Identification of chalcone-based antileishmanial agents targeting trypanothione reductase

Article abstract of DOI:10.1016/j.ejmech.2018.04.057

All currently used first-line and second-line drugs for the treatment of leishmaniasis exhibit several drawbacks including toxicity, high costs and route of administration. Furthermore, some drugs are associated with the emergence of drug resistance. Thus

Full text of DOI:10.1016/j.ejmech.2018.04.057

Accepted Manuscript
Identification of chalcone-based antileishmanial agents targeting trypanothione
reductase
Margherita Ortalli, Andrea Ilari, Gianni Colotti, Ilenia De Ionna, Theo Battista,
Alessandra Bisi, Silvia Gobbi, Angela Rampa, Rita M.C. Di Martino, Giovanna A.
Gentilomi, Stefania Varani, Federica Belluti
PII:
S0223-5234(18)30399-4
10.1016/j.ejmech.2018.04.057
EJMECH 10406
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 12 February 2018
Revised Date: 27 April 2018
Accepted Date: 28 April 2018
Please cite this article as: M. Ortalli, A. Ilari, G. Colotti, I. De Ionna, T. Battista, A. Bisi, S. Gobbi, A.
Rampa, R.M.C. Di Martino, G.A. Gentilomi, S. Varani, F. Belluti, Identification of chalcone-based
antileishmanial agents targeting trypanothione reductase, European Journal of Medicinal Chemistry
(2018), doi: 10.1016/j.ejmech.2018.04.057.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
1
2
Identification of Chalcone-based Antileishmanial Agents Targeting Trypanothione
Reductase
3
4
5
Margherita Ortalli1, Andrea Ilari2, Gianni Colotti2, Ilenia De Ionna3, Theo Battista3, Alessandra
Bisi4, Silvia Gobbi4, Angela Rampa4, Rita M. C. Di Martino4§, Giovanna A. Gentilomi4, Stefania
Varani1,5^ and Federica Belluti4^*
6
7
8
9
1Department of Experimental, Diagnostic and Specialty Medicine, Alma Mater Studiorum
University of Bologna, Bologna, Italy
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
2CNR-Institute of Molecular Biology and Pathology, Rome, Italy
3Department of Biochemical Sciences “A. Rossi Fanelli”, Sapienza University, Rome, Italy
4Department of Pharmacy and Biotechnology, Alma Mater Studiorum University of Bologna,
Bologna, Italy. § Present address: Department. of Drug Discovery and Development, Istituto
Italiano di Tecnologia,via Morego n.30, 16163 Genova, Italy
5Unit of Clinical Microbiology, Regional Reference Centre for Microbiological Emergencies
(CRREM), St. Orsola-Malpighi University Hospital, Bologna, Italy
^ these authors contributed equally to this work
*Corresponding author: federica.belluti@unibo.it
Abstract
All currently used first-line and second-line drugs for the treatment of leishmaniasis exhibit several
drawbacks including toxicity, high costs and route of administration. Furthermore, some drugs are
associated with the emergence of drug resistance. Thus, the development of new treatments for
leishmaniasis is a priority in the field of neglected tropical diseases. The present work highlights the
1

ACCEPTED MANUSCRIPT
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
use of natural derived products, i.e. chalcones, as potential source of antileishmanial agents. Thirty-
one novel chalcone compounds have been synthesized and their activity has been evaluated against
promastigotes of Leishmania donovani; 16 compounds resulted active against L. donovani in a
range from 3.0 to 21.5 µM, showing low toxicity against mammalian cells. Among these molecules,
6 and 16 showed good inhibitory activity on both promastigotes and intracellular amastigotes,
coupled with an high selectivity index. Furthermore, compounds 6 and 16 inhibited the
promastigote growth of other leishmanial species, including L. tropica, L. major and L. infantum.
Finally, 6 and 16 interacted with high affinity with trypanothione reductase (TR), an essential
enzyme for the leishmanial parasite and compound 6 inhibited TR with sub-micromolar potency.
Thus, the effective inhibitory activity against Leishmania, the lack of toxicity on mammalian cells
and the ability to block a crucial parasite’s enzyme, highlight the potential for compound 6 to be
optimized as novel drug candidate against leishmaniasis.
Keywords
Chalcone, drug discovery, leishmaniasis, natural products, neglected tropical disease, trypanothione
reductase
1. INTRODUCTION
Leishmaniases are vector-borne infections caused by protozoan parasites belonging to the genus
Leishmania that are transmitted through the bite of phlebotomine sand flies of the genus
Phlebotomus in the Old World [1]. Most forms of the diseases are zoonotic and only 21 of the 30
Leishmania species that infect mammals may cause human infection.
Most infections caused by Leishmania parasites are asymptomatic, but in symptomatic patients
clinical manifestations range from cutaneous leishmaniasis (CL), which may result in disfiguring
2

ACCEPTED MANUSCRIPT
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
scars if left untreated, to the potentially fatal visceral leishmaniasis (VL), which is characterized by
fever, splenomegaly, pancytopenia and weight loss [2].
Leishmaniases are distributed in Asia, Africa, Latin America and Southern Europe, with an
estimation of 1.5-2.0 million new cases per year of CL and 0.5 million cases of VL [3]. Despite this,
leishmaniases are classified among the most neglected diseases, based on their strong association
with poverty and on the limited resources invested in their diagnosis, treatment and control [2].
Chemotherapy is the only method for protection against leishmaniasis, since there is currently no
approved vaccine for humans [4]. Treatment of leishmaniasis comprises liposomal amphotericin B,
pentavalent antimonials, paromomycin and miltefosine; these drugs are plagued by several
limitations including high costs, toxicity, route of administration and poor efficacy [5]. Moreover,
the increasing emergence of Leishmania parasites that are resistant to antimonial drugs is a serious
problem in several endemic regions [6]. This scenario emphasizes the need of developing novel
effective, safe and economically feasible antileishmanial agents.
The implementation of the genome project for many trypanosomatid species lead to the
identification of several drug targets suitable for gaining parasite selective inhibition [7]. Indeed,
targeting a unique and essential parasite metabolic pathway, which is absent in mammals is
generally considered a successful therapeutic strategy.
In this context, the thiol-dependent redox polyamine metabolism represents an essential
detoxifying system by means of which the parasite eliminates its toxic endogenous metabolites [8].
While the mammalian redox defense machinery is based on glutathione (GSH), the protozoan
parasites from the Trypanosomatidae family, including Trypanosoma and Leishmania, strictly
depend on a different pathway for supporting their intracellular redox homeostasis; they employ
trypanothione (N1,N8-bis–glutathionyl-spermidine) in its reduced thiol form T(SH)2 [9].
Trypanothione disulfide (TS2) is obtained by means of two consecutive steps, each involving the
conjugation of GSH to N1 and N8 amino groups of spermidine by the ATP-dependent C-N ligase
trypanothione synthetase (TryS). Trypanothione reductase (TR), a NADPH-dependent flavoprotein,
3

ACCEPTED MANUSCRIPT
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
100
101
102
103
reduces TS2 to T(SH)2, thus ensuring an intracellular reducing environment. The inhibition of TryS
and/or TR is known to disrupt the parasite redox balance [10, 11]; these two enzymes can be
regarded as validated molecular targets for the development of effective and selective
antileishmanial drugs [9, 12].
TR inhibition may be achieved by competing with trypanothione binding to the active site; for
example, trivalent antimony Sb(III), the active form of the antimonial drug sodium stibogluconate
(SSG) [13], a number of metals, such as Ag(I) and Au(I) [14-16] and some TR inhibitors, such as
azole and diaryl compounds [17, 18] have been reported to directly bind the trypanothione binding
site.
Natural products (NPs) including flavonoids, isoflavonoids, saponins, alkaloids, tannins and
indoles have been shown to exert antileishmanial effects [19, 20]. Among them, chalcones (1,3-
diaryl-2-propen-1-ones), prominent secondary metabolites and precursors of flavonoids, can be
considered “privileged structures”, i.e. evolutionary-chosen molecules that have evolved to achieve
an inherent affinity for diverse biological macromolecules in the natural selection process [21].
Indeed, chalcones display a wide range of pharmacological effects, including antioxidant,
antimutagenic, antimitotic, antimetastatic and antiinflammatory activity [22, 23]. The
antileishmanial potential of chalcones has also been demonstrated [20]; naturally occurring
chalcones such as licochalcone A and isocordoin (Figure 1), isolated from Glycyrrhiza glabra and
Lonchocarpus xuul, respectively, were able to efficiently inhibit the proliferation of different
Leishmania species [24-26]. Unfortunately, the intrinsic cytotoxicity of these molecules may
represent an undesired aspect. In this study, the chalcone framework was selected as main scaffold
to develop a new series of effective and safe antileishmanial agents and targeting a key enzyme in
the polyamine-trypanothione pathway, ie TR.
Therefore, we have synthesized 31 novel chalcones, evaluated their activity against Leishmania
vs. mammalian cells and tested both interaction and ability to inhibit L. donovani TR.
4

ACCEPTED MANUSCRIPT
104
105
106
107
108
109
110
111
112
113
114
115
116
117
118
119
120
121
122
123
124
125
126
127
128
129
2. RESULTS
2.1 Design strategy
A small library of 31 chalcone-based analogues was designed and synthesized (Figure 1). In
particular, the A-ring of the main scaffold was properly functionalized at the positions 2 and 4: the
C-4 position was occupied by a suitable alkoxy function (O-R), namely 3,3-dimethylallyoxy (or
prenyloxy) and propargyloxy affording Series 1 and 2, respectively. The C-2 position was
differently functionalized by introduction of hydroxy, methoxy, prenyloxy and propargyloxy groups
(O-R1). In order to perform a Structure Activity Relationship (SAR) study, different moieties were
introduced as B-ring, namely pyridine or aryl functions bearing methoxy, bromo, nitro, and fluorine
substituents.
2.2. Synthesis
All the tested chalcones were readily synthesized through the classic base-catalyzed Claisen-
Schmidt procedure (as shown in Scheme 1). In details, the selected acetophenone was reacted at
room temperature with the appropriate aldehyde in ethyl alcohol and in the presence of a 50%
KOH/H2O solution, to give the desired final compounds (1-31, Table 1). The acetophenone
intermediates were obtained by reaction of 2,4-dihydroxyacetophenone with the appropriate alkyl
halide to obtain the 2-OH,4-alkoxyacetophenones (32, 33) and 2,4-bi-functionalyzed-acetophenones
(34-37).
2.3. Biological and Enzymatic Assays
First, 31 chalcone-based derivatives were investigated for their antileishmanial effect on the
promastigotes of L. donovani. Then, for 16 analogues that inhibited parasite growth at micromolar
level, cytotoxicity against mammalian kidney epithelial cells and affinity for TR enzyme (SPR-
based assay) were also evaluated. This allowed us to identify two promising molecules in terms of
activity and selectivity that were then further investigated for their ability to inhibit promastigote
5

