Antiparasitic activity of synthetic curcumin monocarbonyl analogues against Trichomonas vaginalis

Article abstract of DOI:10.1016/j.biopha.2018.12.058

Trichomoniasis is a parasitic infection caused by Trichomonas vaginalis and it is considered to be the most common non-viral sexually transmitted infection in the world. Since the 1960s, nitroimidazoles such as metronidazole are the drugs of choice for the treatment of trichomoniasis, but many adverse effects and allergic reactions may result from their use. Reports of metronidazole-resistant infections also highlight the importance for the search of new anti-T. vaginalis agents. Considering this, herein we report the anti-T. vaginalis evaluation of 21 synthetic monocarbonyl analogues of curcumin, which itself has been reported to possess antiparasitic potential. From the in vitro analysis of the synthetic molecules, untreated trophozoites, and metronidazole at 100 μM, it was observed that three curcumin analogues (3a, 3e, and 5e) exhibited anti-T. vaginalis activity comparable to metronidazole (no significant statistical difference). Optimal antiparasitic concentrations were determined to be 80 μM and 90 μM for propanone derivatives 3a and 3e, respectively, and 200 μM for cyclohexanone derivative 5e. Kinetic growth curves showed that, after 24 h, the trophozoites were completely inhibited. At the tested concentrations, natural curcumin did not significantly inhibit the growth of trophozoites, therefore demonstrating that the designed synthetic molecules not only have better chemical stability, but also higher anti-T. vaginalis potential. Cytotoxicity analysis, performed on VERO cells, demonstrated low, moderate and high cytotoxic effects for analogues 3e, 5e and 3a, respectively. This study suggests that these analogues possess chemical features of interest to be further explored as alternatives for the treatment of trichomoniasis.

Full text of DOI:10.1016/j.biopha.2018.12.058

Biomedicine&Pharmacotherapy111(2019)367–377
Contents lists available at ScienceDirect
Biomedicine & Pharmacotherapy
journal homepage: www.elsevier.com/locate/biopha
Antiparasitic activity of synthetic curcumin monocarbonyl analogues
against Trichomonas vaginalis
Caroline Carapina da Silvaa,⁎, Bruna Silveira Pachecoa, Raquel Nascimento das Nevesb,
Mirna Samara Dié Alvesb, Ângela Sena-Lopesb, Sidnei Mourac, Sibele Borsukb,
Claudio Martin Pereira de Pereiraa
a Laboratory of Lipidomics and Bioorganic, Bioforensics Research Group, Federal University of Pelotas, RS, 96010-900, Brazil
b Laboratory of Infecto-parasitic Biotechnology, Federal University of Pelotas, RS, 96010-900, Brazil
c Laboratory of Biotechnology of Natural and Synthetic Products, Biotechnology Institute, University of Caxias do Sul, RS, 95020260, Brazil
A R T I C L E I N F O
A B S T R A C T
Keywords:
C5-curcuminoids
Antiparasitic
Metronidazole
Arylideneketones
Dibenzylideneacetone
Trichomoniasis is a parasitic infection caused by Trichomonas vaginalis and it is considered to be the most
common non-viral sexually transmitted infection in the world. Since the 1960s, nitroimidazoles such as me-
tronidazole are the drugs of choice for the treatment of trichomoniasis, but many adverse effects and allergic
reactions may result from their use. Reports of metronidazole-resistant infections also highlight the importance
for the search of new anti-T. vaginalis agents. Considering this, herein we report the anti-T. vaginalis evaluation of
21 synthetic monocarbonyl analogues of curcumin, which itself has been reported to possess antiparasitic po-
tential. From the in vitro analysis of the synthetic molecules, untreated trophozoites, and metronidazole at 100
μM, it was observed that three curcumin analogues (3a, 3e, and 5e) exhibited anti-T. vaginalis activity com-
parable to metronidazole (no significant statistical difference). Optimal antiparasitic concentrations were de-
termined to be 80 μM and 90 μM for propanone derivatives 3a and 3e, respectively, and 200 μM for cyclo-
hexanone derivative 5e. Kinetic growth curves showed that, after 24 h, the trophozoites were completely
inhibited. At the tested concentrations, natural curcumin did not significantly inhibit the growth of trophozoites,
therefore demonstrating that the designed synthetic molecules not only have better chemical stability, but also
higher anti-T. vaginalis potential. Cytotoxicity analysis, performed on VERO cells, demonstrated low, moderate
and high cytotoxic effects for analogues 3e, 5e and 3a, respectively. This study suggests that these analogues
possess chemical features of interest to be further explored as alternatives for the treatment of trichomoniasis.
1. Introduction
inflammatory disease, AIDS and prostate cancer [3,4]. If patients in-
fected by T. vaginalis report symptoms, they are not specifically char-
Trichomonas vaginalis is the causative agent of trichomoniasis, the
most common non-viral sexually transmitted infection (STI) worldwide.
According to the World Health Organization latest report on global
sexually transmitted infections, the estimated number of new cases
caused by the protozoan T. vaginalis was of 142.6 million, followed by
Chlamydia trachomatis (130.9 million), Neisseria gonorrhoeae (78.3 mil-
lion), and Treponema pallidum (5.6 million) [1,2]. Since it is considered
to be a curable infection, not much attention has been drawn to it
compared to other STIs, but nevertheless it can cause great impact on
human health because the majority of trichomoniasis cases are
asymptomatic. This fact makes it a disease difficult to diagnose and
increases the number of infected sexual partners. Additionally, tricho-
moniasis is associated with other afflictions, such as infertility, pelvic
acteristic but rather similar to the symptoms experienced with other
STIs. The main symptoms in women and men are discharge from the
vagina or urethra (with an unpleasant odor), genital burning, itching,
redness, warts, and frequent urge to urinate (painful or not) [4,5].
The standard drug primarily chosen for the treatment of trichomo-
niasis is the nitroimidazole derivative metronidazole (brand name
Flagyl), which acts as a pro-drug in anaerobic microorganisms where it
suffers reduction and produces nitro radicals which, in turn, inhibit
DNA synthesis of the microorganism. Metronidazole was approved by
the Food and Drug Administration in the early 1960s and is generally
administered orally as a single-dose regimen or as a topical gel, with
significant cure rates [6,7]. On the other hand, there are some limita-
tions associated with the use of metronidazole, including many
⁎ Corresponding author.
E-mail addresses: carapina7@hotmail.com, lahbbioufpel@gmail.com (C. Carapina da Silva).
https://doi.org/10.1016/j.biopha.2018.12.058
Received 15 July 2018; Received in revised form 25 November 2018; Accepted 14 December 2018
0753-3322/©2018ElsevierMassonSAS.ThisisanopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/BY-NC-ND/4.0/).

C. Carapina da Silva et al.
Biomedicine&Pharmacotherapy111(2019)367–377
Scheme 1. Chemical structure of naturally occurring curcumin and the design of synthetic analogues.
reported side effects (convulsive seizures, epigastric distress, abdominal
cramping), and disulfiram-like reaction in alcoholic patients, which
lead to non-compliance to the full period of treatment [8,9]. Several
patients are allergic to metronidazole and require alternative treatment
such as treatment with paromomycin cream [10], and there has been an
increase on the reports of metronidazole-resistant infections along with
a resistance mechanism of reductive inactivation proposed recently
[11,12].
In this way, it is crucial to explore the discovery or development of
new anti-T. vaginalis molecules that can be used as safe alternatives to
metronidazole [4,9,13–15]. In this sense, this research was focused on
the antiparasitic evaluation of synthetic analogues of curcumin due to
their relatively easy obtainment and reproducibility. Curcumin was
chosen as a natural prototype because it is the major constituent of the
plant Curcuma longa L. and has been associated with countless biolo-
gical activities [16–21], including anti-T. vaginalis activity [22]. How-
ever, curcumin’s chemical structure also presents some disadvantages
due to highly reactive sites that favor degradation reactions and lower
its bioavailability (Scheme 1), so it is important to develop compounds
equally bioactive, but more stable [23,24].
catalyst was neutralized with a diluted hydrochloric acid solution, va-
cuum-filtered, and dried at ambient temperature. The solid was purified
by recrystallization with hexane/ethyl acetate, ethanol, or ethanol/
acetonitrile (for series C).
2.1.1. Chemical identification
The synthesis of monocarbonyl analogues of curcumin has been
reported for many years as an alternative to the use of natural cur-
cumin, so the chemical structures and complete characterization of
products are well-established and confirmed [23,26–30]. In this way,
the purified crystals of synthetic curcumin analogues were identified
and checked for agreement with the literature by their melting points
(MP), infrared spectra (IR), mass spectra (MS), and nuclear magnetic
resonance (NMR). Structural information such as molecular weight and
CLog P were calculated in ChemDraw software version 8.0. Melting
points were recorded with a Fisatom 430 apparatus and mercury
thermometer. Chromatographic analysis was performed with a Shi-
madzu GC–MS-QP 2010 SE Standard Gas Chromatograph-Mass Spec-
trometer equipped with an AOC-20 automatic injector and a RTx-5MS
column with the dimensions of 30 m × 0.25 mm × 0.25 μm, using he-
lium as carrier gas. Mass spectra were acquired using a hybrid high-
resolution and high accuracy (5 μL L−1) microTof (Q-TOF) mass spec-
trometer (Bruker® Scientific) by atmospheric pressure chemical ioni-
zation (MS APCI) and tandem APCI(+)-MS/MS. Data were collected in
the m/z range of 70–1000 at the speed of two scans per second. Infrared
spectra were recorded directly from the crystal on a Shimadzu Infrared
Spectrophotometer with Fourier Transformer (FTIR-Prestige 21) using
attenuated total reflectance (ATR) at a 4.0 cm−1 resolution of 12 scans,
and reported in wave numbers (ν). NMR spectra of 1H and 13C were
recorded on a Bruker DPX 300 (1H at 300.13 MHz and 13C at
With this in mind, the aim of this work was to evaluate the in vitro
anti-T. vaginalis activity of a series of synthetic curcumin analogues as
possible alternative antiparasitic agents.
2. Materials and methods
2.1. Chemistry: synthesis of monocarbonyl analogues of curcumin
All reagents and solvents employed in the synthesis were of analy-
tical grade and were purchased from Sigma-Aldrich Co. (St. Louis, MO,
USA). Reactions were monitored by thin layer chromatography (TLC)
using hexane/ethyl acetate 7:3, viewed under ultraviolet light. The
curcumin analogues were synthesized based on a previously described
procedure [25]. A series of 21 molecules was obtained, which were
subdivided into three series according to the ketone used for con-
densation with different substituted aldehydes: propanone (series A),
cyclohexanone (series B) or cyclopentanone (series C) and a fourth
series was obtained by condensing cinnamaldehyde with the ketones
mentioned (Series D) (Scheme 2). Briefly, to a mixture of the respective
aldehyde (20 mmol) and ketone (10 mmol; ratio of 2:1), in cold ethanol
(15 mL), an aqueous solution of sodium hydroxide (40%; 10 mL) was
added and kept under magnetic stirring until reaction was completed
(monitored by TLC; around 2 h for most of the products). Then, the
75.48 MHz) in 5 mm sample tubes at 298 K (digital resolution
0.01
ppm). Molecules were solubilized in deuterated chloroform (CDCl3),
and chemical shifts were recorded in ppm. Identification data is dis-
played below and detailed spectral information is given as supple-
mental files.
