Novel diaryl-2H-azirines: Antitumor hybrids for dual-targeting tubulin and DNA

Article abstract of DOI:10.1016/j.ejmech.2021.113256

Multiple-target drugs may achieve better therapeutic effect via different pathways than single-target ones, especially for complex diseases. Tubulin and DNA are well-characterized molecular targets for anti-cancer drug development. A novel class of diaryl substituted 2H-azirines were designed based on combination of pharmacophores from Combretastatin A-4 (CA-4) and aziridine-type alkylating agents, which are known tubulin polymerization inhibitor and DNA damaging agents, respectively. The antitumor activities of these compounds were evaluated in vitro and 6h showed the most potent activities against four cancer cell lines with IC₅₀ values ranging from 0.16 to 1.40 μM. Further mechanistic studies revealed that 6h worked as a bifunctional agent targeting both tubulin and DNA. In the nude mice xenograft model, 6h significantly inhibited the tumor growth with low toxicity, demonstrating the promising potential for further developing novel cancer therapy with a unique mechanism.

Full text of DOI:10.1016/j.ejmech.2021.113256

European Journal of Medicinal Chemistry 214 (2021) 113256
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Novel diaryl-2H-azirines: Antitumor hybrids for dual-targeting
tubulin and DNA
,
,
, b, *
Shibo Lin a 1, Yuru Liang a 1, Jiayi Cheng a, Feng Pan a, Yang Wang a
a School of Pharmacy, Fudan University, Shanghai, 201203, China
b Shanghai Key Laboratory for Molecular Engineering of Chiral Drugs, Shanghai Jiao Tong University, Shanghai, 200240, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Multiple-target drugs may achieve better therapeutic effect via different pathways than single-target
ones, especially for complex diseases. Tubulin and DNA are well-characterized molecular targets for
anti-cancer drug development. A novel class of diaryl substituted 2H-azirines were designed based on
combination of pharmacophores from Combretastatin A-4 (CA-4) and aziridine-type alkylating agents,
which are known tubulin polymerization inhibitor and DNA damaging agents, respectively. The anti-
tumor activities of these compounds were evaluated in vitro and 6h showed the most potent activities
Received 16 November 2020
Received in revised form
28 January 2021
Accepted 30 January 2021
Available online 3 February 2021
against four cancer cell lines with IC50 values ranging from 0.16 to 1.40 mM. Further mechanistic studies
Keywords:
revealed that 6h worked as a bifunctional agent targeting both tubulin and DNA. In the nude mice
xenograft model, 6h significantly inhibited the tumor growth with low toxicity, demonstrating the
promising potential for further developing novel cancer therapy with a unique mechanism.
© 2021 Elsevier Masson SAS. All rights reserved.
2,3-Diaryl-2H-azirine
Tubulin polymerization
DNA damage
Antitumor
1. Introduction
is inactive against tumor cell proliferation [9]. A number of efforts
have been devoted to resolve this problem, including the synthesis
Microtubules play a critical role in various cellular processes
(e.g., formation of the mitotic spindle, intracellular transport, cell
division, cell signaling and migration), and thus have generally
been considered as an excellent target for development of anti-
tumor agents [1,2]. Antimitotic agents are known to inhibit
microtubule function by the dynamic balance of tubulin stabiliza-
tion or destabilization, leading to mitotic arrest [3,4]. Com-
bretastatin A-4 (CA-4, Fig. 1), a cis-stilbene-type natural compound
isolated from the African willow tree Combretum caffrum, is a
typical molecule that can bound tightly to the colchicine binding
site of tubulin, leading to closed vessels and dead cells in neoplastic
tissues. CA-4 has demonstrated strong cytotoxicity against many
different types of neoplastic cells, including some multidrug-
resistant ones [5,6]. The structure-activity relationship (SAR) of
CA-4 and its derivatives revealed that the presence of a 3,4,5-
trimethoxyphenyl as A-ring and the cis-form of the double bond
are crucial for its potent antitumor activity [7,8]. Unfortunately,
stability studies revealed that CA-4 is highly susceptible to cis/trans
isomerization under physiological conditions, and its trans isomer
and evaluation of various CA-4 analogues whose pharmacophores
are spatially confined by more rigid cores, typically via the substi-
tution of the olefinic bond in CA-4 with heterocyclic moieties, such
as b-lactam [10,11], hydantoin [12], thiazole [13], furan [14], imi-
dazo[4,5-c]pyridin-2-one [15], pyrazole [16], etc, to maintain a
suitable spatial orientation of two aromatic rings to achieve optimal
bioactivity.
On the other hand, the DNA alkylating agents have also been
widely applied clinically in cancer chemotherapy for many years
[17]. One type of the earliest and perhaps most commonly used
DNA cross-linking agents are derivatives of nitrogen mustard
[18e20], in which the N,N-bis(2-chloroethyl)-amino pharmaco-
phore can act as reactive electrophiles through the intramolecular
transformation into aziridine ring [20]. As shown in Fig. 1, a similar
pharmacophore also occurs in some clinical drugs, such as Thiotepa
and Mitomycin C (MCC). Over the last several decades, they have
generally been applied as constituents of cancer drug combinations
against a number of cancers, including gastric, colorectal, breast,
ovarian, bladder and non-small cell lung carcinoma [21,22].
* Corresponding author. School of Pharmacy, Fudan University, Shanghai, 201203, China.
E-mail address: wangyang@shmu.edu.cn (Y. Wang).
1
contributed equally to this work.
https://doi.org/10.1016/j.ejmech.2021.113256
0223-5234/© 2021 Elsevier Masson SAS. All rights reserved.

S. Lin, Y. Liang, J. Cheng et al.
European Journal of Medicinal Chemistry 214 (2021) 113256
Fig. 1. Representative antitumor agents CA-4, Thiotepa and MCC, targeting tubulin and DNA, respectively, and the designed 2H-azirine and aziridine skeletal structures 6 and 7.
MCC undergoes intracellular activation by interaction with cy-
tochrome P450 reductase [23] or other 1- or 2-electron reductases
[24], and the subsequent leaving of methoxy group generates MCC-
leucoaziridinomitosene, which consequently incurs DNA alkylation
under acidic condition [25]. In addition, the MCC-DNA monoadduct
located within the DNA minor groove causes steric hindrance to
further enzymatic reduction of the quinone ring. All these factors
facilitate carbamate departure during subsequent DNA attachment
and tend to produce MCC-DNA interstrand crosslinks (ICLs, Fig. 2)
[26]. Moreover, researches showed that aziridine-bearing anti-
tumor agents including thiotepa can generate covalent adducts
with DNA double-helix strands via the alkylation of the N7 atom of
guanine [27,28], resulting in the obstruction on DNA replication and
transcription, and causing cell death.
Recent studies have indicated that DNA damaging agents,
mainly platinum drugs, could enhance the apoptosis-inducing ac-
Fig. 3. The dual-target compounds acting on microtubules and DNA.
tivity of tubulin inhibitors (Fig. 3) [29,30]. Compounds targeting
both tubulin and DNA can in principle merge the two distinct anti-
tubulin and DNA synergistically, we designed and investigated a
cancer pharmocophores into the structure of one agent, and thus
new series of diaryl-2H-azirine derivatives based on the combina-
may create potential synergy by itself. The dual tubulin polymeri-
tion strategy for multitarget drug (Fig. 1). Among these compounds,
zation inhibition and DNA damage functions might lead to an
compound 6h exhibited satisfactory antineoplastic activity against
improvement in targeting and therapeutic efficacy, and this may
represent an avenue towards the development of new anti-cancer
agents [31e35].
several different cell lines. Further mechanism studies and in vivo
activities demonstrated the promising potential of 6h as a novel
dual-targeting anticancer agent.
In an effort to search for more effective compounds that target
Fig. 2. Mechanism for MCC activation that induces DNA ICLs.
2

S. Lin, Y. Liang, J. Cheng et al.
European Journal of Medicinal Chemistry 214 (2021) 113256
2. Results and discussion
2.2. Anti-proliferation activities against four human cancer cell
lines in vitro
2.1. Chemistry
The newly synthesized 2,3-diaryl-2H-azirines 6a-r and azir-
idines 7a-b were evaluated for anti-proliferative activities in vitro
against A2780, HCT-116, A549 and HeLa human cancer cell lines,
respectively, using CA-4 and Mitomycin C (MMC) as the reference
compounds.
As shown in Table 1, most target compounds 6 and 7 exhibited
moderate to excellent cytotoxicity against these tumor cells,
especially on A2780. Among them, 2,3-diaryl-2H-azirines (6a, 6b,
6e, 6h and 6m) showed good activities with IC50 values in the range
The target compounds were synthesized by following the gen-
eral procedures as outlined in Scheme 1. The reactions of
commercially available aryl acetonitriles (1a-r) and arylboronic
acids (2a-r) proceeded smoothly under Pd(II) catalysis [36], giving a
variety of aryl alkyl ketones (3a-r) in good to excellent yields
(75e87%). Condensation of these ketones (3a-r) with NH2OH$HCl/
K2CO3 gave corresponding ketoximes (4a-r), respectively. Subse-
quent esterification with acetic anhydride afforded the corre-
sponding ketoxime acetates (5a-r), which were then cyclized by
treatment with cesium carbonate [37] into 2H-azirines (6a-r) in
good yields (67e81%). Reduction of 6h with sodium borohydride in
tetrahydrofuran produced 2-(3-fluoro-4-methoxyphenyl)-3-(3,4,5-
trimethoxyphenyl)-aziridine (7a) in excellent yield (91%), and N-
methylation of 7a with methyl iodide gave 7b in 81% yield. The
relative configuration of 7a was predicted to be cis-isomers, based
on Feng group’s work that the chiral diaryl-2H-azirines (93% ee)
could be converted into corresponding cis-aziridines (93% ee) in
presence of NaBH4 with excellent diastereo- and enantio-
selectivity [38].
of 0.16e1.54
decrease in the antiproliferative activity (IC50 ¼ 1.09e8.74
M). These results
mM, while the aziridines 7a and 7b demonstrated a
mM)
compared with that of 6h (IC50 ¼ 0.16e1.40
m
suggested that the rigid 2H-azirine core is likely to be more
appropriate for fixing the orientation of two aryl substituents, so
that the molecule can better bind to the colchicine site of b-tubulin.
The exchange of A and B aromatic rings on the 2H-azirines
exhibited few influence on the antiproliferative activity (6a and 6h,
6b and 6i). In addition, it was found that compounds with 4-
methoxy (6a-b, 6h-i and 6g), 4-methyl (6f and 6m) or 4-
methylthio (6n) substituents on the B ring exhibit enhanced po-
tency compared to those of 4-ethoxy compounds (6e and 6l), which
implied that bulkier groups will compromise the activity to a
Scheme 1. Synthesis of 2H-azirines (6a-r) and aziridines (7a-b). Reagents and conditions: (i) Pd(OAc)2, 2,20-Bipyridine, TFA, KF, THF/H2O, 80 ꢀC, 2 h; (ii) NH2OH$HCl, K2CO3, MeOH,
50 ꢀC, 5 h; (iii) Ac2O, TEA, DCM, r.t., 30 min; (iv) K2CO3, DMF, 80 ꢀC, 1 h; (v) NaBH4, THF, r.t., 2 h; (vi) MeI, r.t., 3 h.
3

