Novel prenyloxy chalcones as potential leishmanicidal and trypanocidal agents: Design, synthesis and evaluation

Article abstract of DOI:10.1016/j.ejmech.2019.02.028

The available drugs for treating Leishmaniasis and American trypanosomiasis have high toxicity and multiple side effects, among other problems. More effective and less toxic treatments are urgently needed. A series of chalcones that contained a prenyloxy or geranyloxy substituent was synthesized and characterized. Each substituent was attached to the A ring in some compounds and to the B ring in others, with additional substituents placed on the chalcone moiety. The present aim was to evaluate the effect of the substitution pattern on leishmanicidal and trypanocidal activity. When tested at a single concentration, the compounds exerting a metabolic inhibition close to or exceeding 50% for Leishmania mexicana were 11, 17 and 12, and for Trypanosoma cruzi were 11, 17, 15 and 26. Upon determining the selectivity index (SI =IC₅₀/CC₅₀), the values were 80.9, 1.24 and 55.12 for 11, 17 and 12 (respectively) versus L. mexicana, and 75.1, 1.43, 27.36 and 33.52 for 11, 17, 15 and 26 (respectively) versus T. cruzi. Structural isomers 11 and 17 showed activity for both the L. mexicana and T. cruzi strains, though the greater cytotoxic activity of 17 led to a lower SI. Compounds 12, 15 and 26 were species specific. For T. cruzi, the SI was higher for 11, 15 and 26 than for the reference drugs nifurtimox and benznidazole. The examination of promastigote morphology after exposing L. mexicana and T. cruzi to 11 revealed a decrease in cell density. The current findings suggest that 11 could be a useful lead compound for further SAR studies.

Full text of DOI:10.1016/j.ejmech.2019.02.028

Accepted Manuscript
Novel prenyloxy chalcones as potential leishmanicidal and trypanocidal agents:
Design, synthesis and evaluation
José C. Espinoza-Hicks, Karla Fabiola Chacón-Vargas, Jessica L. Hernández-Rivera,
Benjamín Nogueda-Torres, Joaquín Tamariz, Luvia Enid Sánchez-Torres, Alejandro
Camacho-Dávila
PII:
S0223-5234(19)30142-4
DOI:
https://doi.org/10.1016/j.ejmech.2019.02.028
EJMECH 11120
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 17 November 2018
Revised Date: 16 January 2019
Accepted Date: 8 February 2019
Please cite this article as: José.C. Espinoza-Hicks, K.F. Chacón-Vargas, J.L. Hernández-Rivera,
Benjamí. Nogueda-Torres, Joaquí. Tamariz, L.E. Sánchez-Torres, A. Camacho-Dávila, Novel prenyloxy
chalcones as potential leishmanicidal and trypanocidal agents: Design, synthesis and evaluation,
European Journal of Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.ejmech.2019.02.028.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Novel Prenyloxy Chalcones as Potential Leishmanicidal and
Trypanocidal Agents: Design, Synthesis and Evaluation
José C. Espinoza-Hicks 1§; Karla Fabiola Chacón-Vargas 2,3§; Jessica L. Hernández-Rivera 1;
Benjamín Nogueda-Torres 3; Joaquín Tamariz 4; Luvia Enid Sánchez-Torres 2*; Alejandro
Camacho-Dávila 1*.
1
Facultad de Ciencias Químicas, Universidad Autónoma de Chihuahua, Circuito Universitario, Campus Universitario,
Apartado Postal 669, Chihuahua, Chih., México.
2Departamento de Inmunología, 3Departamento de Parasitología, 4Departamento de Química Orgánica, Escuela Nacional
de Ciencias Biológicas, Instituto Politécnico Nacional, Prolongación de Carpio y Plan de Ayala, s/n, 11340 Ciudad de
México, México.
* Corresponding author’s acamach@uach.mx; luviasanchez@hotmail.com
§ Equivalent contribution
Abstract
The available drugs for treating Leishmaniasis and American trypanosomiasis have high
toxicity and multiple side effects, among other problems. More effective and less toxic
treatments are urgently needed. A series of chalcones that contained a prenyloxy or
geranyloxy substituent was synthesized and characterized. Each substituent was attached to
the A ring in some compounds and to the B ring in others, with additional substituents
placed on the chalcone moiety. The present aim was to evaluate the effect of the
substitution pattern on leishmanicidal and trypanocidal activity. When tested at a single
concentration, the compounds exerting a metabolic inhibition close to or exceeding 50% for
Leishmania mexicana were 11, 17 and 12, and for Trypanosoma cruzi were 11, 17, 15 and
26. Upon determining the selectivity index (SI =IC50/CC50), the values were 80.9, 1.24 and
55.12 for 11, 17 and 12 (respectively) versus L. mexicana, and 75.1, 1.43, 27.36 and 33.52
for 11, 17, 15 and 26 (respectively) versus T. cruzi. Structural isomers 11 and 17 showed
activity for both the L. mexicana and T. cruzi strains, though the greater cytotoxic activity
of 17 led to a lower SI. Compounds 12, 15 and 26 were species specific. For T. cruzi, the SI
was higher for 11, 15 and 26 than for the reference drugs nifurtimox and benznidazole. The
examination of promastigote morphology after exposing L. mexicana and T. cruzi to 11

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revealed a decrease in cell density. The current findings suggest that 11 could be a useful
lead compound for further SAR studies.
Keywords: Chalcones, Leishmania, Trypanosoma cruzi, structure-activity relationship,
metabolic inhibition, selectivity index.
1. Introduction
Leishmaniasis and American trypanosomiasis are among the neglected tropical diseases
(NTDs), which are overlooked because the world pharmaceutical industry does not
consider it profitable to invest in the development of new medicines for their treatment [1].
NTDs prevail in regions with a tropical and subtropical climate where most of the affected
population is living in extreme poverty.
Leishmaniasis consists of a variety of diseases resulting from infection by flagellated
protozoan parasites of the Leishmania genus. This disease is transmitted to humans by the
bite of Lutzomiya or Phlebotomus mosquitoes carrying the protozoa [2]. According to the
World Health Organization (WHO) estimate, there are between 700,000 and 1,000,000 new
cases each year [3]. Depending on the species of Leishmania involved, the disease can be
manifested as cutaneous leishmaniasis (with cutaneous ulcers), mucocutaneous
leishmaniasis (with massive destruction of nasopharyngeal mucosa), and visceral
leishmaniasis (involving organs such as the liver, leading to a high mortality rate if not
properly treated). Whereas the most prevalent condition is cutaneous leishmaniasis in
America, itis visceral leishmaniasis in India and Africa [2, 3].
The first line treatments for leishmaniasis usually involve drugs derived from pentavalent
antimonium, including meglumine antimoniate (Glucantime) and sodium stibogluconate
(Pentostam). Some alternative treatments are amphotericin B (Amph B), pentamidine and
paromomycin. All currently available drugs require prolonged periods of administration
and produce high toxicity with several side effects, which limits their clinical use [4].
On the other hand, the potentially lethal American trypanosomiasis (Chagas disease) is
caused by the protozoan parasite Trypanosoma cruzi. It is transmitted to humans when an
infected hematophagous triatomine vector deposits its feces after a blood meal. The

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parasite-infected feces enter the organism through lesions or mucous membranes [1b, 5].
Additionally, T. cruzi can be transmitted through transfusions with infected blood, during
pregnancy or orally. The WHO estimates that more than 8 million people are infected
worldwide, resulting in a death rate of around 12,000 people per year in Latin America
[1b]. The pharmacological treatment for Chagas disease is based on the only medications
available for this purpose: nifurtimox (Nfx) and benznidazole (Bnz). These drugs not only
show limited activity during the acute stage of Chagas disease, but also have high toxicity
and multiple side effects [6].
Thus, novel treatments for leishmaniasis and American trypanosomiasis are urgently
needed [7, 8]. The WHO and the Special Program for Research and Training in Tropical
Diseases (TDR) have prioritized the search for new drugs that have cost-effective benefits
for combatting the causal agents of NTDs [9]. In the effort of scientists to develop potential
agents for treating Leishmania spp and T. cruzi, novel compounds have been synthesized
and natural products isolated [8, 10-13].
Substituted phenols are commonly studied in order to develop novel antileishmanial agents
[14]. Among these compounds are chalcones, the structural framework of which is a 1,3-
diphenyl-2-propen-1-one with one or more free hydroxyl groups or an ether function
(Figure 1) [15].
Figure 1. General structure of the chalcone system.
Natural chalcones are ubiquitous substances existing in various plant species (e.g.,
Angelica, Glycyrrhiza, Piper and Ruscus) [15-17]. They are considered precursors to
diverse compounds, especially polyphenols such as flavonoids [16]. The numerous natural
chalcones contain different substituents in their phenyl rings, especially hydroxyl and ether
groups. A wide spectrum of activity has been found for some of these compounds,

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including antibacterial, antiviral, cytotoxic, antifungal and antiprotozoal [17, 18]. The
structural unit of chalcones is easily synthesized by several procedures, especially by
Claisen-Schmidt condensation between properly substituted aromatic aldehydes and
acetophenones [15]. The combination of diverse biological activity, relatively simple
synthesis and readily available precursors has made chalcones a target for the development
of novel compounds that can potentially act as medicinal drugs [19-23].
Among naturally occurring chalcones, those with a prenyl derivative attached to either an
oxygen (O-prenyl) or carbon (C-prenyl) atom are reported to display interesting biological
activity (e.g., anti-inflammatory, antioxidant, antifungal, antimicrobial and cytotoxic) [23,
24]. The synthesis of all these chalcones is based on a common metabolic pathway
involving the isoprene route [25]. For the O-prenyl substituted compounds, the type of
derivative depends on the size of the prenyl group, which can be the C-5 (prenyl), C-10
(geranyl) or C-15 (farnesyl) unit.
Licochalcone A (1), one of the most widely studied C-prenyl substituted chalcones, is
known for its antileishmanial activity [26]. This compound produces growth inhibition of
the promastigote form of L. donovani by damaging the ultrastructure of its mitochondria
[26]. It also blocks the promastigote respiratory chain by inhibiting the activity of the
fumarate reductase enzyme in the mitochondria [27, 28]. Also exhibiting antileishmanial
activity are O-allyl-containing chalcones, such as substituted chalcones with a 4-allyloxy
group on ring A and dimethoxy groups on ring B (2 and 3, respectively; Figure 3). The
latter compounds have demonstrated strong antileishmanial activity (IC50 1.4 – 88.8 µM)
both in vitro and in vivo in L. major and L. donovani amastigotes, apparently by interfering
with mitochondrial function [29]. In addition, the geranyloxy chalcone 4 (Figure 2) at a 50
mg/kg dose administered for 10 days showed over 80% inhibition of the L. donovani
parasite in vivo [26]. Another study synthesized and evaluated some chalcones substituted
with prenyloxy on the B ring, of the same type as 5-10 in the present work (Figure 2). The
prenyl groups increased inhibitory activity compared to the unsubstituted chalcones when
tested against L. amazonensis and L. infantum promastigotes. Overall, these results suggest
that lipophilicity and structural changes may be an important factor in the development of
novel antileishmanial agents [30].

