Tunable Electrochemical C?N versus N?N Bond Formation of Nitrogen-Centered Radicals Enabled by Dehydrogenative Dearomatization: Biological Applications

Article abstract of DOI:10.1002/anie.202001510

Herein, an environmentally friendly electrochemical approach is reported that takes advantage of the captodative effect and delocalization effect to generate nitrogen-centered radicals (NCRs). By changing the reaction parameters of the electrode material and feedstock solubility, dearomatization enabled a selective dehydrogenative C?N versus N?N bond formation reaction. Hence, pyrido[1,2-a]benzimidazole and tetraarylhydrazine frameworks were prepared through a sustainable transition-metal- and exogenous oxidant-free strategy with broad generality. Bioactivity assays demonstrated that pyrido[1,2-a]benzimidazoles displayed antimicrobial activity and cytotoxicity against human cancer cells. Compound 21 exhibited good photochemical properties with a large Stokes shift (approximately 130 nm) and was successfully applied to subcellular imaging. A preliminary mechanism investigation and density functional theory (DFT) calculations revealed the possible reaction pathway.

Full text of DOI:10.1002/anie.202001510

A Journal of the Gesellschaft Deutscher Chemiker
Angewandte
Chemie
International Edition
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Accepted Article
Title: Tunable Electrochemical C-N versus N-N Bond Formation
of Nitrogen-Centered Radicals Enabled by Dehydrogenative
Dearomatization: Biological Applications
Authors: Shide Lv, Xiaoxin Han, Jian-Yong Wang, Mingyang Zhou,
Yanwei Wu, Li Ma, Liwei Niu, Wei Gao, Jianhua Zhou, Wei
Hu, Yuezhi Cui, and Jianbin Chen
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.202001510
Angew. Chem. 10.1002/ange.202001510
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10.1002/anie.202001510
Angewandte Chemie International Edition
RESEARCH ARTICLE
Tunable Electrochemical C-N versus N-N Bond Formation of
Nitrogen-Centered Radicals Enabled by Dehydrogenative
Dearomatization: Biological Applications
Shide Lv,[a],† Xiaoxin Han,[a],† Jian-Yong Wang,[b] Mingyang Zhou,[a] Yanwei Wu,[a] Li Ma,[a] Liwei Niu,[a]
Wei Gao,[a] Jianhua Zhou,[a] Wei Hu,[a] Yuezhi Cui,[a] and Jianbin Chen*[a]
Dedication ((optional))
Abstract: Herein, an environmentally friendly electrochemical
approach is reported that takes advantage of the captodative effect
and delocalization effect to generate nitrogen-centered radicals
(NCRs). By changing the reaction parameters of the electrode
compared with the well-established traditional ionic reactions for
the synthesis of nitrogen-containing skeletons, the
corresponding nitrogen-centered radical (NCR) strategy is in its
infancy due to the highly reactive nature and short lifetime of
those radicals.[3] Notably, most of the success of this strategy
has strongly depended on the assistance of stannyl radicals
and/or photochemistry. In sharp contrast, electrochemical
material and feedstock solubility, dearomatization enabled
selective dehydrogenative C-N versus N-N bond formation reaction.
Hence, pyrido[1,2-a]benzimidazole and tetraarylhydrazine
a
4]
frameworks were prepared via a sustainable transition-metal- and
exogenous oxidant-free strategy with broad generality. Bioactivity
assays demonstrated that pyrido[1,2-a]benzimidazoles displayed
antimicrobial activity and cytotoxicity against human cancer cells.
Compound 21 exhibited good photochemical properties with a large
Stokes shift (approximately 130 nm) and was successfully applied to
subcellular imaging. A preliminary mechanism investigation and
density functional theory (DFT) calculations revealed the possible
reaction pathway. Interestingly, the hydrazine products could be
studies based on NCRs have been limited thus far.[3f, Two
strategies have been employed to stabilize the aromatic aminyl
radicals, namely, the use of captodative effects in the presence
of a strong electron-withdrawing group such as an amide[3f, 4a-d]
or a sulfamide group[4k-n] (Scheme 1a, A) and delocalization
effects originating from diaryl amine motifs[4e-j] (Scheme 1a, B).