S. Lin, Y. Liang, J. Cheng et al.
European Journal of Medicinal Chemistry 214 (2021) 113256
Table 1
Fig. 5AeD, significant DNA lesions were observed for the sample
treated with the positive control drug MMC at the concentration of
200 nM. Noteworthily, DNA damaged by 6h emerged comparable
tail moments at the same concentration as that of MCC, and the
extent of DNA lesion was dose-dependent. In addition, DNA dam-
age related proteins was characterized by proapoptotic Bax protein
Antiproliferative activities of compounds 6 and 7 on different cell lines.
Compound
IC50 (m
M)a
A2780b
HCT-116c
HeLad
A549e
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
6m
6n
6o
6p
6q
6r
0.19
0.44
1.54
2.66
0.58
0.17
0.58
0.16
0.33
1.74
2.92
0.78
0.24
0.40
3.479
0.927
8.333
3.479
1.90
4.68
0.004
0.18
0.32
0.76
0.97
12.40
0.50
0.79
3.59
0.63
1.10
1.89
8.72
1.10
0.58
1.51
2.218
2.058
4.103
2.218
1.84
5.44
0.004
0.56
0.73
1.54
1.52
12.66
1.14
2.07
2.65
1.07
1.97
2.02
4.17
1.96
0.78
2.19
4.498
1.933
11.82
4.498
1.09
4.39
0.004
0.20
0.63
1.12
9.16
17.57
0.76
2.30
6.33
1.40
1.49
2.21
11.05
3.61
1.40
4.16
4.227
1.820
7.031
4.227
1.20
8.74
0.004
0.63
and growth arrest GADD45a that play an essential role in cell cycle
checkpoint after exposure to certain DNA lesions such as alkylating
agents and UV irradiation [40]. As shown in Fig. 5E, the compound
6h significantly up-regulate expression of proapoptotic protein Bax
and GADD45a in a dose-dependent manner. These observations
suggested that 6h could induce DNA damage.
2.5. Induction of cellular apoptosis
To assess whether compound 6h could induce apoptosis of cell,
the 6h-treated HeLa cells were stained with Annexin V-FITC and
propidium iodide (PI), which could be used to distinguish among
live cells (annexin-Vꢁ/PIꢁ), early apoptotic cells (annexin-Vþ/PIꢁ),
late apoptotic cells (annexin-Vþ/PIþ), and necrotic cells (annexin-
Vꢁ/PIþ). The flow cytometric data (Fig. 6A and B) demonstrated a
potent activity of 6h on the apoptosis induction.
In addition, we examined the expression levels of apoptosis
associated proteins via immunoblotting analysis. As indicated in
Fig. 6C, proapoptotic protein p53 was up-regulated by 6h in a dose-
dependent manner. Additionally, 6h was found to dose-
dependently promote cleavage of poly(ADP-ribose) polymerase-1
(PARP-1), a marker of cells undergoing apoptosis. These results
implied that the azirine analogues exhibited their antitumor ac-
tivity through induction of cellular apoptosis.
7a
7b
CA-4
MMC
a
The anti-proliferation activities of individual compound to tumor cells were
determined by the MTT assay. The data are the mean of triplicate determinations.
b
A2780 is a human ovarian cancer cell line.
HCT-116 is a human colon carcinoma cell line.
HeLa is a human cervical carcinoma cell line.
A549 is a human lung adenocarcinoma cell line.
c
d
e
certain extent presumably due to steric factors. Compared with 3-
methoxy (6d, 6k) or 3,4-(methylenedioxy) (6c, 6j) analogues, the
potency could be promoted for compounds with no substituent (6b,
6e-f, 6i, 6l-n) or fluorine (6a, 6h) existing at the 3-position of B ring.
Among the target compounds synthesized, 6h was selected to
further investigate its pharmacological properties and action
mechanism.
2.6. Cell cycle analysis
To explore the influence of 6h on cell cycle arrest, a cell cycle
analysis was conducted by quantitiating the DNA content. The HeLa
cells were treated with 6h at various concentrations for 24 h,
respectively, followed by being stained with propidium iodide, and
the samples were analyzed by flow cytometry. As expected, cell
cycle arrest was found to be induced by 6h at G2/M phase (Fig. 7A
and B). In addition, the treatment with 6h also up-regulated the
expression of the phosphorylation of Histone H3, Cyclin B1 and
BubR1, further supporting that 6h could trigger cellular mitotic
arrest in protein level (Fig. 7C).
2.3. Inhibition of tubulin polymerization
Inhibitory effects of tubulin polymerization by the representa-
tive compound 6h was tested using isolated pig brain tubulin. As
shown in Fig. 4A and Table 2, tubulin polymerization was sup-
pressed dependent on the concentration of 6h with an IC50 value of
3.3 mM, which exhibited better inhibitory activity than the positive
control colchicine.
2.7. Inhibition of colony formation
Furthermore, cellular immunofluorescence assay was per-
formed to confirm the inhibitory effects of 6h on microtubule or-
ganization. As shown in Fig. 4B, the microtubule networks (green)
possessed the characteristic of a normal arrangement with the cell
nucleus were surrounded by slim and fibrous microtubules, indi-
cating tubulin assembly was not affected. However, when HeLa
cells were treated with 6h, the microtubule networks were found
to be disorganized and shrunk to the cell border (as the red arrows
indicated), indicating that the presence of 6h effectively induced
the disruption of the microtubule networks, and eventually resul-
ted in cell apoptosis.
Further studies were performed to investigate the cytotoxic
effects of 6h by colony formation assays. As shown in Fig. 8, the
colonies formed by HeLa were inhibited by exposure to 6h in the
dose dependent manner.
2.8. Anti-angiogenic effects in vitro
The tumor vascular disrupting activity of microtubule binding
agents can significantly disturb the microtubule dynamics, which
would in turn affect angiogenesis. The anti-angiogenic effect of 6h
was evaluated in a tube formation assay, based on the ability of
human umbilical vein endothelial cells (HUVECs) to form cord-like
and tubular networks on Matrigel, an extracellular matrix pos-
sessing fertile proangiogenic cytokines. As shown in Fig. 9, the
HUVEC cells treated with only the DMSO control produced a
complete network structure, while the introduction of 6h into the
system obviously altered the capillary-like structures in a dose-
dependent manner. These results showed that 6h effectively
restrained the tube formation of HUVECs.
2.4. Alkaline single cell gel electrophoresis (comet) assay and the
detection of DNA damage inducing factor
The comet assay is a commonly used, sensitive method to
investigate DNA damage in individual cells [39]. To test whether the
2H-azirine-based 6h could induce DNA damage, Jurkat cells were
treated with 6h at gradient concentrations, and afterwards alkaline
comet assay was used to evaluate the DNA damage. As shown in
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S. Lin, Y. Liang, J. Cheng et al.
European Journal of Medicinal Chemistry 214 (2021) 113256
Fig. 4. (A) Effects of 6h on tubulin polymerization in vitro. (B) Effects of 6h on the organization of cellular microtubule networks.
Table 2
2.11. Absolute configuration
IC50 values of 6h on inhibition of tubulin polymerization.
In order to confirm the absolute configuration of compound 6h,
(þ)-6h was synthetically elaborated by borohydride reduction and
sulfonylation, and the absolute configuration of the corresponding
sulfonamide derivative 8 was determined as (2R, 3S) by X-ray single
crystal diffraction, indicating that (þ)-6h was (R)-configuration
(Scheme 2).
Compound
IC50 M)
6h
Colchicine
10.4
(
m
3.3
2.9. Anti-tumor activities in vivo
We established A2780 cells xenograft model to evaluate the
anti-tumor activity of compound 6h. When the tumor volume
reached 100 mm3, the nude mice were placed in four groups
randomly (n ¼ 10 for each group), control, Paclitaxel (PTX) and 6h
with two doses. As shown in Fig. 10A, in contrast to the fast tumor
growth of the control group, treatment of 6h dose-dependently
inhibited tumor growth, and the efficacy of 50 mg/kg 6h was
found to be similar to that of PTX group. After the observation time
period, the weight of stripped tumor was collected and the analysis
results were shown in Fig. 10C and D, which demonstrated the
potent activity of 6h. Moreover, compared with the control group,
6h caused no obvious weight loss (Fig. 10B), which implied that 6h
displayed no indications of toxicity or other adverse reactions in
these mice, even at high dosage. These data strongly demonstrated
that compound 6h has potent anti-tumor efficacy with a reasonable
therapeutic window in vivo.
3. Conclusion
In this research, a novel series of 2,3-diaryl-2H-azirines were
designed and synthesized based on the combination principles,
among which compound 6h exhibited a remarkably potent anti-
proliferative activity against four cancer cell lines. Further phar-
macological studies demonstrated that 6h could target both tubulin
and DNA via tubulin polymerization assay and the comet assay,
respectively. Moreover, 6h could block cell cycle progression at the
G2/M phase, induce cell apoptosis, inhibit angiogenic activity,
possess favorable metabolic stability, and significantly suppress
xenografts tumor growth and show low toxicity. The anti-
proliferative activity between enantiomers (R)-(þ)-6h and (S)-
(ꢁ)-6h demonstrated no significant difference. Consequently, such
strategy by merging two different types of pharmacophores into
one agent can provide an effective therapy.
2.10. Optical resolution and antitumor evaluation
4. Experimental section
In order to clarify the impact of stereochemistry on the activity,
racemic 6h was submitted to optical resolution by chiral HPLC,
yielding enantiopure (ꢁ)-6h (100% ee) and (þ)-6h (100% ee). As
shown in Table 3, further antiproliferative evaluation indicated that
there is no significant difference in the activities between (þ)-6h
and (ꢁ)-6h.
4.1. General
Melting points were measured on an SGW X-4 apparatus which
was uncorrected. 1H and 13C spectra were obtained by a Varian
400 MHz or a Bruker 600 MHz NMR spectrometer at 303 K, using
5

S. Lin, Y. Liang, J. Cheng et al.
European Journal of Medicinal Chemistry 214 (2021) 113256
Fig. 5. The effect of 6h on DNA damage response in Jurkat cells. Jurkat cells were treated with a DMSO solution 6h (at the specified concentrations), MCC or DMSO for 48 h,
respectively. Representative fluorescent images of Jurkat cells following the modified alkaline comet assay and EB staining (A). Throught Comet Assay Software Pect (CASP 1.2.3 beta
1), Tail DNA% (B), Tail length (C), Tail moment (D) were calculated, respectively. (E) Western blot analysis of DNA damage-related proteins.
CDCl3 or DMSO‑d6 as the solvent with tetramethylsilane (TMS) as
an internal standard at room temperature. High-resolution mass
spectra were collected on an Agilent 6224 TOF LC/MS system.
Commercially obtained reagents and solvents were used without
further purification. Reactions were monitored by thin layer chro-
matography (TLC) by using silica gel (60, GF 254) coated glass slides
and detected using UV light (254 nm). Flash chromatography was
performed with standard silica gel 60 (200e300 mesh). The purity
of all the compounds for bioactivity testing was confirmed to
be ꢂ 95% by HPLC detection.
4.2.1. 1-(3-fluoro-4-methoxyphenyl)-2-(3,4,5-trimethoxyphenyl)
ethan-1-one (3a)
White solid. Yield 85%. Mp 106.5e109.3 ꢀC. 1H NMR (400 MHz,
CDCl3)
J ¼ 8.3 Hz, 1H), 6.46 (s, 2H), 4.16 (s, 2H), 3.96 (s, 3H), 3.84 (s, 6H),
3.83 (s, 3H). 13C NMR (150 MHz, CDCl3)
195.32, 153.37, 152.81,
d
7.81 (d, J ¼ 8.4 Hz, 1H), 7.76 (d, J ¼ 11.9 Hz, 1H), 7.00 (t,
d
152.05, 151.98, 151.16, 136.94, 130.03, 129.75, 129.72, 126.00, 125.98,
116.27, 116.14, 112.37, 106.35, 60.83, 56.30, 56.11, 45.47. ESI-HRMS
(m/z): calcd for C18H19FO5Na (M þ Naþ), 357.1108; found, 357.1119.
4.2.2. 1-(4-methoxyphenyl)-2-(3,4,5-trimethoxyphenyl)ethan-1-
one (3b)
4.2. General procedure for the synthesis of ketones (3a-r)
Light yellow solid. Yield 82%. Mp 84.9e87.5 ꢀC. 1H NMR
(400 MHz, CDCl3)
6.48 (s, 2H), 4.18 (s, 2H), 3.87 (s, 3H), 3.83 (s, 9H). 13C NMR
(150 MHz, CDCl3) 196.20, 163.62, 153.29, 136.80, 130.92, 130.52,
129.57, 113.83, 106.40, 60.83, 56.09, 55.50, 45.46. ESI-HRMS (m/z):
calcd for C18H20O5Na (M þ Naþ), 339.1202; found, 339.1199.
d
8.01 (d, J ¼ 8.5 Hz, 2H), 6.94 (d, J ¼ 8.4 Hz, 2H),
Under a nitrogen atmosphere, aryl acetonitrile 1 (3 mmol),
arylboronic acids 2 (6 mmol), palladium acetate (5 mol %), 2,20-
Bipyridine (10 mol %), trifluoroacetic acid (30 mmol), potassium
fluoride (6 mmol), tetrahydrofuran (20 mL), and water (10 mL)
were successively added to a Schlenk reaction tube. The reaction
mixture was stirred vigorously at 80 ꢀC for 2 h. After the reaction
mixture was cooled to room temperature, saturated sodium bi-
carbonate (30 mL) was added into the solution, and the mixture
was extracted with ethyl acetate (10 mL) for 3 times. The combined
organic layer was concentrated in vacuo. The residue was purified
by flash column chromatography to afford desired product 3.
d
4.2.3. 1-(benzo[d] [1,3]dioxol-5-yl)-2-(3,4,5-trimethoxyphenyl)
ethan-1-one (3c)
White solid. Yield 76%. Mp 117.4e119.0 ꢀC. 1H NMR (400 MHz,
CDCl3)
d
7.64 (d, J ¼ 8.1 Hz, 1H), 7.48 (s, 1H), 6.86 (d, J ¼ 8.1 Hz, 1H),
6.46 (s, 2H), 6.05 (s, 2H), 4.15 (s, 2H), 3.84 (s, 6H), 3.83 (s, 3H). 13
C
6

