Synthesis and evaluation of salicylanilide derivatives as potential epidermal growth factor receptor inhibitors

Article abstract of DOI:10.1111/cbdd.12383

Two series of novel salicylanilide were synthesized as potential epidermal growth factor receptor (EGFR) inhibitors. The enzyme inhibitory activity against EGFR of all compounds was carried out, and their antiproliferative activities against the A549 and A431 cell lines were also evaluated. Of the compounds studied, majority of them exhibited high antiproliferative activities compared with gefitinib; especially, 12a and 12b exhibited stronger inhibitory activity against EGFR with IC₅₀ values of 10.4 ± 2.25 and 15.4 ± 2.33 nM, respectively, which were comparable to the positive control of gefitinib (IC₅₀ = 12.1 ± 2.21 nM). Compound 12b also showed outstanding inhibitory activity against A431 and A549 cell lines with the IC₅₀ values of 0.42 ± 0.43 μM and 0.57 ± 0.43 μM, which was better than the positive controls. In the molecular modeling study, compound 12b was bound into the active pocket of EGFR with two hydrogen bond and with minimum binding free energy ΔG_b = -25.1125 kcal/mol. The result also suggested that compound 12b could bind the EGFR kinase well.

Full text of DOI:10.1111/cbdd.12383

Received Date : 23-Mar-2014
Revised Date : 05-Jun-2014
Accepted Date : 07-Jun-2014
Article type : Research Article
Synthesis and evaluation of salicylanilide derivatives as potential
epidermal growth factor receptor inhibitors
. Minhua Hu,a Wenfeng Ye, a Jiaming Li, a* Guochen Zhong, a Guangwei He,b Qinlong Xu, b Yanchun Zhang, a
a Anhui Key Laboratory of Traditional Chinese Medicine, Anhui University of Chinese Medicine, Hefei
230031(P.R. China)
b Hefei medical engineering pharmaceutical Co., Ltd. Hefei 230088, (P.R. China)
Corresponding author mail id: ahtcm8888@sina.com
[Abstract] Two series of novel salicylanilide were synthesized as potential EGFR
inhibitors. The enzyme inhibitory activity against EGFR of all compounds was carried
out, and their anti-proliferative activities against the A549 and A431 cell lines were
also evaluated. Of the compounds studied, majority of them exhibited high anti-
proliferative activities compared to gefitinib. Especially, 12a and 12b exhibited
stronger inhibitory activity against EGFR with IC50 values of 10.4 2.25 and
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15.4 2.33 nM, respectively, which were comparable to the positive control of
gefitinib (IC50=12.1 2.21 nM). Compound 12b also showed outstanding inhibitory
activity against A431 and A549 cell lines with the IC50 values of 0.42 0.43 μM and
0.57 0.43 μM, which was better than the positive controls. In the molecular
modelling study, compound 12b was bound into the active pocket of EGFR with two
hydrogen bond and with minimum binding free energy △Gb=-25.1125 kcal/mol. The
result also suggested that compound 12b could bind the EGFR kinase well.
Keywords: salicylanilide derivatives; EGFR inhibitor; anti-proliferative effects;
molecular modeling
1. Introduction
The protein tyrosine kinase (PTK) plays critical roles in many signal transduction
processes. Deregulated PTK activity has been observed in many proliferative diseases
[1]. Among the PTKs, the ErbB family, particularly epidermal growth factor receptor
(EGFR) kinase has been identified as being important in cancer. The EGFR has been
implicated in various tumors of epithelial origin (e.g., breast, aquamous cell
carcinoma, ovarian and non-small cell lung cancer) [2-3]. Thus, the EGFR has emerged
as promising anticancer target in pharmaceutical studies and a lot of EGFR inhibitors
have been used in clinical. Representative small-molecular inhibitors of EGFR are the
quinazoline derivatives, such as gefitinib, erlotinib and canertinib (Fig 1). Among
them, 4-anilinoquinazoline moiety, plays a significant role in their inhibiting activity.
This sort of inhibitors generally exhibit high potency in vitro, inhibiting EGFR at low-
molar concentration [4]
.
Figure 1. Representative EGFR inhibitors
The salicylanilide molecule may be via OH…O=C hydrogen bond to form a pseudo
six-membered ring [5]. Thus, salicylanilide is supposed to have the same
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pharmacophore as 4-anilinoquinazoline, and widely used as EGFR inhibitors [6-7]
.
Along with a large number of salicylanilide derivatives reported, the pharmacological
activity of it has been confirmed. Furthermore, their structure-activity relationship
(SAR) has been clearly revealed (Fig 2): (1) Aniline fragment in the hydrophobic
pocket of the kinase, which could accommodate large groups. (2) Introduction of the
electron-donating substituent into the 4′-position showed a better activity than
electron-withdrawing substituent [8-9]. (3) At the 3′-position, small, lipophilic and
electron-withdrawing substituent was both well tolerated [6]. (4) The presence of
electron-donating group at 4-position or 5-position of the salicylanilide moiety was
favorable to the improvement of anti-tumor activity.
On this background we have recently studied a series of salicylanilide derivatives
which were evaluated for their anti-tumor activities (D) (Fig 3) [10]. These compounds
have been synthesized by a structural modification of 4-position, and the preliminary
pharmacological showed that some of them displayed considerable anti-proliferative.
Owing to our continued studies on salicylanilide as an attractive molecular skeleton as
anti-tumor agents, we have designed a number of new salicylanilide derivatives
containing two kinds of side chain at the 4-position and biologically evaluated them in
vitro anti-proliferation activities and anti-EGFR activities, wish to communicate their
potential as EGFR inhibitors (Series a and Series b) (Fig 3). In the present study, the
substitution pattern at the 4-substituted salicylic acid was selected based on different
electronic environment which would affect the hydrophobicity. The objective of
forming these target compounds is an attempt to obtain potential EGFR inhibitors.
Figure 2. The structure of the salicylanilide
Figure 3. The salicylanilide derivatives reported as anti-tumor inhibitors and proposed as
EGFR inhibitors
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2. Chemistry
The general synthetic method for the title compound was shown in Scheme 1.
Esterification of the starting material 2, 4-dihydroxybenzoic acid 1 in the presence of
NaHCO3, KI and methanol at 40 °C gave methyl 2, 4-dihydroxybenzoate 2.
Alkylation of intermediates 2 with 1, 3-dibromopropane in acetone afforded methyl 4-
(3-bromopropoxy)-2-hydroxylbenzoate 3 which was then condensed with
morpholine/piperidine 4a-4b to obtain compounds 5a-5b. Subsequent hydrolysis in
basic condition afforded compounds 6a-6b, and both were transformed into the
corresponding acid chloride using SOCl2 without purification, then reacted with an
appropriate substituted amine 8-13 in the presence of triethylamine to give the title
compounds 8a-13a and 8b-13b, respectively.
