Synthesis and activity evaluation of the cyclic dipeptides arylidene N-alkoxydiketopiperazines

Article abstract of DOI:10.1016/j.bmc.2016.08.038

A series of arylidene N-alkoxydiketopiperazines was designed and stereoselectively synthesized via oxime-ether formation and intramolecular acylation. Possible cyclization and acid-catalyzed rearrangement-fragmentation mechanisms were discussed. The crystal structure of the novel diketopiperazine further confirmed the rearrangement mechanism. Most compounds exhibited antitumor activity. Several compounds were more potent against caspase-3. Specifically, compounds 6e, 6g, and 6f inhibited caspase-3 at IC₅₀values lying within the low micromolar range and demonstrated good selectivity. The binding modes of alkoxydiketopiperazines in the active center of caspase-3 were also discussed based on the molecular docking results.

Full text of DOI:10.1016/j.bmc.2016.08.038

Accepted Manuscript
Synthesis and activity evaluation of the cyclic dipeptides arylidene N-alkoxy-
diketopiperazines
Xia Tian, Juan Feng, Shi-min Fan, Xiao-li zhen, Jian-rong Han, Shouxin Liu
PII:
S0968-0896(16)30645-9
DOI:
http://dx.doi.org/10.1016/j.bmc.2016.08.038
BMC 13221
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
29 June 2016
21 August 2016
22 August 2016
Please cite this article as: Tian, X., Feng, J., Fan, S-m., zhen, X-l., Han, J-r., Liu, S., Synthesis and activity evaluation
of the cyclic dipeptides arylidene N-alkoxydiketopiperazines, Bioorganic & Medicinal Chemistry (2016), doi: http://
dx.doi.org/10.1016/j.bmc.2016.08.038
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Synthesis and activity evaluation of the
cyclic dipeptides arylidene N-
alkoxydiketopiperazines
Xia Tiana, Juan Fengc, Shi-min Fanb, Xiaoli Zhena Jian-rong Hana, Shouxin Liub
a College of Sciences, Hebei Univers ity of Science
& Technology, Shijiazhuang;50018, People’s Republic of China.
b State Key Laboratory Breeding Base-Hebei Province Key Laboratory
of Molecular Chemistry for Drug, Hebei University of Science &
Technology, Shijiazhuang 050018, People’s Republic of China
b College of Chemical & Pharmaceutical Engineering,
Hebei University of Science &Technology,
Shijiazhuang 050018, People’s Republic of China,
O
O
COOEt
Ar
N
Ar
Ar
OEt
N
COOEt
N
RO
OH
O

Bioorganic & Medicinal Chemistry
Synthesis and activity evaluation of the cyclic dipeptides arylidene N-
alkoxydiketopiperazines
Xia Tiana, Juan Fengc, Shi-min Fanb, Xiao-li zhena, Jian-rong Hana*, Shouxin Liub†
a College of Sciences, Hebei University of Science & Technology, Shijiazhuang 050018, People’s
Republic of China
b State Key Laboratory Breeding Base-Hebei Province Key Laboratory of Molecular Chemistry for Drug, Hebei University of Science &Technology,
Shijiazhuang 050018, People’s Republic of China
c College of Chemical & Pharmaceutical Engineering, Hebei University of Science &
Technology, Shijiazhuang 050018, People’s Republic of China
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
A series of arylidene N-alkoxydiketopiperazines was designed and stereoselectively synthesized via
oxime-ether formation and intramolecular acylation. Possible cyclization and acid-catalyzed
rearrangement-fragmentation mechanisms were discussed. The crystal structure of the novel
diketopiperazine further confirmed the rearrangement mechanism. Most compounds exhibited
antitumor activity. Several compounds were more potent against caspase-3. Specifically, compounds
6e, 6g, and 6f inhibited caspase-3 at IC50 values lying within the low micromolar range and
demonstrated good selectivity. The binding modes of alkoxydiketopiperazines in the active center of
Available online
Keywords:
Arylidene N-alkoxydiketopiperazines
Synthesis
Caspase-3
caspase-3 were also discussed based on the molecular docking results.
.
Antitumor activities
Inhibitory activity
Molecular docking
2015 Elsevier Masson SAS, All rights reserved.
O
O
O
O
O
1. Introduction
Ar
R
H
R
H
N
N
N
Diketopiperazines (DKPs) (I) are the smallest cyclic peptides
isolated from microorganisms and sponges, as well as from a
variety of tissues and body fluids [1–3]; they represent an
important class of biologically active natural products that have
been used as versatile intermediates in asymmetric synthesis of
amino acid derivatives and related natural products [4, 5]. These
heterocyclic compounds exhibit remarkable biological and
pharmacological activities, such as antimicrobial, antitumor,
antiviral, and plant growth regulation [6, 7]. Moreover,
investigations show that many DKP derivatives are cytotoxic
against an array of human cancer cells [8]. Different proline-
containing DKPs exhibit a number of bioactivities, such as
inhibition of Serratia marcescens and Vibrio anguillarum and
antihyperglycemic activity [9–11]. Among DKPs, ylidene DKPs
II are natural products often produced by fungi, such as
Actinomyces strains and Penicillium species. Some of these
compounds possess antibacterial activity and inhibit tumor
growth in mice [12].
N
N
N
H
O
II
arylidene diketopiperazines
I
diketopiperazines
Phenylahistin (PLH) [12, 13], an arylidene DKP produced by
Aspergillus ustus, is a cell cycle inhibitor. (−)PLH demonstrates
antitumor activity against eight tumor cell lines at IC50 values
that ranged from 1.8 × 10−7 M to 3.7 × 10−6 M. It also shows
antitumor activity against P388 leukemia and Lewis lung
cancinoma cells in vivo. Total synthesis of PLH was investigated
[14] to understand its precise biological functions and develop
more potent antitumor agents based on DKP structures. In
addition,
a
dehydro
product
of
(−)-PLH
CDP
dehydropenylahistin (△ (−)-PLH) demonstrates potent cytotoxic
activity higher than that of the known anticancer drugs taxol,
vinblastine, and vincristine, indicating that this novel compound
is a candidate anticancer drug; in addition, △ (−)-PLH
demonstrates an inhibitory activity toward the first cleavage of
sea urchin embryos by more than 1000 times that of (−)-PLH
Sell
*
Corresponding author. Tel.: +86 0311 88632227.
E-mail address:han_jianrong@126.com
†
Corresponding author. Tel.: +86 0311 88632254.
E-mail address: chlsx@263.net

