Structure-based design and synthesis of novel furan-diketopiperazine-type derivatives as potent microtubule inhibitors for treating cancer

Article abstract of DOI:10.1016/j.bmc.2020.115435

Plinabulin, a synthetic analog of the marine natural product “diketopiperazine phenylahistin,” displayed depolymerization effects on microtubules and targeted the colchicine site, which has been moved into phase III clinical trials for the treatment of non-small cell lung cancer (NSCLC) and the prevention of chemotherapy-induced neutropenia (CIN). To develop more potent anti-microtubule and cytotoxic derivatives, the co-crystal complexes of plinabulin derivatives were summarized and analyzed. We performed further modifications of the tert-butyl moiety or C-ring of imidazole-type derivatives to build a library of molecules through the introduction of different groups for novel skeletons. Our structure–activity relationship study indicated that compounds 17o (IC₅₀ = 14.0 nM, NCI-H460) and 17p (IC₅₀ = 2.9 nM, NCI-H460) with furan groups exhibited potent cytotoxic activities at the nanomolar level against various human cancer cell lines. In particular, the 5-methyl or methoxymethyl substituent of furan group could replace the alkyl group of imidazole at the 5-position to maintain cytotoxic activity, contradicting previous reports that the tert-butyl moiety at the 5-position of imidazole was essential for the activity of such compounds. Immunofluorescence assay indicated that compounds 17o and 17p could efficiently inhibit microtubule polymerization. Overall, the novel furan-diketopiperazine-type derivatives could be considered as a potential scaffold for the development of anti-cancer drugs.

Full text of DOI:10.1016/j.bmc.2020.115435

Journal Pre-proofs
Structure-based Design and Synthesis of Novel Furan-Diketopiperazine-Type
Derivatives as Potent Microtubule Inhibitors for Treating Cancer
Zhongpeng Ding, Feifei Li, Changjiang Zhong, Feng Li, Yuqian Liu, Shixiao
Wang, Jianchun Zhao, Wenbao Li
PII:
S0968-0896(20)30245-5
https://doi.org/10.1016/j.bmc.2020.115435
BMC 115435
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
4 January 2020
10 March 2020
10 March 2020
Please cite this article as: Z. Ding, F. Li, C. Zhong, F. Li, Y. Liu, S. Wang, J. Zhao, W. Li, Structure-based
Design and Synthesis of Novel Furan-Diketopiperazine-Type Derivatives as Potent Microtubule Inhibitors for
Treating Cancer, Bioorganic & Medicinal Chemistry (2020), doi: https://doi.org/10.1016/j.bmc.2020.115435
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover
page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version
will undergo additional copyediting, typesetting and review before it is published in its final form, but we are
providing this version to give early visibility of the article. Please note that, during the production process, errors
may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
© 2020 Published by Elsevier Ltd.

Graphical Abstract
To create your abstract, type over the instructions in the template box below.
Structure-based Design and Synthesis of Novel
Furan-Diketopiperazine-Type Derivatives as Potent
Microtubule Inhibitors for Treating Cancer
Leave this area blank for abstract info.
Zhongpeng Ding a, Feifei Li a, Changjiang Zhong a, Feng Li c,
Yuqian Liu a, Shixiao Wang d, Jianchun Zhao a, d and
Wenbao Li a, b, d
*
a School of Medicine and Pharmacy, Ocean University of China, Qingdao 266003, China
b Innovation Center for Marine Drug Screening and Evaluation, Qingdao National Laboratory for Marine
Science and Technology, Qingdao 266071, China
c Shandong Peninsula Engineering Research Center of Comprehensive Brine Utilization, Weifang University of
Science and Technology, Weifang 262700, Shandong, China.
d Marine Biomedical Research Institute of Qingdao, Qingdao 266071, China
* Corresponding author: Tel: 86-532-85906861; E-mail: wbli92128@ouc.edu.cn (Wenbao Li)
O
X
NH
N
NH
HN
F
O
1
2
: X = C=O, IC50 =3.8 nM
: X = O, IC50 =4.0 nM
O
O
O
NH
O
HN
F
17f
: X = O, IC50 = 154.5 nM
SAR
O
O
X
NH
O
HN
O
F
17o
17p
: X = O, IC50 =14.0 nM
: X = C=O, IC50 =2.9 nM
Fonts or abstract dimensions should not be changed or altered.

Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com
Structure-based Design and Synthesis of Novel Furan-Diketopiperazine-Type
Derivatives as Potent Microtubule Inhibitors for Treating Cancer
Zhongpeng Ding a, Feifei Li a, Changjiang Zhong a, Feng Li c, Yuqian Liu a, Shixiao Wang d, Jianchun
Zhao a, d and Wenbao Li a, b, d
*
a School of Medicine and Pharmacy, Ocean University of China, Qingdao 266003, China
b Innovation Center for Marine Drug Screening and Evaluation, Qingdao National Laboratory for Marine Science and Technology, Qingdao 266071, China
c Shandong Peninsula Engineering Research Center of Comprehensive Brine Utilization, Weifang University of Science and Technology, Weifang 262700,
Shandong, China.
d Marine Biomedical Research Institute of Qingdao, Qingdao 266071, China
* Corresponding authors: Tel: 86-532-85906861; E-mail: wbli92128@ouc.edu.cn (Wenbao Li)
ARTICLE INFO
ABSTRACT
Article history:
Received
Plinabulin, a synthetic analog of the marine natural product “diketopiperazine
phenylahistin,” displayed depolymerization effects on microtubules and targeted the colchicine
site, which has been moved into phase III clinical trials for the treatment of non-small cell lung
cancer (NSCLC) and the prevention of chemotherapy-induced neutropenia (CIN). To develop
more potent anti-microtubule and cytotoxic derivatives, the co-crystal complexes of plinabulin
derivatives were summarized and analyzed. We performed further modifications of the tert-butyl
moiety or C-ring of imidazole-type derivatives to build a library of molecules through the
introduction of different groups for novel skeletons. Our structure-activity relationship study
indicated that compounds 17o (IC50 = 14.0 nM, NCI-H460) and 17p (IC50 = 2.9 nM, NCI-H460)
with furan groups exhibited potent cytotoxic activities at the nanomolar level against various
human cancer cell lines. In particular, the 5-methyl or methoxymethyl substituent of furan group
could replace the alkyl group of imidazole at the 5-position to maintain cytotoxic activity,
contradicting previous reports that the tert-butyl moiety at the 5-position of imidazole was
essential for the activity of such compounds. Immunofluorescence assay indicated that
compounds 17o and 17p could efficiently inhibit microtubule polymerization. Overall, the novel
furan-diketopiperazine-type derivatives could be considered as a potential scaffold for the
development of anti-cancer drugs.
Received in revised form
Accepted
Available online
Keywords:
Structure-based Drug Design
Furan-Diketopiperazine-Type
SAR
Microtubule Inhibitor
Anticancer
2009 Elsevier Ltd. All rights reserved.
1. Introduction
Cancer has become the primary cause of human death in
the world. There were 17.0 million reported cancer cases and
9.5 million cancer-related deaths globally in 2018, and it is
estimated that the number of new cancer cases will reach 27.5
million by 20401. There were 3.93 million cancer cases and
2.34 million cancer-related deaths reported in China in 2015.
Microtubule targeting agents were discovered through in-depth
and systematic research and currently are divided into four
categories based on their binding sites (i.e. taxanes,
vinblastines, colchicines and laulimalids)2-4
. Microtubule
inhibitors exhibited highly potent anti-microtubule and
cytotoxic activities by stabilizing or disaggregating
microtubule polymerization in the process of microtubule
assembly, a property which could disrupt the dynamic balance

of microtubules, interfere with spindle formation, and prevent
cell division5-7. Vinca alkaloid site-binding agents and taxane
site-binding agents have been widely used to treat various
two additional hydrogen bonds with GLH198 and VAL236,
respectively18-21. In crystal structures (5YL4 and 5XHC), the
benzene ring of the benzoyl group on the A ring could form an
additional π-π interaction, which could be beneficial to anti-
proliferation18,20. The pocket at the colchicine site crossed the
α/β-tubule subunit and extended to the boundary of the GTP
pocket, which was formed from the hydrophilic and
hydrophobic amino acid residues19-21. Among these amino acid
residues around the binding pocket of plinabulin derivatives,
the conformation of the T7 loop (amino acid residue number:
244-251) and H7 helix (amino acid residue number: 224-243)
in β-tubulin was changed in comparison with the structure of
tubulin-apo, which may primarily contribute to the inhibition
of microtubule polymerization19. Therefore, the position of 5-
tert-butyl-1H-imidazole of the C-ring, which was close to the
tubulin heterodimer interface, had an important influence on
tubulin conformation.
cancers8,9.
However,
severe
neurotoxicity,
adverse
myelosuppression effects, and multidrug resistance have also
been observed in their clinical applications8-9. In contrast,
molecules binding at colchicine sites have not yet been
developed into clinical cancer therapies.
Plinabulin, a micro-tubulin inhibitor derived from the
marine natural product “phenylahistin,” has been moved into
phase Ⅲ clinical trials paired with docetaxel as a combination
agent for treating non-small cell lung cancer (NSCLC) and for
the prevention of chemotherapy-induced neutropenia (CIN).
The treatment could block cells in the G2/M phase of the cell
cycle and induce apoptosis through caspase-3, caspase-8,
caspase-9, and PARP (poly ADP-ribose polymerase)
cleavage10-13. Importantly, plinabulin was also shown to inhibit
tumor cell proliferation through the disruption of tumor
vascular endothelial cells14. Recent studies reported that
plinabulin binds at the colchicine site of tubulin to prevent the
assembly of α/β-tubulin heterodimers and induces GEF-H1
release to increase dendritic cells from driving a distinct cell
A systematic structure-activity relationship study (SAR)
of plinabulin derivatives were performed by Hayashi’s group,
in which compound 1 (IC50 = 3.8 nM, NCI-H460) was reported
to have the most potent anti-proliferation activity13,22-23. Our
group also designed and synthesized three series of A/B/C-ring
plinabulin derivatives based on the co-crystal structures. So
far, compound 2 (IC50 = 4.0 nM, NCI-H460) was the optimally
active compound, and the theoretically calculated LogPo/w
and PCaco were superior to other compounds20. In general,
plinabulin derivatives exhibited optimal activities when the C-
rings had 5-tert-butyl 1H-imidazole and 2-pyridine (KPU-300,
IC50=7.0 nM, HT-29, IC50= 6.3 nM, BxPC-3). In particular, the
tert-butyl group at the 5-position of imidazole was essential for
the activity of such compounds. To further understand the SAR
of plinabulin derivatives and to obtain more potent
compounds, we further analyzed the co-crystal structure of
compound 1 with tubulin, then designed and modified the C
ring in this study.
signaling program15,16
.
The co-crystal complex of plinabulin with tubulin (PDB
code: 5C8Y) was cultured and resolved at different resolutions
by Yang et. al and Cavalli et. al, which could elucidate the
detailed interactions between ligands and tubulin16,17. The
crystal structures of the tubulin complex with plinabulin
derivatives (KPU-105, MBRI-001, compound 1) were also
solved and reported previously, for which PDB codes were
5YL4, 5XI5, and 5XHC, respectively. These tubulin–ligand
complex structures revealed that plinabulin derivatives resided
in a deeper position in β-tubulin, which primarily contributed
to the binding affinity of ligands and tubulin. The carbonyl
oxygen atom and the hydrogen atom on the nitrogen of
diketopiperazine (DKP) of plinabulin derivatives could form

O
O
O
O
NH
N
NH
N
NH
NH
HN
HN
Plinabulin
(-)-phenylahistin
C
O
O
A
B
O
O
X
NH
N
NH
HN
N
NH
HN
F
O
1
2
: X = C=O; : X = O
KPU-300
Fig. 1. Chemical structures of (-)-phenylahistin, plinabulin and its representative derivatives.
2.1. Analysis of Co-crystal complex of compound 1 with tubulin
2. Results and discussion
and design strategy
Fig. 2. Co-crystal structure of tubulin with compound 1 in different visual display.
The co-crystal complex of compound 1 at 2.75 Å resolution
was reported and placed in a protein data bank (PDB,
https://www.rcsb.org/structure/5XHC), which brought new
insight to the interaction of microtubules and the molecule20. In
this study, the co-crystal complex was further analyzed by
computer calculation using Maestro software (Fig. 2). Compound
1 could form favorable interactions with tubulin, including
hydrogen bonding and π-π interactions. The majority
hydrophobic residues included ALA314, ALA315, ILE316,
LEU246, LEU240, VAL236, MET233, ILE4, PHE20, LEU135,
PHE167, LEU250, and MET257, and the majority hydrophilic
residues included THR179, HTE6, THR136, GLN134, THR166,
ASN165, and ASN256 from α tubulin or β tubulin, both of which
could form a protein pocket. Moreover, the pocket that crossed
the α/β-tubule subunit and extended to the boundary of the GTP
pocket could provide more space for further modification of 2, 5-
diketopiperazine derivatives.
According to the previous report, the pharmacophore of the
A ring was optional to the para-fluorobenzoylphenyl or para-
fluorophenoxyphenyl group, and the diketopiperazine core
structure of the B ring was immutable if their favorable
interactions were to be maintained20, 22. In this study, we
expanded upon these findings and designed four series of
compounds with different C ring substitutions with which to
explore the new scaffolds and structure-activity relationship
(SAR). The 5-position of imidazole was replaced by different
alkyl groups in series 1 to confirm the function of the tert-butyl
group, and series 2-4 were constructed to explore different types
of aromatic rings at the C ring.

