The discovery and optimization of novel dual inhibitors of topoisomerase ii and histone deacetylase

Article abstract of DOI:10.1016/j.bmc.2013.09.023

A novel class of podophyllotoxin derivatives have been designed and synthesized based on the synergistic antitumor effects of topoisomerase II and histone deacetylase inhibitors. Their inhibitory activities towards histone deacetylases and Topo II and their cytotoxicities in cancer cell lines were evaluated. The aromatic capping group connection, linker length and zinc-binding group were systematically varied and preliminary conclusions regarding structure-activity relationships are discussed. Among all of the synthesized hybrid compounds, compound 24d showed the most potent HDAC inhibitory activity at a low nanomolar level and exhibited powerful antiproliferative activity towards HCT116 colon carcinoma cells at a low micromolar level. Further exploration of this series led to the discovery of potent dual inhibitor 32, which exhibited the strongest in vitro cytotoxic activity.

Full text of DOI:10.1016/j.bmc.2013.09.023

Bioorganic & Medicinal Chemistry 21 (2013) 6981–6995
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
The discovery and optimization of novel dual inhibitors of
topoisomerase ii and histone deacetylase
Xuan Zhang a, Bin Bao a, Xiuhua Yu a, Linjiang Tong b, Yu Luo d, Qingqing Huang a, Mingbo Su c, Li Sheng c,
Jia Li c, Hong Zhu e, Bo Yang e, Xiongwen Zhang a, Yi Chen b, , Wei Lu a,
⇑
⇑
a Institute of Drug Discovery and Development, Shanghai Engineering Research Center of Molecular Therapeutics and New Drug Development, East China Normal University,
3663 North Zhongshan Road, Shanghai 200062, PR China
b Division of Antitumor Pharmacology, Shanghai Institute of Materia Medica, Chinese Academy of Science, Shanghai 201203, PR China
c State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, SIBS, Chinese Academy of Sciences, Shanghai 201203, PR China
d Department of Chemistry, East China Normal University, 3663 North Zhongshan Road, Shanghai 200062, PR China
e Institute of Pharmacology and Toxicology, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 26 July 2013
Revised 7 September 2013
Accepted 7 September 2013
Available online 18 September 2013
A novel class of podophyllotoxin derivatives have been designed and synthesized based on the synergistic
antitumor effects of topoisomerase II and histone deacetylase inhibitors. Their inhibitory activities
towards histone deacetylases and Topo II and their cytotoxicities in cancer cell lines were evaluated.
The aromatic capping group connection, linker length and zinc-binding group were systematically varied
and preliminary conclusions regarding structure–activity relationships are discussed. Among all of the
synthesized hybrid compounds, compound 24d showed the most potent HDAC inhibitory activity at a
low nanomolar level and exhibited powerful antiproliferative activity towards HCT116 colon carcinoma
cells at a low micromolar level. Further exploration of this series led to the discovery of potent dual inhib-
itor 32, which exhibited the strongest in vitro cytotoxic activity.
Keywords:
Podophyllotoxin
HDAC
Topoisomerase II
Cancer
Ó 2013 Elsevier Ltd. All rights reserved.
Hybrid
Synergistic effect
1. Introduction
Combination therapies can create synergistic antitumor effects
by directly blocking the key signaling pathway as well as attenuat-
For decades, cytotoxic drugs have been used in a variety of can-
cer chemotherapy treatments. However, almost all traditional
cytotoxic agents are severely toxic and have other undesirable side
effects. In the last decade, the development of targeted medicines
as well as monoclonal antibodies has brightened the lives of cancer
patients. What was unexpected is that this targeted approach has
only proven to be partially successful. The effectiveness of these
agents is always hindered by poor response rates and acquired
drug resistance. Two decades ago, using a genetic model for colo-
rectal tumorigenesis, Fearon and Vogelstein reported that there is
at least one additional mutation in each stage of colorectal cancer.1
Single target agents can perform excellent during the treatment of
diseases with linear pathways. However, systems biology and net-
work analysis have shown that multi-factorial diseases, such as
cancer, with complex signaling networks are robust and require
the simultaneous perturbation of multiple targets.2.
ing multiple compensatory pathways.3–11 As a result, the efficacy
of the individual agents will be improved in combination therapy.
However, different drugs always have different pharmacokinetics,
including half-lives and distributions. These complex factors have
hindered clinical research on combination therapies. Furthermore,
these agents may introduce adverse effects associated with drug
interactions.
A new strategy to overcome these limitations is the design of
multi-target drugs. There are three types of multi-target agents:
chimeras, fused molecules and hybrids. Though all of these agents
can simultaneously act on several cancer targets and exhibit sim-
pler pharmacokinetics, the hybrid strategy is more likely to result
in suitable molecules with lower formula weights and proper lipo-
some–water partition co-efficients.
The histone deacetylase (HDAC) class of epigenetic enzymes is a
popular drug target. Together with histone acetyl transferase
(HAT), HDAC determines the acetylation status of histones. Inhibi-
tion of HDAC can cause growth arrest, differentiation, and apopto-
sis in tumor cells by inducing histone hyperacetylation and p21
expression.12–15 Two HDAC inhibitors (HDACi), vorinostat (SAHA)
and romidepsin (FK228) were approved by the FDA for the
⇑
Corresponding authors. Fax: +86 2162602475.
E-mail addresses: ychen@jding.dhs.org (Y. Chen), wlu@chem.ecnu.edu.cn (W.
Lu).
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.09.023

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X. Zhang et al. / Bioorg. Med. Chem. 21 (2013) 6981–6995
treatment of cutaneous T-cell lymphoma (CTCL).16–18 A typical
HDAC inhibitor consists of a capping group, a zinc-binding group
(ZBG) and an appropriate linker (Fig. 1). The simple SAR and
effectiveness of these molecules in oncotherapy has led many
researchers to explore HDAC-involved dual inhibitors.19–27
Topoisomerase II (Topo II) is a classical target for cancer chemo-
therapy treatments.28–31 Structural modifications of podophyllo-
toxin (PPT) have significantly contributed to the development of
clinically valuable anticancer drugs. Etoposide (VP-16), teniposide
(VM-26) and the water-soluble prodrug, etoposide phosphate
(Fig. 2) were approved by the FDA.32,33 These small molecules act
on the DNA–enzyme complex and cause errors in DNA synthesis,
which promotes apoptosis in cancer cells. However, chromatin
DNA is tightly coiled, which may hamper the ability of drug mole-
cules to interact with their DNA targets (Fig. 3). Kim et al., reported
the synergistic antitumor effect of HDAC and Topo II inhibitors.3
HDAC inhibitors enable histones to maintain a high degree of acet-
ylation. The resulting looser state of chromatin DNA may increase
the accessibility of DNA drug targets and consequently improve the
efficiency of anticancer drugs targeting DNA, such as Topo II inhib-
itors.3,34 Recently, Guerrant et al., reported their efforts on Topo-
HDAC dual inhibitors and yielded satisfactory results.35,36
under an atmosphere of hydrogen yielded target molecules 7a–c
and 8a–c.
Considering the stability of the complex structure found in PPT
derivatives, a convergent synthesis strategy was introduced for the
synthesis of SAHA-like dual inhibitors (Scheme 2). Preparation of
key intermediates 17–19 began with the esterification of bromo-
carboxylic acids 9a–e. Subsequently, esters 10a–e were treated
with different nitrophenols in the presence of K2CO3 followed by
hydrolysis with LiOH to afford acids 11–13. Treatment of these
compounds with thionyl chloride followed by amidation with N-
Boc-O-TBS hydroxylamine gave compounds 14–16.46 Reduction
of the nitro groups of these compounds using Pd/C under an
atmosphere of hydrogen yielded amines 17–19. Treatment of the
synthesized amines with freshly prepared 4-b-iodo-4’-desoxypod-
ophyllotoxin afforded 20–22. Finally, target molecules 23–25 were
obtained after removal of the protecting groups.
To investigate the effect of the zinc-binding group on biologi-
cal activity, we designed and synthesized a new class of dual
inhibitors using aromatic anilides as ZBGs. As shown in Scheme 3,
acids 11d and 12d were treated with BnBr in the presence of
Cs2CO3 to afford benzyl esters 26 and 27. Reduction of these
compounds using Fe/NH4Cl gave corresponding amines 28 and
29, which were treated with 4-b-iodo-40-desoxypodophyllotoxin
to afford compounds 30 and 31. Hydrogenation of these
compounds gave the corresponding acids, which were subjected
to amidation followed by acidification to afford target
compounds 32 and 33.
Herein, we report novel podophyllotoxin derivatives as dual
inhibitors of Topo II and HDAC (Fig. 4). The aromatic capping group
connection, linker length and zinc-binding group were systemati-
cally varied. Their biological evaluation included determination
of their inhibitory activities towards HDAC and Topo II and their
cytotoxicities in cancer cell lines.
2.2. Biological assays
2. Results and discussion
2.1. Chemistry
2.2.1. In vitro HDAC inhibition and cell growth inhibition
activities of PPT–SAHA hybrids
We first tested the HDAC inhibition activity of the PPT–SAHA
hybrids towards recombinant human HDAC-1, HDAC-3 and
HDAC-6 enzymes using SAHA as the positive control compound
(Table 1). Overall, the first generation of designed dual inhibitors
exhibited good inhibitory activity against a wide range of HDAC
subtypes. A similar trend has been observed, in which the HDAC
inhibition activity increased with increasing carbon chain length
(7c > 7b > 7a and 8c > 8b > 8a).47 The para- and meta-substituted
compounds showed similar anti-HDAC activities (7a vs 8a, 7b vs
8b and 7c vs 8c). Subsequently, we evaluated their antiprolifera-
tive activities towards the HCT-116 human colon cancer cell line
using the MTT assay. As shown in Table 1, most of the hybrids
exhibited powerful antiproliferative activity against the HCT116
cell line. Interestingly, all para-substituted compounds showed
stronger antiproliferative activities than the meta-substituted ana-
logs despite similar anti-HDAC activities. Among the hybrids
tested, compound 7c showed powerful HDAC inhibitory activity
The structure of podophyllotoxin was first elucidated in the
1930s and its structure–activity relationship (SAR) has been thor-
oughly studied.37–44 The biological activities of PPT derivatives
are affected by their configurations at the C-4 and C-40 positions.
In general, C-4-b-substituted 40-O-desmethyl-podophyllotoxins
show more potent biological activities. As a typical example, eto-
poside was approved by the FDA in 1983 and used in the chemo-
therapy of various cancers. By introducing a hydroxamic acid
segment at the C-4 position, we speculated that the hydrophilicity
of the parent agents could be maintained. Additionally, the podo-
phyllotoxin moiety was expected to enhance the affinity between
the cap and the HDAC enzymes. Based on the above theory, we de-
signed three series of PPT derivatives.
The general route used for the synthesis of the PPT–SAHA hy-
brids is depicted in Scheme 1. Acids 2a–c were easily synthesized
from anhydrides 1a–c. PPT derivatives 3 and 4 were prepared
according to Hu’s method.45 Subsequent amidation of amine 3 or
4 with the corresponding carboxylic acid afforded compounds
5a–c and 6a–c. Hydrogenation of these compounds using Pd/C
against HDAC-1 (IC50 = 0.21 lM), HDAC-3 (IC50 = 0.23 lM) and
HDAC-6 (IC50 = 0.09
lM) and the strongest antiproliferative activ-
ity (IC50 = 1.27 M).
l
Cap
Linker
ZBG
Cap
Linker
ZBG
O
N
H
N
OH
N
H
N
N
H
NH2
H
N
O
O
N
MGCD0103
SAHA
Figure 1. Representative structures of HDAC inhibitors.

X. Zhang et al. / Bioorg. Med. Chem. 21 (2013) 6981–6995
6983
O
O
O
O
O
S
O
HO
HO
OH
4
HO
O
HO
O
3
2
O
O
O
O
O
O
O
O
O
1
O
O
O
MeO
OMe
MeO
OMe
MeO
OMe
4'
OMe
OH
OH
Podophyllotoxin
Etoposide (VP-16)
Teniposide (VM-26)
NO2
O
O
O
HO
HO
O
HN
O
O
O
O
O
O
O
O
MeO
OMe
OH
MeO
OMe
OPO3H2
GL-331
Etoposide phosphate
Figure 2. Representative structures of Topo II inhibitors.
Topo II inhibitors
Topo II inhibitors
HAT
HDAC
compacted chromatin
disfavorite
relaxed chromatin
favorite
Figure 3. Synergistic antitumor effect of HDAC and Topo II inhibitors.
2.2.2. In vitro HDAC inhibition and cell growth inhibition
activities of the SAHA-like series
23e and 25e). The weaker observed anti-HDAC activities of the
ortho-substituted hybrids most likely caused by different spatial
orientation. In addition, a parallel trend was observed, in which
the HDAC inhibition activity increased with increasing carbon
chain length. The structure-activity relationship of the linker
length was consistent with what has been previously reported.48
Among this series of hybrids, compound 24d showed the most po-
tent anti-HDAC activity against HDAC-1 (IC50 = 11 nM), HDAC-3
(IC50 = 9.6 nM) and HDAC-6 (IC50 = 5.6 nM) and was 10- to 20 fold
more potent than the reference compound SAHA.
Evaluation of the antiproliferative activities of these compounds
was performed using the MTT assay against the HCT116 cancer cell
line. As shown in Table 2, the majority of the designed molecules
exhibited powerful antiproliferative activity against HCT116 cells
at a micromolar level. Interestingly, all para-substituted com-
pounds displayed potent antiproliferative activity at the same level
despite their different anti-HDAC activities (23a–e). In contrast,
While it is true that the amide linkage is suitable for SAHA, we
hypothesized that a more flexible connection type might increase
the affinities of the hybrids for the surfaces of HDAC enzymes.
Thus, we designed a new series of hybrids containing an ether link-
age.48,49 The anti-HDAC activities of these compounds were evalu-
ated (Table 2). Because of the different test batches, the HDAC
inhibition activity of SAHA slightly fluctuated (Table 2 vs Table 1).
However, most of the dual inhibitors exhibited good to excellent
inhibitory activity toward HDAC enzymes, which suggests that a
larger capping group such as PPT with a flexible connection may
promote the affinity between these compounds and the HDAC en-
zymes. Additionally, the aromatic capping group connection ap-
peared to contribute to the activity. The meta-substituted
compounds showed better activities when compared to the para-
and ortho-substituted analogs with similar linker lengths (24e vs

