Synthesis and biological evaluation of N-aryl salicylamides with a hydroxamic acid moiety at 5-position as novel HDAC-EGFR dual inhibitors

Article abstract of DOI:10.1016/j.bmc.2012.05.034

A novel series of N-aryl salicylamides with a hydroxamic acid moiety at 5-position were synthesized efficiently. Their activities against EGFR kinase and HDACs were evaluated. All compounds displayed inhibitory activity against EGFR and HDACs. The antiproliferative activities of synthesized compounds were evaluated by MTT method against human cancer cell lines A431, A549 and HL-60. Compound 1o showed the most potent inhibitory activity against A431 and A549. Compounds 1k and 1n exhibited higher potency against HL-60 than gefitinib and SAHA. N-Aryl salicylamides with a hydroxamic acid moiety at 5-position is another new HDAC-EGFR dual inhibitors.

Full text of DOI:10.1016/j.bmc.2012.05.034

Bioorganic & Medicinal Chemistry 20 (2012) 4405–4412
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological evaluation of N-aryl salicylamides with a hydroxamic
acid moiety at 5-position as novel HDAC–EGFR dual inhibitors
Miao Zuo a, Yue-Wen Zheng b, She-Min Lu b, Yan Li a, San-Qi Zhang a,
⇑
a Department of Pharmacy, School of Medicine, Xi’an Jiaotong University, Xi’an 710061, PR China
b Department of Genetics and Molecular Biology, School of Medicine, Xi’an Jiaotong University, Xi’an 710061, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel series of N-aryl salicylamides with a hydroxamic acid moiety at 5-position were synthesized effi-
ciently. Their activities against EGFR kinase and HDACs were evaluated. All compounds displayed inhib-
itory activity against EGFR and HDACs. The antiproliferative activities of synthesized compounds were
evaluated by MTT method against human cancer cell lines A431, A549 and HL-60. Compound 1o showed
the most potent inhibitory activity against A431 and A549. Compounds 1k and 1n exhibited higher
potency against HL-60 than gefitinib and SAHA. N-Aryl salicylamides with a hydroxamic acid moiety
at 5-position is another new HDAC–EGFR dual inhibitors.
Received 22 March 2012
Revised 14 May 2012
Accepted 15 May 2012
Available online 29 May 2012
Keywords:
N-Aryl salicylamides
HDAC inhibitor
EGFR inhibitor
Antiproliferative activity
Synthesis
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
catalyzed by histone deacetylases (HDACs), thus promoting a more
closed chromatin structure where transcription is repressed.10
Cancer is a disease characterized by uncontrolled cell growth
and proliferation, which usually results from activation of some en-
zymes. Receptor tyrosine kinases (RTK) have been observed having
over-expression and/or constitutive activation in numerous types
of tumor, including colon, breast, ovarian, head and neck, and
non-small cell lung cancers. RTKs play key roles in the processes
of governing cellular proliferation, differentiation and evasion from
apoptosis as well. Among the known RTKs, the ErbB family, in
particular epidermal growth factor receptor (EGFR) and human
epidermal growth factor receptor 2 (HER2), have been extensively
studied and clinically validated as targets for cancer therapies.1–4
The ErbB family consists of four receptors, including epidermal
growth factor receptor (EGFR/ErbB-1/HER1), HER2 (ErbB-2/neu),
HER-3 and HER-4. Current therapies using either monoclonal anti-
bodies (cetuximab and trastuzumab) or small organic molecules
(gefitinib, erlotinib) to selectively target EGFR or HER2, or recently,
small molecules that target both receptors (lapatinib), have been
found successful clinically.5,6 New compounds for inhibiting both
EGFR and HER2 have been constantly discovered.7
HDAC inhibitors have been applied to treatment of human
cancers.11 Zolinza (vorinostat, SAHA) was approved by the FDA
for the treatment of advanced cutaneous T-cell lymphoma (CTCL).12
Several small molecule HDAC inhibitors have entered clinical trials
for the treatment of a variety haematological and solid tumors.13,14
New scaffolds of small molecular inhibitors against HDAC have
been constantly reported.15–17
However, the efficacy of small organic molecules inhibitors such
as gefitinib, etc. is restricted to a small subset of patients due to
molecular heterogeneity among and within tumors.18,19 Their effec-
tiveness is also limited by the drug resistance that frequently
emerges following treatment.20 HDAC inhibitors have been shown
to synergize with other agents, including RTK inhibitors, to sup-
press proliferation and induce apoptosis in tumor cells.21–24 To
overcome the low response rate and acquired resistance to RTK
inhibitors, a number of strategies have been tested, including com-
bination therapies and multitargeted inhibitors to inhibit multiple
pathogenic pathways.25 One particularly promising approach is
incorporating the pharmacophore of HDAC inhibitor (hydroxamic
acid) and the pharmacophore of EGFR inhibitor (4-arylaminoqui-
nazoline) into one molecule to obtain new chemical entity. A novel
series of compounds with potent, multiacting HDAC, EGFR, and
HER2 inhibition were synthesized and evaluated.26 Meanwhile,
CUDC-101 was identified as a drug candidate, which is now in
clinical development. Almost simultaneously compound A was syn-
thesized and evaluated by combining the pharmacological activity
The reversible acetylation of lysine residues in histone tails plays
a critical role in transcriptional activation and repression.8,9 Histone
acetyltransferases (HATs) catalyze the acetylation of lysine residues
on histone and non-histone proteins. The reverse reaction is
⇑
Corresponding author. Tel./fax: +86 29 82657040.
E-mail address: sqzhang@xjtu.edu.cn (S.-Q. Zhang).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.05.034

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M. Zuo et al. / Bioorg. Med. Chem. 20 (2012) 4405–4412
Figure 1. Incorporation of 4-arylaminoquinazoline and SAHA.
freshly prepared hydroxylamine in methanol to produce target
compounds 1a–1j.
To expand the structural diversity of the salicylic acid moiety,
we synthesized 4-methoxy substituted N-aryl salicylamides 1k
and 1l from 4-methoxysalicylic acid (Scheme 2) .
The commercial available 4-methoxysalicylic acid was nitrated
with concentrated nitric acid in acetic acid to give 5. Compounds
1k and 1l were prepared according to the same procedure de-
scribed in Scheme 1.
To compare the different substituted group at 2-position of ben-
zoic acid under the influence of activity of final compound, com-
pound 1m was synthesized from 3-nitrobenzoic acid, compound
1n was synthesized from 3f, respectively, (Scheme 3).
In the structure of compounds 1a–1n, hydroxamic acid moiety
is linked with N-aryl salicylamides through a amide structure.
Preparation of ether linker compound 1o was shown in Scheme
4. Commercially available 2,5-dihydroxybenzoic acid was con-
verted into amide 6 according to the similar method described in
Scheme 1. The refluxing of 6 with ethyl 7-bromoheptanoate in
the presence of anhydrous K2CO3 and tetrabutylammonium iodide
in acetone afforded intermediate 7. There are two hydroxy groups
in compound 6. Tetrabutylammonium iodide was added to reac-
tion mixture to improve the selectivity and the yield of 7.31 Final
compound 1o was obtained by the treatment of 7 with freshly pre-
pared hydroxylamine.
Figure 2. Incorporate strategy of N-aryl salicylamide and SAHA.
of EGFR/HER2 kinase inhibition with inhibition of HDAC enzymes
(Fig. 1).27 Thus, chimeric HDAC-kinase inhibitors was able to consti-
tute a new class of experimental cancer drugs worth to be studied
in more detail.
The salicylanilide molecule may construct an intramolecular
hydrogen bond and form a pseudo six-membered ring. Thus, N-aryl
salicylamide is supposed to have the same pharmacophore as 4-
arylaminoquinazoline, and has been studied as EGFR inhibitors.28,29
In this study, we selected the scaffold of N-aryl salicylamide to
incorporate the pharmacophore of HDAC inhibitor (hydroxamic
acid) to present a novel class of HDAC–EGFR dual inhibitors. Our
strategy was shown in Figure 2. Here N-aryl salicylamides with a
hydroxamic acid moiety at 5-position were synthesized and their
biological activities against EGFR and HDACs and their antiprolifer-
ative activities against human carcinoma cell lines A431, A549 and
HL-60 were evaluated.
All the synthesized compounds 1a–1o are reported for the first
time. Their structures were characterized by 1H NMR and HRMS.
