Site selective synthesis of cytotoxic 1,3,6-trisubstituted 3,6-diunsaturated (3Z,6Z)-2,5-diketopiperazines via a one-pot multicomponent method

Article abstract of DOI:10.1016/j.tet.2015.12.073

A one-pot multicomponent approach was established for site selective synthesis of novel 1,3,6-trisubstituted 3,6-diunsaturated (3Z,6Z)-2,5-diketopiperazine derivatives with high stereoselectivity. The computational studies revealed that the steric hindran

Full text of DOI:10.1016/j.tet.2015.12.073

Accepted Manuscript
Site Selective Synthesis of Cytotoxic 1,3,6-Trisubstituted 3,6-Diunsaturated
(3Z,6Z)-2,5-Diketopiperazines via a One-Pot Multicomponent Method
Sheng-Rong Liao, Li-Juan Du, Xiao-Chu Qin, Liang Xu, Jun-Feng Wang, Xue-Feng
Zhou, Zheng-Chao Tu, Juan Li, Yong-Hong Liu
PII:
S0040-4020(15)30321-5
10.1016/j.tet.2015.12.073
TET 27399
DOI:
Reference:
To appear in: Tetrahedron
Received Date: 2 November 2015
Revised Date: 18 December 2015
Accepted Date: 28 December 2015
Please cite this article as: Liao S-R, Du L-J, Qin X-C, Xu L, Wang J-F, Zhou X-F, Tu Z-C, Li J,
Liu Y-H, Site Selective Synthesis of Cytotoxic 1,3,6-Trisubstituted 3,6-Diunsaturated (3Z,6Z)-2,5-
Diketopiperazines via a One-Pot Multicomponent Method, Tetrahedron (2016), doi: 10.1016/
j.tet.2015.12.073.
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ACCEPTED MANUSCRIPT
Graphical Abstract
Site selective synthesis of cytotoxic 1,3,6-
trisubstituted 3,6-diunsaturated (3Z,6Z)-2,5-
diketopiperazines via a one-pot
Leave this area blank for abstract info.
multicomponent method
Sheng-Rong Liaoa, †, Li-Juan Dub, †, Xiao-Chu Qinc, Liang Xud, Jun-Feng Wanga, Xue-Feng Zhoua, Zheng-Chao
Tuc, Juan Lib,*, and Yong-Hong Liua,*
a CAS Key Laboratory of Tropical Marine Bio-resources and Ecology, Guang dong Key Laboratory of Marine
Materia Medica, Research Center for Marine Microbes, South China Sea Institute of Oceanology, Chinese
Academy of Sciences, Guangzhou 510301, China
b Department of Chemistry, Jinan University, Guangzhou 510632, China.
c Laboratory of Molecular Engineering and Laboratory of Natural Product Synthesis, Guangzhou Institutes of
Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China
d Enantiotech Corp., Ltd., Zhongshan Torch Hi-Tech, Industrial Development Zone, Zhongshan 528437, China

ACCEPTED MANUSCRIPT
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Tetrahedron
journal homepage: www.elsevier.com
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Site Selective Synthesis of Cytotoxic 1,3,6-Trisubstituted 3,6-Diunsaturated (3Z,6Z)-
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2,5-Diketopiperazines via a One-Pot Multicomponent Method
Sheng-Rong Liaoa,†, Li-Juan Dub, †, Xiao-Chu Qinc, Liang Xud, Jun-Feng Wanga, Xue-Feng Zhoua, Zheng-
Chao Tuc, Juan Lib,*, and Yong-Hong Liua,*
a CAS Key Laboratory of Tropical Marine Bio-resources and Ecology, Guang dong Key Laboratory of Marine Materia Medica, Research Center for Marine
Microbes, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou 510301, China
b Department of Chemistry, Jinan University, Guangzhou 510632, China.
cLaboratory of Molecular Engineering and Laboratory of Natural Product Synthesis, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of
Sciences, Guangzhou 510530, China
d Enantiotech Corp., Ltd., Zhongshan Torch Hi-Tech, Industrial Development Zone, Zhongshan 528437, China
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
A one-pot multicomponent approach was established for site selective synthesis of novel 1,3,6-
trisubstituted 3,6-diunsaturated (3Z,6Z)-2,5-diketopiperazine derivatives with high
stereoselectivity. The computational studies revealed that the steric hindrances between the 2-
hydrogen atoms on the aromatic rings and the carbonyl, as well as the steric repulsions between
the hydrogen atoms of the CH group in the benzylidene and the CH2 group in the N-alkylative
part might be responsible for the Z/E selectivity. Compound (3Z,6Z)-3h (IC50 = 11 nM) has a
close activity to the positive compound plinabulin (IC50 = 15 nM) against the cancer cell line
HL60.
Available online
Keywords:
2,5-diketopiperazines
one-pot
2009 Elsevier Ltd. All rights reserved.
multicomponent
siteselectivity
Adol condensation
1. Introduction
Natural or synthetic N-Monoalkylated 3,6-diunsaturated 2,5-
diketopiperazine derivatives (2,5-DKPs) represent a unique kind
of bioactive compounds1 (Figure 1) as they can serve as
modulators against multidrug resistance (MDR),2 inhibitors
against plasminogen activator inhibitor-1 (PAI-1),3 agents against
biofouling4 or cancer,5 etc. The general protocol for synthesis of
the above 2,5-DKPs often consists of three independent steps
(Scheme 1) via Aldol condensations of 1,4-diacetyl-2,5-DPK
with aromatic aldehydes and the alkylations of halohydrocarbons
(such as CH3I or allyl bromide) on the nitrogen atoms.3,5a,4,6
However, these operations are complicated and give low total
yields of target 3. Additionally, the purification of intermediate 1
is troublesome due to the enolization of amide group at 4-
positon.7 Therefore, exploring new convenient ways for efficient
synthesis of these N-monoalkylated 2,5-DKPs with structural and
bioactive diversities are necessary.
Figure 1. Representative 3,6-Diunsaturated 2,5-Diketopiperazines.
One-pot synthetic strategy has become a powerful tool for
rapid access to natural product derivatives in recent years.8
However, employing this strategy to the synthesis of these N-
monoalkylated 3,6-diunsaturated 2,5-DKPs might come across
difficulties: for instance the methylene groups at 3- or 6-positions
on the 2,5-DKP ring would probably compete to react with
different aromatic aldehydes, and also with the halohydrocarbons;
and the nitrogen and oxygen atoms of the amide could possibly
react with halohydrocarbons due to enolization of amide.9
Theoretically, there will be four possible stereoisomers with two
double bonds formed during reaction. Interestingly, only one or
two isomers were often observed and isolated from the natural
* Corresponding author. E-mail address: yonghongliu@scsio.ac.cn
(Y.-H. Liu); tchjli@jnu.edu.cn (J. Li)
† Both authors contributed equally to this work.

2
Tetrahedron
product 2,5-DPKs.10 This phenomenon inspired us that higher
computational study was carried out. Our computational
ACCEPTED MANUSCRIPT
stereoselectivity could be achieved in 2,5-DKPs through one-pot
synthesis, possibly, by controlling the reaction temperature or
changing the order of the starting material addition.
calculations rationalized that the steric
Table 1. Optimization of the reaction conditionsa.
