Triggering the Antitumor Activity of Acyclic Enediyne through Maleimide-Assisted Rearrangement and Cycloaromatization

Article abstract of DOI:10.1021/acs.joc.0c01124

Acyclic enediynes are generally inactive under physiological conditions to be used as antitumor agents like their natural enediyne counterparts. A new mechanism named as maleimide-assisted rearrangement and cycloaromatization (MARACA) is uncovered to trigger the reactivity of acyclic enediynes. Through this mechanism, cascade 1,3-proton transfer processes are accelerated with the maleimide moiety at the ene position to enable the acyclic enediynes to undergo cycloaromatization and generate reactive radicals under physiological conditions. Computational studies suggest that the highest energy barrier for MARACA is 26.0 kcal/mol, much lower than that of Bergman cyclization pathway (39.6 kcal/mol). Experimental results show that maleimide-based enediynes exhibit low onset temperature, fast generation of radical species at 37 °C, and much faster reaction in aqueous solution than in nonpolar solvent, which is beneficial to achieve both high reactivity in physiological environment and high stability for storage and delivery in nonpolar media. The generated radical species are capable of causing high percentage of double-strand (ds) DNA cleavage, leading to significant cytotoxicity toward a panel of cancer cell lines with half inhibition concentration down to submicromolar level. Overall, the discovery of the MARACA mechanism provides a platform for designing novel acyclic enediynes with high potency for antitumor applications.

Full text of DOI:10.1021/acs.joc.0c01124

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Article
Triggering the Antitumor Activity of Acyclic Enediyne through
Maleimide Assisted Rearrangement and Cycloaromatization
mengsi zhang, Baojun Li, Huimin Chen, Haotian Lu, Hailong Ma,
Xiaoyu Cheng, Wenbo Wang, Yue Wang, Yun Ding, and Aiguo Hu
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.0c01124 • Publication Date (Web): 13 Jul 2020
Downloaded from pubs.acs.org on July 13, 2020
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The Journal of Organic Chemistry
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Triggering the Antitumor Activity of Acyclic Enediyne through Malei-
mide Assisted Rearrangement and Cycloaromatization
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Mengsi Zhang, Baojun Li, Huimin Chen, Haotian Lu, Hailong Ma, Xiaoyu Cheng, Wenbo Wang, Yue
Wang, Yun Ding*, Aiguo Hu*
Shanghai Key Laboratory of Advanced Polymeric Materials, School of Materials Science and Engineering, East China
University of Science and Technology, Shanghai 200237, China.
KEYWORDS. Acyclic enediyne, Cycloaromatization, Rearrangement, Mechanism study, Antitumor agents.
ABSTRACT: Acyclic enediynes are generally inactive under physiological conditions to be used as antitumor agents like
their natural enediyne counterparts. A new mechanism named as maleimide assisted rearrangement and cycloaromatiza-
tion (MARACA) is uncovered to trigger the reactivity of acyclic enediynes. Through this mechanism, cascade 1,3-proton
transfer processes are accelerated with the maleimide moiety at ene position to enable the acyclic enediynes to undergo
cycloaromatization and generate reactive radicals under physiological conditions. Computational studies suggest that the
highest energy barrier for MARACA is 26.0 kcal/mol, much lower than that of Bergman cyclization pathway (39.6 kcal/mol).
Experimental results show that maleimide-based enediynes exhibit low onset temperature, fast generation of radical spe-
cies at 37 °C and much faster reaction in aqueous solution than in nonpolar solvent, which is beneficial to achieve both high
reactivity in physiological environment and high stability for storage and delivery in nonpolar media. The generated radical
species are capable of causing high percentage of double strand (ds) DNA cleavage, leading to significant cytotoxicity to-
ward a panel of cancer cell lines with half inhibition concentration down to sub-micromolar level. Overall, the discovery of
the MARACA mechanism provides a platform for designing novel acyclic enediynes with high potency for antitumor applica-
tions.
Myers-Saito cyclization (MSC) of enyne-allene15,16 is an-
other type of thermal cycloaromatization besides BC that
plays fundamental role in the activation mode of natural
enediyne antibiotics25,26, such as the chromophore of neo-
carzinostatin (NCS)17. Both reactions share significant
similarity in producing highly reactive diradicals (or dirad-
icaloids) from closed-shell compounds, however MSCs
typically take place at much lower temperature. The de-
creased repulsion of in-plane π orbitals in the transoid-
orientation is believed to be the key for this facile and low-
INTRODUCTION
Since their discovery in the 1980s, enediynes become
one of the most fascinating classes of natural products1.
The unique enediyne architectures that prone to produce
aromatic diradicals through Bergman cyclization (BC)2
endow these compounds with extremely high cytotoxicity
against a broad range of cancer cells, leading to great po-
tentials for the development of highly efficient antitumor
agents. Indeed, one of the naturally occurring enediynes,
calicheamicin, has been attached to monoclonal antibodies
to form antibody-drug-conjugates for clinical curing leu-
kemia3-5 and several others are undergoing preclinical
evaluations6. Unfortunately, natural enediynes are difficult
to prepare in good yield and preparation of the highly
strained cyclic and complicated architecture of the unsta-
ble enediynes requires Herculean effort7-9, severely limit-
ing their broader applications10. Employing synthetic ene-
diynes in anticancer applications also constitutes signifi-
cant difficulties11. The cyclic enediynes (especially those
with small ring sizes) typically suffer difficulty in synthesis,
and short half-lives for derivatization or long-term storage,
while the acyclic ones are generally inactive at physiologi-
er activation energy demanding cycloaromatization18-21
.
Although some of the enyne-allene compounds have been
isolated and characterized, due to their high reactivity,
they were typically formed from stable precursors through
various kinds of transformations. The sharp difference
between the MSC reactivity of enyne-allene and the BC
reactivity of acyclic enediyne provides a straightforward
way for novel molecular design by taking advantages of
cascade propargyl-allene rearrangements and thermal
cycloaromatization. Many efforts have been devoted to
facilitate this kind of transformation in the presence of acid,
base, oxidant, transition metal complexes or under high
temperature2,22-27. However, most of these conditions do
not resemble the biological systems.
cal temperature12-14
.
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In pursuit of uncovering the antitumor potencies of acy- Another kind of tert-butyl group was installed in the ma-
1
clic enediynes, we recently found that several kinds of acy-
clic enediyne compounds exhibited unprecedented high
reactivity to generate radical species under relatively mild
conditions28-32. All of these enediyne compounds share the
same functionality at ene position, maleimide. The malei-
mide moiety was firstly found to play an important role in
lowering the onset temperature of cycloaromatization28.
Later on, we found that when the steric and electronic
properties of the substituents at the alkyne termini were
finely tuned, the highly reactive acyclic enediynes were
able to produce radical species under physiological condi-
tions29-32. The energy barriers however were comparable
or even higher than those of the nonreactive acyclic ene-
diynes if calculated through BC pathways (close to 40
kcal/mol)33, implying the possibility of a new mechanism
involved. To this end, we herein report in depth investiga-
tions on this kind of intriguing reactions. We confirmed
that the facile cycloaromatization of the acyclic enediynes
with maleimide moieties originated from a “maleimide
assisted rearrangement and cycloaromatization” mecha-
nism (abbreviated as MARACA). The propargyl-allene tau-
tomerization was greatly facilitated by maleimide group to
enable the otherwise nonreactive acyclic enediynes to un-
dergo MSC in physiologically relevant environments. Fol-
lowing this line, several acyclic enediynes were designed
and synthesized. They were stable in nonpolar solvents but
quickly generated radical species in aqueous solutions to
cause high percentage of double strand (ds) DNA cleavage
and lead to apoptosis of tumor cells at sub-micromolar
concentrations.
leimide moiety (2) for the same reason. Compounds 140
and 241 were synthesized according to literature proce-
dures, while 1,2-diiodobenzene was obtained from com-
mercial resource. The Sonogashira coupling reaction be-
tween alkyne 1 and 1,2-diiodobenzene proceeded smooth-
ly to produce EDY-A in good yield at room temperature
(Scheme 1). Interestingly, when the similar experimental
procedure was subjected to the Sonogashira coupling reac-
tion between alkyne 1 and compound 2, EDY-B was not
isolated as the only product. Compound 3 with distinct
bright blue fluorescence was isolated in a comparable yield
to EDY-B. Moreover, the transformation of EDY-B to com-
pound 3 was clearly observed with prolonged reaction
time or at elevated reaction temperature, suggesting the
possibility of converting EDY-B to compound 3 in the reac-
tion mixture. Indeed, when pure EDY-B was heated in
methanol, compound 3 was also obtained together with
some polymeric products. The molecular structures of two
enediynes were confirmed with NMR and high resolution
mass spectra (HR-MS) analysis (supporting information).
The structure of compound 3 was characterized by 2D
NMR, HR-MS, and single crystal X-ray diffraction analysis
(supporting information). The sharp difference between
the molecular structures of compound 3 and the possible
BC product (see Scheme 2) unambiguously indicates the
involvement of rearrangement and ring closure reactions,
suggesting that a new reaction mechanism hides behind
the cyclization of maleimide-based EDY-B.
