Synthesis of piplartine analogs and preliminary findings on structure–antimicrobial activity relationship

Article abstract of DOI:10.1007/s00044-016-1774-9

In this work it is described the synthesis, characterization and antimicrobial and toxicity evaluation of a series of analogs of piplartine, a piperamide found in Piper sp. The compounds structures were confirmed by infrared spectroscopy, ¹H, ¹³C nuclear magnetic resonance, high resolution mass spectroscopy and were evaluated against strains of Candida spp., Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa. Derivative 24 was almost four-fold more potent (IC₅₀: 48.83 μM) and five-fold less toxic (SI > 3) than piplartine (IC₅₀: 189.2 μM; SI: 0.21) against Candida krusei, as well as two-fold more potent than fluconazole (IC₅₀: 104.48 μM). This compound was also active against Candida tropicalis at 97.67 μM. Benzoyl derivative 17 was three-fold more potent (IC₅₀: 85.2 μM) and more than five-fold less toxic (CC₅₀: 231.71 μM) than piplartine (IC₅₀: 315.33 μM and CC₅₀: 41.14 μM) against Staphylococcus aureus. Given these findings, we have found analogs of piplartine which can be assumed as prototypes for the optimization and the development of new antimicrobial (compounds 24 and 17) agents.

Full text of DOI:10.1007/s00044-016-1774-9

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-016-1774-9
ORIGINAL RESEARCH
Synthesis of piplartine analogs and preliminary findings on
structure–antimicrobial activity relationship
Antonio Maciel Fregnan1 Guilherme Andrade Brancaglion2
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Alexandre Francisco Cerqueira Galvão3 Cinara Oliveira D’Sousa Costa3
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Diogo Rodrigo Magalhães Moreira3,4 Milena Botelho Pereira Soares3,4
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Daniel Pereira Bezerra3 Naiara Chaves Silva5 Stella Maria de Souza Morais5
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Josidel Conceição Oliver5 Amanda Latercia Tranches Dias5 Luiz Felipe Leomil Coelho5
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Diogo Teixeira Carvalho1 Danielle Ferreira Dias2 Thiago Belarmino de Souza2
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Received: 23 May 2016 / Accepted: 30 December 2016
© Springer Science+Business Media New York 2017
Abstract In this work it is described the synthesis, char-
acterization and antimicrobial and toxicity evaluation of a
series of analogs of piplartine, a piperamide found in Piper
sp. The compounds structures were confirmed by infrared
spectroscopy, 1H, 13C nuclear magnetic resonance, high
resolution mass spectroscopy and were evaluated against
strains of Candida spp., Staphylococcus aureus, Escher-
ichia coli and Pseudomonas aeruginosa. Derivative 24 was
almost four-fold more potent (IC50: 48.83 μM) and five-fold
less toxic (SI > 3) than piplartine (IC50: 189.2 μM; SI: 0.21)
against Candida krusei, as well as two-fold more potent
than fluconazole (IC50: 104.48 μM). This compound was
also active against Candida tropicalis at 97.67 μM. Benzoyl
derivative 17 was three-fold more potent (IC50: 85.2 μM)
and more than five-fold less toxic (CC50: 231.71 μM) than
piplartine (IC50: 315.33 μM and CC50: 41.14 μM) against
Staphylococcus aureus. Given these findings, we have
found analogs of piplartine which can be assumed as pro-
totypes for the optimization and the development of new
antimicrobial (compounds 24 and 17) agents.
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Keywords Piplartine Analogs Antifungal activity
Antibacterial activity
Introduction
Fungal and bacterial infections represent a serious problem
of public health and, especially among immunocompro-
mised patients related to AIDS, cancer, organ transplants,
old age, and others factors. The increase in the cases of
microbial resistance to available drugs has contributed to
the rise in mortality rates associated with those infections.
Thus, these factors justify the need for research and
development of new antibacterial and antifungal agents as
an alternative to improve the therapeutic arsenal. (Ling et al.
2015; Low and Rotstein 2011).
Natural products are one of the most rational sources of
new drugs. They evolved naturally to perform specialized
functions in plants and other organisms, so their refined
structural backbones often allow the possibility for biolo-
gical activity or structural modification to improve potency
and pharmacokinetics (Chen et al. 2015). Many plant spe-
cies produce secondary metabolites that are naturally toxic
to bacteria, fungi and other parasitic organisms, as a defense
response and fight for survival in competitive environments
(Chin et al. 2006; Balunas and Kinglorn 2005; Paterson and
Anderson 2005). Moreover, edible plant species often
contain bioactive compounds, which justify the medicinal
effects resulting from observations of their intake (Lampe
2003; Cragg and Newman 2005).
Electronic supplementary material The online version of this article
(doi:10.1007/s00044-016-1774-9) contains supplementary material,
which is available to authorized users.
* Thiago Belarmino de Souza
thiagobs83@yahoo.com.br
1
Faculdade de Ciências Farmacêuticas, Universidade Federal de
Alfenas, Alfenas 37130-000 MG, Brazil
2
Instituto de Química, Universidade Federal de Alfenas, Alfenas
37130-000 MG, Brazil
3
Centro de Pesquisas Gonçalo Moniz, Fundação Oswaldo Cruz,
Rio de Janeiro 40296-710 BA, Brazil
4
Centro de Biotecnologia e Terapia Celular, Hospital São Rafael,
São Marcos 41253-190 BA, Brazil
5
Instituto de Ciências Biomédicas, Universidade Federal de
Alfenas, Alfenas 37130-000 MG, Brazil