7,9
1
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3
4
5
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Temp(°C)/
Time(h)
rt/24
Benzaldehyde Yield(%)b
Entry
Base (equiv)
(equiv)
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.5
3.0
1a/2a/3a/4a
21/24/0/0
33/45/0/0
0/39/0/3
0/59/0/5
0/53/0/5
0/47/0/7
0/0/39/4
0/77/0/0
0/68/0/0
0/49/0/7
0/0/49/0
0/0/48/0
0/0/38/0
0/0/55/0
0/0/48/0
1c
2c
3d
4
NaOH(3.0)
Cs2CO3(3.0)
NaOH(3.0)
Cs2CO3(3.0)
K2CO3(3.0)
DBU(3.0)
rt/24
20 Scheme 1. Routes to N-monoalkylated unsaturated 2,5-diketopiperazines.
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rt/3
rt/4
2. Results and discussion
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5
rt/6
7,9
With the above questions and the previous findings
in
6
rt/4
mind, the reaction was carried out by reacting 1,4-diacetyl-2,5-
DPK with allyl bromide and benzaldehyde in one pot using
NaOH or Cs2CO3 as the condensation base. When the molar ratio
of allyl bromide to 1,4-diacetyl-2,5-DPK was 1.8:1, the reaction
was incomplete and the products were the mixture of
intermediates (3Z)-1a and (3Z)-2a. No improvement was
observed even with the prolonged reaction time (24 h) (Table 1,
entries 1 and 2). Therefore, larger amount of the allyl bromide
(2.5 equiv.) was used but still no target compound was measured.
The products were isolated and identified to be major (3Z)-2a
which was accompanied by an orange compound 4a, while
Cs2CO3 had the best efficacy to promote the formation of (3Z)-2a
(entries 3-6). Surprisingly, compound 4a (identified by X-ray
analysis) was a site isomer of the target compound 3a, that is, the
allyl group was alkylated on the oxygen atom, not on the nitrogen
atom, similar to the natural products neihumicin,
methoxyneihumicin,11 and nocazines A and B.12 Then the (3Z)-2a
contained mixture was moved to heat at 95 °C and we were
pleased to find that the target compound 3a was obtained
smoothly (entry 7), nevertheless, a small amount of compound 4a
was still observed. Effort was made to improve the yield of the
target compound 3a and achieve a higher N-site selectivity. The
reaction was performed firstly under a low temperature (- 10 °C,
entries 8-10) and we found that Cs2CO3 still had the most
efficacy to produce compound (3Z)-2a as the sole product (entry
8). According to these results, the reaction mixture was stirred at
- 10 °C using Cs2CO3 as the base, until the completion of the 1st
Adol condensation and the alkylation of allyl bromide, and then
heated at 95 °C with the target compound 3a as the only product
(entry 11). These results doubtlessly indicated that low
temperature could realize the high site selectivity and was
beneficial for the N-alkylation in this one-pot reaction, thus
resulted in higher yield of the target compound. Meanwhile
compound 3a possessed a (3Z,6Z)-configuration whereas other
stereoisomers of (3Z,6Z)-3a were not measured, and this
unexpected outcome pushed us to make clear the reasons and a
7
Cs2CO3(3.0)
Cs2CO3(3.0)
K2CO3(3.0)
DBU(3.0)
rt/4; 95/3
-10/4
8
9
-10/16
10
11
12
13
14
15
-10/6
Cs2CO3(3.0)
Cs2CO3(3.0)
Cs2CO3(2.0)
Cs2CO3(2.5)
Cs2CO3(2.5)
-10/4; 95/3
-20/4; 95/3
-10/4; 95/3
-10/4; 95/3
-10/4; 95/3
a Reaction conditions: 1.0 equiv. of 1,4-diacetyl-2,5-DPK (0.25 mmol) was
used. The reaction was mixed with 4Å MS in 2 mL dry DMF under a N2
atmosphere. The reaction was firstly stirred at -10 °C until the completion of
the 1st Aldol condensation and the alkylation of the allyl bromide, and then
heated at 95 °C for about 4 h.
b Isolated yield based on themselves as the products.
c 1.8 equiv. of allyl bromide was used.
d 2.5 equiv. of allyl bromide was used in entries 3-15.
hindrance between the 2-hydrogen of the aromatic group and the
carbonyl might be responsible for the origin of the E/Z selectivity
(2.243 Å for O1···H2 in complex (3Z,6Z)-13A, while 1.988 Å for
O1···H1 in complex (3E,6Z)-13A, Figure 2, or see the
Supporting Information Figure S3), in accordance with Ando S.
& co-worker’s study.9 And our further study demonstrated that
the steric repulsion between the hydrogen atoms in benzylidene
(the CH group) and in the N-alkylative part (the CH2 group)
might be another reason (1.952 Å for H2···H5 in complex
(3E,6Z)-13A while 2.304 Å for H1···H5 in complex (3Z,6Z)-13A,
Figure 2, or see the Supporting Information Figure S3). In
addition, the product of halohydrocarbon substituted on 3- or 6-
position was also not monitored. We speculated that the
contribution of the acetyl group resulted in the condensation of
aromatic aldehydes, other than the alkylation of the allyl group,

on the 3- or 6-position more easily.9 And the optimal reaction
conditions were established: using Cs2CO3 as the base, the molar
(3Z,6Z)-3k
4-CF3Bn
4-Cl
47
ACCEPTED MANUSCRIPT
a Isolated yield
ratios of Cs2CO3, allyl bromide, and benzaldehyde to 1,4-
diacetyl-2,5-DPK were 2.5:1, 2.5:1, and 2.5:1, respectively, and
the reaction was firstly stirred at -10 °C until the completion of
1st Aldol condensation and the alkylation of the allyl bromide on
(3Z)-1a, then heated at 95 °C in DMF with the product (3Z,6Z)-
3a 55% of yield (entry 14).
Table 3. Ratios screening experiments on the synthesis of the compound
(3Z,6Z)-3la.
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Benzaldehyde
(equiv)
Yield(%)b
Entry
(3Z,6Z)-3l
1.0
1
2
3
4
39
38
49
36
1.5
2.0
2.5
Figure 2. The distances (Å) for the selected atoms in the complex
(3Z,6Z)-13A (for compound (3Z,6Z)-3a) and in complex (3E,6Z)-13A
(for compound (3E,6Z)-3a) calculated by DFT.
A variety of aromatic aldehydes or halohydrocarbons were
subjected to the above one-pot method, and the results were
summarized in Table 2. Using 3- or 4-substituted aromatic
aldehydes, the reactions showed good site- and stereoselectivities
to form (3Z,6Z)-3 with yields ranging from 31-64%. However,
when used 2-substituted aromatic aldehydes (3b, 3c, 3j) as the
modifying functions, the products were (3Z,6Z)-3 with trace
amount of site isomers (3Z,6Z)-4, which indicated that 2-
substituted aromatic aldehydes had a negligible steric hindrance
which influenced the alkylation of halohydrocarbon selectively
a Reaction conditions: 1,4-diacetyl-2,5-DPK (0.25 mmol), benzaldehyde (0.25
mmol), allyl bromide (0.63 mmol), Cs2CO3 (0.63 mmol), and 4Å MS were
mixed in 2 mL dry DMF under a N2 atmosphere. The reaction was firstly
stirred at -10 ºC until the completion of the 1st Aldol condensation and the
alkylation of the allyl bromide, and then 3-bromobenzaldehyde (1.0 – 2.5
equiv.) was added and the mixture was heated at 95 ºC for about 4 h.
b Isolated yield.