2.2. EGFR and HDAC inhibitory activity
To study biological activity, compounds 1a–1o were evaluated
against EGFR kinase activity assays with gefitinib as positive con-
trol and HDACs enzyme assay with SAHA as positive control. EGFR
was prepared from human A431 carcinoma cell. HDACs were pre-
pared from HeLa cell extracts. Compounds 1a–1o were initially
2. Results and discussion
2.1. Chemistry
The target compounds 1a–1j were synthesized from 5-nitrosal-
icylic acid. The synthetic route was outlined in Scheme 1. The mix-
ture of 5-nitrosalicylic acid, aryl amine, triphenyl phosphite and
toluene was refluxed for hours to yield salicylamides 3 with high
yield. Hydroxy group was not necessary to protect in this reac-
tion.30 Reduction of nitro group in compound 3 with iron powder
in the acidic condition produced corresponding amines with
quantitive yield. The reaction of the amine with acyl chloride, such
as 7-ethyoxyl-7-oxoheptanoyl chloride or 8-ethyoxyl-8-oxoocta-
noyl chloride, afforded compound 4 in the presence of TEA. The
yield range from 40% to 60% in the step because the hydroxy group
at 2-position may react with acyl chloride in the presence of TEA.
Finally, the ethyl ester group in compounds 4 was treated with
screened at final concentrations of 10.0, 1.0 and 0.1 lM by using
ELISA-based EGFR-TK assay and an enzymatic assay measuring to-
tal HDACs activity. IC50s of compounds 1a–1o were calculated
according to inhibitory rates, the in vitro enzymatic inhibition as-
say results are summarized in Table 1.
The length of the hydroxamic acid side chain is important for
EGFR and HDAC inhibition. The optimal carbon chain length is
six (n = 5 or 6) in the structure of 4-arylaminoquinazolines.26 For
this reason, short chain analogues were not synthesized in our
work. The data in Table 1 demonstrated that compounds 1a–1o
exhibited the inhibitory activity against EGFR and HDACs. The
activities of compounds 1b, 1h, 1i, 1k, 1l and 1o against EGFR were

M. Zuo et al. / Bioorg. Med. Chem. 20 (2012) 4405–4412
4407
Scheme 1. Reagents and conditions: (a) ArNH2, P(OPh)3, reflux; (b) Fe, HOAc; (c) RCOCl, TEA; (d) NH2OH, MeOH.
Scheme 2. Reagents and conditions: (a) HNO3, HOAc; (b) ArNH2, P(OPh)3, reflux; (c) Fe, HOAc; (d) EtO2C(CH2)5COCl, TEA; (e) NH2OH, MeOH.
Scheme 3. Reagents and conditions: (a) 3-EthynylPhNH2, P(OPh)3, reflux; (b) Fe, HOAc; (c) EtO2C(CH2)5COCl, TEA; (d) NH2OH, MeOH.
Scheme 4. Reagents and conditions: (a) 3-EthynylPhNH2, P(OPh)3, reflux; (b) ethyl 7-bromoheptylate, K2CO3; (c) NH2OH, MeOH.
close to that of gefitinib. The activities of compounds 1m and 1n
against EGFR were found to be less potent than gefitinib and other
synthesized compounds. Since there is no substituted group at 2-
position of benzoic acid ring of 1m and there is a methoxy group
at 2-position of 1n, a pseudo six-membered ring can not construct
in 1m and 1n. Consequently the activity of 1m and 1n declined in
EGFR inhibition. Compounds 1k and 1l relatively exhibited higher
inhibitory activities than that of 1d and 1f against EGFR. These re-
sults suggest that a methoxy group at 4-position of salicylic acid is
favorable for EGFR inhibition. When n is 5 (compound 1f) or 6
(compound 1j), EGFR inhibition was close to each other. For EGFR
inhibition, an ether linker (compound 1o IC50 = 0.90
EGFR) was more potent than an amide linker (compound 1f
l
M against
IC50 = 3.31 M against EGFR). The same result was observed in
l
HDAC inhibition.
As shown in Table 1, compounds 1a–1o displayed inhibitory
activity against HDACs. Compared with reference compounds, all
the synthesized compounds inhibit HDAC activity with reduced
potency except compound 1n. Compound 1n displayed the most
potent activity (IC50 = 0.56 lM against HDAC). Contrary to EGFR
inhibition, a methoxy group at 2-position of benzoic acid ring im-
proved HDACs inhibition. It seems that the effect of substituted

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M. Zuo et al. / Bioorg. Med. Chem. 20 (2012) 4405–4412
Table 1
tested compounds, including positive drugs, did not show a strong
inhibitory activities (IC50 > 10 M). When tested against HL-60,
most compounds were less potent. However, compounds 1k
(IC50 = 9.83 M against HL-60) and 1n (IC50 = 7.10 M for HL-60)
The structures and inhibitory activities of compounds 1a–1o, SAHA and Gifitinib
against EGFR and HDACs (n = 3, x Æ s)
l
ꢀ
Compounds
IC50 (lM)
l
l
displayed a higher potency than SAHA. This may be related to their
EGFR
HDACs
high inhibitory activities against HDACs.
1a
1b
1c
1d
1e
1f
1g
1h
1i
1j
1k
1l
1m
1n
1o
SAHA
Gifitinib
2.34 0.21
1.41 0.11
2.22 0.18
3.53 0.32
4.20 0.29
3.31 0.60
1.80 0.20
1.41 0.16
1.10 0.13
2.25 0.22
1.56 0.20
1.12 0.15
6.17 0.42
5.16 0.39
0.90 0.11
Nt
1.89 0.30
3.59 0.31
6.02 0.56
3.02 0.28
3.19 0.32
4.09 0.40
3.65 0.51
3.48 0.43
3.01 0.19
2.03 0.27
1.09 0.09
1.86 0.20
3.27 0.55
0.56 0.08
1.21 0.19
0.98 0.11
Nt
3. Conclusions
In summary, a novel series of N-aryl salicylamides with a
hydroxamic acid moiety at 5-position were synthesized efficiently
as novel HDAC–EGFR dual inhibitors. As expected, these compounds
exhibited distinct inhibitory activity against EGFR and HDACs and
potent antiproliferative activities in vitro, especially against A431.
Compound 1o showed the most potent inhibitory activity against
A431 and A549. Compounds 1k and 1n exhibited higher potency
against HL-60 than gefitinib and SAHA. By combining two distinct
pharmacophores into one molecule, HDAC–EGFR dual inhibitors
were postulated to represent a novel approach to cancer therapy.
1.08 0.12
Nt: not tested.
4. Experimental section
4.1. Chemistry
groups on N-phenyl ring (R in Scheme 1) do not obviously affect
EGFR and HDACs inhibitory activity.
Unless specified otherwise, all starting materials, reagents and
solvents were commercially available. All reactions were moni-
tored by thin-layer chromatography on silica gel plates (GF-254)
and visualized with UV light. All the melting points were deter-
mined on a Beijing micromelting-point apparatus and thermome-
ter was uncorrected. 1H NMR spectra were recorded in DMSO-d6
on a 300 MHz Bruker NMR spectrometer with tetramethylsilane
(TMS) as an internal reference. All chemical shifts are reported in
parts per million (ppm). High-resolution exact mass measure-
ments were performed using electrospray ionization (positive
mode) on a quadrupole time-of-flight (Q-TOF) mass spectrometer
(Maxis Q-TOF, Bruker Inc.).
2.3. Antiproliferative activity
To test the anticancer activities of the synthesized compounds
we evaluated antiproliferative activities of compounds1a–1o
against human epithelial carcinoma cell line (A431), lung adeno-
carcinoma epithelial cell line (A549) and promyelocytic leukemia
cell line (HL-60) by applying the MTT colorimetric assay. The re-
sults are summarized in Table 2. Compounds were tested over a
range of concentrations from 10À4 to 10À8 M, and the calculated
IC50 values, that is, the concentration (lM) of a compound that
was able to cause 50% cell death with respect to the control culture,
were reported differently according to different cancer cells.
As expected, compounds 1a–1o exhibited remarked effects
on antiproliferative activities in vitro, especially against A431.
Compound 1o showed the most potent inhibitory activity
4.1.1. N-Aryl-2-hydroxy-5-nitrobenzamide (3)
4.1.1.1. N-Phenyl-2-hydroxy-5-nitrobenzamide (3a). A mixture
containing 2-hydroxy-5-nitrobenzoic acid (1.83 g, 10 mmol), ani-
line (1.21 g, 13 mmol), triphenyl phosphite (3.6 mL) and toluene
(30 mL) was strirred under refluxing for 8 h, cooled to room temper-
ature. The resulted solid was collected by filtration under reduced
pressure, washed with cooled dichlormethane (5 mL Â 2), crystal-
lized from ethanol (20 mL) to produce 2.17 g of 3a as a yellow solid.
Yield 84%. Mp 219–221 °C. MS m/z 258.9 [M+H]+.