Med Chem Res
As members of Piperaceae family, species of the genus
Piper sp. are widely consumed around the world, especially
among those populations that often use peppers in the
preparation of traditional menus. Different biological
activities have been reported for extracts of these species,
their isolated constituents and derivatives thereof, such as
anti-inflammatory (Sun et al. 2015), antiplatelet (Park et al.
2008), anti-diabetic (Rao et al. 2012), anti-microbial
(Bezerra et al. 2013), anti-parasitary (Moraes et al. 2012;
Cotinguiba et al. 2009), antitumor (Bezerra et al. 2006), and
insecticide actions (Alécio et al. 1998). For a long time, it
has been known that the main bioactive components found
in these species, mainly in the roots, are alkaloid amides
called piperamides (Okwute and Egharevba 2013). Piplar-
tine, also known as piperlongumine, is an important piper-
amide found in Piper sp., accounting for many of the
biological actions associated with the consumption of these
species or their extracts.
Fig. 1 Chemical structure of piplartine
compounds and their antifungal, antibacterial and cytotoxic
activities. (Fig. 1)
Materials and methods
Chemistry
Piplartine discretely inhibits the phytopathogenic fungus
Pyricularia grisea (Lee et al. 2001), has antifungal activities
against Cladosporium sphaerospermum and C. cladospor-
ioides, comparable to nystatin and miconazole (Navickiene
et al. 2006; Silva et al. 2012) and is also able to inhibit the
production of mycotoxins by some species of Aspergillus
and Penicillium in agricultural products (Lee et al. 2001,
2007). An antibacterial study of isolates from P. longum
showed that piplartine was four times more potent than
benzylpenicillin against Gram-positive bacteria Bacillus
subtilis (Reddy et al. 2011). Another study found that
piplartine could also inhibit the growth of Klebsiella
pneumoniae, Pseudomonas aeruginosa and Staphylococcus
aureus in a concentration-dependent manner (Naika et al.
2010). Some derivatives showed promising activities of a
set of piplartine-like piperamides against S. aureus, B.
subtilis, Aspergillus flavus and Candida albicans (Prashanth
et al. 2012). Analogs of piplartine with a 4-alkoxy cinnamic
moiety have exhibited submicromolar minimum inhibitory
concentrations against Mycobacterium tuberculosis
(Prithwiraj et al. 2011). More recently, derivatives of
piplartine were synthesized and evaluated as antibacterial
and cytotoxic agents (Kumar et al. 2013; Adams et al.
2012); those derivatives that possessed substituents such as
phenyl, 2-nitrophenyl and 2-thiophenyl at the dihydropyr-
idone ring showed cytostatic effects in bacteria and cancer
cell lines. In another recent work, the synthesis of many
structural analogs of piplartine was also described and the
need for electrophilic sites in these compounds for the
maintenance of this biological action was proven by cyto-
toxicity evaluations (Adams et al. 2012).
Melting point of the compounds was determined on
Microquímica MOAs 301 apparatus and was uncorrected.
IR spectroscopy was performed on a Shimadzu a FTIR-
Affinity-1 spectrometer. 1H-NMR and 13C-NMR spectra
were obtained on a Bruker AC-300 spectrometer (300 MHz
1
for H-NMR and 75 MHz for 13C spectra) in deuterated
chloroform, dimethylsulfoxide or methanol. Chemical shifts
(δ) were reported in parts per million (ppm) with reference
to tetramethylsilane as internal standard and coupling con-
stants (J) were reported in Hertz (Hz). The following
abbreviations were used for the 1H multiplicities: singlet (s),
doublet (d) and multiplet (m). High resolution mass spectra
(HRMS) were acquired using a LCMS-IT-TOF mass
spectrometer and the samples were solubilized in MeOH +
0.1% formic acid, following manual injection. Reaction
courses and product mixtures were monitored by thin-layer
chromatography (TLC) on commercial silica gel 60 plates.
For chromatography, column grade silica gel (0.040–0.063
mm mesh size) was employed. The logP values of the
compounds were determined by ChemBioDraw Ultra 12.0
program.
General procedure for the preparation of acyl chlorides
5–8
To a solution of 2.5 mmol of cinnamic acid in 10 mL of
dichloromethane were added 5 mmol of thionyl chloride
and 10% mmol of N,N-dimethylformamide. After this
addition, the mixture was stirred at room temperature until
completion of the reaction, as noted by TLC. The crude
solution of the acyl chlorides in dichloromethane were
used in further steps without prior treatment or
purification.
In view of these findings and as part of our interest in
synthesizing natural analogs for bioactivity and selectivity
optimization (Abrão et al. 2015; Souza et al. 2014, 2015),
we report herein the preparation of new piplartine-like

