Design, synthesis and biological evaluation of N-phenylsulfonylnicotinamide derivatives as novel antitumor inhibitors

Article abstract of DOI:10.1016/j.bmc.2012.01.004

A series of novel N-phenylsulfonylnicotinamide derivatives (1-24) have been synthesized and evaluated as potential EGFR tyrosine kinase (TK) inhibitors. Among all the compounds, compound 10 (5-bromo-N-(4-chlorophenylsulfonyl) nicotinamide) showed the most

Full text of DOI:10.1016/j.bmc.2012.01.004

Bioorganic & Medicinal Chemistry 20 (2012) 1411–1416
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis and biological evaluation of N-phenylsulfonylnicotinamide
derivatives as novel antitumor inhibitors
⇑
⇑
Hui Zhang , Xiang Lu , Li-Rong Zhang, Jia-Jia Liu, Xian-Hui Yang, Xiao-Ming Wang , Hai-Liang Zhu
State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210093, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel N-phenylsulfonylnicotinamide derivatives (1–24) have been synthesized and evaluated
as potential EGFR tyrosine kinase (TK) inhibitors. Among all the compounds, compound 10 (5-bromo-N-
(4-chlorophenylsulfonyl)nicotinamide) showed the most potent growth inhibitory activity against EGFR
Received 14 December 2011
Revised 31 December 2011
Accepted 2 January 2012
Available online 9 January 2012
TK and antiproliferative activity of MCF-7 cancer cell line in vitro, with IC50 value of 0.09 and 0.07 lM.
Docking simulation was performed to insert compound 10 into the EGFR TK active site to determine
the probable binding model. Based on the preliminary results, compound 10 with potent inhibitory activ-
ity to tumor growth may be a potential anticancer agent.
Keywords:
N-Phenylsulfonylnicotinamide
Anticancer
Ó 2012 Elsevier Ltd. All rights reserved.
MCF-7
EGFR tyrosine kinase
1. Introduction
the target of many drugs for treating breast cancer is not VEGF
but EGFR.19 These results promoted us to hypothesize that N-
Epidermal growth factor receptor (EGFR) kinase, a member of
tyrosine kinases family is a transmembrane protein with an exter-
nal ligand binding receptor domain and an intracellular tyrosine
kinase activity domain. Once a native ligand (e.g., EGF) binds to
the extracellular domain, it results in receptor homo- or heterodi-
merization, activation of intrinsic TK activity, and autophosphory-
lation of the receptors, which regulates cell growth, differentiation,
mitosis, and death.1–6
ion, mutation, or coexpression of the ligand and the receptor,
these receptors can become hyperactivated; the result of this is
uncontrolled cell proliferation.7,8 The abnormal phosphorylation
of EGFR tyrosine kinase (TK) has been studied in nearly all kinds
of cancers.9,10 However breast cancer is most thoroughly studied,
which is overexpressed in 25–30% of cases and is correlated with
a poor prognosis.11–14 Compounds that inhibit the kinase activity
of EGFR are of potential interest as new therapeutic antitumor
agents.
(phenylsulfonyl)benzamide derivatives may also inhibit the over-
expression of EGFR. In addition, Kim et al. have reported bioisoster-
es of terpyridine with considerable protein kinase
C (PKC)
inhibitory activity and antitumor cytotoxicities against several hu-
man cancer cell lines.20 On the other hand, pyridine which has sim-
ilar structure with benzene, a small bioactive molecule, is an
important pharmacophore that can form hydrogen-bonded struc-
tures similar to those encountered with the base-pairing mecha-
nism in DNA and RNA.21,22 In view of their importance as drugs,
biologically active natural products, and in other related applica-
tions, extensive studies have been carried out on the synthesis of
pyridine compounds in recent years. Therefore, we try to replace
the benzene ring with the pyridine ring to improve the biological
activity. All of these encouraged us to synthesize and biologically
In previous studies, N-(phenylsulfonyl)benzamide derivatives
(Fig. 1) revealed remarkable growth inhibitory activity against
VEGF and antitumor effects against a broad range of human tumor
xenografts in SCID mice.15 Historically, the VEGF and EGFR path-
ways are closely related, sharing common downstream. Inhibition
of VEGF-related pathways is proved to contribute to the mecha-
nism of action of agents targeting the EGFR.16–18 Furthermore,
⇑
Corresponding authors. Tel.: +86 25 8359 2572; fax: +86 25 8359 2672.
E-mail address: zhuhl@nju.edu.cn (H.-L. Zhu).
Figure 1. The structure of N-(phenylsulfonyl)benzamide derivatives.
These two authors equally contributed to this paper.