The strategy was further employed to the synthesis of
derivatives with two different side aromatic groups substituted at
3- and 6-positions. In order to realize the site selectivity of
different aromatic aldehydes, the amount of Cs2CO3 and allyl
bromide were kept unchangeable according to the above optimal
conditions, while the addition order of the two different
aldehydes was revised by adding the second aldehyde after the
first one had completely reacted with 1,4-diacetyl-2,5-DPK. The
amount of the first aldehyde was maintained at 1.0 equivalent in
avoidance of the formation of the by-products which condensed
the same aldehydes at the 3- and 6-positions, and the molar ratios
of the second aromatic aldehyde were investigated for high yield
of the derivatives. This goal was achieved based on the template
synthesis of compound (3Z,6Z)-3l which used benzaldehyde and
3-bromobenzaldehyde as the first and the second aldehydes,
respectively (Table 3).
30 on the nitrogen or oxygen atom. And the results also showed that
distinct N-alkylative groups had little influence on the stereo- or
siteselectivity in the synthesis of the alkylated 2,5-DKPs.
Similarly, different aromatic aldehydes seemed to have little
influence on the yields of the derivatives. This one-pot strategy
was then used to synthesize the natural product piperafizine A,
and the formation of this cytotoxic compound was accomplished
easily in 47% yield under the mild conditions.
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Table 2. Scope of the reaction containing three components.
Table 4. Scope of the reaction containing four components.
Compounds
(3Z,6Z)-3a
(3Z,6Z)-3b
(3Z,6Z)-3c
(3Z,6Z)-3d
(3Z,6Z)-3e
(3Z,6Z)-3f
(3Z,6Z)-3g
(3Z,6Z)-3h
(3Z,6Z)-3i
Piperafizine A
(3Z,6Z)-3j
R
R1
Yielda(%)
Allyl
Allyl
Allyl
Allyl
Allyl
Allyl
Allyl
Me
H
55
53
46
44
45
58
64
56
31
47
45
2-Me
2-F
3-F
Compounds
(3Z,6Z)-3m
(3Z,6Z)-3n
(3Z,6Z)-3o
(3Z,6Z)-3p
(3Z,6Z)-3q
(3Z,6Z)-3r
(3Z,6Z)-3s
R
R1
R2
Yield(%)a
3-Cl
3-Br
4-CF3
3-MeO
4-MeO
H
Allyl
Allyl
Allyl
Me
2-F
3-F
2-MeO
H
2,3-(Cl)2
2,3-(Cl)2
2,3-(Cl)2
3-Cl
43
51
45
50
49
60
38
Me
Me
H
3-Br
Me
Me
H
2,3-(Cl)2
3-Cl
Bn
2-Cl
Bn
H

4
Tetrahedron
ACCEPTED MANUSCRIPT
c “-”: Not examined.
(3Z,6Z)-3t
4-CF3Bn
4-Br
3-Me
52
a Isolated yield
4. Conclusion
Following these changes, another eight 2,5-diketopiperazine
derivatives (3Z,6Z)-3m-t were synthesized smoothly with
moderate yields (38-60%) (Table 4). Likewise, trace amount of
O-site isomer (3Z,6Z)-4o was also observed in the preparation of
the known compound (3Z,6Z)-3o,5a which was consistent with
the above examples that 2-substituted aromatic aldehydes
showed a negligible steric hindrance which influenced the site
selectivity of the alkylation.
1
2
3
4
5
6
7
8
9
In conclusion,
a
facile and efficient approach for
multicomponent one-pot synthesis of N-monoalkylated 2,5-DPKs
was developed with total yields highly improved (for example
the total yield of compound (3Z,6Z)-3o was improved from
17%5a to 45%). This method could achieve three or more site
selective modifications on the 2,5-DPK ring, and the synthetic
compounds were more preferred to form Z-configuration at 3, 6-
positions. The compound (3Z,6Z)-3h might be a new template for
further development as anticancer agent.
3. Cytotoxic study
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The cytotoxic results were summarized in Table 5. As shown
in Table 5, nearly half of these compounds ((3Z,6Z)-3a–c, 3h, 3j,
3n–q) displayed strong cytotoxic activities against one to five of
the cancer cell lines, and different compounds exhibited distinct
inhibitory activities to the cancer cells. From the results, methyl
was the best favorable substitutive group for the cytotoxicities of
compounds (such as (3Z,6Z)-3h, (3Z,6Z)-3p–r, and piperafizine
A). Allyl was also a better one (such as (3Z,6Z)-3n, (3Z,6Z)-3a–
c) but less effective than the methyl group, however benzyl
groups (benzyl, 4-trifluoromethylbeznyl) seemed to be not
suitable substitutive functions. The structure － activity
relationship was ambiguous because the substitutive phenyl rings
or the positions of the substituents on the phenyl rings for the
derivatives resulted in inconsistent cytotoxicities. Compared to
plinabulin, all the bioactive compounds showed less potency,
except compound (3Z,6Z)-3h (IC50 = 11 nM) which has a close
activity to plinabulin (IC50 = 15 nM)5e against the cancer cell line
HL60. Nevertheless compared to the compound TSA
(trichostatin A), compound (3Z,6Z)-3h (11 and 104 nM) has
stronger cytotoxicities than TSA (42 and 120 nM) against the
cancer lines HL60 and K562, respectively.
5. Experimental Section
5.1. General Chemical Methods and Materials
All reactions were carried out with 4Å molecular sieve (4Å
MS, activated at 500 °C before to use) in dry solvent under a
nitrogen atmosphere. The anhydrous DMF were purchased from
Acros Organics and stored under nitrogen. Unless otherwise
noted, other chemicals obtained from commercial suppliers were
used without further purification. Melting points (m.p.) were
determined by using a SGW-X4 melting point instrument
(INESA Com. Ltd.) without correction. Analytical thin layer
chromatography was performed on Polygram SIL HSGF254
plates. Visualization was accomplished with short wave UV
light, or I2 staining. Flash column chromatography was
performed using silica gel (200-300 mesh). The NMR spectra
were recorded on a Bruker AC 500 NMR spectrometer with TMS
as an internal standard. The data are reported as follows:
chemical shift, integration, multiplicity (s = singlet, d = doublet, t
= triplet, q = quartet, and m = multiplet), and coupling constant in
Hz. HR-ESI-MS data were measured on AQUITY UPLC/Q-TOF
mass spectrometer.
Table 5. Cytotoxicities of compounds (3Z,6Z)-3a–t, Piperafizine A ,
plinabulin, and TSA against cancer cell linesa
5.2. General procedure for the synthesis of products (3Z,6Z)-3a–
k.
Cancer Cell Lines: IC50 (µM)
Compound
(3Z,6Z)-3a
(3Z,6Z)-3b
(3Z,6Z)-3c
(3Z,6Z)-3d
(3Z,6Z)-3e
(3Z,6Z)-3f
(3Z,6Z)-3g
(3Z,6Z)-3h
U937
HL60
6.9±2.5
1.7±0.0
DU145
NA
4.3±1.3
10.4±0.1
NA
NA
NA
NA
0.272±
0.047
NA
NA
NA
HT29
NA
2.6±0.6
11.5±1.4
NA
NA
NA
NA
0.22±
0.05
NA
NA
NA
K562
2.1±0.3
NA
NA
NA
NA
NA
NA
0.104±
0.012
NA
26.1±0.0
NA
In a 25 mL two-neck flask saturated with nitrogen, 1,4-
diacetyl-2,5-diketopiperazine (50 mg, 0.25 mmol, 1.0 equiv.),
aldehydes (0.63 mmol, 2.5 equiv.), halohydrocarbons (0.63
mmol, 2.5 equiv.), Cs2CO3 (205 mg, 0.63 mmol, 2.5 equiv.), and
4Å MS (200mg) in 2 mL dry DMF were added. The reaction was
firstly stirred at -10 °C until the completion of the 1st Aldol
condensation and the alkylation of the halohydrocarbons, and
then heated at 95°C for about 4 h (monitored with TLC analysis).
