Synthesis and evaluation of paramagnetic caffeic acid phenethyl ester (CAPE) analogs

Article abstract of DOI:10.1007/s00706-019-02458-8

Abstract: The structure–activity relationships of newly prepared caffeates, containing nitroxide moieties as potential antioxidants have been investigated and compared to those of known natural (caffeic acid phenethyl ester, CAPE) and unnatural (N-acetylcysteine, paramagnetic alcohols) antioxidants. The in vitro antioxidant activity was tested by 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) radical scavenging, while assays of cell protection against reactive oxygen species were carried out in the presence of 2′,7′-dichlorofluorescein diacetate and H₂O₂. Paramagnetic esters without phenol motifs exhibited the lowest antioxidant activity, followed by paramagnetic alcohols with moderate activity. Among the compounds investigated, paramagnetic phenolic compounds were the best antioxidants. As the new paramagnetic CAPE analogs were less cytotoxic than CAPE at 10?μM in NIH3T3 fibroblast cells but had similar antioxidant activity, they can be considered promising antioxidants. Graphic abstract: [Figure not available: see fulltext.].

Full text of DOI:10.1007/s00706-019-02458-8

Monatshefte für Chemie - Chemical Monthly
https://doi.org/10.1007/s00706-019-02458-8
ORIGINAL PAPER
Synthesis and evaluation of paramagnetic cafeic acid phenethyl ester
(CAPE) analogs
Masaki Nagane2 · Tadashi Yamashita2 · Patrik Vörös1 · Tamás Kálai1,3 · Kálmán Hideg1 · Balázs Bognár1
Received: 16 March 2019 / Accepted: 12 June 2019
© The Author(s) 2019
Abstract
The structure–activity relationships of newly prepared cafeates, containing nitroxide moieties as potential antioxidants have
been investigated and compared to those of known natural (cafeic acid phenethyl ester, CAPE) and unnatural (N-acetyl-
cysteine, paramagnetic alcohols) antioxidants. The in vitro antioxidant activity was tested by 2,2′-azinobis(3-ethylbenzothia-
zoline-6-sulfonic acid) radical scavenging, while assays of cell protection against reactive oxygen species were carried out in
the presence of 2′,7′-dichlorofuorescein diacetate and H2O2. Paramagnetic esters without phenol motifs exhibited the lowest
antioxidant activity, followed by paramagnetic alcohols with moderate activity. Among the compounds investigated, para-
magnetic phenolic compounds were the best antioxidants. As the new paramagnetic CAPE analogs were less cytotoxic than
CAPE at 10 µM in NIH3T3 fbroblast cells but had similar antioxidant activity, they can be considered promising antioxidants.
Graphic abstract
Keywords Heterocycles · Radicals · Structure–activity relationships · Nitroxides · Antioxidant
Introduction
possesses many biological activities, such as antioxidant
[3–5], anti-infammatory [6, 7], immunomodulatory [8],
antiviral, and neuroprotective activities (Fig. 1) [9, 10].
The phenylpropanoid scafold of cafeic acid (3,4-dihy-
droxycinnamic acid) is commonly used as a template to
develop cafeic acid derivatives and new chemical entities
possessing antioxidant properties. Wang et al. investigated
the relationship between catechol ring modifcations (fuori-
nation, altered numbers of hydroxyl groups) and the cytopro-
tective, cytotoxic, and antioxidant properties of CAPE [11,
12]. Their results showed that at least one hydroxyl group
is important for cytoprotection and that the catechol ring is
required for antioxidant activity. CAPE inhibits the HIV-1
integrase enzyme, and this activity was enhanced with the
insertion of a third hydroxyl group into the catechol ring
[13]. The modifcation of the phenethyl side also may be
benefcial, as cafeic acid 4-aminophenethyl ester showed
Cafeic acid phenethyl ester [2-phenylethyl (2E)-3-(3,4-dihy-
droxyphenyl)acrylate; CAPE, 1] is a phenolic compound
isolated from the propolis of honeybee hives [1]. Although
CAPE is hydrolyzed to cafeic acid in vivo, it has a better
pharmacokinetic profle than that of cafeic acid [2]. CAPE
* Balázs Bognár
balazs.bognar@aok.pte.hu; bognar.balazs83@gmail.com
1
Institute of Organic and Medicinal Chemistry, University
of Pécs, Szigeti st. 12, Pécs 7624, Hungary
2
Laboratory of Biochemistry, School of Veterinary Medicine,
Azabu University, Sagamihara 252-5201, Japan
3
Szentágothai Research Centre, Ifúság st. 20, 7624 Pécs,
Hungary
Vol.:(0123456789)
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M. Nagane et al.
alcohols 3-(hydroxymethyl)-2,2,5,5-tetramethyl-2,5-dihy-
dro-1H-pyrrol-1-oxyl (2), 4-(hydroxymethyl)-2,2,6,6-tetra-
methyl-5,6-dihydropyridin-1(2H)-oxyl (3) (Fig. 2) were also
evaluated [31, 32]. To obtain new paramagnetic analogs of
CAPE, three strategies were followed: (1) replacement of the
catechol ring with nitroxides, (2) replacement of the phene-
thyl group with nitroxide rings, and (3) incorporation the
pyrroline radical in the cafeic acid scafold.