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Thermal Reactivity of EDY-A and EDY-B. The thermal
reactivities of two model enediyne compounds were inves-
tigated with differential scanning calorimetry (DSC) analy-
sis. This method allows recording the onset temperature of
thermal cyclizations of enediynes and enyne-allenes over a
range of temperatures42,43. As shown in Figure 1, EDY-A
holds a sharp endothermal melting peak at 129 °C, fol-
lowed by an exothermal peak at 253 °C which corresponds
to the polymerization of free radical intermediates44
formed from cycloaromatization of enediynes. Basak et al.
demonstrated that a series of structurally similar acyclic
enediyne possessed high onset temperatures, typically
beyond 200 °C, attributing to BC mode45. In contrast, EDY-
B reveals a very low onset temperature at 56 °C, much
closer to physiological temperature and nearly 200 °C low-
er than that of EDY-A despite bearing the identical alkyne
functionalities. The computed activation barriers for EDY-
A and EDY-B through BC pathway are almost the same,
close to 40 kcal/mol (Figure S1), which fits well with the
low thermal reactivity of EDY-A, but clearly has discrepan-
cy with the facile reaction of EDY-B. Therefore, we can
conclude that when maleimide moiety is installed at the
ene position of an enediyne compound, a new pathway
with much lower activation barrier is created, leading to
much faster reaction at low temperature. Taking the fact
that compound 3 is formed from EDY-B into consideration,
rearrangement reactions are also involved in this new
pathway and they are facilitated by the maleimide moiety
as well. Altogether, a new mechanism named as MARACA
is believed to guide the cyclization of EDY-B and other
kinds of maleimide-based enediynes to enable them to
RESULTS AND DISCUSSION
Scheme 1. Synthesis of two model enediyne com-
pounds and schematic illustration of formation com-
pound 3 from EDY-B: (a) (PPh3)2PdCl2, CuI, n-BuNH2,
tetrahydrofuran (THF), r.t., 18 h; (b) (PPh3)2PdCl2, CuI,
N,N-diisopropylethylamine (DIPEA), toluene/THF, r.t.,
12 h.
Synthesis of Model Enediynes. To clarify the unique
role of maleimide moiety in the cyclization of enediynes,
two model compounds equipped with the same alkyne
functionalities were designed. EDY-A features a benzene
group at the ene position, representing common acyclic
enediynes34-39, while EDY-B features a maleimide group at
the same position. The two tert-butyl groups of different
types were installed in the terminal alkyne, tert-butyl tert-
butyl(prop-2-yn-1-yl)carbamate (1), as chemical shift indi-
cators for nuclear magnetic resonance (NMR) analysis.
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generate reactive radical species at low temperature for pathway. A new mechanism thus is proposed after careful
antitumor applications as discussed below.
1
comparison of the molecular structures of compound 3
and its precursor EDY-B, involving cascade rearrange-
ments and cycloaromatization as shown in Scheme 2. The
first step in this multistep transformation is the tautomeri-
zation of one of the propargyl groups to form an enyne-
allene isomer 4, which is known as an MSC precursor to
produce the α, 3-didehydrotoluene diradical 5. On the oth-
er hand, a similar enyne-allene intermediate 8 is formed
starting from 4 via further 1,3-proton transfer processes,
which proceeds through another MSC to give diradical 9.
The aryl radical in 9 immediately undergoes 5-endo ring
closure/radical cyclization and subsequent hydrogen ab-
straction to afford compound 3, which is considered as a
thermodynamically favored product. The proposed
MARACA mechanism is verified by means of density func-
tional theory, where the calculated energy barriers suggest
that the rearrangement and cyclization processes of ene-
diynes are feasible at room temperature (see below). It is
noteworthy that although the conversion of EDY-B to
compound 3 was achieved at 37 °C in methanol without
any additive, the addition of trace amount of weak base
like triethylamine, or sodium acetate indeed accelerated
this transformation. On the contrary, EDY-A was stable at
37 °C even in the presence of excess strong base like 1,8-
diazabicyclo[5.4.0]undec-7-ene. In other words, when
enediynes are exposed to physiologically relevant envi-
ronments, the presence of maleimide group is essential to
facilitate the isomerization, driving enediynes away from
the one step BC mechanism and directing them to multi-
step rearrangements and MSC reaction, triggering the free
radical generation ability of acyclic enediynes at low tem-
perature.
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Figure 1. DSC curves of EDY-A and EDY-B. The baselines are
marked in red for guide of view.
Figure 2. (A) EPR spectra of control, EDY-A (20 mM) and EDY-
B (20 mM) in the presence of PBN (100 mM) in DCE at 37 °C
for 12 h. (B) Time-dependent EPR signal intensities at 3520 G
of control, EDY-A (20 mM) and EDY-B (10 and 20 mM) in the
presence of PBN (100 mM) in DCE at 37 ℃ for 12, 24, and 48 h
respectively.
Detection of Radical Intermediates. The possibility of
generating free radicals from enediynes was verified using
electron paramagnetic resonance (EPR) spectroscopy46. In
general, it is difficult to monitor the transient radical in-
termediates formed from cycloaromatization due to their
very low equilibrium concentration relative to that of ene-
Computational study. To understand the preference of
EDY-B reacting through cascade rearrangements and cy-
cloaromatization to generate compound 3 rather than to
BC product, the reaction mechanism was investigated in
detail with structurally simplified models (the structures
of these models are shown in Figure 3 and Scheme S1).
Acetate anion, acting as the proton shuttle, was chosen to
accomplish the reaction pathways when proton transfers
were involved in the rearrangement reactions. Density
functional theory (DFT)51,52 studies have been carried out
with Gaussian 09 program53 using the (U)B3LYP exchange
and correlation functional and the 6-31G(d) basis set in gas
phase. A restricted approach was employed during geome-
try optimizations for the closed-shell structures, while for
the optimizations of the transition states (TS) of the BC,
MSC and the biradical products, an unrestricted broken-
symmetry ansatz has been utilized. This computational
setup has been successfully applied in the mechanistic
diynes47,48
.
Thus,
a
spin trap, N-tert-butyl-alpha-
phenylnitrone (PBN), was exploited to capture the
short-lived radicals by converting them into stable radical
adducts49,50. EDY-A and EDY-B were dissolved in 1,2-
dichloroethane (DCE) in the presence of excess PBN re-
spectively, then kept at 37 °C for 12, 24, and 48 h for the
EPR studies. As shown in Figure 2A, the spin-trapped PBN
adducts with a typical triplet peak is observed with EDY-B,
uncovering the generation of radicals that probably origi-
nate from the smooth cycloaromatization at physiological
temperature. Figure 2B shows that the intensity of the rec-
orded signals for EDY-B at 3520 G increases in both time
and concentration dependent manners. In contrast, EPR
measurement of EDY-A shows negligible signal, consistent
with its high BC onset temperature and extremely low re-
activity at 37 °C. The maleimide-assisted formation of the
reactive radical species is of critical importance for the
biological activity of maleimide-based enediynes.
studies of rearrangement and cyclization reactions33,45
.
Harmonic vibration frequency calculations were carried
out at 298.15 K and the optimized structures are all shown
to be either minima (with no imaginary frequency) or TSs
(with only one imaginary frequency). Intrinsic reaction
coordinate (IRC) calculations were performed to confirm
the connection of each TS to its corresponding reactant
and product. For each hydrogen transfer intermediate
connecting two adjacent TSs, structure optimizations are
General Scheme of MARACA. According to the above
findings, the two model enediynes exhibit striking differ-
ences in thermal reactivity and the performance of free
radical generation, which is related to the fact that EDY-A
undergoes BC mode, while EDY-B goes through a different
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Scheme 2. Schematic illustration of the proposed MARACA mechanism. The formation of compound 3 and the un-
likely formation of BC product are presented as well.
gyl-allenyl rearrangement by maleimide moiety clearly
stands out. In addition, it is noteworthy that the first dirad-
ical P2 is calculated to lie 8.1 kcal/mol below enyne-allene
R1 in Gibbs free energy, while the latter diradical P3 is
calculated to be exothermic by only 4.2 kcal/mol compared
to R4. Meanwhile, the facile 5-endo attack of phenyl radical
in 9 (similar to P3) to the t-Boc group allows the formation
of thermodynamically stable compound 3 as found in our
experiments (Scheme 1). Combining the experimental and
computational findings, it is unambiguously confirmed that
the presence of maleimide group enables acyclic enediynes
to transform into reactive enyne-allenes via one or multi-
step rearrangement and undergo MSC in physiologically
relevant environments through the MARACA mechanism.
carried out based on both IRC calculations, where the con-
figuration with lower Gibbs free energy is chosen to repre-
sent the intermediate.
As shown in Figure 3, an extremely high activation ener-
gy for BC of the acyclic enediyne R is obtained, at 39.6
kcal/mol. Such a high barrier requires high temperature
(over 200 ℃) to trigger this reaction, which is unfavorable
for the application in biological systems. Alternatively, a
low energy-demanding route to generate diradical prod-
ucts arises, involving isomerization through 1,3-proton
transfer assisted by acetate anion and subsequent MSC.