(IC50 = 1.88
l
M for A431 and IC50 = 15.5
l
M for A549), and compa-
M against A431
lM against A549). In the case of against A549, all
rable to the positive control erlotinib (IC50 = 1.41
and IC50 = 11.8
l
Table 2
The antiproliferative activities of compounds 1a–1o and reference compounds (n = 6,
ꢀ
x Æ s)
Compounds 3b–3l and 6 were synthesized according to the pro-
Compounds
IC50
(l
M)
cedure described above.
A431
A549
HL-60
4.1.1.2. N-(3-Fluorophenyl)-2-hydroxy-5-nitrobenzamide (3b).
Yield 94%. Mp 208–210 °C. 1H NMR: d 10.68 (s, 1H, NH), 8.70 (s,
1H, Ar-H), 8.30 (q, 1H, Ar-H), 7.74 (d, 1H, J = 11.37 Hz, Ar-H), 7.41–
7.50 (m, 2H, Ar-H), 7.19 (d, 1H, J = 9.09 Hz, Ar-H), 6.99 (t, 1H,
J = 8.86 Hz, Ar-H). HRMS Calcd for
299.0444. Found 299.0441.
1a
1b
1c
1d
1e
1f
1g
1h
1i
1j
1k
1l
1m
1n
1o
SAHA
Gifitinib
5.85 0.66
4.28 0.58
4.01 0.51
3.48 0.36
2.75 0.44
5.75 0.27
3.80 0.61
4.14 0.32
2.60 0.18
6.11 0.98
6.46 0.12
4.68 0.81
7.08 0.57
4.20 0.39
1.88 0.09
5.13 0.40
1.41 0.12
14.6 2.2
37.2 3.5
70.1 8.3
21.1 6.5
25.7 3.1
76.2 9.6
17.8 1.9
24.1 4.3
12.4 2.3
43.6 5.0
24.1 3.9
18.4 1.7
37.6 2.9
40.7 8.0
15.5 1.9
27.8 2.6
11.8 1.1
20.3 3.6
—
—
—
—
—
—
—
C
13H9FN2NaO4 [M+Na]+:
—
—
4.1.1.3. N-(3, 4-Difluorophenyl)-2-hydroxy-5-nitrobenzamide
(3c).
Yield 93%. Mp 257–258 °C. 1H NMR: d 10.68 (s, 1H, NH),
8.69 (s, 1H, Ar-H), 8.30 (q, 1H, Ar-H), 7.44–7.94 (m, 3H, Ar-H),
7.19 (d, 1H, J = 9.06 Hz, Ar-H). HRMS Calcd for C13H9F2N2O4
[M+H]+: 295.0530. Found 295.0522.
9.83 2.6
28.6 5.5
—
7.10 1.8
30.9 6.7
76.5 5.6
54.5 8.6
4.1.1.4. N -(3-Chloro-4-fluorolphenyl)-2-hydroxy-5-nitrobenza-
mide (3d).
C
Yield: 95%. Mp 259–260 °C. HRMS Calcd for
13H8ClFN2NaO4 [M+Na]+: 333.0054. Found 333.0048.
‘—’ Means IC50 > 100 lM.

M. Zuo et al. / Bioorg. Med. Chem. 20 (2012) 4405–4412
4409
4.1.1.5. N -(3-Trifluoromethylphenyl)-2-hydroxy-5-nitrobenza-
mide (3e). Yield: 97%. Mp 181–182 °C. HRMS Calcd for C14H9
F3N2NaO4 [M+Na]+: 349.0407. Found 349.0405.
water and dried to give 1.46 g of 3n as a yellow solid. Yield
98%. Mp 194–196 °C. 1H NMR d 10.38 (s, 1H, NH), 8.39–8.43
(m, 2H, Ar-H), 7.90 (s, 1H, Ar-H), 7.23–7.74 (m, 4H, Ar-H), 4.19
(s, 1H, „CH), 4.03 (s, 3H, O-CH3). HRMS Calcd for C16H13N2O4
[M+H]+: 297.0875. Found 297.0877.
4.1.1.6. N-(3-Ethynylphenyl)-2-hydroxy-5-nitrobenzamide (3f).
Yield: 92%. Mp 231–233 °C. 1H NMR: d 10.64 (s, 1H, NH), 8.74 (s,
1H, Ar-H), 8.30 (m, 1H, Ar-H), 7.91 (s, 1H, Ar-H), 7.15–7.42 (m, 4H,
Ar-H), 4.20 (s, 1H, „CH). HRMS Calcd for C15H10N2NaO4 [M+Na]+:
305.0533. Found 305.0535.
4.1.1.14. N-(3-Ethynylphenyl)-2, 5-dihydroxybenzamide (6).
Yield 73%. Mp 199–201 °C. 1H NMR d 10.44 (s, 1H, NH), 9.05 (s,
1H, OH), 7.90 (s, 1H, Ar-H), 7.67 (d, 1H, J = 8.17 Hz, Ar-H), 6.81–7.39
(m, 5H, Ar-H), 4.15 (s, 1H, „CH). HRMS Calcd for C15H12NO3
[M+H]+: 254.0817. Found 254.0813.
4.1.1.7. N-(3, 4-Dichlorophenyl)-2-hydroxy-5-nitrobenzamide
(3g). Yield: 94%. Mp 270–271 °C. 1H NMR: d 10.73 (s, 1H, NH),
8.69 (s, 1H, Ar-H), 8.30 (d, 1H, J = 8.94 Hz, Ar-H), 8.11 (s, 1H, Ar-H),
7.64 (m, 2H, Ar-H), 7.18 (d, 1H, J = 9.03 Hz, Ar-H). HRMS Calcd for
4.1.1.15. Ethyl 7-(4-hydroxy-3-(N-phenylcarbamoyl)phenylamino)-
7-oxoheptanoate (4a).
Reduction of N-aryl-2-hydroxy-5-
C
13H8Cl2N2NaO4 [M+Na]+: 348.9753. Found 348.9763.
nitrobenzamide: A mixture of N-aryl-2-hydroxy-5-nitrobenzamide
(10 mmol), iron powder (100 mmol), acetic acid (10 mL), ethanol
(40 mL) and THF (40 mL) was stirred under refluxing for 7 h. The
solvent was evaporated off under reduced pressure and the residue
was adjusted to pH 10 by adding saturated Na2CO3 solution. The
mixture was extracted with ethyl acetate (50 mL Â 3). The com-
bined organic layer was washed with H2O and brine, dried over
anhydrous Na2SO4, filtered and evaporated to give a grey solid with
a quantitive yield. The products were not characterized and di-
rectly used for the next step.
4.1.1.8.
N -(4-Chloro-3-trifluoromethylphenyl)-2-hydroxy-5-
nitrobenzamide (3h). Yield: 85%. Mp 241–242 °C. 1H NMR: d
10.88 (s, 1H, NH), 8.69 (s, 1H, Ar-H), 8.31 (s, 1H, Ar-H), 8.28 (d,
1H, J = 9.30 Hz, Ar-H), 8.01 (d, 1H, J = 8.40 Hz, Ar-H), 7.25 (d, 1H,
J = 8.40 Hz, Ar-H), 7.19 (d, 1H, J = 9.30 Hz, Ar-H). HRMS Calcd for
C
14H8ClF3N2NaO4 [M+Na]+: 383.0017. Found 383.0027.
4.1.1.9. N-[3-Chloro-4-(3-fluorobenzyloxy)phenyl]-2-hydroxy-5-
nitrobenzamide (3i). Yield: 92%. Mp 242–243 °C. 1H NMR: d
10.63 (s, 1H, NH), 8.76 (s, 1H, Ar-H), 8.30 (d, 1H, J = 8.40 Hz, Ar-
H), 7.91 (s, 1H, Ar-H), 7.58 (d, 1H, J = 8.40 Hz, Ar-H), 7.14–7.47
(m, 6H, Ar-H), 5.24 (s, 2H, CH2). HRMS Calcd for C20H15ClF3N2O6
[M+H]+: 417.0654. Found 417.0660.
7-ethyoxyl-7-oxoheptanoyl chloride (12 mmol) was prepared
from 7-ethyoxyl-7-oxoheptanoic acid (12 mmol) and thionyl
chlororide (6 mL) according to general procedure.