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2012.01.004

1412
H. Zhang et al. / Bioorg. Med. Chem. 20 (2012) 1411–1416
R4
O
O
O
NH2
R2
R3
S
S
R2
R3
COOH
N
H
EDC
O
O
+
DMAP
N
R1
R4
N
R1
13: = Cl, = H, R =H, R = F
1: R1= H, R2= H, R3=H, R4=H
R1
R2
3
4
14: = Cl, = H, R =H, R = Br
R1
R2
2: R1= H, R2= H, R3=H, R4= Me
3: R1= H, R2= H, R3=H, R4= F
4: R1= H, R2= H, R3=H, R4= Br
5: R1= H, R2= H, R3=H, R4= Cl
3
4
15: = Cl, = H, R =H, R = Cl
R1
R2
3
4
16: = H, = H, R =Cl, R = H
R1
R2
3
4
17: = H, = H, R =Cl, R = Me
R1
R2
3
4
18: = H, = H, R =Cl, R = F
6: = H, = Br, R3=H, R4=H
R1
R2
R1
R2
3
4
7: = H, = Br, R3=H, R4= Me
R1
R2
19: = H, = H, R =Cl, R = Br
R1
R2
3
4
8: = H, = Br, R3=H, R4= F
R1
R2
20: = H, = H, R =Cl, R = Cl
3 4
R1
R2
21: R1= Cl, R2= H, R3=Me, R4=H
9: = H, = Br, R3=H, R4= Br
R1
R2
22: R1= Cl, R2= H, R3=Me, R4= Me
23: R1= Cl, R2= H, R3=Me, R4= Br
24: R1= Cl, R2= H, R3=Me, R4= Cl
10: = H, = Br, R3=H, R4=Cl
R1
R2
11: = Cl, = H, R =H, R = H
R1
R2
3
4
12: = Cl, = H, R =H, R = Me
R1
R2
3
4
Scheme 1. Synthesis route of N-phenylsulfonylnicotinamide derivatives (compounds 1–24). Reagents and conditions: EDCÁHCl/DMAP, CH2Cl2, Reflux, 6–8 h
evaluate of a novel series of N-phenylsulfonylnicotinamide deriva-
tives as anti-EGFR TK agents and antiproliferative activity of MCF-7
cancer cell.
Table 1
Inhibition (IC50) of human breast cell lines MCF-7 proliferation and EGFR kinase
phosphorylation of compounds 1–24
2. Results and discussion
2.1. Chemistry
Compounds
IC50
(l
M)
MCF-7
EGFR inhibition IC50
1
2
3
4
5
6
7
8
14.42 0.81
12.04 0.15
15.97 0.62
14.69 0.57
12.97 0.43
12.89 0.61
9.37 0.14
0.98 0.012
0.34 0.016
0.07 0.002
14.02 0.45
12.52 0.61
12.49 0.58
11.37 0.36
10.22 0.41
14.11 0.67
17.21 0.51
16.83 0.67
9.05 0.12
8.07 0.14
13.42 0.79
8.52 0.15
1.97 0.05
1.35 0.08
0.02 0.001
>30
24.83 0.61
>30
The synthesis of N-phenylsulfonylnicotinamide derivatives fol-
lowed the general reaction pathway outlined in Scheme 1. Com-
pounds 1–24 were synthesized by coupling substituted nicotinic
acid with equimolar quantities of substituted benzenesulfonamide
amides using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide
hydrochloride (EDCÁHCl) and 4-dimethylaminopyridine (DMAP)
as coupling reagent. Most of the target compounds 1–24 were re-
ported for the first time. All of the synthetic compounds gave sat-
isfactory analytical and spectroscopic data. 1H NMR and ESI-MS
spectra were consistent with the assigned structures.
>30
25.36 0.29
20.19 0.32
12.11 0.14
3.27 0.23
2.08 0.06
0.09 0.004
>30
27.05 0.23
28.66 0.19
15.68 0.52
19.83 0.21
>30
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
Erlotinib
2.2. Biological activity and molecular modeling
>30
>30
The synthesized N-phenylsulfonylnicotinamide derivatives
1–24 were evaluated for their ability to inhibit the autophosphory-
lation of EGFR TK using ELISA kits and western blotting. The half
maximal inhibitory concentration (IC50) of the compounds 1–24
are summarized in Table 1. As shown in Table 1, it was observed
that some N-phenylsulfonylnicotinamide derivatives (such as com-
pounds 8, 9, 10, 23 and 24) were found to exhibit good inhibitory
29.07 0.08
18.15 0.82
21.56 0.09
15.02 0.38
2.64 0.09
1.19 0.11
0.03 0.001
activity, displaying IC50 values between 0.09 and 3.27 lM.

H. Zhang et al. / Bioorg. Med. Chem. 20 (2012) 1411–1416
1413
structure with the inhibitor excluded from the coordinates (PDB
accession code 1M1723) as a receptor for compound binding. Resi-
dues around Erlotinib at a radius of 6 Å were isolated for the con-
struction of the grid for docking screening. As depicted in Figure 3,
the oxygen atom of carboxyl group of the compound 10 is potently
bound to hydrogen atom of hydroxyl group of Thr 766 in EGFR TK
(distance = 2.76 Å). Nitrogen atom of pyridine ring exhibits a
hydrogen bond with Met 769 in EGFR TK (distance = 1.80 Å). In
addition, the MeSO2–phenyl moiety is oriented towards the EGFR
TK secondary pocket (Lys 721 and Thr 830). One of the O-atom
of p-MeSO2 substituent forms a hydrogen binding interaction with
amino group of Lys 721 (distance = 2.34 Å) whereas the other
O-atom is about 2.42 Å away from hydroxy of this amino acid of
Thr 830, which can provide a good explanation for the high po-
tency of compound 10 towards EGFR TK. This molecular docking
result, along with the enzyme assay data, suggesting that com-
pound 10 is a potential inhibitor of EGFR.
