Dehydrodieugenol B derivatives as antiparasitic agents: Synthesis and biological activity against Trypanosoma cruzi

Article abstract of DOI:10.1016/j.ejmech.2019.05.001

Chagas disease is a neglected protozoan disease that affects more than eight million people in developing countries. Due to the limited number and toxicity profiles of therapies in current use, new drugs are urgently needed. In previous studies, we report

Full text of DOI:10.1016/j.ejmech.2019.05.001

Accepted Manuscript
Dehydrodieugenol B derivatives as antiparasitic agents: Synthesis and biological
activity against Trypanosoma cruzi
Daiane D. Ferreira, Fernanda S. Sousa, Thais A. Costa-Silva, Juliana Q. Reimão,
Ana C. Torrecilhas, Deidre M. Johns, Claire E. Sear, Kathia M. Honorio, João
Henrique G. Lago, Edward A. Anderson, Andre G. Tempone
PII:
S0223-5234(19)30404-0
DOI:
https://doi.org/10.1016/j.ejmech.2019.05.001
EJMECH 11311
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 26 February 2019
Revised Date: 30 April 2019
Accepted Date: 1 May 2019
Please cite this article as: D.D. Ferreira, F.S. Sousa, T.A. Costa-Silva, J.Q. Reimão, A.C. Torrecilhas,
D.M. Johns, C.E. Sear, K.M. Honorio, Joã.Henrique.G. Lago, E.A. Anderson, A.G. Tempone,
Dehydrodieugenol B derivatives as antiparasitic agents: Synthesis and biological activity against
Trypanosoma cruzi, European Journal of Medicinal Chemistry (2019), doi: https://doi.org/10.1016/
j.ejmech.2019.05.001.
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ACCEPTED MANUSCRIPT

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ACCEPTED MANUSCRIPT
Dehydrodieugenol B derivatives as antiparasitic agents: Synthesis and
biological activity against Trypanosoma cruzi
1δ
2δ
3
Daiane D. Ferreira , Fernanda S. Sousa , Thais A. Costa-Silva ,
4
2
5
6
Juliana Q. Reimão , Ana C. Torrecilhas , Deidre M. Johns , Claire E. Sear ,
7
3
6
Kathia M. Honorio , João Henrique G. Lago , Edward A. Anderson *,
1
Andre G. Tempone *
1
Centre for Parasitology and Mycology, Instituto Adolfo Lutz, São Paulo, 01246-000,
Brazil.
2
Institute of Environmental, Chemical and Pharmaceutical Sciences, Federal University
of São Paulo, Diadema 09972-270, Brazil
3
Center of Natural and Human Sciences, Federal University of ABC, Santo André
09210-580, Brazil
4
Faculdade de Medicina de Jundiaí, Rua Francisco Telles, 250 Vila Arens II 13202550
Jundiaí, SP – Brazil
-
5
Department of Biomedical Sciences, Carlson College of Veterinary Medicine, Oregon
State University, Corvallis, Oregon, 97331 USA
6
Chemistry Research Laboratory, University of Oxford, 12 Mansfield Road, Oxford,
OX1 3TA, UK
7
Escola de Artes, Ciências e Humanidades, Universidade de São Paulo, São Paulo,
03828-000, Brazil
*
Corresponding authors. Andre G. Tempone (andre.tempone@ial.sp.gov.br), Edward
A. Anderson (edward.anderson@chem.ox.ac.uk).
δ
These authors contributed equally to the work.

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ABSTRACT
Chagas disease is a neglected protozoan disease that affects more than eight
million people in developing countries. Due to the limited number and toxicity profiles of
therapies in current use, new drugs are urgently needed. In previous studies, we
reported the isolation of two related antitrypanosomal neolignans from Nectandra
leucantha (Lauraceae). In this work, a semi-synthetic library of twenty-three neolignan
derivatives was prepared to explore synthetically accessible structure activity
relationships (SAR) against Trypanosoma cruzi. Five compounds demonstrated activity
against trypomastigotes (IC values from 8 to 64 µM) and eight showed activity against
50
intracellular amastigotes (IC50 values from 7 to 16 µM). Eighteen derivatives
demonstrated no mammalian cytotoxicity up to 200 µM. The phenolic acetate derivative
of natural dehydrodieugenol B was effective against both parasite forms and eliminated
100% of amastigotes inside macrophages. This compound caused rapid and intense
depolarization of the mitochondrial membrane potential, with decreased levels of
intracellular reactive oxygen species being observed. Fluorescence assays
demonstrated that this derivative affected neither the permeability nor the electric
potential of the parasitic plasma membrane, an effect also corroborated by scanning
electron microscopy studies. Structure-activity relationship studies (SARs)
demonstrated that the presence of at least one allyl side chain on the biaryl ether core
was important for antitrypanosomal activity, and that the free phenol is not essential.
This set of neolignan derivatives represents a promising starting point for future
Chagas disease drug discovery studies.
Keywords: neolignans, drugs, therapy, Trypanosoma cruzi, Chagas disease

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Abbreviations
ANOVA, analysis of variance;
BALB/c, isogenic murine lineage;
CCCP, carbonylcyanidem-chlorophenylhydrazone
CC , 50% cytotoxic concentration;
50
DiSBAC2(3), bis-(1,3-diethylthiobarbituric acid) trimethine oxonol;
DMAP, 4-dimethylaminopyridine
DMF, dimethylformamide
DMSO, dimethylsulfoxide
FDA, Food and Drug Administration
FBS, fetal bovine serum;
H2DCf-DA, 2 ,7 -dichlorodihydrofluorescein diacetate;
HBSS, Hank’s Balanced Salt Solution;
HPLC, high performance liquid chromatography
HRESIMS, high resolution electrospray ionization mass spectra
IC , 50% inhibitory concentration;
50
JC-1,
5,5,6,6-tetrachloro-1,1,3,3-tetraethylbenzimidazole
carbocyanide
iodide;
LLCMK2, Kidney Rhesus monkey cells;
MS, mass spectrometry;
MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide;
NCTC cells-clone L929, murine conjunctive cells;
NMR, nuclear magnetic resonance;
PAINS – Pan-Assay Interference Compounds
PBS, phosphate buffered saline;
ROS, reactive oxygen species;
RPMI-1640, Roswell Park Memorial Institute medium
SAR, structure activity relationships;
SI, selectivity index;

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SYTOX®
Green,
amino(4-(6-(amino(iminio)methyl)-1Hindol-2-yl)
phenyl)
methaniminium chloride;
TLC, thin layer chromatography.
THF, tetrahydrofuran
TMS, tetramethylsilane

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1. INTRODUCTION
Chagas disease is a major public health issue in Latin America, where it is
endemic in more than twenty countries [1,2]. Migrant populations, and modes of
transmission such as blood, food and organ donation, have further led to the spread of
this disease to Europe, North America, Japan and Australia [3-5]. Approximately eight
million people are infected worldwide, causing approximately 7,000 deaths annually
[
6,7]. Current treatments for Chagas disease comprise two nitro-substituted
heterocyclic drugs, benznidazole and nifurtimox [8]. These compounds have shown
successful clinical, parasitological, and serological treatment outcomes during the
acute phase and in congenital Chagas disease [9]. However, these drugs are far from
ideal: efficacy diminishes with disease chronification [10], and both exhibit undesirable
toxicity profiles leading to adverse side-effects. Furthermore, parasite resistance to
these treatments is becoming more frequent [11-13]. Collectively, these facts highlight
an urgent need for safer and more efficacious drugs.
Natural products are a proven source of new chemical scaffolds for drug
discovery, as evidenced by the large number of FDA-approved drugs that are derived
from or are inspired by natural products [14]. Compounds isolated from the Nectandra
genus (Lauraceae), have been shown to exhibit antibacterial, anti-inflammatory, arterial
hypertension control, antitumor, and antiparasitic activity [15]. We recently reported the
first phytochemical study of Nectandra leucantha, which led to the isolation of the
neolignans dehydrodieugenol B (1) and methyldehydrodieugenol B (2) (Figure 1).
These compounds were found to display bioactivity against the intracellular forms of
Leishmania donovani [16] and Trypanosoma cruzi [17]. In light of these results, and
due to the urgent need for novel bioactive lead compounds for drug development
against Chagas disease, we performed a semi-synthetic derivatization of compounds 1
and 2 to explore the effects of structural modifications on their biological activity. Here
we describe the results of these studies, which involved the preparation of 23 semi-