[15].
reaction. Therefore, we used oxime ether as a masked amine
group.
O
O
O
O
O
O
NH
*
NH
N
A: Ar=3-Pyridyl
N
N
N
O
N
NH
HN
H
NH
COOR1
H
B: Ar= phenyl
a
b
Br
N
Ar
Ar
OR1
H
Ar
Cl
C: Ar=3,4,5-trimethyloxy phenyl
D: Ar= -Naphthyl
E: Ar=-Quinolyl
R1=Me or Et
COOR1
2A-E
N
Barettin
OH
△ (-)-PLH
(-)-PLH
Phenylahistin
O
1A-E
3A-E
HN
O
N
O
O
O
N
H
O
f
c
d
Ar
N
OH
Ar
OR1
Ar
N
N
O
NH
O
e
N
N
N
N
N
NH
HO
OR
COOH
RO
O
6a-q
O
O
RO
4a-q
5a-q
mycelianamide
brevianamide S
a: Ar=3-Pyridyl, R=n-Bu
b: Ar=3-Pyridyl, R=c-hexyl
c: Ar=3-Pyridyl, R=benzyl
d: Ar= phenyl, R=Methyl
e: Ar= phenyl, R=Ethyl
j: Ar=3,4,5-trimethyloxy phenyl, R=Methyl
K: Ar=3,4,5-trimethyloxy phenyl, R=n-Bu
Synthesis of ylidene DKPs as versatile building blocks of
bicyclic N-heterocycles has attracted a considerable amount of
attention. The various reactive functional groups of ylidene
DKPs allow reactions to occur at different positions of the ring
and at the exocyclic carbon atom of C=C. Therefore, ylidene
DKPs have been synthesized following various substituent
patterns in the non-chiral, racemic, and optically active series via
different synthetic pathways. Liebscher et al. [16, 17] reviewed
the synthesis, properties, and applications of 3-ylidene-DKPs;
other synthesis methods have also been reported [18–20].
l: Ar=3,4,5-trimethyloxy phenyl, R=c-hexyl
m: Ar=3,4,5-trimethyloxy phenyl, R=benzyl
n: Ar=3,4,5-trimethyloxy phenyl, R=4-fluoro benzyl,
f: Ar= phenyl, R=n-Bu
g: Ar= phenyl, R=c-hexyl
h: Ar= phenyl, R=benzyl
o: Ar=3,4,5-trimethyloxy phenyl, R=4-chloro benzyl,
p: Ar= -Naphthyl , R=c-hexyl ,
I: Ar=3,4,5-trimethoxyl benzyl, R=3-Pyridyl
q: Ar=-Quinolyl , R=c-hexyl ,
Proline-containing
arylidene
bicyclo-DKPs,
such
as
Scheme 1 Synthesis of E-arylidene bicyclo-DKPs
(a) CH2(CO2Et)2, nano-K2CO3; (b) EtONO, nano-K2CO3; (c) RX, K2CO3; (d) (1) NaOH,
H2O; (2) (C2H5)3N, HBTU, DMF, proline methyl ester, (e) LiOH, THF:H2O; and (f) SOCl2
brevianamides and barettin, have been found in natural products,
and they exhibit antibacterial and antiviral activities [21, 22].
The characteristic structure of N-hydroxyl DKPs found in
mycelianamide, which is isolated from Penicillium griseofulvum
mycelium [23], caught our interest. To determine the bioactivity
of arylidene bicyclo-DKPs that contain proline and the N-alkoxy
structure fragment, we investigated a novel, efficient, and
stereoselective synthesis and the relative chemical characteristics
of N-alkoxy derivatives of E-arylidene bicyclo-DKPs [24]. In
this work, we synthesized several new E-arylidene bicyclo-DKPs
and investigated their bioactivity against certain tumor cell lines
and their inhibitory effect against caspase-3.
The oxime ether ester of 4 is hydrolyzed with NaOH followed
by coupling with proline methyl ester in the presence of HBTU
and Et3N. The resulting products were deprotected with LiOH in
THF, and H2O produced pseudodipeptides 5 containing free
carboxyl group. Compound 5 was treated with SOCl2 to produce
acyl chloride, which directly underwent intramolecular N-
acylation with the oxime ether to stereoselectively generate
compound 6 at good overall yields.
2.2. Chemistry
The method described above to prepare cyclic dipeptides
involves six synthetic steps. In steps 2, 4, and 5, the resulting
products 3 and 5 were used in the subsequent reaction without
purification. Rearrangement cyclization (step 6) is a key step in
the synthesis process. The mechanism of cyclization of the
pseudodipeptides was studied. First, the carboxyl of
pseudodipeptides 5 react with SOCl2, producing acyl chloride 5-
1. A nucleophile was subsequently added into the carbonyl C
with the N of oxime ether as nucleophilic reagent, and the
leaving group chloride was eliminated to produce the ring-
closing product iminium intermediate 5-2. The hydrogen of
O
Ar
2. Results and discussion
2.1. Synthesis
H
N
N
RO
H
O
O
6
E-Arylidene bicyclo-DKPs were synthesized according to the
general methods described in Scheme 1. We initially used
benzylic halides 1 as raw material. The benzylic halides 1 was
dropped into N,N-dimethylformamide (DMF) dilution of diethyl
malonate and nano-K2CO3, producing the substitute product 2 at
75% yield. However, nucleophilic substitution reaction occurs in
different bases when the aromatic ring varies. Treatment of 2
with EtONO at 0 °C formed a high yield (>90%) of α-oximino
esters 3 in the presence of nano-K2CO3. The subsequent
formation of oxime ether bond with RX by using K2CO3
produced compound 4. The oxime or oxime ether was an
excellent substrate to form an amino group via reduction
Ar
O
O
Ar
Ar
O
H
H
N
H
N
N
H
RO
N
N
RO
N
O
Ar
O
Ar
O
RO
C
H
O
O
Cl
N
SOCl2
N
5-2
N
O
N
Ar
RO
C
H
RO
O
O
HOOC
5
Ar
Ar
Cl
H
N
Et3N
5-1
H
N
N
N
H
RO
RO
N
H
N
O
+
6
RO
C
O
O
O
Ar
H
N
5-4
5-3
N
RO
H
6`
O
Scheme 2. Possible cyclization reaction

OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
O
O
H
O
O
O
O
H
N
N
N
benzylic group can then be fragmented to produce the cyclic
dipeptides 6a–6q that contain exocyclic double bond, and the
stereochemistry of L-proline was maintained. During cyclization,
the configuration of proline depended on the reaction conditions.
In the presence of Et3N, the acyl chloride, which was prepared
through reaction of compound 5 with SOCl2, can be converted
into ketene 5-3. Finally, mixtures of compounds 6 and 6′ were
produced by losing the configuration of proline in the cyclization
products (Scheme 2).
H
+
N
N
N
OCH3
O
O
Ar
Ar
7
7`
O
OH
OCH3
OCH3
OCH3
N
N
H
O
8
Scheme 3. Possible mechanism of rearrangement-
fragmentation reaction
The configurations of C=C in the final products were
confirmed by single-crystal X-ray diffraction (XRD). The XRD
studies revealed that the dipeptide 6b crystallized in the
orthorhombic space group P212121. The conformation of the
molecule is illustrated in the stereo diagram in Fig. 1. Figure 1
shows that E-configuration of the exocyclic C(1)=C(14) in
compound 6b is formed. In addition to the conformation of the
DKP ring, the conformation of the pyrrolidine ring is interesting.
In L-proline, the five-membered ring of the pyrrolidine displays
the envelope conformation. The substituted cyclohexyl group on
nitrogen atom N(2) exhibited a chair conformation. Otherwise,
The molecular structure of dipeptide 8 was determined
through single-crystal XRD analysis. The XRD studies revealed
that the dipeptide 8 crystallized in the triclinic space group P-1.
The conformation of the molecule is illustrated in the stereo
diagram in Figure 2. The diketopiperazine ring of 8 also exists in
the boat conformation ( = −17°) in solid state with axial
dispositions for the O4 oxygen atoms. The dihedral angle
between the average plane of the DKP ring and the plane of the
benzyl residue is 78.4. The hydroxy group on C11 and the H
atoms on C16 are in the axial positions and located on the same
side of the DKP ring, whereas the aromatic rings of the side
chains are in quasi-equatorial positions.
1
the similar chemical shifts of the H(14) singlet in H NMR
suggest the same configuration for the other compounds.
Investigation on the molecular structure further confirmed that
this method can stereoselectively synthesize arylidene N-alkoxy
DKPs.
Fig.1. Structure of 6b with ellipsoid displacement drawn at 30%
probability level.
Fig. 2. Structure of 8 with displacement ellipsoids drawn at
30% probability level
The chemistry of N-alkoxy E-arylidene bicyclo-DKPs was
also investigated. Under acidic condition, the rearrangement-
fragmentation and addition products of 6i, 6j, 6n, and 6o are
similar to those reported in reference [14], whereas 6a, 6c, 6e,
6p, and 6q were stable under the same condition. When the alkyl
in N-alkoxy groups was arylmethyl, the rearrangement-
fragmentation obviously strongly depended on Ar structure in
the N-alkoxy E-arylidene bicyclo-DKPs. The electron-rich Ar,
such as trimethyloxy phenyl 6i-6o, is favorable for these
reactions. The rearrangement-fragmentation proceeds through a
concerted retro-ene reaction mechanism contained heteroatom,
which occurred between aryl formaldehyde and substituted
imine (Scheme 3). Tautomerism of the intermediate 7 cyclic acyl
imine easily occurs to form the balance with 7′, and the
configuration of proline is lost. Thus, the reactions of the
rearrangement-fragmentation of 6i, 6j, 6n, and 6o and H2O
addition produced a mixture containing four isomers of 8.
2.3. Biological evaluations
2.3.1 Antitumor activities
To characterize the biological potential of E-arylidene
bicyclo-DKPs, we determined the antitumor activities of some
compounds against the cell lines K562-1, HCT-15, HT-29, HCT-
8, HePG2, MDA-MB-231, A549, and Bre-04. The results
presented in Table 1 show that most of the DKPs at 20 µg/mL
concentration inhibited the cell lines K562-1, HCT-15, HT-29,
HCT-8, and HePG2, and the activity against MDA-MB-231,
A549, and Bre-04 was weak. These results indicate that the
group on N atom played a critical role in antiproliferative
potency. When the alkyl on nitrogen atom is the same, such as in
6b, 6g and 6l; 6f and 6k; 6h and 6m, the compounds containing
different aryl showed varying antiproliferative activities against
the cell lines. Compared with other compounds, the compound
6f shows a high activity in the K562-1, HCT-15, and A549 cell
lines (Table 1); 6b demonstrates a high activity in the HT-29 and
HCT-8 cell lines; and 6m displays a high activity in the HePG2
and MDA-MB-231 cell lines. When the aryl group is
trimethyloxy phenyl, the chlorinated analogue 6o was more