Scheme 1. Reagents and conditions: (a) CH3I, NaHCO3, DMF, 40 °C,10 h; (b) 1, 3
dibromopropane, K2CO3, TEBAC, acetone, 60 °C,8 h; (c) NaHCO3, NaI, isopropanol, 85
°C,6 h; (d) NaOH,CH3OH, H2O, r.t, 6 h; (e) SOCl2, DMF, 85 °C, 6 h; (f) Et3N, CH2Cl2, ice
bath, 40 min.
3. Results and discussion
3.1. EGFR kinase inhibitory assay
The inhibitory activity of the novel salicylanilide derivatives against EGFR was
evaluated, and gefitinib used as employed as positive control. IC50s of the compounds
were calculated according to inhibitory ratios, the in vitro enzymatic inhibition assay
results was summarized in Table 1.
The data demonstrated that tested compounds exhibited potent inhibitory activity
against EGFR. As shown in Table 1, compounds 8b-13b showed the higher
inhibitory activities than compounds 8a-13a, possibly because the morpholine ring in
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4-position side chain alkyl was high hydrophilic than piperidine ring, which decreased
its interaction with EGFR protein. The IC50 values were different among 8a-13a and
8b-13b, because substituent was different in aniline. But many compounds without
electron-withdrawing substituents on the 3-position of the aniline showed poor EGFR
inhibitory activity which demonstrated that electron-withdrawing substituent was
essential for this kind of EGFR inhibitors. Among them, compounds 8a, 13a, 9b, 8b
showed the moderate inhibitory activities, while 9a, 10a with the lowest inhibitory
activities. On the other hand, replacement of the electron-donating (CH3) by CF3 was
favorable for the EGFR inhibitory (compounds 11a and 11b). Whereas, the
compound 10b was not following the rule, which suggested the substituent at the 4-
position played a significant role in anti-EGFR activity. Furthermore, although the
compounds of 8a-13a exhibited poor activities to EGFR, it was quite interesting to
note that 12a exhibited the potent activity to EGFR (IC50= 10.4 2.25 nM) and more
comparable to the positive control gefitinib (IC50= 12.1 2.21 nM). Meanwhile,
compound 12b displayed moderate inhibitory activity (IC50= 15.4 2.33 nM). The
most possible reason was that the 3-Cl, 4-F aniline moieties increased EGFR
inhibition activities.
x s
Table 1. EGFR inhibition of the salicylanilide derivatives 8a-13a and 8b-13b (n=3,
)
X
R
IC50(nM)
Compounds
O
O
O
O
3-Br
3-Cl
85.8 6.41
195.8 8.76
142.1 8.34
34.6 3.22
8a
9a
3-CH3
3-CF3
10a
11a
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O
O
C
C
C
C
C
C
-
3-Cl,4-F
2-Cl,4-NO2
3-Br
10.4 2.25
84.8 4.23
45.7 2.66
63.9 6.44
22.4 2.49
35.2 4.68
15.4 2.33
23.0 3.65
12.1 2.21
12a
13a
8b
3-Cl
9b
3-CH3
10b
3-CF3
11b
3-Cl,4-F
2-Cl,4-NO2
-
12b
13b
Gefitinib
3.2 Inhibitory activity on A549 and A431
The anti-proliferative effects of the novel salicylanilide derivatives on human
pulmonary carcinoma A549 and squamous cell carcinoma cell line A431 were
investigated with gefitinib as controls by applying the 3-(4, 5-dimethylthylthiazol-2-
yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. The A431 and A549 cell lines
were known to over express EGFR, which was related to abnormal activity of EGFR
pathway in many carcinogenesis. Generally, the activity of inhibiting A431 and A549
cell proliferation were determined after 72 h of treatment with various concentrations
(0.003-50 μM) of the tested compounds. The results were expressed as IC50 values
(Table 2).
As shown in Table 2, when treated of A431 cells and A549 with compounds 8a-13a
and 8b-13b, which made the cell proliferation decrease. Among the two series of
novel salicylanilide derivatives, SAR studies demonstrated that almost all the
compounds belonging to the series b showed good activity both with anti-
proliferative activity and EGFR inhibition. The results also indicated that the
substituented piperidine ring substituent at the 4-position could significantly increase
the activity. Here, compound 12b displayed the most potent inhibitory activity (IC50=
0.42 0.43 μM for A431 and IC50= 0.57 0.43 μM for A549), and comparable to the
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positive control gefitinib (IC50= 4.28 3.93 μM against A431 and IC50= 15.56 3.93
μM against A549).
In the assay, anti-proliferative activities of some compounds were inconsistent
with their kinase assay data. Compound 8b (IC50= 2.07 1.47 μM against A431 and
IC50= 6.96 1.47 μM against A549) was found to have about 2-3 times than gefitinib
in activity against all of the tested cancer cells. These implied that compound 8b may
exert inhibition on the proliferation not only via blocking the EGFR signaling, but
also via other pathways. Compound 12a did not exhibit excellent anti-proliferative
potency even though it had the most potent inhibitory activity against EGFR.
Nonetheless, it also displayed equivalent inhibition activity compared to gefitinib.
Once again, the excellent anti-proliferation activity of compounds 12a and 12b
proved the positive impact of the 3-Cl, 4-F aniline moieties. In the case of all the
compounds, compounds 8a-10a and 9b showed almost no inhibitory activities
(IC50>50 μM). Results for the 11a (37.53 4.77 μM against A549) have shown
equivalent potencies in inhibition compared to the 11b (16.28 1.82 μM against
A549). Whereas the compound 11b showed weaker inhibitory activity against A431,
comparing stronger inhibitory activity against A549. Consistent with their kinase
inhibition, compounds 10b and 13b displayed moderate anti-proliferative activities on
A549 and A431.
