Design and synthesis of novel soluble 2,5-diketopiperazine derivatives as potential anticancer agents

Article abstract of DOI:10.1016/j.ejmech.2014.06.030

Non-protected 2,5-diketopiperazine derivatives have poor solubility thus with negative impact on their bioavailability. In the present study, twenty-one novel soluble mono-protected, and three non-protected 2,5-diketopiperazine derivatives were designed and synthesized. Their anticancer activity to ten cell lines were evaluated by using CCK8 assay, and the results showed that about half of the mono-protected derivatives had broad-spectrum anticancer activity. Among allyl-protected derivatives, compound 4m had strong activity to all the cell lines (IC₅₀ = 0.5-4.5 μM), especially to the cancer cell lines U937 (IC₅₀ = 0.5 μM) and K562 (IC₅₀ = 0.9 μM). Compound 4m could become a lead compound for further development for anticancer agents.

Full text of DOI:10.1016/j.ejmech.2014.06.030

European Journal of Medicinal Chemistry 83 (2014) 236e244
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Design and synthesis of novel soluble 2,5-diketopiperazine derivatives
as potential anticancer agents
Shengrong Liao a, Xiaochu Qin b, Ding Li c, Zhengchao Tu b, Jinsheng Li a, Xuefeng Zhou a,
,
*
Junfeng Wang a, Bin Yang a, Xiuping Lin a, Juan Liu a, Xianwen Yang a, Yonghong Liu a
a CAS Key Laboratory of Tropical Marine Bio-resources and Ecology/Guangdong Key Laboratory of Marine Materia Medica/RNAM Center for Marine
Microbiology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou 510301, PR China
b Laboratory of Molecular Engineering and Laboratory of Natural Product Synthesis, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of
Sciences, Guangzhou 510530, PR China
c School of Pharmaceutical Sciences, Sun Yat-Sen University, Guangzhou 510006, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Non-protected 2,5-diketopiperazine derivatives have poor solubility thus with negative impact on their
bioavailability. In the present study, twenty-one novel soluble mono-protected, and three non-protected
2,5-diketopiperazine derivatives were designed and synthesized. Their anticancer activity to ten cell lines
were evaluated by using CCK8 assay, and the results showed that about half of the mono-protected
derivatives had broad-spectrum anticancer activity. Among allyl-protected derivatives, compound 4m
Received 5 October 2013
Received in revised form
5 February 2014
Accepted 13 June 2014
Available online 14 June 2014
had strong activity to all the cell lines (IC50 ¼ 0.5e4.5
mM), especially to the cancer cell lines U937
M). Compound 4m could become a lead compound for further
(IC50 ¼ 0.5
m
M) and K562 (IC50 ¼ 0.9
m
Keywords:
2,5-Diketopiperazine derivative
Solubility
development for anticancer agents.
© 2014 Elsevier Masson SAS. All rights reserved.
Anticancer agent
Protective group
1. Introduction
while most have complicated chemical structures. Therefore, many
2,5-diketopiperazine derivatives with simple structures have been
synthesized based on 2,5-diketopiperazine scaffold and show good
activities [10,11]. One significant example is plinabulin (NPI-2358/
KPU-2, Fig. 1), which has been derived from natural phenylahistin
(Fig. 1) and first developed as a vascular disrupting agent (VDA)
[12], and is now under phase II clinical trials as an anticancer drug
[13]. Some recent studies have shown that plinabulin is also a
potent anti-microtubule agent with colchicine-like tubulin depo-
lymerization activity [14]. A few of its derivatives modified at the
aromatic moieties have also been synthesized with good efficacy
[14,15]. Another study has shown that 2,5-diketopiperazine de-
Cancer is one of the most serious diseases in the world. Despite
huge effort has been made by researchers during past several de-
cades, the search of effective clinical approaches for the treatment
of cancer is still a tough challenge. Apart from surgery, immuno-
therapy, and radiotherapy, chemotherapy using anticancer agents is
another useful option for the cancer treatment [1]. For a long time,
the development of novel anticancer agents is a highly active
research field, and much effort has been focused on natural prod-
ucts because of their fewer side effects [2]. However, solubility is
one major problem for some active natural or synthetic compounds
in the early stage study of these compounds [3,4]. One classic
instance is about the natural product Camptothecin (CPT, Fig. 1),
whose low solubility limits its broad use as cancer therapeutic
agent, and some optimized derivatives have been synthesized for
its improvement in the following years [5e8].
rivatives have weak activity (100 m
g mLꢀ1) in inhibiting the nauplii
movement without causing their death [16]. But for most non-
protected derivatives, their solubility is poor [17], presumably due
to the combination of intermolecular hydrogen bonds and
pep
stacking interactions from lines or networks of 2,5-
diketopiperazine templates (Fig. 2) [9,18], thus with negative in-
fluence on their solubility and purification. A useful solution to this
problem is to interrupt the formation of hydrogen bonds and
2,5-Diketopiperazine (Fig. 1) is an important scaffold in many
natural products which have a variety of biological activities [9],
disturb the
groups to replace one or two of the amide hydrogen atoms,
resulting in non-planar structure of 2,5-diketopiperazine
pep stacking interactions by introducing protective
* Corresponding author.
E-mail address: yonghongliu@scsio.ac.cn (Y. Liu).
a
http://dx.doi.org/10.1016/j.ejmech.2014.06.030
0223-5234/© 2014 Elsevier Masson SAS. All rights reserved.

S. Liao et al. / European Journal of Medicinal Chemistry 83 (2014) 236e244
237
that for the synthesis of intermediate 2, or with intermediate 2 in
the presence of Cs2CO3 at 80 ꢁC. All the protected compounds can
be easily dissolved in normal solvents, such as ethyl acetate
(AcOEt), dichloromethane (DCM), chloroform, methanol (MeOH),
ethanol (EtOH), N,N-dimethylmethanamide (DMF), dimethylsulf-
oxide (DMSO), etc. The non-protected compound 4v and 4w had
poor solubility in all above solvents, while compound 4x and pli-
nabulin could be dissolved in DMF, DMSO, a mixture of MeOH with
DCM or AcOEt.
2.2. Biological activity
We first investigated the anticancer activity of the allyl-
protected 2,5-diketopiperazine derivatives (4aer) to six cancer
cell lines (BGC-823, Hela, Huh-7, MCF-7, H1975, A549), and their
IC50 values are listed in Table 1. It was found that their inhibitory
activities to these cancer cell lines were different. When Ar1 was the
phenyl (Ph) or 3-bromo phenyl (3-BrPh) group, the derivatives (4a,
4b, 4c, and 4d, Table 1) had no activities, indicating that Ph and 3-
BrPh were not good substitutive groups for their biological activity.
However, when Ar1 was 2-MeOPh, most of the derivatives had high
activity against the cell lines. Therefore, we continued our further
synthesis of derivatives with 2-MeOPh as Ar1, and with other
phenyl groups as Ar2 in the following studies. As shown in Table 1,
when Ar2 was 3-BrPh (4e), 3-ClPh (4f), 2-ClPh (4i), 2-CF3Ph (4k), or
5-F-2-MePh (4q), the derivatives had high activity, except com-
pound 4j with 2-FPh as Ar2. Among all these derivatives, compound
4m with 2,3-ClPh as Ar2 showed the highest inhibitory activity to
all cancer cell lines. In comparison, when its chlorine atom at ortho-
position was moved to para-position, compound 4n lost its activity
to all these cell lines. To our surprise, compound 4l with 2-MeOPh
as Ar2 had high activity only against cell line Hela, while 4g with 3-
FPh as Ar2 had high activity only against cell lines BGC-823 and
Hela. On the other hand, compound 4h with 3-MePh and com-
pound 4o with 5-Br-2-FPh as Ar2, both had low activity, while
compound 4p with 3-Br-4-FPh and 4r with 4-MePh as Ar2, had no
activity at all.
Fig. 1. The structures of Camptothecin, 2,5-diketopiperazine and its derivatives.
template [19,20]. Interestingly, some previous articles [21e23] have
shown that garlic derivatives, e.g. S-allylcysteine [24,25] with an
allyl-protection on the sulfur atom, can well suppress the growth of
a broad spectrum of tumors, and their allyl groups play important
roles for their inhibitory activities [26]. Therefore, in the present
study, a novel series of allyl-protected 2,5-diketopiperazine de-
rivatives were designed and synthesized, and their anticancer ac-
tivities against ten cell lines were evaluated by using CCK8 assay
[27]. Besides, several methyl-protected or non-protected de-
rivatives were also synthesized for comparative studies.
2. Results and discussion
2.1. Chemistry
The synthesis of the target 2,5-diketopiperazine derivatives is
shown in Scheme 1. The purchased compound 2,5-
diketopiperazine was heated under reflux in acetic anhydride
overnight to yield product 1 [28], which was treated with aromatic
aldehydes (Ar1CHO) and Cs2CO3 in dry DMF at room temperature to
afford the intermediate 2 [15]. Compound 2 was then protected
with allyl or methyl group to give compound 3. The final target
compound 4 was obtained from aromatic aldehydes (Ar2CHO) by
reacting either with intermediate 3 under the same condition as
The methyl-protected compounds 4t and 4u showed slightly
higher anticancer activity than their corresponding allyl-protected
compounds 4i and 4m. The methyl-protected compound 4s had
low or no anticancer activity, just like its corresponding allyl-
protected compound 4l. For unprotected compounds, 4v and 4w
had no activity, while 4x and the control compound plinabulin had
higher anticancer activity even than the protected derivatives. This
might indicate that imidazole was beneficial to the solubility of 4x
and plinabulin, resulting in their improved anticancer activity,
similar to those reported previously [15].
Based on the above results, their structureeactivity re-
lationships were still not very clear, because the aromatic
groups, the positions for the substituents, and the protective
groups of the derivatives had somehow different impacts on
their bioactivities. However, some of the allyl-protected de-
rivatives, especially 4m, should be worthwhile for further study
as lead compounds in searching for strong and broad-spectrum
anticancer agents.
