Experimental and Computational Study on the Intramolecular Hydrogen Atom Transfer Reactions of Maleimide-Based Enediynes after Cycloaromatization

Article abstract of DOI:10.1021/acs.joc.0c02401

The follow-up reaction pathways of the diradical species formed from cycloaromatization of enediynes or enyne-allenes determine their ability of H-abstraction from DNA, significantly affecting their biological activity performance. To gain a deeper unders

Full text of DOI:10.1021/acs.joc.0c02401

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Article
Experimental and Computational Study on the Intramolecular
Hydrogen Atom Transfer Reactions of Maleimide-Based Enediynes
After Cycloaromatization
Mengsi Zhang, Haotian Lu, Baojun Li, Hailong Ma, Wenbo Wang, Xiaoyu Cheng, Yun Ding,*
and Aiguo Hu*
Cite This: J. Org. Chem. 2021, 86, 1549−1559
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ABSTRACT: The follow-up reaction pathways of the diradical
species formed from cycloaromatization of enediynes or enyne−
allenes determine their ability of H-abstraction from DNA,
significantly affecting their biological activity performance. To
gain a deeper understanding of subsequent reaction pathways of
the diradical intermediates formed from acyclic enediynes based on
maleimide-assisted rearrangement and cycloaromatization
(MARACA), a maleimide-based enediyne featuring methylene
groups adjacent to the propargyl sites of the terminal alkynes was
synthesized through the Sonogashira coupling reaction. Three
thermal cyclization products after intramolecular hydrogen atom
transfer (HAT) were obtained from the thermolysis experiment
and their structures were confirmed by 1D and 2D nuclear
magnetic resonance spectroscopic analysis. Density functional theory was employed to analyze the important elementary steps
including rearrangement, cycloaromatization, and intramolecular HAT processes toward the formation of the cyclized products,
where the low-energy barriers of HAT pathways relative to the formation of diradicals from cycloaromatization were successfully
identified. Overall, the HAT processes to consume diradicals intramolecularly have become competitive with that of intermolecular
H-abstraction, implying that the DNA-cleavage ability of enediynes can be further boosted once the HAT processes are halted. This
study offers a promising direction for designing novel and potent acyclic enediynes for antitumor applications.
hampered by the scarcity of isolation from natural resources
and the difficulty of their construction for a complicated 9 or
10-membered cyclic architecture through total synthesis.10−12
Many endeavors that explore analogues of natural enediynes
with simple structures and high efficiency have been made so
far and are still ongoing.1,13−16 Among them, a variety of
activation strategies such as cycle constriction,17 strain
release,18 and protecting/deprotecting of a triple bond19
were considered in the design of enediyne structures in
order to balance the stability and reactivity. On the other hand,
Myers−Saito cyclization (MSC)20,21 of enyne−allene plays a
key role in the bioactivity of natural enediynes like neo-
carzinostatin (NCS).22 Moreover, the highly reactive enyne−
allene can be transformed from the enediyne unit via
INTRODUCTION
■
Since their discovery in the 1980s from natural resources,
enediynes have drawn intensive attention because of their high
potential as a class of cytotoxic antitumor agents.1,2 The special
feature of cycloaromatization of the enediyne structural
scaffold, generating reactive radical species from closed-shell
molecules,3 endows it with extraordinary cytotoxicity against a
broad range of tumor cells. The 1,4-benzene diradicals
generated from the 1,6-cyclization of enediynes, known as
Bergman cyclization (BC),4−6 are able to abstract H or
halogen atoms from suitable small molecules, for example, 1,4-
cyclohexadiene (CHD) and haloalkanes,7 which is the
fundamental mechanism of their biological activity when the
carbohydrate moieties in the DNA backbone behave as the H-
donors. As a result, the naturally occurring enediynes with
profound DNA-damaging activities are hailed as the most
active molecules among all the known cytotoxic agents. Indeed,
one of the natural enediynes, calicheamicin, has been used as
the “warhead” of chemotherapeutic agents (Mylotarg and
Besponsa) for the clinical treatment of leukemia after
conjugation with the monoclonal antibody.8,9 Unfortunately,
the development of enediyne-based chemotherapy remains
Received: October 10, 2020
Published: December 29, 2020
https://dx.doi.org/10.1021/acs.joc.0c02401
© 2020 American Chemical Society
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Scheme 1. Structure of EDY-B and the Synthesis of Enediyne Compound EDY-A: (a) LiNPr2 , HMPA, CHCCH2Br,
Tetrahydrofuran (THF), −78 °C, 3 h; (b) BocCH2NH2·HCl, Potassium Acetate, Acetic Acid, 40 °C, 40 h; (c) NaI,
Acetonitrile, Reflux, 12 h; and (d) (PPh3)2PdCl2, CuI, N,N-Diisopropylethylamine (DIPEA), Toluene/THF, r.t., 6 h
propargyl−allene rearrangement, and immediately undergoes
MSC to afford α,3-didehydrotoluene diradicals at relatively
lower temperature. Notably, the isomerization process of
enediynes to enyne−allenes occurring under mild conditions is
of special importance for their biological and medical
applications. However, this transformation typically requires
an acid, a base, an oxidant, transition metal complexes, or
heating at high temperature,23−26 many other organocatalytic
approaches are also possible for this tautomerization,27,28 and
these activation conditions are not accessible to the biological
environments. Recently, a new strategy, maleimide-assisted
rearrangement and cycloaromatization (MARACA) triggering
the antitumor activity of acyclic enediynes, has been uncovered
in our work.29 This approach allows the propargyl−allene
isomerization to be greatly promoted by installing a maleimide
group at the ene position of enediynes, making the otherwise
inactive acyclic enediynes undergoing MSC under physio-
logical conditions.