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synthetic derivatives, and evaluation of their antiparasitic activity against Trypanosoma
cruzi, and their mammalian cytotoxicity. Of these derivatives, compound 14 (see below)
was selected for phenotypic studies of drug-treated parasites to identify possible target
organelles.
OH
OMe
OMe
OMe
MeO
O
MeO
O
1
2
Figure 1. Structures of compounds 1 and 2 isolated from Nectandra leucantha.
. MATERIAL AND METHODS
2
2.1. General experimental procedures
NMR spectra were recorded on a Bruker AVIIIHD 400 nanobay spectrometer,
1
13
operating at 400 and 101 MHz for H and C nuclei, respectively. CDCl was used as
3
solvent with tetramethylsilane as internal standard, or by reference to the residual
solvent peak (δ 7.26; δ 77.10 ppm). Chemical shifts (δ) are reported in ppm and
H
C
coupling constants (J) in Hz. High resolution electrospray ionization mass spectra
HRESIMS) were obtained using a Bruker Daltonics MicrOTOF-Q IITM ESI-Qq-TOF in
(
positive mode. Silica gel 60 (Merck, 63 – 210 mesh) was used for the column
chromatographic separation procedures, while silica gel 60 PF254 (Merck) was used for
TLC (0.25 mm). For all extraction and chromatography procedures, analytical grade
solvents were used.

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2.2. Isolation of compounds 1 and 2 from N. leucantha leaves
As described previously [18], the n-hexane extract from twigs of N. leucantha (7.4 g)
was subjected to successive column chromatography over silica gel and Sephadex LH-
2
2
2
0 to afford compounds 1 (1.58 g) and 2 (1.15 g) in 99% purity as indicated by HPLC.
.3. Hydrogenation procedures for preparation of compounds 3 and 25.
-Methoxy-6-(2-methoxy-4-propylphenoxy)-4-propylphenol (3). To
a
solution of
compound 1 (0.30 mmol) in EtOH (5 mL) was added 10% wt Pd/C (0.09 mmol). The
reaction flask was purged with hydrogen and the reaction mixture was stirred for 1 h
under H (1 atm). After filtration through Celite®, the filtrate was concentrated under
2
1
reduced pressure to afford compounds 3 (98.7 mg, 0.29 mmol,99%) as a yellow oil; H
NMR (CDCl ) δ 6.87 (1H, d, J = 8.1 Hz), 6.79 (1H, d, J = 1.9 Hz), 6.69 (1H, dd, J = 8.1
3
H
and 1.9 Hz), 6.48 (1H, d, J = 1.9 Hz), 6.40 (1H, d, J = 1.9 Hz), 5.85 (1H, s, OH), 3.89
(
3H, s), 3.87 (3H, s), 2.60-2.52 (2H, m), 2.49-2.39 (2H, m), 1.70-1.60 (2H, m), 1.60-
1
3
1
1
5
.50 (2H, m), 0.95 (3H, t, J = 7.3 Hz), 0.89 (3H, t, J = 7.3 Hz). C NMR (CDCl ) δC
3
50.2, 147.6, 144.3, 143.9, 139.1, 134.8, 133.8, 120.7, 119.3, 112.8, 111.7, 107.1,
+
6.2, 56.0, 37.9, 37.8, 24.7, 24.7, 13.9, 13.7. HRESIMS m/z 353.1720 [M+Na] (calc.
for C H NaO 353.1723).
20
26
4
1,2-Dimethoxy-3-(2-methoxy-4-propylphenoxy)-5-propylbenzene (25). To a solution of
compounds 2 (0.088 mmol) in EtOH (2 mL) was added 10% wt Pd/C (0.028 mmol).
The reaction flask was purged with hydrogen and the reaction mixture was stirred for 1
h under H (1 atm). After filtration through Celite®, the filtrate was concentrated under
2
reduced pressure to afford compound 25 (36.0 mg, 0.104 mmol, 98%) as a yellow oil;
1
H NMR (CDCl ) δ 6.78 (2H, m), 6.68 (1H, dd, J = 8.1 and 1.8 Hz), 6.47 (1H, d, J = 1.8
3
H

8
ACCEPTED MANUSCRIPT
Hz), 6.26 (1H, d, J = 1.8 Hz), 3.87 (6H, s), 3.83 (3H, s), 2.61-2.50 (2H, m), 2.48-2.37
(
2H, m), 1.65 (2H, m), 1.59-1.47 (2H, m), 0.95 (3H, t, J = 7.3 Hz), 0.88 (3H, t, J = 7.3
13
Hz). C NMR (CDCl ) δ 153.3, 150.5, 150.4, 143.8, 138.7, 138.2, 137.7, 120.6, 119.2,
3
C
112.9, 111.2, 107.1, 61.0, 56.1, 56.0, 38.1, 37.9, 24.7, 24.5, 13.9, 13.8. HRESIMS m/z
+
3
67.1876 [M+Na] (calc. for C H NaO 367.1880).
21
28
4
2.4. Alkylation procedure for preparation of compounds 4–13.
To a suspension of NaH (3.6 mg, 60% wt, 0.090 mmol) in DMF (300 µL) at 0 ºC
was added compound 1 or 3 (0.060 mmol). To this solution was added the respective
alkyl halide (see specific procedure for quantities) via microsyringe, and the resulting
mixture was stirred at 0 ºC for 2 h. After addition of saturated aq. NH Cl (1 mL) and
4
extraction using EtOAc (3 x 1 mL), the organic layers were washed with H O (4 x 1
2
mL), brine (1 mL) and dried over MgSO After concentration under reduced pressure,
4.
the crude product was purified by flash column chromatography on silica gel
petroleum ether/EtOAc, 8:2) to afford the alkylated products 4–13.
(
5-Allyl-1-(4-allyl-2-methoxyphenoxy)-2-ethoxy-3-methoxybenzene (4). Prepared from 1
using ethyl iodide (0.10 mmol, 1.7 equiv.) as electrophile. Yellow oil, 18.1 mg, 0.051
1
mmol, 85% yield; H NMR (CDCl ) δ 6.80-6.77 (2H, m), 6.68 (1H, dd, J = 8.1, 1.9 Hz),
3
H
6
.48 (1H, d, J = 1.9 Hz), 6.30 (1H, d, J = 1.9 Hz), 6.04-5.83 (2H, m), 5.13-4.99 (4H, m),
.08 (2H, q, J = 7.1 Hz), 3.85 (3H, s), 3.83 (3H, s), 3.36 (2H, d, J = 6.7 Hz), 3.24 (2H, d,
4
13
J = 6.7 Hz), 1.28 (3H, t, J = 7.1 Hz). C NMR (CDCl ) δ 153.8, 150.7, 150.4, 144.4,
3
C
137.5, 137.1, 137.0, 135.7, 135.4, 120.7, 119.1, 115.9, 115.8, 113.0, 111.7, 107.4,
+
6
9.1, 56.1, 56.0, 40.1, 39.9, 15.5. HRESIMS m/z 377.1719 [M+Na] (calc. for
C H NaO 377.1723).
22
26
4

