Caffeic acid phenethyl ester (CAPE)-derivatives act as selective inhibitors of acetylcholinesterase

Article abstract of DOI:10.1016/j.ejmech.2019.05.059

Unexpected inhibitory effects against eeAChE could be found for a newly synthesized class of caffeic acid phenethyl ester (CAPE)derivatives. Thus, phenethyl-(E)-3-(3,5-dimethoxy-4-phenethoxyphenyl)-acrylate (K_i = 1.97 ± 0.38 μM, K_i′ = 2.44 ± 0.07 μM)and 4-(2-(((E)-3-(3,4-bis(benzyloxy)phenyl)acryloyl)oxy)ethyl)-1,2-phenylene (2E,2′E)-bis(3-(3,4-bis(benzyloxy)phenyl)acrylate)(K_i = 0.72 ± 0.31 μM, K_i′ = 1.80 ± 0.21 μM)showed very good inhibition of eeAChE, while being non cytotoxic for malignant human cancer cells and non-malignant mouse fibroblasts. Also, they are weak inhibitors for BChE (from equine serum).

Full text of DOI:10.1016/j.ejmech.2019.05.059

Accepted Manuscript
Caffeic acid phenethyl ester (CAPE)-derivatives act as selective inhibitors of
acetylcholinesterase
Josephine M. Gießel, Anne Loesche, René Csuk
PII:
S0223-5234(19)30469-6
DOI:
https://doi.org/10.1016/j.ejmech.2019.05.059
EJMECH 11369
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 2 May 2019
Revised Date: 20 May 2019
Accepted Date: 20 May 2019
Please cite this article as: J.M. Gießel, A. Loesche, René. Csuk, Caffeic acid phenethyl ester (CAPE)-
derivatives act as selective inhibitors of acetylcholinesterase, European Journal of Medicinal Chemistry
(2019), doi: https://doi.org/10.1016/j.ejmech.2019.05.059.
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ACCEPTED MANUSCRIPT
Graphical abstract:
Caffeic acid phenethyl ester (CAPE)-derivatives act as selective inhibitors of
acetylcholinesterase
Josephine M. Gießel, Anne Loesche, René Csuk*
Martin-Luther-University Halle-Wittenberg, Organic Chemistry, Kurt-Mothes-Str. 2, D-
06120 Halle (Saale), Germany

ACCEPTED MANUSCRIPT
Caffeic acid phenethyl ester (CAPE)-derivatives act as selective inhibitors of
acetylcholinesterase
Josephine M. Gießel, Anne Loesche, Immo Serbian, René Csuk*
Martin-Luther-University Halle-Wittenberg, Organic Chemistry, Kurt-Mothes-Str. 2, D-
06120 Halle (Saale), Germany
* Corresponding author: email: rene.csuk@chemie.uni-halle.de; Tel.: +49 345 5525660; Fax.:
+49 345 27030
Keywords: cinnamic acid phenethyl esters; Caffeic acid phenethyl ester;
acetylcholinesterase; butyrylcholinesterase; inhibitors
Abstract
Unexpected inhibitory effects against eeAChE could be found for a newly synthesized class
of caffeic acid phenethyl ester (CAPE) derivatives. Thus, phenethyl-(E)-3-(3,5-dimethoxy-4-
phenethoxyphenyl)-acrylate (Ki = 1.97 ± 0.38 µM, Ki´ = 2.44 ± 0.07 µM) and 4-(2-(((E)-3-
(3,4-bis(benzyloxy)phenyl)acryloyl)oxy)ethyl)-1,2-phenylene
(2E,2'E)-bis(3-(3,4-
bis(benzyloxy)phenyl)acrylate) (Ki = 0.72 ± 0.31 µM, Ki´ = 1.80 ± 0.21 µM) showed very
good inhibition of eeAChE, while being non cytotoxic for malignant human cancer cells and
non-malignant mouse fibroblasts. Also, they are weak inhibitors for BChE (from equine
serum).
1. Introduction
For thousands of years, people have been learning by observing nature, and many modern
drugs are derived from secondary natural compounds produced by microorganism, plants or
even animals. The latter, however, also often make use of the potential of herbal ingredients.
Thus, honey-bees do not only have a sophisticated way to communicate and an elaborate
sharing of their tasks, they also hold techniques to protect their beehives against diseases.
Thereby, bees seal little holes and gaps around their beehive with propolis. This resin contains
many different compounds which protect the beehives from diseases and infections caused by
bacteria, fungi and other microorganism.[1] One of these ingredients is caffeic acid phenethyl
ester (CAPE, Fig. 1).
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Fig. 1: Structure of caffeic acid phenethyl ester (CAPE)
Thus, CAPE is a well-known compound holding a broad range of different biological
properties, and this molecule - as well as derivatives thereof - has been studied quite often.
Next to inhibitory effects against HIV-1 integrase, cyclooxygenase and lipoxygenase [2], also
anti-inflammatory [3], antioxidant and anti-proliferative [4] effects have been shown.
Furthermore, there are several studies about the cytotoxic and anticancer effect of CAPE [4-
7], and several publications by Lee et al. [8, 9] describe for CAPE and derivatives cytotoxic
impact. Besides to their cytotoxic activity, Gülçin et al. [10] reported CAPE as a picomolar
inhibitor of acetylcholinesterase and butyrylcholinesterase. During our ongoing projects, we
became interested in the cytotoxic activity of these compounds but also in their ability to
inhibit cholinesterases AChE and BChE. Inhibitors of both enzymes are nowadays standard
medication for people suffering from neurodegenerative disorders like dementia especially
Alzheimer’s disease. As a consequence, our products have been tested for their ability to act
as inhibitors of AChE (from electric eel) and BChE (from equine serum) inhibition by
Ellman´s and their cytotoxicity by SRB assays.
2. Results and discussion
2.1 Chemistry
In this project, we investigated the synthesis of CAPE analogues and their biological
properties. First of all, esters of 2-phenylethanol with 16 different cinnamic acids were
prepared using Mitsunobu reaction [11] conditions. Not unexpectedly, the substitution pattern
of the aromatic ring affected the yields of these reactions (Scheme 1, Table 1). Thus, yields
were low to moderate for ortho- and meta-chlorine substituted cinnamic acids, while for
cinnamic acids holding electron-donating substituents yields increased.
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Scheme 1: Mitsunobu reaction of cinnamic acid derivatives 1-16: a) corresponding acid,
PPh3, THF, 2-phenylethanol, DIAD, 0 °C → rt, overnight, 14 – 99%.
Table 1: substitution pattern for compounds 1-16
Compound R1
R2
R3
R4
H
Yield
57
39
17
34
35
46
73
88
73
88
77
39
89
99
14
31
H
H
H
1
H
H
F
H
2
H
F
H
H
3
H
Br
H
H
4
Cl
H
H
H
H
5
Cl
H
H
6
H
H
Cl
H
7
H
Me
H
Me
H
8
OMe
H
H
H
9
OMe
H
H
H
10
11
12
13
14
15
16
H
OMe
OMe
H
H
OMe
H
H
H
OMe
OMe
OMe
OMe
OMe
OMe
OMe
H
H
OMe
O-C2H4Ph
OAc
H
H
Using standard conditions, 3 and 15 were exceptions to this rule: For the former compound
the reaction was very slow while the low yield observed in the reaction of 15 was due to side
reactions caused by the p-hydroxyl-group. Yields, however, could be improved (67%) for this
system by using an excess of 2-phenylethanol. As an alternative, reaction conditions as
described by Xie et al. [4] were applied (Scheme 2), but yields remained low (9%) for the
preparation of 17.
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Scheme 2: Synthesis of CAPE derivatives 17-23: a) Yb(OTF3), CH3NO2, reflux, 1.5 h, 9%;
b) DCC, DMAP, DCM, -20°C → rt, 53%; c) K2CO3, benzyl chloride, EtOH, reflux, 5 h, 57%;
d) K2CO3, allyl bromide, EtOH, reflux, 5 h, 22%; e) NaOH, EtOH, rt, 51%; f) 2-(o-
hydroxyphenyl)-ethanol, DCC, DMAP, DCM, -20°C → rt, 22 = 36%; g) 2-(o-
hydroxyphenyl)-ethanol, DCC, DMAP, DCM, -20°C → rt, 23 = 27%.
Steglich esterification as described by Eicher et al. [12] was used for the synthesis of a 3,4-
dimethoxy cinnamic acid derivative, and compound 18 was obtained with a moderate yield of
53%.
Since methoxy groups are not the preferred protective groups for phenols, as their removal
often proceeds with low yields, benzyl and allyl protective groups were chosen for the
synthesis of several derivatives.
These experiments showed that for benzyl protected caffeic acid higher yields were obtained
than for the allyl protected analogues. The possibility to prepare compound 19 in a one-pot-
reaction (and the known inhibitory effect of compound 22 for AChE) was decisive to use 19
instead of 21 as a starting material for further derivatizations. As a by-product from these
coupling reactions, “dimeric” structures 24-27 were obtained.
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Fig. 2: “Dimeric” CAPE structures 24-27.
2.2 Biological evaluation
2.2.1 Inhibition of ChE
Six compounds were found to be good to excellent inhibitors for eeAChE (Tab. 2). They are
mixed type inhibitors for this enzyme. Their inhibition constants range from moderate (Ki =
14.21 ± 1.40 µM, Ki´ = 56.58 ± 7.80) for 6 to very good (Ki = 0.72 ± 0.31 µM, Ki´ = 1.80 ±
0.21 µM) for compound 26.
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Table 2: Ellman’s assay; %-inhibition at a concentration of 10 µM of compounds 1-27
(compounds not mentioned in the table were insoluble under the conditions of the assay) with
AChE (from electrophorus electricus) and BChE (from equine serum); the inhibition[11]
constants Ki and Ki´ are given in µM and galantamine hydrobromide (GH) was used as a
standard; the values are averaged from experiments each performed in triplicate.
Compound
AChE
BChE
Inhibition [%]
53.00
18.31
3.44
Inhibition [%]
87.70
16.21
13.75
11.09
89.89
19.02
39.82
35.30
14.76
20.26
22.16
16.91
91.49
11.32
9.48
Ki [µM]
0.54 ± 0.01
Ki´ [µM]
--
GH
1
2
13.59
14.70
4.91
3
8.82 ± 1.04
30.92 ± 2.05
56.58 ± 7.80
4
5
14.21 ± 1.40
30.77
7.07
6
7
11.66
9.27
8
9
5.12
10
11
12
13
14
15
16
17
18
19
20
21
22
23
26
6.23
11.89 ± 0.26
1.97 ± 0.38
34.77 ± 1.92
2.44 ± 0.07
11.90
7.16
4.87
98.48
9.59
10.71
8.72
35.00
14.96
26.64
23.95
14.92
79.01
9.24
32.38
10.13
11.67
8.88
2.80
8.55 ± 0.39
0.72 ± 0.31
12.10 ± 0.31
1.80 ± 0.21
23.58
10.14
4.11
73.34
6

