Retro-aza-Michael reaction of an o-aminophenol adduct in protic solvents inspired by natural products

Article abstract of DOI:10.1016/j.bmc.2021.116059

α,β-Unsaturated carbonyls are reactive group often found in bioactive small molecules. Their non-specific reaction with biomolecules can be the cause of the low efficacy and unexpected side-effects of the molecule. Accordingly, unprotected α,β-unsaturated carbonyls are not often found in drugs. Here, we report that o-aminophenol is a new masking group of α,β-unsaturated ketone, which is inspired by natural products saccharothriolides. o-Aminophenol adduct of α,β-unsaturated ketone, but not those of α,β-unsaturated amide or ester, undergoes a retro-Michael reaction to yield o-aminophenol and the Michael acceptor. This reaction was observed only in protic solvents, such as MeOH and aqueous MeOH. In contrast, o-anisidine was not eliminated from its Michael adduct. o-Aminophenol may be a promising masking tool of highly-reactive bioactive α,β-unsaturated carbonyl compounds.

Full text of DOI:10.1016/j.bmc.2021.116059

Bioorg. Med. Chem. 35 (2021) 116059
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Retro-aza-Michael reaction of an o-aminophenol adduct in protic solvents
inspired by natural products
,b,c
Kei Takenakaa, Kensuke Kanekoa, Nobuaki Takahashia, Shinichi Nishimuraa
,
,
*
Hideaki Kakeyaa
a Department of System Chemotherapy and Molecular Sciences, Division of Bioinformatics and Chemical Genomics, Graduate School of Pharmaceutical Sciences, Kyoto
University, Kyoto 606-8501, Japan
b Department of Biotechnology, The University of Tokyo, Tokyo 113-8657, Japan
c Collaborative Research Institute for Innovative Microbiology, The University of Tokyo, Tokyo 113-8657, Japan
A R T I C L E I N F O
A B S T R A C T
Keywords:
α
,β-Unsaturated carbonyls are reactive group often found in bioactive small molecules. Their non-specific re-
Retro-aza-Michael reaction
Protic solvents
action with biomolecules can be the cause of the low efficacy and unexpected side-effects of the molecule.
Accordingly, unprotected α,β-unsaturated carbonyls are not often found in drugs. Here, we report that o-ami-
o-Aminophenol
nophenol is a new masking group of α,β-unsaturated ketone, which is inspired by natural products saccharo-
Natural products
Saccharothriolides
thriolides. o-Aminophenol adduct of α,β-unsaturated ketone, but not those of α,β-unsaturated amide or ester,
undergoes a retro-Michael reaction to yield o-aminophenol and the Michael acceptor. This reaction was observed
only in protic solvents, such as MeOH and aqueous MeOH. In contrast, o-anisidine was not eliminated from its
Michael adduct. o-Aminophenol may be a promising masking tool of highly-reactive bioactive α,β-unsaturated
carbonyl compounds.
1. Introduction
from the Michael reaction between the precursor molecule, pre-
saccharothriolide X (preSTL-X, 2), and nucleophiles (Figure 1a). The
structure–activity relationship study using STL congeners revealed that
STL-B (1) and preSTL-X (2) were most potent cytotoxic STL congeners
against human fibrosarcoma HT1080 cells. PreSTL-X (2) seems to be the
active substance since STL-B (1) released o-aminophenol and thereby
generated preSTL-X (2) in aqueous conditions (Figure 1a).17 This retro-
Michael reaction was not observed in the case of STL-A, -G, or -K, which
had anthranilic acid, o-anisidine, or m-aminophenol at the C-7 position,
respectively (Figure S1). However, the mechanism and the scope of this
elimination was not known.
Michael acceptors in bioactive small molecules can form covalent
bonds with nucleophiles in target proteins.1–4 They are often found in
natural products and have potent biological activities.5,6 Recently,
Michael acceptors were embedded in synthetic anticancer drugs, such as
afatinib and ibrutinib. Both compounds have an α,β-unsaturated amide
moiety and show irreversible binding with their target proteins.7,8 In
general, however, the high chemical reactivity of the Michael acceptors
can lead to the risk of side effects by binding to off-target molecules.1 To
overcome this problem, chemical protection of the Michael acceptor or
substitution with alternative functionalities have been investigated.9–13
We have investigated the potential of o-aminophenol as a protective
group for Michael acceptors using simple model compounds. Among the
For example, installing sulfoxides at the β position of the α,β-unsaturated
ester in brefeldin A furnished a prodrug of brefeldin A.9 3-Aminopropa-
Michael adducts tested, the o-aminophenol adduct of an α,β-unsaturated
namide, which is stable in vitro, was found to be converted to acrylamide
ketone showed retro-Michael reactivity. This reaction was not observed
when o-anisidine was used. In contrast, ,β-unsaturated amides and
esters were not tolerated by this reversible protection by o-aminophenol.
