Synthesis of cinnamic acid derivatives and leishmanicidal activity against Leishmania braziliensis

Article abstract of DOI:10.1016/j.ejmech.2019.111688

Leishmania braziliensis is one of the pathogenic agents of cutaneous and mucocutanoeous leishmaniasis. There are no validated vaccines to prevent the infection and the treatment relies on drugs that often present severe side effects, which justify the efforts to find new potential antileishmanial drugs. An alternative to promote the discovery of new drugs would be the association of different chemical groups of bioactive compounds. Here we describe the synthesis and bioactivity evaluation against L. braziliensis of cinnamic acid derivatives possessing isobenzofuranone and 1,2,3-triazole functionalities. We tested 25 compounds at 10 μM concentration against extracellular promastigotes and intracellular amastigotes during macrophage infection. Most compounds were more active against amastigotes than to promastigotes. The derivatives (E)-3-oxo-1,3-dihydroisobenzofuran-5-yl-(3,4,5-trimethoxy) cinnamate (5c), (1-(3,4-difluorobenzyl)-1H-1,2,3-triazol-4-yl)methyl cinnamate (9g), and (1-(2-bromobenzyl)-1H-1,2,3-triazol-4-yl)methyl cinnamate (9l) were the most effective presenting over 80% toxicity on L. braziliensis amastigotes. While compound 5c is a cinnamate with an isobenzofuranone portion, 9g and 9l are triazolic cinnamic acid derivatives. The action of these compounds was comparable to amphotericin B used as positive control. Ultrastructural analysis revealed that 5c-treated parasites showed impaired cytokinesis and apoptosis triggering. Taken together, these results highlight the potential of cinnamic acid derivatives in development of novel anti-leishmanial drugs.

Full text of DOI:10.1016/j.ejmech.2019.111688

Journal Pre-proof
Synthesis of cinnamic acid derivatives and leishmanicidal activity against Leishmania
braziliensis
Michelle Peixoto Rodrigues, Deborah Campos Tomaz, Luciana Ângelo de Souza,
Thiago Souza Onofre, Wemerson Aquiles de Menezes, Juliana Almeida-Silva, Ana
Márcia Suarez-Fontes, Márcia Rogéria de Almeida, Adalberto Manoel da Silva,
Gustavo Costa Bressan, Marcos André Vannier-Santos, Juliana Lopes Rangel Fietto,
Róbson Ricardo Texeira
PII:
S0223-5234(19)30838-4
DOI:
https://doi.org/10.1016/j.ejmech.2019.111688
EJMECH 111688
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 15 April 2019
Revised Date: 21 August 2019
Accepted Date: 6 September 2019
Please cite this article as: M.P. Rodrigues, D.C. Tomaz, L. Ângelo de Souza, T.S. Onofre, W. Aquiles
de Menezes, J. Almeida-Silva, Ana.Má. Suarez-Fontes, Má. Rogéria de Almeida, A. Manoel da Silva,
G.C. Bressan, Marcos.André. Vannier-Santos, J.L. Rangel Fietto, Ró.Ricardo. Texeira, Synthesis of
cinnamic acid derivatives and leishmanicidal activity against Leishmania braziliensis, European Journal
of Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.ejmech.2019.111688.
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Synthesis of cinnamic acid derivatives and leishmanicidal activity
against Leishmania braziliensis
1
,†
1,†
Michelle Peixoto Rodrigues , Deborah Campos Tomaz , Luciana Ângelo de
2
,†
3,†
2
Souza , Thiago Souza Onofre , Wemerson Aquiles de Menezes , Juliana
4
4
2
Almeida-Silva , Ana Márcia Suarez-Fontes , Márcia Rogéria de Almeida ,
5
2
Adalberto Manoel da Silva , Gustavo Costa Bressan , Marcos André Vannier-
4
2
1,
Santos , Juliana Lopes Rangel Fietto and Róbson Ricardo Texeira *
1
Departamento de Química, Universidade Federal de Viçosa, 36570-900, Viçosa, Minas
Gerais, Brazil.
2
Biochemistry and Molecular Biology Department, Universidade Federal de Viçosa,
Viçosa, Minas Gerais, Brazil.
3
Escola Paulista de Medicina, Universidade Federal de São Paulo, Campus São Paulo,
São Paulo, SP, Brazil
4
Instituto Oswaldo Cruz, Fiocruz, Rio de Janeiro, Brazil.
5
Instituto Federal de Educação, Ciência e Tecnologia Catarinense, Araquari, Santa
Catarina, Brazil.
†
These authors contributed equally to this work.
*
Author to whom correspondence should be addressed; e-mail: robsonr.teixeira@ufv.br

Abstract
Leishmania braziliensis is one of the pathogenic agents of cutaneous and
mucocutanoeous leishmaniasis. There are no validated vaccines to prevent the infection
and the treatment relies on drugs that often present severe side effects, which justify the
efforts to find new potential antileishmanial drugs. An alternative to promote the
discovery of new drugs would be the association of different chemical groups of
bioactive compounds. Here we describe the synthesis and bioactivity evaluation against
L. braziliensis of cinnamic acid derivatives possessing isobenzofuranone and 1,2,3-
triazole functionalities. We tested 25 compounds at 10 µM concentration against
extracellular promastigotes and intracellular amastigotes during macrophage infection.
Most compounds were more active against amastigotes than to promastigotes. The
derivatives (E)-3-oxo-1,3-dihydroisobenzofuran-5-yl-(3,4,5-trimethoxy) cinnamate (5c),
(1-(3,4-difluorobenzyl)-1H-1,2,3-triazol-4-yl)methyl cinnamate (9g), and (1-(2-
bromobenzyl)-1H-1,2,3-triazol-4-yl)methyl cinnamate (9l) were the most effective
presenting over 80% toxicity on L. braziliensis amastigotes While compound 5c is a
cinnamate with an isobenzofuranone portion 9g, and 9l are triazolic cinnamic acid
derivatives. The action of these compounds was comparable to amphotericin B used as
positive control. Ultrastructural analysis revealed that 5c-treated parasites showed
impaired cytokinesis and apoptosis triggering. Taken together, these results highlight
the potential of cinnamic acid derivatives in development of novel anti-leishmanial
drugs.
Key Words: Cinnamic acid derivatives, Leishmanicidal activity, Leishmania
braziliensis, isobenzofuranone, 1,2,3-triazole

Introduction
Leishmaniasis encompasses a group of important tropical neglected parasitic
diseases (WHO, 2017). They are caused by several pathogenic protozoa species of
Leishmania genus and are transmitted to humans through the bite of infected female
phlebotomine sand flies. There are four main clinical manifestations of leishmaniasis
known as cutaneous (CL), mucocutaneous (MCL), visceral infection (VL, also known
as kala-azar) and post-kala-azar dermal leishmaniasis (PKDL) [2,3].
The absence of effective vaccines for humans makes the use of
chemotherapeutics not only important to treat the patients with leishmaniasis, but also to
control the spread of the disease. There is no single treatment to all forms of the
infection and even though there are different possibilities of treatment, they can not be
effective to achieve the cure and can also lead to undesired side effects [4–6]. The
treatment of leishmaniasis can be done with pentavalent antimonials, namely N-
®
®
methylglucamine antimoniate (Glucantime ) and sodium stibogluconate (Pentostan ),
which are the first-line drugs of choice in Brazil. Other drugs that are also used as a
second alternative correspond to amphotericin B and pentamidine. However, these
drugs show limited efficacy causing serious side effects, associated to renal and cardiac
toxicity [7,8]. For these reasons, the development of new drugs for leishmaniasis is a
pressing demand, preferably employing compounds effective in low concentrations,
non-toxic and selective against the parasite.
Natural products have served as important source of novel useful compounds
that have been used directly as medicinal agents [9], as model compounds for synthetic
or semisynthetic structure modifications and optimization [10,11], as biochemical
and/or pharmacological probes [12,13], and as source of inspiration for generations of
synthetic organic medicinal chemists [14,15].