X = C=O, O
Y = C, N
Y' = C, N
Z = O, S
W = CH, N
Z' = O, N
W' = C, N
C
A
B
O
O
X
NH
HN
R
F
R=
R"
N
Y
Z
NH
R'
Y'
Series 1:
Series 3:
Series 4:
W
Z'
Series 2:
W'
R' = H, methyl, isopropyl, cyclopropyl, tert-butyl
R" = H, methyl, methoxymethyl, hydroxymethyl
Fig. 3. General design structures of compounds 14a-14h and compounds 17a-17r.
reaction using MnO2. The intermediate 8c was prepared through
substitution and cyclization reactions in two consecutive steps.
The pure 8c was precipitated from water by adjusting the pH
using aqueous sodium carbonate solution.
2.2. Chemistry
To synthesize the plinabulin derivatives 1, 2, 14a-14h, and
17a-17r, we explored and adopted two synthetic strategies to
condense the A ring and C ring with DKP. The two routes could
be used for different aldehydes, dependent upon improved yield
and fewer by-products of the reaction. The chemical structures of
the compounds were characterized by nuclear magnetic
resonance (NMR) and high-resolution mass spectrometer
(HRMS) analysis (see Supporting Information).
Consecutive aldol reactions with two different aldehydes
onto the diacetyl-2, 5-piperazinedione ring were then carried out
in the presence of Cs2CO3 in N, N-dimethylformamide (DMF).
Namely, the various imidazole aldehydes were condensed with
diacetyl-2, 5-piperazinedione for 20 h at room temperature.
When the alkyl group was tert-butyl, cyclopropyl or isopropyl,
the diacetyl-2, 5-piperazinedione was reacted with a single
imidazole aldehyde. However, the reaction can produce double
imidazol-3, 6-yl piperazine-2, 5-dione as a by-product when
hydrogen or methyl occupies the 5- position of imidazole
because of the resulting small steric hindrance. The by-product of
the unsubstituted imidazole was greater than that of methyl
imidazole and was difficult to purify through silica gel column
chromatography. Therefore, the results indicated that the bulky
group at the 5-position was important for preventing the
formation of by-products. The final condensation reaction was
performed in dark conditions under an N2 atmosphere to avoid
yielding a product of E configuration13. In general, the total
yields of the synthesis of compounds 14a-14h were above 1% in
six steps.
2.2.1. Synthesis of plinabulin derivatives 1, 2 and 14a-14h
The plinabulin derivatives 1, 2, and 14a-14h were
synthesized via a sequence of six linear steps. Firstly, the
colorless liquid oxazole esters 5a and 5b were synthesized
through
a [3 + 2] cyclization reaction using ethyl 2-
cyanoacetoacetate and pivalic anhydride or isobutyric anhydride
as the starting materials in the presence of 1, 8-
diazabicyclo[5.4.0]undec-7-ene (DBU) for 48
h at room
temperature (approximately 20oC), and then purified by silica gel
column chromatography. The oxazole ester was converted into
the imidazole ester by the solvolysis reaction in formamide for 24
o
h at 175 C, which was purified by slurry using water. Then, the
imidazole ester was reduced to alcohol using LiAlH4 as the
reducing agent, and the aldehyde was generated by an oxidation

R
COOEt
O
O
a
c
CN
COOEt
O
O
N
O
N
R
O
R
R
R
COOEt
R
OH
e
H
f
3
5a, 5b
4a, 4b
HN
N
HN
N
HN
d
O
O
O
b
8a-8c
9a-9c
10a-10e
O
O
Cl
7
6
O
O
O
O
O
Ac
X
X
h
g
N
NH
N
NH
N
O
NH
NH
N
N
HN
Ac
F
Ac
F
R
R
O
11
13a, 13b
12a-12e
14a-14h
OH
B
4a, 5a
4b, 5b
1
2
: R = t-Butyl
: R = Isopropyl
: X = C=O, R = t-Butyl
: X = O, R = t-Butyl
: X = O, R = Isopropyl
: X = C=O, R = Isopropy
: X = O, R = Cyclopropyl
: X = C=O, R = Cyclopropyl
: X = O, R = Methyl
: X = C=O, R = Methyl
: X = O, R = Hydrogen
: X = C=O, R = Hydrogen
O
OH
j
O
HO
O
14a
14b
14c
14d
14e
14f
14g
14h
8a 9a 10a 12a
:
,
,
,
F
F
F
R = t-Butyl
8b 9b 10b 12b
:
13a
,
,
,
R = Isopropyl
8c 9c 10c 12c
:
O
,
,
,
O
O
R = Cyclopropyl
10d 12d
i
O
O
Br
N
,
: R = Methyl
: R = Hydrogen
O
F
10e 12e
,
13b
Scheme 1. Synthesis of plinabulin derivatives 1, 2, 14a-14h and intermediate aldehydes 13a-13b. Regents and conditions: (a) DBU, THF, rt, 48 h, 95-99%;
(b) SOCl2, CHCl3, 0-65oC (reflux), 3 h, 97%; (c) formamide, 175oC, 36 h, 50-55%; (d) formamide, H2O, 145oC, 20 h, 4%; (e) LiAlH4, THF, 0oC to rt, 6 h, 94%-
99%; (f) MnO2, DCM, rt, 48 h, 43-72%; (g) Cs2CO3, Na2SO4, DMF, rt, 20 h, 39-50%; (h) Cs2CO3, Na2SO4, DMF, 45-50oC, 20 h, 23-70%; (i) 1) n-BuLi, THF, -
78oC, 5 h; 2) HCl (1 M), THF, rt-40oC, 24 h; two steps yield 67%; (j) Cu(OAc)2, Et3N, DCM, rt, 48 h, 22%
2.2.2. Synthesis of plinabulin C-ring derivatives 17a-17r
reaction temperature was thus raised to 80oC to obtain the
white solid compound 17d. The reaction yield of compound
17j (7 %) was very low in all reaction conditions because the
strong electron-withdrawing action of its nitro group reduced
the reactivity of furanaldehyde. Compound 17n could not be
Route 2 details the synthesis of compounds 17a-17r,
which were produced according to the previous methods and
strategies. The yield of by-products was high, and pure
intermediates in quantity were difficult to obtain. We changed
the synthesis strategy accordingly. Namely, the first aldehydes
of ring A were reacted with DKP under nitrogen at room
temperature for 20 h. In order to avoid the formation of
isomers, the reactions were carried out in the absence of light
to yield intermediates 15a and 15b. Both intermediates were
used directly in the next reaction without further purification.
obtained through
a direct condensation reaction of 5-
hydroxymethylfurfural with intermediate 15a, which could be
explained by the active hydroxyl group. To obtain the
compound
17n,
the
hydroxyl
was protected
and the
group
of
5-
hydroxymethylfurfural
butyldimethylsilyl
with
tert-
5-(tert-
butyldimethylsilyloxymethyl) furfural (16m) was prepared in
the presence of imidazole as a base at room temperature3; after
reacting with 15a to yield compound 17m, the compound 17n
was obtained through the removal of the protective group by
tetrabutylammonium fluoride (TBAF).
The second aldehydes were reacted with the intermediate
o
15a or 15b at 45-50 C in the presence of Cs2CO3 in N, N-
dimethylformamide (DMF). During the preparation of
compound 17d, less product was produced at 50 oC because of
the lower reactivity of the cyclohexanecarboxaldehyde. The

O
O
O
Ac
X
Ac
X
15a
15b
: X = O
: X = C=O
N
N
O
k
O
X
N
HN
R
Ac
F
F
O
16a-16k, 16m, 16o, 16q, 16r
13a, 13b
15a, 15b
11
O
O
O
OH
O
NH
l
m
NH
O
17m
HN
R
HN
F
F
O
17n
17a-17m, 17o-17r
O
O
O
16a, 17a
16b, 17b
16c, 17c
16d, 17d
16e, 17e
: X = O, R = Pyridine-2-yl
: X = O, R = Pyridine-3-yl
: X = O, R = Phenyl
: X = O, R = Cyclohexyl
: X = O, R = 2,5-Imidazole-yl
: X = O, R = Furan-2-yl
16j, 17j
: X = O, R = 5-Nitrofuran-2-yl
16k, 17k
n
o
HO
HO
TBDMSO
: X = O, R = 5-Methylfuran-2-yl
: X = C=O, R = 5-Methylfuran-2-yl
O
O
O
17l
16m, 17m
16m
: X = O, R = 5-(((tert-butyldimethylsilyl)oxy)
methyl)furan-2-yl
: X = O, R = 5-(methoxymethyl)furan-2-yl
16f, 17f
16o, 17o
O
16g, 17g
16h, 17h
16i, 17i
: X = O, R = Thiophene-2-yl
: X = O, R = 5-Bromofuran-2-yl
: X = O, R = 5-Chlorofuran-2-yl
17p
: X = C=O, R = 5-(methoxymethyl)furan-2-yl
O
O
16q, 17q
16r, 17r
: X = O, R = 3-1H-Benzo[d]imidazol-2-yl
: X = O, R = 3-Benzofuran-2-yl
16o
Scheme 2. Synthesis of plinabulin C-ring derivatives 17a-17r. Regents and conditions: (k) Cs2CO3, Na2SO4, DMF, rt, 20 h, 47-77%; (l) Cs2CO3, Na2SO4, DMF,
45-80oC, 24 h, 7-75%; (m) TBAF (1 M), THF, rt, 5 h, 74%. (n) tert-butyldimethylsilyl chloride, imidazole, DCM, rt, overnight, 99%; (o) Methyl iodide, Cs2CO3,
CH3CN, 50oC, 24 h, 50%.
2.3. Cytotoxic activity
fluorobenzoylphenyl group were 3.8 nM, 3.2 nM, and 2.8 nM,
respectively. These results showed that the isopropyl and
cyclopropyl moieties had the same activity as tert-butyl, and
that the para-fluorobenzoylphenyl group and the para-
fluorophenoxyphenyl group had similar contributions.
However, when the tert-butyl was replaced by a methyl group,
the activity of compound 14e (IC50 = 47.6 nM) was reduced 2-
2.3.1. Biological activities of the synthesized plinabulin C-ring
derivatives 1, 2 and 14a-14h.
In order to explore the effect of tert-butyl moiety at the 5-
position of imidazole, we synthesized a series of compounds in
which the moiety was replaced by different alkyl groups. The
activities of compounds 1, 2, and 14a-14h were evaluated by
3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide
(MTT) assay against the human lung cancer NCI-H460 cell
line (Table 1).
fold. A similar decrease was found in compound 14f (IC50
=
15.9 nM), the activity of which was less than that of
compounds 1, 14b, and 14d. Further, when the 5-position of
the imidazole was occupied by a hydrogen atom, the activities
of the compounds 14g (IC50 = 478.5 nM) and 14h (IC50 = 277.5
nM) were decreased significantly. These results suggest that
the large sterically hindered groups at the 5-position of
imidazole were important for maintaining their activities,
consistent with previously reported findings13.
When the A ring was a para-fluorophenoxyphenyl group,
compounds 2 (IC50 = 4.0 nM), 14a (IC50 = 8.8 nM), or 14c
(IC50 = 15.2 nM), which possessed tert-butyl, isopropyl, and
cyclopropyl moiety at the 5-position of imidazole,
respectively, displayed high cytotoxic activities. The IC50
values of compounds 1, 14b, and 14d with the para-
Table 1. Biological activities of the synthesized plinabulin C-ring derivatives 1, 2 and 14a-14h
O
X
NH
N
NH
HN
F
R
O

IC50 (nM) a
NCI-H460
26.2 ± 3.2
Docking
score
Compounds
Plinabulin
2
X
R
QpLogPo/w
QpPCaco
377.8
2.5
4.2
11.7
14.0
O
4.0 ± 0.4
3.8 ± 1.2
533.1
1
C=O
3.6
265.7
15.0
14a
14b
O
8.8±0.7
3.2±0.9
4.0
3.3
451.0
137.9
14.0
14.6
C=O
14c
14d
O
15.2±2.8
2.8±1.1
3.8
3.1
438.5
214.1
13.7
14.8
C=O
14e
14f
O
47.6±12.8
15.9±4.8
3.2
2.6
384.5
191.1
13.1
13.8
C=O
14g
14h
O
H
H
478.5±15.8
277.5±2.0
2.8
2.1
390.7
168.2
14.8
13.3
C=O
a Values represent mean ± SD from three independent dose response curves with NCI-H460 cancer cell line.
The absolute values of the docking scores of these
derivatives ranged from 13 to 15, indicating that they might
have similar activities. Table 1 displays the calculated PCaco
of these diketopiperazine-type derivatives possessing para-
fluorophenoxyphenyl groups, which were superior to the Pcaco
values of compounds with para-fluorobenzoylphenyl
structures (i.e. 533.1 for compound 2 and 265.7 for compound
1) 21.
non-aromatic group, i.e. compound 17a, 17b, 17c, or 17d. The
cytotoxic activity of compound 17a (IC50 = 49.1 nM) with 2-
pyridine structure exhibited 10-fold less potency than
compound 2 against the human lung cancer NCI-H460 cell
line. Compounds 17b and 17d showed significantly decreased
anti-proliferative activity, and the IC50 values of these
derivatives were higher than 1000 nM. In contrast, the IC50
value of compound 17c was 389.1 nM, which can be attributed
to greater transmembrane activity in the cells due to this
molecule’s superior PCaco (1010.6). These results indicated
that the six-membered ring substituents were not functionally
equivalent to the tert-butyl-imidazole group.
2.3.2. Biological activities of the synthesized plinabulin C-ring
derivatives 17a-17r.
To explore the effects of the new scaffold, the C-ring
imidazole was replaced by a six-membered ring aromatic or