potent than the corresponding nonhalogenated 6m against the
K562-1, HCT-15, A549, and Bre-04 cancer cells. The
compounds 6d, 6f, and 6o are worth investigating as potential
agents against the K562-1 and HCT-15 cells. At 10 µM
concentration, the inhibitory effects of 6d and 6f toward K562-1,
HCT-15, HT-29, and A549 were lower than 50%, whereas the
activities of 6l against K562-1 and that of 6o against K562-1 and
HCT-15 remained higher than 50% (Table 2). The IC50 values of
the four compounds against K562-1, HCT-15, HT-29, and A549
cells are shown in Table 3. The compound 6o is more potent
against K562-1, HCT-15, and A549 cells, with corresponding
IC50 values of 7.3, 8.6, and 11.4 µM.
Table 1 In vitro % inhibition of compound 6 (20 µg mL−1) against tumor cell lines
Compound
K562-1
HCT-15
HT-29
HCT-8
HePG2
MDA-MB-231
16.1±4.1 a
23.5±3.2 a
A549
Bre-04
69.3±2.1a 62.1±3.5a 70.3±2.6 a 80.1 ±3.8a 60.4±1.5 a
90.4±2.7 a 88.8±1.5a
35.2 ±3.2a 35.3 ±2.9b
26.6±4.0 a 24.5±3.5 b
6b
6d
65.4 ±4.1a 78.8±2.4 a 62.2±1.9 a
62.8 ±1.1b
42.6
-
-
-
-
25.3 ±3.7b 21.9±4.6 b
6e
±3.1b
96.2±1.9 a 90.6±2.0 a 64.9 ±2.8a 68.4 ±3.2a 59.9±2.2 a
78.2±2.2 a 72.3±1.8a 69.4±5.1 a 76.3 ±3.4a 68.5±6.1 a
65.6±2.1 a 69.7±2.1a 76.2±2.1 a 66.8±2.1 a 69.2±2.1 a
16.3 ±2.6a
17.3±5.1 a
32.6 ±2.1a
-
45.6±4.6 a
42.5±6.3 a
43.2 ±1.5a
50.8±1.7 a
33.8 ±5.3a
41.3±2.1 a 25.0 ±2.1b
27.1±2.6 b
43.4±3.9 a
26.4 ±1.1a
8.9±0.9 a
-
-
6f
6g
6h
6i
-
78.7±4.4 b
30.7 ±3.8b
-
-
-
-
-
64.4 ±6.1a 61.6±4.3a 61.6±1.1 a 60.6 ±2.3a 64.3±3.6a
80.1 ±1.2 67.9±2.2a 60.5±2.8 a 75.2±6.1 a 76.1±3.1 a
73.2 ±2.8a 74.2±1.7a 70.1±4.0 a 79.1 ±3.1a 75.0 ±4.5a
6k
6l
6m
95.7±1.
92.6 ±3.3b 53.2±2.4b
-
-
-
76.6 ±3.6b 50.3 ±2.3b
6o
0b
a: reference[24] b this work
Table 2. Cytotoxic activities of some compounds against tumor cell lines
Compounds
Dose
(μM)
100
50
20
10
5
100
50
20
10
5
100
50
20
10
5
100
50
20
10
5
Cytotoxic activities (%)
HT-29
K562-1
98.6±2.1
87.3±1.5
55.4±3.0
36.2±2.8
24.4±2.0
99.6±3.1
92.7±4.3
46.5±3.2
39.2±5.1
30.2±2.7
96.4±3.1
83.6±5.4
61.3±1.6
50.7±3.5
30.2±2.2
97.8±3.1
96.5±1.6
73.9±3.6
58.1±2.9
36.7±1.5
HCT-15
96.5±1.9
82.6±2.4
60.5±3.6
46.8±1.7
37.3±4.1
99.1±4.1
87.5±1.1
78.3±4.0
48.8±3.7
4.04±0.9
89.7±4.6
69.3±2.1
47.6±1.7
39.2±2.4
40.4±2.9
98.6±3.9
94.4±2.6
81.6±1.1
60.5±4.3
42.4±3.2
A549
88.6±4.4 50.5±3.1
56.4±3.5 22.4±1.2
34.9±1.1
28.1±1.6
-
80.6±6.0 91.1±4.3
61.6±4.1 48.4±3.9
39.8±3.5 16.2±2.8
6d
-
-
-
6f
6l
21.2±2.7
-
-
-
-
88.5±6.1
56.6±5.8 33.4±6.0
42.9±3.9 20.5±4.1
48.8±2.6
-
86.8±8.3 91.7±3.0
55.7±5.1 81.8±3.6
39.2±5.7 63.4±4.2
24.2±4.5 48.1±2.1
-
-
6o
-
4.03±0.5
Table 3. Antiproliferative activity of 6d, 6f, 6l, and 6o
6o
2.3.2 Inhibitory activity of capase-3
7.3±2.6
8.6±2.7
11.4±3.1
against three human tumor cell lines
IC50 μM
HCT-15
Compounds
K562-1
A549
-
During high-throughput screening of enzyme inhibiting
activities, 6e, 6f, and 6j exhibited good inhibition activities
against caspase-3. The corresponding inhibition rates at 50 µM
concentration were 93.7%, 77.3%, and 85.6% (Figure 3).
However, when the concentration was reduced to 20 µM, 6e still
6d
6f
6l
17.6±2.1
14.1± 2.8
-
-
27.3±3.5
9.8±2.3
11.8±1.5
23.7±3.4

Table 4. Inhibitory activities of 6e against caspase-3
Dose (μM) inhibition for caspase-3 (%)
showed
a high inhibitory activity against caspase-3 at
approximately 89% inhibition ratio, whereas the activities of 6f
(25.5%) and 6j (34.3%)were lower than 50%. The dose-
dependent activity of 6e against viable caspase-3 was further
evaluated, and the result shows that the IC50 was 7.6 µM (Table
4).
100
50
20
10
5
96.2
93.7
88.7
57.4
40.6
100
80
60
40
20
0
To obtain some insight into the binding mode of cyclic
dipeptide, we docked 6e into the active site of caspase-3.
Computer docking was performed with the X-ray crystal
structure of caspase-3 (PDB identification code: 3H0E) by using
CDOKER from Discovery Studio version 3.5 (Accelrys, San
Diego, USA). The molecular surface of the caspase-3 catalytic
domain was generated from the surrounding residues of the co-
crystallized inhibitor within 5 Å. The -CDOCKER energies of
the top10 docked conformations were ranked, and the lowest one
was recorded.
6d
6e
6f
6h
6j
Fig. 3. Caspase-3 inhibitory activity of some compounds (50
µM)
Fig.4. (a) Proposed binding mode of 6e to caspase-3 enzyme based on the X-ray co-crystal structure of caspase-3 complexed with a
small molecule inhibitor (left). (b) Interaction of the chain cyclic peptide 6e with adjacent amino acid residues (right).
The cyclic dipeptide 6e can interact with S1, S2, and S3 pockets of caspase-3. One hydrogen bond formed between the NH of
guanidyl of Arg 207 and the O of the amide group. π–π interaction between phenyl ring and HIS 121 formed in the S1 pocket. In
addition, the hydrophobic side chain of 6e plunges into the S2 pocket.
3. Conclusion
DKPs are important in drug discovery because they contain constrained amino acids embedded within their structures. In this work,
we designed and synthesized a series of arylidene N-alkoxyDKPs (6) bearing various substituted chains. Different DKPs (6a–6q)
demonstrate some activities against different tumor cells. 6d, 6f, 6l and 6o showed higher anti-tumor activity, wherein, the compound
6o is more potent against K562-1, HCT-15, and A549 cells, with corresponding IC50 values of 7.3, 8.6, and 11.4 µM. In addition, 6e, 6f,
and 6j show moderate and good caspase-3 inhibitory activities, and 6e at 50μM concentration shows an inhibition rate of 93.7%.
Further experiments are currently conducted to better investigate the mechanism of action of such compounds and to confirm the
improvement in their biological activity.
4. Experimental
4.1. Chemistry
5.1.1. Materials and measurements
1H NMR spectra were obtained using a Bruker Advance II 500 instrument in CDCl3 solution, with tetramethylsilane as internal
reference and operated at 500 for 1 h. Elemental analyses were performed on a Perkin-Elmer 2400 C instrument. Infrared spectra were
recorded on a Shimadzu Bio-Rad FTS 135 instrument. Most of the reagent-grade chemicals were commercially available and used
without further purification unless otherwise noted. DMF was dried over CaH2 for 2 days and then distilled under a reduced pressure