Cinnamic acid is a natural aromatic carboxylic acid, composed of a phenyl ring
substituted with an acrylic acid group, which commonly occurs in the trans
configuration (Fig. 1). This phenyl-alanine deaminated product, produced in plant
tissues, has a long history of human use as a component of plant-derived scents and
flavoring [16]. Cinnamic acid and its ester derivatives exhibit a broad spectrum of
biological activities, including anti-inflammatory [17,18], antimicrobial [19,20],
antifungal [21,23], antioxidant [24] and anticarcinogenic [25–27]. It has been shown
that these compounds have significant leishmanicidal activity [28].
A class of compounds called isobenzofuranones, also commonly termed as
phthalides and isobenzofuran-1(3H)-ones, are characterized by a bicyclic nucleus (Fig.
1
) derived from the fusion of a γ-lactone (ring A) with a benzene (ring B). Phthalides
are widely present in plants, fungi and liverworts, and exhibit a broad spectrum of
biological activities, including antifungal [29,30], antimicrobial [31], insecticidal [32],
cytotoxic [33-35], anti-inflammatory[36], and leishmanicidal [37,38] .
Heterocyclic compounds are of great importance for the pharmaceutical industry
since about 90% of the new drugs possess at least one heterocyclic ring in its structure
[
39,40]. There are reports of the usefulness of heterocycles in the treatment of diseases
since antiquity, such as quinine, used in the 16th century to prevent and treat malaria.
The 1,2,3-triazoles (Fig. 1) are heterocycles of synthetic origin and with a wide range of
applications [43]. They exhibit a broad variety of biological activities, including anti-
HIV [44], antimicrobial [45], antibacterial [46], leishmanicidal [47,48], antiplatelet [49],
antiproliferative [50], trypanocidal [51,52], antitumoral [53], among others.

Fig. 1. Chemical structures of compounds mentioned in the text.
Considering the need to develop new compounds potentially useful, and with
lower undesired effects for the treatment of leishmaniasis, this paper describes the
synthesis of cinnamic acid derivatives containing isobenzofuranone and 1,2,3-triazole
portions and the evaluation of their activities against Leishmania (Viannia) braziliensis
which is the main causative agent of cutaneous and mucocutaneous leishmaniais in New
World.
Results and discussion
Syntheses of cinnamic acid derivatives
The synthesis of cinnamates 5a─5f (Scheme 1) required the preparation of
isobenzofuranone 2 and cinnamic derivatives 4a─4f. Substance 2 was obtained in two
steps from 3-methoxy benzoic acid. This carboxylic acid was initially treated with HCl,
HCHO, CH COOH under microwave irradiation. This procedure afforded
3
isobenzofuranone 1 in 80% yield after purification by silica gel column chromatography.
The demethylation of 1 with BBr gave the hydroxylated isobenzofuranone 2 in 54% yield.
3
The cinnamic acid derivatives 4a─4f were prepared via microwave assisted Knoevenagel
condensation of malonic acid and different aromatic aldehydes (3a─3f). Finally, the
cinnamates containing the isobenzofuranone nucleus 5a─5f were synthesized using the
microwave assisted Steglich esterification between cinnamic acid derivatives 4a─4f and

hydroxylated isobenzofuranone 2. The esters 5a─5f were obtained in yields ranging
from 54 to 76% within 40─45 minutes.
Scheme 1. Synthesis of cinnamic acid derivatives 5a─5f.
To prepare the cinnamates containing triazolic portions, it was required the
synthesis of esters 6 and 7 as well as a series of azides 8 (Scheme 2). Thus, the Steglich
esterification [54] between the cinnamic acid and different alcohols afforded
compounds 6 and 7 in good yields (68 and 83%, respectively) after purification by silica
gel column chromatography. The azides 8 were prepared from the corresponding
benzylic alcohols utilizing methodology previously described [55]. The click reaction
corresponded to the key step involved in the synthesis of cinnamates containing
triazolic functionalities 9a─9m and 10a─10m. Hence, the reactions between esters 6

and 7 and azides 8 afforded the cinnamic acid derivatives containing 1,2,3-triazolic
portions in yields ranging from 73 to 99% (Scheme 2).
Scheme 2. Synthesis of ester cinnamates 9a─9m and 10a─10m.
Evaluation of cytotoxicity of cinnamic acid derivatives
To be used in biological assays, all the cinnamic acid derivatives were
suspended in aqueous solution containing 5% DMSO and then diluted with RPMI to 10
µM (the DMSO final concentration corresponded to 0.1%). The solubilization of the
compounds was evaluated by light microscopy. Those compounds that formed crystals,

namely 5b, 5f, 9c, 9d, 9f, 9j, were considered insoluble and they were not used in the
following assays.
Macrophages are the main host cells of Leishmania and so are used in the
evaluation of in vitro infection assays with L. braziliensis. Thus, all the soluble
compounds were evaluated for their cytotoxicity on macrophages using the resazurin
viability assay and the results are shown as survival rates after 48h of treatment with the
compounds (Fig. 2). Amphotericin B was used as anti-leishmanial control drug. As
expected, the amphotericin B (3.125 µg/mL) and the negative control (0.1% DMSO)
were not toxic to macrophages. In addition, as highlighted by the absence of significant
differences between the controls and cinnamic acid derivatives, the evaluated
compounds were not toxic to macrophages at 10 µM concentration.
Fig. 2. Evaluation of cytotoxicity of compounds upon macrophage cells. Each bar
shows the mean of percentage of survival of macrophage cells determined by the
resazurin viability assay with respective standard deviation. Each mean represents the
results from three independent experiments that were performed with internal
quadruplicates. The open bar (positive control) represents cells treated with
amphotericin B (3.125 µg/mL) and the closed bar (negative control) represents cells
treated with DMSO at the same concentration used on dilution of compounds (0.1%).
All data were statistically evaluated in relation to the control samples by One-way
ANOVA, with post-test of Dunnett´s post test (p≤0.05) and no significant differences
were observed.

Evaluation of leishmanicidal effect of cinnamic acid derivatives against L. braziliensis
The first strategy used during the investigation of leishmanicidal effects of new
compounds is the in vitro assessment of them against different developmental forms of
Leishmania parasites. Consequently, we tested the leishmanicidal activity of the
compounds against total promastigotes forms of L. braziliensis, the insect dwelling
stage. The assays were done using the resazurin method and the results are shown in
Fig. 3 as survival levels in relation to DMSO. The compounds 5a and 5c-e, which are
derivatives containing an isobenzofuranone portion, displayed significant
leishmanicidal activity against promastigotes forms. Also, 10a─f and 10i─j, which are
derivatives containing triazolic portions, also displayed leishmanicidal activity, albeit
presenting lower efficiency as compared to the derivatives containing the
isobenzofuranone moiety. Interestingly, the series of compounds 9a─b, 9g─i, and
9
k─m did not present leishmanicidal activity against promastigote, as compared with
negative control. The synthesis of the compounds 9a─9m and 10a─10m was planned
so that the influence on the leishmanicidal activity of different benzylic groups attached
to triazole ring as well as the length of the chain connecting the triazole ring to the
cinnamic acid portion could be assessed. The evaluation of leishmanicidal activity
against promastigote form revealed that the length of the connecting chain is important
since derivatives 9a-b, 9g-i, 9k-m, presenting a shorter connecting carbon chain, were
ineffective against L. braziliensis promastigote form.

