Synthesis, biological evaluation and molecular docking studies of amide-coupled benzoic nitrogen mustard derivatives as potential antitumor agents

Article abstract of DOI:10.1016/j.bmc.2009.11.037

A series of amide-coupled benzoic nitrogen mustard derivatives as potential EGFR and HER-2 kinase inhibitors were synthesized and reported for the first time. Some of them exhibited significant EGFR and HER-2 inhibitory activity. Of all the studied compounds, compounds 5b and 5t exhibited the most potent inhibitory activity, which was comparable to the positive control erlotinib. Docking simulation was performed to position compounds 5b and 5t into the EGFR active site to determine the probable binding model. Antiproliferative assay results indicated that some of the benzoic nitrogen mustard derivatives possessed high antiproliferative activity against MCF-7. In particular, compounds 5b and 5t with potent inhibitory activity in tumor growth inhibition may function as potential antitumor agents.

Full text of DOI:10.1016/j.bmc.2009.11.037

Bioorganic & Medicinal Chemistry 18 (2010) 880–886
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis, biological evaluation and molecular docking studies of
amide-coupled benzoic nitrogen mustard derivatives as potential
antitumor agents
Qing-Zhong Zheng, Fei Zhang, Kui Cheng, Ying Yang, Yu Chen, Yong Qian, Hong-Juan Zhang,
*
*
*
Huan-Qiu Li, Chang-Fang Zhou, Shu-Qing An , Qing-Cai Jiao , Hai-Liang Zhu
State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210093, People’s Republic of 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 amide-coupled benzoic nitrogen mustard derivatives as potential EGFR and HER-2 kinase
inhibitors were synthesized and reported for the first time. Some of them exhibited significant EGFR
and HER-2 inhibitory activity. Of all the studied compounds, compounds 5b and 5t exhibited the most
potent inhibitory activity, which was comparable to the positive control erlotinib. Docking simulation
was performed to position compounds 5b and 5t into the EGFR active site to determine the probable
binding model. Antiproliferative assay results indicated that some of the benzoic nitrogen mustard deriv-
atives possessed high antiproliferative activity against MCF-7. In particular, compounds 5b and 5t with
potent inhibitory activity in tumor growth inhibition may function as potential antitumor agents.
Ó 2009 Elsevier Ltd. All rights reserved.
Received 13 October 2009
Revised 13 November 2009
Accepted 17 November 2009
Available online 22 November 2009
Keywords:
Benzoic nitrogen mustard derivatives
EGFR
HER-2
Molecular docking
Antitumor
1. Introduction
Nitrogen mustards (chlorambucil, melphalan) are widely used
in the treatment of a variety of cancer.12–14 But because of the high
Receptor protein tyrosine kinases play a key role in signal trans-
duction pathways that regulate cell division and differentiation.
Among the growth factor receptor kinases that have been identified
as being important in cancer is epidermal growth factor receptor
(EGFR) kinase (also known as erb-B1 or HER-1) and the related hu-
man epidermal growth factor receptor HER-2 (also known as erbB-
2). Deregulation of growth-factor signaling due to hyperactivation
of the ErbB receptors (primarily EGFR and HER-2) is seen in several
cancer types.1,2 Activation of EGFR may be because of overexpres-
sion, mutations resulting in constitutive activation, or autocrine
expression of ligand. In contrast, activation of HER-2 occurs mainly
by overexpression, which leads to spontaneous homodimerization
and activation of downstream signaling events in a ligand-indepen-
dent manner.3,4 The role of EGFR and HER-2 has been most
thoroughly studied in breast cancer, where it is overexpressed in
25–30% of cases and is correlated with a poor prognosis. EGFR and
HER-2 overexpression is also seen in ovarian cancer,5 lung cancer
(especially lung adenocarcinomas)6–8 and in hormone-refractory
prostate cancer.9 Compounds that inhibit the kinase activity of EGFR
and/or HER-2 after binding of its cognate ligand are of potential
interest as new therapeutic antitumor agents.10,11
reactivity of nitrogen mustards, they are chemically unstable and
produce many unwanted side effects including bone marrow tox-
icity and genotoxicity.15,16 Moreover, a good many nitrogen mus-
tards
are
typical
bifunctional
DNA-alkylating
agents,
nevertheless, their EGFR and/or HER-2 inhibitory activity has been
less widely documented.17 However, benzoic nitrogen mustard
possessing relatively low toxicity is one of the earliest antitumor
drugs in antibody-directed enzyme prodrug therapy (ADEPT).18 It
is reported that benzoic nitrogen mustard derivatives (e.g., CMDA,
CJS 1050) (Fig. 1) possess favorable anticancer activity.19
In view of these above mentioned facts and an attempt to
achieve new potent antitumor agents with good bioavailability
and low toxicity and develop new benzoic nitrogen mustard deriv-
atives as potential inhibitors of EGFR and HER-2, herein, we first
describe the synthesis and the SAR of a series of amide-coupled
benzoic nitrogen mustard derivatives. Biological evaluation indi-
cated that some of the synthesized compounds exhibited favorable
EGFR and HER-2 inhibitory activity and displayed significant inhib-
itory activity in tumor growth inhibition. Docking simulations
were performed using the X-ray crystallographic structure of the
EGFR in complex with an inhibitor to explore the binding modes
of these compounds at the active site. To the best of our knowl-
edge, the synthesis and biological evaluation of these compounds
have not been reported so far.
* Corresponding author. Tel./fax: +86 25 8359 2672.