ACCEPTED MANUSCRIPT
Thus, compound 26 was the best inhibitor of this series of compounds, and with a Ki-value of
0.72 ± 0.31 µM, this compound had similar properties on AChE as the gold-standard GH.
Some molecular modelling calculations were performed to gain some insight in the mode of
action of this inhibitor. The results from “blind docking” suggest that compound 26 is
blocking the entrance of the active site of AChE. No other possible binding sites were found
within 15 GA runs (only top 5 are shown, Fig. 3). To further evaluate these positions, the Grid
parameters were changed to give more accurate docking poses for the active site of AChE.
The results from these calculations are depicted in Fig. 3.
Blind Docking
Focused Docking
26_1 yellow; 26_2 pink; 26_3 turkis; 26_4
green; 26_5 orange
26_1 green; 26_2 yellow; 26_3 blue; 26_4
grey; 26_5 violett
Fig. 3. Results from the molecular modelling calculations (top 5 GA runs shown) revealing
that 26 is not able to enter the active site but rather blocks the entrance to it.
7

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2.2.2 Cytotoxicity
Table 3: Cytotoxicity of compounds 17 and 18; SRB assay EC50 values (in µM) after 96 h of
treatment; averaged from three independent experiments performed each in triplicate;
confidence interval CI = 95%. Human cancer cell lines: FaDu (hypopharyngeal carcinoma),
A2780 (ovarian carcinoma), HT29 (colorectal carcinoma), MCF7 (breast carcinoma),
SW1736 (thyroid carcinoma) and non-malignant mouse fibroblasts (NIH 3T3).
EC50[µM]
FaDu
A2780
HT29
> 30
MCF7
SW1736
4.7 ± 0.3
NIH 3T3
12.1 ± 1.5
3.5 ± 0.6
12.1 ± 1.7
21.4 ± 4.2
17
18
17.2 ± 2.4 17.0 ± 0.3 16.7 ± 4.9 16.2 ± 4.0 16.7 ± 3.7 17.7 ± 1.5
All compounds were screened for their cytotoxic activity, but only compounds 17 and 18
were cytotoxic for five tested malignant and one non-malignant cell line (NIH 3T3).
3. Conclusion
In this study, a small library of 27 derivatives of CAPE was synthesized, and the compounds
were tested for their biochemical properties. Furthermore, 4 unexpected “dimeric structures”
were found and characterized. Due to the insolubility of these molecules, only 26 coiuld be
tested in Ellman´s assay and showed very good mixed type inhibitory effects for AChE,
holding Ki = 0.72 ± 0.31 µM and Ki´ = 1.80 ± 0.21 µM, while being non-cytotoxic for all
tested cell lines. Because of the molecule size we supposed that 26 did not fit into the active
site of the enzyme, and its mode of action may be to block the entrance to the active side. Out
of the synthesized CAPE derivatives, five substances were shown to be good to excellent and
selective mixed type inhibitors for the enzyme acetylcholinesterase. We assume that the
presence of two benzylic or two 2-phenylethanol groups in these molecules might be the
reason for an increased inhibitory effect for AChE, hence paralleling previous results reported
by Takao et al. [13] All derivatives holding unprotected hydroxyl groups, however, were no
inhibitors for cholinesterases. Furthermore, the presence of halogen substituents at the
cinnamic moiety did not significantly affect the biological activity of these compounds. These
promising results call for further investigations that are presently carried out in our
laboratories.
4. Experimental part
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4.1 General
The reagents were bought from commercial suppliers and used without further purification.
The solvents were dried according to usual procedures. Melting points were determined on
Büchi Melting Point M-565 or LEICA hot stage microscope and are uncorrected, NMR
spectra were recorded on a Varian spectrometer Unity 500 (δ given in ppm, J in Hz), mass
spectra were obtained on a Finnigan MAT LCQ 7000 (electrospray, voltage 4.1 kV, sheath
gas nitrogen) instrument. The optical rotations were measured on a Perkin-Elmer polarimeter
at 20 °C. Macherey-Nagel ALUGRAM® Xtra SIL G/UV254 pre-coated silica gel 60 F254
plates were used for thin layer chromatography (detection with cerium molybdate spray
reagent and UV absorption). IR spectra were recorded on a Perkin-Elmer FT-IR spectrometer
Spectrum 1000, and wave numbers are expressed in cm-1. The absorption spectra were
measured on Perkin Elmer Lambda14 spectrometer. Microanalyses were performed with a
Foss-Heraeus Vario EL (CHNS) instrument. The screening assays were performed as
previously described. [14-16] Blind Docking was performed on eeAChE (PDB = 1C2O). Grid
Center: 35.180, 72.374, -87.078, Grid Points 126,126,126 with spacing of 0.45 Angstroms.
Docking parameters are from Autodock 4.2 with GA parameters are: population size = 250;
number of evaluations = 25000000; number of generations 27000; GA runs = 15.
Focused Docking was performed on eeAChE (PDB = 1C2O). Grid Center: 35.184, 72.374, -
87.072, Grid Points 126,126,126 with spacing of 0.25 Angstroms. Docking parameters are the
same as described for the blind docking.
4.2 Syntheses
4.2.1 General procedure A
Under Argon atmosphere and at 0 °C, the appropriate carboxylic acid (1 eq.) and
triphenylphosphane (1.1 eq.) were dissolved in dry THF (10 mL). 2-Phenylethanol (1 eq.) and
DIAD (1.1 eq.) were added. [11] The reaction mixture was stirred overnight at room
temperature. The solvent was removed under reduced pressure, the residue was dissolved in
ethyl acetate (30 mL) and washed with sodium hydrogen carbonate solution (30 mL) and
brine (30 mL). The organic phase was dried (Na2SO4), and the solvent was removed in vacuo.
The residue was purified by column chromatography (silica gel, n-hexane/ethyl acetate, 4:1).
Compounds 1 – 16 were synthesized following procedure A.
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4.2.1.1 Phenethyl cinnamate (1)
Compound 1 was obtained as a white solid (0.48 g, 57%); RF = 0.60 (silica gel, n-
hexane/ethyl acetate, 4:1); m.p. 56 °C (lit.:[17] 54 °C); IR (ATR): ν = 3029w, 1708s, 1636m,
1578w, 1497w, 1450w, 1383w, 1327w, 1309m, 1255m, 1202m, 1163s, 979m, 766m, 750m,
1
698s, 683m, 494m cm-1; UV-vis (CHCl3): λ (log ε) = 297 (4.15) nm; H NMR (400 MHz,
CDCl3):
δ = 7.68 (d, J = 16.0 Hz, 1H, 3-H), 7.52 (dd, J = 6.6, 3.1 Hz, 2H, 2´-H) , 7.39 (d, J =
3.5 Hz, 2H, 3´-H), 7.38 (d, J = 1.5 Hz, 1H, 4´-H), 7.32 (d, J = 7.2 Hz, 2H, 3´´´-H), 7.29 – 7.27
(m, 2H, 2´´´-H), 7.26 (d, J = 6.0 Hz, 1H, 4´´´-H), 6.43 (d, J = 16.0 Hz, 1H, 2-H), 4.44 (t, J =
13
7.1 Hz, 2H, 1´´-H), 3.03 (t, J = 7.1 Hz, 2H, 2´´-H) ppm; C NMR (100 MHz, CDCl3):
δ =
167.0 (C-1), 145.0 (C-3), 138.0 (C-1´´´), 134.6 (C-1´), 130.4 (C-4´), 129.1 (C-2´´´), 129.0 (C-
3´), 128.7 (C-3´´´), 128.2 (C-2´), 126.7 (C-4´´´), 118.2 (C-2), 65.2 (C-1´´), 35.4 (C-2´´) ppm;
MS (ESI, MeOH): m/z (%) = 252.9 ([M+H]+, 86), 275.1 ([M+Na]+, 100), 397.9 ([3M+Ca]2+,
33), 524.0 ([4M+Ca]2+, 24), 526.9 ([2M+Na]+, 24); analysis calcd for C17H16O2 (252.31): C
80.93, H 6.39; found: C 80.71, H 6.51.
4.2.1.2 Phenethyl (E)-3-(4-fluorophenyl)-acrylate (2)
Compound 2 was obtained as a white solid (0.32 g, 39%); RF = 0.73 (silica gel, n-
hexane/ethyl acetate, 4:1); m.p. 54 °C; IR (ATR): ν = 1713m, 1637m, 1597m, 1507m, 1454w,
1414w, 1311m, 1280w, 1228m, 1199m, 1156s, 1096m, 995s, 831s, 770m, 739m, 700s, 512s,
495s cm-1; UV-vis (CHCl3): λ (log ε) = 280 (4.23) nm; 1H NMR (500 MHz, CDCl3):
δ = 7.63
(d, J = 16.0 Hz, 1H, 3-H), 7.53 – 7.48 (m, 2H, 2´-H) , 7.34 – 7.30 (m, 2H, 2´´´-H), 7.25 (d, J =
7.0 Hz, 2H, 3´´´-H), 7.24 (t, J = 7.2 Hz, 1H, 4´´´-H), 7.08 (t, J = 8.6 Hz, 2H, 3´-H), 6.35 (d, J
13
= 16.0 Hz, 1H, 2-H), 4.4 (t, J = 7.1 Hz, 2H, 1´´-H), 3.02 (t, J = 7.1 Hz, 2H, 2´´-H) ppm; C
NMR (125 MHz, CDCl3):
δ = 166.9 (C-1), 165.1 (C-4´), 143.7 – 143.6 (m, C-3), 138.0 (C-
1´´´), 130.8 (d, J = 3.6 Hz, C-1´), 130.1 (d, J = 8.6 Hz, C-2´), 129.1 (C-3´´´), 128.7 (C-2´´´),
126.7 (C-4´´´), 118.0 (d, J = 2.1 Hz, C-2), 116.2 (d, J = 22.0 Hz, C-3´), 65.2 (C-1´´), 35.4 (C-
2´´) ppm; 19F NMR (470 MHz, CDCl3): δ = -109.66 (tt, J = 8.5, 5.4 Hz) ppm; MS (ESI,
MeOH): m/z (%) = 293.3 ([M+Na]+, 100), 325.3 ([M+Na+MeOH]+, 29); analysis calcd for
C17H15FO2 (270.30): C 75.54, H 5.59; found: C 75.39, H 5.84.
4.2.1.3 Phenethyl (E)-3-(3-fluorophenyl)-acrylate (3)
Compound 3 was obtained as a colourless oil (0.14 g, 17%); RF = 0.71 (silica gel, n-
hexane/ethyl acetate, 4:1); IR (ATR): ν = 3030w, 1710s, 1640m, 1611w, 1583m, 1486w,
1447m, 1383w, 1318m, 1271m, 1242m, 1224s, 1161s, 1076w, 1054w, 1003m, 980m, 857m,
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1
784m, 698s, 672s, 495m cm-1; UV-vis (CHCl3): λ (log ε) = 273 (4.19) nm; H NMR (400
MHz, CDCl3): = 7.63 (d, J = 16.0 Hz, 1H, 3-H), 7.39 – 7.35 (m, 1H, 5´-H) , 7.34 – 7.31 (m,
δ
2H, 2´´´-H), 7.30 – 7.29 (m, 1H, 6´-H), 7.28 (d, J = 2.2 Hz, 2H, 3´´´-H), 7.26 (t, J = 1.7 Hz,
1H, 4´´´-H), 7.21 (dt, J = 9.7, 2.2 Hz, 1H, 2´-H), 7.08 (tdd, J = 8.3, 2.6, 1.0 Hz, 1H, 4´-H),
6.42 (d, J = 16.0 Hz, 1H, 2-H), 4.44 (t, J = 7.1 Hz, 2H, 1´´-H), 3.03 (t, J = 7.1 Hz, 2H, 2´´-H)
ppm; 13C NMR (125 MHz, CDCl3):
δ = 166.6 (C-1), 163.1 (d, J = 246.9 Hz, C-3´), 143.5 (d, J
= 2.7 Hz, C-3), 137.9 (C-1´´´), 136.8 (d, J = 7.7 Hz, C-1´), 130.6 (d, J = 8.2 Hz, C-5´), 129.0
(C-3´´´), 128.7 (C-2´´´), 126.7 (C-4´´´), 124.2 (d, J = 2.9 Hz, C-6´), 119.6 (C-2), 117.3 (d, J =
19
21.4 Hz, C-4´), 114.4 (d, J = 21.9 Hz, C-2´), 65.3 (C-1´´), 35.3 (C-2´´) ppm; F NMR (470
MHz, CDCl3): δ = -112.56 (td, J = 9.0, 5.9 Hz) ppm; MS (ESI, MeOH): m/z (%) = 293.3
([M+Na]+, 100), 309.3 ([M+K]+, 13), 325.4 ([M+Na+MeOH]+, 35); analysis calcd for
C17H15FO2 (270.30): C 75.54, H 5.59; found: C 75.40, H 5.73.
4.2.1.4 Phenethyl (E)-3-(3-bromophenyl)-acrylate (4)
Compound 4 was obtained as a white solid (0.25 g, 34%); RF = 0.70 (silica gel, n-
hexane/ethyl acetate, 4:1); m.p. 63 °C; IR (ATR): ν = 3056w, 1699s, 1632m, 1561m, 1497w,
1475w, 1453w, 1418w, 1309m, 1300m, 1203m, 1174s, 1164s, 1071s, 1031w, 996s, 862m,
785s, 739s, 700s, 674s, 667s, 578m, 500s cm-1; UV-vis (CHCl3): λ (log ε) = 228 (4.02), 276
(4.07) nm; 1H NMR (400 MHz, CDCl3):
δ = 7.66 (t, J = 1.8 Hz, 1H, 4´-H), 7.58 (d, J = 16.0
Hz, 1H, 3-H), 7.50 (ddd, J = 8.0, 2.0, 1.0 Hz, 1H, 2´-H) , 7.43 (dt, J = 7.8, 1.3 Hz, 1H, 6´-H),
7.33 – 7.30 (m, 2H, 2´´´-H), 7.27 (d, J = 2.1 Hz, 2H, 3´´´-H), 7.26 – 7.24 (m, 1H, 5´-H), 7.24
– 7.22 (m, 1H, 4´´´-H), 6.41 (d, J = 16.0 Hz, 1H, 2-H), 4.43 (t, J = 7.0 Hz, 2H, 1´´-H), 3.02 (t,
J = 7.1 Hz, 2H, 2´´-H) ppm; 13C NMR (100 MHz, CDCl3):
δ = 166.6 (C-1), 143.2 (C-3),
137.9 (C-1´´´), 136.7 (C-1´), 133.2 (C-2´), 130.9 (C-4´), 130.5 (C-5´), 129.1 (C-3´´´), 128.7
(C-2´´´), 126.8 (C-4´´´), 126.7 (C-6´), 123.2 (C-3´), 119.7 (C-2), 65.3 (C-1´´), 35.3 (C-2´´)
ppm; MS (ESI, MeOH): m/z (%) = 355.3 ([M+Na]+, 100), 387.4 ([M+Na+MeOH]+, 71);
analysis calcd for C17H15BrO2 (331.21): C 61.65, H 4.57; found: C 61.36, H 4.71.
4.2.1.5 Phenethyl (E)-3-(2-chlorophenyl)-acrylate (5)
Compound 5 was obtained as a white solid (0.28 g, 35%); RF = 0.70 (silica gel, n-
hexane/ethyl acetate, 4:1); m.p. 35 °C; IR (ATR): ν = 1706s, 1441w, 1379w, 1293m, 1271s,
1244s, 1202m, 1172m, 1083w, 1052m, 1017w, 986m, 953m, 753s, 699s, 571m, 494m cm-1;
UV-vis (CHCl3): λ (log ε) = 230 (4.13), 276 (4.31) nm; 1H NMR (500 MHz, CDCl3):
δ = 8.10
(d, J = 16.0 Hz, 1H, 3-H), 7.61 (dd, J = 7.5, 2.0 Hz, 1H, 6´-H) , 7.44 – 7.41 (m, 1H, 3´-H),
11