9
ACCEPTED MANUSCRIPT
5
1
0
-Allyl-1-(4-allyl-2-methoxyphenoxy)-3-methoxy-2-propoxybenzene (5). Prepared from
using n-propyl iodide (0.10 mmol, 1.7 equiv.) as electrophile. Yellow oil, 21.1 mg,
1
.057 mmol, 95%; H NMR (CDCl ) δ 6.78 (1H, d, J = 1.8 Hz), 6.75 (1H, d, J = 8.1
3
H
Hz), 6.67 (1H, dd, J = 8.1, 1.8 Hz), 6.49 (1H, d, J = 1.8 Hz), 6.33 (1H, d, J = 1.8 Hz),
.03-5.83 (2H, m), 5.13-4.99 (4H, m), 3.96 (2H, t, J = 6.7 Hz), 3.85 (3H, s), 3.84 (3H,
s), 3.36 (2H, d, J = 6.7 Hz), 3.25 (2H, d, J = 6.7 Hz), 1.68 (2H, sextet, J = 7.5 Hz), 0.90
6
13
(
3H, t, J = 7.5 Hz). C NMR (CDCl ) δ 153.8, 150.4, 150.2, 144.6, 137.5, 137.5,
3 C
1
4
3
37.1, 135.5, 135.3, 120.7, 118.7, 115.9, 115.8, 112.9, 112.1, 107.6, 75.2, 56.1, 56.0,
+
0.1, 39.9, 23.3, 10.3. HRESIMS m/z 391.1877 [M+Na] (calc. for C H NaO
23
28
4
91.1880).
5-Allyl-1-(4-allyl-2-methoxyphenoxy)-2-butoxy-3-methoxybenzene (6).Prepared from 1
using n-butyl iodide (0.10 mmol, 1.7 equiv.) as electrophile. Yellow oil, 26.4 mg, 0.069
1
mmol, 97%; H NMR (CDCl ) δ 6.79 (1H, d, J = 1.9 Hz), 6.75 (1H, d, J = 8.1 Hz), 6.68
3
H
(
(
1H, dd, J = 8.1, 1.9 Hz), 6.49 (1H, d, J = 1.9 Hz), 6.33 (1H, d, J = 1.9 Hz), 6.04-5.83
2H, m), 5.13-5.00 (4H, m), 3.99 (2H, t, J = 6.7 Hz), 3.84 (3H, s), 3.84 (3H, s), 3.36 (2H,
d, J = 6.7 Hz), 3.25 (2H, d, J = 6.7 Hz), 1.68-1.59 (2H, m), 1.36 (2H, sextet, J = 7.5 Hz),
1
3
0
1
4
4
.87 (3H, t, J = 7.5 Hz). C NMR (CDCl ) δc 153.8, 150.4, 150.2, 144.6, 137.6, 137.5,
3
37.2, 135.5, 135.3, 120.7, 118.6, 115.9, 115.8, 112.9, 112.1, 107.6, 73.3, 56.1, 56.0,
+
0.1, 40.0, 32.1, 19.0, 13.9. HRESIMS m/z 405.2030 [M+Na] (calc. for C H NaO
24
30
4
05.2036).
5-Allyl-1-(4-allyl-2-methoxyphenoxy)-2-(allyloxy)-3-methoxybenzene (7).Prepared from
1
using allyl bromide (0.12 mmol, 2.0 equiv.) as electrophile. Yellow oil, 21.9 mg, 0.059
1
mmol, 99%; H NMR (CDCl ) δ 6.79 (1H, d, J = 8.1 Hz), 6.79 (1H, d, J = 1.9 Hz), 6.69
3
H
(
1H, dd, J = 8.1 and 1.9 Hz), 6.48 (1H, d, J = 1.9 Hz), 6.29 (1H, d, J = 1.9 Hz), 6.11-
5
.81 (3H, m), 5.24 (1H, dq, J = 17.2 and 1.5 Hz), 5.15-4.99 (5H, m), 4.55 (2H, dt, J =
.0 and 1.5 Hz), 3.85 (3H, s), 3.83 (3H, s), 3.37 (2H, d, J = 6.7 Hz), 3.24 (2H, d, J = 6.7
6

1
0
ACCEPTED MANUSCRIPT
13
Hz). C NMR (CDCl ) δ 153.8, 150.7, 150.5, 144.3, 137.4, 137.1, 136.7, 135.8, 135.6,
3
C
134.6, 120.7, 119.2, 117.4, 115.9, 115.8, 113.1, 111.5, 107.4, 74.3, 56.1, 56.0, 40.1,
+
4
0.0. HRESIMS m/z 389.1720 [M+Na] (calc. for C H NaO 389.1723).
23
26
4
5-Allyl-1-(4-allyl-2-methoxyphenoxy)-2-(benzyloxy)-3-methoxybenzene (8). Prepared
from 1 using benzyl bromide (0.087 mmol, 1.45 equiv.) as electrophile. Yellow oil, 24.2
1
mg, 0.058 mmol, 96%; H NMR (CDCl ) δ 7.40-7.36 (2H, m), 7.28-7.25 (3H, m), 6.79-
3
H
6
.74 (2H, m), 6.69 (1H, d, J = 1.9 Hz), 6.49 (1H, d, J = 1.9 Hz), 6.33 (1H, d, J = 1.9 Hz),
.03-5.85 (2H, m), 5.14-5.00 (6H, m), 3.82 (3H, s), 3.80 (3H, s), 3.36 (2H, d, J = 6.7
6
1
3
13
Hz), 3.25 (2H, d, J = 6.7 Hz). C NMR (CDCl3) C NMR (101 MHz, CDCl ) δ 153.9,
3
C
150.6, 150.4, 144.4, 138.0, 137.5, 137.1, 137.0, 135.7, 128.3, 128.0, 127.8, 127.6,
120.8, 119.0, 115.9, 115.8, 113.0, 111.8, 107.6, 75.0, 56.1, 56.0, 40.1, 40.0. HRESIMS
+
m/z 439.1877 [M+Na] (calc. for C H NaO 439.1880).
27
28
4
2-Ethoxy-1-methoxy-3-(2-methoxy-4-propylphenoxy)-5-propylbenzene (9). Prepared
from 3 using ethyl iodide (0.10 mmol, 1.7 equiv.) as electrophile. Pale yellow oil, 19.7
1
mg, 0.055 mmol, 92%; H NMR (CDCl ) δ 6.78 (1H, d, J = 1.9 Hz), 6.76 (1H, d, J = 8.1
3
H
Hz), 6.66 (1H, dd, J = 8.1 and 1.9 Hz), 6.47 (1H, d, J = 1.9 Hz), 6.29 (1H, d, J = 1.9
Hz), 4.08 (2H, q, J = 7.1 Hz), 3.85 (3H, s), 3.84 (3H, s), 2.59-2.52 (2H, m), 2.48-2.40
(
2H, m), 1.70-1.61 (2H, m), 1.59-1.50 (2H, m), 1.27 (3H, t, J = 7.1 Hz), 0.95 (3H, t, J =
1
3
7
1
2
3
.3 Hz), 0.89 (3H, t, J = 7.3 Hz). C NMR (CDCl ) δ 153.6, 150.6, 150.2, 144.1,
3 C
38.4, 138.1, 136.7, 120.6, 118.9, 112.9, 111.5, 107.3, 69.0, 56.1, 56.0, 38.0, 37.8,
+
4.7, 24.5, 15.5, 13.9, 13.8. HRESIMS m/z 381.2036 [M+Na] (calc. for C H NaO
22
30
4
81.2036).
1-Methoxy-3-(2-methoxy-4-propylphenoxy)-2-propoxy-5-propylbenzene (10). Prepared
from 3 using n-propyl iodide (0.10 mmol, 1.7 equiv) as electrophile. Yellow oil, 19.0 mg,
1
0
.051 mmol, 85%; H NMR (CDCl ) δ 6.78 (1H, d, J = 1.9 Hz), 6.73 (1H, d, J = 8.1
3
H