2.1.2. Data of monocarbonyl analogues of curcumin
The main data related to the identification of synthetic curcumin
analogues are displayed in Tables 1–4. Detailed information and se-
lected spectra can be found as supplemental files.
368

C. Carapina da Silva et al.
Biomedicine&Pharmacotherapy111(2019)367–377
Scheme 2. Synthesis of monocarbonyl analogues of curcumin 3a-f, 5a-f, 7a-f and 9a-c.
Table 1
Data of Series A products (monocarbonyl analogues with propanone 3a-f).
Structure IUPAC Name (article code)
Exact Mass (EM); Molecular Weight (MW); Yield; aspect; Melting Point (mp) (Literature: [ref]); CLog Pa; IR νmax / cm−1 1H NMR (300 MHz,
;
CDCl3) δ (ppm); 13C NMR (75 MHz, CDCl3) δ (ppm); MS (APCI) m/z: (M + 1)+1
.
(1E,4E)-1,5-diphenylpenta-1,4-dien-3-one (3a)
C
17H14O. EM: 234.10; MW: 234.29; Yield: 60%; yellow solid; mp (°C): 113 – 115 (Lit.: 113/110 – 112 [31,32]); CLog Pa: 4.278; IR νmax / cm−1
3052, 3025 (C-H), 1654 (C = O), 1626, 1589 (C = C), 1571 (C = C); 1H NMR (300 MHz, CDCl3) δ (ppm): 7.73 (d, 2H, J = 15.9 Hz; 2x = C-H);
7.58 – 7.61 (m, 10 H, 2x Ph); 7.07 (d, 2H, J = 15.9 Hz, 2x = C-H); 13C NMR (75 MHz, CDCl3) δ (ppm): 189.01; 143.4; 134.8; 130.6; 129.0; 128.5;
125.4.; MS (APCI) m/z: 235.11 (M + 1)+1
.
(1E,4E)-1,5-di-p-tolylpenta-1,4-dien-3-one (3b)
19H18O. EM: 262.14; MW: 262.35; Yield: 56%; yellow needle crystals; mp (°C): 175-177 (Lit.: 174 – 176 [32]); CLog Pa: 5.276; IR νmax / cm−1
3026, 2917 (C-H), 1646 (C = O), 1617, 1582, 1565 (C = C), 1183 (C-C). 1H NMR (300 MHz, CDCl3) δ (ppm); 13C NMR (75 MHz, CDCl3) δ (ppm):
same pattern as 3c. MS (APCI) m/z: 263.14 (M + 1)+1
(1E,4E)-1,5-bis(4-methoxyphenyl)penta-1,4-dien-3-one (3c)
C
.
C
19H18O3. EM: 294.13; MW: 294.34; Yield: 63%; pale yellow crystals; mp (°C): 129 – 132 (Lit.: 128 – 130 [32]); CLog Pa: 4.116; IR νmax / cm−1
3048, 2920, 2906 (C-H), 1687 (C = O), 1626, 1591, 1584 (C = C), 1228 (C-O), 1180, 988 (C-C); 1H NMR (300 MHz, CDCl3) δ (ppm): 7.69 (d, 2H,
J = 15.6 Hz, 2x = CH); 7.55 (d, 4H, J = 8.7 Hz, 2x Ar); 6.94 (d, 2H, J = 15.9 Hz, 2x = C-H); 6.91 (d, 4H, J = 8.7 Hz, 2x Ar); 3.83 (s, 6H, 2x
OCH3); 13C NMR (75 MHz, CDCl3,) δ (ppm): 188.9; 161.15; 142.7; 130.2; 127.6; 123.5; 114.4; 55.4. MS (APCI) m/z: 295.13 (M + 1)+1
(1E,4E)-1,5-bis(4-chlorophenyl)penta-1,4-dien-3-one (3d)
.
C
17H12Cl2O. EM: 302.03; MW: 303.18; Yield: 40%; yellow crystals; mp (°C): 184 – 186 (Lit.: 184 – 186 [32]); CLog Pa: 5.704; IR νmax / cm−1 3052,
3017 (C-H), 1646 (C = O), 1628, 1587, 1563 (C = C), 1187, 1086 (C-C), 809, 706 (C-Cl); 1H NMR (300 MHz, CDCl3) δ (ppm): 7.67 (d, 2H,
J = 15.9 Hz, 2x = CH); 7.55 (d, 4H, J = 9.0 Hz, 2x Ar); 7.39 (d, 4H, J = 9.0 Hz, 2x Ar); 7.02 (d, 2H, J = 15.9 Hz, 2x = CH); 13C NMR (75 MHz,
CDCl3) δ (ppm): 188.4; 142.2; 136.6; 133.2; 129.7; 129.4; 125.7. MS (APCI) m/z: 303.03 (M + 1)+1
(1E,4E)-1,5-bis(2-chlorophenyl)penta-1,4-dien-3-one (3e)
.
C
17H12Cl2O. EM: 302.03; MW: 303.18; Yield: 73%; yellow crystals; mp (°C): 118 – 120 (Lit.: 114 – 116 [32]); CLog Pa: 5.704; IR νmax / cm−1 3052,
3026 (C-H), 1646 (C = O), 1624, 1587, 1563 (C = C), 1082 (C-C), 763, 730 (C-Cl); 1H NMR (300 MHz, CDCl3) δ (ppm): 8.13 (d, 2H, J = 15.9 Hz;
2x = C-H); 7.7 – 7.2 (m, 8H, 2x Ar); 7.07 (d, 2H, J = 15.9 Hz; 2x = C-H); 13C NMR (75 MHz, CDCl3) δ (ppm): 188.8; 139.4; 135.5; 133.0; 131.3;
130.3; 127.8; 127.5; 127.2. MS (APCI) m/z: 303.03 (M + 1)+1
.
(1E,4E)-1,5-bis(4-fluorophenyl)penta-1,4-dien-3-one (3f)
C
17H12F2O. EM: 270.09; MW: 270.27; Yield: 74%; orange crystals; mp (°C): 150 – 154 (Lit.: 150 – 152 [33]); CLog Pa: 4.564; IR νmax / cm−1 3074,
3052 (C-H), 1650 (C = O), 1626, 1582 (C = C), 1226, 1154 (C-F); 1187 (C-C); 1H NMR (300 MHz, CDCl3) δ (ppm): 7.70 (d, 2H, J = 15.9 Hz,
2x = C-H); 7.61 (d, 4H, J = 9.0 Hz, 2x Ar); 7.12 (d, 4H, J = 9.0 Hz, 2 x Ar); 6.99 (d, 2H, J = 15.9 Hz; 2x = C-H); 13C NMR (75 MHz, CDCl3) δ
(ppm): 188.5; 164.10 (d, 1C, J1CF = 255.5 Hz, Ar-F); 142.2; 131.03 (d, 1C, JC4F = 3.76 Hz, Ar-F); 130.38 (d, 2C, JC3F = 8.77 Hz, Ar-F); 125.11;
125.08; 116.2; (d, 2C, J2CF = 21.3 Hz, Ar-F); MS (APCI) m/z: 271.09 (M + 1)+1
.
a
Theoretical value of CLog P determined by Chemdraw software 8.0, CambridgeSoft, Cambridge, MA.
369

C. Carapina da Silva et al.
Biomedicine&Pharmacotherapy111(2019)367–377
Table 2
Data of Series B products (monocarbonyl analogues with cyclohexanone 5a-f).
Structure
IUPAC Name (article code)
Exact Mass (EM); Molecular Weight (MW); Yield; aspect; Melting Point (mp) (Literature: [ref]); CLog Pa; IR νmax / cm−1 1H NMR (300 MHz,
;
CDCl3) δ (ppm); 13C NMR (75 MHz, CDCl3) δ (ppm); MS (APCI) m/z: (M + 1)+1
.
(2E,6E)-2,6-dibenzylidenecyclohexanone (5a)
C
20H18O. EM: 274.14; MW: 274.36; Yield: 64%; yellow hexagonal crystals; mp (°C): 116 – 118 (Lit.:116 – 120 [31]); CLog Pa: 5.331; IR νmax /
cm−1 3022, 2932, 2919 (C-H), 1659 (C = O), 1604 (C = C), 1272 (C-C); 1H NMR (300 MHz, CDCl3) δ (ppm): 7.80 (s, 2H, 2x = C-H), 7.78 – 7.47
(m, 10 H. 2x Ph), 2.90 (t, 4H, 2x CH2), 1.76 (quint., 2H, CH2); 13C NMR (75 MHz, CDCl3) δ (ppm): 190.3; 137.0; 136.2; 135.9; 130.4; 128.6; 128.4;
28.5; 23.0. MS (APCI) m/z: 275.14 (M + 1)+1
.
(2E,6E)-2,6-bis(4-methylbenzylidene)cyclohexanone (5b)
C
22H22O. EM: 302.17; MW: 302.41; Yield: 24%; yellow crystals; mp (°C): 171-174 (Lit.: 170 – 171 [34]); CLog Pa: 5.61; IR νmax / cm−1 2938,
2914, 2858, 2834(C-H), 1661 (C = O), 1595, 1565 (C = C), 818 (C-C); 1H NMR (300 MHz, CDCl3) δ (ppm): 7.78 (s, 2x = C-H); 7.40 (d, 2H,
J = 8.7 Hz, 2x Ar); 7.2 (d, 4H, J = 8.7 Hz, 2x Ar); 2.90 (t, 4H, 2x CH2) 2.37 (s, 6H, 2x CH3); 1.77 (quint., 2H, CH2); 13C NMR (75 MHz, CDCl3) δ
(ppm): 190.5; 138.9; 136.9; 135.5; 133.2; 130.6; 129. 2; 28.6; 23.1; 21.5. MS (APCI) m/z: 303.17 (M + 1)+1
.
(2E,6E)-2,6-bis(4-methoxybenzylidene)cyclohexanone (5c)
C
22H22O3. EM: 334.16; MW: 334.41; Yield: 80%; yellow solid; mp (°C): 201 – 203 (Lit.: 202 – 203 [35]); CLog Pa: 5.169; IR νmax / cm−1 2938,
2827 (C-H), 1657 (C = O), 1589, 1554 (C = C), 1248 (C-O), 1021, 831 (C-C); 1H NMR (300 MHz, CDCl3) δ (ppm): 7.76 (s, 2x = C-H); 7.42 (d, 2H,
J = 8.7 Hz, 2x Ar); 6.91 (d, 4H, J = 8.7 Hz, 2x Ar); 3.82 (s, 6H, 2x OCH3); 2.90 (t, 4H, 2x CH2); 1.78 (quint., 2H, CH2); 13C NMR (75 MHz, CDCl3) δ
(ppm): 190.2; 159.9; 136.5; 134.3; 132.2; 128.7; 113.9; 55.3; 28.5; 23.0. MS (APCI) m/z: 335.16 (M + 1)+1
.
