Constructing novel dihydrofuran and dihydroisoxazole analogues of isocombretastatin-4 as tubulin polymerization inhibitors through [3+2] reactions

Article abstract of DOI:10.1016/j.bmc.2017.07.048

[3+2] reactions play a key role in constructing various pharmaceutical moleculars. In this study, using Mn(OAc)₃ mediated and 1,3-dipolar [3+2] cyclization reactions, 38 novel dihydrofuran and dihydroisoxazole analogues of isoCA-4 were synthesized as inhibitors of tubulin polymerization. Among them, compound 6g was found to be the most potent cytotoxic agents against PC-3 cells with IC₅₀ value of 0.47 μM, and compound 5p exhibted highest activity on HeLa cells with IC₅₀ vaule of 2.32 μM. Tubulin polymerization assay revealed that 6g was a dose-dependent and effective inhibitor of tubulin assembly. Immunohistochemistry studies and cell cycle distribution analysis indicated that 6g severely disrupted microtubule network and significantly arrested most cells in the G2/M phase of the cell cycle in PC-3 cells. In addition, molecular docking studies showed that two chiral isomers of 6g can bind efficiently and similarly at colchicine binding site of tubulin.

Full text of DOI:10.1016/j.bmc.2017.07.048

Accepted Manuscript
Constructing novel dihydrofuran and dihydroisoxazole analogues of isocom-
bretastatin-4 as tubulin polymerization inhibitors through [3+2] reactions
Ming-Yu Song, Chen-Yu Cao, Qiu-Rui He, Qing-Miao Dong, Ding Li, Jiang-
Jiang Tang, Jin-Ming Gao
PII:
S0968-0896(17)31283-X
http://dx.doi.org/10.1016/j.bmc.2017.07.048
BMC 13885
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
22 June 2017
25 July 2017
26 July 2017
Please cite this article as: Song, M-Y., Cao, C-Y., He, Q-R., Dong, Q-M., Li, D., Tang, J-J., Gao, J-M., Constructing
novel dihydrofuran and dihydroisoxazole analogues of isocombretastatin-4 as tubulin polymerization inhibitors
through [3+2] reactions, Bioorganic & Medicinal Chemistry (2017), doi: http://dx.doi.org/10.1016/j.bmc.
2017.07.048
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. 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.

Bioorganic & Medicinal Chemistry
Constructing novel dihydrofuran and dihydroisoxazole analogues of
isocombretastatin-4 as tubulin polymerization inhibitors through [3+2] reactions
Ming-Yu Song a, §, Chen-Yu Cao a, §, Qiu-Rui He a, Qing-Miao Dong a, Ding Lia, Jiang-Jiang Tang a,  and
Jin-Ming Gao a, 
a Shaanxi Key Laboratory of Natural Products & Chemical Biology, College of Chemistry & Pharmacy, Northwest A&F University, Yangling 712100, P.R.
China
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
[3+2] reactions play a key role in constructing various pharmaceutical moleculars. In this study,
using Mn(OAc)3 mediated and 1,3-dipolar [3+2] cyclization reactions, 38 novel dihydrofuran
and dihydroisoxazole analogues of isoCA-4 were synthesized as inhibitors of tubulin
polymerization. Among them, compound 6g was found to be the most potent cytotoxic agents
against PC-3 cells with IC50 value of 0.47 μM, and compound 5p exhibted highest activity on
HeLa cells with IC50 vaule of 2.32 µM. Tubulin polymerization assay revealed that 6g was a
dose-dependent and effective inhibitor of tubulin assembly. Immunohistochemistry studies and
cell cycle distribution analysis indicated that 6g severely disrupted microtubule network and
significantly arrested most cells in the G2/M phase of the cell cycle in PC-3 cells. In addition,
molecular docking studies showed that two chiral isomers of 6g can bind efficiently and
similarly at colchicine binding site of tubulin.
Available online
Keywords:
Isocombretastatin-4
Combretastatin-4
Dihydrofuran
Dihydroisoxazole
[3+2] reactions
2017 Elsevier Ltd. All rights reserved.
Tubulin polymerization inhibitors
 Corresponding authors: JJT (main corresponding author): e-mail: tangjiang11@nwsuaf.edu.cn; JMG: e-mail: jin-minggao@nwsuaf.edu.cn
§ These authors contributed equally to this work.

1. Introduction
analogues have been synthesized. Most derivatization of CA-4
laid their emphasis on the modifications for firming the unstable
Z-isomerization with various linkers, including silicon13,
butadiene14, cyclopropane15, cyclopropyl16, chiral‑ lactam17,
furan18, furazan19, imidazole20, 2,3-diarylthiophene21, isoxazole
and terphenyl22, triazoles23, 24, thiazole25, 26 and 1,2,4-triazolo-4,3-
b-pyridazines27.
Due to privileged structures and mechanisms, natural products
always attract chemists much attention to discovering and
developing the final drug entity1. It is statisticed that almost 50%
commercial drugs in the area of cancer are natural products or
their direct derivatives since 1940s2. Combretastatin A-4 (CA-4,
Figure 1), isolated from the stem bark of the African bush
Besides, an unexpectedly obtained substance phenstatin28, and
isoCA-4, one isomer of CA-4 found by M. Alami29, share a very
interesting structure of the 1,1-diaryl double bond. What’s more,
both of them have a very strong inhibition on cancer cell growth.
The synthetic methods of them are simpler than CA-4 without the
control of cis-configuration30, 31. Hence, M. Alami and co-
workers have completed plentiful studies on the modification of
1,1-diaryl double bond scaffold using naphthenic32,
willow Combretum caffrum in 19893,
, has a strong
4
depolymerization effect on tubulin by combining with the
colchicine binding site5. As the extremely significant status of
tubulin in cell mitosis and search of anticancer drugs6, CA-4 with
a small molecular weight and briefly structure has fascinated a
vast amount of further research. Additionally, several clinical
used anticancer prodrugs have been carried out, such as CA-4P
(fosbretabulin, zybrestat)7, a water-soluble prodrug synthesized
with the 3-O-phosphate form and attained the orphan drug status
in both USA and Europe for neuroendocrine neoplasm and
glioblastoma multiforme8, and AVE8062 (ombrabulin), which
can induce tumour blood flow, acquired from stasis serine amino
naphthalene33,
arylchromenes34,
azaisoerianin35,
arylbenzoxepins36 etc. Besides, ring-A and ring-B were also
transformed to find more active compounds by introductions of
fluorine37, quinolines38, quinazolines39, benzofurans and
Scheme 1. Synthesis of 5a-q. Reagents and conditions: (a) TBSCl, Et3N, DMF, rt., 1.5 h; (b) 3,4,5-trimethoxybenzaldehyde, n-BuLi (1.6 M in
o
hexane), THF, -78 oC, 1 h; -78 oC→ rt., 24 h; (c) PDC, 4 Åms, DCM, 0 C, 34 h; (d) Ph3PCH3I, n-BuLi (1.6 M in Hexane), THF, -78 oC, 1 h; -
78 oC→ rt., 24 h; (e) i) acylacetonitriles or β-diketones, Mn(OAc)3, AcOH, 80 oC, 10-90 min; ii) TBAF, THF, 0.5 h.
indoles40. Of course, there are also some researches worked out
by other medicinal chemists, such as oxindole41, arylcoumarin42,
Figure 1. The structures of CA-4 and synthetic prodrugs, phenstation, isoCA-
4, and dihydrofuran, dihydroisoxazole analogues
43, benzosuberene44, 45 analogues of isoCA-4.
Dihydrofuran, as a pharmacophore, has gained prominence
due to exhibiting excellent and various biological activities. A
acid modifying CA-49.
number of pharmaceuticals (such as aflatoxin B1 and clerodin46,
The structure-activity relationship (SAR) studies of CA-4
have made clear the indispensability of ring-A (3,4,5-trimethoxy
groups) and cis-configuration for inhibiting tubulin assembly10.
Nevertheless, when suffering from heat, light and protic media,
the Z-restricted configuration can convert rapidly into 100-fold
less active trans-isomerization11. Meanwhile, the poor metabolic
stability caused by the olefinic bridge and the weak water
solubility of CA-4 resulted in restrictions for its exploitation12.
Thus, it is clearly that CA-4 and those prodrugs still have a huge
development space, and for this reason, an impressive number of
47
)
and
natural
products
(1-O-acetylbritannilactone48,
fraxinellone49 and azadirachtin50) have been explored for clinical
applications and further lucubration, respectively. In addition,
dihydroisoxazole also has been studied as a vital pharmacophore
for inhibitors of HIV51 and transglutaminase 252, 53. Heretofore,
there were few studies for the synthesis of anticancer compounds
via introducing dihydrofuran and dihydroisoxazole to isoCA-4.
[3+2] reactions including Mn(OAc)3 mediated radical
cyclization and 1,3-dipolar cyclization are considered as feasible

measures for the construction of dihydrofuran and
dihydroisoxazole compounds. Mn(OAc)3 has drew a great deal of
attention in the field of oxidative free-radical chemistry as a
single-electron-transfer (SET) reagent with 1.51 V of oxidative
potential54. Varieties of unique molecules, such as triptolide55,
indolo[1,2-alpha]quinolines56, polysubstituted pyrroles57, 58, have
been constructed by using its higher selectivity and moderate
reactivity. Similarly, 1,3-dipolar cycloaddition reactions also
fascinate us due to their superiority of synthetic ability on five-
membered heterocycles59, 60. That encourages us to adopt these
strategies for the constructions of dihydrofuran and
dihydroisoxazole analogues of isoCA-4.
no difference in this reaction regardless of the existence or
absence of Ar in short time (< 1.5 h). Notably, if the reaction was
not terminated in time in the absence of Ar, the main products
would convert back into exceedingly oxidic accessory substance
(3). It is perhaps due to the higher single electron oxidative
potential of Mn(III) with 1.51 V in AcOH.
It should be noted that all compounds 5a-q were synthesized
with a chiral carbon atom. The chiral HPLC analysis of 5b and
5n revealed that the ratio of chiral isomers is about 1:1. To
explore different absolute configurations for biological activities,
Therefore, using Mn(OAc)3 mediated and 1,3-dipolar [3+2]
cyclization reactions, we herein present the synthesis of two
series of novel isoCA-4 analogues through the modification of
olefin scaffold with dihydrofuran and dihydroisoxazole.
Subsequently, their cytotoxic activities were evaluated on PC-3
and HeLa cells. The most potent cytotoxic compound 6g was
selected for tubulin polymerization assay and further probes in
mechanism studies, such as immunohistochemistry studies, cell
cycle analysis and molecular docking studies.
Figure 2. The X-ray crystal structure of (S)-5b (CCDC: 1548952).
5b and 5n were separated to two couples of isomers as the
representatives. Fortunately, X-ray crystal structure of (S)-5b,
depicted in Figure 2, was acquired to verify this geometric
structure. Whereafter, the absolute configurations of other
components were confirmed with the help of specific rotations
(shown in Table S1).
2. Results and discussion
2.1. Chemistry
The synthetic route of target compounds 5a-q was outlined in
Scheme 1. As a starting material, 5-bromo-2-methoxyphenol was
protected with tert-butyldimethylsilyl chloride (TBSCl) in N,N-
dimethylformamide (DMF) and then was reacted with 3,4,5-
trimethoxybenzaldehyde to aff ord the desired alcohol 2.
Subsequently, 2 was oxidized by pyridinium dichromate (PDC)
to yield 3, which produced the key intermediate 4 in 91% yield
via methyltriphenylphosphonium iodide (Ph3PCH3I) Wittig
methylenation. Afterwards, 4 was treated with acylacetonitriles
or β-diketones by Mn(OAc)3 mediated radical [3+2] cyclization
under argon (Ar) atmosphere in glacial acetic acid, followed
upon tetrabutylammonium fluoride (TBAF) deprotection to target
compounds 5a-q. In this case, the majority of these compounds
were generated in modest to good yields (37-94%), except 5l in
15% yield.
Subsequently, the dihydroisoxazole analogues 6a-n were
carried out by the [3+2] cycloaddition of intermediate 4 with
excess new-made 1,3-dipole congeners and further deprotection
(Scheme 2). As an intention of enhancing the target compounds
competence, 3,4,5-trimethoxybenzene and
3-hydroxy-4-
methoxybenzene were retained in ring-A or ring-B. Moreover, 4-
hydroxybenzene and 3,4-dihydroxybenzene were also brought in
to enlarge the polar in modest yields (35-36%). In this case,
compounds 6d, 6f and 6i whose R3 fragment bearing an electron-
withdrawing group of ortho-NO2 or ortho-Cl benzene were
synthesized with low yields (18-26%). Yet, 6h was carried out
with a much better yield (52%) with an electron-donor group of
methoxyl on ortho-position of benzene belonging to R3,
suggesting that the conjugation effect of ortho-OMe on R3 may
dominate the yield in this reaction.
Worsely, we failed in isolating any desired products with
some substrates such as 1-(cyanoacetyl)pyrrolidine and methyl
acetoacetate. Nonetheless, it was interesting to find that there is
Scheme 2. Synthesis of 6a-n. Reagents and conditions: (a) 50% NH2OH in H2O, Et2O, 10 min; (b) NCS, DMF, rt. 0.5 h; (c) 1,3-
dipole congeners, Et3N, DCM, 0 oC, 1 h; TBAF, THF, 0.5 h.