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R6
O
R6'
R5
R4
R5'
R4'
R2 R2'
R3
R3'
1, R2 = R3 = R5 = R6 = R3' = R6' = H, R4 = OH, R2´ = OCH3, R4´ = OH, R5´ = -C(CH3)2CH=CH2
2, R2 = R3 = R5 = R6 = R2' = R3' = R5' = R6' = H, R4 = OCH2CH=CH2, R4´ = OCH3
3, R2 = R3 = R5 = R6 = R2' = R4' = R6' = H, R4 = OCH2CH=CH2, R3´ = R5´ = OCH3
4, R2 = R3 = R5 = R6 = R2' = R3' = R5' = R6' = H, R4 = geranyl, R4´ = OCH3
5, R2 = R3 = R4 = R5 = R6 = H, R2´ = O-prenyl, R3´ = R4´ = R5´ = R6´ = H
6, R2 = R3 = R4 = R5 = R6 = R2´ = H, R3´ = O-prenyl, R4´ = R5´ = R6´ = H
7, R2 = R3 = R4 = R5 = R6 = H, R2´ = O-geranyl, R3´ = R4´ = R5´ = R6´ = H
8, R2 = R3 = R4 = R5 = R6 = R2´ = H, R3´ = O-geranyl, R4´ = R5´ = R6´ = H
9, R2 = R3 = R4 = R5 = R6 = H, R2´ = O-farnesyl, R3´ = R4´ = R5´ = R6´ = H
10, R2 = R3 = R4 = R5 = R6 = H, R2´ = H, R3´ = O-farnesyl, R4´ = R5´ = R6´ = H
Figure 2. Structures of diversely substituted prenyloxy chalcones here in examined for
their antileishmanial activity.
Since chalcones containing O-prenyl groups are a proven source of new antileishmanial
drugs, the present effort was based on synthesizing chalcones with an O-5 (prenyl) or an O-
10 (geranyl) unit on the A ring, and in another compound the same unit on the B ring.
Other substituents were added to the chalcone moiety. The aim of this study was to evaluate
the activity of these compounds against L. mexicana and T. cruzi.
2. Chemistry
For the preparation of a series of substituted chalcones, a substituent was attached to the A
ring in one compound and to the B ring in another. This procedure was carried out for
various substituents to determine whether their respective position has an effect on
biological activity. The structures of the synthesized chalcones and their antileishmanial
activity are summarized in Table 1. The key precursors used herein were vanillin (31) and
acetovanillone (32), which were prenylated by reaction with geranyl chloride or prenyl
bromide, respectively (Scheme 1).

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Scheme 1. Synthesis of O-prenyl and O-geranyl precursors 33-36.
Additional hydroxyl- and methoxy-substituted aldehydes and ketones are commercially
available. The prenylated chalcones, with substituents on either the A or B ring, were
synthesized by Claisen-Schmidt condensation between the appropriately substituted
aromatic aldehydes 33, 34, 37-40 and acetophenones 35, 36, 41-44. The 20 chalcones
obtained (Scheme 2, Table 1) were all appropriately characterized by spectral data (NMR,
IR and HRMS).
Scheme 2. Claisen-Schmidt condensation between substituted
benzaldehydes and acetophenones.

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3. Biological Assays
3.1. Chemical compounds
The synthesized chalcones were evaluated for their in vitro leishmanicidal and trypanocidal
effects, as well as for cytotoxic effects on mammalian cells, as described previously [13a-b,
31]. For each compound, a known amount (50-100 mg) was dissolved in dimethyl
sulfoxide (DMSO; Sigma-Aldrich, St. Louis, MO, USA) to reach a concentration of 10
mg/mL. From this stock solution, the corresponding dilutions were prepared for biological
assays in phosphate-buffered saline (PBS).
3.2. Evaluation of the compound-induced metabolic inhibition in parasites
L. mexicana promastigotes (MNYC-BZ/62/M379 ATCC) and T. cruzi INC-5 epimastigotes
(MHOM/MX/1994/INC5) were examined by using the MTT colorimetric method. In each
case, parasites where used in stationary phase (7-day culture). For L. mexicana, 5 x 105
metacyclic promastigotes were culture in 100 µL supplemented RPMI 1640 medium
(Gibco, Carlsbad, CA, USA) containing 10% heat-inactivated fetal bovine serum (FBS;
Gibco, Grand Island, NY, USA) and 100 U/mL penicillin-streptomycin (In vitro S.A.,
Mexico City, Mexico) and seed in a sterile 96-well microplate. All compounds were
assayed in triplicate at a 10 µg/mL concentration. Amphotericin B (Amph B) was
employed as the reference compound and untreated parasites as the positive viability
control [32, 13b].
For T. cruzi, 1 x 106 epimastigotes were seeded in a 96-well plate in 100 µL of Brain Heart
Infusion (BHI) medium (Becton Dickinson; Heidelberg, Germany) supplemented with 10%
heat-inactivated FBS and 100 U/mL penicillin-streptomycin. Each compound was assayed
in triplicate at a 10 µg/mL concentration. The reference drugs, Nfx and Bnz, served as the
negative viability control and untreated parasites as the positive viability control [13a].
Microplates were incubated at 27°C in the dark for 24 h. Subsequently, 10 µL of 5 mg/mL
MTT were added to each well and incubated for another 24 h under the same conditions.
Upon completion of this time, 100 µL of a solution of SDS (10%) and HCl (0.01N) were
added to each well to dissolve the formazan salts. The plates were read on a

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spectrophotometer (Spectramax Plus 384; Molecular Devices Corp., Sunnyvale, CA, USA)
at 570 nm. The absorbance of each treated well was compared to that of the blank and is
expressed as a percentage of metabolic inhibition [13a].
Compounds inhibiting the metabolic activity of the parasite by at least 50% were assessed
at different concentrations. The same methodology was employed, obtaining the
concentrations by serial dilutions starting from 20 µg/mL. Finally, the 50% inhibitory
concentration (IC50) for the active compounds was determined with Probit [13a-b].
3.3. Evaluation of compound-induced cytotoxic activity on murine macrophages
From the murine macrophage cell line J774A.1 (TIB-61 ATCC), 5x104 cells were placed in
each well of a 96-well microplate, with a final volume of 100 µL. The plates were
incubated at 37 °C in a 5% CO2 atmosphere for 24 h. Each compound was tested at distinct
concentrations (in triplicate), utilizing untreated cells as the negative cytotoxicity control.
After adding the corresponding compound, incubation was carried out for another 20 h
under the same conditions. Then10 µL of MTT (5 mg/mL) were added and the plates were
incubated for an additional 4 h before a solution (100 µL) of 10% SDS and 0.01N HCl was
added to each well to dissolve the formazan crystals. The concentration-response analysis
was conducted as described in the previous section to determine the 50% cytotoxic
concentration (CC50) for each compound [33].
3.4. Selectivity Index (SI)
The SI was calculated as the ratio between the CC50 for mammalian cells and the CI50 for
parasites (CC50/ CI50). A good biological activity is considered when the SI ≥ 10 [34, 35].
3.5. Morphological analysis
The morphology of L. mexicana promastigotes and T. cruzi epimastigotes was analyzed
with light microscopy. Parasites treated under different conditions were washed and
resuspended in PBS, then embedded on a slide using cystopin (LaboFuge 400, Thermo
Scientific, USA). Following the staining of the samples with Giemsa (dilution 1:10; Merck,

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Darmstadt, Germany) during 30 min, the slides were examined on a light microscope at
100x (Primo Star, Zeiss). Images were captured with an Axiocam ER 5s camera [13b].
4. Results and discussion
The synthesis of the targeted chalcones was performed by Claisen-Schmidt condensation
between the appropriately substituted benzaldehydes and benzophenones to afford the
corresponding chalcones in fair to good yields (11-92 %). There was a preliminary
evaluation of the compound-induced metabolic inhibition on L. mexicana promastigotes
and T. cruzi epimastigotes at a concentration of 10 µg/mL (Table 1). The most active
compounds, those producing metabolic inhibition near to or exceeding 50%, were subjected
to further testing at various concentrations.
The most active compounds for L. mexicana were 11 and 17, giving rise to 71.3% and
59.1% inhibition, respectively, while compound 12 showed close to 50% inhibition. None
of the test compounds exceeded the inhibition produced by Amph B, the reference drug.
Compounds 11 and 17 were also the most active for T. cruzi, with 84% and 80.3%
inhibition, respectively. Both exceeded the activity of Bnz, the reference drug. The
inhibition afforded by compounds 15 (43.9%) and 26 (45%) evidenced a lesser degree of
trypanocidal activity. Interestingly, compounds possessing medium-level activity (12, 15
and 26) were species-specific.

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Table 1. Structures of the synthesized chalcones and a preliminary in vitro biological evaluation.
% Metabolic inhibition
µM equivalent
to 10µg/mL
Cpd.
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
R3
R4
R5
R3'
R4'
R5'
L. mexicana
T. cruzi
24.2 µM
26.1 µM
28.4 µM
29.6 µM
19.8 µM
22.9 µM
24.2 µM
20.7 µM
28.4 µM
29.6 µM
19.8 µM
20.8 µM
22.2 µM
23.8 µM
24.6 µM
17.5 µM
20.8 µM
22.2 µM
23.8 µM
OCH3 OCH3 OCH3
H
H
H
H
H
H
O-pre OCH3
O-pre OCH3
O-pre OCH3
O-pre OCH3
O-pre OCH3
O-pre OCH3
71.3 ± 0.8
84.0 ± 1.1
H
H
H
H
H
H
H
H
H
H
OCH3 OCH3
48.3 ± 0.8
25.5 ± 4.7
17.7 ± 1.0
-8.0 ± 3.8
3.9 ± 4.4
59.1 ± 0.5
15.6 ± 1.7
1.0 ± 2.6
4.6 ± 2.0
-3.2 ± 1.4
11.3 ± 0.6
13.5 ± 1.7
0.70 ± 1.9
17.6 ± 0.4
1.0 ± 1.7
2.1 ± 3.6
27.3 ± 0.7
0.0 ± 2.8
11.8 ± 1.9
2.6 ± 1.8
OCH3
OH
H
H
23.8 ± 2.7
43.9 ± 3.4
20.0 ± 4.5
80.3 ± 1.2
25.3 ± 4.7
7.9 ± 0.7
O-ger OCH3
O-pre OCH3
O-pre OCH3 OCH3 OCH3 OCH3
O-pre OCH3
O-pre OCH3
O-pre OCH3
O-pre OCH3
H
H
H
H
H
H
H
H
H
OCH3 OCH3
OCH3
OH
H
H
16.3 ± 1.3
33.2 ± 5.2
31.6 ± 3.0
22.0 ± 0.7
13.7 ± 3.1
20.4 ± 1.7
45.0 ± 3.3
-0.8 ± 7.1
28.3 ± 1.6
-11.6 ± 3.2
O-ger OCH3
O-ger OCH3
O-ger OCH3
O-ger OCH3
O-ger OCH3
O-ger OCH3
OCH3 OCH3 OCH3
H
H
H
H
H
H
H
OCH3 OCH3
OCH3
OH
H
H
O-ger OCH3
O-ger OCH3 OCH3 OCH3 OCH3
O-ger OCH3
O-ger OCH3
O-ger OCH3
H
H
OCH3 OCH3
OCH3
H
24.6 µM
H
-
H
-
OH
-
H
-
4.9 ± 3.7
-5.5 ± 4.0
77.4 ± 3.8
38.42 µM
-
-
Bnz
Amph
B
-
0.54 µM
-
-
-
-
-
-
99.0 ± 1.2
-
(0.5
µg/mL)
Bnz= Benznidazole; Amph B = Amphotericn

