(±)-trans-2-phenyl-2,3-dihydrobenzofurans as leishmanicidal agents: Synthesis, in vitro evaluation and SAR analysis

Article abstract of DOI:10.1016/j.ejmech.2020.112493

Leishmaniasis, a neglected tropical disease caused by parasites of the genus Leishmania, causes a serious burden of disease around the world, represents a threat to the life of millions of people, and therefore is a major public health problem. More effective and non-toxic new treatments are required, especially for visceral leishmaniasis, the most severe form of the disease. On the backdrop that dihydrobenzofurans have previously shown antileishmanial activity, we present here the synthesis of a set of seventy trans-2-phenyl-2,3-dihydrobenzofurans and evaluation of their in vitro activity against Leishmania donovani as well as a discussion of structure-activity relationships. Compounds 8m-o and 8r displayed the highest potency (IC₅₀ < 2 μmol/L) and interesting selectivity of the antileishmanial activity over cytotoxicity against mammalian cells (SI > 4.6). Nonetheless, structural optimization as further requirement was inferred from the high clearance of the most potent compound (8m) observed during determination in vitro of its metabolic stability. On the other hand, chiral separation of 8m and subsequent biological evaluation of its enantiomers demonstrated no effect of chirality on activity and cytotoxicity. Holistic analysis of in silico ADME-like properties and ligand efficiency metrics by a simple scoring function estimating druglikeness highlighted compounds 16c, 18 and 23 as promising candidates for further development. Overall, the potential of trans-2-phenyl-2,3-dihydrobenzofurans as leishmanicidal agents was confirmed.

Full text of DOI:10.1016/j.ejmech.2020.112493

Journal Pre-proof
(±)-trans-2-phenyl-2,3-dihydrobenzofurans as leishmanicidal agents: synthesis, in
vitro evaluation and SAR analysis
Freddy A. Bernal, Marcel Gerhards, Marcel Kaiser, Bernhard Wünsch, Thomas J.
Schmidt
PII:
S0223-5234(20)30465-7
DOI:
https://doi.org/10.1016/j.ejmech.2020.112493
EJMECH 112493
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 12 March 2020
Revised Date: 6 May 2020
Accepted Date: 20 May 2020
Please cite this article as: F.A. Bernal, M. Gerhards, M. Kaiser, B. Wünsch, T.J. Schmidt, (±)-trans-2-
phenyl-2,3-dihydrobenzofurans as leishmanicidal agents: synthesis, in vitro evaluation and SAR
analysis, European Journal of Medicinal Chemistry, https://doi.org/10.1016/j.ejmech.2020.112493.
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(±)-trans-2-phenyl-2,3-dihydrobenzofurans as leishmanicidal agents: synthesis, in vitro
evaluation and SAR analysis
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a
a
b,c
d
a,*
Freddy A. Bernal , Marcel Gerhards , Marcel Kaiser , Bernhard Wünsch , and Thomas J. Schmidt
a
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Institut für Pharmazeutische Biologie und Phytochemie (IPBP); Westfälische Wilhelms Universität–
Münster; Corrensstraße 48, D-48149, Münster, Germany
b
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Swiss Tropical and Public Health Institute (Swiss TPH); Socinstr. 57, Basel CH-4051, Switzerland
c
University of Basel; Petersplatz 1, Basel CH-4003, Switzerland
d
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Institut für Pharmazeutische und Medizinische Chemie; Westfälische Wilhelms Universität–
Münster; Corrensstraße 48, D-48149, Münster, Germany
*
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E-Mail address: thomschm@uni-muenster.de
Abstract
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Leishmaniasis, a neglected tropical disease caused by parasites of the genus Leishmania, causes a
serious burden of disease around the world, represents a threat to the life of millions of people, and
therefore is a major public health problem. More effective and non-toxic new treatments are
required, especially for visceral leishmaniasis, the most severe form of the disease. On the backdrop
that dihydrobenzofurans have previously shown antileishmanial activity, we present here the
synthesis of a set of seventy trans-2-phenyl-2,3-dihydrobenzofurans and evaluation of their in vitro
activity against Leishmania donovani as well as a discussion of structure-activity relationships.
Compounds 8m-o and 8r displayed the highest potency (IC < 2 µmol/L) and interesting selectivity of
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the antileishmanial activity over cytotoxicity against mammalian cells (SI > 4.6). Nonetheless,
structural optimization as further requirement was inferred from the high clearance of the most
potent compound (8m) observed during determination in vitro of its metabolic stability. On the other
hand, chiral separation of 8m and subsequent biological evaluation of its enantiomers demonstrated
no effect of chirality on activity and cytotoxicity. Holistic analysis of in silico ADME-like properties and
ligand efficiency metrics by a simple scoring function estimating druglikeness highlighted compounds
16c, 18 and 23 as promising candidates for further development. Overall, the potential of trans-2-
phenyl-2,3-dihydrobenzofurans as leishmanicidal agents was confirmed.
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Keywords: 2-phenyl-2,3-dihydrobenzofurans; antileishmanial agents; structure-activity relationships;
Leishmania donovani; neolignan analogues.
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1. Introduction
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Visceral leishmaniasis (VL) threatens the life of millions of people around the world, mainly in tropical
and subtropical regions, where poverty and lack of good sanitary conditions are common
characteristic issues. The different forms of leishmaniasis (VL as well as the other clinical forms,
cutaneous and mucocutaneous leishmaniasis) are part of the so-called neglected tropical diseases, as
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currently defined by WHO [1]. Leishmaniasis causes the third-highest burden of disease among
parasitic diseases [2], which reflects its high relevance as a public health problem.
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Leishmaniasis is a vector-borne disease transmitted by hematophagous female sand flies, mainly
from the genera Phlebotomus and Lutzomyia, in the Old and New World, respectively [3,4]. Although
there are approximately 20 Leishmania species that are able to parasitize humans [3,5], only two of
them are responsible for VL, the most severe form of leishmaniasis [6]: L. donovani and L. infantum.
Typical symptoms of VL include anemia, progressive cachexia, intermittent fever, hepatomegaly and
splenomegaly, but most importantly, it can lead to death if it remains untreated [6,7].
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The number of currently available treatments for leishmaniasis is quite limited, pentavalent
antimonials, amphotericin B, paromomycin and miltefosine being those employed as first-line drugs
[7–9]. Unfortunately, these drugs suffer from several disadvantages, including poor efficacy and
major side effects, as well as increasing resistance. Moreover, many of them require parenteral
administration, which is often not suitable or even impossible in the setting of remote rural areas in
low-income countries where the highest burden of disease is found. Additionally, the number of
compounds entering clinical trials is still low despite increasing efforts in recent years [8]. Thus, the
search for new effective antileishmanial agents remains as a fundamental need.
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A group of synthetic analogues of dihydrobenzofuran neolignans (formally trans-(E)-2-phenyl-5-
propenyl-2,3-dihydrobenzofurans; Fig. 1) has previously demonstrated potential as antitumoral and
antileishmanial agents [10,11]. Furthermore, the antileishmanial activity for such compounds was
related to structural features using a Quasar model (quasi-atomistic receptor surface modelling) [11],
as summarized in simplified form in Fig. 1. On this background, the present paper describes the
synthesis and biological evaluation of seventy trans-2-phenyl-2,3-dihydrobenzofurans with varying
substitution patterns, in order to generate a better understanding of the structural requirements for
improving the antileishmanial activity of this kind of compounds. Structure-activity relationships are
discussed in a qualitative manner. Results of an investigation on the in vitro metabolism of the
congener with the most potent antileishmanial activity, compound 8m, as well as the chiral
separation and activity testing of its pure enantiomers, are reported. A comprehensive in silico
evaluation of bioavailability and pharmacokinetic properties in this set of compounds is also
presented to provide a rationale for further optimization of such neolignan analogues.
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Fig. 1. General chemical structure of previously reported synthetic dihydrobenzofuran neolignans
and key structural features for antileishmanial activity [11].
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2. Results and Discussion
2.1. Chemistry
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8
8
8
8
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The preparation of the 2,3-dihydrobenzofurans has been reported using several synthetic strategies
[12]. Two main approaches were chosen here for most of the products based on the previously
reported findings and aiming furthermore at changes on C-3 and C-5 (Fig. 1), as shown in Schemes 1
and 2. Oxidative coupling of cinnamate ester analogues 1a-r using Ag O [13–15] or H O /Horseradish
2
2
2
peroxidase (HRP) [16,17] afforded compounds 8a-r (Scheme 1). The same coupling using H O /HRP
2
2
on the simple phenylpropenes 2a,b led to 9a,b. This dehydrodimerization reaction was further
extended to amides 4a-d and ketones 7a-c to obtain the 2-phenyl-2,3-dihydrobenzofurans 10a-d and
11a-c, respectively. Amides 4a-c were readily prepared from the corresponding cinnamic acid
derivatives 3a,b using BOP as coupling agent [18], while 4d was obtained from 1f by direct amidation
with 2-aminoethanol [19]. Ketones 7a-c were prepared by aldol condensation [20,21] between the
aliphatic ketones 5a,b and 3,4-dimethoxybenzaldehyde, with subsequent total [22] or partial
demethylation [23]. The oxidative coupling follows a radical pathway [12,14,16,24–26], affording
complex mixtures of products with different polymerization degrees. The purification of the 2-
phenyl-2,3-dihydrobenzofurans was therefore typically challenging, resulting in overall low yields. On
the other hand, the non-dimeric compounds 13a-l were synthesized by Heck oxyarylation [27]
between the corresponding diazonium salts prepared in situ from o-aminophenols 12a-c and the
phenylpropene analogues 2a-h (Scheme 2). These alkene derivatives were mainly obtained by
reduction of the corresponding ethyl esters 1b,s-u with DIBAL-H [28]. The use of 3-phenylprop-2-en-
1-ols (2b,d-f,h) as substrates for Heck oxyarylation is herein presented for the first time. In general,
the reaction yields were comparable to those for simple phenylpropenes like 2a (used for
development of the general reaction [27] and its enantioselective version [29]), but formation of 4-
phenyl-chroman-2-ols as side products was evidenced. The obtained 2-phenyl-2,3-
dihydrobenzofurans 13a-l were then further structurally modified by halogenation with the
corresponding halosuccinimides to afford 14a-d,m, bearing a halogen atom on C-5, and 15a-d,m-s as
products of substitution on the 2-phenyl ring (typically side products; Scheme 2). Compounds 14c,d
and 13k were submitted to Heck reaction for introduction of a tert-butyl acrylate unit on C-5
affording 16c,d,k (Scheme 2). Moreover, compounds 14b,c gave 17b,c by demethylation. The same
demethylation procedure on 16c was accompanied by hydrolysis of the ester to give 18 (Scheme 2).
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01
02
03
04
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1
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1
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Scheme 1. Synthesis of trans-2-phenyl-2,3-dihydrobenzofurans by oxidative coupling (dimeric
3
products). Reagents and conditions: a) ethanolamine, Na CO , MeOH, 80 °C, 46 h; b) R NH , BOP,
2
3
2
Et N, DCM, DMF, 0 °C, 3 h; c) 3,4-dimethoxybenzaldehyde, NaOH, EtOH, rt, 24 h; d) BBr , DCM, 0 °C, 2
3
3
h; e) AlCl , DCM, 0 °C to rt, 23 h; f) Ag O, toluene/acetone (2:1), rt, 28 h; g) HRP, H O ,
3
2
2
2
phosphate/citrate buffer (pH 3.1), MeOH, rt, 3 h.
1
11
4

R2
2
3
4
R4
2a: R = H, R = OH, R = 3-OMe
2
3
4
2
2
2
b: R = OH, R = OH, R = 3-OMe
2
3
4
c: R = H, R = OMe, R = H
R3
2
3
4
d: R = OH, R = OMe, R = 3-OMe
2
a-h
2
3
4
R2
2e: R = OH, R = OH, R = H
2
3
4
R4
R1
2f: R = OH, R = OMe, R = H
NH2
R1
a
b
2
3
4
2
2
g: R = OAc, R = OMe, R = H
R3
2
3
4
h: R = OH, R = H, R = 2-Br
OH
O
1
1
1
1
2a: R = H
1
3a-l
d
1
2b: R = Br
1
2c: R = CHO
c
R2
R2
O
R2
O
X
R1
X
d
O
R3
R4
+
R3
R4
R3
O
O
R4
16c: R = R = H, R = OMe
2
4
3
1
5a-d,m-s
14a-d,m
2
3
4
1
6d: R = OH, R = R = OMe
2
3
4
e
16k: R = R = OH, R = H
e
R2
O
Br
O
Me
OH
HO
OH
R4
O
2
4
1
1
7b: R = R = OH
1
8
2
4
7c: R = R = H
1
2
3
4
1
4
2
3
1
3a: R = R = H, R = OH, R = 3-OMe
13g: R = R = H, R = OAc, R = OMe
1
2
3
4
1
3
2
4
1
1
1
1
1
3b: R = H, R = R = OH, R = 3-OMe
13h: R = R = H, R = OH, R = 2-Br
1
2
4
3
1
2
4
3
3c: R = R = R = H, R = OMe
13i: R = CHO, R = R = H, R = OMe
1
2
3
4
1
2
4
3
3d: R = H, R = OH, R = OMe, R = 3-OMe
13j: R = CH=CHCOMe, R = R = H, R = OMe
1
4
2
3
1
2
3
4
3e: R = R = H, R = R = OH
13k: R = Br, R = R = OH, R = H
1
4
2
3
1
2
3
4
3f: R = R = H, R = OH, R = OMe
13l: R = Br, R = OAc, R = OH, R = H
2
3
4
1
2
3
4
1
1
1
1
1
4a: X = Br, R = H, R = OH, R = OMe
15a: R = Br, R = H, R = OH, R = OMe, X = 2-Br
2
3
4
1
2
3
4
4b: X = Br, R = R = OH, R = OMe
15b: R = Br, R = R = OH, R = OMe, X = 2-Br
2
4
3
1
2
4
3
4c: X = Br, R = R = H, R = OMe
15c: R = Br, R = R = H, R = OMe, X = 3-Br
2
3
4
1
2
3
4
4d: X = Br, R = OH, R = R = OMe
15d: R = Br, R = OH, R = R = OMe, X = 2-Br
2
3
4
1
2
3
4
4m: X = Cl, R = H, R = OH, R = OMe
15m: R = Cl, R = H, R = OH, R = OMe, X = 3-Cl
1
2
3
4
1
1
1
1
1
1
5n: R = Br, R = R = OH, R = H, X = 3-Br
1
2
3
4
5o: R = Br, R = H, R = OH, R = OMe, X = 3-Br
1
2
3
4
5p: R = Br, R = R = OH, R = OMe, X = 3-Br
1
2
3
4
5q: R = H, R = R = OH, R = Br, X = 5-Br
1
2
3
4
5r: R = H, R = R = OH, R = H, X = 3-Br
1
2
3
4
5s: R = R = H, R = OH, R = OMe, X = 3-I
1
12
1
1
1
1
13
14
15
16
Scheme 2. Synthesis of trans-2-phenyl-2,3-dihydrobenzofurans by Heck oxyarylation (non-dimeric
products) and derivatization. Reagents and conditions: a) NOPF , ACN, rt, 2 h; b) Pd (dba) , ZnCO , rt,
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2
3
3
20 h; c) N-halosuccinimide, ACN, rt, overnight; d) PdCl , PPh or P(o-tol) , tert-butyl acrylate, K CO ,
2
3
3
2
3
DMF, 100 °C, 24 h; e) BBr , DCM, 0 °C, 2 h.
3
5