Figure 2. Inhibitory effect of compound 10 on EGFR kinase phosphorylation level.
MCF-7 cells were seeded into 6-well plates at a concentration of 1 Â 105 cells/ml.
After 16 h of growth in serum-containing media, cells were incubated in serum-free
media for 24 h. Compound 10 was then added for 1 h at the concentration of 0.75,
1.5 and 3 lM, then stimulated for 15 min with 100 ng/mL EGF. After incubation,
cells were lysed, and protein was applied on SDS–polyacrylamide gel. The level of
EGFR kinase phosphorylation level was examined by western blotting analysis.
Compound 10 displayed the most potent inhibitory activity with
IC50 of 0.09
The results of western blotting assay further confirmed that com-
pound 10 at the concentration above 1.5 M could inhibit EGF-in-
lM (the IC50 of positive control Erlotinib = 0.03 lM).
Besides, in vitro antiproliferative activities of compounds 1–24
were studied in the human breast cancer cell line MCF-7, which
overexpresses EGFR TK, by applying the MTT colorimetric assay.11
As shown in Table 1, most compounds exhibited similar trends
with inhibitory activities against EGFR TK. Compound 10 was
found to exhibit the most potent activity with IC50 values of
l
duced EGFR autophosphorylation in the MCF-7 cell line (Fig. 2).
Subsequently SAR (structure–activity relationship) studies were
performed by modification of the parent compound to determine
how the substituents of the subunits affected the EGFR TK inhibi-
tory activities. As shown in Table 1, structure–activity relation-
ships for compounds 1–24 demonstrated that compounds 6–10
with meta-bromide substituents on pyridine ring showed potent
EGFR TK inhibitory activities. Compounds 21–24 with both
ortho-methyl and chloro groups on the pyridine ring were more
active than compounds 11–20 that contain single ortho-chloro
group on the pyridine ring. Compounds bearing the same substit-
uents on the pyridine ring exhibited distinct EGFR TK inhibitory
activities due to the difference of the substituents on the phenyl
ring. The EGFR TK inhibitory activities of compounds 1–24 with
different para-substituents on phenyl ring increased in the follow-
ing order: CH3 < F < Br < Cl. This result indicated that compounds
1–24 with electronic-withdrawing substituents (halogen) on the
phenyl ring showed more potent EGFR inhibitory activities than
compounds contains donating methyl substituents.
0.07
lM (the IC50 of positive control Erlotinib = 0.02 lM).
3. Conclusions
In summary, a novel series of N-phenylsulfonylnicotinamide
derivatives that may function as inhibitors of EGFR TK have been
prepared, and some of the synthesized compounds displayed effi-
cient EGFR inhibitory activity. Compound 10 displayed the most
potent inhibitory activity (IC50 = 0.09
tive assay results indicated that compound 10 showed promising
inhibitory activity (IC50 = 0.07 M) against the human breast can-
lM). Moreover, antiprolifera-
l
cer cell line, MCF-7, which overexpresses EGFR TK. After analysis
of the binding model of compound 10 with EGFR, it was found that
four hydrogen bonds interaction with the protein residues in the
ATP binding site might play a crucial role in its EGFR and antipro-
liferative activities. Therefore, compound 10 could be a promising
lead for the further development of a potential anticancer agent.
In order to further understand the SARs observed for EGFR TK
and to guide additional SAR studies, molecular docking of the most
potent inhibitor (10) into ATP binding site of EGFR TK was per-
formed. Molsoft ICM-Pro software was chosen as the virtual dock-
ing tool that used the active site of the EGFR/ Erlotinib crystal
4. Experimental
4.1. Chemistry
The synthesis route of compounds 1–24 followed the general
pathway outlined in Scheme 1. The substituted nicotinic acid
(1 mmol) mixed with benzenesulfonamide (1 mmol) through by
using 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochlo-
ride (EDCÁHCl) (1.2 mmol) and 4-dimethylaminopyridine (DMAP)
(1.2 mmol) in anhydrous CH2Cl2 for 6–8 h at 70–80 °C. The reaction
was monitored by TLC. The products are extracted with ethyl ace-
tate. The extract is washed successively with 1 N HCl, water, 1 M
NaHCO3, and water, dried over MgSO4, filtered and evaporated.
The residue is purified by column chromatography using petroleum
ether and ethyl acetate (1:1).
4.2. Spectral properties of N-phenylsulfonylnicotinamide
derivatives
4.2.1. N-(Phenylsulfonyl)nicotinamide (1)
White solid, yield 81%, mp: 242–244 °C. 1H NMR (500 MHz,
DMSO-d6) d: 7.53–7.56 (m, 1H), 7.63–7.66 (m, 2H), 7.72 (t,
J = 7.5 Hz, 1H), 8.00–8.02 (m, 2H), 8.23–8.25 (m, 1H), 8.76–8.78
(m, 1H), 9.00 (m, 1H). MS (ESI): 263.0 (C12H11N2O3S, [M+H]+). Anal.