6984
X. Zhang et al. / Bioorg. Med. Chem. 21 (2013) 6981–6995
Cap
Linker
ZBG
H
N
H
N
OH
n
O
O
HN
O
O
O
1st series of dual inhibitors
PPT-SAHA hybrids
O
O
O
O
HO
O
HO
MeO
MeO
MeO
OMe
O
O
OH
O
O
H
N
O
OH
n
O
HN
MeO
OMe
O
O
OH
2nd series of dual inhibitors
SAHA-like series
O
O
VP-16
Linker
O
OMe
O
H
N
OH
OH
N
H
NH2
H
O
N
n
O
HN
Cap
ZBG
O
O
SAHA
3rd series of dual inhibitors
O
O
OMe
OH
Figure 4. Design of dual inhibitors of Topo II and HDAC.
n
O
HOOC
OBn
a
N
H
n
42-45%
O
O
O
2
2a
n = 2,
1
1a
n = 2,
n = 3, 2b
n = 4, 2c
n = 3, 1b
n = 4, 1c
H
N
H
N
H
N
H
N
NH2HCl
OBn
OH
n
O
O
n
O
O
HN
HN
HN
O
O
O
O
O
b
c
O
O
O
57-73%
28-52%
O
O
O
O
MeO
OMe
MeO
OMe
MeO
OMe
OH
OH
3: (p)
4: (m)
OH
7a-c
8a-c
5: (p)
6: (m)
7: (p)
8: (m)
5a-c
6a-c
Reagents and conditions: a) BnONH2, CH3CN; b) HATU, DIPEA, DCM, c)
Pd/C, H2, MeOH.
Scheme 1. Synthesis of PPT–SAHA hybrids.
ortho-substituted conjugates exhibited lower cytotoxicity due to
steric effects (25b vs 23b and 24b). The most powerful HDAC
inhibitor in this series, 24d, also showed the strongest antiprolifer-
ative activity (IC50 = 3.33 M).
l

X. Zhang et al. / Bioorg. Med. Chem. 21 (2013) 6981–6995
6985
O
O
O
O
a
c
b
OH
Br
Br
O2N
OH
O
n
37-90%
38-88%,
2 steps
n
n
n = 3, 9a
n = 4, 9b
n = 3, 10a
n = 4, 10b
10
9
11a-e
12a-e
13a-e
11: (p)
9c
n = 5,
10c
n = 5,
12: (m)
n = 6, 9d
n = 7, 9e
n = 6, 10d
n = 7, 10e
13: (
)
o
O
O
O
OTBS
O
OTBS
e
d
N
N
O2N
H2N
n
n
41-84%,
2 steps
Boc
Boc
14a-e
15a-e
16a-e
17a-e
18a-e
19a-e
17: (p)
18: (m)
19: (
14: (p)
15: (m)
16: (
)
o
)
o
O
O
O
OTBS
O
OH
N
N
H
n
n
Boc
HN
HN
f
O
O
O
O
O
28-67%
O
O
O
MeO
OMe
MeO
OMe
OH
OH
20 a-e
21a-e
22a-e
23a-e
24a-e
25a-e
23: (p)
20: (p)
24: (m)
21: (m)
25: ( )
o
22: (
)
o
Reagents and conditions: a) SOCl2, MeOH; b) nitrophenol, K2CO3, DMF,
80 oC; LiOH, MeOH, H2O; c) SOCl2, toluene, 60 °C; N-Boc-O-TBS
hydroxylamine, TEA, CH3CN, 0 °C; d) Pd/C, H2, MeOH; e) 4-β-iodo-4'-
desoxypodophyllotoxin, triethylamine, CH3CN; f) TFA, TBAF, DCM.
Scheme 2. Synthesis of the SAHA-like Topo II-HDAC dual inhibitor series.
2.2.3. In vitro HDAC inhibition and cell growth inhibition
activities of the aromatic anilide series
2.2.4. In vitro topoisomerase II relaxation assay
Using pBR322 DNA, we performed a cell-free DNA relaxation as-
say to determine the Topo II inhibition activities of the synthesized
compounds (see Fig. S1 of the Supporting information). Treatment
of pBR322 DNA with Topo II resulted in extensive DNA decatena-
tion. A reaction containing only pBR322 DNA was used as the cat-
enated control and reaction containing pBR322 DNA and Topo II
was used as the decatenated control. As expected, addition of
We speculated that the introduction of a PPT moiety as a cap-
ping group with an ether connection might facilitate the interac-
tions between the cap and the amino acid side chains at the
entrance of the HDAC active site. Thus, we investigated a new ser-
ies of dual inhibitors characterized by aromatic anilides as ZBGs.
When compared with compounds containing hydroxamic acid
moieties, compounds containing anilides as ZBGs always displayed
weaker in vitro HDAC inhibitory activity. However, we hypothe-
sized that introduction of the arylamine moiety might affect the
hydrophile–lipophile balance (HLB) of the dual inhibitors. We also
hypothesized that salification of the amine might increase the sol-
ubility of the hybrids. Compound 7c showed powerful antiprolifer-
ative activity towards HCT116 cells, but exhibited weaker
cytotoxicity in the A549 cell line (Table 3). In contrast, the newly
designed hybrids showed more potent antiproliferative activities
towards these cell lines. The stronger HDAC inhibitor, 32, appeared
to have better cytotoxicity than its meta-substituted analog. Com-
pound 32 showed more potent antiproliferative activity against
HCT116 and A549 cells than the reference compounds MGCD0103
and etoposide.
100
vere impairment of DNA decatenation. In contrast, the addition of
100 M SAHA or MGCD0103 had no effect on Topo II inhibitory
lM etoposide to the decatenation experiment resulted in a se-
l
activity. However, all selected hybrids showed obvious anti-Topo
II activity. The selected compounds are genuine HDAC-Topo II dual
inhibitors.
3. Conclusion
A novel class of PPT derivatives was designed based on the re-
ported synergistic effects of Topo II and HDAC inhibitors. The aro-
matic capping group connection, linker length and zinc-binding
group type were systematically varied. PPT–SAHA hybrids
exhibited potent HDAC inhibitory and antiproliferative activities.

6986
X. Zhang et al. / Bioorg. Med. Chem. 21 (2013) 6981–6995
b
a
O
OH
O
OBn
85%
99%
O2N
O2N
O
O
26 (p)
27 (m)
11d
12d
O
OBn
HN
O
O
O
O
c
O
COOBn
60-70%
O
H2N
MeO
OMe
OH
30 (p)
31 (m)
28 (p)
29 (m)
NH2
H
N
O
HN
O
O
O
d
O
35-41%
O
32 (p)
33 (m)
MeO
OMe
OH
Reagents and conditions: a) BnBr, Cs2CO3, DMF; b) Fe, NH4Cl, EtOH,
H2O, room temperature; c) 4-β-iodo-4'-desoxypodophyllotoxin, triethylamine,
CH3CN; d) Pd(OH)2, H2, MeOH, EtOAc; benzene-1,2-diamine, HATU,
DIPEA, DCM.
Scheme 3. Synthesis of the third generation of Topo II–HDAC dual inhibitors.
Table 1
Table 2
In vitro HDAC inhibition and cell viability assay of PPT–SAHA hybrids
In vitro HDAC inhibition cell viability assay of the SAHA-like series
Compound
IC50
HDAC1
(
l
M) SD
Compound
IC50 (nM) SD
HDAC-1
IC50
(
l
M)
HDAC3
HDAC6
HCT116a
a
HDAC-3
HDAC-6
HCT116
SAHA
7a
7b
7c
8a
0.09 0.01
2.83 0.57
0.59 0.00
0.21 0.02
1.43 0.46
0.38 0.04
0.10 0.00
0.11 0.01
1.69 0.53
0.52 0.04
0.23 0.02
2.11 0.01
0.59 0.06
0.13 0.00
0.07 0.00
1.06 0.10
0.13 0.01
0.09 0.02
0.52 0.08
0.16 0.03
0.04 0.00
0.80
3.76
2.94
SAHA
23a
24a
25a
23b
24b
25b
23c
24c
25c
23d
24d
25d
23e
24e
25e
221 23
243 44
131 15
193 35
767 194
1444 424
242 35
111 21
899 208
69 16
129 26
214 34
405 68
357 49
0.81
5.37
29.45
28.88
5.26
6.29
16.46
4.92
5.72
8.10
5.03
3.33
385
135
1.27
578 208
125 31
93 31
369 119
207 73
14.14
19.62
8.57
88
42
8
3
8b
8c
84 10
a
26
15
20
2
2
2
2
Each value is the result of three separate experiments.
45
8
81
188 41
49
6
145 35
29
11
3
3
5
16.0
9.6 3.2
155 23
5.6 0.0
Further optimization of this series resulted in the PPT–SAHA-like
hybrid series, which had remarkably improved anti-HDAC activity.
Introduction of the PPT moiety as a capping group might facilitate
interactions between the cap and the surfaces of HDAC enzymes.
The optimum linker length might promote binding of the hydroxa-
mic acid moiety with the zinc ion at the bottom of the active site.
In this series of hybrids, compound 24d displayed the best HDAC
inhibitory activity against HDAC-1 (IC50 = 11 nM), HDAC-3
(IC50 = 9.6 nM) and HDAC-6 (IC50 = 5.6 nM) and was 10- to 20 fold
more potent than the reference compound SAHA. Compound 24d
also displayed powerful antiproliferative activity towards the
72 15
87 20
27
53
69
2
8
9
11.34
3.72
5.19
53
69
8
9
39
8
175 32
217 40
217 40
11.35
bIC50 > 20
lg/mL.
a
Each value is the result of three separate experiments.
presented here suggest the potential for further optimization of
this series of compounds.
HCT116 cell line at micromolar concentrations (IC50 = 3.33
l
M).
4. Experimentals
4.1. Chemistry
In addition, the zinc-binding group was found to play a significant
role in the cytotoxicity. The introduction of anilides as ZBGs re-
sulted in increased antiproliferative activity against HCT116 and
A549 cells. Compound 32 showed the most powerful cytotoxicity
against these two cancer cell lines. The encouraging results
Melting points were taken on a Fisher–Johns melting point
apparatus, uncorrected and reported in degrees Centigrade. 1H