Fig. 1 Covalent structure of cafeic acid phenethyl ester (CAPE)
Results and discussion
The Horner–Wadsworth–Emmons reaction of paramagnetic
aldehydes 1-oxyl-2,2,5,5-tetramethyl-2,5-dihydro-1H-pyr-
role-3-carbaldehyde (4) [31] with triethyl phosphonoacetate
and 1-oxyl-2,2,6,6-tetramethyl-1,2,3,6-tetrahydropyridine-
4-carbaldehyde (5) [32] with trimethyl phosphonoacetate
led to the formation of paramagnetic α,β-unsaturated
esters ethyl (E)-3-(1-oxyl-2,2,5,5-tetramethyl-2,5-dihydro-
1H-pyrrol-3-yl)acrylate (6) [33] and methyl (E)-3-(1-oxyl-
2,2,6,6-tetramethyl-1,2,3,6-tetrahydropyridin-4-yl)acrylate
(7), respectively. Their hydrolysis was conducted at room
temperature for 2 h with a 10% aq. NaOH/methanol solu-
tion (2.0 equiv. NaOH) to yield, after acidifcation with aq.
H2SO4, carboxylic acids (E)-3-(1-oxyl-2,2,5,5-tetramethyl-
2,5-dihydro-1H-pyrrol-3-yl)acrylic acid (8) [31] and (E)-
3-(1-oxyl-2,2,6,6-tetramethyl-1,2,3,6-tetrahydropyridin-
4-yl)acrylic acid (9), respectively. Then, the paramagnetic
carboxylic acids were treated with 2-phenethyl bromide in
dry acetonitrile in the presence of 1,8-diazabicyclo[5.4.0]-
undec-7-ene (DBU) to obtain compounds phenethyl (E)-
3-(1-oxyl-2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrol-3-yl)
acrylate (10) and phenethyl (E)-3-(1-oxyl-2,2,6,6-tetrame-
thyl-1,2,3,6-tetrahydropyridin-4-yl)acrylate (11), in which
the catechol ring of CAPE is replaced with dihydropyrrole-
or tetrahydropyridine-type nitroxides (Scheme 1).
Fig. 2 Structure of paramagnetic alcohols to be investigated
better cytoprotection and 4-nitrophenethyl ester showed
better cardioprotection, while the octyl ester showed better
antileukemic activity than CAPE itself [14–16].
The unpaired electron of nitroxide stable free radicals
allows them to take part in one-electron reactions such as
oxidations and reductions, which makes nitroxides potent
unnatural antioxidants [17]. Nitroxides may also function as
intra- and extracellular superoxide dismutase (SOD) mimics
or nonthiol radioprotectants [18–20]. Studies indicate that
as antioxidants, nitroxides can exert cytoprotective action
against oxidative stress induced by cytotoxic drugs or patho-
logical processes such as ischemia–reperfusion and infam-
mation [21–23]. However, some piperidine-type nitroxides
also exert pro-oxidative and cytotoxic efects, in particular
on cancer cells [24, 25].
Several studies indicated that biomolecules (resveratrol,
curcumin) modifed with nitroxides had a benefcial infu-
ence on their activity as supplemented by ‘in statu nascendi
acting’ antioxidants and radical scavengers [26–30]. Herein,
we report the synthesis, antioxidant and cytoprotective activ-
ity of new CAPE analogs compared with those of the par-
ent compound (CAPE) and regular antioxidant standards,
such as N-acetylcysteine and Trolox. The antioxidant and
cell protection activity of paramagnetic building block
For the replacement of the phenethyl moiety of CAPE,
the frst step was to alkylate (E)-3-(3,4-diacetoxyphenyl)
acrylic acid (12) [34] with paramagnetic allylic bro-
mides 3-(bromomethyl)-2,2,5,5-tetramethyl-2,5-dihy-
dro-1H-pyrrol-1-yloxy (13) [35] and 4-(bromomethyl)-
2,2,6,6-tetramethyl-5,6-dihydropyridin-1-yloxy (14) [36].
Scheme 1
1 3

Synthesis and evaluation of paramagnetic cafeic acid phenethyl ester (CAPE) analogs
The alkylation was conducted in CH3CN in the presence
of DBU base at ambient temperature to give compounds
(1-oxyl-2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrole-3-yl)-
methyl (E)-3-(3,4-diacetoxyphenyl)acrylate (15) and
(1-oxyl-2,2,6,6-tetramethyl-1,2,3,6-tetrahydropyridin-4-yl)-
methyl (E)-3-(3,4-diacetoxyphenyl)acrylate (16) in 60–71%
yield. To obtain paramagnetic cafeic acid esters (1-oxyl-
2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrole-3-yl)methyl
(E)-3-(3,4-dihydroxyphenyl)acrylate (17) and (1-oxyl-
2,2,6,6-tetramethyl-1,2,3,6-tetrahydropyridin-4-yl)methyl
(E)-3-(3,4-dihydroxyphenyl)acrylate (18), the acetyl protect-
ing groups were removed by the addition of three equiva-
lents K2CO3 in CH2Cl2/MeOH mixture (Scheme 2).
on the reduction of green-colored 2,2′-azinobis(3-ethyl-
benzothiazoline-6-sulfonic acid) radical (ABTS·+), which
is detected at 734 nm.
