	204–205	85
30	Br	H	Br	H	Cl	C13H8Br2ClNO2	200–202	73
31	Br	H	Br	H	Br	C13H8Br3NO2	166–168	71
32	Br	H	Br	H	OCH3	C14H11Br2NO3	153–155	82

Table 2.  Antiproliferative activity data and inhibition of EGFR kinase for the salicylanilide derivatives 1–32.^a

Compound	Antiproliferative effects against Hep-G2IC50 (μM)	EGFR inhibitionIC50 (μM)
1	NA	>50
2	80 ± 28	24.83
3	52 ± 17	14.18
4	61 ± 10	21.92
5	246 ± 69	>50
6	137 ± 37	41.76
7	140 ± 38	>50
8	NA	>50
9	114 ± 28	45.29
10	84 ± 26	25.74
11	111 ± 21	39.98
12	82 ± 31	29.62
13	NA	>50
14	56 ± 13	22.19
15	110 ± 49	40.17
16	NA	>50
17	NA	>50
18	78 ± 17	31.09
19	104 ± 33	34.67
20	NA	>50
21	5.9 ± 1.3	4.37
22	1.7 ± 0.28	0.92
23	NA	>50
24	24 ± 3.5	13.04
25	24 ± 7.1	13.7
26	19 ± 2.5	9.66
27	5.1 ± 0.85	2.68
28	1.3 ± 0.2	0.63
29	154 ± 44	>50
30	22 ± 5.2	12.19
31	47 ± 16	28.53
32	37 ± 1	22.39
5-fluorouracil	1.8 ± 0.23	0.32

^aNA, not active, the IC₅₀ is greater than the maximal concentration tested (300 μM).

Compounds 1–32 were evaluated for their ability to inhibit the autophosphorylation of EGFR kinases using a solid-phase ELISA assay. A number of the synthesised compounds showed potent EGFR inhibitory activities. The results showed the same trends for antiproliferative activities against Hep-G2 as EGFR was an important factor in liver cancer. (Table 2). Here, again, compounds 22 and 28 showed the most potent inhibitory activities (IC₅₀ = 1.7 μM and 1.3 μM, respectively), and these were comparable to the positive control 5-fluorouracil (IC₅₀ = 1.8 μM).

Among the four salicylic acid series, SAR studies demonstrated that almost all the compounds belonging to the 5-iodo salicylic acid series showed the best activity both in the antiproliferative activities against Hep-G2 cell line and for EGFR inhibition. The results indicated that the iodine atom substituent at the 5-position could significantly increase the activity. Accordingly, compounds 22 and 28, which contained an iodinated substituent at the 5-position, showed the best activity.

In every salicylic acid series, for instance a) salicylic acid series and c) 5-iodo salicylic acid series, substituents at the 2′-position could significantly increase the activity compared with substituents at the 4′-position [Table 1].

Furthermore, among these compounds where substituents took place at the 4′-position, electron-donating groups such as Me and iPr groups, showed better activity than electron-withdrawing groups such as OMe, F, Cl and Br.

However, the SAR result summarised above was a brief overview of the whole 32 compounds synthesised, and for compound 28, was a little different and seemed not to obey the SAR rule stated above. Docking simulations suggested that compound 28 had a good binding activity with EGFR kinase, and due to its chemical structure 28 was also a good EGFR inhibitor, and thus had good antiproliferative activity against Hep-G2.

Binding Mode of 22 and 28 into EGFR Kinase

To give an structural insight into the ligand/enzyme interactions, and to give an explanation and understanding of good activity observed, molecular docking of the most active compounds 22 and 28 into the ATP binding site of EGFR kinase, the binding model based on the EGFR complex structure (PDB code: 1M17, [10]) was performed using the automated docking tools AutoDock (version 4.0) [21–23]. The binding models of compounds 22 and 28 into EGFR were depicted in Figures 1 and 2.

Figure 1.  Binding mode of compound 22 with EGFR kinase. For clarity only the interacting residues were displayed. Ligand (green) and interacting key residues (white) were represented as stick models, while the proteins (white) were represented as ribbons. The H-bond was displayed as spherical surface, and the cation-π interaction was displayed as coniform surface.

Figure 2.  Binding mode of compound 28 with EGFR kinase. For clarity only the interacting residues were displayed. Ligand (green) and interacting key residues (white) were represented as stick models, while the proteins (white) were represented as ribbons. The H-bond was displayed as spherical surface, and the cation-π interaction was displayed as coniform surface.

Docking studies of both compounds 22 and 28 into the active site of EGFR provided well clustered solutions. In the binding model of compound 22 and EGFR, there was a hydrogen bond between the carbonyl oxygen of 22 and the N-H of the Lys828 side chain. Moreover, a cation-π interaction between the Lys828 side chain and the aniline ring (C1′, C2′, C3′, C4′, C5′, C6′ [Table 1]) of 22 was also observed. Cation-π interaction is a noncovalent molecular interaction between the face of an electron-rich π system (e.g. benzene, ethylene) with an adjacent cation. This unusual interaction is an example of noncovalent bonding between a monopole (cation) and a quadrupole (π system). Cation-π interaction energies are of the same order of magnitude as hydrogen bonds or salt bridges and play an important role in molecular recognition [24]. Cation-π interaction made the 22/EGFR kinase complex more stable.