7-ethyoxyl-7-oxoheptanoyl chloride (about 12 mmol, freshly
prepared) diluted with dry THF (15 mL) was added dropwise to
the solution of N-phenyl-2-hydroxy-5-aminobenzamide (10 mmol)
and TEA (1.7 mL, 12 mmol) in dry THF (40 mL) cooled by an ice-
water bath under stirring. After addition, the mixture was stirred
at the same temperature for another 3 h. Then the solvent was evap-
orated. The residue was dissolved in ethyl acetate (150 mL). The or-
ganic phase was washed with saturated NaHCO3 solution, H2O and
brine, dried over anhydrous Na2SO4, filtered and evaporated. The
residue was purified by silica gel column chromatographic using
CHCl3:MeOH (40:1, v/v) as eluent to give 1.95 g of 4a as a pale yellow
solid. Yield 49%. Mp 199–200 °C. 1H NMR d 10.44 (s, 1H, NH), 9.75 (s,
1H, NH), 8.02 (s, 1H, Ar-H), 6.90–7.70 (m, 7H, Ar-H), 4.03 (q, 2H, O-
CH2), 2.27 (m, 4H, 2ÂCO-CH2), 1.56 (m, 4H, 2ÂCH2), 1.31 (m, 2H,
CH2), 1.17 (t, 3H, J = 7.68 Hz,CH3). HRMS Calcd for C22H26N2NaO5
[M+Na]+: 421.1739. Found 421.1736.
4.1.1.10. N-(3-Ethynylphenyl)-2-hydroxy-5-nitrobenzamide (3j@
3f) and N-(3-chloro-4-fluorophenyl)-2-hydroxy-4-methoxy-5-
nitrobenzamide (3k). 2-Hydroxy-4-methoxybenzoic acid (8.4 g,
50 mmol) was added portionwise into a mixture of glacial acetic
acid (31 mL) and nitric acid (31 mL) cooled by ice-water bath.
The obtained reaction mixture was stirred overnight, poured into
ice-water (about 200 mL), filtered. The solid was washed with
water, dried, crystallized from ethanol and water (1:1, v/v) to give
6.40 g of 2-hydroxy-4-methoxy-5-nitrobenzoic acid (5) as a yellow
solid. Yield 60%. Mp 233–235 °C.
Compounds 3k was synthesized according to the procedure de-
scribed in 3a.
Yield 88%. Mp 250–252 °C. 1H NMR: d 10.59 (s, 1H, NH), 8.71 (s,
1H, Ar-H), 7.41–8.00 (m, 3H, Ar-H), 6.80 (s, 1H, Ar-H), 3.96 (s, 3H,
OCH3). HRMS Calcd for C14H10ClFN2NaO5 [M+Na]+: 363.0154.
Found 363.0162.
Compounds 4b–4m were synthesized according to the proce-
dure described above.
4.1.1.16. Ethyl 7-[4-hydroxy-3-(N -3-fluorophenylcarbamoyl)
4.1.1.11. N -(3-Ethynylphenyl)-2-hydroxy-4-methoxy-5-nitrob-
enzamide (3l). Yield 78%. Mp 207–209 °C. 1H NMR: d 10.53 (s,
1H, NH), 8.74 (s, 1H, Ar-H), 7.87 (s, 1H, Ar-H), 7.25–7.72 (m, 3H,
Ar-H), 6.81 (s, 1H, Ar-H), 4.20 (s, 1H, „CH), 3.96 (s, 3H, O-CH3).
HRMS Calcd for C16H13N2O5 [M+H]+: 313.0824. Found 313.0826.
phenylamino]-7-oxoheptanoate (4b).
Yield 45%. Mp 203–
204 °C. 1H NMR: d 11.16 (s, 1H, OH), 10.55 (s, 1H, NH), 9.84 (s,
1H, NH), 8.00 (s, 1H, Ar-H), 7.76 (d, 1H, J = 11.49 Hz, Ar-H), 7.65
(d, 1H, J = 8.43 Hz, Ar-H), 6.93–7.46 (m, 4H, Ar-H), 4.03 (q, 2H, O-
CH2), 2.28 (m, 4H, 2ÂCO-CH2), 1.56 (m, 4H, 2ÂCH2), 1.31 (m, 2H,
CH2), 1.17 (t, 3H, J = 8.08 Hz, CH3). HRMS Calcd for C22H25FN2NaO5
[M+Na]+: 439.1645. Found 439.1648.
4.1.1.12. N-(3-Ethynylphenyl)-5-nitrobenzamide (3m).
The reaction of 3-nitrobenzoyl chloride (12 mmol) with 3-ethy-
nylbenzenamine (1.4 g, 12 mmol) in dry THF (30 ml) and TEA
(0.5 mL, 14 mmol) gave a light-yellow product (3.1 g). Yield 97%.
Mp 177–178 °C. HRMS Calcd for C15H11N2O3 [M+H]+: 267.0770.
Found 267.0773.
4.1.1.17. Ethyl 7-[4-hydroxy-3-(N-3,4-difluorophenylcarbamoyl)
phenylamino]-7-oxoheptanoate (4c). Yield 43%. Mp 208–209
°C. 1H NMR: d 10.54 (s, 1H, NH), 9.84 (s, 1H, NH), 8.00 (s, 1H, Ar-
H), 7.9 (m, 1H, Ar-H), 7.64 (1H, J = 8.49 Hz, Ar-H), 7.41–7.47 (m,
2H, Ar-H), 6.96 (d, 1H, J = 8.85 Hz, Ar-H), 4.05 (q, 2H, O-CH2),
2.28 (m, 4H, 2ÂCO-CH2), 1.57 (m, 4H, 2ÂCH2), 1.31 (m, 2H, CH2),
1.17 (t, 3H, J = 7.66 Hz, CH3). HRMS Calcd for C22H24F2N2NaO5
[M+Na]+: 457.1551. Found 457.1552.
4.1.1.13. N-(3-Ethynylphenyl)-2-methoxy-5-nitrobenzamide
(3n). The mixture of 3f (1.4 g, 5 mmol), Me2SO4 (0.96 g,
10 mmol), K2CO3 (2.1 g, 15 mmol) and acetone (50 mL) was
heated to reflux for 3 h with strirring, cooled to room tempera-
ture, added water (30 mL). Then acetone was evaporated. The
formed precipitate was collected by filtration, washed with
4.1.1.18. Ethyl 7-[4-hydroxy-3-(N -3-chloro-4-fluorophenylcar-
bamoyl)phenylamino]-7-oxoheptanoate (4d). Yield 59%. Mp

4410
M. Zuo et al. / Bioorg. Med. Chem. 20 (2012) 4405–4412
183–184 °C. 1H NMR: d 11.06 (s, 1H, OH), 10.50 (s, 1H, NH), 9.83 (s,
1H, NH), 6.95–8.06 (m, 6H, Ar-H), 4.03 (q, 2H, O-CH2), 2.27 (m, 4H,
2ÂCO-CH2), 1.57 (m, 4H, 2ÂCH2), 1.31 (m, 2H, CH2), 1.17 (t, 3H,
J = 7.76 Hz,CH3). HRMS Calcd for C22H24ClFN2NaO5 [M+Na]+:
473.1255. Found 473.1262.
J = 8.08 Hz, CH3). HRMS Calcd for C23H26ClFN2NaO6 [M+Na]+:
503.11361. Found 503.1333.
4.1.1.26. Ethyl 7-[5-(N -3-ethynylphenylcarbamoyl)-4-hydroxy-
2-methoxy-phenylamino]-7-oxoheptanoate (4l).
Yield 60%.
Mp 96–98 °C. 1H NMR: d 10.35 (s, 1H, NH), 9.10 (s, 1H, NH), 8.22
(s, 1H, Ar-H), 7.87 (s, 1H, Ar-H), 7.71 (d, 1H, J = 7.88 Hz, Ar-H),
7.38 (t, 1H, Ar-H), 7.24 (d, 1H, J = 7.30 Hz, Ar-H), 6.61 (s, 1H, Ar-
H), 4.22 (s, 1H, „CH), 4.08 (q, 2H, O-CH2), 3.84 (s, 3H, O-CH3),
2.32 (m, 4H, 2ÂCO-CH2), 1.56 (m, 4H, 2ÂCH2), 1.34 (m, 2H, CH2),
1.17 (t, 3H, J = 7.87 Hz,CH3). HRMS Calcd for C25H28N2NaO6
[M+Na]+: 475.1845. Found 475.1815.
4.1.1.19. Ethyl 7-[4-hydroxy-3-(N -3-trifluoromethylphenylcar-
bamoyl)phenylamino]-7-oxoheptanoate (4e). Yield 40%. Mp
186–187 °C. 1H NMR: d 11.07 (s, 1H, OH), 10.65 (s, 1H, NH), 9.85
(s, 1H, NH), 8.25 (s, 1H, Ar-H), 8.03 (s, 1H, Ar-H), 7.46–7.94 (m,
4H, Ar-H), 6.96 (d, 1H, J = 8.73 Hz, Ar-H), 4.04 (q, 2H, O-CH2), 2.28
(m, 4H, 2ÂCO-CH2), 1.56 (m, 4H, 2ÂCH2), 1.31 (m, 2H, CH2), 1.18
(t, 3H, J = 8.02 Hz,CH3). HRMS Calcd for C23H25F3N2NaO5 [M+Na]+:
489.1613. Found 489.1617.
4.1.1.27. Ethyl 7-[3-(N-3-ethynylphenylcarbamoyl)phenylami-
no]-7-oxoheptanoate (4m).