The solvent was removed under the reduced pressure, and water
(50 mL) and EtOAc (20 mL) were added. The mixture was
extracted with EtOAc (20 mL × 3). The organic layer was dried
over Na2SO4, filtered, and removed. The residues were purified
by flash column chromatography on silica to afford the pure
product (3Z,6Z)-3 as a slightly yellow oil or solid.
NAb
3.1±0.3
16.0±7.9 13.4±1.5
NA
NA
NA
NA
0.091±
0.006
NA
NA
NA
NA
NA
0.011±
0.001
NA
(3Z,6Z)-3i
(3Z,6Z)-3j
(3Z,6Z)-3k
(3Z,6Z)-3l
20.3±7.4 2.5±0.1
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
(3Z,6Z)-3m NA
(3Z,6Z)-3n
(3Z,6Z)-3o
(3Z,6Z)-3p
(3Z,6Z)-3q
(3Z,6Z)-3r
(3Z,6Z)-3s
(3Z,6Z)-3t
(3Z,6Z)-4a
Piperafizine
A
15.5±1.1 1.5±0.0
0.5 ± 0.0 2.0 ± 0.2
NA
-
0.9±0.2
1.3±0.7
4.2±0.1
NA
NA
-
NA
0.9 ± 0.1
c
0.2±0.0
0.2±0.0
2.7±0.4
NA
3.2±0.1
1.2±0.0
3.1±0.1
NA
0.71±0.05 0.28±0.01
1.04±0.07 0.38±0.04
2.6±0.1
NA
NA
NA
5.2.1. (3Z,6Z)-4-allyl-3,6-dibenzylidenepiperazine-
2,5-dione ((3Z,6Z)-3a)
Following the general procedure, the compound (3Z,6Z)-3a
was obtained in 55% yield as a slightly yellow oil and later
1.2±0.2
NA
NA
NA
NA
NA
NA
NA
NA
NA
1
changed into solid in about one week. mp = 106 – 108 ˚C. H
1.4±0.0
NA
9.4±0.9
6.5±0.9
2.9±0.2
NMR (500 MHz, CDCl3) δ: 8.22 (s, 1H), 7.44 (d, J = 5.0 Hz,
4H), 7.40 (t, J = 5.0 Hz, 2H), 7.36 – 7.34 (m, 2H), 7.32 (d, J =
5.0 Hz, 2H), 7.26 (s, 1H), 7.08 (s, 1H), 5.58 – 5.50 (m, 1H), 5.01
(dd, J = 10.2, 1.1 Hz, 1H), 4.77 (dd, J = 17.1, 1.2 Hz, 1H), 4.28
(d, J = 5.0 Hz, 2H). 13C NMR (125 MHz, CDCl3) δ: 160.3, 159.7,
134.0, 133.1, 131.6, 129.6, 129.1, 128.75, 128.68, 128.4, 126.2,
122.5, 118.5, 118.0, 48.2. NOE correlations between hydrogen
atoms in allyl group (δ: 4.28, d, J = 5.0 Hz, 2H, CH2) and in
Plinabulin
0.0068±
0.0003
0.047±
0.004
0.015±
0.001
0.042±
0.001
0.0140±
0.0005
0.044±
0.003
0.013±
0.001
0.056±
0.003
0.0063±
0.0007
0.12±
0.02
TSA
a Each value represents mean ± SD of three experiments. Plinabulin and TSA
(trichostatin A) are used as positive controls.
b NA: Not active.

phenyl group (δ: 7.36 – 7.34, m, 2H, Ph–H), as well as hydrogen
4.79 (d, J = 17.1 Hz, 1H), 4.27 (d, J = 10.0 Hz, 2H). 13C NMR
ACCEPTED MANUSCRIPT
atoms in amide (δ 8.22, s, 1H, NH-1) and in phenyl group (δ:
7.44, d, J = 5.0 Hz, 2H, Ph–H) were observed. HRMS (ESI): m/z
calcd for C21H19N2O2 [M+H]+ 331.1441, found 331.1445; for
C21H18N2O2Na [M+Na]+ 353.1260, found 353.1264.
(126 MHz, CDCl3) δ: 163.3 (d, JFC = 246.3 Hz), 162.7 (d, JFC
247.5 Hz,), 160.1, 159.4, 136.2 (d, JFC = 8.0 Hz), 135.1 (d, JFC
=
=
8.0 Hz), 131.4, 131.2 (d, JFC = 8.4 Hz), 130.3 (d, JFC = 8.3 Hz),
129.1, 126.8 , 125.4 (d, JFC = 2.8 Hz), 124.6 (d, JFC = 2.8 Hz),
121.0, 118.6, 116.9, 116.4 (d, JFC = 22.0 Hz), 116.1 (d, JFC = 8.1
Hz), 116.0 (d, JFC = 8.1 Hz), 115.8 (d, JFC = 22.0 Hz), 48.3.
HRMS (ESI): m/z calcd for C21H17F2N2O2 [M+H]+ 367.1253,
found 367.1266; for C21H16F2N2O2Na [M+Na]+ 389.1072, found
389.1083.
1
2
3
4
5
6
7
8
9
5.2.2. (3Z,6Z)-5-(allyloxy)-3,6-dibenzylidene-1,6-
dihydropyrazin-2(3H)-one ((3Z,6Z)-4a)
Following the general procedure but changing the firstly
stirred temperature of the reaction from -10 °C to room
temperature, and then the reaction mixture was heated at 95 °C
for about 4 h. the compound (3Z,6Z)-4a accompanied with the
compound (3Z,6Z)-3a was obtained as an orange crystal in 5%
5.2.6. (3Z,6Z)-4-allyl-3,6-bis(3-
chlorobenzylidene)piperazine-2,5-dione ((3Z,6Z)-
3e)
1
yield. mp = 147 – 149 ˚C. H NMR (500 MHz, DMSO) δ: 10.14
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
Following the general procedure, the product (3Z,6Z)-3e was
obtained in 45% yield as a slightly yellow solid. mp = 132 – 134
˚C. H NMR (500 MHz, CDCl3) δ: 8.62 (s, 1H), 7.44 (s, 1H),
(s, 1H), 8.10 (d, J = 10.0 Hz, 2H), 7.53 (d, J = 5.0 Hz, 2H), 7.42
(q, J = 10.0 Hz, 4H), 7.36 – 7.30 (m, 2H), 7.13 (s, 1H), 6.57 (s,
1H), 6.20 – 6.12 (m, 1H), 5.51 (d, J = 15.0 Hz, 1H), 5.33 (d, J =
10.0 Hz, 1H), 4.94 (d, J = 5.0 Hz, 2H). 13C NMR (125 MHz,
DMSO) δ: 159.4, 153.9, 134.8, 133.2, 132.8, 132.1, 131.3, 129.3,
128.8, 128.7, 128.4, 128.1, 126.0, 123.7, 118.2, 111.3, 67.3.
HRMS (ESI): m/z calcd for C21H19N2O2 [M+H]+ 331.1441, found
331.1441; for C21H18N2O2Na [M+Na]+ 353.1260, found
353.1259.