in cells.10 Very recently,
α
-chlorofluoroacetamide with weak intrinsic
α
reactivity was reported as an effective warhead in cells.13
Saccharothriolides (STLs) are 10-membered macrolides, isolated
from the culture broth of an actinomycete Saccharothrix sp. by our group
(Figure 1a, S1).14–17 The variety of the substitution at C-7 is derived
Herein we report that the transient protection of α,β-unsaturated ke-
tones by o-aminophenol is a general protecting group strategy and not
limited to STL-B.
* Corresponding author.
E-mail address: scseigyo-hisyo@pharm.kyoto-u.ac.jp (H. Kakeya).
https://doi.org/10.1016/j.bmc.2021.116059
Received 4 December 2020; Received in revised form 30 January 2021; Accepted 2 February 2021
Available online 13 February 2021
0968-0896/© 2021 Elsevier Ltd. All rights reserved.

K. Takenaka et al.
Bioorganic & Medicinal Chemistry 35 (2021) 116059
2. Results and discussion
2.1. Preference of Michael acceptors
The retro-Michael reaction of STL-B (1) proceeded spontaneously in
aqueous solvents to yield preSTL-X (2) and o-aminophenol (Figure 1a).17
To investigate the scope of this reaction, we examined three Michael
adducts (3, 4 and 5) which were synthesized from α,β-unsaturated ke-
tone (6), ester (7), and amide (8), respectively (Figure 1b & 1c). o-
Aminophenol adducts 3, 4, and 5 were left in aqueous MeOH at 37 ◦C for
two days and then analyzed by HPLC (Fig 2, S2). We found that amount
of keto-type adduct 3 decreased while o-aminophenol and Michael
acceptor 6 appeared. Conversely, o-aminophenol and the corresponding
Michael acceptors (7 or 8) were not detected when the ester or amide-
type adducts (4 and 5, respectively) were incubated. These results
revealed that the spontaneous retro-Michael reaction of the o-amino-
phenol adduct is not limited to STL-B. Instead, this reaction was spe-
cifically observed for the o-aminophenol adduct of
α,β-unsaturated
ketones. The acidity of the α-proton of the carbonyl is likely important
for this reaction. Amides and esters reduce the acidity of the
and thus hamper the spontaneous retro-Michael reaction.
α-protons
2.2. Effect of solvent on the retro-Michael reaction
Fig. 2. Retro-Michael reaction of the o-aminophenol adducts (3). Compound 3
was dissolved in MeOH/H2O (4:1), incubated at 37 ◦C, and analyzed by HPLC.
Top panel: the sample just after preparation (0 h); second from the top: the
sample after 48 h incubation. Chromatograms for the Michael acceptor (6) and
o-aminophenol are also shown. The asterisks indicate caffeine, which was used
as an internal standard. See the experimental section for details.
In the case of STL-B (1), the retro-Michael reaction proceeded in
aqueous MeOH, but not in neat MeOH.17 We examined the effect of
solvents on the reactivity of compound 3 in an NMR tube. When 3 was
dissolved in CD3CN or DMSO‑d6 and incubated at 37 ◦C for two days, the
1H NMR spectra showed no change (Figure S3). In contrast, the signals
corresponding to the Michael acceptor 6 appeared when 3 was incu-
bated in CD3OD or CD3OD/D2O (4:1) (Figure 3). These results suggested
that protic solvents promoted this retro-Michael reaction.
MeCN or DMSO, which suggested that the elimination reaction requires
a protic solvent.