Scheme 3. Synthesis of 8a-c and 9a-c. Reagents and conditions: (a) n-BuLi (1.6 M in hexane), THF, -78 oC, 1 h; -78 oC→ rt., 24 h;
(b) PDC, 4 Åms, DCM, 0 oC, 34 h; (c) Ph3PCH3I, n-BuLi (1.6 M in hexane), THF, -78 oC, 1 h; -78 oC→ rt., 24 h; (d)
acylacetonitriles or β-diketones, Mn(OAc)3, AcOH, 80 oC, 0.5 h; (e) 1,3-dipole congeners, Et3N, DCM, 0 oC, 1 h; for 9c, then TBAF
in THF, 0.5 h.
Accidentally, compound 7 is a byproduct of 4 resulted from a
previous competing reaction between 3,4,5-
with IC50 values of 7.57 µM against HeLa, while with IC50 value
more than 10 µM against PC-3, implying the HeLa cells may be
more sensitive to 6j which is different from other analogues.
trimethoxybenzaldehyde and excess n-BuLi with 1. Next, several
products replacing ring-B with butyl were synthesized in Scheme
3. Except the sole distinction of butyl, compound 9c is extremely
similar to a compound produced by Daniele Simoni et al.22,
which exhibited antiproliferative activity against HeLa cells.
For dihydrofuran and dihydroisoxazole analogues 8a-c and
9a-c with n-butyl-substituted B-ring, all of them displayed
unpromising cytotoxic activity over 10 µM on PC-3 cells. The
results suggested that B-ring with benzene ring might partly be
responsible for cytotoxic activity of these compounds.
2.2. Biological evaluation
2.2.2. Tubulin polymerization assay
2.2.1. Cytotoxic activity
To investigate whether there is such an interaction of these
novel compounds with tubulin responsible for the cytotoxic
activity, we evaluated the effects of the most cytotoxic compound
New synthesized compounds 5a-p, 6a-n, 8a-c and 9a-c were
screened for anticancer activity against PC-3 (human prostate
cancer cell line) and HeLa ( human cervical cancer cell line) with
isoCA-4 and CA-4 as reference compounds using the
sulforhodamine B (SRB) assay61. The results of in vitro cytotoxic
activities (IC50) were summarized in Table 1.
o
6g on tubulin polymerization in vitro for 80 minutes at 37 C. In
this assay, paclitaxel was employed as a positive control while
isoCA-4 and CA-4 as negative controls. The fluorescence
increase was monitored every minute for characterization of
tubulin polymerization and the results were shown in Figure 3. A
blank assay of only 6g (40 µM) was also performed to exclude
the jeopardie of a high concentration of 6g on the results (Figure
S1). Paclitaxel enhanced the assembly of tubulin, while isoCA-4
and CA-4 inhibited it well. The results indicated that compound
6g displayed a dose-dependent manner on inhibiton of tubulin
polymerization with an IC50 value of 30.7 μM. From the tubulin
polymerization assay, we can conclude that 6g could inhibit the
assembly of tubulin and the cytotoxic activity might be generated
from this interaction with tubulin.
For dihydrofuran analogues 5a-p, four compounds 5e (R1=4-
FC6H5, R2=CN), 5f (R1=2-ClC6H5, R2=CN), 5g (R1=4-ClC6H5,
R2=CN) and 5p (R1=C6H5, R2=COC6H5) exhibited a good
cytotoxic activity (IC50=0.78-0.86 µM). Compound 5p displayed
best activity against HeLa with IC50 vaule of 2.32 µM, fourfold
less than CA-4. However, compared with PC-3 cells, HeLa cells
showed a much less sensitivity to the dihydrofuran analogues and
CA-4, with IC50 values of over 10 µM for majority analogues.
Notably, the IC50 results of (R/S)-5b (9.47 µM for R-5b, 10.2 µM
for S-5b) and (R/S)-5n (17.0 µM for R-5n, 15.1 µM for S-5n) on
PC-3 cells indicated no obvious gap in cytotoxic activity between
enantiomers.
For dihydroisoxazole analogues 6a-n, compound 6g (R3=4-
ClC6H5) exhibited the best ability to inhibit PC-3 cells growth
with IC50 value of 0.47 µM and a better cytotoxicity on HeLa
with IC50 value of 3.29 µM. In addition, 6b and 6f also displayed
cytotoxic activity on PC-3 cells with IC50 value of 0.85 and 0.81
µM, respectively, and on HeLa with IC50 value of 4.45 and 3.27
µM, respectively. Moreover, 6d, 6i and 6m displayed modest
cytotoxic activity on PC-3 below 10 µM (IC50=8.21–9.56 µm).
Interestingly, 6j (R3=4-HOC6H5) exhibited potential cytotoxicity
Figure 3. Effects of compound 6g, paclitaxel, CA-4 and isoCA-4 on
tubulin polymerization in vitro. Tubulin (10 mg/mL) was incubated with
6g (10, 20 and 40 µM), paclitaxel (3 µM), CA-4 (3 µM), isoCA-4 (3
o
µM) and DMSO for 80 minutes at 37 C. The fluorescence increase was
monitored every minute for characterization of tubulin polymerization at
360 nm excitation wavelength and 460 nm emission wavelength.
Table 1.
Cytotoxic activities (IC50) of 5a-q, 6a-n, 8a-c and 9a-c.
IC50a (µM)
IC50a (µM)
Compd.
Compd.
PC-3
HeLa
PC-3
HeLa
>10
9.47 ± 0.11
>10
>10
>10
8.21± 1.05
>10
>10
5a
(R)-5b
6c
6d

(S)-5b
5c
10.2 ± 0.07
6.99 ± 0.38
>10
>10
>10
>10
0.81 ± 0.22
0.47 ± 0.11
>10
>10
3.27 ± 0.65
3.29 ± 0.44
>10
6e
6f
>10
5d
6g
6h
6i
0.81 ± 0.06
0.79 ± 0.03
0.86 ± 0.10
>10
7.21 ± 0.28
>10
5e
8.63 ± 0.08
>10
>10
5f
>10
7.57 ± 1.75
>10
5g
6j
>10
>10
5h
6k
6l
>10
>10
>10
>10
5i
4.80 ± 0.17
6.37 ± 0.26
9.45 ± 0.50
8.47 ± 0.10
>10
9.56 ± 0.86
>10
>10
5j
6m
6n
8a
8b
>10
>10
5k
>10
>10
>10
5l
>10
>10
>10
5m
(R)-5n
(S)-5n
5o
17.0 ± 0.08
15.1 ± 0.06
>10
>10
>10
>10
>10
>10
>10
8c
9a
>10
>10
>10
9b
0.78 ± 0.09
>10
2.32 ± 0.04
>10
>10
>10
5p
9c
0.001 ± 0.001
0.007 ± 0.005
0.085 ± 0.02
0.53 ± 0.14
6a
isoCA-4
CA-4
0.85 ± 0.11
4.45 ± 0.54
6b
a The IC50 values represent the concentration that causes 50% inhibition of cell viability. Cells were treated with the compounds for 48 h. All data (mean ± SD)
are the average of three determinations. Cancer cell lines: PC-3 (human prostate cancer), HeLa (human cervix cancer).
(0.02 μM) on microtubule network in PC-3 cells by
immunofluorescence. As shown in Figure 4, control cells
displayed a normal distribution of microtubular network with a
slim and fibrous shape. However, cells treated with 6g and CA-4
showed a disrupted microtubule network becoming short and
wrapped around cell nucleus, demonstrating that 6g would inhibit
the tubulin polymerization of cells.
Figu
CA-4
with
2.2.4. Cell cycle analysis
Surro
are s
6g, is
To confirm the biological mechanism of these novel
compounds, we further used flow cytometry to analyze the cell
cycle distribution of PC-3 cells after treatment with 6g (0.5 and
1.0 µM) and CA-4 (0.05 µM) for 24 h. Representative FACS
Figure 4. Effects of compound 6g (1.0 μM) and CA-4 (0.02 μM) on
inhibition of tubulin polymerization in PC-3 cells by
immunofluorescence. The cell nucleus was visualized with DAPI (blue,
left panel), and microtubules were visualized with an anti-α-tubulin
antibody (TRITC, red, middle panel). The right panel represents a merge
of the corresponding left and middle panels. Images were acquired with
a fluorescence microscope using a 20× objective.
measurements from a standard propidium iodide (PI) procedure
were depicted in Figure 5. A distinct cell cycle arrest at G2/M
phase (71.9%) was observed in PC-3 cells treated with CA-4,
compared to control group (14.0%). Similarly, the cell
accumulation percentage at the G2/M phase increased from
63.5% to 84.5% with a concentration rising from 0.5 to 1.0 µM
of 6g. The observed effects of 6g on cell cycle progression
correlated well with its cytotoxic and anti-tubulin activities, in
agreement with the similar properties reported previously for the
majority of antimitotic agents.
2.2.3. Immunohistochemistry studies
To visualize the tubulin polymerization inhibition of these
novel compounds in cells, we investigated immunocytochemistry
studies to examine effects of compound 6g (1.0 μM) and CA-4
2.3. Molecular modeling

with isoCA-4 and CA-4 in similar distorted conformations.
Although the anticancer activity evaluation of the two series
of novel compounds did not perform as excellently as
isoCA-4, our work expanded the research of olefin
modification of tubulin polymerization agent isoCA-4 and
provided fundamental data and additional insight for new
developments of tubulin inhibitors.
4. Experimental section
4.1. Chemistry
4.1.1. General
Figure 6. Flow cytometric analysis of cell cycle distribution in PC-3
cells. The cultured cells were treated with CA-4 and 6g at the indicated
concentrations for 24 h, then analyzed by a standard propidium iodide
(PI) procedure using a FACS Calibur (BD Biosciences).
All the compounds were inspected by a 500 MHz Bruker
NMR spectrometer with H, 13C and dept135 NMR in CDCl3
1
solution (δ: 7.26). Chemical shift values are given in δ (ppm) and
coupling constants (J) are mentioned in Hz. MS (ESI) was
measured on an ESI-Thermo Fisher LTQ Fleet instrument
spectrometer. HR-ESI-MS spectra were recorded on a LTQ
Orbitrap (Thermo Scientific). Analytical and preparative chical
HPLC were performed on Waters 515-2996 or Shimadzu
Essentia Prep LC-16 with columns of S-Chiral A/B or Daicel
OD-H. The reaction progress was supervised by TLC (Qingdao
Marine Chemical Group, Co.), spots were observed under UV
light (254, 365 nm) and colorated with 5% phosphomolybdic
acid. Silica gels (300-400 mesh, Qingdao Marine Chemical Ltd.)
were used for column chromatography (CC). All reagents were
purchased from commercial sources and used without further
refinement. While the purifications and desiccations of
commercially available solvents were accomplished by standard
techniques before using. Positive control drug isoCA-4 was
obtained by the deprotection of 4, and it’s 1H NMR and MS (ESI)
data were carried out for supporting the structure.
To elucidate the binding modeling of these novel analogues
with the tubulin colchicine binding site, the X-ray crystal
structure (PDB: 1SA0) was employed for performing the docking
studies of the most potent compound 6g using the Surflex-Dock
program in the Sybyl-X 2.0 software. Taking into consideration
the mixed enantiomers of 6g in cytotoxic activity, we executed
the docking studies of both enantiomers of (S)-6g and (R)-6g.
Figure 6a exhibits (S)-6g and (R)-6g can bind well with the
colchicines binding site of tubulin. The meta-methoxy groups on
ring-A of both (S)-6g and (R)-6g formed two hydrogen bonds
with the key amino acid Cys241 about 2.0 Å and 2.1 Å bond
distance, respectively. Meanwhile, another two hydrogen bonds
formed between the oxygen atoms of the hydroxyl groups and
amino acid Val181 about 2.0 Å and 1.8 Å bond distance,
respectively. It is noteworthy that (R)-6g and (S)-6g bear almost
the same overlay form at the colchicine binding site with a sole
difference on the direction of dihydroisoxazole which have little
influence on binding with colchicine binding site. The similar
results (Figure S1) also were carried out on R/S enantiomers of
5g, a dihydrofuran analogue exhibiting a good cytotoxic activity
against PC-3 cells. That will be a possible explanation for the few
distinction in cytotoxic activity between R/S enantiomers of 5b
and 5n. Figure 6b shows that (S)-6g, (R)-6g superimposed well
with isoCA-4 and CA-4 at the colchicine binding site with
similar distorted conformations. These docking studies suggested
that the mechanism of 6g inhibition tubulin may be the same as
that of isoCA-4 and CA-4.
4.1.2.
Synthesis
of
(3-((tert-butyldimethylsilyl)oxy)-4-
methoxyphenyl)(3,4,5-trimethoxyphenyl)methanone (3)
Tert-butyldimethylsilyl chloride (TBSCl, 6.03 g, 40 mmol)
was added to a solution of 5-bromo-2-methoxyphenol (4.06 g, 20
mmol) in N,N-dimethylformamide (DMF, 20 mL), followed by
triethylamine (5.6 mL, 40 mmol). After 1.5 h, the mixture was
extracted with diethyl ether (3×20 mL), and the combined organic
phases were evaporated under reduced pressure to give crude
product of 1. Next, 1.6 mol/L n-butyl lithium in hexane (27.5
mL, 44 mmol) was added to a stirred suspension of crude
o
products of 1 in dry tetrahydrofuran (THF, 20 mL) at -78 C
under
argon
atmosphere.
After
1
h,
3,4,5-
trimethoxybenzaldehyde (5.47 g, 26 mmol, 20 mL) in dry THF
was slowly added to the resulting yellowish solution, allowing
the reaction to slowly reach room temperature. After 24 h, the
mixture was poured over ice and extracted with CH2Cl2. The
organic phases were dried over anhydrous Na2SO4, filtered, and
evaporated under reduced pressure to give crude product of 2.
3. Conclusions
In the current work, based on Mn(OAc)3 mediated and
1,3-dipolar [3+2] cycloaddition reactions, we synthesized
thirty-eight isoCA-4 analogues belonging to two series of
novel dihydrofuran and dihydroisoxazole and evaluated their
activities as inhibitors of tubulin polymerization. Most
compounds exhibited potential cytotoxic activity. Among
them, 6g was found to be the most potent cytotoxic agents
against PC-3 cells with IC50 value of 0.47 μM, and 5p
exhibted best activity on HeLa cells with IC50 vaule of 2.32
µM. Tubulin polymerization assay revealed that 6g was a
dose-dependent and effective inhibitor of tubulin assembly.
As probes in mechanism studies, immunohistochemistry
studies and cell cycle analysis indicated that 6g disrupted
microtubule network severely and significantly arrested
most cells in the G2/M phase of the cell cycle in PC-3 cells.
In addition, molecular docking studies showed that two
chiral isomers of 6g binded similarly and efficiently at
colchicine binding site on tubulin and superimposed well
Pyridinium dichromate (PDC, 11.3 g, 30 mmol) was added to
a solution of crude products of 2 and 4 Å molecular sieves in
o
CH2Cl2 (50 mL) at 0 C. After 24 h, the reaction mixture was
filtered through pads of celite. The filtrates were evaporated
under reduced pressure, and crude products were purified by
column chromatography on silica gel using petroleum ether/ethyl
acetate (8:1) as eluent.
1
That gave compound 3 as a white solid (4.30 g, 49%); H
NMR (500 MHz, CDCl3): δ = 7.42 (dd, J = 8.3, 1.8 Hz, 1H), 7.35
(d, J = 1.8 Hz, 1H), 7.01 (s, 2H), 6.88 (d, J = 8.4 Hz, 1H), 3.91
(s, 3H), 3.87 (s, 3H), 3.86 (s, 6H), 0.98 (s, 9H), 0.16 ppm (s, 6H);
13C NMR (125 MHz, CDCl3): δ = 194.53, 154.94, 152.80 (2C),
144.69, 141.55, 133.39, 130.44, 125.31, 122.34, 110.75, 107.44