With the above findings, we next studied compound 4m, along
with the other twenty-three compounds against four leukemic cell
lines HL60, K562, MOLT-4, and U937, which are related to acute or
chronic malignant diseases, and the results are shown in Table 2. It
was found that the allyl-protected derivatives 4a, 4b, 4c, 4d, 4n, 4p,
and 4r had no activity to these cell lines, while compound 4m and
compounds 4e, 4f, 4g, 4i, 4k, and 4q, had high activity to these cell
lines, and in comparison, 4h, 4j, 4l, and 4o showed high activity
against cell lines K562 and U937 only. The methyl-protected com-
pounds 4t and 4u had high activity in suppressing the cancer cell
Fig. 2. Schematic view of H-bonding patterns for 2,5-diketopiperazine derivatives.

238
S. Liao et al. / European Journal of Medicinal Chemistry 83 (2014) 236e244
Scheme 1. Synthesis of the 2,5-diketopiperazine derivatives.
growth, whereas 4s had moderate activity. The unprotected com-
pounds 4v and 4w had no activity, while 4x and plinabulin had high
anticancer activity (Table 2). These results were similar to those for
their suppressing the growth of other six cancer cell lines.
Combining the results in Tables 1 and 2, we found that almost half
of the derivatives had high and broad-spectrum activity against
these ten cancer cell lines, and all active derivatives seemed to be
more effective against cell line U937, especially the allyl-protected
compound 4m with IC50 value of 0.5 mM.
The IC50 values of compound 4m for ten cancer cell lines were
compared as shown in Fig. 3. It was found that 4m had high
inhibitory activity to cell lines U937 (IC50 ¼ 0.5
mM) and K562
Table 1
The IC50 values (
m
M) of derivatives against cancer cell lines.
Cell linesa
Compd.
BGC-823
Hela
Huh-7
MCF-7
H1975
A549
4a
NAb
NA
NA
NA
NA
NA
4b
NA
NA
NA
NA
NA
NA
4c
NA
NA
NA
NA
NA
NA
4d
NA
NA
NA
NA
NA
NA
4e
4f
4g
4h
4i
4j
4k
4l
5.9 0.6
4.6 0.7
5.6 0.1
20.4 0.1
4.6 0.1
18.9 3.5
5.1 0.2
13.4 2.4
2.2 0.9
NA
5.4 0.3
5.5 0.9
9.1 0.3
19.3 2.0
5.1 0.8
45 12.9
4.6 0.2
6.9 1.2
1.6 0.2
NA
2.3 0.7
8.9 0.9
27.2 9.5
21.4 0.7
6.4 0.7
31.3 0.5
9.5 1.7
21.2 2.9
1.2 0.2
NA
5.4 0.2
5.2 0.1
13.7 1.7
20.7 0.3
4.7 0.3
23.0 4.0
6.5 0.9
41.1 8.0
2.1 0.6
NA
NA
5.4 0.2
5.2 0.1
19.7 6.0
22.7 1.1
5.3 0.3
NA
7.0 0.6
22.4 3.7
2.3 0.2
NA
5.1 0.1
25.6 5.2
32.8 5.4
9.2 0.5
25.1 3.9
14.5 4.1
21.5 2.8
4.5 0.5
NA
4m
4n
4o
4p
NA
NA
34.6
NA
4
NA
NA
28.1 5.0
NA
NA
NA
NA
NA
4q
4r
1.6 0.8
NA
2.9 0.7
NA
5.6 0.5
NA
3.1 0.7
NA
7.3 0.5
NA
4.9 0.1
NA
4s
4t
NA
NA
NA
9.0 6.0
0.2 0.1
0.2 0.1
NA
16.9 9.0
0.2 0.1
0.3 0.1
NA
7.0 3.0
0.3 0.1
0.2 0.1
NA
0.3 0.1
0.3 0.1
NA
0.3 0.1
0.2 0.1
NA
0.3 0.1
0.3 0.1
NA
4u
4vc
4wc
4x
NA
NA
NA
NA
NA
NA
0.09 0.04
0.009 0.005
0.080 0.007
0.08 0.02
0.010 0.001
0.11 0.02
0.09 0.02
0.031 0.006
0.10 0.01
0.10 0.01
0.010 0.002
0.060 0.001
0.10 0.02
0.011 0.001
0.21 0.02
0.09 0.02
0.010 0.002
0.050 0.001
Plinabulind
TSAd
a
Each value represents mean SD of three experiments.
NA means no activity.
These results might not be accurate because of the compounds' poor solubility.
b
c
d
Plinabulin and TSA (trichostatin A) are used as positive controls.

S. Liao et al. / European Journal of Medicinal Chemistry 83 (2014) 236e244
239
Table 2
growth of cell lines K562 and H1975 treated with 5.0 or 10.0
m
M of
The IC50 values (mM) of derivatives against four leukemic cell lines.
4m for 24 h, while the cell growth was significantly suppressed
after 48 h incubation. In comparison, the growth of cell line U937
was significantly suppressed even after 24 h incubation with 4m at
5.0 mM concentration. These results are consistent with the IC50
values as shown in Tables 1 and 2
Compd.
Cell linesa
HL60
K562
MOLT-4
U937
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
4o
4p
4q
4r
NAb
NA
NA
NA
5.2 0.5
4.5 0.3
8.89 1.0
16.7 2.1
4.2 0.9
18.5 1.7
4.6 0.4
15.7 3.0
2.0 0.2
NA
17.4 1.7
NA
2.6 0.7
NA
16.2 6.4
0.20 0.02
0.20 0.02
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
3. Conclusion
2.4 0.1
2.7 0.4
4.1 0.2
8.8 0.9
2.5 0.1
5.6 1.2
2.7 0.4
7.4 0.8
0.9 0.1
NA
5.1 0.1
NA
1.8 0.3
NA
2.2 0.2
0.11 0.01
0.11 0.01
NA
5.0 0.1
4.9 0.1
6.5 0.9
16.0 1.9
4.4 0.8
20.0 0.5
4.4 0.6
13.1 0.5
1.2 0.1
NA
1.1 0.0
1.2 0.3
2.5 0.2
5.1 0.3
0.7 0.1
5.5 0.8
1.5 0.1
5.1 0.3
0.5 0.0
NA
4.7 0.3
NA
1.0 0.0
NA
1.50 0.08
0.10 0.01
0.10 0.01
NA
A novel series of mono-protected 2,5-diketopiperazine de-
rivatives were synthesized and showed good solubility, and the
non-protected 2,5-diketopiperazine derivatives with imidazole
group as the substituent also showed good solubility. Our CCK8
assay results demonstrated that most of the active derivatives had
high and broad-spectrum anticancer activity. Among the allyl-
protected derivatives, compound 4m might become a promising
lead compound for further development for anticancer agents.
Further studies for the effects of different protective groups on the
anticancer activity of 2,5-diketopiperazine derivatives for their
quantitative structureeactivity relationship are currently in
progress.
12.9 1.8
NA
3.8 0.8
NA
4s
4t
4u
4vd
4wd
4x
ec
e
e
e
4. Experimental
NA
0.07 0.02
NA
e
NA
0.060 0.001
e
0.050 0.003
0.0060 0.0004
Plinabuline 0.010 0.001 0.0080 0.0006
e
4.1. Materials and synthetic methods
TSAe
0.08 0.01
0.41 0.01
0.030 0.002 0.10 0.01
Melting points (m.p.) were determined by using a SRSO-ptiMelt
automated melting point instrument without correction. NMR
spectra were recorded with Bruker Avance spectrometers operating
at 500 MHz for 1H, and at 125 MHz for 13C by using TMS as internal
standard. IR spectra were recorded on a Shimadzu IR Affinity-1
fourier transform infrared spectrometer. Mass spectrometry data
were collected with an HRMS-TOF instrument or a low-resolution
MS instrument by using ESI ionization. Silica gel (200e300 mesh)
and TLC plates (25 ꢂ 10 ꢂ 0.04 cm) from Qingdao Mar. Chem. Ind.
Co. Ltd were used for chromatography. All solvents were analytical
grade. DMF was dried in CaH2 and distilled. Glycine anhydride was
purchased from Alfa Aesar, trichostatin A (TSA, ꢃ98%) from Sigma
Aldrich, and plinabulin (>98%) and others from Aladdin. All
chemicals were used without further purification.
a
Each value represents mean SD of three experiments.
NA means no activity.
“e” means that the date are not obtained.
These results might not be accurate because of the compounds' poor solubility.
Plinabulin and TSA (trichostatin A) are used as positive controls.
b
c
d
e
(IC50 ¼ 0.9
lines. The relationship for the inhibitory activity versus the con-
centrations (0.01, 0.03, 0.1, 0.3, 1.0, 5.0, and 20.0 M) of compound
4m against cell lines U937, K562, and H1975 are shown in Fig. 4.
Compound 4m exhibited good concentration-dependent activity
on these three cell lines, and almost completely suppressed the cell
growth when its concentration was higher than 20
The viability assays of cancer cell lines U937, K562 and H1975
treated with compound 4m at different concentrations (5 or 10 M)
mM), without obvious activity difference to other cell
m
mM.
m
4.1.1. Synthesis of intermediate 1,4-diacetylpiperazine-2,5-dione (1)
Glycine anhydride (500 mg, 2.5 mmol) and acetic anhydride
(20 mL) were heated under reflux overnight, and the excess acetic
anhydride were removed under reduce pressure. The residue was
purified by using silica gel chromatography and intermediate 1 was
obtained as a white solid with yield of 97%. 1H NMR (500 MHz,
for 24 h or 48 h were also carried out, and the results are sum-
marized as shown in Table 3. No obvious change was found for the
CDCl3)
CDCl3)
d
¼ 4.56 (s, 4H, CH2), 2.54 (s, 6H, CH3); 13C NMR (125 MHz,
d
¼ 170.65, 165.83, 47.04, 26.58 ppm; MS (ESI) m/z
(%) ¼ 197.2 (100) [MꢀH]ꢀ.