E-mail address: zhuhl@nju.edu.cn (H.-L. Zhu).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.11.037

Q.-Z. Zheng et al. / Bioorg. Med. Chem. 18 (2010) 880–886
881
Figure 1. The chemical structures of CMDA and CJS 1050.
afforded ethyl 4-aminobenzoate (2). Treatment of compound 2
with ethylene oxide in acetic acid aqueous solution at room tem-
perature gave ethyl 4-(bis(2-hydroxyethyl)amino)benzoate (3).
Reaction of compound 3 with thionyl chloride in benzene under re-
flux for 3 h, and then acidation with concentrated HCl under reflux
for 3 h provided compound 4. Target compounds 5a–t were
synthesized by the coupling reaction between compound 4 and
corresponding arylamines or fatty amines or 3-morpholinopro-
pan-1-amine using N,N0-2 dicyclohexylcarbodiimide (DCC) and 4-
dimethylaminopyridine (4-DMAP) in anhydrous dichloromethane.
2. Results and discussion
2.1. Chemistry
The synthetic route for the new amide-coupled benzoic nitro-
gen mustard derivatives 5a–t is outlined in Schemes 1 and 2. The
synthesis of these nitrogen mustard derivatives started from
4-(bis(2-chloroethyl)amino)benzoic acid (4). Compound 4 was
prepared according to modified procedures of Taylor et al.20 Con-
densation of 4-aminobenzoic acid (1) with anhydrous ethanol
Scheme 1. Synthesis of compounds 5a–5m. Reagents and conditions: (i) H2SO4 (concd), ethanol, reflux, 6 h; (ii) ethylene oxide, 25% aqueous acetic acid, rt, 24 h; (iii) (1)
SOCl2, benzene, reflux, 3 h; (2) HCl (concd), reflux, 3 h; (iv) DCC/4-DMAP, DCM, rt or reflux, 24 h.

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Q.-Z. Zheng et al. / Bioorg. Med. Chem. 18 (2010) 880–886
Scheme 2. Synthesis of compounds 5n–5t. Reagents and conditions: (iv) DCC/DMAP, DCM, rt or reflux, 24 h.
All of the synthetic compounds gave satisfactory analytical and
spectroscopic data, which were in full accordance with their de-
picted structures.
evident that there is also a reasonable correlation between the
EGFR and HER-2 inhibitory activities; thus, this is not surprising
in view of the high sequence homology of the catalytic domains
of these two kinases. As shown in Table 1, some of nitrogen mus-
tard derivatives displayed significant EGFR and HER-2 inhibitory
activity. Of all the studied compounds, compounds 5b and 5t
exhibited the most potent inhibitory activity (as for 5b,
2.2. Biological activity
All new synthesized compounds 5a–t were evaluated for their
ability to inhibit the autophosphorylation of EGFR and HER-2 ki-
nases using a solid-phase ELISA assay. The results were shown in
Table 1. For the studied compounds, we observed that the IC50 va-
lue for inhibition of HER-2 kinase is, in general, higher than that
observed for EGFR kinase but have the same trends. This is possibly
due, in part, to the fact that in the enzyme assays we used higher
concentration of the purified HER-2 kinase than EGFR kinase. It is
IC50 = 0.08
IC50 = 0.09
l
l
M for EGFR and IC50 = 0.41
M for EGFR and IC50 = 0.35
l
M for HER-2; as for 5t,
l
M for HER-2), which was
comparable to the positive control erlotinib (IC50 = 0.02
lM for
EGFR).
Structure–activity relationships in these nitrogen mustard
derivatives demonstrated that compounds containing arylamine
moiety (e.g., 5a–j, 5m, 5s, 5t) showed better inhibitory activity
than those containing fatty amine moiety (e.g., 5k, 5l, 5n–q). More-
over, compounds with electron-withdrawing groups on the ortho
or para position of benzene ring (e.g., 5d–f, 5g–j) displayed less
inhibitory activity than those with electron-donating groups on
the para position (e.g., 5a–c). Meanwhile, a comparison of the sub-
stitution on benzene ring demonstrated that para position-halo-
gen-substituted derivatives (e.g., 5g–i) have more potent EGFR
inhibitory activity than that of ortho position-halogen-substituted
derivatives (e.g., 5d–f), and the potency order is F < Cl < Br. Besides,
introduction of a heterocycle, as for compound 5r, resulted in inac-
Table 1
Inhibition (IC50) of EGFR and HER-2 kinases and inhibition (IC50) of cell proliferation
Compounds
Enzyme assays (IC50
,
l
M)
MCF-7
(IC50, lM)
EGFR
HER-2
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
5m
5n
5o
5p
5q
5r
5s
5t
Erlotinib
0.58
0.08
0.75
11.57
9.46
7.82
1.72
1.49
0.94
19.26
29.83
31.18
14.72
22.81
31.43
>50
35.29
>50
17.51
0.09
1.88
0.41
2.32
15.39
12.87
9.95
3.89
3.33
0.18 0.03
0.05 0.02
0.27 0.04
4.52 0.28
4.08 0.39
3.45 0.41
2.24 0.18
1.71 0.22
0.62 0.14
11.07 1.13
15.57 2.32
18.45 2.27
8.19 0.56
14.68 1.15
17.73 1.21
44.2 3.55
22.75 2.93
36.5 3.22
9.82 1.37
0.06 0.02
0.02 0.004
tive inhibitory activity with IC50 values of more than 50 lM. Fur-
thermore, variation of the aromatic ring moiety was also
explored. Compared with compound 5b, compound 5t bearing
naphthalen-1-amine moiety was compatible with the most potent
inhibitory activity. That is to say, compounds 5b and 5t exhibited
the most potent EGFR inhibitory activity with IC50 of 0.08 and
2.58
27.19
39.64
44.51
22.75
30.06
46.10
>50
48.35
>50
25.87
0.35
0.09
control.