Our results suggest that the catechol functionality is nec-
essary for good antioxidant activity, as the TEAC values
of paramagnetic alcohols 2 and 3 nonphenolic esters 7, 10,
and 11; and acid 9 were below 0.2, while the cafeic acid
derivatives CAPE (1), 17, 18, and 23 showed good anti-
oxidant activity. Compounds 17 and 18 have even greater
proton- and electron-donating properties than Trolox and
CAPE. Nevertheless, in the case of compound 23, the vicin-
ity of nitroxide and catechol had adverse efect on antioxi-
dant activity (Fig. 3), which can be the consequence of an
intramolecular redox reaction between the nitroxide and
catechol rings [41].
To keep both catechol and phenethyl groups, the car-
bon–carbon double bond of CAPE seemed to be an accessi-
ble point for the synthesis of a new paramagnetic derivative.
4-Bromo-1-oxyl-2,2,5,5-tetramethyl-2,5-dihydro-1H-pyr-
role-3-carboxylic acid (19) [37] was alkylated with 2-phene-
thyl bromide to yield phenethyl 4-bromo-1-oxyl-2,2,5,5-
tetramethyl-2,5-dihydro-1H-pyrrol-3-carboxylate (20). The
Suzuki reaction of 20 and 2-[3,4-bis(methoxymethoxy)-
phenyl]-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (21) [38]
in the presence of Pd(PPh3)4 and Na2CO3 under a N2 atmos-
phere afforded phenethyl 4-[3,4-bis(methoxymethoxy)-
phenyl]-1-oxyl-2,2,5,5-tetramethyl-2,5-dihydro-1H-pyr-
role-3-carboxylate (22) in moderate yield. The methoxy-
methyl protecting groups were removed by refuxing in
MeOH containing a catalytic amount of aq. HCl, resulting
in nitroxide phenethyl 4-(3,4-dihydroxyphenyl)-1-oxyl-
2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrole-3-carboxylate
(23) (Scheme 3).
Cytotoxicity of CAPEs was analyzed by the WST assay.
As shown in Fig. 4, 10 µM CAPE (1) decreased cellular
viability (0.63 0.09, relative to DMSO), and the ref-
erence compound N-acetylcysteine (NAC) increased it
(1.36 0.24). We did not observe any statistical signifcance
in the diference in cellular viability between the CAPE
analogs and DMSO; thus, these new compounds have less
cytotoxic activity than CAPE. Compounds 1, 17, 18, and 23
exerted some toxicity toward fbroblasts, while the paramag-
netic alcohols and nonphenolic esters were not toxic. This
result suggests the responsibility of the catechol ring for
cytotoxicity (Fig. 4), but this is still tolerable.
The intracellular oxidative stress within NIH3T3 cells was
evaluated by 2′,7′-dichlorofuorescein diacetate (DCFDA)
[42, 43], which is taken up by the cells, wherein nonspecifc
esterases cleave the acyl groups, forming 2′,7′-dichlorodi-
hydrofuorescein (H2DCF), which can be oxidized to fuo-
rescent DCF by reactive oxygen species (ROS). Therefore,
the limited formation of DCF (and fuorescence intensity)
The new nitroxides were tested in a Trolox-equivalent
antioxidant capacity assay (TEAC), which monitors electron
and proton donor activity [39, 40]. The method is based
Scheme 2
1 3

M. Nagane et al.
Scheme 3
Fig. 3 Trolox equivalent
antioxidant capacity (TEAC)
presents the antioxidant activ-
ity of nitroxides measured by
ABTS radical scavenging assay.
Data represent the mean SD
(n=6)
indicates the enhanced ROS scavenging activity of the inves-
tigated compound. All of the studied compounds decreased
the ROS formation in cells: CAPE (1), 17, 18, and NAC
signifcantly decreased intracellular oxidative stress (Fig. 5).
This activity cannot be attributed to the presence of nitroxide
functionality, as compounds 2, 3, and 23 were not active, but
retaining the original cafeic acid ester scafold was advanta-
geous, as in the case of compounds 1, 17, and 18.
confrm the antioxidant properties of new cafeic acid phene-
thyl ester analogs, the cytoprotective efect of the paramag-
netic CAPEs against hydrogen peroxide (H2O2) was tested.
H2O2 treatment decreased cellular viability (0.41 0.08), but
CAPE (1), 17, and 23, containing cafeic acid ester scafold,
prevented H2O2-induced cytotoxicity. It is interesting to note
that compounds 7, 9, 10, and 11 did not exhibit antioxidant
protection in this assay, while alcohols 2, 3 and compound 18
produced a moderate protective efect (Fig. 6).