Once the diradical species are produced from facile MSC of
enyne−allenes, another important aspect concerning the
consumption pathways of these reactive species should be
paid attention to, because the fate of the diradicals is closely
related to their DNA cleavage activity and the ultimate
cytotoxic performance. Diradicals that have no self-quenching
mechanism would undergo H-abstraction from external
sources (such as DNA backbones). On the contrary, the
chances of diradicals to interact with external sources would be
severely reduced if any kind of intramolecular radical transfer
pathways exist. Indeed, a variety of intramolecular radical self-
quenching cases have been found in different enyne−allene
systems.30−32 Among them, the intramolecular hydrogen atom
transfer (HAT) reactions were generally involved as the
elementary step, which lead to the consumption of diradicals.33
On the other hand, the intramolecular capture of transient
phenyl radicals via HAT reactions has been proved as an
activation mechanism of natural enediyne antibiotics prodrug
depending on a favorable conformational change.34 The
resulting irreversible cycloaromatization renders the newly
formed radical center a longer lifetime for abstracting H from
external sources.
directing the diradicals to stable products involving intra-
molecular HAT steps are elaborated experimentally and
computationally. A deeper understanding of these intra-
molecular reaction pathways would provide a guideline for
exploring and designing novel enediyne compounds with H-
abstraction ability close or even comparable to natural
enediynes like calicheamicin and high potential for antitumor
applications.
RESULTS AND DISCUSSION
■
Synthesis of Enediynes. Encouraged by the MARACA
strategy demonstrated in our previous work,29 the same
maleimide functionality was installed at the ene position to
assist the transformation of the inert enediyne core into the
highly reactive enyne−allene structure. Because the substituent
effect at the propargyl site plays a vital role in the reactivity of
the rearrangement and cycloaromatization processes,3,39 two
terminal alkynes, tert-butyl pent-4-ynoate (1) and prop-2-yn-1-
yl acetate (2), featuring carbon and oxygen atoms at the
propargyl site, respectively, were used in the construction of
enediyne compounds, which is distinct from EDY-B29 that has
a nitrogen atom at the same position (Scheme 1). Compounds
140 and 341 were synthesized using the literature-reported
procedures without any modification. The Sonogashira
coupling reaction between alkyne 1 and alkene 3 occurred to
give EDY-A with a moderate yield at ambient temperature
(Scheme 1). Meanwhile, compound 4 with bright blue
fluorescence was detected by thin layer chromatography
(TLC) (vide infra) even at the early stage of this reaction.
Similar to the in situ enyne−allene tautomerization strategy
described in our previous work,29 the transformation of EDY-A
into compound 4 and a trace amount of mixed compounds 5
and 6 was also observed with increased reaction time.
Interestingly, when the transformation process was carried
out at an elevated temperature, the mixtures of compounds 5
and 6 were isolated as the major products. In contrast to the
transformation of EDY-B into the corresponding cyclized
product, a longer reaction time or a higher reaction
temperature was needed in the EDY-A system. The molecular
structure of EDY-A was confirmed with nuclear magnetic
resonance spectroscopy (NMR) and high-resolution mass
spectra (HRMS) analysis (Supporting Information). The
structures of compounds 4, 5, and 6 were characterized by
1D, 2D NMR, and HRMS analyses. More detailed analysis of
these cyclized products would be discussed below. On the
other hand, when similar experimental procedures were
subjected to the Sonogashira coupling between alkyne 2 and
diiodomaleimide 2S, the only separable small molecular
product was the monosubstituted maleimide by the terminal
alkyne 2 (Scheme S1). As demonstrated earlier by our group
In our early efforts of screening maleimide-based acyclic
enediynes with antitumor potencies, the focus was mainly on
the steric and electronic effects of the functionalities close to
the alkyne termini to tune the cycloaromatization reactiv-
ity.35−38 As for the new MARACA mechanism guiding the
design of various enediynes with potent anticancer activity, it is
essential to gain insight into the fate of the diradical species
after their cycloaromatization. Herein, the follow-up intra-
molecular radical transfer processes of enediynes after
MARACA have been investigated. Three possible pathways
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Figure 1. (A) DSC curve of EDY-A determined at a heating rate of 10 °C/min. The baseline is marked in red. (B) EPR spectra of control and
EDY-A (20 mM) in the presence of PBN (100 mM) in DMSO at 37 °C for 12 h. (C) EPR spectra of the samples in DCE under the same
experimental conditions. (D) EPR signal intensities at 3520 G of samples in DMSO and DCE, respectively.
and others, the enediynes with propargyl groups could be
smoothly synthesized under the Sonogashira conditions when
the benzene or vinyl group functions as the alkene source,42−46
meanwhile, asymmetric maleimide-based enediynes with
propargyl ester at one of the two arms were synthesized in a
good yield.38 Therefore, the failure of synthesizing maleimide-
based enediynes with symmetrical propargyl substituents
implies the significant effect of the maleimide moiety on the
relationship between the structure and reactivity of the
enediynes. A minor modification of the substituents at the
yne termini of maleimide-based enediynes would lead to a
dramatic change in the reactivity of rearrangement and
cycloaromatization. Nonetheless, deeper experimental and
computational studies are needed to clarify these issues.