ACCEPTED MANUSCRIPT
130
131
132
133
134
135
136
137
138
139
140
141
142
143
144
145
146
147
148
149
150
151
152
153
154
155
growth of different parasitic species, including L.tropica, L.major and L.infantum and to affect the
growth L.donovani amastigotes. Finally, the mechanism of TR inhibition was also studied.
2.3.1. In vitro inhibition of Leishmania promastigote growth
To assess the antileishmanial potential of the synthesized compounds, a reference strain of L.
donovani (MHOM/NP/02/BPK282/0cl4) was employed in two different stages of the parasitic life
cycle; the extracellular promastigote form is found in the sandfly vector, while intracellular
amastigote form is specifically found in the host cell.
In a first experiment, 31 chalcone-based derivatives were investigated for their antileishmanial
efficacy on the promastigote forms of L. donovani (Table 1). Amphotericin B was employed as
reference compound. Data were expressed as IC50, i.e. the concentration of compound that is
required to inhibit growth by 50%.
Among the tested chalcones, compounds 1-16 turned out to effectively inhibit the promastigote
growth with micromolar potency and IC50/72h values ranged from 21.5 µM (compound 12) to 3.0
µM (compound 6). Concerning analogues 17-31, no antileishmanial effect was observed at the
maximal dose of 40.0 µM and were then discarded from further evaluation. Among derivatives with
a simple phenyl function as B-ring included in Series 1 (7, 19-21), the substituent at the 2-position
of the A-ring markedly affected the inhibitory behaviour against Leishmania. In details, compound
7, bearing a methoxy function, showed an IC50/72h of 15.0 µM, while the presence of hydroxy,
acethoxy, and prenyloxy (19, 20 and 21, respectively) rendered the derivatives almost inactive. By
keeping the most favourable 2-methoxy-4-prenyloxy substitution pattern in the A-ring, further
modifications were applied to the B-aryl ring. The presence of electron-donating moieties, such as
methoxy groups, on several positions of the B-ring (compounds 8, 23, 24) gave different results, as
only the bulky 3,4,5-trimethoxylated analogue 8 proved to inhibit parasite growth with an IC50/72h
of 11.0 µM. The effect of different electron-withdrawing groups, namely bromo, nitro, and fluoro,
on the para position of the B-ring (compounds 5, 6, and 22, respectively) was also investigated:
6

ACCEPTED MANUSCRIPT
156
157
158
159
160
161
162
163
164
165
166
167
168
169
170
171
172
173
174
175
176
177
178
179
180
181
compound 6, with the nitro substituent, resulted to be the most active among the series, showing an
IC50/72h of 3.0 µM, followed by the bromo-derivative 5 (IC50/72h = 16.0 µM). The introduction of
a heterocyclic furyl group (18) led to a loss of activity, while a 4- and 3-pyridyl moiety (3 and 4,
respectively) allowed to retain good activities (IC50/72h = 10.5 µM). The corresponding pyridyl-
based analogues (1 and 2), characterized by a 2-hydroxyl function, retained leishmanicidal activity
(IC50/72h = 5.0 µM and 8.5 µM, respectively).
A different trend of potency was observed in the 4-propargyloxylated Series 2 upon applying
different B-ring functionalization. The insertion on B-ring of methoxy groups (27, 28 and 29-31)
and para-NO2 functions (analogues 25 and 26) led to inactive compounds. Interestingly, the
presence of a para-F phenyl B-ring and of a 2-propargyloxylated A-ring (compound 16) conferred
an effective ability to inhibit Leishmania growth (IC50/72h = 12.5 µM). Moreover, the para-Br
derivatives 12 and 15 retained a moderate antileishmanial activity (IC50/72h = 21.5 µM and 15.0
µM, respectively) that was comparable to the corresponding Br-derivative 5 of Series 1. The
pyridine-based analogues 10, 11, and 13, 14 with a 2-methoxy-4-propargyloxy and 2,4-bis-
propargyloxy A-ring, respectively, showed low micromolar potencies (IC50/72h values ranging
from 4.0 µM to 9.5 µM), similar to that of the corresponding Series 1 analogues (1-4). A reduction
of activity was observed for compound 9, designed as the 2-hydroxy congener of 11 and 14
derivatives.
Compound 6 and 16 were further investigated for their antileishmanial efficacy on the promastigote
forms of three other parasitic species, ie L. tropica, L. major and L. infantum. Interestingly, the
results obtained highlight diverse susceptibilities of different parasitic species to the compounds., In
detail, compound 6 exhibited an IC50/72h of 5.2 µM, 3.3 µM and 1.6 µM on L. tropica , L. major
and L. infantum, respectively, while compound 16 showed an IC50/72h of 13 µM, 10 µM and 1.6
µM on L. tropica, L. major and L. infantum, respectively. Thus, both compound 6 and 16 exhibited
the highest inhibitory activity on L. infantum, revealing L. infantum as the most susceptible species
to the examined chalcones.
7

ACCEPTED MANUSCRIPT
182
183
184
185
186
187
188
189
190
191
192
193
194
195
196
197
198
199
200
201
202
203
204
205
206
2.3.2 In vitro mammalian cell toxicity
The cytotoxic effect against mammalian cells was evaluated for the most active chalcones (1-16)
by using Vero cell line (mammalian kidney epithelial cells) (Table 1). Data were expressed as 50%
cytotoxic concentration (CC50). The tested compounds generally displayed moderate to low
cytotoxicity, with CC50/72h values above 30 µM. In particular, analogues 5, 6 and 16 were
characterized by low toxicity, with cytotoxic effect detected only at 600 µM. On the contrary, the
pyridine-based chalcones showed an unfavorable SI due to their moderate cytotoxic effects. It is
noteworthy that CC50/72h value of the reference compound amphotericin B was 200 µM, lower
than those of compound 6 and 16. The cytotoxic effect against human acute monocytic leukemia
cell line (THP-1) was also evaluated (Table 1) and the selectivity index (SI) was calculated as
CC50/IC50 ratio. Analogue 5 showed a higher cytotoxic effect on THP-1 than on the Vero cell line.
On the other hand, compound 6 and 16, the most promising of the series, showed the same low
cytotoxic effect on the Vero cell line and on THP-1. The cytotoxicity assays allowed to identify
compounds 6 and 16 as the most promising of the series. Indeed they showed a remarkable
antileishmanial potency against the extracellular form of the parasite (IC50/72h = 3.0 µM and 12.5
µM, for compound 6 and 16, respectively) that was coupled with a good SI on mammalian cells
(200 and 48, for compound 6 and 16, respectively).
2.3.3 Inhibition of L. donovani amastigote growth
Considering the promising results obtained for 6 and 16, these compounds were selected to be
tested for their efficacy against the amastigote stage of L. donovani. The amastigote assay was
performed by using metacyclic promastigotes to infect differentiated THP-1 macrophagic cells;
amastigotes transformed from metacyclic promastigotes proliferated inside host macrophages. An
inversion of potencies was observed when focusing on inhibitory activity of 6 and 16 on
amastigotes growth, with respect to activities recorded on promastigotes (Figure 2a-
8

ACCEPTED MANUSCRIPT
207
208
209
210
211
212
213
214
215
216
217
218
219
220
221
222
223
224
225
226
227
228
229
230
231
2b). Indeed, compound 16 exhibited a higher inhibitory effect on amastigotes than on
promastigotes (4.5 µM vs 12.5 µM, IC50/72h calculated on amastigote and promastigote cultures,
respectively), while compound 6 showed a lower potency on amastigotes than on promastigotes
(14.0 µM vs 3.0 µM, IC50/72h detected on amastigote and promastigote cultures, respectively).
2.3.4. Evaluation of activity toward trypanothione reductase
2.3.4.1. Surface Plasmon Resonance screening
For Surface Plasmon Resonance (SPR) experiments, TR from Leishmania spp. has been
immobilized by amine coupling on COOH5 sensorchips, while chalcones were analytes; FastStep
SPR experiments were performed by stepped analyte gradient injections (ranging between 1.5 and
100 µM). Screening of the 16 most active chalcones (compounds 1-16) demonstrated that two
molecules, compounds 6 and 16, interacted directly and with high affinity with TR. KD values
calculated for these compounds were KD (compound 6) = 0.6 ± 0.2 µM and KD (compound 16)= 2.4
± 0.5 µM (Figure 3).
2.3.4.2. Enzymatic assays
Kinetic studies were performed on compound 6, endowed with the highest activity against the
promastigote forms of L. donovani and affinity toward TR. Steady state kinetic experiments were
carried out at various concentrations of TS2 and compound 6, while fixed TR and NADPH
concentrations (10 nM and 40 µM, respectively) were maintained. After starting the reaction by the
addition of NADPH, the absorbance decrease at 340 nm, indicative of NADPH oxidation, was
measured. As shown in Figure 4, compound 6 competitively inhibited the binding of TS2 to TR.
Each line in the Dixon plot represents linear regression analysis of reciprocal of average fitted rates
of TS2 reduction for different substrate concentrations, as a function of inhibitor concentration. The
KM and kcat of TR used for the Ki calculation were 23.0 ± 1.0 µM and 11.4 ± 0.3 s-1 respectively
9

ACCEPTED MANUSCRIPT
232
233
234
235
236
237
238
239
240
241
242
243
244
245
246
247
248
249
250
251
252
253
254
255
256
257
[17]. The value of Ki calculated from the Dixon plot analysis was 0.45 ± 0.11 µM, about three times
lower than that of Sb(III) (1.5 µM) [13].
2.4. Docking studies
The crystal structure of TR from L. infantum revealed two crucial cysteine residues (Cys52 and
Cys57) in the active site, involved in a concerted nucleophilic attack to the TS2 disulfide bridge to
produce the reduced substrate T(SH)2. In order to gain functional and structural insight into the
mechanism of inhibition, molecular docking simulations of compound 6 to TR were performed
using the x-ray structures of the enzyme in both reduced and oxidized states. Figure 5a illustrates
the most probable and energetically favourable binding modes of compound 6 at the active site of
TR in oxidized state (PDB code: 2JK6) and Figure 5b shows the conformation with the lowest
energy in the most populated cluster. As shown in TableS1, the most populated cluster contains
32/100 poses and the lowest energy pose in this cluster displays a binding energy of -7.63 kcal/mol
corresponding to a Ki=2.56 µM. Figure 5c displays the conformation with the lowest energy in the
most populated cluster, resulting from the docking procedure performed using TR in reduced state
(PDB code: 4ADW); the clusters and the energies of the poses are reported in TableS2. In this case,
the most populated cluster contains 16/100 poses and the lowest energy pose in this cluster displays
a binding energy of -6.66 kcal/mol, corresponding to a Ki=13.19 µM.
As shown in Figure 5, both the docking procedures performed using the TR structures in the
oxidized and reduced state gave similar results. Interestingly, in both the procedures the most
populated clusters occupy the same portion of the trypanothione cavity volume. Compound 6 binds
to TR in both oxidized and reduced states at the same hydrophobic pocket close to the two catalytic
cysteines lined by the following residues: Ile339, Ile458’, His461’, E466’, E467’, S470’, F396’,
L399’. Compound 6 establishes electrostatic interactions with and K61, E467’ and S470’.
Interestingly, compound 6 appears to have a higher affinity for the oxidized form of TR.
10

