Coordination-Accelerated Radical Formation from Acyclic Enediynes for Tumor Cell Suppression

Article abstract of DOI:10.1002/asia.201901182

A maleimide-based acyclic enediyne with salicylaldiminato substituents at the alkyne termini was synthesized, which was further chelated with three kinds of metal-ions, Cu^II, Zn^II, and Mg^II, and form metalloenediynes. The cycloaromatization of this thermally inactive enediyne ligand was greatly accelerated through the coordination with metal ions. Specifically, the Cu^II-metalloenediyne showed an extremely low onset temperature of 55 °C and underwent spontaneous cycloaromatization at ambient temperature to produce free radicals, followed by generation of reactive oxygen species in the physiological environment. The metalloenediyne exhibited excellent DNA cleavage ability and high cytotoxicity towards HeLa cells, with half-maximal inhibitory concentration values comparable to many commercial antitumor agents. The combination of the electron-withdrawing effect of the maleimide moiety at the ene position and metal coordination at the yne termini provides a new inspiration for designing and synthesizing highly efficient enediyne antitumor agents.

Full text of DOI:10.1002/asia.201901182

DOI: 10.1002/asia.201901182
Full Paper
Coordination-Accelerated Radical Formation from Acyclic
Enediynes for Tumor Cell Suppression
Baojun Li, Mengsi Zhang, Haotian Lu, Hailong Ma, Yue Wang, Huimin Chen, Yun Ding,* and
Aiguo Hu*[a]
Abstract: A maleimide-based acyclic enediyne with salicylal-
diminato substituents at the alkyne termini was synthesized,
which was further chelated with three kinds of metal-ions,
CuII, ZnII, and MgII, and form metalloenediynes. The cycloaro-
matization of this thermally inactive enediyne ligand was
greatly accelerated through the coordination with metal
ions. Specifically, the CuII-metalloenediyne showed an ex-
tremely low onset temperature of 558C and underwent
spontaneous cycloaromatization at ambient temperature to
produce free radicals, followed by generation of reactive
oxygen species in the physiological environment. The metal-
loenediyne exhibited excellent DNA cleavage ability and
high cytotoxicity towards HeLa cells, with half-maximal in-
hibitory concentration values comparable to many commer-
cial antitumor agents. The combination of the electron-with-
drawing effect of the maleimide moiety at the ene position
and metal coordination at the yne termini provides a new
inspiration for designing and synthesizing highly efficient
enediyne antitumor agents.
has been considered as the most important one.[6] Density
functional theory studies suggested a CD distance around
3.4 ꢁ is needed for a spontaneous cycloaromatization to take
place at ambient temperatures. Several strategies have been
proposed to shorten the CD distance of enediynes, including
converting acyclic enediynes to highly strained cyclic ene-
diynes[7] and coordinating enediyne ligand with metal-ions.[1d,8]
Metal chelation is a simple and straightforward way to bring
the two alkyne termini closer, in consequence lowering the
cyclization barrier and the onset temperature of acyclic ene-
diynes.[9] Moreover, the valence and crystal field of central
metal-ion significantly affect the activation barrier of the cyclo-
aromatization of an enediyne ligand.[10] For example, the ther-
mal cyclization of a CuII-metalloenediyne is more active than
its CuI-metalloenediyne analogues.[9b,11] In the molecular-level
design of highly reactive metalloenediynes, on the other hand,
the structure of the enediyne ligand should also be sophisticat-
edly modulated to enable the cycloaromatization occurring at
low temperature.[9b,c,12] Recently, we have demonstrated that
the onset temperature of an acyclic enediyne was significantly
lowered with the installation of an electron-withdrawing malei-
mide moiety at the ene position.[13] We also found that the cy-
cloaromatization activities of the enediyne compounds could
be regulated by simply adjusting the functional groups at the
alkyne termini in order to realize the generation of free diradi-
cals at low temperature.[14] These maleimide-based enediynes
showed strong DNA-cleavage and tumor cell suppression acti-
vities.[14b,c,15] Furthermore, substituent modifications at the mal-
eimide moiety were applied in tailored synthesis of enediyne
compounds employed in drug conjugates[16] and drug deliv-
ery.[15,17] By combining the electron-withdrawing effect of male-
imide moiety at the ene position and the metal coordination