Thermal Reactivity of Enediynes. The thermal reactivity
of enediynes was evaluated using differential scanning
calorimetry (DSC). It could provide the onset temperatures
of the enediynes or enyne−allenes corresponding to radical
polymerization after their thermal cycloaromatization pro-
cesses,47,48 which is the alternative method for qualitative
evaluation of the reactivity in solution. For example, EDY-A
and its analogue EDY-C (replacement of the Boc group in
EDY-A with a phenyl substituent, Scheme S2) equipped with
the same alkyne functionalities share a similar onset temper-
ature at ∼80 °C (Figures 1A and S1), indicating that the onset
temperatures determined by DSC are not affected by the N-
substituents of maleimide. In contrast, EDY-B, bearing the
identical structure of EDY-A except for the N atom at the
propargyl site, showed a relatively low onset temperature at 56
°C.29 The higher reactivity of EDY-B may be attributed to the
hyperconjugative effects of σ-acceptors (donation from alkyne
to the σ*C−N orbitals).49 As described in the MARACA
mechanism, the propargyl−allene tautomerization is greatly
assisted when the maleimide moiety functions as the ene
source in the acyclic enediynes, ultimately shifting the reaction
pathway from BC to MSC. Thus, the cyclization mode of both
enediynes is attributed to the MSC behavior, dramatically
lowering the cycloaromatization reaction temperatures in
comparison to that of BC mode. Moreover, it is worth noting
that the electronic effect of the substituents at the propargyl
position influences the MSC reactivity significantly, according
to DSC experiments performed at different onset temper-
atures. This observation of the relative reactivity of two
enediynes using DSC is in good agreement with the activation
barriers obtained using density functional theory (DFT)50,51
calculations, where the value of activation energy for EDY-B in
the MSC is 1.6 kcal/mol lower than that of EDY-A (see in
Table 1).
Table 1. Activation Energy Values (kcal/mol) for the MSC
of Enediynes
ΔG
entry
X
MSC1
MSC2
1
2
3
C
N
O
26.3
24.0
24.4
27.6
26.0
20.5
Detection of Radical Intermediates. As mentioned
above, EDY-A undergoes thermal cycloaromatization at a
relatively low temperature. Next, the electron paramagnetic
resonance (EPR) spectroscopy52 was used to examine the
possibility of producing radicals from enediynes.
Generally, it is hard to directly detect these radical species in
situ because of the short lifetime and high reactivity53,54 in this
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Figure 2. (A) 1H−1H COSY spectrum of compound 4. Inset is the enlarged 1H NMR spectrum. (B) 1H−1H COSY spectrum of compounds 5 and
1
1
6. (C) H NMR analysis of compounds 5 and 6. (D) Key H−1H correlations for compound 6.
special type of reactions. Thus, N-tert-butyl-alpha-phenyl-
nitrone (PBN) was exploited as a spin trap agent to convert
the transient species into stable radical adducts.55,56 For the
preparation of samples for EPR studies, EDY-A (1 equiv) was
dissolved in dimethyl sulfoxide (DMSO) and 1,2-dichloro-
ethane (DCE) with the addition of excess PBN (5 equiv) to
each sample. After that, the samples were placed at 37 °C for
12 h before EPR measurement. As can be seen in Figure 1B,
strong and typical triplet-doublet signals with a hyperfine
splitting constants of 14.2 and 2.3 G were observed, suggesting
the formation of nitroxide radicals relative to spin-trapped
PBN adducts with diradicals generated from the cyclo-
aromatization of enediynes. This value is similar to that
reported for the calicheamicin phenyl radical adduct with PBN
(AN = 14.2 G, AH = 2.6 G).56 On the contrary, when the
mixture of EDY-A and PBN was warmed at 37 °C in a
nonpolar solvent DCE, the EPR signals appear much weaker
(Figure 1C). Figure 1D displays the recorded intensity at 3520
G for EDY-A in two different solvents, indicating that the
generation of free radicals is strongly dependent on the solvent
polarity. This observation is because of the existence of polar
intermediates in the propargyl−allene tautomerization pro-
cesses, and the polar solvent is beneficial to the rearrangement
behavior.
tautomerization, cycloaromatization, and eventually intra-
molecular HAT processes. When EDY-A was dissolved in
CHD to a final concentration of 10 mM and the reaction
system was heated at 80 °C for 3 d, a mixture of these three
cyclized products was equally formed as those from the tandem
reactions. No relevant radical H-abstraction product was
isolated even if EDY-A was exposed to a large excess of
CHD, which suggests that when an intramolecular radical
cyclization/H-abstraction path is involved in the fate of the
diradical intermediate, the intermolecular H-abstraction may
became less efficient regardless of the cycloaromatization
modes.47,57,58 On the other hand, because of the similarities of
the cyclized products in molecular structure and polarity, it is
very difficult if not impossible to separate out each compound
in pure form by column chromatography. To characterize the
accurate structures from the isolated mixed compounds, 2D
NMR correlation experiments, including heteronuclear single
quantum coherence (HSQC), heteronuclear multiple bond
1
correlation (HMBC), and H−1H correlation spectroscopy
(COSY), have been carried out (Figure 2 and Supporting
Information).