ACCEPTED MANUSCRIPT
7.34 – 7.31 (m, 2H, 2´´´-H), 7.31 (d, J = 1.9 Hz, 1H, 5´-H), 7.30 – 7.29 (m, 2H, 3´´´-H), 7.28
– 7.26 (m, 1H, 4´-H), 7.26 – 7.23 (m, 1H, 4´´´-H), 6.42 (d, J = 16.0 Hz, 1H, 2-H), 4.45 (t, J =
13
7.0 Hz, 2H, 1´´-H), 3.04 (t, J = 7.0 Hz, 2H, 2´´-H) ppm; C NMR (125 MHz, CDCl3):
δ =
166.5 (C-1), 140.7 (C-3), 138.0 (C-1´´´), 135.1 (C-1´), 132.8 (C-2´), 131.2 (C-5´), 130.3 (C-
3´), 129.1 (C-3´´´), 128.7 (C-2´´´), 127.7 (C-6´), 127.2 (C-4´), 126.7 (C-4´´´), 120.8 (C-2),
65.3 (C-1´´), 35.3 (C-2´´) ppm; MS (ESI, MeOH): m/z (%) = 355.3 ([M+Na]+, 100), 387.4
([M+Na+MeOH]+, 60); analysis calcd for C17H15BrO2 (331.21): C 61.65, H 4.57; found: C
61.31, H 4.75.
4.2.1.6 Phenethyl (E)-3-(3-chlorophenyl)-acrylate (6)
Compound 6 was obtained as a white solid (0.36 g, 46%); RF = 0.75 (silica gel, n-
hexane/ethyl acetate, 4:1); m.p. 53 °C; IR (ATR): ν = 3066w, 1703m, 1636m, 1562w, 1471w,
1455w, 1421w, 1301m, 1206m, 1175s, 1161s, 1077m, 993s, 861s, 786s, 745s, 697s, 684m,
672s, 500s cm-1; UV-vis (CHCl3): λ (log ε) = 230 (4.13), 274 (4.30) nm; 1H NMR (500 MHz,
CDCl3):
δ = 7.60 (d, J = 16.0 Hz, 1H, 3-H), 7.50 (t, J = 1.9 Hz, 1H, 6´-H) , 7.38 (tt, J = 6.2,
1.7 Hz, 2H, 2´-H+5´-H), 7.35 (t, J = 1.7 Hz, 2H, 2´´´-H), 7.33 (dd, J = 6.7, 1.9 Hz, 2H, 3´´´-
H), 7.27 (q, J = 2.8, 2.2 Hz, 1H, 4´-H), 7.26 – 7.23 (m, 1H, 4´´´-H), 6.42 (d, J = 16.0 Hz, 1H,
13
2-H), 4.44 (t, J = 7.1 Hz, 2H, 1´´-H), 3.03 (t, J = 7.0 Hz, 2H, 2´´-H) ppm; C NMR (125
MHz, CDCl3):
δ = 166.6 (C-1), 143.3 (C-3), 137.9 (C-1´´´), 136.4 (C-1´), 135.0 (C-3´), 130.3
(C-5´), 130.2 (C-4´), 129.0 (C-3´´´), 128.7 (C-2´´´), 127.9 (C-6´), 126.7 (C-4´´´), 126.4 (C-2´),
119.7 (C-2), 65.3 (C-1´´), 35.3 (C-2´´) ppm; MS (ESI, MeOH): m/z (%) = 309.3 ([M+Na]+,
100), 341.4 ([M+Na+MeOH]+, 56); analysis calcd for C17H15ClO2 (286.76): C 71.21, H 5.27;
found: C 70.97, H 5.39.
4.2.1.7 Phenethyl (E)-3-(4-chlorophenyl)-acrylate (7)
Compound 7 was obtained as a white solid (0.58 g, 73%); RF = 0.77 (silica gel, n-
hexane/ethyl acetate, 4:1); m.p. 63 °C (lit.:[18] 62 – 64 °C); IR (ATR): ν = 1711s, 1634m,
1590w, 1490m, 1455w, 1406w, 1308s, 1203w, 1184w, 1156s, 1110m, 1086s, 995s, 824s,
1
742m, 701s, 518m, 494s cm-1; UV-vis (CHCl3): λ (log ε) = 229 (3.77), 286 (4.14) nm; H
NMR (500 MHz, CDCl3):
δ = 7.61 (d, J = 16.0 Hz, 1H, 3-H), 7.46 – 7.43 (m, 2H, 2´-H) , 7.38
– 7.35 (m, 2H, 3´-H), 7.34 – 7.30 (m, 2H, 2´´´-H), 7.27 (t, J = 2.2 Hz, 2H, 3´´´-H), 7.26 – 7.23
(m, 1H, 4´´´-H), 6.39 (d, J = 16.0 Hz, 1H, 2-H), 4.43 (t, J = 7.1 Hz, 2H, 1´´-H), 3.02 (t, J = 7.0
13
Hz, 2H, 2´´-H) ppm; C NMR (125 MHz, CDCl3):
δ = 166.8 (C-1), 143.5 (C-3), 138.0 (C-
1´´´), 136.4 (C-4´), 133.0 (C-1´), 129.4 (C-2´), 129.3 (C-3´), 129.1 (C-3´´´), 128.7 (C-2´´´),
12