(2C), 60.96, 56.28 (2C), 55.51, 25.68 (3C), 18.44, -4.54 ppm
(2CC-Si); MS (ESI): m/z calcd for C23H33O6Si+ [M+H]+ 433.60,
found 433.18.
disappeared (in 10-90 min). Acetic acid was evaporated under
reduced pressure, and water (10 mL) was added to the residue.
Then the mixture was neutralized with sated NaHCO3 solution
and extracted with CHCl3 (3×10 mL). The combined organic
phases were evaporated under reduced pressure.
4.1.3.
Synthesis
of
tert-butyl(2-methoxy-5-(1-(3,4,5-
trimethoxyphenyl)vinyl)phenoxy)dimethylsilane (4)
The residues from reactions of 4 and acylacetonitriles or β-
diketones were dissolved in THF (2 mL), followed by 1.0 mol/L
TBAF (175 µL, 0.175 mmol) in THF in 0.5 h. Water (10 mL)
was added to this solution and extracted with ethyl acetate (3×10
mL). The combined organic phases were dried over anhydrous
Na2SO4, filtered and evaporated. Crude products were purified by
column chromatography on silica gel using petroleum ether/ethyl
acetate as eluent.
A solution of methyltriphenylphosphonium iodide (Ph3PCH3I,
11.32 g, 28 mmol) in dry THF (50 mL) was cooled to -78 C
o
under argon atmosphere. Then, 1.6 mol/L n-butyl lithium in
hexane (12.5 mL, 20 mmol) was added dropwise, and the
resulting yellow solution was stirred for 1 h. Then a solution of 3
(4.30 g, 10 mmol) in dry THF was added and warmed to room
o
temperature. Once completed, the mixtures were cooled to 0 C,
stopped with saturated NH4Cl solution and extracted with CH2Cl2
(3×50 mL). The organic phases were washed with brine, dried
over anhydrous Na2SO4, filtered, and evaporated under reduced
pressure. Crude products were purified by column
chromatography on silica gel using petroleum ether/ethyl acetate
(10:1) as eluent. That gave compound 4 as a pale green oil (4.0 g,
93%); 1H NMR (500 MHz, CDCl3): δ = 6.91 (dd, J = 8.3, 2.2 Hz,
1H), 6.85 (d, J = 2.2 Hz, 1H), 6.80 (d, J = 8.4 Hz, 1H), 6.55 (s,
2H), 5.34 (d, J = 1.1 Hz, 1H), 5.30 (d, J = 1.1 Hz, 1H), 3.87 (s,
3H), 3.81 (s, 3H), 3.80 (s, 6H), 0.98 (s, 9H), 0.15 ppm (s, 6H);
13C NMR (125 MHz, CDCl3): δ = 152.84 (2C), 150.88, 149.56,
144.55, 137.79, 137.52, 134.11, 121.78, 120.95, 112.45, 111.55,
105.69 (2C), 60.90, 56.09(2C), 55.49, 25.75 (3C), 18.47, -4.54
ppm (2CC-Si); MS (ESI): m/z calcd for C24H35O5Si+ [M+H]+
431.62, found 431.31.
4.1.6.1. 2-(tert-butyl)-5-(3-hydroxy-4-methoxyphenyl)-5-(3,4,5-
trimethoxyphenyl)-4,5-dihydrofuran-3-carbonitrile (5a)
This compound was obtained from reaction of 4 with 4,4-
dimethyl-3-oxopentanenitrile after 50 min, followed by TBAF
0.5 h. Eluent: petroleum ether/ethyl acetate = 5:1. Reddish brown
1
solid. Yield: 35 mg, 68%; H NMR (500 MHz, CDCl3): δ = 6.90
(d, J = 1.8 Hz, 1H), 6.80 (dd, J = 6.9, 5.2 Hz, 2H), 6.55 (s, 2H),
5.69 (s, 1H), 3.88 (s, 3H), 3.84 (s, 3H), 3.81 (s, 6H), 3.56 (d, J =
14.3 Hz, 1H), 3.49 (d, J = 14.3 Hz, 1H), 1.38 ppm (s, 9H); 13C
NMR (125 MHz, CDCl3): δ = 177.12, 153.10 (2C), 146.31,
145.54, 140.14, 137.49, 137.41, 117.36, 117.02, 112.43, 110.20,
102.87 (2C), 91.95, 77.66, 60.87, 56.18 (2C), 56.00, 46.01,
+
34.84, 28.11 ppm (3C); MS (ESI): m/z calcd for C25H30NO6
[M+H]+ 440.52, found 440.28.
4.1.4. Synthesis of isocombretastatin-4
4.1.6.2.
5-(3-hydroxy-4-methoxyphenyl)-2-phenyl-5-(3,4,5-
Compound
4
(50 mg, 116 µM) were dissolved in
trimethoxyphenyl)-4,5-dihydrofuran-3-carbonitrile (5b)
tetrahydrofuran (5 mL), followed by 1.0 mol/L TBAF (175 µL,
0.175 mmol) in THF for 0.5 h. Water (10 mL) was added to this
solution and extracted with ethyl acetate (3×10 mL). The
combined organic phases were dried over anhydrous Na2SO4,
filtered and evaporated. Crude products were purified by column
chromatography on silica gel using petroleum ether/ethyl acetate
(3:1).That gave the compound isoCA-4 as a white soild (34 mg,
92%); 1H NMR (500 MHz, CDCl3): δ = 6.97 (d, J = 1.8 Hz, 1H),
6.84 (dd, J = 8.4, 1.9 Hz, 1H), 6.80 (d, J = 8.3 Hz, 1H), 6.55 (s,
2H), 5.37 (s, 1H), 5.30 (s, 1H), 3.89 (s, 3H), 3.87 (s, 3H), 3.81
This compound was obtained from reaction of 4 with 3-oxo-3-
phenylpropanenitrile after 1 h, followed by TBAF 0.5 h. Eluent:
petroleum ether/acetone = 10:1. Yellow solid. (R)-5b and (S)-5b
were prepared by chical HPLC performed on Waters 515-2996
with columns of S-Chiral B. (R)-5b: yield: 18 mg, 34%; mp: 148-
150 oC; [α]D30= -74.99o (c 0.036 in CHCl3); (S)-5b: yield: 17 mg,
31%; mp: 147-149 C; [α]D30= +98.22o (c 0.036 in CHCl3); H
NMR (500 MHz, CDCl3): δ = 8.07 (dd, J = 7.7, 1.7 Hz, 2H), 7.48
(s, 2H), 7.47 (s, 1H), 7.00 (d, J = 2.1 Hz, 1H), 6.90 (dd, J = 8.4,
2.2 Hz, 1H), 6.82 (d, J = 8.5 Hz, 1H), 6.64 (s, 2H), 5.82 (s, 1H),
3.85 (s, 3H), 3.84 (s, 3H), 3.81 (s, 6H), 3.75 (d, J = 14.9 Hz, 1H),
3.70 ppm (d, J = 14.8 Hz, 1H); 13C NMR (125 MHz, CDCl3): δ =
165.25, 153.19 (2C), 146.52, 145.65, 139.62, 137.71, 136.83,
131.57, 128.85 (2C), 127.98, 127.13 (2C), 117.44, 117.39,
112.60, 110.34, 103.17 (2C), 92.84, 79.22, 60.87, 56.27 (2C),
o
1
-
ppm (s, 6H); MS (ESI): m/z calcd for C18H19O5 [M-H]- 315.35,
found 315.09.
4.1.5. Synthesis of 5-(hex-1-en-2-yl)-1,2,3-trimethoxybenzene (7)
The preparation of 7 shared a same method with intermediate 4
except the absent of purification during process in the second
preparation of 4. That gave compound 7 as a pale green liquild
(1.0 g, 20%); 1H NMR (500 MHz, CDCl3): δ = 6.60 (s, 2H), 5.19
(s, 1H), 5.01 (s, 1H), 3.85 (s, 6H), 3.84 (s, 3H), 2.45 (t, J = 7.5
Hz, 2H), 1.47 – 1.40 (m, 2H), 1.38 – 1.30 (m, 2H), 0.89 ppm (t, J
= 7.3 Hz, 3H); 13C NMR (125 MHz, CDCl3): δ = 152.95 (2C),
148.81, 137.52, 137.50, 111.75, 103.45 (2C), 60.86, 56.09 (2C),
35.22, 30.47, 22.43, 13.95 ppm; MS (ESI): m/z calcd for
C15H23O3+ [M+H]+ 251.35, found 251.18.
-
56.01, 45.61 ppm. MS (ESI): m/z calcd for C27H24NO6 [MￚH]-
+
458.49, found 458.15; HRMS (ESI): m/z calcd for C27H26NO6
[M+H]+ 460.17546, found 460.17460; Chiral HPLC: (S)-5b: tR =
10.2 min, purity = 99.7%; (R)-5b: tR = 10.9 min, purity =
98.6%@ 220 nm, 25% ethanol in hexane.
4.1.6.3.
5-(3-hydroxy-4-methoxyphenyl)-2-(p-tolyl)-5-(3,4,5-
trimethoxyphenyl)-4,5-dihydrofuran-3-carbonitrile (5c)
This compound was obtained from reaction of 4 with 3-oxo-3-
(p-tolyl)propanenitrile after 1.5 h, followed by TBAF 0.5 h.
Eluent: petroleum ether/ ethyl acetate = 3:1. Yellow solid. Yield:
4.1.6. General procedure for the synthesis of compounds 5a-p
and 8a-c
1
52 mg, 94%; H NMR (500 MHz, CDCl3): δ = 7.96 (d, J = 8.3
A solution of manganese(III) acetate dehydrate (109 mg, 0.41
mmol or 156 mg, 0.58 mmol) in glacial acetic acid (2 mL) was
heated under argon atmosphere at 80 oC until it dissolved
completely. After the solution was cooled down to 70 oC, a
solution of 4 (50 mg, 0.12 mmol) or 7 (30 mg, 0.12 mmol) and
-diketones (0.18 mmol) in acetic acid were
Hz, 2H), 7.27 (d, J = 8.2 Hz, 2H), 6.99 (d, J = 2.3 Hz, 1H), 6.89
(dd, J = 8.5, 2.3 Hz, 1H), 6.81 (d, J = 8.5 Hz, 1H), 6.62 (s, 2H),
5.73 (s, 1H), 3.87 (d, J = 2.0 Hz, 3H), 3.83 (s, 3H), 3.81 (s, 6H),
3.72 (d, J = 14.7 Hz, 1H), 3.67 (d, J = 14.7 Hz, 1H), 2.41 ppm (s,
3H); 13C NMR (125 MHz, CDCl3): δ = 165.42, 153.17 (2C),
146.44, 145.61, 142.14, 139.66, 137.73, 136.96, 129.51 (2C),
127.11 (2C), 125.25, 117.59, 117.45, 112.60, 110.29, 103.22
o
added to this mixture, and the temperature rised to 80 C. The
reaction completed when the dark brown colour of the solution