(2E,6E)-2,6-bis(4-chlorobenzylidene)cyclohexanone (5d)
C
20H16Cl2O. EM: 342.06; MW: 343.25; Yield: 62%; yellow crystals; mp (°C): 147 – 148 (Lit.: 147 – 148 [34]); CLog Pa: 6.757; IR νmax / cm−1 2967,
2942 2928 (C-H), 1665 (C = O), 1604 (C = C), 1161, 1145 (C-C), 798 (C-Cl); 1H NMR (300 MHz, CDCl3) δ (ppm): 7.72 (d, 2H, 2x Ar); 7.37 (s,
2x = C-H); 7.3 – 7.41 (m, 8H, 2x Ar); 2.8 (t, 4H, 2 x CH2); 1.78 (quint., 2H, CH2); 13C NMR (75 MHz, CDCl3) δ (ppm): 189.7; 136.3; 135.7; 134.5;
134.2; 131.5; 128.6; 28.3; 22.7. MS (APCI) m/z: 343.05 (M + 1)+1
.
(2E,6E)-2,6-bis(2-chlorobenzylidene)cyclohexanone (5e)
C
20H16Cl2O. EM: 342.06; MW: 34,325; Yield: 83%; yellow crystals; mp (°C): 111 – 114 (Lit.: 103 – 105 [36]); CLog Pa: 6.757; IR νmax / cm−1 3063,
2972, 2917 (C-H), 1663 (C = O), 1587, 1576 (C = C), 1143 (C-C), 765, 733 (C-Cl); 1H NMR (300 MHz, CDCl3) δ (ppm): 7.91 (s, 2x = C-H); 7.2 –
7.46 (m, 8H, 2x Ar); 2.7 (t, 4H, 2x CH2); 1.74 (quint., 2H, CH2); 13C NMR (75 MHz, CDCl3) δ (ppm): 189.8; 137.8; 135.1; 134.4; 134.1; 130.6;
129.8; 129.7; 126.3; 28.5; 23.2. MS (APCI) m/z: 343.06 (M + 1)+1
.
(2E,6E)-2,6-bis(4-fluorobenzylidene)cyclohexanone (5f)
C
20H16F2O. EM: 310.12; MW: 310.34; Yield: 36%; pale yellow solid; mp (°C): 154 – 157 (Lit.: 156 [37]); CLog Pa: 5.167; IR νmax / cm−1 2954,
2932, 2872 (C-H), 1661 (C = O), 1609, 1598 (C = C), 1143 (C-C), 1220, 1268 (C-F); 1H NMR (300 MHz, CDCl3) δ (ppm): 7.74 (s, 2x = C-H); 7.45
(d, 4H, J = 8.7 Hz, 2x Ar); (7.08 d, 4H, J = 8.7 Hz, 2x Ar); 2.8 (t, 4H, 2x CH2); 1.78 (quint., 2H, CH2); 13C NMR (75 MHz, CDCl3) δ (ppm): 190.6;
162.7 (d, 1C, JC1F = 246.7 Hz, Ar-F); 135.96; 135.7 (d, 1C, JC4F = 3.76 Hz, Ar-F); 132.3 (d, 2C, JC3F = 8.24 Hz, Ar-F); 132.1; 132.06; 115.6 (d, 2C,
J2CF = 25.3 Hz, Ar-F); 28.4; 22.9. MS (APCI) m/z: 311.12 (M + 1)+1
.
a
Theoretical value of CLog P determined by Chemdraw software 8.0, CambridgeSoft, Cambridge, MA.
Table 3
Data of Series C products (monocarbonyl analogues with cyclopentanone 7a-f).
Structure
IUPAC Name (article code)
Exact Mass (EM); Molecular Weight (MW); Yield; aspect; Melting Point (mp) (Literature: [ref]); CLog Pa; IR νmax / cm−1 1H NMR (300 MHz, CDCl3)
;
δ (ppm); 13C NMR (75 MHz, CDCl3) δ (ppm); MS (APCI) m/z: (M + 1)+1
.
(2E,5E)-2,5-dibenzylidenecyclopentanone (7a)
C
19H16O. EM: 260.12; MW: 260.33; Yield: 74%; yellow solid; mp (°C): 189 –193 (Lit.: 194 – 195 [31]); CLog Pa: 4.772; IR νmax / cm−1 3054, 3019,
2910 (C-H), 1685 (C = O), 1622, 1598 (C = C), 1178 (C-C); 1H NMR (300 MHz, CDCl3) δ (ppm): 7.60 (d, 4H, J = 6.7 Hz, 2x Ar); 7.43 (s, 2H,
2x = CH); 7.33 – 7.47 (m, 8 H, 2x Ph); 3.08 (s, 4H, 2x CH2); 13C NMR (75 MHz, CDCl3) δ (ppm): 196.42; 137.3; 135.8; 133.9; 130.8; 129.4; 128.8;
26.6. MS (APCI) m/z: 261.12 (M + 1)+1
.
(2E,5E)-2,5-bis(4-methylbenzylidene)cyclopentanone (7b)
C
21H20O. EM: 288.15; MW: 288.38; Yield: 60%; yellow solid; mp (°C): 243 – 244 (Lit.: 243 – 245 [36]); CLog Pa: 5.77; IR νmax / cm−1 2909, 2850
(C-H), 1687 (C = O), 1615, 1584 (C = C), 1169, 809 (C-C); 1H NMR (300 MHz, CDCl3) δ (ppm): 7.57 (s, 2H, 2x = CH); 7.50 (d, 4H, J = 8 Hz, 2x Ar);
7.24 (d, 4H, J = 8 Hz, 2x Ar); 3.08 (s, 4H, 2x CH2); 2.4 (s, 6H, CH3); 13C NMR (75 MHz, CDCl3) δ (ppm): 196.62; 139.89; 136.62; 133.84; 130.23;
130.12; 130.06; 133.81; 133.20; 130.91; 129.65; 26.55; 21.65. MS (APCI) m/z: 289.15 (M + 1)+1
.
(2E,5E)-2,5-bis(4-methoxybenzylidene)cyclopentanone (7c)
C
21H20O3. EM: 320.14; MW: 320.38; Yield: 75%; yellow solid; mp (°C): 215 – 217 (Lit.: 210 – 211 [38]); CLog Pa: 4.61; IR νmax / cm−1 2966, 2842
(C-H), 1694 (C = O), 1630, 1595 (C = C), 1246, 1032 (C-O), 1167 (C-C); 1H NMR (300 MHz, CDCl3) δ (ppm): 7.56 (d, 4H, J = 9.0 Hz, 2x Ar); 7.37
(s, 2H, 2x = CH); 6.95 (d, 4H, J = 9.0 Hz, 2x Ar); 3.8 (s, 6H, 2x OCH3); 3.07 (s, 4H, 2x CH2); 13C NMR (75 MHz, CDCl3) δ (ppm): 196.4; 160.5; 135.3;
133.3; 132.6; 128.8; 114.3; 55.4; 26.5. MS (APCI) m/z: 321.14 (M + 1)+1
.
(2E,5E)-2,5-bis(4-chlorobenzylidene)cyclopentanone (7d)
C
19H14Cl2O. EM: 328.04; MW: 329.22; Yield: 82%; yellow crystals; mp (°C): 225 – 228 (Lit.: 227 – 229 [36]); CLog Pa: 6.198; IR νmax / cm−1 2933,
2914 (C-H), 1694 (C = O), 1606, 1580 (C = C), 927 (C-C), 816, 682 (C-Cl); 1H NMR (300 MHz, CDCl3) δ (ppm): 13C NMR (75 MHz, CDCl3) δ (ppm):
same pattern as 7f. MS (APCI) m/z: 329.04 (M + 1)+1
(2E,5E)-2,5-bis(2-chlorobenzylidene)cyclopentanone (7e)
.
C
19H14Cl2O. EM: 328.04; MW: 329.22; Yield: 69%; yellow crystals; mp (°C): 158 – 160 (Lit.: 155 – 158 [28]); CLog Pa: 6.198; IR νmax / cm−1 3067,
3049, 2926, 2911 (C-H), 1685 (C = O), 1600, 1587 (C = C), 1180, 1036 (C-C), 750 (C-Cl); 1H NMR (300 MHz, CDCl3) δ (ppm): 7.92 (s, 2H,
2x = CH); 7.30 – 7.56 (m, 8H, 2x Ar); 2.9 (s, 4H, 2x CH2); 13C NMR (75 MHz, CDCl3) δ (ppm): 195.5; 139.3; 136.0; 133.8; 130.12; 130.1; 126.6; 26.5.
MS (APCI) m/z: 329.05 (M + 1)+1
.
(2E,5E)-2,5-bis(4-fluorobenzylidene)cyclopentanone (7f)
C
19H14F2O. EM: 296.10; MW: 296.31; Yield: 80%; pale yellow solid; mp (°C): 237 – 239 (Lit.: 239 – 240 [37]); CLog Pa: 5.058; IR νmax / cm−1 n;
2921, 2907 (C-H), 1689 (C = O), 1587 (C = C), 1226 (C-F), 1183, 827 (C-C);); 1H NMR (300 MHz, CDCl3) δ (ppm): 7.6 (d, 4H, J = 8.9 Hz, 2x Ar);
7.57 (s, 2H, 2x = CH); 7.14 (d, 4H, J = 9.0 Hz, 2x Ar); 3.09 (s, 4H, 2x CH2); 13C NMR (75 MHz, CDCl3) δ (ppm): 196.25; 163.2 (d, 1C,
J1CF = 249.6 Hz, Ar-F); 136.8; 132.84 (d, 1C, JC4F = 3.10 Hz, Ar-F); 132.7 (d, 2C, J3CF = 8.17 Hz, Ar-F); 132.13; 132.08; 116.15 (d, 2C, JC2F = 24.0 Hz,
Ar-F); MS (APCI) m/z: 297.10 (M + 1)+1
.
a
Theoretical value of CLog P determined by Chemdraw software 8.0, CambridgeSoft, Cambridge, MA.
370

C. Carapina da Silva et al.
Biomedicine&Pharmacotherapy111(2019)367–377
Table 4
Data of Series D products (derivatives of cinnamaldehyde 9a-c).
Structure
IUPAC Name (article code)
Exact Mass (EM); Molecular Weight (MW); Yield; aspect; Melting Point (mp) (Literature: [ref]); CLog Pa; IR νmax / cm−1 1H NMR (300 MHz, CDCl3) δ
;
(ppm); 13C NMR (75 MHz, CDCl3) δ (ppm); MS (APCI) m/z: (M + 1)+1
.
(1E,3E,6E,8E)-1,9-diphenylnona-1,3,6,8-tetraen-5-one (9a)
C
21H18O. EM: 286.14; MW: 286.37; Yield: 42%; orange crystals; mp (°C): 141 – 142 (Lit.: 142 [37]); CLog Pa: 5.336; IR νmax / cm−1 3029, 3019 (C-H),
1654 (C = O), 1589, 1563 (C = C), 1067, 995 (C-C); 1H NMR (300 MHz, CDCl3) δ (ppm): 7.60 (d, 4H, J = 7.2 Hz, 2x Ar); 7.52 (q, 2 H, J = 15 Hz,
2x = C-Hβ); 7.40 (t, 4H, J = 7.2 Hz, 2x Ar); 7.33 (t, 2H, J = 7.2 Hz; 2x Ar); 7.02 (d, 2H, J = 16.5 Hz, 2x = C-H-Ar); 6.71 (d, 2H, J = 16.5 Hz,
2x = CH); 6.69 (d, 2H, J = 15 Hz, 2x = C-Hα); 13C NMR (75 MHz, CDCl3) δ (ppm): 188.9; 142.9; 141.4; 136.1; 129.1; 128.9; 128.8; 127.2; 126.9. MS
(APCI) m/z: 287.13 (M + 1)+1
.