1
7.38 – 7.34 (m, 3H), 7.31 – 7.28 (m, 3H), 7.20 – 7.18 (m, 1H),
7.10 (s, 1H), 7.01 (s, 1H), 5.57 – 549 (m, 1H), 5.04 (dd, J = 10.2,
0.8 Hz, 1H), 4.78 (dd, J = 17.1, 0.9 Hz, 1H), 4.26 (d, J = 5.0 Hz,
2H). 13C NMR (125 MHz, CDCl3) δ: 160.2, 159.3, 135.9, 135.4,
134.8, 134.7, 131.3, 130.7, 129.9, 129.4, 129.2, 129.1, 129.0,
128.8, 127.6, 127.0, 126.9, 120.8, 118.6, 116.8, 48.3. HRMS
(ESI): m/z calcd for C21H17Cl2N2O2 [M+H]+ 399.0662, found
399.0664; for C21H16Cl2N2O2Na [M+Na]+ 321.0481, found
421.0480.
5.2.3. (3Z,6Z)-4-allyl-3,6-bis(2-
methylbenzylidene)piperazine-2,5-dione ((3Z,6Z)-
3b)
5.2.7. (3Z,6Z)-4-allyl-3,6-bis(3-
bromobenzylidene)piperazine-2,5-dione ((3Z,6Z)-
3f)
Following the general procedure, the product (3Z,6Z)-3b was
obtained in 53% yield as a slightly yellow oil and changed into
solid in about two weeks. mp = 80 – 83 ˚C. 1H NMR (500 MHz,
DMSO) δ: 10.36 (s, 1H), 7.48 (t, J = 5.0 Hz, 1H), 7.33 – 7.19 (m,
7H), 7.10 (s, 1H), 6.92 (s, 1H), 5.52 – 5.44 (m, 1H), 4.97 (d, J =
10.2 Hz, 1H), 4.55 (d, J = 17.1 Hz, 1H), 4.08 (d, J = 5.0 Hz, 2H),
Following the general procedure, the product (3Z,6Z)-3f was
obtained in 58% yield as a slightly yellow solid. mp = 114 – 116
1
˚C. H NMR (500 MHz, CDCl3) δ: 8.72 (s, 1H), 7.64 (s, 1H),
29 2.28 (s, 3H), 2.23 (s, 3H). 13C NMR (125 MHz, DMSO) δ: 159.6,
7.52 (d, J = 10.0 Hz, 1H), 7.45 (d, J = 15.0 Hz, 2H), 7.38 (d, J =
10.0 Hz, 1H), 7.34 – 7.29 (m, 2H), 7.26 (d, J = 10.0 Hz, 1H),
7.10 (s, 1H), 7.03 (s, 1H), 5.59 – 5.52 (m, 1H), 5.07 (d, J = 10.2
Hz, 1H), 4.81 (d, J = 17.1 Hz, 1H), 4.28 (d, J = 10.0 Hz, 2H). 13C
NMR (125 MHz, CDCl3) δ: 160.3, 159.3, 136.1, 135.2, 132.2,
132.0, 131.8, 131.6, 131.3, 130.9, 130.2, 129.2, 128.0, 127.6,
127.0, 123.5, 122.8, 120.6, 118.6, 116.9, 48.3. HRMS (ESI): m/z
calcd for C21H17Br2N2O2 [M+H]+ 486.9651, found 486.9654; for
C21H16Br2N2O2Na [M+Na]+ 508.9471, found 508.9480.
159.4, 137.0, 136.7, 133.5, 132.2, 132.0, 130.3, 130.1, 129.4,
129.1, 128.7, 128.6, 128.3, 126.8, 126.0, 125.7, 118.9, 117.1,
115.9, 45.8, 19.75, 19.71. HRMS (ESI): m/z calcd for
C23H23N2O2 [M+H]+ 359.1754, found 359.1757; for
C23H22N2O2Na [M+Na]+ 381.1573, found 381.1577.
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
5.2.4.(3Z,6Z)-4-allyl-3,6-bis(2-
fluorobenzylidene)piperazine-2,5-dione ((3Z,6Z)-
3c)
5.2.8. (3Z,6Z)-4-allyl-3,6-bis(4-
(trifluoromethyl)benzylidene)piperazine -2,5-dione
((3Z,6Z)-3g)
Following the general procedure, the product (3Z,6Z)-3c was
1
obtained in 46% yield as a slightly yellow oil. H NMR (500
MHz, CDCl3) δ: 8.37 (s, 1H), 7.43 (t, J = 10.0 Hz, 1H), 7.39 –
7.31 (m, 2H), 7.27 (d, J = 10.0 Hz, 1H), 7.23 – 7.17 (m, 2H),
7.15 (s, 1H), 7.13 – 7.10 (m, 1H), 7.06 (s, 1H), 5.58 – 5.47 (m,
1H), 5.02 (d, J = 10.2 Hz, 1H), 4.74 (d, J = 17.1 Hz, 1H), 4.28 (d,
Following the general procedure, the product (3Z,6Z)-3g was
obtained in 64% yield as a slightly yellow solid. mp = 130 – 133
1
˚C. H NMR (500 MHz, CDCl3) δ: 8.64 (s, 1H), 7.71 (d, J = 5.0
J = 5.0 Hz, 2H). 13C NMR (125 MHz, CDCl3) δ: 160.3 (d, JFC
=
Hz, 2H), 7.67 (d, J = 10.0 Hz, 2H), 7.57 (d, J = 5.0 Hz, 2H), 7.42
(d, J = 5.0 Hz, 2H), 7.17 (s, 1H), 7.10 (s, 1H), 5.56 – 5.48 (m,
1H), 5.04 (dd, J = 10.2, 0.6 Hz, 1H), 4.75 (dd, J = 17.1, 0.7 Hz,
1H), 4.24 (d, J = 5.0 Hz, 2H). 13C NMR (125 MHz, CDCl3) δ:
160.0, 159.2, 137.7, 136.6, 131.1, 130.9 (dd, J FC = 32.7, 11.3
Hz), 129.8, 129.7, 129.3, 127.3, 126.4 (d, J FC = 3.6 Hz), 125.7 (d,
J FC = 3.7 Hz), 124.0 (d, J FC = 271.0 Hz), 120.6, 118.8, 116.7,
48.4. HRMS (ESI): m/z calcd for C23H17F6N2O2 [M+H]+
467.1189, found 467.1197; for C23H16F6N2O2Na [M+Na]+
489.1008, found 489.1015.
250.0 Hz), 160.1 (d, JFC = 247.5 Hz), 159.6, 158.9, 131.3, 131.1,
131.0 (d, JFC = 5.0 Hz), 130.9, 130.3 (d, JFC = 5.0 Hz), 129.9,
127.3, 125.0 (d, JFC = 3.4 Hz), 124.2 (d, JFC = 3.4 Hz), 122.3 (d,
JFC = 14.8 Hz), 120.8 (d, JFC = 14.9 Hz), 118.5, 116.7 (d, JFC
=
22.0 Hz), 116.0 (d, JFC = 21.3 Hz), 115.1, 111.5, 47.6. HRMS
(ESI): m/z calcd for C21H17F2N2O2 [M+H]+ 367.1253, found
367.1262; for C21H16F2N2O2Na [M+Na]+ 389.1072, found
389.1081.