(2C), 92.66, 78.25, 60.85, 56.26 (2C), 56.02, 45.61, 21.65 ppm;
117.45, 117.08, 112.53, 110.29, 103.22 (2C), 93.07, 79.78, 60.88,
56.29 (2C), 56.03, 45.61 ppm. MS (ESI): m/z calcd for
C27H24ClNNaO6+ [M+Na]+ 516.93, found 516.20.
+
MS (ESI): m/z calcd for C28H27NNaO6 [M+Na]+ 496.51, found
496.32.
4.1.6.4. 5-(3-hydroxy-4-methoxyphenyl)-2-(4-methoxyphenyl)-5-
(3,4,5-trimethoxyphenyl)-4,5-dihydrofuran-3-carbonitrile (5d)
4.1.6.8. 2-(3,4-dichlorophenyl)-5-(3-hydroxy-4-methoxyphenyl)-
5-(3,4,5-trimethoxyphenyl)-4,5-dihydrofuran-3-carbonitrile (5h)
This compound was obtained from reaction of 4 with 3-(4-
methoxyphenyl)-3-oxopropanenitrile after 10 min, followed by
TBAF 0.5 h. Eluent: petroleum ether/ethyl acetate = 3:1. Yellow
This compound was obtained from reaction of 4 with 3-(3,4-
dichlorophenyl)-3-oxopropanenitrile after 10 min, followed by
TBAF 0.5 h. Eluent: petroleum ether/ethyl acetate = 3:1. Yellow
1
1
solid. Yield: 39 mg, 68%; H NMR (500 MHz, CDCl3): δ = 8.04
solid. Yield: 33 mg, 53%; H NMR (500 MHz, CDCl3): δ = 8.07
(d, J = 8.9 Hz, 2H), 6.99 (d, J = 2.3 Hz, 1H), 6.97 (d, J = 8.9 Hz,
2H), 6.89 (dd, J = 8.5, 2.3 Hz, 1H), 6.81 (d, J = 8.5 Hz, 1H), 6.62
(s, 2H), 5.71 (s, 1H), 3.87 (s, 3H), 3.86 (s, 3H), 3.83 (s, 3H), 3.81
(s, 6H), 3.71 (d, J = 14.6 Hz, 1H), 3.66 ppm (d, J = 14.6 Hz, 1H);
13C NMR (125 MHz, CDCl3): δ = 165.16, 162.08, 153.16 (2C),
146.41, 145.59, 139.68, 137.72, 137.00, 128.98 (2C), 120.60,
117.91, 117.46, 114.19 (2C), 112.60, 110.27, 103.23 (2C), 92.58,
76.95, 60.86, 56.26 (2C), 56.02, 55.49, 45.55 ppm. MS (ESI):
m/z calcd for C28H27NNaO7+ [M+Na]+ 512.51, found 512.23.
(d, J = 2.0 Hz, 1H), 7.95 (dd, J = 8.5, 2.1 Hz, 1H), 7.55 (d, J =
8.5 Hz, 1H), 6.95 (d, J = 2.0 Hz, 1H), 6.85 – 6.81(m, 2H), 6.58
(s, 2H), 5.72 (s, 1H), 3.89 (s, 3H), 3.84 (s, 3H), 3.81 (s, 6H), 3.74
(d, J = 11.7 Hz, 1H), 3.70 ppm (d, J = 12.3 Hz, 1H); 13C NMR
(125 MHz, CDCl3): δ = 162.64, 153.25 (2C), 146.60, 145.68,
139.02, 137.95, 136.38, 135.82, 133.42, 130.97, 128.76, 127.74,
126.26, 117.50, 116.61, 112.52, 110.32, 103.23 (2C), 93.40,
81.02, 60.88, 56.31 (2C), 56.04, 45.62 ppm; MS (ESI): m/z calcd
for C27H24Cl2NO6+ [M+H]+ 529.39, found 528.13.
4.1.6.5.
2-(4-fluorophenyl)-5-(3-hydroxy-4-methoxyphenyl)-5-
4.1.6.9.
2-(4-bromophenyl)-5-(3-hydroxy-4-methoxyphenyl)-5-
(3,4,5-trimethoxyphenyl)-4,5-dihydrofuran-3-carbonitrile (5e)
(3,4,5-trimethoxyphenyl)-4,5-dihydrofuran-3-carbonitrile (5i)
This compound was obtained from reaction of 4 with 3-(4-
fluorophenyl)-3-oxopropanenitrile after 1 h, followed by TBAF
0.5 h. Eluent: petroleum ether/ethyl acetate = 4:1. Yellow solid.
Yield: 45 mg, 82%; 1H NMR (500 MHz, CDCl3): δ = 8.11 – 8.05
(m, 2H), 7.18 – 7.13 (m, 2H), 6.98 (d, J = 2.3 Hz, 1H), 6.87 (dd,
J = 8.5, 2.3 Hz, 1H), 6.82 (d, J = 8.5 Hz, 1H), 6.61 (s, 2H), 5.76
(s, 1H), 3.87 (s, 3H), 3.83 (s, 3H), 3.81 (s, 6H), 3.73 (d, J = 14.8
Hz, 1H), 3.68 ppm (d, J = 14.8 Hz, 1H). 13C NMR (125 MHz,
CDCl3): δ = 164.41, (JC-F = 254.1 Hz), 164.17, 153.22 (2C),
146.53, 145.67, 139.39, 137.89, 136.73, 129.50, 129.43, 124.30
(JC-F = 3.3 Hz), 117.44, 117.26, 116.16, 115.98, 112.56, 110.33,
103.30 (2C), 93.02, 78.98 (JC-F = 1.3 Hz), 60.84, 56.29, 56.01
This compound was obtained from reaction of 4 with 3-(4-
bromophenyl)-3-oxopropanenitrile after 10 min, followed by
TBAF 0.5 h. Eluent: petroleum ether/ethyl acetate = 3:1. Yellow
1
solid. Yield: 31 mg, 49%; H NMR (500 MHz, CDCl3): δ = 7.93
(d, J = 8.6 Hz, 2H), 7.61 (d, J = 8.6 Hz, 2H), 6.97 (d, J = 2.2 Hz,
1H), 6.86 (dd, J = 8.5, 2.3 Hz, 1H), 6.82 (d, J = 8.5 Hz, 1H), 6.59
(s, 2H), 5.69 (s, 1H), 3.88 (s, 3H), 3.84 (s, 3H), 3.81 (s, 6H), 3.73
(d, J = 15.0 Hz, 1H), 3.67 ppm (d, J = 14.9 Hz, 1H); 13C NMR
(125 MHz, CDCl3): δ = 164.09, 153.22 (2C), 146.52, 145.65,
139.29, 137.88, 136.64, 132.15 (2C), 128.55 (2C), 126.83,
126.11, 117.45, 117.05, 112.53, 110.29, 103.23 (2C), 93.08,
79.92, 60.88, 56.29 (2C), 56.04, 45.63 ppm; MS (ESI): m/z calcd
for C27H24BrNNaO6+ [M+Na]+ 561.38, found 560.15.
-
(2C), 45.53 ppm. MS (ESI): m/z calcd for C27H23FNO6 [M-H]-
476.48, found 476.30.
4.1.6.10.
5-(3-hydroxy-4-methoxyphenyl)-2-(thiophen-2-yl)-5-
4.1.6.6. 2-(2-chlorophenyl)-5-(3-hydroxy-4-methoxyphenyl)-5-
(3,4,5-trimethoxyphenyl)-4,5-dihydrofuran-3-carbonitrile (5f)
(3,4,5-trimethoxyphenyl)-4,5-dihydrofuran-3-carbonitrile (5j)
This compound was obtained from reaction of 4 with 3-oxo-3-
(thiophen-2-yl)propanenitrile after 1.5 h, followed by TBAF 0.5
h. Eluent: petroleum ether/ethyl acetate = 2:1. Yellow solid.
Yield: 41 mg, 76%; 1H NMR (500 MHz, CDCl3): δ = 7.93 (d, J =
3.7 Hz, 1H), 7.54 (d, J = 4.9 Hz, 1H), 7.17 (dd, J = 4.8, 4.0 Hz,
1H), 6.98 (d, J = 2.3 Hz, 1H), 6.88 (dd, J = 8.5, 2.3 Hz, 1H), 6.82
(d, J = 8.5 Hz, 1H), 6.63 (s, 2H), 5.65 (s, 1H), 3.89 (s, 3H), 3.83
(s, 3H), 3.82 (s, 6H), 3.73 (d, J = 14.7 Hz, 1H), 3.66 ppm (d, J =
14.7 Hz, 1H); 13C NMR (125 MHz, CDCl3): δ = 160.65, 153.18
(2C), 146.47, 145.59, 139.21, 137.77, 136.57, 130.20, 130.05,
129.75, 128.30, 117.56, 117.00, 112.60, 110.27, 103.12 (2C),
93.68, 77.66, 60.86, 56.25 (2C), 56.03, 45.38 ppm; MS (ESI):
m/z calcd for C25H24NO6S+ [M+H]+ 466.53, found 466.23.
This compound was obtained from reaction of 4 with 3-(2-
chlorophenyl)-3-oxopropanenitrile after 10 min, followed by
TBAF 0.5 h. Eluent: petroleum ether/ethyl acetate = 4:1. Yellow
1
solid. Yield: 26 mg, 45%; H NMR (500 MHz, CDCl3): δ = 7.55
(dd, J = 7.6, 1.4 Hz, 1H), 7.49 (dd, J = 8.0, 0.7 Hz, 1H), 7.42 (td,
J = 7.8, 1.5 Hz, 1H), 7.35 (td, J = 7.5, 1.1 Hz, 1H), 6.99 (d, J =
2.2 Hz, 1H), 6.90 (dd, J = 8.5, 2.2 Hz, 1H), 6.83 (d, J = 8.5 Hz,
1H), 6.64 (s, 2H), 5.68 (s, 1H), 3.89 (s, 3H), 3.85 (s, 3H), 3.82 (s,
6H), 3.78 (d, J = 14.9 Hz, 1H), 3.70 ppm (d, J = 14.9 Hz, 1H);
13C NMR (125 MHz, CDCl3): δ = 165.63, 153.17 (2C), 146.52,
145.59, 139.60, 137.69, 136.83, 133.16, 132.10, 130.96, 130.61,
127.71, 127.00, 117.70, 115.83, 112.69, 110.25, 103.29 (2C),
94.41, 84.75 , 60.89, 56.27 (2C), 56.03, 45.26 ppm. MS (ESI):
m/z calcd for C27H25ClNO6+ [M+H]+ 494.95, found 494.07.
4.1.6.11. 1-(5-(3-hydroxy-4-methoxyphenyl)-2-methyl-5-(3,4,5-
trimethoxyphenyl)-4,5-dihydrofuran-3-yl)ethan-1-one (5k)
4.1.6.7. 2-(4-chlorophenyl)-5-(3-hydroxy-4-methoxyphenyl)-5-
(3,4,5-trimethoxyphenyl)-4,5-dihydrofuran-3-carbonitrile (5g)
This compound was obtained from reaction of 4 with pentane-
2,4-dione after 1 h, followed by TBAF 0.5 h. Eluent: petroleum
1
This compound was obtained from reaction of 4 with 3-(4-
chlorophenyl)-3-oxopropanenitrile after 10 min, followed by
TBAF 0.5 h. Eluent: petroleum ether/ethyl acetate = 3:1. Yellow
ether/acetone = 2:1. Yellow solid. Yield: 30 mg, 63%; H NMR
(500 MHz, CDCl3): δ = 6.90 (d, J = 1.9 Hz, 1H), 6.77 (dd, J =
8.4, 2.0 Hz, 1H), 6.73 (d, J = 8.5 Hz, 1H), 6.56 (s, 2H), 6.27 (s,
1H), 3.77 (s, 6H), 3.77 (s, 6H), 3.58 (d, J = 14.5 Hz, 1H), 3.53 (d,
J = 14.4 Hz, 1H), 2.32 (s, 3H), 2.10 ppm (s, 3H); 13C NMR (125
MHz, CDCl3): δ = 194.35, 165.96, 152.99 (2C), 146.36, 145.62,
140.69, 138.01, 137.26, 117.18, 112.52, 112.45, 110.37, 103.08
(2C), 91.48, 60.77, 56.17 (2C), 55.91, 44.75, 29.32, 15.27 ppm;
1
solid. Yield: 36 mg, 62%; H NMR (500 MHz, CDCl3): δ = 8.01
(d, J = 8.6 Hz, 2H), 7.45 (d, J = 8.7 Hz, 2H), 6.97 (d, J = 2.2 Hz,
1H), 6.87 (dd, J = 8.5, 2.2 Hz, 1H), 6.82 (d, J = 8.5 Hz, 1H), 6.59
(s, 2H), 3.88 (s, 3H), 3.84 (s, 3H), 3.81 (s, 6H), 3.74 (d, J = 14.9
Hz, 1H), 3.68 ppm (d, J = 15.0 Hz, 1H); 13C NMR (125 MHz,
CDCl3): δ = 164.03, 153.22 (2C), 146.51, 145.64, 139.30,
137.86, 137.65, 136.65, 129.18 (2C), 128.42 (2C), 126.40,
-
MS (ESI): m/z calcd for C23H25O7 [M-H]- 413.45, found 413.50.