ACCEPTED MANUSCRIPT
258
259
260
261
262
263
264
265
266
267
268
269
270
271
272
273
274
275
276
277
278
279
280
281
282
283
3. DISCUSSION
To support the urgent need of safe and effective agents against leishmaniasis. a plethora of
bioactive compounds with antileishmanial activity and acting through different mechanisms have
been synthetized [27, 28]. Evidence indicates that a number of natural and synthetic chalcones
exhibit antileishmanial activity [25, 29-32]. In this study, we evaluated the antileishmanial effect of
a small library of synthetic chalcones and we investigated the mechanism of action of selected
compounds. Among the 31 tested compounds, 16 (1-16) turned out to inhibit the promastigote form
of L.donovani with micromolar potency, and among them, two (6 and 16) showed good potency
(IC50/72h values of 3.0 and 12.5 µM, respectively), even if lower with respect to the reference drug
amphotericin B. Furthermore compounds 6 and 16 maintained a good inhibitory activity when
tested on amastigotes of L.donovani (IC50 values of 14.0 and 4.5 µM, respectively). Interestingly,
these derivatives were characterized by low toxicity when tested on Vero cells and THP-1 cells,
being three times less toxic than amphotericin B and thus exhibiting a very favorable SI.
Moreover, compounds 6 and 16 efficiently inhibited the promastigote growth of other leishmanial
species, including L.tropica, L.major and L.infantum, being particularly active on L.infantum.
In an effort to assess the mechanism by which 6 and 16 inhibit Leishmania growth, we evaluated
their affinity and activity against TR, a pivotal enzyme involved in the parasite detoxification. The
enzymes of the trypanothione pathway are not present in mammals, and are often considered among
the most promising antileishmanial targets. However, it was previously demonstrated that at least
90% of TR inactivation needs to be obtained by inhibitor compounds to kill the parasite; therefore,
effective TR inhibitors should have submicromolar inhibition activity [12]. Here, we observed that
compound 6 showed a submicromolar Ki value vs. TR (0.45 ± 0.11 µM) that is about 6 times lower
than the IC50/72h value vs. promastigotes and 30 times lower than the IC50/72h vs. the amastigotes.
This result is in agreement with numerous results present in literature showing that specific and
efficient trypanosomatid TR inhibitors have been found to be less active on the parasites growth.
This apparent paradox was shown by Krieger and coworkers who produced conditioned TR
11

ACCEPTED MANUSCRIPT
284
285
286
287
288
289
290
291
292
293
294
295
296
297
298
299
300
301
302
303
304
305
306
307
308
309
knockout in T. brucei [33], demonstrating that the redox metabolism of the parasite was affected
only when TR was titrated down to less than 5% of normal. Thus, compound 6 is a good TR
inhibitor, with a Ki value remarkably lower than Sb(III) and azole-based compounds and in the
same order of magnitude of RDS 777, a diaryl sulphide derivative [17, 18].
The docking experiments furnished a possible binding mode of compound 6 to the catalytic site.
Indeed, compound 6 binds to TR in both the oxidized and reduced states to a hydrophobic pocket
close to the catalytic site, which was already shown to be part of RDS 777 binding site in the TR
trypanothione cleft. Interestingly, F396’ and E467’ lining the compound 6 binding site have been
already identified as important residues to establish interaction with other TR inhibitors [18].
This study has some limitations, including the employment of a small library of chalcones for the
screening of antileishmanial activity and the lack of in vivo pharmacokinetic and pharmacodynamic
testing of the selected compounds.
4. CONCLUSIONS
A small library of 31 chalcone-based analogues were synthetized and tested for their
antileishmanial activity. Among tested compounds, 6 and 16 were found to be significantly active in
in vitro evaluation against L. donovani promastigotes and amastigotes without eliciting cytotoxic
effects towards human cells, thus showing optimal performance in terms of potency and selectivity.
Furthermore, compounds 6 and 16 inhibited the promastigote growth of other leishmanial species,
including L. tropica, L. major and L. infantum. Finally, compounds 6 and 16 interacted with TR and
compound 6 effectively inhibited TR activity, providing evidence that TR inhibition could represent
one of the possible mechanisms of action of this molecule. In conclusion, the effective inhibitory
activity against different Leishmania species, the lack of toxicity on mammalian cells and the ability
to block a crucial parasite enzyme target, highlight the potential for the chalcone 6 to be further
optimized and to develop novel drug candidates against leishmaniasis.
12

ACCEPTED MANUSCRIPT
310
311
312
313
314
315
316
317
318
319
320
321
322
323
324
325
326
327
5. EXPERIMENTAL SECTION
5.1. Chemistry.
Starting materials, unless otherwise specified, were used as high grade commercial products.
Solvents were of analytical grade. Reaction progress was followed by thin layer chromatography
(TLC) on precoated silica gel plates (Merck Silica Gel 60 F254) and then visualized with a UV254
lamplight. Chromatographic separations were performed on Merck silica gel columns by flash
method (Kieselgel 40, 0.040-0.063 mm, 240 – 400 mesh). Melting points were determined in open
glass capillaries, using a Büchi apparatus and are uncorrected. 1H NMR and 13C NMR spectra were
recorded on a Varian Gemini spectrometer 400 MHz and 101 MHz, respectively, and chemical
shifts (δ) are reported as parts per million (ppm) values relative to tetramethylsilane (TMS) as
internal standard; standard abbreviations indicating spin multiplicities are given as follows: s
(singlet), d (doublet), t (triplet), br (broad), q (quartet) or m (multiplet); coupling constants (J) are
reported in Hertz (Hz). Mass spectra were recorded on a Waters ZQ 4000 apparatus operating in
electrospray mode (ES). Analyses indicated by the symbols of the elements were within ± 0.4 % of
the theoretical values. Compounds were named relying on the naming algorithm developed by
CambridgeSoft Corporation and used in Chem-BioDraw Ultra 14.0.
328
5.2. Williamson Reaction: General Procedure
329
330
331
332
333
A mixture of selected hydroxylated acetophenone (1.0 eq), alkyl halide (1.1-1.5 eq), K2CO3 (1.1
eq) in acetone, was heated for 6-10 h at 80 °C; reaction progress was monitored by TLC. Upon
reaction completion, the mixture was hot filtered and the solvent was evaporated under reduced
pressure. The resulting crude product was purified by column chromatography over a silica gel
using a mixture of petroleum ether/EtOAc as the eluent to give the desired pure product.
13

ACCEPTED MANUSCRIPT
334
335
5.2.1. 1-(2-hydroxy-4-((3-methylbut-2-en-1-yl)oxy)phenyl)ethan-1-one (32) Reaction of 2,4-di-
hydroxyacetophenone (5.0 mmol, 0.75 g) and 3,3-dimethylallyl bromide (5.5 mmol, 0.82 g) gave
336
337
338
339
the crude 32 that was purified by flash chromatography (petroleum ether/EtOAc 9:1), 97% yield,
mp 42-44 °C. 1H-NMR (CDCl3) δ 1.88 (s, 3H, CH3), 1.91 (s, 3H, CH3), 2.55 (s, 3H, COCH3), 4.65
(d, 2 H, J = 6.6 Hz, OCH2), 5.55 (t, 1H, J = 6.6 Hz, CH), 6.44 (d, J = 1.8 Hz, 1H, H-3), 6.54 (dd, J
= 1.8 and 8.6 Hz, 1H, H-5), 7.78 (d, J = 8.6 Hz, 1H, H-6).
340
341
342
343
344
345
5.2.2.
1-(2-hydroxy-4-(prop-2-yn-1-yloxy)phenyl)-1-ethanone
(33).
Reaction
of
2,4-
dihydroxyacetophenone (5.0 mmol, 0.75 g) and propargyl bromide solution 80 wt. % in toluene (5.5
mmol, 0.80 g) gave the crude final product 33 that was purified by flash chromatography
(petroleum ether/EtOAc 7:3), 93% yield, mp 64-66 °C. 1H-NMR (CDCl3) δ 2.55 (s, 3H, CH3), 2.60
(s, 1H, CH), 4.72 (s, 2H, OCH2), 6.44 (d, J = 1.8 Hz, 1H, H-3), 6.55 (dd, J = 1.8 and 8.6 Hz, 1H, H-
5), 7.70 (d, J = 8.6 Hz, 1H, H-6).
346
347
348
349
350
351
5.2.3. 1-(2-methoxy-4-((3-methylbut-2-en-1-yl)oxy)phenyl)ethan-1-one (34). Reaction of 32 (4.2 g,
20.1 mmol) with methyl iodide (4.18 g, 30.25 mmol) gave the crude final product 34 that was
purified by flash chromatography (petroleum ether/EtOAc 9.75:0.25), 93% yield, mp 74-76 °C. 1H-
NMR (CDCl3) δ 1.77 (s, 3H, CH3), 1.81 (s, 3H, CH3), 2.57 (s, 3H, COCH3), 3.00 (s, 3H, OCH3),
4.58 (d, 2 H, J = 6.6 Hz, OCH2), 5.48 (t, 1H, J = 6.6 Hz, CH), 6.42 (d, J = 1.8 Hz, 1H, H-3), 6.53
(dd, J = 1.8 and 8.6 Hz, 1H, H-5), 7.83 (d, J = 8.6 Hz, 1H, H-6).
352
353
354
355
356
5.2.4. 1-(2,4-bis((3-methylbut-2-en-1-yl)oxy)phenyl)ethan-1-one (35). Reaction of 32 (4.2 g, 20.1
mmol) with 3,3-dimethylallyl bromide (4.41 g, 30.25 mmol) gave the crude final product 35 that
was purified by flash chromatography (petroleum ether/EtOAc 9.75:0.25) as transparent oil, 56 %
1
yield. H NMR (CDCl3) δ 1.75 (s, 3H, CH3), 1.80 (s, 3H, CH3), 1.84 (s, 3H, CH3), 1.88 (s, 3H,
CH3), 2.55 (s, 3H, COCH3), 4.50 (d, 2H, J = 6.6 Hz, OCH2), 4.57 (d, 2H, J = 6.6 Hz, OCH2), 5.44
14