The activity in the presence of H2O in this reaction seems to be higher
than that of MeOH. In the 1H NMR spectra, deuterium exchange in
Michael acceptor 6 and adduct 3 was observed both in CD3OD/D2O
(4:1) and in CD3OD, but the degree of the exchange was higher in the
When we found that STL-B (1) released o-aminophenol in an aqueous
MeOH, but not in neat MeOH, we could not explain the molecular
mechanism.17 The simple model compound 3 underwent the retro-
Michael reaction both in neat MeOH and aqueous MeOH but not in
Fig. 1. Chemical structures of STLs and model Michael acceptors and adducts. (a) STL-B (1) releases o-aminophenol to generate preSTL-X (2). (b, c) Chemical
structures of model compounds: keto-type (3, 6, 9), ester-type (4, 7), and amide-type (5, 8).
2

K. Takenaka et al.
Bioorganic & Medicinal Chemistry 35 (2021) 116059
Fig. 3. Effect of solvents on the retro-Michael
reaction of compound 3 (500 MHz 1H NMR). o-
Aminophenol adduct 3 was dissolved in CD3OD
(a) or CD3OD/D2O (4:1) (b). Top blue: compound
3 just after being dissolved in the solvent; middle
brown; compound 3 after two days at 37 ◦C;
bottom green: Michael acceptor 6. (c) Enlarged
view of the 1H NMR spectra shown in (b). The
proton signals highlighted in blue and pink
correspond to the α-protons in compounds 3 and
6, respectively. (For interpretation of the refer-
ences to colour in this figure legend, the reader is
referred to the web version of this article.)
presence of D2O (Figure 3c, S4). In CD3OD, the peak area of the
α
2.3. Quantitative analyses of elimination of o-aminophenol in protic
environments
methylene protons was 1.27H, indicating more than 35% of the protons
were replaced by deuterium. Conversely, in the presence of D2O, the
peak area was 0.67H, which indicated more than 65% of the protons
were replaced by deuterium (Figure S4c). These deuterium exchanges
seem to couple with the retro-Michael reaction, as this exchange was not
observed when the o-anisidine adduct was examined (Figure S6, see
below).
The NMR analyses of compound 3 indicated that aqueous MeOH has
higher catalytic activity in the retro-Michael reaction than that of neat
MeOH. To investigate the solvent effect in a quantitative manner, we
conducted time-dependent analyses of the retro-Michael reaction of
compound 3 in MeOH or MeOH/water (4:1) (Figure 4). In MeOH, the
Fig. 4. Quantitative analyses of the elimination of o-aminophenol from compound 3. Reaction was monitored in MeOH (a) or MeOH/H2O (4:1) (b). Compound 3 (1
mM) was incubated at 37 ◦C for 48 h. Data represent the mean ± SD of three independent experiments. See the experimental section for details.
3

K. Takenaka et al.
Bioorganic & Medicinal Chemistry 35 (2021) 116059
amount of compound 3 linearly decreased, whereas the amounts of o-
aminophenol and the Michael acceptor 6 increased at a similar speed.
After 48 h incubation, the remaining amount of compound 3 was esti-
mated to be 50 (±7.7) %. The amount of o-aminophenol and compound
6 were 54 (±3.9) and 50 (±1.7) % relative to starting material 3,
respectively. In the presence of water, the reaction was faster than that
in neat MeOH, and it took 22 h to reach the equilibrium. At that time, the
amount of compound 3, o-aminophenol, and compound 6 were 58
(±9.8), 50 (±8.3), and 50 (±5.8) %, respectively. These results unveiled
that aqueous MeOH is a more effective solvent for the retro-Michael
reaction of the o-aminophenol Michael adduct than neat MeOH.
Although the promotion of Michael addition with anilines by polar
protic solvents, without any promoting agent, has been reported,18 this
is the first report of a retro-Michael reaction promoted by protic solvents
and no additional reagents. Additionally, the effects of pH for this re-
action were tested. The reactions were monitored in MeOH/NaOAc aq.
(4:1) and MeOH/Tris-HCl aq. (4:1) as models of acidic (pH 4.0) and
basic (pH 8.8) conditions. The results were not different from that of
neutral condition, they took ca. 24 h to reach the equilibrium
(Figure S5). It is noted that compound 3 gave no detectable byproducts
in these conditions.
protic solvents can generate a hydrogen-bonding network to form the
enol, followed by deuteration or elimination (Figure S7). However, the
difference between neat MeOH and aqueous MeOH remains to be
explained.
3. Conclusion
In summary, we demonstrated that the retro-Michael reaction of the
o-aminophenol Michael adduct is observed not only in STL-B (1) but also
in the simple model compound 3.