4.1.6.12.
2,2,2-trifluoro-1-(5-(3-hydroxy-4-methoxyphenyl)-2-
(2C), 128.50 (2C), 117.36, 115.06, 112.45, 110.25, 103.14 (2C),
91.60, 60.84, 56.24 (2C), 56.00, 45.29, 28.80 ppm; MS (ESI):
m/z calcd for C28H28NaO7+ [M+Na]+ 499.51, found 499.10.
methyl-5-(3,4,5-trimethoxyphenyl)-4,5-dihydrofuran-3-yl)ethan-
1-one (5l)
This compound was obtained from reaction of 4 with 1,1,1-
trifluoropentane-2,4-dione after 1 h, followed by TBAF 0.5 h.
Eluent: petroleum ether/ethyl acetate = 4:1. Yellow solid. Yield:
4.1.6.16.
(5-(3-hydroxy-4-methoxyphenyl)-2-phenyl-5-(3,4,5-
trimethoxyphenyl)-4,5-dihydrofuran-3-yl)(phenyl)methanone (5p)
This compound was obtained from reaction of 4 with 1,3-
diphenylpropane-1,3-dione after 1.5 h, followed by TBAF 0.5 h.
Eluent: petroleum ether/ ethyl acetate = 2:1. Yellow solid. Yield:
1
8 mg, 15%; H NMR (500 MHz, CDCl3): δ = 6.91 (s, 1H), 6.81
(s, 2H), 6.56 (s, 2H), 5.71 (s, 1H), 3.88 (s, 3H), 3.84 (s, 3H), 3.82
(s, 6H), 3.74 (d, J = 14.4 Hz, 1H), 3.68 (d, J = 14.2 Hz, 1H), 2.46
ppm (s, 3H); 13C NMR (125 MHz, CDCl3): δ = 176.17, 175.89,
153.20 (2C), 146.46, 145.62, 139.40, 137.80, 136.93, 117.37,
116.71 (JC-F = 291.7), 112.42, 110.28 , 105.69, 103.20 (2C),
94.51, 60.85, 56.27 (2C), 56.00, 42.16 (JC-F = 2.8 Hz), 15.98
1
23 mg, 37%; H NMR (500 MHz, CDCl3): δ = 7.38 (d, J = 7.3
Hz, 2H), 7.30 (d, J = 7.3 Hz, 2H), 7.20 (t, J = 7.4 Hz, 2H), 7.12 –
6.99 (m, 6H), 6.85 (d, J = 8.5 Hz, 1H), 6.74 (s, 2H), 5.68 (s, 1H),
3.95 – 3.86 (m, 5H), 3.84 ppm (s, 9H); 13C NMR (125 MHz,
CDCl3): δ = 193.42, 164.24, 153.11 (2C), 146.14, 145.52,
140.53, 138.93, 138.01, 137.37, 131.23, 130.19, 129.96, 129.53
(2C), 128.96 (2C), 127.74 (2C), 127.69 (2C), 117.45, 112.61,
112.18, 110.27, 103.22 (2C), 91.46, 60.88, 56.25 (2C), 56.01,
-
ppm; MS (ESI): m/z calcd for C23H22F3O7 [M-H]- 467.42, found
467.10.
4.1.6.13.
1-(2-ethyl-5-(3-hydroxy-4-methoxyphenyl)-5-(3,4,5-
+
trimethoxyphenyl)-4,5-dihydrofuran-3-yl)propan-1-one (5m)
46.47 ppm; MS (ESI): m/z calcd for C33H30NaO7 [M+Na]+
561.59, found 561.32.
This compound was obtained from reaction of 4 with heptane-
3,5-dione after 20 min, followed by TBAF 0.5 h. Eluent:
petroleum ether/ethyl acetate = 4:1. Yellow solid. Yield: 44 mg,
86%; 1H NMR (500 MHz, CDCl3): δ = 6.96 (d, J = 2.2 Hz, 1H),
6.84 (dd, J = 8.4, 2.2 Hz, 1H), 6.79 (d, J = 8.5 Hz, 1H), 6.61 (s,
2H), 5.64 (s, 1H), 3.87 (s, 3H), 3.83 (s, 3H), 3.82 (s, 6H), 3.64 (d,
J = 14.3 Hz, 1H), 3.56 (d, J = 14.3 Hz, 1H), 2.92-2.83 (m, 1H),
2.80 – 2.71 (m, 1H), 2.48 (q, J = 7.3 Hz, 2H), 1.26 (t, J = 7.5 Hz,
3H), 1.08 ppm (t, J = 7.3 Hz, 3H); 13C NMR (125 MHz, CDCl3):
δ = 197.14, 169.96, 153.03 (2C), 146.07, 145.42, 141.03, 138.38,
137.24, 117.44, 112.49, 110.14, 110.06, 102.98 (2C), 91.00,
60.86, 56.19 (2C), 56.00, 45.11, 34.64, 22.14, 11.32, 7.95 ppm;.
4.1.6.17. 2-(tert-butyl)-5-butyl-5-(3,4,5-trimethoxyphenyl)-4,5-
dihydrofuran-3-carbonitrile (8a)
This compound was obtained from reaction between 7 and
4,4-dimethyl-3-oxopentanenitrile after 30 min. Eluent: petroleum
ether/ ethyl acetate = 8:1. Yellow liquid. Yield: 12 mg, 47%; H
NMR (500 MHz, CDCl3): δ = 6.47 (s, 2H), 3.85 (s, 6H), 3.84 (s,
3H), 3.05 (s, 2H), 1.94 – 1.81 (m, 2H), 1.36 (s, 9H), 1.34 – 1.19
(m, 4H), 0.86 ppm (t, J = 7.2 Hz, 3H); 13C NMR (125 MHz,
CDCl3): δ = 177.49, 153.24 (2C), 140.90 , 137.12 , 117.33,
101.58 (2C), 91.41, 77.31, 60.87, 56.16 (2C), 44.68, 42.19,
34.92, 28.13 (3C), 25.61, 22.68, 13.95 ppm; MS (ESI): m/z calcd
for C22H31NNaO4+ [M+Na]+ 396.48, found 396.20.
1
+
MS (ESI): m/z calcd for C25H30NaO7 [M+Na]+ 465.50, found
465.27.
4.1.6.18. 5-butyl-2-(4-chlorophenyl)-5-(3,4,5-trimethoxyphenyl)-
4,5-dihydrofuran-3-carbonitrile (8b)
4.1.6.14.
2-(3-hydroxy-4-methoxyphenyl)-2-(3,4,5-
trimethoxyphenyl)-3,5,6,7-tetrahydrobenzofuran-4(2H)-one (5n)
This compound was obtained from reaction between 7 and 3-
(4-chlorophenyl)-3-oxopropanenitrile after 30 min. Eluent:
petroleum ether/ethyl acetate = 8:1. White viscous liquid. Yield:
This compound was obtained from reaction of 4 with
cyclohexane-1,3-dione after 20 min, followed by TBAF 0.5 h.
Eluent: petroleum ether/ ethyl acetate = 4:1. Yellow solid. (R)-5n
and (R)-5n were prepared by chical HPLC performed on Waters
515-2996 with columns of S-Chiral A. (R)-5n: yield: 9 mg, 18%;
mp: 62– 63 oC, [α]D30 =-56.36o (c 0.036 in CHCl3); (S)-5n: yield:
10 mg, 20%; mp: 62– 63 oC, [α]D30=+65.66o (c 0.036 in CHCl3);
1H NMR (500 MHz, CDCl3): δ = 6.83 (dd, J = 8.5, 2.3 Hz, 1H),
6.75 (dd, J = 6.6, 5.6 Hz, 2H), 6.54 (s, 2H), 3.79 (s, 3H), 3.77 (s,
6H), 3.74 (s, 3H), 3.47 (s, 2H), 2.54 (t, J = 5.9 Hz, 2H), 2.34 (t, J
= 6.4 Hz, 2H), 2.08 – 2.02 (m, 2H), 0.91 (s, 9H), 0.07 ppm (s,
6H); 13C NMR (125 MHz, CDCl3): δ = 195.70, 175.88, 153.02
(2C), 146.47, 145.65, 140.11, 137.68, 137.43, 117.31, 112.90,
112.60, 110.37, 103.24 (2C), 95.48, 60.80, 56.19 (2C), 55.95,
40.56, 36.32, 24.04, 21.72 ppm. HRMS (ESI): m/z calcd for
1
11 mg, 39%; H NMR (500 MHz, CDCl3): δ = 7.98 (d, J = 8.6
Hz, 2H), 7.45 (d, J = 8.6 Hz, 2H), 6.53 (s, 2H), 3.85 (s, 6H), 3.84
(s, 3H), 3.26 (s, 2H), 2.08 – 1.94 (m, 2H), 1.44 – 1.13 (m, 4H),
0.86 ppm (t, J = 7.3 Hz, 3H); 13C NMR (125 MHz, CDCl3): δ =
164.34, 153.35 (2C), 140.01, 137.50, 137.39, 129.17 (2C),
128.33 (2C), 126.62, 117.36, 101.70 (2C), 92.71, 79.60, 60.90,
56.27 (2C), 44.70, 42.23, 25.81, 22.72, 13.98 ppm; MS (ESI):
m/z calcd for C24H26ClNNaO4+ [M+Na]+ 450.91, found 450.10.
4.1.6.19.
(5-butyl-2-phenyl-5-(3,4,5-trimethoxyphenyl)-4,5-
dihydrofuran-3-yl)(phenyl)methanone (8c)
This compound was obtained from reaction between 7 and
+
C24H27O7 [M+H]+ 427.17513, found 427.17480. Chiral HPLC:
1,3-diphenylpropane-1,3-dione after 30 min. Eluent: petroleum
1
ether/ethyl acetate = 8:1. Yellow solid. Yield: 10 mg, 31%; H
(S)-5n: tR = 9.65 min, purity = 98.2%; (R)-5n: tR = 12.8 min,
purity = 95.1%@ 220 nm, 50% isopropanol in hexane.
NMR (500 MHz, CDCl3): δ = 7.39 (s, 1H), 7.37 (s, 1H), 7.27 (d,
J = 7.1 Hz, 2H), 7.20 (t, J = 7.4 Hz, 2H), , 7.11 – 7.04 (m, 4H),
6.69 (s, 2H), 3.89 (s, 6H), 3.87 (s, 3H), 3.55 (d, J = 14.9 Hz, 1H),
3.51 (d, J = 14.9 Hz, 1H), 2.16 – 1.99 (m, 2H), 1.50 – 1.26 (m,
4H), 0.90 ppm (t, J = 7.1 Hz, 3H); 13C NMR (125 MHz, CDCl3):
δ = 193.58, 164.91, 153.18 (2C), 141.15, 139.12, 137.03, 131.08,
130.34, 130.05, 129.37 (2C), 128.87 (2C), 127.74 (2C), 127.65
(2C), 112.13, 102.10 (2C), 91.24, 60.92, 56.27 (2C), 45.24,
42.69, 26.09, 22.87, 14.06 ppm; MS (ESI): m/z calcd for
C30H32NaO5+ [M+Na]+ 495.57, found 495.35.
4.1.6.15.
(5-(3-hydroxy-4-methoxyphenyl)-2-methyl-5-(3,4,5-
trimethoxyphenyl)-4,5-dihydrofuran-3-yl)(phenyl)methanone (5o)
This compound was obtained from reaction of 4 with 1-
phenylbutane-1,3-dione after 1.5 h, followed by TBAF 0.5 h.
Eluent: petroleum ether/ ethyl acetate = 2:1. Yellow solid. Yield:
22 mg, 40%; 1H NMR (500 MHz, CDCl3): δ = 7.61 (dd, J = 8.0,
1.5 Hz, 2H), 7.52 – 7.45 (m, 3H), 7.04 (d, J = 2.3 Hz, 1H), 6.91
(dd, J = 8.4, 2.3 Hz, 1H), 6.81 (d, J = 8.5 Hz, 1H), 6.68 (s, 2H),
5.64 (s, 1H), 3.88 (s, 3H), 3.84-3.78 (m, 11H), 1.93 ppm (s, 3H);
13C NMR (125 MHz, CDCl3): δ = 194.47, 164.42, 153.11 (2C),
146.12, 145.52, 140.69, 138.27, 137.40, 130.98, 130.69, 129.29
4.1.7. General procedure for the synthesis of the residue of 1,3-
dipole congeners.