Over TS1 (6.8 kcal/mol), the propargyl proton of R is ab-
stracted by the acetate anion, leading to the intermediate
INT1. Subsequently, the protonation of alkynyl carbon
occurs to yield the enyne-allene R1 over a barrier of 8.7
kcal/mol. The MSC of R1 might take place to generate
diradical P2 via TS-MSC1 with the activation energy of
24.0 kcal/mol. These results clearly reveal the preference
of MSC over BC if the rearrangement reaction is achieved
under mild conditions. The isomerization of R1 may con-
tinue with further 1,3-proton transfer to yield retro-
propargyl R2 via deprotonation and protonation processes
with the barrier of 1.5 and 16.4 kcal/mol respectively, cor-
responding to the TS3 and TS4. With two more 1,3-proton
transfer steps, a second enyne-allene R4 is formed. At this
point, the diradical P3 is generated through MSC over TS-
MSC2 (26.0 kcal/mol). Throughout this MARACA mecha-
nism, the MSC steps are the rate-limiting steps, while the
proton transfer steps are all with low energy barriers, and
even a very weak base may act as an efficient proton shut-
tle in the 1,3-proton transfer processes. In comparison
with much high barriers of 1,3-proton shifts observed by
Alabugin et al.54, the dramatic acceleration of the propar-
Synthesis of Maleimide-Based Enediyne Derivatives.
According to the above findings, only one propargyl struc-
ture in the enediyne core is needed to realize the desired
MARACA mechanism for maleimide-based enediynes.
Therefore, we rationally designed and synthesized four
more enediynes in view of three adjustable sites, the one
propargyl group necessary for rearrangement reaction, the
substituents at maleimide moiety and the other alkyne
both ready for modulating the LogP value regarding to the
quantitative estimate of drug-likeness (QED)55. The struc-
tures of these enediynes (Figure 4) have been character-
ized by NMR and HR-MS analysis. DSC study showed that
these enediynes had similar onset temperatures ranging
from 56~70 °C (Figure S3 and Table S1), implying similar
thermal reactivity as EDY-B at physiological temperature
for antitumor applications.
Solution Phase Reactivity of Enediynes. The chemical-
ly synthesized enediynes typically face the dilemma of high
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Figure 3. DFT computed Gibbs free energy profile (kcal/mol) for BC (blue lines), cascade 1,3-proton transfer in the presence of
acetate anion (black lines) and MSC (red lines) pathways of model enediyne R calculated at (U)B3LYP/6-31G(d) level. Optimized
structures of selected important stationary points in the potential energy surface shown below. Color code: C, green; O, red; N, blue;
H, white. The rest of the structures can be found in the supporting information.
Figure 4. Molecular structures of EDY-C, D, E, F.
tions like BC and MSC are intramolecular processes and
are insensitive to their environments56. Encouragingly, the
MARACA mechanism provides a solution to this issue as
the 1,3-proton shift exhibits significant solvent polarity
dependence57-59. To investigate the effect of solvent polari-
ty on their reactivity, EDY-F that has considerable solubili-
ty in both organic and aqueous solutions for facile UV-Vis
spectroscopic study was chosen as a model enediyne. Pure
water and DCE were selected to represent polar and non-
polar media, respectively. The solution phase kinetic ex-
periments were conducted at 37±0.1 °C and the concentra-
tion changes of EDY-F were monitored with UV-Vis spec-
troscopy. The clear decrease of absorption intensity at 365
nm for EDY-F has been observed within 9 h in aqueous
solution (Figure 5A). Accordingly, the absorbance repre-
sentative of concentration versus time is plotted (Figure
Figure 5. (A) UV-Vis absorption spectral changes of EDY-F
(10-4 M) in water. (B) Solution phase kinetic plots for EDY-F in
water at 365 nm.
reactivity in antitumor application and low stability for
long-term storage or derivatization since cycloaromatiza-
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Figure 6. (A) Schematic presentation of the DNA cleavage activity of EDY-C and EDY-F. Agarose gel electrophoretic images (B) and
quantified cleavage data (C) for DNA cleavage assay. Lane 1 and Lane 8: pUC19 (10 μg/ml) alone; Lane 2 to Lane 7: pUC19 (10
μg/ml) was incubated with 1.25 mM (Lane 2), 2.5 mM (Lane 3), 3.25 mM (Lane 4), 5 mM (Lane 5), 7.25 mM (Lane 6), 10 mM (Lane
7) of EDY-C at 37 °C for 48 h; Lane 9 to Lane 13: pUC19 (10 μg/ml) was incubated with 0.625 mM (Lane 9), 1.25 mM (Lane 10), 2.5
mM (Lane 11), 3.25 mM (Lane 12), 5 mM (Lane 13) of EDY-F at 37 °C for 48 h. Code: intact DNA (Form I, green block), single-
strand scission (Form II, yellow block), and double-strand scission (Form III, red block).
for chemotherapy. For example, calicheamicin possessed
25 % of ds DNA cleavage (~1:3 ds:ss ratio)63 and a similar
ds:ss ratio (1:5) was found with NCS64, attributing to their
outstanding antitumor performance. For the maleimide-
based enediynes, highly reactive diradicals are generated
through the MARACA mechanism, endowing them with
DNA cleavage ability. To this end, EDY-C and EDY-F were
selected to evaluate their DNA cleavage performance and
agarose gel electrophoresis experiments were carried out
to differentiate DNA in supercoiled circular (Form I), circu-
lar (Form II), linear (Form III) forms or even fragments
(Figure 6A). pUC19 plasmid DNA were incubated with var-
ied concentrations of EDY-C or EDY-F at 37 °C for 48 h,
respectively and the relative amounts of the three DNA
forms (Figure 6B) were analyzed with densitometric anal-
ysis on the stained electrophoretic images. As the concen-
tration of EDY-C or EDY-F increases, a decrease of Form I
(and even Form II) and concurrent increase of Form III of
pUC19 is observed. The high percentage of ds DNA cleav-
age (Figure 6C) originates from binding of enediyne mole-
cules in the minor groove of DNA62 and cutting both
strands of DNA in a single event. Meanwhile, the possibility
that multiple binding/cleavage events take place with
more than one drug molecules involved should not be
ruled out either65. In order to differenciate the direct ds
cleavage from the random path due to multiple ss breaks,
further statistical analysis was conducted by using the
5B), which shows a first order reaction kinetics with the
rate constant (k) of 3.83×10-2 h-1 and the half-life of 18 h, a
balanced reactivity and stability in the respect of biological
applications60,61. Interestingly, the situation in DCE solu-
tion is entirely different with extremely slow changes on
the concentration of EDY-F for the initial 12 h (Figure S4),
corresponding to a half-life of nearly two months. Such a
huge difference of reactivity in two kinds of solvents
stresses that the stability of enediynes largely depends on
the solvent polarity that dictates the propargyl-allenyl re-
arrangement, providing a promising strategy to balance
the reactivity and stability for acyclic enediynes, i.e. to pro-
ceed the derivation, storage and drug delivery in nonpolar
media and to exert their high reactivity in physiological
environments, rendering the potential application as clini-
cal drugs.
DNA Cleavage Activity of Enediynes. One crucial fea-
ture of enediynes is their ability to abstract H atoms from
the deoxyribose backbone and cause DNA cleavage. For
natural enediynes, even at low concentrations, they can
significantly lead to double-stranded (ds) DNA cleavage62.
The ds DNA cleavage mode, which is much more difficult
for cells to repair than the single-strand (ss) DNA cleavage,
can culminate in efficient self-programmed cell death
(apoptosis), achieving the high cytotoxicity to tumor cells
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Table 1. In vitro cytotoxicity of enediynes against three kinds of tumor cell lines and their physicochemical data.
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9
IC50 (μM)
MCF-7
>50
Physicochemical properties
Compound
Hela
>50
5.8
HepG2
>50
8.2
LogP
tPSA (Å2)
59.1
MW (Da)
EDY-A
EDY-B
EDY-C
EDY-D
EDY-E
EDY-F
5.7
3.2
1.6
3.4
1.4
0.9
496
629
502
500
488
331
7.3
122.8
113.4
93.2
1.6
2.5
2.1
0.96
2.3
1.4
1.2
3.0
3.0
113.4
83.9
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0.54
1.2
1.1
Figure 7. (A) Cellular uptake CLSM analysis of Hela cells treated with EDY-B (6 μM) or EDY-F (1 μM) after 4 or 8 h incubation. Cell
nuclei are stained with PI (red). Scale bar is 10 μm. (B) γ-H2AX foci immunofluorescence staining of Hela cells treated with EDY-A
(6 μM), EDY-B (6 μM), or EDY-C (2 μM). Cell nuclei are stained with PI (red). Scale bar is 10 μm. (C) The cell cycle distribution of
Hela cells treated with EDY-F of varied concentrations for 24 h and then analyzed by flow cytometry. (D) Cell apoptosis assay of
Hela cells treated with EDY-F of varied concentrations for 24 h and then analyzed by flow cytometry.
tinct from many kinds of small molecular enediyne com-
Poisson distribution f(III) = n2 exp (-n2) and the Freifeld-
er−Trumbo equation66,67 f(I) = exp [-(n1+n2)]. According to
the fraction of supercoiled f(I) and linear f(III) DNA from
each experiment, the average number of single strand
breaks (n1) and double strand breaks (n2) per DNA mole-
cule were calculated from the above equations. The n1/n2
ratio shown in Table S2 (ranging from 0.1 to 6.3) are much
lower than 100, clearly indicated that both enediynes
caused ds cleavage through a concerted mechanism rather
than the random pattern. This surprisingly finding is dis-
pounds, suggesting the high possibility of the placement of
these enediynes in the minor groove of DNA. The mecha-
nism of this intriguing binding is still under investigation.