1
1
ACCEPTED MANUSCRIPT
Hz), 6.65 (1H, dd, J = 8.1 and 1.9 Hz), 6.48 (1H, d, J = 1.9 Hz), 6.32 (1H, d, J = 1.9
Hz), 3.95 (2H, t, J = 6.8 Hz), 3.85 (3H, s), 3.84 (3H, s), 2.59 - 2.51 (2H, m), 2.48 - 2.41
(
2H, m), 1.72-1.60 (4H, m), 1.59-1.50 (2H, m), 0.94 (3H, t, J = 7.3 Hz), 0.93 (3H, t, J =
1
3
13
7
1
7
.3 Hz), 0.89 (3H, t, J = 7.3 Hz). C NMR (CDCl ) δC C NMR (101 MHz, CDCl ) δC
3
3
53.6, 150.3, 150.0, 144.3, 138.1, 138.0, 137.2, 120.5, 118.4, 112.8, 111.9, 107.5,
5.2, 56.1, 56.0, 38.0, 37.8, 24.7, 24.5, 23.3, 13.8, 13.8, 10.3. HRESIMS m/z 395.2190
+
[
M+Na] (calc. for C H NaO 395.2193).
23
32
4
2-Butoxy-1-methoxy-3-(2-methoxy-4-propylphenoxy)-5-propylbenzene (11). Prepared
from 3 using n-butyl iodide (0.10 mmol, 1.7 equiv.) as electrophile. Yellow oil, 18.2 mg,
1
0
6
3
.047mmol, 78%; H NMR (CDCl ) δ 6.78 (1H, d, J = 1.9 Hz), 6.72 (1H, d, J = 8.1 Hz),
3
H
.65 (1H, dd, J = 8.1 and 1.9 Hz), 6.48 (1H, d, J = 1.9 Hz), 6.32 (1H, d, J = 1.9 Hz),
.98 (2H, t, J = 6.7 Hz), 3.84 (6H, s), 2.58-2.53 (2H, m), 2.47-2.42 (2H, m), 1.68-1.61
(
4H, m), 1.59-1.51 (2H, m), 1.41-1.31 (2H, m), 0.95 (3H, t, J = 7.3 Hz), 0.95 (3H, t, J =
1
3
7
1
3
.3 Hz), 0.87 (3H, t, J = 7.3 Hz). C NMR (CDCl ) δ 153.6, 150.2, 150.0, 144.3,
3 C
38.1, 138.0, 137.2, 120.5, 118.4, 112.8, 111.9, 107.5, 73.3, 56.1, 56.0, 38.0, 37.8,
+
2.1, 24.7, 24.5, 19.0, 13.9, 13.8, 13.8. HRESIMS m/z 409.2345 [M+Na] (calc. for
C H NaO 409.2349).
24
34
4
2-(Allyloxy)-1-methoxy-3-(2-methoxy-4-propylphenoxy)-5-propylbenzene
(12).
Prepared from 3 using allyl bromide (0.12 mmol, 2.0 equiv.) as electrophile. Pale yellow
1
oil, 19.2 mg, 0.052 mmol, 86%; H NMR (CDCl ) δ 6.80-6.75 (2H, m), 6.67 (1H, dd, J
3
H
=
8.1 and 1.9 Hz), 6.47 (1H, d, J = 1.9 Hz), 6.28 (1H, d, J = 1.9 Hz), 6.10-5.98 (1H, m),
.23 (1H, dq, J = 17.2 and 1.5 Hz), 5.11 (1H, ddd, J = 10.3, 2.9 and 1.1 Hz), 4.54 (2H,
5
dt, J = 6.0 and 1.5 Hz), 3.85 (3H, s), 3.83 (3H, s), 2.59-2.53 (2H, m), 2.46-2.41 (2H, m),
1
0
1
.64 (2H, sextet, J = 7.4 Hz), 1.54 (2H, sextet, J = 7.4 Hz), 0.95 (3H, t, J = 7.4 Hz),
13
.88 (3H, t, J = 7.4 Hz). C NMR (CDCl ) δ 153.6, 150.5, 150.3, 144.0, 138.5, 138.3,
3
C
36.4, 134.7, 120.6, 119.0, 117.3, 113.0, 111.3, 107.2, 74.2, 56.1, 56.0, 38.0, 37.8,

1
2
ACCEPTED MANUSCRIPT
+
2
4.7, 24.5, 13.9, 13.8. HRESIMS m/z 393.2034 [M+Na] (calc. for C H NaO
23 30
4
393.2036).
2-(Benzyloxy)-1-methoxy-3-(2-methoxy-4-propylphenoxy)-5-propylbenzene
(13).
Prepared from 3 using benzyl bromide (0.10 mmol, 1.7 equiv.) as electrophile. Pale
1
yellow oil, 19.9 mg, 0.048 mmol, 80%; H NMR (CDCl ) δ 7.39-7.37 (2H, m), 7.28-7.26
3
H
(
3H, m), 6.77 (1H, d, J = 1.9 Hz), 6.74 (1H, d, J = 8.1 Hz), 6.66 (1H, dd, J = 8.1 and 1.9
Hz), 6.48 (1H, d, J = 1.9 Hz), 6.33 (1H, d, J = 1.9 Hz), 5.04 (2H, s), 3.83 (3H, s), 3.80
(
3H, s), 2.59-2.53 (2H, m), 2.48-2.43 (2H, m), 1.70-1.61 (2H, m), 1.61-1.51 (2H, m),
1
3
0
1
1
4
.96 (3H, t, J = 7.3 Hz), 0.89 (3H, t, J = 7.3 Hz). C NMR (CDCl ) δ 153.7, 150.4,
3 C
50.1, 144.2, 138.4, 138.4, 138.0, 136.7, 128.3, 128.0, 127.5, 120.6, 118.7, 112.9,
11.7, 107.5, 75.0, 56.1, 55.9, 38.1, 37.9, 24.7, 24.5, 13.9, 13.8. HRESIMS m/z
+
43.2191 [M+Na] (calc. for C H NaO 443.2193).
27
32
4
2.5. Acetylation procedure for preparation of compounds 14 and 15.
Compounds 1 or 3 (0.060 mmol), acetic anhydride (0.45 mmol) and pyridine
(
1.38 mmol) were heated to 100 ºC for 2 h. The cooled mixture was diluted with iced
H O, acidified dropwise with 1 M HCl and extracted with EtOAc (3 x 1 mL). The
2
combined organic extracts were dried over MgSO and concentrated under reduced
4
pressure. The crude product was purified by flash chromatography (petroleum
ether/EtOAc, 8:2), to afford products 14 and 15, respectively.
4-Allyl-2-(4-allyl-2-methoxyphenoxy)-6-methoxyphenyl acetate (14). Prepared from 1;
1
Yellow oil, 21.2 mg, 0.058 mmol, 96%; H NMR (CDCl ) δ 6.87 (1H, d, J = 8.1 Hz),
3
H
6
.79 (1H, d, J = 1.9 Hz), 6.70 (1H, dd, J = 8.1 and 1.9 Hz), 6.50 (1H, d, J = 1.9 Hz),
.26 (1H, d, J = 1.9 Hz), 6.04-5.81 (2H, m), 5.14-5.00 (4H, m), 3.82 (3H, s), 3.81 (3H,
6
13
s), 3.37 (2H, d, J = 6.7 Hz), 3.26 (2H, d, J = 6.7 Hz), 2.25 (3H, s). C NMR (CDCl ) δ
3
168.7, 152.3, 150.9, 150.2, 143.5, 138.4, 137.3, 136.8, 136.8, 128.5, 121.0, 120.6,