In the binding model of compound 28 and EGFR, there was also a cation-π interaction between the Lys828 side chain and the salicylic ring (C1, C2, C3, C4, C5 and C6 shown in Table 1) of 28. Unexpectedly, there were two H-bonds observable for compound 28: one between the N-H of 28 and the carboxylate of Gln767, and the other between the phenolic hydroxyl of 28 and the carboxylate of Gln767.

In both cases, the binding was further stabilised by hydrophobic interactions between the phenyl group and a hydrophobic region, comprised of the side chains of Leu754, Thr766 and the Met769 side chains.

Conclusions

A series of salicylanilide derivatives were synthesised by reacting substituted salicylic acids and anilines then evaluated for antiproliferative activities against the human cancer cell line Hep-G2. Compounds 22 and 28 showed the most potent antiproliferative activities with IC₅₀ values of 1.7 and 1.3 μM. The EGFR inhibitory ability of these synthesised salicylanilide derivatives were also evaluated using a solid-phase ELISA assay, which had almost the same trend as the antiproliferative activities assay. SAR results suggested that the substituents at the 5-position and 2′-position could increase the activity significantly. Docking simulations were performed to give the probable binding modes of compounds 22 and 28 into the ATP binding site of EGFR kinase. Compounds 22 and 28 both formed a cation-π interaction with Lys828 through their phenyl rings. However, compound 28 formed two H-bonds with Gln767 through its N-H and phenolic hydroxyl into the EGFR binding site, and compound 22 formed only one H-bond with Lys828 through its carbonyl oxygen. Still, both 22 and 28 could bind the EGFR kinase well. The result indicated that compounds 22 and 28, in particular, had significant Hep-G2 antiproliferative activities and EGFR inhibitory activity, and are promising potential agents for the treatment of liver cancer.

Experimental Section

Chemistry

All chemicals (reagent grade) used in the experiment were purchased from Aldrich (St. Louis, USA) and Sinopharm Chemical Reagent (Shanghai, China). Melting points (uncorrected) were determined on a XT4 MP apparatus (Taike, Beijing). TLC was run on the silica gel coated aluminum sheets (silica gel 60 GF₂₅₄,E. Merk, Darmstadt, Germany) and visualised in UV light (254 nm). EI spectra were obtained on a Waters GCT mass spectrometer (Illinois, USA), and ¹H NMR spectra were recorded on a Bruker DPX-300, AV-300 or AV-500 spectrometer (Rheinstetten, Germany) at 25 °C with TMS and solvent signals allotted as internal standards. Chemical shifts were reported in ppm (δ). Elemental analyses were performed on a CHN-O-Rapid instrument (Hanau, Germany) and were within ± 0.4 % of the theoretical values.

General method of synthesis 1–32

Equimolar quantities (1 mmol) of substituted salicylic acid and aniline were dissolved in CH₂Cl₂ (5 mL), and 1 mmol EDC (1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide) were also added to the solution as catalyst. The solution was then stirred under refluxing for approximately 8 hours. The solvent was evaporated and the residue purified by column chromatography on silica gel, eluting with petroleum ether/EtOAc (3:1) to give a white, light yellow or brown solid of high purity with a good to high yield (70–86%).

2-Hydroxy-N-phenylbenzamide (1)

Light yellow powder, mp 127–129°C ¹H NMR (300 MHz, d₆-DMSO): 6.97 (t, J = 8.3 Hz, 2H); 7.15 (t, J = 7.5 Hz, 1H); 7.35–7.47 (m, 3H); 7.71 (d, J = 7.7 Hz, 2H); 7.97 (dd, J₁ = 1.3 Hz, J₂ = 7.7 Hz, 1H); 10.37 (s, 1H); 11.79 (s, 1H). MS (ESI): 214.1 (C₁₃H₁₂NO₂, [M+H]⁺). Anal. Calcd for C₁₃H₁₁NO₂: C, 73.23; H, 5.2; N, 6.57%; Found: C, 73.31; H, 5.17; N, 6.54%.

N-(2-fluorophenyl)-2-hydroxybenzamide (2)

White powder, mp 120–122°C ¹H NMR (300 MHz, d₆-DMSO): 7.03 (d, J = 8.8 Hz, 2H); 7.16–7.36 (m, 3H); 7.43–7.48 (m, 1H); 8.03 (d, J = 7.9 Hz, 1H); 8.18–8.24 (m, 1H); 10.69 (s, 1H); 11.91 (s, 1H). MS (ESI): 232.1 (C₁₃H₁₁FNO₂, [M+H]⁺). Anal. Calcd for C₁₃H₁₀FNO₂: C, 67.53; H, 4.36; F, 8.22; N, 6.06%; Found: C, 67.42; H, 4.34; F, 8.26; N, 6.08%.