1
Take the H−1H COSY analysis for example, it provides
clear and crucial proton correlations, allowing the assignment
of vital structural fragment when combining with the J-
1
1
Scheme of Intramolecular HAT after MARACA and
Cyclized Products. The cyclized products 4, 5, and 6 were
initially obtained in a one-pot manner through a tandem
Sonogashira coupling reaction, followed by propargyl−allenyl
coupling data from H NMR. In the H−1H COSY spectrum
(Figure 2A), the correlation information of Ha/Hb and Hc/Hb,
together with HMBC correlations of Ha/Cb, Cc, C1, C2, and C3
(Supporting Information), confirmed the connection of the
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Scheme 2. Outline of Intramolecular HAT Pathways to Form the Cyclized Products 4, 5, and 6
ester moiety to a newly formed aromatic ring subunit as
depicted in structure 4. Similarly, Figure 2B,C illustrates the
proton correlations, which help us to distinguish structures
between two mixed compounds 5 and 6. For instance, the
distinct Ha/Hb and Hc/Hd cross peaks of compound 5 could
accordingly. Pathway A proceeds through an intramolecular
1,5-HAT from P1 to give another diradical intermediate A1,
which intermediately undergoes 1,3-HAT to afford the closed-
shell product 5. Apart from forming compound 5, there is a
possibility of the formation of a cyclobutane ring counterpart
by the radical coupling from intermediate A1. To this end, the
simulated NMR spectrum of the possible cyclobutane ring
structure (Figure S3) indicated that the H1 signal should locate
between 7.5−8.0 ppm as a sharp singlet peak. Meanwhile,
there are some unidentified signals after assignment of these
major peaks to compounds 5 and 6, probably accounting for
other unseparated/unidentified compounds under these
experimental conditions because of the complexity of free
radical-involved reaction pathways. Altogether, the formation
of a cyclobutane ring product from A1 cannot be ruled out, but
the amount of this compound could be very small. For the
cases starting from P2, pathway B involves the 1,4-HAT to
yield intermediate B1 followed by 5-endo radical cyclization to
form compound 4. A second path from P2 is the pathway C
that leads to the formation of compound 6 via HAT assisted by
the carbonyl group and subsequent self-quenching process of
diradical intermediate C1. As a consequence, the three
pathways dominate the consumption of reactive free radical
species intramolecularly by means of various HAT routes.
Computational Study. DFT calculations were carried out
to examine the possible pathways to understand the proton
transfer processes and the reactivity of the cycloaromatization
reaction as well as the intramolecular hydrogen shift reactions
to explain the formation of the cyclized products. The
structurally simplified models have been used to carry out
the computational studies (Scheme S3 and Figure S4), in
which the acetate anion serves as the proton shuttle to assist
the rearrangement processes. DFT calculations have been
performed in the Gaussian 09 package61 at the (U)B3LYP/6-
31G(d) level. Specifically, a restricted method was used for
geometry optimization of the closed-shell molecules, while for
a variety of other geometries possessing the open-shell
character, including the transition states (TS) of the MSC
and HAT processes and all biradical intermediates, an
unrestricted broken-symmetry approach with the “guess =
(mix, always)” designation has been utilized. The optimized
geometries are all verified to be either minima (no imaginary
frequencies) or TSs (one imaginary frequency) via frequency
1
be observed in the H−1H COSY spectrum (Figure 2B). The
coupling constant between Ha and Hb (7.6 Hz) indicates that
the two protons are adjacent in position (Figure 2C). Besides,
the large vicinal coupling constant (15.7 Hz) between Hc and
Hd reveals that the double bond is in trans configuration as
depicted in compound 5, identifying from the coupling
constant range of 3−13 Hz for cis and 12−20 Hz for trans
alkenes.59 In this case, once the configuration of compound 5
was determined by interpretation of its COSY spectrum, the
other structure is prone to be recognized by using the
remaining information of the 1D and 2D NMR data. First, an
obvious He/Hf cross peak with a vicinal coupling constant (7.5
Hz) strongly indicates that these protons belong to a tetra
substituted phenyl ring (Figure 2B,C). Apart from He and Hf,
another two tertiary hydrogen H1 and H2 were assigned to
construct the cyclobutane part, on the basis of DEPT, HSQC,
and HMBC analyses (Figure 2D and Supporting Information).
Figure 1C gives the detailed coupling information between
correlative protons, in which the small coupling constant (1.1
Hz) between H1 and Hf means the presence of long-range
coupling. Moreover, the rotation along single bonds is
probably restricted in the cyclobutane subunit, resulting in
1
the complexity of H NMR signals in H3, H4, H5, and H6,
evidenced in the displayed spectrum of a similar benzofused
cyclobutane moiety.60 H2 has correlations with H1, H3, and H4,
corresponding to coupling constants of 2.8, 6.6, and 9.0 Hz,
respectively, matching well the assigned structure 6. The
sample of mixed compounds 5 and 6 was further evaluated by
the high-performance liquid chromatography−mass spectrom-
etry (HPLC−MS) method, accessible to two main peaks
belonging to compounds 5 and 6 (Figure S2).