A stirred solution of aldehyde (3.6 mmol) in diethyl ether (3
mL) added 50% hydroxylamine in H2O (440 µL, 7.2 mmol) in
one portion. After 10 min, the reaction mixture dried over
MgSO4, filtered, and the ether was removed under reduced
pressure to give the residue of oximes. Subsequently, the residue
was dissolved in DMF (3 mL), and N-chlorosuccinimide (NCS,
529 mg, 3.96 mmol) was added in one portion. The resulting
solution was stirred for 0.5 h at room temperature. Then, the
solution was diluted with water (30 mL) and extracted with ethyl
acetate (3×30 mL). The organic layers were combined, washed
with water (2×30 mL) and brine (2×30 mL), dried over
anhydrous Na2SO4, filtered and evaporated under reduced
pressure to give the residue of 1, 3-dipole.
This compound was obtained from reaction of 4 with the
residue of N-hydroxy-4-methoxybenzimidoyl chloride after 1 h,
followed by TBAF 0.5 h. Eluent: petroleum ether/ethyl acetate =
5:1 to 2:1. Yellow solid. Yield: 34 mg, 62%; 1H NMR (500 MHz,
CDCl3): δ = 7.62 (d, J = 8.9 Hz, 2H), 7.01 (d, J = 2.3 Hz, 1H),
6.95 (dd, J = 8.4, 2.3 Hz, 1H), 6.90 (d, J = 8.9 Hz, 2H), 6.80 (d, J
= 8.5 Hz, 1H), 6.70 (s, 2H), 3.94 (d, J = 16.4 Hz, 1H), 3.87 –
3.84 (m, 4H), 3.83 (s, 6H), 3.83 (s, 3H), 3.82 ppm (s, 3H); 13C
NMR (125 MHz, CDCl3): δ = 161.16, 155.93, 153.08 (2C),
146.13, 145.45, 140.05, 137.40, 137.33, 128.25 (2C), 122.14,
117.92, 114.17 (2C), 112.90, 110.30, 103.62 (2C), 91.42, 60.82,
56.28 (2C), 56.00, 55.39, 48.63 ppm; MS (ESI): m/z calcd for
-
C26H26NO7 [M-H]- 464.49, found 464.25.
4.1.8. General procedure for the synthesis of compounds 6a-n
and 9a-c
4.1.8.4.
2-methoxy-5-(3-(2-nitrophenyl)-5-(3,4,5-
trimethoxyphenyl)-4,5-dihydroisoxazol-5-yl)phenol (6d)
A mixture of 4 (50 mg, 0.12 mmol) or 7 (30 mg, 0.12 mmol)
and the residue of 1,3-dipole congeners (about 0.7 mmol) in
CH2Cl2 (2 mL) was cooled to 0 °C, followed by triethylamine
(Et3N, 114 µL, 0.82 mmol). After the mixture was stirred at 25
°C for 1 h, water (10 mL) was added. The organic phase was
washed with water and brine, then, evaporated under reduced
pressure.
This compound was obtained from reaction of 4 with the
residue of N-hydroxy-2-nitrobenzimidoyl chloride after 1 h,
followed by TBAF 0.5 h. Eluent: petroleum ether/ethyl acetate =
1
3:1. Yellow solid. Yield: 12 mg, 22%; H NMR (500 MHz,
CDCl3): δ = 8.00 (d, J = 8.1 Hz, 1H), 7.64 (t, J = 7.5 Hz, 1H),
7.58 (t, J = 7.6 Hz, 2H), 6.98 (dd, J = 11.5, 3.0 Hz, 2H), 6.85 (d,
J = 8.3 Hz, 1H), 6.66 (s, 2H), 5.67 (s, 1H), 3.89 (s, 3H), 3.86 –
3.81 ppm (m, 11H); 13C NMR (125 MHz, CDCl3): δ = 154.83,
153.10 (2C), 148.05, 146.26, 145.45, 139.47, 137.47, 136.57,
133.35, 131.25, 130.69, 125.41, 124.74, 118.06, 112.97, 110.34,
103.63 (2C), 92.91, 60.87, 56.26 (2C), 56.01, 50.02 ppm; MS
(ESI): m/z calcd for C25H25N2O8+ [M+H]+ 481.48, found 481.15.
The residues from reactions of 4 and 1,3-dipole congeners
were dissolved in THF (2 mL), followed by 1.0 mol/L TBAF in
THF (175 µL, 0.175 mmol) for 0.5 h. Water (10 mL) was added
to this solution and extracted with ethyl acetate (3×10 mL). The
combined organic phases were dried over anhydrous Na2SO4,
filtered and evaporated under reduced pressure. Crude products
were purified by column chromatography on silica gel using
petroleum ether/ethyl acetate as eluent.
4.1.8.5.
2-methoxy-5-(3-(4-nitrophenyl)-5-(3,4,5-
trimethoxyphenyl)-4,5-dihydroisoxazol-5-yl)phenol (6e)
This compound was obtained from reaction of 4 with the
residue of N-hydroxy-4-nitrobenzimidoyl chloride after 1 h,
followed by TBAF 0.5 h. Eluent: petroleum ether/ethyl acetate =
4.1.8.1. 2-methoxy-5-(3-phenyl-5-(3,4,5-trimethoxyphenyl)-4,5-
dihydroisoxazol-5-yl)phenol (6a)
1
5:1-2:1. Yellow solid. Yield: 33 mg, 60%; H NMR (500 MHz,
This compound was obtained from reaction of 4 with the
residue of N-hydroxybenzimidoyl chloride after 1 h, followed by
TBAF 0.5 h. Eluent: petroleum ether/ethyl acetate = 3:1. White
CDCl3): δ = 8.25 (d, J = 8.9 Hz, 2H), 7.85 (d, J = 8.9 Hz, 2H),
7.00 (d, J = 2.3 Hz, 1H), 6.92 (dd, J = 8.4, 2.3 Hz, 1H), 6.82 (d, J
= 8.5 Hz, 1H), 6.68 (s, 2H), 5.64 (s, 1H), 3.99 (d, J = 16.5 Hz,
1H), 3.91 (d, J = 16.6 Hz, 1H), 3.88 (s, 3H), 3.84 (s, 6H), 3.83
ppm (s, 3H); 13C NMR (125 MHz, CDCl3): δ = 154.76, 153.24
(2C), 148.54, 146.38, 145.61, 139.23, 137.71, 136.55, 135.68,
127.40 (2C), 124.03 (2C), 117.85, 112.73, 110.35, 103.53 (2C),
93.13, 60.85, 56.33 (2C), 56.03, 47.83 ppm; MS (ESI): m/z calcd
1
solid. Yield: 25 mg, 50%; H NMR (500 MHz, CDCl3): δ = 7.69
(dd, J = 6.6, 3.0 Hz, 2H), 7.40 (d, J = 2.6 Hz, 2H), 7.39 (s, 1H),
7.01 (d, J = 2.3 Hz, 1H), 6.95 (dd, J = 8.4, 2.3 Hz, 1H), 6.81 (d, J
= 8.5 Hz, 1H), 6.70 (s, 2H), 3.97 (d, J = 16.5 Hz, 1H), 3.89 (d, J
= 16.5 Hz, 1H), 3.87 (s, 3H), 3.83 (s, 6H), 3.82 ppm (s, 3H); 13C
NMR (125 MHz, CDCl3): δ = 156.37, 153.09 (2C), 146.18,
145.47, 139.93, 137.34, 137.13, 130.22, 129.56, 128.76 (2C),
126.72 (2C), 117.92, 112.88, 110.30, 103.50 (2C), 91.78, 60.85,
56.26 (2C), 56.01, 48.41 ppm; MS (ESI): m/z calcd for
C25H26NO6+ [M+H]+ 436.48, found 436.26.
-
for C25H23N2O8 [M-H]- 479.47, found 479.25.
4.1.8.6.
5-(3-(2-chlorophenyl)-5-(3,4,5-trimethoxyphenyl)-4,5-
dihydroisoxazol-5-yl)-2-methoxyphenol (6f)
This compound was obtained from reaction of 4 with the
residue of 2-chloro-N-hydroxybenzimidoyl chloride after 1 h,
followed by TBAF 0.5 h. Eluent: petroleum ether/ethyl acetate =
4.1.8.2. 2-methoxy-5-(3-(p-tolyl)-5-(3,4,5-trimethoxyphenyl)-4,5-
dihydroisoxazol-5-yl)phenol (6b)
1
4:1. Yellow solid. Yield: 14 mg, 26%; H NMR (500 MHz,
This compound was obtained from reaction of 4 with the
residue of N-hydroxy-4-methylbenzimidoyl chloride after 1 h,
followed by TBAF 0.5 h. Eluent: petroleum ether/ethyl acetate =
CDCl3): δ = 7.64 (dd, J = 7.7, 1.5 Hz, 1H), 7.40 (dd, J = 7.9, 0.9
Hz, 1H), 7.35 – 7.27 (m, 2H), 7.03 (d, J = 2.2 Hz, 1H), 6.97 (dd,
J = 8.4, 2.2 Hz, 1H), 6.82 (d, J = 8.5 Hz, 1H), 6.69 (s, 2H), 5.68
(s, 1H), 4.13 (d, J = 16.9 Hz, 1H), 4.04 (d, J = 16.9 Hz, 1H), 3.88
(s, 3H), 3.84 (s, 6H), 3.83 ppm (s, 3H); 13C NMR (125 MHz,
CDCl3): δ = 156.51, 153.08 (2C), 146.20, 145.46, 139.69,
137.37, 136.77, 132.80, 130.98, 130.68, 130.57, 129.06, 127.04,
117.97, 112.92, 110.30, 103.49 (2C), 92.47, 60.85, 56.26 (2C),
56.00, 50.41 ppm; MS (ESI): m/z calcd for C25H25ClNO6+
[M+H]+ 470.93, found 470.21.
1
5:1-3:1. Reddish brown solid. Yield: 44 mg, 85%; H NMR (500
MHz, CDCl3): δ = 7.57 (d, J = 8.2 Hz, 2H), 7.19 (d, J = 8.1 Hz,
2H), 7.01 (d, J = 2.3 Hz, 1H), 6.95 (dd, J = 8.4, 2.3 Hz, 1H), 6.80
(d, J = 8.5 Hz, 1H), 6.70 (s, 2H), 5.66 (s, 1H), 3.95 (d, J = 16.6
Hz, 1H), 3.88 – 3.86 (m, 4H), 3.83 (s, 6H), 3.82 (s, 3H), 2.36
ppm (s, 3H); 13C NMR (125 MHz, CDCl3): δ = 156.32, 153.08
(2C), 146.15, 145.47, 140.46, 140.03, 137.40, 137.28, 129.43
(2C), 126.76, 126.66 (2C) 117.92, 112.91, 110.31, 103.61 (2C),
91.56, 60.82, 56.27 (2C), 56.00, 48.51, 21.45 ppm; MS (ESI):
m/z calcd for C26H28NO6+ [M+H]+ 450.51, found 450.23.
4.1.8.7.
5-(3-(4-chlorophenyl)-5-(3,4,5-trimethoxyphenyl)-4,5-
dihydroisoxazol-5-yl)-2-methoxyphenol (6g)
4.1.8.3.
2-methoxy-5-(3-(4-methoxyphenyl)-5-(3,4,5-
This compound was obtained from reaction of 4 with the
residue of 4-chloro-N-hydroxybenzimidoyl chloride after 1 h,
trimethoxyphenyl)-4,5-dihydroisoxazol-5-yl)phenol (6c)

followed by TBAF 0.5 h. Eluent: petroleum ether/ethyl acetate =
4:1. Yellow solid. Yield: 35 mg, 65%; H NMR (500 MHz,
This compound was obtained from reaction of 4 with the
residue of N,3-dihydroxy-4-methoxybenzimidoyl chloride after 1
h, followed by TBAF 0.5 h. Yellow solid. Eluent: petroleum
1
CDCl3): δ = 7.62 (d, J = 8.6 Hz, 2H), 7.36 (d, J = 8.6 Hz, 2H),
7.00 (d, J = 2.3 Hz, 1H), 6.93 (dd, J = 8.4, 2.3 Hz, 1H), 6.81 (d, J
= 8.5 Hz, 1H), 6.68 (s, 2H), 5.64 (s, 1H), 3.93 – 3.83 (m, 11H),
3.82 ppm (s, 3H); 13C NMR (125 MHz, CDCl3): δ = 155.41,
153.14 (2C), 146.23, 145.51, 139.70, 137.50, 136.99, 136.16,
129.02 (2C), 128.10, 127.93 (2C), 117.87, 112.81, 110.31,
103.53 (2C), 92.11, 60.83, 56.29 (2C), 56.01, 48.24 ppm; MS
1
ether/ethyl acetate = 3:1. Yield: 29 mg, 52%; H NMR (500
MHz, CDCl3): δ = 7.28 (d, J = 1.9 Hz, 1H), 7.18 (dd, J = 8.4, 1.9
Hz, 1H), 7.00 (d, J = 2.1 Hz, 1H), 6.94 (dd, J = 8.4, 2.2 Hz, 1H),
6.84 (d, J = 8.4 Hz, 1H), 6.79 (d, J = 8.5 Hz, 1H), 6.69 (s, 2H),
5.73 (s, 1H), 5.67 (s, 1H), 3.93 – 3.90 (m, 4H), 3.86 – 3.83 (m,
10H), 3.81 ppm (s, 3H); 13C NMR (125 MHz, CDCl3): δ =
156.02, 153.05 (2C), 148.26, 146.13, 145.72, 145.42, 140.04,
137.28, 137.24, 122.92, 119.28, 117.91, 112.89, 112.84, 110.51,
110.29, 103.49 (2C), 91.51, 60.83, 56.25 (2C), 56.03, 56.00,
48.57 ppm; MS (ESI): m/z calcd for C26H28NO8+ [M+H]+ 482.51,
found 482.26.
-
(ESI): m/z calcd for C25H23ClNO6 [M-H]- 468.91, found 468.24.
4.1.8.8.
5-(3-(4-chloro-2-methoxyphenyl)-5-(3,4,5-
trimethoxyphenyl)-4,5-dihydroisoxazol-5-yl)-2-methoxyphenol
(6h)
This compound was obtained from reaction of 4 with the
residue of 4-chloro-N-hydroxy-2-methoxybenzimidoyl chloride
after 1 h, followed by TBAF 0.5 h. Eluent: petroleum ether/ethyl
4.1.8.12.
4-(5-(3-hydroxy-4-methoxyphenyl)-5-(3,4,5-
trimethoxyphenyl)-4,5-dihydroisoxazol-3-yl)-2-methoxyphenol
(6l)
1
acetate = 3:1. Yellow solid. Yield: 30 mg, 52%; H NMR (500
MHz, CDCl3): δ = 7.74 (d, J = 2.6 Hz, 1H), 7.30 (dd, J = 8.9, 2.7
Hz, 1H), 7.02 (d, J = 2.2 Hz, 1H), 6.95 (dd, J = 8.4, 2.3 Hz, 1H),
6.85 (d, J = 8.9 Hz, 1H), 6.81 (d, J = 8.5 Hz, 1H), 6.68 (s, 2H),
5.61 (s, 1H), 4.05 (d, J = 17.3 Hz, 1H), 3.93 (d, J = 17.3 Hz,
1H), 3.88 (s, 3H), 3.84 (s, 9H), 3.83 ppm (s, 3H); 13C NMR (125
MHz, CDCl3): δ = 156.07, 155.04, 153.05 (2C), 146.10, 145.38,
139.97, 137.37, 137.15, 130.89, 128.99, 126.03, 120.08, 118.08,
112.94, 112.70, 110.26, 103.62 (2C), 91.86, 60.84, 56.29 (2C),
56.00, 55.95, 50.44 ppm; MS (ESI): m/z calcd for C26H27ClNO7+
[M+H]+ 500.95, found 500.21.
This compound was obtained from reaction of 4 with the
residue of N,4-dihydroxy-3-methoxybenzimidoyl chloride after 1
h, followed by TBAF 0.5 h. Eluent: petroleum ether/ethyl acetate
1
= 3:1. Yellow solid. Yield: 22 mg, 40%; H NMR (500 MHz,
CDCl3): δ = 7.42 (d, J = 1.7 Hz, 1H), 7.01 (d, J = 2.3 Hz, 1H),
6.99 (dd, J = 8.2, 1.9 Hz, 1H), 6.94 (dd, J = 8.4, 2.3 Hz, 1H),
6.89 (d, J = 8.2 Hz, 1H), 6.80 (d, J = 8.5 Hz, 1H), 6.69 (s, 2H),
5.71 (s, 2H), 3.95 – 3.91 (m, 4H), 3.88 – 3.83 (m, 10H), 3.82
ppm (s, 3H); 13C NMR (125 MHz, CDCl3): δ = 156.26, 153.07
(2C), 147.81, 146.84, 146.14, 145.44, 139.97, 137.34, 137.25,
121.82, 121.03, 117.92, 114.10, 112.90, 110.27, 108.25, 103.55
(2C), 91.53, 60.84, 56.26 (2C), 56.07, 56.00, 48.59 ppm; MS
(ESI): m/z calcd for C26H28NO8+ [M+H]+ 482.51, found 482.23.
4.1.8.9.
5-(3-(2-chloro-3,4,5-trimethoxyphenyl)-5-(3,4,5-
trimethoxyphenyl)-4,5-dihydroisoxazol-5-yl)-2-methoxyphenol
(6i)
4.1.8.13.
4-(5-(3-hydroxy-4-methoxyphenyl)-5-(3,4,5-
This compound was obtained from reaction of 4 with the
residue of 2-chloro-N-hydroxy-3,4,5-trimethoxybenzimidoyl
chloride after 1 h, followed by TBAF 0.5 h. Eluent: petroleum
trimethoxyphenyl)-4,5-dihydroisoxazol-3-yl)benzene-1,2-diol
(6m)
1
ether/ethyl acetate = 3:1. Yellow solid. Yield: 12 mg, 18%; H
This compound was obtained from reaction of 4 with the
residue of N,3,4-trihydroxybenzimidoyl chloride after 1 h,
followed by TBAF 0.5 h. Eluent: petroleum ether/ethyl acetate =
NMR (500 MHz, CDCl3): δ = 7.03 (s, 1H), 7.03 (d, J = 2.3 Hz,
1H), 6.96 (dd, J = 8.4, 2.1 Hz, 1H), 6.83 (d, J = 8.4 Hz, 1H), 6.69
(s, 2H), 5.62 (s, 1H), 4.14 (d, J = 17.0 Hz, 1H), 4.07 (d, J = 17.0
Hz, 1H), 3.91 (s, 3H), 3.89 (s, 3H), 3.89 (s, 3H), 3.86 (s, 3H),
3.84 ppm (s, 9H); 13C NMR (125 MHz, CDCl3): δ = 156.64,
153.09 (2C), 152.17, 150.39, 146.19, 145.46, 144.82, 139.66,
136.90, 129.51, 124.32, 119.66, 117.99, 112.92, 110.29, 108.62,
103.63 (2C), 92.58, 61.20 (2C), 60.84, 56.29 (3C), 56.01, 50.46
1
3:1. Yellow solid. Yield: 20 mg, 36%; H NMR (500 MHz,
CDCl3): δ = 7.39 (d, J = 1.9 Hz, 1H), 7.03 – 6.99 (m, 2H), 6.92
(dd, J = 8.4, 2.3 Hz, 1H), 6.86 (d, J = 8.3 Hz, 1H), 6.79 (d, J =
8.5 Hz, 1H), 6.66 (s, 2H), 5.75 (s, 1H), 3.92 (d, J = 16.5 Hz, 1H),
3.86 – 3.84 (m, 4H), 3.82 (s, 3H), 3.80 ppm (s, 6H); 13C NMR
(125 MHz, CDCl3): δ = 156.68, 153.05 (2C), 146.64, 146.23,
145.41, 143.91, 139.78, 137.30, 136.87, 121.79, 120.56, 117.98 ,
115.16, 113.30, 112.91, 110.35, 103.55 (2C), 91.61, 60.87, 56.26
+
ppm; MS (ESI): m/z calcd for C28H31ClNO9 [M+H]+ 561.00,
found 560.26.
+
(2C), 56.00, 48.59 ppm; MS (ESI): m/z calcd for C25H26NO8
4.1.8.10. 5-(3-(4-hydroxyphenyl)-5-(3,4,5-trimethoxyphenyl)-4,5-
dihydroisoxazol-5-yl)-2-methoxyphenol (6j)
[M+H]+ 468.48, found 468.24.
4.1.8.14.
2-methoxy-5-(3-(pyridin-2-yl)-5-(3,4,5-
This compound was obtained from reaction of 4 with the
residue of N,4-dihydroxybenzimidoyl chloride after 1 h, followed
by TBAF 0.5 h. Eluent: petroleum ether/ethyl acetate = 3:1.
trimethoxyphenyl)-4,5-dihydroisoxazol-5-yl)phenol (6n)
This compound was obtained from reaction of 4 with the
residue of N-hydroxypicolinimidoyl chloride after 1 h, followed
by TBAF 0.5 h. Eluent: petroleum ether/ethyl acetate = 3:1.
1
Reddish brown solid. Yield: 19 mg, 35%; H NMR (500 MHz,
CDCl3): δ = 7.55 (d, J = 8.7 Hz, 2H), 7.00 (d, J = 2.3 Hz, 1H),
6.94 (dd, J = 8.4, 2.3 Hz, 1H), 6.84 (d, J = 8.7 Hz, 2H), 6.80 (d, J
= 8.5 Hz, 1H), 6.69 (s, 2H), 6.05 (s, 1H), 5.66 (s, 1H), 3.93 (d, J
= 16.4 Hz, 1H), 3.87 – 3.83 (m, 4H), 3.82 ppm (s, 9H); 13C NMR
(125 MHz, CDCl3): δ = 157.79, 156.15, 153.05 (2C), 146.16,
145.43, 140.03, 137.29, 137.28, 128.49 (2C), 121.87, 117.95,
115.77 (2C), 112.89, 110.32, 103.57 (2C), 91.42, 60.85, 56.26
1
White solid. Yield: 29 mg, 58%; H NMR (500 MHz, CDCl3): δ
= 8.61 – 8.56 (m, 1H), 8.06 (d, J = 8.0 Hz, 1H), 7.72 (td, J = 7.8,
1.7 Hz, 1H), 7.28 (ddd, J = 7.5, 4.9, 1.0 Hz, 1H), 7.02 (d, J = 2.3
Hz, 1H), 6.94 (dd, J = 8.4, 2.3 Hz, 1H), 6.80 (d, J = 8.5 Hz, 1H),
6.71 (s, 2H), 4.15 (d, J = 17.3 Hz, 1H), 4.09 (d, J = 17.3 Hz, 1H),
3.85 (s, 3H), 3.83 (s, 6H), 3.82 ppm (s, 3H); 13C NMR (125
MHz, CDCl3): δ = 158.24, 153.13, 153.09 (2C), 149.36, 149.28,
146.17, 145.50, 139.87, 137.29, 136.50, 124.36, 121.79, 117.75,
112.85, 110.29, 103.38 (2C), 92.61, 60.83, 56.22 (2C), 55.97,
+
(2C), 56.01, 48.64 ppm; MS (ESI): m/z calcd for C25H26NO7
[M+H]+ 452.48, found 452.25.
4.1.8.11. 5,5'-(5-(3,4,5-trimethoxyphenyl)-4,5-dihydroisoxazole-
3,5-diyl)bis(2-methoxyphenol) (6k)
47.79 ppm; MS (ESI): m/z calcd for C24H25N2O6 [M+H]+
437.47, found 437.16.
+