N-(2-chlorophenyl)-2-hydroxybenzamide (3)

White powder, mp 161–162°C ¹H NMR (500 MHz, d₆-DMSO): 7–7.05 (m, 2H); 7.17–7.2 (m, 1H); 7.37–7.41 (m, 1H); 7.44–7.48 (m, 1H); 7.56 (dd, J₁ = 1.5 Hz, J₂ = 8 Hz, 1H); 8.05 (dd, J₁ = 1.8 Hz, J₂ = 8 Hz, 1H); 8.39 (dd, J₁ = 1.5 Hz, J₂ = 8.2 Hz, 1H); 10.88 (s, 1H); 11.91 (s, 1H). MS (ESI): 248 (C₁₃H₁₁ClNO₂, [M+H]⁺). Anal. Calcd for C₁₃H₁₀ClNO₂: C, 63.04; H, 4.07; Cl, 14.31; N, 5.66%; Found: C, 63.13; H, 4.02; Cl, 14.28; N, 5.69%.

N-(2-bromophenyl)-2-hydroxybenzamide (4)

White powder, mp 155–157°C ¹H NMR (500 MHz, d₆-DMSO): 6.99–7.05 (m, 2H); 7.11–7.14 (m, 1H); 7.41–7.48 (m, 2H); 7.71 (dd, J₁ = 1.6 Hz, J₂ = 8.3 Hz, 1H); 8.05 (dd, J₁ = 1.9 Hz, J₂ = 8 Hz, 1H); 8.3 (dd, J₁ = 1.6 Hz, J₂ = 8.3 Hz); 10.76 (s, 1H); 11.91 (s, 1H). MS (ESI): 293.9 (C₁₃H₁₁BrNO₂, [M+H]⁺). Anal. Calcd for C₁₃H₁₀BrNO₂: C, 53.45; H, 3.45; Br, 27.35; N, 4.79%; Found: C, 53.52; H, 3.42; Br, 27.36; N, 4.75%.

N-(4-fluorophenyl)-2-hydroxybenzamide (5)

White powder, mp 157–158°C ¹H NMR (500 MHz, d₆-DMSO): 6.95–6.99 (m, 2H); 7.19–7.23 (m, 2H); 7.42–7.46 (m, 1H); 7.71–7.74 (m, 2H); 7.95 (dd, J₁ = 1.5 Hz, J₂ = 7.8 Hz, 1H); 10.39 (s, 1H); 11.75 (s, 1H). MS (ESI): 232.1 (C₁₃H₁₁FNO₂, [M+H]⁺). Anal. Calcd for C₁₃H₁₀FNO₂: C, 67.53; H, 4.36; F, 8.22; N, 6.06%; Found: C, 67.61; H, 4.32; F, 8.24; N, 6.01%.

N-(4-chlorophenyl)-2-hydroxybenzamide (6)

Light yellow powder, mp 157–159°C ¹H NMR (300 MHz, d₆-DMSO): 6.99 (d, J = 8.4 Hz, 2H); 7.43 (dd, J₁ = 1.8 Hz, J₂ = 7 Hz, 3H); 7.76 (dd, J₁ = 2 Hz, J₂ = 6.9 Hz, 2H); 7.93 (dd, J₁= 1.7 Hz, J₂ = 7.9 Hz, 1H); 10.44 (s, 1H); 11.63 (s, 1H). MS (ESI): 248.7 (C₁₃H₁₁ClNO₂, [M+H]⁺). Anal. Calcd for C₁₃H₁₀ClNO₂: C, 63.04; H, 4.07; Cl, 14.31; N, 5.66%; Found: C, 62.98; H, 4.1; Cl, 14.29; N, 5.7%.

N-(4-bromophenyl)-2-hydroxybenzamide (7)

White powder, mp 159–161°C ¹H NMR (300 MHz, d₆-DMSO): 6.94–7 (m, 2H); 7.41–7.47 (m, 1H); 7.56 (d, J = 8.8 Hz, 2H); 7.71 (d, J = 8.8 Hz, 2H); 7.93 (d, J = 7.9 Hz, 1H); 10.45 (s, 1H); 11.63 (s, 1H). MS (ESI): 293.1 (C₁₃H₁₁BrNO₂, [M+H]⁺). Anal. Calcd for C₁₃H₁₀BrNO₂: C, 53.45; H, 3.45; Br, 27.35; N, 4.79%; Found: C, 53.5; H, 3.48; Br, 27.28; N, 4.77%.

N-(2,4-difluorophenyl)-2-hydroxybenzamide (8)

Brown powder, mp 183–185°C ¹H NMR (500 MHz, d₆-DMSO): 6.98–7.02 (m, 2H); 7.12–7.15 (m, 1H); 7.37–7.42 (m, 1H); 7.44–7.48 (m, 1H); 8.01 (dd, J₁ = 1.8 Hz, J₂ = 8.0 Hz, 1H); 8.09–8.13 (m, 1H); 10.58 (s, 1H); 11.89 (s, 1H). MS (ESI): 250.2 (C₁₃H₁₀F₂NO₂, [M+H]⁺). Anal. Calcd for C₁₃H₉F₂NO₂: C, 62.65; H, 3.64; F, 15.25; N, 5.62%; Found: C, 62.58; H, 3.66; F, 15.19; N, 5.64%.