Med Chem Res
General procedure for the preparation of hydrazides 9–12
mmol of 11 and 1.5 mmol of phthalic anhydride, according
to the general procedure. The product was obtained in 70%
yield as white crystals; mp 189–190 °C; IR (ῡ/cm−1): 3213,
2980, 1739, 1654, 1619, 1596, 1581, 1512, 1467. 1H-NMR
(300 MHz, DMSO-d6) δ: 10.73 (1H, s, N-H), 7.99–7.93
(4H, m, Ar–H), 7.57 (1H, d, olefin, J3 = 12.0 Hz),
7.27–7.22 (2H, m, Ar–H), 7.02 (1H, d, Ar–H, J5 = 6,2 Hz),
6.69 (1H, d, olefin, J3 = 12.0 Hz), 3.81 (3H, s, methylic),
3.80 (3H, s, methylic). 13C-NMR (75 MHz, DMSO-d6) δ:
165.3 (1C, C-9), 164.6 (2C, C-10), 150.8 (1C, C-3), 148.9
(1C, C-4), 142.3 (1C, C-7), 135.2 (2C, C-13), 129.5 (2C, C-
11), 126.9 (1C, C-1), 123.7 (2C, C-12), 122.1 (1C, C-6),
115.2 (1C, C-8), 111.7 (1C, C-5), 110.4 (1C, C-2), 55.5
(1C, OCH3), 55.4 (1C, OCH3). HRMS-ESI: m/z calcd. for
C19H16N2O5 (M+H)+: 353.1132; found: 353.0984.
To a solution of 12.5 mmol of hydrazine hydrate in 10 mL
of acetonitrile at 5 °C was added dropwise 2.5 mmol of acid
chlorides 5–8. After this addition, the mixture was allowed
to warm up to room temperature until completion of the
reaction, as noted by TLC. Then, 50 mL of water were
added to the mixture and the product was extracted with
ethyl acetate (3 × 25 mL). The organic layer was dried by
anhydrous sodium sulfate, filtered, and the solvent was
removed under reduced pressure. The hydrazide derivatives
were used without previous purification.
General procedure for the preparation of phthalimides
13–16
(E)-N-(1,3-dioxoisoindolin-2-yl)-3-(3,4,5-trimethox-
To a solution of 9–12 (1.5 mmol) in glacial acetic acid (30
mL) was added 1.5 mol of phthalic anhydride and this
mixture was heated under reflux for 3 h. The mixture was
cooled and poured into iced water and the formed solid was
filtered.
yphenyl)acrylamide (16) This compound was prepared by
1.5 mmol of 12 and 1.5 mmol of phthalic anhydride,
according to the general procedure. The product was
obtained in 85% yield as white crystals; mp >230 °C; IR (ῡ/
cm−1): 3187, 2979, 1791, 1682, 1651, 1624, 1588. 1H-
NMR (300 MHz, DMSO-d6) δ: 10.78 (1H, s, N–H),
8.00–7.93(4H, m, Ar–H), 7.58 (1H, d, olefin, J3 = 12.0 Hz),
7.02 (2H, s, Ar–H), 6.78 (1H, d, olefin, J3 = 12.0 Hz), 3.83
(6H, s, methylic), 3.71 (3H, s, methylic). 13C-NMR (75
MHz, DMSO-d6) δ: 165.6 (1C, C-9), 164.8 (2C, C-10),
153.5 (2C, C-3/C-5), 142.3 (1C, C-7), 139.7 (1C, C-4),
135.6 (2C, C-13), 130.2 (1C, C-1), 129.9 (2C, C-11), 124.1
(2C, C-12), 117.5 (1C, C-8), 105.9 (2C, C-2/C-6), 60.5 (1C,
OCH3), 56.3 (2C, OCH3). HRMS-ESI: m/z calcd. for
C20H18N2O6 (M+Na)+: 405.1057; found: 405.0894.
(E)-N-(1,3-dioxoisoindolin-2-yl)cinnamamide (13) This
compound was prepared by 1.5 mmol of 9 and 1.5 mmol of
phthalic anhydride, according to the general procedure. The
product was obtained in 90% yield as white crystals; mp
181–182 °C; IR (ῡ/cm−1): 3376, 2887, 1738, 1677, 1633,
1566, 1496, 1467. 1H-NMR (300 MHz, CDCl3) δ:
7.86–7.79 (4H, m, Ar–H), 7.63(1H, d, olefin, J3 = 12.0 Hz),
7.55–7.53 (2H, m, Ar–H), 7.34–7.32 (3H, Ar–H), 6.68 (1H,
d, olefin, J3 = 12.0 Hz), 5.38 (1H, s, N–H). 13C-NMR (75
MHz, CDCl3) δ: 166.1 (1C, C-9), 165.2 (2C, C-10), 143.5
(1C, C-7); 134.7 (2C, C-13), 134.3 (1C, C-1), 130.1 (2C, C-
11), 130.0 (1C, C-4), 128.6 (2C, C-3/C-5), 127.8 (2C, C-2/
C-6), 123.3 (2C, C-12), 116.3 (1C, C-8). HRMS-ESI: m/z
calcd. for C17H12N2O3 (M+Na)+: 315.0740; found:
315.0564.
(E)-N-(1,3-dioxoisoindolin-2-yl)-3-(4-methoxyphenyl)
acrylamide (14) This compound was prepared by 1.5 mmol
of 10 and 1.5 mmol of phthalic anhydride, according to the
general procedure. The product was obtained in 85% yield
as white crystals; mp 233–234 °C; IR (ῡ/cm−1): 3223, 3008,
1737, 1655, 1623, 1599, 1570, 1520.1H-NMR (300 MHz,
DMSO-d6) δ: 10.77 (1H, s, N–H), 8.00–7.92(4H, m, Ar–H),
7.64–7.56 (3H, m, olefin, Ar–H), 7.02 (2H, d, Ar–H, J3 =
8,8 Hz), 6.66 (1H, d, olefin, J3 = 12.0 Hz), 3.80 (3H, s,
methylic). 13C-NMR (75 MHz, DMSO-d6) δ: 165.7 (1C, C-
9), 165.0 (2C, C-10), 161.4 (1C, C-4), 142.4 (1C, C-7),
135.6 (2C, C-13); 130.1 (2C, C-11), 129.9 (2C, C-2/C-6),
127.1 (1C, C-1), 124.1 (2C, C-12), 115.4 (1C, C-8), 114.9
(2C, C-3/C-5), 55.7 (1C, OCH3). HRMS-ESI: m/z calcd. for
C18H14N2O4 (M+Na)+: 345.0846; found: 345.0678.
(E)-3-(3,4-dimethoxyphenyl)-N-(1,3-dioxoisoindolin-2-
yl)acrylamide (15) This compound was prepared by 1.5
General procedure for the preparation of
benzoylhydrazides 17–20
To a solution of 9–12 (1.5 mmol) in 5 mL of pyridine at 5 °
C was added dropwise 1.5 mmol of benzoyl chloride and
after addition, the mixture was allowed to warm up to room
temperature until completion of the reaction, as reported by
TLC. Then 1 M HCl was added to the mixture until pH 1
and the precipitate formed was filtered affording the pure
products.
(E)-N′-cinnamoylbenzohydrazide (17) This compound
was prepared by 1.5 mmol of 9 and 1.5 mmol of benzoyl
chloride, according to the general procedure. The product
was obtained in 94% yield as white crystals; mp 210–211 °
C; IR (ῡ/cm−1): 3290, 3213, 3023, 1692, 1641, 1630, 1601,
1587, 1511, 1487. 1H-NMR (300 MHz, DMSO-d6) δ: 10.51
(1H, d, N–H, J3 = 1.5 Hz), 10.21 (1H, d, N–H, J3 = 1.5
Hz), 7.92–7.89(2H, m, Ar–H), 7.64–7.42 (9H, m, Ar–H,
olefin), 6.75 (1H, d, olefin, J3 = 12.0 Hz). 13C-NMR (75
MHz, DMSO-d6) δ: 165.4 (1C, C-9), 164.4 (1C, C-10),
140.3 (1C, C-7), 134.5 (1C, C-1), 132.4 (1C, C-14), 131.8