ACCEPTED MANUSCRIPT
357
358
(t, 1H, J = 6.6 Hz, CH), 5.60 (t, 1H, J = 6.6 Hz, CH), 6.45 (d, J = 1.8 Hz, 1H, H-3), 6.58 (dd, J =
1.8 and 8.6 Hz, 1H, H-5), 7.81 (d, J = 8.6 Hz, 1H, H-6).
359
360
361
362
363
5.2.5. 1-(2-methoxy-4-(prop-2-yn-1-yloxy)phenyl)ethan-1-one (36). Reaction of 33 (3.16 g, 17.7
mmol) with methyl iodide (3.75 g, 26.55 mmol) gave the crude final product 36 that was purified by
1
flash chromatography (petroleum ether/EtOAc 9.5:0.5), 92%, yield, mp 93-95 °C. H NMR
(CDCl3) δ 2.59-2.61 (m, 4H, COCH3 and CH), 3.95 (s, 3H, OCH3), 4.78 (s, 2H, OCH2), 6.57 (d, J =
1.8 Hz, 1H, H-3), 6.59 (dd, J = 1.8 and 8.6 Hz, 1H, H-5), 7.82 (d, J = 8.6 Hz, 1H, H-6).
364
365
366
367
368
369
5.2.6. 1-(2,4-bis(prop-2-yn-1-yloxy)phenyl)ethan-1-one (37). Reaction of 33 (3.16 g, 17.7 mmol)
with propargyl bromide solution 80 wt. % in toluene (4.97 g, 26.55 mmol) gave the crude final
product 37 that was purified by flash chromatography (petroleum ether/EtOAc 9.5/0.5), 75 % yield,
mp 101-103 °C. 1H NMR (CDCl3) δ 2.59-2.61 (m, 5H, COCH3 and CH2), 4.78 (s, 2H, OCH2), 4.81
(s, 2H, OCH2), 6.57 (d, J = 1.8 Hz, 1H, H-3), 6.57 (dd, J = 1.8 and 8.6 Hz, 1H, H-5), 7.80 (d, J =
8.6 Hz, 1H, H-6).
370
5.3. Claisen–Schmidt reaction: General Procedure
371
372
373
374
375
To an ethanol solution of acetophenone (32-37, 1.0 eq) and the selected benzaldehyde (1.1 eq), a
KOH aqueous solution (50 % p/v, 6 eq) was added dropwise. The reaction mixture was stirred at
room temperature overnight, then diluted with water and acidified with 6N HCl. The separated solid
was collected by vacuum filtration and was purified by flash chromatography using petroleum
ether/EtOAc as eluent. The final products were crystallized from suitable solvent.
376
377
378
379
380
5.3.1. (E)-1-(2-hydroxy-4-((3-methylbut-2-en-1-yl)oxy)phenyl)-3-(pyridin-4-yl)prop-2-en-1-one (1).
Starting from 32 (0.22 g, 1.0 mmol) and 4-pyridinecarboxaldehyde (0.19 g, 1.1 mmol) the crude
final product 1 was obtained and was purified by crystallization from EtOH to obtain a red-orange
solid (0.14 g), 45% yield, mp 102-103 °C. 1H NMR (CDCl3) δ 1.77 (s, 3H, CH3), 1.82 (s, 3H, CH3),
4.59 (d, J = 6.4 Hz, 2H, OCH2), 5.49-5.53 (m, 1H, CH=C), 6.50 (d, J = 1.8 Hz, 1H, H-3), 6.52 (dd,
15

ACCEPTED MANUSCRIPT
381
382
383
384
385
J = 1.8 and J= 8.4 Hz, 1H, H-5), 7.49 (d, J = 4.4 Hz, 2H, H-2’ and H-6’), 7.71 (d, J = 15.6 Hz, 1H,
=CH), 7.73 (d, J = 15.6 Hz, 1H, CH=), 7.81 (d, J = 8.8 Hz, 1H, H-6), 8.70 (d, J = 4.4 Hz, 2H, H-3’
and H-5’). 13C NMR (CDCl3) δ 18.2, 25.7, 65.3, 101.7, 108.6, 113.8, 118.5, 122.0, 124.7, 131.2,
138.5, 141.0, 142.0, 148.8, 149.5, 166.0, 166.9, 192.1. ESI-MS (m/z): 310 (M + H); Anal.
C19H19NO3 (C, H, N).
386
387
388
389
390
391
392
393
394
395
5.3.2. (E)-1-(2-hydroxy-4-((3-methylbut-2-en-1-yl)oxy)phenyl)-3-(pyridin-3-yl)prop-2-en-1-one (2).
Starting from 32 (0.22 g, 1.0 mmol) and 3-pyridinecarboxaldehyde (0.19 g, 1.1 mmol) gave the
crude final product 2 that was purified by crystallization from EtOH to obtain a orange solid (0.21
g), 67% yield, mp 108-109 °C. 1H NMR (CDCl3) δ 1.77 (s, 3H, CH3), 1.82 (s, 3H, CH3), 4.59 (d, J
= 6.4 Hz, 2H, OCH2), 5.49-5.53 (m, 1H, CH=C), 6.51 (d, J = 2.0 Hz 1H, H-3), 6.53 (dd, J = 8.8 and
2.0 Hz, 1H, H-5), 7.32-7.36 (m, 1H, H-5’), 7.62 (d, J = 15.6 Hz, 1H,=CH), 7.82 (d, J = 16.0 Hz, 1H,
CH=), 7.84 (d, J = 8.8 Hz, 1H, H-6’), 7.79 (d, J = 7.6 Hz, 1H, H-6), 8.61 (d, J = 4.8 Hz, 1H, H-4’),
8.94 (s, 1H, H-2’). 13C NMR (CDCl3) δ 18.66, 25.6, 65.2, 102.7, 107.6, 113.8, 119.2, 123.5, 125.7,
131.2, 138.4, 144.0, 144.1, 149.8, 149.5, 166.0, 166.7, 192.8. ESI-MS (m/z): 310 (M + H); Anal.
C19H19NO3 (C, H, N).
396
397
398
399
400
401
402
403
404
405
5.3.3. (E)-1-(2-methoxy-4-((3-methylbut-2-en-1-yl)oxy)phenyl)-3-(pyridin-4-yl)prop-2-en-1-one (3).
Starting from 34 (0.23 g, 1.0 mmol) and 4-pyridinecarboxaldehyde (0.19 g, 1.1 mmol) gave the
crude final product 3 that was purified by flash chromatography (DCM/MeOH 9.75:0.25) and then
1
crystallized from AcOEt/n-hexane to obtain a solid(0.09 g), 30% yield, mp 102-103 °C. H NMR
(CDCl3) δ 1.79 (s, 3H, CH3), 1.82 (s, 3H, CH3), 3.96 (s, 3H, OCH3), 4.51 (d, J = 6.4 Hz, 2H,
OCH2), 5.44-5.51 (m, 1H, CH=C), 6.30 (d, J = 1.8 Hz, 1H, H-3), 6.42 (dd, J = 1.8 and 8.4 Hz, 1H,
H-5), 7.49 (d, J = 4.4 Hz, 2H, H-2’ and H-6’), 7.70 (d, J = 15.6 Hz, 1H, =CH), 7.72 (d, J = 15.6 Hz,
13
1H, CH=), 7.80 (d, J = 8.8 Hz, 1H, H-6), 8.65 (d, J = 4.4 Hz, 2H, H-3’ and H-5’). C NMR
(CDCl3) δ 18.5, 24.5, 55.2, 64.3, 101.9, 107.6, 113.8, 119.1, 123.0, 125.7, 131.2, 138.5, 144.1,
144.6, 148.8, 149.3, 162.1, 166.5, 192.5. ESI-MS (m/z): 324 (M + H); Anal. C20H21NO3 (C, H, N).
16

ACCEPTED MANUSCRIPT
406
407
408
409
410
411
412
413
414
415
416
5.3.4. (E)-1-(2-methoxy-4-((3-methylbut-2-en-1-yl)oxy)phenyl)-3-(pyridin-3-yl)prop-2-en-1-one (4).
Starting from 34 (0.23 g, 1.0 mmol) and 3-pyridinecarboxaldehyde (0.19 g, 1.1 mmol) gave the
crude final product 4 that was purified by flash chromatography (DCM/MeOH 9.75:0.25) and then
crystallized from AcOEt/n-hexane to obtain an orange solid (0.14 g), 45% yield, mp 98-100 °C. 1H
NMR (CDCl3) δ 1.78 (s, 3H, CH3), 1.83 (s, 3H, CH3), 3.93 (s, 3H, OCH3), 4.78 (d, J = 4 Hz, 2H,
OCH2) 5.49-5.53 (m, 1H, CH=C), 6.61 (s, 1H, H-3), 6.66 (d, J = 8.8 Hz, 1H, H-5) 7.34-7.37 (m,
1H, H-5’), 7.56 (d, J = 15.6 Hz, 1H,=CH), 7.62 (d, J = 16 Hz, 1H, CH= ), 7.80 (d, J = 8.8 Hz, 1H,
13
H-6’), 7.89 (d, J = 7.6 Hz, 1H, H-6), 8.60 (d, J = 4.8 Hz, 1H, H-4’), 8.84 (s, 1H, H-2’). C NMR
(CDCl3) δ 18.4, 24.7, 55.8, 64.9, 101.5, 107.6, 119.3, 123.5, 127.1, 129.7, 131.3, 132.7, 138.4,
141.1, 148.0, 148.6, 149.3, 163.3, 168.3, 192.2. ESI-MS (m/z): 324 (M + H); Anal. C20H21NO3 (C,
H, N).
417
418
419
420
421
422
423
424
425
426
5.3.5. (E)-3-(4-bromophenyl)-1-(2-methoxy-4-((3-methylbut-2-en-1-yl)oxy)phenyl)prop-2-en-1-one
(5). Starting from 34 (0.23 g, 1.0 mmol) and 4-bromobenzaldehyde (0.20 g, 1.1 mmol) gave the
crude final product 5 that was purified by flash chromatography (petroleum ether/AcOEt 4:1) and
then crystallized from AcOEt/n-hexane to obtain a yellow solid (0.32 g), 81% yield, mp 96-98 °C.
1H NMR (CDCl3) δ 1.78 (s, 3H, CH3), 1.83 (s, 3H, CH3), 3.91 (s, 3H, OCH3), 4.59 (d, J = 6.4 Hz,
2H, OCH2), 5.49-5.53 (m, 1H, CH=C), 6.64 (d, J = 2.0 Hz, 1H, H-3), 6.72 (dd, J = 2.0 and 8.8 Hz,
1H, H-5), 7.62 (d, J = 15.6 Hz, 1H, =CH), 7.66 (d, J = 15.8 Hz, 1H, CH= ), 7.76 (d, J = 8.8 Hz, 2H,
13
H-2’ and H-6’), 7.80 (d, J = 8.4 Hz, 1H, H-6), 8.24 (d, J = 8.8 Hz, 2H, H-3’ and H-5’). C NMR
(CDCl3) δ 18.3, 24.9, 54.2, 65.1, 100.3, 106.6, 118.7, 119.9, 123.4, 128.2, 128.4, 131.8, 133.5,
138.9, 145.4, 162.4, 168.8, 191.2. ESI-MS (m/z): 402 (M + H); Anal. C21H21BrO3 (C, H,).
427
428
429
430
5.3.6. (E)-1-(2-methoxy-4-((3-methylbut-2-en-1-yl)oxy)phenyl)-3-(4-nitrophenyl)prop-2-en-1-one
(6). Starting from 34 (0.23 g, 1.0 mmol) and 4-nitrobenzaldehyde (0.17 g, 1.1 mmol) gave the crude
final product 6 that was purified by crystallization from EtOH to obtain a yellow solid (0.28 g),
1
77% yield, mp 147-147 °C. H NMR (CDCl3) δ 1.77 (s, 3H, CH3), 1.82 (s, 3H, CH3), 3.93 (s, 3H,
17