N-(2,4-dichlorophenyl)-2-hydroxybenzamide (9)

Light yellow powder, mp 191–193°C ¹H NMR (300 MHz, d₆-DMSO): 6.99–7.06 (m, 2H); 7.44–7.5 (m, 2H); 7.74 (d, J = 2.4 Hz, 1H); 8.04 (d, J = 7.9 Hz, 1H); 8.46 (d, J = 8.8 Hz, 1H); 10.94 (s, 1H); 11.95 (s, 1H). MS (ESI): 283.1 (C₁₃H₁₀Cl₂NO₂, [M+H]⁺). Anal. Calcd for C₁₃H₉Cl₂NO₂: C, 55.34; H, 3.22; Cl, 25.13; N, 4.96%; Found: C, 55.24; H, 3.23; Cl, 25.19; N, 4.99%.

2-Hydroxy-N-p-tolylbenzamide (10)

Light yellow powder, mp 145–147°C ¹H NMR (300 MHz, d₆-DMSO): 2.29 (s, 3H); 6.98 (d, J = 7.7 Hz, 2H); 7.18 (d, J = 8.2 Hz, 2H); 7.41-7.47 (m, 1H); 7.59 (d, J = 8.4 Hz, 2H); 7.97 (d, J = 8.2 Hz, 1H); 10.32 (s, 1H); 11.89 (s, 1H). MS (ESI): 228.3 (C₁₄H₁₄NO₂, [M+H]⁺). Anal. Calcd for C₁₄H₁₃NO₂: C, 73.99; H, 5.77; N, 6.16%; Found: C, 73.87; H, 5.79; N, 6.21%.

2-Hydroxy-N-(4-methoxyphenyl)benzamide (11)

White powder, mp 156–157°C ¹H NMR (500 MHz, d₆-DMSO): 3.76 (s, 3H); 6.94–6.97 (m, 4H); 7.42–7.45 (m, 1H); 7.6 (d, J = 9.2 Hz, 2H); 7.98 (d, J = 7.7 Hz, 1H); 10.27 (s, 1H); 11.99 (s, 1H). MS (ESI): 244.3 (C₁₄H₁₄NO₃, [M+H]⁺). Anal. Calcd for C₁₄H₁₃NO₃: C, 69.12; H, 5.39; N, 5.76%; Found: C, 69.24; H, 5.34; N, 5.80%.

2-Hydroxy-N-(4-isopropylphenyl)benzamide (12)

White powder, mp 90–92°C ¹H NMR (300 MHz, d₆-DMSO): 1.21 (d, J = 7.0 Hz, 6H); 2.84–2.93 (m, 1H); 6.97 (d, J = 7.9Hz, 2H); 7.24 (d, J = 8.4 Hz, 2H); 7.41–7.46 (m, 1H); 7.61 (d, J = 8.4 Hz, 2H); 7.98 (d, J = 8.2 Hz, 1H); 10.31 (s, 1H); 11.89 (s, 1H). MS (ESI): 256.3 (C₁₆H₁₈NO₂, [M+H]⁺). Anal. Calcd for C₁₆H₁₇NO₂: C, 75.27; H, 6.71; N, 5.49%; Found: C, 75.35; H, 6.68; N, 5.46%.

2-Hydroxy-4-methyl-N-phenylbenzamide (13)

White powder, mp 193–195°C ¹H NMR (300 MHz, d₆-DMSO): 2.31 (s, 3H); 6.79 (d, J = 4.9 Hz, 2H); 7.14 (t, J = 7.5 Hz, 1H); 7.37 (t, J = 8 Hz, 2H); 7.69 (d, J = 7.5 Hz, 2H); 7.91 (d, J = 8.4 Hz, 1H); 10.31 (s, 1H); 11.93 (s, 1H). MS (ESI): 228.3 (C₁₄H₁₄NO₂, [M+H]⁺). Anal. Calcd for C₁₄H₁₃NO₂: C, 73.99; H, 5.77; N, 6.16%; Found: C, 73.87; H, 5.81; N, 6.19%.

N-(2-bromophenyl)-2-hydroxy-4-methylbenzamide (14)

White powder, mp 165–167°C ¹H NMR (500 MHz, d₆-DMSO): 2.31 (s, 3H); 6.81–6.84 (m, 2H); 7.1–7.13 (m, 1H); 7.4–7.43 (m, 1H); 7.70 (dd, J₁ = 1.5 Hz, J₂ = 8.3 Hz, 1H); 7.93 (dd, J₁ = 1.5 Hz, J₂ = 8 Hz, 1H); 8.29 (dd, J₁ = 1.5 Hz, J₂ = 8.3 Hz, 1H); 10.68 (s, 1H); 11.83 (s, 1H). MS (ESI): 307.2 (C₁₄H₁₃BrNO₂, [M+H]⁺). Anal. Calcd for C₁₄H₁₂BrNO₂: C, 54.92; H, 3.95; Br, 26.1; N, 4.58%; Found: C, 55.01; H, 3.94; Br, 26.07; N, 4.59%.