Med Chem Res
(1C, C-11), 129.8 (1C, C-4), 129.0 (2C, C-3/C-5), 128.5
(2C, C-2/C-6), 127.7 (2C, C-13), 127.4 (2C, C-12), 119.4
(1C, C-8). HRMS-ESI: m/z calcd. for C16H14N2O2 (M
+Na)+: 289.0947; found: 289.0750.
118.8 (1C, C-8), 105.2 (2C, C-2/C-6), 60.1 (1C, OCH3),
55.9 (2C, OCH3). HRMS-ESI: m/z calcd. for C19H20N2O5
(M+Na)+: 379.1264; found: 379.1096.
(E)-N′-(3-(4-methoxyphenyl)acryloyl)benzohydrazide
(18) This compound was prepared by 1.5 mmol of 10 and
1.5 mmol of benzoyl chloride, according to the general
procedure. The product was obtained in 86% yield as white
crystals; mp 182–183 °C; IR (ῡ/cm−1): 3203, 2999, 1693,
General procedure for the preparation of amides 21–24
To a solution of 5.6 mmol of morpholine in 10 mL of
dichloromethane at 5 °C was added dropwise 1.4 mmol of
acid chlorides 5–8 and after the addition, the mixture was
allowed to warm up to room temperature until completion
of the reaction, as noticed by TLC. Then, 50 mL de water
were added to the mixture and the product was extracted
with dichloromethane (3 × 25 mL). The organic layer was
dried by anhydrous sodium sulfate, filtered and the solvent
was removed under reduced pressure, affording the pure
amides.
(E)-1-morpholino-3-phenylprop-2-en-1-one (21) This
compound was prepared by 1.4 mmol of 5 and 5.6 mmol of
morpholine, according to the general procedure. The pro-
duct was obtained in 72% yield as white crystals; mp 90–91
°C; IR (ῡ/cm−1): 3080, 3026, 2964, 2922, 2856, 1644,
1595, 1577, 1494. 1H-NMR (300 MHz, CDCl3) δ: 7.69
(1H, d, olefin, J3 = 12.0 Hz), 7.53–7.33(5H, m, Ar–H), 6.84
(1H, d, olefin, J3 = 12.0 Hz), 3.71 (8H, s, methylenic). 13C-
NMR (75 MHz, CDCl3) δ: 165.5 (1C, C-9), 143.1 (1C, C-
7), 135.0 (1C, C-1), 129.7 (1C, C-4), 128.7 (2C, C-3/C-5),
127.7 (2C, C-2/C-6), 116.5 (1C, C-8), 66.8 (4C, C-10, C-
11). HRMS-ESI: m/z calcd. for C13H15NO2 (M+Na)+:
240.0995 found: 240.0846.
1
1642, 1627, 1601, 1577, 1508, 1479. H-NMR (300 MHz,
DMSO-d6) δ: 10.47 (1H, d, N–H, J3 = 1.5 Hz), 10.10 (1H,
d, N–H, J3 = 1.5 Hz), 7.90 (2H, d, Ar–H, J3 = 8.5 Hz),
7.59–7.50 (6H, m, Ar–H, olefin), 7.00 (2H, d, Ar–H, J3 =
8.5 Hz), 6.60 (1H, d, olefin, J3 = 12.0 Hz), 3.80 (3H, s,
methylic). 13C-NMR (75 MHz, DMSO-d6) δ: 165.4 (1C, C-
9), 164.7 (1C, C-10), 160.6 (1C, C-4), 140.0 (1C, C-7),
132.4 (1C, C-14); 131.8 (1C, C-11), 129.3 (2C, C-2/C-6),
128.4 (2C, C-13), 127.4 (2C, C-12), 127.1 (1C, C-1), 116.9
(1C, C-8), 114.4 (2C, C-3/C-5), 55.3 (1C, OCH3). HRMS-
ESI: m/z calcd. for C17H16N2O3 (M+Na)+: 319.1053;
found: 319.0862.
(E)-N′-(3-(3,4-dimethoxyphenyl)acryloyl)benzohy-
drazide (19) This compound was prepared by 1.5 mmol of
11 and 1.5 mmol of benzoyl chloride, according to the
general procedure. The product was obtained in 82% yield
as white crystals; mp 188–189 °C; IR (ῡ/cm−1): 3292, 3188,
1
1667, 1642, 1622, 1564, 1513, 1467. H-NMR (300 MHz,
DMSO-d6) δ: 10.48 (1H, d, N–H, J3 = 1.5 Hz), 10.06 (1H,
d, N–H, J3 = 1.5 Hz), 7.92–7.88 (1H, m, Ar–H), 7.58–7.45
(3H, m, Ar–H, olefin), 7.22–7.17 (2H, m, Ar–H), 7.02–6.98
(1H, m, Ar–H), 6.64 (1H, d, olefin, J3 = 12.0 Hz), 3.81 (3H,
s, methylic), 3.79 (3H, s, methylic). 13C-NMR (75 MHz,
DMSO-d6) δ: 165.7 (1C, C-9), 164.8 (1C, C-10), 150.4 (1C,
C-5), 148.9 (1C, C-4), 140.4 (1C, C-7), 140.1 (1C, C-1),
132.5 (1C, C-14), 131.8 (1C, C-11), 128.5 (2C, C-13),
127.4 (2C, C-12), 121.6 (1C, C-2), 117.1 (1C, C-8), 111.8
(2C, C-3), 110.3 (1C, C-6), 55.6 (1C, OCH3), 55.5 (1C,
OCH3). HRMS-ESI: m/z calcd. for C18H18N2O4 (M+Na)+:
349.1159; found: 349.1004.
(E)-3-(4-methoxyphenyl)-1-morpholinoprop-2-en-1-one
(22) This compound was prepared by 1.4 mmol of 6 and
5.6 mmol of morpholine, according to the general proce-
dure. The product was obtained in 75% yield as white
crystals; mp 105–106 °C; IR (ῡ/cm−1): 2963, 2909, 2860,
1
1644, 1593, 1573, 1510. H-NMR (300 MHz, CDCl3) δ:
7.65 (1H, d, olefin, J3 = 12.0 Hz), 7.46 (2H, dd, Ar–H, J3 =
8,50 J4 = 4,7 Hz), 6.88 (2H, dd, Ar–H, J3 = 8,50 J4 = 4,7
Hz), 6.70 (1H, d, olefin, J3 = 12.0 Hz), 3.82 (3H, s,
methylic), 3.70 (8H, s, methylenic). 13C-NMR (75 MHz,
CDCl3) δ: 165.8 (1C, C-9), 160.9 (1C, C-4), 142.9 (1C, C-
7), 129.3 (2C, C-2/C-6), 127.8 (1C, C-1), 114.2 (2C, C-3/C-
5), 113.9 (1C, C-8), 66.8 (4C, C-10, C-11), 55.3 (1C,
OCH3),. HRMS-ESI: m/z calcd. for C14H17NO3 (M+Na)+:
270.1101 found: 270.0921.
(E)-N′-(3-(3,4,5-trimethoxyphenyl)acryloyl)benzohy-
drazide (20) This compound was prepared by 1.5 mmol of
12 and 1.5 mmol of benzoyl chloride, according to the
general procedure. The product was obtained in 87% yield
as white crystals; mp 183–184 °C; IR (ῡ/cm−1): 3445, 3326,
3069, 2998, 1665, 1644, 1641, 1602, 1581, 1478. 1H-NMR
(300 MHz, DMSO-d6) δ: 10.50 (1H, d, N–H, J3 = 1.5 Hz),
10.10 (1H, d, N–H, J3 = 1.5 Hz), 7.92–7.89 (2H, m, Ar–H),
7.54–7.49 (4H, m, Ar–H, olefin), 6.69 (2H, s, Ar–H), 6.60
(1H, d, olefin, J3 = 12.0 Hz), 3.83 (6H, s, methylic), 3.69
(3H, s, methylic). 13C-NMR (75 MHz, DMSO-d6) δ: 165.3
(1C, C-9), 164.5 (1C, C-10), 153.1 (2C, C-3/C-5), 140.4
(1C, C-7), 138.9 (1C, C-4); 132.4 (1C, C-14), 131.8 (1C, C-
11), 130.2 (1C, C-1), 128.4 (2C, C-13), 127.4 (2C, C-12),
(E)-3-(3,4-dimethoxyphenyl)-1-morpholinoprop-2-en-1-
one (23) This compound was prepared by 1.4 mmol of 7
and 5.6 mmol of morpholine, according to the general
procedure. The product was obtained in 72% yield as white
crystals; mp 120–121 °C; IR (ῡ/cm−1): 2980, 2937, 2857,
1
1643, 1591, 1514, 1455. H-NMR (300 MHz, CDCl3) δ:
7.63 (1H, d, olefin, J3 = 12.0Hz), 7.10 (1H, dd, Ar–H, J3 =
8,50 Hz, J4 = 2,0 Hz), 7.02 (1H, d, Ar–H, J4 = 2,0 Hz), 6.84
(1H, d, Ar–H, J3 = 8.5 Hz), 6.68 (1H, d, olefin, J3 = 12.0