S. Lin, Y. Liang, J. Cheng et al.
European Journal of Medicinal Chemistry 214 (2021) 113256
Fig. 6. (A) Flow cytometry profiles indicated that 6h does-dependently promoted Hela cells apoptosis. (B) Statistic showed that 6h caused apoptosis of Hela cells. (C) Western blot
analysis of cell apoptosis-related proteins.
Fig. 7. Compound 6h regulated HeLa cell cycle progression. (A) Compound 6h could dose-dependently arrest cell cycle. (B) Statistic showed that 6h induced G2/M phase arrest in
HeLa cells. (C) Cell cycle-related proteins were analyzed by western blot analysis.
NMR (151 MHz, CDCl3)
131.34, 130.34, 124.99, 108.34, 107.94, 106.35, 101.92, 60.84, 56.10,
d
195.71, 153.32, 151.94, 148.27, 136.85,
45.54. ESI-HRMS (m/z): calcd for C18H18O6Na (M þ Naþ), 353.0995;
found, 353.0996.
7

S. Lin, Y. Liang, J. Cheng et al.
European Journal of Medicinal Chemistry 214 (2021) 113256
Fig. 8. Compound 6h dose-dependently inhibited cell colony formation of HeLa cells.
Fig. 9. Effects on the formation of a HUVEC capillary-like tubular network.
Fig. 10. Antitumor effect of 6h on the A2780 xenograft models. At the end of observation, tumor volumes (A), body weights (B), tumor weights (C) and representative isolated
tumors (D) were measured, respectively. Tumor volumes were gauged by vernier caliper every other day. Results represented the means SD. *p < 0.05, ***p < 0.001 vs untreated
control.
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European Journal of Medicinal Chemistry 214 (2021) 113256
Table 3
4.2.7. 1-(4-Methoxy-3-methylphenyl)-2-(3,4,5-trimethoxyphenyl)
ethan-1-one (3g)
Enantiomers of 6h and their antiproliferative activities.
Red solid. Yield 82%. Mp 83.4e86.2 ꢀC.1H NMR (400 MHz, CDCl3)
Compound
[
a
]25 D
ee
IC50 (mM)
d
7.89 (dd, J ¼ 8.5, 1.9 Hz, 1H), 7.84 (s, 1H), 6.85 (d, J ¼ 8.6 Hz, 1H),
6.48 (s, 2H), 4.17 (s, 2H), 3.89 (s, 3H), 3.84 (s, 6H), 3.82 (s, 3H), 2.25
(s, 3H). 13C NMR (150 MHz, CDCl3)
196.47, 161.92, 153.27, 136.77,
131.19, 130.69, 129.06, 128.84, 126.94, 109.25, 106.41, 60.83, 56.08,
A2780
HCT-116
HeLa
A549
(þ)-6h
(ꢁ)-6h
6h
þ356.18
ꢁ355.87
100%
100%
0.12
0.20
0.16
0.57
0.66
0.63
0.89
0.72
0.87
1.18
1.02
1.19
d
55.55,
C
45.40,
16.32.
ESI-HRMS
(m/z):
calcd
for
19H22O5Na(M þ Naþ), 353.1359; found, 353.1353.
4.2.4. 1-(3,4-dimethoxyphenyl)-2-(3,4,5-trimethoxyphenyl)ethan-
1-one (3d)
4.2.8. 2-(3-fluoro-4-methoxyphenyl)-1-(3,4,5-trimethoxyphenyl)
ethan-1-one (3h)
Light yellow solid. Yield 79%. Mp 151.2e156.1 ꢀC. 1H NMR
(400 MHz, CDCl
)
d
7.68 (d, J ¼ 8.3 Hz, 1H), 7.57 (s, 1H), 6.90 (d,
J ¼ 8.3 Hz, 1H), 6.49 (s, 2H), 4.19 (s, 2H), 3.95 (s, 3H), 3.93 (s, 3H),
3.84 (s, 6H), 3.83 (s, 3H). 13C NMR (150 MHz, CDCl3)
196.25,153.44,
White solid. Yield 79%. Mp 125.3e127.8 ꢀC.1H NMR (400 MHz,
3
DMSO)
2H), 3.85 (s, 6H), 3.81 (s, 3H), 3.74 (s, 3H). 13C NMR (150 MHz,
DMSO) 196.30, 152.94, 152.68, 151.87, 150.26, 145.66, 145.59,
d 7.33 (s, 2H), 7.16e7.07 (m, 2H), 7.04e7.02 (m, 1H), 4.35 (s,
d
153.32, 149.11, 136.85, 130.58, 129.72, 123.44, 110.61, 110.00, 106.36,
60.84, 56.10, 55.97, 45.37. ESI-HRMS (m/z): calcd for C19H22O6Na
(M þ Naþ), 369.1308; found, 369.1302.
d
141.82, 131.45, 128.03, 127.98, 125.74, 117.08, 116.96, 113.58, 105.90,
104.47, 60.06, 59.96, 56.01, 55.95, 55.81, 43.32. ESI-HRMS (m/z):
calcd for C18H19FO5Na(M þ Naþ), 357.1108; found, 357.1112.
4.2.5. 1-(4-ethoxyphenyl)-2-(3,4,5-trimethoxyphenyl)ethan-1-one
(3e)
4.2.9. 2-(4-methoxyphenyl)-1-(3,4,5-trimethoxyphenyl)ethan-1-
one (3i)
Light yellow solid. Yield 87%. Mp 99.6e101.1 ꢀC.1H NMR
White solid. Yield 83%. Mp 87.2e89.0 ꢀC. 1H NMR (400 MHz,
(400 MHz, CDCl3)
d
7.99 (d, J ¼ 8.4 Hz, 2H), 6.93 (d, J ¼ 8.4 Hz, 2H),
CDCl3)
4.19 (s, 2H), 3.90 (s, 3H), 3.89 (s, 6H), 3.78 (s, 3H). 13C NMR
(150 MHz, CDCl3) 196.76, 158.55, 153.00, 142.52, 131.71, 130.27,
d
7.26 (s, 2H), 7.19 (d, J ¼ 8.2 Hz, 2H), 6.87 (d, J ¼ 8.1 Hz, 2H),
6.48 (s, 2H), 4.17 (s, 2H), 4.10 (q, J ¼ 6.6 Hz, 2H), 3.83 (s, 6H), 3.82 (s,
3H), 1.44 (t, J ¼ 6.8 Hz, 3H). 13C NMR (150 MHz, CDCl3)
d 196.18,
d
163.06, 153.28, 136.80, 130.92, 130.56, 129.38, 114.25, 106.41, 63.79,
60.83, 56.09, 45.42, 14.67. ESI-HRMS (m/z): calcd for C19H22O5Na
(M þ Naþ), 353.1359; found, 353.1355.
126.76, 114.20, 106.20, 60.93, 56.24, 55.25, 44.72. ESI-HRMS (m/z):
calcd for C18H20O5Na (M þ Naþ), 339.1202; found, 339.1198.
4.2.10. 2-(benzo[d] [1,3]dioxol-5-yl)-1-(3,4,5-trimethoxyphenyl)
ethan-1-one (3j)
4.2.6. 1-(p-tolyl)-2-(3,4,5-trimethoxyphenyl)ethan-1-one (3f)
Red solid. Yield 84%. Mp 86.4e89.5 ꢀC.1H NMR (400 MHz, CDCl3)
White solid. Yield 75%. Mp 98.9e101.1 ꢀC. 1H NMR (400 MHz,
d
7.92 (d, J ¼ 8.2 Hz, 2H), 7.27 (d, J ¼ 8.2 Hz, 2H), 6.48 (s, 2H), 4.20 (s,
2H), 3.83 (s, 6H), 3.82 (s, 3H), 2.41 (s, 3H). 13C NMR (150 MHz, CDCl3)
197.28, 153.29, 144.16, 136.83, 134.06, 130.30, 129.38, 128.73,
CDCl3)
d
7.26 (s, 2H), 6.82e6.67 (m, 3H), 5.94 (s, 2H), 4.16 (s, 2H),
196.50,153.03,
3.91 (s, 3H), 3.90 (s, 6H). 13C NMR (150 MHz, CDCl3)
d
d
147.93, 146.60, 142.62, 131.62, 128.32, 122.37, 109.69, 108.49, 106.18,
101.05, 60.95, 56.28, 45.17. ESI-HRMS (m/z): calcd for C18H18O6Na
(M þ Naþ), 353.0995; found, 353.0989.
106.45, 60.83, 56.09, 45.62, 21.68. ESI-HRMS (m/z): calcd for
C
18H20O4Na (M þ Naþ), 323.1253; found, 323.1251.
Scheme 2. Synthesis of compound 8 and its X-ray single crystal structure.
9