(2E,6E)-2,6-bis((E)-3-phenylallylidene)cyclohexanone (9b)
C
22H22O. EM: 326.17; MW: 326.43; Yield: 44%; orange solid; mp (°C): 177 – 179 (Lit.: 178 – 179 [39]); CLog Pa: 6.389; IR νmax / cm−1 3007, 2935,
2869 (C-H), 1652 (C = O), 1580 (C = C), 1150, 975, 744 (C-C); 1H NMR (300 MHz, CDCl3) δ (ppm): 7.51 (d, 4H, J = 7.2 Hz, 2x Ar); 7.46 (t, 4H, 2x
Ar); 7.34 (t, 2H, 2x Ar) 7.29 (d, 2H, 2x = C-Hβ); 7.10 (d, 2H, J = 15 Hz, 2x = CH-Ar), 7.06 (d, 2H, J = 15 Hz, 2x = CH); 2.9 (t, 4H, 2x CH2), 1.9
(quint., 2H, CH2). 13C NMR (75 MHz, CDCl3) δ (ppm): 188.8; 140.7; 136.7; 136.2; 135.3; 128.75; 128.7; 127.1; 123.6; 26.5. 21.9. MS (APCI) m/z:
327.16 (M + 1)+1
.
(2E,5E)-2,5-bis((E)-3-phenylallylidene)cyclopentanone (9c)
C
23H20O. EM: 312.15; MW: 312.40; Yield: 61%; orange solid; mp (°C): 228 – 229 (Lit.: 229 [37]); CLog Pa: 5.83; IR νmax / cm−1 3025(C-H), 1670
(C = O), 1611, 1580 (C = C), 966, 748 (C-C); 1H NMR (MHz, CDCl3) δ (ppm): 7.51 (d, 4H, J = 7.2 Hz, 2x Ar); 7.4 (t, 4H, J = 7.2 Hz, 2x Ar), 7.34 (t,
2H, J = 7.2 Hz, 2x Ar), 7.26 (d, 2H, 2x = CHβ); 7.2 (d, 2H, J = 7 Hz, 2x = CH-Ar), 6.94 (d, 2H, J = 7 Hz, 2x = CH); 2.88 (s, 4H, 2x CH2); 13C NMR (75
CDCl3, MHz) δ (ppm): 194.9; 141.2; 139.7; 136.5; 132.6; 128.9; 128.7; 127.2; 124.7; 23.8. MS (APCI) m/z: 313.14 (M + 1)+1
.
a
Theoretical value of CLog P determined by Chemdraw software 8.0, CambridgeSoft, Cambridge, MA.
2.2. Parasite culture conditions
(400x), where the viable trophozoites, with negative staining, were
counted in Neubauer chamber, and the motility and morphology of
trophozoites were analyzed. All assays were performed independently
at least three times in triplicate and results were expressed as the per-
centage of viable trophozoites in comparison with the negative control.
T. vaginalis 30236 isolates from American Type Culture Collection
(ATCC) were axenically cultured in tubes filled with pre-warmed
trypticase-yeast extract-maltose (TYM) medium without agar (pH 6.0),
supplemented with 10% sterile bovine serum, previously inactivated at
56 °C, and 5 mg/mL streptomycin and incubated at 37 °C (aerobic at-
mosphere) [40]. Cultures were analyzed through observation of moti-
lity, morphology, and trypan blue (0.4%) dye exclusion assay under
light microscope at 400x magnification and those with viability equal
or higher than 95% were used for the anti-T. vaginalis assay.
2.4. Cytotoxicity assay
This assay was performed following methodologies previously de-
scribed with some modifications [9,13,41]. Active compounds were
evaluated for their cytotoxicity against VERO cells (African green
monkey kidney epithelial cells). VERO cells were grown in RPMI-1640
medium supplemented with 10% fetal bovine serum, 1% L-glutamine,
and 1% penicillin/streptomycin at 37 °C in an atmosphere with 95%
humidified air and 5% CO2. For the assay, cells were seeded in 96-well
plates in a concentration of 2 × 104 cells/well and incubated for 24 h.
After incubation, cells were treated with five concentrations of each
compound tested, including MIC, for 24 h at 37 °C, 5% CO2. Three
controls were carried out: untreated cells as a negative control, vehicle
for solubilization (0.6% DMSO), and 20% DMSO as a positive control.
After incubation, medium was removed and 100 μL of 3-(4,5-di-
methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution
(5 mg/mL) was added and the wells kept in the dark for three hours at
37 °C, 5% CO2. Subsequently, the medium was removed and 100 μL/
well of DMSO were added to solubilize formazan crystals. The reduc-
tion of MTT to formazan, directly proportional to the number of living
cells, was measured on a microplate reader at a wavelength of 492 nm.
Results were expressed as the percentage of viable cells in comparison
with untreated cells. The CC50 was calculated using GraphPad Prism 7.0
Software. The assay was performed at least three independent times in
triplicate. The selectivity index (SI) was calculated as SI = CC50 cells/
IC50 trophozoites. A SI ≥ 10 normally indicates that the biological ef-
ficacy is not a result of in vitro cytotoxicity [13].
2.3. Antiparasitic assay
Anti-T. vaginalis evaluation of curcumin analogues, minimum in-
hibitory concentration (MIC), inhibitory concentration of 50% of
growth (IC50) and kinetic growth assay were performed following the
methodology of Sena-Lopes et al. [9]. Briefly, the anti- T. vaginalis ac-
tivity was evaluated in 96-well microtiter plates, where 2.6 × 105 tro-
phozoites/mL were treated with the curcumin analogues, initially at
100 μM, previously diluted in DMSO. Three controls were applied: a
negative control containing only trophozoites, a 0.6% DMSO control
(vehicle for solubilization) and a positive control containing 100 μM of
metronidazole (MTZ) (Sigma-Aldrich). The microtiter plates were in-
cubated at 37 °C with 5% CO2 (aerobic atmosphere) for 24 h, and after
statistical analysis, only the analogues that did not show significant
statistical difference from the positive control were evaluated in the
next experiments.
The MIC and IC50 values against T. vaginalis were established in the
same conditions described above, with variations on different con-
centrations of the curcumin analogues, which ranged from 6.25 μM to
300 μM. After the MIC was determined, the culture solution from MIC
wells and from the concentrations directly below and above, as well as
controls, were inoculated in fresh TYM medium and re-incubated at
37 °C with 5% CO2. Trophozoites were counted in a Neubauer chamber
every 24 h during 96 h to confirm MIC.
2.5. Statistical analysis
A kinetic growth curve was established to obtain a more accurate
activity profile for the curcumin compounds against T. vaginalis.
Trophozoites viability was observed by light microscopy for 96 h after
incubation at respective MIC, and growth analysis was performed at 1,
6, 12, 24, 48, 72, and 96 h.
Statistical analysis was carried out by one-way analysis of variance
(ANOVA) using a probability value of p < 0.05 using the GraphPad
Prism 5.0 software. Tukey’s test was also conducted to identify sig-
nificant differences between the means of different treatments.
After all assays, a preparation containing trophozoites and trypan
blue (0.4%) dye at a ratio of 1:1, was analyzed under light microscopy
371

C. Carapina da Silva et al.
Biomedicine&Pharmacotherapy111(2019)367–377
3. Results and discussion
the most prominent because all of them induced some percentage of
inhibition of the trophozoites at 100 μM (Fig. 1). The influence of ring
substituent in Series A could be ranked in the sequence (from most to
least active): -H (no substituent) (3a) ≈ -2-Cl (3e) > -4-F (3f) > -4-Cl
(3d) > -4−CH3 (3b) > -4−OCH3 (3c). From Series B (cyclohexanone
derivatives), analogues 5e (-2-Cl) and 5b (-4−CH3) inhibited tropho-
zoites growth. Analogues from Series C (cyclopentanone) and Series D
(derived from cinnamaldehyde) did not show statistical difference from
negative control, that is, they did not reduce trophozoites’ viability at
the tested concentrations.
From these results, it could be inferred that every structural aspect
of the curcumin analogues has an influence on their antiparasitic po-
tential. First, it is worth citing the importance of the length of the hy-
drocarbon linker between aromatic rings, analogue 3a (5-carbon linker)
was the most potent inhibitor of the assessed molecules, while analogue
9a (9-carbon linker) similarly lacking substituents in the aromatic rings
did not inhibit trophozoites’ growth, showing that a shorter linker can
have a more prominent antiparasitic activity. Aguilera et al. reported
similar results, in which both these analogues (3a and 9a) were tested
against the parasite Trypanosoma cruzi and, the lower the length of the
hydrocarbon linker, the higher the antiparasitic activity [39]. Never-
theless, the α-β-unsaturated system plays an important role for the
biological activity of curcumin and its analogues, and other authors
have reported that curcuminoids carrying a conjugated ketone motif
3.1. Chemistry
Firstly, in relation to the synthesis of monocarbonyl analogues of
curcumin, it can be seen from the Tables 1–4 that 21 products were
obtained with a good variety of substituents and in moderate to good
yields. Also, chromatographic monitoring of reactions development
indicated that most of them took less than two hours to be completed,
while others required an average of four hours although there was no
degradation observed if stirring was kept overnight. Analogues derived
from condensation of aromatic aldehydes with acetone within the
Series A were obtained in yields ranging from 40% (3d; -4-Cl) to 74%
(3f; -4-F). Series B analogues derived from cyclohexanone were ob-
tained in 24% (5b; -4−CH3) to 83% (5e; -2-Cl) yields; Series C from
cyclopentanone presented the highest reaction yields [60% for 7b
(4−CH3) to 82% (7d; -4-Cl)], and derivatives of cinnamaldehyde
(Series D) had an overall 49% yield. The aldol condensation is an in-
teresting tool in organic chemistry because it combines two carbonyl
compounds to give rather stable α-β-unsaturated products, especially
condensations between ketones and aromatic aldehydes, such as ben-
zaldehyde, which avoid crossed aldol condensations since benzalde-
hyde does not have any α-hydrogen. The synthetic methodology is re-
latively easier than others as it does not require many work up steps nor
does involve the use of reagents and solvents of high toxicity since the
solvent of choice for the reaction is ethanol [42]. Molecules obtained by
synthetic methodologies also surpass some of the barriers often related
to the use of natural products, such as mixture of compounds and
seasonal quantity variation, low yield and commercial availability
[30,43–45]. In fact, many of the molecules described here are com-
mercially available (e.g. analogues 3a, 3d, 5a), can be obtained in
macroscale under mild conditions [46], and some of them are already
present in commercial products, such as analogue 3a which is used in
sunscreen formulations [47–49].
(enone) particularly contribute for
a higher antiparasitic activity
(against protozoa of the Trypanosoma and Leishmania species) [50,51].