Figure 3. Binding mode of compound 10 with EGFR kinase; Ligand is represented
as wire, the interacting residues are depicted as stick and ball. The H-bond (green) is
displayed as line.

1414
H. Zhang et al. / Bioorg. Med. Chem. 20 (2012) 1411–1416
Calcd for C12H10N2O3S: C, 54.95; H, 3.84; N, 10.68. Found: C, 54.74;
H, 3.85; N, 10.72.
4.2.10. 5-bromo-N-(4-chlorophenylsulfonyl)nicotinamide (10)
White solid, yield 87%, mp: 230 °C. 1 NMR (300 MHz, DMSO-d6)
d: 7.69 (d, J = 8.6 Hz, 2H), 7.99 (d, J = 8.8 Hz, 2H), 8.45 (t, J = 2.0 Hz,
1H), 8.89 (d, J = 2.2 Hz, 1H), 8.94 (d, J = 1.9 Hz, 1H). MS (ESI): 375.9
(C12H9BrClN2O3S, [M+H]+). Anal. Calcd for C12H8BrClN2O3S: C,
38.37; H, 2.15; N, 7.46. Found: C, 38.48; H, 2.16; N, 7.43.
4.2.2. N-Tosylnicotinamide (2)
1
White solid, yield 83%, mp: 225–227 °C.
NMR (300 MHz,
DMSO-d6) d: 2.40 (s, 3H), 7.44 (d, J = 8.0 Hz, 2H), 7.52–7.57 (m,
1H), 7.90 (d, J = 8.2 Hz, 2H), 8.23 (d, J = 8.0 Hz, 1H), 8.77–8.79 (m,
1H), 8.99 (d, J = 1.8 Hz, 1H). MS (ESI): 277.1 (C13H13N2O3S,
[M+H]+). Anal. Calcd for C13H12N2O3S: C, 56.51; H, 4.38; N, 10.14.
Found: C, 56.38; H, 4.39; N, 10.10.
4.2.11. 2-Chloro-N-(phenylsulfonyl)nicotinamide (11)
1
White solid, yield 75%, mp: 170–172 °C.
NMR (300 MHz,
DMSO-d6) d: 7.48–7.52 (m, 1H), 7.68 (t, J = 7.4 Hz, 2H), 7.77 (t,
J = 7.3 Hz, 1H), 7.97–8.02 (m, 3H), 8.50–8.52 (m, 1H), 12.90 (s,
1H). MS (ESI): 297.0 (C12H10ClN2O3S, [M+H]+). Anal. Calcd for
4.2.3. N-(4-Fluorophenylsulfonyl)nicotinamide (3)
White solid, yield 85%, mp: 245–246 °C. 1H NMR (500 MHz,
DMSO-d6) d: 7.46–7.49 (m, 2H), 7.56–7.58 (m, 1H), 8.06–8.09 (m,
2H), 8.26–8.28 (m, 1H), 8.77–8.79(m, 1H), 9.01 (d, J = 1.6 Hz, 1H).
MS (ESI): 281.0 (C12H10FN2O3S, [M+H]+). Anal. Calcd for
C12H9FN2O3S: C, 51.42; H, 3.24; N, 9.99. Found: C, 51.24; H, 3.25;
N, 9.95.
C12H9ClN2O3S: C, 48.57; H, 3.06; N, 9.44. Found: C, 48.75; H,
3.07; N, 9.47.
4.2.12. 2-Chloro-N-tosylnicotinamide (12)
White solid, yield 73%, mp: 182 °C. 1 NMR (300 MHz, DMSO-d6)
d: 2.92 (s, 3H), 7.92–7.96 (m, 3H), 8.40–8.46 (m, 3H), 8.94–8.96 (m,
1H), 11.59 (s, 1H). MS (ESI): 311.0 (C13H12ClN2O3S, [M+H]+). Anal.
Calcd for C13H11ClN2O3S: C, 50.24; H, 3.57; N, 9.01. Found: C,
50.42; H, 3.58; N, 9.03.
4.2.4. N-(4-Bromophenylsulfonyl)nicotinamide (4)
1
White solid, yield 79%, mp: 255–256 °C.
NMR (300 MHz,
DMSO-d6) d: 7.51–7.61 (m, 1H), 7.82–7.85 (m, 2H), 7.90–7.93 (m,
2H), 8.28–8.32 (m, 1H), 8.77–8.79 (m, 1H), 9.01 (d, J = 2.2 Hz,
1H). MS (ESI): 343.0 (C12H10BrN2O3S, [M+H]+). Anal. Calcd for
4.2.13. 2-Chloro-N-(4-fluorophenylsulfonyl)nicotinamide (13)
White solid, yield 86%, mp: 182 °C. 1 NMR (300 MHz, DMSO-d6)
d: 7.92–7.96 (m, 3H), 8.40–8.46 (m, 3H), 8.94–8.96 (m, 1H), 11.59
(s, 1H). MS (ESI): 315.0 (C12H9ClFN2O3S, [M+H]+). Anal. Calcd for
C12H9BrN2O3S: C, 42.24; H, 2.66; N, 8.21. Found: C, 42.37; H,
2.67; N, 8.23.