To reveal more potent diketopiperazine derivatives, a
series of five-membered aromatic ring groups were substituted
for the imidazole C ring. The IC50 values of compounds 17e,
17f, and 17g were >1000 nM, 154.5 nM, and >1000 nM,
respectively. Compound 17e (with 1H-5-imidazolyl group)
exhibited significant inactivity and its theoretical calculated
PCaco was inferior to the other two compounds. Although
compound 17g had a large transmembrane coefficient, it had
almost no activity because the sulfur atom on thiophene cannot
form a strong hydrogen bond with the NH of DKP. In contrast,
compound 17f (with furan group) could establish and maintain
more potent cytotoxic activity with IC50 at 154.2 nM, in
comparison with compound 14g, with IC50 of 478.5 nM. These
results indicate that the oxygen atom could form a hydrogen
of furan) showed more potent activity than compound 17f. In
contrast, the anti-proliferative activity of compound 17m,
which had a large protective group, reduced about two-fold in
potency. When the TBDMS protective group was removed
from compound 17m, the resulting compound (17n) possessed
a hydrophilic hydroxyl structure and exhibited improved
cytotoxic activity, equivalent to that of compound 17k. The
cytotoxic activity of compound 17o (with methoxymethyl
substituent group at the 5- position of furan) was increased ten-
fold, displaying an activity (IC50 =14.0 nM) that was similar to
previously reported compounds 2, 14a, and 14c. These results
indicate that the alkyl group of imidazole at the 5-position
could be replaced by the methyl or methoxymethyl substituent
of furan group, which is contradicting previous reports. The
para-fluorophenoxyphenyl groups of Compounds 17k and 17o
were substituted by para-fluorobenzoylphenyl structures to
yield compounds 17l and 17p. Their IC50 values were 5.3 nM
and 2.9 nM, respectively, which confirmed these activities.
These results indicate that an appropriate electron-donating
group occupying the 5-position of furan was important for
maintaining the activity of novel furan-diketopiperazine-type
derivatives.
bond interaction with the NH of DKP in
a planar
pseudopentacyclic conformation, and the imidazole group
could be replaced by a furan group to build a novel scaffold.
To further explore the structure-activity relationship, the
electron-withdrawing substituents (such as bromo, chloro, and
nitro) were synthesized to occupy the 5-position of furan,
yielding compounds 17h-17j. Compound 17h (IC50 = 361.1
nM), with a bromine atom at the 5-position, exhibited an
approximately 2-fold decrease in antiproliferative activity in
comparison with compound 17f. The IC50 value of compound
17i (with chloro atom) was 180.3 nM, similar to that of
compound 17f. However, the cytotoxic activity of compound
17j (with nitro group) was significantly decreased. This might
be due to the strong electron-withdrawing group affecting the
electron cloud distribution of the furyl oxygen atom, resulting
in impeded hydrogen bond formation.
In light of the promising results described above, we
designed and synthesized derivatives of imidazophenyl and
furanophenyl (compounds 17q and 17r, respectively). In
comparison with compound 17f, compound 17r decreased the
potency significantly with an IC50 of 347.2 nM. Compound
17q displayed a total loss of activity, with IC50 greater than
1000 nM.
In summary, compounds 17o and 17p had the best anti-
proliferative activities in the novel scaffold derivatives. In
addition, as reported in Table 1, the cytotoxic activities of
compounds 2, 1, 14a, 14b, 14c, and 14d were also potent at the
nanomolar level. We further explored their biological activities
in different cancer cell lines.
Compounds 17k-17p were obtained with the substitution
of an electron-donating group for the hydrogen atom at the 5-
position of furan. The IC50 values of compounds 17k, 17m,
17n, and 17o were 30.0 nM, 377.7 nM, 41.5 nM, and 14.0 nM,
respectively. Compound 17k (with methyl group at 5-position
Table 2. Biological activities of the synthesized plinabulin C-ring derivatives 17a-17r
O
X
NH
HN
R
F
O

IC50 (nM) a
NCI-H460
26.2 ± 3.2
Docking
score
Compounds
Plinabulin
X
R
QLogPo/w
2.5
QPCaco
377.8
11.7
N
NH
NH
2
1
O
4.0 ± 0.4
3.8 ± 1.2
4.2
3.6
533.1
265.7
14.0
N
N
C=O
15.0
17a
17b
O
O
49.1±4.7
3.9
3.6
799.8
649.5
13.3
13.0
N
>1000 nM
17c
17d
O
O
389.11±5.55
>1000 nM
4.5
4.6
1010.6
515.3
13.1
12.7
N
17e
17f
17g
O
O
O
>1000 nM
154.5±3.2
>1000 nM
3.0
3.9
4.4
383.1
12.7
12.7
12.9
N
H
O
S
1012.9
1015.1
Br
Cl
O
O
17h
17i
O
O
361.1±8.3
180.3±5.2
4.5
4.4
1031.8
1062.7
12.8
12.6

NO2
O
17j
O
O
>1000
3.2
4.2
118.3
983.1
13.0
13.0
O
17k
30.0±1.2
O
17l
C=O
5.3±0.3
3.5
486.4
13.7
Si
O
17m
17n
17o
O
O
O
377.7±20.3
41.5±4.9
14.0±0.2
6.4
3.2
4.0
1619.8
333.7
10.4
13.0
13.3
O
OH
O
O
O
1303.2
O
17p
C=O
O
N
2.9±1.1
3.4
274.1
13.9
17q
17r
O
O
>1000 nM
347.2±30.8
3.9
4.9
441.3
979.9
13.1
9.8
N
H
O
a Values represent mean ± SD from three independent dose response curves with NCI-H460 cancer cell line.
2.3.3. Biological activities of plinabulin and different
derivatives in various cancer cell lines
cancer HT-29 cell line, by MTT assay. As shown in Table 3,
compounds 1, 2, 14a-14d, 14h, 17l, 17o, and 17p exhibited
very potent activities against different cancer cell lines at
nanomolar levels. For example, the IC50 were 6.6 and 7.0 nM
for compound 17o, and 2.0 and 2.2 nM for compound 17p.
The cytotoxic activities of derivatives 1, 2, 14a-14d, 14h,
17l, 17o, and 17p were further evaluated against various tumor
cells, such as the pancreatic cancer BxPC-3 cell line and colon
Table 3. Biological activities of plinabulin and different derivatives 1, 2, 14a-14d, 14h, 17o and 17p in various cancer cell lines
Compds
IC50 a (nM) /BxPC-3
5.8±0.2
IC50 (nM)/ HT-29
6.6±1.6
Plinabulin

2
3.2±0.4
0.6±0.1
3.4±0.3
0.6±0.02
9.4±1.5
1.4±0.02
4.3±0.2
3.7±0.02
6.6±1.1
2.0±1.5
2.1±0.3
0.6±0.2
3.7±0.3
0.8±0.02
7.4±0.01
1.4±0.03
5.1±0.6
3.3±0.08
7.0±0.9
2.2±0.3
1
14a
14b
14c
14d
14h
17l
17o
17p
a Values represent mean ± SD from three independent dose response curves with four human cancer cell lines.
2.4. Immunofluorescent assay
To further explore the effects of derivatives 1, 2, 14a,
14b, 17o, and 17p on microtubules in cancer cells, an
immunofluorescence assay was performed. As shown in Figure
4, NCI-H460 cells were treated individually with Plinabulin
(10 nM), compounds 2 (10 nM), 1 (2 nM), 14a (10 nM), 14b
(10 nM), 17o (10 nM), or 17p (10 nM) for 24 h and then
stained for β-tubulin and DAPI. In comparison to the control,
the microtubule networks had been damaged (Figure 4). The
semi-quantitative calculations were performed through the
software Image Pro Plus 6.0, and is shown in Figure 5. The
inhibition activities were consistent with the anti-proliferative
activities detailed previously.
Fig. 4. Immunofluorescence assays of Plinabulin, compounds 1, 2, 14a, 14b, 17o and 17p. (a) NCI-H460 cells were treated with Plinabulin (10 nM),
compounds 2 (10 nM), 1 (2 nM), 14a (10 nM), 14b (10 nM), 17o (10 nM) and 17p (10 nM) for 24 h and then stained for β-tubulin and DAPI. (i) Nuclear (blue);
(ii) tubulin (red); (iii) (i) and (ii) were overlapped.

Fig. 5. Semi-quantitative analysis of the inhibition of tubulin polymerization plinabulin, compounds 1, 2, 14a, 14b, 17o and 17p.
2.5. Theoretical calculations and molecular docking
Theoretical calculations of the physical properties of these
synthesized compounds were performed using the Qikprop
Module of Maestro software. The interaction modes of
compounds 1, 2, 14a-14h, and 17a-17r were investigated by
molecular docking using Maestro software. The oil-water
partition coefficient (LogPo/w), cell permeability (Pcaco), and
docking score were calculated, and are shown in Table 1 and
Table 2, respectively. The LogPo/w values of the derivatives
were within the reasonable range of -2.0-6.5. The cell
permeability of some compounds was greater than 100, which
was beneficial for the improvement of activity.
important to maintaining the activity of such compounds, and
could support experiments investigating biological activities.
Figures 7A and 7B illustrate the similarity of compounds
2, 17a, and 17f in terms of interaction and configuration. Table
2 shows in detail that compound 17f (without a favorable alkyl
group at 5-position of C ring) exhibited favorable biological
activity in a series of compounds, which can be attributed to its
better cell permeability (PCaco = 1012.86) in comparison with
that of compound 2 (PCaco = 533.05). The volume of
compound 17f at the C ring was slightly smaller than
compound 17a, resulting in reduced cytotoxic activity.
Compound 17g had no cytotoxic activity because there was no
strong intra-ligand hydrogen bond in the docking mode shown
in Fig. 7C.
The co-crystal structure of compound 1 is shown in Fig.
6B. The binding modes of compounds 14a and 14c with para-
fluorophenoxyphenyl group were similar to that of compound
2, which formed hydrogen bonds with amino acid residues (the
carbonyl oxygen atom of VAL236 with the NH of DKP, and
the hydrogen atom of the carboxylic acid of GLH198 with the
oxygen atom of the carbonyl group of DKP in Fig. 6A). In
contrast, compounds 1, 14b, and 14d with para-
fluorobenzoylphenyl structure were able not only to form the
same hydrogen bond with amino acid residues, but also to
form π-π interaction with the benzene ring of amino acid
PHE20, as shown in Fig. 6B. In general, compounds 1, 2, and
14a-14h had highly similar molecular conformations in the
tubulin-binding pocket in Fig. 2A, 6C, 6D, 6E, 6F, 6G,
respectively, which was able to form intramolecular hydrogen
bonds. These results show that favorable interactions were
In addition, we found that the hydroxyl hydrogen atom of
compound 17n could form a hydrogen bond with the oxygen
atom of ASN256, while the hydroxyl group occupied the
interface of α/β-tubulin heterodimers, which could cause
potent cytotoxic activity. This result indicates that the
hydrophilic group could exist in the extension pocket of the
interface. In contrast, the methoxymethyl substituent group at
the 5-position of the furan of compound 17p might have
influenced the H8 and T7 loops to interfere with the
microtubule assembly, which could perform the same function
as the tert-butyl imidazole of compound 1, shown in Fig. 8B19.

Fig. 6. Binding poses of compounds 1, compounds 2 and 14a-14d (stick-model) with co-crystal structure (PDB: 5XHC). A) Compounds 2 (faded-green),
14a (faded-red-orange) and 14c (violet) are shown as sticks. B) Compounds 1 (green), 14b (faded-red) and 14d (blue) are shown as sticks. C) Compound 14a
(faded-red-orange) are shown as sticks. D) Compound 14c (violet) are shown as sticks. E) Compound 2 (faded-green) are shown as sticks. F) Compound 14b
(faded-red) are shown as sticks. G) Compound 14d (blue) are shown as sticks. Yellow dashed: hydrogen bonds of ligand-receptor; green dashed: hydrogen bonds
of intra-ligand; blue dashed: π-π interaction.

Fig. 7. Binding poses of compounds 2, 17a, 17f and 17g with co-crystal structure (PDB: 5XHC). A) Compounds 2 (teal), and 17f (orange) are shown as
sticks. B) Compounds 17a (green) and 17f (orange) are shown as sticks. C) Compounds 17f (orange) and 17g (azure) are shown as sticks. Yellow dashed:
hydrogen bonds of ligand-receptor; green dashed: hydrogen bonds of intra-ligand.
Fig. 8. Binding poses of compounds 1, 17n and 17p with co-crystal structure (PDB: 5XHC). A) 17n (green) are shown as sticks. B) Compounds 1 (teal) and
17p (violet) are shown as sticks. Yellow dashed: hydrogen bonds of ligand-receptor; green dashed: hydrogen bonds of intra-ligand.
group could be considered as potential agents in the treatment of
cancer. The subsequent pharmaceutical in vivo and SAR studies
are forthcoming.
3. Conclusion
We designed and synthesized a total of 26 novel plinabulin
C ring derivatives based on the co-crystal structure of tubulin
with compound 1. Among them, the derivatives 17o and 17p
with the novel furan group displayed potent cytotoxicity against
several human cancer cell lines, which could effectively inhibit
tubulin polymerization, as observed in immunofluorescence
assay. The docking models of compounds 17o and 17p were
similar to the co-crystal structure of compound 1. Based on the
SAR study, we found that the appropriate electron-donating
group containing the 5-position of furan was important for
maintaining the activity of novel furan-diketopiperazine-type
derivatives. Therefore, the derivatives 17o and 17p with the furan
4. 4. Experimental section
4.1. General
All starting materials were purchased from commercial
suppliers and were used without further purification. Column
chromatography was performed on silica gel (200−300 mesh,
Yantai Chemical Industry Research Institute). Thin-layer
chromatography (TLC) was performed using silica gel GF-254
plates (Qing-Dao Chemical Company, Qingdao, China) with
detection by UV (254 nm or 365 nm). Melting points were