ACCEPTED MANUSCRIPT
431
432
433
434
435
436
OCH3), 4.57 (d, J = 6.4 Hz, 2H, OCH2), 5.49-5.53 (m, 1H, CH=C), 6.52 (d, J = 1.6 Hz, 1H, H-3),
6.58 (dd, J = 1.6 and 8.0 Hz, 1H, H-5), 7.68 (d, J = 16.0 Hz, 1H, =CH), 7.59 (d, J = 16.0 Hz, 1H,
CH=), 7.72 (d, J = 8.4 Hz, 1H, H-2’ and H-6’), 7.81 (d, J = 8.4 Hz, 1H, H-6), 8.26 (d, J = 8.4 Hz,
13
1H, H-3’ and H-5’). C NMR (CDCl3) δ 18.2, 25.8, 55.7, 65.1, 105.8, 118.9, 121.3, 124.1, 128.6,
131.0, 133.2, 138.2, 141.9, 160.7, 164.2, 183.2. ESI-MS (m/z): 368 (M + H); Anal. C21H21NO5 (C,
H, N).
437
438
439
440
441
442
443
444
445
446
5.3.7.
(E)-1-(2-methoxy-4-((3-methylbut-2-en-1-yl)oxy)phenyl)-3-phenylprop-2-en-1-one
(7).
Starting from 34 (0.23 g, 1.0 mmol) and benzaldehyde (0.11 g, 1.1 mmol) gave the crude final
product 7 that was purified by flash chromatography (petroleum ether/AcOEt 9.5:0.5) to obtain a
1
yellow solid (0.14 g), 42% yield, mp 147-149 °C. H NMR (CDCl3) δ 1.78 (s, 3H, CH3), 1.82 (s,
3H, CH3), 3.90 (s, 3H, OCH3), 4.58 (d, J = 6.4 Hz, 2H, OCH2), 5.49-5.53 (m, 1H, CH=C), 6.53 (d, J
= 1.6 Hz, 1H, H-3), 6.57 (dd, J = 1.6 and 8.0 Hz, 1H, H-5), 7.30-7.43 (m, 3H, H-3’-H-5’), 7.53 (d, J
= 15.6 Hz, 1H, CH=), 7.61-7.67 (m, 2H, H-2’ and H-6’), 7.68 (d, J = 15.6 Hz, 1H, CH=), 7.77 (d, J
13
= 8.4 Hz, 1H, H-6). C NMR (CDCl3) δ 18.2, 25.8, 55.7, 65.0, 99.3, 105.8, 118.9, 122.0, 127.2,
128.2, 128.8, 129.9, 132.8, 135.5, 141.9, 160.4, 163.5, 190.6. ESI-MS (m/z): 323 (M + H); Anal.
C20H22O3 (C, H).
447
448
449
450
451
452
453
454
5.3.8. (E)-1-(2-methoxy-4-((3-methylbut-2-en-1-yl)oxy)phenyl)-3-(3,4,5-trimethoxyphenyl)prop-2-
en-1-one (8). Starting from 34 (0.23 g, 1.0 mmol) and 3,4,5-trimethoxybenzaldehyde (0.21 g, 1.1
mmol) gave the crude final product 8 that was purified by crystallization from EtOH to obtain a
yellow solid (0.29 g), 71% yield, mp 62-64 °C. 1H NMR (CDCl3) δ 1.78 (s, 3H, CH3), 1.82 (s, 3H,
CH3), 3.90 (s, 3H, OCH3), 3.89 (s, 9H, OCH3), 4.57 (d, J = 6.4 Hz, 2H, OCH2), 5.49-5.53 (m, 1H,
CH), 6.52 (d, J = 1.6 Hz, 1H, H-3), 6.57 (dd, J = 1.6 and 8.0 Hz, 1H, H-5), 6.81 (s, 2H, H-2’ and H-
6’), 7.34 (d, J = 15.6 Hz, 1H, CH=), 7.54 (d, J = 15.6 Hz, 1H, CH=), 7.70 (d, J = 8.4 Hz, 1H, H-6).
13C NMR (CDCl3) δ 18.7, 25.7, 55.6, 56.7, 56.9, 60.5, 63.8, 99.9, 103.8, 107.4, 118.5, 118.9, 123.0,
18

ACCEPTED MANUSCRIPT
455
456
126.6, 131.3, 132.8, 138.5, 138.7, 145.9, 153.1, 153.8, 162.2, 168.8, 190.6. ESI-MS (m/z): 413 (M +
H); Anal. C24H28O6 (C, H).
457
458
459
460
461
462
463
464
465
5.3.9. (E)-1-(2-hydroxy-4-(prop-2-yn-1-yloxy)phenyl)-3-(pyridin-3-yl)prop-2-en-1-one (9). Starting
from 33 (0.19 g, 1.0 mmol) and 3-pyridinecarboxaldehyde (0.19 g, 1.1 mmol) gave the crude final
product 9 that was purified by crystallization from EtOH to obtain an orange solid (0.22 g), 79%
1
yield, mp 122-124 °C. H NMR (CDCl3) δ 2.58 (s, 1H, C≡CH), 4.77 (d, J = 1.6 Hz, 2H, OCH2),
6.61 (d, J = 1.8 Hz, 1H, H-3), 6.66 (dd, J = 8.8 and 1.8 Hz, 1H, H-5), 7.34-7.37 (m, 1H, H-5’), 7.56
(d, J = 15.6 Hz, 1H, =CH), 7.64 (d, J = 15.6 Hz, 1H, CH=), 7.81 (d, J = 8.4 Hz, 1H, H-6’), 7.89 (d,
J = 4.8 Hz, 1H, H-6), 8.60 (d, J = 4.8 Hz, 1H, H-4’), 8.84 (s, 1H, H-2’). 13C NMR (CDCl3) δ 55.8,
76.3, 76.8, 101.6, 106.7, 127.6, 129.6, 131.3, 132.6, 133.8, 134.9, 145.6, 147.9, 149.8, 160.3, 162.4,
190.1. ESI-MS (m/z): 280 (M + H); Anal. C17H13NO3 (C, H, N).
466
467
468
469
470
471
472
473
474
475
5.3.10.
(E)-1-(2-methoxy-4-(prop-2-yn-1-yloxy)phenyl)-3-(pyridin-4-yl)prop-2-en-1-one
(10).
Starting from 36 (0.20 g, 1.0 mmol) and 4-pyridinecarboxaldehyde (0.19 g, 1.1 mmol) gave the
crude final product 10 that was purified by flash chromatography (DCM/MeOH 9.75:0.25) and then
1
crystallized from ethanol to obtain a brown solid (0.11 g), 36% yield, mp 98-100 °C. H NMR
(CDCl3): δ 2.56 (s, 1H, C≡CH), 3.91 (s, 3H, OCH3), 4.75 (d, J = 2.0 Hz, 2H, OCH2), 6.58 (d, J =
1.6 Hz, 1H, H-3), 6.64 (dd, J = 2 and 8.4 Hz, 1H, H-5), 7.40 (d, J = 5.6 Hz, 2H, H-2’ and H-6’),
7.54 (d, J = 16.0 Hz, 1H, =CH), 7.65 (d, J = 16.0 Hz, 1H, CH=), 7.79 (d, J = 8.4 Hz, 1H, H-6), 8.63
(d, J = 5.6 Hz, 2H, H-3’ and H-5’). 13C NMR (CDCl3) δ 55.7, 56.6, 76.3, 77.7, 102.5, 107.7, 113.8,
118.2, 123.5, 125.7, 130.2, 138.7, 144.5, 146.3, 149.9, 150.5, 166.0, 166.7, 192.6. ESI-MS (m/z):
294 (M + H); Anal. C18H15NO3 (C, H, N).
476
477
478
5.3.11.
(E)-1-(2-methoxy-4-(prop-2-yn-1-yloxy)phenyl)-3-(pyridin-3-yl)prop-2-en-1-one
(11).
Starting from 36 (0.20 g, 1.0 mmol) and 3-pyridinecarboxaldehyde (0.19 g, 1.1 mmol) gave the
crude final product 11 that was purified by flash chromatography (DCM/MeOH 9.75:0.25) and then
19

ACCEPTED MANUSCRIPT
1
479
480
481
482
483
484
485
crystallized from ethanol to obtain a yellow solid (0.21 g), 72% yield, mp 120-122 °C. H NMR
(CDCl3): δ 2.58 (s, 1H, C≡CH), 3.93 (s, 3H, OCH3), 4.78 (d, J = 1.6 Hz, 2H, OCH2), 6.62 (s, 1H,
H-3), 6.66 (d, J = 8.8 Hz, 1H, H-5), 7.32-7.36 (m, 1H, H-5’), 7.56 (d, J = 15.6 Hz, 1H, =CH), 7.62
(d, J = 16 Hz, 1H, CH= ), 7.81 (d, J = 8.8 Hz, 1H, H-6’), 7.89 (d, J = 8 Hz, 1H, H-6), 8.61 (d, J =
13
4.4 Hz, 1H, H-4’), 8.84 (s, 1H, H-2’). C NMR (CDCl3) δ 55.7, 56.6, 76.3, 77.7, 101.7, 108.5,
113.3, 122.3, 125.7, 131.2, 136.5, 141.0, 142.0, 148.8, 149.5, 151.2, 166.5, 166.9, 192.0. ESI-MS
(m/z): 294 (M + H); Anal. C18H15NO3 (C, H, N).
486
487
488
489
490
491
492
493
494
495
5.3.12. (E)-3-(4-bromophenyl)-1-(2-methoxy-4-(prop-2-yn-1-yloxy)phenyl)prop-2-en-1-one (12).
Starting from 36 (0.20 g, 1.0 mmol) and 4-bromobenzaldheyde (0.20 g, 1.1 mmol) gave the crude
final product 12 that was purified by flash chromatography (petroleum ether/EtOAc 9:1) and then
1
crystallized from ethanol to obtain a yellow solid (0.35 g), 95% yield, mp 131-133 °C. H NMR
(CDCl3) δ 2.59 (t, J = 2.4 Hz, 1H, C≡CH), 3.92 (s, 3H, OCH3), 4.77 (d, J = 1.6 Hz, 2H, OCH2),
6.61 (s, 1H, H-3), 6.65 (d, J = 8.4 Hz, 1H, H-5), 7.49 (d, J = 17.2 Hz, 1H, =CH), 7.53 (d, J = 8.4
Hz, 2H, H-2’ and H-6’) 7.61 (d, J = 15.6 Hz, 1H, CH=), 7.77 (d, J = 8.4 Hz, 1H, H-6) 8.26 (d, J =
13
8.8 Hz, 2H, H-3’ and H-5’). C NMR (CDCl3) δ 55.9, 56.6, 76.2, 77.9, 99.7, 106.2, 122.3, 124.9,
127.1, 129.6, 130.0, 132.1, 132.0, 134.9, 140.6, 160.8, 162.0, 190.2. ESI-MS (m/z): 372 (M + H);
Anal. C19H15BrO3 (C, H).
496
497
498
499
500
501
502
5.3.13. (E)-1-(2,4-bis(prop-2-yn-1-yloxy)phenyl)-3-(pyridin-4-yl)prop-2-en-1-one (13). Starting
from 37 (0.23 g, 1.0 mmol) and 4-pyridinecarboxaldehyde (0.19 g, 1.1 mmol) gave the crude final
product 13 that was purified by crystallization from EtOH to obtain a yellow solid (0.08 g), 27%
1
yield, mp 156-158 °C. H NMR (CDCl3) δ 2.58-2.60 (m, 2H, C≡CH), 4.78 (d, J = 2.0 Hz, 2H,
OCH2) 4.80 (d, J = 2.4 Hz, 2H, OCH2), 6.69 (d, J = 1.6 Hz, 1H, H-3), 6.72 (dd, J = 2.0 and 8.4 Hz,
1H, H-5), 7.46 (d, J = 5.2 Hz, 2H, H-2’ and H-6’), 7.57 (d, J = 15.6 Hz, 1H, =CH), 7.73 (d, J = 15.6
13
Hz, 1H, CH=), 7.84 (d, J = 8.8 Hz, 1H, H-6), 8.66 (d, J = 4.4 Hz, 2H, H-3’ and H-5’). C NMR
20