Table 2. Anti-proliferative activity of title salicylanilide compounds
IC50(μM)
Compounds
A431
A549
8a
9a
﹥
﹥
﹥
50
50
50
﹥
﹥
﹥
50
50
50
10a
11a
12a
9.32 4.77
6.06 1.24
37.53 4.77
14.42 1.24
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29.86 4.77
2.07 1.47
13a
8b
﹥
50
6.96 1.47
50
9b
﹥
50
59.71 2.87
50
﹥
18.15 2.87
16.28 1.82
10b
11b
﹥
0.42 0.43
10.58 1.32
4.28 3.93
0.57 0.43
21.12 1.32
15.56 3.93
12b
13b
Gefitinib
3.3 Molecular docking study with EGFR
To give structural insight into the ligand/enzyme interactions, and to give an
explanation and understanding of good activity observed, we proceeded to examine
the interaction of those with EGFR complex structure (PDB code: 2ITO) by
molecular docking, of compounds into the ATP binding site in EGFR. The binding
free energy of all the compounds was mentioned in Table 3. Of the compounds
studied, compound 12b was nicely bound into the active site of EGFR with minimum
binding free energy △Gb= -25.1125 kcal/mol. The binding model of compound 12b
with EGFR was depicted in Figures 4a and 4b. In general, the aniline of compound
12b could fit well into the hydrophobic pocket. In the binding mode, compound 12b
was nicely bound to the ATP binding site of EGFR through hydrophobic interaction
and the binding was stabilized by two hydrogen bonds. Among them one hydrogen
bond formed H atom of -OH group and MET793, second one between O atom of
carbonyl group and MET793. From this binding model, it could be concluded that
two hydrogen bonds were responsible for the effective EGFR inhibition.
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Table 3. Binding free energy of compounds 8a-13a and 8b-13b with EGFR
Compounds
Binding free energy
Gb (kcal/mol)
Compounds
Binding free energy
Gb (kcal/mol)
△
△
-21.1105
-19.0346
-18.1034
-17.5341
-24.1109
-18.0346
-24.1523
-19.1034
-16.1552
-22.5462
-22.1031
-25.1125
-21.1325
8a
9a
8b
9b
10a
11a
12a
10b
11b
12b
13b
13a
Gefitinib
Figure 4. (a) Compound 12b (light blue) docked into the EGFR binding domain. (b) Dock
structure of compound 12b with EGFR. Hydrogen bonds towards MET793 were highlighted.
4. Conclusion
In general, the aim of the present investigation was to design two series of new
salicylanilide derivatives by introduction of substituted side chain at the 4-position for
evaluating their anti-EGFR activities. The anti-proliferative activities against the
human cancer cell line A431 and A549 were evaluated by MTT assay, which had
almost the same trend as the anti-EGFR activities assay. Some magnificent biological
results have been obtained and it has been concluded that majority of the compounds
showed effective both against anti-EGFR and anti-proliferative activity. EGFR kinase
assay and MTT assay showed that an electron-withdrawing group in 3′-position or
pyridine ring in 4-position would maintain the inhibitory activity of the salicylanilides
pharmacophore at a high level. In the case of inhibitory activities, compounds 12a and
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12b against EGFR had been found to be the most effective members of the two series.
But, the compound 12a did not show excellent activity against EGFR. Compound 12b
demonstrated the most potent inhibitory activity that inhibited the growth of tumor
cell, which was almost 10 times compared to the positive control gefitinib. Molecular
docking of the most potent inhibitor 12b into ATP binding site of EGFR kinase was
performed. The docking study also revealed that compound 12b nicely bound into the
active site of EGFR with minimum binding free energy. So, it is worthy of elucidate
the molecular mechanism of compound 12b and 12a in detail. These results are of
help in the rational design of higher selectivity EGFR inhibitors in further.
5. Experimental section
5.1 General
All analytical grade chemicals and solvents were purchased from commercial sources
and used without further purification in our laboratory. Melting points were
determined by a WRS-1B apparatus and were reported without any correction. IR
spectra were recorded on a Nicolet Avatar 370DTGS spectrometer using KBr film.
The 1H and 13C NMR spectra were collected on Bruker Anance-400 MHz instruments
using deuterated solvents with tetramethylsilane (TMS) as internal standard. EI-MS
was recorded on FINNIGA LCQ ADVANTAGE MAX apparatus. TLCs and
preparative TLC were performed on silica gel GF/UV 254, and the chromatograms
were conducted on silica gel (10-20 mesh) and visualized under UV light at 254 and
365 nm.
5.1.1 Synthesis of 2, 4-dihydroxy benzoic acid methyl ester (2) [11]
2, 4-dihydroxy benzoic acid (10 g, 0.065 mol) was dissolved in 100 mL of dry
DMF, NaHCO3 (6.5 g, 0.077 mol) was added and stirred for several minutes at room
temperature. Then, CH3I (13.8 g, 0.097 mol) was added. The reaction mixture was
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stirred at 40 °C and monitored by TLC. Upon completion, 100 mL of water was
poured into the reaction mixture and extracted with ethyl acetate (3×100 mL). The
organic layer was subsequently washed with 5 % of NaHCO3 (3×50 mL) and 5 % of
NaCl (3×50 mL) solution, and dried over anhydrous Na2SO4, filtered and
concentrated under reduced pressure to afford compound 8 as a white powder (9.2 g,
84.4 %): Rf=0.6 (Pe/EtOAc 3:1); mp:110.9-113.2 °C. The melting point for
compound 8 is consistent with previous reports [12]
.
5.1.2 Synthesis of 4-(3-bromo-propoxy)-2-hydroxy benzoic acid methyl ester (3)
[13]
To a solution of compound 2 (8.0 g, 0.047 mol) in 80 mL of acetone was added
potassium carbonate (6.5 g, 0.047 mol) and, benzyl triethyl ammonium chloride
(BTEAC) (0.1 g) followed by stirring at 60 °C for 0.5 h. Then 1, 3-dibromopropane
(19.0 g, 0.095 mol) was added and stirred for another 8 h. After completion of the
reaction (monitored by TLC), the reaction mixture was cooled and the solid inside
was removed by filtration. The filtrate was concentrated to afford a crude product,
which was further purified with a silica gel column eluted with petroleum ether-
chloroform(2:1, v/v) give compound 3 as a white solid (4.8 g, 55.5 %): Rf=0.8
(Pe/EtOAc 10:1); mp: 72.8-74.5 °C; 1H-NMR (400 MHz, CDCl3) δ: 10.97 (s, 1H),
7.77 (d, J=8.8 Hz, 1H), 6.48 (d, J=2.4 Hz, 1H), 6.46 (dd, J=8.8 Hz, 2.4 Hz, 1H), 4.16
(t, J=5.6 Hz, 2H), 3.93 (s, 3H), 3.62 (t, J=6.4 Hz, 2H), 2.37-2.31 (m, 2H); 13C-NMR
(100 MHz, CDCl3) δ: 170.3, 164.6, 163.7, 161.4, 131.3, 107.7, 101.3, 65.5, 52.0,
32.0, 29.7; IR (KBr) υ,==3016, 2949, 1673, 1623, 1585, 1509, 1467, 1438.3, 1351,
1256, 1222, 1187, 1138, 1022, 986, 952, 917, 832 cm-1; MS (ESI) m/z 294.26
[M+H]+.