1
17
1
1
1
1
1
18
19
20
21
22
Further derivatization attempts were carried out as depicted in Scheme 3. Acetylation and tert-butyl
ether formation were successfully accomplished on 8e, affording 19 and 20, respectively.
Deoxyfluorination [30] attempts were mainly unsuccessful, leading only to traces of 21 (from 16d)
and the undesired intermediate 22 (from 13e). Hydrolysis of 8g produced 23. In addition, compounds
24a-d were obtained from 13k by Suzuki reaction.
1
23
O
O
O
O
O
O
O
O
O
O
b
O
O
O
1
9
2
0
a
R2
R1
R3
e
O
c
R4
R6
O
R5
OH
8
e,g
Ar
R1
1
1
3e,k
6d
OH
R3
O
R4
2
4a-d
2
1,22
d
1
2
1: R = CH=CHCO tBu,
2
COOH
3
4
6
2
2
2
2
4a: Ar =
HOOC
R = R = OMe, R = F
1
4
OH
22: R = R = H,
OH
OMe
O
3
6
R = R = OSO C H N
2
5 4
OMe
4b: Ar =
4c: Ar =
4d: Ar =
CHO
2
3
1
2
3
4
5
8
8
1
e: R = CH=CHCO tBu, R = CO tBu, R = OH, R = R = H
2
2
F
N
g: R = CH=CHCO Et, R = CO Et, R = OH, R = R = OMe
1
2
3
4
5
2
2
1
2
3
4
5
3e: R = H, R = CH OH, R = OH, R = R = H
2
S
1
2
3
4
5
13k: R = Br, R = CH OH, R = OH, R = R = H
2
1
2
3
4
5
1
6d: R = CH=CHCO tBu, R = CH OH, R = R = OMe, R = H
2 2
1
24
1
1
1
1
25
26
27
28
Scheme 3. Further derivatization of trans-2-phenyl-2,3-dihydrobenzofurans. Reagents and
conditions: a) Ac O, NaHCO , toluene, rt, 24 h; b) Sc(OTf) , Boc O, DCM, rt, 48 h; c) PyFluor, DBU,
2
3
3
2
toluene, rt or 0 °C, 6-24 h; d) NaOH, EtOH, rt, overnight; e) PdCl , PPh , ArB(OH) , H O/toluene/DMF
2
3
2
2
(1:4:2), 100 °C, 24 h.
1
29
1
1
1
1
1
1
1
30
31
32
33
34
35
36
All the isolated products from the reactions shown in Schemes 1-3 were effectively characterized as
trans-configured compounds using NMR spectroscopy. Although it is well known that the coupling
constant between 2-H and 3-H is not sufficient to distinguish trans- from cis-dihydrobenzofurans
[31], the chemical shifts of the carbon and hydrogen atoms in 2-position and those from the
substituent on 3-position (e.g. methyl/hydroxymethyl in non-dimeric compounds 13a-l and alkoxy
groups in dimeric compounds 8a-r) have demonstrated significant differences among isomers [31–
35] and were therefore used here as a viable means of identification. The relative configuration
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1
1
37
38
39
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assignment agreed with the typically observed and reported diastereoselectivity for both synthetic
pathways (i.e. oxidative coupling and Heck oxyarylation) [12,14,16,17,27,29,36–39]. However, the
employed reactions do not offer any enantioselectivity and thus, all synthesized compounds were
obtained as racemates.
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2.2. Biological activity and structure-activity relationships
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1
1
1
1
1
1
43
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45
46
47
48
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All compounds obtained as described in the previous section were evaluated for their antileishmanial
potential against axenically grown amastigotes of L. donovani and toxicity against rat skeletal
myoblasts (L-6 cell line). The results are shown in Table 1 and 2. The corresponding confidence
intervals for the activity values are compiled in the Supporting Information. Overall, 34% of the
compounds demonstrated to be active against the parasite with IC50 values below 10 µmol/L.
Moreover, around half of those active compounds were found to be selective against the parasite as
compared to mammalian cells (selectivity index, SI > 5).
1
50
1
1
51
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Table 1. Antileishmanial and cytotoxic activity of dimeric compounds (all compounds tested as
racemic mixtures unless stated otherwise).
IC (µmol/L)
1
R
2
50
Compound
R
SI
Ld
L-6
8
a
b
COOCH3
H
H
7.84
7.45
4.21
10.8
6.07
10.6
9.10
3.95
3.11
2.10
12.6
3.87
0.54
0.65
0.53
1.66
1.66
23.5
6.32
1.97
6.45
23.2
26.3
14.3
14.3
13.3
2.43
4.42
10.3
8.18
11.4
0.37
9.99
10.2
10.6
10.2
9.34
9.05
19.1
6.41
9.22
6.21
3.0
3.5
3.4
1.3
2.2
0.2
0.5
2.6
2.6
5.4
0.03
2.6
18.9
16.4
19.3
5.6
5.5
0.8
1.0
4.7
1.0
8
COOCH CH
2
3
8
c
COOCH(CH3)2
COO(CH ) CH
3
H
8
d
e
H
2
3
8
COOC(CH3)3
COOCH3
H
8
f
OCH3
OCH3
OCH3
OCH3
OCH3
OH
OH
OH
OH
OH
OH
OH
OH
OH
F
8
g
COOCH CH
2
3
8
h
COOCH(CH3)2
COO(CH ) CH
3
8
i
j
2
3
8
COOC(CH3)3
COOCH3
8
k
8
l
COOCH CH
2
3
8
m
COOCH(CH3)2
COOCH(CH3)2
COOCH(CH3)2
COO(CH ) CH
(
2R,3R)-8m†
2S,3S)-8m†
(
8
8
8
8
n
o
p
q
2
3
3
COOC(CH3)3
COO(CH ) CH
2
7
3
CH CH=CH
2
2
8
r
COOC(CH3)3
CH3
9
a
OCH3
7

IC (µmol/L)
1
R
2
50
Compound
R
SI
Ld
L-6
9
b
CH OH
OCH3
OCH3
OCH3
OH
160
74.2
73.8
77.2
55.5
212
0.5
8.3
1.4
0.7
1.0
1.4
1.1
0.7
37.6
2
1
0a
0b
CONH(CH ) CH
3
8.88
53.6
82.3
212
2
2
1
CONHCH(CH3)2
CONHCH(CH3)2
CONH(CH ) OH
1
0c
10d
11a
11b
OCH3
OCH3
OH
2
2
COCH CH(CH )
3.81
4.42
3.42
6.89
0.77
5.29
5.00
2.45
259
2
3 2
COCH CH(CH )
2
3 2
1
1c
COCH C(CH )
OH
2
3 3
2
3
COOH
OCH3
Miltefosine
Podophyllotoxin
0.017
1
1
1
53
54
55
† Data from a separate determination scaled by the relative IC value of the positive control,
50
miltefosine. Absolute values for miltefosine and the tested compounds in this experiment were
higher by factor 6.6.
1
1
56
57
1
1
58
59
Table 2. Antileishmanial and cytotoxic activity of non-dimeric compounds (all compounds tested as
racemic mixtures).
IC (µmol/L)
1
R
2
3
50
Compound
R
R
SI
Ld
L-6
1
3a
3b
H
H
CH3
59.0
185
84.4
1.4
1
CH OH
58.0
154
0.3
1.6
2
1
3c
H
CH3
99.9
1
3d
3e
H
H
H
CH OH
136
158
70.3
53.7
217
0.4
1.4
0.6
2
1
CH OH
2
1
3f
CH OH
43.6
2
8

IC (µmol/L)
1
R
2
3
50
Compound
R
R
SI
Ld
L-6
1
3g
H
H
CH OAc
58.4
43.9
0.8
2
1
3h
CH OH
21.7
30.5
25.4
43.8
1.2
1.4
2
1
3i
CHO
CH3
CH3
O
1
3j
17.4
9.35
0.5
Me
1
3k
Br
Br
Br
Br
CH OH
18.8
13.7
24.8
30.5
22.0
55.5
13.1
32.3
1.2
4.0
0.5
1.1
2
1
3l
CH OAc
2
1
4a
4b
CH3
1
CH OH
2
1
4c
Br
Br
CH3
31.8
33.8
60.3
21.2
1.9
0.6
1
4d
CH OH
2
1
4m
Cl
Br
Br
CH3
CH3
16.1
15.0
10.7
55.4
18.2
40.6
3.4
1.2
3.8
1
5a
1
5b
CH OH
2
1
5c
Br
Br
CH3
29.7
11.4
39.7
21.0
1.3
1.8
1
5d
CH OH
2
1
5m
Cl
CH3
13.4
64.2
4.8
9

IC (µmol/L)
1
R
2
3
50
Compound
R
R
SI
Ld
L-6
1
1
5n
5o
Br
Br
CH OH
21.8
64.8
3.0
2
CH3
17.2
12.1
44.8
45.4
2.6
3.8
1
1
5p
5q
Br
CH OH
2
H
H
H
CH OH
10.1
43.8
13.4
109
115
46.9
10.8
2.6
2
1
5r
5s
CH OH
2
1
CH3
CH3
3.5
1
6c
4.01
10.7
17.1
40.1
22.9
33.8
10.0
2.1
1
6d
CH OH
2
1
6k
7b
CH OH
2.0
2
1
Br
Br
CH OH
17.8
32.5
5.64
3.93
56.6
73.9
0.2
1.7
2
1
7c
CH3
CH3
O
1
8
9
13.1
HO
1
2.06
11.8
5.7
2
2
0
1
13.1
13.0
202
15.5
2.6
CH2F
34.0
1
0

IC (µmol/L)
1
R
2
3
50
Compound
R
R
SI
Ld
L-6
2
2
H
6.31
189
29.9
2
4a
4b
CH OH
16.5
37.7
18.8
44.5
55.9
52.4
15.8
2.7
1.5
2.8
1.0
2
2
CH OH
2
2
4c
CH OH
2
2
4d
CH OH
16.1
0.77
2
Miltefosine
Podophyllotoxin
0.017
1
60
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
Structure-activity relationships (SARs) were separately analyzed for the dimeric (Table 1) and the
non-dimeric (Table 2) compounds as disclosed below. Within the former group (Table 1), the
antileishmanial activity showed dependence on the lateral chain of the esters 8a-p. Thus, larger and
bulkier lateral chains typically led to higher activities, isopropyl and tert-butyl being those conferring
better leishmanicidal potential (compounds 8c,j,m-o). Further increase of length of the alkyl chain
(8p), however, led to marked loss of activity accompanied by toxicity (SI < 1). Unsaturation on the
2
lateral chain seemed not to benefit the activity (8q). Hydroxy groups on the aromatic rings (R = OH,
Table 1; 8k-o) provided significantly better activity values than both the corresponding methoxy
derivatives (8f-j) and the non-substituted compounds (8a-e). Despite compound 8m showed the best
activity among the whole set of products, 8n and 8o displayed comparable values. Their SIs were
lower than that of 8m, though. Replacement of OH by F (8o vs 8r) led to similar antileishmanial and
cytotoxic activities. Surprisingly, the amide analogues 10b-d were inactive against the parasite. Only
10a exhibited some activity, indicating that the general trends observed for the esters are no longer
valid for the amides (e.g. OH did not improve the activity; 10b vs 10c). Although the presence of OH
groups for the ketone 11b seemed not appear to play a role as important as for the esters (when
compared to 11a bearing OMe groups instead), all the ketones (11a-c) were actually active against L.
donovani. Unfortunately, those compounds showed higher toxicity against the mammalian cells than
the corresponding esters (8h,m,o), which resulted in significantly lower SIs. On the other hand, the
carboxylic acid 23 displayed antileishmanial activity lower than the most active esters and ketones.
Yet its almost inexistent toxicity against the L-6 cells rendered it the most selective compound within
this group. Compound 9b, the alcohol corresponding to the carboxylic acid 23, was totally inactive.
Additionally, the less polar 9a, bearing a methyl group instead of the hydroxymethyl group of 9b, was
as active as the acid 23. However, the replacement of COOH by CH was not useful in terms of
3
cytotoxicity, 9a being quite toxic and non-selective.
1
1