ACCEPTED MANUSCRIPT
126.8 (C-4´´´), 118.8 (C-2), 65.3 (C-1´´), 35.4 (C-2´´); MS (ESI, MeOH): m/z (%) = 309.3
([M+Na]+, 100), 341.4 ([M+Na+MeOH]+, 50); analysis calcd for C17H15ClO2 (286.76): C
71.21, H 5.27; found: C 70.96, H 5.50.
4.2.1.8 Phenethyl (E)-3-(3,4-dimethylphenyl)-acrylate (8)
Compound 8 was obtained as a white solid (0.35 g, 88%); RF = 0.76 (silica gel, n-
hexane/ethyl acetate, 4:1); m.p. 40 °C; IR (ATR): ν = 3028w, 2945w, 1707s, 1634m, 1608w,
1571w, 1498w, 1454w, 1407w, 1384w, 1315m, 1260m, 1233m, 1205m, 1154s, 1124w, 1087w,
1054w, 1020w, 982m, 817m, 697m cm-1; UV-vis (MeOH): λ (log ε) = 224 (3.92), 290 (4.12)
1
nm; H NMR (400 MHz, CDCl3):
δ = 7.65 (d, J = 16.0 Hz, 1H, 3-H), 7.34 – 7.32 (m, 2H,
2´´´-H), 7.31 (d, J = 2.0 Hz, 1H, 2´-H), 7.29 (d, J = 1.9 Hz, 2H, 3´´´-H), 7.28 – 7.26 (m, 1H,
6´-H), 7.26 – 7.23 (m, 1H, 4´´´-H), 7.15 (d, J = 7.8 Hz, 1H, 5´-H), 6.39 (d, J = 16.0 Hz, 1H, 2-
H), 4.44 (t, J = 7.1 Hz, 2H, 1´´-H), 3.03 (t, J = 7.1 Hz, 2H, 2´´-H), 2.29 (s, 6H, 7´-H+8´-H)
13
ppm; C NMR (100 MHz, CDCl3):
δ = 167.3 (C-1), 145.2 (C-3), 139.6 (C-3´), 138.1 (C-
1´´´), 137.2 (C-4´), 132.2 (C-1´), 130.3 (C-5´), 129.4 (C-2´), 129.1 (C-3´´´), 128.6 (C-2´´´),
126.7 (C-4´´´), 125.9 (C-6´), 116.9 (C-2), 65.0 (C-1´´), 35.4 (C-2´´), 19.9 (C-7´), 19.8 (C-8´)
ppm; MS (ESI, MeOH): m/z (%) = 280.9 ([M+H]+, 44), 303.1 ([M+Na]+, 77), 440.0
([3M+Ca]2+, 34), 582.9 ([2M+Na]+, 100); analysis calcd for C19H20O2 (280.37): C 81.40, H
7.19; found: C 81.17, H 6.89.
4.2.1.9 Phenethyl (E)-3-(2-methoxyphenyl)-acrylate (9)
Compound 9 was obtained as a colourless oil (0.58 g, 73%); RF = 0.61 (silica gel, n-
hexane/ethyl acetate, 4:1); IR (ATR): ν = 1705s, 1629m, 1598m, 1489m, 1464m, 1437w,
1319m, 1246s, 11159s, 1107m, 1050m, 1024m, 989m, 749s, 699m cm-1; UV-vis (CHCl3): λ
(log ε) = 232 (4.12), 276 (4.25), 323 (4.01) nm; 1H NMR (400 MHz, CDCl3):
δ = 8.00 (d, J =
16.2 Hz, 1H, 3-H), 7.51 (dd, J = 7.7, 1.5 Hz, 1H, 4´-H) , 7.36 (dd, J = 10.3, 2.0 Hz, 1H, 6´-H),
7.33 – 7.30 (m, 2H, 3´´´-H), 7.29 – 7.26 (m, 2H, 2´´´-H), 7.25 – 7.22 (m, 1H, 4´´´-H), 6.96 (t,
J = 7.5 Hz, 1H, 5´-H), 6.92 (d, J = 8.3 Hz, 1H, 3´-H), 6.52 (d, J = 16.2 Hz, 1H, 2-H), 4.43 (t, J
= 7.1 Hz, 2H, 1´´-H), 3.89 (s, 3H, 7´-H), 3.03 (t, J = 7.1 Hz, 2H, 2´´-H) ppm;13C NMR (100
MHz, CDCl3):
δ = 167.5 (C-1), 158.5 (C-2´), 140.4 (C-3), 138.2 (C-1´´´), 131.6 (C-6´), 129.1
(C-2´´´), 129.0 (C-4´), 128.6 (C-3´´´), 126.7 (C-4´´´), 123.6 (C-1´), 120.8 (C-5´), 118.7 (C-2),
111.3 (C-3´), 65.0 (C-1´´), 55.6 (C-7´), 35.4 (C-2´´) ppm; MS (ESI, MeOH): m/z (%) = 282.9
([M+H]+, 67), 305.1 ([M+Na]+, 100), 443.0 ([3M+Ca]2+, 14), 586.8 ([2M+Na]+, 33); analysis
calcd for C18H18O3 (282.34): C 76.57, H 6.43, found: C 76.37, H 6.61.
13

ACCEPTED MANUSCRIPT
4.2.1.10 Phenethyl (E)-3-(3-methoxyphenyl)-acrylate (10)
Compound 10 was obtained as a yellowish oil (0.70 g, 88%); RF = 0.68 (silica gel, n-
hexane/ethyl acetate, 4:1); IR (ATR): ν = 1708s, 1637m, 1579m, 1488w, 1454m, 1433w,
1308m, 1247s, 1230s, 1161s, 1043m, 981m, 782m, 749m, 699m, 679m, 495m cm-1; UV-vis
(CHCl3): λ (log ε) = 236 (4.20), 279 (4.34) nm; 1H NMR (400 MHz, CDCl3):
δ = 7.65 (d, J =
16.0 Hz, 1H, 3-H), 7.36 – 7.32 (m, 2H, 3´´´-H) , 7.32 – 7.31 (m, 1H, 5´-H), 7.28 (d, J = 1.4
Hz, 2H, 2´´´-H), 7.24 (d, J = 7.1 Hz, 1H, 4´´´-H), 7.12 (dt, J = 7.5, 1.2 Hz, 1H, 6´-H), 7.04
(dd, J = 2.5, 1.7 Hz, 1H, 2´-H), 6.94 (ddd, J = 8.3, 2.6, 1.0 Hz, 1H, 4´-H), 6.42 (d, J = 16.0
Hz, 1H, 2-H), 4.44 (t, J = 7.1 Hz, 2H, 1´´-H), 3.84 (s, 3H, 7´-H), 3.03 (t, J = 7.1 Hz, 2H, 2´´-
H) ppm; 13C NMR (100 MHz, CDCl3):
δ = 166.9 (C-1), 160.0 (C-3´), 144.9 (C-3), 138.0 (C-
1´´´), 135.9 (C-1´), 130.0 (C-5´), 129.1 (C-2´´´), 128.7 (C-3´´´), 126.7 (C-4´´´), 120.9 (C-6´),
118.5 (C-2), 116.3 (C-4´), 113.1 (C-2´), 65.2 (C-1´´), 55.4 (C-7´), 35.4 (C-2´´) ppm; MS (ESI,
MeOH): m/z (%) = 282.9 ([M+H]+, 44), 305.1 ([M+Na]+, 100), 443.0 ([3M+Ca]2+, 21), 586.8
([2M+Na]+, 30); analysis calcd for C18H18O3 (282.34): C 76.57, H 6.43; found: C 76.32, H
6.60.
4.2.1.11 Phenethyl-(E)-3-(4-methoxyphenyl)-acrylate (11)
Compound 11 was obtained as a white solid (0.61 g, 77%); RF = 0.62 (silica gel, n-
hexane/ethyl acetate, 4:1); IR (ATR): ν = 1702m, 1634w, 1600s, 1511s, 1456w, 1444w,
1422w, 1314m, 1250m, 1204m, 1171s, 1028m, 986s, 962m, 830s, 816s, 742m, 701s, 539m,
520s, 506s, 490m cm-1; UV-vis (CHCl3): λ (log ε) = 232 (4.07), 311 (4.42) nm; 1H NMR (400
MHz, CDCl3):
δ = 7.64 (d, J = 16.0 Hz, 1H, 3-H), 7.49 – 7.44 (m, 2H, 2´-H), 7.35 – 7.31 (m,
2H, 3´´´-H) , 7.29 – 7.26 (m, 2H, 2´´´-H), 7.26 – 7.22 (m, 1H, 4´´´-H), 6.92 – 6.89 (m, 2H, 3´-
H), 6.31 (d, J = 16.0 Hz, 1H, 2-H), 4.42 (t, J = 7.1 Hz, 2H, 1´´-H), 3.84 (s, 3H, 5´-H), 3.02 (t,
13
J = 7.1 Hz, 2H, 2´´-H) ppm; C NMR (100 MHz, CDCl3):
δ = 167.2 (C-1), 161.4 (C-4´),
144.5 (C-3), 138.0 (C-1´´´), 129.7 (C-2´), 128.9 (C-2´´´), 128.5 (C-3´´´), 127.2 (C-1´), 126.5
(C-4´´´), 115.5 (C-2), 114.3 (C-3´), 64.8 (C-1´´), 55.4 (C-5´), 35.3 (C-2´´) ppm; MS (ESI,
MeOH): m/z (%) = 282.9 ([M+H]+, 89), 305.1 ([M+Na]+, 100), 443.0 ([3M+Ca]2+, 16), 586.8
([2M+Na]+, 44); analysis calcd for C18H18O3 (282.34): C 76.57, H 6.43; found: C 76.26, H
6.70.
4.2.1.12 Phenethyl (E)-3-(2,4-dimethoxyphenyl)-acrylate (12)
14

ACCEPTED MANUSCRIPT
Compound 12 was obtained as a white solid (0.27 g, 39%); RF = 0.32 (silica gel, n-
hexane/ethyl acetate, 4:1); m.p. 52 °C; IR (film): ν = 1702m, 1602s, 1504m, 1455m, 1300s,
1251m, 1210s, 1153s, 1117s, 1029s, 824m, 699m cm-1; UV-vis (CHCl3): λ (log ε) = 252
(3.99), 318 (4.10), 358 (4.22) nm; 1H NMR (400 MHz, CDCl3):
δ = 7.91 (d, J = 16.1 Hz, 1H,
3-H), 7.44 (d, J = 8.6 Hz, 1H, 3´-H), 7.34 – 7.31 (m, 1H, 4´´´-H), 7.29 (dd, J = 9.2, 2.0 Hz,
2H, 2´´´-H), 7.25 – 7.21 (m, 2H, 3´´´-H), 6.50 (dd, J = 8.6, 2.4 Hz, 1H, 6´-H), 6.45 (d, J = 2.3
Hz, 1H, 5´-H), 6.42 (d, J = 16.1 Hz, 1H, 2-H), 4.41 (t, J = 7.1 Hz, 2H, 1´´-H), 3.87 (s, 3H, 8´-
H), 3.84 (s, 3H, 7´-H), 3.02 (t, J = 7.1 Hz, 2H, 2´´-H) ppm; 13C NMR (100 MHz, CDCl3):
δ =
167.9 (C-1), 162.9 (C-4´), 160.0 (C-2´), 140.3 (C-3), 138.2 (C-1´´´), 130.5 (C-3´), 129.1 (C-
4´´´), 128.6 (C-2´´´), 126.6 (C-3´´´), 116.8 (C-1´), 116.0 (C-2), 105.4 (C-6´), 98.6 (C-5´), 64.9
(C-1´´), 55.6 (C-8´), 55.5 (C-7´), 35.5 (C-2´´) ppm; MS (ESI, MeOH): m/z (%) = 313.0
([M+H]+, 46), 335.1 ([M+Na]+, 67), 488.1 ([3M+Ca]2+, 21), 646.9 ([2M+Na]+, 100); analysis
calcd for C19H20O4 (312.36): C 73.06, H 6.45; found: C 72.77, H 6.69.
4.2.1.13 Phenethyl (E)-3-(3,5-dimethoxyphenyl)-acrylate (13)
Compound 13 was obtained as a colourless oil (0.34 g, 89%); RF = 0.54 (silica gel, n-
hexane/ethyl acetate, 4:1); IR (ATR): ν = 2957w, 2839w, 1708m, 1638w, 1591s, 1498w,
1455m, 1427m, 1386w, 1302w, 1278s, 1239m, 1205m, 1152s, 1062m, 1002w, 979m, 835m,
1
699m, 674m cm-1; UV-vis (CHCl3): λ (log ε) = 226 (4.12), 287 (4.03) nm; H NMR (400
MHz, CDCl3): δ = 7.59 (d, J = 16.0 Hz, 1H, 3-H), 7.36 – 7.30 (m, 2H, 2´´´-H), 7.27 (d, J = 5.7
Hz, 2H, 3´´´-H), 7.25 (d, J = 4.5 Hz, 1H, 4´´´-H), 6.66 (d, J = 2.3 Hz, 2H, 2´-H), 6.50 (t, J =
2.2 Hz, 1H, 4´-H), 6.39 (d, J = 16.0 Hz, 1H, 2-H), 4.43 (t, J = 7.1 Hz, 2H, 1´´-H), 3.81 (s, 3H,
5´-H), 3.84 (s, 3H, 7´-H), 3.03 (t, J = 7.1 Hz, 2H, 2´´-H) ppm; 13C NMR (100 MHz, CDCl3):
δ
= 166.9 (C-1), 161.2 (C-3´), 145.0 (C-3), 138.0 (C-1´´´), 136.4 (C-1´), 129.1 (C-3´´´), 128.7
(C-2´´´), 126.7 (C-4´´´), 118.7 (C-2), 106.1 (C-2´), 102.7 (C-4´), 65.2 (C-1´´), 55.6 (C-5´),
35.4 (C-2´´) ppm; MS (ESI, MeOH): m/z (%) = 313.1 ([M+H]+, 52), 335.0 ([M+Na]+, 82),
488.0 ([3M+Ca]2+, 35), 646.8 ([2M+Na]+, 100); analysis calcd for C19H20O4 (312.36): C
73.06, H 6.45; found: C 72.84, H 6.65.
4.2.1.14 Phenethyl (E)-3-(3,4,5-trimethoxyphenyl)-acrylate (14)
Compound 14 was obtained as a white solid (0.36 g, 99%); RF = 0.21 (silica gel, n-
hexane/ethyl acetate, 4:1); m.p. 97°C; IR (ATR): ν = 1705m, 1631w, 1582m, 1505w, 1451w,
1419w, 1305w, 1269m, 1248m, 1165m, 1148m, 1127s, 991m, 975m, 842m, 817m, 754m,
1
703m cm-1; UV-vis (CHCl3): λ (log ε) = 310 (4.27) nm; H NMR (400 MHz, CDCl3):
δ
=
15