prior to use. Ethanol was refluxed over sodium turnings and then distilled fractionally. Flash chromatography was performed using
silica gel (200–400 mesh).
4.1.2 Synthesis
General procedure to prepare substituent diethyl malonate 2 [25]:
Nano-K2CO3 (0.65 mol, 0.90 g), diethyl malonate solution (0.5 mol, 80.1 g), and benzylic halide 1 (1.1 mol) in absolute ethanol (250
ml) were added into a round-bottomed flask equipped with a water-cooled reflux condenser and a thermometer. The mixture was heated
to 65 °C on oil bath and stirred for 8 h. The reaction was monitored by TLC. The mixture was filtered, and the solvents were removed
in vacuo. The residue obtained was added into an equal volume of the mixture of water and ice, and the organic layer was separated.
Water layer was extracted with ethyl acetate (4 × 30 ml), mixed with organic the layer, and dried over MgSO4. Concentration under
reduced pressure followed by column chromatography over silica gel produced the title compound 2.
3-Pyridine methyl malonic acid diethyl ester 2A: 2A was obtained as colorless oil at 75% yield (94.2g) after purification through
1
column chromatography (EtOAc/hexane, 1:1). H NMR (500 MHz, CDCl3)  1.25 (t, J=7.0Hz, 6H), 3.22 (d, J=7.5Hz, 2H), 3.36 (t,
J=7.5Hz, 1H), 4.17 (q, J=7.0Hz, 4H), 7.56 (d, J=7.5Hz, 1H), 8.47 (d, J=7.5Hz, 1H), 8.49 (d, J=7.5Hz, 2H), ppm; IR (KBr)  1732,
1576, 1479, 1445 cm−1; Anal. Calcd for C13H17NO4: C, 62.14; H, 6.82; N, 5.57. Found: C, 62.32; H, 7.01; N, 5.46. HR-MS (ESI, m/s)
calcd. for C13H17NO4 (M+1)+: 252.1230, found: 252.1243
Benzyl malonic acid diethyl ester 2B: 2B was obtained as colorless oil at 90% yield (112.6 g) after purification through column
1
chromatography (EtOAc/hexane, 1:1). H NMR (500 MHz, CDCl3)  1.25 (t, J=7.0Hz, 6H), 3.22 (d, J=8.5Hz 2H), 3.63 (t, J=8.5Hz
1H), 4.14 (q, J=7.0Hz, 4H), 7.45 (m, 5H) ppm; IR (KBr)  1733, 1606, 1585, 1490, 1466 cm−1; Anal. Calcd for C14H18O4: C, 67.18; H,
7.25; Found: C, 67.22; H, 7.19. HR-MS (ESI, m/s) calcd. for C14H18O4 (M+1)+: 251.1278, found: 251.1260
3-(3,4,5-trimethyloxy)benzylmalonic acid diethyl ester 2C: 2C was obtained as white solid at 80% yield (136.1 g) (m.p. 77 °C–78
1
°C) after purification through column chromatography (EtOAc/hexane, 5:1) H NMR (500 MHz, CDCl3)  1.22 (t, J=7.0Hz, 3H), 1.27
(t, J=7.0Hz 3H), 3.79 (t, J=8.5Hz, 2H), 3.86(s, 9H), 4.12 (d, J=8.5Hz, 1H), 4.16 (q, J=7.0Hz, 4H), 6.64 (s, 2H) ppm; IR (KBr)  1734,
1568, 1479, 1485 cm−1; Anal. Calcd for C17H24O7: C, 59.99; H, 7.11; Found: C, 59.78; H, 7.26. HR-MS (ESI, m/s) calcd. for C17H24O7
(M+1)+:341.1595, found: 341.1622
2-Naphthylmethyl malonic acid dimethyl ester 2D: 2D was obtained as viscous liquid at 75% yield(102.0g) after purification
through column chromatography (EtOAc/hexane, 3:1) 1H NMR (500 MHz, CDCl3)  3.70 (s, 6H), 4.23 (d, J=9.0Hz 2H), 4.40 (t,
J=9.0Hz, 1H), 7.44 (m, 3H), 7.76 (m, 4H) ppm; IR (KBr)  1738, 1564, 1480, 1486 cm−1; Anal. Calcd for C16H16O4: C, 70.57; H, 5.92;
Found: C, 70.53; H, 5.98. HR-MS (ESI, m/s) calcd. for C16H16O4 (M+1)+: 273.1121, found: 273.1102
2-(3-Quinolylmethyl malonic acid dimethyl ester 2E: 2E was obtained as colorless oil at 71% yield(97.0g) after purification through
column chromatography (EtOAc/hexane, 3:1), 1H NMR (500 MHz, CDCl3)  3.61 (d, J=7.5Hz, 2H), 3.75 (s, 6H), 4.36 (t, J=7.5Hz,
1H), 7.30 (d, J=8.5Hz, 1H), 7.47 (t, J=7.5Hz, 1H), 7.66 (t, J=7.5Hz, 1H), 7.77 (d, J=8.5Hz, 1H), 7.99 (d, J=8.5Hz, 1H), 8.07 (d,
J=8.5Hz, 1H) ppm; IR (KBr)  1738, 1564, 1480, 1486 cm−1; Anal. Calcd for C15H15NO4: C, 65.92; H, 5.53; N,5.13; Found: C, 65.88;
H, 5.5050; N, 5.20. HR-MS (ESI, m/s) calcd. for C17H24O7 (M+1)+: 274.1074, found:274.1088
General procedure to prepare ethyl 2-alkyloxyimine-3-arylpropionate 4:
Diethyl or dimethyl arylmethylmalonate (0.1 mol) was added into a 250 ml reaction flask equipped with a mechanical stirrer, reflux
condenser with drying tube, and dropping funnel. After cooling to −15 °C, ethyl nitrite (0.105 mol) was added into the reaction solution.
Subsequently, EtONa/EtOH solution (0.1 mol) was slowly added dropwise under mechanical stirring, and the mixture was refrigerated
for 12 h. The solution was concentrated, and the residue obtained was added into equal volume of water. The pH of the mixture was
adjusted to 6 with diluted hydrochloric acid, extracted with ethyl acetate (4 × 50 ml), and dried over anhydrous Na2SO4. After the
solvent was removed under reduced pressure, the crude product 3A–E was obtained without purification (yield >90%).
The above crude product (0.05 mol), 50 ml of acetone, and anhydrous K2CO3 (0.055 mol) were added into a 250 ml reaction flask
equipped with a mechanical stirrer, reflux condenser with dry tube, and dropping funnel. Alkyl halide (0.1 mol) was subsequently added
dropwise into the reaction solution. The mixture was stirred for 4 h at 35 °C–40 °C. After the precipitates were filtered, the solution was
concentrated to produce a residue, which was purified through column chromatography over silica gel to obtain the title compound 4.
Ethyl 2-butyloxyimine-3--pyridylpropionate 4a: 4a was obtained (with n-BuBr) at 81% yield (10.7g) according to general
1
procedure after purification through column chromatography (EtOAc/hexane, 1:1). H NMR (500 MHz, CDCl3) 0.94 (t, J=7.5Hz, 3H),
1.33 (t, J=7.0Hz, 3H), 1.33 (m, 2H), 1.35 (m, 2H), 3.91 (s, 2H), 3.91 (t, J=7.5Hz 2H), 4.32 (q, J=7.0Hz, 2H), 7.20 (m, 1H), 7.66 (d,
J=7.0Hz, 1H), 8.43 (d, J=4.5Hz, 1H), 8.53 (d, J=2.0Hz, 1H) ppm; IR (KBr)  1716, 1603, 1576, 1477 cm−1; Anal. Calcd for
C14H20N2O3: C, 63.62; H, 7.63; N, 10.60; Found: C, 63.75; H, 7.41; N, 10.73. HR-MS (ESI, m/s) calcd. for C14H20N2O3 (M+1)+:
265.1552, found: 265.1538
Ethyl 2-benzyloxyimine-3--pyridylpropionate 4c: 4c was obtained (with benzyl chloride) at 85% yield (12.7g) according to general
1
procedure after purification through column chromatography (EtOAc/hexane, 1:1). H NMR (500 MHz, CDCl3) 1.33 (t, J=7.0Hz, 3H),
3.92 (s, 2H), 4.31 (q, J=7.0Hz, 2H), 5.33 (s, 2H), 7.15 (m, 1H), 7.53 (m, 1H), 8.44 (m, 1H), 8.51 (s, 1H), 7.32–7.37 (m, 5H) ppm; IR
(KBr)  1715, 1574, 1496 cm−1; Anal. Calcd for C17H18N2O3: C, 68.44; H, 6.08; N, 9.39; Found: C, 68.22; H, 6.23; N, 9.50. HR-MS
(ESI, m/s) calcd. for C17H18N2O3 (M+1)+: 299.1396, found: 299.1411
Ethyl 2-ethyloxyimine-3-phenyl propionate 4e: 4e was obtained (with C2H5Br) at 80% yield(9.4g) according to general procedure
1
after purification through column chromatography (EtOAc/hexane, 1:1). H NMR (500 MHz, CDCl3) 1.30 (t, J=7.0Hz, 6H), 3.93 (s,
2H), 4.20 (q, J=7.0Hz, 2H), 4.28 (q, J=7.0Hz, 2H), 7.14–7.16 (m, 2H), 7.23–7.25 (m, 3H) ppm; IR (KBr)  1717, 1578, and 1495 cm−1;