1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
00
01
02
03
04
05
06
07
08
09
In general, most of the non-dimeric 2-phenyl-2,3-dihydrobenzofurans were significantly less active
than the dimers as clearly observed in Table 2. Substitution on C-5 was found to be essential for the
leishmanicidal potential as inferred from the lack of activity in all the compounds without any
substitution on this position (13a-g). Although introduction of Br on C-5 (13k,l and 14a-d) led to
improved antileishmanial activity (three- to four-fold more active than unsubstituted congeners), the
IC values for those compounds were still too high to be considered interesting candidates (only
50
three of them have IC < 20 µmol/L). For these brominated compounds, changes from methyl to
50
hydroxymethyl on C-3 did not lead to any significant difference. Acetylation of the hydroxymethyl
moiety of 13k did not lead to higher activity (13l) but improved selectivity. Moreover, only 4-
hydroxyphenyl groups on C-2 seemed to confer better activity (13k,l). However, this was not a
defined rule, as 17c was not more active than its respective congeners 14a,c. Interestingly, the
presence of a second OH group on the pending phenyl ring (3,4-dihydroxyphenyl) did not increase
the activity in this series (17b vs 13k) as would be expected by comparison with the dimeric products,
where catechol units led to improved activity (8l-o). Use of Cl instead of Br (14m vs 14a) provided not
only slightly better potency but also a marked reduction in cytotoxicity (increasing the SI 6.5 times).
Introduction of another halogen atom on the 2-phenyl ring afforded products with variable
antileishmanial potential (15a-d,m-s). While the second halogen caused only slight changes in activity
for compounds with methyl groups on C-3 (15a,c,m,o vs 14a,c,m,a, respectively), this modification
typically improved the activity by a factor of about 2 for those substances bearing a hydroxymethyl
moiety on C-3 (with exception of 15n; 15b,d,p vs 14b,d,b, respectively). Regardless of the group on
C-3, a clear improvement in selectivity was observed in most of dihalogenated cases. A marked
increase in antileishmanial activity was also found for compounds with halogen substituents only on
the 2-phenyl ring (15q-s). This was accompanied by a rise of selectivity, as highlighted by the
significant SI of 15q. This compound was not as active as many of the dimeric ones in Table 1 but its
selectivity was far better than that of most dimers.
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
No difference in activity was observed when Br (14c) was replaced by CHO (13i; Table 2), whereas the
presence of an α,β-unsaturated carbonyl moiety (13j) enhanced the activity. A similar effect was
generally observed when an acrylate unit was introduced on C-5. Thus, compound 16c was eight
times more active than the corresponding brominated analogue 14c, making it part of the group of
most active compounds. The acrylate moiety in 16c also rendered it 5.2-fold more selective than 14c.
A similar improvement of activity and selectivity was also observed with 16d in comparison with 14d,
although at lesser extent. Surprisingly, introduction of the acrylate moiety on 13k showed little effect
on either activity or selectivity (16k). Direct comparison of 16k (Table 2) with 8e (Table 1) would
indicate that the presence of a tert-butylcarboxylate on C-3 might be important for the activity.
Nevertheless, only an insignificant difference in selectivity was observed between these two
compounds. It should be noted that 16k did not follow the trend described above for 16c,d so that a
clear conclusion cannot be drawn. Regarding changes on C-3, replacement of OH by F from 16d to 21
did not improve the activity (Table 2). Presence of a free acrylic acid moiety instead of Br led to a 5.8-
fold increase of activity and a more than seven-fold improvement of selectivity (18 vs 17c; SI = 1.74
and 13.10, respectively). Insertion of a third aromatic ring on C-5 (24a-d) instead of the acrylate
moiety (16k) did not lead to an improvement but rather a decrease of the leishmanicidal potential.
However, 24a,c were less cytotoxic than 16k and 13k. The undesired product 22, obtained after an
unsuccessful deoxyfluorination attempt, was found to be the only compound without substitution on
C-5 with an IC value below 10 µmol/L, which also showed one of the best SI values among the
50
analyzed compounds.
2
2
30
31
Additional interesting findings were obtained from blocking the phenol group on 8e (Table 1).
Etherification with a tert-butyl group led to 20 (Table 2), which was only half as potent but seven
1
2

2
2
32
33
times more selective than 8e. In contrast, acetylation of 8e gave the three-fold more potent 19, the
most potent within the series of non-dimeric compounds, whose selectivity was also improved.
2
2
2
34
35
36
2.3. Effect of chirality on the bioactivity of 8m
2
2
2
2
2
2
2
2
2
2
2
2
2
37
38
39
40
41
42
43
44
45
46
47
48
49
The compound with the highest antileishmanial potency also showing promising selectivity, 8m, was
investigated in more detail. As mentioned above, all compounds of this study were synthesized in
racemic form, so that it was of special interest to investigate whether the two enantiomers of 8m
would differ in activity. To study the effect of chirality at C-2 and C-3 on the biological activity, 8m
was subjected to preparative chiral HPLC separation (Fig. 2A). Measurement of the electronic circular
dichroism (ECD) spectra for both pure enantiomers (2R,3R)-8m and (2S,3S)-8m, and subsequent
comparison with the corresponding calculated spectra obtained by time-dependent density
functional theory (TDDFT) computation (Fig. 2B) allowed to establish their absolute configuration.
Thus, the first eluted enantiomer was defined as (2R,3R)-8m. Conversely, the second eluted
enantiomer corresponded to (2S,3S)-8m. These results were in agreement with previously reported
CD assignments [10,15]. Moreover, all the conformers for (2R,3R)-8m optimized at DFT level
effectively showed M helicity (Fig. 2C), following the empirical rule proposed by Antus et al. [40],
1
which is connected to a negative Cotton effect for the L band (appearing over 280 nm).
b
2
50
1
3

2
51
2
2
2
2
2
52
53
54
55
56
Fig. 2. Chiral HPLC profile for 8m (A), calculated ECD spectra for (2R,3R)-8m and experimental ECD for
both (2R,3R)-8m and (2S,3S)-8m (B), and lowest energy conformer for (2R,3R)-8m showing M helicity
(C). The racemic mixture was separated on a Chiralpak IA column (5 μm, 250 mm × 20 mm) eluted at
a flow rate of 10 mL/min with isohexane-EtOH (9:1). TDDFT calculated ECD spectrum for the
weighted ensemble of five conformers at B3LYP/6-31G(d,p) level of theory.
2
57
2
2
2
2
2
58
59
60
61
62
Both pure enantiomers were tested for antileishmanial and cytotoxic activity as shown in Table 1.
Although the antileishmanial activity of (2R,3R)-8m seems slightly lower than that for its enantiomer,
statistically significant differences were not observed as indicated by ANOVA (p = 0.569 from
triplicates). This result might be seen as an advantage, offering then the possibility of avoiding chiral
separations or enantioselective synthetic routes.
1
4

2
2
63
64
2.4. In vitro metabolism of 8m in mouse liver microsomes
2
2
2
2
2
2
2
2
2
65
66
67
68
69
70
71
72
73
The usefulness of liver microsomes for in vitro studies of metabolic stability of potential drugs has
been demonstrated [41]. Therefore, the metabolism of the most active neolignan analogue, 8m, was
studied using a method previously described and optimized [42]. After 90 min of incubation of
compound 8m (25 μmol/L) with mouse liver microsomes at 37 °C, quantification of the amount of
residual parent compound was conducted by LC-MS. Only 41.8% (±1.1%) of 8m remained unchanged
after treatment under phase I conditions. Combination of phase I + II conditions led to 31.4% (±0.2%)
of residual 8m. These results indicated that 8m is easily transformed when incubated with the
microsomes, which represents an important limitation for further development as leishmanicidal
agent.
2
2
2
2
2
2
2
2
74
75
76
77
78
79
80
81
Extensive analysis of the LC-MS profiles using higher concentration of 8m (100 μmol/L) revealed that
its high in vitro metabolization rate was mainly due to hydrolysis of one ester group (early hydrolysis
under phase I conditions) and glucuronidation (under phase II). Thus, the major metabolites detected
were the glucuronidated 8m-A, and the two partially hydrolyzed 8m-B and 8m-C (Fig. 3). The exact
position of the glucuronic acid unit in 8m-A as well for the residual isopropyl group in 8m-B and 8m-C
were not determined so the structures are shown only for illustrative purposes. Additionally, the
minor metabolite 8m-D was also found. It could come from hydrolysis of 8m-A or glucuronidation of
8m-B or 8m-C.
2
82
1
5

2
2
83
84
Fig. 3. Proposed metabolism pathway for 8m under in vitro conditions using mouse liver microsomes.
Numbers in parenthesis represent the relative percentage of each metabolite in the profile.
2
2
85
86
2.5. Efficiency metrics and in silico ADME
2
2
2
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
87
88
89
90
91
92
93
94
95
96
97
98
99
00
01
02
03
04
05
06
07
08
09
10
11
12
13
14
The low metabolic stability of 8m suggested that it might be useful to assess the potential of the
more selective compounds from our set of synthesized 2-phenyl-2,3-dihydrobenzofurans as possible
candidates for further development using theoretical predictions of their ADME properties with
established computational methods. To this end, topological descriptors related to ADME properties
were calculated for the more selective compounds of our series (SI ≥ 3; Table 3). Two calculated
water/octanol distribution coefficient (cLogP) values were independently obtained and included (one
representing the consensus calculation of five different methods employed by the SwissADME
webtool [43] and another calculated by the Molecular Software Environment, MOE [44]).
Furthermore, calculated ligand efficiency metrics are known to be useful in drug discovery pipelines
[45–48]. Therefore, the ligand efficiency (LE), lipophilic efficiency (LipE), binding efficiency index (BEI),
and surface efficiency index (SEI) were also calculated using the number of Hydrogen bond acceptors
(HBA), cLogP, molecular weight (MW), and the topological polar surface area (TPSA), respectively
[46,48]. The latter was also obtained from MOE. The results obtained for these theoretical
descriptors are shown together with the activity and selectivity data in Table 3. As can be seen, the
most selective compounds covered a narrow activity range compared to the whole range of activity
found (Table 1 and 2). The table entries are traffic light color-coded using different thresholds, with
green representing a suitable property, yellow for acceptable values, and red in cases where the
value is unfavorable for drug development. In case of MW, HBA, Hydrogen bond donors (HBD), and
cLogP, compliance with the rule of five (Ro5; compliance: green) [49] and the rule of three (Ro3;
compliance: yellow) [50] were used to delimit them. The thresholds for the ligand efficiency metrics
were arbitrarily defined taking into account that representative values for such metrics are typically
calculated rather using pK (for target-specific drugs). SI and activity values were also ranked using
i
arbitrarily selected cutoffs. The use of these metrics and ranges were considered to give an easy
overview to the compounds’ characteristics, and to represent a tool to rank interesting candidates
beyond a purely potency-based criterion. From the selected group of compounds, only four (8r, 19,
20, and 22) violated the Ro5. Those compounds exceeded the threshold for cLogP (> 5 in both cases
for 8r and 20 and in one case for 19 and 22). In case of 22, it has also a MW exceeding the Ro5
threshold.
1
6

Table 3. Ligand efficiency metrics and in silico ADME-related properties for selected compounds.
IC Ld
50
a
cLogP 1 cLogP 2
b
c
d
e
f
g
h
Compound
SI
MW (Da)
HBA
HBD
LE
BEI
SEI
LipE 1
LipE 2
Score
(
µmol/L)
7.45
4.21
2.10
0.54
1.66
1.66
1.97
8.88
13.7
16.1
10.7
13.4
12.1
10.1
13.4
4.01
5.64
2.06
13.1
6.31
6.89
8
b
3.5
3.4
382.412
410.466
498.572
442.464
470.518
470.518
474.500
468.550
363.207
290.746
430.092
325.191
430.092
400.066
382.197
366.457
296.322
480.557
494.628
524.574
6
6
8
8
8
8
8
6
4
3
4
3
4
3
3
4
4
7
6
9
8
1
1
1
3
3
3
1
3
1
1
2
1
2
2
1
0
2
0
0
0
3.41
4.01
4.53
3.28
3.88
3.77
5.14
3.33
3.34
3.60
3.56
4.11
3.47
3.48
3.72
4.59
2.92
4.84
5.72
3.43
1.97
3.75
4.27
4.69
3.32
4.36
3.85
5.13
3.58
3.52
3.45
3.90
4.18
3.92
4.04
3.90
4.67
3.00
5.45
5.95
5.37
0.25
0.25
0.22
0.27
0.23
0.23
0.23
0.20
0.30
0.33
0.31
0.32
0.31
0.34
0.33
0.27
0.33
0.22
0.19
0.20
13.4
13.1
11.4
14.2
12.3
12.3
12.0
10.8
13.4
16.5
11.6
15.0
11.4
12.5
12.8
14.7
17.7
11.8
9.9
6.2
6.6
1.72
1.37
1.15
2.99
1.90
2.01
0.57
1.72
1.52
1.19
1.41
0.76
1.45
1.51
1.15
0.81
2.33
0.85
-0.84
1.77
1.38
1.11
0.99
2.94
1.42
1.93
0.57
1.47
1.34
1.34
1.07
0.69
1.00
0.95
0.97
0.73
2.24
0.24
-1.07
-0.17
3.03
9
10
11
16
9
8
c
8
j
5.4
5.6
8
m
18.9
5.6
5.1
8
n
o
4.7
8
5.5
4.7
11
6
8
r
4.7
7.0
1
0a
3l
4m
8.3
4.8
8
1
4.0
8.7
8
1
1
3.4
12.4
8.4
11
8
1
5b
3.8
5m
4.8
12.6
8.3
11
8
1
5p
5q
3.8
1
10.8
3.5
10.0
12.6
12.1
7.9
11
10
13
19
9
1
5s
6c
1
10.0
13.1
5.7
1
8
9
0
2
3
1
6.5
2
2
2
15.5
29.9
6.9
0
9.9
4.3
2
37.6
b
386.356
c
3
2.13
e
0.25
ꢃꢄꢅꢆꢇ
13.4
4.2
3.19
g
15
a
h
d
f
Consensus from SwissADME; from MOE; LE ≈ 1.37ꢀpIC /HA; BEI = pIC /MW; SEI =
; LipE = pIC − cLogP 1; LipE = pIC − cLogP 2;
ꢁꢂ
ꢁꢂ
ꢁꢂ
ꢁꢂ
ꢈꢉꢊ⁄ꢋꢂꢂꢌ
green = +2 points, yellow = +1 point, red = - 2 points
1
7