Considering of the intriguing structure of pyridine, both the
captodative effect and the delocalization effect might be
achieved in one molecule. Interestingly, dearomatization of the
pyridine motif can effectively proceed under electrolytic
conditions.[4q, 5, 16e] The aforementioned meaningful discoveries
promoted us to question whether a tunable behavior of species
C could be achieved via the dearomative strategy (Scheme 1a,
C).
converted into pyrido[1,2-a]benzimidazole cores under
a
complementary acidic conditions, although the former was not the
intermediate en route to the latter. Consequently, this divergent
synthetic route will stimulate the NCRs development in
electroorganic synthesis and related biomedical sciences.
Pyrido[1,2-a]benzimidazoles are an important class of
heterocyclic compounds, and they display a myriad of biological
activities, such as antimalarial,[6] antifungal,[7] antitumor,[8]
antibacterial,[9] and antiviral,[10] as well as applications in
fluorescence and dyes[11] (Scheme 1b). Thus, remarkable
attention has been focused on their construction. For instance,
Introduction
Nitrogen-containing compounds are frequently found in
natural products, pharmaceuticals, agrochemicals, and materials
science. Considerable efforts have been devoted to the
construction of privileged manifolds, especially via transition-
metal-catalyzed
cross-coupling
and/or
dehydrogenative
amination strategies.[1] Free radicals play a pivotal role in
biological systems and can be employed as versatile
intermediates in novel reactions that would otherwise be difficult
to achieve with typical ionic transformations.[2] However,
[a]
S. Lv, X. Han, Prof. Dr. M. Zhou, Y. Wu, Dr. L. Ma, Dr. L. Niu, W.
Gao, Prof. Dr. J. Zhou, Prof. Dr. W. Hu, Prof. Dr. Y. Cui, Prof. Dr. J.
Chen
Shandong Provincial Key Laboratory of Molecular Engineering,
School of Chemistry and Pharmaceutical Engineering
Qilu University of Technology (Shandong Academy of Sciences),
Jinan, 250353, P R China
E-mail: jchen@qlu.edu.cn
[b]
Prof. Dr. J.-Y. Wang
School of Light Industry and Engineering
Qilu University of Technology (Shandong Academy of Sciences),
Jinan, 250353, P R China
[†]
These authors contributed equally to this work.
Supporting information for this article is given via a link at the end of
the document.((Please delete this text if not appropriate))
Scheme 1. Representative scaffolds of aminyl radicals, pyrido[1,2-
a]benzimidazoles and tetraarylhydrazines.
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10.1002/anie.202001510
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RESEARCH ARTICLE
Scheme 2. Tunable electrochemical C-N vs N-N formation.
copper-catalyzed and/or hypervalent iodine (III)-mediated
intramolecular C-H amination has been independently reported
by the Zhu, Maes, and Dasgroups.[12] In addition,
tetraarylhydrazines are unique motifs in the natural product
intermolecular N-N oxidative coupling reactions with N-(4-
chlorophenyl)-5-fluoropyridin-2-amine
1
as the feedstock
(Scheme 2, see Table S2 and S3). First, the generality of the
cyclization product was tested (Table 1, standard condition A).
As shown, a broad spectrum of functional groups was tolerated
with good to quantitative efficiency. Specifically, electron-
donating groups, such as alkyl (4, 5, 15, 16), ether (6, 7, 17, 18),
and thioether groups (9), and electron-withdrawing groups
including fluoro (8, 22, 23, 25, 28, 29), chloro (2, 22, 26, 28),
bromo (27), and trifluoromethoxy (10, 19) moieties reacted
smoothly in the aqueous system. Interestingly, the groups that
was electrochemical oxidative and reductive labile were intact in
moderate yields. This observation was exemplified by the amino
(20, 21), and thioether groups for the electrochemical oxidative
moieties and by the aldehyde (32), ketone (31), and ester (11,
33) groups for the reductive lable moieties. Regarding the steric
hindrance, the meta-substituted starting materials exclusively
favored the less steric position (16, 22, 23, 28, 29). Moreover,
ortho-phenyl (24) was formed selectively in our system
accomplishing the desired pyrido[1,2-a]benzimidazole core
instead of the carbazole unit. The naphthalene site could also be
cyclized with moderate efficiency (34). In some cases, methyl
thioglycolate[18] or NaI[5] was added to improve the yields.