α,β-Unsaturated ketones were
compatible with this reaction, which was promoted in protic solvents,
especially aqueous methanol. It is noted that this elimination reaction
can be controlled by protecting the phenolic alcohol. This phenomenon
may be applied as a drug-delivery system consisting of labile bioactive
compounds possessing an
α,β-unsaturated ketone. Protection of the
phenolic alcohol will be crucial for tissue- or microenvironment-
selective release of the bioactive compounds, which is currently being
investigated in our group.
4. Experimental section
4.1. General procedures
2.4. Inert Michael adduct with o-anisidine
All commercially available reagents were purchased from Nacalai,
Fujifilm-Wako or Tokyo-Kasei unless described. Silica gel column was
performed using Kanto Silica Gel 60 N (63–210 mm) (Kanto Chemical).
TLC was performed using glass-backed silica gel 60F254 (Merck). NMR
spectra were recorded on a JOEL 500 MHz instrument (JEOL). 1H and
13C chemical shifts are shown relative to the residual solvent: δH 7.26
and δ 77.16 for CDCl3; δ 2.50 for (CD3)2SO; δ 1.94 for CD3CN; δ 3.31
STL-G, an STL derivative that has o-anisidine at the C-7 position, did
not show any retro-Michael reactivity (Figure S1).17 We finally exam-
ined the requirement of the phenolic hydroxy group on o-aminophenol.
To examine this phenomenon using a model compound, compound 9
was synthesized, and the reactivity was monitored by NMR and HPLC
(Figure 1c, S6). As expected, compound 9 showed no change in the 1H
NMR spectrum in CD3OD/D2O (4:1) (Figure S6a). Quantitative analyses
by HPLC confirmed that compound 9 is too stable to yield Michael
acceptor 6 both in MeOH and MeOH/H2O (4:1) (Figure S6b).
C
H
H
and δC 49.00 for CD3OD. Chemical shifts (δ) are shown in parHts per
million (ppm) and coupling constants (J) are in hertz (Hz). High-
resolution mass spectra were recorded on an ESI-ITTOF-MS (Shi-
madzu) or a FAB-MS JEOL JMS-700 double-focusing mass spectrometer
(JEOL).
2.5. Plausible mechanism of retro-Michael reaction
We found that compound 9 neither released the Michael acceptor nor
showed deuterium exchange in protic solvents. This indicated that the
phenolic alcohol is required not only for the keto-enol equilibrium but
also for the elimination reaction. Enol formation can be accomplished by
forming the zwitterion, as shown in Figure 5. The zwitterion is more
stable in protic solvents than in non-protic solvents, which is consistent
with the retro-Michael reaction occurring protic solvents. The enol form
of compound 3 undergoes deuteration or elimination. The proposed
elimination mechanism of o-aminophenol is similar to that of the E1cB
reaction by which a Mannich base can be converted to an enone.19 In
this case, however, alkylation of the amine to form a quaternary amine
and a base to start the deamination reaction are required. Alternatively,
4.2. Synthesis of the compounds
4.2.1. Dodec-1-en-3-one (6)
To a stirred solution of decanal (0.83 g, 5.3 mmol) in THF (10.6 mL)
was added vinylmagnesium bromide (1.0 M in THF, 6.4 mL, 6.4 mmol)
at 0 ◦C. After being stirred for 1 h, the mixture was warmed up to room
temperature and stirred for an additional five days. The reaction was
quenched with 3 M HCl aqueous solution and the layers were separated.
The aqueous layer was extracted six times with Et2O, and the combined
organic layers were washed with sat aq NaHCO3, dried over Na2SO4, and
evaporated. The residue was subjected to silica gel column chromatog-
raphy (n-hexane/EtOAc) to afford a fraction containing 1-dodecen-3-ol
Fig. 5. Proposed mechanism of the retro-Michael reaction. Compound 3 (form A) exists as a zwitterion (form B), which may promote enol formation (form C to E and
F). Generation of form E and F can be followed by deuteration or elimination of aminophenol.