Aiming at a simple ranking of the compounds based on the properties presented in Table 3 and
considering the “desirability” criteria published by Bickerton [51] as fundamental guide, the
qualitative analysis represented by the color code in Table 3 was employed to afford a quality score.
It was arbitrarily defined assigning two points for each parameter in green, one point for each
parameter in yellow and penalizing with two points any parameter in red. The quality score of each
compound is presented in the rightmost column of Table 3. High scores indicate low incidence of
parameters within the unfavorable red-colored range and prevalence of good or acceptable
parameters (green and yellow, respectively), which would represent an overall estimate of better
candidates. A look at the score for the selected compounds makes clear the extremely poor quality
of 20 and 22 as possible candidates for further development. Many of the other compounds were
ranked in an intermediate range. However, four compounds (8m, 16c, 18, and 23) were indicated to
be the best candidates through this estimation of quality. These compounds were structurally rather
dissimilar so that, besides 8m (with the limitations mentioned above), 16c, 18 and 23 would appear
interesting candidates for further optimization despite their somewhat lower activity. Based on its
highest score value in this assessment, 18 could be considered as the best candidate. Due to its low
MW and cLogP (fulfilling the Ro3), structural optimization of 18 appears more than feasible. The four
mentioned compounds were moreover predicted to be able to pass the gastrointestinal barrier
according to the brain or intestinal estimated permeation method implemented in the SwissADME
[43]. However, 18 was anticipated by the SwissADME calculations to inhibit two out of five major CYP
isoenzymes, CYP1A2 and CYP2C9. Thus, attempts to optimize this compound should not only pursue
an increase of antileishmanial potency but also strive to avoid this interaction. In this regard, 23,
even though somewhat less active and presenting a lower score in the above-mentioned
investigation, might be similarly suitable for optimization since it was not predicted to inhibit any of
the CYP enzymes included in the calculations.
3
. Conclusion
In the present research, a medium size set of structurally diverse trans-2-phenyl-2,3-
dihydrobenzofurans was successfully prepared and evaluated against L. donovani. The reasonably
high activity (IC < 10 µmol/L) of more than 30% of the analyzed compounds demonstrated their
50
potential as leishmanicidal agents. Furthermore, the wide range of activity values observed indicates
that the desired biological activity strongly depends on the molecular structure. Qualitative SAR
analysis, even though rather complex, confirmed the previously reported importance of the lateral
chain for the antileishmanial activity of dehydrodimers bearing ester functionalities. Structurally
related ketones, synthesized to avoid possible instability due to ester hydrolysis, were less active and
more toxic than the corresponding esters, while amide analogues resulted to be inactive. In general,
dehydrodimers were significantly more active than the corresponding non-dimeric congeners,
highlighting the fundamental role of acrylate moieties on C-5 for the antileishmanial activity. Double
halogenation on non-dimeric compounds usually increased activity and selectivity. It was also found
that the presence of catechol moieties represented an activity-improving unit in case of
dehydrodimers. Biological testing of both enantiomers of 8m revealed no effect of chirality on
activity. The limited metabolic stability of 8m (evaluated in vitro with the racemate) represents an
impediment for further development. However, a ranking of the more selective compounds (SI > 3)
using a simple quality approach based on in silico determined properties, such as compliance with
Ro5 and Ro3, as well as activity, selectivity and ligand efficiency metrics, pointed towards compounds
1
6c, 18 and 23 as interesting candidates for future attempts at structural optimization.
1
8

4
. Experimental
4
.1. Chemistry: General methods and instrumentation
All the solvents and chemicals were used as obtained from Merck, Sigma-Aldrich, Thermofisher Acros
Organics, and Fluorochem. NMR spectra were recorded on an Agilent DD2 400 MHz and an Agilent
DD2 600 MHz spectrometers (the latter equipped with a cryo-probe). Exact mass determinations
were accomplished by UHPLC/+ESI-QqTOF-MS as follows: Chromatographic separations were
performed on a Dionex Ultimate 3000 RS Liquid Chromatography System (UHPLC) on a Dionex
Acclaim RSLC 120, C18 column (2.1 x 100 mm, 2.2 µm) at 40 °C with a binary gradient (A: water with
0
0
.1% formic acid; B: acetonitrile with 0.1% formic acid) at 0.8 mL/min: 0 to 0.4 min: isocratic at 5% B;
.4 to 9.9 min: linear from 5% B to 100% B; 9.9 to 15.0 min: isocratic at 100% B; 15.0 to 15.1 min:
linear from 100% B to 5% B; 15.1 to 20 min: isocratic at 5% B. The injection volume was 2 µL. Eluted
compounds were detected using a Dionex Ultimate DAD-3000 RS over a wavelength range of 200-
4
00 nm and a Bruker Daltonics micrOTOF-QII time-of-flight mass spectrometer equipped with an
Apolo electrospray ionization source in positive mode at 3 Hz over a mass range of m/z 50-1500 using
the following instrument settings: nebulizer gas nitrogen, 4 bar; dry gas nitrogen, 9 L/min, 220 °C;
capillary voltage 4500 V; end plate offset -500 V; transfer time 100 µs; collision gas nitrogen; collision
energy and collision RF settings were combined to each single spectrum of 1250 summations as
follows: 624 summations with 80 eV collision energy and 130 Vpp + 313 summations with 16 eV
collision energy and 200 Vpp + 313 summations with 16 eV collision energy and 200 Vpp. Internal
dataset calibration (HPC mode) was performed for each analysis using the mass spectrum of a 10
mmol/L solution of sodium formiate in 50% isopropyl alcohol that was infused during LC re-
equilibration using a divert valve equipped with a 20 µL sample loop. The purity of all compounds
was assessed by this method. With exception of 11b, the purity of all compounds was at least 95%.
The preparation of the intermediate cinnamate ester analogues 1a-u and 2b was carried out as
recently described [52]. The synthesis of 8a,f,g,k-o, 9a,b, 13a,c, 14c, and 17c has been published
before [10,11,15,16,27,29,39].
4
.2. Synthetic procedures
4
.2.1. Synthesis of cinnamic amides (General procedure A)
The procedure was adapted from literature [18]. To a solution of ferulic or caffeic acid (3a,b; 1 mmol)
in dry DMF (2.0 mL), Et N (1 mmol) was added and the mixture cooled to 0 °C. The corresponding
3
amine (1 mmol) was added to the solution, followed by addition of solution of BOP (1 mmol) in dry
CH Cl (2.0 mL) over a period of 30 min. The mixture was stirred (from 0 °C to rt) until full conversion
2
2
by TLC was observed. Afterwards, the volatile solvent was evaporated, and the residue was diluted
with H O (15 mL) and extracted with EtOAc (3x). The combined organic layers were successively
2
washed with 1 mol/L HCl (2x), 1 mol/L NaHCO (2x), H O (3x), and afterwards dried over anhydrous
3
2
Na SO . The organic solvent was removed under reduced pressure and the product purified as
2
4
indicated.
.2.2. Synthesis of cinnamic ketones (General procedure B)
4
The procedure was based on previous reports in aldol condensation of benzaldehyde derivatives
20,21]. 3,4-dimethoxybenzaldehyde (9 mmol), EtOH (15.0 mL), and the appropriate ketone
[
1
9

derivative (27 mmol) were mixed. A 5% NaOH aqueous solution (720 µL) was then slowly added to
the mixture over a period of 15 min. The resulting solution was stirred at rt until full conversion
observed by TLC. Afterwards, the solvent was evaporated under reduced pressure and the resulting
yellow resin was taken up in H O (75 mL) and extracted EtOAc (3x). The combined organic layers
2
were washed with H O, dried over anhydrous Na SO , filtered and concentrated under reduced
2
2
4
pressure. The pure product was obtained by chromatographic separation as indicated.
.2.3. Demethylation of cinnamic ketones (General procedure C)
4
The procedure was adapted from literature [23]. To a solution of the corresponding compound 6a,b
1.5 mmol) in dry CH Cl (9.0 mL) at 0 °C, a dilution of 1 mol/L BBr (6.0 mL, 6.0 mmol) in CH Cl (12.0
(
2
2
3
2
2
mL) was dropwise added. The resulting mixture was stirred at rt for 2 h and then carefully quenched
with cold water. The aqueous layer was extracted with CH Cl (3x). The combined organic layers were
2
2
dried over anhydrous MgSO and the solvent removed under reduced pressure. The product was
4
purified as indicated.
4
.2.4. Oxidative coupling with Ag O (General procedure D)
2
The protocol was adapted from literature [15]. The corresponding cinnamate analogue (0.5 mmol),
silver(I) oxide (0.25 mmol), dry toluene (1.7 mL) and dry acetone (1.0 mL) were mixed together under
nitrogen. The mixture was stirred for 28 h at rt in absence of light. The inorganic material was then
filtered off and the organic solvent removed under reduced pressure. The product was purified out
of the residue by chromatography as indicated.
4
.2.5. Oxidative coupling with HRP (General procedure E)
The procedure was carried out according to literature [16]. The corresponding cinnamate analogue
(
(
0.5 mmol) was dissolved in acetone (2.5 mL) and diluted under stirring with citrate-phosphate buffer
19 mmol/L, pH 3.1, 22.5 mL). Horseradish peroxidase in buffer (1.48 mg/mL, 707 μL, ∼ 210 U) and 1
mol/L H O (250 μL, 0.25 mmol) were portion-wise added over a period of 20 min. The reaction was
2
2
stirred for 3 h at rt. Afterwards 5% NaCl (5 mL) was added and the organic solvent carefully removed.
The residual mixture was extracted with EtOAc (3x) and the product purified as indicated.
4
.2.6. Synthesis of o-aminophenols (General procedure F)
The o-aminophenols 12b,c were prepared by nitration of the corresponding phenol [53] and
subsequent reduction [54]. The corresponding phenol (10 mmol) was dissolved in acetone (35 mL)
and Bi(NO ) •5H O (5 mmol) was added. The mixture was stirred at 50 °C. Afterwards, the inorganic
3
3
2
material was filtered off over a Celite® pad and the solvent removed under reduced pressure. The
residue was chromatographed as indicated to afford the respective nitrophenol.
Tin(II) chloride dihydrate (20 mmol) was dissolved in MeOH (17.2 mL) with conc. HCl (9.2 mL) and the
resulting solution cooled to 0 °C. The corresponding purified nitrophenol (4 mmol) was then added in
one portion and the mixture stirred at rt overnight to end with a colorless solution. The mixture was
diluted with EtOAc and neutralized with saturated NaHCO solution. The inorganic material was
3
removed by filtration and the residue washed with EtOAc. Filtrates were combined and phases
separated. The residual aqueous phase was further extracted with EtOAc (3x). The combined organic
layers were dried over magnesium sulfate and the organic solvent removed under reduced pressure.
The product was used without additional purification.
4
.2.7. Synthesis of 3-phenylprop-2-en-1-ols (General procedure G)
2
0

Prepared following a procedure adapted from literature [28,55]. The corresponding cinnamate ester
1 mmol) was dissolved in dry toluene (10 mL) under nitrogen. The solution was cooled to 0 °C and
.2 mol/L DIBAL-H (in toluene, 3.3 mL, 4 mmol) was dropwise added (via syringe). Afterwards, the
(
1
stirring was continued for 15 min at 0 °C and for 2 h more at rt. The reaction was then quenched by
carefully adding EtOH. The organic solvent was partially removed under reduced pressure. Water was
then added, and the gelatinous precipitate was filtered off. The precipitate was washed with EtOAc.
The filtrate was extracted with EtOAc (3x). All the organic layers were combined and dried over
anhydrous Na SO . After removing the solvent under reduced pressure, the product was purified as
2
4
indicated.
.2.8. Heck oxyarylation (General procedure H)
4
One-pot diazotization and Heck oxyarylation between 12a-c and 2a-h was performed using optimized
reaction conditions reported in literature [27,56]. In brief, the corresponding o-aminophenol (1
mmol) was dissolved in ACN (9 mL) and cooled to 0 °C. Then, NOPF (1 mmol) was added in one
6
portion. Immediate formation of brown gas was observed. After 2 h of stirring at that temperature,
ZnCO (2 mmol), Pd (dba) (5 mol%) and phenylpropene derivative (1.2 mmol) were added. The
3
2
3
resulting darkened mixture was stirred at rt for 20 h and then 10% NaHCO (30 mL) was added. The
3
mixture was filtered over a Celite® pad and the filtrate extracted with EtOAc (3x). The combined
organic layers were dried over anhydrous Na SO and the solvent removed under reduced pressure.
2
4
The product was purified by chromatography as indicated.
.2.9. Halogenation (General procedure I)
4
A solution of the corresponding compound (0.5 mmol) in ACN (1.8 mL) was cooled to 0 °C and NBS,
NCS, or NIS (0.5 mmol) was then added in one portion. The mixture was stirred overnight. The
product was purified as indicated.
4
.2.10. Heck coupling (General procedure J)
The corresponding bromide (0.3 mmol) was dissolved in dry DMF (0.9 mL) under nitrogen. Tert-butyl
acrylate (0.6 mmol), anhydrous potassium carbonate (0.6 mmol), palladium dichloride (1 mol%) and
PPh or P(o-tol) (2 mol%) were added. The mixture was heated at 100 °C under nitrogen for 24 h.
3
3
After that period, the mixture was cooled down and filtered over a Celite® pad. The pad was washed
several times with ethyl acetate and the layers separated. The organic one was washed with 1 mol/L
HCl (2x), 10% NaCl (2x), and water (3x), and afterwards dried over anhydrous Na SO . The solvent
2
4
was removed under reduced pressure and the product purified as indicated.
.2.11. Demethylation of 2-phenyl-2,3-dihydrobenzofurans (General procedure K)
To a solution of the corresponding 2-phenyl-2,3-dihydrobenzofurans (0.2 mmol) in dry CH Cl (1.28
4
2
2
mL) at 0 °C, a dilution of 1 mol/L BBr (0.43 mL, 0.4 mmol) in CH Cl (0.85 mL) was dropwise added.
3
2
2
The resulting mixture was stirred at rt for 2 h and then carefully quenched with cold water. The
aqueous layer was extracted with CH Cl (3x). The combined organic layers were dried over
2
2
anhydrous MgSO and the solvent removed under reduced pressure. The product was purified as
4
indicated.
4
.2.12. Attempts of deoxyfluorination (General procedure L)
The procedure was adopted from the original report [30]. In an HPLC vial the corresponding
compound (0.1 mmol), toluene (0.1 mL), DBU (0.2 mmol) and PyFluor (0.11 mmol) were added.
PyFluor was dispensed as liquid. The mixture was stirred at rt or 0 °C for 24-36 h. The product was
purified as indicated.
2
1