dixiamycins
A
and B[13] and can be employed as
electrocatalysts[14] (Scheme 1c). Conventional transition-metal-
catalyzed oxidative coupling of secondary amines with oxidants
is the primary procedure for their preparation.[15] In view of these
privileged structures and our interests[16] in organic
electrochemistry,[17] we envision that by controlling one of the
key factors in electrochemistry, namely, the current density, the
distinctive target compound might be furnished in a tunable
manner via transition-metal- and exogenous oxidant-free
electrochemical dehydrogenative NCR strategies. The
hypothesis was based on the principle that the current density is
proportional to the reaction rate at the electrode surface, and
hence correlates to the concentration of the reactive
intermediate species.
Results and discussion
With this idea in mind, we identified two separate optimal
systems for electrochemical intramolecular C-N and
Next, we moved to the substrate scope of the N-N bond
Table 1. Generality of the intramolecular C-N bond cyclization.
Reaction condition A: Substance (0.2 mmol), KPF6 (2 equiv.), NaOAc3H2O (2 equiv.), a graphite rod ( 10 mm) as the anode, a Pt sheet (1  1 cm) as the
cathode, H2O (5 mL), 10 mA (j = 4.95  10-4 mA/cm2), N2, 100 C, 16 h. [a] with HSCH2CO2Me (10 mol%) and [b] with NaI (1.2 equiv.).
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10.1002/anie.202001510
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RESEARCH ARTICLE
Table 2. Generality of the intermolecular N-N bond formation reaction.
Reaction condition B: Substance (0.25 mmol), TBAI (2 equiv.), a Pt sheet (1  1 cm) as the anode and cathode, MeCN-MeOH (7-0.5 mL), 5 mA (j = 5 mA/cm2), N2,
60 C, 10 h.
formation part (Table 2, standard condition B). Once again, the
electronic and steric effects on the benzene ring were examined.
Electron-rich groups such as an alkyl group (35, 36, 46, 47, 57,
58, 59) and electron-deficient groups such as fluoro (39, 40, 41,
50, 55, 56, 62), chloro (3, 40, 42, 48, 54, 56, 63), bromo (49, 52,
53), and trifluoromethyl (38, 61) moieties were compatible.
Electrochemically active thioether (43) was also furnished in
moderate yield. Particularly, the intact bromo group provides an
orthogonal reactive handle for further transition-metal-catalyzed
cross-coupling reactions. In terms of the substitutents on the
pyridine ring, the fluoro (3, 35-45), chloro (46-51), bromo (52),
and methyl (64) groups worked successfully. Additionally, diaryl
amine (65) produced the corresponding hydrazine with
acceptable efficiency. Fortunately, a single crystal of 53 was
obtained, and thus, all the structures of the N-N coupling
products were unambiguously determined. Considering the
practicability of these newly developed protocols, gram-scale
reactions were conducted, although diminished yields were
observed [19] (see supporting information Figure S4 and S5).
substituted amine resulted in almost no product (Eq. 1). This
observation highlighted the importance of the free N-H moiety.
Importantly, deprotonation of the free N-H starting material by a
base i.e., hydroxide, was validated by the nuclear magnetic
resonance (NMR, Figure S6) and CV results (green line 1 +
tetrabutylammonium hydroxide (TBAOH) vs pink line 1, Figure
2a). Clearly, the oxidative peak potential for the nitrogen anion
(green line, Ep = 0.44 V vs SCE) was remarkably lower than that
of the corresponding starting material 1 (pink line, Ep = 1.22 V vs
SCE, Figure 2a). The addition of radical scavengers such as
2,2,6,6-teramethyl-1-piperidinyloxy (TEMPO) or butylated
hydroxytoluene (BHT) under the standard conditions A or B led
to significant inhibition (Eq. 2 and 4). Remarkably, cross-
coupling product 66 was accompanied by two other
homocoupling products 36 and 55, in a ratio of approximately of
1: 1:
1 when an equal molar substance was present.