S. Lin, Y. Liang, J. Cheng et al.
European Journal of Medicinal Chemistry 214 (2021) 113256
4.2.11. 2-(3,4-dimethoxyphenyl)-1-(3,4,5-trimethoxyphenyl)ethan-
1-one (3k)
4.2.18. 2-Phenyl-1-(3,4,5-trimethoxyphenyl)ethan-1-one(3r)
Yellow oil. Yield 81%.1H NMR (400 MHz, CDCl3)
d
7.33 (t,
J ¼ 7.2 Hz, 2H), 7.29e7.22 (m, 5H), 4.25 (s, 2H), 3.90 (s, 3H), 3.88 (s,
6H).13C NMR (150 MHz, CDCl3)
196.45, 153.01, 142.57, 134.84,
Yellow solid. Yield 81%. Mp 92.5e93.5 ꢀC. 1H NMR (400 MHz,
CDCl3)
d
7.27 (s, 2H), 6.83 (s, 2H), 6.79 (s, 1H), 4.20 (s, 2H), 3.91 (s,
196.71,
d
3H), 3.90 (s, 6H), 3.86 (s, 6H). 13C NMR (150 MHz, CDCl3)
d
131.69, 129.27, 128.76, 126.94, 106.24, 60.93, 56.24, 45.65.ESI-HRMS
153.02, 149.11, 148.04, 142.60, 131.70, 127.21, 121.43, 112.32, 111.35,
(m/z): calcd for C17H19O4 (M þ Hþ), 287.1278; found, 287.1279.
106.23, 60.95, 56.27, 55.89, 55.86, 45.20. ESI-HRMS (m/z): calcd for
C
19H22O6Na (M þ Naþ), 369.1308; found, 369.1311.
4.3. General procedure for the synthesis of ketoximes (4a-r)
The mixture of diaryl ethyl ketone 3 (3 mmol), hydroxylamine
hydrochloride (4.5 mmol) and potassium carbonate (6 mmol) in
anhydrous methanol (20 mL) was heated at 50 ꢀC until starting
materials (3a-r) had been completely consumed. The solvent was
removed under reduced pressure and the residue was directly
purified by flash column chromatography to give the title com-
pound 4.
4.2.12. 2-(4-ethoxyphenyl)-1-(3,4,5-trimethoxyphenyl)ethan-1-
one (3l)
White solid. Yield 84%. Mp 62.8e64.9 ꢀC. 1H NMR (400 MHz,
CDCl3)
d
7.26 (s, 2H), 7.17 (d, J ¼ 7.6 Hz, 2H), 6.86 (d, J ¼ 7.4 Hz, 2H),
4.18 (s, 2H), 4.01 (q, J ¼ 6.4 Hz, 2H), 3.90 (s, 3H), 3.89 (s, 6H), 1.40 (t,
J ¼ 6.9 Hz, 3H). 13C NMR (150 MHz, CDCl3)
d 196.82, 157.94, 153.00,
142.51, 131.73, 130.24, 126.62, 114.78, 106.23, 63.43, 60.94, 56.25,
44.79, 14.84. ESI-HRMS (m/z): calcd for C19H22O5Na (M þ Naþ),
353.1359 found, 353.1357.
4.3.1. 1-(3-fluoro-4-methoxyphenyl)-2-(3,4,5-trimethoxyphenyl)
ethan-1-one oxime (4a)
White solid. Yield 86%. Mp 123.5e126.0 ꢀC. 1H NMR (400 MHz,
4.2.13. 2-(p-tolyl)-1-(3,4,5-trimethoxyphenyl)ethan-1-one (3m)
White solid. Yield 87%. Mp 75.1e76.3 ꢀC. 1H NMR (400 MHz,
CDCl3)
d
7.43 (d, J ¼ 12.6 Hz, 1H), 7.34 (d, J ¼ 7.6 Hz, 1H), 6.91 (t,
J ¼ 8.6 Hz, 1H), 6.46 (s, 2H), 4.10 (s, 2H), 3.89 (s, 3H), 3.80 (d,
J ¼ 2.6 Hz, 9H). 13C NMR (150 MHz, CDCl3)
d
156.16, 153.34, 152.99,
CDCl3)
d
7.26 (s, 2H), 7.15 (dd, J ¼ 11.6, 8.4 Hz, 4H), 4.21 (s, 2H), 3.90
(s, 3H), 3.88 (s, 6H), 2.32 (s, 3H). 13C NMR (151 MHz, CDCl3)
d
196.66,
151.36, 148.68, 148.61, 136.58, 131.90, 128.65, 128.61, 122.71, 114.16,
114.03, 112.90, 105.40, 60.85, 56.20, 56.08, 31.97. ESI-HRMS (m/z):
calcd for C18H20FNO5Na (M þ Naþ), 372.1223; found, 372.1228.
152.99, 142.51, 136.55, 131.71, 129.48, 129.11, 106.24, 60.93, 56.24,
45.28, 21.07. ESI-HRMS (m/z): calcd for C18H20O4Na (M þ Naþ),
323.1253; found, 323.1247.
4.3.2. 1-(4-methoxyphenyl)-2-(3,4,5-trimethoxyphenyl)ethan-1-
4.2.14. 2-(4-(methylthio)phenyl)-1-(3,4,5-trimethoxyphenyl)
ethan-1-one (3n)
one oxime (4b)
Yellow oil. Yield 82%. 1H NMR (600 MHz, CDCl3)
d
7.57 (d,
J ¼ 8.9 Hz, 2H), 6.87 (d, J ¼ 8.9 Hz, 2H), 6.47 (s, 2H), 4.12 (s, 2H), 3.81
(s, 3H), 3.80 (s, 3H), 3.79 (s, 6H). 13C NMR (150 MHz, CDCl3)
160.55,
157.10, 153.28, 136.50, 132.27, 128.14, 127.87, 113.94, 105.50, 60.83,
Pink solid. Yield 87%. Mp 104.0e106.8 ꢀC. 1H NMR (400 MHz,
CDCl3)
d
7.25 (s, 2H), 7.21 (q, J ¼ 8.3 Hz, 4H), 4.21 (s, 2H), 3.91 (s, 3H),
d
3.89 (s, 6H), 2.47 (s, 3H). 13C NMR (151 MHz, CDCl3)
d
196.33, 153.03,
142.64, 137.04, 131.61, 131.54, 129.77, 127.01, 106.19, 60.95, 56.27,
44.99, 15.92. ESI-HRMS (m/z): calcd for C18H20O4SNa (M þ Naþ),
355.0974; found, 355.0971.
56.06, 55.30, 32.22. ESI-HRMS (m/z): calcd for
C18H21NO5Na
(M þ Naþ), 354.1317; found, 354.1313.
4.3.3. 1-(benzo[d] [1,3]dioxol-5-yl)-2-(3,4,5-trimethoxyphenyl)
ethan-1-one oxime (4c)
4.2.15. 1-(Naphthalen-2-yl)-2-(3,4,5-trimethoxyphenyl)ethan-1-
White solid. Yield 83%. Mp 143.3e146.6 ꢀC. 1H NMR (400 MHz,
one (3o)
Yellow oil. Yield 84%. 1H NMR (400 MHz, CDCl3)
d 8.55 (s, 1H),
CDCl3)
6.47 (s, 2H), 5.97 (s, 2H), 4.10 (s, 2H), 3.80 (s, 9H). 13C NMR
(150 MHz, CDCl3) 157.04, 153.30, 148.68, 147.96, 136.54, 132.12,
d
7.17 (s, 1H), 7.10 (d, J ¼ 7.9 Hz, 1H), 6.78 (d, J ¼ 8.0 Hz, 1H),
8.06 (d, J ¼ 8.6 Hz,1H), 7.96 (d, J ¼ 8.0 Hz,1H), 7.88 (t, J ¼ 8.9 Hz, 2H),
7.58 (m, 2H), 6.53 (s, 2H), 4.35 (s, 2H), 3.83 (d, J ¼ 3.7 Hz, 9H).13
C
d
129.81, 120.91, 108.15, 106.62, 105.45, 101.34, 60.84, 56.09, 32.30.
ESI-HRMS (m/z): calcd for C18H19NO6Na (M þ Naþ), 368.1104;
found, 368.1101.
NMR (150 MHz, CDCl3)
d 197.54, 153.35, 136.91, 135.62, 133.89,
132.48, 130.35, 130.19, 129.59, 128.64, 128.57, 127.80, 126.88, 124.19,
106.52, 60.83, 56.10, 45.76.ESI-HRMS (m/z): calcd for C21H21O4
(M þ Hþ), 337.1434; found, 337.1432.
4.3.4. 1-(3,4-dimethoxyphenyl)-2-(3,4,5-trimethoxyphenyl)ethan-
1-one oxime (4d)
Yellow solid. Yield 78%. Mp 118.2e120.6 ꢀC. 1H NMR (400 MHz,
4.2.16. 1-Phenyl-2-(3,4,5-trimethoxyphenyl)ethan-1-one (3p)
Yellow oil. Yield 79%. 1H NMR (400 MHz, CDCl3)
d
8.07e7.95 (m,
2H), 7.56 (d, J ¼ 7.3 Hz, 1H), 7.47 (t, J ¼ 7.5 Hz, 2H), 6.48 (s, 2H), 4.22
(s, 2H), 3.83 (s, 6H), 3.83 (s, 3H).13C NMR (150 MHz, CDCl3)
197.59,
153.33, 136.90, 136.57, 133.29, 130.04, 128.69, 128.57, 106.47, 60.83,
CDCl3)
1H), 6.83 (d, J ¼ 8.4 Hz,1H), 6.49 (s, 2H), 4.12 (s, 2H), 3.89 (s, 3H), 3.88
(s, 3H), 3.80 (s, 3H), 3.80 (s, 6H). 13C NMR (150 MHz, CDCl3)
157.15,
d
7.88 (s, 1H), 7.29 (d, J ¼ 1.9 Hz, 1H), 7.15 (dd, J ¼ 8.4, 2.0 Hz,
d
d
153.29, 150.21, 148.90, 136.49, 132.44, 128.36, 119.72, 110.60, 108.92,
105.46, 60.85, 56.07, 55.89, 55.84, 32.10, 29.33. ESI-HRMS (m/z): calcd
for C19H23NO6Na (M þ Naþ), 384.1416; found, 384.1417.
56.09, 45.71.ESI-HRMS (m/z): calcd for
287.1278; found, 287.1275.
C17H19O4 (M
þ
Hþ),
4.2.17. 2-(Naphthalen-2-yl)-1-(3,4,5-trimethoxyphenyl)ethan-1-
4.3.5. 1-(4-ethoxyphenyl)-2-(3,4,5-trimethoxyphenyl)ethan-1-one
oxime (4e)
one (3q)
Yellow oil. Yield 87%.1H NMR (400 MHz, CDCl3)
d
7.80 (m, 3H),
Red solid. Yield 86%. Mp 112.1e115.3 ꢀC. 1H NMR (400 MHz,
7.72 (s, 1H), 7.50e7.42 (m, 2H), 7.40 (d, J ¼ 8.4 Hz, 1H), 7.30 (s, 1H),
CDCl3)
d
7.56 (d, J ¼ 8.6 Hz, 2H), 6.86 (d, J ¼ 8.6 Hz, 2H), 6.48 (s, 2H),
4.41 (s, 2H), 3.89 (s, 3H), 3.88 (s, 6H).13C NMR (150 MHz, CDCl3)
4.13 (s, 2H), 4.03 (q, J ¼ 7.0 Hz, 2H), 3.80 (s, 3H), 3.78 (s, 6H), 1.41 (t,
d
196.44, 153.03, 142.62, 133.57, 132.36, 131.67, 128.42, 127.94,
J ¼ 6.9 Hz, 3H). 13C NMR (150 MHz, CDCl3)
d 159.94, 157.04, 153.25,
127.68,127.57, 127.40,126.21, 125.80, 106.27, 60.93, 56.25, 45.81.ESI-
HRMS (m/z): calcd for C21H21O4 (M þ Hþ), 337.1434; found,
337.1432.
136.44, 132.28, 127.91, 127.84, 114.45, 105.47, 63.50, 60.83, 56.05,
32.26, 14.75. ESI-HRMS (m/z): calcd for C19H23NO5Na (M þ Naþ),
368.1468; found, 368.1469.
10

