An acyclic enediyne anticancer compound attributed to a Bergman cyclization at physiological temperature

Article abstract of DOI:10.1016/j.tet.2018.08.042

Enediyne cytotoxic drugs have attracted much attention because of their unique structure and potent anticancer activity. However, acyclic enediynes are long considered as inactive at physiological temperature due to their long C1-C6 distance. By adjusting the steric bulkiness of the functional groups at the alkynyl termini and the electron-withdrawing effect at the ene moiety, herein, a simple acyclic enediyne was designed and synthesized to achieve the onset of thermal Bergman cyclization at physiological temperature in polar solvents. The spontaneous generation of diradical intermediates was confirmed through EPR analysis and further supported by spin trapping experiment, radical indicator experiment, and high resolution MS analysis. The reactive diradicals generated in aqueous media induced single and double stranded cleavage of DNA, and showed high cytotoxicity to Hela cells. The IC₅₀ value of the enediyne compound is comparable to many clinical used anti-tumor agents.

Full text of DOI:10.1016/j.tet.2018.08.042

Accepted Manuscript
An acyclic enediyne anticancer compound attributed to a Bergman cyclization at
physiological temperature
Baojun Li, Bing Duan, Jing Li, Mengsi Zhang, Yuan Yuan, Yun Ding, Aiguo Hu
PII:
S0040-4020(18)31024-X
10.1016/j.tet.2018.08.042
TET 29762
DOI:
Reference:
To appear in: Tetrahedron
Received Date: 17 May 2018
Revised Date: 20 August 2018
Accepted Date: 23 August 2018
Please cite this article as: Li B, Duan B, Li J, Zhang M, Yuan Y, Ding Y, Hu A, An acyclic enediyne
anticancer compound attributed to a Bergman cyclization at physiological temperature, Tetrahedron
(2018), doi: 10.1016/j.tet.2018.08.042.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT
An Acyclic Enediyne Anticancer Compound Attributed to a Bergman
Cyclization at Physiological Temperature
Baojun Lia, Bing Duanb, Jing Lia, Mengsi Zhanga, Yuan Yuanb, Yun Dinga* and Aiguo Hua*
An acyclic enediyne functions possibly through spontaneous Bergman cyclization to induce DNA cleavage,
inhibit cancer cell growth, and lead cancer cells to apoptosis pathway.

ACCEPTED MANUSCRIPT
An Acyclic Enediyne Anticancer Compound Attributed to a Bergman
Cyclization at Physiological Temperature
Baojun Lia, Bing Duanb, Jing Lia, Mengsi Zhanga, Yuan Yuanb, Yun Dinga* and Aiguo Hua*
a Shanghai Key Laboratory of Advanced Polymeric Materials, School of Materials Science and Engineering, East
China University of Science and Technology, Shanghai 200237, China. b The State Key Laboratory of Bioreactor
Engineering, East China University of Science and Technology, Shanghai, 200237, China.
Abstract Enediyne cytotoxic drugs have attracted much attention because of their unique structure and
potent anticancer activity. However, acyclic enediynes are long considered as inactive at physiological
temperature due to their long C1-C6 distance. By adjusting the steric bulkiness of the functional groups at the
alkynyl termini and the electron-withdrawing effect at the ene moiety, herein, a simple acyclic enediyne was
designed and synthesized to achieve the onset of thermal Bergman cyclization at physiological temperature in
polar solvents. The spontaneous generation of diradical intermediates was confirmed through EPR analysis
and further supported by spin trapping experiment, radical indicator experiment, and high resolution MS
analysis. The reactive diradicals generated in aqueous media induced single and double stranded cleavage of
DNA, and showed high cytotoxicity to Hela cells. The IC50 value of the enediyne compound is comparable to
many clinical used anti-tumor agents.
Keywords: Anticancer Agents • Bergman cyclization • Cytotoxicity • DNA cleavage • Enediyne
Introduction
Cancer is one of the major diseases seriously threatening the lives of human beings. Searching for
medicines employed in cancer treatment is therefore of great importance in medicinal chemistry.1-3
A
number of anticancer agents have been used for clinical practice,4-8 and there have been many ways to
load these drugs.9-11 Unfortunately, many conventional anticancer agents face failures in their
development due to the insurgence of multidrug resistance.12-14 Enediyne antitumor agents, on the other
hand, are promising to solve this problem thanks to their unique radiomimetic cytotoxic mechanism.15-18
Indeed, enediyne compounds are acknowledged as the strongest antitumoral agents, with their half
maximal inhibitory concentration (IC50) ranging from submicromolar to picomolar towards a wide
spreading of cancer cell lines.
Neocarzinostatin (NCS) is the first member of natural enediyne family secreted from Streptomyces
macromomyceticus in 1965.19 Afterwards, more than 20 kinds of natural enediyne compounds have been
discovered, such as Calicheamicins,20 Dynemicins,21 and Esperamicins.22 These compounds are named for
an enediyne structure, which induce DNA cleavage in cancer cells through diradicals generated from a
Bergman or Bergman-like cyclization. At present, NCS and Calicheamicins have been employed in
clinical treatment; studies on the modification of natural enediynes also emerge.23,24 However, due to the
complicated molecular structure, the difficulty in chemical synthesis of most natural enediyne compounds
isolated from microorganisms brings a great obstacle to the development of new types of enediynes
antitumoral drugs. For example, Nicolaou et al. calls for 35 steps of organic reactions to the total synthesis
of Calicheamicins.25 Besides, the yield of natural enediynes or analogues is also problematic due to the
tedium synthetic procedure,24 hindering the industrialization of enediyne anticancer agents. To this end,
1