Furthermore, selective cross-coupling of free radicals is highly
desirable.[20] Under standard condition B, the cross-coupling of
compound 66 could be significantly improved by simply adding 5
equivalents of one substance (Eq. 5). Because of the relatively
low peak potential (red line, Ep = 0.75V vs SCE, Figure 2a) in
MeCN/MeOH, the direct oxidation of the iodide anion is
operative. Interestingly, only compound 3 was afforded in 73%
yield when the constant potential electrolysis was set at 0.75 V
vs SCE, corresponding to the anodic oxidation of iodide anions
(Eq. 6). Taken together, these data substantially corroborated
that the nitrogen-centered radical intermediate was involved in
Preliminary mechanism investigation
To obtain insight into the reaction mechanism, a number of
control experiments (Figure 1) and cyclic voltammetry (CV,
Figure 2) analyses were conducted. Electrolysis of a methyl-
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10.1002/anie.202001510
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RESEARCH ARTICLE
the process. Notably, an inverse secondary kinetic isotope effect
(KIE = 0.89) under condition A was measured (Eq. 3). The data
were consistent with the configuration change in the carbon
center from sp2 to sp3 and demonstrated that C-H bond
cleavage was not involved in the amination step. Markedly, this
result was completely different from the Cu-catalyzed C-H
amination
reaction.[12a]
Then,
we
investigated
the
electrochemical behavior of compounds 1, 2, and 3 via CV.
Interestingly, a reversible cyclic voltammogram for 3 was
observed whereas a completely irreversible result was found for
1; however, almost no oxidative peak was observed for 2 in the
potential window of interest (Figure 2b).
Density functional theory (DFT)[21] was used to explain the
differences in enegy values for the reaction steps of interest
(Scheme 3). Once nitrogen radical B was formed, -91.06 kJ/mol
(at the B3LYP/6-311++G(d,p) level) was released when the
intermolecular N-N homocoupling reaction took place.
Nevertheless, the intramolecular C-N annulation was
complicated. The energy barrier for the transition state (TS1)
indicated that nitrogen radical attack of the aryl ring was an
endothermic step and generated radical D. At this point, the
enegy for the anodic heterogenous oxidation at the electrode
surface of D affording species E was significantly lower than that
of the homogenous oxidation reaction in bulk solution (pink line
vs blue line, Scheme 3). Deprotonation of E in the presence of
hydroxide furnished final product 14 in an exothermic step.
Despite the evidence for the homogenous bulk chemical
process, the selectivity was still not explained. Hence, we
focused on the electrode materials. A commercially available
graphite rod was employed as the anode in condition A and the
surface area was determined to be 2.022 m2/g (see Figure S13).
The mesoporous structure allows the organic component to
easily diffuse to the electrode surface. Thus, the corresponding
current density was calculated to be 4.95  10-4 mA/cm2, which
was approximately one ten thousandth to that of the platinum
anode, as in condition B. Moreover, the starting material has
poor solubility in water whereas it is totally soluble in an organic
medium. The above reaction parameters ultimately led to a
difference in the concentration of NCR B. In addition, the
graphite electrode exhibited a greater paramagnetic nature than
the other tested electrode, resulting in increased attraction of the
radical species to the graphite surface, thereby promoting
oxidation of the second electron.[17a, r, 22]
Figure 1. Control experiments.
Based on the preliminary mechanism investigation, two
putative reaction pathways were proposed, as shown in
Scheme 3. The first pathway for generating NCR B is the
halide-mediated pathway, namely, the anodic oxidation of iodide
anions accompanied with the following homolytic cleavage of the
N-I bond. The second one is the direct oxidation of the nitrogen
anion species A originating from the deprotonation of the free N-
H feedstock by an electrochemically generated base. Under
condition B, the good substrate solubility and the relatively high
Figure 2. CV analysis. Substrate (0.01 mol/L), LiClO4 (0.1 mol/L), graphite
carbon (GC) as the working electrode, Pt wire as the counter electrode, SCE
as the reference electrode, scan rate: 100 mV/s. a) in MeCN/MeOH (14:1); b)
in MeCN.