S. Lin, Y. Liang, J. Cheng et al.
European Journal of Medicinal Chemistry 214 (2021) 113256
4.3.6. 1-(p-tolyl)-2-(3,4,5-trimethoxyphenyl)ethan-1-one oxime
(4f)
4.3.13. 2-(p-tolyl)-1-(3,4,5-trimethoxyphenyl)ethan-1-one oxime
(4m)
Yellow solid. Yield 79%. Mp 124.1e126.9 ꢀC. 1H NMR (400 MHz,
Colorless oil. Yield 86%. 1H NMR (400 MHz, CDCl3)
d
7.16 (d,
J ¼ 8.0 Hz, 2H), 7.08 (d, J ¼ 7.9 Hz, 2H), 6.86 (s, 2H), 4.14 (s, 2H), 3.84
(s, 3H), 3.81 (s, 6H), 2.30 (s, 3H). 13C NMR (150 MHz, CDCl3)
157.50,
CDCl3)
4.14 (s, 2H), 3.80 (s, 3H), 3.78 (s, 6H), 2.35 (s, 3H). 13C NMR
(150 MHz, CDCl3) 157.43, 153.24, 139.48, 136.45, 132.80, 132.20,
d
7.52 (d, J ¼ 8.2 Hz, 2H), 7.16 (d, J ¼ 8.0 Hz, 2H), 6.47 (s, 2H),
d
d
153.09, 139.08, 135.98, 133.59, 131.14, 129.36, 128.38, 103.88, 60.87,
129.28, 126.38, 105.48,60.83, 56.22, 56.04, 32.29, 21.27. ESI-HRMS
60.44, 56.09, 31.82, 21.07, 21.02, 14.20. ESI-HRMS (m/z): calcd for
C
(m/z): calcd for C18H21NO4Na (M þ Naþ), 338.1362; found, 338.1360.
18H21NO4Na (M þ Naþ), 338.1362; found, 338.1360.
4.3.7. 1-(4-Methoxy-3-methylphenyl)-2-(3,4,5-trimethoxyphenyl)
ethan-1-one oxime (4g)
4.3.14. 2-(4-(methylthio)phenyl)-1-(3,4,5-trimethoxyphenyl)
ethan-1-one oxime (4n)
Yellow solid. Yield 85%. Mp 117.1e120.4 ꢀC. 1H NMR (400 MHz,
White solid. Yield 79%. Mp 95.6e98.9 ꢀC. 1H NMR (400 MHz,
CDCl3)
1H), 6.49 (s, 2H), 4.13 (s, 2H), 3.83 (s, 3H), 3.80 (s, 3H), 3.79 (s, 6H),
2.20 (s, 3H). 13C NMR (150 MHz, CDCl3)
158.82, 157.22, 153.23,
136.42, 132.41, 128.63, 127.57, 126.80, 125.43, 109.63, 105.49, 60.83,
56.04, 55.35, 32.26, 16.37. ESI-HRMS (m/z): calcd for C19H23NO5Na
(M þ Naþ),368.1468; found,368.1459.
d
7.46 (s, 1H), 7.42 (dd, J ¼ 8.5, 2.1 Hz, 1H), 6.78 (d, J ¼ 8.5 Hz,
CDCl3)
d
7.19 (m, 4H), 6.84 (s, 2H), 4.13 (s, 2H), 3.84 (s, 3H), 3.81 (s,
157.24, 153.13, 139.19,
6H), 2.45 (s, 3H). 13C NMR (150 MHz, CDCl3)
d
d
136.31, 133.59, 130.94, 129.02, 127.10, 103.87, 60.89, 56.12, 31.68,
16.02. ESI-HRMS (m/z): calcd for C18H21NO4SNa (M þ Naþ),
370.1083; found, 370.1081.
4.3.15. 1-(Naphthalen-2-yl)-2-(3,4,5-trimethoxyphenyl)ethan-1-
4.3.8. 2-(3-fluoroxyphenyl)etha-4-methoxyphenyl)-1-(3,4,5-
trimethon-1-one oxime (4h)
one oxime (4o)
Colourless oil. Yield 73%. 1H NMR (400 MHz, CDCl3)
d
8.04 (s,
1H), 7.84 (m, 4H), 7.55e7.44 (m, 2H), 6.53 (s, 2H), 4.27 (s, 2H), 3.80
(s, 3H), 3.78 (s, 6H).13C NMR (150 MHz, CDCl3)
157.54, 153.32,
136.52, 133.70, 133.05, 132.98, 132.19, 128.50, 128.27, 127.65, 126.82,
126.49,126.45,123.65,105.48,105.00, 60.83, 56.22, 56.06, 32.11.ESI-
HRMS (m/z): calcd for C21H22NO4 (M þ Hþ), 352.1543; found,
352.1537.
White solid. Yield 85%. Mp 96.9e99.1 ꢀC. 1H NMR (400 MHz,
CDCl3)
J ¼ 9.1 Hz, 1H), 6.84 (s, 2H), 4.10 (s, 2H), 3.85 (d, J ¼ 2.6 Hz, 6H), 3.83
(s, 6H). 13C NMR (150 MHz, CDCl3)
157.09, 153.18, 151.54, 146.23,
d
7.04 (d, J ¼ 12.2 Hz, 1H), 6.97 (d, J ¼ 8.2 Hz, 1H), 6.87 (d,
d
d
146.16, 139.27, 130.83, 129.59, 129.55, 124.14, 124.12, 116.46, 116.34,
113.51, 103.84, 60.90, 56.28, 56.14, 31.29. ESI-HRMS (m/z): calcd for
C
18H20FNO5Na (M þ Naþ), 372.1217; found, 372.1216.
4.3.16. 1-Phenyl-2-(3,4,5-trimethoxyphenyl)ethan-1-one oxime
4.3.9. 2-(4-methoxyphenyl)-1-(3,4,5-trimethoxyphenyl)ethan-1-
one oxime (4i)
(4p)
Yellow oil. Yield 77%. 1H NMR (400 MHz, CDCl3)
d
7.67e7.57 (m,
2H), 7.36 (s, 3H), 6.47 (s, 2H), 4.15 (s, 2H), 3.80 (s, 3H), 3.78 (s,
6H).13C NMR (150 MHz, CDCl3)
157.50, 153.25, 136.49, 135.68,
White solid. Yield 72%. Mp 123.4e125.3 ꢀC. 1H NMR (400 MHz,
CDCl3)
d
8.97 (s, 1H), 7.19 (d, J ¼ 7.7 Hz, 2H), 6.85 (s, 2H), 6.81 (d,
d
J ¼ 7.5 Hz, 2H), 4.11 (s, 2H), 3.84 (s, 3H), 3.81 (s, 6H), 3.76 (s, 3H). 13
C
132.03, 129.39, 128.56, 126.51, 105.49, 60.83, 56.04, 32.39.ESI-HRMS
NMR (150 MHz, CDCl3)
d
158.16, 157.62, 153.10, 139.08, 131.14,
(m/z): calcd for C17H20NO4 (M þ Hþ), 302.1387; found, 302.1385.
129.53, 128.65, 114.08, 103.90, 60.88, 56.10, 55.24, 31.41. ESI-HRMS
(m/z): calcd for C18H21NO5Na (M þ Naþ), 354.1311; found, 354.1305.
4.3.17. 2-(Naphthalen-2-yl)-1-(3,4,5-trimethoxyphenyl)ethan-1-
one oxime (4q)
4.3.10. 2-(benzo[d] [1,3]dioxol-5-yl)-1-(3,4,5-trimethoxyphenyl)
ethan-1-one oxime (4j)
Yellow oil. Yield 79%. 1H NMR (400 MHz, CDCl3)
d
7.74 (m, 4H),
7.43 (s, 3H), 6.90 (s, 2H), 4.34 (s, 2H), 3.82 (s, 3H), 3.77 (s, 6H).13
NMR (150 MHz, CDCl3) 157.26, 153.13, 139.17, 134.22, 133.61,
C
White solid. Yield 76%. Mp 96.3e98.7 ꢀC. 1H NMR (400 MHz,
d
CDCl3)
2H), 4.09 (s, 2H), 3.85 (s, 3H), 3.83 (s, 6H). 13C NMR (150 MHz,
CDCl3) 157.44, 153.12, 147.91, 146.14, 139.16, 130.97, 130.36, 121.46,
d
8.17 (s, 1H), 6.86 (s, 2H), 6.78 (s, 1H), 6.73 (s, 2H), 5.91 (s,
132.17, 131.05, 128.36, 127.62, 127.49, 126.98, 126.85, 126.12, 125.55,
103.92, 60.86, 56.08, 32.47.ESI-HRMS (m/z): calcdfor C21H22NO4
(M þ Hþ), 352.1543; found, 352.1540.
d
109.07, 108.34, 103.86, 100.94, 60.89, 56.13, 31.73. ESI-HRMS (m/z):
calcd for C18H19NO6Na (M þ Naþ), 368.1104; found, 368.1109.
4.3.18. 2-Phenyl-1-(3,4,5-trimethoxyphenyl)ethan-1-one oxime
(4r)
4.3.11. 2-(3,4-dimethoxyphenyl)-1-(3,4,5-trimethoxyphenyl)ethan-
1-one oxime (4k)
Yellow oil. Yield 75%. 1H NMR (400 MHz, CDCl3)
d
7.27 (t,
J ¼ 4.6 Hz, 4H), 7.20 (dd, J ¼ 8.2, 4.0 Hz, 1H), 6.85 (s, 2H), 4.18 (s, 2H),
3.84 (s, 3H), 3.80 (s, 6H).13C NMR (150 MHz, CDCl3)
157.30, 153.10,
White solid. Yield 83%. Mp 137.4e139.9 ꢀC. 1H NMR (400 MHz,
d
cdcl3)
4.12 (s, 2H), 3.84 (s, 6H), 3.81 (s, 9H). 13C NMR (150 MHz, CDCl3)
157.58, 153.11, 149.06, 147.65, 139.13, 131.22, 129.16, 120.49, 111.86,
d
8.73 (s, 1H), 6.86 (s, 2H), 6.83 (s, 1H), 6.79 (d, J ¼ 5.6 Hz, 2H),
139.12, 136.73, 131.07, 128.68, 128.52, 126.46, 103.89, 60.88, 56.08,
32.31.ESI-HRMS (m/z): calcd for C17H20NO4 (M þ Hþ), 302.1387;
found, 302.1386.
d
111.28, 103.93, 60.89, 56.12, 55.88, 55.81, 31.86. ESI-HRMS (m/z):
calcd for C19H23NO6Na (M þ Naþ), 384.1417; found, 384.1421.
4.4. General procedure for the synthesis of 2H-azirines (6a-r)
4.3.12. 2-(4-ethoxyphenyl)-1-(3,4,5-trimethoxyphenyl)ethan-1-
one oxime (4l)
A solution of ketoxime 4a-r (1 mmol), acetic anhydride
(1.5 mmol) and triethylamine (2 mmol) in dichloromethane (15 mL)
was stirred at room temperature for 30 min. Water (20 mL) was
then added into the solution, and the mixture was extracted with
dichloromethane (10 mL) for 3 times. The organic layer was sepa-
rated, washed with brine, dried over anhydrous sodium sulfate and
filtered. After removing the solvent, the crude ketoxime acetates
5a-r were used directly for the next step.
White solid. Yield 81%. Mp 148.0e150.3 ꢀC. 1H NMR (400 MHz,
CDCl3)
d
9.01 (s, 1H), 7.18 (d, J ¼ 8.6 Hz, 2H), 6.85 (s, 2H), 6.80 (d,
J ¼ 8.7 Hz, 2H), 4.11 (s, 2H), 3.98 (q, J ¼ 7.0 Hz, 2H), 3.84 (s, 3H), 3.80
(s, 6H), 1.38 (t, J ¼ 7.0 Hz, 3H). 13C NMR (150 MHz, CDCl3)
d 157.63,
157.53, 153.08, 139.06, 131.17, 129.51, 128.51, 114.65, 103.90, 63.40,
60.88, 56.09, 31.45, 14.84. ESI-HRMS (m/z): calcd for C19H23NO5Na
(M þ Naþ), 368.1468; found, 368.1469.
Under a nitrogen atmosphere, ketoxime acetate 5a-r (0.5 mmol)
11

S. Lin, Y. Liang, J. Cheng et al.
European Journal of Medicinal Chemistry 214 (2021) 113256
and cesium carbonate (1 mmol) were stirred in N,N-dime-
thylformamide (DMF, 10 mL) at 80 ꢀC for 1 h. The reaction mixture
was then cooled to room temperature, extracted with ethyl acetate
(15 mL) for 3 times and washed with brine (15 mL). The combined
organic layer was dried over anhydrous sodium sulfate and
concentrated in vacuo. The residue was purified by flash column
chromatography to afford corresponding compounds 6a-r.
4.4.7. 3-(4-Methoxy-3-methylphenyl)-2-(3,4,5-trimethoxyphenyl)-
2H-azirine (6g)
Yellow solid. Yield 67%. Mp 96.6e98.8 ꢀC. 1H NMR (400 MHz,
CDCl3)
6.35 (s, 2H), 3.92 (s, 3H), 3.81 (s, 3H), 3.81 (s, 6H), 3.19 (s, 1H), 2.27
(s, 3H). 13C NMR (151 MHz, CDCl3)
162.36, 161.83, 153.28, 137.22,
137.10, 131.98, 129.91, 128.08, 115.64, 110.21, 102.75, 60.87, 56.05,
d
7.73 (d, J ¼ 8.7 Hz, 1H), 7.70 (s, 1H), 6.96 (d, J ¼ 8.3 Hz, 1H),
d
55.61, 34.37, 16.18. ESI-HRMS (m/z): calcd for
C19H21NO4Na
(M þ Naþ), 350.1362; found, 350.1357.
4.4.1. 3-(3-fluoro-4-methoxyphenyl)-2-(3,4,5-trimethoxyphenyl)-
2H-azirine (6a)
Yellow solid. Yield 78%. Mp 121.1e123.8 ꢀC. 1H NMR (400 MHz,
4.4.8. 2-(3-fluoro-4-methoxyphenyl)-3-(3,4,5-trimethoxyphenyl)-
2H-azirine (6h)
CDCl3)
3.98 (s, 3H), 3.81 (s, 9H), 3.24 (s, 1H). 13C NMR (150 MHz, CDCl3)
162.43, 153.34, 153.27, 151.98, 151.91, 151.62, 137.32, 136.46, 127.31,
d
7.66 (d, J ¼ 9.5 Hz, 2H), 7.11 (t, J ¼ 7.9 Hz, 1H), 6.34 (s, 2H),
Yellow solid. Yield 78%. Mp 84.3e86.5 ꢀC. 1H NMR (400 MHz,
CDCl3)
J ¼ 11.9 Hz, 1H), 3.95 (s, 3H), 3.92 (s, 6H), 3.87 (s, 3H), 3.28 (s, 1H).
13C NMR (150 MHz, CDCl3)
163.28, 153.89, 153.25, 151.62, 146.99,
d
7.12 (s, 1H), 6.91 (s, 1H), 6.90 (d, J ¼ 11.9 Hz, 1H), 6.84 (d,
d
116.91, 116.79, 116.65, 116.60, 113.40, 102.78, 60.88, 56.38, 56.06,
34.93. ESI-HRMS (m/z): calcd for C18H18FNO4Na (M þ Naþ),
354.1118; found, 354.1113.
d
146.91, 142.33, 134.14, 134.10, 121.97, 118.84, 113.87, 113.74, 113.37,
106.74, 61.05, 56.39, 34.3. ESI-HRMS (m/z): calcd for C18H18FNO4Na
(M þ Naþ), 354.1112; found, 354.1107.
4.4.2. 3-(4-methoxyphenyl)-2-(3,4,5-trimethoxyphenyl)-2H-
azirine (6b)
4.4.9. 2-(4-methoxyphenyl)-3-(3,4,5-trimethoxyphenyl)-2H-
azirine (6i)
Yellow solid. Yield 71%. Mp 117.5e120.6 ꢀC. 1H NMR (400 MHz,
Yellow solid. Yield 72%. Mp 88.8e91.5 ꢀC. 1H NMR (400 MHz,
CDCl3)
3.90 (s, 3H), 3.81 (s, 3H), 3.80 (s, 6H), 3.21 (s, 1H). 13C NMR
(150 MHz, CDCl3) 163.58, 162.38, 153.29, 137.15, 137.02, 131.91,
d
7.86 (d, J ¼ 8.2 Hz, 2H), 7.06 (d, J ¼ 8.2 Hz, 2H), 6.35 (s, 2H),
CDCl3)
3.94 (s, 3H), 3.91 (s, 6H), 3.79 (s, 3H), 3.32 (s, 1H). 13C NMR
(150 MHz, CDCl3) 163.80, 159.04, 153.84, 142.11, 132.89, 127.38,
d
7.14 (s, 2H), 7.09 (d, J ¼ 7.8 Hz, 2H), 6.84 (d, J ¼ 7.8 Hz, 2H),
d
116.28, 114.81, 102.75, 60.87, 56.05, 55.58, 34.41. ESI-HRMS (m/z):
d
calcd for C18H19NO4Na (M þ Naþ), 336.1212; found, 336.1204.
119.38, 113.87, 106.64, 61.04, 56.38, 55.33, 34.84. ESI-HRMS (m/z):
calcd for C18H19NO4Na (M þ Naþ), 336.1206; found, 336.1208.
4.4.3. 3-(benzo[d] [1,3]dioxol-5-yl)-2-(3,4,5-trimethoxyphenyl)-
2H-azirine (6c)
4.4.10. 2-(benzo[d] [1,3]dioxol-5-yl)-3-(3,4,5-trimethoxyphenyl)-
2H-azirine (6j)
Yellow solid. Yield 79%. Mp 113.6e115.5 ꢀC. 1H NMR (400 MHz,
CDCl3)
3.81 (s, 9H), 3.22 (s, 1H), 1.65 (s, 2H). 13C NMR (150 MHz, CDCl3)
162.73, 153.30, 152.01, 148.59, 137.22, 136.71, 126.38, 117.82,
d
7.40 (s, 2H), 6.96 (d, J ¼ 7.1 Hz, 1H), 6.34 (s, 2H), 6.10 (s, 2H),
Yellow solid. Yield 68%. Mp 89.2e91.5 ꢀC. 1H NMR (400 MHz,
CDCl3)
d
7.13 (s, 2H), 6.73 (m, 2H), 6.56 (s, 1H), 5.93 (s, 2H), 3.94 (s,
163.69,
d
3H), 3.92 (s, 6H), 3.29 (s, 1H). 13C NMR (150 MHz, CDCl3)
d
109.04, 108.69, 102.76, 102.09, 60.87, 56.05, 34.93. ESI-HRMS (m/z):
153.86, 147.86, 147.00, 142.21, 134.96, 119.84, 119.11, 108.22, 106.69,
calcd for C18H17NO5Na (M þ Naþ), 350.1004; found, 350.0990.
106.39, 101.05, 61.04, 56.39, 35.17. ESI-HRMS (m/z): calcd for
C
18H17NO5Na (M þ Naþ), 350.0998; found, 350.0991.
4.4.4. 3-(3,4-dimethoxyphenyl)-2-(3,4,5-trimethoxyphenyl)-2H-
azirine (6d)
4.4.11. 2-(3,4-dimethoxyphenyl)-3-(3,4,5-trimethoxyphenyl)-2H-
azirine (6k)
Yellow solid. Yield 75%. Mp 101.5e103.6 ꢀC. 1H NMR (400 MHz,
Yellow solid. Yield 77%. Mp 121.5e124.7 ꢀC. 1H NMR (400 MHz,
CDCl 3)
J ¼ 8.3 Hz, 1H), 6.36 (s, 2H), 3.97 (s, 6H), 3.82 (s, 3H), 3.81 (s, 6H),
3.24 (s, 1H). 13C NMR (150 MHz, CDCl3)
162.81, 153.32, 153.26,
d
7.48 (d, J ¼ 8.6 Hz, 1H), 7.43 (dd, J ¼ 8.2, 1.7 Hz, 1H), 6.99 (d,
CDCl 3)
6.66 (s, 1H), 3.94 (s, 3H), 3.92 (s, 6H), 3.87 (s, 3H), 3.84 (s, 3H), 3.31
(s, 1H). 13C NMR (150 MHz, CDCl3)
163.78, 153.85, 149.01, 148.49,
142.17, 133.44, 119.26, 118.56, 111.18, 109.18, 106.67, 61.04, 56.39,
d
7.14 (s, 2H), 6.82 (d, J ¼ 8.0 Hz, 1H), 6.74 (d, J ¼ 7.7 Hz, 1H),
d
d
149.69, 137.23, 136.92, 124.83, 116.41, 111.06, 110.91, 102.85, 60.89,
56.18, 56.16, 56.07, 34.97. ESI-HRMS (m/z): calcd for C19H21NO5Na
(M þ Naþ), 366.1317; found, 366.1312.
55.99, 55.89, 35.16. ESI-HRMS (m/z): calcd for
C19H21NO5Na
(M þ Naþ), 366.1311; found, 366.1312.
4.4.5. 3-(4-ethoxyphenyl)-2-(3,4,5-trimethoxyphenyl)-2H-azirine
(6e)
4.4.12. 2-(4-ethoxyphenyl)-3-(3,4,5-trimethoxyphenyl)-2H-azirine
(6l)
Yellow solid. Yield 81%. Mp 99.8e102.1 ꢀC. 1H NMR (400 MHz,
Yellow solid. Yield 74%. Mp 103.1e105.7 ꢀC. 1H NMR (400 MHz,
CDCl3)
d
7.84 (d, J ¼ 8.6 Hz, 2H), 7.04 (d, J ¼ 8.6 Hz, 2H), 6.35 (s, 2H),
CDCl3)
d
7.14 (s, 2H), 7.08 (d, J ¼ 8.6 Hz, 2H), 6.83 (d, J ¼ 8.6 Hz, 2H),
4.12 (q, J ¼ 6.9 Hz, 2H), 3.81 (s, 3H), 3.80 (s, 6H), 3.20 (s, 1H), 1.46 (t,
4.01 (q, J ¼ 7.0 Hz, 2H), 3.94 (s, 3H), 3.91 (s, 6H), 3.31 (s, 1H), 1.40 (t,
J ¼ 6.9 Hz, 3H). 13C NMR (150 MHz, CDCl3)
d 163.00, 162.34, 153.28,
J ¼ 7.0 Hz, 3H). 13C NMR (150 MHz, CDCl3)
d 163.83, 158.41, 153.84,
137.13, 137.07, 131.91, 116.03, 115.22, 102.74, 63.90, 60.87, 56.04,
34.37, 14.66. ESI-HRMS (m/z): calcd for C19H21NO4Na (M þ Naþ),
350.1368; found, 350.1364.
142.09, 132.72, 127.38, 119.43, 114.44, 106.63, 63.50, 61.04, 56.39,
34.89, 14.83. ESI-HRMS (m/z): calcd for C19H21NO4Na (M þ Naþ),
350.1362; found, 350.1354.
4.4.6. 3-(p-tolyl)-2-(3,4,5-trimethoxyphenyl)-2H-azirine (6f)
Yellow solid. Yield 73%. Mp 79.8e82.6 ꢀC. 1H NMR (400 MHz,
4.4.13. 2-(p-tolyl)-3-(3,4,5-trimethoxyphenyl)-2H-azirine (6m)
Yellow solid. Yield 80%. Mp 89.6e92.4 ꢀC. 1H NMR (400 MHz,
CDCl3)
3.81 (s, 3H), 3.80 (s, 6H), 3.23 (s, 1H), 2.46 (s, 3H). 13C NMR
(150 MHz, CDCl3) 163.26, 153.30, 144.22, 137.21, 136.86, 130.04,
d
7.81 (d, J ¼ 7.9 Hz, 2H), 7.37 (d, J ¼ 7.8 Hz, 2H), 6.35 (s, 2H),
CDCl3)
3.94 (s, 3H), 3.91 (s, 6H), 3.32 (s, 1H), 2.33 (s, 3H). 13C NMR
(150 MHz, CDCl3) 163.44, 153.83, 142.13, 137.84, 136.92, 129.05,
d
7.14 (s, 2H), 7.11 (d, J ¼ 8.0 Hz, 1H), 7.06 (d, J ¼ 8.1 Hz, 1H),
d
d
129.92, 121.16, 102.79, 60.87, 56.05, 34.55, 21.92. ESI-HRMS (m/z):
126.18, 119.28, 106.70, 61.04, 56.38, 35.08, 21.14. ESI-HRMS (m/z):
calcd for C18H19NO3Na (M þ Naþ), 320.1257; found, 320.1250.
calcd for C18H19NO3Na (M þ Naþ), 320.1257; found, 320.1254.
12