4.1.2. General procedure for synthesis of intermediates
1-Acetyl-3-benzylidenepiperazine-2,5-dione (2a),
1-Acetyl-3-(3-bromobenzylidene)piperazine-2,5-dione (2b),
1-Acetyl-3-(2-methoxybenzylidene)piperazine-2,5-dione (2c),
and
1-Acetyl-3-((5-(tert-butyl)-1H-imidazole-4-yl)methylene)
piperazine-2,5-dione (2d).
Cs2CO3 (1.5 equiv., 4.9 g) was added into the solution containing
intermediate 1 (1.5 equiv., 3.0 g) and aromatic aldehyde (1.0 equiv.,
10 mmol) in dry DMF (20 mL), and the mixture was stirred at room
temperature for about 5 h. After the reaction was completed, the
mixture was poured into crashed ice and the solid was filtered,
washed with water for three times and dried. The target in-
termediates were obtained as white solids.
Fig. 3. A comparison for the effect of compound 4m to ten cancer cell lines.

240
S. Liao et al. / European Journal of Medicinal Chemistry 83 (2014) 236e244
4.1.3. General procedure for synthesis of intermediates
1-Acetyl-4-allyl-3-benzylidenepiperazine-2,5-dione (3a),
1-Acetyl-4-allyl-3-(3-bromobenzylidene)piperazine-2,5-dione
(3b),
1-Acetyl-4-allyl-3-(2-methoxybenzylidene)piperazine-2,5-
dione (3c), and
1-Acetyl-3-(2-methoxybenzylidene)-4-methylpiperazine-2,5-
dione (3d).
K2CO3 (2 equiv., 1.38 g) was added into the solution containing
intermediate 2 (1 equiv., 5.0 mmol) and allyl bromide (1.2 equiv.,
0.5 mL) or CH3I (1.2 equiv., 0.37 mL) in dry DMF (20 mL), and the
mixture was stirred at room temperature overnight. After the re-
action was completed, the mixture was poured into crashed ice and
the solid was filtered, washed with water for three times and dried.
The target intermediates were obtained as white solids.
4.1.3.1. 1-Acetyl-4-allyl-3-benzylidenepiperazine-2,5-dione
(3a).
¼ 7.42e7.37 (m, 3H, AreH),
Yield: 45%. 1H NMR (500 MHz, CDCl3)
d
Fig. 4. The suppression of cancer cell line growth with varying concentrations (0.01,
0.03, 0.1, 0.3, 1.0, 5.0, and 20.0 mM) of compound 4m.
7.34 (d, J ¼ 10.0 Hz, 2H, AreH), 7.29 (s, 1H, ]CeH), 5.54e5.47 (m,
1H, ]CeH), 5.01 (d, J ¼ 10.0 Hz, 1H, ]CHeH), 4.72 (d, J ¼ 15.0 Hz,
1H, ]CHeH), 4.52 (s, 2H, CH2), 4.09 (d, J ¼ 5.0 Hz, 2H, CH2), 2.61 (s,
4.1.2.1. 1-Acetyl-3-benzylidenepiperazine-2,5-dione (2a). Yield: 87%.
1H NMR (500 MHz, CDCl3)
¼ 7.99 (s, 1H, NeH), 7.46 (t, J ¼ 10.0 Hz,
2H, AreH), 7.40e7.38 (m, 3H, AreH), 7.18 (s, 1H, ]CeH), 4.51 (s, 2H,
CH2), 2.66 (s, 3H, CH3); 13C NMR (125 MHz, CDCl3)
3H, CH3); 13C NMR (125 MHz, CDCl3)
d
¼ 171.29, 164.80, 164.35,
d
132.63, 131.02, 129.67, 129.57, 129.26, 128.71, 126.64, 118.84, 46.35,
45.22, 26.61 ppm; MS (ESI) m/z (%) ¼ 285.2 (10) [MþH]þ, 307.1 (10)
[MþNa]þ, 241.3 (5).
d
¼ 172.47,
162.71, 159.95, 132.49, 129.56, 129.38, 128.52, 125.66, 119.93, 46.11,
27.19; MS (ESI) m/z (%) ¼ 245.1 (5) [MþH]þ, 267.1 (4) [MþNa]þ,
243.3 (40) [MꢀH]ꢀ, 201.3 (5).
4.1.3.2. 1-Acetyl-4-allyl-3-(3-bromobenzylidene)piperazine-2,5-
dione (3b). Yield: 28%. 1H NMR (500 MHz, CDCl3)
d
¼ 7.57 (d,
J ¼ 5.0 Hz, 1H, AreH), 7.53 (s, 1H, AreH), 7.36e7.31 (m, 2H, AreH),
7.30 (s, 1H, ]CeH), 5.61e5.53 (m, 1H, ]CeH), 5.10 (d, J ¼ 10.0 Hz,
1H, ]CHeH), 4.81 (d, J ¼ 20.0 Hz, 1H, ]CHeH), 4.58 (s, 2H, CH2),
4.14 (d, J ¼ 5.0 Hz, 2H, CH2), 2.68 (s, 3H, CH3); 13C NMR (125 MHz,
4.1.2.2. 1-Acetyl-3-(3-bromobenzylidene)piperazine-2,5-dione (2b).
Yield: 88%. 1H NMR (500 MHz, CDCl3)
d
¼ 8.02 (s, 1H, NeH), 7.52 (d,
J ¼ 5.0 Hz, 2H, AreH), 7.36e7.31 (m, 2H, AreH), 7.09 (s, 1H, ]CeH),
4.52 (s, 2H, CH2), 2.66 (s, 3H, CH3); 13C NMR (125 MHz, CDCl3)
CDCl3)
d
¼ 171.25, 164.68, 163.91, 134.82, 132.58, 132.24, 130.89,
d
¼ 172.40, 162.82, 159.59, 134.54, 132.32, 131.41, 130.99, 126.98,
130.49, 130.23, 127.99, 124.56, 122.87, 119.08, 46.63, 45.25, 26.70;
MS (ESI) m/z (%) ¼ 263.3 (15), 365.1 (14) [MþH]þ, 385.1 (15), 387.1
(15) [MþNa]þ.
126.62, 123.66, 118.1, 46.1, 27.2 ppm; MS (ESI) m/z (%) ¼ 321.2 (100),
323.2 (98) [MꢀH]ꢀ.
4.1.2.3. 1-Acetyl-3-(2-methoxybenzylidene)piperazine-2,5-dione
4.1.3.3. 1-Acetyl-4-allyl-3-(2-methoxybenzylidene)piperazine-2,5-
(2c). Yield: 69%. 1H NMR (500 MHz, CDCl3)
d
¼ 8.48 (s, 1H, NeH),
dione (3c). Yield: 44%. 1H NMR (500 MHz, CDCl3)
d
¼ 7.44 (s, 1H, ]
7.38 (t, J ¼ 10.0 Hz, 1H, AreH), 7.30 (d, J ¼ 10.0 Hz, 1H, AreH), 7.16 (s,
1H, ]CeH), 7.03 (t, J ¼ 10.0 Hz, 1H, AreH), 6.99 (d, J ¼ 5.0 Hz, 1H,
AreH), 4.46 (s, 2H, CH2), 3.93 (s, 3H, OCH3), 2.64 (s, 3H, CH3); 13C
CeH), 7.38 (t, J ¼ 10.0 Hz, 1H, AreH), 7.23 (d, J ¼ 10.0 Hz, 1H, AreH),
6.99 (t, J ¼ 10.0 Hz, 1H, AreH), 6.93 (d, J ¼ 10.0 Hz, 1H, AreH),
5.55e5.47 (m, 1H, ]CeH), 5.01 (d, J ¼ 10.0 Hz, 1H, ]CHeH), 4.73
(d, J ¼ 15.0 Hz,1H, ]CHeH), 4.51 (s, 2H, CH2), 4.02 (d, J ¼ 5.0 Hz, 2H,
CH2), 3.85 (s, 3H, OCH3) 2.63 (s, 3H, CH3); 13C NMR (125 MHz,
NMR (125 MHz, CDCl3)
d
¼ 172.59, 162.50, 160.28, 156.29, 131.10,
131.05, 125.63, 121.67, 121.56, 117.25, 111.99, 55.97, 46.11, 27.13 ppm;
MS (ESI) m/z (%) ¼ 273.2 (70) [MꢀH]ꢀ, 231.2 (35).
CDCl3)
d
¼ 171.44, 164.61, 164.43, 157.52, 131.30, 131.05, 130.08,
129.34, 122.45, 121.69, 120.41, 118.64, 110.81, 55.57, 45.77, 45.23,
26.62 ppm; MS (ESI) m/z (%) ¼ 313.2 (45), 315.2 (14) [MþH]þ, 337.2
(50) [MþNa]þ.
4.1.2.4. 1-Acetyl-3-((5-(tert-butyl)-1H-imidazole-4-yl)methylene)
piperazine-2,5-dione (2d). Yield: 42%. 1H NMR (500 MHz, CDCl3)
d
¼ 12.13 (s, 1H, NeH), 9.17 (s, 1H, NeH) 7.57 (s, 1H, AreH), 7.18 (s,
1H, ]CeH), 4.47 (s, 2H, CH2), 2.65 (s, 3H, CH3), 1.46 (s, 9H, 3CH3);
13C NMR (125 MHz, CDCl3)
4.1.3.4. 1-Acetyl-3-(2-methoxybenzylidene)-4-methylpiperazine-2,5-
d
¼ 172.81,162.45,160.53,140.82,132.31,
dione (3d). Yellow solid, yield: 60.4%. 1H NMR (500 MHz, CDCl3)
131.58, 123.90, 108.83, 46.46, 31.83, 30.70, 27.33 ppm; MS (ESI) m/z
(%) ¼ 313.3 (80) [MþNa]þ, 603.1 (50) [2 MþNa]þ, 289.4 (100)
[MꢀH]ꢀ.
d
¼ 7.44 (s, 1H, ]CeH), 7.36 (t, J ¼ 10 Hz, 1H, AreH), 7.19 (d,
J ¼ 10 Hz, 1H, AreH), 6.98 (t, J ¼ 10 Hz, 1H, AreH), 6.92 (d, J ¼ 5 Hz,
1H, AreH), 4.52 (s, 2H, CH2), 3.86 (s, 3H, OCH3), 2.83 (s, 3H, NeCH3),
Table 3
The viability of cancer cell lines treated with compound 4m.