The type and position of the aryl substituents indicated that the pre-
sence of electron-withdrawing groups such as halogens may enhance
antiparasitic activity (3e, 5e), specially at the ortho position of the rings
(3e with chlorine at the ortho position presented higher inhibition than
3d with chlorine at the para position). Another point worth mentioning
is the central structure of the carbonyl group derived from the ketone
reagent: comparing analogues 3e, 5e, and 7e, all containing chlorine as
substituent, it could be seen that analogue 3e with a central open-chain
presented the highest anti-T. vaginalis activity, followed by analogue 5e
with cyclohexanone and 7e with cyclopentanone (no significant ac-
tivity). The presence of the cyclic enone can relatively increase mole-
cular stiffness and lipophilicity of a molecule (higher values of Clog P),
and so, reduce its inhibitory ability against a parasite. These results are
in agreement with the recent reports from Aguilera et al. and Álvarez
et al., who tested some of the same molecules (3a, 5a, 7a, 9a, 9b, 9c)
reported here and others with higher ring sizes (e.g. with cyclohepta-
none). It was observed that these molecules not only were more difficult
to synthesize but were also less biologically active [52]. On the other
hand, similar analogues containing heterocycles derived from fur-
ylaldehyde (O) or thienylaldehyde (S) presented high percentages of
parasite inhibition [39,53]. Finally, it can be seen from Fig. 1 that
natural curcumin (acquired commercially) did not significantly inhibit
T. vaginalis growth at the concentration tested (100 μM). As mentioned
before, natural curcumin is reported to present a wide range of biolo-
gical activities, including antiparasitic activity [18–20,29], despite its
low water solubility and stability, as it can be degraded when exposed
to light and mild room temperature for long periods of time. Wachter
et al. reported the antiparasitic activity of natural curcumin against
three strains of T. vaginalis with different metronidazole susceptibilities
(ATCC 30001, ATCC 30,236 and ATCC 50,138), in which the effective
concentrations (EC50) ranged from 73.0 to 105.8 μg/mL (≈ 198 μM –
287 μM considering the molecular weight of curcumin as 368.38 g/mol)
[22]. Our results are in agreement with the aforementioned report
because, in our assessment, the maximum tested concentration was 100
μM, a concentration that indeed would not completely inhibit the ATCC
30,236 strain. Regardless of the relatively high EC50 concentrations of
natural curcumin, it showed antiparasitic effect against strains with
different metronidazole susceptibilities. Natural curcumin is currently
being tested in some clinical trials in high doses with good tolerance
and no toxicity related observations [16,54].
3.2. Antiparasitic assay
Data analysis obtained from the anti- T. vaginalis assay showed that
curcumin analogues 3a, 3e and 5e were the most effective against T.
vaginalis after 24 h of exposure. Compounds 3a and 3e reduced parasite
viability by 98.8%, and compound 5e reduced 70% when compared
with the negative control (untreated trophozoites), showing absence of
significant statistical difference when compared with the positive con-
trol (MTZ). As expected, the positive control completely reduced
parasite’s viability determined by a lack of motility and positive
staining with trypan blue (0.4%), whereas the negative control and
0.6% DMSO control exhibited motility and negative staining with
trypan blue (0.4%) dye, and did not present a significant statistical
difference when compared with each other. Therefore, this assures that
the vehicle for solubilization did not interfere in the trophozoites’ via-
bility (Fig. 1).
Considering the absence of significant statistical difference of cur-
cumin analogues 3a, 3e and 5e in the anti-T. vaginalis activity assay, the
consecutive experimental procedures were performed using these
compounds. The assay to determinate the MIC and IC50 values against
T. vaginalis established that compounds 3a, 3e and 5e showed optimal
anti-T. vaginalis activity at the concentration of 80 μM, 90 μM and 200
μM, respectively. Both analogues 3a and 3e presented an IC50 value of
50 μM while 5e presented an IC50 value of 70 μM (Fig. 2).
From the kinetic growth curves, it could be determined that ana-
logues 3a and 3e prevented proliferation at the first 12 h of incubation,
displaying a reduction of 95% and 99% in trophozoite’s viability, re-
spectively. Curcumin analogue 5e reduced trophozoite’s viability in
70% after 12 h and reached 100% of reduction 12 h later (Fig. 3).
Taking all synthesized molecules into consideration, it could be seen
that monocarbonyl analogues derived from propanone (Series A) were
372

C. Carapina da Silva et al.
Biomedicine&Pharmacotherapy111(2019)367–377
Fig. 1. Trichomonacidal activity of monocarbonyl curcumin analogues at 100 μM against Trichomonas vaginalis 30236 isolate confirmed by trypan blue dye exclusion
assay (0.4%) after 24 h of exposure. Vehicle for solubilization at 0.6% (DMSO), metronidazole at 100 μM (MTZ), and untreated trophozoites (NC). Data is represented
as mean
standard deviation of at least three independent experiments in triplicate. Different letters indicate significant statistical difference considering
p < 0.05.
3.3. Cytotoxicity assay
compound 3e at any of the tested concentrations (5.625 μM to 500 μM)
(Fig. 4). As expected, 0.6% DMSO did not alter cell growth and the
positive control (20% DMSO) induced 100% of cell mortality. CC50 was
calculated by nonlinear regression and determined to be 34.8 μM and
613.1 μM for 3a and 5e, respectively. Since the highest concentration
tested for compound 3e was not cytotoxic to VERO cells, it was not
possible to calculate a specific CC50 for this compound, but results
showed it is higher than 500 μM (Table 5).
The in vitro cytotoxicity of the active curcumin analogues 3a, 3e and
5e was evaluated through an MTT assay performed using mammalian
VERO cell line. Results showed a cell viability of 2.8%, 100% and
791%, for 3a, 3e and 5e at their respective MIC after 24 h incubation,
showing a high cytotoxic effect for compound 3a and a moderate effect
for 5e at their MIC, while no cytotoxic effect was observed for
Fig. 2. Determination of MIC and IC50 of curcumin analogues 3a and 3e at 40–100 μM, and 5e at 70–300 μM for treatment of Trichomonas vaginalis 30236 isolate in a
24 h exposure period. Vehicle for solubilization at 0.6% (DMSO), metronidazole at 100 μM (MTZ), untreated trophozoites (NC). Data is represented as mean
standard deviation of at least three independent experiments in triplicate. Different letters indicate significant statistical difference considering p < 0.05.
373

C. Carapina da Silva et al.
Biomedicine&Pharmacotherapy111(2019)367–377
Fig. 3. Kinetic growth curve of Trichomonas vaginalis 30236 isolate after treatment with curcumin analogues 3a at 80 μM, 3e at 90 μM, 5e at 200 μM and me-
tronidazole at 100 μM during 1, 6, 12, 24, 48, 72 and 96 h. Data is represented as mean
Different letters indicate significant statistical difference considering p < 0.05.
standard deviation of at least three independent experiments in triplicate.
Fig. 4. Cytotoxicity effect of curcumin analogues 3a, 3e and 5e at different concentrations, including MIC (highlighted), on VERO cells analyzed through MTT assay
after 24 h of exposure. NC (negative control – untreated cells), DMSO (vehicle for solubilization at 0.6%), PC (positive control – 20% DMSO). Data is represented as
mean
standard deviation of at least three independent experiments. Different letters indicate significant statistical difference considering p < 0.05.
Several authors have suggested that a measure called selectivity
(Table 5). Therefore, according to the abovementioned parameters,
compound 3e is the one with the most significant antiparasitic biolo-
gical activity and selectivity for T. vaginalis, being a promising mole-
cule. Nonetheless, although SIs are a good criterion for selecting com-
pounds, they must not be the single parameter for deciding if a
compound should be excluded from biological trials and animal model
assays [57]. Therefore, there are still methodologies that can be applied
index (SI) is an efficient way to analyze antiparasitic biological efficacy
by comparing it with cytotoxicity [13,55,56]. Normally, an index equal
to or greater than 10 (SI = CC50 VERO cells / IC50 T. vaginalis) would be
considered good, because it would indicate that the in vitro antiparasitic
activity is not a result of in vitro cytotoxicity. Our results showed SIs
lower than 10 for 3a (0.7) and 5e (8.8) and higher for 3e (> 10)
374

C. Carapina da Silva et al.
Biomedicine&Pharmacotherapy111(2019)367–377
2.6–98.3 μM), which indicates a broad-spectrum bioactivity of these
Table 5
Anti-Trichomonas vaginalis and mammalian cell effects of monocarbonyl cur-
cumin analogues 3a, 3e and 5e.
compounds [28].
Ohori et al. screened curcumin analogues, which are similar to the
compounds reported here, for anticancer activity (e.g. compounds GO-
Y012 = 3a; GO-Y017 = 3e; GO-Y008 = 5a), also attempting to find
molecules with biological properties similar to the one’s from natural
curcumin. The suggestions of the authors are in agreement with ours in
the sense that the 5-carbon linker of monocarbonyl analogues showed
higher anticancer potential, while the conformational restriction of the
central cyclohexanone (like the compounds of Series B here) attenuated
the biological activity, with IC50 values ranging from 0.25 to over
100 μM. Safety of curcumin analogues was assessed in vitro on normal
human hepatocyte hNHeps cells, and in vivo in mice, which were fed
with the synthetic compounds (0.1% w/w in a 5 g/d high fat diet).
Based on their body weight, no adverse effects were observed for 45
days, showing one more example of safe curcumin analogues applica-
tion [63]. No other research papers with in vivo assays were found
specifically for the 1,5-diaryl-pentadienones in the present work, but
the reports just described indicate a general good safety profile for these
compounds [39,58–61,63]. Our studies are at preliminary stages, but
the antiparasitic potential and cytotoxicity assays revealed at least two
good candidates (3e and 5e) for further in vitro and in vivo assays.
A database search showed no reports of combinations of the com-
pounds described here with standard drugs. The Drug Combination
Database (DCDB, version 2.0) also does not have records on the com-
bination of metronidazole with synthetic compounds approved for
clinical use. However, Rani et al. reported the combination of α,β-un-
saturated carbonyl compounds (including curcumin analogue 3a) and
natural products (neem oil or nicotinic acid) with insecticidal activity
against Phenacoccus solenopsis [64,65]. This year, la Puente et al. re-
ported the combination of Tris (Dibenzylideneacetone) dipalladium
(Tris DBA) with proteasome inhibitors bortezomib or carfilzomib as a
strategy to reverse hypoxia-induced drug resistance in multiple mye-
loma. Tris DBA has the same structure of analogue 3a, only it is com-
plexed with palladium [Puente et al., 2018] [66]. Also, curcumin
analogue 3a is commonly used as an “UV Filter” in photoprotective
compositions in order to prevent skin damage from sun’s ultraviolet
rays. Examples of the combination of 3a (1–40 wt. %) with other
sunscreen agents to be applied on the skin can be found in patents
US20090185991A1 (see “dibenzalacetone” in claims #15 and #23) and
WO2004110366A2 (see “dibenzalacetone” in claims #2 and #13). Our
research group has started to develop projects which include the eva-
luation of synthetic compounds with standard drugs, such as the recent
published work by Saueressig et al. [67], and compounds 3a, 3e and 5e
are in the list of compounds to be tested in possible synergic formula-
tions.