Anal. Calcd for C13H17NO3: C, 66.36; H, 7.28; N, 5.95; Found: C, 66.48; H, 6.13; N, 6.16. HR-MS (ESI, m/s) calcd. for C13H17NO3
(M+1)+:236.1287, found: 236.1305
Ethyl 2-(3-pyridylmethyloxyimine-3-(3,4,5-trimethoxy)phenyl propionate 4i: 4i was obtained(with 3-pyridylmethyl chloride) ( at
1
79% yield(15.3g) according to general procedure after purification through column chromatography (EtOAc/hexane, 1:1). H NMR
(500 MHz, CDCl3) 1.29 (t, J=7.0Hz, 3H), 3.63 (s, 3H), 3.84 (s, 3H), 3.86 (s, 3H), 4.03 (s, 2H), 4.28 (q, J=7.0Hz, 2H), 5.31 (s, 2H), 6.41
(s, 2H), 7.26 (m, 1H), 7.60 (m, 1H), 8.55 (d, J=7.5Hz, 1H), 8.59 (s, 1H) ppm; IR (KBr)  1719, 1592, 1568, 1484 cm−1; Anal. Calcd for
C20H24N2O6: C, 61.68; H, 6.47; N, 7.19; Found: C, 61.45; H, 6.52; N, 7.60. HR-MS (ESI, m/s) calcd. for C20H24N2O6 (M+1)+:389.1713,
found:389.1736
Ethyl 2-(methyloxyimine-3-(3,4,5-trimethoxy)phenyl prop-ionate 4j: 4j (yellow liquid) was obtained (with CH3I) at 65%
1
yield(10.1g) according to general procedure after purification through column chromatography (EtOAc/hexane, 1:5). H NMR (500
MHz, CDCl3) 1.33 (t, J=7.0Hz, 3H), 3.81 (s, 3H) 3.82 (s, 6H), 3.87 (s, 2H), 4.11 (s, 3H), 4.31 (q, J=7.0Hz 2H), 6.49 (s,2H) ppm; IR
(KBr)  2941, 1718, 1591, 1507, and 1460 cm−1; Anal. Calcd for C15H21NO6: C, 57.87; H, 6.80; N,4.50; Found: C, 57.35; H, 7.15;
N,4.81. HR-MS (ESI, m/s) calcd. for C15H21NO6 (M+1)+:312.1447, found:312.1425
Ethyl 2-(4-fluorobenzyloxy imine)-3-(3,4,5-trimethyloxy) phenyl propionate 4n: 4n: (yellow liquid) was obtained (with 4-
fluorobenzyl chloride) at 65% yield(13.2g) according to general procedure after purification through column chromatography
(EtOAc/hexane, 1:5). 1H NMR (500 MHz, CDCl3) 1.20 (t, J=7.0Hz, 3H), 3.52 (s, 3H), 3.75 (s, 3H), 3.77 (s, 3H), 3.98 (s,2H), 4.20 (q,
J=7.0Hz, 2H), 5.15 (s,2H), 6.28 (s, 2H), 7.10 (m, 2H), 7.19 (m, 2H) ppm; IR (KBr)  2960,1718, 1597, 1575, 1478 cm−1; Anal. Calcd
for C21H24FNO6: C, 62.21, H, 5.97; N, 3.45; Found: C, 62.18; H, 5.92; N, 3.52. HR-MS (ESI, m/s) calcd. for C21H24FNO6
(M+1)+:406.1666, found:406.1688
Ethyl 2-(4-chlorobenzyloxy imine)-3-(3,4,5-trimethyloxy) phenyl propionate 4o: 4o (yellow liquid) was obtained (with 4-
chlorobenzyl chloride)at 70% yield(14.7g) according to general procedure after purification through column chromatography
(EtOAc/hexane, 1:5). 1H NMR (500 MHz, CDCl3) 1.20 (t, J=7.0Hz, 3H), 3.52 (s, 3H), 3.75 (s, 3H), 3.77 (s, 3H), 3.98 (s, 2H), 4.20 (q,
J=7.0Hz, 2H), 5.15 (s, 2H), 6.28 (s, 2H), 7.10 (m, 2H), 7.19 (m, 2H) ppm; IR (KBr)  1718, 1597, 1575, 1478 cm−1; Anal. Calcd for
C21H24ClNO6: C, 62.21, H, 5.97; N, 3.45; Found: C, 62.22; H, 5.99; N, 3.49. HR-MS (ESI, m/s) calcd. for C21H24ClNO6
(M+1)+:422.1370, found:422.1359
Methyl 2-cyclohexyloxyimine-3--Naphthylpropionate 4p: 4p was obtained(with cyclohexyl bromide) at 72% yield(11.7g)
according to general procedure after purification through column chromatography (EtOAc/hexane, 1:5). 1H NMR (500 MHz, CDCl3)
1.28–1.37 (m, 3H), 1.51–1.56 (m, 3H), 1.72 (m, 2H), 1.98 (m, 2H), 3.56(s,3H), 3.94 (s, 2H), 4.34 (m, 1H), 7.25–7.45 (m, 3H), and
7.72–7.79 (m, 4H) ppm; IR (KBr)  1718, 1597, 1575, and 1478 cm−1; Anal. Calcd for C21H23NO3: C, 73.82, H, 7.12; N, 4.30; Found:
C, 74.35; H, 7.38; N, 4.20. HR-MS (ESI, m/s) calcd. for C21H23NO3 (M+1)+:326.1756, found:326.1790
Methyl 2-cyclohexyloxyimine-3--Quinolyl propionate 4q: 4q was obtained(with cyclohexyl bromide) at 72% yield(11.7g)
1
according to the general procedure after purification through column chromatography (EtOAc/hexane, 1:5). H NMR (500 MHz,
CDCl3), 1.16–1.20 (m, 1H), 1.24–1.32 (m, 2H), 1.40–1.47 (m, 3H), 1.58–1.61 (m, 2H), 1.86–1.92 (m, 2H), 3.85 (s, 3H), 4.34 (m, 1H),
4.34 (s, 2H), 7.37 (d, J=8.5Hz, 1H), 7.51 (t, J=7.5Hz, 1H), 7.69 (t, J=7.5Hz,1H), 7.78 (d, J=8.0Hz, 1H), and 8.03 (m, J=8.0Hz,1H),
8.10 (m, J=8.5Hz, 1H) ppm; IR (KBr)  1717, 1600, 1574, 1478 cm−1; Anal. Calcd for C19H22N2O3: C, 69.92, H, 6.79; N, 8.58; Found:
C, 70.51; H, 7.17; N, 8.27. HR-MS (ESI, m/s) calcd. for C19H22N2O3 (M+1)+:327.1709, found:327.1732
General procedure to prepare N-(2-alkyloxyimine-3-arylpropionyl)proline 5
The above preparation product 4 (0.02 mol) and 60 ml of 2N NaOH were placed in a 100 ml reaction flask equipped with a
mechanical stirrer, reflux condenser, and a thermometer. The mixture was stirred for 2 h at 95 °C. After cooling, the solution was
acidified with hydrochloric acid to pH 3. The mixture was subsequently extracted with EtOAc (5 × 30 ml), and the extract was dried
over MgSO4. The solution was concentrated under reduced pressure to produce the corresponding crude product carboxylic acid, which
was used in the subsequent reaction without purification.
(C2H5)3N (1.5 ml) and HBTU (2.6 g) were added into DMF solution (30 ml, 6.5 mmol) to the above corresponding product under
stirring, and methyl proline hydrochlorinate (6.8 mmol) was subsequently added dropwise. The mixture was stirred for 24 h at 35 °C–
40°C, then 10 ml of H2O was added. The pH of the mixture was adjusted to 6 with diluted hydrochloric acid and then extracted with
CH2Cl2 (2 × 50 ml). The organic layer was dried over anhydrous MgSO4, filtered, and concentrated to produce solid residue. The crude
product was purified through flash chromatography on silica gel to produce coupling products.
LiOH (5 eq) was added under stirring in a 100 ml reaction flask containing 0.1 mol of the above reaction products and solvent
(THF:H2O = 7:1). The mixture was stirred for 12 h at room temperature. The solution was concentrated, and then equal volume of water
was added. The pH of the solution was adjusted to 3.0 with hydrochloric acid and then extracted with ethyl acetate (30 ml × 5). Organic
phase was dried over anhydrous MgSO4 and concentrated under reduced pressure to produce the crude product 5 (5a-5q) (yield >95%),
which was used in the subsequent reaction without purification.
General procedure to prepare N-alkyloxy DKP 6
In a 100 ml reaction flask, 0.1 mol compound 5(a–q) and 40 ml of anhydrous benzene were added and cooled to 0 °C. SOCl2 (0.15 ml)
was added dropwise under stirring. The reaction lasted for 4 h at room temperature. The solution was concentrated in vacuo to produce
ropy residue and then equal volume of water was added under stirring. The pH of solution was adjusted to 8 with hydrochloric acid and
then extracted with CH2Cl2 (30 ml × 5). The organic phase was dried over anhydrous MgSO4, filtered, and concentrated under reduced
pressure to produce crude product, which was purified through flash chromatography on silica gel to produce the title compound 6. The
racemic product 6 were obtained , if equivalent Et3N was added to reaction system, after adding SOCl2