4
.2.13. Suzuki-Miyaura coupling (General procedure M)
A mixture of compound 13k (0.32 mmol), PdCl (5 mol%), PPh (20 mol%), the corresponding boronic
2
3
acid (0.53 mmol), water (0.5 mL), toluene (2 mL), and DMF (1 mL) was heated at 100 °C under
nitrogen for 24 h. The resulting mixture was diluted with EtOAc and filtered over a Celite® pad. The
filtrate was washed with water, dried over anhydrous Na SO and the solvent was then removed
2
4
under reduced pressure. The product was purified as indicated.
.2.14. (E)-3-(3,4-dimethoxyphenyl)prop-2-en-1-ol (2d)
4
Prepared according to the general procedure G from ethyl (E)-3-(3,4-dimethoxyphenyl)acrylate (1t,
36 mg, 1 mmol); isolated by column chromatography (31 g silica gel, hexane/EtOAc 65:35); colorless
2
1
liquid; yield: 144 mg (74%). H NMR (600 MHz, Chloroform-d) δ 6.94 (d, J = 2.1 Hz, 1H), 6.92 (dd, J =
8
4
1
.2, 2.0 Hz, 1H), 6.82 (d, J = 8.2 Hz, 1H), 6.55 (dt, J = 15.8, 1.6 Hz, 1H), 6.24 (dt, J = 15.8, 5.9 Hz, 1H),
13
.30 (dd, J = 6.0, 1.6 Hz, 2H), 3.89 (s, 3H), 3.88 (s, 3H). C NMR (151 MHz, Chloroform-d) δ 149.2,
49.0, 131.3, 129.9, 126.7, 119.8, 111.2, 109.0, 64.0, 56.0, 55.9. HRMS (ESI): m/z calcd. for
+
C H NaO : 217.0835 [M+Na] ; found: 217.0821.
11
14
3
4
.2.15. (E)-4-(3-hydroxyprop-1-en-1-yl)phenol (2e)
Prepared according to the general procedure G from ethyl 4-hydroxycinnamate (1b, 192 mg, 1
mmol); isolated by flash chromatography (30 g silica gel, CH Cl /EtOAc 85:15). White solid; yield: 120
2
2
1
mg (98%). H NMR (600 MHz, Acetone-d ) δ 8.34 (br s, 1H), 7.27 (d, J = 8.6 Hz, 2H), 6.79 (d, J = 8.6 Hz,
6
2
2
H), 6.51 (dt, J = 15.8, 1.7 Hz, 1H), 6.20 (dt, J = 15.9, 5.6 Hz, 1H), 5.61 (s, 1H), 4.19 (dd, J = 5.7, 1.8 Hz,
13
H). C NMR (151 MHz, Acetone-d ) δ 157.8, 130.2, 129.8, 128.4, 127.9, 116.2, 63.5. HRMS (ESI): m/z
6
+
calcd. for C H NaO : 173.0573 [M+Na] ; found: 173.0585.
9
10
2
4
.2.16. (E)-3-(4-methoxyphenyl)prop-2-en-1-ol (2f)
Prepared according to the general procedure G from ethyl (E)-3-(4-methoxyphenyl)acrylate (1s, 206
mg, 1 mmol); isolated by column chromatography (16 g silica gel, hexane/EtOAc 9:1 → 7:3); colorless
1
liquid; yield: 80 mg (48%). H NMR (600 MHz, Chloroform-d) δ 7.32 (d, J = 8.8 Hz, 2H), 6.86 (d, J = 8.8
Hz, 2H), 6.55 (dt, J = 15.8, 1.6 Hz, 1H), 6.24 (dt, J = 15.8, 6.0 Hz, 1H), 4.29 (dd, J = 6.0, 1.6 Hz, 2H), 3.81
1
3
(
s, 3H). C NMR (151 MHz, Chloroform-d) δ 159.4, 131.1, 129.5, 127.8, 126.4, 114.1, 64.0, 55.4.
+
HRMS (ESI): m/z calcd. for C H NaO : 187.0730 [M+Na] ; found: 187.0752.
10
12
2
4
.2.17. (E)-3-(4-methoxyphenyl)allyl acetate (2g)
Obtained as side product by the reaction of synthesis of compound 13f and purified therefore out of
1
the same chromatographic separation; colorless liquid; yield: 49 mg (24%). H NMR (600 MHz,
Chloroform-d) δ 7.33 (d, J = 8.8 Hz, 2H), 6.86 (d, J = 8.8 Hz, 2H), 6.60 (dd, J = 15.9, 1.5 Hz, 1H), 6.15
1
3
(
dt, J = 15.8, 6.6 Hz, 1H), 4.70 (dd, J = 6.7, 1.3 Hz, 2H), 3.81 (s, 3H), 2.09 (s, 3H). C NMR (151 MHz,
Chloroform-d) δ 171.0, 159.7, 134.2, 129.1, 128.0, 121.0, 114.1, 65.5, 55.4, 21.2. HRMS (ESI): m/z
+
calcd. for C H NaO : 229.0835 [M+Na] ; found: 229.0861.
12
14
3
4
.2.18. (E)-3-(2-bromophenyl)prop-2-en-1-ol (2h)
Prepared according to the general procedure G from ethyl (E)-3-(2-bromophenyl)acrylate (1u, 255
mg, 1 mmol); isolated by flash chromatography (23 g silica gel, hexane/EtOAc 85:15); colorless liquid;
1
yield: 188 mg (88%). H NMR (600 MHz, Chloroform-d) δ 7.55 (dd, J = 8.0, 1.3 Hz, 1H), 7.52 (dd, J =
7
.8, 1.7 Hz, 1H), 7.30 – 7.24 (m, 1H), 7.10 (td, J = 7.6, 1.7 Hz, 1H), 6.96 (dt, J = 15.8, 1.7 Hz, 1H), 6.31
13
(
dt, J = 15.8, 5.6 Hz, 1H), 4.36 (dd, J = 5.7, 1.7 Hz, 2H). C NMR (151 MHz, Chloroform-d) δ 136.7,
2
2

1
33.1, 131.8, 129.9, 129.1, 127.7, 127.3, 123.8, 63.7. HRMS (ESI): m/z calcd. for C H BrNaO: 234.9729
9 9
+
[M+Na] ; found: 234.9726.
4
.2.19. (E)-3-(4-hydroxy-3-methoxyphenyl)-N-propylacrylamide (4a)
Prepared according to general procedure A from ferulic acid (3a, 194 mg, 1 mmol) and propylamine
85 µL, 1 mmol); isolated by column chromatography (60 g silica gel, hexane/EtOAc/MeOH 1:1:0.1);
(
1
yellow oil; yield: 135 mg (58%). H NMR (600 MHz, Methanol-d ) δ 7.42 (d, J = 15.7 Hz, 1H), 7.10 (d, J
4
=
3
2.0 Hz, 1H), 7.01 (dd, J = 8.2, 2.0 Hz, 1H), 6.78 (d, J = 8.1 Hz, 1H), 6.42 (d, J = 15.6 Hz, 1H), 3.87 (s,
13
H), 3.24 (t, J = 7.1 Hz, 2H), 1.60 – 1.53 (m, 2H), 0.95 (t, J = 7.4 Hz, 3H). C NMR (151 MHz, Methanol-
d ) δ 170.4, 151.1, 150.5, 143.2, 129.5, 124.4, 120.0, 117.7, 112.8, 57.6, 43.6, 25.0, 13.0. HRMS (ESI):
4
+
m/z calcd. for C H NO : 236.1208 [M+H] ; found: 236.1311.
13
18
3
4
.2.20. (E)-3-(4-hydroxy-3-methoxyphenyl)-N-isopropylacrylamide (4b)
Prepared according to general procedure A from ferulic acid (3a, 194 mg, 1 mmol) and
isopropylamine (85 µL, 1 mmol); isolated by column chromatography (60 g silica gel,
1
hexane/EtOAc/MeOH 1:1:0.1); yellow oil; yield: 172 mg (73%). H NMR (600 MHz, Methanol-d ) δ
4
7
6
.41 (d, J = 15.7 Hz, 1H), 7.09 (d, J = 2.0 Hz, 1H), 7.01 (dd, J = 8.3, 1.9 Hz, 1H), 6.78 (d, J = 8.1 Hz, 1H),
13
.39 (d, J = 15.7 Hz, 1H), 4.07 (hept, J = 6.6 Hz, 1H), 3.86 (s, 3H), 1.18 (d, J = 6.6 Hz, 6H). C NMR (151
MHz, Methanol-d ) δ 169.5, 151.0, 150.5, 143.1, 129.6, 124.3, 120.3, 117.7, 112.8, 57.6, 43.8, 24.0.
4
+
HRMS (ESI): m/z calcd. for C H NO : 236.1242 [M+H] ; found: 236.1270.
13
18
3
4
.2.21. (E)-3-(3,4-dihydroxyphenyl)-N-isopropylacrylamide (4c)
Prepared according to general procedure A from caffeic acid (3b, 180 mg, 1 mmol) and
isopropylamine (85 µL, 1 mmol); isolated by column chromatography (60 g silica gel,
1
hexane/EtOAc/MeOH 1:1:0.1); yellow oil; yield: 86 mg (39%). H NMR (600 MHz, Methanol-d ) δ 7.38
4
(
(
d, J = 15.7 Hz, 1H), 7.01 (d, J = 2.1 Hz, 1H), 6.91 (dd, J = 8.2, 2.1 Hz, 1H), 6.77 (d, J = 8.2 Hz, 1H), 6.35
13
d, J = 15.7 Hz, 1H), 4.08 (hept, J = 6.6 Hz, 1H), 1.20 (d, J = 6.6 Hz, 6H). C NMR (151 MHz, Methanol-
d ) δ 168.3, 148.7, 146.7, 142.0, 128.4, 122.0, 118.7, 116.4, 115.0, 42.5, 22.7. HRMS (ESI): m/z calcd.
4
+
for C H NO : 222.1085 [M+H] ; found 222.1148.
12
16
3
4
.2.22. (E)-3-(4-hydroxy-3-methoxyphenyl)-N-(2-hydroxyethyl)acrylamide (4d)
The procedure was adapted from literature [19]. To a solution of methyl ferulate (1f, 536 mg, 2.6
mmol) in MeOH (3 mL), ethanolamine (780 μL, 12.9 mmol) and Na CO (272 mg, 2.6 mmol) were
2
3
added. The mixture was heated at 80 °C for 46 h. The resulting mixture was then diluted with MeOH
and filtered. The filtrate was concentrated under reduced pressure. The product was purified by flash
1
chromatography (60 g silica gel, CHCl /MeOH 9:1); white solid; yield: 255 mg (42%). H NMR (600
3
MHz, Methanol-d ) δ = 7.45 (d, J = 15.7 Hz, 1H), 7.12 (d, J = 1.9 Hz, 1H), 7.03 (dd, J = 8.1, 2.0 Hz, 1H),
4
6
2
6
.80 (d, J = 8.0 Hz, 1H), 6.46 (d, J = 15.7 Hz, 1H), 3.88 (s, 3H), 3.66 (t, J = 5.8 Hz, 2H), 3.42 (t, J = 5.8 Hz,
13
H). C NMR (151 MHz, Methanol-d ) δ 169.5, 149.9, 149.3, 142.2, 128.2, 123.2, 118.7, 116.5, 111.6,
4
+
1.7, 56.4, 43.1. HRMS (ESI): m/z calcd. for C H NO : 238.1074 [M+H] ; found: 238.1113.
12
16
4
4
.2.23. (E)-1-(3,4-dimethoxyphenyl)-5-methylhex-1-en-3-one (6a)
Prepared according to general procedure B using 4-methylpentan-2-one (3.4 mL, 27 mmol); the
1
resulting yellow resin was used without further purification; yield: 2.26 g (quantitative). H NMR (600
MHz, Chloroform-d) δ 7.48 (d, J = 16.1 Hz, 1H), 7.13 (dd, J = 8.3, 2.0 Hz, 1H), 7.07 (d, J = 2.0 Hz, 1H),
6
.87 (d, J = 8.3 Hz, 1H), 6.62 (d, J = 16.1 Hz, 1H), 3.92 (s, 3H), 3.91 (s, 3H), 2.52 (d, J = 7.1 Hz, 2H), 2.24
13
(
m, 1H), 0.97 (d, J = 6.7 Hz, 6H). C NMR (151 MHz, Chloroform-d) δ 200.4, 151.4, 149.4, 142.6,
2
3