ACCEPTED MANUSCRIPT
7.59 (d, J = 15.9 Hz, 1H, 3-H), 7.31 (d, J = 2.8 Hz, 2H, 2´´´-H), 7.24 (d, J = 4.1 Hz, 2H, 3´´´-
H), 7.23 – 7.22 (m, 1H, 4´´´-H), 6.74 (s, 2H, 2´-H), 6.33 (d, J = 15.9 Hz, 1H, 2-H), 4.44 (t, J =
7.1 Hz, 2H, 1´´-H), 3.89 (s, 6H, 5´-H), 3.88 (s, 3H, 6´-H), 3.03 (t, J = 7.1 Hz, 2H, 2´´-H) ppm;
13C NMR (100 MHz, CDCl3):
δ = 167.0 (C-1), 153.6 (C-3´), 145.0 (C-3), 140.3 (C-4´), 138.0
(C-1´´´), 130.1 (C-1´), 129.1 (C-3´´´), 128.7 (C-2´´´), 126.7 (C-4´´´), 117.4 (C-2), 105.5 (C-
2´), 65.1 (C-1´´), 61.1 (C-6´), 56.3 (C-5´), 35.4 (C-2´´) ppm; MS (ESI, MeOH): m/z (%) =
221.1 ([M-C8H9]+), 343.0 ([M+H]+, 60), 365.1 ([M+Na]+); analysis calcd for C20H22O5
(342.39): C 70.16, H 6.48; found: C 69.82, H 6.63.
4.2.1.15 Phenethyl (E)-3-(3,5-dimethoxy-4-phenethoxyphenyl)-acrylate (15)
Compound 15 was obtained as a colourless oil (0.13 g, 14%); RF = 0.33 (silica gel, n-
hexane/ethyl acetate, 4:1); IR (ATR): ν = 1707m, 1635m, 1581m, 1500m, 1454m, 1418m,
1271s, 1241s, 1150s, 1124s, 999m, 827m, 745m, 698s, 492m cm-1; UV-vis (CHCl3): λ (log ε)
= 250 (4.20), 337 (4.33) nm; 1H NMR (400 MHz, CDCl3):
δ = 7.59 (d, J = 15.9 Hz, 1H, 3-H),
7.35 – 7.32 (m, 2H, 4´´-H+4´´´´-H), 7.30 (dd, J = 6.7, 2.1 Hz, 2H, 2´´´´-H), 7.28 (d, J = 1.6
Hz, 4H, 2´´-H+3´´´´-H), 7.25 – 7.18 (m, 2H, 3´´-H), 6.73 (s, 2H, 2´-H), 6.32 (d, J = 15.9 Hz,
1H, 2-H), 4.44 (t, J = 7.1 Hz, 2H, 1´´´-H), 4.23 (t, J = 7.6 Hz, 2H, 6´-H), 3.84 (s, 6H, 5´-H),
13
3.10 (t, J = 7.6 Hz, 2H, 7´-H), 3.03 (t, J = 7.0 Hz, 2H, 2´´´-H) ppm; C NMR (100 MHz,
CDCl3):
δ =167.0 (C-1), 153.8 (C-3´), 145.0 (C-3), 139.4 (C-4´), 138.4 (C-1´´), 138.0 (C-
1´´´´), 130.0 (C-1´), 129.2 (C-4´´), 129.1 (C-4´´´´), 128.7 (C-2´´), 128.5 (C-2´´´´), 126.7 (C-
3´´), 126.4 (C-3´´´´), 117.3 (C-2), 105.5 (C-2´), 74.2 (C-6´), 65.1 (C-1´´´), 56.3 (C-5´), 36.7
(C-7´), 35.4 (C-2´´´) ppm; MS (ESI, MeOH): m/z (%) = 433.0 ([M+H]+, 56), 450.0
([M+NH4]+, 14), 455.1 ([M+Na]+, 41), 470.9 ([M+K]+, 2), 668.1 ([3M+Ca]2+, 16), 886.9
([2M+Na]+, 100); analysis calcd for C27H28O5 (432.52): C 74.98, H 6.53; found: C 74.59, H
6.77.
4.2.1.16 Phenethyl (E)-3-(4-acetoxy-3-methoxyphenyl)-acrylate (16)
Compound 16 was obtained as a white solid (0.11 g, 31%); RF = 0.37 (silica gel, n-
hexane/ethyl acetate, 4:1); m.p. 79 °C; IR (ATR): ν = 2956w, 1758m, 1707m, 1634m, 1601w,
1510m, 1466w, 1454w, 1419m, 1372w, 1333m, 1302w, 1261m, 1214m, 1200m, 1172s, 1149s,
1123m, 1030m, 1012m, 983m, 906m, 868m, 831m, 695m, 648m, 492m cm-1; UV-vis (MeOH):
λ (log ε) = 211 (4.45), 280 (4.47) nm; 1H NMR (400 MHz, CDCl3):
δ = 7.62 (d, J = 16.0 Hz,
1H, 3-H), 7.32 (d, J = 7.2 Hz, 1H, 6´-H), 7.27 (d, J = 1.8 Hz, 2H, 2´´´-H), 7.26 – 7.24 (m, 2H,
3´´´-H), 7.11 (d, J = 1.2 Hz, 1H, 5´-H), 7.09 (t, J = 1.9 Hz, 1H, 2´-H), 7.05 (d, J = 7.9 Hz, 1H,
16

ACCEPTED MANUSCRIPT
4´´´-H), 6.37 (d, J = 16.0 Hz, 1H, 2-H), 4.43 (t, J = 7.1 Hz, 2H, 1´´´-H), 3.86 (s, 3H, 7´-H),
13
3.03 (t, J = 7.1 Hz, 2H, 2´´´-H), 2.32 (s, 3H, 9´-H) ppm; C NMR (100 MHz, CDCl3):
δ =
168.9 (C-8´), 166.9 (C-1), 151.6 (C-3´) 144.3 (C-3), 141.6 (C-4´), 138.0 (C-1´´´), 133.6 (C-
1´), 129.1 (C-2´´´), 128.7 (C-6´), 126.7 (C-3´´´), 123.4 (C-4´´´), 121.4 (C-5´), 118.7 (C-2),
111.4 (C-2´), 65.2 (C-1´´), 56.1 (C-7´), 35.4 (C-2´´), 20.8 (C-9´) ppm; MS (ESI, MeOH): m/z
(%) = 340.9 ([M+H]+, 23), 357.9 ([M+NH4]+, 5), 363.0 ([M+Na]+, 40), 702.8 ([2M+Na]+,
100); analysis calcd for C20H20O5 (340.38): C 70.58, H 5.92; found: C 70.36, H 6.18.
4.2.1.17 Phenethyl (E)-3-(3,4-dihydroxyphenyl)-acrylate (17)
Caffeic acid (0.5 g, 2.78 mmol) and 2-phenylethanol (0.35 mL, 2.78 mmol) were dissolved in
nitromethane (62.5 mL). Yb(OTf)3 (0.017 g, 0.028 mmol) was added, and the suspension was
stirred for 5 min in an ultrasonic bath followed by an additional 1.5 h stirring under reflux.[4]
The reaction mixture was stirred at room temperature overnight and washed with NaHCO3-
solution (2%, 15 mL) and brine (15 mL). The organic phase was dried over Na2SO4 and
concentrated under reduced pressure. The crude product was purified by column
chromatography (silica gel; chloroform/methanol, 99:1), and compound 17 was obtained as a
white solid (0.07 g, 9%); RF = 0.04 (silica gel; chloroform/methanol, 99:1); m.p. 128 – 130 °C
(lit.:[19] 126 – 128 °C); IR (KBr): ν = 3480s, 1685m, 1636m, 1602s, 1535w, 1442w, 1363w,
1302m, 1279s, 1182s cm-1; UV-vis (CHCl3): λ (log ε) = 250 (4.06), 320 (4.21), 353 (4.28)
1
nm; H NMR (500 MHz, CDCl3):
δ = 7.56 (d, J = 15.9 Hz, 1H, 3-H), 7.34 – 7.30 (m, 2H,
2´´´-H), 7.27 – 7.22 (m, 3H, 3´´´-H+4´´´-H), 7.07 (d, J = 2.0 Hz, 1H, 5´-H), 7.01 (dd, J = 8.2,
2.0 Hz, 1H, 2´-H), 6.87 (d, J = 8.2 Hz, 1H, 6´-H), 6.25 (d, J = 15.9 Hz, 1H, 2-H), 4.42 (t, J =
13
7.1 Hz, 2H, 1´´-H), 3.01 (t, J = 7.1 Hz, 2H, 2´´-H); C NMR (125 MHz, CDCl3):
δ = 167.8
(C-1), 146.5 (C-4´), 145.2 (C-3´), 143.9 (C-3), 138.0 (C-1´´´), 129.1 (C-3´´´), 128.7 (C-2´´´),
127.7 (C-1´), 126.8 (C-4´´´), 122.7 (C-6´), 115.7 (C-2), 115.6 (C-5´), 144.6 (C-2´), 65.3 (C-
1´´), 35.4 (C-2´´) ppm; MS (ESI, MeOH): m/z (%) = 285.0 ([M+H]+, 45), 302.2 ([M+NH4]+,
[
23), 307.1 ([M+Na]+, 100), 446.1 ([3M+K+H]2+, 44), 580.1 ( 4M+Na+H]2+, 23), 588.0
([4M+K+H]2+, 50), 590.8 ([2M+Na]+, 42), 599.9 ([4M+Zn]2+, 48); analysis calcd for
C17H16O4 (284.31): C 71.82, H 5.67; found: C 71.69, H 5.83.
4.2.1.18 Phenethyl (E)-3-(3,4-dimethoxyphenyl)-acrylate (18)
At -20 °C (E)-3,4-dimethoxy cinnamic acid (1.00 g, 4.8 mmol) und 2-phenylethanol
(0.44 mL, 3.69 mmol) were dissolved in dry DCM (18.5 mL). DCC (0.953 g, 4.62 mmol) and
DMAP (0.067 g, 0.55 mmol) were added, and the reaction mixture was stirred at room
17