measured on a Shenguang WRS-3 melting point instrument
Na2CO3 (60 mL), extracted with petroleum ether, and then
extracted with EtOAc. The petroleum ether layer was given up,
and the EtOAc layers were combined and washed with saturated
brine, and dried over anhydrous MgSO4. The solvent was
evaporated in vacuo, and the crude product was purified by slurry
using H2O to produce 31.49 g of compound 8a with a yield at
55%. 1H NMR (500 MHz, CDCl3) δ 7.50 (s, 1H), 4.34 (q, J = 7.1
Hz, 2H), 1.46 (s, 9H), 1.36 (t, J = 7.1 Hz, 3H). MS (ESI) m/z: [M
+ H]+ Calcd for C10H17N2O2: 197.13, Found: 196.81.
1
(China). H and 13C NMR spectra were obtained using a JEOL
400 spectrometer (400 MHz) or Agilent Pro pulse 500 MHz
spectrometer with TMS as an internal standard. The following
abbreviations were used to explain the multiplicities: s = singlet,
d = doublet, t = triplet, q = quartet, b = broad, td = triple doublet,
dt = double triplet, dq = double quartet, and m = multiplet. High-
resolution mass spectra (ESI or EI) were recorded on an Agilent
1290 Infinity II UHPLC/6530 Q-TOF mass spectrometer.
4.2. Synthesis
4.2.4. Preparation of ethyl 5-(isopropyl)-1H-imidazole-4-
carboxylate (8b)
4.2.1. Preparation of ethyl 5-(tert-butyl) oxazole-4-
carboxylate (5a)
Compound 5b (5.00 g, 27.29 mmol) and formamide (49.17
g (43 ml), 1.09 mmol), 2.97 g faded orange solid with a yield at
60%. 1H NMR (500 MHz, CDCl3) δ 7.60 (s, 1H), 4.31 (q, J = 7.1
Hz, 2H), 3.77 (s, 1H), 1.30 (t, 9H). MS (ESI) m/z: [M + H]+
Calcd for C9H15N2O2: 183.11, Found: 182.77.
To a solution of ethyl isocyanoacetate (100 g, 884.02 mmol)
in THF, 1, 8-diazabicyclo[5.4.0]undec-7-ene (DBU) (161.50 g,
1.06 mmol) and pivalic anhydride (197.58 g, 1.06 mmol) were
added dropwise. The mixture was stirred for 48 h at room
temperature. The reaction solution was removed by evaporation
under reduced pressure. The residue was extracted with EtOAc,
then washed with 10% Na2CO3 and 10% citric acid. The
combined organic layer was washed with saturated brine, and
dried over anhydrous MgSO4. The solvent was concentrated in
vacuo, and the crude product was purified by column
chromatography (EtOAc–petroleum ether, 4:1) to produce 5a
(172.50 g) as yellow oil with a yield at 99%. 1H NMR (500 MHz,
CDCl3) δ 7.70 (s, 1H), 4.37 (q, J = 7.1 Hz, 2H), 1.45 (s, 9H), 1.40
(t, J = 7.1 Hz, 3H). MS (ESI) m/z: [M + H]+ Calcd for
C10H16NO3: 198.11, Found: 197.82 .
4.2.5. Preparation of ethyl-5-(cyclopropyl)-1H-imidazole-4-
carboxylate (8c)
1) A solution of ethyl 2-cyclopropylacetoacetate (20 g,
128.06 mmol) in CHCl3, the mixture was stired at 0 °C for 10
min, and sulfonyl chloride (20.73 g, 153.66 mmol) was added.
The reaction solution was moved to room temperature, stirred for
10 min, and refluxed at 65 °C for 3 h. The solution was extracted
with EtOAc, then washed with 10% NaHCO3 and water. The
combined organic layer was washed with saturated brine, and
dried over anhydrous sodium sulfate. The solvent was
concentrated in vacuo to produce compound 8c (24 g) as an
orange oil with a yield at 97%; this compound was used without
further purification.
4.2.2. Preparation of ethyl 5-(isopropyl) oxazole-4-
carboxylate (5b)
Ethyl isocyanoacetate (50.00 g, 441.66 mmol), 1, 8-
diazabicyclo[5.4.0]undec-7-ene (DBU) (80.69 g, 530.00 mmol)
and dimethylacetic anhydride (83.84 g, 530.00 mmol), THF (200
2) A mixture of ethyl 2-chloro-cyclopropylacetoacetate (67
g, 351.48 mmol), formamide (316 g, 7.03 mol), and water (25 g,
1.40 mmol) was stirred at 145°C for 20 h. The reaction was
monitored by TLC (DCM: MeOH = 20: 1). The mixture was then
cooled to room temperature. Then, sodium carbonate aqueous
solution was added to separate out a gray solid. The mixture was
filtered, and the filter cake was washed with water and dried in
vacuo at 50 °C to produce 2.90 g of brown solid, with a yield of
4%. 1H NMR (500 MHz, CDCl3) δ = 7.54 (s, 1H), 4.37 (d,
J=7.1, 2H), 2.65 – 2.53 (m, 1H), 1.38 (t, J=7.1, 3H), 0.99 (d,
J=7.4, 4H). MS (ESI) m/z: [M + H]+ Calcd for C9H13N2O2:
181.10, Found: 181.35.
1
ml), 73 g colourless oil with a yield at 95%. H NMR (500 MHz,
1
CDCl3) H NMR (500 MHz, CDCl3) δ 7.73 (s, 1H), 4.37 (q, J =
7.1 Hz, 2H), 3.79 (dt, J = 14.0, 7.0 Hz, 1H), 1.38 (t, J = 7.1 Hz,
3H), 1.28 (d, J = 7.0 Hz, 6H). MS (ESI) m/z: [M + H]+ Calcd for
C9H14NO3: 184.10, Found: 183.74.
4.2.3. Preparation of ethyl 5-(tert-butyl)-1H-imidazole-4-
carboxylate (8a)
A mixture of compound 5a (27.50 g, 139.43 mmol) and
formamide (157.03 g, 485.78 mmol) was heated at 175 °C for 36
h. After cooling, the reaction mixture was added with 10%

4.2.6. Preparation of (5-(tert-butyl)-1H-imidazol-4-yl)
methanol (9a)
(m, 1H), 1.37 (d, J = 6.9 Hz, 6H). MS (ESI) m/z: [M + H]+ Calcd
for C7H11N2O: 139.09, Found: 138.65.
4.2.11. Preparation of 5-(cyclopropyl)-1H-imidazole-4-
carbaldehyde (10c)
Ethyl 5-(tert-butyl)-1H-imidazole-4-carboxylate (9.92 g,
261.28 mmol) in THF (80 ml) was added dropwise to a solution
of LiAlH4 (17.07 g, 87.09 mol) in THF (80 mL) at 0 °C under
nitrogen. The mixture was stirred for 4 h at room temperature. To
this solution, water was added and the resulting precipitate was
removed by celite filtration. The filtrate was evaporated in vacuo
to produce 15.36 g of white solid with a yield of 95 %. The crude
product was used in the next step without further purification.
MS (ESI) m/z: [M + H]+ Calcd for C8H15N2O: 155.12, Found:
154.64.
Compound 9c (2.22 g, 16.11 mmol), MnO2 (13.86 g, 161.11
mmol), DCM (80 ml), 951.2 mg white solid with a yield at 43%.
1H NMR (400 MHz, CDCl3) δ 9.87 (s, 1H), 7.74 (s, 1H), 2.27 (s,
1H), 1.09 (d, J = 8.3 Hz, 4H). MS (ESI) m/z: [M + H]+ Calcd for
C7H9N2O: 137.07, Found: 136.62.
4.2.12. Preparation of (Z)-1-acetyl-3-((5-(tert-butyl)-1H-
imidazol-4-yl) methylene) piperazine-2, 5-dione (12a)
Under nitrogen, a mixture of compound 10a (4.88 g, 32.11
mmol), 1, 4-diacetylpiperazine-2, 5-dione (12.73 g, 64.21 mmol),
and Cs2CO3 (15.69 g, 48.16 mmol) in DMF (50 mL) was stirred
for 20 h at room temperature. The solution was poured into cool
water to precipitate a solid. The mixture was filtered to produce
4.43 g of orange solid with a yield of 47 %. 1H NMR (500 MHz,
DMSO-d6) δ 12.36 (s, 1H), 12.01 (s, 1H), 7.85 (s, 1H), 7.04 (s,
1H), 4.30 (s, 2H), 2.49 (s, 3H), 1.39 (s, 9H). MS (ESI) m/z: [M +
H]+ Calcd for C14H19N4O3: 291.15, Found: 290.79.
4.2.7. Preparation of (5-(isopropyl)-1H-imidazol-4-yl)
methanol (9b)
LiAlH4 (1.56 g, 41.10 mmol), ethyl 5-(isopropyl)-1H-
imidazole-4-carboxylate (2.50 g, 13.70 mmol), THF (20 ml), 1.9
g white solid with a yield at 99%. MS (ESI) m/z: [M + H]+ Calcd
for C7H13N2O: 141.10, Found: 140.68.
4.2.8. Preparation of (5-(cyclopropyl)-1H-imidazol-4-yl)
methanol (9c)
4.2.13. Preparation of (Z)-1-acetyl-3-((5-(isopropyl)-1H-
imidazol-4-yl) methylene) piperazine-2, 5-dione (12b)
LiAlH4 (1.83 g ， 48.33 mmol), ethyl 5-(cyclopropyl)-1H-
imidazole-4-carboxylate (2.90 g ， 16.11 mmol), THF (20 ml),
2.09 g orange solid with a yield at 94%. MS (ESI) m/z: [M + H]+
Calcd for C7H11N2O: 139.09, Found: 138.64.
Compound 10b (500.00 mg, 3.70 mmol), 1, 4-
diacetylpiperazine-2, 5-dione (1.47 g, 7.40 mmol) and Cs2CO3
(1.81 g, 5.55 mmol), DMF (4 mL). 367.0 mg orange solid with a
1
4.2.9. Preparation of 5-(tert-butyl)-1H-imidazole-4-
carbaldehyde (10a)
yield at 36%. H NMR (500 MHz, DMSO-d6) δ 12.60 (s, 1H),
11.78 (s, 1H), 7.90 (s, 1H), 6.80 (s, 1H), 4.31 (s, 2H), 3.30 – 3.16
(m, 1H), 2.50 (d, J = 3.9 Hz, 3H), 1.24 (d, J = 6.9 Hz, 6H). MS
(ESI) m/z: [M + H]+ Calcd for C13H17N4O3: 277.13, Found:
276.83.
MnO2 (60.59 g, 696.72 mol) was added to a solution of
compound 9a (13.41 g, 87.09 mmol) in DCM and the mixture
was stirred for 48 h at 25 °C. The reaction was monitored by
TLC. The solution was filtrated using celite filtration. The
solvent was evaporated in vacuo, and the crude product was
purified by column chromatography (EtOAc–petroleum ether,
4:1) to obtain 9.04 g of white solid with a yield of 68%. 1H NMR
(500 MHz, CDCl3) δ 10.06 (s, 1H), 7.73 (s, 1H), 1.48 (s, 9H).
MS (ESI) m/z: [M + H]+ Calcd for C8H13N2O: 153.10, Found:
152.66.
4.2.14. Preparation of (Z)-1-acetyl-3-((5-(cyclopropyl)-1H-
imidazol-4-yl) methylene) piperazine-2, 5-dione (12c)
Compound 10c (1.53 g, 11.24 mmol), 1, 4-
diacetylpiperazine-2, 5-dione (4.45 g, 22.48 mmol) and Cs2CO3
(1.81 g, 5.55 mmol), DMF (15 mL). 1.2 g orange solid with a
yield at 39%. 1H NMR (500 MHz, DMSO-d6) δ 12.34 (s, 1H),
11.69 (s, 1H), 7.81 (s, 1H), 6.88 (s, 1H), 4.30 (s, 2H), 2.49 (s,
3H), 2.15 – 1.94 (m, 1H), 1.07 – 0.91 (m, 2H), 0.76 (t, J = 3.2
Hz, 2H). MS (ESI) m/z: [M + H]+ Calcd for C13H15N4O3: 275.11,
Found: 274.78.
4.2.10. Preparation of 5-(isopropyl)-1H-imidazole-4-
carbaldehyde (10b)
Compound 9b (1.92 g, 13.70 mmol), MnO2 (11.91 g, 0.14
mmol), DCM (20 ml), 1.2 g white solid with a yield at 61%. 1H
NMR (500 MHz, CDCl3) δ 9.88 (s, 1H), 7.79 (s, 1H), 3.56 – 3.43
4.2.15. Preparation of (Z)-1-acetyl-3-((5-(methyl)-1H-
imidazol-4-yl) methylene-d) piperazine-2, 5-dione (12d)

Compound 10d (1.00 g, 9.08 mmol), 1, 4-
diacetylpiperazine-2, 5-dione (13.60 g, 18.16 mmol) and Cs2CO3
(4.44 g, 13.62 mmol), DMF (18 mL). 1.06 g orange solid with a
yield at 47%. 1H NMR (500 MHz, DMSO-d6) δ 12.56 (s, 1H),
11.74 (s, 1H), 7.87 (s, 1H), 6.76 (s, 1H), 4.31 (s, 2H), 2.50 (s,
3H), 2.33 (s, 3H). MS (ESI) m/z: [M + H]+ Calcd for
C11H13N4O3: 249.10, Found: 248.81.
added dropwise, and the mixture was stirred at -78 °C for 2 h.
The reaction mixture was quenched using a saturated ammonium
chloride solution. The residue was dissolved in methanol and
hydrochloric acid (2 mol/L) was added. The mixture was stirred
at room temperature. The solvent was moved under pressure,
extracted with EtOAc (100 ml * 3), washed with saturated NaCl
(50 ml * 3), dried over anhydrous sodium sulfate, and
concentrated in vacuo. The residue was purified by silica gel
column chromatography using petroleum ether/ethyl acetate
(10:1/8:1) to produce 2.51 g of white solid with a yield of 67%.
1H NMR (500 MHz, DMSO-d6) δ 10.11 (s, 1H), 8.24 – 8.18 (m,
2H), 8.07 – 8.03 (m, 1H), 7.90 – 7.85 (m, 2H), 7.80 (t, J = 7.7
Hz, 1H), 7.46 – 7.39 (m, 2H). MS (ESI) m/z: [M + H]+ Calcd for
C14H10FO2: 229.07, Found: 228.75.
4.2.16. Preparation of (Z)-1-acetyl-3-((1H-imidazol-4-yl)
methylene-d) piperazine-2, 5-dione (12e)
Compound 10e (1.00 g, 10.41 mmol), 1, 4-
diacetylpiperazine-2, 5-dione (4.13 g, 20.82 mmol) and Cs2CO3
(5.09 g, 15.62 mmol), DMF (10 mL). 1.0 g orange solid with a
1
yield at 41%. H NMR (500 MHz, DMSO-d6) δ 12.68 (s, 1H),
11.70 (s, 1H), 7.99 (s, 1H), 7.63 (s, 1H), 6.83 (s, 1H), 4.32 (s,
2H), 2.57 – 2.37 (s, 3H). MS (ESI) m/z: [M + H]+ Calcd for
C10H11N4O3: 235.08, Found: 234.79.
4.2.19. Preparation of (Z)-N-acetyl-3-(4-fluorobenzoyl)
benzylidene) piperazine-2, 5-dione (15a)
A mixture of 3-para-fluorophenoxybenzaldehyde (156.77
mg, 0.72 mmol), 1, 4-diacetylpiperazine-2, 5-dione (150 mg,
0.60 mmol), cesium carbonate (295.32 mg, 0.91) mmol), and
anhydrous sodium sulfate (171.66 mg, 1.21 mmol) was stirred in
DMF (3 mL) under nitrogen at room temperature for 20 h. The
reaction was detected by thin-layer chromatography. The reaction
solution was poured into cold water. The mixture solution was
filtered, then the filter cake was washed with water and dried in
vacuum drying equipment at 50 °C to obtain 115.0 mg of faded-
orange solid with a yield of 47 %. The product was directly used
4.2.17. Preparation of 3-(4-fluorobenzoyl) benzaldehyde
(13a)
To a solution of 4-fluorophenol (500 mg，4.46 mol) in dry
DCM (10 mL), (3-formylphenyl)boronic acid(1.00 g，6.69 mol),
Cu(OAc)2 (811 mg，4.46 mol) and Et3N (135.6 mg，1.34 mol)
were added under O2 atmosphere and the mixture was stirred at
25oC for 48 h. The mixture was diluted with brine and extracted
with EtOAc. The solvent was removed under reduced pressure.
The residue was purified by silica gel column chromatography
using petroleum ether/ethyl acetate (10:1) to produce 0.22 g of
0.22 g with a yield of 22%.
1
in the next reaction without further purification. H NMR (500
MHz, DMSO-d6) δ 10.41 (s, 1H), 7.43 (t, J = 7.9 Hz, 1H), 7.32
(d, J = 8.1 Hz, 1H), 7.27 – 7.20 (m, 3H), 7.11 (ddd, J = 6.8, 5.4,
3.2 Hz, 2H), 6.96 – 6.91 (m, 2H), 4.35 (s, 2H), 2.49 (s, 3H). MS
(ESI) m/z: [M + H]+ Calcd for C19H16FN2O4: 355.11, Found:
354.90.
1H NMR (500 MHz, CDCl3) δ 9.96 (s, 1H), 7.59 (d, J = 7.5
Hz, 1H), 7.50 (t, J = 7.8 Hz, 1H), 7.41 (dd, J = 2.2, 1.4 Hz, 1H),
7.25 (dt, J = 3.5, 1.4 Hz, 1H), 7.10 – 7.04 (m, 2H), 7.02 (ddd, J =
6.8, 5.2, 3.0 Hz, 2H). MS (ESI) m/z: [M + H]+ Calcd for
C13H10FO2: 217.07, Found: 217.27.
4.2.20. Preparation of (Z)-N-acetyl-3-(4-fluorophenoxy)
benzylidene) piperazine-2, 5-di one (15b)
4.2.18. Preparation of 3-(4-fluorophenoxy) benzaldehyde
3-para-fluorobenzoylbenzaldehyde (1.06 g, 4.63 mmol), 1,
4-diacetylpiperazine-2, 5-dione (1.83 g, 9.25 mmol), Cs2CO3
(2.01 g, 6.94 mmol), Na2SO4 (1.31 g, 9.25 mmol), DMF (15 mL),
(13b)
The tetrahydrofuran solution (12 mL) of 2-(3-
bromophenyl)-1, 3-dioxolane (4.88 g ， 21.29 mmol)was added
dropwise to a solution of n-BuLi (20.48 ml (1.6 M THF)), 32.75
mmol) in dry THF (20 mL) at -78oC under nitrogen atmosphere.
The solution was then stirred at -78oC under nitrogen atmosphere
1
1.3 g faded-orange solid with a yield at 77%. H NMR (500
MHz, DMSO-d6) δ 10.53 (s, 1H), 7.94 – 7.88 (m, 3H), 7.81 (d, J
= 7.7 Hz, 1H), 7.68 (d, J = 7.8 Hz, 1H), 7.61 (t, J = 7.7 Hz, 1H),
7.40 (dd, J = 12.3, 5.4 Hz, 2H), 7.02 (s, 1H), 4.36 (s, 2H), 2.50 (s,
3H). MS (ESI) m/z: [M + H]+ Calcd for C20H16FN2O4: 367.11,
Found: 366.93.
for 50 min.
A
solution of 4-fluoro-N-methyl-N-
methoxybenzamide (3.0 g, 16.38 mmol) in THF (10 ml) was