X. Zhang et al. / Bioorg. Med. Chem. 21 (2013) 6981–6995
6987
(m, 4H); HRMS (ESI): m/z calcd for C40H41N3O10Na+ (M+Na+):
746.2684, found: 746.2696.
Table 3
In vitro HDAC inhibition and cell viability assay
Compound
IC50 (lM) SD
4.1.2.2. Compound 5b.
Starting from 2b and 3, 58% of 5b was
HDAC1
HDAC2
HDAC3
HCT116a A549a
obtained as a yellow oil according to above-mentioned general
procedure. 1H NMR (500 MHz, CDCl3) d 8.66 (br s, 1H), 7.57 (br s,
1H), 7.38–7.36 (m, 7H), 6.74 (s, 1H), 6.52 (s, 1H), 6.48–6.46 (m,
2H), 6.34 (s, 2H), 5.96 (d, J = 9.8 Hz, 2H), 5.50 (s, 1H), 4.94–4.87
(m, 2H), 4.61–4.57 (m, 2H), 4.34–4.31 (m, 1H), 3.96–3.84 (m,
2H), 3.80 (s, 6H), 3.17–3.13 (m, 1H), 2.99–2.95 (m, 1H), 2.35–2.30
(m, 2H), 2.10–2.04 (m, 2H), 1.70–1.65(m, 4H), 1.39–1.34 (m, 2H);
HRMS (ESI): m/z calcd for C41H43N3O10Na+ (M+Na+): 760.2841,
found: 760.2835.
MGCD0103
Etoposide
7c
32
33
0.95 0.16
—
0.21 0.02
0.28 0.02
—
0.14 0.01
1.67 0.01 1.57
1.65
1.71
18.64
1.16
b
—
0.87
0.23 0.02 1.27
14.08 5.41 10.39 1.51 11.62 0.67 0.52
53.52 6.56 0.92
—
—
1.11
a
Each value is the result of three separate experiments.
IC50 > 20 lg/mL.
b
NMR spectra and 13C NMR were recorded in CDCl3, CD3OD and ace-
tone-d6 on a Bruker DRX-500 (500 MHz) or a Bruker DRX-400
(400 MHz) using TMS as internal standard. Chemical shifts were
reported as a d (ppm) and spin–spin coupling constants as J (Hz)
values. The mass spectra (MS) were recorded on a Finnigan MAT-
95 mass spectrometer. The purity of all tested compounds was
established by HPLC to be >95.0%. HPLC analyses were performed
on Agilent 1200 series instrument using an Agilent Eclipse XDB-
C18 (250 ꢀ 4.6 mm).
4.1.2.3. Compound 5c.
Starting from 2c and 3, 61% of 2c was
obtained as a yellow oil according to above-mentioned general
procedure. 1H NMR (300 MHz, CDCl3) d 8.65 (br s, 1H), 7.58 (br s,
1H), 7.40–7.32 (m, 7H), 6.74 (s, 1H), 6.52–6.45 (m, 3H), 6.33 (s,
2H), 5.96 (d, J = 9.6 Hz, 2H), 5.45 (s, 1H), 4.92–4.87 (m, 2H), 4.62–
4.57 (m, 2H), 4.33 (t, J = 8.1 Hz, 1H), 3.95 (t, J = 9.3 Hz, 1H), 3.78
(s, 6H), 3.15 (dd, J = 14.1, 4.8 Hz, 1H), 3.02–2.96 (m, 1H), 2.32–
2.27 (m, 2H), 2.08–2.00 (m, 2H), 1.58–1.59 (m, 4H), 1.34–1.24
(m, 4H); HRMS (ESI): m/z calcd for C42H45N3O10Na+ (M+Na+):
774.2997, found: 774.3005.
4.1.1. Representative procedure for 2a–c
Anhydride 2a–c (1.0 equiv) and BnONH2 (1.0 equiv) were stir-
red in CH3CN for 12 h. The mixture was extracted with EtOAc,
washed with brine and concentrated to dryness. The crude product
was crystallized in a mixture of EtOAc and petroleum ether.
4.1.2.4. Compound 6a.
Starting from 2a and 4, 70% of 6a was
obtained as a brown oil according to above-mentioned general pro-
cedure. 1H NMR (500 MHz, CDCl3) d 8.64 (br s, 1H), 7.92(br s, 1H),
7.35–7.29 (m, 6H), 7.09 (t, J = 8.1 Hz, 1H), 6.74 (s, 1H), 6.65–6.63
(m, 1H), 6.50 (s, 1H), 6.32 (s, 2H), 6.29–6.27 (m, 1H), 5.95 (d,
J = 10.5 Hz, 2H), 5.49 (s, 1H), 4.87–4.80 (m, 2H), 4.69–4.68 (m,
1H), 4.57 (d, J = 4.9 Hz, 1H), 4.44 (t, J = 8.3 Hz, 1H), 3.98–3.94 (m,
2H), 3.78(s, 6H), 3.14–3.10(m, 1H), 3.02–2.95(m, 1H), 2.35–2.30
(m, 2H), 2.10–2.04 (m, 2H), 1.70–1.65(m, 4H); HRMS (ESI): m/z
calcd for C40H41N3O10Na+ (M+Na+): 746.2684, found: 746.2681.
4.1.1.1. 6-((Benzyloxy)amino)-6-oxohexanoic acid (2a).
Start-
ing from 1a, 52% of 2a was obtained as a white solid according to
above-mentioned general procedure. mp 76–78 °C, 1H NMR
(400 MHz, CDCl3) d 8.20 (br s, 1H), 7.46–7.31 (m, 5H), 5.00–4.73
(m, 2H), 2.43–1.97 (m, 4H), 1.75–1.56 (m, 4H); HRMS (ESI): m/z
calcd for C13H17NO4Na+ (M+Na+): 274.1055, found: 274.1042.
4.1.1.2. 7-((Benzyloxy)amino)-7-oxoheptanoic acid (2b).
Start-
4.1.2.5. Compound 6b.
Starting from 2b and 4, 57% of 6b was
ing from 1b, 45% of 2b was obtained as a white solid according to
above-mentioned general procedure. mp 61–63 °C, 1H NMR
(400 MHz, CDCl3) d 8.07 (br s, 1H), 7.49–7.30 (m, 5H), 5.08–4.69
(m, 2H), 2.44–1.94 (m, 4H), 1.75–1.52 (m, 4H), 1.43–1.24 (m,
2H); HRMS (ESI): m/z calcd for C14H19NO4Na+ (M+Na+): 288.1212,
found: 288.1204.
obtained as a yellow oil according to above-mentioned general
procedure. 1H NMR (500 MHz, CDCl3) d 8.67 (br s, 1H), 7.86(br s,
1H), 7.39–7.32 (m, 6H), 7.12 (t, J = 8.0 Hz, 1H), 6.78 (s, 1H), 6.66–
6.65 (m, 1H), 6.55 (s, 1H), 6.38 (s, 2H), 6.33 (d, J = 7.6 Hz, 1H),
5.99 (d, J = 11.1 Hz, 2H), 5.56 (m, 1H), 4.93–4.87 (m, 2H), 4.75–
4.70 (m, 1H), 4.62 (d, J = 4.9 Hz, 1H), 4.50–4.46 (m, 1H), 4.02–
3.97 (m, 2H), 3.83 (s, 6H), 3.18–3.03 (m, 2H), 2.40–2.32 (m, 2H),
2.13–2.07 (m, 2H), 1.71–1.65 (m, 4H), 1.41–1.37 (m, 2H); HRMS
(ESI): m/z calcd for C41H43N3O10Na+ (M+Na+): 760.2841, found:
760.2820.
4.1.1.3. 8-((Benzyloxy)amino)-8-oxooctanoic acid (2c).
Start-
ing from 1c, 42% of 2c was obtained as a white solid according to
above-mentioned general procedure. mp 73–76 °C, 1H NMR
(400 MHz, CDCl3) d 7.91 (br s, 1H), 7.47–7.31 (m, 5H), 5.27–4.64
(m, 2H), 2.47–1.95 (m, 4H), 1.70–1.54 (m, 4H), 1.42–1.21 (m, 4H);
HRMS (ESI): m/z calcd for C15H21NO4Na+ (M+Na+): 302.1368, found:
302.1354.
4.1.2.6. Compound 6c.
Starting from 2c and 4, 73% of 6c was
obtained as a yellow oil according to above-mentioned general
procedure. 1H NMR (500 MHz, CDCl3) d 8.56 (br s, 1H), 7.78 (br s,
1H), 7.38–7.32 (m, 6H), 7.08 (t, J = 7.0 Hz, 1H), 6.74 (s, 1H), 6.58–
6.56 (m, 1H), 6.50 (s, 1H), 6.32 (s, 2H), 6.28–6.27 (d, J = 6.6 Hz,
1H), 5.96 (d, J = 10.6 Hz, 2H), 5.50 (s, 1H), 4.92–4.85 (m, 2H),
4.71–4.67 (m, 1H), 4.57 (d, J = 4.9 Hz, 1H), 4.43 (t, J = 7.9 Hz, 1H),
3.97–3.93 (m, 2H), 3.78 (s, 6H), 3.13–3.09 (m, 1H), 3.05–3.00 (m,
1H), 2.31–2.27 (m, 2H), 2.04–2.00 (m, 2H), 1.68–1.64 (m, 2H),
1.62–1.58 (m, 2H), 1.33–1.29 (m, 4H); HRMS (ESI): m/z calcd for
4.1.2. Representative procedure for 5a–c and 6a–c
A
mixture of amine (1.0 equiv), acid (1.0 equiv), HATU
(1.0 equiv) and DIPEA (4.0 equiv) were stirred in DCM for 12 h
and then extracted with DCM. Concentration in vacuo gave crude
product, which was further purified by column chromatography.
C
42H45N3O10Na+ (M+Na+): 774.2997, found: 774.3009.
4.1.2.1. Compound 5a.
Starting from 2a and 3, 65% of 5a was
obtained as a yellow oil according to above-mentioned general
procedure. 1H NMR (500 MHz, CDCl3) d 8.73 (br s, 1H), 7.66 (br s,
1H), 7.41–7.37 (m, 7H), 6.75 (s, 1H), 6.53–6.49 (m, 3H), 6.34 (s,
2H), 5.97 (d, J = 9.2 Hz, 2H), 5.49 (s, 1H), 4.92–4.89 (m, 2H),
4.65–4.58 (m, 2H), 4.35 (t, J = 8.3 Hz, 1H), 3.97(t, J = 9.5 Hz, 1H),
3.85–3.83 (m, 1H), 3.80 (s, 6H), 3.15 (dd, J = 19.0, 5.0 Hz, 1H),
3.02–2.98 (m, 1H), 2.36–2.32 (m, 2H), 2.13–2.09 (m, 2H), 1.72–1.68
4.1.3. Representative procedure for 7a–c and 8a–c
A mixture of compound 5a–c/6a–c and 10% Pd/C (10% w/w) in
methanol was hydrogenated at room temperature for 10 h. After
filtration, the organic layer was evaporated to dryness to give the
crude product, which was further purified by column
chromatography.

6988
X. Zhang et al. / Bioorg. Med. Chem. 21 (2013) 6981–6995
4.1.3.1. Compound 7a.
Starting from 5a, 43% of 7a was ob-
(m, 4H); HPLC: room temperature; eluent, CH3OH/H2O (80:20);
tained as a yellow foam according to above-mentioned general
procedure. 1H NMR (500 MHz, CD3OD) d 7.28 (d, J = 8.6 Hz, 2H),
6.74 (s, 1H), 6.62 (d, J = 8.7 Hz, 2H), 6.47 (s, 1H), 6.36 (s, 2H), 5.91
(s, 2H), 5.48 (s, 1H), 4.79–4.76 (m, 1H), 4.57–4.53 (m, 1H), 4.38
(t, J = 7.8 Hz, 1H), 3.93–3.89 (m, 1H), 3.73 (s, 6H), 3.30–3.26 (m,
1H), 3.08–3.04 (m, 1H), 2.36–2.32 (m, 2H), 2.16–2.12 (m, 2H),
1.71–1.67 (m, 4H); HPLC: room temperature; eluent, CH3OH/H2O
(80:20); flow rate, 1.0 mL/min; tR = 3.60 min, UV254 = 98.3%; HRMS
(ESI): m/z calcd for C33H35N3O10Na+ (M+Na+): 656.2215, found:
656.2227.
flow rate, 1.0 mL/min; tR = 5.43 min, UV254 = 99.7%; HRMS (ESI):
m/z calcd for
684.2543.
C
35H39N3O10Na+ (M+Na+): 684.2528, found:
4.1.4. Representative procedure for 10a–e
SOCl2 (15.0 mmol) was added dropwise into a solution of bromo
carboxylic acid (10.0 mmol) in methanol (30 mL). The mixture was
stirred at room temperature for 2 h. The solution was concentrated
to give the crude product, which was used directly for the next
step.
4.1.3.2. Compound 7b.
Starting from 5b, 37% of 7b was ob-
4.1.5. Representative procedure for 11–13
tained as a yellow foam according to above-mentioned general
procedure. 1H NMR (500 MHz, CD3OD) d 7.29 (d, J = 8.8 Hz, 2H),
6.74 (s, 1H), 6.63 (d, J = 8.8 Hz, 2H), 6.47 (s, 1H), 6.36 (s, 2H), 5.91
(s, 2H), 5.48 (s, 1H), 4.78–4.76 (m, 1H), 4.56–4.53 (m, 1H), 4.42–
4.37 (m, 1H), 3.93–3.88 (m, 1H), 3.73 (s, 6H), 3.30–3.26 (m, 1H),
3.05–3.00 (m, 1H), 2.33 (t, J = 7.4 Hz, 2H), 2.11 (t, J = 7.4 Hz, 2H),
1.73–1.64 (m, 4H), 1.42–1.35 (m, 2H); HPLC: room temperature;
eluent, CH3OH/H2O (80:20); flow rate, 1.0 mL/min; tR = 3.85 min,
UV254 = 99.6%; HRMS (ESI): m/z calcd for C34H37N3O10Na+
(M+Na+): 670.2371, found: 670.2382.
A mixture of bromo ester (10.0 mmol), nitrophenol (9.1 mmol)
and K2CO3 (11.0 mmol) in DMF (30 mL) was stirred at 90 °C for
16 h. The reaction mixture was extracted with EtOAc, and the or-
ganic layer was washed with brine and dried over anhydrous
Na2SO4. Concentration in vacuo gave crude product, which was
crystallized from DCM and diethyl ether. After filtration, the filter
cake was dissolved in a mixed solvent of THF and water followed
by addition of NaOH (600 mg, 15.0 mmol). The mixture was re-
fluxed for 2 h and acidified with 1 N aqueous HCl till pH 2.0. Then
it was extracted with EtOAc, washed with brine, dried over anhy-
drous Na2SO4 and evaporated to dryness.
4.1.3.3. Compound 7c.
Starting from 5c, 52% of 7c was ob-
tained as a yellow foam according to above-mentioned general
procedure. 1H NMR (300 MHz, CD3OD) d 7.29 (d, J = 8.4 Hz, 2H),
6.74 (s, 1H), 6.62 (d, J = 8.4 Hz, 2H), 6.47(s, 1H), 6.36 (s, 2H),
5.91(s, 2H), 4.78–4.76 (m, 1H), 4.57–4.53 (m, 1H), 4.40–4.35 (m,
1H), 3.94–3.88 (m, 1H), 3.73 (s, 6H), 3.30–3.27 (m, 1H), 3.08–3.04
(m, 1H), 2.35–2.30 (m, 2H), 2.12–2.07 (m, 2H), 1.75–1.55(m, 4H),
1.48–1.38 (m, 4H); HPLC: room temperature; eluent, CH3OH/H2O
(80:20); flow rate, 1.0 mL/min; tR = 3.91 min, UV254 = 99.8%; HRMS
(ESI): m/z calcd for C35H39N3O10Na+ (M+Na+): 684.2528, found:
684.2515.
4.1.5.1. 4-(4-Nitrophenoxy)butanoic acid (11a).
Starting
from 10a and 4–nitrophenol, 60% of 11a was obtained as a white
solid according to above-mentioned general procedure. mp 122–
124 °C.
4.1.5.2. 5–(4-Nitrophenoxy)pentanoic acid (11b).
Starting
from 10b and 4-nitrophenol, 88% of 11b was obtained as a white
solid according to above-mentioned general procedure. mp 100–
101 °C.
4.1.5.3. 6-(4-Nitrophenoxy)hexanoic acid (11c).
Starting
4.1.3.4. Compound 8a.
Starting from 6a, 35% of 8a was ob-
from 10c and 4-nitrophenol, 47% of 11c was obtained as a white
solid according to above-mentioned general procedure. mp 103–
104 °C.
tained as a yellow foam according to above-mentioned general
procedure. 1H NMR (500 MHz, CD3OD) d 7.12–7.07 (m, 2H), 6.79
(s, 1H), 6.73–6.67 (m, 1H), 6.51–6.45 (m, 2H), 6.39 (s, 2H), 5.95
(m, 2H), 4.82–4.80 (m, 1H), 4.61–4.57 (m, 1H), 4.47–4.44 (m,
1H), 3.97–3.92 (m, 1H), 3.77 (s, 6H), 3.30–3.27 (m, 1H), 3.08–3.05
(m, 1H), 2.42–2.38 (m, 2H), 2.19–2.15 (m, 2H), 1.77–1.70 (m,
4H); HPLC: room temperature; eluent, CH3OH/H2O (80:20); flow
rate, 1.0 mL/min; tR = 4.43 min, UV254 = 98.9%; HRMS (ESI): m/z
calcd for C33H35N3O10Na+ (M+Na+): 656.2215, found: 656.2223.
4.1.5.4. 7-(4-Nitrophenoxy)heptanoic acid (11d).
Starting
from 10d and 4-nitrophenol, 50% of 11d was obtained as a yellow
solid according to above-mentioned general procedure. mp 84–
86 °C, 1H NMR (400 MHz, CDCl3) d 8.19 (d, J = 8.1 Hz, 2H), 6.94
(d, J = 8.2 Hz, 2H), 4.05 (t, J = 6.2 Hz, 2H), 2.38 (t, J = 7.2 Hz, 2H),
1.88–1.77 (m, 2H), 1.74–1.64 (m, 2H), 1.56–1.36 (m, 4H); MS
(ESI) m/z = 266.0 (MꢁH)ꢁ.
4.1.3.5. Compound 8b.
Starting from 6b, 43% of 8b was ob-
tained as a yellow foam according to above-mentioned general
procedure. 1H NMR (500 MHz, CD3OD) d 7.08–7.04 (m, 2H), 6.75
(s, 1H), 6.68 (d, J = 7.5 Hz, 1H), 6.47 (s, 1H), 6.43 (d, J = 7.9 Hz,
1H), 6.36 (s, 2H), 5.91(s, 2H), 4.78–4.76 (m, 1H), 4.57–4.55 (m,
1H), 4.43–4.41 (m, 1H), 3.91–3.87 (m, 1H), 3.73 (s, 6H), 3.30–
3.27 (m, 1H), 3.08–3.00 (m, 1H), 2.36–2.32 (m, 2H), 2.12–2.08
(m, 2H), 1.71–1.62 (m, 4H), 1.41–1.36 (m, 2H); HPLC: room tem-
perature; eluent, CH3OH/H2O (80:20); flow rate, 1.0 mL/min;
tR = 4.72 min, UV254 = 99.5%; HRMS (ESI): m/z calcd for C34H37N3-
O10Na+ (M+Na+): 670.2371, found: 670.2386.
4.1.5.5. 8-(4-Nitrophenoxy)octanoic acid (11e).
Starting
from 10e and 4-nitrophenol, 50% of 11e was obtained as a white
solid according to above-mentioned general procedure. mp 94–
95 °C, 1H NMR (400 MHz, CDCl3) d 8.19 (d, J = 8.3 Hz, 2H), 6.94
(d, J = 8.3 Hz, 2H), 4.04 (t, J = 6.3 Hz, 2H), 2.37 (t, J = 7.3 Hz, 2H),
1.91–1.76 (m, 2H), 1.73–1.60 (m, 2H), 1.56–1.30 (m, 6H); MS
(ESI) m/z = 280.1 (MꢁH)ꢁ.
4.1.5.6. 4-(3-Nitrophenoxy)butanoic acid (12a).
Starting
from 10a and 3-nitrophenol, 65% of 12a was obtained as a yellow
solid according to above-mentioned general procedure. mp 114–
115 °C.
4.1.3.6. Compound 8c.
Starting from 6c, 28% of 8c was ob-
tained as a yellow foam according to above-mentioned general
procedure. 1H NMR (500 MHz, CD3OD) d 7.13–7.07 (m, 2H), 6.78–
6.71 (m, 2H), 6.50–6.44 (m, 2H), 6.39 (s, 2H), 5.94 (s, 2H), 4.81–
4.79 (m, 1H), 4.59–4.56 (m, 1H), 4.46–4.42 (m, 1H), 3.96–3.92
(m, 1H), 3.76 (s, 6H), 3.30–3.27 (m, 1H), 3.10–3.07 (m, 1H), 2.39–
2.35 (m, 2H), 2.14–2.10 (m, 2H), 1.75–1.62 (m, 4H), 1.43–1.38
4.1.5.7. 5-(3-Nitrophenoxy)pentanoic acid (12b).
Starting
from 10b and 3-nitrophenol, 67% of 12b was obtained as a yellow
solid according to above-mentioned general procedure. mp 77–
78 °C.