Med Chem Res
Hz), 3.90 (6H, s, methylic), 3.70 (8H, s, methylenic). 13C-
NMR (75 MHz, CDCl3) δ: 165.7 (1C, C-9), 150.6 (1C, C-
3), 149.1 (1C, C-4), 143.2 (1C, C-7), 128.0 (1C, C-1), 121.8
(1C, C-6), 114.1 (1C, C-8), 111.0 (1C, C-5), 109.8 (1C, C-
2), 66.8 (4C, C-10, C-11), 55.9 (2C, OCH3). HRMS-ESI:
m/z calcd. for C15H19NO4 (M+H)+: 278.1387 found:
278.1215.
as white crystals after purified by recrystallization from
ethanol/water solution 80:20; mp 148–149 °C; IR (ῡ/cm−1):
3061, 2937, 1757, 1725, 1623, 1595. H-NMR (300 MHz,
1
CDCl3) δ: 7.65 (1H, d, olefin, J3 = 12.0 Hz), 7.51 (2H, d,
Ar–H, J3 = 8,50 Hz), 6.91 (2H, d, Ar–H, J3 = 8,50 Hz),
6.43 (1H, d, olefin, J3 = 12.0 Hz), 3.84 (3H, s, methylic),
2.86 (4H, s, methylenic). 13C-NMR (75 MHz, CDCl3) δ:
169.4 (1C, C-9), 162.4 (2C, C-10), 162.3 (1C, C-4), 149.7
(1C, C-7), 130.5 (2C, C-2/C-6), 126.2 (1C, C-1), 114.5 (2C,
C-3/C-5), 108.5 (1C, C-8), 55.4 (1C, OCH3), 25.5 (2C, C-
11). HRMS-ESI: m/z calcd. for C14H13NO5 (M+Na)+:
298.0686 found: 298.0526.
(E)-1-morpholino-3-(3,4,5-trimethoxyphenyl)prop-2-en-
1-one (24) This compound was prepared by 1.4 mmol of 8
and 5.6 mmol of morpholine, according to the general
procedure. The product was obtained in 86% yield as white
crystals; mp 127–128 °C; IR (ῡ/cm−1): 2951, 2896, 2846,
1
1640, 1580, 1504, 1498. H-NMR (300 MHz, CDCl3) δ:
(E)-2,5-dioxopyrrolidin-1-yl
3-(3,4-dimethoxyphenyl)
7.59 (1H, d, olefin, J3 = 12.0 Hz), 6.72–6.67 (3H, m, Ar–H,
olefin), 3.87 (9H, s, methylic), 3.70 (8H, s, methylenic).
13C-NMR (75 MHz, CDCl3) δ: 165.5 (1C, C-9), 153.4 (2C,
C-3/C-5), 143.3 (1C, C-7), 139.7 (1C, C-4), 130.6 (1C, C-
1), 115.6 (1C, C-8), 105.0 (2C, C-2, C-6), 66.8 (4C, C-10,
C-11), 60.9 (1C, OCH3), 56.1 (2C, OCH3). HRMS-ESI: m/z
calcd. for C16H21NO5 (M+H)+: 308.1492 found: 308.1294.
acrylate (27) This compound was prepared by 1.4 mmol of 7
and 2.8 mmol of N-hydroxysuccinimide, according to the
general procedure. The product was obtained in 88% yield as
white crystals after purified by recrystallization from ethanol/
water solution 80:20; mp 149–150 °C; IR (ῡ/cm−1): 3074,
1
2967, 1759, 1728, 1621, 1598. H-NMR (300 MHz, CDCl3)
δ: 7.84 (1H, d, olefin, J3 = 12.0 Hz), 7.15 (1H, d, Ar–H, J3 =
8,50 Hz), 7.06 (1H, s, Ar–H), 6.88 (1H, d, Ar–H, J3 = 8.5
Hz), 6.43 (1H, d, olefin, J3 = 12.0 Hz), 3.91 (6H, s, methylic),
2.86 (4H, s, methylenic). 13C-NMR (75 MHz, CDCl3) δ:
169.4 (1C, C-9), 162.2 (2C, C-10), 152.2 (1C, C-5), 149.9
(1C, C-7), 149.3 (1C, C-4), 126.4 (1C, C-1), 123.8 (1C, C-2),
111.3 (1C, C-8), 109.8 (1C, C-3), 108.8 (1C, C-6), 55.9 (2C,
OCH3), 25.6 (2C, C-11). HRMS-ESI: m/z calcd. for
C15H15NO6 (M+Na)+ 328.0792 found: 328.0600.
General procedure for the preparation of derivatives N-
hydroxysuccinimides 25–28
To a solution of 1.4 mmol of 5–8 in 10 mL of dichlor-
omethane were added 2.1 mmol of N-hydroxysuccinimide
and 2.1 mmol of pyridine. The mixture was stirred at room
temperature until completion of the reaction, noticed by
TLC. Then 2 M HCl was added to the mixture until pH 1
and the product was extracted with ethyl acetate (3 × 25
mL). The organic layer was washed with NaOH 10% (3 ×
25 mL), dried with anhydrous sodium sulfate, filtered and
removed under reduced pressure affording the pure acyl
derivatives after purified by recrystallization from ethanol/
water solution 80:20.
(E)-2,5-dioxopyrrolidin-1-yl 3-(3,4,5-trimethoxyphenyl)
acrylate (28) This compound was prepared by 1.4 mmol of
8 and 2.8 mmol of N-hydroxysuccinimide, according to the
general procedure. The product was obtained in 70% yield
as white crystals after purified by recrystallization from
aqueous ethanol (80% v/v); mp 176–177 °C; IR (ῡ/cm−1):
1
3009, 2940, 1758, 1728, 1625, 1578. H-NMR (300 MHz,
(E)-2,5-dioxopyrrolidin-1-yl cinnamate (25) This com-
pound was prepared by 1.4 mmol of 5 and 2.8 mmol of N-
hydroxysuccinimide, according to the general procedure.
The product was obtained in 83% yield as white crystals
after purified by recrystallization from ethanol/water solu-
tion 80:20; mp 177–178 °C; IR (ῡ/cm−1): 3065, 2943,
CDCl3) δ: 7.82 (1H, d, olefin, J3 = 12.0 Hz), 6.78 (2H, s,
Ar–H), 6.48 (1H, d, olefin, J3 = 12.0 Hz), 3.89 (9H, s,
methylic), 2.86 (4H, s, methylenic). 13C-NMR (75 MHz,
CDCl3) δ: 169.3 (1C, C-9), 162.0 (2C, C-10), 153.4 (2C, C-
3/C-5), 149.9 (1C, C-7), 141.2 (1C, C-4), 128.8 (1C, C-1),
110.5 (1C, C-8), 105.8 (2C, C-2/C-6), 60.9 (1C, OCH3),
56.1 (2C, OCH3), 25.6 (2C, C-11). HRMS-ESI: m/z calcd.
for C16H17NO7 (M+Na)+: 358.0897 found: 358.0721.
1
1755, 1723, 1625, 1574. H-NMR (300 MHz, CDCl3) δ:
7.92 (1H, d, olefin, J3 = 12.0 Hz), 7.58–7.25(5H, m, Ar–H),
6.68 (1H, d, olefin, J3 = 12.0 Hz), 2.88 (4H, s, methylenic).
13C-NMR (75 MHz, CDCl3) δ: 169.2 (1C, C-9), 162.0 (2C,
C-10), 150.0 (1C, C-7), 133.4 (1C, C-1), 131.5 (1C, C-4),
129.0 (2C, C-3/C-5), 128.6 (2C, C-2/C-6), 111.5 (1C, C-8),
25.6 (2C, C-11). HRMS-ESI: m/z calcd. for C13H11NO4 (M
+Na)+: 268.0580 found: 268.0433.
In vitro bioassays
Antibacterial and antifungal activities evaluation
The piplartine analogs were evaluated in vitro for their
antimicrobial activities against the fungi through a Mueller
Hinton broth microdilution method and with the metho-
dology and interpretative criteria proposed by document
M27A3 (CLSI 2008) and through a standard Mueller
(E)-2,5-dioxopyrrolidin-1-yl 3-(4-methoxyphenyl)acry-
late (26) This compound was prepared by 1.4 mmol of 6
and 2.8 mmol of N-hydroxysuccinimide, according to the
general procedure. The product was obtained in 75% yield