ACCEPTED MANUSCRIPT
developing new enediyne anticancer compounds with simple structure and high tumor cell suppression
efficiency is of great significance.
In most cases, the chemically synthesized enediynes with simple molecular structure are inert towards
thermal Bergman cyclization at physiological temperature. Nicolaou26 has pointed out that the C1-C6
distance of an enediyne compound should be shorter than 3.35 Å (which was later on expanded by
Schreiner et al. to be 3.4 Å27) for a spontaneous Bergman cyclization to take place at ambient temperature.
Many strategies have been adopted to lower down the onset temperature of enediyne compounds, such as
introducing strong electron-withdrawing groups,28-31 coordinating with metal ions32-36 or embedding the
enediyne moiety into a highly strained 9- or 10-member ring.37,38 Recently, our work revealed that by
incorporating the ene moiety of enediyne into electron-deficient maleimide framework, the onset
temperature of thermal Bergman cyclization is drastically lowered down.39 The maleimide-based enediyne
compounds with triethoxy group at alkyne termini are active at around 100 ºC.40,41 When the orthoester
groups were hydrolyzed in an acidic environment, the enediyne was activated towards Bergman
cyclization at low temperature, leading to the formation of reactive diradicals for DNA-cleavage and
tumor cell suppression.
Herein, by sophisticated adjusting the steric bulkiness of the substituents at the alkyne termini, the
onset temperature of an acyclic enediyne was modulated to close to physiological temperature.
Spontaneous Bergman cyclization which was attributed has arisen in polar solvents, leading to diradical
formation. The acyclic enediyne showed remarkable cytotoxicity towards Hela cell line, and induced
apoptosis in the cancer cells.
Experimental
Materials
Toluene and tetrahydrofuran (THF) were dried over calcium hydride and distilled before use. Other
chemicals were of commercial grade and used as received. 3,4-Diiodo-N-benzylmaleimide (1
),42
3,3,3-triethoxy-1-propyne,43 and catalyst NHC-PdCl2-3-chloropyridine44 were synthesized according to
literature procedures with minor modification. The detailed synthesis of these precursors is presented in
the ESI.
Synthesis of enediyne compounds
2-Ethoxy-2-ethynyl-[1,3]dioxolane (compound 2). 3,3,3-triethoxy-1-propyne (4.776 g, 27.76 mmol),
succinic acid (0.147 g, 1.24 mmol), and ethylene glycol (3.923 g, 63.20 mmol) are successively weighed
in a 50 ml sealed bottle. The solution system was stirred for 2 h at 120 ºC and purified by silica
1
chromatography with hexane/ethyl acetate = 10/1 as eluent to give a colorless liquid (2.01 g 51.2%). H
NMR (CDCl3, ppm): 4.03-4.07 (m, 4 H, -O-CH2-CH2-), 3.63-3.69 (q, 2 H, J = 7.2 Hz, -CH2), 2.55 (s, 1 H,
C≡CH), 1.15-1.19 (t, 3 H, J = 7.2 Hz, CH3) 13C NMR (CDCl3, ppm): 113.1, 77.5, 71.1, 64.8, 59.6, 14.8.
HR-MS (ESI): m/z calcd. for C7H10O3Na (M + Na)+: 165.0528; found: 165.0525.
1-Benzyl-3,4-bis-(2-ethoxy-[1,3]dioxolan-2-ylethynyl)-pyrrole-2,5-dione (enediyne 3). Compound
1 (263.4
mg, 0.6 mmol), CuI (45.7 mg, 40%), NHC-PdCl2-3-chloropyridine (102.0 mg, 25%), DIPEA (244.4 mg,
1.8 mmol) were successively added to a solvent mixture of dry THF (3 mL) and toluene (6 mL) under
nitrogen. Then, compound
2 (0.2558 g, 1.8 mmol) was added dropwisely. The mixture was stirred at 20 ºC
for 15 h. After the completion of the reaction as detected by TLC, the mixture was directly purified by
column chromatography over magnesium silicate (hexane/ethyl acetate = 4 : 1) to yield a red viscous
liquid (0.12 g, 42.8%). The product was stored at low temperature. 1H NMR (CDCl3, ppm): 7.29-7.32 (m,
2