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5.1.3 Synthesis of 4-(3-morpholinopropoxy)-2-hydroxy benzoic acid methyl ester
(5a) [14]
A 50 mL of isopropanol solution of compound 3 (7.0 g, 0.024 mol), morpholine (3.1
g, 0.036 mol) and NaHCO3 (3.1 g, 0.037 mol) was heated at 85 °C. The progress of
the reaction was followed by TLC. After completion of the reaction, the reaction
mixture was cooled and the solid was filtered. The filtrate was concentrated to afford
mixture. The mixture was purified through silica gel column separation (ethyl acetate)
to obtain compound 5a as light yellow oil (4.8 g, 67.6 %): Rf=0.3 (EtOAc); 1H-NMR
(400 MHz, CDCl3) δ: 10.95 (s, 1H, OH), 7.73 (d, J=8.8 Hz, 1H, ArH), 6.45 (d, J=2.4
Hz, 1H, ArH), 6.44 (dd, J=8.8 Hz, 2.4 Hz, 1H, ArH), 4.07 (t, J=6.4 Hz, 2H,
NCH2CH2CH2O), 3.91 (s, 3H, COOCH3), 3.73 (t, J=4.8 Hz, 4H, morpholine ring
CH2OCH2), 2.52-2.46 (m, 6H, morpholine ring CH2NCH2 and NCH2CH2CH2O),
2.00-1.93 (m, 2H, NCH2CH2 CH2O); 13C-NMR (100 MHz, CDCl3) δ: 170.4, 165.0,
163.7, 131.2, 107.8, 105.3，101.2, 67.0, 66.3, 55.3, 53.7, 51.9, 26.2; IR (KBr)
υ,==3416, 3066, 2956, 2861, 2824, 2782, 1663, 1626, 1582, 1508, 1470, 1436, 1402,
1354, 1306, 1259, 1222, 1191, 1144, 1113, 1028, 999, 973, 951, 915, 860, 832, 779
cm-1; MS (ESI) m/z 294.26 [M-H]+.
Compound 5b was synthesized according to the procedure described above.
5.1.4 4-(3-(piperidin-1-yl)propoxy)-2-hydroxy benzoic acid methyl ester (5b)
Compound 5b was obtain as a light yellow oil (4.9 g, 70.5 %): Rf=0.3 (EtOAc); 1H-
NMR (CDCl3, 400 MHz) δ: 10.93 (s, 1H, OH), 7.71 (d, J=8.8 Hz, 1H, ArH), 6.42 (d,
J=9.2 Hz, 1H, ArH), 4.03 (t, J=6.4 Hz, 2H, NCH2 CH2CH2O), 3.89 (s, 3H, COOCH3),
2.51-2.43 (m, 6H, piperidine ring NCH2CH2CH2O), 2.02~1.96 (m, 2H,
NCH2CH2CH2O), 1.64~1.58 (m, 4H, piperidine ring), 1.46-1.44 (m, 2H, piperidine
ring); 13C-NMR (CDCl3, 100 MHz) δ: 170.3, 165.0, 163.7, 131.1, 107.7, 105.3，
101.2, 66.6, 55.6, 54.5, 51.8, 26.4, 25.7, 24.2; IR (KBr) υ,==3430, 2955, 2874, 2647,
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1627, 1587, 1502, 1439, 1364, 1227, 1188, 1149, 1089, 975, 905, 865 cm-1; MS (ESI)
m/z 294.19 [M+H]+.
5.1.5 Synthesis of 4-(3-morpholinopropoxy)-2-hydroxy benzoic acid (6a) [15]
To a solution of 6a (0.014 mol) in methanol (20 mL) and pure water (10 mL)
was added NaOH (0.8 g, 0.020 mmol). The reaction mixture was stirred at room
temperature. After the reaction was completed (monitored by TLC), the mixture was
concentrated and acidified with HCl solution (3 mol·L-1) till pH to be 5~6, the white
precipitation appeared immediately. Then precipitation was filtered and washed with
water, dried to afford the 6a (3.1 g, 81.6 %): Rf=0.58 (CHCl3); white solid; mp: 199.8-
201.1 °C; 1H-NMR (400MHz, DMSO-d6) δ: 7.65 (d, J=8.8 Hz, 1H, ArH), 6.38 (s, 2H,
ArH), 4.08 (t, J=5.6 Hz, 2H, NCH2CH2CH2O), 3.78 (s, 4H, morpholine ring
CH2OCH2), 2.98 (s, 6H morpholine ring CH2NCH2 and NCH2CH2CH2O), 2.07 (s, 2H,
NCH2CH2CH2O); 13C-NMR (100 MHz, DMSO-d6) δ: 172.4, 164.0, 163.8, 131.9,
106.8, 106.7，101.5, 65.7, 64.6, 54.2, 52.1, 24.2; IR (KBr) υ,==2949, 2872, 1591,
1504, 1436, 1369, 1263, 1184, 1112, 1083, 983, 932, 866, 830, 779 cm-1; MS (ESI)
m/z 280.19 [M-H]+.
Compounds 6b was synthesized according to the procedure described above.
5.1.6 4-(3-(piperidin-1-yl)propoxy)-2-hydroxy-benzoic acid (6b) Compound 6a
was obtained as white solid (3.2 g, 82.1 %): Rf=0.58 (CHCl3); mp: 180.5-182.2 °C;
1H-NMR (DMSO-d6, 300 MHz) δ: 12.89 (s, 1H, COOH), 10.87 (s, 1H, OH), 7.71 (d,
J=9.6 Hz, 1H, ArH), 6.47 (d, J=9.6 Hz, 2H, ArH), 4.12 (t, J=6.0 Hz, 2H,
NCH2CH2CH2O), 3.42 (s, 2H, NCH2CH2CH2O), 3.17 (t, J=7.8 Hz, 2H, piperidine
ring CH2N), 2.87 (s, 2H, piperidine ring CH2N), 2.23-2.18 (m, 2H, NCH2CH2CH2O),
1.79 (s, 4H, piperidine ring CH2), 1.69~1.40 (m, 2H,piperidine ring CH2); 13C-NMR
(DMSO-d6, 100 MHz) δ: 172.4, 164.0, 163.8, 131.9, 106.8, 106.7，101.5, 65.7, 64.6,
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54.2, 52.1, 24.2; IR (KBr) υ,==3373, 2954, 2873, 1591, 1505, 1438, 1363, 1265,
1185, 1089, 1049, 978, 907, 830, 786 cm-1; MS (ESI) m/z 278.40 [M-H]+.