5.2.5. (3Z,6Z)-4-allyl-3,6-bis(3-
fluorobenzylidene)piperazine-2,5-dione ((3Z,6Z)-
3d)
Following the general procedure, the product (3Z,6Z)-3d was
obtained in 44% yield as a slightly yellow oil and changed into
solid in about two days. mp = 110 – 113 ˚C. 1H NMR (500 MHz,
CDCl3) δ: 8.47 (s, 1H), 7.43 – 7.35 (m, 2H), 7.21 (d, J = 5.0 Hz,
1H), 7.15 (d, J = 15.0 Hz, 2H), 7.09 (d, J = 10.0 Hz, 1H), 7.06 –
7.00 (m,4H ), 5.57 – 5.50 (m, 1H), 5.04 (d, J = 10.2 Hz, 1H),
5.2.9. (3Z,6Z)-3,6-bis(3-methoxybenzylidene)-4-
methylpiperazine-2,5-dione ((3Z,6Z)-3h)
Following the general procedure, the product (3Z,6Z)-3h was
obtained in 56% yield as a slightly yellow solid. mp = 141 – 143
˚C. 1H NMR (500 MHz, CDCl3) δ: 8.30 (s, 1H), 7.35 (t, J = 10.0
Hz, 1H), 7.29 (t, J = 10.0 Hz, 1H), 7.23 (s, 1H), 7.02 (s, 1H),
7.01 (d, J = 5.0 Hz, 1H), 6.92 (s, 1H), 6.89 – 6.85 (m, 3H), 6.81
(s, 1H), 3.81 (s, 6H), 3.01 (s, 3H). 13C NMR (125 MHz, CDCl3)

6
Tetrahedron
ACCEPTED MANUSCRIPT
δ: 160.4, 159.52, 159.46, 135.4, 134.4, 130.6, 129.5, 126.2,
In a 25 mL two-neck flask saturated with nitrogen, 1,4-
122.1, 121.0, 120.7, 117.3, 115.0, 114.5, 114.4, 114.3, 55.49,
55.47, 36.8. HRMS (ESI): m/z calcd for C21H21N2O4 [M+H]+
365.1496, found 365.1505; for C21H20N2O4Na [M+Na]+
387.1315, found 387.1322.
diacetyl-2,5-diketopiperazine (50 mg, 0.25 mmol, 1.0 equiv.), the
first aldehydes (0.25 mmol, 1.0 equiv.), halohydrocarbons (0.63
mmol, 2.5 equiv.), Cs2CO3 (205 mg, 0.63 mmol, 2.5 equiv.), and
4Å MS (200mg) in 2 mL dry DMF were added. The reaction was
stirred at -10 °C until the completion of the 1st Aldol
condensation and the alkylation of the halohydrocarbons, and
then the second aldehydes (0.5 mmol, 2.0 equiv.) was added and
the mixture was heated at 95°C for about 4 h. The solvent was
removed under the reduced pressure, and water (50 mL) and
EtOAc (20 mL) were added. The mixture was extracted with
EtOAc (20 mL × 3). The organic layer was dried over Na2SO4,
filtered, and removed. The residues were purified by flash
column chromatography on silica to afford the pure product
(3Z,6Z)-3 as a slightly yellow solid.
1
2
3
4
5
6
7
8
9
5.2.10. (3Z,6Z)-3,6-bis(4-methoxybenzylidene)-4-
methylpiperazine-2,5-dione ((3Z,6Z)-3i)
Following the general procedure, the product (3Z,6Z)-3i was
obtained in 31% yield as a slightly yellow solid. mp = 148 – 150
˚C. 1H NMR (500 MHz, CDCl3) δ: 8.16 (s, 1H), 7.38 (d, J = 10.0
Hz, 2H), 7.23 (d, J = 10.0 Hz, 2H), 7.20 (s, 1H), 6.99 (s, 1H),
6.95 (d, J = 10.0 Hz, 2H), 6.91 (d, J = 5.0 Hz, 2H), 3.83 (s, 3H),
3.82 (s, 3H), 3.02 (s, 3H). 13C NMR (125 MHz, CDCl3) δ: 160.2,
160.1, 160.0, 131.3, 130.3, 129.4, 126.2, 125.5, 124.8, 121.1,
117.3, 115.0, 114.0, 55.6, 55.5, 36.8. HRMS (ESI): m/z calcd for
10
11
12
13 C21H21N2O4 [M+H]+ 365.1496, found 365.1506; for
5.3.1. (3Z,6Z)-4-allyl-3-benzylidene-6-(3-
bromobenzylidene)piperazine-2,5-dione ((3Z,6Z)-3l)
14 C21H20N2O4Na [M+Na]+ 387.1315, found 387.1323.
Following the general procedure, the product (3Z,6Z)-3l was
obtained in 49% yield as a slightly yellow solid. mp = 87 – 90 ˚C.
1H NMR (500 MHz, CDCl3) δ: 8.67 (s, 1H), 7.62 (s, 1H), 7.44 –
7.39 (m, 3H), 7.37 – 7.35 (m, 2H), 7.32 – 7.27 (m, 3H), 7.17 (s,
1H), 6.99 (s, 1H), 5.57 – 5.49 (m, 1H), 5.01 (d, J = 10.0 Hz, 1H),
15
16
17
18
5.2.11. (3Z,6Z)-4-benzyl-3,6-bis(2-
chlorobenzylidene)piperazine-2,5-dione ((3Z,6Z)-
3j)
Following the general procedure, the product (3Z,6Z)-3j was
obtained in 45% yield as a slightly yellow solid. mp = 170 – 172
19
4.76 (dd, J = 17.1, 1.0 Hz, 1H), 4.27 (t, J = 10.0 Hz, 2H). 13
C
˚C. 1H NMR (500 MHz, DMSO) δ: 10.80 (s, 1H), 7.69 (d, J = 5.0
Hz, 1H), 7.53 (t, J = 5.0, 2H), 7.46 (d, J = 5.0 Hz, 1H), 7.43 –
7.35 (m, 4H), 7.21 (dd, J = 4.9, 1.5 Hz, 3H), 7.02 (s, 1H), 6.96 (s,
1H), 6.75 (t, J = 5.0 Hz, 2H), 4.70 (s, 2H). 13C NMR (125 MHz,
DMSO) δ: 159.4, 159.3, 135.7, 133.31, 133.26, 132.4, 131.5,
131.3, 130.6, 130.4, 130.0, 129.6, 129.3, 128.4, 128.1, 127.9,
127.44, 127.38, 127.0, 126.8, 116.5, 113.5, 47.3. NOE
correlations between hydrogen atoms in benzyl group (δ: 4.7, s,
2H, CH2) and in phenyl group (δ: 6.75, t, J = 5.0 Hz, 1H, Ph–H),
as well as hydrogen atoms in amide (δ: 10.80, s, 1H, NH-1) and
in phenyl group (δ 7.69, d, J = 5.0 Hz, 1H, Ph–H) were observed.
HRMS (ESI): m/z calcd for C25H19Cl2N2O2 [M+H]+ 449.0818,
found 449.0823; for C25H18Cl2N2O2Na [M+Na]+ 471.0638, found
471.0604.
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
NMR (125 MHz, CDCl3) δ: 160.7, 159.5, 135.3, 133.9, 131.8,
131.6, 131.5, 130.9, 129.6, 129.2, 128.7, 128.2, 127.5, 127.2,
123.6, 122.9, 118.5, 116.4, 48.2. HRMS (ESI): m/z calcd for
C21H18BrN2O2 [M+H]+ 409.0546, found 409.0553; for
C21H17BrN2O2Na [M+Na]+ 431.0366, found 431.0367.
5.3.2. (3Z,6Z)-4-allyl-6-(2,3-dichlorobenzylidene)-
3-(2-fluorobenzylidene)piperazine-2,5-dione
((3Z,6Z)-3m)
Following the general procedure, the product (3Z,6Z)-3m was
obtained in 43% yield as a slightly yellow solid. mp = 67 – 68 ˚C.