ACCEPTED MANUSCRIPT
5 H, Ph), 4.69 (s, 2 H, CH2), 4.16-4.14 (m, 8 H, CH2), 3.76-3.82 (q, 4 H, J = 7.1 Hz, CH2), 1.24-1.27 (t, 6
H, J = 7.1 Hz, CH3). 13C NMR (CDCl3, ppm): 165.6, 135.5, 129.3, 128.9, 128.7, 128.6, 128.3, 128.0,
114.2, 103.3, 72.0, 65.4, 60.5, 42.8, 15.2. HR-MS (EI): m/z calcd. for C25H25NO8 (M + 2H): 469.1738;
found: 469.1739.
Characterizations
1H NMR (500 MHz) and 13C NMR (125 MHz) spectra were recorded in chloroform (CDCl3) on an Ultra
Shield 500 spectrometer (BRUKER BIOSPIN AG, Magnet System 500 MHz/54 mm) and referenced to
Me4Si. Mass spectra were recorded on a Micromass LCTTM mass spectrometer using the ESI method or
EI method. Differential scanning calorimetry (DSC) was carried out with a Pyris Diamond thermal
analysis workstation equipped with a model 822e DSC module under a constant nitrogen flow. The
electron paramagnetic resonance (EPR) study was carried out with the enediynes dissolved in DMSO or
chloroform at a concentration of 8 mM. Measurements were performed with an X-band EMX-8/2.7C EPR
spectrometer (Bruker, Germany). The settings of the spectrometer were as follows: sweep width, 200G;
time constant, 163.84 ms; conversion time, 40.96 ms; resolution, 1024 points; modulation frequency,
100.00 kHz; modulation amplitude, 1.00 G; and microwave power, 6.370 mW.
DNA cleavage experiments
The freshly prepared enediyne
3 was respectively dissolved in DMSO (2 ꢀL) at concentrations of 200
mM, 100 mM and 50 mM, and added to a solution of supercoiled plasmid FX174 RF1 DNA (0.5 mg/mL)
in TE buffer (pH 7.6, 4 ꢀL). Control samples were separately incubated with pure DMSO and DNA. All
the systems were incubated at 37 ºC for 48 h. After incubation, each system (6 ꢀL) was mixed with a
6×loading buffer (1 ꢀL) and subjected to a 0.8% agarose gel electrophoresis at 128 V (101 mA) for 30
min, stained by ethidium bromide and then the gel was photographed on the UV transilluminator
(FR-200A) and analyzed by scanning densitometry.
Cell viability
The cell viability tests were investigated by an MTT assay.45 Human uterine cervix carcinoma HeLa cells
were respectively cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine
serum (FBS) at 37 ºC under a humidified atmosphere containing 5% carbon dioxide. The cells were
trypsinized until it reaching 70% confluence in the tissue culture flasks with buffered saline solution
containing 0.25% trypsin and 0.03% EDTA.
Cells were seeded into a 96-well plate at a density of 5000 cells per well in 200 ꢀL of the cell culture
medium. After 24 h of incubation, the culture medium was removed and the cells were then exposed to
0.75, 1.5, 3.13, 6.25, 12.5, 25 and 50 ꢀM concentrations of enediyne 3. The enediyne 3 solution was
sterilized by a 0.22 ꢀm membrane filter (Sartorius Stedim Biotech, France). The concentration of DMSO
was 0.1 %. The culture medium (0.05% DMSO) was used as a blank control. After incubation for another
determined
period
of
48
h,
20
ꢀL
of
sterile
filtered
MTT
(3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazoliumbromide) stock solution (5 mg/mL) in PBS (pH 7.4)
was added to each well and the cells were further incubated for 4 h at 37 ºC to allow the yellow dye to be
transformed into blue crystals. Then, the unreacted dye was removed by aspiration, and 200 ꢀL of DMSO
was added to each well to dissolve the dark blue crystals. Finally, the optical density was measured by a
microtitre reader at a wavelength of 492 nm. The spectrophotometer was normalized using culture
medium without cells. Cell viability (%) related to control wells containing cell culture medium without
enediyne was calculated by [A]test/[A]control
.
Flow cytometric analysis
3

ACCEPTED MANUSCRIPT
Cell death was analyzed by fluorescence-activated cell sorting (FACS) using Annexin V and propidium
iodide (PI) staining assay. HeLa cells were seeded in a 6-well plate at a density of 1×105 cells per well and
incubated in DMEM/FBS incubation medium and allowed to settle for 24 h. The medium was replaced
with the fresh one containing enediyne
3 (30ꢀM). After incubation for 48 h, the cells were washed twice
with cold PBS, trypsinized and centrifuged. Then the cells were collected for Annexin V and propidium
iodide (PI) staining and analyzed by flow cytometry.
Results and discussion
O
O
O
OH
+
HO
a
O
O
O
O
O
O
I
I
N
O
O
N
b
+
O
O
O
O
O
1
2
3
Scheme 1. Synthesis of enediyne 3. a) succinic acid, 120 ºC, 2h; b) CuI, NHC-PdCl2-3-chloropyridine, DIPEA,
THF/ toluene, 20 ºC, 15 h.
The terminal alkyne with dioxolane-type orthoester substituent (
exchange reaction between 3,3,3-triethoxy-1-propyne and ethylene glycol with succinic acid as the
catalyst. The Sonogashira reaction between and to produce was achieved at 20 ºC using an air-stable
and highly reactive Pd catalyst, NHC-PdCl2-3-chloropyridine, developed by Michael et al.44 It should be
noted that this reaction should be run at low temperature to reduce the consumption of the enediyne
through the reactions like thermal Bergman cyclization and the formation of side products (see below) in
the reaction mixture. The obtained enediyne was characterized by NMR spectroscopy and high
2) was firstly synthesized from orthoester
1
2
3
3
3
resolution mass spectroscopy (ESI† Figure S1 and S2), and stored in a refrigerator.
O
O
O
O
O
O
O
O
N
N
O
O
O
O
O
O
O
O
4
Large Steric Hindrance
Onset at 105 °C
Similar Critical Range
Similar Electronic Structure
O
O
O
O
O
O
O
O
O
N
N
O
O
O
O
O
O
O
Small Steric Hindrance
Onset at 49 °C
3
Figure 1. Comparison of enediyne 3 and another acyclic enediyne (4) with a similar molecular structure.
4