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Compounds exhibiting medium and high inhibitory activity were selected for evaluation at
various concentrations to determine their IC50 for L. mexicana promastigotes and T. cruzi
epimastigotes. Additionally, the CC50 for murine macrophages was ascertained to calculate
the selectivity index (SI). As previously mentioned [35, 36], selectivity is considered for
compounds with an SI ≥10 (Table 2).
Table 2. IC50 and CC50 for the most active compounds.
Macrophages
J774
T. cruzi
INC-5
SI
L. mexicana
IC50 (µM)
IC50 (µM)
CC50 (µM)
L. mexicana
80.94
55.12
Nd
T. cruzi
75.11
Nd
16.2
(13.4-19.2)
31.8
(23.4-40.2)
17.5
(13.5-20.6)
1312.9
(1080.4-1544.8)
1752.6
11
12
Nd
(1638.7-1866.5)
30.1
(23.9-36.3)
17.8
824.8
(718.7-931.1)
25.6
Nd
27.36
1.43
15
20.7
(13.4-32.5)
1.24
17
(14.9-20.7)
(11.5-45.3)
30.4
(22.9-37.9)
1018.4
(875.5-1161.2)
60.7
(52.6-68.9)
352.2
(334.9-369.1)
201.1
(188.6-213.5)
Nd
Nd
33.52
Nd
26
0.28
(0.26-0.29)
Nd
215.84
Nd
Amph B
Bnz
Nfx
42.3
(36.6-48.1)
8.7
Nd
Nd
8.31
Nd
23.10
(4.8-12.5)
Nd = Not determined
The values in parentheses represent the confidence interval determined by the Probit method with
95% confidence
Interestingly, there was a lower cytotoxic effect (CC50) produced by compounds 11, 12, 15
and 26 than by the reference drugs. When applied to the L. mexicana strain, compounds 11
and 12 showed a high SI (80.94 and 55.12, respectively), though they were below the value
for Amph B. The lower cytotoxic activity of 11 and 12 than Amph B makes them
candidates for lead compounds. Compound 17 exhibited a good IC50 value, but generated
cytotoxic activity against mammalian macrophages at reduced concentrations, resulting in
an SI value below that of 11 (Table 2).
For the T. cruzi epimastigotes, compound 11 also displayed the highest SI (75.11), followed
by compounds 15 (27.36) and 26 (33.52). The SI of these three chalcones was better than

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reference drugs, Bnz (8.31) and Nfx (23.10). As found with L. mexicana, compound 17
displayed a low SI value with T. cruzi (Table 2).
Since compound 11 showed the best SI for both trypanosomatids, its effect on parasite cell
morphology was examined at concentrations higher and lower than the IC50 value. Typical
promastigote morphology was found when Leishmania parasites were incubated without
treatment (0 µg/mL), being elongated, thin, flagellated, and with a defined nucleus and
kinetoplast. When 11 was added in increasing concentrations, its leishmanicidal activity
was evidenced by the corresponding decrease in parasite cell density (Figure 3). At the
highest concentration (equivalent to three times the IC50), only a few promastigotes were
observed, which were rounded, dead and with a short flagella.
There was also a dose-dependent effect on the cell density of T. cruzi epimastigotes
induced by 11 as of 2.5 µg/mL (Figure 4). Although a clear parasiticidal activity was
evident for compound 11 at concentrations near its IC50 (5 and 10 µg/mL), judging by the
metabolic inhibition and reduced cell density, the parasites maintained their general
structure (Table 2), indicating that light microscopy is not enough sensitive for detecting
modifications induced by the compound.

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Figure 3. Effect of compound 11 on the morphology of L. mexicana promastigotes.
Parasites were exposed to different concentrations of compound 11 for 24 h, stained with
Giemsa and analyzed by light microscopy at 100x.
Figure 4. Effect of compound 11 on the morphology of T. cruzi epimastigotes. Parasites
were exposed to different concentrations of compound 11 for 24 h, stained with Giemsa
and examined by light microscopy at 100x.
According to the analysis of the structure-activity relationship (SAR) of chalcones for L.
mexicana, the greatest inhibition corresponded to compounds containing the 3,4,5-
trimethoxyphenyl and 3,4-dimethoxyphenyl groups as substituents. Contrarily, the
compounds with geranyloxy groups were not active. For T. cruzi, the 3,4-dimethoxyphenyl
group did not show activity, but inhibition was produced by the geranyloxy and prenyloxy
groups when located on either side of the chalcone ring system.
The inhibitory effects were compared for two distinct positions of each substituent, on the
A ring and on the B ring. Two structural isomers, compounds 11 and 17, showed very
similar IC50 values when tested on both L. mexicana and T. cruzi. However, significantly
greater cytotoxic activity against macrophages was caused by 17 than 11. The only
difference between the two molecules is that the prenyloxy group is attached to the A ring

ACCEPTED MANUSCRIPT
of 17 and to the B ring of 11. Hence, 17 is characterized by a lower selectivity (Figure 5,
Table 2). This highlights the importance of structural modifications on the A and B rings of
the chalcone scaffold, suggesting that the nature and position of the substituents probably
influences biological activity by an electronic and/or steric interaction with the parasite. In
particular, the lipophilicity of the chain length (prenyl vs. geranyl) may affect the
absorption of the compound into the parasite and therefore its biological activity.
Figure 5. Chemical structures of the most active synthetized analogs.
In addition, an in silico analysis was carried out to gain further insights into the drug
likeness properties of the synthesized compounds (Table 3).

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Table 3. In silico results of the synthesized chalcones.
Molecular Properties
cLogP cLogS TPSA HBA HBD logSw
R3
R4
R5
R3`
R4`
R5`
Comp
MW
-4.69
-4.79
-4.74
-4.67
-7.24
-5.7
Nd
Nd
-4.56
Nd
Nd
OCH3 OCH3 OCH3
H
H
H
H
H
H
O-pre OCH3 412.48
O-pre OCH3 382.45
O-pre OCH3 352.42
4.47
4.49
4.51
4.11
7.12
5.64
4.47
4.48
4.51
4.1
7.08
5.95
5.93
5.97
5.57
8.51
5.92
5.96
5.97
5.53
-6.12
-5.95
-5.78
-5.68
-9.43
-7.5
-6.12
-5.95
-5.78
-5.68
-9.43
-8.04
-7.88
-7.71
-7.6
63.22
53.99
44.76
55.76
53.99
53.99
63.22
53.99
44.76
55.76
53.99
63.22
53.99
44.76
55.76
53.99
63.22
53.99
44.76
55.76
6
5
4
4
5
5
6
5
4
4
5
6
5
4
4
5
6
5
4
4
0
0
0
1
0
0
0
0
0
1
0
0
0
0
1
0
0
0
0
1
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
H
H
H
H
H
H
H
H
H
H
OCH3 OCH3
OCH3
OH
H
H
O-pre OCH3
338.4
O-ger OCH3
O-pre OCH3
O-pre OCH3 OCH3 OCH3 OCH3 412.48
O-pre OCH3
O-pre OCH3
O-pre OCH3
O-pre OCH3
O-pre OCH3 504.66
O-pre OCH3 436.54
H
H
H
H
H
H
H
H
H
OCH3 OCH3 382.45
OCH3
OH
H
H
352.42
338.4
O-ger OCH3 504.66
O-ger OCH3 480.59
O-ger OCH3 450.57
O-ger OCH3 420.54
O-ger OCH3 406.51
O-ger OCH3 572.77
-5.87
OCH3 OCH3 OCH3
-5.67
Nd
Nd
-8.31
Nd
Nd
H
H
H
H
H
H
H
H
OCH3 OCH3
OCH3
OH
H
H
O-ger OCH3
O-ger OCH3 OCH3 OCH3 OCH3 480.59
O-ger OCH3
O-ger OCH3
O-ger OCH3
-11.36
-8.04
-7.88
-7.71
-7.6
H
H
H
OCH3 OCH3 450.57
-6.07
-5.61
OCH3
OH
H
H
420.54
406.51
Predictions were made by using the web tool SwissADME [36]. MW, molecular weight; cLogP, average predicted
octanol/water partition coefficient; cLogS, predicted aqueous solubility; TPSA, calculated topological surface polar
area; HBA, number of H-Bond acceptors; HBD, number of H-Bond donors. Prediction of Log Sw were made using
the general solubility equation reported in [37].
The biological activity of 15 and 26 was specific to T. cruzi. These compounds have some
physicochemical characteristics that violate L+6ipinski’s rule of five, which could possibly
be reflected in their pharmacokinetic and pharmacodynamic properties, such as limited
drug availability.
According to the in silico results, all the synthesized compounds show lipophilicity and low
water solubility. These properties may be advantageous for the delivery of the active

ACCEPTED MANUSCRIPT
compounds in topical form, specifically as an ointment for the treatment of cutaneous
leishmaniasis. However, further structural analysis is required in order to develop analogs
with more equilibrated cLogP and cLogS properties for application in other drug delivery
forms.
5. Conclusions
In summary, a series of chalcones containing a prenyloxy or geranyloxy unit were
synthesized and characterized. Compounds 11 and 17 displayed the greatest parasiticidal
activity against both trypanosomatids herein tested (L. mexicana and T. cruzi). For chalcone
12 and derivatives 15 and 26, a selective activity was found for L. mexicana and T. cruzi,
respectively. The highest SI against L. mexicana and T. cruzi corresponded to compound
11, owing itself to lower cytotoxic activity on mammalian macrophages than that found
with its structural isomer 17.
The SAR study demonstrated that the position of the substituents in the molecule, being
either on the A or B ring of the chalcone system, has an influence on the biological activity
and the selectivity index of these compounds. Although the mechanism of action of the
compounds on each parasite is still unclear, the present biological results justify further
research on chalcone derivatives, especially by using 11 as a lead compound. The synthesis
of chalcones with attracting and donating functional groups other than prenyloxy or
geranyloxy substituents is currently underway and will be reported in due time.
6. Experimental section
General: Melting points (uncorrected) were determined on a capillary melting point
apparatus. The NMR spectra were recorded on a Varian (at 300 MHz) or Bruker (at 400
MHz) spectrophotometer, using TMS as internal standard. High-resolution mass spectra
were obtained in the electron impact (EI) mode (70 eV) (Jeol JSM-GCMate-II). Infrared
spectra were recorded by the ATR technique from 4000 to 400 cm-1 (Agilent Cary-600-
Series-FTIR).
3-Methoxy-4-((3-methylbut-2-en-1-yl)oxy)benzaldehyde (33). A mixture of 2.28 g (15.0
mmol) vanillin (31), 3.04 g (22.0 mmol) potassium carbonate and 30 mL acetone was