C12H8ClFN2O3S: C, 45.80; H, 2.56; N, 8.90. Found: C, 45.65; H,
4.2.5. N-(4-Chlorophenylsulfonyl)nicotinamide (5)
2.55; N, 8.85.
1
White solid, yield 82%, mp: 241–243 °C.
NMR (300 MHz,
DMSO-d6) d: 7.58–7.62 (m, 1H), 7.84 (d, J = 8.6 Hz, 2H), 7.93 (d,
J = 9.1 Hz, 2H), 8.29–8.33 (m, 1H), 8.78–8.81 (m, 1H), 9.02 (d,
J = 2.0 Hz, 1H). MS (ESI): 298.1 (C12H10ClN2O3S, [M+H]+). Anal.
Calcd for C12H9ClN2O3S: C, 48.57; H, 3.06; N, 9.44. Found: C,
48.31; H, 3.31; N, 9.22.
4.2.14. N-(4-bromophenylsulfonyl)-2-chloronicotinamide (14)
White solid, yield 87%, mp: 202 °C. 1 NMR (300 MHz, DMSO-d6)
d: 7.49–7.53 (m, 1H), 7.89–7.95 (m, 4H), 7.99–8.02 (dd, J1 = 2.0,
J2 = 7.5, 1H), 8.51–8.53 (dd, J1 = 2.0, J2 = 5.0, 1H). MS (ESI): 375.9
(C12H9BrClN2O3S, [M+H]+). Anal. Calcd for C12H8BrClN2O3S: C,
38.37; H, 2.15; N, 7.46. Found: C, 38.28; H, 2.16; N, 7.48.
4.2.6. 5-bromo-N-(phenylsulfonyl)nicotinamide (6)
White solid, yield 86%, mp: 206 °C. 1 NMR (300 MHz, DMSO-d6)
d: 7.64 (t, J = 7.0 Hz, 2H), 7.73 (d, J = 7.3 Hz, 1H), 8.01 (t, J = 4.4 Hz,
2H), 8.48 (t, J = 2.0 Hz, 1H), 8.91–8.95 (dd, J1=1.8, J2=8.6 Hz, 2H).
MS (ESI): 342.9 (C12H10BrN2O3S, [M+H]+). Anal. Calcd for
4.2.15. 2-Chloro-N-(4-chlorophenylsulfonyl)nicotinamide (15)
1
White solid, yield 76%, mp: 196–198 °C.
NMR (300 MHz,
DMSO-d6) d: 7.49–7.53 (m, 1H), 7.75–7.78 (d, J = 10.3 Hz, 2H),
7.99–8.03 (m, 3H), 8.51–8.53 (dd, J1 = 1.8, J2 = 4.9 Hz, 1H), 12.94
(s, 1H). MS (ESI): 330.9 (C12H9Cl2N2O3S, [M+H]+). Anal. Calcd for
C12H9BrN2O3S: C, 42.24; H, 2.66; N, 8.21. Found: C, 42.40; H,
C12H8Cl2N2O3S: C, 43.52; H, 2.43; N, 8.46. Found: C, 43.36; H,
2.65; N, 8.23.
2.42; N, 8.48.
4.2.7. 5-bromo-N-tosylnicotinamide (7)
White solid, yield 67%, mp: 250 °C. 1 NMR (300 MHz, DMSO-d6)
d: 2.20–2.62 (m, 3H), 8.08–8.18 (m, 2H), 8.19–8.23 (m, 1H), 8.56–
8.59 (m, 1H), 8.90 (t, J = 2.1 Hz, 1H), 9.33 (d, J = 2.2 Hz, 1H), 9.46
(d, J = 1.8 Hz, 1H), 11.65 (s, 1H). MS (ESI): 357.2 (C13H12BrN2O3S,
[M+H]+). Anal. Calcd for C13H11BrN2O3S: C, 43.96; H, 3.12; N,
7.89. Found: C, 43.80; H, 3.11; N, 7.85.
4.2.16. 6-Chloro-N-(phenylsulfonyl)nicotinamide (16)
White solid, yield 80%, mp: 191 °C. 1 NMR (300 MHz, DMSO-d6)
d: 8.04–8.17 (m, 3H), 8.18–8.23 (m, 1H), 8.56–8.59 (m, 2H), 8.73–
8.76 (dd, J1 = 2.6 Hz, J2 = 8.43 Hz, 1H), 9.35–9.36 (d, J = 2.6 Hz,
1H), 11.65 (s, 1H). MS (ESI): 297.0 (C12H10ClN2O3S, [M+H]+). Anal.
Calcd for C12H9ClN2O3S: C, 48.57; H, 3.06; N, 9.44. Found: C,
48.68; H, 3.05; N, 9.41.
4.2.8. 5-bromo-N-(4-fluorophenylsulfonyl)nicotinamide (8)
1
4.2.17. 6-Chloro-N-tosylnicotinamide (17)
White solid, yield 78%, mp: 165–166 °C.
NMR (300 MHz,
1
White solid, yield 81%, mp: 193–194 °C.
NMR (300 MHz,
DMSO-d6) d: 7.48 (t, J = 8.8 Hz, 2H), 8.06–8.10 (m, 2H), 8.48 (s,
1H), 8.91–8.94 (m, 2H). MS (ESI): 361.2 (C12H9BrFN2O3S, [M+H]+).