N-(4-fluorophenyl)-2-hydroxy-4-methylbenzamide (15)

White powder, mp 135–137°C ¹H NMR (300 MHz, d₆-DMSO): 2.30 (s, 3H); 6.79 (d, J = 4.4 Hz, 2H); 7.21 (t, J = 9 Hz, 2H); 7.69–7.73 (m, 2H); 7.89 (d, J = 8.2 Hz, 1H); 10.34 (s, 1H); 11.9 (s, 1H). MS (ESI): 246.3 (C₁₄H₁₃FNO₂, [M+H]⁺). Anal. Calcd for C₁₄H₁₂FNO₂: C, 68.56; H, 4.93; F, 7.75; N, 5.71%; Found: C, 68.69; H, 4.89; F, 7.72; N, 5.69%.

N-(4-chlorophenyl)-2-hydroxy-4-methylbenzamide (16)

White powder, mp 158–160°C ¹H NMR (300 MHz, d₆-DMSO): 2.3 (s, 3H); 6.79 (d, J = 6.4 Hz, 2H); 7.43 (dd, J₁ = 2 Hz, J₂ = 6.8 Hz, 2H); 7.75 (dd, J₁ = 2 Hz, J₂ = 6.8 Hz, 2H); 7.87 (d, J = 8.4 Hz); 10.4 (s, 1H); 11.79 (s, 1H). MS (ESI): 262.7 (C₁₄H₁₃ClNO₂, [M+H]⁺). Anal. Calcd for C₁₄H₁₂ClNO₂: C, 64.25; H, 4.62; Cl, 13.55; N, 5.35%; Found: C, 64.31; H, 4.64; Cl, 13.49; N, 5.37%.

N-(2,4-difluorophenyl)-2-hydroxy-4-methylbenzamide (17)

Light yellow powder, mp 196–198°C ¹H NMR (300 MHz, d₆-DMSO): 2.3 (s, 3H); 6.81 (d, J = 5 Hz, 2H); 7.13 (t, J = 8.6 Hz, 1H); 7.35-7.43 (m, 1H); 7.91 (d, J = 8.4 Hz, 1H); 8.05-8.13 (m, 1H); 10.52 (s, 1H); 11.87 (s, 1H). MS (ESI): 264.2 (C₁₄H₁₂F₂NO₂, [M+H]⁺). Anal. Calcd for C₁₄H₁₁F₂NO₂: C, 63.88; H, 4.21; F, 14.43; N, 5.32%; Found: C, 63.97; H, 4.18; F, 14.38; N, 5.29%.

N-(2,4-dichlorophenyl)-2-hydroxy-4-methylbenzamide (18)

White powder, mp 171–173°C ¹H NMR (500 MHz, d₆-DMSO): 2.31 (s, 3H); 6.85–6.87 (m, 2H); 7.2 (s, 1H); 7.26 (d, J = 8.0 Hz, 1H); 7.88–7.90 (m, 2H); 10.1 (s, 1H); 12.88 (br s, 1H). MS (ESI): 297.2 (C₁₄H₁₂Cl₂NO₂, [M+H]⁺). Anal. Calcd for C₁₄H₁₁Cl₂NO₂: C, 56.78; H, 3.74; Cl, 23.94; N, 4.73%; Found: C, 56.7; H, 3.78; Cl, 23.99; N, 4.75%.

2-Hydroxy-4-methyl-N-p-tolylbenzamide (19)

Light yellow powder, mp 154–156°C ¹H NMR (300 MHz, d₆-DMSO): 2.29 (s, 3H); 2.3 (s, 3H); 6.78 (d, J = 4.8 Hz, 2H); 7.18 (d, J = 8.3 Hz, 2H); 7.57 (d, J = 8.4 Hz, 2H); 7.91 (d, J = 8.4 Hz, 1H); 10.24 (s, 1H); 12.02 (s, 1H). MS (ESI): 242.3 (C₁₅H₁₆NO₂, [M+H]⁺). Anal. Calcd for C₁₅H₁₅NO₂: C, 74.67; H, 6.27; N, 5.81%; Found: C, 74.8 H, 6.22; N, 5.78%.

2-Hydroxy-N-(4-isopropylphenyl)-4-methylbenzamide (20)

White powder, mp 173–175°C ¹H NMR (300 MHz, d₆-DMSO): 1.21 (d, J = 7 Hz, 6H); 2.3 (s, 3H); 2.86–2.9 (m, 1H); 6.77–6.79 (m, 2H); 7.24 (d, J = 8.4 Hz, 2H); 7.6 (d, J = 8.6 Hz, 2H); 7.91 (d, J = 8.4 Hz, 1H); 10.28 (s, 1H); 12.04 (s, 1H). MS (ESI): 270.3 (C₁₇H₂₀NO₂, [M+H]⁺). Anal. Calcd for C₁₇H₁₉NO₂: C, 75.81; H, 7.11; N, 5.20%; Found: C, 75.7; H, 7.16; N, 5.23%.