In an effort to elucidate the structure–activity relationships ob-
lM, respectively, which was comparable to the positive
served at the EGFR and guide further SAR studies, molecular dock-
ing of the most potent inhibitors 5b and 5t into ATP binding site of
EGFR kinase was performed on the binding model based on the
EGFR complex structure (1M17.pdb). The binding models of com-
pounds 5b and 5t and EGFR are depicted in Figures 2 and 3, respec-
0.02
0.16

Q.-Z. Zheng et al. / Bioorg. Med. Chem. 18 (2010) 880–886
883
Encouraged by these promising results, the in vitro antiprolifer-
ative activity of compounds 5a–t was evaluated on a panel of one
human tumor cell line (MCF-7), which over expresses EGFR and, to
a less extent HER-2. To our delight, compounds 5a–c, 5i and 5t,
which have potent inhibitory activity of EGFR and HER-2 showed
high antiproliferative activity against MCF-7 with IC50 ranging
from 0.05 lM to 0.62 lM, which indicated that these amide-cou-
pled benzoic nitrogen mustard derivatives were potent inhibitor
of EGFR and HER-2 as antitumor agents. In particular, compounds
5b and 5t displayed significant inhibitory activity in tumor growth
inhibition and exhibited favorable EGFR and HER-2 inhibitory
activity.
Figure 2. Molecular docking modeling of compound 5b with EGFR kinase: The
carbonyl group of 5b forms hydrogen bond (O–Hꢀ ꢀ ꢀO: 2.073 Å, 159.6°) with
hydroxyl group of Thr 766, and the nitrogen atom of 4-(bis(2-chloroethyl)amino)
group of 5b also forms hydrogen bond (N–Hꢀ ꢀ ꢀN: 1.967 Å, 134.8°) with amino
3. Conclusion
In summary, a series of new amide-coupled benzoic nitrogen
mustard derivatives were synthesized, some of which displayed
potent EGFR and HER-2 inhibitory. Of all the studied compounds,
compounds 5b and 5t exhibited the most potent inhibitory activity
hydrogen of Lys 828, and there is a p-cation interaction between the benzene ring
of 4-(bis(2-chloroethyl)amino)benzoic acid moiety of 5b and Lys 828.
(as for 5b, IC50 = 0.08
lM for EGFR and IC50 = 0.41
lM for HER-2; as
for 5t, IC50 = 0.09 M for EGFR and IC50 = 0.35
l
lM for HER-2),
which was comparable to the positive control erlotinib. Docking
simulation was performed to position compounds 5b and 5t into
the EGFR active site to determine the probable binding model.
Antiproliferative assay results indicated that some of the benzoic
nitrogen mustard derivatives possessed high antiproliferative
activity against MCF-7. In particular, compounds 5b and 5t with
potent inhibitory activity in tumor growth inhibition would be po-
tential antitumor agents.
4. Experimental
4.1. Chemistry general
Separation of the compounds by column chromatography was
carried out with Silica Gel 60 (200–300 mesh ASTM, E. Merck).
The quantity of silica gel used was 50–100 times the weight
charged on the column. Then, the eluates were monitored using
thin-layer chromatography (TLC). TLC was carried out on silica
GF254 plates (Qingdao Haiyang Chemical Co., Ltd, China). All the
melting points were determined on a WRS-1B digital melting point
apparatus and are uncorrected. ESI mass spectra were recorded on
a Mariner System 5304 mass spectrometer, and 1H NMR spectra
were recorded on a Bruker DRX 500 or DPX 300 model Spectrom-
eter in chloroform-d (CDCl3) and tetramethylsilane (TMS) was used
as an internal standard. Elemental analyses were performed on a
CHN–O-Rapid instrument and were within 0.4% of the theoretical
values. All chemicals were used as received without further purifi-
cation unless otherwise stated.
Figure 3. Molecular docking modeling of compound 5t with EGFR kinase: The
carbonyl group of 5t forms hydrogen bond (N–Hꢀ ꢀ ꢀO: 2.179 Å, 155.6°) with amino
hydrogen of Lys 828. There is one
p-cation interaction between the naphthalene
ring of 5t and Lys 828, and there is the other
p-cation interaction between the
benzene ring of 4-(bis(2-chloroethyl)amino)benzoic acid moiety of 5t and Lys 822.
tively. In the binding model, compound 5b is nicely bound to the
region with the carbonyl group project toward the hydroxyl group
of Thr 766, with the carbonyl group forming a more optimal H-
bond (O–Hꢀ ꢀ ꢀO: 2.073 Å, 159.6°) interaction, and nitrogen atom
of 4-(bis(2-chloroethyl)amino) group of 5b also forms hydrogen
bond (N–Hꢀ ꢀ ꢀN: 1.967 Å, 134.8°) with amino hydrogen of Lys
828. The modeling also suggested that there is a
tion between the benzene ring of 4-(bis(2-chloroethyl)amino)ben-
zoic acid moiety of 5b and Lys 828. The -cation interaction is a
noncovalent molecular interaction between the face of an elec-
p-cation interac-
p
4.2. General procedure for the preparation of target
compounds 5a–t
tron-rich
ion. The
p
system (e.g., benzene, ethylene) with an adjacent cat-
p
-cation interaction energies are of the same order of
Compound 2: To a solution of compound 1 (17 g) in ethanol
(50 mL) was added dropwise concentrated sulfuric acid (12 mL).