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10.1002/anie.202001510
Angewandte Chemie International Edition
RESEARCH ARTICLE
current density from the platinum anode resulted in a sufficient
concentration of NCR B which benefited the N-N coupling
pathway. However, under condition A, an extremely low
concentration of nitrogen radicals was generated. To meet the
current demand, the formal dearomative isomerization of B to C
along with the ensuing intramolecular nitrogen radical attack of
the aryl ring supplied species D. Further heterogeneous
oxidation of D on the graphite anode surface took place, thereby
giving cation species E; subsequent deprotonation in the
presence of a base eventually delivered the formal 2e oxidation
scaffold 14.[23] Finally, hydrogen evolution was responsible for
the cathodic half reaction which was confirmed by gas
chromatography (GC) analysis (see Figure S14).
N
H
N
H
00
N
I
N
N
E
00
00
00
00
0
-311.49
-311.49
374.75
250.22
1/2 I2
N
E
CV
‡
N
-
N
I
N
N
N
N
N
N
-50.04
H
TS1
115.17
N
N
14
D
-
B
-91.06
C
CV
N
00
00
00
00
00
N
N
A
N
-
NMR
OH
N
H
N
N
60
N
GC
-
ROH
1/2H2 +
OR
Scheme 3. Putative mechanism.
Table 4. Bioactivity of the pyrido[1,2-a]benzimidazole compounds.
Conversion of compound 3 to 2
Compounds
Inhibition Ratio/%
Staphylococcus aureus ATCC-6538
55.31 ± 1.49
We questioned whether the N-N product was the
intermediate en route to the C-N cyclized product. This
possibility was ruled out based on the control experiment in Eq.
7a. Stimulated by the fact that potassium hexafluorophosphate
(KPF6) is hydrolyzed in water to make an acidic solution, we
controversially performed electrolysis in an acidic medium (Eq.
7c)[24]. Surprisingly, compound 2 was furnished with moderate
efficiency at 2 V vs SCE by the electrolysis of N-N product 3 in a
strong acidic environment (standard condition C). This discovery
elucidated the reason for the failure, as exhibited in Eq. 7a
which was ascribed to the weak acidity. Inspired by this result,
several transformations were set up, as shown in Table 3, to
consider a complementary route from the N-N structure to C-N
fragments.
15
34
Saccharomyces cerevisiae
49.37 ± 5.39
Hepatocarcinoma cell line HepG2
38.24 ± 4.99
2
8
49.16 ± 5.97
29
42.99 ± 17.57
Melanoma cell line A375
53.31 ± 3.57
8
Table 3. Transformation of N-N compounds to C-N products.
30
35.76 ± 11.61
Cervix adenocarcinoma HeLa cells
43.90 ± 2.05
12
Photochemical properties and biological
applications
Reaction condition C: Substance (0.1-0.2 mmol), TBABF4 (5 equiv.), carbon
felt (1  1  0.3 cm) as the anode, a Pt sheet (1  1 cm) as the cathode, DCM-
TFA (4.5-0.5 mL), 2 V vs SCE, air, r.t., 18 h.
In terms of the biological activity and fluorescence
properties
of
pyrido[1,2-a]benzimidazole
heterocyclic
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10.1002/anie.202001510
Angewandte Chemie International Edition
RESEARCH ARTICLE
compounds,[6-11] applications in related areas were also Conclusion
evaluated. First, the antimicrobial activity and cytotoxicity against
In summary, based on the captodative effect and
delocalization effect, an electrochemical tunable methodology
from readily available feedstock was presented, providing easy
access to intermolecular N-N coupling and intramolecular C-N
bond formation architectures. By changing the reaction
human cancer cells were tested. Several compounds showed
high activity. For instance, compounds 15, 34, and 12 could
inhibit the growth of cells by 55%, 49%, and 44%, respectively.