5.1.7 Synthesis of 4-(3-morpholinopropoxy)-2-hydroxy benzoyl chloride (7a) [16]
To a solution of 6a (7.1 mmol) in excess thionyl chloride (15 mL), DMF (3 drops)
was added. Then, the solution stirred at 50 °C for 3 h. After completion of the reaction,
the solvent was evaporated and the crude was dissolved with CH2Cl2 (10mL) and
cooled for the next step.
Compounds 7b was synthesized according to the procedure described above.
5.1.8 Synthesis of 4-(3-morpholinopropoxy)-N-(3-bromophenyl)-2-hydroxy-benz-
amide (8a)
The compound 7a was added dropwise to a cooled solution of dichloromethane (20
mL) (ice bath) of 3-bromoaniline (7.6 mmol), triethylamine (2 mL, 14.4 mmol), and
stirred for 10 minutes at 0-5 °C. After completion of the reaction and monitored by
TLC, the resulted mixture was added to 10mL of water and extracted with ethyl
dichloromethane (3×20 mL). The organic phase was dried over Na2SO4 and filtered to
afford the mixture of 8a, which was concentrated and purified by column
chromaogrtaphy (CHCl3:CH3OH=20:1) to afford 8a as a white solid (1.0 g, 36.7 %):
Rf=0.38 (CHCl3:CH3OH 15:1); mp: 167.3-168.4 °C; 1H-NMR (400 MHz, DMSO-d6)
δ: 12.15 (s, 1H, NH), 10.39 (s, 1H, OH), 8.04 (s, 1H, ArH), 7.95 (d, J=8.8 Hz, 1H,
ArH), 7.65 (s, 1H, ArH), 7.32 (s, 1H, ArH), 7.31 (s, 1H, ArH), 6.55 (d, J=8.8 Hz, 1H,
ArH), 6.49 (s, 1H, ArH), 4.06 (t, J=6.4 Hz, 2H, NCH2CH2CH2O), 3.57 (s, 4H,
morpholine ring CH2OCH2), 2.44-2.37 (m, 6H, morpholine ring CH2NCH2 and
NCH2CH2CH2O), 1.91-1.84 (m, 2H, NCH2CH2CH2O); 13C-NMR (100MHz, DMSO-
d6) δ: 167.5, 163.7, 162.0, 140.3, 131.1, 130.8, 126.9, 123.6, 121.8, 120.1, 109.5,
107.1, 102.2, 66.6, 66.5, 55.1, 53.7, 26.1; IR (KBr) υ,==3438, 2923, 2867, 1648,
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1588, 1530, 1470, 1439, 1234, 1211, 1113, 1058, 988, 781 cm-1; MS (ESI) m/z
433.39 [M-H]+.
Compound 9a-13a and 8b-13b were synthesized according to the procedure described
above.
5.1.9 4-(3-morpholinopropoxy)-N-(3-chlorophenyl)-2-hydroxy-benzamide (9a)
Compound 9a was obtained as a white solid (0.95, 36.5 %): Rf=0.39 (CHCl3:CH3OH
15:1); mp: 167.3-168.4 °C; 1H-NMR (DMSO-d6, 400 MHz) δ: 12.15 (s, 1H, NH),
10.36 (s, 1H, OH), 7.95 (d, J=8.8 Hz, 1H, ArH), 7.90 (s, 1H, ArH), 7.62 (t, J=7.6 Hz,
1H, ArH), 7.41 (t, J=8.4 Hz, 1H, ArH), 7.19 (d, J=7.6 Hz, 1H, ArH), 6.57 (dd, J=8.8
Hz, 1.6 Hz, 1H, ArH), 6.49 (d, J=1.6 Hz, 1H, ArH), 4.07 (t, J=6.4 Hz, 2H,
NCH2CH2CH2O), 3.58 (s, 4H, morpholine ring CH2OCH2 ), 2.45-2.39 (m, 6H,
morpholine ring CH2NCH2 and NCH2CH2CH2O), 1.93-1.85 (m, 2H, NCH2CH2CH2-
O); 13C NMR (DMSO-d6, 100 MHz) δ: 167.5, 163.7, 161.9, 140.2, 133.4, 130.8,
124.1, 120.8, 119.7, 119.6, 109.5, 107.2, 102.2, 66.5, 55.1, 55.0, 53.7, 26.0; IR (KBr)
υ,==3422, 2867, 1650, 1588, 1534, 1469, 1432, 1236, 1112, 1056, 990, 689 cm-1; MS
(ESI) m/z 389.23 [M-H]+.
5.1.10 4-(3-morpholinopropoxy)-N-(m-tolyl)-2-hydroxy-benzamide (10a)
Compound 10a was obtained as a white solid (1.1 g, 37.9 %): Rf=0.37
(CHCl3:CH3OH 15:1); mp: 166.8-168.7°C; 1H-NMR (DMSO-d6, 400 MHz) δ: 12.41
(s, 1H, NH), 10.17 (s, 1H, OH), 7.99 (d, J=8.8 Hz, 1H, ArH), 7.51 (s, 1H, ArH), 7.48
(d, J=8.0 Hz, 1H, ArH), 7.26 (t, J=7.6 Hz, 1H, ArH), 6.96 (d, J=7.6 Hz, 1H, ArH),
6.55 (d, J=8.8 Hz, 1H, ArH), 6.48 (d, J=1.6Hz, 1H, ArH), 4.07 (t, J=6.0 Hz, 2H,
NCH2CH2CH2O), 3.58 (s, 4H, morpholine ring CH2OCH2), 2.45-2.39 (m, 6H,
morpholine ring CH2NCH2 and NCH2CH2CH2O), 2.31 (s, 3H, CH3),1.93-1.85 (m,
2H, NCH2CH2CH2O); 13C-NMR (DMSO-d6, 100 MHz) δ: 167.5, 163.5, 162.1, 138.4,
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138.3, 130.5, 128.9, 125.2, 122.1, 118.8, 109.4, 107.0, 102.2, 66.6, 66.5, 55.1, 53.7,
26.1, 21.6; IR (KBr) υ,==3423, 2921, 2871, 1649, 1592, 1555, 1489, 1439, 1240.9,
1216, 1169, 1110, 1056, 986, 900, 840, 782 cm-1; MS (ESI) m/z 369.37 [M-H]+.