at alkyne termini, herein, we propose a new strategy for the
synthesis of highly reactive metalloenediyne. The enediyne
Introduction
Natural enediyne antibiotics with potent antitumor activities
and inhibition effects on various tumor cells are acknowledged
as the strongest antitumor agents discovered up to date.[1] The
cyclic enediyne chromophores in these enediyne antibiotics
undergo Bergman-type[2] or Myers-type[3] cycloaromatization in
physiological environment to generate highly reactive free rad-
icals and cause DNA damage, providing unique radiomimetic
cytotoxic mechanisms. The extremely strong cytotoxicity
endows natural enediynes the promising payloads for anti-
body-drug conjugates (ADC).[4] Indeed, two ADCs with Cali-
cheamicin as the “warhead”, Mylotargꢀ and Besponsaꢀ, have
been approved for clinical treatment of leukemia in recent
years. Unfortunately, owing to the complicated structures of
Calicheamicin and other natural enediynes, the total synthesis
and structural modification of them are severely hindered for
large-scale preparation.[5]
To this end, the development of non-natural enediyne com-
pounds with potent biological activities has attracted tremen-
dous attentions over the last two decades. The reactivity of an
enediyne compound towards cycloaromatization is dictated by
many factors. Among them, the separation between two ter-
minal alkyne groups, also known as the critical distance (CD),
[a] B. Li, M. Zhang, H. Lu, H. Ma, Y. Wang, H. Chen, Dr. Y. Ding, Prof. Dr. A. Hu
Shanghai Key Laboratory of Advanced Polymeric Materials
School of Materials Science and Engineering
East China University of Science and Technology
Shanghai 200237 (China)
E-mail: yunding@ecust.edu.cn
hagmhsn@ecust.edu.cn
Supporting information and the ORCID identification number(s) for the au-
thor(s) of this article can be found under:
https://doi.org/10.1002/asia.201901182.
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Scheme 1. Synthesis of ligand EDY and metalloenediynes EDY-M. (a) PhCH2NH2, acetic acid, 408C, 40 h; (b) NaI, acetonitrile, 1008C, 6 h; (c) MgSO4, dichlorome-
thane, 208C, 24 h; (d) Pd(PPh3)2Cl2, CuI, N,N-diisopropylethylamine (DIPEA), tetrahydrofuran/toluene, 208C, 18 h; (f) dichloromethane/methanol, 1 equiv of
Cu(NO3)2, Zn(NO3)2 and Mg(NO3)2, respectively.
compound with salicylaldiminato groups showed low cycloaro-
matization activity and no DNA-cleavage ability. On the contra-
ry, once chelated with metal ions at the alkyne termini, the
thermal reaction was accelerated as demonstrated by differen-
tial scanning calorimetry (DSC) experiment. In particular, the
cycloaromatization was greatly enhanced to produce radicals
when enediyne was chelated with CuII (Scheme 1), leading to
the generation of reactive oxygen species at ambient tempera-
ture and showing high DNA-cleavage ability and cytotoxicity.
Results and Discussion
Synthesis and Coordination of Enediyne
Figure 1. Infrared spectra of ligand EDY (yellow line); EDY-Zn (green line);
3,4-Diiodo-N-benzylmaleimide (1) was synthesized by a two-
EDY-Cu (red line); and EDY-Cu after 24 hours storage (blue line).
step reaction according to previous reports with minor modifi-
cation (ESI).[18] The terminal alkyne (2) with a chelating salicylal-
diminato moiety was formed from salicylaldehyde and 1,1-di-
methylpropargylamine (ESI). The Sonogashira reaction be-
tween 1 and 2 was catalyzed by palladium(II)bis(triphenylphos-
phine) dichloride and cuprous iodide to produce the enediyne
ligand (EDY) at ambient temperature (Scheme 1). The obtained
EDY was characterized by NMR spectroscopy (Figure S2) and
high-resolution mass spectroscopy (Figure S3). It is a yellow
powder and can be stored stably at room temperature.
ing that the hydroxyl groups were involved in the chelation
with ZnII. The peaks corresponding to the imine groups are
split, implying the coordination of the imine groups with metal
ions. Interestingly, while EDY and EDY-Zn are stable at ambient
temperature, the EDY-Cu is highly reactive. After 24 h storage
at room temperature, the acetylene peak (2216 cmÀ1) disap-
peared completely, corresponding to the spontaneous cycloar-
omatization of the metalloenediyne EDY-Cu. Further monitor-
ing the spontaneous cyclization of EDY-Cu with FT-IR spectros-
copy (Figure S7) showed that the half-life was 27 min. In other
words, the thermal cycloaromatization of the salicylaldiminato-
enediyne (EDY) is significantly accelerated by the chelation
with CuII.