ACCEPTED MANUSCRIPT
503
504
(CDCl3) δ 55.9, 56.0, 76.2, 77.9, 100.7, 107.6, 113.8, 118.4, 122.4, 124.7, 131.2, 138.5, 140.9,
142.0, 150.5, 166.7, 167.9, 192.1. ESI-MS (m/z): 318 (M + H); Anal. C20H15NO3 (C, H, N).
505
506
507
508
509
510
511
512
513
514
5.3.14. (E)-1-(2,4-bis(prop-2-yn-1-yloxy)phenyl)-3-(pyridin-3-yl)prop-2-en-1-one (14). Starting
from 37 (0.23 g, 1.0 mmol) and 3-pyridinecarboxaldehyde (0.19 g, 1.1 mmol) gave the crude final
product 14 that was purified by crystallization from ethanol to obtain a yellow solid (0.23 g), 72%
1
yield, mp 156-158 °C. H NMR (CDCl3): δ 2.58-2.60 (m, 2H, C≡CH), 4.78 (d, J = 2.0 Hz, 2H,
OCH2), 4.80 (d, J = 2.4 Hz, 2H, OCH2), 6.51 (s, 1H, H-3), 6.53 (d, J = 8.8 Hz, 1H, H-5), 7.32-7.36
(m, 1H, H-5’), 7.62 (d, J = 15.6 Hz, 1H, =CH), 7.82 (d, J = 16 Hz, 1H, CH=), 7.84 (d, J = 8.8 Hz,
13
1H, H-6’), 7.79 (d, J = 7.6 Hz, 1H, H-6), 8.61 (d, J = 4.8 Hz, 1H, H-4’), 8.94 (s, 1H, H-2’). C
NMR (CDCl3) δ 56.3, 56.9, 76.4, 76.7, 77.9, 101.1, 107.2, 123.4, 127.6, 128.5, 130.5, 131.6, 131.7,
132.9, 141.0, 158.0, 159.2, 160.7, 165.0, 190.0. ESI-MS (m/z):318 (M + H); Anal. C20H15NO3 (C,
H, N).
515
516
5.3.15. (E)-1-(2,4-bis(prop-2-yn-1-yloxy)phenyl)-3-(4-bromophenyl)prop-2-en-1-one (15)
Starting from 37 (0.23 g, 1.0 mmol) and 4-bromobenzaldheyde (0.20 g, 1.1 mmol) gave the crude
517
518
519
520
521
522
523
final product 15 that was purified by flash chromatography (petroleum ether/EtOAc 9:1) and then
1
crystallized from EtOH to obtain a yellow solid (0.37 g), 95% yield, mp 123-125 °C. H-NMR
(CDCl3) δ 2.57-2.59 (m, 2H, 2 C≡CH), 4.78 (d, J = 8.8 Hz, 4H, 2 OCH2), 6.70 (s, 1H, H-3), 6.73 (d,
J = 8.4 Hz, 1H, H-5), 7.46-7.54 (m, 4H, H-2’-H-6’), 7.56 (d, J = 15.6 Hz, 1H,=CH), 7.62 (d, J = 16
13
Hz, 1H, CH=), 7.80 (d, J = 8.4 Hz, 1H, H-6). C NMR (CDCl3) δ 55.8, 56.0, 76.1, 76.3, 99.6,
106.1, 122.8, 124.2, 127.6, 129.6, 131.0, 132.1, 132.8, 134.9, 140.6, 160.3, 162.1, 190.1. ESI-MS
(m/z): 396 (M + H); Anal. C21H15BrO3 (C, H).
524
525
526
5.3.16. (E)-1-(2,4-bis(prop-2-yn-1-yloxy)phenyl)-3-(4-fluorophenyl)prop-2-en-1-one (16). Starting
from 37 (0.23 g, 1.0 mmol) and 4-fluorobenzaldheyde (0.14 g, 1.1 mmol) gave the crude final
product 16 that was purified by flash chromatography (petroleum ether/EtOAc 4:1) and then
21

ACCEPTED MANUSCRIPT
1
527
528
529
530
531
532
533
crystallized from EtOH to obtain a yellow solid (0.30 g), 91% yield, mp 159-161 °C. H NMR
(CDCl3) δ 2.57-2.59 (m, 2H, 2 C≡CH), 4.77 (d, J = 2.4 Hz, 2H, OCH2), 4.79 (d. J = 2.4 Hz, 2H,
OCH2) 6.71 (s, 1H, H-3), 6.72 (d, J = 2.4 Hz, 1H, H-5), 7.06-7.11 (m, 2H, H-2’ and H-6’), 7.48 (d,
J = 15.6 Hz, 1H, =CH), 7.59-7.62 (m, 2H, H-3’ and H-5’), 7.65 (d, J = 16 Hz, 1H, CH=), 7.79 (d, J
= 9.6 Hz, 1H, H-6). 13C NMR (CDCl3) δ 56.0, 56.6, 76.2, 76.3, 77.7, 77.8, 101.1, 107.2, 115.8 (d, J
= 22 Hz), 123.4, 126.8, 126.9, 130.1 (d, J = 8.5 Hz), 131.6, 131.7, 132.9, 141.0, 158.1, 161.7, 165.0,
190.0. ESI-MS (m/z): 335 (M + H); Anal. C21H15FO3 (C, H, N).
534
535
536
537
538
539
540
541
542
543
5.3.17.
(E)-N-(4-(3-(2-methoxy-4-((3-methylbut-2-en-1-yl)oxy)phenyl)-3-oxoprop-1-en-1-
yl)phenyl)acetamide (17). Starting from 34 (0.23 g, 1.0 mmol) and 4-acetamidobenzaldehyde (0.18
g, 1.1 mmol) gave the crude final product 17 that was purified by crystallization from EtOH to
1
obtain a yellow solid (0.12 g), 58% yield, mp 131-133 °C. H NMR (CDCl3) δ 1.78 (s, 3H, CH3),
1.82 (s, 3H, CH3), 2.21 (s, 3H, COCH3), 3.90 (s, 3H, OCH3), 4.57 (d, J = 6.4 Hz, 2H, OCH2), 5.49-
5.54 (m, 1H, CH), 6.52 (d, J = 2.0 Hz, 1H, H-3), 6.57 (dd, J = 8.0 and 2.0 Hz, 1H, H-5), 7.23 (br,
1H, NH), 7.46 (d, J = 16.0 Hz, 1H, =CH), 7.53 (d, J = 8.0 Hz, 2H, H-2’ and H-6’), 7.57 (d, J = 8.0
13
Hz, 2H, H-3’ and H-5’), 7.64 (d, J = 16.0 Hz, 1H, CH=), 7.75 (d, J = 8.2 Hz, 1H, H-6). C NMR
(CDCl3) δ 18.2, 23.3, 25.8, 55.7, 65.1, 105.8, 118.9, 121.3, 124.5, 129.6, 131.0, 134.2, 138.6, 145.9,
161.5, 163.2, 169.9, 188.2. ESI-MS (m/z): 380 (M + H); Anal. C19H20O4 (C, H).
544
545
546
547
548
549
550
551
5.3.18.
(E)-3-(furan-2-yl)-1-(2-methoxy-4-((3-methylbut-2-en-1-yl)oxy)phenyl)prop-2-en-1-one
(18). Starting from 34 (0.23 g, 1.0 mmol) and 2-furaldehyde (0.11 g, 1.1 mmol) gave the crude final
product 18 that was purified by purified by flash chromatography (petroleum ether/EtOAc 4:1) to
1
obtain a brown oil (0.16 g), 51% yield. H NMR (CDCl3) δ 1.77 (s, 3H, CH3), 1.82 (s, 3H, CH3),
3.90 (s, 3H, OCH3), 4.58 (d, J = 6.4 Hz, 2H, OCH2), 5.47-5.50 (m, 1H, CH), 6.48-6.51(m, 2H,
furfuryl), 6.56 (d, J = 1.8 Hz, 1H, H-3), 6.64 (dd, J = 1.8 and 8.4 Hz, 1H, H-5), 7.26-7.27 (m, 1H,
furfuryl), 7.42 (d, J = 15.6 Hz, 1H, =CH), 7.48 (d, J = 15.6 Hz, 1H, CH=), 7.76 (d, J = 8.2 Hz, 1H,
13
H-6). C NMR (CDCl3) δ 18.2, 24.3, 55.7, 64.2, 101.2, 107.9, 112.5, 113.9, 119.5, 120.8, 123.4,
22