5.1.11 4-(3-morpholinopropoxy)-N-(3-(trifluoromethyl)phenyl)-2-hydroxy-benz -
amide (11a) Compound 11a was obtained as a white solid (1.0 g, 38.9 %): Rf=0.38
(CHCl3:CH3OH 15:1); mp: 159.2-160.8 °C; 1H-NMR (DMSO-d6, 400 MHz) δ: 12.07
(s, 1H, NH), 10.48 (s, 1H, OH), 8.17 (s, 1H, ArH), 7.97 (t, J=8.8 Hz, 2H, ArH), 7.62
(t, J=8.0 Hz, 1H, ArH), 7.48 (d, J=7.6 Hz, 1H, ArH), 6.58 (d, J=8.8 Hz, 1H, ArH),
6.50 (s, 1H, ArH), 4.07 (t, J=6.0 Hz, 2H, NCH2CH2CH2O), 3.58 (s, 4H, morpholine
ring CH2OCH2), 2.45-2.38 (m, 6H, morpholine ring CH2NCH2 and NCH2CH2CH2O),
1.90~1.87 (m, 2H, NCH2CH2CH2O); 13C-NMR (DMSO-d6, 100 MHz) δ: 167.7,
163.8, 162.0, 139.5, 130.8, 130.3, 124.9, 120.8, 120.7, 117.5, 117.4, 109.4, 107.2,
102.2, 66.6, 66.5, 55.1, 53.7, 26.1; IR (KBr) υ,==3298, 2963, 2862, 1662, 1601, 1558,
1508, 1446, 1332, 1289, 1244, 1192, 1161, 1120, 1068, 998, 759, 705 cm-1; MS (ESI)
m/z 423.37 [M-H]+.
5.1.12 4-(3-morpholinopropoxy)-N-(3-chloro-4-fluorophenyl)-2-hydroxy-
benzam-
ide (12a) Compound 12a was obtained as a white solid (1.2 g, 37.2 %): Rf=0.36
(CHCl3:CH3OH 15:1); mp: 168.3-170.1 °C; 1H NMR (DMSO-d6, 400 MHz) δ: 12.14
(s, 1H, NH), 10.39 (s, 1H, OH), 8.01 (dd, J=6.8 Hz, 2.0 Hz, 1H, ArH), 7.94 (d, J=9.2
Hz, 1H, ArH), 7.65~7.61 (m, 1H, ArH), 7.44 (t, J=8.8 Hz, 1H, ArH), 6.56 (dd, J=9.2
Hz, 2.0 Hz, 1H, ArH), 6.49 (d, J=2.0 Hz, 1H, ArH), 4.06 (t, J=6.4 Hz, 2H,
NCH2CH2CH2O), 3.57 (s, 4H, morpholine ring CH2OCH2), 2.44-2.38 (m, 6H,
morpholine ring CH2NCH2 and NCH2CH2CH2O), 1.91-1.84 (m, 2H,
NCH2CH2CH2O); 13C-NMR (DMSO-d6, 100 MHz) δ: 167.5, 163.7, 162.0, 135.9,
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130.7, 129.7, 123.0, 121.9, 121.8, 117.4, 117.1, 107.2, 102.2, 66.5, 55.1, 55.0, 53.7,
26.0; IR (KBr) υ,==3413, 2955, 2862, 1658, 1600, 1546, 1502, 1460, 1433, 1393,
1325.3, 1238, 1115, 1052, 997, 817 cm-1; MS (ESI) m/z 407.33 [M-H]+.
5.1.13 4-(3-morpholinopropoxy)-N-(2-chloro-4-nitrophenyl)-2-hydroxy-benzami-
de (13a) Compound 13a was obtained as a white solid (1.2 g, 39.2 %): Rf=0.38
(CHCl3:CH3OH 15:1); mp: 176.9-178.2 °C; 1H-NMR (DMSO-d6, 400 MHz) δ: 11.65
(s, 1H, NH), 8.87 (d, J=9.2 Hz, 1H, ArH), 8.41 (d, J=2.0 Hz, 1H, ArH), 8.28 (dd,
J=9.2 Hz, 2.0 Hz, 1H, ArH), 7.96 (d, J=9.2 Hz, 1H, ArH), 6.58 (d, J=8.8 Hz, 1H,
ArH), 6.52 (s, 1H, ArH), 4.04 (t, J=6.4 Hz, 2H, NCH2CH2CH2O), 3.58 (s, 4H,
morpholine ring CH2OCH2), 2.47-2.43 (m, 6H, morpholine ring CH2NCH2 and
NCH2CH2CH2O), 1.92-1.86 (m, 2H, NCH2CH2CH2O); 13C-NMR (DMSO-d6, 100
MHz) δ: 164.4, 163.8, 159.6, 142.5, 142.3, 133.1, 125.2, 124.3, 122.4, 120.8, 111.4,
107.3, 102.3, 66.4, 66.3, 55.0, 53.6, 25.8; IR (KBr) υ,==3408, 3288, 2918, 1678.6,
1607, 1546, 1503, 1468, 1397, 1337, 1236, 1114 cm-1; MS (ESI) m/z 434.22 [M-H]+.
5.1.14 4-(3-(piperidin-1-yl)propoxy)-N-(3-bromophenyl)-2-hydroxy-benzamide
(8b) Compound 8b was obtained as a white solid (1.0 g, 32.7 %): Rf=0.36
(CHCl3:CH3OH 15:1); mp: 220.5-222.3 °C; 1H-NMR (DMSO-d6, 400 MHz) δ: 10.74
(s, 1H, OH), 8.03 (s, 1H, ArH), 7.90 (d, J=9.2 Hz, 1H, ArH), 7.61-7.59 (m, 1H, ArH),
7.30~7.26 (m, 2H, ArH), 6.48-6.46 (m, 1H, ArH), 6.43 (d, J=2.4 Hz, 1H, ArH), 4.02
(t, J=6.0 Hz, 2H, NCH2CH2CH2O), 3.37~3.29 (m, 6H, piperidine ring CH2NCH2 and
NCH2CH2CH2O), 1.91~1.84 (m, 2H, NCH2CH2CH2O), 1.53-1.48 (m, 4H, piperidine
ring CH2), 1.38 (s, 2H, piperidine ring CH2); 13C-NMR (DMSO-d6, 100 MHz) δ:
166.9, 163.0, 162.1, 138.1, 137.8, 130.1, 128.4, 124.5, 121.4, 118.1, 109.2, 106.1,
101.8, 65.8, 55.3, 44.8, 26.4, 21.1; IR (KBr) υ,==2947, 1647, 1578.2, 1528, 1473,
1435, 1225, 986, 779.7 cm-1; MS (ESI) m/z 431.37 [M-H]+.