Fig. 3. Leishmanicidal activity against the promastigotes of L. braziliensis.
Promastigotes from log phase were treated during 48 h with each of compound and the
rate of survive was evaluated by resazurin method. Each bar shows the mean of
percentage of survival of promastigotes and the respective standard deviation. The
results are at least two or three independent experiments, performed with internal
quadruplicates. The white bar (positive control) represents cells treated with
amphotericin B (3.125 µg/mL) and the black bar (negative control) represents cells
treated with DMSO at the same concentration used on dilution of derivatives (0.1%).
*
Means statically difference from negative control by One-way ANOVA, with
Dunnett´s post-test (p≤0.05).
Then, the next step was to evaluate the action of the compounds against
intracellular amastigotes of L. braziliensis during in vitro macrophage infection. The
Fig. 4 shows the rate of survival of the parasite after 48 h of treatment with the
compounds. It is possible to observe that several compounds were significantly active
against intracellular amastigotes with different levels. In general, the effectiveness of
the compounds against the amastigote forms was higher than on promastigotes (Fig. 3).
The most active compounds against the amastigote forms were 5c, 9b, 9g, 9i, 9k, 9l, 9m
(Fig. 4).

Fig. 4. Leishmanicidal activity against the L. braziliensis intracellular amastigotes.
Promastigotes at log growth phase were used to infect macrophages. After the
establishment of the infection, the infected cultures were treated for 48 h with each
compound. Then, the macrophages were lysed and the released amastigotes were
cultivated until their differentiated to promastigotes which were subsequently
quantitated by resazurin method. Each bar shows the mean of percentage of viable
promastigotes ± standard deviation. The results are at least two or three independent
experiments that were performed with internal quadruplicate. The open bar (positive
control) represents cells treated with amphotericin B (3.125 µg/mL) and the closed bar
(negative control) represents cells treated with DMSO at the same concentration used in
the dilution of derivatives (0.1%). *Indicates statically difference from negative control
by One-way ANOVA, with Dunnett´s post-test (p≤0.05).
In general, the cinnamic acid derivatives 9b, 9g, 9i, 9k, 9l and 9m showed better
results than the others. Even though this series of compounds did not show good results
against promastigote form (Fig. 2), it was very effective against intracellular form of L.
braziliensis. The highest activity corresponded to compound 9g that presented 86% of
toxicity on Leishmania amastigote forms. Table 1 highlights the activity of the
compounds on intracellular amastigotes.

Table 1 - Relationship between the toxicity on Leishmania intracellular amastigotes and
chemical structures of the compounds
%
toxicity
43.87
80.47
59.54
Compound
*
Structure
5
5
5
a
c
d
O
Ar
O
5a: Ar = 4-fluorophenyl
5
5
5
c: Ar = 3,4,5-trimetoxyphenyl
d: Ar = 4-nitrophenyl
e: Ar = 4-bromophenyl
O
O
5
e
50.14
Leishmanicidal activity 5c > 5d > 5e > 5a
9
9
9
9
9
9
9
9
a
b
g
h
i
19.91
78.98
86.32
59.10
77.39
76.00
80.12
70.38
k
l
m
Leishmanicidal activity 9g > 9l > 9b > 9i > 9k > 9m > 9h > 9a
1
1
1
1
1
1
1
1
1
1
1
1
1
0a
0b
0c
0d
0e
0f
39.73
45.74
52.07
20.58
45.10
49.01
47.85
67.84
65.84
69.24
60.48
57.32
19.67
0g
0h
0i
0j
0k
0l
0m
1
1
0j > 10h > 10i > 10k> 10l> 10c > 10f > 10g >
0b,10e>10a>10d>10m
Leishmanicidal activity
*
Amphotericin B: 98.25%; DMSO: 10.71%

Since compounds 5c and 9g were non-toxic to macrophages and displayed the
most significant leishmanicidal effect upon L. braziliensis intracellular amastigotes,
they were selected to be used in further assays. Thus, the IC for the activities of these
5
0
compounds against Leishmania promastigote, macrophage, and intracellular amastigote
form were determined. For promastigote forms, the IC of 5c and 9g were, respectively,
5
0
7
.58 µM and 3.05 µM. Considering macrophages, IC values were over than 80 µM for
50
both compounds (Table 2). In the infection assays, the IC of 9g was undetermined
5
0
(the compound was tested at different concentrations but the data were not consistent
enough to lead to an IC determination by the PRISMA program), although in the
50
screening assays its efficacy against intracellular amastigotes was superior to 80% at 10
µM (Fig. 4 and Table 1). In contrast, the IC of 5c was determined as being equal to
5
0
8
.27 µM. Thus, it is possible to infer that the compound 5c is more effective against
intracellular amastigotes than 9g. This conclusion is based on the fact that the behavior
of 9c at different concentrations was not consistent with its previous action at the 10 µM
screening assay. The Selectivity Index (SI) is an indication of how much a compound is
most effective against the parasite instead of to the host mammalian cells. The higher
the SI, the more selective the drug is on the parasite and less toxic to mammalian cells.
For 5c, the SI is greater than 9.67.

Table 2 – IC of different activities of compounds 5c and 9g on L. braziliensis
5
0
IC L. braziliensis
Compound promastigote forms
IC Raw 264.7
macrophage
(48 hours) (µM)
IC50
Infection Assay
(48 hours) (µM)
5
0
50
Selectivity index
SI)
(
(
48 hours) (µM)
5
c
7.58
> 80
> 8.27
> 9.67
*
*
9
g
3.05
>80
N
N
Compounds were tested at the concentrations of 80 µM, 40 µM, 30 µM, 20 µM, 10 µM,
µM, 2.5 µM and 1 µM for IC determination after 48 hours by resazurin method.
5
5
0
Assay controls were amphotericin B (positive control) and DMSO (negative control).
The IC50 was calculated using GraphPad Prism Version 5.0. For each test, three
*
independent experiments were performed in quadruplicates. N = Not determined
Considering that during the natural infection only intracellular amastigotes are
important to maintenance of the infection, we proceed only with compound 5c and
evaluated the ultrastructural effects of it on the macrophage infection. Ultrastructural
approaches may be useful in the elucidation of antiparasitic agent mechanisms of action
[
(
56,57]. Transmission electron microscopy analysis of infected macrophage cultures
Fig. 5) indicates that contrary to DMSO-treated cells, 5c-treated parasites displayed
lobulated cell bodies, which may be due to the truncated cytokinesis, as reported in
vinblastine-treated L. amazonensis [58]. Images indicating autophagy, apoptosis and
presumably necrosis were also observed. Autophagy may comprise an escape
mechanism under stress conditions as well as to cause parasite cell death and it also be
associated to apoptosis in Leishmania [59, 60]. These processes may be related to
necrotic cell death in Trypanosoma cruzi [61] as necrosis may follow apoptosis onset
[
62]. The electrolucent areas seem to indicate loss of cytoplasmic content i.e. necrosis.
Thus, the compound 5c may have impaired the cell cycle and induced autophagy,
ensuing triggered programmed cell death machineries.