Cyclo[N-butyloxy-2-[(-pyridyl) methylene]glycyl-prolyl] 6a: 6a was obtained at 80% yield according to general procedure after
purification through flash chromatography (EtOAc/hexane, 1:2). IR (KBr)  1701, 1665, 1628, 1586, 1475 cm−1; 1H NMR (500 MHz,
CDCl3) 0.67–0.70 (t, J=7.5Hz，3H), 0.92–0.97 (m, 2H), 1.02–1.16 (m, 2H), 1.97–2.03 (m, 1H), 2.11–2.19 (m, 2H), 2.49 (m, 1H),
3.47–3.51 (q, J=7.0Hz, 1H), 3.66–3.78 (m, 3H), 4.31 (q, J=7.0Hz, 1H), 7.11 (s, 1H), 7.27 (dd, J=5.0Hz, J=3.0Hz, 1H), 7.75 (d, J=8.0
Hz, 1H), 8.51 (dd, J=6.5 Hz, J=3.0Hz, 1H), 8.65 (s, 1H) ppm; Anal. Calcd for C17H21N3O3: C, 64.74; H, 6.71; N, 13.32; Found: C,
64.81; H, 6.73; N, 13.35. HR-MS (ESI, m/s) calcd. for C17H21N3O3 (M+1)+:316.1661, found:316.1653
Cyclo[N-cyclohexyloxy-2-[(-pyridyl)methylene]glycyl-prolyl] 6b
The product 6b was obtained as a white solid according to the general procedure in 88% yield after purification by silica gel flash
chromatography (EtOAc–hexane, 1:2). Crystals suitable for single crystal X-ray analysis were grown by slow evaporation from CHCl3.
Mp 129.5~131 ◦C; IR (KBr)  1712, 1666, 1631, 1586,1480 cm-1; 1H NMR (500 MHz, CDCl3) 0.90–1.16 (m, 6H), 1.38–1.41 (m, 1H),
1.47–1.49 (m, 1H), 1.59–1.62 (m, 1H), 1.67–1.69 (m,1H), 1.97–2.02 (q, J=8.5 Hz, 1H), 2.09–2.17 (m, 2H), 2.50–2.54(m, 1H), 3.66–
3.76 (m, 4H), 4.30 (q, J= 7.0 Hz, 1H), 7.05 (s, 1H),7.76 (d, J= 9.0 Hz, 1H), 8.50 (d, J=5.0 Hz, 1H), 8.65 (s, 1H) ppm;13C NMR (125
MHz, CDCl3) 162.1, 157.8, 151.9, 150.1, 137.0,128.6, 128.3, 121.4, 114.0, 81.9, 57.5, 44.5, 29.0, 28.9, 27.7, 24.1,22.8, 22.6, 20.0; Anal.
Calcd for C19H23N3O3: C, 66.84; H, 6.79; N, 12.31; Found: C, 66.88; H, 6.85; N, 12.33. HR-MS (ESI, m/s) calcd. for C19H23N3O3
(M+1)+:342.1818, found:342.1829
Cyclo[N-benzyloxy-2-[(-pyridyl)methylene]glycyl-prolyl] 6c: 6c was obtained at 83% yield according to the general procedure after
purification through flash chromatography (EtOAc/hexane, 1:2). IR (KBr)  1705, 1671, 1631, 1589, 1479 cm−1; 1H NMR (500 MHz,
CDCl3), 1.69 (s, 1H), 1.96–2.08 (m, 2H), 2.10 (m, 1H), 2.44 (m, 1H), 3.67 (m, 2H), 4.28 (q, J = 7.0 Hz, 1H), 4.52 (d, J = 9.5 Hz, 1H),
4.63(d, J = 9.5 Hz, 1H), 6.88 (d, J = 7.5 Hz, 2H), 7.14–7.2 (m, 3H), 7.26 (d, J = 7.0 Hz, 1H), 7.73 (d, J = 8.0 Hz, 1H), 8.46 (d, J = 4.5
Hz, 1H), and 8.70 (d, J = 1.5 Hz, 1H) ppm; Anal. Calcd for C20H19N3O3: C, 68.75; H, 5.48; N, 12.03; Found: C, 68.68; H, 5.45; N,
12.08. HR-MS (ESI, m/s) calcd. for C20H19N3O3 (M+1)+:350.1505, found:350.1531
Cyclo[N-methyloxy-2-(phenylmethylene)glycyl-prolyl] 6d
The product 6d was obtained according to the general procedure in 87% yield after purification by silica gel flash chromatography
(EtOAc–hexane, 1:2). Chiral HPLC analyses of the product showed amajor peak for the (S)-enantiomer at Rf 14.5min (99.3% e.e.)
compared to racemic 6d (synthesized by using racemic 5d) which showed two equal peaks with Rfs of 14.5 min and 15.5 min for the
(S)- and (R)-enantiomers respectively. IR (KBr) 1698, 1674, 1622, 1578, 1506 cm-1; 1H NMR (500 MHz, CDCl3) 1.85–2.02 (m, 1H),
2.11–2.21 (m, 2H), 2.45–2.49 (m, 1H), 3.39 (s, 3H),3.67–3.72 (m, 2H), 4.29 (q, J=7.0 Hz, 1H), 7.21 (s, 1H), 7.31–7.33(m, 3H), 7.45 (d,
J=7.0Hz, 2H) ppm; 13C NMR(125 MHz, CDCl3)162.1, 159.4, 132.5, 130.7 (2), 128.6, 127.6 (2), 126.0, 120.3, 61.5, 58.1, 45.6, 28.2,
22.4; Anal. Calcd for C15H16N2O3: C, 66.16; H,5.92; N, 10.29; Found: C,66.20; H, 5.97; N, 10.33. HR-MS (ESI, m/s) calcd. for
C15H16N2O3 (M+1)+:273.1239, found:273.1256.
Cyclo[N-ethyloxy-2-(phenylmethylene)glycyl-prolyl] 6e: 6e was obtained at 85% yield according to the general procedure after
purification through column chromatography (EtOAc/hexane, 1:2). 1H NMR (500 MHz, CDCl3) 1.01 (t, J = 7.0 Hz, 3H), 1.87–1.98 (m,
1H), 2.11–2.20 (m, 2H), 2.46–2.48 (m, 1H), 3.47 (q, J = 7.0 Hz, 2H), 3.67–3.72 (m, 2H), 4.29 (q, J = 7.0 Hz, 1H), 7.20 (s, 1H), 7.30–
7.33 (m, 3H), 7.44 (d, J = 7.0 Hz, 2H) ppm; IR (KBr)  1702, 1678, 1620, 1577, and 1505 cm−1; Anal. Calcd for C16H18N2O3: C, 67.12;
H, 6.34; N, 9.78; Found: C,67.25; H, 6.25; N, 9.83. HR-MS (ESI, m/s) calcd. for C16H18N2O3 (M+1)+:287.1396, found:287.1412.
Cyclo[N-butyloxy-2-(phenylmethylene)glycyl-prolyl] 4f
The product 4f was obtained according to the general procedure in 80% yield after purification by silica gel flash chromatography
(EtOAc–hexane, 1 : 2). IR (KBr)  1705, 1680, 1618, 1577, 1503 cm-1; 1H-NMR (500 MHz, CDCl3) 0.65 (t, J=7.0 Hz, 3H), 0.91–0.95
(m, 2H), 1.03–1.13 (m, 2H), 1.96–1.99 (m, 1H), 2.10–2.18 (m,2H), 2.46–2.48 (m, 1H), 3.49 (q, J=7.0 Hz, 1H), 3.66–3.74 (m, 3H), 4.29
(q, J=6.5 Hz, 1H), 7.18 (s, 1H), 7.28–7.33 (m, 3H), 7.44 (d, J=7.0 Hz, 2H) ppm; 13C NMR (125 MHz, CDCl3) 162.2, 159.3, 132.8,
130.7(2), 128.5, 127.5(2), 126.6, 120.0, 74.1, 58.1, 45.6, 29.0, 28.3, 22.4, 18.5, 13.6; Anal. Calcd for C18H22N2O3: C, 68.77; H, 7.05; N,
8.91; Found: C,68.81; H, 7.09; N, 8.94. HR-MS (ESI, m/s) calcd. for C18H22N2O3 (M+1)+:315.1709, found:315.1688.
Cyclo[N-cyclohexyloxy-2-(phenylmethylene)glycyl-prolyl] 6g
The product 6g was obtained according to the general procedure in 85% yield after purification by silica gel flash chromatography
(EtOAc–hexane, 1 : 2). IR (KBr)  1701, 1670, 1624, 1585, 1503 cm-1; 1H NMR (500 MHz, CDCl3) 0.88–1.08 (m, 5H), 1.18–1.20(m,
1H), 1.36–1.39 (m, 1H), 1.46–1.47 (m, 1H), 1.63–1.66 (m, 2H),1.97–2.00 (m, 1H), 2.10–2.15 (m, 2H), 2.59–2.51 (m, 1H), 3.63–3.74
(m, 3H), 4.29 (q, J=6.5 Hz, 1H), 7.12 (s, 1H), 7.28–7.33 (m,3H), 7.43 (d, J=7.0 Hz, 2H) ppm; 13C NMR (125 MHz, CDCl3):163.2,
159.6, 132.7, 130.9(2), 128.4, 127.4(2), 119.8, 82.2, 58.4,45.4, 30.0, 29.5, 28.7, 25.2, 23.8, 23.6, 22.6, 22.4; Anal. Calcd forC20H24N2O3:
C, 70.56; H, 7.11; N, 8.23; Found: C, 70.59; H, 7.15;N, 8.26. HR-MS (ESI, m/s) calcd. for C20H24N2O3 (M+1)+:341.1865,
found:341.1842
Cyclo[N-benzyloxy-2-(phenylmethylene)glycyl-prolyl] 6h
The product 6h was obtained according to the general procedure in 88% yield after purification by silica gel flash chromatography
1
(EtOAc–hexane, 1 : 2). IR (KBr) 1706, 1681, 1618, 1589, 1577, 1511, 1503 cm-1; H NMR (500 MHz, CDCl3) 1.92–2.08 (m, 3H),
2.39–2.44 (m, 1H), 3.63–3.66 (m, 2H), 4.26 (q, J=6.5 Hz, 1H), 4.56(s, 2H), 6.83–6.85 (m, 2H), 7.14–7.17 (m, 2H), 7.23–7.25 (m,
2H),7.30–7.32 (m, 3H), 7.48–7.49 (m, 2H) ppm; 13C NMR (125 MHz, CDCl3) 162.4, 159.2, 132.7, 132.5, 131.0(2), 130.1(2), 128.9,
128.7, 128.1(2), 127.7(2), 126.5, 120.4, 75.8, 58.0, 45.6, 29.5, 22.4; Anal. Calcd for C21H20N2O3: C, 72.40; H, 5.79; N, 8.04; Found:
C,72.44; H, 5.82; N, 8.08. HR-MS (ESI, m/s) calcd. for C21H20N2O3 (M+1)+:349.552, found:349.1568
Cyclo[N-(3-pyridylmethyloxy)-2-[(3,4,5-trimethyloxy) phenyl -methylene] glycyl-prolyl] 6i: 6i was obtained at 75% yield according
to the general procedure after purification through column chromatography (EtOAc/hexane, 1:1). IR (KBr)  1696, 1668, 1622, 1590,