4.1.8.15. 5-butyl-3-(4-chlorophenyl)-5-(3,4,5-trimethoxyphenyl)-
4,5-dihydroisoxazole (9a)
KU/L) and streptomycin (100 KU/L) at 37 oC in a 5% CO2
humidified incubator.
This compound was obtained from reaction between 7 and the
residue of 4-chloro-N-hydroxybenzimidoyl chloride after 1 h.
Eluent: petroleum ether/ethyl acetate = 8:1. Yellow solid. Yield:
9 mg, 31%; 1H NMR (500 MHz, CDCl3): δ = 7.58 (d, J = 8.5 Hz,
2H), 7.35 (d, J = 8.5 Hz, 2H), 6.65 (s, 2H), 3.88 (s, 6H), 3.83 (s,
3H), 3.46 (s, 2H), 2.05 – 1.97 (m, 2H), 1.42 – 1.17 (m, 4H), 0.87
ppm (t, J = 7.2 Hz, 3H); 13C NMR (125 MHz, CDCl3): δ =
155.25, 153.24 (2C), 140.66, 137.00, 135.96, 128.97 (2C),
128.31, 127.80 (2C), 102.13 (2C), 91.06, 60.86, 56.28 (2C),
47.16, 41.60, 26.19, 22.83, 13.99 ppm; MS (ESI): m/z calcd for
C22H27ClNO4+ [M+H]+ 404.91, found 404.17.
4.2.2. Cytotoxic activity (SRB)
In vitro antiproliferative activity was evaluated by using the
SRB colorimetric assay as previous methods61, 65
.
4.2.3. Tubulin polymerization assay
Effects of compounds on tubulin polymerization in vitro were
performed in the light of the fluorescence based tubulin
polymerization assay kit’s protocol (Cat. #BK011P, Cytoskeleton
Inc.). Tubulin (10 mg/mL) containing buffer 1, tubulin glycerol
buffer and GTP stock (100 mM) was incubated with 6g (10, 20
and 40 µM), paclitaxel (3 µM), CA-4 (3 µM), isoCA-4 (3 µM)
and DMSO for 80 minutes at 37 oC. Paclitaxel, CA-4 and isoCA-
4 were used as reference compounds in this assay. The
fluorescence increase was monitored every minute for
characterization of tubulin polymerization at 360 nm excitation
wavelength and 460 nm emission wavelength by microplate
spectrophotometer (Synergy HTX, BioTek Instruments, Inc). The
IC50 value of compound 6g was determined by five
concentrations (3, 5, 10, 20, 40 µM) against tubulin
polymerization.
4.1.8.16.
5-butyl-3-(4-chloro-2-methoxyphenyl)-5-(3,4,5-
trimethoxyphenyl)-4,5-dihydroisoxazole (9b)
This compound was obtained from reaction between 7 and the
residue of 4-chloro-N-hydroxy-2-methoxybenzimidoyl chloride
after 1 h. Eluent: petroleum ether/ethyl acetate = 10:1. Yellow
solid. Yield: 13 mg, 42%; 1H NMR (500 MHz, CDCl3): δ = 7.71
(d, J = 2.6 Hz, 1H), 7.28 (dd, J = 8.9, 2.6 Hz, 1H), 6.83 (d, J =
8.9 Hz, 1H), 6.64 (s, 2H), 3.88 (s, 6H), 3.83 (s, 3H), 3.82 (s, 3H),
3.58 (d, J = 17.3 Hz, 1H), 3.51 (d, J = 17.3 Hz, 1H), 2.02 – 1.95
(m, 2H), 1.40 – 1.20 (m, 4H), 0.87 ppm (t, J = 7.2 Hz, 3H); 13C
NMR (125 MHz, CDCl3): δ = 156.04, 154.80, 153.14 (2C),
140.93, 136.87, 130.71, 128.87, 125.91, 120.49, 112.70, 102.24
(2C), 90.78, 60.86, 56.28 (2C), 55.90, 49.53, 41.37, 26.21, 22.88,
4.2.4. Immunohistochemistry studies
PC-3 cells were incubated in 12-well plates in the presence or
absence of tested compounds (6g: 1.0 μM and CA-4: 0.02 μM)
for 48 h. After 4% paraformaldehyde was used to fix cells for 30
min and rinsed thrice with PBS containing 5% bovine serum
albumin (BSA), 0.1% Triton X-100 in PBS was employed for
+
14.01 ppm; MS (ESI): m/z calcd for C23H29ClNO5 [M+H]+
434.94, found 434.22.
o
4.1.8.17.
4-(5-butyl-5-(3,4,5-trimethoxyphenyl)-4,5-
permeabilization at 4 C in the dark for 30 min and rinsed again
dihydroisoxazol-3-yl)-2-methoxyphenol (9c)
with PBS containing 5% BSA. Then the cells were blocked with
PBS containing 5% BSA for 30 min. Anti-α-tubulin antibodies
(1: 500, Abcam) and the secondary antibody (goat anti-rabbit
IgG, 1: 500, ZSGB-BIO) were added sequentially to each well
This compound was obtained from reaction between 7 and the
residue of N,3-dihydroxy-4-methoxybenzimidoyl chloride after 1
h, followed by TBAF 0.5 h. Yellow solid. Eluent: petroleum
o
and rinsed with PBS before incubating for overnight at 4 C and
1
ether/ethyl acetate = 4:1. Yield: 12 mg, 41%; H NMR (500
1 h at room temperature, respectively. At last, the cells were
stained with DAPI for 5 min at room temperature. Images of
results were acquire using a fluorescence microscope with a 20×
objective
MHz, CDCl3): δ = 7.24 (d, J = 1.9 Hz, 1H), 7.14 (dd, J = 8.4, 1.8
Hz, 1H), 6.82 (d, J = 8.4 Hz, 1H), 6.65 (s, 2H), 5.77 (s, 1H), 3.88
(s, 3H), 3.87 (s, 6H), 3.82 (s, 3H), 3.43 (s, 2H), 2.02 – 1.95 (m,
2H), 1.42 – 1.18 (m, 4H), 0.85 ppm (t, J = 7.2 Hz, 3H); 13C NMR
(125 MHz, CDCl3): δ = 155.83, 153.15 (2C), 148.15, 145.70,
141.01, 136.86, 123.16, 119.10, 112.73, 110.48, 102.22 (2C),
90.42, 60.85, 56.25 (2C), 56.01, 47.52, 41.54, 26.22, 22.86, 13.99
4.2.5. Cell cycle analysis
2.5 mL aliquots of PC-3 cells containing 1.0×105 cells/mL
o
-
ppm; MS (ESI): m/z calcd for C23H28NO6 [M-H]- 414.48, found
were plated in 6-well plates and incubated at 37 C for 24 h.
Propidium iodide staining assay (Sigma) was used for the cell
cycle arrest of PC-3 cells as described previously. The cultured
cells were treated with 6g and CA-4 at the indicated
concentrations (6g: 0.5, 1.0 µM; CA-4: 0.05 µM) for 48 h. After
that, centrifugation, fixation using 70% ethanol at 4 oC overnight
and resuspension process in PBS containing 100 μL RNase A
and 400 μL PI were carried out in sequence. For cell cycle
distribution analysis, cellular DNA content was collected twenty
thousand events per sample by a FACS Calibur flow cytometer
(BD Biosciences). The results were analyzed using Modfit LT
3.0 software.
414.21.
4.1.9. X-ray experimental
Single crystals of (S)-5b obtained from solvent volatilization in
CH3OH. An appropriate crystal was selected and analysed on a
SuperNova, Dual, Cu at zero, Eos diffractometer. The crystal was
held at 293(2) K in the period of data collection. Using Olex262,
the structure was solved with the Superflip63 structure solution
64
program using Charge Flipping and refined with the ShelXL
refinement package using Least Squares minimisation.
4.2. Biological evaluation
4.2.6. Statistics analysis
4.2.1. Cell Culture
All the data reported were shown as the arithmetic mean of
date from three separate determinations where each group owns
three parallels. The form of results presented as mean ± standard
deviation (SD). Statistical analysis was performed using
GraphPad software with the 2-tailed Student t-test.
PC-3 cell line was originally acquired from Shanghai Institute
of Biochemistry and Cell Biology, Chinese Academy of
Sciences. HeLa cell line was donated by Prof. Lei group of
college of life sciences, Northwest A&F University. The two cell
lines were grown in RPMI-1640 (Gibco) including 10% (v/v)
thermally inactivated fetal bovine serum (FBS), penicillin (100
4.3. Molecular modeling