Nevertheless, the efficient DNA cleavage and especially
high percentage of ds DNA cleavage of both enediynes at
millimolar concentration in physiologically relevant envi-
ronments endows their high cytotoxicity towards tumor
cells.
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cells were treated with EDY-F in different concentrations
Cytotoxicity Assays. The ability of enediynes to induce
cell death was evaluated by the 3-(4,5-dimethyl-2-
thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT)
assay (Figure S5). Three kinds of cancer cell lines, human
cervical cancer (Hela), human breast adenocarcinoma
(MCF-7), and human liver carcinoma (HepG2) were used
for these experiments. The half maximum inhibition con-
centrations (IC50) of the enediynes towards cancer cells are
listed in Table 1. The extremely high IC50 values of EDY-A
indicate its low toxicity due to its incapability to generate
radical species in physiological environments. On the other
hand, taking advantage of the MARACA mechanism, all the
maleimide-based enediynes exhibit significant cytotoxicity
towards all three kinds of cancer cells. The IC50 values
range from micromolar to sub-micromolar levels, compa-
rable or even superior to many commercial antitumor
agents like cisplatin68,69, doxorubicin70. As essential prop-
erties for small molecular drugs, solubility and permeabil-
ity are measured by logP and topological polar surface
area (tPSA) values71. The differences in potency among
maleimide-enediynes are the result of substituent effect to
modulate relevant physicochemical properties as stated by
Lipinski’s Rule of Five (Ro5)72 for drug-likeness criteria.
1
2
3
4
5
6
7
8
(1, 2, and 4 μM respectively) for 24 h. The cell cycle distri-
bution was determined by flow cytometry as shown in
Figure 7C. EDY-F arrests the cell cycle at the S phase in a
dose-dependent manner, which reveals that the EDY-F
may display its cytotoxicity through the inhibition of DNA
replication process75 and that the generated diradicals
greatly contribute to this effect via direct DNA interaction
and damage. To further evaluate the cell death pathways
induced by EDY-F, Annexin V-FITC/PI staining assay is
carried out. Hela cells were treated with EDY-F of 1, 2, and
4 μM respectively. The fractions of viable, early apoptotic,
late apoptotic, and necrotic cells were quantified by flow
cytometry. Figure 7D shows that EDY-F causes cell apop-
tosis at the concentration of 1 μM (Q2+Q3=26%) and sig-
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nificant apoptosis behavior is observed at
4 μM
(Q2+Q3=89%). All these results confirm that the generated
diradicals through MARACA mechanism lead to effective S
phase block and promote apoptotic response, eventually
triggering the antitumor potency of acyclic enediynes.
CONCLUSIONS
In summary, we have uncovered a new mechanism
named as MARACA to trigger the antitumor potency of
acyclic enediynes. The presence of maleimide moiety was
proved to be essential for the cascade 1,3-proton shift pro-
cesses and for transforming the otherwise nonreactive
acyclic enediynes to reactive enyne-allenes in physiologi-
cally relevant environments. The first order kinetics of the
enediyne compounds suggest the intramolecular cy-
cloaromatization (MSC) being the rate-limiting step in
MARACA mechanism, which was further confirmed with
theoretical computation. The distinct effect of solvent po-
larity on the overall reactivity of these enediyne com-
pounds endows them with capability for further derivati-
zation, long-term storage and drug delivery without com-
promising their high reactivity in polar media. The gener-
ated free radicals lead to high percentage of ds DNA dam-
age, and cytotoxicity to all tested cancer cell lines with IC50
values of micromolar to sub-micromolar level. Intracellular
studies of enediynes indicated that the radical-involved
DNA damage activity induced cell death via apoptosis
mechanism. Overall, this MARACA mechanism provides a
strategy to design simple yet robust acyclic enediyne anti-
tumor agents for potential clinical applications.
Cellular Uptake. Based on the considerably high ther-
mal reactivity of enediynes in polar solvent and the unique
free-radical-involved direct DNA interaction/damage
mechanism, the efficiency of enediynes entering the cells
or even the cell nucleus is crucial to their cytotoxicity per-
formance. Taking advantages of the inherent fluorescent
property of these maleimide-based enediynes (Figure S6),
their cellular uptake was directly visualized by confocal
laser scanning microscopy (CLSM). Figure 7A shows that
the EDY-F mainly enriches in cell nuclei whereas the EDY-
B in cell cytoplasm for the most of HeLa cells after 4 to 8 h
incubation. The difference in cellular accumulation loca-
tions might be responsible for the distinct efficacy of cyto-
toxicity, as observed from EDY-B and EDY-F (Table 1).
Intracellular DNA Damage Assay. Given the DNA-
targeted cytotoxic mechanism of enediynes, the intracellu-
lar enediyne-induced DNA-damaging activity was investi-
gated using γ-H2AX foci analysis. γ-H2AX is a DNA double-
strand break (DSB) marker that can create γ-H2AX foci at
or near the DNA damage site and can be visualized by im-
munofluorescence73. Comparing with the negligible γ-
H2AX foci formation in EDY-A sample, the green fluores-
cent spots of γ-H2AX foci in EDY-B and EDY-C are obvious
(Figure 7B), indicating the generated diradicals via
MARACA, which resulted in DNA damaging activity in liv-
ing cells. Combining with the extracellular DNA cleavage
study above, these results corroborate the ds DNA break
effect stemming from maleimide-based enediynes.
EXPERIMENTAL SECTION
General experimental methods. Toluene, tetrahydro-
furan (THF) and dichloromethane (DCM) were dried over
calcium hydride (CaH2) and distilled before use. 3-[4,5-
dimethylthiazol-2-yl]-2,5-diphenyltetrazolium-bromide
(MTT) was obtained from Macklin. Annexin V-FITC/PI
apoptosis detection kit, phospho-histone H2A.X (Ser139)
rabbit monoclonal antibody and Alexa Fluor 488-labeled
Goat Anti-Rabbit IgG(H+L) were purchased from Beyotime
Biotechnology. Dulbecco’s modified Eagle’s medium
(DMEM), fetal bovine serum (FBS), and phosphate-
buffered saline (PBS) were provided by BBI Life Sciences
Corporation. Other reagents were purchased at commer-
cial grade and used without further purification. So-
Cell Cycle Arrest and Apoptosis Assay. Intracellular
DNA damage resulting from environmental toxicants may
activate cell cycle checkpoint arrest and induce cell apop-
tosis74. Encouraged by cell viability inhibition of EDY-F
with an IC50 value of 0.54 μM against Hela cells, EDY-F was
used to examine the cell cycle arrest ability and to investi-
gate the cell death pathway of Hela cells. To this end, Hela
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nogashira reactions were performed with dry Schlenk between DCM (50 ml) and H2O (50 ml). The resulting or-
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techniques under an atmosphere of nitrogen.
ganic phase was washed with H2O (2 × 50 ml), dried over
MgSO4, filtered and concentrated. The crude product was
separated by column chromatography on silica gel (hex-
ane/ethyl acetate = 10:1) to give the product as white solid
(11.5 g, 62%). All spectroscopic data were in accordance
with the literature.
1H NMR and 13C{1H} NMR spectra were recorded on
Bruker DRX-400, DRX-500 or DRX-600 instruments and
calibrated using residual undeuterated solvent (CDCl3: δH =
7.26 ppm, δC = 77.2 ppm; CD3OD: δH = 3.31 ppm, δC = 49.0
ppm) as an internal reference. High resolution mass spec-
tra (HR-MS) were obtained on a Micromass LCTTM mass
spectrometer using the ESI or EI method. The electron
paramagnetic resonance (EPR) measurements were per-
formed with an X-band EMX-8/2.7C EPR spectrometer
(Bruker). Fluorescence spectra were recorded on a Perki-
nElmer LS-55 (excited at 350 nm). Absorbance spectra
were collected using a UV-vis spectrophotometer (Lambda
Bio40, Perkin-Elmer). Differential scanning calorimetry
(DSC) was carried out with a Pyris Diamond thermal anal-
ysis workstation equipped with a model 822e DSC module
under a constant nitrogen flow. Cytotoxicity assay was
measured by microplate reader (Thermo Scientific). Fluo-
rescence microscopy images were taken using a confocal
laser scanning microscopy (CLSM, Nikon). Quantitative
flow cytometry was taken by Flow Cytometry (Beckman).