Med Chem Res
Scheme 1 Synthesis of
piplartine analogs. i SOCl2,
DMF, CH2Cl2, r.t.; ii NH2NH2.
H2O, CH3CN, 0 °C; iii phthalic
anhydride, HAc, reflux; iv
benzoyl chloride, piridine, 0 °C;
v morpholine, CH2Cl2, r.t.; vi
NHS, CH2Cl2, r.t
Hinton broth microdilution method for bacteria proposed by
document M7A6 (CLSI 2012). The stock solutions of all
the compounds were prepared in DMSO 1% at final con-
centration and tested at concentrations (µg/mL) 100, 60, 30,
15, 7.5, 3.75, 1.875, 0.468, 0.23, 0.06. The standard drug
fluconazole was applied as control of fungistatic action at
concentrations from 64 to 0.031 g/mL and the standard drug
chloramphenicol as a control of bacteriostatic action at
concentrations from 125 to 0.06 g/mL. The microplates
were incubated at 35 °C for 24 h for bacteria and 37 °C and
for 24 h for fungi. Results were visualized and analyzed at
530 nm in an Anthos Zenyth 200rt Microplate Reader. The
inhibitory concentrations of microbial growth were deter-
mined at 50% (IC50) and 90% (IC90) in µmol/mL and
compared among the microorganisms. The tests were all
done in duplicates.
mg/mL) was added and the cells were incubated again for
an additional 4 h period. Then, the medium was carefully
removed and added to each well 100 μL of DMSO. The
plates were shaken for 5 min and absorbance for each
sample was measured in a spectrophotometric microplate
reader at 560 nm. The percentage of cytotoxicity was cal-
culated as [(A−B)/A × 100)], where A and B are the
absorbance of control and treated cells, respectively. Data
were analyzed using linear regression to obtain values for
CC50 and CC90 (cytotoxic concentration for 50 and 90% of
cells, respectively). Selectivity indexes were expressed as
the ratio CC50/IC50.
Results and discussion
Chemistry
Toxicity evaluation
The proposed piplartine analogs were obtained by a simple
and objective synthetic route (Scheme 1); the compounds
were achieved in good yields and high purity and char-
The toxicity of the compounds (200–1.5 μg/mL) to non-
tumor cell line, BHK-21 cells (kidney cells of hamster), was
determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyl tetrazolium bromide (MTT) method (Souza et al.
2015). The BHK-21 cells at a concentration of 1.0 × 105
cells/mL were distributed in a 96-well plate, 98 μL in each
well with 2 μL of test compounds at different concentra-
tions. The plate was incubated at 37 °C in an incubator at
5% CO2 for additional 48 h. After, 10 μL of MTT dye (5
1
acterized by infrared, H and 13C nuclear magnetic reso-
nance and high resolution mass spectroscopy. Here in,
several classes of compounds containing different groups
have been synthesized in substitution for the heterocyclic
ring of piplartin, such as α,β-unsaturated carbonyl hetero-
cyclic derivatives (phthalimides), α,β-saturated carbonyl
heterocyclic derivatives (succinimides), non-carbonylated

Med Chem Res
Table 1 Chemical structures, yields and logP values of the synthesized compounds
Compound
R1
R2
R3
X
Y
cLogPa
Yield (%)
90
13
H
H
H
NH
2.45
14
15
16
H
OCH3
OCH3
OCH3
H
NH
NH
NH
2.33
2.2
85
70
85
OCH3
OCH3
H
OCH3
2.08
17
18
19
20
H
H
H
NH
NH
NH
NH
2.64
2.52
2.39
2.26
94
86
82
87
H
OCH3
OCH3
OCH3
H
OCH3
OCH3
H
OCH3
21
22
23
24
25
H
H
H
–
–
–
–
O
1.35
1.23
1.1
72
75
72
86
83
H
OCH3
OCH3
OCH3
H
H
OCH3
OCH3
H
H
OCH3
H
0.97
2.1