According to the MARACA mechanism for maleimide-based
enediynes, two kinds of α,3-didehydrotoluenes derivatives P1
and P2 could be generated from EDY-A, and these diradicals
would further undergo either an intramolecular or an
intermolecular consumption pathway. After the formation of
diradical species, the possible pathways A−C shown in Scheme
2 would explain the mixture of obtained compounds 4−6
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Figure 3. Gibbs free energy profile (kcal/mol) for rearrangement (black lines) and MSC (red lines) processes in Scheme S3. All the optimized
structures for stationary points can be found from Figure S4.
Scheme 3. Two MSC Pathways From Their Corresponding Enyne−Allene Precursors and the Initial Enediyne Model R
calculations at 298.15 K. Moreover, intrinsic reaction
coordinate (IRC) calculations were carried out to confirm
the connection of these TSs to two directions of reactants and
products. The solvent effects of toluene and THF were also
considered by employing a polarizable continuum model
(PCM)62 with the self-consistent reaction field (SCRF)
method63 based on the optimized structures in the gas phase
at the (U)B3LYP/6-31G(d) level. However, the solution-
phase single-point energy calculations were accomplished at a
higher level ((U)M06/6-311++G(d,p)). This method suited
for application in the mechanistic studies of proton transfer
reaction64 and the resulting Gibbs free energy in solvents
(toluene and THF) was computed with reference to the
known method in the literature.
As shown in Figure 3, prior to the formation of diradical
intermediates via a cycloaromatization reaction (red line), the
substrate R (similar to EDY-A) would proceed isomerization
to enyne−allene R1 and R4, respectively, via a 1,3-proton shift
with the help of acetate anion (black lines). Take the first
tautomerization of the propargyl group in enediyne R to form
the enyne-allene R1 as an example. With the assistance of the
proton shuttle, the abstraction of propargyl protons from R
occurs over TS1 (2.1 kcal/mol), causing the formation of
INT1. Immediately, the isomerization to enyne−allene R1 is
completed by protonation with a small barrier of 7.9 kcal/mol.
Once the precursor R1 for MSC is formed, the cyclo-
aromatization would take place to give diradical P1* through
TS-MSC1. Similarly, the other path to afford P2* needs to
undergo further 1,3-proton transfer steps and cycloaromatiza-
tion reaction. In the whole MARACA reaction, the MSC step
has the highest energy barrier of 27.6 kcal/mol, and the proton
transfer processes are accessible because of the low activation
barriers. Meanwhile, it is noteworthy that the involved polar
intermediates in the rearrangement processes should be
responsible for the observed solvent dependence in the EPR
performance.
DFT Calculations for the Evaluation of MSC Reac-
tivity for Enediynes. Judging from the above computational
results, the highest energy barrier in the MARACA mechanism
lies in the MSC step. To estimate the relative stability/
reactivity balance of enediynes, the DFT computed activation
energies for MSCs (directly formed from structure R1 as
MSC1 and from structure R4 as MSC2, Scheme 3) are given in
Table 1. Calculations revealed that different atoms at the
propargyl site affect the MSC reactivity significantly especially
for the latter case. When the heteroatoms (N, O) are placed at
the propargyl position of enediynes, the energy barrier values
are close and only ∼2 kcal/mol lower than that of the C atom
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Figure 4. Gibbs free energy profile (kcal/mol) of HAT pathways A, B, and C to form products 5*, 4*, and 6* starting from P1* and P2*,
respectively. The values of Gibbs free energy calculated in the gas phase are shown in the normal text, while the ones calculated in toluene are in
parentheses, and the ones calculated in THF are in brackets.
in the MSC1 situation. However, the activation energy values
calculated for MSC2 show relatively large differences. While
the structure bearing the methylene group at the propargyl site
has the highest activation barrier value of 27.6 kcal/mol (Table
1, entry 1), the other two structures have the values of 26.0 and
20.5 kcal/mol, respectively (Table 1, entries 2 and 3).
Reference to the results from Table 1, we can conclude that
the MSC reactivity order of enediynes is C < N < O according
to the activation energy values. Meanwhile, these results are
consistent with what has been observed in the DSC
experiments that the onset temperature of EDY-A is 26 °C
higher than that of EDY-B. These findings could prove the
relative stability/reactivity scale of maleimide-based enediynes,
providing an “optimal structure” with a good reactivity along
with a reasonable stability for exploring compounds for
biological applications.
Upon the formation of diradicals P1* and P2* through the
MARACA strategy, the follow-up reaction pathways of the two
kinds of diradicals toward 5* and 4* were examined by
computational studies, respectively (Figure 4). First of all, the
intramolecular 1,5-HAT relative to the benzyl radical may
occur over TS-A1 (21.6 kcal/mol) through an open-shell
singlet diradical pathway yielding intermediate A1*, followed
by 1,4-HAT with a barrier of 19.1 kcal/mol to give the closed-
shell product 5* (Pathway A). Indeed, the 1,3-HAT process is
unfavorable and relevant examples are rather scarce because
the process occurs accompanying the highly strained and
distorted four-membered-ring transition structure.65,66 The
1,3-HAT barrier of a similar structure A1** (the absence of
the carbon radical adjacent to the carbonyl group) has also
been calculated for comparison (Figure S5). Unsurprisingly,
the activation energy of 1,3-HAT (36.8 kcal/mol) for A1** is
high. Thus, the presence of one radical adjacent to the
carbonyl group in A1* seems to accelerate the 1,3-HAT
process, making it possible to be a thermal process in this
special case. Likewise, pathway B also discloses the two-step
radical transfer mechanism involving the 1,4-HAT and 5-endo
cyclization processes, eventually leading to the formation of 4*.