ACCEPTED MANUSCRIPT
The attributed thermal Bergman cyclization of enediyne
3
is characterized with differential scanning
calorimetry (DSC) experiment (ESI† Fig. S3). A strong exothermal peak was found starting at 49 ºC,
which, to the best of our knowledge, is among the lowest onset temperature for thermal Bergman
cyclization of an acyclic enediyne, and is very close to physiological temperature, implying a spontaneous
Bergman cyclization would occur in physiological environment. Comparing the molecular structure of
enediyne
3 with a previously reported enediyne 4
(Fig. 1),40 both with an electron-deficient maleimide
moiety and two orthoester substituents at the alkynyl termini, it is striking to find out that by simply
releasing the steric hindrance raised by the relatively bulky triethoxy groups, the thermal cyclization is
drastically activated, which is indispensable for further applications in DNA-cleavage and tumor cell
suppression.
Figure 2. EPR spectra of enediyne 3 (A) and PBN adduct 5 (B) in DMSO at ambient temperature.
Electron paramagnetic resonance (EPR) spectroscopy is typically used to verify the radical nature of
Bergman cyclization. Freshly prepared enediyne
3 was dissolved in dimethylsulfoxide (DMSO) at
ambient temperature and immediately subjected to EPR analysis. As shown in figure 2a, an intense single
peak at 3520 G is clearly observed, indicating the formation of carbon-centered free radicals.39 Even after
the sample was stored for one week, the EPR peak corresponding to free radicals had not decayed
significantly (ESI† Fig. S4). The spectrum became slightly complicated, probably due to the generation of
polymeric species46 through the coupling of the free diradicals. Alabugin et al. showed that if there was an
alkoxyl group at the ortho-position to the free radical, intra-molecular H-transfer would occur, helping to
stabilize the free radical.47 At the same time, this process renders the cyclization step essentially
irreversible and produce other kinds of free radicals.48 Interestingly, when enediyne
3 was dissolved in
chloroform, no free radical signal was observed in EPR spectrum (ESI† Fig. S5), implying that the
polarity of the solvent also shows strong influences on the thermal cyclization of this kind of enediyne
compounds. This unexpected solvent effect is still under investigation, we currently believe that it is
related to the polarization of enediyne system by the solvent molecules.49 Nevertheless, this phenomena
suggests a simple yet efficient drug loading approach, in other words, the enediyne is safely preserved in
nonpolar environments (for example, in the nonpolar core of a micellar drug delivery system) and then
activated when they are released in cancer cell with highly polar cytosol microenvironments.
The reactive free diradicals generated from thermal cyclization can be converted into stable free
radical in the presence of spin traps.50 To this end, freshly prepared enediyne
N-tert-butyl- -phenylnitrone (PBN) were dissolved in DMSO and kept at 37 ºC for 1 h (Scheme 2). The
3 and excess of
α
diradical intermediates might interact with 1 or 2 equivalent of PBN to produce two different types of
nitroxide radicals. ESI-MS analysis showed an intense peak at m/z of 645.2811 (ESI† Figure S6),
corresponding to the radical molecular ion (M·+) of the single PBN-adduct. Meanwhile, EI-MS analysis
showed a peak at m/z of 821.3881 (ESI† Figure S7), corresponding to the double PBN-adduct. The single
PBN-adduct of enediyne (
5
) was separated out and subjected to EPR analysis. As shown in figure 2b,
5

ACCEPTED MANUSCRIPT
three sets of peaks with an intensity ratio of 1 : 1 : 1 and hyperfine splitting constant (AN) of 14.05 G,
similar to that of the calicheamicin-derived phenyl radical PBN monoadduct (AN = 14.6),51 confirming the
conversion of carbon-free radicals to nitrogen oxygen radicals. The hyperfine splitting of each set of peaks
is probably originated from the neighboring methine group.
O
Ph
O
N
O
O
O
O
O
O
O
O
O
O
O
Bergman cyclization
PBN and [H]
O
O
N
N
N
O
O
O
O
O
O
O
O
O
O
5
3
Scheme 2. Bergman cyclization of enediyne 3 and related PBN addition (5).
It is difficult to monitor the generation of free radicals in aqueous solution with EPR experiment due to
the absorption of microwave irradiation by water molecules. To this end, a free-radical-indicator recently
developed by Li. et al.52 was adopted. A mixture of enediyne
3 (1 mg) and the indicator (2 mg) in DMSO
(1 ml) was suspended in water (5 ml) and maintained at 25 ºC. The color of the mixture slowly turned into
pink in 10 min (ESI† Fig. S8), indicating the formation of free radicals through the reaction that was
attributed to Bergman cyclization. This color change originated from the attack of the spirocyclic
rhodamine amide (the indicator) by the diradical species, resulting in the restoring of the
π
-conjugation
system.52 In comparison, in the absence of the indicator, the color of the enediyne
3 in aqueous media
stayed unchanged under the same condition.
The high reactivity of enediyne
3
was indirectly evidenced by ESI-MS analysis of an unexpected
. In this Sonogashira reaction, the temperature was not
reaction mixture during the synthesis of enediyne
3
restrictively controlled below room temperature (roughly 25 ºC), and a complicated mixture of various
radical addition and abstraction products was obtained. As indicated in the high resolution mass
spectrometry spectrum (ESI† Figure S9), hydrogen donors in the reaction system and the raw material
alkynes were attacked by the highly reactive free diradicals generated from spontaneous Bergman
cyclization of enediyne
3
, generating at least three different kinds of addition products (ESI† Scheme S4).
under mild conditions is fascinating and puzzling as most
The radical-forming processes of enediyne
3
of the other enediyne compounds with similar structural features, like acceptor substitution the ene part,
small cycle annealed at the ene part, and bulky terminal groups, were considered to be inactive at low
temperature. We acknowledge that our findings go against the current understanding of electronic effects
in Bergman cyclization, and we have also investigated other possible mechanisms of generating radical
species from a close shell enediyne compound, like C1-C5 cyclization,53 enediyne dimerization.54 Since
the calculated barrier for C1-C5 cyclization is even higher and the dimerization of enediyne is not
supported by mass spectroscopy analysis in this work, we tentatively consider the generation of radical
species is most likely from the Bergman cyclization of enediyne compounds. The discrepancy between
theoretical calculation and experimental results are common. In an early report by P. Schreiner et al.,55 the
authors found that while the carbonyl-substituted enediynes showed high tendency to polymerize under
room temperature, the calculated energy barrier (28.8 kcal/mol) of these compounds for Bergman
cyclization were comparable or even higher than those of many stable enediyne compounds. Therefore, at
the current stage, we prefer to base more on the experimental results. The high activity of enediyne
3 is
probably due to the contribution of the electron-withdrawing effect action by maleimide group and OR
substituents which facilitate Bergman cyclization by helping the enediyne system to distort and also by
decreasing electron-electron repulsion between the in-plane alkyne
π
-bonds.56 Herein, we tentatively
6