1
2
27.6, 124.9, 123.1, 111.2, 109.8, 56.1, 56.0, 49.8, 25.5, 22.9. HRMS (ESI): m/z calcd. for C H O :
15
21
3
+
49.1446 [M+H] ; found: 249.1454.
4
.2.24. (E)-1-(3,4-dimethoxyphenyl)-5,5-dimethylhex-1-en-3-one (6b)
Prepared according to general procedure B using 4,4-dimethylpentan-2-one (3.8 mL, 27 mmol);
yellow needles crystalized over time and were used without further purification; yield: 2.36 g
1
(
quantitative). H NMR (400 MHz, Chloroform-d) δ 7.45 (d, J = 16.0 Hz, 1H), 7.13 (dd, J = 8.3, 2.0 Hz,
1
2
1
H), 7.06 (d, J = 2.0 Hz, 1H), 6.87 (d, J = 8.3 Hz, 1H), 6.61 (d, J = 16.0 Hz, 1H), 3.92 (s, 3H), 3.91 (s, 3H),
13
.53 (s, 2H), 1.06 (s, 9H). C NMR (101 MHz, Chloroform-d) δ 200.3, 151.4, 149.4, 142.3, 127.7,
26.0, 123.1, 111.2, 109.9, 56.1, 56.0, 53.6, 31.7, 30.2. HRMS (ESI): m/z calcd. for C H O : 263.1602
16
23
3
+
[M+H] ; found: 263.1640.
4
.2.25. (E)-1-(4-hydroxy-3-methoxyphenyl)-5-methylhex-1-en-3-one (7a)
The procedure was adapted from literature [23]. Anhydrous AlCl (3.19 g, 27.7 mmol) was added
3
carefully to a solution of 6a (1.96 g, 7.9 mmol) in dry CH Cl (20 mL), and the resulting mixture stirred
2
2
at rt. After full conversion as indicated by TLC, the mixture was quenched by H O (40 mL) and the
2
solvent removed under reduced pressure. The residue was then extracted with EtOAc (3x). The
combined organic layers were dried over anhydrous Na SO and the solvent removed under reduced
2
4
pressure. The mixture of regioisomers was separated by column chromatography (100 g silica gel,
1
hexane/EtOAc/MeOH 80:20:2.5); yellow oil; yield: 557 mg (30%). H NMR (400 MHz, Chloroform-d) δ
7
6
6
1
.46 (d, J = 16.1 Hz, 1H), 7.08 (dd, J = 8.2, 2.0 Hz, 1H), 7.04 (d, J = 2.0 Hz 1H), 6.91 (d, J = 8.2 Hz, 1H),
.59 (d, J = 16.1 Hz, 1H), 3.91 (s, 3H), 2.51 (d, J = 7.0 Hz, 2H), 2.28 – 2.26 (m, 1H), 0.96 (d, J = 6.7 Hz,
13
H). C NMR (101 MHz, Chloroform-d) δ 200.6, 148.4, 147.0, 142.9, 127.1, 124.5, 123.5, 115.0,
+
09.6, 56.0, 49.7, 25.5, 22.8. HRMS (ESI): m/z calcd. for C H O : 235.1289 [M+H] ; found: 235.1396.
14
19
3
4
.2.26. (E)-1-(3,4-dihydroxyphenyl)-5-methylhex-1-en-3-one (7b)
Prepared according to general procedure C from 6a (374 mg, 1.51 mmol); isolated by column
1
chromatography (50 g silica gel, hexane/EtOAc 6:4); greenish crystals; yield: 96 mg (29%). H NMR
(
400 MHz, Methanol-d ) δ 7.50 (d, J = 16.0 Hz, 1H), 7.08 (d, J = 2.1 Hz, 1H), 7.00 (dd, J = 8.2, 2.1 Hz,
4
1
H), 6.79 (d, J = 8.2 Hz, 1H), 6.60 (d, J = 16.1 Hz, 1H), 2.53 (d, J = 7.0 Hz, 2H), 2.22 – 2.09 (m, 1H), 0.96
13
(
d, J = 6.7 Hz, 6H). C NMR (101 MHz, Methanol-d ) δ 203.4, 149.9, 146.9, 145.6, 127.8, 124.3, 123.5,
4
+
1
2
16.6, 115.3, 50.2, 26.7, 23.0. HRMS (ESI): m/z calcd. for C H O : 221.1133 [M+H] ; found:
13 17 3
21.1179.
4
.2.27. (E)-1-(3,4-dihydroxyphenyl)-5,5-dimethylhex-1-en-3-one (7c)
Prepared according to general procedure C from 6b (397 mg, 1.51 mmol); isolated by column
1
chromatography (50 g silica gel, CHCl /MeOH 93:7); yellow crystals; yield: 258 mg (73%). H NMR
3
(
400 MHz, Methanol-d ) δ 7.46 (d, J = 16.0 Hz, 1H), 7.08 (d, J = 2.1 Hz, 1H), 7.00 (dd, J = 8.2, 2.1 Hz,
4
13
1
H), 6.80 (d, J = 8.2 Hz, 1H), 6.62 (d, J = 16.0 Hz, 1H), 2.55 (s, 2H), 1.05 (s, 9H). C NMR (151 MHz,
Methanol-d ) δ 203.1, 149.9, 146.9, 145.3, 127.9, 125.6, 123.5, 116.6, 115.3, 53.9, 32.4, 30.4. HRMS
4
+
(
ESI): m/z calcd. for C H O : 235.1289 [M+H] ; found: 235.1382.
14 19 3
4
.2.28. (±)-Methyl trans-(E)-2-(4-hydroxyphenyl)-5-(-3-methoxy-3-oxoprop-1-en-1-yl)-2,3-dihy-
drobenzofuran-3-carboxylate (8a)
Prepared according to the general procedure E from methyl 4-hydroxycinnamate (1a, 89 mg, 0.5
mmol); isolated by column chromatography (16 g silica gel, hexane/EtOAc 7:3); white solid; yield: 19
1
mg (10%). H NMR (400 MHz, Acetone-d ) δ 8.52 (br s, 1H), 7.71 (t, J = 1.6 Hz, 1H), 7.65 (d, J = 16.0
6
Hz, 1H), 7.60 (dd, J = 8.4, 1.9 Hz, 1H), 7.28 (d, J = 8.6 Hz, 2H), 6.91 (d, J = 8.4 Hz, 1H), 6.87 (d, J = 8.6
2
4

Hz, 2H), 6.41 (d, J = 15.9 Hz, 1H), 6.03 (d, J = 7.4 Hz, 1H), 4.39 (d, J = 7.4 Hz, 1H), 3.81 (s, 3H), 3.73 (s,
1
3
3
1
3
H). C NMR (101 MHz, Acetone-d ) δ 171.7, 167.8, 162.3, 158.8, 145.2, 132.0, 131.8, 128.8, 128.6,
6
26.9, 126.2, 116.4, 116.2, 110.9, 87.9, 55.7, 53.1, 51.7. HRMS (ESI): m/z calcd. for C H O :
20
19
6
+
55.1176 [M+H] ; found: 355.1204.
4
.2.29. (±)-Ethyl trans-(E)-5-(3-ethoxy-3-oxoprop-1-en-1-yl)-2-(4-hydroxyphenyl)-2,3-di-hydro-
benzofuran-3-carboxylate (8b)
Prepared according to the general procedure E from ethyl 4-hydroxycinnamate (1b, 96 mg, 0.5
mmol); isolated by column chromatography (16 g silica gel, hexane/EtOAc 7:3); white solid; yield: 23
1
mg (12%). H NMR (600 MHz, Acetone-d ) δ 8.56 (br s, 1H), 7.71 (br s, 1H), 7.65 (d, J = 16.0 Hz, 1H),
6
7
.60 (br d, J = 8.8 Hz, 1H), 7.29 (d, J = 8.5 Hz, 2H), 6.91 (d, J = 8.4 Hz, 1H), 6.87 (d, J = 8.6 Hz, 2H), 6.39
(
d, J = 16.0 Hz, 1H), 6.03 (d, J = 7.5 Hz, 1H), 4.36 (d, J = 7.5 Hz, 1H), 4.34 – 4.21 (m, 2H), 4.20 (q, J = 7.0
13
Hz, 2H), 1.30 (t, J = 6.9 Hz, 3H), 1.28 (t, J = 7.4 Hz, 3H). C NMR (151 MHz, Acetone-d ) δ 171.1, 167.3,
6
1
5
62.2, 158.7, 145.0, 132.0, 131.6, 128.8, 128.6, 126.9, 126.1, 116.5, 116.4, 110.9, 87.8, 62.3, 60.7,
+
5.8, 14.7, 14.6. HRMS (ESI): m/z calcd. for C H O : 383.1489 [M+H] ; found: 383.1494.
22
23
6
4
.2.30. (±)-Isopropyl trans-(E)-2-(4-hydroxyphenyl)-5-(3-isopropoxy-3-oxoprop-1-en-1-yl)-2,3-di-
hydrobenzofuran-3-carboxylate (8c)
Prepared according to the general procedure E from isopropyl 4-hydroxycinnamate (1c, 103 mg, 0.5
mmol); isolated by column chromatography (31 g silica gel, hexane/Et O 6:4); white solid; yield: 20
2
1
mg (10%). H NMR (600 MHz, Acetone-d ) δ 8.57 (br s, 1H), 7.70 (t, J = 1.6 Hz, 1H), 7.64 (d, J = 15.9
6
Hz, 1H), 7.61 (dd, J = 8.3, 1.8 Hz, 1H), 7.28 (d, J = 8.5 Hz, 2H), 6.91 (d, J = 8.4 Hz, 1H), 6.87 (d, J = 8.6
Hz, 2H), 6.36 (d, J = 15.9 Hz, 1H), 6.02 (d, J = 7.5 Hz, 1H), 5.16 – 5.04 (m, 1H), 5.09 – 4.99 (m, 1H), 4.32
1
3
(
d, J = 7.5 Hz, 1H), 1.30 (d, J = 6.3 Hz, 6H), 1.27 (d, J = 6.3 Hz, 6H). C NMR (151 MHz, Acetone-d ) δ
6
1
7
4
70.7, 166.9, 162.2, 158.8, 144.9, 132.0, 131.5, 128.8, 128.6, 127.0, 126.0, 117.0, 116.4, 110.9, 87.9,
+
0.0, 68.0, 56.0, 22.2, 22.1, 22.0. HRMS (ESI): m/z calcd. for C H O : 411.1802 [M+H] ; found:
24
27
6
11.1800.
4
.2.31. (±)-Butyl trans-(E)-5-(3-butoxy-3-oxoprop-1-en-1-yl)-2-(4-hydroxyphenyl)-2,3-dihydro-
benzofuran-3-carboxylate (8d)
Prepared according to the general procedure E from butyl 4-hydroxycinnamate (1d, 110 mg, 0.5
mmol); isolated by flash chromatography (SVF D26-RP18 column, 25-40 μm, gradient MeOH/H O, 9
2
1
mL/min flow); white solid; yield: 10 mg (5%). H NMR (600 MHz, Acetone-d ) δ 8.57 (br s, 1H), 7.72 (t,
6
J = 1.6 Hz, 1H), 7.65 (d, J = 16.0 Hz, 1H), 7.60 (dd, J = 8.4, 2.0 Hz, 1H), 7.28 (d, J = 8.6 Hz, 2H), 6.92 (d, J
=
8.3 Hz, 1H), 6.87 (d, J = 8.6 Hz, 2H), 6.39 (d, J = 16.0 Hz, 1H), 6.04 (d, J = 7.4 Hz, 1H), 4.39 (d, J = 7.4
Hz, 1H), 4.29 – 4.18 (m, 2H), 4.16 (t, J = 6.4 Hz, 2H), 1.74 – 1.62 (m, 4H), 1.47 – 1.39 (m, 4H), 0.95 (t, J
1
3
=
1
3
7.4 Hz, 3H), 0.94 (t, J = 7.4 Hz, 3H). C NMR (151 MHz, Acetone-d ) δ 171.2, 167.4, 162.2, 158.8,
6
45.0, 132.0, 131.8, 130.2, 128.8, 128.6, 127.0, 126.0, 116.5, 116.4, 110.9, 87.8, 66.1, 64.6, 55.8,
+
1.7, 31.5, 19.9, 14.1, 14.0. HRMS (ESI): m/z calcd. for C H O : 439.2115 [M+H] ; found: 439.2110.
26
31
6
4
.2.32. (±)-tert-butyl trans-(E)-5-(3-(tert-butoxy)-3-oxoprop-1-en-1-yl)-2-(4-hydroxyphen-yl)-2,3-
dihydrobenzofuran-3-carboxylate (8e)
Prepared according to the general procedure E from tert-butyl 4-hydroxycinnamate (1e, 110 mg, 0.5
mmol); isolated by preparative RP-HPLC (gradient MeOH/H O, 10 mL/min flow); white solid; yield: 20
2
1
mg (9%). H NMR (400 MHz, Acetone-d ) δ 8.49 (s, 1H), 7.67 (br s, 1H), 7.64 – 7.54 (m, 2H), 7.27 (d, J
6
=
8.4 Hz, 2H), 6.89 (d, J = 8.3 Hz, 1H), 6.87 (d, J = 8.6 Hz, 2H), 6.30 (d, J = 16.0 Hz, 1H), 5.98 (d, J = 7.5
13
Hz, 1H), 4.26 (d, J = 7.5, 1H), 1.52 (s, 9H), 1.50 (s, 9H). C NMR (101 MHz, Acetone-d ) δ 170.4, 166.9,
6
2
5