Tert-butyl 2-(3,4-diiodo-2,5-dioxo-2,5-dihydro-1H-
pyrrol-1-yl)acetate (2)41. A solution of sodium iodide
(15.0 g, 100 mmol) and tert-butyl 2-(3,4-dichloro-2,5-
dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetate (2s, 7.0 g, 25
mmol) in acetonitrile (50 ml) was heated at reflux in an oil
bath for 12 h. Then the mixture was concentrated under
reduced pressure. The crude product was purified by col-
umn chromatography on silica gel (hexane/ethyl acetate =
10:1) to afford a pale yellow solid (10.6 g, 92 %). All spec-
troscopic data were in accordance with the literature.
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3,4-dichloro-1-neopentyl-1H-pyrrole-2,5-dione (3s).
Dichloromaleic anhydride (6.7 g, 40 mmol) was dissolved
in acetic acid (70 ml) with slow addition of neopentyla-
mine (3.2 g, 36 mmol). The mixture was then heated at
reflux in an oil bath for 8 h. After removal of the solvent,
the resulting crude residue was separated by column
chromatography on silica gel (hexane/ethyl acetate = 10:1)
N-(tert-butyl)prop-2-yn-1-amine (1s)40. To a stirred
solution of 2-methylpropan-2-amine (52.5 ml, 500 mmol)
in DCM (100 mL), 3-bromo-1-propyne (8.6 mL, 100 mmol)
was added dropwise at room temperature. After stirring
for 36 hours, the reaction mixture was washed three times
with water. The resulting organic phase was dried over
MgSO4, filtered and the solvent was evaporated under re-
duced pressure. The product was purified by distillation to
give a pale yellow oil (8.8 g, 79%). All spectroscopic data
were in accordance with the literature.
1
to give the product as yellow solid (7.0 g, 83 %); H NMR
(400 MHz, CD3OD) δ 3.37 (s, 2H), 0.93 (s, 9H); 13C{1H} NMR
(101 MHz, CD3OD) δ 165.1, 134.1, 51.6, 34.2, 28.2; HRMS
(EI) m/z: [M]+ Calcd for C9H11Cl2NO2 235.0167; Found
235.0170.
3,4-diiodo-1-neopentyl-1H-pyrrole-2,5-dione (4s). A
solution of sodium iodide (6.0 g, 40 mmol) and 3,4-
dichloro-1-neopentyl-1H-pyrrole-2,5-dione (3s, 2.4 g, 10
mmol) in acetonitrile (50 ml) was stirred at reflux in an oil
bath for 12 h. Then the solution was concentrated under
reduced pressure. The crude product was purified by col-
umn chromatography on silica gel (hexane/ethyl acetate =
Tert-butyl tert-butyl(prop-2-yn-1-yl)carbamate (1).
N-(tert-butyl)prop-2-yn-1-amine (1s, 4.0 g, 36 mmol) was
dissolved in DCM (40 ml) with subsequent addition of
(Boc)2O (12.4 ml, 54 mmol). The mixture was stirred at
reflux in an oil bath for 36 h, then cooled down to room
temperature. Imidazole (6.0 g, 36 mmol) was added to
consume the excess (Boc)2O completely76. After removal of
the solvent under vacuum, the crude residue was parti-
tioned between ethyl acetate (50 ml) and 1% HCl (50 ml).
The resulting organic phase was washed with 1% HCl (2 ×
50 ml) and brine (2 × 50 ml), dried over MgSO4, filtered
and concentrated. The crude product was purified by col-
umn chromatography on silica gel (hexane/ethyl acetate =
50:1) to give the compound 1 as pale yellow oil (6.4 g,
1
10:1) to afford a yellow solid (3.4 g, 82 %); H NMR (400
MHz, CDCl3) δ 3.40 (s, 2H), 0.90 (s, 9H); 13C{1H} NMR (101
MHz, CDCl3) δ 167.1, 117.5, 51.6, 33.7, 28.0; HRMS (EI)
m/z: [M]+ Calcd for C9H11I2NO2 418.8879; Found 418.8884.
Di-tert-butyl
diyl))bis(tert-butylcarbamate)
(1,2-phenylenebis(prop-2-yne-3,1-
(EDY-A). 1,2-
Diiodobenzene (165.0 mg, 0.5 mmol), CuI (38.1 mg, 40 %),
(PPh3)2PdCl2 (35.1 mg, 10 %), and n-BuNH2 (109.7 mg, 1.5
mmol) were successively added to dry THF (4 mL) solution
under nitrogen. Then, compound 1 (316.5 mg, 1.5 mmol)
in THF (1 mL) was added dropwise. The mixture was
stirred at ambient temperature for 18 h. After the comple-
tion of the reaction as detected by TLC, the mixture was
directly purified by column chromatography on silica gel
(hexane/ethyl acetate = 20 : 1) to yield a yellow oil (136
mg, 55 %); 1H NMR (600 MHz, CDCl3) δ 7.38 (dd, J = 5.7, 3.4
Hz, 2H), 7.21 (dd, J = 5.8, 3.3 Hz, 2H), 4.34 (s, 4H), 1.51 (s,
18H), 1.49 (s, 18H); 13C{1H} NMR (151 MHz, CDCl3) δ 155.5,
132.3, 127.8, 125.6, 91.5, 81.2, 80.1, 56.1, 35.3, 29.5, 28.7;
HRMS (ESI-TOF) m/z: [M + Na]+ Calcd for C30H44N2O4Na
519.3199; Found 519.3198.
1
84%); H NMR (500 MHz, CDCl3) δ 4.05 (d, J = 2.1 Hz, 2H),
2.13 (t, J = 2.2 Hz, 1H), 1.45 (s, 9H), 1.42 (s, 9H); 13C{1H}
NMR (126 MHz, CDCl3) δ 155.2, 82.2, 79.9, 70.2, 55.9, 34.1,
29.2, 28.4; HRMS (ESI-TOF) m/z: [M + Na]+ Calcd for
C12H21NO2Na 234.1470; Found 234.1471.
Tert-butyl
2-(3,4-dichloro-2,5-dioxo-2,5-dihydro-
1H-pyrrol-1-yl)acetate (2s)41. Glycine tert butyl ester
hydrochloride (11.0 g, 66 mmol) was added to a solution of
dichloromaleic anhydride (12.0 g, 72 mmol) and potassi-
um acetate (6.5 g, 66 mmol) in acetic acid (80 ml). The
mixture was heated in an oil bath to 40 °C for 40 h. After
removal of the solvent, the crude residue was partitioned
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Tert-butyl
2-(3,4-bis(3-((tert-butoxycarbonyl)(tert-
9H), 1.32 (s, 9H); 13C{1H} NMR (151 MHz, CDCl3) δ 166.9,
1
2
3
4
5
6
7
8
butyl)amino)prop-1-yn-1-yl)-2,5-dioxo-2,5-dihydro-1H-
pyrrol-1-yl)acetate (EDY-B). Compound 2 (231.4 mg, 0.5
mmol), CuI (38.1 mg, 40 %), (PPh3)2PdCl2 (35.1 mg, 10 %),
and DIPEA (193.9 mg, 1.5 mmol) were successively added
to a solvent mixture of dry THF (3 mL) and toluene (6 mL)
under nitrogen. Then, compound 1 (316.6 mg, 1.5 mmol)
in THF (1 mL) was added dropwise. The mixture was
stirred at ambient temperature for 12 h. After the comple-
tion of the reaction as detected by TLC, the mixture was
directly purified by column chromatography on silica gel
(hexane/ethyl acetate = 10 : 1) to yield a yellow oil (32 mg,
166.7, 166.0, 155.2, 129.4, 127.4, 120.6, 108.2, 83.1, 80.5,
73.1, 70.4, 56.4, 40.3, 35.7, 30.5, 29.6, 29.2, 28.6, 28.1;
HRMS (ESI-TOF) m/z: [M + Na]+ Calcd for C28H40N2O6Na
523.2784; Found 523.2783.
Tert-butyl
2-(3-(3-((tert-butoxycarbonyl)(tert-
butyl)amino)prop-1-yn-1-yl)-4-(4-hydroxybut-1-yn-1-yl)-
2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetate
(EDY-E):
1
pale yellow oil, 20% (49 mg); H NMR (600 MHz, CDCl3) δ
4.42 (s, 2H), 4.16 (s, 2H), 3.83 (t, J = 6.2 Hz, 2H), 2.79 (t, J =
6.2 Hz, 2H), 1.49 (s, 9H), 1.48 (s, 9H), 1.44 (s, 9H); 13C{1H}
NMR (151 MHz, CDCl3) δ 166.8, 166.6, 165.9, 155.4, 129.3,
128.5, 109.4, 109.1, 83.2, 80.7, 73.1, 72.9, 60.5, 56.5, 40.4,
35.8, 29.6, 28.6, 28.1, 25.0; HRMS (ESI-TOF) m/z: [M + Na]+
Calcd for C26H36N2O7Na 511.2420; Found 511.2421.