4.2.21. Preparation of 5-(((tert-butyldimethylsilyl) oxy)
methyl) furan-2-carbaldehyde (16a)
12a (300.00 mg, 1.09 mmol), 13a (282.00 mg, 1.30 mmol),
Cs2CO3 (531.00 mg, 1.63 mmol), 300 mg yellow solid with a
yield at 64%. MP: 260-262C. 1H NMR (500 MHz, DMSO-d6) δ
12.29 (d, J = 33.1 Hz, 2H), 10.10 (s, 1H), 7.85 (s, 1H), 7.41 (t, J
= 7.9 Hz, 1H), 7.31-7.07 (m, 6H), 6.90 (d, J = 8.1 Hz, 1H), 6.86
(s, 1H), 6.71 (s, 1H), 1.38 (s, 9H). 13C NMR (125 MHz, DMSO-
d6) δ 159.1, 157.5, 157.2, 156.9 (JC-F = 113.1 Hz), 152.6, 140.4,
A mixture of 5-hydroxymethylfurfural (1.26 g, 10.00
mmol), potassium carbonate (748.88 mg, 11.00 mmol), and tert-
butyldimethylchlorosilane (1.66 g, 11.00 mmol) was stirred in
DCM at room temperature for 12 h. The reaction solution was
filtered using silica gel and washed with petroleum ether. The
solvent was removed under reduced pressure to obtain 2.4 g of
brown oil with a yield of 99%. 1H NMR (500 MHz, DMSO-d6) δ
9.56 (s, 1H), 7.49 (d, J = 3.5 Hz, 1H), 6.63 (d, J = 3.6 Hz, 1H),
4.73 (s, 2H), 0.87 (s, 9H), 0.08 (s, 6H). MS (ESI) m/z: [M + H]+
Calcd for C12H21O3Si: 241.13, Found: 240.85.
135.2, 134.4, 130.7, 130.3, 127.3, 124.4, 123.7, 120.5*2 (JC-F
=
8.5 Hz), 118.9, 117.7, 116.5*2 (JC-F = 23.2 Hz), 113.0, 105.2,
31.9, 30.6*3. MS (ESI) m/z: [M + H]+ Calcd for C25H24FN4O3:
447.1827, Found: 447.18。
4.2.24. (3Z, 6Z)-3-(4-fluorobenzoyl) benzylidene)-6-((5-
(tert-butyl)- 1H-imidazol-4-yl) methylene)- piperazine-2, 5-dione
(1)
4.2.22. Preparation of 5-(methoxymethyl) furan-2-
carbaldehyde (16b)
12a (200.00 mg, 0.69 mmol), 13b (188.67 mg, 0.83 mmol),
Cs2CO3 (336.67 mg, 1.03 mmol), 221.4 mg yellow solid with a
yield at 70%. MP: 255-257C. 1H NMR (400 MHz, DMSO-d6) δ
12.33 (s, 1H), 12.29 (s, 1H), 10.34 (s, 1H), 7.95–7.88 (m, 2H),
7.85 (s, 1H), 7.82 (s, 1H), 7.75 (d, J = 7.55 Hz, 1H), 7.64 (d, J =
7.70 Hz, 1H), 7.58 (t, J = 7.58 Hz, 1H), 7.40 (t, J = 8.82 Hz, 2H),
6.86 (s, 1H), 6.80 (s, 1H), 1.38 (s, 9H). MS (ESI) m/z: [M + H]+
Calcd for C26H24FN4O3: 459.1827, Found 459.15.
A mixture of 5-hydroxymethylfurfural (500.00 mg, 3.96
mmol), methyl iodide (1.69 g, 11.89 mmol), and cesium
carbonate (1.94 g, 5.95 mmol) was stirred in acetonitrile (20 ml)
o
at 50 C for 24 h. The reaction was monitored by LC-MS. The
residue was purified by silica gel column chromatography using
ethyl acetate/petroleum ether (1: 20, 1:15, 1:10, 1: 8, 1: 5) to
produce 280.0 mg of yellow liquid with a yield of 50%. 1H NMR
(500 MHz, DMSO-d6) δ 9.58 (s, 1H), 7.51 (d, J = 3.5 Hz, 1H),
6.73 (d, J = 3.6 Hz, 1H), 4.47 (s, 2H), 3.30 (s, 3H). MS (ESI)
m/z: [M + H]+ Calcd for C7H9O3: 141.05, Found: 140.88.
4.2.25. (3Z, 6Z)-3-(4-fluorophenoxy) benzylidene)-6-((5-
(isopropyl)- 1H-imidazol-4-yl) methylene)- piperazine-2, 5-dione
(14a)
General procedure for synthesis of 1, 2 and 14a-14h, and
synthesis of 14a as an example.
12b (211.83 mg, 0.77 mmol)), 13a (150.00 mg, 0.69
mmol), Cs2CO3 (282.55 mg，0.87 mmol), Na2SO4 (164.23 mg，
1.16 mmol), 231.4 mg yellow solid with a yield at 70%. MP:
A mixture of (Z)-1-acetyl-3-((5-(isopropyl)-1H-imidazol-4-
yl) methylene) piperazine-2, 5-dione (211.83 mg, 0.77 mmol), 3-
(4-fluorobenzyloxy) benzaldehyde (150.0 mg, 0.69 mmol),
Cs2CO3 (282.55 mg, 0.87 mmol), and Na2SO4 (164.23 mg, 1.16
mmol) was stirred in DMF (4 ml) under nitrogen at 60 °C for 24
h. The resulting solution was poured into cold water (40 ml) and
the filter cake was re-dissolved using methanol and
dichloromethane (1:3) then filtered. The solvent was combined
and concentrated under reduced pressure. The filtrate was stirred
in methanol at room temperature for 2 h, then moved to 0 °C.
The solution was filtered, washed with methanol, and dried in
vacuo at 50 °C to produce 231.4 mg of yellow solid with a yield
of 70 %.
1
246-248C. H NMR (500 MHz, DMSO-d6) δ 12.58 (s, 1H),
12.02 (s, 1H), 10.12 (s, 1H), 7.90 (s, 1H), 7.40 (t, J = 7.9 Hz,
1H), 7.24 (m, 3H), 7.17 (s, 1H), 7.14 – 7.09 (m, 2H), 6.90 (dd, J
= 8.2, 2.1 Hz, 1H), 6.71 (s, 1H), 6.61 (s, 1H), 3.25 (dt, J = 13.9,
6.9 Hz, 1H), 1.23 (d, J = 6.9 Hz, 6H). 13C NMR (125 MHz,
DMSO-d6) δ 159.14, 157.43, 157.08, 156.11, 152.58, 139.02,
135.33, 135.22, 130.70, 130.29, 127.33, 124.39, 123.68,
120.57*2 (JC-F = 8.5 Hz), 118.85, 117.71, 116.54*2 (JC-F = 23.3
Hz), 113.00, 104.02, 24.00, 22.53*2. HRMS (ESI) m/z: [M + H]+
Calcd for C24H22FN4O3: 433.1670, Found 433.1673.
4.2.26. (3Z, 6Z)-3-(4-fluorobenzoyl) benzylidene)-6-((5-
(isopropyl)- 1H-imidazol-4-yl) methylene)- piperazine-2, 5-dione
(14b)
4.2.23. (3Z, 6Z)-3-(4-fluorophenoxy) benzylidene)-6-((5-
(tert-butyl)- 1H-imidazol-4-yl) methylene)- piperazine-2, 5-dione
(2)