1
3
ACCEPTED MANUSCRIPT
116.2, 116.0, 113.1, 110.5, 106.7, 56.1, 56.1, 40.2, 40.0, 20.5. HRESIMS m/z
+
3
91.1518 [M+Na] (calc. for C H NaO 391.1516).
22
24
5
2
3
6
6
-Methoxy-6-(2-methoxy-4-propylphenoxy)-4-propylphenyl acetate (15). Prepared from
1
; yellow oil, 19.7 mg, 0.053 mmol, 62%; H NMR (CDCl ) δ 6.84 (1H, d, J = 8.1 Hz),
3
H
.78 (1H, d, J = 1.9 Hz), 6.68 (1H, dd, J = 8.1 and 1.9 Hz), 6.48 (1H, d, J = 1.9 Hz),
.25 (1H, d, J = 1.9 Hz), 3.82 (3H, s), 3.82 (3H, s), 2.60-2.52 (2H, m), 2.48-2.42 (2H,
m), 2.24 (3H, s), 1.70-1.60 (2H, m), 1.58-1.49 (2H, m), 0.95 (3H, t, J = 7.3 Hz), 0.89
13
(
3H, t, J = 7.3 Hz). C NMR (CDCl ) δ 168.8, 152.1, 150.7, 150.1, 143.2, 141.1,
3 C
139.4, 128.1, 120.8, 120.4, 113.1, 110.4, 106.6, 56.1, 56.1, 38.3, 37.9, 24.6, 24.4, 20.5,
+
1
3.8, 13.8. HRESIMS m/z 395.1826 [M+Na] (calc. for C H NaO 395.1829).
22
28
5
2
4
1
.6. Propionoylation procedure for preparation of compounds 16 and 17.
-Allyl-2-(4-allyl-2-methoxyphenoxy)-6-methoxyphenyl propionate (16). To a solution of
(23.7 mg, 0.071mmol) in 120 µL of CH Cl at 0 ºC were added Et N (19 µL, 0.14
2
2
3
mmol), DMAP (0.8 mg, 0.007 mmol) and propionyl chloride (6.2 µL, 0.071 mmol). The
reaction mixture was allowed to warm to room temperature and was stirred for 3 h.
After concentration under reduced pressure, the resulting oily residue was redissolved
in EtOAc (1 mL), washed with H O and the aqueous phase extracted with EtOAc (4 x 1
2
mL). The combined organic phases were dried over MgSO , filtered and evaporated to
4
dryness under reduced pressure. The crude product was purified by flash
chromatography (petroleum ether/EtOAc, 8:2), to afford 16 (17.5 mg, 0.046 mmol,
1
6
4%) as a pale yellow oil; H NMR (CDCl ) δ 6.85 (1H, d, J = 8.1 Hz), 6.78 (1H, d, J =
3
H
1.8 Hz), 6.69 (1H, dd, J = 8.1 and 1.8 Hz), 6.50 (1H, d, J = 1.8 Hz), 6.29 (1H, d, J = 1.8
Hz), 6.03-5.81 (2H, m), 5.14-5.00 (4H, m), 3.81 (6H, s), 3.36 (2H, d, J = 6.7 Hz), 3.26
13
(
2H, d, J = 6.7 Hz), 2.53 (2H, q, J = 7.5 Hz), 1.18 (3H, t, J = 7.5 Hz). C NMR (CDCl )
3
δC 172.1, 152.4, 150.8, 150.1, 143.7, 138.2, 137.3, 136.8, 136.5, 128.7, 120.9, 120.3,

1
4
ACCEPTED MANUSCRIPT
116.2, 115.9, 113.1, 110.9, 106.9, 56.2, 56.1, 40.2, 40.0, 27.1, 9.2. HRESIMS m/z
+
4
05.1669 [M+Na] (calc. for C H NaO 405.1672).
23
26
5
2-Methoxy-6-(2-methoxy-4-propylphenoxy)-4-propylphenyl propionate (17). To
a
solution of 3 (20 mg, 0.06 mmol) in 120 µL of CH Cl at 0 ºC were added Et N (16 µL,
2
2
3
0.12 mmol), DMAP (0.7 mg, 0.006 mmol) and propionyl chloride (5.8 µL, 0.066 mmol).
The reaction mixture was allowed to warm to room temperature and was stirred for 3 h.
After concentration under reduced pressure, the resulting oily residue was redissolved
in EtOAc (1 mL), washed with H O and the aqueous phase extracted with EtOAc (4 x 1
2
mL). The combined organic phases were dried over MgSO , filtered and evaporated to
4
dryness under reduced pressure. The crude product was purified by flash
chromatography (petroleum ether/EtOAc, 8:2), to afford 17 (14.3 mg, 0.037 mmol,
1
5
6%) as a yellow oil; H NMR (CDCl ) δ 6.82 (1H, d, J = 8.1 Hz), 6.77 (1H, d, J = 1.9
3
H
Hz), 6.67 (1H, dd, J = 8.1 and 1.9 Hz), 6.49 (1H, d, J = 1.9 Hz), 6.28 (1H, d, J = 1.9
Hz), 3.82 (6H, s), 2.58-2.54 (2H, m), 2.54-2.50 (2H, m), 2.50-2.43 (2H, m), 1.69-1.60
(
2H, m), 1.59-1.50 (2H, m), 1.17 (3H, t, J = 7.3 Hz), 0.95 (3H, t, J = 7.3 Hz), 0.89 (3H, t,
13
J = 7.3 Hz). C NMR (CDCl ) δ 172.1, 152.2, 150.6, 149.9, 143.5, 140.9, 139.1,
3
C
128.4, 120.8, 120.0, 113.1, 110.7, 106.8, 56.1, 56.1, 38.2, 37.8, 27.1, 24.7, 24.4, 13.8,
+
1
3.8, 9.2. HRESIMS m/z 409.1984 [M+Na] (calc. for C H NaO 409.1985).
23
30
5
2.7. Isomerization procedure for preparation of compounds 18 and 22.
2-Methoxy-6-(2-methoxy-4-prop-1-en-1-yl)phenoxy)-4-prop-1-en-1-yl)phenol (18).
A
solution of 1 (50 mg, 0.153 mmol) and KOH (85 mg, 1.53 mmol) in ethylene glycol (200
µL) was refluxed at 150 ºC for 16 h. The reaction mixture was cooled to room
temperature, diluted with H O and extracted with EtOAc (4 x 1 mL). The combined
2
organic extracts were washed with H O (3 x 1 mL), brine (1 mL), dried over MgSO and
2
4
concentrated under reduced pressure. The crude product was purified by flash

1
5
ACCEPTED MANUSCRIPT
chromatography (petroleum ether/EtOAc, 7:3), to afford 18 (33.2 mg, 0.102 mmol, 66
%
yield) as a yellow oil. A mixture of diastereomers (5:1) was formed at each alkene.
Data is reported for the major (E,E) diastereomer, which was assigned by analogy to
1
literature precedents. H NMR (CDCl ) δ 6.95 (1H, d, J = 1.8 Hz), 6.89 (1H, d, J = 8.2
3
H
Hz), 6.84 (1H, dd, J = 8.2 and 1.8 Hz), 6.66 (1H, d, J = 1.8 Hz), 6.53 (1H, d, J = 1.8
Hz), 6.40-6.31 (1H, m), 6.25-6.12 (2H, m), 6.06-5.95 (1H, m), 3.91 (3H, s), 3.87 (3H, s),
13
1
.88 (3H, dd, J = 6.6 and 1.5 Hz), 1.81 (3H, dd, J = 6.6 and 1.5 Hz). C NMR (CDCl )
3
δC153.6, 150.5, 144.7, 144.5, 135.9, 134.7, 130.5, 130.4, 129.6, 129.4, 125.3, 124.1,
+
119.6, 109.7, 109.4, 104.3, 56.2, 56.0, 18.4, 18.3. HRESIMS m/z 349.1407 [M+Na]
(
calc. for C H NaO 349.1410).
20
22
4
1,2-Diethoxy-3-(2-methoxy-4-prop-1-en-1-yl)phenoxy)-5-prop-1-en-1-yl)benzene (22).
A solution of 2 (50 mg, 0.146 mmol) and KOH (81 mg, 1.46 mmol) in ethylene glycol
200 µL) was refluxed at 150 ºC for 16 h. The reaction mixture was cooled to room
(
temperature, diluted with H O and extracted with EtOAc (4 x 1 mL). The combined
2
organic extracts were washed with H O (3 x 1 mL), brine (1 mL), dried over MgSO and
2
4
concentrated under reduced pressure. The crude product was purified by flash
chromatography (petroleum ether/EtOAc, 7:3), to afford 22 (49 mg, 0.144 mmol, 98%)
as a pale yellow oil. A mixture of diastereomers (5:1) was formed at each alkene. Data
is reported for the major (E,E) diastereomer, which was assigned by analogy to
literature precedents, and by a coupling constant of 15.7 Hz for one of the alkene
1
protons. H NMR (CDCl ) δ 6.96 (1H, d, J = 1.9 Hz), 6.86-6.80 (2H, m), 6.64 (1H, d, J
3
H
=
1.9 Hz), 6.41 (1H, d, J = 1.9 Hz), 6.37 (1H, dd, J = 15.7 and 1.9 Hz), 6.25-6.13 (2H,
m), 6.11-5.97 (1H, m), 3.89 (3H, s), 3.87 (3H, s), 3.85 (3H, s), 1.88 (3H, dd, J = 6.6 and
1
3
1
1
1
.5 Hz), 1.81 (3H, dd, J = 6.5 and 1.5 Hz). C NMR (CDCl ) δ 153.7, 150.7, 150.7,
3 C
44.9, 138.9, 134.4, 133.7, 130.6, 130.5, 125.6, 125.2, 119.7, 118.7, 110.0, 109.0,
+
04.6, 61.2, 56.2, 56.1, 18.6, 18.4. HRESIMS m/z 363.1563 [M+Na] (calc. for
C H NaO 363.1567).
21
24
4