*
Fig. 5. Transmission electron microscopy of DMSO (A) and 5c-treated (B, C) cultures.
Control macrophages presented parasites (p) with well-preserved structure and electron
density. 5c-treated parasite within parasitophorous vacuole displaying large lobules (B,
arrows) with cytoplasmic core containing organelles such as mitochondria, radiating
from the central cell body, indicated by the flagellar pocket with two flagella (F). A
vesicle containing amorphous material (V) presumably may comprise an
autophagosome for the presence of multiple membranes (inset arrowhead) as well as
cytoplasmic contents. Parasite attached to the parasitophorous vacuole in a 5c-treated
macrophage (C). Electronense structures (white asterisk) at the cell periphery consist of
detached fragments of pycnotic chromatin; the nucleus is unusually-shaped
(arrowheads) with evidence of initial pycnosis (arrow). Some electrolucent areas were
also observed (black asterisks).
Previously,
our
group
demonstrated
leishmanicidal
activity
of
isobenzofuranones containing aromatic and alicyclic functionalities as represented by
general structure shown in Fig. 6. However, those compounds presented leishmanicidal
action at 100 µM and the decreasing in the concentration drastically reduced the

leishmanicidal effect [37]. It should be noticed that those phtalides previously evaluated
by us presented functionalization at position C-3 of the isobenzofuranone nucleus.
Fig. 6. General structures of isobenzofuranones investigated by Pereira et al. [37] as
leishmanicidal compounds.
In the present work, functionalization made at C-6 position of the
isobenzofuranone nucleus, namely introduction of cinnamoyl groups, rendered
compounds with significant leishmanicidal activity at ten times lower concentration (10
µM) in comparison with the phtalides previously reported by us [37]. In other words,
the functionalized isobenzofuranones herein described are ten times more potent. This
means that isobenzofuranone as well as cinnamic acid derivatives may comprise lead
compounds in the research and development of leishmanicidal agents. It should be
mentioned that Pereira et al [37] used the isobenzofuranones against Leishmania
infantum; thus isobenzofuranones could be per se effective against other species.
Material and Methods
Synthesis
Generalities
Solvents and formaldehyde 37% (w/v) aqueous solution were purchased from
Vetec (Rio de Janeiro, Brazil). Piperidine, boron tribromide 1.0 M solution in
dichloromethane, 3-methoxybenzoic acid, malonic acid, 4-chlorobenzaldehyde, 4-

methoxybenzaldehyde, 3,4,5-trimethoxybenzaldehyde, 4-nitrobenzaldehyde, 4-
bromobenzaldehyde, 4-chlorobenzaldehyde, pent-4-yn-1-ol, prop-2-yn-1-ol, triethyl
amine, dimethyl sulfoxide (DMSO), N,N’-dicyclohexylcarbodiimide (DCC), 4-N,N’-
dimethylaminopyridine (DMAP), trans-cinnamic acid, mesylate chloride, benzyl
alcohol, 4-nitrobenzyl alcohol, 4-bromobenzyl alcohol, 2,5-dichlorobenzyl alcohol,
2
,4,6-trichlorobenzyl alcohol, 4-chlorobenzyl alcohol, 3,4-difluorobenzyl alcohol, 4-
fluorobenzyl alcohol, 4-trifluoromethoxybenzyl alcohol, 4-trifluoromethylbenzyl
alcohol, 4-methoxybenzyl alcohol, 4-iodobenzyl alcohol, 2-bromobenzyl alcohol, 2-
fluorobenzyl alcohol, sodium ascorbate, sodium azide and copper(II) sulfate
pentahydrate were purchased from Sigma Aldrich (St. Louis, MO, USA) and used
without further purification. The NMR spectra were recorded on a Varian Mercury 300
1
13
instrument (Varian, Palo Alto, CA, US) at 300 MHz ( H) and 75 MHz ( C),
1
respectively, using CDCl or DMSO-d as solvents. The H-NMR data are presented as
3
6
follows: chemical shift (
δ
) in ppm, multiplicity, number of protons, J values in Hertz
(Hz). Multiplicities are indicated by the following abbreviations: s (singlet), d (doublet),
dd (double of doublets), t (triplet), m (multiplet), q (quartet), quint (quintet), td (double
of triplets), dap (apparent doublet), tap (apparent triplet). For fluorine-containing
derivatives, the multiplicity of some carbon signals are described along with J values in
Hertz. IR spectra were obtained using Varian 660-IR equipped with GladiATR (Varian,
−
1
Palo Alto, CA, US) scanning from 4000 to 500 cm . Melting points were determined
using a MQAPF-302 melting point apparatus (Microquimica, Santa Catarina, Brazil)
and are uncorrected. The progress of the reactions was monitored by thin layer
chromatography (TLC). Flash column chromatography was performed using silica gel
(60−230 mesh).

Procedure for synthesis of 6-methoxyisobenzofuran-1(3H)-one (1)
To a 100 mL round bottom flask, it was added 3-methoxybenzoic acid (5.00 g,
3
2.9 mmol), 3.50 mL of aqueous solution of formaldehyde (37% w/v), 5.50 mL of
concentrated HCl, and 12.0 mL of acetic acid. The flask containing the mixture was
inserted at the cavity of the CEM Discover System microwave apparatus and the pre-
programmed method of the equipment was used for an open system with magnetic
stirring for 20 minutes at 150 W power. After this period, the TLC analysis of the
reaction mixture showed that 3-methoxybenzoic acid had been completely consumed.
The volume of the solution was then reduced on rotary evaporator and a saturated
potassium bicarbonate solution was added (pH 6.0). The aqueous phase was extracted
with ethyl acetate (3 x 30.0 mL). The organic extracts were combined and the resulting
organic phase was dried over sodium sulfate sodium, filtered and concentrated under
reduced pressure. The product was purified by recrystallization using acetone as the
solvent. The compound 1 was obtained in 80% yield (4.31 g, 26.3 mmol). White solid,
-1
m.p. 118.0─119.5 °C, TLC: R = 0.31(hexane-ethyl acetate 2:1 v/v), IR (ATR) v max/cm :
f
-1
3
481, 3085, 2953, 2848, 1754, 1620, 1277, 1247, 1054, 1018, 992, 725, 692, 545 cm .
1
13
H NMR (300 MHz, CDCl3)
75 MHz, CDCl3)
δ
: 3.86 (s, 3H), 5.26 (s, 2H), 7.22─7.38 (m, 3H). C NMR
(
δ
: 55.7, 69.5, 107.4, 122.8, 123.0, 126.9, 138.8, 160.5, 171.2.
Synthesis of 6-hydroxyisobenzofuran-1(3H)-one (2)
Compound 1 (0.600 g, 3.65 mmol) was added to a 50.0 mL flask followed by
addition of 7.00 mL of HBr (48% w/v) and the mixture was submitted to microwave
irradiation (20 minutes at 200 W) similar to what was described for compound 1. The
volume of the solution was then reduced in a rotary evaporator. Hydrobromic acid
(HBr) was neutralized with sodium bicarbonate saturated solution. The resulting
mixture was transferred to a separatory funnel and the aqueous phase was extracted with