1H NMR (500 MHz, DMSO) δ: 10.77 (s, 1H), 7.60 (dd, J = 15.0,
5.0 Hz, 2H), 7.48 – 7.40 (m, 3H), 7.31 – 7.27 (m, 2H), 7.02 (s,
1H), 6.89 (s, 1H), 5.55 – 5.51 (m, 1H), 4.97 (dd, J = 10.2, 1.0 Hz,
1H), 4.57 (dd, J = 17.1, 1.2 Hz, 1H), 4.18 (d, J = 10.0 Hz, 2H).
13C NMR (125 MHz, DMSO) δ: 159.5 (JC-F = 246.3 Hz), 159.4,
158.7, 134.2, 132.0, 131.9, 131.2, 131.1, 131.0, 130.6, 130.0,
129.3, 128.8, 128.3, 124.4, 121.8 (JC-F = 13.8 Hz), 117.3, 115.6
(JC-F = 21.3 Hz), 113.1, 112.6, 46.3. NOE correlations between
hydrogen atoms in allyl group (δ: 4.18, d, J = 10.0 Hz, 2H, CH2)
and in phenyl group (δ: 7.48 – 7.40, m, 1H, Ph–H), as well as
hydrogen atoms in amide (δ: 10.77, s, 1H, NH-1) and in phenyl
group (δ: 7.60, dd, J = 15.0, 5.0 Hz, 1H, Ph–H) were observed.
HRMS (ESI): m/z calcd for C21H16Cl2FN2O2 [M+H]+ 417.0567,
found 417.0575; for C21H15Cl2FN2O2Na [M+Na]+ 439.0387,
found 439.0388.
5.2.12. (3Z,6Z)-3,6-bis(4-chlorobenzylidene)-4-(4-
(trifluoromethyl)benzyl)piperazine-2,5-dione
((3Z,6Z)-3k)
Following the general procedure, the product (3Z,6Z)-3k was
obtained in 47% yield as a slightly yellow solid. mp = 190 – 192
˚C. 1H NMR (500 MHz, CDCl3) δ: 8.52 (s, 1H), 7.46 (d, J = 10.0
Hz, 2H), 7.41 – 7.35 (m, 6H), 7.22 (d, J = 10.0 Hz, 2H), 7.12 (s,
1H), 7.03 (s, 1H), 7.00 (d, J = 10.0 Hz, 2H), 4.86 (s, 2H). 13C
NMR (125 MHz, CDCl3) δ: 159.9, 159.8, 140.0, 135.4, 135.1,
132.0, 131.3, 130.9, 130.2, 129.8, 129.1, 127.8, 126.1, 125.8,
125.7, 121.3, 117.6, 49.0. HRMS (ESI): m/z calcd for
C26H18Cl2F3N2O2 [M+H]+ 517.0692, found 517.0702; for
C26H17Cl2F3N2O2Na [M+Na]+ 539.0511, found 539.0517.
5.3.3. (3Z,6Z)-4-allyl-6-(2,3-dichlorobenzylidene)-
3-(3-fluorobenzylidene)piperazine-2,5-dione
((3Z,6Z)-3n)
5.2.13. Piperafizine A
Following the general procedure, the natural product
piperafizine A was obtained in 47% yield as a slightly yellow
solid. mp = 174 – 177 ˚C (lit: 181 – 182 ˚C).1 1H NMR (500
MHz, CDCl3) δ: 8.06 (s, 1H), 7.47 – 7.41 (m, 4H), 7.39 (d, J =
5.0 Hz, 2H), 7.36 – 7.32 (m, 2H), 7.30 (s, 2H), 7.29 (s, 1H), 7.08
(s, 1H), 3.01 (s, 3H). 13C NMR (125 MHz, CDCl3) δ: 159.7,
159.6, 134.1, 133.1, 130.5, 129.7, 129.1, 128.8, 128.7, 128.5,
126.1, 121.3, 117.4, 37.0. HRMS (ESI): m/z calcd for
C19H17N2O2 [M+H]+ 305.1285, found 305.1281; for
C19H16N2O2Na [M+Na]+ 327.1104, found 327.1100.
Following the general procedure, the product (3Z,6Z)-3n was
obtained in 51% yield as a slightly yellow solid. mp = 152 – 155
˚C. 1H NMR (500 MHz, CDCl3) δ: 8.71 (s, 1H), 7.43 (d, J = 10.0
Hz, 1H), 7.40 – 7.36 (m, 1H), 7.34 (d, J = 5.0 Hz, 1H), 7.25 (t, J
= 10.0 Hz, 1H), 7.12 (s, 1H), 7.09 – 7.04 (m, 2H), 7.03 (s, 1H),
7.01 (d, J = 10.0 Hz, 1H), 5.57 – 5.49 (m, 1H), 5.04 (dd, J = 10.2,
0.6 Hz, 1H), 4.77 (dd, J = 17.1, 0.8 Hz, 1H), 4.28 (d, J = 5.0 Hz,
2H). 13C NMR (126 MHz, CDCl3) δ: 162.7 (JC-F = 246.3 Hz),
160.1, 158.9, 136.1 (JC-F = 8.8 Hz), 134.5, 133.8, 132.9, 131.3,
130.6, 130.3 (JC-F = 8.8 Hz), 128.8, 127.91, 127.89, 125.35,
125.33, 121.1, 118.7, 116.3 (JC-F = 21.3 Hz), 116.1 (JC-F = 20.0
Hz), 115.0, 48.2. HRMS (ESI): m/z calcd for C21H16Cl2FN2O2
5.3 General procedure for the synthesis of products (3Z,6Z)-3l–t

[M+H]+ 417.0567, found 417.0575; for C21H15Cl2FN2O2Na
134.9, 133.8, 130.6, 129.7, 129.3, 128.9, 128.8, 128.74, 128.65,
128.5, 127.8, 127.7, 127.12, 127.06, 123.0, 116.7, 49.1. HRMS
(ESI): m/z calcd for C25H20ClN2O2 [M+H]+ 415.1208, found
415.1209; for C25H19ClN2O2Na [M+Na]+ 437.1027, found
437.1032.
ACCEPTED MANUSCRIPT
[M+Na]+ 439.0387, found 439.0391.
5.3.4. (3Z,6Z)-4-allyl-6-(2,3-dichlorobenzylidene)-
3-(2-methoxybenzylidene)piperazine-2,5-dione
((3Z,6Z)-3o)
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2
3
4
5
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8
9
10
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5.3.9. (3Z,6Z)-3-(4-bromobenzylidene)-6-(3-
methylbenzylidene)-4-(4-
(trifluoromethyl)benzyl)piperazine-2,5-dione
((3Z,6Z)-3t)
Following the general procedure, the product (3Z,6Z)-3o was
obtained in 45% yield as a slightly yellow solid. mp = 139 – 141
˚C (lit: 134 – 136 ˚C).2 1H NMR (500 MHz, CDCl3) δ: 7.97 (s,
1H), 7.46 (d, J = 5.0 Hz, 1H), 7.37 (t, J = 10.0 Hz, 2H), 7.29 (m,
7.31 – 7.27, 2H), 7.22 (d, J = 10.0 Hz, 1H), 7.11 (s, 1H), 6.99 (t,
J = 10.0 Hz, 1H), 6.93 (d, J = 5.0 Hz, 1H), 5.62 – 5.49 (m, 1H),
5.01 (d, J = 10.2 Hz, 1H), 4.76 (d, J = 17.1 Hz, 1H), 4.26 (d, J =
5.0 Hz, 2H), 3.86 (s, 3H). 13C NMR (125 MHz, CDCl3) δ: 160.0,
158.5, 157.4, 134.6, 133.7, 132.8, 131.3, 130.8, 130.6, 130.5,
128.0, 127.9, 127.4, 122.7, 120.3, 119.1, 118.4, 113.6, 110.7,
55.54, 47.49. HRMS (ESI): m/z calcd for C22H19Cl2N2O3 [M+H]+
429.0767, found 429.0769; for C22H19Cl2N2O3Na [M+Na]+
451.0587, found 451.0586.