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1
10 %); H NMR (500 MHz, CDCl3) δ 4.40 (s, 4H), 4.14 (s,
2H), 1.47 (s, 18H), 1.46 (s, 18H), 1.43 (s, 9H); 13C{1H} NMR
(126 MHz, CDCl3) δ 166.4, 166.0, 155.2, 128.3, 109.1, 83.2,
80.6, 73.1, 56.4, 40.3, 35.6, 29.5, 28.6, 28.1; HRMS (ESI-TOF)
m/z: [M + Na]+ Calcd for C34H51N3O8Na 652.3574; Found
652.3573.
3-(4-(4-hydroxybut-1-yn-1-yl)-1-neopentyl-2,5-
dioxo-2,5-dihydro-1H-pyrrol-3-yl)prop-2-yn-1-yl ace-
tate (EDY-F). Compound 10 (209.4 mg, 0.5 mmol), CuI
(38.1 mg, 40 %), (PPh3)2PdCl2 (35.1 mg, 10 %), and DIPEA
(193.9 mg, 1.5 mmol) were successively added to a solvent
mixture of dry THF (2 mL) and toluene (4 mL) under ni-
trogen. Then, prop-2-yn-1-yl acetate (73.5 mg, 0.75 mmol)
and but-3-yn-1-ol (52.5 mg, 0.75 mmol) in THF (1 mL) was
added dropwise. The mixture was stirred at ambient tem-
perature overnight. After the completion of the reaction as
detected by TLC, the mixture was directly purified by col-
umn chromatography on silica gel (hexane/ethyl acetate =
2 : 1) to give the product as pale yellow oil, 26 % (43 mg);
1H NMR (600 MHz, CDCl3) δ 4.95 (s, 2H), 3.85 (t, J = 6.3 Hz,
2H), 3.33 (s, 2H), 2.83 (t, J = 6.3 Hz, 2H), 2.12 (s, 3H), 0.89
(s, 9H); 13C{1H} NMR (151 MHz, CDCl3) δ 170.3, 167.6,
130.4, 127.0, 110.5, 102.2, 76.2, 73.0, 60.4, 52.6, 50.3, 33.7,
28.0, 25.1, 20.8; HRMS (ESI-TOF) m/z: [M + Na]+ Calcd for
C18H21NO5Na 354.1317; Found 354.1316.
Tert-butyl
2-(4-(2-((tert-butoxycarbonyl)(tert-
butyl)amino)ethyl)-3-(tert-butyl)-2,5,7-trioxo-2,3,5,7-
tetrahydro-6H-oxazolo[4,5-f]isoindol-6-yl)acetate (com-
pound 3): isolated as a yellow solid (35 mg, 12 %) from the
synthesis procedure of EDY-B; 1H NMR (500 MHz, CDCl3) δ
7.45 (s, 1H), 4.29 (s, 2H), 3.59 (t, J = 6.5 Hz, 2H), 3.45 (t, J =
6.5 Hz, 2H), 1.74 (s, 9H), 1.45 (s, 9H), 1.37 (s, 9H), 1.31 (s,
9H); 13C{1H} NMR (126 MHz, CDCl3) δ 168.1, 166.6, 166.4,
156.0, 153.4, 147.7, 136.0, 128.4, 127.1, 126.0, 103.3, 83.0,
80.0, 60.6, 55.8, 46.3, 39.9, 34.1, 30.2, 30.0, 28.6, 28.1;
HRMS (ESI-TOF) m/z: [M + Na]+ Calcd for C30H43N3O8Na
596.2948; Found 596.2949.
General Procedure for the Synthesis of EDY-C, D, and
E. Compound 2 (231.4 mg, 0.5 mmol), CuI (38.1 mg, 40 %),
(PPh3)2PdCl2 (35.1 mg, 10 %), and DIPEA (193.9 mg, 1.5
mmol) were successively added to a solvent mixture of dry
THF (2 mL) and toluene (4 mL) under nitrogen. Then,
compound 1 (158.3 mg, 0.75 mmol) and the corresponding
another terminal alkyne (0.75 mmol, commercially availa-
ble) in THF (1 mL) was added dropwise. The mixture was
stirred at ambient temperature overnight. After the com-
pletion of the reaction as detected by TLC, the mixture was
directly purified by column chromatography on silica gel
(eluted with 75-90 % hexane/ethyl acetate) to give the
product.
ASSOCIATED CONTENT
Supporting Information
The following files are available free of charge.
Experimental procedure and fundamental characterization
(PDF)
AUTHOR INFORMATION
Corresponding Author
* yunding@ecust.edu.cn (YD)
* hagmhsn@ecust.edu.cn (AH)
Tert-butyl
2-(3-(3-((tert-butoxycarbonyl)(tert-
butyl)amino)prop-1-yn-1-yl)-4-(3-hydroxy-3 methylbut-1-
yn-1-yl)-2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetate
(EDY-C): pale yellow oil, 18 % (45 mg); H NMR (600 MHz,
CDCl3) δ 4.44 (s, 2H), 4.17 (s, 2H), 1.60 (s, 6H), 1.49 (s, 9H),
1.49 (s, 9H), 1.45 (s, 9H); 13C{1H} NMR (151 MHz, CDCl3) δ
166.5, 166.4, 165.9, 155.4, 128.9, 128.5, 114.4, 109.7, 83.2,
80.8, 73.0, 72.5, 65.9, 56.5, 40.4, 35.8, 31.0, 29.7, 28.7, 28.1;
HRMS (ESI-TOF) m/z: [M + Na]+ Calcd for C27H38N2O7Na
525.2577; Found 525.2576.
ORCID
1
Yun Ding: 0000-0002-7012-5621
Aiguo Hu: 0000-0003-0456-7269
Author Contributions
The manuscript was written through contributions of all au-
thors.
Notes
Tert-butyl
2-(3-(3-((tert-butoxycarbonyl)(tert-
The authors declare no competing financial interests.
butyl)amino)prop-1-yn-1-yl)-4-(3,3-dimethylbut-1-yn-1-
yl)-2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetate (EDY-D):
pale yellow oil, 26 % (65 mg); H NMR (600 MHz, CDCl3) δ
ACKNOWLEDGMENT
1
4.43 (s, 2H), 4.15 (s, 2H), 1.49 (s, 9H), 1.48 (s, 9H), 1.44 (s,
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(17) Ishida, N.; Miyazaki, K.; Kumagai, K.; Rikimaru, M.
The authors gratefully acknowledge the financial support
"Neocarzinostatin, antitumor antibiotic of high molecular weight;
isolation, physicochemical properties, and biological activities". J.
Antibiot., Ser. A 1965, 18, 68-76.
1
2
3
4
5
6
7
8
from National Natural Science Foundation of China
(21871080, 21503078), the Fundamental Research Funds for
the Central Universities (22221818014), and Shanghai Lead-
ing Academic Discipline Project (B502). AH thanks the “East-
ern Scholar Professorship” support from Shanghai local gov-
ernment. AH thanks Profs. Runhui Liu and Junyou Wang for
their kindly help in cellular experiments.
(18) Koga, N.; Morokuma, K. "Comparison of biradical formation
between enediyne and enyne-allene. Ab initio CASSCF and
MRSDCI study". J. Am. Chem. Soc. 1991, 113, 1907-1911.
(19) 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.
(20) 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. Soc. 2017, 139, 3406-3416.
(21) Mohamed, R. K.; Peterson, P. W.; Alabugin, I. V. "Concerted
Reactions That Produce Diradicals and Zwitterions: Electronic,
Steric, Conformational, and Kinetic Control of Cycloaromatization
Processes". Chem. Rev. 2013, 113, 7089-7129.
(22) 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.
REFERENCES
9
(1) Chang, C.-Y.; Yan, X.; Crnovcic, I.; Annaval, T.; Chang, C.; Nocek,
B.; Rudolf, J. D.; Yang, D.; Hindra; Babnigg, G.; Joachimiak, A.;
Phillips, G. N.; Shen, B. "Resistance to Enediyne Antitumor
Antibiotics by Sequestration". Cell Chem. Biol. 2018, 25, 1075-
1085.
(2) Nicolaou, K. C.; Dai, W. M. "Chemistry and Biology of the
Enediyne Anticancer Antibiotics". Angew. Chem., Int. Ed. Eng.
1991, 30, 1387-1416.
(3) DiJoseph, J. F.; Dougher, M. M.; Armellino, D. C.; Kalyandrug, L.;
Kunz, A.; Boghaert, E. R.; Hamann, P. R.; Damle, N. K. "CD20-
specific antibody-targeted chemotherapy of non-Hodgkin's B-cell
lymphoma using calicheamicin-conjugated rituximab". Cancer
Immunol. Immunother. 2007, 56, 1107-1117.
(4) DiJoseph, J. F.; Armellino, D. C.; Boghaert, E. R.; Khandke, K.;
Dougher, M. M.; Sridharan, L.; Kunz, A.; Hamann, P. R.; Gorovits, B.;
Udata, C.; Moran, J. K.; Popplewell, A. G.; Stephens, S.; Frost, P.;
Damle, N. K. "Antibody-targeted chemotherapy with CMC-544: a
CD22-targeted immunoconjugate of calicheamicin for the
treatment of B-lymphoid malignancies". Blood 2004, 103, 1807-
1814.