X. Zhang et al. / Bioorg. Med. Chem. 21 (2013) 6981–6995
6989
4.1.5.8. 6-(3-Nitrophenoxy)hexanoic acid (12c).
Starting
crude product, which was further purified by column chromatog-
raphy (EtOAc/petroleum ether = 1:10).
from 10c and 3-nitrophenol, 79% of 12c was obtained as a yellow
solid according to above-mentioned general procedure. mp 75–
78 °C, 1H NMR (400 MHz, CDCl3) d 7.81 (d, J = 8.0 Hz, 1H), 7.71 (s,
1H), 7.41 (t, J = 8.1 Hz, 1H), 7.21 (d, J = 8.2 Hz, 1H), 4.04 (t,
J = 6.1 Hz, 2H), 2.42 (t, J = 7.2 Hz, 2H), 1.95–1.80 (m, 2H), 1.81–
1.67 (m, 2H), 1.64–1.47 (m, 2H); MS (ESI) m/z = 252.0 (MꢁH)ꢁ.
4.1.6.1. tert-Butyl (tert-butyldimethylsilyl)oxy(4-(4-nitrophen-
oxy)butanoyl)carbamate (14a).
Starting from 11a, 45% of
14a was obtained as a yellow solid according to above-mentioned
general procedure. mp 45–48 °C, 1H NMR (400 MHz, CDCl3) d 8.19
(d, J = 8.5 Hz, 2H), 6.94 (d, J = 8.6 Hz, 2H), 4.13 (t, J = 6.1 Hz, 2H),
3.01 (t, J = 6.9 Hz, 2H), 2.19 (p, J = 6.3 Hz, 2H), 1.54 (s, 9H), 1.00
(s, 9H), 0.14 (s, 6H); MS (ESI) m/z = 477.1 (M+Na+).
4.1.5.9. 7-(3-Nitrophenoxy)heptanoic acid (12d).
Starting
from 10d and 3-nitrophenol, 60% of 12d was obtained as a yellow
solid according to above-mentioned general procedure. mp 65–
68 °C, 1H NMR (400 MHz, CDCl3) d 7.80 (d, J = 8.1 Hz, 1H), 7.71 (s,
1H), 7.41 (t, J = 8.2 Hz, 1H), 7.21 (d, J = 8.2 Hz, 1H), 4.03 (t,
J = 6.2 Hz, 2H), 2.39 (t, J = 7.3 Hz, 2H), 1.91–1.78 (m, 2H), 1.77–
1.62 (m, 2H), 1.59–1.35 (m, 4H); MS (ESI) m/z = 266.0 (MꢁH)ꢁ.
4.1.6.2. tert-Butyl (tert-butyldimethylsilyl)oxy(5-(4-nitrophen-
oxy)pentanoyl)carbamate (14b).
Starting from 11b, 47% of
14b was obtained as a yellow solid according to above-mentioned
general procedure. mp 87–90 °C, 1H NMR (400 MHz, CDCl3) d 8.19
(d, J = 8.4 Hz, 2H), 6.93 (d, J = 8.4 Hz, 2H), 4.14–4.03 (m, 2H), 2.89 (t,
J = 6.2 Hz, 2H), 2.04–1.73 (m, 4H), 1.55 (s, 9H), 1.00 (s, 9H), 0.15 (s,
6H); MS (ESI) m/z = 491.1 (M+Na+).
4.1.5.10. 8-(3-Nitrophenoxy)octanoic acid (12e).
Starting
from 10e and 3-nitrophenol, 64% of 12e was obtained as a white
solid according to above-mentioned general procedure. mp 86–
88 °C, 1H NMR (400 MHz, CDCl3) d 7.80 (d, J = 8.1 Hz, 1H), 7.71 (s,
1H), 7.41 (t, J = 8.1 Hz, 1H), 7.21 (d, J = 8.2 Hz, 1H), 4.03 (t,
J = 6.2 Hz, 2H), 2.37 (t, J = 7.3 Hz, 2H), 1.88–1.77 (m, 2H), 1.73–
1.61 (m, 2H), 1.56–1.35 (m, 6H); MS (ESI) m/z = 280.0 (MꢁH)ꢁ.
4.1.6.3. tert-Butyl (tert-butyldimethylsilyl)oxy(6-(4-nitrophen-
oxy)hexanoyl)carbamate (14c).
Starting from 11c, 69% of
14c was obtained as a yellow solid according to above-mentioned
general procedure. mp 36–38 °C, 1H NMR (400 MHz, CDCl3) d 8.19
(d, J = 7.9 Hz, 2H), 6.93 (d, J = 8.0 Hz, 2H), 4.05 (t, J = 6.2 Hz, 2H),
2.84 (t, J = 7.2 Hz, 2H), 1.95–1.66 (m, 4H), 1.56–1.53 (m, 11H),
1.00 (s, 9H), 0.15 (s, 6H); MS (ESI) m/z = 505.1 (M+Na+).
4.1.5.11. 4-(2-Nitrophenoxy)butanoic acid (13a).
Starting
from 10a and 2-nitrophenol, 58% of 13a was obtained as a white
solid according to above-mentioned general procedure. mp 103–
104 °C.
4.1.6.4. tert-Butyl (tert-butyldimethylsilyl)oxy(7-(4-nitrophen-
oxy)heptanoyl)carbamate (14d).
Starting from 11d, 47% of
14d was obtained as a yellow solid according to above-mentioned
general procedure. mp 55–57 °C, 1H NMR (400 MHz, CDCl3) d 8.19
(d, J = 8.2 Hz, 2H), 6.93 (d, J = 8.3 Hz, 2H), 4.04 (t, J = 6.4 Hz, 2H),
2.81 (t, J = 7.2 Hz, 2H), 1.91–1.65 (m, 4H), 1.55 (s, 9H), 1.52–1.38
(m, 4H), 1.00 (s, 9H), 0.15 (s, 6H); MS (ESI) m/z = 519.1 (M+Na+).
4.1.5.12. 5-(2-Nitrophenoxy)pentanoic acid (13b).
Starting
from 10b and 2-nitrophenol, 69% of 13b was obtained as a white
solid according to above-mentioned general procedure. mp 80–
81 °C.
4.1.5.13. 6-(2-Nitrophenoxy)hexanoic acid (13c).
Starting
4.1.6.5. tert-Butyl (tert-butyldimethylsilyl)oxy(8-(4-nitrophen-
from 10c and 2-nitrophenol, 77% of 13c was obtained as a white
solid according to above-mentioned general procedure. mp 75–
76 °C, 1H NMR (400 MHz, CDCl3) d 7.81 (d, J = 8.0 Hz, 1H), 7.50 (t,
J = 7.9 Hz, 1H), 7.11–6.96 (m, 2H), 4.11 (t, J = 6.1 Hz, 2H), 2.41 (t,
J = 7.3 Hz, 2H), 1.94–1.81 (m, 2H), 1.81–1.66 (m, 2H), 1.64–1.50
(m, 2H); MS (ESI) m/z = 252.0 (MꢁH)ꢁ.
oxy)octanoyl)carbamate (14e).
Starting from 11e, 90% of 14e
was obtained as a yellow solid according to above-mentioned gen-
eral procedure. mp 54–55 °C, 1H NMR (400 MHz, CDCl3) d 8.19 (d,
J = 8.6 Hz, 2H), 6.93 (d, J = 8.6 Hz, 2H), 4.03 (t, J = 6.4 Hz, 2H), 2.79
(t, J = 7.3 Hz, 2H), 1.86–1.76 (m, 2H), 1.71–1.63 (m, 2H), 1.54 (s,
9H), 1.50–1.36 (m, 6H), 0.99 (s, 9H), 0.14 (s, 6H); MS (ESI) m/
z = 533.2 (M+Na+).
4.1.5.14. 7-(2-Nitrophenoxy)heptanoic acid (13d).
Starting
from 10d and 2-nitrophenol, 38% of 13d was obtained as a white
solid according to above-mentioned general procedure. mp 46–
48 °C, 1H NMR (400 MHz, CDCl3) d 7.81 (d, J = 8.0 Hz, 1H), 7.50 (t,
J = 7.8 Hz, 1H), 7.12–6.96 (m, 2H), 4.10 (t, J = 6.0 Hz, 2H), 2.38 (t,
J = 7.2 Hz, 2H), 1.90–1.77 (m, 2H), 1.74–1.61 (m, 2H), 1.61–1.38
(m, 4H); MS (ESI) m/z = 266.0 (MꢁH)ꢁ.
4.1.6.6. tert-Butyl (tert-butyldimethylsilyl)oxy(4-(3-nitrophen-
oxy)butanoyl)carbamate (15a).
Starting from 12a, 49% of
15a was obtained as a yellow solid according to above-mentioned
general procedure. mp 44–47 °C, 1H NMR (400 MHz, CDCl3) d 7.80
(d, J = 8.0 Hz, 1H), 7.72 (s, 1H), 7.41 (t, J = 8.1 Hz, 1H), 7.21 (d,
J = 8.3 Hz, 1H), 4.11 (t, J = 6.0 Hz, 2H), 3.02 (t, J = 7.0 Hz, 2H), 2.19
(p, J = 6.3 Hz, 2H), 1.55 (s, 9H), 1.00 (s, 9H), 0.15 (s, 6H); MS (ESI)
m/z = 477.1 (M+Na+).
4.1.5.15. 8-(2-Nitrophenoxy)octanoic acid (13e).
Starting
from 10e and 2-nitrophenol, 71% of 13e was obtained as a white
solid according to above-mentioned general procedure. mp 87–
88 °C, 1H NMR (400 MHz, CDCl3) d 7.81 (d, J = 8.1 Hz, 1H), 7.50 (t,
J = 7.9 Hz, 1H), 7.06 (d, J = 8.4 Hz, 1H), 7.00 (t, J = 7.6 Hz, 1H), 4.09
(t, J = 6.1 Hz, 2H), 2.36 (t, J = 7.3 Hz, 2H), 1.91–1.76 (m, 2H), 1.73–
1.59 (m, 2H), 1.56–1.30 (m, 6H); MS (ESI) m/z = 279.8 (MꢁH)ꢁ.
4.1.6.7. tert-Butyl (tert-butyldimethylsilyl)oxy(5-(3-nitrophen-
oxy)pentanoyl)carbamate (15b).
Starting from 12b, 43% of
15b was obtained as a yellow solid according to above-mentioned
general procedure. mp 56–57 °C, 1H NMR (400 MHz, CDCl3) d 7.80
(d, J = 8.1 Hz, 1H), 7.71 (s, 1H), 7.41 (t, J = 8.2 Hz, 1H), 7.20 (d,
J = 8.3 Hz, 1H), 4.12–4.01 (m, 2H), 2.94–2.80 (m, 2H), 1.96–1.80
(m, 4H), 1.55 (s, 9H), 1.00 (s, 9H), 0.15 (s, 6H); MS (ESI) m/
z = 491.1 (M+Na+).
4.1.6. Representative procedure for 14–16
Nitro acid (1.0 equiv) in toluene was treated with SOCl2
(3.0 equiv), the mixture was stirred at 60 °C for 2 h. After removal
of solvent by evaporation, the residue was dissolved in CH3CN and
added dropwise into a solution of triethylamine (6.0 equiv), DMAP
(0.1 equiv) and N-BOC-O-TBS hydroxylamine (2.0 equiv) at 0 °C.
The mixture was stirred for 0.5 h. Concentration in vacuo gave
4.1.6.8. tert-Butyl (tert-butyldimethylsilyl)oxy(6-(3-nitrophen-
oxy)hexanoyl)carbamate (15c).
Starting from 12c, 39% of
15c was obtained as a yellow solid according to above-mentioned
general procedure. mp 33–35 °C, 1H NMR (400 MHz, CDCl3) d 7.80