S. Lin, Y. Liang, J. Cheng et al.
European Journal of Medicinal Chemistry 214 (2021) 113256
4.4.14. 2-(4-(methylthio)phenyl)-3-(3,4,5-trimethoxyphenyl)-2H-
azirine (6n)
4.5.2. 2-(3-fluoro-4-methoxyphenyl)-1-methyl-3-(3,4,5-
trimethoxyphenyl)aziridine (7b)
Yellow solid. Yield 71%. Mp 84.7e86.0 ꢀC. 1H NMR (400 MHz,
7a (25 mg, 0.075 mmol) and MeI (2 mL) were charged into a
round-bottom bottle. The mixture was allowed to be stirred at
room temperature for 3 h, which was then concentrated under
reduced pressure. The resulting residue was purified by silica gel
column chromatography (elute with ethyl acetate/hexane ¼ 2:1, v/
v) to give 7b as white solid (21 mg, yield 81%), Mp 110.6e112.8 ꢀC.
CDCl3)
3.94 (s, 3H), 3.91 (s, 6H), 3.31 (s, 1H), 2.47 (s, 3H). 13C NMR
(150 MHz, CDCl3) 163.28, 153.86, 142.25, 137.92, 137.22, 126.72,
d
7.20 (d, J ¼ 8.3 Hz, 2H), 7.13 (s, 2H), 7.09 (d, J ¼ 8.3 Hz, 2H),
d
119.01, 106.73, 61.05, 56.40, 34.84, 16.05. ESI-HRMS (m/z): calcd for
C
18H19NO3SNa (M þ Naþ), 352.0977; found, 352.0981.
1H NMR (400 MHz, CDCl3)
d
6.97 (d, J ¼ 12.4 Hz, 1H), 6.87 (d,
J ¼ 8.4 Hz, 1H), 6.75 (t, J ¼ 8.5 Hz, 1H), 6.35 (s, 2H), 3.81 (s, 3H), 3.75
4.4.15. 3-(Naphthalen-2-yl)-2-(3,4,5-trimethoxyphenyl)-2H-
(s, 3H), 3.73 (s, 6H), 2.76 (d, J ¼ 6.48 Hz,1H), 2.72 (d, J ¼ 6.48 Hz,1H),
azirine (6o)
2.68 (s, 3H). 13C NMR (150 MHz, CDCl3)
d 152.67, 151.08, 146.30,
Yellow oil. Yield 67%. 1H NMR (400 MHz, CDCl3)
d 8.32 (s, 1H),
146.22, 136.60, 131.97, 129.68, 129.64, 123.47, 115.82, 115.69, 112.79,
104.63, 60.80, 56.25, 55.95, 50.54, 49.70, 47.77.ESI-HRMS (m/z):
calcd for C19H22FNO4Na (M þ Naþ), 370.1403; found, 370.1425.
8.03 (q, J ¼ 8.5 Hz, 2H), 7.93 (t, J ¼ 6.9 Hz, 2H), 7.60 (m, 2H), 6.41 (s,
2H), 3.82 (s, 3H), 3.80 (s, 6H), 3.35 (s, 1H).13C NMR (150 MHz, CDCl3)
d
191.09, 163.79, 153.64, 153.36, 137.32, 136.67, 135.62, 132.84,
132.05, 129.39, 129.08, 128.75, 128.11, 127.23, 124.56, 121.23, 106.70,
102.87, 60.87, 56.05, 35.05.ESI-HRMS (m/z): calcd for C21H20NO3
(M þ Hþ), 334.1438; found, 334.1435.
4.6. Procedure for the synthesis of chiral aziridine derivatives
4.6.1. (2R,3S)-2-(3-fluoro-4-methoxyphenyl)-3-(3,4,5-
trimethoxyphenyl)aziridine ((þ)-7a)
A mixture of (þ)-6h (115 mg, 0.345 mmol) and sodium boro-
hydride (50 mg, 1.39 mmol) in tetrahydrofuran (5 mL) and meth-
anol (5 mL) was stirred at room temperature for 2 h. The solvent
was removed under reduced pressure and the residue was directly
purified by flash column chromatography (elute with ethyl acetate/
hexane ¼ 2:1, v/v) to give the title compound (þ)-7a as yellow solid
(105 mg, yield 91%), Mp 117.4e120.3 ꢀC. 1H NMR (400 MHz, CDCl3)
4.4.16. 3-Phenyl-2-(3,4,5-trimethoxyphenyl)-2H-azirine (6p)
Yellow oil. Yield 64%. 1H NMR (400 MHz, CDCl3)
d
7.92 (d,
J ¼ 7.0 Hz, 2H), 7.66e7.48 (m, 3H), 6.36 (s, 2H), 3.82 (s, 3H), 3.80 (s,
6H), 3.26 (s, 1H).13C NMR (150 MHz, CDCl3)
163.80, 153.32, 137.29,
d
136.61, 133.29, 129.88, 129.29, 124.00, 102.81, 60.86, 56.05,
34.79.ESI-HRMS (m/z): calcd for C17H18NO3 (M þ Hþ), 284.1281;
found, 284.1279.
d
6.99 (dd, J ¼ 12.4, 1.8 Hz, 1H), 6.91 (d, J ¼ 8.4 Hz, 1H), 6.75 (t,
J ¼ 8.5 Hz, 1H), 6.39 (s, 2H), 3.81 (s, 3H), 3.76 (s, 3H), 3.74 (s, 6H),
3.50 (q, J ¼ 6.5 Hz, 2H). 13C NMR (150 MHz, CDCl3)
d 152.65, 151.06,
4.4.17. 2-(Naphthalen-2-yl)-3-(3,4,5-trimethoxyphenyl)-2H-
azirine (6q)
146.36, 146.29, 136.67, 132.06, 129.81, 129.77, 123.58, 123.56, 115.86,
115.74, 112.73, 104.73, 60.80, 56.21, 55.96, 39.90, 39.17. ESI-HRMS
(m/z): calcd for C18H20FNO4 (M þ Hþ), 334.1449; found, 334.1445.
Yellow oil. Yield 76%. 1H NMR (400 MHz, CDCl3)
d 7.86e7.72 (m,
3H), 7.67 (s, 1H), 7.45 (d, J ¼ 6.1 Hz, 2H), 7.23 (d, J ¼ 8.5 Hz, 1H), 7.17
(s, 2H), 3.94 (s, 3H), 3.90 (s, 6H), 3.50 (s, 1H).13C NMR (150 MHz,
CDCl3)
d 163.21, 153.88, 142.28, 138.44, 133.25, 132.79, 128.08,
4.6.2. (2R,3S)-2-(3-fluoro-4-methoxyphenyl)-1-((4-nitrophenyl)
sulfonyl)-3-(3,4,5-trimethoxyphenyl)aziridine (8)
127.73, 127.53, 126.30, 125.62, 125.05, 124.08, 119.05, 106.82, 61.04,
56.37, 35.41.ESI-HRMS (m/z): calcd for C21H20NO3 (M þ Hþ),
334.1438; found, 334.1435.
A
mixture of (þ)-7a (20 mg, 0.06 mmol), 4-
nitrobenzenesulphonyl chloride (20 mg, 0.09 mmol), triethyl-
amine (17 L, 0.12 mmol) and 4-dimethylaminopyridine (1 mg,
m
0.01 mmol) in dichloromethane (5 mL) was stirred at room tem-
perature for 1 h. The solvent was removed under reduced pressure
and the residue was directly purified by flash column chromatog-
raphy (elute with ethyl acetate/petroleum ether ¼ 1:5, v/v) to give
the title compound 8 as yellow solid (27 mg, yield 87%), Mp
4.4.18. 2-Phenyl-3-(3,4,5-trimethoxyphenyl)-2H-azirine (6r)
Yellow oil. Yield 73%. 1H NMR (400 MHz, CDCl3)
d 7.34e7.21 (m,
3H), 7.20e7.15 (m, 2H), 7.14 (s, 2H), 3.94 (d, J ¼ 1.6 Hz, 3H), 3.90 (d,
J ¼ 1.6 Hz, 6H), 3.33 (s, 1H).13C NMR (150 MHz, CDCl3)
d 163.12,
153.85, 142.21, 140.89, 128.33, 127.15, 126.23, 119.08, 106.76, 61.03,
133.1e134.2 ꢀC. 1H NMR (400 MHz, CDCl3)
d
8.42 (d, J ¼ 8.9 Hz, 2H),
56.39, 35.17.ESI-HRMS (m/z): calcd for
C
17H18NO3(M
þ
Hþ),
8.27 (d, J ¼ 9.0 Hz, 2H), 6.85e6.72 (m, 3H), 6.22 (s, 2H), 4.28e4.20
284.1281; found, 284.1279.
(m, 2H), 3.80 (s, 3H), 3.75 (s, 3H), 3.65 (s, 6H).13C NMR (150 MHz,
CDCl3) d153.06, 152.72, 151.08, 150.83, 147.70, 147.63, 143.69, 138.03,
4.5. Procedure for the synthesis ofaziridinederivatives
129.29, 126.38, 124.52, 124.11, 124.07, 123.70, 115.59, 115.46, 113.08,
104.84, 60.82, 56.19, 56.02, 48.34, 47.44. ESI-HRMS (m/z): calcd for
4.5.1. 2-(3-fluoro-4-methoxyphenyl)-3-(3,4,5-trimethoxyphenyl)
aziridine (7a)
C
24H23FN2O8S (M þ Naþ), 541.1051; found, 541.1050.
Single crystals of 8 suitable for X-ray crystallographic analysis
A mixture of 6h (23 mg, 0.069 mmol) and sodium borohydride
(10 mg, 0.278 mmol) in tetrahydrofuran (1.5 mL) was stirred at
room temperature for 2 h. The solvent was removed under reduced
pressure and the residue was directly purified by flash column
chromatography (elute with ethyl acetate/hexane ¼ 2:1, v/v) to give
the title compound 7a as yellow solid (21 mg, yield 91%), Mp
were obtained by slow evaporation of a solution of 8 in petroleum
ether/dichloromethane (Table 4).
CIF file of 8 can be obtained from the Cambridge Crystallo-
graphic Data Centre using deposition number CCDC2036521.
Copies of the data can be obtained, free of charge, on application to
the CCDC, 12 Union Road, Cambridge CB2 1EZ, UK [fax: þ44(1223)
336033; e-mail: deposit@ccdc.cam.ac.uk].
117.4e120.3 ꢀC. 1H NMR (400 MHz, CDCl3)
d
6.99 (dd, J ¼ 12.4,
1.8 Hz, 1H), 6.91 (d, J ¼ 8.4 Hz, 1H), 6.75 (t, J ¼ 8.5 Hz, 1H), 6.39 (s,
2H), 3.81 (s, 3H), 3.76 (s, 3H), 3.74 (s, 6H), 3.51 (d, J ¼ 6.56 Hz, 1H),
4.7. In vitro cell growth inhibition assay
3.49 (d, J ¼ 6.56 Hz, 1H). 13C NMR (150 MHz, CDCl3)
d 152.65, 151.06,
146.36, 146.29, 136.67, 132.06, 129.81, 129.77, 123.58, 123.56, 115.86,
115.74, 112.73, 104.73, 60.80, 56.21, 55.96, 39.90, 39.17. ESI-HRMS
(m/z): calcd for C18H20FNO4 (M þ Hþ), 334.1449; found, 334.1445.
Human ovarian carcinoma (A2780), human colonic adenoma
(HCT-116), human lung adenocarcinoma (A549), human cervical
carcinoma (HeLa) and immortalized T-lymphocyte (Jurkat) were
13