Med Chem Res
Table 1 continued
Compound
R1
R2
R3
X
Y
cLogPa
Yield (%)
75
26
H
OCH3
H
O
2.0
27
28
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
H
O
O
–
1.7
1.4
1.6
88
70
–
OCH3
OCH3
Piplartine
a
The logP values were determined by ChemBioDraw Ultra 12.0 program
heterocyclic derivatives (amides) and finally non-
heterocyclic compounds (acylhydrazides). With these che-
mical variations combined with the variations of the tri-
methoxylated ring of piplartine, preliminary information
about structure–antimicrobial activity relationship could be
determined.
benzoyl derivatives 17–20 in yields higher than 82%. In the
1H-NMR spectra, it was possible to observe a coupling con-
stant (J3) of 1.5 Hz for the two NH protons and these doublets
were registered near 10.5 and 10.2 ppm. Two signals corre-
sponding to the two carbonyl groups were observed between
165.7–164.4 ppm, confirming the successful benzoylation of
hydrazides. The amides 21–24 were smoothly synthesized by
the reaction of acyl chlorides 5–8 with morpholine and were
obtained in high yields (>72%). It was possible to notice a
broad signal at 3.7 ppm relative to the eight methylene pro-
The initial step of the synthetic route involved the synthesis
of acyl chlorides (5–8) from four commercial cinnamic acids
(cinnamic acid, 4-methoxycinnamic acid, 3,4-dimethox-
ycinnamic acid and 3,4,5-trimethoxycinnamic acid) (Carvalho
et al. 2012). The crude dichloromethane solution of these acyl
chlorides was employed in the synthesis of hydrazides 9–12
which were also used without prior purification in the fol-
lowing steps. Phthalimides 13–16 were obtained in yields
higher than 70% from the reaction of hydrazides 9–12 with
phthalic anhydride in glacial acetic acid under heating (Joshi
et al. 2014). The analysis of 13C-NMR spectra of phthali-
mides showed two signals corresponding to the carbonyl
carbons of the compounds between 166.1–164.6 ppm, con-
firming the cyclization reaction and formation of phthalimide
derivatives. The IR spectra of these compounds also showed
bands corresponding to carbonyl groups between 1737 and
1791 cm−1. The singlet corresponding to the NH proton was
1
tons of the morpholine moiety in amides 21–24 in their H-
NMR spectra and one signal near 165 ppm corresponding to
the amidic carbonyl in their 13C-NMR spectra. The bands
referring to the amidic carbonyl were recorded near 1640
cm−1 in their infrared spectra. The reaction of acyl chlorides
5–8 with N-hydroxysuccinimide afforded the pure acylated
derivatives 25–28 in yields higher than 70% after recrys-
1
tallization from ethanol/water. In their H-NMR spectra, it
was possible to observe a singlet at 2.8 ppm relative to the
four methylene protons and two signals between 169.4–162.0
ppm in their 13C-NMR spectra, corresponding to the carbonyl
groups of the compounds. Finally, it is important to note that
the protons related to the cinnamic subunit olefin have been
preserved (J3 = 12 Hz), which proves the absence of
1
observed between 11 and 10 ppm in H-NMR spectra. The
reaction of hydrazides with benzoyl chloride provided the

Med Chem Res
conjugate addition products and the maintenance of E con-
figuration. (Table 1).
Among the synthesized piplartine analogs, morpholine
3,4,5-trimethoxylated derivative 24 was the most promising
analog showing activity against C. krusei at 48.83 μM, and
a selectivity index greater than three. This compound was
almost four-fold more potent and about five-fold less toxic
than piplartine (IC50: 189.2 μM; selectivity index of 0.21).
Furthermore, derivative 24 was more than two-fold more
effective than fluconazole (IC50: 104.48 μM) against this
strain. Although less active than fluconazole, derivative 24
also showed activity against C. tropicalis at 97.67 μM,
while piplartine was inactive up to the highest evaluated
concentration. It is also worth noting that piperazine or
morpholine bioisosteric rings play a crucial role in anti-
microbial activity, as in drugs such as linezolid, eperezolid
and itraconazole or in morpholine–eugenol derivatives
reported in our recent paper (26). Moreover, it is important
to note that derivative 24 showed the lowest logP value
(0.97) among all the synthesized compounds, suggesting an
interesting value for anti-Candida activity (Table 1).
Eleven other analogs of piplartine were moderately
active against different Candida species. Phthalimide deri-
vatives 13, 14, and 15 showed some activity against C.
tropicalis; for these three compounds, there is a clear and
direct correlation between the number of methoxyl sub-
stituents on the aromatic ring and the activity against this
strain (IC50 of 13: 342.32 μM; IC50 of 14: 310.47 μM; IC50
of 15: 170.40 μM), which may suggest that the investigation
of other ring substitution patterns may lead to the discovery
of more active antifungal agents. Again, it can be seen that a
lower logP value is related to a better antifungal activities,
since derivative 15 has a logP 2.2. On the other hand, only
the phthalimide derivative 16 (3,4,5-trimethoxy-substituted)
showed some activity against C. krusei and it was not active
against C. tropicalis, suggesting that more than two meth-
oxyl groups at this aromatic ring is unfavorable for activity
against this species.
Among the benzoylhydrazides (17–20), derivative 17
showed a broader spectrum of action, presenting activity
against all species evaluated, except C. krusei, although at a
low potency (IC50: 375.7 μM). Derivative 19 was the most
potent of the four acylhydrazides, showing IC50 of 183.9
μM against C. glabrata, and once again it was noticed that a
3,4-disubstituted cinnamic ring may be related to a better
antifungal activity. Tri-substituted acylhydrazide 20 was
also moderately active at 280.7 μM against C. albicans, C.
tropicalis, and C. krusei.
It was also observed that the increasing in the number of
methoxyl groups in the cinnamic ring of the O-acyl-succinic
derivatives and the decreasing of their logP values led to an
increase in activity against C. tropicalis, as noted for the
phthalic derivatives. Succinic unsubstituted derivative 25
(logP 2.1) was inactive against C. tropicalis, while the
three-substituted (26; logP 2.0), 3,4-disubstituted (27; logP
Antimicrobial and toxic assays
The toxicity action of piplartine and its analogs and refer-
ence drugs on healthy hamster kidney cells (BHK-21) was
evaluated and the results are shown in Table 2. This eva-
luation was necessary to establish the indices of selectivity
with respect to antifungal and antibacterial actions.
Piplartine and its analogs were evaluated against Can-
dida albicans, C. tropicalis, C. krusei, C. glabrata and C.
parapsilosis; the results of the investigation on these yeasts
are reported here for the first time, along with their selec-
tivity indexes (Table 3).
Fluconazole was used as a reference fungistatic drug and
the results were estimated as the minimum concentration
required to inhibit 50% (IC50, related to fungistatic activity)
and 90% (IC90, related to fungicidal activity) of fungal cell
growth. Piplartine presented the broadest spectrum of anti-
fungal activity, and was shown to be fungistatic against all
fungi stains evaluated, except C. tropicalis, in the range of
94.60–189.20 μM. However, piplartine was the most toxic
compound to BHK-21 cell culture with the lowest selective
index range, between 0.2 and 0.4. This high toxicity pre-
vents the substances for future in vivo testing.
Table 2 In vitro cytotoxicity (μM) against hamster kidney cell culture
(BHK-21)
Compound
CC50
CC90
13
285.12
252.91
239.98
156.73
231.71
212.75
188.18
125.20
354.75
295.28
281.96
185,55
259.93
249.93
196.34
174.99
40.14
452.13
396.65
379.21
427.23
439.04
402.75
361.64
333.19
586.79
506.59
458.91
384.38
462.70
429.70
397.76
415.13
80.31
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
Piplartine
Fluconazole
Chloramphenicol
382.60
269.18
521.13
486.52