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stirred at room temperature for 10 minutes. Subsequently, 1.88 g (18.0 mmol) prenyl
chloride were added dropwise and then the mixture was stirred at reflux for 24 hours. Upon
completion of this time, the mixture was filtered and diluted with EtOAc (25 mL), the
organic layer washed with NaOH 3N (2x25 mL), and finally the solvent removed under
vacuum and purified by column chromatography over silica gel (10 g/g crude,
hexane/EtOAc, 9:1) to give 1.58 g (48%) as a whitish-lemon solid. Rf 0.40 (hexane/EtOAc,
8:2); mp47-49 °C. IR (film) ν
2935, 1679, 1583, 1506, 1464, 1424, 1260, 1232, 1134,
1032, 979, 865, 730 cm-1. 1H NMR (300 MHz, CDCl3)
δ
= 1.76 (s, 3H, CH=C(CH3)2), 1.79
(s, 3H, CH=C(CH3)2), 3.93 (s, 3H, OCH3), 4.68 (d, J = 6.7 Hz, 2H, H-1’’),5.52 (dddt, J =
6.7, 5.6, 2.8, 1.4 Hz, 1H, H-2’’), 6.98 (d, J = 8.1 Hz, 1H, H-5’), 7.41 (d, J = 1.8 Hz, 1H, H-
2’), 7.44 (dd, J = 8.1, 1.9 Hz, 1H, H-6’), 9.84 (s, 1H, CHO). 13C NMR (75.4 MHz, CDCl3)
δ
= 18.3 (CH=C(CH3)2), 25.9 (CH=C(CH3)2), 56.0 (OCH3), 65.9 (C-1’’), 108.9 (C-2’),
111.5 (C-5’), 118.9 (C-2’’), 126.8 (C-6’), 129.8 (C-1’), 138.8 (C-3’’), 149.8 (C-3’), 153.8
(C-4’), 191.0 (CHO). HRMS (EI) m/z [M+] calculated for C13H16O3: 220.1100. Found:
220.1105.
1-(3-Methoxy-4-((3-methylbut-2-en-1-yl)oxy)phenyl)ethan-1-one (35). To a mixture of
2.50 g (15.0 mmol) acetovanillone (32) in 30 mL acetone was added 3.04 g (22.0 mmol) of
K2CO3 and the mixture was stirred at room temperature for 10 minutes. Afterwards, 1.88 g
(18.0 mmol) prenyl bromide was added dropwise and the mixture stirred at reflux for 24
hours. The crude was filtered and the solvent removed under vacuum and dissolved in 25
mL of EtOAc, followed by washing the organic layer with NaOH 3N (2x25 mL). The
solvent was removed under vacuum and dissolved in hexane/EtOAc (9:1), the insoluble
solid removed by filtration and crystalized to afford 1.58 g (40%) of 35 as a whitish-lemon
ν
solid. Rf 0.35 (hexane/EtOAc, 8:2); mp 42-43 °C. IR (film)
1464, 1424, 1260, 1232, 1134, 1032, 979, 865, 730 cm-1. 1H NMR (300 MHz, CDCl3)
2935, 1679, 1583, 1506,
δ
=
1.76 (s, 3H, CH=C(CH3)2), 1.79 (s, 3H, CH=C(CH3)2), 2.57 (s, 3H, C=OCH3), 3.93 (s, 3H,
OCH3), 4.66 (d, J = 6.7 Hz, 2H, H-1’’), 5.51 (dddt, J = 6.7, 5.6, 2.8, 1.4 Hz, 1H, H-2’’),
6.89 (d, J = 8.3 Hz, 1H, H-5’), 7.53 (d, J = 2.0 Hz, 1H, H-2’), 7.56 (dd, J = 8.3, 2.0 Hz, 1H,
H-6’). 13C NMR (75.4 MHz, CDCl3)
(C=OCH3), 56.0 (OCH3), 65.9 (C-1’’), 110.1 (C-2’), 111.2 (H-5’), 119.2 (C-2’’), 123.3 (C-
δ = 18.4 (CH=C(CH3)2), 26.0 (CH=C(CH3)2), 26.3

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6’), 130.3 (C-1’), 138.6 (C-3’’), 149.3 (C-3’), 152.7 (C-4’), 197.0 (C=O). HRMS (EI) m/z
[M+] calculated for C14H18O3: 234.1256. Found: 234.1258.
4-((3,7-Dimethylocta-2,6-dien-1-yl)oxy)-3-methoxybenzaldehyde (34). A mixture of
2.28 g (15.0 mmol) vanillin (31) and 3.04 g (22.0 mmol) potassium carbonate in 30 mL
acetone was stirred at room temperature for 10 minutes. Subsequently, 3.11 g (18.0 mmol)
geranyl chloride was added dropwise and the mixture stirred at reflux for 24 hours. It was
then filtered and diluted with EtOAc (25 mL) and the organic layer washed with 2 x 25 mL
NaOH 3N. The solvent was removed under vacuum and purified by column
chromatography over silica gel (10 g/g crude, hexane/EtOAc, 9:1) to furnish 0.21 g (33%)
ν
of 34 as a pale-yellow oil. Rf 0.56 (hexane/EtOAc, 8:2). IR (film)
1682, 1541, 1503, 1440, 1377, 1303, 1264, 1175, 1028, 847, 791, 666 cm-1. 1H NMR (400
MHz, CDCl3) = 1.60 (s, 3H, CH=C(CH3)2), 1.66 (s, 3H, CH=C(CH3)2), 1.76 (s, 3H,
2968, 2915, 1683,
δ
CH=C(CH3)2), 2.03 – 2.17 (m, 4H, H-4’’ and H-5’’), 3.94 (s, 3H, OCH3), 4.72 (d, J = 6.5
Hz, 2H, H-1’’), 5.07 (t, J = 6.0 Hz, 1H, H-6’’), 5.51 (t, J = 6.4 Hz, 1H, H-2’’), 6.97 (d, J =
8.1 Hz, 1H, H-5’), 7.41 (d, J = 1.7 Hz, 1H, H-2’), 7.44 (dd, J = 8.2, 1.7 Hz, 1H, H-6’), 9.85
13
(s, 1H, CHO). C NMR (101 MHz, CDCl3)
δ = 16.9 (CH=C(CH3)2), 17.8 (CH=C(CH3)2),
25.8 (CH=C(CH3)2), 26.3 (C-4’’ or C-5’’), 39.6 (C-4’’ or C-5’’), 56.1 (OCH3), 66.1 (C-
1’’), 109.0 (C-2’), 111.7 (C-5’), 118.8 (C-2’’), 123.7 (C-6’’), 126.9 (C-6’), 129.9 (C-1’),
132.0 (C-7’’), 141.8 (C-3’’), 149.9 (C-3’), 153.9 (C-4’), 191.1 (CHO). HRMS (EI) m/z
[M+] calculated for C18H24O3: 288.1726. Found: 288.1723.
1-(4-((3,7-Dimethylocta-2,6-dien-1-yl)oxy)-3-methoxyphenyl)ethan-1-one
(36).
A
mixture of 2.50 g (15.0 mmol) acetovanillone (32) and 3.04 g (22.0 mmol) potassium
carbonate were stirred in 30 mL of acetone at room temperature for 10 minutes.
Afterwards, 3.11 gr (18.0 mmol) geranyl chloride was added dropwise and the mixture
stirred at reflux for 24 hours before filtration followed by dilution with 25 mL EtOAc. The
solution was washed with 2 x 25 mL 3N NaOH, the organic layer dried, and the solvent
removed under vacuum and purified by column chromatography over silica gel (10 g/g
crude, hexane/EtOAc, 9:1) to provide 1.78 g (40%) of 36 as a pale-yellow oil. Rf 0.49
ν
(hexane/EtOAc, 8:2). IR (film)
2924, 1674, 1586, 1509, 1417, 1356, 1267, 1218, 1148,
1033, 987, 876, 807, 642, 576 cm-1. H NMR (300 MHz, CDCl3)
δ = 1.59 (s, 3H,
1

ACCEPTED MANUSCRIPT
CH=C(CH3)2), 1.66 (s, 3H, CH=C(CH3)2), 1.75 (s, 3H, CH=C(CH3)2), 2.05 – 2.17 (m, 4H,
H-4’’ and H-5’’), 2.56 (s, 3H, C=OCH3), 3.91 (s, 3H, OCH3), 4.69 (d, J = 6.4 Hz, 2H, H-
1’’), 5.02 – 5.11 (m, 1H, H-6’’), 5.50 (tt, J = 5.2, 2.7 Hz, 1H, H-2’’), 6.88 (d, J = 8.2 Hz,
1H, H-5’), 7.52 (d, J = 1.9 Hz, 1H, H-2’), 7.45 (dd, J = 8.1, 2.1 Hz, 1H, H-6’). 13C NMR
(75.4 MHz, CDCl3)
δ 16.6 (CH=C(CH3)2), 17.6 (CH=C(CH3)2), 25.6 (CH=C(CH3)2), 26.1
(C=OCH3 and C-4’’ or C-5’’), 39.4 (C-4’’ or C-5’’), 55.8 (OCH3), 65.8 (C-1’’), 110.0 (C-
2’), 111.2 (C-5’), 118.9 (C-2’’), 123.1 (C-6’’), 123.6 (C-6’), 130.1 (C-1’), 131.7 (C-7’’),
141.3 (C-3’’), 149.1 (C-3’), 152.5 (C-4’), 196.7 (C-1). HRMS (EI) m/z [M+] calculated for
C19H26O3: 302.1882. Found: 302.1881.
General procedure for the preparation of chalcones 11–30. (E)-3-(3-Methoxy-4-((3-
methylbut-2-en-1-yl)oxy)phenyl)-1-(3,4,5-trimethoxyphenyl)prop-2-en-1-one (11). A
mixture of 0.220 g (1.00 mmol) 33, 0.210 g (1.00 mmol) 3,4,5-trimethoxyacetophenone
(41) and 0.680 g (17.0 mmol) NaOH in 40 mL H2O/EtOH (2:1) was stirred at room
temperature for 48 hours. Upon completion of this time, the solvent was removed under
vacuum and the residue purified by column chromatography over silica gel (10 g/g of
crude, hexane/EtOAc, 9:1) to yield 0.050 g (12%) of 11 as a yellow powder. Rf 0.10
ν
(hexane/EtOAc, 8:2); mp 96-98 °C. IR (film)
2936, 1732, 1655, 1578, 1505, 1463,
= 1.76 (s,
1414, 1337, 1253, 1127, 1001, 808, 702, 573 cm-1. 1H NMR (400 MHz, CDCl3)
δ
3H, CH=C(CH3)2), 1.79 (s, 3H, CH=C(CH3)2), 3.94 (s, 6H, OCH3), 3.95 (s, 6H, OCH3),
4.65 (d, J = 6.7 Hz, 2H, OCH2CH=C), 5.48 – 5.58 (m, 1H, OCH2CH=C), 6.91 (d, J = 8.3
Hz, 1H, H-5’’), 7.15 (d, J = 1.8 Hz, 1H, H-2’’), 7.24 (dd, J = 8.3, 1.8 Hz, 1H, H-6’’), 7.27
(s, 2H, H-2’ and H-6’), 7.33 (d, J = 15.6 Hz, 1H, H-2), 7.77 (d, J = 15.6 Hz, 1H, H-3). 13C
NMR (101 MHz, CDCl3)
δ = 18.4 (CH=C(CH3)2), 26.0 (CH=C(CH3)2), 56.1 (OCH3), 56.5
(OCH3), 61.1 (OCH3), 65.9 (C-1’’’), 106.1 (C-6’ and C-2’), 110.7 (C-2’’), 112.6 (C-5’’),
119.4 (C-2’’’), 119.8 (C-2), 122.9 (C-6’’), 127.8 (C-1’’), 134.0 (C-1’), 138.4 (C-3’’’),
142.3 (C-4’), 145.2 (C-3), 149.6 (C-3’’), 150.9 (C-4’’), 153.2 (C-3’ and C-5’), 189.6 (C-1).
HRMS (EI) m/z [M+] calculated for C24H28O6: 412.1886. Found: 412.1896.
(E)-1-(3,4-Dimethoxyphenyl)-3-(3-methoxy-4-((3-methylbut-2-en-1-
yl)oxy)phenyl)prop-2-en-1-one (12). Following the general procedure for the preparation
of chalcones, a mixture of 0.220 g (1.00 mmol) 33, 0.180 g (1.00 mmol) 42 and 0.680 g