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5.1.15 4-(3-(piperidin-1-yl)propoxy)-N-(3-chlorophenyl)-2-hydroxy-benzamide
(9b) Compound 9b was obtained as a white solid (1.1 g, 36.8 %): Rf=0.39
(CHCl3:CH3OH 15:1); mp:215.4-217.3 °C; 1H NMR (DMSO-d6, 300 MHz) δ: 11.59
(s, 1H, NH), 10.75 (s, 1H, OH), 7.93(s, 1H, ArH), 7.91 (t, J=1.8 Hz, 1H, ArH), 7.59
(t, J=8.1 Hz, 1H, ArH), 7.40 (t, J=8.1 Hz, 1H, ArH), 7.18 (d, J=7.5 Hz, 1H, ArH),
6.52 (dd, J=9.0, 2.4 Hz, 1H, ArH), 6.54 (d, J=2.1 Hz, 1H, ArH), 4.05 (t, J=6.3 Hz,
2H, NCH2CH2CH2O), 3.34 (m, 6H, piperidine ring CH2NCH2 and NCH2CH2CH2O),
1.92-1.87 (m, 2H, NCH2CH2CH2O), 1.54-1.51 (m, 4H, piperidine ring CH2), 1.41 (s,
2H, piperidine ring CH2); 13C-NMR (DMSO-d6, 75 MHz) δ: 167.6, 163.6, 162.4,
160.1, 136.5, 133.4, 132.1, 130.8, 124.0, 120.8, 119.7, 106.9, 102.4, 66.4, 55.0, 54.1,
25.8, 25.3, 23.8; IR (KBr) υ,==3412, 2945, 1646, 1588, 1532, 1475, 1433, 1327,
1230, 1135, 1096, 986, 871, 690 cm-1; MS (ESI) m/z 387.29 [M-H]+.
5.1.16 4-(3-(piperidin-1-yl)propoxy)-N-(m-tolyl)-2-hydroxy-benzamide (10b)
Compound 10b was obtained as a white solid (0.9 g, 35.6 %): Rf=0.37
(CHCl3:CH3OH 15:1); mp: 169.8-171.6 °C; 1H-NMR (DMSO-d6, 300 MHz) δ: 11.48
(s, 1H, NH), 10.26 (s, 1H, OH), 8.02 (d, J=8.7 Hz, 1H, ArH), 7.51 (t, J=8.7 Hz, 2H,
ArH), 7.27 (t, J=7.5 Hz, 1H, ArH), 6.96 (d, J=7.5 Hz, 1H, ArH), 6.56 (t, J=8.7 Hz,
2H, ArH), 4.07 (t, J=5.7 Hz, 2H, NCH2CH2CH2O), 2.83 (m, 6H, piperidine ring
CH2NCH2 and NCH2CH2CH2O), 2.31 (s, 3H, CH3), 2.09~2.03 (m, 2H,
NCH2CH2CH2O), 1.66~1.64 (m, 4H, piperidine ring CH2), 1.47 (s, 2H, piperidine
ring CH2); 13C NMR (DMSO-d6, 75 MHz) δ: 167.4, 163.2, 162.1, 138.4, 138.3, 130.7,
129.0, 125.3, 122.1, 118.8, 109.7, 106.9, 102.3, 65.8, 54.0, 52.8, 24.1, 23.4, 22.2,
21.6; IR (KBr) υ,==2945, 1645, 1597, 1549, 1488, 1438, 1380, 1330, 1244, 1145,
1093, 982, 850, 788, 694 cm-1; MS (ESI) m/z 367.33 [M-H]+.
5.1.17 4-(3-(piperidin-1-yl)propoxy)-N-(3-(trifluoromethyl)phenyl)-2-hydroxybe-
nzamide (11b) Compound 11b was obtained as a white solid (1.1 g, 37.5 %):
Rf=0.37 (CHCl3:CH3OH 15:1); mp: 196.7-198.0 °C; 1H-NMR (DMSO-d6, 400 MHz)
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δ: 11.31 (s, 1H, NH), 10.61 (s, 1H, OH), 8.20 (s, 1H, ArH), 8.05 (d, J=8.4 Hz, 1H,
ArH), 7.97 (d, J=8.4 Hz, 1H, ArH), 7.62 (t, J=7.6 Hz, 1H, ArH), 7.48 (d, J=7.6 Hz,
1H, ArH), 6.58 (d, J=9.2 Hz, 2H, ArH), 4.12 (t, J=5.6 Hz, 2H, NCH2CH2CH2O), 3.08
(m, 6H, piperidine ring CH2NCH2 and NCH2CH2CH2O), 2.18-2.14 (m, 2H,
NCH2CH2CH2O), 1.77-1.74 (m, 4H, piperidine ring CH2), 1.52 (s, 2H, piperidine ring
CH2); 13C-NMR (DMSO-d6, 100 MHz) δ: 167.7, 163.4, 161.9, 139.5, 131.0, 130.3,
125.0, 120.8, 120.7, 117.6, 117.5, 109.8, 107.0, 102.4, 65.8, 53.8, 52.7, 23.9, 23.1,
22.1; IR (KBr) υ,==3283, 2947, 2635, 1648, 1598, 1554, 1494, 1447, 1379, 1332,
1249, 1154, 1122, 1071, 801 cm-1; MS (ESI) m/z 421.38 [M-H]+.
5.1.18 4-(3-(piperidin-1-yl)propoxy)-N-(3-chloro-4-fluorophenyl)-2-hydroxy-ben-
zamide (12b) Compound 12b was obtained as a white solid (1.0 g, 37.2 %): Rf=0.37
(CHCl3:CH3OH 15:1); mp: 214.4-215.7 °C; 1H NMR (DMSO-d6, 300 MHz) δ:
10.81(s, 1H, OH), 8.03(m, 1H, ArH), 7.91(d, J=8.7 Hz, 1H, ArH), 7.63-7.58(m, 1H,
ArH), 7.44(t, J=8.7 Hz, 1H, ArH), 6.51(dd, J=8.7, 2.1 Hz, 1H, ArH), 6.45(s, 1H,
ArH), 4.05(t, J=6.0 Hz, 2H, NCH2CH2CH2O), 3.36(m, 6H, piperidine ring CH2NCH2
and NCH2CH2CH2O), 1.93~1.85(m, 2H, NCH2CH2CH2O), 1.54-1.51(m, 4H,
piperidine ring CH2), 1.41(s, 2H, piperidine ring CH2); 13C-NMR (DMSO-d6, 75
MHz) δ: 180.6, 169.4, 167.5, 163.6, 136.1, 130.7, 125.4, 122.8, 121.7, 117.4, 117.1,
106.7, 102.4, 66.4, 55.1, 54.2, 26.1, 25.5, 24.0; IR (KBr) υ,==3416, 2944, 2867, 1646,
1594, 1546, 1498, 1436, 1324, 1243, 1212, 1140, 1051, 982, 859 cm-1; MS (ESI) m/z
405.39 [M-H]+.