Reactive oxygen species are involved in the pathogenesis
of many diseases. The hydroxyl radical is the most dangerous
ROS and can be generated from H2O2 in the presence of transi-
tion metal ions including Fe2+ and Cu+ in certain processes. To
1 3

Synthesis and evaluation of paramagnetic cafeic acid phenethyl ester (CAPE) analogs
Fig. 4 Cytotoxicity of CAPEs were analyzed by WST assay. Cellular viability of NIH3T3 cells treated with nitroxides, CAPE or NAC (10 μM)
for 24 h. Data represent the mean SD (n=6). *Statistically signifcant (p<0.05) when compared to control
Fig. 5 Intracellular oxidative stress was evaluated by the fuorescence
intensity of 2′,7′-dichlorofuorescein. Cells were treated with 10 µM
each compound for 24 h. After treatment, cells were stained with
20 µM DCFDA for 30 min at 37 °C. Data represent the mean SD
(n=6). *Statistically signifcant (p<0.05) when compared to control
modifcations were the replacement of the catechol ring
or phenethyl group with 5- and 6-membered nitroxides
and the incorporation of pyrrolidine type of nitroxide into
the CC double bond of the acrylate. The toxicity and anti-
oxidant properties of the new compounds were compared
to those of CAPE (1) and regular antioxidant standards.
Conclusion
Three structural fragments of cafeic acid phenethyl ester
were modifed with nitroxide free radicals to study their
antioxidant structure–activity relationships. The main
1 3

M. Nagane et al.
Fig. 6 To determine the cytoprotective activity of CAPEs toward H2O2 were analyzed using WST assay. Cells were treated with H2O2 (500 µM)
with CAPEs (10 µM), respectively, for 24 h. Data represent the mean SD (n=6). *Statistically signifcant (p<0.05) when compared to control
Regarding the ABTS assay, paramagnetic nonphenolic
esters 7, 10, and 11 and alcohols 2 and 3 have poor ABTS·+
scavenging activity, which is not surprising because esters
can be electron donors but not proton donors. As expected,
compounds 1, 17, 18, and 23 were found to be good proton
and electron donor antioxidants.
Experimental
Elemental analyses were performed on a Fisons EA 1110
CHNS elemental analyzer, and the results were found to
be in good agreement ( 0.3%) with the calculated values.
Melting points were determined with a Boetius micro-
melting point apparatus. Mass spectra were recorded on a
Thermoquest Automass Multi in EI mode (70 eV). 1H NMR
spectra were recorded with Bruker Avance 3 Ascend 500 at
500 MHz and 13C NMR spectra at 125 MHz in CDCl3 or
DMSO-d6 at 298 K in the presence of fve equivalents of
hydrazobenzene (DPPH/radical). The IR spectra were taken
with a Bruker Alpha FT-IR instrument with ATR support on
a ZnSe plate. Spectroscopic measurements were performed
on Specord 40 UV/VIS Spectrophotometer at 732 nm. Flash
column chromatography was performed on Merck Kieselgel
60 (0.040–0.063 mm). Qualitative TLC was carried out on
commercially available plates (20 × 20 × 0.02 cm) coated
with Merck Kieselgel GF254. Compounds 1 [34], 2 [31], 3
[32], 4 [31], 5 [32], 12 [33], 13 [34], 14 [35], 19 [36], and
21 [37] were synthesized according to published procedures.
All other reagents were purchased from Aldrich or Molar
Chemicals.
The results of the cytotoxicity assays suggest that the cat-
echol ring motif increases cytotoxicity, but not signifcantly.
The most toxic compounds were CAPE (1) and compound
23, but when nitroxides were the esterifying groups, as in
the case of compounds 17 and 18, the toxicity of the result-
ing compound was lower than that of 1 and 23. In DCF
assays, compounds 1, 17, and 18 were the most active in
intracellular ROS scavenging and had the same or slightly
greater antioxidant efects than NAC. In cell viability assays
performed under H2O2-induced stress, compounds 1, 17, and
23 were the most protective molecules, as all showed better
activity than the standard (NAC). However, the results of
the assays of nitroxide derivatives were contradictory, as
compounds 17 and 18 with nitroxide and catechol structural
units were the best proton and electron donor antioxidants
and the most protective against H2O2-induced cytotoxicity.
These fndings support our hybrid drug design strategy
and are in good accordance with our previous fndings. The
cafeic acid moiety should remain intact, and proper esteri-
fcation, in our case with nitroxides, decreases its cytotoxic-
ity while simultaneously improving its protective activity
against H2O2−-induced oxidative stress. Our results can be
a good starting point in the search for new cafeic acid-based
lead compounds, which is in progress in our laboratories.
(E)‑Methyl 3‑(1‑oxyl‑2,2,6,6‑tetramethyl‑1,2,3,6‑tetrahy‑
dropyridin‑4‑yl)acrylate (7, C13H20NO3) To a stirred sus-
pension of 0.3 g NaH (12.5 mmol) in 10 cm3 THF 910 mg
trimethyl phosphonoacetate (5.0 mmol) was added at 0 °C
under a N2 atmosphere. After stirring for 10 min, 910 mg
aldehyde 5 (5.0 mmol) was added, and the mixture was
1 3

Synthesis and evaluation of paramagnetic cafeic acid phenethyl ester (CAPE) analogs
stirred and refuxed for another 30 min. After cooling to
room temperature, 20 cm3 brine and 20 cm3 ether were
added, the organic phase was separated, the aqueous phase
was washed with 2×20 cm3 ether, and then the combined
organic phase was dried (MgSO4), filtered, evaporated,
and the residue was purified by flash column chroma-
tography to give 655 mg (55%) 7 as a pale orange solid.