Anal. Calcd for C12H8BrFN2O3S: C, 40.13; H, 2.25; N, 7.80. Found:
C, 40.28; H, 2.26; N, 7.76.
DMSO-d6) d: 2.89 (s, 3H), 7.88 (d, J = 8.6 Hz, 2H), 8.03–8.06
(J = 8.4 Hz, 1H), 8.42–8.45 (J = 8.4 Hz, 2H), 8.72–8.75 (dd, J1 = 2.6,
J2 = 8.4 Hz, 1H), 9.34–9.35 (m, 1H), 11.61 (s, 1H). MS (ESI): 312.6
(C13H12ClN2O3S, [M+H]+). Anal. Calcd for C13H11ClN2O3S: C, 50.24;
H, 3.57; N, 9.01. Found: C, 50.48; H, 3.58; N, 9.03.
4.2.9. 5-bromo-N-(4-bromophenylsulfonyl)nicotinamide (9)
1
White solid, yield 82%, mp: 249–250 °C.
NMR (300 MHz,
4.2.18. 6-Chloro-N-(4-fluorophenylsulfonyl)nicotinamide (18)
DMSO-d6) d: 7.44–7.50 (t, J = 8.9 Hz, 2H), 8.05–8.09 (m, 2H), 8.46
(t, J = 2.1 Hz, 1H), 8.89 (d, J = 2.2 Hz, 1H), 8.94 (d, J = 2.0 Hz, 1H).
MS (ESI): 422.6 (C12H9Br2N2O3S, [M+H]+). Anal. Calcd for
1
White solid, yield 83%, mp: 199–200 °C.
NMR (300 MHz,
DMSO-d6) d: 7.83–7.91 (m, 2H), 8.04–8.07 (d, J = 8.4 Hz, 1H),
8.61–8.65 (m, 2H), 8.67–8.77 (dd, J1 = 2.6, J2 = 8.4 Hz, 1H), 9.35
(d, J = 2.0 Hz, 1H), 11.70 (s, 1H). MS (ESI): 314.9 (C12H9ClFN2O3S,
C12H8Br2N2O3S: C, 34.31; H, 1.92; N, 6.67. Found: C, 34.44; H,
1.93; N, 6.65.

H. Zhang et al. / Bioorg. Med. Chem. 20 (2012) 1411–1416
1415
[M+H]+). Anal. Calcd for C12H8ClFN2O3S: C, 45.80; H, 2.56; N, 8.90.
Found: C, 45.58; H, 2.55; N, 8.88.
50 mM NaCl, 20 mM HEPES, 10% glycerol, and 1
l
g/mL each of
aprotinin, leupeptin, and pepstatin for 2 h. The entire purification
procedure was performed at 4 °C or on ice.24
4.2.19. N-(4-bromophenylsulfonyl)-6-chloronicotinamide (19)
The EGFR kinase assay was set up to assess the level of autophos-
phorylation based on DELFIA/Time-Resolved Fluorometry. Com-
pounds 1–24 were dissolved in 100% DMSO and diluted to the
appropriate concentrations with 25 mM HEPES at pH 7.4. In each
1
White solid, yield 77%, mp: 235–237 °C.
NMR (300 MHz,
DMSO-d6) d: 8.04–8.07 (d, J = 8.4 Hz, 1H), 8.28–8.33 (m, 2H),
8.47–8.51 (m, 2H), 8.73–8.77 (dd, J1 = 2.6, J2 = 8.4 Hz, 1H), 9.35–
9.36 (d, J = 2.6 Hz, 1H), 11.73 (s, 1H). MS (ESI): 375.9
(C12H9BrClN2O3S, [M+H]+). Anal. Calcd for C12H8BrClN2O3S: C,
38.37; H, 2.15; N, 7.46. Found: C, 38.48; H, 2.16; N, 7.44.
well, 10
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 L of
5 mM buffer (containing 20 mM HEPES, 2 mM MnCl2, 100
Na3VO4, and 1 mM DTT) and 20 L of 0.1 mM ATP-50 mM MgCl2
lL of compound was incubated with 10 lL (12.5 ng for
l
lM
4.2.20. 6-Chloro-N-(4-chlorophenylsulfonyl)nicotinamide (20)
l
1
White solid, yield 78%, mp: 221–222 °C.
NMR (300 MHz,
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 incubation, liquid was aspirated, and plates were
DMSO-d6) d: 7.65 (d, J = 8.2 Hz, 1H), 7.71–7.75 (d, J = 8.67 Hz,
2H), 7.99–8.04 (d, J = 8.76 Hz, 2H), 8.23–8.27 (dd, J1 = 2.6 Hz,
J2 = 8.43 Hz, 1H), 8.84 (d, J = 2.6 Hz, 1H), 11.60 (s, 1H). MS (ESI):
333.8 (C12H9Cl2N2O3S, [M+H]+). Anal. Calcd for C12H8Cl2N2O3S: C,
43.52; H, 2.43; N, 8.46. Found: C, 43.38; H, 2.41; N, 8.43.
washed three times with wash buffer. A 75 lL (400 ng) sample of
europium labeled anti-phosphotyrosine antibody was added to
each well for another 1 h of incubation. After washing, enhance-
ment solution was added and the signal was detected by Victor
(Wallac Inc.) with excitation at 340 nm and emission at 615 nm.