5.1.19 4-(3-(piperidin-1-yl)propoxy)-N-(2-chloro-4-nitrophenyl)-2-hydroxy-ben-
zamide (13b) Compound 13b was obtained as a yellow solid (1.1 g, 37.2 %):
Rf=0.37 (CHCl3:CH3OH 15:1); mp:.231.5-232.7; 1H-NMR (DMSO-d6, 400 MHz) δ:
8.90 (d, J=9.2 Hz, 1H, ArH), 8.38 (d, J=2.8 Hz, 1H, ArH), 8.26 (dd, J=9.2, 2.4 Hz,
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1H, ArH), 7.93 (d, J=9.2 Hz, 1H, ArH), 6.47 (s, 1H, ArH), 6.45 (s, 1H, ArH), 4.04 (t,
J=6.0 Hz, 2H, NCH2CH2CH2O), 2.96 (m, 6H, piperidine ring CH2NCH2 and
NCH2CH2CH2O), 2.06-2.02 (m, 2H, NCH2CH2CH2O), 1.69-1.65 (m, 4H, piperidine
ring CH2), 1.49 (s, 2H, piperidine ring CH2); 13C-NMR (DMSO-d6, 100 MHz) δ:
170.0, 168.2, 148.1, 146.7, 137.5, 129.9, 129.0, 127.1, 125.5, 116.6, 115.9, 110.6,
107.5, 70.3, 59.0, 57.8, 29.2, 28.5, 27.2; IR (KBr) υ,==2966, 2703, 1658, 1618, 1587,
1513, 1470, 1413, 1344, 1296, 1220, 1155, 1098, 981, 846 cm-1; MS (ESI) m/z
434.11[M-H]+.
5.2 Pharmacology
5.2.1 In vitro EGFR tyrosine kinase activity.
In vitro EGFR-TK inhibition assays were carried out as described in reference.
EGFR and EGF were purchased from Wuhan Boster Biological Technology, Ltd, and
the ADP-GloTM Kinase was purchased from Promega. The experiments were
performed according to the instructions of the manufacturer. The EGFR tyrosine
kinase activity was performed using Kinase-Glo Plus luminescence kinase assay kit
(Promega). It measures kinase activity by quantitating the amount of ATP remaining
in solution following a kinase reaction. The luminescent signal from the assay is
correlated with the amount of ATP present and is inversely correlated with the
amount of kinase activity. Briefly, 96-well plates were precoated with EGFR, EGF
and tested compound in a ratio of 2:2:1 were added. The reaction mixtures were
incubated for 40 min at room temperature while being shaken. After completion of the
reaction, 20 μL of ADP-GloTM Plus Luminescence kinase assay solution were added,
incubate the plate for 30 min at room temperature. The corrected activity for each
protein kinase target was determined by removing the blank control value.
Luminescence signal was measured using a Tecan Infinite Spectramax M2e
microplate reader.
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5.2.2 Experimental method for cell proliferation assay
The anti-proliferative activities of salicylanilide derivatives 8a-13a and 8b-13b were
determined using a standard (MTT)-based colorimetric assay. Briefly, cell lines were
seeded at a density 104 cells/well in 96-well microtiter plates. The cells were then
treated in triplicate with various concentration of each compound and cultured in 5 %
medium for 72 h. Control cells were treated with vehicle alone. During the last 4 h of
incubation, the cells were exposed to tetrazolium dye (MTT) Solution (5 mg/mL, 20
μL per well). The generated formazan crystals were dissolved in 100 μL of dimethyl
sulfoxide (DMSO), and the absorbance was read spectrophotometrically at 570 nm
using an enzyme-linked immunosorbent assay plate reader. The data was calculated
using Graph Pad Prism version 5.0. The IC50 were fitted using a non-linear regression
model with a sigmodial dose response.
5.2.3 Molecular modeling study
The docking was performed using a receptor from the 2ITO structure from
RCSB Protein Data Bank [17], and Auto Dock Vina. The co-crystal inhibitor (gefitinib)
was removed from the initial X-ray structure, water molecules were then removed and
polar hydrogens and gastieger charges were added by using Autodock Tool. Ligands
were optimized for the energy and geometry using MM2 force fields. In the structures
of the ligands, all bonds were treated as rotatable, except for the aromatic bonds. The
force field for protein is “Semiempirical Free Energy Force Field with Charge-Based
Desolvation”. Lamarckian genetic algorithmwas used for all molecular docking
simulations. Population size of 300, mutation rate of 0.02, and crossover rate of 0.8
were set as the parameters. Simulations were performed using up to 2.5 million
energy evaluations with a maximum of 27,000 generations. Docking proceeded with
an exhaustiveness value of 100 and a maximum output of 10 structures. The lowest
energy conformations were regarded as the binding conformations between the
ligands and the proteins. Visualization was done using the PyMOL software (The
PyMOL Molecular Graphics System, Version 1.3, Schrödinger, LLC).
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Acknowledgement
This study was supported by National Drug Innovation Incubator
Base Project (2012ZX09401006) and Natural Science Foundation of
Anhui Province (1208085MH128).
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Figure 1. Representative EGFR inhibitors
Figure 2. The structure of the salicylanilide
Figure 3. The salicylanilide derivatives reported as anti-tumor inhibitors and proposed as
EGFR inhibitors
Scheme 1. Reagents and conditions: (a) CH3I, NaHCO3, DMF, 40 °C,10 h; (b) 1, 3
dibromopropane, K2CO3, TEBAC, acetone, 60 °C,8 h; (c) NaHCO3, NaI, isopropanol, 85
°C,6 h; (d) NaOH,CH3OH, H2O, r.t, 6 h; (e) SOCl2, DMF, 85 °C, 6 h; (f) Et3N, CH2Cl2, ice
bath, 40 min.
x s
Table 1. EGFR inhibition of the salicylanilide derivatives 8a-13a and 8b-13b (n=3,
)
Figure 4. (a) Compound 12b (light blue) docked into the EGFR binding domain. (b) Dock
structure of compound 12b with EGFR. Hydrogen bonds towards MET793 were highlighted.
Figure 1. Representative EGFR inhibitors
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Figure 2. The structure of the salicylanilide
Figure 3. The salicylanilide derivatives reported as anti-tumor inhibitors and
proposed as EGFR inhibitors
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Figure 4. (a) Compound 12b (light blue) docked into the EGFR binding domain. (b) Dock
structure of compound 12b with EGFR. Hydrogen bonds towards MET793 were
highlighted.
This article is protected by copyright. All rights reserved.