(5) Hamann, P. R.; Hinman, L. M.; Beyer, C. F.; Lindh, D.; Upeslacis,
J.; Flowers, D. A.; Bernstein, I. "An Anti-CD33 Antibody-
Calicheamicin Conjugate for Treatment of Acute Myeloid
Leukemia. Choice of Linker". Bioconjugate Chem. 2002, 13, 40-46.
(6) Sapra, P.; Shor, B. "Monoclonal antibody-based therapies in
cancer: Advances and challenges". Pharmacol. Ther. 2013, 138,
452-469.
(7) Siegel, R.; Ma, J.; Zou, Z.; Jemal, A. "Cancer statistics, 2014". CA
Cancer J. Clin. 2014, 64, 9-29.
(8) Torre, L. A.; Bray, F.; Siegel, R. L.; Ferlay, J.; Lortet-Tieulent, J.;
Jemal, A. "Global cancer statistics, 2012". CA Cancer J. Clin. 2015,
65, 87-108.
(9) Cao, Y.; DePinho, R. A.; Ernst, M.; Vousden, K. "Cancer research:
past, present and future". Nat. Rev. Cancer 2011, 11, 749-754.
(10) Cragg, G. M.; Kingston, D. G.; Newman, D. J. Anticancer agents
from natural products; CRC Press, 2011.
(11) Porter, M. R.; Lindahl, S. E.; Lietzke, A.; Metzger, E. M.; Wang,
Q.; Henck, E.; Chen, C.-H.; Niu, H.; Zaleski, J. M. "Metal-mediated
diradical tuning for DNA replication arrest via template strand
scission". Proc. Natl. Acad. Sci. 2017, 114, E7405-E7414.
(12) Baker, J. R.; Woolfson, D. N.; Muskett, F. W.; Stoneman, R. G.;
Urbaniak, M. D.; Caddick, S. "Protein–Small Molecule Interactions
in Neocarzinostatin, the Prototypical Enediyne Chromoprotein
Antibiotic". ChemBioChem 2007, 8, 704-717.
(13) Jones, G. B.; Plourde, G. W.; Wright, J. M. "Understanding
Enediyne-Protein Interactions:ꢀ Diyl Atom Transfer Results in
Generation of Aminoacyl Radicals". Org. Lett. 2000, 2, 811-813.
(14) Kawata, S.; Ashizawa, S.; Hirama, M. "Synthetic Study of
Kedarcidin Chromophore:ꢀ Revised Structure". J. Am. Chem. Soc.
1997, 119, 12012-12013.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(23) Cunico, R. F.; Nair, S. K. "Cycloaromatization of
a
solvolytically generated ene-yne-allene". Tetrahedron Lett. 1997,
38, 25-28.
(24) Ferri, F.; Brückner, R.; Herges, R. "Straightforward syntheses
of biradical-producing bicyclic dienediynes-dienediyne ketones
cycloaromatize ia the Saito-Myers and not ia the neocarzinostatin
pathway". New J. Chem. 1998, 22, 531-546.
(25) Li, Y.; Kirillov, A. M.; Fang, R.; Yang, L. "Effect of Substituent
on the Mechanism and Chemoselectivity of the Gold(I)-Catalyzed
Propargyl Ester Tandem Cyclization". Organometallics 2017, 36,
1164-1172.
(26) Raviola, C.; Protti, S.; Ravelli, D.; Fagnoni, M.
"(Hetero)aromatics from dienynes, enediynes and enyne-allenes".
Chem. Soc. Rev. 2016, 45, 4364-4390.
(27) Grissom, J. W.; Huang, D. "Tandem Eneyne Allene-Radical
Cyclization via [3,3] Sigmatropic Rearrangements". J. Org. Chem.
1994, 59, 5114-5116.
(28) 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.
(29) Li, J.; Li, B.; Sun, L.; Duan, B.; Huang, S.; Yuan, Y.; Ding, Y.; Hu,
A. "Self-delivery nanoparticles of an amphiphilic irinotecan–
enediyne conjugate for cancer combination chemotherapy". J.
Mater. Chem. B 2019, 7, 103-111.
(30) 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, 74,
6419-6425.
(31) 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.
(32) Lu, H.; Ma, H.; Li, B.; Zhang, M.; Chen, H.; Wang, Y.; Li, X.; Ding,
Y.; Hu, A. "Facilitating Myers-Saito cyclization through acid-
triggered tautomerization for the development of maleimide-
based antitumor agents". J. Mater. Chem. B 2020, 8, 1971-1979.
(33) Li, J.; Zhang, M.; Huang, S.; Ding, Y.; Hu, A. "Concurrent
Radical Addition and [3+2] Cycloaddition between Nitrones and
Maleimide-Based Acyclic Enediynes". Asian J. Org. Chem. 2018, 7,
2076-2081.
(15) Dedon, P. C.; Jiang, Z. W.; Goldberg, I. H. "Neocarzinostatin-
mediated DNA damage in
a model AGT.cntdot.ACT site:
(34) Das, E.; Basak, S.; Anoop, A.; Chand, S.; Basak, A. "How To
Achieve High Regioselectivity in Barrier-less Nucleophilic
Addition to p-Benzynes Generated via Bergman Cyclization of
Unsymmetrical Cyclic Azaenediyne?". J. Org. Chem. 2019, 84,
2911-2921.
mechanistic studies of thiol-sensitive partitioning of C4' DNA
damage products". Biochemistry 1992, 31, 1917-1927.
(16) Kappen, L.; Goldberg, I. "DNA conformation-induced
activation of an enediyne for site-specific cleavage". Science 1993,
261, 1319-1321.
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 12 of 14
(35) Borie, C.; Mondal, S.; Arif, T.; Briand, M.; Lingua, H.; Dumur, F.;
(52) Kohn, W.; Sham, L. J. "Self-Consistent Equations Including
Exchange and Correlation Effects". Phys. Rev. 1965, 140, A1133-
A1138.
Gigmes, D.; Stocker, P.; Barbarat, B.; Robert, V.; Nicoletti, C.; Olive,
D.; Maresca, M.; Nechab, M. "Enediynes bearing polyfluoroaryl
sulfoxide as new antiproliferative agents with dual targeting of
microtubules and DNA". Eur. J. Med. Chem. 2018, 148, 306-313.
(36) Valenzuela, S. A.; Cortés, A. J.; Tippins, Z. J. E.; Daly, M. H.;
Keel, T. E.; Gherman, B. F.; Spence, J. D. "Effect of Extended
Benzannelation Orientation on Bergman and Related Cyclizations
of Isomeric Quinoxalenediynes". J. Org. Chem. 2017, 82, 13297-
13312.
(37) Das, J.; Roy, S.; Halnor, S.; Das, A. K.; Basak, A. "Enediyne-
based protein capture agents: demonstration of an enediyne
moiety acting as a photoaffinity label". Org. Biomol. Chem. 2017,
15, 1122-1129.
(38) Peterson, P. W.; Shevchenko, N.; Breiner, B.; Manoharan, M.;
Lufti, F.; Delaune, J.; Kingsley, M.; Kovnir, K.; Alabugin, I. V.
"Orbital Crossings Activated through Electron Injection: Opening
Communication between Orthogonal Orbitals in Anionic C1–C5
Cyclizations of Enediynes". J. Am. Chem. Soc. 2016, 138, 15617-
15628.
1
2
3
4
5
6
7
8
(53) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.;
Robb, M. A.; Cheeseman, J. R.; Scalmani, G.; Barone, V.; Mennucci,
B.; Petersson, G. A.; Nakatsuji, H.; Caricato, M.; Li, X.; Hratchian, H.
P.; Izmaylov, A. F.; Bloino, J.; Zheng, G.; Sonnenberg, J. L.; Hada, M.;
Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.;
Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.; Vreven, T.;
Montgomery, J. A., Jr.; Peralta, J. E.; Ogliaro, F.; Bearpark, M.; Heyd,
J. J.; Brothers, E.; Kudin, K. N.; Staroverov, V. N.; Kobayashi, R.;
Normand, J.; Raghavachari, K.; Rendell, A.; Burant, J. C.; Iyengar, S.
S.; Tomasi, J.; Cossi, M.; Rega, N.; Millam, J. M.; Klene, M.; Knox, J. E.;
Cross, J. B.; Bakken, V.; Adamo, C.; Jaramillo, J.; Gomperts, R.;
Stratmann, R. E.; Yazyev, O.; Austin, A. J.; Cammi, R.; Pomelli, C.;
Ochterski, J. W.; Martin, R. L.; Morokuma, K.; Zakrzewski, V. G.;
Voth, G. A.; Salvador, P.; Dannenberg, J. J.; Dapprich, S.; Daniels, A.
D.; Farkas, Ö.; Foresman, J. B.; Ortiz, J. V.; Cioslowski, J.; Fox, D. J.;
Gaussian, Inc.: Wallingford CT: 2009.
(54) dos Passos Gomes, G.; Morrison, A. E.; Dudley, G. B.; Alabugin,
I. V. "Optimizing Amine-Mediated Alkyne–Allene Isomerization to
Improve Benzannulation Cascades: Synergy between Theory and
Experiments". Eur. J. Org. Chem. 2019, 2019, 512-518.