Besides, along the reaction paths, the transition states TS-A1,
TS-A2, TS-B1, and TS-B2 all exhibit the open-shell character-
istics, and the typical diradical characteristics in the
intermediates A1* and B1* were observed from the spin
density distributions (Figure 5), confirming the free radical-
involved pathways.
For diradical P2*, a competitive consumption pathway to
give 6* is proposed apart from the path to 4*. After careful
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enediynes (IC50 values of submicromolar vs nanomolar
level). By combining the experimental and computational
results, this investigation on intramolecular radical transfer
pathways of maleimide-based acyclic enediynes would provide
a guidance for the discovery of novel structurally simple yet
more powerful enediyne antitumor agents, which is underway
in our group.
EXPERIMENTAL SECTION
Figure 5. Spin density plots for intermediates A1* and B1*.
■
General Methods. Toluene and THF were dried over calcium
hydride (CaH2) and distilled before use. Other reagents were
purchased from commercial sources. Sonogashira reactions were
conducted under a nitrogen atmosphere. 1H and 13C{1H} NMR
spectra of all compounds were reported in ppm at 400 or 600 MHz
(Bruker) using deuterated solvents (CDCl3: δH = 7.26, δC = 77.2
ppm; CD3OD: δH = 3.31, δC = 49.0 ppm) as reference. Structural
assignments for compounds 4, 5, and 6 were made with additional
information from gHSQC and gHMBC experiments. HRMS data
were recorded on the Micromass LCTTM mass spectrometer using
the ESI method. EPR studies were performed with an X-band EMX-
8/2.7C spectrometer (Bruker). DSC experiments were investigated
with the Pyris Diamond thermal analysis workstation. DSC samples
were studied with the heating rate of 10 °C/min ranging from −10 to
250 °C under a flow of nitrogen. The new compounds (EDY-A, 3S,
and EDY-C) were synthesized following our previous general
procedures for the Sonogashira coupling reaction with minor
modifications.29
consideration of the structure of compound 6, a unimolecular
1,3-H shift is taken into account by DFT calculations (Figure
S6), leading to the radical relocation from the phenyl to benzyl
position. However, the one-step radical transfer should be
ruled out because of the high activation energy of 35.4 kcal/
mol. Alternatively, the possibility of the radical transfer assisted
by the carbonyl group was examined. As expected, the
activation energy of direct keto-enol tautomerization induced
by proton transfer is calculated to be as high as 68.5 kcal/mol,
comparable to reported cases.67 Given the ability of water
molecules serving as the proton donor and acceptor
simultaneously,68 single or two water molecules are considered
as the effective proton shuttle in such a reaction system to
catalyze the keto-enol transformation, respectively. Calcula-
tions indicate that one water decreases the activation energy to
40.4 kcal/mol, and the latter case of involving two water for
the interconversion shows a far lower barrier of 27.6 kcal/mol,
attributing to the acetoxy group around the reaction active site
functioning as a proton acceptor to stabilize the hydrogen
bond network, which is the lowest energy pathway identified
toward intermediate C1**. It is noticeable that the enol
formed from keto-enol tautomerization of the above three
cases cannot be obtained from structural optimization. DFT
calculations only afford the further H-abstraction structure
C1* with the absence of any unpaired electron. In fact, the
intermediate C1* and its diradical resonance structure similar
to the Garratt−Braverman cyclization69 intermediate would
undergo intramolecular reaction to form 6* via either diradical
self-quenching or formal [2+2] cycloadditions70 with a barrier
of 16.2 kcal/mol over TS-C2. Overall, the DFT calculation
results suggest that the diradicals formed from MSC could
become diamagnetic through various HAT pathways depend-
ing on the substrates, eventually yielding the thermodynami-
cally stable products.
Tert-butyl Pent-4-ynoate (1).40 N-Butyllithium (2.7 M in
hexane, 9.6 mL, 26 mmol) was added to Pri2NH (4.2 mL, 30
mmol) in THF (120 mL) under N2 at −78 °C. After 30 min, t-butyl
acetate (2.6 g, 22 mmol) was added and the mixture was stirred for 1
h at −78 °C. After that, HMPA (10.4 mL, 60 mmol) was added and
stirred for 10 min followed by the addition of 3-bromo-1-propyne
(2.4 g, 20 mmol) dropwise. The reaction system was held at −78 °C
for another 1 h, then warmed to room temperature. In the end, the
reaction was quenched with the saturated NH4Cl solution (5.0 mL)
before the mixture was diluted with hexane (100 mL), and then
washed with HCl (1.0 M, 2 × 50 mL) and brine (2 × 50 mL). The
resulting organic phase was dried over MgSO4, filtered, and the
solvent was evaporated under reduced pressure to give 1 as a yellow
oil (2.4 g, 79%). 1H NMR (400 MHz, CDCl3): δ 2.43 (m, 4H), 1.94
(m, 1H), 1.43 (s, 9H); 13C{1H} NMR (151 MHz, CDCl3): δ 171.2,
82.9, 81.0, 68.9, 34.6, 28.2, 14.6.