Compd. 4m
Cells
U937
K562
H1975
Treatment
24 h
48 h
0.9 0.3
1.3 0.2
24 h
104.4 0.5
103.2 4.6
48 h
24.2 0.7
14.9 0.4
24 h
107.4 1.2
98.9 3.0
48 h
42.7 1.4
42.6 0.8
5
m
M
7.0 4.1a
8.7 3.2
10 mM
a
Each value represents mean SD of three experiments.

S. Liao et al. / European Journal of Medicinal Chemistry 83 (2014) 236e244
241
2.63 (s, 3H, CH3); 13C NMR (125 MHz, CDCl3)
d
¼ 171.58, 164.71,
CHeH), 4.29 (d, J ¼ 10.0 Hz, 2H, CH2); 13C NMR (125 MHz, CDCl3)
163.81, 157.48, 131.79, 131.05, 130.21, 121.73, 121. 42, 120.36, 110.66,
55.57, 45.30, 33.76, 26.71 ppm; MS (ESI) m/z (%) ¼ 289.1 (100)
[MþH]þ, 311.1 (50) [MþNa]þ, 599.0 (50) [2 MþNa]þ.
d
¼ 159.63, 158.98, 135.94, 134.90, 132.06, 131.88, 131.36, 131.10,
130.90, 129.98, 129.01, 127.86, 127.03, 126.97, 126.71, 122.61, 120.50,
118.43, 116.32,116.26, 48.17 ppm; FT-IR (neat): 3179, 1682, 1620,
1589, 1558, 1474, 1373, 1246 cmꢀ1; HRMS (ESI) (m/z) [MþH]þ for
(C21H17Br2N2O2) calculated 486.9651 found 486.9652.
4.1.4. General procedure for synthesis of the target 2,5-
diketopiperazine derivatives (4)
Method A: Cs2CO3 (1.5 equiv.) was added into the solution
containing intermediate 3 (1.0 equiv., 50.0 mg) and aromatic
aldehyde (1.2 equiv.) in dry DMF (1.5 mL), and the mixture was
stirred at room temperature for about 5 h. After the reaction was
completed, the mixture was slowly poured into water and extrac-
ted with AcOEt for three times, and the organic layer was dried,
filtered, and purified with silica gel column to afford the target
compound.
Method B: Cs2CO3 (1.5 equiv.) was added into the solution
containing intermediate 2 (1.0 equiv., 50.0 mg) and aromatic
aldehyde (1.2 equiv.) in dry DMF (1.5 mL), and the mixture was
stirred at 80 ꢁC for about 5 h. The purification of the target com-
pound was described as follows: compounds 4v and 4w were
recrystallized in hot DMF, and compound 4x was purified according
to a previously reported procedure by using a semi-preparative
4 .1. 4 . 4 . 1 - A l l y l - 6 - ( 3 - b r o m o b e n z y l i d e n e ) - 3 - ( 3 , 4 , 5 -
trimethoxybenzylidene)piperazine-2,5-dione (4d). According to
method A, compound 4d was synthesized as a yellow solid with a
yield of 68.3%. m.p. 163e165 ꢁC. 1H NMR (500 MHz, CDCl3)
d
¼ 8.25
(s, 1H, NeH), 7.56 (d, J ¼ 5.0 Hz, 1H, AreH), 7.54 (s, 1H, AreH),
7.37e7.32 (m, 2H, AreH,1H, ]CeH), 7.10 (s,1H, ]CeH), 6.69 (s, 2H,
AreH), 5.66e5.58 (m, 1H, ]CeH), 5.13 (d, J ¼ 10.0 Hz, 1H, ]CHeH),
4.87 (d, J ¼ 15.0 Hz, 1H, ]CHeH), 4.34 (d, J ¼ 5.0 Hz, 2H, CH2), 3.961
(s, 3H, OCH3), 3.957 (s, 6H, 2CHO3); 13C NMR (125 MHz, CDCl3)
d
¼ 159.52, 159.33, 153.84, 138.61, 136.04, 132.05, 131.74, 131.22,
129.91, 129.28, 128.12, 127.84, 125.50, 122.55, 120.02, 118.39, 118.25,
105.64, 60.91, 56.22, 48.08 ppm; FT-IR (neat): 3224, 1682, 1620,
1582, 1504, 1454, 1373, 1319, 1246 cmꢀ1; HRMS (ESI) (m/z) [MþH]þ
for (C24H24BrN2O5) calculated 499.0863 found 499.0873.
HPLC column (ODS-AQ C18, 250 ꢂ 10 mm, d.s-5
m
M, 12 nm), and
4.1.4.5. 1-Allyl-3-(3-bromobenzylidene)-6-(2-methoxybenzylidene)
piperazine-2,5-dione (4e). According to method A, compound 4e
was synthesized as a yellow solid with a yield of 52.3%. m.p.
eluted with a mixture of solvent MeOH:H2O ¼ 60:40 in 0.1%
aqueous TFA over 27 min at a flow rate of 2 mL minꢀ1. The desired
fraction was collected, concentrated through evaporation, and then
lyophilized to give the target compound.
167e169 ꢁC. 1H NMR (500 MHz, CDCl3)
d
¼ 8.01 (s,1H, NeH), 7.56 (s,
1H, AreH), 7.47 (d, J ¼ 5.0 Hz, 1H, AreH), 7.37e7.30 (m, 3H, AreH;
1H, ]CeH), 7.21 (d, J ¼ 5.0 Hz, 1H, AreH), 6.98 (d, J ¼ 5.0 Hz, 1H,
AreH), 6.97 (s, 1H, ]CeH), 6.92 (d, J ¼ 5.0 Hz, 1H, AreH), 5.58e5.50
(m, 1H, ]CeH), 5.01 (d, J ¼ 10.0 Hz, 1H, ]CHeH), 4.76 (d,
J ¼ 15.0 Hz, 1H, ]CHeH), 4.23 (d, J ¼ 5.0 Hz, 2H, CH2), 3.86 (s, 3H,
4.1.4.1. 1-Allyl-6-benzylidene-3-(3,4-dichlorobenzylidene)piperazine-
2,5-dione (4a). According to method A, compound 4a was syn-
thesized as a yellow solid with a yield of 46.3%. m.p. 155e157 ꢁC. 1H
NMR (500 MHz, CDCl3)
d
¼ 7.96 (s, 1H, NeH), 7.52 (t, J ¼ 5.0 Hz, 1H,
OCH3); 13C NMR (125 MHz, CDCl3)
d
¼ 160.20,158.85,157.35,135.16,
AreH), 7.41 (t, J ¼ 5.0 Hz, 1H, AreH), 7.37 (d, J ¼ 5.0 Hz, 1H, AreH),
7.31 (d, J ¼ 10.0 Hz,1H, AreH), 7.29 (s,1H, AreH,1H, ]CeH), 6.94 (s,
1H, ]CeH), 5.58e5.50 (m, 1H, ]CeH), 5.03 (d, J ¼ 10.0 Hz, 1H, ]
CHeH), 4.77 (d, J ¼ 15.0 Hz, 1H, ]CHeH), 4.27 (d, J ¼ 5.0 Hz, 2H, ]
131.60, 131.39, 131.32, 130.74, 130.65, 130.36, 128.09, 127.12, 127.03,
123.41, 122.70, 120.24, 118.85, 118.16, 115.38, 110.65, 55.47,
47.41 ppm; FT-IR (neat): 3186, 1682, 1620, 1558, 1485, 1458, 1381,
1353, 1246 cmꢀ1; HRMS (ESI) (m/z) [MþH]þ for (C22H20BrN2O3)
calculated 439.0652 found 439.0651.
CHeH); 13C NMR (125 MHz, CDCl3)
d
¼ 160.14, 159.03, 133.83,
133.75, 132.97, 131.37, 131.29, 130.34, 129.45, 129.06, 128.56, 127.92,
127.67, 127.32, 122.97, 118.41, 114.76, 48.09 ppm; FT-IR (neat): 3179,
1682, 1620, 1474, 1380, 1354, 1265 cmꢀ1; HRMS (ESI) (m/z) [MþH]þ
for (C21H17N2O2) calculated 399.0662 found 399.0662.
4.1.4.6. 1-Allyl-3-(3-chlorobenzylidene)-6-(2-methoxybenzylidene)
piperazine-2,5-dione (4f). According to method A, compound 4f
was synthesized as a yellow solid with a yield of 62.4%. m.p.
143e145 ꢁC. 1H NMR (500 MHz, CDCl3)
d
¼ 8.10 (s, 1H, NeH), 7.41 (s,
4.1.4.2. 1-Allyl-6-benzylidene-3-(3,4,5-trimethoxybenzylidene)piper-
azine-2,5-dione (4b). According to method A, compound 4b was
synthesized as a yellow solid with a yield of 62.3%. m.p. 151e153 ꢁC.