Compounds
MIC (μM)
IC50 (μM)
CC50 (μM)
Selectivity Index
T. vaginalis
T. vaginalis
VERO cells
3a
3e
5e
80
90
200
50
50
70
34.8
> 500
613.1
0.70
> 10
8.8
to explore the biological/antiparasitic potential of analogues 3a and 5e.
A study by Aguilera et al. also evaluated curcumin derivatives with
different carbon chain lengths for their selective trypanosomicidal ac-
tivity, including one compound structurally equal to analogue 3a,
which was determined to have a CC50 of 22 μM against J774.1 mouse
macrophages and a SI of 3 for its activity against T. cruzi, corroborating
with our findings for such compound [39]. In another study, mono-
carbonyl analogues of curcumin had their cytotoxicity evaluated
against VERO cells and, for the ten compounds tested, the CC50 varied
between 3.94 and 16.15 μM with SIs lower than one for the antitumor
activity tested [58]. These studies show similar results to what was
found here for compound 3a and also highlight the importance of the
findings for compounds 3e and 5e, in the sense that these compounds’
structures hold characteristics that lower their cytotoxicity in compar-
ison with other curcumin analogues. These facts can help establishing a
rational parameter for the synthesis of biologically active and selective
molecules.
Alternatively, a study showed two monocarbonyl analogues of
curcumin with low cytotoxicity in macrophages [59] and, another
study showed three curcumin analogues with SIs greater than 30 for
their antitumor activity against oral cancer cells (in comparison with
CC50 for VERO cells) [60]. Such studies demonstrate that curcumin
analogues have a high potential as novel drug candidates, and data
from this study reaffirms that.
Lee et al. analyzed the in vivo activity of curcumin analogues and, in
their work, the molecular mechanism by which dibenzylideneacetone
(DBA), which has the same structure as curcumin analogue 3a in our
study, exerts its antitumoral activity on human mucoepidermoid car-
cinoma (MEC) cell lines was investigated. DBA was administered in vivo
in an athymic nude mouse xenograft model in doses of 20 mg/kg/day
three times per week for 36 days. Authors determined that DBA induced
apoptosis in MEC cells, however in the concentrations tested (up to 15
μM for 48 h), DBA had no significant histopathological effect on the
organs studied when compared with the vehicle, which indicates that
there was no systemic toxicity. These results show that there are safe
concentrations of this compound that can be used for in vivo assays
[61]. In this sense, compounds 3a and 5e could be tested in lower (non
or less cytotoxic) concentrations for their synergic activity with me-
tronidazole or even with other curcumin analogues, as it has been
previously done with other molecules [13,57,62] and still have a sui-
table application as an alternative treatment for trichomoniasis. Ad-
ditionally, Bitencourt et al. showed that half of the MIC (6.25 μM) of a
ursolic acid derivative tested in their study associated with 15 μM of
metronidazole, which alone inhibited only about 25% of T. vaginalis
growth, was able to completely inhibit trophozoites’ growth [62]. Thus,
synergistic associations of compounds can lead to an improved biolo-
gical activity and this could also be applied for analogues 3a and 5e.
Liang et al. evaluated the in vitro stability and in vivo pharmacoki-
netic profiles of curcumin analogues (compounds B34 and C34 in their
study are equal to the structures of analogues 3e and 5e in our study,
respectively) and showed that they exhibited higher peak plasma con-
centrations and higher stability under pH 7.4 phosphate buffer than
natural curcumin, indicating their potential as biologically active mo-
lecules. Also, in this study, compound C34 (= 5e) and B34 (= 3e)
Our research group has been successfully designing synthetic mo-
lecules based on naturally occurring bioactive molecules [67–71] which
demonstrates that the search for optimized molecules through struc-
tural changes is an important approach. Additionally, it would be in-
teresting to determine a possible mechanism of action of the anti-T.
vaginalis activity of these curcumin analogues. In silico studies have
demonstrated that natural curcumin along with other phytochemicals
show low-energy docked poses with T. vaginalis protein purine nu-
cleoside phosphorylase (TvPNP), although natural curcumin also
docked well with human PNP isozyme [72,73]. Alkhaldi et al. estab-
lished that monocarbonyl analogues of curcumin act differently from
their parent compound and from metronidazole by forming an adduct
with trypanothione and depleting thiol levels in trypanosomes from
Trypanosoma brucei [51]. The alternative modes of action from these
molecules suggests a promising anti-T. vaginalis activity and different
mechanisms to be explored when analyzing the analogues presented in
this work.
In conclusion, this study reveals that monocarbonyl analogues of
curcumins 3a, 3e and 5e have a highly significant antiparasitic activity
against T. vaginalis, with 3e showing a great selectivity for the parasite
exhibited cytotoxic activity against five different tumor line cells (IC50
:
375

C. Carapina da Silva et al.
Biomedicine&Pharmacotherapy111(2019)367–377
over normal human cells. Although results for 3a and 5e were not as
promising as the ones for 3e, there are strategies that can be applied to
explore the biological potential of these molecules. Therefore, this
study presents promising molecules for studies on new trichomonacidal
agents.
Am. J. Obstet. Gynecol. 174 (1996) 934–936, https://doi.org/10.1016/S0002-
9378(96)70329-0.
[9] Â. Sena-Lopes, R.N. das Neves, F.S.B. Bezerra, M.T. de Oliveira Silva, P.C. Nobre,
G. Perin, D. Alves, L. Savegnago, K.R. Begnini, F.K. Seixas, T. Collares, S. Borsuk,
Antiparasitic activity of 1,3-dioxolanes containing tellurium in Trichomonas vagi-
nalis, Biomed. Pharmacother. 89 (2017) 284–287, https://doi.org/10.1016/j.
biopha.2017.01.173.
[10] P. Nyirjesy, J.D. Sobel, M.V. Weitz, D.J. Leaman, S.P. Gelone, Difficult-to-treat
trichomoniasis: results with paromomycin cream, Clin. Infect. Dis. 26 (1998)
986–988, https://doi.org/10.1086/513951.
4. Conclusion
[11] S.A. Dingsdag, N. Hunter, Metronidazole: an update on metabolism, structure–-
cytotoxicity and resistance mechanisms, J. Antimicrob. Chemother. 73 (2018)
265–279, https://doi.org/10.1093/jac/dkx351.
[12] R.D. Kirkcaldy, P. Augostini, L.E. Asbel, K.T. Bernstein, R.P. Kerani,
C.J. Mettenbrink, P. Pathela, J.R. Schwebke, W. Evan Secor, K.A. Workowski,
D. Davis, J. Braxton, H.S. Weinstock, Trichomonas vaginalis antimicrobial drug
resistance in 6 US cities, STD surveillance network, 2009-2010, Emerg. Infect. Dis.
18 (2012) 939–943, https://doi.org/10.3201/eid1806.111590.
[13] D.P.G. Hübner, P. de Brum Vieira, A.P. Frasson, C.B. Menezes, F.R. Senger,
G.N. Santos da Silva, S.C. Baggio Gnoatto, T. Tasca, Anti-Trichomonas vaginalis
activity of betulinic acid derivatives, Biomed. Pharmacother. 84 (2016) 476–484,
https://doi.org/10.1016/j.biopha.2016.09.064.
[14] M. Mehriardestani, A. Aliahmadi, T. Toliat, R. Rahimi, Medicinal plants and their
isolated compounds showing anti-Trichomonas vaginalis- activity, Biomed.
Pharmacother. 88 (2017) 885–893, https://doi.org/10.1016/j.biopha.2017.01.
149.
[15] G.V. Rigo, M.R. Trein, D. da Silva Trentin, A.J. Macedo, B.A. de Oliveira, A.M. de
Almeida, R.B. Giordani, M.V. de Almeida, T. Tasca, Diamine derivative anti-
Trichomonas vaginalis and anti-Tritrichomonas foetus activities by effect on poly-
amine metabolism, Biomed. Pharmacother. 95 (2017) 847–855, https://doi.org/10.
1016/j.biopha.2017.09.007.
[16] H. Zhou, C.S. Beevers, S. Huang, The targets of curcumin, Curr. Drug Targets 12
(2011) 332–347, https://doi.org/10.2174/138945011794815356.
[17] S. Prakobwong, S.C. Gupta, J.H. Kim, B. Sung, P. Pinlaor, Y. Hiraku, B. Sripa,
S. Pinlaor, B.B. Aggarwal, Curcumin suppresses proliferation and induces apoptosis
in human biliary cancer cells through modulation of multiple cell signaling path-
ways, Carcinogenesis 32 (2011) 1372–1380, https://doi.org/10.1093/carcin/
bgr032.
[18] S.Z. Moghadamtousi, H.A. Kadir, P. Hassandarvish, H. Tajik, S. Abubakar, K. Zandi,
A review on antibacterial, antiviral, and antifungal activity of curcumin, Biomed
Res. Int. 2014 (2014) 1–12, https://doi.org/10.1155/2014/186864.
[19] Y.M. Liu, Q.Z. Zhang, D.H. Xu, Y.W. Fu, D.J. Lin, S.Y. Zhou, Antiparasitic efficacy of
commercial curcumin against Ichthyophthirius multifiliis in grass carp
(Ctenopharyngodon idellus), Aquaculture. 480 (2017) 65–70, https://doi.org/10.
1016/j.aquaculture.2017.07.041.
[20] E.R. Morais, K.C. Oliveira, L.G. Magalhães, É.B.C. Moreira, S. Verjovski-Almeida,
V. Rodrigues, Effects of curcumin on the parasite Schistosoma mansoni: a tran-
scriptomic approach, Mol. Biochem. Parasitol. 187 (2013) 91–97, https://doi.org/
10.1016/j.molbiopara.2012.11.006.
[21] T. Arablou, R. Kolahdouz-Mohammadi, Curcumin and endometriosis: review on
potential roles and molecular mechanisms, Biomed. Pharmacother. 97 (2018)
91–97, https://doi.org/10.1016/j.biopha.2017.10.119.
[22] B. Wachter, M. Syrowatka, A. Obwaller, J. Walochnik, In vitro efficacy of curcumin
on Trichomonas vaginalis, Wien. Klin. Wochenschr. 126 (2014) 32–36, https://doi.
org/10.1007/s00508-014-0522-8.
[23] D. Shetty, Y.J. Kim, H. Shim, J.P. Snyder, Eliminating the heart from the curcumin
molecule: monocarbonyl curcumin mimics (MACs), Molecules 20 (2015) 249–292,
https://doi.org/10.3390/molecules20010249.
[24] K.M. Nelson, J.L. Dahlin, J. Bisson, J. Graham, G.F. Pauli, M.A. Walters, The es-
sential medicinal chemistry of curcumin, J. Med. Chem. (2017), https://doi.org/10.
1021/acs.jmedchem.6b00975 acs.jmedchem.6b00975.
[25] K.H. Lee, F.H. Farida, A. Syahida, F. Abas, K. Shaari, D.A. Israf, N.H. Lajis, Synthesis
and biological evaluation of curcumin-like diarylpentanoid analogues for anti-in-
flammatory, antioxidant and anti-tyrosinase activities, Eur. J. Med. Chem. 44
(2009) 3195–3200, https://doi.org/10.1016/j.ejmech.2009.03.020.