12b (200.00 mg, 0.72 mmol), 13b (198.23 mg, 0.87 mmol),
Cs2CO3 (353.78 mg, 1.09 mmol), Na2SO4 (205.63 mg, 1.45
mmol), DMF (4 ml), 115.6 mg yellow solid with a yield at 36%.
MP: 156-157C. 1H NMR (500 MHz, DMSO-d6) δ12.56 (s, 1H),
12.01 (s, 1H), 10.37 (s, 1H), 7.90 (m, 3H), 7.83 (s, 1H), 7.77 (d,
J=7.1, 1H), 7.62 (d, J = 7.1 Hz, 1H), 7.60-7.53 (m, 1H), 7.40 (t,
J=8.6, 2H), 6.78 (s, 1H), 6.60 (s, 1H), 3.27-3.22 (m, 1H), 1.23 (d,
J=6.6, 6H). 13C NMR (125 MHz, DMSO-d6) δ 194.18, 165.74,
163.74, 157.57, 156.03, 139.03, 137.28, 135.33, 133.57, 133.32,
132.78*2 (JC-F = 9.4 Hz), 130.69, 130.10, 128.85, 128.68, 127.76,
123.70, 115.70*2 (JC-F = 21.9 Hz), 112.59, 104.04, 23.98,
22.52*2. HRMS (ESI) m/z: [M + H]+ Calcd for C25H22FN4O3:
445.1670, Found: 445.1672.
HRMS (ESI) m/z: [M + Na]+ Calcd for C25H19FN4O3Na:
465.1339, Found 465.1328.
4.2.29. (3Z, 6Z)-3-(4-fluorophenoxy) benzylidene)-6-((5-
(methyl)- 1H-imidazol-4-yl) methylene)- piperazine-2, 5-dione
(14e)
12d (150.00 mg, 0.60 mmol), 13a (156.77 mg, 0.73 mmol),
Cs2CO3 (295.32 mg, 0.91 mmol), Na2SO4 (171.66 mg ， 1.21
mmol), DMF (2 ml), 115.0 mg yellow solid with a yield at 47%.
MP: 219-221C. 1H NMR (500 MHz, DMSO-d6) δ 12.54 (s, 1H),
11.99 (s, 1H), 10.11 (s, 1H), 7.87 (s, 1H), 7.41 (t, J = 7.9 Hz,
1H), 7.29 – 7.21 (m, 3H), 7.17 (s, 1H), 7.14 – 7.10 (m, 2H), 6.90
(dd, J = 8.2, 2.2 Hz, 1H), 6.71 (s, 1H), 6.59 (s, 1H), 2.32 (s, 3H).
13C NMR (125 MHz, DMSO-d6) δ 159.15, 157.41, 157.09,
156.10, 152.59, 135.22, 135.12, 132.48, 130.30, 129.16, 127.34,
124.39, 123.48, 120.58*2 (JC-F = 8.5 Hz), 118.85, 117.71,
116.55*2 (JC-F = 23.3 Hz), 112.96, 104.19, 9.05. HRMS (ESI)
m/z: [M + H]+ Calcd for C22H18FN4O3: 405.1357, Found
405.1351.
4.2.27. (3Z, 6Z)-3-(4-fluorophenoxy) benzylidene)-6-((5-
(cyclopropyl)- 1H-imidazol-4-yl) methylene)- piperazine-2,
5-dione (14c)
12c (150.00 mg, 0.55 mmol), 13a (141.90 mg, 0.66 mmol),
Cs2CO3 (267.27 mg, 0.42 mmol), Na2SO4(155.36 mg ， 1.09
mmol), DMF (3 ml), 101.1 mg yellow solid with a yield at 43%.
MP: 257-259C. 1H NMR (500 MHz, DMSO-d6) δ 12.31 (s, 1H),
11.94 (s, 1H), 10.12 (s, 1H), 7.82 (s, 1H), 7.41 (t, J = 7.9 Hz,
1H), 7.29 – 7.20 (m, 3H), 7.17 (s, 1H), 7.14 – 7.08 (m, 2H), 6.90
(dd, J = 8.2, 2.2 Hz, 1H), 6.72 (s, 2H), 2.14 – 2.06 (m, 1H), 1.04
– 0.95 (m, 2H), 0.80 – 0.73 (m, 2H). 13C NMR (125 MHz,
DMSO-d6) δ 159.13, 157.39, 157.06, 156.08, 152.59, 135.22,
135.05, 134.88, 133.09, 130.28, 127.33, 124.38, 123.54,
120.55*2 (JC-F = 8.5 Hz), 118.86, 117.70, 116.53*2 (JC-F = 23.3
Hz), 112.94, 104.18, 7.38*2, 5.43. HRMS (ESI) m/z: [M + H]+
Calcd for C24H20FN4O3: 431.1514, Found 431.1514.
4.2.30. (3Z, 6Z)-3-(4-fluorobenzoyl) benzylidene)-6-((5-
(methyl)- 1H-imidazol-4-yl) methylene)- piperazine-2, 5-dione
(14f)
12d (200.00 mg, 0.81 mmol), 13b (220.64 mg, 0.97 mmo),
Cs2CO3 (393.77 mg, 1.21 mmol), DMF (3 ml), 96.25 mg yellow
1
solid with a yield at 29%. MP: 243-245C. H NMR (500 MHz,
DMSO-d6) δ 12.52 (s, 1H), 12.00 (s, 1H), 10.30 (s, 1H), 7.91 (dd,
J = 5.65, 8.44 Hz, 2H), 7.87 (s, 1H), 7.82 (s, 1H), 7.75 (d, J =
7.62 Hz, 1H), 7.64 (d, J = 7.70 Hz, 1H), 7.58 (t, J = 7.63 Hz,
1H), 7.40 (t, J = 8.72 Hz, 2H), 6.80 (s, 1H), 6.60 (s, 1H), 2.32 (s,
3H). 13C NMR (125 MHz, DMSO-d6) δ 194.18, 165.74, 163.74,
4.2.28. (3Z, 6Z)-3-(4-fluorobenzoyl) benzylidene)-6-((5-
(cyclopropyl)- 1H-imidazol-4-yl) methylene)- piperazine-2,
5-dione (14d)
157.55, 156.02, 137.28, 135.11, 133.56, 133.32, 132.78*2 (JC-F
=
9.4 Hz), 132.47, 130.11, 129.16, 128.86, 128.68, 127.76, 123.49,
115.70*2 (JC-F = 21.9 Hz), 112.56, 104.22, 9.04. HRMS (ESI)
m/z: [M + Na]+ Calcd for C23H17FN4O3Na: 439.1182, Found:
439.1181.
12c (70.00 mg, 0.26 mmol), 13b (69.90 mg, 0.31 mmol),
Cs2CO3 (124.72 mg, 0.38 mmol), Na2SO4 (72.50 mg, 0.51 mmol),
DMF (3 ml), 79.5 mg yellow solid with a yield at 70%. MP: 217-
219C. 1H NMR (500 MHz, DMSO-d6) δ 12.30 (s, 1H), 11.95 (s,
1H), 10.30 (s, 1H), 7.96–7.87 (m, 2H), 7.81 (s, 2H), 7.75 (d, J =
7.21 Hz, 1H), 7.67–7.53 (m, 2H), 7.40 (t, J = 8.49 Hz, 2H), 6.80
(s, 1H), 6.71 (s, 1H), 2.10 (s, 1H), 0.99 (d, J = 7.05 Hz, 2H), 0.77
(d, J = 4.08 Hz, 2H). 13C NMR (125 MHz, DMSO-d6) δ 194.17,
165.74, 163.74, 157.54, 156.01, 137.28, 135.08, 134.88, 133.56,
133.32, 132.78*2 (JC-F = 9.4 Hz), 130.10, 128.86, 128.68, 127.75,
123.56, 115.70*2 (JC-F = 21.9 Hz), 112.57, 104.22, 7.39*2, 5.43.
4.2.31. (3Z, 6Z)-3-(4-fluorophenoxy)
benzylidene)-6-
((1H-imidazol-4-yl) methylene)- piperazine-2, 5-dione (14g)
12e (150.00 mg, 0.64 mmol), 13a (166.16 mg, 0.77 mmol),
Cs2CO3 (312.98 mg, 0.96 mmol), Na2SO4 (181.93 mg ， 1.28
mmol, DMF (3 ml), 56.9 mg yellow solid with a yield at 23%.
MP: 202-204C. 1H NMR (500 MHz, DMSO-d6) δ 12.65 (s, 1H),
11.90 (s, 1H), 10.14 (s, 1H), 7.99 (s, 1H), 7.58 (s, 1H), 7.40 (t, J
= 7.9 Hz, 1H), 7.27 (d, J = 8.0 Hz, 1H), 7.25 – 7.20 (m, 2H), 7.17
(s, 1H), 7.14 – 7.07 (m, 2H), 6.90 (dd, J = 8.2, 2.3 Hz, 1H), 6.72

(s, 1H), 6.66 (s, 1H). 13C NMR (125 MHz, DMSO-d6) δ 159.13,
157.32, 157.07, 156.17, 152.57, 136.64, 136.38, 135.19, 130.29,
127.25, 124.49, 124.39, 120.55*2 (JC-F = 8.5 Hz), 119.71, 118.90,
117.75, 116.53*2 (JC-F = 23.3 Hz), 113.20, 105.27. HRMS (ESI)
m/z: [M + H]+ Calcd for C21H16FN4O3: 391.1201, Found
391.1194.
7.37 (ddd, J = 7.6, 5.0, 1.0 Hz, 1H), 7.29 (d, J = 7.7 Hz, 1H),
7.26 – 7.19 (m, 3H), 7.15 – 7.10 (m, 2H), 6.92 (dd, J = 8.1, 2.0
Hz, 1H), 6.81 (s, 1H), 6.72 (s, 1H). 13C NMR (125 MHz, DMSO-
d6) δ 159.14, 157.24, 157.05, 156.72, 156.66, 154.60, 152.56,
148.52, 137.76, 134.87, 131.00, 130.29, 126.70, 126.53, 124.61,
122.54, 120.56 (JC-F = 8.4 Hz), 119.07, 118.03, 116.53 (JC-F
=
4.2.32. (3Z, 6Z)-3-(4-fluorobenzoyl) benzylidene)-6-((1H-
23.3 Hz), 114.78, 107.73. HRMS (ESI) m/z: [M + H]+ Calcd for
C23H17FN3O3: 402.1248, Found 402.1249.
imidazol-4-yl) methylene)- piperazine-2,
5-dione (14h)
4.2.34. (3Z, 6Z)-3-(4-fluorophenoxy) benzylidene-6-(3-
pyridylmethylene) piperazine-2, 5-dione (17b)
12e (150.00 mg, 0.64 mmol), 13b (141.42 mg, 0.77 mmol),
Cs2CO3 (312.98 mg, 0.96 mmol), Na2SO4 (181.93 mg ， 1.28
mmol, DMF (3 ml), 115.9 mg yellow solid with a yield a 45%.
MP: 150-152C. 1H NMR (500 MHz, DMSO-d6) δ 12.67 (s, 1H),
11.92 (s, 1H), 10.36 (s, 1H), 8.00 (s, 1H), 7.95 – 7.87 (m, 2H),
7.82 (s, 1H), 7.75 (d, J = 7.7 Hz, 1H), 7.64 (d, J = 7.7 Hz, 1H),
7.58 (dd, J = 8.9, 6.3 Hz, 2H), 7.40 (t, J = 8.8 Hz, 2H), 6.81 (s,
1H), 6.67 (s, 1H). 13C NMR (125 MHz, DMSO-d6) δ 194.17,
165.74, 163.74, 157.47, 156.10, 137.29, 136.65, 136.38, 133.54,
133.32, 132.78*2 (JC-F=9.4 Hz), 130.14, 128.87, 128.72, 127.68,
124.51, 119.73, 115.70*2 (JC-F = 22.0 Hz), 112.84, 105.32.
HRMS (ESI) m/z: [M + Na]+ Calcd for C22H15FN4O3Na:
425.1026, Found 425.1026.
15a (100.00 mg, 0.28 mmol), 3-pyridinaldehyde (45.34 mg,
0.42 mmol), Cs2CO3 (137.90 mg, 0.42 mmol), Na2SO4 (80.20
mg, 0.56 mmol), DMF (3 ml), 84.2 mg yellow solid with a yield
at 74%. MP: 263-266C. 1H NMR (500 MHz, DMSO-d6) δ 10.68
(s, 1H), 10.42 (s, 1H), 8.69 (s, 1H), 8.48 (s, 1H), 7.93 (d, J = 7.1
Hz, 1H), 7.42 (d, J = 7.0 Hz, 2H), 7.32 – 7.18 (m, 4H), 7.13 (s,
2H), 6.92 (d, J = 7.0 Hz, 1H), 6.76 (s, 2H). 13C NMR (125 MHz,
DMSO-d6) δ 159.20, 157.97, 157.65, 157.04, 152.53, 150.17,
148.44, 136.18, 135.02, 130.27, 128.11, 127.36, 127.08, 124.53,
123.49, 120.56 (JC-F = 8.5 Hz), 118.91, 117.93, 116.54 (JC-F
=
23.3 Hz), 114.38, 111.39. HRMS (ESI) m/z: [M + H]+ Calcd for
C23H17FN3O3: 402.1248, Found 402.1243.
General procedure for synthesis of 17a-17m and 17o-17r,
and synthesis of 17a as an example.
4.2.35. (3Z, 6Z)-3-(4-fluorophenoxy) benzylidene-6-
(benzylidene) piperazine-2, 5-dione (17c)
A mixture of (Z)-N-acetyl-3-((3-para-fluorophenoxyphenyl)
methylene) piperazine-2, 5-dione 15a (100 mg, 0.28 mmol), 2-
pyridinaldehyde (45.34 mg, 0.42 mmol), cesium carbonate (137.9
mg, 0.42 mmol), and anhydrous sodium sulfate (80.2 mg, 0.56
mmol), was stirred in DMF (3 ml) under nitrogen at 45oC for 24
h. The resulting solution was dropped into cold water (4oC, 60
ml), then filtered, and the filter cake was washed with cold water,
then dried in vacuo at 50 °C. The filtration was stirred in
methanol at room temperature for 2 h, then moved to 0 °C. The
solution was filtered, washed with methanol, and dried in
vacuum at 50°C to obtain 80.8 mg of yellow solid with a yield of
71%.
15a (100.00 mg, 0.28 mmol), benzaldehyde (44.92 mg ，
0.42 mmol), Cs2CO3 (137.90 mg, 0.42 mmol), Na2SO4 (80.20
mg, 0.56 mmol), DMF (3 ml), 67.4 mg yellow solid with a yield
at 60%. MP: 275-277C. 1H NMR (500 MHz, DMSO-d6) δ 10.32
(d, J = 26.6 Hz, 2H), 7.54 (s, 2H), 7.42 (s, 3H), 7.34 – 7.08 (m,
7H), 6.92 (s, 1H), 6.76 (d, 2H). 13C NMR (125 MHz, DMSO-d6)
δ 158.00, 157.03, 135.07, 133.05, 130.27, 129.33*2, 128.67*2,
128.19, 126.31, 124.51, 120.59, 120.52, 118.92, 117.90, 116.63,
116.45, 115.13, 114.14. HRMS (ESI) m/z: [M + H]+ Calcd for
C24H18FN2O3: 401.1296, Found 401.1294.
4.2.36. (3Z, 6Z)-3-(4-fluorophenoxy) benzylidene-6-
(cyclohexylmethylene) piperazine-2, 5-dione (17d)
4.2.33. (3Z, 6Z)-3-(4-fluorophenoxy) benzylidene-6-(2-
pyridylmethylene) piperazine-2, 5-dione (17a)
15a (100.00 mg, 0.28 mmol), cyclohexane formaldehyde
(47.48 mg ， 0.42 mmol), Cs2CO3 (137.90 mg, 0.42 mmol),
Na2SO4 (80.20 mg, 0.56 mmol), DMF (3 ml), 13.8 mg yellow
15a (100.00 mg, 0.28 mmol), 2-pyridinaldehyde (45.34 mg,
0.42 mmol), Cs2CO3 (137.90 mg, 0.42 mmol), Na2SO4 (80.20
mg, 0.56 mmol), DMF (3 ml), 80.8 mg yellow solid with a yield
at 71%. MP: 198-200C. 1H NMR (500 MHz, DMSO-d6) δ 12.59
(s, 1H), 10.48 (s, 1H), 8.73 (t, J = 5.9 Hz, 1H), 7.91 (td, J = 7.8,
1.8 Hz, 1H), 7.68 (d, J = 8.0 Hz, 1H), 7.42 (t, J = 7.9 Hz, 1H),
1
solid with a yield at 12%. MP: 227-229C. H NMR (400 MHz,
DMSO-d6) δ 10.43 (s, 1H), 10.11 (s, 1H), 7.39 (t, J = 7.9 Hz,
1H), 7.23 (dt, J = 5.9, 2.7 Hz, 3H), 7.15 – 7.05 (m, 3H), 6.89 (dd,
J = 8.1, 2.0 Hz, 1H), 6.69 (s, 1H), 5.70 (d, J = 10.4 Hz, 1H), 2.67