1H, AreH), 7.38 (t, J ¼ 10.0 Hz, 2H, AreH), 7.33e7.31 (m, 2H, AreH;
1H, ]CeH), 7.21 (d, J ¼ 5.0 Hz, 1H, AreH), 6.98 (t, J ¼ 10.0 Hz, 1H,
AreH; 1H, ]CeH), 6.92 (d, J ¼ 10.0 Hz, 1H, AreH), 5.58e5.51 (m,
1H, ]CeH), 5.01 (d, J ¼ 10.0 Hz, 1H, ]CHeH), 4.76 (d, J ¼ 15.0 Hz,
1H, ]CHeH), 4.23 (d, J ¼ 5.0 Hz, 2H, CH2), 3.86 (s, 3H, OCH3); 13C
1H NMR (500 MHz, CDCl3)
d
¼ 7.99 (s, 1H, NeH), 7.41 (t, J ¼ 10.0 Hz,
2H, AreH), 7.36 (d, J ¼ 5.0 Hz, 1H, AreH), 7.32 (d, J ¼ 5.0 Hz, 2H,
AreH), 7.29 (s, 1H, ]CeH), 7.01 (s, 1H, ]CeH), 6.62 (s, 2H, AreH),
5.58e5.51 (m, 1H, ]CeH), 5.02 (d, J ¼ 10.0 Hz, 1H, ]CHeH), 4.77
(d, J ¼ 15.0 Hz, 1H, ]CHeH), 4.28 (d, J ¼ 5.0 Hz, 2H, CH2), 3.89 (s,
NMR (125 MHz, CDCl3)
d
¼ 160.18, 158.84, 157.37, 135.37, 134.83,
131.32, 130.69, 130.60, 130.38, 128.78, 128. 51, 128.08, 127.07, 126.54,
122.69, 120.26, 118.92, 118.22, 115.50, 110.67, 55.49, 47.45 ppm; FT-
9H, 3OCH3); 13C NMR (125 MHz, CDCl3)
d
¼ 160.02, 159.51, 153.95,
IR (neat): 3232,1682, 1620, 1578, 1489, 1462, 1381, 1354, 1246 cmꢀ1
;
138.64, 133.86, 131.43, 129.44, 128.94, 128.52, 128.32, 128.24,
125.84, 122.35, 118.27, 117.90, 105.64, 60.98, 56.31, 48.03 ppm; FT-IR
HRMS (ESI) (m/z) [MþH]þ for (C22H20ClN2O3) calculated 395.1157
found 395.1163.
(neat): 3210, 1682, 1624, 1581, 1504, 1454, 1373, 1323, 1246 cmꢀ1
;
HRMS (ESI) (m/z) [MþH]þ for (C24H25N2O5) calculated 421.1758
4.1.4.7. 1-Allyl-3-(3-fluorobenzylidene)-6-(2-methoxybenzylidene)
piperazine-2,5-dione (4g). According to method A, compound 4g
was synthesized as a yellow solid with a yield of 54.2%. m.p.
found 421.1763.
4.1.4.3. 1-Allyl-3,6-bis(3-bromobenzylidene)piperazine-2,5-dione
(4c). According to method A, compound 4c was synthesized as a
yellow solid with a yield of 56.7%. m.p. 141e143 ꢁC. 1H NMR
108e110 ꢁC. 1H NMR (500 MHz, CDCl3)
d
¼ 8.56 (s, 1H, NeH), 7.42 (t,
J ¼ 5.0 Hz, 1H, AreH), 7.37 (t, J ¼ 10.0 Hz, 1H, AreH), 7.29 (s, 1H, ]
CeH), 7.24 (t, J ¼ 10.0 Hz, 2H, AreH), 7.17 (d, J ¼ 10.0 Hz, 1H, AreH),
7.05e6.98 (m, 2H, AreH; 1H, ]CeH), 6.94 (d, J ¼ 10.0 Hz, 1H,
AreH), 5.59e5.52 (m,1H, ]CeH), 5.01 (d, J ¼ 10.0 Hz,1H, ]CHeH),
4.77 (d, J ¼ 15.0 Hz, 1H, ]CHeH), 4.24 (d, J ¼ 5.0 Hz, 2H, CH2), 3.87
(500 MHz, CDCl3)
d
¼ 8.53 (s, 1H, NeH), 7.62 (s, 1H, AreH), 7.51 (t,
J ¼ 10.0 Hz, 2H, AreH), 7.47 (d, J ¼ 5.0 Hz, 1H, AreH), 7.38 (d,
J ¼ 5.0 Hz, 1H, AreH), 7.35e7.31 (m, 2H, AreH), 7.28 (t, J ¼ 10.0 Hz,
1H, AreH), 7.17 (s, 1H, ]CeH), 7.07 (s, 1H), 5.60e5.53 (m, 1H, ]
CeH), 5.08 (d, J ¼ 10.0 Hz, 1H, ]CHeH), 4.82 (d, J ¼ 20.0 Hz, 1H, ]
(s, 3H, OCH3); 13C NMR (125 MHz, CDCl3)
d
¼ 163.00 (d,
JFC ¼ 246.3 Hz),160.26,158.93,157.28,135.10 (d, JFC ¼ 7.5 Hz),131.30,

242
S. Liao et al. / European Journal of Medicinal Chemistry 83 (2014) 236e244
130.77 (d, JFC ¼ 8.8 Hz), 130.54, 130.30, 128.09, 126.91, 124.26,
122.69, 120.17, 118.61, 118.03, 115.77, 115.60 (d, JFC ¼ 3.8 Hz), 115.42
(d, JFC ¼ 2.5 Hz), 110.60, 55.39, 47.32 ppm; FT-IR (neat): 3197, 1682,
1620, 1581, 1489, 1381, 1350, 1242 cmꢀ1; HRMS (ESI) (m/z) [MþH]þ
for (C22H20FN2O3) calculated 379.1452 found 379.1455.
NMR (125 MHz, CDCl3)
d
¼ 160.03, 158.42, 157.33, 132.43, 131.44,
131.31, 130.62, 130.40, 129.64, 129.37, 128.60, 127.99, 127.92, 126.91
(q, JFC ¼ 10.0, 5.0 Hz), 123.64 (d, JFC ¼ 272.5 Hz), 122.77, 120.22,
118.88, 118.20, 113.45, 110.63, 55.45, 47.33 ppm; FT-IR (neat): 3156,
1686, 1620, 1578, 1485, 1381, 1367, 1312, 1254 cmꢀ1; HRMS (ESI) (m/
z) [MþH]þ for (C23H20N2O3) calculated 429.1421 found 429.1427.
4.1.4.8. 1-Allyl-6-(2-methoxybenzylidene)-3-(3-methylbenzylidene)
piperazine-2,5-dione (4h). According to method A, compound 4h
was synthesized as a yellow solid with a yield of 50.7%. m.p.
4.1.4.12. 1-Allyl-3,6-bis(2-methoxybenzylidene)piperazine-2,5-dione
(4l). According to method A, compound 4l was synthesized as a
yellow solid with a yield of 74.4%. m.p. 124e126 ꢁC. 1H NMR
146e148 ꢁC. 1H NMR (500 MHz, CDCl3)
d
¼ 8.05 (s, 1H, NeH),
7.36e7.31 (m, 3H, AreH), 7.22 (d, J ¼ 10.0 Hz, 2H, AreH; 1H, ]
CeH), 7.16 (d, J ¼ 10.0 Hz, 1H, AreH), 7.04 (s, 1H, ]CeH), 6.98 (t,
J ¼ 10.0 Hz, 1H, AreH), 5.59e5.51 (m, 1H, ]CeH), 5.00 (d,
J ¼ 10.0 Hz, 1H, ]CHeH), 4.76 (d, J ¼ 20.0 Hz, 1H, ]CHeH), 4.23 (d,
J ¼ 5.0 Hz, 2H, CH2), 3.85 (s, 3H, OCH3), 2.38 (s, 3H, CH3); 13C NMR
(500 MHz, CDCl3)
d
¼ 8.50 (s, 1H, NeH), 7.36e7.32 (m, 3H, AreH),
7.31 (s, 1H, ]CeH), 7.20 (d, J ¼ 5.0 Hz, 1H, AreH), 7.06 (s, 1H, ]
CeH), 7.02 (t, J ¼ 10.0 Hz, 1H, AreH), 7.00e6.95 (m, 2H, AreH), 6.90
(d, J ¼ 5.0 Hz, 1H, AreH), 5.60e5.52 (m, 1H, ]CeH), 4.99 (d,
J ¼ 10.0 Hz, 1H, ]CHeH), 4.76 (d, J ¼ 20.0 Hz, 1H, ]CHeH), 4.24 (d,
J ¼ 5.0 Hz, 2H, CH2), 3.94 (s, 3H, OCH3), 3.84 (s, 3H, OCH3); 13C NMR
(125 MHz, CDCl3)
d
¼ 160.01, 159.21, 157.35, 139.18, 132.97, 131.47,
130.52, 130.38, 129.59, 129.27, 129.02, 128.38, 126.00, 125.55,
122.87, 120.22, 118.29, 118.03, 117.40, 110.63, 55.46, 47.36,
21.44 ppm; FT-IR (neat): 3201, 1682, 1620, 1578, 1489, 1458, 1381,
1350, 1246 cmꢀ1; HRMS (ESI) (m/z) [MþH]þ for (C23H23N2O3)
calculated 375.1703 found 375.1709.
(125 MHz, CDCl3)
d
¼ 159.70, 159.44, 157.31, 156.31, 131.60, 130.93,
130.35, 128.63, 126.18, 123.02, 122.27, 121.41, 120.14, 117.89, 117.84,
114.30, 111.89, 110.58, 55.94, 55.42, 47.35 ppm; FT-IR (neat): 3271,
1682, 1624,1597,1489, 1462, 1378, 1358, 1246 cmꢀ1; HRMS (ESI) (m/
z) [MþH]þ for (C23H23N2O4) calculated 391.1652 found 391.1650.
4.1.4.9. 1-Allyl-3-(2-chlorobenzylidene)-6-(2-methoxybenzylidene)
piperazine-2,5-dione (4i). According to method A, compound 4i
was synthesized as a yellow solid with a yield of 57.9%. m.p.