The mixture was heated under reflux for 6 h. The resulting solution
was made neutral by addition of concentrated ammonia water. The
precipitation was collected by filtration and then washed with
water and subsequently dried to afford compound 2 (yield: 90.0%).
Compound 3: To a chilled suspension of compound 2 (15.3 g,
93 mmol) prepared above in 25% aqueous acetic acid (120 mL)
was added ethylene oxide (50 mL, 1.0 mol) with stirring. The reac-
tion was left to stir for 24 h at room temperature to yield a clear
solution. The solution was made basic by addition of concentrated
ammonia water and extracted with ethyl acetate (200 mL ꢁ 3).
magnitude as hydrogen bonds or salt bridges and play an impor-
tant role in stabilizing the three-dimensional structure of a pro-
tein.21,22 In the binding model, compound 5t is nicely bound to
the region with the carbonyl group project toward the amino
group of Lys 828, with the carbonyl group forming a more optimal
H-bond (N–Hꢀ ꢀ ꢀO: 2.179 Å, 155.6°) interaction. Furthermore, there
is one
p
-cation interaction between the naphthalene ring of 5t and
-cation interaction between the
Lys 828, and there is the other
p
benzene ring of 4-(bis(2-chloroethyl)amino)benzoic acid moiety
of 5t and Lys 822. The enzyme assay data and the molecular dock-
ing results indicated that compounds 5b and 5t were potential
inhibitors of EGFR.

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Q.-Z. Zheng et al. / Bioorg. Med. Chem. 18 (2010) 880–886
Pooled organic extracts were dried over anhydrous MgSO4, filtered
and excess solvent removed under reduced pressure to yield the
crude product. Flash chromatography (ethyl acetate/petroleum
ether = 1:2) gave the desired compound 3 (yield: 84.5%).
1H); 8.47–8.53 (m, 1H). MS (ESI): 372.7 (C17H17Cl3N2O, [M+H]+).
Anal. Calcd for C17H17Cl3N2O: C, 54.93; H, 4.61; N, 7.54. Found: C,
54.81; H, 4.75; N, 7.68.
Compound 4: Compound 3 (6.9 g, 27 mmol) prepared above was
added to benzene (80 mL) containing thionyl chloride (10 mL,
137 mmol). The solution was heated at reflux for 3 h and after
cooling to room temperature, the solvent was removed under re-
duced pressure to yield yellow oil. To the resulting oil, concen-
trated HCl (55 mL) was added and the solution heated at reflux
for 3 h. After the reaction mixture cooled overnight, the precipitate
was filtered, washed with cold 50% aqueous ethanol to obtain com-
pound 4 (yield: 45.0%).
4.2.6. 4-(Bis(2-chloroethyl)amino)-N-(2-bromophenyl)benzamide
(5f)
White powder. Yield: 58%; mp: 104–106 °C. 1H NMR (300 MHz,
CDCl3): 3.68 (t, J = 6.6 Hz, 4H); 3.82 (t, J = 6.6 Hz, 4H); 6.74 (d,
J = 9.0 Hz, 2H); 7.07–7.20 (m, 3H); 7.85 (d, J = 8.9 Hz, 2H); 7.91 (s,
1H); 8.46–8.53 (m, 1H). MS (ESI): 417.1 (C17H17BrCl2N2O,
[M+H]+). Anal. Calcd for C17H17BrCl2N2O: C, 49.07; H, 4.12; N,
6.73. Found: C, 49.26; H, 4.32; N, 6.54.
To a stirred solution of 4 (0.26 g, 1 mmol) in anhydrous dichlo-
romethane (12 mL) was added N,N0-dicyclohexylcarbodiimide
(DCC, 0.21 g, 1 mmol), corresponding arylamines or fatty amines
or 3-morpholinopropan-1-amine and 4-dimethylaminopyridine
(4-DMAP, 0.12 g, 0.2 mmol). The reaction mixture was left stirring
at room temperature (for target compounds 5a–c, 5k, 5l and 5n–q)
or at reflux (for target compounds 5d–j, 5m, and 5r–t) for 24 h.
The resulting mixture was filtered, washed with dichloromethane,
dried over anhydrous Na2SO4 and concentrated. The residue was
purified with a silica gel column and was eluted with ethyl ace-
tate/petroleum ether = 1:3–1:8 to afford target compounds 5a–t.
4.2.7. 4-(Bis(2-chloroethyl)amino)-N-(4-fluorophenyl)benzamide
(5g)
White powder. Yield: 63%; mp: 125–127 °C. 1H NMR (300 MHz,
CDCl3): 3.66 (t, J = 6.6 Hz, 4H); 3.82 (t, J = 6.6 Hz, 4H); 6.65 (d,
J = 8.9 Hz, 2H); 6.73 (d, J = 8.9 Hz, 1H); 7.06 (t, J = 8.7 Hz, 1H);
7.51–7.58 (m, 3H); 7.67 (s, 1H); 7.80 (d, J = 9.0 Hz, 1H). MS (ESI):
356.2 (C17H17Cl2FN2O, [M+H]+). Anal. Calcd for C17H17Cl2FN2O: C,
57.48; H, 4.82; N, 7.89. Found: C, 57.69; H, 5.01; N, 7.68.
4.2.8. 4-(Bis(2-chloroethyl)amino)-N-(4-chlorophenyl)benzamide
(5h)
White powder. Yield: 54%; mp: 116–118 °C. 1H NMR (300 MHz,
CDCl3): 3.66 (t, J = 6.8 Hz, 4H); 3.80 (t, J = 6.8 Hz, 4H); 6.68 (d,
J = 8.9 Hz, 2H); 6.73 (d, J = 8.9 Hz, 1H); 7.02 (t, J = 8.9 Hz, 1H);
7.49–7.56 (m, 3H); 7.64 (s, 1H); 7.73 (d, J = 8.9 Hz, 1H). MS (ESI):
372.7 (C17H17Cl3N2O, [M+H]+). Anal. Calcd for C17H17Cl3N2O: C,
54.93; H, 4.61; N, 7.54. Found: C, 54.72; H, 4.73; N, 7.41.