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(17.0 mmol) NaOH produced 0.15 g (39%) of 12 as a yellow powder. Rf 0.07
ν
(hexane/EtOAc, 8:2); mp 104-106 °C. IR (film)
2934, 1651, 1594, 1509, 1420, 1259,
= 1.75 (s, 3H,
1
1199, 1140, 1025, 982, 804, 766 cm-1. H NMR (400 MHz, CDCl3)
δ
CH=C(CH3)2), 1.78 (s, 3H, CH=C(CH3)2), 3.94 (s, 3H, OCH3), 3.96 (s, 3H, OCH3), 3.97 (s,
3H, OCH3), 4.64 (d, J = 6.7 Hz, 2H, OCH2CH=C), 5.48 – 5.57 (m, 1H, OCH2CH=C), 6.91
(t, J = 8.8 Hz, 2H, H-5’ and H-5’’), 7.17 (d, J = 1.6 Hz, 1H, H-2’’), 7.22 (dd, J = 8.3, 1.6
Hz, 1H, H-6’’), 7.43 (d, J = 15.5 Hz, 1H, H-2), 7.63 (d, J = 1.5 Hz, 1H, H-2’), 7.66 – 7.72
(dd, J = 8.4, 1.2 Hz, 1H, H-6’), 7.77 (d, J = 15.5 Hz, 1H, H-3). 13C NMR (101 MHz,
CDCl3)
δ = 18.4 (CH=C(CH3)2), 25.9 (CH=C(CH3)2), 56.0 (OCH3), 56.1 (OCH3), 56.1
(OCH3), 65.8 (C-2’’’), 109.9 (C-5’’), 110.4 (C-2’’), 110.8 (C-2’), 112.6 (C-5’), 119.4 (C-
2’’’), 119.5 (C-2), 122.9 (C-6’’), 122.9 (C-6’), 127.9 (C-1’’), 131.6 (C-1’), 138.3 (C-3’’’),
144.3 (C-3), 149.2 (C-3’), 149.6 (C-3’’), 150.7 (C-4’’), 153.1 (C-4’), 188.7 (C-1). HRMS
(EI) m/z [M+] calculated for C23H26O5: 382.1780. Found: 382.1771.
(E)-3-(3-Methoxy-4-((3-methylbut-2-en-1-yl)oxy)phenyl)-1-(4-methoxyphenyl)prop-2-
en-1-one (13). Following the general procedure for the preparation of chalcones, a mixture
of 0.220 g (1.00 mmol) 33, 0.150 g (1.00 mmol) 43 and 0.680 g (17.0 mmol) NaOH
generated 0.14 g (39%) of 13 as a yellow powder. Rf 0.20 (hexane/EtOAc, 8:2); mp 97-98
1
2929, 1734, 1655, 1599, 1508, 1255, 1167, 1024, 983, 833, 596 cm-1. H
ν
°C. IR (film)
NMR (400 MHz, CDCl3)
δ = 1.75 (s, 3H, CH=C(CH3)2), 1.79 (s, 3H, CH=C(CH3)2), 3.89
(s, 3H, OCH3), 3.94 (s, 3H, OCH3), 4.64 (d, J = 6.7 Hz, 2H, OCH2CH=C), 5.48 – 5.56 (m,
1H, OCH2CH=C), 6.90 (d, J = 8.3 Hz, 1H, H-5’’), 6.95 – 7.02 (m, 2H, H-3’ and H-5’), 7.16
(d, J = 1.7 Hz, 1H, H-2’’), 7.21 (dd, J = 8.3, 1.8 Hz, 1H, H-6’’), 7.41 (d, J = 15.5 Hz, 1H,
13
H-2), 7.76 (d, J = 15.5 Hz, 1H, H-3), 8.01 – 8.07 (m, 2H, H-2’ and H-6’). C NMR (101
MHz, CDCl3)
δ = 18.4 (CH=C(CH3)2), 26.0 (CH=C(CH3)2), 55.6 (OCH3), 56.1 (OCH3),
65.9 (C-1’’’), 110.3 (C-2’’), 112.6 (C-5’’), 113.9 (C-3’ and C-5’), 119.5 (C-2’’’), 119.7 (C-
2), 123.0 (C-6’’), 128.0 (C-1’’), 130.8 (C-2’ and C-6’), 131.4 (C-1’), 138.3 (C-3’’’), 144.4
(C-3), 149.6 (C-3’’), 150.7 (C-4’’), 163.4 (C-4’), 188.9 (C-1). HRMS (EI) m/z [M+]
calculated for C22H24O4: 352.1675. Found: 352.1668.
(E
)-1-(4-Hydroxyphenyl)-3-(3-methoxy-4-((3-methylbut-2-en-1-yl)oxy)phenyl)prop-2-
en-1-one (14). Following the general procedure for the preparation of chalcones, a mixture

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of 0.220 g (1.00 mmol) 33, 0.136 g (1.00 mmol) 44 and 0.680 g (17.0 mmol) NaOH
resulted in 0.14 g (40%) of 14 as a yellow powder. Rf 0.23 (hexane/EtOAc, 8:2); mp 130-
ν
131 °C. IR (film)
2939, 1733, 1655, 1599, 1508, 1421, 1251, 1166, 1035, 957, 918,
= 1.75 (s, 3H, CH=C(CH3)2), 1.78 (s, 3H,
835, 804 cm-1. H NMR (400 MHz, CDCl3)
δ
1
CH=C(CH3)2), 3.93 (s, 3H, OCH3), 4.64 (d, J = 6.7 Hz, 2H, OCH2CH=C), 5.51 (td, J = 6.1,
5.4, 3.4 Hz, 1H, OCH2CH=C), 6.90 (d, J = 8.3 Hz, 1H, H-5’’), 6.95 – 7.00 (m, 2H, H-3’
and H-5’), 7.15 (d, J = 1.8 Hz, 1H, H-2’’), 7.21 (dd, J = 8.3, 1.9 Hz, 1H, H-6’’), 7.41 (d, J =
15.5 Hz, 1H, H-2), 7.77 (d, J = 15.5 Hz, 1H, H-3), 7.98 - 8.00 (m, 2H, H-2’ and H-6’). 13C
NMR (101 MHz, CDCl3)
δ = 18.4 (CH=C(CH3)2), 26.0 (CH=C(CH3)2), 56.1 (OCH3), 65.9
(C-1’’’), 110.4 (C-2’’), 112.7 (C-5’’), 115.7 (C-3’ and C-5’), 119.4 (C-2’’’), 119.7 (C-2),
123.2 (C-6’’), 127.9 (C-1’’), 131.1 (C-1’), 131.3 (C-2’ and C-6’), 138.5 (C-3’’’), 144.9 (C-
3), 149.6 (C-3’’), 150.8 (C-4’’), 160.6 (C-4’), 189.5 (C-1). HRMS (EI) m/z [M+] calculated
for C21H22O4: 338.1518. Found: 338.1520.
(E)-1-(4-((3,7-Dimethylocta-2,6-dien-1-yl)oxy)-3-methoxyphenyl)-3-(3-methoxy-4-((3-
methylbut-2-en-1-yl)oxy)phenyl)prop-2-en-1-one (15). Following the general procedure
for the preparation of chalcones, a mixture of 0.220 g (1.00 mmol) 33, 0.302 g (1.00 mmol)
36 and 0.680 g (17.0 mmol) NaOH promoted the formation of 0.25 g (50%) of 15 as a
ν
yellow powder. Rf 0.27 (hexane/EtOAc, 8:2); mp 86-87 °C. IR (film)
2929, 1653, 1594,
= 1.60 (s, 3H,
1509, 1258, 1141, 983, 803, 615 cm-1. H NMR (400 MHz, CDCl3)
δ
1
CH=C(CH3)2), 1.67 (s, 3H, CH=C(CH3)2), 1.76 (s, 6H, CH=C(CH3)2), 1.79 (s, 3H,
CH=C(CH3)2), 2.04 – 2.17 (m, 4H, H-4’’’ and H-5’’’), 3.94 (s, 3H, OCH3), 3.96 (s, 3H,
OCH3), 4.64 (d, J = 6.7 Hz, 2H, H-1’’’’), 4.72 (d, J = 6.4 Hz, 2H, H-1’’’), 5.04 – 5.12 (m,
1H, H-6’’’), 5.53 (dt, J = 6.6, 3.2 Hz, 2H, H-2’’’ and H-2’’’’), 6.91 (t, J = 8.6 Hz, 2H, H-5’
and H-5’’), 7.16 (d, J = 1.8 Hz, 1H, H-2’’), 7.22 (dd, J = 8.3 Hz, 1.8, 1H, H-6’’), 7.42 (d, J
= 15.5 Hz, 1H, H-2), 7.63 (d, J = 1.9 Hz, 1H, H-2’), 7.66 (dd, J = 8.4, 1.9 Hz, 1H, H-6’),
7.77 (d, J = 15.5 Hz, 1H, H-3). 13C NMR (101 MHz, CDCl3)
δ = 16.9 (CH=C(CH3)2), 17.8
(CH=C(CH3)2), 18.4 (CH=C(CH3)2), 25.8 (CH=C(CH3)2), 26.0 (CH=C(CH3)2), 26.3 (C-
4’’’ or C-5’’’), 39.6 (C-4’’’ or C-5’’’), 56.1 (OCH3), 56.2 (OCH3), 65.8 (C-1’’’ or C-1’’’’),
66.0 (C-1’’’ or C-1’’’’), 110.4 (C-2’’), 111.0 (C-2’), 111.4 (C-5’’), 112.6 (C-5’), 119.2 (C-
2’’’), 119.5 (C-2), 119.6 (C-2’’’’), 122.9 (C-6’), 122.9 (C-6’’), 123.8 (C-6’’’), 128.0 (C-
1’’), 131.4 (C-1’), 132.0 (C-7’’’), 138.3 (C-3’’’’), 141.4 (C-3’’’), 144.3 (C-3), 149.6 (C-