1
(dt, J = 13.6, 10.7 Hz, 1H), 1.63 (dd, J = 26.5, 12.4 Hz, 5H), 1.36
– 1.20 (m, 3H), 1.12 – 0.99 (m, 2H). 13C NMR (125 MHz,
DMSO-d6) δ 159.13, 157.60, 157.47, 157.22, 157.04, 152.56,
135.19, 130.25, 125.45, 124.35, 124.19, 120.55*2 (JC-F = 8.5 Hz),
118.80, 117.69, 116.52*2 (JC-F = 23.4 Hz), 113.23, 33.14, 31.78,
25.34, 24.96. HRMS (ESI) m/z: [M - H]- Calcd for C24H22FN2O3:
405.1620, Found 405.1615.
solid with a yield at 55%. MP: 230-232C. H NMR (500 MHz,
DMSO-d6) δ 10.40 (s, 1H), 9.91 (s, 1H), 7.73 (d, J = 4.9 Hz, 1H),
7.56 (d, J = 3.3 Hz, 1H), 7.41 (t, J = 7.9 Hz, 1H), 7.29 (d, J = 7.6
Hz, 1H), 7.26 – 7.17 (m, 4H), 7.12 (dd, J = 8.9, 4.4 Hz, 2H), 6.95
(s, 1H), 6.92 (d, J = 8.0 Hz, 1H), 6.76 (s, 1H). 13C NMR (125
MHz, DMSO-d6) δ 159.15, 157.86, 157.24, 157.04, 152.56,
135.54, 135.03, 130.27, 129.99, 128.42, 128.32, 127.03, 124.54,
4.2.37. (3Z, 6Z)-3-(4-fluorophenoxy) benzylidene-6-(1H-
imidazol-2-yl) methylene) piperazine-2, 5-dione (17e)
124.46, 120.56 (JC-F = 8.5 Hz), 118.96, 117.94, 116.54 (JC-F
=
23.4 Hz), 114.45, 108.62. HRMS (ESI) m/z: [M + H]+ Calcd for
C22H16FN2O3S: 407.0860, Found 407.0866.
15a (100.00 mg, 0.28 mmol), imidazole-2-formaldehyde
(32.5 mg ， 0.34 mmol), Cs2CO3 (137.90 mg, 0.42 mmol),
Na2SO4 (80.20 mg, 0.56 mmol), DMF (3 ml), 20.8 mg yellow
4.2.40. (3Z, 6Z)-3-(4-fluorophenoxy) benzylidene-6-(5-
bromofuran-2-yl) methylene) piperazine-2, 5-dione (17h)
1
15a (100.00 mg, 0.28 mmol), 5-bromo-2-furan
formaldehyde (74.07 mg, 0.42 mmol), Cs2CO3 (137.9 mg, 0.42
mmol), Na2SO4 (80.20 mg, 0.56 mmol), DMF (3 ml), 90.9 mg
yellow solid with a yield at 68%. MP: 211-214C. 1H NMR (500
MHz, DMSO-d6) δ 10.42 (s, 1H), 9.65 (s, 1H), 7.41 (t, J = 7.9
Hz, 1H), 7.29 (d, J = 7.7 Hz, 1H), 7.26 – 7.21 (m, 2H), 7.20 (s,
1H), 7.14 – 7.08 (m, 2H), 6.95 – 6.90 (m, 2H), 6.79 – 6.75 (m,
2H), 6.58 (s, 1H). 13C NMR (125 MHz, DMSO-d6) δ 159.14,
157.23, 157.08, 157.03, 152.54, 151.87, 134.95, 130.27, 126.79,
124.55, 124.23, 123.60, 120.55*2 (JC-F = 8.5 Hz), 119.00, 117.98,
116.63, 116.44, 114.54, 101.05. HRMS (ESI) m/z: [M - H]-
Calcd for C22H13BrFN2O4: 467.0048, Found 467.0054.
solid with a yield at 19%. MP: 243-245C. H NMR (500 MHz,
DMSO-d6) δ 12.62 (s, 1H), 12.04 (s, 1H), 10.38 (s, 1H), 7.42 (t, J
= 7.9 Hz, 1H), 7.37 (s, 1H), 7.30 – 7.21 (m, 4H), 7.19 (s, 1H),
7.14 – 7.09 (m, 2H), 6.92 (dd, J = 8.1, 2.2 Hz, 1H), 6.79 (s, 1H),
6.54 (s, 1H). 13C NMR (125 MHz, DMSO-d6) δ 159.14, 157.23,
157.05, 156.73, 156.60, 152.56, 143.55, 134.98, 130.29, 129.45,
128.07, 126.89, 124.55, 120.54*2 (JC-F = 8.5 Hz), 119.04, 118.01,
117.96, 116.54*2 (JC-F = 23.4 Hz), 114.42, 98.56. HRMS (ESI)
m/z: [M + H]+ Calcd for C21H16FN4O3: 391.1201, Found
391.1204.
4.2.38. (3Z, 6Z)-3-(4-fluorophenoxy) benzylidene-6-(furan-
2-ylmethylene) piperazine-2, 5-dione (17f)
4.2.41. (3Z, 6Z)-3-(4-fluorophenoxy) benzylidene-6-(5-
chlorofuran-2-yl) methylene) piperazine-2, 5-dione (17i)
15a (100.00 mg, 0.28 mmol), 2-furanaldehyde (40.67 mg，
0.42 mmol), Cs2CO3 (137.90 mg, 0.42 mmol), Na2SO4 (80.20
mg, 0.56 mmol), DMF (3 ml), 51.4 mg yellow solid with a yield
at 46%. MP: 212-214C. 1H NMR (500 MHz, DMSO-d6) δ 10.38
(s, 1H), 9.55 (s, 1H), 7.91 (s, 1H), 7.41 (t, J = 7.9 Hz, 1H), 7.28
(d, J = 7.6 Hz, 1H), 7.24 (t, J = 8.7 Hz, 2H), 7.19 (s, 1H), 7.12
(dd, J = 8.8, 4.4 Hz, 2H), 6.90 (dd, J = 11.3, 5.8 Hz, 2H), 6.77 (s,
1H), 6.66 (s, 2H). 13C NMR (125 MHz, DMSO-d6) δ 159.15,
157.25, 157.12, 157.05, 156.83, 152.57, 149.89, 144.93, 134.98,
130.30, 126.82, 124.53, 123.72, 120.56*2 (JC-F = 8.5 Hz), 118.99,
117.96, 116.55*2 (JC-F = 23.3 Hz), 114.47, 114.33, 112.45,
101.89. HRMS (ESI) m/z: [M + Na]+ Calcd for C22H15FN2O4Na:
413.0914, Found 413.0906.
15a (100.00 mg, 0.28 mmol), 5-methylfurfural (46.61 mg,
0.42 mmol), Cs2CO3 (137.9 mg, 0.42 mmol), Na2SO4 (80.20 mg,
0.56 mmol), DMF (3 ml), 44.0 mg yellow solid with a yield at
1
38%. MP: 214-216C. H NMR (500 MHz, DMSO-d6) δ 10.42
(s, 1H), 9.69 (s, 1H), 7.41 (t, J = 7.9 Hz, 1H), 7.29 (d, J = 7.7 Hz,
1H), 7.26 – 7.21 (m, 2H), 7.20 (s, 1H), 7.14 – 7.09 (m, 2H), 6.98
(d, J = 3.5 Hz, 1H), 6.92 (dd, J = 8.1, 2.0 Hz, 1H), 6.77 (s, 1H),
6.68 (d, J = 3.6 Hz, 1H), 6.58 (s, 1H). 13C NMR (125 MHz,
DMSO-d6) δ 159.14, 157.23, 157.07, 157.03, 152.56, 149.71,
136.43, 134.96, 130.28, 126.81, 124.55, 124.23, 120.55 (JC-F
=
8.5 Hz), 119.00, 117.98, 116.54 (JC-F = 23.4 Hz), 116.17, 114.55,
109.72, 101.13. HRMS (ESI) m/z: [M - H]- Calcd for
C22H13ClFN2O4: 423.0553, Found 423.0558.
4.2.39. (3Z, 6Z)-3-(4-fluorophenoxy) benzylidene-6-
(thiophen-2-ylmethylene) piperazine-2, 5-dione (17g)
4.2.42. (3Z, 6Z)-3-(4-fluorophenoxy) benzylidene-6-(5-
nitrofuran-2-yl) methylene) piperazine-2, 5-dione (17j)
15a (100.00 mg, 0.28 mmol), 2-thiophene formaldehyde
(47.89 mg, 0.42 mmol), Cs2CO3 (137.92 mg, 0.42 mmol),
Na2SO4 (80.17 mg，0.56 mmol), DMF (3 ml), 63.2 mg yellow
15a (100.00 mg, 0.28 mmol), 5-nitro-2-furaldehyde (59.70
mg, 0.42 mmol), Cs2CO3 (137.90 mg, 0.42 mmol), Na2SO4

(80.20 mg, 0.56 mmol), DMF (3 ml), 8.4 mg yellow solid with a
15a
(100.00
mg,
0.28
mmol),
5-tert-
1
butyldimethylsilylmethylolfurfural (101.80 mg, 0.56 mmol),
Cs2CO3 (137.90 mg, 0.42 mmol), Na2SO4 (80.20 mg, 0.56
mmol), DMF (3 ml), 45.1 mg yellow solid with a yield at 30%.
MP: 176-178C. 1H NMR (500 MHz, DMSO-d6) δ 10.33 (s, 1H),
9.42 (s, 1H), 7.42 (t, J = 7.9 Hz, 1H), 7.29 (d, J = 7.7 Hz, 1H),
7.26 – 7.21 (m, 2H), 7.19 (s, 1H), 7.14 – 7.09 (m, 2H), 6.92 (dd,
J = 8.1, 2.1 Hz, 1H), 6.84 (d, J = 3.4 Hz, 1H), 6.78 (s, 1H), 6.63
(s, 1H), 6.52 (d, J = 3.4 Hz, 1H), 4.75 (s, 2H), 0.88 (s, 9H), 0.10
(s, 6H). 13C NMR (125 MHz, DMSO-d6) δ 159.14, 157.67,
157.23, 157.15, 157.04, 156.86, 156.69, 155.95, 152.57, 149.53,
149.26, 135.01, 134.97, 130.28, 126.86, 126.77, 124.52, 123.50,
123.12, 120.58, 120.51, 118.98, 117.93, 116.63, 116.44, 115.48,
115.28, 114.31, 114.14, 110.01, 109.46, 102.06, 101.76, 57.55,
55.71, 25.80, 25.74, 17.96, 17.78, -3.20, -5.27. HRMS (ESI) m/z:
[M + H]+ Calcd for C29H32FN2O5Si: 535.2059, Found 535.2054.
4.2.46. (3Z, 6Z)-3-(4-fluorophenoxy) benzylidene-6-((5-
(hydroxymethyl) furan-2-yl) methylene) piperazine-2, 5-dione
(17n)
yield at 7%. MP: 237-240C. H NMR (500 MHz, DMSO-d6) δ
10.53 (s, 1H), 10.08 (s, 1H), 7.80 (s, 1H), 7.42 (s, 1H), 7.33 –
7.09 (m, 7H), 6.94 (s, 1H), 6.84 (s, 1H), 6.68 (s, 1H). 13C NMR
(125 MHz, DMSO-d6) δ 159.19, 157.32, 157.05, 156.40, 152.83,
152.57, 151.22, 134.81, 130.34, 129.58, 126.55, 124.77,
120.60*2 (JC-F = 8.5 Hz), 119.19, 118.25, 116.61*2 (JC-F = 23.4
Hz), 115.95, 115.81, 115.18, 98.86. HRMS (ESI) m/z: [M - H]-
Calcd for C22H13FN3O6: 434.0794, Found 434.0797.
4.2.43. (3Z, 6Z)-3-(4-fluorophenoxy) benzylidene-6-(5-
methylfuran-2-yl) methylene) piperazine-2, 5-dione (17k)
15a (100.00 mg, 0.28 mmol), 5-methyl-2-furanaldehyde
(59.70 mg, 0.42 mmol), Cs2CO3 (137.90 mg, 0.42 mmol),
Na2SO4 (80.20 mg, 0.56 mmol), DMF (3 ml), 37.7 mg yellow
1
solid with a yield at 71%. MP: 184-186C. H NMR (500 MHz,
DMSO-d6) δ 10.32 (s, 1H), 9.60 – 9.35 (m, 1H), 7.41 (t, J = 7.9
Hz, 1H), 7.28 (d, J = 7.8 Hz, 1H), 7.26 – 7.21 (m, 2H), 7.19 (s,
1H), 7.14 – 7.09 (m, 2H), 6.91 (dd, J = 8.1, 2.1 Hz, 1H), 6.78 (d,
J = 3.3 Hz, 1H), 6.75 (s, 1H), 6.59 (s, 1H), 6.29 (d, J = 2.5 Hz,
1H), 2.41 (s, 3H). 13C NMR (125 MHz, DMSO-d6) δ 159.14,
157.24, 157.05, 156.74, 154.64, 152.55, 148.53, 135.03, 130.28,
126.91, 124.49, 122.40, 120.55*2 (JC-F = 8.5 Hz), 118.95, 117.90,
116.54*2 (JC-F = 23.4 Hz), 116.09, 113.98, 108.96, 102.30, 13.71.
HRMS (ESI) m/z: [M + Na]+ Calcd for C23H17FN2O4Na:
427.1065, Found 427.1063.
The tetrabutylammonium fluoride (TBAF, 1M) (1.35 mL,
0.14 mmol) was added to a solution of ((3Z, 6Z)-3-(3-(para-
fluorobenzyloxy)
benzene)
methylene-6-((5-tert-
butyldimethylsilyl)
hydroxymethylfuran)-2-methylene)
piperazine-2, 5-dione (120 mg, 0.22 mmol) in dry THF (3 mL),.
The mixture was stirred at room temperature in the absence of
light. The reaction solution was removed under reduced pressure.
The filtration was stirred in methanol at room temperature for 2
h, then moved to 0 C. The solution was filtered, washed with
methanol, and dried in vacuum equipment at 50 °C to obtain 70.2
4.2.44. (3Z, 6Z)-3-(4-fluorobenzoyl) benzylidene-6-(5-
methylfuran-2-yl) methylene) piperazine-2, 5-dione (17l)
15b (100.00 mg, 0.27 mmol), 5-methyl-2-furanaldehyde
(45.10 mg, 0.41 mmol), Cs2CO3 (133.40 mg, 0.41 mmol),
Na2SO4 (77.50 mg, 0.55 mmol), DMF (3 ml), 50.6 mg yellow
1
mg of yellow solid with a yield of 74%. MP: 215-218C. H
NMR (500 MHz, DMSO-d6) δ 10.32 (s, 1H), 9.58 (s, 1H), 7.41
(t, J = 7.9 Hz, 1H), 7.29 (d, J = 7.7 Hz, 1H), 7.26 – 7.21 (m, 2H),
7.19 (s, 1H), 7.15 – 7.09 (m, 2H), 6.92 (dd, J = 8.1, 2.0 Hz, 1H),
6.80 (d, J = 3.3 Hz, 1H), 6.78 (s, 1H), 6.64 (s, 1H), 6.47 (d, J =
3.3 Hz, 1H), 5.52 (t, J = 6.0 Hz, 1H), 4.49 (d, J = 6.0 Hz, 2H).
13C NMR (125 MHz, DMSO-d6) δ 159.14, 157.67, 157.24,
157.15, 157.05, 156.86, 152.56, 149.27, 135.01, 130.29, 126.87,
124.52, 123.13, 120.56*2 (JC-F = 8.5 Hz), 118.98, 117.94,
116.54*2 (JC-F = 23.4 Hz), 115.49, 114.15, 109.47, 102.07, 55.72.
HRMS (ESI) m/z: [M + Na]+ Calcd for C23H17FN2O5Na:
443.1014, Found 443.1020.
1
solid with a yield at 44%. MP: 232-234C. H NMR (500 MHz,
DMSO-d6) δ 10.51 (s, 1H), 9.49 (s, 1H), 7.91 (dd, J = 8.7, 5.6
Hz, 2H), 7.84 (s, 1H), 7.77 (d, J = 7.7 Hz, 1H), 7.65 (d, J = 7.7
Hz, 1H), 7.59 (t, J = 7.6 Hz, 1H), 7.40 (t, J = 8.8 Hz, 2H), 6.84
(s, 1H), 6.78 (d, J = 3.3 Hz, 1H), 6.60 (s, 1H), 6.29 (d, J = 2.6
Hz, 1H), 2.41 (s, 3H). 13C NMR (125 MHz, DMSO-d6) δ 194.14,
165.75, 163.74, 157.38, 156.66, 154.65, 148.54, 137.27, 133.39,
132.77*2 (JC-F = 9.4 Hz), 130.19, 128.87, 127.36, 122.42, 116.11,
115.71*2 (JC-F = 22.0 Hz), 113.57, 108.97, 102.32, 13.71. HRMS
(ESI) m/z: [M + H]+ Calcd for C24H18FN2O4: 417.1245, Found
417.1236.
4.2.47. (3Z, 6Z)-3-(4-fluorophenoxy) benzylidene-6-((5-
(methoxymethyl) furan-2-yl) methylene) piperazine-2, 5-dione
(17o)
4.2.45. (3Z, 6Z)-3-(4-fluorophenoxy) benzylidene-6-(((tert-
butyldimethylsilyl) oxy) methyl) furan-2-yl) methylene)
piperazine-2, 5-dione (17m)