Di-tert-butyl 5,5′-(1-(2-(Tert-butoxy)-2-oxoethyl)-2,5-dioxo-
2,5-dihydro-1H-pyrrole-3,4-diyl)bis(pent-4-ynoate) (EDY-A).
The EDY-A was synthesized by Sonogashira coupling between
compounds 3 and 1. The mixture was stirred at ambient temperature
until completion of the reaction as detected by TLC (6 h). After that,
the mixture was subjected to silica gel chromatography (hexane/ethyl
1
acetate = 6:1) to give a yellow oil (150 mg, 58%). H NMR (400
CONCLUSIONS
MHz, CDCl3): δ 4.12 (s, 2H), 2.79 (t, J = 7.5 Hz, 4H), 2.54 (t, J = 7.5
Hz, 4H), 1.43 (s, 18H), 1.40 (s, 9H); 13C{1H} NMR (151 MHz,
CDCl3): δ 170.6, 166.7, 165.9, 128.6, 110.2, 83.0, 81.2, 71.8, 40.2,
33.9, 28.1, 28.0, 16.5; HRMS (ESI-TOF) m/z: [M + Na]+ calcd for
C28H37NO8Na, 538.2417; found 538.2416.
■
In summary, the maleimide-based acyclic enediynes are able to
generate diradical species through the MARACA strategy
under mild conditions. The effect of solvent polarity on the
cascade rearrangement and cycloaromatization was revealed,
and the polar solvent was found to accelerate the generation of
free radicals from the EPR studies. Subsequent thermolysis
experiment of enediynes provided three kinds of intra-
molecular HAT products. The formation pathways of these
products from open-shell diradical intermediates have been
verified using theoretical calculations. During the radical
transformation, the highest energy barrier for intramolecular
H-abstraction steps is 27.6 kcal/mol, posing a powerful
competition with external H-abstraction behavior from either
thermodynamic or kinetic aspect. This probably is the reason
for the relatively low cytotoxicity of currently developed
maleimide-based enediynes in comparison with natural
Tert-butyl 4-(2-(Tert-butoxy)-6-(2-(tert-butoxy)-2-oxoeth-
yl)-5,7-dioxo-6,7-dihydro-5H-furo[2,3-f]isoindol-4-yl)-
butanoate (4). Isolated by silica gel chromatography (hexane/ethyl
acetate = 6:1) to give a yellow solid (24 mg, 9%) from the synthesis
procedure of EDY-A in which the reaction system was further heated
to 37 °C in an oil bath for a week. 1H NMR (600 MHz, CD3OD): δ
7.82 (s, 1H), 7.07 (s, 1H), 4.32 (s, 2H), 3.35 (t, J = 7.7 Hz, 2H), 2.32
(t, J = 7.2 Hz, 2H), 1.98 (m, 2H), 1.49 (s, 9H), 1.47 (s, 9H), 1.43 (s,
9H); 13C{1H} NMR (151 MHz, CD3OD): δ 174.4, 169.4, 168.9,
168.5, 158.2, 157.8, 137.5, 135.9, 130.1, 124.3, 106.5, 106.0, 83.7,
83.2, 81.6, 40.5, 35.7, 28.7, 28.3, 28.2, 28.2, 26.8; HRMS (ESI-TOF)
m/z: [M + Na]+ calcd for C28H37NO8Na, 538.2417; found, 538.2415.
Tert-butyl (E)-3-(2-(2-(Tert-butoxy)-2-oxoethyl)-6-(3-(Tert-
butoxy)-3-oxopropyl)-1,3-dioxoisoindolin-5-yl)acrylate (5).
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Article
Compounds 5 and 6 were isolated by silica gel chromatography
Authors
(hexane/ethyl acetate = 6:1) to give a mixture (with a molar ratio of
Mengsi Zhang − Shanghai Key Laboratory of Advanced
Polymeric Materials, School of Materials Science and
Engineering, East China University of Science and
Technology, Shanghai 200237, China
Haotian Lu − Shanghai Key Laboratory of Advanced
Polymeric Materials, School of Materials Science and
Engineering, East China University of Science and
Technology, Shanghai 200237, China
Baojun Li − Shanghai Key Laboratory of Advanced Polymeric
Materials, School of Materials Science and Engineering, East
China University of Science and Technology, Shanghai
200237, China
Hailong Ma − Shanghai Key Laboratory of Advanced
Polymeric Materials, School of Materials Science and
Engineering, East China University of Science and
Technology, Shanghai 200237, China
1
roughly 0.7:1 as estimated from H NMR analysis) as a yellow solid
(32 mg, 12%) from the synthesis procedure of EDY-A in which the
reaction system was further heated to 50 °C in an oil bath for a week.