[26] S. Sudha, N. Sundaraganesan, K. Vanchinathan, K. Muthu, S. Meenakshisundaram,
Spectroscopic (FTIR, FT-Raman, NMR and UV) and molecular structure investiga-
tions of 1,5-diphenylpenta-1,4-dien-3-one: a combined experimental and theore-
tical study, J. Mol. Struct. 1030 (2012) 191–203, https://doi.org/10.1016/j.
molstruc.2012.04.030.
[27] K. Vanchinathan, G. Bhagavannarayana, K. Muthu, S.P. Meenakshisundaram,
Synthesis, crystal growth and characterization of 1,5-diphenylpenta-1,4- dien-3-
one: an organic crystal, Phys. B Condens. Matter. 406 (2011) 4195–4199, https://
doi.org/10.1016/j.physb.2011.07.055.
[28] G. Liang, L. Shao, Y. Wang, C. Zhao, Y. Chu, J. Xiao, Y. Zhao, X. Li, S. Yang,
Exploration and synthesis of curcumin analogues with improved structural stability
both in vitro and in vivo as cytotoxic agents, Bioorg. Med. Chem. 17 (2009)
2623–2631, https://doi.org/10.1016/j.bmc.2008.10.044.
[29] P. Anand, S.G. Thomas, A.B. Kunnumakkara, C. Sundaram, K.B. Harikumar,
B. Sung, S.T. Tharakan, K. Misra, I.K. Priyadarsini, K.N. Rajasekharan,
B.B. Aggarwal, Biological activities of curcumin and its analogues (Congeners)
made by man and Mother Nature, Biochem. Pharmacol. 76 (2008) 1590–1611,
https://doi.org/10.1016/j.bcp.2008.08.008.
From the aforementioned results, it could be concluded that a range
of monocarbonyl curcumin analogues was obtained by reproductive
protocols, in moderate to good yields, with high purity and stability.
From the twenty-one synthetic analogues, two derivatives of propa-
none, namely: 1,5-diphenylpenta-1,4-dien-3-one (3a), and 1,5-bis(2-
chlorophenyl)penta-1,4-dien-3-one (3e); and one cyclohexanone deri-
vative, named 2,6-bis(2-chlorobenzylidene)cyclohexanone (5e) pre-
sented anti-T. vaginalis activity statistically similar to the standard drug
metronidazole at the tested concentrations. Analogue 3e did not show
cytotoxicity on mammalian cells, while 5e and 3a presented moderate
and high levels of cytotoxicity, respectively. The effective concentra-
tions of the designed synthetic analogues were lower than natural
parent compound curcumin. Therefore, it would be interesting to per-
form further studies with this class of compounds and these analogues
should be further explored as possible alternatives for the treatment of
trichomoniasis and other parasitic infections.
Conflict of interest
The authors declare that there is no conflict of interest.
Acknowledgement
The authors are grateful to the Federal University of Pelotas and its
post-graduate programs in Biochemistry and Bioprospection and in
Biotechnology for the opportunity to develop studies and scientific re-
search. We also thank CAPES (Finance Code 001), FAPERGS (17/2551-
0000) and CNPq (310415/2015-2) for the financial support, and all
laboratories involved in the synthesis, identification and biological
evaluation of the molecules.
Appendix A. Supplementary data
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.biopha.2018.12.058.
References
[1] L. Newman, J. Rowley, S. Vander Hoorn, N.S. Wijesooriya, M. Unemo, N. Low,
G. Stevens, S. Gottlieb, J. Kiarie, M. Temmerman, Global estimates of the pre-
valence and incidence of four curable sexually transmitted infections in 2012 based
on systematic review and global reporting, PLoS One 10 (2015) 1–17, https://doi.
org/10.1371/journal.pone.0143304.
[2] World Health Organization, Report on Global Sexually Transmitted Infection
Surveillance 2015, (2015) doi:ISBN 978 92 4 150740 0..
[3] V. Mundodi, A.S. Kucknoor, D.J. Klumpp, T.H. Chang, J.F. Alderete, Silencing the
ap65 gene reduces adherence to vaginal epithelial cells by Trichomonas vaginalis,
Mol. Microbiol. 53 (2004) 1099–1108, https://doi.org/10.1111/j.1365-2958.2004.
04192.x.
[4] P. de B. Vieira, R.B. Giordani, A.J. Macedo, T. Tasca, Natural and synthetic com-
pound anti-Trichomonas vaginalis: an update review, Parasitol. Res. 114 (2015)
1249–1261, https://doi.org/10.1007/s00436-015-4340-3.
[5] A. Shahesmaeili, M. Karamouzian, M. Shokoohi, K. Kamali, N. Fahimfar, S.A. Nadji,
H. Sharifi, A.A. Haghdoost, A. Mirzazadeh, Symptom-based versus laboratory-based
diagnosis of five sexually transmitted infections in female sex workers in Iran, AIDS
Behav. (2018) 1–7, https://doi.org/10.1007/s10461-018-2130-5.
[6] G. Schmid, E. Narcisi, D. Mosure, W.E. Secor, J. Higgins, H. Moreno, Prevalence of
metronidazole-resistant trichomonas vaginalis in a gynecology clinic, Obstet.
Gynecol. Surv. 56 (2001) 693–694.
[7] S.L. Cudmore, K.L. Delgaty, F. Shannon, D.P. Petrin, G.E. Garber, S.F. Hayward-
McClelland, Treatment of infections caused by metronidazole-resistant trichomonas
vaginalis, Clin. Microbiol. Rev. 17 (2004) 783–793, https://doi.org/10.1128/CMR.
17.4.783.
[30] A. Amalraj, A. Pius, S. Gopi, S. Gopi, Biological activities of curcuminoids, other
biomolecules from turmeric and their derivatives – a review, J. Tradit.
Complement. Med. 7 (2017) 205–233, https://doi.org/10.1016/j.jtcme.2016.05.
005.
[31] P.C. Leow, P. Bahety, C.P. Boon, C.Y. Lee, K.L. Tan, T. Yang, P.L.R. Ee,
Functionalized curcumin analogs as potent modulators of the Wnt/beta-catenin
signaling pathway, Eur. J. Med. Chem. 71 (2014) 67–80, https://doi.org/10.1016/
[8] M.D. Pearlman, C. Yashar, S. Ernst, W. Solomon, An incremental dosing protocol for
women with severe vaginal trichomoniasis and adverse reactions to metronidazole,
376

C. Carapina da Silva et al.
Biomedicine&Pharmacotherapy111(2019)367–377
[54] E. Pagano, B. Romano, A.A. Izzo, F. Borrelli, The clinical efficacy of curcumin-
containing nutraceuticals: An overview of systematic reviews, Pharmacol. Res. 134
(2018) 79–91, https://doi.org/10.1016/j.phrs.2018.06.007.
[55] G. Navarrete-Vázquez, F. Chávez-Silva, B. Colín-Lozano, S. Estrada-Soto, S. Hidalgo-
Figueroa, J. Guerrero-Álvarez, S.T. Méndez, H. Reyes-Vivas, J. Oria-Hernández,
J. Canul-Canché, R. Ortiz-Andrade, R. Moo-Puc, Synthesis of nitro(benzo)thiazole
acetamides and in vitro antiprotozoal effect against amitochondriate parasites
Giardia intestinalis and Trichomonas vaginalis, Bioorg. Med. Chem. 23 (2015)
2204–2210, https://doi.org/10.1016/j.bmc.2015.02.059.
[56] C. Nava-Zuazo, S. Estrada-Soto, J. Guerrero-Álvarez, I. León-Rivera, G.M. Molina-
Salinas, S. Said-Fernández, M.J. Chan-Bacab, R. Cedillo-Rivera, R. Moo-Puc,
G. Mirón-López, G. Navarrete-Vazquez, Design, synthesis, and in vitro anti-
protozoal, antimycobacterial activities of N-{2-[(7-chloroquinolin-4-yl)amino]
ethyl}ureas, Bioorg. Med. Chem. 18 (2010) 6398–6403, https://doi.org/10.1016/j.
bmc.2010.07.008.
[57] P. de B. Vieira, N.L.F. Silva, G.N.S. da Silva, D.B. Silva, N.P. Lopes, S.C.B. Gnoatto,
M.V. da Silva, A.J. Macedo, J. Bastida, T. Tasca, Caatinga plants: natural and semi-
synthetic compounds potentially active against Trichomonas vaginalis, Bioorg.
Med. Chem. Lett. 26 (2016) 2229–2236, https://doi.org/10.1016/j.bmcl.2016.03.
061.
[58] P.W. Prasetyaningrum, A. Bahtiar, H. Hayun, Synthesis and cytotoxicity evaluation
of novel asymmetrical mono-carbonyl analogs of curcumin (AMACs) against vero,
HeLa, and MCF7 cell lines, Sci. Pharm. 86 (2018), https://doi.org/10.3390/
scipharm86020025.
[59] C. Zhao, Y. Cai, X. He, J. Li, L. Zhang, J. Wu, Y. Zhao, S. Yang, X. Li, W. Li, G. Liang,
Synthesis and anti-inflammatory evaluation of novel mono-carbonyl analogues of
curcumin in LPS-stimulated RAW 264.7 macrophages, Eur. J. Med. Chem. 45
(2010) 5773–5780, https://doi.org/10.1016/j.ejmech.2010.09.037.
[60] T. Chuprajob, C. Changtam, R. Chokchaisiri, W. Chunglok, N. Sornkaew,
A. Suksamrarn, Synthesis, cytotoxicity against human oral cancer KB cells and
structure-activity relationship studies of trienone analogues of curcuminoids,
Bioorg. Med. Chem. Lett. 24 (2014) 2839–2844, https://doi.org/10.1016/j.bmcl.
2014.04.105.
j.ejmech.2013.10.073.
[32] W.M. Weber, L.A. Hunsaker, S.F. Abcouwer, L.M. Deck, D.L. Vander Jagt, Anti-
oxidant activities of curcumin and related enones, Bioorg. Med. Chem. Lett. 13
(2005) 3811–3820, https://doi.org/10.1016/j.bmc.2005.03.035.
[33] W.M. Weber, L.A. Hunsaker, C.N. Roybal, E.V. Bobrovnikova-Marjon,
S.F. Abcouwer, R.E. Royer, L.M. Deck, D.L. Vander Jagt, Activation of NFkB is in-
hibited by curcumin and related enones, Bioorg. Med. Chem. Lett. 14 (2006)
2450–2461, https://doi.org/10.1016/j.bmc.2005.11.035.
[34] M.A. Bigdeli, G.H. Mahdavinia, S. Jafari, H. Hazarkhani, Wet 2,4,6-trichloro[1,3,5]
triazine (TCT) an efficient catalyst for synthesis of α, α′-bis(substituted-benzyli-
dene) cycloalkanones under solvent-free conditions, Catal. Commun. 8 (2007)
2229–2231, https://doi.org/10.1016/j.catcom.2007.05.010.
[35] Y.W. Zhu, W. Bin Yi, C. Cai, A recyclable fluoroalkylated 1,4-disubstituted [1,2,3]-
triazole organocatalyst for aldol condensation of aldehydes and ketones, J. Fluor.