ACCEPTED MANUSCRIPT
temperature overnight. The precipitate was filtrated off, and the solvent was removed in
vacuo. The residue was purified by column chromatography (silica gel; dichloromethane),
and compound 18 was obtained as a white solid (0.798 g, 53%); RF = 0.7 (silica gel;
dichloromethane); m.p. 97 – 99 °C; IR (KBr): ν = 3455w, 3062w, 3003m, 2959m, 2937m,
2886m, 2838m, 1852w, 1702s, 1636s, 1597s, 1582m, 1515s, 1472m, 1462m, 1447m, 1415w,
1378w, 1340m, 1312m, 1290w, 1258s, 1236s, 1202w, 1178s, 1162s, 1139s, 1075m, 1040m,
1021s, 1000m, 854m, 810m, 757m, 706m cm-1; UV-vis (CHCl3): λ (log ε) = 253 (4.13), 320
(4.20), 355 (4.33) nm; 1H NMR (400 MHz, CDCl3):
δ = 7.62 (d, J = 15.9 Hz, 1H, 3-H), 7.35
– 7.22 (m, 5H, 2´´´-H+3´´´-H+4´´´), 7.10 (dd, J = 8.3, 1.9 Hz, 1H, 2´-H), 7.05 (d, J = 1.8 Hz,
1H, 5´-H), 6.87 (d, J = 8.3 Hz, 1H, 6´-H), 6.30 (d, J = 15.9 Hz, 1H, 2-H), 4.43 (t, J = 7.1 Hz,
2H, 1´´-H), 3.91 (s, 6H, 7´-H+8´-H), 3.02 (t, J = 7.1 Hz, 2H, 2´´-H) ppm; 13C NMR (100
MHz, CDCl3):
δ = 167.3 (C-1), 151.3 (C-4´), 149.4 (C-3´), 144.9 (C-3), 138.0 (C-1´´´), 129.0
(C-3´´´), 128.7 (C-2´´´), 127.6 (C-1´), 126.7 (C-4´´´), 122.8 (C-6´), 115.9 (C-2), 111.2 (C-5´),
109.8 (C-2´), 65.0 (C-1´´), 56.1 (C-8´), 56.0 (C-7´), 35.4 (C-2´´) ppm; MS (ESI, MeOH): m/z
(%) = 313.1 ([M+H]+, 30), 335.1 ([M+Na]+, 100); analysis calcd for C19H20O4 (312.36): C
73.06, H 6.45; found: C 72.88, H 6.64.
4.2.1.19 (E) 3-(3,4-Bis(benzyloxy)phenyl)-acrylic acid (19)
Caffeic acid (2.00 g, 11.1 mmol) and K2CO3 (9.14 g, 66 mmol) were dissolved in ethanol (74
mL), and benzyl chloride (3.84 mL, 4.22 g, 33.3 mmol) was added carefully. After 5 h of
stirring under reflux, the reaction was cooled to room temperature, and KOH (1.85 g,
33 mmol) was added, and the mixture was stirred overnight. The solvent was removed under
reduced pressure; the residue was solved in water (200 mL) and washed with ethyl acetate (70
mL). The aqueous phase was acidified with hydrochloric acid to pH = 5 and washed with
ethyl acetate (3 x 100 mL). The combined organic phases were washed with brine (100 mL),
dried (MgSO4), and the solvent was removed until a precipitate was formed. This suspension
was stored in the fridge overnight and filtrated. Compound 19 was obtained as an off-white
solid (2.28 g, 57%); RF = 0.04 (silica gel; chloroform/methanol, 99:1); m.p. 199 – 202 °C
(lit.:[20] 201 – 202 °C); IR (KBr): ν = 3442m, 3036w, 1670s, 1623m, 1596m, 1510m, 1453w,
1433m, 1382w, 1308m, 1268s, 1206m, 1169w, 1140m, 1021m, 695m cm-1; UV-vis (CHCl3): λ
(log ε) = 252 (3.87), 322 (3.92), 352 (3.99) nm; 1H NMR (400 MHz, CDCl3):
δ = 7.66 (d, J =
15.8 Hz, 1H, 3-H), 7.48 – 7.42 (m, 4H, 3´´´-H+4´´´-H), 7.41 – 7.29 (m, 6H, 3´´-H+4´´-H+5´´-
H+5´´´-H), 7.14 (d, J = 1.9 Hz, 1H, 5´-H), 7.09 (dd, J = 8.3, 2.0 Hz, 1H, 2´-H), 6.93 (d, J =
8.3 Hz, 1H, 6´-H), 6.24 (d, J = 15.9 Hz, 1H, 2-H), 5.20 (d, J = 8.4 Hz, 4H, 1´´-H+1´´´-H)
18

ACCEPTED MANUSCRIPT
ppm; 13C NMR (100 MHz, CDCl3):
δ = 171.2 (C-1), 151.6 (C-4´), 149.2 (C-3´), 146.9 (C-3),
137.0 (C-2´´´), 136.8 (C-2´´), 128.7 (C-4´´´), 128.6 (C-4´´), 128.1 (C-5´´´), 127.6 (C-1´),
127.5 (C-5´´), 127.4 (C-3´´´), 127.3 (C-3´´), 123.5 (C-2), 115.0 (C-6´), 114.4 (C-5´), 114.1
(C-2´), 71.6 (C-1´´´), 71.1 (C-1´´) ppm; MS (ESI, MeOH): m/z (%) = 361.1 ([M+H]+, 10),
383.1 ([M+Na]+, 100), 742.8 ([2M+Na]+, 13); analysis calcd for C23H20O4 (360.41): C 76.65,
H 5.59; found: C 76.37, H 5.81.
4.2.1.20 Allyl (E)-3-(3,4-bis(allyloxy)phenyl)-acrylate (20)
Caffeic acid (1.00 g, 5.55 mmol) and K2CO3 (0.92 g, 6.66 mmol) were dissolved in ethanol
(8 mL). Allyl bromide (1.73 mL, 2.42 g, 19.98 mmol) was added dropwise. After stirring for
5 h under reflux, the mixture was cooled to room temperature, and the solvent was removed in
vacuo. The residue was purified by column chromatography (silica gel; dichloromethane),
and compound 20 was obtained as white needles (0.37 g, 22%); RF = 0.84 (silica gel;
dichloromethane); m.p. 48 – 51 °C; IR (KBr): ν = 3449m, 3067w, 3018w, 2927w, 1706s,
1649w, 1629w, 1596m, 1512s, 1458m, 1424m, 1411w, 1342w, 1270s, 1223s, 1174m, 1143s,
1020m, 996s, 924m, 810m cm-1; UV-vis (CHCl3): λ (log ε) = 253 (4.09), 323 (4.17), 354
1
(4.26) nm; H NMR (400 MHz, CDCl3):
δ = 7.62 (d, J = 15.9 Hz, 1H, 3-H), 7.09 (d, J = 2.0
Hz, 1H, 6´-H), 7.07 (dd, J = 3.1, 2.0 Hz, 1H, 2´-H), 6.87 (d, J = 8.8 Hz, 1H, 5´-H), 6.30 (d, J
= 15.9 Hz, 1H, 2-H), 6.13 – 5.94 (m, 3H, 2´´-H+2´´´-H+2´´´´-H), 5.46 – 5.38 (m, 3H, 3´´trans
-
H+3´´´trans-H+3´´´´trans-H)), 5.36 – 5.24 (m, 3H, 3´´cis-H+3´´´cis-H+3´´´´cis-H), 4.70 (dt, J = 5.7,
13
1.4 Hz, 2H, 1´´´´-H), 4.63 (dtd, J = 4.0, 2.8, 1.5 Hz, 4H, 1´´-H+1´´´-H) ppm; C NMR (100
MHz, CDCl3):
δ = 166.9 (C-1), 150.8 (C-4´), 148.7 (C-3´), 145.0 (C-3), 133.2 (C-2´´´), 133.0
(C-2´´), 132.5 (C-2´´´´), 127.7 (C-1´), 122.8 (C-2), 118.3 (C-3´´´), 118.1 (C-3´´), 118.0 (C-
3´´´´), 115.7 (C-5´), 113.6 (C-6´), 112.9 (C-2´), 70.1 (C-1´´´), 69.9 (C-1´´), 65.2 (C-1´´´´)
ppm; MS (ESI, MeOH): m/z (%) = 301.0 ([M+H]+, 81), 323.1 ([M+Na]+, 100), 622.9
([2M+Na]+, 21); analysis calcd for C18H20O4 (300.35): C 71.98, H 6.71; found: C 71.77, H
6.96.
4.2.1.21 (E) 3-(3,4-Bis(allyloxy)phenyl) acrylic acid (21)
Compound 20 (0.66 g, 2.2 mmol) and NaOH (0.18 g, 4.4 mmol) were dissolved in ethanol (5
mL). The mixture was stirred at room temperature overnight. The solvent was removed under
reduced pressure, the crude product was purified by column chromatography (silica gel;
dichloromethane), and compound 21 was obtained as a white solid (0.29 g, 51%); RF = 0.06
(silica gel; dichloromethane); m.p. 95 – 98 °C (lit.:[21] 159 – 160 °C); IR (KBr): ν = 3448m,
19