N-(2-chlorophenyl)-2-hydroxy-5-iodobenzamide (21)

Brown powder, mp 194–195°C ¹H NMR (500 MHz, d₆-DMSO): 7.02 (d, J = 8.5 Hz, 1H); 7.2 (t, J = 7.7 Hz, 1H); 7.4 (t, J = 7.7 Hz, 1H); 7.57 (d, J = 8 Hz, 1H); 7.62 (dd, J₁ = 2.5 Hz, J₂ = 8.5 Hz, 1H); 8.13 (d, J = 2.5 Hz, 1H); 8.38 (d, J = 8.2 Hz, 1H); 10.85 (s, 1H); 12.25 (s, 1H). MS (ESI): 374.6 (C₁₃H₁₀ClINO₂, [M+H]⁺). Anal. Calcd for C₁₃H₉ClINO₂: C, 41.8; H, 2.43; Cl, 9.49; I, 33.97; N, 3.75%; Found: C, 41.72; H, 2.45; Cl, 9.53; I, 33.92; N, 3.77%.

N-(2-bromophenyl)-2-hydroxy-5-iodobenzamide (22)

Brown powder, mp 181–182°C ¹H NMR (500 MHz, d₆-DMSO): 7.02 (d, J = 8.8 Hz, 1H); 7.15 (t, J = 7.7 Hz, 1H); 7.44 (t, J = 7.7 Hz, 1H); 7.62 (dd, J₁ = 2.5 Hz, J₂ = 8.8 Hz, 1H); 7.72 (d, J = 8 Hz, 1H); 8.13 (d, J = 2.7 Hz, 1H); 8.29 (d, J = 8.2 Hz, 1H); 10.74 (s, 1H); 12.24 (s, 1H). MS (ESI): 419 (C₁₃H₁₀BrINO₂, [M+H]⁺). Anal. Calcd for C₁₃H₉BrINO₂: C, 37.35; H, 2.17; Br, 19.11; I, 30.36; N, 3.35%; Found: C, 37.44; H, 2.16; Br, 19.08; I, 30.31; N, 3.37%.

N-(4-fluorophenyl)-2-hydroxy-5-iodobenzamide (23)

White powder, mp 230–232°C ¹H NMR (500 MHz, d₆-DMSO): 6.96 (d, J = 8.8 Hz, 1H); 7.22 (t, J = 8.8 Hz, 2H); 7.58 (dd, J₁ = 2.5 Hz, J₂ = 8.8 Hz, 1H); 7.7–7.73 (m, 2H); 8.07 (d, J = 2.5 Hz, 1H); 10.42 (s, 1H); 11.81 (s, 1H). MS (ESI): 358.1 (C₁₃H₁₀FINO₂, [M+H]⁺). Anal. Calcd for C₁₃H₉FINO₂: C, 43.72; H, 2.54; F, 5.32; I, 35.54; N, 3.92%; Found: C, 43.83; H, 2.52; F, 5.31; I, 35.59; N, 3.9%.

N-(4-chlorophenyl)-2-hydroxy-5-iodobenzamide (24)

White powder, mp 238–240°C ¹H NMR (500 MHz, d₆-DMSO): 6.96 (d, J = 8.8 Hz, 1H); 7.43 (d, J = 8.8 Hz, 2H); 7.58 (dd, J₁ = 2.5 Hz, J₂ = 8.8 Hz, 1H); 7.74 (d, J = 8.8 Hz, 2H); 8.03 (d, J = 2.5 Hz, 1H); 10.46 (s, 1H); 11.7 (s, 1H). MS (ESI): 374.6 (C₁₃H₁₀ClINO₂, [M+H]⁺). Anal. Calcd for C₁₃H₉ClINO₂: C, 41.8; H, 2.43; Cl, 9.49; I, 33.97; N, 3.75%; Found: C, 41.72; H, 2.43; Cl, 9.52; I, 34.03; N, 3.78%.

N-(4-bromophenyl)-2-hydroxy-5-iodobenzamide (25)

White powder, mp 236–237°C ¹H NMR (500 MHz, d₆-DMSO): 6.97 (d, J = 8.5 Hz, 1H); 7.55–7.59 (m, 3H); 7.69 (d, J = 8.8 Hz, 2H); 8.03 (d, J = 2.5 Hz, 1H); 10.45 (s, 1H); 11.7 (s, 1H). MS (ESI): 419 (C₁₃H₁₀BrINO₂, [M+H]⁺). Anal. Calcd for C₁₃H₉BrINO₂: C, 37.35; H, 2.17; Br, 19.11; I, 30.36; N, 3.35%; Found: C, 37.49; H, 2.16; Br, 19.07; I, 30.4; N, 3.32%.