M.p.: 66–68 °C; Rf =0.55 (hexane–EtOAc, 2:1); 1H NMR
(500 MHz, CDCl3 +(PhNH)2): δ=7.38 (d, J=15.8 Hz, 1H),
5.93 (s, 1H), 5.86 (d, J=15.7 Hz, 1H), 3.82 (s, 3H), 2.26 (s,
2H), 1.35 (s, 6H), 1.26 (s, 6H) ppm; 13C NMR (125 MHz,
CDCl3 + (PhNH)2): δ = 167.7, 146.8, 144.3, 128.5, 116.1,
60.1, 57.2, 51.6, 38.9, 25.9, 25.1 ppm; IR (neat): ̄ꢀ =1712,
1642, 1624 cm−1; MS (70 eV): m/z (%)=238 (M+, 93), 208
(28), 193 (30), 149 (100).
(t, J=7.1 Hz, 2H), 1.41 (s, 6H), 1.32 (s, 6H) ppm; 13C NMR
(125 MHz, CDCl3 + (PhNH)2): δ = 166.9, 141.2, 140.1,
138.6, 137.9, 129.4, 128.6, 126.7, 119.9, 69.7, 67.7, 65.1,
35.3, 25.5, 24.9 ppm; IR (neat): ̄ꢀ =1698, 1603, 1455 cm−1;
MS (70 eV): m/z (%)=314 (M+, 6), 299 (5), 284 (18), 105
(100), 91 (44).
Phenethyl (E)‑3‑(1‑oxyl‑2,2,6,6‑tetramethyl‑1,2,3,6‑tetrahy‑
dropyridin‑4‑yl)acrylate (11, C20H26NO3) Orange solid; yield
215 mg (65%); m.p.: 60–61 °C; Rf =0.60 (hexane–EtOAc,
1
2:1); H NMR (500 MHz, CDCl3 + (PhNH)2): δ = 7.37
(d, J = 6.6 Hz, 2H), 7.27 (t, J= 7.3 Hz, 3H), 5.91 (s, 1H),
5.83 (d, J=15.8 Hz, 1H), 4.44 (t, J=7.1 Hz, 2H), 3.04 (t,
J=7.1 Hz, 2H), 2.24 (s, 2H), 1.34 (s, 6H), 1.25 (s, 6H) ppm;
13C NMR (125 MHz, CDCl3 +(PhNH)2): δ=167.2, 146.8,
144.3, 137.9, 129.4, 128.55, 128.53, 126.6, 116.3, 64.9,
60.1, 57.2, 38.9, 35.3, 26.1, 25.0 ppm; IR (neat): ̄ꢀ =1705,
1636, 1617 cm−1; MS (70 eV): m/z (%)=328 (M+, 2), 179
(5), 149 (31), 105 (100).
(E)‑3‑(1‑Oxyl‑2,2,6,6‑tetramethyl‑1,2,3,6‑tetrahydropyri‑
din‑4‑yl)acrylic acid (9, C12H18NO3) To a solution of 1.19 g
7 (5.0 mmol) in 30 cm3 MeOH, 4 cm3 aq. 10% NaOH
(10.0 mmol) was added, and the mixture stirred for 2 h at
ambient temperature. The mixture was diluted with 10 cm3
water, the methanol was evaporated of, and the residue pH
was adjusted to 2 at 0 °C by adding aq. 5% H2SO4 solution.
The acid formed was extracted with 2×2 cm3 CHCl3, and
the combined organic phase was dried (MgSO4), fltered,
and evaporated. The residue was purifed by fash column
chromatography (hexane–EtOAc, 2:1) to give 870 mg (78%)
compound 9 as a yellow solid. M.p.: 156–158 °C; Rf =0.25
(CHCl3–Et2O, 2:1); 1H NMR (500 MHz, CDCl3 +(PhNH)2):
δ=7.25 (d, J=16 Hz, 1H), 6.01 (d, J=15.7 Hz, 1H), 5.84
(s, 1H), 2.59 (s, 2H), 1.49 (s, 6H), 1.42 (s, 6H) ppm; 13C
NMR (125 MHz, CDCl3 + (PhNH)2): δ = 173.6, 142.1,
136.5, 125.5, 123.6, 64.4, 62.3 36.5, 23.9 (brs, 4C) ppm; IR
(neat): ̄ꢀ =3250–2500, 1671, 1613 cm−1; MS (70 eV): m/z
(%)=224 (M+, 76), 194 (32), 179 (72), 149 (100).