ethyl acetate (3 x 30.0 mL). The organic extracts were combined and the resulting
organic phase was dried over anhydrous sodium sulfate, filtered and concentrated under
reduced pressure. The compound 2 was obtained in 54% yield (0.297 g, 1.98 mmol).
White solid, m.p. 197.1 198.7 ºC, TLC: R = 0.26 (hexane-ethyl acetate 2:1 v/v),
─
f
_
IR (ATR) ν max: 3214 (broad band), 1722, 1598, 1505, 1449, 1227, 1192, 1055, 773, 539
-1 1
cm . H RMN (300 MHz, CD OD)
δ
: 5.25 (s, 2H), 7.15─7.18 (m, 2H), 7.40 (d, 1H,
J =
3
13
9
1
.0 Hz). C NMR (75 MHz, CD OD)
δ
: 70.1, 109.6, 122.7, 123.2, 126.5, 138.3, 158.6,
3
72.5.
General procedure for the synthesis of cinnamic acid derivatives 4a─4f
A 25 mL round-bottom flask was charged with 1.37 g (13.2 mmol) of malonic
acid, an aldehyde (6.60 mmol, compounds 3a─3f, Scheme 1), pyridine (5.00 mL), and
piperidine (1.00 mL, 13.0 mmol). The resulting mixture was submitted to microwave
irradiation (20 minutes at 150 W), similar to compounds 1 and 2. Then, the mixture was
poured into an ice bath and 10.0 mL of HCl 16 M. The resulting mixture was vacuum
filtered on sintered funnel. The residue was washed with portions of cold water (4 x
5
.00 mL). All the cinnamic acid derivatives 4a─4f were purified by silica gel column
chromatography. The structures of the compounds are supported by the following data.
Data for (E)-4-fluorocinnamic acid (4a)
White solid, obtained in 95% yield, m.p. 207.0─209.0 °C, purified by silica gel
column chromatography eluted with hexane/ethyl acetate (1:2 v/v), TLC: R = 0.35
f
-1
(
hexane-ethyl acetate 1:2 v/v), IR (ATR) v max/cm : 3500─3200 (broad band), 2980,
1
2
1
δ
1
600, 1688, 1625, 1227, 820. H NMR (300 MHz, DMSO-d6
)
δ
: 6.44 (d, 1H, Jtrans
=
1
3
5.9 Hz), 7.14 (t, 2H,
J
= 8.7 Hz), 7.68─
7
.63 (m, 3H). C NMR (75 MHz, DMSO-d6)
: 115.5 (d, JC-F = 21.8 Hz), 117.9, 129.9 (d, JC-F = 8.3 Hz), 130.0 (d, JC-F = 3.8 Hz),
43.4, 163.8 (d, JC-F = 248.3 Hz), 168.6.

Data for (E)-4-methoxycinnamic acid (4b)
White solid, obtained in 87% yield, m.p. 174.0─177.2 °C, purified by silica gel
column chromatography eluted with hexane/ethyl acetate (1:2 v/v), TLC: R = 0.35
f
-1
(
hexane-ethyl acetate 1:2 v/v). IR (ATR) v max/cm : 3602─3200 (broad band), 2938,
1
2
842, 1680, 1598, 1252, 826. H NMR (300 MHz, DMSO-d6
H, Jtrans = 15.9 Hz), 6.96 (d, 2H, = 8.7 Hz), 7.55 (d, 2H,
: 54.5, 114.1, 115.4, 127.0, 129.5,
)
δ
: 3.83 (s, 3H), 6.34 (d,
1
J
J = 8.7 Hz), 7.62 (d, 1H,
1
3
Jtrans = 15.9 Hz). C NMR (75 MHz, DMSO-d6
44.6, 161.6, 169.1.
Data for (E)-(3,4,5)-trimethoxycinnamic acid (4c)
) δ
1
White solid, obtained in 81% yield, m.p. 215.3─219.0 °C, purified by silica gel
column chromatography eluted with hexane/ethyl acetate (1:2 v/v), TLC: R = 0.30
f
-
1
(
hexane-ethyl acetate 1:2 v/v). IR (ATR) v max/cm : 3134
─
2905 (broad band), 2836,
654, 1689, 1627, 1684, 1121, 827. H NMR (300 MHz, DMSO-d6 : 3.78 (s, 3H), 3.87
s, 6H), 6.42 (d, 1H, Jtrans = 16.2 Hz), 6.92 (s, 2H), 7.60 (d, 1H, Jtrans = 16.2 Hz). C
NMR (75 MHz, DMSO- 6 : 52.2, 59.8, 105.3, 117.3, 130.1, 139.8, 144.9, 153.6,
69.2.
Data for (E)-4-nitrocinnamic acid (4d)
1
2
) δ
13
(
d
) δ
1
o
White solid, obtained in 71% yield, m.p. 284.3─285.9 C, purified by silica gel
column chromatography eluted with hexane/ethyl acetate (1:2 v/v), TLC: R = 0.30
f
-1
hexane/ethyl acetate (1:2 v/v), IR (ATR) v max/cm : 3100─2500
(broad band), 1692,
1
1
7
1
526, 1349, 847. H NMR (300 MHz, DMSO-d6
)
δ
: 6.68 (d, 1H, Jtrans = 16.2 Hz),
: 124.1, 124.3, 129.7, 141.2,
13
.62─8.13 (m, 5H). C NMR (75 MHz, DMSO-d6
)
δ
41.6, 148.4, 167.3.
Data for (E)-4-bromocinnamic acid (4e)
White solid, obtained in 84% yield, m.p. 264.0─265.0 °C purified by silica gel

column chromatography eluted with hexane/ethyl acetate (1:2 v/v), TLC: R = 0.30
f
-1
hexane/ethyl acetate (1:2 v/v), IR (ATR) v max/cm : 2970─2760
(broad band), 1886,
1
1
7
692, 1622, 1227, 814. H NMR (300 MHz. DMSO-d6
)
δ
: 6.49 (d, 1H, Jtrans = 16.1 Hz),
13
.43 (d, 2H,
J
= 8.3 Hz), 7.54 (d, 2H, Jtrans = 16.1 Hz), 7.65 (d, 1H,
: 20.7, 129.7, 130.3, 133.6, 135.0, 143.0, 167.6.
Data for (E)-4chlorocinnamic acid (4f)
J = 8.3 Hz). C
NMR (75 MHz, DMSO-d6) δ
White solid, obtained in 77% yield, m.p. 241.2─242.0 °C, purified by silica gel
column chromatography eluted with hexane/ethyl acetate (1:2 v/v), TLC: R = 0.30. IR
f
-1
1
(
ATR) v max/cm : 2928─2520 (broad band), 1902, 1701, 1630, 1400, 821. H NMR (300
1
3
MHz, DMSO- 6 : 6.51 (d, 1H, Jtrans = 15.9 Hz), 7.49─7.59 (m, 5H). C NMR (75
d
)
δ
MHz, DMSO-d6) δ : 120.7, 123.8, 130.5, 132.2, 134.0, 142.7, 167.7.
General procedure for the microwave-assisted synthesis of cinnamates 5a─5f
To a 25.0 mL round-bottom flask, it was added 1.00 mmol of a cinnamic acid
derivative (4a─4f), along with 6-hydroxyisobenzofuran-1(3H)-one (2) (0.459 g, 2.20
mmol), DCC (0.460 g, 2.20 mmol), DMAP (0.0200 g, 0.200 mmol), and 12.0 mL of
methanol. Then, the reaction mixture was submitted to microwave irradiation (40
minutes at 200 W). After this time, the resulting mixture was filtered and washed with
an aqueous solution of citric acid 10% (w/v) (2 x 6.00 mL), distilled water (5.00 mL)
and brine (10.0 mL). The aqueous phase was then extracted with ethyl acetate (3 x 15.0
mL). The organic extracts were combined and the organic phase was dried over
anhydrous magnesium sulfate, filtered and concentrated under reduced pressure. The
resulting material was purified by silica gel column chromatography eluted with
hexane-dichloromethane-ethyl acetate (3:2:1 v/v). The structures of the compounds
5
a─5f are supported by the following data.