N-(2,4-dichlorophenyl)-2-hydroxy-5-iodobenzamide (26)

Brown powder, mp 209–211°C ¹H NMR (300 MHz, d₆-DMSO): 7.3 (d, J = 8.8 Hz, 1H); 7.49 (dd, J₁ = 2.4 Hz, J₂ = 8.8 Hz, 1H); 7.63 (dd, J₁ = 2.7 Hz, J₂ = 8.6 Hz, 1H); 7.75 (d, J = 2.4 Hz, 1H); 8.11 (d, J = 2.6 Hz, 1H); 8.43 (d, J = 8.8 Hz, 1H); 10.91 (s, 1H); 12.3 (s, 1H). MS (ESI): 409 (C₁₃H₉Cl₂INO₂, [M+H]⁺). Anal. Calcd for C₁₃H₈Cl₂INO₂: C, 38.27; H, 1.98; Cl, 17.38; I, 31.1; N, 3.43%; Found: C, 38.41; H, 1.96; Cl, 17.35; I, 31.07; N, 3.44%.

2-Hydroxy-5-iodo-N-p-tolylbenzamide (27)

Light yellow powder, mp 241–243°C ¹H NMR (300 MHz, d₆-DMSO): 2.29 (s, 3H); 6.96 (d, J = 8.8 Hz, 1H); 7.18 (d, J = 8.2 Hz, 2H); 7.58 (dd, J₁ = 2.2 Hz, J₂ = 8.8 Hz, 3H); 8.1 (d, J = 2.4 Hz, 1H); 10.34 (s, 1H); 11.95 (s, 1H). MS (ESI): 354.2 (C₁₄H₁₃INO₂, [M+H]⁺). Anal. Calcd for C₁₄H₁₂INO₂: C, 47.61; H, 3.42; I, 35.93; N, 3.97%; Found: C, 47.72; H, 3.41; I, 35.87; N, 3.95%.

2-Hydroxy-5-iodo-N-(4-methoxyphenyl)benzamide (28)

White powder, mp 232–234°C ¹H NMR (300 MHz, d₆-DMSO): 3.76 (s, 3H); 6.95 (d, J = 9 Hz, 3H); 7.56–7.61 (m, 3H); 8.12 (d, J = 2.6 Hz, 1H); 10.31 (s, 1H); 12.04 (s, 1H). MS (ESI): 370.2 (C₁₄H₁₃INO₃, [M+H]⁺). Anal. Calcd for C₁₄H₁₂INO₃: C, 45.55; H, 3.28; I, 34.38; N, 3.79%; Found: C, 45.43; H, 3.3; I, 34.45; N, 3.82%.

3,5-Dibromo-N-(4-fluorophenyl)-2-hydroxybenzamide (29)

Brown powder, mp 204–205°C ¹H NMR (500 MHz, d₆-DMSO): 7.25 (t, J = 7.9 Hz, 2H); 7.68 (dd, J₁ = 5.2 Hz, J₂ = 7.9 Hz, 2H); 8.23 (s, 1H); 8.39 (s, 1H); 10.66 (s, 1H); 13.16 (br s, 1H). MS (ESI): 390 (C₁₃H₉Br₂FNO₂, [M+H]⁺). Anal. Calcd for C₁₃H₈Br₂FNO₂: C, 40.14; H, 2.07; Br, 41.08; F, 4.88; N, 3.6%; Found: C, 40.22; H, 2.06; Br, 41.01; F, 4.86; N, 3.59%.

3,5-Dibromo-N-(4-chlorophenyl)-2-hydroxybenzamide (30)

Brown powder, mp 200–202°C ¹H NMR (500 MHz, d₆-DMSO): 7.47 (dd, J₁ = 2.1 Hz, J₂ = 6.8 Hz, 2H); 7.72 (dd, J₁ = 2.2 Hz, J₂ = 6.7 Hz, 2H); 8.02 (d, J = 2.4 Hz, 1H); 8.26 (d, J = 2.1 Hz, 1H); 10.72 (s, 1H); 12.72 (br s, 1H). MS (ESI): 406.5 (C₁₃H₉Br₂ClNO₂, [M+H]⁺). Anal. Calcd for C₁₃H₈Br₂ClNO₂: C, 38.51; H, 1.99; Br, 39.41; Cl, 8.74; N, 3.45%; Found: C, 38.6; H, 1.99; Br, 39.37; Cl, 8.72; N, 3.44%.

3,5-Dibromo-N-(4-bromophenyl)-2-hydroxybenzamide (31)

Brown powder, mp 166–168°C ¹H NMR (500 MHz, d₆-DMSO): 7.46 (d, J = 8.9 Hz, 2H); 7.71 (d, J = 8.6 Hz, 2H); 8.24 (d, J = 1.8 Hz, 1H); 8.37 (d, J = 1.8 Hz, 1H); 10.71 (s, 1H); 13 (br s, 1H). MS (ESI): 450.9 (C₁₃H₉Br₃NO₂, [M+H]⁺). Anal. Calcd for C₁₃H₈Br₃NO₂: C, 34.7; H, 1.79; Br, 53.28; N, 3.11%; Found: C, 34.81; H, 1.78; Br, 53.24; N, 3.09%.