Phenethyl 4‑bromo‑1‑oxyl‑2,2,5,5‑tetramethyl‑2,5‑dihy‑
dro‑1H‑pyrrol‑3‑carboxylate (20, C17H21BrNO3) Yellow
solid; yield 256 mg (70%); m.p.: 77–79 °C; Rf =0.71 (hex-
ane–EtOAc, 2:1); 1H NMR (500 MHz, CDCl3 +(PhNH)2):
δ=7.32 (t, J=7.4 Hz, 2H), 4.46 (t, J=7.1 Hz, 2H), 3.04 (t,
J=7.1 Hz, 2H), 1.34 (s, 6H), 1.32 (s, 6H) ppm; 13C NMR
(125 MHz, CDCl3 + (PhNH)2): δ = 163.1, 137.6, 136.9,
134.3, 129.4, 128.9, 128.6, 126.7, 72.0, 70.6, 65.3, 35.0,
24.5, 24.4 ppm; IR (neat): ̄ꢀ =1704, 1624, 1592 cm−1; MS
(70 eV): m/z (%)=368 (M+, 15), 262 (5), 232 (6), 105 (100),
91 (83).
General procedure for the synthesis of protected,
paramagnetic cafeic acids 15, 16
Cafeic acid diacetate 12 (2.64 g, 10.0 mmol), 1.67 g DBU
(11.0 mmol), and 2.33 g 13 or 2.47 g 14 (10.0 mmol) were
dissolved in 15 cm3 CH3CN and the mixture was allowed to
react for 24 h at ambient temperature. The solvent was then
evaporated, and the residue was purifed by fash column
chromatography (hexane–EtOAc, 4:1) to give the title com-
pounds in 60–71% yield.
General procedure for the synthesis of phenethyl
esters 10, 11, 20
To the solution of 212 mg 8 or 224 mg 9 or 263 mg 19
(1.0 mmol) in 5 cm3 CH3CN, 167 mg DBU (1.1 mmol), and
277 mg (2-bromoethyl)benzene (1.1 mmol) were added, and
the mixture was allowed to react for 24 h at room tempera-
ture. Then, the solvent was evaporated, and the residue was
purifed by fash column chromatography (hexane–EtOAc,
4:1) to give the title compounds in 65–70% yield.
(1‑Ox yl‑2,2,5,5‑tetramethyl‑2,5‑ dihydro‑1H‑pyr‑
role‑3‑yl)methyl (E)‑3‑(3,4‑diacetoxyphenyl)acrylate (15,
C22H26NO7) Pale yellow solid; yield 2.95 g (71%); m.p.:
132–135 °C; Rf = 0.59 (CHCl3-Et2O, 2:1); 1H NMR
(500 MHz, CDCl3 +(PhNH)2): δ=7.73 (d, J=16 Hz, 1H),
7.48–7.45 (m, 2H), 6.48 (d, J= 16 Hz, 1H), 5.69 (s, 1H),
4.81 (s, 2H), 2.37 (2 s, 6H), 1.37 (s, 6H), 1.33 (s, 6H) ppm;
13C NMR (125 MHz, CDCl3 +(PhNH)2): δ=168.0, 167.9,
166.2, 143.7, 143.3, 142.6, 139.3, 133.2, 132.4, 126.5,
123.9, 122.8, 119.9, 69.9, 67.8, 60.8, 25.6, 24.6, 20.6 ppm;
Phenethyl (E)‑3‑(1‑oxyl‑2,2,5,5‑tetramethyl‑2,5‑dihy‑
dro‑1H‑pyrrol‑3‑yl)acrylate (10, C19H24NO3) Dark yellow
solid; yield 233 mg (70%); m.p.: 61–63 °C; Rf =0.59 (hex-
ane–EtOAc, 2:1); 1H NMR (500 MHz, CDCl3 +(PhNH)2):
δ = 7.37 (t, J = 6.9 Hz, 2H), 7.26–7.22 (m, 3H), 6.15 (d,
J=16.3 Hz, 1H), 5.99 (s, 1H), 4.46 (t, J=7.1 Hz, 2H), 3.05
1 3

M. Nagane et al.
IR (neat): ̄ꢀ = 1766, 1713, 1634 cm−1; MS (70 eV): m/z
114.4, 67.1, 61.5, 59.5, 39.7, 25.6, 24.5 ppm; IR (neat):
̄ꢀ =3500–2800, 1694, 1597 cm−1; MS (70 eV): m/z (%)=346
(M+, 4), 307 (5), 234 (26), 163 (100).
(%)=416 (M+, 10), 317 (2), 205 (16), 105 (75), 43 (100).