ACCEPTED MANUSCRIPT
552
553
131.6, 138.9, 143.2, 152.5, 163.4, 168.8, 191.5. ESI-MS (m/z): 313 (M + H); Anal. C20H15NO3 (C,
H, N).
554
555
556
557
558
559
560
561
562
563
5.3.19. (E)-1-(2-hydroxy-4-((3-methylbut-2-en-1-yl)oxy)phenyl)-3-phenylprop-2-en-1-one (19) [34].
Reaction of 32 (1.0 mmol, 0.17 g) and benzaldehyde (1.1 mmol, 0.12 g) gave the crude 19 that was
purified by crystallization from EtOH to obtain a yellow solid (0.2 g), 64% yield, mp 92-95 °C. 1H
NMR (CDCl3) δ 1.76 (s, 3H, CH3), 1.81 (s, 3H, CH3), 4.56 (d, J = 6.0 Hz, 2H, OCH2), 5.47 (t, J =
6.0 Hz, 1H, CH), 6.49 (dd, J = 2.0 and 8.4 Hz, 1H, H-5), 6.52 (d, J = 1.8 Hz, 1H, H-3), 7.43-7.55
(m, 3H, H-3’-H-5’), 7.60 (d, J = 15.6 Hz, 1H, =CH), 7.63-7.65 (m, 2H, H-2’and H-6’), 7.82 (d, J =
13
8.4 Hz, 1H, H-6), 7.89 (d, J = 15.3 Hz, 1H, CH=), 10.45 (br, 1H, OH). C NMR (CDCl3) δ 18.6,
19.5, 65.0, 101.8, 108.2, 113.9, 118.6, 120.5, 128.4, 128.8, 130.7, 130.5, 130.0, 134.2, 138.6, 143.7,
165.0, 166.1, 191.7, (In accordance with previously published spectroscopic data). MS (ESI+) m/z:
309 (M + H); Anal. C20H20O3 (C, H).
564
565
566
567
568
569
570
571
572
573
574
575
576
5.3.20. (E)-1-(2,4-bis((3-methylbut-2-en-1-yl)oxy)phenyl)-3-phenylprop-2-en-1-one (21). Reaction
of 35 (1.0 mmol, 0.29 g) and benzaldehyde (1.1 mmol, 0.12 g) gave the crude 21 that was purified
by flash chromatography (petroleum ether/EtOAc 9.75:0.25) to obtain a white oil (0.04 g), 10%
1
yield. H NMR (CDCl3) δ 1.76 (s, 3H, CH3), 1.82 (s, 3H, CH3), 1.85 (s, 3H, CH3), 1.89 (s, 3H,
CH3), 4.51 (d, J = 6.0 Hz, 2H, OCH2), 4.58 (d, J = 6.0 Hz, 2H, OCH2), 5.47-5.61 (m, 2H, CH), 6.47
(d, J = 2.0, 1H, H-3), 6.61 (dd, J = 2.0 and 8.4 Hz, 1H, H-5), 7.38-7.42 (m, 3H, H-3’-H-5’), 7.63-
7.67 (m, 2H, H-2’and H-6’), 7.69 (d, J = 15.6 Hz, 1H, =C), 7.70 (d, J = 8.4 Hz, 1H, H-6), 7.81 (d, J
= 15.3 Hz, 1H, C=). 13C NMR (CDCl3) δ 18.6, 18.7, 20.5, 20.7, 24.5, 24.8, 65.1, 65.4, 101.8, 107.3,
118.6, 119.6, 123.5, 128.1, 128.4, 128.6, 135.5, 138.0, 145.2, 162.4, 168.7, 191.9. ESI-MS (m/z):
378 (M + H); Anal. C25H28O3 (C, H).
5.3.21. (E)-3-(4-fluorophenyl)-1-(2-methoxy-4-((3-methylbut-2-en-1-yl)oxy)phenyl)prop-2-en-1-one
(22). Starting from 34 (0.23 g, 1.0 mmol) and 4-fluorobenzaldheyde (0.14 g, 1.1 mmol) gave the
crude final product 22 that was purified by flash chromatography (petroleum ether/EtOAc 9:1) and
23

ACCEPTED MANUSCRIPT
1
577
578
579
580
581
582
583
then crystallized from EtOH to obtain a white solid (0.12 g), 35% yield, mp 81-83 °C. H NMR
(CDCl3) δ 1.78 (s, 3H, CH3), 1.83 (s, 3H, CH3), 3.91 (s, 3H, OCH3), 4.59 (d, J =6.4 Hz, 2H, OCH2),
5.49-5.53 (m, 1H, CH), 6.53 (d, J = 1.6 Hz, 1H, H-3), 6.58 (dd, J = 1.6 Hz and 8.0 Hz, 1H, H-5),
7.06-7.11 (m, 2H, H-2’ and H-6’), 7.46 (d, J = 16.0 Hz, 1H, =CH), 7.57-7.60 (m, 2H, H-3’ and H-
5’), 7.65 (d, J = 16.0 Hz, 1H, CH=), 7.76 (d, J = 8.8 Hz, 1H, H-6). 13C NMR (CDCl3) δ 18.2, 25.8,
55.7, 65.1, 103.8, 107.4, 118.9, 119.3 (d, J = 22 Hz), 124.6, 130.0 (d, J = 8.5 Hz), 131.2, 138.4,
145.9, 162.7, 168.2, 193.4. ESI-MS (m/z): 341 (M + H); Anal. C21H21FO3 (C, H).
584
585
586
587
588
589
590
591
592
593
5.3.22. (E)-1-(2-methoxy-4-((3-methylbut-2-en-1-yl)oxy)phenyl)-3-(4-methoxyphenyl)prop-2-en-1-
one (23). Starting from 34 (0.23 g, 1.0 mmol) 4-methoxybenzaldheyde (0.14 g, 1.1 mmol) gave the
crude final product 23 that was purified by flash chromatography (petroleum ether/EtOAc 4:1) and
1
then crystallized from EtOH to obtain a yellow solid (0.23 g), 66% yield, mp 62-64 °C. H NMR
(CDCl3) δ 1.78 (s, 3H, CH3), 1.83 (s, 3H, CH3), 3.86 (s, 3H, OCH3), 3.90 (s, 3H, OCH3), 4.58 (d, J
= 7.2 Hz, 2H, OCH2), 5.49-5.53 (m, 1H, CH), 6.53 (d, J = 2.0 Hz, 1H, H-3), 6.57 (dd, J = 2.0 Hz
and 8.8 Hz, 1H, H-5), 6.90-6.95 (m, 2H, H-3’ and H-5’), 7.40 (d, J = 16.0 Hz, 1H, =CH), 7.54-7.57
13
(m, 2H, H-2’ and H-6’), 7.66 (d, J = 15.6 Hz, 1H, CH=), 7.74 (d, J = 8.8 Hz, 1H, H-6). C NMR
(CDCl3) δ 18.2, 24.8, 55.7, 64.8, 64.0, 100.9, 107.8, 118.9, 122.5, 126.2, 127.8, 128.7, 132.8, 135.5,
141.9, 159.4, 160.4, 162.5, 190.1. ESI-MS (m/z): 353 (M + H); Anal. C22H24O4 (C, H).
594
595
596
597
598
599
600
601
5.3.23. (E)-3-(3,4-dimethoxyphenyl)-1-(2-methoxy-4-((3-methylbut-2-en-1-yl)oxy)phenyl)prop-2-en-
1-one (24). Starting from 34 (0.23 g, 1.0 mmol) and 3,4-dimethoxybenzaldheyde (0.18 g, 1.1 mmol)
gave the crude final product 24 that was purified by flash chromatography (petroleum ether/EtOAc
4:1) and then crystallized from DCM/petroleum ether to obtain a yellow solid (0.29 g), 77% yield,
mp 83-85 °C. 1H NMR (CDCl3) δ 1.78 (s, 3H, CH3), 1.83 (s, 3H, CH3), 3.90 (s, 6H, OCH3), 3.93 (s,
3H, OCH3), 4.59 (d, J = 7.4 Hz, 2H, OCH2), 5.48-5.52 (m, 1H, CH), 6.54 (s, 1H, H-3), 6.58 (d, J =
8.4 Hz, 1H, H-5), 6.89 (d, J = 8.4 Hz, 1H, H-5’), 7.13 (d, J = 2.1, 1H, H-2’), 7.20 (dd, J = 8.4 and
2.1 Hz, 1H, H-6’), 7.37 (d, J = 15.6 Hz, 1H, =CH), 7.63 (d, J = 15.6 Hz, 1H, CH=), 7.73 (d, J = 8.8
24

ACCEPTED MANUSCRIPT
13
602
603
604
Hz, 1H, H-6). C NMR (CDCl3) δ 18.2, 24.8, 55.7, 56.2, 64.8, 64.0, 100.9, 107.8, 118.5, 122.5,
125.3, 127.8, 132.1, 134.4, 141.9, 159.4, 160.4, 161.4, 162.5, 190.4. ESI-MS (m/z): 383 (M + H)¸
Anal. C23H26O5 (C, H).
605
606
607
608
609
610
611
612
613
5.3.24. (E)-1-(2-methoxy-4-(prop-2-yn-1-yloxy)phenyl)-3-(4-nitrophenyl)prop-2-en-1-one (25).
Starting from 36 (0.20 g, 1.0 mmol) and 4-nitrobenzaldheyde (0.17 g, 1.1 mmol) gave the crude
final product 25 that was purified by crystallization from ethanol to obtain a white solid (0.22 g),
67% yield, mp 178-180°C. 1H NMR (CDCl3) δ 2.59 (t, J = 2.4 Hz, 1H, C≡CH), 3.94 (s, 3H, OCH3),
4.78 (d, J = 2.4 Hz, 2H, OCH2), 6.61 (d, J = 2.0 Hz, 1H, H-3), 6.67 (dd, J = 2.0 and 8.4 Hz, 1H, H-
5), 7.64 (d, J = 16.0 Hz, 1H, =CH), 7.70 (d, J = 16.0 Hz, 1H, CH=), 7.73 (d, J = 8.4 Hz, 2H, H-2’
13
and H-6’), 7.82 (d, J = 8.4 Hz, 1H, H-6), 8.26 (d, J = 8.8 Hz, 2H, H-3’ and H-5’). C NMR
(CDCl3) δ 55.7, 56.5, 77.7, 105.8, 121.3, 124.1, 128.6, 131.0, 133.2, 138.2, 160.7, 164.2, 183.2.
ESI-MS (m/z): 338 (M + H); Anal. C19H15NO5 (C, H, N).
614
615
616
617
618
619
620
621
622
623
5.3.25. (E)-1-(2,4-bis(prop-2-yn-1-yloxy)phenyl)-3-(4-nitrophenyl)prop-2-en-1-one (26). Starting
from 37 (0.23 g, 1.0 mmol) and 4-nitrobenzaldheyde (0.17 g, 1.1 mmol) gave the crude final
product 26 that was purified by flash chromatography (petroleum ether/EtOAc 9:1) and then
1
crystallized from ethanol to obtain a yellow solid (0.37 g), 95% yield, mp 186-188 °C. H NMR
(CDCl3) δ 2.58-2.60 (m, 2H, C≡CH), 4.75 (d, J = 2.4 Hz, 2H, OCH2), 4.78 (d, J = 2.4 Hz, 2H,
OCH2) 6.66 (d, J = 2.0 Hz, 1H, H-3), 6.70 (dd, J = 2.0 and 8.8 Hz, 1H, H-5), 7.60 (d, J = 15.6 Hz,
1H,=CH), 7.66 (d, J = 16 Hz, 1H, CH= ), 7.74 (d, J = 8.8 Hz, 2H, H-2’ and H-6’), 7.83 (d, J = 8.4
Hz, 1H, H-6), 8.23 (d, J = 8.8 Hz, 2H, H-3’ and H-5’). 13C NMR (CDCl3) δ 55.7, 55.9, 76.2, 76.6,
77.7, 77.8, 101.8, 107.3, 123.1, 127.7, 130.5, 133.2, 138.2, 162.8, 164.4, 187.2. ESI-MS (m/z): 362
(M + H) Anal. C21H15NO5 (C, H, N).
624
625
5.3.26. (E)-1-(2-methoxy-4-(prop-2-yn-1-yloxy)phenyl)-3-(3,4,5-trimethoxyphenyl)prop-2-en-1-one
(27) Starting from 36 (0.20 g, 1.0 mmol) and 3,4,5-trimethoxybenzaldheyde (0.21 g, 1.1 mmol)
25