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X. Zhang et al. / Bioorg. Med. Chem. 21 (2013) 6981–6995
(d, J = 8.1 Hz, 1H), 7.71 (s, 1H), 7.41 (t, J = 8.1 Hz, 1H), 7.20 (d,
J = 8.2 Hz, 1H), 4.03 (t, J = 6.3 Hz, 2H), 2.84 (t, J = 7.2 Hz, 2H),
1.94–1.68 (m, 4H), 1.60–1.56 (m, 2H), 1.55 (s, 9H), 1.00 (s, 9H),
0.15 (s, 6H); MS (ESI) m/z = 505.1 (M+Na+).
2H), 1.42–1.33 (m, 4H), 0.99 (s, 9H), 0.15 (s, 6H); MS (ESI) m/
z = 533.2 (M+Na+).
4.1.7. Representative procedure for 17–19
A mixture of nitro compound and 10% Pd/C (10% w/w) in meth-
anol was hydrogenated at room temperature for 10 h. The mixture
was filtered and the filtrate was evaporated to dryness to give the
corresponding compound, which was used directly in the next
step.
4.1.6.9. tert-Butyl (tert-butyldimethylsilyl)oxy(7-(3-nitrophen-
oxy)heptanoyl)carbamate (15d).
Starting from 12d, 42% of
15d was obtained as a yellow solid according to above-mentioned
general procedure. mp 27–29 °C, 1H NMR (400 MHz, CDCl3) d 7.80
(d, J = 8.0 Hz, 1H), 7.71 (s, 1H), 7.41 (t, J = 8.2 Hz, 1H), 7.21 (d,
J = 8.1 Hz, 1H), 4.02 (t, J = 6.3 Hz, 2H), 2.81 (t, J = 7.3 Hz, 2H),
1.92–1.65 (m, 4H), 1.55 (s, 9H), 1.52–1.37 (m, 4H), 1.00 (s, 9H),
0.15 (s, 6H); MS (ESI) m/z = 519.1 (M+Na+).
4.1.8. Representative procedure for 20–22
TMSCl (143 mg, 1.32 mmol) was slowly dropped into a mixture
of VP-16 (194 mg, 0.33 mmol) and NaI (198 mg, 1.32 mmol) in
CH3CN (10 mL) under the protection of nitrogen. After stirring at
room temperature for 1 h, triethylamine (117 mg, 1.16 mmol)
and Ba2CO3 (197 mg, 0.99 mmol) was added into the flask. Then
corresponding aromatic amine in CH3CN (3 mL) was added drop-
wise into the mixture and stirred for 12 h. Subsequently, the mix-
ture was filtered and extracted with EtOAc. Concentration in vacuo
gave crude product, which was further purified by column chroma-
tography (EtOAc/petroleum ether = 1:2).
4.1.6.10. tert-Butyl (tert-butyldimethylsilyl)oxy(8-(3-nitrophen-
oxy)octanoyl)carbamate (15e).
Starting from 12e, 40% of 15e
was obtained as a yellow solid according to above-mentioned gen-
eral procedure. mp 26–28 °C, 1H NMR (400 MHz, CDCl3) d 7.80 (d,
J = 8.0 Hz, 1H), 7.71 (s, 1H), 7.41 (t, J = 8.2 Hz, 1H), 7.20 (d,
J = 8.3 Hz, 1H), 4.02 (t, J = 6.3 Hz, 2H), 2.79 (t, J = 7.3 Hz, 2H),
1.90–1.62 (m, 4H), 1.55 (s, 9H), 1.43 (d, J = 34.5 Hz, 6H), 1.00 (s,
9H), 0.15 (s, 6H); MS (ESI) m/z = 533.1 (M+Na+).
4.1.8.1. Compound 20a.
Starting from 17a, 40% of 20a was
4.1.6.11. tert-Butyl (tert-butyldimethylsilyl)oxy(4-(2-nitrophen-
obtained as a brown foam according to above-mentioned general
procedure. 1H NMR (400 MHz, CDCl3) d 6.83–6.74 (m, 3H), 6.52
(s, 1H), 6.47 (d, J = 7.3 Hz, 2H), 6.33 (s, 2H), 5.96 (s, 1H), 5.95 (s,
1H), 5.41 (s, 1H), 4.62–4.53 (m, 2H), 4.36 (t, J = 7.7 Hz, 1H), 4.07–
3.92 (m, 3H), 3.79 (s, 6H), 3.20–3.13 (m, 1H), 3.03–2.90 (m, 3H),
2.18–2.07 (m, 2H), 1.55 (s, 9H), 1.00 (s, 9H), 0.15 (s, 6H); MS
(ESI) m/z = 829.3 (M+Na+).
oxy)butanoyl)carbamate (16a).
Starting from 13a, 50% of
16a was obtained as a yellow solid according to above-mentioned
general procedure. mp 40–43 °C, 1H NMR (400 MHz, CDCl3) d 7.82
(d, J = 8.0 Hz, 1H), 7.50 (t, J = 7.9 Hz, 1H), 7.06 (d, J = 8.4 Hz, 1H),
7.00 (t, J = 7.7 Hz, 1H), 4.19 (t, J = 5.9 Hz, 2H), 3.04 (t, J = 6.8 Hz,
2H), 2.19 (p, J = 6.3 Hz, 2H), 1.55 (s, 9H), 0.99 (s, 9H), 0.13 (s,
6H); MS (ESI) m/z = 477.1 (M+Na+).
4.1.8.2. Compound 20b.
Starting from 17b, 59% of 20b was
4.1.6.12. tert-Butyl (tert-butyldimethylsilyl)oxy(5-(2-nitrophen-
obtained as a brown foam according to above-mentioned general
procedure. 1H NMR (400 MHz, CDCl3) d 6.83–6.74 (m, 3H), 6.55–
6.45 (m, 3H), 6.33 (s, 2H), 5.96 (s, 1H), 5.95 (s, 1H), 5.41 (s, 1H),
4.58 (s, 2H), 4.36 (t, J = 7.4 Hz, 1H), 4.03 (t, J = 10.1 Hz, 1H), 3.95–
3.89 (m, 2H), 3.79 (s, 6H), 3.21–3.12 (m, 1H), 3.03–2.91 (m, 1H),
2.91–2.83 (m, 2H), 1.87–1.79 (m, 4H), 1.55 (s, 9H), 1.00 (s, 9H),
0.15 (s, 6H); MS (ESI) m/z = 843.2 (M+Na+).
oxy)pentanoyl)carbamate (16b).
Starting from 13b, 51% of
16b was obtained as a yellow solid according to above-mentioned
general procedure. mp 56–58 °C, 1H NMR (400 MHz, CDCl3) d 7.81
(d, J = 8.0 Hz, 1H), 7.50 (t, J = 7.9 Hz, 1H), 7.06 (d, J = 8.4 Hz, 1H),
7.00 (t, J = 7.7 Hz, 1H), 4.12 (t, J = 5.2 Hz, 2H), 2.88 (t, J = 6.3 Hz,
2H), 1.97–1.82 (m, 4H), 1.55 (s, 9H), 1.00 (s, 9H), 0.15 (s, 6H); MS
(ESI) m/z = 491.1 (M+Na+).
4.1.8.3. Compound 20c.
Starting from 17c, 68% of 20c was
4.1.6.13. tert-Butyl (tert-butyldimethylsilyl)oxy(6-(2-nitrophen-
obtained as a brown foam according to above-mentioned general
procedure. 1H NMR (400 MHz, CDCl3) d 6.82–6.74 (m, 3H), 6.54–
6.45 (m, 3H), 6.33 (s, 2H), 5.96 (s, 1H), 5.95 (s, 1H), 5.41 (s, 1H),
4.58 (s, 2H), 4.36 (t, J = 7.4 Hz, 1H), 4.03 (t, J = 9.6 Hz, 1H), 3.93–
3.87 (m, 2H), 3.79 (s, 6H), 3.24–3.10 (m, 1H), 3.03–2.91 (m, 1H),
2.82 (t, J = 7.1 Hz, 2H), 1.84–1.68 (m, 4H), 1.55 (s, 9H), 1.53–1.48
(m, 2H), 1.00 (s, 9H), 0.15 (s, 6H); MS (ESI) m/z = 857.4 (M+Na+).
oxy)hexanoyl)carbamate (16c).
Starting from 13c, 46% of
16c was obtained as a yellow solid according to above-mentioned
general procedure. mp 36–38 °C, 1H NMR (400 MHz, CDCl3) d 7.81
(d, J = 8.1 Hz, 1H), 7.50 (t, J = 8.0 Hz, 1H), 7.06 (d, J = 8.4 Hz, 1H),
7.00 (t, J = 7.7 Hz, 1H), 4.10 (t, J = 6.3 Hz, 2H), 2.82 (t, J = 7.3 Hz,
2H), 1.96–1.65 (m, 4H), 1.60–1.56 (m, 2H), 1.55 (s, 9H), 1.00 (s,
9H), 0.15 (s, 6H); MS (ESI) m/z = 505.1 (M+Na+).
4.1.8.4. Compound 20d.
Starting from 17d, 61% of 20d was
4.1.6.14. tert-Butyl (tert-butyldimethylsilyl)oxy(7-(2-nitrophen-
obtained as a brown foam according to above-mentioned general
procedure. 1H NMR (400 MHz, CDCl3) d 6.83–6.74 (m, 3H), 6.56–
6.45 (m, 3H), 6.34 (s, 2H), 5.96 (s, 1H), 5.95 (s, 1H), 5.41 (s, 1H),
4.63–4.55 (m, 2H), 4.36 (t, J = 7.5 Hz, 1H), 4.03 (t, J = 9.5 Hz, 1H),
3.89 (t, J = 5.9 Hz, 2H), 3.79 (s, 6H), 3.17 (dd, J = 14.2, 3.5 Hz, 1H),
3.03–2.91 (m, 1H), 2.80 (t, J = 7.1 Hz, 2H), 1.82–1.64 (m, 4H), 1.55
(s, 9H), 1.51–1.37 (m, 4H), 1.00 (s, 9H), 0.15 (s, 6H); MS (ESI) m/
z = 871.3 (M+Na+).
oxy)heptanoyl)carbamate (16d).
Starting from 13d, 37% of
16d was obtained as a yellow solid according to above-mentioned
general procedure. mp 58–60 °C, 1H NMR (400 MHz, CDCl3) d 7.81
(d, J = 8.1 Hz, 1H), 7.50 (t, J = 8.0 Hz, 1H), 7.06 (d, J = 8.5 Hz, 1H),
7.00 (t, J = 7.8 Hz, 1H), 4.09 (t, J = 6.4 Hz, 2H), 2.80 (t, J = 7.3 Hz,
2H), 1.89–1.63 (m, 4H), 1.55 (s, 9H), 1.53–1.38 (m, 4H), 1.00 (s,
9H), 0.15 (s, 6H); MS (ESI) m/z = 519.0 (M+Na+).
4.1.6.15. tert-Butyl (tert-butyldimethylsilyl)oxy(8-(2-nitrophen-
4.1.8.5. Compound 20e.
Starting from 17e, 84% of 20e was
oxy)octanoyl)carbamate (16e).
Starting from 13e, 40% of 16e
obtained as a brown foam according to above-mentioned general
procedure. 1H NMR (400 MHz, CDCl3) d 6.80 (d, J = 7.6 Hz, 2H),
6.76 (s, 1H), 6.52 (s, 1H), 6.48 (d, J = 7.9 Hz, 2H), 6.33 (s, 2H), 5.96
(s, 1H), 5.95 (s, 1H), 5.41 (s, 1H), 4.61–4.55 (m, 2H), 4.36 (t,
J = 8.0 Hz, 1H), 4.04 (t, J = 9.7 Hz, 1H), 3.89 (t, J = 5.9 Hz, 2H), 3.79
(s, 6H), 3.20–3.13 (m, 1H), 3.02–2.91 (m, 1H), 2.79 (t, J = 7.3 Hz,
was obtained as a colourless oil according to above-mentioned
general procedure. 1H NMR (400 MHz, CDCl3) d 7.80 (d, J = 8.1 Hz,
1H), 7.50 (t, J = 7.9 Hz, 1H), 7.05 (d, J = 8.5 Hz, 1H), 6.99 (t,
J = 7.7 Hz, 1H), 4.08 (t, J = 6.4 Hz, 2H), 2.78 (t, J = 7.4 Hz, 2H),
1.87–1.78 (m, 2H), 1.72–1.61 (m, 2H), 1.54 (s, 9H), 1.51–1.46 (m,