1H NMR (600 MHz, CDCl3): δ 8.01 (s, 1H), 7.90 (d, J = 15.7 Hz,
1H), 7.75 (s, 1H), 6.45 (d, J = 15.7 Hz, 1H), 4.32 (s, 2H), 3.15 (t, J =
7.6 Hz, 2H), 2.56 (t, J = 7.6 Hz, 2H), 1.54 (s, 9H), 1.45 (s, 9H), 1.42
(s, 9H); 13C{1H} NMR (151 MHz, CD3OD): δ 171.0, 167.1, 166.4,
166.2, 165.3, 146.8, 139.6, 138.6, 132.3, 130.6, 125.8, 124.8, 121.8,
82.9, 81.8, 81.1, 39.8, 35.7, 28.7, 28.1, 28.0, 28.0; HRMS (ESI-TOF)
m/z: [M + Na]+ calcd for C28H37NO8Na, 538.2417; found, 538.2418.
Tert-butyl 2-(2-(Tert-butoxy)-2-oxoethyl)-7-(3-(tert-bu-
toxy)-3-oxopropyl)-1,3-dioxo-2,3,6,7-tetrahydro-1H-
cyclobuta[e]isoindole-6-carboxylate (6). 1H NMR (600 MHz,
CDCl3): δ 7.80 (d, J = 7.5 Hz, 1H), 7.47 (dd, J = 7.5, 1.1 Hz, 1H),
4.34−4.26 (m, 2H), 4.01 (dd, J = 2.8, 1.1 Hz, 1H), 3.93 (ddd, J = 9.0,
6.6, 2.8 Hz, 1H), 2.66−2.51 (m, 2H), 2.33−2.28 (m, 1H), 2.14−2.08
(m, 1H), 1.47 (s, 9H), 1.46 (s, 9H), 1.45 (s, 9H); 13C{1H} NMR
(151 MHz, CDCl3): δ 172.3, 169.4, 169.4, 167.8, 166.3, 148.9, 144.8,
132.2, 127.5, 126.5, 123.8, 82.8, 81.3, 80.4, 54.2, 48.3, 39.6, 33.6, 28.3,
28.1, 28.0, 28.0; HRMS (ESI-TOF) m/z: [M + Na]+ calcd for
C28H37NO8Na, 538.2417; found, 538.2418.
Wenbo Wang − Shanghai Key Laboratory of Advanced
Polymeric Materials, School of Materials Science and
Engineering, East China University of Science and
Technology, Shanghai 200237, China
3-(4-Iodo-1-neopentyl-2,5-dioxo-2,5-dihydro-1H-pyrrol-3-
yl)prop-2-yn-1-yl Acetate (3S). The compound 3S was synthesized
by Sonogashira coupling between compound 2S29 and 2. The mixture
was stirred at ambient temperature until completion of the reaction as
detected by TLC (13 h). After that, the mixture was subjected to silica
gel chromatography (hexane/ethyl acetate = 10:1) to give a yellow oil
(35 mg, 18%). 1H NMR (600 MHz, CDCl3): δ 4.94 (s, 2H), 3.39 (s,
2H), 2.14 (s, 3H), 0.91 (s, 9H); 13C{1H} NMR (151 MHz, CDCl3):
δ 170.2, 167.1, 166.9, 135.9, 108.9, 102.6, 77.6, 52.6, 51.1, 33.8, 28.0,
20.8; HRMS (ESI-TOF) m/z: [M + Na]+ calcd for C14H16INO4Na,
412.0022; found, 412.0023.
Xiaoyu Cheng − Shanghai Key Laboratory of Advanced
Polymeric Materials, School of Materials Science and
Engineering, East China University of Science and
Technology, Shanghai 200237, China
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.joc.0c02401
Author Contributions
This article was written through contributions of all authors.
Ditert-butyl 5,5′-(1-benzyl-2,5-dioxo-2,5-dihydro-1H-pyr-
role-3,4-diyl)bis(pent-4-ynoate) (EDY-C). The EDY-C was
synthesized by Sonogashira coupling between compound 4S13 and
1. The mixture was stirred at ambient temperature until completion of
the reaction as detected by TLC (6 h). After that, the mixture was
subjected to silica gel chromatography (hexane/ethyl acetate = 6:1) to
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
Dedicated to the 100th anniversary of Chemistry at Nankai
University. The authors gratefully acknowledge the financial
support from the National Natural Science Foundation of
China (21871080 and 21503078), the Fundamental Research
Funds for the Central Universities (22221818014), and
Shanghai Leading Academic Discipline Project (B502). A.H.
thanks the “Eastern Scholar Professorship” support from
Shanghai local government.
1
give a yellow oil (147 mg, 60%). H NMR (600 MHz, CDCl3): δ
7.34−7.25 (m, 5H), 4.66 (s, 2H), 2.81 (t, J = 7.5 Hz, 4H), 2.57 (t, J =
7.5 Hz, 4H), 1.46 (s, 18H); 13C{1H} NMR (151 MHz, CDCl3): δ
170.6, 167.1, 135.9, 128.8, 128.7, 128.4, 128.1, 110.0, 81.2, 71.9, 42.4,
34.0, 28.2, 16.6; HRMS (ESI-TOF) m/z: [M + Na]+ calcd for
C29H33NO6Na, 514.2206; found, 514.2205.
ASSOCIATED CONTENT
■
sı
* Supporting Information
REFERENCES
■
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J. Org. Chem. 2021, 86, 1549−1559