Yield 83%. Mp 148–149 °C. 1H
4.1.1.20. Ethyl 7-[4-hydroxy-3-(N -3-ethynylphenylcarbamoyl)
phenylamino]-7-oxoheptanoate (4f). Yield 56%. Mp 187–188
°C. 1H NMR: d 11.12 (s, 1H, OH), 10.44 (s, 1H, NH), 9.81 (s, 1H,
NH), 8.00 (s, 1H, Ar-H), 7.91 (s, 1H, Ar-H), 6.93–7.70 (m, 5H, Ar-
H), 4.20 (s, 1H, „CH), 4.05 (q, 2H, O-CH2), 2.28 (m, 4H, 2ÂCO-
CH2), 1.56 (m, 4H, 2ÂCH2), 1.30 (m, 2H, CH2), 1.18 (t, 3H,
J = 7.77 Hz,CH3). HRMS Calcd for C24H26N2NaO5 [M+Na]+:
445.1739. Found 445.1738.
NMR: d 10.36 (s, 1H, NH), 10.10 (s, 1H, NH), 8.10 (s, 1H, Ar-H),
7.96 (s, 1H, Ar-H), 7.84 (d, 1H, J = 8.10 Hz, Ar-H), 7.81 (d, 1H,
J = 7.50 Hz, Ar-H), 7.62 (d, 1H, J = 7.20 Hz, Ar-H), 7.46 (t, 1H, Ar-
H), 7.37 (t, 1H, Ar-H), 7.22 (d, 1H, J = 7.50 Hz, Ar-H), 4.20 (s,1H,
„CH), 4.05 (q, 2H, O-CH2), 2.29 (m, 4H, 2ÂCO-CH2), 1.58 (m, 4H,
2ÂCH2), 1.32 (m, 2H, CH2), 1.17 (t, 3H, J = 7.88 Hz, CH3). HRMS
Calcd for C24H26N2NaO4 [M+Na]+: 429.1790. Found 429.1753.
4.1.1.28. 7-[3-(N -Ethynylphenylcarbamoyl)-4-methoxyphenyl-
4.1.1.21. Ethyl 7-[4-hydroxy-3-(N-3,4-dichlorophenylcarbamoyl)
phenylamino]-7-oxoheptanoate (4g). Yield: 49%. Mp 193–194
°C. 1H NMR: d 10.60 (s, 1H, NH), 9.83 (s, 1H, NH), 8.13 (s, 1H, Ar-
H), 7.98 (s, 1H, Ar-H), 7.59–7.68 (m, 3H, Ar-H), 6.95 (d, 1H,
J = 8.71 Hz, Ar-H), 4.04 (q, 2H, O-CH2), 2.28 (m, 4H, 2ÂCO-CH2),
1.57 (m, 4H, 2ÂCH2), 1.31 (m, 2H, CH2), 1.17 (t, 3H, J = 8.00 Hz,CH3).
amino]-7-oxoheptanoic acid (4n).
The mixture of 5-amino-
N-(3-ethynylphenyl)-2-methoxybenzamide (4.3 mmol), pimelic
acid anhydride (0.74 g, 5.1 mmol), anhydrous THF (20 mL) and
TEA (0.9 mL) was stirred overnight. Then the solvent was evapo-
rated and the product was purified by silica gel column chromato-
graphic using CHCl3:MeOH (50:1, v/v) as eluent to give 1.20 g of 4n
as a white solid. Yield 68%. Mp > 260 °C. 1H NMR d 11.95 (s, 1H,
OH), 10.21 (s, 1H, NH), 9.89 (s, 1H, NH), 7.90 (s, 1H, Ar-H), 7.84
(s, 1H, Ar-H), 7.11–7.77 (m, 5H, Ar-H), 4.20 (s, 1H, „CH), 3.87 (s,
3H, O-CH3), 2.18–2.30 (m, 4H, CO-CH2), 1.52–1.58 (m, 4H,
2ÂCH2), 1.30 (m, 2H, CH2). HRMS Calcd for C23H24N2NaO5
[M+Na]+: 431.1583. Found 431.1559.
HRMS Calcd for
489.0968.
C
22H24Cl2N2NaO5 [M+Na]+: 489.0960. Found
4.1.1.22. Ethyl 7-[4-hydroxy-3-(N-4-chloro-3-trifluoromethylphe-
nylcarbamoyl) phenylamino]-7-oxoheptanoate (4h). Yield: 40%.
Mp 151–152 °C. 1H NMR: d 10.69 (s, 1H, NH), 9.80 (s, 1H, NH), 8.69
(s, 1H, Ar-H), 8.33 (s, 1H, Ar-H), 8.00 (m, 2H, Ar-H), 6.94–7.72 (m, 3H,
Ar-H), 4.03 (q, 2H, O-CH2), 2.28 (m, 4H, 2ÂCO-CH2), 1.57 (m, 4H,
2ÂCH2), 1.31 (m, 2H, CH2), 1.17 (t, 3H, J = 7.84 Hz,CH3). HRMS Calcd
for C23H24ClF3N2NaO5 [M+Na]+: 523.1224. Found 523.1220.
4.1.1.29. Ethyl 7-[3-(N-ethynylphenyl)carbamoyl-4-hydroxy-
phenoxy]heptanoate (7).
The mixture of 6 (0.51 g, 2 mmol),
Ethyl 7-bromoheptanoate (0.47 g, 2 mol), K2CO3 (0.83 g, 6 mmol),
tetrabutylammonium iodide (1.48 g, 4 mmol) and acetone
(15 mL) was strirred under refluxing for 4 h. The solvent was evap-
orated. The residue was dissolved in water (30 mL) and ethyl ace-
tate (100 mL). The organic phase was washed with brine, dried
over anhydrous sodium sulfate, filtered and evaporated. The resi-
due was purified by silica gel column chromatographic using
CHCl3:MeOH (50:1, v/v) as eluent to produce 0.53 g of 7 as a pale
yellow solid. Yield 65%. Mp 117–118 °C. 1H NMR d 10.17 (s, 1H,
NH), 9.26 (s, 1H, OH), 7.87 (s, 1H, Ar-H), 7.13 (s, 1H, Ar-H), 6.86–
7.68 (m, 5H, Ar-H), 4.15 (s, 1H, „CH), 4.02 (m, 4H, 2ÂO-CH2),
2.20 (m, 2H, CH2), 1.73 (m, 2H, CH2), 1.42 (m, 4H, 2ÂCH2), 1.30
(m, 2H, CH2), 1.16 (t, 3H, J = 7.79 Hz, CH3). HRMS Calcd for
4.1.1.23. Ethyl 7-[4-hydroxy-3-(N-3-chloro-4-(3-fluorobenzyloxy)
phenyl)carbamoyl] phenylamino)-7-oxoheptanoate (4i). Yield
54%. Mp 173–174 °C. 1H NMR: d 11.16 (s, 1H, OH), 10.36 (s, 1H,
NH), 9.81 (s, 1H, NH), 8.01 (s, 1H, Ar-H), 7.93 (s, 1H, Ar-H), 7.17–
7.63 (m, 7H, Ar-H), 6.95 (d, 1H, J = 8.37 Hz, Ar-H), 5.24 (s, 2H,
CH2), 4.05 (q, 2H, O-CH2), 2.28 (m, 4H, 2ÂCO-CH2), 1.56 (m, 4H,
2ÂCH2), 1.31 (m, 2H, CH2), 1.17 (t, 3H, J = 8.02 Hz,CH3). HRMS Calcd
for C29H30ClFN2NaO6 [M+Na]+: 579.1674. Found 579.1676.
4.1.1.24. Ethyl 8-[4-hydroxy-3-(N -3-ethynylphenyllcarbamoyl)
phenylamino]-8-oxooctanoate (4j). The reaction of N-3-ethyn-
ylphenyl-2-hydroxy-5-aminobenzamide with 8-ethyoxyl-8-oxo-
octanoyl chloride gave 4j. Yield 38%. Mp 179–180 °C. 1H NMR: d
11.09 (s, 1H, OH), 10.46 (s, 1H, NH), 9.78 (s, 1H, NH), 8.01 (s, 1H,
Ar-H), 7.91 (s, 1H, Ar-H), 6.92–7.70 (m, 5H, Ar-H), 4.18 (s, 1H,
„CH), 4.05 (q, 2H, O-CH2), 2.27 (m, 4H, 2ÂCO-CH2), 1.56 (m, 4H,
2ÂCH2), 1.30 (m, 4H, 2ÂCH2), 1.19 (t, 3H, J = 7.94 Hz, CH3). HRMS
Calcd for C25H28N2NaO5 [M+Na]+: 459.1896. Found 459.1894.