(1‑Oxyl‑2,2,6,6‑tetramethyl‑1,2,3,6‑tetrahydropyri‑
din‑4‑yl)methyl (E)‑3‑(3,4‑diacetoxyphenyl)acrylate
(16, C23H28NO7) Orange solid; yield 2.58 g (60%);
Phenethyl 4‑[3,4‑bis(methox ymethox y)phenyl]‑
1‑oxyl‑2,2,5,5‑tetramethyl‑2,5‑dihydro‑1H‑pyrrole‑3‑
carboxylate (22, C27H34NO7) To a deoxygenated solution of
545 mg 20 (1.5 mmol) in 20 cm3 dioxane/water (4:1), 75 mg
Pd(PPh3)4 (0.067 mmol), 490 mg pinacolate 21 (1.5 mmol),
and 5 cm3 10% Na2CO3 solution were added under N2 gas,
and then the reaction mixture was stirred and refuxed for
3 h. After consumption of the starting materials (followed by
TLC), dioxane was removed by vacuum evaporation, 10 cm3
H2O was added, and the aqueous phase was extracted with
2×20 cm3 CHCl3. The combined organic phase was dried
(MgSO4), fltered, and evaporated. The residue was puri-
fed by fash column chromatography (hexane–EtOAc, 2:1),
to ofer 280 mg (58%) 22 as a brown oil. Rf = 0.38 (hex-
ane–EtOAc 2:1); 1H NMR (500 MHz, CDCl3 +(PhNH)2):
δ = 7.33 (t, J = 7.5 Hz, 2H), 7.21 (d, J = 8.3 Hz, 1H), 7.14
(d, J=7.2 Hz, 2H), 7.07 (s, 1H), 6.82 (d, J=6.4 Hz, 1H),
5.30 (2 s, 4H), 4.24 (t, J=7.2 Hz, 2H), 3.58 (d, J=3.3 Hz,
6H), 2.71 (t, J=7.2 Hz, 2H), 1.54 (s, 6H), 1.35 (s, 6H) ppm;
13C NMR (125 MHz, CDCl3 +(PhNH)2): δ=164.7, 154.4,
147.1, 146.6, 137.8, 133.3, 129.9, 128.8, 128.5, 126.5,
121.2, 117.4, 116.1, 95.8, 95.5, 71.4, 69.2, 64.6, 56.28, 34.7,
24.8, 24.3 ppm; IR (neat): ̄ꢀ =1707, 1602, 1506 cm−1; MS
(70 eV): m/z (%)=484 (M+, 6), 470 (1), 274 (30), 105 (100).
1
m.p.: 57–59 °C; Rf = 0.64 (CHCl3–Et2O, 2:1); H NMR
(500 MHz, CDCl3 + (PhNH)2): δ = 7.72 (d, J = 15.9 Hz,
1H), 7.46–7.43 (m, 2H), 6.48 (d, J=15.9 Hz, 1H), 5.61 (s,
1H), 4.65 (s, 2H), 2.34 (2 s, 6H), 2.18 (s, 2H), 1.34 (s, 6H),
1.28 (s, 6H) ppm; 13C NMR (125 MHz, CDCl3 +(PhNH)2):
δ=168.1, 167.9, 166.4, 143.6, 143.1, 142.5, 133.5, 133.3,
126.6, 126.5, 123.9, 122.8, 119.2, 67.7, 59.5, 57.4, 40.8,
26.17, 25.12 ppm; 20.66, 20.63 IR (neat): ꢀ̄ =1765, 1709,
1503 cm−1; MS (70 eV): m/z (%)=430 (M+, 2), 247 (9), 205
(36), 163 (38), 121 (79), 43 (100).
General procedure for the synthesis
of paramagnetic cafeic acids 17, 18
15 (2.08 g) or 16 (2.15 g) (5.0 mmol) was dissolved in
20 cm3 MeOH–CH2Cl2 (1:1), 2.07 g K2CO3 (15.0 mmol)
was added, and the mixture was stirred at 20 °C for 1 h.
The solvent was evaporated, and the residue was parti-
tioned between 10 cm3 5% H2SO4 and 20 cm3 EtOAc. After
separation, the water phase was extracted with additional
2×20 cm3 EtOAc. The combined organic phases were dried
over MgSO4, fltered, and evaporated, and the residue was
purifed by column chromatography (hexane–EtOAc) to
obtain paramagnetic cafeic acids in 67–72% yield.
Phenethyl 4‑(3,4‑dihydroxyphenyl)‑1‑oxyl‑2,2,5,5‑
tetramethyl‑2,5‑dihydro‑1H‑pyrrole‑3‑carboxylate (23,
C23H26NO5) 484 mg 22 (1.0 mmol) was dissolved in 10 cm3
MeOH, then concentrated 0.1 cm3 aq. HCl was added,
and the mixture was refuxed for 10 min. After removal
of the solvent, 10 cm3 water was added, and the residue
was neutralized with K2CO3. EtOAc (20 cm3) was added,
the phases were separated, and the aqueous phase was
extracted with 2 × 20 cm3 EtOAc. The combined organic
phase was dried (MgSO4), fltered, evaporated, and puri-
fed by fash chromatography (hexane–EtOAc 2:1) to give
310 mg (78%) title compound 23 as a brown oil. Rf =0.57
(CHCl3-Et2O, 2:1); 1H NMR (500 MHz, CDCl3 +(PhNH)2):
δ=7.09 (d, J=7.3 Hz, 2H), 6.69 (s, 1H), 6.61 (s, 1H), 4.21
(t, J=6.8 Hz, 2H), 2.69 (t, J=6.7 Hz, 2H), 1.51 (s, 6H), 1.31
(2 s, 6H) ppm; 13C NMR (125 MHz, CDCl3 + (PhNH)2):
δ=164.3, 154.7, 144.2, 143.2, 137.6, 132.7, 128.8, 128.5,
127.3, 126.5, 119.5, 115.7, 114.9, 71.7, 69.7, 64.8, 34.7,
24.8, 23.8 ppm; IR (neat): ꢀ̄ = 3600–2850, 1703, 1601,
1515 cm−1; MS (70 eV): m/z (%)=396 (M+, 49), 381 (5),
366 (6), 105 (100).