1569, 1518, 1490 cm−1; 1H NMR (500 MHz, CDCl3), 1.95 (m, 1H), 2.10 (m, 2H), 2.41 (m, 1H), 3.67 (m, 2H), 3.85 (s, 3H), 3.89 (s, 3H),
3.90 (s, 3H), , 4.27 (q, J=6.5 Hz, 1H), 4.63(s, 2H) 6.75 (s, 2H),7.14(s,1H), 7.28 (m, 1H), 7.58 (m, 1H), 8.52 (d, J=7.5Hz, 1H), 8.60 (s,
1H) ppm; Anal. Calcd for C23H25N3O6: C, 62.86; H, 5.73; N, 9.56; Found: C, 62.79; H, 5.76; N, 9.64. HR-MS (ESI, m/s) calcd. for
C23H25N3O6 (M+1)+:440.1822, found:440.1806
Cyclo[N-methyloxy-2-[(3,4,5-trimethyloxy) phenylmethylene] glycyl-prolyl] 6j: 6j was obtained at 86% yield according to the
general procedure after purification through flash chromatography (EtOAc/hexane, 1:2). IR (KBr)  1697, 1670, 1624, 1580, 1505
cm−1; 1H NMR (500 MHz, CDCl3) 1.99 (m, 1H), 2.13 (m, 1H), 2.22 (m, 1H), 2.46(m,1H), 3.48 (s, 3H), 3.68(m,2H) 3.88 (s, 3H), 3.89
(s, 3H), 3.91 (s, 3H), 4.29 (q, J=6.5Hz, 1H), 6.75 (s, 2H), 7.13(s, 1H) ppm; Anal. Calcd for C18H22N2O6: C, 64.12; H, 5.81; N, 9.84;
Found: C, 64.10; H, 5.78; N, 9.92. HR-MS (ESI, m/s) calcd. for C18H22N2O6 (M+1)+:363.1556, found:363.1570
Cyclo[N-butyloxy-2-[(3,4,5-trimethyloxy)phenylmethylene] glycylprolyl] 6k
The product 6k was obtained as a white solid according to the general procedure in 85% yield after purification by silica gel flash
1
chromatography (EtOAc–hexane, 1:2). Mp. 138.5~141.5 ◦C; IR(KBr)  1701, 1676, 1608, 1583, 1099 cm-1; H NMR (500 MHz,
CDCl3) 0.71 (t, J=7.0 Hz, 3H), 0.96–1.05 (m, 2H), 1.19–1.28 (m, 2H), 1.99–2.05 (m, 1H), 2.11–2.18 (m, 2H), 2.44–2.47 (m, 1H), 3.55
(dd, J=7.0 and J=7.5 Hz, 1H), 3.67–3.70 (m, 2H), 3.77–3.80(m, 1H), 3.85 (s, 9H), 4.27–4.31 (m, 1H), 6.69 (s, 2H), 7.10 (s, 1H) ppm;
Anal. Calcd for C21H28N2O6: C, 62.36; H, 6.98; N, 6.93; Found: C, 62.39; H, 7.02; N, 6.98. HR-MS (ESI, m/s) calcd. for C21H28N2O6
(M+1)+:405.2026, found:405.2019
Cyclo[N-cyclohexyloxy-2-[(3,4,5-trimethyloxy)phenyl methyl-ene]glycyl-prolyl] 6l
The product 6l was obtained according to the general procedure in 83% yield after purification by silica gel flash chromatography
(EtOAc–hexane, 1:2). IR (KBr)  1693, 1678, 1621, 1583, 1503,1099 cm-1; 1H-NMR (500 MHz, CDCl3): 1.01–1.14 (m, 4H), 1.19 (br,
1H), 1.28–1.29 (m, 2H), 1.42 (br, 1H), 1.65–1.72 (m, 2H),1.99–2.01 (m, 1H), 2.13–2.19 (m, 2H), 2.49–2.51 (m, 1H), 3.69–3.77 (m, 3H),
3.86–3.90 (m, 9H), 4.29 (q, J 7.0 Hz, 1H), 6.77 (s,2H), 7.04 (s, 1H) ppm; 13C NMR(125 MHz, CDCl3) 163.2, 159.9, 152.2, 138.8, 127.9,
126.9, 119.9, 108.5(2), 82.28, 65.9, 61.0, 58.4, 56.3(2), 45.5, 30.8, 30.1, 28.6, 25.2, 23.8, 23.6, 22.5; Anal. Calcd for C23H30N2O6: C,
64.17; H, 7.02; N, 6.51; Found: C, 64.21; H, 7.07; N, 6.48. HR-MS (ESI, m/s) calcd. for C23H30N2O6 (M+1)+:431.2182, found:431.2155
Cyclo[N-benzyloxy-2-[(3,4,5-trimethyloxy)phenylmethylene] glycyl-prolyl] 6m:The product 6m was obtained according to the general
procedure in 87% yield after purification by silica gel flash chromatography (EtOAc/hexane, 1 : 2). IR (KBr)  1698, 1677, 1621, 1608,
1
1583, 1503, 1099 cm-1; H NMR (500 MHz, CDCl3) 1.67 (s, 2H), 1.95-1.98 (m, 1H), 2.05–2.09 (m, 2H), 2.41–2.43 (m, 1H), 3.75 (s,
6H),3.87(s, 3H), 4.25–4.28 (m, 1H), 4.58 (d, J=9.0 Hz, 1H), 4.68 (d,J=9.0 Hz, 1H), 6.77 (s, 2H), 6.95 (d, J=7.5 Hz, 2H), 7.16 (s,
1H),7.19–7.26 (m, 3H) ppm; Anal. Calcd for C24H26N2O6: C, 65.74; H,5.98; N, 6.39; Found: C, 65.75; H, 6.01; N, 6.42. HR-MS (ESI,
m/s) calcd. for C24H26N2O6 (M+1)+:439.1869, found:439.1888
Cyclo[N-4-fluorobenzyoxyl-2-[(3,4,5-trimethyloxy) phenyl-methylene]glycyl-prolyl] 6n: The product 6n was obtained according to
the general procedure in 80% yield after purification by flash chromatography (EtOAc/hexane, 1:2). IR (KBr)  1736, 1688, 1632,
1591, 1510, 1457 cm−1; 1H NMR (500 MHz, CDCl3) 1.96 (m, 1H), 2.08 (m, 2H), 2.42 (m, 1H), 3.65 (m, 2H), 3.84 (s, 3H), 3.86 (s, 3H),
3.88 (s, 3H), 4.12 (m, 1H), 5.14 (s, 2H), 6.75 (s, 2H), 7.16 (s, 1H), 7.36 (m, 2H), 7.58 (m, 2H) ppm; Anal. Calcd for C24H25FN2O6: C,
63.15; H, 5.52; N, 6.14; Found: C, 63.18; H, 5.55; N, 6.22. HR-MS (ESI, m/s) calcd. for C24H25FN2O6 (M+1)+:457.1775,
found:457.1763
Cyclo[N-4-clorobenzyoxyl-2-[(3,4,5-trimethyloxy) phenyl-methyl-ene]glycyl-prolyl] 6o: 6o was obtained at 83% yield according to
the general procedure after purification through flash chromatography (EtOAc/hexane, 1:2). IR (KBr)  1739, 1686, 1635, 1590, 1507,
1461 cm−1; 1H NMR (500 MHz, CDCl3) 2.00 (m, 1H), 2.16 (m, 2H), 2.42 (m, 1H), 3.64 (m, 2H), 3.84 (s, 3H) 3.86 (s, 3H), 3.88 (s, 3H),
4.13 (m, 1H), 5.12(s, 2H), 6.75 (s, 2H), 7.36 (m, 2H), 7.16 (s, 1H), 7.58(m, 2H) ppm; Anal. Calcd for C24H25ClN2O6: C, 63.15; H, 5.52;
N, 6.14; Found: C, 63.09; H, 5.51; N, 6.20. HR-MS (ESI, m/s) calcd. for C24H25ClN2O6 (M+1)+:473.1479, found:473.1482
Cyclo[N-cyclohexyloxy-2-(-naphthylmethylene)glycyl-prolyl] 6p: 6p was obtained at 75% yield according to the general procedure
after purification through flash chromatography (EtOAc/hexane, 1:2). IR (KBr)  1715, 1668, 1628, 1586, 1509, 1480 cm−1; 1H NMR
(500 MHz, CDCl3) 0.86–0.91 (m, 4H), 1.00–1.05 (m, 1H), 1.13–1.18 (m, 1H), 1.31–1.38 (m, 2H), 1.59–1.67 (m, 2H), 1.85 (m, 1H),
2.04–2.11 (m, 2H), 2.48–2.52 (m, 1H), 3.61–3.66 (m, 1H), 3.69–3.73 (m, 2H), 4.28(m, 1H), 7.12 (s, 1H), 7.44–7.47(m, 3H), 7.71–7.84
(m, 4H) ppm. Anal. Calcd for C23H26N2O3: C, 73.82; H, 6.71; N, 7.17; Found: C, 73.77; H, 6.80; N, 7.25. HR-MS (ESI, m/s) calcd. for
C23H26N2O3 (M+1)+:391.2022, found:391.2009
Cyclo[N-cyclohexyloxy-2-(-quinolylmethylene)glycyl-prolyl] 6q: 6q was obtained at 70% yield according to the general procedure
after purification through flash chromatography (EtOAc/hexane, 1:2). IR (KBr)  1710, 1670, 1624, 1583, 1500, 1475 cm−1; 1H NMR
(500 MHz, CDCl3) 0.85–0.91 (m, 4H), 1.26–1.35 (m, 3H), 1.57–1.60 (m, 2H), 1.85 (m, 1H), 2.00–2.10 (m, 2H), 2.44–2.50 (m, 1H),
3.59–3.64 (m, 1H), 3.66–3.70 (m, 2H), 4.29 (q, J = 7.0 Hz, 1H), 7.15 (s, 1H), 7.33 (dd, J = 3.0 Hz, J = 5.5 Hz, 1H), 7.47 (t, J = 7.5 Hz,
1H), 7.65 (t, J = 7.5 Hz, 1H), 7.75 (d, J = 8.0 Hz, 1H), 7.93 (t, J = 9.0 Hz, 1H), 8.03 (dd, J = 3.0 Hz, J = 5.5 Hz, 4H). Anal. Calcd for
C23H25N3O3: C, 70.57; H, 6.44; N, 10.73; Found: C, 70.63; H, 6.37; N, 10.81. HR-MS (ESI, m/s) calcd. for C23H25N3O3
(M+1)+:392.1974, found:392.1960.
4.2. Molecular modeling
Computer docking was performed using the X-ray crystal structure of human caspase-3 (PDB Identification Code: 3H0E) by utilizing
DOKER of the Discovery Studio version 3.5 (Accelrys, San Diego, USA). The molecular surface of the caspase-3 catalytic domain was
generated from the surrounding residues of co-crystallized inhibitor within 5 Å. The Docker energies of the top 10 docked
conformations were ranked, and the lowest energy was recorded.
Acknowledgment