The presence of the salicylaldiminato groups at the alkyne
termini enables the ligand EDY to coordinate with metal-ions
to form metalloenediyne complexes (EDY-M).[19] Three metal-
loenediynes were synthesized by mixing EDY with Cu(NO3)2,
Zn(NO3)2 and Mg(NO3)2 in a solvent mixture of methanol and
dichloromethane, respectively, followed by removal of the sol-
vent (Scheme 1). Zaleski et al. reported that the chelation of
MgII,[12c,20] CuII,[21] and ZnII[21] with an enediyne ligand, (Z)-N,N¢-
bis[1-pyridin-2-yl-meth-(E)-ylidene]oct-4-ene-2,6-diyne-1,8-dia-
mine, took place at the four N atoms in imines and pyridines.
Basak et al proved that[12a] the two N atoms in imines and the
two ortho-phenolic hydroxyls in salicylaldiminato-enediyne
were the coordination sites to form a tetra-coordination com-
plex with a metal-ion. The coordination processes of EDY with
metal ions were investigated by NMR spectroscopy (Figure S8),
electron paramagnetic resonance (EPR) spectroscopy (Fig-
ure S9), and infrared (IR) spectroscopy (Figure 1). As shown in
Figure 1, the hydroxyl peak (3450 cmÀ1) in the IR spectrum of
ligand EDY is absent in that of ZnII complex (EDY-Zn), indicat-
Thermal Reactivity of Enediyne Ligand and Metalloene-
diynes
The thermal reactivities of the ligand EDY and metalloenediyne
complexes (EDY-M) were evaluated by differential scanning
calorimetry (DSC) analysis. As shown in Figure 2, the EDY ex-
hibits a sharp endothermal melting peak at 1598C, followed by
an exothermal peak corresponding to the cycloaromatization
and the polymerization of free radical intermediates.[22] The
onset temperature of ligand EDY is 1638C, suggesting that the
ligand EDY is thermally inert at room temperature. After the
chelation with CuII, ZnII or MgII, the onset temperatures signifi-
cantly decreased to 558C (Figure 2B), 1258C (Figure S4A), and
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Figure 2. DSC curves of ligand EDY (A) and EDY-Cu (B). The baselines are marked in red.
1008C (Figure S4B), respectively. The greatly accelerated cyclo-
aromatization and the reduction of onset temperature are con-
sistent with the reduction of CD distance after metal coordina-
tion with enediyne compounds.[21b] Meanwhile, the charge sep-
aration assistance in the metal-ion-catalyzed Bergman cycliza-
tion should not be excluded as well.[23] Nevertheless, this study
confirmed that the metalloenediyne EDY-Cu is highly reactive
towards spontaneous cycloaromatization at physiological tem-
perature to produce free diradicals,[14b,15] endowing it with the
ability of DNA cleavage and tumor cell suppression as dis-
cussed below.
shown in Figure 3, both of the samples exhibit strong radical
signals at 3520 G. In particular, for the reaction system at 08C,
there are two sets of triplet peaks with an intensity ratio of
1:1:1 and a hyperfine splitting constant (AN) of 14.78 G, similar
to that of the PBN adduct of calicheamicin-derived phenyl radi-
cal (AN =14.6 G),[25] suggesting the formation of two kinds of
radical species either from asymmetric cycloaromatization or
partial reaction of symmetric diradical intermediates with some
donor groups (like solvent molecules). The EPR spectrum of
the sample obtained at 378C is even more complicated as at
least one more set of triplet peaks with similar hyperfine split-
ting constant shows up. The formation of a complex mixture
of PBN-adducts is another evidence of the high reactivity of
the diradical intermediates, which might abstract hydrogen or
chlorine atoms from the solvent molecules and turn to differ-
ent kinds of radical species before meeting with PBN. For com-
parison, no EPR signal was found in EDY (Figure S5A) or EDY-
Zn (Figure S5B) under the same conditions. Accordingly, the
ligand EDY is thermally inactive at ambient temperature, while
its chelate with CuII is capable of undergoing spontaneous
thermal cycloaromatization to generate diradicals at 378C
(physiological temperature) and even at 08C, shedding some
light on the fabrication of chelation-triggered enediyne antitu-
mor antibiotics system.
Generation of free diradicals
The radical nature of cycloaromatization is typically verified by
electron paramagnetic resonance (EPR) spectroscopy. It is diffi-
cult to monitor the free diradicals produced by cycloaromatiza-
tion in situ because of their high reactivity and short lifetime.