ACCEPTED MANUSCRIPT
attribute the formation of radicals to the Bergman cyclization but we cannot exclude other radical-forming
processes. Deeper investigations into the reaction mechanism of this kind of enediyne compounds are still
underway in our group. Nevertheless, the high reactivity of enediyne
3 under low temperature to produce
free radicals is essential for its DNA-cleavage ability and tumor cell cytotoxicity.
Figure 3. Schematic illustration of the cleavage of DNA by enediyne 3 (left) and agarose gel electrophoresis of supercoiled
plasmid DNA (right). Lane 1, free DNA; Lane 2 DNA + DMSO; Lane 3, DNA + enediyne 3 (66.7 mM); Lane 4, DNA +
enediyne 3 (33.3 mM); Lane 5, DNA + enediyne 3 (16.7 mM). The solvent system is [Water : DMSO = 2 : 1].
The DNA-cleavage ability of enediyne
3 was estimated by measuring the conversion of supercoiled
plasmid DNA from native supercoiled form (Form I) to circular relaxed form (Form II, single-strand
cleavage).57 The DNA samples (ΦX174 supercoiled plasmid RF1 DNA 0.5 ꢀg/ꢀL in TE buffer) with
different concentration of enediyne
gel electrophoresis to analyze the conversion of DNA by photographed scanning. As shown in figure 3,
the supercoiled DNA is significantly converted to Form II in the presence of 16.7 mM of enediyne (Lane
3 were incubated for 48 h at 37 °C, which were subjected to agarose
3
5). In comparison, the control samples were not affected (Lane 1, free DNA; Lane 2 DNA+DMSO). When
the concentration of enediyne increased, the DNA was completely fractured into strips (33.3 mM, Lane 4;
66.7 mM, Lane 3), demonstrating the strong DNA-cleavage performance of enediyne in physiological
environments.
Figure 4. MTT assay of the cell viability of Hela cell in the presence of enediyne 3.
The cytotoxicity of enediyne
3
was investigated by using the MTT method45 in a tumor model line
was dissolved in DMSO, diluted with cell culture medium to a
cell. Firstly, freshly prepared enediyne
3
series of concentrations and then respectively cultured with Hela cells for 48 h. A control sample was
involved with the addition of the same amount of DMSO to the cell culture media. As shown in figure 4,
the cell viabilities of Hela cell are lower than 90% when the concentrations of enediyne
3
3
are beyond 0.75
. In contrast, the
ꢀM. In addition, the cell viability clearly shows concentration dependence of enediyne
previously reported maleimide based enediynes with high onset temperature (150 ºC) exhibited no
7

ACCEPTED MANUSCRIPT
cytotoxicity, suggesting that the cytotoxicity of enediyne
3
is derived from the free radicals generated
probably through Bergman cyclization. Specifically, the IC50 value of enediyne
3 fit from the cell
variability curve is 8.3 ꢀM, comparable to many clinical anticancer drugs, like cisplatin,58 Irinotecan,59
and doxorubicin.60 It is speculated that enediyne
with a low molecular weight shows great potential of
3
being a bullet in a drug carrier to increase cytotoxicity like nature enediyne antibiotics with
multi-functional groups.
Flow cytometry analysis is a common method of detecting drug cytotoxic pathways. Hela cells were
used as model cancer cells and co-cultured with enediyne
3 (30 ꢀM) for 48 h, followed by staining with
annexin V-FITC and propidium iodide (PI) to evaluate the percentage of live (annexin V-/PI-), early
apoptotic (annexin V+/PI-), late apoptotic (annexin V+/PI+) and necrotic (annexin V-/PI+) cells. The
cellular density plots that treated with enediyne
control group, enediyne exhibits the ability to induce apoptosis. Similar to commonly used anti-tumor
agents such as cisplatin and its biotin-tagged complexes61. This result unambiguously confirms that
3 are shown in figure 5. Compared with the untreated
3
enediyne
3
acts on DNA-damage in cells to cause cancer cell death through apoptosis pathway.
Figure 5. Flow cytometry analysis of enediyne 3.
Conclusions
A novel acyclic enediyne functions at physiological temperature was designed and synthesized with subtle
balancing of the steric and electronic effects in molecular design. Spontaneous Bergman cyclization which
was attributed of the enediyne has arisen to produce diradical in a polar solvent DMSO at ambient
temperature. The free radicals are highly reactive towards spin trapping agents and hydrogen donors,
showing effective DNA-cleavage abilities and high cytotoxicity towards tumor cells. Flow cytometry
analysis showed that the inhibition of the cancer cell variability by the enediyne is mainly through
apoptosis pathway. In conclusion, thanks to its spontaneous thermal cyclization at physiological
temperature, this highly reactive enediyne is promising to act as a radiomimetic agent with strong activity
on DNA-cleavage and high cytotoxicity to cancer cells.
Conflicts of interest
There are no conflicts to declare.
Acknowledgements
8