Dubois, J.; Brion, J. D.; Alami, M. J Med Chem. 2009; 52: 4538-4542.
30. Tréguier, B.; Hamze, A.; Provot, O.; Brion, J.-D.; Alami, M. Tetrahedron
Lett. 2009; 50: 6549-6552.
31. Stocker, V.; Ghinet, A.; Leman, M.; Rigo, B.; Millet, R.; Farce, A.;
Desravines, D.; Dubois, J.; Waterlot, C.; Gautret, P. Rsc Adv. 2013; 3:
3683-3696.
32. Aziz, J.; Brachet, E.; Hamze, A.; Peyrat, J. F.; Bernadat, G.; Morvan, E.;
Bignon, J.; Wdzieczak-Bakala, J.; Desravines, D.; Dubois, J.; Tueni, M.;
Yassine, A.; Brion, J. D.; Alami, M. Org Biomol Chem. 2013; 11: 430-442.
33. Rasolofonjatovo, E.; Provot, O.; Hamze, A.; Rodrigo, J.; Bignon, J.;
Wdzieczak-Bakala, J.; Desravines, D.; Dubois, J.; Brion, J. D.; Alami, M.
Eur J Med Chem. 2012; 52: 22-32.
All docking studies were performed by using the Surflex-
Dock program in the Sybyl-X 2.0 software. The X-ray crystal
structure of tubulin protein (PDB ID: 1SA0) was acquired from
RSCB Protein Data Bank (http:// www.rcsb.org/pdb). The
analysis of results was carried out by USCF Chimera software,
which was downloaded from http://www.cgl.ucsf.edu/chimera.
Acknowledgments
This work was supported by the National Natural Science
Foundation of China (21542016).
34. Renko, D.; Provot, O.; Rasolofonjatovo, E.; Bignon, J.; Rodrigo, J.; Dubois,
J.; Brion, J. D.; Hamze, A.; Alami, M. Eur J Med Chem. 2015; 90: 834-844.
35. Soussi, M. A.; Provot, O.; Bernadat, G.; Bignon, J.; Wdzieczak-Bakala, J.;
Desravines, D.; Dubois, J.; Brion, J. D.; Messaoudi, S.; Alami, M. Eur J
Med Chem. 2014; 78: 178-189.
36. Rasolofonjatovo, E.; Provot, O.; Hamze, A.; Rodrigo, J.; Bignon, J.;
Wdzieczak-Bakala, J.; Lenoir, C.; Desravines, D.; Dubois, J.; Brion, J.-D.;
Alami, M. Eur J Med Chem. 2013; 62: 28-39.
References and notes
1. Bauer, A.; Bronstrup, M. Nat Prod Rep. 2014; 31: 35-60.
2. Newman, D. J.; Cragg, G. M. J Nat Prod. 2016; 79: 629-661.
3. Pettit, G. R.; Singh, S. B.; Hamel, E.; Lin, C. M.; Alberts, D. S.;
Garciakendall, D. Experientia. 1989; 45: 209-211.
37. Hamze, A.; Giraud, A.; Messaoudi, S.; Provot, O.; Peyrat, J. F.; Bignon, J.;
Liu, J. M.; Wdzieczak-Bakala, J.; Thoret, S.; Dubois, J.; Brion, J. D.; Alami,
M. ChemMedChem. 2009; 4: 1912-1924.
4. Hamel, E. Med Res Rev. 1996; 16: 207-231.
5. Woods, J. A.; Hadfield, J. A.; Pettit, G. R.; Fox, B. W.; McGown, A. T. Brit J
Cancer. 1995; 71: 705-711.
38. Khelifi, I.; Naret, T.; Renko, D.; Hamze, A.; Bernadat, G.; Bignon, J.;
Lenoir, C.; Dubois, J.; Brion, J.-D.; Provot, O.; Alami, M. Eur J Med Chem.
2017; 127: 1025-1034.
6. Jordan, M. A.; Wilson, L. Nat Rev Cancer. 2004; 4: 253-265.
7. Young, S. L.; Chaplin, D. J. Expert Opin Investig Drugs. 2004; 13: 1171-
1182.
39. Soussi, M. A.; Provot, O.; Bernadat, G.; Bignon, J.; Desravines, D.; Dubois,
J.; Brion, J.-D.; Messaoudi, S.; Alami, M. ChemMedChem. 2015; 10: 1392-
1402.
40. Tréguier, B.; Rasolofonjatovo, E.; Hamze, A.; Provot, O.; Wdzieczak-
Bakala, J.; Dubois, J.; Brion, J.-D.; Alami, M. Eur J Org Chem. 2011; 3:
3683-3696.
8. www.mateon.com.
9. Hori, K.; Saito, S. Brit J Cancer. 2003; 89: 1334-1344.
10. Tron, G. C.; Pirali, T.; Sorba, G.; Pagliai, F.; Busacca, S.; Genazzani, A. A. J
Med Chem. 2006; 49: 3033-3044.
11. Nam, N. H. Curr Med Chem. 2003; 10: 1697-1722.
12. Aprile, S.; Del Grosso, E.; Tron, G. C.; Grosa, G. Drug Metab Dispos. 2007;
41. Kumar, G. B.; Nayak, V. L.; Bin Sayeed, I.; Reddy, V. S.; Shaik, A. B.;
Mahesh, R.; Baig, M. F.; Shareef, M. A.; Ravikumar, A.; Kamal, A. Bioorg
Med Chem. 2016; 24: 1729-1740.
35: 2252-2261.
13. Nakamura, M.; Kajita, D.; Matsumoto, Y.; Hashimoto, Y. Bioorg Med
Chem. 2013; 21: 7381-7391.
42. Combes, S.; Barbier, P.; Douillard, S.; McLeer-Florin, A.; Bourgarel-Rey,
V.; Pierson, J. T.; Fedorov, A. Y.; Finet, J. P.; Boutonnat, J.; Peyrot, V. J
Med Chem. 2011; 54: 3153-3162.
14. Pang, Y. Q.; Yan, J.; An, B. J.; Huang, L.; Li, X. S. Bioorg Med Chem. 2017;
25: 3059-3067.
15. Chen, H.; Li, Y.; Sheng, C.; Lv, Z.; Dong, G.; Wang, T.; Liu, J.; Zhang, M.; Li,
L.; Zhang, T.; Geng, D.; Niu, C.; Li, K. J Med Chem. 2013; 56: 685-699.
16. Ty, N.; Pontikis, R.; Chabot, G. G.; Devillers, E.; Quentin, L.; Bourg, S.;
Florent, J. C. Bioorg Med Chem. 2013; 21: 1357-1366.
17. Zhou, P.; Liu, Y.; Zhou, L.; Zhu, K.; Feng, K.; Zhang, H.; Liang, Y.; Jiang, H.;
Luo, C.; Liu, M.; Wang, Y. J Med Chem. 2016; 59: 10329-10334.
18. Theeramunkong, S.; Caldarelli, A.; Massarotti, A.; Aprile, S.; Caprioglio,
D.; Zaninetti, R.; Teruggi, A.; Pirali, T.; Grosa, G.; Tron, G. C.; Genazzani,
A. A. J Med Chem. 2011; 54: 4977-4986.
19. Pirali, T.; Busacca, S.; Beltrami, L.; Imovilli, D.; Pagliai, F.; Miglio, G.;
Massarotti, A.; Verotta, L.; Tron, G. C.; Sorba, G.; Genazzani, A. A. J Med
Chem. 2006; 49: 5372-5376.
20. Assadieskandar, A.; Amini, M.; Ostad, S. N.; Riazi, G. H.; Cheraghi-Shavi,
T.; Shafiei, B.; Shafiee, A. Bioorg Med Chem. 2013; 21: 2703-2709.
21. Wang, Z.; Yang, Q.; Bai, Z.; Sun, J.; Jiang, X.; Song, H.; Wu, Y.; Zhang, W.
Med Chem Commun. 2015; 6: 971-976.
22. Simoni, D.; Grisolia, G.; Giannini, G.; Roberti, M.; Rondanin, R.; Piccagli,
L.; Baruchello, R.; Rossi, M.; Romagnoli, R.; Invidiata, F. P.; Grimaudo, S.;
Jung, M. K.; Hamel, E.; Gebbia, N.; Crosta, L.; Abbadessa, V.; Di Cristina,
A.; Dusonchet, L.; Meli, M.; Tolomeo, M. J Med Chem. 2005; 48: 723-
736.
43. Bailly, C.; Bal, C.; Barbier, P.; Combes, S.; Finet, J. P.; Hildebrand, M. P.;
Peyrot, V.; Wattez, N. J Med Chem. 2003; 46: 5437-5444.
44. Tanpure, R. P.; George, C. S.; Strecker, T. E.; Devkota, L.; Tidmore, J. K.;
Lin, C. M.; Herdman, C. A.; MacDonough, M. T.; Sriram, M.; Chaplin, D.
J.; Trawick, M. L.; Pinney, K. G. Bioorg Med Chem. 2013; 21: 8019-8032.
45. Herdman, C. A.; Devkota, L.; Lin, C. M.; Niu, H.; Strecker, T. E.; Lopez, R.;
Liu, L.; George, C. S.; Tanpure, R. P.; Hamel, E.; Chaplin, D. J.; Mason, R.
P.; Trawick, M. L.; Pinney, K. G. Bioorg Med Chem. 2015; 23: 7497-7520.
46. Dean, F. M. Adv Heterocycl Chem. 1982; 30: 167-238.
47. Mordue, A. J.; Blackwell, A. J Insect Physiol. 1993; 39: 903-924.
48. Dong, S.; Tang, J. J.; Zhang, C. C.; Tian, J. M.; Guo, J. T.; Zhang, Q.; Li, H.;
Gao, J. M. Eur J Med Chem. 2014; 80: 71-82.
49. Lü, M.; Wu, W.; Liu, H. Pestic Biochem Physiol. 2010; 98: 263-268.
50. Ruscoe, C. N. Nature: New biology. 1972; 236: 159-160.
51. Xiang, Y. J.; Chen, J.; Schinazi, R. F.; Zhao, K. Bioorg Med Chem Lett.
1996; 6: 1051-1054.
52. Watts, R. E.; Siegel, M.; Khosla, C. J Med Chem. 2006; 49: 7493-7501.
53. Choi, K.; Siegel, M.; Piper, J. L.; Yuan, L.; Cho, E.; Strnad, P.; Omary, B.;
Rich, K. M.; Khosla, C. Chem Biol. 2005; 12: 469-475.
54. Mondal, M.; Bora, U. RSC Adv. 2013; 3: 18716-18754.
55. Yang, D.; Ye, X. Y.; Xu, M.; Pang, K. W.; Cheung, K. K. J Am Chem Soc.
2000; 122: 1658-1663.
23. Li, Y. H.; Zhang, B.; Yang, H. K.; Li, Q.; Diao, P. C.; You, W. W.; Zhao, P. L.
Eur J Med Chem. 2017; 125: 1098-1106.
56. Lee, H. S.; Kim, S. H.; Kim, Y. M.; Kim, J. N. Tetrahedron Lett. 2010; 51:
5071-5075.
57. Sajna, K. V.; Kumara Swamy, K. C. J Org Chem. 2012; 77: 8712-8722.
58. Zeng, J.-C.; Xu, H.; Yu, F.; Zhang, Z. Tetrahedron Lett. 2017; 58: 674-678.
59. Feng, J.-J.; Zhang, J. ACS Catal. 2016; 6: 6651-6661.
24. Subba Rao, A. V.; Swapna, K.; Shaik, S. P.; Lakshma Nayak, V.; Srinivasa
Reddy, T.; Sunkari, S.; Shaik, T. B.; Bagul, C.; Kamal, A. Bioorg Med Chem.
2017; 25: 977-999.
25. Salehi, M.; Amini, M.; Ostad, S. N.; Riazi, G. H.; Assadieskandar, A.;
Shafiei, B.; Shafiee, A. Bioorg Med Chem. 2013; 21: 7648-7654.
26. Wang, F.; Yang, Z.; Liu, Y.; Ma, L.; Wu, Y.; He, L.; Shao, M.; Yu, K.; Wu,
W.; Pu, Y.; Nie, C.; Chen, L. Bioorg Med Chem. 2015; 23: 3337-3350.
27. Xu, Q.; Wang, Y.; Xu, J.; Sun, M.; Tian, H.; Zuo, D.; Guan, Q.; Bao, K.; Wu,
Y.; Zhang, W. ACS Med Chem Lett. 2016; 7: 1202-1206.
28. Pettit, G. R.; Toki, B.; Herald, D. L.; Verdier-Pinard, P.; Boyd, M. R.;
Hamel, E.; Pettit, R. K. J Med Chem. 1998; 41: 1688-1695.
29. Messaoudi, S.; Treguier, B.; Hamze, A.; Provot, O.; Peyrat, J. F.; De
Losada, J. R.; Liu, J. M.; Bignon, J.; Wdzieczak-Bakala, J.; Thoret, S.;
60. Osborn, H. M. I.; Gemmell, N.; Harwood, L. M. J Chem Soc, Perkin 1.
2002: 2419-2438.
61. Skehan, P.; Storeng, R.; Scudiero, D.; Monks, A.; McMahon, J.; Vistica,
D.; Warren, J. T.; Bokesch, H.; Kenney, S.; Boyd, M. R. J Natl Cancer I.
1990; 82: 1107-1112.
62. Dolomanov, O. V.; Bourhis, L. J.; Gildea, R. J.; Howard, J. A.; Puschmann,
H. J Appl Crystallogr. 2009; 42: 339-341.
63. Palatinus, L.; Chapuis, G. J Appl Crystallogr. 2007; 40: 786-790.

64. Sheldrick, G. M. Acta Crystallogr Sect A. 2007; 64: 112-122.
65. Dong, S.; Tang, J.-J.; Zhang, C.-C.; Tian, J.-M.; Guo, J.-T.; Zhang, Q.; Li, H.;
Gao, J.-M. Eur J Med Chem. 2014; 80: 71-82.

Constructing novel dihydrofuran and dihydroisoxazole analogues of
isocombretastatin-4 as tubulin polymerization inhibitors through [3+2] reactions
Ming-Yu Song a, §, Chen-Yu Cao a, §, Qiu-Rui He a, Qing-Miao Dong a, Ding Lia, Jiang-Jiang Tang a,  and
Jin-Ming Gao a, 
a Shaanxi Key Laboratory of Natural Products & Chemical Biology, College of Chemistry & Pharmacy, Northwest A&F University, Yangling 712100, P.R.
China
Graphical abstract:
 Corresponding authors: JJT (main corresponding author): e-mail: tangjiang11@nwsuaf.edu.cn; JMG: e-mail: jin-minggao@nwsuaf.edu.cn
§ These authors contributed equally to this work.