However, they can be converted into stable free radicals in the
presence of a spin trap like N-tert-butyl-alpha-phenylnitrone
(PBN), 5,5-dimethyl-1-pyrroline N-oxide (DMPO), or 2-methyl-2-
nitrosopropane (MNP).[24] In order to verify the generation of
free radical intermediates, the ligand EDY and PBN were dis-
solved in DCM, followed by the addition of a methanol solu-
tion of cupric nitrate and stirred at 08C or 378C for 24 h. As
Figure 3. (A) EPR spectrum of EDY-Cu (14 mm) in the presence of PBN (140 mm) in DCM:MeOH=1:1 at 08C. (B) EPR spectrum of EDY-Cu (14 mm) in the pres-
ence of PBN (140 mm) in DCM:MeOH=1:1 at 378C.
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Figure 4. (A) Schematic illustration of the ROS generated by diradicals of EDY-Cu. (B) Fluorescence intensities of DCF in PBS (pH 7.4) with control (gray),
ligand EDY (75 mm, yellow line), EDY-Cu (75 mm, red line), and cupric nitrate (75 mm, blue line). [DCFH]=50 mm. (C) The rates of fluorescence intensity calculat-
ed by linear fitting.
Assessment of ROS
pristine DNA molecule exhibits a double-stranded closed heli-
cal structure and is further twisted to form a super-tertiary
structure. Once the scissions of one or two strands appear, the
supercoiled plasmid DNA becomes open circular forms. Three
different DNA forms: native supercoiled form (Form I, intact
DNA), circular relaxed form (Form II, single-strand scissions),
and linear form (Form III, double-strand scissions) which can
be distinguished by gel electrophoresis. The DNA samples
(FX174 supercoiled plasmid RF1 DNA, 16.7 mgmLÀ1) in PBS
buffer (pH 7.4) were, respectively incubated with EDY
(1.25 mm), metalloenediynes (1.25 mm), and nitrates (1.25 mm)
for 24 hours at 378C, and then subjected to agarose gel elec-
trophoresis for photographed scanning analysis. A control
sample was prepared with the addition of the same amount of
DMSO to the DNA solution. As shown in Figure 5, most of the
samples have no obvious DNA cleavage compared with con-
trol except EDY-Cu and Cu. While the native DNA was partially
converted to Form II and Form III in the presence of copper ni-
trate (Cu) for an unknown reason, EDY-Cu leads to nearly com-
plete scission of DNA into Form II (79%) and Form III (11%)
owing to the spontaneous cycloaromatization and the genera-
tion of radical species and ROS.
Reactive oxygen species (ROS), including singlet oxygen, super-
oxide, hydrogen peroxide, and hydroxyl radical,[26] are generat-
ed from ground state oxygen either by energy or electron
transfer reactions. ROS causes oxidative damages to DNA,
lipids, and proteins. They are a class of important mediators in-
ducing cytotoxicity of some antitumor agents.[27] To evaluate
the level of ROS induced by enediyne, dichlorodihydrofluores-
cein (DCFH) was used as an indicator in PBS buffer solutions
(Figure 4A). The non-fluorescent DCFH is converted to fluores-
cent dichlorofluorescein (DCF) in the presence of ROS,[28] and
the increase of fluorescence intensity of DCF at 525 nm indi-
cates the production of ROS in the system. As shown in
Figure 4, after the addition of EDY or Cu (cupric nitrate) to
DCFH, the fluorescence intensity of DCF slowly increases in
12 min. The calculated increasing rates are 6.26minÀ1 (EDY)
and 5.05minÀ1 (Cu), respectively, slightly higher than that of
control (2.28minÀ1). Interestingly, the fluorescence intensity of
DCF in the presence of EDY-Cu increases significantly with a
rate of 20.72minÀ1, suggesting that the radicals formed from
the coordination-accelerated cycloaromatization transform O2
into ROS, which further turns DCFH into fluorescent DCF.
These results indicate that the EDY-Cu is able to produce ROS
in physiological environment, which is essential for DNA-cleav-
age and tumor cell suppression.