C
24H27NNaO5 [M+Na]+: 432.1787. Found 432.1759.
4.1.1.30. N -Phenyl-2-hydroxy-5-(7-hydroxyamino-7-oxohepta-
namido)benzamide (1a). KOH (2.64 g, 40 mmol) was added
to a solution of hydroxyamine hydrochloride (1.88 g, 27 mmol) in
methanol (15 mL) cooled by an ice bath. The mixture was stirred
for another 1 h. The resulting precipitate was filtered off, and the
solution of free hydroxylamine was prepared.
4.1.1.25. Ethyl 7-[5-(N -3-chloro-4-fluorophenylcarbamoyl)-4-
hydroxy-2-methoxy-phenylamino]-7-oxoheptanoate (4k). Yield
43%. Mp 82–83 °C. 1H NMR: d 12.09 (s, 1H, OH), 10.38 (s, 1H, NH),
9.10 (s, 1H, NH), 8.20 (s, 1H, Ar-H), 7.39–8.00 (m, 3H, Ar-H), 6.61
(s, 1H, Ar-H), 4.06 (q, 2H, O-CH2), 3.84 (s, 3H, O-CH3), 2.32 (m, 4H,
2ÂCO-CH2), 1.56 (m, 4H, 2ÂCH2), 1.35 (m, 2H, CH2), 1.17 (t, 3H,
The above freshly prepared hydroxyamine solution was placed
in a round bottom flask cooled by an ice bath. 4a (1.08 g, 2.7 mmol)
was added to the solution. Then the mixture was stirred over night,
water (50 mL) and AcOH were added to adjust pH to 4–5, kept for
3 h in a refrigerator. The precipitate was collected by filtration,
washed with water, then crystallized from acetone/THF (1:1, v/v)

M. Zuo et al. / Bioorg. Med. Chem. 20 (2012) 4405–4412
4411
to afford 0.65 g of 1a as a carneous solid. Yield 63%. Mp 249–
251 °C. 1H NMR d 10.68 (s, 1H, NH), 10.35 (s, 1H, NH), 9.81 (s,
1H, NH), 8.04 (s, 1H, Ar-H), 6.92–7.70 (m, 7H, Ar-H), 2.27 (m, 2H,
CO-CH2), 1.95 (m, 2H, CO-CH2), 1.55 (m, 4H, 2ÂCH2), 1.29 (m,
2H, CH2). HRMS Calcd for C20H23N3NaO5 [M+Na]+: 408.1535. Found
408.1540.
1.58 (m, 4H, 2ÂCH2), 1.28 (m, 2H, CH2). HRMS Calcd for
21H21ClF3N3NaO5 [M+Na]+: 5710.1020. Found 510.1001.
C
4.1.1.38. N-[3-Chloro-4-(3-fluorobenzyloxy)phenyl]-2-hydroxy-
5-(7-hydroxyamino-7-oxoheptanamido)benzamide (1i). Yield
69%. Mp 228–231 °C. 1H NMR d 10.38 (s, 1H, NH), 9.85 (s, 1H,
NH), 8.04 (s, 1H, Ar-H), 7.94 (s, 1H, Ar-H), 7.17–7.64 (m, 7H, Ar-
H), 6.97 (d, 1H, J = 8.37 Hz, Ar-H), 5.24 (s, 2H, CH2), 2.28 (m, 2H,
CO-CH2), 1.97 (m, 2H, CO-CH2), 1.56 (m, 4H, 2ÂCH2), 1.29 (m,
2H, CH2). HRMS Calcd for C27H27ClFN3NaO6 [M+Na]+: 566.1470.
Found 566.1445.
Compounds 1b–1m and 1o were synthesized according to the
procedure described above.
4.1.1.31. N -(3-Fluorophenyl)-2-hydroxy-5-(7-hydroxyamino-7-
oxohep-tanamido) benzamide (1b).
Yield 63%. Mp 260–
262 °C. 1H NMR d 10.76 (s, 1H, NH), 10.34 (s, 1H, NH), 9.81 (s,
1H, NH), 8.00 (s, 1H, Ar-H), 7.76 (d, 1H, J = 11.37 Hz, Ar-H), 7.64
(d, 1H, J = 8.37 Hz, Ar-H), 6.92–7.42 (m, 4H, Ar-H), 2.26 (m, 2H,
CO-CH2), 1.95 (m, 2H, CO-CH2), 1.55 (m, 4H, 2ÂCH2), 1.29 (m,
2H, CH2). HRMS Calcd for C20H23FN3NaO5 [M+Na]+: 404.1622.
Found 404.1632.
4.1.1.39. N-(3-Ethynylphenyl)-2-hydroxy-5-(8-hydroxyamino-8-
oxooctanamido)benzamide (1j).
Yield 72%. mp > 260 °C. 1H
NMR d 10.61 (s, 1H, NH), 10.31 (s, 1H, NH), 9.79 (s, 1H, NH), 8.65
(s, 1H, OH), 8.00 (s, 1H, Ar-H), 7.91 (s, 1H, Ar-H), 6.91–7.69 (m,
5H, Ar-H), 4.19 (s, 1H, „CH), 2.26 (m, 2H, CO-CH2), 1.94 (m, 2H,
CO-CH2), 1.49–1.57 (m, 4H, 2ÂCH2), 1.29 (m, 4H, 2ÂCH2). HRMS
Calcd for C23H25N3NaO5 [M+Na]+: 466.1692. Found 466.1675.
4.1.1.32. N-(3, 4-Difluorophenyl)-2-hydroxy-5-(7-hydroxyami-
no-7-oxoheptanamido) benzamide (1c).
Yield 71%. Mp 254–
256 °C. 1H NMR d 10.74 (s, 1H, NH), 10.34 (s, 1H, NH), 9.81 (s, 1H,
NH), 8.00 (s, 1H, Ar-H), 7.43–7.94 (m, 4H, Ar-H), 6.94 (d, 1H,
J = 8.76 Hz, Ar-H), 2.26 (m, 2H, CO-CH2), 1.96 (m, 2H, CO-CH2),
1.56 (m, 4H, 2ÂCH2), 1.29 (m, 2H, CH2). HRMS Calcd for
4.1.1.40. N-(3-Chloro-4-fluorophenyl)-2-hydroxy-5-(7-hydroxya-
mino-7-oxoheptanamido)-4-methoxybenzamide (1k). Yield
89%. Mp 193–195 °C [crystallized from ethyl acetate/THF (3:1, v/
v)]. 1H NMR d 10.47 (s, 1H, NH), 10.38 (s, 1H, NH), 9.09 (s, 1H,
NH), 8.20 (s, 1H, Ar-H), 7.39–8.01 (m, 3H, Ar-H), 6.60 (s, 1H, Ar-
H), 3.84 (s, 3H, O-CH3), 2.32 (m, 2H, CO-CH2), 1.97 (m, 2H, CO-
CH2), 1.56 (m, 4H, 2ÂCH2), 1.29 (m, 2H, CH2). HRMS Calcd for
C
20H21F2N3NaO5 [M+Na]+: 444.1347. Found 444.1354.
4.1.1.33. N -(3-Chloro-4-fluorophenyl)-2-hydroxy-5-(7-hydrox-
yamino-7-oxoheptanamido)benzamide (1d).
Yield 63%. Mp
C
21H23ClFN3NaO6 [M+Na]+: 490.1157. Found 490.1135.
219–222 °C. 1H NMR d 11.07 (s, 1H, Ar-OH), 10.51 (s, 1H, NH),
10.33 (s, 1H, NH), 9.82 (s, 1H, NH), 8.65 (s, 1H, N-OH), 6.92–8.06
(m, 6H, Ar-H), 2.26 (m, 2H, CO-CH2), 1.95 (m, 2H, CO-CH2), 1.55
(m, 4H, 2ÂCH2), 1.28 (m, 2H, CH2). HRMS Calcd for
4.1.1.41. N-(3-Ethynylphenyl)-2-hydroxy-5-(7-hydroxyamino-7-
oxoheptanamido)-4-methoxybenzamide (1l). Yield 85%.