S. Lin, Y. Liang, J. Cheng et al.
European Journal of Medicinal Chemistry 214 (2021) 113256
Table 4
Crystal data for 8.
4.10. Cell cycle analysis
HeLa cells were seeded into a six-well plate and being cultured
at 37 ꢀC for 24 h. After treatment with gradient concentrations of 6h
or DMSO (negative control) for 24 h, cells were collected, centri-
fuged and fixed with 75% ethanol at ꢁ20 ꢀC overnight. Fixed cells
were treated with RNase A solution and stained with a propidium
iodide containing PBS solution at room temperature in dark con-
ditions. Cell cycle analysis was determined with Multicycle AV (for
Windows, version 320) software.
Temperature
170.01 K
Wavelength
Crystal system
Space group
1.34139 Å
Monoclinic
C 1 2 1
Unit cell dimensions
b ¼ 7.7471(2) Å
a ¼ 21.7846(5) Åa ¼ 90ꢀ
b ¼ 123.5070(10)ꢀ.
g ¼ 90ꢀ.
c ¼ 18.7739(4) Å
Volume
2641.89(11) Å3
4
Z
Density (calculated)
Absorption coefficient
F(000)
Crystal size
Theta range for data collection
Index ranges
Reflections collected
Independent reflections
Completeness to theta ¼ 53.594ꢀ
Absorption correction
Max. and min. transmission
Refinement method
Data/restraints/parameters
Goodness-of-fit on F2
Final R indices [I > 2sigma(I)]
R indices (all data)
Absolute structure parameter
Extinction coefficient
Largest diff. peak and hole
1.454 Mg/m3
1.969 mmꢁ1
4.11. Cell apoptosis analysis
1196
0.12 ꢃ 0.06 ꢃ 0.05 mm3
3.532e54.971ꢀ.
ꢁ26 ꢄ h<¼23, ꢁ6 ꢄ k<¼9, ꢁ22 ꢄ l<¼22
11981
HeLa cells growth was seeded into each well of six-well plate.
After incubation overnight, cells were treated with gradient con-
centrations of 6h or DMSO (negative control) for 48 h. Then the
cells were collected, centrifuged and resuspended in staining so-
lution (containing Annexin V-FITC and propidium iodide in Binding
Buffer) in the dark place for 15 min. Samples were analyzed on flow
cytometer (Cytomics FC 500 MPL, Beckman Coulter) analysis.
4427 [R(int) ¼ 0.0454]
99.7%
Semi-empirical from equivalents
0.7508 and 0.5628
Full-matrix least-squares on F2
4427/24/365
1.043
4.12. Western blot analysis
R1 ¼ 0.0461, wR2 ¼ 0.0975
R1 ¼ 0.0572, wR2 ¼ 0.1050
0009(15)
After treatment with gradient concentrations of 6h or DMSO
(negative control), HeLa cells were collected, washed twice with
phosphate buffer saline and lysed with cell lysis buffer. After
centrifugation for 30 min at 4 ꢀC, supernatant was obtained. Protein
concentrations were determined by BCA protein assay kit. Then
protein samples were separated by SDS-PAGE electrophoresis and
transferred onto polyvinylidene fluoride (PVDF) membranes. After
being blocked with 5% BSA in TBST, membranes were interacted
with diluted primary antibodies at 4 ꢀC overnight and incubated
with horseradish peroxidase (HRP) labeled secondary antibodies at
room temperature. Finally, protein was detected by chemilumi-
nescent reagent.
n/a
0.604 and ꢁ0.417 e.Åꢁ3
cultivated in Dulbecco’s modified Eagle medium (DMEM) con-
taining 100 units/mL penicillin G, 100 mg/mL streptomycin and 10%
(v/v) fetal bovine serum in 37 ꢀC and 5% carbon dioxide
atmosphere.
The anti-proliferative of compounds were evaluated by
employing MTT assay. Cells were seeded in 96-well plates for 24 h.
Then cells were treated with different concentrations of com-
pounds for 48 h. 20
well, followed by incubating for another 4 h at 37 ꢀC. Then, the
suspension was discarded and DMSO (150 L) was added to each
mL MTT solution (5 mg/mL) was added to each
4.13. Immunofluorescent analysis
m
well. The plates were read by microplate reader (Biotech ELx800) at
the wavelength of 570 nm and half maximal inhibitory concen-
tration (IC50) was counted using software GraphPad Prism (version
6.0).
HeLa cells were cultured with DMSO (negative control) or the
tested compound with different concentrations overnight, HeLa
cells were fixed by methanol, permeabilized with 0.1% Triton X-100
and incubated in 3% BSA for 1 h. Then cells were incubated with a-
tubulin monoclonal antibody overnight at 4 ꢀC and stained with
Alexa 488 labeled secondary antibody dilution. Then the nucleus of
cells were stained with DAPI for 10 min at room temperature. Im-
4.8. Colony formation assay
ages were collected by
microscope.
a Leica SP5 co-focal fluorescence
HeLa cells were seeded into a six-well plate. After being cultured
at 37 ꢀC for 24 h, the cells were treated with gradient concentra-
tions of 6h or DMSO (negative control) for 48 h, followed by
replacing the agents by fresh medium and incubation at 37 ꢀC for
additional 7e10 days. Then the cells were dyed with gentian violet
staining solution for 30 min and washed twice with fresh phos-
phate buffer saline. The stained colonies were counted and
compared with the control cells.
4.14. Tube formation assay
Matrigel was added into a 96-well plate and incubated at 37 ꢀC
for 2 h to emerge its polymerization morphology, and human
umbilical vein endothelial cells (HUVECs) suspended in DMEM
were seeded afterwards. They were then treated with The tested
compound with different concentrations or DMSO (negative con-
trol). After incubation for 12 h at 37 ꢀC, capillary-like tube formed,
and cells were photographed with a CCD Sensicam camera attached
to an inverted microscope.
4.9. In vitro tubulin polymerization assay
Pig brain microtubule protein was commercially available.
Compound 6h with a range of concentrations, colchicine (positive
control) or DMSO (negative control) were treated with a solution of
tubulin protein in G-PEM buffer (100 mM PIPES, 10 mM MgCl2,
1 mM EGTA, 1 mM GTP and 3.4 M glycerol). Then the mixtures were
transferred to 37 ꢀC and detected light scattering at 340 nm using a
SPECTRA MAX 190 (Molecular Device) spectrophotometer. And the
plateau absorbance values were used for calculations.
4.15. Comet assay
The following processes should be operated in the darkness to
avoid additional DNA damage. Jurkat cells were seeded into a six-
well plate and then exposed to various concentrations of tested
compounds, MMC (positive control), or DMSO (negative control)
14