1
6
ACCEPTED MANUSCRIPT
2.8. Hydroboration/oxidation procedure for preparation of compounds 19 and 23.
4-(3-Hydroxypropyl)-2-(4-(3-hydroxypropyl)-2-methoxyphenoxy)-6-methoxyphenol (19).
To a stirred solution of 1 (25 mg, 0.076 mmol) in THF (200 µL) under argon was added
BH •THF (152 µL, 1M in THF, 0.152 mmol). After 2 h, 3 M NaOH (150 µL, 0.23 mmol)
3
and H O (160 µL, 30% aq., 0.76 mmol) were added, and the reaction mixture was
2
2
stirred at room temperature for 1.5 h. Brine (1 mL) was added, and the product was
extracted with EtOAc (3 x 1 mL). The organic layer was washed with brine (1 mL),
dried over MgSO and the solvent removed under reduced pressure. The crude
4
product was purified by flash chromatography (petroleum ether/EtOAc 1:1 and 3:7) to
1
afford 19 (22 mg, 0.061 mmol, 80%) as a pale yellow oil; H NMR (CDCl ) δ 6.86 (1H,
3
H
d, J = 8.1 Hz), 6.81 (1H, d, J = 1.8 Hz), 6.71 (1H, dd, J = 8.1 and 1.8 Hz), 6.50 (1H, d, J
=
3
1
1.8 Hz), 6.38 (1H, d, J = 1.8 Hz), 3.89 (3H, s), 3.86 (3H, s), 3.68 (2H, t, J = 6.4 Hz),
.62 (2H, t, J = 6.4 Hz), 2.72-2.64 (2H, m), 2.60-2.51 (2H, m), 1.95-1.85 (2H, m), 1.84-
13
.75 (2H, m), 1.26 (1H, d, J = 6.2 Hz, OH), 1.19 (1H, d, J = 6.2 Hz, OH). C NMR
(
CDCl ) δ 150.3, 147.8, 144.4, 144.0, 138.3, 135.0, 132.9, 120.7, 119.5, 112.8, 111.4,
3 C
+
107.1, 62.2, 62.1, 56.2, 56.0, 34.3, 34.2, 31.9, 31.8. HRESIMS m/z 385.1618 [M+Na]
(
calc. for C H NaO 385.1622).
20 26 6
1-(4-(5-(1-Hydroxypropyl)-2,3-dimethoxyphenoxy)-3-methoxyphenyl)propan-1-ol (23).
To a stirred solution of 22 (25 mg, 0.073 mmol) in THF (200 µL) under argon was
added BH •THF (146 µL, 1M in THF, 0.146 mmol). After 2 h, 3 M NaOH (140 µL, 0.22
3
mmol) and H O (180 µL, 30% aq., 0.73 mmol) were added, and the reaction mixture
2
2
was stirred at room temperature for 1.5 h. Brine (1 mL) was added, and the product
was extracted with EtOAc (3 x 1 mL). The organic layer was washed with brine (1 mL),
dried over MgSO and the solvent removed under reduced pressure. The crude
4
product was purified by flash chromatography (petroleum ether/EtOAc 1:1 and 3:7) to

1
7
ACCEPTED MANUSCRIPT
afford 23 (22 mg, 0.058 mmol, 80%, unassigned mixture of stereoisomers) as a yellow
1
oil; H NMR (CDCl ) δ 6.99 (1H, s), 6.80 (2H, s), 6.69 (1H, d, J = 1.7 Hz), 6.41 (1H, d,
3
H
J = 1.7 Hz), 4.56 (1H, t, J = 6.6 Hz), 4.43 (1H, t, J = 6.6 Hz), 3.89 (3H, s), 3.86 (3H, s),
.85 (3H, s), 1.76-1.59 (4H, m), 1.25 (1H, d, J = 6.2 Hz, OH), 1.19 (1H, d, J = 6.2 Hz,
3
13
OH), 0.92 (3H, t, J = 7.4 Hz), 0.86 (3H, t, J = 7.4 Hz). C NMR (CDCl ) δ 153.7,
3
C
150.6, 150.2, 145.1, 140.6, 140.3, 119.2, 118.9, 118.4, 110.2, 109.2, 104.6, 75.8, 75.8,
+
6
1.0, 56.1, 56.0, 31.9, 31.8, 10.2, 10.1. HRESIMS m/z 399.1773 [M+Na] (calc. for
C H NaO 399.1778).
21
28
6
2.9.
1-(3,4-Dimethoxy-5-(2-methoxy-4-(2-oxopropyl)phenoxy)phenyl)propan-2-one
(
20). A vial charged with a mixture of PdCl (1.0 mg, 0.0073 mmol), CuCl (7.2 mg,
2
0
.073 mmol) and DMF / H O 7:1 (100 µL) was stirred under an oxygen atmosphere (1
2
atm) for 1 h. 2 (25 mg, 0.073 mmol) was added and the solution was stirred for 16 h. 1
M HCl (1 mL) was added and the product was extracted with EtOAc (3 x 1 mL). The
organic layers were combined and washed successively with sat. NaHCO (1 mL),
3
brine (1 mL) and dried over MgSO . After concentration under reduced pressure, the
4
crude product was purified by flash chromatography (petroleum ether/EtOAc gradient
1
8
:2, 7:3 and 1:1 v/v) to afford 20 (12.8 mg, 0.034 mmol, 47%) as a yellow oil; H NMR
(
CDCl ) δ 6.83 (1H, d, J = 8.1 Hz), 6.80 (1H, d, J = 1.9 Hz), 6.71 (1H, dd, J = 8.1 and
3 H
1
3
1
6
.9 Hz), 6.49 (1H, d, J = 1.9 Hz), 6.30 (1H, d, J = 1.9 Hz), 3.87 (6H, s), 3.83 (3H, s),
13
.67 (2H, s), 3.54 (2H, s), 2.18 (3H, s), 2.11 (3H, s). C NMR (CDCl ) δ 206.5, 206.2,
3
C
53.8, 150.7, 150.5, 144.8, 138.9, 130.2, 129.7, 121.8, 119.5, 113.6, 112.5, 108.2,
+
1.0, 56.1, 56.0, 50.9, 50.7, 29.3, 29.1. HRESIMS m/z 395.1468 [M+Na] (calc. for
C H NaO 395.1465).
21
24
6
2.10. 1-(4-(5-(2-Hydroxypropyl)-2,3-dimethoxyphenoxy)-3-methoxyphenyl)propan-2-ol
(
(
21). To a solution of 20 (12.0 mg, 0.032 mmol) in MeOH (100 µL) was added NaBH4
3.0 mg, 0.083 mmol) and the reaction was stirred for 40 min at room temperature. The