ACCEPTED MANUSCRIPT
Dedicated to Prof. Ji-tao Wang on the occasion of his 100th birthday. The authors gratefully acknowledge
the financial support from National Natural Science Foundation of China (21474027, 21503078,
21871080), the Fundamental Research Funds for the Central Universities (22221818014), and Shanghai
Leading Academic Discipline Project (B502). AH thanks the “Eastern Scholar Professorship” support
from Shanghai local government.
Notes and references
(1) Hanahan, D.; Weinberg, R. A. "Hallmarks of Cancer: The Next Generation". Cell 2011, 144,
646-674.
(2) Ruffell, B.; Coussens, Lisa M. "Macrophages and Therapeutic Resistance in Cancer". Cancer
Cell, 27, 462-472.
(3) Dawson, M. A.; Kouzarides, T. "Cancer Epigenetics: From Mechanism to Therapy". Cell 2012,
150, 12-27.
(4) Connors, T. A. "Platinum coordination complexes in cancer chemotherapy". Platinum Met.
Rev. 1973, 17, 98-101.
(5) Pendyala, L.; Kidani, Y.; Perez, R.; Wilkes, J.; Bernacki, R. J.; Creaven, P. J. "Cytotoxicity,
cellular accumulation and DNA binding of oxaliplatin isomers". Cancer Lett. 1995, 97, 177-184.
(6) Hsiang, Y. H.; Hertzberg, R.; Hecht, S.; Liu, L. F. "Camptothecin induces protein-linked DNA
breaks via mammalian DNA topoisomerase I". J. Biol. Chem. 1985, 260, 14873-14878.
(7) Nicolaou, K. C.; Yang, Z.; Liu, J. J.; Ueno, H.; Nantermet, P. G.; Guy, R. K.; Claiborne, C. F.;
Renaud, J.; Couladouros, E. A.; Paulvannan, K.; Sorensen, E. J. "Total synthesis of taxol". Nature 1994,
367, 630.
(8) Giaccone, G.; Herbst, R. S.; Manegold, C.; Scagliotti, G.; Rosell, R.; Miller, V.; Natale, R. B.;
Schiller, J. H.; von Pawel, J.; Pluzanska, A.; Gatzemeier, M.; Grous, J.; Ochs, J. S.; Averbuch, S. D.; Wolf,
M. K.; Rennie, P.; Fandi, A.; Johnson, D. H. "Gefitinib in combination with gemcitabine and cisplatin in
advanced non-small-cell lung cancer: A phase III trial-INTACT1". J Clin Oncol 2004, 22, 777-784.
(9) Jones, M.-C.; Leroux, J.-C. "Polymeric micelles – a new generation of colloidal drug carriers".
Eur. J. Pharm. Biopharm. 1999, 48, 101-111.
(10) Uchegbu, I. F.; Vyas, S. P. "Non-ionic surfactant based vesicles (niosomes) in drug delivery".
Int. J. Pharm. 1998, 172, 33-70.
(11) Nishiyama, N.; Kataoka, K. "Current state, achievements, and future prospects of polymeric
micelles as nanocarriers for drug and gene delivery". Pharmacol. Ther. 2006, 112, 630-648.
(12) Ambrosini, G.; Adida, C.; Sirugo, G.; Altieri, D. C. "Induction of Apoptosis and Inhibition of
Cell Proliferation bysurvivin Gene Targeting". J. Biol. Chem. 1998, 273, 11177-11182.
(13) Perez, R. P. "Cellular and molecular determinants of cisplatin resistance". Eur J Cancer 1998,
34, 1535-1542.
(14) Sobot, D.; Mura, S.; Couvreur, P. "How can nanomedicines overcome cellular-based
anticancer drug resistance?". J. Mater. Chem. B 2016, 4, 5078-5100.
(15) Povirk, L. F. "DNA damage and mutagenesis by radiomimetic DNA-cleaving agents:
bleomycin, neocarzinostatin and other enediynes". Mutat. Res., Fundam. Mol. Mech. Mutagen. 1996,
355, 71-89.
(16) Beerman, T. A.; Gawron, L. S.; Shin, S.; Shen, B.; McHugh, M. M. "C-1027, A Radiomimetic
Enediyne Anticancer Drug, Preferentially Targets Hypoxic Cells". Cancer Res. 2009, 69, 593-598.
(17) Kennedy, D. R.; Gawron, L. S.; Ju, J.; Liu, W.; Shen, B.; Beerman, T. A. "Single Chemical
9