4 .1. 4 .13 . 1 - A l l y l - 3 - ( 2 , 3 - d i c h l o r o b e n z y l i d e n e ) - 6 - ( 2 -
methoxybenzylidene)piperazine-2,5-dione
method A, compound 4m was synthesized as a yellow solid with a
yield of 58.2%. m.p. 134e136 ꢁC. 1H NMR (500 MHz, CDCl3)
(4m). According
to
167e169 ꢁC. 1H NMR (500 MHz, CDCl3)
d
¼ 7.86 (s, 1H, NeH), 7.49
d
¼ 8.21
(d, J ¼ 5.0 Hz, 1H, AreH), 7.44 (d, J ¼ 5.0 Hz, 1H, AreH), 7.37e7.30
(m, 3H, AreH; 1H, ]CeH), 7.22 (d, J ¼ 5.0 Hz, 1H, AreH), 7.15 (s, 1H,
]CeH), 6.98 (t, J ¼ 10.0 Hz, 1H, AreH), 6.92 (d, J ¼ 10.0 Hz, 1H,
AreH), 5.60e5.52 (m,1H, ]CeH), 5.01 (d, J ¼ 10.0 Hz,1H, ]CHeH),
4.76 (d, J ¼ 20.0 Hz, 1H, ]CHeH), 4.26 (d, J ¼ 5.0 Hz, 2H, CH2), 3.86
(s, 1H, NeH), 7.44 (d, J ¼ 5.0 Hz, 1H, AreH), 7.36 (t, J ¼ 10.0 Hz, 2H,
AreH), 7.29e7.26 (m, 1H, AreH; 1H, ]CeH), 7.21 (d, J ¼ 5.0 Hz, 1H,
AreH), 7.11 (s, 1H, ]CeH), 6.99 (d, J ¼ 10.0 Hz, 1H, AreH), 6.92 (d,
J ¼ 5.0 Hz,1H, AreH), 5.58e5.51 (m,1H, ]CeH), 5.01 (d, J ¼ 10.0 Hz,
1H, ]CHeH), 4.76 (d, J ¼ 15.0 Hz, 1H, ]CHeH), 4.25 (d, J ¼ 5.0 Hz,
(s, 3H, OCH3); 13C NMR (125 MHz, CDCl3)
d
159.97, 158.65, 157.37,
2H, CH2), 3.86 (s, 3H, OCH3); 13C NMR (125 MHz, CDCl3)
d
¼ 160.08,
134.47, 131.46, 131.37, 130.64, 130.58, 130.42, 129.97, 129.12, 128.02,
127.44, 127.39, 122.78, 120.23, 118.82, 118.25, 114.01, 110.64, 55.49,
47.40 ppm; FT-IR (neat): 3183, 1682, 1620, 1489, 1381, 1354,
1246 cmꢀ1; HRMS (ESI) (m/z) [MþH]þ for (C22H20ClN2O3) calcu-
lated 395.1157 found 395.1165.
158.48, 157.34, 134.42, 133.68, 132.69, 131.28, 130.68, 130.44, 130.39,
127.96, 127.87, 127.76, 127.45, 122.70, 120.24, 119.01, 118.28, 113.68,
110.65, 55.49, 47.41 ppm; FT-IR (neat): 3179, 1682, 1620, 1489, 1462,
1381, 1358, 1246 cmꢀ1
(C22H19Cl2N2O3) calculated 429.0767 found 429.0775.
;
HRMS (ESI) (m/z) [MþH]þ for
4.1.4.10. 1-Allyl-3-(2-fluorobenzylidene)-6-(2-methoxybenzylidene)
piperazine-2,5-dione (4j). According to method A, compound 4j
was synthesized as a yellow solid with a yield of 51.3%. m.p.
4 .1. 4 .14 . 1 - A l l y l - 3 - ( 3 , 4 - d i c h l o r o b e n z y l i d e n e ) - 6 - ( 2 -
methoxybenzylidene)piperazine-2,5-dione
method A, compound 4n was synthesized as a yellow solid with a
yield of 64.2%. m.p. 166e168 ꢁC. 1H NMR (500 MHz, CDCl3)
(4n). According
to
126e128 ꢁC. 1H NMR (500 MHz, CDCl3)
d
¼ 7.99 (s,1H, NeH), 7.43 (t,
d
¼ 8.17
J ¼ 10.0 Hz, 1H, AreH), 7.38e7.33 (m, 2H, AreH; 1H, ]CeH), 7.22 (t,
J ¼ 5.0 Hz, 2H, AreH), 7.16 (t, J ¼ 10.0 Hz, 1H, AreH), 7.04 (s, 1H, ]
CeH), 6.98 (t, J ¼ 10.0 Hz, 1H, AreH), 6.92 (d, J ¼ 10.0 Hz, 1H, AreH),
5.59e5.52 (m, 1H, ]CeH), 5.01 (d, J ¼ 10.0 Hz, 1H, ]CHeH), 4.76
(d, J ¼ 15.0 Hz,1H, ]CHeH), 4.24 (d, J ¼ 5.0 Hz, 2H, CH2), 3.86 (s, 3H,
(s, 1H, NeH), 7.53 (s, 1H, AreH), 7.51 (d, J ¼ 10.0 Hz, 1H, AreH), 7.36
(t, J ¼ 10.0 Hz, 1H, AreH), 7.31 (s, 1H, ]CeH), 7.26 (d, J ¼ 5.0 Hz, 1H,
AreH), 7.21 (d, J ¼ 5.0 Hz, 1H, AreH), 6.98 (t, J ¼ 10.0 Hz, 1H, AreH),
6.93 (s,1H, ]CeH), 6.92 (d, J ¼ 5.0 Hz,1H, AreH), 5.58e5.50 (m, 1H,
]CeH), 5.01 (d, J ¼ 10.0 Hz,1H, ]CHeH), 4.76 (d, J ¼ 15.0 Hz,1H, ]
CHeH), 4.23 (d, J ¼ 5.0 Hz, 2H, CH2), 3.86 (s, 3H, OCH3); 13C NMR
OCH3); 13C NMR (125 MHz, CDCl3)
d
¼
160.00, 159.89 (d,
JFC ¼ 247.5 Hz), 158.82, 157.35, 131.38, 130.66, 130.59, 130.38, 129.95
(125 MHz, CDCl3)
d
¼ 160.22, 158.73, 157.39, 133.64, 133.06, 132.73,
(d, JFC ¼ 2.5 Hz), 128.13, 127.49, 124.82 (d, JFC ¼ 2.5 Hz), 122.79,
131.25, 130.75, 130.39, 130.33, 127.98, 127.72, 127.35, 122.61, 120.27,
119.13, 118.29, 114.33, 110.67, 55.50, 47.46 ppm; FT-IR (neat): 3167,
1678, 1620, 1578, 1458, 1381, 1358, 1250 cmꢀ1; HRMS (ESI) (m/z)
[MþH]þ for (C22H19Cl2N2O3) calculated 429.0767 found 429.0774.
120.82 (d, JFC
¼
13.8 Hz), 120.23, 118.74, 118.14, 116.48 (d,
JFC ¼ 22.5 Hz), 110.65, 110.37, 55.47, 47.43 ppm; FT-IR (neat): 3190,
1682,1620,1578,1489, 1454,1381,1354,1246 cmꢀ1; HRMS (ESI) (m/
z) [MþH]þ for (C22H20FN2O3) calculated 379.1452 found 379.1454.
4.1.4.15. 1-Allyl-3-(5-bromo-2-fluorobenzylidene)-6-(2-
4.1.4.11. 1-Allyl-6-(2-methoxybenzylidene)-3-(2-(trifluoromethyl)
benzylidene)piperazine-2,5-dione (4k). According to method A,
compound 4k was synthesized as a yellow solid with a yield of
methoxybenzylidene)piperazine-2,5-dione
method A, compound 4o was synthesized as a yellow solid with a
yield of 67.2%. m.p. 158e161 ꢁC. 1H NMR (500 MHz, CDCl3)
¼ 8.38
(4o). According
to
d
57.1%. m.p. 134e136 ꢁC. 1H NMR (500 MHz, CDCl3)
d
¼ 7.93 (s, 1H,
(s, 1H, NeH), 7.58 (d, J ¼ 5.0 Hz,1H, AreH), 7.41e7.39 (m, 1H, AreH),
7.36 (d, J ¼ 10.0 Hz, 1H, AreH), 7.27 (s, 1H, ]CeH), 7.22 (d,
J ¼ 5.0 Hz, 1H, AreH), 7.21 (d, J ¼ 5.0 Hz, 1H, AreH), 7.04 (t,
J ¼ 10.0 Hz, 1H, AreH), 6.99 (t, J ¼ 10.0 Hz, 1H, AreH), 6.94 (s, 1H, ]
CeH), 6.92 (d, J ¼ 10.0 Hz, 1H, AreH), 5.59e5.51 (m, 1H, ]CeH),
5.02 (d, J ¼ 10.0 Hz, 1H, ]CHeH), 4.76 (d, J ¼ 15.0 Hz, 1H, ]CHeH),
4.24 (d, J ¼ 5.0 Hz, 2H, CH2), 3.87 (s, 3H, OCH3); 13C NMR (125 MHz,
NeH), 7.77 (d, J ¼ 10.0 Hz,1H, AreH), 7.62 (d, J ¼ 10.0 Hz,1H, AreH),
7.51e7.46 (m, 2H, AreH), 7.36 (t, J ¼ 10.0 Hz, 1H, AreH), 7.29 (s, 1H,
]CeH), 7.22 (t, J ¼ 10.0 Hz, 1H, AreH; 1H, ]CeH), 6.99 (t,
J ¼ 10.0 Hz,1H, AreH), 6.92 (d, J ¼ 10.0 Hz,1H, AreH), 5.59e5.51 (m,
1H, ]CeH), 5.01 (d, J ¼ 10.0 Hz, 1H, ]CHeH), 4.75 (d, J ¼ 15.0 Hz,
1H, ]CHeH), 4.26 (d, J ¼ 10.0 Hz, 2H, CH2), 3.86 (s, 3H, OCH3); 13
C

S. Liao et al. / European Journal of Medicinal Chemistry 83 (2014) 236e244
243
CDCl3)
d
¼ 160.17, 158.99 (d, JFC ¼ 248.8 Hz), 158.50, 157.37, 133.22
OCH3), 3.86 (s, 3H, OCH3), 2.97 (s, 3H, NCH3); 13C NMR (125 MHz,
CDCl3)
130.04, 126.03, 123.17, 122.28, 121.45, 120.04, 116.87, 113.92, 111.91,
110.39, 56.96, 55.42, 35.95 ppm; FT-IR (neat): 3251, 2943, 2835,
1678, 1620, 1489, 1338, 1242 cmꢀ1; HRMS (ESI) (m/z) [MþH]þ for
(C21H21N2O4) calculated 365.1496 found 395.1491.