4

K. Takenaka et al.
Bioorganic & Medicinal Chemistry 35 (2021) 116059
(1.17 g). To a stirred solution of oxalyl chloride (0.75 mL, 9.5 mmol) in
CH2Cl2 (34 mL) was added DMSO (0.90 mL, 12.7 mmol) at ꢀ 78 ◦C. The
resulting solution was stirred at ꢀ 78 ◦C for 30 min, and then a solution
containing dodec-1-en-3-ol (1.17 g in 2.5 mL CH2Cl2) was added. After
being stirred at ꢀ 78 ◦C for 45 min, triethylamine (4.4 mL, 31.8 mmol)
was added to the mixture. After 10 min the reaction mixture was
warmed up to room temperature. The reaction mixture was diluted with
CH2Cl2, washed with water and brine, dried over Na2SO4, and evapo-
rated. The residue was purified by silica gel open column chromatog-
raphy (n-hexane/EtOAc) to afford compound 6 as a colorless oil (557.9
mg, 58% over two steps): 1H NMR (CD3OD, 500 MHz) δ 6.38 (dd, J =
17.8, 10.3 Hz, 1H), 6.27 (dd, J = 17.8, 1.3 Hz, 1H), 5.88 (dd, J = 10.3,
1.3 Hz, 1H), 2.63 (t, J = 7.5 Hz, 2H), 1.54–1.64 (m, 2H), 1.36–1.24
(overlapped, 12H), 0.90 (t, J = 7.0 Hz, 3H); 13C NMR (CD3OD, 125 MHz)
δ 203.4, 137.7, 129.2, 40.3, 33.1, 30.61, 30,59, 30.4, 30.3, 25.2, 23.7,
14.4; HR-MS (FAB) [M + H]+ m/z 183.1746 (calcd. for C12H23O,
183.1749).
= 6.8 Hz, 2H); 13C NMR (CD3OD, 125 MHz) δ 174.4, 146.3, 139.8,
138.0, 129.5 (2C), 128.5 (2C), 128.1, 121.2, 118.7, 114.7, 112.6, 44.1,
41.5, 36.7 ; HR-MS (ESI) [M + H]+ m/z 271.1440 (calcd. for
C16H19N2O2, 271.1441).
4.2.6. 1-((2-Methoxyphenyl) amino) dodecan-3-one (9)
Compound 6 (10.4 mg, 0.057 mmol) and o-anisidine (35.0 mg, 0.28
mmol) were dissolved in CH3CN (57.0 μL) and stirred at room temper-
ature overnight. The mixture was purified by silica gel column chro-
matography (n-hexane/EtOAc) to afford compound 9 as a white solid
(10.0 mg, 57%): 1H NMR (CD3OD, 500 MHz) δ 6.84–6.79 (m, 2H),
6.69–6.63 (m, 2H), 3.81 (s, 3H), 3.38 (t, J = 6.8 Hz, 2H), 2.76 (t, J = 6.3
Hz, 2H), 2.45 (t, J = 7.3 Hz, 2H), 1.57–1.50 (m, 2H), 1.35–1.21 (over-
lapped, 12H), 0.90 (t, J = 7.0 Hz, 3H); 13C NMR (CD3OD, 125 MHz) δ
212.9, 149.2, 138.3, 122.3, 119.1, 112.5, 111.0, 56.0, 43.8, 42.3, 40.0,
33.1, 30.60, 30.58, 30.4, 30.2, 24.8, 23.7, 14.5; HR-MS (ESI) [M + H]+
m/z 306.2435 (calcd. for C19H32NO2, 306.2428).
4.2.2. 1-((2-Hydroxyphenyl) amino) dodecan-3-one (3)
4.2.7. Monitoring of the conversion of o-aminophenol Michael adduct
Compound 6 (10.4 mg, 0.057 mmol) and o-aminophenol (31.1 mg,
Compounds (3, 4, 5, and 9) (1.0 mM) and caffeine (100 μM) were
0.28 mmol) were dissolved in CH3CN (57.0
μL) and stirred at room
dissolved in MeOH, MeOH/milliQ water (4:1), MeOH/0.1 M NaOAc aq.