X. Zhang et al. / Bioorg. Med. Chem. 21 (2013) 6981–6995
6991
2H), 1.79–1.62 (m, 4H), 1.55 (s, 9H), 1.42 (m, 6H), 1.00 (s, 9H), 0.15
(s, 6H); MS (ESI) m/z = 885.3 (M+Na+).
4.1.8.12. Compound 22b.
Starting from 19b, 67% of 22b was
obtained as a white foam according to above-mentioned general
procedure. 1H NMR (400 MHz, CDCl3) d 6.87 (t, J = 7.0 Hz, 1H),
6.82–6.68 (m, 3H), 6.52 (s, 1H), 6.47 (d, J = 7.8 Hz, 1H), 6.36 (s,
2H), 5.97 (s, 1H), 5.95 (s, 1H), 5.41 (s, 1H), 4.63 (s, 2H), 4.37 (t,
J = 7.7 Hz, 1H), 4.07–3.93 (m, 3H), 3.80 (s, 6H), 3.20 (dd, J = 14.1,
3.1 Hz, 1H), 3.06–2.93 (m, 1H), 2.81 (t, J = 6.6 Hz, 2H), 1.86–1.69
(m, 4H), 1.54 (s, 9H), 1.00 (s, 9H), 0.13 (s, 6H); MS (ESI) m/
z = 843.2 (M+Na+).
4.1.8.6. Compound 21a.
Starting from 17a, 59% of 20a was
obtained as a yellow foam according to above-mentioned general
procedure. 1H NMR (400 MHz, CDCl3) d 7.09 (t, J = 7.3 Hz, 1H),
6.77 (s, 1H), 6.52 (s, 1H), 6.37–6.27 (m, 3H), 6.19–6.06 (m, 2H),
5.97 (s, 1H), 5.95 (s, 1H), 5.41 (s, 1H), 4.66 (s, 1H), 4.61–4.57 (m,
1H), 4.39 (t, J = 7.8 Hz, 1H), 4.05–3.96 (m, 3H), 3.80 (s, 6H), 3.16–
2.93 (m, 4H), 2.25–2.06 (m, 2H), 1.54 (s, 9H), 1.02–0.90 (m, 9H),
0.17–0.08 (m, 6H); MS (ESI) m/z = 829.2 (M+Na+).
4.1.8.13. Compound 22c.
Starting from 19c, 65% of 22c was
obtained as a white foam according to above-mentioned general
procedure. 1H NMR (400 MHz, CDCl3) d 6.87 (t, J = 8.1 Hz, 1H),
6.82–6.69 (m, 3H), 6.53 (s, 1H), 6.47 (d, J = 7.3 Hz, 1H), 6.36 (s,
2H), 5.98 (s, 1H), 5.95 (s, 1H), 5.41 (s, 1H), 4.62 (s, 2H), 4.40–4.34
(m, 1H), 4.07–3.91 (m, 3H), 3.80 (s, 6H), 3.24–3.14 (m, 1H), 3.10–
2.91 (m, 1H), 2.76 (t, J = 7.0 Hz, 2H), 1.85–1.74 (m, 2H), 1.72–1.65
(m, 2H), 1.55 (s, 9H), 1.46–1.39 (m, 2H), 1.00 (s, 9H), 0.14 (s, 6H);
MS (ESI) m/z = 857.2 (M+Na+).
4.1.8.7. Compound 21b.
Starting from 18b, 78% of 21b was
obtained as a yellow foam according to above-mentioned general
procedure. 1H NMR (400 MHz, CDCl3) d 7.09 (t, J = 7.9 Hz, 1H),
6.78 (s, 1H), 6.52 (s, 1H), 6.36–6.29 (m, 3H), 6.15 (d, J = 8.1 Hz,
1H), 6.09 (s, 1H), 5.97 (s, 1H), 5.95 (s, 1H), 5.41 (s, 1H), 4.67 (s,
1H), 4.60 (d, J = 3.0 Hz, 1H), 4.39 (t, J = 7.6 Hz, 1H), 4.01 (t,
J = 9.6 Hz, 1H), 3.97–3.91 (m, 2H), 3.80 (s, 6H), 3.13 (dd, J = 14.0,
3.6 Hz, 1H), 3.04–2.94 (m, 1H), 2.91–2.82 (m, 2H), 1.88–1.80 (m,
4H), 1.54 (s, 9H), 1.00 (s, 9H), 0.15 (s, 6H); MS (ESI) m/z = 843.2
(M+Na+).
4.1.8.14. Compound 22d.
Starting from 19d, 75% of 22d was
obtained as a white foam according to above-mentioned general
procedure. 1H NMR (400 MHz, CDCl3) d 6.87 (t, J = 7.4 Hz, 1H),
6.82–6.68 (m, 3H), 6.53 (s, 1H), 6.46 (d, J = 7.7 Hz, 1H), 6.36 (s,
2H), 5.99 (s, 1H), 5.95 (s, 1H), 5.41 (s, 1H), 4.63 (s, 2H), 4.37 (t,
J = 7.9 Hz, 1H), 4.06–3.90 (m, 3H), 3.80 (s, 6H), 3.23–3.15 (m, 1H),
3.07–2.93 (m, 1H), 2.76 (t, J = 7.2 Hz, 2H), 1.80–1.69 (m, 2H),
1.67–1.59 (m, 2H), 1.54 (s, 9H), 1.41–1.33 (m, 4H), 1.00 (s, 9H),
0.14 (s, 6H); MS (ESI) m/z = 871.3 (M+Na+).
4.1.8.8. Compound 21c.
Starting from 18c, 76% of 21c was
obtained as a yellow foam according to above-mentioned general
procedure. 1H NMR (400 MHz, CDCl3) d 7.09 (t, J = 7.7 Hz, 1H),
6.78 (s, 1H), 6.52 (s, 1H), 6.37–6.28 (m, 3H), 6.14 (d, J = 7.8 Hz,
1H), 6.09 (s, 1H), 5.97 (s, 1H), 5.95 (s, 1H), 5.41 (s, 1H), 4.67 (s,
1H), 4.59 (d, J = 4.0 Hz, 1H), 4.39 (t, J = 7.9 Hz, 1H), 4.02 (t,
J = 9.8 Hz, 1H), 3.96–3.89 (m, 2H), 3.79 (s, 6H), 3.13 (dd, J = 14.5,
4.3 Hz, 1H), 3.05–2.91 (m, 1H), 2.82 (t, J = 7.0 Hz, 2H), 1.84–1.69
(m, 4H), 1.55 (s, 9H), 1.53–1.47 (m, 2H), 0.99 (s, 9H), 0.15 (s, 6H);
MS (ESI) m/z = 857.3 (M+Na+).
4.1.8.15. Compound 22e.
Starting from 19e, 41% of 22e was
obtained as a white foam according to above-mentioned general
procedure. 1H NMR (400 MHz, CDCl3) d 6.87 (t, J = 7.3 Hz, 1H),
6.82–6.69 (m, 3H), 6.54 (s, 1H), 6.47 (d, J = 7.6 Hz, 1H), 6.36 (s,
2H), 5.97 (s, 1H), 5.95 (s, 1H), 5.41 (s, 1H), 4.63 (s, 2H), 4.37 (t,
J = 7.6 Hz, 1H), 4.05–3.91 (m, 3H), 3.80 (s, 6H), 3.19 (dd, J = 13.9,
3.5 Hz, 1H), 3.05–2.93 (m, 1H), 2.76 (t, J = 7.1 Hz, 2H), 1.78–1.69
(m, 2H), 1.66–1.60 (m, 2H), 1.55 (s, 9H), 1.42–1.27 (m, 6H), 1.00
(s, 9H), 0.14 (s, 6H); MS (ESI) m/z = 885.3 (M+Na+).
4.1.8.9. Compound 21d.
Starting from 18d, 67% of 21d was
obtained as a yellow foam according to above-mentioned general
procedure. 1H NMR (400 MHz, CDCl3) d 7.09 (t, J = 8.0 Hz, 1H),
6.78 (s, 1H), 6.52 (s, 1H), 6.36–6.28 (m, 3H), 6.14 (d, J = 7.9 Hz,
1H), 6.09 (s, 1H), 5.97 (s, 1H), 5.95 (s, 1H), 5.41 (s, 1H), 4.67 (s,
1H), 4.59 (d, J = 4.2 Hz, 1H), 4.39 (t, J = 7.8 Hz, 1H), 4.02 (t,
J = 9.6 Hz, 1H), 3.91 (t, J = 6.1 Hz, 2H), 3.79 (s, 6H), 3.13 (dd,
J = 14.0, 4.5 Hz, 1H), 3.03–2.92 (m, 1H), 2.80 (t, J = 7.1 Hz, 2H),
1.82–1.64 (m, 4H), 1.54 (s, 9H), 1.49–1.37 (m, 4H), 0.99 (s, 9H),
0.14 (s, 6H); MS (ESI) m/z = 871.3 (M+Na+).
4.1.9. Representative procedure for 23–25
Compound 20–22 (1.0 equiv) and CsF (2.5 equiv) were added
into a mixed solvent of TFA and DCM (1:4 v/v). The mixture was
stirred at room temperature for 24 h. Then it was extracted with
EtOAc, washed with 5% NaHCO3 (aq) followed by brine and dried
over anhydrous Na2SO4. Concentration in vacuo gave crude prod-
uct, which was further purified by column chromatography
(DCM/methanol = 1:25) to give pure target compound.
4.1.8.10. Compound 21e.
Starting from 18e, 54% of 21e was
obtained as a yellow foam according to above-mentioned general
procedure. 1H NMR (400 MHz, CDCl3) d 7.10 (t, J = 7.9 Hz, 1H),
6.78 (s, 1H), 6.52 (s, 1H), 6.36–6.30 (m, 3H), 6.14 (d, J = 8.1 Hz,
1H), 6.10 (s, 1H), 5.97 (s, 1H), 5.95 (s, 1H), 5.41 (s, 1H), 4.67 (s,
1H), 4.59 (d, J = 4.1 Hz, 1H), 4.39 (t, J = 7.7 Hz, 1H), 4.02 (t,
J = 9.4 Hz, 1H), 3.94–3.88 (m, 2H), 3.79 (s, 6H), 3.13 (dd, J = 13.9,
3.9 Hz, 1H), 3.03–2.92 (m, 1H), 2.78 (t, J = 7.0 Hz, 2H), 1.81–1.72
(m, 2H), 1.71–1.64 (m, 2H), 1.54 (s, 9H), 1.47–1.36 (m, 6H), 0.99
(s, 9H), 0.14 (s, 6H); MS (ESI) m/z = 885.3 (M+Na+).
4.1.9.1. Compound 23a.
Starting from 20a (100 mg,
0.12 mmol), 23a (25 mg, 34%) was obtained as a yellow foam
according to above-mentioned general procedure. 1H NMR
(500 MHz, acetone-d6) d 6.65–6.85 (m, 5H), 6.47 (s, 1H), 6.39 (s,
2H), 5.97 (s, 1H), 5.96 (s, 1H), 4.80–4.98 (m, 2H), 4.55 (d,
J = 5.0 Hz, 1H), 4.37 (t, J = 7.8 Hz, 1H), 3.85–4.00 (m, 3H), 3.64 (s,
6H), 3.26 (dd, J = 14.2, 5.0 Hz, 1H), 3.05–3.15 (m, 1H), 2.20–2.35
(m, 2H), 1.95–2.05 (m, 2H); HPLC: room temperature; eluent, CH3-
OH–H2O (65:35); flow rate, 1.0 mL/min; tR = 4.43 min,
UV254 = 97.7%; HRMS (ESI): m/z calcd for C31H32N2O10Na+
(M+Na+): 615.1949, found: 615.1934.
4.1.8.11. Compound 22a.
Starting from 17b, 55% of 20b was
obtained as a yellow foam according to above-mentioned general
procedure. 1H NMR (400 MHz, CDCl3) d 6.88 (t, J = 7.9 Hz, 1H),
6.82–6.68 (m, 3H), 6.53 (s, 1H), 6.47 (d, J = 7.6 Hz, 1H), 6.36 (s,
2H), 5.96 (s, 2H), 5.41 (s, 1H), 4.65 (s, 1H), 4.62 (d, J = 3.9 Hz, 1H),
4.36 (t, J = 7.4 Hz, 1H), 4.04 (t, J = 6.3 Hz, 2H), 3.96 (t, J = 9.5 Hz,
1H), 3.80 (s, 6H), 3.19 (dd, J = 14.0, 3.7 Hz, 1H), 3.04–2.87 (m,
3H), 2.17–2.07 (m, 2H), 1.54 (s, 9H), 0.99 (s, 9H), 0.13 (s, 6H); MS
(ESI) m/z = 807.2 (M+H+).
4.1.9.2. Compound 23b.
Starting from 20b (150 mg,
0.19 mmol), 23b (35 mg, 32%) was obtained as a yellow foam
according to above-mentioned general procedure.1H NMR
(500 MHz, acetone-d6) d 6.70–6.80 (m, 5H), 6.52 (s, 1H), 6.39 (s,
2H), 5.97 (s, 1H), 5.96 (s, 1H), 4.80–5.00 (m, 2H), 4.30–4.55 (m,