(1‑Oxyl‑2,2,5,5‑tetramethyl‑2,5‑dihydro‑1H‑pyrrole‑3‑yl)‑
methyl (E)‑3‑(3,4‑dihydroxyphenyl)acrylate (17,
C18H22NO5) Yellow solid; yield 1.19 g (72%); m.p.: 123–
125 °C; Rf =0.36 (CHCl3-Et2O, 2:1); 1H NMR (500 MHz,
DMSO-d6 +(PhNH)2): δ=7.03 (d, J=5.1 Hz, 1H), 6.81 (d,
J=6.2 Hz, 1H), 6.6 (s, 1H), 6.34 (d, J=15.3 Hz, 1H), 5.60
(s, 1H), 5.83 (d, J = 15.8 Hz, 1H), 4.69 (s, J = 7 Hz, 2H),
1.16, 1.12 (2 s, 12H) ppm; 13C NMR (125 MHz, DMSO-
d6 +(PhNH)2): δ=166.6, 150.4, 146.1, 145.9, 140.3, 125.9,
121.9, 118.2, 116.3, 115.4, 114.2, 68.9, 66.9, 60.4, 26.1,
25.0 ppm; IR (neat): ̄ꢀ =3480–2700, 1717, 1603 cm−1; MS
(70 eV): m/z (%)=332 (M+, 3), 317 (1), 182 (72), 107 (100).
(1‑Oxyl‑2,2,6,6‑tetramethyl‑1,2,3,6‑tetrahydropyri‑
din‑4‑yl)methyl (E)‑3‑(3,4‑dihydroxyphenyl)acrylate (18,
C19H24NO5) Orange-brownish solid; yield 1.16 g (67%);
1
m.p.: 74–76 °C; Rf = 0.37 (CHCl3-Et2O, 2:1); H NMR
(500 MHz, CDCl3 + (PhNH)2): δ = 7.12 (s, 1H), 7.1 (d,
J = 7.5 Hz, 1H), 6.28 (d, J = 15.8 Hz, 1H), 5.63 (s, 1H),
4.61 (s, 2H), 1.73 (s, 2H), 1.40 (s, 6H), 1.32 (s, 6H) ppm;
13C NMR (125 MHz, CDCl3 +(PhNH)2): δ=167.4, 147.3,
145.8, 144.7, 131.5, 127.3, 127.1, 122.5, 115.6, 114.7,
1 3

Synthesis and evaluation of paramagnetic cafeic acid phenethyl ester (CAPE) analogs
30 min at 37 °C. Then, the cells were trypsinized, and their
ABTS scavenging assay
fuorescence intensity was analyzed using an EC800 Ana-
lyzer (Sony Corporation, Tokyo, Japan).
The measurements were taken on a Specord 40 instru-
ment. ABTS was dissolved in PBS bufer (0.136 M NaCl,
0.0027 M KCl, 0.01 M Na2HPO4, 0.00176 M KH2PO4) to
a 7-mM concentration. ABTS radical cation (ABTS•+) was
produced by reacting ABTS stock solution with potassium
persulfate at a fnal concentration of 2.45 mM and allowing
the mixture to stand in the dark at room temperature for
16 h before use. For the study of compounds, the ABTS·+
solution was diluted with ethanol to an absorbance of 0.70
( 0.02) at 734 nm and equilibrated at 37 °C. Stock solutions
of new compounds and Trolox in DMSO were added to the
diluted ABTS·+ solution, in fnal concentrations of 12.5, 10,
7.5, 2.5 µM. After addition, the mixtures were incubated for
6 min at 37 °C before measuring their absorbance at 734 nm.
All determinations were carried out three times. The per-
centage inhibition of absorbance at 734 nm is calculated
with the usual formula: (A0−Aantioxidant)/A0, where A0 is the
absorbance of the diluted ABTS·+ solution. The concentra-
tion–response curves of new compounds were compared
with the curve of Trolox.
Statistical analyses were done by Dunnett’s test.
Acknowledgements Open access funding provided by University of
Pécs (PTE). We are grateful to Gergely Gulyás-Fekete (Department
of Biochemistry and Medical Chemistry) for NMR measurements.
The project was supported by Hungarian National, Research, Devel-
opment and Innovation Ofce (OTKA 124331) and Grant GINOP
(GINOP-2.3.2-15-2016-00049).
Open Access This article is distributed under the terms of the Crea-
tive Commons Attribution 4.0 International License (http://creativeco
mmons.org/licenses/by/4.0/), which permits unrestricted use, distribu-
tion, and reproduction in any medium, provided you give appropriate
credit to the original author(s) and the source, provide a link to the
Creative Commons license, and indicate if changes were made.
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1 3