ACCEPTED MANUSCRIPT
626
627
628
629
630
631
632
gave the crude final product 27 that was purified by crystallization from EtOH to obtain a yellow
1
solid (0.27 g), 73% yield, mp 106-108 °C. H NMR (CDCl3) δ 2.58 (s, 1H, C≡CH), 3.86 (s, 3H,
OCH3), 3.94 (s, 3H, OCH3), 4.75 (d, J = 2.0 Hz, 2H, OCH2), 6.65 (s, 1H, H-3), 6.67 (d, J = 8.4 Hz,
1H, H-5), 6.81 (s, 2H, H-3’ e H-5’), 7.37 (d, J = 15.6 Hz, 1H, =CH), 7.58 (d, J = 8.8 Hz, 2H, H-2’
and H-6’), 7.64 (d, J = 15.6 Hz, 1H, CH=), 7.78 (d, J = 8.8 Hz, 1H, H-6). 13C NMR (CDCl3) δ 55.8,
55.9, 56.0, 56.2, 56.3, 76.2, 76.6, 77.8. 77.9. 100.9, 107.8, 110.4, 118.5, 123.0, 123.6, 125.8, 131.0,
132.8, 142.5, 9 149.2, 157.8, 162.2, 190.5. ESI-MS (m/z): 382 (M + H); Anal. C22H22O6 (C, H, N).
633
634
635
636
637
638
639
640
641
642
5.3.27.
(E)-3-(3,4-dimethoxyphenyl)-1-(2-methoxy-4-(prop-2-yn-1-yloxy)phenyl)prop-2-en-1-one
(28). Starting from 37 (0.23 g, 1.0 mmol) and 3,4-dimethoxybenzaldheyde (0.18 g, 1.1 mmol) gave
the crude final product 28 that was purified by flash chromatography (petroleum ether/EtOAc 9:1)
1
and then crystallized from EtOH to obtain a yellow solid (0.22 g), 69% yield, mp 130-132 °C. H
NMR (CDCl3) δ 2.58 (s, 1H, C≡CH), 3.90 (s, 6H, OCH3), 3.93 (s, 3H, OCH3), 4.76 (s, 2H, OCH2),
6.60 (s, 1H, H-3) 6.64 (d, J = 8.4 Hz, 1H, H-5), 6.88 (d, J = 8.4 Hz, 1H, H-5’), 7.12 (d, J = 2.2 Hz,
1H, H-2’), 7.19 (dd, J = 8.0 and 2.2 Hz, 1H, H-6’), 7.33 (d, J = 15.6 Hz, 1H, =CH), 7.62 (d, J =
15.6 Hz, 1H, CH=), 7.72 (d, J = 8.0 Hz, 1H, H-6). 13C NMR (CDCl3) δ 55.7, 55.8, 56.2, 56.5, 76.7,
77.9, 101.1, 107.1, 110.0, 111.4, 123.6, 123.9, 125.8, 127.3, 131.8, 142.7, 147.1, 154.5, 157.8,
161.4, 190.4. ESI-MS (m/z): 353 (M + H); Anal. C21H20O5 (C, H).
643
644
645
646
647
648
649
5.3.28. (E)-1-(2,4-bis(prop-2-yn-1-yloxy)phenyl)-3-(3,4,5-trimethoxyphenyl)prop-2-en-1-one (29).
Starting from 37 (0.23 g, 1 mmol) and 3,4,5-trimethoxybenzaldheyde (0.21 g, 1.1 mmol) gave the
crude final product 29 that was purified by crystallization from ethanol to obtain a yellow solid
1
(0.27 g), 67% yield, mp 147-138 °C. H NMR (CDCl3) δ 2.55-2.58 (m, 2H, C≡CH), 3.94 (s, 6H,
OCH3), 3.97 (s, 3H, OCH3), 4.77-4.79 (m, 4H, OCH2), 6.71 (d, J = 2.2 Hz, 1H, H-3) 6.88 (dd, J =
8.4 and 2.0 Hz, 1H, H-5), 6.81 (s, 2H, H-2’ and H-6’), 7.42 (d, J = 15.6 Hz, 1H, =CH), 7.63 (d, J =
16.0 Hz, 1H, CH=), 7.81 (d, J = 8.8 Hz, 1H, H-6). 13C NMR (CDCl3) δ 55.8, 55.9, 56.0, 56.4, 56.5,
26

ACCEPTED MANUSCRIPT
650
651
76.2, 76.7, 77.8, 77.9, 101.1, 107.1, 110.2, 111.3, 123.7, 125.4, 128.3, 132.5, 142.7, 149.1, 150.8,
151.1, 157.8, 161.4, 190.3. ESI-MS (m/z): 407 (M + H); Anal. C24H22O6 (C, H).
652
653
654
655
656
657
658
659
660
661
5.3.29. (E)-1-(2,4-bis(prop-2-yn-1-yloxy)phenyl)-3-(3,4-dimethoxyphenyl)prop-2-en-1-one (30).
Starting from 37 (0.23 g, 1.0 mmol) and 3,4-dimethoxybenzaldheyde (0.18 g, 1.1 mmol) gave the
crude final product 30 that was purified by flash chromatography (petroleum ether/EtOAc 4:1) and
then crystallized from EtOH to obtain a yellow solid (0.24 g), 65% yield, mp 137-139 °C. 1H NMR
(CDCl3) δ 2.55-2.58 (m, 2H, C≡CH), 3.94 (s, 6H, OCH3), 4.77-4.79 (m, 4H, OCH2), 6.71 (d, J =
2.2 Hz, 1H, H-3) 6.88 (d, J = 8.4 and 2.2 Hz, 1H, H-5), 7.16 (d, J = 8.2 Hz, 1H, H-5’), 7.20 (d, J =
2.0 Hz, 1H,H-2’), 7.22 (dd, J = 8.0 and 2.2 Hz, 1H, H-6’), 7.40 (d, J = 15.6 Hz, 1H, =CH), 7.64 (d,
13
J = 16.0 Hz, 1H, CH=), 7.75 (d, J = 8.8 Hz, 1H, H-6). C NMR (CDCl3) δ 55.8, 55.9, 56.0, 56.5,
76.2, 76.7, 77.8, 77.9, 101.1, 107.1, 110.0, 111.1, 122.9, 123.8, 125.1, 128.3, 132.6, 142.7, 149.1,
151.1, 157.8, 161.4, 190.4. ESI-MS (m/z): 377 (M + H); Anal. C23H20O5 (C, H).
662
663
664
665
666
667
668
669
670
671
5.3.30.
(E)-1-(2,4-bis(prop-2-yn-1-yloxy)phenyl)-3-(4-methoxyphenyl)prop-2-en-1-one
(31).
Starting from 37 (0.23 g, 1.0 mmol) and 4-methoxybenzaldheyde (0.14 g, 1.1 mmol) gave the crude
final product 31 that was purified by flash chromatography (petroleum ether/EtOAc 9:1) and then
1
crystallized from EtOH to obtain an orange solid (0.20 g), 59% yield; mp 120-122 °C. H NMR
(CDCl3) δ 2.57-2.60 (m, 2H, C≡CH), 3.86 (s, 3H, OCH3), 4.76 (d, J = 1.6 Hz, 2H, OCH2), 4.78 (d,
J = 2.0 Hz, 2H, OCH2) 6.70 (d, J = 2.2 Hz, 1H, H-3), 6.72 (d, J = 8.2 Hz, 1H, H-5), 6.92 (d, J = 8.4
Hz, 2H, H-3’ and H-5’), 7.42 (d, J = 15.6 Hz, 1H, =CH), 7.58 (d, J = 8.4 Hz, 2H, H-2’ and H-6’),
13
7.66 (d, J = 15.6 Hz, 1H, CH= ), 7.76 (d, J = 8.8 Hz, 1H, H-6). C NMR (CDCl3) δ 55.9, 56.0,
56.5, 76.2, 76.7, 77.7, 77.9, 101.1, 107.1, 110.0, 111.1, 122.4, 123.7, 125.2, 128.9, 132.7, 132.8,
142.7, 149.8, 157.8, 161.2, 190.5. ESI-MS (m/z): 345 (M + H); Anal. C20H15NO3 (C, H, N).
672
673
5.3.32. 2-cinnamoyl-5-((3-methylbut-2-en-1-yl)oxy)phenyl acetate (20).Compound 19 (0.31 g, 1.0
mmol) was reacted with acetic anhydride (10 mL) and the mixture was heated under reflux for 4 hr
27

ACCEPTED MANUSCRIPT
674
675
676
677
678
679
680
681
and then poured into ice/water. The solid was collected and crystallized from EtOH to give 19 as
1
white solid (0.23 g), 66 % yield, mp 121-122 °C. H NMR (CDCl3) δ 1.79 (s, 3H, CH3), 1.82 (s,
3H, CH3), 2.35 (s, 3H, COCH3), 4.56 (d, J = 6.0 Hz, 2H, OCH2), 5.47-5.50 (m, 1H, =CH), 6.41 (d, J
= 2.0, 1H, H-5), 6.60 (dd, J = 1.8 and 8.4 Hz, 1H, H-3), 7.27 (d, J = 15.6 Hz, 1H, =CH), 7.33-7.45
(m, 3H, H-3’, H-4’, H-5’), 7.53-7.61 (m, 2H, H-2’and H-6’), 7.65 (d, J = 15.3 Hz, 1H, =CH), 7.78
13
(d, J = 8.4 Hz, 1H, H-6). C NMR (CDCl3) δ 18.61, 20.5, 24.5, 65.1, 104.8, 118.6, 113.6, 118.6,
118.9, 120.5, 127.7, 128.2, 139.7, 135.9, 138.0, 145.2, 154.4, 154.7, 166.0, 169.11, 191.7. ESI-MS
(m/z): 351 (M + H); Anal. C20H20O3 (C, H).
682
5.4. Parasitology.
683
684
685
686
687
688
5.4.1.
Parasites.
Promastigote
forms
of
a
L.
donovani
reference
strain
(MHOM/NP/02/BPK282/0cl4), L. major reference strain (MHOM/SU/73/5-ASKH), L. tropica
reference strain (MHOM/SU/74/K27), L. infantum reference strain (MHOM/ TN/ 80/ IPT1) were
cultured at 26°C in HOMEM (Gibco Thermo Fisher Scientific Inc., Waltham, USA), a liquid
custom made medium supplemented with 20% foetal bovine serum (FBS, EuroClone SpA, Milan,
Italy) and 1% penicillin-streptomycin (EuroClone SpA).
689
690
691
692
693
694
695
696
697
698
5.4.2. Cell cultures. THP-1 cells (human leukemia monocytic cell line) were cultured at 37 °C in
RPMI-1640 (EuroClone SpA) liquid medium supplemented with 10% FBS (EuroClone SpA), 1%
levoglutamine (EuroClone SpA), Mercaptoethanol (Gibco) 50 µM , 1% penicillin-streptomycin.
Vero cells (kidney of African green monkey epithelial cell line) were cultured at 37 °C in MEM
liquid medium supplemented with 10% FBS (EuroClone SpA), 1% levoglutamine (EuroClone
SpA), 1% penicillin-streptomycin (EuroClone SpA).
5.4.3. Promastigote growth inhibition assay. The late log/stationary phase of promastigotes were
seeded with complete HOMEM medium at 106/mL in 96-well plates and incubated with tested
compounds at a range concentration of 40 µM – 1.6 µM in a 26 °C incubator for 72 h. The
antileishmanial drug amphotericin B was used as standard drug (positive control). Each experiment
28