6992
X. Zhang et al. / Bioorg. Med. Chem. 21 (2013) 6981–6995
2H), 3.80–4.00 (m, 3H), 3.70 (s, 6H), 3.05–3.30 (m, 2H), 2.15–2.25
(m, 2H), 1.75–1.80 (m, 4H); HPLC: room temperature; eluent, CH3-
OH–H2O (60:40); flow rate, 1.0 mL/min; tR = 8.00 min,
UV254 = 100.0%; HRMS (ESI): m/z calcd for C32H34N2O10Na+
(M+Na+): 629.2106, found: 629.2112.
rate, 1.0 mL/min; tR = 9.83 min, UV254 = 98.0%; HRMS (ESI): m/z
calcd for C32H34N2O10Na+ (M+Na+): 629.2106, found: 629.2097.
4.1.9.8. Compound 24c.
Starting from 21c (95 mg,
0.11 mmol), 24c (20 mg, 28%) was obtained as a yellow foam
according to above-mentioned general procedure. 1H NMR
(500 MHz, acetone-d6) d 10.00 (br s, 1H), 8.00–8.25 (br s, 1H),
7.10 (br s, 1H), 7.02 (t, J = 8.0 Hz, 1H), 6.82 (s, 1H), 6.52 (s, 1H),
6.39 (s, 2H), 6.13–6.30 (m, 3H), 5.98 (s, 1H), 5.97 (s, 1H), 5.29 (d,
J = 7.7 Hz, 1H), 4.90–4.95 (m, 1H), 4.30–4.45 (m, 2H), 3.90–4.00
(m, 3H), 3.70 (s, 6H), 3.05–3.35 (m, 2H), 2.05–2.13 (m, 2H), 1.60–
1.80 (m, 4H), 1.40–1.50 (m, 2H); HPLC: room temperature; eluent,
CH3OH–H2O (60:40); flow rate, 1.0 mL/min; tR = 14.00 min,
UV254 = 98.1%; HRMS (ESI): m/z calcd for C33H36N2O10Na+
(M+Na+): 643.2262, found: 643.2274.
4.1.9.3. Compound 23c.
Starting from 20c (150 mg,
0.18 mmol), 23c (40 mg, 36%) was obtained as a yellow foam
according to above-mentioned general procedure.1H NMR
(500 MHz, acetone-d6) d 10.01 (br s, 1H), 8.30–8.45 (br s, 1H),
7.14 (br s, 1H), 6.65–6.80 (m, 5H), 6.51 (s, 1H), 6.39 (s, 2H), 5.97
(s, 1H), 5.96 (s, 1H), 4.80–4.90 (m, 2H), 4.54 (d, J = 4.6Hz, 1H),
4.36 (t, J = 7.9 Hz, 1H), 3.75–3.95 (m, 3H), 3.70 (s, 6H), 3.26 (dd,
J = 14.2, 4.8, 1H), 3.10–3.15 (m, 1H), 2.05–2.15 (m, 2H), 1.60–1.80
(m, 4H), 1.40–1.50 (m, 2H); HPLC: room temperature; eluent, CH3-
OH–H2O (60:40); flow rate, 1.0 mL/min; tR = 11.37 min,
UV254 = 97.3%; HRMS (ESI): m/z calcd for C33H36N2O10Na+
(M+Na+): 643.2262, found: 643.2274.
4.1.9.9. Compound 24d.
Starting from 21d (80 mg,
0.09 mmol), 24d (26 mg, 44%) was obtained as a yellow foam
according to above-mentioned general procedure. 1H NMR
(400 MHz, acetone-d6) d 9.98 (br s, 1H), 8.14 (br s, 1H), 7.11 (br
s, 1H), 7.02 (t, J = 8.4 Hz, 1H), 6.81 (s, 1H), 6.52 (s, 1H), 6.39 (s,
2H), 6.34–6.30 (m, 2H), 6.26–6.21 (m, 1H), 5.97 (s, 1H), 5.96 (s,
1H), 5.29 (d, J = 7.7 Hz, 1H), 4.92 (dd, J = 7.4, 4.1 Hz, 1H), 4.54 (d,
J = 4.9 Hz, 1H), 4.37 (t, J = 7.7 Hz, 1H), 3.96–3.88 (m, 3H), 3.70 (s,
6H), 3.26 (dd, J = 14.2, 5.0 Hz, 1H), 3.20–3.10 (m, 1H), 2.14–2.07
(m, 2H), 1.77–1.69 (m, 2H), 1.66–1.58 (m, 2H), 1.50–1.42 (m,
2H), 1.42–1.33 (m, 2H); 13C NMR (100 MHz, acetone-d6) d 175.5,
170.8, 161.5, 150.8, 148.7, 148.1, 148.0, 136.1, 133.1, 132.5,
131.6, 130.8, 110.3, 110.2, 109.8, 105.9, 103.9, 102.3, 99.5, 69.6,
68.2, 56.7, 52.4, 44.4, 42.3, 39.8, 33.2, 30.6, 30.0, 26.6, 26.2; HPLC:
room temperature; eluent, CH3OH–H2O (60:40); flow rate, 1.0 mL/
min; tR = 21.58 min, UV254 = 97.7%; HRMS (ESI): m/z calcd for C34-
H38N2O10Na+ (M+Na+): 657.2419, found: 657.2415.
4.1.9.4. Compound 23d.
Starting from 20d (120 mg,
0.14 mmol), 23d (30 mg, 39%) was obtained as a yellow foam
according to above-mentioned general procedure. 1H NMR
(400 MHz, acetone-d6) d 6.81–6.76 (m, 3H), 6.73–6.67 (m, 2H),
6.52 (s, 1H), 6.40 (s, 2H), 5.97 (s, 1H), 5.96 (s, 1H), 4.81 (d,
J = 3.9 Hz, 1H), 4.55 (d, J = 4.8 Hz, 1H), 4.38 (t, J = 7.7 Hz, 1H),
3.99–3.86 (m, 3H), 3.71 (s, 6H), 3.27 (dd, J = 14.2, 4.9 Hz, 1H),
3.17–3.10 (m, 1H), 2.23–2.10 (m, 2H), 1.76–1.68 (m, 2H), 1.67–
1.59 (m, 2H), 1.50–1.44 (m, 2H), 1.42–1.35 (m, 2H); HPLC: room
temperature; eluent, CH3OH–H2O (60:40); flow rate, 1.0 mL/min;
tR = 17.88 min, UV254 = 100.0%; HRMS (ESI): m/z calcd for C34H38N2-
O10Na+ (M+Na+): 657.2419, found: 657.2423.
4.1.9.5. Compound 23e.
Starting from 20e (100 mg,
0.12 mmol), 23e (25 mg, 33%) was obtained as a yellow foam
according to above-mentioned general procedure. 1H NMR
(400 MHz, acetone-d6) d 9.96 (br s, 1H), 8.19 (br s, 1H), 7.11 (br
s, 1H), 6.82–6.66 (m, 5H), 6.52 (s, 1H), 6.39 (s, 2H), 5.97 (d,
J = 3.5 Hz, 2H), 4.92–4.78 (m, 2H), 4.55 (d, J = 4.2 Hz, 1H), 4.37 (t,
J = 7.7 Hz, 1H), 3.98–3.86 (m, 3H), 3.70 (s, 6H), 3.27 (dd, J = 14.2,
4.3 Hz, 1H), 3.18–3.07 (m, 1H), 2.15–2.07 (m, 2H), 1.76–1.56 (m,
4H), 1.50–1.31 (m, 6H); HPLC: room temperature; eluent, CH3-
OH–H2O (60:40); flow rate, 1.0 mL/min; tR = 30.15 min,
UV254 = 98.3%; HRMS (ESI): m/z calcd for C35H40N2O10Na+
(M+Na+): 671.2575, found: 671.2574.
4.1.9.10. Compound 24e.
Starting from 21e (80 mg,
0.09 mmol), 24e (28 mg, 46%) was obtained as a yellow foam
according to above-mentioned general procedure. 1H NMR
(400 MHz, acetone-d6) d 7.03 (t, J = 7.9 Hz, 1H), 6.82 (s, 1H), 6.53
(s, 1H), 6.40 (s, 2H), 6.33 (s, 2H), 6.24 (d, J = 7.8 Hz, 1H), 5.97 (s,
2H), 4.93 (s, 1H), 4.56 (d, J = 4.1 Hz, 1H), 4.39 (t, J = 7.9 Hz, 1H),
3.97–3.89 (m, 3H), 3.71 (s, 6H), 3.30–3.22 (m, 1H), 3.22–3.10 (m,
1H), 2.14–2.07 (m, 2H), 1.79–1.69 (m, 2H), 1.64–1.56 (m, 2H),
1.52–1.30 (m, 6H); HPLC: room temperature; eluent, CH3OH–H2O
(60:40); flow rate, 1.0 mL/min; tR = 35.99 min, UV254 = 98.3%;
HRMS (ESI): m/z calcd for C35H40N2O10Na+ (M+Na+): 671.2575,
found: 671.2568.
4.1.9.6. Compound 24a.
Starting from 21a (100 mg,
0.12 mmol), 24a (22 mg, 30%) was obtained as a yellow foam
according to above-mentioned general procedure. 1H NMR
(500 MHz, acetone-d6) d 7.02 (m, 1H), 6.81 (s, 1H), 6.52 (s, 1H),
6.40 (s, 2H), 6.20–6.35 (m, 3H), 5.97 (s, 1H), 5.96 (s, 1H), 4.85–
4.95 (m, 1H), 4.53–4.55 (m, 1H), 4.37–4.41 (m, 1H), 3.85–4.04
(m, 3H), 3.70 (s, 6H), 3.05–3.30 (m, 2H), 2.00–2.50 (m, 4H); HPLC:
room temperature; eluent, CH3OH–H2O (60:40); flow rate, 1.0 mL/
min; tR = 7.86 min, UV254 = 98.4%; HRMS (ESI): m/z calcd for C31-
H32N2O10Na+ (M+Na+): 615.1949, found: 615.1934.
4.1.9.11. Compound 25a.
Starting from 22a (100 mg,
0.12 mmol), 25a (18 mg, 24%) was obtained as a yellow foam
according to above-mentioned general procedure. 1H NMR
(500 MHz, acetone-d6) d 6.60–6.80 (m, 5H), 6.52 (s, 1H), 6.41 (s,
2H), 5.98 (s, 1H), 5.97 (s, 1H), 4.85–4.99 (m, 2H), 4.40–4.55 (m,
2H), 3.70–4.05 (m, 3H), 3.64 (s, 6H), 3.44 (dd, J = 13.7, 4.2 Hz,
1H), 3.14–3.20 (m, 1H), 1.95–2.25 (m, 4H); HPLC: room tempera-
ture; eluent, CH3OH–H2O (60:40); flow rate, 1.0 mL/min; tR = 7.53 -
min, UV254 = 100.0%; HRMS (ESI): m/z calcd for C31H32N2O10Na+
(M+Na+): 615.1949, found: 615.1943.
4.1.9.7. Compound 24b.
Starting from 21b (90 mg,
0.11 mmol), 24b (29 mg, 44%) was obtained as a yellow foam
according to above-mentioned general procedure. 1H NMR
(500 MHz, acetone-d6) d 10.03 (br s, 1H), 8.25–8.40 (br s, 1H),
7.11 (br s, 1H), 7.02 (t, J = 8.0 Hz, 1H), 6.81 (s, 1H), 6.52 (s, 1H),
6.39 (s, 2H), 6.23–6.35 (m, 3H), 5.97 (s, 1H), 5.96 (s, 1H), 5.30 (d,
J = 8.0 Hz, 1H), 4.90–4.95 (m, 1H), 4.54 (d, J = 4.5Hz, 1H), 4.38 (t,
J = 8.0 Hz, 1H), 3.80–4.00 (m, 3H), 3.70 (s, 6H), 3.25 (dd, J = 14.5,
5.0 Hz, 1H), 3.11–3.20 (m, 1H), 2.07–2.15 (m, 2H), 1.70–1.80 (m,
4H); HPLC: room temperature; eluent, CH3OH–H2O (60:40); flow
4.1.9.12. Compound 25b.
Starting from 22b (90 mg,
0.11 mmol), 25b (38 mg, 57%) was obtained as a yellow foam
according to above-mentioned general procedure. 1H NMR
(500 MHz, acetone-d6) d 10.00 (br s, 1H), 8.25–8.50 (br s, 1H),
7.12 (br s, 1H), 6.60–6.90 (m, 5H), 6.51 (s, 1H), 6.41 (s, 2H), 5.98
(s, 1H), 5.95 (s, 1H), 4.80–4.90 (m, 2H), 4.57 (d, J = 5.0 Hz, 1H),
4.38 (t, J = 8.0 Hz, 1H), 3.89–4.02 (m, 2H), 3.85 (t, J = 9.5Hz, 1H),
3.71 (s, 6H), 3.34 (dd, J = 14.5, 5.0 Hz, 1H), 3.13–3.20 (m, 1H),