(4:1) or MeOH/50 mM Tris-HCl aq. (4:1) and incubated at 37 ◦C. A
portion of the reaction mixture and the authentic samples were analyzed
by HPLC. HPLC conditions for compounds 3 and 9: Cosmosil MS II Φ 4.6
× 250 mm, 0.8 mL/min, 5–100% aq MeOH; HPLC conditions for com-
pounds 4 and 5: Cosmosil PBr Ф 4.6 × 250 mm, 0.8 mL/min, 5–100% aq
MeOH. Chromatograms observed at 210 nm (3–9, o-aminophenol, o-
anisidine, and caffeine) were used for measuring the amount of the
compounds.
temperature for 17 h. The mixture was subjected to silica gel column
chromatography (n-hexane/EtOAc) to afford compound 3 as an orange
solid (11.2 mg, 67%): 1H NMR (CDCl3, 500 MHz) δ 6.87–6.80 (m, 1H),
6.78–6.66 (overlapped, 3H), 3.39 (t, J = 6.0 Hz, 2H), 2.73 (t, J = 6.0 Hz,
2H), 2.42 (t, J = 7.5 Hz, 2H), 1.62–1.52 (m, 2H), 1.34–1.18 (overlapped,
12H), 0.88 (t, J = 6.8 Hz, 3H); 13C NMR (CDCl3, 125 MHz) δ 211.1,
145.3, 136.2, 121.4, 119.3, 114.8, 114.2, 43.5, 42.0, 39.8, 32.0, 29.6,
29.5, 29.4, 29.3, 23.9, 22.8, 14.3; HR-MS (ESI) [M + H]+ m/z 292.2278
(calcd. for C18H30NO2, 292.2271).
4.2.8. 1H NMR-based monitoring
4.2.3. Benzyl 3-((2-hydroxyphenyl) amino) propanoate (4)
Compounds 3 or 9 (3.7–4.6 mM) were dissolved into CD3OD or
CD3OD/D2O (4:1). The mixtures in NMR tubes were incubated at 37 ◦C
sealing with parafilm. The chemical shifts of 1H NMR were recorded at 1
day and 2 days after the incubation.
Benzyl acrylate (7) (210.8 mg, 1.3 mmol), o-aminophenol (709.3 mg,
6.5 mmol) and silica gel (700.8 mg) were dissolved in CH3CN (1.3 mL)
and stirred at 86 ◦C for 15.5 h. The mixture was subjected to silica gel
column chromatography (n-hexane/EtOAc) to afford compound 4 as a
dark brown oil (202.5 mg, 57%): 1H NMR (CD3OD, 500 MHz) δ
7.37–7.28 (overlapped, 5H), 6.73–6.62 (overlapped, 3H), 6.57–6.51 (m,
1H), 5.14 (s, 2H), 3.44 (t, J = 6.5 Hz, 2H), 2.67 (t, J = 6.3 Hz, 2H); 13C
NMR (CD3OD, 125 MHz) δ 173.9, 146.4, 137.8, 137.5, 129.5 (2C),
129.18 (2C), 129.16, 121.2, 118.9, 114.8, 112.7, 67.4, 40.8, 35.0; HR-
MS (ESI) [M + H]+ m/z 272.1283 (calcd. for C16H18NO3, 272.1281).
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgements
4.2.4. N-benzylacrylamide (8)
To a stirred solution of benzylamine (0.22 mL, 2.0 mmol) in CH2Cl2
(5 mL) were added Et3N (0.31 mL, 2.2 mmol) and acryloyl chloride
(0.18 mL, 2.2 mmol) at 0 ◦C. After being stirred at room temperature for
1 h, the reaction was quenched with sat. NaHCO3 and extracted with
CH2Cl2 three times. The combined organic layers were dried over
Na2SO4, and concentrated in vacuo. The residue was purified by silica gel
column chromatography (n-hexane/EtOAc) to afford N-benzylacryla-
mide (8) as a white solid (221.1 mg, 69%): 1H NMR (CDCl3, 500 MHz) δ
7.37–7.26 (overlapped, 5H), 6.33 (dd, J = 17.0, 1.5 Hz, 1H), 6.11 (dd, J
= 17.0, 10.5 Hz, 1H), 5.88 (br. s, 1H), 5.67 (dd, J = 10.3, 1.8 Hz, 1H),
4.52 (d, J = 6.0 Hz, 2H); 13C NMR (CDCl3, 125 MHz) δ 165.5, 138.1,
130.7, 128.9 (2C), 128.1 (2C), 127.8, 127.0, 43.9 ; HR-MS (ESI) [M +
H]+ m/z 162.0916 (calcd. for C10H12NO, 162.0913).
We thank Drs. Shan Lu and Takefumi Kuranaga for helpful discus-
sions. This work was supported in part by a Grant-in-Aid for Scientific
Research from the Ministry of Education, Culture, Sports, Science and
Technology, Japan (17H06401 (HK and SN), 19H02840 (HK)).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bmc.2021.116059.
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