ACCEPTED MANUSCRIPT
Modifications of the C-1027 Enediyne Core, a Radiomimetic Antitumor Drug, Affect Both Drug Potency
and the Role of Ataxia-Telangiectasia Mutated in Cellular Responses to DNA Double-Strand Breaks".
Cancer Res. 2007, 67, 773-781.
(18) Kennedy, D. R.; Beerman, T. A. "The Radiomimetic Enediyne C-1027 Induces Unusual DNA
Damage Responses to Double-Strand Breaks". Biochemistry 2006, 45, 3747-3754.
(19) Nicolaou, K. C.; Dai, W. M. "Chemistry and Biology of the Enediyne Anticancer Antibiotics".
Angew. Chem. Int. Ed. Eng. 1991, 30, 1387-1416.
(20) Lee, M. D.; Dunne, T. S.; Chang, C. C.; Ellestad, G. A.; Siegel, M. M.; Morton, G. O.; McGahren,
W. J.; Borders, D. B. "Calichemicins, a novel family of antitumor antibiotics. 2. Chemistry and structure
of calichemicin .gamma.1I". J. Am. Chem. Soc. 1987, 109, 3466-3468.
(21) Konishi, M.; Ohkuma, H.; Matsumoto, K.; Tsuno, T.; Kamei, H.; Miyaki, T.; Oki, T.; Kawaguchi,
H.; Vanduyne, G. D.; Clardy, J. "Dynemicin A, a novel antibiotic with the anthraquinone and
1,5-diyn-3-ene subunit". J. Antibiot. 1989, 42, 1449-52.
(22) Golik, J.; Clardy, J.; Dubay, G.; Groenewold, G.; Kawaguchi, H.; Konishi, M.; Krishnan, B.;
Ohkuma, H.; Saitoh, K.; Doyle, T. W. "Esperamicins, a novel class of potent antitumor antibiotics. 2.
Structure of esperamicin X". J. Am. Chem. Soc. 1987, 109, 3461-3462.
(23) Groneberg, R. D.; Miyazaki, T.; Stylianides, N. A.; Schulze, T. J.; Stahl, W.; Schreiner, E. P.;
Suzuki, T.; Iwabuchi, Y.; Smith, A. L.; Nicolaou, K. C. "Total synthesis of calicheamicin .gamma.1I. 1.
Synthesis of the oligosaccharide fragment". J. Am. Chem. Soc. 1993, 115, 7593-7611.
(24) Nicolaou, K. C.; Wang, Y.; Lu, M.; Mandal, D.; Pattanayak, M. R.; Yu, R.; Shah, A. A.; Chen, J.
S.; Zhang, H.; Crawford, J. J.; Pasunoori, L.; Poudel, Y. B.; Chowdari, N. S.; Pan, C.; Nazeer, A.; Gangwar,
S.; Vite, G.; Pitsinos, E. N. "Streamlined Total Synthesis of Uncialamycin and Its Application to the
Synthesis of Designed Analogues for Biological Investigations". J. Am. Chem. Soc. 2016, 138, 8235-46.
(25) Nicolaou, K. C.; Hummel, C. W.; Pitsinos, E. N.; Nakada, M.; Smith, A. L.; Shibayama, K.;
Saimoto, H. "Total synthesis of calicheamicin .gamma.1I". J. Am. Chem. Soc. 1992, 114, 10082-10084.
(26) Nicolaou, K. C.; Ogawa, Y.; Zuccarello, G.; Schweiger, E. J.; Kumazawa, T. "Cyclic conjugated
enediynes related to calicheamicins and esperamicins: calculations, synthesis, and properties". J. Am.
Chem. Soc. 1988, 110, 4866-4868.
(27) Schreiner, P. R. "Monocyclic Enediynes:ꢀ Relationships between Ring Sizes, Alkyne Carbon
Distances, Cyclization Barriers, and Hydrogen Abstraction Reactions. Singlet−Triplet Separations of
Methyl-Substituted p-Benzynes". J. Am. Chem. Soc. 1998, 120, 4184-4190.
(28) Nicolaou, K. C.; Liu, A.; Zeng, Z.; McComb, S. "Redox-controlled Bergman
cycloaromatizations. Designed enediynes with DNA-cleaving properties and antitumor activity". J. Am.
Chem. Soc. 1992, 114, 9279-9282.
(29) Semmelhack, M. F.; Gallagher, J. "Cyclic conjugated enediynes via elimination of a
thionocarbonate in a latent Z-hex-3-ene-1,5-diyne unit". Tetrahedron Lett. 1993, 34, 4121-4124.
(30) Schmittel, M.; Kiau, S. "Polar Effects in the Transition State of the Bergman Cyclization".
Chem. Lett. 1995, 1995, 953-954.
(31) Maier, M. E.; Greiner, B. "Synthesis and Reactivity of a p-Methoxyphenyl-Substituted
Enediyne. A Case of Electronic Influence on the Rate of the Bergman Cycloaromatization". Liebigs Ann.
Chem. 1992, 1992, 855-861.
(32) Warner, B. P.; Millar, S. P.; Broene, R. D.; Buchwald, S. L. "Controlled Acceleration and
Inhibition of Bergman Cyclization by Metal Chlorides". Science 1995, 269, 814-816.
(33) Benites, P. J.; Rawat, D. S.; Zaleski, J. M. "Metalloenediynes: Ligand field control of thermal
10