Moreover, Staphylococcus aureus, Saccharomyces cerevisiae,
and cervix adenocarcinoma HeLa compounds 8 and 29 inhibited
the hepatocarcinoma cell line HepG2 by more than 42%.
Furthermore, compound 8 could also inhibit melanoma cell line
A375 by 53%.
parameters including the anode material and solvent,
a
considerable difference in the nitrogen radical concentration
could be established. Generally, a high concentration of NCR
benefits the radical N-N coupling route, while
a
low
Second, compound 21 exhibited strong fluorescence
enhancement with a large Stokes shift[25] (approximately 130
nm) at different concentrations, making it potentially applicable
in subcellular imaging (Figure 3). Morpholine was treated as a
target group of lysosomes, thus increasing the compound 21
distribution in lysosomes. To investigate the subcellular
localization ability, compound 21 with the commercial lysosomal
tracker LysoTracker red was applied in HeLa cells. As shown in
Figure 4, the fluorescence signal from the green channel
overlaid very well with that of LysoTracker red. Moreover, the
fluorescent intensity of the green channel and red channel is
well correlated with a highly overlapping Pearson´s coefficient of
0.80 and an overlapping Mander´s coefficient of 0.81. Therefore,
the compound 21 was well located in the lysosomes of living
cells.
concentration of NCRs is further oxidized to formal
dearomatization 2e oxidation products. Alternatively, conversion
of the hydrazine product to pyrido[1,2-a]benzimidazole cores
was also viable under acidic conditions, although the former was
not the intermediate en route to the latter. The transition-metal-
and exogenous-free features of this protocol highlight the user-
friendly and sustainable nature of the newly developed organic
electrolysis method. Biological evaluation demonstrated the
potential application of our products, including the use of their
antimicrobial and antitumor activities. The photochemical
properties revealed that compound 21 was suitable for cell
labeling with a large Stokes shift (approximately 130 nm);
furthermore, lysosomal fluorescence imaging was realized in
living cells. On the basis of these advantages, the newly
developed approach represents an ideal strategy for divergent
synthesis approaches, and could potentially be useful in
chemistry, biomedicine and materials science.
Acknowledgements
We greatly appreciate the financial support from the National
Natural Science Foundation of China (Nos. 21801144,
81872744, 51602164), the Youth Innovative Talents
Recruitment and Cultivation Program of Shandong Higher
Education, the Natural Science Foundation of Shandong
Provincie (No. ZR2018BB017), Qilu University of Technology
(Shandong Academy of Sciences, No. 0412048811), and the
Program for Scientific Research Innovation Team in Colleges
and Universities of Shandong Province.
Figure 3. UV-vis absorption spectra and fluorescence emission spectra of
compound 21 in DMF.
Keywords: nitrogen-centered-radicals •dearomatization •
dehydrogenative • electrosynthesis • tunable
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10.1002/anie.202001510
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RESEARCH ARTICLE
RESEARCH ARTICLE
Shide Lv,† Xiaoxin Han,† Jian-Yong
Wang, Mingyang Zhou, Yanwei Wu, Li
Ma, Liwei Niu, Wei Gao, Jianhua Zhou,
Wei Hu, Yuezhi Cui, Jianbin Chen*
Page No. – Page No.
Tunable Electrochemical C-N versus
N-N Bond Formation of Nitrogen-
Centered Radicals Enabled by
Dehydrogenative Dearomatization:
Biological Applications
Tunable C-N versus N-N bond formation of nitrogen-centered radicals was
achieved by an electrochemical dehydrogenative dearomatization strategy. Control
experiments, DFT calculations, and investigations of the electrode material
elucidated the origin of the chemoselectivity. The N-N product could be transformed
into the C-N scaffold under complementary conditions, although the former was not
the intermediate en route to the latter. Bioactivity assays demonstrated that
pyrido[1,2-a]benzimidazoles displayed antimicrobial activity and cytotoxicity against
human cancer cells. A large Stokes shift (approximately130 nm) and subcellular
imaging were also demonstrated.
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