ACCEPTED MANUSCRIPT
1666s, 1623s, 1597s, 1510s, 1426s, 1370m, 1307m, 1263s, 1204m, 1173w, 1136m, 1005m
1
cm-1; UV-vis (MeOH): λ (log ε) = 315 (4.13), 350 (4.17) nm; H NMR (400 MHz, DMSO-
d6):
δ = 7.48 (d, J = 15.9 Hz, 1H, 3-H), 7.32 (d, J = 1.9 Hz, 1H, 5´-H), 7.17 (dd, J = 8.3, 1.9
Hz, 1H, 2´-H), 6.98 (d, J = 8.4 Hz, 1H, 6´-H), 6.41 (d, J = 15.9 Hz, 1H, 2-H), 6.10 – 5.98 (m,
2H, 2´´-H+2´´´-H), 5.40 (ddq, J = 17.3, 5.3, 1.7 Hz, 2H, 3´´trans-H+3´´´trans-H), 5.25 (dp, J =
10.5, 1.5 Hz, 2H, 3´´cis-H+3´´´cis-H), 4.60 (ddt, J = 6.6, 5.3, 1.6 Hz, 4H, 1´´-H+1´´´-H) ppm;
13C NMR (100 MHz, DMSO-d6):
δ = 167.7 (C-1), 149.8 (C-4´), 147.9 (C-3´), 143.9 (C-3),
133.8 (C-2´´´), 133.5 (C-2´´), 127.3 (C-1´), 122.6 (C-2), 117.5 (C-3´´´), 117.4 (C-3´´), 116.9
(C-5´), 113.4 (C-6´), 112.4 (C-2´), 68.9 (C-1´´´), 68.8 (C-1´´) ppm; MS (ESI, MeOH): m/z
(%) = 259.0 ([M-H]-, 100), 519.1 ([2M-H]-, 56), 541.1 ([2M-2H+Na]-, 11); analysis calcd for
C15H16O4 (260.29): C 69.22, H 6.20; found: C 68.97, H 6.48.
4.2.1.22 General procedure B
Under Argon atmosphere and at -20 °C top of a solution of the carboxylic acid (1.3 eq.) in dry
DCM (5 mL / mmol), the corresponding phenethyl alcohol (1 eq.), DCC (1.25 eq.) and
DMAP (0.15 eq.) were added. The reaction mixture was stirred overnight at room
temperature. The precipitate was filtrated off, and the solvent was removed in vacuo; the
residue was purified by column chromatography (silica gel; dichloromethane). [11, 12]
Compounds 22 – 27 were synthesized following procedure B.
4.2.1.23 2-Hydroxyphenethyl (E)-3-(3,4-bis(benzyloxy)phenyl)-acrylate (22)
Compound 22 was obtained as a colourless oil (0.18 g, 36%); RF = 0.44 (silica gel;
dichloromethane); m.p. 97 °C; IR (film): ν = 3440m, 1682m, 1633s, 1596m, 1510m, 1455m,
1384w, 1263s, 1168m, 1136m, 1015w cm-1; UV-vis (CHCl3): λ (log ε) = 240 (4.27), 323
(4.32), 355 (4.41) nm; 1H NMR (400 MHz, CDCl3):
δ = 7.60 (d, J = 15.9 Hz, 1H, 3-H), 7.48
– 7.39 (m, 5H, 3´´´-H+4´´´-H+5´´´-H), 7.39 – 7.29 (m, 5H, 3´´-H+4´´-H+5´´-H), 7.18 – 7.13
(m, 2H, 4v-H+6v-H), 7.12 (d, J = 2.1 Hz, 1H, 6´-H), 7.07 (dd, J = 8.3, 2.1 Hz, 1H, 2´-H),
6.92 (d, J = 8.3 Hz, 1H, 5´-H), 6.88 (dd, J = 11.4, 4.3 Hz, 2H, 3v-H+5v-H), 6.24 (d, J = 15.9
Hz, 1H, 2-H), 5.20 (s, 2H, 1´´´-H), 5.18 (s, 2H, 1´´-H), 4.38 (t, J = 7.1 Hz, 2H, 1´´´´-H), 3.02
(t, J = 7.1 Hz, 2H, 2´´´´-H) ppm; 13C NMR (100 MHz, CDCl3):
δ
= 167.8 (C-1), 154.7 (C-2v),
151.4 (C-4´), 149.1 (C-3´), 145.5 (C-3), 137.0 (C-2´´´), 136.9 (C-2´´), 131.0 (C-6v), 128.7 (C-
4´´´), 128.5 (C-4´´), 128.1 (C-5´´+C-5´´´), 127.8 (C-1´), 127.5 (C-3´´+C-3´´´), 127.3 (C-4v),
123.7 (C-1v), 123.2 (C-6´), 120.8 (C-5v), 116.2 (C-3v), 115.6 (C-2), 114.4 (C-5´), 114.0 (C-
2´), 71.6 (C-1´´´), 71.1 (C-1´´), 64.6 (C-1´´´´), 30.5 (C-2´´´´) ppm; MS (ESI, MeOH): m/z (%)
20

ACCEPTED MANUSCRIPT
= 481.0 ([M+H]+, 5), 503.2 ([M+Na]+, 100), 983.0 ([2M+Na]+, 51); analysis calcd for
C31H28O5 (480.56): C 77.48, H 5.87; found: C 77.21, H 6.03.
4.2.1.24 2-Hydroxyphenethyl (E)-3-(3,4-bis(allyloxy)phenyl)-acrylate (23)
Compound 23 was obtained as a white solid (0.09 g, 27%); RF = 0.32 (silica gel;
dichloromethane); m.p. 119 °C; IR (KBr): ν = 3383s, 2927w, 1684s, 1628s, 1594m, 1578w,
1509s, 1458m, 1426m, 1348w, 1307m, 1264s, 1230m, 1196s, 1170s, 1132s, 1114w, 1064w,
1
1008m, 974m cm-1; UV-vis (CHCl3): λ (log ε) = 254 (3.99), 355 (4.19) nm; H NMR (400
MHz, CDCl3):
H+6v-H), 7.09 – 7.07 (m, 2H, 3v-H+5v-H), 6.90 – 7.84 (m, 3H, 2´-H+5´-H+6´-H), 6.27 (d, J =
15.9 Hz, 1H, 2-H), 6.14 – 6.02 (m, 2H, 2´´-H+2´´´-H), 5.46 – 5.41 (m, 2H, 3´´trans-H+3´´´trans
δ
= 7.63 (d, J = 15.9 Hz, 1H, 3-H), 7.14 (ddd, J = 8.0, 4.5. 1.7 Hz, 2H, 4v-
-
H), 5.32 – 5.29 (m, 2H, 3´´cis-H+3´´´cis-H), 4.64 (ddd, J = 5.5, 2.8, 1.4 Hz, 4H, 1´´-H+1´´´-H),
4.39 (t, J = 7.1 Hz, 2H, 1´´´´-H), 3.03 (t, J = 7.1 Hz, 2H, 2´´´´-H) ppm; 13C NMR (100 MHz,
CDCl3):
δ
= 167.9 (C-1), 154.7 (C-2v), 151.0 (C-4´), 148.8 (C-3´), 145.6 (C-3), 133.2 (C-
2´´´), 133.0 (C-2´´), 131.0 (C-6v), 128.5 (C-4v), 127.6 (C-1´), 123.7 (C-1v), 123.0 (C-5v),
120.8 (C-5´), 118.2 (C-3´´´), 118.1 (C-3´´), 116.2 (C-6´), 115.4 (C-2), 113.6 (C-2´), 113.0 (C-
3v), 70.2 (C-1´´´), 69.9 (C-1´´), 64.6 (C-1´´´´), 30.5 (C-2´´´´) ppm; MS (ESI, MeOH): m/z (%)
= 381.0 ([M+H]+, 19), 403.1 ([M+Na]+, 100), 783.1 ([2M+Na]+, 83); analysis calcd for
C23H24O5 (380.44): C 72.61, H 6.36; found: C 72.44, H 6.50.
4.2.1.25 4-{2-[((E)-3-(3,4-Bis(benzyloxy)phenyl)acryloyl)oxy]ethyl}phenyl (E)-3-(3,4-
bis(benzyloxy)phenyl)-acrylate (24)
Compound 24 was obtained after column chromatography (silica gel; dichloromethane) as a
white solid (0.14 g, 15%); RF = 0.51 (silica gel; dichloromethane); m.p. 172 – 175 °C; IR
(KBr): ν = 3446w, 3033w, 1731m, 1710m, 1630s, 1596m, 1578w, 1508s, 1454m, 1432m,
1383w, 1350w, 1303m, 1264s, 1199m, 1161s, 1128s, 1006m, 741m, 694m cm-1; UV-vis
(CHCl3): λ (log ε) = 248 (4.33), 356 (4.48) nm; 1H NMR (400 MHz, CDCl3):
δ = 7.74 (d, J =
15.9 Hz, 1H, 3-H), 7.56 (d, J = 15.9 Hz, 1H, 3v´-H), 7.49 – 7.42 (m, 8H), 7.41 – 7.35 (m, 8H),
7.33 (dd, J = 6.9, 3.9 Hz, 4H), 7.28 (d, J = 8.5 Hz, 2H), 7.19 – 7.05 (m, 6H), 6.93 (dd, J =
11.8, 8.4 Hz, 2H), 6.42 (d, J = 15.9 Hz, 1H, 2-H), 6.23 (d, J = 15.9 Hz, 1H, 2v´-H), 5.20 (t, J =
7.1 Hz, 8H, 1´´-H+1´´´-H+1v´´´-H+1´x), 4.41 (t, J = 7.0 Hz, 2H, 1´v-H), 3.02 (t, J = 7.1 Hz,
13
2H, 2´v-H) ppm; C NMR (100 MHz, CDCl3):
δ
= 167.5 (C-1v´), 166.0 (C-1), 154.7 (C-4´),
151.6 (C-4v´´), 149.7 (C-3´), 149.1 (C-3v´´), 146.5 (C-3v´), 144.9 (C-3), 136.9 (C-2´´+C-
2´´´+C-2v´´´+C-2´v), 130.0, 128.8, 128.7, 128.7, 128.2, 128.1, 127.5, 127.5, 127.4, 127.3,
21

ACCEPTED MANUSCRIPT
123.1, 121.8, 115.3 (C-2v´), 115.2 (C-2), 114.5 + 114.4 (C-5´+C-5v´´), 114.1 + 114.0 (C-
2´+C-2v´´), 71.5 + 71.1 (C-1´´+C-1´´´+C-1v´´´+C-1´v), 64.9 (C-1v´), 34.8 (C-2v´) ppm; MS
(ESI, MeOH/CH2Cl2): m/z (%) = 823.1 ([M+H]+, 32), 840.1 ([M+NH4]+, 24), 845.3
([M+Na]+, 100), 1668.3 ([2M+Na]+, 94); analysis calcd for C54H46O8 (822.95): C 78.81, H
5.63; found: C 78.59, H 5.89.
4.2.1.26 3-{2-[((E)-3-(3,4-Bis(benzyloxy)phenyl)acryloyl)oxy]ethyl}phenyl (E)-3-(3,4-
bis(benzyloxy)phenyl)-acrylate (25)
After column chromatography (silica gel; dichloromethane) compound 25 was obtained as a
white solid (0.42 g, 48%); RF = 0.48 (silica gel; dichloromethane); m.p. 80 °C; IR (KBr): ν =
3448w, 3034w, 2864w, 1725m, 1699s, 1629m, 1596m, 1511s, 1454m, 1434m, 1383m, 1306m,
1270s, 1241s, 1150s, 1134s, 1016m, 984m, 800m, 739m, 695s cm-1; UV-vis (CHCl3): λ (log
ε) = 248 (4.38), 358 (4.53) nm; 1H NMR (400 MHz, CDCl3):
δ = 7.74 (d, J = 15.9 Hz, 1H, 3-
H), 7.56 (d, J = 15.9 Hz, 1H, 3v´-H), 7.46 (ddd, J = 8.8, 4.4, 1.7 Hz, 6H), 7.43 – 7.40 (m, 2H),
7.37 (dd, J = 7.1, 1.2 Hz, 6H), 7.35 – 7.32 (m, 4H), 7.32 (d, J = 5.1 Hz, 2H), 7.18 – 7.13 (m,
3H), 7.11 (dd, J = 8.4, 2.1 Hz, 1H), 7.09 – 7.03 (m, 4H), 6.94 (d, J = 8.4 Hz, 1H), 6.90 (d, J =
8.3 Hz, 1H), 6.42 (d, J = 15.9 Hz, 1H, 2-H), 6.24 (d, J = 15.9 Hz, 1H, 2v´-H), 5.29 + 5.21 +
5.19 + 5.17 (s, 8H, 1´´-H+1´´´-H+1v´´´-H+1´x), 4.43 (t, J = 6.9 Hz, 2H, 1´v-H), 3.03 (t, J = 6.9
Hz, 2H, 2´v-H) ppm; 13C NMR (100 MHz, CDCl3):
δ
= 167.2 (C-1v´), 165.7 (C-1), 151.6 (C-
4´), 151.1 (C-4v´´), 149.2 (C-3´), 149.1 (C-3v´´), 146.5 (C-3v´), 144.8 (C-3), 139.8, 137.0 +
136.9 + 136.8 + 136.8 (C-2´´+C-2´´´+C-2v´´´+C-2´v), 129.6, 128.7, 128.7, 128.7, 128.6,
128.2, 128.1, 128.0, 127.7, 127.5, 127.4, 127.3, 126.4, 123.4, 123.1, 120.0, 116.1 (C-2v´),
115.2 (C-2), 114.4 + 114.3 (C-5´+C-5v´´), 114.1 + 114.0 (C-2´+C-2v´´), 71.5 + 71.1 (C-
1´´+C-1´´´+C-1v´´´+C-1´v), 64.6 (C-1v´), 35.2 (C-2v´) ppm; MS (ESI, MeOH/CH2Cl2): m/z
(%) = 822.9 ([M+H]+, 30), 840.2 ([M+NH4]+, 13), 845.3 ([M+Na]+, 100), 1667.1 ([2M+Na]+,
35); analysis calcd for C54H46O8 (822.95): C 78.81, H 5.63; found: C 78.69, H 5.91.
4.2.1.27
4-{2-[((E)-3-(3,4-bis(benzyloxy)phenyl)acryloyl)oxy]ethyl}-1,2-phenylene
(2E,2'E)-bis(3-(3,4-bis(benzyloxy)phenyl)-acrylate) (26)
Compound 26 was obtained as a white solid (0.07 g, 6%); RF = 0.27 (silica gel;
dichloromethane); m.p. 100 °C; IR (KBr): ν = 3448w, 3031w, 2864w, 1719s, 1629m, 1597m,
1509s, 1454m, 1431m, 1382m, 1304m, 1259s, 1168s, 1133s, 1007m, 982m, 840m, 802m,
734m, 695s cm-1; UV-vis (CHCl3): λ (log ε) = 248 (4.53), 357 (4.66) nm; 1H NMR (400 MHz,
CDCl3):
δ
= 7.70 (dd, J = 15.9, 3.4 Hz, 2H), 7.57 (d, J = 15.9 Hz, 1H, 3-H), 7.47 – 7.27 (m,
22