(d, JFC ¼ 8.8 Hz), 132.52 (d, JFC ¼ 3.8 Hz), 131.28, 130.70, 130.39,
128.47, 127.93, 123.04 (d, JFC ¼ 16.3 Hz), 122.69, 120.27, 119.18,
118.24, 118.12 (d, JFC ¼ 23.8 Hz), 117.27 (d, JFC ¼ 3.8 Hz), 110.68,
108.69, 55.49, 47.46 ppm; FT-IR (neat): 3179, 1682, 1620, 1578, 1485,
1462, 1381, 1358, 1242 cmꢀ1; HRMS (ESI) (m/z) [MþH]þ for
(C22H19BrFN2O3) calculated 457.0558 found 457.0564.
d
¼ 159.47, 158.98, 157.29, 156.33, 130.96, 130.51, 130.37,
4.1.4.20. 3-(2-chlorobenzylidene)-6-(2-methoxybenzylidene)-1-
methylpiperazine-2,5-dione (4t). According to method A, com-
pound 4t was synthesized as a yellow solid with a yield of 42.6%.
4.1.4.16. 1-Allyl-3-(3-bromo-4-fluorobenzylidene)-6-(2-
methoxybenzylidene)piperazine-2,5-dione
method A, compound 4p was synthesized as a yellow solid with a
yield of 67.4%. m.p. 172e174 ꢁC. 1H NMR (500 MHz, CDCl3)
(4p). According
to
m.p. 185e187 ꢁC. 1H NMR (500 MHz, CDCl3)
d
¼ 7.93 (s, 1H, NeH),
d
¼ 7.91
7.49 (d, J ¼ 10 Hz,1H, AreH), 7.43 (d, J ¼ 10 Hz,1H, AreH), 7.36e7.31
(m, 4H, AreH, ]CeH), 7.16 (d, J ¼ 5.0 Hz, 1H, AreH), 7.13 (s, 1H, ]
CeH), 6.98 (t, J ¼ 10 Hz, 1H, AreH), 6.92 (d, J ¼ 10 Hz, 1H, AreH),
3.87 (s, 3H, OCH3), 2.99 (s, 3H, NCH3); 13C NMR (125 MHz, CDCl3)
(s, 1H, NeH), 7.62 (d, J ¼ 5.0 Hz, 1H, AreH), 7.38e7.34 (m, 2H, AreH;
1H, ]CeH), 7.22e7.18 (m, 2H, AreH), 6.98 (t, J ¼ 10.0 Hz,1H, AreH),
6.94 (s, 1H, ]CeH), 6.92 (d, J ¼ 10.0 Hz, 1H, AreH), 5.58e5.50 (m,
1H, ]CeH), 5.01 (d, J ¼ 10.0 Hz, 1H, ]CHeH), 4.77 (d, J ¼ 20.0 Hz,
1H, ]CHeH), 4.23 (d, J ¼ 5.0 Hz, 2H, CH2), 3.86 (s, 3H, OCH3); 13C
d
¼ 159.19, 158.65, 157.31, 134.44, 131.47, 130.54, 130.52, 130.35,
130.29, 129.94, 129.15, 127.39, 127.28, 122.90, 120.10, 117.77, 113.61,
110.45, 55.45, 36.00 ppm; FT-IR (neat): 3175, 3005, 2936, 1681,
1620, 1431, 1381, 1342, 1253 cmꢀ1; HRMS (ESI) (m/z) [MþH]þ for
(C20H18ClN2O3) calculated 369.1000 found 369.1005.
NMR (125 MHz, CDCl3)
d
¼ 160.21,158.84 (d, JFC ¼ 248.8 Hz),158.80,
157.39, 133.61, 131.30, 130.73, 130.67 (d, JFC ¼ 3.8 Hz), 130.37, 129.23
(d, JFC ¼ 7.5 Hz), 128.04, 126.99, 122.64, 120.27, 119.01, 118.25, 117.46,
117.28, 114.45, 110.68, 55.50, 47.45 ppm; FT-IR (neat): 3190, 1682,
1620, 1578, 1492, 1462, 1381, 1354, 1246 cmꢀ1; HRMS (ESI) (m/z)
[MþH]þ for (C22H19BrFN2O3) calculated 457.0558 found 457.0557.
4.1.4.21. 3-(2,3-dichlorobenzylidene)-6-(2-methoxybenzylidene)-1-
methylpiperazine-2,5-dione (4u). According to method A, com-
pound 4u was synthesized as a yellow solid with a yield of 39.0%.
4.1.4.17. 1-Allyl-3-(5-fluoro-2-methylbenzylidene)-6-(2-
m.p. 176e178 ꢁC. 1H NMR (500 MHz, CDCl3)
d
¼ 7.89 (s, 1H, NeH),
methoxybenzylidene)piperazine-2,5-dione
method A, compound 4q was synthesized as a yellow solid with a
yield of 55.2%. m.p. 205e207 ꢁC. 1H NMR (500 MHz, CDCl3)
(4q). According
to
7.48 (d, J ¼ 10 Hz, 1H, AreH), 7.36 (t, 2H, J ¼ 5.0 Hz, AreH), 7.32 (s,
1H, ]CeH), 7.29 (t, J ¼ 10 Hz, 1H, AreH), 7.16 (d, J ¼ 10 Hz, 1H,
AreH), 7.10 (s, 1H, ]CeH), 6.98 (t, J ¼ 10 Hz, 1H, AreH), 6.92 (d,
J ¼ 10 Hz,1H, AreH), 3.87 (s, 3H, OCH3), 2.99 (s, 3H, NCH3); 13C NMR
d
¼ 7.83
(s, 1H, NeH), 7.36 (d, J ¼ 5.0 Hz, 1H, AreH), 7.33 (s, 1H, ]CeH),
7.25e7.21 (m, 2H, AreH), 7.04 (d, J ¼ 10.0 Hz, 1H, AreH), 7.02 (s, 1H,
]CeH), 6.98 (t, J ¼ 10.0 Hz, 2H, AreH), 6.92 (d, J ¼ 10.0 Hz, 1H,
AreH), 5.59e5.51 (m,1H, ]CeH), 5.01 (d, J ¼ 10.0 Hz,1H, ]CHeH),
4.77 (d, J ¼ 20.0 Hz, 1H, ]CHeH), 4.25 (d, J ¼ 5.0 Hz, 2H, CH2), 3.86
(s, 3H, OCH3), 2.29 (s, 3H, CH3); 13C NMR (125 MHz, CDCl3)
(125 MHz, CDCl3)
d
¼ 159.17, 157.33, 134.60, 133.68, 132.75, 130.55,
130.52, 130.40, 130.18, 127.86, 127.78, 127.28, 122.80, 120.13, 118.13,
113.18, 110.48, 55.48, 36.06 ppm; FT-IR (neat): 3178, 3005, 2943,
1682, 1624, 1435, 1373, 1346, 1250 cmꢀ1; HRMS (ESI) (m/z) [MþH]þ
for (C20H17Cl2N2O3) calculated 403.0611 found 403.0605.
d
¼ 161.08 (d, JFC ¼ 245.0 Hz), 159.93, 158.74, 157.34, 133.24, 133.15
(t, JFC ¼ 3.8 Hz), 132.32 (d, JFC ¼ 8.8 Hz), 131.36, 130.57, 130.37,
128.04, 127.21, 128.81, 120.20, 118.74, 118.12, 115.52 (d,
JFC ¼ 21.3 Hz), 115.08, 115.50 (d, JFC ¼ 21.3 Hz), 110.64, 55.45, 47.38,
19.23 ppm; FT-IR (neat): 3182, 1682, 1620, 1582, 1489, 1458, 1381,
1354, 1246 cmꢀ1; HRMS (ESI) (m/z) [MþH]þ for (C23H22FN2O3)
calculated 393.1609 found 393.1616.
4.1.4.22. 3,6-Bis(2-methoxybenzylidene)piperazine-2,5-dione (4v).
According to method B, compound 4v was synthesized as a yellow
solid with a yield of 20.1%. m.p. 279e281 ꢁC. 1H NMR (500 MHz,
DMSOd6)
d
¼ 9.99 (s, 1H, NeH), 7.48 (d, J ¼ 10.0 Hz, 1H, AreH), 7.35
(t, J ¼ 10.0 Hz, 1H, AreH), 7.07 (d, J ¼ 10.0 Hz, 1H, AreH), 7.01 (t,
J ¼ 10.0 Hz, 1H, AreH), 6.84 (s, 1H, ]CeH), 3.85 (s, 3H, OCH3); 13
C
NMR (125 MHz, CDCl3)
d
¼ 157.39, 156.84, 129.99, 126.28, 121.72,
4.1.4.18. 1-Allyl-6-(2-methoxybenzylidene)-3-(4-methylbenzylidene)
piperazine-2,5-dione (4r). According to method A, compound 4r
was synthesized as a yellow solid with a yield of 46.7%. m.p.
120.61, 117.33, 111.42, 110.81, 55.56 ppm; FT-IR (neat): 3209, 1674,
1628, 1597, 1492, 1462, 1396, 1354, 1246 cmꢀ1; HRMS (ESI) (m/z)
[MþH]þ for (C20H19N2O4) calculated 351.1339 found 351.1344.