The percentage of autophosphorylation inhibition by the com-
pounds was calculated using the following equation: 100%À[
(negative control)/(positive controlÀnegative control)]. The IC50
was obtained from curves of percentage inhibition with eight con-
centrations of compound. As the contaminants in the enzyme prep-
aration are fairly low, the majority of the signal detected by the anti-
phosphotyrosine antibody is from EGFR.
4.2.21. 2-Chloro-6-methyl-N-(phenylsulfonyl)nicotinamide (21)
Yellow solid, yield 66%, mp: 90–92 °C. 1 NMR (500 MHz, DMSO-
d6) d: 1.99–2.60 (m, 3H), 7.34 (d, J = 7.9 Hz, 1H), 7.66–7.69 (m, 2H),
7.74–7.78 (m, 1H), 7.84 (d, J = 7.6 Hz, 1H), 7.99–8.01 (m, 2H), 12.82
(s, 1H). MS (ESI): 311.0 (C13H12ClN2O3S, [M+H]+). Anal. Calcd for
C13H11ClN2O3S: C, 50.24; H, 3.57; N, 9.01. Found: C, 50.40; H,
3.56; N, 9.04.
4.2.22. 2-Chloro-6-methyl-N-tosylnicotinamide (22)
1
Yellow solid, yield 68%, mp: 96 °C. NMR (300 MHz, DMSO-d6)
4.4. Western immunoblot analysis
d: 1.15–1.19 (s, 3H), 2.40 (s, 3H), 7.74 (d, J = 8.0 Hz, 2H), 7.89 (d,
J = 8.2 Hz, 2H), 8.47 (s, 1H), 8.92 (d, J = 5.3 Hz, 2H). MS (ESI):
325.0 (C14H14ClN2O3S, [M+H]+). Anal. Calcd for C14H13ClN2O3S: C,
51.77; H, 4.03; N, 8.63. Found: C, 51.68; H, 4.01; N, 8.66.
MCF-7 cells were pretreated with compounds 10 at the concen-
trations of 0.75, 1.5 and 3 lM for 15 min before treatment with
100 ng/mL EGF and examining the expression of EGFR protein.
Cells were lysed with lysis buffer. Western immunoblot analysis
was performed using a method described by Cheenpracha, et al.25
4.2.23. N-(4-bromophenylsulfonyl)-2-chloro-6-methylnicotin
amide (23)
Yellow solid, yield 72%, mp: 121 °C. 1H NMR (300 MHz, DMSO-
d6) d: 2.04 (s, 3H), 7.35 (d, J = 7.9 Hz, 1H), 7.88 (t, J = 8.6 Hz, 1H),
7.91–7.94 (t, J = 4.8 Hz, 4H). MS (ESI): 391.9 (C13H11BrClN2O3S,
[M+H]+). Anal. Calcd for C13H10BrClN2O3S: C, 40.07; H, 2.59; N,
7.19. Found: C, 40.33; H, 2.60; N, 7.16.
4.5. Antiproliferative activities assay
The antiproliferative activities of compounds 1–24 were deter-
mined using a standard (MTT)-based colorimetric assay (Sigma).
Briefly, cell lines were seeded at a density of 7 Â 103 cells/well in
96-well microtiter plates (Costar). After 24 h, exponentially grow-
ing cells were exposed to the indicated compounds at final concen-
4.2.24. 2-Chloro-N-(4-chlorophenylsulfonyl)-6-methylnicotin
amide (24)
Yellow solid, yield 69%, mp: 125 °C. 1 NMR (500 MHz, DMSO-d6) d:
2.00 (s, 3H), 7.35 (d, J = 7.9 Hz, 1H), 7.70–7.77 (d, J = 8.8 Hz, 2H), 7.88
(d, J = 8.8 Hz, 1H), 8.00 (d, J = 8.8 Hz, 2H), 12.98 (s, 1H). MS (ESI):
344.9 (C13H11Cl2N2O3S, [M+H]+). Anal. Calcd for C13H10Cl2N2O3S: C,
45.23; H, 2.92; N, 8.12. Found: C, 45.43; H, 2.93; N, 8.15.
trations ranging from 0.1 to 100
determined by the addition of an MTT solution (10
mL MTT in PBS). After 4 h, 100 L of 10% SDS in 0.01 N HCl was
lM. After 48 h, cell survival was
l
L of 5 mg/
l
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. Survival ratios are expressed in per-
centages with respect to untreated cells. IC50 values were deter-
mined from replicates of six wells from at least two independent
experiments.