S. Lin, Y. Liang, J. Cheng et al.
European Journal of Medicinal Chemistry 214 (2021) 113256
and incubated at 37 ꢀC for 48 h, followed by combination with
molten LM Agarose and were immediately pipette onto Comet
Slide, which were immersed in Lysis buffer successively and incu-
bated for 2 h at 4 ꢀC. Slides were dipped into alkaline unwinding
solution (1 mM EDTA, 200 mM NaOH) for 30 min, followed by
electrophoresis solution (1 mM EDTA, 200 mM NaOH) at 25 V for
15 min, and washed twice in water. The slides were stained with
ethidium bromide (EB) and washed twice with water. The results
were visualized by microscopy (OLYMPOS BX51). Under these
conditions the formation of “comet tail” is indicative of DBSs, SSDs,
and/or active excision repair of DNA cross-links.
Acknowledgements
This work was supported by grants from the National Natural
Science Foundation of China (21877015) and Science and Technol-
ogy Commission of Shanghai Municipality (20S11902500).
Appendix A. Supplementary data
Supplementary data related to this article can be found at
https://doi.org/10.1016/j.ejmech.2021.113256.
References
4.16. Xenografts tumor growth assay in nude mice
[1] A. Jordan, J.A. Hadfield, N.J. Lawrence, A.T. Mcgown, Tubulin as a target for
anticancer drugs: agents which interact with the mitotic spindle, Med. Res.
Rev. 18 (1998) 259e296.
[2] R. Heald, E. Nogales, Microtubule dynamics, J. Cell Sci. 115 (2002) 3e4.
[3] J.R. McIntosh, E. Grishchuk, R.R. West, Chromosome-microtubule interactions
during mitosis, Annu. Rev. Cell Dev. Biol. 18 (2002) 193e219.
[4] M. Kavallaris, Microtubules and resistance to tubulin-binding agents, Nat. Rev.
Canc. 10 (2010) 194e204.
[5] G.G. Dark, S.A. Hill, V.E. Prise, G.M. Tozer, G.R. Pettit, D. J Chaplin, Com-
bretastatin A-4, an agent that displays potent and selective toxicity toward
tumor vasculature, Canc. Res. 57 (1997) 1829e1834.
[6] R. Mikstacka, T. Stefanski, J. Rozanski, Tubulin-interactive stilbene derivatives
as anticancer agents, Cell. Mol. Biol. Lett. 18 (2013) 368e397.
[7] G.C. Tron, T. Pirali, G. Sorba, F. Pagliai, S. Busacca, A.A. Genazzani, Medicinal
chemistry of combretastatin A4: present and future directions, J. Med. Chem.
49 (2006) 3033e3044.
We purchased 6 weeks old female Balb/C nude mice from
Shanghai Slac Laboratory Animal Co. Ltd. (Shanghai, China), and
implanted A2780 cells suspended in PBS in the right flank of mice.
When tumor volumes reached 100 mm3, mice were treated with
compound 6h (25, 50 mg/kg), PTX (10 mg/kg) or vehicle (con-
taining 10% castor oil and 10% DMSO in PBS) every two days. Tumor
volumes and body weights were gauged when administrated with
a digital caliper each time (calculated volume (mm3) ¼ (
p
/
6) ꢃ length ꢃ width ꢃ width). The mice were sacrificed and the
tumors were isolated and weighed subsequently when the average
tumor volume reached 2000 mm3. The animal experimental pro-
tocols were approved by the Animal Ethics Committee of School of
Pharmacy, Fudan University.
[8] L. Li, S. Jiang, X. Li, Y. Liu, J. Su, J. Chen, Recent advances in trimethoxyphenyl
(TMP) based tubulin inhibitors targeting the colchicine binding site, Eur. J.
Med. Chem. 151 (2018) 482e494.
[9] Y. Lu, J. Chen, M. Xiao, W. Li, D.D. Miller, Anoverview of tubulin inhibitors that
interact with the colchicine binding site, Pharm. Res. (N. Y.) 29 (2012)
2943e2971.
4.17. Enantiomeric separation
[10] N.M. O’Boyle, J.K. Pollock, M. Carr, A.J.S. Knox, S.M. Nathwani, S. Wang,
L. Caboni, D.M. Zisterer, M.J. Meegan,
and dual-targeting estrogen receptor/tubulin ligand, J. Med. Chem. 57
(2014) 9370e9382.
[11] P. Zhou, Y. Liu, L. Zhou, K. Zhu, K. Feng, H. Zhang, Y. Liang, H. Jiang, C. Luo,
M. Liu, Y. Wang, Potent antitumor activities and structure basis of the chiral
lactam bridged analogue of combretastatin A-4 binding to tubulin, J. Med.
Chem. 59 (2016) 10329e10334.
[12] M. Zhang, Y.R. Liang, H. Li, M. Liu, Y. Wang, Design, synthesis, and biological
evaluation of hydantoin bridged analogues of combretastatin A-4 as potential
anticancer agents, Bioorg. Med. Chem. 25 (2017) 6623e6634.
b-Lactam estrogen receptor antagonists
The separation was performed on the Themo Fisher Ultimate
3000 HPLC equipped with Daicel Chiralpak IC. The mobile phase
was n-hexane/i-PrOH (80/20, v/v) with flow rate of 1.0 mL/min. The
UV response was detected under 230 nm at 25 ꢀC.
The specific rotation data were recorded at 25 ꢀC on a Rudolph
Autopol IV polarimeter with 1.0 mg/mL of each enantiomer in
CHCl3.
a
b-
[13] R. Romagnoli, P.G. Baraldi, A. Brancale, A. Ricci, E. Hamel, R. Bortolozzi,
G. Basso, G. Viola, Convergent synthesis and biological evaluation of 2-amino-
4-(3’,4’,5’-trimethoxyphenyl)-5-aryl thiazoles as microtubule targeting
agents, J. Med. Chem. 54 (2011) 5144e5153.
[14] S. Theeramunkong, A. Caldarelli, A. Massarotti, S. Aprile, D. Caprioglio,
R. Zaninetti, A. Teruggi, T. Pirali, G. Grosa, G.G. Tron, Regioselectivesuzuki
coupling of dihaloheteroaromatic compounds as a rapid strategy to synthesize
potent rigid combretastatin analogues, J. Med. Chem. 54 (2011) 4977e4986.
[15] F. Gao, Y. Liang, P. Zhou, J. Cheng, K. Ding, Y. Wang, Design, synthesis, anti-
tumor activities and biological studies of novel diaryl substituted fused het-
erocycles as dual ligands targeting tubulin and katanin, Eur. J. Med. Chem. 178
(2019) 177e194.
4.18. Preliminary stability study
The solution of 6h (5 mg/mL in methanol, 20
phosphate buffers (480 L, pH 7.4). The resulted solutions were
filtered by 0.22 m microfiltration membrane. The filtrate were
m;
mL) was mixed with
m
m
analyzed by HPLC (Poroshell 120 EC-C18 3.0 ꢃ 50 mm, 2.7
m
mobile phase methanol/H2O, 60:40, 1 mL/min) after 4 h, 8 h, 12 h,
24 h and 48 h under pH 7.4 condition, respectively. 95.77% of
compound 6h was recovered at pH 7.4 media after 48 h.
[16] Y.N. Liu, J.J. Wang, Y.T. Ji, G.D. Zhao, L.Q. Tang, C.M. Zhang, X.L. Guo, Z.P. Liu,
Design, synthesis, and biological evaluation of 1-methyl-1,4-dihydroindeno
[1,2-c]pyrazole analogues as potential anticancer agents targeting tubulin
colchicine binding site, J. Med. Chem. 59 (2016) 5341e5355.
Author contributions
[17] A. Stornetta, M. Zimmermann, G.D. Cimino, P.T. Henderson, S.J. Sturla, DNA
adducts from anticancer drugs as candidate predictive markers for precision
medicine, Chem. Res. Toxicol. 30 (2017) 388e409.
The manuscript was written through contributions of all au-
thors./All authors have given approval to the final version of the
manuscript.
[18] M.D. Urbaniak, J.P. Bingham, J.A. Hartley, D.N. Woolfson, S. Caddick, Design
and synthesis of
a nitrogen mustard derivative stabilized by apo-neo-
carzinostatin, J. Med. Chem. 47 (2004) 4710e4715.
[19] A.A. Nazarov, S.M. Meier, O. Zava, Y.N. Nosova, E.R. Milaeva, C.G. Hartinger,
P.J. Dyson, Protein ruthenation and DNA alkylation: chlorambucil-
functionalized RAPTA complexes and their anticancer activity, Dalton Trans.
44 (2015) 3614e3623.
[20] B.S. Iyengar, R.T. Dorr, D.S. Alberts, E.M. Hersh, S.E. Salmon, W.A. Remers,
Novel antitumor 2-cyanoaziridine-1-carboxamides, J. Med. Chem. 42 (1992)
510e514.
Notes
The authors declare no competing financial interest.
Declaration of competing interest
[21] E. Wall, J.H. Beijnen, S. Rodenhuis, High-dose chemotherapy regimens for
solid tumors, Canc. Treat Rev. 21 (1995) 105e132.
[22] W.T. Bradner, Mitomycin C: a clinical update, Canc. Treat Rev. 27 (2001)
35e50.
[23] S.S. Pan, P.A. Andrews, C.J. Glover, N.R. Bachur, Reductive activation of mito-
mycin C and mitomycin C metabolites catalyzed by NADPH-cytochrome P-
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
15

S. Lin, Y. Liang, J. Cheng et al.
European Journal of Medicinal Chemistry 214 (2021) 113256
450 reductase and xanthine oxidase, J. Biol. Chem. 259 (1984) 959e966.
[24] V.J. Spanswick, J. Cummings, J.F. Smyth, Enzymology of mitomycin C meta-
bolic activation in tumour tissue: characterization of a novel mitochondrial
reductase, Biochem. Pharmacol. 51 (1998) 405.
[25] M. Sastry, R. Fiala, R. Lipman, M. Tomasz, D.J. Patel, Solution structure of the
monoalkylated mitomycin C-DNA complex, J. Mol. Biol. 247 (1995) 338.
[26] C.M. Mallory, R.P. Carfi, S.P. Moon, K.A. Cornell, D.L. Warner, Modification of
cellular DNA by synthetic aziridinomitosenes, Bioorg. Med. Chem. 23 (2015)
7378e7385.
microtubule inhibitors displayed potent antitumor activity and ability to
overcome cisplatin resistance, Eur. J. Med. Chem. 156 (2018) 666e679.
[33] X. Huang, R. Huang, Z. Wang, L. Li, S. Gou, Z. Liao, H. Wang, Pt(IV) complexes
conjugating with chalcone analogue as inhibitors of microtubule polymeri-
zation exhibited selective inhibition in human cancer cells, Eur. J. Med. Chem.
146 (2018) 435e450.
[34] X. Huang, R. Huang, S. Gou, Z. Wang, Z. Liao, H. Wang, Combretastatin A-4
analogue: a dual-targeting and tubulin inhibitor containing antitumor Pt(IV)
moiety with
a unique mode of action, Bioconjugate Chem. 27 (2016)
[27] X. Qin, L. Fang, F. Chen, S. Gou, Conjugation of platinum (IV) complexes with
chlorambucil to overcome cisplatin resistance via a “joint action” mode to-
ward DNA, Eur. J. Med. Chem. 137 (2017) 167e175.
[28] S.M. Musser, S.S. Pan, M.J. Egorin, D. J Kyle, P. S Callery, Alkylation of DNA with
aziridine produced during the hydrolysis of N,N’,N’’-triethylenethiophos-
phoramide, Chem. Res. Toxicol. 5 (1992) 95e99.
[29] H. Kostrhunova, J. Zajac, V. Novohradsky, J. Kasparkova, J. Malina, J.R. Aldrich-
Wright, E. Petruzzella, R. Sirota, D. Gibson, V. Brabec, A subset of new plat-
inum antitumor agents kills cells by a multimodal mechanism of action also
involving changes in the organization of the microtubule cytoskeleton, J. Med.
Chem. 62 (2019) 5176e5190.
2132e2148.
[35] Y. Zhang, M. Zhai, Z. Chen, X. Han, F. Yu, Z. Li, X. Xie, C. Han, L. Yu, Y. Yang,
X. Mei, Dual-modified liposome codelivery of doxorubicin and vincristine
improve targeting and therapeutic efficacy of glioma, Drug Deliv. 24 (2017)
1045e1055.
[36] S. Yu, L. Qi, K. Hu, J. Gong, T. Cheng, Q. Wang, J. Chen, H. Wu, The development
of a palladium-catalyzed tandem addition/cyclization for the construction of
indole skeletons, J. Org. Chem. 82 (2017) 3631e3638.
[37] M.N. Zhao, W. Zhang, X.C. Wang, Y. Zhang, D. Yang, Z. Guan, Modular 2,3-
diaryl-2H-azirine synthesis from ketoxime acetates via Cs2CO3-mediated
cyclization, Org. Biomol. Chem. 16 (2018) 4333e4337.
[30] R. Schobert, B. Biersack, A. Dietrich, S. Knauer, M. Zoldakova, A. Fruehauf,
T. Mueller, Pt (II) complexes of a combretastatin A-4 analogous chalcone:
effects of conjugation on cytotoxicity, tumor specificity, and long-term tumor
growth suppression, J. Med. Chem. 52 (2009) 241e246.
[31] P. Diana, A. Martorana, P. Barraja, A. Montalbano, G. Dattolo, G. Cirrincione,
F.D. Acqua, A. Salvador, D. Vedaldi, G. Basso, G. Viola, Isoindolo[2,1-a] qui-
noxaline derivatives, novel potent antitumor agents with dual inhibition of
tubulin polymerization and topoisomerase I, J. Med. Chem. 51 (2008)
2387e2399.
[38] H. Hu, Y. Liu, L. Lin, Y. Zhang, X. Liu, X. Feng, Kinetic resolution of 2H-azirines
by asymmetric imine amidation, Angew. Chem. Int. Ed. 55 (2016)
10098e10101.
[39] G. Speit, A. Hartmann, The comet assay: a sensitive genotoxicity test for the
detection of DNA damage and repair, Methods Mol. Biol. 314 (2006) 79e90.
[40] Q. Cui, J. Yu, J. Wu, S. Tashiro, S. Onodera, M. Minami, T. Ikejima, P53-mediated
cell cycle arrest and apoptosis through a caspase-3-independent, but caspase-
9-dependent pathway in oridonin-treated MCF-7 human breast cancer cells,
Acta Pharmacol. Sin. 28 (2007) 1057e1066.
[32] L. Li, X. Huang, R. Huang, S. Gou, Z. Wang, H. Wang, Pt(IV) prodrugs containing
16