4.2.1. 4-(Bis(2-chloroethyl)amino)-N-p-tolylbenzamide (5a)
White powder. Yield: 86%; mp: 130–132 °C; 1H NMR (300 MHz,
CDCl3): 2.32 (s, 3H); 3.66 (t, J = 7.0 Hz, 4H); 3.80 (t, J = 7.1 Hz, 4H);
6.70 (d, J = 8.9 Hz, 2H); 7.15 (d, J = 8.9 Hz, 2H); 7.50 (d, J = 8.4 Hz,
2H); 7.71 (s, 1H); 7.79 (d, J = 8.9 Hz, 2H). MS (ESI): 352.3
(C18H20Cl2N2O, [M+H]+). Anal. Calcd for C18H20Cl2N2O: C, 61.55;
H, 5.74; N, 7.97. Found: C, 61.70; H, 5.60; N, 7.81.
4.2.9. 4-(Bis(2-chloroethyl)amino)-N-(4-bromophenyl)benzamide
(5i)
4.2.2. 4-(Bis(2-chloroethyl)amino)-N-(4-methoxyphenyl)
benzamide (5b)
White powder. Yield: 51%; mp: 120–122 °C. 1H NMR (300 MHz,
CDCl3): 3.65 (t, J = 6.6 Hz, 4H); 3.80 (t, J = 6.6 Hz, 4H); 6.69 (d,
J = 8.9 Hz, 2H); 6.77 (d, J = 8.9 Hz, 1H); 7.10 (t, J = 8.8 Hz, 1H);
7.55–7.62 (m, 3H); 7.66 (s, 1H); 7.83 (d, J = 9.0 Hz, 1H). MS (ESI):
417.1 (C17H17BrCl2N2O, [M+H]+). Anal. Calcd for C17H17BrCl2N2O:
C, 49.07; H, 4.12; N, 6.73. Found: C, 49.25; H, 4.31; N, 6.56.
White powder. Yield: 88%; mp: 140–141 °C. 1H NMR (300 MHz,
CDCl3): 3.67 (t, J = 6.4 Hz, 4H); 3.79–3.83 (m, 7H); 6.72 (d,
J = 7.2 Hz, 2H); 6.90 (d, J = 7.2 Hz, 2H); 7.52 (d, J = 9.0 Hz, 2H);
7.59 (s, 1H); 7.79 (d, J = 9.0 Hz, 2H). MS (ESI): 368.3
(C18H20Cl2N2O2, [M+H]+). Anal. Calcd for C18H20Cl2N2O2: C, 58.86;
H, 5.49; N, 7.63. Found: C, 58.67; H, 5.68; N, 7.81.
4.2.10. 4-(Bis(2-chloroethyl)amino)-N-(4-nitrophenyl)benzamide
(5j)
4.2.3. 4-(Bis(2-chloroethyl)amino)-N-(4-isopropylphenyl)
benzamide (5c)
White powder. Yield: 49%; mp: 127–129 °C. 1H NMR (300 MHz,
CDCl3): 3.66 (t, J = 6.6 Hz, 4H); 3.82 (t, J = 6.6 Hz, 4H); 6.73 (d, J
=9.0 Hz, 2H); 7.22 (d, J = 9.0 Hz, 2H); 7.68 (s, 1H); 7.72 (d,
J = 9.0 Hz, 2H); 8.08 (d, J = 9.0 Hz, 2H). MS (ESI): 383.2
(C17H17Cl2N3O3, [M+H]+). Anal. Calcd for C17H17Cl2N3O3: C, 53.42;
H, 4.48; N, 10.99. Found: C, 53.25; H, 4.27; N, 11.19.
White powder. Yield: 84%; mp: 130–131 °C. 1H NMR (300 MHz,
CDCl3): 1.22 (d, J = 6.9 Hz, 6H); 2.84–2.89 (m, 1H); 3.67 (t,
J = 6.6 Hz, 4H); 3.81 (t, J = 6.6 Hz, 4H); 6.73 (d, J = 9.0 Hz, 2H);
7.22 (d, J = 9.0 Hz, 2H); 7.53 (d, J = 9.0 Hz, 2H); 7.63 (s, 1H); 7.80
(d, J = 9.1 Hz, 2H). MS (ESI): 380.3 (C20H24Cl2N2O, [M+H]+). Anal.
Calcd for C20H24Cl2N2O: C, 63.33; H, 6.38; N, 7.39. Found: C,
63.42; H, 6.49; N, 7.21.
4.2.11. 4-(Bis(2-chloroethyl)amino)-N,N-dipropylbenzamide (5k)
White powder. Yield: 78%; mp: 88–90 °C. 1H NMR (300 MHz,
CDCl3): 0.92 (t, J = 7.3 Hz, 6H); 1.36–1.46 (m, 4H); 3.56 (t,
J = 6.6 Hz, 4H); 3.68 (t, J = 6.6 Hz, 4H); 3.78 (t, J = 6.6 Hz, 4H);
6.65 (d, J = 8.9 Hz, 2H); 7.68 (d, J = 8.9 Hz, 2H). MS (ESI): 346.3
(C17H26Cl2N2O, [M+H]+). Anal. Calcd for C17H26Cl2N2O: C, 59.13;
H, 7.59; N, 8.11. Found: C, 59.02; H, 7.40; N, 8.32.