4.3. Preparation, and purification of EGFR and inhibitory assay
A 1.6 kb cDNA encoded for the EGFR cytoplasmic domain
(EGFR-CD, amino acids 645–1186) were cloned into baculoviral
expression vector pFASTBacHTc. A sequence that encodes (His)6
was located at the 50 upstream to the EGFR sequence. Sf-9 cells
were infected for 3 days for protein expression. Sf-9 cell pellets
were solubilized at 0 °C in a buffer at pH 7.4 containing 50 mM
4.6. Docking simulations
Molecular docking of compounds 10 into the three-dimensional
EGFR complex structure (PDB code: 1M17, download from the
PDB) was carried out using the Molsoft ICM-Pro software package
(version 3.5-0a).26
HEPES, 10 mM NaCl, 1% Triton, 10
100 M sodium vanadate, 10 g/mL aprotinin, 10
10 g/mL pepstatin, and 16 g/mL benzamidine HCl for 20 min fol-
l
M ammonium molybdate,
l
l
lg/mL leupeptin,
l
l
Acknowledgements
lowed by 20 min centrifugation. Crude extract supernatant was
passed through an equilibrated Ni–NTA superflow packed column
and washed with 10 mM and then 100 mM imidazole to remove
nonspecifically bound material. Histidine tagged proteins were
eluted with 250 and 500 mM imidazole and dialyzed against
This work was supported by Jiangsu National Science Founda-
tion (No. BK2009239) and the Fundamental Research Funds for
the Central Universities (No. 1092020804 & 1106020824).

1416
H. Zhang et al. / Bioorg. Med. Chem. 20 (2012) 1411–1416
15. Lobb, K. L.; Hipskind, P. A.; Aikins, J. A.; Alvarez, E.; Cheung, Y. Y., et al J. Med.
References and notes
Chem. 2004, 47, 5367.
16. Josep, Tabernero Mol. Cancer Res. 2007, 5, 203–220.
17. Herbst, R. S.; Johnson, D. H.; Mininberg, E., et al J. Clin. Oncol. 2005, 23, 2544–
2555.
18. Ellis, L. Hematol. Oncol. Clin. North Am. 2004, 18, 1007–1021.
19. Johnston, S. R. D. Clin. Cancer Res. 2006, 12, 1061s–1068s.
20. Kim, D. S. H. L.; Ashendel, C. L.; Zhou, Q.; Chang, C. T.; Lee, E. S.; Chang, C. J.
Bioorg. Med. Chem. Lett. 1998, 8, 2695.
21. Tipparaju, S. K.; Joyasawal, S. Bioorg. Med. Chem. Lett. 2008, 18, 3565.
22. Hanessian, S.; Simard, D.; Bayrakdarian, M.; Therrien, E.; Nilsson, I. Bioorg. Med.
Chem. Lett. 2008, 18, 1972.
23. Stamos, J.; Sliwkowski, M. X.; Eigenbrot, C. J. Biol. Chem. 2002, 277, 46265–
46272.
24. Tsou, H. R.; Mamuya, N.; Johnson, B. D.; Reich, M. F.; Gruber, B. C.; Ye, F.;
Nilakantan, R.; Shen, R.; Discafani, C.; DeBlanc, R.; Davis, R.; Koehn, F. E.;
Greenberger, L. M.; Wang, Y. F.; Wissner, A. J. Med. Chem. 2001, 44, 2719.
25. Cheenpracha, S.; Park, E. J.; Yoshida, W. Y.; Barit, C.; Wall, M. Bioorg. Med. Chem.
2010, 18, 6598.
1. Kamath, S.; Buolamwini, J. K. J. Med. Chem. 2003, 46, 4657.
2. Workman, P. New Mol. Targets Cancer Chemother. 1994, 1–44.
3. Ullrich, A.; Schlessinger, J. Cell 1991, 61, 203.
4. Aaronson, S. A. Science 1991, 254, 1146.
5. Mu, F.; Stephanie; Coffing, S. L.; Riese, D. J.; Geahlen, R. L. J. Med. Chem. 2001, 44,
441.
6. Wu, C. H.; Coumar, M. S.; Chu, C. Y.; Lin, W. H.; Chen, Y. R. J. Med. Chem. 2010,
53, 7316.
7. Li, H. Q.; Yan, T.; Yang, Y.; Shi, L.; Zhou, C. F. Bioorg. Med. Chem. 2010, 18, 305.
8. Hickey, K.; Grehan, D.; Reid, I. M.; O’Briain, S.; Walsh, T. N.; Hennessy, T. P. J.
Cancer 1994, 74, 1693.
9. Mendelsohn, J. J. Clin. Oncol. 2002, 07, 121.
10. Vlahovic, G.; Crawford, J. Oncologist 2003, 8, 531–538.
11. Lv, P. C.; Li, H. Q.; Sun, J.; Zhou, Y.; Zhu, H. L. Bioorg. Med. Chem. 2010, 18, 4606.
12. Scheurle, D.; Jahanzeb, M.; Aronsohn, R. S.; Watzek, L.; Narayanan, R. H.
Anticancer. Res. 2000, 20, 2091.
13. Cox, G.; Vyberg, M.; Melgaard, B.; Askaa, J.; Oster, A.; O’Byrne, K. J. Int. J. Cancer.
2001, 92, 480.
26. Abagyan, R.; Totrov, M.; Kuznetsov, D. J. Comput. Chem. 1994, 15, 488.
14. Wikstrand, C. J.; McLendon, R. E.; Friedman, A.; Bigner, D. D. Cancer Res. 1997,
57, 4130.