Data for (E)-3-oxo-1,3-dihydroisobenzofuran-5-yl-(4-fluoro)-cinnamate (5a
)
Yellow solid, obtained in 71% yield, m.p. 189.1─193.6 °C, purified by silica gel
column chromatography eluted with hexane/dichloromethane/ethyl acetate (3:2:1 v/v),
-1
TLC: R = 0.40 (hexane-dichloromethane-ethyl acetate (3:2:1 v/v), IR (ATR) v max/cm :
f
1
3
2
7
026, 2932, 1767, 1740, 1636, 1238, 829. H NMR (300 MHz, DMSO-d6
)
δ
:
5.42 (s,
= 8.4 Hz),
: 70.5, 116.5
= 2.3 Hz), 118.4, 124.7, 126.8, 128.8, 130.9 (d, JC-F = 3.1
H), 6.87 (d, 1H, Jtrans = 16.0 Hz), 7.30 (t, 2H,
J
= 9.0 Hz), 7.62 (dd, 1H,
J
13
.60-7.60 (m, 2H), 7.87─7.92 (m, 3H). C NMR (75 MHz, DMSO-d6
)
δ
(d, JC-F = 21.8 Hz), 117.1 (d,
J
Hz), 131.6 (d, JC-F = 8.7 Hz), 145.1, 146.1, 151.2, 164.2 (d, JC-F = 248.3 Hz), 165.7,
+
1
70.3. HRMS (M+H ): Calculated for C H FO , 299.0720; found: 299.0719. HRMS
1
7
12
4
+
(
M+Na ): Calculated for C H FNaO : 321.0539; found: 321.0542.
17 11 4
Data for (E)-3-oxo-1,3-dihydroisobenzofuran-5-yl-(4-methoxy)–cinnamate (5b
)
White solid, obtained in 76% yield, m.p. 244.0─247.1 °C, purified by silica gel
column chromatography eluted with hexane/dichloromethane/ethyl acetate (3:2:1 v/v),
-1
TLC: R = 0.35 (hexane-dichloromethane-ethyl acetate (3:2:1 v/v), IR (ATR) v max/cm :
f
1
2
3
5
1
1
934, 2848, 1756, 1728, 1604, 1136, 822. H NMR (300 MHz, DMSO-d6
)
δ
: 3.81 (s,
= 8.7 Hz), 7.58─7.78 (m,
H), 7.84 (d, 1H, Jtrans = 15.9 Hz). C NMR (75 MHz, DMSO-d6 : 55.8, 70.2, 55.7,
H), 5.41 (s, 1H), 6.68 (d, 1H, Jtrans = 15.9 Hz), 7.00 (d, 2H,
J
13
)
δ
14.5, 115.0, 124.5, 126.7, 126.9, 128.9, 130.9, 144.8, 147.1, 151.5, 162.1, 165.4,
+
70.2. HRMS (M+H ): Calculated for C H O , 311.0919; found: 311.0914.
1
8
15
5
Data for (E)-3-oxo-1,3-dihydroisobenzofuran-5-yl-(3,4,5-trimethoxy)-cinnamate (5c
)
White solid, obtained in 66% yield, m.p. 221.0─222.1 °C, purified by silica gel
column chromatography eluted with hexane/dichloromethane/ethyl acetate (3:2:1 v/v),
-1
TLC: R = 0.35 (hexane-dichloromethane-ethyl acetate (3:2:1 v/v), IR (ATR) v max/cm :
f
1
3
062, 2944, 2840, 1768, 1720, 1632, 1130, 838. H NMR (300 MHz, DMSO-d6
)
δ
:

3
7
1
.67 (s, 1H), 3.90 (s, 1H), 5.42 (s, 1H), 6.92 (d, 2H, Jtrans = 16.0 Hz), 7.17 (s, 2H),
1
3
.60─7.84 (m, 4H). C NMR (75 MHz, DMSO-d6
) δ: 55.8, 70.2, 55.7, 114.5, 115.0,
24.5, 126.7, 126.9, 128.9, 130.9, 144.8, 147.1, 151.5, 162.1, 165.4, 170.2. HRMS
+
+
(
M+H ): Calculated for C H O , 371.1131; found: 371.1127. HRMS (M+Na ):
20 19 7
Calculated for C H NaO : 393.0950; found: 393.0949.
2
0
18
7
Data for (E)-3-oxo-1,3-dihydroisobenzofuran-5-yl-(4-nitro)-cinnamate (5d
)
Yellow solid, obtained in 54% yield, m.p. 290.0─292.0 °C, purified by silica
gel column chromatography eluted with hexane/dichloromethane/ethyl acetate (3:2:1
v/v), TLC: R = 0.40 (hexane-dichloromethane-ethyl acetate (3:2:1 v/v), IR (ATR)
f
-1
1
v max/cm : 3074, 2923, 2851, 1752, 1726, 1520, 1146, 842. H NMR (300 MHz, DMSO-d6
)
δ
:
5.43 (s, 2H), 7.12 (d, 1H, Jtrans = 16.2 Hz), 7.72─7.63 (m, 3H), 8.09 (d, 1H, Jtrans =
1
3
1
6.2 Hz), 8.10 (d, 2H, J = 8.9 Hz), 8.27 (d, 2H, J = 8.9 Hz). C NMR (75 MHz,
DMSO- 6
d
)
δ
: 70.3, 118.2, 121.5, 124.4, 124.7, 126.6, 128.7, 130.1, 140.5, 144.4, 145.2,
+
1
48.8, 151.3, 164.6, 170.3. HRMS (M+H ): Calculated for C H NO , 326.0665;
1
7
12
6
+
found: 326.0668 HRMS (M+Na ): Calculated for C H NaNO : 348.0484; found:
1
7
11
6
3
48.0479.
Data for (E)-3-oxo-1,3-dihydroisobenzofuran-5-yl-(4-bromo)-cinnamate (5e
)
White solid, obtained in 75% yield, m.p. 273.4─272.0 °C, purified by silica gel
column chromatography eluted with hexane/dichloromethane/ethyl acetate (3:2:1 v/v),
-1
TLC: R = 0.40 (hexane-dichloromethane-ethyl acetate (3:2:1 v/v), IR (ATR) v max/cm :
f
1
3
2
028, 2926, 2852, 1752, 1715, 1264, 811. H NMR (300 MHz, DMSO-d6
)
δ
: 5.42 (s,
1
3
H), 7.94 (d, 1H, Jtrans = 16.1 Hz), 7.78─7.60 (m, 7H), 7.86 (d, 1H, Jtrans = 16.1 Hz). C
NMR (75 MHz, DMSO-d6
)
δ
: 70.1, 118.1, 118.4, 124.6, 124.8, 126.7, 128.7, 130.9,
+
1
32.4, 133.5, 145.3, 146.3, 151.2, 165.4, 170.4. HRMS (M+H ): Calculated for
+
C H BrO , 358.9919; found: 358.9918. HRMS (M+Na ): Calculated for
17
12
4