ACCEPTED MANUSCRIPT
3’’), 149.6 (C-3’), 150.7 (C-4’’), 152.5 (C-4’), 188.8 (C-1). HRMS (EI) m/z [M+] calculated
for C32H40O5: 504.2876. Found: 504.2869.
(E
)-1,3-Bis(3-methoxy-4-((3-methylbut-2-en-1-yl)oxy)phenyl)prop-2-en-1-one
(16).
Following the general procedure for the preparation of chalcones, a mixture of 0.220 g
(1.00 mmol) 33, 0.234 g (1.00 mmol) 35 and 0.680 g (17.0 mmol) NaOH delivered 0.188 g
ν
(43%) of 16 as a yellow powder. Rf 0.18 (hexane/EtOAc, 8:2); mp 76-78 °C. IR (film)
1
2933, 1736, 1652, 1593, 1508, 1420, 1257, 1140, 982, 804 cm-1. H NMR (400 MHz,
CDCl3)
δ = 1.77 (s, 3H, CH=C(CH3)2), 1.78 (s, 3H, CH=C(CH3)2), 1.81 (s, 6H,
CH=C(CH3)2), 3.96 (s, 3H, OCH3), 3.98 (s, 3H, OCH3), 4.66 (d, J = 6.7 Hz, 2H, H-1’’’’),
4.70 (d, J = 6.7 Hz, 2H, H-1’’’), 5.50 – 5.59 (m, 2H, H2’’’ and H-2’’’’), 6.93 (dd, J = 10.2,
8.4 Hz, 2H, H-5’ and H-5’’), 7.18 (d, J = 1.8 Hz, 1H, H-2’’), 7.24 (dd, J = 8.3, 1.8 Hz, 1H,
H-6’’), 7.44 (d, J = 15.5 Hz, 1H, H-2), 7.64 (d, J = 1.9 Hz, 1H, H-2’), 7.68 (dd, J = 8.4, 1.9
Hz, 1H, H-6’), 7.78 (d, J = 15.5 Hz, 1H, H-3). 13C NMR (101 MHz, CDCl3)
δ = 18.4
(CH=C(CH3)2), 18.4 (CH=C(CH3)2), 26.0 (CH=C(CH3)2), 56.1 (OCH3), 56.2 (OCH3), 65.9
(C-1’’’ or C-1’’’’), 65.9 (C-1’’’ or C-1’’’’), 110.4 (C-2’’), 111.0 (C-2’), 111.3 (C-5’), 112.6
(C-5’’), 119.3 (C-2’’’ or C-2’’’’), 119.5 (C-2), 119.6 (C-2’’’ or C-2’’’’), 122.9 (C-6’ or C-
6’’), 122.9 (C-6’ or C-6’’), 128.0 (C-1’’), 131.5 (C-1’), 138.3 (C-3’’’ or C-3’’’), 138.5 (C-
3’’’ or C-3’’’), 144.3 (C-3), 149.6 (C-3’ or C-3’’), 149.6 (C-3’ or C-3’’), 150.7 (C-4’’),
152.6 (C-4’), 188.8 (C-1). HRMS (EI) m/z [M+] calculated for C27H32O5: 436.2250. Found:
436.2252.
(E)-1-(3-Methoxy-4-((3-methylbut-2-en-1-yl)oxy)phenyl)-3-(3,4,5-
trimethoxyphenyl)prop-2-en-1-one (17). Following the general procedure for the
preparation of chalcones, with a mixture of 0.234 g (1.00 mmol) 35, 0.196 g (1.00 mmol)
37 and 0.680 g (17.0 mmol) NaOH, 0.272 g (66%) of 13 were obtained as a yellow oil. Rf
ν
0.07 (hexane/EtOAc, 8:2). IR (film)
2937, 1716, 1655, 1579, 1504, 1419, 1317, 1271,
1166, 1126, 1092, 980, 812, 615 cm-1. H NMR (400 MHz, CDCl3)
δ = 1.77 (s, 3H,
1
CH=C(CH3)2), 1.79 (s, 3H, CH=C(CH3)2), 3.90 (s, 3H, OCH3), 3.93 (s, 6H, OCH3), 3.96 (s,
3H, OCH3), 4.68 (d, J = 6.6 Hz, 2H, H-1’’’), 5.44 – 5.61 (m, 1H, H-2’’’), 6.88 (s, 2H, H2’’
and H-6’’), 6.93 (d, J = 8.4 Hz, 1H, H-5’), 7.45 (d, J = 15.5 Hz, 1H, H-2), 7.62 (d, J = 1.8,
13
1H, H-2’), 7.67 (dd, J = 8.4, 1.9 Hz, 1H, H-6’), 7.73 (d, J = 15.5 Hz, 1H, H-3). C NMR

ACCEPTED MANUSCRIPT
(101 MHz, CDCl3)
δ
= 18.4 (CH=C(CH3)2), 26.0 (CH=C(CH3)2), 56.2 (OCH3), 56.3
(OCH3), 61.1 (OCH3), 65.9 (C-1’’’), 105.6 (C-2’’ and C-6’’), 111.0 (C-2’), 111.3 (C-5’),
119.2 (C-2’’’), 121.2 (C-2), 123.0 (C-6’), 130.7 (C-1’’), 131.2 (C-1’), 138.6 (C-3’’’), 140.3
(C-4’’), 144.1 (C-3), 149.6 (C-3’), 152.7 (C-4’), 153.5 (C-3’’ and C-5’’), 188.7 (C-1).
HRMS (EI) m/z [M+] calculated for C24H28O6: 412.1886. Found: 412.1886.
(E)-3-(3,4-Dimethoxyphenyl)-1-(3-methoxy-4-((3-methylbut-2-en-1-
yl)oxy)phenyl)prop-2-en-1-one (18). Following the general procedure for the preparation
of chalcones, a mixture of 0.234 g (1.00 mmol) 35, 0.166 g (1.00 mmol) 38 and 0.680 g
(17.0 mmol) NaOH gave 0.25 g (52%) of 18 as a yellow oil. Rf 0.06 (hexane/EtOAc, 8:2).
ν
IR (film)
2935, 1716, 1651, 1593, 1578, 1509, 1420, 1309, 1259, 1142, 1025, 981, 805,
615 cm-1. H NMR (400 MHz,CDCl3)
δ
= 1.77 (s, 3H, CH=C(CH3)2), 1.79 (s, 3H,
1
CH=C(CH3)2), 3.94 (s, 3H, OCH3), 3.96 (s, 6H, OCH3), 4.68 (d, J = 6.7 Hz, 2H, H-1’’’),
5.53 (dt, J = 6.6, 3.3 Hz, 1H, H-2’’’), 6.90 (d, J = 8.4 Hz, 1H, H-5’’), 6.92 (d, J = 8.4 Hz,
1H, H-5’) 7.17 (d, J = 1.8 Hz, 1H, H-2’’), 7.25 (dd, J = 8.3, 1.8 Hz, 1H, H-6’’), 7.43 (d, J =
15.5 Hz, 1H, H-2), 7.62 (d, J = 1.9 Hz, 1H, H-2’), 7.67 (dd, J = 8.4, 1.9 Hz, 1H, H-6’), 7.77
(d, J = 15.5 Hz, 1H, H-3). 13C NMR (101 MHz, CDCl3)
δ = 18.4 (CH=C(CH3)2), 26.0
(CH=C(CH3)2), 31.1 (OCH3), 56.1 (OCH3), 56.1 (OCH3), 56.2 (OCH3), 65.9 (C-1’’’),
110.2 (C-2’’), 111.0 (C-2’), 111.2 (C-5’ or C-5’’), 111.3 (C-5’ or C-5’’), 119.3 (C-2’’’),
119.8 (C-2), 122.9 (C-6’ or C-6’’), 123.0 (C-6’ or C-6’’), 128.2 (C-1’’), 131.4 (C-1’), 138.6
(C-3’’’), 144.2 (C-3), 149.3 (C-3’’), 149.6 (C-3’), 151.3 (C-4’’), 152.6 (C-4’), 188.9 (C-1).
HRMS (EI) m/z [M+] calculated for C23H26O5: 382.1780. Found: 382.1790.
(E)-1-(3-Methoxy-4-((3-methylbut-2-en-1-yl)oxy)phenyl)-3-(4-methoxyphenyl)prop-2-
en-1-one (19). Following the general procedure for the preparation of chalcones, a mixture
of 0.234 g (1.00 mmol) 35, 0.136 g (1.00 mmol) 39 and 0.680 g (17.0 mmol) NaOH
afforded 0.324 g (92%) of 19 as a yellow powder. Rf 0.18 (hexane/EtOAc, 8:2); mp 82-83
ν
°C. IR (film)
829, 799 cm-1. H NMR (400 MHz, CDCl3)
2933, 1735, 1652, 1595, 1572, 1509, 1422, 1252, 1172, 1148, 1030, 982,
1
δ
= 1.78 (s, 3H, CH=C(CH3)2), 1.80 (s, 3H,
CH=C(CH3)2), 3.86 (s, 3H, OCH3), 3.97 (s, 3H, OCH3), 4.69 (d, J = 6.8 Hz, 2H, H-1’’’),
5.54 (t, J = 6.6 Hz, 1H, H-2’’’), 6.91 – 6.99 (m, 3H, H-5’ and H-3’’), 7.47 (d, J = 15.5 Hz,
13
1H, H-2), 7.59 – 7.70 (m, 4H, H-2’, H-6’ and H-2’’), 7.80 (d, J = 15.6 Hz, 1H, H-3). C