Med Chem Res
Table 3 In vitro antifungal
activity (μM) and selectivity
index (SI) of piplartine and
synthesized analogs
Compound
C. albicans
ATCC 10231
(SI)
C. tropicalis
ATCC 750 (SI) ATCC 6258
(SI)
C. krusei
C. glabrata
ATCC 90030
(SI)
C. parapsilosis
ATCC 22019
(SI)
a
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
Pip.
Fluc.
IC50
IC90
IC50
IC90
IC50
IC90
IC50
IC90
–
342.32 (0.83)
–
–
–
–
–
–
–
–
–
–
–
–
–
–
310.47 (0.81)
–
–
–
–
–
–
–
170.40 (1.4)
–
–
–
–
–
–
–
–
261.70 (0.59)
–
–
–
–
–
–
IC50 375.79 (0.61)
375.79 (0.61)
–
375.79 (0.61)
375.79 (0.61)
IC90
IC50
IC90
IC50
IC90
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
183.98 (1.02)
–
–
–
–
–
IC50 280.79 (0.44)
280.79 (0.44)
280.79 (0.44)
–
–
IC90
IC50
IC90
IC50
IC90
IC50
IC90
IC50
IC90
IC50
IC90
IC50
IC90
IC50
IC90
IC50
IC90
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
280.79 (1.18)
280.79 (1.18)
460.59 (0.77)
460.59 (0,77)
–
460.59 (0.77)
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
97.67 (1.89)
48.83 (3.79)
–
–
–
–
–
–
–
–
–
–
–
–
–
–
218.12 (1.14)
363.54 (0.68)
–
–
–
–
–
–
196.66 (0.99)
–
–
–
–
–
–
–
179.05 (0.97)
–
–
–
–
–
189.20 (0.21)
–
–
189.20 (0.21)
–
–
94.60 (0.42)
–
IC50 94.60 (0.42)
–
–
IC90
IC50
–
1.63 (234)
3.26 (117)
104.48 (3.6)
52.24 (7.3)
3.26 (117)
Pip. Piplartine, Fluc. Fluconazole
a
Inactive at highest evaluated concentration
1.7) and 3,4,5-trisubstituted (28, logP 1.4) derivatives were
active at 218.12, 196.66, and 179.05 μM, respectively,
against this strain.
Piplartine, as well as its synthetic analogs, were also
assessed for their antibacterial potential against Staphylo-
coccus aureus, Eschericha coli and Pseudomonas aerugi-
nosa strains using the same concentrations and dilutions
employed in the antifungal assays.
The results and selectivity indexes on BHK-21 cells are
shown in Table 4. Three piplartine analogs were active
against the same strain and it was noticed that all of these
active derivatives bear an unsubstituted cinnamic ring (13,
17 and 25 compounds). Among them, benzyl hydrazide 17
was more than three-fold more potent than the prototype
piplartine, showing an IC50 value of 85.20 μM and a greater
selectivity index (2.71). This suggests that new

Med Chem Res
Table 4 In vitro antibacterial activity (μM) and selectivity index (SI)
acylhydrazides can be exploited as interesting potential
antibacterial agents. Phthalic derivative 13 was moderately
active at 205 μM (selectivity index of 1.38) and the succinic
derivative 25 at 408.06 μM (0.63 selectivity index). All of
the compounds were inactive against Gram-negative E. coli
or P. aeruginosa strains up to the highest concentration
employed; however, piplartine and some synthetic analogs
showed potential against Gram-positive S. aureus. Piplar-
tine was the least active and the most toxic compound
against this strain, showing IC50 value of 315.33 μM and a
low selectivity index of 0.12. Again, synthetic analogs
showed less toxicity than piplartine. Different from what
was noted in the results of antifungal assessment, higher
logP values contributed to better antibacterial activity of the
compounds in each synthesized series (Tables 1 and 4).
Based on the results found in antimicrobial tests it was
possible to realize the major structural requirements for
antifungal and antibacterial activities among the synthesized
piplartine analogs (Fig. 2).
of piplartine and synthesized analogs
Compound
S. aureus
ATCC 6538 (SI) ATCC 25922 ATCC 9027
(SI) (SI)
E. coli
P. aeruginosa
a
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
Pip.
Chl.
IC50 205.42 (1.38)
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
IC90
IC50
IC90
IC50
IC90
IC50
IC90
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
IC50 85.20 (2.71)
IC90
IC50
IC90
IC50
IC90
IC50
IC90
IC50
IC90
IC50
IC90
IC50
IC90
IC50
IC90
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
Conclusion
Sixteen piplartine analogs were synthesized and these
compounds were assessed for their antimicrobial potential
and toxicity. Derivative 24 showed the best results against
C. krusei (more than twice as potent as fluconazole, four-
fold more potent and five-fold less toxic than piplartine).
Other analogs had moderate activity against different fungal
strains and a direct correlation was seen between the
number of aromatic methoxyl groups and antifungal activ-
ity. Furthermore, benzoyl hydrazide 17 was more than
three-fold more potent than piplartine in antibacterial eva-
luation against S. aureus and five-fold less toxic than
piplartine. It was possible to note that an aromatic ring
lacking methoxyl groups is important for the antibacterial
activity of these compounds. These findings encourage the
design and synthesis of new piplartine analogs as potential
and safer candidates to new antimicrobial agents.
IC50 408.06 (0.63)
IC90
IC50
IC90
IC50
IC90
IC50
IC90
–
–
–
–
–
–
–
IC50 315.33 (0.12)
IC90
IC50
–
0.975 (276)
0.975 (276)
16.5 (16.3)
Pip. Piplartine, Chl. Choramphenicol
a
Inactive at highest evaluated concentration
Fig. 2 Structure–antimicrobial
activity relationships for
piplartine analogs

Med Chem Res
Acknowledgments This work was supported by FAPEMIG (APQ-
01209-13), CAPES, CNPq and FINEP.
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Compliance with Ethical Standards
Conflict of Interest The authors declare that they have no compet-
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