97e99 ꢁC. 1H NMR (500 MHz, CDCl3)
d
¼ 8.00 (s, 1H, NeH),
7.36e7.32 (m, 3H, AreH, 1H, ]CeH), 7.24 (d, J ¼ 5.0 Hz, 2H, AreH),
7.22 (d, J ¼ 10.0 Hz, 1H, AreH), 7.04 (s, 1H, ]CeH), 6.97 (t,
J ¼ 10.0 Hz, 1H, AreH), 6.91 (d, J ¼ 5.0 Hz, 1H, AreH), 5.59e5.51 (m,
1H, ]CeH), 5.00 (d, J ¼ 10.0 Hz, 1H, ]CHeH), 4.77 (d, J ¼ 20.0 Hz,
1H, ]CHeH), 4.23 (d, J ¼ 5.0 Hz, 2H, CH2), 3.85 (s, 3H, OCH3), 2.38
4.1.4.23. (2,3-dichlorobenzylidene)-6-(2-methoxybenzylidene)piper-
azine-2,5-dione (4w). According to method B, compound 4w was
synthesized as a yellow solid with a yield of 32.8%. m.p. >300 ꢁC. 1H
NMR (500 MHz, CDCl3)
d
¼ 7.60 (d, J ¼ 10.0 Hz, 1H, AreH), 7.53 (d,
J ¼ 5.0 Hz, 1H, AreH), 7.48 (d, J ¼ 5.0 Hz, 1H, AreH), 7.40 (t,
J ¼ 10.0 Hz, 1H, AreH), 7.36 (t, J ¼ 10.0 Hz, 1H, AreH), 7.07 (d,
J ¼ 5.0 Hz, 1H, AreH), 7.01 (t, J ¼ 5.0 Hz, 1H, AreH), 6.86 (s, 1H, ]
CHeH), 6.74 (s, 1H, ]CHeH), 3.85 (s, 3H, OCH3); Attempt to gather
13C NMR was failure; FT-IR (neat): 3190, 1678, 1632, 1574, 1492,
1466, 1400, 1354, 1250 cmꢀ1; Attempt to obtain HRMS (ESI) was
failure because of its poor solubility.
(s, 3H, CH3); 13C NMR (125 MHz, CDCl3)
d
¼ 160.07, 159.33, 157.34,
138.98, 131.48, 130.50, 130.37, 130.08, 130.05, 128.47, 128.40, 125.53,
122.86, 120.20, 118.21, 118.01, 117.44, 110.61, 55.45, 47.35,
21.33 ppm; FT-IR (neat): 3217, 1682, 1620, 1578, 1489, 1458, 1377,
1354, 1246 cmꢀ1; HRMS (ESI) (m/z) [MþH]þ for (C23H23N2O3)
calculated 375.1703 found 375.1695.
4.1.4.19. 3,6-Bis(2-methoxybenzylidene)-1-methylpiperazine-2,5-
dione (4s). According to method A, compound 4s was synthesized
as a yellow solid with a yield of 65.6%. m.p. 115e117 ꢁC. 1H NMR
4.1.4.24. 3-((5-(tert-butyl)-1H-imidazole-4-yl)methylene)-6-(2-
methoxybenzylidene)piperazine-2,5-dione
method B, compound 4x was synthesized as a yellow solid with a
yield of 48%. m.p. 264e267 ꢁC. 1H NMR (500 MHz, CDCl3)
(4x). According
to
(500 MHz, CDCl3)
d
¼ 8.53 (s, 1H, NeH), 7.37e7.32 (m, 3H, AreH),
d
¼ 11.79
7.30 (s,1H, ]CeH), 7.14 (d, J ¼ 10 Hz,1H, AreH), 7.05 (s,1H, ]CeH),
7.03 (t, J ¼ 10 Hz, 1H, AreH), 6.99 (d, J ¼ 10 Hz, 1H, AreH), 6.96 (t,
J ¼ 10 Hz, 1H, AreH), 6.90 (d, J ¼ 10 Hz, 1H, AreH), 3.95 (s, 3H,
(s, 1H, NeH), 9.90 (s, 1H, NeH), 8.17 (s, 1H, AreH), 7.42 (d, J ¼ 5.0 Hz,
1H, AreH), 7.31 (t, J ¼ 10.0 Hz, 1H, AreH), 7.03 (d, J ¼ 10.0 Hz, 1H,
AreH), 6.96 (t, J ¼ 10.0 Hz, 1H, AreH), 6.79 (s, 1H, ]CeH), 6.72 (s,

244
S. Liao et al. / European Journal of Medicinal Chemistry 83 (2014) 236e244
1H, ]CeH), 3.82 (s, 3H, OCH3), 1.33 (s, 9H, 3CH3); 13C NMR
(125 MHz, CDCl3)
129.95, 126.29, 121.75, 120.65, 119.26, 111.46, 110.69, 110.26, 99.55,
55.57, 31.81, 30.27 ppm; FT-IR (neat): 3178, 2954, 1666, 1624, 1412,
1369, 1342, 1246 cmꢀ1; HRMS (ESI) (m/z) [MþH]þ for (C20H23N4O3)
calculated 367.1765 found 367.1757.
[5] G. Rodriguez-Berna, M.J.D. Cabanas, V. Mangas-Sanjuan, M. Gonzalez-Alvarez,
I. Gonzalez-Alvarez, I. Abasolo, S. Schwartz, M. Bermejo, A. Corma, Semisyn-
thesis, cytotoxic activity, and oral availability of new lipophilic 9-substituted
camptothecin derivatives, ACS Med. Chem. Lett. 4 (2013) 86e90.
[6] S.M. Arnold, J.J. Rinehart, E. Tsakalozou, J.R. Eckardt, S.Z. Fields, B.J. Shelton,
P.A. DeSimone, B.K. Kee, J.A. Moscow, M. Leggas, A phase I study of 7-t-
butyldimethylsilyl-10-hydroxycamptothecin in adult patients with refractory
or metastatic solid malignancies, Clin. Cancer Res. 16 (2010) 673e680.
[7] J. Horn, M. Milewska, S.M. Arnold, M. Leggas, Metabolic pathways of the
camptothecin analog AR-67, Drug. Metab. Dispos. 39 (2011) 683e692.
[8] A.Y. Chen, S.J. Shih, L.N. Garriques, M.L. Rothenberg, M. Hsiao, D.P. Curran,
Silatecan DB-67 is a novel DNA topoisomerase I-targeted radiation sensitizer,
Mol. Cancer Ther. 4 (2005) 317e324.
[9] A.D. Borthwick, 2,5-Diketopiperazines: synthesis, reactions, medicinal chem-
istry, and bioactive natural products, Chem. Rev. 112 (2012) 3641e3716.
[10] A. Maity, A. Hazra, P. Palit, S. Mondal, S. Lala, N.B. Mondal, The cytotoxic effects
of diketopiperaizes against Leishmania donovani promastigotes and amasti-
gotes, Med. Chem. Res. 22 (2013) 3452e3458.
[11] C. Cornacchia, I. Cacciatore, L. Baldassarre, A. Mollica, F. Feliciani, F. Pinnen,
2,5-Diketopiperazines as neuroprotective agents, Mini Rev. Med. Chem. 12
(2012) 2e12.
d
¼ 158.45, 158.16, 156.83, 139.91, 134.17, 130.08,
4.2. Cell culture and cell viability assay
Cell lines, Hela, HL60, MCF-7, BGC-823, A549, Huh-7, K562,
U937, H1975, and Molt-4 were purchased from Shanghai Cell Bank,
Chinese Academy of Sciences. Cells were routinely grown and
maintained in mediums RPMI or DMEM with 10% FBS and 1%
penicillin/streptomycin. All cell lines were incubated in a Thermo/
Forma Scientific CO2 Water Jacketed Incubator with 5% CO2 in air at
37 ꢁC. Cell viability assay was determined by using the CCK8
[12] Y. Yamazaki, Y. Mori, A. Oda, Y. Okuno, Y. Kiso, Y. Hayashi, Acid catalyzed
monodehydro-2,5-diketopiperazine formation from N-alpha-ketoacyl amino
acid amides, Tetrahedron 65 (2009) 3688e3694.
[13] A.V. Singh, M. Bandi, N. Raje, P. Richardson, M.A. Palladino, D. Chauhan,
K.C. Anderson, A novel vascular disrupting agent plinabulin triggers JNK-
mediated apoptosis and inhibits angiogenesis in multiple myeloma cells,
Blood 117 (2011) 5692e5700.
[14] Y. Yamazaki, M. Sumikura, Y. Masuda, Y. Hayashi, H. Yasui, Y. Kiso, T. Chinen,
T. Usui, F. Yakushiji, B. Potts, S. Neuteboom, M. Palladino, G.K. Lloyd,
Y. Hayashi, Synthesis and structure-activity relationships of benzophenone-
bearing diketopiperazine-type anti-microtubule agents, Bioorg. Med. Chem.
20 (2012) 4279e4289.
(DOjinDo, Japan) assay. Cells were seeded at
a density of
400e800 cells/well in 384 well plates and treated with varying
concentrations of compounds or solution as control. After 72 h
incubation, CCK8 reagent was added, and absorbance was
measured at 450 nm using Envision 2104 multi-label Reader (Per-
kin Elmer, USA). Dose response curves were plotted to determine
the IC50 values using Prism 5.0 (GraphPad Software Inc., USA).
Conflict of interest
[15] Y. Yamazaki, K. Tanaka, B. Nicholson, G. Deyanat-Yazdi, B. Potts, T. Yoshida,
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We declare that we have no conflict of interest.
Acknowledgments
This study was supported by grants from the National Key Basic
Research Program of China (973)'s Project (2010CB833800,
2011CB915503), the National High Technology Research and
Development Program (863 Program, 2012AA092104), National
Natural Science Foundation of China (31270402, 21172230,
30973679, 41176148, 21002110), Guangdong Province-CAS Joint
Research Program (2011B090300023 and 2012B091100264), and
Guangdong Marine Economic Development and Innovation of
Regional Demonstration Project (GD2012-D01-001 and GD2012-
D01-002).
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.06.030.
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