Following the general procedure, the product (3Z,6Z)-3t was
obtained in 52% yield as a slightly yellow solid. mp = 176 – 179
1
˚C. H NMR (500 MHz, CDCl3) δ: 8.11 (s, 1H), 7.52 (d, J = 5.0
Hz, 2H), 7.46 (d, J = 5.0 Hz, 2H), 7.34 (t, J = 10.0 Hz, 1H), 7.22
(s, 2H), 7.17 (d, J = 10.0 Hz, 2H), 7.14 (d, J = 10.0 Hz, 2H), 7.08
(s, 1H), 7.01 (d, J = 5.0 Hz, 2H), 4.87 (s, 2H), 2.38 (s, 3H). 13C
NMR (126 MHz, CDCl3) δ: 160.0, 159.6, 140.0, 139.6, 132.7,
132.6, 132.0, 131.1, 130.2, 129.6, 129.32, 129.31, 127.8, 125.8,
125.75, 125.72, 125.5, 123.5, 121.0, 119.2, 49.0, 21.7. HRMS
(ESI): m/z calcd for C27H21BrF3N2O2 [M+H]+ 541.0733, found
541.0741; for C27H20BrF3N2O2Na [M+Na]+ 563.0552, found
563.0558.
5.3.5. (3Z,6Z)-3-benzylidene-6-(3-
chlorobenzylidene)-4-methylpiperazine-2,5-dione
((3Z,6Z)-3p)
5.4. Cytotoxic experiments5a
Following the general procedure, the product (3Z,6Z)-3p was
obtained in 50% yield as a slightly yellow solid. mp = 137 – 139
1
Cell lines, U937, HL60, DU145, HT29, and K562 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 ℃. Cell
viability assay was determined by using the CCK8 (DOjinDo,
Japan) assay. Cells were seeded at a density of 400–800
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
(Perkin Elmer, USA). Dose response curves were plotted to
determine the IC50 values using Prism 5.0 (GraphPad Software
Inc., USA).
˚C. H NMR (500 MHz, CDCl3) δ: 8.51 (s, 1H), 7.43 (s, 1H),
7.41 – 7.34 (m, 4H), 7.32 – 7.28 (m, 4H), 7.23 (s, 1H), 6.99 (s,
1H), 3.00 (s, 3H). 13C NMR (125 MHz, CDCl3) δ: 159.9, 159.4,
135.5, 135.0, 134.0, 130.7, 130.3, 129.6, 129.0, 128.83, 128.76,
128.5, 127.0, 126.9, 121.7, 115.9, 37.0. HRMS (ESI): m/z calcd
for C19H16ClN2O2 [M+H]+ 339.0895, found 339.0895; for
27 C19H15ClN2O2Na [M+Na]+ 361.0714, found 361.0716.
28
5.3.6. (3Z,6Z)-3-benzylidene-6-(3-
29
bromobenzylidene)-4-methylpiperazine-2,5-
dione((3Z,6Z)-3q)
30
31
32
Following the general procedure, the product (3Z,6Z)-3q was
obtained in 49% yield as a slightly yellow solid. mp = 159 – 161
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
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52
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61
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1
˚C. H NMR (500 MHz, CDCl3) δ: 8.73 (s, 1H), 7.60 (s, 1H),
7.46 (d, J = 10.0 Hz, 1H), 7.41 – 7.37 (m, 2H), 7.35 (d, J = 5.0
Hz, 2H), 7.33 (d, J = 5.0 Hz, 1H), 7.29 (d, J = 10.0 Hz, 3H), 7.01
(s, 1H), 3.00 (s, 3H). 13C NMR (126 MHz, CDCl3) δ: 160.2,
159.4, 136.5, 135.2, 134.0, 131.9, 131.7, 131.0, 130.3, 129.7,
128.9, 128.6, 127.5, 126.9, 121.8, 116.2, 37.0. HRMS (ESI): m/z
calcd for C19H16BrN2O2 [M+H]+ 383.0390, found 383.0393; for
C19H15BrN2O2Na [M+Na]+ 405.0209, found 405.0213.
Acknowledgments
This work was supported by a grant from NSFC (21402218),
the Fundamental Research Funds for the Central Universities
(Grant No. 21615405) and the high-performance computing
platform of Jinan University, MOST (973 project2011CB915503
and 863 Program 2012AA092104), the Strategic Priority
Research Program of the Chinese Academy of Sciences
5.3.7. (3Z,6Z)-3-benzylidene-6-(2,3-
dichlorobenzylidene)-4-methylpiperazine-2,5-dione
(3Z,6Z)-3r)
(XDA11030403),
and
Guangdong
Marine
Economic
Following the general procedure, the product (3Z,6Z)-3r was
obtained in 60% yield as a slightly yellow solid. mp = 189 – 190
˚C. 1H NMR (500 MHz, CDCl3) δ: 8.60 (s, 1H), 7.46 (d, J = 10.0
Hz, 1H), 7.43 – 7.39 (m, 2H), 7.38 – 7.35 (m, 2H), 7.29 (t, J =
10.0 Hz, 3H), 7.16 (s, 1H), 7.12 (s, 1H), 3.01 (s, 3H). 13C NMR
(125 MHz, CDCl3) δ: 159.7, 158.9, 134.5, 134.0, 133.9, 132.9,
130.6, 130.1, 129.6, 128.8, 128.5, 127.93, 127.86, 121.7, 114.1,
37.0. HRMS (ESI): m/z calcd for C19H15Cl2N2O2 [M+H]+
373.0505, found 373.0507.
Development and Innovation of Regional Demonstration Project
(GD2012-D01-001). We also thank the foundation of the
innovation team of Zhongshan City.
Supplementary Material
Supplementary data (1H, 13C NMR spectra for all compound,
NOESY NMR spectra for compounds (3Z,6Z)-3a, (3Z,6Z)-3j,
and (3Z,6Z)-3m, X-ray analysis data for compounds (3Z,6Z)-3l
and (3Z,6Z)-4a, and computational calculations) associated with
this article can be found in the online version, at
http://xxxxxxxxxxxxxxxxxxx
5.3.8. (3Z,6Z)-4-benzyl-3-benzylidene-6-(3-
chlorobenzylidene)piperazine-2,5-dione (3Z,6Z)-3s)
Following the general procedure, the product (3Z,6Z)-3s was
obtained in 38% yield as a slightly yellow solid. mp = 121 – 123
1
˚C. H NMR (500 MHz, CDCl3) δ: 8.75 (s, 1H), 7.46 – 7.37 (m,
References and notes
4H), 7.33 – 7.29 (m, 4H), 7.24 (d, J = 5.0 Hz, 1H), 7.19 – 7.18
(m, 3H), 7.14 (s, 1H), 7.02 (s, 1H), 6.90 – 6.88 (m, 2H), 4.84 (s,
2H). 13C NMR (126 MHz, CDCl3) δ: 160.7, 159.8, 136.2, 135.3,
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