4.2.4. 4-(Bis(2-chloroethyl)amino)-N-(2-fluorophenyl)benzamide
(5d)
White powder. Yield: 65%; mp: 80–82 °C. 1H NMR (300 MHz,
CDCl3): 3.68 (t, J = 6.8 Hz, 4H); 3.83 (t, J = 6.8 Hz, 4H); 6.75 (d,
J = 9.0 Hz, 2H); 7.04–7.19 (m, 3H); 7.83 (d, J = 8.9 Hz, 2H); 7.96 (s,
1H); 8.45–8.51 (m, 1H). MS (ESI): 356.2 (C17H17Cl2FN2O, [M+H]+).
Anal. Calcd for C17H17Cl2FN2O: C, 57.48; H, 4.82; N, 7.89. Found:
C, 57.63; H, 4.70; N, 7.71.
4.2.12. 4-(Bis(2-chloroethyl)amino)-N,N-dibutylbenzamide (5l)
White powder. Yield: 76%; mp: 91–93 °C. 1H NMR (300 MHz,
CDCl3): 0.95 (t, J = 7.3 Hz, 6H); 1.33–1.49 (m, 8H); 3.56 (t,
J = 6.6 Hz, 4H); 3.68 (t, J = 6.6 Hz, 4H); 3.78 (t, J = 6.6 Hz, 4H);
6.66 (d, J = 9.0 Hz, 2H); 7.67 (d, J = 9.0 Hz, 2H). MS (ESI): 374.4
(C19H30Cl2N2O, [M+H]+). Anal. Calcd for C19H30Cl2N2O: C, 61.12;
H, 8.10; N, 7.50. Found: C, 61.31; H, 7.97; N, 7.32.
4.2.5. 4-(Bis(2-chloroethyl)amino)-N-(2-chlorophenyl)benzamide
(5e)
White powder. Yield: 60%; mp: 111–113 °C. 1H NMR (300 MHz,
CDCl3): 3.66 (t, J = 6.8 Hz, 4H); 3.81 (t, J = 6.8 Hz, 4H); 6.73 (d,
J = 9.0 Hz, 2H); 7.06–7.17 (m, 3H); 7.84 (d, J = 8.9 Hz, 2H); 7.88 (s,

Q.-Z. Zheng et al. / Bioorg. Med. Chem. 18 (2010) 880–886
885
4.2.13. 4-(Bis(2-chloroethyl)amino)-N-methyl-N-
phenylbenzamide (5m)
1H); 7.90–7.94 (d, J = 8.0 Hz, 2H); 8.04 (d, J = 8.0 Hz, 2H). MS
(ESI): 388.3 (C21H20Cl2N2O, [M+H]+). Anal. Calcd for C21H20Cl2N2O:
C, 65.12; H, 5.20; N, 7.23. Found: C, 65.29; H, 5.35; N, 7.12.
White powder. Yield: 72%; mp: 107–109 °C. 1H NMR (300 MHz,
CDCl3): 3.16 (s, 3H); 3.66 (t, J = 7.0 Hz, 4H); 3.82 (t, J = 7.0 Hz, 4H);
6.73 (d, J = 8.8 Hz, 2H); 7.12 (t, J = 7.7 Hz, 1H); 7.36 (t, J = 8.3 Hz,
2H); 7.62 (d, J = 8.3 Hz, 2H); 7.80 (d, J = 8.8 Hz, 2H). MS (ESI):
352.3 (C18H20Cl2N2O, [M+H]+). Anal. Calcd for C18H20Cl2N2O: C,
61.55; H, 5.74; N, 7.97. Found: C, 61.38; H, 5.90; N, 7.83.
4.3. Preparation, purification of HER-2 and EGFR and inhibitory
assay
A 1.7 Kb cDNA encoded for human HER-2 cytoplasmic domain
(HER-2-CD, amino acids 676–1245) and 1.6 kb cDNA encoded for
the EGFR cytoplasmic domain (EGFR-CD, amino acids 645–1186)
were cloned into baculoviral expression vectors pBlueBacHis2B
and pFASTBacHTc (Huakang Company China), separately. A se-
quence that encodes (His)6 was located at the 50 upstream to the
HER-2 and EGFR sequences. 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 HEPES, 10 mM NaCl, 1% Triton,
4.2.14. 4-(Bis(2-chloroethyl)amino)-N-butylbenzamide (5n)
White powder. Yield: 85%; mp: 52–54 °C. 1H NMR (300 MHz,
CDCl3): 0.95 (t, J = 7.3 Hz, 3H); 1.36–1.46 (m, 2H); 1.54–1.63 (m,
2H); 3.39–3.46 (m, 2H); 3.67 (t, J = 6.6 Hz, 4H); 3.77 (t, J = 6.6 Hz,
4H); 6.07 (s, 1H); 6.66 (d, J = 8.8 Hz, 2H); 7.69 (d, J = 8.8 Hz, 2H).
MS (ESI): 318.3 (C15H22Cl2N2O, [M+H]+). Anal. Calcd for
C15H22Cl2N2O: C, 56.79; H, 6.99; N, 8.83. Found: C, 56.68; H,
6.90; N, 8.94.
10
l
M ammonium molybdate, 100
g/mL leupeptin, 10
l
M sodium vanadate, 10
l
l
g/mL
g/mL
aprotinin, 10
l
lg/mL pepstatin, and 16
4.2.15. 4-(Bis(2-chloroethyl)amino)-N-tert-butylbenzamide
(5o)
benzamidine HCl for 20 min followed 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.