X. Zhang et al. / Bioorg. Med. Chem. 21 (2013) 6981–6995
6993
2.05–2.20 (m, 2H), 1.70–1.80 (m, 4H); HPLC: room temperature;
eluent, CH3OH–H2O (60:40); flow rate, 1.0 mL/min; tR = 9.64 min,
UV254 = 100.0%; HRMS (ESI): m/z calcd for C32H34N2O10Na+
(M+Na+): 629.2106, found: 629.2094.
(400 MHz, CDCl3) d 7.86–7.77 (m, 1H), 7.70 (t, J = 2.3 Hz, 1H),
7.45–7.27 (m, 6H), 7.22–7.17 (m, 1H), 5.12 (s, 2H), 4.01 (t,
J = 6.4 Hz, 2H), 2.38 (t, J = 7.5 Hz, 2H), 1.86–1.65 (m, 4H), 1.55–
1.35 (m, 4H); HRMS (ESI): m/z calcd for C20H23NO5Na+ (M+Na+):
380.1474, found: 380.1477.
4.1.9.13. Compound 25c.
Starting from 22c (80 mg,
0.10 mmol), 25c (40 mg, 67%) was obtained as a yellow foam
according to above-mentioned general procedure. 1H NMR
(500 MHz, acetone-d6) d 9.97 (s, 1H), 8.25–8.50 (br s, 1H), 7.02–
7.15 (br s, 1H), 6.60–6.85 (m, 5H), 6.52 (s, 1H), 6.41 (s, 2H), 5.99
(s, 1H), 5.96 (s, 1H), 4.75–4.85 (m, 2H), 4.59 (d, J = 4.9 Hz, 1H),
4.40 (t, J = 7.7 Hz, 1H), 3.85–4.00 (m, 3H), 3.71 (s, 6H), 3.32 (dd,
J = 14.2, 4.9 Hz, 1H), 3.15–3.25 (m, 1H), 2.00–2.10 (m, 2H), 1.55–
1.75 (m, 4H), 1.35–1.50 (m, 2H); HPLC: room temperature; eluent,
CH3OH–H2O (60:40); flow rate, 1.0 mL/min; tR = 14.24 min,
UV254 = 100.0%; HRMS (ESI): m/z calcd for C33H36N2O10Na+
(M+Na+): 643.2262, found: 643.2277.
4.1.11. Representative procedure for 28–29
A mixture of nitro compounds 26/27 (1.0 equiv), iron powder
(5.0 equiv) and NH4Cl (5.0 equiv) in EtOH–H2O was stirred at
60 °C for 1 h. Then the solution was cooled to room temperature
and filtered. Subsequently, the filtrate was extracted with EtOAc
and the organic layer was evaporated to dryness to give the crude
product.
4.1.11.1. Benzyl 7-(4-aminophenoxy)heptanoate hydrochloride
(28).
Starting from 26, 82% of compound 28 was obtained as a
white solid according to above-mentioned general procedure. The
resulting oil was dissolved in EtOH and concentrated hydrochloric
acid was dropped into the solution. The crystals are collected by
vacuum filtration and washed by EtOH. mp 138–141 °C, 1H NMR
(400 MHz, DMSO) d 9.87 (br s, 2H), 7.44–7.30 (m, 5H), 7.27 (d,
J = 8.7 Hz, 2H), 7.01 (d, J = 8.8 Hz, 2H), 5.08 (s, 2H), 3.95 (t,
J = 6.4 Hz, 2H), 2.36 (t, J = 7.3 Hz, 2H), 1.78–1.64 (m, 2H), 1.63–
1.53 (m, 2H), 1.45–1.26 (m, 4H); HRMS (ESI): m/z calcd for
4.1.9.14. Compound 25d.
Starting from 22d (90 mg,
0.11 mmol), 25d (31 mg, 44%) was obtained as a yellow foam
according to above-mentioned general procedure. 1H NMR
(400 MHz, acetone-d6) d 6.91–6.81 (m, 2H), 6.79 (s, 1H), 6.72 (d,
J = 7.6 Hz, 1H), 6.66 (t, J = 7.7 Hz, 1H), 6.55 (s, 1H), 6.41 (s, 2H),
6.02 (s, 1H), 5.97 (s, 1H), 4.88–4.75 (m, 2H), 4.60 (d, J = 3.7 Hz,
1H), 4.42 (t, J = 7.7 Hz, 1H), 4.09–3.93 (m, 2H), 3.87 (t, J = 9.4 Hz,
1H), 3.71 (s, 6H), 3.31 (dd, J = 14.1, 4.1 Hz, 1H), 3.19 (s, 1H), 2.08–
2.06 (m, 2H), 1.74–1.65 (m, 2H), 1.59–1.50 (m, 2H), 1.42–1.26
(m, 4H); HPLC: room temperature; eluent, CH3OH–H2O (60:40);
flow rate, 1.0 mL/min; tR = 25.58 min, UV254 = 100.0%; HRMS
(ESI): m/z calcd for C34H38N2O10Na+ (M+Na+): 657.2419, found:
657.2427
C
20H26NO3 (M+H+): 328.1913, found: 328.1917.
4.1.11.2. Benzyl 7-(3-aminophenoxy)heptanoate (29).
Start-
ing from 27, 95% of compound 29 was obtained as a brown oil
according to above-mentioned general procedure. 1H NMR
(400 MHz, CDCl3) d 7.43–7.28 (m, 5H), 7.04 (t, J = 8.0 Hz, 1H),
6.29 (t, J = 9.5 Hz, 2H), 6.23 (s, 1H), 5.12 (s, 2H), 3.89 (t, J = 6.5 Hz,
2H), 3.63 (br s, 2H), 2.37 (t, J = 7.4 Hz, 2H), 1.79–1.63 (m, 4H),
1.50–1.33 (m, 4H); HRMS (ESI): m/z calcd for C20H26NO3 (M+H+):
328.1913, found: 328.1901.
4.1.9.15. Compound 25e.
Starting from 22e (104 mg,
0.12 mmol), 25e (34 mg, 44%) was obtained as a yellow foam
according to above-mentioned general procedure. 1H NMR
(400 MHz, acetone-d6) d 6.90–6.81 (m, 2H), 6.79 (s, 1H), 6.71 (d,
J = 7.6 Hz, 1H), 6.65 (t, J = 7.5 Hz, 1H), 6.54 (s, 1H), 6.42 (s, 2H),
5.99 (s, 1H), 5.96 (s, 1H), 4.87–4.77 (m, 2H), 4.60 (d, J = 3.5 Hz,
1H), 4.41 (t, J = 7.5 Hz, 1H), 4.08–3.92 (m, 2H), 3.86 (t, J = 9.5 Hz,
1H), 3.71 (s, 6H), 3.29 (dd, J = 14.3, 3.8 Hz, 1H), 3.23–3.12 (m,
1H), 2.07–2.02 (m, 2H), 1.73–1.63 (m, 2H), 1.59–1.49 (m, 2H),
1.39–1.21 (m, 6H); HPLC: room temperature; eluent, CH3OH–H2O
(60:40); flow rate, 1.0 mL/min; tR = 45.36 min, UV254 = 100.0%;
HRMS (ESI): m/z calcd for C35H40N2O10Na+ (M+Na+): 671.2575,
found: 671.2578.
4.1.12. Representative procedure for 30–31
TMSCl (4.0 equiv) was slowly dropped into a mixture of VP-16
(1.0 equiv) and NaI (4.0 equiv) in CH3CN (10 mL) under nitrogen
atmosphere. After stirring at room temperature for 1 h, TEA
(3.5 equiv) and Ba2CO3 (3.0 equiv) was added into the flask. Then
corresponding aromatic amine 28/29 was dissolved in CH3CN
and added into the mixture dropwise. The mixture was stirred
for 12 h and extracted with EtOAc. Concentration in vacuo gave
crude product, which was further purified by column
chromatography.
4.1.10. Representative procedure for 26–27
4.1.12.1. Compound 30.
Starting from 28, 70% of compound
A
mixture of acid compound 11d/12d (1.0 equiv), BnBr
30 was obtained as a colourless oil according to above-mentioned
general procedure. 1H NMR (400 MHz, CDCl3) d 7.41–7.29 (m, 5H),
6.84–6.74 (m, 3H), 6.53–6.46 (m, 3H), 6.33 (s, 2H), 5.99–5.93 (m,
2H), 5.43 (s, 1H), 5.12 (s, 2H), 4.64–4.53 (m, 2H), 4.36 (t,
J = 7.9 Hz, 1H), 4.03 (dd, J = 10.6, 8.6 Hz, 1H), 3.88 (t, J = 6.5 Hz,
2H), 3.79 (s, 6H), 3.63–3.52 (m, 1H), 3.16 (dd, J = 14.0, 5.0 Hz,
1H), 3.05–2.88 (m, 1H), 2.37 (t, J = 7.5 Hz, 2H), 1.81–1.64 (m, 4H),
1.52–1.34 (m, 4H); HRMS (ESI): m/z calcd for C40H43NO10Na+
(M+Na+): 732.2785, found: 732.2761.
(1.0 equiv) and Cs2CO3 (1.05 equiv) in DMF was stirred at room
temperature for 1 h. Then the solution was poured into water
and extracted with EtOAc. The organic layer was concentrated to
give the crude product, which was further purified by column
chromatography.
4.1.10.1. Benzyl 7-(4-nitrophenoxy)heptanoate (26).
Start-
ing from 11d, 93% of compound 26 was obtained as a yellow solid
according to above-mentioned general procedure. mp 57–60 °C. 1H
NMR (400 MHz, CDCl3) d 8.19 (d, J = 9.2 Hz, 2H), 7.39–7.33 (m, 5H),
6.92 (d, J = 9.2 Hz, 2H), 5.12 (s, 2H), 4.03 (t, J = 6.4 Hz, 2H), 2.38 (t,
J = 7.4 Hz, 2H), 1.87–1.76 (m, 2H), 1.75–1.63 (m, 2H), 1.53–1.36 (m,
4H); HRMS (ESI): m/z calcd for C20H23NO5Na+ (M+Na+): 380.1474,
found: 380.1464.
4.1.12.2. Compound 31.
Starting from 29, 70% of compound
31 was obtained as a colourless oil according to above-mentioned
general procedure. 1H NMR (400 MHz, CDCl3) d 7.42–7.29 (m, 5H),
7.10 (t, J = 8.1 Hz, 1H), 6.78 (s, 1H), 6.52 (s, 1H), 6.38–6.26 (m, 3H),
6.19–6.12 (m, 1H), 6.12–6.07 (m, 1H), 5.96 (d, J = 7.0 Hz, 2H), 5.43
(s, 1H), 5.11 (s, 2H), 4.70–4.65 (m, 1H), 4.59 (d, J = 4.8 Hz, 1H), 4.38
(t, J = 8.0 Hz, 1H), 4.05–3.97 (m, 1H), 3.91 (t, J = 6.4 Hz, 2H), 3.80 (s,
6H), 3.13 (dd, J = 14.0, 4.9 Hz, 1H), 3.04–2.92 (m, 1H), 2.37 (t,
J = 7.5 Hz, 2H), 1.80–1.64 (m, 4H), 1.50–1.35 (m, 4H); HRMS
4.1.10.2. Benzyl 7-(3-nitrophenoxy)heptanoate (27).
Start-
ing from 12d, 98% of compound 27 was obtained as a yellow oil
according to above-mentioned general procedure. 1H NMR

6994
X. Zhang et al. / Bioorg. Med. Chem. 21 (2013) 6981–6995
(ESI): m/z calcd for C40H43NO10Na+ (M+Na+): 732.2785, found:
4.2.2. Cell culture and cytotoxicity/proliferation assay
732.2794.
Cells were kept at logarithmic growth phase in 5% CO2 at 37 °C
with the corresponding medium supplemented with 10% fetal bo-
vine serum and 100 units/mL each of penicillin G and streptomy-
cin. Cells were seeded onto a 96-well plate at a concentration of
2000–3000 cells/well and incubated at 37 °C in 5% CO2 for 24 h.
A range of concentrations of the test compounds were added and
4.1.13. Representative procedure for 32–33
A mixture of benzyl ester (71 mg, 0.1 mmol) and 7 mg Pd(OH)2
(20% w/w) in MeOH–EtOAc was hydrogenated at room tempera-
ture for 48 h. After filtration, the organic layer was evaporated to
dryness to give the corresponding acids. The intermediate was dis-
solved in 5 mL DCM and benzene-1,2-diamine (33 mg, 0.3 mmol),
HATU (40 mg, 0.105 mmol), DIPEA (39 mg, 0.3 mmol) were added
into the solution. The mixture was stirred for 12 h and extracted
with DCM. Concentration in vacuo gave crude product, which
was further purified by column chromatography (DCM/
MeOH = 30:1).
the plate was incubated at 37 °C for 72 h before 20
(5 mg/mL)/well was added. After 3 h of incubation, the medium
was removed and 100 L DMSO was added to each well. The absor-
lL MTT
l
bance was measured using a SpectraMax 340 microplate reader
(Molecular Devices, Sunnyvale, CA, USA) at 550 nm with a refer-
ence at 690 nm. The optical density of the result of the MTT assay
was directly proportional to the number of viable cells.24
4.2.3. In vitro topoisomerase II relaxation assay
The relaxation of pBR322 DNA was assayed according to Topo-
Gen protocol in order to determine topoisomerase II activity. The
4.1.13.1. Compound 32.
Starting from 30, compound 32
(29 mg, 41%) of was obtained as a yellow solid according to
above-mentioned general procedure. mp 119–122 °C, 1H NMR
(400 MHz, CDCl3) d 7.21–7.03 (m, 3H), 6.86–6.73 (m, 5H), 6.56–
6.44 (m, 3H), 6.33 (s, 2H), 5.96 (d, J = 6.6 Hz, 2H), 5.43 (s, 1H),
4.66–4.55 (m, 2H), 4.37 (t, J = 7.8 Hz, 1H), 4.03 (t, J = 9.5 Hz, 1H),
3.97–3.78 (m, 10H), 3.60–3.53 (m, 1H), 3.22–3.13 (m, 1H), 3.04–
2.92 (m, 1H), 2.43 (t, J = 7.3 Hz, 2H), 1.86–1.74 (m, 4H), 1.55–1.43
(m, 4H); HPLC: room temperature; eluent, CH3OH–H2O (70:30);
flow rate, 1.0 mL/min; tR = 10.49 min, UV254 = 95.0%; HRMS (ESI):
m/z calcd for C40H43N3O9Na+ (M+Na+): 732.2897, found: 732.2889.
substrate pBR322 DNA (200 ng) and 100
in assay buffer (50 mM TrisꢁHCl, pH 8.0, 120 mM KCl,
10 mM MgCl2, 0.5 mM ATP, 0.5 mM dithiothreitol, 300 g/mL bo-
lM drug were combined
l
vine serum albumin (BSA)) and incubated for 30 min on ice. Subse-
quently, 4 units of topoisomerase II (TopoGEN, Port Orange,
Florida, USA) were added and the reaction was allowed to proceed
for 10 min at 37 °C. The reaction was quenched via the addition of
loading buffer (1% sarkosyl, 0.025% bromophenol blue, and 5% glyc-
erol) and was analyzed by electrophoresis on a 1% agarose gel in
TBE buffer (89 mM Tris, 89 mM borate, and 2 mM Na-EDTA, pH
8.3) for 3.5 h at 40 V. The gel was stained with ethidium bromide
(EB) for 30 min and was visualized under UV illumination.
4.1.13.2. Compound 33.
Starting from 31, compound 33
(25 mg, 35%) was obtained as a yellow solid according to above-
mentioned general procedure. mp 126–129 °C, 1H NMR
(400 MHz, CDCl3) d 7.20–7.02 (m, 4H), 6.84–6.75 (m, 3H), 6.52 (s,
1H), 6.37–6.31 (m, 3H), 6.19–6.07 (m, 2H), 5.96 (d, J = 6.9 Hz,
2H), 5.44 (s, 1H), 4.70–4.64 (m, 1H), 4.58 (d, J = 4.0 Hz, 1H), 4.39
(t, J = 8.0 Hz, 1H), 4.01 (t, J = 9.7 Hz, 1H), 3.97–3.91 (m, 2H), 3.90–
3.76 (m, 9H), 3.13 (dd, J = 13.9, 4.0 Hz, 1H), 3.03–2.90 (m, 1H),
2.42 (t, J = 7.2 Hz, 2H), 1.88–1.69 (m, 4H), 1.56–1.41 (m, 4H); HPLC:
room temperature; eluent, CH3OH–H2O (70:30); flow rate, 1.0 mL/
min; tR = 14.32 min, UV254 = 97.0%; HRMS (ESI): m/z calcd for
Acknowledgments
This work was supported by the Youth Scientific Innovation
Foundation of East China Normal University (Nos. 78210157 and
78210198), the State Key Laboratory of Drug Research (No.
SIMM1203KF–10), the Grants of The National Natural Science
Foundation of China (Nos. 81172936 and 21102046), and Grants
of The Fundamental Research Funds for the Central Universities.
C
40H44N3O9 (M+H+): 710.3078, found: 710.3078.
Supplementary data
4.2. Biological assays
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2013.09.023.
4.2.1. HDAC enzymatic assay in vitro
Recombinant human HDAC1, HDAC3 and HDAC6 were cloned
and expressed in High5 insect cells using a baculovirus expression
system and purified using Ni-NTA (QIAGEN). The histone deacetyl-
ase inhibitory activity of HDAC1 and HDAC3 was determined using
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