+
C H NaBrO : 380.9738; found: 380.9738. HRMS (M+K ): Calculated for C H NO ,
17
11
4
17 12
6
+
3
26.0665; found: 326.0668 HRMS (M+K ): Calculated for C H KBrO : 396.9478;
17 11 4
found: 396.9465.
Data for (E)-3-oxo-1,3-dihydroisobenzofuran-5-yl-(4-chloro)-cinnamate (5f
)
White solid, obtained in 69% yield, m.p. 260.1─264.2 ºC purified by silica gel
column chromatography eluted with hexane/ethyl acetate (3:2:1 v/v ), TLC: R = 0.26
f
-1
(
hexane-dichloromethane-ethyl acetate (3:2:1 v/v), IR (ATR) v max/cm : 3062, 3030,
1
2
968, 2852, 1753, 1715, 1057, 816. H NMR (300 MHz, DMSO-d6
)
δ
: 5.42 (s, 2H),
= 8.6 Hz, = 2.1
: 70.4,
18.0, 118.4, 124.7, 126.7, 128.6, 129.4, 130.8, 133.1, 135.9, 145.0, 145.2, 151.4,
6
.92 (d, 1H, Jtrans = 15.9 Hz), 7.51 (d, 2H, = 8.4 Hz), 7.62 (dd, 1H,
J
J
J
13
Hz), 7.69─7.75 (m, 3H), 7.83─7.91 (m. 3H). C NMR (75 MHz, DMSO-d6) δ
1
1
+
65.5, 170.5. HRMS (M+H ): Calculated for C H ClO , 315.0424; found: 315.0421.
1
7
12
4
+
HRMS (M+Na ): Calculated for C H NaClO : 337.0244; found: 337.0236. HRMS
1
7
11
4
+
(
M+K ): Calculated for C H NO , 326.0665; found: 326.0668.
17 12 6
General procedure for the synthesis of cinnamates
6 and 7, exemplified by the synthesis
of prop-2-yn-1-yl cinnamate (6)
Propargyl alcohol (0.300 g, 5.35 mmol) was added to a 50 mL round-bottom
flask containing 25.0 mL of dichloromethane, 0.720 g (4.86 mmol) of cinnamic acid
(1), 1.10 g (5.35 mmol) of DCC and 0.0594 g (0.486 mmol) of DMAP. After 25
minutes of stirring at room temperature, the reaction mixture was filtered and the
resulting solution washed with 10% citric acid (2 x 20.0 mL), water (15.0 mL) and brine
(30.0 mL). The resulting organic phase was reserved and the aqueous phase was
extracted with dichloromethane (3 x 20.0 mL). The organic extracts were combined,
and the resulting organic phase was dried over MgSO , filtered and concentrated under
4
reduced pressure. The resulting material was purified by silica gel column

chromatography eluted with hexane-ethyl acetate (4:1 v/v). The described procedure
afforded compound 6 in 68% yield (0.613 g, 3.30 mmol). Yellow oil, TLC: R = 0.41
f
-1
(
hexane-ethyl acetate 4:1 v/v). IR (ATR) v max/cm : 3291, 3062, 2919, 2852, 2127, 1713,
1
1
634, 1446, 1307, 1152. H NMR (300 MHz, CDCl )
δ
: 2.51 (t, 1H,
J
= 2.5 Hz), 4.82
3
(d, 2H, J = 2.5 Hz), 6.47 (d, 1H, Jtrans = 16.0 Hz), 7.38─7.40 (m, 3H), 7.51─7.55 (m,
1
3
2
H), 7.75 (d, 1H, Jtrans = 16.0 Hz). C NMR (75 MHz, CDCl )
δ
: 52.0, 74.9, 77.8,
3
1
17.0, 128.2, 128.9, 130.5, 134.1, 145.9, 166.0.
Pent-4-yn-1-yl cinnamate (7) was prepared as described for 6. The structure of
is supported by the following data.
7
Yellow oil, obtained in 86% yield, purified by silica gel column chromatography
eluted with hexane-ethyl acetate (6:1 v/v), TLC: R = 0.53 (hexane-ethyl acetate 6:1 v/v). IR
f
-
1
1
(
ATR) v max/cm : 3296, 3058, 2926, 2118, 1707, 1636, 1446, 1308, 1160. H NMR (300
MHz, CDCl3) : 1.90─2.00 (m, 3H), 2.35 (td, 2H, J1 = 7.0 Hz and J2 = 2.6 Hz), 4.32 (t,
= 6.3 Hz), 6.44 (d, 1H, Jtrans = 16.0 Hz), 7.37─7.40 (m, 3H), 7.51─7.54 (m, 2H),
δ
2
7
8
H, J
13
.69 (d, 1H, Jtrans = 16.0 Hz). C NMR (75 MHz, CDCl )
δ
: 15.3, 27.7, 63.0, 69.0,
3
3.1, 117.9, 128.1, 128.9, 130.3, 134.4, 144.9, 166.9.
Synthesis of compounds 9a, 9c─9m, 10a─10m, exemplified by the synthesis of (1-(2,5-
dichlorobenzyl)-1H-1,2,3-triazol-4-yl)methyl cinnamate (9b)
To a 10.0 mL round-bottom flask, it was added 2,5-(dichloro)benzyl azide
(0.109 g, 0.540 mmol), water (1.00 mL), dichloromethane (1.00 mL), sodium ascorbate
(0.0427 g, 0.216 mmol), prop-2-yn-1-yl cinnamate (6) (0.100 g, 0.540 mmol) and
CuSO ⋅5H O (0.0270 g, 0.108 mmol). The reaction mixture was vigorously stirred at
4
2
room temperature for 30 minutes. Subsequently, saturated Na CO solution (10.0 mL)
2
3
was added and the resulting mixture was extracted with dichloromethane (3 x 20.0 mL).
The organic extracts were combined, and the resulting organic phase was dried over

MgSO , filtered and concentrated under reduced pressure. The resulting material was
4
purified by silica gel column chromatography eluted with hexane-ethyl acetate (2:1 v/v).
The described procedure gave compound 9b in 87% yield (0.181 g, 0.466 mmol). White
solid, m.p. 97.9─98.4 °C, TLC: R = 0.58 (hexane-ethyl acetate-dichloromethane 3:1:3
f
-1
1
v/v). IR (ATR) v max/cm : 3113, 3073, 2974, 1713, 1634, 1493, 1304, 1146. H NMR
300 MHz, CDCl3) : 5.36 (s, 2H), 5.62 (s, 2H), 6.43 (d, 1H, Jtrans = 16.0 Hz),
.17─7.51 (m, 8H), 7.67─7.73 (m, 2H). C NMR (75 MHz, CDCl )
(
δ
1
3
7
1
1
3
4
4
δ: 51.0, 57.6,
3
17.4, 124.1, 128.1, 128.9, 130.2, 130.4, 130.5, 131.0, 131.6, 133.5, 133.8, 134.2,
+
43.5, 145.6, 166.7. HRMS (M+H ): Calculated for C H Cl N O , 388.0620; found:
1
9
16
2
3
2
+
88.0607. HRMS (M+Na ): Calculated for C H Cl NaN O : 410.0439; found:
1
9
15
2
3
2
+
10.0412. HRMS (M+K ): Calculated for C H Cl KN O , 426.0178; found:
1
9
15
2
3
2
26.0166.
Compounds 9a, 9c─9m and 10a─10m were prepared from the corresponding
azides and cinnamates (6 or 7) as described for compound 9b. All the compounds were
1
13
fully characterized by IR and NMR ( H and C). Structures of the remaining
compounds are supported by the following data.
Data for (1-benzyl-1H-1,2,3-triazol-4-yl)methyl cinnamate (9a
)
o
White solid, obtained in 98% yield, m.p. 123.6─124.5 C, purified by silica gel
column chromatography eluted with hexane-ethyl acetate (2:1 v/v), TLC: R = 0.28
f
-1
(
hexane-ethyl acetate-dichloromethane 3:1:3 v/v). IR (ATR) v max/cm : 3117, 3053, 2948,
1
1
707, 1637, 1577, 1493, 1160. H NMR (300 MHz, CDCl )
δ
: 5.33 (s, 2H), 5.52 (s,
3
2
H), 6.42 (d, 1H, Jtrans = 16.0 Hz), 7.29─7.48 (m, 10H), 7.57 (s, 1H), 7.69 (d, 1H,
13
Jtrans = 16.0 Hz). C NMR (75 MHz, CDCl3)
δ
: 54.2, 57.6, 117.4, 123.7, 128.10,
+
1
28.14, 128.8, 128.9, 129.1, 130.4, 134.2, 134.4, 143.4, 145.5, 166.7. HRMS (M+H ):
Calculated for C H N O , 320.1399; found: 320.1390.
1
9
18
3
2