15a (100.00 mg, 0.28 mmol), 5-methoxymethylaldehyde
(59.30 mg, 0.42 mmol), Cs2CO3 (137.90 mg, 0.42 mmol),
Na2SO4 (80.20 mg, 0.56 mmol), DMF (3.5 ml), 44.5 mg yellow
126.69, 124.67, 123.51, 122.64, 120.56*2 (JC-F = 8.4 Hz), 119.13,
118.51, 118.12, 116.55*2 (JC-F = 23.3 Hz), 115.36, 111.64, 97.33.
HRMS (ESI) m/z: [M + H]+ Calcd for C25H18FN4O3: 441.1357,
Found 441.1350.
1
solid with a yield at 36%. MP: 150-152C. H NMR (500 MHz,
4.2.50. (3Z, 6Z)-3-(4-fluorophenoxy) benzylidene-6-
(benzofuran-2-yl methylene) piperazine-2, 5-dione (17r)
DMSO-d6) δ 10.35 (s, 1H), 9.49 (s, 1H), 7.41 (t, J = 7.9 Hz, 1H),
7.28 (d, J = 7.7 Hz, 1H), 7.26 – 7.21 (m, 2H), 7.19 (s, 1H), 7.14 –
7.08 (m, 2H), 6.91 (dd, J = 8.1, 2.1 Hz, 1H), 6.85 (d, J = 3.4 Hz,
1H), 6.77 (s, 1H), 6.63 (s, 1H), 6.60 (d, J = 3.4 Hz, 1H), 4.48 (s,
2H), 3.29 (s, 3H). 13C NMR (125 MHz, DMSO-d6) δ 159.14,
157.24, 157.07, 157.04, 156.83, 153.59, 152.57, 149.93, 134.97,
130.28, 126.81, 124.53, 123.73, 120.55*2 (JC-F = 8.5 Hz), 118.99,
117.97, 116.54*2 (JC-F = 23.3 Hz), 115.16, 114.33, 111.96,
101.79, 65.43, 57.36. HRMS (ESI) m/z: [M + Na]+ Calcd for
C24H19FN2O5Na: 457.1170, Found 457.1171.
15a (100.00 mg, 0.28 mmol), benzofuran-2-formaldehyde
(61.90 mg ， 0.42 mmol), Cs2CO3 (137.90 mg, 0.42 mmol),
Na2SO4 (80.20 mg, 0.56 mmol), DMF (3 ml), 76.5 mg yellow
1
solid with a yield at 61%. MP: 254-256C. H NMR (500 MHz,
DMSO-d6) δ 10.51 (s, 1H), 9.91 (s, 1H), 7.74 (d, J = 8.5 Hz, 1H),
7.68 (d, J = 7.7 Hz, 1H), 7.43 (t, J = 7.9 Hz, 1H), 7.40 – 7.36 (m,
1H), 7.33 – 7.28 (m, 3H), 7.24 (d, J = 8.7 Hz, 3H), 7.15 – 7.11
(m, 2H), 6.93 (dd, J = 8.0, 2.1 Hz, 1H), 6.82 (s, 1H), 6.80 (s, 1H).
13C NMR (125 MHz, DMSO-d6) δ 159.15, 157.25, 157.05,
156.92, 156.86, 154.64, 152.56, 151.88, 134.94, 130.28, 127.76,
4.2.48. (3Z, 6Z)-3-(4-fluorophenzoyl) benzylidene-6-((5-
(methoxymethyl) furan-2-yl)methylene)piperazine-2, 5-dione
(17p)
126.77, 126.36, 125.72, 124.58, 123.64, 121.43, 120.57*2 (JC-F
=
15b (100.00 mg, 0.27 mmol), 5-methoxymethylaldehyde
(98.40 mg, 0.41 mmol), Cs2CO3 (133.40 mg, 0.41 mmol),
Na2SO4 (77.50 mg，0.55 mmol), DMF (3.5 ml), 91.0 mg yellow
8.5 Hz), 119.03, 118.02, 116.54*2 (JC-F = 23.3 Hz), 114.79,
111.59, 110.05, 101.43. HRMS (ESI) m/z: [M + H]+ Calcd for
C26H18FN2O4: 441.1245, Found 441.1243.
1
solid with a yield at 75%. MP: 214-216C. H NMR (500 MHz,
4.3. Biology
DMSO-d6) δ 10.57 (s, 1H), 9.52 (s, 1H), 7.94 – 7.89 (m, 2H),
7.84 (s, 1H), 7.77 (d, J = 7.7 Hz, 1H), 7.65 (d, J = 7.7 Hz, 1H),
7.59 (t, J = 7.7 Hz, 1H), 7.40 (t, J = 8.8 Hz, 2H), 6.86 (d, J = 4.3
Hz, 2H), 6.64 (s, 1H), 6.61 (d, J = 3.4 Hz, 1H), 4.49 (s, 2H), 3.29
(s, 3H). 13C NMR (125 MHz, DMSO-d6) δ 194.13, 165.75,
163.75, 157.20, 156.75, 153.60, 149.93, 137.28, 133.42, 133.33,
132.77*2 (JC-F = 9.4 Hz), 130.22, 128.87, 127.24, 123.74,
115.71*2 (JC-F = 22.0 Hz), 115.18, 113.93, 111.96, 101.81, 65.44,
57.36. HRMS (ESI) m/z: [M + H]+ Calcd for C25H20FN2O5:
447.1351, Found 447.1354.
4.3.1. Anticancer activities
Human cancer cell lines were purchased from American
Type Cell Culture Collection (ATCC, USA). Cells were
maintained in DMEM medium supplemented with 10% (v/v)
heat-inactivated fetal bovine serum, penicillin-streptomycin (100
U/mL-100 g/mL) and 2 mM glutamine at 37°C in a humidified
atmosphere (5% CO2-95% air). Cells (5 × 103 per well) were
seeded in 96-well plates for 24 h. All test derivatives were
dissolved in 100% cell culture grade DMSO. After incubation
cells were treated with test compounds for 72 h. Cell viability
was assessed by MTT assay. The absorbance at 490 nm was
measured with a Microplate Reader (Molecular Devices, Silicon
Valley, USA). The data was analyzed by Origin 8.5.
4.2.49. (3Z, 6Z)-3-(4-fluorophenoxy) benzylidene-6-(1H-
benzo[d] imidazol-2-yl)methylene) piperazine-2, 5-dione (17q)
15a (100.00 mg, 0.28 mmol), 2-formyl benzimidazole
(61.90 mg ， 0.42 mmol), Cs2CO3 (137.90 mg, 0.42 mmol),
Na2SO4 (80.20 mg, 0.56 mmol), DMF (3 ml), 42.3 mg yellow
4.3.2. Immunofluorescence assay
1
solid with a yield at 34%. MP: 245-247C. H NMR (500 MHz,
NCI-H460 cells were seeded in a 24-well plate (with
coverslips plated) at a density of 5 × 104 cells. After overnight
adherence, the cells were exposed to compounds at 5 nM or 10
nM for 24 h. Then, the cells were fixed with 4% cold
paraformaldehyde at 4 °C for 15 min and then permeabilized in
0.05 % Triton X-100 for 10 min. The cells were blocked in 1 %
BSA for 30 min. Microtubules were detected by incubation with
DMSO-d6) δ 12.89 (s, 1H), 12.25 (s, 1H), 10.58 (s, 1H), 7.77 (dd,
J = 6.5, 2.0 Hz, 1H), 7.60 – 7.55 (m, 1H), 7.43 (t, J = 7.9 Hz,
1H), 7.33 – 7.20 (m, 6H), 7.13 (ddd, J = 10.5, 5.3, 3.1 Hz, 2H),
6.94 (dd, J = 8.1, 2.1 Hz, 1H), 6.87 (s, 1H), 6.65 (s, 1H). 13C
NMR (125 MHz, DMSO-d6) δ 159.15, 157.25, 157.05, 156.83,
156.26, 152.55, 149.19, 142.87, 134.85, 132.93, 132.46, 130.30,

7.
8.
9.
Prota A E, Bargsten K, Zurwerra D, et al. Science, 2013,
a monoclonal anti-β-tubulin at 4 °C for 12 h. Then, the cells were
washed with PBS and incubated with a FITC-conjugated anti-
mouse IgG antibody. Nuclei were stained with DAPI (G1012,
Servicebio, China). The coverslips were visualized under a
fluorescence microscope (Nikon Eclipse C1, Nikon, Japan) and
image-forming system (Nikon DS-U3, Nikon, Japan).
339(6119):587-590.
Daniele F, Giuseppe B, Francesco P, et al. Analytical Cellular
Pathology, 2015, 1-19.
Raffaele D F, Luigi A, Salvo D M, et al. Frontiers in Pharmacology,
2017, 8:797.
10. Kanoh K, Kohno S, Katada J, Hayashi Y, Muramatsu M, Uno I.
Bioscience Biotechnology and Biochemistry, 1999, 63(6):1130-1133.
11. Kanoh K, Kohno S, Katada J, et al. Bioorganic & Medicinal Chemistry,
1999, 7(7):1451-1457.
4.4. Molecular modeling
Ligands were prepared using the QuickPrep module in
12. Singh A V, Bandi M, Raje N, et al. Blood, 2011, 117(21):5692-5700.
13. Yamazaki Y, Tanaka K, Nicholson B, et al. Journal of Medicinal
Chemistry, 2012, 55(3):1056-71.
Maestro and energy was minimized through the general method.
Theoretical calculations of the physical properties (QpLogPo/w
and QpPCaco) of these synthesized compounds were performed
using the Qikprop Module of Maestro software. The X-ray
crystallographic structure was retrieved from the protein data
bank (PDB: 5XHC) at a resolution of 2.75 Å. The protein domain
subunits C and D and all water molecules were removed using
Maestro 11.5 using the protein preparation refinement module. A
subsequent energy minimization was carried out using the
OPLS_2005 force field. Then, molecules were docked into the
co-crystal structure of tubulin-compound 1.
14. Nicholson B, Lloyd G K, Miller B R, et al. Anti-Cancer Drugs, 2006,
17(1):25-31.
15. Giuseppina L S, Natacha O, Ashwani S, et al. Nature Chemistry,
2019, 5(11):2969-2986.
16. Kashyap A S, Fernandez-Rodriguez L, Zhao Y, et al. Cell Reports,
2019, 28(13):3367-3380.e8
17. Wang Y X, Zhang H, Gigant B, et al. FEBS Journal, 2016, 283(1):102-
111.
18. Fu Z Y, Hou Y W, Ji C P, et al. Bioorganic & Medicinal Chemistry,
2018, 26(8):2061-2072.
19. Tian Z H, Chu Y Y, Wang H, et al. RSC Advances, 2018, 8(2):1055-
1064.
The docking position was constrained by hydrogen bond using
the receptor grid generation module. Molecular modeling was
made by the ligand docking module according to imports from
the pretreatment ligand and the protein. At least 5 poses for each
compound were retained, and the best poses of rigid docking and
induced fit docking were refined.
20. Ding Z P, Ma M X, Zhong C J, et al. Bioorganic & Medicinal
Chemistry, 2019. DOI: 10.1016/j.bmc.2019.115186.
21. Ma M X, Ding Z P, Wang S X, et al. Bioorganic & Medicinal
Chemistry, 2019, 17:1836-1844.
22. Yamazaki Y, Sumikura M, Masuda Y, et al. Bioorganic & Medicinal
Chemistry, 2012, 20(14):4279-4289.
23. Hayashi Y, Takeno H, Chinen T, et al. ACS Medicinal Chemistry
Letters, 2014, 5(10):1094-1098.
Acknowledgments
This work was supported by “Zhufeng Scholar Program” of
Ocean University of China (841412016), and Aoshan Talents
Cultivation Program of Qingdao National Laboratory for Marine
Science and Technology (No. 2017ASTCP-OS08) to Dr.
Wenbao Li. We also acknowledge that the supports provided by
Ms Xiaoxiao Xu, Zhenhua Tian and Mr. Sifeng Zhu.
24. Xu G Y G., Pagare P P., Ghatge M S., et al. Molecular Pharmaceutics,
2017, 14(10):3499-3511.
References
1.
2.
3.
4.
5.
6.
AACR
cancer
progress
report
2019.
2019.
https://cancerprogressreport.org/Documents/AACR_CPR_2019.pdf
Zhou J, Giannakakou P. Current Medicinal Chemistry-Anti-Cancer
Agents, 2005, 5(1):65-71.
Dumontet C, Jordan M A. Nature Reviews Drug Discovery, 2010,
9(10):790-803.
Guo H, Li X, Guo Y, et al. Medicinal Chemistry Research, 2019,
28(7):927-937.
Downing K H. Current Opinion in Structural Biology, 1998, 8(6):785-
91.
Nogales E. Annual Review of Biochemistry, 2000, 69(1):277-302.

Dear Editors:
This is a manuscript entitled “Structure-based
design and synthesis of novel furan-
diketopiperazine-type derivatives as potent
microtubule inhibitors for treating cancer”.
There is no conflict of interest exists in submission
of this manuscript. I would like to declare on behalf
of my co-authors that the work described is original
research that has not been published in whole or in
part previously. And, this manuscript is approved
by all authors for publication.