1
5
62.2, 158.8, 144.3, 132.3, 131.3, 128.9, 128.6, 127.2, 126.0, 118.3, 116.5, 110.9, 87.8, 82.7, 80.4,
+
6.8, 28.5, 28.3. HRMS (ESI): m/z calcd. for C H O : 439.2115 [M+H] ; found: 439.2139.
26
31
6
4
.2.33. (±)-Methyl trans-(E)-2-(4-hydroxy-3-methoxyphenyl)-7-methoxy-5-(3-methoxy-3-oxoprop-1-
en-1-yl)-2,3-dihydrobenzofuran-3-carboxylate (8f)
Prepared according to the general procedure D from methyl ferulate (1f, 105 mg, 0.50 mmol);
isolated by column chromatography (15 g silica gel, hexane/EtOAc 7:3); white solid; yield: 34 mg
1
(
16%). H NMR (600 MHz, Acetone-d ) δ 7.72 (s, 1H), 7.63 (d, J = 16.0 Hz, 1H), 7.33 (d, J = 1.5 Hz, 1H),
6
7
1
3
1
.29 (br s, 1H), 7.10 (d, J = 2.0 Hz, 1H), 6.92 (dd, J = 8.2, 2.0 Hz, 1H), 6.84 (d, J = 8.1 Hz, 1H), 6.44 (d, J =
5.9 Hz, 1H), 6.04 (d, J = 7.9 Hz, 1H), 4.47 (d, J = 7.9 Hz, 1H), 3.92 (s, 3H), 3.84 (s, 3H), 3.81 (s, 3H),
13
.73 (s, 3H). C NMR (151 MHz, Acetone-d ) δ 171.8, 167.9, 151.2, 148.7, 148.2, 146.0, 145.6, 132.2,
6
29.6, 127.5, 120.4, 119.1, 116.5, 116.0, 113.7, 110.9, 88.5, 56.7, 56.5, 56.1, 53.2, 51.7. HRMS (ESI):
+
m/z calcd. for C H O : 415.1387 [M+H] ; found: 415.1366.
22
23
8
4
.2.34. (±)-Ethyl trans-(E)-5-(3-ethoxy-3-oxoprop-1-en-1-yl)-2-(4-hydroxy-3-methoxyphen-yl)-7-
methoxy-2,3-dihydrobenzofuran-3-carboxylate (8g)
Prepared according to the general procedure E from ethyl ferulate (1g, 112 mg, 0.5 mmol); isolated
1
by column chromatography (24 g silica gel, hexane/EtOAc 7:3); white solid; yield: 40 mg (18%). H
NMR (600 MHz, Acetone-d ) δ 7.76 (s, 1H), 7.63 (d, J = 15.9 Hz, 1H), 7.34 (d, J = 1.6 Hz, 1H), 7.29 (t, J =
6
1
1
2
.3 Hz, 1H), 7.09 (d, J = 2.0 Hz, 1H), 6.92 (dd, J = 8.0, 2.0 Hz, 1H), 6.85 (d, J = 8.1 Hz, 1H), 6.42 (d, J =
5.9 Hz, 1H), 6.04 (d, J = 8.0 Hz, 1H), 4.44 (d, J = 7.9 Hz, 1H), 4.36 – 4.20 (m, 2H), 4.19 (q, J = 7.1 Hz,
13
H), 3.93 (s, 3H), 3.84 (s, 3H), 1.30 (t, J = 7.1 Hz, 3H), 1.28 (t, J = 7.1 Hz, 3H). C NMR (151 MHz,
Acetone-d ) δ 171.2, 167.4, 151.1, 148.7, 148.1, 145.9, 145.3, 132.2, 129.6, 127.5, 120.3, 119.0,
6
1
16.8, 115.9, 113.3, 110.8, 88.5, 62.3, 60.7, 56.6, 56.4, 56.2, 14.8, 14.6. HRMS (ESI): m/z calcd. for
+
C H O : 443.1700 [M+H] ; found: 443.1709.
24
27
8
4
.2.35. (±)-Isopropyl trans-(E)-2-(4-hydroxy-3-methoxyphenyl)-5-(3-isopropoxy-3-oxo-prop-1-en-1-
yl)-7-methoxy-2,3-dihydrobenzofuran-3-carboxylate (8h)
Prepared according to the general procedure E from isopropyl ferulate (1h, 118 mg, 0.5 mmol);
isolated by column chromatography (22 g silica gel, hexane/EtOAc 7:3); white solid; yield: 33 mg
1
(
14%). H NMR (600 MHz, Acetone-d ) δ 7.75 (s, 1H, OH), 7.62 (d, J = 15.9 Hz, 1H, H-1''), 7.34 (d, J =
6
1
.6 Hz, 1H, H-6), 7.27 (t, J = 1.4 Hz, 1H, H-4), 7.09 (d, J = 2.0 Hz, 1H, H-2'), 6.92 (dd, J = 8.2, 2.0 Hz, 1H,
H-6'), 6.85 (d, J = 8.1 Hz, 1H, H-5'), 6.39 (d, J = 15.9 Hz, 1H, H-2''), 6.03 (d, J = 8.1 Hz, 1H, H-2), 5.09
hept, J = 6.3 Hz, 1H, OCHcarboxylate), 5.06 (hept, J = 6.3 Hz, 1H, OCHacrylate), 4.40 (d, J = 8.1 Hz, 1H, H-3),
(
3
1
.93 (s, 3H, 7-OCH ), 3.84 (s, 3H, 3'-OCH ), 1.30 (d, J = 6.3 Hz, 3H, CH ), 1.30 (d, J = 6.3 Hz, 3H, CH ),
3 3 3 3
1
3
.27 (d, J = 6.3 Hz, 6H, 2xCH ). C NMR (151 MHz, Acetone-d ) δ 170.7 (3-C=O), 167.0 (C-3''), 151.1
3
6
(
C-7a), 148.7 (C-3'), 148.1 (C-4'), 146.0 (C-7), 145.2 (C-1''), 132.2 (C-1'), 129.6 (C-5), 127.6 (C-3a),
20.3 (C-6'), 119.0 (C-4), 117.2 (C-2''), 115.9 (C-5'), 113.2 (C-6), 110.8 (C-2'), 88.5 (C-2), 70.0
OCHcarboxylate), 67.9 (OCHacrylate), 56.6 (7-OCH ), 56.4 (3'-OCH ), 56.3 (C-3), 22.3 (2xCH ), 22.1 (CH ),
1
(
3
3
3
3
+
2
2.0 (CH ). HRMS (ESI): m/z calcd. for C H O : 471.2013 [M+H] ; found: 471.2023.
3 26 31 8
4
.2.36. (±)-Butyl trans-(E)-5-(3-butoxy-3-oxoprop-1-en-1-yl)-2-(4-hydroxy-3-methoxyphen-yl)-7-
methoxy-2,3-dihydrobenzofuran-3-carboxylate (8i)
Prepared according to the general procedure E from butyl ferulate (1i, 125 mg, 0.5 mmol); isolated
by column chromatography (21 g silica gel, hexane/EtOAc 8:2 → 7:3); white solid; yield: 26 mg (10%).
1
H NMR (600 MHz, Acetone-d ) δ 7.75 (s, 1H), 7.63 (d, J = 15.9 Hz, 1H), 7.33 (br s, 1H), 7.29 (br s, 1H),
6
7
6
.09 (d, J = 2.0 Hz, 1H), 6.92 (dd, J = 8.1, 2.0 Hz, 1H), 6.85 (d, J = 8.2 Hz, 1H), 6.42 (d, J = 15.9 Hz, 1H),
.04 (d, J = 7.9 Hz, 1H), 4.46 (d, J = 7.9 Hz, 1H), 4.25 (dt, J = 10.8, 6.6 Hz, 1H), 4.20 (dt, J = 10.8, 6.5 Hz,
2
6

1
=
1
5
H), 4.16 (t, J = 6.6 Hz, 2H), 3.93 (s, 3H), 3.84 (s, 3H), 1.72 – 1.62 (m, 4H), 1.47 – 1.38 (m, 4H), 0.95 (t, J
13
7.4 Hz, 3H), 0.93 (t, J = 7.4 Hz, 3H). C NMR (151 MHz, Acetone-d ) δ 171.2, 167.4, 151.1, 148.7,
6
48.1, 145.9, 145.3, 132.2, 129.5, 127.5, 120.3, 119.0, 116.8, 115.9, 113.5, 110.8, 88.4, 66.1, 64.6,
6.6, 56.4, 56.2, 31.7, 31.5, 20.0, 19.9, 14.1, 14.0. HRMS (ESI): m/z calcd. for C H O : 499.2326
28
35
8
+
[M+H] ; found: 499.2349.
4
.2.37. (±)-tert-butyl trans-(E)-5-(3-(tert-butoxy)-3-oxoprop-1-en-1-yl)-2-(4-hydroxy-3-methoxy-
phenyl)-7-methoxy-2,3-dihydrobenzofuran-3-carboxylate (8j)
Prepared according to the general procedure E from tert-butyl ferulate (1j, 125 mg, 0.5 mmol);
isolated by flash chromatography (24 g silica gel, hexane/EtOAc 85:15 → 80:20); white solid; yield: 27
1
mg (11%). H NMR (600 MHz, Chloroform-d) δ 7.55 (d, J = 15.9 Hz, 1H), 7.15 (d, J = 1.5 Hz, 1H), 7.02
(
d, J = 1.5 Hz, 1H), 6.91 – 6.89 (m, 3H), 6.23 (d, J = 15.8 Hz, 1H), 6.05 (d, J = 8.4 Hz, 1H), 5.65 (br s, 1H),
13
4
.22 (d, J = 8.4 Hz, 1H), 3.91 (s, 3H), 3.87 (s, 3H), 1.54 (s, 9H), 1.51 (s, 9H). C NMR (151 MHz,
Chloroform-d) δ 169.4, 166.8, 149.9, 146.8, 146.1, 144.8, 143.8, 131.9, 128.8, 126.4, 119.7, 118.2,
17.8, 114.6, 111.5, 108.9, 87.6, 82.6, 80.5, 56.5, 56.2, 56.1, 28.4, 28.3. HRMS (ESI): m/z calcd. for
1
+
C H O : 499.2326 [M+H] ; found: 499.2350.
28
35
8
4
2
.2.38. (±)-Methyl trans-(E)-2-(3,4-dihydroxyphenyl)-7-hydroxy-5-(3-methoxy-3-oxoprop-1-en-1-yl)-
,3-dihydrobenzofuran-3-carboxylate (8k)
Prepared according to the general procedure D from methyl caffeate (1k, 97 mg, 0.5 mmol); isolated
1
by column chromatography (9.8 g silica gel, hexane/acetone 7:3); white foam; yield: 33 mg (17%). H
NMR (600 MHz, Acetone-d ) δ 8.15 (br s, 3H), 7.58 (d, J = 15.9 Hz, 1H), 7.21 (t, J = 1.4 Hz, 1H), 7.15 (d,
6
J = 1.7 Hz, 1H), 6.90 (d, J = 2.0 Hz, 1H), 6.84 (d, J = 8.1 Hz, 1H), 6.80 (dd, J = 8.1, 2.1 Hz, 1H), 6.34 (d, J =
1
3
1
6.0 Hz, 1H), 5.98 (d, J = 7.3 Hz, 1H), 4.36 (d, J = 7.3 Hz, 1H), 3.80 (s, 3H), 3.72 (s, 3H). C NMR (151
MHz, Acetone-d ) δ 170.8, 166.8, 149.2, 145.5, 145.3, 144.5, 141.7, 131.8, 128.5, 126.5, 117.8, 116.9,
6
1
16.5, 115.3, 115.2, 113.0, 87.0, 55.4, 52.1, 50.7. HRMS (ESI): m/z calcd. for C H O : 387.1074
20 19 8
+
[M+H] ; found: 387.1063.
4
.2.39. (±)-Ethyl trans-(E)-2-(3,4-dihydroxyphenyl)-5-(3-ethoxy-3-oxoprop-1-en-1-yl)-7-hydroxy-2,3-
dihydrobenzofuran-3-carboxylate (8l)
Prepared according to the general procedure D from ethyl caffeate (1l, 104 mg, 0.5 mmol); isolated
by flash chromatography (20 g silica gel, CH Cl /acetone 9:1 → 8:2); white solid; yield: 29 mg (14%).
2
2
1
H NMR (600 MHz, Acetone-d ) δ 8.16 (s, 3H), 7.58 (d, J = 15.9 Hz, 1H), 7.21 (t, J = 1.5 Hz, 1H), 7.15 (d,
6
J = 1.6 Hz, 1H), 6.90 (d, J = 2.1 Hz, 1H), 6.84 (d, J = 8.1 Hz, 1H), 6.80 (dd, J = 8.2, 2.1 Hz, 1H), 6.32 (d, J =
1
2
1
6
5.9 Hz, 1H), 5.98 (d, J = 7.4 Hz, 1H), 4.33 (d, J = 7.4 Hz, 1H), 4.30 – 4.23 (m, 2H), 4.19 (q, J = 7.1 Hz,
13
H), 1.30 (t, J = 7.1 Hz, 3H), 1.28 (t, J = 7.1 Hz, 3H). C NMR (151 MHz, Acetone-d ) δ 171.3, 167.4,
6
50.1, 146.5, 146.3, 145.4, 142.7, 132.9, 129.6, 127.6, 118.9, 117.8, 117.4, 116.6, 116.3, 114.1, 88.0,
+
2.3, 60.7, 56.6, 14.8, 14.6. HRMS (ESI): m/z calcd. for C H O : 415.1387 [M+H] ; found: 415.1403.
22
23
8
4
.2.40. (±)-Isopropyl trans-(E)-2-(3,4-dihydroxyphenyl)-7-hydroxy-5-(3-isopropoxy-3-oxo-prop-1-en-1-
yl)-2,3-dihydrobenzofuran-3-carboxylate (8m)
Prepared according to the general procedure D from isopropyl caffeate (1m, 111 mg, 0.5 mmol);
isolated by flash chromatography (15 g silica gel, hexane/acetone 7:3); yellowish oil; yield: 13 mg
1
(
6%). H NMR (600 MHz, Acetone-d ) δ 8.16 (br s, 3H, 3xOH), 7.57 (d, J = 15.9 Hz, 1H, H-1''), 7.19 (t, J
6
=
5
1.4 Hz, 1H, H-4), 7.15 (d, J = 1.7 Hz, 1H, H-6), 6.90 (d, J = 2.1 Hz, 1H, H-2'), 6.84 (d, J = 8.1 Hz, 1H, H-
'), 6.80 (dd, J = 8.1, 2.1 Hz, 1H, H-6'), 6.29 (d, J = 15.9 Hz, 1H, H-2''), 5.97 (d, J = 7.4 Hz, 1H, H-2), 5.09
(
hept, J = 6.3 Hz, 1H, OCHcarboxylate), 5.05 (hept, J = 6.4 Hz, 1H, OCH
), 4.29 (d, J = 7.5 Hz, 1H, H-3),
acrylate
13
1
.30 (d, J = 6.2 Hz, 6H, CH3,carboxylate), 1.27 (d, J = 6.3 Hz, 6H, CH3,acrylate). C NMR (151 MHz, Acetone-d )
6
2
7