The authors express their gratitude for the financial assistance extended by the National Natural Science Foundation of China (Grant
Nos. 1272052 and 21472034), the National Basic Research Program of China (Grant Nos. 2011CB512007 and 2012CB723501), the
Hebei Province Natural Science Foundation (Grant Nos. B2014208138 and 12966737D), the Hebei Province Science and Technology
Support Program (Grant No. 14272604D), and the Foundation of the Education Department of Hebei Province (Grant Nos. ZH2012025
and ZD2014017).
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O
O
O
O
O
O
NH
*
NH
N
N
N
N
N
NH
HN
H
NH
H
Br
N
H
Barettin
△ (-)-PLH
(-)-PLH
Phenylahistin
O
HN
N
O
N
O
H
OH
N
O
NH
O
N
N
NH
HO
O
O
mycelianamide
brevianamide S
A: Ar=3-Pyridyl
B: Ar= phenyl
O
COOR1
COOR1
a
b
Ar
Ar
OR1
Ar
Cl
C: Ar=3,4,5-trimethyloxy phenyl
D: Ar= -Naphthyl
E: Ar=-Quinolyl
N
OH
1A-E
2A-E
3A-E
R1=Me or Et
O
O
O
N
f
c
d
e
Ar
N
Ar
OR1
Ar
N
N
N
OR
COOH
RO
O
6a-q
RO
4a-q
5a-q
a: Ar=3-Pyridyl, R=n-Bu
b: Ar=3-Pyridyl, R=c-hexyl
c: Ar=3-Pyridyl, R=benzyl
d: Ar= phenyl, R=Methyl
e: Ar= phenyl, R=Ethyl
j: Ar=3,4,5-trimethyloxy phenyl, R=Methyl
K: Ar=3,4,5-trimethyloxy phenyl, R=n-Bu
l: Ar=3,4,5-trimethyloxy phenyl, R=c-hexyl
m: Ar=3,4,5-trimethyloxy phenyl, R=benzyl
n: Ar=3,4,5-trimethyloxy phenyl, R=4-fluoro benzyl,
f: Ar= phenyl, R=n-Bu
g: Ar= phenyl, R=c-hexyl
h: Ar= phenyl, R=benzyl
o: Ar=3,4,5-trimethyloxy phenyl, R=4-chloro benzyl,
p: Ar= -Naphthyl , R=c-hexyl ,
I: Ar=3,4,5-trimethoxyl benzyl, R=3-Pyridyl
q: Ar=-Quinolyl , R=c-hexyl ,
Scheme 1 Synthesis of E-arylidene bicyclo-DKPs

O
Ar
H
N
N
RO
H
O
O
6
Ar
O
O
Ar
Ar
O
H
N
H
N
N
H
RO
H
RO
N
N
N
O
Ar
O
Ar
O
RO
C
H
O
O
Cl
N
SOCl2
N
5-2
N
O
O
N
Ar
RO
C
H
RO
O
HOOC
5
Ar
Ar
Cl
H
N
Et3N
5-1
H
N
N
N
H
RO
RO
N
H
N
O
+
6
RO
C
O
O
O
Ar
H
N
5-4
5-3
N
RO
H
6`
O
Scheme 2. Possible cyclization reaction
Fig.1. Structure of 6b with ellipsoid displacement drawn at 30% probability level.
Fig. 2. Structure of 8 with displacement ellipsoids drawn at 30% probability level
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
O
O
H
O
O
O
O
H
N
N
N
H
+
N
N
N
OCH3
O
O
Ar
Ar
7
7`
O
OH
OCH3
OCH3
OCH3
N
N
H
O
8
Scheme 3. Possible mechanism of rearrangement-fragmentation reaction

100
80
60
40
20
0
6d
6e
6f
6h
6j
Fig. 3. Caspase-3 inhibitory activity of some compounds (50 µM)
Fig.4. (a) Proposed binding mode of 6e to caspase-3 enzyme based on the X-ray co-crystal structure of caspase-3 complexed with a
small molecule inhibitor (left). (b) Interaction of the chain cyclic peptide 6e with adjacent amino acid residues (right).