ACCEPTED MANUSCRIPT
NMR (101 MHz, CDCl3)
δ
= 18.4 (CH=C(CH3)2), 26.0 (CH=C(CH3)2), 55.5 (OCH3), 56.1
(OCH3), 65.9 (C-1’’’), 110.9 (C-2’), 111.3 (C-5’), 114.5 (C-3’’), 119.3 (C-2’’’), 119.4 (C-
2), 122.8 (C-6’), 127.9 (C-1’’), 130.2 (C-2’’), 131.4 (C-1’), 138.5 (C-3’’’), 143.8 (C-3),
149.5 (C-3’), 152.5 (C-4’), 161.6 (C-4’’), 188.7 (C-1). HRMS (EI) m/z [M+] calculated for
C22H24O4: 352.1675. Found: 352.1675.
(E)-3-(4-Hydroxyphenyl)-1-(3-methoxy-4-((3-methylbut-2-en-1-yl)oxy)phenyl)prop-2-
en-1-one (20). Following the general procedure for the preparation of chalcones, a mixture
of 0.234 g (1.00 mmol) 35, 0.122 g (1.00 mmol) 40 and 0.680 g (17.0 mmol) NaOH
ν
furnished 0.149 g (44%) of 20 as a yellow oil. Rf 0.20 (hexane/EtOAc, 8:2). IR (film)
1
2938, 1716, 1654, 1595, 1508, 1423, 1243, 1171, 1035, 960, 918, 830, 748 cm-1. H NMR
(400 MHz, CDCl3)
δ = 1.76 (s, 3H, CH=C(CH3)2), 1.78 (s, 3H, CH=C(CH3)2), 3.94 (s, 3H,
OCH3), 4.68 (d, J = 6.7 Hz, 2H, H-1’’’), 5.52 (ddd, J = 6.7, 5.4, 1.3 Hz, 1H, H-2’’’), 6.89 –
6.96 (m, 3H, H-5’ and H-3’’), 7.44 (d, J = 15.6 Hz, 1H, H-2), 7.52 – 7.57 (m, 2H, H-2’’),
7.62 (d, J = 1.9 Hz, 1H, H-2’), 7.67 (dd, J = 8.4, 2.0 Hz, 1H, H-6’), 7.79 (d, J = 15.5 Hz,
1H, H-3). 13C NMR (101 MHz, CDCl3)
δ = 18.4 (CH=C(CH3)2), 26.0 (CH=C(CH3)2), 56.1
(OCH3), 66.0 (C-1’’’), 111.1 (C-2’), 111.4 (C-5’), 116.2 (C-3’’), 119.1 (C-2), 123.1 (C-6’),
127.5 (C-1’’), 130.6 (C-2’’), 131.3 (C-1’), 138.7 (C-3’’’), 144.6 (C-3), 149.6 (C-3’), 152.7
(C-4’), 158.8 (C-4’’), 189.5 (C-1). HRMS (EI) m/z [M+] calculated for C21H22O4: 338.1518.
Found: 338.1518.
(E)-3-(4-((3,7-Dimethylocta-2,6-dien-1-yl)oxy)-3-methoxyphenyl)-1-(3-methoxy-4-((3-
methylbut-2-en-1-yl)oxy)phenyl)prop-2-en-1-one (21). Following the general procedure
for the preparation of chalcones, a mixture of 0.234 g (1.00 mmol) 35, 0.288 g (1.00 mmol)
34 and 0.680 g (17.0 mmol) NaOH provided 0.242 g (48%) of 21 as a yellow oil. Rf 0.21
ν
(hexane/EtOAc, 8:2). IR (film)
1031, 983, 844, 803, 612 cm-1. 1H NMR (400 MHz, CDCl3)
2931, 1716, 1653, 1593, 1508, 1420, 1340, 1258, 1140,
= 1.59 (s, 3H, CH=C(CH3)2),
δ
1.66 (s, 3H, CH=C(CH3)2), 1.73 (s, 3H, CH=C(CH3)2), 1.74 (s, 3H, CH=C(CH3)2), 1.77 (s,
3H, CH=C(CH3)2), 1.99 – 2.19 (m, 4H, H-4’’’’ and H-5’’’’), 3.92 (s, 3H, OCH3), 3.93 (s,
3H, OCH3), 4.61 – 4.69 (m, 4H, H-1’’’ and H-1’’’’), 4.99 – 5.11 (m, 1H, H-6’’’’), 5.50 (dt,
J = 5.2, 2.7 Hz, 2H, H-2’’’ and H-2’’’’), 6.89 (t, J = 8.1 Hz, 2H, H-5’ and H-5’’), 7.17 (d, J
= 1.8 Hz, 1H), 7.20 (dd, J = 8.3, 1.9 Hz, 1H, H-6’’), 7.43 (d, J = 15.5 Hz, 1H, H-2), 7.61 (d,

ACCEPTED MANUSCRIPT
J = 1.9 Hz, 1H, H-2’), 7.66 (dd, J = 8.4, 2.0 Hz, 1H, H-6’), 7.76 (d, J = 15.5 Hz, 1H, H-3).
13C NMR (101 MHz, CDCl3)
δ = 16.7 (CH=C(CH3)2), 17.7 (CH=C(CH3)2), 18.3
(CH=C(CH3)2), 25.7 (CH=C(CH3)2), 25.8 (CH=C(CH3)2), 26.2 (C-4’’’’ or C-5’’’’), 39.5
(C-4’’’’ or C-5’’’’), 55.9 (OCH3), 56.0 (OCH3), 65.8 (C-1’’’ or C-1’’’’), 65.8 (C-1’’’ or C-
1’’’’), 110.3 (C-2’’), 110.8 (C-2’), 111.2 (C-5’), 112.6 (C-5’’), 119.2 (C-2’’’ and C-3’’’’),
119.4 (C-2), 122.8 (C-6’ or C-6’’), 122.8 (C-6’ or C-6’’), 123.7 (C-6’’’’), 127.9 (C-1’’),
131.3 (C-1’), 131.7 (C-7’’’’), 138.4 (C-3’’’), 141.1 (C-3’’’’), 144.1 (C-3), 149.5 (C-3’ and
C-3’’), 150.5 (C-4’’), 152.4 (C-4’), 188.6 (C-1). HRMS (EI) m/z [M+] calculated for
C32H40O5: 504.2876. Found: 504.2884.
(E)-3-(4-((3,7-Dimethylocta-2,6-dien-1-yl)oxy)-3-methoxyphenyl)-1-(3,4,5-
trimethoxyphenyl)prop-2-en-1-one (22). Following the general procedure for the
preparation of chalcones, a mixture of 0.288 g (1.00 mmol) 34, 0.210 g (1.00 mmol) 41 and
0.680 g (17.0 mmol) NaOH yielded 0.159 g (33%) of 22 as a yellow powder. Rf 0.15
ν
(hexane/EtOAc, 8:2); mp 66-67 °C. IR (film)
1334, 1253, 1231, 1157, 1127, 1001, 840, 766 cm-1. H NMR (400 MHz, CDCl3)
2937, 1655, 1578, 1505, 1463, 1414,
1
δ = 1.60
(s, 3H, CH=C(CH3)2), 1.67 (s, 3H, CH=C(CH3)2), 1.75 (s, 3H, CH=C(CH3)2), 2.03 – 2.16
(m, 4H, H-4’’’ and H-5’’’), 3.94 (s, 3H, OCH3), 3.94 (s, 3H, OCH3), 3.95 (s, 6H, OCH3),
4.69 (d, J = 6.4 Hz, 2H, H-1’’’), 5.08 (td, J = 6.7, 6.0, 3.3 Hz, 1H, H-6’’’), 5.51 (dt, J = 6.3,
3.2 Hz, 1H, H-2’’’), 6.91 (d, J = 8.3 Hz, 1H, H-5’’), 7.16 (d, J = 1.9 Hz, 1H, H-2’’), 7.23
(dd, J = 8.5, 2.0 Hz, 1H, H-6’’), 7.27 (d, J = 2.2 Hz, 2H, H-2’ and H-6’), 7.33 (d, J = 15.6
Hz, 1H, H-2), 7.77 (d, J = 15.6 Hz, 1H, H-3). 13C NMR (101 MHz, CDCl3)
δ = 16.9
(CH=C(CH3)2), 17.8 (CH=C(CH3)2), 25.8 (CH=C(CH3)2), 26.3 (C-4’’’ or C-5’’’), 39.7 (C-
4’’’ or C-5’’’), 56.2 (OCH3), 56.5 (OCH3), 61.1 (OCH3), 66.0 (C-1’’’), 106.2 (C-2’ and C-
6’), 110.8 (C-2’’), 112.8 (C-5’’), 119.3 (C-2’’’), 119.8 (C-2), 122.9 (C-6’’), 123.8 (C-6’’’),
127.8 (C-1’’), 132.0 (C-7’’’), 134.0 (C-1’), 141.4 (C-3’’’), 142.4 (C-4’), 145.2 (C-3), 149.7
(C-3’’), 150.9 (C-4’’), 153.2 (C-3’ and C-5’), 189.6 (C-1). HRMS (EI) m/z [M+] calculated
for C29H36O6: 480.2512. Found: 480.2510.
(E)-1-(3,4-Dimethoxyphenyl)-3-(4-((3,7-dimethylocta-2,6-dien-1-yl)oxy)-3-
methoxyphenyl)prop-2-en-1-one (23). Following the general procedure for the
preparation of chalcones, a mixture of 0.288 g (1.00 mmol) 34, 0.180 g (1.00 mmol) 42 and

ACCEPTED MANUSCRIPT
0.680 g (17.0 mmol) NaOH produced 0.167 g (37%) of 23 as a yellow powder. Rf 0.10
ν
(hexane/EtOAc, 8:2); mp 48-49 °C. IR (film)
1420, 1259, 1160, 1139, 1024, 984, 803, 766 cm-1. 1H NMR (400 MHz, CDCl3)
2933, 1652, 1594, 1578, 1509, 1463,
= 1.60 (s,
δ
3H, CH=C(CH3)2), 1.67 (s, 3H, CH=C(CH3)2), 1.75 (s, 3H, CH=C(CH3)2), 2.02 – 2.19 (m,
4H, H-4’’’ and H-5’’’), 3.95 (s, 3H, OCH3), 3.97 (s, 3H, OCH3), 3.98 (s, 3H, OCH3), 4.68
(d, J = 6.4 Hz, 2H, H-1’’’), 5.08 (t, J = 6.0 Hz, 1H, H-6’’’), 5.45 – 5.59 (m, 1H, H-2’’’),
6.90 (d, J = 8.3 Hz, 1H, H-5’’), 6.94 (d, J = 8.4 Hz, 1H, H-5’), 7.17 (d, J = 1.7 Hz, 1H, H-
2’’), 7.22 (dd, J = 8.3, 1.7 Hz, 1H, H-6’’), 7.42 (d, J = 15.5 Hz, 1H, H-2), 7.63 (d, J = 1.8
Hz, 1H, H-2’), 7.69 (dd, J = 8.4, 1.9 Hz, 1H, H-6’), 7.77 (d, J = 15.5 Hz, 1H, H-3). 13C
NMR (101 MHz, CDCl3)
δ = 16.9 (CH=C(CH3)2), 17.8 (CH=C(CH3)2), 25.8
(CH=C(CH3)2), 26.3 (C-4’’’ or C-5’’’), 39.7 (C-4’’’ or C-5’’’), 56.1 (OCH3), 56.2 (OCH3),
56.2 (OCH3), 66.0 (C-1’’’), 110.0 (C-5’), 110.5 (C-2’’), 110.9 (C-2’), 112.8 (C-5’’), 119.4
(C-2’’’), 119.6 (C-2), 122.9 (C-6’ and C-6’’), 123.9 (C-6’’’), 128.0 (C-1’’), 131.7 (C-1’),
132.0 (C-7’’’), 141.3 (C-3’’’), 144.4 (C-3), 149.3 (C-3’), 149.6 (C-3’’), 150.7 (C-4’’),
153.2 (C-4’), 188.8 (C-1). HRMS (EI) m/z [M+] calculated for C28H34O5: 450.2406. Found:
450.2406.
(E)-3-(4-((3,7-Dimethylocta-2,6-dien-1-yl)oxy)-3-methoxyphenyl)-1-(4-
methoxyphenyl)prop-2-en-1-one (24). Following the general procedure for the
preparation of chalcones, a mixture of 0.288 g (1.00 mmol) 34, 0.150 g (1.00 mmol) 43 and
0.680 g (17.0 mmol) NaOH generated 0.130 g (31%) of 24 as a yellow oil. Rf 0.25
ν
(hexane/EtOAc, 8:2). IR (film)
2930, 1736, 1655, 1599, 1508, 1463, 1421, 1308, 1252,
= 1.60 (s, 3H, CH=C(CH3)2),
1212, 1165, 1138, 983 cm-1. H NMR (400 MHz, CDCl3)
δ
1
1.67 (s, 3H, CH=C(CH3)2), 1.74 (s, 3H, CH=C(CH3)2), 2.03 – 2.15 (m, 4H, H-4’’’ and H-
5’’’), 3.87 (s, 3H, OCH3), 3.94 (s, 3H, OCH3), 4.67 (d, J = 6.4 Hz, 2H, H-1’’’), 5.04 – 5.12
(m, 1H, H-6’’’), 5.47 – 5.55 (m, 1H, H-2’’’), 6.89 (d, J = 8.3 Hz, 1H, H-5’’), 6.94 – 7.01
(m, 2H, H-3’), 7.17 (d, J = 1.8 Hz, 1H, H-2’’), 7.20 (dd, J = 8.3, 1.9 Hz, 1H, H-6’’), 7.41
13
(d, J = 15.5 Hz, 1H, H-2), 7.76 (d, J = 15.5 Hz, 1H, H-3), 8.01 – 8.08 (m, 2H, H-2’). C
NMR (101 MHz, CDCl3)
δ 16.8 (CH=C(CH3)2), 17.8 (CH=C(CH3)2), 25.8 (CH=C(CH3)2),
26.3 (C-4’’’ or C-5’’’), 39.6 (C-4’’’ or C-5’’’), 55.5 (OCH3), 56.0 (OCH3), 65.9 (C-1’’’),
110.3 (C-2’’), 112.7 (C-5’’), 113.8 (C-3’), 119.3 (C-2’’’), 119.6 (C-2), 123.0 (C-6’’), 123.8
(C-6’’’), 127.9 (C-1’), 130.8 (C-2’), 131.4 (C-1’’), 131.9 (C-7’’’), 141.2 (C-3’’’), 144.3 (C-