δ 170.8 (3-C=O), 167.0 (C-3''), 150.2 (C-7a), 146.6 (C-4'), 146.3 (C-3'), 145.2 (C-1''), 142.8 (C-7), 133.0
(
(
C-1'), 129.7 (C-5), 127.8 (C-3a), 118.9 (C-6'), 117.7 (C-4), 117.3 (C-6), 117.1 (C-2''), 116.3 (C-5'), 114.1
C-2'), 88.1 (C-2), 70.0 (OCHcarboxylate), 68.0 (OCHacrylate), 56.8 (C-3), 22.3 (2xCH ), 22.1 (CH ), 22.1 (CH ).
3
3
3
+
HRMS (ESI): m/z calcd. for C H O : 443.1700 [M+H] ; found: 443.1723.
24
27
8
4
.2.41. (±)-Butyl trans-(E)-5-(3-butoxy-3-oxoprop-1-en-1-yl)-2-(3,4-dihydroxyphenyl)-7-hydroxy-2,3-
dihydrobenzofuran-3-carboxylate (8n)
Prepared according to the general procedure D from butyl caffeate (1n, 118 mg, 0.5 mmol); isolated
by flash chromatography (30 g silica gel, CH Cl /acetone 10:0 → 9:1); white solid; yield: 19 mg (8%).
2
2
1
H NMR (400 MHz, Methanol-d ) δ 7.56 (d, J = 15.9 Hz, 1H), 7.11 (d, J = 1.6 Hz, 1H), 7.02 (d, J = 1.6 Hz,
4
1
1
1
H), 6.81 (d, J = 2.0 Hz, 1H), 6.76 (d, J = 8.1 Hz, 1H), 6.72 (dd, J = 8.2, 2.1 Hz, 1H), 6.29 (d, J = 15.9 Hz,
H), 5.95 (d, J = 7.2 Hz, 1H), 4.28 (d, J = 7.2 Hz, 1H), 4.30 – 4.17 (m, 2H), 4.18 (t, J = 6.5 Hz, 2H), 1.76 –
13
.60 (m, 4H), 1.52 – 1.34 (m, 4H), 0.98 (t, J = 7.4 Hz, 3H), 0.95 (t, J = 7.4 Hz, 3H). C NMR (101 MHz,
Methanol-d ) δ 172.4, 169.2, 150.7, 146.9, 146.7, 146.3, 143.2, 133.1, 129.8, 127.7, 118.7, 117.8,
4
1
17.4, 116.4, 116.1, 113.9, 88.5, 66.6, 65.4, 57.1, 32.0, 31.8, 20.2, 14.1, 14.0. HRMS (ESI): m/z calcd.
+
for C H O : 471.2013 [M+H] ; found: 471.1982.
26
31
8
4
.2.42. (±)-tert-butyl trans-(E)-5-(3-(tert-butoxy)-3-oxoprop-1-en-1-yl)-2-(3,4-dihydroxy-phenyl)-7-
hydroxy-2,3-dihydrobenzofuran-3-carboxylate (8o)
Prepared according to the general procedure D from tert-butyl caffeate (1o, 118 mg, 0.5 mmol);
isolated by flash chromatography (31 g silica gel, hexane/acetone 8:2 → 7:3); further purificaꢀon by
1
preparative RP-HPLC; colorless oil; yield: 29 mg (12%). H NMR (600 MHz, Methanol-d ) δ 7.47 (d, J =
4
1
1
1
1
2
5.9 Hz, 1H), 7.07 (t, J = 1.4 Hz, 1H), 7.00 (d, J = 1.7 Hz, 1H), 6.80 (d, J = 2.0 Hz, 1H), 6.76 (d, J = 8.1 Hz,
H), 6.72 (dd, J = 8.2, 2.1 Hz, 1H), 6.20 (d, J = 15.8 Hz, 1H), 5.90 (d, J = 7.3 Hz, 1H), 4.16 (d, J = 7.4 Hz,
13
H), 1.53 (s, 9H), 1.51 (s, 9H). C NMR (151 MHz, Methanol-d ) δ 171.4, 168.6, 150.6, 146.9, 146.7,
4
45.4, 143.1, 133.2, 129.8, 128.0, 118.7, 117.9, 117.7, 117.0, 116.4, 113.9, 88.5, 83.4, 81.6, 58.1,
+
8.5, 28.3. HRMS (ESI): m/z calcd. for C H O : 471.2013 [M+H] ; found: 471.2049.
26
31
8
4
2
.2.43. (±)-Octyl trans-(E)-2-(3,4-dihydroxyphenyl)-7-hydroxy-5-(3-(octyloxy)-3-oxoprop-1-en-1-yl)-
,3-dihydrobenzofuran-3-carboxylate (8p)
Prepared according to the general procedure D from octyl caffeate (1p, 146 mg, 0.35 mmol); isolated
1
by flash chromatography (50 g silica gel, CHCl /MeOH 97:3); white solid; yield: 27 mg (9%). H NMR
3
(
600 MHz, Chloroform-d) δ 7.53 (d, J = 15.9 Hz, 1H), 7.07 (br s, 1H), 6.98 (d, J = 1.5 Hz, 1H), 6.86 (d, J =
2
1
1
.0 Hz, 1H), 6.82 (d, J = 8.1 Hz, 1H), 6.77 (dd, J = 8.2, 2.0 Hz, 1H), 6.23 (d, J = 15.9 Hz, 1H), 6.11 (br s,
H), 6.02 (d, J = 7.5 Hz, 1H), 5.95 (br s, 1H), 5.84 (br s, 1H), 4.26 (d, J = 7.4 Hz, 1H), 4.24 – 4.13 (m, 4H),
13
.72 – 1.63 (m, 4H), 1.44 – 1.18 (m, 20H), 0.88 (t, J = 7.0 Hz, 3H), 0.86 (t, J = 7.1 Hz, 3H). C NMR (151
MHz, Chloroform-d) δ 170.8, 168.1, 148.5, 144.9, 144.5, 144.1, 140.5, 132.4, 129.0, 125.7, 119.0,
1
2
5
17.6, 116.5, 116.0, 115.7, 113.2, 87.4, 66.4, 65.1, 55.9, 31.9, 31.9, 29.4, 29.3, 29.3, 29.3, 28.9, 28.7,
+
6.1, 26.0, 22.8, 22.7, 14.2, 14.2. HRMS (ESI): m/z calcd. for C H O : 583.3265 [M+H] ; found:
34
47
8
83.3256.
4
.2.44. (±)-Allyl trans-(E)-5-(3-(allyloxy)-3-oxoprop-1-en-1-yl)-2-(3,4-dihydroxyphenyl)-7-hydroxy-2,3-
dihydrobenzofuran-3-carboxylate (8q)
Prepared according to the general procedure D from allyl caffeate (1q, 110 mg, 0.5 mmol); isolated
by flash chromatography (31 g silica gel, hexane/acetone 7:3 → 6:4); white solid; yield: 11 mg (5%).
1
H NMR (600 MHz, Methanol-d ) δ 7.58 (d, J = 15.9 Hz, 1H), 7.11 (br s, 1H), 7.04 (d, J = 1.7 Hz, 1H),
4
6
6
.81 (d, J = 2.0 Hz, 1H), 6.76 (d, J = 8.1 Hz, 1H), 6.73 (dd, J = 8.2, 2.1 Hz, 1H), 6.31 (d, J = 15.9 Hz, 1H),
.04 – 5.94 (m, 2H), 5.97 (d, J = 7.1 Hz, 1H), 5.34 (ddd, J = 17.2, 11.2, 1.6 Hz, 2H), 5.27 – 5.22 (m, 2H),
2
8

1
3
4
.77 – 4.67 (m, 2H), 4.69 – 4.65 (m, 2H), 4.32 (dd, J = 7.2, 1.0 Hz, 1H). C NMR (151 MHz, Methanol-
d4) δ 171.9, 168.6, 150.8, 146.9, 146.7, 146.6, 143.1, 133.9, 133.3, 133.0, 129.7, 127.5, 119.0, 118.7,
1
4
18.3, 118.1, 117.3, 116.4, 115.8, 113.9, 88.5, 67.2, 66.1, 57.0. HRMS (ESI): m/z calcd. for C H O :
24 23 8
+
39.1387 [M+H] ; found: 439.1417.
4
.2.45. (±)-tert-butyl trans-(E)-5-(3-(tert-butoxy)-3-oxoprop-1-en-1-yl)-7-fluoro-2-(3-fluoro-4-hy-
droxyphenyl)-2,3-dihydrobenzofuran-3-carboxylate (8r)
Prepared according to the general procedure
E
from tert-butyl (E)-3-(3-fluoro-4-
hydroxyphenyl)acrylate (1r, 119 mg, 0.5 mmol); isolated by flash chromatography (25 g silica gel,
1
hexane/EtOAc 85:15); white solid; yield: 14 mg (6%). H NMR (600 MHz, Chloroform-d) δ 7.49 (d, J =
1
5.9 Hz, 1H), 7.29 (t, J = 1.5 Hz, 1H), 7.22 (dd, J = 11.0, 1.6 Hz, 1H), 7.15 (dd, J = 11.1, 2.1 Hz, 1H), 7.09
(
(
(
dd, J = 8.3, 2.0 Hz, 1H), 7.01 (t, J = 8.5 Hz, 1H), 6.22 (d, J = 15.9 Hz, 1H), 6.09 (d, J = 8.0 Hz, 1H), 4.18
13
d, J = 7.9 Hz, 1H), 1.53 (s, 9H), 1.52 (s, 9H). C NMR (151 MHz, Chloroform-d) δ 168.8, 166.5, 151.1
d, J = 238.7 Hz), 147.7 (d, J = 11.6 Hz), 147.4 (d, J = 247.9 Hz), 144.1 (d, J = 14.4 Hz), 142.5 (d, J = 2.2
Hz), 132.6 (d, J = 5.7 Hz), 129.2 (d, J = 5.2 Hz), 128.4 (d, J = 3.4 Hz), 122.7 (d, J = 3.3 Hz), 120.8 (d, J =
2
8
–
4
.9 Hz), 119.2, 117.7 (d, J = 2.3 Hz), 116.1 (d, J = 17.3 Hz), 113.6 (d, J = 19.6 Hz), 87.2 (d, J = 1.6 Hz),
19
3.2, 80.8, 56.4 (d, J = 2.2 Hz), 28.3, 28.2. F NMR (376 MHz, Chloroform-d) δ –137.1 (d, J = 11.1 Hz),
+
139.7 (dd, J = 11.2, 8.6 Hz). HRMS (ESI): m/z calcd. for C H F O : 475.1927 [M+H] ; found:
26
29
2
6
75.1944.
4
2
.2.46. (±)-trans-(E)-2-methoxy-4-(7-methoxy-3-methyl-5-(prop-1-en-1-yl)-2,3-dihydro-benzo-furan-
-yl)phenol (9a)
Prepared according to the general procedure E from isoeugenol (2a, 82 mg, 0.5 mmol); isolated by
1
column chromatography (25 g silica gel, hexane/EtOAc 8:2); white solid; yield: 31 mg (19%). H NMR
(
(
600 MHz, Chloroform-d) δ 6.98 (d, J = 1.7 Hz, 1H), 6.92 – 6.88 (m, 2H), 6.80 (d, J = 1.6 Hz, 1H), 6.77
d, J = 1.4 Hz, 1H), 6.37 (dd, J = 15.7, 1.8 Hz, 1H), 6.11 (dq, J = 15.6, 6.6 Hz, 1H), 5.67 (d, J = 1.4 Hz, 1H),
5
1
1
.11 (d, J = 9.5 Hz, 1H), 3.90 (s, 3H), 3.88 (s, 3H), 3.50 – 3.42 (m, 1H), 1.88 (dd, J = 6.7, 1.8 Hz, 3H),
13
.38 (d, J = 6.9 Hz, 3H). C NMR (151 MHz, Chloroform-d) δ 146.8, 146.7, 145.9, 144.3, 133.4, 132.3,
32.2, 131.0, 123.6, 120.1, 114.2, 113.4, 109.3, 109.0, 93.9, 56.1, 56.0, 45.7, 18.5, 17.7. HRMS (ESI):
+
m/z calcd. for C H O : 327.1591 [M+H] ; found: 327.1621.
20
23
4
4
.2.47. (±)-trans-(E)-4-(3-(hydroxymethyl)-5-(3-hydroxyprop-1-en-1-yl)-7-methoxy-2,3-di-hydro-
benzofuran-2-yl)-2-methoxyphenol (9b)
Prepared according to the general procedure E from coniferyl alcohol (2b, 90 mg, 0.5 mmol); isolated
by preparative RP-HPLC (gradient H O/MeOH) and further purification by column chromatography
2
1
(
Sephadex LH-20, MeOH); white solid; yield: 24 mg (13%). H NMR (600 MHz, Methanol-d ) δ 6.96 (t,
4
J = 1.2 Hz, 1H), 6.95 (d, J = 2.0 Hz, 1H), 6.94 (d, J = 1.6 Hz, 1H), 6.82 (dd, J = 8.2, 2.0 Hz, 1H), 6.77 (d, J =
.1 Hz, 1H), 6.54 (dt, J = 15.7, 1.6 Hz, 1H), 6.22 (dt, J = 15.8, 5.9 Hz, 1H), 5.52 (d, J = 6.3 Hz, 1H), 4.20
8
13
(
(
dd, J = 5.9, 1.6 Hz, 2H), 3.87 (s, 3H), 3.90 – 3.75 (m, 2H), 3.81 (s, 3H), 3.49 (q, J = 6.2 Hz, 1H). C NMR
151 MHz, Methanol-d ) δ 149.2, 149.1, 147.6, 145.5, 134.5, 132.6, 132.0, 130.3, 127.5, 119.8, 116.5,
4
1
16.2, 112.1, 110.5, 89.3, 64.8, 63.9, 56.7, 56.4, 55.1. HRMS (ESI): m/z calcd. for C H O : 359.1489
20 23 6
+
[M+H] ; found: 359.1491.
4
1
.2.48. (±)-trans-(E)-2-(4-hydroxy-3-methoxyphenyl)-7-methoxy-5-(3-oxo-3-(propylamino)prop-1-en-
-yl)-N-propyl-2,3-dihydrobenzofuran-3-carboxamide (10a)
Prepared according to general procedure E from 4a (118 mg, 0.5 mmol); isolated by column
chromatography (50 g aluminium oxide, CHCl /MeOH 97:3) and subsequent purification by HPLC;
3
1
white solid; yield: 8 mg (14%). H NMR (400 MHz, Methanol-d ) δ 7.45 (d, J = 15.7 Hz, 1H), 7.14 (d, J =
4
2
9