Histidinetagged proteins were eluted with 250 and 500 mM imid-
azole and dialyzed against 50 mM NaCl, 20 mM HEPES, 10% glyc-
White powder. Yield: 80%; mp: 141–143 °C. 1H NMR (300 MHz,
CDCl3): 1.28 (s, 9 H); 3.63 (t, J = 6.6 Hz, 4H); 3.76 (t, J = 6.6 Hz, 4H);
5.83 (s, 1H); 6.68 (d, J = 8.9 Hz, 2H); 7.70 (d, J = 8.9 Hz, 2H); MS
(ESI): 318.3 (C15H22Cl2N2O, [M+H]+). Anal. Calcd for C15H22Cl2N2O:
C, 56.79; H, 6.99; N, 8.83. Found: C, 56.88; H, 6.72; N, 8.70.
erol, and 1 lg/mL each of aprotinin, leupeptin, and pepstatin for
2 h. The entire purification procedure was performed at 4 °C or
4.2.16. 4-(Bis(2-chloroethyl)amino)-N-dodecylbenzamide (5p)
White powder. Yield: 75%; mp: 60–62 °C. 1H NMR (300 MHz,
CDCl3): 0.90 (t, J = 7.2 Hz, 3H); 1.19–1.31 (m, 18 H); 1.41–1.48
(m, 2 H); 3.12–3.16 (m, 2 H); 3.67 (t, J = 6.6 Hz, 4H); 3.81 (t,
J = 6.7 Hz, 4H); 5.96 (s, 1H); 6.70 (d, J = 8.9 Hz, 2H); 7.71 (d,
J = 8.8 Hz, 2H). MS (ESI): 430.5 (C23H38Cl2N2O, [M+H]+). Anal. Calcd
for C23H38Cl2N2O: C, 64.32; H, 8.92; N, 6.52. Found: C, 64.51; H,
8.73; N, 6.34.
on ice.23
Both EGFR and HER-2 kinase assays were set up to assess the le-
vel of autophosphorylation based on DELFIA/Time-Resolved Fluo-
rometry. Target compounds 5a–t were dissolved in 100% DMSO
and diluted to the appropriate concentrations with 25 mM HEPES
at pH 7.4. In each well, 10
10 L (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
L of 5 ꢁ buffer (containing 20 mM HEPES, 2 mM MnCl2,
100 M Na3VO4, and 1 mM DTT) and 20 L of 0.1 mM ATP-
lL of compound was incubated with
l
l
4.2.17. 4-(Bis(2-chloroethyl)amino)-N-cyclopentylbenzamide
(5q)
l
l
50 mM MgCl2 was added for 1 h. Positive and negative controls
were included in each plate by incubation of enzyme with or with-
out ATP-MgCl2. At the end of incubation, liquid was aspirated, and
White powder. Yield: 81%; mp: 84–86 °C. 1H NMR (300 MHz,
CDCl3): 1.67–1.69 (m, 4H); 1.70–1.73 (m, 4H); 3.64 (t, J = 6.8 Hz,
4H); 3.78 (t, J = 6.8 Hz, 4H); 4.33–4.45 (m, 1H); 5.90 (s, 1H); 6.67
(d, J = 8.9 Hz, 2H); 7.68 (d, J = 8.9 Hz, 2H). MS (ESI): 330.3
(C16H22Cl2N2O, [M+H]+). Anal. Calcd for C16H22Cl2N2O: C, 58.36;
H, 6.73; N, 8.51. Found: C, 58.19; H, 6.58; N, 8.64.
plates were washed three times with wash buffer. A 75 lL (400 ng)
sample of europiumlabeled anti-phosphotyrosine antibody was
added to each well for another 1 h of incubation. After washing,
enhancement 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
compounds was calculated using the following equation:
100% ꢂ [(negative control)/(positive control ꢂ negative control)].
The IC50 was obtained from curves of percentage inhibition with
eight concentrations of compound. As the contaminants in the en-
zyme preparation are fairly low, the majority of the signal detected
by the anti-phosphotyrosine antibody is from EGFR or HER-2.
4.2.18. 4-(Bis(2-chloroethyl)amino)-N-(3-morpholinopropyl)
benzamide (5r)
White powder. Yield: 61%; mp: 93–95 °C. 1H NMR (300 MHz,
CDCl3): 1.68–1.71 (m, 2H); 2.33–2.36 (t, J = 6.6 Hz, 4H); 2.44–
2.46 (m, 2H); 3.36–3.39 (m, 2H); 3.62–3.69 (m, 12H); 6.26 (s,
1H); 6.67 (d, J = 8.8 Hz, 2H); 7.37 (d, J = 8.8 Hz, 2H). MS (ESI):
389.3 (C18H27Cl2N3O2, [M+H]+). Anal. Calcd for C18H27Cl2N3O2: C,
55.67; H, 7.01; N, 10.82. Found: C, 55.79; H, 7.21; N, 10.63.
4.4. Cell proliferation assay
4.2.19. N-Benzyl-4-(bis(2-chloroethyl)amino)benzamide (5s)
White powder. Yield: 81%; mp: 105–107 °C. 1H NMR (300 MHz,
CDCl3): 3.64 (t, J = 6.6 Hz, 4H); 3.78 (t, J = 6.6 Hz, 4H); 4.64 (d,
J = 5.7 Hz, 2H); 6.26 (s, 1H); 6.67 (d, J = 9.0 Hz, 2H); 7.30–7.36(m,
5H); 7.72 (d, J = 9.0 Hz, 2H). MS (ESI): 352.3 (C18H20Cl2N2O,
[M+H]+). Anal. Calcd for C18H20Cl2N2O: C, 61.55; H, 5.74; N, 7.97.