ACCEPTED MANUSCRIPT
Bergman cyclization reactions". J. Am. Chem. Soc. 2000, 122, 7208-7217.
(34) Bhattacharyya, S.; Pink, M.; Huffman, J. C.; Zaleski, J. M. "Thioether coordinated
metalloenediynes: Syntheses, structures and thermal reactivity comparison". Polyhedron 2006, 25,
550-558.
(35) Bhattacharyya, S.; Dye, D. F.; Pink, M.; Zaleski, J. M. "A geometric switching approach
toward thermal activation of metalloenediynes". Chem. Commun. 2005, 5295-7.
(36) Lindahl, S. E.; Park, H.; Pink, M.; Zaleski, J. M. "Utilizing Redox-Mediated Bergman
Cyclization toward the Development of Dual-Action Metalloenediyne Therapeutics". J. Am. Chem. Soc.
2013, 135, 3826-3833.
(37) Nicolaou, K.; Ogawa, Y.; Zuccarello, G.; Kataoka, H. "DNA cleavage by a synthetic mimic of
the calicheamicin-esperamicin class of antibiotics". J. Am. Chem. Soc. 1988, 110, 7247-7248.
(38) Boger, D. L.; Zhou, J. "CDPI3-enediyne and CDPI3-EDTA conjugates: a new class of DNA
cleaving agents". J. Org. Chem. 1993, 58, 3018-3024.
(39) Sun, S.; Zhu, C.; Song, D.; Li, F.; Hu, A. "Preparation of conjugated polyphenylenes from
maleimide-based enediynes through thermal-triggered Bergman cyclization polymerization". Polym.
Chem. 2014, 5, 1241-1247.
(40) Song, D.; Sun, S.; Tian, Y.; Huang, S.; Ding, Y.; Yuan, Y.; Hu, A. "Maleimide-based acyclic
enediyne for efficient DNA-cleavage and tumor cell suppression". J. Mater. Chem. B 2015, 3,
3195-3200.
(41) Song, D.; Tian, Y.; Huang, S.; Li, B.; Yuan, Y.; Hu, A. "An acyclic enediyne with a furyl tethering
group for efficient inhibition of tumor cell viability". J. Mater. Chem. B 2015, 3, 8584-8588.
(42) Dubernet, M.; Caubert, V.; Guillard, J.; Viaud-Massuard, M.-C. "Synthesis of substituted
bis(heteroaryl)maleimides". Tetrahedron 2005, 61, 4585-4593.
(43) Mames, A.; Stecko, S.; Mikołajczyk, P.; Soluch, M.; Furman, B.; Chmielewski, M. "Direct,
Catalytic Synthesis of Carbapenams via Cycloaddition/Rearrangement Cascade Reaction: Unexpected
Acetylenes’ Structure Effect". J. Org. Chem. 2010, 75, 7580-7587.
(44) O'Brien, C. J.; Kantchev, E. A. B.; Valente, C.; Hadei, N.; Chass, G. A.; Lough, A.; Hopkinson, A.
C.; Organ, M. G. "Easily Prepared Air- and Moisture-Stable Pd–NHC (NHC=N-Heterocyclic Carbene)
Complexes: A Reliable, User-Friendly, Highly Active Palladium Precatalyst for the Suzuki–Miyaura
Reaction". Chem.-Eur. J. 2006, 12, 4743-4748.
(45) Lu, Y. J.; Yang, S. H.; Chien, C. M.; Lin, Y. H.; Hu, X. W.; Wu, Z. Z.; Wu, M. J.; Lin, S. R.
"Induction of G2/M phase arrest and apoptosis by a novel enediyne derivative, THDB, in chronic
myeloid leukemia (HL-60) cells". Toxicol. in Vitro 2007, 21, 90-98.
(46) Mifsud, N.; Mellon, V.; Perera, K. P. U.; Smith, D. W.; Echegoyen, L. "In situ EPR spectroscopy
of aromatic diyne cyclopolymerization". J. Org. Chem. 2004, 69, 6124-6127.
(47) Zeidan, T. A.; Manoharan, M.; Alabugin, I. V. "Ortho effect in the Bergman cyclization:
Interception of p-benzyne intermediate by intramolecular hydrogen abstraction". J. Org. Chem. 2006,
71, 954-961.
(48) Baroudi, A.; Mauldin, J.; Alabugin, I. V. "Conformationally Gated Fragmentations and
Rearrangements Promoted by Interception of the Bergman Cyclization through Intramolecular
H-Abstraction: A Possible Mechanism of Auto-Resistance to Natural Enediyne Antibiotics?". J. Am.
Chem. Soc. 2010, 132, 967-979.
(49) dos Passos Gomes, G.; Alabugin, I. V. "Drawing Catalytic Power from Charge Separation:
Stereoelectronic and Zwitterionic Assistance in the Au(I)-Catalyzed Bergman Cyclization". J. Am. Chem.
11

ACCEPTED MANUSCRIPT
Soc. 2017, 139, 3406-3416.
(50) Janzen, E. G.; Blackburn, B. J. "Detection and identification of short-lived free radicals by an
electron spin resonance trapping technique". J. Am. Chem. Soc. 1968, 90, 5909-5910.
(51) Usuki, T.; Kawai, M.; Nakanishi, K.; Ellestad, G. A. "Calicheamicin gamma(I)(1) and phenyl
tert-butyl nitrone (PBN): observation of a kinetic isotope effect by an ESR study". Chem. Commun.
2010, 46, 737-739.
(52) Yao, Q.; Li, C.-J. "Development of an indicator for the direct visualization of radical
intermediates in organic reactions". Chem. Commun. 2017, 53, 11225-11228.
(53) Vavilala, C.; Byrne, N.; Kraml, C. M.; Ho, D. M.; Pascal, R. A. "Thermal C1-C5 Diradical
Cyclization of Enediynes". J. Am. Chem. Soc. 2008, 130, 13549-13551.
(54) Haberhauer, G.; Gleiter, R.; Fabig, S. "Enediyne Dimerization vs Bergman Cyclization". Org.
Lett. 2015, 17, 1425-1428.
(55) Konig, B.; Pitsch, W.; Klein, M.; Vasold, R.; Prall, M.; Schreiner, P. R. "Carbonyl- and
carboxyl-substituted enediynes: synthesis, computations, and thermal reactivity". J. Org. Chem. 2001,
66, 1742-1746.
(56) Alabugin, I. V.; Manoharan, M. "Reactant Destabilization in the Bergman Cyclization and
Rational Design of Light- and pH-Activated Enediynes". J. Phys. Chem. A 2003, 107, 3363-3371.
(57) Kar, M.; Basak, A. "Design, Synthesis, and Biological Activity of Unnatural Enediynes and
Related Analogues Equipped with pH-Dependent or Phototriggering Devices". Chem. Rev. 2007, 107,
2861-2890.
(58) Dhar, S.; Lippard, S. J. "Mitaplatin, a potent fusion of cisplatin and the orphan drug
dichloroacetate". Proc. Natl. Acad. Sci. 2009, 106, 22199-22204.
(59) Huang, P.; Wang, D.; Su, Y.; Huang, W.; Zhou, Y.; Cui, D.; Zhu, X.; Yan, D. "Combination of
Small Molecule Prodrug and Nanodrug Delivery: Amphiphilic Drug–Drug Conjugate for Cancer
Therapy". J. Am. Chem. Soc. 2014, 136, 11748-11756.
(60) He, H. Y.; Zhao, J. N.; Jia, R.; Zhao, Y. L.; Yang, S. Y.; Yu, L. T.; Yang, L. "Novel Pyrazolo[3,4-d]
pyrimidine Derivatives as Potential Antitumor Agents: Exploratory Synthesis, Preliminary
Structure-Activity Relationships, and in Vitro Biological Evaluation". Molecules 2011, 16, 10685-10694.
(61) Muhammad, N.; Sadia, N.; Zhu, C.; Luo, C.; Guo, Z.; Wang, X. "Biotin-tagged platinum(iv)
complexes as targeted cytostatic agents against breast cancer cells". Chem. Commun. 2017, 53,
9971-9974.
12