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37H), 7.24 – 7.13 (m, 4H), 7.10 – 6.98 (m, 4H), 6.90 (d, J = 8.4 Hz, 1H), 6.85 (dd, J = 8.4, 4.6
Hz, 2H), 6.37 (dd, J = 15.9, 2.3 Hz, 2H), 6.25 (d, J = 15.9 Hz, 1H, 2-H), 5.30 (s, 1H), 5.19 –
13
5.04 (m, 5H), 4.44 (t, J = 6.8 Hz, 2H, 1´v-H), 3.04 (t, J = 6.8 Hz, 2H, 2´v-H) ppm; C NMR
(100 MHz, CDCl3):
δ = 167.2, 164.9, 151.6, 151.2, 149.1, 147.1, 147.0, 144.9, 142.5, 141.3,
137.0, 136.9, 136.8, 136.7, 128.7, 128.6, 128.1, 128.0, 127.5, 127.4, 127.4, 127.3, 127.2,
124.2, 123.6, 123.5, 123.1, 116.0, 114.4, 114.2, 114.0, 113.9, 71.5, 71.4, 71.1, 71.0, 64.5 (C-
1v´), 34.8 (C-2v´) ppm; MS (ESI, MeOH/CH2Cl2): m/z (%) = 1199.3 ([M+NH4]+, 19), 1203.4
([M+Na]+, 100), 1219. 4 ([M+K]+, 41); analysis calcd for C77H64O12 (1181,35): C 78.29, H
5.46; found: C 77.99, H 5.64.
4.2.1.28 2-{2-[((E)-3-(3,4-bis(benzyloxy)phenyl)acryloyl)oxy]ethyl}phenyl (E)-3-(3,4-
bis(benzyloxy)phenyl)-acrylate (27)
Compound 27 was obtained as a white solid (0.35 g, 40%); RF = 0.49 (silica gel;
dichloromethane); m.p. 96 – 99 °C; IR (KBr): ν = 3448w, 3030w, 1714m, 1631s, 1596m,
1511s, 1492m, 1454m, 1433m, 1383m, 1303m, 1263s, 1217m, 1168s, 1134s, 1018m, 1000m,
1
750m, 695m cm-1; UV-vis (CHCl3): λ (log ε) = 248 (4.43), 354 (4.57) nm; H NMR (400
MHz, CDCl3):
δ
= 7.77 (d, J = 15.9 Hz, 1H, 3-H), 7.54 (d, J = 15.9 Hz, 1H, 3v´-H), 7.49 –
7.41 (m, 8H), 7.41 – 7.28 (m, 11H), 7.23 (td, J = 7.4, 1.2 Hz, 2H), 7.19 – 7.14 (m, 4H), 7.10
(dt, J = 4.7, 2.2 Hz, 2H), 7.02 (dd, J = 8.3, 2.0 Hz, 1H), 6.90 (dd, J = 14.3, 8.4 Hz, 2H), 6.47
(d, J = 15.9 Hz, 1H, 2-H), 6.22 (d, J = 15.9 Hz, 1H, 2v´-H), 5.20 + 5.18 + 5.17 + 5.15 (s, 8H,
1´´-H+1´´´-H+1v´´´-H+1´x), 4.42 (t, J = 6.9 Hz, 2H, 1´v-H), 2-99 (t, J = 6.9 Hz, 2H, 2´v-H)
13
ppm; C NMR (100 MHz, CDCl3):
δ
= 167.2 (C-1v´), 165.6 (C-1), 151.6 (C-4´), 151.2 (C-
4v´´), 149.6 (C-3´), 149.2 (C-3v´´), 146.8 (C-3v´), 144.7 (C-3), 137.0 + 136.9 + 136.8 + 136.8
(C-2´´+C-2´´´+C-2v´´´+C-2´v), 130.9, 130.2, 128.7, 128.7, 128.1, 128.0, 127.5, 127.4, 127.3,
126.2, 123.5, 123.0, 122.7, 116.1 (C-2v´), 114.9 (C-2), 114.4 + 114.0 (C-5´+C-5v´´), 113.9 +
113.8 (C-2´+C-2v´´), 71.5 + 71.1 (C-1´´+C-1´´´+C-1v´´´+C-1´v), 63.9 (C-1v´), 30.0 (C-2v´)
ppm; MS (ESI, MeOH/CH2Cl2): m/z (%) = 823.1 ([M+H]+, 5), 840.1 ([M+NH4]+, 18), 845.2
([M+Na]+, 100), 1668.1 ([2M+Na]+, 86); analysis calcd for C54H46O8 (822.95): C 78.81, H
5.63; found: C 78.60, H 5.81.
4.3 Biological assays
4.3.1 Solutions
Preparation of 50 mM Tris-HCl buffer solutions: Tris(hydroxymethyl)-amino-methane (606
mg) was dissolved in bi-distilled water (100 mL) and adjusted with HCl to a pH of 8.0±0.1.
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The buffer was freshly prepared and stored in the refrigerator. AChE solution 2.005 U/mL:
the enzyme (271 U/mg, 0.037 mg) was dissolved in freshly prepared buffer pH 8.0 (5 mL)
containing NaN3 (0.98 mg). BChE solution 2.040 U/mL: the enzyme (7.54 U/mg, 1.353 mg)
was dissolved in freshly prepared buffer pH 8.0 (5 mL) containing NaN3 (0.98 mg). DTNB
solution 3 mM: DTNB (23.8 mg) was dissolved in freshly prepared buffer pH 8.0 (20 mL)
containing NaCl (116.8 mg) and MgCl2 (38.0 mg). ATChI solution 15 mM: ATChI (43.4 mg)
was dissolved in bi-distilled water (10 mL). All solutions were stored in Eppendorf caps in the
refrigerator or freezer, if necessary. The pure compounds were initially dissolved in DMSO,
galantamine hydrobromide as standard for AChE and BChE was dissolved in bi-distilled
water. The final concentrations for the enzymatic assay were obtained by diluting the stock
solution with bi-distilled water. No inhibition was detected by residual DMSO or methanol
(<0.5%).
4.2.2 Ellman´s assay
A mixture of the DTNB solution (125 mL), enzyme (25 mL) and compounds solutions (25
mL, 3 different concentrations and once water) was prepared and incubated at 30 °C for 20
min. The substrate (25 mL, 4 different concentrations) was added to start the enzymatic
reaction. The absorbance data were recorded under a controlled temperature of 30 °C for 30
min at 1 min intervals. The used substrate concentrations in the test were as follows:
[ATChI]=[BTChI]=0.9375 mM, 0.625 mM, 0.325 mM, 0.1875 mM. The mode of inhibition
as well as Ki and Ki’ were determined using Lineweaver-Burk, Dixon and Cornish-Bowden
plots. Data were fitted using the Origin 6.1G software (Origin-Lab Corp., Northampton, MA,
U.S.A.).
4.2.3 Cell lines and culture conditions
The cell lines used are human cancer cell lines: 518A2 (melanoma), A549 (alveolar basal
epithelial adenocarcinoma), A2780 (ovarian carcinoma), MCF-7 (breast adenocarcinoma),
and non-malignant mouse fibroblasts NIH 3T3. Cultures were maintained as monolayers in
RPMI 1640 medium with L-glutamine (Capricorn Scientific GmbH, Ebsdorfergrund,
Germany) supplemented with 10% heat inactivated fetal bovineserum (Sigma-Aldrich
Chemie GmbH, Steinheim, Germany) and penicillin/streptomycin (Capricorn Scientific
GmbH, Ebsdorfergrund, Germany) at 37 °C in a humidified atmosphere with 5% CO2.
4.2.4 Cytotoxic assay
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The cytotoxicity of the compounds was evaluated using the sulforhodamine-B (Kiton-Red S,
ABCR) micro culture colorimetric assay. Cells were seeded into 96-well plates on day 0 at
appropriate cell densities to prevent confluence of the cells during the period of experiment.
After 24 h, the cells were treated with six different concentrations (1, 3, 7, 12, 20, and 30 µM)
minimum. The final concentration of DMSO/DMF never exceeded 0.5%, which was non-
toxic to the cells. After a 96h treatment, the supernatant medium from the 96-well plates was
discarded, the cells were fixed with 10% trichloroacetic acid (TCA) and allowed to rest at 4
°C. After 24 h fixation, the cells were washed in a strip washer and dyed with SRB solution
(100 µL, 0.4% in 1% acetic acid) for about 20 min. After dying, the plates were washed four
times with 1% acetic acid to remove the excess of the dye and allowed to air-dry overnight.
Tris base solution (200 µL, 10 mM) was added to each well and absorbance was measured at
λ = 570 nm using a 96-well plate reader (Tecan Spectra, Crailsheim, Germany). The EC50
values were averaged from three independent experiments performed each in triplicate
calculated from semi logarithmic dose response curves applying a non-linear 4 P Hills-slope
equation (GraphPad Prism5; variables top and bottom were set to 100 and 0, respectively).
Acknowledgments
Financial support by “Stiftung der Deutschen Wirtschaft” (PhD grant to JMG) is gratefully
acknowledged. Many thanks are due to Dr. D. Ströhl and his team for the NMR spectra and to
Dr. R. Kluge for the MS-measurements. IR- and UV-vis spectra were recorded by Ms V.
Simon. Cytotoxic screening was done by S. Hoenke.
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Highlights
* Novel caffeic acid phenethyl ester (CAPE) derivatives were synthesized
* Ellman’s assays showed several of them as inhibitors of AChE
* Thus, the best inhibitor gave Ki = 0.72 ± 0.31 µM, Ki´ = 1.80 ± 0.21 µM) for AChE
* Almost no inhibition was observed for BChE
* The compounds were not cytotoxic for human tumor and non-malignant NIH 3T3 cells