1
8
ACCEPTED MANUSCRIPT
reaction was diluted with water (1 mL), and the product was extracted with EtOAc (3 x
1
mL). The combined organic layers were washed with HCl (1 mL, 10% aq.), brine (1
mL), dried over MgSO and concentrated to afford 21 (9.6 mg, 0.025 mmol, 80%,
4
1
unassigned mixture of stereoisomers) as a yellow oil; H NMR (CDCl ) δ 6.78-6.74
3
H
(
2H, m), 6.65 (1H, dd, J = 8.1 and 1.9 Hz), 6.45 (1H, d, J = 1.9 Hz), 6.22 (1H, d, J = 1.9
Hz), 4.07-3.98 (1H, m), 3.88-3.83 (1H, m), 3.81 (3H, s), 3.81 (3H, s), 3.77 (3H, s), 2.71
(
1H, dd, J = 13.6 and 4.5 Hz), 2.62-2.55 (2H, m), 2.45 (1H, dd, J = 13.6 and 8.3 Hz),
1
3
1
1
5
.20 (3H, d, J = 6.2 Hz), 1.13 (3H, d, J = 6.2 Hz). C NMR (CDCl ) δ 153.7, 150.6,
3 C
50.6, 144.3, 138.4, 134.6, 134.0, 121.6, 119.6, 113.8, 111.9, 108.1, 68.8, 68.7, 61.0,
+
6.1, 56.0, 45.7, 45.5, 22.8, 22.6. HRESIMS m/z 399.1776 [M+Na] (calc. for
C H NaO 399.1778).
21
28
6
2.11. 3-(4-Formyl-2-methoxyphenoxy)-4,5-dimethoxybenzaldehyde (24). To a solution
of 22 (42 mg, 0.123 mmol) in 1,4-dioxane (1 mL) and H O (200 µL) was added 2,6-
2
lutidine (58 µL, 0.492 mmol), NaIO (210 mg, 0.984 mmol) and OsO (32 µL, 4.0 wt %
4
4
in H O, 0.005 mmol). The reaction was stirred for 4 h at room temperature and then
2
quenched with aq. Na SO (1 mL) and EtOAc (4 x 1 mL). The combined organic layers
2
3
were washed with 1 M HCl (1 mL), dried over MgSO and concentrated in vacuo to
4
1
afford 24 (26.7 mg, 0.084 mmol, 70%) as a yellow oil; H NMR (CDCl ) δ 9.90 (1H, s),
3
H
9
=
3
1
3
.80 (1H, s), 7.53 (1H, d, J = 1.8 Hz), 7.39 (1H, dd, J = 8.2 and 1.8 Hz), 7.31 (1H, d, J
1.8 Hz), 7.11 (1H, d, J = 1.8 Hz), 6.88 (1H, d, J = 8.2 Hz), 3.96 (3H, s), 3.95 (3H, s),
13
.93 (3H, s). C NMR (CDCl ) δ 190.9, 190.3, 154.3, 151.4, 150.6, 148.8, 146.0,
3
C
32.7, 131.8, 125.7, 117.3, 116.2, 110.7, 107.7, 61.3, 56.4, 56.2. HRESIMS m/z
+
17.1020 [M+Na] (calc. for C H O 317.1020).
17
17
6

1
9
ACCEPTED MANUSCRIPT
2.12. General bioassays procedures
BALB/c mice were obtained from the animal breeding facility at the Instituto
Adolfo Lutz, Brazil. The animals were maintained in sterilized cages under a controlled
environment, and received water and food ad libitum. All procedures performed were
previously approved by the Animal Care and Use Committee from Instituto Adolfo Lutz
–
Secretary of Health of Sao Paulo State (Project number CEUA 04/2016) in
agreement with the Guide for the Care and Use of Laboratory Animals from the
National Academy of Sciences.
2.13. Parasites and mammalian cell maintenance. Macrophages were collected from
the peritoneal cavity of BALB/c mice by washing them with RPMI-1640 medium
supplemented with 10% fetal calf serum, and were maintained at 37ºC in a 5% CO2
humidified incubator. Trypomastigotes of T. cruzi (Y strain) were maintained in Rhesus
monkey kidney cells (LLC-MK2 - ATCC CCL 7), cultivated in RPMI-1640 medium
supplemented with 2% fetal calf serum at 37ºC in 5% CO humidified incubator. The
2
murine conjunctive cells (NCTC clone 929, ATCC) were maintained in RPMI-1640
o
supplemented with 10% FBS at 37 C in a humidified atmosphere containing 5% CO .
2
2
.14. Determination of the 50% inhibitory concentration (IC )
50
2.14.1. Extracellular forms. Trypomastigotes were counted in a hemocytometer
6
chamber and seeded at 1 x 10 cells per well in 96 well microplates. The compounds
were dissolved in DMSO and diluted in RPMI-1640 medium and incubated for 24h at
3
7ºC in a 5% CO humidified incubator. The parasite viability was determined using the
2
resazurin (0.011% in PBS). The optical density was read at 570 nm using control wells
without drugs (100% viability) and without cells (blank). The control group consisted of
trypomastigotes incubated with 0.5% (v/v) DMSO. Benznidazole was used as a

2
0
ACCEPTED MANUSCRIPT
positive control. Compounds were tested to the highest concentration of 30 µM and
were reported as NA (not active) when the IC value was above this concentration.
50
2.14.2. Intracellular amastigotes. Peritoneal macrophages were dispensed (1 x
5
1
0 /well) in 16 well chamber slides (NUNC, Thermo, USA) and maintained for 24 h in
the same medium at 37ºC in a 5% CO humidified incubator for attachment. Non-
2
adherent cells were removed by two step washings with medium. After 24 h, these
5
cells were infected with 5 x 10 culture trypomastigotes for 4 h. Benznidazole was used
as the standard drug. Subsequently, infected cells were incubated with the compounds
for 48 h. Finally, the slides were fixed with methanol, stained with Giemsa, and
observed in light microscopy (100X magnification). The parasite load was defined by
the number of infected macrophages X number of intracellular amastigotes/number of
total macrophages.
2.15. Cytotoxicity in mammalian cells
The murine conjunctive cells (NCTC clone 929, ATCC) were counted in a
4
hemocytometer chamber, seeded at 6 x 10 /well and incubated in highest
concentrations to 200 µM for 48 h at 37ºC in a 5% CO humidified incubator. The cell
2
viability was determined using the MTT assay. Benznidazole was used as a positive
control. The selectivity index (SI) was determined using the relationship, CC against
50
NCTC/IC against amastigotes parasites.
50
2.16. Evaluation of plasma membrane permeability
6
Late growth-phase trypomastigotes of T. cruzi (2 x 10 /well) were washed and
incubated in the dark with 1 µM SYTOX Green probe (Molecular Probes) in HANKS'
balanced salts solution (HBSS; Sigma-Aldrich) supplemented with 10 mM D-Glucose

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(
HBSS+Glu). The test compound was added (t=0) at the IC50 and fluorescence was
measured every 20 minutes for 1 h. The maximum permeabilization was obtained with
.5% Triton X-100. Fluorescence intensity was determined using a fluorimetric
0
microplate reader (FilterMax F5 Multi-Mode Microplate Reader-Molecular Devices) with
excitation and emission wavelengths of 485 and 520 nm, respectively. The following
internal controls were used in the evaluation: i) the background fluorescence of the
compound at the respective wavelengths, ii) the possible interference of DMSO.
Samples were tested in duplicate [19].
2.17. Assessment of plasma membrane electric potential (∆Ψp)
Estimation of ∆Ψ was monitored by measuring the increase in absorbance of
p
bis-(1,3-diethylthiobarbituric acid) trimethineoxonol [DiSBAC (3)] (Invitrogen) as
2
6
described by [19]. Briefly, trypomastigotes of T. cruzi (2 x 10 ) were washed with
HBSS+Glc, added into flow cytometry tubes and treated with compound 14 (IC ) in a
50
final volume of 150 µL and incubated at 37ºC for 60 min. Next, 0.2 µM of DiSBAC (3)
2
were add in a final volume of 500 µL/ tube and incubated at 37ºC for 10 minutes. The
samples were read in an Attune NxT Flow Cytometer (Thermo Fisher Scientific).
Gramicidin D (0.5 µg/mL) (Sigma-Aldrich) was used as a positive control. Untreated
parasites were used as negative controls. Two independent experiments were
performed, each one with duplicate samples.
2.18. Evaluation of mitochondrial membrane potential (ꢀψm)
6
Late growth-phase trypomastigotes of T. cruzi (2 x 10 /well) were treated with
the test compound at the IC50 in HBSS+Glu during 2 h incubation at 37ºC. After
incubation, parasites were washed and co-stained with JC-1(0.3 µg/mL) for 10 minutes
under the absence of light at 37ºC in order to monitor the mitochondrial membrane

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potential (ꢀψm) and cell viability, respectively. Flow cytometry was performed using an
Attune NxT Acoustic Focusing Cytometer (ThermoFisher Scientific) by analyzing
10,000 gated events using forward/side scatter (FSC/SSC), JC-1 fluorescence (BL1-A,
filter 530/30 nm), PI fluorescence (BL2-A, filter 574/26 nm) and Attune Nxt® software
included with the equipment. Unstained, JC-1-stained (untreated) parasites were used
to set background fluorescence and compensation. Trypomastigotes treated with
CCCP (10 µM) and untreated were used to obtain maximal and minimal mitochondrial
depolarization, respectively [20].
2.19. Statistical analysis
The determination of the CC and IC values was obtained from sigmoid dose-
50
50
response curves. The statistical significance (p value) between the samples was
evaluated through the One-way ANOVA method using the Tukey's Multiple
Comparison test. All analyzes were performed using Graph Pad Prism 5.0 software.
The samples were tested in duplicate or triplicate and the assays were repeated at
least twice.