ACCEPTED MANUSCRIPT
3), 149.6 (C-3’’), 150.6 (C-4’’), 163.3 (C-4’), 188.8 (C-1). HRMS (EI) m/z [M+] calculated
for C27H32O4: 420.2301. Found: 420.2305.
(E)-3-(4-((3,7-Dimethylocta-2,6-dien-1-yl)oxy)-3-methoxyphenyl)-1-(4-
hydroxyphenyl)prop-2-en-1-one (25). Following the general procedure for the preparation
of chalcones, a mixture of 0.288 g (1.00 mmol) 34, 0.136 g (1.00 mmol) 44 and 0.680 g
(17.0 mmol) NaOH resulted in 0.126 g (31%) of 25 as a yellow oil. Rf 0.29 (hexane/EtOAc,
ν
8:2). IR (film)
2938, 1655, 1599, 1508, 1421, 1308, 1252, 1212, 1166, 1035, 957, 918,
= 1.59 (s, 3H, CH=C(CH3)2), 1.66 (s, 3H,
803 cm-1. H NMR (400 MHz, CDCl3)
δ
1
CH=C(CH3)2), 1.74 (s, 3H, CH=C(CH3)2), 2.02 – 2.16 (m, 4H, H-4’’’ and H-5’’’), 3.93 (s,
3H, OCH3), 4.67 (d, J = 6.4 Hz, 2H, H-1’’’), 5.02 – 5.11 (m, 1H, H-6’’’), 5.46 – 5.57 (m,
1H, H-2’’’), 6.89 (d, J = 8.3 Hz, 1H, H-5’’), 6.97 (d, J = 8.7 Hz, 2H, H-3’), 7.15 (d, J = 1.8
Hz, 1H, H-2’’), 7.20 (dd, J = 8.3, 1.8 Hz, 1H, H-6’’), 7.42 (d, J = 15.5 Hz, 1H, H-2), 7.77
(d, J = 15.5 Hz, 1H, H-3), 7.99 (d, J = 8.8 Hz, 2H, H-2’). 13C NMR (101 MHz, CDCl3)
δ =
16.9 (CH=C(CH3)2), 17.8 (CH=C(CH3)2), 25.8 (CH=C(CH3)2), 26.3 (C-4’’’ or C-5’’’), 39.6
(C-4’’’ or C-5’’’), 56.1 (OCH3), 66.0 (C-1’’’), 110.4 (C-2’’), 112.7 (C-5’’), 115.7 (C-3’),
119.2 (C-2’’’), 119.7 (C-2), 123.2 (C-6’’), 123.9 (C-6’’’), 127.9 (C-1’’), 130.8 (C-1’),
131.3 (C-2’), 132.0 (C-7’’’), 141.4 (C-3’’’), 144.9 (C-3), 149.6 (C-3’), 150.8 (C-4’’), 161.1
(C-4’), 189.6 (C-1). HRMS (EI) m/z [M+] calculated for C26H30O4: 406.2144. Found:
406.2148.
(E)-1,3-Bis(4-((3,7-dimethylocta-2,6-dien-1-yl)oxy)-3-methoxyphenyl)prop-2-en-1-one
(26). Following the general procedure for the preparation of chalcones, a mixture of 0.288 g
(1.00 mmol) 34, 0.302 g (1.00 mmol) 36 and 0.680 g (17.0 mmol) NaOH promoted the
formation of 0.309 g (54%) of 26 as a yellow powder. Rf 0.47 (hexane/EtOAc, 8:2); mp 54-
1
2916, 1654, 1594, 1509, 1420, 1259, 1141, 1032, 985, 801 cm-1. H
ν
55 °C. IR (film)
NMR (400 MHz, CDCl3)
δ = 1.60 (s, 6H, CH=C(CH3)2), 1.67 (s, 6H, CH=C(CH3)2), 1.75
(d, J = 4.1 Hz, 6H, CH=C(CH3)2), 2.03 – 2.16 (m, 8H, H-4’’’, H-5’’’, H-4’’’’ and H-5’’’’),
3.94 (s, 3H, OCH3), 3.96 (s, 3H, OCH3), 4.68 (d, J = 6.4 Hz, 2H, H-1’’’’), 4.72 (d, J = 6.4
Hz, 2H, H-1’’’), 5.02 – 5.15 (m, 2H, H-6’’’ and H-6’’’’), 5.52 (q, J = 5.0 Hz, 2H, H-2’’’
and H-2’’’’), 6.91 (dd, J = 10.5, 8.4 Hz, 2H, H-5’ and H-5’’), 7.17 (d, J = 1.8 Hz, 1H, H-
2’’), 7.22 (dd, J = 8.3, 1.8 Hz, 1H, H-6’’), 7.42 (d, J = 15.5 Hz, 1H, H-2), 7.63 (d, J = 1.9

ACCEPTED MANUSCRIPT
Hz, 1H, H-2’), 7.66 (dd, J = 8.4, 1.9 Hz, 1H, H-6’), 7.77 (d, J = 15.5 Hz, 1H, H-3). 13C
NMR (101 MHz, CDCl3)
δ = 16.9 (CH=C(CH3)2), 17.8 (CH=C(CH3)2), 25.8
(CH=C(CH3)2), 26.3 (C-4’’’ or C-5’’’ and C-4’’’’ or C-5’’’’), 39.6 (C-4’’’ or C-5’’’ and C-
4’’’’ or C-5’’’’), 56.1 (OCH3), 56.2 (OCH3), 66.0 (C-1’’’ or C-1’’’’), 66.0 (C-1’’’ or C-
1’’’’), 110.4 (C-2’’), 111.0 (C-2’), 111.4 (C-5’), 112.7 (C-5’’), 119.2 (C-2’’’ or C-2’’’’),
119.3 (C-2’’’ or C-2’’’’), 119.6 (C-2), 122.8 (C-6’ or C-6’’), 122.9 (C-6’ or C-6’’), 123.8
(C-6’’’ or C-6’’’’), 123.8 (C-6’’’ or C-6’’’’), 128.0 (C-1’’), 131.4 (C-1’), 131.9 (C-7’’’ or
C-7’’’’), 131.9 (C-7’’’ or C-7’’’’), 141.3 (C-3’’’ or C-3’’’’), 141.4 (C-3’’’ or C-3’’’’),
144.3 (C-3), 149.6 (C-3’ or C-3’’), 149.6 (C-3’ or C-3’’), 150.7 (C-4’’), 152.5 (C-4’), 188.8
(C-1). HRMS (EI) m/z [M+] calculated for C37H48O5: 572.3502. Found: 572.3493.
(E)-1-(4-((3,7-Dimethylocta-2,6-dien-1-yl)oxy)-3-methoxyphenyl)-3-(3,4,5-
trimethoxyphenyl)prop-2-en-1-one (27). Following the general procedure for the
preparation of chalcones, a mixture of 0.302 g (1.00 mmol) 36, 0.196 g (1.00 mmol) 37 and
0.680 g (17.0 mmol) NaOH delivered 0.187 g (39%) of 27 as a yellow oil. Rf 0.11
ν
(hexane/EtOAc, 8:2). IR (film)
2933, 1653, 1594, 1578, 1463, 1420, 1341, 1309, 1259,
1196, 1143, 1025, 982, 913 cm-1. H NMR (400 MHz, CDCl3)
δ = 1.59 (s, 3H,
1
CH=C(CH3)2), 1.66 (s, 3H, CH=C(CH3)2), 1.75 (s, 3H, CH=C(CH3)2), 2.02 – 2.20 (m, 4H,
H-4’’’ and H-5’’’), 3.90 (s, 3H, OCH3), 3.92 (s, 6H, OCH3), 3.96 (s, 3H, OCH3), 4.71 (d, J
= 6.4 Hz, 2H, H-1’’’), 5.07 (ddd, J = 6.7, 4.0, 1.3 Hz, 1H, H-6’’’), 5.46 – 5.56 (m, 1H, H-
2’’’), 6.87 (s, 2H, H-2’’ and H-6’’), 6.92 (d, J = 8.4 Hz, 1H, H-5’), 7.45 (d, J = 15.5 Hz,
1H, H-2), 7.62 (d, J = 1.9 Hz, 1H, H-2’), 7.67 (dd, J = 8.4, 2.0 Hz, 1H, H-6’), 7.72 (d, J =
15.5 Hz, 1H, H-3). 13C NMR (101 MHz, CDCl3)
δ = 16.8 (CH=C(CH3)2), 17.8
(CH=C(CH3)2), 25.7 (CH=C(CH3)2), 26.2 (C-4’’’ or C-5’’’), 39.6 (C-4’’’ or C-5’’’), 56.1
(OCH3), 56.2 (OCH3), 61.0 (OCH3), 66.0 (C-1’’’), 105.5 (C-2’’ and C-6’’), 110.9 (C-2’),
111.3 (C-5’), 119.0 (C-2’’’), 121.1 (C-2), 123.0 (C-6’), 123.7 (C-6’’’), 130.7 (C-1’’), 131.1
(C-1’), 131.9 (C-7’’’), 140.2 (C-4’’), 141.5 (C-3’’’), 144.1 (C-3), 149.6 (C-3’), 152.6 (C-
4’), 153.5 (C-3’’ and C-5’’), 188.6 (C-1). HRMS (EI) m/z [M+] calculated for C29H36O6:
480.2512. Found: 480.2505.
(E
)-3-(3,4-Dimethoxyphenyl)-1-(4-((3,7-dimethylocta-2,6-dien-1-yl)oxy)-3-
methoxyphenyl)prop-2-en-1-one (28). Following the general procedure for the