(55) Bickerton, G. R.; Paolini, G. V.; Besnard, J.; Muresan, S.;
Hopkins, A. L. "Quantifying the chemical beauty of drugs". Nature
Chem. 2012, 4, 90-98.
(56) Cremeens, M. E.; Hughes, T. S.; Carpenter, B. K. "Mechanistic
Studies on the Cyclization of (Z)-1,2,4-Heptatrien-6-yne in
Methanol:ꢀ A Possible Nonadiabatic Thermal Reaction". J. Am.
Chem. Soc. 2005, 127, 6652-6661.
(57) Yu, M.; Liu, X.; Jiang, Q.; Du, H. "Expeditious synthesis of 9-
allenylpurines via cesium carbonate catalyzed isomerization of 9-
alkynylpurines". Phosphorus, Sulfur Silicon Relat. Elem. 2018, 193,
451-458.
(58) Li, Y.; Chen, J.; Qiu, R.; Wang, X.; Long, J.; Zhu, L.; Au, C.; Xu, X.
"Cesium hydroxide-catalyzed isomerization of terminal alkynes
for the synthesis of O-allenes and N-allenes". Tetrahedron Lett.
2015, 56, 5504-5507.
(59) Chen, W.; Li, J.; Zhu, Y.; Hu, W.; Mo, W. "Et3N-promoted
sequential reactions for the synthesis of 6H-benzo[c]chromenes".
Arkivoc 2011, 2012.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(39) Campolo, D.; Arif, T.; Borie, C.; Mouysset, D.; Vanthuyne, N.;
Naubron, J. V.; Bertrand, M. P.; Nechab, M. "Double Transfer of
Chirality
in
Organocopper-Mediated
bis(Alkylating)
Cycloisomerization of Enediynes". Angew. Chem., Int. Ed. 2014, 53,
3227-3231.
(40) Brunel, P.; Monot, J.; Kefalidis, C. E.; Maron, L.; Martin-Vaca,
B.; Bourissou, D. "Valorization of CO2: Preparation of 2-
Oxazolidinones by Metal–Ligand Cooperative Catalysis with SCS
Indenediide Pd Complexes". ACS Catal. 2017, 7, 2652-2660.
(41) Li, B.; Wu, Y.; Wang, Y.; Zhang, M.; Chen, H.; Li, J.; Liu, R.; Ding,
Y.; Hu, A. "Light-Cross-linked Enediyne Small-Molecule Micelle-
Based Drug-Delivery System". ACS Appl. Mater. Interfaces 2019,
11, 8896-8903.
(42) Basak, A.; Kar, M. "Benzofused N-substituted cyclic
enediynes: Activation and DNA-cleavage potential". Bioorg. Med.
Chem. 2008, 16, 4532-4537.
(43) Schmittel, M.; Maywald, M. "Electronic effects in the thermal
C2–C6 biradical cyclisation of enyne-allenes". Chem. Commun.
2001, 155-156.
(44) Smith, D. W.; Shah, H. V.; Perera, K. P. U.; Perpall, M. W.; Babb,
D. A.; Martin, S. J. "Polyarylene networks via Bergman
cyclopolymerization of bis-ortho-diynyl arenes". Adv. Funct. Mater.
2007, 17, 1237-1246.
(45) Roy, S.; Bag, S. S.; Basak, A. "Synthesis and reactivity of
enediyne–nucleobase hybrids: effect of intramolecular π-
stacking". Tetrahedron 2012, 68, 8600-8611.
(60) Lyapunova, A. G.; Danilkina, N. A.; Rumyantsev, A. M.;
Khlebnikov, A. F.; Chislov, M. V.; Starova, G. L.; Sambuk, E. V.;
Govdi, A. I.; Bräse, S.; Balova, I. A. "Relative Reactivity of
Benzothiophene-Fused Enediynes in the Bergman Cyclization". J.
Org. Chem. 2018, 83, 2788-2801.
(46) 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.
(47) Iida, K.-i.; Hirama, M. "Synthesis and Characterization of
Nine-Membered Cyclic Enediynes, Models of the C-1027 and
(61) Kraka, E.; Cremer, D. "Computer design of anticancer drugs.
A new enediyne warhead". J. Am. Chem. Soc. 2000, 122, 8245-
8264.
(62) Zhang, H.; Li, R.; Ba, S.; Lu, Z.; Pitsinos, E. N.; Li, T.; Nicolaou,
K. C. "DNA Binding and Cleavage Modes of Shishijimicin A". J. Am.
Chem. Soc. 2019, 141, 7842-7852.
(63) Elmroth, K.; Nygren, J.; Mårtensson, S.; Ismail, I. H.;
Hammarsten, O. "Cleavage of cellular DNA by calicheamicin γ1".
DNA Repair 2003, 2, 363-374.
(64) Hatayama, T.; Goldberg, I. H. "DNA damage and repair in
relation to cell killing in neocarzinostatin-treated HeLa cells".
Biochim. Biophys. Acta, Nucleic Acids Protein Synth. 1979, 563, 59-
71.
(65) Goodisman, J.; Kirk, C.; Dabrowiak, J. C. "Kinetic analysis of
drug cleavage of closed-circular DNA". Biophys. Chem. 1997, 69,
249-268.
(66) Freifelder, D.; Trumbo, B. "Matching of single-strand breaks
to form double-strand breaks in DNA". Biopolymers 1972, 11,
1116-1116.
(67) Joyner, J. C.; Reichfield, J.; Cowan, J. A. "Factors Influencing
the DNA Nuclease Activity of Iron, Cobalt, Nickel, and Copper
Chelates". J. Am. Chem. Soc. 2011, 133, 15613-15626.
Kedarcidin Chromophores: Equilibration with
a p-Benzyne
Biradical and Kinetic Stabilization". J. Am. Chem. Soc. 1995, 117,
8875-8876.
(48) 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.
(49) Usuki, T.; Nakanishi, K.; Ellestad, G. A. "Spin-trapping of the
p-benzyne intermediates from ten-membered enediyne
calicheamicin gamma1I". Org. Lett. 2006, 8, 5461-5463.
(50) 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.
(51) Hohenberg, P.; Kohn, W. "Inhomogeneous Electron Gas".
Phys. Rev. B. 1964, 136, 864-871.
ACS Paragon Plus Environment

Page 13 of 14
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(68) Zheng, Y. R.; Suntharalingam, K.; Johnstone, T. C.; Lippard, S.
J. "Encapsulation of Pt(IV) Prodrugs within a Pt(II) Cage for Drug
Delivery". Chem. Sci. 2015, 6, 1189-1193.
(73) Rothkamm, K.; Barnard, S.; Moquet, J.; Ellender, M.; Rana, Z.;
Burdak-Rothkamm, S. "DNA damage foci: Meaning and
significance". Environ. Mol. Mutagen. 2015, 56, 491-504.
(74) Chao, H. X.; Poovey, C. E.; Privette, A. A.; Grant, G. D.; Chao, H.
Y.; Cook, J. G.; Purvis, J. E. "Orchestration of DNA Damage
Checkpoint Dynamics across the Human Cell Cycle". Cell Systems
2017, 5, 445-459.e445.
(75) Pan, Z.; Luo, Y.; Xia, Y.; Zhang, X.; Qin, Y.; Liu, W.; Li, M.; Liu, X.;
Zheng, Q.; Li, D. "Cinobufagin induces cell cycle arrest at the S
phase and promotes apoptosis in nasopharyngeal carcinoma
cells". Biomed. Pharmacother. 2020, 122, 109763.
1
2
3
4
5
6
7
8
(69) Dhar, S.; Lippard, S. J. "Mitaplatin, a potent fusion of cisplatin
and the orphan drug dichloroacetate". Proc. Natl. Acad. Sci. 2009,
106, 22199-22204.
(70) Chen, K.; Liao, S.; Guo, S.; Zhang, H.; Cai, H.; Gong, Q.; Gu, Z.;
Luo, K. "Enzyme/pH-sensitive dendritic polymer-DOX conjugate
for cancer treatment". Sci. China Mater. 2018, 61, 1462-1474.
(71) Rankovic, Z. "CNS Drug Design: Balancing Physicochemical
Properties for Optimal Brain Exposure". J. Med. Chem. 2015, 58,
2584-2608.
(72) Lipinski, C. A.; Lombardo, F.; Dominy, B. W.; Feeney, P. J.
"Experimental and computational approaches to estimate
solubility and permeability in drug discovery and development
settings". Adv. Drug Delivery Rev. 1997, 23, 3-25.
9
(76) Basel, Y.; Hassner, A. "Imidazole and Trifluoroethanol as
Efficient and Mild Reagents for Destruction of Excess Di-tert-butyl
Dicarbonate [(BOC)2O]". Synthesis 2001, 2001, 0550-0552.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
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1
2
3
4
5
6
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Triggering the Antitumor Activity of Acyclic Enediyne through Malei-
mide Assisted Rearrangement and Cycloaromatization
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14
ACS Paragon Plus Environment