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3. RESULTS
3.1. Preparation of semi-synthetic derivatives 3–25 from natural compounds 1
and 2.
Natural products 1 and 2 present a number of sites that permit synthetic
modification, including the phenol residue in the case of compound 1, and the two
unsaturated allyl side chains. The modifications selected were designed with several
purposes in mind: to discover where potential exists for structural diversification without
compromising bioactivity, to investigate derivatives that are easily accessible from the
natural products, and to explore modifications that would address potential
pharmacological concerns. Targeted modifications included transformations of the aryl
ethers and phenols, double bond functionalization (such as hydrogentaion, addition of
heteroatoms, and oxidation). The synthetic routes employed to access the 23
derivatives studied in this work are shown in Figures 2 and 3.
Figure 2. Conversion of dehydrodieugenol B (1) to derivatives 3–19.

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Figure 3. Conversion of methyl dehydrodieugenol B (2) to derivatives 20–25.
.2. In silico analysis of semi-synthetic derivatives
3
In silico predictions of drug safety and physiochemical properties associated
with clinical success were performed for the semi-synthetic library (compounds 3–25)
using FAF-Drugs4 web server [21]. This analysis includes multiple predictive models
including the Lilly MedChem Rules, phospholipidosis, aqueous solubility, and oral
property space prediction. The series was predicted to have good oral bioavailability
based on a comparison of 15 physiochemical descriptors of oral drugs. In general, the
series has higher lipophilicity than optimal, which may result in low aqueous solubility
and a higher probability of exhibiting toxicity. No analogs were identified as potential
Pan Assay Interference (PAINS) compounds based on the three available filters; nor is
the series predicted to induce phospholipidosis. Compound 14, which proved promising
in bioassays has good predicted oral bioavailability based on the VeberRules, Egan
Rules, and Bayer Oral Property Space Rules, despite not meeting Lipinski’s R05.
Overall, in silico analysis indicated the primary risks for the series are associated with

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high lipophilicity, which could in the future be addressed by structural modifications that
decrease lipophilicity.
3.3. In vitro efficacy and cytotoxicity
Twenty-three neolignan derivatives (compounds 3–25) were prepared and
tested in vitro against T. cruzi trypomastigotes and intracellular amastigotes, using the
colorimetric resazurin assay and light microscopy counting, respectively. Among them,
eight compounds (5, 8, 12–14, 16, 22, and 25) showed activity against the intracellular
amastigotes, with IC50 values between 7 and 16 µM; five compounds (3, 14–16, and
18) demonstrated activity against trypomastigotes, with IC50 values between 8 and 64
µM (Table 1). Two compounds (14 and 16) exhibited activity against both forms of the
parasite. Among the twenty-three compounds, eighteen presented no mammalian
cytotoxicity (NCTC cells) to the highest tested concentration of 200 µM, while five
derivatives (3, 14-17), demonstrated CC50 values between 58.0 and 156.0 µM.
Benznidazole was used as standard, giving IC50 values of 17.7± 1.9 µM against
trypomastigotes and 5.0 ± 1.5 µM against intracellular amastigotes.

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Table 1. Anti-T.cruzi activity (trypomastigote and amastigote forms) and mammalian
cytotoxicity (NCTC cells) of natural compounds 1 and 2, semisynthetic derivatives 3 –
25 and benznidazole.
compound
IC (µM) (± S.D.)
CC (µM) (± S.D.)
S.I.
5
0
50
trypomastigotes
38.6 ± 8.3
>100
amastigotes
86.5 ± 16.2
>30
NCTC
>200
amastigotes
1
2
3
4
5
6
7
8
9
> 2.3
>200
-
7.9 ± 0.7
>100
>30
57.7 ± 1.1
>200
-
>30
-
>100
16.4 ± 0.4
>30
>200
> 12.2
>100
>200
-
>100
>30
>200
-
>100
9.5 ± 1.3
>30
>200
> 21.1
>100
>200
-
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
>100
>30
>200
-
>100
>30
>200
-
>100
9.4 ± 3.9
7.5 ± 1.1
8.0 ± 5.8
>30
>200
> 21.2
>100
>200
> 26.6
64.2 ± 8.2
21.5 ± 2.9
30.5 ± 10.4
>100
64.4 ± 4.2
66.3 ± 6.0
75.0 ± 13.8
156.1 ± 15.0
>200
8.1
-
10.0 ± 0.8
>30
7.5
-
26.6 ± 5.3
>100
>30
-
>30
>200
-
>100
>30
>200
-
>100
>30
>200
-
> 16.4
-
>100
12.2 ± 3.5
>30
>200
>100
>200
>100
>30
>200
-
>100
13.3 ± 1.2
5.0 ± 1.5
>200
> 15.4
38.1
benznidazole
17.7 ± 1.9
190.6 ± 13.4
IC : 50% Inhibitory Concentration; CC : 50% Cytotoxic Concentration; S.I. : Selectivity
50
50
Index, given by the ratio between CC50 in NCTC cells and IC50 in intracellular
amastigotes; S.D. : Standard Deviation.

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Additionally, light microscopy images of T. cruzi-infected macrophages (Figure
4A), demonstrated that compound 14 eliminated 100% of intracellular amastigotes,
without affecting the morphology of host cells at the highest tested concentration of 30
µM (Figure 4B).
Figure 4. Treatment of Trypanosoma cruzi-infected macrophages with compound 14.
Light microscopy images of Giemsa-stained slides. A. Untreated group with
intracellular amastigotes. B. Group treated with compound 14 for a period of 48 h at 30
µM. Magnification 100X (oil immersion).
3.4. Mechanism of action studies
3.4.1. Plasma membrane permeability. In light of the in vitro potency of
compound 14 and its capacity to eliminate 100% of parasites, its plasma membrane
permeability was investigated using the fluorescent probe SYTOX Green. 14 induced
no significant alteration in plasma membrane permeability within 60 minutes of
incubation when compared to untreated parasites (Figure 5A). Triton X-100 was used
as the positive control and was also included at the end of the assay to all groups. 14
was also incubated with macrophages, and the fluorescence monitored for 60 min.
When compared to the untreated control, 14 showed no alteration of plasma
membrane permeability (Figure 5B). Scanning electron microscopy studies of
trypomastigotes incubated with 14 corroborated the SYTOX Green assay, as
demonstrated by the normal morphology of T. cruzi trypomastigotes, including the

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plasma membrane of treated parasites (Figure 5D), when compared to untreated group
(
Figure 5C).
Figure 5. Evaluation of the plasma membrane of T. cruzi trypomastigotes. SYTOX
Green assay for the determination of the plasma membrane permeability of
trypomastigotes (A) and peritoneal macrophages (B), both treated with compound 14.
Triton X-100 was used as the 100% of permeabilization (positive control) and a control
group (untreated) was also included for both cells. C. Scanning electron microscopy T.
cruzi untreated trypomastigotes (control group). D Scanning electron microscopy T.
cruzi 14-treated trypomastigotes (bar = 2.5 µm).
3.4.2. Plasma membrane electric potential (∆Ψp). Using flow cytometry
analysis, the electric potential of plasma membrane of T. cruzi trypomastigotes was
investigated in the presence of compound 14. After 60 min incubation, no significant
alteration was observed when compared to untreated control (Figure 6). Gramicidin, (a
commercial antibacterial polypeptide) was used as positive control, and induced the
depolarization of potential as shown in Figure 6.