Mp 157–159 °C. 1H NMR d 10.63 (s, 1H, NH), 10.36 (s, 1H, NH),
9.06 (s, 1H, NH), 8.70 (s, 1H, OH), 8.19 (s, 1H, Ar-H), 7.87 (s, 1H,
Ar-H), 7.70 (d, 1H, J = 7.95 Hz, Ar-H), 7.37 (t, 1H, Ar-H), 7.23 (d,
1H, J = 7.35 Hz, Ar-H), 6.58 (s, 1H, Ar-H), 4.22 (s, 1H, „CH), 3.83
(s, 3H, O-CH3), 2.31 (m, 2H, CO-CH2), 1.96 (m, 2H, CO-CH2), 1.56
(m, 4H, 2ÂCH2), 1.28 (m, 2H, CH2). HRMS Calcd for C23H25N3NaO6
[M+Na]+: 462.1641. Found 462.1620.
C
20H21ClFN3NaO5 [M+Na]+: 460.1051. Found 460.1062.
4.1.1.34. N-(3-Trifluoromethylphenyl-2-hydroxy-5-(7-hydroxya-
mino-7-oxoheptanamido)benzamide (1e). Yield 60%. Mp
212–214 °C. 1H NMR d 10.70 (s, 1H, NH), 10.36 (s, 1H, NH), 9.85
(s, 1H, NH), 8.25 (s, 1H, Ar-H), 8.03 (s, 1H, Ar-H), 7.46–7.94 (m,
4H, Ar-H), 6.96 (d, 1H, J = 8.55 Hz, Ar-H), 2.28 (m, 2H, CO-CH2),
1.97 (m, 2H, CO-CH2), 1.56 (m, 4H, 2ÂCH2), 1.29 (m, 2H, CH2).
HRMS Calcd for C21H22F3N3NaO5 [M+Na]+: 476.1409. Found
476.1424.
4.1.1.42. N -(3-Ethynylphenyl)-3-(7-hydroxyamino-7-oxohepta-
namido)benzamide (1m).
Yield 81%. Mp > 250 °C (crystal-
lized from ethyl acetate). 1H NMR d 10.36 (s, 1H, NH), 10.11 (s,
1H, NH), 9.76 (s, 1H, NH), 8.69 (s, 1H, OH), 8.09 (s, 1H, Ar-H),
7.95 (s, 1H, Ar-H), 7.86 (d, 1H, J = 8.10 Hz, Ar-H), 7.80 (d, 1H,
J = 7.80 Hz, Ar-H), 7.61 (d, 1H, J = 7.50 Hz, Ar-H), 7.48 (t, 1H, Ar-
H), 7.37 (t, 1H, J = 6.82 Hz,Ar-H), 7.22 (d, 1H, J = 7.20 Hz, Ar-H),
4.21 (s, 1H, „CH), 2.32 (m, 2H, CO-CH2), 1.95 (m, 2H, CO-CH2),
1.57 (m, 4H, 2ÂCH2), 1.28 (m, 2H, CH2). HRMS Calcd for
4.1.1.35. N-(3-Ethynylphenyl)-2-hydroxy-5-(7-hydroxyamino-7-
oxoheptanamido) benzamide (1f).
Yield 73%. Mp > 260 °C.
1H NMR d 10.60 (s, 1H, NH), 10.34 (s, 1H, NH), 9.80 (s, 1H, NH),
8.02 (s, 1H, Ar-H), 7.92 (s, 1H, Ar-H), 6.93–7.70 (m, 5H, Ar-H),
4.18 (s, 1H, „CH), 2.27 (m, 2H, CO-CH2), 1.96 (m, 2H, CO-CH2),
1.56 (m, 4H, 2ÂCH2), 1.28 (m, 2H, CH2). HRMS Calcd for
C22H23N3NaO5 [M+Na]+: 432.1535. Found 432.1519.
C
22H23N3NaO4 [M+Na]+: 416.1586. Found 416.1568.
4.1.1.43. N -(3-Ethynylphenyl)-5-(7-hydroxyamino-7-oxohepta-
namido)-2-methoxy benzamide (1n). A solution of methyl
4.1.1.36. N -(3,4-Dichlorophenyl)-2-hydroxy-5-(7-hydroxyami-
no-7-oxoheptanamido) benzamide (1g).
Yield 67%. Mp
chloroformate (2 drops) in anhydrous THF (1 mL) was added drop-
wise to the solution of 4n (0.2 g, 0.49 mmol) and TEA (0.1 mL) in
dry THF (15 mL) cooled by an ice-water bath under N2 atmosphere
with stirring. The mixture was stirred at the same temperature for
another 1 h, added the freshly prepared hydroxyamine (10 equiv)
solution, then stirred for 1 h, adjusted pH to 4–5 by adding AcOH.
Then the solvent was evaporated and the residue was purified by
silica gel column chromatographic using CHCl3/MeOH (30:1, v/v)
as eluent to give 0.11 g of 1n as a pale yellow solid. Yield 53%.
Mp 154–155 °C. 1H NMR d 10.62 (s, 1H, NH), 10.28 (s, 1H, NH),
9.86 (s, 1H, NH), 7.91 (s, 1H, Ar-H), 7.85 (s, 1H, Ar-H), 7.1,5–7.76
(m, 5H, Ar-H), 4.19 (s, 1H, „CH), 3.85 (s, 3H, O-CH3), 2.27 (m,
2H, CO-CH2), 1.96 (m, 2H, CO-CH2), 1.56 (m, 4H, 2ÂCH2), 1.28
225–227 °C. 1H NMR d 10.63 (s, 1H, NH), 10.35 (s, 1H, NH), 9.84
(s, 1H, NH), 8.71 (s, 1H, OH), 8.14 (s, 1H, Ar-H), 7.99 (s, 1H, Ar-
H), 7.59–7.68 (m, 3H, Ar-H), 6.95 (d, 1H, J = 8.76 Hz, Ar-H), 2.25
(m, 2H, CO-CH2), 1.96 (m, 2H, CO-CH2), 1.55 (m, 4H, 2ÂCH2),
1.28 (m, 2H, CH2). HRMS Calcd for C20H21Cl2N3NaO5 [M+Na]+:
476.0756. Found 476.0737.
4.1.1.37. N-(4-Chloro-3-trifluoromethylphenyl)-2-hydroxy-5-(7-
hydroxyamino-7-oxoheptanamido)benzamide (1h).
Yield
67%. Mp 197–198 °C. 1H NMR d 10.82 (s, 1H, NH), 10.30 (s, 1H,
NH), 9.78 (s, 1H, NH), 8.32 (s, 1H, Ar-H), 8.00 (s, 1H, Ar-H), 6.92–
7.97 (m, 4H, Ar-H), 2.28 (m, 2H, CO-CH2), 1.96 (m, 2H, CO-CH2),

4412
M. Zuo et al. / Bioorg. Med. Chem. 20 (2012) 4405–4412
(m, 2H, CH2). HRMS Calcd for C23H26N3O5 [M+H]+: 424.1872. Found
424.1853.
in the exponential phase throughout the 48 h incubation period.
Cells were treated with final concentrations of 100.0, 10.0, 1.0,
0.1 and 0.01
lM of tested compounds simultaneously and incu-
4.1.1.44. N-(3-Ethynylphenyl)-2-hydroxy-5-(7-hydroxyamino-7-
oxoheptyloxy) benzamide (1o). Yield 80%. Mp 149–151 °C
(crystallized from ethyl acetate). 1H NMR d 10.33 (s, 1H, NH),
10.21 (s, 1H, NH), 8.71 (s, 1H, OH), 7.89 (s, 1H, Ar-H), 7.67 (d, 1H,
J = 8.07 Hz, Ar-H), 7.37 (t, 1H, Ar-H), 7.20 (d, 1H, J = 7.41 Hz, Ar-
H), 7.13 (s, 1H, Ar-H), 7.00 (d, 1H, J = 8.73 Hz, Ar-H), 6.90 (d, 1H,
J = 8.07 Hz, Ar-H), 4.20 (s, 1H, „CH), 4.02 (t, 2H, O-CH2), 1.90 (t,
2H, CO-CH2), 1.73 (m, 2H, CH2), 1.42 (m, 4H, 2ÂCH2), 1.26 (m,
2H, CH2). HRMS Calcd for C22H24N2NaO5 [M+Na]+: 419.1583. Found
419.1565.
bated for 48 h and then 50
l
L of MTT solution (5 mg/ml in med-
ium) was added to each well and incubated for 2.5 h. The formed
blue formazan crystals were pelleted to the bottom of the well
by centrifugation, separated from the supernatant, and dissolved
in 150 lL of DMSO. The optical density at 570 nm was determined
by an ELISA reader. Two separate experiments with triplicate data
were performed to obtain mean cell viability. The IC50 values were
calculated according to the inhibition ratios.
Acknowledgment
4.2. Biological materials and methods
Financial support from National Natural Science Foundation of
China (Grant No. 21072156) is gratefully acknowledged.