3,5-Dibromo-2-hydroxy-N-(4-methoxyphenyl)benzamide (32)

Brown powder, mp 153–155°C ¹H NMR (500 MHz, d₆-DMSO): 3.77 (s, 3H); 6.98 (dd, J₁ = 2.2 Hz, J₂ = 6.8 Hz, 2H); 7.57 (dd, J₁ = 2.2 Hz, J₂ = 6.8 Hz, 2H); 8.01 (d, J = 2.5 Hz, 1H); 8.31 (d, J = 2.5 Hz, 1H); 10.56 (s, 1H); 13.25 (br s, 1H). MS (ESI): 402.1 (C₁₄H₁₂Br₂NO₃, [M+H]⁺). Anal. Calcd for C₁₄H₁₁Br₂NO₃: C, 41.93; H, 2.76; Br, 39.85; N, 3.49%; Found: C, 42.01; H, 2.75; Br, 39.81; N, 3.47%.

Antiproliferative activities assay

The antiproliferative activities of the salicylanilide derivatives 1–32 were determined using a standard 3-(4,5-dimethylthylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay (Sigma, St. Louis, USA). Briefly, cell lines were seeded at a density of 7 × 10³ cells/well in 96-well microtiter plates (Costar, Bethesda, USA). After 24 h, exponentially growing cells were exposed to the indicated compounds at final concentrations ranging from 0.5 to 300 μΜ, 5-fluorouracil was used as a positive control [25]. After 48 h, cell survival was determined by the addition of an MTT solution (10 μL of 5 mg/mL MTT in PBS). After 4 h, 100 μL of 10% SDS in 0.01 N HCl was added, and the plates were incubated at 37°C for a further 18 h; optical absorbance was measured at 570 nm on an LX300 Epson Diagnostic microplate reader (Offenburg, Germany). Survival ratios were expressed as a percentage with respect to untreated cells. IC₅₀ values were determined from replicates of six wells from at least two independent experiments.

EGFR Inhibitory Assay

A 1.6 kb cDNA encoded for the EGFR cytoplasmic domain (EGFR-CD, amino acids 645–1186) were cloned into the baculoviral expression vector pFASTBacHTc. A sequence that encodes (His)₆ was located at the 5′ upstream to the EGFR sequence and Sf-9 cells were infected for three days for protein expression. The Sf-9 cell pellets were solubilised at 0°C in a buffer at pH 7.4 containing 50 mM HEPES, 10 mM NaCl, 1% Triton, 10 μM ammonium molybdate, 100 μM sodium vanadate, 10 μg/mL aprotinin, 10 μg/mL leupeptin, 10 μg/mL pepstatin, and 16 μg/mL benzamidine HCl for 20 min followed by 20 min centrifugation. The crude extract supernatant was passed through an equilibrated Ni-NTA superflow packed column (Qiagen, Hilden, Germany) and washed with 10 mM and then 100 mM imidazole to remove any nonspecifically bound material. The histidine tagged proteins were eluted with 250 and 500 mM imidazole and dialysed against 50 mM NaCl, 20 mM HEPES, 10% glycerol, and 1 μg/mL each of aprotinin, leupeptin, and pepstatin for 2 h. The entire purification procedure was performed at 4°C or on ice [26].

The EGFR kinase assay was set up to assess the level of autophosphorylation based on DELFIA/Time-Resolved Fluorometry. Compounds 1–32 were dissolved in 100% DMSO and diluted to the appropriate concentrations with 25 mM HEPES at pH 7.4. In each well, 10 μL of compound was incubated with 10 μL (12.5 ng for HER-2 or 5 ng for EGFR) of recombinant enzyme (1:80 dilution in 100 mM HEPES) for 10 min at room temperature. Then, 10 μL of 5 mM buffer (containing 20 mM HEPES, 2 mM MnCl₂, 100 μM Na₃VO₄, and 1 mM DTT) and 20 μL of 0.1 mM ATP-50 mM MgCl₂ was added for 1 h. Positive and negative controls were included in each plate by incubation of enzyme with or without ATP-MgCl₂. At the end of the incubation period, the liquid was aspirated, and plates were washed three times with wash buffer. A 75 μL (400 ng) sample of europium labeled anti-phosphotyrosine antibody was added to each well for a further 1 h of incubation. After washing, enhancement solution was added and the signal was detected by Victor (Wallac, Massachusetts, USA) with excitation at 340 nm and emission at 615 nm. The percentage of autophosphorylation inhibition by the compounds was calculated using the following equation: 100% - [(negative control)/(positive control- negative control)]. The IC₅₀ was obtained from the percentage inhibition curves with eight concentrations of the compound. As the contaminants in the enzyme preparation are fairly low, the majority of the signal detected by the anti-phosphotyrosine antibody was from EGFR.

Docking Simulations

Molecular docking of compounds 22 and 28 into the three-dimensional EGFR complex structure (download from the Protein data Bank (PDB) PDB code: 1M17) was carried out using the AutoDock software package (version 4.0) as implemented through the graphical user interface AutoDockTools (ADT 1.4.6).

Declaration of Interest

This work was supported by the Jiangsu National Science Foundation (No. BK2009239) and Anhui National Science Foundation (No. 070416274X).