Data for (1-(4-iodobenzyl)-1H-1,2,3-triazol-4-yl)methyl cinnamate (9c)
o
White solid, obtained in 81% yield, m.p. 200.8─201.7 C, purified by silica gel
column chromatography eluted with hexane-ethyl acetate (2:1 v/v), TLC: R = 0.33
f
-1
(
hexane-ethyl acetate-dichloromethane 3:1:3 v/v). IR (ATR) v max/cm : 3132, 3080,
1
3
5
7
025, 2967, 1709, 1632, 1576, 1482, 1304, 1160. H NMR (300 MHz, DMSO-d6
.25 (s, 2H), 5.55 (s, 2H), 6.59 (d, 1H, Jtrans = 16.0 Hz), 7.12 (d, 2H, = 8.3 Hz),
.39─7.41 (m, 3H), 7.62─7.69 (m, 3H), 7.73 (d, 2H, = 8.3 Hz), 8.19 (s, 1H). C
) δ:
J
13
J
NMR (75 MHz, DMSO-d6
)
δ
: 52.8, 57.7, 94.7, 118.2, 125.3, 128.8, 129.3, 130.7, 130.9,
+
1
34.4, 136.1, 138.0, 142.8, 145.4, 166.3. HRMS (M+H ): Calculated for C H IN O ,
19
17
3
2
+
4
46.0365; found: 446.0354. HRMS (M+Na ): Calculated for C H INaN O :
19 16 3 2
+
4
68.0185; found: 468.0178. HRMS (M+K ): Calculated for C H IKN O , 483.9924;
19 16 3 2
found: 483.9910.
Data for (1-(4-nitrobenzyl)-1H-1,2,3-triazol-4-yl)methyl cinnamate (9d)
White solid, obtained in 85% yield, m.p. 194.1─195.0 °C, purified by silica gel
column chromatography eluted with hexane-ethyl acetate (2:1 v/v), TLC: R = 0.18
f
-1
(
hexane-ethyl acetate-dichloromethane 3:1:3 v/v). IR (ATR) v max/cm : 3127, 3083,
1
2
5
J
966, 2852, 1704, 1625, 1523, 1433, 1341, 1154. H NMR (300 MHz, DMSO-d6
) δ:
.28 (s, 2H), 5.78 (s, 2H), 6.59 (d, 1H, Jtrans = 16.0 Hz), 7.39─7.41 (m, 3H), 7.55 (d, 2H,
1
3
= 8.7 Hz), 7.63─7.68 (m, 3H), 8.21 (d, 2H,
J = 8.7 Hz), 8.28 (s, 1H). C NMR (75
MHz, DMSO- 6
d
)
δ
: 52.5, 57.7, 118.2, 124.3, 125.6, 128.8, 129.3, 129.6, 130.9, 134.4,
+
1
3
42.9, 143.6, 145.4, 147.9, 166.3. HRMS (M+H ): Calculated for C H N O ,
19
17
4
4
+
65.1250; found: 365.1232. HRMS (M+Na ): Calculated for C H NaN O : 387.1069;
1
9
16
4
4
+
found: 387.1059. HRMS (M+K ): Calculated for C H KN O , 403.0809; found:
1
9
16
4
4
4
03.0786.

Data for (1-(4-bromobenzyl)-1H-1,2,3-triazol-4-yl)methyl cinnamate (9e)
White solid, obtained in 94% yield, m.p. 182.0─183.2 °C, purified by silica gel
column chromatography eluted with hexane-ethyl acetate (2:1 v/v), TLC: R = 0.30
f
-1
(
hexane-ethyl acetate-dichloromethane 3:1:3 v/v). IR (ATR) v max/cm : 3136, 3082,
1
2
5
7
922, 2852, 1706, 1632, 1577, 1486, 1305, 1162. H NMR (300 MHz, DMSO-d6
.24 (s, 2H), 5.58 (s, 2H), 6.63 (d, 1H, Jtrans = 16.0 Hz), 7.28 (d, 2H, = 8.4 Hz),
.39─7.41 (m, 3H), 7.57 (d, 2H, = 8.4 Hz), 7.63─7.71 (m, 3H), 8.25 (s, 1H). C
) δ:
J
13
J
NMR (75 MHz, DMSO-d6
)
δ
: 52.5, 57.7, 118.0, 121.9, 125.5, 128.9, 129.4, 130.7,
+
1
31.0, 132.2, 134.3, 135.8, 142.7, 145.5, 166.3. HRMS (M+H ): Calculated for
+
C H BrN O , 398.0504; found: 398.0495. HRMS (M+Na ): Calculated for
19
17
3
2
C H BrNaN O : 420.0324; found: 420.0319.
19
16
3
2
Data for (1-(4-chlorobenzyl)-1H-1,2,3-triazol-4-yl)methyl cinnamate (9f)
White solid, obtained in 78% yield, m.p. 171.2─172.6 °C, purified by silica gel
column chromatography eluted with hexane-ethyl acetate (2:1 v/v), TLC: R = 0.37
f
-1
(
hexane-ethyl acetate-dichloromethane 3:1:3 v/v). IR (ATR) v max/cm : 3139, 3058,
1
2
5
1
1
922, 2852, 1709, 1632, 1577, 1486, 1305, 1160. H NMR (300 MHz, DMSO-d6
) δ:
.24 (s, 2H), 5.60 (s, 2H), 6.63 (d, 1H, Jtrans = 16.0 Hz), 7.33─7.71 (m, 10H), 8.25 (s,
1
3
H). C NMR (75 MHz, DMSO-d6
)
δ
: 52.5, 57.7, 118.0, 125.5, 128.9, 129.2, 129.4,
+
30.4, 131.0, 133.4, 134.3, 135.4, 142.7, 145.5, 166.3. HRMS (M+H ): Calculated for
C H ClN O , 354.1009; found: 354.1002.
19
17
3
2
Data for (1-(3,4-difluorobenzyl)-1H-1,2,3-triazol-4-yl)methyl cinnamate (9g)
°
White solid, obtained in 77% yield, m.p. 149.7─150.8 C, purified by silica gel
column chromatography eluted with hexane-ethyl acetate (2:1 v/v), TLC: R = 0.28
f
-1
(
hexane-ethyl acetate-dichloromethane 3:1:3 v/v). IR (ATR) v max/cm : 3132, 3064,
1
2
968, 1710, 1632, 1518, 1434, 1282, 1160. H NMR (300 MHz, CDCl )
δ
:5.34 (s, 2H),
3