Found: C, 61.36; H, 5.91; N, 7.80.
The antiproliferative activity was determined using a standard
(MTT)-based colorimetric assay (Sigma). Briefly, cell lines were
seeded at a density of 7 ꢁ 103 cells/well in 96-well microtiter
plates (Costar). After 24 h, exponentially growing cells were ex-
posed to the indicated compounds at final concentrations ranging
from 0.1 to 100
by the addition of an MTT solution (10
PBS). After 4 h, 100 L of 10% SDS in 0.01 N HCl was added, and
the plates were incubated at 37 °C for a further 18 h; optical absor-
bance was measured at 570 nm on an LX300 Epson Diagnostic
microplate reader. Survival ratios are expressed in percentages
l
g/mL. After 48 h, cell survival was determined
lL of 5 mg/mL MTT in
4.2.20. 4-(Bis(2-chloroethyl)amino)-N-(1-naphthyl)benzamide
(5t)
l
White powder. Yield: 72%; mp: 107–109 °C. 1H NMR (300 MHz,
CDCl3): 3.66 (t, J = 6.6 Hz, 4H); 3.83 (t, J = 6.6 Hz, 4H); 6.74 (d,
J = 8.8 Hz, 2H); 7.35 (d, J = 8.8 Hz, 2H); 7.56–7.61 (m, 3H); 7.86 (s,

886
Q.-Z. Zheng et al. / Bioorg. Med. Chem. 18 (2010) 880–886
5. Scheurle, D.; Jahanzeb, M.; Aronsohn, R. S.; Watzek, L.; Narayanan, R. H.
Anticancer Res. 2000, 20, 2091.
6. Cox, G.; Vyberg, M.; Melgaard, B.; Askaa, J.; Oster, A.; O’Byrne, K. J. Int. J. Cancer
2001, 92, 480.
with respect to untreated cells. IC50 values were determined from
replicates of 6 wells from at least three independent experiments.
7. Gullick, W. J. Br. Med. Bull. 1991, 47, 87.
4.5. Molecular docking modeling
8. Moscatello, D. K.; Holgado-Mudruga, M.; Godwin, A. K.; Ramirez, G.; Gunn, G.;
Zoltick, P. W.; Biegel, J. A.; Hayes, R. L.; Wong, A. J. Cancer Res. 1995, 55, 5536.
9. Wikstrand, C. J.; McLendon, R. E.; Friedman, A.; Bigner, D. D. Cancer Res. 1997,
57, 4130.
10. Bridges, A. J. Curr. Med. Chem. 1999, 6, 825.
11. Boschelli, D. H. Drugs Future 1999, 24, 515.
Molecular docking of compounds 5b and 5t into the three-
dimensional EGFR complex structure (1M17.pdb, downloaded
from the PDB) was carried out using the AutoDock software pack-
age (version 4.0) as implemented through the graphical user inter-
face AutoDockTools (ADT 1.4.6).
12. Denny, W. A. Curr. Med. Chem. 2001, 8, 533.
13. Alexanian, R.; Dimopoulos, M. A. N. Eng. J. Med. 1994, 330, 484.
14. Hansson, J.; Lewensohn, R.; Ringborg, U.; Nilsson, B. Cancer Res. 1987, 47, 2631.
15. Rodney, M.; Carney, J. P.; Kelley, M. R.; Glassner, B. J.; Williams, D. A.; Samson,
L. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 206.
Acknowledgement
16. Brendal, M.; Ruhland, A. Mutat. Res. 1984, 133, 51.
17. Zakaria, R.; Fouad, B.; Qiyu, Q.; Christopher, W.; Janet, M. H.; John, A. H.;
Bertrand, J. J. C. J. Med. Chem. 2007, 50, 2605.
18. Springer, C. J.; Antoniw, P.; Bagshawe, K. D.; Searle, F.; Bisset, G. M.; Jarman, M.
J. Med. Chem. 1990, 33, 677.
19. Niculescu-Duvaz, I.; Scanlon, I.; Niculescu-Duvaz, D.; Friedlos, F.; Martin, J.;
Marais, R.; Springer, C. J. J. Med. Chem. 2004, 47, 2651.
This work was supported by National Basic Research Programs
(973) of China (No. 2008CB418004 & No. 2008CB418201) and the
Major Projects on Control and Rectification of Water Body Pollu-
tion (Water Special Project) (No. 2008ZX07526-001).
20. Taylor, C. K.; Abel, P. W.; Hulce, M.; Smith, D. D. Chem. Biol. Drug Des. 2007, 70,
216.
References and notes
21. Jeffrey, D. S.; Christopher, G. V. S.; Caldwell, W. S. J. Med. Chem. 1999, 42, 3066.
22. Pless, S. A.; Millen, K. S.; Hanek, A. P.; Lynch, J. W.; Lester, H. A.; Lummis, S. C. R.;
Dougherty, D. A. J. Neurosci. 2008, 28, 10937.
23. 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.
1. Yarden, Y.; Sliwkowski, M. X. Mol. Cell Biol. 2001, 2, 127.
2. Hynes, N. E.; Stern, D. F. Biochim. Biophys. Acta 1994, 1198, 165.
3. Slamon, D. J.; Clark, G. M.; Wong, S. G.; Levin, W. J.; Ullrich, A.; McGuire, W. L.
Science 1987, 235, 177.
4. Slamon, D. J.; Godolphin, W.; Jones, L. A. Science (Wash. DC) 1989, 244, 707.