4.2.1. ELISA-based EGFR-TK assay
In vitro EGFR-TK inhibition assays were carried out as described
in reference.32 EGFR-TK was prepared from shed membrane vesi-
cles of human A431 carcinoma cell. Briefly, 96-well plates were
References and notes
1. Sergina, N. V.; Moasser, M. M. Trends Mol. Med. 2007, 13, 527.
2. Hynes, N. E.; Lane, H. A. Nat. Rev. Cancer 2005, 5, 341.
3. Moasser, M. M. Oncogene 2007, 26, 6577.
precoated with
0.25 mg/mL) overnight at 37 °C. Ten microlitres of EGFR-TK ex-
tract, reaction medium and tested compound (20 L) were added.
a synthetic substrate poly-Glu-Tyr (Sigma,
4. Moasser, M. M. Oncogene 2007, 26, 6469.
5. Geyer, C.; Forster, J.; Lindquist, D.; Chan, S.; Romieu, C.; Pienkowski, T.; Jagiello-
Gruszfeld, A.; Crown, J.; Chan, A.; Kaufman, B.; Skarlos, D.; Campone, M.;
Davidson, N.; Berger, M.; Oliva, C.; Rubin, S.; Stein, S.; Cameron, D. N. Eng. J.
Med. 2006, 355, 2733.
l
The reaction mixtures were incubated for 30 min at room temper-
ature while being shaken. Kinase reaction was quenched by re-
moval of the reaction mixture, then the wells were washed with
washing buffer for three times. Phosphorylated tyrosine substrate
was blocked in PBS containing 3% BSA for 30 min, washed with
washing buffer (PBS containing 0.1% Tween 20) for three times, de-
tected by adding anti-phosphotyrosine antibody for 1 h. Then anti-
body was removed, and wells were washed with washing buffer
6. Press, M. F.; Lenz, H.-J. Drugs 2007, 67, 2045.
7. Ishikawa, T.; Seto, M.; Banno, H.; Kawakita, Y.; Oorui, M.; Nakayama, A.; Miki,
H.; Kamiguchi, H.; Tanaka, T.; Habuka, N.; Sogabe, S.; Yano, J.; Aertgeerts, K.;
Kamiyama, K. J. Med. Chem. 2011, 54, 8030.
8. Minucci, S.; Pelicci, P. G. Nat. Rev. Cancer 2006, 6, 38.
9. Bertrand, P. Eur. J. Med. Chem. 2010, 45, 2095.
10. Witt, O.; Deubzer, H. E.; Milde, T.; Oehm, I. Cancer Lett. 2009, 277, 8.
11. Pandolfi, P. P. Cancer Chemother. Pharmacol. 2001, 48(Suppl 1), S17.
12. Mann, B. S.; Johnson, J. R.; Cohen, M. H.; Justice, R.; Pazdur, R. Oncologist 2007,
12, 1247.
13. Paris, M.; Porcelloni, M.; Binaschi, M.; Fattori, D. J. Med. Chem. 2008, 51,
1505.
14. Ma, B. B. Y.; Sung, F.; Tao, Q.; Poon, F. F.; Lui, V. W.; Yeo, W.; Chan, S. L.; Chan, A.
T. C. Invest. New Drugs 2010, 28, 107.
15. Zhang, Y.; Feng, J.; Jia, Y.; Xu, Y.; Liu, C.; Fang, H.; Xu, W. Eur. J. Med. Chem. 2011,
46, 5387.
16. Zhang, Y.; Feng, J.; Liu, C.; Fang, H.; Xu, W. Bioorg. Med. Chem. 2011, 19, 4437.
17. Shultz, M.; Fan, J.; Chen, C.; Cho, Y. S.; Davis, N.; Bickford, S.; Buteau, K.; Cao, X.;
Holmqvis, M.; Hsu, M.; Jiang, L.; Liu, G.; Lu, Q.; Patel, C.; Suresh, J. R.; Selvaraj,
M.; Urban, L.; Wang, P.; Yan-Neale, Y.; Whitehead, L.; Zhang, H.; Zhou, L.; Atadj,
P. Bioorg. Med. Chem. Lett. 2011, 21, 4909.
18. Sharma, S. V.; Bell, D. W.; Settleman, J.; Haber, D. A. Nat. Rev. Cancer 2007, 7,
169.
19. Zhang, H.; Berezov, A.; Wang, Q.; Zhang, G.; Drebin, J.; Murali, R.; Greene, M. I. J.
Clin. Invest. 2007, 117, 2051.
20. Avizienyte, E.; Ward, R. A.; Garner, A. P. Biochem. J. 2008, 415, 197.
21. Qian, D. Z.; Wang, X.; Kachhap, S. K.; Kato, Y.; Wei, Y.; Zhang, L.; Atadja, P.; Pili,
R. Cancer Res. 2004, 64, 6626.
22. Bali, P.; Pranpat, M.; Swaby, R.; Fiskus, W.; Yamaguchi, H.; Balasis, M.; Rocha,
K.; Wang, H.; Victoria, R.; Kapil, B. Clin. Cancer Res. 2005, 11, 6382.
23. Edwards, A.; Li, J.; Atadja, P.; Bhalla, K.; Haura, E. B. Mol. Cancer Ther. 2007, 6,
2515.
24. Yu, C.; Friday, B. B.; Lai, J.-P.; McCollum, A.; Atadja, P.; Roberts, L. R.; Adjei, A. A.
Clin. Cancer Res. 2007, 13, 1140.
25. Chen, L.; Petrelli, R.; Gao, G.; Wilson, D. J.; McLean, G. T.; Jayaram, H. N.; Sham,
Y. Y.; Pankiewicz, K. W. Bioorg. Med. Chem. 2010, 18, 5950.
26. Cai, X.; Zhai, H.; Wang, J.; Forrester, J.; Qu, H.; Yin, L.; Lai, C.; Bao, R.; Qian, C. J.
Med. Chem. 2000, 2010, 53.
27. Mahboobi, S.; Sellmer, A.; Winkler, M.; Eichhorn, E.; Pongratz, H.; Ciossek, T.;
Baer, T.; Maier, T.; Beckers, T. J. Med. Chem. 2010, 53, 8546.
28. Liechti, C.; Sequin, U.; Bold, G.; Furet, P.; Meyer, T.; Traxler, P. Eur. J. Med. Chem.
2004, 39, 11.
29. Deng, W.; Guo, Z.; Guo, Y.; Feng, Z.; Jiang, Y.; Chu, F. Bioorg. Med. Chem. Lett.
2006, 16, 469.
for three times. Anti-mouse lgG (ZSGB-BIO; ZB-2305; 100
coupled with horseradish peroxidase (HRP) was added and incu-
bated for 1 h. HRP substrate was added (100 L per well) and incu-
bated for 10À20 min. The TMB reaction was quenched by addition
of 50 L of 0.1 M H2SO4. The optical density was measured at
lL /well)
l
l
450 nm by an ELISA reader. Experiment with triplicate data were
performed. IC50 values were calculated for test compounds by
using a regression analysis of the concentration/inhibition data.
4.2.2. In vitro HDACs inhibition fluorescence assay
In vitro HDACs inhibition assays were conducted as described in
reference.15 Briefly, 10
l
L of HeLa nuclear extract was mixed with
L). Five minutes later, fluorogenic substrate
Boc-Lys (acetyl)-AMC (40 L) was added, and the mixture was
incubated at 37 °C for 30 min and then stopped by addition of
100 L of developer containing trypsin and TSA. After incubation
tested compound (50
l
l
l
at 37 °C for 20 min, fluorescence intensity was measured using a
microplate reader at excitation and emission wavelengths of 390
and 460 nm, respectively. The inhibition ratios were calculated
from the fluorescence intensity readings of tested wells relative
to those of control wells. Experiment with triplicate data were per-
formed. The IC50 values were calculated using a regression analysis
of the concentration/inhibition data.
4.2.3. In vitro antiproliferative assay
Cellular chemosensitivity was determined by using a modified
MTT (3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyl-2H-tetrazolium
bromide) method assay in vitro. In brief, A431, A549 and HL-60
cells in 100 lL culture medium were seeded into 96-well micro-
plates, respectively, and incubated at 37 °C for 24 h prior to drug
30. Scott, G.; Aaron, D. W.; Jeffrey, A. Org. Lett. 2005, 7, 3167.
31. Bruce, J. M.; Roshan-Ali, Y. J. Chem. Res. Synop. 1981, 7, 193.
32. Varkondi, E.; Schaefer, E.; Boekoenyi, G.; Gyoekeres, T.; Oerfi, L.; Petak, I.; Pap,
A.; Szokoloczi, O.; Keri, G.; Schwab, R. J. Recept. Signal Transduct. 2005, 25, 45.
exposure. Cell numbers were titrated to keep control cells growing