Cytotoxicity Test
Cytotoxicity of ligand EDY and metalloenediyne complexes
was evaluated by measuring the IC50 on HeLa cells via the 3-
(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide
(MTT) method.[30] The DMSO solution of EDY, three EDY-M, and
three nitrates solutions were diluted to the test concentrations
with culture medium and cultured with HeLa cells for 24
Cleavage of DNA
Supercoiled plasmid DNA was used to evaluate the DNA cleav-
age activities of EDY, metalloenediynes, and metal-ions.[29] The
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Figure 5. (A) Agarose gel electrophoresis of FX174 supercoiled plasmid RF1 DNA. The DNA samples (16.7 mgmLÀ1) in PBS buffer (pH 7.4) were, respectively in-
cubated with EDY (1.25 mm), nitrates (1.25 mm), and metalloenediynes (1.25 mm) at 378C for 24 h. (B) Percentage of (i) uncleaved DNA (Form I, supercoiled
circular, green block), (ii) single-strand scission (Form II, circular, yellow block), and (iii) double-strand scission (Form III, linear, red block).
Figure 6. Cell viability of HeLa cells in the presence of EDY, EDY-Cu, EDY-Zn, and EDY-Mg after cultured for 24 h at 378C with a series of concentrations. HeLa
cells incubated without any materials were used as the control. The IC50 values are expressed in parentheses. A significant difference in the cell viability com-
pared with the ligand EDY is denoted as “*” (P<0.05), “**” (P<0.01).
hours, and then subjected to MTT analysis. The cell viabilities
in the presence of EDY or EDY-M show clear concentration de-
pendence (Figure 6), while the nitrates exhibit negligible cyto-
toxicity even at the concentration up to 100 mm (Figure S6).
The Schiff base structure of the EDY ligand might be hydro-
lyzed in the intracellular environment to give a diamine com-
pound (EDY 2) which might endow the ligand with cytotoxici-
ty (Scheme S7). Indeed, the hydrolysate EDY 2 is a more active
enediyne in thermal aromatization (onset temperature=
1248C, Figure S11A) and shows a comparable cytotoxicity to
the free ligand EDY (IC50 =47 mm, Figure S11B). The IC50 levels
of EDY, EDY-Zn, and EDY-Mg are all around 40 mm, implying
that the coordination of Zn or Mg barely affects the cycloaro-
matization of the enediyne ligand, which is consistent to the
results from EPR and DNA cleavage experiments. It is notewor-
thy that the cytotoxicity of EDY-Cu is much greater than those
of the other groups, and the differences between EDY-Cu and
the ligand EDY are statistically significant when the concentra-
tion is above 6.25 mm. Almost all Hela cells are destroyed when
the concentration of EDY-Cu is 100 mm. The EDY-Cu shows ex-
cellent cytotoxicity with an IC50 value of 22 mm, As a control,
the cytotoxicity of the cyclized products was much lower (Fig-
ure S12), suggesting that the cytotoxicity originates from the
spontaneous generation of free radicals and reactive oxygen
species at physiological temperature thanks to the coordina-
tion effect of CuII to enediyne ligand.
Conclusions
A maleimide-based acyclic enediyne, which is capable of coor-
dinating with metal ions, has been designed and synthesized
with salicylaldiminato substituent groups at the alkyne termini.
The ligand EDY is inert towards thermal cycloaromatization,
while the metalloenediyne EDY-Cu spontaneously undergoes
cycloaromatization at ambient temperature to produce highly
reactive free radicals. The active free radicals induce the gener-
ation of reactive oxygen species, causing DNA cleavage by the
scissions of single and double strands, resulting in cytotoxicity
against HeLa cells. The combination of the electron-withdraw-
ing effect at the ene position and metal coordination effect at
the yne termini greatly facilitates the cycloaromatization of an
enediyne compound, which would be potentially applied in
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The authors gratefully acknowledge the financial support from
National Natural Science Foundation of China (21871080,
21503078, 21474027), the Fundamental Research Funds for the
Central Universities (22221818014), and Shanghai Leading Aca-
demic Discipline Project (B502). AH thanks the “Eastern Scholar
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Keywords: Anticancer agents · Cytotoxicity · DNA cleavage ·
Enediynes · Metalloenediynes
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Manuscript received: August 22, 2019
Revised manuscript received: October 21, 2019
Version of record online: && &&, 0000
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ÝÝ These are not the final page numbers!

Full Paper
FULL PAPER
Baojun Li, Mengsi Zhang, Haotian Lu,
Hailong Ma, Yue Wang, Huimin Chen,
Yun Ding,* Aiguo Hu*
&& – &&
Coordination-Accelerated Radical
Formation from Acyclic Enediynes for
Tumor Cell Suppression
An acyclic enediyne functions via coor-
dination-accelerated radicals to induce
ROS and DNA cleavage, thereby inhibit-
ing cancer cell growth.
Chem. Asian J. 2019, 00, 0 – 0
www.chemasianj.org
7
ꢂ 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
&
&
These are not the final page numbers! ÞÞ