Chem. 132 (2011) 71–74, https://doi.org/10.1016/j.jfluchem.2010.11.004.
[36] A. Hasaninejad, A. Zare, L. Balooty, H. Mehregan, M. Shekouhy, Solvent-free, cross-
aldol condensation reaction using silica-supported, phosphorus-containing reagents
leading to α,α′-Bis(arylidene) cycloalkanones, Synth. Commun. 40 (2010)
3488–3495, https://doi.org/10.1080/00397910903457282.
[37] G. Liang, S. Yang, L. Jiang, Y. Zhao, L. Shao, J. Xiao, F. Ye, Y. Li, X. Li, Synthesis and
anti-bacterial properties of mono-carbonyl analogues of curcumin, Chem. Pharm.
Bull. (Tokyo) 56 (2008) 162–167, https://doi.org/10.1248/cpb.56.162.
[38] M. Zheng, L. Wang, J. Shao, Q. Zhong, A facile synthesis of α,α’-bis(substituted
benzylidene)cycloalkanones catalyzed by bis(p-ethoxyphenyl)telluroxide(BMPTO)
under microwave irradiation, Synth. Commun. 27 (1997) 351–354, https://doi.
org/10.1080/00397919708005039.
[39] E. Aguilera, J. Varela, E. Birriel, E. Serna, S. Torres, G. Yaluff, N.V. de Bilbao,
B. Aguirre-López, N. Cabrera, S. Díaz Mazariegos, M.T. de Gómez-Puyou, A. Gómez-
Puyou, R. Pérez-Montfort, L. Minini, A. Merlino, H. Cerecetto, M. González,
G. Alvarez, Potent and selective inhibitors of Trypanosoma cruzi triosephosphate
isomerase with concomitant inhibition of cruzipain: inhibition of parasite growth
through multitarget activity, ChemMedChem 11 (2016) 1328–1338, https://doi.
org/10.1002/cmdc.201500385.
[40] L.S. Diamond, The establishment of various trichomonads of animals and man in
axenic cultures, J. Parasitol. 43 (1957) 488–490, https://doi.org/10.2307/
3274682.
[41] S. Mashjoor, M. Yousefzadi, M.A. Esmaeili, R. Rafiee, Cytotoxicity and anti-
microbial activity of marine macro algae (Dictyotaceae and Ulvaceae) from the
Persian Gulf, Cytotechnology 68 (2016) 1717–1726, https://doi.org/10.1007/
s10616-015-9921-6.
[42] G.B. Hill, J.B. Sweeney, Reaction workup planning: a structured flowchart ap-
proach, exemplified in difficult aqueous workup of hydrophilic products, J. Chem.
Educ. 92 (2015) 488–496, https://doi.org/10.1021/ed500580p.
[43] D. Angelopoulou, C. Demetzos, D. Perdetzoglou, Diurnal and seasonal variation of
the essential oil labdanes and clerodanes from Cistus monspeliensis L. leaves,
Biochem. Syst. Ecol. 30 (2002) 189–203, https://doi.org/10.1016/S0305-1978(01)
00074-6.
[61] H.E. Lee, E.S. Choi, J.Y. Jung, M.J. You, L.H. Kim, S.D. Cho, Inhibition of specificity
protein 1 by dibenzylideneacetone, a curcumin analogue, induces apoptosis in
mucoepidermoid carcinomas and tumor xenografts through Bim and truncated Bid,
Oral Oncol. 50 (2014) 189–195, https://doi.org/10.1016/j.oraloncology.2013.11.
006.
[62] F.G. Bitencourt, P.D.B. Vieira, L.C. Meirelles, G.V. Rigo, Anti- Trichomonas vagi-
nalis activity of ursolic acid derivative : a promising alternative, Parasitol. Res.
(2018) 1573–1580.
[63] H. Ohori, H. Yamakoshi, M. Tomizawa, M. Shibuya, Y. Kakudo, A. Takahashi,
S. Takahashi, S. Kato, T. Suzuki, C. Ishioka, Y. Iwabuchi, H. Shibata, Synthesis and
biological analysis of new curcumin analogues bearing an enhanced potential for
the medicinal treatment of cancer, Mol. Cancer Ther. 5 (2006) 2563–2571, https://
doi.org/10.1158/1535-7163.MCT-06-0174.
[64] A. Rani, S. Jain, P. Dureja, P.K. Tripathi, K. Singh, Combination of synthetic and
natural products as pesticides (CSYNAP): a new class of antifungal agents, Res.
Commun. Mol. Pathol. Pharmacol. 120–121 (2007) 15–21 (accessed September 24,
2018), http://www.ncbi.nlm.nih.gov/pubmed/21469500.
[44] T. Siatka, M. Kašparová, Seasonal variation in total phenolic and flavonoid contents
and DPPH scavenging activity of Bellis perennis L. flowers, Molecules. 15 (2010)
9450–9461, https://doi.org/10.3390/molecules15129450.
[45] C. Monton, L. Charoenchai, J. Suksaeree, L. Sueree, Quantitation of curcuminoid
contents, dissolution profile, and volatile oil content of turmeric capsules produced
at some secondary government hospitals, J. Food Drug Anal. 24 (2016) 493–499,
https://doi.org/10.1016/j.jfda.2016.01.007.
[46] R.C.C. Duarte, M.G.T.C. Ribeiro, A.A.S.C. Machado, Reaction scale and green
chemistry: microscale or macroscale, which is greener? J. Chem. Educ. 94 (2017)
1255–1264, https://doi.org/10.1021/acs.jchemed.7b00056.
[47] P. Aline, E. Dias, E. Vinícius, V. Varejão, V.B. Martins, A. Lúcia, T. Gois, M.A. De
Resende-stoianoff, J.E. De Carvalho, Â. De Fátima, S.A. Fernandes, Antiproliferative
and Antifungal Activities of 1, 3-Diarylpropane-1, 3-Diones Commonly Used as
Sunscreen Agents 1789 (2013), pp. 661–665.
[48] S. Handayani, Synthesis and activity test of Two asymmetric dibenzalacetone as
potential sunscreen material, in: K. Li (Ed.), Chem. Biol. Environ. Eng. Proc. Int.
Conf. CBEE 2009 (2009) 119–122, https://doi.org/10.1142/9789814295048_0024.
[49] L.A. Huck, W.J. Leigh, A better sunscreen: structural effects on spectral properties,
J. Chem. Educ. 87 (2010) 10–13.
[65] A. Rani, S. Jain, R. Gautam, Investigation of insecticidal activity of some α,β-un-
saturated carbonyl compounds and their synergistic combination with natural
products against phenacoccus solenopsis tinsley, J. Plant Prot. Res. 52 (2012)
146–155, https://doi.org/10.2478/v10045-012-0023-6.
[66] P. de la Puente, F. Azab, B. Muz, M. Luderer, J. Arbiser, A.K. Azab, Tris DBA pal-
ladium overcomes hypoxia-mediated drug resistance in multiple myeloma, Leuk.
Lymphoma 57 (2016) 1677–1686, https://doi.org/10.3109/10428194.2015.
1099645.
[67] S. Saueressig, J. Tessmann, R. Mastelari, L.P. da Silva, J. Buss, N.V. Segatto,
K.R. Begnini, B. Pacheco, C.M.P. de Pereira, T. Collares, F.K. Seixas, Synergistic
effect of pyrazoles derivatives and doxorubicin in claudin-low breast cancer sub-
type, Biomed. Pharmacother. 98 (2018) 390–398, https://doi.org/10.1016/j.
biopha.2017.12.062.
[68] M. Ritter, R.M. Martins, S.A. Rosa, J.L. Malavolta, R.G. Lund, A.F.C. Flores,
C.M.P. Pereira, Green synthesis of Chalcones and microbiological evaluation, J.
Braz. Chem. Soc. 26 (2015) 1201–1210.
[50] C. Changtam, H.P. de Koning, H. Ibrahim, M.S. Sajid, M.K. Gould, A. Suksamrarn,
Curcuminoid analogs with potent activity against Trypanosoma and Leishmania
species, Eur. J. Med. Chem. 45 (2010) 941–956, https://doi.org/10.1016/j.ejmech.
2009.11.035.
[51] A.A.M. Alkhaldi, D.J. Creek, H. Ibrahim, D.-H. Kim, N.B. Quashie, K.E. Burgess,
C. Changtam, M.P. Barrett, A. Suksamrarn, H.P. de Koning, Potent trypanocidal
curcumin analogs bearing a monoenone linker motif act on trypanosoma brucei by
forming an adduct with trypanothione, Mol. Pharmacol. 87 (2015) 451–464,
https://doi.org/10.1124/mol.114.096016.
[52] R.J. Cremlyn, M.J. Frearson, S. Graham, The synthesis and chlorosulfonation of
some diarylidene and heteroarylidene ketones with varying alicyclic ring size,
Phosphorus Sulfur Silicon Relat. Elem. 107 (1995) 205–217, https://doi.org/10.
1080/10426509508027936.
[53] G. Álvarez, C. Perdomo, C. Coronel, E. Aguilera, J. Varela, G. Aparicio, F.R. Zolessi,
N. Cabrera, C. Vega, M. Rolón, A.R. De Arias, R. Pérez-Montfort, H. Cerecetto,
M. González, Multi-anti-parasitic activity of arylidene ketones and thiazolidene
hydrazines against Trypanosoma cruzi and Leishmania spp, Molecules 22 (2017)
1–26, https://doi.org/10.3390/molecules22050709.
[69] R.M. Martins, M.D.A. Farias, F. Nedel, C.M.P. de Pereira, C. Lencina, R.G. Lund,
Antimicrobial and cytotoxic evaluation of eugenol derivatives, Med. Chem. Res. 25
(2016) 2360–2367, https://doi.org/10.1007/s00044-016-1682-z.
[70] T.B. Fogaça, R.M. Martins, K.R. Begnini, C. Carapina, M. Ritter, C.M.P. de Pereira,
F.K. Seixas, T. Collares, Apoptotic effect of chalcone derivatives of 2-acet-
ylthiophene in human breast cancer cells, Pharmacol. Rep. 69 (2017) 156–161,
https://doi.org/10.1016/j.pharep.2016.10.003.
[71] J.W. Tessmann, J. Buss, K.R. Begnini, L.M. Berneira, F.R. Paula, C.M.P. de Pereira,
T. Collares, F.K. Seixas, Antitumor potential of 1-thiocarbamoyl-3,5-diaryl-4,5-di-
hydro-1H-pyrazoles in human bladder cancer cells, Biomed. Pharmacother. 94
(2017) 37–46, https://doi.org/10.1016/j.biopha.2017.07.060.
[72] M.S. Setzer, K.G. Byler, I.V. Ogungbe, W.N. Setzer, Natural products as new treat-
ment options for trichomoniasis: a molecular docking investigation, Sci. Pharm. 85
(2017), https://doi.org/10.3390/scipharm85010005.
[73] V. Zannis, D. Doyle, D.W.J. Martin, Purification and characterization of human
erythrocyte purine nucleoside phosphorylase and its subunits, J. Biol. Chem. 253
(1978) 504–510.
377